WO1991009047A1 - Amphotericin b derivatives, their production and use - Google Patents

Abstract

A compound of formula (I), or a pharmaceutically acceptable salt thereof, wherein R1 is a carboxylic acid group, a derivative thereof, or hydroxymethyl; R2 is optionally substituted C1-8 alkoxy; and R3 is an amino group or a derivative thereof; with the proviso that when R1 is hydroxymethyl, R2 is not an unsubstituted C1-8 alkoxy group.

Description

Amphotericin B derivatives, their production and use.

The present invention relates to compounds having pharmacological activity, their preparation, compositions containing them and their use in the treatment of fungal infections in animals, including humans.

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

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

Derivatives of amphotericin B are reported in the literature. Nicolaou et al. (J. American. Chem. Soc, 110, 4660, 1988) describe the synthesis of a compound of formula (B) :

wherein R_a is acetyl, R_b is methyl, R_c is methyl, R_d and R_e together are isopropylidene, and either R_f is hydrogen and

SUBSTITUTESHEET R and R_h together are isopropylidene,. or _Rf and R_ together are isopropylidene and R^ is hydrogen.

European Patent Application Publication No. 0 350 164 5 (Beecham) discloses compounds of formula (B) wherein R_a is acetyl or 9-fluorenylmethoxycarbonyl, R_b is hydrogen, R_c is methyl and R^, R_e, Rf, ^~R_Q and R^ are all hydrogen, their preparation and their use as chemical intermediates.

10 No pharmacological activity has been attributed to the aforementioned compounds of formula (B) .

It has now been found that a group of derivatives of amphotericin B having an alkoxy group at the anomeric 1513-ρosition have 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: 20

wherein R^ is a carboxylic acid group, a derivative thereof, or hydroxymethyl; R₂ is optionally substituted C_1-8 alkoxy; 30 and R3 is an amino group or a derivative thereof; with the proviso that when R.^ is hydroxymethyl, R₂ is not an unsubstituted C-^_g alkoxy group.

TITUTESHEET As used herein, the term carboxylic acid group derivative includes esters and amides. Included within the term ester are alkoxycarbonyl, alkenyloxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl, aralkyloxycarbonyl and heteroaralkyloxycarbonyl esters. Amides include primary, secondary and tertiary amides. For example the amine moiety may be substituted by one or two alkyl groups.

Unless otherwise specified, each alkyl or alkenyl group is preferably a C-^_g group, more preferably a C-^-_zj group and may be straight chain or branched. Where R₂ is substituted ^cl-8 ^alkoχy_> suitable substituents include hydroxy and cyano.

The term aryl includes both monocyclic and bicyclic carbocyclic moieties, for example phenyl and naphthyl. An aryl moiety may be mono-, di-, or tri-substituted by groups including carboxy, alkoxyca " ">onyl, hydroxy, alkyl, alkoxy, halogen, and amino optional. substituted by alkyl, and is preferably mono- or di-substituted.

The term halogen includes fluorine, chlorine, bromine and iodine.

he term h eroaryl includes 5- or 6- membered monocyc c and 9- or 10- membered bicyclic heteroaryl.

In addition, 5- or 6- membered monocyclic and 9- or 10- membered bicyclic heteroaryl 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. When 9- or 10- membered bicyclic heteroaryl, the t ?o rings are fused, preferably with one 5- or 6- membered ring containing a single heteroatom.

SUBSTITUTESHEET When used herein, the term amino group derivative includes acyl derivatives, in particular 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 suitable amino group derivatives is described in European Patent Publication 0 010 297 (Schering) , European Patent Publication 0 031 722 (Dumex) and US 4,195,172.

It will be appreciated for the avoidance of doubt that the compounds of formula (B) , disclosed in EP-A 0 350

164 (Beecham) , wherein R_a is acetyl or

9-fluorenylmethoxycarbonyl are not included within the scope of the invention.

Compounds of formula (I) and salts thereof may also form solvates such as hydrates and the invention also extends to these forms. When referred to herein it is understood that a compound of the invention or a salt thereof includes solvates.

Pharmaceutically acceptable salts of compounds of formula (I) may be formed conventionally, for example by reaction with the appropriate acid or base.

Compounds of formula (I) 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 invention also extends to quaternary salts.

Compounds of formula (I) wherein R^_^ is hydroxycarbonyl can form basic addition salts with bases, such as conventional pharmaceutically acceptable bases, for example sodium hydrogen carbonate, potassium carbonate, lithium hydroxide.

ET triethylamine, pyridine, lutidine and.N-methylglucamine.

Suitable values for R-^ include hydroxycarbonyl, methoxycarbonyl, hydroxymethyl, and prop-2-enyloxycarbonyl.

Suitable values for R₂ include methoxy, 2-hydroxyethoxy, 2-cyanoethoxy and n-propyloxy.

Preferably R3 is an amino group.

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

wherein R^' is a carboxylic acid group, a derivative thereof or hydroxymethyl; R₂' is hydroxy and R₂'' is hydrogen, or R₂' and R₂" together are a bond; and R₃' is a protected amino group; with an optionally substituted C^.g alkyl alcohol under acid-catalysed conditions, and thereafter, optionally or as necessary and in any appropriate order converting R-^' when other than R-^, to R^, converting R3' to an R3 amino group, forming an amino group derivative,

SUBSTITUTE SHEET interconverting R-L, interconverting R₂-, and worming a pharmaceutically acceptable salt.

When used herein with respect to R^' , the term carboxylic acid group derivative includes those groups hereinbefore described as carboxylic acid group derivatives for R-^ in formula (I) .

Suitable values for amine protection groups in R3' include trifluoroacetyl, 9-fluorenylmethoxycarbonyl, 2,2,2-trichloroethoxycarbonyl, 2-methylsulphonylethoxy-carbonyl and 2-trimethylsilylethoxycarbonyl. A preferred amine protection group is 9-fluorenylmethoxycarbonyl.

The reaction of a compound of formula (II) with an optionally substituted C-ι_g alkyl alcohol is suitably carried out at ambient temperature under anhydrous conditions in the presence of an acid catalyst such as pyridinium p_-toluene-sulphonate or 10-camphorsulphonic acid. The reaction may be carried out in an inert solvent such as tetrahydrofuran and the C-_j^g alkyl alcohol may act either wholly or partially as the solvent. The reaction may be conveniently carried out in the presence of an H₂0-scavenger such as molecular sieves and under an inert atmosphere.

Preferably, derivatisation of the anomeric 13-position of a compound of formula (II) is carried out using a compound of formula (II) in which R₂' is hydroxy and R₂'' is hydrogen.

Conversion of R-^' to R^ and interconversion of R-^ to further values of R- may be carried out using functional group interconversion procedures suitable for use in the field of polyene macrolide chemistry. For example, an R-^ carboxylic acid may be esterified to give an R^ methyl ester using

SUBSTITUTESHEET diazomethane in an ether solvent at reduced temperatures, or to give an allyl ester using allyl bromide in dimethylformamide in the presence of methanol and a base such as diisopropylethylamine. An allyl ester may be converted to a carboxylic acid by treatment with an amine such as pyrrolidine or morpholine in the presence of a catalytic amount of a palladium (0) catalyst, for example tetrakis (triphenylphosphine)palladium (0) . A methyl ester may be converted to hydroxymethyl by treatment with a reducing agent such as sodium borohydride.

It may be convenient or necessary to carry out these functional group interconversions using compounds of formula (I) in which R is in protected form.

Conversion of a protected amino group R3' to R3 amino may be carried out under basic conditions.

An amine protection group such as trifluoroacetyl may be removed using a base such as ammonia or potassium carbonate in anhydrous methanol.

An amine protection group, such as 9-fluorenylmethoxy-carbonyl, 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 1,8-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

EET 50°C and preferably from 0°C to room temperature, over a time period ranging from 1 minute to 5 hours and preferably from 30 minutes to 2.5 hours.

5 Intermediate compounds of formula (II) in which R₂' is hydroxy and R₂' ' is hydrogen may be prepared from the natural product amphotericin B by protecting the amine function of the 19-position sugar moiety with an amine protection group and, as necessary, derivatising the 10 16-position carboxy group.

Amine protection groups may be introduced by standard procedures. For example, a trifluoroacetyl amine protection group may be introduced by reaction of the primary amine 15 with ethyl trifluoroacetate in the presence of a base such as diisopropylethylamine in amethanol-dimethyl sulphoxide or methanol-dimethylformamide solvent mixture at reduced to normal temperatures, for example at 0°C.

0 A 9-fluorenylmethoxycarbonyl group may be introduced by addition of N-(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. 5

Alternatively, a 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 0 the presence of a base such as potassium carbonate.

Intermediate compounds of formula (II) in which R₂' and R '' together are a bond may be prepared using procedures described in EP-A 0 350 164 (Beecham Group) . 5 Certain precursors to compounds of formula (I) in which the amino group (R3) is in protected form are novel compounds and as such form part of the invention.

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, icrosporum or

Epidermop yton, 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, Cryptococcus neoformans, Aspercrillus 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.

The invention also provides a process for the preparation of a pharmaceutical composition which comprises admixing a compound of formula (I) or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

For human use, an antifungal compound of the formula (I) or a pharmaceutically acceptable salt thereof can be administered alone, but will generally be administered in

SUBSTITUTESHEET admixture with a pharmaceutical carrier selected with regard to the intended route of administration and standard pharmaceutical practice. For example, it 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. A compound may be injected parenterally, for example, intravenously, intramuscularly or subcutaneously. For parenteral administration, a compound is 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 an antifungal compound 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 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, an antifungal compound of formula (I) can be administered in the form of a suppository or pessary, or it may be applied topically in the form of a lotion, solution, cream, ointment or dusting powder. For example, a compound can be incorporated into a cream consisting of an aqueous emulsion of polyethylene glycols or liquid paraffin; or can

S B T be incorporated, at a concentration between 1 and 10%, into an o. ;tment 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.

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 in need thereof.

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 for use in the treatment of fungal infections in animals, including humans.

SUBSTITUTESHEET The following Examples illustrate the-preparation of compounds of formula (I) and the following Descriptions describe the preparation of intermediates thereto. The abbreviations 'TES' and 'Fmoc' are used to represent triethylsilyl and 9-fluorenylmethoxycarbonyl groups respectively.

SUBST Description 1

N-(9-Fluorenylmethoxycarbonyl)amphotericin B (Dl)

Method A

To a solution of amphotericin B (5.0g, 5.4mmol) in dry dimethylsulphoxide (50ml) and dry methanol (15ml) was added dry pyridine(0.53ml, 6.5mmol) . Under a nitrogen atmosphere was added solid N- (9-fluorenylmethoxy-carbonyloxy)succinimide (2.59g, 7.6mmol). After stirring for 1 hr a further portion of - (9-fluorenyl-methoxycarbonyloxy)succinimide (0.28g, O.δmmol) was added. After a further 0.25 hrs glacial acetic acid (0.5ml, 8.7mmol) was added, the solution was diluted with methanol (35ml) and poured into diethyl ether (5L) . The precipitate was filtered, washed with diethyl ether and dried to give the title compound (Dl) which was used without further purification.

Method B

To a solution of amphotericin B (0.50g, 0.54mmol) and anhydrous potassium carbonate (0.17g, 1.2mmol) in dry dimethylsulphoxide (10ml) and dry methanol (2ml) under a

SUBSTITUTE SHEET nitrogen atmosphere at 0°C, was added solid 9-fluorenylmethyl chloroformate (0.21g, O.δlmmol). After stirring for 1 hour a further portion of 9-fluorenylmethyl chloroformate (0.04g, 0.17mmol) was added. After 0.25 hours the reaction mixture was poured into distilled water

(200ml) . The precipitate was collected by centrifugation, dissolved in methanol and evaporated in vacuo. The residue was dissolved in the minimum volume of a mixture of tetrahydrofuran and methanol (1:1) and poured into distilled water (200ml, adjusted to pH 3.2 by the addition of glacial acetic acid) . The preciptate was centrifuged, washed with water and dried n vacuo to give the title compound (Dl) which was used without further purification.

Description 2

N-(9-Fluorenylmethoxycarbonyl)amphotericin B methyl ester (D2)

(D2)

Method A

Crude N-(9-fluorenylmethoxycarbonyl)amphotericin B (Dl) (0.36g, 0.31mmol) was dissolved in 1:1 dimethylsulphoxide and methanol (20ml) . At 0°C and with stirring, a solution of diazomethane in diethyl ether (25ml) was added over 0.3 hours. The diazomethane was generated from; Diazald^R (0.39g, l.δmmol); potassium hydroxide (0.18g, 3.2mmol); water (1ml) and 2-(2-ethoxyethoxy)ethanol (2ml). The reaction was stirred for a further 1.5 hours and then quenched cautiously with glacial acetic acid. The product was precipitated by pouring into diethyl ether. It was collected by centrifugation, washed with diethyl ether, dissolved in methanol and evaporated in vacuo.

5

The crude material was purified by means of medium pressure column chromatography on silica-gel eluting with ethyl acetate/methanol mixtures. The title compound (D2) was obtained as a yellow solid.

10 δ¹H (270MHz) (dg HF/d₄-MeOH) 7.78 (2H,d,J 6.9Hz), 7.70 (2H,d,J 7.4Hz), 7.35 (2H,dd,J 7.4 and 6.3Hz), 7.28 (2H,t,7.4 Hz), 6.63-5.93 (13H, complex), 5.50 (lH,m) , 5.32 (lH,dd,J 10.2 and 14.8Hz), 4.75-4.04 (10H, complex), 3.90-3.0 (8H,

15 complex) , 3.74 (3H,s) 2.50-1.15 (19H, complex), 1.28 (3H,d,J 6.2Hz), 1.20 (3H,d,J 6.3Hz), 1.10 (3H,d,J 6.3Hz) and 0.99 (3H,d,J 7.2Hz)ppm.

Hplc : Reverse phase using : ODS 5μ 250x4.6mm column; eluant 20 80% methanol - 20% pH 3 phosphate buffer - lml/min; detection wavelength 350nm; Retention time 18.8 minutes.

Method B

N-(9-Fluorenylmethoxycarbonyl) amphotericin B (3.11g,

252.7mmol) in dimethylformamide (90ml) was sequentially treated with methanol (10ml), diisopropylethylamine (2.0ml, 11.3mmol) and methyl iodide (10.5ml), and the mixture stirred in a sealed flask for 4 hours. The solution was added to ether (41) , and the precipitate filtered and washed

30 with ether. The solids were then vigorously stirred for 5 minutes in water and refiltered to give, after drying under vacuum, the title product (D2) (2.17g), data as for method A.

SUBSTITUTESHEET Description 3

N-(9-Fluorenylmethoxycarbonyl)-13-0-methyl- amphotericin B (D3)

(D3)

N-(9-Fluorenylmethoxycarbonyl)amphotericin B (Dl) (1.85g, l.δlmmol) and d-10-camphorsulphonic acid (156mg, 0.67mmol) were stirred in dry tetrahydrofuran (10ml) /methanol (60ml) at room temperature under nitrogen. After 15 minutes, triethylamine (0.14ml, 102mg, l.Olmmol) was added, the mixture was filtered, concentrated to ca. 10ml and poured into diethylether/ ri-hexane (800ml 1:1). The precipitated product was collected by centrifugation, washed with diethylether/ ethylacetate (1:1) and dried to give the title compound (D3) as a yellow powder.

HPLC: Reverse phase ODS 5μ 250 x 4.6 mm column; eluent 80% methanol-20% pH 3 phosphate buffer - 1ml.min-¹; detection wavelength 350nm; retention time: 7.6 minutes.

SUBSTITUTESHEET Description 4

N- (9-Fluorenylmethoxycarbonyl)-13-0-methylamphotericin B methyl ester (D4)

(D4)

Method A

N-(9-Fluorenylmethoxycarbonyl) amphotericin B methyl ester (D2) (10.25g, 8.94mmol) suspended in a 3:1 mixture of dry methanol/tetrahydrofuran (1.06L) was treated with pyridininium p_-toluenesulphonate (12.42g, 49.4mmol) . After 2.Oh the reaction mixture was treated with triethylamine (8.26ml, 59.3mmol), concentrated and poured into saturated sodium hydrogen carbonate solution (5 litres) . The yellow product, N-(9- fluorenylmethoxycarbonyl)-13-0-methylamphotericin B methyl ester (D4) was filtered, washed with water and dried under vacuum.

Method B

N-(9-Fluorenylmethoxycarbonyl)amphotericin B methyl ester (D2) (7.38g, 6.36 x 10-^mol) suspended in a 4:1 mixture of dry methanol/tetrahydrofuran (400ml) was treated with

SUBSTITUTESHEET 10-camphorsulphonic acid (1.42g, 5.66-x 10-^mol) , to lower the pH of the reaction mixture to ca. 2.5. After 1.2h the reaction mixture was neutralised with triethylamine (700μl, 5.02 x 10-^mol) , concentrated and poured into diethyl ether. 5 The yellow product, N-(9-fluorenylmethoxycarbonyl)-13-0- methylamphotericin B methyl ester (D4) was filtered, washed with diethyl ether and dried under vacuum.

λmax (EtOH) 407 (εll9,000), 384 (εll2,000), 364 (ε69,000).

10 vmax (nujol) 1720, 1710, 1700cm-¹. δ^-H (400MHz) (C₅H₅D: CD₃0D;1:1) Characteristic signals include:-

1.15 (3H,d,J7.1HZ) , 1.24 (3H,d,J6.5Hz) , 1.34 (3H,d,J6.4Hz) , 1.48 (3H,d,J6.1Hz) , 2.39 (lH,dd,J16.6,3.6Hz) , 2.52

15 (lH,dd,J16.6, 8.7Hz), 3.25 (3H,s), 3.58 (lH,m) , 3.80 (3H,s), 3.97 (lH,m), 4.05 (lH,dd,J10.2, 2.8Hz), 4.22 (lH,br s) , 4.38 (2H,d,J7.2Hz) , 4.45 (lH,m) , 4.55 (lH,m) , 4.78 (lH,s), 4.82 (lH,m), 5.44 (lH,m), 5.63 (lH,dd,J14.2, 9.6Hz), 6.10 (lH,dd,J14.0, 6.6Hz), 7.30 (2H,t,J7.4Hz) , 7.42

20 (2H,t,J7.4Hz), 7.70 (2H,d,J7.4Hz) , 7.85 (2H,d,J7.5Hz) . δ¹³C (67.8MHz) (C₅H₅D:CD₃0D;1:1) 12.39, 17.75, 18.57, 18.89, 30.59, 36.17, 37.85, 41.71, 42.02, 42.91, 43.00, 43.38, 43.65, 44.82, 48.19, 48.82, 52.27, 57.42, 58.54, 67.37, 67.22, 67.66, 68.42, 71.11, 71.43, 71.71, 71.95, 74.58,

2575.06, 75.67, 75.86, 78.40, 99.26, 102.22, 120.76, 126.09, 126.15, 127.91, 128.50, 130.64-134.75(m) , 137.00, 137.64, 142.10, 144.93, 145.10, 158.18, 171.96,174.22.

Hplc: Reverse phase ODS 5μ 250 x 4.6mm column; eluant 78% 30 methanol-22% pH 3 phosphate buffer - lmlmin-¹; detection wavelength 350nm; Retention time: 12.7 minutes.

Mass spectrum FAB (thioglycerol matrix) observed mass 1196 - calculated mass for C₆₄Hg₇N0₁₉Na,1196.6.

ET Mass spectrum (FAB; 3-NOBA:Na) :Found, MNa⁺, 1196.6; ^C64^H87^N018 ^reΦ^jires M, 1173.6.

Description 5

N-(9-Fluorenylmethoxycarbonyl)-13-0-(2-hydroxyethyl)- amphotericin B, methyl ester (D5)

N-(9-Fluorenylmethoxycarbonyl) amphotericin B, methyl ester (D2) (0.69g, 0.60mmol) in tetrahydrofuran (6ml): ethylene glycol (6ml) was stirred till a solution was obtained. This was cooled to 0° and camphorsulphonic acid (45mg, O.lδmmol) added. After 10 minutes stirring at 0°, ethyl acetate (200ml) and sodium bicarbonate solution (100ml) were added. The organic layer was shaken and separated from the aqueous layer, which was twice back-extracted with ethyl acetate (200ml) . The organic layers were washed with water, combined, dried (sodium sulphate) and evaporated to give 0.45g mixture of two main components (tic) . These were separated by chromatography on silica (30g) , eluting with methylene chloride:methanol (10:1). The faster running component was starting material (0.12g); the slower component was the title product (D5) (O.lδg) .

SUBSTITUTE SHEET λmax (MeOH) 402, 381, 362nm. vmax (THF) 3400-3500, 1700-1720cm-¹. δ¹H (270 MHz) (CD3OD) . Characteristic signals include:- 1.00 (3H,d,J 7.15 Hz), 1.11 (3H,d,J 6.6Hz) , 1.20 (3H,d,J 56.3Hz), 1,28 (3H,d), 3.74 (3H,s), 4.36 (2H,d,J 6.9 Hz), 4.61 (lH,s), 5.13 (lH,m), 5.53 (lH,m), 5.90 (lH,m), 7.31 (2H,t,J 7.3Hz), 7.39 (2H,t,J 7.3 Hz) , 7.68 (2H,d,J 7.4Hz) , 7.80 (2H,d,J 7.4Hz), MS (FAB; TDE Na matrix). Found: M+Na⁺, 1226. C₆₅H₈₉NO₂₀ requires M, 1203.6. 0

Description 6

N- (9-Fluorenylmethoxycarbonyl)-13-0-(2-cyanoethyl)- amphotericin B, methyl ester (D6) 5

(D6)

A solution of N-(9-fluorenylmethoxycarbonyl)-amphotericin B methyl ester (D2) (0.516g, 0.44mmol) and 2-hydroxypropionitrile (3.3ml) in anhydrous ,N-dimethylformamide (10ml) containing d-10-camphorsulphonic acid (0.3g) was stirred for three hours at 25°C under a nitrogen atmosphere.

The mixture was partitioned between saturated aqueous potassium hydrogen carbonate solution (100ml) and ethyl acetate (200ml) . The ethyl acetate extract was dried

SUBSTITUTESHEET (Na₂S0₄) and concentrated under reduced pressure. Short-path silica gel chromatography (8:1 dichloromethane-methanol) afforded pure product (30mg) . vmax (KBr) 3420, and 1720cm-¹. 5 δ¹H (270 MHz) (CD3OD) . Characteristic signals include: 1.01 (3H,d,J7.15 Hz) , 1.12 (3H,d,J6.3 Hz) , 1.21 (3H,d,J6.3 Hz), 1.30 (3H,d,J5.8 Hz) , 3.74 (3H,s), 4.35 (2H,d,J6.8Hz) , 4.60 (lH,s), 5.15 (lH,m), 5.44 (lH,m) , 5.87 (lH,dd), 7.30 (2H,t,J7.42 Hz), 7.36 (2H,t,J7.01 Hz), 7.67 (2H,d,J7.42 Hz),

107.78 (2H,d,J7.15 Hz) . δ¹³C (68 MHz, CD3OD) 12.70, 17.62, 18.34, 19.37, 21.38, 26.51, 29.17, 34.36, 35.40, 37.08, 39.20, 42.13, 43.76, 43.88, 44.45, 49.95, 52.81, 58.26, 58.45, 63.53, 68.00, 68.53, 68.δδ, 69.11, 69.51, 71.59, 71.92, 73.22, 74.46,

1575.06, 75.30, 76.09, 80.11, 99.33, 100.79, 120.97, 126.15, 126.30, 128.23, 128.82, 129.65, 132.71, 133.10, 133.39, 133,50, 133.73, 133.85, 134.05, 134.95, 135.21, 135.33, 137.08, 137.15, 142.61, 145.31, 145.43, 158.71, 172.77, 174.91.

20

Description 7

N- (9-Fluorenylmethoxycarbonyl) amphotericin B, allyl ester (D7)

(D7)

35

SUBSTITUTE SHEE N- (9-Fluorenylmethoxycarbonyl) amphotericin B (Dl) (1.24g, l.Oδmmol) in dimethylformamide (40ml) was sequentially treated with methanol (4ml), diisopropylethylamine (0.80ml, 4.52mmol) and allyl bromide (4ml), and the mixture stirred 5 in a sealed flask for 4 hours. The solution was added to ether (21) , and the precipitate filtered and washed with ether. The solids were then stirred thoroughly in water for 5 minutes and refiltered to give a residue which was purified by chromatography on silica, eluting with methylene 10 chloride:methanol (10:1) to give the title product (D7) , (0.53g) .

λmax (MeOH) 404 (£130,000), 381 (εll6,000), 362 (ε72,000). vmax (nujol) 3400, 1725, 1705cm-¹.

15 δ¹H (270MHz) (CD3OD) . Characteristic signals include:- 1.12 (3H,d,J 7.15 Hz), 1.20 (3H,d,J 6.3 Hz), 1.31 (3H,d,J 6.6Hz), 1.42 (3H,d), 4.33 (2H,d,J 6.3Hz) , 4.69 (lH,s), 5.55 (lH,m), 6.01 (lH,m), 7.30 (2H,t), 7.41 (2H,t,J 7.4 Hz) , 7.69 (2H,m), 7.83 (2H,d,J 7.4 Hz).

20 δ¹³C (68 MHz) (CD₃OD:C₅D₅N, 1:1) 12.57, 17.33, 18.49, 19.18, 31.52, 36.35, 38.93, 40.89, 41.41, 42.81, 44.08, 44.δ3, 45.26, 47.57, 46.25, 5δ.32, 5δ.46, 67.58, 68-50, 66.79, 67.22, 68.87, 69.84, 70.64, 71.50, 71.95, 72.47, 74.98, 75.42, 76.29, 77.45, 79.33, 98.60, 99.04, 118.63, 120.83,

25 126.23, 128 .03, 128 . 61, 130. 61, 133.10, 133.39, 133.53, 133.71 , 133. 94 , 134.00, 134.13, 134.29, 134.40 , 134 . 95, 135.03, 137.35, 137.59, 142.26, 145.10 , 145.20, 158.30, 172.52, 173. 93. Mass spectrum (FAB; NOBA-Na matrix) . Found: M+Na⁺, 1208 .

30 C₆₅H₈₇N0₁₉ requires M, 1185. 6.

UBSTITUTE SHEET Description 8

N- (9-Fluorenylmethoxycarbonyl)-13-0- (2-hydroxyethyl)- amphotericin B, allyl ester (D8)

(D8)

N-(9-Fluorenylmethoxycarbonyl) amphotericin B, allyl ester (D7) (0.35g, 0.30mmol) in tetrahydrofuran: ethylene glycol (1:1) (6ml) was cooled to 0°. To the stirred solution was added camphorsulphonic acid (20mg, O.Oδmmol). After 15 minutes, ethyl acetate was added and the mixture washed with sodium bicarbonate solution and water. Drying (Na₂S0₄) and evaporation gave a mixture comprising starting material and a more polar (tic) product which was purified by chromatography on silica (methylene chloride:methanol, 10:1) to give the title compound (D8) , (O.llg). λmax (MeOH) 406 (ε96,000), 383 (ε8δ,000), 363 (ε5δ,000) nm. vmax (THF) 3400-3500, 1725 cm-¹.

Characteristic nmr signals include:- δ^-H (270 MHz) (CD₃OD) 1.00 (3H,d,J 7.15 Hz), 1.12 (3H,d,J 6.6 Hz), 1.20 (3H,d,J 6.3 Hz), 1.29 (3H,d,J 5.0 Hz), 4.36

(2H,d,J 6.9 Hz), 4.59 (1H,S,) 5.14 (lH,m) , 5.25 (lH,d,J 10.4 Hz), 5.39 (lH,dd,J max 17.3 Hz), 5.53 (lH,m) , 5.92 (2H, complex), 7.31 (2H,t,J 7.1 Hz), 7.39 (2H,t,J 7.2 Hz), 7.6δ (2H,d,J 7.2 Hz), 7.79 (2H,d,J 7.2 Hz).

STITUTE SHEET δ¹³C (68 MHz) (CD₃OD) 12.21, 17.91, 18.29, 18.96, 30.13, 35.95, 38.3, 57.57, 58.11, 62.34, 63.01, 99.99, 102.36, 119.09, 120.94, 126.27, 126.21, 126.81, 137.16, 137.55, 142.59, 145.31, 145.40, 158.71, 172.55, 174.08.

Description 9

N- (9-Fluorenylmethoxycarbonyl)-16-decarboxy-13-Q- (2-hydroxyethyl)-16-hvdroxymethyl amphotericin B (D9)

N-(9-Fluorenylmethoxycarbonyl)-16-decarboxy-13-Q- (2-hydroxyethyl)-16-hvdroxymethyl amphotericin B (D9)

N-(9-Fluorenylmethoxycarbonyl)-13-0-(2-hydroxyethyl) amphotericin B, methyl ester (D5) (80mg, 0.066mmol) in methanol (5ml) was treated with sodium borohydride (50mg, 1.32mmol) and the solution warmed to 30°C. Effervescence ceased after 10 min. After 5 minutes further, the cooled (20°C) solution was treated with saturated aqueous ammonium chloride (50ml) . Ethyl acetate (100ml) was added and the mixture shaken. The organic layer was separated, washed with water (50ml) , dried (sodium sulphate) and evaporated to give the crude product which was purified by chromatography on silica (lOg) , eluting with methylene chloride:methanol (8:1) .

5 The title product (D9) (12mg) possessed:- λmax (MeOH) 402, 382, 362nm. δ¹H (270MHz) (CD3OD: C₅D₅N; 1:1). Characteristic signals include:-

101.16 (3H,d, J7.15HZ), 1.25 (3H,d, J6.6Hz), 1.34 (3H,d,

J6.3HZ), 1.47 (3H,d, Jδ.OHz), 2.40 (lH,d d, J16.6, 3.2Hz), 4.37 (2H,d, Jδ.OHz), 4.81 (lH,br t) , 4.89 (lH,s), 5.64 (lH,m), 6.18 (lH,dd, J14.2, 7.2Hz), 7.29 (2H,d, J7.4Hz), 7.42 (2H,d, J7.6HZ), 7.69 (2H,d), 7.85 (2H,d, J7.4Hz).

15 Mass spectrum (FAB; TDE Na matrix). Found: MNa⁺, 1198.5. ^64^H89°19^N requires M, 1175.6.

Description 10

N-(9-Fluorenylmethoxycarbonyl)-13-0-(n-propyl) amphotericin 20 B, allyl ester (DIP)

SUBSTITUTE SHEET N-(9-Fluorenylmethoxycarbonyl) amphotericin B, allyl ester (D7) (2.02g, l.δlmmol) in tetrahydrofuran (200ml): n- propanol (100ml) was treated at room temperature with stirring with camphorsulphonic acid (0.41g, 1.6mmol). After 575 minutes, the solution was added to ethyl acetate and the organic solution washed with sodium bicarbonate solution and water. Drying (Na S0₄) and evaporation gave a mixture comprising starting material and a less polar (tic) product which was purified by chromatography on silica (methylene 10 chloride:methanol; 15:1) to give the title compound (D10) , 0.71g.

λmax (MeOH) 406 (εl37,000), 362 (εl25,000), 363 (ε76,000)nm. Characteristic nmr signals include:-

15 δ'H (400MHz) (CD₃OD-C₅D₅N, 1:1) 0.96 (3H, t, J 7.4Hz), 1.16 (3H, d, J 7.1 Hz), 1.24 (3H, d, J 6.5 Hz), 1.34 (3H, d, J 6.9 Hz), 1.48 (3H, d, J 6.1 Hz), 2.40 (IH, dd, J 16.6, 3.4 Hz), 2.52 (IH, dd, J 16.7, 8.65 Hz), 2.66 (IH, t, J 10.5 Hz), 3.76 (IH, t, J 9.7 Hz), 4.06 (IH, dd, J 10.2, 2.8 Hz),

204.63 (IH, td, J 10.7, 4.5 Hz), 4.80 (IH, s) , 5.31 (IH, d, J 10.7 Hz), 5.50 (IH, dd, J 17.2, 1.35 Hz), 5.61 (IH, dd, J 14.2, 9.6 Hz), 7.30 (2H, t, J 7.4 Hz), 7.42- (2H, t, J 7.4 Hz), 7.71 (2H, m) , 7.84 (2H, d, J 7.5 Hz) ppm.

25 δ¹³C (100 MHz) (CD₃OD:C₅D₅N,l:l) 11.30, 12.41, 17.66, 18.56, 18.9δ, 23.88, 30.58, 36.21, 38.07, 41.63, 41.86, 43.04, 43.14, 43.34, 44.51, 44.84, 48.19, 57.21, 58.53, 62.55, 65.99, 67.39, 67.27, 67.98, 68.03, 68.49, 71.06, 71.52,

30 71 .79, 72.01, 74.72, 75.01, 75.71, 76.19, 78.51 , 99.35, 102.12, 118 .73, 120.73, 126.12 , 127. 90, 128 .49, 130-135 (complex) , 136.95, 137. 64, 142.11, 144. 98 , 145. 09, 158 .14, 172. 01, 173.48 ppm. Mass spectrum (FAB; TDE Na matrix) . Found: M+Na+, 1250 .7 .

35 Cg₈H_g3 0₁₉ requires M, 1227. 6.

SUBSTITUTE SHEET Description 11

N-(9-Fluorenylmethoxycarbonyl)-13-0-methyl amphotericin B, allyl ester (DID

N-(9-Fluorenylmethoxycarbonyl) amphotericin B, allyl ester (D7) (7.0g, 5.9 mmol) was dissolved in dry methanol and dry tetrahydrofuran (3:1, 320 ml) . Under nitrogen, solid pyridinium p_-toluenesulphonate (13.3g, 53.1 mmol) was added and the reaction was stirred for 4 hours. Triethylamine (7.3g, 72.1 mmol) was then added and the reaction was precipitated in saturated sodium bicarbonate (5L) . The precipitate was filtered, washed with water and dried. The crude product was purified by chromatography on flash silica (155g) , eluting with methylene chloride:methanol (10:1) to give the title compound, 2.4g.

vmax (KBr) 3432 and 1718 cm^-1.

Characteristic nmr signals include: δ^-H (400 MHz) [CD₃0D: C₅D₅N; 1:1]

SUBSTITUTE SHEET 7.84 (2H, d, J 7.5 Hz), 7.71 (2H, t, J 7.3 Hz), 7.42 (2H, t, J 7.4 Hz), 7.30 (2H, t, J 7.5 Hz), 6.43 (12H, complex), 6.06 (2H, complex), 5.63 (IH, dd, J 14.2 and 9.5 Hz), 5.51-5.28 (3H, complex including a dd at 5.48, J 17.3 and 1.4 Hz), 54.82-4.73 (4H, complex including a singlet at 4.79), 4.58 (IH, dt, J 10.6 and 4.6 Hz), 4.47-4.37 (3H, complex including a doublet at 4.38, J 7.2 Hz), 4.33 (IH, multiplet), 4.22 (2H, complex), 4.06 (IH, dd, J 10.2 and 2.8 Hz), 3.97 (IH, complex), 3.81-3.74 (2H, complex including a 10 triplet at 3.76, J 9.6 Hz), 3.61-3.46 (3H, complex including a dd at 3.5δ, J 9.1 and 6.2 Hz), 3.24 (3H, s) , 2.64 (IH, t, J 10.5 Hz), 2.52 (IH, dd, J 16.6 and 8.9 Hz), 2.39 (IH, dd, J 16.6 and 3.5 Hz), 2.22 (IH, complex), 1.48 (3H, d, J 6.1 Hz), 1.34 (3H, d, J 6.4 Hz), 1.24 (3H, d, J 6.5 Hz) and 1.15 15 (3H, d, J 7.1 Hz) ppm.

δ¹³C (100 MHz) [CD3OD: C₅D₅N; 1:1]

173.84, 172.36, 158.53, 145.48, 145.35, 142.49, 138.03,

137.32, 135.11, 135.07, 134.64, 134.59, 134.17, 134.04,

20 133.95, 133.68, 133.47, 133.36, 132.81, 130.95, 128.66,

128.28, 126.50, 121.11, 119.22, 102.63, 99.88, 78.81, 76.53, 76.06, 75.37, 75.01, 72.37, 72.17, 71.85, 71.51, 68.84, 68.25, 68.11, 67.76, 67.61, 66.41, 58.87, 57.72, 50.07, 49.20, 48.56, 45.19, 44.11, 43.75, 43.41, 43.26, 42.36,

25 42. 05, 38.41, 36.55, 30.99, 19.32, 18. 92, 18 .10 and 12.76 ppm.

Mass spectrum (FAB : thiodiethanol/Na matrix) . Found: MNa⁺ 1223. 30 C₆₆H₈₉N0₁₉ requires M⁺ 1199.60. -29-

Example 1

13-O-Methyl amphotericin B, methyl ester (El)

(El)

15

N- (9-Fluorenylmethoxycarbonyl)-13-O-methyl amphotericin B, methyl ester (E4) (58mg, 0.05mmol) was dissolved in dimethylsulphoxide (1ml) and methanol (0.3ml) under nitrogen at room temperature and treated with piperidine (lOμl, 20 O.lOmmol) . After stirring for 2 hours, the solution was poured into ether (200ml) and the precipitate filtered and washed with ether. Drying under vacuum gave the title product (El) , (29mg) .

25 vmax (nujol) 1725cm-¹. λmax (MeOH) 406 (εl30,000), 382 (εll9,000), 363 (ε73,100)nm δ¹!! (270MHz) (CD₃OD:C₅D₅N;l:l) 1.15 (3H,d,J 6.9Hz) , 1.25 (3H,d,J 6.6Hz) , 1.34 (3H,d,J 6.3Hz) , 1.46 (3H,d,J 5.5Hz) , 2.61 (lH,t,J 10.7Hz), 2.87 (lH,dd,J 8.8, 2.75Hz), 3.24 30 (3H,s), 3.79 (3H,s), 3.97 (lH,m) , 4.17 (lH,d,J 2.75Hz) , 4.73 (lH,s), 4.83 (lH,m), 5.63 (lH,dd,J 14.0, 10.0Hz), 6.10 (lH,m) .

δ¹³C (67.8MHz) (CD₃OD: C₅D₅N; 1:1) 12.37, 17.72, 18.32, 3518.96, 30.57, 36.15, 37.74, 41.71, 42.99, 43.36, 43.63,

SUBSTITUTESHEET 44.81, 42.00, 42.92, 48.40, 52.13, 57.26, 57.75, 67.24, 67.64, 67.75, 68.40, 71.08, 71.49, 71.93, 74.35, 74.56, 74.76, 75.65, 78.41, 99.50, 102.20, 130.64-134.70 (complex), 137.01, 137.62, 171.94, 174.37. 5 Mass spectrum (FAB; 3NOBA:Na) : Found, M+Na⁺, 974; M⁺ requires 951.

Example 2

10 13-O-Methyl amphotericin B (E2)

20 (E2 )

N-(9-Fluorenylmethoxycarboxyl)-13-O-methyl amphotericin B (D3) (0.26g, 0.22mmol) in dimethyl sulphoxide (4ml) and methanol (1ml) was treated under nitrogen at room 25 temperature with piperidine (50μl, 0.50mmol) . After stirring for 90 minutes, the solution was poured into ether (400ml) and the precipitate filtered and washed with ether.

Drying under vacuum gave the title product (E2) , (0.21g) .

0 vmax (nujol) 1720cm-¹. λmax (MeOH) 406, 382, 363 nm. δ-'-H (270MHz) ((CD₃)₂SO) Characteristic signals include 0.92 (3H,d,J 6.9Hz), 1.04 (3H,d,J 6.05Hz), 1.13 (3H,d,J 6.3Hz), 1.22 (3H,d), 3.05 (3H,s), 4.62 (lH,s), 5.01 (lH,m) , 5.59 (lH,dd), 5.93 (lH,br) (lOOMHz) (C₅H₅D:CD₃OD;l:l) 12.39, 17.67, 18.22, 19.04, 30.67, 36.23, 40.51, 41.52, 41.95, 43.15, 43.34, 43.60, 44.81, 45.29, 49.04, 57.33, 60.8, 68-76 (m), 78.54, 99.4, 5 102.29, 131-140(m), 172.02, 180.0.

Mass spectrum (FAB; 3-NOBA:Na) : Found, M+Na⁺, 960; M⁺ requires 937.

0 Example 2

Alternative synthesis:

13-.0-Methyl amphotericin B, allyl ester (E10) (1.75g, 1.8 mmol) was dissolved in dry tetrahydrofuran and dry methanol (2:1, 30ml). Under nitrogen, pyrrolidine (0.64ml, 7.7mmol) 5 was added followed by solid tetrakis (triphenylphosphine) palladium (0) (0.21g, O.lδmmol) . After 1 hour, the reaction was poured into diethyl ether (5L) . The precipitate was filtered, washed with acetone, washed with diethyl ether and dried. The product was taken up in tetrahydrofuran, ethanol 0 and dimethylsulphoxide and reprecipitated in diethyl ether (2L) . The solid was filtered, washed and dried to give the title compound, (1.26g) . Data was identical to that described above.

5 Example 3

13-0- (2-Hydroxyethyl) amphotericin B, methyl ester (E3;

(E3)

SUBSTITUTE SHEET N-(9-Fluorenylmethoxycarbonyl)-13-0- (2-hydroxyethyl) amphotericin B, methyl ester (D5) (92mg, 0.075mmol) in dimethylsulphoxide (1ml) : methanol (0.3ml) was stirred at room temperature and treated with piperidine (15μl, 50.15mmol) . After two hours at room temperature, the solution was poured into ether (200ml) and the precipitate filtered and washed with ether. Drying under vacuum gave the title product (E3) , (63mg) .

10 λmax (MeOH) 406 (εl04,000), 382 (ε98,000), 363 (ε62,000); vmax (nujol) 3400 (br) , 1720cm-¹.

δ¹H (400MHz) (CD₃OD:C₅D₅N, 1:1). Characteristic signals include 1.06 (3H,d,J 7.1 Hz), 1.17 (3H,d,J 6.4 Hz), 1.25

15 (3H,d,J 6.3 Hz), 1.34 (3H,d,J 5.7Hz), 2.41 (2H,t,J 10.5Hz), 2.63 (lH,dd,J 9.2, 2.9 Hz), 3.76 (3H,s), 3.94 (lH,d,J 2.9 Hz), 4.39 (lH,dt,J 10.8, 4.4 Hz), 4.57 (lH,s), 4.67 (lH,m), 5.24 (lH,m), 5.60 (lH,dd,J 14.0, 9.1Hz), 6.00 (lH,dd,J 14.2, 6.1Hz) .

20 δ¹³C (68MHz) (CD3OD: C₅D₅N,1:1) 12.24, 17.91, 18.18, 18.92, 30.41, 36.00, 37.97, 41.68, 42.62, 42.91, 43.38, 44.34, 44.72, 52.35, 57.56, 57.62, 62.22, 62.97, 67.60, 67.74, 67.80, 68.65, 71.69, 71.91, 72.18, 74.46, 74.84, 75.79, 76.31, 78.77, 100.13, 102.27, 130.43, 132.61, 133.10,

25133.35, 133.77, 133.90, 134.04, 134.49, 134.59, 134.97, 135.01, 137.34, 137.60, 172.34, 174.76.

Mass spectrum (FAB; TDE Na matrix). Found: M+Na⁺, 1004. ^C50^H79°18^N requires M, 961.5. Example 4

13-0- (2-Cvanoethyl) amphotericin B, methyl ester (E4 )

(E4 )

15 A solution of N-(9-fluorenylmethoxycarbonyl)-13-0- (2-cyanoethyl)amphotericin B methyl ester (D6) (30mg, 0.025mmol) in dimethylsulphoxide (0.3ml) and methanol (0.1ml) containing piperidine (5μl) was stirred at 25°C for 1.5 hours under a nitrogen atmosphere.

20

The mixture was poured into ether (100ml) , the solid filtered and washed thoroughly with ether (5 x 100ml) . The solid was dissolved off the filter pad in tetrahydrofuran/methanol (1:1) and the filtrate concentrated

25 giving product (E4) (llmg, 42%) . λmax (MeOH) 402,381, and 362nm. δ¹H (400 MHz) (CD3OD) Characteristic signals include: 1.01 (3H,d7.14 Hz), 1.11 (3H,d,J6.52 Hz) , 1.19 (3H,d,J6.35 Hz), 1.26 (3H,d,J6 Hz), 2.34 (lH,m) , 2.58 (lH,dd,J4.7 Hz and

3012 Hz), 3.72 (3H,s) 4.35 (1H,S), 4.58 (lH,m) , 5.16 (lH,dd, J6.35 Hz and 2.4 Hz), 5.46 (lH,dd,J10 Hz and 14.5 Hz), 5.87 (lH,dd,J15 Hz and 6.15 Hz).

SUBSTITUTESHEET Example 5

13-0- (2-Hydroxyethyl) amphotericin B, allyl ester (E5)

(E5)

N-(9-Fluorenylmethoxycarbonyl)-13-_-(2-hydroxyethyl)-amphote ricin B, allyl ester (D8) (0.13g, 0.105mmol) in dimethylsulphoxide (1ml) and methanol (0.3ml) was treated at room temperature with piperidine (21μl, 0.21mmol) . After 90 minutes, further methanol (1ml) was added and the solution poured into ether (200ml) . The precipitate was filtered and washed with ether to give the title product (E5) (0.10g) . λmax (MeOH) 404, 3δ2, 364nm. ^vmax ^{nu3^{o1) 340}° ⁽br⁾ , 1720 cm-¹. δ¹H (400 MHz) (CD₃OD: C₅D₅N; 1:1). Characteristic signals include:-

1.14 (3H,d,J 7.1 Hz), 1.24 (3H,d,J 6.55 Hz), 1.33 (3H,d,J 6.3 Hz), 1.44 (3H,d,J 5.7 Hz), 2.40 (lH,dd,J 16.6, 3.6 Hz), 2.53 (lH,dd,J 16.6, 8.7 Hz), 2.80 (lH,dd,J 9.2, 3.1 Hz), 3.87 (2H, complex), 3.98 (lH,m) , 4.14 (lH,d,J 3.1 Hz) , 4.32 (lH,m), 4.65 (lH,m), 4.71 (lH,s), 4.76 (2H,d,J 5.5 Hz) , 4.81 (lH,t,J 6.7 Hz), 5.28 (lH,dq,J 10.5, 1.3 Hz), 5.47 (lH,dq,J 17.2, 1.6 Hz), 5.68 (lH,dd,J 14.2, 9.3 Hz), 6.03 (lH,m),

6.15 (lH,dd,J 14.4, 6.1 Hz), 6.3-6.5 (complex).

Mass spectrum (FAB; TDE-Na matrix) . Found: M+Na⁺, 1030 . C₅₂H₈₁0₁₈ requires M, 1007.5. Example 6

13-0- (2-Hvdroxyethyl)amphotericin B (E6)

10

(E6)

15 Under nitrogen in the dark,

13-0-(2-hydroxyethyl)-amphotericin B, allyl ester (E5)

(87mg, 0.0δ6mmol) in tetrahydrofuran (5ml) was treated with pyrrolidine (llμl, 0.13mmol) and tetrakis

(triphenylphosphine) palladium (0) (13mg, 0.012mmol). The 20 solution was stirred for 1 hour, and the precipitate filtered and washed with THF: ether. Drying under vacuum gave the title product (17mg) . λmax (MeOH) 406 (εll4,000), 383 (εl06,000), 364 (δ67,000) nm. 25 vmax (nujol) 3500-2600, 1720, 1565cm-¹. δ-'-H (400MHz) (CD₃OD:C₅D₅N; 1:1) . Characteristic signals include:-

1.15 (3H,d,J7.1HZ), 1.24 (3H,d,J6.5Hz) , 1.27 (3H,d,J6.3Hz) ,

1.44 (3H,d,J6.1Hz), 2.40 (IH,dd,J16.6, 3.5Hz), 3.72 30 (lH,t,J9.6HZ) , 4.40 (lH,d,J3.OHz) , 4.67 (lH,m) , 4.83 (lH,m) ,

4.92 (lH,s), 5.43 (lH,m), 5.65 (lH,m) , 6.19 (lH,dd,J14.0,

6.3Hz) . δ¹³C (66MHz) 12.39, 17.63, 16.29, 18.99, 30.59, 36.21,

38.61, 41.61, 42.13, 42.74, 43.36, 44.37, 44.75, 45.96, 3557.21, 60.75, 62.13, 63.38, 67.49, 67.81, 68.22, 68.45,

69.78, 71.13, 71.48, 72.08, 74.38, 74.51, 75.64, 77.07,

78.34, 99.48, 102.04, 129.66-134.98 (m) , 137.40, 137.58,

171.93, 180.12.

SUBSTITUTESHEET Mass spectrum (FAB; TDE-Na matrix) . Found: MNa⁺, 990. ^C49^H77^N018 retires M, 967.5.

Example 7

16-Decarboxy-13-0- (2-hydroxethyl) -16-hydroxymethyl amphotericin B (E7)

N- (9-Fluroenylmethoxycarbonyl)-16-decarboxy-13-0- (2-hydroxyethyl)-16-hydroxymethyl amphotericin B (D9) (12mg, O.Olmmol) in dimethylsulphoxide (0.3ml): methanol (0.1ml) was treated at room temperature with piperidine (2μl,

0.02mmol). After l³/₄ hours, further methanol (0.2ml) was added and the solution poured into ether (50ml) . The precipitate was collected by centrifugation. Drying gave the title product (E7) (7mg) . λmax (MeOH) 402, 380, 362nm. δ'H (400MHz) (CD₃OD: C₅D₅N; 1:1). Characteristic signals include:-

1.15 (3H,d, J7.1Hz), 1.24 (3H,d, J6.5Hz), 1.34 (3H,d, J6.4Hz), 1.43 (3H,d, J5.7Hz), 2.40 (lH,dd, J16.5, 3.4Hz),

T 2.82 (lH,dd, J9.2, 2.8Hz), 4.05 (lH,dd_y J11.0, 3.7Hz), 4.15 (lH,d, J2.9HZ), 4.19 (lH,dd, J11.0, 3.6Hz), 4.80 (IH, br t) , 4.84 (1H,S), 5.44 (lH,m) , 5.64 (lH,dd, J14.4, 9.4Hz), 6.17 (lH,dd, J14.4, 6.9Hz) . Mass spectrum (FAB; TDE Na matrix) . Found: MNa⁺, 976.

^49^H7 ^17^N retires ^M 953.5.

Example 8

13-0- (n-Propyl) amphotericin B, allyl ester (E8)

N-(9-Fluorenylmethoxycarbonyl)-13-0 (rv-propyl) amphotericin B, allyl ester (D10) (0.66g, 0.54mmol) in dimethylsulphoxide (5ml) : methanol (1.5ml) was treated at room temperature with piperidine (0.11ml, l.lmmol), and the solution stirred for 2 hours. Further methanol (5ml) was added and the solution poured into ether (11) . Solids were fitted, washed with ether and dried under vacuum to give the title product (E8) , 0.42g.

TE SHEET λmax (MeOH) 406 (εl42,000), 382 (εl30,-000) , 363 (ε78,000)nm. Characteristic nmr signals include:- δ'H (400 MHz) (CD₃OD:C₅D₅N, 1:1) 0.99 (3H, t) , 1.16 (3H, d, J 7.15 Hz), 1.24 (3H, d, J 6.5 Hz), 1.34 (3H, d, J 6.4 Hz), 51.45 (3H, d, J 5.7 Hz), 2.22 (IH, dd, J 13.8, 7.5 Hz), 2.40 (IH, dd, J 16.6, 3.5 Hz), 2.53 (IH, dd, J 16.55, 8.55 Hz), 2.66 (IH, t, J 10.4 Hz), 2.80 (IH, dd, J 9.1, 3.0 Hz), 4.13 (IH, d, J 3.3 Hz), 4.74 (IH, s) , 5.29 (IH, brd, J 10.5 Hz), 5.48 (IH, brd, J 17.2 Hz), 5.62 (IH, dd, J 14.2, 9.5 Hz)

10 ppm. δ¹³C (100 MHz) (CD₃OD:C₅D₅N, 1:1) 11.31, 12.39, 17.68, 18.36, 18.96, 23.89, 30.63, 36.21, 37.835, 41.64, 41.91, 42.98, 43.16, 43.37, 44.49, 44.86, 57.21, 57.90, 62.52, 65.91, 67.32, 67.95, 6δ.47, 71.08, 71.82, 71.9δ, 74.70,

15 74.76, 75.70, 78.47, 99.50, 102.10, 118.50, . 130-135 (complex) , 136. 94 , 137. 65, 172. 00, 173.595 ppm.

v ax (KBr) 1725, 3430 (br)cm .-^"1 Mass spectrum (FAB; TDE Na matrix) . Found: M+Na+, 1028 20 C₅3H₈3 0₁₇ requires M, 1005.6.

Example 9

13-0- (n-Propyl) amphotericin B (E9)

25

35

SUBSTITUTE SHEET The title compound was prepared from 13-0-(n-propyl) amphotericin B, allyl ester (E8) in a manner analogous to the preparation of 13-0-(2-hydroxyethyl) amphotericin B (E6) from 13-0-(2-hydroxethyl) amphotericin B, allyl ester (E5) . The product (E9) possessed:- λmax (MeOH) 406, 383, 364nm. Characteristic nmr signals include:- δ'H (400 MHz) (CD₃OD:C₅D₅N, 1:1) 0.99 (3H, t, J 7.4 Hz), 1.16 (3H, d, J 7.1 Hz), 1.24 (3H, d, J 6.4 Hz), 1.34 (3H, d, J 6.3 Hz), 2.40 (IH, dd, J 16.5, 3.1 Hz) 4.92 (IH, s) , 5.59 (IH, dd, J 14.2, 9.7 Hz) ppm.

Mass spectrum (FAB; TDE Na matrix). Found: M+Na+, 988. ^c50^H79^NO17 retires ^M/ 965.6.

Example 10

13-0-Methyl amphotericin B, allyl ester (E10)

9-(N-Fluorenylmethoxycarbonyl)-13-0.-methyl amphotericin B, allyl ester (Dll) (2.38g, 2.0mmol) was dissolved in dimethylsulphoxide and methanol (3:1, 99ml). Under

TESHEET nitrogen, piperidine (0.51g, 5.9mmol)-was added and the reaction was stirred for 1.5 hours. The mixture was precipitated in diethyl ether (5L) and the solid was filtered, washed with diethyl ether and dried to give the 5 title compound, (1.75g).

λmax (MeOH) 406 (εl36,800), 3δ3 (εl26,600)and 364 (ε77,700) mn.

10 Characteristic nmr signals include:- δ^-H (400 MHz) [CD₃0D: C₅D₅N; 1:1]

6.54-6.32 (12H, complex), 6.12-6.00 (2H, complex), 5.63 (IH, dd, J 14.2 and 9.5 Hz), 5.50-5.30 (3H, complex), 4.84-4.72 (3H, complex including a singlet at 4.72), 4.57 (IH, dd, J

1510.8 and 4.5 Hz), 4.45 (IH, multiplet), 4.29 (IH, multiplet), 4.23 (IH, multiplet), 4.15 (IH, d, J 3.0 Hz), 3.97 (IH, multiplet), 3.80 (IH, multiplet), 3.24 (3H, s) , 2.81 (IH, dd, J 9.1 and 3.0 Hz), 2.70-2.49(4H, complex including a dd at 2.52, J 16.6 and 8.δHz), 2.40 (IH, dd, J

20 16.6 and 3.5 Hz) 2.21 (IH, complex), 1.45 (3H, d, J 5.6 Hz), 1.34 (3H, d, J 6.4 Hz), 1.24 (3H, d, J 6.5 Hz) and 1.15 (3H, d, J 7.1 Hz) ppm.

δ¹³C (100 MHz) [CD3OD : C₅D₅N; 1 : 1]

25 173. 98, 172.36, 138. 04, 137.46, 135.12, 135.07, 134. 66, 134.59, 134.18, 134.04, 133.93, 133. 68 , 133.47 , 133.32, 132.80 , 130. δδ , llδ . 99, 102. 62, 100.09, 78. δO , 76.16, 76. 06, 75.14, 75. 03, 72.36, 72 .11, 71.52, 6δ . 83, 68 .24 , 68. 09, 67. 65, 66.32, 5δ .22, 57. 64, 48. 99, 45.20, 44 .09, 43.76,

30 43.41, 43.25, 42.36, 42.05, 40.89, 38.23, 36.54, 30. 97, 19.33, 18 .73, 18 .11 and 12.77 ppm.

Mass spectrum (FAB : thiodiethanol/Na matrix) . Found: MNa⁺, 1000. 35 C₅₁H₇₉N0₁₇ requires M⁺, 977.53.

SUBSTITUTE SHEET

Claims

-41- Claims

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

wherein R^ is a carboxylic acid group, a derivative thereof, or hydroxymethyl; R₂ is optionally substituted C^_ alkoxy; and R is an amino group or a derivative thereof; with the proviso that when R-^ is hydroxymethyl, R₂ is not an unsubstituted C-,_ alkoxy group.

2. A compound according to claim 1 wherein R-^ is a C^_g alkoxy ester group, a C₂_g alkenyloxy ester group, a carboxylic acid group or an hydroxymethyl group.

3. A compound according to claims 1 or 2 wherein R-, is methoxycarbonyl, hydroxycarbony1, or prop-2-enyloxy-carbonyl.

4. A compound according to any one of claims 1 to 3 wherein R₂ is methoxy,2-hydroxy-ethoxy, 2-cyanoethoxy or n- propyloxy.

5. A compound according to any one of claims 1 to 4 wherein R3 is amino.

SUBSTITUTESHEET 6. 13-O-Methyl amphotericin B, methyl ester,

13-O-methyl amphotericin B,

513-0-(2-hydroxyethyl) amphotericin B, methyl ester,

13-0-(2-cyanoethyl) amphotericin B, methyl ester,

13-0- (2-hydroxyethyl) amphotericin B, allyl ester,

10

13-0-(2-hydroxyethyl)amphotericin B,

16-decarboxy-13-0-(2-hydroxethyl)-16-hydroxymethyl amphotericin B, 15

13-0-(n-propyl) amphotericin B, allyl ester,

13-0-(n-propyl) amphotericin B or

2013-0-methyl amphotericin B, allyl ester.

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

25

-43- . wherein R-^' is a carboxylic acid group, a derivative thereof or hydroxymethyl; R₂' is hydroxy and R '' is hydrogen, or R ' and 2 together are a bond; and R3' is a protected amino group; with an optionally substituted C_1- alkyl alcohol under acid-catalysed conditions, and thereafter, optionally or as necessary and in any appropriate order converting Ri' when other than R-^, to R-^, converting R3' to an R3 amino group, forming an amino group derivative, interconverting R-^, interconverting R₂, and forming a pharmaceutically acceptable salt.

8. A pharmaceutical composition comprising a compound of the 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 in the treatment of fungal infections.

10. 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) as defined in claim 1 or a pharmaceutically acceptable salt thereof to the animal in need thereof.

11. 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 for use in the treatment of fungal infections in animals, including humans.

SUBSTITUTESHEET