CA2176893A1 - Antibiotics - Google Patents
AntibioticsInfo
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- CA2176893A1 CA2176893A1 CA002176893A CA2176893A CA2176893A1 CA 2176893 A1 CA2176893 A1 CA 2176893A1 CA 002176893 A CA002176893 A CA 002176893A CA 2176893 A CA2176893 A CA 2176893A CA 2176893 A1 CA2176893 A1 CA 2176893A1
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- alkyl
- group
- derivative
- polyene macrolide
- antibiotics
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H17/00—Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
- C07H17/04—Heterocyclic radicals containing only oxygen as ring hetero atoms
- C07H17/08—Hetero rings containing eight or more ring members, e.g. erythromycins
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- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
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- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Saccharide Compounds (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention relates to an N-alkyl-N-glycosyl derivative of antifungal antibiotics of the polyene macrolide group of general formula 1(a), wherein M represents polyene macrolide antibiotics residues, R represents a variable part of sugar residue, R1 represents a C1-4 alkyl group and R2 represents hydrogen or a C1-4 alkyl group; their salts of general formula 2(a), wherein R, R1, R2 and M are as herein defined and A represents an anion of organic or inorganic acid. Compounds of formula 1(b) and 2(b) wherein the N-alkyl and alkyl ester groups are represented by methyl are of particular interest.
The invention relates also to processes for preparation of the compounds, to compositions containing these compounds and to their use in therapy and the treatment of external and internal fungal infections in humans and animals.
(see fig. 1a) (see fig. 1b) (see fig. 2a) (see fig. 2b)
The invention relates also to processes for preparation of the compounds, to compositions containing these compounds and to their use in therapy and the treatment of external and internal fungal infections in humans and animals.
(see fig. 1a) (see fig. 1b) (see fig. 2a) (see fig. 2b)
Description
1.
A4s6rLsrEQCANADAol 2 1 7 6 8 9 3 ANTiP~lOTlCS
The invention relates to N-alkyl-N-glycosyl derivatives of antifungal antibiotics of the polyene macrolide group of general formula l(a), wherein M represents polyene macrolide antibiotics residues, R represents a variable part of sugar residue, Rl represents a Cl 4 alkyl group and R2 represents hydrogen or a Cl 4 alkyl group; to salts of compound S I(a), IC~ ClliCd by the general formula 2(a), wherein M, R, Rl and R2 are as defined above and A represents an anion of an inorganic or organic ~id, to methods of preparation of ~,u~ uul~d~ of general formula l(a) arld 2(a) and their use in medicine.
~'omrollndc of particular interest include N-methyl-N-glycosyl derivatives of metbyl esters of antifungal antibiotics of polyene macrolide group of general formula l(b), whereirl 10 M represents polyene macrolide antibiotics residues, wherein R represents a variable part of sugar residue, and their salts of general formula 2(b), wherein M represents polyene macrolide antibiotics residues, wherein R represents a variable part of sugar residue, and A represents an anion of inorganic or organic acid, and to methods of their preparation, and use in medicine.
N-alkyl derivatives of antibiotics of polyene macrolide group wherein the amino group of the parent antibiotic is substituted by an alkyl group are known.
N-glycosyl derivatives of polyene macrolides whereirl amino group of the parent antibiotic is substituted with a residue of sugar are also known (J. Antibiotics ;~, 244 (1975), L. Falkowski, J. Golik, P. Kolori7iPj~7yk, J. Pawlak, J. Zieliriski, T. Zimiriski, E.
20 Borowski; Acta Polon. Pharm. 37, 517 (1980), L. Falkowski, J. Pawlak, J. Golik, P.
Kolodziejczyk, B. Stefanska, E. Bylec, E. Borowski). Examples of sugars used in the preparation of these derivatives irlclude D-glucose, D-mannose, L-rhamnose, D-ribose and maltose. Upon reaction of polyene macrolides with the appropriate sugar a ~ u~l~Amadori Ic~lallg~ .lL occurs to give the ~ullc~u~ N-glycosyl derivatives. The 25 compounds have the advantage that they exhibit a biological activity similar to those of the st~rting antibiotics and form water soluble salts. However, the high toxicity associated therewith represents a significant disadvantage.
From papers J. Antibiot;cs 28, 244 (1975), L. Falkowski, J. Golik, P. Kolodziejc~yk, J. Pawlak, J. Zielinski, T. Zimiriski, E. Borowski; Acta Polon. Pharm. ~, 517 (1980), 30 L. Falkowski, J. Pawlak~ J. Golik, P. K~ rl7iPj~7yk, B. Stefa~ska, E. Bylec, E. Borowski), I
~ r~l3714sorLsrEccANADAol 2 1 7 6 ~ 9 3 there are known N-glycosyl derivatives of polyene macrolides, in which the amino group of the parent antibiotic ;s substituted with a residue of sugar, such as D-glucose, D-mannose, L-rhamnose, D-ribose, maltose. The compounds are prepared by reaction of polyene macrolides with the listed sugars, and ~;., ...11,1, ,~,...~ Amadori rP~rr~n~pmpnt The S ~,u~ uu~lds exhibit the biological activity similar to those of the starting antibiotics and forln water soluble salts. However, they exhibit high toxicity.
Trim~llyl,.,..,,.o.,;ll,.l derivatives of polyene mac{olides methyl esters wherein the amino group of the parent antibiotic is fully methylated to give a quaternary ammonium salt are also known (J. Antibiotics ~, 1080 (1979), L. Falkowski, B. Stefatiska, J. Zieliliski, E.
10 Bylec, J. Golik, P. Krl~-~17iPjl7yk, E. Borowski). The compounds are prepared by exhaustive methylation of the parent antibiotic with dimethyl sulphate. Advantageous properties of the derivatives include their solubility in water and an antifungal activity similar to that of the starting antibiotics. ullrul LullaL~ly they are very toxic and unstable.
Another type of derivatives are trim~ilyl~,.,l,l,-l,;l,.,. derivatives of polyene 15 macrolides methyl esters, which are known from a paper - J. Antibiotics ~, 1080 (1979), L. Falkowski, B. Stefatiska, J. Zielitiski, E. Bylec, J. Golik, P. Kolorl7iPj~7yk, E. Borowski and in these compounds the amino group of the antibiotic methyl esters is fully methylated to give quaternary ammonium salt. The compounds are prepared by exhaustive methylation of the parent antibiotic with dimethyl sulphate. The derivatives are soluble in 20 water and are .1."~ rd by antifungal activity similar to the activity of the starting antibiotics, but they are very toxic and umstable.
Other types of N-alkyl derivatives include theN-~u~,.,;lulll;dyl derivatives formed by Michael addition reaction of the antibiotics and N-substituted 1~ , such as:
N-ethyl...~ , N,N'-h~A~Il~.llylrl.Pl~ lPimi~lp~ N-(3-dilll1~1lyla llillOIJIu,uyl)-25 m~lleimi(3,P; the l,Ulll~UUlld~ are known from a paper - J. Antibiotics, 44, 979 (1991), A. C~rwitiski, W.A. Konig, T. Zieniawa, P. Sowitiski, V. Sinnwell, S. Milewski, E. Borowski. Such compounds are less toxic than the parent antibiotics, but their antifungal activity is ~3,imin;chP~I
Finally, the last known group of N-alkyl derivatives of polyene macrolides are 30 N-enamine and amidine derivatives, fornled by reaction of the antibiotics with au~Lyla~ull~, ethyl ~r~yla~ ~LaL~, dimethylacetal or dimethylform:~mi-lP; the LUIII~)UUIU
A4s6rLsrEQCANADAol 2 1 7 6 8 9 3 ANTiP~lOTlCS
The invention relates to N-alkyl-N-glycosyl derivatives of antifungal antibiotics of the polyene macrolide group of general formula l(a), wherein M represents polyene macrolide antibiotics residues, R represents a variable part of sugar residue, Rl represents a Cl 4 alkyl group and R2 represents hydrogen or a Cl 4 alkyl group; to salts of compound S I(a), IC~ ClliCd by the general formula 2(a), wherein M, R, Rl and R2 are as defined above and A represents an anion of an inorganic or organic ~id, to methods of preparation of ~,u~ uul~d~ of general formula l(a) arld 2(a) and their use in medicine.
~'omrollndc of particular interest include N-methyl-N-glycosyl derivatives of metbyl esters of antifungal antibiotics of polyene macrolide group of general formula l(b), whereirl 10 M represents polyene macrolide antibiotics residues, wherein R represents a variable part of sugar residue, and their salts of general formula 2(b), wherein M represents polyene macrolide antibiotics residues, wherein R represents a variable part of sugar residue, and A represents an anion of inorganic or organic acid, and to methods of their preparation, and use in medicine.
N-alkyl derivatives of antibiotics of polyene macrolide group wherein the amino group of the parent antibiotic is substituted by an alkyl group are known.
N-glycosyl derivatives of polyene macrolides whereirl amino group of the parent antibiotic is substituted with a residue of sugar are also known (J. Antibiotics ;~, 244 (1975), L. Falkowski, J. Golik, P. Kolori7iPj~7yk, J. Pawlak, J. Zieliriski, T. Zimiriski, E.
20 Borowski; Acta Polon. Pharm. 37, 517 (1980), L. Falkowski, J. Pawlak, J. Golik, P.
Kolodziejczyk, B. Stefanska, E. Bylec, E. Borowski). Examples of sugars used in the preparation of these derivatives irlclude D-glucose, D-mannose, L-rhamnose, D-ribose and maltose. Upon reaction of polyene macrolides with the appropriate sugar a ~ u~l~Amadori Ic~lallg~ .lL occurs to give the ~ullc~u~ N-glycosyl derivatives. The 25 compounds have the advantage that they exhibit a biological activity similar to those of the st~rting antibiotics and form water soluble salts. However, the high toxicity associated therewith represents a significant disadvantage.
From papers J. Antibiot;cs 28, 244 (1975), L. Falkowski, J. Golik, P. Kolodziejc~yk, J. Pawlak, J. Zielinski, T. Zimiriski, E. Borowski; Acta Polon. Pharm. ~, 517 (1980), 30 L. Falkowski, J. Pawlak~ J. Golik, P. K~ rl7iPj~7yk, B. Stefa~ska, E. Bylec, E. Borowski), I
~ r~l3714sorLsrEccANADAol 2 1 7 6 ~ 9 3 there are known N-glycosyl derivatives of polyene macrolides, in which the amino group of the parent antibiotic ;s substituted with a residue of sugar, such as D-glucose, D-mannose, L-rhamnose, D-ribose, maltose. The compounds are prepared by reaction of polyene macrolides with the listed sugars, and ~;., ...11,1, ,~,...~ Amadori rP~rr~n~pmpnt The S ~,u~ uu~lds exhibit the biological activity similar to those of the starting antibiotics and forln water soluble salts. However, they exhibit high toxicity.
Trim~llyl,.,..,,.o.,;ll,.l derivatives of polyene mac{olides methyl esters wherein the amino group of the parent antibiotic is fully methylated to give a quaternary ammonium salt are also known (J. Antibiotics ~, 1080 (1979), L. Falkowski, B. Stefatiska, J. Zieliliski, E.
10 Bylec, J. Golik, P. Krl~-~17iPjl7yk, E. Borowski). The compounds are prepared by exhaustive methylation of the parent antibiotic with dimethyl sulphate. Advantageous properties of the derivatives include their solubility in water and an antifungal activity similar to that of the starting antibiotics. ullrul LullaL~ly they are very toxic and unstable.
Another type of derivatives are trim~ilyl~,.,l,l,-l,;l,.,. derivatives of polyene 15 macrolides methyl esters, which are known from a paper - J. Antibiotics ~, 1080 (1979), L. Falkowski, B. Stefatiska, J. Zielitiski, E. Bylec, J. Golik, P. Kolorl7iPj~7yk, E. Borowski and in these compounds the amino group of the antibiotic methyl esters is fully methylated to give quaternary ammonium salt. The compounds are prepared by exhaustive methylation of the parent antibiotic with dimethyl sulphate. The derivatives are soluble in 20 water and are .1."~ rd by antifungal activity similar to the activity of the starting antibiotics, but they are very toxic and umstable.
Other types of N-alkyl derivatives include theN-~u~,.,;lulll;dyl derivatives formed by Michael addition reaction of the antibiotics and N-substituted 1~ , such as:
N-ethyl...~ , N,N'-h~A~Il~.llylrl.Pl~ lPimi~lp~ N-(3-dilll1~1lyla llillOIJIu,uyl)-25 m~lleimi(3,P; the l,Ulll~UUlld~ are known from a paper - J. Antibiotics, 44, 979 (1991), A. C~rwitiski, W.A. Konig, T. Zieniawa, P. Sowitiski, V. Sinnwell, S. Milewski, E. Borowski. Such compounds are less toxic than the parent antibiotics, but their antifungal activity is ~3,imin;chP~I
Finally, the last known group of N-alkyl derivatives of polyene macrolides are 30 N-enamine and amidine derivatives, fornled by reaction of the antibiotics with au~Lyla~ull~, ethyl ~r~yla~ ~LaL~, dimethylacetal or dimethylform:~mi-lP; the LUIII~)UUIU
~ F \1~ 5~ r~ErEClCANADA 01 2 1 7 6 8 9 3 are presented in a paper - Acta Polonica Phann. ~, 71 (1988), B. Stefariska, J. Zieliriski, E. Borowski, L. Falkowski. The derivatives exhibit antifungal activity similar to those of the parent antibiotics and improved solubility in organic solvents, however, they are still significantly toxic and very unstable.
The present inventors have now prepared mixed N-alkyl-N-glycosyl derivatives of alkyl esters of polyene macrolide antibiotics as well as the free acid. Methods of preparation of these mixed compounds have also been established. These novel compounds have been found to have high anti-fungal activity, similar to those of the parent antibiotics;
form water soluble salts with acids; and are significantly less toxic. These properties are unexpected since all of the N-alkyl derivatives of polyene macrolides of the prior art exhibit a high toxicity, which is a I ~ lF~ di:~ad~ . The ~,U~ lUlllld~ comprised by theinvention are devoid of this di~al~lla~,.
Until now, N-methyl-N-glycosyl derivatives of methyl esters of polyene macrolideantibiotics and methods of their preparation were unknown. Surprisingly, such compounds preserve high antifungal activity, similar to those of the parent antibiotics, they form water soluble salts with acids, and are dramatically less toxic. These compounds do not exhibit the same toxicity as the N-alkyl derivatives of the prior art. A high toxicity constitutes the basic drawback of all known before N-alkyl derivatives of polyene macrolides, and the .~,,,,ll,.ll,,.l~comprisedbytheinventionaredevoidofthisdisadvantage.
A first aspect of the invention provides an N-alkyl-N-glycosyl derivative of antibiotics of the polyene macrolide group of general formula I (a), wherein M represents residue of an antibiotic of polyene macrolide group, R represents a part of sugar residue formed by reaction of the antibiotic with a mono or oligr~c~rrh~n~ R I represents a C 1-4 alkyl group arld R2 represents hydrogen or a C 1_4 alkyl group.
Rl--N--CH
\C/~) Formula I (a) F~137~15~FLSrEC\CANADA~I
2 1 76~93 Preferably the residue of the antibiotic of polyene macrolide group M is selected from amphotericin B, candidin, candidoin, candidinin, mycoheptin, nystatin, polyfungin, aureofacin, vacidin, llicllolllycill or candicidin.
It is preferred that the mono or nli~oC~f ~hAride from which the sugar residue R is 5 derived is selected from D-glucose, or L-glucose, or D-mannose, or D-galactose, or lactose, ormaltose. Uponreactionofpolyenemacrolideswiththeappropriatesugf~ra~.il,l,lll...,...,1~
Amadori 1l ,.. .", IJ~,r~I If 11 occurs to give the N-glycosyl precursors to the compounds of the invention.
A preferred clllb()dilllcllL of the frst aspect of the invention comprises an N-methyl-10 N-glycosyl derivative of a methyl ester of antibiotics of the polyene macrolide group presented by general formula I (b), wherein M represents residue of an antibiotic of polyene macrolide group, wherein R represents a part of sugar residue formed by reaction of the antibiotic with mono or oli~ , preferably with D-glucose, or L-glucose, or D-mannose, or D-galactose, or lactose, or maltose, and by eiml~lt~nPollC Amadori 15 I~;,- l~lll~,f,.l.rlll H 3 C--N--C~2 C/ ) Formula l(b) The invention in its preferred form relates to tne derivatives wherein the antibiotic of polyene macrolide group is ~ t- ,. .; 1 1 B, or candidi4 or candidoin, or candidinin, or Illy~fjllf~ , or nystatin, or polyfumgin, or aureofacin, or vacidin, or l~ y~ or20 candicidin.
A second aspect of the invention provides a salt of an N-alkyl-N-glycosyl derivative of antibiotics of the polyene macrolide group of general formula 2(a) wherein M, R, Rl and R2 are as defined above for the first aspect of tbe invention and A represents an anion of an organic or Inorganic acid. It is preferred that the salt is a physiologically acceptable salt 25 and f -~mr~llnfiC wherein A is the anion of L-aspartic acid are especially preferred. Salts r\l~ 5~rLSrEClCANADA.OI
2 1 76P~93 wherein Rl and R2 are methyl groups are especially preferred.
Rl--N--CH2 ~3 H ,,C~ ) A
HO R
Formula 2(a) A preferred embodiment of the second aspect of the invention comprises a salt of an N-methyl-N-glycosyl derivative of antibiotics of the polyene macrolide group presented S by general formula 2(b) wherein M represents residue of an antibiotic of polyene macrolide group, wherein R represents a part of sugar residue formed by reaction of the arltibiotic with mono or oli~,u~a~,ullalid~, preferably with D-glucose, or L-glucose, or D-mannose, or D-galactose, or lactose, or maltose, and by ~;IIIIIIlhll('Ull~ Amadori ~ , and A
represents an anion of organic or inorganic acid.
--H 3 C--N--C H2 A~
H ~
HO R
Formula 2(b) The invention in its preferred form relates to tlle salts wherein the antibiotic ofpolyene macrolide group is ~" "~ B, or candidin, or candidoin, or candidinin, or ,ol~ , or nystatin, or polyfungin, or aureofacin, or vacidin, or l~;~,llulll~ orcandicidin, and also preferably A relates to the anion of L-aspartic acid.
A tllird a~pect of the invention comprises a method of preparation of C.U~ UL lld::l of formula I (a) wherein M, R, Rl and R2 are as defined above for the first and second aspects of the invention and which comprises the steps of reacting a polyene macrolide antibiotic ~, } ~1~56\r.5PE~AN~D~ 01 2 1 7 6 8 9 3 with a mono or oli~ ,~,.. 1~,., ;~lr, the reaction being ~1,,.. ,.. ;~r~l by the occurrence of a ~;1"ll1l,.... ~.1l~ Amadori IrA~ ,..,,g...,~ .~1 to give the N-glycosyl derivatives of the polyene macrolide antibiotics; isolating the product of the Amadori lr~m~ ; treating theproduct with an alkylating agent and purifying the crude product.
In one r~ vdil~ of the third aspect of the invention the product of the Amadori Jrl I lr~ Il is isolated in the forrn of a suspension by precipitation from tbe solution in which the Icallall~ occurs. Organic solvents such as N,N-dimethylform~lmi~ are preferred to support the Amadori Ir ~ Organic solvents such as diethyl ether aresuitable to effect formation of a suspension by ~IC~ iOI~ of the product of the Amadori 1~ ,rl l ~ .1 l In a second embodiment of the third aspect of the invention alkylation of the product of the Amadori Ir,lllall~r.llrlll is carried out at reduced Ltlll~laLulc;~ Tclllv~,~a~ulc~ of between -5~C and +S''C are preferred. Alkylating agents such as diazo alkanes may be used An ethereal solution of.l;,.~. "". ~ r is the preferred alkylating agent.
Crude N alkyl-N-glycosyl products can be isolated upon removal of the solvent and v;~Livll from diethyl ether. The pure product may be isolated using known purification procedures.
A preferred embodiment of the third aspect of the invention comprises a process for preparation of an N-methyl-N-glycosyl derivative of a methyl ester of antibiotics of the 20 polyene macrolide group presented by general formula l(b), wherein M represents residue of an antibiotic of polyene macrolide group, wherein R represents a part of sugar residue formed by reaction of the antibiotic with mono or oli~,,-~,.. .1,,.. ;~lr, preferably with D-glucose, or L-glucose, or D-mannose, or D-galactose, or lactose, or maltose, and by ~;,.,,.ll5.,.~ v ~ Amadori ICallallg~,lllc;ll~, wherein the product obtained by Amadori 25 I~ " of the N-glycosyl derivative of antibiotics of the polyene macrolide group is ~ rl., .,.. ;~ into suspension by precipitation with solvent, preferably diethyl ether, from asolutionofthederivativesinorganicsolvent,preferablyinN,N-d;lll~,;l.ylr(.,...,.,..;.lr,and ~l,s. .l..rl,lly treated with ethereal solution of ~II,.,~IIIlrlll,.~l~ at lowered ~tlll~v~,laLulc, preferably in the ran~e from -s ~c to +5 oc~ stirred~ and isolated by ~ vula~iull of solvents~
30 and precipitation from the concentrated solution, preferably by an excess of diethyl ether, then, the crude product is purifled according to known procedures 7\456 r~SrEOl:lANADA 01 . 2 1 76893 A fourth aspect of the present invention provides an alternative method for the preparation compounds of formula I (a) wherein M, R, Rl and R2 are as defined above for the first and second aspects of the invention and which comprises the steps of reacting a polyene macrol;de antibiotic with a species which provides a protecting group to give a first 5 N-protected (amino) derivative; reacting said first N-protected derivative with a further protecting group providing species thereby protecting the carboxy group of the macrolide to give a second N-protected-carboxy-protected derivative; reacting said second derivative with an alkylating agent to give the N-alkyl N-protected-carboxy-protected derivative, removing the N-protecting group and re~ting the resulting N-alkyl derivative with a mono-10, di- or oli~ r, the reaction being ~ rl;~ by the occurrence of a simultaneous ~ rl~ , to give the N-alkyl-N-glycosyl derivative; removal of the carboxy protecting group from said N-alkyl-N-glycosyl derivative followed by the required derivatisation of said carboxy group.
The choice of amino protecting group will be apparent to those skilled in the art.
Species which provide ~liLluulvac~yl N-protecting groups are of most interest. Typical examples include Llinuulua~,~Lic anhydride and ~linuulu~ ic acid.
The nature of tlle carboxy protecting groups will also be apparent to a skilled person Species giving rise to labile ester groups at the carboxy centre are preferred. Typical groups used for protection include benzyl esters, p-methoxybenzyl ester and t-butyl ester.
The choice of alkylating agents will also be apparent to persons skilled in the art.
Preferred alkylating agents include the appropriate dialkyl sulphate and alkyl iodides.
Dimethyl sulphate and methyl iodide are of particular interest.
Conditions used for removal of the N-protecting group will depend upon the nature of the group itself and will be apparent to a person skilled in the art. Typically the ~5 irltroduction of mildly acidic conditions will suffice. This is particularly the case when N-inuuludC~,.yl protecting groups are used.
Similarly, removal of the carboxy protecting group will likewise depend upon thenature of the protecting group employed. Typical conditions will be apparent to a person skilled in the art.
3û The introduction of mildly acidic conditions will result in the production of the carboxylic acid where Rl is hydrogen. Reaction of this carboxylic acid form with C 1-4 F\~ 4s6\rLEl~EclcANADAol 2 1 7 6 ~ 9 3 alcohols will give the ~U~ .V.ILI;Il~ ester form It should therefore be understood that the preparation of mixed N-alkyl-N-glycosyl derivatives wherein Rl and R2 are different is provided by the present invention.
A fifth aspect of the invention provides a metbod of preparation of a salt of an N-5 alkyl-N-glycosyl derivative of the polyene macrolide antibiotics of general formula 2(a) wherein M, R, Rl and R2 are as defined above for the first to fourth aspects of the invention and which comprises the steps of suspending an N-alkyl-N-glycosyl derivative prepared according to the third or fourth aspects of the invention in sufficient water to effect formation of a 1~-", Ih~ suspension, acidifying the resulting suspension and 10 isolating the product. The preparation of salts of N-methyl-N-glycosyl derivatives of methyl esters of polyene macrolide antibiotics is especially preferred.
Organic or inorganic acids may be used to acidify the S-lerPneihn L-aspartic acid is preferred Isolation of the pure product may be effected by precipitation of the crude product 15 with an organic solvent which is then washed with an additional appropriate solvent and dried It is preferred that the solvent used to precipitate the crude product is miscible with water; acetone is preferred. Typical solvents employed for washing the product include acetone and diethyl ether. It is preferred that the product is dried under reduced pressure.
~ preferred rl..khLl;..l.,l.L of the fifth aspect of the invention comprises a process for 20 preparation of salts of N-met_yl-N-glycosyl derivatives of methyl esters of antibiotics of polyene macrolide group presented by general formula 2(b), wherein M represents residue of an antibiotic of polyene macrolide group, wherein R represents a part of sugar residue formed by reaction of the antibiotic with mono or olighc~ h~ri~P, preferably with D-glucose, or L-glucose, or D-mannose, or D-galactose, or lactose, or maltose, and by 25 ~ ulla.~.,vu~ Amadori l~ g~ , wherein the obtained by A}nadori l r~ g. . ~ N-glycosyl derivatives of antibiotics of polyene macrolide group are trr1ncfornnPd into suspension by precipitation with solvent, preferably diethyl ether, from a solution of the derivative in organic solvent, preferably in N,N-dimethylformamide, and ~ y treated with ethereal solution Of.l;,.~ P at lowered t~ pv.aLu~, preferably in the 3 0 range from -5 ~C to +5 ~C, stirred, and isolated by evaporation of solvents, and precipitation from the ~ "~ r~l solution, preferably by an excess of diethyl etller, then the crude P\I~A45~PLSPECICA~ADA.01 2 1 7 6 8 9 3 product is purifed according to known procedures, ~ub~LIL~ Lly7 the obtained derivative, as a solid, is suspended in small amount of water, and ~ , amount of organic or inorganic acid is added, next the product is ~IG~ ,d from the formed solution by an excess of organic solvent miscible with water, preferably acetone, the solid is then washed, S preferably with acetone and ~ ly preferably with diethyl ether, and dried, preferably under reduced pressure.
A sixth aspect of the present invention comprises an N-alkyl-N-glycosyl derivative of formula I (a) or a salt thereof for use in therapy.
A seventh aspect of the invention provides a method for the treatment of fungal infections in humans and animals which comprises the A~ thereto of an N-alkyl-N-glycosyl derivative of formula I (a) or a salt thereof as herein before defined. N-methyl-N-glycosyl derivatives of fommula I (b) or the salts thereof are of particular interest.
A first preferred embodiment of the seventh aspect of the invention comprises a method for treatment of e~ternal and intemal fungal infections in humans and animals, wherein an N-methyl-N-glycosyl derivative of a methyl ester of antibiotics of the polyene macrolide group presented by general fommula l(b), wherein M represents residue of an antibiotic of polyene macrolide group, wherein R represents a part of sugar residue fommed by reaction of the antibiotic with mono or oli,~ , preferably with D-glucose, orL-glucose, or D-mannose, or D-galactose, or lactose, or maltose, and by qimlllt~nPollc Amadori n'~ ~"lrl~l are used to treat the infections.
A second preferred emhorlim~nt of the seventh aspect of the invention comprises a method for treatment of extemal and intemal fungal infections in humans and animals, wherein salts of N-methyl-N-glycosyl derivatives of methyl esters of antibiotics of polyene macrolide group presented by general fommula 2(b), wherein M represents residue of an antibiotic of polyene macrolide group, wherein R represents a part of sugar residue formed by reaction ofthe antibiotic with mono or nli~ne:lerh:~ride, preferably with D-glucose, or L-glucose, or D-mannose, or D-galactose, or lactose, or maltose, amd by simultaneous Amadori l~allal~ , and A represents an anion of organic or inorganic acid are used to treat the infections.
An eighth aspect of the invention provides an N-alkyl-N-glycosyl derivative of formula I (a) or a salt thereof as herein before defined for use in the treatment of fungal r~ ~s6rLsr~NAD~ rl 2 1 7 6 8 9 3 infections. N-methyl-N-glycosyl derivatives of formula l(b) or the salts thereof are of particular interest.
The infections for which treatment is provided may be intemal or external. The mode of ~ wi~l depend upon the nature of the infection. Thus the ~v~ vu ld~ of tlle S invention may be formulated for intravenous, intra peritoneal, oral, topical, ~ub~,uLa~ Ju~, rectal or vaginal ,~I".;"~ n For intemal infections perfusion of the therapeuticcompound is the preferred form of ~ In some instances the use of perfusion in the treatment of external fungal infections will be advantageous.
A ninth aspect of the invention provides a method of treatment of fungal infections 10 which comprises the perfusion of compounds of formula l(a) or salts thereof into the body of a patient suffering therefrom.
A tenth aspect of tlle invention provides compositions for use in the treatment of fullgal infections comprising an N-alkyl-N-glycosyl derivative of formula l(a) or a salt thereof and a physiologically acceptable carrier. The nature of the N-alkyl-N-glycosyl 15 derivative and the carrier will depend upon the mode of ,.~1",;,.;~1.,.l;~l The composition may be formulated from one or more compourlds according to the invention, optionally in combination with other known antifungal agents, according to lcsu u~ . " "~
containing N-methyl-N-glycosyl derivatives of formula l(b) or the salts thereof are of particular interest.
An eleventh aspect of the invention provides a unit dosage form comprising one or more N-alkyl-N-glycosyl derivatives of formula l(a) or salts thereof and a physiologically acceptable carrier formulated for ~ allllàc.,u~i-al or veterinary use. The unit dosage form may be formulated from one or more compounds according to the invention optionally in rnmhin~tinn with other known antifurlgal agents. By analogy with the previous aspects of ~5 the invention unit dosage forms containing N-methyl-N-glycosyl derivatives of formula I (b) are of particular interest.
As mentioned above the nature of the unit dosage form will depend upon the mode of",l",;";~ ;..,. Typicallytabletsandcapsulesaresuitablefororal~.l.,.;~,;~l,.~;l", creams and patches are suitable for topical ,~ " with pessaries being suitable for rectal 30 and vaginal ddlllill;~Lla~ion.
A twelfth aspect of the ulvention provides the use of an N-alkyl-N-glycosyl derivative ;
~ F~l~S6\rL6F~C~CANAD~01 2 1 7 6 8 9 3 of formula l (a) or a salt thereof ror the preparation of a ~ for use in the treatrnent of fungal infections. Compounds wherein the N-alkyl and alkyl ester ~ are both methyl groups are of particular interest.
Structural rlr~ ;"~ carried out on the compounds of the invention using S ~ LIua. u~;c methods indicate that the integrity of the parent antibiotic is preserved during the reaction.
rhe invented process for p}eparation of N-methyl-N-glycosyl derivatives of methyl esters of antibiotics of polyene macrolide group gives the desired products without changes in structure of the parent antibiotic. Structure of the obtained compounds was proved using 10 ~ ,LIu~.,u,u;c methods. The proof is illustrated by drt~nnin~t;r)n of t~le structure of N-methyl-N-D-rl u.,Lu~yla~ ul~l ;c;ll B methyl ester, of formula 3.
1' /~ CH3 H3C ,~ ~ ~ ~ ~ I H3 CN~<~OH
~CH3 OH OH OH OH o~cooCH3 OH
H3CJ~O~OH
OH
OH
Formula 3 Electronic spectrum of N-methyl-N-D-rlu~u~yl~ . B methyl ester is identical with that of the parent antibiotic, that means ~ uLtl ;~ ;11 B, and dc~lllull~LI~lL~
15 that the invented method does not lead to ~ l of the polyene uluulllu~llul~, and a bigh value of extinction (E 1% 1 cm = 13 00 at 3 82 nm) conflrms a high purity of the obtained product. Absorption infrared spect~um of the N-methyl-N-D-fructosylamphotericin B
methyl ester d~ u-l~LI.lLt~ the band related to stretching vibrations of the ester carbonyl group at 1730 cm~l, and lack of band of free carboxylic group, what means that the 20 carboxylic group was completely l,~"~r.~",,.rl into methyl ester group. Complete -Il-~ r \~ 456 pLspEc cANADA ol 2 1 7 6 8 9 3 irlformation on structure of the N-methyl-N-D-rlu~,lu~yl~ utericin B methyl ester was provided by nuclear magnetic resonance spectra (NMR) upon I H (DQF-COSY, ROESY),13c (DEPT) and ll~ u~oll~ldled spectra (Varian 300 MHZ spectrometer) allowed an :~cei~nm~nt of the formula 3 for the compound.
The most significant IH and 13c inform~tinn are listed in Tables I and 2, respect;vely. The NMR data for aglycone of " ~ B are in full agreement with literature data - Magn. Reson. Chem. ~Q 275, (1992), P. Sowinski, J. Pawlak, E. Borowski, P. Gariboldi. IH chemical shifts (in DMSO/MeOD solvent system) of N-CH3 (o = 2.35 ppm) and H-l" (2.30 and 3.15 ppm) are . ~.,,.,,. I~, ;~lir for influence of an amino substituent. After r4~i~1ifir:1tinn ~ changes to 2.92 ppm for N-CH3, and to 3 .64 for H- I " due to the protonation of the amino group (o for H-3' changes to 3.19 ppm). These data are supported also by ROE effects between protons NCH3/H3', NCH3/H2', NCH3/Hl"b and I "a/H3'. Coupling constants and ROE indicate 4C 1 ~;ulL~IIllaLiull of the mycosamine moiety, as it i~ras found before for free a~ llu~ ,;ll B.
Table I presents chemical shifts IH and ROE effects of the rlie~--rh~4ri~l.o fragment of the N-methyl-N-D-fructosylamphotericin B methyl ester.
~ r~l3~s5\rLsrE~ANADAol 2 1 7 6 8 9 3 Table I
proton o[ppm] ROE for protons o[ppm] ROE for protons pyridine-d5 methanol-d4 9: I DMSO-d6: methanol-d4 4: 6 1' 4 77 2', 3', 5', 18b, COOMe 4.48 3', 5' 2' 4.41 1', 3', NMe, 17, COOMe, I"a? 4.04 3', NMe, COOMe 3' 2.06 1', 2', 5', NMe, I"a, COOMe 1.89 1', 2', NMe, I"b 4' 4.38 6' 3.80 6' 5' 3.61 1',3'76' 3.45 1',3',6' 6' 1.23 4',5' 1.27 4', 5' I"a2.56 3', 3" (2') 2.30 I"b3 58 NMe 3.15 3", 3' 3" 4.41 I"a? 3.65 I"b 4" 4.76 3.97 5 "4.4 1 3.63 6"a4 21 3.57 6"b4.36 3.76 NMe2.29 2', 3', I"b, COOMe 2.35 2', 3', I"a, I"b. COOMe COOMe 3.70 1', 2', 3', NMe, 16 3.77 2', NMe Table 2 presents l 3C-NMR chemical shifts of the ~ fragment of the N-methyl-N-D-fructo~ykullul~u~ ;ll B methyl ester and their ~u~ u~ ull with data fûr D-fructose.
~ r\ll~s6rLsrE~ANADAol 2 ~ 7 6 ~ 9 3 Trlble 2 data for fructoses~
~-D-r~u~Lu~y~ D-furanose ~-D-fructofuranose carbon ¦ o [ppm] o [ppm] o [ppm] o [ppm]
1'98.2 2'72.4 3'66.4 4'698 S'72.3 6'18.2 1"62.2 64.1 62.1 63.9 2"98.1 99 1 lû5.3 lû2 4 3"66.9 70.5 83.0 76.5 4"71.2 68.4 77.0 75.5 S"72.2 7û.0 82.2 8 1 .S
6"64.6 64.7 62. 1 63.3 NMe40.9 COOMeS 1.6 S. N. Rosenthal & J.~I. Fendler, Progr. Pllys. Org. Chem. 1~. 280 (1976).
Comparison of the 13C-NMR data from Table 2 for the fructosyl fragment with literature data for D-fructose indicates the pyranoside forrn ofthe sugar sllh~titllrnt The observed coupling constants, presented in Table 3, evidence a boat ~,Ol~lllla~iull of the rru~,~u~yl~osid~ ring presented by formula 4. Only this conformation of the ring is in full 5 agreement with the measured coupling constants.
Table 3 presents coupling constants JH H for protons of the ~ie~ h:-ri~lr fragment of the N-methyl-N-D-fructosylamphotericin B methyl ester (pyridine-ds: methanol-d4 9: 1), coupling constants and chemical shifts of closely coupled spin system 113" - H6" were reflned iteratively by computer simulation.
F~137\.155\rLSPECC~NADA01 2 1 7 6 8 q 3 . .
Table 3 protons J [Hz] protons J [Hz]
1',2' -O I"a, I"b 11.6 2', 3' 2.2 3", 4" I.OS
3',4' 9.5 4",5" 8.09 4', 5' 9.2 5", 6"a 5.76 5', 6' 6.0 5", 6"b 3.61 6"a, 6"b -10.86 4", 6"a -0.1 10 The presented results confirm the ~onforrn~ltil~n of the fructosyl fragment of the N-methyl-N-D-r~u~3:,yku~ 1lu~ B methyl ester presented by formula 4 Other N-methyl-N-glycosyl derivatives of methyl esters of antibiotics of polyene macrolide group were , ,.. t~ d by methods similar to those described above.
OH
OH
Formula 4 The invention ~vill now be described by reference to the following non-limiting examples. Further ~.I.bo~ falling within the scope of the invention will be apparent to a skilled person.
Anti-fungal Activity For all of the prepared compounds antifungal activity and also toxicity in vitro were 20 rl~tPnnin~ ~ For the compound with the best properties, that means for the N-methyl-N-D-fructosyld~ llot~ B methyl ester aspartate, activity and toxicity in vivo were also ~l~tP~rnnin~
1~1 T'ifro Antifungal Activity The antifungal activity of ~ Lllld:~ was deterrnined following the standard for _15_ F~ 56~rLSrEC CANADA 01 2 1 7 6 8 9 3 polyene macrolides procedure. The liquid Sabouraud medium was inoculated with 104 cells/ml of test organism Candida alhicans ATCC 262778 and incubated for 24 hours at 30~C witll tlle tested antibiotic (serial dilutions). Amphotericin B ~vas used as the reference. Compounds ~vere dissolved in DMF and suitable amounts of the solutions were 5 added to the medium. Turbidimetric method (660 nm) was applied to determine the degree of growth inhibition The ...~ ", of antibiotic at which the growth of fungi was inhibited by 50% was determined from the dose response curve. The obtained LCso value r~ d antifungal activity of the compound.
In Vifro Toxicity Toxicity of compounds in vitro against animal cells was determined using standard for polyene macrolides procedure, by the determination of the degree of haemolysis of human ery~rocytes. Human r~ly Ll~lu~,y t-,., isolated from fresh, citrated human blood were washed twice ~vith cold saline. The cells were diluted 250 times with saline andPq--ilihrr tP~l for 30 minutes at 37~C. Samples of erythrocytes were incubated with various 5 COnrPnlrr~ n~ of antibiotics (the base solution in DMF) for 30 minutes at 37r'C. After ~.,1.irl.t,".1;..,.thelysisoferythrocyteswasassessedbyrl~ .,.l;,".ofthehaemoglobin released to the solution. Optical density of the ~ was measured at 550 nm. The results were expressed by EHso value, as rnnrPntr~tion of antibiotic at ~hich 50% of hemolysis occurred. The values of EHso were read from a curve relating the degree of 20 hemolysis with antibiotic dose.
In Vivo Toxicity In vivo toxicity was determined for methyl ester of N-methyl-N-D-r~U~,Lu~rla..l,ul.uLcl;.,;.. B L-aspartate as the maximum tolerated dose (MTD), and as the acute toxicity (LDso). To determine MTD dose the compound was dissolved in 5%
solution of glucose, and a.ll~ d ~llLla~,.luL ~ly and ;llLla~J.l iLull~aly to Balb/c mice in single and multiple doses. .The maximum single tolerated dose was 100 mg/kg for the illLIa~,lluu~ and more than 200 mg/lcg for the ;. .~ .1, ";. ,~ ~ The maximum multiple tolerated dose ror i l~ a~ l; Lul l~al ~ l of l oo mgAcg for s days was much higher. For such dose, toxic effects were not observed during 20 days of observation.
The acute toxicity, LDso of methyl ester of N-methyl-N-D-fructosylamphotericin B
~ F~7\456\FI,SPECCANADA.01 2 1 7 6 8 9 3 L-aspa}tate was determined for Swiss Webster female mice of average weight of 20 g.
Various doses of the tested compound, and for ~,u.~ Vll, amphotericin B in form of Fungizone, dissolved in 5% glucose were given intravenously to the animals. The administered volume of the solution was 0.5 ml. 0.5 ml of 5 % solution of glucose was 5 administered to mice as a control. Every dose of both preparations was ~dlllilli~Lclcd to S mice. The animals were observed for 7 days. Next, the animals were killed, and some serum indexes were ~J~tP~rmin~d No increased level of aspartate al~lilwL-~ulartl~c or creatinine were found in comparison with the control. For methyl ester of N-methyl-N-D-fructosyl~..~ B L-aspartate, the LDso was found to be 400 mg/kg, while ror 10 ampllotericin B in form of Fungizone it was 6 mg/kg.
~t :- , " Efficacy (~hP m~ 11 ;r efficacy of methyl ester of N-methyl-N-D-fructosylamphotericin B L-aspartate was detemmined using systemic murine candidose model. Candida albicans was grown overnight in Sabouraud dextrose broth at room Ltlll~claLulc. The fungal cells 15 were ~ .-ntrifi1~, washed twice with 0.9 % solution of sodium chloride, and suspended in physiological salt solution. Female Swiss Webster mice of 25 g weight, were injected intravenously with 105 cells of Candida in 0.2 ml of 0.9 % sodium chloride solution.
Initially, the infection was systemic, but by 2 to 3 days it was locali~d to the kidneys.
Untreated animals usually died between 7 to 14 days post-infection. Three days 20 post-infection, animals were treated ;~L~ ,ly, twice a day, for 5 consecutive days with a 5 to 6 hours interval. Preparation was ad..l;l.; "I,lcd as a solution in 5% glucose. The animals were observed for 5 weeks starting from the day of infection. Afler this time, the surviving animals were sacrificed, tbeir kidneys were removed, homogenized in sterile water, and the llull~o~ . was plated on Sabouraud dextrose agar, and the grown colonies 25 of Candida were counted. The .1,..,..,l~ rrt~LiV~ a waS represented as a doseof mg kg, which in the above test resulted in a survival of 50 % of animals, and on clearance of Candida from kidneys of half of the mice. The dose, called EDso, was calculated using a method given in J. Hyg. 2:Z 493, (1938). The values of EDso for methyl ester of N-methyl-N-D-Lu~ ylal--pl~oLt-;cin B L-aspartate are 2.3 mg/kg based on the 30 survival, and 6 mg/kg based on the kidneys clearance.
r~ 561rLSrECCANADArl ~ 21 76~93 Preparative Examples N-Methyl-N-glycosyl derivatives of methyl esters of antibiotics of polyene macrolide group, their salts, and methods of preparation are illustrated by the examples given below.
Example 1.
S I g of Amrh~tPririn B (El%lCm = 1350 at 382 nm, MeOlLI) was dissolved in 15 ml of N,N-dimethylform~mi~lP, 0.3 g of D-glucose was added, and the mixture was stirred in darkness at 37'C for 40 hours. Next the reaction was cooled and a solid was precipitated with an excess of diethyl ether. The solid was rPntrifil~pr1~ washed twice with diethyl ether and dried under reduced pressure. To remove an excess of glucose the solid was suspended 10 in 20 ml of water, rPntrjfilgP~I, washed twice with small amount of water, twice with acetone, and next twice with diethyl ether. The product was dried under reduced pressure to give 0.98 g of N-D-fructu~y ~ l )tl "~; " B (El %l Cm = 1 2û0 at 3 82 nm, MeOH). The product was dissolved, with stirring, in 10 ml of N,N-Lli~ Ll~y~r~ lP and 50 ml of diethyl ether was added to the solution, to give a fine sl~irpn~irln The suspension was 15 cooled in ice to 0 - 2'C, and freshly prepared diethyl ether solution of 2.5 mole of 1A~ per I mole of N-D-rlu~u~ t~ . B, was added with vigorous stirring. The reaction was followed by thin layer chr(lm~t~\gr~rlly on silica gel in chlorofomm - methanol - water 10:6:1 v/v solvent system. After completion of the reaction, what took about 2 hours, the excess of 11i,,,..,,,..1,~.,~ and diethyl ether was evaporated 20 under reduced pressure at Lc~ laLul~: not higher than 40'C. The crude product was iLL ~,1 from the residue with an excess of diethyl ether, rPntrifil~P~1, washed twice with diethy~ ether, next with n-hexane, and dried under reduced pressure to give 0 95 g of the crude product. Pure N-methyl-N-D-rlucLu~ . B methyl ester was isolated from the crude product by column chromatography on Merck Silicagel 60, 70 -23û mesh in 25 chloroform - methanol - water 20: 8: I v/v solvent system. Thus, 0.95 g of the crude product was suspended in the mixture of solvents specified above, and if the product dissolved with difticulties, proportions of the same solvents were changed to 10: 6: 1 rhe undissolved part was centrifuged off, and the ~Illlrlll,ahlll charged on chrom~t-.~r~rhy column, next developed in tlle solvent mixture listed above, but in proportion 20: 8: I v/v.
30 The eluate was analysed on silica plates using chlorororm - methanol - water 10: 6: I v/v F\l~S~\rl5FeCCANAD~1 2, 7 6 ~ 9 3 solvent system. The plates were visualized with cerium sulphate reagent. Fractions of Rf = 0.5 - 0.54 containing pure N-methyl-N-D-fructosyl derivative of amphotericin B
methyl ester ~ere collected The combined fractions were evaporated under reducedpressure. The dry residue was dissolved in small amount of N,N-dimethylformamide, and 5 the product was ~u~ );LdL~d with an excess of diethyl ether, the solid was centrifuged, washed twice with diethyl ether and dried in avacuum desiccator. 0.137 g of N-methyl-N-D-rluu~u~ lpllotericin B methyl ester, El%lCm = 1300 on 382 nm in methanol, was obtained. The proof of structure was given above in the descriptive part. Antifungal activity of the compoumd against Cand~da albicans, determined as it was described above, 10 gave ICso = 0.12 llg/ml, and toxicity for human erythrocytes, determined as described above, gave EHso value higher than 350 llglml. For Cu~ ull~ EHs0 value for the starting amphotericin B was 1.5 ~Lg/ml. An exact value of EHso for N-methyl-N-D-fructosylamphotericin B methyl ester could not be irtFrminr~i, as above 350 lag/ml the compound was insoluble under conditions of the experiment.
15 Example II.
0.5 g of candidin (El%lCm = 1175 at 382 nm, MeOH) and 0.15 g of D-glucose were dissolvedinlOm~ofN,N-~;II.cLllylr."".,",~ andstirredat37~Cfor36hours. Further procedure was analogous to this of the Example I, and resulted in 0.43 g of N-D-fructosylcandidin, El%lCm = 1100 at 382 nm in MeOEI. The product was methylated 20 with .1i~ r in diethyl ether, analogously as in tlle Example I to give 0.4 g of crude product. Pure N-methyl-N-D-fructosylcandidin methyl ester was isolated by columnchromatography, by method similar to that given in the Example 1. Fractions containing pure derivative were ..l,",,,. lr"~r~l on thin layer chrf)mAt~rAphy by Rf = 0.49 - 0.52. The fractions were combined, evaporated to dryness, dissolved in small amount of 25 N,N-dimcLllylru~ ulli~lc, and solid was ~Ic~;~;Ld~d with diethyl ether. The solid was rrntrifi~ ri washed with diethyl ether and dried in vacuum desiccator to give 0.05 g of N-methyl-N-D-fructosylcandidin methyl ester; El%lCm = 1200 at 382 nm, in MeOEI, ICso = 0 75 ,ug ml and EHs0 above 300 ~Lg/ml. Structure of &e compound was determined by the same methods as for of N-methyl-N-D-fructosylamphotericin B methyl ester.
r\l3A~s6PLsP~AuADA~1 2 1 7 6 8 9 3 Example III.
2 g of nystatin (El%lcm = 870 at 304 nm, in MeOEI) and 0.6 g of D-glucose were dissolved in 35 ml of N,N-dimethylacetamide and stirred at 37~C for 40 hours. After completion of the reaction the crude product was precipitated with diethyl ether, S centrifuged, and dried umder vacuum. Then, an excess of glucose was washed off with small amount of water - acetone 1: I mixture, next with acetone, diethyl ether, and the product was centrifuged and dried in vacuum desiccator to give 1.2 g of N-D-rlu~,~u~yllly~LdLill (El%lcm = 720 at 304 nm, in MeOH). The product was methylated using-l;",.".,rll,,.,.~asintheExampleltogivel.25gofcrudeproduct. Theproductwas 10 purifled on silica gel column as in the Example 1. Fractions containing pure N-methyl-N-D-fructu~yllly 31d~ill methyl ester were ~1,, "- 1~ ~;, ~l on thin layer LIUl~ y by Rf =
0.49 - 0.52 as in the Example 1. The fractions were combined, evaporated to dryness at 30~C under reduced pressure, dissolved in small amount of N,N-dimethylfnrmAmi-lP, and product was ~ ;,LI;LdL~d with diethyl ether. The product was rPn~rifil~P~I, washed with 15 diethyl et~ler and dried in vacuum desiccator to give 0.17 g of N-methyl-N-D-fructosylnystatin methyl ester; El%lcm = 900 at 304 nm, in MeOEI. Structure of the compound was determined by a method described for N-methyl-N-D-rru~,Lu~ l ~ ., ,1 .l ,. ~tPriri n B methyl ester. For the obtained compound ICso = 6.8 llg/ml and EHs0 above 300 ~Lg/ml were found.
2û Example IV.
0.79 g of vacidin, the main component of antibiotic complex aureofacin, (E 1% 1 cm = 900 at 378 nm, in MeOH) and 0.22 g of D-glucose in 15 ml of N,N-dimethylacetamide were stirred at 37~C for 18 hours. After completion of the reaction the mixture was cooled and solid was precipitated with an excess of diethyl ether. The solid was rPntrifil~P~I, washed 25 with diethyl ether and dried under reduced pressure to give 0.8 g of crude N-D-fructosylvacidin (El%lcm = 720 at 378 nm, in MeOH). The obtained derivative was methylated using ~iiA7nmPthAnP, as in the Example I to give 0.5 g of crude product. Pure N-methyl-N-D-rlu~Lu~ a~;d;.- methyl ester was isolated by chromatography on silica gel column similarly as it was described in the Example 1, but the column was developed with 30 chloroform - methanol - water 30: 8: I solvent system. Fractions containing pure F~l371~6uL5rE~ANllD/~6l 2 1 7 6 8 9 3 derivative of vacidin and having on thin layer chrnm~tr~r~rhy value of Rf = 0.53 - 0.55 in chloroform - methanol - water 13: 8: I solvent system were collected and combined.
Further procedure was as in the Example Ill. 0.07 g of N-methyl-N-D-fructosylvacidin methyl ester was obtailled (El %I cm = 900 at 378 nm, in MeOH). The compound exhibited 5 ICso = 0.01 ,ug/ml and El 150 = 170 ,ug/ml. Structure of the compound was determined by a method described for N-methyl-N-D-fructosylamphotericin B methyl ester.
Example V.
0.79 g of candicidin D, the main component of antibiotic complex candicidin, ~vas treated identically as described in the Example IV. 0.1 g of N-methyl-N-D-fructosylcandicidin D
10 methylesterwasobtained; E1%1cm=920at378nm,inMeOH,Rfofthecompoundin thin layer ~ ".,.I"~,.3l,l.y, under conditions described in the Example IV, was 0.50 - 0.53.
The compound exnibited ICso = 0.01 ,ug/ml and EHs0 = 180 ,ug/ml. Structure of the compoundwasdeterminedbyamethoddescribedforN-methyl-N-D-r~u,~ ,yl~ )trl;l ;"
B methyl ester.
15 Example Vl.
0.79 g of the main component of antibiotic complex lly~llollly~;ll was treated identically as it was described in the Example IV. 0.1 g of N-methyl-N-fructosyltrychomycin methyl esterwas obtained; El%lcm = 910 at 378 nm, in MeOH Rfofthe compound in thin layer chrom~t~r~rhy, under conditions described in the Example IV, was 0.49 - 0.52. The 20 compound exhibited ICso = 0.013 ,ug/ml and Ells0 = 165 ug/ml. Structure of the compound was determined by a method described forN-methyl-N-D-fructosyl~mrh,-t~ ruin B methyl ester.
Example Vll.
0.5 g of a:ll,~JIl(.)~liLill B (El%lCm = 1350 at 382 nm, MeOH) and 0.15 g of L-glucose 25 in 8 ml of N,N-dimetnylformamide was stirred at 37~C for 40 hours. Subsequentoperations were as in the Example 1. 0.065 g of N-methyl-N-L-fructosylamphotericin B
methyl ester was obtained; El%lcm = 1280 at 382 nm, in MeOH. The compound exhibited ICso = 0.42 ug/ml and EHs0 = above 200 ug/ml. Structure of the compound r~ \PLS~E~/~ DAOI 2 1 7 6 8 9 3 was determined by a method described for methyl ester of N-methyl-N-D-rlu~ yla~ uLericin B.
Example VIII.
0.52gofamphotericinB(E1%1cm=1350at382nm,MeOll)andO.153gofD-mannose S was dissolved in 10 ml of N,N-di~ Lll~lr~,,.,,..,.i-lP and stirred at 37''C for 40 hours.
Subsequent operations were as in the Example 1. Pure derivative was isolated by silica gel column chrnm~to~r~rhy, in a manner analogous to that of the Example 1, fractions having in thin layer ~lu~llla~ lly, run according to the method described in the Example 1, value of Rf = 0.53 - 0.55 were collected. Subsequent operations were as in the Example 1.
10 0.08 g of N-methyl-N-D-fructosylall~ o~ ;ll B methyl ester was obtained;
El%lcm = 1280 at 382 nm. in MeOH. The compound exhibited ICs0 = 0.42 ,ug/ml and EHso above 200 ,ug/ml. Structure of tlle compound ~vas determined by a method described for N-methyl-N-D-fructosylamphotericin B methyl ester.
Example IX.
15 0.5 g of amphotericin B (El%lcm = 1350 at 382 nm, MeOH) and 0.3 g of D-lactose in 12 ml of N,N-dimethylr.).l",..,.i.lr was stirred at 37~C for two days. Subsequent operations were as in the Example 1. Pure derivative was isolated by silica gel columri chr ~m~t~-~r~rhy, in a manner analogous to that of the Example 1, fractions having in thin layer chromatography, run according to the method described in the Example 1, value of 20 Rf= 0.25 - 0.30 were collected. 0.08 g of N-methyl-N-D-fructosyl-gald~,Lv~yla.,lL,l.~ericin B methyl ester was obtained; El%lCm = 1000 at 382 nm, in MeOH. The compound exhibited ICso = 6.0 ,ug/ml and EHs0 above 200 ,ug/ml.
Example X.
0.5 g of N-methyl-N-D-rlu~o~y~ Lericin B methyl ester, prepared according to the25 Example 1, was suspended in 10 ml of water and 0.059 g of aspart;c acid dissolved in 2 ml of water was added. The solution of acid was added dropwise, with stirring to effect th~
solution. The solution was filtered to ren1ove a small amount of residual solid amd an excess of acetone was added to the clear filtrate until the whole salt was precipitated. The F\137~53VLSF~CCANADA01 ~ 2 1 76~93 solid was filtered offor r~-n~rifi~ l, washed twice with acetone, twice with diethyl ether and dried under reduced pressure, 0.5 g of methyl ester of N-methyl-N-D-rlu~L~ylcu~ elicin B L-aspartate, El%lcm = 1100 at 382 nrn, in MeOH, was obtained.
Thin layer chromatography under conditions as in the Exarnple I gave Rf= 0.5 - 054.
S Product was soluble in N,N-dimethylf )rm3~nni~, dimethylsulphoxide and ~% water solution of glucose. It was very well soluble in water. The compound exhibited ICso = 0.125 g/ml and EHso above 3~0 ,ug/ml.
The present inventors have now prepared mixed N-alkyl-N-glycosyl derivatives of alkyl esters of polyene macrolide antibiotics as well as the free acid. Methods of preparation of these mixed compounds have also been established. These novel compounds have been found to have high anti-fungal activity, similar to those of the parent antibiotics;
form water soluble salts with acids; and are significantly less toxic. These properties are unexpected since all of the N-alkyl derivatives of polyene macrolides of the prior art exhibit a high toxicity, which is a I ~ lF~ di:~ad~ . The ~,U~ lUlllld~ comprised by theinvention are devoid of this di~al~lla~,.
Until now, N-methyl-N-glycosyl derivatives of methyl esters of polyene macrolideantibiotics and methods of their preparation were unknown. Surprisingly, such compounds preserve high antifungal activity, similar to those of the parent antibiotics, they form water soluble salts with acids, and are dramatically less toxic. These compounds do not exhibit the same toxicity as the N-alkyl derivatives of the prior art. A high toxicity constitutes the basic drawback of all known before N-alkyl derivatives of polyene macrolides, and the .~,,,,ll,.ll,,.l~comprisedbytheinventionaredevoidofthisdisadvantage.
A first aspect of the invention provides an N-alkyl-N-glycosyl derivative of antibiotics of the polyene macrolide group of general formula I (a), wherein M represents residue of an antibiotic of polyene macrolide group, R represents a part of sugar residue formed by reaction of the antibiotic with a mono or oligr~c~rrh~n~ R I represents a C 1-4 alkyl group arld R2 represents hydrogen or a C 1_4 alkyl group.
Rl--N--CH
\C/~) Formula I (a) F~137~15~FLSrEC\CANADA~I
2 1 76~93 Preferably the residue of the antibiotic of polyene macrolide group M is selected from amphotericin B, candidin, candidoin, candidinin, mycoheptin, nystatin, polyfungin, aureofacin, vacidin, llicllolllycill or candicidin.
It is preferred that the mono or nli~oC~f ~hAride from which the sugar residue R is 5 derived is selected from D-glucose, or L-glucose, or D-mannose, or D-galactose, or lactose, ormaltose. Uponreactionofpolyenemacrolideswiththeappropriatesugf~ra~.il,l,lll...,...,1~
Amadori 1l ,.. .", IJ~,r~I If 11 occurs to give the N-glycosyl precursors to the compounds of the invention.
A preferred clllb()dilllcllL of the frst aspect of the invention comprises an N-methyl-10 N-glycosyl derivative of a methyl ester of antibiotics of the polyene macrolide group presented by general formula I (b), wherein M represents residue of an antibiotic of polyene macrolide group, wherein R represents a part of sugar residue formed by reaction of the antibiotic with mono or oli~ , preferably with D-glucose, or L-glucose, or D-mannose, or D-galactose, or lactose, or maltose, and by eiml~lt~nPollC Amadori 15 I~;,- l~lll~,f,.l.rlll H 3 C--N--C~2 C/ ) Formula l(b) The invention in its preferred form relates to tne derivatives wherein the antibiotic of polyene macrolide group is ~ t- ,. .; 1 1 B, or candidi4 or candidoin, or candidinin, or Illy~fjllf~ , or nystatin, or polyfumgin, or aureofacin, or vacidin, or l~ y~ or20 candicidin.
A second aspect of the invention provides a salt of an N-alkyl-N-glycosyl derivative of antibiotics of the polyene macrolide group of general formula 2(a) wherein M, R, Rl and R2 are as defined above for the first aspect of tbe invention and A represents an anion of an organic or Inorganic acid. It is preferred that the salt is a physiologically acceptable salt 25 and f -~mr~llnfiC wherein A is the anion of L-aspartic acid are especially preferred. Salts r\l~ 5~rLSrEClCANADA.OI
2 1 76P~93 wherein Rl and R2 are methyl groups are especially preferred.
Rl--N--CH2 ~3 H ,,C~ ) A
HO R
Formula 2(a) A preferred embodiment of the second aspect of the invention comprises a salt of an N-methyl-N-glycosyl derivative of antibiotics of the polyene macrolide group presented S by general formula 2(b) wherein M represents residue of an antibiotic of polyene macrolide group, wherein R represents a part of sugar residue formed by reaction of the arltibiotic with mono or oli~,u~a~,ullalid~, preferably with D-glucose, or L-glucose, or D-mannose, or D-galactose, or lactose, or maltose, and by ~;IIIIIIlhll('Ull~ Amadori ~ , and A
represents an anion of organic or inorganic acid.
--H 3 C--N--C H2 A~
H ~
HO R
Formula 2(b) The invention in its preferred form relates to tlle salts wherein the antibiotic ofpolyene macrolide group is ~" "~ B, or candidin, or candidoin, or candidinin, or ,ol~ , or nystatin, or polyfungin, or aureofacin, or vacidin, or l~;~,llulll~ orcandicidin, and also preferably A relates to the anion of L-aspartic acid.
A tllird a~pect of the invention comprises a method of preparation of C.U~ UL lld::l of formula I (a) wherein M, R, Rl and R2 are as defined above for the first and second aspects of the invention and which comprises the steps of reacting a polyene macrolide antibiotic ~, } ~1~56\r.5PE~AN~D~ 01 2 1 7 6 8 9 3 with a mono or oli~ ,~,.. 1~,., ;~lr, the reaction being ~1,,.. ,.. ;~r~l by the occurrence of a ~;1"ll1l,.... ~.1l~ Amadori IrA~ ,..,,g...,~ .~1 to give the N-glycosyl derivatives of the polyene macrolide antibiotics; isolating the product of the Amadori lr~m~ ; treating theproduct with an alkylating agent and purifying the crude product.
In one r~ vdil~ of the third aspect of the invention the product of the Amadori Jrl I lr~ Il is isolated in the forrn of a suspension by precipitation from tbe solution in which the Icallall~ occurs. Organic solvents such as N,N-dimethylform~lmi~ are preferred to support the Amadori Ir ~ Organic solvents such as diethyl ether aresuitable to effect formation of a suspension by ~IC~ iOI~ of the product of the Amadori 1~ ,rl l ~ .1 l In a second embodiment of the third aspect of the invention alkylation of the product of the Amadori Ir,lllall~r.llrlll is carried out at reduced Ltlll~laLulc;~ Tclllv~,~a~ulc~ of between -5~C and +S''C are preferred. Alkylating agents such as diazo alkanes may be used An ethereal solution of.l;,.~. "". ~ r is the preferred alkylating agent.
Crude N alkyl-N-glycosyl products can be isolated upon removal of the solvent and v;~Livll from diethyl ether. The pure product may be isolated using known purification procedures.
A preferred embodiment of the third aspect of the invention comprises a process for preparation of an N-methyl-N-glycosyl derivative of a methyl ester of antibiotics of the 20 polyene macrolide group presented by general formula l(b), wherein M represents residue of an antibiotic of polyene macrolide group, wherein R represents a part of sugar residue formed by reaction of the antibiotic with mono or oli~,,-~,.. .1,,.. ;~lr, preferably with D-glucose, or L-glucose, or D-mannose, or D-galactose, or lactose, or maltose, and by ~;,.,,.ll5.,.~ v ~ Amadori ICallallg~,lllc;ll~, wherein the product obtained by Amadori 25 I~ " of the N-glycosyl derivative of antibiotics of the polyene macrolide group is ~ rl., .,.. ;~ into suspension by precipitation with solvent, preferably diethyl ether, from asolutionofthederivativesinorganicsolvent,preferablyinN,N-d;lll~,;l.ylr(.,...,.,..;.lr,and ~l,s. .l..rl,lly treated with ethereal solution of ~II,.,~IIIlrlll,.~l~ at lowered ~tlll~v~,laLulc, preferably in the ran~e from -s ~c to +5 oc~ stirred~ and isolated by ~ vula~iull of solvents~
30 and precipitation from the concentrated solution, preferably by an excess of diethyl ether, then, the crude product is purifled according to known procedures 7\456 r~SrEOl:lANADA 01 . 2 1 76893 A fourth aspect of the present invention provides an alternative method for the preparation compounds of formula I (a) wherein M, R, Rl and R2 are as defined above for the first and second aspects of the invention and which comprises the steps of reacting a polyene macrol;de antibiotic with a species which provides a protecting group to give a first 5 N-protected (amino) derivative; reacting said first N-protected derivative with a further protecting group providing species thereby protecting the carboxy group of the macrolide to give a second N-protected-carboxy-protected derivative; reacting said second derivative with an alkylating agent to give the N-alkyl N-protected-carboxy-protected derivative, removing the N-protecting group and re~ting the resulting N-alkyl derivative with a mono-10, di- or oli~ r, the reaction being ~ rl;~ by the occurrence of a simultaneous ~ rl~ , to give the N-alkyl-N-glycosyl derivative; removal of the carboxy protecting group from said N-alkyl-N-glycosyl derivative followed by the required derivatisation of said carboxy group.
The choice of amino protecting group will be apparent to those skilled in the art.
Species which provide ~liLluulvac~yl N-protecting groups are of most interest. Typical examples include Llinuulua~,~Lic anhydride and ~linuulu~ ic acid.
The nature of tlle carboxy protecting groups will also be apparent to a skilled person Species giving rise to labile ester groups at the carboxy centre are preferred. Typical groups used for protection include benzyl esters, p-methoxybenzyl ester and t-butyl ester.
The choice of alkylating agents will also be apparent to persons skilled in the art.
Preferred alkylating agents include the appropriate dialkyl sulphate and alkyl iodides.
Dimethyl sulphate and methyl iodide are of particular interest.
Conditions used for removal of the N-protecting group will depend upon the nature of the group itself and will be apparent to a person skilled in the art. Typically the ~5 irltroduction of mildly acidic conditions will suffice. This is particularly the case when N-inuuludC~,.yl protecting groups are used.
Similarly, removal of the carboxy protecting group will likewise depend upon thenature of the protecting group employed. Typical conditions will be apparent to a person skilled in the art.
3û The introduction of mildly acidic conditions will result in the production of the carboxylic acid where Rl is hydrogen. Reaction of this carboxylic acid form with C 1-4 F\~ 4s6\rLEl~EclcANADAol 2 1 7 6 ~ 9 3 alcohols will give the ~U~ .V.ILI;Il~ ester form It should therefore be understood that the preparation of mixed N-alkyl-N-glycosyl derivatives wherein Rl and R2 are different is provided by the present invention.
A fifth aspect of the invention provides a metbod of preparation of a salt of an N-5 alkyl-N-glycosyl derivative of the polyene macrolide antibiotics of general formula 2(a) wherein M, R, Rl and R2 are as defined above for the first to fourth aspects of the invention and which comprises the steps of suspending an N-alkyl-N-glycosyl derivative prepared according to the third or fourth aspects of the invention in sufficient water to effect formation of a 1~-", Ih~ suspension, acidifying the resulting suspension and 10 isolating the product. The preparation of salts of N-methyl-N-glycosyl derivatives of methyl esters of polyene macrolide antibiotics is especially preferred.
Organic or inorganic acids may be used to acidify the S-lerPneihn L-aspartic acid is preferred Isolation of the pure product may be effected by precipitation of the crude product 15 with an organic solvent which is then washed with an additional appropriate solvent and dried It is preferred that the solvent used to precipitate the crude product is miscible with water; acetone is preferred. Typical solvents employed for washing the product include acetone and diethyl ether. It is preferred that the product is dried under reduced pressure.
~ preferred rl..khLl;..l.,l.L of the fifth aspect of the invention comprises a process for 20 preparation of salts of N-met_yl-N-glycosyl derivatives of methyl esters of antibiotics of polyene macrolide group presented by general formula 2(b), wherein M represents residue of an antibiotic of polyene macrolide group, wherein R represents a part of sugar residue formed by reaction of the antibiotic with mono or olighc~ h~ri~P, preferably with D-glucose, or L-glucose, or D-mannose, or D-galactose, or lactose, or maltose, and by 25 ~ ulla.~.,vu~ Amadori l~ g~ , wherein the obtained by A}nadori l r~ g. . ~ N-glycosyl derivatives of antibiotics of polyene macrolide group are trr1ncfornnPd into suspension by precipitation with solvent, preferably diethyl ether, from a solution of the derivative in organic solvent, preferably in N,N-dimethylformamide, and ~ y treated with ethereal solution Of.l;,.~ P at lowered t~ pv.aLu~, preferably in the 3 0 range from -5 ~C to +5 ~C, stirred, and isolated by evaporation of solvents, and precipitation from the ~ "~ r~l solution, preferably by an excess of diethyl etller, then the crude P\I~A45~PLSPECICA~ADA.01 2 1 7 6 8 9 3 product is purifed according to known procedures, ~ub~LIL~ Lly7 the obtained derivative, as a solid, is suspended in small amount of water, and ~ , amount of organic or inorganic acid is added, next the product is ~IG~ ,d from the formed solution by an excess of organic solvent miscible with water, preferably acetone, the solid is then washed, S preferably with acetone and ~ ly preferably with diethyl ether, and dried, preferably under reduced pressure.
A sixth aspect of the present invention comprises an N-alkyl-N-glycosyl derivative of formula I (a) or a salt thereof for use in therapy.
A seventh aspect of the invention provides a method for the treatment of fungal infections in humans and animals which comprises the A~ thereto of an N-alkyl-N-glycosyl derivative of formula I (a) or a salt thereof as herein before defined. N-methyl-N-glycosyl derivatives of fommula I (b) or the salts thereof are of particular interest.
A first preferred embodiment of the seventh aspect of the invention comprises a method for treatment of e~ternal and intemal fungal infections in humans and animals, wherein an N-methyl-N-glycosyl derivative of a methyl ester of antibiotics of the polyene macrolide group presented by general fommula l(b), wherein M represents residue of an antibiotic of polyene macrolide group, wherein R represents a part of sugar residue fommed by reaction of the antibiotic with mono or oli,~ , preferably with D-glucose, orL-glucose, or D-mannose, or D-galactose, or lactose, or maltose, and by qimlllt~nPollc Amadori n'~ ~"lrl~l are used to treat the infections.
A second preferred emhorlim~nt of the seventh aspect of the invention comprises a method for treatment of extemal and intemal fungal infections in humans and animals, wherein salts of N-methyl-N-glycosyl derivatives of methyl esters of antibiotics of polyene macrolide group presented by general fommula 2(b), wherein M represents residue of an antibiotic of polyene macrolide group, wherein R represents a part of sugar residue formed by reaction ofthe antibiotic with mono or nli~ne:lerh:~ride, preferably with D-glucose, or L-glucose, or D-mannose, or D-galactose, or lactose, or maltose, amd by simultaneous Amadori l~allal~ , and A represents an anion of organic or inorganic acid are used to treat the infections.
An eighth aspect of the invention provides an N-alkyl-N-glycosyl derivative of formula I (a) or a salt thereof as herein before defined for use in the treatment of fungal r~ ~s6rLsr~NAD~ rl 2 1 7 6 8 9 3 infections. N-methyl-N-glycosyl derivatives of formula l(b) or the salts thereof are of particular interest.
The infections for which treatment is provided may be intemal or external. The mode of ~ wi~l depend upon the nature of the infection. Thus the ~v~ vu ld~ of tlle S invention may be formulated for intravenous, intra peritoneal, oral, topical, ~ub~,uLa~ Ju~, rectal or vaginal ,~I".;"~ n For intemal infections perfusion of the therapeuticcompound is the preferred form of ~ In some instances the use of perfusion in the treatment of external fungal infections will be advantageous.
A ninth aspect of the invention provides a method of treatment of fungal infections 10 which comprises the perfusion of compounds of formula l(a) or salts thereof into the body of a patient suffering therefrom.
A tenth aspect of tlle invention provides compositions for use in the treatment of fullgal infections comprising an N-alkyl-N-glycosyl derivative of formula l(a) or a salt thereof and a physiologically acceptable carrier. The nature of the N-alkyl-N-glycosyl 15 derivative and the carrier will depend upon the mode of ,.~1",;,.;~1.,.l;~l The composition may be formulated from one or more compourlds according to the invention, optionally in combination with other known antifungal agents, according to lcsu u~ . " "~
containing N-methyl-N-glycosyl derivatives of formula l(b) or the salts thereof are of particular interest.
An eleventh aspect of the invention provides a unit dosage form comprising one or more N-alkyl-N-glycosyl derivatives of formula l(a) or salts thereof and a physiologically acceptable carrier formulated for ~ allllàc.,u~i-al or veterinary use. The unit dosage form may be formulated from one or more compounds according to the invention optionally in rnmhin~tinn with other known antifurlgal agents. By analogy with the previous aspects of ~5 the invention unit dosage forms containing N-methyl-N-glycosyl derivatives of formula I (b) are of particular interest.
As mentioned above the nature of the unit dosage form will depend upon the mode of",l",;";~ ;..,. Typicallytabletsandcapsulesaresuitablefororal~.l.,.;~,;~l,.~;l", creams and patches are suitable for topical ,~ " with pessaries being suitable for rectal 30 and vaginal ddlllill;~Lla~ion.
A twelfth aspect of the ulvention provides the use of an N-alkyl-N-glycosyl derivative ;
~ F~l~S6\rL6F~C~CANAD~01 2 1 7 6 8 9 3 of formula l (a) or a salt thereof ror the preparation of a ~ for use in the treatrnent of fungal infections. Compounds wherein the N-alkyl and alkyl ester ~ are both methyl groups are of particular interest.
Structural rlr~ ;"~ carried out on the compounds of the invention using S ~ LIua. u~;c methods indicate that the integrity of the parent antibiotic is preserved during the reaction.
rhe invented process for p}eparation of N-methyl-N-glycosyl derivatives of methyl esters of antibiotics of polyene macrolide group gives the desired products without changes in structure of the parent antibiotic. Structure of the obtained compounds was proved using 10 ~ ,LIu~.,u,u;c methods. The proof is illustrated by drt~nnin~t;r)n of t~le structure of N-methyl-N-D-rl u.,Lu~yla~ ul~l ;c;ll B methyl ester, of formula 3.
1' /~ CH3 H3C ,~ ~ ~ ~ ~ I H3 CN~<~OH
~CH3 OH OH OH OH o~cooCH3 OH
H3CJ~O~OH
OH
OH
Formula 3 Electronic spectrum of N-methyl-N-D-rlu~u~yl~ . B methyl ester is identical with that of the parent antibiotic, that means ~ uLtl ;~ ;11 B, and dc~lllull~LI~lL~
15 that the invented method does not lead to ~ l of the polyene uluulllu~llul~, and a bigh value of extinction (E 1% 1 cm = 13 00 at 3 82 nm) conflrms a high purity of the obtained product. Absorption infrared spect~um of the N-methyl-N-D-fructosylamphotericin B
methyl ester d~ u-l~LI.lLt~ the band related to stretching vibrations of the ester carbonyl group at 1730 cm~l, and lack of band of free carboxylic group, what means that the 20 carboxylic group was completely l,~"~r.~",,.rl into methyl ester group. Complete -Il-~ r \~ 456 pLspEc cANADA ol 2 1 7 6 8 9 3 irlformation on structure of the N-methyl-N-D-rlu~,lu~yl~ utericin B methyl ester was provided by nuclear magnetic resonance spectra (NMR) upon I H (DQF-COSY, ROESY),13c (DEPT) and ll~ u~oll~ldled spectra (Varian 300 MHZ spectrometer) allowed an :~cei~nm~nt of the formula 3 for the compound.
The most significant IH and 13c inform~tinn are listed in Tables I and 2, respect;vely. The NMR data for aglycone of " ~ B are in full agreement with literature data - Magn. Reson. Chem. ~Q 275, (1992), P. Sowinski, J. Pawlak, E. Borowski, P. Gariboldi. IH chemical shifts (in DMSO/MeOD solvent system) of N-CH3 (o = 2.35 ppm) and H-l" (2.30 and 3.15 ppm) are . ~.,,.,,. I~, ;~lir for influence of an amino substituent. After r4~i~1ifir:1tinn ~ changes to 2.92 ppm for N-CH3, and to 3 .64 for H- I " due to the protonation of the amino group (o for H-3' changes to 3.19 ppm). These data are supported also by ROE effects between protons NCH3/H3', NCH3/H2', NCH3/Hl"b and I "a/H3'. Coupling constants and ROE indicate 4C 1 ~;ulL~IIllaLiull of the mycosamine moiety, as it i~ras found before for free a~ llu~ ,;ll B.
Table I presents chemical shifts IH and ROE effects of the rlie~--rh~4ri~l.o fragment of the N-methyl-N-D-fructosylamphotericin B methyl ester.
~ r~l3~s5\rLsrE~ANADAol 2 1 7 6 8 9 3 Table I
proton o[ppm] ROE for protons o[ppm] ROE for protons pyridine-d5 methanol-d4 9: I DMSO-d6: methanol-d4 4: 6 1' 4 77 2', 3', 5', 18b, COOMe 4.48 3', 5' 2' 4.41 1', 3', NMe, 17, COOMe, I"a? 4.04 3', NMe, COOMe 3' 2.06 1', 2', 5', NMe, I"a, COOMe 1.89 1', 2', NMe, I"b 4' 4.38 6' 3.80 6' 5' 3.61 1',3'76' 3.45 1',3',6' 6' 1.23 4',5' 1.27 4', 5' I"a2.56 3', 3" (2') 2.30 I"b3 58 NMe 3.15 3", 3' 3" 4.41 I"a? 3.65 I"b 4" 4.76 3.97 5 "4.4 1 3.63 6"a4 21 3.57 6"b4.36 3.76 NMe2.29 2', 3', I"b, COOMe 2.35 2', 3', I"a, I"b. COOMe COOMe 3.70 1', 2', 3', NMe, 16 3.77 2', NMe Table 2 presents l 3C-NMR chemical shifts of the ~ fragment of the N-methyl-N-D-fructo~ykullul~u~ ;ll B methyl ester and their ~u~ u~ ull with data fûr D-fructose.
~ r\ll~s6rLsrE~ANADAol 2 ~ 7 6 ~ 9 3 Trlble 2 data for fructoses~
~-D-r~u~Lu~y~ D-furanose ~-D-fructofuranose carbon ¦ o [ppm] o [ppm] o [ppm] o [ppm]
1'98.2 2'72.4 3'66.4 4'698 S'72.3 6'18.2 1"62.2 64.1 62.1 63.9 2"98.1 99 1 lû5.3 lû2 4 3"66.9 70.5 83.0 76.5 4"71.2 68.4 77.0 75.5 S"72.2 7û.0 82.2 8 1 .S
6"64.6 64.7 62. 1 63.3 NMe40.9 COOMeS 1.6 S. N. Rosenthal & J.~I. Fendler, Progr. Pllys. Org. Chem. 1~. 280 (1976).
Comparison of the 13C-NMR data from Table 2 for the fructosyl fragment with literature data for D-fructose indicates the pyranoside forrn ofthe sugar sllh~titllrnt The observed coupling constants, presented in Table 3, evidence a boat ~,Ol~lllla~iull of the rru~,~u~yl~osid~ ring presented by formula 4. Only this conformation of the ring is in full 5 agreement with the measured coupling constants.
Table 3 presents coupling constants JH H for protons of the ~ie~ h:-ri~lr fragment of the N-methyl-N-D-fructosylamphotericin B methyl ester (pyridine-ds: methanol-d4 9: 1), coupling constants and chemical shifts of closely coupled spin system 113" - H6" were reflned iteratively by computer simulation.
F~137\.155\rLSPECC~NADA01 2 1 7 6 8 q 3 . .
Table 3 protons J [Hz] protons J [Hz]
1',2' -O I"a, I"b 11.6 2', 3' 2.2 3", 4" I.OS
3',4' 9.5 4",5" 8.09 4', 5' 9.2 5", 6"a 5.76 5', 6' 6.0 5", 6"b 3.61 6"a, 6"b -10.86 4", 6"a -0.1 10 The presented results confirm the ~onforrn~ltil~n of the fructosyl fragment of the N-methyl-N-D-r~u~3:,yku~ 1lu~ B methyl ester presented by formula 4 Other N-methyl-N-glycosyl derivatives of methyl esters of antibiotics of polyene macrolide group were , ,.. t~ d by methods similar to those described above.
OH
OH
Formula 4 The invention ~vill now be described by reference to the following non-limiting examples. Further ~.I.bo~ falling within the scope of the invention will be apparent to a skilled person.
Anti-fungal Activity For all of the prepared compounds antifungal activity and also toxicity in vitro were 20 rl~tPnnin~ ~ For the compound with the best properties, that means for the N-methyl-N-D-fructosyld~ llot~ B methyl ester aspartate, activity and toxicity in vivo were also ~l~tP~rnnin~
1~1 T'ifro Antifungal Activity The antifungal activity of ~ Lllld:~ was deterrnined following the standard for _15_ F~ 56~rLSrEC CANADA 01 2 1 7 6 8 9 3 polyene macrolides procedure. The liquid Sabouraud medium was inoculated with 104 cells/ml of test organism Candida alhicans ATCC 262778 and incubated for 24 hours at 30~C witll tlle tested antibiotic (serial dilutions). Amphotericin B ~vas used as the reference. Compounds ~vere dissolved in DMF and suitable amounts of the solutions were 5 added to the medium. Turbidimetric method (660 nm) was applied to determine the degree of growth inhibition The ...~ ", of antibiotic at which the growth of fungi was inhibited by 50% was determined from the dose response curve. The obtained LCso value r~ d antifungal activity of the compound.
In Vifro Toxicity Toxicity of compounds in vitro against animal cells was determined using standard for polyene macrolides procedure, by the determination of the degree of haemolysis of human ery~rocytes. Human r~ly Ll~lu~,y t-,., isolated from fresh, citrated human blood were washed twice ~vith cold saline. The cells were diluted 250 times with saline andPq--ilihrr tP~l for 30 minutes at 37~C. Samples of erythrocytes were incubated with various 5 COnrPnlrr~ n~ of antibiotics (the base solution in DMF) for 30 minutes at 37r'C. After ~.,1.irl.t,".1;..,.thelysisoferythrocyteswasassessedbyrl~ .,.l;,".ofthehaemoglobin released to the solution. Optical density of the ~ was measured at 550 nm. The results were expressed by EHso value, as rnnrPntr~tion of antibiotic at ~hich 50% of hemolysis occurred. The values of EHso were read from a curve relating the degree of 20 hemolysis with antibiotic dose.
In Vivo Toxicity In vivo toxicity was determined for methyl ester of N-methyl-N-D-r~U~,Lu~rla..l,ul.uLcl;.,;.. B L-aspartate as the maximum tolerated dose (MTD), and as the acute toxicity (LDso). To determine MTD dose the compound was dissolved in 5%
solution of glucose, and a.ll~ d ~llLla~,.luL ~ly and ;llLla~J.l iLull~aly to Balb/c mice in single and multiple doses. .The maximum single tolerated dose was 100 mg/kg for the illLIa~,lluu~ and more than 200 mg/lcg for the ;. .~ .1, ";. ,~ ~ The maximum multiple tolerated dose ror i l~ a~ l; Lul l~al ~ l of l oo mgAcg for s days was much higher. For such dose, toxic effects were not observed during 20 days of observation.
The acute toxicity, LDso of methyl ester of N-methyl-N-D-fructosylamphotericin B
~ F~7\456\FI,SPECCANADA.01 2 1 7 6 8 9 3 L-aspa}tate was determined for Swiss Webster female mice of average weight of 20 g.
Various doses of the tested compound, and for ~,u.~ Vll, amphotericin B in form of Fungizone, dissolved in 5% glucose were given intravenously to the animals. The administered volume of the solution was 0.5 ml. 0.5 ml of 5 % solution of glucose was 5 administered to mice as a control. Every dose of both preparations was ~dlllilli~Lclcd to S mice. The animals were observed for 7 days. Next, the animals were killed, and some serum indexes were ~J~tP~rmin~d No increased level of aspartate al~lilwL-~ulartl~c or creatinine were found in comparison with the control. For methyl ester of N-methyl-N-D-fructosyl~..~ B L-aspartate, the LDso was found to be 400 mg/kg, while ror 10 ampllotericin B in form of Fungizone it was 6 mg/kg.
~t :- , " Efficacy (~hP m~ 11 ;r efficacy of methyl ester of N-methyl-N-D-fructosylamphotericin B L-aspartate was detemmined using systemic murine candidose model. Candida albicans was grown overnight in Sabouraud dextrose broth at room Ltlll~claLulc. The fungal cells 15 were ~ .-ntrifi1~, washed twice with 0.9 % solution of sodium chloride, and suspended in physiological salt solution. Female Swiss Webster mice of 25 g weight, were injected intravenously with 105 cells of Candida in 0.2 ml of 0.9 % sodium chloride solution.
Initially, the infection was systemic, but by 2 to 3 days it was locali~d to the kidneys.
Untreated animals usually died between 7 to 14 days post-infection. Three days 20 post-infection, animals were treated ;~L~ ,ly, twice a day, for 5 consecutive days with a 5 to 6 hours interval. Preparation was ad..l;l.; "I,lcd as a solution in 5% glucose. The animals were observed for 5 weeks starting from the day of infection. Afler this time, the surviving animals were sacrificed, tbeir kidneys were removed, homogenized in sterile water, and the llull~o~ . was plated on Sabouraud dextrose agar, and the grown colonies 25 of Candida were counted. The .1,..,..,l~ rrt~LiV~ a waS represented as a doseof mg kg, which in the above test resulted in a survival of 50 % of animals, and on clearance of Candida from kidneys of half of the mice. The dose, called EDso, was calculated using a method given in J. Hyg. 2:Z 493, (1938). The values of EDso for methyl ester of N-methyl-N-D-Lu~ ylal--pl~oLt-;cin B L-aspartate are 2.3 mg/kg based on the 30 survival, and 6 mg/kg based on the kidneys clearance.
r~ 561rLSrECCANADArl ~ 21 76~93 Preparative Examples N-Methyl-N-glycosyl derivatives of methyl esters of antibiotics of polyene macrolide group, their salts, and methods of preparation are illustrated by the examples given below.
Example 1.
S I g of Amrh~tPririn B (El%lCm = 1350 at 382 nm, MeOlLI) was dissolved in 15 ml of N,N-dimethylform~mi~lP, 0.3 g of D-glucose was added, and the mixture was stirred in darkness at 37'C for 40 hours. Next the reaction was cooled and a solid was precipitated with an excess of diethyl ether. The solid was rPntrifil~pr1~ washed twice with diethyl ether and dried under reduced pressure. To remove an excess of glucose the solid was suspended 10 in 20 ml of water, rPntrjfilgP~I, washed twice with small amount of water, twice with acetone, and next twice with diethyl ether. The product was dried under reduced pressure to give 0.98 g of N-D-fructu~y ~ l )tl "~; " B (El %l Cm = 1 2û0 at 3 82 nm, MeOH). The product was dissolved, with stirring, in 10 ml of N,N-Lli~ Ll~y~r~ lP and 50 ml of diethyl ether was added to the solution, to give a fine sl~irpn~irln The suspension was 15 cooled in ice to 0 - 2'C, and freshly prepared diethyl ether solution of 2.5 mole of 1A~ per I mole of N-D-rlu~u~ t~ . B, was added with vigorous stirring. The reaction was followed by thin layer chr(lm~t~\gr~rlly on silica gel in chlorofomm - methanol - water 10:6:1 v/v solvent system. After completion of the reaction, what took about 2 hours, the excess of 11i,,,..,,,..1,~.,~ and diethyl ether was evaporated 20 under reduced pressure at Lc~ laLul~: not higher than 40'C. The crude product was iLL ~,1 from the residue with an excess of diethyl ether, rPntrifil~P~1, washed twice with diethy~ ether, next with n-hexane, and dried under reduced pressure to give 0 95 g of the crude product. Pure N-methyl-N-D-rlucLu~ . B methyl ester was isolated from the crude product by column chromatography on Merck Silicagel 60, 70 -23û mesh in 25 chloroform - methanol - water 20: 8: I v/v solvent system. Thus, 0.95 g of the crude product was suspended in the mixture of solvents specified above, and if the product dissolved with difticulties, proportions of the same solvents were changed to 10: 6: 1 rhe undissolved part was centrifuged off, and the ~Illlrlll,ahlll charged on chrom~t-.~r~rhy column, next developed in tlle solvent mixture listed above, but in proportion 20: 8: I v/v.
30 The eluate was analysed on silica plates using chlorororm - methanol - water 10: 6: I v/v F\l~S~\rl5FeCCANAD~1 2, 7 6 ~ 9 3 solvent system. The plates were visualized with cerium sulphate reagent. Fractions of Rf = 0.5 - 0.54 containing pure N-methyl-N-D-fructosyl derivative of amphotericin B
methyl ester ~ere collected The combined fractions were evaporated under reducedpressure. The dry residue was dissolved in small amount of N,N-dimethylformamide, and 5 the product was ~u~ );LdL~d with an excess of diethyl ether, the solid was centrifuged, washed twice with diethyl ether and dried in avacuum desiccator. 0.137 g of N-methyl-N-D-rluu~u~ lpllotericin B methyl ester, El%lCm = 1300 on 382 nm in methanol, was obtained. The proof of structure was given above in the descriptive part. Antifungal activity of the compoumd against Cand~da albicans, determined as it was described above, 10 gave ICso = 0.12 llg/ml, and toxicity for human erythrocytes, determined as described above, gave EHso value higher than 350 llglml. For Cu~ ull~ EHs0 value for the starting amphotericin B was 1.5 ~Lg/ml. An exact value of EHso for N-methyl-N-D-fructosylamphotericin B methyl ester could not be irtFrminr~i, as above 350 lag/ml the compound was insoluble under conditions of the experiment.
15 Example II.
0.5 g of candidin (El%lCm = 1175 at 382 nm, MeOH) and 0.15 g of D-glucose were dissolvedinlOm~ofN,N-~;II.cLllylr."".,",~ andstirredat37~Cfor36hours. Further procedure was analogous to this of the Example I, and resulted in 0.43 g of N-D-fructosylcandidin, El%lCm = 1100 at 382 nm in MeOEI. The product was methylated 20 with .1i~ r in diethyl ether, analogously as in tlle Example I to give 0.4 g of crude product. Pure N-methyl-N-D-fructosylcandidin methyl ester was isolated by columnchromatography, by method similar to that given in the Example 1. Fractions containing pure derivative were ..l,",,,. lr"~r~l on thin layer chrf)mAt~rAphy by Rf = 0.49 - 0.52. The fractions were combined, evaporated to dryness, dissolved in small amount of 25 N,N-dimcLllylru~ ulli~lc, and solid was ~Ic~;~;Ld~d with diethyl ether. The solid was rrntrifi~ ri washed with diethyl ether and dried in vacuum desiccator to give 0.05 g of N-methyl-N-D-fructosylcandidin methyl ester; El%lCm = 1200 at 382 nm, in MeOEI, ICso = 0 75 ,ug ml and EHs0 above 300 ~Lg/ml. Structure of &e compound was determined by the same methods as for of N-methyl-N-D-fructosylamphotericin B methyl ester.
r\l3A~s6PLsP~AuADA~1 2 1 7 6 8 9 3 Example III.
2 g of nystatin (El%lcm = 870 at 304 nm, in MeOEI) and 0.6 g of D-glucose were dissolved in 35 ml of N,N-dimethylacetamide and stirred at 37~C for 40 hours. After completion of the reaction the crude product was precipitated with diethyl ether, S centrifuged, and dried umder vacuum. Then, an excess of glucose was washed off with small amount of water - acetone 1: I mixture, next with acetone, diethyl ether, and the product was centrifuged and dried in vacuum desiccator to give 1.2 g of N-D-rlu~,~u~yllly~LdLill (El%lcm = 720 at 304 nm, in MeOH). The product was methylated using-l;",.".,rll,,.,.~asintheExampleltogivel.25gofcrudeproduct. Theproductwas 10 purifled on silica gel column as in the Example 1. Fractions containing pure N-methyl-N-D-fructu~yllly 31d~ill methyl ester were ~1,, "- 1~ ~;, ~l on thin layer LIUl~ y by Rf =
0.49 - 0.52 as in the Example 1. The fractions were combined, evaporated to dryness at 30~C under reduced pressure, dissolved in small amount of N,N-dimethylfnrmAmi-lP, and product was ~ ;,LI;LdL~d with diethyl ether. The product was rPn~rifil~P~I, washed with 15 diethyl et~ler and dried in vacuum desiccator to give 0.17 g of N-methyl-N-D-fructosylnystatin methyl ester; El%lcm = 900 at 304 nm, in MeOEI. Structure of the compound was determined by a method described for N-methyl-N-D-rru~,Lu~ l ~ ., ,1 .l ,. ~tPriri n B methyl ester. For the obtained compound ICso = 6.8 llg/ml and EHs0 above 300 ~Lg/ml were found.
2û Example IV.
0.79 g of vacidin, the main component of antibiotic complex aureofacin, (E 1% 1 cm = 900 at 378 nm, in MeOH) and 0.22 g of D-glucose in 15 ml of N,N-dimethylacetamide were stirred at 37~C for 18 hours. After completion of the reaction the mixture was cooled and solid was precipitated with an excess of diethyl ether. The solid was rPntrifil~P~I, washed 25 with diethyl ether and dried under reduced pressure to give 0.8 g of crude N-D-fructosylvacidin (El%lcm = 720 at 378 nm, in MeOH). The obtained derivative was methylated using ~iiA7nmPthAnP, as in the Example I to give 0.5 g of crude product. Pure N-methyl-N-D-rlu~Lu~ a~;d;.- methyl ester was isolated by chromatography on silica gel column similarly as it was described in the Example 1, but the column was developed with 30 chloroform - methanol - water 30: 8: I solvent system. Fractions containing pure F~l371~6uL5rE~ANllD/~6l 2 1 7 6 8 9 3 derivative of vacidin and having on thin layer chrnm~tr~r~rhy value of Rf = 0.53 - 0.55 in chloroform - methanol - water 13: 8: I solvent system were collected and combined.
Further procedure was as in the Example Ill. 0.07 g of N-methyl-N-D-fructosylvacidin methyl ester was obtailled (El %I cm = 900 at 378 nm, in MeOH). The compound exhibited 5 ICso = 0.01 ,ug/ml and El 150 = 170 ,ug/ml. Structure of the compound was determined by a method described for N-methyl-N-D-fructosylamphotericin B methyl ester.
Example V.
0.79 g of candicidin D, the main component of antibiotic complex candicidin, ~vas treated identically as described in the Example IV. 0.1 g of N-methyl-N-D-fructosylcandicidin D
10 methylesterwasobtained; E1%1cm=920at378nm,inMeOH,Rfofthecompoundin thin layer ~ ".,.I"~,.3l,l.y, under conditions described in the Example IV, was 0.50 - 0.53.
The compound exnibited ICso = 0.01 ,ug/ml and EHs0 = 180 ,ug/ml. Structure of the compoundwasdeterminedbyamethoddescribedforN-methyl-N-D-r~u,~ ,yl~ )trl;l ;"
B methyl ester.
15 Example Vl.
0.79 g of the main component of antibiotic complex lly~llollly~;ll was treated identically as it was described in the Example IV. 0.1 g of N-methyl-N-fructosyltrychomycin methyl esterwas obtained; El%lcm = 910 at 378 nm, in MeOH Rfofthe compound in thin layer chrom~t~r~rhy, under conditions described in the Example IV, was 0.49 - 0.52. The 20 compound exhibited ICso = 0.013 ,ug/ml and Ells0 = 165 ug/ml. Structure of the compound was determined by a method described forN-methyl-N-D-fructosyl~mrh,-t~ ruin B methyl ester.
Example Vll.
0.5 g of a:ll,~JIl(.)~liLill B (El%lCm = 1350 at 382 nm, MeOH) and 0.15 g of L-glucose 25 in 8 ml of N,N-dimetnylformamide was stirred at 37~C for 40 hours. Subsequentoperations were as in the Example 1. 0.065 g of N-methyl-N-L-fructosylamphotericin B
methyl ester was obtained; El%lcm = 1280 at 382 nm, in MeOH. The compound exhibited ICso = 0.42 ug/ml and EHs0 = above 200 ug/ml. Structure of the compound r~ \PLS~E~/~ DAOI 2 1 7 6 8 9 3 was determined by a method described for methyl ester of N-methyl-N-D-rlu~ yla~ uLericin B.
Example VIII.
0.52gofamphotericinB(E1%1cm=1350at382nm,MeOll)andO.153gofD-mannose S was dissolved in 10 ml of N,N-di~ Lll~lr~,,.,,..,.i-lP and stirred at 37''C for 40 hours.
Subsequent operations were as in the Example 1. Pure derivative was isolated by silica gel column chrnm~to~r~rhy, in a manner analogous to that of the Example 1, fractions having in thin layer ~lu~llla~ lly, run according to the method described in the Example 1, value of Rf = 0.53 - 0.55 were collected. Subsequent operations were as in the Example 1.
10 0.08 g of N-methyl-N-D-fructosylall~ o~ ;ll B methyl ester was obtained;
El%lcm = 1280 at 382 nm. in MeOH. The compound exhibited ICs0 = 0.42 ,ug/ml and EHso above 200 ,ug/ml. Structure of tlle compound ~vas determined by a method described for N-methyl-N-D-fructosylamphotericin B methyl ester.
Example IX.
15 0.5 g of amphotericin B (El%lcm = 1350 at 382 nm, MeOH) and 0.3 g of D-lactose in 12 ml of N,N-dimethylr.).l",..,.i.lr was stirred at 37~C for two days. Subsequent operations were as in the Example 1. Pure derivative was isolated by silica gel columri chr ~m~t~-~r~rhy, in a manner analogous to that of the Example 1, fractions having in thin layer chromatography, run according to the method described in the Example 1, value of 20 Rf= 0.25 - 0.30 were collected. 0.08 g of N-methyl-N-D-fructosyl-gald~,Lv~yla.,lL,l.~ericin B methyl ester was obtained; El%lCm = 1000 at 382 nm, in MeOH. The compound exhibited ICso = 6.0 ,ug/ml and EHs0 above 200 ,ug/ml.
Example X.
0.5 g of N-methyl-N-D-rlu~o~y~ Lericin B methyl ester, prepared according to the25 Example 1, was suspended in 10 ml of water and 0.059 g of aspart;c acid dissolved in 2 ml of water was added. The solution of acid was added dropwise, with stirring to effect th~
solution. The solution was filtered to ren1ove a small amount of residual solid amd an excess of acetone was added to the clear filtrate until the whole salt was precipitated. The F\137~53VLSF~CCANADA01 ~ 2 1 76~93 solid was filtered offor r~-n~rifi~ l, washed twice with acetone, twice with diethyl ether and dried under reduced pressure, 0.5 g of methyl ester of N-methyl-N-D-rlu~L~ylcu~ elicin B L-aspartate, El%lcm = 1100 at 382 nrn, in MeOH, was obtained.
Thin layer chromatography under conditions as in the Exarnple I gave Rf= 0.5 - 054.
S Product was soluble in N,N-dimethylf )rm3~nni~, dimethylsulphoxide and ~% water solution of glucose. It was very well soluble in water. The compound exhibited ICso = 0.125 g/ml and EHso above 3~0 ,ug/ml.
Claims (56)
1. An N-alkyl-N-glycosyl derivative of antibiotics of the polyene macrolide group of general formula 1(a), wherein M represents residue of an antibiotic of polyene macrolide group, R represents a part of sugar residue formed by reaction of the antibiotic with a mono or oligosaccharide, R1 represents a C1-4 alkyl group and R2 represents hydrogen or a C1-4 alkyl group.
Formula 1(a)
Formula 1(a)
2. An N-alkyl-N-glycosyl derivative of antibiotics of the polyene macrolide group according to claim 1 wherein M is selected from amphotericin B, candidin, candidoin, candidinin, mycoheptin, nystatin, polyfungin, aureofacin, vacidin, trichomycin or candicidin.
3. An N-alkyl-N-glycosyl derivative of antibiotics of the polyene macrolide group according to either claim 1 or 2 wherein the mono or oligosaccharide from which the sugar residue R is derived is selected from D-glucose, or L-glucose, or D-mannose, or D-galactose, or lactose, or maltose.
4. An N-alkyl-N-glycosyl derivative of antibiotics of the polyene macrolide group according to any one of claims 1 to 3 wherein R1 and R2 are methyl groups.
5. An N-alkyl-N-glycosyl derivative of formula 1(a) according to any one of claims 1 to 3 wherein R1 is hydrogen.
6. An N-alkyl-N-glycosyl derivative according to claim 5 wherein R2 is a methyl group.
7. A Salt of an N-alkyl-N-glycosyl derivative of antibiotics of the polyene macrolide group of general formula 2(a) wherein M, R, R1 and R2 are defined according to any one of claims 1 to 6 and A represents an anion of an organic or inorganic acid.
Formula 2(a)
Formula 2(a)
8. A Salt of an N-alkyl-N-glycosyl derivative of antibiotics of the polyene macrolide group according to claim 7 wherein A is the anion of L-aspartic acid.
9. A method of preparation of an N-alkyl-N-glycosyl derivative of antibiotics of the polyene macrolide group as defined in any one of claims 1 to 6 which comprises the steps of reacting a polyene macrolide antibiotic with a mono or oligosaccharide, the reaction being characterised by the occurrence of a simultaneous Amadori rearrangement, to give the N-glycosyl derivative of the polyene macrolide antibiotics;
isolating the product of the Amadori rearrangement; treating the product with analkylating agent and purifying the crude product.
isolating the product of the Amadori rearrangement; treating the product with analkylating agent and purifying the crude product.
10. A method of preparation according to claim 9 wherein product of the Amadori rearrangement is isolated in the form of a suspension by precipitation from the solution in which the rearrangement occurs.
11. A method of preparation according to claim 9 or claim 10 wherein N,N-dimethylformamide is used to support the Amadori rearrangement.
12. A method of preparation according to any one of claims 9 to 11 wherein diethyl ether is used to effect formation of a suspension by precipitation of the product of the Amadori rearrangement.
13. A method of preparation according to any one of claims 9 to 12 wherein alkylation of the product of the Amadori rearrangement is carried out at reduced temperature.
14. A method of preparation according to claim 12 wherein alkylation is carried out at temperatures of between -5°C and +5°C.
15. A method of preparation according to any one of claims 9 to 14 wherein the alkylating agent is an ethereal solution of diazomethane.
16. A method of preparation compounds of formula 1 (a) wherein M, R, R1 and R2 are as defined above for the first to third aspects of the invention and which comprises the steps of reacting a polyene macrolide antibiotic with a species which provides a protecting group to give a first N-protected (amino) derivative; reacting said first N-protected derivative with a further protecting group providing species therebyprotecting the carboxy group of the macrolide to give a second N-protected-carboxy-protected derivative; reacting said second derivative with an alkylating agent to give the N-alkyl N-protected-carboxy-protected derivative; removing the N-protecting group and reacting the resulting N-alkyl derivative with a mono-, di- or oligosaccharide, the reaction being characterised by the occurrence of a simultaneous rearrangement, to give the N-alkyl-N-glycosyl derivative; removal of the carboxyprotecting group from said N-alkyl-N-glycosyl derivative followed by the required derivatisation of said carboxy group.
17. A method according to claim 17 wherein the N-protecting (amino) group is a trifluoroacetyl group.
18. A method according to claim 18 wherein the N-protecting trifluoroacetyl group is derived from trifluoroacetic anhydride and trifluoroacetic acid.
19. A method according to any one of claims 17 to 19 wherein carboxy protecting groups are labile ester groups.
20. A method according to claim 20 wherein the carboxy protecting group is derived from benzyl esters, p-methoxybenzyl esters and t-butyl ester.
21. A method according to any one of claims 17 to 21 wherein the alkylating agents are selected from dialkyl sulphates and alkyl iodides.
22. A method according to claim 22 wherein the alkylating agents are selected from dimethyl sulphate and methyl iodide.
23. A method according to any one of claims 17 to 23 wherein removal of the N-protecting group is effected by mildly acidic conditions.
24. A method according to any one of claims 17 to 24 wherein removal of the carboxy protecting groups is effected by mildly acidic condition to give the corresponding carboxylic acid.
25. A method according to any one of claims 17 to 25 wherein derivatisation of the carboxy group is effected by reaction of the carboxylic acid formed according toclaim 24 with a corresponding C1-4 alcohol.
26. A method of preparation of a salt of an N-alkyl-N-glycosyl derivative of the polyene macrolide antibiotics of general formula 2(a) as defined in claim 7 or claim 8 which comprises the steps of suspending an N-alkyl-N-glycosyl derivative prepared according to any one of claims 9 to 25 in sufficient water to effect formation of a homogeneous suspension, acidifying the resulting suspension and isolating the product.
27. A method of preparation of a salt of an N-alkyl-N-glycosyl derivative of the polyene macrolide antibiotics according to claim 26 wherein organic or inorganic acids are used to acidify the suspension.
28. A method of preparation of a salt of an N-alkyl-N-glycosyl derivative of the polyene macrolide antibiotics according to claim 27 wherein L-aspartic acid is used to acidify the suspension.
29. A method of preparation of a salt of an N-alkyl-N-glycosyl derivative of the polyene macrolide antibiotics according to any one of claims 26 to 28 wherein isolation of the pure product is effected by precipitation of the crude product with an organic solvent followed by subsequent washing with an additional appropriate solvent and drying.
30. A method of preparation of a salt of an N-alkyl-N-glycosyl derivative of the polyene macrolide antibiotics according to any one of claims 26 to 29 wherein the solvent used to precipitate the crude product is miscible with water.
31. A method of preparation of a salt of an N-alkyl-N-glycosyl derivative of the polyene macrolide antibiotics according to claim 30 wherein the solvent is acetone.
32. A method of preparation of a salt of an N-alkyl-N-glycosyl derivative of the polyene macrolide antibiotics according to any one of claims 29 to 31 wherein the additional appropriate solvents employed for washing the product are selected from acetone and diethyl ether.
33. A method of preparation of a salt of an N-alkyl-N-glycosyl derivative of the polyene macrolide antibiotics according to any one of claims 26 to 32 wherein the product is dried under reduced pressure.
34. An N-alkyl-N-glycosyl derivative of formula 1 (a) or a salt thereof according to any one of claims 1 to 8 for use in therapy.
35. A method for the treatment of fungal infections in humans and animals which comprises the administration thereto of an N-alkyl-N-glycosyl derivative of formula 1 (a) or a salt thereof according to any one of claims 1 to 8.
36. A method for the treatment of fungal infections in humans and animals which comprises the administration thereto of an N-methyl-N-glycosyl derivative of formula 1(a) or a salt thereof according to any one of claims 1 to 8.
37. A composition for use in the treatment of fungal infections comprising an N-alkyl-N-glycosyl derivative of formula 1(a) or a salt thereof according to any one of claims 1 to 8 and a physiologically acceptable carrier.
38. A unit dosage form comprising one or more N-alkyl-N-glycosyl derivatives of formula 1(a) or salts thereof according to any one of claims 1 to 8 and a physiologically acceptable carrier formulated for pharmaceutical or veterinary use.
39. A composition for use in the treatment of fungal infections according to claim 37 or a unit dosage form according to claim 38 wherein the N-alkyl-N-glycosyl derivative of formula 1(a) or the salt thereof includes N-methyl-N-glycosyl derivative of the methyl esters of formula 1(b) or the salts thereof.
40. A composition or unit dosage form according to any one of claims 37 to 39 which further comprises a known anti-fungal agent.
41. A composition or unit dosage form according to any one of claims 37 to 40 which is formulated for intravenous, intra peritoneal, oral, topical, subcutaneous, rectal or vaginal administration.
42. Use of an N-alkyl-N-glycosyl derivative of formula 1(a) or a salt thereof according to any one of claims 1 to 8 for the preparation of a medicament for use in the treatment of fungal infections.
43. Use according to claim 42 wherein the N-alkyl and alkyl ester substitutents are both methyl groups.
44. Use of an N-alkyl-N-glycosyl derivative of formula 1(a) or a salt thereof according to any one of claims 1 to 8 for the treatment of fungal infections.
45. Use according to claim 44 wherein the N-alkyl and alkyl ester substituents are both methyl groups.
46. Use according to claim 44 or claim 45 wherein the fungal infection is external or internal.
47. An N-Methyl-N-glycosyl derivative of a methyl ester of antibiotics of the polyene macrolide group presented by general formula 1(b), wherein M represents residue of an antibiotic of polyene macrolide group, wherein R represents a part of sugar residue formed by reaction of the antibiotic with mono or oligosaccharide, preferably with D-glucose, or L-glucose, or D-mannose, or D-galactose, or lactose, or maltose, and by simultaneous Amadori rearrangement.
Formula 1(b)
Formula 1(b)
48. Derivative according to claim 47, wherein the antibiotic of polyene macrolide group is amphotericin B, or candidin, or candidoin, or candidinin, or mycoheptin, or nystatin, or polyfungin, or aureofacin, or vacidin, or trichomycin or candicidin.
49. A Salt of an N-methyl-N-glycosyl derivative of antibiotics of the polyene macrolide group presented by general formula 2(b) wherein M represents residue of an antibiotic of polyene macrolide group, wherein R represents a part of sugar residue formed by reaction of the antibiotic with mono or oligosaccharide, preferably with D-glucose, or L-glucose, or D-mannose, or D-galactose, or lactose, or maltose, and by simultaneous Amadori rearrangement, and A represents an anion of organic or inorganic acid.
Formula 2(b)
Formula 2(b)
50. Salts, according to claim 49, wherein the antibiotic of polyene macrolide group is amphotericin B, or candidin, or candidoin, or candidinin, or mycoheptin, or nystatin, or polyfungin, or aureofacin, or vacidin, or trichomycin or candicidin.
51. Salts according to claims 49 or 50, wherein A relates to the anion of L-aspartic acid.
52. A process for preparation of an N-methyl-N-glycosyl derivative of a methyl ester of antibiotics of the polyene macrolide group presented by general formula 1(b), wherein M represents residue of an antibiotic of polyene macrolide group, wherein R
represents a part of sugar residue formed by reaction of the antibiotic with mono or oligosaccharide, preferably with D-glucose, or L-glucose, or D-mannose, or D-galactose, or lactose, or maltose, and by simultaneous Amadori rearrangement, wherein the obtained by Amadori rearrangement N-glycosyl derivative of antibiotics of polyene macrolide group are transformed into suspension by precipitation withsolvent, preferably diethyl ether, from a solution of the derivative in organic solvent, preferably in N,N-dimethylformamide, and subsequently treated with ethereal solution of diazomethane at lowered temperature, preferably in the range from -5°C
to +5°C, stirred, and isolated by evaporation of solvents, and precipitation from the concentrated solution, preferably by excess of diethyl ether, the crude product is purified according to known procedures.
represents a part of sugar residue formed by reaction of the antibiotic with mono or oligosaccharide, preferably with D-glucose, or L-glucose, or D-mannose, or D-galactose, or lactose, or maltose, and by simultaneous Amadori rearrangement, wherein the obtained by Amadori rearrangement N-glycosyl derivative of antibiotics of polyene macrolide group are transformed into suspension by precipitation withsolvent, preferably diethyl ether, from a solution of the derivative in organic solvent, preferably in N,N-dimethylformamide, and subsequently treated with ethereal solution of diazomethane at lowered temperature, preferably in the range from -5°C
to +5°C, stirred, and isolated by evaporation of solvents, and precipitation from the concentrated solution, preferably by excess of diethyl ether, the crude product is purified according to known procedures.
53. A process for preparation of a salt of an N-methyl-N-glycosyl derivative of a methyl ester of antibiotics of the polyene macrolide group presented by general formula 1(b), wherein M represents residue of an antibiotic of polyene macrolide group, wherein R represents a part of sugar residue formed by reaction of the antibiotic with mono or oligosaccharide, preferably with D-glucose, or L-glucose, or D-mannose, or D-galactose, or lactose, or maltose, and by simultaneous Amadori rearrangement, wherein the obtained by Amadori rearrangement N-glycosyl derivative of antibiotics of polyene macrolide group are transformed into suspension by precipitation withsolvent, preferably diethyl ether, from a solution of the derivative in organic solvent, preferably N,N-dimethylformamide, and subsequently treated with ethereal solution of diazomethane at lowered temperature, preferably in the range from -5°C to +5°C, stirred, and isolated by evaporation of solvents, and precipitation from the concentrated solution, preferably by an excess of diethyl ether, the crude product is purified according to known procedures, subsequently, the obtained derivative, as a solid, is suspended in small amount of water, and stoichiometric amount of organic or inorganic acid is added, next the product is precipitated from the formed solution by an excess of organic solvent miscible with water, preferably acetone, the solid is washed, preferably with acetone and subsequently preferably with diethyl ether, and dried, preferably under reduced pressure.
54. A method for treatment of external and internal fungal infections in humans and animals, wherein an N-methyl-N-glycosyl derivative of a methyl ester of antibiotics of the polyene macrolide group presented by general formula 1(a) 1, wherein M
represents residue of an antibiotic of polyene macrolide group, wherein R represents a part of sugar residue formed by reaction of the antibiotic with mono or oligosaccharide, preferably with D-glucose, or L-glucose, or D-mannose, or D-galactose, or lactose, or maltose, and by simultaneous Amadori rearrangement are used to treat the infections.
represents residue of an antibiotic of polyene macrolide group, wherein R represents a part of sugar residue formed by reaction of the antibiotic with mono or oligosaccharide, preferably with D-glucose, or L-glucose, or D-mannose, or D-galactose, or lactose, or maltose, and by simultaneous Amadori rearrangement are used to treat the infections.
55. A method for treatment of external and internal fungal infections in humans and animals, wherein a salt of an N-methyl-N-glycosyl derivative of a methyl esters of antibiotics of the polyene macrolide group presented by general formula 2(b), wherein M represents residue of an antibiotic of polyene macrolide group, wherein R
represents a part of sugar residue formed by reaction of the antibiotic with mono or oligosaccharide, preferably with D-glucose, or L-glucose, or D-mannose, or D-galactose, or lactose, or maltose, and by simultaneous Amadori rearrangement, and A represents an anion of organic or inorganic acid are used to treat the infections.
represents a part of sugar residue formed by reaction of the antibiotic with mono or oligosaccharide, preferably with D-glucose, or L-glucose, or D-mannose, or D-galactose, or lactose, or maltose, and by simultaneous Amadori rearrangement, and A represents an anion of organic or inorganic acid are used to treat the infections.
56. A method of treatment of fungal infection which comprises the perfusion of a compound of formula 1(a) or a salt thereof into the body of a patient suffering therefrom.
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| CA002176893A CA2176893A1 (en) | 1996-05-17 | 1996-05-17 | Antibiotics |
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-
1996
- 1996-05-17 CA CA002176893A patent/CA2176893A1/en not_active Abandoned
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| FZDE | Discontinued |