CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of all of the following copending applications: Serial No. 07/392,753, filed August 11, 1989; Serial No. 07/392,793, filed August 11, 1989; Serial No. 07/392,796, filed August 11, 1989; and Serial

No. 07/392,803, filed August 11, 1989.

The U.S. Government has a paid-up license in this invention and the right in limited circumstances to require the patent owner to license others on reasonable terms as provided for by the terms of PHS Grant No. Al 16943 awarded by the

National Institutes of Health.

This invention relates to antibiotics, and particularly to glycoside antibiotics, as well as to methods of synthesizing carbohydrates.

BACKGROUND OF THE INVENTION

The various roles of oligosaccharides in biophysiology are well known. These include acting as reservoirs for energy storage, as structural components for biological synthesis, and as immunological determinants. 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. tied. 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 . , Proc. Natl . Acad. Sci. U. S.A. 77:7204 (1980); and Wang, et al . , Biochemistry 26:1152 (1987). Anthracycline antibiotics are a prime example. As in the case of most antibiotics, however, there has been extensive research on the bioorganic and

_SUBS TITUTESHEET

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. There have been attempts to synthesize the oligosaccharides of anthracyclines and other antibiotics. Reports of investigations in this area include those of Thiem, J. , "Trends in Synthetic Organic Chemistry," Horton, D. , et al . , eds., ACS Symposium Series 386, American Chemical Society Washington, D.C. (1989); Thiem, J., et al . , Carbohydrate Research 174:201 (1988); Heyns, K. , et al . , Chem. Ber. 114:232 (1981); Thiem, J. , et al . , Carbohydrate Research 136:325 (1985); Monneret, C. , et al . , J. Carbohydrate Chem. 7:417 (1988); and Abbaci, B. , et al. , J. Chem. Soc. Chem. Commun. 1989:1896. None of these attempts, however, include glycosylation of an anthracycline with a fully synthetic oligosaccharide.

SUMMARY OF THE INVENTION 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

SUBSTITUTESHEET

either entirely or substantially pure in terms of stereospecific configuration.

Included among the synthetic methods for preparing the carbohydrate domains to be conjugated to the aglycones are the following non-mutually exclusive methods:

(i) methods in which mixtures of stereoisomers of saccharide monomers, either naturally derived or synthetically prepared from non-saccharide starting materials, are resolved and, when appropriate, joined to other such monomers, likewise resolved and either naturally derived or synthetically prepared from non- saccharide starting materials, or to stereospecific multimers similarly or otherwise prepared.

(ii) methods in which naturally derived saccharides are joined in a stereospecific manner, in the case of carbohydrate domains which are saccharide multimers;

(iii) methods involving the synthetic preparation of saccharide monomers from non-saccharide starting materials, followed by the joinder of such monomers to other such monomers or to naturally derived monomers or multimers in controlled stereospecific manner; and The first of these methods in itself forms a further part of this invention. 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. In the reaction which occurs, 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, however, 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. While the particular enantiomer which is acetylated will vary with the ring substituents and their orientation, certain types of consistent behavior are observed among species which bear common features in their configurations. For example, in preferred embodiments 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. Among glycals, those forming five- and six-membered rings are particularly preferred, with six-membered rings the most preferred. In the class of such glycals in which 3-position bears the hydroxyl group and thus forms the chiral center which defines the enantiomers, the enantiomer which is acetylated is generally that in which the hydroxyl group is in the β-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. In each case, the resulting saccharide multimers have a predetermined stereochemical configuration both in terms of

SU

glycosidic bonds and in terms of individual saccharide substituents.

According to one such method, 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. In the reaction, 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. The terms "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.

In this particular method, the substituents on the add¬ on 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. When trisaccharides or higher saccharide multimers are desired, 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. To permit this new add-on to react, however, 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 add¬ on 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.

According to another such method, 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. In the reaction, 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.

In this reaction, as opposed to that of the first method, the 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) .

Using terminology parallel to that used in describing the first method, 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.

SUBSTITUTE

To permit use of the same reaction for such subsequent add-ons, 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. With a suitable add-on glycal at each stage (i.e., one with a selectively removable protecting moiety) , 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.

Like the first method, 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. According to this method, 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.

In a variation on this method, the "starting" species is the species bearing the reactive hydroxyl group which takes part in the glycosylation, and 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.

In both methods in which the reaction proceeds through a 1,2-anhydrosugar intermediate, 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. In the variation where 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. Examples of such 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.

In all methods involving the use of a solid phase, particles are preferred as the solid phase, due to their high surface area useful for linkage and liquid phase contact, and the ease in which they can be separated from the liquid phase of the reaction medium and washed. Linkage to the solid phase is preferably achieved by a bond which is selectively severable, to permit severance of the completed saccharide multimer in intact form from the solid phase. Materials and methods for achieving such linkage and severance are known to those skilled in the art.

DEFINITIONS While all words and phrases herein are used in accordance with meanings recognized by those skilled in the art, the following is offered for purposes of convenience and clarification.

The terms "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.

The terms "monosaccharide," "saccharide unit" and "saccharide residue" are equivalent to the term "simple sugar,"

S ^UBST ITUTESHEET

and indicate sugars or sugar moieties that cannot be hydrolyzed into smaller units.

An "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.

The term "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 iε usually drawn, whereas in beta (β) anomers, the substituent extends above the plane of the ring when so drawn. The alpha and Beta designations apply in likewise manner to substitutions at other ring positions as well. The terms "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

SUBSTIT

the oxygen of the hydroxyl substituent at the 4-position of a six-membered cyclic saccharide residue.

The term "glycoside" refers to a molecule to which one or more saccharide residues, including oligo- and polysaccharides, are covalently bonded through a glycoside bond.

The term "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."

The term "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:

6

The term "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 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.

S ^U B ^STI TUTESHEET

The glycosyl donor and glycosyl acceptor terminology explained above may also be applied to oligosaccharides. Thus, 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. The last saccharide unit that is not itself a glycosyl donor to another saccharide unit is referred to herein as the "terminal" saccharide. Thus, 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.

The term "1,2-halonium species intermediate" refers to an adduct formed by combining an electrophilic halogenating reagent (electrophile) and a glycal double bond. The reaction to form this adduct is generally referred to as a "haloglycosylation."

When two substituents or bonds are on the same side of the plane of a sugar ring as drawn in a two-dimensional representation, those substituents are referred to as being "syn" or "coequatorial" to each other. When two substituents are on opposite sides of a similarly drawn sugar ring plane, they are in the "anti" or "transequatorial" configuration. The εyn and anti nomenclature is usually utilized for substituents separated in a ring by at least one ring carbon atom. For adjacent carbons, "ciε" and "trans" are used for two substituents on the same and opposite sides of the ring plane, respectively.

The term "aglycone" is used herein to denote antibiotics which do not contain carbohydrate domains. This term includes, but iε not limited to, nonsugar productε of the hydrolysis of a glycoside. For anthracyclinones (a subclasε of aglyconeε) , the following numbering system, which iε the

numbering system used in the anthracycline literature, is used herein:

The terms "aliphatic," "aryl," "alkyl" and all related terms used herein are used in accordance with their conventional meanings. The term "aliphatic" includes all hydrocarbyl moieties which are neither cyclic nor aromatic, but does include straight- and branched-chain groups. The term "alkyl" refers to saturated aliphatics and also includes both straight- and branched-chain groups, and "lower alkyl" designates ^c ~ ^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.

DETAILED DESCRIPTION OF THE INVENTION

I. Carbohydrate-Modified Antibiotics

In accordance with this invention, 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.

SUBSTITUTE SHEET

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.

Examples of anthracyclinones are adriamycinones, rhodomycinones, isorhodomycinones, pyrromycinones, aklavinones, daunomycinones, nogalamycinones, steffimycinones, isoquinocyclinones, and trypanomycinones, each type including isomers and analogε differing by the presence and position of substituents on the anthracyclinone structure. Among 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. In preferred embodiments, the carbohydrate domain consists of one or more saccharide units in cyclic ether form. In the case of multiple units, or saccharide multimers, 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.

Examples of 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

SUBSTITUTESHEET

(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.

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. With three or more rings in the chain, ring-

SUBSTITϋTE SHEET

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.

II. Carbohydrates Prepared by Synthetic Means

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.

SUBSTITUTE SHEET

A. Glycal Synthesis and Scope

Certain glycals are available commercially. Methods of synthesis for glycals are known in the art.

For 6-membered ring glycals, 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. Examples of Lewis acids useful in the reaction are A1C1 ₃ , AlBr ₃ , BeCl ₂ , CdCl ₂ , ZnCl-., BF ₃ , BF ₃ -0(C ₂ H ₅ ) ₂ , BF ₃ -0(C ₂ H ₅ ) ₂ -Ce(OCOCH ₃ ) ₃ , BCI3, BBr ₃ , TiCl ₄ , ZrCl., MgBr ₂ , tris{3-(heptafluoropropyl)hydroxymethylene- d-camphorat0}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 .

Reaction conditions and procedures for both steps are extensively described in the literature. For such descriptions, one is referred to: Danishefsky, S.J., et al . , J. Jim. Chem. Soc. 104:3585

(1982) , Danishefsky, S.J., et al . , J. Org. Chem. 50:4650

(1985), Danishefsky, S.J., et al . , J. Am. Chem. Soc. 104:360 (1982),

Danishefsky, S.J., et al . , J. Am. Chem. Soc. 107:1256

(1985), Bednarski, M. , et al . , Tetrahedron Lett. 24:3451 (1983) , Bednarski, et al . , J. Am. Chem. Soc. 105:6968 (1983) ,

Danishefsky, S.J., et al . , J. Org. Chem. 50:3672 (1985),

SUBSTfTU E S T

Danishefsky, S.J., et al . , J. Am. Chem. Soc. 107:6647

(1985), Danishefsky, S.J., et al . , Aldrichim. Acta 19:59 (1986) , and Danishefsky, S.J., et al . , J. Am. Chem. Soc. 110:3929

(1988), each of which is specifically incorporated herein by reference. Glycals useful in the present invention will generally have substituents, i.e., groups other than hydrogen atoms, at various positions on the ring. 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 -C ₆ alkylsilyl groups such as diphenylmethylsilyl, aryl-di-C»-Cg alkylsilyl groups such as a phenyldimethylsilyl ether, and tri-C ₁ -C ₈ alkylsilyl groups such as trimethylsilyl and t-butyldimethylsilyl. Acetals and ketalε are also considered to contain ether linkages since each contains the C-O-C bond of an ether. Ether-forming protecting groups

SUBSTITUTE SHEET

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.

Removal of these protecting groups ("deprotection") is accomplished by methods well known in the art. For example, 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.

Selectivity in deprotection is achieved by appropriate selection of the protecting groups. For example, a hydroxyl protected by a benzyl protecting group can be deprotected selectively over silyl ether, acetal and ketal protecting groups. Tri-C,-Cg 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.

Certain glycals used in the practice of this invention will require substituents which are not readily removable. Included among such substituents are ether-type substituents such _as —o—R in which R is C^-C^g alkyl, Cg-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. Saccharides units themselves, bonded to the glycal through an ether-type bond such as an O-glycoside bond, are further substituents that are not readily removed.

SUBSTITUTE SHEET

Both electron-withdrawing and electron-donating substituents are used in the various synthetic methods used in connection with this invention. In addition, the substitution of an electron-donating group for an electron-withdrawing group is frequently an important step in the preparation of a saccharide multimer. Examples of substituents which are relatively electron-withdrawing are C ₁ -C _lg 0-acyl groups and C.-C,. O-carbamyl ester groups, specific examples being such groups formed from ethyl, tolyl isocyanate and l-(l-naphthyl)ethyl isocyanate. Examples of substituents which are relatively electron-donating are O-ether groups. These groups are particularly useful in reaction schemes involving haloglycosylation.

In reaction schemes which involve the conversion of a glycal to a 1,2-anhydrosugar, it is important that 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. Further examples are 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.

In many cases, one must prevent substituents from participating anchimerically in reactions involved in the synthesis scheme. Examples of such "participating" substituents are O-acyl and N-acyl protected substituents, particularly when present at the 4- and 6-positionε 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

HEET

group prior to the oxidative conversion and glycosylation steps. For example, prior to the conversion of a glycal to a 1,2-anhydrosugar, participating ^c _~ ^c _g acyl substituents are replaced with non-participating silyl ether substituents. Likewise, prior to the haloglycosylation step, participating groups are replaced with non-participating substituents such as ether linkages or cyclic imido groups, for example phthalimido groups.

When two glycals are joined in such a manner that one functions as a "glycosyl donor" and the other as a "glycosyl acceptor," O-acyl substituents are to be avoided on the "donor," εince these substituents are relatively electron-withdrawing and as a result will tend to diminish the distinction between the donor and acceptor character of the glycals, thereby slowing the rate of reaction. Substituents on the "acceptor," on the other hand, are preferably relatively electron-withdrawing in character. Examples are C^-C-g acyl groups present as O-acyl esters. Further examples are O-carbamate esters formed from isocyanates having 1 to about 14 carbon atoms, such as methyl, phenyl, tolyl and l-(l-naphthyl)ethyl isocyanates.

Whether 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. These relative sums are thus an indication of the electron- donating or -withdrawing character of the substituents. A partial listing of published Hammett sigma constants for para- substituents is provided by Hine, J. , Physical Organic Chemistry, 2d ed. , McGraw-Hill Book Company, Inc., New York, page 87 (1982). For substituents whose sigma values are not available in the literature, the skilled technician can approximate the value and its sign (positive or negative) my techniques known in the art. Hydrogen and N-acyl groups are each normally considered to be neither electron-withdrawing nor -donating since the Hammett sigma value system arbitrarily places hydrogen at zero and N- acyl groups typically also have zero sigma constant values for

SUBSTITUTE SHEET

the para-position. Relative to an O-acyl group, however, both hydrogen and N-acyl are electron donators.

A general formula for glycals serving as glycosyl donors in the practice of the present invention is as follows:

in which:

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^-Cg alkyl, 2-furyl, OR ⁴ , NR ⁵ R ⁶ or SR ⁶ ; or R 1 and R2 together form a cyclic acetal or ketal prepared from an aldehyde or ketone containing 1 to 12 carbon atoms;

R ³ is H, C^-Cg alkyl, OR ⁴ , (CH ₂ ) _m OR ⁴ , 2-furyl, NR ⁵ R ⁶ ,

SR CH(OR ⁴ )CH(OR ⁴ ) (CH,OR ⁴ )

HC CH,

or R ^J and R together, or R ^J and R ^*1 together, form a cyclic acetal or ketal prepared from an aldehyde or ketone containing 1 to 12 carbon atoms; m iε 1, 2, 3 or 4, such that the total number of carbon atoms in the ring plus the group is not greater than 9; n iε zero or 1;

R ⁴ iε C ₁ -C ₁₈ alkyl, Cg-C ₁₀ aryl, C ₇ -C ₁₀ aralkyl, tri- C^Cg alkylεilyl, diaryl-C^Cg alkylsilyl, aryl

SUBSTITUTESHEET

di-C -Cg alkylsilyl, or a substituted mono- or oligosaccharide; R ⁵ is C ₁ -C ₁₈ alkyl, Cg-C ₁₀ aryl, C ₇ -c ₁₀ aralkyl, tri- C^ _^ -Cg alkylsilyl, aryl di-C^Cg alkylsilyl, diaryl ^c ι~ ^c ₆ ^alk y ^lsil y ¹ / ^{or c} i ^_c i8 ^{ac 1 ;}

R ⁶ is H, C ₁ -C ₁₈ alkyl, C ₇ -C ₁₀ aralkyl, and C^-C^ 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 _lg 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 atomε;

R ⁷ and R ⁸ are independently H or C.,-C ₈ alkyl εuch that the number of carbon atoms in R plus those of R is nine or fewer; and

R ⁹ is selected from the group consisting of hydrogen (H) , or C ₁ -C ₆ alkyl, C0 ₂ R ⁴ , CN, CH-.OR ⁴ , 2-furyl,

R ¹⁰ is hydrogen (H) , 2-furyl, or C-^-Cg 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 0R ⁴ , and CH(OR ⁴ )CH ₂ 0H, 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 , R and R ³ includes an OH group.

B. Cyclic Ether Resolution

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

SUB ^S TITUTE SHE

ring is considered in a planar representation) , and the oth orientation being on the other side.

The types of stereoisomers thus resolved include isom pairε differing in relative configuration, such as diastereomer as well as those having the same relative configuration b differing in absolute configuration, such as enantiomers (al referred to as antipodes) . Cyclic ethers which are diastereome will differ at the hydroxyl substitution site but not at oth substitution sites on the ring, and will thus not be mirr images of each other. The relative orientation of the hydrox group and at least one other ring substituent will thus coequatorial in one diastereomer and transequatorial in anothe

As a result, diastereomers will generally display differences chemical and/or physical properties. Cyclic ethers which a enantiomers, however, are mirror images of each other, i.e., t various substituents will have the same equatorial orientati relative to each other in both isomers, but the number and/ location or the substituents, and/or other feature(s) of the ri such as the double bond of a glycal, render the molecu incapable of being superimposed over its mirror image. Su isomers display identical chemical and physical properties exce for the direction of rotation of polarized light.

This aspect of the invention is of particular intere in resolving enantiomers, particularly enantiomeric cyclic en ethers, and more particularly enantiomers of εix-membered ri glycals. Further preferred characteristics of the enantiomer 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 no hydroxyl substituent(s) ; and that the ring contain at least t non-hydroxyl substituents, all of which are at positions oth than position 1, preferably one being at position 4 or Examples of non-hydroxyl substituents are lower alkyl (i.e C^C _g alkyl, preferably ^{c _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.

Known 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.

Vinyl acetate and isoprenyl acetate are preferred. The particular lipase used is also subject to variation and thereby noncritical to the practice of the invention. Lipases εuch as

Lipaεe P and Lipase PS-30, both from species such as Pseudomonas cepacia , axe 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.

Among the various preferences to be expected are those involving glycals in which the acetylated hydroxyl group is at position 3 of the glycal. When the glycal ring is viewed such that the double bond carbons (i. e. , those at positions 1 and 2) immediately succeed the ring oxygen in the clockwise direction and the hydroxyl group is at position 3, 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 β-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. Once 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.

C. Saccharide Multimer Preparation

Of the various synthetic methods which may be used in the preparation of saccharide multimers in the practice of this invention, 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. 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, and preferred halonium reagents are (2,4,6-collidine) ₂ BrC10. and (2,4,6-collidine)IC10 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.

Since the reaction occurε under oxidative conditionε 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 groupε, and particularly aliphatic hydroxyl groupε, 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 siteε when thiε reaction iε used.

In the joining of two saccharide moieties through an

O-glycoside bond, the nucleophilic atom is preferably a hydroxyl oxygen. Preferably, a single hydroxyl oxygen atom is present on the nucleophile saccharide unit. For saccharide units that have more than one hydroxyl group, however, selectivity is readily achieved by protection of the hydroxyls where reaction is not desired. Once the haloglycosylation reaction has occurred, the resulting 2-position halo group can be inverted in orientation or substituted with another substituent. For example, a

2-position iodide can be reacted with phthalimide to form a nitrogen-containing substituent. As another example, 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.

In the reaction, 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 subεtituents, however, can be modified or controlled by steric hindrance. Thus, 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. For example, large substituentε at the 4- and 5- positions of a six-membered glycal tend to influence the side

SUBSTITUTE SHEET

toward which the nucleophile will successfully approach. Placing such substituents on the upper side thereby permits glycoside formation from the lower side, resulting in α-anomer formation. Haloglycosylation is generally conducted in a solvent that iε inert to the reaction conditionε. Exampleε 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., 22 ^β C.

The alternative synthesis method which proceeds through a 1,2-anhydrosugar intermediate avoids the 1,2-halonium species intermediate. Methods for forming 1,2-anhydrosugars are known in the literature. One such reaction is a dehydrohalogenation reaction aε reported by Sondheimer, et al . , Carbohydr. Res.

74:327 (1979). Such a reaction doeε not involve converεion of a glycal. Preferred methodε, however, are thoεe involving the converεion of a glycal by epoxidation using a dialkyl dioxirane.

Regardless of its method of preparation, a subεtituted 1,2-anhydrosugar can be represented by the general formula

1 _? 3 9 10 wherein R , R , R , and R are as described above in connection with the general glycal formula. Substituted 1,2-

SHEET

anhydrosugars useful in glycosylations according to this invention are those that are free of participating subεtituent groupε.

Dialkyl dioxiraneε 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 groupε. Exampleε are dimethyldioxirane, diethyldioxirane, methyl iεopropyldioxirane, methyl propyldioxirane, ethyl sec- butyldioxirane. Dimethyldioxirane (technically, 3,3-dimethyl- dioxirane) iε 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. In the formation of the 1,2-anhydrosugar by this reaction, 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. Thus, by selection of the size, location and orientation of the neighboring substituent groups, the 1,2-epoxide ring can be directed to the o>- or β-side of the ring. The conversion of a glycal to the corresponding 1,2- anhydrosugar is generally conducted at a temperature of from about -40 ^e C to about +20 " 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 100"C to facilitate isolation and recovery of the resulting 1,2-anhydrosugar by εolvent removal. Thiε 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 20"C so that the otherwise reactive epoxide ring does not react prematurely.

When the starting glycal contains one or more participating substituents, such subεtituentε εhould be removed and replaced with non-participating substituents prior to the reaction of the substituted 1,2-anhydrosugar with the nucleophile to be glycosylated. Preferably, this replacement is done prior to conversion of the glycal to the 1,2-anhydrosugar. The 1,2- anhydroεugar to be used in the subsequent glycosylation step will then contain only non-participating subεtituentε.

SUBSTITUTE SHEET

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) . The most nucleophilic hydroxyl groups are generally primary hydroxyls as compared to secondary or tertiary hydroxyls. Reducing sugarε contain a εingle 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 atomε of a pentose, hexose and heptose, respectively. Nonreducing sugarε (ketoεeε) contain two primary hydroxyl groups, one at the 1-position and the other at the 6-, 7-, 8-, and 9-positions, respectively, for hexuloseε, heptuloses, octuloses and nonuloses, εuch aε εialic acid derivativeε. Selective glycosylation at a particular hydroxyl group when more than one are present is readily achieved by using protecting groups at the hydroxylε other than that where the reaction iε to occur. The protecting groups can be participating or non-participating. The substituents on the 1,2-anhydrosugar, however, must be of the non-participating type.

The reaction is preferably conducted in the presence of a Lewiε 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 -78 ^C C to about room temperature (about 22 ^β C). Lewis acids uεeful in thiε reaction include SnCl ₄ , AgCl0 ₄ , BF ₃ , trimethylεilyl trifluoromethanesulfonate (triflate) , Zn(triflate) _? , Mg(triflate) ₂ , MgCl ₂ , A1C1 ₃ , ZnBr ₂ and ZnCl ₂ . Tri-n-butyltin salts of the glycal alcohol have also been successfully utilized. Lewis acid catalystε are typically utilized in ethereal or chlorinated solvents. Examples are diethylether, tetrahydrofuran, dimethoxyethane, methylene chloride and chloroform. In addition, reaction conditions are εelected or controlled to either prevent or minimize polymerization of the 1,2-anhydroεugar. Care iε also taken to avoid conditions in which the Lewis acid catalyst removes a protecting group, aε can occur with BC1 ₃ . Meanε of avoiding theεe occurrenceε will be

readily apparent to those skilled in the art. See Sharkey, et al . , Carbohydr. Res. 96:223 (1981).

Subsequent to the glycosylation reaction, one can proceed with manipulations of the εubεtituentε, including inversions and exchanges, by conventional methods known in the art, as needed for subsequent reactions.

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, εhape or form. Particularly convenient solid phases, however, are particulate materials.

In solid phase methods, 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 εupport.

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 groupε are alεo known. One εuch group iε 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 aε discussed herein or by other well known means. Terminal glycosides thuε linked are readily cleavable from the solid phase by known methods.

Suitable solid supportε 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 resinε of hydrophobic polymerized styrene crosε-linked with divinylbenzene, typically at about 0.5 to about 2 weight percent. Such resins can be

^S U ^B STITU ^T ESHEET

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 resinε. Although the reactive benzyl moietieε are typically added after the resin bead has been synthesized by reaction of a polymerized styrene moiety, such resinε are generally described as polymerized styrene crosεlinked with divinyl benzene and including a known amount of polymerized vinyl benzyl moiety.

Further exampleε of solid supports are silica- containing particles such as porous glasε beads and silica gel. Reactive benzyl moieties can be used in conjunction with these supports as well. Further examples are glaεε particleε 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 groupε 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. The particle-linked reactant is thereafter ready to use.

III. Conjugation Methods

Conjugation of the carbohydrate domain to the antibiotic nucleuε is achieved through one or more covalent bonds, either directly or through linking agents. For purposeε of convenience, the following diεcussion addreεses 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 specieε to the next, depending on εuch factorε 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.

SHEET

Owing to the general structure of carbohydrates, 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. Included among the atoms which can serve as nucleophilic atoms are 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. Thus, 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-anhydroεugar. The reaction then proceeds in essentially the same manner and under essentially the same conditions described above for the corresponding reactionε in the joining of εaccharideε, including the use of protecting groups where necessary.

As those knowledgeable in the technology of nucleophilic reactions will appreciate, certain types of nucleophiles exhibit improved reactivity under certain conditions. For example, a Lewis acid catalyst appearε to be required for most neutral nucleophiles such as alcoholε, amineε and mercaptans, whereas negatively charged nucleophiles such as azide ion or trimethylsilyl thiophenoxide do not require Lewis acidε. Also, when the reaction is conducted in the presence of methanol as a solvent, a Lewiε acid catalyst is generally not necesεary.

The reaction to form the carbohydrate and the reaction to conjugate the carbohydrate to the aglycone are generally performed εeparately. In preferred practice, the carbohydrate domain iε 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.

SUBSTITUTE SHEET

IV. Novel Anthracycline Antibiotics

Glycosylated anthracyclines represented by the following formula are novel:

R ¹ L (A)

R ^* ^

In this formula represents an anthracycline aglycone, and R represents one of the two enantiomers

in which R ³ is either aliphatic, aryl or aralkyl. The subclasε in which R 2 is the enantiomer (B) are

L-sugar species whereas those with enantiomer (C) are D-sugar species. The L-sugar subclass is preferred. In both subclasseε, the iodine atom iε in an orientation which is commonly termed "axial."

In addition to the L-sugar subclasε, further εubclaεεeε and embodimentε are preferred. Theεe include εubclasεes in which R ³ is lower alkyl, aryl or aryl-(lower alkyl), and particularly lower alkyl or aryl. The term "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. The term "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. The term "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. The term "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.

Of the aglycone sector, a preferred subclaεε is that which includes adriamycinone, daunomycinone, aklavinone, e-pyrromycinone, ---pyrroiiiycinone, β-rhodomycinone, δ-rhodomycinone, e-rhodomycinone, nogalamycinone, and steffimycinone. A further preferred subclass is that of adriamycinone, daunomycinone, aklavinone, e-pyrromycinone, ⁿ -pyrromycinone, β-rhodomycinone, δ-rhodomycinone, and e-rhodomycinone, and a still further preferred subclass is that of adriamycinone, daunomycinone, aklavinone, and e-pyrromycinone.

Examples of specific compoundε within preferred subclasses of the generic formula (A) above are aε followε:

Compound D:

Compound E:

SUBSTITUTE SHEET

Compound F:

Compound G:

Compound H:

Compound I:

B TITUTE SHEET

Compound J:

Compound K:

The aglycones in Compounds D through K are as follows: D, E: daunomycinone F, G: adriamycinone

H, I: e-pyrromycinone J, K: aklavinone The symbol "Ph" in these compounds denotes a phenyl group.

Compounds within the scope of generic formula (A) are useful as antibiotics in application to a variety of disease conditions, particularly those for which known anthracyclines are active, and particularly those which are resistant to other antibiotics, including conditions of multiple drug resiεtance.

Of particular interest is the use of these compounds as antineoplastics. Administration of the drugs to human subjects is done in accordance with the same dosages, formulations, and methods and frequency of administration which are used and known to be effective with known anthracycline antibiotics. These amounts and means of administration will be apparent to those knowledgeable in cancer therapy. In many cases, lesεer doεages

^S UBSTITUTESHEET

than those normally used for known anthracyclines will be sufficient since the activity of these new compounds is higher.

The following examples are offered for purposes of illustration, and are intended neither to define nor to limit the invention in any manner.

The reagents used in the following syntheseε were, unleεε otherwise indicated, obtained from commercial sources and used without further purification, and all reactions were carried out under inert atmosphere. Lipase PS-30 (from Pseudomonas cepacia) was obtained from Amano. The εolventε used were, unless otherwise indicated, freshly distilled under dry nitrogen. Tetrahydrofuran and ether were distilled from deep blue solutions of benzophenone ketyl, benzene from calcium hydride, dichloromethane from either calcium hydride or phosphorus pentoxide, BF ₃ ~etherate from calcium hydride, and ethanol from Na-diethyl phthalate. Benzaldehyde was distilled under reduced presεure. All reactionε requiring anhydrous conditions were conducted under a blanket of dry nitrogen in flame- or oven- dried glasεware. Column chromatography waε performed on silica gel 60

(E. Merck 9285, 230-400 mesh) . Thin layer chromatographic analyseε were conducted on 0.25mm silica gel plates with a 254nm ultraviolet indicator (E. Merck silica gel 60 F-254) . Spectroscopic analyεeε were performed on the following instruments: IR, Perkin Elmer 1420; UV-vis, Cary 219; NMR, 500MHz, Brucker WM500; NMR, 250MHz, Brucker WM 250. High resolution (El, CI and FAB) mass spectrometric analyses were conducted on a Kratos MS 80 RFA. Low resolution mass εpectrometric analyses were conducted on a Hewlett Packard 5985 masε εpectrometer. Melting pointε were taken on a Hoover capillary melting point apparatuε and are uncorrected. Optical rotations were taken on a Perkin Elmer 241 polarimeter. Elemental analyses were performed by Oneida Research Serviceε, Inc. Abbreviationε are uεed in theεe exampleε as follows:

Bz = benzoyl

benzyl = tert-butyldimethylεilyl trimethylsilyl = methyl ethyl acetyl trifluoroacetic acid 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 εtereoεpecific configuration.

SUBSTITUTE SHB

EXAMPLE 1

Preparation of 5,6-Dihydropyral

From Diene and Aldehyde

This example illustrateε the first step of the preparation. The reaction scheme for this stage is shown below.

(la.b.c) ((+,-)-2a) ((+,-)-2b)

2a: (2R ,3S )-3-Benzoyloxy-2-phenyl-2,3-dihydro-4H-pyran-4-one 2b: (2R ,3R )-3-Benzoyloxy-2-phenyl-2,3-dihydro-4H-pyran-4-one

(Note that the formulas for (+,-)-2a and (+,-)-2b shown above actually show only one enantiomer each, although the "(+,-)" notation indicates the racemate, which is intended in each case. The formulaε are shown in this manner to indicate the relative orientation of the substituents and to distinguish between the diastereomerε, each 2a being a diastereomer of each 2b. This method of indicating racemates of species which are also diastereomers is followed in the succeeding examples as well.)

To a solution of the diene mixture la,b,c (8.5g, 25.5mmol, prepared according to Danishefsky, S.J., et al . , J. Am. Chem. Soc. 107:1269-74 (1985)) and benzaldehyde (2.16mL, 21.2mmol) in methylene chloride (lOOmL) was added BF ₃ -etherate (2.16mL, 21.2mmol), dropwise and with stirring. The reaction mixture waε allowed to warm slowly to -40 ^β C over a period of 2 hours, after which time the reaction was quenched by the addition of NaHC0 ₃ (aqueous, 15mL) . The resulting two-phase mixture waε allowed to warm to room temperature, then poured into methylene chloride (250mL)/NaHCO ₃ (aqueous, 85mL) . The aqueous layer waε extracted once more with methylene chloride (lOOmL) , and the combined organic layers were washed with brine (lOOmL) , dried (MgS0 ₄ ) and evaporated.

The crude cycloaddition product mixture was disεolved in carbon tetrachloride (50mL) , and trifluoroacetic acid (2.13mL, 27.6mmol) was added dropwise with stirring at room temperature. After one hour, the reaction was quenched by the addition of NaHC0 ₃ (aqueouε, lOmL) , and then poured into methylene chloride (200mL)/NaHC0 ₃ (aqueous, 90mL) . The aqueous layer was extracted with a second portion of methylene chloride (lOOmL) , and the combined organic layers were washed with brine (50mL) , dried (MgS0 ₄ ) , and evaporated. Flash chromatography (25-30% diethylether/hexane) yielded, in order of elution, Compound 2b (401mg, 6%) as a slightly yellow oil, and Compound 2a (3.39g, 54%) as an off-white solid.

The structural formula of Compound 2a (melting point 89-91°C) was verified by ¹ H-NMR, IR, MS, HRMS and elemental analysiε. The structural formula of Compound 2b was verified by --Η-NMR, IR, MS, and HRMS.

EXAMPLE 2

Conversion of 5, 6-Dihydropyral to

Glycal Racemate

The reaction in this step is illustrated below.

((+,-)-2a) ((+.-)-3)

(+,-)-3: (2R ,3R , 4R ) -3-Benzoyloxy-4-hydroxy-2-phenyl-2 , 3- dihydro-4Jf-pyran

To a solution of the diastereomer Compound 2a (ll.95g, 6.63mmol) and CeCl ₃ -7H ₂ 0 (2.47g, 6.63mmol) in methylene chloride (57mL)/ethanol (28mL) at -78 °C was added, via syringe pump, a solution of NaBH ₄ (276mg, 7.30mmol) in ethanol (28mL) . The addition took 75 minutes. After an additional 1.5 hours at -78 °C, the reaction was complete (aε indicated by thin layer chromatography) , and was quenched by addition of NaHC0 ₃ (aqueous, 40mL) . The two-phase mixture was stirred and allowed to slowly

warm to 0°C, and was then poured into ethyl acetate (250mL)/ diethylether (250mL)/NaHC0 ₃ (aqueous, 200mL) . The organic layer was washed with NaHC0 ₃ (aqueous, 2 x 150mL) and brine (lOOmL) , dried (MgS0 ₄ ) and evaporated. Flash chromatography (20-40% ethyl acetate/hexane) yielded Compound 3 (1.82g, 93%) aε a racemic mixture of enantiomers in the form of a white solid, with melting point 129 ^β C. The structural formula of the product was verified aε that shown above by ¹ H-NMR, IR, MS, and HRMS.

EXAMPLE 3 Separation of Glycal Enantiomers by Selective

Acetylation, Followed by Conversion to 2,3-Diprotected Glycals

The reaction scheme illustrated in this step is as follows.

^^^OAc, Lipase PS-30

((-)-4) (( + )-3)

K ₂ C0 ₃ , MeOH I K ₂ C0 ₃ , MeOH i

((+)-5) I TMS-imidazole

((-)-6) (( + )-6)

(+)-3: (2S , 3S , 4S) -3-Benzoyloxy-4-hydroxy-2-phenyl-2 , 3- dihydro-4if-pyran

(-)-4: (2S , 3S , 4R) -4-Acetoxy-3-benzoyloxy-2-phenyl-2 , 3- dihydro-4i-pyran

(-)-5: (2R, 3R, 4R) -3 , 4-Dihydroxy-2-phenyl-2 , 3-dihydro-4fJ-pyran (+)-5: (2S , 3S , 4S) -3 , 4-Dihydroxy-2-phenyl-2 , 3-dihydro-4H-pyran (-)-6: (2R,3S,4R) -2-Phenyl-3 , 4-Jis- (trimethylεilyloxy) -2, 3- dihydro-4H-pyran (+)-6: (2S,3R,4S) -2-Phenyl-3 , 4-Jbis- (trimethylsilyloxy) -2, 3- dihydro-4#-pyran

To the racemic mixture of Compound 3 (l.OOg, 3.38mmol) in vinyl acetate (50mL, 542mmol)/dimethoxyethane (25mL) was added lipase PS-30 (6.0g), and the resulting εuspension was εtirred vigorouεly in a stoppered round-bottom flask for 7 days. The reaction was stopped by addition of diethylether (50mL) and filtration through a medium (ASTM 10-15) fritted funnel. The filter cake was washed with diethylether (3 x 15mL) and ethyl acetate (3 x 15mL) , and the combined filtrates concentrated in vacuo . Flash chromatography (30-80% diethylether/hexane) yielded, in order of elution, (-)-4 (568mg, 50%), melting point 99 ^β C, [α.] _D ²¹ -147" (c 1.16, CHCl ₃ ) , as a slightly yellow solid, and (+)-3, melting point 115-117 ^β C, [α] _D ²¹ +221° (c 1.06, CHC1 ₃ ), as a white εolid. The structure of the former was verified as that shown above by ¹ H-NMR, IR, MS, and HRMS. The latter displayed 1H-NMR and IR spectra identical to racemic Compound 3 (Example 2 above) , and was judged to be greater than 97% ee based upon the H-NMR spectrum of its Mosher ester(s) generated from (+)-α-methoxy-α-trifluoromethylphenylacetyl chloride.

To a solution of the diester (-)-4 (413mg, 1.22mmol) in methanol (15mL) was added ₂ C0 ₃ (lOlmg, 0.733mmol) . The reaction mixture was stirred at room temperature for 3 hours, then concentrated in vacuo and chromatographed (1-4% methanol/ methylene chloride) to yield (-)-5 (219mg, 85%) as a white solid, melting point 94-96 ^β C, [α] _D ²¹ -103" (c 1.16, CHC1 ₃ ) . The structure was verified as that shown above by H-NMR, IR, MS, and HRMS.

To a solution of the monoester (+)-3 (400mg, 1.35mmol) in methanol (lOmL) was added ₂ C0 ₃ (112mg, O.δlOmmol). The reaction mixture was stirred at room temperature for 2 hours, then concentrated in vacuo and chromatographed (2-4% methanol/methylene chloride) to give (+)-5 (221mg, 85%) as a foam, [α.] _D ²¹ +96.3" (c 0.865, CHC1 ₃ ) . This compound displayed

H-NMR and IR spectra identical to that of Compound (-)-5 above.

Returning to Compound (-)-5, a solution of the compound (185mg, 0.964mmol) in methylene chloride (6mL) was added TMS- imidazole (311μL, 2.12mmol), dropwise, and with stirring. After one hour, the reaction was complete, as indicated by thin-layer chromatography. The solvent was removed in vacuo , and the

residue chromatographed directly (2-3% diethylether/hexane) to give (-)-6 (272mg, 84%) as a clear, colorlesε oil, [α] _D ²¹ -68.3" (c 1.15, CHC1 ₃ ), with structure verified as that shown above by ¹ H-NMR, IR, MS, and HRMS, as follows: ^■ '"H-NMR (250MHz, CDC1 ₃ ): δ -0.209 (s, 9H) , 0.163 (s,

9H) , 3.81-3.83 (dd, .7=1.9, 4.1Hz, 1H) , 4.58-4.62 (apparent dt, .7=1.9, 6.2Hz, 1H) , 4.64-4.66 ( , 1H) , 4.95 (far S, IK), 6.44-6.47 (dd, J ^" =1.8, 6.2Hz, 1H) , 7.27-7.36 (m, 5H) ; IR (film): 3050, 3020, 2940, 2880, 1645, 1490, 1450,

1390, 1250, 1085cm ^"1 ; MS (CI) : m/Z 337 (1.7, MH ⁺ ) , 247 (100, -OTMS), 217 (22), 193 (19), 192 (62), 187 (13), 145 (31), 91(10) , 73 (16) ; HRMS (CI) : m/z (MH ⁺ ) calculated for C ₁₇ H ₂₈ 0 ₃ Si ₂

337.1655, observed 337.1647. Returning to Compound (+)-5, this compound (192mg, l.OOmmol) was treated with TMS-imidazole (352μL, 2.40mmol), just as for the antipode (-)-5, to give (+)-6 (301mg, 90%) as a clear, colorleεε oil, [QJ] _D ²¹ +69.4° (c 1.25, CHC1 ₃ ) . This compound displayed ¹ H-NMR and IR spectra identical to that of Compound (-)-6 above.

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.

SUBSTITW £SHE!

EXAMPLE 4

Conjugation to Aglycone —

Formation of D-Sugar Analog of Compound E

The reaction illustrated in this example is as follows.

D aun omy cinone

(7: R = TMS) (8: R = H)

7: (2*S,3 ^, S,4 ^, S,5*S,6 ^, R) -7-0-{2 •-(3 ^• -iodo-6 •-phenyl- 4 * ,5 *-Jis(trimethylsilyloxy) )tetrahydropyranosyl}- daunomycinone

8: (2 ^, S,3'S,4 ^, S,5 ^, R,6 ^« R)-7-0-{2 *-(4 ^• ,5 ^« -dihydroxy-3 '- iodo-6'-phenyl)tetrahydropyranoεyl}daunomycinone

A suspension of Compound (-)-6 (I32g, 0.393mmol), daunomycinone (188mg, 0.471mmol) and 4A molecular sieve powder (Aldrich, hand-ground, freshly activated, lOOmg) in CH ₂ C1 ₂ (18mL) was stirred for 1 hour at room temperature and then for 0.5 hour at 0°C in the dark. To the mixture was then added l (sym- collidine) ₂ C10 ₄ (205mg, 0.393mmol) in one portion, and stirring continued for 1 hour at 0°C in the dark. The reaction mixture was then diluted (lOmL CH ₂ C1 ₂ ) and filtered (Celite) . The filtrate was then washed with 10% Na ₂ S ₂ 0 ₃ (aqueous, 5mL) , saturated CuS0 ₄ (aqueous, 2 x 5mL) and brine (5mL) , then dried (MgS0 ₄ ), filtered and evaporated. Chromatography (25-35% ethyl

acetate/hexane) gave a mixture of isomeric coupling products (238mg, 70%) , greater than 90% of which was Compound 7, as established by ¹ H-NMR.

The mixture of isomeric coupling products (115mg, 0.134mmol) was dissolved in THF (12mL) , and HF-pyridine (600μL) 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 C1 ₂ (75mL) and quenched with NaHC0 ₃ (aqueous, 50mL) . The aqueous layer was extracted once more with CH-C1 ₂ (75mL) and the combined organic layers washed with CuS0 ₄

(aqueous, 75mL) and brine (75mL) . After drying (MgS0 ₄ ) , filtering and evaporating the solvent in vacuo , the product mixture was chromatographed (0.5-1.5% CH ₃ 0H/CH ₂ C1 ₂ ) to give

Compound 8 (the 2'S,3*S isomer) aε an orange powder. Later fractionε contained the (2-S,3*R) (daunomycinone axial, iodine eq.) iεomer (~5mg, 5%) and the (2'R,3 ^» R) (daunomycinone axial, iodine eq. ) isomer (~2mg, 2%), both established by ¹ H-NMR.

SUBST, _{TUTE SHEeτ}

EXAMPLE 5

Conjugation to Aglycone —

Formation of Compound E

The reaction illustrated in this example iε aε followε,

((+)-6) Daunomvcinone

(9: R = TMS) (10: R = H)

9: (2'R,3'R,4 ^I R _I 5 ^, R,6 ^, S) -7-0-{2 ^! -(3 ^• -iodo-6 •-phenyl- 4 ' ,5'-ibis(trimethylsilyloxy) )tetrahydropyranosyl}- daunomycinone

10: (2*R,3*R,4*R,5'S,6*S)-7-0-{2 •-( ' ,5>-dihydroxy-3 ^» - iodo-6 ^• -phenyl)tetrahydropyranosyl}daunomycinone

A suεpension of Compound (+)-6 (252g, 0.750mmol), daunomycinone (358mg, 0.750mmol) and 4A molecular sieve powder (310mg) in CH ₂ C1 ₂ (34mL) was treated with I(sym-collidine) ₂ C10 ₄ (390mg, 0.750mmol) in the same manner as in Example 4 to give a mixture of isomeric coupling products (412mg, 64%) , greater than 90% of which was Compound 9, as established by H-NMR.

A solution of the mixture of products (341mg, 0.400mmol) in THF (34mL) was desilylated with HF-pyridine (1.8mL) as in Example 4, over 11 hours. Chromatography yielded Compound

10 (the 2-R,3*R isomer) as an orange powder. Later fractions contained the (2*R,3*S) (daunomycinone axial, iodine eq. ) isomer (~10mg, 4%) and the (2*S,3'S) (daunomycinone axial, iodine eq.)

^• 1 isomer (~5mg, 2%) , both establiεhed by H-NMR.

EXAMPLE 6

Preparation of Additional Glycal

This example illustrates the preparation of a further glycal enantiomer. The overall reaction scheme is aε follows.

^OAc, Lipase PS-30

d Si0 ₉ )

L^ 'OTMS OTMS ((-)-15)

(+, -) -ll: (2R* , 3S*) -3-Acetoxy-2-methyl-2 , 3-dihydro-4#-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-4#- pyran; 3,4-Diacety1fucal

(+)-13: (2S,3R,4S)-3,4-Diacetoxy-2-methyl-2 ,3-dihydro-4H- pyran; 3,4-DiacetyIfucal

(-)-14: (2R,3R,4R) -3,4-Dihydroxy-2-methyl-2, 3-dihydro-4H- pyran; D-fucal

(-)-15: (2R,3S,4R)-2-Methyl-3,4-bis(trimethylsilyloxy)-2,3- dihydro-4#-pyran; 3,4-bis(Trimethylsilyl)-D-fucal

Following a known procedure with the conditionε indicated in the reaction scheme, according to Danishefsky, S.J., et al . , Tetrahedron Lett. 26:3411-12 (1985), and Danishefsky, S.J., et al., J. Am. Chem. Soc. 109:8119-20 (1987), the racemic mixture (+,-)-ll was prepared, resulting in an off-white solid, melting point 78-79°C, structure confirmed by H-NMR and IR.

The racemic mixture (200mg, l.lδmmol) was then combined with CeCl ₃ -7H ₂ 0 (438mg, l.lδmmol) in a CH ₂ C1 ₂ (12mL)/ethanol (6mL) solution. To the solution was added a solution of NaBH ₄ (49.0mg, 130mmol) in ethanol (6mL) at -78°C, via syringe pump over two hours. After an additional 2.5 hours at -78 ^β C, the reaction was complete according to TLC, and was accordingly quenched by addition of pH 7 buffer (KP0 ₄ , aqueous, lOmL) . The resulting two-phase mixture was stirred and allowed to warm to -50 ^β C, then poured into diethyl ether (60mL)/brine (10mL) . The aqueous layer waε extracted with diethyl ether (3 x 40mL) , and the combined organic layers were washed with brine (20mL) , dried (MgS0 ₄ ) and evaporated to give the racemic mixture (+,-)-12

(160mg, 79%) as a white solid, melting point 101°C, of sufficient purity to be used directly in the next step. The structure waε confirmed by H-NMR and IR. (Exposure of this compound to silica gel resulted in partial migration of the acetyl group from the 4- to the 3-position.)

To a solution of the racemic mixture (+,-)-12 (1.40mg, δ.lOmmol) in vinyl acetate (105mL, 1.14mol)/dimethoxyethane (52mL) was added lipase PS-30 (1.40g), and the resulting

suspension was εtirred vigorouεly in a εtoppered round-bottom flaεk for 33 hours. The reaction was then stopped by addition of diethyl ether (lOOmL) and filtration through a medium (ASTM 10- 15) fritted funnel. The filter cake was washed with diethyl ether (lOOmL) and ethyl acetate (2 x lOOmL) , and the combined filtrates were concentrated in vacuo . Flash chromatography (70% diethyl ether/pentane) yielded Compound (-)-13 (650mg, 37%) as a colorless solid, melting point 49-51°C, [Q!] _D ²¹ -8.53" (σ 0.950, acetone) . The structure was confirmed by ¹ H-NMR and IR as follows:

^■ "-H-NMR (250MHz, CDC1 ₃ ): δ 1.25-1.28 (d, -7=6.6Hz, 3H) , 2.01 (S, 3H) , 4.16-4.24 (br q, 7=6.6Hz, 1H) , 5.26- 5.29 (br d, 7=4.7Hz. 1H) , 5.55-5.59 (ddd, 7=1.0, 2.0, 4.7Hz), 1H) , 6.43-6.47 (dd, 7=1.9, 6.3Hz, 1H; IR (CHC1 ₃ ): 3020, 3000, 2930, 1740 (br) , 1650, 1375,

1200 (br) , 1095, lOδOcm ^"1 . This compound was judged to be *>97% ee based upon its ¹ H-NMR spectrum (250MHz, CCl ₄ :d _g -benzene (4:1)) in the presence of the chiral shift reagent (+)-Eu(hfc) ₃ . Upon further elution, a mixture of (optically enriched)

Compound (+)-12 and the corresponding 3-acetyl isomer (total of 712mg, 51%) was obtained. Thiε mixture (4.14mmol) waε dissolved in CH_C1 ₂ (40mL) , and 4-dimethylaminopyridine (51mg, 0.41mmol), triethylamine (6.92mL, 49.7mmol) and acetic anhydride (2.35mL, 24.8mmol) were sequentially added. After 3 hours at room temperature, the reaction mixture was concentrated in vacuo and chromatographed (30% diethyl ether/hexane) directly to give (optically enriched) Compound (+)-13 (876mg, 99%) as a colorlesε solid, melting point 47-50 ^β C, [of] _D ²¹ +5.72" (c 1.35, acetone). The H-NMR spectrum was identical to that of Compound (-)-l3, and the compound was judged to be 64% ee based upon its ¹ H-NMR spectrum (250MHz, CCl ₄ :d _g -benzene (4:1)) in the presence of the chiral shift reagent (+)-Eu(hfc) ₃ .

The diacetate (-)-13 was converted to the diol (-)-14 by dissolving the diacetate (500mg, 2.34mmol) in methanol (20mL) and adding K-.C0 ₃ (194mg, 1.40mmol). The reaction mixture waε stirred at room temperature for 2 hours, then concentrated in vacuo and chromatographed (5% methanol/CH ₂ Cl ₂ ) to yield the diol

(-)-14 (2δ0mg, 92%) as a white solid, melting point 72-73°C, [Q] _D ²¹ -lδ.9° (c 1.25, CHC1 ₃ ), with structure confirmed by ¹ H-NMR and IR.

The diol (-)-14 (235mg, l.δlmmol) was then dissolved in CH ₂ C1 ₂ (13mL) , and a first portion of TMS-imidazole (637μL, 4.34mmol) was added, followed after 5 hours by a second portion (239μL, 1.63mmol). After a total of six hours, the solvent was removed in vacuo, and the residue chromatographed directly (5% diethyl ether/hexane) to give Compound (-)-15 (447mg, 90%) as a clear, colorlesε oil, [Q!] _D ²¹ -39.2° (σ 1.59, CHC1 ₃ ) , with structure confirmed by H-NMR and IR as follows:

¹ H-NMR (250MHz, CDC1 ₃ ): δ 0.135 (s, 9H) , 0.145 (s, 9H) ,

1.25-1.27 (d, 7=6.5Hz, 3H) , 3.64-3.65 (br d,

7=4.3Hz, 1H) , 3.96-4.03 (q, 7=6.5Hz, 1H) , 4.38- 4.40 (m, 1H) , 4.47-4.51 (apparent dt, 7=1.8,

6.3HZ, 1H) , 6.26-6.29 (dd, 7=1.δ, 6.3Hz, 1H) IR (film): 3060, 2950, 2680, 1645, 1395, 1370, 1250, 1195, 1110cm ^"1 .

EXAMPLE 7 Conjugation to Aglycone —

Formation of D-Sugar Analog of Compound D

The examples illustrateε the conjugation of the glycal prepared in Example 6 to daunomycinone. The reaction iε aε followε.

SUBSTITUTE SHEET

((-)-15) Daunomycinone

(16: R = TMS) (17: R = H)

16: 7 - O - ( 2 ^• , 6 * -D ideoxy- 2 ' - i odo-3 ' , 4 ' - bi s (trimethylsilyloxy) -C--D-talo-pyranosyl) - daunomycinone

17: 7-0- (2 ^■ , 6 ' -Dideoxy-2 * -iodo-oi-D- talo-pyranoεy1) - daunomycinone

A suspension of Compound (-)-l5 (300mg, 1.09mmol), daunomycinone (523mg, 1.31mmol) and 4A molecular sieve powder (450mg) in CH ₂ C1 ₂ (50mL) was treated with I(sym-collidine) _C10 ₄ (570mg, 1.09mmol) in the same manner as that described in Example 4, to give a mixture of isomeric coupling products (358mg, 41%), of which greater than 90% was Compound 16, as verified by ^• '-H-NMR.

A solution of the isomeric mixture (29δmg, 0.373mmol) in THF (34mL) was deεilylated with HF-pyridine (1.7mL), alεo aε in Example 4, for eighteen hours total. Chromatography (1-4%

CH ₃ 0H/CH ₂ C1 ₂ ) gave Compound 17 as a red glass, [c] _D 21 -30.1° (c 0.095, CHC1 ₃ ) . Its structure was verified by ¹ H-NMR and IR as follows:

¹ H-NMR (250MHz, CDC1 ₃ ): δ 1.31-1.34 (d, 7=6.6Hz, 3H) , 1.84-1.69 (d, 7=11HZ, 1H) , 1.93-2.00 (dd, 7=3.6,

15HZ, IH) , 2.39 (s, 3H) , 2.39-2.45 (br d, IH) , 2.73-2.77 (d, 7=10Hz, IH) , 2.96-3.06 (d, 7 ^" =19Hz, IH) , 3.21-3.29 (br d, 7=19Hz, IH) , 3.27-3.35 (m, IH) , 3.73-3.78 (dd, 7=1.8, 11Hz, IH) , 4.09 (ε, 3H) , 4.26 (S, IH) , 4.27-4.29 (d, 7=4.9Hz, IH) ,

4.48-4.58 (br q, 7=6.6Hz, IH) , 5.52-5.54 (m, IH) , 5.74 (br 8, IH) , 7.38-7.42 (d, 7=δ.5Hz, IH) , 7.76- 7.82 (apparent t, 7=8Hz, IH) , δ.02-δ.06 (dd, 7=0.8, 7.4HZ, IH) , 13.27 (ε, IH) , 14.14 (s, IH) ; IR (KBr) : 3150-3600 (br, OH), 2980, 2940, 1715, 1620,

1580, 1415, 1385, 1355, 1290, 1240, 1215,

1000cm .

A later fraction contained the corresponding a-D-galacto- pyranoεyl (daunomycinone eq. , iodine eq.) iεomer (~3mg, 1%), verified by ¹ H-NMR (250MHz, CDC1 ₃ ) : anomeric proton δ 5.02-5.06

(d, 7=8.6Hz) .

^S UB ^S T ^I TUTE SHEET

EXAMPLE 8 Formation of Compound D

The reaction illustrated in this example is as follows.

(21) Daunomycinone

(22: R = TMS) (23: R = H)

21: di-silylfucal 22: (2 ^» R,3 ^« R,4'R,5 ^» R,6 ^« S)-7-0-{2'-(3 ^, -iodo-6'-methyl- 4 ' , 5 ' -bis (trimethylsilyloxy) )tetrahydropyranosyl}- daunomycinone

23: (2'R,3'R,4 ^* R,5 ^* S,6 ^* S)-7-0-{2'-(4 ^, ,5'-dihydroxy-3'- iodo-6'-methyl)tetrahydropyranosyl}daunomycinone

To a solution of daunomycinone (199mg, 0.500mmol) and di-silylfucal 21 (137mg, 0.500mmol) in dichloromethane (15mL) waε added 4A-εieveε (150mg) . The reεulting mixture waε cooled to 0°C in an ice bath and εtirred for 0.5 hour. The mixture was then treated with I(sym-collidine) ₂ C10 ₄ (90%, 240mg) . After εtirring at 0°C for 0.5 hour, the mixture was diluted with dichloromethane (30mL) and filtered through celite. The filtrate was washed with 10% sodium thiosulfate (lOmL) , 10% copper sulfate

(10mL) , water (lOmL) , and brine (lOmL) . The organic layer was dried (MgS0 ₄ ) and concentrated. Flash chromatography on 2% KH ₂ P0 ₄ -silica gel (1:1 hexane/ethyl acetate) furnished 211mg (53%) of a 7:1 mixture of Compound No. 22 and its diequatorial isomer. The structures were confirmed by ¹ H-NMR, IR, and HRMS. To a stirred solution of Compound No. 22 (211mg, 0.264mmol) at 0°C was added HF-pyridine complex (0.7mL) . The mixture was stirred at room temperature for 2.0 hours, neutralized with saturated sodium bicarbonate, and extracted with dichloromethane. The combined extracts were washed with 10% copper sulfate (15mL) , water (lOmL) and brine (lOmL) . Flash chromatography on 2% KH ₂ P0 ₄ ~silica gel (30:1 chloroform/methanol) furnished Compound No. 23 (122mg, 71%) as a red solid. Its structure was verified by ¹ H-NMR, IR, and HRMS, as follows. "-"H-NMR (250MHz, CDCl ₃ ) : δ 14.02 (s, IH) , 13.24 (ε, IH) ,

8.02 (dd, 7=7.8, O.δHZ, IH) , 7.79 (app t, 7=δ.2Hz, IH) , 7.39 (d, 7=6.5Hz, IH) , 5.96 (br s, IH) , 5.25 (br d, 7=3.6Hz, IH) , 4.37 (br d, 7=4.8Hz, IH) , 4.29 (q, J=6.6Hz, IH) , 4.09 (s, 3H) , 4.06 (s, IH, 9-0H) , 3.82 (d, 7=11.0Hz, IH) . 3.30 (m, IH) , 3.23

(d, 7=18.8Hz, IH) , 2.92 (d, 7=18.9Hz, IH) , 2.72 (d, 7=10.1Hz, IH, -OH), 2.41 (s, 3H) , 2.33 (d, T-=15.1HZ, IH) , 2.14 (dd, 7=15.0, 4.3Hz, IH) , 1.93 (d, 7=10.9HZ, IH, -OH), 1.37 (d, 7=6.6Hz, 3H) ; IR (CHC1-) : 3510, 2910, 1715, 1620, 1580, 1450, 1415,

^J -1 1400, 1290cm ,*

HRMS (FAB) jn/e 677.0488 (M+Na) ⁺ calculated for

C ₂₇ H ₂₇ 0 _1]L NaI: 677.0496.

Examples 9 through 11 illustrate the preparation of a trisaccharide of a particular stereospecific configuration, and the coupling of the trisaccharide to anthracyclines.

EXAMPLE 9 Trisaccharide Synthesis

The trisaccharide prepared in this example is 1,5- anhydro-2 , 6-dideoxy-4-0-{4-0- (2,3, 6-trideoxy-α.-L-ςrlycero- hexopyranosid-4-ulose)-2,6-dideoxy-3-0-trimethylsilyl-α.-L- lyxo- hexopyranosyl}-3-O-trimethylsilyl-L-lyxO-hex-1-enitol. The reaction εcheme used for its preparation is shown in two stageε below.

(41 - 43)

59

10

15

50 TMS TMS

A. Preparation of l,5-Anhydro-4-o-(4-0-benzyl-2,3,6- trideoxy-α-L-araJ ino-hexapyranosyl) -3 -O-tert - butyldimethylεilyl-2,6-dideoxy-L-Iyxo-hex-l-enitol (Compound 43)

A solution of 2,6-diacetoxy-l,5-anhydro-L-2y_s:o-hex-l- enitol (506mg, 2.36mmol) in methanol (106mL) was treated with potasεium carbonate (33mg, 0.236mmol) . The resulting mixture was stirred for 3h at room temperature. Evaporation of the solvent afforded crude product as a slightly yellow oil. This was purified by flash chromatography (9δ:2 chloroform/methanol) to give the diol (297mg 97%) as colorless needles, m.p. 71-72°C, [Q!] _D ²² = +lδ.0°(σ 0.77, CHC1 ₃ ) . The structure was verified by "•"H-NMR and IR.

A solution of the diol (2.0g, 15.4mmol) in dichloromethane (lOOmL) , triethylamine (10.4mL, 74.6mmol) was treated with benzoyl chloride (4.3g, 30.6mmol). The resulting mixture was stirred at room temperature for 4h. At that time excess benzoyl chloride was quenched by the addition of methanol (lmL) . Saturated sodium bicarbonate solution (200mL) was then added. The resulting mixture was extracted (3 x 200mL) with dichloromethane. The combined organic extracts were washed with brine and dried over anhydrous magnesium sulfate. Filtration and concentration in vacuo followed by flash chromatography (95:5 hexane/ethyl acetate) afforded 2.80g (78%) of l,5-anhydro-3-0- benzoyl-2,6-dideoxy-L-IyxO-hex-l-enitol (referred to below as the "3-benzoate") ; 805mg (16%) of 3,4-dibenzoate; and 210mg (6%) of the 4-benzoate, as colorless oils. The structure of the 3- benzoate, with [α] _D ²² = +66.3° (c 0.54, CHC1 ₃ ), was verified by - ^• -H-NMR, IR and HRMS. The benzyl ether reactant, l,5-anhydro-4-θ-benzyl-

2,3,6-trideoxy-L-erythro-hex-l-enitol, waε then formed as follows.

To a cold (0°C) solution of l,5-anhydro-4-0-hydroxy- 2,3 , 6-trideoxy-L-erythro-hex-l-enitol (597mg, 5.30mmol) in tetrahydrofuran (20mL) was added solid sodium hydride (60% in oil, 314mg, 7.δ5mmol) . After stirring 30min, benzyl bromide

(0.93mL, 7.8mmol) was added. This mixture was stirred at room temperature for 14h. At that time excess sodium hydride was quenched by addition of water (50mL) . The resulting mixture was then extracted (3 x lOOmL) with ether. The combined ethereal extracts were washed with brine and dried over anhydrous magnesium εulfate. Filtration and concentration furniεhed the crude benzyl ether. The residue was purified by flash chromatography (25:1 hexane/ether) . The benzyl ether was further purified by fractional distillation under vacuum (130-140'C, 15 mm Hg) to afford pure l,5-anhydro-4-0-benzyl-2,3,6-trideoxy-L- erythro-hex-1-enitol (902mg, 96%) as a colorless oil, [α_] _D ²³ = - 120.0° (c 1.05, CHC1 ₃ ), its εtructure verified by ¹ H-NMR, IR and HRMS.

To a solution of the benzyl ether, which may also be viewed as a glycal (7.0g, 34.3mmol), and the benzoate, which may also be viewed as an alcohol (8.85g, 37.8mmol), in dichloromethane (600mL) was added 4A-sieveε (12.0g). The resulting mixture was stirred at room temperature for 30min, cooled to 0° in an ice bath and stirred for an additional 30min. The cold reaction mixture was then treated with I(sym-collidine) ₂ C10 ₄ (4) (90%, 23.2g, 44.5mmol). After stirring at 0" for 30min, 10% sodium thiosulfate solution (200mL) was added and the mixture filtered through celite. The two-phase mixture was then extracted (3 x 500mL) with dichloromethane, the organic phases were combined, dried (MgS0 ₄ ) and concentrated. Flash chromatography (9:1 hexane/ethyl acetate) afforded the iodide (Compound 41) (12.8g).

Without further purification 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. Evaporation of the solvent provided crude product which was purified by flash chromatography (9:1 hexane/ethyl acetate) to furnish the benzoate, l,5-anhydro-3-0-benzyl-4-0-(4-0-benzyl-2,3,6-trideoxy- α-L-araJino-hexapyranosyl) -2, 6-dideoxy-L-lyxO-hex-l-enitol (Compound 42) (δ.51g, 57%), as a colorless oil, [α] _D ²³ = +43.9° (c 0.46, CHC1 ₃ ) , itε structure verified by ¹ H-NMR, IR and HRMS.

A solution of Compound 42 (5.26g, 12.0mmol) in tetrahydrofuran (250mL) was cooled to 0° in an ice bath and treated with lithium aluminum hydride (1M solution in ether, 24.7mL, 24.7mmol), which was added in a dropwise manner. The mixture was stirred at 0" for 30min. Saturated ammonium chloride solution (2mL) and anhydrous magnesium sulfate were then added and the resulting mixture filtered, and concentrated in vacuo to afford a slightly yellow oil. Flash chromatography (9:1 hexane/ether) provided the corresponding alcohol (2.98g, 75%) as a colorless oil, [α] _D ²³ = -85.6° (c 1.08, CHC1 ₃ ) , its structure verified by "-"H-NMR, IR and HRMS.

A solution of the alcohol (3.38g, lO.lmmol) in dichloromethane (300mL) was treated with tert-butyldimethylsilyl chloride (6.1g, 40.5mmol) and imidazole (4.13g, 60.7mmol) . The mixture was then heated at reflux for 29h and concentrated in vacuo to afford crude product. The residue was purified by flash chromatography (95:5 hexane/ethyl acetate) to afford the ether, 1, 5-anhydro-4-0- (4-0-benzyl-2 , 3 , 6-trideoxy-a_-L-araJbi.no- hexapyranoεy1) -3-0-tert-butyldimethylεilyl-2, 6-dideoxy-L-lyχ-o- hex-1-enitol (Compound 43) (3.21g, 80%) as a colorless oil.

Compound 43 Verification:

[C-] _D ²³ = -39.9° (c 0.72, CHC1 ₃ );

"--H-NMR (CDC1 ₃ , 250 MHz) δ 7.33 (m, 5H) , 6.23 (d,

7-=6.1Hz, IH) , 4.92 (d, 7=2.8Hz, IH) , 4.73 (dd, J=4.8, 6.1HZ, IH) , 4.67 (d, 7-=11.9Hz, IH) , 4.48

(d, 7-=11.9Hz, IH) , 4.38-4.26 (m, 2H) , 3.95 (t,

T-=4.1HZ, IH) , 3.91 (m, IH) , 3.08 (dt, 7 ^" =4.0,

10.0Hz, IH) , 2.04-1.69 (m, 4H) , 1.39 (d, 7=6.8Hz,

3H) , 1.25 (d, 7=6.2HZ, 3H) , 0.91 (s, 9H) , 0.12 (s, 3H) , 0.11 (s, 3H) ;

IR (CHC1 ₃ ) 3018, 3010, 1648, 1460, 1390, 1360, 1255,

1130, 1110, 1080cm .- ^" 1.

HRMS m m//ee 444499..:2706 (M+H) ⁺ ; Calculated for C ₂₅ H ₄₁ 0 ₅ Si: 449.2724.

B. Preparation of 1,5-Anhydro-2,6-dideoxy-4-0-{4-0- (2,3,6-trideoxy-o;-L-glyσero-hexopyranosid-4-ulose)-2,6- dideoxy-3-0-trimethylsilyl-o;-L-lyxo-hexopyranosyl}-3- O-trimethylsilyl-L-lyxo-hex-l-enitol (Compound 50)

To a solution of Compound 43 (338mg, 0.753mmol) and

1 ,5-anhydro-3-0-benzoyl-2,6-dideoxy-L-lyxo-hex-l-enitol (the "3- benzoate" also used as a starting material in the preceding section) (194mg, 0.828mmol) in dichloromethane (20mL) was added 4A-sieveε (300mg) . The mixture waε εtirred at room temperature for 30min, cooled to -78" in a dry ice/acetone bath and stirred for an additional 30min. The cold reaction mixture was then treated with I(sym-collidine) ₂ C10 ₄ (90%, 510mg, 0.979mmol). After stirring at -78°C for 30min, 10% sodium thiosulfate solution (20mL) was added and the mixture filtered through celite. The resulting two-phase mixture was then extracted (3 x 30mL) with dichloromethane, the organic phaseε were combined, dried (MgS0 ₄ ) and concentrated. Flash chromatography (95:5 hexane/ethyl acetate) afforded the iodide (332mg, 54%) as a colorless oil, [α] _D ²² = -8.7" (σ 0.31, CHC1 ₃ ) , its structure verified by - ^* Η-NMR, IR and elemental analysiε.

A solution of the iodide (279mg, 0.345mmol) in benzene (lOmL) was treated with triphenyltin hydride (145mg, 0.413mmol) and azobisiεobutyronitrile (lOmg, 0.06mmol). The mixture was then heated at reflux for 30min. The reaction mixture waε then cooled to room temperature and concentrated to afford crude product. Flash chromatography (95:5 hexane/ethylacetate) afforded Compound 44 (220mg, 93%) as a colorlesε glaεε, [α.] _D ²³ = -30.0° (c 0.24 CHC1 ₃ ) , itε structure verified by ¹ H-NMR, IR and elemental analysiε. A εolution of Compound 44 (1.70g, 2.49mmol) in ether

(120mL) was cooled to 0 ^β in an ice bath and treated with lithium aluminum hydride (lΛ solution in ether, 2.5mL, 2.5mmol), which was added in a dropwise manner. The mixture was stirred at 0° for 1.5h. Saturated ammonium chloride solution (0.5mL) and anhydrous magnesium sulfate were then added and the resulting mixture filtered, and concentrated in vacuo to afford crude

SUBSTITUTE SHEET

alcohol. The alcohol was dissolved in dimethylformamide (50mL) and tert-butyldimethylsilyl chloride (1.12g, 7.43mmol) and imidazole (678mg, 9.96mmol) were added. The mixture was then heated to 50" for 4h. Water (200mL) was added and the mixture was extracted (3 x lOOmL) with benzene. The combined organic extracts were then washed with brine, dried over anhydrous magnesium sulfate, filtered, and concentrated in vacuo. Flash chromatography (95:5 hexane/ethyl acetate) furnished the silylether, 1,5-anhydro-4-0-{4-0-(4-0-benzyl-2,3,6-trideoxy-α;- L-arabino-hexopyranosyl)-3 -O-tert-butyldimethy1silyl-cu-L-lyxO- hexopyranosyl}-3-0-tert-butyldimethylsilyl-2,6-dideooxy-L-ly xo- hex-1-enitol (Compound 45) (1.37 g, 79%) as a colorless oil, [α;] _D ²³ = -89.6° (c 0.43, CHC1 ₃ ), its structure verified by ^■■ -H-NMR, IR and elemental analysiε. Liquid ammonia (ca. 130mL) waε collected via a dry ice condenεer in a 500mL 3-necked flaεk cooled to -78° in a dry ice/acetone bath and sodium metal (445mg, 19.35mmol) was added. To the resulting dark blue solution was then added a solution of the silylether 45 (1.34g, 1.93mmol) in tetrahydrofuran (60mL) . After stirring for lOmin, solid ammonium chloride was added at which time the solution became colorless. The dry ice condenser and dry ice/acetone bath were removed and the liquid ammonia was allowed to evaporate as the mixture εlowly warmed to room temperature. The resulting slurry waε diluted with chloroform (200mL) and dried over anhydrous magnesium sulfate, filtered, and concentrated in vacuo to afford the crude alcohol, Compound 46. The alcohol was then dissolved in dichloromethane (lOOmL) and treated with triethylamine (1.35mL, 9.69mmol), 4-dimethylamino pyridine (47mg, 0.385mmol), and acetic anhydride (0.92mL, 9.75mmol). The resulting mixture was stirred at room temperature for 12h. Saturated sodium bicarbonate solution (lOOmL) was then added and the mixture was extracted (3 x 150mL) with dichloromethane. The combined organic extracts were dried over anhydrous magnesium sulfate, filtered, and concentrated in vacuo to provide the crude acetate, 4-θ-{4-θ- (4-0-Acetoxy-2,3,6-trideoxy-αι-L-arajbino-hexopyranosyl)-3 -O-tert- butyldimethylεilyl-cϋ-L-lyxO-hexopyranoεyl}-l,5-anhydro-3 -0-tert- butyldimethylεilyl-2,6-dideoxy-L-2yxO-hex-l-enitol (Compound47) .

Flash chromatography (95:5 hexane/ethyl acetate) furnished the acetate (1.09g, 87%) as a colorless oil, [α] _D ²² = -67.8° (c 0.18, CHC1 ₃ ) , its structure verified by H-NMR, IR and elemental analysis. A solution of Compound 47 (2.90g, 4.50mmol) in tetrahydrofuran (200mL) was cooled to 0° in an ice bath. To this cooled solution was added tetrabutylammonium fluoride (lΛf solution in tetrahydrofuran, 18.0mL, lδ.Ommol) . The resulting mixture was stirred at room temperature for 5h. Evaporation of the solvent afforded crude product which was purified by flash chromatography (9:1 hexane/ethyl acetate) to furnish the diol, 4-0-{4-0-(4-0-acetyl-2,3,6-trideoxy-α;-L-arajbino-hexopyran osyl)- 2,6-dideoxy-α-L-lyxO-hexopyranosyl}-l,5-anhydro-2,6-dideoxy -L- IyxO-hex-1-enitol (Compound 48) (l.δ7g, 100%) as a colorlesε oil, [α] _D ²² ~ -119.5° (σ 0.55, CHC1 ₃ ), its structure verified by ^• *Η-NMR, ¹³ C-NMR, IR and elemental analysis.

A solution of Compound 48 (590mg, 1.42mmol), triethylamine (1.19mL, 8.54mmol), and 4-dimethylamino pyridine

(17.3mg, 0.142mmol) in dichloromethane (60mL) was cooled to 0° in an ice bath. The cooled solution was treated with trimethylsilyl chloride (0.72mL, 5.67mmol) which waε added in a dropwise manner. The mixture was stirred at 0° for 30min. Water

(50mL) was then added and resulting mixture was extracted (3 x lOOmL) with dichloromethane. The combined organic extracts were then dried over anhydrous magneεium sulfate, filtered, and concentrated in vacuo to furnish crude product. Flash chromatography (9:1 hexane/ethyl acetate) afforded 4-0-{4-0-4-

O-acetyl-2,3,6-trideoxy-α_-L-arajb±no-hexopyranosyl)-2, 6-dideoxy-

3-0-trimethylsilyl-0!-L-lyχ-o-hexopyranosyl}-l,5-anhydro -2,6- dideoxy-3-0-trimethylsilyl-L-Iy_XO-hex-l-enitol (Compound 49)

(785mg, 99%) as a colorless oil, [α] _D ²² = -164.2° (c 0.19,

CHC1 ₃ ), its structure verified by - ^* Η-NMR, IR and HRMS.

A solution of Compound 49 (726mg, 1.29mmol) in tetrahydrofuran (76mL) was cooled to 0° in an ice bath. The solution was treated with lithium aluminum hydride (1M solution in ether, 0.97mL, 0.97mmol) which was added in a dropwise manner. The resulting mixture waε stirred at 0° for 30min. Saturated ammonium chloride solution (0.5mL) and anhydrous magnesium

εulfate were added. The mixture waε then filtered, and concentrated in vacuo to afford crude product. Flaεh chromatography (3:1 hexane/ethyl acetate) provided the correεponding alcohol (614mg, 82%) as a colorless glasε, [α.] _D = -123.6° (c 0.28, CHC1 ₃ ), itε structure verified by -"-H-NMR, IR and elemental analysis.

A solution of the alcohol (300mg, 0.578mmol) in dichloromethane (45mL) was treated with εodium bicarbonate (1.75g, 20.8mmol) and the Dess-Martin periodinate (590mg, 1.39mmol). The mixture was stirred at room temperature for 50min, 10% sodium thiosulfate solution (50mL) and saturated sodium bicarbonate solution (50mL) were then added. The resulting biphasic mixture was stirred for an additional 30min. The mixture was then extracted (3 x lOOmL) with dichloromethane. The combined organic extracts were dried over anhydrous magnesium sulfate, filtered and concentrated in vacuo to afford crude ketone. Flash chromatography (9:1 hexane/ethyl acetate) furnished the ketone, l,5-anhydro-2,6-dideoxy-4-0-{4-0-(2,3,6- trideoxy-α_-L-g_lycero-hexopyranoεid-4-ulose)-2,6-dideoxy- 3-0- trimethylsilyl-c--L-lyxo-hexopyranosyl}-3-0-trimethylsilyl-L - lyxo-hex-l-enitol (Compound 50) (286mg, 95%) aε a colorless solid. Recrystallization from ethanol-H ₂ 0 gave colorlesε needleε.

Compound 50 Structure Verification: mp 104-105 ^β C;

[α!] _D ²² = -271.4° (c 0.28 CHC1 ₃ );

^■ • ^■ H-NMR (CDC1 ₃ , 250MHZ) δ 6.24 (dd, -7=1.6, 6.2Hz, IH) ,

5.12-5.06 (m, 2H) , 4.90 (q, 7=6.6Hz, IH) , 4.60

(dd, 7-=3.0, 6.2Hz, IH) , 4.40 (br ε, IH) , 4.33 (q, J-=6.7HZ, IH) , 4.28-4.15 (m, IH) , 4.15 (q, 7=6.7Hz ,

IH) , 3.78-3.70 (m, 2H) , 2.69 (dt, J=7.7 , 15.7Hz, IH) , 2.42 (dt, 7-=6.3, 15.7Hz, IH) , 2.28-2.17 (m, 2H) , 2.06 (dt, 7=3.6, 13.5Hz, IH) , 1.76 (dd, .7=4.2, 13.5HZ, IH) , 1.33 (d, 7=6.7Hz, 3H) , 1.26 (d, 7=6.7Hz, 3H) , 1.22 (d, 7=6.6Hz , 3H) , 0.16 (ε,

9H) , 0.13 (ε, 9H) ;

IR (CHC1 ₃ ) 2960, 2920, 1714, 1632, 1245, 1160, 1100,

1018cm ^"1 ; Elemental Analysis: Calculated for C ₂₄ H ₄₄ 0 _g Si ₂ : C,

55.78%; H, 8.58%; Si, 10.87%. Found: C, 55.72%; H, 8.69; Si, 11.24%.

EXAMPLE 10 Synthesis of Ciclamycin 0 and Epimer

This example illustrates the coupling of the trisaccharide (Compound 50) to e-pyrromycinone. The εtructure of e-pyrromycinone and the reaction εchemeε followed in thiε example are εhown below.

SUBSTITUTE SHEET

Compound No. R'

51 H I TMS TMS

52 I H TMS TMS

53 H I TMS H

54 H I H H

55 I H H H

56 H H H H (Ciclamycin O)

To a εolution of e-pyrromycinone (886mg, 2.07mmol) and ketone (Compound 50) (1.07g, 2.07mmol) in dichloromethane (200mL) waε added 4A - sieves (1.5g) . The mixture was stirred at room temperature for 30min, cooled to 0° in an ice bath and stirred for an additional 30min. The cold reaction mixture was then treated with I(sym-collidine) ₂ C10 ₄ (90%, 1.40g, 2.69mmol). After stirring at 0° for 20min, 10% sodium thiosulfate solution (200mL) was added and the mixture waε filtered through celite. The reεulting mixture waε then extracted (3 x 200mL) with dichloromethane, the organic phases were combined, dried (MgS0 ₄ ) and concentrated. Flash chromatography on 2% KH_P0 ₄ -silica gel (8:2 hexane/ethyl acetate) furnished 770mg (35%) of Compound 51, melting point 159.5-160° (with decomposition), [α] _D ²² = +208.90 (c 0.27, CHC1 ₃ ) ; 562mg (25%) of Compound 52, [α] _D ²² = +125.9 (c 0.54, CHC1 ₃ ); and 20δmg (9%) of the 2'-epimer of Compound 52, melting point 156-157°C, [α] _D ²² = -6.1° (c 0.44, CHC1 ₃ ) . The structures of all three were verified by ¹ H-NMR, IR and elemental analysis, as follows.

7-0-[4-0-{4-0-2, 3 , 6-Trideoxy-oi-L- lycero-hexopyranosid-4- uloεe) -2 , 6-dideoxy-3 -O-trimethy1s ily1 - a -L - l yxo - hexopyranosyl}-2, 6-dideoxy-2-iodo-3-0-trimethylsilyl-β-L- galactopyranosyl]-e-pyrromycinone (Compound 51) :

"•"H NMR (CDC1 ₃ , 250MHz) δ 9.81 (ε, IH) , 9.31 (s, IH) ,

9.10 (s, IH) , 7.71 (ε, IH) , 7.31 (d, J = 9.5Hz, 1 H) , 7.27 (d, 7=9.5HZ, IH) , 5.59 (d, 7=1.5Hz, IH) ,

5.05 (t, 7=3.4HZ, IH) , 4.99 (d, 7 ^" =9.1Hz, IH) , 4.93 (br s, IH) , 4.84 (q, 7=6.-6Hz, IH) 4.24 (s, IH) , 4.25-4.10 (m, IH) , 4.15 (ε, IH) , 3.99 (dd, 7=9.1, 10.5Hz, IH) , 4.78-4.65 (m, 3H) , 3.66 (s, 3H) , 3.59 (q, 7=6.2HZ, IH) , 3.45 (d, 7^2.7Hz, IH) , 2.71-

2.15 (m, 6H) , 2.01 (dt, 7=3.1, 10.2Hz, IH) , 1.80- 1.46 (m, 3H) , 1.22 (d, 7=6.6Hz, 3H) , 1.19 (d, 7=6.2Hz, 3H) , 1.16 (d, 7=6.6Hz, 3H) , 1.10 (t, 7-=7.3Hz, 3H) , 0.24 (s, 9H) , 0.08 (ε, 9H) ; IR (CHC1 ₃ ) 3490, 2928, 1718, 1590, 1442, 1290, 1248,

1152, 1096, 1010, 1002, 900, 890, 840cm ^"1 ;

Elemental Analysis: Calculated for C ₄₆ H ₆₃ 0 ₁₇ ISi ₂ : C, 51.58%; H, 5.93%; Si, 5.24%. Found: C, 50.99%; H, 5.87%; Si, 5.28%.

7-0- [4-0-{4-0- (2 , 3 , 6-Trideoxy-α.-L-gr2ycero-hexopyranoεid-4- uloεe) -2, 6 -dideoxy- 3 -O-tr imethy 1 s i ly 1 -a.-L-2 .XO- hexopyranosyl}-2,6-dideoxy-2-iodo-3-0-trimethylsilyl-α_-L- galactopyranosyl] -e-pyrromycinone (Compound 52):

^• -Η NMR (CDC1 ₃ 250MHZ) δ 9.74 (s, IH) , 9.08 (ε, IH) ,

9.01 (s, IH) , 7.74 (s, IH) , 7.31 (d, J = 9.5Hz, IH) , 7.27 (d, 7=9.5HZ, IH) , 5.60 (d, 7=3.1Hz , IH) ,

5.38 (br ε, IH) , 5.06 (t, 7=3.7Hz, IH) , 4.99 (d, 7 ^" =1.2Hz, IH) , 4.85 (q, 7=6.7Hz , IH) , 4.29-4.11 (m, 6H) , 4.17 (ε, IH) , 3.80-3.62 (m, 2H) , 3.68 (ε, 3H) , 2.74-2.16 (m, 6H) , 2.02 (dt, 7=3.4, 11.8Hz, IH) , 1.86-1.45 (m, 3H) , 1.27 (d, 7=6.7Hz , 3H) ,

1.24 (d, 7=6.7HZ, 3H) , 1.20 (d, 7=6.5Hz, 3H) , 1.08 (t, 7-=7.2Hz, 3H) , 0.15 (s, 18H) ; IR (CHC1 ₃ ) 3480, 2930, 1718, 1590, 1443, 1310, 1285, 1245, 1150, 1110, 1095, 1000, 900, 870, 840cm ^"1 ; Elemental Analysis: Calculated for C ₄₆ H ₆₃ 0 ₁₇ ISi ₂ : C,

51.58%; H, 5.93%; Si; 5.24%. Found: C, 51.80%; H, 6.07%; Si, 5.30%.

7-0-[4-0-{4-0-2 , 3 , 6-Trideoxy-α_-L-grlycero-hexopyranosid-

4-uloεe) -2 , 6 -dideoxy-3 -O-trimethylε ilyl-a.-L-2y.XO- hexopyranoεyl } -2 , 6-dideoxy-2-iodo-3-0-trimethylεilyl-α_-L- talopyranosyl] -e-pyrromycinone (2'-epimer of Compound 52):

"""H NMR (CDC1 ₃ , 250MHZ) δ 9.76 (S, IH) , 9.13 (s, IH) ,

9.04 (S, IH) , 7.75 (S, IH) , 7.36 (d, 7=9.0Hz , IH) ,

7.32 (d, 7=9.0Hz, IH) , 5.91 (br s, IH) , 5.30 (br ε, IH) , 5.11 (t, 7=3.6Hz, IH) , 4.95-4.78 (m, 2H) ,

4.4δ (br ε, 2H) , 4.21-4.10 (m, IH) , 4.14 (ε, IH) ,

3.9δ (br ε, IH) , 3.60-3.60 (m, IH) , 3.78 (d,

T-=2.7HZ, IH) , 3.71 (s, 3H) , 3.33 (br S, IH) , 2.70

(dt, .7=7.3, 15.9HZ, IH) , 2.5δ-2.1δ (m, 6H) , 2.06 (dt, 7=4.1, 11.4HZ, IH) , 1.85-1.43 (m, 3H) , 1.32

(br ε, 3H), 1.27 (d, 7=6.6Hz, 3H) , 1.22 (d,

7=6 . 2HZ , 3H) , 1. 10 (t , 7=7 . 4Hz , 3H, 0 . 17 ( s , 9H) ,

0 . 12 (br s , 9H) ;

IR (CHC1 ₃ ) 3495 2960, 2930, 1720, 1591, 1445, 1396,

1310, 1290, 124δ, 1156, 1120, 1100, 1010, 979cm ^"1 ; Elemental Analyεiε: Calculated for C ₄₆ H ₆₃ 0 ₁₇ ISi ₂ : C,

51.58%; H, 5.93%; Si, 5.24%. Found: C, 51.51%; H, 5.92%; Si, 5.17%.

A. Preparation of Ciclamycin 0

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) .

A εolution of Compound 52 (121mg, 0.113mmol) in a 3:2:2 mixture of acetic acid, tetrahydrofuran, and methanol (20mL) was stirred at room temperature for 42h. Saturated sodium bicarbonate solution (200mL) was then added and the mixture was extracted (3 x lOOmL) with dichloromethane. The combined organic extracts were dried over anhydrous magnesium sulfate, filtered and concentrated in vacuo to afford crude diol. The residue waε chromatographed on 2% KH ₂ P0 ₄ -silica gel (99.7:0.3 dichloromethane/methanol) to furnish the diol, 7-0-[4-0-{4-0- (2,3,6-trideoxy-α.-L-g2yσero-hexopyranosid-4-ulose)-2,6-di deoxy- α;-L-2yx ^, o-hexopyranosyl}-2 , 6-dideoxy-2-iodo-α-L- galactopyranosyl]-e-pyrromycinone (Compound 55) (83mg, 79%) aε a red solid. Recrystallization from iso-propyl alcohol gave red needles, melting point 155°C (with decomposition) ,[ c ] _D = +170.6° (c 0.33, CHC13) , its structure verified by ¹ H-NMR, IR and elemental analysis.

A solution of Compound 55 (18.5mg, 0.02mmol) in benzene (18mL) was treated with triphenyltin hydride (210mg, 0.598mmol) and azobisisobutyronitrile (2.5mg, 0.015mmol). The resulting mixture was then heated at reflux for 30min. Evaporation of the solvent afforded crude product which was chromatographed on 2% KH ₂ P0 ₄ ~silica gel (99.5:0.5 dichloromethane/methanol) to afford 4.1mg (22%) of iodide 20 and 8.8mg (55%) of ciclamycin 0 (Compound 56) aε a red solid. Structural verification waε identical to that shown in the succeeding section.

B. Alternate Preparation of Ciclamycin O

The section illustrates an alternate route for the preparation of ciclamycin 0, by first converting Compound 51 to Compound 53, followed by conversion of the latter to Compound 54, and finally to ciclamycin O (Compound 56) .

A solution of Compound 51 (514mg, 0.480mmol) in a 3:2:2 mixture of acetic acid, tetrahydrofuran, and methanol (80mL) waε εtirred at room temperature for lh. Saturated εodium bicarbonate εolution (400mL) was then added and the mixture was extracted (3 x 200mL) with dichloromethane. The combined organic extracts were dried over anhydrous magnesium εulfate, filtered and concentrated in vacuo to afford crude alcohol. Thiε waε chromatographed on 2% KH ₂ P0 ₄ ~εilica gel (2:1 hexane/ethyl acetate) to furnish 7-0-[4-0-{4-0-(2,3,6-trideoxy-α;-L- 2yσero- hexopyranosid-4-ulose)-2,6-dideoxy-α.-L-2yxo-hexopyranosyl} -2,6- dideoxy-2-iodo-3-0-trimethylsilyl-α_-L-talopyranosyl] -e- pyrromycinone (Compound 53) (464mg, 97%) . Recrystallization from iso-propyl alcohol gave red needles, melting point 164-165.5 ^β C, [CU] _D ²² = +10.7° (c 0.46, CHC1 ₃ ), its structure verified by ¹ H-NMR, IR and HRMS.

A solution of Compound 53 (5δmg, 0.058mmol) in tetrahydrofuran (20mL) was treated with tetrabutylammonium fluoride (0.02M solution in tetrahydrofuran, 3.7mL, 0.074mmol). The resulting mixture was stirred at room temperature for 40min. Water (50mL) was then added and the mixture waε extracted (3 x lOOmL) with dichloromethane. The combined organic extractε were dried over anhydrous magnesium sulfate, filtered and concentrated in vacuo to give crude diol. Flash chromatography on 2% KH-PO.- εilica gel (99:1 dichloromethane/methanol) afforded 21mg (36.2%) of alcohol 18 and 22mg (41%) of 7-0-[4-0-{4-0-(2,3,6-trideoxy- α;-L-g2ycero-hexopyranosid-4-ulose) -2 ,6-dideoxy-α;-L-2yxO- hexopyranoεyl}-2 , 6-dideoxy-2-iodo-α.-L-talopyranoεyl] -e- pyrromycinone (Compound 54) . Recryεtallization from methanol gave red needleε, melting point 149°C (with decomposition), [α] _D ²² = -25.8° (c 0.36, CHC1 ₃ ), its structure verified by H-NMR, IR and elemental analysiε.

To a solution of Compound 54 (55mg, 0.059mmol) in benzene (55mL) was added triphenyltin hydride (412mg, 1.17mmol) and azobisisobutyronitrile (7mg, 0.043mmol) . The mixture was then heated at reflux for 2.5h. Removal of the solvent gave crude product which was chromatographed on 2% KH ₂ P0 ₄ ~silica gel (99.5:0.5 dichloromethane/methanol) to furnish 14mg (25.5%) of iodide 19 and 32mg (72%) of ciclamycin O (Compound 56) as a red solid. Recrystallization from methanol gave ciclamycin 0 as red needles.

Ciclamycin 0 Product Verification: mp 165.5-166.0°C;

[0!] _D ²² = +64.8° (c 0.29, CHC1 ₃ ) ;

- ^* Η-NMR (CDC1-, 500MHz) δ 12.99 (s, IH) , 12.84 (S, IH) , 12.26 (S, IH) , 7.74 (s, IH) , 7.34 (d, 7=9.4Hz, IH) , 7.31 (d, 7=9.4Hz, IH) , 5.50 (d, 7 ^" =3.6Hz, IH) ,

5.26 (d, 7=4.2HZ, IH) , 5.16 (t, J=6.1Hz, IH) , 4.99

(d, 7=3.1Hz, IH) , 4.49 (q, 7=6.7Hz, IH) , 4.23 (q,

7=6.5HZ, IH) , 4.15 (q, 7=6.4Hz, IH) , 4.14 (m, IH) ,

4.11 (ε, IH) , 3.77 (m, IH) , 3.75 (ε, IH) , 3.71 (s, 3H) , 3.60 (S, IH) , 2.53 (dd, 7 ^" =4.2 , 15.2Hz, IH) ,

2.50-2.44 (m, 3H) , 2.31 (d, 7=15.2Hz, 1H) ,2.17 (m, IH) , 2.09 (dd, 7=4.0, 12.2HZ, IH) , 1.93 (dd, 7=3.6, 12.7HZ, IH) , 1.92 (dt, 7=4.0, 12.2Hz, IH) , 1.79 (dt, 7=4, 12.7HZ, IH) , 1.75 (m, IH) , 1.57 (ε, 3H) , 1.52 (m, IH) , 1.34 (d, 7=6.7Hz, 3H) , 1.31 (d,

7-=6.4HZ, 3H) , 1.25 (d, 7=6.5Hz, 3H) , 1.09 (t, -=7.3HZ, 3H) ; IR (CHC1 ₃ ) 3450, 3410, 2940, 2910, 2850, 1720, 1590, 1442, 1310, 1286, 1151, 1110, 1095, 1030, 1000cm ^"1 HRMS (FAB) m/e 823.2811 (M+Na) ⁺ ; Calculated for

^C 40 ^H 48°17 ^{Na: 823} - ²⁷⁸⁹ -

SUBSTITUTE SHEET,

C. Preparation of C-, '-Epiciclamycin O

This example illustrates the coupling of the 2'-epimer of Compound 52 to e-pyrromycinone through a route analogous to that of Section A of this example. A solution of the trans-diequatorial subεtituted compound (the 2'-epimer) (120mg, 0.112mmol) in a 3:3:2 mixture of acetic acid, tetrahydrofuran, and methanol (20mL) waε εtirred at room temperature for 36h. Saturated sodium bicarbonate solution (200mL) was then added and the mixture waε extracted (3 x lOOmL) with dichloromethane. The combined organic extractε were dried over anhydrouε magnesium sulfate, filtered, and concentrated in vacuo to afford crude diol. The residue was chromatographed on 2% KH ₂ P04-silica gel (99.5:0.5 dichloromethane/methanol) to furnish a diol analogous to Compound 55 (86mg, 83%) as a red solid. The diol was then recrystallized from methanol to produce red needles, melting point 148.5-149.0°C (with decomposition), [α] _D ²² ~ +263.5° (c 0.40, CHC1 ₃ ) , its structure verified by ¹ H-NMR, IR and HRMS.

A solution of the diol (50mg, 0.054mmol) in benzene (50mL) was treated with triphenyltin hydride (455mg, 1.30mmol) and azobisisobutyronitrile (5.5mg, 0.034mmol). This mixture was then heated at reflux for 40min. Evaporation of the solvent gave crude product which was purified by flash chromatography on 2% KH ₂ P0 ₄ ~silica gel (99.6:0.4 dichloromethane/methanol) to afford C, •-epiciclamycin O, 7-0-[4-0-{4-0-(2,3,6-trideoxy-c.-L-gr2 ycero- hexopyranosid-4-ulose)-2,6-dideoxy-α-L-2yxo-hexopyranosyl}- 2,6- dideoxy-β-L-2yxO-pyranoεyl]-e-pyrromycinone (34mg, 79%), aε a red εolid.

C, '-Epiciclamycin O Product Verification: melting point 143-144°C (decomp.);

[0!] _D ²² = +213.2° (c 0.25, CHC1 ₃ ) ;

" ^■ Η-NMR (CDC1 ₃ , 250MHZ) δ 9.71 (s, IH) , 9.14 (s, IH) ,

9.01 (S, IH), 7.70 (ε, IH), 7.32 (d, 7=9.5Hz, IH) ,

7.27 (d, .7=9.5HZ, IH) , 5.51 (t, =1.9Hz, IH) , 5.05 (t, 7-=5.6Hz, IH) , 4.92 (d, 7=2.5Hz, IH) , 4.86 (dd,

7=1.9, 8.41HZ, IH) , 4.64 (s, IH) , 4.44 (q,

7 ^" =6.7HZ, IH) , 4.21-4.03 (m, 3H) , 4.17 (s, IH) ,

3.75-3.60 ( , 1 H) , 3.67 (s, 3H) , 3.53 (q, 7=6.2HZ, IH) , 3.45 (d, 7-=0.9Hz, IH) , 2.52-2.36 (m, 5H) , 2.20-1.96 (m, 3H) , 1.86 (dt, .7=1.4, 12.5Hz, IH) , 1.75-1.45 (m, 5H) , 1 .28 (d, J=6.7Kz, 3H) , 1.21 (d, 7=6.8HZ, 3H) , 1.18 (d, J=6.2Hz , 3H) , 1.08

(t, 7=7.2Hz, 3H) ; IR (CHC1 ₃ ) 3410, 2920, 1721, 1591, 1444, 1310, 1290,

1156, 1095, 1035, 1010, 905cm ^"1 ; HRMS (FAB) m/e 823.2652 (M+Na) ⁺ ; Calculated for C ₄₀ H ₄₈ 0 ₁₇ Na: 823.2789.

EXAMPLE 11 Syntheεiε of Trisaccharide-Modified Daunomycinone

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.

Me o-i Me7 ^■ o- ¹ θ ^' 0 ^''

(57 - 61) [62)

To a solution of daunomycinone (53mg, 0.132mmol) and Compound 50 (60mg, 0.120mmol) in dichloromethane (7mL) was added

4A-sieves (60mg) . The resulting mixture was stirred at room temperature for 30min, cooled to 0° in an ice bath and stirred

ET

for an additional lh. The mixture was then treated with I(sym- collidine) ₂ C10 ₄ (90%, 70mg, 0.156mmol). After stirring at 0° for lh, the mixture was diluted with dichloromethane (30mL) and filtered through celite. The filtrate was washed with 10% sodium thiosulfate (lOmL) , 10% copper sulfate (lOmL) , water (lOmL) and brine (lOmL) . The organic layer was dried (MgS0 ₄ ) and concentrated. Flash chromatography on 2% KH ₂ P04-silica gel (2:1 hexane/ethyl acetate) furnished 54mg (44%) of a 7:1 mixture of Compound 57, [α!] _D ²² = -5.0° (c 0.04, CHC1 ₃ ) , and its diequatorial isomer, and 20mg (16%) of Compound 58, [α] _D ²² = +36.9° (σ 0.13, CHC1 ₃ ) . The structureε of each were verified by ¹ H-NMR, IR and HRMS, aε followε:

Compound 57 Verification:

7-0-[4-0-{4-0-(2,3,6-Trideoxy-o!-L-g2ycer ^* o-hexopyranoεid-4- ulose) -2 , 6-dideoxy-3 -O-tr imethylε ily 1-oi-L-lyxo- hexopyranosyl}-2,6-dideoxy-2-iodo-3-0-trimethylsilyl-α.-L- talopyranoεyl ] -daunomycinone :

"•"H-NMR (CDC1 ₃ , 250MHz) δ 13.95 (s, IH) , 13.24 (s, IH) ,

8.00 (d, 7=7.8HZ, IH) , 7.76 (t, 7=8.2Hz , IH) , 7.38 (d, 7 ^r =8.3Hz, IH) , 5.88 (br ε, IH) , 5.30 (br s,

IH) , 5.07 (at, 7=5.8HZ, IH) , 4.92 (m, 2H) , 4.48- 4.12 (m, 5H) , 4.08 (s, 3H) , 3.87-3.60 (m, 2H) , 3.20 (d, 7=19.0Hz, IH) , 2.93 (d, 7=19.0Hz, IH) , 2.65 (m, IH) , 2.56-2.40 (m, 2H) , 2.40 (s, 3H) , 2.28-2.00 (m, 6H) , 1.35-1.20 (m, 9H) , 0.13 (s,

18H) ; IR (CHC1 ₃ ) 3500, 3000, 2930, 2650, 1715, 1620, 1580,

1415, 1385, 1305, 1125, 1100cm ^"1 ; HRMS (FAB) m/e 1063.2401 (M+Na) ⁺ ; Calculated for C ₄₅ H ₆₁ 0 _lg INaSi ₂ : 1063.2441.

Compound 58 Verification:

7-0- [4-0- {4-0- (2 , 3 , 6-Trideoxy-α.-L-g2ycero-hexopyranosid-4- uloεe) -2 , 6-dideoxy-3 -O-tr imethyls ily 1 -a-L-lyxo- hexopyranosyl }-2 , 6-dideoxy-2-iodo-3- -trimethylsilyl-α!-L- galactopyranosyl ] -daunomycinone :

SUBSTITUTE SHEET

76

"•"H-NMR (CDC1 ₃ , 250MHZ) δ 14.08 (s, IH) , 13.30 (S,1H),

8.04 (d, .7=7.0Hz, IH) , 7.78 (t, 7 ^" =8.3Hz, IH) , 7.40

(d, 7=8.4HZ, IH) , 5.59 (d, 7 ^" =3.1Hz, IH) , 5.48 (br s, IH) , 5.09 (at, 7=3.7Hz, IH) , 5.00 (d, 7=2.3Hz, IH) , 4.87 (q, 7=6.7Hz, IH) , 4.56, (s, IH, -OH),

4.30-4.10 (m, 4H) , 4.09 (s, 3H) , 3.8δ (dd, 7 ^" =2.6, 10.8HZ, IH) , 3.80-3.68 (m,2H), 3.31 (d, 7=19.3Hz, IH) , 3.02 (d, 7=19.2HZ, IH) , 2.77-2.58 (m, IH) , 2.42 (S, 3H) , 2.50-2.00 (m, 6H) , 1.81 (dd, 7=4.2, 12.0HZ, IH) , 1.30 (d, J=β .5Hz, 3H) , 1.26 (d,

7 ^" =6.5HZ, 3H) , 1.22 (d, 7=6.8Hz, 3H) , 0.20 (s, 9H) , 0.16 (S, 9H) ,* IR (CHC1 ₃ ) 3480, 2940, 1715, 1610, 1565, 1400, 1270, 1250, 1090, 1000, 850, 620cm ^"1 ; HRMS (FAB) m/e 1063.2434 (M+Na) ⁺ ; Calculated for

^C 45 ^H 61°16 ^INaSi 2 ^{: 1063 *2441*}

A. Preparation of Trisaccharide-Modified Daunomycinone

The route followed in this synthesis proceeds from Compound 57 through Compound 59 to the product mixture. To a stirred solution of Compound 57 (30mg, 0.029mmol) in THF (4.0mL) at 0° was added HF-pyridine complex (0.2mL) . The mixture was stirred at room temperature for 3h, neutralized with saturated sodium bicarbonate and extracted with dichloromethane. The combined extracts were washed with 10% copper sulfate (15mL) , water (lOmL) and brine (lOmL) . The organic layer was then dried (MgS0 ₄ ) and concentrated. Flash chromatography on 2% KH_P0 ₄ - silica gel (20:1 chloroform/methanol) furnished the diol, 7-0- [4-0-{4-0-(2,3,6-trideoxy-α!-L-g2ycero-hexopyranosid-4-ulos e)- 2,6-dideoxy-α;-L-2yxO-hexopyranosyl}-2,6-dideoxy-2-iodo-c-L - talopyranosyl]-daunomycinone (Compound 59) (lδmg, 69%), as a red εolid, melting point 163.0-165.0°C, [Q!] _D ²² = -87.5° (c 0.16,

CHC1 ₃ ) , itε εtructure confirmed by ¹ H-NMR, ¹³ C-NMR, IR, and HRMS.

Nitrogen was passed through a solution of Compound 59

(14mg, 0.016mmol) and triphenyltin hydride (82mg, 0.234mmol) in benzene (3mL) for 10 minutes. To the reaction mixture was then added azobisisobutyronitrile (2.5mg, O.Olδmmol). The resulting mixture waε then heated to 55° for 12h. Additional

azobiεisobutyronitrile (2mg) was added every lh. The reaction mixture was diluted with acetonitrile (20mL) and washed with hexaneε (3 x lOmL) . The acetonitrile layer waε concentrated and the crude product chromatographed on 2% KH ₂ P0 ₄ ~silica gel (30:1 chloroform/methanol) to afford 8mg (65%) of the trisaccharide- modified daunomycinone, 7-0-[4-0-{4-0-(2,3,6-Trideoxy-α_-L- g2ycero-hexopyranosid-4-ulose) -2 , 6-dideoxy-c.-L-2 xO- hexopyranosyl}-2,6-dideoxy-α_-L-galactopyranosyl]-daunomyci none (Compound 61) , aε a red εolid, and 2mg (16%) of the leuco isomer (Compound 62) as a yellow residue. Verification of the products is shown in the following section.

B. Alternate Preparation of Trisaccharide-Modified Daunomycinone The route followed in this section proceeds from Compound 58 through Compound 60 to the product mixture.

To a stirred solution of Compound 58 (42mg, 0.040mmol) in THF (lO.OmL) at 0° was added HF-pyridine complex (0.3mL). The mixture was stirred at room temperature for 3h, neutralized with saturated sodium bicarbonate and extracted with dichloromethane. The combined extracts were washed with 10% copper sulfate (15mL) , water (lOmL) and brine (lOmL) . The organic layer was then dried (MgS0 ₄ ) and concentrated. Flash chromatography on 2% KH ₂ P0 ₄ -εilica gel (20:1 chloroform/methanol) furnished the diol, 7-0-[4-0-{4-0-(2,3,6-trideoxy-α.-L-g-2ycero- hexopyranosid-4-ulose)-2,6-dideoxy-α.-L-2y.XO-hexopyranosyl }-2,6- dideoxy-2-iodo-αf-L-galactopyranoεyl]-daunomycinone (Compound 60) (22mg, 60%), aε a red εolid, melting point 164.5-165.5°C, [α:] _D ²² = +62.2° (c 0.06, CHC1 ₃ ) , itε structure verified by ¹ H-NMR, ¹³ C-NMR, IR, UV-vis, and HRMS. Nitrogen was passed through a solution of Compound 60

(12mg, 0.013mmol) and triphenyltin hydride (45mg, O.l30mmol) in benzene (2mL) for 10 minuteε. To the reaction mixture was then added azobisisobutyronitrile (2.5mg, 0.015mmol) . The reεulting mixture was then heated to 55° for lOh. Additional azobisiεobutyronitrile (2mg) waε added every lh. The reaction mixture waε diluted with acetonitrile (20mL) and washed with hexaneε (3 x lOmL) . The acetonitrile layer waε concentrated and

the crude product chromatographed on 2% H ₂ P0 ₄ -silica gel (30:1 chloroform/methanol) to afford 4mg (40%) of Compound 61 as a red solid, melting point 234.0-235.0 ^β C, [α] _D ²² = -24.1° (c 0.06, CHC1 ₃ ) ; and 2mg (15%) of the leuco isomer, Compound 62, as a yellow residue. The structure of the two products was verified aε followε.

Product Verification:

7-0-[4-0-{4-0-(2,3,6-Trideoxy-α_-L-glycero-hexopyranoεi d-4- ulose) -2,6-dideoxy-α.-L-2y ^χ -o-hexopyranosyl}-2, 6-dideoxy-α;-L- galactopyranosyl]-daunomycinone (Compound 61) :

-•-H-NMR (CDC1 ₃ , 250MHz) δ 13.98 (s, IH) , 13.29 (S, IH) , δ.03 (d, 7 ^" =6.9Hz, IH) , 7.78 (t, 7=8.0Hz, IH) , 7.39

(d, 7 ^" =7.8HZ, IH) , 5.54 (d, 7=3.2Hz, IH) , 5.28 (br

S, IH) , 5.09 (t, 7-=6.2Hz, IH) , 4.98 (d, 7=2.7Hz, IH) , 4.60 (s, IH, -OH), 4.48 (q, ,7=6.7Hz, IH) ,

4.19-4.11 (m, 3H) , 4.09 (ε, 3H) , 3.90-3.58 (m, 5H) , 3.24 (d, T-=18.9HZ, IH) , 2.96 (d, 7=16.9Hz, IH) , 2.60-2.40 (m,3H), 2.40 (ε,3H), 2.30 (d, 7-=14.1Hz, IH) , 2.21-2.05 (m, 4H) , 1.92 (dt, 7=3.7, 12.2HZ, IH) , 1.75 (dt, 7=3.8, 12.4Hz, IH) , 1.32

(d, 7=6.7Hz, 3H) , 1.29 (d, 7=6.4Hz, 3H) , 1.24 (d, -=6.4HZ, 3H) ;

¹³ C NMR (CDC1 ₃ , 62.9MHz) δ 211.4, 209.2, 187.0, 186.8,

163.2, 156.0, 135.8, 135.6, 134.6, 134.3, 123.0, 119.9, 118.7, 111.7, 111.5, 106.0, 101.5, 100.1,

82.7, 81.9, 76.9, 72.0, 69.6, 68.0, 67.7, 65.6, 65.1, 56.8, 35.2, 34.4, 34.1, 33.6, 33.4, 27.9, 25.0, 17.1, 17.0, 14.8; IR (CHC1 ₃ ) 3580-3230, 3000, 2940, 1730, 1715, 1620, 1580, 1450, 1435, 1415, 1290 cm ^"1 ;

UV-viε λ _maχ (MeOH) 540 (e 4,212) 500 (9,500), 480

(9,300) , 293 (7,000), 254 (22,000) 235 (32,000) ; HRMS (FAB) m/e 793.2654 (M+Na) ⁺ ; Calculated for ^C 39 ^H 46°16 ^{Na: 793} - ²⁶⁸⁴ -

Leuco isomer (Compound 62) :

-•"H-NMR (CDC1 ₃ , 250MHZ) δ 14.29 (s, IH) , 13.39 (ε, IH) ,

8.06 (d, 7=7.9HZ, IH) , 7.71 (t, .7=8.0Hz, IH) , 7.19

(d, 7=7.8Hz, IH) , 5.29 (d superimposed on a br s, 7-=4.5Hz, 2H) , 4.84 (t, 7=6.4Hz, IH) , 4.79 (d,

J=2.7Hz, IH) , 4.41-4.36 (m, 2H) , 4.12-4.00 (m, 3H) , 4.08 (s, 3H) , 3.67 (s, IH, -OH), 3.50 (m, 3H) , 3.34 (br s, IH) , 3.24 (m, IH) , 2.73 (dd, 7=15.7, 5.6Hz, IH) , 2.51-2.25 (m, 6H) , 2.32 (s, 3H) , 2.18-1.88 (m, 5H) , 1.77 (dt, 7=2.8, 8.5Hz,

IH) , 1.23 (at, 6.5HZ, 6H) , 0.54 (d, 7=6.4Hz, 3H) ;

IR (CHC1-) 3600-3100, 3000, 2920, 1725, 1705, 1575,

^J - ₁

1450, 1390, 1110, 1100cm " ^L ;

UV-viε λ _maχ (MeOH) 448 (e 17,220), 421 (15,773), 397 (9,760), 266 (24,283), 241 (25,533).

EXAMPLE 11 Cytotoxicity Tests

Eight 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 5 _Q ^of *he 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 The 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. The lower the IC ₅₀ , the higher the activity of the compound. To verify the resultε, each compound waε tested in triplicate tests on separate test plates. The results are listed in the following table.

CYTOTOXICITY TEST RESULTS

The 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.

The foregoing is offered primarily for purposes of illustration. It will be readily apparent to those skilled in the art that further embodiments, variations and equivalentε beyond thoεe deεcribed herein will fall within the spirit and scope of the invention.