SACCHARIDES LINKED TO COMPOUNDS THAT BIND
CELL-SURFACE PEPTIDES OR PROTEINS
1. Field of the Invention
The present invention relates to saccharide compounds having transglycosylase inhibitory activity linked to non-saccharide compounds that bind to molecules located at the bacterial cell surface (e.g., cell-surface peptides or proteins). The compounds of this invention are useful as antibacterial agents.
2. 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 any stage of peptidoglycan synthesis are potentially useful as antibacterial agents. The only recognized commercialized class of compounds that inhibit the transglycosylation step comprises moenomycin and its derivatives. A disaccharide fragment of moenomycin inhibits transglycosylase activity. 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. Furthermore, there has been no suggestion that a new class of antibacterial agents could be made by coupling a saccharide that inhibits transglycosylase activity to a non-saccharide compound that binds to molecules located at the bacterial cell surface.
The glycopeptide antibacterial agents, of which vancomycin is the best known example, are believed to inhibit peptidoglycan synthesis by functioning as peptide binders. These compounds bind D-alanyl-D-alanine, preventing transpeptidation by sequestering the peptide substrates. The structural formula of vancomycin is shown below and is characterized by a disaccharide moiety covalently linked to a heptapeptide structure. The structure of vancomycin places it in a class of molecules referred to as the "dalbaheptides." [Malabarba A., et al. (1997)] Dalbaheptides in general are characterized by the presence of seven amino acids linked together by peptide bonds and held in a rigid conformation by cross-links through the aromatic substituent groups of at least five of the amino acid residues. In the heptapeptide structure of vancomycin, which is commonly referred to as the "aglycone" of vancomycin, the aromatic side-chains of amino acids 2, 4, and 6 are fused together through ether linkages. The side-chains of amino acids 5 and 7 are joined via a carbon-carbon bond. Amino acids 1 and 3 are leucine and asparagine, respectively. Other naturally-occurring glycopeptide antibacterial agents are similar to vancomycin in that they have a glucose residue linked to the aromatic substituent on amino acid 4 through formation of a bond with a phenolic hydroxyl group. The glucose residue in vancomycin and some other glycopeptides is linked through its vicinal hydroxyl position to the amino sugar, L-vancosamine. The sugars have been separately removed from vancomycin, and it has been found that the presence of both sugars enhances the activity of this class of antibacterial agents. [Nagarajan
R. (1988), (1991), (1993] However, there is no suggestion in the literature that the sugars themselves have any activity as antibacterial agents.
(I)
In addition to the glycopeptide antibacterial agents, other compounds are known that bind to molecules located at the cell surface. Compounds that bind to Lipid II include nisin, mersacidin, actagardine, and other antibiotics, as well as ramoplanin. Compounds that bind to proteins located at the cell surface include the beta lactams and related antibiotics such as cephalosporins, carbapenems, and imipenems. Other compounds thought to bind to molecules located at the bacterial cell surface include daptamycin and bacitracin. There has been no suggestion that linking these molecules or portions thereof to saccharide compounds having transglycosylase inhibitory activity would improve antibacterial efficacy.
In 1994, Wennemers and Still showed that several commercially available dyes (e.g., rhodamine, see Depiction 1) can bind selectively to components of a resin-bound peptide library. Hence, some of these dyes could have affinity for peptides located at the bacterial cell surface. [Wennemers and Still Tetrahedron Letters (1994) 35:6413-6416] Still and
others have also shown that synthetic peptide-binding host molecules can be made from simple building blocks. There has been no suggestion that these peptide binding dyes or synthetic host molecules could be linked to saccharides having transglycosylase activity to produce compounds with better antibacterial activity than either the saccaride or the dye alone. (Depiction I).
Depiction I
Co-pending application serial number 60/115,595, filed January 12, 1999 and entitled "Substituted Alpha-Linked Disaccharides," which is incorporated herein by reference, discloses disaccharide compounds comprising two hexose residues joined by an alpha glycosidic linkage. These compounds display activity as transglycosylase inhibitors.
International (PCT) application number PCT US99/15845, filed July 14, 1999 (publication date on or about January 20, 2000) entitled "Glycopeptide Antibiotics, Combinatorial Libraries of Glycopeptide Antibiotics and Methods of Producing Same," and which is incorporated herein by reference, discloses glycopeptide compounds in which the substituted sugar residues are attached to the peptide core through glycosidic linkages. There is no suggestion that compounds in which the sugar residues are linked to the peptide through non- glycosidic linkages, or through glycosidic linkages to a linker attached to the peptide core, would be useful as antibacterial agents.
3. Summary of the Invention
This invention is directed to a compound which comprises: (i) a saccharide compound having transglycosylase inhibitory activity; and (ii) a second compound that is capable of binding a protein or enzyme involved in cell wall biosynthesis; a precursor used in cell wall biosynthesis; and/or the cell wall surface. The saccharide compound is linked, directly or through a linker (e.g., a difunctional linker), to the second compound. In a preferred
embodiment, the second compound is a non-saccharide compound, provided that: when the non-saccharide compound is a hexapeptide or a heptapeptide and the saccharide compound does not contain a phosphate or phosphonate ester, then the saccharide compound is not linked directly to the non-saccharide compound through a glycosidic linkage at A4.
In a preferred embodiment of the invention, the saccharide compound is a disaccharide comprising two hexose residues joined by an alpha glycosidic linkage, and the non- saccharide compound is a peptide.
In a second preferred embodiment, this invention is directed to a glycopeptide in which a disaccharide comprising two hexose residues joined by an alpha glycosidic linkage is linked to a peptide directly through a non-glycosidic linkage, or through a difunctional linker. The peptide has the formula Aι-A2-A3-A4-A5-A6-A , in which each dash represents a covalent bond; wherein the group Ai comprises a modified or unmodified -am ino acid residue, hydrogen, alkyl, aryl, aralkyl, alkanoyl, aroyl, aralkanoyl, heterocyclic, heterocyclic- carbonyl, heterocyclic-alkyl, heterocyclic-alkyl-carbonyl, alkylsulfonyl, arylsulfonyl, guanidinyl, carbamoyl, or xanthyl; wherein each of the groups A2 to A7 comprises a modified or unmodified -amino acid residue, whereby (i) the group Aj is linked to an amino group on the group A2, (ii) each of the groups A2, } and A6 bears an aromatic side chain, which aromatic side chains are cross-linked together by two or more covalent bonds, and (iii) the group A bears a terminal carboxyl, ester, thioester, amide, or N-substituted amide group.
The disaccharide in the second preferred embodiment has the formula
wherein R Y
2Yι is bonded to a ring carbon atom adjacent to the alpha glycosidic linkage; Rj, R
2 and R
3 are independently hydrogen, alkyl, aryl, aralkyl, alkylsulfpnyl, arylsulfonyl, aralkylsulfonyl, alkanoyl, aroyl, aralkanoyl, heterocyclic, heterocyclic-alkyl, heterocyclic- carbonyl or heterocyclic-alkyl-carbonyl; t, R
5, R$ and R
7 are independently hydrogen, or a hydroxyl, amino or thiol protecting group; Wi, W
2, W
3 and W
4 are independently O, NH or S; Re 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, NR
9 or S; X
2 is O, NR
12, S, C(O)O, C(O)S, C(S)O, C(S)S, C(NR
12)O or C(O)NR
12; Yi is a single bond, O, NR
10 or S; Y
2 is O, NR
13, S, C(O)O, C(O)S, C(S)O, C(S)S, C(NR
13)O or C(O)NR
13; Z> is a single bond, O, NR
n or S; Z
2 is O, NR
14, S, C(O)O, C(O)S, C(S)O, C(S)S, C(NR
14)O or C(O)NR
14; R
9, Rι
0, R„, R
12, R
13 and Rι
4 are independently hydrogen, alkyl or aralkyl; none of the pairs Xi and X
2, Yi and Y , and Zi and Z
2 comprises O and O, S and O, or O and S, respectively;
provided that when X2 is C(O)O, C(O)S, C(S)O, C(S)S or C(NRι2)O, then R. is not hydrogen; when Y2 is C(O)O, C(O)S, C(S)O, C(S)S or C(NRj2)O, then R2 is not hydrogen; and when Z2 is C(O)O, C(O)S, C(S)O, C(S)S or C(NRι2)O, then R3 is not hydrogen.
Hence, in a particular embodiment of the invention, a saccharide compound exhibiting transglycosylase inhibitory activity (e.g., the vancomycin disaccharide bearing a lipid-like substituent on the vancosamine nitrogen and analogs and derivatives thereof) is covalently bound, via a linker, to non-saccharide compounds that bind to molecules located at a bacterial cell surface (e.g., the vancomycin aglycone). In a preferred embodiment, the lipid-like substituent comprises a biaryl moiety in which the aromatic groups are joined directly or via a substituted or unsubstituted linker comprising one or more atoms, including heteroatoms.
One preferred biaryl moiety is a chlorophenylbenzyl group. In yet another preferred embodiment, covalent attachment of the disaccharide is accomplished directly or via an amine (or diamine) linker to a carboxyl group of an aglycone.
In still other embodiments of the invention, "unnatural" aglycones are utlized, such as certain peptide-binding dyes, e.g., rhodamine and the like. A disaccharide coupled to a peptide-
binding dye, e.g., rhodamine, via a linker provides rhodamine conjugates 5a and 5b. It is contemplated that such rhodamine conjugates could target peptides found, for example, in the developing bacterial cell wall. In doing so, the rhodamine conjugate would bring the inhibitory disaccharide moiety closer to enzymes involved in cell wall biosynthesis.
In preliminary MIC assays, compounds 5a,b displayed greatly improved activity against both vancomycin sensitive and resistant enteroccoci. In contrast, Rhodamine B alone showed little or no activity. A related disaccharide alone has tenfold poorer activity. [Ge, M. et al. Science (1999) 284:507-511]. Clearly, there is a distinct advantage to coupling the disaccharide to the Rhodamine.
This invention is also directed to a chemical library comprising a plurality of these compounds. The invention is further directed to a method for preparing compounds in which a functionalized dalbaheptide aglycone is linked to a disaccharide transglycosylase inhibitor.
4. Brief Description of the Drawings
Figure 1 is a graph showing the effects on macromolecular synthesis in Bacillus megaterium MB410 of known antibacterial agents.
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.
Figure 4 is a table presenting results obtained on selected compounds disclosed herein for synthesis of RNA, DNA, protein and peptidoglycan, and for the site of inhibition of peptidoglycan synthesis in ether treated bacteria.
Figure 5 is a table presenting results obtained on selected compounds disclosed herein for synthesis of RNA, DNA, protein and peptidoglycan, and for the site of inhibition of peptidoglycan synthesis in ether treated bacteria.
Figure 6 is a table presenting results obtained on selected compounds disclosed herein for synthesis of RNA, DNA, protein and peptidoglycan, and for the site of inhibition of peptidoglycan synthesis in ether treated bacteria.
Figure 7 is a table presenting results obtained on selected known substances of RNA, DNA, protein and peptidoglycan, and for the site of inhibition of peptidoglycan synthesis in ether treated bacteria.
Figure 8 is a table presenting the MIC values of selected conjugates of the invention.
Figure 9 exhibits a preferred synthetic scheme for the preparation of rhodamine linked disaccharides (or rhodamine conjugates) of the invention.
Figure 10 is a table presenting the MIC values of selected rhodamine conjugates of the invention.
5. Detailed Description of the Preferred Embodiments of the Invention
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, trialkylarnmonium, 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, trialkylarnmonium, 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 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 "difunctional linker" is a group with two points of attachment, one of which is attached to the non-saccharide compound and the other to the saccharide compound. The attachment to the non-saccharide compound joins the difunctional linker to, e.g., a hydroxyl, amino or carboxyl group on the non-saccharide compound. The attachment to the saccharide compound joins the difunctional linker to a hydroxyl, amino or carboxyl group on the saccharide compound. Exemplary linkers are derived from dicarboxylic acids, including carboxyl-substituted amino acids, which form ester or amide linkages at the points of attachment, diols, which form ethers or ester linkages at the points of attachment, or from compounds of mixed functionality, e.g., hydroxy acids. Other exemplary linkers are derived from reductive alkylation with a compound bearing an aldehyde group and a carboxyl or hydroxyl group, or from alkylation of a hydroxyl or amino group with an α-halocarboxylate and subsequent esterification at the carboxyl group.
A "glycopeptide" is a compound comprising a peptide linked to at least one carbohydrate. The term "peptide," as used in this invention, refers to a substance containing from 2 to 20 amino acid residues linked by peptide bonds. The term "protein," as used in this invention, refers to a substance containing more than 20 amino acid residues linked by peptide bonds.
An "aglycone" is the result of removing the carbohydrate residues from a glycopeptide, leaving only a peptide core. A "pseudoaglycone" is the result of removing only one of two sugar residues of a disaccharide residue linked to residue AA of a glycopeptide. Thus, a pseudoaglycone comprises an aglycone in which A is linked to a monosaccharide residue.
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
antibacterial agents. 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; "MeOH" refers to methanol; "MeCN" refers to acetonitrile; "Tf ' refers to the trifluoroacetyl group; "DMSO" refers to dimethyl sulfoxide; "DTBMP" refers to 2,6-di-tert-butyl-4-methylpyridine; "DIEA" refers to diisopropylethylamine; "AH" 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 hexafiuorophosphate; "Su" refers to the succinimidyl group; "HBTU" refers to O-benzotriazol-l-yl-N,N,N',N'-tetramethyluronium hexafiuorophosphate; "aloe" refers to allyloxycarbonyl; and "CBz" refers to benzyloxycarbonyl.
Consistent with the objectives of the present invention a compound is provided, which comprises: (i) a saccharide compound having transglycosylase inhibitory activity; and (ii) a non-saccharide compound that is capable of binding a molecule located at a bacterial cell surface, the saccharide compound being linked directly, or indirectly through a linker, to the non-saccharide compound, provided that when the non-saccharide compound is a hexapeptide or a heptapeptide and the saccharide compound does not contain a phosphate or phosphonate ester, then the saccharide compound is not covalently bound directly to the non- saccharide compound via a glycosidic linkage. Non-saccharide compounds according to the invention include, but are not limited to, "natural" and unnatural" aglycones. In particular, the unnatural aglycone can be selected from peptide-binding dyes.
In a particular embodiment of the invention compounds are provided, which comprise: (i) a saccharide compound having transglycosylase activity; and (ii) a non-saccharide compound that is capable of binding a cell-surface peptide or protein. The saccharide compound is linked directly, through a glycosidic or non-glycosidic linkage, or indirectly, through a difunctional linker, to the non-saccharide compound; provided that: when the non-saccharide compound is a hexapeptide or a heptapeptide, e.g., the peptide cores of the naturally occurring glycopeptide antibacterial agents, and the saccharide compound does not contain a phosphate or phosphonate ester, then the saccharide compound is not linked directly to the non-saccharide compound through a glycosidic linkage. Non-glycosidic linkages include, without limitation, ether linkages between non-anomeric saccharide hydroxyl groups and hydroxyl groups on the non-saccharide compound, and ester linkages between saccharide hydroxyl groups and carboxyls on the non-saccharide compound or between saccharide carboxyls and hydroxyl groups on the non-saccharide compound.
For the purposes of this invention, to be considered capable of binding a cell-surface peptide or protein, a compound must have an association constant of at least 103. Methods for determining association constants by NMR, fluorescence, uv or CD techniques are well known in the art. See, e.g., D.H. Williams et al. (1991).
Suitable saccharide compounds include, without limitation, moenomycin, the moenomycin disaccharide fragment shown hereinabove, other moenomycin derivatives having transglycosylase activity, and a disaccharide comprising two hexose residues joined by an alpha glycosidic linkage. In a preferred embodiment of the invention, the saccharide compound is a disaccharide comprising two hexose residues joined by an alpha glycosidic linkage.
Suitable non-saccharide compounds that bind cell-surface molecules include, without limitation, synthetic receptors capable of binding cell-surface peptides, e.g., those reported in Still (1996), and natural peptides such as the dalbaheptides that are capable of binding cell- surface peptides or proteins, including but not limited to the terminal D-alanyl-D-alanine or D-alanyl-D-lactate units of immature cell-surface peptidoglycan. In a preferred embodiment, the non-saccharide compound is a peptide. There is no fixed upper limit on the number of
amino acid residues in the peptide because only that portion of the peptide which acts as a binding site is significant in determining activity of the compounds. Suitable non-saccharide compounds that bind cell-surface proteins include, without limitation, non-saccharide inhibitors of penicillin binding proteins or other enzymes displayed on the bacterial surface. In another preferred embodiment of the invention, the cell-surface peptide comprises D- alanyl-D-alanine or D-alanyl-D-lactate, preferably as a terminal unit, i.e., the non-saccharide compound is capable of binding D-alanyl-D-alanine or D-alanyl-D-lactate.
In another preferred embodiment, this invention is directed to a glycopeptide in which a disaccharide comprising two hexose residues joined by an alpha glycosidic linkage is linked to a peptide directly through a non-glycosidic linkage, or through a difunctional linker. The peptide has the formula
in which each dash represents a covalent bond; wherein the group Ai comprises a modified or unmodified -am ino acid residue, hydrogen, alkyl, aryl, aralkyl, alkanoyl, aroyl, aralkanoyl, heterocyclic, heterocyclic- carbonyl, heterocyclic-alkyl, heterocyclic-alkyl-carbonyl, alkylsulfonyl, arylsulfonyl, guanidinyl, carbamoyl, or xanthyl; wherein each of the groups A
2 to A
7 comprises a modified or unmodified -amino acid residue, whereby (i) the group Ai is linked to an amino group on the group A
2, (ii) each of the groups A
2,
t and A
6 bears an aromatic side chain, which aromatic side chains are cross-linked together by two or more covalent bonds, and (iii) the group A
7 bears a terminal carboxyl, ester, amide, or N-substituted amide group.
In another preferred embodiment, the disaccharide 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. The disaccharide thus substituted possesses transglycosylase inhibitory activity.
Preferably, the disaccharide compound has the formula (II)
(II)
wherein R2Y2Yι is bonded to a ring carbon atom adjacent to the alpha glycosidic linkage; Rj, R2 and R3 are independently hydrogen, alkyl, aryl, aralkyl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, alkanoyl, aroyl, aralkanoyl, heterocyclic, heterocyclic-alkyl, heterocyclic- carbonyl or heterocyclic-alkyl-carbonyl; t, R5, R$ and R are independently hydrogen, or a hydroxyl, amino or thiol protecting group; Wj, W2, W3 and W are independently O, NH or
S; Rs 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(O)O, C(O)S, C(S)O, C(S)S, C(NR12)O or C(O)NR12; Yx is a single bond, O, NR]0 or S; Y2 is O, NR , S, C(O)O, C(O)S, C(S)O, C(S)S, C(NR,3)O or C(O)NR13; Zx is a single bond, O, NRn or S; Z2 is O, NR14, S, C(O)O, C(O)S, C(S)O, C(S)S, C(NRM)O or C(O)NR,4; R9, R10, Rn, R12, Rι3 and Rj4 are independently hydrogen, alkyl or aralkyl; none of the pairs Xi and X2, Yi and Y2, and Zj and Z2 comprises O and O, S and O, or O and S, respectively;
provided that 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; and when Z2 is C(O)O, C(O)S, C(S)O, C(S)S or C(NRj2)O, then R3 is not hydrogen.
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, At and A6, may be cross- linked through their aromatic substituent groups.
Preferably, residues A2 to A7 of the glycopeptide are linked sequentially by peptide bonds and are cross-linked as in a dalbaheptide, as defined hereinabove. The preferred glycopeptides thus have a peptide core in which the residues are linked as in the natural glycopeptide antibacterial agents, including, e.g., vancomycin, teicoplanin, ristocetin, avoparicin and chloroeremomycin. Substitution of different amino acids at A3 is permitted, as are modified amino acid residues at all positions, as described hereinabove. In a preferred embodiment of this invention, residue Ai is an -amino acid, which may be substituted on the terminal amino group by alkyl, aryl, aralkyl, alkanoyl, aroyl, aralkanoyl, heterocyclic, heterocyclic-carbonyl, heterocyclic alkyl, alkylsulfonyl, arylsulfonyl, guanidinyl, carbamoyl, or xanthyl, and the structures and interconnections of Ai to A are those of vancomycin, i.e., the glycopeptide has the heptapeptide core of vancomycin, subject to the amino acid modifications and substitutions on Ai and A7 described hereinabove.
It is preferred that the R Y2Yι 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 substituent on the disaccharide, not including the non-saccharide compound, is 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 Xi is a single bond and X2 is NRι . In another embodiment of the invention, it is preferred that Zi is a single bond, Z2 is O, S or NRι4, and Rt, R5 and Re are hydrogen, further preferred that Xx is a single bond, X2 is NR12, Yi is a single bond and Y2 is O. In another embodiment, it is preferred that XιX2Rι 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 11, 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 11, 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
6a 11
The compounds of formula (II) are prepared by allowing a first monosaccharide having the formula
wherein R
2Y
2Yι is bonded to a ring carbon atom adjacent to a free hydroxyl group; and none of R
2Y
2Yι, WiRt, W
2R
5 and ZιZ
2R
3 is 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 R«, RόW3 and XjX2Rι is 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 XjX2Rι substituent after deprotection is an amino or alkylamino group, i.e., when Xi is a single bond, X2 is NR12 and Ri 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.
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 (trifiic anhydride, Tf2O). 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
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, YOMe 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 to react with a hexose bearing an anomeric sulfoxide substituent, 2, in the presence of Tf O to produce 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 animation gives products 6a-6c, as shown. This approach may be used to introduce a variety of XιX2Rι and R2Y2Y1 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 desmethylvancosamine 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 ZιZ2R3 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 a preferred embodiment of the invention, a disaccharide is attached, directly or through a linker, to any one of several positions on the non-saccharide compound. When the non- saccharide compound is a peptide,
the positions of attachment include benzylic hydroxyl groups, phenolic hydroxyl groups, the terminal group on residue A , and the amino group of Ai or A
2. In a preferred embodiment of the invention, Ai is hydrogen and one carboxyl group of a protected dicarboxylic acid is allowed to react with the terminal amino group of A
2. Following deprotection of the other carboxyl group, the disaccharide is allowed to react through a hydroxyl or amino group to form an ester or amide linkage, respectively. This linkage is glycosidic or non-glycosidic, depending on the position of the hydroxyl or amino group on the disaccharide. This strategy is shown in Figure 12 of 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. In another preferred embodiment of the invention, the disaccharide is linked directly to the
t phenolic hydroxyl group through a non-glycosidic linkage.
Disaccharides are also attached via linkers, for example, by forming an ester, amide or thioester of a carboxylic acid on a linker group attached to a peptide with a free hydroxyl, amino or thiol, respectively, on the saccharide compound. Disaccharides are also attached by reaction of haloalkyl groups, or other groups containing a leaving group, with a nucleophilic group on the peptide, e.g., an unprotected amino or hydroxyl group. Preferably, the peptide has the structure of the vancomycin aglycone.
In another preferred embodiment of the invention, a sugar bearing a haloalkyl group is coupled to the A4 phenolic hydroxyl group of an aglycone by displacement of halide. This method is illustrated in Example 22. In another preferred embodiment of the invention, a sugar bearing one free amino group, e.g., an aminoalkyl group, is coupled to a carboxymethyl
group attached via an ether linkage to the t phenolic hydroxyl group of an aglycone. This method is illustrated in Example 23.
The chemical library of compounds of this invention is prepared to explore the effects on biological activity of varying the substituents on different parts of the molecule. 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 the molecule, resulting in a library containing a large number of substituted compounds, 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 compounds may be prepared relatively quickly. Methods for performing combinatorial synthesis are well known and are described in several review articles. [Thompson (1996), Gallop (1994), Gordon (1994), Terrett (1995)]
One strategy for combinatorial synthesis to produce the chemical library of this invention comprises sequential attachment to the non-saccharide compound of two or more sugar residues bearing the desired substituent groups via glycosylation reactions. Preferably, the glycosylation reactions are performed with glycosyl donors bearing an activated anomeric sulfoxide group, and more preferably, they are carried out on a polymeric support. A strategy for constructing a library of substituted saccharides using solid-phase glycosylation reactions is described in Liang et al. (1996). A suitable resin is a cross-linked polymer insoluble in the reaction solvent which is suitably functionalized for attachment, e.g., SASRTN (Wang's resin). Once coupled to the resin, the differentially protected hydroxyl group on the attached sugar is deprotected. Alternatively, this hydroxyl group is freed before attachment to the resin, since the hydroxyl group does not interfere with the coupling reaction. The free hydroxyl group then serves as the nucleophile in a second glycosylation reaction. In this second glycosylation, the hydroxyl is glycosylated, preferably in a solid phase reaction, with
a variety of azido sugars. Following the glycosylation reaction, the azido groups are reduced and the resulting amino groups are then derivatized. The solid phase portion of the library construction can be carried out using a parallel synthesis or a mix and split strategy. The carbohydrate-modified glycopeptide derivatives would then be deprotected and cleaved from the resin. This set of compounds would then be assayed for peptide binding and anti- bacterial activity.
Another strategy for combinatorial synthesis to produce the chemical library of this invention comprises introduction of substituent groups at various positions on the non-saccharide compound. When the non-saccharide compound is a peptide, Aι-A -A3-A4-A5-A6-A7, the preferred positions for introduction of substituent groups are the terminal amino group of Ai or A2, the terminal carboxyl group of A7 and the phenolic hydroxyl groups present on A5 and A . Substituent groups may be introduced directly at these positions or indirectly, via linkers, as described hereinabove.
The following examples are presented to illustrate various aspects of the present invention, but are not intended to limit it.
6. Examples
EXAMPLE 1 : 3-(N-benzyloxy-carbonyloxy)-4-O-acetyl-2,3,6-trideoxy-3-C-methyl-α-L- lyxo-hexopyranosyl-(l— >2)-3,4,6-tri-O-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 Et2O. 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 Et2O 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 NaHCO3 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% EtO Ac/petroleum ether to give 22 mg (71%) compound 3a as a white solid. Rf 0.45 (40%EtO Ac/petroleum ether).
1H NMR (CDC13, 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-ι, 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 = 11.9 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-s, 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 ΝMR (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 C52H59ΝOι3Νa [M+Na+]: 928.3884, found
928.3918.
EXAMPLE 2: Phenyl 2-(3-N-Cbz-4-O-acetyl-2,3,6-trideoxy-3-C-methyl-α-L-lyxo- hexopyranosyl)-3,4,6~tri-O-benzyl-l -thio-β-D-glucopyranoside (3 b).
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 Et2O. 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 1 mL Et2θ over 10 minutes. The reaction is warmed up to 0°C in 1 hour and then quenched with 10 mL of saturated aqueous NaHCO3 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% EtO Ac/petroleum ether to give 50 mg (85%) compound 3b as a white solid. Rf 0.35 (30%EtO Ac/petroleum ether).
1H NMR (500MHz, CDCI3) 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-S), 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-O-acetyl-2,3,6-trideoxy-3-C-methyl-α-L-lyxo- hexopyranosyl)-3 ,4,6~tri-O-benzyl-α-D-glucopyranoside (3 c).
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 CH2CI2 layer is separated and the aqueous layer is further extracted with CH2CI2 (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 % EtO Ac/petroleum ether) to give 20 mg (44%) of compound 3c as a white solid. Rf 0.2
(30% EtOAc/petroleum ether).
1H NMR (500MHz, CDCI3) δ 7.39 (m, 20H), 5.15-4.41 (m, 11H), 4.24-4.20 (m, 1H, VH-s), 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, 1 H, 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- -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 mL dichloromethane and 10 mL saturated NH CI aqueous 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 40% EtO Ac/petroleum ether to give 17.5 mg (92%) of compound 4a as a white solid. R 0.35 (40% EtO Ac/petroleum ether).
Η NMR (CDCl3j 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-ι, 1 H), 5.08 (d, J = 7.6 Hz, GH-ι, 1 H), 5.05 (s, 2 H), 4.90 (d, J = 11.0 Hz, 1 H), 4.88 - 4.76 (m, 2 H), 4.67 (q, J = 6.4 Hz, NH-5, 2 H), 4.59 (d, J = 11.0 Hz, 1 H), 4.52 (d, J = 11.9 Hz, 1 H), 4.43 (d, J = 11.9 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-s, 1 H), 3.20 (s, VH-., 1 H), 2.24 (d, J = 14.0 Hz, NH- , 1 H), 1.78 (s, CH3, 3 H), 1.72 (dd, J = 4.9, 14.0 Hz, NH-2S 1 H),
1.07 (d, J = 6.4 Hz, VH-6, 3 H). I3C ΝMR (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 C5oH57ΝO12Νa [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 NF fCl solution. The aqueous layer is separated and the organic layer is further extracted with NH CI 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% EtO Ac/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 H> 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 UN 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).
Η ΝMR (CD3OD, 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-I, 1 H), 5.16 (J = 7.6 Hz, GH-ι, 1 H), 4.55-4.52 (m, VH-5, 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^, 1 H), 3.20 (s, VH^, 1 H), 3.14-3.11 (m, GH.5, 1 H), 2.04 - 1.93 (m, VH-2, VH-2s 2 H), 1.65
(s, CH3, 3 H), 1.07 (d, J = 6.7 Hz, CH3, 3 H). 13C ΝMR (CD3OD, 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 C21H33ΝOι0Νa [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).
1H NMR (CD3OD, 500 MHz) δ 5.05 (d, J=4Hz, 1H, VHι), 4.78 (d, J=4Hz, 1H, GH-ι), 4.13 (q,
J=6Hz, 1H, VH-s), 3.81 (dd, J=2, 12Hz, 1H, GH-6), 3.70-3.66 (m, 2H, GH-6-, GH-3), 3.52-3.49 (m, 1H, GH-5), 3.40 (s, 3H, OCH3), 3.38-3.30 (m, 2H, GH , VH- 2.05 (dd, J=4.13Hz, 1H, VH-2), 1.96 (d, J=13Hz, 1H, VH-r), 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 NaCNBF t (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 UN detection at
270 run. 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).
1H ΝMR (CD3OD, 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, 1H), 6.70 (d, J=8.5Hz, 2H), 5.41 (bs,
1H, NH-i), 5.21 (d, J=7.6Hz, 1H, GH-ι), 4.58 (m, 1H, VH-5), 4.03 (d, J=12.2Hz, 1H), 3.96 (d, J=l 1.9Hz, 1H), 3.86 (s, 6H), 3.75-3.70 (m, 2H, GH-2, GH-6), 3.65 (dd, J=4.9, 11.9Hz, 1H, GH- 6>), 3.55 (t, J=9.1Hz, 1H, GH-3), 3.49 (s, 1H, VH^), 3.45 (t, J=9.4Hz, 1H, GH ), 3.18-3.15 (m, 1H, GH-5), 2.00 (s, 2H, VH.2, VH-2\), 1-75 (s, 3H), 1.13 (d, J=6.7Hz, 3H, NH-6). 13C ΝMR (CD3OD, 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 C^H^ΝClO^Νa [M+Νa+]: 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 (11 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 NaCNBFLt (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 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 UN 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.
1H ΝMR (500MHz, CDC13) δ 7.05 (t, J=8.5Hz, 1H), 6.09 (d, J=8.5Hz, 2H), 5.44 (d, J=4.3Hz, 1H, NH-i), 5.18 (d, J=7.6Hz, 1H, GH-ι), 4.52 (m, 1H, VH.5), 3.83 (s, 6H), 3.74-3.69 (m, 2H,
GH-2, GH-6')> 3.66-3.62 (dd, J=4.9, 11.9Hz, 1H, GH-6), 3.53 (t, J=9.2Hz, 1H, GH-3), 3.44 (t, J=9.5Hz, 1H, GH- 3.39 (s, 1H, VH ), 3.16-3.13 (m, 1H, GH-5), 3.00-2.91 (m, 2H), 2.10 (dd, J=4.6, 13.4Hz, 1H, VH-2), 2.00 (d, J=14.1Hz, 1H, VH-2-)> 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 ΝMR (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-Ν-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 NaCNBH* (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 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 UN detection at 270 nm. The fractions containing the pure products are combined and evaporated to give 11 mg (69%) of compound 6c as a white solid. Rf 0.65 (CHCl3/MeOH/H2O=3:2:0.5).
1H ΝMR (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, 1H, NH-ι), 4.83 (d, J=3.7Hz, 1H, GH- 1), 4.21-4.15 (m, 3H), 3.85 (dd, J=2.1H, 11.9Hz, 1H, GH-6), 3.73-3.68 (m, 3H), 3.55-3.51 (m, 1H, GH-S), 3.44 (s, 3H, OHe), 3.41-3.35 (m, 2H), 2.13 (dd, J=4.6, 13.4Hz, 1H, VH-2), 2.04 (d,
J=13.4Hz, 1H, VH-2-X 1.77 (s, 3H), 1.33 (d, J=6.4Hz, 3H, NH-6); )• 13C ΝMR (500MHz, CDCI3) 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-O-acetyl-2,3,6- trideoxy-1-thio-α-L-galactopyranoside as a clear oil. Rf 0.65 (25% EtO Ac/petroleum ether).
1H ΝMR (500MHz, CDCI3) δ 7.84 (m, 2H), 7.35-7.27 (m, 3H), 5.76 (d, J=5.5Hz, 1H, H-l), 5.23 (t, J=1.2Hz, 1H, H-4), 4.51-4.48 (m, 1H, H-5), 3.88-3.84 (m, 1H, H-3), 2.50 (dt, J=5.8,
13.4Hz, 1H, H-2), 2.20 (s, 3H), 2.15 (dd, J=4.6, 13.6Hz, 1H, H-2'), 1.16 (d, J=6.7Hz, 3H, H- 6). 13C ΝMR (500MHz, CDC13) 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.
- 5 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 NaHCO3 solution. The aqueous layer is further extracted with CH2CI2 (20
10 mLx3). The CH2CI2 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).
15 EXAMPLE 12: Compound (8).
Compound la (65 mg, 0.111 mmol) and DTBMP (91 mg, 0.444 mmol) are azeotroped with toluene 3 times and then dissolved in 6 mL Et2O and 2 mL CH2CI2. The reaction solution is cooled to -78 C. Trifiic anhydride (19 μL, 0.111 mmol) is added to the reaction solution.
20 The sulfoxide 7 (71 mg, 0.222 mmol) in lmL Et2O 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 NaHCO3 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
25 (lOmmxδcm) 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).
1H NMR (500MHz, CDC13) δ 7.41-7.24 (m, 15H), 7.09 (t, J=8.2Hz, 1H), 6.63 (d, J=8.6Hz, 2H), 5.49 (d, J=2.8Hz, 1H), 5.10 (s, 1H), 5.01-4.98 (m, 2H), 4.84-4.80 (m, 2H), 4.69 (q,
30 J=6.4Hz, 1H), 4.65 (d, J=11.0Hz, 1H), 4.60 (d, J=11.9Hz, 1H), 4.51 (d, J=11.9Hz, 1H), 4.05
(t, 1H, J=7.6Hz, 1H, GH-2), 3.98-3.94 (m, 1H), 3.84 (s, 6H), 3.70-3.67 (m, 4H), 3.44-3.42 (m, 1H, GH-5),2.18 (S, 3H), 2.00 (dt, J=3.7, 12.8 Hz, 1H), 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,
35 75.8, 75.6, 75.0, 73.8, 71.0, 69.0, 65.5, 56.4, 54.9, 29.7, 21.0, 16.5.
- 5 EXAMPLE 13: Compound (9).
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
10 residue is partitioned between 10 mL dichloromethane and 10 mL saturated aqueous NFLtCl 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 (lOmmxδcm) and eluted with 25% EtOAc/petroleum ether to give 35.7 mg (47%) of
15 compound 9 as a clear oil and 9.6 mg (12%) of recovered 8. Rf 0.3 (25% EtOAc/petroleum ether).
1H NMR (500MHz, CDCI3) δ 7.40-7.24 (m, 15H), 7.07 (t, J=8.5Hz, 1H), 6.62 (d, J=8.5Hz, 2H), 5.43 (d, J=4.4Hz, 1H), 5.03 (d, J=7.3Hz, 1H, GH-ι), 4.98 (d, J=llHz, 1H), 4.84-4.79 (m,
20 2H), 4.65 (d, J=10.7Hz, 1H), 4.59-4.49 (m, 3H), 4.03 (dt, J=2.5, 7.4Hz, 1H, GH-2), 3.83 (s,
6H), 3.77-3.72 (m, 4H), 3.67 (dd, J=4.9, 11.3Hz, 1H, GH-6), 3.63 (s, 1H), 3.46-3.43 (m, 1H, GH-S), 2.00 (dt, J=3.5, 12.8Hz, 1H), 1.80 (dd, J=4.9, 13.1Hz, 1H), 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.δ, 70.6, 69.0,
25 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 30 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- 35 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 UN detection at 270 run.
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).
1H ΝMR (500MHz, CDC13) δ 7.02 (t, J=8.6Hz, 1H), 6.67 (d, J=δ.2Hz, 2H), 5.45 (br s, 1H), 5.04 (d, J=7.6Hz, 1H, GH-ι), 4.53 (q, J=6.7Hz, 1H), 3.83 (s, 6H), 3.77-3.67 (m, 3H), 3.63 (dd,
J=4.9, 12.2Hz, 1H, GH-6), 3.59 (s, 1H), 3.52 (t, J=9.1Hz, GH.3), 3.42 (t, J=9.4Hz, 1H, GH-4), 3.15-3.12 (m, 1H, GH-5), 2.04-2.00 (m, 2H), 1.05 (d, J=6.4Hz, 3H). 13C ΝMR (500MHz, CDCI3) 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 ΝaCΝBF t (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 18 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 UN 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).
1H ΝMR (500MHz, CDCI3) 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, 1H), 6.66 (d, J=8.7Hz, 2H), 5.50 (d, J=2.7Hz, 1H), 5.10 (d, J=7.6Hz, 1H, GH-ι), 4.54-4.51 (m, 1H), 4.34 (d, J=13.1Hz, 1H), 4.25 (d, J=13.4Hz, 1H), 3.81 (s, 1H), 3.75 (s, 6H), 3.73-3.62 (m, 4H), 3.53 (t, J=δ.9Hz, 1H, GH-3), 3.46 (t, J=9.4Hz, 1H, GH-0, 3.17-3.14 (m, 1H, GH-5), 2.20 (dd, J=4.3, 12.2Hz, 1H), 2.10 (dt,
J=3.6, 12.5Hz, 1H), 1.12 (d, J=6.7Hz, 3H). 13C NMR (500MHz, CDC13) 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: Process for Introducing a Linker at the N-methyl Leucine Position.
(a) deleucine-vancomycin (B)
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 CH2CI2 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 H2O. 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:H2O=3:5:1.5). Mass Spec. [M+H]+, 1322; [M-V]+,
1178.
(b) MeO-gly-deleucine-vancomycin (C) Compound B (162 mg, 0.117 mmol) and glycine methyl ester hydrochloride (74 mg, 0.585 mmol) are dissolved in 0.8 mL DMSO and 0.8 mL DMF and stirred at 0°C in an ice bath.
Diisopropyl ethyl amine (204 μL, 0.585 mmol) is added to the reaction vessel via syringe followed by HOBt/HBTU (1.17 mL 0.45M DMF solution, 0.526 mmol). The ice bath is removed after the addition is complete. After 10 minutes, the reaction is completed and the reaction solution is directly loaded onto a poly(divinylbenzene) column (30mmx8cm, 50-100 micron particle size) and eluted with methanol/water (0, 10%, 20%, 30%, 40%, 50% of 100 mL each). The fractions containing the pure products are combined and evaporated to give
160 mg white powder, 95%. Rf=0.1 (CHCl3:MeOH:H2O=3:3:l). Mass Spec. [M+H]+, 1393 ;
[M-V]+, 1249.
(c) AllOC-MeO-gly-deleucine-vancomycin (D)
Compound C (647 mg, 0.465 mmol) is dissolved in 10 mL water and 10 mL dioxane mixture. FMOC-succinimide (172 mg, 0.511 mmol) in 5 mL dioxane is added to the solution over 10 hours via syringe pump. The reaction is stirred for additional 5 hours after addition. Then the solution is rotovaped to dryness under reduced pressure. This gum is used in next reaction without further purification.
The crude oil from last reaction is dissolved in 10 mL DMF. To this clear solution is added diisopropyl ethyl amine (406 μL, 2.32 mmol) followed by AUOC-OBt (102 mg, 0.465 mmol) in 1 mL DMF. The reaction is stirred at room temperature for 30 minutes. 2 mL piperidine is added to the reaction flask at this time. After stirring for another 5 minutes, the solution is suspended into 160 mL of acetone and stirred, centrifuged, and decanted. The white precipitate is collected, loaded onto a Clδ reverse phase column (50mmxl2cm, particle size
40μm, pore size 60 A, from J. T. Baker) and eluted with iPrOH/water (0, 10%, 20%, 30%, 40%, 50%, 60% of 100 mL each). The fractions containing the pure products are combined and evaporated to give 309 mg white power, 58% over 3 steps. Rf=0.4 (CHC1- 3:MeOH:H2O=3:2:0.5). Mass Spec. [M+2H]+,1478; [M-V+H]+, 1250.
(d) MeO-gly-Nancomycin-Asp-COOH (E)
Compound D (102 mg, 0.0691 mmol) and AllOC-Asp(OFm)-OH (55 mg, 0.138 mmol) are premixed and azeotroped with toluene 3 times, taken in 1.5 mL DMF and then cooled to 0°C in an ice bath. Diisopropylethylamine (48 μL, 0.276 mmol) is added to reaction vessel followed by HOBt (19 mg, 0.138 mmol) and PyBOP (72 mg, 0.138 mmol). After stirring for
15 minutes, 200 μL piperidine is added to the reaction. The ice bath is removed and reaction is stirred at room temperature for 5 minutes. The clear solution is suspended in 45 mL acetone and stirred, centrifuged, and decanted. The solid is dried under reduced pressure and purified by reverse-phase HPLC using a PHENOMENEX LUNA C18 column (21.2x250mm), 5μm particle, eluting with a 30 min. linear gradient of 0.1 % acetic acid in water to 70% acetonitrile/0.1% acetic acid in water; flow rate of 7 mL/min. and ultraviolet
(UV) detection at 285 nm. The fractions containing the product are combined and evaporated to give 71 mg product, 62% over 2 steps. Rf=0.5 (CHCl3:MeOH:H2O=3:2:0.5). Mass Spec. [M+Na]+,1698; [M-V+Na]+, 1472.
EXAMPLE 17: Process for Introducing a Linker on Vancosamine:
(a) MeO-gly -vancomycin (P)
Vancomycin hydrochloride (317 mg, 0.213 mmol) and glycine methyl ester hydrochloride (54 mg, 0.426 mmol) is dissolved in 2 mL DMSO and 2 mL DMF and stirred at 0°C. Diisopropylethylamine (186 μL, 0.3195 mmol) is added to the reaction vessel via syringe followed by HOBt HBTU (710 mL 0.45M DMF solution, 0.319 mmol). The ice bath is removed after the addition is complete. After 10 minutes, the reaction is completed and the reaction solution is directly loaded onto a poly(divinylbenzene) column (30mmx8cm, 50-100 micron particle size) and eluted with methanol water (0, 10%, 20%, 30%, 40%, 50% of 100 mL each). The fractions containing the pure products are combined and evaporated to give 249 mg white powder, 77%. Rf=0.15 (CHCl3:MeOH:H2O=3:2:0.5). Mass Spec. [M+H]+,
1521 ; [M-V]+, 1377.
(b) MeO-Gly-Vancomycin-leucine-AUOC (Q)
Compound P (110 mg, 0.0723 mmol) is dissolved in 3 mL DMF. AllOC-OBt (17 mg, 0.0795 mmol) in 0.5 mL DMF is added to the solution over 10 hours via syringe pump. The reaction is stirred for an additional 5 hours after addition. The solution is then suspended into 160 mL of acetone and stirred, centrifuged, and decanted. The white solid is directly loaded onto a poly(divinylbenzene) column (30mmx8cm, 50-100 micron particle size) and eluted with methanol/water (0, 10%, 20%, 30%, 40%, 50%) of 100 mL each). The fractions containing the pure product are combined and evaporated to give 115 mg white powder, 62%. Rf=0.4
(CHCl3:MeOH:H2O=3:2:0.5). Mass Spec. [M+H]+, 1605; [M-V]+,1461.
(c) MeO-Gly-vancomycin-vancosamine-linker ( R)
Compound P (32 mg, 0.0202 mmol) and glyoxylic acid monohydrate (2 mg, 0.0222 mmol) are dissolved in 400 μL methanol and stirred at 40°C for 2 hours. A large amount of white precipitate is generated. Then the suspension is cooled back to room temperature and 100 μL
DMF is added followed by 61 μL of NaCNBH3 in THF (1M solution). After 20 minutes, the resulting clear solution is directly purified by reverse-phase HPLC using a PHENOMENEX LUNA Clδ column (21.2x250mm), 5μm 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 7 mL/min. and ultraviolet (UV) detection at 285 nm. The fractions containing the product are combined and evaporated to give 18 mg product, 54% . Rf=0.4 (CHC1-
3:MeOH:H2O=3:2:0.5 ). Mass Spec. [M+H]+, 1662 ; [M-V]+, 1460.
EXAMPLE 18: Process for Introducing a Linker at the Glucose C-6 Position.
All-dialloc-C-6-NH2-linker-COOH (N)
Compound M (17 mg, 0.0103 mmol) and glyoxylic acid monohydrate (0.95 mg, 0.103 mmol) are dissolved in 1 mL methanol and stirred at 40°C for 2 hours. A large amount of white precipitate is generated. Then the suspension is cooled back to room temperature and 250 μL DMF is added followed by 200 μL of NaCNBH3 in THF (1M solution). After 20 minutes, the resulting clear solution is directly purified by reverse-phase HPLC using a
PHENOMENEX LUNA C18 column (21.2x250mm), 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 7 mL/min. and ultraviolet (UV) detection at 285 nm. The fractions containing the product are combined and evaporated to give 6 mg product, 33% . Rf=0.28 (CHCl3:MeOH:H2O=3:2:0.5 ). Mass Spec. [M+H]+, 1716; [M-V]+, 1488.
EXAMPLE 19: Process for Introducing a Linker Acid on a Sugar
MeO-gly-Vanco-Asp-CONH-glucosamine (I) Compound E (20 mg, 0.0119 mmol) and glucosamine.HCl (8 mg, 0.0358 mmol) are premixed and azeotroped with toluene 3 times, taken in 240 μL DMF and then cooled to 0°C. Diisopropylethylamine (21 μL, 0.119 mmol) is added to reaction vessel followed by HOBt (4.8 mg, 0.0357 mmol) and pyBOP (18 mg, 0.0358 mmol). After stirring for 15 minutes, the clear solution is suspended in 45 mL acetone and stirred, centrifuged, and decanted. The solid is dried under reduced pressure and purified by reverse-phase HPLC using a
PHENOMENEX LUNA C18 column (21.2x250mm), 5μm particle, eluting with a 40 min.
linear gradient of 0.1% acetic acid in water to 40% acetonitrile/0.1% acetic acid in water; flow rate of 7 mL/min. and ultraviolet (UV) detection at 285 nm. The fractions containing the product are combined and evaporated to give 13 mg product, 60% over 2 steps. Rf=0.15 (CHCl3:MeOH:H2O=3:2:0.5). Mass Spec. [M+Na]+,1859; [M-V+Na]+, 1632.
EXAMPLE 20: Linking a Disaccharide to A \ via a Linker.
This process is illustrated in Scheme 4, as shown below:
Compound A (20 mg, 0.0501 mmol) and DTBMP (32 mg, 0.158 mmol) are azeotroped with toluene 3 times and then dissolved in 3 mL Et
2O. The reaction solution is cooled to -78°C and 0.5 mL toluene is added. Triflic anhydride (10 μL, 0.100 mmol) is added to the reaction solution. The sulfoxide B (37 mg, 0.100 mmol) in lmL Et
2O 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 NaHCO
3 aqueous solution. The ether layer is separated and the aqueous layer is further extracted with EtOAc (3 x 5 mL). The organic layers are combined and dried over anhydrous sodium sulfate, filtered, concentrated to give a clear oil. This oil is loaded onto a silica gel column (10 mm x 8 cm) and eluted with 35% EtOAc/petroleum ether to give 15 mg (45%) compound C as a clear oil. R 0.35 (40%EtOAc/petroleum ether).
To a solution of sulfide C (50 mg, 0.0749 mmol) and DTBMP (24 mg, 0.116 mmol) in 2 mL CH2C1 and L (143 mg, 0.749 mmol) is added Hg(OOCCF3)2 (35 mg, 0.0823 mmol) in one portion. The reaction is stirred at room temperature for 10 minutes and then quenched by addition of addition of 20 mL saturated NaHCO3 aqueous solution. The CH2CI2 layer is separated and the aqueous layer is further extracted with CH2CI2 (3 x 15 mL). The organic layers are combined and dried over anhydrous sodium sulfate, filtered, concentrated to a clear oil. This oil is purified by flash chromatography (45 % EtOAc/petroleum ether) to give 25 mg (44 %) of compound D as a clear oil.
Compound D (20 mg, 0.0268 mmol) is dissolved in a mixture of 100 μL allyl alcohol, 330 μL THF and 660 μL methanol. 100 μL anhydrous hydrazine is added. The reaction is stirred at room temperature for 10 hours and then quenched by addition of 0.5 mL acetic acid. All the solvents are removed and the residue is purified on a Cl reverse-phase column (10 mm x δ cm) (eluting with 0%, 10%, 20%, 30%, 40% of CH3CN/H2O/0.1% AcOH 20 mL each) to give 7 mg (59%) of compound E as a white solid. R 0.1 (CHC13/MeOH H2O = 3/2/0.5).
To a solution of F (3.7g, 2.50mmol) in 35 mL water is added 3 mL concentrated HC1 aqueous solution. The reaction is refluxed for 5 minutes and then cooled to room temperature. The white suspension is suction filtered through a glass funnel equipped with a medium frit. After filtration, the precipitate is washed with acetone and dried to give a brown solid. The crude product G is used without further purification.
To a solution of crude compound G (105 mg) in 5 mL 1:1 mixture of water and dioxane is added FMOC-succinimide (46 mg, 0.136 mmol) followed by NaHCO3 (23 mg, 0.273 mmol). The reaction is stirred for 2 hours and all solvents are removed under reduced pressure. The residue is azeotroped with toluene (3 x 20 mL) and then dissolved in 2 mL DMF. NaHCO3 (20 mg) is added followed by allyl bromide (12 μL, 0.136 mmol). The reaction is stirred for
1 hour and then 0.5 mL piperidine is added. The reaction is further stirred for 5 minutes. The reaction mixture is suspended in 45 mL acetone, stirred, centrifuged, and decanted. The solid is dried under reduced pressure and purified by reverse-phase HPLC using a PHENOMENEX LUNA Clδ column (21.2 x 250 mm), 5μm particle, eluting with a 30 min. linear gradient of 20% CH3CN/0.1% trifluoroacetic acid in water to 100% acetonitrile/0.1% trifluoroacetic acid in water; flow rate of 7 mL/min. and ultraviolet (UV) detection at 2δ5 nm. The fractions containing the product are combined and evaporated to give 38 mg (35%) product H as a white solid.
Compound H (38 mg, 0.036 mmol) and AUOC-Asp(OFm)-OH (28 mg, 0.069 mmol) are premixed and azeotroped with toluene 3 times, dissolved in 1.5 mL DMF and then cooled to 0°C. Diisopropylethylamine (24 μL, 0.138 mmol) is added to reaction vessel followed by HOBt (10 mg, 0.069 mmol) and PyBOP (36 mg, 0.069 mmol). After stirring for 15 minutes, 200 μL piperidine is added to the reaction. The ice bath is removed and the reaction is stirred at room temperature for 5 minutes. The clear solution is suspended in 45 mL acetone, stirred, centrifuged, and decanted. The solid is dried under reduced pressure and purified by reverse- phase HPLC using a PHENOMENEX LUNA C18 column (21.2 x 250 mm), 5μm particle size, eluting with a 30 min. linear gradient of 20% CH3CN/0.1% trifluoroacetic acid in water to 100% acetonitrile/0.1% trifluoroacetic acid in water; flow rate of 7 mL/min. and ultraviolet (UV) detection at 285 nm. The fractions containing the product are combined and evaporated to give 36 mg (80%) product I, 62% over 2 steps.
Compounds I (15 mg, 0.0119 mmol) and E (16 mg, 0.0358 mmol) are premixed and azeotroped with toluene 3 times, dissolved in 240 μL DMF and then cooled to 0°C. Diisopropylethylamine (21 μL, 0.119 mmol) is added to reaction vessel followed by HOBt
(4.8mg, 0.0357mmol) and pyBOP (18 mg, 0.0358 mmol). After stirring 15 minutes, the clear
solution is suspended in 45 mL acetone and stirred, centrifuged, decanted. The solid is dried under reduced pressure and purified by reverse-phase HPLC using a PHENOMENEX LUNA Clδ column (21.2 x 250 mm), 5μm particle size, eluting with a 40 min. linear gradient of 0.1% acetic acid in water to 80% acetonitrile/0.1%) acetic acid in water; flow rate of 7 mL/min. and ultraviolet (UV) detection at 285 nm. The fractions containing the product are combined and evaporated to give 17 mg (δ5%) product J.
Compound J (3.9 mg, 0.00229 mmol) is dissolved in 0.5 mL DMF/0.5 mL acetic acid. A small amount of palladium dichloride-bis-triphenylphosphine (~1 mg) is added and the reaction vessel is filled with nitrogen. To this mixture is added, with vigorous stirring, tributyltin hydride in 50 μL portions every 5 minutes until all starting materials and intermediates have disappeared by TLC. The crude reaction mixture is precipitated with 30 mL acetone in a 50 mL centrifuge tube. The mixture is centrifuged and decanted to give a white solid. This white solid is taken in 5 mL water and kept in 0°C overnight. Next day the suspension is filtered through a disposable 13 mm syringe filter (Whatman Inc.) and the resulted filtrate is concentrated and purified by reverse-phase HPLC using a
PHENOMENEX LUNA Clδ column (21.2 x 250 mm), 5μm particle, eluting with 0.1% acetic acid in water for 5 minutes and then a 30 min. linear gradient of 0.1 % acetic acid in water to 40% acetonitrile/0.1% acetic acid in water; flow rate of 7 mL/min. and ultraviolet (UV) detection at 2δ5 nm. The fractions containing the product are combined and concentrated to give K as acetic acid salt (2.6 mg, 79%).
EXAMPLE 21: Preparation of Phenyl 3-Azido-2,3,6-trideoxy-l-thio- -L- galactopyranoside.
The steps in the preparation of this compound, the precursor to compound (7), are outlined in
Scheme 5, as shown below:
(a) 1 ,4-Di-O-acetyl-3-azido-2,3,6-trideoxy-α,β-L-glucopyranoside (A).
To a solution of 4-0-acetyl-3-azido-2,3,6-trideoxy-α,β-L-glucopyranoside (J. Carbohyd.
Chem., 1990, 9:δ73; J.C.S. Chem. Commun. 19δ7, 1171) (350 mg, 1.63 mmol) in 16 mL of CH2CI2 at room temperature are added acetic anhydride (307 μL, 332 mg, 3.25 mmol), pyridine (526 μL, 515 mg, 6.51 mmol), and 4-dimethylaminopryridine (20 mg, 0.163 mmol).
The reaction is stirred at room temperature for 40 min and then poured into saturated
NaHCO3 solution (20 mL). The organic and aqueous layers are separated, and the organic layer is washed with IN HC1 (20 mL) and saturated NaHCO3 solution (20 mL), dried over Na2SO4, filtered, and concentrated to afford a yellow oil. Purification is accomplished by flash chromatography (20% EtOAc/petroleum ether) to yield 41 δ mg (100%) of the product
A as a clear oil: Rβ.51 (40% EtOAc/petroleum ether).
1H NMR (CDCI3, 270 MHz) (« anomer) δ 6.13 (d, J= 2.6 Hz, 1H, H-l), 4.70 (app t, J= 9.9 Hz, 1H, H-4), 3.δ8 -3.78 (m, 2H, H-3 and H-5), 2.19 - 2.13 (m, 1H, H-2), 2.11 (s, 3H,
COCH3), 2.09 (s, 3H, COCH3), 1.88 - 1.77 (m, 1H, H-2'), 1.14 (d, J= 5.9 Hz, 3H, H-6). 13C NMR (CDCI3, 67.9 MHz) (α anomer) δ 169.9, 169.2, 90.6, 75.1, 68.5, 57.4, 34.2, 21.2, 20.9, 17.6. HRMS: Calculated for C10Hι9N4O5 (MNILt÷): 275.1355; Found: 275.1357.
(b) Phenyl 4-O-acetyl-3-azido-2,3,6-trideoxy-l-thio-α,β-L-glucopyranoside (3.51).
To a solution of l,4-di-0-acetyl-3-azido-2,3,6-trideoxy-α, β-L-glucopyranoside A (410 mg, 1.59 mmol) in 16 mL of CH2C12 at -78°C are added thiophenol (200 μL, 215 g, 1.98 mmol)
and boron trifluoride diethyl etherate (1.0 mL, 1.15 g, 8.13 mmol). The reaction is stirred at -
78°C and is allowed to warm to -70°C over 1 h. The reaction mixture is poured into saturated NaHCO3 solution (20 mL). The organic and aqueous layers are separated, and the organic layer is washed once more with saturated NaHCO3 solution (20 mL). The aqueous layers are combined and extracted with CH2CI2 (2 x 20 mL). The organic layers are combined and dried over Na2SO4, filtered, and concentrated to give a yellow oil. Purification is accomplished by flash chromatography (10% EtOAc/petroleum ether) to afford both α and β anomers of the product B (490 mg total, 100%; α:β, 3:1): R/(α anomer) 0.55, R/ (β anomer) 0.45 (20% EtOAc/petroleum ether).
1H NMR (CDCI3, 270 MHz) (oc anomer) δ 7.46 - 7.25(m, 5H, ArH), 5.57 (d, J= 5.3 Hz, 1H,
H-l), 4.72 (app t, J = 9.9 Hz, 1H, H-4), 4.31 (qd, J = 9.6, 6.3 Hz, 1H, H-5), 3.δδ (ddd, J = 12.5, 9.6, 4.9 Hz, 1H, H-3), 2.36 (dd, J= 13.2, 5.3 Hz, 1H, H-2), 2.17 - 2.06 (m, 1H, H-2'), 2.14 (s, 3H, COCH3), 1.17 (d, J = 5.9 Hz, 3H, H-6). 13C NMR (CDCI3, 67.9 MHz) (α anomer) δ 170.0, 134.2, 131.3. 129.1, 127.5, 83.1, 75.7, 66.9, 58.4, 36.1, 20.9, 17.4. HRMS: Calc'd for Cι2Hι9N4O2S (MNHt÷): 325.1334; Found: 325.1322.
(c) Phenyl 3-azido-2,3,6-trideoxy-l-thio-α,β-L-glucopyranoside (C).
To a solution of phenyl-4-0-acetyl-3-azido-2,3,6-trideoxy-l-thio-α,β-L-glucopyranoside B
(276 mg, 0.89δ mmol) in 9 mL of methanol at room temperature is added potassium carbonate (74 mg, 0.449 mmol). The reaction is stirred at room temperature for 12 h. The reaction mixture is then poured into saturated NH4CI (10 mL) solution. The organic and aqueous layers are separated, and the aqueous layer is extracted with CH2CI 2 (2 x 5 mL). The organic layers are combined, washed with saturated NH CI solution (10 mL), saturated NaHCO3 solution (10 mL), and saturated NaCl solution (10 mL), dried over Na2SO4, filtered and concentrated. Purification is accomplished by flash chromatography (gradient elution with 10-20%) EtOAc/petroleum ether) to afford 235 mg of the product C as a white solid: Rf 0.45 (20% EtOAc/petroleum ether).
1H NMR (CDC13, 270 MHz) ( anomer) δ 7.47 - 7.27 (m, 5H ArH), 5.58 (d, J= 5.3 Hz, 1H, H-l), 4.21 (qd, J = 9.2, 6.6 Hz, 1H, H-5), 3.77 (ddd, J = 12.7, 9.4, 4.8 Hz, 1H, H-3), 3.20
(app t, J = 9.2 Hz, 1H, H-4), 2.39 (dd, J = 13.5, 5.0 Hz, 1H, H-2), 2.26 (bs, 1H, OH), 2.13
(app dt, J= 13.0, 5.7 Hz, 1H, H-2'), 1.30 (d, J= 6.6 Hz, 3H, H-6). 13C NMR (CDC13, 67.9 MHz) (α anomer) δ 134.4, 131.5, 129.0, 127.4, 83.4, 76.4, 68.δ, 61.1, 36.0, 17.7. HRMS: Calc'd for C12H19N4O2S (MNH4+): 2δ3.1229; Found: 2δ3.1233.
(d) Phenyl 3 -azido-2,3 ,6-trideoxy-l-thio-α-L-galactopyranoside.
To a solution of phenyl 3-azido-2,3,6-trideoxy-l-thio-α,β-L-glucopyranoside C (231 mg, 0.δ71 mmol) in 9 mL of CH2CI2 at -25°C (ethanol/wet ice/dry ice) is added triflic anhydride (293 μL, 491 mg, 1.74 mmol) dropwise and pyridine (155 μL, 152 mg, 1.92 mmol). The reaction is stirred for 4 h between -25°Cand -5°C (temperature of cold bath is not stable). More Tf2O (200 μL, 335 mg, 1.19 mmol) and pyridine (100 μL, 97.δ mg, 1.24 mmol) are added, and the reaction is allowed to proceed for 1 h more. The reaction mixture is poured into saturated NaHCO3 solution (10 mL). The organic and aqueous layers are separated, and the aqueous layer is extracted with CH2C12 (3 x 5 mL). The organic layers are combined and washed with IN HC1 (10 mL) and saturated NaHCO3 (10 mL), dried over Na2SO4, filtered, and concentrated. Purification is accomplished by flash chromatography (7%
EtOAc/petroleum ether) to afford 190 mg (71% yield) of the desired product D: R 0.69 (20% EtOAc/petroleum ether).
To a solution of phenyl 3 -azido-2,3 ,6-trideoxy-l-thio-4-0-trifloyl-α-L-glucopyranoside D (190 mg, 0.616 mmol) in dimethylformamide (6.2 mL) at room temperature are added potassium benzoate (118 mg, 0.739 mmol) and 18-crown-6 (195 mg, 0.739 mmol). The reaction appears to be complete after 1 hour, but is stirred overnight (15 h) at room temperature. The reaction mixture is diluted with 10 mL of EtOAc and poured into saturated NaHCO3 solution (10 mL). The organic and aqueous layers are separated, and the aqueous layer is extracted with EtOAc (2 x 5 mL). The organic layers are combined, washed with saturated NaHCO3 solution (10 mL) and saturated NaCl solution (10 mL), dried over Na2SO4, filtered, and concentrated: R/0.62 (20% EtOAc/petroleum ether).
To a solution of phenyl 3 -azido-2,3 ,6-trideoxy-l-thio-4-0-benzoyl-α-L-galactopyranoside (22δ mg, 0.616 mmol, theoretical yield from previous reaction) in 6 mL of methanol at room temperature is added lithium hydroxide monohydrate (259 mg, 6.16 mmol). The reaction is
stirred at room temperature for lδ h. The reaction mixture is diluted with 10 mL of EtOAc and poured into saturated NELtCl solution. The organic and aqueous layers are separated, and the organic layer is washed with saturated NaHCO3 solution (5 mL) and saturated NaCl solution (5 mL), dried over Na2SO4, filtered, and concentrated. The crude product is purified by flash chromatography (2% MeOH/CH2Cl2) to afford 91 mg (55%, 2 steps) of the desired product (α anomer): R 0.33 (20% EtOAc/petroleum ether).
The β anomer of this product is prepared in exactly the same way; spectroscopic data for this compound are identical to those given for the anomer.
1H NMR (CDC13, 270 MHz) (α anomer) δ 7.51 - 7.23 (m, 5H ArH), 5.70 (d, J= 5.9 Hz, 1H,
H-l), 4.42 (q, J= 6.6 Hz, 1H, H-5), 3.81 - 3.75 (m, 2H, H-3 and H-4), 2.4δ (app dt, J= 13.4, 5.7 Hz, 1H, H-2), 2.11 (dd, J= 14.1, 3.5 Hz, 1H, H-2'), 1.97 (bs, 1H, OH), 1.28 (d, J = 6.6 Hz, 3H, H-6). 13C NMR (CDCI3, 67.9 MHZ) (α anomer) δ 134.6, 131.4, 129.2, 127.5, 83.7, 70.0, 67.2, 57.9, 29,7, 16.9. HRMS: Calc'd for C12Hι9N4O2S (M H4+): 283.1229; Found: 283.1221.
EXAMPLE 22: Linking a Disaccharide to At via a Haloalkyl-Substituted Saccharide.
This process is illustrated in Schemes 6 to 8, as shown below:
Scheme 6
(a) i. 4 eq. Alloc-succinimide, 3 eq. NaHC03, H20/dioxane, r.t., 3h; ii. 5 eq. allyl bomide, 2 eq. NaHC03, DMF, r.t., 2 h; iii. 10 eq. allyl bromide, 5 eq. Cs2C03, DMF, r.t., 6h;
(b) HBr, HOAc, PhSH, 30 min.
Scheme 7
o
SPh SPh SPh
I NHalloc I NHalloc I NHalloc
HO ACO ACO
(c) Ac20, Et3N, DMAP, CH2C12, r.t., Ih; (d) mCPBA, CH2C12, -78°Cto -20°C, lh; (e) mCPBA, 2-chloroethanol, CH2C12, r.t., 24h; (f) E, Tf20, 2,6-di-t-butyl-4- methylpyridine, -78°Cto -10°C, lh; (g) NaOMe, MeOH, r.t., 30 min.; (h) Nal, acetone, reflux, 20h.
Scheme 8
(i) I, Cs2C03, DMF, r.t., 5h; 0) PdCl2(PPh3)2, Bu3SnH, DMF/AcOH 1:1, 20 min.; (k) 4,4'-chlorobiphenylaldehyde, DIEA, NaBH3CN, 65°C, 5h.
EXAMPLE 23: Linking a Disaccharide to t via an Amide Linkage.
This process is illustrated in Scheme 9, as shown below. Any saccharide compound having one free amino group can be attached by this method.
Scheme 9
(a) i. 4 eq. Alloc-succinimide, 3 eq. NaHC03, H20/dioxane, r.t., 3h; ii. 5 eq. allyl bomide, 2 eq. NaHC03, DMF, r.t., 2 h; iii. 10 eq. allyl bromide, 5 eq. Cs2C03, DMF, r.t., 6h;
(b) 3% HBr/HOAc, PhSH, r.t., 10 min., 63% over 4 steps; (c) i. BrCH2COOTMSE, K2C03, DMF; ii. TBAF, DMF; (d) PyBOP, HOBt, DIEA, DMF; (e) Pd(PPh3)2Cl2, Bu3SnH, DMF/AcOH.
EXAMPLE 24: 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 antibacterial agents 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 NFLtOAc 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 + [14C]-Lipid Intermediate II
(ii) Transglycosylase step: product retained by PVDF filter Reaction run in the presence of 1 mg/mL pemcillin G to inhibit transpeptidation
[I4C]-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 + [14C]-peptidoglycan cross-linked [,4C]-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 incoφoration 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 incoφoration 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 W7. 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 VCδ. 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 antibacterial agents 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.
EXAMPLE 25: Preparation of a Disaccharide Coupled to a "Natural" Aglycone.
The preparation of disaccharide aglycone conjugate (9) is illustrated in Scheme 10, below:
Scheme 10
1 2. NxaBH3CcNι 3. TBAF
Methods of preparing compound (9) of Example 25 are provided, below:
4-(3-azido-propyl)-2,6-dimethoxyphenol ( 1 ) To a solution of allyl dimethoxyphenol (900 mg, 4.6 mmol) in THF at 0 C is added
BH3 THF (12 ml, IM in THF). After stirring at room temperature for 30 min, water is added
slowly followed by 12 ml 3N NaOH and 12 ml 30% H2O2. The mixture is stirred for 3h, then
38g K2CO3 are added and the aqueous/THF layers separated. The THF layer is collected and concentrated. The product is purified by flash chromatography (20%-60% EtOAc/ petroleum ether) to give 450 mg. The alcohol (415 mg, 1.9 mmol) is dissolved in acetonitrile and added to a solution of PPh3 (772 mg, 2.9 mmol) and Br2 (146 μl, 2.84 mmol) in acetonitrile at 4 C. The mixture is stirred at room temperature for 2h, concentrated and purified by flash chromatography (10%-40% EtOAc/ petroleum ether) to give 470 mg. The bromide (1.7 g, 6.2 mmol) is dissolved in DMF, NaN3 (0.8 g) is added and the mixture heated to 80 C overnight. The product is purified by flash chromatography (10%-40% EtOAc/ petroleum ether) to give 1.5 g of a pale brown oil.
1H NMR (270 MHz, CDC13): δ 6.42 (s, 2H), 5.65 (s, IH), 3.84 (s, 6H), 3.27 (t, 2H), 2.63 (t, 2H), 1.88 (m, 2H)
4-(3-azido-propyl)-2,6-dimethoxyphenyl 3,4,6-tri-0-benzyl-β-D-glucopyranoside (3) To 3-azido-propyl-2, 6-dimethoxyphenol (1) (1.5 g, 6.3 mmol) in benzene is added bis
(tributyl tin) oxide (1.6 ml, 3.15 mmol). The mixture is heated to reflux with a Dean-Stark trap overnight.
2-O-pivaloyl-3,4,6-tri-O-benzyl glucose sulfoxide (2) (2.1 g, 3.27 mmol) and DTBMP (2 g, 9.81 mmol) are azeotroped 3x with toluene, dissolved in 40 ml EtO Ac/3 ml CH2C12 and stirred over molecular sieves for lh. The mixture is then cooled to -78 C and Tf2O (330 μl, 1.96 mmol) is added. The mixture is warmed to -60 C, kept there for 15 min and cooled to -78 C. The phenol is dissolved in 10 ml EtO Ac/1 ml CH2Cl2and added dropwise to the activated sulfoxide. The reaction is warmed to -50 C over 1.5h and quenched with Et2NH. The product is partially purified by flash chromatography (10%-25% EtOAc/ petroleum ether). The semi-pure product is dissolved in 6 ml water/8 ml MeOH/ 12 ml THF (two layers). LiOH (1.5 g) is added and the mixture stirred at 35 C for 25h. The product is purified by flash chromatography (35% Et2O/ petroleum ether) to give 1.6 g.
4-(3-azido-propyl)-2,6-dimethoxyphenyl, 2-(3-N-Cbz-2,3,6,trideoxy-3-C-methyl-α-L-lyxo- hexapyranosyl)-3,4,6-tri-0-benzyl-β-D-glucopyranoside (5)
5δ 3-N-Cbz-4-0-acetyl vancosamine phenyl sulfide (2.7 g, 6.29 mmol) is dissolved in CH2C12 and cooled to -40 C. wCPBA (1.87 g, 0.7 mmol) is added. After lh dimethylsulfide is added and the mixture extracted with aqueous ΝaHC03. It is then dried over Na2SO4 and azeotroped 3x with toluene. The nucleophile (3) (2.0 g, 3.4 mmol) and DTBMP (2.6 g, 12.6 mmol) are azeotroped 3x with toluene, dissolved in 40 ml Et2O/ 8 ml CH2C12, molecular sieves are added and the mixture is cooled to -78 C. Tf2O (5δ2 μl, 3.46 mmol) is added.
The sulfoxide (4) is dissolved in 10 ml Et2O/ 2 ml CH2C12 and added dropwise over 20 min to the nucleophile. The mixture is warmed to -10 C over 3 h and poured into aqueous NaHCO3. The product is purified by flash chromatography (10%-40% EtOAc/petroleum ether) to give 2.3 g. ESI-MS calculated for C55H64N4O13 9δδ [M + Na]+: 1011
The disaccharide is then dissolved in MeOH and NaOMe (10 mg) is added. After stirring at room temperature for 3 h, 10 mg NaOMe are added and stirred for 15 min. Amberlite is added and the product is purified by flash chromatography (15%-35% EtOAc/petroleum ether) to give 1.5 g. 1H NMR (500 MHz, CDC13): δ 7.34-7.71 (m, 20H), 6.3δ (s, 2H), 5.51 (s, IH), 5.27 (d, J=4.5δ Hz, IH), 5.04-5.06 (m, 3H), 4.43-4.90 (m, δH), 3.94 (t, J=δ.24 Hz,
IH), 3.60-3.75 (m, 10H), 3.39-3.3δ (m, IH), 3.26 (t, J=6.72 Hz, 2H), 3.20 (d, J=9.16 Hz, IH), 2.62 (t, =7.63 Hz, 2H), 2.25-2.15 (m, 2H), 1.89-1.78 (m, 2H), 1.78 (s, 3H), 1.07 (d, J=6.4, 3H)
4-(3-(2-trimethylsilylethyl carbamate) propyl)-2,6-dimethoxyphenyl 2-vancosaminyl-β-D- glucopyranoside (6)
A solution of disaccharide (5) (1.5 g, 1.55 mmol) and triphenylphosphine (1.2 g, 4.5 mmol) in THF/water (3:1) is stirred at 50 C overnight. The mixture is concentrated and purified by flash chromatography in 5% to 12% MeOH/CH2Cl2/0.1%Et3N to give 1.15 g.
To a solution this disaccharide amine (1.15 g, 1.25 mmol) in 10 ml DMF is added Hunig's base (1.1 ml) and 2-trimethylsilylethyl nitrophenol carbonate (391 mg, 1.38- mmol). After lh, the bright yellow mixture is concentrated and purified by flash chromatography (20%-50% EtOAc/petroleum ether) to give 1.23 g.
This product (490 mg, 0.46 mmol) is dissolved in EtOH, Pearlman's catalyst and 1,4 cyclohexadiene (1.7 ml, 18.5 mmol) are added. The mixture is refluxed for 8h, more catalyst and cyclohexadiene are added and the mixture is refluxed overnight. The sample is filtered through celite which is then washed with 200 ml MeOH. The product is then concentrated and purified by reverse-phase HPLC using a Phenomenex LUNA Clδ column (21.2 x 250 mm, 5 μm particle size), eluting with a linear gradient (15% to δ0% CH3CN H2O with 0.1%
HOAc over 30 min, retention time 14 min) to give 190 mg of the desired product. ESI-MS calculated for C30H52N2O12Si 660 [M + H]+: 661
4-(3-aminopropyl)-2,6-dimethoxyphenyl, 2-(3-N-chlorobiphenyl-vancosaminyl)-β-D- glucopyranoside (7)
To a solution of disaccharide (6) (90 mg, 0.14 mmol) in 5 ml DMF is added chlorobiphenyl aldehyde (3δ mg, 0.18 mmol) and Hunig's base (122 μl, 0.7 mmol). The mixture is stirred at 55 C for 3h, NaBH3CN (0.7 ml, IM in THF) is added and the mixture is stirred at 55 C for 3h 0.4 ml HOAc are added to quench and the product is partially purified by reverse-phase HPLC using a Phenomenex LUNA Cl 8 column (21.2 x 250 mm, 5 μm particle size), eluting with a linear gradient (15% to 80% CH3CN/H2O with 0.1%HOAc over 30 min, retention time 25 min). The lyophilized powder is dissolved in 1 ml DMF and TBAF (1 ml, IM in THF) is added. The mixture is heated at 55 C overnight. The product is purified by reverse-phase HPLC using a Phenomenex LUNA C18 column (21.2 x 250 mm, 5 μm particle size), eluting with a linear gradient (10% to 60% CH3CN/H2O with 0.1% HOAc over 30 min, retention time 17 min) to yield 5δ mg. ESI-MS calcd for CS^CIN^K) 716.3 [M + H]+: 717
Carboxy-linked aglycone (9)
To a solution of vancomycin aglycone (δ) (16 mg, 0.014 mmol) and disaccharide (7) (5 mg, 0.007 mmol) in 1 ml DMF is added HOBt (3 mg, O.Olδ mmol) and TBTU (6 mg, O.Olδ mmol) followed by N-methyl moφholine (4 1, 0.035 mmol). The reaction is stirred overnight at room temperature. The solution is purified by reverse-phase HPLC using a Phenomenex LUNA Clδ column (21.2 x 250 mm, 5 μm particle size), eluting with a linear gradient (10% to 60% CH3CN/H2O with 0.1% TFA over 60 min, retention time 43 min) to give 3 mg of the desired product. ESI-MS calculated for C90H99Cl3Nι0O26.lδ40.6 [M + H]+: lδ42
The aglycone (8) is prepared as described by Nagarajan, R. and Schnabel, A. A. J. Chem. Soc, Chem. Commun. (1988) 1306-1307. The vancosamine sulfide is prepared similarly to Thompson, C, Ge, M. and Kahne, D. J. Am. Chem. Soc. (1999) 121: 1237-1244. For a route to the glucose sulfoxide (2): See, C. Thompson, Ph.D. Thesis, Princeton University, 1999.
EXAMPLE 26: Minimum Inhibitory Concentrations for Selected Compounds.
Referring now to Figure 8, MIC values selected conjugates of the present invention are presented. In these examples, the disaccharide moiety is linked either to the carboxyl group of the residue A7 or to the phenyl group of the residue t.
EXAMPLE 27: Preparation of a Representative Rhodamine Conjugate.
An exemplary synthetic procedure for the preparation of a rhodamine disaccharide conjugate is presented in Fig. 9. Individual steps in the synthesis is described in further detail, below.
3,4,6-Tri-0-acetyl-l-(l-chloroethoxy)-β-L-glucopyranoside (1)
To a flame dried, argon purged 100 mL round bottom flask is added 3,4,6-tri-O-acetyl-glucal (1.5 g, 5.5 mmol), and the glucal is azeotroped thrice from toluene. The glucal is dissolved in dry CH2C12 (20 mL) and 2-chloroethanol (10 mL) and 4 A molecular sieves are added. mCPBA (1.9 g, 6.6 mmol, predried under high vacuum for two days) is added and the reaction is stirred for 40 hours at room temperature under argon. Methylsulfide (500 μL) is added to quench unreacted oxidant and stirred for 1 hour. The reaction is concentrated in vacuo to a tan residue. The crude residue is partitioned between CH2C12 and saturated NaHCO3 (60 mL). The cloudy aqueous layer is extracted thrice with CH2C12 (60 mL). The organic layers are pooled, washed with brine, dried over Na2SO4, filtered, and concentrated in
vacuo to yield a yellow oil. The oil is applied to a silica flash column (5 x 12 cm) and eluted with 75%, 80%) and 90% diethylether/petroleum ether. Fractions containing a 3:1 mixture of glucose and mannose are concentrated in vacuo to a white solid. The white solid is dissolved in toluene with heating and allowed to cool slowly to room temperature, resulting in the formation of colorless crystals of 1 (576 mg, 1.56 mmol, 28%)
1H-NMR (270 MHz, CDC13): δ 5.15 (t, J= 9.23 Hz, IH), 5.05 (t, J= 9.56 Hz, IH), 4.44 (d, J = 7.91 Hz, IH), 4.32-4.10 (m, 3H), 3.86 (dt, J= 11.20, 5.94 Hz, IH), 3.74-3.60 (m, 4H), 2.10 (s, 6H), 2.04 (s, 3H).
3,4,6-Tri-O-acetyl-2-(4-0-acetyl-3-N-allyloxycarbonyl-2,3,6-tridexoy-3-C-methyl-α-L-/y o- hexopyranosido)- 1 -( 1 -chloroethoxy)-β-L-glucopyranoside (2)
To a flame dried, argon purged 50 mL round bottom flask is added glucose 1 (200 mg, 0.54 mmol) and 2,6-di-tert-butyl-4-methyl-pyridine (336 mg, 1.63 mmol). The glucose and base are azeotroped thrice from toluene and dissolved in dry CH C12 (2 mL) and Et2O (6 mL). Oven dried 4A molecular sieves are added, and the clear solution stirred at -78° C for 30 minutes. Triflic anhydride (0.5 mL of a 1.31 M stock solution in CH2C12) is then added, yielding a cloudy solution.
A 10 mL tear drop flask is charged with phenyl-3-(N-allyloxycarbonyl)-4-O-acetyl-l- sulfinyl-2,3,6-trideoxy-3-C-methyl-L- yxo-hexopyranoside [Thompson, C. et al. J. Am. Chem. Soc. (1999) 121 :1237-1244] (276 mg, 0.70 mmol) and azeotroped thrice from toluene. The sulfoxide is dissolved in dry Et2O (2 mL) and added dropwise to the nucleophile 1 solution over 10 minutes at -78° C. The sulfoxide flask is washed once with CH2C12 (1 mL) and added dropwise to the glucose over 5 minutes. The reaction is stirred for 30 minutes at -
74° C and then allowed to warm to -15° C over 2.5 hours. Most of the starting material is consumed once the reaction reaches -60° C. After stirring at -15° C for 30 minutes, the reaction is quenched by adding saturated NaHCO3 to the reaction and warming to room temperature. The reaction is filtered through a cotton plug directly into a separatory funnel and extracted thrice with CH2C12. The pooled organic layers are dried over Na2SO4, filtered, and concentrated in vacuo to a light yellow oil. Purified by silica flash chromatography (2 x
9 cm) eluting with 27% EtOAc/CH2Cl2 to yield 2 as an oil (0.128 g, 0.20 mmol, 37%).
1H-NMR (270 MHz, CDCl3):δ 5.94-5.80 (m, IH), 5.28 (broad s, IH), 5.27-5.16 (m, 2H), 5.06 (d, J = 3.96 Hz, IH), 4.96 (t, J = 9.89 Hz, IH), 4.89 (s, IH), 4.77 (broad s, IH), 4.53- 4.39 (m, 4H), 4.28-4.02 (m, 3H), 3.83-3.59 (m, 5H), 2.22-1.91 (m, 14H), 1.64 (s, 3H), 1.09
(d, J= 6.59 Hz, 3H).
3,4,6-Tri-O-acetyl-2-(4-( -acetyl-3-N-allyloxycarbonyl-2,3,6-tridexoy-3-C-methyl-α-L-/ cco- hexopyranosido)- 1 -( 1 -aminoethoxy)- β -L-glucopyranoside (3)
Disaccharide 2 (143 mg, 0.22 mmol) is dissolved in dry DMF (2 mL) in a 25 mL round bottom flask. To the clear solution is added ΝaΝ3 (50 mg, 0.77 mmol) and KI (24 mg, 0.14 mmol). The cloudy solution is stirred at 80° C under argon for 20 hours. The reaction is concentrated in vacuo and partitioned between H2O/CH2Cl2 (10 mL). The aqueous layer is extracted thrice with CH2C12 (10 mL). The pooled organic layers are dried over Na2SO4, filtered, and concentrated in vacuo. The residue is applied to a silica flash column (2 x 6 cm) and eluted with 40% EtOAc/petroleum ether to yield the azide as a white foam (99 mg, 0.15 mmol, 69%).
To a 25 mL round bottom flask containing the tetraacetyl disaccharide (71 mg, 0.11 mmol) is added dry MeOH (4 mL) and NaOMe (2.0 mg, 0.035 mmol). The reaction is stirred at room temperature under argon for 1.2 hours at which point all the starting material has been consumed. Add methanol washed acidic Amberlite to quench the reaction and stirr for 30 minutes. The Amberlite is removed by filtration, the filtrate neutralized with NHUOac (200 mg) and concentrated in vacuo to an oil. The oil is applied to a flash silica column (2 x 12 cm) eluting with 10% MeOH/CH2Cl2 to yield the deacetylated disaccharide (3 mg, 0.0δ0 mmol, 72%o).
To a 25 mL round bottom flask containing the disaccharide azide (36 mg, 0.076 mmol) is added THF (1.2 mL) and H2O (O.δ mL). Triphenylphosphine (161 mg, 0.61 mmol) is added to the slightly turbid solution. The reaction is brought to 60° C and stirred for 4.5 hours, at which point all starting material has been consumed yielding product and unwanted oxazolidinone. Remove THF in vacuo and partition between H2O and Et2O (5 mL). Extract aqueous layer thrice with Et2O (5 mL). Collect aqueous layer, freeze, and lyophillize to yield a beige residue. Apply to a C-lδ reverse phase flash column (1 x 9 cm) eluting in a stepwise gradient with 10%-50% MeCN/H2O (+0.1%) AcOH). Concentrate in vαcuo fractuions eluting in 20-50%, freeze, and lyophillize to yield 3 as a glassy residue (19 mg, 0.046 mmol, 56%).
1H-NMR (500 MHz, CD3OD): δ 5.95-5.δ9 (m, IH), 5.30 (broad s, IH), 5.2δ (d, J= 12.8 Hz, IH), 5.17 (d, J = 10.38 Hz, IH), 4.48 (broad s, 2H), 4.40 (d J = 7.63 Hz, IH), 4.29 (q, J =
6.41 Hz, IH), 4.02-3.99 (m, IH), 3.95-3.90 (m, IH), 3.66-3.63 (m, IH), 3.50-3.40 (m, 3H),
3.34-3.23 (m, 3H), 3.20-3.17 (m, IH), 3.08-3.06 (m, IH), 2.06 (d, J = 13.74 Hz, IH), 1.87
(dd, J= 13.89, 4.5δ Hz, IH), 1.60 (s, 3H), 1.20 (d, J = 6.41 Hz, 3H). "c-NMR (125 MHz, CD3OD): δ 134.72, 117.52, 103.77, 99.10, 79.12, 78.58, 78.05, 73.35, 71.76, 68.14, 66.04, 65.33, 62.70, 54.62, 41.45, 36.06, 24.14, 17.83. ESI-MS: 923 (M+472, 20%), 901 (M+450,
33%), 659 (M+208, 13%), 591 (M+140, 20%), 540 (M+89, 57%), 473 (M+22, Na+, 63%), 451 (M, 57%); 228 (M-223, 100%).
Disaccharide-rhodamine B conjugate (4)
To a flame dried, argon purged 10 mL tear drop flask is added disaccharide 3 (11 mg, 0.023 mmol). The disaccharide is azeotroped thrice from toluene. The disaccharide is then dissolved in dry DMF (1 mL) and chilled to 0° C under argon. Rhodamine B (11 mg, 0.023 mmol), HOBT (8.4 mg, 0.053 mmol), and TBTU (17 mg, 0.053 mmol) is added, and the reaction is stirred for 1 hour at 0° C. N-methylmoφholine (13 μL) is then added, and the reaction is allowed to warm to room temperature. After stirring for 46 hours, the reaction is concentrated in vacuo to a puφle residue. The residue is first applied to a LH-20 sephadex gel filtration column (2 x 18 cm) and eluted with MeOH to remove some of the unreacted Rhodamine B. After concentration in vacuo, the crude residue is applied to a silica flash column (1.5 x 14 cm) and eluted with 6.5-8% MeOH/CH2Cl2.to yield 4 as a puφle residue (6.5 mg, 0.0074 mmol, 32%).
1H-ΝMR (500 MHz, CDCl3):δ 7.87 (t, J= 4.12 Hz, IH), 7.44 (t, J= 3.66 Hz, 2H), 7.10-7.09 (m, IH), 6.40-6.37 (m, 4H), 6.26 (dd, J= 8.85, 1.83 Hz, IH), 5.93-5.88 (m, IH), 5.29 (dd, J=
17.25, 0.92 Hz, IH), 5.23 (d, J = 4.27 Hz, IH), 5.19 (d, J = 10.38 Hz, IH), 4.50 (broad s, 2H), 4.17 (q, J= 6.72 Hz, IH), 4.09 (d J = 7.63 Hz, IH), .3.80-3.73 (m, 4H), 3.52-3.15 (m, 16H), 2.23 (d, J= 14.65 Hz, IH), 1.71 (dd, J= 14.20, 4.58 Hz, IH), 1.33 (s, 3H), 1.16 (t, J= 7.02 Hz, 12H), 1.0 (d, J = 6.41 Hz, 3H). ESI-MS: 875 (M, 100%), 648 (M-227, 13%); 511 (M-364, 58%).
Chlorobiphenyl-disaccharide-Rodamine B conjugate (5)
To a 25 mL round bottom flask is added disaccharide conjugate 4 (36 mg, 0.04 mmol), dry DMF (1.0 mL), and glacial AcOH (1.0 mL). The clear solution is then degassed under vacuum for 8 minutes. PdCl2(PPh3)2 (11 mg, 0.015 mmol) is added and the reaction is stirred at room temperature under argon for 10 minutes. Tributyltin hydride (0.5 mL) is added in 0.1 mL portions every 10 minutes and then stirred for an additional 20 minutes. The reaction is diluted with H2O (1.0 mL) and stored overnight in the refrigerator to promote tin salt precipitation. The suspension is filtered through a 0.2 μ Nylon syinge filter and purified by C18 reverse phase HPLC (Phenomenex Luna C-18 (2) lOμ semi-preparative column; λ = 2δ5 nm; gradient: 0% MeCN/H2O (+0.1% HOAc) for 10 minutes, then 0 to 60% MeCN/H2O
(+0.1% HOAc) over 30 minutes, 7.5 mL/min; product elutes after 36 minutes). The collected fractions are concentrated in vacuo to yield the free amine conjugate as a puφle glass (19 mg, 0.024 mmol, 60%).
To a flame dried, argon purged 10 mL tear drop flask is added the amino conjugate (10 mg,
0.011 mmol), dry DMF (0.75 mL), diisopropylethylamine (14 μL, 0.066 mmol), and 4'- chlorobiphenyl aldehyde [J. Heterocyclic Chem. (1985) 22:δ73] (110 μL of a 100 rnM stock solution in DMF, 0.011 mmol). The reaction was warmed to 65° C and stirred for 2.75 hours under argon. NaCNBH3 (70 μL of a IM solution in THF, 0.07 mmol) is added to the red solution, and the reaction is stirred for 11 hours at 65° C under argon. The reaction becomes a dark yellow color. The reaction is cooled to room temperature and filtered through a 0.2 μ Nylon syringe filter. The crude product is purified by Clδ reverse phase HPLC (Phenomenex Luna C-lδ (2) lOμ semi-preparative column; λ = 2δ5 nm; gradient: 0% MeCN/H2O (+0.1% HOAc) for 10 minutes, then 0 to 85% MeCN/H2O (+0.1% HOAc) over 40 minutes, 7.5 mL/min; product elutes after 41 minutes). The MeCN is removed in vacuo
from the collected fractions, the sample frozen and lyophillized to yield 5 (4 mg, 0.004 mmol,
37%).
1H-NMR (500 MHz, CD3OD):δ 7.δ9 (d, J= 6.72 Hz, IH), 7.73 (d, J= 7.94 Hz, 2H), 7.65 (d, J= δ.55 Hz, 2H), 7.60-7.54 (m, 4H), 7.4δ (d J= δ.55 Hz, 2H), 7.11 (d, J= 7.33 Hz, 2H), 6.4δ (s, IH), 6.43-6.37 (m, 4H), 5.34 (d, J= 4.27 Hz, IH), 4.20 (q, J= 6.41 Hz, IH), 4.02 (dd, J=
43.35, 12.21 Hz, 2H), 4.01 (d, J= 7.63 Hz, IH), 3.76 (d, J = 10.08 Hz, IH), 3.68-3.64 (m, IH), 3.46-3.25 (m, 17H), 3.12-3.00 (m, IH), 1.95 (s, AcOH), 1.84 (d, J= 13.43 Hz, IH), 1.48
(s, 3H), 1.3 (broad s, IH), 1.18 (q, J= 7.02, Hz, 10H), 1.11 (d, J= 6.41 Hz, 3H). 13C-NMR (125 MHz, CD3OD): δ 170.15, 155.02, 150.66, 141.98, 140.30, 135.07, 134.43, 132.24, 131.72, 130.25, 129.84, 129.65, 128.66, 125.34, 123.73, 109.91, 106.28, 105.39, 103.20,
99.17, 98.11, 79.47, 77.89, 71.44, 70.63, 67.25, 67.16, 64.96, 62.53, 45.59, 44.66, 40.79, 21.07, 17.42, 13.09. ESI-MS: 991 (M, 15%), 496 (M-495, 100%).
EXAMPLE 28: MIC Values for Rhodamine Conjugates.
The results of assays to determine MIC values for selected rhodamine conjugates are presented in Figure 10, which demonstrated their potential as antibacterial agents.
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.
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