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WO1992007862A1 - Antibiotiques ameliores par conjugaison avec des glucides stereospecifiques et procedes de stereoselectivite de glucides - Google Patents

Antibiotiques ameliores par conjugaison avec des glucides stereospecifiques et procedes de stereoselectivite de glucides Download PDF

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WO1992007862A1
WO1992007862A1 PCT/US1991/008288 US9108288W WO9207862A1 WO 1992007862 A1 WO1992007862 A1 WO 1992007862A1 US 9108288 W US9108288 W US 9108288W WO 9207862 A1 WO9207862 A1 WO 9207862A1
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Samuel J. Danishefsky
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Yale University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/20—Carbocyclic rings
    • C07H15/24—Condensed ring systems having three or more rings
    • C07H15/252—Naphthacene radicals, e.g. daunomycins, adriamycins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D309/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings
    • C07D309/16—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member
    • C07D309/28—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D309/30—Oxygen atoms, e.g. delta-lactones
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D309/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings
    • C07D309/32—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members
    • 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

Definitions

  • This invention relates to antibiotics, and particularly to glycoside antibiotics, as well as to methods of synthesizing carbohydrates.
  • Oligosaccharides are also an integral part of the molecular structure of drugs such as certain classes of antibiotics and steroidal hormones. Investigations of such drugs and their possible mechanisms have been reported by Arcamone, F., et al . , J. Med . Chem. 19:733 (1976); Acton, E.M., et al . , J. Med. Chem. 29:2074 (1986); Israel, M., et al . , J. Med. Chem. 25:24 (1982); Quigley, G.J., et al .
  • Anthracycline antibiotics are a prime example. As in the case of most antibiotics, however, there has been extensive research on the bioorganic and biophysical chemistry of the aglycone sectors of anthracyclines and on the interaction between the aglycone sectors and oligonucleotides, and little or none on the carbohydrate domains.
  • the present invention resides in part in the discovery that antibiotics, and particularly anthracycline antibiotics, can be improved, controlled or modulated in terms of their efficacy, drug resistance, and other properties directed to or related to their therapeutic utility, by the addition, selection and/or control of carbohydrate domains.
  • Carbohydrate domains of preselected structure and stereospecificity to be joined to the aglycones of interest are either naturally derived or are made available by the use of synthetic chemical methods which produce the isolated species of interest in high yields.
  • These methods include the placement of carbohydrate ring substituents at preselected locations and in preselected stereochemical orientations in high yields when desired, and the joinder of carbohydrate rings at preselected linkage sites in high yields to form saccharide multimers when desired.
  • This discovery is implemented in accordance with this invention by conjugating aglycones with carbohydrate domains, the aglycones being either naturally derived, chemically synthesized from non-antibiotic starting materials, or prepared from naturally occurring glycosides by hydrolytic removal of the glycoside portions, and the carbohydrate domains being of preselected structure which are either entirely or substantially pure in terms of stereospecific configuration.
  • the invention resides still further in a novel class of anthracycline antibiotics bearing stereospecific glycoside domains which generate improvements in beneficial properties to the antibiotics, in terms of either cytotoxicity, drug resistance, or other properties directed to or related to their therapeutic utility, or combinations of such properties. Detailed descriptions of this class are given below.
  • the resolution of stereoisomers of saccharide monomers referred to above as synthetic method (i) is, in more specific terms, the isolation of a selected enantiomer from a mixture of enantiomers of a cyclic ether, the cyclic ether being one which bears a hydroxyl group substitution on the ring in alternate orientations which define the enantiomeric forms.
  • Isolation of the selected enantiomer from the enantiomeric mixture is achieved by first contacting the mixture with an acetylating agent in the presence of a lipase.
  • the acetylating agent selectively acetylates the hydroxyl group on one of the enantiomers, resulting in an acetylation product mixture which contains one of the enantiomers in acetylated form and the other in nonacetylated form.
  • the desired enantiomer is then recovered from the acetylation product mixture by any of a wide variety of conventional separation methods based on the difference in properties attributable to the acetylation.
  • the resolution is of particular utility when the ring bears substituents in addition to the hydroxyl substituent.
  • the particular enantiomer which is acetylated in the acetylation reaction will vary depending on the substituents of the cyclic ether and the orientation of these substituents, including that of the hydroxyl group, with respect to each other and the ring.
  • the method of isolation is independent of which enantiomer is acetylated, since once the enantiomers are separated from each other and identified by conventional analytical methods, the desired enantiomer is readily selected.
  • the cyclic ether is a glycal, i.e., a cyclic enol ether in which the double bond joins the carbons at positions 1 and 2 of the ring.
  • glycals those forming five- and six-membered rings are particularly preferred, with six-membered rings the most preferred.
  • the enantiomer which is acetylated is generally that in which the hydroxyl group is in the ⁇ -orientation relative to the ring. Other patterns of consistent behavior are detectable by routine experimentation.
  • Synthetic methods (ii) and (iii) listed above may be conducted according to a variety of reaction schemes or protocols.
  • the resulting saccharide multimers have a predetermined stereochemical configuration both in terms of glycosidic bonds and in terms of individual saccharide substituents.
  • two glycals are joined in conjunction with a halogen substitution to form a halo- substituted glycosyl glycal, and, depending on the length of the multimer ultimately sought, the reaction is repeated a sufficient number of times to add further glycals in sequence.
  • one glycal (which may be termed the "add-on” glycal) has a plurality of substituent groups and a single reactive (nucleophilic) hydroxyl group, while the other (which may be termed the "starting" glycal) has non-participating electron donating groups.
  • starting and add-on are used herein in descriptions of all of the methods, as indications of the direction of chain growth for saccharide multimers of three or more units, and for differentiation between reacting species in reaction systems in which one such species is immobilized by linkage to a solid phase prior to the reaction.
  • the substituents on the addon glycal are electron-withdrawing relative to the substituents on the starting glycal, and the haloglycosylation is carried out in the presence of a halonium ion reagent and in the absence of water.
  • the reactive hydroxyl of the add-on glycal is joined to the starting glycal across the double bond of the starting glycal, thereby forming the glycoside bond.
  • the halo-substituted glycosyl glycal product becomes the new starting glycal (which is actually a glycal-terminated dimer), and is reacted with a new add-on glycal having a plurality of substituent groups and a single reactive hydroxyl group.
  • a new add-on glycal having a plurality of substituent groups and a single reactive hydroxyl group.
  • one of the electron-withdrawing substituents is removed from the glycal portion of the new starting glycal and is replaced with an electron-donating substituent.
  • the substituents on the new addon glycal will then be electron-withdrawing relative to the substituents on the glycal portion of the new starting glycal, and the haloglycosylation reaction will proceed as before.
  • the cycle is repeated a sufficient number of times to achieve the desired number of saccharide units, and in each case the halogen will assume the position on the carbon atom adjacent to the glycoside bond (formerly the no. 2 carbon terminus of the double bond of the glycal moiety), and will be in a trans relation (i . e. , transequatorial) to the glycoside bond.
  • Any of the products or intermediates can be dehalogenated as (and if) desired by conventional techniques.
  • an add-on glycal is joined to a nucleophilic molecule linked to a solid phase.
  • the nucleophilic molecule has a single reactive hydroxyl group, and the linkage reaction is performed in a liquid medium free from other nucleophilic groups, including water.
  • the add-on glycal and a halonium ion reagent both dissolved or dispersed in the liquid medium in about equimolar amounts and in excess relative to the moles of nucleophilic hydroxyl groups linked to the solid phase, are contacted with the solid phase.
  • the product of the reaction is a newly formed glycoside bond joining the solid phase with what was formerly the add-on glycal and is now a saccharide residue.
  • This newly formed solid-phase glycoside has a halide group at the 2-position of the added glycosyl ring that is trans to the adjacent (newly formed) glycoside bond.
  • the solid and liquid phases are readily separated.
  • reaction occurs at the double bond of the add-on glycal, and the reacting hydroxyl group is on the starting species to which the add-on glycal is added (i.e., the solid phase-linked species).
  • the solid phase-linked nucleophilic molecule assumes the role of the "starting" material, and the liquid- phase glycal is the "add-on.”
  • the solid phase-linked nucleophile molecule may itself be a saccharide residue (and hence a "starting" saccharide), and the above-described reaction will then result in a solid phase-linked disaccharide.
  • This solid phase-linked disaccharide may then serve as a new starting saccharide, to which further add-on glycals may be joined, thereby forming trisaccharides and higher saccharide multimers.
  • the first add-on glycal preferably includes a protected hydroxyl group whose protecting moiety is selectively removable over any other substituent present, i . e. , without affecting such other substituents.
  • exemplary of selectively removable protecting moieties are acyl and trisubstituted silyl groups. Selective removal of such a protecting moiety (deprotection) forms a particle-linked glycoside having a free nucleophilic hydroxyl group suitable for reaction with the new add-on glycal and halonium ion reagent.
  • the reaction proceeds in the same manner as the first stage glycosylation, followed by phase separation.
  • deprotection can then be performed, followed once again by haloglycosylation and phase separation steps, and the cycle repeated until a solid phase-linked saccharide multimer of the desired length is achieved.
  • this method results in a halogen substitution on each saccharide unit, adjacent to and trans to the glycoside bond.
  • the halogen may be removed as and if desired at any stage of the procedure.
  • a third method avoids the halogen substitution in the formation of the glycoside bond.
  • the starting glycal is first converted to a 1,2-anhydrosugar, which is defined as a glycal in which the double bond has been converted to a 1,2-epoxide linkage. Conversion of the glycal to the corresponding 1,2-anhydrosugar is achieved by known methods, preferably by epoxidation with a dialkyl dioxirane having a total of two to about six carbon atoms in the two alkyl groups.
  • the 1,2-anhydrosugar is one which contains only non- participating substituents, and, once formed, is reacted with a hydroxyl group on the add-on glycal, the hydroxyl group being the sole reactive, nucleophilic hydroxyl group on the add-on glycal.
  • Other nucleophilic atoms such as nitrogen or sulfur may be used in place of the hydroxyl group. In either case, this reaction occurs in the presence of a Lewis acid catalyst and in the absence of water.
  • the result is the opening of the epoxide ring to form a glycoside bond between the add-on glycal and the starting species, and thus a new glycal-terminated starting species suitable for a new cycle of conversion to a 1,2- anhydrosugar followed by glycosylation to attach a new add-on glycal.
  • the cycle is repeated a sufficient number of times to result in a glycal-terminated saccharide multimer of the desired number of saccharide units.
  • Substituted sugar derivatives other than glycals can also be used in place of the add-on glycal.
  • the result will be a saccharide multimer in which the terminal unit is a saccharide residue other than a glycal.
  • One of various ways of implementing this method is to first link the starting glycal to a solid phase, and then converting the resultant solid phase-linked glycal to a solid phase-linked 1,2-anhydrosugar.
  • the glycosylation reaction is then performed by contacting the solid phase with a liquid medium containing an add-on species meeting the above description.
  • the use of an add-on glycal at each stage which is appropriately substituted will permit the cycle of conversion and glycosylation to be repeated a sufficient number of times to achieve the resulting saccharide multimer.
  • the final add-on species need not be a glycal.
  • the "starting" species is the species bearing the reactive hydroxyl group which takes part in the glycosylation
  • the "add-on” species is the glycal which is converted to the 1,2-anhydrosugar to prepare for the glycosylation.
  • This has significance not only in the direction of multimer growth in repeated cycles of the reaction, but also in the use of a solid phase.
  • the solid phase-linked species in this variation is the species bearing the reactive hydroxyl group rather than the glycal to be converted to the 1,2-anhydrosugar. Otherwise, the reaction is conducted in the same manner in both cases.
  • a hydroxyl group is formed at the 2-position of the glycosyl ring which is formed from the 1,2- anhydrosugar, and that hydroxyl group is preferably protected prior to carrying out any further glycosylation reactions, and maintained in the protected state during subsequent deprotection reactions which may be conducted to permit further glycosylations.
  • the 1,2-anhydrosugar intermediate is the add-on species, it is preferred that one of the non-participating substituents of the substituted 1,2- anhydrosugar is a protected nucleophilic hydroxyl group whose protecting moiety can be removed in a selective manner with respect to any other substituent present.
  • protecting moieties are trisubstituted silyl moieties such as trimethylsilyl.
  • An example of a protecting moiety which can be left intact during deprotection of a trisubstituted silyl moiety is a benzyl group. Thus deprotected, the nucleophilic hydroxyl group is available for reaction with a new 1,2-anhydrosugar in a new glycosylation cycle.
  • sugar and sugar derivative are used generically to indicate a carbohydrate or carbohydrate derivative consisting of one or more units linked together, each unit containing 5-9 atoms in its backbone chain.
  • Single unit sugars are referred to as “simple sugars,” while multi-unit sugars are referred to as “compound sugars.”
  • Simple sugars of interest herein include pentoses, hexoses, heptoses, octuloses and nonuloses.
  • oligosaccharide is a compound sugar or sugar moiety that yields two to ten monosaccharides upon hydrolysis.
  • a "polysaccharide” is a compound sugar or sugar moiety that yields more than ten monosaccharides on hydrolysis.
  • saccharide multimer encompasses both oligosaccharides and polysaccharides.
  • Saccharide residues of greatest interest in this invention are those which are in cyclic form as cyclic ethers, and are identified herein by nomenclature conventionally used among carbohydrate chemists. Position numbering for these cyclic ethers begins with the ring carbon adjacent to the ethereal oxygen, and in cases where a non-cyclic carbon is included, such as in the common cyclic simple sugars, the starting carbon is on the side opposite that of the non-cyclic carbon.
  • An example of the numbering scheme for a six-membered ring is as follows:
  • the same numbering scheme is used for non-cyclic residues, the starting carbon being the aldehydic carbon for aldoses and the terminal carbon closest to the keto group for ketoses.
  • the carbon atom at position 1 is also referred to as the "anomeric atom” or “anomeric carbon atom” due to the possible formation of anomers at that position.
  • Alpha (a) anomers bear a hydroxyl group or substituent extending below the plane of the ring as it is usually drawn, whereas in beta ( ⁇ ) anomers, the substituent extends above the plane of the ring when so drawn.
  • beta ( ⁇ ) anomers bear in likewise manner to substitutions at other ring positions as well.
  • glycoside bond and "glycosyl bond” are used equivalently herein to designate a covalent single bond between the anomeric carbon of a saccharide residue and an oxygen, nitrogen or sulfur atom of another moiety.
  • the latter may be a substituent of another saccharide residue, and in many cases is the oxygen of the hydroxyl substituent at the 4-position of a six-membered cyclic saccharide residue.
  • glycoside refers to a molecule to which one or more saccharide residues, including oligo- and polysaccharides, are covalently bonded through a glycoside bond.
  • glycosylation refers to the formation of a glycosyl bond. In reactions to form such bonds, one molecule provides the anomeric carbon atom of the glycosyl bond, and another, a nucleophile molecule, provides a nucleophilic atom that bonds to the anomeric carbon atom.
  • the molecule providing the anomeric atom is referred to as a "glycosyl donor,” while the nucleophile molecule is referred to as a "glycosyl acceptor.”
  • glycal designates a cyclic enol ether derivative of a sugar having a double bond between carbon atoms at positions 1 and 2 of the ring.
  • Preferred glycals are those containing a chain of 5-9 carbon atoms, 4, 5 or 6 of which are members of the ring together with the ethereal oxygen.
  • An example of a glycal with a six-membered ring structure is as follows:
  • 1,2-anhydrosugar is a sugar derivative containing a cyclic saccharide residue having an epoxide linkage between the carbon atoms of the 1- and 2-positions of a cyclic residue.
  • Disaccharides are named as glycosyl glycosides for nonreducing disaccharides, and as glycosyl glycoses for reducing disaccharides. Larger oligosaccharides are similarly named -- hence the terms glycosyl glycosyl glycoside for a nonreducing trisaccharide, glycosyl glycosyl glycosyl glycoside for a nonreducing tetrasaccharide, glycosyl glycosyl glycose for a reducing tetrasaccharide, and the like.
  • the product of a glycosylation reaction is often referred to as a "derivative" such as a glycosyl derivative or a glycoside derivative.
  • glycosyl donor and glycosyl acceptor terminology explained above may also be applied to oligosaccharides.
  • a disaccharide is named by first reciting the glycosyl donor and then the glycosyl acceptor.
  • a longer oligosaccharide is named by first reciting the first glycosyl donor and then the first acceptor.
  • the first glycosyl acceptor or the glycosylated first glycosyl acceptor can also be viewed as a glycosyl donor for the third saccharide unit and so on through the chain to the last saccharide unit.
  • a "glycal-terminated" oligosaccharide is an oligosaccharide containing a glycal derivative as its last saccharide unit.
  • the anomeric carbon atom is thus free from a glycosidic bond to another saccharide unit.
  • a substituted 1,2-anhydrosugar or sugar derivative can also occupy a "terminal" position of an oligosaccharide.
  • 1,2-halonium species intermediate refers to an adduct formed by combining an electrophilic halogenating reagent (electrophile) and a glycal double bond.
  • electrophilic halogenating reagent electrophilic halogenating reagent
  • glycal double bond The reaction to form this adduct is generally referred to as a "haloglycosylation.”
  • antibiotics which do not contain carbohydrate domains. This term includes, but is not limited to, nonsugar products of the hydrolysis of a glycoside.
  • anthracyclinones a subclass of aglycones
  • the following numbering system which is the numbering system used in the anthracycline literature, is used herein:
  • aliphatic includes all hydrocarbyl moieties which are neither cyclic nor aromatic, but does include straight- and branched-chain groups.
  • alkyl refers to saturated aliphatics and also includes both straight- and branched-chain groups, and "lower alkyl” designates C 1 -C 4 alkyl groups. Examples are methyl, ethyl, propyl, isopropyl, n-butyl and t-butyl.
  • Prime examples of "aryl” groups are phenyl and naphthyl.
  • Prime examples of aralkyl groups are benzyl and phenylethyl. Other examples will be readily apparent to the skilled chemist.
  • the role of the carbohydrate domain in glycoside antibiotics is established. Antibiotics of improved and controlled properties are thus obtained by selection of a specific carbohydrate domain, whether it be a monosaccharide or a saccharide multimer, and conjugation of the carbohydrate to an appropriate nucleus, whether it be a naturally occurring aglycone, an aglycone synthesized from a naturally occurring glycoside by hydrolysis, or an aglycone synthesized entirely from simpler starting materials, or any other type of nonsugar-containing drug.
  • the exact nature of the nucleus, which term is used herein to refer to the nonsugar antibiotic molecule to which the carbohydrate domain is attached, is not critical and may vary widely. Nuclei of. particular interest are anthracyclinone nuclei, which include both naturally occurring or otherwise known anthracyclinones as well as aglycones of naturally occurring or otherwise known anthracyclines.
  • anthracyclinones examples include adriamycinones, rhodomycinones, isorhodomycinones, pyrromycinones, aklavinones, daunomycinones, nogalamycinones, steffimycinones, isoquinocyclinones, and trypanomycinones, each type including isomers and analogs differing by the presence and position of substituents on the anthracyclinone structure.
  • those which have been found to be particularly susceptible to improvement by carbohydrate conjugation are adriamycinone and n -pyrromycinone.
  • the carbohydrate domains to be conjugated to the aglycones may vary widely in structure and character.
  • the carbohydrate domain consists of one or more saccharide units in cyclic ether form.
  • preferred structures are linear chains linked by O-glycoside bonds.
  • Preferred carbohydrate domains contain from 1 to 3 saccharide units, inclusive. Preferred such units are those containing 5- or 6-membered rings, a 5-membered ring containing one oxygen atom and four carbon atoms, and a 6-membered ring containing one oxygen atom and five carbon atoms. Most preferred are 6-membered rings.
  • saccharide units which serve individually as the carbohydrate domain or combined with other saccharide units as a saccharide multimer carbohydrate domain are glucose, galactose, altrose, arabinose, ribose, xylose, fructose, mannose, allose, talose, idose, gulose, lyxose, threose, and rhodinose, in either the D- or L- configuration for those units capable of both.
  • the saccharide units will have a stereospecific configuration selected to provide optimal activity enhancement of the antibiotic. The configuration will be stereospecific either in terms of the saccharide unit itself (due to substitutions on the ring), or between two saccharide units relative to each other (due to the orientation of the glycoside bond joining the units), or both.
  • the saccharide units will in some cases bear substituents which are not present on naturally occurring analogs of the units. The identity and orientation of these substituents will be selected to provide optimal activity enhancement of the antibiotic. Examples of such unnatural substituents are alkyl groups, halogen atoms, amino groups (optionally substituted), and oxo substitutions. Carbohydrates which have been found to be particularly active are those in which at least one substituent is a halogen atom, preferably either a chlorine, bromine or iodine atom, and most preferably an iodine atom.
  • Preferred substituents, both natural and unnatural, on the carbohydrate domain in addition to the halogen atom include lower alkyl, phenyl and hydroxyl.
  • Preferred alkyl groups are methyl and ethyl, with methyl the most preferred.
  • the carbohydrate domain is joined to the antibiotic nucleus through a covalent bond, preferably a glycoside bond, and most preferably an O-glycoside bond utilizing an oxygen atom which was the oxygen of a nucleophilic hydroxyl group on the aglycone prior to the conjugation.
  • the ring substituents in cyclic saccharide units, the ring substituents, whether natural or unnatural, which are joined to the ring by single bonds may be either coequatorial or transequatorial with respect to each other.
  • Preferred structures are those in which the substituents on a single ring are coequatorial. This refers to substituents other than the glycoside bond which connects the ring to the aglycone portion of the molecule, and likewise to substituents other than glycoside bonds which connect adjacent rings. It is preferred that the ring substituents on the ring nearest the aglycone portion are transequatorial with the glycoside bond between that ring and the aglycone portion.
  • Analogous orientations are preferred for the substituents on any single ring in a multi-ring structure, i . e .
  • the ring substituents on a second or higher order ring in the chain are preferably transequatorial with the glycoside bond connecting that ring with the ring adjacent to it which is closer to the aglycone portion.
  • ring- connecting glycoside bonds on a non-terminal ring are preferably transequatorial with each other.
  • the nature and degree of the improvement in properties achieved by the conjugation will vary, depending on both the antibiotic nucleus and the carbohydrate domain.
  • the improvement will generally be an improvement in antibiotic activity.
  • the mechanism of the improvement may for example be a result of improved delivery of the antibiotic to the target cells, or improved contacts for interaction with nucleic acids, or both.
  • Conventional screening methods may be used for determining the effect of the carbohydrate domain on the antibiotic activity, and thus for selecting the optimal carbohydrate domain for any given antibiotic nucleus.
  • Such screening procedures will generally involve the measurement of cell viability on cells incubated with the candidate conjugate.
  • the measurement may for example be a determination of the conjugate concentration which gives rise to a 50% level of cell inhibition (IC 50 ) as compared with untreated controls.
  • the cells used may for example be mammalian tumor cells when testing for anti-tumor activity, or any other type of cell depending on the type of cell growth or antibiotic activity targeted.
  • Saccharide units for use in preparing the compounds of the present invention are obtainable from natural sources and commercial suppliers, and alternatively may be synthesized by known techniques. Included among the synthetic techniques, as indicated above, are techniques involving glycals as intermediate structures. Section A below describes the glycals useful in this invention, and methods for their preparation. Succeeding sections address further aspects of the overall synthesis. A. Glycal Synthesis and Scope
  • glycals are available commercially. Methods of synthesis for glycals are known in the art.
  • a particularly useful synthetic method is a cyclocondensation between a diene and an aldehyde.
  • This reaction has been extensively reported in the literature and is well known. The reactivity of the species is greater when electron-donating groups are present on the diene, or when electron-withdrawing groups are present on the aldehyde, or both. Regardless of such activating groups, however, the reaction may be catalyzed by Lewis acids, which is particularly useful when such groups are not present.
  • Lewis acids useful in the reaction are AlCl 3 , AlBr 3 , BeCl 2 , CdCl 2 , ZnCl 2 , BF 3 , BF 3 .O(C 2 H 5 ) 2 , BF 3 .O(C 2 H 5 ) 2 -Ce(OCOCH 3 ) 3 , BCI 3 , BBr 3 , TiCl 4 , ZrCl 4 , MgBr 2 , tris ⁇ 3-(heptafluoropropyl)hydroxymethylene- d-camphorato ⁇ europium, tris(6,6,7,7,8,8,8-heptafluoro- 2,2-dimethyl-3,5-octanedionato)europium, and tris(6,6,7,7,8,8,8- heptafluoro-2,2-dimethyl-3,5-octanedionato)europium.
  • the 5,6-dihydropyral formed by this reaction is readily reduced to the corresponding 3-hydroxy glycal by conventional reducing agents.
  • a prime example is NaBH 4 /CeCl 3 .
  • Glycals useful in the present invention will generally have substituents, i.e., groups other than hydrogen atoms, at various positions on the ring.
  • substituents i.e., groups other than hydrogen atoms
  • the most common examples of such substituents are hydroxyl and C 1 -C 6 alkyl groups, as well as protected hydroxyl, protected mercaptan and protected amine groups.
  • Protected substituents are those terminated in moieties which remain intact, i. e. , prevent reaction at the position occupied by the substituent when exposed to conditions of conversion during any of the various steps of the synthesis, including the conversion of the glycal to a 1,2-anhydrosugar, during a glycosylation reaction, or during a haloglycosylation. These moieties are referred to herein as "protecting groups.” Protecting groups which are readily removable are preferred, and in certain syntheses, protecting groups which are selectively removable, i. e. , removable in preference over other protecting groups, are preferred for specific locations on the ring. Removable groups useful herein are those which can be removed with little or no alteration of the stereochemical configuration of the protected substituent or the glycoside bonds.
  • Preferred protecting groups for hydroxyl substituents are ether-forming groups, various types of which are readily removable.
  • the substituents when thus protected are ethers.
  • Preferred readily removable ether-forming protecting groups are benzyl or ring-substituted benzyl groups having 7-10 carbon atoms, diaryl-C 1 -C 6 alkylsilyl groups such as diphenylmethylsilyl, aryl-di-C 1 -C 6 alkylsilyl groups such as a phenyldimethylsilyl ether, and tri-C 1 -C 6 alkylsilyl groups such as trimethylsilyl and t-butyldimethylsilyl.
  • Acetals and ketals are also considered to contain ether linkages since each contains the C-O-C bond of an ether.
  • Ether-forming protecting groups further those which form acetals and ketals.
  • the protecting reactants are aldehydes and ketones, respectively, preferably containing 1 to about 12 carbon atoms. Examples are formaldehyde, acetone, cyclohexanone, 1-decanal and 5-nonanone or an aromatic aldehyde such as benzaldehyde or naphthaldehyde or an aromatic ketone such as acetophenone. Acetone, formaldehyde and benzaldehyde are preferred. Additional useful readily removable protecting groups are discussed in Kunz, Angew. Chem. Int. Ed. Engl . 26:294 (1988), whose disclosures are incorporated by reference.
  • benzyl ether-type protecting groups are removed by hydrogenolysis over a palladium catalyst or by sodium or lithium in liquid ammonia.
  • Silyl ethers are removed by reaction with tetrabutylammonium fluoride.
  • Acetals and ketals can be removed with mild acids.
  • a hydroxyl protected by a benzyl protecting group can be deprotected selectively over silyl ether, acetal and ketal protecting groups.
  • Tri-C 1 -C 6 alkylsilyl protecting groups such as trimethylsilyl, are preferred protecting moieties for selective deprotection of a nucleophilic hydroxyl group after a glycosylation or haloglycosylation step has been completed, to prepare the hydroxyl group for a subsequent glycosylation or haloglycosylation.
  • substituents which are not readily removable. Included among such substituents are ether-type substituents such as —O—R in which R is C 1 -C 18 alkyl, C 6 -C 10 aryl or substituted aryl, or non-benzyl C 7 -C 10 aralkyl. Examples of such R groups are methyl, ethyl, isopropyl, cyclohexyl, lauryl and stearyl, as well as phenyl, p-tolyl, 2-naphthyl, ethylphenyl, and 4-t-butylphenyl.
  • R is C 1 -C 18 alkyl
  • non-benzyl C 7 -C 10 aralkyl examples of such R groups are methyl, ethyl, isopropyl, cyclohexyl, lauryl and stearyl, as well as phenyl,
  • glycal substituents or protecting groups at locations other than the 1- and 2-carbon atoms remain intact without undergoing oxidation during the epoxidation reaction.
  • Groups which will not undergo oxidation include acyl protecting groups, preferably those formed from C 1 -C 18 alkanoic acids, such as for example acetic, stearic, cyclohexanoic, benzoic, and 1-naphthaleneacetic acid, and those formed from anhydrides, acid chlorides or activated esters such as N-hydroxysuccinimido ester of such acids.
  • Such groups may be bonded to the ring through hydroxyl, thiol or amine groups on the ring.
  • cyclic imides containing a total of 4 to 10 carbon atoms with 5 to 7 atoms in the imido ring such as succinimido, methylsuccinimido, phthalimido and 4-chloro- phthalimido.
  • Still further examples are cyclic amides having 4 to 10 carbon atoms with 5 to 7 atoms in the amido ring, such as pyrrolidinyl, valerolactamyl and caprolactamyl.
  • substituents from participating anchimerically in reactions involved in the synthesis scheme are O-acyl and N-acyl protected substituents, particularly when present at the 4- and 6-positions of glucopyranose derivatives. Further examples are the unprotected 3-, 4- and 6-hydroxyl groups themselves.
  • a participating group may be removed and replaced with a "non-participating" substituent such as a protecting ether group prior to the oxidative conversion and glycosylation steps.
  • participating C 1 -C 18 acyl substituents are replaced with non-participating silyl ether substituents.
  • participating groups are replaced with non-participating substituents such as ether linkages or cyclic imido groups, for example phthalimido groups.
  • a glycal reacts cleanly as a glycosyl donor or a glycosyl acceptor in a haloglycosylation or any other reaction in which these roles are assumed can be predicted by comparing the sums of the Hammett sigma constants for para-substituents for each glycal.
  • the sum of such sigma constants for the donor is negative relative to the corresponding sum for the acceptor.
  • a general formula for glycals serving as glycosyl donors in the practice of the present invention is as follows:
  • R 1 is OH, H, C 1 -C 6 alkyl, 2-furyl, OR 4 , NR 5 R 6 or SR 6 ;
  • R 2 is H, C 1 -C 6 alkyl, 2-furyl, OR 4 , NR 5 R 6 or SR 6 ;
  • R 1 and R 2 together form a cyclic acetal or ketal prepared from an aldehyde or ketone containing 1 to 12 carbon atoms;
  • R 3 is H, C 1 -C 6 alkyl, OR 4 , (CH 2 ) m OR 4 , 2-furyl, NR 5 R 6 , V
  • n 1, 2, 3 or 4, such that the total number of carbon atoms in the ring plus the R 3 group is not greater than 9;
  • n zero or 1;
  • R 4 is C 1 -C 18 alkyl, C 6 -C 10 aryl, C 7 -C 10 aralkyl, tri- C 1 -C 6 alkylsilyl, diaryl-C 1 -C 6 alkylsilyl, aryl di-C 1 -C 6 alkylsilyl, or a substituted mono- or oligosaccharide;
  • R 5 is C 1 -C 18 alkyl, C 6 -C 10 aryl, C 7 -C 10 aralkyl, tri- C 1 -C 6 alkylsilyl, aryl di-C 1 -C 6 alkylsilyl, diaryl C 1 -C 6 alkylsilyl , or C 1 -C 18 acyl ;
  • R 6 is H, C 1 -C 18 alkyl, C 7 -C 10 aralkyl, and C 1 -C 18 acyl such that (a) at least one of R 5 or R 6 of NR 5 R 6 and SR 6 is C 1 -C 18 acyl, or (b) NR 4 R 5 together form a cyclic amide or imide containing 5 to 7 atoms in the ring and a total of 4 to 10 carbon atoms;
  • R 7 and R 8 are independently H or C 1 -C 9 alkyl such that the number of carbon atoms in R 7 plus those of R 8 is nine or fewer;
  • R 9 is selected from the group consisting of hydrogen (H), or C 1 -C 6 alkyl, CO 2 R 4 , CN, CH 2 OR 4 , 2-furyl,
  • R 10 is hydrogen (H), 2-furyl, or C 1 -C 6 alkyl.
  • Glycals serving as glycosyl acceptors are also represented by the above general formula with the additional features that R 4 also includes H and C 1 -C 18 acyl, R 3 also includes CN, O-carbamyl ester having a total of 1-14 carbon atoms, CH(OH)CH 2 OR 4 , and CH(OR 4 )CH 2 OH, R 1 and R 2 also include CN and O-carbamyl ester having a total of 1-14 carbon atoms, and the limitation that at least one of R 1 , R 2 and R 3 includes an OH group.
  • Cyclic ethers resolved in accordance with this invention are those containing a hydroxyl group at a chiral center on the ring.
  • the resolution is between stereoisomers defined by alternate orientations of the hydroxyl group, one such orientation being on one side of the plane of the ring (when the ring is considered in a planar representation), and the other orientation being on the other side.
  • stereoisomers thus resolved include isomer pairs differing in relative configuration, such as diastereomers, as well as those having the same relative configuration but differing in absolute configuration, such as enantiomers (also referred to as antipodes).
  • Cyclic ethers which are diastereomers will differ at the hydroxyl substitution site but not at other substitution sites on the ring, and will thus not be mirror images of each other. The relative orientation of the hydroxyl group and at least one other ring substituent will thus be coequatorial in one diastereomer and transequatorial in another.
  • diastereomers will generally display differences in chemical and/or physical properties.
  • Cyclic ethers which are enantiomers, however, are mirror images of each other, i.e., the various substituents will have the same equatorial orientation relative to each other in both isomers, but the number and/or location or the substituents, and/or other feature(s) of the ring such as the double bond of a glycal, render the molecule incapable of being superimposed over its mirror image.
  • Such isomers display identical chemical and physical properties except for the direction of rotation of polarized light.
  • This aspect of the invention is of particular interest in resolving enantiomers, particularly enantiomeric cyclic enol ethers, and more particularly enantiomers of six-membered ring glycals.
  • Further preferred characteristics of the enantiomers, taken either individually or together, are that the hydrox substitution site be at position 3 on the glycal ring; that least one of the positions 2, 4, and 5 on ring contain non- hydroxyl substituent(s); and that the ring contain at least two non-hydroxyl substituents, all of which are at positions other than position 1, preferably one being at position 4 or 5.
  • non-hydroxyl substituents are lower alkyl (i.e., C 1 -C 6 alkyl, preferably C 1 -C 4 alkyl), phenyl, benzoy phenylalkyl (in which the alkyl group is lower alkyl), low alkanoyloxy, furyl, thienyl, pyrrolyl, pyridyl, quinolyl, a isoquinolyl.
  • the resolution is achieved by acetylation, which when conducted in the presence of a lipase, is found to proceed in selective manner, only one such stereoisomer being acetylated.
  • acetylating agents may be used; the choice of acetylating agent is noncritical to the practice of the invention, permitting the use of a wide variety of such agents. Examples are vinyl acetate, isoprenyl acetate, acetyl chloride and acetic anhydride.
  • Lipase P and Lipase PS-30 both from species such as Pseudomonas cepacia , are available commercially and may be used.
  • the reaction is conducted at room temperature in the presence of a suitable solvent, under otherwise conventional acetylation conditions well described in the literature.
  • the particular enantiomer which is acetylated in preference over the other will vary with the ring size, the location and orientation of the hydroxyl group which is acetylated in the reaction, and the number, identity and orientation of the other substituents on the ring.
  • the degree of selectivity will likewise vary.
  • the separation of the acetylated from the nonacetylated enantiomers, however, and the determination of which enantiomer has been preferentially acetylated are achieved by conventional means. Spectroscopic and chemical methods commonly used to differentiate enantiomers are effective in this regard.
  • the acetylated hydroxyl group is at position 3 of the glycal.
  • the preferentially acetylated enantiomer is that in which the hydroxyl is forward of the glycal ring plane, i.e., between the ring and the viewer, which is the ⁇ -orientation.
  • Other patterns and preferences are readily determinable by the routine technician.
  • the degree of selectivity may be varied or enhanced by the attachment of protecting groups of selected configurations, preferably removable in subsequent steps of the synthesis.
  • Such protecting groups include many of the non-hydroxy substituents listed above. Others will be readily apparent to those skilled in the art.
  • acetylation has been performed, the acetylated and nonacetylated species are separated by conventional separation methods.
  • the actual method used is not critical, and the optimal choice in any particular embodiment will depend on the scale of the process and the particular species being separated. Two common examples are column chromatography and crystallization, each using conventional materials and equipment and performed using conventional procedures, the optimal materials, equipment and procedures in any given case being either readily apparent to the skilled technician or readily determinable by routine experimentation.
  • one such method is that of haloglycosylation, which occurs by the reaction among an electrophilic halogenating (“halonium”) reagent and two saccharide units, one of which is a glycal, and the other of which contains a nucleophilic hydroxyl group at which the glycoside bond is formed.
  • halonium electrophilic halogenating
  • Such reactions proceed through a 1,2-halonium species intermediate, which is the adduct formed by reacting a halonium reagent at the glycal double bond.
  • Halonium reagents useful in this reaction are well known in the art.
  • Preferred halogens are bromine and iodine
  • preferred halonium reagents are (2,4,6-collidine) 2 BrClO 4 and (2,4,6-collidine)IClO 4 , respectively.
  • the reaction is generally conducted in a solvent which is both inert to the reaction conditions and readily removable. Examples of such solvents are dichloromethane or chloroform.
  • the moiety 2,4,6-collidine is also referred to as "sym-collidine.”
  • Other halonium reagents are N-bromosuccinimide and N-iodosuccinimide. Still others known to those skilled in the art may be used.
  • the nucleophile Since the reaction occurs under oxidative conditions and in the presence of the nucleophile, the nucleophile must be one which remains substantially inert under such conditions. Since the same reaction may be used to conjugate a saccharide unit of multimer to an antibiotic nucleus, this will affect the choice of conjugation sites and methods of conjugation. Alcohol hydroxyl groups, and particularly aliphatic hydroxyl groups, are substantially inert to the conditions encountered in the haloglycosylation reaction. Many amines and most mercaptans are not sufficiently inert, however, and as a result, amines and mercaptans are not useful nucleophilic sites when this reaction is used.
  • the nucleophilic atom is preferably a hydroxyl oxygen.
  • a single hydroxyl oxygen atom is present on the nucleophile saccharide unit.
  • selectivity is readily achieved by protection of the hydroxyls where reaction is not desired.
  • the resulting 2-position halo group can be inverted in orientation or substituted with another substituent.
  • a substituent for example, a
  • 2-position iodide can be reacted with phthalimide to form a nitrogen-containing substituent.
  • a 2-position halide can be exchanged for a hydrogen substituent by reaction of the haloglycoside with tributyltin hydride in the presence of azobis(isobutyro)nitrile in refluxing benzene.
  • the nucleophilic hydroxyl oxygen and the halonium ion generally add in a trans diaxial manner, i . e . , transequatorially, across the double bond of the substituted glycal.
  • the product is a 2-deoxy-2-(halo-substituted) glycoside.
  • the orientation of the nucleophile with respect to the other ring substituents can be modified or controlled by steric hindrance.
  • by placing large substituent groups on one side of the ring while leaving the other side relatively unhindered one can favor an approach by the nucleophile from the other, less hindered side.
  • Haloglycosylation is generally conducted in a solvent that is inert to the reaction conditions. Examples are dichloromethane, chloroform, diethyl ether and tetrahydrofuran.
  • the reaction mixture is maintained free of water, and consequently the solvents are used in dry form. It is preferred that an excess of glycal and halonium ion reagent be present relative to the nucleophilic hydroxyl group. While the actual amounts are not critical, best results are usually obtained using ratios in the range of about 2:1 to about 10:1 (glycal:nucleophilic hydroxyl), with the halonium ion reagent and glycal being present in about equimolar amounts.
  • Anhydrous conditions may be maintained, for example, by the use of 4A-molecular sieves in the reaction medium.
  • the reaction temperature is likewise not critical, but best results are usually achieved in reactions conducted at about -20 to about +40 degrees C, and preferably from about zero to about ambient room temperature, i.e., 22oC.
  • a substituted 1,2-anhydrosugar can be represented by the general formula
  • Substituted 1,2- anhydrosugars useful in glycosylations according to this invention are those that are free of participating substituent groups.
  • Dialkyl dioxiranes used in forming these 1,2- anhydrosugars from glycals are preferably those having a total of two to about six carbon atoms in the dialkyl groups. Examples are dimethyldioxirane, diethyldioxirane, methyl isopropyldioxirane, methyl propyldioxirane, ethyl sec- butyldioxirane. Dimethyldioxirane (technically, 3 , 3-dimethyl- dioxirane) is a particularly preferred dialkyl dioxirane because its reaction product, acetone, is relatively readily removable from the reaction mixture, and because side reactions with peroxides such as peracetic acid do not occur.
  • the oxygen atom of the formed epoxide ring is formed cis to the olefinic unsaturation, on the less sterically hindered side of the glycal ring.
  • the 1,2-epoxide ring can be directed to the ⁇ - or ⁇ -side of the ring.
  • the conversion of a glycal to the corresponding 1,2- anhydrosugar is generally conducted at a temperature of from about -40oC to about +20o C, and typically at about 0°C.
  • the reaction is generally conducted in an inert solvent such as acetone, methylene chloride-acetone.
  • Preferred solvents are those which boil at less than about 100oC to facilitate isolation and recovery of the resulting 1,2-anhydrosugar by solvent removal. This is accomplished by conventional means, such as for example by use of a stream of dry nitrogen or under reduced pressure, at a temperature below about 20oC so that the otherwise reactive epoxide ring does not react prematurely.
  • the starting glycal contains one or more participating substituents
  • substituents should be removed and replaced with non-participating substituents prior to the reaction of the substituted 1,2-anhydrosugar with the nucleophile to be glycosylated.
  • this replacement is done prior to conversion of the glycal to the 1,2-anhydrosugar.
  • the 1,2- anhydrosugar to be used in the subsequent glycosylation step will then contain only non-participating substituents.
  • Glycosylation proceeds by reaction of the 1,2- anhydrosugar (serving as the glycosyl acceptor) with a nucleophilic hydroxyl group on another saccharide unit (serving as the glycosyl donor).
  • nucleophilic hydroxyl groups are generally primary hydroxyls as compared to secondary or tertiary hydroxyls.
  • Reducing sugars contain a single primary hydroxyl group located on the carbon atom furthest along the chain from the aldehydic (anomeric) carbon atom, i.e., on the 5- , 6- and 7-position carbon atoms of a pentose, hexose and heptose, respectively.
  • Nonreducing sugars contain two primary hydroxyl groups, one at the 1-position and the other at the 6-, 7-, 8-, and 9-positions, respectively, for hexuloses, heptuloses, octuloses and nonuloses, such as sialic acid derivatives.
  • the reaction is preferably conducted in the presence of a Lewis acid catalyst in an appropriate solvent that is inert to the reaction, and at a temperature of from about -100°C to about +40°C, and preferably at a temperature of from about -78oC to about room temperature (about 22oC).
  • Lewis acids useful in this reaction include SnCl 4 , AgClO 4 , BF 3 , trimethylsilyl trifluoromethanesulfonate (triflate), Zn(triflate) 2 , Mg(triflate) 2 , MgCl 2 , AlCl 3 , ZnBr 2 and ZnCl 2 .
  • Tri-n-butyltin salts of the glycal alcohol have also been successfully utilized.
  • Lewis acid catalysts are typically utilized in ethereal or chlorinated solvents. Examples are diethylether, tetrahydrofuran, dimethoxyethane, methylene chloride and chloroform.
  • reaction conditions are selected or controlled to either prevent or minimize polymerization of the 1,2-anhydrosugar. Care is also taken to avoid conditions in which the Lewis acid catalyst removes a protecting group, as can occur with BCl 3 . Means of avoiding these occurrences will be readily apparent to those skilled in the art. See Sharkey, et al . , Carbohydr. Res. 96:223 (1981).
  • Glycosylations utilizing a solid phase are analogous to solid phase syntheses of oligo- and polypeptides or oligo- and polynucleotides.
  • the solid phase may assume any size, shape or form. Particularly convenient solid phases, however, are particulate materials.
  • one of the reacting saccharide units is linked directly to the solid phase support.
  • the linkage can be through a direct covalent bond or through a linking agent, and the linkage must be inert to the reaction conditions, but is preferably capable of being cleaved or severed when desired so that the resulting saccharide multimer can be separated from the support.
  • Benzyl ether linkages are preferred routes of bonding to the solid phase support, and reactions used to form such linkages in particles are well known.
  • Linking groups are also known.
  • One such group is the 3-aminopropanol group.
  • THis group can be reacted with a benzyl halide-containing particle to provide a primary hydroxyl group that can be haloglycosylated with a glycal as discussed herein or by other well known means. Terminal glycosides thus linked are readily cleavable from the solid phase by known methods.
  • Suitable solid supports will be ones which are insoluble in the reaction medium, and all solvents utilized, and which are substantially chemically inert to the reaction conditions encountered.
  • the solid support preferably swells in the solvent during synthesis due to physical, rather than chemical processes.
  • the solid support may be fabricated from a wide variety of materials.
  • Polymerized resins in the form of porous beads are notable examples.
  • a preferred subclass are resins of hydrophobic polymerized styrene cross-linked with divinylbenzene, typically at about 0.5 to about 2 weight percent.
  • Such resins can be further reacted to provide a known quantity of a benzyl moiety on the solid phase surface.
  • the benzyl moiety contains a reactive functional group through which the glycal or substituted 1,2- anhydrosugar can be covalently linked by a selectively severable bond.
  • the before-noted linkers are also readily utilized with benzyl halide-substituted resins.
  • the reactive benzyl moieties are typically added after the resin bead has been synthesized by reaction of a polymerized styrene moiety, such resins are generally described as polymerized styrene crosslinked with divinyl benzene and including a known amount of polymerized vinyl benzyl moiety.
  • solid supports are silica- containing particles such as porous glass beads and silica gel. Reactive benzyl moieties can be used in conjunction with these supports as well. Further examples are glass particles coated with hydrophobic, polymerized, cross-linked styrene containing reactive chloromethyl groups.
  • the saccharide unit or linking group is joined to the particulate support under usual benzylation conditions to form a particulate support-linked substituted nucleophile. Any remaining reactive groups on the support surface are then protected as, for example by reaction with a primary alcohol such as methanol or a tertiary amine such as triethylamine.
  • a primary alcohol such as methanol or a tertiary amine such as triethylamine.
  • the particle-linked reactant is thereafter ready to use.
  • Conjugation of the carbohydrate domain to the antibiotic nucleus is achieved through one or more covalent bonds, either directly or through linking agents.
  • covalent bonds either directly or through linking agents.
  • the following discussion addresses aglycones, but is equally applicable to other types of antibiotic nuclei.
  • the optimal type of covalent bond or linking agent in any particular case will vary from one species to the next, depending on such factors as the structure and character of the aglycone, and the sensitivity of the antibiotic activity of the glycosylated product to the location of the linkage site on the aglycone.
  • the most convenient type of bond is a glycoside bond.
  • the bond is formed between a terminal saccharide unit on the carbohydrate domain (the unit serving as a glycosyl donor) and a nucleophilic atom on the aglycone.
  • the nucleophilic atom is one which is either native to the aglycone or one which has been appended to the aglycone as a linking group.
  • oxygen oxygen, nitrogen and sulfur.
  • Oxygen atoms and hence the formation of O-glycoside bonds, are preferred.
  • the most preferred are the oxygen atoms of nucleophilic hydroxyl groups which are native to the aglycone.
  • the bond is most conveniently formed by any of the methods described previously in this specification for the joining of two saccharide units.
  • the terminal saccharide unit is preferably in the form of a glycal, which is joined to the nucleophilic atom either in the presence of a halonium reagent or through the intermediate stage of a 1,2-anhydrosugar.
  • the reaction then proceeds in essentially the same manner and under essentially the same conditions described above for the corresponding reactions in the joining of saccharides, including the use of protecting groups where necessary.
  • nucleophiles exhibit improved reactivity under certain conditions.
  • a Lewis acid catalyst appears to be required for most neutral nucleophiles such as alcohols, amines and mercaptans, whereas negatively charged nucleophiles such as azide ion or trimethylsilyl thiophenoxide do not require Lewis acids.
  • a Lewis acid catalyst is generally not necessary.
  • the reaction to form the carbohydrate and the reaction to conjugate the carbohydrate to the aglycone are generally performed separately.
  • the carbohydrate domain is fully formed first, leaving the terminal unit (to be joined to the aglycone) in glycal form, then once formed, the carbohydrate is conjugated to the aglycone.
  • Glycosylated anthracyclines represented by the following formula are novel:
  • R 1 represents an anthracycline aglycone
  • R 2 represents one of the two enantiomers
  • R 3 is either aliphatic, aryl or aralkyl.
  • L-sugar species whereas those with enantiomer (C) are D-sugar species.
  • the L-sugar subclass is preferred. In both subclasses, the iodine atom is in an orientation which is commonly termed "axial.”
  • R 3 is lower alkyl, aryl or aryl-(lower alkyl), and particularly lower alkyl or aryl.
  • aliphatic is used herein to denote saturated and unsaturated hydrocarbon groups, the unsaturated groups including those with one or more double bonds, a triple bond, or a combination, and includes both straight- chain and branched-chain groups.
  • alkyl is used herein to designate a saturated hydrocarbon group, again including both straight-chain and branched-chain structures.
  • Preferred alkyl groups are those containing six carbon atoms or less, and "lower alkyl” generally refers to four carbon atoms or less. Examples are methyl, ethyl, isopropyl, n-butyl and t-butyl.
  • aryl denotes aromatic structures, including both single and multiple ring structures, and further including substituted aromatic rings, with substituents such as alkyl, hydroxyl, and halogen. Examples are phenyl, naphthyl, hydroxylphenyl, chlorophenyl, iodophenyl, and methylphenyl.
  • aralkyl denotes an aryl group linked to the sugar ring through an alkyl group, both within the definitions given above. Examples are benzyl and phenylethyl. The most preferred groups for R 3 are methyl and phenyl.
  • a preferred subclass is that which includes adriamycinone, daunomycinone, aklavinone, ⁇ -pyrromycinone, n - pyrromycinone, ⁇ -rhodomycinone, ⁇ -rhodomycinone, ⁇ -rhodomycinone, nogalamycinone, and steffimycinone.
  • a further preferred subclass is that of adriamycinone, daunomycinone, aklavinone, ⁇ -pyrromycinone, n -pyrromycinone, ⁇ -rhodomycinone, ⁇ -rhodomycinone, and ⁇ -rhodomycinone, and a still further preferred subclass is that of adriamycinone, daunomycinone, aklavinone, and ⁇ -pyrromycinone.
  • TBS tert-butyldimethylsilyl
  • Ph phenyl
  • Eu(hfc) 3 tris ⁇ 3-(heptafluoropropyl)- hydroxymethylene-d-camphorato ⁇ europium
  • Examples 1, 2 and 3 illustrate the preparation of glycal enantiomer of a particular stereospecific configuration.
  • This example illustrates the first step of the preparation.
  • the reaction scheme for this stage is shown below.
  • the resulting two-phase mixture was allowed to warm to room temperature, then poured into methylene chloride (250mL)/NaHCO 3 (aqueous, 85mL). The aqueous layer was extracted once more with methylene chloride (100mL), and the combined organic layers were washed with brine (100mL), dried (MgSO 4 ) and evaporated.
  • the crude cycloaddition product mixture was dissolved in carbon tetrachloride (50mL), and trifluoroacetic acid (2.13mL, 27.6mmol) was added dropwise with stirring at room temperature.
  • (+,-)-3 (2R * ,3R * ,4R * )-3-Benzoyloxy-4-hydroxy-2-phenyl-2,3- dihydro-4H-pyran
  • the reaction scheme illustrated in this step is as follows.
  • (+)-6 (2S,3R,4S)-2-Phenyl-3,4-bis-(trimethylsilyloxy)-2,3- dihydro-4H-pyran
  • vinyl acetate 50mL, 542mmol
  • lipase PS-30 6.0g
  • the reaction was stopped by addition of diethylether (50mL) and filtration through a medium (ASTM 10-15) fritted funnel.
  • Examples 4 and 5 illustrate the conjugation of the enantiomers (-)-6 and (+)-6 individually to daunomycinone.
  • the enantiomer (-)-6 is used in Example 4 and the enantiomer (+)-6 is used in Example 5.
  • the reaction illustrated in this example is as follows.
  • the mixture of isomeric coupling products (115mg, 0.134mmol) was dissolved in THF (12mL), and HF-pyridine (600 ⁇ L) was added at 0°C. The reaction mixture was then slowly allowed to reach room temperature. After nine hours, the reaction mixture was diluted with CH 2 Cl 2 (75mL) and quenched with NaHCO 3 (aqueous, 50mL). The aqueous layer was extracted once more with CH 2 Cl 2 (75mL) and the combined organic layers washed with CuSO 4
  • Compound 8 (the 2'S,3'S isomer) as an orange powder. Later fractions contained the (2'S,3'R) (daunomycinone axial, iodine eq.) isomer ( ⁇ 5mg, 5%) and the (2'R,3'R) (daunomycinone axial, iodine eq.) isomer ( ⁇ 2mg, 2%), both established by 1 H-NMR.
  • (+, -) -11 (2R * , 3S * ) -3-Acetoxy-2-methyl-2 , 3-dihydro-4H-pyran-4- one
  • (+,-)-12 (2R * ,3R * ,4R * )-3-Acetoxy-4-hydroxy-2-methyl-2,3- dihydro-4H-pyran; alternatively: 4-Acetylfucal
  • (-)-13 (2R, 3S,4R) -3 , 4-Diacetoxy-2-methyl-2,3-dihydro-4H- pyran; 3,4-Diacetylfucal
  • (+)-13 (2S,3R,4S)-3,4-Diacetoxy-2-methyl-2,3-dihydro-4H- pyran; 3,4-DiacetyIfucal
  • the diacetate (-)-13 was converted to the diol (-)-14 by dissolving the diacetate (500mg, 2.34mmol) in methanol (20mL) and adding K 2 CO 3 (194mg, 1.40mmol). The reaction mixture was stirred at room temperature for 2 hours, then concentrated in vacuo and chromatographed (5% methanol/CH 2 Cl 2 ) to yield the diol (-)-14 (280mg, 92%) as a white solid, melting point 72-73°C, [ ⁇ ] D 21 -18.9° (c 1.25, CHCl 3 ), with structure confirmed by 1 H-NMR and IR.
  • the examples illustrates the conjugation of the glycal prepared in Example 6 to daunomycinone.
  • the reaction is as follows.
  • the reaction illustrated in this example is as follows.
  • Examples 9 through 11 illustrate the preparation of a trisaccharide of a particular stereospecific configuration, and the coupling of the trisaccharide to anthracyclines.
  • the trisaccharide prepared in this example is 1,5- anhydro-2,6-dideoxy-4-0- ⁇ 4-0-(2,3,6-trideoxy- ⁇ -L-glycero- hexopyranosid-4-ulose)-2,6-dideoxy-3-0-trimethylsilyl- ⁇ -L-lyxo- hexopyranosyl ⁇ -3-0-trimethylsilyl-L-lyxo-hex-1-enitol.
  • the reaction scheme used for its preparation is shown in two stages below.
  • the iodide was dissolved in benzene (500mL) and treated with triphenyltin hydride (9.55g, 27.2mmol) and azobisisobutyronitrile (120mg, 0.73mmol). The mixture was then heated at reflux for 30min.
  • Liquid ammonia (ca. 130mL) was collected via a dry ice condenser in a 500mL 3-necked flask cooled to -78° in a
  • This example illustrates the coupling of the trisaccharide (Compound 50) to ⁇ -pyrromycinone.
  • the structure of ⁇ -pyrromycinone and the reaction schemes followed in this example are shown below.
  • the section illustrates one route for the preparation of ciclamycin O, by first converting Compound 52 to Compound 55, followed by conversion of the latter to ciclamycin O (Compound 55).
  • the section illustrates an alternate route for the preparation of ciclamycin O, by first converting Compound 51 to Compound 53, followed by conversion of the latter to Compound 54, and finally to ciclamycin O (Compound 56).
  • This example illustrates the coupling of the 2'-epimer of Compound 52 to ⁇ -pyrromycinone through a route analogous to that of Section A of this example.
  • This example illustrates the coupling of the trisaccharide (Compound 50) to daunomycinone.
  • the structure of daunomycinone and the reaction schemes followed in this example are shown below.
  • IR (CHCl 3 ) 3580-3230, 3000, 2940, 1730, 1715, 1620, 1580, 1450, 1435, 1415, 1290 cm -1 ;
  • UV-vis ⁇ max (MeOH) 448 ( ⁇ 17,220), 421 (15,773), 397 (9,760), 266 (24,283), 241 (25,533).
  • anthracycline compounds were tested in vitro on mammalian tumor cells. Of the eight compounds, six were L-sugar species, and of these six, one was adriamycin (doxorubicin) and three of the remaining five were iodinated. The three were Compounds D, E and F shown in Section IV of this specification, and each of the remainder (except for adriamycin) were identical to one of these except for the isomeric form of the sugar moiety or the presence of a hydrogen atom in place of the iodine atom, or both, as indicated below. Adriamycin is included for comparison as a well-known structurally similar commercial analog.
  • the test consisted of incubating mammalian tumor cells with the drugs for 72 hours according to standard and well-known in vitro testing techniques, and determining the cell viability using a vital stain.
  • a variety of cell lines were used:
  • HCT116 human colon tumor cell line
  • HCT/VM46 a drug-resistant variant of HCT116, derived by culturing the HCT116 in the presence of teniposide, to achieve a resistance ratio, i.e., the ratio of IC 50 of the drug resistant HCT116/VM46 to that of the parent HCT116, of about 4 with teniposide, etoposide and adriamycin
  • HCT/VM35 a drug-resistant variant of HCT116, derived by culturing the HCT116 in the presence of VP-16, to achieve a resistance ratio of about 13 with teniposide, etoposide and adriamycin
  • HCT/VP35 a further drug-resistant variant of HCT116, similarly derived by culturing the HCT116 in the presence of VP-16
  • A2780S human ovarian cell line
  • A2780DDP a DDP-resistant variant of A2780S, derived by culturing A2780S in the presence of DDP
  • test results are expressed in terms of the IC 50 , which is the drug concentration in micrograms per milliliter required to inhibit cell growth by 50% compared to non-drug- treated cells on the same plate.
  • IC 50 the drug concentration in micrograms per milliliter required to inhibit cell growth by 50% compared to non-drug- treated cells on the same plate.
  • the lower the IC 50 the higher the activity of the compound.
  • each compound was tested in triplicate tests on separate test plates. The results are listed in the following table.
  • test data show that L-sugar species are superior to D-sugar species, and iodinated species are superior to non- iodinated species under all test conditions. Compounds D, E and F are also superior to adriamycin.

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Abstract

Nouvelles anthracyclines dont les domaines glucidiques constituent une des deux formes énantiomères du groupe 2'-{4', 5'-dihydroxy-3'-iodo-6'-(aryle ou aliphatique)} tétrahydropyranosyle, présentant des propriétés antibiotiques améliorées. L'invention concerne également un procédé de résolution d'énantiomères d'éthers cycliques, notamment des glycaux, par acétylation sélective d'une substitution hydroxyle dans le noyau, à l'aide d'une lipase associée à un agent d'acétylation. En outre, l'invention concerne une nouvelle approche permettant d'améliorer, de commander ou de moduler l'activité cytotoxique d'un antibiotique ayant pour origine la découverte du rôle du domaine des glucides.
PCT/US1991/008288 1990-10-30 1991-10-28 Antibiotiques ameliores par conjugaison avec des glucides stereospecifiques et procedes de stereoselectivite de glucides Ceased WO1992007862A1 (fr)

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Publication number Priority date Publication date Assignee Title
WO1995009173A1 (fr) * 1993-09-30 1995-04-06 A. Menarini Industrie Farmaceutiche Riunite S.R.L. Disaccharides d'anthracycline, leur procede de preparation et compositions pharmaceutiques les contenant
EP1634887A3 (fr) * 2004-07-23 2006-06-21 Zaklad Badawczo-Produkcyjny Procédé de préparation de 2-déoxy-2-iodo-pyranosides complexes de haute pureté, approprié pour la fabrication d'annamycin de pureté pharmaceutique
WO2007003236A1 (fr) * 2005-06-30 2007-01-11 Zaclad Badawczo-Produkcyjny 'syntex' Processus de synthese de 2-desoxy-2-iodo pyranosides complexes de grande pureté, convenant en particulier à la fabrication d’annamycine pharmaceutiquement pure
US12161380B2 (en) 2016-06-15 2024-12-10 Arrinex, Inc. Devices and methods for treating a lateral surface of a nasal cavity

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995009173A1 (fr) * 1993-09-30 1995-04-06 A. Menarini Industrie Farmaceutiche Riunite S.R.L. Disaccharides d'anthracycline, leur procede de preparation et compositions pharmaceutiques les contenant
EP1634887A3 (fr) * 2004-07-23 2006-06-21 Zaklad Badawczo-Produkcyjny Procédé de préparation de 2-déoxy-2-iodo-pyranosides complexes de haute pureté, approprié pour la fabrication d'annamycin de pureté pharmaceutique
WO2007003236A1 (fr) * 2005-06-30 2007-01-11 Zaclad Badawczo-Produkcyjny 'syntex' Processus de synthese de 2-desoxy-2-iodo pyranosides complexes de grande pureté, convenant en particulier à la fabrication d’annamycine pharmaceutiquement pure
US12161380B2 (en) 2016-06-15 2024-12-10 Arrinex, Inc. Devices and methods for treating a lateral surface of a nasal cavity

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