N-substituted second generation derivatives of antifungal antibiotic amphotericin B and methods of their preparation and application

Abstract

The invention provides semisynthetic N-substituted derivatives of the antifungal antibiotic Amphotericin B and water soluble salts and complexes, pharmaceutical compositions and plant and building treatment products comprising the derivatives and their use as antifungal antibiotics. ##STR00001##

Claims

1. A compound of Formula 1a ##STR00068## or a salt, hydrate or complex thereof; wherein R.sub.1 is: a) a thioureidyl residue of structure ##STR00069##  wherein R.sub.5 is —W—Z; W is an optionally substituted alkyl linker or a bond; Z is an optionally substituted aryl, heteroaryl, nitrogen-containing heterocycloaliphatic, NR*.sub.2, NH.sub.2 or NHR*; and R* is an optionally substituted aliphatic moiety, an optionally substituted carbocyclic moiety, or an optionally substituted heterocyclic moiety; or alternatively two R*, together with the nitrogen atom to which they are bound, form an optionally substituted heterocycle, wherein Z is optionally substituted with one or more of alkyl or halo; or b) benzyl substituted with one or more substituents selected from optionally substituted carbocycle, optionally substituted heterocycle, branched C.sub.3-6alkyl, or dialkylamino; R.sub.2 is a hydrogen atom, an unsubstituted non-branched alkyl, a substituted alkyl, or one of the substituents as defined in R.sub.1; R.sub.3 is a hydroxyl group, alkoxyl group, alkylamino, or —NR.sub.14—(C.sub.1-C.sub.6alkyl)-NR.sub.15R.sub.16, wherein R.sub.14 is a hydrogen atom or methyl, and R.sub.15 and R.sub.16 are independently chosen from hydrogen or optionally substituted aliphatic.

2. A compound according to claim 1, wherein R.sub.2 is a hydrogen atom, a non-branched alkyl, or alkyl substituted with alkylamino or an optionally substituted carbocyclic or N-containing heterocyclic moiety.

3. A compound according to claim 1, wherein R.sub.3 is hydroxyl, methoxy, or —NR.sub.14—(C.sub.1-C.sub.6alkyl)-NR.sub.15R.sub.16, wherein R.sub.14 is a hydrogen atom or methyl, R.sub.15 and R.sub.16 are independently chosen from hydrogen or optionally substituted aliphatic.

4. A compound according to claim 1, selected from: N-[3-(2-piperidin-1-ylethyl)-thioureidyl]amphotericin B, N-[(3-phenyl)thioureidyl]amphotericin B, N-[3-(2-morpholin-1-ylethylo)thioureidyl]amphotericin B, N-{3-[2-(N,N-diethylamino)ethyl]thioureidyl}amphotericin B, N-[3-(pyridin-3-yl)thioureidyl]amphotericin B, N-[3-(2-pirrolidin-1-ylethyl)thioureidyl]amphotericin B, N-{3-[2-(N,N-dimethylamino)ethyl]thioureidyl}-amphotericin B, N-[3-(pyridin-4-ylmethyl)thioureidyl]amphotericin B, N-(4-N,N-diethylaminobenzyl)amphotericin B, N-[(4-biphenyl)-methyl]amphotericin B, N-(4-tert-butylbenzyl)amphotericin B, N-[3-(2-piperidin-1-yl)ethyl]thioureidyl}amphotericin B methyl ester, N-(4-N,N-diethylaminobenzyl)amphotericin B methyl ester, N-{[3-(2-piperidin-1-yl)ethyl]thioureidyl]amphotericin B 3-(N,N-dimethylamino)propylamide, N-(4-N,N-diethylaminobenzyl)amphotericin B 3-(N,N-dimetyloamino)propylamide or a salt, hydrate or complex thereof.

5. A compound, according to claim 1, in the form of a salt with an inorganic or organic base or an inorganic or organic acid.

6. A compound, according to claim 1, in the form of a complex with an inorganic or organic complexing compound.

7. A pharmaceutical composition comprising a compound of claim 1.

8. A method for treating fungal infection in a patient comprising administering to the patient a therapeutically effective amount of a compound according to claim 1, wherein the patient is a human or animal.

9. A method of treating fungal infection in a plant, comprising administering a compound according to claim 1 to a plant.

10. A method of treating a building comprising administering a compound according to claim 1 to a building.

11. A method of treatment as claimed in claim 8, wherein the fungal infection is caused by pathogenic fungi from the group of yeasts and filamentous fungi or a strain of the genus Candida.

12. A compound selected from the group consisting of: N-[N-(2,4,6-trimethylphenyl)succinimidyl]amphotericin B, N-(N-benzylsuccinimidyl)amphotericin B, N-[N-(4-bromophenyl)succinimidyl]amphotericin B, N-[N-(2-tert-butylphenyl)succinimidyl]amphotericin B, N-[N-(4-nitrophenyl)succinimidyl]amphotericin B, N-[N-(2-piperidn-1-ylethyl)succinimidyl]amphotericin B, N-{N-[3-(N,N-dimethylamino)-2,2-dimethylpropyl]succinimidyl}amphotericin B, N-[N-(2-hydroxyethyl)succinimidyl]amphotericin B, N,N-dimethylamphotericin B, N,N-diethylamphotericin B, N,N-di-n-propylamphotericin B, N,N-di[3-(N-piperidin-1-yl)propyl]amphotericin B, N,N-di[3-(4-ethylpiperazin-1-yl)propyl]amphotericin B, N-ethyl-N-fructosylamphotericin B, N-fructosyl-N-n-propylamphotericin B, N-fructosyl-N-(N,N-dimethyl-3-aminopropyl)amphotericin B, N-fructosyl-N[3-(piperidin-1-yl)aminopropyl]amphotericin B, N-L-phenylalanyloamphotericin B, N-L-p-iodophenylalanylamphotericin B, N-D-β-naphtylalanylamphotericin B, N-L-p-nitrophenylalanylamphotericin B, N-methyl-L-(O.sup.γ-tert-butyl)glutamylamphotericin B, N-D-(O.sup.β-tert-butyl)asparagylamphotericin B, N-D-β-(pyridin-3-yl)alanylamphotericin B, N-L-(S-tert-butyl)cystylamphotericin B, N-o-fluorophenylalanylamphotericin B, N-D-(O.sup.γ-tert-butyl)glutamylamphotericin B, N-D-(O-tert-butyl)serylamphotericin B, N-D-phenylglycylamphotericin B, N-(L-N,N-diethylphenylalanyl)amphotericin B, N-D-β-(pyridin-3-yl)alanylamphotericin B methyl ester, and N-D-β-(pyridin-3-yl)alanylamphotericin B 3 -(N,N-dimethylamino)propylamide, or a salt, hydrate or complex thereof.

Description

DETAILED DESCRIPTION OF INVENTION

(1) In a first aspect, the invention provides sterically hindered N-substituted derivatives of Amphotericin B. Accordingly, the invention provides a compound according to the Formula 1a:

(2) ##STR00005##
or a salt, hydrate or complex thereof; wherein R.sub.1 is chosen from a hydrogen atom, optionally substituted alkyl (preferably a non-branched alkyl or a substituted alkyl), a succinimidyl derivative, a glycosyl residue, an optionally substituted aminoacyl residue, or an optionally substituted thioureidyl residue; R.sub.2 is a hydrogen atom or a substituent such as defined for R.sub.1; R.sub.3 is a hydroxyl group, alkoxy group or an alkylamino or aminoalkyl derivative; wherein R.sub.1 and R.sub.2 are not both a hydrogen atom.

(3) In some embodiments a compound of Formula 1a as defined above may be a compound according to Formula 1b:

(4) ##STR00006##
wherein R.sub.1, R.sub.2 and R.sub.3 are as defined in respect of formula 1a; X is absent or present and, when present, X is one or more molecules of base or acid, or complexing compound.

(5) In some embodiments a compound of the invention is a compound according to Formula 1:

(6) ##STR00007##
wherein R.sub.1 is chosen from a hydrogen atom, a non-branched alkyl with 1 to 15 carbon atoms, a spatially extended alkyl with 1 to 15 carbon atoms, including a succinimidyl derivative, an alkyl derivative containing cyclic carbo- or heterocyclic ring moieties with 5 to 8 atoms, a glycosyl residue, an optionally substituted or spatially branched aminoacyl residue, a dialkylaminoacyl residue with 1 to 5 of carbon atoms in the alkyl substituent, a thioureidyl residue optionally substituted with a bulky aliphatic or cyclic substituent containing at least one basic nitrogen atom; R.sub.2 is hydrogen atom or substituents as defined for R.sub.1; R.sub.3 is hydroxyl group, alkoxy or an aminoalkyl derivative; and its water-soluble salts and complexes, where X is one or more molecule of base acid or complexing compound.

(7) In further embodiments, the invention provides a compound of formula 1a, 1b or 1, wherein one or both of R.sub.1 or R.sub.2 (preferably one) is a bulky substituent, represented by an optionally substituted thioureidyl residue, substituted alkyl, a succinimidyl derivative, a glycosyl residue, or an optionally substituted aminoacyl residue, for example of the structure as defined in respect of any of sub-classes (a) to (f) below:

(8) (a) In some embodiments, the invention provides a compound wherein one or both (preferably one) of R.sub.1 or R.sub.2 is a thioureidyl residue of structure

(9) ##STR00008##
and wherein R.sub.5 is —W—Z, wherein W is an optionally substituted alkyl linker or a single bond; and Z is an optionally substituted carbocycle or heterocycle (preferably aryl, heteroaryl or nitrogen-containing heterocycloaliphatic (preferably N-linked heterocycloalkyl)), or NR*.sub.2, NH.sub.2, NHR*, where R* is an optionally substituted aliphatic (preferably lower alkyl), an optionally substituted carbo- or heterocyclic moiety, or two R* form, together with the nitrogen atom to which they are bound, an optionally substituted heterocycle. W may be a single bond, branched or non-branched alkyl, e.g. lower alkyl. In some embodiments, Z is unsubstituted or substituted with one or more of alkyl or halo. Preferably Z is phenyl, piperidinyl, morpholinyl, pyrrolidinyl, pyridinyl or alkylamino, all of which may be substituted.

(10) (b) In some embodiments, the invention provides a compound wherein one or both of R.sub.1 and R.sub.2 is -alkyl (for example, C.sub.1-6alkyl, C.sub.2-6alkyl, or C.sub.3-6alkyl) substituted with an optionally substituted alkylamino or an optionally substituted carbo- or heterocyclic moiety. In some embodiments, one or both of R.sub.1 and R.sub.2 is, independently, -alkyl (for example C.sub.1-6alkyl, preferably C.sub.2-6alkyl, more preferably C.sub.3-6alkyl, C.sub.3alkyl or C.sub.4alkyl) substituted with a dialkylamino or an optionally substituted N-containing heterocycle (preferably heterocycloalkyl and more preferably piperidinyl or piperazinyl). In some embodiments, the heterocycle is N-linked and is unsubstituted or substituted with alkyl. In some other embodiments, one or both of R.sub.1 or R.sub.2 (preferably one) is alkyl (for example C.sub.1-6alkyl, preferably C.sub.1-3alkyl, more preferably C.sub.1alkyl) substituted with an optionally substituted carbocycle (preferably aryl), wherein the carbocycle, when substituted, is preferably substituted with one or more substituents selected from optionally substituted carbocycle or heterocycle, aliphatic (preferably branched C.sub.3-6alkyl), alkylamino, alkoxy nitro, halo (preferably bromo), or alkoxycarbonyl. In some embodiments one of R.sub.1 or R.sub.2 is an optionally substituted benzyl group. Optionally substituted benzyl may preferably be substituted with one or more substituents selected from optionally substituted carbocycle or heterocycle, aliphatic (preferably branched C.sub.3-6alkyl), alkylamino, alkoxy nitro, halo (preferably bromo), or alkoxycarbonyl. In some embodiments, benzyl is substituted with alkylamino (preferably dialkylamino), carbocycle (for example aryl), or branched C.sub.3-6alkyl (preferably tert-butyl, any of which may be optionally substituted.

(11) (c) In some embodiments, the invention provides a compound wherein one or both (preferably one) of R.sub.1 or R.sub.2 is a succinimidyl derivative of structure

(12) ##STR00009##
wherein R.sub.4 is —X—Y, wherein X is an optionally substituted alkyl linker or a single bond; and Y is an optionally substituted carbo- or heterocyclic moiety or —OH, —OR*, —NR*.sub.2, —NH.sub.2, —NHR*, where R* is an optionally substituted aliphatic, an optionally substituted carbo- or heterocyclic moiety or two R* form, together with the nitrogen atom to which they are bound, an optionally substituted heterocycle. X may be a single bond or a branched or non-branched alkyl, e.g. lower alkyl. Preferably, Y is an optionally substituted carbo- or heterocyclic moiety, hydroxyl or a dialkylamino. In some embodiments, R.sub.4 is optionally substituted aryl (preferably phenyl), alkyl substituted with optionally substituted aryl (preferably benzyl), alkyl substituted with optionally substituted N-linked heterocycle or hydroxyl, or alkyl (preferably branched alkyl) substituted with alkylamino (preferably dialkylamino). In any of the above embodiments, where Y is a carbo- or heterocyclic moiety, Y is unsubstituted or substituted and in some embodiments Y may be substituted with one or more of alkyl (preferably non-branched lower alkyl, e.g. methyl or ethyl, or branched C.sub.3-6alkyl, e.g. tert-butyl), nitro or halo (preferably bromo).

(13) (d) In some embodiments, the invention provides a compound wherein one or both (preferably one) of R.sub.1 or R.sub.2 is an aminoacyl residue of structure:

(14) ##STR00010##
wherein R.sub.6 and R.sub.7 are independently chosen from a hydrogen atom or an optionally substituted alkyl (preferably lower alkyl), or R.sub.6 and R.sub.7 can be taken, together with the atom to which they are joined, to form an optionally substituted nitrogen-containing cyclic moiety; R.sub.8 and R.sub.9 are, independently, hydrogen or —U—V, wherein U is an optionally substituted alkyl linker (preferably lower alkyl and preferably unsubstituted) or a single bond and V is an optionally substituted aliphatic, carbocyclic (preferably aryl or napthyl), heterocyclic (preferably heteroaryl or heterocycloalkyl), alkoxy, alkylthio moiety, or ester moiety, any of which may be optionally substituted. In some embodiments, one of R.sub.8 and R.sub.9 is hydrogen. In some embodiments, one of R.sub.8 and R.sub.9 is hydrogen and the other of R.sub.8 and R.sub.9 is —U—V, wherein U is an optionally substituted alkyl linker (preferably lower alkyl) or a single bond and V is an optionally substituted carbo- or heterocycle (preferably aryl, for example phenyl or napthyl, heteroaryl or heterocycloalkyl), —OR.sup.9a, —SR.sup.9a or —C(O)OR.sup.9a, wherein R.sup.9a is optionally substituted branched alkyl (preferably C.sub.3-6alkyl, for example tert-butyl) or optionally substituted carbo- or heterocycle and R.sub.6 and R.sub.7 are as defined above, preferably lower alkyl or hydrogen. In any of the above embodiments, where V is a carbo- or heterocyclic moiety, V may be unsubstituted or substituted, for example, with one or more of alkyl, nitro or halo.

(15) (e) In some embodiments, the invention provides a compound wherein one or both (preferably one) of R.sub.1 or R.sub.2 is an aminoacyl of structure:

(16) ##STR00011##
wherein; R.sub.10 and R.sub.11 independently chosen from a hydrogen atom or an optionally substituted alkyl or R.sub.10 and R.sub.11 can be taken, together with the atom to which they are joined, to form an optionally substituted nitrogen-containing cyclic moiety; R.sub.12 and R.sub.13 are, independently, hydrogen or —U—V, wherein U is an optionally substituted alkyl linker or a single bond and V is a hydrogen atom, or an optionally substituted aliphatic, carbocyclic, heterocyclic, alkoxy, alkylthio moiety or ester moiety, any of which may be optionally substituted; and R.sub.12′ and R.sub.13′ are, independently, hydrogen or alkyl (preferably lower alkyl). Preferably V is a carbocyclic (preferably aryl or napthyl), heterocyclic (preferably heteroaryl or heterocycloalkyl), alkoxy, alkylthio moiety, or ester moiety, any of which may be optionally substituted. In some embodiments one of R.sub.10 and R.sub.11 is hydrogen and the other of R.sub.10 and R.sub.11 is —U—V, wherein U is an optionally substituted alkyl linker (preferably lower alkyl) or a single bond and V is an optionally substituted carbo- or heterocycle (preferably aryl, for example phenyl or napthyl, heteroaryl or heterocycloalkyl), —OR.sup.9a, —SR.sup.9a or —C(O)OR.sup.9a, wherein R.sup.9a is optionally substituted branched alkyl (preferably C.sub.3-6alkyl, for example tert-butyl) or optionally substituted carbo- or heterocycle and R.sub.12, R.sub.12′, R.sub.13 and R.sub.13′ are as defined above, preferably lower alkyl or hydrogen. In any of the above embodiments, where V is a carbo- or heterocyclic moiety, V may be unsubstituted or substituted, for example, with one or more of alkyl, nitro or halo.

(17) (f) In some embodiments, the invention provides a compound wherein one or both (preferably one) of R.sub.1 or R.sub.2 is a glycosyl residue, preferably a fructosyl residue (more preferably fructopyranose residue of structure

(18) ##STR00012##
In some embodiments, wherein one of R.sub.1 or R.sub.2 is a glycosyl residue and the other of R.sub.1 or R.sub.2 is an unsubstituted alkyl or hydrogen, R.sub.3 is hydroxyl. In some other embodiments, one of R.sub.1 or R.sub.2 is a glycosyl residue and the other of R.sub.1 or R.sub.2 is a substituted alkyl as defined in subclass (b), preferably an alkyl substituted with an optionally substituted alkylamino (preferably dialkylamino) or an optionally substituted heterocycle (preferably N-containing heterocycle, and more preferably N-linked heterocycloalkyl).

(19) In some embodiments, one of R.sub.1 or R.sub.2 is as defined in respect of any of subclasses (a) to (f), preferably (a) to (d) and (f), and the other of R.sub.1 or R.sub.2 is hydrogen, unsubstituted alkyl (preferably non-branched), substituted alkyl or a substituent as defined in respect of any of subclasses (a) to (f).

(20) In some embodiments, the invention provides a compound wherein one of R.sub.1 and R.sub.2 is a hydrogen atom or an unsubstituted alkyl (preferably non-branched) or a substituted alkyl (for example as defined in respect of subclass (b)); and the other of R.sub.1 and R.sub.2 is an unsubstituted non-branched alkyl, a substituted alkyl, or a succinimidyl derivative, a glycosyl residue, an optionally substituted aminoacyl residue, or an optionally substituted thioureidyl residue, preferably as defined in respect of any of subclasses (a) to (f). In some preferred embodiments, one of R.sub.1 and R.sub.2 is a hydrogen atom, a non-branched alkyl or alkyl substituted with alkylamino (preferably dialkylamino) or an optionally substituted carbo- or heterocyclic moiety (preferably a N-linked heterocycloalkyl, optionally substituted with alkyl); and the other of R.sub.1 and R.sub.2 is a substituted alkyl (preferably substituted with an optionally substituted carbo- or heterocyclic moiety), a succinimidyl derivative, a glycosyl residue, an optionally substituted aminoacyl residue, or an optionally substituted thioureidyl residue as described in respect of any of subclasses (a) to (f), preferably (a) to (d).

(21) In some embodiments, the invention provides a compound wherein one of R.sub.1 and R.sub.2 is an optionally substituted glycosyl (preferably as defined in respect of subclass (f)) and the other of R.sub.1 and R.sub.2 is a substituted alkyl, a succinimidyl derivative, an optionally substituted aminoacyl residue, or an optionally substituted thioureidyl residue, preferably as described in respect of any of subclasses (a) to (e) above. Preferably one of R.sub.1 and R.sub.2 is an alkyl substituted with an optionally substituted alkylamino (preferably dialkylamino) or carbo- or heterocyclic moiety (preferably a N-linked heterocycloalkyl, optionally substituted with alkyl).

(22) In some embodiments, the invention provides a compound wherein one of one of R.sub.1 and R.sub.2 is a hydrogen atom; the other of R.sub.1 and R.sub.2 is a succinimidyl derivative, an optionally substituted benzyl, an optionally substituted thioureidyl residue an optionally substituted aminoacyl residue, preferably as described above in respect of subclasses (a) to (e), preferably (a) to (d).

(23) In some embodiments, the invention provides a compound wherein one of one of R.sub.1 and R.sub.2 is a hydrogen atom, an unsubstituted alkyl or an alkyl substituted with a carbo- or heterocyclic moiety (preferably a N-linked heterocycloalkyl, optionally substituted with alkyl); the other of R.sub.1 and R.sub.2 is an alkyl substituted with a carbo- or heterocyclic moiety (preferably a N-linked heterocycloalkyl, optionally substituted with alkyl), preferably as defined in respect of subclass (b).

(24) In any of the compounds of the invention as described herein R.sub.3 is a hydroxyl group, alkoxy group or an alkylamino or aminoalkyl derivative. In some embodiments, the invention provides a compound as defined in respect of any of the above embodiments, wherein R.sub.3 is hydroxyl, methoxy, or —NR.sub.14—(C.sub.1-C.sub.6alkyl)-NR.sub.15R.sub.16, wherein R.sub.14 is a hydrogen atom or methyl, R.sub.15 and R.sub.16 are independently chosen from optionally substituted aliphatic, preferably lower alkyl.

(25) In some embodiments, a compound of the invention is not a compound wherein:

(26) (i) R.sub.3 is methoxy, one of R.sub.1 or R.sub.2 is hydrogen, and the other of R.sub.1 or R.sub.2 is:

(27) ##STR00013##

(28) (ii) one of R.sub.1 or R.sub.2 is hydrogen, the other of R.sub.1 or R.sub.2 is

(29) ##STR00014##
and R.sub.3 is

(30) ##STR00015##

(31) (iii) one of R.sub.1 or R.sub.2 is hydrogen or unsubstituted alkyl, and the other of R.sub.1 or R.sub.2 is glycosyl;

(32) (iv) R.sub.3 is OH and both of R.sub.1 and R.sub.2 are 2-aminoethyl, 3-aminopropyl, 3-(Fmoc-amino)propyl, 3-hydroxypropyl, 2,6-diaminohexyl, 3-carboxypropyl, 3-(methyoxycarbonyl)propyl or 2-guanidinoethyl;

(33) (v) both of R.sub.1 and R.sub.2 are 3-aminopropyl or 3-(Fmoc-amino)propyl and R.sub.3 is methoxy, 2-aminoethylamino, 2-(dimethylamino)ethylamino or 3-(4-morpholino)propylamino; or

(34) (vi) R.sub.3 is hydroxyl, one of R.sub.1 or R.sub.2 is hydrogen or 2-aminoethyl, and the other of R.sub.1 or R.sub.2 is 3-aminopropyl or 3(Fmoc-amino)propyl;

(35) or a salt, hydrate or complex thereof.

(36) In some embodiments a compound of the invention is not N-succinyl Amphotericin B, or a salt, hydrate or complex thereof.

(37) In some embodiments, a compound of the invention is not a compound wherein one of R.sub.1 or R.sub.2 is:

(38) ##STR00016##
or a salt, hydrate or complex thereof.

(39) In some embodiments, the invention provides a compound of any of the subclasses described above wherein R.sub.1 is any of the groups as listed for R.sub.1 in Table 1.

(40) In some embodiments, the invention provides a compound of any of the subclasses described above wherein R.sub.2 is any of the groups as listed for R.sub.2 in Table 1.

(41) In some embodiments, the invention provides a compound of any of the subclasses described above wherein R.sub.3 is any of the groups as listed for R.sub.3 in Table 1.

(42) In further embodiments, a compound as described herein may be provided in the form of a salt with an inorganic or organic base, preferably as salt with N-methylglucamine.

(43) In further embodiments, a compound as described herein may be provided in the form of a complex with an inorganic or organic complexing compound, preferably as a complex with calcium salt, succinic acid, sodium deoxycholate or a sterol (most preferably with sodium deoxycholate).

(44) In further embodiments, a compound as described herein may be provided in form of salt with an inorganic or organic acid, preferably with aspartic acid.

(45) Any of the salts or complexes as described above may be water soluble.

(46) In another embodiment, the invention provides a compound of Formula 1

(47) ##STR00017##
where R.sub.1 is hydrogen atom, or alkyl substituent simple or spatially enlarged with 1 to 15 carbon atoms in chain, advantageously as a residue of succinimidyl derivatives or alkyl containing carbo- or heterocyclic ring moieties with ring size 5 to 8 atoms, glycosyl residue, or spatially branched aminoacyl residue or dialkylaminoacyl residue with 1 to 5 carbon atoms in alkyl substituent, thioureidyl residue containing basic nitrogen atom and spatially branched aliphatic or cyclic substituents; R.sub.2 is hydrogen atom or substituents such as designed for R.sub.1 R.sub.3 is hydroxyl group or alkoxyl or alkylamino or an aminoalkyl derivative and their water soluble salts or complexes, where X is one or more molecules of base or acid or complexing compound.

(48) According to the invention, advantageous N-substituted derivatives, of Formula 1, 1a or 1b, characterized by the presence of bulky substituent at amino group of mycosamine moiety, able to induce steric hindrance effect, were obtained in several exemplary versions, all of which are exemplary embodiments of the invention:

(49) Succinimidyl derivatives of Amphotericin B, exemplary including: N—[N-(2,4,6-trimethylphenyl)succinimidyl]amphotericin B, N—(N-benzylsuccinimidyl)amphotericin B, N—[N-(4-bromophenyl)succinimidyl]amphotericin B, N—[N-(2-tert-butylphenyl)succin imidyl]amphotericin B, N—[N-(4-nitrophenyl)succynimidyl]amphotericin B, N—[N-(2-piperidin-2-ylethyl)succinimidyl]amphotericin B, N-{N-[3-(N,N-dimethylamino)-2,2-dimethylpropyl]succinimidyl}amphotericin B, N—[N-(2-hydroxy-ethyl)succynimidyl]amphotericin B.

(50) In another version of the invention, N-substituted derivatives can be N-thioureidyl derivatives of Amphotericin B, exemplary including: N-[3-(2-piperidin-1-yl)ethylthioureidyl]amphotericin B, N-[(3-phenyl)-thioureidyl]amphotericin B, N-[3-(2-morpholin-1-yl)ethylthioureidyl]amphotericin B, N-{3-[2-(N,N-diethylamino)ethyl]thioureidyl}amphotericin B, N-[3-(pyridin-3-yl)thioureidyl]amfotericin B, N-[3-(2-pirrolidin-1-yl ethyl)thioureidyl]amphotericin B, N-{3-[2-(N,N-dimethylamin)ethyl]thioureidyl}amphotericin B, N-[(3-(pyrydin-4-ylmethyl)thioureidyl]amphotericin B.

(51) In further version according to the invention, N-substituted derivatives can be N,N-dialkyl derivatives of Amphotericin B containing carbocyclic ring, exemplary including: (N,N-dialkylaminobenzyl)amphotericin B or N-alkyl derivatives not containing ring system chosen from the group including: N,N-dimethylamphotericin B, N,N-diethylamphotericin B, N,N-di-n-propylamphotericin B, N,N-di[3-(N-piperidin-1-yl)propyl]amphotericin B, N,N-di[3-(4-ethylpiperazin-1-yl)propyl]amphotericin B, N-(4-N,N-diethylaminobenzyl)amphotericin B, N-[(4-biphenyl)-methyl]amphotericin B, N-(4-tert-butylbenzyl)amphotericin B.

(52) According to another version of the invention, N-substituted derivatives can be N-alkyl derivatives of N-fructosylamphotericin B, exemplary including: N-fructosyl-N-methylamphotericin B, N-fructosyl-N-ethylamphotericin B, N-fructosyl-N-n-propylamphotericin B, N-fructosyl-N—(N,N-dimethyl-3-aminopropyl)amphotericin B, N-fructosyl-N-[3-(piperidin-1-yl)aminopropyl]amphotericin B.

(53) N-substituted derivatives according to the invention can also be N-aminoacyl or N,N-dialkylaminoacyl derivatives of amphotericin B, exemplary including: N-L-phenylalanylamphotericin B, N-L-p-iodophenylalanylamphotericin B, N-D-β-naphtoalanylamphotericin B, N-L-p-nitrophenylalanylamphotericin B, N-methyl-L-(O.sup.γ-tert-butyl)glutamylamphotericin B, N-D-(O.sup.β-tert-butyl)asparagyl amphotericin B, N-D-β-(3-(pyridin-3-yl)alanylamphotericin B, N-L-(S-tert-butyl)cystylamphotericin B, N-o-fluorophenylalanylamphotericin B, N-D-(O.sup.γ-tert-butyl)glutamylamphotericin B, N-D-(O-tert-butyl)-serylamphotericin B, N-D-phenylglycylamphotericin B, N-(L-N,N-diethylphenylalanyl)amphotericin B, N-(L-N,N-dimethylphenyl-alanyl)amphotericin B.

(54) According to the invention N-substituted derivatives of Amphotericin B can be their esters, exemplary including: N-D-β-(pyridin-3-yl)alanylamphotericin B methyl ester, N-[3-(2-piperidin-1-ylethyl)thioureidyl]amphotericin B methyl ester, N-(4-N,N-diethylaminobenzyl)amphotericin B methyl ester.

(55) In another version of the invention N-substituted derivatives can be amide derivatives of Amphotericin B, exemplary including: N-D-β-(pyridin-3-yl)alanylamphotericin B 3-(N,N-dimethylamin)propylamide, N-[3-(2-piperidin-1-ylethyl)thioureidyl]amphotericin B 3-(N,N-dimethylamin)propylamide, N-(4-N,N-diethylaminobenzyl) amphotericin B 3-(N,N-dimethylamino)propylamide.

(56) In further version according to the invention N-substituted derivatives can be water soluble salts with inorganic or organic bases, advantageously salts with N-methylglucamine. In another advantageous solution according to the invention, N-substituted derivatives can be water soluble complexes with inorganic or organic complexing compounds.

(57) N-substituted derivatives according to the invention can also be water soluble salts with inorganic or organic acids, advantageously with aspartic acid.

(58) According to the invention the application of above described N-substituted derivatives concerns production of drugs for the treatment of diseases caused by fungal microorganism such as pathogenic yeasts or filamentous fungi or a strain of the genus Candida, especially by multidrug resistant (MDR) strains with overexpression of protein transporters MDR1p, as Cdr1p and Cdr2p.

(59) The application of N-substituted derivatives according to the invention concerns also the production of specimens for the control of fungal infections in veterinary, plant protection as well as for protection of buildings from fungal invasion.

(60) The subject of the invention is evidenced below in the examples. In advantageous examples according to the invention, the compounds of structure presented in figure 1, characterized by the presence of bulky moieties, linked to amino group of mycosamine residue which can induce steric hindrance effect, were exemplary obtained in a number of versions presented below.

(61) N-succinimidyl derivatives of the specification from A1 to A8, exemplary include: N—[N-(2,4,6-trimethylphenyl)succinimidyl]amphotericin B (A1), N—(N-benzylsuccinimidyl)amphotericin B (A2), N—[N-(4-bromophenyl)succinimidyl]amphotericin B (A3), N—[N-(2-tert-butylophenyl)succinimidyl]amphotericin B (A4), N—[N-(4-nitrophenyl)succinimidyl]amphotericin B (A5), N—[N-(2-piperidin-1-ylethyl)succinimidyl]amphotericin B (A6), N-{N-[3-(N,N-dimethylamino)-2,2-dimethylpropyl]succinimidyl}amphotericin B (A7), N—[N-(2-hydroxyethyl)succinimidyl]amphotericin B (A8);

(62) N,N-dialkyl derivatives are from A9 to A13 and exemplary including: N,N-dimethylamphotericin B (A9), N,N-diethylamphotericin B (A10), N,N-di-n-propylamphotericin B (A11), N,N-di[3-(N-piperidin-1-yl)propyl]amphotericin B (A12), N,N-di[3-(4-ethylpiperazin-1-yl)propyl]amphotericin B (A13);

(63) N-alkyl derivatives of N-fructosylamphotericin B are from A14 to A18 and exemplary including: N-fructosyl-N-methylamphotericin B (A14), N-ethyl-N-fructosylamphotericin B (A15), N-fructosyl-N-n-propylamphotericin B (A16), N-fructosyl-N—(N,N-dimethyl-3-aminopropyl)amphotericin B (A17), N-fructosyl-N-[3-(piperidin-1-yl)aminopropyl]amphotericin B (A18);

(64) N-benzyl derivatives are from A19 to A21 and exemplary including: N-(4-N,N-diethylaminobenzyl)amphotericin B (A19), N-[(4-biphenyl)methyl]amphotericin B (A20), N-(4-tert-butylbenzyl)amphotericin B (A21);

(65) N-thioureidyl derivatives are from A22 to A29 and exemplary including: N-{[3-(2-piperidin-1-yl)ethyl]thioureidyl}amphotericin B (A22), N-[(3-phenyl)-thioureidyl]amphotericin B (A23), N-{[3-(2-morpholin-1-yl)ethyl]thioureidyl}amphotericin B (A24), N-{3-[2-(N,N-diethylamino)ethyl]thioureidyl}amphotericin B (A25), N-[3-(pyridin-3-yl)thioureidyl]amphotericin B (A26), N-{[3-(2-pyrrolidin-1-yl)ethyl]thioureidyl}amphotericin B (A27), N-{3-[2-(N,N-dimethylamino)ethyl]thioureidyl}amphotericin B (A28), N-{[3-(pyridin-4-yl)methyl]thioureidyl}amphotericin B (A29);

(66) N-aminoacyl derivatives from A30 to A41 and exemplary including: N-L-phenylalanylamphotericin B (A30), N-L-p-iodophenylalanylamphotericin B (A31), N-D-β-naphtylalanylamphotericin B (A32), N-L-p-nitrophenylalanylamphotericin B (A33), N-methyl-L-(O.sup.γ-tert-butyl)glutamylamphotericin B (A34), N-D-(O.sup.β-tert-butyl)asparagylamphotericin B (A35), N-D-β-(pyridin-3-yl)alanylamphotericin B (A36), N-L-(S-tert-butyl)cystylamphotericin B (A37), N-o-fluorophenylalanylamphotericin B (A38), N-D-(O.sup.γ-tert-butyl)glutamylamphotericin B (A39), N-D-(O-tert-butyl)serylamphotericin B (A40), N-D-phenylglycylamphotericin B (A41);

(67) N,N-dialkyloaminoacyl derivatives of Amphotericin B from A42 to A43 and exemplary including: N-(L-N,N-diethylphenylalanyl)amphotericin B (A42), N-(L-N,N-dimethylphenylalanyl)amphotericin B (A43);

(68) Esters of N-substituted derivatives of Amphotericin B from A44 to A46 and exemplary including: N-D-β-(pyridin-3-yl)alanylamphotericin B methyl ester (A44), N-{[3-(2-piperidin-1-yl)ethyl]thioureidyl}amphotericin B methyl ester (A45), N-(4-N,N-diethylaminobenzyl)amphotericin B methyl ester (A46).

(69) Amides of N-substituted derivatives of Amphotericin B from A47 to A49 and exemplary including: N-D-β-(pyridin-3-yl)alanylamphotericin B 3-(N,N-dimethylamino) propylamide (A47), N-{[3-(2-piperidin-1-yl)ethyl]thioureidyl}amphotericin B 3(N,N-dimethylamino)propylamide (A48), N-(4-N,N-diethylaminobenzyl)amphotericin B 3-(N,N-dimethylamino)propylamide (A49).

(70) Structure of Amphotericin B derivatives, according to the invention, are presented below in Table 1. Amphotericin B structure is provided for reference purposes.

(71) TABLE-US-00001 TABLE 1 Structure of Amphotericin B derivatives. Lp R1 R2 R3 Symbol —H —H —H AmB N-succinimidyl derivatives  1. —H —OH A1  2. —H —OH A2  3. 0 —H —OH A3  4. —H —OH A4  5. —H —OH A5  6. —H —OH A6  7. —H —OH A7  8. —H —OH A8 N,N-dialkyl derivatives  9. —CH.sub.3 —CH.sub.3 —OH A9 10. —CH.sub.2CH.sub.3 —CH.sub.2CH.sub.3 —OH A10 11. —CH.sub.2CH.sub.2CH.sub.3 —CH.sub.2CH.sub.2CH.sub.3 —OH A11 12. —OH A12 13. —OH A13 N-alkyl-N-fructosyl derivatives 14. 0 —CH.sub.3 —OH A14 15. —CH.sub.2CH.sub.3 —OH A15 16. —CH.sub.2CH.sub.2CH.sub.3 —OH A16 17. —OH A17 18. —OH A18 N-benzyl derivatives 19. —H —OH A19 20. —H —OH A20 21. —H —OH A21 Thioureidyl derivatives 22. 0 —H —OH A22 23. —H —OH A23 24. —H —OH A24 25. —H —OH A25 26. —H —OH A26 27. —H —OH A27 28. —H —OH A28 29 —H —OH A29 N-aminoacyl derivatives 30. —H —OH A30 31. —H —OH A31 32. 0 —H —OH A32 33. —H —OH A33 34. —H —OH A34 35. —H —OH A35 36. —H —OH A36 37. —H —OH A37 38. —H —OH A38 39. —H —OH A39 40. —H —OH A40 41. —H —OH A41 N,N-dialkylaminoacyl derivatives 42. 0 —H —OH A42 43. —H —OH A43 Esters and amides of Amphotericin B derivatives 44. —H —OCH.sub.3 A44 45. —H —OCH.sub.3 A45 46. —H —OCH.sub.3 A46 47. —H —NHCH.sub.2CH.sub.2CH.sub.2N(CH.sub.3).sub.2 A47 48. —H —NHCH.sub.2CH.sub.2CH.sub.2N(CH.sub.3).sub.2 A48 49. —H —NHCH.sub.2CH.sub.2CH.sub.2N(CH.sub.3).sub.2 A49

(72) The subject of the invention concerns also application of the compounds being sterically hindered derivatives of antifungal antibiotic Amphotericin B of Formula 1, 1a or 1b, where R.sub.1 is hydrogen atom or alkyl substituent simple or bulky one, advantageously as a residue of succinimidyl derivative or alkyl containing cyclic moieties carbo- or heterocyclic or glycosyl residue or bulky thioureidyl residue (preferably containing basic nitrogen atom and bulky aliphatic or cyclic substituents), R.sub.2 is hydrogen atom or substituents such as defined for R.sub.1, while R.sub.3 is hydroxyl group or alkoxyl or alkylamino or aminoalkyl derivative also their salts and complexes being water soluble forms, where X is one or more molecules of base or acid or complexing compound, for the combat of fungi, preferably multidrug resistant ones directly or as active components of various formulation of antifungal drugs.

(73) In a second aspect, the invention provides a pharmaceutical composition comprising a compound according to the invention as defined herein. As referenced throughout, a compound according to the invention includes salts, hydrates and complexes thereof.

(74) In a third aspect, the invention provides a compound as defined herein, for use in the treatment of fungal infection. The compound may also be for use in the treatment of diseases caused by fungal infection. Treatment may be in humans or in veterinary medicine.

(75) In a fourth aspect, the invention provides use of a compound in the manufacture of a medicament for the treatment of fungal infection, diseases caused by fungal infection, including treatment in humans or in veterinary medicine.

(76) In a fifth aspect, the invention provides a method of treating diseases caused by fungal infection in a patient comprising administering the patient a therapeutically effective amount of a compound of the invention as defined herein, wherein the patient is a human or animal.

(77) In a sixth aspect, the invention provides the use of a compound of the invention as defined herein for treating a fungal infection in a plant.

(78) In a seventh aspect, the invention provides a plant protection product comprising a compound of the invention as defined herein.

(79) In an eighth aspect, the invention provides a method of treating a fungal infection in a building comprising administering to the building a compound of the invention as defined herein. The compound of the invention may be administered to the building in the form of a solution (preferably an aqueous solution). In some embodiments, the solution may be prepared, for example, by dissolving the compound of the invention (preferably in the form of a powder or granules) in a solvent (preferably water or a water-miscible solvent). In some embodiments, the method of treating a fungal infection in a building comprises applying the solution to the infected area by spraying or brushing.

(80) In a ninth aspect, the invention provides an antifungal building treatment product comprising a compound of the invention as defined herein. The product may be in the form of powder or granules of the compound of the invention, or a solution containing the compound of the invention (preferably an aqueous solution).

(81) Embodiments described herein in relation to the first aspect of the invention (i.e. a compound of the invention) apply mutatis mutandis to the second to ninth aspects of the invention.

(82) In some embodiments, treatment as referred to herein relates to treatment of fungal infections caused by pathogenic fungi from the group of yeasts and filamentous fungi or a strain of the genus Candida, optionally wherein the yeasts or fungi have multidrug resistance (MDR), optionally with overexpression of protein transporters MDR1p as Cdr1p and Cdr2p.

(83) Compounds of the invention, when used for preventing or treating a disease, may be administered in an “effective amount”. By an “effective amount” it is meant a “therapeutically effective amount”, namely an amount of compound sufficient, upon single dose or multiple dose administration, to cause a detectable decrease in disease severity, to prevent advancement of a disease or alleviate disease symptoms beyond that expected in the absence of treatment.

(84) Compounds of the invention are useful for reducing the severity of symptoms of any of the above disorders to be treated. Compounds of the invention are also useful for administration to patients susceptible to, at risk of or suffering from any of the above disorders. Compounds useful for prevention of the above disorders are not required to absolutely prevent occurrence of the disorder in all cases, but may prevent or delay onset of the disorder when administered to a patient susceptible to or at risk of the disorder.

(85) The compounds of the invention may be provided as the free compound or as a suitable salt or hydrate thereof. Salts should be those that are pharmaceutically acceptable and salts and hydrates can be prepared by conventional methods, such as contacting a compound of the invention with an acid or base whose counterpart ion does not interfere with the intended use of the compound. Examples of pharmaceutically acceptable salts include hydrohalogenates, inorganic acid salts, organic carboxylic acid salts, organic sulfonic acid salts, amino acid salt, quaternary ammonium salts, alkaline metal salts, alkaline earth metal salts and the like.

(86) The compounds of the invention can be provided as a pharmaceutical composition. The pharmaceutical composition may additionally comprise a pharmaceutically acceptable excipient for example a pharmaceutically acceptable carrier and/or a pharmaceutically acceptable diluent. Suitable carriers and/or diluents are well known in the art and include pharmaceutical grade starch, mannitol, lactose, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose (or other sugar), magnesium carbonate, gelatin oil, alcohol, detergents, emulsifiers or water (preferably sterile).

(87) A pharmaceutical composition may be provided in unit dosage form, will generally be provided in a sealed container and may be provided as part of a kit. Such a kit would normally (although not necessarily) include instructions for use. It may include a plurality of said unit dosage forms.

(88) A pharmaceutical composition may be adapted for administration by any appropriate route, for example by the oral (including buccal or sublingual), rectal or topical (including buccal, sublingual or transdermal) route. Such compositions may be prepared by any method known in the art of pharmacy, for example by admixing the active ingredient with a carrier(s) or excipient(s) under sterile conditions.

(89) Pharmaceutical compositions adapted for oral administration may be presented as discrete units such as capsules or tablets; as powders or granules; as solutions, syrups or suspensions (in aqueous or non-aqueous liquids; or as edible foams or whips; or as emulsions). Suitable excipients for tablets or hard gelatine capsules include lactose, maize starch or derivatives thereof, stearic acid or salts thereof. Suitable excipients for use with soft gelatine capsules include for example vegetable oils, waxes, fats, semi-solid, or liquid polyols etc. For the preparation of solutions and syrups, excipients which may be used include for example water, polyols and sugars. For the preparation of suspensions oils (e.g. vegetable oils) may be used to provide oil-in-water or water in oil suspensions.

(90) Pharmaceutical compositions adapted for topical administration may be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols or oils. For infections of the eye or other external tissues, for example mouth and skin, the compositions are preferably applied as a topical ointment or cream. When formulated in an ointment, the active ingredient may be employed with either a paraffinic or a water-miscible ointment base. Alternatively, the active ingredient may be formulated in a cream with an oil-in-water cream base or a water-in-oil base. Pharmaceutical compositions adapted for topical administration to the eye include eye drops wherein the active ingredient is dissolved or suspended in a suitable carrier, especially an aqueous solvent. Pharmaceutical compositions adapted for topical administration in the mouth include lozenges, pastilles and mouth washes. Pharmaceutical compositions adapted for rectal administration may be presented as suppositories or enemas.

(91) Pharmaceutical compositions adapted for nasal administration wherein the carrier is a solid include a coarse powder having a particle size for example in the range 20 to 500 microns which is administered in the manner in which snuff is taken, i.e. by rapid inhalation through the nasal passage from a container of the powder held close up to the nose. Suitable compositions wherein the carrier is a liquid, for administration as a nasal spray or as nasal drops, include aqueous or oil solutions of the active ingredient.

(92) Pharmaceutical compositions adapted for administration by inhalation include fine particle dusts or mists which may be generated by means of various types of metered dose pressurised aerosols, nebulizers or insufflators. Pharmaceutical compositions adapted for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations.

(93) Pharmaceutical compositions adapted for parenteral administration include aqueous and non-aqueous sterile injection solution which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation substantially isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. Excipients which may be used for injectable solutions include water, alcohols, polyols, glycerine and vegetable oils, for example. The compositions may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carried, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets.

(94) The pharmaceutical compositions may contain preserving agents, solubilising agents, stabilising agents, wetting agents, emulsifiers, sweeteners, colourants, odourants, salts, buffers, coating agents or antioxidants. They may also contain an adjuvant and/or therapeutically active agents in addition to the substance of the present invention.

(95) Dosages of the substance of the present invention can vary between wide limits, depending upon a variety of factors including the disease or disorder to be treated, the age, weight and condition of the individual to be treated, the route of administration etc. and a physician will ultimately determine appropriate dosages to be used.

(96) Compositions adapted for the treatment of buildings having a fingal infection can be in the form of a powder or granules to be dissolved in water before use and may be applied to the infected area by spraying or brushing.

(97) Below are described methods for obtaining compounds which are bulky, spatially hindered amphoteric or basic N-alkyl or N-aminoacyl derivatives of antifungal antibiotic of polyene macrolides group, Amphotericin B, of general formula 1, where R.sub.1 is hydrogen atom or simple or bulky alkyl substituent, the last one advantageously as residue of succinimide derivative or as branched alkyl or containing carbo- or heterocyclic moieties or glycoyl residue or spatially extended aminoacyl residue or thioureidyl residue containing basic nitrogen atom and bulky aliphatic or cyclic substituents; R.sub.2 is hydrogen atom or substituents such as are defined for R.sub.1, while R.sub.3 is hydroxyl group or alkoxyl or aminoalkyl derivative, also their salts and complexes being their water soluble forms, where X is one or more basic molecules, advantageously N-methyl-D-glucamine one or acid molecule, preferably aspartic acid or complexing agent.

(98) To obtain N-succinimidyl derivatives the reaction of Michael's addition is performed as follows: solution of Amphotericin B in dimethyl formamide, in the presence of triethylamine, is reacted with suitable derivative of maleimide. The obtained product is precipitated with ethyl ether, centrifugated, dried and purified by column chromatography (Silica Gel).

(99) N-aminosuccinimidyl derivatives are obtained in similar manner as N-succinimidyl derivatives but double excess of maleimide derivatives is used. N-thioureidyl derivatives are obtained in the reaction of Amphotericin B, in dimethyl formamide solution, and in the presence of triethylamine, with proper derivatives of isothiocynate. The obtained product is purified by column chromatography (Silica Gel).

(100) N-benzyl derivatives are obtained in the reaction of reductive alkylation in solution of Amphotericin B in dimethyl formamide and methanol, with benzaldehyde or its derivatives, using sodium cyanoborohydride as reducing agent and catalytic amount of acetic acid. The reaction mixture is neutralized with solution of methylamine in tetrahydrofuran, then the final product is precipitated with ethyl ether and purified by column chromatography (Silica Gel).

(101) N-alkyl derivatives of Amphotericin B are obtained in the reaction of reductive alkylation of Amphotericin B, in solution of dimethyl formamide, with aliphatic aldehyde using sodium cyanoborohydride as reducing agent and catalytic amount of acetic acid. The reaction mixture is neutralized with solution of methylamine in tetrahydrofuran, the reaction product is precipitated with ethyl ether and then purified by column chromatography (Silica Gel).

(102) Obtaining of N-alkyl derivatives of N-fructosylamphotericin B is based on method of reductive alkylation of N-fructosylamphotericin B, in solution of dimethyl formamide, with suitable aliphatic aldehyde using sodium cyanoborohydride as reducing agent and catalytic amount of acetic acid. The reaction mixture is neutralized with solution of methylamine in tetrahydrofuran, product is precipitated with ethyl ether and purified by column chromatography (Silica Gel).

(103) N-aminoacyl and N—(N′-alkylamino)acyl derivatives of Amphotericin B are obtained in the reaction of N-acylation of antibiotic by appropriate N-protected aminoacids. First, the reaction of N-(fluorenylmetoxycarbonyl)-aminoacid with N-hydroxysuccinimide in the presence of N,N-′dicyclohexylcarbodiimide is performed in solution of dimethyl formamide. Precipitated N,N′-dicyclohexylurea is removed, then to the rection mixture Amphotericin B and triethylamine are added. Progress of the reaction is monitored by thin layer chromatography. The final product is precipitated with ethyl ether and next purified by column chromatography (Silica Gel).

(104) Synthesis of N—(N,N-dialkylamino)aminoacyl derivatives of Amphotericin B is performed by activation of aminoacid by N-hydroxysuccinimide and N,N-dicyclohexylcarbodiimide in dimethyl formamide solution. Precipitated solid of N,N-dicyclohexylurea is removed, and to the reaction mixture amphotericin B is added. Crude product of the reaction is precipitated with excess of ethyl ether and purified by column chromatography (Silica Gel).

(105) Synthesis of methyl esters of N-substituted Amphotericin B derivatives is performed in the reaction of the antibiotic, in dimethyl formamide solution, with diazomethane which is added to the reaction mixture in ethyl ether solution. Then, excess of diazomethane is removed with acetic acid, the formed product is precipitated by excess of ethyl ether and purified by column chromatography (Silica Gel).

(106) Synthesis of amides of N-substituted Amphotericin B derivatives, for example N-fructosyl-N-propylamphotericin B 3-(N,N-dimethylamino)propyl amide, is performed in the reaction of N-substituted antibiotic derivative, in dimethyl formamide solution, with respective amine in the presence of diphenyl azidephosphate and triethylamine. Product of the reaction is precipitated by excess of ethyl ether and purified by column chromatography (Silica Gel).

(107) Obtaining of salts of amphoteric Amphotericin B derivatives, advantageously with N-methyl-D-glucamine, consist of addition to aqueous suspension of Amphotericin B derivatives of a small excess of N-methyl-D-glucamine diluted in water and precipitation of product by excess of acetone.

(108) Water-soluble complex of amphoteric Amphotericin B derivatives is obtained according to conventional methods.

(109) Water-soluble salts of basic Amphotericin B derivatives, advantageously with aspartic acid, are synthesized by adding to aqueous suspension of antibiotic a slight molar excess of L-aspartic acid, then obtained salt is precipitated by excess of acetone. The method for obtaining sterically hindered Amphotericin B derivatives, according to the invention, univocally leads to obtain the desired product. All obtained compounds have been characterized with respect to their chemical structure and biological properties. Identification of the compounds includes their spectroscopic data as λ.sub.max determination, extinction value of E.sub.1cm.sup.1%, molecular weight determined by mass spectrometry MS-ESI, thin layer chromatography with indicated of R.sub.F value. The biological properties of the compounds, according to the invention, were determined using the obligatory standards. There was determined activities of the compounds in vitro against a number of fungal strains, primarily of the genus Candida and filament fungi, activity towards multidrug resistance fungal strains with overexpression of protein transporters of both ABC and MFS type. Also hemotoxicity of the compounds was determined by measurement of their hemolytic activity for human erythrocytes as well as their cytotoxicity determined in tissue culture for several mammalian cell lines. The obtained results show that, depending on the kind of steric hindering moieties introduced to Amphotericin B molecule, the compounds exhibit reduced to different extent, in relation to the native antibiotic, hemotoxic activity and are characterized by low cytotoxicity, good antifungal activity and are also active against multidrug resistant strains (MDR).

(110) Compounds according to the invention which are so far unknown sterically hindered derivatives of Amphotericin B, is that they fulfill the basic requirements for antifungal chemotherapeutics. They are characterized by very low hemotoxicity, exhibit low toxicity for mammalian somatic cells, are active against multidrug resistant fungal strains (MDR). Moreover, they form with acids or bases water-soluble salts and also soluble complexes with complexing compounds. The advantage of the compounds according to the invention is also simple and efficient method of their preparation.

(111) The subject of the invention is shown in the below examples, where are presented methods for the preparation and properties of the compounds according to the invention and of their water-soluble salts and complexes.

EXAMPLES

Example 1

Synthesis of N-succinimidyl Derivatives of Amphotericin B

(112) 200 mg (0.22 mmol) of Amphotericin B is dissolved in 4 ml of dimethyl formamide (DMF) in 25 ml round-bottomed flask equipped with a magnetic stirrer. The solution is cooled to 0° C. and 0.029 ml (0.21 mmol) of triethylamine (TEA) is slowly added. After 10 minutes 0.25 mmol of the appropriate maleimide is added and the reaction mixture is warmed to room temperature. The reaction progress is monitored by thin layer chromatography (TLC) on Silica Gel (60 F254, Merck) in chloroform: methanol: water (20:8:1 v/v) solvent system. After then, the reaction mixture is added dropwise to 150 ml of diethyl ether. The resulting, pale yellow precipitate is filtered under reduced pressure on a Millipore funnel. The crude product is twice washed with diethyl ether (2×50 ml) and then dried in a vacuum desiccator. The residue is purified by column chromatography on normal phases, where the solid phase is Silica Gel and solvent system is chloroform: methanol: water (25:8:1 v/v). The fractions with pure product were collected and combined, then evaporated under reduced pressure at temperature not exceeding 35° C. Using in the reaction below indicated maleimides, the following derivatives of Amphotericin B are obtained: a) In the reaction with N-(2,4,6-trimethylphenyl)maleimide is obtained 30 mg of N—[N-(2,4,6-trimethylphenyl)succinimidyl]amphotericin B (A1)

(113) TLC R.sub.f=0.32; UV-vis: λ.sub.max (MeOH) 406; 382; 363 nm; E.sub.1cm.sup.1% (MeOH, λ=406 nm)=1210 (theoretically for C.sub.60H.sub.86N.sub.2O.sub.19 is 1300); MS-ESI found m/z: 1137.4 [M−H.sup.+].sup.−; calculated for C.sub.60H.sub.86N.sub.2O.sub.19 [M−H].sup.− 1137.6 b) In the reaction with N-benzylmaleimide is obtained 60 mg of N—(N-benzyl-succinimidyl)amphotericin B (A2)

(114) TLC R.sub.f=0.27; UV-vis: λ.sub.max (MeOH) 406; 382; 363 nm; E.sub.1cm.sup.1% (MeOH, λ=406 nm)=1180 (theoretically for C.sub.58H.sub.82N.sub.2O.sub.19 is 1330); MS-ESI found m/z: 1109.3 [M−H.sup.+].sup.−; calculated for C.sub.58H.sub.82N.sub.2O.sub.19 [M−H].sup.− 1109.6 c) In the reaction with N-(4-bromophenyl)maleimide is obtained 52 mg of N—[N-(4-bromophenyl)-succinimidyl]amphotericin B (A3)

(115) TLC R.sub.f=0.21; UV-vis: λ.sub.max (MeOH) 406; 382; 363 nm; E.sub.1cm.sup.1% (MeOH, λ=406 nm)=1141 (theoretically for C.sub.57H.sub.79BrN.sub.2O.sub.19 is 1260); MS-ESI found m/z: 1207.2 [M+CH.sub.3OH].sup.−; calculated for C.sub.57H.sub.79BrN.sub.2O.sub.19 [M].sup.+ 1174.5 d) In the reaction with N-(2-tert-butylophenyl)maleimide is obtained 36 mg of N—[N-(2-tert-butylphenyl)succinimidyl]amphotericin B (A4)

(116) TLC R.sub.f=0.22; UV-vis: λ.sub.max (MeOH) 406; 382; 363 nm; E.sub.1cm.sup.1% (MeOH, λ=406 nm)=1236 (theoretically for C.sub.61H.sub.88N.sub.2O.sub.19 is 1283); MS-ESI found m/z: 1151.4 [M−H.sup.+].sup.−; calculated for C.sub.1H.sub.88N.sub.2O.sub.19 [M].sup.+• 1152.6 e) In the reaction with N-(4-nitrofenylo)maleimid is obtained 40 mg of N—[N-(4-nitro phenyl)succinimidyl]amphotericin B (A5)

(117) TLC R.sub.f=0.42; UV-vis: λ.sub.max (MeOH) 406; 382; 363 nm; E.sub.1cm.sup.1% (MeOH, λ=406 nm)=1261 (theoretically for C.sub.57H.sub.79N.sub.3O.sub.21 is 1295); MS-ESI found m/z: 1172.3 [M+CH.sub.3OH].sup.−; calculated for C.sub.57H.sub.79N.sub.3O.sub.21 [M].sup.+ 1141.5 f) In the reaction with N-(2-hydroxyethyl)maleimid is obtained 56 mg of N—[N-(2-hydroxy-ethyl)succinimidyl]amphotericin B (A8)

(118) TLC R.sub.f=0.56; UV-vis: λ.sub.max (MeOH) 406; 382; 363 nm; E.sub.1cm.sup.1% (MeOH, λ=406 nm)=1350 (theoretically for C.sub.53H.sub.80N.sub.2O.sub.20 is 1390); MS-ESI found m/z 1047.5 [M−H.sub.2O].sup.+; calculated for C.sub.53H.sub.80N.sub.2O.sub.20 [M].sup.+ 1064.5

Example 2

Synthesis of N-aminosuccinimidyl Derivatives of Amphotericin B

(119) 200 mg (0.22 mmol) of Amphotericin B is dissolved in 4 ml of dimethyl formamide (DMF) in 25 ml round-bottomed flask equipped with a magnetic stirrer. The solution is cooled to 0° C. and 0.030 ml (0.22 mmol) of triethylamine (TEA) is slowly added. After 10 minutes 0.44 mmol of the appropriate basic maleimide is added and the reaction mixture is warmed to room temperature. The reaction progress is monitored by thin layer chromatography (TLC) on Silica Gel (60 F254, Merck) in chloroform: methanol: water (10:6:1 v/v) solvent system. After then, the reaction mixture is added dropwise to 150 ml of diethyl ether. The resulting, pale yellow precipitate is filtered under reduced pressure on a Millipore funnel. The crude product is twice washed with diethyl ether (2×50 ml) and then dried in a vacuum desiccator. The residue is purified by column chromatography on normal phases, where the solid phase is Silica Gel and solvent system is chloroform: methanol (gradient from 20% to 80% of methanol) or chloroform: methanol: water (10:6:1 v/v). The fractions with pure product were collected and combined, then evaporated under reduced pressure at temperature not exceeding 35° C. Using in the reaction below indicated basic maleimides, the following derivatives of Amphotericin B are obtained: g) In the reaction with N-(2-piperidin-1-ylethyl)maleimide is obtained 48 mg of N—[N-(2-piperidin-1-ylethyl)succinimidyl]amphotericin B (A6)

(120) TLC R.sub.f=0.18; UV-vis: λ.sub.max (MeOH) 406; 382; 363 nm; E.sub.1cm.sup.1% (MeOH, λ=406 nm)=1210 (theoretically for C.sub.58H.sub.90N.sub.4O.sub.19 is 1290); MS-ESI found m/z: 1147.6 [M+H].sup.+; calculated for C.sub.58H.sub.90N.sub.4O.sub.19 [M].sup.+• 1146.6 h) In the reaction with N-[3-(N,N-dimethylamino)-2,2-dimethylpropyl]maleimide is obtained 65 mg of N-{N-[3-(N,N-dimethylamino)-2,2-dimethylpropyl]succinimidyl}-amphotericin B (A7)

(121) TLC R.sub.f=0.23; UV-vis: λ.sub.max (MeOH) 406; 382; 363 nm; E.sub.1cm.sup.1% (MeOH, λ=406 nm)=1210 (theoretically for C.sub.58H.sub.91N.sub.3O.sub.19 is 1304); MS-ESI found m/z: 1134.6 [M+H].sup.+; calculated for C.sub.58H.sub.91N.sub.3O.sub.19 [M].sup.+• 1133.6

Example 3

Synthesis of N,N-dialkyl Derivatives of Amphotericin B

(122) 200 mg (0.22 mmol) of Amphotericin B is dissolved in 3 ml of dimethyl formamide (DMF) in 25 ml round-bottomed flask equipped with a magnetic stirrer. Next, 0.63 mmol of appropriate aliphatic aldehyde is added and solution is stirred at room temperature for 1 hour. After 1 hour 3 ml of anhydrous methanol, 0.63 mmol of sodium cyanoborohydride (NaBH.sub.3CN) and catalytic amount of acetic acid (0.015 ml) are added. The reaction progress is monitored by thin layer chromatography (TLC) on Silica Gel (60 F254, Merck) in chloroform:methanol:water (10:6:1 v/v) solvent system. The reaction mixture is cooled at −5° C., and then 0.015 ml of methylamine in tetrahydrofurane is added. The reaction mixture is left for 10 minutes and then added dropwise to 150 ml of diethyl ether. The resulting pale yellow precipitate is filtered under reduced pressure on a Millipore funnel. The crude product is twice washed with diethyl ether (2×50 ml) and then dried in a vacuum desiccator. The residue is purified on column chromatography on normal phase, where the solid phase is Silica Gel and solvent system is chloroform: methanol: water (10:6:1 v/v) or chloroform:methanol (gradient from 20% to 60% of methanol). The fractions with pure product were collected and combined, then evaporated under reduced pressure at temperatures not exceeding 35° C. The following derivatives of Amphotericin B are obtained: a) In the reaction with methanal is obtained 40 mg of N,N-dimethylamphotericin B (A9)

(123) TLC R.sub.f=0.22; UV-vis: λ.sub.max (MeOH) 406; 382; 363 nm; E.sub.1cm.sup.1% (MeOH, λ=406 nm)=1290 (theoretically for C.sub.49H.sub.77NO.sub.17 is 1554); MS-ESI found m/z: 950.5 [M−H].sup.−; calculated for C.sub.49H.sub.77NO.sub.17 [M].sup.+• 951.5 b) In the reaction with ethanal is obtained 37 mg of N,N-diethylamphotericin B (A10)

(124) TLC R.sub.f=0.31; UV-vis: λ.sub.max (MeOH) 406; 382; 363 nm; E.sub.1cm.sup.1% (MeOH, λ=406 nm)=1300 (theoretically for C.sub.51H.sub.81NO.sub.17 is 1514); MS-ESI found m/z: 978.5 [M−H.sup.+].sup.−; calculated for C.sub.51H.sub.81NO.sub.17 [M].sup.+• 979.3 c) In the reaction with propanal is 42 mg of N,N-di-n-propylamphotericin B (A11)

(125) TLC R.sub.f=0.27; UV-vis: λ.sub.max (MeOH) 406; 382; 363 nm; E.sub.1cm.sup.1% (MeOH, λ=406 nm)=1250 (theoretically for C.sub.53H.sub.87NO.sub.17 is 1468); MS-ESI found m/z: 1106.6 [M−H.sup.+].sup.−; calculated for C.sub.53H.sub.85NO.sub.17 [M].sup.+•1007.6 d) In the reaction with 3-(N-piperidin-1-ylo)propanal is obtained 20 mg N,N-di[3-(N-piperidin-1-yl)propyl]amphotericin B (A12)

(126) TLC R.sub.f=0.31; UV-vis: λ.sub.max (MeOH) 406; 382; 363 nm; E.sub.1cm.sup.1% (MeOH, λ=406 nm)=1180 (theoretically for C.sub.63H.sub.103N.sub.3O.sub.17 is 1258); MS-ESI found m/z: 1175.5 [M+H].sup.+; calculated for C.sub.63H.sub.103N.sub.3O.sub.17 [M+H].sup.+ 1175.4 e) In the reaction with 3-(4-ethylpiperazin-1-yl)propanal is obtained 31 mg of N,N-di[3-(4-ethylpiperazin-1-yl)propyl]amphotericin B (A13)

(127) TLC R.sub.f=0.26; UV-vis: λ.sub.max (MeOH) 406; 382; 363 nm; E.sub.1cm.sup.1% (MeOH, λ=406 nm)=1150 (theoretically for C.sub.63H.sub.105N.sub.5O.sub.17 is 1226); MS-ESI found m/z: 1205.8 [M+H].sup.+•; calculated for C.sub.63H.sub.105N.sub.5O.sub.17 [M+H].sup.+ 1205.5

Example 4

Synthesis of N-alkyl Derivatives of N-fructosylamphotericin B

(128) 200 mg (0.18 mmol) of N-fructosylamphotericin B is dissolved in 3 ml of dimethyl formamide (DMF) in 25 ml round-bottomed flask equipped with a magnetic stirrer. Next, 0.63 mmol of appropriate aliphatic aldehyde is added and solution is stirred at room temperature for 1 hour. After 1 hour 3 ml of anhydrous methanol, 0.63 mmol of sodium cyanoborohydride and catalytic amount of acetic acid (0.015 ml) are added. The reaction progress is monitored by thin layer chromatography (TLC) on Silica Gel (60 F254, Merck) in chloroform: methanol: water (7:6:1 v/v) or n-butanol:acetic acid:water (4:1:1 v/v) solvent system. The reaction mixture is cooled to −5° C., and then 0.015 ml (2M) of methylamine in tetrahydrofurane is added. The reaction mixture is left for 10 minutes and then is added dropwise to 150 ml of diethyl ether. The resulting pale yellow precipitate is filtered under reduced pressure on a Millipore funnel. The crude product is twice washed with diethyl ether (2×50 ml) and then dried in a vacuum desiccator. The residue is purified by column chromatography on normal phase where the solid phase is Silica Gel and solvent system is chloroform: methanol: water (7:6:1 v/v). The fractions with pure product were collected and combined, then evaporated under reduced pressure at temperatures not exceeding 35° C. The following derivatives of N-fructosylamphotericin B are obtained: a) In the reaction with methanal is obtained 32 mg of N-fructosyl-N-methylamphotericin B (A14)

(129) TLC R.sub.f=0.13; UV-vis: λ.sub.max (MeOH) 406; 382; 363 nm; E.sub.1cm.sup.1% (MeOH, λ=406 nm)=1280 (theoretically for C.sub.54H.sub.87NO.sub.22 is 1345); MS-ESI found m/z: 1098.3 [M−H].sup.−; calculated for C.sub.54H.sub.85NO.sub.22 [M].sup.+ 1099.6 b) In the reaction with ethanal is obtained 21 mg of N-ethyl-N-fructosylamphotericin B (A15)

(130) TLC R.sub.f=0.11; UV-vis: λ.sub.max (MeOH) 406; 382; 363 nm; E.sub.1cm.sup.1% (MeOH, λ=406 nm)=1230 (theoretically for C.sub.55H.sub.89NO.sub.22 is 1328); MS-ESI found m/z: 1112.5 [M−H].sup.−; calculated for C.sub.55H.sub.87NO.sub.22 [M].sup.+ 1113.4 c) In the reaction with propanal is obtained 28 mg of N-fructosyl-N-n-propylamphotericin B (A16)

(131) TLC R.sub.f=0.14; UV-vis: λ.sub.max (MeOH) 406; 382; 363 nm; E.sub.1cm.sup.1% (MeOH, λ=406 nm)=1210 (theoretically for C.sub.56H.sub.91NO.sub.22 is 1311); MS-ESI found m/z: 1126.4 [M−H].sup.−; calculated for C.sub.56H.sub.89NO.sub.22 [M].sup.+• 1127.6 d) In the reaction with N,N-dimethyl-3-aminopropanal is obtained 15 mg of N-fructosyl-N—(N,N-dimethyl-3-aminopropyl)amphotericin B (A17)

(132) TLC R.sub.f=0.11; UV-vis: λ.sub.max (MeOH) 406; 382; 363 nm; E.sub.1cm.sup.1% (MeOH, λ=406 nm)=1120 (theoretically for C.sub.58H.sub.94N.sub.2O.sub.22 is 1261); MS-ESI found m/z: 1172.7 [M+H].sup.+; calculated for C.sub.58H.sub.94N.sub.2O.sub.22 [M+H].sup.+ 1172.5 e) In the reaction with 3-(piperidin-1-yl)propanal is obtained 28 mg N-fructosyl-N-[3-(piperidin-1-yl)aminopropyl]amphotericin B (A18)

(133) TLC R.sub.f=0.15; UV-vis: λ.sub.max (MeOH) 406; 382; 363 nm; E.sub.1cm.sup.1% (MeOH, λ=406 nm)=1180 (theoretically for C.sub.61H.sub.98N.sub.2O.sub.22 is 1221); MS-EI found m/z: 1211.9 [M+H].sup.+; calculated for C.sub.61H.sub.98N.sub.2O.sub.22 [M+H].sup.+ 1211.5

Example 5

Synthesis of N-benzyl Derivatives of Amphotericin B

(134) 200 mg (0.22 mmol) of Amphotericin B is dissolved in 3 ml of dimethyl formamide (DMF) in 25 ml round-bottomed flask equipped with a magnetic stirrer. Next, 0.3 mmol of aromatic aldehyde is added and stirred at room temperature for 1 hour. After 1 hour 3 ml of anhydrous methanol, 0.3 mmol of sodium cyanoborohydride (NaBH.sub.3CN) and catalytic amount (0.015 ml) of acetic acid are added. The reaction progress is monitored by thin layer chromatography (TLC) on Silica Gel (60 F254, Merck) in chloroform: methanol: water (20:6:1 v/v) solvent system. The reaction mixture is cooled to −5° C., and then 0.015 ml of methylamine in tetrahydrofurane is added. The reaction mixture is left for 10 minutes and then added dropwise to 150 ml of diethyl ether. The resulting pale yellow precipitate is filtered under reduced pressure on a Millipore funnel. The crude product is twice washed with diethyl ether (2×50 ml) and then dried in a vacuum dessicator. The residue is purified by column chromatography on normal phase, where the solid phase is Silica Gel and solvent system is chloroform: methanol: water (20:6:1 v/v). The fractions with pure product were collected and combined, then evaporated under reduced pressure at temperatures not exceeding 35° C. The following derivatives of Amphotericin B are obtained: a) In the reaction with z 4-(N,N-diethylamino)benzaldehyde is obtained 15 mg of N-(4-N,N-diethylaminobenzyl)amphotericin B (A19)

(135) TLC R.sub.f=0.75; UV-vis: λ.sub.max (MeOH) 406; 382; 363 nm; E.sub.1cm.sup.1% (MeOH, λ=406 nm)=1150 (theoretically for C.sub.58H.sub.88N.sub.2O.sub.17 is 1363); MS-ESI found m/z: 1083.3 [M−H].sup.−; calculated for C.sub.58H.sub.88N.sub.2O.sub.17 [M].sup.+• 1084.6 b) In the reaction with 4-phenylbenzaldehyde is obtained 45 mg of N-[(4-biphenyl)-methyl]amphotericin B (A20)

(136) TLC R.sub.f=0.86; UV-vis: λ.sub.max (MeOH) 406; 382; 363 nm; E.sub.1cm.sup.1% (MeOH, λ=406 nm)=1230 (theoretically for C.sub.60H.sub.83NO.sub.17 is 1357); MS-ESI found m/z: 1088.8 [M−H].sup.−; calculated for C.sub.60H.sub.83NO.sub.17 [M].sup.+•1089.6 c) In the reaction with z 4-tert-butylbenzaldehyde is obtained 47 mg of N-(4-tert-butylobenzylo)amphotericin B (A21)

(137) TLC R.sub.f=0.87; UV-vis: λ.sub.max (MeOH) 406; 382; 363 nm; E.sub.1cm.sup.1% (MeOH, λ=406 nm)=1200 (theoretically for C.sub.54H.sub.81NO.sub.17 is 1382); MS-ESI found m/z: 1068.5 [M−H].sup.−; calculated for C.sub.58H.sub.87NO.sub.17 [M].sup.+• 1069.6

Example 6

Synthesis of N-thioureidyl Derivatives of Amphotericin B

(138) 200 mg (0.22 mmol) of Amphotericin B is dissolved in 4 ml of dimethyl formamide (DMF) in 100 ml round-bottomed flask equipped with a magnetic stirrer. The solution is cooled to 0° C. and 0.029 ml (0.21 mmol) of triethylamine (TEA) is slowly added. After 10 minutes 0.25 mmol of the appropriate isothiocyanate is added and the reaction mixture is warmed to room temperature. The reaction progress is monitored by thin layer chromatography (TLC) on Silica Gel (60 F254, Merck) in chloroform: methanol: water (10:6:1 v/v) solvent system. After then the reaction mixture is added dropwise to 150 ml of diethyl ether. The resulting, pale yellow precipitate is filtered under reduced pressure on a Millipore funnel. The crude product is twice washed with diethyl ether (2×50 ml) and then dried in a vacuum desiccator. The residue is purified by column chromatography on normal phase, where the solid phase is Silica Gel and solvent system is chloroform: methanol (gradient from 20% to 55% of methanol). The fractions with pure product were collected and combined, then evaporated under reduced pressure at temperature not exceeding 35° C. The following derivatives of Amphotericin B are obtained: a) In the reaction with 2-piperidin-1-yl-ethylisothiocyanate is obtained 35 mg of N-{[3-(2-piperidin-1-yl)ethyl]thioureidyl]amphotericin B (A22)

(139) TLC R.sub.f=0.4; UV-vis: λ.sub.max (MeOH) 406; 382; 363 nm; E.sub.1cm.sup.1% (MeOH, λ=406 nm)=1230 (theoretically for C.sub.55H.sub.87N.sub.3O.sub.17S is 1352); MS-ESI found m/z: 1094.5 [M+H].sup.+; calculated for C.sub.55H.sub.87N.sub.3O.sub.17S [M].sup.+• 1093.6 b) In the reaction with phenylisothiocyanate is obtained 10 mg of N-[(3-phenyl)-thioureidyl]amphotericin B (A23)

(140) TLC R.sub.f=0.85; UV-vis: λ.sub.max (MeOH) 406; 382; 363 nm; E.sub.1cm.sup.1% (MeOH, λ=406 nm)=1320 (theoretically for C.sub.54H.sub.78N.sub.2O.sub.17S is 1397); ESI-MS found m/z: 1057.5 [M−H].sup.−; calculated for C.sub.54H.sub.78N.sub.2O.sub.17S [M].sup.+• 1058.5 c) In the reaction with 2-(1-morpholin-1-yl)ethylisothiocyanate is obtained 23 mg of N-{[3-(2-morpholin-1-yl)ethyl]thioureidyl}amphotericin B (A24)

(141) TLC R.sub.f=0.5; UV-vis: λ.sub.max (MeOH) 406; 382; 363 nm; E.sub.1cm.sup.1% (MeOH, λ=406 nm)=1290 (theoretically for C.sub.54H.sub.87N.sub.3O.sub.18S is 1348); MS-ESI found m/z: 1096.4[M+H].sup.+; calculated for C.sub.54H.sub.87N.sub.3O.sub.18S [M].sup.+• 1095.6 d) In the reaction with N,N-diethyl-2-aminoethylisothiocyanate is obtained 44 mg of N-{3-[2-(N,N-diethylamino)ethyl]thioureidyl}amphotericin B (A25)

(142) TLC R.sub.f=0.32; UV-vis: λ.sub.max (MeOH) 406; 382; 363 nm; E.sub.1cm.sup.1% (MeOH, λ=406 nm)=1240 (theoretically for C.sub.55H.sub.89N.sub.3O.sub.17S is 1350); MS-ESI found m/z: 1096.5 [M+H].sup.+; calculated for C.sub.55H.sub.89N.sub.3O.sub.17S [M].sup.+• 1095.6 e) In the reaction with (pyridin-3-yl)isothiocyanate is 130 mg of N-[3-(pyridn-3-yl)thioureidyl]amphotericin B (A26)

(143) TLC R.sub.f=0.83; UV-vis: λ.sub.max (MeOH) 406; 382; 363 nm; E.sub.1cm.sup.1% (MeOH, λ=406 nm)=1280 (theoretically for C.sub.53H.sub.77N.sub.3O.sub.17S is 1396); MS-ESI found m/z: 1060.5 [M+H].sup.+; calculated for C.sub.53H.sub.77N.sub.3O.sub.17S [M].sup.+• 1059.5 f) In the reaction with 2-(pyrrolidin-1-yl)ethyloisothiocyanate is obtained 25 mg of N-{[3-(2-pyrrolidin-1-yl)ethyl)thioureidyl]amphotericin B (A27)

(144) TLC R.sub.f=0.38; UV-vis: λ.sub.max (MeOH) 406; 382; 363 nm; E.sub.1cm.sup.1% (MeOH, λ=406 nm)=1270 (theoretically for C.sub.54H.sub.85N.sub.3O.sub.17S is 1370); MS-ESI found m/z: 1080.1 [M+H].sup.+; calculated for C.sub.54H.sub.85N.sub.3O.sub.17S [M].sup.+• 1079.6 g) In the reaction with 2-(N,N-dimethylamino)ethylisothiocyanate is obtained 38 mg of N-{3-[2-(N,N-dimethylamino)ethyl]thioureidyl}amphotericin B (A28)

(145) TLC R.sub.f=0.33; UV-vis: λ.sub.max (MeOH) 406; 382; 363 nm; E.sub.1cm.sup.1% (MeOH, λ=406 nm)=1320 (theoretically for C.sub.52H.sub.83N.sub.3O.sub.17S is 1400); MS-ESI found m/z: 1054.4 [M+H].sup.+; calculated for C.sub.52H.sub.83N.sub.3O.sub.17S [M].sup.+• 1053.5 h) In the reaction with (pyridin-4-yl)methylisothiocyanate is obtained 40 mg N-{[3-(pyridin-4-yl)methyl)thioureidyl]amphotericin B (A29)

(146) TLC R.sub.f=0.73; UV-vis: λ.sub.max (MeOH) 406; 382; 363 nm; E.sub.1cm.sup.1% (MeOH, λ=406 nm)=1190 (theoretically for C.sub.53H.sub.77N.sub.3O.sub.17S is 1377); MS-ESI found m/z: 1074.6 [M+H].sup.+; calculated for C.sub.54H.sub.79N.sub.3O.sub.17S [M].sup.+• 1073.5

Example 7

Synthesis of N-aminoacyl and N—(N′-alkylamino)Acyl Derivatives of Amphotericin B

(147) 0.26 mmol of N-(9-fluorenylmethoxycarbonyl)aminoacid (Fmoc-aminoacid), 0.26 mmol of N-hydroxysuccinimide (HONSu), 53 mg (0.26 mmol) of dicyclohexylcarbodiimide (DCC) is dissolved in 3 ml of dimethyl formamide (DMF) in 25 ml round-bottomed flask equipped with a magnetic stirrer. The reaction mixture is stirred at 37° C. for 1 hour. The reaction progress is monitored by thin layer chromatography (TLC) on Silica Gel (60 F254, Merck) in ethyl acetate:hexane (7:3 v/v) solvent system. During the reaction, the precipitated N,N-dicyclohexylurea is filtered and washed with 1 ml of DMF. To the filtrate 200 mg (0.22 mmol) of Amphotericin B and 0.04 ml (0.22 mmol) of triethylamine (TEA) are added. Stirring is continued at 37° C. for 3 hours. After the reaction, another portion of 0.04 ml (0.22 mmol) of TEA is added and the reaction mixture is left for 2 hours at room temperature, and then added dropwise to 150 ml of diethyl ether. The resulting, pale yellow precipitate is filtered under reduced pressure on a Millipore funnel. The crude product is washed twice with diethyl ether (2×50 ml) and then dried in a vacuum desiccators. The residue is purified on column chromatography on normal phase, where the solid phase is Silica Gel and solvent system is chloroform: methanol: water (15:8:1 v/v). The fractions with pure product were collected and combined, then evaporated under reduced pressure at temperature not exceeding 35° C. The following derivatives of Amphotericin B are obtained in the reaction with the corresponding protected amino acids: a) In the reaction with N-Fmoc-L-phenylalanine is obtained 56 mg of N-L-phenylalanylamphotericin B (A30)

(148) TLC R.sub.f=0.41; UV-vis: λ.sub.max (MeOH) 406; 382; 363 nm; E.sub.1cm.sup.1% (MeOH, λ=406 nm)=1280 (theoretically for C.sub.56H.sub.84N.sub.2O.sub.18 is 1380); MS-ESI found m/z: 1073.9 [M+H].sup.+; 1096 [M+Na].sup.+; calculated for C.sub.56H.sub.84N.sub.2O.sub.18 [M].sup.+• 1072.6 b) In the reaction with N-Fmoc-L-p-iodophenylalanine is obtained 35 mg of N-L-p-iodophenylalanylamphotericin B (A31)

(149) TLC R.sub.f=0.53; UV-vis: λ.sub.max (MeOH) 406; 382; 363 nm; E.sub.1cm.sup.1% (MeOH, λ=406 nm)=1130 (theoretically for C.sub.56H.sub.84N.sub.2O.sub.18 is 1233); MS-ESI found m/z: 1199.9 [M+H].sup.+; calculated for C.sub.56H.sub.83IN.sub.2O.sub.18 [M].sup.+• 1198.5 c) In the reaction with N-Fmoc-D-β-naphtylalanine is obtained 30 mg N-D-β-naphtylalanylamphotericin B (A32)

(150) TLC R.sub.f=0.40; UV-vis: λ.sub.max (MeOH) 406; 382; 363 nm; E.sub.1cm.sup.1% (MeOH, λ=406 nm)=1150 (theoretically for C.sub.60H.sub.86N.sub.2O.sub.18 is 1317); MS-ESI found m/z: 1124.9 [M+H].sup.+; 1189.9 [M+2MeOH].sup.+; calculated C.sub.60H.sub.86N.sub.2O.sub.18 [M].sup.+• 1122.6 d) In the reaction with N-Fmoc-L-p-nitrophenylalanine is obtained 23 mg of N-L-p-nitrophenylalanylamphotericin B (A33)

(151) TLC R.sub.f=0.41; UV-vis: λ.sub.max (MeOH) 406; 382; 363 nm; E.sub.1cm.sup.1% (MeOH, λ=406 nm)=1240 (theoretically for C.sub.56H.sub.83N.sub.3O.sub.20 is 1323); MS-ESI found m/z: 1119.9 [M+H].sup.+; calculated for C.sub.56H.sub.83N.sub.3O.sub.20 [M].sup.+• 1117.6 e) In the reaction with N-Fmoc-N-methyl-L-(O.sup.γ-tert-butyl)glutamic acid is obtained 20 mg of N-methyl-L-(O.sup.γ-tert-butyl)glutamylamphotericin B (A34)

(152) TLC R.sub.f=0.24; UV-vis: λ.sub.max (MeOH) 406; 382; 363 nm; E.sub.1cm.sup.1% (MeOH, λ=406 nm)=1230 (theoretically for C.sub.57H.sub.92N.sub.2O.sub.20 is 1315); MS-ESI found m/z 1126: [M+H].sup.+; 1158 [M+MeOH].sup.+; calculated for C.sub.57H.sub.92N.sub.2O.sub.20 [M].sup.+• 1124.6 f) In the reaction with N-Fmoc-D-(O.sup.β-tert-butyl)asparagine is obtained 30 mg of N-D-(O.sup.β-tert-butyl)asparagylamphotericin B (A35)

(153) TLC R.sub.f=0.35; UV-vis: λ.sub.max (MeOH) 406; 382; 363 nm; E.sub.1cm.sup.1% (MeOH, λ=406 nm)=1270 (theoretically for C.sub.55H.sub.88N.sub.2O.sub.20 is 1348); MS-ESI found m/z: 1098 [M+H].sup.+; 1164 [M+MeOH].sup.+; calculated for C.sub.55H.sub.88N.sub.2O.sub.20 [M].sup.+• 1096.6 g) In the reaction with N-Fmoc-D-β-(pyridin-3-yl)alanine is obtained 70 mg of N-D-β-(pyridin-3-yl)alanylamphotericin B (A36)

(154) TLC R.sub.f=0.37; UV-vis: λ.sub.max (MeOH) 406; 382; 363 nm; E.sub.1cm.sup.1% (MeOH, λ=406 nm)=1240 (theoretically for C.sub.55H.sub.81N.sub.3O.sub.18 is 1380); MS-ESI found m/z: 1070.3 [M−H].sup.−; calculated for C.sub.55H.sub.81N.sub.3O.sub.18 [M].sup.+• 1071.6 h) In the reaction with N-Fmoc-L-(S-tert-butyl)cysteine is obtained 80 mg N-L-(S-tert-butyl)cystylamphotericin B (A37)

(155) TLC R.sub.f=0.34; UV-vis: λ.sub.max (MeOH) 406; 382; 363 nm; E.sub.1cm.sup.1% (MeOH, λ=406 nm)=1280 (theoretically for C.sub.54H.sub.86N.sub.2O.sub.18S is 1366); MS-ESI found m/z: 1081.5 [M−H].sup.−; calculated for C.sub.54H.sub.86N.sub.2O.sub.18S [M].sup.+• 1082.5 i) In the reaction with rac-N-Fmoc-o-fluorophenylalanine is obtained 23 mg N-o-fluorophenylalanylamphotericin B (A38)

(156) TLC R.sub.f=0.41; UV-vis: λ.sub.max (MeOH) 406; 382; 363 nm; E.sub.1cm.sup.1% (MeOH, λ=406 nm)=1290 (theoretically for C.sub.56H.sub.81FN.sub.2O.sub.18 is 1360); MS-ESI found m/z: 1087.2 [M−H].sup.−; calculated for C.sub.56H.sub.81FN.sub.2O.sub.18 [M].sup.+• 1188.5 j) In the reaction with N-Fmoc-D-(O.sup.γ-tert-butyl)glutamic acid is obtained 56 mg of N-D-(O.sup.γ-tert-butyl)glutamylamphotericin B (A39)

(157) TLC R.sub.f=0.26; UV-vis: λ.sub.max (MeOH) 406; 382; 363 nm; E.sub.1cm.sup.1% (MeOH, λ=406 nm)=1270 (theoretically for C.sub.56H.sub.88N.sub.2O.sub.20 is 1334); MS-ESI found m/z: 1107.4 [M−H].sup.−; calculated for C.sub.56H.sub.88N.sub.2O.sub.20 [M].sup.+• 1108.6 k) In the reaction with N-Fmoc-D-(O-tert-butyl)serine is obtained 106 mg N-D-(O-tert-butyl)serylamfotericin B (A40)

(158) TLC R.sub.f=0.35; UV-vis: λ.sub.max (MeOH) 406; 382; 363 nm; E.sub.1cm.sup.1% (MeOH, λ=406 nm)=1310 (theoretically for C.sub.54H.sub.86N.sub.2O.sub.19 is 1386); MS-ESI found m/z: 1065.7 [M−H].sup.−; calculated for C.sub.54H.sub.86N.sub.2O.sub.19 [M].sup.+• 1066.6 l) In the reaction with N-Fmoc-D-phenylglycyne is obtained 82 mg N-D-phenyloglycylamphotericin B (A41)

(159) TLC R.sub.f=0.35; UV-vis: λ.sub.max (MeOH) 406; 382; 363 nm; E.sub.1cm.sup.1% (MeOH, λ=406 nm)=1280 (theoretically for C.sub.55H.sub.82N.sub.2O.sub.18 is 1400); MS-ESI found m/z: 1081.3 [M+Na].sup.+; calculated for C.sub.55H.sub.80N.sub.2O.sub.18 [M].sup.+• 1056.5

Example 8

Synthesis of N—(N′,N′-dialkyl)Aminoacyl Derivatives of Amphotericin B

(160) 0.44 mmol of aminoacid, 0.44 mmol of N-hydroxysuccinimide (HONSu), 0.44 mmol (0.26 mmol) of dicyclohexylcarbodiimid (DCC) is dissolved in 3 ml of dimethyl formamide (DMF) in 25 ml round-bottomed flask equipped with a magnetic stirrer. The reaction mixture is stirred at 37° C. for 1 hour. The reaction progress is monitored by thin layer chromatography (TLC) on Silica Gel (60 F254, Merck) in ethyl acetate:hexane (7:3 v/v) solvent system. During the reaction precipitated N,N′-dicyclohexylurea is filtered and washed with 1 ml DMF. To the filtrate 200 mg (0.22 mmol) of Amphotericin B and 0.04 ml (0.22 mmol) of triethylamine are added. The reaction progress is monitored by thin layer chromatography (TLC) on Silica Gel (60 F254, Merck) in chloroform: methanol: water (10:6:1 v/v) solvent system. Stirring is continued at 37° C. for 6-16 hours. After then the reaction mixture is added dropwise to 150 ml of diethyl ether. The resulting pale yellow precipitate is filtered under reduced pressure on a Millipore funnel. The crude product is twice washed with diethyl ether (2×50 ml) and then dried in a vacuum desiccator. The residue is purified by column chromatography on normal phase, where the solid phase is Silica Gel and solvent system is chloroform: methanol: water (10:6:1 v/v). The fractions containing pure product were collected and combined, then evaporated under reduced pressure at temperature not exceeding 35° C. The following derivatives of Amphotericin B are obtained in the reaction with the corresponding amino acids: a) In the reaction with N,N-diethyl-L-phenylalanine is obtained 48 mg of N-(L-N,N-diethylphenylalanyl)amphotericin B (A42)

(161) TLC R.sub.f=0.49; UV-vis: λ.sub.max (MeOH) 406; 382; 363 nm; E.sub.1cm.sup.1% (MeOH, λ=406 nm)=1210 (theoretically for C.sub.60H.sub.90N.sub.2O.sub.18 is 1312); MS-ESI found m/z: 1125.4 [M−H].sup.−; calculated for C.sub.60H.sub.90N.sub.2O.sub.18 [M].sup.+• 1126.6 b) In the reaction with N,N-dimethyl-L-phenylalanine is obtained 155 mg of N-(L-N,N-dimethylphenylalanyl)amphotericin B (A43)

(162) TLC R.sub.f=0.42; UV-vis: λ.sub.max (MeOH) 406; 382; 363 nm; E.sub.1cm.sup.1% (MeOH, λ=406 nm)=1210 (theoretically for C.sub.58H.sub.86N.sub.2O.sub.18 is 1346); MS-ESI found m/z: 1097.6 [M−H].sup.−; calculated for C.sub.58H.sub.86N.sub.2O.sub.18[M].sup.+• 1098.6

Example 9

Synthesis of Methyl Esters of N-substituted Derivatives of Amphotericin B

(163) 0.1 mmol of Amphotericin B derivative is dissolved in a mixture of dimethyl formamide/methanol (3 ml/1 ml), next mixture was cooled to 5 C and excess of diazomethane (ether solution) in a molar ratio of 1:2.5 is added. The reaction mixture is left for 2 hour at 0° C. The reaction progress is monitored by thin layer chromatography (TLC) on Silica Gel (60 F254, Merck) in chloroform: methanol: water (10:6:1 v/v) solvent system. After then the excess of diazomethane is decomposed with acetic acid, and the reaction mixture is added dropwise to 150 ml of diethyl ether. The resulting pale yellow precipitate is filtered under reduced pressure on a Millipore funnel. The crude product is twice washed with diethyl ether (2×50 ml) and then dried in a vacuum desiccator. The residue is purified by column chromatography on normal phase, where the solid phase is Silica Gel and solvent system is chloroform: methanol: water (15:6:1 v/v). The fractions containing product were collected and combined, then evaporated under reduced pressure at temperature not exceeding 35° C. In the case of an ester derivatives of Amphotericin B with the protected amino group, the purified product was dissolved in DMF and treated with equimolar amounts of DBN (1,5-diazabicyclo[4.2.0]non5-en) in order to remove the protected group. The reaction progress is monitored by thin layer chromatography (TLC) on Silica Gel (60 F254, Merck) in chloroform: methanol: water (10:6:1 v/v) solvent system. After the reaction excess of diethyl ether is added, the resulting pale yellow precipitate is filtered under reduced pressure on a Millipore funnel. In manner described above, exemplary is obtained 40 mg of N-D-β-(pyridin-3-yl)alanylamphotericin B methyl ester (A44)

(164) TLC R.sub.f=0.57; UV-vis: λ.sub.max (MeOH) 406; 382; 363 nm; E.sub.1cm.sup.1% (MeOH, λ=406 nm)=1250 (theoretically is C.sub.56H.sub.83N.sub.3O.sub.18: is 1362); MS-ESI found m/z: 1084.3 [M−H].sup.−; calculated for C.sub.56H.sub.83N.sub.3O.sub.18 [M].sup.+• 1085.6

Example 10

Synthesis of Amides of N-substituted Derivatives of Amphotericin B

(165) 0.1 mmol of Amphotericin B derivative is dissolved in 5 ml of dimethyl formamide (DMF) in the round-bottomed flask equipped with a magnetic stirrer. The mixture was cooled to 0° C. and 102 mg (1 mmol) of 3-N, N-dimethylpropyldiamine, 275 (1 mmol) of diphenyl azidephosphate (DPPA), and 14 ml (1 mmol) triethyl amine (TEA) are added. The reaction mixture is left for 24 hours. The reaction progress is monitored by thin layer chromatography (TLC) on Silica Gel (60 F254, Merck) in chloroform: methanol: water (10:6:1 v/v) solvent system. The reaction mixture is added dropwise to 100 ml of diethyl ether. The resulting pale yellow precipitate is filtered under reduced pressure on a Millipore funnel. The crude product is twice washed with diethyl ether (2×50 ml) and then dried in a vacuum desiccator. The residue is purified by column chromatography on normal phase, where the solid phase is Silica Gel and solvent system is chloroform: methanol: water (15:6:1 v/v). The fractions containing product were collected and combined, then evaporated under reduced pressure at temperature not exceeding 35° C. In the case of an amide derivative of Amphotericin B with the protected amino group, the purified product was dissolved in DMF and treated with equimolar amounts of DBN (1,5-diazabicyclo[4.2.0]non5-en) in order to remove the protected group. The reaction progress is monitored by thin layer chromatography (TLC) on Silica Gel (60 F254, Merck) in chloroform: methanol: water (10:6:1 v/v) solvent system. After the reaction excess of diethyl ether is added, the resulting pale yellow precipitate is filtered under reduced pressure on a Millipore funnel. In manner described above, exemplary is obtained 21 mg of N-D-β-(pyridn-3-yl)alanylamphotericin B 3-(N,N-dimethylamino)propylamide (A47)

(166) TLC R.sub.f=0.52; UV-vis: λ.sub.max (MeOH) 406; 382; 363 nm; E.sub.1cm.sup.1% (MeOH, λ=406 nm)=1150 (theoretically for C.sub.59H.sub.91N.sub.5O.sub.17 is 1220); MS-ESI found m/z: 1210.3 [M−H].sup.−; calculated for C.sub.59H.sub.91N.sub.5O.sub.17 [M].sup.+• 1211.6

Example 11

Preparation of Salts with N-methyl-D-glucamine of Amphoteric Amphotericin B Derivatives

(167) 0.1 mmol derivative of Amphotericin B in 2 ml of deionized water is suspended in round-bottomed flask equipped with a magnetic stirrer and then 0.11 mmol of N-methyl-D-glucamine dissolved in 1 ml of water is added. Next, to the solution excess of acetone is added. The resulting pale yellow precipitate is filtered under reduced pressure on a Millipore funnel. The crude product is twice washed with diethyl ether (2×50 ml) and then dried in a vacuum desiccator. In the manner described above exemplary is obtained 89 mg of N-methyl-D-glucamine salt of N-D-β-(pyridin-3-yl)alanylamphotericin B (A50)

(168) UV-vis: λ.sub.max (MeOH) 406; 382; 363 nm; E.sub.1cm.sup.1% (MeOH, λ=406 nm)=1190 (theoretically for C.sub.62H.sub.100N.sub.4O.sub.23 is 1267.9).

Example 12

Preparation of Salts with L-aspartic Acids of Basic or Amphoteric Derivatives of Amphotericin B

(169) 0.1 mmol derivative of Amphotericin B (A47) is suspended in 2 ml of deionized water in round-bottomed flask equipped with a magnetic stirrer. Next, to the reaction mixture 0.3 mmol of L-aspartic acid dissolved in 2 ml of water is added. The solution is filtered and to clear filtrate excess of acetone is added to precipitate. The resulting pale yellow precipitate is filtered under reduced pressure on a Millipore funnel. The crude product is twice washed with diethyl ether (2×50 ml) and then dried in a vacuum desiccator. In the manner described above exemplary is obtained 89 mg of 3-dimethylaminopropylamide N-D-β-(pirydyn-3-yl)alanylamphotericin B L-aspartate (A51)

(170) UV-vis: λmax (MeOH) 406; 382; 363 nm; (MeOH, λ=406 nm)=1060 (theoretically for C.sub.64H.sub.100N.sub.6O.sub.21 is 1148)

(171) Below are shown the result of in vitro antifungal activity and hemotoxicity of Amphotericin B derivatives.

(172) For the determination of antifungal activity in vitro we used method of serial dilution in buffered medium RPMI 1640, pH 7.0, in a 96-wells microplates, according to the standard procedure (National Committee for Clinical Laboratory Standards. Reference method for broth dilution antifungal susceptibility testing of yeast, approved standard, 2nd ed. M27-A2 vol. 22 Wayne, Pa., 2002). The optical density of cells suspension was measured using a microplates reader (Victor.sup.3, Perkin-Elmer) at the wavelength λ=531 nm (A.sub.531). On the basis of obtained results the diagrams of relation between A.sub.531 values and concentration of examined compound were made. From these graphs, the IC.sub.50 values were read, which were the interpolated concentrations of a tested compound, at which the A.sub.531 value was exactly 50% of the A.sub.531 value for the control sample. Moreover, MIC values, that are the lowest concentration of tested compound at which the A.sub.531 value were at most 20% of the A.sub.531 value measured for the control sample.

(173) The hemotoxicity determination was carried out by the serial dilutions method, according to the procedure described earlier (Slisz, M., et al., E., J Antibiot 57: 669-678 (2004). Human erythrocytes were suspended in the solution of saline to obtain a cell density of suspension 2×10.sup.7/ml. Suitable amounts of diluted solutions of compounds were added to the cell suspension in tubes and were incubated at 37° C. for 30 minutes and then centrifuged (1700×g, 10 min, 4° C.). The concentration of hemoglobin in supernatant after centrifugation of erythrocytes suspension were determined by measuring the absorbance at wavelength λ=540 nm (A.sub.540). The maximum level of hemolysis was obtained after incubation of cells suspension in the presence of 0.1% Tritone X-100 (control sample). On the basis of obtained results the diagrams on relation between the A.sub.540 value and concentration of examined compound were made. From these graphs, the EH.sub.50 values were read which are the interpolated concentrations of compound, for which the A.sub.540 value is exactly 50% of the A.sub.540 value measured for the control sample. Maximum concentrations of tested derivatives could not exceed 100 μg/ml, to maintain full solubility in experimental conditions. At this maximum concentration of compounds which exhibited especially low hemotoxicity, it was not possible to determine the EH.sub.50 value and in such cases was hemotoxicity specified as EH.sub.50>100 μg/ml.

(174) Obtained results are presented below in tables 2A and 2B. Table 2A presents the antifungal and hemolytic activity of a large series of Amphotericin B derivatives, additionally introducing factor describing the improvement of Amphotericin B derivatives selectivity in regard to the native antibiotic. WS factor determines the selectivity data of the derivatives, and WE factor shows the above selectivity data in regard to the same of the native Amphotericin B. Table 2B presents data concerning the broader antifungal spectrum of examined compounds.

(175) TABLE-US-00002 TABLE 2A Antifungal activity [μg ml.sup.−1] Saccharomyces Candida Candida Candida Candida Candida cerevisiae albicans tropicalis glabrata krusei lusitaniae ATCC ATCC KKP DSM DSM DSM Hemotoxi WS WE 9763 10231 334 11226 6128 70102 [μg ml.sup.−1] EH.sub.50/ WS.sub.(z)/ No. Symbol MIC IC.sub.50 MIC MIC MIC MIC MIC EH.sub.50 IC.sub.50 WS.sub.(AmB) 1. AmB 0.25 0.116 0.125 0.25 1 0.5 0.125 2.06 17.76 1 2. A1 2 0.98 4 4 8 8 2 >100 >102.04 >5.74 3. A2 4 2.22 4 4 8 8 2 >100 >45.04 >2.54 4. A3 4 2.85 4 4 8 8 4 >100 >35.09 >1.97 5. A4 4 3.11 4 4 8 8 4 >100 >32.15 >1.81 6. A5 4 1.94 4 4 4 4 2 >100 >51.55 >2.90 7. A6 2 0.64 2 2 4 8 1 >100 >156.25 >8.80 8. A7 2 0.70 4 2 4 4 1 >100 >142.86 >8.04 9. A8 4 1.36 4 4 8 8 4 >100 >73.53 >4.14 10. A19 1 0.53 1 1 2 2 1 >100 >188.68 >10.62 11. A20 4 1.63 4 4 4 4 4 >100 >61.35 >3.45 12. A21 4 1.58 4 4 4 4 2 >100 >63.29 >3.56 13. A22 1 0.71 1 1 4 2 1 >100 >140.84 >7.93 14. A23 4 2.54 4 4 8 8 4 >100 >39.37 >2.22 15. A24 2 1.13 2 2 4 4 2 >100 >88.49 >4.98 16. A25 2 0.85 2 2 4 4 2 >100 >117.65 >6.62 17. A26 4 2.55 4 4 8 8 4 >100 >39.21 >2.21 18. A27 2 0.75 2 2 4 4 2 >100 >133.33 >7.51 19. A28 1 0.50 1 1 4 2 1 81.73 163.46 9.20 20. A29 4 2.56 4 4 8 8 4 >100 >39.06 >2.20 21. A30 2 0.90 2 2 2 2 2 >100 >111.11 >6.26 22. A31 4 2.52 4 4 8 8 4 >100 >39.68 >2.23 23. A32 4 3.01 8 8 8 8 4 >100 >33.22 >1.87 24. A33 2 1.39 2 4 4 4 2 >100 >71.94 >4.05 25. A34 2 1.22 4 4 4 4 2 >100 >81.97 >4.61 26. A35 4 1.40 4 4 4 4 2 >100 >71.42 >4.02 27. A36 1 0.53 2 2 4 4 2 >100 >188.68 >10.62 28. A37 2 1.28 2 2 4 4 1 >100 >78.12 >4.40 29. A38 4 1.92 2 4 4 4 2 >100 >52.08 >2.93 30. A40 2 1.35 2 4 4 4 2 >200 >148.15 >8.34 31. A41 4 1.46 2 4 4 4 2 >100 >68.49 >3.86 32. A42 4 2.75 4 4 8 8 4 >100 >36.36 >2.05 33. A43 4 1.43 4 4 4 4 2 >100 >69.93 >3.94 WS— selectivity coefficient WE—effectivity factor WS.sub.(z)—selectivity coefficient for derivative WS.sub.(AmB)—selectivity coefficient for native antibiotic

(176) TABLE-US-00003 TABLE 2B Antifungal activity MIC [μg ml.sup.−1] Candida Candida pseudo- Candida Candida Candida Candida Candida Candida Aspergillus Trichoderma Sym- albicans tropicalis stellatoidea parapsilosis dubliniensis quilliermondii arborea lipolytica niger LOCK viride LOCK L.p. bol SC 5314 KKP 324 CBS 1905 DSM 5784 CBS 7987 DSM 11947 KKP 319 KKP 322 E201 E159 1. AmB 0.5 0.25 0.25 0.5 0.25 0.25 1 0.5 0.25 0.5 2. A19 0.5 0.5 1 2 0.5 2 2 2 1 4 3. A22 2 2 1 2 1 1 2 1 2 2 4. A30 1 1 1 2 1 1 4 2 2 4 5. A33 1 1 1 1 1 1 2 1 1 4 6. A34 1 1 1 1 1 2 4 1 2 4 7. A36 1 1 2 2 1 1 4 2 0.5 4 8. A41 1 2 2 2 1 1 4 2 2 4 9. A42 4 4 4 4 2 4 4 4 1 2 10. A43 1 2 1 1 1 1 8 1 1 4
Antifungal in vitro activity of Amphotericin B derivatives against multidrug resistant (MDR) fungal strains was also examined. The results are presented in table 3.

(177) TABLE-US-00004 TABLE 3 Antifungal activity MIC [μg ml.sup.−1] Candida albicans clinical strains Gu5 5674 B4 (CaCdr1p, F5 STY7 (CaCdr1p, L.p. Symbol B3 (CaMdr1p) Gu4 CaCdr2p) F2 (CaMdr1p) STY31 (CaCdr1p) CaCdr2p) 1. AmB 0.125 0.25 0.125 0.5 0.5 0.5 1 1 1 2. A19 1 2 1 1 2 2 1 1 1 3. A22 0.25 4 0.25 2 4 8 2 2 1 4. A30 0.5 2 1 2 2 4 1 1 0.5 5. A33 0.5 1 1 1 4 8 0.5 1 2 6. A34 1 2 1 2 4 8 2 2 4 7. A36 0.5 4 1 4 4 4 0.25 0.5 0.5 8. A41 1 1 1 1 2 4 1 1 1 9. A42 2 8 2 4 1 2 4 4 4 10. A43 0.5 1 1 2 4 4 1 0.5 1

(178) TABLE-US-00005 Strains Description Reference Candida albicans clinical isolates B3 fluconazole sensitive, parent strain for B4 1 B4 fluconazole-resistant due to the overexpression 1 of CaMDR1 Gu4 fluconazole sensitive, parent strain for Gu5 1 Gu5 fluconazole-resistant due to the overexpression 1 of CDR1 and CDR2 F2 fluconazole sensitive, parent strain for F5 2 F5 fluconazole-resistant due to the overexpression 2 of CaMDR1 and ERG11 STY7 C. albicans 5674 overexpressing CDR1 and CDR2 3 mutant derivative, deletion of CDR2 STY31 C. albicans 5674 overexpressing CDR1 and CDR2 3 mutant derivative, deletion of CDR1 and CDR2 References: 1. Franz, R., Ruhnke M, Morschhäuser J. 1999 Molecular aspects of fluconazole resistance development in Candida albicans. Mycoses, 42, 453-458. 2. Franz, R., Kelly S. L., Lamb D. C., Kelly D. E., Ruhnke M., Morschhäuser J. 1998. Multiple molecular mechanisms contribute to a stepwise development of fluconazole resistance in Clinical Candida albicans strains. Antimicrobial Agents and Chemotherapy 42: 3065-3072. 3. Tsao S., Rahkhoodaee F., Raymond M. 2009. Relative contributions of the Candida albicans ABC transporters Cdr1p and Cdr2p to clinical azole resistance. Antimicrobial Agents and Chemotherapy 53: 1344-1352. Cytotoxic activity of Amphotericin B derivatives against mammalian cells was determined in tissue culture. For examinations were used selected cell lines: CCRF-CEM—human acute lymphoblastic leukemia; HepG2—human malignant hepatoma; LLC-PK1—epithelial cell of pig kidney; All lines were from ATCC collection. The study was conducted using described below methods of culturing and determination of cytotoxic activity. CCRF-CEM cells were cultured in medium RPMI 1640+10% fetal bovine serum (FBS), LLC-PK1 cells in medium Medium 199+3% FBS, HepG2 cells in medium MEM+10% FBS. All media contained 100 μg/ml of penicillin G and streptomycin. 24-wells microplates containing appropriate medium were inoculated with the cells in amount of 1.2×10.sup.4 cells/well and allowed to stand overnight. Next, tested compounds as solution in dimethylsulfoxide (DMSO) were added in volume of 10 μl (serial 2× dilutions). To control well 10 μl of DMSO was added. Microplates with cell suspensions were incubated for 120 h at temperature 37° C. at atmosphere of 95%/5% CO.sub.2. After incubation, to all wells 200 μl of solution of 3-(4,5-dimethyltiazole-2-yl)-2,5-diphenylotetrazole bromide (MTT) in PBS (4 mg/ml) was added and plates were further incubated for 4 h at 37° C. Next, to dissolve crystals of formazane 1 ml of DMSO was added and absorption of solutions was measured at the wavelength λ=540 nm (A.sub.540), using a microplates reader (Victor.sup.3, Perkin-Wallac). On the basis of received results the diagrams on the relation between the A.sub.540 value and concentration of examined compound were prepared. From these graphs IC.sub.50 values were read, that is concentration of tested compound in the presence at which A.sub.540 value is half of A.sub.540 value measured in the control sample.
The obtained results are presented in Table 4.

(179) TABLE-US-00006 TABLE 4 IC.sub.50 [μg/ml] Compound HepG2 LLC-PK1 CCRF-CEM Amfotericin B 5.40 ± 1.05 19.7 ± 8.05  4.30 ± 0.86 (Fungizon) A-6 >100 >100 >100 A-16 73.3 ± 1.20 >100 50.1 ± 1.3 A-19 >100 >100 >100 A-22 >100 >100 >100 A-24 >100 >100 80.9 ± 2.3 A-25 >100 >100 >100 A-27 >100 >100 >100 A-28 >100 >100 >100 A-30 >100 >100 >100 A-33 >100 >100 >100 A-34 >100 >100 >100 A-35 >100 70.7 ± 2.3  >100 A-36 >100 >100 >100 A-41 >100 >100 >100 A-42 >100 >100 >100 A-43 >100 >100 >100