# Patent

## Title
Semisynthetic derivatives of Nystatin A1

## Abstract

The invention provides semisynthetic derivatives of Nystatin A1, water soluble salts and complexes, pharmaceutical compositions and plant protection products comprising the derivatives and their use, as antifungal antibiotics (Formula 1a).

## Background
The invention concerns water soluble and reduced toxicity, sterically hindered derivatives of the antifungal antibiotic, Nystatin A1 which contain bulky fragments on the substituent linked to the amino group of the antibiotic, which may cause steric hindering effects, as well as esters and amides of such derivatives and their salts with acids or bases or formulations containing complexing compounds, as water-soluble forms, and their use for the production of antifungal preparations primarily for medical and veterinary needs and for plant protection, as well as for other applications like overcoming the fungal invasion in buildings.
BACKGROUND TO THE INVENTION
Antibiotic Nystatin A1 is the main component of antibiotic complex Nystatin, a group of tetraeno-diene polyene macrolides produced by Streptomyces noursei (E. L. Hazen, R. Brown, Science 112, 112, 1950; E. L. Hazen, R. Brown, Proc. Soc. Exptl. Biol., 76, 93, 1951; A. H. Thomas and in., J. Chemother., 216, 367, 1981; A. H. Thomas et al., Analyst 107, 849, 1982) which in addition to Nystatin A1 (Borowski et al., Tetrahedron Lett. 8, 685, 1971) also contains Nystatin A2 (J. Pawlak et al., Polish J. Chem. 79, 1673, 2005) and Nystatin A3 (J. Zielinski et al., J. Antibiot. 41, 1289, 1988). Nystatin A1 is currently commercially available in acceptably pure form, manufactured by Bristol-Myers-Squibb in USA, as well as by other companies. Nystatin A1 and other components of antibiotic complex Nystatins are also produced by Streptomyces noursei var. Polyfungini named polyfungin (N. Porowska et al., Rec. Tray. Chem. 91, 780, 1971). This complex contains additionally a new component called polyfungin B (Zielinski et al., J. Antibiot. 32, 565, 1979). Among tetraeno-diene polyene macrolides, well identified compound is also Amphotericin A (P. Sowinski et al., J. Antibiot. 38, 175, 1985), produced together with Amphotericin B by Streptomyces nodosus. According to our invention, spatially hindered derivatives of Nystatin A1 and process of their preparation also apply to the other above-mentioned tetraeno-dienes.
Chemotherapy of fungal infections is one of the most difficult and not yet successfully solved problems in modern medicine. This is a consequence of the fact that both, pathogenic fungal organisms, and the humans are eukaryotic organisms and this is the reason of essential difficulties in developing the selectively acting drugs with low toxicity for the patients. This difficulty has been omitted in the treatment of topical fungal infections. This includes such areas of clinical mycology as gynecology, dermatology, gastroenterology, pulmomology, urology and ophthalmology, where the problem of compounds toxicity occurs to be less dramatic (C. P. Schaffner, in Macrolide antibiotics, S. Omura (ed.), Academic Press. Inc., Orlando, p. 457, 1984). The most popular drugs from polyene group for such treatment are Nystatin, Pimaricin and Amphotericin B, which due to the lack of resorption in oral administration are practically non-toxic. However, invasive mycoses concerning the infection of internal organs and fungemia are still problems far from successful solution. Current epidemiological statistics concerning the mortality in such types of illnesses are not satisfactory (M. A. Pfaller, D. J. Diekem, Clin. Microbiol. Rev. 20, 133, 2007; T. F. Patterson, Lancet 366, 1013, 2005; S. K Fridkin, Clin. Infect. Dis. 41, 240, 2005). Especially dangerous are invasive candidoses and aspergilloses and infections caused by certain other fungal pathogenes. In the case of invasive candidosis the mortality is in the range 30-70%, aspergillosis more than 50%, the frequency of invasive mycoses in oncology/hematology is approximately 50%, in the case of mycoses of children with leukemia is 29-39% (S. E. Soloviera et al., Rus. Chem. Rev. 80, 103, 2011: A. L. Demain, S. Sanchez, J. Antibiot., 62, 5, 2009; G. O. Bronin et al., Pediatryia 4, 31, 2004). Over 90% of HIV-positive patients suffer from mycoses, while pneumonia caused by Pneumocystis carinii is the main reason of death in patients with AIDS. Systemic mycoses are common reason of death in adult patients with leukemia. Candida spp., in regard to frequency of incidences, are the fourth etiological factor of hospital infections and is a cause of 8-11% of all general infection with mortality up to 40%. The frequency of fungal infections in patients after organ transplants is in the range 5-40% depending on the type of organ transplanted. Invasive aspergilosis of lungs is main reason of patients death after transplant of bone marrow. Blastomycosis, histoplasmosis and coccidiomycosis are endemic mycoses with high frequency of appearance in many regions of the world.
The unfavourable situation in the clinical mycology since over 20 years is constantly getting worse for several reasons. One of them is steady increase of infections caused by species of fungal microorganisms previously being non-pathogenic (D. A. Enoch et al., J. Med. Microbiol. 55, 809, 2006; N. Nucii, K. A. Man, Clin. Infect Dis. 41, 521, 2005). The increase of fungal infections is also caused by the use of antibacterial chemotherapeutical agents with broad spectrum and by the use of steroids, and above all by decreasing the immune system activity in an increasing number of patients, connected with the development of transplantology, which required the use of immunosuppressive drugs, and also with the increase of cancer cases and thus usage of immunosuppressive cytostatics (N. Siugh, Med. Mycol. 43, suppl. 1, 267, 2005; A. L. Demain, S. Sanchez, J. Antibiot. 62, 5, 2009).
Especially alarming is the steady decrease of therapeutic values of currently available antifungal chemotherapeutics used for the treatment of systemic infections, as a result of the rapid development of resistance of pathogenic fungal strains (D. Sanglard, Curr. Opinion Microbiol. 5, 378, 2002; D. P. Kontoyiannis, R. E. Lewis, Lancet 359, 1135, 2002). The most dangerous type of fungal resistance is the multidrug resistance (MDR), which affects antifunagals activity towards systemic as well as topical administration. The MDR strains overexpress transporter proteins which export a drug from the cells, thus not allowing to retain in fungal cells its therapeutic concentration.
Enzyme inhibitor, 5-fluorocytosine, often used in combination with Amphotericin B to increase its uptake by membrane permeabilisation, as an antimetabolite avoids the exporting activity of MDR transporting proteins, but enhances the development of specific type of resistance mainly by the loss of cytosine permease and deaminase. Particularly clinically valuable fungicides of “azoles” group, mostly triazoles, such as flucanazole, voriconazole, posaconazole and others, are partially susceptible to their removal from the cells by MDR exporting proteins (R. Franz et al., Antimicrob. Ag. Chemother., 42, 3065, 1998; R. Wakiec et al., Mycoses, 50, 109, 2007). However, being the inhibitors of lanosterol demethylase, interacting with the enzyme, also induce the changes in the structure of enzymatic protein leading to the loss of inhibitory activity of these compounds. Valuable and very promising fungicide caspofungin, although with narrow antifungal spectrum but with excellent selectivity, as inhibitor of β-D-glucan synthase, interacts with the enzyme, which unfortunately leads to the induction of changes in the structure of enzyme protein and in consequence the loss of inhibitory activity of compound. The reports on growing resistance to the action of this drug are starting to be published. Therefore, concerning the MDR problem, there practically remains only one effective group of fungicides, polyene macrolides, which do not induce the development of resistant strains. These antibiotics as not being the substrates of MDR exporting proteins, retain full activity against multidrug resistant strains (M. Ślisz i in., J Antibiot. 60, 436, 2007). Although there is data on the appearance of strains with reduced sensitivity to these antibiotics, as a result of certain changes in the lipid composition of cytoplasmic membrane, but these changes are rather phenotypic and regressing after discontinued contact with the drug.
Presented situation in clinical mycology points to the necessity of further search for antifungal drugs. One of the intensively developing research projects concerned study on the modifications of polyene macrolides aimed at removing of their main shortcomings, which are high toxicity and lack of water solubility of native compounds. However, until now none of the products of this antibiotics modification have been introduced to clinical practice. The only practical progress in this area was the introduction to clinical use of Amphotericin B complexes with lipids or liposomal formulations as Abalcet®, Amphotec® and AmBisome®. However, these formulations of Amphotericin B are only a little less toxic in comparison to the native antibiotic.
Due to the high toxicity of Amphotericin B, as well as its very high cost, the attention has been drawn by another clinical antibiotic from polyenes group, Nystatin, so far used only topically. This antibiotic exhibits all advantageous features of that type of compounds such as: broad antifungal spectrum, fungicidal effect, high antifungal activity, lack of induction of resistance and lack of interaction with overexpressed protein transporters in multidrug resistant strains (MDR). Furthermore, Nystatin is much cheaper than Amphotericin B. Studies have been undertaken on the application of Nystatin also in the treatment of systemic fungal infections, using soluble lipidic and lyposomal formulations (R. Semis et al., Mycopathologia, 169, 333, 2010; R. Semis et al., J. Antimicr. Ag., 38, 336, 2011; R. Semis et al., J. Antimicrob. Chemother., 67, 1716, 2012; R. Semis et al., Med. Mycol. Month Early on line 1, 2012).
The studies on chemical modifications of Nystatin have been also carried out. Earlier obtained Nystatin A1 derivatives, are compounds with the modified amino group of the mycosamine moiety and also the carboxyl group of aglycone. The attempts to modify compounds by genetic manipulations of antibiotic-producing organism have also been performed. Chemical modifications of Nystatin A1 have been intended to improve the solubility and to reduce toxicity of the compounds. However, no significant progress has been achieved in this matter, because no theoretical background for the rational modifications has been worked out. The synthesis of derivatives had accidental character and were based rather on random screening.
The prior art on Nystatin A1 derivatives includes 1) derivatives at amino group; 2) derivatives at carboxyl group; 3) double derivatives including amino and carboxyl group; 4) analogues with genetically modified aglycone fragment.
Among the derivatives at amino group many N-acyl derivatives have been synthesized. There are simple derivatives (U.S. Pat. No. 3,244,590; C. P. Schaffner, E. Borowski, Antibiot. Chemother. 11, 724, 1961), more complicated ones (L. Silva et al., J. Photochem. Photobiol. B. Biol. 72, 17, 2003), compounds including fluoroorganic derivatives (Yu. Shenin et al., Pharmac. J. 32, 109, 1996), amino acyl derivatives (A. Czerwiński., J. Antibiot. 39, 1025, 1986; Polish Patent 142847). Other derivatives at amino group comprise guanidine derivative (U.S. Pat. No. 4,396,610), N-enamine and amidine derivatives (Stefańska et al., Acta Polon. Pharm., XLV, 71, 1988; Polish Patent 120111), N-trialkylsilyl derivatives (V. V. Balakhov et al., Khim. Farm. Zh. 11, 45, 1977) and Schiff bases (V. V. Balakhov et al., Khim. Farm. Zh. 11, 45, 1977). A large group constitutes N-alkyl derivatives. These are N-alkylhydrofosforyl derivatives (V. V. Belakhov et al., Khim. Farm. Zh. 25, 45, 1991), N,N,N-trimethyl derivative (U.S. Pat. No. 4,144,328), products of reductive N-alkylation (V. Paquet, E. M. Carreira, Org. Lett. 8, 1807, 2006; U.S. Pat. No. 6,664,241 B2). Particular attention is drawn to N-alkyl derivatives which are N-glycosyl products of Amadori reaction rearrangement (L. Falkowski et al., J. Antibiot. 28, 244, 1975; L. Falkowski et al., Acta Polon. Pharm. 37, 517, 1980; L. Falkowski et al., Polish J. Chem. 56, 123, 1982; Polish Patent 82224; U.S. Pat. No. 6,664,241 B2) and their water-soluble salts with N-methyl-D-glucamine (U.S. Pat. No. 4,195,172).
Nystatin A1 derivatives with modified carboxyl group include a number of compounds. These are esters (T. Bruzzesse et al., Experientia 28, 1515, 1972; T. Bruzzesse et al., J. Pharm. Sci. 64, 462, 1975; U.S. Pat. No. 3,780,173; P. Schaffner, W. Mechlinski, J. Antibiot. 25, 259, 1972; D. P. Bonner et al., J. Antibiot. 25, 261, 1972; B. Stefańska et al., Acta Polon. Pharm. 40, 71, 1983; U.S. Pat. No. 5,981,721), hydrazides (J. Grzybowska, E. Borowski J. Antibiot. 43, 907, 1990) and amides (Polish Patent 138831; U.S. Pat. No. 6,664,241 B2), as well their soluble salts (Polish Patent 138831).
There are also known Nystatin A1 derivatives modified in both amino and carboxyl groups of the antibiotic. These compounds combine above described types of chemical modification at both groups. They include the methyl ester of N,N,N-trimethyl derivative (L. Falkowski et al., J. Antibiot. 32, 1080, 1979; L. Falkowski et al., Acta Polon. Pharm., 37, 631, 1980; Polish Patent 122884), methyl esters of N-enamine and amidine derivatives (Polish Patent 120035), esters of guanidine derivatives (U.S. Pat. No. 4,396,610), methyl ester of N-fructosyl-N-methyl Nystatin A1 (Polish patent 199213), esters of glycosyl derivatives (U.S. Pat. No. 6,562,796 B2), methyl esters of aminoacyl derivatives (Polish Patent 142848) and amides of N-mono and di-alkyl, as well of N-glycosyl derivatives.
Efforts have been also made to modify the macrolide ring of Nystatin A1 by the genetic manipulation of Streptomyces noursei, which produces this antibiotic. That way two new hydroxyl groups have been introduced to that ring, which slighty increase the hydrophilicity of the molecule and thus improve its solubility in water (S. F. E. Borgos et al., J. Med. Chem. 49, 2431, 2006). However, these derivatives cannot be regarded as derivatives of Nystatin A1, but as its analogs. Similarly, other genetic modifications of the antibiotic “producer” also lead to obtaining novel antibiotics and their derivatives, which cannot be classified as a semi-synthetic derivatives of the original native product, Nystatin A1 (International Patent Application PCT/GB2008/002238). Other known Nystatin derivatives are presented in the patent specification WO 01/68102, and in U.S. Pat. No. 6,413,537.
Among above mentioned Nystatin A1 derivatives, there are no compounds which could combine features, particularly important for practical application, such as water solubility and essentially reduced toxicity.
The advantage of new semisynthetic Nystatin A1 derivatives, according to the invention, is that they exhibit low hemotoxicity, which is a common toxicity test for compounds of polyene macrolides group, and form water soluble salts. They also exhibit antifungal activity towards broad spectrum of Candida species, filamentous fungi and dermatophytes and against strains with multidrug resistance (MDR) with overexpression of MDR protein transporters MDR1p, as well as Cdr1p and Cdr2p.
Unexpected novelty enabling to obtain according to the invention, advantageous effect of hemotoxicity reduction and water solubility, is introduction to substituents at amino group of Nystatin A1 of bulky moieties, which induce steric hindrance effects. It appeared that such steric hindrance factors decrease lethal permeabilising activity of Nystatin A1 derivatives a greater degree towards mammalian than fungal cells, which increases their selectively of action and essentially reduces the hemotoxicity of these compounds. The presence of a bulky moiety attached to the amino group of Nystatin A1 also disturbs the zwitterionic structure of the antibiotic thus enabling the formation of soluble salts.
Bulky moieties which may give the effect of steric hindrance include ring systems carbo-, as well as heterocyclic, alicyclic and aromatic, bulky substituents as tert-butyl, nitro group, bromine atom and also aliphatic fragments, which, due to their flexibility, can form voluminous conformational structures. The introduction of the steric hindering moieties to the molecule of a biologically active compounds can influence in a different way the affinity to its molecular target, which in the case of Nystatin A1 and its derivatives is ergosterol in fungal cells and cholesterol in mammalian cells. Until now, for Nystatin A1 derivatives, the influence of steric hindrance on the differentiation of lethal effects towards both types of cells has not been known. There is no general rule concerning the influence of steric hindrance of biologically active compounds on their properties. This effect is associated with a defined compound with a specific structure and its conformational dynamics. Nystatin A1 molecule, because of its flexible structure is very dynamic in regard to conformational changes. Effect of steric hindrance of derivatives on their biological properties can be confronted only with other polyene macrolides of the same group which are tetraeno-dienes. In that respect, there is no data available prior to the invention.
SUMMARY OF INVENTION
Accordingly, a first aspect, the invention provides sterically hindered derivatives of Nystatin A1 according to the formula 1:




or a salt or complex thereof;

    wherein R1 is independently chosen from a hydrogen atom, optionally substituted alkyl, a succinimidyl derivative, a glycosyl residue, an optionally substituted aminoacyl residue, or an optionally substituted thioureidyl residue;
    R2 is a hydrogen atom or a substituent such as defined for R1;
    R3 is a hydroxyl group, alkoxyl group or an alkylamino or aminoalkyl derivative.


In certain embodiments, when R3 is hydroxyl, R1 and R2 are not both hydrogen.
Definitions
The term “halogen” or “halo” as used herein means fluorine, chlorine, bromine or iodine.
The term “heteroatom” as used herein refers to one or more of oxygen, sulfur, nitrogen, phosphorus or silicon.
The term “aliphatic” as used herein refers to a straight or branched chain hydrocarbon which is completely saturated or contains one or more units of unsaturation. Thus, aliphatic may be alkyl, alkenyl or alkynyl, preferably having up to 20 carbon atoms, up to 12 carbon atoms or up to 6 carbon atoms.
The terms “bulky” or “spatially expanded”, which can be used interchangeably, as used herein refer to a group or moiety which may give the effect of steric hindrance and include ring systems carbo-, as well as heterocyclic, alicyclic and aromatic, bulky substituents as tert-butyl, nitro group, bromine atom and also aliphatic fragments, which, due to their flexibility, can form voluminous conformational structures. In the context of the invention a bulky alkyl may, for example be optionally substituted branched alkyl of 4 or more carbon atoms or an optionally substituted alkyl or alkyl-comprising moiety, also comprising an optionally substituted carbo- or heterocyclic, aminoacyl, thiureidyl or succinimdyl moeity.
The term “alkyl” as used herein refers to a straight or branched chain alkyl group. Preferably, an alkyl group as referred to herein is a C1-C20alkyl group, preferably a C1-C12alkyl group. More preferably, an alkyl group as referred to herein is a lower alkyl having 1 to 6 carbon atoms.
The term “non-branched”, used interchangebly with “simple”, as used herein refers to a straight chain alkyl group. Preferably, a simple alkyl group as referred to herein is a lower alkyl having 1 to 6 carbon atoms.
The terms “carbocycle” or “carbocyclic moiety” as used herein refer to a saturated or partially unsaturated mono-, bi- or tri-cyclic group having 3 to 14, preferably 3 to 8 and more preferably 3 to 6, ring carbon atoms or a mono-, bi- or tri-cyclic aromatic ring having 6 to 14, preferably 6 to 10, carbon atoms. A carbocycle is a cycloaliphatic, preferably a “cycloalkyl” which as used herein refers to a fully saturated hydrocarbon cyclic group, or an “aryl”. Preferably, a cycloalkyl group is a C3-C6 cycloalkyl group and preferably, an aryl is phenyl or napthyl. Bi- or tri-cyclic groups may contain fused aromatic, saturated and/or partially unsaturated rings.
The terms “heterocycle” or “heterocyclic moiety” as used herein refer to a saturated or partially unsaturated mono-, bi- or tri-cyclic group having 3 to 14, preferably 3 to 10, ring atoms or a mono-, bi- or tri-cyclic aromatic ring having 6 to 14, preferably 6 to 10, ring atoms and having, in addition to carbon ring atoms, one or more ring heteroatoms selected from O, N, P and S (preferably O, N and S). A heterocycle is cycloheteroaliphatic, preferably a “heterocycloalkyl”, which as used herein refers to a saturated heterocyclic group, or a “heteroaryl”, which refers to a monocyclic or bicyclic aromatic ring system. A heterocycle preferably has 3 to 7 ring atoms or if aromatic, 5 to 10 ring atoms and may contain fused aromatic, satuated and/or partially unsaturated rings. Preferably a heterocycle is piperidine, morpholine, piperazine, pyrrolidine, pyridine or imidazole.
An aliphatic, alkyl, carbocycle, heterocycle, cycloalkyl, aryl, heteroaryl or aminoacyl group as referred to in respect or any of the chemical moeities described herein, may be unsubstituted or may be substituted by one or more substituents independently selected from the group consisting of halo, aliphatic, —ORo, —Ro, —SRo, NHRo, —NRO2, —CORo, —COORo, —NH2, —NO2, —OH, —COOH, —CN, hydroxyalkyl, alkylcarbonyloxy, alkoxycarbonyl, alkylcarbonyl or alkylsulfonylamino, wherein Ro is an optionally substituted aliphatic (preferably alkyl), carbocycle (preferably aryl or cycloalkyl) or heterocycle (preferably hereoaryl or heterocycloalkyl) optionally substituted with or with any one or more of substituents independently selected from halo, aliphatic, —OR, —R, —SR, NHR, —NR2, —COR, —COOOR, —NH2, —NO2, —OH, —COOH, —CN, hydroxyalkyl, alkylcarbonyloxy, alkoxycarbonyl, alkylcarbonyl or alkylsulfonylamino, wherein R is as defined for Ro, substituted or unsubstituted. Preferred substituents include halo, lower alkyl, alkylamino, —NH2, NO2, —OH, —CN, or alkoxycarbonyl.
An alkylamino or aminoalkyl derivative is a moiety comprising an alkylamino or an aminoalkyl moiety, the alkyl portion of which may be optionally substituted with any substituent as described for alkyl above.
The term “alkylamino” as used herein includes “monoalkylamino” and “dialkylamino”.
The term “succinimidyl derivative” as used herein refers to a moiety containing a succinimide residue of structure




Preferably, a succinimidyl residue is of structure





wherein R′′ is an optionally substituted aliphatic or an optionally substituted carbo- or heterocyclic moiety.

The term “glycosyl” as used herein refers to a cyclic monosaccharide or oligosaccharide. Preferably, glycosyl is fructosyl.
The term “thioureidyl residue” as used herein refers to a substituent bonded to a nitrogen atom to form a —N—C(S)—NR′2 group, wherein each R′ is independently selected from a hydrogen atom or R′′ as defined above. Preferably R′ is a hydrogen atom or an optionally substituted aliphatic and more preferably one R′ is a hydrogen atom and the other R′ is an optionally substituted aliphatic.
The term “ester” refers to a group —C(O)O—R, wherein R is, for example, optionally substituted aliphatic, carbocycle or heterocycle.
The term “alkoxy” as used herein refers to a group of the form —O—R, wherein R is alkyl, preferably lower alkyl.
The term “aminoacyl residue” refers to a moeity comprising an optionally substituted aminoacyl group, wherein an amino acyl group as used herein refers to an acyl group substituted with an amine, monoalkylamine or dialkylamine at the α or β position relative to the carbonyl. Aminoacyl may be substituted by one or more substituents as described above. In some embodiments, aminoacyl may be substituted by one or more substituents independently selected from the group consisting of optionally substituted aliphatic, alkoxy, aralkyl, heteroaralkyl, carbocyclo, heterocyclo, alkylcarbonyloxy, alkoxycarbonyl or alkylcarbonyl. Preferred substituents include optionally substituted lower alkyl, alkylamino (monoalkylamino or dialkylamino), aralkyl, heteroaralkyl, carbocyclo and heterocyclo.
The terms “aralkyl” and “heteroaralkyl” as used herein refers to an alkyl group as defined above substituted with an aryl or heteroaryl group as defined above. The alkyl component of an “aralkyl” or “heteroaralkyl” group may be substituted with any one or more of the substituents listed above for an aliphatic group and the aryl or heteroaryl component of an “aralkyl” or “heteroaralkyl” group may be substituted with any one or more of the substituents listed above for aryl, heteroaryl, carbocycle or heterocycle groups. Preferably, aralkyl is benzyl.
In compounds of the invention, one or more asymmetric carbon atoms may be present. For such compounds, the invention is understood to include all isomeric forms (e.g. enantiomers and diastereoisomers) of the compounds as well as mixtures thereof, for example racemic mixtures.
A compound of the invention, including salts, hydrates and complexes thereof, may in some embodiments be provided in a water soluble form. A compound can be considered to be water soluble, for example, if it will dissolve in water at room temperature (20° C.), optionally with heating, agitation or sonication. In some embodiments, a compound of the invention (e.g. in a salt form) may be considered water soluble if it is soluble in water at room temperature at a concentration of at least 10 mg/ml, preferably at least 20 mg/ml. As described herein, a compound of the invention may also be provided in the form of a complex, with a complexing agent. In the context of the invention, a complex may be considered to be water soluble if it forms a transparent colloidal suspension in water, for example under the conditions mentioned above.
The term “complexing compound” as used herein refers to a compound with which a compound of the invention can form a non-covalent complex. Complexing compounds can include, for example, calcium salts, succinic acid, sodium deoxycholate or sterols.


DETAILED DESCRIPTION OF INVENTION
In a first aspect, the invention provides sterically hindered derivatives of Nystatin A1 according to the Formula 1a:




or a salt, hydrate or complex thereof;

    wherein R1 is independently chosen from a hydrogen atom, optionally substituted alkyl (preferably a non-branched alkyl or a substituted alkyl), an optionally substituted succinimidyl derivative, a glycosyl residue, an optionally substituted aminoacyl residue, or an optionally substituted thioureidyl residue;
    R2 is a hydrogen atom or a substituent such as defined for R1;
    R3 is a hydroxyl group, alkoxyl group or an alkylamino or aminoalkyl derivative.


In a preferred embodiment of a compound of Formula 1a as defined above, when R3 is hydroxyl, R1 and R2 are not both a hydrogen atom.
In some embodiments a compound of Formula 1a as defined above may be a compound according to Formula 1b




or a salt or complex thereof;

    wherein R1, R2 and R3 are as defined in claim 1;
    X is absent or present and, when present, X is one or more molecules of base or acid, or complexing compound.


In some embodiments a compound of the invention is a compound according to Formula 1b




    
    
        wherein R1 is chosen from the group comprising: a hydrogen atom, a non-branched alkyl, an optionally substituted or spatially expanded alkyl, including a succinimidyl derivative, an alkyl derivative containing carbo- or heterocyclic moieties, a glycosyl residue, an optionally substituted or spatially expanded aminoacyl residue or thioureidyl residue optionally substituted with a bulky aliphatic or cyclic substituent containing at least one basic nitrogen atom;
    
    
    R2 is hydrogen atom or substituents such as defined for R1,
    R3 is hydroxyl group, or alkoxyl group or an aminoalkyl derivative;
    and their water-soluble salt or complexes, where X is one or more molecules of base or acid, or complexing compound.


In further embodiments, the invention provides a compound wherein one or both of R1 and R2 is alkyl substituted with an optionally substituted alkylamino or an optionally substituted carbo- or heterocyclic moiety. In some embodiments one or both of R1 and R2 is, independently, dialkylamino or optionally substituted N-linked heterocycle (preferably heterocycloalkyl). In some embodiments, N-linked heterocycle is unsubstituted or substituted with alkyl.
In further embodiments, the invention provides a compound wherein one or both (preferably one) of R1 or R2 is a succinimidyl derivative of structure




and

    wherein R4 is X—Y, wherein X is an optionally substituted alkyl or a single bond; and Y is an optionally substituted carbo- or heterocyclic moiety or NR*2, NH2, NHR*, where R* is an optionally substituted aliphatic, or 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. Preferably, Y is an optionally substituted carbo- or heterocyclic moiety or a dialkylamino. X may be branched or non-branched alkyl, e.g. lower alkyl. In some embodiments, Y is unsubstituted or substituted with one or more of alkyl or halo.


In further embodiments, the invention provides a compound wherein one or both (preferably one) of R1 or R2 is a thioureidyl or structure




and wherein R5 is in the form W—Z, wherein W is an optionally substituted alkyl; and Z is an optionally substituted nitrogen-containing heterocycle, preferably nitrogen-linked, or NR*2, NH2, NHR*, where R* is an optionally substituted aliphatic, or 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 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.

In further embodiments, the invention provides a compound wherein one or both (preferably one) of R1 or R2 is a aminoacyl of structure




and wherein;

    R6 and R7 are independently chosen from a hydrogen atom or an optionally substituted alkyl, or R6 and R7 can be taken, together with the atom to which they are joined, to form an optionally substituted nitrogen-containing cyclic moiety; R8 and R9 are, independently, hydrogen or —U—V, wherein U is an optionally substituted alkyl linker (preferably lower alkyl) or a single bond and V is a hydrogen atom, or an optionally substituted aliphatic, carbocyclic (preferably aryl), heterocyclic (preferably heteroaryl or heterocycloalkyl), alkoxy, or ester moiety. In some embodiments, one of R8 and R9 is hydrogen.


In further embodiments, the invention provides a compound wherein one or both (preferably one) of R1 or R2 is a aminoacyl of structure




and wherein;

    R10 and R11 independently chosen from a hydrogen atom or an optionally substituted alkyl or R10 and R11 can be taken, together with the atom to which they are joined, to form an optionally substituted nitrogen-containing cyclic moiety;
    R12, and R13 are, independently, hydrogen or —U—V, wherein U is an optionally substituted alkyl linker (preferably lower alkyl) or a single bond and V is a hydrogen atom, or an optionally substituted aliphatic, carbocyclic (preferably aryl), heterocyclic (preferably heteroaryl or heterocycloalkyl), alkoxy, or ester moiety; and R12′ and R13′ are, independently, hydrogen or alkyl (preferably lower alkyl), preferably both R12′ and R13′ are hydrogen.


In further embodiments, the invention provides a compound wherein one or both (preferably one) of R1 or R2 is a glycosyl residue, preferably a fructosyl residue.
In some embodiments, the invention provides a compound wherein one of R1 and R2 is a hydrogen atom or an unsubstituted alkyl (preferably non-branched) or a substituted alkyl; and the other of R1 and R2 is an unsubstituted non-branched alkyl, a substituted alkyl, a succinimidyl derivative, a glycosyl residue, an optionally substituted aminoacyl residue, or an optionally substituted thioureidyl residue. In some preferred embodiments, one of R1 and R2 is a hydrogen atom or a non-branched alkyl or alkyl substituted with alkylamino or an optionally substituted carbo- or heterocyclic moiety (preferably a N-linked heterocycloalkyl, optionally substituted with alkyl); and the other of R1 and R2 is an unsubstituted non-branched alkyl, a substituted alkyl, a succinimidyl derivative, a glycosyl residue, an optionally substituted aminoacyl residue, or an optionally substituted thioureidyl residue as described in respect of any of the classes or subclasses of compounds above.
In preferred embodiments, the invention provides a compound wherein R3 is hydroxyl, methoxy, or —NR14—(C1-C6alkyl)-NR15R16, wherein R14 is a hydrogen atom or methyl, R15 and R16 are independently chosen from optionally substituted aliphatic, preferably lower alkyl.
In preferred embodiments, the invention provides a compound wherein, the hemotoxicity expressed by EH50 value is greater than 200 μg/ml, preferably greater than 300 μg/ml.
In some embodiments, the invention provides a compound of any of the subclasses described above wherein R1 is any of the groups as listed for R1 in Table 1.
In some embodiments, the invention provides a compound of any of the subclasses described above wherein R2 is any of the groups as listed for R2 in Table 1.
In some embodiments, the invention provides a compound of any of the subclasses described above wherein R3 is any of the groups as listed for R3 in Table 1.
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.
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 a calcium salt, succinic acid, sodium deoxycholate or a sterol.
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.
Any of the salts or complexes as described above may be water soluble.
In another embodiment, the invention provides a compound of Formula 1:




    
    
        where R1 is hydrogen atom or alkyl substituent simple or spatially enlarged, advantageously as residue of succinimidyl derivative or alkyl containing carbo- or heterocyclic ring moieties or glycosyl residue or spatially branched aminoacyl residue or thioureidyl residue containing basic nitrogen atom and spacially branched aliphatic or cyclic substituents;

R2 is hydrogen atom or substituents such as designated for R1, while R3 is hydroxyl group, alkoxyl, or aminoalkyl, and their salts and complexes being water soluble forms, where X is one or more molecules of base or acid, or complexing compound.

    
    


According to the invention in an advantageous variant, the compounds of structure presented by Formula 1, 1a or 1b characterized by the presence of bulky moieties which could induce steric hindrance effect, attached to amino group of mycosamine residue have been obtained in several versions, all of which are exemplary embodiments of the invention:
N-succinimidyl derivatives from N1 to N11, exemplary including: N—(N′-benzylsuccinimidyl)nystatin A1 (N1), N—(N′-4-bromophenylsuccinimidyl)nystatin A1 (N2), N—(N′-2,4,6-trimethylphenylsuccinimidyl)nystatin A1 (N3), N—(N′-2,6-dimethyl-phenylsuccinimidyl)nystatin A1 (N4), N—(N′-cyclohexylsuccinimidyl)nystatin A1 (N5); N—(N′-amino)succinimidyl derivatives exemplary including: N—[N′-3-(N′′,N′′-dimethylamino)propylsuccinimidyl]nystatin A1 (N6), N—[N′-2-(N′′,N′′-dimethylamino) ethylsuccinimidyl]nystatin A1 (N7), N—[N′-2-(piperidin-1-yl)ethylsuccinimidyl]nystatin A1 (N8), N—[N′-2-(4-methylpiperazin-1-yl)ethylsuccinimidyl]nystatin A1 (N9), N—[N′-3-(N′′,N′′-dimethylamino)-2,2-dimethylpropylsuccinimidyl]nystatin A1 (N10), N—[N′-2-(pyrrolidin-1-yl)ethylsuccinimidyl]nystatin A1 (N11);
Thioureidyl derivatives from N12 to N20, exemplary including: N-{3-[2-(piperidin-1-yl)ethyl]thioureidyl}nystatin A1 (N12), N-{3-[2-(morpholin-1-yl)ethyl]thioureidyl}nystatin A1 (N13), N-{3-[2-(N,N-diethylamino)ethyl]thioureidyl}nystatin A1 (N14), N-{3-[2-(4-methylpiperazin-1-yl)ethyl]thioureidyl}nystatin A1 (N15), N-{3-[2-(pyrrolidin-1-yl)ethyl]thioureidyl}nystatin A1 (N16), N-{3-[3-(N,N-dimethylamino)propyl]thioureidyl}nystatin A1 (N17), N-{3-[2-(N,N-dimethylamino)ethyl]thioureidyl}nystatin A1 (N18), N-{3-[3-(N,N-dimethylamino)-2,2-dimethylpropyl]thioureidyl}nystatin A1 (N19), N-{3-[3-(imidazo-1-yl)propyl]thioureidyl}nystatin A1 (N20);
N-benzyl derivatives exemplary including: N-benzylnystatin A1 (N21);
N-alkyl derivatives from N22 to N26, exemplary including: N,N-di-n-propylnystatin A1 (N22), N,N-dimethylnystatin A1 (N23), N,N-diethylnystatin A1 (N24), N,N-di[3-(piperidin-1-yl)propyl]nystatin A1 (N25), N,N-di[3-(4-ethylpiperazin-1-yl)propyl]nystatin A1 (N26);
N-alkyl derivatives of N-fructosylnystatin A1 from N27 to N32, exemplary including: N-fructosyl-N-n-propylnystatin A1 (N27), N-fructosyl-N-methylnystatin A1 (N28), N-fructosyl-N-ethylnystatin A1 (N29), N-fructosyl-N-[3-(N′,N′-dimethylamin) propyl]nystatin A1 (N30), N-fructosyl-N-[3-(piperidin-1′-yl)propyl]nystatin A1 (N31), N-fructosyl-N-[3-(4-ethylpiperazin-1-yl)propyl)]nystatin A1 (N32);
N-aminoacyl and N—(N′-alkylamino)acyl Nystatin A1 derivatives from N33 to N43, exemplary including: N-D-phenylglycylnystatin A1 (N33), N-L-phenylalanylnystatin A1 (N34), N-L-(O-tert-butyl)glutamylnystatin A1 (N35), N-L-(O-tert-butyl)serylnystatin A1 (N36), N-D-(O-tert-butyl)glutamylnystatin A1 (N37), N-D-(O-tert-butyl)serylnystatin A1 (N38), N-D-β-naphtylalanylnystatin A1 (N39), N-L-(4-nitrophenyl)alanylnystatin A1 (N40), N-D-(O-tert-butyl)asparagylnystatin A1 (N41), N-D-β-(pyridin-3-yl)alanylnystatin A1 (N42), N-[2-(N-methylamino)-2-methylpropan]nystatin A1 (N43); N—(N,N-dialkylamino)alkylacyl derivatives of Nystatin A1 from N44 to N50, exemplary including: N-[3-(piperidin-1-yl)propionyl]nystatin A1 (N44), N-[L-(N,N-dimethyl)phenylalanyl]nystatin A1 (N45); N—[L-(N,N-diethyl)phenylalanyl]nystatin A1 (N46), N—[(N,N-di-n-propyl)glycyl]nystatin A1 (N47), N-[3-(N,N-di-n-propylamino)propionyl]nystatin A1 (N48), N-[3-(N,N-diethylamino) propionyl]nystatin A1 (N49), N-[3-(N,N-dimethylamino)propionyl]nystatin A1 (N50);
Methyl esters of N-substituted derivatives of Nystatin A1 from NM to NM exemplary including: N-{3-[3-(N,N-dimethylamino)-2,2-dimethylpropyl]thioureidyl}nystatin A1 methyl ester (N51), N,N-di-n-propylnystatin methyl ester A1 (N52), N-fructosyl-N-n-propylnystatin A1 methyl ester (N53), N-D-phenylglycylnystatin A1 methyl ester (N54);
Amides of N-substituted derivatives of Nystatin A1 from N55 to N58, exemplary including: N-{3-[3-(N,N-dimethylamino)-2,2-dimethylpropyl]tioureidyl}nystatin A1 3-(N,N-dimethyl-amino)propylamide (N55), N,N-di-n-propylnystatin A1 3-(N,N-dimethylamino)propylamide (N56), N-fructosyl-N-n-propylnystatin A1 3-(N,N-dimethylamino)propylamide (N57), N-D-phenylglycylnystatin A1 3-(N,N-dimethyl-amino)propylamide (N58);
According to the invention, water soluble salts of amphoteric derivatives of Nystatin A1 with bases, exemplary including N-methyl-D-glucamine salt of N-fructosyl-N-n-propylnystatin A1 were obtained (N59);
According to the invention, water soluble complexes of amphoteric derivatives of Nystatin A1, can be obtained according to know procedure for native Nystatin A1;
Water soluble salts of basic Nystatin A1 derivatives with acids, exemplary L-aspartate of N-fructosyl-N-n-propylnystatin A1 methyl ester, were obtained (N60).
In some embodiments, a compound as described herein has hemotoxicity expressed by EH50 value of greater than 200 μg/ml, preferably greater than 300 μg/ml.
Structures of Nystatin A1 derivatives, according to the invention, are presented below in Table 1. Nystatin structure is provided for reference purposes.







TABLE 1







Structures of Nystatin A1 derivatives.











Lp
Symbol
R1
R2
R3






Nystatin
—H
—H
—OH







N-succinimidyl derivatives











1
N1
—H




—OH


 


2
N2
—H




—OH


 


3
N3
—H




—OH


 


4
N4
—H




—OH


 


5
N5
—H




—OH


 


6
N6
—H




—OH


 


7
N7
—H




—OH


 


8
N8
—H




—OH


 


9
N9
—H




—OH


 


10
N10
—H




—OH


 


11
N11
—H




—OH


 







Thioureidyl derivatives











12
N12
—H




—OH


 


13
N13
—H




—OH


 


14
N14
—H




—OH


 


15
N15
—H




—OH


 


16
N16
—H




—OH


 


17
N17
—H




—OH


 


18
N18
—H




—OH


 


19
N19
—H




—OH


 


20
N20
—H




—OH


 







N-alkyl derivatives











21
N21
—H




—OH


 


22
N22
—CH2CH2CH3
—CH2CH2CH3
—OH


23
N23
—CH3
—CH3
—OH


24
N24
—CH2CH3
—CH2CH3
—OH


 


25
N25








—OH


 


26
N26








—OH


 


27
N27




—CH2CH2CH3
—OH


 


28
N28




—CH3
—OH


 


29
N29




—CH2CH3
—OH


 


30
N30








—OH


 


31
N31








—OH


 


32
N32








—OH


 







N-aminoacyl derivatives











33
N33
—H




—OH


 


34
N34
—H




—OH


 


35
N35
—H




—OH


 


36
N36
—H




—OH


 


37
N37
—H




—OH


 


38
N38
—H




—OH


 


39
N39
—H




—OH


 


40
N40
—H




—OH


 


41
N41
—H




—OH


 


42
N42
—H




—OH


 


43
N43
—H




—OH


 


44
N44
—H




—OH


 


45
N45
—H




—OH


 


46
N46
—H




—OH


 


47
N47
—H




—OH


 


48
N48
—H




—OH


 


49
N49
—H




—OH


 


50
N50
—H




—OH


 







di-modified ester derivatives











51
N51
—H




—OCH3


 


52
N52
—CH2CH2CH3
—CH2CH2CH3
—OCH3


 


53
N53




—CH2CH2CH3
—OCH3


 


54
N54
—H




—OCH3


 







di-modified amide derivatives











55
N55
—H




—NHCH2CH2CH2N(CH3)2


 


56
N56
—CH2CH2CH3
—CH2CH2CH3
—NHCH2CH2CH2N(CH3)2


 


57
N57




—CH2CH2CH3
—NHCH2CH2CH2N(CH3)2


 


58
N58
—H




—NHCH2CH2CH2N(CH3)2









The idea of the invention includes also application of the compounds being sterically hindered derivatives of antifungal antibiotic Nystatin A1 of Formula 1, 1a or 1b, where R1 is a hydrogen atom or simple non-branched or bulky alkyl, advantageously as residue of succinimide derivative or alkyl containing cyclic moieties carbo- or heterocyclic, glycosyl residue or bulky aminoacyl residue or thioureidyl residue containing basic nitrogen atom and bulky aliphatic or cyclic substituents, R2 is hydrogen atom or substituents such as are defined for R1, while R3 is hydroxyl group or alkoxyl or aminoalkyl group 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 control of fungal microorganisms, preferably of multidrug resistance once directly or as active components for various formulations of antifungal drugs.
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.
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.
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.
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, wherein the patient is a human or animal.
In a sixth aspect, the invention provides the use of a compound of the invention for treating a fungal infection in a plant.
In a seventh aspect, the invention provides a plant protection product comprising a compound of the invention.
In some embodiments, treatment as referred to herein relates to treatment of fungal infections caused by a strain of the genus Candida. This may be a strain of the genus Candida having multidrug resistance (MDR).
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.
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.
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.
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).
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.
A pharmaceutical composition may be adapted for administration by any appropriate route, for example by the oral (including buccal or sublingual), rectal, nasal, topical (including buccal, sublingual or transdermal), vaginal or parenteral (including subcutaneous, intramuscular, intravenous or intradermal) 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.
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.
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.
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.
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.
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.
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.
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.
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, Nystatin A1, of general Formula 1, 1a or 1b, where R1 is hydrogen atom or simple or bulky alkyl substituent, the last one preferably as a residue of succinimide derivative, or branched alkyl or containing cyclic carbo- or heterocyclic moieties or glycosyl residue, or bulky, spatially extended aminoacyl residue or thioureidyl residue containing basic nitrogen atom and bulky aliphatic or cyclic substituents; R2 is hydrogen atom or substituents such as are defined for R1, while R3 is hydroxyl group or alkoxyl or aminoalkyl group, also their salts and complexes being their water soluble forms, where X is one or more basic molecules, advantageously N-methyl-D-glucamine or acid molecule, preferably aspartic acid, or complexing agent, preferably sodium deoxycholate.
To obtain N-succinimidyl derivatives, reaction of Michael's addition is performed as follows: solution of Nystatin A1 in dimethyl formamide, in the presence of triethylamine, is reacted with suitable derivative of maleimide. The product is precipitated by ethyl ether, centrifuged, dried and purified by column chromatography (Silica Gel).
N-aminosuccinimidyl derivatives are obtained in similar manner as N-succinimidyl derivatives, but a two-fold excess of maleimide derivative is used.
N-thioureidyl derivatives are obtained in the reaction of Nystatin A1 in dimethyl formamide solution and in the presence of triethylamine with a proper derivative of isothiocyanate, then the reaction product is purified by column chromatography (Silica Gel).
N-benzyl derivatives are obtained in the reaction of reductive alkylation of Nystatin A1, in solution of dimethyl formamide and methanol, with benzaldehyde or its derivatives, using sodium cyanoborohydride as a reducing agent and a catalytic amount of acetic acid. The reaction mixture is neutralized with solution of methylamine in tetrahydrofuran, then the final product is precipitated by ethyl ether and purified by column chromatography (Silica Gel).
N-alkyl derivatives of Nystatin A1 are obtained in the reaction of reductive alkylation of Nystatin A1, in solution of dimethyl formamide, with aliphatic aldehyde using sodium cyanoborohydride as a reducing agent and a catalytic amount of acetic acid. The reaction mixture is neutralized with solution of methylamine in tetrahydrofuran, the product is precipitated by ethyl ether, and then purified by column chromatography (Silica Gel).
The synthesis of N-alkyl derivatives of N-fructosylnystatin A1 is based on method of reductive alkylation of N-fructosylnystatin A1, in solution of dimethyl formamide, with suitable aliphatic aldehyde and sodium cyanoborohydride as a reducing agent and a catalytic amount of acetic acid. The neutralization of reaction mixture is performed by adding of methylamine in tetrahydrofuran, then reaction product is precipitated by ethyl ether and purified by column chromatography (Silica Gel).
N-aminoacyl and N—(N′ alkylamino)acyl derivatives of Nystatin A1 are obtained in the reaction of N-acylation of antibiotic by appropriate N-protected aminoacid. First, the reaction of N-(9-fluorenylmetoxycarbonyl)-aminoacid with N-hydroxysuccinimide in the presence of N,N′-dicyclohexylcarbodiimide is performed in solution of dimethyl formamide. Precipitated solid of N,N′-dicyclohexylurea is removed, then to the reaction mixture Nystatin A1 and triethyamine are added, monitoring progress of the reaction by thin layer chromatography. The final product is precipitated with ethyl ether and next purified by column chromatography (Silica Gel);
Synthesis of N—(N′,N′-dialkyloamino)aminoacyl derivatives of Nystatin A1 is performed by activation of an amino acid by N-hydroxysuccinimide and N,N′-dicyclohexylcarbodiimide in dimethyl formamide. Precipitated solid of N,N′-dicyclohexylurea is removed and Nystatin A1 is added to the reaction mixture. Crude product of the reaction is precipitated by an excess of ethyl ether and purified by column chromatography (Silica Gel);
Synthesis of methyl esters of N-substituted Nystatin A1 derivatives is performed in the reaction of antibiotic, in dimethyl formamide solution, with diazomethane which is added to the reaction mixture in ethyl ether solution. Then, an excess of diazomethane is removed with acetic acid, the formed product is precipitated by an excess of ethyl ether and purified by column chromatography (Silica Gel);
Synthesis of amides of N-substituted Nystatin A1 derivatives, on example of 3-(N,N-dimethylamino)propylamide of N-fructosyl-N-propylnystatin, is performed in the reaction of N-substituted antibiotic derivatives, in dimethyl formamide solution, with appropriate amine in the presence of diphenyl azidephosphate and triethylamine. The product of the reaction is precipitated by an excess of ethyl ether and purified by column chromatography (Silica Gel).
It will be appreciated that in the syntheses described above, the reagents used may be selected to prepare a compound of Formula 1, 1a or 1b, for example a compound having any of the substituents as illustrated for exemplary compounds of the invention in Table 1, in any combination.
Obtaining of salts of amphoteric Nystatin A1 derivatives, advantageously with N-methyl-D-glucamine, consist in addition to aqueous suspension of Nystatin A1 derivatives a small excess of N-methyl-D-glucamine diluted in water and precipitation of product by excess of acetone.
Water-soluble complex of amphoteric Nystatin A1 derivatives with sodium deoxycholate can be obtained by the interacting the derivative with deoxycholate in phosphate buffer according to the standard procedure.
Water-soluble salts of basic Nystatin A1 derivatives, advantageously with aspartic acid, are obtained 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 Nystatin A1 derivatives, according to the invention, leads to the desired products. All obtained compounds have been characterized with respect to their chemical structure and biological properties. Identification data of the compounds include their spectroscopic data as λmax determination, extinction value of E1 cm1%, molecular weight determined by mass spectrometry MS-ESI, thin layer chromatographic characterization with indicated RF value. The biological properties of the compounds, according to the invention, were determined using the obligatory standards. There were determined activities of the compounds in vitro against a number of fungal strains, primarily of the genus Candida, and activity towards multidrug resistant 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 hindrance moieties introduced to Nystatin A1 molecule, the compounds exhibit, reduced to different extent, in relation to native antibiotic, hemotoxic activity and are characterized by low cytotoxicity, good antifungal activity and are also active against multidrug resistant strains (MDR).
The advantage of the compounds, according to the invention, which are previously unknown sterically hindered derivatives of Nystatin A1, is that they fulfill the essential requirements for antifungal chemotherapeutics. They are characterized by very low hemotoxicity, exhibit low toxicity towards mammalian cells, are active towards multidrug resistant fungal strains (MDR). Moreover, they form with acids and bases water-soluble salts and also soluble complexes with solubilizing complexing compounds. The advantage according to the invention is also simple and efficient method of their preparation.
According to the invention, can also be synthesized analogous derivatives of other antibiotics of tetraeno-diene polyene macrolides, like other components of Nystatin complex, namely Nystatin A2 and A3, components of Polyfungin complex including Polyfungin B and antibiotic Amphotericin A.
The subject of the invention is shown in the examples below, where are presented methods for the preparation and demonstrated properties of the compounds according to the invention, and of their water-soluble salts and complexes.
EXAMPLE 1
Synthesis of N-Succinimidyl Derivatives of Nystatin A1 
200 mg (0.21 mmol) of Nystatin A1 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 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 Nystatin A1 are obtained:

    a) In the reaction with N-benzylmaleimide is obtained 80 mg of N—(N′-benzylsuccinimidyl)nystatyn A1 (N1)


TLC Rf=0.62; UV-vis: λmax (MeOH) 319; 304; 291 nm; E1 cm1% (MeOH, λ=304 nm)=500 (theoretically for C58H84N2O19 is 658); MS-ESI calculated for C58H84N2O19 [M+K]+ 1150.6. found: 1150.1.

    b) In the reaction with N-(4-bromophenyl)maleimide is obtained 92 mg of N—(N′-4-bromo phenylsuccinimidyl)nystatin A1 (N2)


TLC Rf=0.55; UV-vis: λmax (MeOH) 319; 304; 291 nm; E1 cm1% (MeOH, λ=304 nm)=522 (theoretically for C57H81BrN2O19 is 621); MS-ESI calculated for C57H81BrN2O19 [M+H]+ 1177.5; found: 1177.8.

    c) In the reaction with N-(2,4,6-trimethylphenyl)maleimide is obtained 95 mg of N—(N′-2,4,6-trimethylphenylsuccinimidyl)nystatin A1 (N3)


TLC Rf=0.77; UV-vis: λmax (MeOH) 319; 304; 291 nm; E1 cm1% (MeOH, λ=304 nm)=620 (theoretically for C60H88N2O19 is 641); MS-ESI calculated for C60H88N2O19 [M+H]+ 1141.5; found: 1142.0.

    d) In the reaction with N-(2,6-dimethylphenyl)maleimide is obtained 86 mg of N—(N′-2,6-dimethylphenylsuccinimidyl)nystatin A1 (N4)


TLC Rf=0.58; UV-vis: λmax (MeOH) 319; 304; 291 nm; E1 cm1% (MeOH, λ=304 nm)=620 (theoretically for C59H86N2O19 is 649); MS-ESI calculated for C59H86N2O19 [M+H]+ 1127.6; found: 1128.0.

    e) In the reaction with N-cyclohexylmaleimide is obtained 60 mg of N—(N′-cyclohexyl succinimidyl)nystatin A1 (N5)


TLC Rf=0.65; UV-vis: λmax (MeOH) 319; 304; 291 nm; E1 cm1% (MeOH, λ=304 nm)=630 (theoretically for C57H88N2O19 is 661); MS-ESI calculated for C57H88N2O19 [M+H]+ 1105.6; found: 1106.0.
EXAMPLE 2
Synthesis of N—(N′-Alkyloamino)Succinimidyl Derivatives of Nystatin A1 
200 mg (0.21 mmol) of Nystatin A1 in 4 ml of dimethyl formamide (DMF) is dissolved 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 0.42 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 phase, where the solid phase is Silica Gel and solvent system is chloroform:methanol (gradient from 20% to 80% of methanol). 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 maleimide, the following derivatives of Nystatin A1 are obtained:

    a) In the reaction with N-3-(N′,N′-dimethylamino)propylmaleimide is obtained 45 mg of N—[N′-3-(N′′,N′′-dimethylamino)propylsuccinimidyl]nystatin A1 (N6)


TLC Rf=0.14; UV-vis: λmax (MeOH) 319; 304; 291 nm; E1 cm1% (MeOH, λ=304 nm)=600 (theoretically for C56H89N3O19 is 660); MS-ESI calculated for C56H89N3O19 [M+H]+ 1108.3. found: 1108.6.

    b) In the reaction with N-2-(N′,N′-dimethylamino)ethylmaleimide is obtained 38 mg of N-[N′-2-(N′′N′′-dimethylamino)ethylsuccinimidyl]nystatin A1 (N7)


TLC Rf=0.18; UV-vis: λmax (MeOH) 319; 304; 291 nm; E1 cm1% (MeOH, λ=304 nm)=540 (theoretically for C55H87N3O19 is 669); MS-ESI calculated for C55H87N3O19 [M+H]+ 1094.3. found: 1094.6.

    c) In the reaction with N-2-(piperidin-1-yl)ethylmaleimide is obtained 49 mg of N—(N′-2-(piperidin-1-yl)ethylsuccimidyl)nystatin A1 (N8)


TLC Rf=0.34; UV-vis: λmax (MeOH) 319; 304; 291 nm; E1 cm1% (MeOH, λ=304 nm)=500 (theoretically for C58H91N3O19 is 645); MS-ESI calculated for C58H91N3O19 [M+H]+ 1134.3. found: 1134.6.

    d) In the reaction with N-2-(4-methylpiperazin-1-yl)ethylmaleimide is obtained 52 mg of N—[N′-2-(4-methylpiperazin-1-yl)ethylsuccinimidyl)nystatin A1 (N9)


TLC Rf=0.11; UV-vis, (MeOH) 319; 304; 291 nm; E1 cm1% (MeOH, λ=304 nm)=570 (theoretically for C58H92N4O19 is 636); MS-ESI calculated C58H92N4O19 [M+H]+ 1149.3. found: 1149.6.

    e) In the reaction with N-3-(N′,N′-dimethylamino)-2,2-dimethylpropylmaleimide is obtained 58 mg of N—[N′-3-(N′′,N′′-dimethyloamino)-2,2-dimethylpropylsuccinimidyl]nystatin A1 (N10)


TLC Rf=0.25; UV-vis: λmax (MeOH) 319; 304; 291 nm; E1 cm1% (MeOH, λ=304 nm)=540 (theoretically for C58H93N3O19 is 644); MS-ESI calculated for C58H93N3O19 [M+H]+: 1136.3. found: 1136.6.

    f) In the reaction with N-2-(pyrrolidin-1-yl)ethylmaleimide is obtained 56 mg of N—[N′-2-(pyrrolidin-1-yl)ethylsuccinimidyl]nystatin A1 (N11)


TLC Rf=0.19; UV-vis: λmax (MeOH) 319; 304; 291 nm; E1 cm1% (MeOH, λ=304 nm)=550 (theoretically for C57H89N3O19 is 653); MS-ESI calculated C57H89N3O19 [M+H]+ 1120.3. found: 1120.6.
EXAMPLE 3
Synthesis of N-Thioureidyl Derivatives of Nystatin
200 mg (0.21 mmol) of Nystatin A1 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 washed twice 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. Using in the reaction below indicated isothiocyanates, the following derivatives of Nystatin A1 are obtained:

    a) In the reaction with 2-(piperidin-1-yl)ethylisothiocyanate is obtained 50 mg of N-{3-[2-(piperidin-1-yl)ethyl]thioureidyl}nystatin A1 (N12)


TLC Rf=0.32; UV-vis: λmax (MeOH) 319; 304; 291 nm; E1 cm1% (MeOH, λ=304 nm)=600 (theoretically for C55H89N3O17S is 667); MS-ESI calculated for C55H89N3O17S [M+H]+ 1096.3; found: 1096.5.

    b) In the reaction with 2-(morpholin-1-yl)ethylisothiocyanate is obtained 110 mg of N-{3-[2-(morpholin-1-yl)ethyl]thioureidyl}nystatin A1(N13)


TLC Rf=0.2; UV-vis: λmax (MeOH) 319; 304; 291 nm; E1 cm1% (MeOH, λ=304 nm)=402 (theoretically for C55H89N3O18S is 657); MS-ESI calculated for C55H89N3O18S [M+H]+ 1112.3; found: 1112.5.

    c) In the reaction with N,N-diethyl-2-aminoethylisothiocyanate is obtained 20 mg of N-{3-[2-(N,N-diethylamino)ethyl]thioureidyl}nystatin A1 (N14)


TLC Rf=0.12; UV-vis: λmax (MeOH) 319; 304; 291 nm; E1 cm1% (MeOH, λ=304 nm)=608 (theoretically for C55H91N3O17S is 666); MS-ESI calculated for C55H91N3O17S [M+H]+ 1098.3; found: 1098.5.

    d) In the reaction with 2-(4-methylpiperazin-1-yl)ethylisothiocyanate is obtained 50 mg of N-{3-[2-(4-methylpiperazin-1-yl)ethyl]thioureidyl}nystatin A1 (N15)


TLC Rf=0.08; UV-vis: λmax (MeOH) 319; 304; 291 nm; E1 cm1% (MeOH, λ=304 nm)=662 (theoretically for C55H90N4O17S is 658); MS-ESI calculated for C55H90N4O17S [M+H]+ 1111.3; found 1111.6.

    e) In the reaction with z 2-(pyrrolidin-1-yl)ethylisothiocyanate is obtained 57 mg of N-{3-[2-(pyrrolidin-1-yl)ethyl]thioureidyl}nystatin A1 (N16)


TLC Rf=0.26; UV-vis: λmax (MeOH) 319; 304; 291 nm; E1 cm1% (MeOH, λ=304 nm)=670 (theoretically for C54H87N3O17S is 676); MS-ESI calculated for C54H87N3O17S [M+H]+ 1082.3; found 1082.1.

    f) In the reaction with 3-(N,N-dimethylamino)propylisothiocyanate is obtained 44 mg of N-{3-[3-(N′,N′-dimethylamino)propyl]thioureidyl}nystatin A1 (N17)


TLC Rf=0.12; UV-vis: λmax (MeOH) 319; 304; 291 nm; E1 cm1% (MeOH, λ=304 nm)=641 (theoretically for C53H87N3O17S is 683); MS-ESI calculated for C53H87N3O17S [M+H]+ 1070.3; found 1070.8.

    g) In the reaction with 2-(N,N-dimethylamino)ethylisothiocyanate is obtained 53 mg of N-{3-[2-(N′,N′-dimethylamino)ethyl]thioureidyl}nystatin A1 (N18)


TLC Rf=0.19; UV-vis: λmax (MeOH) 319; 304; 291 nm; E1 cm1% (MeOH, λ=304 nm)=623 (theoretically for C52H85N3O17S is 692); MS-ESI calculated for C52H85N3O17S [M+H]+ 1056.3; found 1055.9.

    h) In the reaction with 3-(N,N-dimethylamino)-2,2-dimethylopropylisothiocyanate is obtained 124 mg of N-{3-[3-(N′,N′-dimethylamino)-2,2-dimethylpropyl]thioureidyl}nystatin A1 (N19)


TLC Rf=0.17; UV-vis λmax (MeOH) 319; 304; 291 nm, E1 cm1% (MeOH, λ=304 nm)=540 (theoretically for C55H91N3O17S is 666); MS-ESI calculated for C55H91N3O17S [M+H]+ 1098.4; found 1098.2.

    i) In the reaction with 3-(imidazo-1-yl)propylisothiocyanate is obtained 30 mg N-{3-[3-(imidazo-1-yl)propyl]thioureidyl}nystatin A1 (N20)


TLC Rf=0.2; UV-vis: λmax (MeOH): 319; 304; 291 nm; E1 cm1% (MeOH, λ=304 nm)=560 (theoretically for C54H84N4O17S is 669); MS-ESI calculated for C54H84N4O17S [M+H]+ 1093.3; found 1093.7.
EXAMPLE 4
Synthesis of N-Benzyl Derivatives of Nystatin A1 
200 mg (0.21 mmol) of Nystatin A1 is dissolved in 3 ml of dimethyl formamide (DMF) in 100 ml round-bottomed flask equipped with a magnetic stirrer. Next, 0.3 mmol of benzaldehyde is added and solution is stirred at room temperature for 1 hour. After 1 hour 3 ml of anhydrous methanol, 0.3 mmol of sodium cyanoborohydride 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 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 (20:6:1 v/v). The fractions with pure product were collected and combined, then evaporated under reduced pressure at temperature not exceeding 35° C. For example, in the reaction with benzaldehyde is obtained 55 mg of N-benzylnystatin A1 (N21).
TLC Rf=0.83; UV-vis: λmax (MeOH) 319; 304; 291 nm; E1 cm1% (MeOH, λ=304 nm)=420 (theoretically for C54H81NO17 is 680); MS-ESI calculated for C54H81NO17 [M−H]− 1114.6; found 1114.6.
EXAMPLE 5
Synthesis of N-Alkyl Derivatives of Nystatin
200 mg (0.21 mmol) of Nystatin A1 is dissolved in 3 ml of dimethyl formamide (DMF) in 100 ml round-bottomed flask equipped with a magnetic stirrer. Next, 0.63 mmol of appropriate aliphatic aldehyde is added and solution is tirred 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 (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 temperature not exceeding 35° C. The following derivatives of Nystatin A1 are obtained:

    a) In the reaction with propanal is obtained 60 mg of N,N-di-n-propylnystatin A1 (N22)


TLC Rf=0.35; UV-vis: λmax (MeOH) 319; 304; 291 nm; E1 cm1% (MeOH, λ=304 nm)=640 (theoretically for C53H87NO17 is 659); MS-ESI calculated for C53H87NO17 [M+H]+ 1110.6; found: 1110.6.

    b) In the reaction with methanal is obtained 90 mg N,N-dimethylnystatin A1 (N23)


TLC Rf=0.18; UV-vis: λmax (MeOH) 319; 304; 291 nm; E1 cm1% (MeOH, λ=304 nm)=660 (theoretically for C49H79NO17 is 766); MS-ESI calculated for C49H79NO17 [M+H]+ 954.7; found: 954.5.

    c) In the reaction with ethanal is obtained 40 mg of N,N-diethylnystatin A1 (N24)


TLC Rf=0.23; UV-vis: λmax (MeOH) 319; 304; 291 nm; E1 cm1% (MeOH, λ=304 nm)=570 (theoretically for C51H83NO17 is 745); MS-ESI calculated for C51H83NO17 [M+H]+ 982.3; found: 982.5.

    d) In the reaction with 3-(piperidin-1-yl)propanal is obtained N,N-di[3-(piperidin-1-yl) propyl]nystatin A1 (N25)


TLC Rf=0.55; UV-vis: λmax (MeOH) 319; 304; 291 nm; E1 cm1% (MeOH, λ=304 nm)=540 (theoretically for C63H105N3O17 is 622); MS-ESI calculated for C63H105N3O17 [M+H]+ 1177.4; found: 1177.5.

    e) In the reaction with 3-(4-ethylpiperazin-1-yl)propanal is obtained N,N-di[3-(4-ethyl piperazin-1-yl)propyl]nystatin A1 (N26)


TLC Rf=0.43; UV-vis: λmax (MeOH) 319; 304; 291 nm; E1 cm1% (MeOH, λ=304 nm)=520 (theoretically for C63H107N5O17 is 606); MS-ESI calculated for C63H107N5O17 [M+H]+ 1207.5; found: 1207.8.
EXAMPLE 6
Synthesis of N-Alkyl Derivatives of N-Fructosylnystatin A1 
200 mg (0.18 mmol) of N-fructosylnystatin A1 is dissolved in 3 ml of dimethyl formamide (DMF) in 100 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 temperature not exceeding 35° C. The following derivatives of N-fructosylnystatin A1 are obtained:

    a) In the reaction with propanal is obtained 40 mg of N-fructosyl-N-n-propylnystatin A1 (N27)


TLC Rf=0.15; UV-vis λmax (MeOH) 319; 304; 291 nm; E1 cm1% (MeOH, λ=304 nm)=590 (theoretically for C56H91NO22 is 647); MS-ESI calculated for C56H91NO22 [M+H]+ 1130.6; found: 1131.0.

    b) In the reaction with metanal is obtained 60 mg of N-fructosyl-N-methylnystatin A1 (N28)


TLC Rf=0.16; UV-vis: λmax (MeOH) 319; 304; 291 nm; E1 cm1% (MeOH, λ=304 nm)=580 (theoretically for C54H87NO22 is 664); MS-ESI calculated for C54H87NO22 [M+H]+ 1102.5; found: 1102.5.

    c) In the reaction with ethanal is obtained 39 mg of N-fruktosyl-N-ethylnystatin A1 (N29)


TLC Rf=0.18; UV-vis: λmax (MeOH) 319; 304; 291 nm; E1 cm1% (MeOH, λ=304 nm)=540 (theoretically for C55H89NO22 is 655); MS-ESI calculated for C55H89NO22 [M+H]+ 1116.5; found: 1116.5.

    d) In the reaction with 3-(N,N-dimethylamino)propanal is obtained N-fructosyl-N-[3-(N′,N′-dimethylamino)propyl]nystatin A1 (N30)


TLC Rf=0.12; UV-vis: λmax (MeOH) 319; 304; 291 nm; E1 cm1% (MeOH, λ=304 nm)=550 (theoretically for C58H96N2O22 is 624); MS-ESI calculated for C58H96N2O22 [M+H]+ 1174.5; found: 1174.7.

    e) In the reaction with 3-(piperidin-1-yl)propanal is obtained N-fructosyl-N-[3-(piperidin-1-yl)propyl]nystatin A1 (N31)


TLC Rf=0.17; UV-vis: λmax (MeOH) 319; 304; 291 nm; E1 cm1% (MeOH, λ=304 nm)=520 (theoretically for C61H100N2O22 is 603); MS-ESI calculated for C61H100N2O22 [M+H]+ 1213.5; found: 1213.9.

    f) In the reaction with 3-(4-ethylpiperazin-1-yl)propanal is obtained N-fructosyl-N-[3-(4-ethylpiperazin-1-yl)propyl]nystatin A1 (N32)


TLC Rf=0.1; UV-vis: λmax (MeOH) 319; 304; 291 nm; E1 cm1% (MeOH, λ=304 nm)=510 (theoretically for C61H101N3O22 is 595); MS-ESI calculated for C61H101N3O22 [M+H]+ 1229.5; found: 1229.9.
EXAMPLE 7
Synthesis of N-Aminoacyl and N—(N′-Alkylamino)Acyl Derivatives of Nystatin A1 
0.26 mmol of N-(9-fluorenylomethoxycarbonyl)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 50 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 aceteate:hexan (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 Nystatin A1 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 Nystatin A1 are obtained in the reaction of the corresponding protected amino acids:

    a) In the reaction with N-Fmoc-D-phenylglycine is obtained 92 mg of N-D-phenylglycylnystatin A1 (N33)


TLC Rf=0.38; UV-vis: λmax (MeOH) 319; 304; 291 nm; E1 cm1% (MeOH, λ=304 nm)=610 (theoretically for C55H82N2O18 is 691); MS-ESI calculated for C55H82N2O18 [M+H]+: 1059.6; found 1059.6.

    b) In the reaction with N-Fmoc-L-phenylalanine is obtained 140 mg of N-L-phenylalanylnystatin A1 (N34)


TLC Rf=0.42; UV-vis: λmax (MeOH) 319; 304; 291 nm; E1 cm1% (MeOH, λ=304 nm)=600 (theoretically for C56H84N2O18 is 681); MS-ESI calculated for C56H84N2O18 [M+H]+ 1073.6; found 1073.9.

    c) In the reaction with N-Fmoc-L-(O-tert-butyl)glutamic acid is obtained 30 mg of N-L-(O-tert-butyl)glutamylnystatin A1 (N35)


TLC Rf=0.26; UV-vis: λmax (MeOH) 319; 304; 291 nm; E1 cm1% (MeOH, λ=304 nm)=610 (theoretically for C56H90N2O20 is 658); MS-ESI calculated for C56H90N2O20 [M+H]+ 1111.7; found 1112.0.

    d) In the reaction with N-Fmoc-L-(O-tert-butyl)serine is obtained 80 mg of N-L-(O-tert-butyl)serylnystatin A1 (N36)


TLC Rf=0.35; UV-vis: λmax (MeOH) 319; 304; 291 nm; E1 cm1% (MeOH, λ=304 nm)=610 (theoretically for C54H88N2O19 is 684); MS-ESI calculated for C54H88N2O19 [M+H]+ 1069.6; found 1069.7.

    e) In the reaction with N-Fmoc-D-(O-tert-butyl)glutamic acid is obtained 40 mg of N-D-(O-tert-butyl)glutamylnystatine A1 (N37)


TLC Rf=0.26; UV-vis: λmax (MeOH) 319; 304; 291 nm; E1 cm1% (MeOH, λ=304 nm)=510 (theoretically for C56H90N2O20 is 658); MS-ESI calculated for C56H90N2O20 [M+H]+ 1111.7; found 1112.

    f) In the reaction with N-Fmoc-D-(O-tert-butyl)serine is obtained 95 mg of N-D-(O-tert-butyl)serylnystatin A1 (N38)


TLC Rf=0.35; UV-vis: λmax (MeOH) 319; 304; 291 nm; E1 cm1% (MeOH, λ=304 nm)=540 (theoretically for C54H88N2O19 is 684); MS-ESI calculated for C54H88N2O19 [M+H]+ 1069.6; found 1069.7.

    g) In the reaction with N-Fmoc-D-β-(1-naphtyl)alanine is obtained 30 mg of N-D-β-(1-naphtyl)alanylnystatin A1 (N39)


TLC Rf=0.41; UV-vis: λmax (MeOH) 319; 304; 291 nm; E1 cm1% (MeOH, λ=304 nm)=640 (theoretical for C60H86N2O18 is 651); MS-ESI calculated for C60H86N2O18 [M+H]+ 1123.6; found 1124.3.

    h) In the reaction with N-Fmoc-L-(4-nitrophenyl)alanine is obtained 56 mg of N-L-(4-nitrophenyl)alanylnystatin A1 (N40)


TLC Rf=0.42; UV-vis: λmax (MeOH) 319; 304; 291 nm; E1 cm1% (MeOH, λ=304 nm)=560 (theoretically for C56H83N3O20 is 654); MS-ESI calculated for C56H83N3O20 [M+H]+ 1118.6; found 1119.2.

    i) In the reaction with N-Fmoc-D-(O-tert-butyl)asparagine is obtained 40 mg of N-D-(O-tert-butyl)asparagylnystatin A1 (N41)


TLC Rf=0.35; UV-vis: λmax (MeOH) 319; 304; 291 nm; E1 cm1% (MeOH, λ=304 nm)=620 (theoretically C55H88N2O20 is 667); MS-ESI calculated for C55H88N2O20 [M+H]+ 1097.7; found 1098.0.

    j) In the reaction with N-Fmoc-D-β-(pyridin-3-yl)alanine is obtained 80 mg of N-D-β-(pyridin-3-yl)alanylnystatin A1 (N42)


TLC Rf=0.37; UV-vis: λmax (MeOH) 319; 304; 291 nm; E1 cm1% (MeOH, λ=304 nm)=520 (theoretically for C55H83N3O18 is 681); MS-ESI calculated for C55H83N3O18 [M−H]− 1073; found 1072.9.

    k) In the reaction with N-Fmoc-N-methyl-2-amino-2-methylpropanoic acid is obtained 50 mg of N-[2-(N-methylamino)-2-methylpropan]nystatin A1 (N43)


TLC Rf=0.13; UV-vis: λmax (MeOH) 319; 304; 291 nm; E1 cm1% (MeOH, λ=304 nm)=610 (theoretically for C52H84N2O18 is 715); MS-ESI calculated for C52H84N2O18 [M−H]− 1023.6; found 1023.7.
EXAMPLE 8
Synthesis of N—(N′,N′-Dialkylamino)Aminoacyl Derivatives of Nystatin A1 
0.44 mmol of aminoacid, 0.44 mmol of N-hydroxysuccinimide (HONSu), 0.44 mmol (0.26 mmola) of dicyclohexylcarbodiimid (DCC) is dissolved in 3 ml of dimethyl formamide (DMF) in 50 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 Nystatin A1 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 Nystatin A1 are obtained in the reaction with the corresponding amino acids:

    a) In the reaction with 3-(piperidin-1-yl)propionic acid is obtained 50 mg of N-[3-(piperidin-1-yl)propionyl]nystatin A1 (N44)


TLC Rf=0.09; UV-vis: λmax (MeOH) 319; 304; 291 nm; E1 cm1% (MeOH, λ=304 nm)=680 (theoretically for C55H83N3O18 is 688); MS-ESI calculated for C55H88N2O18 [M−H]−: 1063.6; found 1063.4.

    b) In the reaction with N,N-dimethyl-L-phenylalanine is obtained 110 mg of N—[L-(N,N-dimethylphenylalanyl]nystatin A1 (N45)


TLC Rf=0.43; UV-vis: λmax (MeOH) 319; 304; 291; E1 cm1% (MeOH, λ=304 nm)=590 (theoretically for C58H88N2O18 is 664); MS-ESI calculated for C58H88N2O18 [M−H]−: 1101.6; found 1101.6.
EXAMPLE 9
Synthesis of Methyl Esters of N-Substituted Derivatives of Nystatin A1 
0.1 mmol of Nystatin A1 derivative is dissolved in a mixture of dimethyl formamide/methanol (3 ml/l ml), next mixture was cooled to 0° C. and excess of diazomethane (ether solution) in a molar ratio of 2.5:1 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 the reaction, 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 manner described above, exemplary is obtained 40 mg of N-fructosyl-N-n-propylnystatin A1 methyl ester (N53)
TLC Rf=0.55; UV-vis λmax (MeOH) 319; 304; 291 nm; E1 cm1% (MeOH, λ=304 nm)=550 (theoretically for C57H93NO22 is 639); MS-ESI calculated for C57H93NO22 [M+H]+ 1144.6; found: 1145.0.
EXAMPLE 10
Synthesis of Amides of N-Substituted Derivatives of Nystatin A1 
0.1 mmol of Nystatin A1 derivative is dissolved in 5 ml of DMF in the round-bottomed flask equipped with a magnetic stirrer. The mixture was cooled to 0° C. and 1 mmol of 3-N,N-dimethylpropyldiamine, 1 mmol of diphenyl azidephosphate (DPPA), and 1 mmol 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 manner described above, exemplary is obtained 25 mg of N-fructosyl-N-n-propylnystatin A1 3-(N′,N′-dimethylamino)propylamide (N57).
TLC Rf=0.45; UV-vis λmax (MeOH) 319; 304; 291 nm; E1 cm1% (MeOH, λ=304 nm)=530 (teoretically for C61H103N3O21 is 603); MS-ESI calculated for C61H103N3O21 [M+H]+ 1213.6; found: 1213.0.
EXAMPLE 11
Preparation of Salts with N-Methyl-D-Glucamine of Amphoteric Nystatin A1 Derivatives
0.1 mmol derivative of Nystatin A1 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 96 mg of N-methyl-D-glucamine salt of N-fructosyl-N-n-propylnystatin A1 (N59).
UV-vis λmax (MeOH) 319; 304; 291 nm; E1 cm1% (MeOH, λ=304 nm)=530 (theoretically for C63H104N2O27 is 553).
EXAMPLE 12
Preparation of Salts with L-Aspartic Acids of Basic Derivatives of Nystatin A1 
0.1 mmol derivative of Nystatin A1 is suspended in 2 ml of deionized water in round-bottomed flask equipped with a magnetic stirrer. Next, to the reaction mixture 0.11 mmol of L-aspartic acid dissolved in 1 ml of water is added. The solution is filtered and to clear filtrate excess of acetone is added to precipitated. 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 105 mg of L-aspartate of N-methyl-N-n-fructosylpropylnystatin A1 methyl ester (N60)
UV-vis λmax (MeOH) 319; 304; 291 nm; E1 cm1% (MeOH, λ=304 nm)=510 (theoretically for C61H100N2O29 is 572).
EXAMPLE 13
Antifungal Activity In Vitro and Hemotoxicity of Nystatin A1 Derivatives
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 (Victor3, Perkin-Elmer) at the wavelength λ=531 nm (A531). On the basis of obtained results the diagrams of relation between A531 values and concentration of examined compound were made. From these graphs, the IC50 values were read, which were the interpolated concentrations of a tested compound, at which the A531 value was exactly 50% of the A531 value for the control sample. Moreover, MIC values, that are the lowest concentration of tested compound at which the A531 value were at most 20% of the A531 value measured for the control sample.
The hemotoxicity determination were carried out by the serial dilutions method, according to the procedure described earlier (Ślisz, M., et al., E., J Antibiot 57: 669-678 (2004)). Human erythrocytes were suspensed in the solution of saline to obtain a cell density of suspension 2×107/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 (A540). 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 A540 value and concentration of examined compound were made. From these graphs, the EH50 values were read which are the interpolated concentrations of compound, for which the A540 value is exactly 50% of the A540 value measured for the control sample. Maximum concentrations of tested derivatives could not exceed 300 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 EH50 value. For this group of derivatives, additionally the value of EH10 (the concentration causing 10% of hemolysis) was determined. In addition for the group of compounds of the lowest hemotoxiciy, % of hemolysis was determined at concentration of examined compound 300 μg/ml, and at concentration 60 μg/ml, at which Nystatin A1 induces 100% hemolysis.
Obtained results are presented below in tables 2A, 2B, 2C and 2D.







TABLE 2A







Antifungal activity and hemotoxicity of compounds.










Antifungal activity [μg ml−1]
















Saccharomyces
Candida
Candida
Candida
Candida
Candida




cerevisiae
albicans
tropicalis
glabrata
krusei
lusitaniae
Hemotoxicity



ATCC 9763
ATCC 10231
KKP 334
DSM 11226
DSM 6128
DSM 70102
[μg ml−1]
















No
Symbol
MIC
MIC
IC50
MIC
MIC
MIC
MIC
BEH50



















1.
Nys
0.5
0.5
0.23
0.5
1
1
  0.25
35.87


2.
N1
8
8
5.36
8
16
32
nt
>300


3.
N2
8
8
4.99
8
16
16
nt
>300


4.
N3
8
16
11.76
16
32
Nt
nt
>300


5.
N4
8
8
6.32
8
16
16
nt
>300


6.
N5
8
8
5.77
8
16
16
nt
>300


7.
N6
8
8
5.52
8
16
16
4
>300


8.
N7
8
8
3.99
8
8
16
4
>300


9.
N8
2
2
1.54
2
4
4
2
175.29


10.
N9
8
8
5.62
8
16
16
4
>300


11.
N10
8
8
5.47
8
16
16
4
>300


12.
N11
8
8
3.91
8
8
8
4
>150


13.
N12
8
8
4.20
8
8
16
4
>300


14.
N13
8
8
3.88
8
16
16
8
>300


15.
N14
4
8
3.21
8
16
16
4
>150


16.
N15
8
8
5.74
8
16
16
4
>300


17.
N16
4
4
3.04
4
8
8
4
146.19


18.
N17
4
4
2.92
4
8
8
4
257.05


19.
N18
4
4
2.97
4
8
8
4
133.37


20.
N19
4
4
2.90
4
8
8
4
280.47


21.
N20
8
8
6.47
8
16
16
8
>200


22.
N21
2
4
2.32
4
4
4
nt
188.56


23.
N22
2
4
3.14
4
4
8
nt
280.41


24.
N23
4
4
2.47
4
8
8
2
282.63


25.
N24
8
8
3.82
8
16
16
4
>300


26.
N28
4
8
5.80
8
16
16
nt
>250


27.
N29
8
8
3.56
8
16
16
8
>300


28.
N30
8
8
5.79
16
16
16
8
>300


29.
N34
4
8
6.01
8
8
8
nt
>300


30.
N35
8
8
6.36
16
16
16
nt
>300


31.
N37
4
4
2.73
4
8
8
nt
>300


32.
N38
8
8
5.95
8
16
16
nt
>300


33.
N39
8
8
6.05
8
16
16
nt
>300


34.
N40
8
8
5.56
8
16
16
nt
>300


35.
N41
4
8
4.69
4
8
8
nt
>300


36.
N42
8
16
10.54
16
32
32
nt
>300


37.
N43
8
8
5.46
8
8
16
nt
>300


38.
N44
4
4
2.95
4
8
8
nt
>300


39.
N45
2
4
2.83
4
4
8
nt
236.19


40.
N46
4
4
2.83
4
8
8
nt
>300
















TABLE 2B







Antifungal activity of selected Nystatin A1 derivatives and reference fluconazole.









MIC [μg/ml]

















Strain
Fluconazole
Nystatin
N19
N22
N28
N33
N37
N41
N45
N46




















Candida albicans ATCC 10231
2
0.5
4
4
8
8
4
8
4
4


Candida pseudotropicalis KKP 324
0.5
1
4
4
8
8
8
4
4
16


Candida stellatoidea CBS 1905
0.5
1
8
4
8
8
8
4
4
8


Candida parapsilosis DSM 5784
8
1
4
8
16
32
16
8
4
32


Candida dubliniensis CBS 7987
2
1
4
2
8
16
8
2
2
16


Candida quilliermondii DSM 11947
8
0.5
4
8
16
16
16
8
4
32


Candida arborea KKP 319
8
1
4
8
32
32
32
8
8
32


Candida lipolytica KKP 322
8
0.5
4
4
8
16
16
8
8
16


Saccharomyces cerevisiae ATCC
2
0.5
4
2
4
4
4
4
2
4


9763


Aspergillus niger LOCK E201
16
1
8
8
16
32
16
8
8
32


Trichoderma viride LOCK E159
8
1
8
8
16
32
32
32
16
16
















TABLE 2C







Hemotoxicity of selected compounds at 300 μg ml−1















% of hemolysis at the



No
Symbol
EH10 [μg ml−1]
concentration 300 μg ml−1
















1.
Nys
22.13
100



2.
N8 
150.73



3.
N17
156.33



4.
N19
209.91



5.
N21
91.60



6.
N22
202.34



7.
N23
214.20



8.
N27
>300
7.34



9.
N33
>300
8.69



10.
N36
>300
8.24



11.
N40
>300
7.45



12.
N43
283.84



13.
N44
209.39



14.
N45
>300
7.11

















TABLE 2D







Hemotoxicity of selected compounds at 60 μg ml−1









No
Symbol
% of hemolysis at the concentration 60 μg ml−1












1.
Nys
100


2.
N28
0.17


3.
N34
0.02


4.
N37
0.04


5.
N41
0.15


6.
N44
0.95


7.
N46
0.37









EXAMPLE 14
Antifungal Activity in vitro of Nystatin A1 Derivatives Against Multidrug Resistant (MDR) Fungal Strains
The determination of antifungal activity was performed by the serial dilutions method in buffered RPMI 1640 medium, 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 (Victor3, Perkin-Elmer) at the wavelength λ=660 nm (A660). On the basis of received results the diagrams on interrelation between the A660 value and the concentration of tested compound were made. From these graphs MIC values were read, that are the concentrations of tested compounds, in the presence at which A660 value is at most 20% of A660 value measured in control sample.
The results are presented in table 3.







TABLE 3





Antifungal activity of compounds towards resistant strains.

















MIC [μg/ml]

















Candida albicans
Fluconazole
Nystatin
N19
N22
N28
N33
N37
N41
N45
N46





B3
0.5
0.5
4
8
8
4
8
8
4
8


B4
16
1
4
8
8
8
8
16
4
8


Gu4
0.5
0.5
4
8
8
8
8
8
4
8


Gu5
256
1
4
8
16
8
8
8
4
16


F2
32
1
4
4
8
16
16
16
8
16


F5
128
1
4
4
16
32
16
16
8
16


5674
2048
1
4
4
16
16
32
16
8
32


STY7
512
1
4
4
16
16
32
16
8
32


STY19
64
1
4
4
16
16
32
0.5
4
32


STY31
64
1
4
4
16
16
32
16
8
32












Strains
Description
Reference










Candida albicans clinical isolates









B3
fluconazole sensitive, parent strain for B4
1


B4
fluconazole-resistant due to the overexpression of CaMDR1
1


Gu4
fluconazole sensitive, parent strain for Gu5
1


Gu5
fluconazole-resistant due to the overexpression of CDR1 and CDR2
1


F2
fluconazole sensitive, parent strain for F5
2


F5
fluconazole-resistant due to the overexpression of CaMDR1 and ERG11
2


STY7
C. albicans 5674 overexpressing CDR1 and CDR2 mutant derivative, deletion
3



of CDR2


STY19
C. albicans 5674 overexpressing CDR1 and CDR2 mutant derivative, deletion
3



of CDR1


STY31
C. albicans 5674 overexpressing CDR1 and CDR2 mutant derivative, deletion
3



of CDR1 and CDR2





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 albicansABC transporters Cdr1p and Cdr2p to clinical azole resistance. Antimicrobial Agents and Chemotherapy 53: 1344-1352.






EXAMPLE 15
Cytotoxic Activity of Nystatin A1 Derivatives Against Mammalian Cells 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 are 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 1.2×104 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% CO2. After incubation, to all wells 200 μl of solution of 3-(4,5-dimethylthiazole-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 (A540), using a microplates reader (Victor3, Perkin-Wallac). On the basis of received results the diagrams on the relation between the A540 value and concentration of examined compound were prepared. From these graphs IC50 values were read, that is concentration of tested compound in the presence at which A540 value is half of A540 value measured in the control sample.
The obtained results are presented in Table 4.







TABLE 4







Cytotoxicity of compounds towards mammalian cells.









IC50 [μg/ml]












Compound
HepG2
LLC-PK1
CCRF-CEM







Nystatin
24.9 ± 2.332
>100
>100



N14
>100
>100
>100



N17
>100
>100
>100



N19
>100
>100
>100



N21
68.3 ± 8.06 
>100
91.9 ± 4.35



N22
82.9 ± 19.87
>100
>100



N28
>100
>100
>100



N34
>100
nt
>100



N37
>100
85.5 ± 4.30
>100



N39
>100
>100
>100



N41
>100
>100
>100



N44
>100
>100
>100



N45
>100
>100
>100



N46
>100
>100
>100

## Claims

1. A compound of Formula 1a



or a salt, hydrate or complex thereof;
wherein R1 selected from the group consisting of:
a) alkyl substituted with an optionally substituted alkylamino or an optionally substituted N-linked heterocycle;
b) a succinimidyl derivative;
c) an optionally substituted thioureidyl residue;
d) an aminoacyl residue of structure:






 wherein R6 and R7 are independently chosen from a hydrogen atom or an optionally substituted alkyl, or R6 and R7 can be taken, together with the atom to which they are joined, to form an optionally substituted nitrogen-containing cyclic moiety; one of R8 and R9 is —U—V, wherein U is an optionally substituted alkyl linker or a single bond and V is an optionally substituted carbocyclic, heterocyclic, alkoxy, or ester moiety, and the other of R8 and R9 is 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, or ester moiety; and
e) an aminoacyl of structure:






 wherein R10 and R11 can be taken, together with the atom to which they are joined, to form an optionally substituted nitrogen-containing cyclic moiety; R12 and R13 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, or ester moiety; and R12′ and R13′ are, independently, hydrogen or alkyl;

R2 is a hydrogen atom, optionally substituted alkyl, a succinimidyl derivative, a glycosyl residue, an optionally substituted aminoacyl residue, or an optionally substituted thioureidyl residue;
R3 is a hydroxyl group, alkoxyl group or an alkylamino or aminoalkyl derivative.

2. A compound according to claim 1, of Formula 1b



or a salt or complex thereof;
X is absent or present and, when present, X is one or more molecules of base or acid, or complexing compound.

3. The compound of claim 1, wherein, when one or both of R1 and R2 is, independently, selected from the group consisting of:
a) dialkylamino, or an optionally substituted N-linked heterocycle;
b) a succinimidyl derivative of structure




 wherein R4 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 —NR*2, —NH2, or —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;
c) a thioureidyl residue of structure




 wherein R5 is —W—Z, wherein W is an optionally substituted alkyl linker; and Z is an optionally substituted nitrogen-containing heterocycle, NR*2, NH2, or 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;
d) an aminoacyl residue of structure:




 wherein R6 and R7 are independently chosen from a hydrogen atom or an optionally substituted alkyl, or R6 and R7 can be taken, together with the atom to which they are joined, to form an optionally substituted nitrogen-containing cyclic moiety; one of R8 and R9 is hydrogen and the other of R8 and R9 is —U—V, wherein U is an optionally substituted alkyl linker or a single bond and V is a hydrogen atom, or an optionally substituted carbocyclic, heterocyclic, alkoxy, or ester moiety; and
e) an aminoacyl of structure:




 wherein R10 and R11 can be taken, together with the atom to which they are joined, to form an optionally substituted nitrogen-containing cyclic moiety; R12 and R13 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, or ester moiety; and R12′ and R13′ are, independently, hydrogen or alkyl.

4. The compound according to claim 3, wherein Y is an optionally substituted carbo- or heterocyclic moiety or a dialkylamino.

5. The compound according to claim 1, wherein R2 is a hydrogen atom, an unsubstituted non-branched alkyl or a substituted alkyl.

6. The compound according to claim 1, wherein R3 is hydroxyl, methoxy, or —NR14—(C1-C6alkyl)-NR15R16, wherein R14 is a hydrogen atom or methyl, and R15 and R16 are independently selected from the group consisting of hydrogen and optionally substituted aliphatic.

7. A compound according to claim 1, selected from the group consisting of: N-(N′-benzylsuccinimidyl)nystatin A1, N-(N′-4-bromophenylsuccinimidyl)nystatin A1, N-(N′-2,4,6-trimethylphenylsuccinimidyl) nystatin A1, N-(N′-2,6-dimethyl-phenylsuccinimidyl)nystatin A1, N-(N′-cyclohexyl succinimidyl)nystatin A1, N-[N′-3-(N′′,N′′-dimethylamino)propylsuccinimidyl]nystatin A1, N-[N′-2-(N′′,N′′-dimethylamino)ethylsuccinimidyl]nystatin A1, N-[N′-2-(piperidin-1-yl)ethylsuccinimidyl] nystatin A1, N-[N′-2-(4-methylpiperazin-1-yl)ethylsuccinimidyl]nystatin A1, N-[N′-3-(N′′,N′′-dimethylamino)-2,2-dimethylpropylosuccinimidyl]nystatin A1 and N-[N′-2-(pyrrolidin-1-yl)ethylsuccinimidyl]nystatin A1 or a salt, hydrate or complex thereof.

8. A compound according to claim 1, selected from the group consisting of: N-{3-[2-(piperidin-1-yl)ethyl]thioureidyl}nystatin A1, N-{3-[2-(mopholin-1-yl)ethyl]thioureidyl}nystatin A1, N-{3-[2-(N′,N′-diethyl amino)ethyl]thioureidyl}nystatin A1, N-{3-[2-(4-methylpiperazin-1-yl)ethyl]thioureidyl} nystatin A1, N-{3-[2-(pyrrolidin-1-yl)ethyl]thioureidyl}nystatin A1, N-{3-[3-(N′,N′-dimethyl amino)propyl]thioureidyl}nystatin A1, N-{3-[2-(N,N -dimethylamine)ethyl]thioureidyl}nystatin A1, N-{3-[3-(N′,N′-dimethylamine)-2,2-dimethylpropyl]thioureidyl}nystatin A1 and N-{3-[3-(imidazole-1-yl)propyl]thioureidyl}nystatin A1 or a salt, hydrate or complex thereof.

9. A compound according to claim 1, selected from the group consisting of: N,N-di[3-(piperidin-1-yl)propyl]nystatin A1 or N,N-di[3-(4-ethylpiperazin-1-yl)propyl]nystatin A1, N-fructosyl-N-[3-(N′,N′-dimethylamine)propyl]nystatin A1, N-fructosyl-N-[3-(piperidin-1′-yl)propyl]nystatin A1, and N-fructosyl-N-[3-(4-ethylpiperazin-1-yl) propyl)]nystatin A1, or a salt, hydrate or complex thereof.

10. A compound selected from the group consisting of: N-benzylnystatin A1, N,N-di-n-propylnystatin A1, N,N-dimethylnystatin A1, N,N-diethylnystatin A1, N,N-di-n-propylnystatin A1 methyl ester, N-fructosyl-N-n-propylnystatin A1 methyl ester, 3-(N′,N′-dimethyl-amino)propylamide of N′′,N′′-di-n-propylonystatin A1, 3-(N′,N′-dimethylamino)propylamide of N-fructosyl-N-n-propylnystatin A1, N-[2-(N-methylamino)-2-methylpropyl]nystatin A1, N-[(N′,N′-di-n-propyl)glycyl]nystatin A1, N-[3-(N′,N′-di-n-propylamino)propionyl]nystatin A1, N-[3-(N,N-diethyl-amino)propionyl]nystatin A1, and N-[3-(N,N-dimethylamino)propionyl]nystatin A1 or a salt, hydrate or complex thereof.

11. A compound, according to claim 1, selected from the group consisting of: N-D-phenyl-glycylnystatin A1, N-L-phenylalanylnystatin A1, N-L-(O-tert-butyl) glutamylnystatin A1, N-L-(O-tert-butyl)serylnystatin A1, N-D-(O-tert-butyl)glutamylnystatin A1, N-D-(O-tert-butyl)serylnystatin A1, N-D-β-naphtylalanylnystatin A1, N-L-(4-nitrophenyl)alanylnystatin A1, N-D-(O-tert-butyl)asparagylnystatin A1, N-D-β-(pyridin-3-yl)alanylnystatin A1N-[3-(piperidin-1-yl)propionyl]nystatin A1, N-[L-(N′,N′-dimethyl) phenylalanyl]nystatinA1, and N-[L-(N′,N′-diethyl)phenylalanyl]nystatin A1 or a salt, hydrate or complex thereof.

12. A compound according to claim 1, selected from the group consisting of:
N-{3-[3-(N′,N′-dimethylamino)-2,2-dimethylpropyl]thioureidyl}nystatin A1 methyl ester, N-D-phenylglycylnystatin A1 methyl ester, 3-(N′,N′-dimethylamino)propylamide of N-{3-[3-(N′′,N′′-dimethylamino)-2,3-dimethylpropyl]thioureidyl}nystatin A1 and 3-(N,N-dimethylamino)propylamide of N-D-pheynylglycylnystatin A1 or a salt, hydrate or complex thereof.

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

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

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

16. 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.

17. A method of treating fungal infection in a plant, comprising administering a composition comprising a compound of claim 1 to the plant.

18. A method of treatment as claimed in claim 16 or 17 wherein the fungal infection is caused by a strain of the genus Candida.