Pleuromutilin derivatives as antimicrobials

The present invention relates to pleuromutilin derivatives, to processes for their preparation, to pharmaceutical compositions containing them and to their use in medical therapy, particularly antibacterial therapy.

CROSS REFERENCES TO RELATED APPLICATIONS

This application is a reissue application of U.S. Pat. No.6,281,226, granted on Aug.28,2001; which is a national phase entry under371of International Application No. PCT/GB98/03211, filed Apr.27,2000; which claims the benefit of priority of Great Britain Application Nos.9722817, filed Oct.29,1997and9813689, filed Jun.25,1998.

The present invention relates to novel compounds, to processes for their preparation, to pharmaceutical compositions containing them and to their use in medical therapy, particularly antibacterial therapy.

Pleuromutilin, the compound of formula (A), is a naturally occurring antibiotic which has antimycoplasmal activity and modest antibacterial activity. It has been shown that the antimicrobial activity can be improved by replacing the glycolic ester moiety at position 14 by an R—X—CH2CO2— group, where R is an aliphatic or aromatic moiety and X is O, S, or NR′ (H Egger and H Reinshagen, J Antibiotics, 1976, 29, 923). Tiamulin, the compound of formula (B), which is used as a veterinary antibiotic, is a derivative of this type (G Hogenauer in Antibiotics, Vol. V, part 1, ed. F E Hahn, Springer-Verlag, 1979, p.344).
In this application, the non-conventional numbering system which is generally used in the literature (G Hogenauer, loc.cit.) is used.

WO 97/25309 (SmithKline Beecham) describes further modification of the acyloxy group, disclosing 14-O-carbamoyl derivatives of mutilin or 19, 20-dihydromutilin, in which the N-atom of the carbamoyl group is unsubstituted, mono- or di-substituted.

WO 98/05659 (SmithKline Beecham) discloses 14-O-carbamoyl derivatives of mutilin or 19, 20-dihydromutilin, in which the N-atom of the carbamoyl group is acylated by a group which includes an azabicyclic moiety.

We have now found that further novel pleuromutilin derivatives have improved antimicrobial properties.

Accordingly, the present invention provides a compound of general formula (IA) or (EB):
in which:each of n and m is independently 0, 1 or 2;X is selected from —O—, —S—, —S(O)—, —SO2—, —CO.O—, —NH—, —CONH—, —NHCONH— and a bond;R1is vinyl or ethyl;R2is a non-aromatic monocyclic or bicyclic group containing one or two basic nitrogen atoms and attached through a ring carbon atom;R3is H or OH; or the moiety R2(CH2))mX(CH2)nCOO at position 14 of (IA) or (IB) is replaced by RaRbC═CHCOO in which one of Raand Rbis hydrogen and the other is R2or Raand Rbtogether form R2, or
a pharmaceutically acceptable salt thereof.

When R2is monocyclic, it typically contains from 4 to 8 ring atoms, and, when bicyclic, it typically contains from 5 to 10 ring atoms in each ring, and is optionally substituted on carbon by up to 3 substituents. Suitable substituents include alkyl, alkyloxy, alkenyl and alkenyloxy, each of which may be carried by either a bridgehead or a non-bridgehead carbon atom. In addition, the or each nitrogen atom may be substituted by oxygen, to form an N-oxide, or by mono- or dialkyl, in which case it will be appreciated that a quaternary cation can be formed. The counterion may be a halide ion such as chloride or bromide, preferably chloride. The aza ring system additionally may contain one or more double bonds.

The compounds of formula (IA) in which R3is hydroxy have the (2S) configuration at the carbon bearing this hydroxy group.

Preferably, n is 0. Preferably, m is 0 or 1.

Preferred compounds are those of formula (IA).

Alkyl and alkenyl groups referred to herein include straight and branched groups containing up to six carbon atoms and are optionally substituted by one or more groups selected from the group consisting of aryl, heterocyclyl, (C1-6)alkoxy, (C1-6)alkylthio, aryl(C1-6)alkoxy, aryl(C1-6)alkylthio, amino, mono- or di-(C1-6)alkylamino, cycloalkyl, cycloalkenyl, carboxy and esters thereof, amides of carboxy, ureido, carbamimidoyl (amidino), guanidino, alkyl-sulfonyl, amino-sulfonyl (C1-6)acyloxy, (C1-6)acylamino, azido, hydroxy, and halogen.

Cycloalkyl and cycloalkenyl groups referred to herein include groups having from three to eight ring carbon atoms and are optionally substituted as described hereinabove for alkyl and alkenyl groups.

When used herein, the term “aryl” means single and fused rings suitably containing from 4 to 7, preferably 5 or 6, ring atoms in each ring, which rings may each be unsubstituted or substituted by, for example, up to three substituents. A fused ring system may include aliphatic rings and need include only one aromatic ring. Representative aryl groups include phenyl and naphthyl such as 1-naphthyl or 2-naphthyl.

When used herein the terms “heterocyclyl” and “heterocyclic” suitably include, unless otherwise defined, aromatic and non-aromatic, single and fused, rings suitably containing up to four heteroatoms in each ring, each of which is selected from oxygen, nitrogen and sulphur, which rings, may be unsubstituted or substituted by, for example, up to three substituents. Each heterocyclic ring suitably has from 4 to 7, preferably 5 or 6, ring atoms. A fused heterocyclic ring system may include carbocyclic rings and need include only one heterocyclic ring.

Depending on the position of attachment of substituents, two or more diastereoisomers may be possible. In that situation the present invention includes the individual diastereoisomers and mixtures thereof.

Preferred examples of compounds of the invention include:Mutilin 14-(quinuclidin-4-yl-sulfanyl)-acetate;Mutilin 14-(quinuclid-4-ylmethylsulfanyl)-acetate;Mutilin-14-(1-methylpiperid-4-ylsulfanyl)-acetate, andMutilin 14-(exo-8-methyl-8-azabicyclo[3.2.1]oct-3-ylsulfanyl)-acetate.

The compounds of this invention may be in crystalline or non-crystalline form, and, if crystalline, may optionally be solvated, especially hydrated. This invention includes within its scope stoichiometric hydrates as well as compounds containing variable amounts of water.

The compounds according to the invention are suitably provided in substantially pure form, for example at least 50% pure, suitable at least 60% pure, advantageously at least 75% pure, preferably at least 85% pure, more preferably at least 95% pure, especially at least 98% pure, all percentages being calculated as weight/weight.

The compounds of the invention may be in the form of free bases or acid addition salts. Compounds carrying a carboxy substituent may be in the form of zwitterions, or alkali metal salts (of the carboxy group). Pharmaceutically acceptable salts are preferred.

Compounds of the present invention may be readily prepared form available starting materials by adapting synthetic processes well known in the art.

Accordingly, in a first aspect, the present invention provides a process for preapring a compound of formula (I) which comprises reacting a compound of formula (IIA) or (IIB):
in which Y is hydrogen or a removable hydroxy-protecting group, and R1Aand R3Aare R1and R3are as defined for formulae (IA) and (IB) or groups convertible to R1and R3,with an active derivative of a carboxylic acid of formula (III):
R2A—(CH2)m—X—(CH2))n—CH2CO2)H  (III)
where R2Ais R2as defined for formulae IA and IB or a group convertible to R2, under ester forming conditions and, where required or desired,converting Y to hydrogen,converting an R1A, R2Aor R3Agroup to a R1, R2or R3group, and/orconverting one R1, R2or R3group to another R1, R2or R3group.

Conventional methods for ester formation are described in the literature, for example in Comprehensive Organic Functional Group Transformations, Vol. 5, ed. C J Moody, p. 123-130, Elsevier Scientific, Oxford, 1995. The active derivative used as an acylating agent may be for example an acid chloride, acid bromide, a mixed anhydride, or an N-acyl-imidazole. The preferred agent is an acid chloride. General methods for forming such acylating agents are described in the chemical literature (see I O Sutherland, Comprehensive Organic Chemistry, Vol. 2, ed. I O Sutherland, pages 875-883 (Pergamon Press, Oxford, 1979), and references therein).

The ester-forming reaction can be carried out in the presence of an organic base, an inorganic base, or an acid. Organic bases include pyridine, 2,6-lutidine, triethylamine, and N,N-dimethylaniline. Inorganic bases include sodium hydride, lithium hydride, potassium carbonate, lithium hexamethyldisilazide, and sodium hexamethyldisilazide. Acids include p-toluenesulphonic acid, benzene sulphonic acid, and sulphuric acid. Optionally, when the reaction is carried out in the presence of a base, an acylation catalyst (G Hofle and W Steglich, Synthesis, 1972, 619) such as 4-dimethyamino-pyridine or 4-pyrrolidino-pyridine may also be added to the reaction mixture. Solvents for the ester forming reaction include tetrahydrofuran, 1,4-dioxane, acetonitrile, N,N-dimethylformamide, diethyl ether, dichloromethane, and chloroform. A preferred solvent is tetrahydrofuran.

Useful methods for acylating the 14-hydroxyl in the present invention include the use of the following: acid chloride in N,N-dimethylformamide at elevated temperature (e.g. 100° C. to 120° C.), acid chloride in the presence of an organic base (e.g. pyridine, 2,6-lutidine, 2,4,6-collidine, di-iso-propylethylamine) or an inorganic base (e.g. sodium or lithium hexamethyldisilazide); carboxylic acid in the presence of dicyclohexylcarbodiimide and an acylation catalyst (e.g. 4-dimethylamino-pyridine, 4-pyrrolidino-pyridine); a mutilin 14-chloroformate derivative plus carboxylic acid, tertiary base (e.g. triethylamine, di-iso-propylethylamine), and an acylation catalyst (e.g. 4-dimethylamino-pyridine, 4-pyrrolidino-pyridine).

Conversions of an R1A, R2Aor R3Agroup to a R1, R2or R3group typically arise when a protecting group is needed during the above coupling reaction or during the preparation of the reactants by the procedures described below. Interconversion of one R1, R2or R3group to another typically arises when one compound of formula IA/B is used as the immediate precursor of another compound of formula IA/B or when it is easier to introduce a more complex or reactive substituent at the end of a synthetic sequence.

Preferably Y is a hydroxyl protecting group such as an acyl group, for example so that —OY is trifluoroacetyl or dichloroacetyl. When the intended R3is also hydroxyl, then R3Ais also preferably acyloxy, for example acetyl or dichloroacetyl. Hydroxyl groups at positions 11 and 2 (as groups OY and R3A) may be protected using, for example, dichloroacetic anhydride and pyridine in tetrahydrofuran or N-trifluoroacetyl-imidazole in tetrahydrofuran at 0° C. After the reaction with the derivative of acid III is complete the protecting acyl groups may be removed to restore the hydroxyl groups by hydrolysis e.g. using NaOH in MeOH.

It may also be necessary to protect substituent groups in the acid component (III) prior to reaction with the the compound of formulae (IIA) or (IIB), for example protecting N atoms with alkoxycarbonyl, for example t-butoxycarbonyl.

Suitable hydroxy, carboxy and amino protecting groups are those well known in the art and which may be removed under conventional conditions and without disrupting the remainder of the molecule. A comprehensive discussion of the ways in which hydroxy, carboxy and amino groups may be protected and methods for cleaving the resulting protected derivatives is given in for example “Protective Groups in Organic Chemistry” (T. W. Greene, Wiley-Interscience, New York, 2nd edition, 1991). Particularly suitable hydroxy protecting groups include, for example, triorganosilyl groups such as, for instance, trialkylsilyl and also organocarbonyl and organooxycarbonyl groups such as, for instance, acetyl, allyloxycarbonyl, 4-methoxybenzyloxycarbonyl and 4-nitrobenzyloxycarbonyl. Particularly suitable carboxy protecting groups include alkyl and aryl groups, for instance methyl, ethyl and phenyl. Particularly suitable amino protecting groups include alkoxycarbonyl, 4-methoxybenzyloxycarbonyl and 4-nitrobenzyloxycarbonyl.

R1Ais typically the R1group vinyl, and this may be converted to the alternative R1ethyl group by hydrogenating the vinyl group to form an ethyl group, typically by hydrogenation over a palladium catalyst (e.g. 10% palladium-on-carbon) in a solvent such as ethyl acetate, ethanol, dioxane, or tetrahydrofuran.

R3Ais typically hydrogen or protected hydroxyl, such as acyloxy. After the coupling reaction, protecting acyl groups may be removed to restore the hydroxyl groups by hydrolysis e.g. using NaOH in MeOH.

Alternatively a compound of formula (IA) in which R3is hydrogen may be prepared by treating a compound of formula (IIC):
where R1Ais as defined for formula (IIA) and (IIB),with an active derivative of the acid of formula (III) under ester forming conditions, andthen treating the product with an acid, and, where required or desired,converting an R1Aor R2Agroup to a R1or R2group, and/orconverting one R1or R2group to another R1or R2group.

The acid treatment indicated above converts the epi-mutilin configuration of formula (IIC) to the usual mutilin nucleus of formula (IIA). Typically this conversion is carried out by treatment with conc. HCl or Lukas reagent (conc. HCl saturated with ZnCl2) in dioxane.

As in formulae (IIA) and (IIB), R2Ais typically the R2group vinyl, and this may be converted to the alternative R2group by hydrogenating the vinyl group to form an ethyl group. Also it may again be necessary to protect substituent groups in the derivative of acid compound (III) prior to reaction, for example protecting N atoms with , for example, t-butoxycarbonyl.

In cases where the intermediate of formula (IIA) and (IIB) (such as Y=acetyl) are used, a base-labile protecting group may conveniently be removed at the same time as the group Y is deprotected. In cases when the intermediate of formula (IIC) is used, an acid-labile protecting group may conveniently be removed at the same time as the acid treatment that converts the epi-mutilin configuration into the desired configuration of the compounds of the invention.

Compounds (IV) and (V) are effectively the compounds of formula (IIA) and (IIC) respectively in which R1Ais vinyl and R3Ais hydrogen (compound IIA). They may be converted into the corresponding compounds in which R1Ais ethyl by hydrogenation, typically by hydrogenation over a palladium catalyst (e.g. 10% palladium-on-carbon) in a solvent such as ethyl acetate, ethanol, dioxane, or tetrahydrofuran.

Copmpounds of formula (IIA) in which R3Ais hydroxyl may be obtained by first preparing 2-hydroxymethylene mutilin from a compound of formula (IV). Using procedures based on that described by A. J. Birch, C. W. Holzapfel and R. W. Rickards (Tet (Suppl) 1996 8 part III 359), a compound of formula (IV) in toluene and methyl formate is treated with sodium methoxide and stirred under argon. The product is a mixture of the desired 2-hydroxymethylene compound and corresponding compounds substituted by formate at position 11 (if OY is OH) and/or position 14. The formate groups may be removed when desired by treatment with potassium hydroxide in methanol.

The product mixture may however be used directly to prepare 2-diazo-mutilin derivatives using the method described by H Berner, G Schulz, and G Fisher, Monatsh. Chem., 1981, 112, 1441, for example reacting a solution of a 2-hydroxymethylene-mutilin and the formate derivatives in dichloromethane at −10° C. under argon with tosylazide and triethylamine. Removal of the formate groups as described above leaves 2-diazo-mutilin. which may be reacted with a carboxylic acid to give a 2-acyloxy-mutilin, effectively a compound of formula (IIA) in which R3Ais protected hydroxyl. Suitably reaction with dichloroacetic acid gives 2-dichloroacetoxy-mutilin, which can be deprotected as described above to provide 2-OH. preferably after coupling with the derivative of acid (III). This reaction produces (2S)-2-hydroxy derivatives.

Compounds of formula (IIB) are either 1,2-didehydro-mutilin or obtainable therefrom by manipulation of OY and R1Aas described above. 1,2-Didehydro-mutilins may be prepared using the method described by G Schulz and H Berner in Tetrahedron, 7, 1984, 40, 905.

The above described modifications to the mutilin nucleus may also be carried out after coupling of compounds of formula (IIA) and (IIC) where R3Ais hydrogen (i.e. based on mutilin an d epi-mutilin) with the active derivative of acid (III).

In another aspect, the present invention provides a method for preparing compounds of the invention in which X is O, S, NH, CO.O or CONH which comprises reacting a compound of formula VIA o r VIB
where Y is hydrogzen or a removable hydroxy-protecting, group. and R1Aand R3Aare R1and R3as defined for formulae IA and IB or groups convertible to R1and R3, n is as defined for formulae IA and IB, and RLis a leaving group or OH or NH2, with a compound of formula (VII):
R2A—(CH2)m—XH  (VII)
where R2Ais R2as defined for formula (IA) and (IB) or a group convegible to R2, and X and m are as defined for formulae IA and IB, orwhen X is CO.O with an active derivative of the acid of formula (VII),by one of the procedures set out below,and where required or desiredconverting Y to hydrogen,converting an R1Aor R3Agroup to an R1, R2or R3group, and/orconverting one R1, R2or R3group to another R1, R2or R3group.

As in the method discussed above startin from compounds (IIA/B/C), preferably Y is a hydroxyl protecting group such as an acyl group, for example so that —OY is trifluoroacetyl or dichloroacetyl. When the intended R3is also hydroxyl then R3Ais also preferably acyloxy, for example acetyl or dichloroacetyl.

It may also be necessary to protect substituent groups in the compound of formula (VII) prior to reaction with the compound (VIA) or (VIB), for example protecting N atoms with alkoxycarbonyl, for example t-butoxycarbonyl.

Suitable hydroxy, carboxy and amino protecting croups are those well known in the art and are discussed above.

R1Ais typically the R1group vinyl, and this may be converted to the alternative R1ethyl group by hydrogenating the vinyl group to form an ethyl group, typically by hydrogenation over a palladium catalyst (e.g. 10% palladium-on-carbon) in a solvent such as ethyl acetate, ethanol, dioxane, or tetrahydrofuran.

R3Ais typically hydrogen or protected hydroxyl, such as acyloxy. After the coupling reaction, protecting acyl groups may be removed to restore the hydroxyl groups by hydrolysis e.g. using NaOH in MeOH.

Procedures for coupling the group RL(CH2)nCH2CO.O— with compound R2A—(CH2)m—XH include the following:(a) when RLis a leaving group, such as 4-MeC6H4SO2O, MeSO2O, F3CSO2O, Br or Cl, and X is O, S or NH:(i) where X=O, the alcohol R2—(CH2)m—OH may be converted into the alkoxide by reaction with an inorganic base, such as sodium hydride, lithium hydride, sodium hexamethyldisilazide, or lithium hexamethyldisilazide, in a non-hydroxylic solvent, such as N,N-dimethylformamide or tetrahydrofuran, prior to reaction with the compound of formula VIA/B;(ii) where X=S, the thiol R2—(CH2))m—SH may be reacted with the compound of formula VIA/B in the presence of an inorganic base, such as sodium methoxide, sodium ethoxide, sodium hydride, sodium hexamethyldisilazide, or lithium hexamethyldisilazide, in a solvent such as 2-propanol, ethanol, methanol, N,N-dimethylformamide, or tetrahydro furan.(iii) where X=NH, the amine R2—(CH2)m—NH2may be reacted with the compound of formula VIA/B in a solvent such as N,N-dimethylformamide or tetrahydrofuran, optionally in the presence of a base such as potassium carbonate, pyridine, N,N-di-(isopropyl)-ethylamine, or triethylamine.(b) when X is CONH, a compound of formula VIA in which RLis amino may be reacted with a compound of formula R2A—(CH2)m—CO2H, or an acylating agent derived therefrom, using one of the general methods for amide formation that are described in the chemical literature. General methods for amide formation are described by B C Challis and J A Challis in Comprehensive Organic Chemistry, Vol. 2, ed. I O Sutherland, pages 959-964 (Pergamon Press, Oxford, 1979).(c) when X is CO.O, a compound of formula VIA/B in which RLis hydroxy may be reacted with an acylating agent derived from a compound of formula R2A—(CH2)m—CO2H, using one of the general methods that are described in the chemical literature, for example treating the acid with oxalyl chloride and reacting with RL=hydroxy in a suitable solvent such as DMF.

Alternatively the above reactions may be carried out using a compound of formula (VIC):
where Y and R1Aare as defined for formulae IIA and IIB and RLis as defined for formulae (VIA) and (VIB)
with the compound (VII) by the procedures (a), (b) or (c) set out above,
and then
treating the product with an acid,
and where required or desired
converting an R1Aor R2Agroup to a R1or R2group, and/or
converting one R1or R2group to another R1or R2group.

As mentioned previously, the acid treatment indicated above converts the epi-mutilin configuration of formula (VIC) to the usual mutilin nucleus of formula (VIA). Typically this conversion is carried out by treatment with conc. HCl or Lukas reagent (conc. HCl saturated with ZnCl2) in dioxane.

As in formulae (VIA) and (VIB), R1Ais typically the R1group vinyl, and this may be converted to the alternative R1group by hydrogenating the vinyl group to form an ethyl group. Also it may again be necessary to protect substituent groups in the compound (VII) prior to reaction, for example protecting N atoms with alkoxycarbonyl, for example t-butoxycarbonyl.

The compounds of formulae (VIA), (VIB) and (VIC) may be prepared by reacting the corresponding compounds of formula (IIA), (IIB) and (IIC) by conventional methodology to introduce acyl groups substituted by hydroxyl or amine or a leaving group.

Reference is directed to the preparation of the chloride and tosylate by K Riedl in J. Antibiotics, 1976, 29, 132; and the tosylate and mesylate described by H Egger and H Reinshagen in J. Antibiotics. 1976, 29, 915; starting from pleuromutilin or 19,20-dihydro-pleuromutilin (n=0) . Also compounds where RLis chloro or bromo may be prepared by reacting Br(CH2))n(CH2)COOCl or Cl(CH2)n(CH2)COOCl with compounds IV and V above. It will be appreciated that when n=0, compounds where RLis hydroxy are pleuromutilin and 19,20-dihydro-pleuromutilin. Compounds where RLis NH2may be prepared from the compound where RLis a leaving group, for example treating a tosylate with sodium azide, followed by treatment with triphenyl phosphine and a base.

Compounds of formula (IA) wherein X is S(O) or SO2may be obtained by preparing the corresponding compound in which X=S and treating it with an oxidising agent; for example, 3-chloroperoxybenzoic acid in chloroform, or catalytic osmium tetroxide plus N-methylmorpholine N-oxide in tetrahydrofuran and tertiary-butanol.

It will be appreciated that it is also possible to carry out the reaction of the compounds VIA/B/C with compound VII with the substituents reversed, i.e. with —CH2(CH2)nXH as a 14-mutilin substituent and RLon the R2A—(CH2)m— residue. For example 22-deoxy-22-sulfanyl-pleuromutilin (U.S. Pat. No. 4,130,709) may be reacted with a compound of formula R2A—(CH2)m—RL, where RLis a leaving group, such as 4-MeC6H4SO2O, MeSO2O, CF3SO2O, or Cl, in the presence of an inorganic base, such as sodium methoxide, sodium ethoxide, or sodium hydride, in a solvent such as 2-propanol, ethanol, methanol, or tetrahydrofuran.

The compounds (III) and (VII) are commercially available or may be formed by conventional methodology from compounds that are commercially available compounds or described in the literature.

Where intermediates disclosed for the above processes are novel compounds, they also form part of this invention.

The compounds of the present invention may contain a chiral centre, and therefore the products of the above processes may comprise a mixture of diastereoisomers or a single diastereoisomer. A single diastereoisomer may be prepared by separating such a mixture of diastereoisomers which has been synthesised using a racemic starting material, or by synthesis using an optically pure starting material.

The products of the processes of this invention may be in crystalline or non-crystalline form, and, if crystalline, may optionally be hydrated or solvated. When some of the compounds of this invention are allowed to crystallise or are recrystallised from organic solvents, solvent of crystallisation may be present in the crystalline product. This invention includes within its scope such solvates. Similarly, some of the compounds of this invention may be crystallised or recrystallised from solvents containing water. In such cases water of hydration may be present in the crystalline product. This invention includes within its scope stoichiometric hydrates as well as compounds containing variable amounts of water that may be produced by processes such as lyophilisation.

The compounds obtained according to the processes of the invention are suitably worked up to a substantially pure form, for example at least 50% pure, suitable at least 60% pure, advantageously at least 75% pure, preferably at least 85% pure, more preferably at least 95% pure, especially at least 98% pure, all percentages being calculated as weight/weight. An impure or less pure form of a compound according to the invention may, for example, be used in the preparation of a more pure form of the same compound or of a related compound (for example a corresponding derivative) suitable for pharmaceutical use.

The present invention also includes pharmaceutically acceptable salts and derivatives of the compounds of the invention. Salt formation may be possible when one of the substituents carries an acidic or basic group. Salts may be prepared by salt exchange in conventional manner

Acid-addition salts may be pharmaceutically acceptable or non-pharmaceutically acceptable. In the latter case, such salts may be useful for isolation and purification of the compound of the invention, or intermediates thereto, and will subsequently be converted into a pharmaceutically acceptable salt or the free base. Pharmaceutically acceptable acid-addition salts include those described by Berge, Bighley, and Monkhouse, J. Pharm. Sci., 1977, 66, 1-19. Suitable salts include the hydrochloride, maleate, and methanesulphonate; particularly the hydrochloride.

It will also be understood that where the compound of the invention contains a free carboxy moiety, it can form a zwitterion.

The compounds of the present invention and their pharmaceutically acceptable salts or derivatives have antimicrobial properties and are therefore of use in therapy, in partiuclar for treating microbial infections in animals, especially mammals, including humans, in particular humans and domesticated animals (including farm animals). The compounds may be used for the treatment of infections caused by, for example, Gram-positive and Gram-negative bacteria and mycoplasmas, including, for example, Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus pneumoniae, Haemophilius sp., Neisseria sp., Legionella sp., Chlamydia sp., Moraxella catarrhalis, Mycoplasma pneumoniae, and Mycoplasma gallisepticum.

The present invention also provides a method of treating microbial infections in animals, especially in humans and in domesticated mammals, which comprises administering a compound of the invention or a pharmaceutically acceptable salt or derivative or solvate thereof, or a composition according to the invention, to a patient in need thereof.

The invention further provides the use of a compound of the invention or a pharmaceutically acceptable salt or derivative or solvate thereof in the preparation of a medicament for use in the treatment of microbial infections.

Compounds of the present invention may be used to treat skin and soft tissue infections and acne, by topical application. Accordingly, in a further aspect the present invention provides the use of a compound of the invention or a pharmaceutically acceptable salt or derivative or solvate thereof in the preparation of a medicament adapted for topical administration for use in the treatment of skin and soft tissue infections and also in the treatment of acne in humans.

Compounds of the present invention may be also used for the elimination or reduction of nasal carriage of pathogenic bacteria such as S. aureus, H. influenzae, S. pneumonia and M. catarrhalis, in particular colonisation of the nasospharynx by such organisms, by the administration of a compound of the present invention thereto. Accordingly, in a further aspect, the present invention provides for the use of a compound of the invention or a pharmaceutically acceptable salt or derivative or solvate thereof in the manufacture of a medicament adapted for administration to the nasal cavity, for reducing or eliminating the nasal carriage of pathogenic organisms. Preferably, the medicament is adapted for focussed delivery to the nasopharynx, in particular the anterior nasopharynx.

Such reduction or elimination of nasal carriage is believed to be useful in prophylaxis of recurrent acute bacterial sinusitis or recurrent otitis media in humans, in particular in reducing the number of episodes experienced by a patient over a given period of time or reducing the time intervals between episodes. Accordingly, in a further aspect, the present invention provides for the use of a compound of the invention or a pharmaceutically acceptable salt or derivative or solvate thereof in the manufacture of a medicament adapted for administration to the nasal cavity, for prophylaxis of recurrent acute bacterial sinusitis or recurrent otitis media.

Compounds of the present invention are also useful in treating chronic sinusitis. Accordingly, in a further aspect, the present invention provides for the use of a compound of the invention or a pharmaceutically acceptable salt or derivative or solvate thereof in the manufacture of a medicament, for treating of chronic sinusitis.

The compounds according to the invention may suitably be administered to the patient at a daily dosage of from 1.0 to 50 mg/kg of body weight. For an adult human (of approximately 70 kg body weight), from 50 to 3000 mg, for example about 1500 mg, of a compound according to the invention may be administered daily. Suitably, the dosage for adult humans is from 5 to 20 mg/kg per day. Higher or lower dosages may, however, be used in accordance with normal clinical practice.

To lessen the risk of encouraging the development of resistant organisms during prophylaxis of recurrent otitis media or recurrent acute bacterial sinusitis, it is preferred to administer the drug on an intermittent, rather than a continual, basis. In a suitable intermittent treatment regimen for prophylaxis of recurrent otitis media or recurrent sinusitis, drug substance is administered on a daily basis, for a small number of days, for instance from 2 to 10, suitably 3 to 8, more suitably about 5 days, the administration then being repeated after an interval, for instance, on a monthly basis over a period of months, for instance up to six months. Less preferably, the drug substance may be administered on a continuing, daily basis, over a prolonged period, for instance several months. Suitably, for prophylaxis of recurrent otitis media or recurrent sinusitis, drug substance is administered once or twice a day. Suitablyv drug substance is administered during the winter months when bacterial infections such as recurrent otitis media and recurrent sinusitis tend to be more prevalent. The drug substance may be administered at a dosage of from 0.05 to 1.00 mg, typically about 0.1 to 0.2mg, in each nostril, once or twice a day.

More generally, the compounds and compositions according to the invention may be formulated for administration in any convenient way for use in human or veterinary medicine, by analogy with other antibiotics.

Accordingly, in a further aspect, the present invention provides a pharmaceutical composition comprising a compound of the invention or a pharmaceutically acceptable salt or derivative or solvate thereof toether with a pharmaceutically acceptable carrier or excipient.

The compounds and compositions according to the invention may be formulated for administration by any route, for example oral, topical or parenteral. The compositions may, for example, be made up in the form of tablets, capsules, powders, granules, lozenges, creams, syrups, sprays or liquid preparations, for example solutions or suspensions, which may be formulated for oral use or in sterile form for parenteral administration by injection or infusion.

Tablets and capsules for oral administration may be in unit dosage form, and may contain conventional excipients including, for example, binding agents, for example, syrup, acacia, gelatin, sorbitol, tragacanth, or polyvinylpyrrollidone; fillers, for example lactose, sugar, maize-starch, calcium phosphate, sorbitol or glycine; tabletting lubricants, for example magnesium stearate, talc, polyethylene glycol or silica; disintegrants, for example potato starch: and pharmaceutically acceptable wetting agents, for example sodium lauryl sulphate. The tablets may be coated according to methods well known in normal pharmaceutical practice.

Oral liquid preparations may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be presented as a dry product for reconstitution with water or another suitable vehicle before use. Such liquid preparations may contain conventional additives, including, for example, suspending agents, for example sorbitol, methyl cellulose, glucose syrup, gelatin, hydroxyethyl cellulose, carboxymethyl cellulose, aluminium stearate gel or hydrogenated edible fats; emulsifying agents, for example lecithin, sorbitan monooleate or acacia; non-aqueous vehicles (which may include edible oils), for example almond oil, oily esters (for example glycerine), propylene glycol, or ethyl alcohol, preservatives, for example methyl or propyl p-hydroxybenzoate or sorbic acid; and, if desired, conventional flavouring and colour agents.

Compositions according to the invention intended for topical administration may, for example, be in the form of ointments, creams, lotions, eye ointments, eye drops, ear drops, nose drops, nasal sprays, impregnated dressings, and aerosols, and may contain appropriate conventional additives, including, for example, preservatives, solvents to assist drug penetration, and emollients in ointments and creams. Such topical formulations may also contain compatible conventional carriers, for example cream or ointment bases, ethanol or oleyl alcohol for lotions and aqueous bases for sprays. Such carriers may constitute from about 1% to about 98% by weight of the formulation; more usually they will constitute up to about 80% by weight of the formulation.

Compositions according to the invention intended for topical administration, in addition to the above, may also contain a steroidal anti-inflammatory agent; for example, betamethasone.

Compositions according to the invention may be formulated as suppositories, which may contain conventional suppository bases, for example cocoa-butter or other glycerides.

Compositions according to the invention intended for parenteral administration may conveniently be in fluid unit dosage forms, which may be prepared utilizing the compound and a sterile vehicle, water being preferred. The compound, depending on the vehicle and concentration used, may be either suspended or dissolved in the vehicle. In preparing solutions, the compound may be dissolved in water for injection and filter-sterilised before being filled into a suitable vial or ampoule, which is then sealed. Advantageously, conventional additives including, for example, local anaesthetics, preservatives, and buffering agents can be dissolved in the vehicle. In order to enhance the stability of the solution, the composition may be frozen after being filled into the vial, and the water removed under vacuum; the resulting dry lyophilised powder may then be sealed in the vial and a accompanying vial of water for injection may be supplied to reconstitute the liquid prior to use. Parenteral suspensions may be prepared in substantially the same manner except that the compound is suspended in the vehicle instead of being dissolved and sterilisation cannot be accomplished by filtration. The compound may instead by sterilised by exposure to ethylene oxide before being suspended in the sterile vehicle. Advantageously, a surfactant or wetting agent is included in such suspensions in order to facilitate uniform distribution of the compound.

A compound or composition according to the invention is suitably administered to the patient in an antimicrobially effective amount.

A composition according to the invention may suitably contain from 0.001% by weight, preferably (for other than spray compositions) from 10 to 60% by weight, of a compound according to the invention (based on the total weight of the composition), dependin on the method of administration.

When the compositions according to the invention are presented in unit dosage form, for instance as a tablet, each unit dose may suitably comprise from 25 to 1000 mg, preferable from 50 to 500 mg, of a compound according to the invention.

Preferred compositions of the present invention include those adapted for intranasal administration, in particular, those that will reach into the nasopharynx. Such compositions are preferably adapted for focussed delivery to, and residence within, the nasopharynx. The term ‘focussed delivery’ is used to mean that the composition is delivered to the nasopharynx, rather than remaining within the nares. The term ‘residence’ within the nasopharynx is used to mean that the composition, once delivered to the nasopharynx, remains within the nasopharynx over a course of several hours, rather than being washed away more or less immediately. Preferred compositions include spray compositions and creams. Representative spray compositions include aqueous compositions, as well as oily compositions which contain amphiphilic agents so that the composition increases in viscosity when in contact with moisture. Creams may also be used, especially creams having a rheology that allows the cream to spread readily in the nasopharynx.

Preferred aqueous spray compositions include, in addition to water, further excipients including a tonicity modifier such as a salt, for instance sodium chloride: preservative, such as benzalkonium salt; a surfactant such as a non-ionic surfactant, for instance a polysorbate; and buffer, such as sodium dihydrogen phosphate; present in low levels, typically less than 1%. The pH of the composition may also be adjusted, for optimum stability of the drug substance during storage. For compounds of the present invention, a pH in the range 5 to 6, preferably about 5.3 to 5.8, typically about 5.5 is optimal

Representative oily spray and cream compositions are described in WO 98/14189 (SmithKline Beecham).

Suitably, the drug substance is present in compositions for nasal delivery in between 0.001 and 5%, preferably 0.005 and 3%, by weight of the composition. Suitable amounts include 0.5% and 1% by weight of the composition (for oily compositions and creams) and from 0.01 to 0.2% (aqueous compositions).

Preferably, an aqueous spray composition is used. Such compositions are found to show similar retention in the target area (nasal cavity and nasopharynx) in gamma scintigraphy studies and have superior release rates in synthetic membrane diffusion studies when compared to an oily composition as described in WO 98/14189. In addition, an aqueous base was found to be preferred to an oily base in sensory analysis studies.

Spray compositions according to the present invention may be delivered to the nasal cavity by spray devices well known in the art for nasal sprays, for instance an air lift pump. Preferred devices include those which are metered to provide a unit volume of composition prefereably about 100 μl, and optionally adpated for nasal administration by addition of a modified nozzle.

The following Examples illustrate the present invention and particularly the preparative procedures outlined above, by reference to the preparation of specific compounds within the scope of the present invention.

Note on Naming of Pleuromutilin Analogues

In the Examples, compound (a), which in the IUPAC system has the systematic name (1S, 2R, 3S, 4S, 6R, 7R, 8R, 14R)-3,6-dihydroxy-2,4,7,14-tetramethyl-4-vinyl-tricyclo[5.4.3.01,8]tetradecan-9-one, is referred to using the trivial name mutilin and with the numbering system described by H Berner, G Schulz, and H Schneider in Tetrahedron, 1981, 37, 915-919.

The preparation of quinuclidin-3-thiol was based on patent literature (J. Barriere. C Cotret and J. Paris, E.P. 248703 [1987]). A solution of triphenylphosphine (12 g) in THF (85 ml) was ice-cooled under argon and treated dropwise with diisopropyl azodicarboxylate (9 ml). After 30 minutes a solution of 3-quinuclidinol (2.9 g) and thiolacetic acid (3.24 ml) in THF (170 ml) was added dropwise over 1 hour. The mixture was stirred overnight at room temperature, evaporated and the residue taken up in ether (250 ml). This solution was extracted with 1M hydrochloric acid (2×40 ml), the combined aqueous extracts washed with ether (100 ml) and evaporated to dryness. The residue was desiccated under vacuum over P2O5for 4 days to provide a pale yellow solid. A portion of this solid (0.443 g) was dissolved in ethanol (10 ml) and treated with sodium methoxide (0.216 g). After 1 hour, mutilin 14-methanesulfonyloxyacetate (0.912 g) was added, the mixture stirred a further 1 hour, diluted with chloroform (30 ml) and water (30 ml), shaken and separated. The organic layer was washed with water (30 ml), dried over MgSO4and evaporated. Chromatography of the residue on silica eluting with chloroform/methanol/35% ammonia solution (19:1:0.1) provided the title compound as a pale yellow foam, 0.62 g (62%);υmax (CHCl3) 3563, 1730 cm−1;1H NMR (CDCl3) inter alia 0.74 (3H, d, J 6 Hz), 0.88 (3H, d, J 7 Hz), 5.1-5.4 (2H, m), 5.76 and 5.77 (1H, 2d, J 8.3 Hz), 6.49 (1H, dd, J 17 and 11 Hz); MS (+ve ion electrospray) m/z 504 (MH+, 100%), 202 (55%).

Quinuclidin-4-thiol hydrochloride (5 g) was stirred with ethanol (110 ml) under argon and solid sodium methoxide (3.15 g) added. After 30 minutes mutilin 14-methanesulfonyloxyacetate (12.7 g) was added, followed by ethanol (30 ml). After a further 30 minutes the mixture was diluted with chloroform (250 ml) and water (250 ml), shaken and separated. The organic layer was washed with water (200 ml), dried over MgSO4and evaporated. Chromatography of the residue on silica, eluting with chloroform/methanol/35% ammonia solution (19:1:0.1) provided the title compound as a pale coloured foam (12.24 g), identical by NMR with the product of Example 1.

To a stirred solution of mutilin 14-toluenesulfonyloxyacetate (5.33 g, 0.01 mole) in acetone (50 ml) was added a solution of sodium azide (0.7 g 0.011 mole) in water (6.5 ml). A solid precipitated briefly then redissolved. The homogenous mixture was stirred for 2 hours at ambient temperature then heated to reflux for 3 hours. The mixture was concentrated in vacuo to low volume then diluted with chloroform. The resulting solution was washed three times with water then dried over magnesium sulfate. Concentration in vacuo gave a pale yellow foam which was purified by chromatography on silica gel. Elution with ethyl acetate/hexane mixtures provided the title compound as a white foam 3.3 g (82%);1H NMR (CDCl3) inter alia 0.73 (3H, d, J 6.8 Hz), 0.89 (3H, d, J 7.1 Hz), 1.23 (3H, s), 1.47 (3H, s), 3.37 (1H, dd, J 10.7 and 6.6 Hz), 3.77 (2H, s), 5.22 (1H, dd, J 17.4 and 1.3 Hz), 5.38 (1H, dd, J 11 and 1.3 Hz), 5.86 (1H, d, J 8.5 Hz), 6.49 (1H, dd, J 17.4 and 11 Hz).

Triphenylphosphine (0.275 g, 0.00105 mole) was added to a stirred solution of mutilin 14-azidoacetate (0.404 g, 0.001 mole) in dichloromethane maintained under an atmosphere of argon. The solution rapidly became homogenous and a gas was evolved. Stirring was continued for 17 hours; the mixture was then concentrated in vacuo to give the title compound as a white solid, obtained by filtration after trituration in petroleum ether 0.638 g (100%); MS (+ve ion electrospray) m/z 638 (MH+, 100%)

Mutilin 14-(triphenylphosphinimino)-acetate (1 g, 0.00157 mole) was suspended in ethanol (25 ml) and potassium hydroxide (0.175 g, 0.00314 mole) was added. The mixture was stirred for 17 hours during which time it became homogenous. 2M hydrochloric acid (1.7 ml) was then added, stirring continued for ten minutes and the mixture concentrated in vacuo. The residue was taken up in 2M hydrochloric acid and the solution washed three times with dichloromethane. The aqueous phase was then layered with dichloromethane and the pH adjusted to 11 by addition of solid potassium carbonate with vigorous stirring The organic phase was then separated, the aqueous phase extracted with dichloromethane, the combined organic extract washed with brine, dried over magnesium sulfate and concentrated in vacuo. The title compound was obtained as a white foam 0.505 g (85%);1H NMR (CDCl3) inter alia 0.71 (3H, d, J 6.5 Hz), 0.89 (3H, d, J 6.9 Hz), 1.17 (3H, s), 1.45 (3H, s), 3.33 (3H, m), 5.21 (1H, d, J 17.4 Hz), 5.36 (1d, J 11 Hz), 5.78 (1H, d, J 8.4 Hz), 6.52 (1H, dd, J 17.4 and 11 Hz).

Quinuclidine-4-carboxylic acid hydrochloride (0.192 g, 0.001 mole) was suspended in dichloromethane (5 ml) and dimethylformamide (1 drop) and oxalyl chloride (0.436 ml. 0.635 g, 0.005 mole) were added. The resulting suspension was heated to reflux under an atmosphere of argon for six hours. Following concentration of the suspension in vacuo the residue was suspended in dichloromethane, concentrated in vacuo and finally dried in vacuo to give the title compound as a pale brown solid.

Quinuclidin-4-ylcarbonyl chloride hydrochloride (0.001 mole theoretical, Step 4) was suspended in dichloromethane (6 ml) and mutilin 14-aminoacetate (0.126 g, 0.00033 mole) was added. To the stirred suspension, under an atmosphere of argon, was added triethylamine (0.278 ml. 0.202 g 0.002 mole) and stirring continued for 18 hours. Chloroform and water were added and the pH of the aqueous phase adjusted to 11 by addition of solid potassium carbonate. After shaking, the phases were separated, the organic phase was washed once with saturated aqueous sodium hydrogen carbonate and once with brine, dried over magnesium sulfate and concentrated in vacuo to give the crude product as an off-white foam. Purification by chromatography on silica gel eluting with chlorofom/methanol/35% ammonia solution provided a pale yellow glass. The product was dissolved in 2M hydrochloric acid, the solution washed twice with dichloromethane, then layered with dichloromethane. The pH of the aqueous phase was adjusted to 11 by addition of solid potassium carbonate. After shaking, the organic phase was separated, dried over magnesium sulfate and concentrated in vacuo. The residue was repeatedly dissolved in chloroform and concentrated in vacuo. Finally the residue was triturated with diethyl ether to give the title compound as a buff solid 0.0019 (11%);1H NMR (CDCl3) inter alia 0.71 (3H, d, J 6.9 Hz), 0.88 (3H, d, J 7 Hz), 1.18 (3H, s), 1.45 (3H, s), 2.96 (6H, m), 3.37 (1H, m), 3.93 (2H, d, J 4.9 Hz), 5.23 (1H, d, J 17.4 Hz), 5.36 (1H, d, J 11 Hz), 5.79 (1H, d, J 8.5 Hz), 6.02 (1H, m(br)), 6.47 (1H, dd, J 17.4 and 11 Hz); MS (+ve ion electrospray) m/z 515 (MH+, 100%).

3R,4R-Azabicyclo[2.2.1]heptane-3-carboxylic acid hydrobromide (0.127 g 0.0005 mole) was suspended in dichloromethane (2 ml) and dimethylformamide (1 drop) and oxalyl chloride (0.131 ml, 0.191 g, 0.0015 mole) were added. The mixture was stirred for 4 hours under an atmosphere of argon. The resulting homogenous solution was concentrated in vacuo, the residue was dissolved in dichloromethane, concentrated in vacuo and finally dried in vacuo to give the title compound as an off-white solid.

(3R,4R)-Azabicyclo[2.2.1]hept-3-ylcarbonyl chloride hydrochloride (0.0005 mole theoretical, Step 1) was dissolved in dichloromethane (4 ml) and mutilin 14-aminoacetate (0.126 g, 0.00033 mole) was added. To the stirred solution under an atmosphere of argon was added triethylamine (0.134 ml. 0.101 g, 0.001 mole). The resulting solution was stirred for 17 hours. Chloroform and water were added and the pH of the aqueous phase adjusted to 11 by addition of solid potassium carbonate. After shaking, the phases were separated, the organic phase was washed once with saturated aqueous sodium hydrogen carbonate and once with brine, dried over magnesium sulfate and concentrated in vacuo to give the crude product as an off-white foam. Purification by chromatography on silica gel eluting with chloroformn/methanol/35% ammonia solution provided the product as a white foam 0.142 a (86%);1H NMR (CDCl3) inter alia 0.72 (3H, d, J 6.9 Hz), 0.89 (3H, d, J 7 Hz), 1.18 (3H, s), 1.46 (3H, s), 3.37 (1H, m(br)), 3.96 (2H, d, J 5.1 Hz), 5.22 (1H d, J 17.4 Hz), 5.36 (1H, d, J 11 Hz), 5.78 (1H, d, 8.4 Hz), 5.96 (1H, m(br)), 6.47 (1H, dd, J 17.4 and 11 Hz),; MS (+ve ion electrospray) m/z 501 (MH+, 40%).

Step 1.1-Methylpiperid-4-ylcarbonyl chloride hydrochloride

1-Methylpiperidine-4-carboxylic acid hydrochloride (0.09 g, 0.0005 mole) was suspended in dichloromethane (5 ml) and dimethylformamide (1 drop) and oxalyl chloride (0.131 ml, 0.19 g, 0.0015 mole) were added. The mixture was stirred for 4 hours under an atmosphere of argon. The resulting homogenous solution was concentrated in vacuo, the residue was dissolved in dichloromethane, concentrated in vacuo and finally dried in vacuo to give the title compound as an off-white solid.

A suspension of 3-(1-methylpiperid-4-yl)propionic acid hydrochloride (WO 9620173 A1, Example 1)(0.33 g, 0.00159 mole) in dry dichloromethane (10 ml) was treated with dimethylformamide (1 drop) and oxalyl chloride (0.416 ml, 0.605 g, 0.00477 mole) under an atmosphere of argon. After stirring for 3½ hours the mixture was concentrated in vacuo. The residue was dissolved in dry dichloromethane and concentrated in vacuo to give the title compound as a white solid.

An ice cooled solution of triphenylphosphine (1.19 g, 0.0042 mole) in dry tetrahydrofuran was treated dropwise with diisopropyl azodicarboxylate (0.85 g, 0.0042 mole). After 30 minutes a solution of quinuclid-4-ylmethanol (0.565 g, 0.004 mole) and thiolacetic acid (0.315 ml, 0.0042 mole) in dry tetrahydrofuran (20 ml) added dropwise over a period of 10 minutes. The mixture was left at 5° for 72 hours then concentrated in vacuo and the residue dissolved in ether (200 ml). The resulting solution was extracted with 1M hydrochloric acid (3×50 ml). The combined extract was concentrated in vacuo and dried in vacuo to give a gummy residue 0.65 g. The residue was dissolved in ethanol (30 ml) and treated under argon with potassium tert butoxide (0.785 g, 0.007 mole) for 30 minutes. Mutilin 14-methanesulfonyloxyacetate 1.38 g. 0.003 mole) was then added to the ethanolic solution and the mixture stirred overnight under argon. The insoluble byproducts were filtered off and the filtrate evaporated to dryness. The residue was partitioned between chloroform and water. The organic layer washed with brine and dried over magnesium sulfate and evaporated to dryness. Chromatography of the residue on silica gel eluting with chloroform/methanol/35% ammonia solution (19:1:0.1) provided the title compound as a white foam, 0.48 g (31%);1H NMR (CDCl3) inter alia 0.74 (3H, d, J 6.6 Hz), 0.88 (3H, d, J 7 Hz), 1.76 (3H, s), 1.44 (6H, t, J 7.7 Hz), 2.47 (2H, s), 2.87 (6H, t, J 7.5 Hz), 3.09 (2H, s), 3.36 (1H, m), 5.1-5.4 (2H, m), 5.75 (1H, d, J 8.3 Hz), 6.48 (1H, m); MS (+ve ion electrospray) m/z 518 (MH+, 100%).

A solution of triphenylphosphine (5.51 g, 0.021 mole) in dry tetrahydrofuran (100 ml) was ice-cooled under argon and treated with dilsopropyl azodicarboxylate (4.25 g, 0.021 mole). After 30 minutes a solution of 4-hydroxy-1-methylpiperidine (2.3 g, 0.02 mole) and thiolacetic acid (1.54 g, 0.02 mole) in dry tetrahydrofuran (50 ml) was added over a period of 30 minutes. The mixture was stirred overnight at room temperature, evaporated in vacuo and the residue taken up in ether (200 ml). The ethereal solution was extracted with 1M hydrochloric acid (50 ml×4). The combined aqueous extract was washed with ether, evaporated to dryness and dried in vacuo to give a yellow gum (2.4 g). A portion of this gum (0.517 g) was dissolved in ethanol and treated with potassium tert-butoxide (0.785 g) under argon for 30 minutes. Mutilin-14-methanesulfonyloxyacetate (0.92 g, 0.002 mole) was added and the mixture stirred overnight, then concentrated in vacuo. The residue was partitioned between chloroform and water. The organic layer was washed with brine, dried over magnesium sulfate and concentrated in vacuo. Chromatography on silica gel eluting with chloroform/methanol/35% ammonia solution provided the title compound as a foam, 0.557 g (57%);1H NMR (CDCl3) inter alia 0.73 (3H, d, J 6.5 Hz), 0.87 (3H, d, J 7 Hz), 1.30 (3H, s), 1.67 (3H, s), 2.25 (3H, s), 3.16 (2H, s), 3.36 (1H, m), 5.28 (2H, m), 5.77 (1H, d, J 8.5 Hz), 6.47 (1H, m); MS (+ve ion electrospray) m/z 492 (MH+, 100%).

A suspension of quinuclidin-3-one hydrochloride (3.23 g) in DMF (20 ml) was treated with sodium methoxide (1.08 g) and stirred vigorously for 30 minutes. A solution of trimethyl phosphonoacetate (4.05 ml) and sodium methoxide (1.35 g) in DMF (20 ml) was added dropwise over 15 minutes and stirred a further 2½ hours. The DMF was evaporated and the residue treated with dry ether (100 ml), triturated and filtered. The filtrate was treated with 1N HCl in ether (30 ml), the resulting solid triturated and the ether decanted. Ether (200 ml) was added, the suspension stirred vigorously for 30 minutes, the solid filtered off and heated at 60° C. under vacuum for 2 days. The resulting methylquinuclidin-3-ylideneacetate hydrochloride (3.93 g) was a ca. 1:1 mixture of geometric isomers;1H NMR (D2O) inter alia 5.84 (broad s) and 5.94 (t, J 2.5 Hz) (vinyl protons of the two geom. isomers).

Methyl quinuclidin-3-ylideneacetate hydrochloride (1 g) was heated in concentrated hydrochloric acid (10 ml) at 60° C. for 18 hrs and the solution evaporated to dryness. The residue was kept under vacuum over P2O5for 3 days to give quinuclidin-3-ylidene acetic acid hydrochloride, 0.91 g (97%) as a white solid;1H NMR (D2O) inter alia 5.77 (broad s) and 5.86 (broad s) (ca. 1:1, vinyl protons of the two geom. isomers).

Quinuclidin-3-ylideneacetic acid hydrochloride (0.204 g) was suspended in chloroform (5 ml), stirred under argon and treated with 1 drop DMF and oxalyl chloride (0.87 ml). After 2 hours the solvent was evaporated, toluene (10 ml) added to the residue and evaporated. The residue was taken up in DMF (2 ml), treated with (3R)-3-deoxo-11-deoxy-3-methoxy-11-oxo-4-epimutilin (0.334 g, prepared according to H. Berner, G. Schulz and H. Schneider, Tetrahedron (1980) 36, 1807) and heated at 100° C. under argon for 3 hours. After leaving at room temperature overnight, the mixture was diluted with chloroform (20 ml), washed with aqueous NaHCO3and water, dried and evaporated to dryness. Chromatography on silica. eluting with chloroform/methanol/35% ammonia solution (19:1:0.1) separated the 2 geometric isomers of the title compound.

A mixture of methyl quinuclidin-3-ylideneacetate hydrochloride (Example 18, Step 1) (2 g), ethanol (50 ml), 2M hydrochloric acid (5 ml) and 10% Pd/C (1 g) was stirred for 24 hours under H2at atmospheric pressure, filtered through celite and evaporated to dryness. The residue was dissolved in concentrated hydrochloric acid (10 ml). heated at 60° C. for 18 hours, treated with a further 10 ml of concentrated hydrochloric acid, heated at 80° C. for 6 hours and evaporated to dryness. The residue was kept under vacuum over P2O5for 3 days to give the title compound as a white solid (1.8 g); MS (+ve ion electrospray) m/z 170 (MH+, 100%).

(±)-Quinuclidine-3-acetic acid hydrochloride (0.206 g) was suspended in chloroform (5 ml) under argon, treated with DMF (1 drop) and oxalyl chloride (0.87 ml) and stirred1 hour. The solution was evaporated, toluene was added and evaporated and the residue taken up in DMF (2 ml). Pleuromutilin (0.378 g) was added and the mixture stirred under argon for 18 hours, then heated at 110° C. for 30 minutes. It was diluted with chloroform (10 ml), washed with aqueous NaHCO3(twice) and water, dried and evaporated. The residue was chromatographed, eluting with chloroform/methanol/35% ammonia solution (9:1:0.1). A chloroform solution of the material obtained was treated with 1M HCl in ether (2 ml) and evaporated. Trituration under ether and filtration gave the title compound as an off-white solid, 0.22 g (42%);1H NMR (CD3SOCD3) interalia 4.5-4.7 (3H, m, reduces to 2H, m on D2O exchange); 5.0-5.2 (2H, m), 5.59 (1H, d, J 8 Hz), 6.10 (1H, dd, J 17.5 and 10.5 Hz), 10.06 (1H, broad s, disappears on D2O exchange); MS (+ve ion electrospray) m/z 530 (MH+, 100%).

Step 1. Mixture of (±)-quinuclidin-3-ylmethylsulfanylacetate hydrochloride and (±)-quinuclidin-3ylmethanethiol hydrochloride

Quinuclidine-4-carboxylic acid hydrochloride (6.0 g, 0.031 mmoles) in tetrahydrofuran (300 ml) was treated with lithium aluminum hydride (5.0 g, 0.137 mmoles) at ambient temperature for 18 hours. Water (20 ml) and 10% aqueous sodium hydroxide (7.5 ml) was added carefully and the mixture filtered, washing with diethyl ether. The combined filtrates were evaporated to dryness to give the title compound as a white solid 4.04 g, (91%): MS (+ve ion electrospray) m/z 142 (MH+, 100%)

Quinuclidin-4-ylmethanol (2.19 g 0.0015 moles) was converted to the corresponding mesylate by treatment with triethylamine/methanesulphonyl chloride in chloroform. Washing the organics with saturated potassium carbonate, drying over sodium sulphate and evaporation to dryness gave the mesylate 3.24 g (95%). The mesylate was dissolved in dry dimethyl formamide (50 ml) and treated with sodium cyanide (2.26 g, 0.0046 moles) and heated to 130° C. for 18 hours. The mixture was evaporated to dryness and the residue partitioned between saturated potassium carbonate and chloroform. The organics were dried (Na2SO4) and chromatographed on silica gel eluting with 0-10% methanol/chloroform. This gave the title compound 1.1 g (50%);1H NMR (CDCl3) 1.45 (6H, t, J 9 Hz), 2.12 (2H, s), 2.85 (6H, t, J 9 Hz); MS (+ve ion electrospray) m/z 151 (MH+, 100%).

Quinuclidine-4-acetic acid hydrochloride (0.5 g, 0.0024 moles) was converted to the title compound using the method of Example 8, Step 4. MS (+ve ion electrospray in methanol) m/z 183 (MH+for methyl ester, 100% showing complete conversion).

Quinuclidin-4-ylcarbonylchloride hydrochloride (Example 8, Step 4) (3.4 g 0.0016 moles) was dissolved in acetonitrile (150 ml) and treated with 35% ammonia solution (50 ml). The mixture was stirred for 18 hours at ambient temperature then concentrated to dryness in vacuo. 1 g of the residue was then treated with phosphoms oxychloride (8 ml) at reflux for 5 hours. The mixture was then concentrated in vacuo and the residue partitioned between saturated potassium carbonate and diethylether (4×50 ml). The combined organic extracts were dried (Na2SO4), filtered and concentrated. Column chromatography on silica gel elutin with 0-5% methanol/chloroform gave the title compound 0.34 g (75%);1H NMR (CDCl3) 1.85 (6H t, J 10 Hz), 2.91 (6H, t, J 10 Hz).

4- Cyanoquinuclidine (0.31 g, 0.0028 moles) was reduced with lithium aluminium hydride (0.45 g, 0.012 moles) in tetrahydrofuran (20 ml) at ambient temperature for 18 hours. Diethyl ether (20 mls) was added followed by water (1.8 ml) and 10%w/v aqueous sodium hydroxide (0.68 ml) and the mixture stirred for 30 minutes. The mixture was then filtered and the filtrate concentrated in vacuo to give the title compound 0.3 g (94%).

The title compound was prepared from 3-(quinuclidin-4-yl)-acrylic acid as in the method of Example 8. Step 4 (0.24g, 100%). MS (+ve ion electrospray) m/z 196 (MH+, 100%-methyl ester from reaction with methanol).

3-(Quinuclidin-4-yl)acryloyl chloride (0.24 g 0.001 moles) and (3R)-3-deoxo-11-deoxy-3-methoxy-11-oxo-4-epimutilin (0.34 g, 0.001 moles) were heated together in dimethylformamide (15 ml) at 110° C. for 18 hours. The mixture was allowed to cool and concentrated in vacuo. The residue was partitioned between chloroform and saturated sodium hydrogen carbonate solution. The organic layer was dried (Na2SO4), filtered and evaporated to dryness. Chromatography on Sep-Pak silica gel 10 g cartridge eluting with 0-10% (9:1 methanol/35% ammonia solution) in chloroform gave the title compound 0.035 g (6.5%): MS (+ve ion electrospray) m/z 498 (MH+, 100%).

3-(Quinuclidin-4-yl)-acrylic acid (Example 26, Step 4) (0.2 g, 0.0009 moles) was hydrogenated at atmospheric pressure and ambient temperature over 10% palladium on charcoal (0.05 g) for 18 hours. The catalyst was filtered off and the filtrate evaporated to dryness to give the title compound 0.18 g (89%); MS (+ve ion electrospray) m/z 184 (MH+, 100%).

The title compound was prepared from 3-(quinuclidin-4-yl)-propionic acid hydrochloride (0.18 g, 0.0008 moles) as in the method of Example 8, Step 4 0.19 g (100%). MS (+ve ion electrospray) m/z 198 (MH+, 100%)-methyl ester from reaction with methanol).

Quinuclidine-4-carboxylic acid hydrochloride (3.0 g, 0.016 moles) was treated with lithium aluminium hydride (2.5 g, 0.066 moles) in tetrahydrofuran (150 ml) at ambient temperature for 18 hours. The reaction was worked up as in the method of Example 25 Step 1 to give the title compound 2.24 g (100%). MS (+ve electrospray) m/z 142 (MH+, 100%).

Quinuclidin-4-ylcarbonylchloride (Example 8, Step 4) (1.0 g, 0.0048 moles) was treated with sodium azide (0.34 g, 0.005 moles) in dimethylformamide (10 ml) at 50° C. for 18 hours. The mixture was concentrated in vacuo and the residue partitioned between saturated potassium carbonate and toluene. The toluene solution was separated, dried (Na2SO4), filtered and the filtrate was heated under reflux for 1 hour to give the isocyanate. The mixture was allowed to cool and then extracted with 5 M hydrochloric acid (3×20 ml). The combined acid extracts were then heated under reflux for 1 hour, cooled then evaporated to dryness. Trituration with acetone gave the title compound as a white solid 0.56 g (60%). M.S. (+ve ion electrospray) m/z 127 (MH+, 100%).

Quinuclidin-4-ylmethanol (1.94 g, 0.014 moles) was converted to the corresponding mesylate by treatment with methane sulphonyl chloride and triethylamine in chloroform. The mesylate was dissolved in dimethylformamide (50 ml) and treated with sodium cyanide (1.4 g, 0.028 moles) at 120° C. for 18 hours. The mixture was cooled and concentrated in vacuo. The residue was partitioned between saturated potassium carbonate and chloroform. The organic layer was separated and dried (Na2SO4), filtered and evaporated to dryness. Chromatography on silica gel eluting with 0-10% methanol/chloroform gave the title compound 1.5 g (72%). M.S. (+ve electrospray) m/z 151 (MH+, 100%).

Quinuclidin-4-ylacetonitrile (3.0 g, 0.02 moles) in dry toluene (100 ml) was treated with 1.5 molar diusobutyl aluminium hydride (19.7 ml, 0.03 moles) at ambient temperature for 5 hours. The mixture was quenched by adding 2 M hydrochloric acid (50 ml) and stirring for 30 minutes. The mixture was then basified with potassium carbonate and extracted with chloroform. The organics were separated, dried (Na2SO4), filtered and evaporated to dryness to give the title compound as an oil 2.2 g (72%). M.S. (+ve ion electrospray) m/z 154 (MH+, 100%).

tert-Butyl (piperidin-4-one-1-yl)-acetate (3 g, 0.014 moles) was treated with sodium borohydride (1.13 g, 0.028 moles) in methanol (150 ml) at ambient temperature for 1 hour. Glacial acetic acid (1.68 g, 0.028 moles) was added and the mixture stirred for 15 minutes. The mixture was concentrated in vacuo and the residue partitioned between saturated sodium carbonate and ethyl acetate. The organics were separated, dried (Na2SO4), filtered and evaporated to dryness to give the title compound (2.9g, 96%). M.S. (+ve ion electrospray) m/z 216 (MH+, 100%).

The title compound was prepared as in the method of Example 15 from 1-(tert-butoxycarbonyl)piperidin-4-ol (2.5 g, 0.012 moles). M.S. (-ve ion electrospray) m/z 576 (M-H, 100%).

The product of Step 2 (0.19 g. 0.001 mole) was dissolved in dry ethanol (10 ml) under argon and treated with sodium methoxide (0.054 g, 0.001 mole). The mixture was stirred for 1 hour and mutilin 14-methanesulfonyloxyacetate (0.456 g, 0.001 mole) was added. The mixture was stirred at room temperature overnight. The solvent was removed in vacuo and the residue partitioned between water and dichloromethane. The organic layer was dried (magnesium sulfate) and evaporated in vacuo. The residue was purified by column chromatography, eluting with dichloromethane to 15% methanol/dichloromethane. The resulting gum was converted to the hydrochloride salt to afford the title compound 0.17 g (34%) as a white foam;1H NMR (CDCl3) inter alia 0.73 (3H, d, J 7 Hz), 0.90 (3H, d, J 7 Hz), 5.23 (1H, dd, J 17 and 3 Hz), 5.35 (1dd, J 13 and 3 Hz), 5.73 (1H, d, J 7 Hz), 6.48 (1H, q, J 17 and 10 Hz), 12.26-12.69 (1H, br s); MS (+ve ion electrospray) m/z 506 (MH+free base).

The product of Step 1 (0.60 g, 0.003 mole) in dry tetrahydrofuran (10 ml) was added dropwise to a suspension of lithium aluminium hydride (0.57 g, 0.015 mole) in dry tetrahydrofuran (20 ml). The mixture was heated under reflux for 2 hours and stirred at room temperature overnight. The reaction mixture was cooled to 0° C. and water (0.5 ml) was added dropwise, followed by 10% sodium hydroxide solution (0.9 ml) and water (1.4 ml). The mixture was stirred for 1 hour, filtered through Celite and the filtrate evaporated in vacuo to afford the title compound 0.31 g (80%) as a pale yellow oil;1H NMR (CDCl3) 1.17-2.00 (8H, m), 2.14 (1H, dt, J 13 and 2 Hz), 2.30 (3H, s), 2.76-2.96 (1H, m), 3.40 (1H, dd, J 13 and 1 Hz), 3.88 (1H, dd, J 12 and 5 Hz); MS (+ve ion electrospray) m/z 130 (MH+).

22-Deoxy-22-sulfanylpleuromutilin (U.S. Pat. No. 4,130,709, 1978) (0.1 g, 0.00025 mole) in ethanol (4 ml) was treated with sodium methoxide (0.014 g, 0.0026 mole) and the resulting mixture stirred for 30 minutes. A solution of endo-3-methanesulfonyloxy-8-methyl-8-azabicyclo[3.2.1]octane (prepared from endo-8-methyl-8-azabicyclo[3.2.1]octan-3-ol and methanesulfonyl chloride) (0.061 g, 0.00028 mole) in ethanol (1 ml) was then added. Stirring was continued for 68 hours; a further portion of endo-3-methanesulfonyloxy-8-methyl-8-azabicyclo[3.2.1]octane (0.061 g, 0.00028 mole) was then added and stirring continued for a further 18 hours. The mixture was then diluted with dichloromethane, washed twice with aqueous potassium carbonate, once with brine, dried over magnesium sulfate and concentrated in vacuo. Chromatography on silica gel eluting with chloroform/methanol/35% ammonia solution (9:1:0:1) gave the title compound 0.035 g (27%), identical to the compound described in Example 50.

Mutilin (1-carboxymethylpiperidin-4-ylsulfanyl)-acetate (Example 37) (0.08 g, 0.00015 mole) in dichloromethane (3 ml) was treated with oxalyl chloride (0.032 ml, 0.00036 moles) and dimethylformamide (1 drop) and stirred at ambient temperature for 2 hours. The mixture was then evaporated to dryness and the residue suspended in tetrahydofuran (3 ml) and treated with 35% aqueous ammonia solution (25 ml) and stirred for 2 hours. The mixture was evaporated to dryness and the residue positioned between saturated sodium bicarbonate and chloroform. The organic layer was separated and dried (Na2SO4), filtered and evaporated to dryness. Chromatography was saturated on silica gel eluting with chloroform/methanol/35% aqueous ammonia solution (90:9:1). Trituration of the residue obtained with methanol/diethyl ether gave the title compound 0.035 g; M.S. (+ve ion electrospray) m/z 535(MH30, 88%).

Antibacterial Activity

The following Table illustrates the antibacterial activities of representative mutilin 14-esters. Activities are given as minimum inhibitory concentrations in micrograms per milliliter (10−6g/ml), and were determined using a standard broth dilution method in microtitre.

PHARMACEUTICAL COMPOSITIONS

Oily Spray Formulation

A carrier for a nasal spray formulation was prepared by forming a blend of 67% w/w fractionated coconut oil (medium chain length)* and 33% w/w of glyceryl monooleate **. To this blend was added 0.2% w/w of powdered lemon juice flavour, followed by 0.5 or 1.0% w/w of drug substance (either in solution or, if insoluble, micronized) ***.

Commercial product Miglyol, obtainable from Condea.

for example, the compound of Example 1 or Example 8.

The resultant formulation has a viscosity which is sprayable at 20° C. or above. When sprayed into the nose of a patient, the liquid coats the nasal passages and contact with moisture inside the nose (from the mucous membranes, and the humid environment generally) causes the carrier to thicken. This prolongs the residence time of the sprayed formulation on the nasal surfaces. A spray volume of about 100 μl contains approximately 0.5 or 1 mg of drug substance.

Aqueous Spray formulation

Hydrochloric acid and sodium hydroxide were used to adjust the pH of the composition to about pH 5.5. The drug molecule shows optimum stability at this pH.