WO1993016090A1

WO1993016090A1 - Amphotericin b derivative

Info

Publication number: WO1993016090A1
Application number: PCT/GB1993/000290
Authority: WO
Inventors: William Skinner Maclachlan
Original assignee: Smithkline Beecham Plc
Priority date: 1992-02-12
Filing date: 1993-02-11
Publication date: 1993-08-19
Also published as: AU3460693A

Abstract

A compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein R1 is a carboxylic acid group, an ester or amide thereof, a group -X-Y where X is a carbonyl group and Y is hydrogen, C1-8alkyl, C2-8alkenyl, or optionally substituted aryl or heteroaryl group -C(J)=NOZ where J is hydrogen, C1-8alkyl, C2-8alkenyl, optionally substituted aryl C1-8alkyl, aryl or heteroaryl; and Z is hydrogen, optionally substituted C1-8alkyl, C2-8alkenyl, aryl C1-8alkyl, heteroaryl C1-8alkyl, aryl or heteroaryl; a group -CH2-E where E is hydrogen, halogen, -CN, -N3, -OC(O)R5, -S(O)nR5, -SH, -OC(O)NHR5, -NHCONHR5 or -NR6R7, where R5 is hydrogen, optionally substituted C1-8alkyl, or aryl, heteroaryl, aryl C1-4alkyl or heteroaryl C1-4alkyl in each of which the aromatic moiety is optionally substituted with the proviso that when E is -S(O)nR5, R5 does not represent hydrogen, R6 and R7 are independently hydrogen or C1-6alkyl, or one of R6 and R7 is hydrogen and the other is formyl, C2-8alkanoyl, dialkoxyphosphoryl, aroyl, heteroaroyl, aryl C1-4alkanoyl, heteroaryl C1-4alkanoyl, C1-8alkylsulphonyl, arylsulphonyl, heteroarylsulphonyl, aryl C1-4alkylsulphonyl or heteroaryl C1-4alkylsulphonyl, where any aromatic moiety in R6 or R7 is optionally substituted, and n is 0, 1 or 2; or R1 is a group CH2OH; R2 is hydrogen; R3 is an amino group or a derivative thereof; and each R4 is hydrogen.

Description

AMPHOTERICIN B DERIVATIVE

The present invention relates to novel compounds, their preparation and their use in the treatment of fungal infections in animals, including humans.

The polyene macrolide amphotericin B, produced by Streptomvces nodosus. is widely used for the treatment of fungal infections.

Amphotericin B is the only complex polyene macrolide whose molecular structure and absolute configuration have been firmly established by x-ray crystallographic analysis. Amphotericin B has the formula (A): ou OH

EP-A-035( .34 (Beecham Group p.l.c.) describes derivatives of amphotericin B having a 13, 14 double bond of formula (B):

wherein: each B_*i is independently hydrogen or a silyl protecting group; B,2 i*^{3 a} carboxylic acid or derivative thereof; a ketone or aldehyde function; hydroxymethyl in which the hydroxyl function is optionally silylated, or substituted by hydroxy C1.4 alkyl, alkoxy C1.4 alkyl, alkoxycarbonyl C1.4 alkyl, hydroxycarbonyl C1.4 alkyl, alkyl, alkanoyl, or optionally substituted aryl or aroyl; one of X and Ϋ is hydrogen, and the other is a sugar residue; hydroxy; optionally substituted Cι_6 alkoxy or C3.8 cycloalkoxy; alkanoyloxy; thioalkanoyloxy; sulphonyloxy; halogen; or optionally substituted aryloxy, aralkyloxy or aroyloxy; or X and Y together with the carbon atom to which they are bonded are a carbonyl group or derivative thereof; with the proviso that when each R-^ is trimethylsilyl and R2 is methoxycarbonyl, one of X and Y is not hydroxy, 4-nitro-phenylcarboxy or N-acetyl-3,4-Q-trimethyl- silyl-mycosamine, or X and Y together with the carbon atom to which they are bonded are not a carbonyl group;when each Rj is hydrogen and R2 is methoxycarbonyl, one of X and Y is not hydroxy or 4-nitro-phenyl carboxy; and when each R is hydrogen and R2 is carboxy, one of X and Y is not hydroxy, which compounds are disclosed as possessing antifungal activity.

EP-A-0375222 (Beecham Group p.l.c.) describes compounds of formula (C):

wherein R} is CH2OH; R2 is hydrogen or Cj.β alkyl; R3 is hydrogen or an amine protection group; and each R4 is hydrogen, which compounds are disclosed as possessing antifungal activity.

EP-A-0375223 (Beecham Group p.l.c.) describes compounds of formula (D):

wherein R is a group -X-Y where X is a carbonyl group and Y is hydrogen, C^*[_g alkyl, C2-8 alkenyl, or optionally substituted aryl or heteroaryl; R2 is hydroxy or C^.g alkoxy; R3 is hydrogen or an amine protection group; and each R4 is hydrogen, which compounds are described as possessing antifungal activity.

EP- Application No.90313106.8 (Beecham Group p.l.c.) describes compounds of formula (E):

wherein: l is a group -CH₂-X where X is halogen, -CN, -N3, OC(0)R₅, -S(0)_nRδ or N R , where R5 is optionally substituted Cι_g alkyl, or aryl, heteroaryl, aryl C1.4 alkyl or heteroaryl C1.4 alkyl in each of which the aromatic moiety is optionally substituted, Rg and R7 are independently hydrogen or C^.g alkyl, or one of Rg and R7 is hydrogen and the other is Cι_g alkoxy, C2-8 alkanoyl, aroyl, heteroaroyl, aryl C1.4 alkanoyl, heteroaryl Cι_4 alkanoyl, C .g alkylsulphonyl, arylsulphonyl, heteroarylsulphonyl, aryl C .4 alkylsulphonyl or heteroaryl C1.4 alkylsulphonyl, where any aromatic moiety in Rg or R7 is optionallt substituted, and n is O, 1 or 2, which compounds are described as possessing antifungal activity. Novel derivatives of amphotericin B have now been prepared which have a 13-position hydrogen atom, and a 14-position hydroxy function which derivatives have been shown to possess anti-fungal activity and have potential utility as anti-fungal agents.

Accordingly, the present invention provides a compound of formula (I) or a pharmaceutically acceptable salt thereof:

wherein R^* is a carboxylic acid group, an ester or amide thereof, a group -X-Y where X is a carbonyl group and Y is hydrogen, Cχ.g alkyl, C2-8 alkenyl, or optionally substituted aryl or heteroaryl a group -C(J)=NOZ where J is hydrogen, Cι_g alkyl, C2-8 alkenyl, optionally substituted aryl Ci.galkyl, aryl or heteroaryl; and Z is hydrogen, optionally substituted Cι_ alkyl, C2-g alkenyl, aryl Cι_g alkyl, heteroaryl Cχ_g alkyl, aryl or heteroaryl; a group -CH2-E where E is hydrogen, halogen, -CN, -N3,

-OC(0)R₅, -S(0)_nR₅, -SH, -OC(0)NHR₅, -NHCONHR5 or -NRgR₇, where R5 is hydrogen, optionally substituted Cι_8 alkyl, or aryl, heteroaryl, aryl Cι_4 alkyl or heteroaryl C1.4 alkyl in each of which the aromatic moiety is optionally substituted with the proviso that when E is -S(0)_nR5, R5 does not represent hydrogen, Rg and R7 are independently hydrogen or C .g alkyl, or one of Rg and R7 is hydrogen and the other is formyl, C2-8 alkanoyl, dialkoxyphosphoryl, aroyl, heteroaroyl, aryl C1.4 alkanoyl, heteroaryl Cχ_ alkanoyl, Cj.g alkylsulphonyl, arylsulphonyl, heteroarylsulphonyl, aryl C .4 alkylsulphonyl or heteroaryl Cχ_4 alkylsulphonyl, where any aromatic moiety in Rg or R7 is optionally substituted, and n is 0, 1 or 2; or R is a group CH2OH; R2 is hydrogen; R3 is an amino group or a derivative thereof; and each R4 is hydrogen. The term ester includes alkoxycarbonyl, alkenyl oxycarbonyl, aryloxycarbonyl, arylalkyloxycarbonyl, heteroaryloxycarbonyl and heteroarylalkyloxycarbonyl groups and the term amide includes primary, secondary and tertiary amides. For example the amine moiety may be substituted by one or two alkyl groups.

An R5 Cι_8 alkyl group and an R5, Rg or R7 aryl or heteroaryl moiety may be mono-, di-, or tri-substituted by groups including carboxy, nitro, alkoxycarbonyl, hydroxy, Cχ_g alkyl, Cj. alkoxy, halogen and NRgR7 where Rg and R7 are as defined in relation to formula (I).

When mentioned in relation to formula (I) suitable halogens include fluorine, chlorine, bromine and iodine.

Unless otherwise specified, an alkyl or alkenyl group or moiety preferably has from 1 to 6 carbon atoms, more preferably 1 to 4 carbon atoms and may be straight-chain or branched and may be optionally substituted.

When used herein, the term aryl includes aromatic carbocyclic groups such as phenyl and naphthyl, preferably phenyl and may be optionally substituted.

The term heteroaryl includes 4-, 5- or 6- membered monocyclic and 9- or 10- membered bicyclic optionally substituted aromatic rings containing at least one heteroatom.

Suitably, 5- or 6- membered monocyclic and 9- or 10- membered bicyclic aromatic rings preferably contain one or two heteroatoms selected from nitrogen, oxygen and sulphur which in the case of there being more than one heteroatom may be the same or different. Suitably 4- membered monocyclic aromatic rings preferably contain one heteroatom selected from nitrogen oxygen and sulphur. When 9- or 10- membered bicyclic heteroaryl, the two rings are fused, preferably with one 5- or 6- membered ring containing a single heteroatom.

Optional substituents for alkyl, alkenyl, aryl and heteroaryl groups or moieties may be selected from hydroxy, acyl, alkyl, alkoxy, a carboxylic acid or derivative thereof, nitro, halogen, and amino optionally substituted by Cχ.g alkyl, Cj. alkanoyl or aryl. Suitable carboxylic acid derivatives include esters and amides such as those described for the variable Rj in compounds of formula (I).

The term acyl includes carboxyl derivatives (CORs) as well as sulphonyl (SO_rR8) derivatives, wherein r is 1 or 2 and Rs is optionally substituted alkyl, alkenyl, aryl, heteroaryl, alkyl aryl and alkyl heteroaryl.

When Rj is an ester group, Rj is preferably an alkoxycarbonyl group in which the alkyl moiety is preferably methyl.

Suitable R3 amine derivatives include acyl derivatives bearing a basic substituent such as N-D-lysyl and N-D-ornithyl derivatives, guanidine derivatives, and N-glycosyl derivatives. The preparation of these and other suitable amino derivatives is described in European Patent

Publication 0 010 297 (Schering), European Patent Publication 0 031 722 (Dumex) and US 4 195 172. Preferably R3 is a primary amine group.

The term pharmaceutically acceptable salt encompasses solvates and hydrates. Thus where compounds of formula (I) or pharmaceutically acceptable salts thereof form solvates or hydrates, these also form an aspect of the invention.

The compounds of formula (I) wherein R3 is a primary amine can form acid addition salts with acids, such as conventional pharmaceutically acceptable acids, for example hydrochloric, hydrobromic, phosphoric, acetic, fumaric, salicylic, citric, lactic, mandelic, tartaric, oxalic, methanesulphonic, aspartic and ascorbic.

The present invention also extends to quaternary salts.

The present invention also provides a process for the preparation of compounds of formula (I) which process comprises the conversion of a compound of formula (II):

wherein R-j is a carboxylic acid, an ester or amide thereof an aldehyde, a group -CH2E wherein E is as defined in relation to formula (I) or an optionally protected hydroxymethyl group, R3' is an amine protecting group, and each R4^* is a silyl protecting group; and thereafter, optionally or as necessary converting R- to R^*j_, interconverting R^*j_, converting NHR3' to R3, removing the R4' silyl protecting groups, and forming a pharmaceutically acceptable salt.

Suitable R]_' carboxylic acid ester and amide groups are as defined in relation to R^*j_, for example, preferably R]_' is methoxycarboxyl or 4-bromophenoxycarbonyl which may be converted to the carboxylic acid by conventional means.

Suitable R,\ -CH2E groups are as defined in relation to Ri in formula (I).

Suitable protecting groups for the protected hydroxymethyl group R\ ) may include those described in Greene, T.W. 'Protective groups in Organic Synthesis' New York, Wiley (1981) as well as silyl protecting groups such as those of R4^*, all of which can be removed conventionally.

It should be appreciated that the conversion of a compound of formula (II) to a compound of formula (I) proceeds via a reduction step followed by an oxidation step.

Any intermediate product of the reduction step may be optionally isolated before performing the oxidation step. The reduction step may be carried out in an inert solvent such as tetrahydrofuran using a suitable reducing agent such as a borane. The oxidation step may be carried out with a suitable oxidising agent such as sodium perborate in the presence of a suitable base such as potassium hydroxide.

It should be appreciated that certain Rj' groups may be reduced in the reduction step therefore necessitating the use of 1 molar equivalent for example, 2 molar equivalents of the reducing agent. Examples of such Ri' groups include carboxylic acid and aldehyde which may be reduced to a group -CH₂OH.

It is preferred, when preparing compounds of formula (I) in which Rj is ketone, carboxylic acid, aldehyde, CH2E, CH2OH and -C(J)=NOZ, that compounds of formula (II) in which R is carboxylic acid ester are used and that the required interconversion of R is carried out using the procedures hereinafter described.

Interconversions of the group are conventional in the art of organic chemistry, for example, a methyl ester function may be converted to a hydroxymethyl function by the methods outlined in EP-A-0375222 (Beecham Group p.l.c.) and a methyl ester function may be converted to carboxylic acid function by the method outlined in Nicholaou et al., J. Am. Chem. Soc. (1988) 110, 14, 4685 - 4696.

An Ri carboxylic acid group may then be converted to an carboxylic acid ester or amide, aldehyde, ketone or -CH2E group by the methods outlined in EP-A-0350164 (Beecham Group p.l.c), EP-A-0375223 (Beecham Group p.l.c) and EP- Application No. 90313106.8 (Beecham Group p.l.c).

An Ri aldehyde or ketone group may then be converted to a group -C(J)=NOZ by conventional reaction with a compound of formula NH^OZ' or an acid addition salt thereof, wherein Z' is Z as defined in relation to formula (I) or a group convertible thereto and thereafter, optionally or as necessary separating any geometric isomers converting Z', when other than Z to Z, and interconverting Z. The reaction of ketone or aldehyde of formula (I) with a compound NH2OZ' or an acid addition salt thereof is preferably carried out in a suitable solvent such as an ethanol/THF mixture in the presence of a conventional buffer or base.

Values for Z' may include protected forms of Z, for example those described in Greene, T.W. 'Protective groups in Organic Synthesis' New York, Wiley (1981).

Interconversion of Z are conventional in the art of organic chemistry.

R3' amine protection groups are chosen such that they are readily removable subsequent to the initial reduction of a compound of formula (II) to provide a compound of formula (I) in which R3 is a primary amine group.

Values for R3' include 9-fluorenylmethoxycarbonyl, trichloroethoxycarbonyl, 2-methylsulphonylethoxycarbonyl and 2-trimethylsilylethoxycarbonyl.

Preferably R3' is 9-fluorenylmethoxycarbonyl.

Suitable R4' silyl protecting groups include trimethylsilyl, triethylsilyl and t-butyldimethylsilyl.

Preferably R4' is triethylsilyl.

Where R3 in compounds of formula (I) is a primary amine, conversion of a readily removable R3' amine protection group to a R3 primary amine may be carried out under basic conditions.

An R3' amine protection group, such as 9-fluorenylmethoxycarbonyl, may be removed under basic conditions in a solvent such as methanolic dimethyl sulphoxide. Suitable bases for amine deprotection include ammonia, dialkylamines such as dimethylamine and diethylamine, trialkylamines such as triethylamine, cyclic amines and especially cyclic secondary amines such as morpholine, piperazine and more especially piperidine, and diazabicyclic bases such as l,5-diazabicyclo[4.3.0]non-5-ene (DBN) and preferably l,8-diazabicyclo[5.4.0]undec-7-ene (DBU).

The amine deprotection may be carried out using from 1-10 equivalents of base, preferably from 1-2 equivalents, at reduced or elevated temperatures, for example from -30°C to 50°C and preferably from 0°C to room temperature, over a time period ranging from 1 minute to 24 hours and preferably from 30 minutes to 5 hours.

R4' silyl protecting groups in compounds of formula (II) may be removed using known deprotection methods, for example using a solution of hydrogen fluoride-pyridine in tetrahydrofuran or tetrahydrofuran/methanol mixtures at normal or reduced temperature, for example from -10°C to 50°C and preferably from 0°C to room temperature, over a time period up to 60 hours and preferably from 4 to 24 hours.

Intermediate compounds of formula (II) in which Ri' is a carboxylic acid group or ester or amide thereof, an aldehyde or CH2E may be prepared from amphotericin B according to the procedures outlined in EP-A-0350164 (Beecham Group p.l.c), EP-A-0375223 (Beecham Group p.l.c.) EP-A-0375222 (Beecham Group p.l.c.) and EP- Application No. 90313106.8 (Beecham Group p.l.c).

Intermediates of formula (II) in which R ' is a protected hydroxymethyl group may be prepared by conventionally protecting the free hydroxymethyl group of intermediates which may be prepared from amphotericin B according to the procedures outlined in EP-A-0375223 (Beecham Group p.l.c).

Certain compounds of formula (II) are novel and as such form a further aspect of the present invention.

An R3' 9-fluorenylmethoxycarbonyl amine protection group may be introduced by addition of 9-fluorenylmethyl chloroformate to a solution of the primary amine in methanol-dimethylformamide under anhydrous conditions, in the presence of a base such as potassium carbonate. Alternatively an R3' 9-fluorenylmethoxycarbonyl group may be introduced by addition of ϋ-(9-fluorenylmethoxycarbonyloxy)succinimide to a slurry of the primary amine in methanol-dimethylformamide under anhydrous conditions, in the presence of a base such as pyridine.

Free hydroxyl groups may be silylated using standard procedures. The reaction with silyating agents such as trimethylsilyl trifluoromethanesulphonate and triethylsilyl trifluoromethanesulphonate may be carried out in an inert solvent, for example dichloromethane, hexane or diethyl ether, under an inert atmosphere at ambient or reduced temperatures, for example from 0°C to 25°C. The reaction is conveniently effected using an excess of the silylating agent in the presence of a base, for example a pyridine derivative such as 2,6-lutidine. Alternatively, when a liquid, the base may replace the solvent. The reaction time is dependent on the size of the silyl group, ranging from a few minutes for a trimethylsilyl group to several hours for larger silyl groups.

The compounds of the formula (I) and their pharmaceutically acceptable salts are anti-fungal agents, potentially useful in combating fungal infections in animals, including humans. For example they are potentially useful in treating topical fungal infections in man caused by, among other organisms, species of Candida. Trichophvton. Microsporum or Epidermophvton. or in mucosal infections caused by Candida albicans (e.g. thrush and vaginal candidiasis). They may also be used in the treatment of systemic fungal infections caused by, for example Candida albicans. Crvptococcus neoformans. Aspergillus fumigatus. Coccidioides. Paracoccidioides. Histoplasma or Blastomvces spp. They may also be of use in treating eumycotic mycetoma, chromoblastomycosis, and phycomycosis.

The invention further provides a pharmaceutical composition comprising a compound of the formula (I) or a pharmaceutically acceptable salt thereof together with a pharmaceutically acceptable diluent or carrier. The composition is preferably for human use in tablet, capsule, injectable or cream form.

For human use, the antifungal compounds of the formula (I) or pharmaceutically acceptable salts thereof can be administered alone, but will generally be administered in admixture with a pharmaceutical carrier selected with regard to the intended route of administration and standard pharmaceutical practice. For example, they may be administered orally in the form of a tablet containing such excipients as starch or lactose, or in a capsule or ovule either alone or in admixture with excipients, or in the form of an elixir or suspension containing a flavouring or colouring agent.

They may be injected parenterally, for example, intravenously, intramuscularly or subcutaneously. For parenteral administration, they are best used in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic

For oral and parenteral administration to human patients, it is expected that the daily dosage level of the antifungal compounds of the formula (I) will be from 0.1 to 1 mg/kg (in divided doses) when administered by either the oral or parenteral route. Thus tablets or capsules of the compounds can be expected to contain from 5 mg to 0.5 g of active compound for administration singly or two or more at a time as appropriate. The physician in any event will determine the actual dosage which will be most suitable for an individual patient and will vary with the age, weight and response of the particular patient. The above dosages are exemplary of the average case. There can, of course, be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention.

Alternatively, the antifungal compounds of formula (I) can be administered in the form of a suppository or pessary, or they may be applied topically in the form of a lotion, solution, cream, ointment or dusting powder. For example, they can be incorporated into a cream consisting of an aqueous emulsion of polyethylene glycols or liquid paraffin; or they can be incorporated, at a concentration between 1 and 10%, into an ointment consisting of a white wax or white soft paraffin base together with such stabilizers and preservatives as may be required.

Within the indicated dose range, no adverse toxicological effects have been observed with the compounds of the invention which would preclude their administration to suitable patients for the treatment of fungal infections. The present invention further provides a compound of formula (I) or a pharmaceutically acceptable salt thereof for use as an active therapeutic substance.

A compound for use as an active therapeutic substance is intended for use in the treatment of disorders in animals including humans. As stated above, compounds of formula (I) and their pharmaceutically acceptable salts have anti-fungal activity and are potentially useful in combating fungal infections in animals including humans.

Accordingly the present invention further provides a compound of formula (I) or a pharmaceutically acceptable salt thereof for use in the treatment of fungal infections.

The present invention additionally provides a method of treatment of fungal infections in animals, including humans, which comprises administering an effective anti-fungal amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof to the animal.

The present invention also provides the use of a compound of formula (I) or a pharmaceutically acceptable salt thereof for the manufacture of a medicament in the treatment of fungal infections in animals, including humans.

The present invention also provides a pharmaceutical composition for use in the treatment of fungal infections in animals, including humans, which comprises a compound of formula (I) or a pharmaceutically acceptable salt thereof admixed with a pharmaceutically acceptable carrier.

The following Example illustrates the preparation of a compound of the invention and the following Descriptions illustrate the preparation of intermediates thereto. Description 1

N-(9-Fluorenylmethoxycarbonyl)-13-dehydroxy-14-(S)-hydroxy- 3,5,8,9, ll,35-2',4'-octa-0- triethylsilylamphotericin B methyl ester (Dl)

N-(9-Fluorenylmethoxycarbonyl)-13,14-anhydro-3,5,8,9,ll,15,35,2',4'- nona-Q-triethylsilylamphotericin B methyl ester (lg, 0.46 mmol) was dissolved in dry tetrahydrofuran (5ml) under nitrogen. In ice, 1.0M borane-tetrahydrofuran solution (0.51ml, O.δlmmol) was added via a syringe. The solution was stirred in ice for 1 hr. Water (0.40ml) was then added at room temperature to give the boronated intermediate. [The boronated intermediate (N-(9-fluorenylmethoxycarbonyl)-13-dehydroxy- 14-(R)-dihydroxyboryl-3,5,8,9,ll,15,35,2',4'-nona-Q- triethylsilylamphotericin B methyl ester) can be isolated at this stage by dilution of the reaction with diethyl ether. The organic solution was then washed with water, dried with magnesium sulphate and evaporated. The crude intermediate was purified by means of flash chromatography on silica gel using methylene chloride/methanol mixtures.] To prepare the title compound, solid sodium perborate tetrahydrate (0.43g, 2.8mmol) and 2.1M potassium hydroxide solution (21ml, 0.044mmol) were added to the unisolated boronate intermediate solution. After 0.75 hr the reaction was diluted with hexane and washed with water to neutrality. The organic solution was dried with magnesium sulphate and evaporated. The crude product was purified by means of flash chromatography on silica gel using diethyl ether/methylene chloride mixtures to give the title compound (0.19g).

The title compound possessed IR υmax (thin film) 3448 and 1733 cm"¹ UV λmax (thin film) 361, 379 and 400nπr¹ δiH (400MHz) [(CD₃)₂CO]:

7.88 (2H, d, J 7.5Hz), 7.70 (2H, multiplet), 7.43 (2H, multiplet), 7.35 (2H, multiplet), 6.37-6.11 (12H, complex), 5.95 (1H, dd, J 14.9 and 7.4 Hz), 5.64 (1H, dd, J 14.7 and 8.9 Hz), 5.32 (1H, d, J 9.9 Hz), 4.93 (1H, multiplet), 4.50 (2H, complex), 4.36-4.31 (2H, complex), 4.25 (1H, multiplet), 3.97- 3.91 (3H, complex), 3.81 (1H, multiplet), 3.76-3.62 (5H, complex with a singlet at 3.72), 3.47-3.39 (3H, complex with a triplet at 3.45, J 9.1 Hz), 3.32 (1H, multiplet), 3.06 (1H, t, J 8.9 Hz), 2.46 (4H, complex), 1.96-1.53

(10H, complex), 1.24 (3H, d, J 6.1 Hz), 1.20 (3H, d, J 6.2 Hz), 1.08-0.90

(80H, complex) and 0.72-0.60 (54H, complex) ppm.

δ¹ C (67.8 MHz) [(CD₃)₂CO] signals include 172.50, 171.14, 156.30, 145.06, 145.01, 142.15, 137.87, 135.86, 134.12, 133.99, 133.87, 133.66, 133.58, 133.35, 133.17, 132.89, 132.39, 132.05, 131.14, 130.25, 128.45, 127.81, 125.82, 125.71, 120.76, 98.59, 78.59, 77.40, 76.66, 76.45, 76.28, 74.40, 74.33, 73.81, 73.62, 73.39, 72.16, 70.70, 67.50, 67.18, 58.10, 57.58, 52.11, 47.98, 46.93, 43.72, 41.72, 39.61, 37.49, 35.74, 27.45, 19.14, 18.89, 18.43, 11.80, 7.49-7.16 (complex) and 6.30-5.43 (complex) ppm.

Mass spectrum: FAB (3-NOBA/Na matrix) observed mass MNa⁺ 2095. Calculated mass for

2072.3.

The boronated intermediate possessed UV lmax (thin film) 360, 380 and 410nm.

δ!3C (67.8 MHz) [(CD₃)₂CO] signals include 173.81, 171.20, 156.33, 145.16, 145.01, 142.15, 138.36, 135.92, 134.97, 134.44, 133.57, 133.50, 132.89, 132.82, 132.54, 132.48, 131.66, 131.12, 130.30, 128.46, 127.85, 126.29, 126.02, 125.83, 125.71, 120.76, 120.70, 98.83, 95.92, 78.73, 76.95, 76.80, 76.20, 74.36, 73.78, 73.41, 73.06, 72.77, 72.20, 72.02, 71.67, 70.76, 67.60, 67.31, 67.19, 60.13, 58.08, 57.56, 51.99, 47.97, 47.33, 44.12, 42.83, 42.07, 40.77, 40.34, 39.09, 37.91, 35.76, 27.95, 18.98, 18.89, 18.24, 12.14 and 7.55-5.41 (complex) ppm.

Description 2

N-(9-Fluorenylmethoxycarbonyl)-13-dehydroxy-14-(S)-hydroxy amphotericin B methyl ester (D2)

N-(9-Fluorenylmethoxycarbonyl)-13-dehydroxy-14-(S)-hydroxy- 3,5,8,9, ll,35,2',4'-octa-_Q-triethylsilylamphoteriein B methyl ester (77mg, 0.037mmol) was dissolved in dry tetrahydrofuran (5ml) in a plastic bottle. Under nitrogen, hydrogen fluoride.pyridine solution (1.6ml of a solution made from 13.85g of 70% hydrogen fluoride.pyridine reagent and 90ml of pyridine and made up to 250ml with dry tetrahydrofuran - equivalent to 1.9M HF-3.0mmol) was added with a plastic syringe. After 22 hours, the reaction was precipitated in diethyl etheπhexane (1:1, 250ml). The solid was collected by centrifugation and was washed with diethyl ether. The product was purified on silica gel by flash chromatography using methylene chloride/methanol mixtures to give the title compound (35mg).

IR υmax (KBr disc) 3430 and 1718 cm-¹.

UV λmax (methanol) 345, 363, 382 and 406 nm. 6 δ!H (400MHz) [d5-pyridine:d -methanol, 1:1]

7.84 (2H, d, J 7.5Hz), 7.71 (2H, multiplet), 7.42 (2H, t, J 7.4Hz), 7.30 (2H, t, 7.4Hz), 6.69-6.23 (13H, complex), 5.60 (1H, multiplet), 5.51 (1H, dd, J 14.8 and 10.1Hz), 4.73-4.70 (2H, complex including a singlet at 4.73), 4.47 (1H, multiplet), 4.38-4.30 (3H, complex including a doublet at 4.37, J

7.0Hz), 4.22 (2H, complex), 4.19-4.05 (3H, complex including a triplet at 4.10, J 10.4Hz), 3.98 (1H, multiplet), 3.91 (1H, d, J 10.7Hz), 3.84-3.71 (6H, complex including a singlet at 3.81), 3.58 (1H, multiplet), 3.39-3.32 (3H, complex), 2.76 (1H, t, J 10.4Hz), 2.61-2.46 (2H, complex including a dd at 2.49, J 16.8 and 9.6Hz), 2.38-2.15 (3H, complex), 2.09-1.99 (2H, complex), 1.89-1.78 (3H, complex), 1.72-1.47 (8H, complex including a doublet at 1.48, J 6.1Hz), 1.35 (3H, d, J 6.4Hz), 1.24 (3H, d, J 6.4Hz) and 1.16 (3H, d, J 7.1Hz) ppm.

δ¹³C (67.8MHz) [d5-pyridine:d4-methanol, 1:1] signals include 173.35, 172.26, 158.13, 145.08, 144.95, 142.09, 137.55, 136.39, 134.88, 134.81, 134.25, 134.19, 133.99, 133.90, 133.64, 133.24, 132.95, 130.89, 128.50, 127.91, 126.15, 126.10, 120.74, 98.83, 79.03, 76.92, 76.46, 76.29, 75.82, 75.39, 74.99, 73.36, 72.23, 71.69, 71.39, 70.49, 68.62, 68.44, 67.34, 58.43,

57.80, 52.41, 48.10, 44.94, 43.87, 42.86, 42.13, 41.33, 38.51, 36.49, 31.66,

19.10, 18.58, 17.39 and 12.64 ppm.

Mass spectrum: FAB (thiodiethanol Na matrix) observed mass MNa+ 1182.5. Calculated mass for CgsHssNOig, 1159.57.

Description 3

N-(9-Fluorenylmethoxycarbonyl)-16-decarboxy-13-dehydroxy-14- (S)-hydroxy-16-hydroxymethylamphotericin B

N-(9-Fluorenylmethoxycarbonyl)- 13-dehydroxy- 14- (S)-hydroxy- amphotericin B methyl ester (0.126 g, O.llmmol) was dissolved in tetrahydrofuran/methanol (3:1, 5.5ml). At room temperature, sodium borohydride (0.06g, 1.59mmol) was added portionwise causing refluxing of the solvent. The reaction was stirred for 0.5 hr when another batch of sodium borohydride (0.04g, l.lmmol) was added portionwise. After stirring for a further O.δhr, the reaction was precipitated in saturated sodium bicarbonate (250ml). The solid was filtered, washed with water and dried. The crude product was purified by means of medium pressure chromatography on silica gel eluting with the lower phase of mixtures of chloroform:methanol:ammonia to give the title compound (0.019g).

The title compound possessed

IR υmax (KBr disc) 3429 and 1707cm-¹

UV λmax (methanol) 347, 363, 382, 406nm

δ-J-H (400MHz) [dg-dimethylsulphoxide: d4-methanol, 5:1]

7.88 (2H, d, J 7.5Hz), 7.7δ (2H, d, J 7.4Hz), 7.42 (2H, t, J 7.4Hz), 7.34 (2H, complex), 6.49-6.10 (12H, complex), 6.00 (IH, dd, J 15.2 and 8.6Hz), 5.44 (IH, dd, J 14.3 and 10.0Hz), 5.23 (IH, complex), 4.56 (IH, singlet), 4.37 (IH, complex), 4.26 (3H, complex), 3.90 (IH, multiplet), 3.69 (IH, d, J 2.5Hz), 3.60-2.45 (8H, complex including a singlet at 3.50), 3.30-3.10 (6H, complexj, 2.82 {lH, t, J 8.9_tiz), 2.39 (IH, multiplet), 2.31 (IH, multiplet),

2.17 (2H, complex), 1.83 (IH, t, J 11.7Hz), 1.76-1.23 (11H, complex), 1.19

(3H, d, J 5.2Hz), 1.13 (3H, d, J 6.3 Hz), 1.05 (3H, d, J 6.3Hz) and 0.94 (3H, d, J 7.1Hz) ppm.

Mass spectrum: FAB (thiodiethanol/Na matrix) observed mass MNa⁺

1154. Calculated mass for C ₂H85NOιg, 1131.6.

Description 4

N-(9-Fluorenylmethoxyearbonyl)amphotericin B (4- bromophenyl)ester

N-(9-Fluorenylmethoxycarbonyl)amphotericin B (0.30g, 0.26mmol) was dissolved in dry dimethylformamide (2.5ml). Under nitrogen, Hunig's base (0.11ml, 0.63mmol) was added followed sequentially by 4- bromophenol (0.054g, 0.31mmol) and bromo-trispyrrolidino-phosphonium hexafluorophosphate (0.13g, 0.27mmol). The reaction was stirred for 2.5 hr and then precipitated in diethyl ether (250ml). The solid was filtered and washed with ether and water. The dried crude product was purified by medium pressure column chromatography on silica gel eluting with methanol/dichloromethane mixtures to give the title compound (0.17g).

The title compound possessed

IR υmax (KBr disc) 3420 and 1708cm-¹

UV λmax (methanol) 345, 363, 382 and 406mm

δ-^-H (400MHz) [d5-pyridine:d -methanol, 1:1]

7.84 (2H, d, J 7.5Hz), 7.71 (2H, complex), 7.59 (2H, d, J 8.7Hz), 7.41 (2H, multiplet), 7.29 (2H, t, J 7.5Hz), 7.26 (2H, d, J 8.8Hz), 6.69-6.63 (2H, complex), 6.49-6.34 (11H, complex), 5.64 (IH, complex), 5.49 (IH, dd, J 14.7 and 10.1Hz), 5.00 (IH, t, J 9.4Hz), 4.86 (IH, singlet), 4.83 (IH, dt, J 10.9 and 4.4Hz), 4.73 (IH, complex), 4.67 (IH, multiplet), 4.48-4.36 (3H, complex), 4.25-4.22 (2H, complex), 4.03 (IH, dd, J 10.3 and 2.6Hz), 3.96 (1H, complex), 3.88 (IH, d, J 10.7Hz), 3.75 (IH, t, J 9.7Hz), 3.56 (IH, multiplet), 3.36 (IH, multiplet), 2.77 (IH, t, J10.4Hz), 2.59-2.33 (5H, complex including a dd at 2.49, J17.0 and 9.8Hz), 2.15-1.99 (5H, complex),

1.89 (IH, multiplet), 1.79-1.51 (8H, complex), 1.44 (3H, d, J 6.0Hz); 1.36 (3H, d, J 6.5Hz), 1.24 (3H, d, J 6.4Hz) and 1.17 (3H, d, J 7.1Hz) ppm.

δ¹³C (67.8MHz) [d5-pyridine:d4-methanol, 1:1]

172.48, 172.36, 158.09, 151.01, 145.07, 145.01, 142.10, 137.58, 137.35, 134.89, 134.81, 134.17, 134.07, 133.94, 133.67, 133.41, 133.24, 132.98,

130.74, 128.49, 127.91, 126.15, 124.78, 120.74, 119.δ2, 99.95, 98.78, 79.07, 78.36, 76.29, 75.2δ, 74.96, 72.30, 71.83, 71.33, 70.47, 69.80, 68.70, 67.40, 67.17, 66.76, δ8.7δ, 58.49, 55.07, 48.09, 47.38, 45.40, 44.89, 44.02, 71.83, 42.80, 41.21, 40.84, 40.11, 36.37, 31.58, 19.13, 18.57, 17.33 and 12.62 ppm.

Mass spectrum: FAB (thiodiethanol/Na matrix) observed mass MNa⁺ 1323. Calculated mass for CgsHsgHOigBr, 1299.5.

Description 5

N-(9-Fluorenylmethoxycarbonyl)-13,14-anhydro- 3,5,8,9,1 l,15,35,2',4'-nona-0-triethylsilylamphotericin B (4- bromophenyDester

N-(9-Fluorenylmethoxycarbonyl)amphotericin B (4-bromophenyl)ester (1.48g, 1.14mmol) was slurried in dry dichloromethane (25ml). Under nitrogen, 2,6-lutidine (2.38ml, 20.50mmol) was added followed by trimethylsilyl trifluoromethanesulphonate (2.86ml, 14.80mmol). After stirring for lhr, the volatiles were removed under vacuum and the residue was triturated with n-hexane (700ml). The hexane solution was filtered through Celite and then evaporated to give crude pertrimethylsilylated product (1.99g).

Without purification the crude pertrimethylsilylated 13,14-anhydro product (1.99g) was dissolved in the minimum of dry tetrahydrofuran in a plastic container. Under nitrogen, hydrogen fluoride.pyridine solution (41ml of a solution made form 12.11g of 70% hydrogen fluoride.pyridine reagent and pyridine (90ml) and made up to 200ml with dry tetrahydrofuran - equivalent to 2.1M HF - 86.1 mmol) was added and the reaction was stirred for 4 hr. The product was then precipitated in diethyl ether (11) and collected by filtration. Washing with diethyl ether and drying gave the crude desilylated 13,14-anhydro polyene (1.23g).

Without purification the crude 13,14-anhydro polyene (1.23g) was slurried in dry dichloromethane (25ml). Under nitrogen, 2,6-lutidine (2.00ml, 17.2mmol) was added followed by triethylsilyl trifluoromethanesulphonate (2.82ml, 12.5mmol). The reaction was stirred for 2hr and then the volatiles were removed under vacuum. The residue was triturated with n-hexane (11) and the hexane solution was filtered through Celite. The hexane was removed under vacuum. The crude product was purified using medium pressure chromatography on silica gel eluting with dichloromethane/diethyl ether mixtures to give the title compound (0.68g).

The title compound possessed

IR υmax (KBr disc) 2955, 2911, 2877 and 1734cm^{" 1}

UV λmax (hexane) 346, 364, 384 and 407nm

δ!H (400MHz) [dg-acetone]

7.87 (2H, d, J 7.6Hz), 7.69 (2H, d, J 7.5Hz), 7.66 (2H, d, J 8.8Hz), 7.42 (2H, t, J 7.4Hz), 7.32 (2H, complex), 7.19 (2H, d, J 8.8Hz), 6.52-6.11 (12H, complex), 6.04 (IH, dd, J 15.3 and 6.3Hz), 5.56 (IH, dd, J 14.9 and 9.2Hz), 5.31 (IH, d, J 9.7Hz), 4.91 (IH, d, J 8.5Hz), 4.71-4.65 (4H, complex), 4.51 (IH, dd, J 10.5 and 6.4Hz), 4.46 (IH, multiplet), 4.36 (IH, dd, J 10.4 and 6.5Hz), 4.24 (2H, complex), 4.14 (IH, multiplet), 4.02 (IH, complex), 3.85 (2H, complex), 3.75 (3H, complex), 3.57 (IH, multiplet), 3.43 (IH, t, J 9.1Hz), 3.24 (IH, multiplet), 2.92 (IH, dd, J 10.4 and 8.6Hz), 2.58-2.37 (4H, complex), 2.22 (2H, complex), 2.08-1.91 (3H, complex), 1.82-1.63 (5H, complex), 1.51 (IH, multiplet), 1.21 (3H, d, J 5.8Hz), 1.19 (3H, d, J 6.0Hz), 1.08-0.89 (87H, complex) and 0.75-0.55 (54H, complex) ppm.

Mass spectrum: FAB (thiodiethanol/Na matrix) observed mass MNa⁺ 2331. Calculated mass for Ci22H2lθBrNOi8Sig, 2308.

Description 6 5

N-(9-Fluorenylmethoxycarbonyl)- 13-dehydroxy- 14-(S)- hydroxyamphotericin B (4-bromophenyl)ester

N-(9-Fluorenylmethoxycarbonyl)-13,14-anhydro-3, δ, 8, 9, 11, lδ, 3δ, 2', 4',

10 -nona-Q-triethylsilylamphotericin B (4-bromophenyl)ester (646mg,

0.29mmol) was dissolved in dry tetrahydrofuran (δml) under nitrogen. In ice, 1.0M borane-tetrahydrofuran solution (0.3δml, 0.3δmmol) was added and the reaction was stirred at room temperature for lhr. Then a further aliquot of 1.0M borane-tetrahydrofuran solution (0.20ml, 0.20mmol) was lδ added and the reaction was stirred for O.δhr. Water (lml) was then added followed by sodium perborate (132mg, 0.86mmol) and potassium hydroxide solution [O.lδml of a solution made from 126mg of potassium hydroxide (>8δ%) in lml of water, 0.29mmol]. After stirring for 0.8hr more sodium perborate (132mg, 0.86mmol) and potassium hydroxide

20 solution (O.lδml, 0.29mmol) were added. After lhr, the reaction was extracted with diethyl ether (200ml) and the ethereal extracts were washed with water to neutrality. The extracts were dried and evaporated. This crude product was purified using medium pressure silica eluting with mixtures of dichloromethane and diethylether to give a pair of 13-

2δ dehydroxy-14-(S)-hydroxy-octa-0-triethylsilyl isomers (34mg) and (14mg).

Both isomers possessed

Mass spectrum: FAB (3-NOBA/Na matrix) observed mass M+2212 and 30 MNa⁺223δ. Calculated mass for CngHigsBrNOi Sis, 2212.

Without further characterisation both isomers were treated as follows.

The major isomer (31mg, 0.014mmol) was dissolved in dry 3δ tetrahydrofuran (2ml) in a plastic flask. Under nitrogen, hydrogen fluoride.pyridine solution [1.2ml of a solution made from 12.11g of 70% hydrogen fluoride.pyridine reagent and pyridine (90ml) and made up to 200ml with dry tetrahydrofuran - equivalent to 2.1M HF - 2.δmmol] was added and the reaction was stirred for 26hr. The solution was precipitated in diethylether (250ml) and the solid gathered by centrifugation. The product was washed with diethyl ether and dried.

The crude material was purified by flash chromatography on silica gel eluting with dichloromethane/methanol mixtures to give the title compound, (δ.δmg).

The minor isomer (11.4mg, O.OOδmmol) was dissolved in dry tetrahydrofuran (2ml) in a plastic flask. Under nitrogen, hydrogen fluoride.pyridine solution [0.41ml of a solution made from 12.11g of 70% hydrogen fluoride.pyridine reagent and pyridine (90ml) and made up to 200ml with dry tetrahydrofuran - equivalent to 2.1M HF -0.87mmol] was added and the reaction was stirred for 26hr. The solution was precipitated in diethyl ether (2δ0ml) and the solid gathered by centrifugation. The product was washed with diethyl ether and dried. The crude material was purified by flash chromatography on silica gel eluting with dichloromethane/methanol mixtures to give the same title compound, (2.5mg), as that obtained from the major isomer.

The title compound possessed

5^*41 (400MHz) [d₅-pyridine: d₄-methanol, 1:1] 7.84 (2H, d, J7.6Hz), 7.71 (2H, complex), 7.61 (2H, d, J8.7Hz), 7.41 (2H, complex), 7.30 (2H, d, J7.6Hz), 7.27 (2H, d, J8.δHz), 6.72-6.63 (2H, complex), 6.53-6.30 (11H, complex), 5.62 (IH, complex), 5.50 (IH, dd, J14.8 and lO.OHz), 4.83 (IH, s), 4.68 (IH, complex), 4.47 (IH, multiplet), 4.41-4.18 (7H, complex), 4.04- 3.91 (3H, complex), 3.88 (IH, multiplet), 3.75 (IH, t, J9.7Hz), 3.55 (IH, multiplet), 3.45-3.36 (3H, complex), 2.95 (IH, t, 10.4Hz), 2.56 (IH, complex), 2.49 (IH, dd, J16.8 and 9.6Hz), 2.38-2.33 (2H, complex), 2.19 (IH, complex), 2.08-1.99 (3H, complex), 1.80-1.52 (8H, complex), 1.44 (3H, d, Jβ.lHz), 1.35 (3H, d, J6.4Hz), 1.24 (3H, d, J6.4Hz) and 1.16 (3H, d, J7.2Hz) ppm. Description 7

N-(9-Fluorenylmethoxycarbonyl)-13-dehydroxy-14-(S)- hydroxyamphotericin B

δ N-(9-Fluorenylmethoxycarbonyl)-13-dehydroxy-14-(S)-- hydroxyamphotericin B (4-bromophenyl)ester (2δ4mg, 0.20mmol) was dissolved in tetrahydrofuran water, (3:1, 9ml). Under nitrogen, lithium hydroxide solution (0.1ml of a solution made form 19.δmg of lithium hydroxide monohydrate in O.δml of water, 0.09mmol) was added and the

10 reaction stirred. Successive aliquots of lithium hydroxide (0.1ml,

0.09mmol) were added after O.δhr, 1.2hr, 1.8hr and 2.3hr. After stirring for a total of 2.8hr, the reaction was acidified with acetic acid (0.1ml, 1.7δmmol) and precipitated in diethyl ether (1L). The precipitate was filtered then washed with diethyl ether and water. Drying gave the crude lδ product (178mg). This product was purified using medium pressure chromatography on silica gel eluting with the lower phase of chloroform, methanol and ammonia mixtures to give the title compound (86mg).

The title compound possessed

20

IR υmax (KBr disc) 3428 and 1707cm"¹

UV λmax (methanol) 34δ, 364, 383 and 406nm δ-tø (400MHz) [dδ-pyridine: d₄-methanol, 1:1] 7.8δ (2H, d, J7.6Hz), 7.71 (2H, t, J7.δHz), 7.42 (2H, t, J7.4Hz), 7.30 (2H, t, J7.δHz), 6.69-6.60 (2H,

2δ complex), 6.δ5-6.27 (11H, complex), 5.62 (IH, complex), δ.δl (IH, dd,

J14.8 and 10.1Hz), 4.84 (IH, s), 4.69 (IH, complex), 4.48 (IH, multiplet), 4.40-4.33 (3H, complex), 4.29-4.20 (4H, complex), 4.04 (IH, dd, J10.0 and 3.0Hz), 3.98 (IH, multiplet), 3.92 (IH, d, JlO.βHz), 3.87 (IH, multiplet), 3.74 (IH, t, J9.7Hz), 3.δ4 (IH, multiplet), 3.44-3.37 (3H, complex), 2.76

30 (IH, t, 10.3Hz), 2.60-2.46 (3H, complex including a dd at 2.49, J16.8 and 9.6Hz), 2.38-2.31 (2H, complex), 2.21 (IH, complex), 2.11-1.92 (3H, complex), 1.81 (IH, complex), 1.73-1.52 (6H, complex), 1.44 (3H, d, Jβ.OHz), 1.36 (3H, d, J6.4Hz), 1.24 (3H, d, J6.4Hz) and 1.17 (3H, d, J7.2Hz) ppm.

3δ δ¹³C (100.6MHz) [dg-dimethylsulphoxide:d4-methanol, 3:1]

175.19, 171.09, 156.δ6„ 144.41, 144.38, 141.19, 137.06, 134.22, 134.09, 133.84, 133.57, 132.97, 132.91, 132.67, 132.36, 132.29, 131.78, 129.28,

127.97, 127.45, 125.77, 125.70, 120.30, 98.08, 77.74, 76.25, 75.7δ, 7δ.08, 74.94, 74.11, 73.δ2, 72.87, 70.48, 70.25, 69.71, 69.46, 67.10, 67.01, 66.26, 57.60, 57.00, 47.19, 44.69, 42.60, 42.30, 41.35, 40.46, 40.13, 38.01, 5.59, 30.09, 18.64, 18.25, 17.12 and 12.20 ppm.

Mass spectrum: FAB (thiodiethanol Na matrix) observed mass MNa⁺1169. Calculated mass for Cg2H83NOχ ' 1145.6.

Example 1

13-Dehydroxy-14-(S)-hydroxyamphotericin B methyl ester (El)

N-(9-Fluorenylmethoxycarbonyl)- 13-dehydroxy- 14-(S)- hydroxyamphotericin B methyl ester (176mg, O.lδmmol) was dissolved in dimethylsulphoxide/methanol (4ml, 3:1). Under nitrogen, piperidine (30ml, 0.31mmol) was added. After stirring for 1.5hr the reaction was poured into diethyl ether (700ml). The precipitate was filtered and washed with ether. Drying gave the title compound (122mg).

IR υmax (KBr disc) 3426 and 1718 cm'¹ UV λmax (MeOH) 34δ, 363, 382 and 40δnm

δ-~B (400MHz) [d5-pyridine:d₄-methanol, 1:1]

6.70-6.19 (13H, complex), 5.61 (IH, multiplet), 5.δ2 (IH, dd, J 14.5 and 10.1Hz) and 4.69 (2H, complex), 4.47 (IH, multiplet), 4.32 (IH, multiplet), 4.22 (IH, multiplet), 4.13 (IH, t, J 9.5 Hz), 4.08 (IH, t, J 9.6 Hz), 3.97 (IH, multiplet), 3.90 (IH, d, J 10.6 Hz), 3.83-3.68 (4H, complex with a singlet at 3.78), 3.59 (IH, t, J 9.3Hz), 3.50-3.32 (4H, complex), 3.02 (IH, multiplet), 2.76 (IH, t, J 10.4Hz), 2.60-2.46 (2H, complex including a dd at 2.49, J 16.9 and 9.6 Hz), 2.37-2.29 (2H, complex), 2.20 (2H, complex), 2.10- 1.99 (2H, complex), 1.89-1.52 (8H, complex), 1.44 (3H, d, J 6.0Hz), 1.35 (3H, d, J 6.4Hz), 1.25 (3H, d, J 6.4Hz) and 1.16 (3H, d, J 7.1Hz) ppm.

δ¹³C (100.6MHz) [d5-pyridine:d4-methanol, 1:1] signals include 173.96, 172.68, 137.98, 136.78, 135.27, 135.21, 134.64, 134.59, 134.35, 134.25, 134.21, 134.00, 133.62, 133.28, 131.31, 99.29, 79.42, 77.21, 76.80, 76.74, 76.28, 75.81, 75.01, 73.78, 73.71, 72.67, 71.21, 70.98, 69.04, 68.84, 58.07,

57.99, 52.58, 45.29,44.17, 43.30, 42.δ0, 41.79, 40.92, 38.87, 36.83, 32.0δ, 19.47, 18.66, 17.93 and 13.00 ppm.

δ Mass spectrum: FAB (thiodiethanol/Na matrix) observed mass MNa⁺ 960. Calculated mass for C4gH75NOi7, 937. δ.

Example 2 10

16-Decarboxy- 13-dehy droxy- 14- (S)-hydroxy- 16- hydroxymethylamphotericin B

N-(9-Fluorenylmethoxycarbonyl)-16-decarboxy-13-dehydroxy-14-(S)- lδ hydroxy- 16-hydroxymethylamphotericin B (12mg, O.Ollmmol) was dissolved in dimethyl sulphoxide/methanol (3:1, l.δml). Under nitrogen, piperidine (4ml/ 0.041mmol) was added and the reaction was stirred for 1.7 hr and then precipitated in diethyl ether. The precipitate was centrifuged and washed with diethyl ether. Drying gave the title 20 compound (lOmg).

The title compound possessed

δlH (400MHz) [dg-dimethylsulphoxide:d4-methanol, δ:l]

25

6.49-6.09 (12H, complex), 6.00 (IH, dd, J 15.2 and 8.6Hz), δ.44 (IH, dd, J 10.0 and 14.4 Hz), δ.24 (IH, complex), 4.51 (IH, s), 4.37 (IH, complex), 3.90 (IH, multiplet), 3.61-3.51 (7H, complex including a singlet at 3.61), 3.31-3.23 (2H, complex), 3.12-3.08 (3H, complex), 2.93 (IH, t, J 9.3Hz),

30 2.82 (IH, t, J 9.0Hz), 2.42-2.40 (2H, complex), 2.31 (IH, multiplet), 2.20- 2.18 (2H, complex), 1.83-1.20 (13H, complex), 1.17 (3H, d, J 6.1Hz), 1.13 (3H, d, J 6.4Hz), 1.05 (3H, d, J 6.4Hz) and 0.94 (3H, d, J 7.1Hz) ppm.

Mass spectrum: FAB (thiodiethanol Na matrix) observed mass MNa⁺ 932. 3δ Calculated mass for C47H75NOιg, 909.δ. Example 3

13-Dehydroxy-14-(S)-hydroxyamphotericin B

N_-(9-Fluorenylmethoxycarbonyl)-13-dehydroxy-14-(S)-- hydroxyamphotericin B (86mg, 0.07δmmol) was dissolved in dimethylsulphoxide methanol (3:1, 3ml) under nitrogen. Piperidine (11ml, O.llmmol) was added and the reaction was stirred for 2hr. The product was precipitated in diethyl ether (O.δl) and the solid gathered by filtration. The solid was washed with diethyl ether and water then dried to give the title compound (67mg).

The title compound possessed

IR υmax (KBr disc) 3398 and 1718cm"¹

UV λmax (methanol) 34δ, 363, 382 and 405nm δ-~ΕL (400MHz) [dg-dimethylsulphoxide: d4-methanol, 5:1] characteristic signals include 6.50-6.00 (13H, complex), δ.43 (IH, multiplet), 5.26 (IH, complex), 4.54 (IH, s), 4.36 (IH, complex), 3.70 (IH, complex), 2.92 (IH, t, J8.8Hz), 1.99 (IH, t, J10.2Hz), 1.85 (IH, complex), 1.20 (3H, d, J5.2Hz), 1.13 (3H, d, J6.4Hz), 1.06 (3H, d, J6.4Hz) and 0.94 (3H, d, J6.8Hz) ppm.

Mass spectrum: FAB (thiodiethanol/Na matrix) observed mass M- H+2Na+, 968. Calculated mass for C47H73NO17. 923.5.

Pharmacological Data

Method

The Minimum Inhibitory Concentration (MIC) was determined by diluting the test compound in a broth medium in a microtitre tray. The organisms, which had been grown previously in a broth medium, were diluted and added to the wells to provide a final incoulum of approximately 10^ colony-forming units per well. The trays were incubated at 37°C and the turbidity of each well noted at intervals. The MIC was taken as the lowest concentration (in μg/ml) which prevented significant growth.

Results

Minimum Inhibitory Concentration (mg/ml)

(determined after 2 and 3 days incubation)

* Inoculum 10-5 cells/ml YNB: Yeast Nitrogen Base Broth SAB: Sabouraud's Dextrose Broth.

Claims

1. A compound of formula (I) or a pharmaceutically acceptable salt thereof:

wherein Ri is a carboxylic acid group, an ester or amide thereof, a group -X-Y where X is a carbonyl group and Y is hydrogen, Cι_g alkyl, C2- alkenyl, or optionally substituted aryl or heteroaryl a group -C(J)=NOZ where J is hydrogen, Cι_g alkyl, C2-8 alkenyl, optionally substituted aryl Ci.galkyl, aryl or heteroaryl; and Z is hydrogen, optionally substituted Cι_ alkyl, C2-g alkenyl, aryl Cι_g alkyl, heteroaryl Cι_8 alkyl, aryl or heteroaryl; a group -CH2-E where E is hydrogen, halogen, -CN, -N3, -OC(0)R₅, -S(0)_nR₅, -SH, -OC(0)NHR₅, -NHCONHR5 or -NRgR₇, where R5 is hydrogen, optionally substituted Cι_8 alkyl, or aryl, heteroaryl, aryl Cι_4 alkyl or heteroaryl C1.4 alkyl in each of which the aromatic moiety is optionally substituted with the proviso that when E is -S(0)_nR5, R5 does not represent hydrogen, Rg and R7 are independently hydrogen or Cι_g alkyl, or one of Rg and R7 is hydrogen and the other is formyl, C2-8 alkanoyl, dialkoxyphosphoryl, aroyl, heteroaroyl, aryl C1.4 alkanoyl, heteroaryl C1.4 alkanoyl, Cι_8 alkylsulphonyl, arylsulphonyl, heteroarylsulphonyi, aryl C1.4 alkylsulphonyl or heteroaryl C1.4 alkylsulphonyl, where any aromatic moiety in Rg or R7 is optionally substituted, and n is 0, 1 or 2; or R is a group CH2OH; R2 is hydrogen; R3 is an amino group or a derivative thereof; and each R4 is hydrogen.

2. A compound according to claim 1 wherein R3 is an amino group.

3. A compound according to claims 1 or 2 wherein i is methylcarbonyl .

4. A compound according to any one of claims 1 to 3 wherein Ri is methyl.

5 δ. A compound according to any one of claim 1 to 4 wherein Ri is a carboxylic acid group.

6. A compound selected from the gorup consisting of

10 13-Dehydroxy-14-(S)-hydroxyamphotericin B methyl ester,

16-Decarboxy-13-dehydroxy-14-(S)-hydroxy-16-hydroxymethyl- amphotericin B, and 13-Dehydroxy-14-(S)-hydroxyamphotericin B or a pharmaceutically acceptable salt thereof.

lδ

7. A process for the preparation of compounds of formula (I) as defined in claim 1 which process comprises the conversion of a compound of formula (II):

^R3'

wherein R is a carboxylic acid, an ester or amide thereof an aldehyde, a 30 group -CH2E wherein E is as defined in relation to formula (I) or an optionally protected hydroxymethyl group, R3' is an amine protecting group, and each R4' is a silyl protecting group; and thereafter, optionally or as necessary converting Ri' to Ri, interconverting Ri, converting NHR3' to R3, removing the R4' silyl protecting groups, and forming a 3δ pharmaceutically acceptable salt.

8. A pharmaceutical composition comprising a compound of formula

(I) as defined in claim 1 or a pharmaceutically acceptable salt thereof together with a pharmaceutically acceptable diluent or carrier.

9. A compound of formula (I) as defined in claim 1 or a pharmaceutically acceptable salt thereof for use as an active therapeutic δ substance.

10. A compound of formula (I) as defined in claim 1 or a pharmaceutically acceptable salt thereof for use in the treatment of fungal infections.

10

11. A method of treatment of fungal infections in animals, which comprises administering an effective anti-fungal amount of a compound of formula (I) as defined in claim 1 or a pharmaceutically acceptable salt thereof to the animal in need thereof. lδ

12. The use of a compound of formula (I) as defined in claim 1 or a pharmaceutically acceptable salt thereof for the manufacture of a medicament in the treatment of fungal infections in animals.

20 13. A pharmaceutical composition for use in the treatment of fungal infections in animals, which comprises a compound of formula (I) as defined in claim 1 or a pharmaceutically acceptable salt thereof admixed with a pharmaceutically acceptable carrier.