EP1147118A1 - Substituted alpha-linked disaccharides - Google Patents

Substituted alpha-linked disaccharides

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Publication number
EP1147118A1
EP1147118A1 EP00906899A EP00906899A EP1147118A1 EP 1147118 A1 EP1147118 A1 EP 1147118A1 EP 00906899 A EP00906899 A EP 00906899A EP 00906899 A EP00906899 A EP 00906899A EP 1147118 A1 EP1147118 A1 EP 1147118A1
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EP
European Patent Office
Prior art keywords
hydrogen
heterocyclic
alkyl
single bond
group
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EP00906899A
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German (de)
French (fr)
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EP1147118A4 (en
Inventor
Daniel Kahne
Min Ge
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Princeton University
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Princeton University
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Publication of EP1147118A1 publication Critical patent/EP1147118A1/en
Publication of EP1147118A4 publication Critical patent/EP1147118A4/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/20—Carbocyclic rings
    • C07H15/203—Monocyclic carbocyclic rings other than cyclohexane rings; Bicyclic carbocyclic ring systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K9/00—Peptides having up to 20 amino acids, containing saccharide radicals and having a fully defined sequence; Derivatives thereof
    • C07K9/006—Peptides having up to 20 amino acids, containing saccharide radicals and having a fully defined sequence; Derivatives thereof the peptide sequence being part of a ring structure
    • C07K9/008—Peptides having up to 20 amino acids, containing saccharide radicals and having a fully defined sequence; Derivatives thereof the peptide sequence being part of a ring structure directly attached to a hetero atom of the saccharide radical, e.g. actaplanin, avoparcin, ristomycin, vancomycin
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the present invention relates to substituted alpha-linked disaccharide compounds comprising two hexose residues. These compounds are useful as antibiotics, and are believed to function as transglycosylase inhibitors.
  • Peptidoglycan synthesis in bacteria is known to proceed in stages, the last of which involves transglycosylation of the disaccharide building blocks and cross-linking of the peptide chains attached thereto.
  • Compounds that inhibit transglycosylation are potentially very useful as antibiotics.
  • a disaccharide fragment of moenomycin inhibits transglycosylase activity with the same potency as moenomycin itself. This disaccharide, as shown below,
  • This invention is directed to a disaccharide compound comprising two hexose residues joined by an alpha glycosidic linkage.
  • the compound has the formula
  • R 2 Y 2 Y ⁇ is bonded to a ring carbon atom adjacent to the alpha glycosidic linkage;
  • and R 3 are independently hydrogen, alkyl, aryl, aralkyl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, alkanoyl, aroyl, aralkanoyl, heterocyclic, heterocyclic-alkyl, heterocyclic-carbonyl or heterocyclic-alkyl-carbonyl;
  • R 2 is hydrogen, alkyl, aryl, aralkyl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, alkanoyl, aroyl, aralkanoyl, heterocyclic, heterocyclic-alkyl, heterocyclic-carbonyl, heterocyclic-alkyl-carbonyl or a peptide comprising 2-6 amino acid residues
  • Rj, R 2 and R 3 are not hydrogen or methyl; when p is 0, Xi is a single bond, and X 2 is NR 12 , then Ri is not benzoyl or methylbenzoyl; when X 2 is C(O)O, C(O)S, C(S)O, C(S)S or C(NR 12 )O, then Ri is not hydrogen; when Y 2 is C(O)O, C(O)S, C(S)O, C(S)S or C(NR 12 )O, then R 2 is not hydrogen; when Z 2 is C(O)O, C(O)S, C(S)O, C(S)S or C(NR 12 )O, then R 3 is not hydrogen; and when R 2 is a peptide comprising 2-6 amino acid residues, then Rj is not hydrogen or methyl.
  • This invention is also directed to a method for preparation of these compounds by allowing a first monosaccharide having the formula
  • R 2 Y 2 Y ⁇ is bonded to a ring carbon atom adjacent to a free hydroxyl group; and none of R 2 Y 2 Y ⁇ , W 1 R 4 , W 2 R5 and Z 1 Z 2 R3 is a free hydroxyl, amino or thiol group, or bears a free hydroxyl, amino or thiol group; to react with a second monosaccharide having the formula
  • Ar is an aryl group, and none of R 8 , R ⁇ W 3 , R 7 W 4 and X)X 2 R ⁇ is a free hydroxyl, amino or thiol group, or bears a free hydroxyl, amino or thiol group; and an activating agent; via a glycosylation reaction in which an alpha glycosidic linkage is formed between the first monosaccharide and the second monosaccharide.
  • This invention is also directed to a method for preparing a disaccharide compound comprising two hexose residues joined by an alpha glycosidic linkage; said compound having the formula
  • Ri and R 3 are independently hydrogen, alkyl, aryl, aralkyl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, alkanoyl, aroyl, aralkanoyl, heterocyclic, heterocyclic-alkyl, heterocyclic-carbonyl or heterocyclic-alkyl-carbonyl;
  • R 2 is hydrogen, alkyl, aryl, aralkyl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, alkanoyl, aroyl, aralkanoyl, heterocyclic, heterocyclic-alkyl, heterocyclic-carbonyl, heterocyclic-alkyl-carbonyl or a peptide comprising 2-6 amino acid residues;
  • Ri is not hydrogen or methyl
  • Ri is not benzoyl or methylbenzoyl
  • X 2 is C(0)0, C(0)S, C(S)0, C(S)S or C(NR ⁇ 2 )0
  • R x is not hydrogen
  • Y 2 is C(0)0, C(0)S, C(S)0, C(S)S or C(NR, 2 )0, then
  • R 2 is not hydrogen; when Z 2 is C(0)0, C(0)S, C(S)0, C(S)S or C(NR ⁇ 2 )0, then R 3 is not hydrogen; and when R 2 is a peptide comprising 2-6 amino acid residues, then R] is not hydrogen or methyl;
  • said method comprising:
  • R 2 Y 2 1 is bonded to a ring carbon atom adjacent to a free hydroxyl group; and none of R 2 Y 2 Yj, W 1 R 4 , W 2 R 5 and Z ⁇ Z 2 R 3 is a free hydroxyl, amino or thiol group, or bears a free hydroxyl, amino or thiol group;
  • Ar is an aryl group, and none of R 8 , R ⁇ W 3 and R 7 W 4 is a free hydroxyl, amino or thiol group, or bears a free hydroxyl, amino or thiol group; and an activating agent; and
  • This invention is further directed to a chemical library comprising a plurality of these compounds, and to a method of treating bacterial infections in humans by administering an effective amount of the compound.
  • Figure 1 is a graph showing the effects on macromolecular synthesis in Bacillus megaterium MB410 of known antibiotics.
  • Figure 2 is a graph showing the effect of compound 6a on synthesis of RNA, DNA, protein and peptidoglycan in comparison with the effects of vancomycin and ampicillin.
  • Figures 3 A and 3B are graphs showing the activity of compound 6a in ether-treated bacteria, and the site of inhibition of peptidoglycan synthesis.
  • Figures 4 - 7 are tables presenting results obtained on several compounds of this invention, along with controls, for synthesis of RNA, DNA, protein and peptidoglycan, and for the site of inhibition of peptidoglycan synthesis in ether treated bacteria.
  • alkyl refers to an acyclic or non-aromatic cyclic group having from one to twenty carbon atoms connected by single or multiple bonds.
  • An alkyl group may be substituted by one or more of halo,
  • hydroxyl protected hydroxyl, amino, nitro, cyano, alkoxy, aryloxy, aralkyloxy, COOH, aroyloxy, alkylamino, dialkylamino, trialkylammonium, alkylthio, alkanoyl, alkanoyloxy, alkanoylamido, alkylsulfonyl, arylsulfonyl, aroyl, aralkanoyl, heterocyclic, CONH 2 , CONH-alkyl, CONH-aryl, CONH- aralkyl, CON(alkyl) 2 , COO-aralkyl, COO-aryl, COO-heterocyclic, COO-alkyl or phosphonium substituted by any combination of alkyl, aryl, aralkyl or heterocyclic.
  • aryl refers to a group derived from a non-heterocyclic aromatic compound having from six to twenty carbon atoms and from one to four rings which may be fused or connected by single bonds.
  • An aryl group may be substituted by one or more of alkyl, aralkyl, heterocyclic, heterocyclic-alkyl, heterocyclic-carbonyl, halo, hydroxyl, protected hydroxyl, amino, hydrazino, alkylhydrazino, arylhydrazino, nitro, cyano, alkoxy, aryloxy, aralkyloxy, aroyloxy, alkylamino, dialkylamino, trialkylammonium, alkylthio, alkanoyl, alkanoyloxy, alkanoylamido, alkylsulfonyl, arylsulfonyl, aroyl, aralkanoyl, COO-alkyl, COO-aralkyl
  • heterocyclic refers to a group derived from a heterocyclic compound having from one to four rings, which may be fused or connected by single bonds; said compound having from three to twenty ring atoms which may be carbon, nitrogen, oxygen, sulfur or phosphorus.
  • a heterocyclic group may be substituted by one or more of alkyl, aryl, aralkyl, halo, hydroxyl, protected hydroxyl, amino, hydrazino, alkylhydrazino, arylhydrazino, nitro, cyano, alkoxy, aryloxy, aralkyloxy, aroyloxy, alkylamino, dialkylamino, trialkylammonium, alkylthio, alkanoyl, alkanoyloxy, alkanoylamido, alkylsulfonyl, arylsulfonyl, aroyl, aralkanoyl, COO-alkyl, COO-aralkyl, COO-aryl, COO-heterocyclic, CONH 2 , CONH- alkyl, CONH-aryl, CONH-aralkyl, CON(alkyl) 2 or phosphonium substituted by any combination of alkyl, aryl
  • alkoxy refers to groups derived from bonding an oxygen atom to an alkyl, aryl or aralkyl group, respectively.
  • alkanoyl refers to groups derived from bonding a carbonyl to an alkyl, aryl or aralkyl group, respectively.
  • heterocyclic-alkyl and “heterocyclic-carbonyl” refer to groups derived from bonding a heterocyclic group to an alkyl or a carbonyl group, respectively.
  • heterocyclic-alkyl-carbonyl refers to a group derived from bonding a heterocyclic-alkyl group to a carbonyl group.
  • hydroxyl protecting group refers to a group bonded to a hydroxyl group which is easily removed to regenerate the free hydroxyl group by treatment with acid or base, by reduction, or by exposure to light.
  • exemplary hydroxyl protecting groups include, without limitation, acetyl, chloroacetyl, pivaloyl, benzyl, benzoyl, p- nitrobenzoyl, tert-butyl-diphenylsilyl, allyloxycarbonyl and allyl.
  • amino protecting group and “thiol protecting group” refer to groups bonded to an amino or thiol group, respectively, which are easily removed to regenerate the free amino or thiol group, respectively, by treatment with acid or base, by reduction, or by exposure to light.
  • amino protecting groups include, without limitation, Fmoc, CBz, aloe and alkanoyl and alkoxycarbonyl groups.
  • thiol protecting groups include, without limitation, alkanoyl and aroyl groups.
  • a “glycopeptide” is a compound comprising a peptide linked to at least one carbohydrate.
  • An “aglycone” is the result of removing the carbohydrate residues from a glycopeptide, leaving only a peptide core.
  • a “dalbaheptide” is a glycopeptide containing a heptapeptide moiety which is held in a rigid conformation by cross-links between the aromatic substituent groups of at least five of the seven ⁇ -amino acid residues, including a cross-link comprising a direct carbon-carbon bond between the aryl substituents of amino acid residues 5 and 7, and aryl ether cross-links between the substituents of amino acid residues 2 and 4, and 4 and 6.
  • Amino acid residues 2 and 4-7 in different dalbaheptides are those found in the naturally occurring glycopeptide antibiotics. These amino acid residues differ only in that residues 2 and 6 do not always have a chlorine substituent on their aromatic rings, and in that substitution on free hydroxyl or amino groups may be present. Amino acid residues 1 and 3 may differ substantially in different dalbaheptides; if both bear aryl substituents, these may be cross-linked. Molecules having a dalbaheptide structure include, e.g., vancomycin and teicoplanin.
  • a “chemical library” is a synthesized set of compounds having different structures. The chemical library may be screened for biological activity to identify individual active compounds of interest.
  • the disaccharide compound of the present invention comprises two hexose residues joined by an alpha glycosidic linkage. At least one of the hexose residues is substituted by a lipid group, i.e., an organic functional group having from 2-30 carbon atoms, preferably 2-20 carbon atoms and may also contain heteroatoms.
  • the lipid group may be linear, branched, or cyclic, and may include aliphatic, aromatic and/or heterocyclic groups.
  • a number of substituents can also be present on the hexose rings, in particular the ring not bearing the lipid group. However, the substituents exclude peptidic moieties having demonstrable transpeptidase inhibitory activity.
  • the disaccharide of the invention exhibits anti-infective, preferably antibiotic activity. Most preferably, the compound of the invention exhibits transglycosylase inhibitory activity.
  • the disaccharide compound has the formula (I)
  • R 2 Y 2 Y ⁇ is bonded to a ring carbon atom adjacent to the alpha glycosidic linkage;
  • R) and R 3 are independently hydrogen, alkyl, aryl, aralkyl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, alkanoyl, aroyl, aralkanoyl, heterocyclic, heterocyclic-alkyl, heterocyclic-carbonyl or heterocyclic-alkyl-carbonyl;
  • R 2 is hydrogen, alkyl, aryl, aralkyl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, alkanoyl, aroyl, aralkanoyl, heterocyclic, heterocyclic-alkyl, heterocyclic-carbonyl, heterocyclic-alkyl-carbonyl or a peptide comprising 2-6 amino acid
  • Rj, R 2 and R 3 are not hydrogen or methyl; when p is 0, X, is a single bond, and X 2 is NRn, then Ri is not benzoyl or methy lbenzoyl; when X 2 is C(0)0, C(0)S, C(S)0, C(S)S or C(NR, 2 )0, then Ri is not hydrogen; when Y 2 is C(0)0, C(0)S, C(S)0, C(S)S or C(NR 12 )0, then
  • R 2 is not hydrogen; when Z 2 is C(0)0, C(0)S, C(S)0, C(S)S or C(NR, 2 )0, then R 3 is not hydrogen; and when R 2 is a peptide comprising 2-6 amino acid residues, then Rj is not hydrogen or methyl.
  • the alpha glycosidic linkage results in a very different relative spatial presentation of the hydroxyls and other functional groups on the two sugars than would be observed for any other linkage.
  • the transglycosylase activity displayed by the compounds of this invention could not have been predicted based on the activity of moenomycin and its derivatives, which have beta glycosidic linkages.
  • Another consequence of the unique shape of the disaccharide compounds of this invention is that a great variety of substituents may be introduced without destroying the antibiotic activity of this compound.
  • R 2 is a peptide comprising 2-6 amino acid residues
  • R is any natural or synthetic peptide in the stated size range. It is preferred that R 2 has the formula A 2 -A 3 -A 4 -A 5 -A 6 -A 7 , in which each dash represents a covalent bond; wherein each of the groups A 2 to A 7 comprises a modified or unmodified ⁇ -amino acid residue, whereby (i) each of the groups A 2 , A 4 and A 6 bears an aromatic side chain, which aromatic side chains are cross-linked together by two or more covalent bonds, and (ii) the group A bears a terminal carboxyl, ester, amide, or N-substituted amide group.
  • R 2 is one of the dalbaheptide aglycones of the natural vancomycin family of antibiotics from which the leucine residue has been removed completely, thereby producing a hexapeptide. It is not intended that R 2 is a glycopeptide.
  • Modified amino acid residues include amino acid residues whose aromatic groups have been substituted by halo, alkyl, alkoxy, alkanoyl, or other groups easily introduced by electrophilic substitution reactions or by reaction of phenolic hydroxyl groups with alkylating or acylating agents; and amino acid residues which have protecting groups or other easily introduced substituents on their hydroxyl or amino groups, including, but not limited to alkyl, alkanoyl, aroyl, aralkyl, aralkanoyl, carbamoyl, alkyloxycarbonyl, aralkyloxycarbonyl, aryloxycarbonyl, alkylsulfonyl, arylsulfonyl, heterocyclic, heterocyclic-alkyl or heterocyclic-carbonyl substituents.
  • Examples of preferred protecting groups include acetyl, allyloxycarbonyl (aloe), CBz, allyl, benzyl, p-methoxybenzyl and methyl. Modifications of hydroxyl groups occur on phenolic hydroxyl groups, benzylic hydroxyl groups, or aliphatic hydroxyl groups. Other amino acid residues, in addition to A 2 , A 4 and A , may be cross-linked through their, aromatic substituent groups.
  • R 2 Y 2 Y ⁇ group is attached to the anomeric position of a monosaccharide and the alpha glycosidic linkage is attached to the 2-position of the same monosaccharide. It is further preferred
  • Wi, W 2 and W 3 are O. It is also preferred that at least one of Ri, R 2 and R 3 is not acetyl, benzyl or benzoyl. It is also preferred that at least two substituents on the disaccharide are not hydroxyl, amino, protected hydroxyl or protected amino. In one embodiment of the invention, it is preferred that R 8 is hydrogen and p is 0, further preferred that k is 1 and m is 0, still further preferred that r is 1, and most preferred that X] is a single bond and X 2 is NR ⁇ 2 .
  • Zj is a single bond
  • Z 2 is O, S or NRj 4
  • Rj, R5 and Rs are hydrogen
  • X) is a single bond
  • X 2 is NR ⁇ 2
  • Yi is a single bond
  • Y 2 is O.
  • X]X 2 Ri and a CH 3 group are both attached to the 3-position of a monosaccharide.
  • the disaccharide is derived from the disaccharide component of vancomycin, which has a glucose residue attached through its 2-position to a vancosamine residue.
  • Examples of such disaccharides are shown below in Scheme 1.
  • the vancosamine residue may lack the methyl group geminal to the amine, as in compound 11.
  • Compounds 1 1 , 6a and 6c are substituted with an N-4-(4-chlorophenyl)benzyl substituent on the vancosamine nitrogen, while compound 6b has an n-decyl substituent on the vancosamine nitrogen.
  • Compounds 1 1, 6a and 6b have an equatorial 2,6- dimethoxyphenyl substituent on the glucose anomeric hydroxyl, while compound 6c has an axial methoxy substituent.
  • the compounds of formula (I) are prepared by allowing a first monosaccharide having the formula
  • R 2 Y 2 Y] is bonded to a ring carbon atom adjacent to a free hydroxyl group; and none of R 2 Y 2 Y ⁇ , W 1 R 4 , W 2 R5 and Z]Z 2 R 3 is a free hydroxyl, amino or thiol group, or bears a free hydroxyl, amino or thiol group; to react with a second monosaccharide having the formula
  • Ar is an aryl group, and none of Rg, 6W3, R 7 W 4 and X ⁇ X 2 Rj is a free hydroxyl, amino or thiol group, or bears a free hydroxyl, amino or thiol group; and an activating agent; via a glycosylation reaction in which an alpha glycosidic linkage is formed between the first monosaccharide and the second monosaccharide.
  • XjX 2 R] substituent after deprotection is an amino or alkylamino group, i.e., when Xi is a single bond, X 2 is NR ⁇ 2 and Rj is hydrogen
  • the disaccharide is contacted with an alkylating agent capable of reacting with the amino or alkylamino group to produce an alkylated substituent.
  • suitable alkylating agents include, without limitation, alkyl halides, alkyl sulfonate esters, and aldehydes or ketones under reactive amination conditions.
  • the X ⁇ X 2 R] substituent is replaced by an azido group, i.e., the second monosaccharide bears a group (CH 2 ) P N 3 .
  • the azido group preferably is reduced to an amino group using one of the suitable reducing agents that are well known in the art. This method is exemplified in Scheme 3.
  • the anomeric aryl sulfoxide group is activated by contacting it with an organic acid anhydride which will react with the sulfoxide.
  • the organic acid anhydride may be an anhydride of a sulfonic acid, of two different sulfonic acids or of a sulfonic acid and a carboxylic acid.
  • the preferred organic acid anhydride is trifluoromethanesulfonic anhydride (triflic anhydride, Tf 2 0).
  • a non-nucleophilic mild base is also added to the reaction mixture.
  • Suitable non-nucleophilic mild bases include, but are not limited to, porphyrins, 2,6-dialkylanilines, acetamides, 2,6-dialkylpyridines and co-solvents such as ethyl acetate or ethers.
  • the preferred base is 2,6-di-tert-butyl-4-methylpyridine (DTBMP).
  • a partially protected glucose, la or lb, having one free hydroxyl group is allowed glycosylated product 3a or 3b, respectively.
  • the ⁇ -thiophenoxy substituent in 3b is converted to an ⁇ - methoxy substituent by treatment with mercury(II) trifluoroacetate and DTBMP to give 3c.
  • Treatment of 3a or 3c with hydrazine gives the partially deprotected product 4a or 4b, respectively.
  • Hydrogenation of 4a or 4b gives completely deprotected product 5a or 5b, respectively.
  • chlorobiphenyl aldehyde 4-(4- chlorophenyl)benzaldehyde
  • 1 -decanal under conditions effective for reductive amination gives products 6a-6c, as shown.
  • This approach may be used to introduce a variety of XiX 2 Ri and R 2 Y 2 Y ⁇ substituent groups at the vancosamine nitrogen and at the glucose anomeric carbon.
  • Scheme 3 shows the reaction of a partially protected glucose la with a hexose bearing an anomeric sulfoxide substituent 7.
  • compound 7 is a desmethyl vancosamine derivative.
  • the same sequence of reactions carried out in Scheme 2 produces compound 11, a desmethyl derivative of compound 6a.
  • Particular preferred compounds of this invention are those derived from the desmethyl vancomycin disaccharide and substituted on the C-6 position of the glucose residue, as well as on the vancosamine nitrogen.
  • Derivatives at the C-6 position are produced from intermediates having a mesitylenesulfonyl group at the C-6 position and a protected vancosamine nitrogen.
  • a method for functionalizing the C-6 position is described in copending application Serial No. 09/115,667, titled “Glycopeptide Antibiotics, Combinatorial Libraries of Glycopeptide Antibiotics and Methods of Producing Same," filed July 14, 1998, and which is incorporated herein by reference.
  • a variety of Z]Z 2 R 3 substituent groups are introduced at the glucose-6 position by using common methods for nucleophilic displacement of primary arylsulfonyl groups directly, or by further synthetic modification of initial displacement products, including azido and iodo groups.
  • the iodo group is displaced by a variety of nucleophiles to produce additional C ⁇ -derivatives.
  • a preferred nucleophile is a thiol compound, especially a heterocyclic thiol.
  • Modification of an azido group at the 6-position is performed, e.g., by reducing the azido group to an amino group, which in turn is functionalized by means of reductive alkylation, nucleophilic substitution, or other amino-group reactions well known to those skilled in the art.
  • the substituent R 2 Y 2 Y ⁇ is a peptide having from 2-6 amino acid residues and linked through an oxygen atom to the anomeric carbon atom of the saccharide ring, i.e., Yi is a single bond, Y 2 is O, and R 2 is a peptide having from 2-6 amino acid residues.
  • Yi is a single bond
  • Y 2 is O
  • R 2 is a peptide having from 2-6 amino acid residues.
  • An example of a compound in this embodiment is compound 12, whose preparation and structure are shown below in Scheme 4: Scheme 4
  • the chemical library of compounds of this invention is prepared to explore the effects on biological activity of introducing a large number of different substituents on disaccharides having an alpha glycosidic linkage.
  • at least two steps are performed, each of which introduces a substituent group.
  • a combinatorial format is established in which many different predetermined substituent groups are introduced independently at each of at least two positions on an alpha-linked disaccharide, resulting in a library containing a large number of substituted alpha-linked disaccharides, wherein each possible combination of the predetermined substituent groups is represented.
  • At least two of the X ⁇ X 2 R ⁇ , R 2 Y 2 Y ⁇ and Z)Z 2 R 3 substituent groups are introduced in a combinatorial format, choosing each substituent from a group of possible substituents, thereby generating a chemical library.
  • the methods used to introduce these substituents are those presented in Scheme 2 for introduction of the and R 2 Y 2 Y ⁇ substituent groups, and the method outlined hereinabove for functionalization of the glucose C-6 position with a Z ⁇ R substituent by means of a C-6 mesitylenesulfonyl group.
  • EXAMPLE 1 3-(N-benzyloxy-carbonyloxy)-4-0-acetyl-2,3,6-trideoxy-3-C-methyl- ⁇ -L-lyxo- hexopyranosyl-(l— »2)-3,4,6-tri-0-benzyl- ⁇ -glucopyranoyl 2,6-dimethoxyphenol (3a).
  • the compound la (20 mg, 0.0315 mmol) and DTBMP (32 mg, 0.158 mmol) are azeotroped with toluene 3 times and then dissolved in 2 mL Et 2 0.
  • the reaction solution is cooled to -78 C and 0.5 mL toluene is added.
  • Triflic anhydride (6 ⁇ L, 0.0347 mmol) is added to the reaction solution, and the sulfoxide 2 (28 mg, 0.0629 mmol) in 1 mL Et 0 is added dropwise over 10 minutes.
  • the reaction is warmed up to 0°C in 1 hour and then quenched with 3 mL of saturated aqueous NaHC0 3 solution.
  • EXAMPLE 2 Phenyl 2-(3-N-Cbz-4-0-acetyl-2,3,6-trideoxy-3-C-methyl- ⁇ -L-lyxo-hexopyranosyl)-3,4,6- -tri-O-benzyl- 1 -thio- ⁇ -D-glucopyranoside (3b).
  • EXAMPLE 3 Methyl 2-(3-N-Cbz-4-0-acetyl-2,3,6-trideoxy-3-C-methyl- ⁇ -L-lyxo-hexopyranosyl)-3,4,6- -tri-O-benzyl- ⁇ -D-glucopyranoside (3c).
  • the combined filtrate is concentrated and the residue is purified by reverse-phase HPLC using a PHENOMENEX LUNA Ci8 column (21.2x250 mm), 5 ⁇ m particle, eluting with a 40 minute linear gradient of 0% acetonitrile/0.1% acetic acid in water to 70% acetonitrile/0.1% acetic acid in water; flow rate of 8 mL/min. and UV detection at 270 nm.
  • the fractions containing the pure product are combined and evaporated to give 7 mg (73%) of compound 5a as a white solid.
  • the solution is cooled back to room temperature, concentrated and purified by reverse-phase HPLC using a PHENOMENEX LUNA Ci8 column (21.2x250 mm), 5 ⁇ m particle, eluting with a 30 min. linear gradient of 20% acetonitrile/0.1% acetic acid in water to 70% acetonitrile/0.1% acetic acid in water; flow rate of 8 mL/min. and UV detection at 270 nm.
  • the fractions containing the pure products are combined and evaporated to give 8 mg (90%) of compound 6a as white solid.
  • the combined filtrate is concentrated and the residue is purified by reverse-phase HPLC using a PHENOMENEX LUNA C]8 column (21.2x250 mm), 5 ⁇ m particle, eluting with a 40 minute linear gradient of 0% acetonitrile/0.1% acetic acid in water to 70% acetonitrile/0.1% acetic acid in water; flow rate of 8 mL/min. and UV detection at 270 nm.
  • the fractions containing the pure product are combined and evaporated to give 15 mg (73%) of compound 10 as a white solid.
  • the clear solution is purified by reverse-phase HPLC using a PHENOMENEX LUNA C18 column (21.2 x 250 mm), 5 ⁇ m particle size, eluting with a 30 minute linear gradient of 0.1% acetic acid in water to 60% acetonitrile/0.1% acetic acid in water; flow rate of 8 mL/min. and ultraviolet (UV) detection at 285 nm to give 8 mg of the desired product. Retention time: 23 minutes.
  • ESI-MS calc. for C 72 H 72 N 8 0 23 Cl3 [M+H * ]: 1522.5, found: 1522.5.
  • Compound 6a selectively inhibited peptidoglycan synthesis and RNA synthesis.
  • the inhibition of RNA synthesis is likely not to be a secondary effect of the inhibition of peptidoglycan synthesis because ampicillin had no effect on RNA synthesis.
  • Rifampicin did not inhibit peptidoglycan synthesis.
  • Vancomycin inhibited peptidoglycan synthesis and RNA synthesis.
  • Lipid intermediate I consists of bactoprenol MurNAc-pentapeptide.
  • Lipid intermediate II consists of bactoprenol-GlcNAc-MurNAc-pentapeptide.
  • ramoplanin is an inhibitor of the transferase step in stage II.
  • the compound inhibits incorporation into all three fractions.
  • Bambermycin is the only known inhibitor of the transglycosylase step and it inhibits incorporation into the material retained by the PVDF filters and into the fraction that is insoluble in hot SDS but not into the butanol-soluble fractions.
  • Cefoxitin inhibits transpeptidation. It only inhibits incorporation of [ 14 C]GlcNAc into the hot SDS-insoluble fraction.
  • Compound 6a is tested for activity in ether-treated bacteria (ETB) prepared from E. coli VC8 and from E. coli Wl. In the test against the ETB prepared from strain VC8, it is not possible to confirm that inhibition of stage II steps would have been observed. The separation scheme that was designed with strain W7 did operate in the same way with ETB from strain VC8. However, there is good evidence for the inhibition of the transglycosylase step by compound 6a, as shown in Figure 3A.
  • ETB ether-treated bacteria
  • Compound 6a is re-tested with ETB prepared from strain W7. The selectivity test with the known antibiotics confirmed that inhibition of stage II steps is observable with this strain. Again, compound 6a displays a pattern of inhibition that suggests inhibition of the transglycosylase step, as shown in Figure 3B.

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Abstract

A disaccharide compound having formula (I); wherein R2Y2Y1 is bounded to a ring carbon atom adjacent to the alpha glycosidic linkage; W1, W2, W3 and W4 are independently O, NH or S; R8 is hydrogen, hydroxyl or a hydroxyl protecting group; k, m, n, p and r are independently 0 or 1; X1 is a single bond, O, NR9 or S; X2 is O, NR12, S, C(O)O, C(O)S, C(S)O, C(S)S, C(NR¿12¿)O or C(O)NR¿12?; Y1 is a single bond, O, NR10 or S; Y2 is O, NR13, S, C(O)O, C(O)S, C(S)O, C(S)S, C(NR13)O or C(O)NR13; Z1 is a single bond, O, NR11 or S; Z2 is O, NR14, S, C(O)O, C(O)S, C(S)O, C(S)S, C(NR14)O or C(O)NR14. A method for preparing the disaccharide compound via an activated anomeric sulfoxide glycosylation reaction. A chemical library comprising the compounds of this invention. A method of treating bacterial infections with the compound.

Description

SUBSTITUTED ALPHA-LINKED DISACCHARIDES
BACKGROUND OF THE INVENTION
Field of the Invention
The present invention relates to substituted alpha-linked disaccharide compounds comprising two hexose residues. These compounds are useful as antibiotics, and are believed to function as transglycosylase inhibitors.
Background of the Invention
Peptidoglycan synthesis in bacteria is known to proceed in stages, the last of which involves transglycosylation of the disaccharide building blocks and cross-linking of the peptide chains attached thereto. Compounds that inhibit transglycosylation are potentially very useful as antibiotics. There is currently only one commercialized class of compounds known to inhibit transglycosylation. This class comprises moenomycin and its derivatives, which are believed to bind to the transglycosylase active site. A disaccharide fragment of moenomycin inhibits transglycosylase activity with the same potency as moenomycin itself. This disaccharide, as shown below,
comprises two hexoses joined by a beta-l,2-glycosidic linkage, and a lipid chain attached to the anomeric carbon of the first sugar residue. There has been no suggestion in the literature that any other type of disaccharide could act as a transglycosylase inhibitor. SUMMARY OF THE INVENTION
This invention is directed to a disaccharide compound comprising two hexose residues joined by an alpha glycosidic linkage. The compound has the formula
wherein R2Y2Yι is bonded to a ring carbon atom adjacent to the alpha glycosidic linkage; | and R3 are independently hydrogen, alkyl, aryl, aralkyl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, alkanoyl, aroyl, aralkanoyl, heterocyclic, heterocyclic-alkyl, heterocyclic-carbonyl or heterocyclic-alkyl-carbonyl; R2 is hydrogen, alkyl, aryl, aralkyl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, alkanoyl, aroyl, aralkanoyl, heterocyclic, heterocyclic-alkyl, heterocyclic-carbonyl, heterocyclic-alkyl-carbonyl or a peptide comprising 2-6 amino acid residues; Rt, R5, Re and R7 are independently hydrogen, or a hydroxyl, amino or thiol protecting group; Wj, W2, W3 and W are independently O, NH or S; R8 is hydrogen, hydroxyl or a hydroxyl protecting group; k, m, n, p and r are independently 0 or 1; Xi is a single bond, O, NR9 or S; X2 is O, NRn, S, C(0)0, C(O)S, C(S)0, C(S)S, C(NRι2)0 or C(0)NR12; Y, is a single bond, O, NRI0 or S; Y2 is O, NR,3, S, C(0)0, C(0)S, C(S)O, C(S)S, C(NR13)0 or C(0)NR13; Z, is a single bond, O, NR„ or S; Z2 is O, NR14, S, C(0)0, C(0)S, C(S)0, C(S)S, C(NR14)0 or C(0)NR14; R9, Rio, R11, R12, R13 and R14 are independently hydrogen, alkyl or aralkyl; none of the pairs Xj and X2, Yi and Y2, and Z) and Z2 comprises O and O, S and O, or O and S, respectively;
provided that: at least one of Rj, R2 and R3 is not hydrogen or methyl; when p is 0, Xi is a single bond, and X2 is NR12, then Ri is not benzoyl or methylbenzoyl; when X2 is C(O)O, C(O)S, C(S)O, C(S)S or C(NR12)O, then Ri is not hydrogen; when Y2 is C(O)O, C(O)S, C(S)O, C(S)S or C(NR12)O, then R2 is not hydrogen; when Z2 is C(O)O, C(O)S, C(S)O, C(S)S or C(NR12)O, then R3 is not hydrogen; and when R2 is a peptide comprising 2-6 amino acid residues, then Rj is not hydrogen or methyl. This invention is also directed to a method for preparation of these compounds by allowing a first monosaccharide having the formula
wherein R2Y2Yι is bonded to a ring carbon atom adjacent to a free hydroxyl group; and none of R2Y2Yι, W1R4, W2R5 and Z1Z2R3 is a free hydroxyl, amino or thiol group, or bears a free hydroxyl, amino or thiol group; to react with a second monosaccharide having the formula
wherein Ar is an aryl group, and none of R8, RόW3, R7W4 and X)X2Rι is a free hydroxyl, amino or thiol group, or bears a free hydroxyl, amino or thiol group; and an activating agent; via a glycosylation reaction in which an alpha glycosidic linkage is formed between the first monosaccharide and the second monosaccharide.
This invention is also directed to a method for preparing a disaccharide compound comprising two hexose residues joined by an alpha glycosidic linkage; said compound having the formula
wherein R2Y2Yι is bonded to a ring carbon atom adjacent to the alpha glycosidic linkage; Ri and R3 are independently hydrogen, alkyl, aryl, aralkyl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, alkanoyl, aroyl, aralkanoyl, heterocyclic, heterocyclic-alkyl, heterocyclic-carbonyl or heterocyclic-alkyl-carbonyl; R2 is hydrogen, alkyl, aryl, aralkyl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, alkanoyl, aroyl, aralkanoyl, heterocyclic, heterocyclic-alkyl, heterocyclic-carbonyl, heterocyclic-alkyl-carbonyl or a peptide comprising 2-6 amino acid residues; Rt, R5, Re and R7 are independently hydrogen, or a hydroxyl, amino or thiol protecting group; Wb W2, W3 and W4 are independently O, NH or S; Rg is hydrogen, hydroxyl or a hydroxyl protecting group; k, m, n, p and r are independently 0 or 1; Xi is a single bond, O, NR9 or S; X2 is O, NR,2, S, C(0)0, C(0)S, C(S)0, C(S)S, C(NR,2)0 or C(0)NRI2; Y, is a single bond, O, NR,0 or S; Y2 is O, NR13, S, C(0)0, C(0)S, C(S)0, C(S)S, C(NR!3)0 or C(0)NR,3; Z, is a single bond, O, NR„ or S; Z2 is O, NR14, S, C(0)0, C(0)S, C(S)0, C(S)S, C(NR14)0 or C(0)NR14; R9, R10, R11, R12, 13 and R,4 are independently hydrogen, alkyl or aralkyl; none of the pairs Xi and X2, Yi and Y2, and Zi and Z2 comprises O and O, S and O, or O and S, respectively;
provided that: at least one of Ri, R2 and R3 is not hydrogen or methyl; when p is 0, Xi is a single bond, and X2 is NR]2, then Ri is not benzoyl or methylbenzoyl; when X2 is C(0)0, C(0)S, C(S)0, C(S)S or C(NRι2)0, then Rx is not hydrogen; when Y2 is C(0)0, C(0)S, C(S)0, C(S)S or C(NR,2)0, then
R2 is not hydrogen; when Z2 is C(0)0, C(0)S, C(S)0, C(S)S or C(NRι2)0, then R3 is not hydrogen; and when R2 is a peptide comprising 2-6 amino acid residues, then R] is not hydrogen or methyl;
said method comprising:
(a) providing a first monosaccharide having the formula
wherein R2Y2 1 is bonded to a ring carbon atom adjacent to a free hydroxyl group; and none of R2Y2Yj, W1R4, W2R5 and ZιZ2R3 is a free hydroxyl, amino or thiol group, or bears a free hydroxyl, amino or thiol group;
(b) contacting said first monosaccharide with a second monosaccharide having the formula
wherein Ar is an aryl group, and none of R8, RβW3 and R7W4 is a free hydroxyl, amino or thiol group, or bears a free hydroxyl, amino or thiol group; and an activating agent; and
(c) allowing a glycosylation reaction to proceed such that an alpha glycosidic linkage is formed between said first monosaccharide and said second monosaccharide.
This invention is further directed to a chemical library comprising a plurality of these compounds, and to a method of treating bacterial infections in humans by administering an effective amount of the compound.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a graph showing the effects on macromolecular synthesis in Bacillus megaterium MB410 of known antibiotics. Figure 2 is a graph showing the effect of compound 6a on synthesis of RNA, DNA, protein and peptidoglycan in comparison with the effects of vancomycin and ampicillin.
Figures 3 A and 3B are graphs showing the activity of compound 6a in ether-treated bacteria, and the site of inhibition of peptidoglycan synthesis.
Figures 4 - 7 are tables presenting results obtained on several compounds of this invention, along with controls, for synthesis of RNA, DNA, protein and peptidoglycan, and for the site of inhibition of peptidoglycan synthesis in ether treated bacteria.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
The term "alkyl" refers to an acyclic or non-aromatic cyclic group having from one to twenty carbon atoms connected by single or multiple bonds. An alkyl group may be substituted by one or more of halo,
hydroxyl, protected hydroxyl, amino, nitro, cyano, alkoxy, aryloxy, aralkyloxy, COOH, aroyloxy, alkylamino, dialkylamino, trialkylammonium, alkylthio, alkanoyl, alkanoyloxy, alkanoylamido, alkylsulfonyl, arylsulfonyl, aroyl, aralkanoyl, heterocyclic, CONH2, CONH-alkyl, CONH-aryl, CONH- aralkyl, CON(alkyl)2, COO-aralkyl, COO-aryl, COO-heterocyclic, COO-alkyl or phosphonium substituted by any combination of alkyl, aryl, aralkyl or heterocyclic.
The term "aryl" refers to a group derived from a non-heterocyclic aromatic compound having from six to twenty carbon atoms and from one to four rings which may be fused or connected by single bonds. An aryl group may be substituted by one or more of alkyl, aralkyl, heterocyclic, heterocyclic-alkyl, heterocyclic-carbonyl, halo, hydroxyl, protected hydroxyl, amino, hydrazino, alkylhydrazino, arylhydrazino, nitro, cyano, alkoxy, aryloxy, aralkyloxy, aroyloxy, alkylamino, dialkylamino, trialkylammonium, alkylthio, alkanoyl, alkanoyloxy, alkanoylamido, alkylsulfonyl, arylsulfonyl, aroyl, aralkanoyl, COO-alkyl, COO-aralkyl, COO-aryl, COO-heterocyclic, CONH2, CONH-alkyl, CONH-aryl, CONH-aralkyl, CON(alkyl) or phosphonium substituted by any combination of alkyl, aryl, aralkyl or heterocyclic. The term "aralkyl" refers to an alkyl group substituted by an aryl group.
The term "heterocyclic" refers to a group derived from a heterocyclic compound having from one to four rings, which may be fused or connected by single bonds; said compound having from three to twenty ring atoms which may be carbon, nitrogen, oxygen, sulfur or phosphorus. A heterocyclic group may be substituted by one or more of alkyl, aryl, aralkyl, halo, hydroxyl, protected hydroxyl, amino, hydrazino, alkylhydrazino, arylhydrazino, nitro, cyano, alkoxy, aryloxy, aralkyloxy, aroyloxy, alkylamino, dialkylamino, trialkylammonium, alkylthio, alkanoyl, alkanoyloxy, alkanoylamido, alkylsulfonyl, arylsulfonyl, aroyl, aralkanoyl, COO-alkyl, COO-aralkyl, COO-aryl, COO-heterocyclic, CONH2, CONH- alkyl, CONH-aryl, CONH-aralkyl, CON(alkyl)2 or phosphonium substituted by any combination of alkyl, aryl, aralkyl or heterocyclic.
The terms "alkoxy," "aryloxy" and "aralkyloxy" refer to groups derived from bonding an oxygen atom to an alkyl, aryl or aralkyl group, respectively. The terms "alkanoyl," "aroyl" and "aralkanoyl" refer to groups derived from bonding a carbonyl to an alkyl, aryl or aralkyl group, respectively. The terms "heterocyclic-alkyl" and "heterocyclic-carbonyl" refer to groups derived from bonding a heterocyclic group to an alkyl or a carbonyl group, respectively. The term "heterocyclic-alkyl-carbonyl" refers to a group derived from bonding a heterocyclic-alkyl group to a carbonyl group. The term "hydroxyl protecting group" refers to a group bonded to a hydroxyl group which is easily removed to regenerate the free hydroxyl group by treatment with acid or base, by reduction, or by exposure to light. Exemplary hydroxyl protecting groups include, without limitation, acetyl, chloroacetyl, pivaloyl, benzyl, benzoyl, p- nitrobenzoyl, tert-butyl-diphenylsilyl, allyloxycarbonyl and allyl. Likewise, the terms "amino protecting group" and "thiol protecting group" refer to groups bonded to an amino or thiol group, respectively, which are easily removed to regenerate the free amino or thiol group, respectively, by treatment with acid or base, by reduction, or by exposure to light. Exemplary amino protecting groups include, without limitation, Fmoc, CBz, aloe and alkanoyl and alkoxycarbonyl groups. Exemplary thiol protecting groups include, without limitation, alkanoyl and aroyl groups.
A "glycopeptide" is a compound comprising a peptide linked to at least one carbohydrate. An "aglycone" is the result of removing the carbohydrate residues from a glycopeptide, leaving only a peptide core. A "dalbaheptide" is a glycopeptide containing a heptapeptide moiety which is held in a rigid conformation by cross-links between the aromatic substituent groups of at least five of the seven α-amino acid residues, including a cross-link comprising a direct carbon-carbon bond between the aryl substituents of amino acid residues 5 and 7, and aryl ether cross-links between the substituents of amino acid residues 2 and 4, and 4 and 6. Amino acid residues 2 and 4-7 in different dalbaheptides are those found in the naturally occurring glycopeptide antibiotics. These amino acid residues differ only in that residues 2 and 6 do not always have a chlorine substituent on their aromatic rings, and in that substitution on free hydroxyl or amino groups may be present. Amino acid residues 1 and 3 may differ substantially in different dalbaheptides; if both bear aryl substituents, these may be cross-linked. Molecules having a dalbaheptide structure include, e.g., vancomycin and teicoplanin.
A "chemical library" is a synthesized set of compounds having different structures. The chemical library may be screened for biological activity to identify individual active compounds of interest. The term "DMF" refers to N,N-dimethylformamide; "THF" refers to tetrahydrofuran; "TFA" refers to trifluoroacetic acid; "EtOAc" refers to ethyl acetate; Et20 refers to diethyl ether; "MeOH" refers to methanol; "MeCN" refers to acetonitrile; "Tf" refers to the trifluoroacetyl group; "DMSO" refers to dimethyl sulfoxide; "DIEA" refers to diisopropylethylamine; "DTBMP" refers to 2,6-di-tert-butyl-4- methylpyridine ; "All" in structural formulas refers to the allyl group; "Fmoc" refers to 9- fluorenylmethyloxycarbonyl; "HOBt" refers to 1 -hydroxybenzotriazole and "OBt" to the 1- oxybenzotriazolyl group; "PyBOP" refers to benzotriazol-1-yl-oxytripyrrolidine-phosphonium hexafluorophosphate; "Su" refers to the succinimidyl group; "HBTU" refers to O-benzotriazol-1-yl- N,N,N',N'-tetramethyluronium hexafluorophosphate; "aloe" refers to allyloxycarbonyl; and "CBz" refers to benzyloxycarbonyl.
The disaccharide compound of the present invention comprises two hexose residues joined by an alpha glycosidic linkage. At least one of the hexose residues is substituted by a lipid group, i.e., an organic functional group having from 2-30 carbon atoms, preferably 2-20 carbon atoms and may also contain heteroatoms. The lipid group may be linear, branched, or cyclic, and may include aliphatic, aromatic and/or heterocyclic groups. A number of substituents can also be present on the hexose rings, in particular the ring not bearing the lipid group. However, the substituents exclude peptidic moieties having demonstrable transpeptidase inhibitory activity. The disaccharide of the invention exhibits anti-infective, preferably antibiotic activity. Most preferably, the compound of the invention exhibits transglycosylase inhibitory activity.
Preferably, the disaccharide compound has the formula (I)
(I) wherein R2Y2Yι is bonded to a ring carbon atom adjacent to the alpha glycosidic linkage; R) and R3 are independently hydrogen, alkyl, aryl, aralkyl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, alkanoyl, aroyl, aralkanoyl, heterocyclic, heterocyclic-alkyl, heterocyclic-carbonyl or heterocyclic-alkyl-carbonyl; R2 is hydrogen, alkyl, aryl, aralkyl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, alkanoyl, aroyl, aralkanoyl, heterocyclic, heterocyclic-alkyl, heterocyclic-carbonyl, heterocyclic-alkyl-carbonyl or a peptide comprising 2-6 amino acid residues; Rt, R5, Re and R7 are independently hydrogen, or a hydroxyl, amino or thiol protecting group; Wi, W2, W3 and W4 are independently O, NH or S; R8 is hydrogen, hydroxyl or a hydroxyl protecting group; k, m, n, p and r are independently 0 or 1; Xj is a single bond, O, NR9 or S; X2 is O, NRj2, S, C(0)0, C(0)S, C(S)0, C(S)S, C(NR12)0 or C(0)NR12; Y, is a single bond, O, NR10 or S; Y2 is O, NR13, S, C(0)0, C(0)S, C(S)0, C(S)S, C(NR13)0 or C(0)NR13; Z, is a single bond, O, NR, , or S; Z2 is O, NR14, S, C(0)0, C(0)S, C(S)0, C(S)S, C(NR14)0 or C(0)NR14; R9, R10, R„, Rj2, R,3 and R14 are independently hydrogen, alkyl or aralkyl; none of the pairs X! and X2, Y, and Y , and Zi and Z2 comprises O and O, S and O, or O and S, respectively;
provided that: at least one of Rj, R2 and R3 is not hydrogen or methyl; when p is 0, X, is a single bond, and X2 is NRn, then Ri is not benzoyl or methy lbenzoyl; when X2 is C(0)0, C(0)S, C(S)0, C(S)S or C(NR,2)0, then Ri is not hydrogen; when Y2 is C(0)0, C(0)S, C(S)0, C(S)S or C(NR12)0, then
R2 is not hydrogen; when Z2 is C(0)0, C(0)S, C(S)0, C(S)S or C(NR,2)0, then R3 is not hydrogen; and when R2 is a peptide comprising 2-6 amino acid residues, then Rj is not hydrogen or methyl.
Since the general shape of a disaccharide comprising two hexose residues is determined by the conformation of the glycosidic linkage and the position of large substituent groups, the alpha glycosidic linkage results in a very different relative spatial presentation of the hydroxyls and other functional groups on the two sugars than would be observed for any other linkage. For this reason, the transglycosylase activity displayed by the compounds of this invention could not have been predicted based on the activity of moenomycin and its derivatives, which have beta glycosidic linkages. Another consequence of the unique shape of the disaccharide compounds of this invention is that a great variety of substituents may be introduced without destroying the antibiotic activity of this compound.
When R2 is a peptide comprising 2-6 amino acid residues, R is any natural or synthetic peptide in the stated size range. It is preferred that R2 has the formula A2-A3-A4-A5-A6-A7, in which each dash represents a covalent bond; wherein each of the groups A2 to A7 comprises a modified or unmodified α-amino acid residue, whereby (i) each of the groups A2, A4 and A6 bears an aromatic side chain, which aromatic side chains are cross-linked together by two or more covalent bonds, and (ii) the group A bears a terminal carboxyl, ester, amide, or N-substituted amide group. It is further preferred that R2 is one of the dalbaheptide aglycones of the natural vancomycin family of antibiotics from which the leucine residue has been removed completely, thereby producing a hexapeptide. It is not intended that R2 is a glycopeptide.
Modified amino acid residues include amino acid residues whose aromatic groups have been substituted by halo, alkyl, alkoxy, alkanoyl, or other groups easily introduced by electrophilic substitution reactions or by reaction of phenolic hydroxyl groups with alkylating or acylating agents; and amino acid residues which have protecting groups or other easily introduced substituents on their hydroxyl or amino groups, including, but not limited to alkyl, alkanoyl, aroyl, aralkyl, aralkanoyl, carbamoyl, alkyloxycarbonyl, aralkyloxycarbonyl, aryloxycarbonyl, alkylsulfonyl, arylsulfonyl, heterocyclic, heterocyclic-alkyl or heterocyclic-carbonyl substituents. Examples of preferred protecting groups include acetyl, allyloxycarbonyl (aloe), CBz, allyl, benzyl, p-methoxybenzyl and methyl. Modifications of hydroxyl groups occur on phenolic hydroxyl groups, benzylic hydroxyl groups, or aliphatic hydroxyl groups. Other amino acid residues, in addition to A2, A4 and A , may be cross-linked through their, aromatic substituent groups.
It is preferred that the R2Y2Yι group is attached to the anomeric position of a monosaccharide and the alpha glycosidic linkage is attached to the 2-position of the same monosaccharide. It is further preferred
that Wi, W2 and W3 are O. It is also preferred that at least one of Ri, R2 and R3 is not acetyl, benzyl or benzoyl. It is also preferred that at least two substituents on the disaccharide are not hydroxyl, amino, protected hydroxyl or protected amino. In one embodiment of the invention, it is preferred that R8 is hydrogen and p is 0, further preferred that k is 1 and m is 0, still further preferred that r is 1, and most preferred that X] is a single bond and X2 is NRι2. In another embodiment of the invention, it is preferred that Zj is a single bond, Z2 is O, S or NRj4, and Rj, R5 and Rs are hydrogen, and further preferred that X) is a single bond, X2 is NRι2, Yi is a single bond and Y2 is O. In another embodiment, it is preferred that X]X2Ri and a CH3 group are both attached to the 3-position of a monosaccharide.
In a particularly preferred embodiment of the invention, the disaccharide is derived from the disaccharide component of vancomycin, which has a glucose residue attached through its 2-position to a vancosamine residue. Examples of such disaccharides are shown below in Scheme 1. The vancosamine residue may lack the methyl group geminal to the amine, as in compound 11. Compounds 1 1 , 6a and 6c are substituted with an N-4-(4-chlorophenyl)benzyl substituent on the vancosamine nitrogen, while compound 6b has an n-decyl substituent on the vancosamine nitrogen. Compounds 1 1, 6a and 6b have an equatorial 2,6- dimethoxyphenyl substituent on the glucose anomeric hydroxyl, while compound 6c has an axial methoxy substituent. Scheme 1
In the method of this invention, the compounds of formula (I) are prepared by allowing a first monosaccharide having the formula
wherein R2Y2Y] is bonded to a ring carbon atom adjacent to a free hydroxyl group; and none of R2Y2Yι, W1R4, W2R5 and Z]Z2R3 is a free hydroxyl, amino or thiol group, or bears a free hydroxyl, amino or thiol group; to react with a second monosaccharide having the formula
wherein Ar is an aryl group, and none of Rg, 6W3, R7W4 and XιX2Rj is a free hydroxyl, amino or thiol group, or bears a free hydroxyl, amino or thiol group; and an activating agent; via a glycosylation reaction in which an alpha glycosidic linkage is formed between the first monosaccharide and the second monosaccharide.
All hydroxyl, amino and thiol groups on both monosaccharides are protected with the exception of the single free hydroxyl on the first monosaccharide. Optionally, these protecting groups are removed from the disaccharide product using conventional techniques for removal of protecting groups. Optionally, when the XjX2R] substituent after deprotection is an amino or alkylamino group, i.e., when Xi is a single bond, X2 is NRι2 and Rj is hydrogen, the disaccharide is contacted with an alkylating agent capable of reacting with the amino or alkylamino group to produce an alkylated substituent. Examples of suitable alkylating agents include, without limitation, alkyl halides, alkyl sulfonate esters, and aldehydes or ketones under reactive amination conditions.
In another embodiment of the invention, the XιX2R] substituent is replaced by an azido group, i.e., the second monosaccharide bears a group (CH2)PN3. After the glycosylation reaction has been performed, the azido group preferably is reduced to an amino group using one of the suitable reducing agents that are well known in the art. This method is exemplified in Scheme 3.
The anomeric aryl sulfoxide group is activated by contacting it with an organic acid anhydride which will react with the sulfoxide. The organic acid anhydride may be an anhydride of a sulfonic acid, of two different sulfonic acids or of a sulfonic acid and a carboxylic acid. The preferred organic acid anhydride is trifluoromethanesulfonic anhydride (triflic anhydride, Tf20). Preferably, a non-nucleophilic mild base is also added to the reaction mixture. Suitable non-nucleophilic mild bases include, but are not limited to, porphyrins, 2,6-dialkylanilines, acetamides, 2,6-dialkylpyridines and co-solvents such as ethyl acetate or ethers. The preferred base is 2,6-di-tert-butyl-4-methylpyridine (DTBMP).
A method for preparation of the compounds shown in Scheme 1 is illustrated below in Schemes 2 and 3.
Scheme 2
3a R=2,6-dimethoxyphenoxy 3b R=β-SPh 3c R=α-OMe
4a X=2,6-dimethoxyphenoxy, 5a X=2,6-dimethoxyphenoxy, 6a X=2,6-dimethoxyphenoxy,
Y=H Y=H Y=H, R=chlorobiphenyl
4b X=H, Y=OMe 5b X=H, Y=OMe 6b X=2,6-dimethoxyphenoxy,
Y=H, R=n-decyl 6c X=H, Y=OMe, R= chlorobiphenyl
As shown in Scheme 2, a partially protected glucose, la or lb, having one free hydroxyl group is allowed glycosylated product 3a or 3b, respectively. The β-thiophenoxy substituent in 3b is converted to an α- methoxy substituent by treatment with mercury(II) trifluoroacetate and DTBMP to give 3c. Treatment of 3a or 3c with hydrazine gives the partially deprotected product 4a or 4b, respectively. Hydrogenation of 4a or 4b gives completely deprotected product 5a or 5b, respectively. Reaction with 4-(4- chlorophenyl)benzaldehyde ("chlorobiphenyl aldehyde") or 1 -decanal under conditions effective for reductive amination gives products 6a-6c, as shown. This approach may be used to introduce a variety of XiX2Ri and R2Y2Yι substituent groups at the vancosamine nitrogen and at the glucose anomeric carbon.
Scheme 3
Scheme 3 shows the reaction of a partially protected glucose la with a hexose bearing an anomeric sulfoxide substituent 7. However, unlike vancosamine derivative 2 in Scheme 2, compound 7 is a desmethyl vancosamine derivative. The same sequence of reactions carried out in Scheme 2 produces compound 11, a desmethyl derivative of compound 6a.
Other particularly preferred compounds of this invention are those derived from the desmethyl vancomycin disaccharide and substituted on the C-6 position of the glucose residue, as well as on the vancosamine nitrogen. An example of a C-6 functionalized compound, which is also functionalized on the vancosamine nitrogen and the glucose anomeric carbon, is shown below.
Derivatives at the C-6 position are produced from intermediates having a mesitylenesulfonyl group at the C-6 position and a protected vancosamine nitrogen. A method for functionalizing the C-6 position is described in copending application Serial No. 09/115,667, titled "Glycopeptide Antibiotics, Combinatorial Libraries of Glycopeptide Antibiotics and Methods of Producing Same," filed July 14, 1998, and which is incorporated herein by reference.
Selectively introducing the mesitylenesulfonyl group at the glucose-6-position differentiates this position from the other hydroxyl groups and allows further reaction to displace the mesitylenesulfonyl group, affording many derivatives. A variety of Z]Z2R3 substituent groups are introduced at the glucose-6 position by using common methods for nucleophilic displacement of primary arylsulfonyl groups directly, or by further synthetic modification of initial displacement products, including azido and iodo groups. For example, the iodo group is displaced by a variety of nucleophiles to produce additional Cό-derivatives. A preferred nucleophile is a thiol compound, especially a heterocyclic thiol. Modification of an azido group at the 6-position is performed, e.g., by reducing the azido group to an amino group, which in turn is functionalized by means of reductive alkylation, nucleophilic substitution, or other amino-group reactions well known to those skilled in the art.
In another preferred embodiment of the invention, the substituent R2Y2Yι is a peptide having from 2-6 amino acid residues and linked through an oxygen atom to the anomeric carbon atom of the saccharide ring, i.e., Yi is a single bond, Y2 is O, and R2 is a peptide having from 2-6 amino acid residues. An example of a compound in this embodiment is compound 12, whose preparation and structure are shown below in Scheme 4: Scheme 4
des-leucine vancomycin
12
The chemical library of compounds of this invention is prepared to explore the effects on biological activity of introducing a large number of different substituents on disaccharides having an alpha glycosidic linkage. In any preparation of a chemical library, at least two steps are performed, each of which introduces a substituent group. A combinatorial format is established in which many different predetermined substituent groups are introduced independently at each of at least two positions on an alpha-linked disaccharide, resulting in a library containing a large number of substituted alpha-linked disaccharides, wherein each possible combination of the predetermined substituent groups is represented. For example, if three positions are to be substituted and 36 different substituent groups (3 sets of 12) are chosen, 1 of each set of 12 to be substituted at each position, the total number of unique compounds (each of which bears 3 substituent groups) in the library will be 12x12x12=1,728. It is readily apparent that, when a combinatorial synthesis is performed in an automated system, a large number of related Methods for performing combinatorial synthesis are well known and are described in several review articles. [Thompson (1996), Gallop (1994), Gordon (1994), Terrett (1995)]
In a preferred embodiment of this invention, at least two of the XιX2Rι, R2Y2Yι and Z)Z2R3 substituent groups are introduced in a combinatorial format, choosing each substituent from a group of possible substituents, thereby generating a chemical library. The methods used to introduce these substituents are those presented in Scheme 2 for introduction of the and R2Y2Yι substituent groups, and the method outlined hereinabove for functionalization of the glucose C-6 position with a Z^ R substituent by means of a C-6 mesitylenesulfonyl group.
The following examples are presented to illustrate various aspects of the present invention, but are not intended to limit it.
EXAMPLES
EXAMPLE 1 : 3-(N-benzyloxy-carbonyloxy)-4-0-acetyl-2,3,6-trideoxy-3-C-methyl-α-L-lyxo- hexopyranosyl-(l— »2)-3,4,6-tri-0-benzyl-β-glucopyranoyl 2,6-dimethoxyphenol (3a).
The compound la (20 mg, 0.0315 mmol) and DTBMP (32 mg, 0.158 mmol) are azeotroped with toluene 3 times and then dissolved in 2 mL Et20. The reaction solution is cooled to -78 C and 0.5 mL toluene is added. Triflic anhydride (6 μL, 0.0347 mmol) is added to the reaction solution, and the sulfoxide 2 (28 mg, 0.0629 mmol) in 1 mL Et 0 is added dropwise over 10 minutes. The reaction is warmed up to 0°C in 1 hour and then quenched with 3 mL of saturated aqueous NaHC03 solution. The ether layer is separated and the aqueous layer is further extracted with EtOAc (5 mLx3). The organic layers are combined and dried over anhydrous sodium sulfate, filtered, and concentrated to a clear oil. This oil is loaded onto a silica gel column (10mmx8cm) and eluted with 25%-35% EtOAc/petroleum ether to give 22 mg (71%) compound 3a as a white solid. Rf 0.45 (40%EtOAc/petroleum ether); 'H NMR (CDCf., 500 MHz) δ 7.36-7.18 (m, 20 H), 7.02 (t, J = 8.2 Hz, 1 H), 6.57 (d, J = 8.2 Hz, 2 H), 5.34 (d, J = 4.3 Hz, VH.,, 1 H), 5.07 (d, J = 7.6 Hz, GH-ι, 1 H), 4.96 - 4.89 (m, 3 H), 4.82-4.74 (m, 3 H), 4.64-4.58 (m, 2 H), 4.52 (d, J = 1 1.9 Hz, 1 H), 4.43 (d, J = 11.9 Hz, 1 H), 3.95 (t, J = 8.5 Hz, GH.2, 1 H), 3.77 (s, OCH3, 6 H), 3.76 - 3.60 (m, 4 H), 3.38 (m, GH.5, 1 H), 2.09 (d, J = 13.4 Hz, VH.2, 1 H), 2.07 (s, COCH3, 3 H), 1.87 (s, CH3, 3 H), 1.82 (dd, J = 4.6, 13.4 Hz, VH.2\ 1 H), 0.95 (d, J = 6.4 Hz, CH3, 3 H); 13C NMR (CDC13, 500 MHz) δ
171.46, 155.00, 153.98, 138.79, 138.68, 138.31, 136.79, 134.01, 128.68, 128.62, 128.58, 128.44, 128.27, 128.10, 128.00, 127.92, 127.75, 127.60, 124.46, 105.73, 100.91, 97.95, 86.23, 78.51, 77.90, 76.02, 75.64, 74.97, 74.53, 73.82, 69.50, 68.97, 66.42, 63.28, 56.24, 53.36, 36.30, 23.79, 21.02, 17.07; HR-MS (FAB) calcd for C52H59NOι3Na [M+Na+]: 928.3884, found 928.3918.
EXAMPLE 2: Phenyl 2-(3-N-Cbz-4-0-acetyl-2,3,6-trideoxy-3-C-methyl-α-L-lyxo-hexopyranosyl)-3,4,6- -tri-O-benzyl- 1 -thio-β-D-glucopyranoside (3b).
Compound lb (37 mg, 0.0685 mmol) and DTBMP (70 mg, 0.342 mmol) are azeotroped with toluene 3 times and then dissolved in 5 mL Et20. The reaction solution is cooled to -78°. Triflic anhydride (11.5 μL, 0.0685 mmol) is added to the reaction solution, followed by dropwise addition of the sulfoxide 2 (61 mg, 0.0137 mmol) in lmL Et20 over 10 minutes. The reaction is warmed up to 0°C in 1 hour and then quenched with 10 mL of saturated aqueous NaHC03 solution. The ether layer is separated and the aqueous layer is further extracted with EtOAc (5 mLx3). The organic layers are combined and dried over anhydrous sodium sulfate, filtered, and concentrated to a clear oil. This oil is loaded onto a silica gel column (10mmx8cm) and eluted with 35% EtOAc/petroleum ether to give 50 mg (85%) compound 3b as a white solid. Rf 0.35 (30%EtOAc/petroleum ether); Η NMR (500MHz, CDC13) δ 7.53-7.51 (m, 2H), 7.34-7.19 (m, 21H), 7.17-7.15 (m, 2H), 5.45 (d, J=4.6Hz, 1H, VH-ι), 5.08 (d, J=12.2Hz, GH-ι), 5.45 (s, 1H), 4.95-4.91 (m, 2H), 4.76-4.72 (m, 4H), 4.63-4.50 (m, 4H), 3.77-3.72 (m, 2H), 3.69-3.60 (m, 3H), 3.49-3.47 (m, 1H, GH-5), 2.09 (s, 3H), 2.06-1.99 (m, 1H, VH.2), 1.84-1.80 (m, 1H, VH.2 , 1.74 (s, 3H), 1.16 (d, J=6.4Hz, 3H).
EXAMPLE 3: Methyl 2-(3-N-Cbz-4-0-acetyl-2,3,6-trideoxy-3-C-methyl-α-L-lyxo-hexopyranosyl)-3,4,6- -tri-O-benzyl-α-D-glucopyranoside (3c).
To a solution of sulfide 3b (50 mg, 0.058 mmol) and DTBMP (24 mg, 0.116 mmol) in 2 mL CH2C12 and 0.5 mL methanol is added Hg(OOCCF3)2 (27 mg, 0.0638 mmol) in one portion. The reaction is stirred at room temperature for 10 minutes and then quenched by addition of 20 mL CH2C12. The CH C12 layer is separated and the aqueous layer is further extracted with CH2C12 (15 mL x 3). The organic layers are combined and dried over anhydrous sodium sulfate, filtered, and concentrated to a clear oil. This oil is purified by flash chromatography (25 % EtOAc/petroleum ether) to give 20 mg (44%) of compound 3c as a white solid. Rf 0.2 (30% EtOAc/petroleum ether); ]H NMR (500MHz, CDC13) δ 7.39 (m, 20H), 5.15- 4.41 (m, 11H), 4.24-4.20 (m, 1H, VH.5), 3.90 (t, J=9.2Hz, 1H), 3.76-3.60 (m, 5H), 3.40 (s, 3H, OCH3), 2.09 (s, 3H), 2.05-2.04 (m, 1H, VH.2), 1.98 (m, 1H, VH-2>), 1.81 (s, 3H), 1.13 (d, J=6.4Hz, 3H, VH.6). EXAMPLE 4: 3-(N-benzyloxy-carbonyloxy)-2,3,6-trideoxy-3-C-methyl-α-L-lyxo-hexopyranosyl-(l- 2)- 3,4,6-tri-O-benzyl-β-glucopyranoyl 2,6-dimethoxyphenol (4a).
Compound 3a (20 mg, 0.022 mmol) is dissolved in 330 μL THF and 660 μL methanol. 100 μL anhydrous hydrazine is added. The reaction is stirred at room temperature for 5 hours and then quenched by addition of 0.5 mL acetic acid. All the solvents are removed and the residue is partitioned between 10 L dichloromethane and 10 mL saturated NH4C1 aqueous solution. The CH2C12 layer is separated and the aqueous layer is further extracted with CH2CI2 (5 mLx3). The organic layers are combined and dried over anhydrous sodium sulfate, filtered, and concentrated to a clear oil. This oil is loaded onto a silica gel column (10mmx8cm) and eluted with 40% EtOAc/petroleum ether to give 17.5 mg (92%) of compound 4a as a white solid. Rf 0.35 (40% EtOAc/petroleum ether); Η NMR (CDC13; 500 MHz) δ 7.19-7.35 (m, 20 H), 7.02 (t, J = 8.5 Hz, 1 H), 6.57 (d, J = 8.5 Hz, 2 H), 5.48 (s, 1 H), 5.27 (d, J = 4.6 Hz, VH.,, 1 H), 5.08 (d, J = 7.6 Hz, GH-I, 1 H), 5.05 (s, 2 H), 4.90 (d, J = 1 1.0 Hz, 1 H), 4.88 - 4.76 (m, 2 H), 4.67 (q, J = 6.4 Hz, VH-5, 2 H), 4.59 (d, J = 1 1.0 Hz, 1 H), 4.52 (d, J = 1 1.9 Hz, 1 H), 4.43 (d, J = 1 1.9 Hz, 1 H), 3.94 (t, J = 8.5 Hz, GH-2, 1 H), 3.78 (s, OCH3, 6 H), 3.74-3.60 (m, 4 H), 3.38 (m, GH.5, 1 H), 3.20 (s, VH.4, 1 H), 2.24 (d, J = 14.0 Hz, VH-2, 1 H), 1.78 (s, CH3, 3 H), 1.72 (dd, J = 4.9, 14.0 Hz, VH.2', 1 H), 1.07 (d, J = 6.4 Hz, VH-6, 3 H); ,3C NMR (CDC13, 500 MHz) δ 155.31, 153.98, 138.77, 138.57, 138.30, 136.89, 134.06, 128.68, 128.61, 128.56, 128.42, 128.20, 128.07, 127.97, 127.91, 127.78, 127.66, 127.58, 124.40, 105.70, 100.85, 97.83, 86.20, 78.46, 77.79, 75.99, 75.66, 74.95, 73.99, 73.79, 68.92, 66.30, 63.42, 56.21, 53.82, 35.80, 22.82, 16.92; HR-MS (FAB) calcd for C50H57NOι2Na [M+Na+]: calcd 886.3778, found 886.3827.
EXAMPLE 5: Methyl 2-(3-N-Cbz-4-hydroxy-vancosaminyl)-3,4,6— tri-O-benzyl-α-D-glucopyranoside (4b).
Compound 3b (20 mg, 0.0255 mmol) is dissolved in 330 μL THF and 660 μL methanol. 100 μL anhydrous hydrazine is added. The reaction is stirred at room temperature for 5 hours and then quenched by addition of 0.5 mL acetic acid. All the solvents are removed and the residue is partitioned between 10 mL EtOAc and 10 mL saturated aqueous NH4CI solution. The aqueous layer is separated and the organic layer is further extracted with NH4CI solution (5 mLx3). The organic layers are separated and dried over anhydrous sodium sulfate, filtered, and concentrated to give 16 mg (85%) of crude compound 4b as a clear oil. This oil is subjected to hydrogenation without further purification. Rf 0.16 (30% EtOAc/petroleum ether). EXAMPLE 6: Vancosaminyl-(l→2)-β-glucopyranosyl 2,6-dimethoxyphenol (5a).
Compound 4a (18 mg, 0.0208 mmol) is dissolved in 8 mL methanol and 25 mg Pearlman's catalyst is added. The suspension is stirred under H2 for 30 minutes, and then another 15 mg Pearlman's catalyst is added. After another 30 minutes stirring under H2, TLC shows completed reaction. 40 mL methanol is added and the suspension is stirred for 30 minutes under argon and then filtered. The catalyst is resuspended in 40 mL methanol and then filtered. The combined filtrate is concentrated and the residue is purified by reverse-phase HPLC using a PHENOMENEX LUNA Ci8 column (21.2x250 mm), 5 μm particle, eluting with a 40 minute linear gradient of 0% acetonitrile/0.1% acetic acid in water to 70% acetonitrile/0.1% acetic acid in water; flow rate of 8 mL/min. and UV detection at 270 nm. The fractions containing the pure product are combined and evaporated to give 7 mg (73%) of compound 5a as a white solid. Rf 0.1 (CHCl3/MeOH/H2O=3/2/0.5); 'H NMR (CD-OD, 500 MHz) δ 7.03 (t, J = 8.2 Hz, 1 H), 6.68 (d, J = 9.2 Hz, 2 H), 5.39 (d, J = 4.0 Hz, VH-ι, 1 H), 5.16 (J = 7.6 Hz, GH_ι, 1 H), 4.55-4.52 (m, VH-s, 1 H), 3.81 (s, OMe, 6 H), 3.68-3.60 (m, GH.2, GH.6', GH.6, 3 H), 3.51 (t, J = 9.1 Hz, GH.3, 1 H), 3.42 (t, J = 9.5 Hz, GH-4, 1 H), 3.20 (s, VH-4, 1 H), 3.14-3.11 (m, GH-s, 1 H), 2.04 - 1.93 (m, VH.2, VH.2, 2 H), 1.65 (s, CH3, 3
H), 1.07 (d, J = 6.7 Hz, CH3, 3 H); 13C NMR (CD-OD, 500 MHz) δ 154.90, 134.00, 125.66, 107.30,
101.95, 98.59, 79.50, 79.27, 78.17, 73.29, 71.65, 64.97, 62.62, 56.91, 55.60, 35.14, 23.68, 17.08; HR-MS (FAB) calcd for C2,H33NO,oNa [M+Na+]: 482.2002, found 482.1991.
EXAMPLE 7: Methyl 2-vancosaminyl-α-D-glucopyranoside (5b).
Compound 4b (16 mg, 0.0216 mmol) is dissolved in 8 mL methanol and 25 mg Pearlman's catalyst is added. The suspension is stirred under H2 for 30 minutes, and then another 15 mg Pearlman's catalyst is added. After another 30 minutes stirring under H2, TLC indicates that the reaction is completed. 40 mL methanol is added and the suspension is stirred for 30 minutes under argon and then filtered. The catalyst is resuspended in 40 mL methanol and then filtered. The combined filtrate is concentrated and the solvent is removed under reduced pressure to give 7 mg (96%) of compound 5b as a clear oil. Rf 0.1 (CHCl3/MeOH/H2O=3/2/0.5); Η NMR (CD-OD, 500 MHz) δ 5.05 (d, J=4Hz, IH, VHι), 4.78 (d, J=4Hz, IH, GH-I), 4.13 (q, J=6Hz, IH, VH.5), 3.81 (dd, J=2, 12Hz, IH, GH.6), 3.70-3.66 (m, 2H, GH-6>, GH- ), 3.52- 3.49 (m, IH, GH.5), 3.40 (s, 3H, OCH3), 3.38-3.30 (m, 2H, GH.4, VH-4), 2.05 (dd, J=4.13Hz, IH, VH-2), 1 -96 (d, J=13Hz, IH, VH-2-), 1 -61 (s, 3H), 1.25 (d, J=6Hz, 3H, VH.6);
13C NMR (500MHz, CDC13) δ 100.7, 100.2, 81.6, 74.2, 73.3, 72.3, 72.0, 65.2, 62.7, 55.9, 55.4, 34.4, 23.22. EXAMPLE 8: 2-(3-N-chlorobiphenyl-vancosaminyl)-β-D-glucopyranosyl 2,6-dimethoxyphenol (6a).
To a solution of disaccharide 5a (6 mg, 0.0131 mmol) in 0.5 mL DMF is added chlorobiphenyl aldehyde (2.55 mg, 0.0117 mmol) in one portion. The reaction is stirred at 70°C for 20 minutes and then NaCNBHt (131 μL of 1M solution in THF, 0.131 mmol) is added at this temperature. The reaction is monitored by analytical HPLC using a PHENOMENEX PRODIGY 5 μm ODS(3) lOOA column (250x4.6 mm), eluting a linear gradient of 0.1% TFA in water to 70% CH3CN/0.1% TFA over 25 minutes. After the reaction is done, the solution is cooled back to room temperature, concentrated and purified by reverse-phase HPLC using a PHENOMENEX LUNA Ci8 column (21.2x250 mm), 5 μm particle, eluting with a 30 min. linear gradient of 20% acetonitrile/0.1% acetic acid in water to 70% acetonitrile/0.1% acetic acid in water; flow rate of 8 mL/min. and UV detection at 270 nm. The fractions containing the pure products are combined and evaporated to give 8 mg (90%) of compound 6a as white solid. Rf 0.7 (CHCl3/MeOH/H2O=3:2:0.5); Η NMR (CD.OD, 500 MHz) δ 7.68 (d, J=8.2Hz, 2H), 7.63 (d, J=8.5Hz, 2H), 7.55 (d, J=7.9Hz, 2H), 7.47 (d, J=8.5Hz, 2H), 7.06 (t, J=8.5Hz, IH), 6.70 (d, J=8.5Hz, 2H), 5.41 (bs, IH, VH.ι), 5.21 (d, J=7.6Hz, IH, GH-I), 4.58 (m, IH, VH.5), 4.03 (d, J=12.2Hz, IH), 3.96 (d, J=l 1.9Hz, IH), 3.86 (s, 6H), 3.75-3.70 (m, 2H, GH.2, GH.6), 3.65 (dd, J=4.9, 11.9Hz, IH, GH.6 ), 3.55 (t, J=9.1Hz, IH, GH.3), 3.49 (s, IH, VH.4), 3.45 (t, J=9.4Hz, IH, GH-4), 3.18-3.15 (m, IH, GH-S), 2.00 (s, 2H, VH-2, VH.2 ), 1.75 (s, 3H), 1.13 (d, J=6.7Hz, 3H, VH-6); 13C NMR (CD-OD, 500 MHz) δ 154.93, 141.64, 140.43, 135.35, 134.94, 131.52, 130.21, 129.61,
128.53, 125.64, 107.30, 102.05, 98.96, 79.53, 79.28, 78.17, 71.67, 71.17, 65.21, 62.65, 54.00, 56.94, 44.70, 35.81, 20.96, 17.31; HR-MS (FAB) calcd for C34H42NCIOιoNa [M+Na+]: 682.2395, found 682.2371.
EXAMPLE 9: 2-(3-N-decyl-vancosaminyl)-β-D-glucopyranosyl 2,6-dimethoxyphenol (6b).
To a solution of disaccharide 5a (1 1 mg, 0.0240 mmol) in 0.5 mL DMF is added n-decyl aldehyde (4 μL, 0.0215 mmol) in one portion. The reaction is stirred at 70°C for 20 minutes and then NaCNBHt (240 μL of 1M solution in THF, 0.24 mmol) is added at this temperature. The reaction is monitored by analytical HPLC using a PHENOMENEX PRODIGY 5 μm ODS(3) lOOA column (250x4.6 mm), eluting a linear gradient of 0.1% TFA in water to 70% CH3CN/0.1% TFA over 25 minutes. After the reaction is done, the solution is cooled back to room temperature, concentrated and purified by reverse-phase HPLC using a PHENOMENEX LUNA C] column (21.2x250 mm), 5 μm particle, eluting with a 30 min. linear gradient of 20% acetonitrile/0.1% acetic acid in water to 70% acetonitrile/0.1% acetic acid in water; flow rate of 8 mL/min. and UV detection at 270 nm. The fractions containing the pure products are combined and evaporated to give 7 mg (49%) of compound 6b as white solid. Η NMR (500MHz, CDC13) δ 7.05 (t, IH, VH-S), 3.83 (s, 6H), 3.74-3.69 (m, 2H, GH-2, GH-6-). 3.66-3.62 (dd, J=4.9, 11.9Hz, IH, GH.6), 3.53 (t, J=9.2Hz, IH, GH.3), 3.44 (t, J=9.5Hz, IH, GH^), 3.39 (s, IH, VH-4), 3.16-3.13 (m, IH, GH.5), 3.00-2.91 (m, 2H), 2.10 (dd, J=4.6, 13.4Hz, 1HNH. ), 2.00 (d, J=14.1Hz, IH, VH. , 1-72 (s, 3H), 1.70-1.66 (m, 2H), 1.44-1.26 (m, 14H), 1.10 (d, J=6.7Hz, 3H, VH-6), 0.92 (t, J=7.0Hz, 3H); 13C NMR (500MHz, CDC13) δ 154.9, 135.2, 125.7, 107.3, 101.9, 98.4, 79.4, 79.3, 78.2, 71.7, 70.3, 64.9, 62.6, 61.0, 56.9, 40.8, 34.6, 33.2, 30.7, 30.6, 30.5, 30.4, 27.9, 23.8, 20.0, 17.0, 14.6.
EXAMPLE 10: Methyl 2-(3-N-chlorobiphenyl-vancosaminyl)-α-D-glucopyranoside (6c).
To a solution of disaccharide 5b (10 mg, 0.0297 mmol) in 0.5 mL DMF is added chlorobiphenyl aldehyde (10 mg, 0.0475 mmol) in one portion. The reaction is stirred at 70°C for 20 minutes and then NaCNBHt (297 μL of 1M solution in THF, 0.297 mmol) is added at this temperature. The reaction is monitored by analytical HPLC using a PHENOMENEX PRODIGY 5 μm ODS(3) lOOA column (250x4.6 mm), eluting a linear gradient of 0.1 % TFA in water to 70% CH3CN/0.1% TFA over 25 minutes. After the reaction is done, the solution is cooled back to room temperature, concentrated and purified by reverse-phase HPLC using a PHENOMENEX LUNA C]8 column (21.2x250 mm), 5 μm particle, eluting with a 30 min. linear gradient of 20% acetonitrile/0.1% acetic acid in water to 70% acetonitrile/0.1% acetic acid in water; flow rate of 8 mL/min. and UV detection at 270 nm. The fractions containing the pure products are combined and evaporated to give 1 1 mg (69%) of compound 6c as a white solid. Rf 0.65 (CHCl3/MeOH/H2O=3:2:0.5); Η NMR (500MHz, CDC13) δ 7.75 (d, J=8.3Hz, 2H), 7.65 (d, J=8.6Hz, 2H), 7.61 (d, J=8.2Hz, 2H), 7.48 (d, J=8.5Hz, 2H), 5.12 (d, J=4.2Hz, IH, VH-ι), 4.83 (d, J=3.7Hz, IH, GH. ,), 4.21-4.15 (m, 3H), 3.85 (dd, J=2.1H, 11.9Hz, IH, GH.6), 3.73-3.68 (m, 3H), 3.55-3.51 (m, IH, GH.5), 3.44 (s, 3H, OHe), 3.41-3.35 (m, 2H), 2.13 (dd, J=4.6, 13.4Hz, IH, VH.2), 2.04 (d, J=13.4Hz, IH, VH.2-), 1.77 (s, 3H), 1.33 (d, J=6.4Hz, 3H, VH.6); ); 13C NMR (500MHz, CDC13) δ 144.0, 141.5, 139.3, 134.3, 131.1, 129.4, 128.8, 127.9, 99.9, 99.5, 80.7, 73.4, 72.5, 71.2, 69.1, 64.5, 61.9, 61.0, 54.6, 43.7, 33.8, 19.4, 16.9.
EXAMPLE 11 : Compound (7).
To phenyl 3-azido-2,3,6-trideoxy-l-thio-α-L-galactopyranoside (100 mg, 0.379 mmol) in 5 mL pyridine is added acetic anhydride (100 μL, 1.06 mmol). The reaction is stirred at room temperature overnight and then quenched by addition of 0.5 mL methanol. The solvent is removed under reduced pressure and the residue is purified by flash chromatography (25% EtOAc/petroleum ether) to give 105 mg (90.5%) of phenyl 3-azido-4-0-acetyl-2,3,6-trideoxy-l-thio-α-L-galactopyranoside as a clear oil. Rf 0.65 (25% EtOAc/petroleum ether); Η NMR (500MHz, CDC13) δ 7.84 (m, 2H), 7.35-7.27 (m, 3H), 5.76 (d, J=5.5Hz, IH, H-l), 5.23 (t, J=1.2Hz, IH, H-4), 4.51-4.48 (m, IH, H-5), 3.88-3.84 (m, IH, H-3), 2.50 (dt, J=5.8, 13.4Hz, IH, H-2), 2.20 (s, 3H), 2.15 (dd, J=4.6, 13.6Hz, IH, H-2'), 1.16 (d, J=6.7Hz, 3H, H-6); 13C NMR (500MHz, CDCl3) δ 170.6, 134.4, 131.3, 129.2, 127.5, 83.7, 70.2, 66.3, 55.6, 30.5, 20.9, 16.8.
The sulfide (105 mg, 0.342 mmol) is dissolved in 7 mL CH2C12 and cooled to -78°C. mCPBA (103 mg of 64% purity, 0.342 mmol) is added and the reaction is slowly warmed up to -20°C in 1 hour. TLC indicates that the reaction is complete. The reaction mixture is quenched by addition of lOOmL dimethyl sulfide, and the mixture is extracted with 20 mL saturated aqueous NaHCU3 solution. The aqueous layer is further extracted with CH C12 (20 mLx3). The CH2C12 layers are combined, dried over anhydrous sodium sulfate, filtered, and concentrated to a clear oil. This oil is loaded onto a silica gel column (30mmxl2cm) and eluted with 60% EtOAc/PE to give 101 mg (92%) compound 7 as a clear oil. Rf=0.1 and 0.08 (25% EtOAc/PE).
EXAMPLE 12: Compound (8).
Compound la (65 mg, 0.11 1 mmol) and DTBMP (91 mg, 0.444 mmol) are azeotroped with toluene 3 times and then dissolved in 6 mL Et20 and 2 mL CH2C12. The reaction solution is cooled to -78 C. Triflic anhydride (19 μL, 0.1 1 1 mmol) is added to the reaction solution. The sulfoxide 7 (71 mg, 0.222 mmol) in lmL Et20 is added dropwise over 10 minutes. The reaction is warmed up to 0°C in 1 hour and then quenched with 10 mL of saturated aqueous NaHC03 solution. The ether layer is separated and the aqueous layer is further extracted with EtOAc (10 mLx3). The organic layers are combined and dried over anhydrous sodium sulfate, filtered, and concentrated to a clear oil. This oil is loaded onto a silica gel column (10mmx8cm) and eluted with 25% EtOAc/petroleum ether to give 52 mg (60%) compound 8 as a white solid. Rf 0.35 (25%EtOAc/petroleum ether); Η NMR (500MHz, CDC13) δ 7.41-7.24 (m, 15H), 7.09 (t, J=8.2Hz, IH), 6.63 (d, J=8.6Hz, 2H), 5.49 (d, J=2.8Hz, IH), 5.10 (s, IH), 5.01-4.98 (m, 2H), 4.84- 4.80 (m, 2H), 4.69 (q, J=6.4Hz, IH), 4.65 (d, J=l 1.0Hz, IH), 4.60 (d, J=l 1.9Hz, IH), 4.51 (d, J=l 1.9Hz,
IH), 4.05 (t, IH, J=7.6Hz, IH, GH.2), 3.98-3.94 (m, IH), 3.84 (s, 6H), 3.70-3.67 (m, 4H), 3.44-3.42 (m, IH, GH.5),2.18 (s, 3H), 2.00 (dt, J=3.7, 12.8 Hz, IH), 1.82 (dd, J=4.6, 12.8Hz, V), 1.01 (d, J=6.4Hz, 3H); 13C NMR (500MHz, CDC13) δ 170.9, 153.8, 138.6, 138.5, 138.1, 134.4, 128.7, 128.6, 128.4, 128.2, 128.0, 127.95, 127.7, 124.7, 105.8, 101.8, 97.4, 86.2, 78.6, 77.0, 75.8, 75.6, 75.0, 73.8, 71.0, 69.0, 65.5, 56.4, 54.9, 29.7, 21.0, 16.5. Compound 8 (50 mg, 0.0627 mmol) is dissolved in 1.6 mL THF and 3.2 mL methanol, and 200 μL anhydrous hydrazine is added. The reaction is stirred at room temperature for 5 hours and then quenched by addition of 0.5 mL acetic acid. All the solvents are removed and the residue is partitioned between 10 mL dichloromethane and 10 mL saturated aqueous NHtCl solution. The CH2C12 layer is separated and the aqueous layer is further extracted with CH2C12 (5 mLx3). The organic layers are combined and dried over anhydrous sodium sulfate, filtered, and concentrated to a clear oil. This oil is loaded onto a silica gel column (10mmx8cm) and eluted with 25% EtOAc/petroleum ether to give 35.7 mg (47%) of compound 9 as a clear oil and 9.6 mg (12%) of recovered 8. Rf 0.3 (25% EtOAc/petroleum ether); ]H NMR (500MHz, CDC13) δ 7.40-7.24 (m, 15H), 7.07 (t, J=8.5Hz, IH), 6.62 (d, J=8.5Hz, 2H), 5.43 (d, J=4.4Hz, IH), 5.03 (d, J=7.3Hz, IH, GH-ι), 4.98 (d, J=l 1Hz, IH), 4.84-4.79 (m, 2H), 4.65 (d, J=10.7Hz, IH), 4.59-4.49 (m, 3H), 4.03 (dt, J=2.5, 7.4Hz, IH, GH.2), 3.83 (s, 6H), 3.77-3.72 (m, 4H), 3.67 (dd, J=4.9, 11.3Hz, IH, GH.6), 3.63 (s, IH), 3.46-3.43 (m, IH, GH-5), 2.00 (dt, J=3.5, 12.8Hz, IH), 1.80 (dd, J=4.9, 13.1Hz, IH), 1.13 (d, J=6.7Hz, 3H,); ); 13C NMR (500MHz, CDC13) δ 153.9, 138.7, 138.4, 138.1, 134.5, 128.7, 128.6, 128.4, 128.0, 127.7, 127.6, 124.5, 105.9, 101.7, 97.3, 86.2, 78.6, 77.0, 75.9, 75.6, 75.0, 73.8, 70.6, 69.0, 66.3, 57.3, 56.4, 28.8, 16.6.
EXAMPLE 14: Compound (10).
Compound 9 (35 mg, 0.0463 mmol) is dissolved in 8 mL methanol and 25 mg Pearlman's catalyst is added. The suspension is stirred under H2 for 30 minutes. Another 15 mg Pearlman's catalyst is added at this time. After another 30 minutes stirring under H2, TLC indicates that the reaction is complete. 40 mL methanol is added and the suspension is stirred for 30 minutes under argon and then filtered. The catalyst is resuspended in 40 mL methanol and then filtered. The combined filtrate is concentrated and the residue is purified by reverse-phase HPLC using a PHENOMENEX LUNA C]8 column (21.2x250 mm), 5 μm particle, eluting with a 40 minute linear gradient of 0% acetonitrile/0.1% acetic acid in water to 70% acetonitrile/0.1% acetic acid in water; flow rate of 8 mL/min. and UV detection at 270 nm. The fractions containing the pure product are combined and evaporated to give 15 mg (73%) of compound 10 as a white solid. Rf 0.1 (CHCl3/MeOH/H2O=3/2/0.5); Η NMR (500MHz, CDC13) δ 7.02 (t, J=8.6Hz, IH), 6.67 (d, J=8.2Hz, 2H), 5.45 (br s, IH), 5.04 (d, J=7.6Hz, IH, GH.ι), 4.53 (q, J=6.7Hz, IH), 3.83 (s, 6H), 3.77-3.67 (m, 3H), 3.63 (dd, J=4.9, 12.2Hz, IH, GH.6), 3.59 (s, IH), 3.52 (t, J=9.1Hz, GH-3), 3.42 (t, J=9.4Hz, IH, GH. 4), 3.15-3.12 (m, IH, GH.5), 2.04-2.00 (m, 2H), 1.05 (d, J=6.4Hz, 3H); ); ,3C NMR (500MHz, CDC13) δ 154.9, 135.9, 125.8, 107.5, 103.0, 98.0, 79.3, 79.1, 78.0, 71.5, 68.3, 67.3, 62.6, 57.2, 53.3, 29.5, 16.8. EXAMPLE 15: Compound (11).
To a solution of disaccharide 10 (10 mg, 0.0225 mmol) in 0.5 mL DMF is added chlorobiphenyl aldehyde (4.9 mg, 0.0225 mmol) in one portion. The reaction is stirred at 70°C for 20 minutes and then NaCNBFLt (225 μL of 1M solution in THF, 0.225 mmol) is added at this temperature. The reaction is monitored by analytical HPLC using a PHENOMENEX PRODIGY 5 μm ODS(3) lOOA column (250x4.6 mm), eluting a linear gradient of 0.1% TFA in water to 70% CH3CN/0.1% TFA over 25 minutes. After the reaction is done, the solution is cooled back to room temperature, concentrated and purified by reverse-phase HPLC using a PHENOMENEX LUNA C]8 column (21.2x250 mm), 5 μm particle, eluting with a 30 min. linear gradient of 20% acetonitrile/0.1% acetic acid in water to 80% acetonitrile/0.1% acetic acid in water; flow rate of 8 mL/min. and UV detection at 270 nm. The fractions containing the pure products are combined and evaporated to give 9.4 mg (65%) of compound 11 as a white solid. Rf 0.6 (CHCl3/MeOH/H2O=3:2:0.5); Η NMR (500MHz, CDC13) δ 7.74 (d, J=8.3Hz, 2H), 7.66 (d, J=8.6Hz, 2H), 7.62 (d, J=8.2Hz, 2H), 7.50 (d, J=8.5Hz, 2H), 7.02 (t, J=8.5Hz, IH), 6.66 (d, J=8.7Hz, 2H), 5.50 (d, J=2.7Hz, IH), 5.10 (d, J=7.6Hz, IH, GH.ι), 4.54-4.51 (m, IH), 4.34 (d, J= 13.1 Hz, IH), 4.25 (d, J=13.4Hz, IH), 3.81 (s, IH), 3.75 (s, 6H), 3.73-3.62 (m, 4H), 3.53 (t, J=8.9Hz, IH, GH.3), 3.46 (t, J=9.4Hz, IH, GH.4), 3.17-3.14 (m, IH, GH.5), 2.20 (dd, J=4.3, 12.2Hz, IH), 2.10 (dt, J=3.6, 12.5Hz, IH), 1.12 (d, J=6.7Hz, 3H); 13C NMR (500MHz, CDCl3) δ 153.9, 141.7, 139.2, 135.8, 131.0, 129.5, 128.8, 128.0, 124.8, 123.5, 106.6, 101.7, 97.8, 78.5, 77.3, 70.7, 66.5, 65.2, 62.1, 61.0, 56.2, 53.9, 27.9, 16.0, 14.0.
EXAMPLE 16: Preparation of compound (12).
(a) deleucine-vancomycin. Vancomycin -HCl (497 mg, 0.335 mmol) is dissolved in 4 mL water, 4 mL distilled pyridine is added, and the mixture is stirred in a 40°C oil bath. To this solution is added phenylisothiocyanate (50 mg, 0.368 mmol). After stirring for 30 minutes the organic solvents are removed from the clear solution under reduced pressure, 100 mL water is added, and the solution is frozen and lyophilized to dryness. To the resulting powder is added 4 mL of CH2C12 and 4 mL of trifluoroacetic acid. This clear solution is stirred at room temperature for 3 minutes and then evaporated under reduced pressure to dryness. The resulting brown oil is partitioned between 100 mL of EtOAc and 100 mL H20. The aqueous layer is collected and the organic layer is extracted twice with water (40 mL each). The aqueous layers are combined and evaporated under reduced pressure to dryness. The white solid is dissolved in methanol, loaded onto a C18 reverse phase column (50mmxl2cm, particle size 40 μm, pore size 60 A, from J. T. Baker) and eluted with 10% acetonitrile/0.1% acetic acid in water. The fractions containing the pure products are combined and evaporated to give 325 mg white powder, 73.5%. Rf=0.1 (CHCl3:MeOH:H2θ=3:5:1.5). Mass Spec. [M+H]+, 1322; [M-V]+, 1178.
(b) Compound (12).
To a solution of des-leucine vancomycin (38 mg) in 2 mL of DMF at 80°C is added DIEA (100 μL) and chlorobiphenyl aldehyde (6.2 mg). The reaction is kept at 80°C for 30 minutes and then NaCNBH3 (287 μL of 1 M solution in THF) is added. After one hour, the reaction mixture is cooled to room temperature and acetic acid (200 μL) is added. The clear solution is purified by reverse-phase HPLC using a PHENOMENEX LUNA C18 column (21.2 x 250 mm), 5 μm particle size, eluting with a 30 minute linear gradient of 0.1% acetic acid in water to 60% acetonitrile/0.1% acetic acid in water; flow rate of 8 mL/min. and ultraviolet (UV) detection at 285 nm to give 8 mg of the desired product. Retention time: 23 minutes. ESI-MS calc. for C72H72N8023Cl3 [M+H*]: 1522.5, found: 1522.5.
EXAMPLE 17: Biological Testing
(a) Effect on macromolecular syntheses in Bacillus megaterium MB410.
Incorporation of labeled precursors into 5% TCA-insoluble fraction is measured. Specificity of radioactive labeling is tested by observing the effects of inhibitors with known modes of action on incorporation.
The results are presented in Figure 1. Inhibition of incorporation of the substrates by antibiotics with known sites of inhibition is used to test the specificity of labeling. The results suggested that the substrates were incorporated into the expected macromolecules. In the case of [3H]Leu incorporation, it is likely that its inhibition by rifampicin is a secondary effect caused by the inhibition of mRNA synthesis.
Compound 6a selectively inhibited peptidoglycan synthesis and RNA synthesis. The inhibition of RNA synthesis is likely not to be a secondary effect of the inhibition of peptidoglycan synthesis because ampicillin had no effect on RNA synthesis. Rifampicin did not inhibit peptidoglycan synthesis. Vancomycin inhibited peptidoglycan synthesis and RNA synthesis. These results are shown in Figure 2.
(b) Effect on peptidoglycan synthesis in ether-treated bacteria prepared from E. coli W7. Compounds are tested in parallel reactions. One reaction is run in the presence of penicillin G (1 mg/mL). The product of this reaction is "immature" peptidoglycan, a polysaccharide chain with peptide side groups, but with no peptide cross-links between polysaccharide chains. Immature peptidoglycan is soluble in 4% SDS heated to 95°C. In the second reaction, which is run without penicillin, the product is cross-linked, "mature" peptidoglycan that is insoluble in hot SDS. Both types of reactions are terminated by the addition of 6M pyridinium acetate, pH 4.2, and n-butanol (1 :4). The residue from the reaction run in the presence of penicillin G is dispersed in DMSO by sonication and filtered through a hydrophilic PVDF filter that is subsequently washed with 0.4M NH4OAc prepared in methanol. The residue from the reaction run in the absence of penicillin G is suspended in 4% SDS and heated at 95°C for 15 minutes. Hot SDS-insoluble material is collected on a mixed cellulose HAWP filter that is then washed with distilled water. The series of reactions observed is summarized below.
(i) Stage II steps, Translocase and Transferase: products soluble in butanol Reactions are resistant to penicillin G. Lipid intermediate I consists of bactoprenol MurNAc-pentapeptide. Lipid intermediate II consists of bactoprenol-GlcNAc-MurNAc-pentapeptide.
ETB + UDP-MurNAc-pentapeptide → UMP + Lipid Intermediate I
Lipid Intermediate I + UDP-[14C]GlcNAc → UDP + [I4C]-Lipid Intermediate II
(ii) Transglycosylase step: product retained by PVDF filter Reaction run in the presence of 1 mg/mL penicillin G to inhibit transpeptidation
[14C]-Lipid Intermediate II + cell wall acceptor → "immature" [14C]-peptidoglycan
(iii) Transpeptidation step: product insoluble in hot 4% SDS
Reaction goes to completion (no penicillin present)
[14C]-peptidoglycan + [,4C]-peptidoglycan — » cross-linked [14C]-peptidoglycan Incorporation into three fractions is measured: (1) butanol-soluble radioactivity; (2) radioactivity retained by hydrophilic PVDF filters from the reaction run in the presence of 1 mg/mL penicillin G; and (3) hot SDS-insoluble radioactivity retained by mixed cellulose HAWP membrane filters from the reaction run in the absence of penicillin G. Since peptidoglycan synthesis occurs sequentially, the site of inhibition can be determined by the pattern of inhibition, as shown in the following table:
In the example shown below, ramoplanin is an inhibitor of the transferase step in stage II. The compound inhibits incorporation into all three fractions. Bambermycin is the only known inhibitor of the transglycosylase step and it inhibits incorporation into the material retained by the PVDF filters and into the fraction that is insoluble in hot SDS but not into the butanol-soluble fractions. Cefoxitin inhibits transpeptidation. It only inhibits incorporation of [14C]GlcNAc into the hot SDS-insoluble fraction.
Compound 6a is tested for activity in ether-treated bacteria (ETB) prepared from E. coli VC8 and from E. coli Wl. In the test against the ETB prepared from strain VC8, it is not possible to confirm that inhibition of stage II steps would have been observed. The separation scheme that was designed with strain W7 did operate in the same way with ETB from strain VC8. However, there is good evidence for the inhibition of the transglycosylase step by compound 6a, as shown in Figure 3A.
Compound 6a is re-tested with ETB prepared from strain W7. The selectivity test with the known antibiotics confirmed that inhibition of stage II steps is observable with this strain. Again, compound 6a displays a pattern of inhibition that suggests inhibition of the transglycosylase step, as shown in Figure 3B.
Compounds 5a, 6a, 6b, 5b, 6c and 11 were tested with ETB prepared from strain W7, along with vancomycin and ampicillin. The results are presented in Figures 4 - 7.
The preceding examples are intended to describe certain preferred embodiments of the invention. It should be appreciated, however, that obvious additions and modifications of the invention will be apparent to one skilled in the art. The invention is not limited except as set forth in the claims. REFERENCES CITED
Gallop, M.A. et al. (1994) "Applications of Combinatorial Technologies to Drug Discovery. 1. Background and Peptide Combinatorial Libraries," J. Med. Chem. 37: 1233-1251. Gordon, E.M. et al. (1994) "Applications of Combinatorial Technologies to Drug Discovery. 2.
Combinatorial Organic Synthesis, Library Screening Strategies, and Future Directions," J. Med. Chem. 37: 1385-1401.
Terrett, N.K. et al. (1995) "Combinatorial Synthesis - The Design of Compound Libraries and their Application to Drug Discovery," Tetrahedron 51:8135-8173. Thompson, L.A. and Ellman, J.A. (1996) "Synthesis and Applications of Small Molecule
Libraries," Chem. Rev. 96:555-600.

Claims

WHAT IS CLAIMED IS:
1. A disaccharide compound comprising two hexose residues joined by an alpha glycosidic linkage; said compound having the formula
wherein R2Y2Yι is bonded to a ring carbon atom adjacent to the alpha glycosidic linkage; R) and R3 are independently hydrogen, alkyl, aryl, aralkyl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, alkanoyl, aroyl, aralkanoyl, heterocyclic, heterocyclic-alkyl, heterocyclic-carbonyl or heterocyclic-alkyl- carbonyl; R2 is hydrogen, alkyl, aryl, aralkyl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, alkanoyl, aroyl, aralkanoyl, heterocyclic, heterocyclic-alkyl, heterocyclic-carbonyl, heterocyclic-alkyl-carbonyl or a peptide comprising 2-6 amino acid residues; R4, R5, R(, and R7 are independently hydrogen, or a hydroxyl, amino or thiol protecting group; Wi, W , W3 and W are independently O, NH or S; R8 is hydrogen, hydroxyl or a hydroxyl protecting group; k, m, n, p and r are independently 0 or 1 ; Xi is a single bond, O, NR9 or S; X2 is O, NR12, S, C(0)0, C(0)S, C(S)0, C(S)S, C(NR,2)0 or C(0)NR12; Y, is a single bond, O, NRI0 or S; Y2 is O, NRI3, S, C(0)0, C(0)S, C(S)0, C(S)S, C(NRι3)0 or C(0)NR13; Z, is a single bond, O, NRn or S; Z2 is O, NR,4, S, C(0)0, C(0)S, C(S)0, C(S)S, C(NR14)0 or C(0)NR,4; R9, R,0, Rπ, R]2, R13 and R) are independently hydrogen, alkyl or aralkyl; none of the pairs X] and X2, Yj and Y2, and Zi and Z2 comprises O and O, S and O, or O and S, respectively;
provided that: at least one of Ri, R2 and R3 is not hydrogen or methyl; when p is 0, Xj is a single bond, and X2 is NR]2, then Rj is not benzoyl or methylbenzoyl; when X2 is C(0)0, C(0)S, C(S)0, C(S)S or C(NRj2)0, then Rj is not hydrogen; when Y2 is C(0)0, C(0)S, C(S)0, C(S)S or C(NR,2)0, then
R2 is not hydrogen; when Z2 is C(0)0, C(0)S, C(S)0, C(S)S or C(NR12)0, then R3 is not hydrogen; and when R2 is a peptide comprising 2-6 amino acid residues, then R] is not hydrogen or methyl.
2. The disaccharide of claim 1 in which R Y2Y] is bonded to an anomeric carbon atom of a monosaccharide ring and the alpha glycosidic linkage is at the 2-position of said monosaccharide ring.
3. The disaccharide of claim 2 in which Wj, W2 and W are O.
4. The disaccharide of claim 3 in which R8 is hydrogen and p is 0.
5. The disaccharide of claim 4 in which k is 1 and m is 0.
6. The disaccharide of claim 5 in which r is 1.
7. The disaccharide of claim 6 in which Xi is a single bond and X2 is NRI2.
8. The disaccharide of claim 3 in which X] is a single bond, X2 is NR]2, Yi is a single bond and Y2 is O.
9. The disaccharide of claim 8 in which Z\ is a single bond, Z2 is O, S or NR]4, and Rt, R5 and Rf, are hydrogen.
10. The disaccharide of claim 9 in which XιX2R] and a CH3 group are both attached to the 3- position of a monosaccharide and p is 0.
11. The disaccharide of claim 10 in which R2 is a peptide comprising 2-6 amino acid residues.
12. The disaccharide of claim 9 which is selected from the group consisting of
13. The disaccharide of claim 1 1 which is:
14. A method for preparing a disaccharide compound comprising two hexose residues joined by an alpha glycosidic linkage; said compound having the formula
wherein R2Y2Yι is bonded to a ring carbon atom adjacent to the alpha glycosidic linkage;
Ri and R3 are independently hydrogen, alkyl, aryl, aralkyl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, alkanoyl, aroyl, aralkanoyl, heterocyclic, heterocyclic-alkyl, heterocyclic-carbonyl or heterocyclic-alkyl- carbonyl; R2 is hydrogen, alkyl, aryl, aralkyl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, alkanoyl, aroyl, aralkanoyl, heterocyclic, heterocyclic-alkyl, heterocyclic-carbonyl, heterocyclic-alkyl-carbonyl or a peptide comprising 2-6 amino acid residues; R4, R5, Re and R7 are independently hydrogen, or a hydroxyl, amino or thiol protecting group; W], W2, W3 and W are independently O, NH or S; R8 is hydrogen, hydroxyl or a hydroxyl protecting group; k, m, n, p and r are independently 0 or 1 ; Xi is a single bond, O, NR9 or S; X2 is O, NR12, S, C(0)0, C(0)S, C(S)0, C(S)S, C(NR]2)0 or C(0)NR,2; Yi is a single bond, O, NR10 or S; Y2 is O, NR,3, S, C(0)0, C(0)S, C(S)0, C(S)S, C(NR13)0 or C(0)NR13; Z, is a single bond, O, NR„ or S; Z2 is O, NR,4, S, C(0)0, C(0)S, C(S)0, C(S)S, C(NRM)0 or C(0)NR!4; R9, Rio, R11, R12, is and R14 are independently hydrogen, alkyl or aralkyl; none of the pairs Xi and X2, Y] and Y2, and Zi and Z2 comprises O and O, S and O, or O and S, respectively;
provided that: at least one of Ri, R2 and R3 is not hydrogen or methyl; when p is 0, Xi is a single bond, and X2 is NR]2, then Ri is not benzoyl or methylbenzoyl; when X2 is C(0)0, C(0)S, C(S)0,
C(S)S or C(NR12)0, then R, is not hydrogen; when Y2 is C(0)0, C(0)S, C(S)0, C(S)S or C(NR]2)0, then
R2 is not hydrogen; when Z2 is C(0)0, C(0)S, C(S)0, C(S)S or C(NRι2)0, then R3 is not hydrogen; and when R2 is a peptide comprising 2-6 amino acid residues, then Rj is not hydrogen or methyl;
said method comprising:
(a) providing a first monosaccharide having the formula
wherein R2Y2Y1 i bonded to a ring carbon atom adjacent to a free hydroxyl group; and none of R2Y2Y1, W1R4, W2R5 and Z1Z2R3 is a free hydroxyl, amino or thiol group, or bears a free hydroxyl, amino or thiol group;
(b) contacting said first monosaccharide with a second monosaccharide having the formula
wherein Ar is an aryl group, and none of R8, R0W3, R7W4 and X,X2Rι is a free hydroxyl, amino or thiol group, or bears a free hydroxyl, amino or thiol group; and an activating agent; and
(c) allowing a glycosylation reaction to proceed such that an alpha glycosidic linkage is formed between said first monosaccharide and said second monosaccharide.
15. The method of claim 14 in which the activating agent is triflic anhydride.
16. The method of claim 15 in which Ar is phenyl.
17. The method of claim 14, further comprising deprotecting all hydroxyl, amino and thiol groups on the disaccharide compound.
18. The method of claim 17 in which Xi is a single bond, X2 is NRι2 and Ri is hydrogen; further comprising contacting the disaccharide compound with an alkylating agent capable of reacting with NR12H.
19. A chemical library comprising a plurality of disaccharide compounds; each of said disaccharide compounds having the formula
wherein R2Y Yj is bonded to a ring carbon atom adjacent to the alpha glycosidic linkage; Ri and R3 are independently hydrogen, alkyl, aryl, aralkyl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, alkanoyl, aroyl, aralkanoyl, heterocyclic, heterocyclic-alkyl, heterocyclic-carbonyl or heterocyclic-alkyl- carbonyl; R2 is hydrogen, alkyl, aryl, aralkyl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, alkanoyl, aroyl, aralkanoyl, heterocyclic, heterocyclic-alkyl, heterocyclic-carbonyl, heterocyclic-alkyl-carbonyl or a peptide comprising 2-6 amino acid residues; t, R5, Re and R7 are independently hydrogen, or a hydroxyl, amino or thiol protecting group; Wi, W2, W3 and W4 are independently O, NH or S; R8 is hydrogen, hydroxyl or a hydroxyl protecting group; k, m, n, p and r are independently 0 or 1; Xi is a single bond, O, NR9 or S; X2 is O, NR,2, S, C(0)0, C(0)S, C(S)0, C(S)S, C(NR,2)0 or C(0)NR]2; Y, is a single bond, O, NR10 or S; Y2 is O, NR,3, S, C(0)0, C(0)S, C(S)0, C(S)S, C(NRι3)0 or C(0)NR,3; Z, is a single bond, O, NRπ or S; Z2 is O, NR14, S, C(0)0, C(0)S, C(S)0, C(S)S, C(NRι4)0 or C(0)NR14; R9, R,0, R„, R12, Rι3 and Ri4 are independently hydrogen, alkyl or aralkyl; none of the pairs Xi and X , Y] and Y2, and Zi and Z2 comprises O and O, S and O, or O and S, respectively;
provided that: at least one of R1} R2 and R3 is not hydrogen or methyl; when p is 0, X] is a single bond, and X2 is NR]2, then Ri is not benzoyl or methylbenzoyl; when X2 is C(0)0, C(0)S, C(S)0, C(S)S or C(NR12)0, then Ri is not hydrogen; when Y2 is C(0)0, C(0)S, C(S)0, C(S)S or C(NR12)O- then R2 is not hydrogen; when Z2 is C(0)0, C(0)S, C(S)0, C(S)S or C(NR12)0, then R3 is not hydrogen; and when R2 is a peptide comprising 2-6 amino acid residues, then R] is not hydrogen or methyl.
20. The chemical library of claim 19 in which R2Y2Yι is bonded to an anomeric carbon atom of a monosaccharide ring and the alpha glycosidic linkage is at the 2-position of said monosaccharide ring.
21. The chemical library of claim 20 in which Wt, W2 and W3 are O.
22. The chemical library of claim 21 in which R8 is hydrogen and p is 0.
23. The chemical library of claim 22 in which k is 1 and m is 0.
24. The chemical library of claim 23 in which r is 1.
25. The chemical library of claim 24 in which X! is a single bond and X2 is NR)2.
26. The chemical library of claim 19 in which Zi is a single bond, Z2 is O, S or NR14, and R4, R5 and e are hydrogen.
27. The chemical library of claim 26 in which Xi is a single bond, X2 is NR] , Yi is a single bond and Y2 is O.
28. The chemical library of claim 27 in which XιX2R] and a CH3 group are both attached to the 3-position of a monosaccharide and p is 0.
29. A method of treating a human suffering from a bacterial infection; said method comprising administering an effective amount of the compound of claim 1 to said human.
30. A method for preparing a disaccharide compound comprising two hexose residues joined by an alpha glycosidic linkage; said compound having the formula
wherein R Y2Yι is bonded to a ring carbon atom adjacent to the alpha glycosidic linkage; Ri and R are independently hydrogen, alkyl, aryl, aralkyl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, alkanoyl, aroyl, aralkanoyl, heterocyclic, heterocyclic-alkyl, heterocyclic-carbonyl or heterocyclic-alkyl- carbonyl; R2 is hydrogen, alkyl, aryl, aralkyl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, alkanoyl, aroyl, aralkanoyl, heterocyclic, heterocyclic-alkyl, heterocyclic-carbonyl, heterocyclic-alkyl-carbonyl or a peptide comprising 2-6 amino acid residues; R», R5, R^ and R7 are independently hydrogen, or a hydroxyl, amino or thiol protecting group; W W2, W3 and W4 are independently O, NH or S; R8 is hydrogen, hydroxyl or a hydroxyl protecting group; k, m, n, p and r are independently 0 or 1; X] is a single bond, O, NR9 or S; X2 is O, NR12, S, C(0)0, C(0)S, C(S)0, C(S)S, C(NRI2)0 or C(0)NR,2; Y, is a single bond, O, NR10 or S; Y2 is O, NR,3, S, C(0)0, C(0)S, C(S)0, C(S)S, C(NR,3)0 or C(0)NR13; Z, is a single bond, O, NRn or S; Z2 is O, NR14, S, C(0)0, C(0)S, C(S)0, C(S)S, C(NR14)0 or C(0)NR14; R9, Rio, Rπ, Rι2, Ris and R!4 are independently hydrogen, alkyl or aralkyl; none of the pairs Xi and X2, Y'ι and Y2, and Z] and Z2 comprises O and O, S and O, or O and S, respectively;
provided that: at least one of Ri, R2 and R3 is not hydrogen or methyl; when p is 0, X] is a single bond, and X2 is NR]2, then Ri is not benzoyl or methylbenzoyl; when X2 is C(0)0, C(0)S, C(S)0, C(S)S or C(NRι2)0, then Ri is not hydrogen; when Y2 is C(0)0, C(0)S, C(S)0, C(S)S or C(NR]2)0, then
R2 is not hydrogen; when Z2 is C(0)0, C(0)S, C(S)0, C(S)S or C(NRπ)0, then R3 is not hydrogen; and when R2 is a peptide comprising 2-6 amino acid residues, then Rj is not hydrogen or methyl;
said method comprising:
(a) providing a first monosaccharide having the formula
wherein R2Y2Yι is bonded to a ring carbon atom adjacent to a free hydroxyl group; and none of R2Y2Yl5 W1R4, W2R5 and Z]Z R3 is a free hydroxyl, amino or thiol group, or bears a free hydroxyl, amino or thiol group;
(b) contacting said first monosaccharide with a second monosaccharide having the formula
wherein Ar is an aryl group, and none of R8, R<;W3 and R7W4 is a free hydroxyl, amino or thiol group, or bears a free hydroxyl, amino or thiol group; and an activating agent; and
(c) allowing a glycosylation reaction to proceed such that an alpha glycosidic linkage is formed between said first monosaccharide and said second monosaccharide.
31. The method of claim 30, further comprising treating a product of step (c) with a reducing agent under conditions effective to reduce an azido group to an amino group.
32. The method of claim 31 in which the activating agent is triflic anhydride.
33. The method of claim 32 in which Ar is phenyl. AMENDED CLAIMS
[received by the International Bureau on 28 June 2000 (28.06.00); original claims 1 and 19 amended remaining claims unchanged (4 pages)]
1. A disaccharide compound comprising two hexose residues joined by an alpha glycosidic linkage; said compound having the formula
wherein R2Y2Yι is bonded to a ring carbon atom adjacent to the alpha glycosidic linkage; Ri and R3 are independently hydrogen, alkyl, aryl, aralkyl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, alkanoyl, aroyl, aralkanoyl, heterocyclic, heterocyclic-alkyl, heterocyclic-carbonyl or heterocyclic-alkyl-carbonyl; R2 is hydrogen, alkyl, aryl, aralkyl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, alkanoyl, aroyl, aralkanoyl, heterocyclic, heterocyclic-alkyl, heterocyclic-carbonyl, heterocyclic-alkyl-carbonyl or a peptide comprising 2-6 amino acid residues; R4, R5, R$ and R7 are independently hydrogen, or a hydroxyl, amino or thiol protecting group; Wi, W2, W3 and W4 are independently O, NH or S; R8 is hydrogen, hydroxyl or a hydroxyl protecting group; k, m, n, p and r are independently 0 or 1; X, is a single bond, O, NR9 or S; X2 is O, NR,2, S, C(0)0, C(0)S, C(S)0, C(S)S,
C(NR,2)0 or C(0)NR!2; Y, is a single bond, O, NR,0 or S; Y2 is O, NR13, S, C(0)0, C(0)S, C(S)0,
C(S)S, C(NR,3)0 or C(0)NRB; Z, is a single bond, O, NRπ or S; Z2 is O, NR,4, S, C(0)0, C(0)S, C(S)0, C(S)S, C(NR,4)0 or C(0)NR14; R9, R,o, R11, R12, 13 and R,4 are independently hydrogen, alkyl or aralkyl; none of the pairs Xi and X2, Yi and Y2, and Zi and Z2 comprises O and O, S and O, or O and
S, respectively;
provided that: at least one of R1? R2 and R3 is not hydrogen or methyl; when p is 0, Xi is a single bond, and X2 is NRι2, then R! is not benzoyl or methylbenzoyl; when X2 is C(0)0, C(0)S,
C(S)0, C(S)S or C(NR,2)0, then R, is not hydrogen; when Y2 is C(0)0, C(0)S, C(S)0, C(S)S or
C(NR!2)0, then R2 is not hydrogen; when Z2 is C(0)0, C(0)S, C(S)0. C(S)S or C(NR,2)0, then R3 is not hydrogen; when R2 is a peptide comprising 2-6 amino acid residues, then Ri is not hydrogen or methyl; and provided that the compound is not
wherein B is hydrogen, acetyl or S03M where M is a nontoxic pharmaceutically acceptable cation; A is S or SO, and D is phenyl mono-, di- or trisubstituted with a moiety selected from the group consisting of hydrogen, OB, alkoxy (C C3), hydroxy, halo, trifluoromethyl, amino, mercapto, acetylamino, COOM, NHCOCOOC2H5 and
C(S)S or C(NR,2)0, then R, is not hydrogen; when Y2 is C(0)0, C(0)S, C(S)0, C(S)S or C(NR,2)0, then R2 is not hydrogen; when Z2 is C(0)0, C(0)S, C(S)0, C(S)S or C(NR12)0, then R3 is not hydrogen; when R2 is a peptide comprising 2-6 amino acid residues, then Ri is not hydrogen or methyl; and provided that the disaccharide is not
wherein B is hydrogen, acetyl or S03M where M is a nontoxic pharmaceutically acceptable cation; A is S or SO, and D is phenyl mono-, di- or trisubstituted with a moiety selected from the group consisting of hydrogen, OB, alkoxy (Cι-C3), hydroxy, halo, trifluoromethyl, amino, mercapto, acetylamino, COOM, NHCOCOOC2H5 and
20. The chemical library of claim 19 in which R2Y2Yι is bonded to an anomeric carbon atom of a monosaccharide ring and the alpha glycosidic linkage is at the 2-position of said monosaccharide ring.
21. The chemical library of claim 20 in which Wi, W2 and W3 are O.
22. The chemical library of claim 21 in which R8 is hydrogen and p is 0.
23. The chemical library of claim 22 in which k is 1 and m is 0.
24. The chemical library of claim 23 in which r is 1.
25. The chemical library of claim 24 in which Xj is a single bond and X2 is NR]2.
26. The chemical library of claim 19 in which Zi is a single bond, Z2 is O, S or NR[4, and Rt, R5 and R5 are hydrogen.
27. The chemical library of claim 26 in which Xi is a single bond, X2 is NRj2, Yi is a single bond and Y2 is O.
28. The chemical library of claim 27 in which and a CH3 group are both attached to the 3-position of a monosaccharide and p is 0.
29. A method of treating a human suffering from a bacterial infection; said method comprising administering an effective amount of the compound of claim 1 to said human.
30. A method for preparing a disaccharide compound comprising two hexose residues joined by an alpha glycosidic linkage; said compound having the formula
EP00906899A 1999-01-12 2000-01-12 Substituted alpha-linked disaccharides Withdrawn EP1147118A4 (en)

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US6699836B2 (en) 1999-04-02 2004-03-02 The Trustees Of Princeton University Vancomycin analogs

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