AU717099B2 - Carbopeptoids and carbonucleotoids - Google Patents

Carbopeptoids and carbonucleotoids Download PDF

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AU717099B2
AU717099B2 AU51882/96A AU5188296A AU717099B2 AU 717099 B2 AU717099 B2 AU 717099B2 AU 51882/96 A AU51882/96 A AU 51882/96A AU 5188296 A AU5188296 A AU 5188296A AU 717099 B2 AU717099 B2 AU 717099B2
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carbon
carbohydrate
anomeric
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equivalents
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Kyriacos C Nicolaou
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Scripps Research Institute
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    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D407/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen atoms as the only ring hetero atoms, not provided for by group C07D405/00
    • C07D407/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen atoms as the only ring hetero atoms, not provided for by group C07D405/00 containing two hetero rings
    • C07D407/04—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen atoms as the only ring hetero atoms, not provided for by group C07D405/00 containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D309/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings
    • C07D309/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings having no double bonds between ring members or between ring members and non-ring members
    • C07D309/08—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings having no double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D309/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings
    • C07D309/16—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member
    • C07D309/20—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member with hydrogen atoms and substituted hydrocarbon radicals directly attached to ring carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D309/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings
    • C07D309/16—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member
    • C07D309/20—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member with hydrogen atoms and substituted hydrocarbon radicals directly attached to ring carbon atoms
    • C07D309/22—Radicals substituted by oxygen atoms
    • C07D309/24—Methylol radicals
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    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/655—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having oxygen atoms, with or without sulfur, selenium, or tellurium atoms, as the only ring hetero atoms
    • C07F9/6552—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having oxygen atoms, with or without sulfur, selenium, or tellurium atoms, as the only ring hetero atoms the oxygen atom being part of a six-membered ring
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    • C40B40/00—Libraries per se, e.g. arrays, mixtures
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    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
    • C40B50/14—Solid phase synthesis, i.e. wherein one or more library building blocks are bound to a solid support during library creation; Particular methods of cleavage from the solid support
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B2200/00—Indexing scheme relating to specific properties of organic compounds
    • C07B2200/11—Compounds covalently bound to a solid support

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Description

WO 96/27379 PCT/US96/03227 1 CARBOPEPTOIDS AND CARBONUCLEOTOIDS Specification Field of the Invention: The invention relates to oligosaccharides and libraries incorporating oligosaccharide. More particularly, the invention relates to oligosaccharides and libraries of oligosaccharides which employ amide and/or phosphodiester linkages for joining adjacent carbohydrate subunits.
Background: Carbohydrates are known to mediate many cellular recognition processes. Carbohydrates can serve directly as binding molecules and, in such instances, are essential to the recognition process. A review of the biological role of carbohydrates with respect to cellular recognition phenomena is provided by Sharon et al.
(Scientific American, January 1993, 82). The emerging importance of glycobiology is further characterized by Mekelburger et al. (Angew. Chem. Int. Ed. Engi. 1992, 31, 1571) and by Dagani et al. (Chem. Eng. News, February 1, 1993, 28).
Dysfunctional mediation of cellular recognition processes can lead to disease states. If a cellular recognition process is mediated by an oligosaccharide, then an absence or excess of such oligosaccharide can lead to a dysfunctional mediation of such process. The SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCTIUS96/03227 2 mediating oligosaccharide may be deficient or absent due to a deficiency of production or due to a high rate of catabolism. If rate of catabolism is excessive, then catabolically resistant analogs of the bioactive oligosaccharide may be preferred as drug candidates as compared to the native bioactive oligosaccharide.
Accordingly, what is needed is a library which includes analogs of known bioactive oligosaccharides.
Such a library may be usefully employed for screening drug candidates.
Central requirements for the design of libraries of oligosaccharide analogs include the following: A need to maximize the potential of the designed oligosaccharides as ligand and drug candidates; A need to capitalize on existing highly sophisticated technology directed to the synthesis of oligopeptides and oligonucleotides in order to facilitate the rapid and efficient design and construction of oligosaccharides; and A need for flexibility with respect to synthesizing either single target molecules or large libraries of target molecules simultaneously.
Methodologies for synthesizing biopolymers are well developed for peptides, nucleic acids, and saccharides.
Segments of oligopeptides and of oligonucleotides can now be routinely synthesized both in solution and in the solid phase, manually and/or on automated systems. The synthesis of such structures is facilitated by the SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCT/US96/03227 3 availability of efficient techniques and sophisticated instrumentation for synthesizing peptide and phosphate bonds with high yields. The synthesis of oligopeptides and oligonucleotides is also facilitated by the absence of stereocenters in these linkages. In contrast, technology for the construction of oligosaccharides is comparatively less sophisticated and efficient.
Synthetic methods for constructing oligosaccharides give comparatively lower yields and are complicated by the two isomer possibilities (a and 9) in glycoside bond formation.
Techniques and chemical methods for simultaneously synthesizing multiple oligopeptides, e.g. 100-150 completely different peptides having lengths of up to amino acid residues, are reviewed by Jung, G. et al.
(Angew. Chem, Int. Ed. Engl. 1992, 31, 367-383 incorporated therein by reference). Such techniques facilitate the construction of oligopeptide libraries.
Simon, et al. (Proc, Natl. Acad. Sci. USA, 1992, 89, 9367-9371) disclose oligopeptide analogs in which amino acid side chain groups are attached not to conventional peptide backbone carbons but to peptide backbone nitrogens. Such analogs are termed peptoids. Simon also discloses the construction of peptoid libraries as a modular approach to drug discovery. Simon's oligopeptoids are shown by calculation to have greater conformational freedom as compared to conventional oligopeptides. Accordingly, oligopeptoids are thought to have greater potential as pharmaceutically useful binding SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCT/US96/03227 4 ligands as compared to conventional oligopeptides having close sequence homology to such oligopeptoids.
Von Roedern et al. disclose a carbohydrate amino acid (Angew. Chem, Int. Ed. Engl. 1994, 31, 687-689).
Although von Roedern discloses that carbohydrate amino acids may be coupled to peptides, he does not disclose that they may also be polymerized so as to form oligosaccharides.
Summary: A first aspect of the invention involves the molecular design and chemical synthesis of a class of carbohydrates designated as carbopeptoids (CPD's).
Glycopeptoids are preferred carbopeptoids. Carbopeptoids and glcopeptoids are oligosaccharides which employ peptide-like amide bonds for linking the various carbohydrate subunits within an oligomer assembly. Amide bond formation may be achieved by employing oligopeptide synthesis technology and instrumentation. The method allows for the design and synthesis of specific compounds for biological and pharmacological investigations. The method also allows for the generation of libraries of compounds for biological and pharmacological screening.
Conventional screening techniques employed with respect to peptide and peptoid libraries (Simon et al., supra) may also be employed with respect to carbopeptoid libraries. The design takes advantage of the multifunctionality of carbohydrate subunits to maximize SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCT/US96/03227 5 the binding properties of the molecules. The ease and high efficiency by which the peptide-like linkages can be constructed make the synthesis of these molecules a practical proposition. Furthermore, non-carbohydrate units may be inserted into the sequence making this approach even more flexible and versatile for the generation of new libraries of organic compounds.
More particularly, the invention is directed to a oligomeric carbopeptoid or glycopeptoid compound having carbohydrate amino acid subunits (CA's) or glycoside amino acid subunits (GA's) coupled to one another via an amide linkage. The amide linkage may be represented by the formula CA!-(CO-NH)-CA 2 The amide linkage (CO-NH) includes a carbonyl carbon and an amido nitrogen. A first carbohydrate amino acid subunit CA, or glycoside amino acid subunit GA 1 has an anomeric carbon bonded to the carbonyl carbon of the amide linkage. The anomeric carbon of the first carbohydrate amino acid subunit CA 1 forms a C-glycosidic bond with the carbonyl carbon of the amide linkage and maintains the carbohydrate in a closed ring configuration. A second carbohydrate amino acid subunit CA 2 has a non-anomeric carbon bonded to the amido nitrogen of the amide linkage. The second carbohydrate amino acid subunit CA 2 like the first amino acid subunit
CA
1 may include an anomeric carbon bonded to the carbonyl carbon of a second amide linkage linking the second carbohydrate amino acid subunit CA 2 to a third carbohydrate amino acid subunit CA3, etc. In this instance, the anomeric carbon of the second SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCT/US96/03227 6 carbohydrate amino acid subunit CA 2 forms a C-glycosidic bond with the carbonyl carbon of the amide linkage and maintains the carbohydrate in a closed ring configuration. On the other hand, if the second carbohydrate amino acid subunit CA 2 is a terminal subunit, then its anomeric carbon may form a hemiacetal, a hemiketal, or a glycoside.
The invention is also directed to a process for synthesizing the above oligomeric carbopeptoid or glycopeptoid compound. The synthetic process involves the coupling of two or more carbohydrate amino acid subunits (CA's) or glycoside amino acid subunits (GA's) to one another by means of amide linkages.
The invention is also directed to libraries of oligomeric carbopeptoid or glycopeptoid compounds. Such libraries are employable for drug screening. Each oligomeric carbopeptoid or glydopeptoid compound includes at least two carbohydrate amino acid subunits (CA's) or glycoside amino acid subunits (GA's) coupled to one another via an amide linkage as indicated above. The invention is also directed to an improved process for synthesizing the above library of oligomers. The process employs an elongation step for coupling the subunits to one another to produce the oligomers. In the elongation step, two carbohydrate amino acid subunits (CA's) or glycoside amino acid subunits (GA's) are coupled to one another via an amide linkage as indicated above.
The invention is also directed to chemical intermediates for producing oligomeric carbopeptoids. A SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCT/US96/03227 7 first chemical intermediate is a derived carbohydrate amino acid having an anomeric carbon and non-anomeric carbons. The anomeric carbon is substituted with a carboxyl radical. Each of the non-anomeric carbons is substituted with a radical selected from the group consisting of blocked hydroxyl, blocked amino, differentially protected amino, and hydrogen, with the proviso that at least one radical is a differentially protected amino. A second chemical intermediate is a derived carbohydrate amino acid similar to the first except that the non-anomeric carbons are substituted with a radical selected from the group consisting of blocked hydroxyl, blocked amino, unprotected amino, and hydrogen, with the proviso that at least one radical is an unprotected amino and at least one radical is a blocked hydroxyl or amino.
A second aspect of the invention involves the molecular design and chemical synthesis of a class of carbohydrates designated as carbonucleotoids (CND's) Carbonucleotoids are oligosaccharides which employ oligonucleotide-like phosphate bonds for linking the various carbohydrate subunits within an oligomer assembly. Phosphate bond formation may be achieved by employing technology and instrumentation developed for oligonucleotide synthesis. The phosphate bonds employed within carbonucleotoids are convenient linkages for coupling these units. The ease and high efficiency by which the oligonucleotide-like linkages can be constructed make the synthesis of these molecules a SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCT/US96/03227 8practical proposition.
The disclosed methods are characterized by their versatility and practicality. The methods may exploit conventional solid phase and automated synthesis techniques for producing carbopeptoids and carbonucleotoids in large scale.
More particularly, the second aspect of the invention is directed to an oligomeric carbonucleotoid molecule comprising carbohydrate C-glycoside subunits (CG's) coupled to one another via a phosphodiester linkage. The phosphodiester linkage may be represented by the structure: CGi-Cl'-(O-PO(OH)-O)-CG 2 The first carbohydrate C-glycoside subunit (CGI-C 1 has an anomeric carbon forming a C-glycosidic bond with a carbon Ci'. In turn the carbon Ci' is bonded to the phosphodiester linkage. The second carbohydrate C-glycoside subunit CG 2 has a non-anomeric carbon bonded to the phosphodiester linkage. The invention is also directed a process for synthesizing the oligomeric carbonucleotoid molecule.
The process employs a coupling step wherein two or more carbohydrate C-glycoside subunits (CG's) are coupled by means of a phosphodiester linkage as indicated above.
The second aspect of the invention is also directed to libraries of oligomeric carbonucleotoid molecules.
The libraries are employable for drug screening. Each oligomeric carbonucleotoid molecule including at least two carbohydrate C-glycoside subunits (CG's) coupled to one another by means of a phosphodiester linkage as indicated above. The invention is also directed to an SUBSTITUTE SHEET (RULE 26) 9 improved process for synthesizing a library of oligomers.
The process employs an elongation step wherein subunits ard coupled to one another to produce the oligomers. The improvement is directed to the use of phosphodiester linkage linkages for linking the C-glycoside subunits as indicated above.
The second aspect of the invention is also directed "to derived carbohydrate C-glycosides having an.anomeric carbon and non-anomeric carbons. The anomeric carbon S 10 forms a C-glycosidic bond with carbon C 1 In turn, the carbon Ci' is bonded to an phosphoramidite. Each of the non-anomeric carbons is substituted with a radical selected from the group consisting of blocked hydroxyl, differentially protected hydroxyl, and hydrogen, with the 15 proviso that at least one radical is a differentially protected hydroxyl. An alternative derived carbohydrate :'*C-glycoside is similar to the above except that each of the non-anomeric carbons is substituted with a radical selected from the group consisting of blocked hydroxyl, unprotected hydroxyl, and hydrogen, with the proviso that at least one radical is an unprotected hydroxyl and at least one radical is a blocked hydroxyl.
In the claims which follow and in the preceding summary of the invention, except where the context requires otherwise due to express language or necessary implication, the word "comprising" is used in the sense of "including", i.e. the features specified may be associated with further features in various embodiments of the invention.
WO 96/27379 WO 9627379PCTIUS96/03227 10 CHL:H Li -H 9H -N 0 N oN
CA
1
CA
2 C, A 3
-CA
4 [1 I carbopeoptoid (CPD)
H
2 N- COOH H 2 N COOH C A 1
CA
H
2 N o COO-I H 2 N o-COOH
CA
3
CA
4 :0 It:G 1 11: IG H H H [1I I carbonucleotoid (CND) HO,, OH HO-,O ,-OH HO-, O CG C G CG_ HO- o ,-OH
CG
Scheme 1. Designed carbopeptoids (CPD's) and carbon ucleotoids (CND's) SUBSTITUTE SHEET (RULE 26) 11 Detailed Description
.EXAMPLES
Retrosynthetic schemes for carbopeptoids (compound I) and carbonucleotoids (compound II) are illustrated in Scheme 1.
The carbopeptoids (CPD's) are oligomers having repeating carbohydrate subunits linked to one another by means of amide linkage units. More particularly, the carbonyl carbon of each amide linkage unit is bonded to the anomeric carbon of a carbohydrate subunit.
Similarly, the amide nitrogen of the amide linkage unit is bonded to a non-anomeric carbon. The retrosynthetic scheme suggests that the amide bond may be split and that the preferred starting materials are carbohydrate amino acids.
Carbonucleotoids (CND's) are oligosaccharides in which carbohydrate C-glycoside subunits (CG's) are linked to one another by means of phosphodiester bonds. More particularly, the retrosynthetic scheme suggests that the phosphate group may be eliminated, yielding hydroxylated starting material.
Scheme 2 illustrates representative carbohydrate amino acid subunits (CA's) and carbohydrate C-glycoside subunits Preferred carbohydrate amino acid subunits (CA's) include the following: D-glucose having an unprotected carboxyl at the anomeric C(1) position, an unprotected amino group at the C(6) position, and blocked hydroxyls at the and C(4) positions; D-mannose having an unprotected carboxyl at the
TC
/NT 0< WO 96/27379 PCT/US96/03227 12 anomeric C(1) position, an unprotected amino group at the C(6) position, and blocked hydroxyls at the and C(4) positions; D-galactose having an unprotected carboxyl at the anomeric C(1) position, an unprotected amino group at the C(6) position, and blocked hydroxyls at the and C(4) positions; SUBSTITUTE SHEET (RULE 26) WO 96/27379 WO 9627379PCrIUS96IO3227 13 0 GCOH
H
2 N TE~d"' '"0TES
OTES
D-glucose (a +1) 2
H
2 N"O~
COOH
TESO"'' OTES
OTES
a D- idlose HOOC,_ _O o COOH
H
2
N
TESO OTES
OTES
D-galactose (a 6 D-mannose (a 13) 4 N-acetyl-D-glucosa midne (a +1) 8 HOOC 0 0-NH 2 TESO:V 'OTES
OTES
D-glucose (a +1) 16 a D- altrose a D- gulose 12 14
OTES
D- mannose (a
H
2
N
TESO 0 D- ribose 24
OCH
2 0 P*
N
C
-"'OTES
OTES
D-glucose (a +1) 28
OCH
0- 0P
CH
2
CN
Pr 2 2
CH
2
CN
I Pr 2 HOOC 0 0N 2 HOOC 0 0-NH 2
TESOX
1 q~OTES TESOV NHAc OTES
OTES
D- galactose (a 1)N-acetyl- D-glucosan-dne(a+3) 2 ~COOH H2 COOH TES TESO OTES D- arabinose 26
OCH
2
CH
2
CN
0P HO jr10 N'Pr 2 TESO OTES
OTES
D-galactose (ax 13) 32 N-acetyl-D-glucosamiie (a 13) D-mannose (ax P Scheme 2. Structures of carbohydrate amainoacids (CA's) and C-glycosides (CG's) SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCT/US96/03227 14 N-acetyl-D-glucosamine having an unprotected carboxyl at the anomeric C(1) position, an unprotected amino group at.the C(6) position, a blocked amino group at the C(2) position, and blocked hydroxyls at the C(3) and C(4) positions; a-D-idose having an unprotected carboxyl at the anomeric C(1) position, an unprotected amino group at the C(6) position, and blocked hydroxyls at the and C(4) positions; a-D-altrose having an unprotected carboxyl at the anomeric C(1) position, an unprotected amino group at the C(6) position, and blocked hydroxyls at the and C(4) positions; a-D-gulose having an unprotected carboxyl at the anomeric C(1) position, an unprotected amino group at the C(6) position, and blocked hydroxyls at the and C(4) positions; a-D-glucose having an unprotected O-glycosidic amino at the anomeric C(l) position, an unprotected carboxyl as the C(6) position, and blocked hydroxyls at the and C(4) positions; D-mannose having an unprotected O-glycosidic amino at the anomeric C(1) position, an unprotected carboxyl as the C(6) position, and blocked hydroxyls at the and C(4) positions; D-galactose having an unprotected O-glycosidic amino at the anomeric C(1) position, an unprotected carboxyl as the C(6) position, and blocked hydroxyls at the and C(4) positions; SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCT/US96/03227 15 N-acetyl-D-glucosamine having an unprotected Oglycosidic amino at the anomeric C(1) position, an unprotected carboxyl as the C(6) position, a blocked amino group at the C(2) position and blocked hydroxyls at the C(3) and C(4) positions; D-ribose having an unprotected carboxyl at the anomeric C(1) position, an unprotected amino group at the C(5) position, and blocked hydroxyls at the C(2) and C(3) positions; and D-arabinose having an unprotected carboxyl at the anomeric C(1) position, an unprotected amino group at the C(5) position, and blocked hydroxyls at the C(2) and C(3) positions.
Preferred carbohydrate amino acid subunits (CA's) include the following: D-glucose having a C(1) C 1 glycosidic carbon bonded to a phosphoramidite, an unprotected hydroxyl at the C(6) position and blocked hydroxyls at the and C(4) positions; D-mannose having a C(1) Ci.-glycosidic carbon bonded to a phosphoramidite, an unprotected hydroxyl at the C(6) position and blocked hydroxyls at the and C(4) positions; D-galactose having a C(1) C 1 glycosidic carbon bonded to a phosphoramidite, an unprotected hydroxyl at the C(6) position and blocked hydroxyls at the and C(4) positions; and N-acetyl-D-glucosamine having a C(1) Cl.-glycosidic SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCT/US96/03227 16 carbon bonded to a phosphoramidite, an unprotected hydroxyl at the C(6) position, a blocked amino at the C(2) position, and blocked hydroxyls at the C(3) and C(4) positions.
Scheme 3 outlines a preferred synthesis of suitably protected carbohydrate amino acid subunits (CA's) from Dglucose, i.e. compound 46.
SUBSTITUTE SHEET (RULE 26) WO 96/27379 WO 9627379PCTIUS96/03227 17 'OOEt 1. TMSCN, SnCL 4 2. EtCH, H 2 S0 4 (cat) (37-38) HO "0 CAc 36 1. PivCl., DIMAP pyridine 2. Et 3 SiOTf, 1 PrEtN, CH,C1 2 1. NaQEt, EtCH 2. DPPA, DIAD, Ph 3 l
THF
(41-42)
OTES
121
H
2 Pd on C
H
2
N
TESO"
1. NaOH, p-dioxane 2. FMOC-Cl, NaHCO 3
H-,O
H 0COOH M OON "O
E
TESO"
"TES
OTES
44 Scheme 3. SNynthesis of glucose-derived carbohydrate amino acid 46 SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCT/US96/03227 18 Scheme 4 outlines the synthesis of suitably protected carbohydrate amino acid subunits (CA's) from Nacetyl-D-glucosamine, i.e. compound 62.
SUBSTITUTE SHEET (RULE 26) WO 96/27379 WO 9627379PCT1US96/03227 19o QAc HO0 COODt 1. TMSCN, SnC1 4
HO
"NP hth 2. EtOH, H 2 S0 4 (cat) HO"' NHAc 3. Ac 2 O, MeOH OAc OH 54 48 1.PiCl, DMAP pyridine 2. EtSiOTf, 'PrEtN, CH 2 Cl 2 O COOD io0tO~ 1. NaQEt, EtOH Pv O~ "NHAc 2. DPPA, DIAD, Ph~ TESO"' N HAc
TFF
OTES (57-58) OTES 58 56 I1. H 2 Pd on C H 0
COOH
o COODt FMOCN 1. NaCH, p-dioxane E ON c "NHAc 2. FMOC-Cl, NaHCO 3
H
2 0
TESH~
OTES (61-62) OE 62 Scheme 4. Synthesis of N-acet-ylgIlucosamine-derived amino acid 62 SUBSTITUTE SHEET (RULE 26) WO 96127379 WO 9627379PCT/US96/03227 20 Scheme 5 suimmarizes the synthesis of hexamer 74, i. e glucose-glucosanine hetero carbopeptoid
(CPD).
SUBSTITUTE SHEET (RULE 26) EMOON -H 2 N COOEt
H
VOTES "NHAc 0 TESO
TESOV
Scheme 5a. Synthesis of mixed carbopeptoid 3 (hexamer) OTES 46 OTES 1 DCC, HOBT, Et 3 N, DMF 2.piperidine, DMF 0 0UOH 0 CO FMOCN 'jH 2 N N "OR "NHAc TESde NHAc RO"O Rd(JV'' COTES 0OR OR C: 62 64 m 1. DCC, HOBT, Et3N, DMF; LA 2. piperidine, DMF; 66 m 0 1 M 0 H 0 COEI 1_ N .'RN "O R R "NHAC RO' W&"NHAc ROdO' O OR OR 66 I1. 1, DCC, H-OBT, Et3N, DMF; 67 2. piperidine, DMF; 68 0 I o 0 0 COOEI 0
H
2 N NN N- )00 Scheme 5b. Synthesis of mixed carbopeptoid 3 (hexamer) 1. 2, DCC, UHOBT, Et 3 N, DMF; 69 2. pip eridirie, DMF; C/3
CO,
C:,
r- P1 1 1, DCC, HOBT, Et 3 N ,DMF; 71 2. piperidiine, DMF; 72 WO 96/27379 PCT/US96/03227 23 Scheme 6 illustrates the construction of suitably protected and activated C-glycoside subunits (CG's) corresponding to glucose.
SUBSTITUTE SHEET (RULE 26) WO 96/27379 WO 9627379PCT1US96/03227 24 1. TMSCN, SnC1 4 2. EtCH, H 2 S0 4 (cat) 1. dimethoxytrityl chloride, DMAP, 2. Et 3 SiOTf, 'Pr 2 EtN, CH 2 C1 2 'OH 'Bu,AIH, CH 2 C1 2
OTES
OCH
2
CH
2
CN
I
C1 P N'Pr 2 1 Pr.EtN, CH-,C1 2 DMTOX\V' 0 1.E 3 Slid.
DMF
2. C1 3 CCOOH, 2C3 2CH NO 2
THF
OCH
2
CH
2
UCN
-N'Pr 2
DMTO'
"OTES
OTES
82
OTES
Scheme 6. Synthesis of glucose-derived C-glycoside (CG) SUBSTITUTE SHEET (RULE 26) WO 96127379 WO 9627379PCr1US96/03227 25 Scheme 7 illustrates the construction of suitably protected and activated C-glycoside subunits (CG's) corresponding to glucosanine.
SUBSTITUTE SHEET (RULE 26) WO 96/27379 WO 9627379PCTfUS96/03227 26 I. TMSCN, SnCI 4 2. EtOH, I- 2 S0 4 (Cat)
I.AC
2 O, MeOH OAc 48 HO COQEt HO" NHAc
OH
86 j1. dimethoxytrityi chloride, DMAP, DMF 2.Et 3 SiOTf, 'Pr 2 EtN, CH 2 C1 2 H 'BuA1H,
CH
2 C1 2
OCH
2
CH
2
CN
I
C! N' Pr 2 1. Et 3 SiCl, imid., DMF 2. C1 3 CCOOH, CH 3
NO
2
THF
PrI 2 t'N, Iri 2 1 OCH 2
CH
2
CN
DMTOr N'Pr 2 TESO%" "jNHAc
OTES
DMTO'
"NHAc 92 94 Scheme 7. Svnthesis of N-acetylglucosamine-derived C-glvcoside (CG) Scheme 8 sunmar 4zes the synthesis of hexainer 116, i.e.
glucose-alucosaine hetero carboriucleotoid
(CND).
SUB13STITUTE S HE ET (R ULE 26)
OCE
0 PF1 0 DMTO 0 N Pr, Flo OTES 0 TESO' "OTES TESO '-4wH Scheme 8a. Synthesis of mixed carbonticleotoid 6 (hexamer) OTES 82 OTES 94' 1 tetrazole, CH1 3
CN
2.12, 2,6-ltidine, TF 3. CI 3 CCOOI-I, CF1 3 NO,, THF tA
OCE
C0 0 0 II0 IM DM TO 0~ "NPr 2 HO o-p-O OR
CC
HOTES OR OR 9 m 92 9 LA 1. tetrazole, C[1 3
CN
x 2.1-2'2,6-Itiiine, TI-IF' t m 3. Cl 3 CCOOI-I, C1-1 3 N0 2
TI-IF
0q 0 0 HO O-P-O O-P-O OR c I I "NHcOE OC ."NH, OR OR 102 1 4,'tetrazole, CI-1 3
CN
2.12, 2,6-lutidine, TI-IF 3. CI 3 CCOOH-, CH 3
,NO
2
TI-IF
0 0 0 0 0 0 0C 0II 0 II11 0 r OCE-.,,OCE OCE- Scheme 8b. Synthesis of mixed carbonucleotoid 6 (hexamer)3 0 0 0 O 01-0- 0 -11- 0 HO- OpO Op-OO I I I OCE OCE *uO OE"N" OR OR OR OR 106 1. 5, tetrazole, CH 3
CN
LA 2. 12, 2,6-lutidine, TI1IF C 3. CICCOOI 1, CII;:NO,, TI IF II0 0 0 0 I 1I111 0 c "N U CE uu OC CE u OE"Nt m OR OR OR OR OR LA 1. 4, tetrazole, CH 3 CN 110 x 2.1,,2,6-lutidine, THF K) m 3. CI 3 CCOOH, Ci1 3 N0 2 TlI- 0 0I 0 0I 00I0II0I CI I I I I "OO 11R OCE O 'VCE RO OC OC C t*
*~H
OR R OR 114
ORO
0 0 0 0 0 OO II It 0 I0I HO op-o -p-OO-P- o-pO O I1 I IO O U,'HH V0HH OH Ac, OH UriO OHc. OH UrOHi.
HO' OH0- HO' HO OHO OH OH Oil1 OH OHl Oil 116-.
WO 96/27379 WO 9627379PCr/US96/03227 29 The chemistries illustrated in Schemes 5 and 8 for synthesizing heterohexainer CPD 74 and heterohexamer cND 116 can also be employed for synthesizing homohexainer CPD's 118 (glucose) and 120 (glucosamine) and homohexazner CND's 122 (glucose) and 124 (glucosamine).
SUBSTITUTE SHEET (RULE 26) In
C
w In
-I
-I
m
U,
I
m m -4
C
I-
m
NJ
0~ WO 96/27379 PCT/US96/03227 31 In analogy with the construction of oligopeptide and oligonucleotide libraries, a oligosaccharide carbopeptoid (CPD) library may be constructed by performing using a split synthesis method of oligomerization as illustrated in Scheme 500 for carbopeptoids and Scheme 550 for carbonucleotoids. For example, the split synthesis may employ beads upon which to build the oligomers. Beads are aliquoted into each of a several reaction vessels, each reacrtion vessel containing-a-different core molecule. The core molecules are-then allowed to attach to the beads. The beads are washed, mixed with one another, and then re-aliquoted (split) into a second set of reaction vessels for addition of a second core molecule to the first added core molecule. The process is then reiterated until the oligomerization process is complete. The resultant library of oligosaccharides may then be screened using conventional methods developed for oligopeptide and oligonucleotide libraries. Screening an oligosaccharide library can lead to the identification of individual oligosaccharide components within the library having binding activity and/or bioactivity.
The above oligosaccharide libraries (CPD and CND) may be enlarged by introducing additional functionalities into the basic CA's and CG's.
The above oligosaccharide libraries (CPD and CND) may be further enlarged by enlarging the pool of free functional groups on the CA's and CG's and employed this enlarged pools of CA's and CG's during the respective split synthesis processes.
SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCT/US96/03227 32 Scheme 20 illustrate a protocol published by Fuchs, E.F. et al. Chem Ber. 1975, 108, 2254) for the synthesis of CA 45 and 46 from glucose pentaacetate.
Additionally, Scheme 20 illustrates a synthetic route for CG 82, also starting from glucose pentaacetate. The reagents and conditions for synthesizing CG 82 are provided as follows: Steps according to Fuchs (supra) Step DMTC1, DMAP, Pyridine; room temperature.
TESTfl; 0°C.
Step DIBAL-H, CH 2 C1 2 -78 0 C; and Step (NCCH 2
CH
2 (NiPr 2 )PC1, tetrazole,
CH
2 C1 2 The reagents and conditions for synthesizing CA 46 from CA 45 are provided in Step M as follows: Step FMOC-C1, K 2 C0 3 THF, H 2 0; 0°C.
SUBSTITUTE SHEET (RULE 26) WO 96/27379 WO 9627379PCTIUS96/03227 33 AcO 0 OAc Ace' *"OAc OAc 36 HO 0 COOMe a -d 0O HO"
H
OH
h-1 76 DMT0 0 COOMe TESO OTES
OTES
78 g [F 45: R=H OTES U'I ZI Scheme 20. Conventional route to CAs and a variation for the synthesis of CG. Reagents and conditions: corresp. Lit.: DMTC1, DMAP, Py; RT. TESTfl; 0 0 DIBAL-H, CR 9 Cl2; -78 (g)
(NCCH
9 ,CH2)(NiPr29PCL tetrazole CH 2 ,C1 9 (mn) FIVOC-Ci, KC0 3
THF,
H
2 0; 0 C.
SUBSTITUTE SHEET (RULE 26) WO 96/27379 WO 96/7379PUS9SIO3227 34 A synthetic route for producing C-glycosides (CG's) with I-configuration at the former anoiner center is illustrated in Scheme 21. The starting material (compound 36) is commercially available. The reagents and conditions for synthesizing CG 181 and CG 185 are as follows: Step Co 2
(CO)
8 HSiEt 2 Me, CO.
Step AcOH, H 2 0, THF; RuCl 3 NalO 4 CH-zCN, H 2 0, CCL 4 room temp~erature; Step NaO~e, MeOH; Step DMTC1, DMAP, Pyridine, room temperature; TESOTf; Step
BH
3
-THF;
Step :(NCCH 2
CH
2 (NiPr 2 PC1, tetrazole, Ch 2 Cl 2 Step 1 equiv TsCI. base; TESOTf; Step NaN 3 Step
H
2 Pd (OH) 2
-C;
Step EMOC-Ci, base.
SUBSTITUTE SHEET (RULE 26) WO 96/27379 WO 9627379PCTUS96O3227 35 AcO 0 QAc AcO" OAC OAc a bo
ACO*"
AcO 0 OH
ACO'
OAc 176 1
V
R =OAc or NPhth or NHAc-
OH
d,e f R OTES or NPhth or NH-Ac R OAc or NPhth or NHAc FMOCNH 0 00H 1 TESV" R
OTES
N
3 0 CO 2 H TsO 0 COH 3 h TESO" R
TESO"R
OTES
OTES
183 182 R OTES or NPhth or NHAc Scheme 21. Synthesis of C-glycosides with 1-configuration at the former anomeric center. Reagents and conditions: Co 2
(CO)
8 HSiEt 2 Me, CO. (1) AcOH, H 2 0, THF; RuC1 3 NaIO 4
CH
3 CN, H 2 0, CCd 4 RT. NaOMe, MeOH. DMTC1, DMAP, Py, RT: TESOTE. BH 3 -THF. (f)
(NCCH,,CH
3 )(NPr 9 tetrazole, CH 2 C1 2 1 equiv TsC1, base: (2) TESOTf. NaN 3 Pd(OH) 2 FMOC-C1, base.
SUBSTITUTE SHEET (RULE 26) WO 96/27379 WO 9627379PCTUS96/03227 36 BnO
O
Bno"' d "OBn OBn 190 *a-b 191-192 TsO 0 O1 TESO"
'OTES
OTES
c, d ,%%C0 2
H
"OTES
TESO"'
a, gh, I e, f DMTO 0 O TESO"
"OTES
OTES
200
FMOCNH'
NHFMOC
j, e,f, k 201-204
OTES
OTES
Scheme 22. Synthesis of C-glycosides with cc-configuration at the former anomeric center. Reagents and conditions: reductive dlebenzvlation. 1 equiv TsC1, base; TESOTf. NaN 3 RuC1 3 NaIO 4
CH
3 CN, H-O, CC1 4
H
2 Pd-C. EMOC-Ci, base. DMTC1, DMAP, Py, RT; TESOTf.
RuC1 3 NaIO 4
CH
3 CN, CC1 4
CH,N
2 DIBAL-H. PPh 3 DIAD, diphenvi phosphoryl azide (DPPIA), THE.- KMnO 4 t-BuOH, buffer.
SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCT/US96/03227 37 Synthetic routes for producing with C-glycosides with a-configurations at the former anomeric center, i.e.
CG 196 and CG 1204, are illustrated in Scheme 22. The common starting material for these synthetic routes (compound 190) is disclosed by Schmidt, R. R. et al.
(Liebigs Ann. Chem. 1987, 825). The reagents and conditions for the reactions leading to CG 196 and CG 204 are as follows: Step reductive debenzylation; Step equiv TsCl. base; Step Step Step Step Step temperature; TESOTf.
NaN 3 RuC13, Nal0 4
CH
3 CN, H 2 0, CC14.
H
2 Pd-C.
FMOC-C1, base.
DMTC1, DMAP, Pyridine, room TESOTf.
RuC13, NalO 4
CH
3 CN, H 2 0, CC1 4
CH
2
N
2
DIBAL-H.
PPh 3 DIAD, diphenyl phosphoryl azide KMnO4, t-BuOH, buffer.
Step Step Step
(DPPA),THF.
Step Reactions for the development of the galactose derived C-glycoside 138 into protected CA's and diols is illustrate in Scheme 23. The common starting material for these synthetic routes (compound 138) is disclosed by SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCrUS96O3227 -38 Petrus, L. et al. (Chem. zvesti. 1982, 36, 103). The reagents and conditions required for the synthesis of compound 209, compound 214, compound 220, and compound 224 are indicated below: Step 1.1 equivalent DMTC1, DMAP, Pyridine, 12 hour, 20 0
C;
TesOTf, CH 2 0 0 C, 1 hour, 83%.
Step LAH, ether, reflux, 2 hour; (2)FMOC-Cl, K 2 C0 3 TEF, H 2 0, 0 0 C, 1 hour, Step :10% HCOOH in CH 2 C1 2 0 0 C, 2 minutes, 100%.
Step :RuCl 3 Nalb 4
CH
3 CN, H 2 0, CCd 4 20'C, minutes, 54%.
Step Ce) 1 equiv. TsCl, base; TESOTf.
Step NaN 3 Sten oxidative NEF.
Step Pd-C, H 2 Step Ci) FNOC-C1, base.
Step Cj): 1 equiv. PivCl, base; TESOTf.
StepD (1C) oxidative Nef; C2) CH 2
N
2 Step DTBAL-H.
Step DMTC1, DMAP, Pvridine.
Step Cn) LAH-.
Step) Nef reaction Step LAH.
SUBSTITUTE SHEET (RULE 26) WO 96/27379 WO 9627379PCrIUS96/03227 39 h(H)FMOC a-d ES 0 e-1 FMOCNH 0 C0 2
H
TESO "OTES
OTES
0*' DMTO 6 r OH TESO "OTES
OTES
a, 0, p 221-224 i-n 215-220
OTES
Scheme 23. Development of the galactose derived C-glycoside 138 to protected CAs and dials. Reagents and conditions: 1.1 equiv DMTC1, DMAP, Py, 12h, 20 TesOTf, CH 2 C1 2 0 1h, LAH, ether, reflux, 2h; FMOC-C1, K 2 C0 3 THE, H 2 0, 0 0 C, 1h, 55%.o 10% HCOOH in
CH-
2 C1 2 0 OC, 2 min', 100%. RuC1 3 NaIO 4
CH
3 CN, H 2 0, CC1 4 20 0 C, min 54%.s 1 equiv TsC1, base; TESOTf. NaN 3 oxidative Nef. (h) Pd-C, H 2 FMOC-Ci, base. 1 equiv PivCl, base; TESOTf. (1) oxidative Nef; CH 2
N
2 DIBAL-H. (in) DMTC1, DMAP, Py. LAH. (o) Nef reaction. LAH SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCT/US96/03227 40 An exemplary protocol for synthesizing a hexamer carbopeptoid (CPD 234) starting from galactose derived CA 214, glucosamine derived CA 62, and glucose derived CA, using standard methods for solid phase peptide synthesis is illustrated in Scheme 24. The reagents and condition for these reactions are as follows: Step 1: DCC, HOBT, Et 3
DMF;
Step 2: Piperidine, DMF SUBSTITUTE SHEET (RULE 26) 00 o COOH 0 FMOGNH H 2 N0 TESO 'OTES TESO "OTES OTES OTES R TS214 1 216 c 2. piperidine, DMF ca0 0 FMOCNH 0 OHH 2 N "RN 0 rn RONHAc R*'OR RO "OR UOR OR OR m 226 m 621. DCC, HOIIT, Et N, DMF c~2. piperidine, DMF N 0 COOH 0 0 H 0 ImFMOCNH HN N0 Ce "OR R d' "NHAC RO OR RO 'O OR OR OR OR 228 Scheme 24a. Synthesis of a CIT, (exemplified ona lear)using standard methods for solid phase peptidlesytei Omf oI:~ Schemec 24b. Synthesis of a CPF (exemplified on a hexarner) Using standard methods for solid phase peptidle synthesis WO 96/27379 PCTIUS96/03227 -43- SYNTHETIC METHODS Preparation of 37 AcO 0O N AcO"' 'OAc AcO 37 To a solution of 3-D-Glucose pentaacetate 36 i n nitromethane from Aldrich company (.13 Molar), is added trimethylsilyl cyanide (3.0 equivalents) and then SnC14 (.02 equivalents). The mixture is stirred for one hour and then an aqueous solution of sodium acetate was added to hydrolyze the remaining trimethylsilyl cyanide. The mixture is evaporated and the remaining oil is resuspended in dichloromethane and washed with sodium acetate solution water brine (IX) and then dried over magnesium sulphate and concentrated. The crude solid is then recrystallized from methanol to yield 37 as a white solid scheme 3 step 1; scheme 9, step a.
SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCTIUS96/03227 -44- Preparation of 38 .COOEt
"OH
OH
The crude product 37 is next dissolved in ethanol (0.15 M) and then concentrated
H
2 S04 (0.01 equivalents-catalytic) is added. The reaction mixture is heated to 85 °C for eight hours.
The solution is next concentrated in vacuo and purification by flash column chromatography affords compound 38. scheme 3 step 2 Preparation of 39 ,COOEt
OH
39 To a solution of 38 (1.0 equivalents) in pyridine Molar), is added trimethylacetyl chloride (pivaloyl chloride) equivalents) at 0 The reaction is stirred for 2 hours and then diluted with diethylether and washed with ammonium SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCT/US96/03227 chloride copper sulfate brine dried over MgSO 4 and concentrated. Purification by flash column chromatography affords compound 39. scheme 3 step 1 Preparation of PivO O COOEt PivO TESO 'OTES
OTES
To a solution of 39 (1.0 equivalents) in methylene chloride Molar), is added diisopropylethylamine (3.3 equivalents) at 0 oC. Subsequent addition of triethylsilyl trifluoromethanesulfonate (3.3 equivalents) is followed by stirring for 2 hours and then the reaction is diluted with diethylether and washed with ammonium chloride brine (lX) and then dried (MgSO4) and concentrated. Purification by flash column chromatography affords compound 40. scheme 3 step 2 SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCT/US96/03227 -46- Preparation of 41 HO COOEt TESO""
'OTES
OTES
41.
To a solution of 40 in ethanol (.13 Molar), is added sodium ethoxide (0.3 equivalents) and the reaction mixture is stirred for two hours at room temperature. The solution is then concentrated in vacuo and purification by flash column chromatography affords compound 41. scheme 3 step 1 Preparation of 42
N
3 0 COOEt TESO""
OTES
OTES
42 A solution of 41 (1.0 equivalents) in tetrahydrofuran (.18 M) is treated with DPPA (diphenylphosphorylazide, equivalents), triphenylphosphine (1.3 equivalents) and DIAD (diisopropyl-azo-dicarboxylate, 1.3 equivalents). The reaction is heated to 80 °C for 3 hours and then diluted with ether (2X) and SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCTIUS96/03227 -47washed with .5 M aqueous NaOH The organic layer is dried over MgSO 4 and evaporated. Purification by flash column chromatography affords compound 42. scheme 3 step 2 Preparation of 44
H
2 N 0 COOEt TESO" 'OTES
OTES
44 A solution of 42 (1.0 equivalents) is dissolved in ethanol (.01 M total) at 25 The mixture is next exposed to 10% Pd/C equivalents) and is then subsequently capped with a hydrogen balloon at 1 atmosphere. The reaction is stirred for 72 hours and is then filtered through celite. The crude mixture is subsequently diluted with ether and washed with NaHCO 3 (3X), brine (lX) and dried (MgSO 4 and concentrated. Purification by flash column chromatography affords compound 44. scheme 3 step 1 SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCTUS96/03227 -48- Preparation of
H
2 N O
COOH
TESO"' 'OTES
OTES
A solution of_.44 (1.0 equivalents) is dissolved in pdioxanes M) and then exposed to a solution 3.0 Molar solution of sodium hydroxide (1.5 equivalents). The reaction is then stirred for 2 hours at 50 °C and is subsequently diluted with ether and washed with a solution of NH 4 C brine (IX) and dried (MgSO 4 and concentrated. Purification by flash column chromatography affords compound 45. scheme 3 step 1 Preparation of 46 To a solution of 45 (1.0 equivalents) in methylene chloride Molar), is added sodium bicarbonate (2.0 equivalents) at 0 0 C. Subsequent addition of 9-fluorenylmethyl chloroformate (FMOC-C1, 1.2 equivalents) is followed by stirring for 2 hours and then the reaction is diluted with diethylether and washed with ammonium chloride brine (lX) and then dried (M gS0 4 and concentrated. Purification by flash column SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCT/US96/03227 -49chromatography affords compound 46. scheme 3 step 2 Preparation of 48 AcO 0 OAc AcO"' NPhth OAc Procedure as described in Methods in Carbohydrate chemistry, Whistler, II, 1963, p. 327. A mixture of anhydrous D-glucosamine hydrochloride or D-galactosamine hydrochloride from Aldrich chemical company, in 200 mL.
methanol and 20g Dowex 50 acidic resin, is stirred at the boiling point in a round bottom flask. After 24-hr. reaction time, the resin is removed by filtration and ished three times with 20 ml. of methanol. The filrate and ishings are combined and concentrated to about 125 ml by rotovap. The concentrate is allowed to cool to room temperature and the product crystallizes overnight.
To a solution of free amine, in chloroform is added phthalic anhydride (1.5 equiv.) and the reaction mixture is allowed to reflux at 70 °C for 4 h. The product is then crystallized and carried onto the next step.
To a solution of triol in methylene chloride is added SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCT/US96/03227 acetic anhydride (3.5 equiv.) and triethyl amine (3.5 equiv.) and the reaction mixture is allowed to stir at 0, C for 4 h. The product 48, is then crystallized or purified by flash column chromatography and carried onto the next step.
SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCT/US96/03227 -51- Preparation of AcO C AcO'" 'NPhth AcO To a solution of N-phthalamido-D-Glucosamine tetraacetae 48 in nitromethane (.13 Molar), is added trimethylsilyl cyanide equivalents) and then SnC1 4 (.02 equivalents). The mixture is stirred for one hour and then an aqueous solution of sodium acetate was added to hydrolyze the remaining trimethylsilyl cyanide. The mixture is evaporated and the remaining oil is resuspended in dichloromethane and washed with sodium acetate solution water brine (IX) and then dried over magnesium sulphate and concentrated. The crude solid is then recrystallized from methanol to yield 50 as a white solid scheme 4 Preparation of 52 The crude product 50 is next dissolved in ethanol (0.15 M) and then concentrated H 2 S0 4 (0.01 equivalents-catalytic) is added. The reaction mixture is heated to 85 °C for eight hours.
The solution is next concentrated in vacuo and purification by SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCT/US96/03227 -52flash column chromatography affords compound 52. scheme 4 Preparation of 54 HO 0 COOEt HO" 'N HAc
OH
54 A solution of 52 (1.0 equivalents) is dissolved in methanol M total). The reaction is then charged with acetic anhydride (1.1 equivalents) and is subsequently stirred for 2 hours at The reaction is next diluted with ether and washed with NaHCO 3 brine (1X) and dried (MgSO 4 and concentrated.
Purification by flash column chromatography affords compound 54. scheme 4 Preparation of Piv COOEt PivO HO" ~'NHAc
OH
SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCT/US96/03227 -53- To a solution of 54 (1.0 equivalents) in pyridine Molar), is added trimethylacetylchloride (pivaloyl chloride) equivalents) at 0 The reaction is stirred for 2 hours and then diluted with diethylether and washed with ammonium chloride copper sulfate brine dried over MgSO 4 and concentrated. Purification by flash column chromatography affords compound 55. scheme 4 Preparation of 56 PivO 0 CO
O
Et PivO TESO"'" 'NHAc
OTES
56 To a solution of 55 (1.0 equivalents) in methylene chloride (.10 Molar), is added diisopropylethylamine (2.2 equivalents) at 0 oC. Subsequent addition of triethylsilyl trifluoromethanesulfonate (2.2 equivalents) is followed by stirring for 2 hours and then the reaction is diluted with diethylether and washed with ammonium chloride brine (lX) and then dried (MgS0 4 and concentrated. Purification by flash column chromatography affords compound 56. scheme 4 SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCT/US96/03227 -54- Preparation of 57 HO, COOEt TESO" "NHAc
OTES
57 To a solution of 56 in ethanol (.13 Molar), is added sodium ethoxide (0.3 equivalents) and the reaction mixture is stirred for two hours at room temperature. The solution is then concentrated in vacuo and purification by flash column chromatography affords compound 57. scheme 4 Preparation of 58
N
3 O COOEt TESO"' 'NHAc
OTES
58 A solution of 57 (1.0 equivalents) in tetrahydrofuran (.18 M) is treated with DPPA (diphenylphosphorylazide, equivalents), triphenylphosphine (1.3 equivalents) and DIAD (diisopropyl-azo-dicarboxylate, 1.3 equivalents). The reaction is SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCT/US96/03227 heated to 80 °C for 3 hours and then diluted with ether (2X) and washed with .5 M aqueous NaOH The organic layer is dried over MgSO 4 and evaporated. Purification by flash column chromatography affords compound 58. scheme 4 Preparation of ,COOEt
H
2
N
TESO" 'NHAc
OTES
A solution of 58 (1.0 equivalents) is dissolved in ethanol (.01 M total) at 25 The mixture is next exposed to 10% Pd/C equivalents) and is then subsequently capped with a hydrogen balloon at 1 atm. The reaction is stirred for 72 hours and is then filtered through celite. The crude mixture is subsequently diluted with ether and washed with NaHCO 3 (3X), brine (lX) and dried (MgSO4) and concentrated. Purification by flash column chromatography affords compound 60. scheme 4 SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCT/US96/03227 -56- Preparation of 61 H2N" O
COOH
TESO" 'NHAc
OTES
61 A solution of 60 (1.0 equivalents) is dissolved in pdioxanes M) and then exposed to a solution 3.0 Molar solution of sodium hydroxide (1.5 equivalents). The reaction is then stirred for 2 hours at 50 °C and is subsequently diluted with ether and washed with a solution of NH 4 C brine (IX) and dried (MgSO 4 and concentrated. Purification by flash column chromatography affords compound 61. scheme 4 Preparation of 62
H
FMOCN 0
COOH
TESO"' 'NHAc
OTES
62 To a solution of 61 (1.0 equivalents) in methylene chloride (.10 Molar), is added sodium bicarbonate (2.0 equivalents) at 0 SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCT/US96/03227 -57- 0 C. Subsequent addition of 9-fluorenylmethyl chloroformate (FMOC-C1, 1.2 equivalents) is followed by stirring for 2 hours and then the reaction is diluted with diethylether and washed with ammonium chloride brine (IX) and then dried (MgS0 4 and concentrated. Purification by flash column chromatography affords compound 62. scheme 4 Preparation of 63 0 FMOCN O N 0 COOEt H H OTSO" 'OTESTESO" ""NHAc OTES OTES 63 To a stirred solution of the acid 46 (1.0 equivalents) and the amine 60 (1.1 equivalents) in dimethylformamide Molar) at 25 is added 1-hydroxybenzotriazole (HOBT; 1.1 equivalents). Next dicyclohexylcarbodiimide (1.2 equivalents) is added and the reaction is stirred for 14 hours. The mixture is diluted with ether, filtered and the filtrate is washed with aqueous NaHCO 3 water and brine The organic phase is dried over MgS0 4 and then concentrated. Purification by flash column chromatography affords compound 63.
SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCTIUS96/03227 -58scheme 5 step 1 Preparation of 64
H
2
N
TESO"'
'''NHAc
OTES
OTES
To a stirred solution of 63 (1.0 equivalents) in dimethylformamide (.10 Molar) at 25 is added piperidine (1.1 equivalents). The reaction is stirred for 1 hour and is then diluted with ether, and washed with aqueous CuSO 4 water and brine The organic phase is dried over MgSO 4 and then concentrated. Purification by flash column chromatography affords compound 64. scheme 5 step 2 Preparation of .COOEt
FMOCN'
H
TESO'
OTES OTES OTES SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCT/US96/03227 -59- To a stirred solution of the acid 62 (1.0 equivalents) and the amine 64 (1.1 equivalents) in dimethylformamide Molar) at 25 is added 1-hydroxybenzotriazole (HOBT; 1.1 equivalents). Note: numerous iterations can be performed using the acid 62 or intermixing with other acids including for example acid 46 to form successive oligomers where n=2 to infinity (a hexamer is shown in scheme 5) to obtain large carbopeptoid libraries. Next dicyclohexylcarbodiimide (1.2 equivalents) is added and the reaction is stirred for 14 hours.
The mixture is diluted with ether, filtered and the filtrate is washed with aqueous NaHCO 3 water and brine (2X).
The organic phase is dried over MgSO4 and then concentrated.
Purification by flash column chromatography affords compound scheme 5 step 1 Preparation of 66 0
O
NH
2 0 0N O N 0 CO
N
Et H
H
TESO .NHATESO' OTESTESO" "NHAc OTES OTES OTES 66 To a stirred solution of 65 (1.0 equivalents) in SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCTIUS96/03227 dimethylformamide (.10 Molar) at 25 oC, is added piperidine (1.1 equivalents). The reaction is stirred for 1 hour and is then diluted with ether, and washed with aqueous CuSO 4 water and brine The organic phase is dried over MgSO 4 and then concentrated. Purification by flash column chromatography affords compound 66. Note: numerous iterations can be performed using variable length oligomers of 66 to form peptoid oligomers where n=2 to infinity (a hexamer is shown in scheme scheme 5 step 2 Preparation of 67 o o MOCN N 0 N 0 N O c o o H H
H
TESO"' TESSOHAc TESO" OTESTESO NHAc OTES OTES OTES
OTES
67 To a stirred solution of the acid 46 (1.0 equivalents) and the amine 66 (1.1 equivalents) in dimethylformamide Molar) at 25 is added 1-hydroxybenzotriazole (HOBT; 1.1 equivalents). Note: numerous iterations can be performed using the acid 46 or intermixing with other acids including for example acid 62, to form successive oligomers where n=2 to infinity (a hexamer is shown in scheme 5) to obtain large carbopeptoid libraries. Next dicyclohexylcarbodiimide (1.2 SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCT/US96/03227 -61equivalents) is added and the reaction is stirred for 14 hours.
The mixture is diluted with ether, filtered and the filtrate is washed with aqueous NaHCO 3 water and brine (2X).
The organic phase is dried over MgSO 4 and then concentrated.
Purification by flash column chromatography affords compound 67. scheme 5 step 1 SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCT/US96/03227 -62- Preparation of 68 o o
H
2 N N o N O CO
O
Et H
H
TESO"' OTESTESO" T NHAcTESO" O 'OTESTESO""
NC
OTES OTES OTES OTES 68 To a stirred solution of 67 (1.0 equivalents) in dimethylformamide (.10 Molar) at 25 OC, is added piperidine (1.1 equivalents). The reaction is stirred for 1 hour and is then diluted with ether, and washed with aqueous CuSO 4 water and brine The organic phase is dried over MgS04 and then concentrated. Purification by flash column chromatography affords compound 68. Note: numerous iterations can be performed using variable length oligomers of 68 to form peptoid oligomers where n=2 to infinity (a hexamer is shown in scheme scheme 5 step 2 Preparation of 69 o 0 0 0 MOCN N N NE 0L. o T N COOEt H H H
H
N HAc OTES OTES OTES OTES OTES 69 To a stirred solution of the acid 62 (1.0 equivalents) and SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCTIUS96/03227 -63the amine 68 (1.1 equivalents) in dimethylformamide Molar) at 25 is added 1-hydroxybenzotriazole (HOBT; 1.1 equivalents). Note: numerous iterations can be performed using the acid 62, or intermixing with other acids including for example acid 46, to form successive oligomers where n=2 to infinity (a hexamer is shown in scheme 5) to obtain large carbopeptoid libraries. Next dicyclohexylcarbodiimide (1.2 equivalents) is added and the reaction is stirred for 14 hours.
The mixture is diluted with ether, filtered and the filtrate is washed with aqueous NaHCO 3 water and brine (2X).
The organic phase is dried over MgSO 4 and then concentrated.
Purification by flash column chromatography affords compound 69. scheme 5 step 1 Preparation of 0 0 0 0 S S O SE 0 N N NE NCOOEt TESO'". ^ESO' 'OTESTESO "*NHAcTESO 'OTESTESON OTES OTES OTES OTES OTES To a stirred solution of 69 (1.0 equivalents) in dimethylformamide (.10 Molar) at 25 is added piperidine (1.1 equivalents). The reaction is stirred for 1 hour and is then SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCT/US96/03227 -64diluted with ether, and washed with aqueous CuSO 4 water and brine The organic phase is dried over MgSO 4 and then concentrated. Purification by flash column chromatography affords compound 70. Note: numerous iterations can be performed using variable length oligomers of to form peptoid oligomers where n=2 to infinity (a hexamer is shown in scheme scheme 5 step 2 Preparation of 71 o o o FMOCN-O N O N COOEt TESO"" OT s T 'NHAc TESO''' TESO 'ES NH c NHAc OTES OTES OTES OTES OTES OTES 71 To a stirred solution of the acid 46 (1.0 equivalents) and the amine 70 (1.1 equivalents) in dimethylformamide Molar) at 25 oC, is added 1-hydroxybenzotriazole (HOBT; 1.1 equivalents). Note: numerous iterations can be performed using the acid 46 or intermixing with other acids including for example acid 62, to form successive oligomers where n=2 to infinity (a hexamer is shown in scheme 5) to obtain large carbopeptoid libraries. Next dicyclohexylcarbodiimide (1.2 equivalents) is added and the reaction is stirred for 14 hours.
The mixture is diluted with ether, filtered and the filtrate is SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCT/US96/03227 washed with aqueous NaHCO 3 water and brine (2X).
The organic phase is dried over MgSO 4 and then concentrated.
Purification by flash column chromatography affords compound 71. scheme 5 step 1 SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCTIUS96/03227 -66- Preparation of 72 H2ooN 0 0 0 ao N COoEt H 2 N E N S 0OA)NE H H H TESO" .OTr^SO 0 'NHAcTESO' TOTES TESO TES 'OTESSO" OTES OTES OTES OTES OTES OTES 72 To a stirred solution of 71 (1.0 equivalents) in dimethylformamide (.10 Molar) at 25 is added piperidine (1.1 equivalents). The reaction is stirred for 1 hour and is then diluted with ether, and washed with aqueous CuSO 4 water and brine The organic phase is dried over MgSO 4 and then concentrated. Purification by flash column chromatography affords compound 72. Note: numerous iterations can be performed using variable length oligomers of 72 to form peptoid oligomers where n=2 to infinity (a hexamer is shown in scheme scheme 5 step 2 Preparation of 74 0 U
COOH
H HH H HO'y"Y "OH HO ''NHAc HO" 'OH "OH HO OH HO NHAc OH OH OH OH OH OH 74 To a stirred solution of 72 (1.0 equivalents) in acetonitrile SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCT/US96/03227 -67- Molar) is added an HF-pyridine solution (.50 M) from Aldrich chemical company. The reaction is allowed to stir for five hours and is then condensed. The crude 73 oligomer is then resuspended in p-dioxane (.50 Molar) to which is added a 3.0 Molar solution of NaOH (3.0 equivalents). The reaction is stirred for 1 hour at 50 °C and is then quenched with aqueous
NH
4 C1 (2X) and subsequently lyophilized. Purification by HPLC chromatography affords compound 74. scheme Preparation of 76 HO C O OEt HO"
'OH
OH
76 To a solution of p-D-Glucose pentaacetate 36 i n nitromethane from Aldrich company (.13 Molar), is added trimethylsilylcyanide (3.0 equivalents) and then tin tetrachloride (.02 equivalents). Note: other pyranose sugars such as P-D-Mannose, P-D-Galactose pentaacetate and other lewis acids such as BF3 OEt 2 may be used for alternative derivatives. The mixture is stirred for one hour and then an aqueous solution of sodium acetate was added to hydrolyze the SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCT/US96/03227 -68remaining trimethylsilylcyanide. The mixture is evaporated and the remaining oil is resuspended in dichloromethane and washed with sodium acetate solution water brine (IX) and then dried over magnesium sulphate and concentrated. The crude product is next dissolved in ethanol (or methanol if the 0methyl glycoside is desired as in scheme 20), (0.15 M) and then concentrated
H
2 S0 4 (0.01 equivalents) is added. The reaction mixture is heated to 85 °C for eight hours. The solution is next concentrated in vacuo and purification by flash column chromatography affords compound 76. scheme 6; 76, scheme (as the O-methyl glycoside).
Preparation of 78 )MTO COOEt TESO"'
OTES
OTES
78 To Tetrol 76 (1.0 equivalents) in pyridine (.10 Molar), is added dimethyoxytritylchloride (DMT chloride) (2.5 equivalents) at 0 The reaction is stirred for 2 hours and then diluted with diethylether and washed with ammonium chloride copper sulfate brine dried over MgSO 4 and concentrated.
SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCTIUS96/03227 -69- Next a solution of the crude intermediate (1.0 equivalents) is dissolved in methylene chloride (.10 Molar) and diisopropylethylamine (4.4 equivalents) is added at 0 °C.
Subsequent addition of triethylsilyl trifluoromethanesulfonate (4.4 equivalents) is followed by stirring for 2 hours and then the reaction is diluted with diethylether and washed with ammonium chloride brine (1X) and then dried (MgSO 4 and concentrated. Purification by flash column chromatography affords compound 78, scheme 6; 78, scheme 20 (as the 0methyl glycoside).
Preparation of DMTO 0 OH TESO'" "OTES
OTES
To a solution of 78 (1.0 equivalents) in methylene chloride Molar) is added a 1.0 M solution of DIBALH in methylene chloride from Aldrich chemical company (1.2 equivalents) at 0 Subsequent stirring for 2 hours is followed by dilution with diethylether and washing with sodium-potassium tartrate (2X), brine (IX) and then MgSO 4 The solution is then concentrated and purification by flash column chromatography affords SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCT/US96/03227 compound 80. scheme 6 Preparation of 82 N(iPr) 2 DMTO O O P O C/ C N TESO" 'OTES
OTES-
82 To a solution of 80 (1.0 equivalents) in methylene chloride is added diisopropylethylamine (4.0 equivalents) at The reaction is stirred for 5 minutes and then 2-cyanoethyl- N,N-diisopropyl-chlorophosphoramidite (1.5 equivalents) is added, as prepared from the procedures of Sinha et al. Nucl.
Acids Res. 1984, 12, 4539. After 15 minutes the reaction is complete and is next diluted with ether and next washed with brine (IX) and is then dried (MgSO4) and concentrated.
Purification by flash column chromatography (silica, 30% ethyl acetate in petroleum ether) affords compound 82 (66% yield).
scheme 6 SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCT/US96/03227 -71- Preparation of 84 HO 0 OTES TESO" 'OTES
OTES
84 To 80 (1.0 equivalents) in methylene chloride (.10 Molar) at 0 is added diisopropylethylamine (1.1 equivalents).
Subsequent addition of triethylsilyl trifluoromethanesulfonate (1.1 equivalents) is followed by stirring for 2 hours and then the reaction is diluted with diethylether and washed with ammonium chloride brine (lX) and then dried (MgSO4) and concentrated. The crude is then resuspended in nitromethane and exposed to 10% C13COOH (1.1 equivalents) in THF Molar). The reaction is stirred at 0 °C for 2 hours and is then diluted with ether and washed with sodium bicarbonate (2X), brine (lX) and then dried (MgSO4) and concentrated. Purification by flash column chromatography affords compound 84.
scheme 6 SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCTIUS96/03227 -72- Preparation of 86 HO 0 COOEt HO' '"NHAc
OH
86 To a solution of N-phthalamido-D-Glucosamine tetraacetate 48 in nitromethane (.13 Molar), is added trimethylsilyl cyanide equivalents) and then SnC1 4 (.02 equivalents). The mixture is stirred for one hour and then an aqueous solution of sodium acetate was added to hydrolyze the remaining trimethylsilyl cyanide. The mixture is evaporated and the remaining oil is resuspended in dichloromethane and washed with sodium acetate solution water brine (IX) and then dried over magnesium sulphate and concentrated. The crude product is next dissolved in ethanol (0.15 M) and then concentrated H2S0 4 (0.04 equivalents) is added. The reaction mixture is heated to °C for eight hours. The solution is next concentrated in vacuo and is then resuspended in methanol (.10 M) and acetic anhydride (1.1 equivalents) from Aldrich company is added in one step. After 2 hours, condensation and purification by flash column chromatography affords compound 86. scheme 7 SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCT/US96/03227 -73- Preparation of 88 )MTO 0 COOEt TESO'" NHAc
OTES
88 To Triol 86 (1.0 equivalents) in pyridine (.10 Molar), is added dimethyoxytritylchloride. (DMT chloride) equivalents) at 0 The reaction is stirred for 2 hours and then diluted with diethylether and washed with ammonium chloride copper sulfate brine dried over MgSO 4 and concentrated. Next a solution of the crude intermediate equivalents) is dissolved in methylene chloride (.10 Molar) and diisopropylethylamine (3.3 equivalents) is added at 0 °C.
Subsequent addition of triethylsilyl trifluoromethanesulfonate (3.3 equivalents) is followed by stirring for 2 hours and then the reaction is diluted with diethylether and washed with ammonium chloride brine (IX) and then dried (MgS04) and concentrated. Purification by flash column chromatography affords compound 88. scheme 7 SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCT/US96/03227 -74- Preparation of 3MTO O OH TESO"' NHAc
OTES
To a solution of 88 (1.0 equivalents) in methylene chloride Molar) is added a 1.0 M solution of DIBALH in methylene chloride from Aldrich chemical company (1.2 equivalents) at 0 OC. Subsequent stirring for 2 hours is followed by dilution with diethylether and washing with sodium-potassium tartrate (2X), brine (IX) and then MgSO 4 The solution is then concentrated and purification by flash column chromatography affords compound 90. scheme 7 Preparation of 92 N(iPr) 2 DMTO 0 P C TESO" "NHAc
OTES
92 To a solution of 90 (1.0 equivalents) in methylene chloride is added diisopropylethylamine (4.0 equivalents) at SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCT1US96/03227 OC. The reaction is stirred for 5 minutes and then 2 -cyanoethyl- N,N-diisopropyl-chlorophosphoramidite (1.5 equivalents) is added, as prepared from the procedures of Sinha et al. Nucl.
Acids Res. 1984, 12, 4539. After 15 minutes the reaction is complete and is next diluted with ether and next washed with brine (IX) and is then dried (MgSO4) and concentrated.
Purification by flash column chromatography (silica, 30% ethyl acetate in petroleum ether) affords compound 92 (66% yield).
scheme 7 Preparation of 94 HO O OTES TESO" 'NHAc
OTES
94 To 90 (1.0 equivalents) in methylene chloride (.10 Molar) at 0 is added diisopropylethylamine (1.1 equivalents).
Subsequent addition of triethylsilyl trifluoromethanesulfonate (1.1 equivalents) is followed by stirring for 2 hours and then the reaction is diluted with diethylether and washed with ammonium chloride brine (IX) and then dried (MgSO 4 and concentrated. The crude is then resuspended in nitromethane and exposed to 10% C13COOH (1.1 equivalents) in THF Molar). The reaction is stirred at 0 °C for 2 hours and is then SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCTIUS96/03227 -76diluted with ether and washed with sodium bicarbonate (2X), brine (IX) and then dried (MgS0 4 and concentrated. Purification by flash column chromatography affords compound 94.
scheme 7 Preparation of 98 (homodimer scheme 8) To a solution of 94 (1.0 equivalents) in methylene chloride is added 1-H-tetrazole from Aldrich company (10.0 equivalents) at 25 OC. Next, a solution of 82 (3.0 equivalents) in methylene chloride (1.0 is added dropwise with stirring at After 25 minutes, the mixture is cooled to 0 OC and 12 equivalents), 2,6 lutidine (4.0 equivalents) in THF (1.0 M) is added to oxidize the phosphoamidate to the phosphate (Alternatively m-chloroperoxybenzoic acid (4.5 equivalents) is added). The reaction is next stirred for an additional 5 minutes and is next diluted with ether and washed with brine (IX) and dried (MgSO 4 and concentrated. Purification by flash column chromatography and then the product is suspended in acetic acid-tetrahydrofuran-water (.01 M) and stirred for 18 hours at 25 The reaction is then diluted with ether and washed with NaHCO 3 brine (lX) and dried (MgSO 4 and concentrated. Purification by flash column chromatography affords compound 98 (scheme 8).
SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCT/US96/03227 -77- Preparation of 102 (heterotrimer scheme 8) To a solution of 98 (1.0 equivalents) in methylene chloride is added 1-H-tetrazole from Aldrich company (10.0 equivalents) at 25 Next, a solution of 92 (3.0 equivalents) in methylene chloride (1.0 is added dropwise with stirring at After 25 minutes, the mixture is cooled to 0 °C and 12 equivalents), 2,6 lutidine (4.0 equivalents) in THF (1.0 M) is added to oxidize the phosphoamidate to the phosphate (Alternatively m-chloroperoxybenzoic acid (4.5 equivalents) is added). The reaction is next stirred for an additional 5 minutes and is next diluted with ether and washed with brine (IX) and dried (MgSO 4 and concentrated. Purification by flash column chromatography and then the product is suspended in acetic acid-tetrahydrofuran-water (.01 M) and stirred for 18 hours at 25 The reaction is then diluted with ether and washed with NaHCO 3 brine (IX) and dried (MgSO 4 and concentrated. Purification by flash column chromatography affords compound 102 (scheme 8).
Preparation of 106 (heterotetramer scheme 8) To a solution of 102 (1.0 equivalents) in methylene chloride (.10 is added 1-H-tetrazole from Aldrich company (10.0 equivalents) at 25 Next, a solution of 82 equivalents) in methylene chloride (1.0 is added dropwise SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCTIUS9603227 -78with stirring at 25 After 25 minutes, the mixture is cooled to 0 °C and I2 (4.0 equivalents), 2,6 lutidine (4.0 equivalents) in THF (1.0 M) is added to oxidize the phosphoamidate to the phosphate (Alternatively m-chloroperoxybenzoic acid equivalents) is added). The reaction is next stirred for an additional 5 minutes and is next diluted with ether and washed with brine (IX) and dried (MgSO 4 and concentrated.
Purification by flash column chromatography and then the product is suspended in acetic acid-tetrahydrofuran-water (.01 M) and stirred for 18 hours at 25 The reaction is then diluted with ether and washed with NaHCO 3 brine (IX) and dried (MgSO 4 and concentrated. Purification by flash column chromatography affords compound 106 (scheme 8).
Preparation of 110 (heteropentamer scheme 8) To a solution of 106 (1.0 equivalents) in methylene chloride (.10 is added l-H-tetrazole from Aldrich company (10.0 equivalents) at 25 Next, a solution of 92 equivalents) in methylene chloride (1.0 is added dropwise with stirring at 25 After 25 minutes, the mixture is cooled to 0 OC and I2 (4.0 equivalents), 2,6 lutidine (4.0 equivalents) in THF (1.0 M) is added to oxidize the phosphoamidate to the phosphate (Alternatively m-chloroperoxybenzoic acid equivalents) is added). The reaction is next stirred for an SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCT/US96/03227 -79additional 5 minutes and is next diluted with ether and washed with brine (lX) and dried (MgSO 4 and concentrated.
Purification by flash column chromatography and then the product is suspended in acetic acid-tetrahydrofuran-water (.01 M) and stirred for 18 hours at 25 The reaction is then diluted with ether and washed with NaHCO 3 brine (1X) and dried (MgSO 4 and concentrated. Purification by flash column chromatography affords compound 110 (scheme 8).
Preparation of 114 (heterohexamer scheme 8) To a solution of 110 (1.0 equivalents) in methylene chloride (.10 is added 1-H-tetrazole from Aldrich company (10.0 equivalents) at 25 Next, a solution of 82 equivalents) in methylene chloride (1.0 is added dropwise with stirring at 25 After 25 minutes, the mixture is cooled to 0 °C and I2 (4.0 equivalents), 2,6 lutidine (4.0 equivalents) in THF (1.0 M) is added to oxidize the phosphoamidate to the phosphate (Alternatively m-chloroperoxybenzoic acid equivalents) is added). The reaction is next stirred for an additional 5 minutes and is next diluted with ether and washed with brine (IX) and dried (MgSO 4 and concentrated.
Purification by flash column chromatography and then the product is suspended in acetic acid-tetrahydrofuran-water (.01 M) and stirred for 18 hours at 25 The reaction is SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCTMS9603227 then diluted with ether and washed with NaHCO 3 brine (1X) and dried (MgSO 4 and concentrated. Purification by flash column chromatography affords compound 114 (scheme 8).
Preparation of 116 (heterohexamer scheme 8) To a solution of 114 (1.0 equivalents) in methylene chloride (.10 is added a solution of HF-pyridine (1.0 M) at 0 The reaction is next stirred for an additional 30 minutes and is next diluted with ether and washed with a saturated solution of sodium bicarbonate copper sulfate solution to remove the pyridine (2X) brine dried (MgSO 4 and concentrated.
Purification by flash column chromatography and then the product is resuspended in concentrated aqueous ammonium hydroxide and acetonitrile M total). The reaction is then stirred for 2 hours at 50 °C and is subsequently diluted with ether and washed with NaHCO 3 brine (lX) and dried (MgSO 4 and concentrated. Purification by flash column chromatography affords compound 116 scheme 8.
SUBSTITUTE SHEET (RULE 26) WO 96/27379 PTU9132 PCrfUS96103227 -81- AcO O a -d HO, O Oe- g ButMe 2 SiO: OH AcO .OAc (126-13O)HO Y "OH (131-134) BnO 'Bn OAc OH OBn 36 130 134 N(i-Pr) 2 c 1 35'N~N N(APr) 2 ButMe 2 SiO", 0roo---,C BnO *-'"OBn OBn 138 j 0 HO 0 BnO"Y 'OBn NN OBn 136 R0 0 OR 3 R O-P-6-O
OR
4
R
2 0"C"OR 2 0
R
2
"OR
2
OR
2 OR 2 kE140: RI SitBuMe 2
R
2 Bn o 142: R, R n 4=Nc 0 OR 3 0 OR 3 0 R,0
O--OR
4 OR 2
R
2 0' "O 2 0R 2 0V R 2
OR
2 OR 2 OR 2 kE 14: R SitBuMe 2
R
2 Bn; R 4 =Nphth 146: R, H; R 2 =Bn; R 4 Nphth 0 OR 3 0 OR 3 0 OR 3 0 R0O-P- u 0-0-0) -rOR 4
R
2 0 "OR 2 R,0'
"OR
2
R
2 0"Y "R R 2 0" OR
OR
2
OR
2
OR
2
OR
2 k 148: R, Sit~uMe 2
R
2 Bn; A 3
CH
2
CH
2 CN; R 4 Nphth 150: R, H; R 2 Bn; R 3
=CH
2
CH
2 CN; R 4 Nphth 152: Rl R 3 H; R 2 Bn; R 4 Nphth mL154: RR 2
R
3
R
4 Nphth Scheme 9. Synthesis of a Carbonucleotoid SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCT/US96/03227 -82- Preparation of 125 O CN AcO AcO'"' 'OAc OAc 125 To a solution of B-D-Glucose pentaacetate in nitromethane from Aldrich company (.13 Molar), is added trimethylsilylcyanide (3.0 equivalents) and then borontrifluoride etherate (.02 equivalents). Note: other pyranose sugars such as 3 -D-Mannose, P-D-Galactose pentaacetate and other lewis acids such as SnCl4, may be used for alternative derivatives. The mixture is stirred for one hour and then an aqueous solution of sodium acetate was added to hydrolyze the remaining trimethylsilylcyanide. The mixture is evaporated and the remaining oil is resuspended in dichloromethane and washed with sodium acetate solution (IX), water brine (IX) and then dried over magnesium sulphate and concentrated. The crude solid is then recrystallized from methanol to yield 125 (also 37) as a white solid scheme 9 step a SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCT/US96/03227 -83- Preparation of 126 HO O
CN
HOO
H 0 0 H HO" 'OH
OH
126 To a solution of 125 in methanol (.13 Molar), is added sodium methoxide (0.3 equivalents) and the reaction mixture is stirred for two hours at room temperature. The dark brown solution is then concentrated in vacuo to give a dark brown syrup of compound 126 which is carried on without purification as a crude oil for the next step. scheme 9 step b Preparation of 127 HO COOH
HO
H 'OH
OH
127 The crude product 126 is dissolved in 25% NaOH (0.5 M) and heated at reflux for 18 hours (vigorous reflux is necessary).
Next, the solution is diluted with an addition of water (0.1 M) and to this solution is added Amberlite 112120 resin (H+-form) and is then stirred. The supernatant is then decanted and the SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCTIUS96/03227 -84resin is washed until the eluate is colorless. The eluate is then collected, condensed and azeotroped with MeOH which yields 127 as a crude, pale yellow syrup Preparation of 130 H 0 O COOMe
HO
H OH
OH
130 The crude product 127 is next dissolved in methanol (0.15 M) and then concentrated HCI (0.01 equivalents) is added. The reaction mixture is heated to 85 °C for eight hours. The solution is next concentrated in vacuo and purification by flash column chromatography (silica, 20% methanol in ethyl acetate), affords compound 130 as a white solid (60% yield). scheme 9 step d Preparation of 131 TBDMSO 0 COOMe H "'OH
OH
131 SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCT/US96/03227 To a solution of 130 (1.0 equivalents) in dimethylformamide (.23 Molar), is added imidazole equivalents) at 0 OC. Subsequent addition of tert-Butyldimethylsilylchloride (2.5 equivalents) is followed by stirring for 2 hours and then the reaction is diluted with diethylether and washed with ammonium chloride brine (IX) and then dried (MgSO 4 and concentrated. Purification by flash column chromatography (silica, 50% ethyl acetate) affords compound 131 as a white solid (93% yield). scheme 9 step e Note: the molecule can be protected with other primary directing protecting groups such as DMT (dimethoxytrityl), and TBDPS tert-butyldiphenlysilyl, etc.
Preparation of 132 o COOMe ButMe 2 SiO CO BnO" 'OBn OBn 132 To a solution of 1 3 1 (1.0 equivalents) in dimethylformamide (.23 is added Ag20 (6.0 equivalents) at Benzyl bromide (9.0 equivalents) is next added and the SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCT/US96103227 -86reaction is allowed to stir for 20 hours. The reaction is diluted with diethylether and washed with ammonium chloride (2X), brine (IX) and then dried (MgSO 4 and concentrated.
Purification by flash column chromatography (silica, 20% ethyl acetate) affords compound 132 (83% yield). scheme 9 step f Note: the choice of the protecting group is relative and the molecule can be protected with other protecting groups at C2, C3, C4, such as PMB (paramethoxybenzyl), TES (triethylsilyl), TBS (tertbutyldimethylsilyl), etc.
Preparation of 134 0 ButMe 2 SiO OH BnO"' 'OBn OBn 134 To a solution of 132 (1.0 equivalents) in tetrahydrofuran (.08 is added diisobutylaluminumhydride (DIBALH) equivalents) at 0 The reaction is stirred for 1 hour and then quenched with methanol and diluted with ether. The reaction is next worked-up with ammonium chloride brine (IX) and is then dried (MgSO4) and concentrated. Purification by flash column chromatography (silica, 20% ethyl acetate) affords compound 134 (66% yield). scheme 9 step g SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCI/US96/03227 -87- Preparation of 136 0 HO O BnO" "OBn OBn 136 To a solution of 134 (1.0 equivalents) in pyridine (10.0 equivalents), is added naphthoyl chloride (3.0 equivalents) from Aldrich company (3.0 equivalents) at 25 The reaction is stirred for 45 minutes and then diluted with ether and workedup with a saturated solution of CuSO 4 brine (IX) and is then dried (MgSO4) and concentrated. The crude product is then exposed to acetic acid/tetrahydrofuran/water at 25 °C and allowed to stir for 15 hours. The reaction is then diluted with ether and worked-up with brine (2X) and is then dried (MgSO4) and concentrated. Purification by flash column chromatography (silica, 20% ethyl acetate) affords compound 136 (95% yield). Note: alternatively, one could originally protect the C7 position as a DMT (dimethoxytrityl) functionality and protect the C1 position as a TES (triethyl silyl) group.
Subsequent mild acid hydrolysis of the DMT group leads to the SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCr/US96/03227 -88above compound with the TES group at the Cl position and a free hydroxyl at the C7 position. scheme 9 step h Preparation of 138 N(i-Pr) 2 ButMe 2 SiO 0 0 O 0 Po CN BnO" '"OBn OBn 138 To a solution of 134 (1.0 equivalents) in methylene chloride (.10 is added diisopropylethylamine equivalents) at 25 The reaction is stirred for 5 minutes and then 2-cyanoethyl-N,N-diisopropyl-chlorophosphoramidite equivalents) is added, as prepared from the procedures of Sinha et al. Nucl. Acids Res. 1984, 12, 4539. After 15 minutes the reaction is complete and is next diluted with ether and next washed with brine (IX) and is then dried (MgSO4) and concentrated. Purification by flash column chromatography (silica, 30% ethyl acetate in petroleum ether) affords compound 138 (66% yield). scheme 9 step i It should be noted that the oligomerization process as shown below in scheme 9, uses the same C-glycoside 138 in an iterative fashion. The process can be SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCTIUS96/03227 -89extended however to include a pool of random or ordered C-glycosides as depicted in scheme 8.
Preparation of 140 0 -CN
O
FBDMSO O-P-O O 0 BnO" "'OBn 0 BnO'" "'OBn OBn OBn 140 To a solution of 136 (1.0 equivalents) in methylene chloride (.10 is added l-H-tetrazole from Aldrich company (10.0 equivalents) at 25 Next, a solution of 138 equivalents) in methylene chloride (1.0 is added dropwise with stirring at 25 After 25 minutes, the mixture is cooled to 0 °C and m-chloroperoxybenzoic acid (4.5 equivalents) is added.
The reaction is stirred for an additional 5 minutes and is next diluted with ether and washed with brine (lX) and dried (MgS04) and concentrated. Purification by flash column chromatography (silica, 50% ethyl acetate in petroleum ether) affords compound 140 (97% yield). scheme 9 step j Note the process can iterate as many times as possible to build large carbonucleotide libraries.
SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCTIUS96/03227 Preparation of 142 BnO"' Y'-OBn 0 BnO"Y.
OBn OBn 142 A solution of 140 (1.0 equivalents) in acetic acidtetrahydrofuran-water (.01 M) is stirred for 18 hours at The reaction is then diluted with ether and washed with NaHCO 3 brine (IX) and dried (MgSO 4 and concentrated.
Purification by flash column chromatography (silica, 60% ethyl acetate in petroleum ether) affords compound 142 (95% yield).
scheme 9 step k Note the process can iterate as many times as possible to build large carbonucleotide libraries.
Preparation of 144 rBDMS Y""OBn ORn OBn OBn 144 To a solution of 138 (1.0 equivalents) in methylene chloride (.10 is added 1-H-tetrazole from Aldrich company SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCT/US96/03227 -91- (10.0 equivalents) at 25 Next, a solution of 142 equivalents) in methylene chloride (1.0 is added dropwise with stirring at 25 After 25 minutes, the mixture is cooled to 0 °C and m-chloroperoxybenzoic acid (4.5 equivalents) is added.
The reaction is stirred for an additional 5 minutes and is next diluted with ether and washed with brine (lX) and dried (MgSO 4 and concentrated. Purification by flash column chromatography (silica, 50% ethyl acetate in petroleum ether) affords compound 144 (97% yield). scheme 9 step j Note the process can iterate as many times as possible to build large carbonucleotide libraries.
SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCT/US96/03227 -92- Preparation of 146 O N O C N
O
HO O-P-O
O
II
II
BnO" "OBn BnO" "OBn O BnO" ""OBn OBn OBn OBn 146 A solution of 144 (1.0 equivalents) in acetic acidtetrahydrofuran-water (.01 M total) is stirred for 18 hours at 25 The reaction is then diluted with ether and washed with NaHCO 3 brine (IX) and dried (MgSO 4 and concentrated. Purification by flash column chromatography (silica, 60% ethyl acetate in petroleum ether) affords compound 146 (95% yield). scheme 9 step k Note the process can iterate as many times as possible to build large carbonucleotide libraries.
Preparation of 148 S C N O CN O' CN BDMSO O-P' O- -0
O-P-
BnO'" 'r OBn BnO" 'OBn BnO" ''OBn BnO''' OBn OBn OBn OBn OBn 148 To a solution of 138 (1.0 equivalents) in methylene SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCT/US96/03227 -93chloride (.10 is added 1-H-tetrazole from Aldrich company (10.0 equivalents) at 25 Next, a solution of 146 equivalents) in methylene chloride (1.0 is added dropwise with stirring at 25 After 25 minutes, the mixture is cooled to 0 °C and m-chloroperoxybenzoic acid (4.5 equivalents) is added.
The reaction is stirred for an additional 5 minutes and is next diluted with ether and washed with brine (lX) and dried (MgSO4) and concentrated. Purification by flash column chromatography (silica, 50% ethyl acetate in petroleum ether) affords compound 148 (97% yield). scheme 9 step j Note the process can iterate as many times as possible to build large carbonucleotide libraries.
Preparation of 150 OCN c CN oCN 0 oo:/O e 6 O. c Bn0 oo "a 0 .o 1 5 0 08 OBn OBn 0150 A solution of 148 (1.0 equivalents) in acetic acidtetrahydrofuran-water (.01 M total) is stirred for 18 hours at 25 oC. The reaction is then diluted with ether and washed with NaHCO 3 brine (IX) and dried (MgSO4) and concentrated. Purification by flash column chromatography (silica. 60% ethyl acetate in petroleum ether) affords compound 150 (95% yield). scheme 9 step k Note the process can iterate as many times as possible to build large carbonucleotide SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCT/US96/03227 -94libraries.
Preparation of 152 BnO- 8[0J 08H 6 08, enO' 0. 152 A solution of 150 (1.0 equivalents) is dissolved in concentrated aqueous ammonium hydroxide and acetonitrile M total). The reaction is then stirred for 2 hours at "C and is subsequently diluted with ether and washed with NaHCO 3 brine (IX) and dried (MgSO 4 and concentrated.
Purification by flash column chromatography (silica, 80% ethyl acetate in petroleum ether) affords compound 152 (88% yield).
scheme 9 step L Preparation of 154 ^o o 0 0 1: a 54 A solution of 152 (1.0 equivalents) is dissolved in a mixture of ethanol-tetrahydrofuran-acetic acid (.01 M total) at 25 The mixture is next exposed to 10% Pd/C equivalents) and is then subsequently capped with a hydrogen balloon at 1 atmosphere. The reaction is stirred for 72 hours and is then filtered through celite. The crude mixture is SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCIUS96O3227 subsequently diluted with ether and washed with NaHCO 3 (3X), brine (IX) and dried (MgS0 4 and concentrated. Purification by flash column chromatography (silica, 100% ethyl acetate in petroleum ether) affords compound 154 (78% yield). scheme 9 step m Preparation of 174 (R group OTES, NPhth or NHAc) To a solution of tetraacetate derived from 36 or 48 (glucose or glucosamine derived) in methylene chloride molar) is added a 1.0 molar solution of Co 2
(CO)
8 (1.5 equivalents in methylene chloride and diethylmethylsilane (1.5 equivalents) at 0 To the stirring reaction mixture, a stream of carbon monoxide is bubbled at 1 ml per 10 seconds for 30 minutes. The reaction mixture is then quenched with water (1.5 equivalents), diluted with ether, washed with sodium bicarbonate brine (Ix) and dried over magnesium sulfate. The crude is purified by column chromatography and affords product 174.
Preparation of 176 (R group OTES, NPhth or NHAc) To a solution of compound 174 in acetonitrile/water (1:1 ratio, .1 molar combined), is added RuCl3 (.03 equiv.) and NaIO4 equiv.) at 25 °C and the muddy black mixture is allowed to stir for 1.5 h. The mixture is then diluted with ether (25 mL), washed with water (2X 5.0 mL) and brine (1X 5 mL). The SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCT/US96/03227 -96aqueous layer is back extracted recombined, and the organic layer was then dried MgSO4 and evaporated.
Purification by flash column chromatography yields the desired product 176.
Preparation of 178 (R group OTES, NPhth or NHAc) A solution of triacetate 176 (1.0 equiv.) in methanol (0.5 is treated with NaOMe (0.4 equiv.) and allowed to stir at 25 °C for 24 h. The reaction mixture is then condensed and purified by flash column chromatography to afford compound 178.
Preparation of 180 (R group OTES, NPhth or NHAc) To triol 178 (1.0 equivalents) in pyridine (.10 Molar), is added dimethyoxytritylchloride (DMT chloride) (1.5 equivalents) at 0 oC. The reaction is stirred for 2 hours and then diluted with diethylether and washed with ammonium chloride copper sulfate brine dried over MgSO 4 and concentrated.
Next a solution of the crude intermediate (1.0 equivalents) is dissolved in methylene chloride (.10 Molar) and diisopropylethylamine (3.3 equivalents) is added at 0 °C.
Subsequent addition of triethylsilyl trifluoromethanesulfonate (3.3 equivalents) is followed by stirring for 2 hours and then the reaction is diluted with diethylether and washed with ammonium chloride brine (lX) and then dried (MgSO4) and SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCT/US96/03227 -97concentrated. Purification by flash column chromatography affords the intermediate acid, which is then resuspended in THF M) and exposed to a 1.0 M solution of BH 3 -THF equivalents) at 0 °C for 1 hour. The reaction is then quenched with methanol for an additional hour and the crude is then diluted with diethylether and washed with ammonium chloride brine (IX) and then dried (MgSO 4 and concentrated.
Purification by flash column chromatography affords the desired tetraprotected alcohol 180.
Preparation of 181 (R group OTES, NPhth or NHAc) To a solution of 180 (1.0 equivalents) in methylene chloride is added tetrazole (4.0 equivalents) at 25 The reaction is stirred for 5 minutes and then 2 -cyanoethyl-N,N-diisopropylchlorophosphoramidite (1.5 equiv.) is added, as prepared from the procedures of Sinha et al. Nucl. Acids Res. 1984, 12, 4539.
After 15 minutes the reaction is complete and is next diluted with ether and next washed with brine (IX) and is then dried (MgS0 4 and concentrated. Purification by flash column chromatography (silica, 30% ethyl acetate in petroleum ether) affords compound 181 (66% yield). scheme 21 Preparation of 182 (R group OTES, NPhth or NHAc) To a solution of triol 178 equiv.) in CH2C12 M) at 0 was SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCT/US96/03227 -98added triethylamine (1.2 equiv.), 4-DMAP (.10 equiv.) and then TOSC1 (1.1 equiv.). .The reaction is stirred for 1 h and then is quenched with saturated ammonium chloride (1.5 mL), diluted with ethyl acetate (25 mL), washed with water (2X 5 mL), brine (IX 5 mL), back-extracted recombined, dried (MgSO4) and evaporated. The compound is purified by flash column chromatography and then a solution of the crude intermediate equivalents) is dissolved in methylene chloride (.10 Molar) and diisopropylethylamine (2.2 equivalents) is added at 0 OC.
Subsequent addition of triethylsilyl trifluoromethanesulfonate (2.2 equivalents) is followed by stirring for 2 hours and then the reaction is diluted with diethylether and washed with ammonium chloride brine (IX) and then dried (MgSO 4 and concentrated. Purification by flash column chromatography affords the protected tosylate/acid 182.
Preparation of 183 (R group OTES, NPhth or NHAc) To a solution of triol 182 equiv.) in CH2C12 M) at 0 oC, is added sodium-azide (1.2 equiv.) from Aldrich chemical company at 0 The reaction is stirred for 1 h and then is quenched with saturated ammonium chloride (1.5 mL), diluted with ethyl acetate (25 mL), washed with water (2X 5 mL), brine (lX 5 mL), back-extracted recombined, dried (MgSO4) and evaporated.
The compound is purified by flash column chromatography and SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCT/US96/03227 -99affords compound 183.
Preparation of 185 (R group OTES, NPhth or NHAc) A solution of 183 (1.0 equivalents) in ethanol (.01 M total) at °C is exposed to 10% Pd(OH) 2 -C (0.1 equivalents) and is then subsequently capped with a hydrogen balloon at 1 atmosphere.
The reaction is stirred for 72 hours and is then filtered through celite. The crude mixture is subsequently diluted with ether and washed with NaHCO 3 brine (lX) and dried (MgSO 4 and concentrated. Purification by flash column chromatography affords compound 185 scheme 21.
Preparation of 191 HO O HO" "'OH
OH
191 A solution of starting material 190 as disclosed by Schmidt, R. R. et al. (Liebigs Ann. Chem. 1987, 825), equivalents) is dissolved in a mixture of ethanoltetrahydrofuran-acetic acid (.01 M total) at 25 OC. The mixture is next exposed to 10% Pd/C (1.0 equivalents) and is SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCT/US96/03227 -100then subsequently capped with a hydrogen balloon at 1 atmosphere. The reaction is stirred for 72 hours and is then filtered through celite. The crude mixture is subsequently diluted with ether and washed with NaHCO 3 brine (IX) and dried (MgSO 4 and concentrated. Purification by flash column chromatography (silica, 100% ethyl acetate in petroleum ether) affords compound 191. scheme 22 step a SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCT/US96/03227 -101- Preparation of 192 TsO O o TESO"
'OTES
OTES
192 To a solution of 191 (1.0 equivalents) in methylene chloride (.10 Molar) is added tosylchloride (1.2 equivalents) at 0 OC. Subsequent addition of triethylamine (1.5 equivalents) is followed by stirring for 2 hours and then the reaction is diluted with diethylether and washed with ammonium chloride (2X), brine (lX) and then dried (MgSO 4 and concentrated to afford the crude tosylate. Next a solution of the crude intermediate equivalents) is dissolved in methylene chloride (.10 Molar) and diisopropylethylamine (3.3 equivalents) is added at 0 °C.
Subsequent addition of triethylsilyl trifluoromethanesulfonate (3.3 equivalents) is followed by stirring for 2 hours and then the reaction is diluted with diethylether and washed with ammonium chloride brine (IX) and then dried (MgSO 4 and concentrated. Purification by flash column chromatography affords compound 192. scheme 22 step b SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCT1US96/03227 -102- Preparation of 193 N 0 N3
O
TESO" 'OTES
OTES
193 To a solution of 192 (1.0 equivalents) in methylene chloride (.10 Molar) is added sodium azide from Aldrich chemical company (1.2 equivalents) at 0 Subsequent stirring for 2 hours is followed by dilution with diethylether and washing with ammonium chloride brine (IX) and then MgS04. The solution is then concentrated and purification by flash column chromatography affords compound 193. scheme 22 step c Preparation of 194
N
3 ~O0 CO 2
H
TESO" "OTES
OTES
194 To solution of 193 in CC14 (.33 CH3CN (.33 M) and water (.22 M) at 0 °C is added RuCl3 (.03 equiv.) and NaIO4 equiv.) and the muddy black mixture is allowed to stir for 1.5 h.
The mixture is then diluted with ether (25 mL), washed with SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCTIUS96103227 -103water (2X 5.0 mL) and brine (IX 5 mL). The aqueous layer is back extracted recombined, and the organic layer iss then dried MgSO4 and evaporated. Purification by flash column chromatography affords the compound 194. scheme 22 step d Preparation of 196 FMOCNH 0
C
0 2
H
TESO" OTES
OTES
196 A solution of 194 (1.0 equivalents) is dissolved in ethanol (.01 M total) at 25 The mixture is next exposed to 10% Pd/C equivalents) and is then subsequently capped with a hydrogen balloon at 1 atmosphere. The reaction is stirred for 72 hours and is then filtered through celite. The crude mixture is subsequently diluted with ether and washed with NaHCO 3 (3X), brine (IX) and dried (MgSO 4 and concentrated. Next, to a solution of crude amine (1.0 equivalents) in methylene chloride (.10 Molar), is added sodium bicarbonate (2.0 equivalents) at 0 0 C. Subsequent addition of 9 -fluorenylmethyl chloroformate (FMOC-C1, 1.2 equivalents) is followed by stirring for 2 hours SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCT/US96/03227 -104and then the reaction is diluted with diethylether and washed with ammonium chloride brine (IX) and then dried (M gS04) and concentrated. Purification by flash column chromatography affords compound 196. scheme 22 steps e-f SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCTIUS96/03227 -105- Preparation of 197 O0 DMTO
O
TESO
'"OTES
OTES
197 To Tetrol 191 (1.0 equivalents) in pyridine (.10 Molar), is added dimethyoxytritylchloride (DMT chloride) (2.5 equivalents) at 0 The reaction is stirred for 2 hours and then diluted with diethylether and washed with ammonium chloride copper sulfate brine dried over MgSO4 and concentrated.
Next a solution of the crude intermediate (1.0 equivalents) is dissolved in methylene chloride (.10 Molar) and diisopropylethylamine (3.3 equivalents) is added at 0 °C.
Subsequent addition of triethylsilyl trifluoromethanesulfonate (3.3 equivalents) is followed by stirring for 2 hours and then the reaction is diluted with diethylether and washed with ammonium chloride brine (lX) and then dried (MgSO 4 and concentrated. Purification by flash column chromatography affords compound 197. scheme 22 step g SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCT/US96/03227 -106- Preparation of 198 DMTO 0 .,CC 2
CH
3 TESO" OTES
OTES
198 To solution of 197 in CC14 (.33 CH3CN (.33 M) and water (.22 M) at 0 °C is added RuCl3 (.03 equiv.) and NaIO4 equiv.) and the muddy black mixture is allowed to stir for 1.5 h.
The mixture is then diluted with ether (25 mL), washed with water (2X 5.0 mL) and brine (IX 5 mL). The crude is then resuspended in a mixture of methylene chloride/water .1 M total) and diazomethane (1.2 equivalents) is gradually dropped into the flask via an addition funnel at the rate of 1 seconds. After complete addition the mixture is diluted with ether, washed with brine (2X) and the aqueous layer is back extracted (2X) recombined, and the organic layer is then dried MgS04 and evaporated. Purification by flash column chromatography affords the compound 198. scheme 22 step h SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCIrUS96/03227 -107- Preparation of 200
"'"OTES
OTES
200 To a solution of 198 (1.0 equivalents) in methylene chloride (.10 Molar) is added a 1.0 M solution of DIBALH in methylene chloride from Aldrich chemical company (1.2 equivalents) at 0 Subsequent stirring for 2 hours is followed by dilution with diethylether and washing with sodiumpotassium tartrate brine (1X) and then MgSO4. The solution is then concentrated and purification by flash column chromatography affords compound 200. scheme 22 step i Preparation of 201
N
3
TESO"
OTES
201 A solution of 200 (1.0 equivalents) in tetrahydrofuran (.18 M) is treated with DPPA (diphenylphosphorylazide, SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCT/US96/03227 -108equivalents), triphenylphosphine (1.3 equivalents) and DIAD (diisopropyl-azo-dicarboxylate, 1.3 equivalents). The reaction is heated to 80 °C for 3 hours and then diluted with ether (2X) and washed with .5 M aqueous NaOH The organic layer is dried over MgSO 4 and evaporated. Purification by flash column chromatography affords compound 201. scheme 22 step j Preparation of 202 DMTO 0 "-'NHFMOC TESO" 'OTES
OTES
202 A solution of 201 (1.0 equivalents) is dissolved in ethanol (.01 M total) at 25 The mixture is next exposed to 10% Pd/C equivalents) and is then subsequently capped with a hydrogen balloon at 1 atmosphere. The reaction is stirred for 72 hours and is then filtered through celite. The crude mixture is subsequently diluted with ether and washed with NaHCO 3 (3X), brine (IX) and dried (MgSO 4 and concentrated. Next, to a solution of crude amine (1.0 equivalents) in methylene chloride (.10 Molar), is added sodium bicarbonate (2.0 equivalents) at 0 0 C. Subsequent addition of 9 -fluorenylmethyl chloroformate (FMOC-C1, 1.2 equivalents) is followed by stirring for 2 hours SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCT/US96/03227 -109and then the reaction is diluted with diethylether and washed with ammonium chloride brine (lX) and then dried (MgS0 4 and concentrated. Purification by flash column chromatography affords compound 202. scheme 22 step e Preparation of 204
HO
2
O~,
0
NHFMOC
TESO' OTES
OTES
204 To a solution of 202 (1.0 equivalents) in methylene chloride (.10 Molar) is added 10% HCOOH from Aldrich chemical company (1.2 equivalents) at 0 OC. Subsequent stirring for 2 hours is followed by dilution with diethylether and washing with sodium bicarbonate brine (IX) and then MgSO4. The solution is then resuspended in t-BuOH (.10 M) and pH 7 buffer M) and is then exposed to KMnO 4 (1.2 equivalents) for 2 hours at 0 OC. The reaction mixture is next washed with sodium bicarbonate brine (IX) and then MgSO 4 The organic layer is then concentrated and purified by flash column chromatography to afford compound 204. scheme 22 step k SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCTrUS96/03227 -110- Preparation of 206 DMTO 0
NO
2 TESO
"OTES
OTES
206 To Tetrol 205 (1.0 equivalents) (as disclosed by Petrus, L.
et al.Chem. zvesti. 1982, 36, 103) in pyridine (.10 Molar), is added dimethyoxytritylchloride (DMT chloride) (2.5 equivalents) at 0 The reaction is stirred for 2 hours and then diluted with diethylether and washed with ammonium chloride copper sulfate brine dried over MgSO4 and concentrated.
Next a solution of the crude intermediate (1.0 equivalents) is dissolved in methylene chloride (.10 Molar) and diisopropylethylamine (3.3 equivalents) is added at 0 °C.
Subsequent addition of triethylsilyl trifluoromethanesulfonate (3.3 equivalents) is followed by stirring for 2 hours and then the reaction is diluted with diethylether and washed with ammonium chloride brine (lX) and then dried (MgSO 4 and concentrated. Purification by flash column chromatography affords compound 206. scheme 23 step a SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCT/US96/03227 -111- Preparation of 207 DMTO 0 NHFMOC TESO "OTES
OTES
207 To a solution of 206 (1.0 equivalents) in diethylether (.08 is added lithiumaluminumhydride (LAH) (1.5 equivalents) at The reaction is refluxed for 2 hours and then quenched with methanol and diluted with ether. The reaction is next worked-up with sodium potassium tartrate brine (IX) and is then dried (MgSO4) and concentrated. The crude mixture is resuspended in methylene chloride (.10 Molar) and to it is added sodium bicarbonate (2.0 equivalents) at 0 0
C.
Subsequent addition of 9-fluorenylmethyl chloroformate (FMOC- Cl, 1.2 equivalents) is followed by stirring for 2 hours and then the reaction is diluted with diethvlether and washed with ammonium chloride brine (lX) and then dried (MgSO4) and concentrated. Purification by flash column chromatography affords compound 207. scheme 23 step b SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCT/US96/03227 -112- Preparation of 208 HO 0 NHFMOC TESO
"OTES
OTES
208 To a solution of 207 (1.0 equivalents) in methylene chloride (.10 Molar) is addedl0% HCOOH (1.1 equivalents). The reaction is stirred at 0 °C for 2 minutes and is then diluted with ether and washed with sodium bicarbonate brine (lX) and then dried (MgSO 4 and concentrated. Purification by flash column chromatography affords compound 208. scheme 23 step c Preparation of 209 HOOC 0
NHFMOC
TESO y "OTES
OTES
209 To solution of 208 in CC14 (.33 CH3CN (.33 M) and water (.22 M) at 20 °C is added RuC13 (.03 equiv.) and NaIO4 equiv.) and the muddy black mixture is allowed to stir for min. The mixture is then diluted with ether (25 mL), washed with water (2X 5.0 mL) and brine (1X 5 mL). The aqueous layer SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCTIUS96/03227 -113is back extracted recombined, and the organic layer iss then dried MgSO4 and evaporated. Purification by flash column chromatography affords the compound 209. scheme 23 step d Preparation of 210 TOSO 0
NO,
TESO
OTES
OTES
210 To a solution of 205 (1.0 equivalents) in methylene chloride (.10 Molar) is added tosylchloride (1.2 equivalents) at 0 Subsequent addition of triethylamine (1.5 equivalents) is followed by stirring for 2 hours and then the reaction is diluted with diethylether and washed with ammonium chloride (2X), brine (lX) and then dried (MgSO4) and concentrated to afford the crude tosylate. Next a solution of the crude intermediate equivalents) is dissolved in methylene chloride (.10 Molar) and diisopropylethylamine (3.3 equivalents) is added at 0 °C.
Subsequent addition of triethylsilyl trifluoromethanesulfonate (3.3 equivalents) is followed by stirring for 2 hours and then the reaction is diluted with diethylether and washed with SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCT/US96/03227 -114ammonium chloride brine (IX) and then dried (MgS0 4 and concentrated. Purification by flash column chromatography affords compound 210. scheme 23 step e Preparation of 211
N
3
NO
2 TESO
OTES
OTES
211 To a solution of 210 (1.0 equivalents) in methylene chloride (.10 Molar) is added sodium azide from Aldrich chemical company (1.2 equivalents) at 0 oC. Subsequent stirring for 2 hours is followed by dilution with diethylether and washing with ammonium chloride brine (lX) and then MgS04. The solution is then concentrated and purification by flash column chromatography affords compound 211. scheme 23 step f SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCT/US96/03227 -115- Preparation of 212
N
3 TESO 'OTES
OTES
212 To solution of 211 in CC14 (.33 CH3CN (.33 M) and water (.22 M) at 20 °C is added RuCl3 (.03 equiv.) and NaIO4 equiv.) and the muddy black mixture is allowed to stir for min. The mixture is then diluted with ether (25 mL), washed with water (2X 5.0 mL) and brine (lX 5 mL). The aqueous layer is back extracted recombined, and the organic layer iss then dried MgSO4 and evaporated. Purification by flash column chromatography affords the compound 212. scheme 23 step g Preparation of 213
H
2 N 0 CO 2
H
TESO' "OTES
OTES
213 A solution of 212 (1.0 equivalents) in ethanol (.01 M total) at 25 "C is exposed to 10% Pd/C (0.1 equivalents) and is then SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCT/US96/03227 -116subsequently capped with a hydrogen balloon at 1 atmosphere.
The reaction is stirred for 72 hours and is then filtered through celite. The crude mixture is subsequently diluted with ether and washed with NaHCO 3 brine (IX) and dried (MgSO 4 and concentrated. Purification by flash column chromatography affords compound 213. scheme 23 step h Preparation of 214 FMOCHN 0 C 0 2
H
TESO'
"OTES
OTES
214 Compound 213 is suspended in methylene chloride Molar) and to it is added sodium bicarbonate (2.0 equivalents) at 0 o C. Subsequent addition of 9 -fluorenylmethyl chloroformate (FMOC-C1, 1.2 equivalents) is followed by stirring for 2 hours and then the reaction is diluted with diethylether and washed with ammonium chloride brine (lX) and then dried (MgSO 4 and concentrated. Purification by flash column chromatography affords compound 214. scheme 23 step i SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCT/US96/03227 -117- Preparation of 215 PIVO O NO 2 TESO 'OTES
OTES
215 To a solution of 205 (1.0 equivalents) in pyridine Molar), is added trimethylacetyl chloride (pivaloyl chloride) equivalents) at 0 The reaction is stirred for 2 hours and then diluted with diethylether and washed with ammonium chloride copper sulfate brine dried over MgSO4 and concentrated. Next a solution of the crude intermediate equivalents) is dissolved in methylene chloride (.10 Molar) and diisopropylethylamine (3.3 equivalents) is added at 0 °C.
Subsequent addition of triethylsilyl trifluoromethanesulfonate (3.3 equivalents) is followed by stirring for 2 hours and then the reaction is diluted with diethylether and washed with ammonium chloride brine (IX) and then dried (MgSO4) and concentrated. Purification by flash column chromatography affords compound 215. scheme 23 step j SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCT/US96/03227 -118- Preparation of 216 PIVO
CO
2 Me TE.SO "OTES
OTES
216 To solution of 215 in CC14 (.33 CH3CN (.33 M) and water (.22 M) at 20 'C is added RuC13 (.03 equiv.) and NaIO4 equiv.) and the muddy black mixture is allowed to stir for min. The mixture is then diluted with ether (25 mL), washed with water (2X 5.0 mL) and brine (1X 5 mL). The aqueous layer is back extracted recombined, and the organic layer is then dried MgSO4 and evaporated. The crude is then resuspended in a mixture of methylene chloride/water .1 M total) and diazomethane (1.2 equivalents) is gradually dropped into the flask via an addition funnel at the rate of 1 drop/10 seconds.
After complete addition the mixture is diluted with ether, washed with brine (2X) and the aqueous layer is back extracted (2X) recombined, and the organic layer is then dried MgSO4 and evaporated. Purification by flash column chromatography affords the compound 216. scheme 23 step k SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCT/US96/03227 -119- Preparation of 217 PIVO
CH
2
OH
TESO
"'OTES
OTES
217 To a solution of 216 (1.0 equivalents) in methylene chloride (.10 Molar) is added a 1.0 M solution of DIBALH in methylene chloride from Aldrich chemical company (1.2 equivalents) at 0 OC. Subsequent stirring for 2 hours is followed by dilution with diethylether and washing with sodiumpotassium tartrate brine (IX) and then MgSO 4 The solution is then concentrated and purification by flash column chromatography affords compound 217. scheme 23 step 1 Preparation of 218 0 TESO
"OTES
OTES
218 To 217 (1.0 equivalents) in pyridine (.10 Molar), is added dimethyoxytritylchloride (DMT chloride) (1.1 equivalents) at 0 o C. The reaction is stirred for 2 hours and then diluted with SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCT/US96/03227 -120diethylether and washed with ammonium chloride copper sulfate brine dried over MgS0 4 and concentrated.
Purification by flash column chromatography affords compound 218. scheme 23 step m Preparation of 220 HO ~0 TESO
"OTES
OTES
220 To a solution of 218 (1.0 equivalents) in diethylether (.08 is added lithiumaluminumhydride (LAH) (1.5 equivalents) at The reaction is refluxed for 2 hours and then quenched with methanol and diluted with ether. The reaction is next worked-up with sodium potassium tartrate brine (IX) and is then dried (MgSO 4 and concentrated. Purification by flash column chromatography affords compound 220. scheme 23 step n SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCT/US96/03227 -121- Preparation of 221 DMTO 0 N 2 TESO
"OTES
OTES
221 To Tetrol 205 (1.0 equivalents) in pyridine Molar), is added dimethyoxytritylchloride (DMT chloride) equivalents) at 0 The reaction is stirred for 2 hours and then diluted with diethylether and washed with ammonium chloride copper sulfate brine dried over MgSO 4 and concentrated. Next a solution of the crude intermediate equivalents) is dissolved in methylene chloride (.10 Molar) and diisopropylethylamine (3.3 equivalents) is added at 0 °C.
Subsequent addition of triethylsilyl trifluoromethanesulfonate (3.3 equivalents) is followed by stirring for 2 hours and then the reaction is diluted with diethylether and washed with ammonium chloride brine (lX) and then dried (MgS04) and concentrated. Purification by flash column chromatography affords compound 221. scheme 23 step a SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCIfUS96/03227 -122- Preparation of 222 DMTO O CO 2
H
TESO 'y"OTES
OTES
222 To solution of 221 in CC14 (.33 CH3CN (.33 M) and water (.22 M) at 20 °C is added RuCl3 (.03 equiv.) and NaIO4 equiv.) and the muddy black mixture is allowed to stir for min. The mixture is then diluted with ether (25 mL), washed with water (2X 5.0 mL) and brine (1X 5 mL). The aqueous layer is back extracted recombined, and the organic layer iss then dried MgSO4 and evaporated. Purification by flash column chromatography affords the compound 222. scheme 23 step Preparation of 224
.CH
2
OH
TESO'
OTES
224 To a solution of 222 (1.0 equivalents) in diethylether (.08 SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCTUS96/03227 -123is added lithiumaluminumhydride (LAH) (1.5 equivalents) at OC. The reaction is refluxed for 2 hours and then quenched with methanol and diluted with ether. The reaction is next worked-up with sodium potassium tartrate brine (IX) and is then dried (MgSO 4 and concentrated. Purification by flash column chromatography affords compound 224. scheme 23 step p Preparation of 216 To a stirred solution of the acid 214 (1.0 equivalents) in dimethylformamide (.10 Molar) at 25 is added 1hydroxybenzotriazole (HOBT; 1.1 equivalents). Next dicyclohexylcarbodiimide (1.2 equivalents) is added and the reaction is stirred for 1 hour in the presence of an appropriately substituted solid support 2 -Aminoethyl)-3-amino-propyl glass; aminopolystyrene resin; aminopropyl glass; isothiocvanato glass, all with or without a linker extending from the amino group on the support etc. from Sigma Company). The mixture is then diluted with ether, filtered and the filtrate is washed with aqueous NaHCO 3 water and brine The organic phase is dried over MgSO 4 and then concentrated.
Preparation of 226; 228; 230 or 232 To a stirred solution of the acid 214; 62; 215 or 62 SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCT/US96/03227 -124equivalents) and the amine 216; 226; 228 or 230 (1.1 equivalents) in dimethylformamide (.10 Molar) at 25 oC, is added 1-hydroxybenzotriazole (HOBT; 1.1 equivalents). Next dicyclohexylcarbodiimide (1.2 equivalents) is added and the reaction is stirred for 14 hours. The mixture is diluted with ether, filtered and the filtrate is washed with aqueous NaHCO 3 water and brine The organic phase is dried over MgS0 4 and then concentrated. Purification by flash column chromatography and then reexposure of the intermediate compound (1.0 equivalents) in dimethyl-formamide (.10 Molar) at 25 oC, is added piperidine (1.1 equivalents). The reaction is stirred for 1 hour and is then diluted with ether, and washed with aqueous CuSO 4 water and brine The organic phase is dried over MgSO 4 and then concentrated.
Purification by flash column chromatography affords compound 226; 228; 230 or 232, respectively, scheme 24 Preparation of 234 To a stirred solution of 232 (1.0 equivalents) in acetonitrile Molar) is added an HF-pyridine solution (.50 M) from Aldrich chemical company. The reaction is allowed to stir for five hours and is then condensed. The crude 234 oligomer is then resuspended in p-dioxane (.50 Molar) to which is added a Molar solution of NaOH (3.0 equivalents). The reaction is stirred SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCT/US96/03227 -125for 1 hour at 50 °C and is then quenched with aqueous NH 4 C I (2X) and subsequently lyophilized. Purification by HPLC chromatography affords compound 234. scheme 24 SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCTrUS96/03227 -126- Preparation of Peptoid Combinatorial libraries (Scheme 500) A depiction of the generation of a combinatorial library for oligopeptoid compounds is shown in scheme 500. The example uses an alphabet of eight D-aldose hexose sugars (other sugars groups such as the D/L ketoses and L-configurations of aldose hexoses, may be used) and carries the synthesis to a degree of three or 512 compounds. (The process can repeat itself to afford the library of desired size). Standard chemistry is shown and follows the reaction conditions as described above herein for peptoid synthesis. The solid support used is the standard N-(2- Aminoethyl)-3-amino-propyl glass support; amino-polystyrene resin; aminopropyl glass; isothiocyanato glass and others as purchased from Sigma company. All supports may be with or without a linker extending from the amino group on the support (eg. succinate linkage, amide, ether, alkyl chain with terminal carbon activated as free alcohol, bromide etc.).
SUBSTITUTE SHEET (RULE 26) A: FMCHHB: FONC D: E: F:CHH2 CO G: H:
FONC
2 0 COOH FONC 2 0 COOH FMOCNHCH 2 0 COOH ONC 2
~CO
OCNHICH
2 0 CO0H E~FMOCNHCH2 0 C0OH r' FMOCNHCH 2 0 .C0OH r FMOCNHCH 2 0 COOH r' TTE S ESO 6 TEHHA TSXHTES ESOTE NH )NHc TETES HOXfNHAc TE0 <NHAc 1 NHEc OTESH TESO NHCOTES TS OTES OTES OTES OTES OTES D-Glucose amine D-Allose amine D-Altrose amine 0-Mannose amine D-Gulose amine 0-Idose amine D-Ghlactose amine D-Talose amine LAderived derived derived derived derived derived derived derived C 1.0DCC. HOBT, E1 3 N, DMF 1. DCC, HOBT, UP 3 DMF 1.000C, HOBT. E1 3 N, DMF 1. DCC, HOBT, EI 3 N. DMF w 1.000, HOBT, EI 3 N, EMF 1. DCC, H0BT, EIN, 'MF 1 1. CC, HOBT. E1 3 N, IMF 1I1. DCC, H0BT, Et 3 N. DMF HNiQ HN-1j HN-RJ HN4Q H 2
H
2 N-RJ H 2 N4Q H 2
N-R
2.8 eiie I piperidineF2-ipriin, M DMF 12. piperidine, DMF 2.8 aeide, 2PrdnDMF H2telie NCH INCHerdie DM 2 In 2. Hip in DMF 2. q~eiddine, DMF
H
2
NCH
2 0 2NCH2 2 H 2
CH
2 0 IA NCI' 2 0 NQ 2o4 H2NCH2 rO,2J% H2NCI2 0~N NHSOc TESO *NH1Ac TESO' NHj-Ic TSO N14c TEOfNHAcTSYNHAc TESO'N NHAC TSV~ NHAc OIES 6TES OTES OTES OTES OTES OTES OTES A-SB=-S =C-s =0-S =E-S =F-S =G-S =H-S Combine, randomize and divide: LIBRARY 1 y _8=8COPNS e.g. Couple with
OE
Scheme 500A Combine, randomize and divide: LIBRARY I (8=8CMPNS Couple with H, NCH, 0
N
e.g. I TSO NHAc
OTES
1: FMOCNHC11 2 0 COOH TESO4XOTES
OTES
0-Glucose derived 1. DCC, HOST, Et 2. piperidine, DMF I-X a
(XI=A.H)
S support
)CNHCH
2 0 -0 TESO IXOTE%
OTES
D-Allose derived ,,1RC HOBT, El I 2. plperidine. DM J- a
(X
1
=A-H)
S support
K:
)CNHCH
2 0 COO1 I TEO' 4
~FM:
TS OTES
OTES
0-Allrose derived N, DMF 1. DCC, HOBT, E 2. piperidine. DM' K-X-
L
(X
1
=A.H)
S =suppo FMc CI
)CNH-CH
2 0 COOH
TESOL
4 X0TES
OTES
D.Mannose derived 1. DCC, HOST, t3 3 N. DMF 2. piperidine, 0 F
(X
1
=A-H)
rt S support
NJHCH
2 0 CooH TESO .0TES
OTES
0-Gulose derived N, DMF 1. DCC. HOST, 2. piperidine.
DI
M-X.
L
(X
1
=A-
S suppc
FMC
'NHCH
2 0 COOH TESOJIqrXTES
OTES
D-Idose derived 1.0DCC, HOST, I E1 3 N. DMF IF 2. piperidine, Ui
CNHCH
2 0 OI TESO';f'OTES
OTES
D-Galactose derived t 3 N, DMF 1. DCC. HOST, E1 3 2. piperidine, DMF NHCH, 0 COOH
TESOAOTES
OTES
D-Talose 1. De 'r-WK. Et, qJ. DMF 2. piperidine. DMF J, DMF
N-X-
1
X=A-H)
S support O--a
(XI=A-H)
S support a-- S support Combine, randomize and divide: LIBRARY2 1 64CtNS 0 01 l- 2
NCH
2 0IO C. N 0 J I TEOTES OTES J Scheme 500B Combine. randomize and divide: LIBRARY2 1 (82 64 COMPUNDS) I 0 0
V
2
NCH
2 0 N 0- N.~ e.g. TESO 0 NHAc OTIES OTES 0
'C
a,' '0
I
Couple wi A: B: I C: D: E: F; G; H: HOBT, E1 3 N, DMF 1. DCC, HOBI, Et 3 N, DMF 1. DCC, HOBT, UP., DMF 1. DCC. HOBT. Et 3 N, DMF 2. piperidine. 0 AF 2. piperidine, 0 VF 2. piperidine. 0 AF 2. piperidine. DI F 2. piperidine, DNF 2. piperidine, 0 F 2. piperidine, 0 AF 2. piperidine. M IF 1.CC IfBT ifN ifF 1. ICfHBtNDF1 CHOT~tN 1. IC. fOT~ 3 N M A-XX,-
U
(Xi=A-H)
(X
2 =i-P) S support
B-X
2
XI
1 I C-X,2lt So D-X 2 Xr- E-X 2 Xl 1
F-X
2
XI
1 E G-X 2
XI
1 H-XaXI-
(X
1
=A-H)
(X
2
=I-P)
S support
(X
2
=I-P)
S support
(X
1
=A-H)
(X
2
=I.P)
S support
(X
1
=A.H)
(X
2 =1.P) S support (X
(X
2
=I.P)
S support
(X
2 =l.P) S support
(X
1
=A.H)
(X
2 =1.P) S support Combine, randomize and divide: LIBRARY 3 L 12COMUN3r 0 9 0
HNCH
2 0 N'0 C-N: 0 N-QJ e.g. r. _HF_ IOTE OTES OTES OTES Scheme 500C 0 A: B; c: D: E: F: G:
H:
)MTOy -rOH DMTO OH DMTO 0 'O DMTO OH DMTOX 0OH DMTO 0 OH DM*TO':O OO OH T E S O N HEc T E ON ~E O N A c E S O H A c T E S O N N H A c T E S O N A H C SNHA c T E O N H A c OTSOE TSOTES OTES OTES OTES OTES 0-Glucose amine 0-Allose amine 0-Altrose amine D-Mannose amine Guoeaie 0doemne -Gllsemne 0Toeaie der ve d ri edder ve d ri edderived derived derived derived kA1. DCC, HOST, EI 3 N, DMF I1.0CC, HOST, Et 3 N, DMF 1. DCC, HOST, Et 3 N, DMF 10C OT tN M C 1*C~HBE 3 N. HOF 1. DCC, HOST, E1 3 N, MF DCC, HOST, E 3 N. IMF 1. D CC, HOST, Et 3 N, DM IM HHOO HCG7 IHooC.--fJ~j o-oOc-1J ElC HO-- 'oc HOO.-J 03, l CH 3 j THFF 2o CICC0 2.CI 3 CCOOHN,, CH 3 2.C 2.C 3 CCOOH CHNJ.TH? H, H3N.T(iFCOOH CN C HCHNTH 2- CI 3 CCDH, CH 3
N
3 HO GOH HN ,CNOTHIP -C1 3 CCOOH, CH 3 NO,, THF H O C J HO\ OCdQ HOy O C4IJ HO OC T f O H uC"rlI. Co H NHA HO HO*4 1 v-o- HOYOr C,;j TESO NHc TESO NHAc TESO*' NHAc TEO TESON TES ,NcTESO INNHAc TESO--INHAc ni OTES 6 TES OTES OTES OTES OTES OTES
OE
=A-S =-SC-S D-S E-S F-S G-S =1-S
M
C
cyl Comb~~Ljin andoe nivide: LIBRARY I Scheme 550A Combine. randomize and divide: LIBRARY 1 I -8=8CMPNS [HO 0 OC(F 1.
ITESO Y NHAc OTES Couple wi~th I: j: OCE OCE 0 P.NIP DMO OPl TE 0' N OTES TESO' 'OTES OTES 0
OTESI
K:
MATO-'O,-OPN
TESO-' T-OTESN
OTES
D-Altrose derived 12 1. telrazole, OH 2 12 2. m-OPBA, OH 2 3. AcOHITHF/H, L: OCE vIO 0
TESO--'-OTEI
OTES 0-Glucose derived 1. tetrazole, CH 2 C1 2. m-CPBA, CH 2 01 3. AcOHITHF/H 29 D-Allose derived 1. leirazole, CH 2
C
2. m-CPBA, CH 2
C
3. AcOHrrHF/H 2 D-Mannose derived 1. lelrazole, OH;C 2. m-CPBA, OH O 3. AcOH/THF/
NO
LX
PF
2
QCE
D-G
9 lW derived 12 1. tetrazole, CH 2 O1 12 2. m-OPBA, CH 2 O1 I3. AcOH/THF/H 2 9 N: 0C )r2TESO TE I
OTES
D-Idose derived 2 1. ltrazole, OH 2 2 2. m-CPBA, OH 2 3. AcOH/TH
F/H
2 N'Pr 2
OCE
)MTO 0 -O..N TESO '-OTES
OTES
D-Galactose derived 12 1. letrazole, CH 2
I;
12 2. m-CPBA, CH 2 OIh I 3. AcOHrTHF/H 2
O,
r. OCE 0MATO> Oy N t Pr 2 rr2 TESOV...<.OTES
OTES
D-Talose derived 1. telrazole, OH 2
OI
2. m-OPBA, OH 2 OI1 3. AcOHfTHF/H 2 0 J-x-
U
(X=A-H)
S support K-X-
US
(X=A-H)
S support N-X- a S support a--E
(X=A-H)
S support P--a
(X=A-H)
S support
(X=A-H)
S support
(X=A-H)
S support
(X=A-H)
S support Combine, randomize and divide: LIBRARY2 1 1132 64 COMpUNS) Scheme 550B Combine, randomize and divide: IRAY Couple Will? HO\ 0 e.g. TESO OTEPTESOV HAc OTES
OTES
A: OCE TO-0 OP.1 ESO NIIAc OMI 0
OTESTE
0-Glucose amine derived I. telrazote, CH- 2
C
2. m-CPBA, CH- 2
C
3. AcOHTHFlH 2
A.X
2 Xj-
U
(X
1
=A-H)
(X
2 =i-P) S support
OCE
S NHAC
OTES
D-Allose amine derived 1. letrazole,
CH
2
C
2. m-OPBA,
CH
2
C
3. AcOHIIFHF/1- 2 C: 0C p'rEO- 0 P Ir-EO NHAc 0
OTES
D-Alirose amino derived 12 1. tetrazole, CH 2 12 2. m-CPBA, OH 2 1 3. AcOHITHF/Hj E 0: qJ Pr 2 OCE \ATO 0*y P TESO-'- 'NHAc
OTES
D-Mannose amine derived 12 1 felrazole, CH; ~2 2. m-CPBA,
CH,
3. AcOH/THF/H DMTO -P F: C I G: OCE H: TESO I )TO IPE O10 WT 0Nr Pr 2 ,NHAc 0 PrIEON'.H MO 0 .Pr OTES TESO NHwAc OTES TESO NHNWc OTES
OTES
0-Gulose amine D-Idose amine D-Galaclose amine 0-Talose amine 1.derived derived derived derived C12 1.tetrazole, CI-12C 2 I. tetrazole OH 2 12 1. telrazole, CH- 2 OI I. telrazole, 0H- 2 01 022-CPA HC 2. mn-CPBA OH 2 12 2. m-CPBA, CH 2 CI 2. m-CPBA, H 2
CI
t3. AcOHfrHFIH 2 4 3. AcOH/THF/H- 2 3. AcOH/THF/H 2 0 3. AcOHITHF/H 2
O
B-X
2 XI- i C-X 2
X
1 Id D-X 2
X
1
E-.X
2
X
1
F-X
2
X
1 Ej G-X 2
X
1
H-X
2 XI- .i
(X
2 =1.P) S support
IAI=A-)
(X
2 =1-P) S support
(X
1
=A-H)
(X
2 =1-P) S support
(X
1
=A-H)
(X
2
=I-P)
S support
(X
1
=A-H)
(X
2 =1-P) S support
(X
2 =l.P) S support
(X
1
=A-H)
(X
2 =1-P) S support Combine, randomize and divide: LIBRARY 3 W =512 CQPU trNrS s Scheme 550c e~.tEOOTES OTES
OTES
WO 96/27379 PCTIUS96/03227 -133- Preparation of Nucleotoid Combinatorial libraries (Scheme 550) A depiction of the generation of a combinatorial library for oligonucleotoid compounds is shown in scheme 550. The example uses an alphabet of eight D-aldose hexose sugars (other sugars groups such as the D/L ketoses and L-configurations of aldose hexoses, may be used) and carries the synthesis to a degree of three or 512 compounds. (The process can repeat itself to afford the library of desired size). Standard chemistry is shown and follows the reaction conditions as described above herein for carbonucleotoid synthesis. The solid support used is the standard N-( 2 -Aminoethyl)-3-amino-propyl glass support; amino-polystyrene resin; aminopropyl glass; isothiocyanato glass and others as purchased from Sigma company. All supports may be with or without a linker extending from the amino group on the support (eg. succinate linkage, amide, ether, alkyl chain with terminal carbon activated as free alcohol, bromide etc.).
Preparation of compound 2000. To a solution of 76 equiv) was added methylene chloride M) and benzaldehyde 1.1 equiv), and the solution was exposed to ZnCl (1.1 equiv) at °C and allowed to stir for 2.5 hour. The solution is then SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCrTUS96/03227 -134diluted with ether and then washed with a saturated solution of sodium bicarbonate water brine (lX) and then dried over MgSO4. The compound is purified by flash column chromatography to yield the desired benzylidene.
SUBSTITUTE SHEET (RULE 26) WO 96/27379 WO 9627379PCTIUS96b03227 -135- 1. Synthesis of a C-2 differentiated sugar HO' a CO 2 Et 1. Ph( H0 VOH HO 2. Bu the 76 BnO 0 4. Ph' H0 -0 JPh 0 OBn 2020 BnBr, NaH, THF ~HO, ZnGI, CH 2 C 0 CO 2 Et
'OH
2 SnO, MeCH OBn ~n BnBr, CsF, DMF 2C00 3. NaCl (3 A sie
IBH
3 (5 eq), OME yes, TFA 10 eq) ~HO, ZnCI. 0H 2
CI
2 BnO 0 OH H0 0-OH OBn 2010 BnO 0 0 BnO h OBn 2030 6. NBS, BaCO 3 0 H BnO OBz CC1 4
-C
2
H
2
CI
4
H
2 0 Bn'OBn 2040 7.
,,C
0H 2
CI
2 ('Pr) 2 EtN BnO- 0 0-PN'(Pr) 2 BnOV Bz OBn 2050 Bz Benzoate Bn Benzyl ether Scheme 2000a SU BSTITUTE S H EET (RU LE 26) WO 96/27379 WO 9627379PCTfUS96/03227 -136- 2.Connection of the C-2 differentiated sugar to a solid support BnO
OH
BnO_ _08z OBn 2040 1. NaH, THF BnO *0 N-JIold uppo 2. then add to B O 0H Br--N ~Poj2060 0 3.1% NaOH, MeOH Scheme 2000b SU BSTITUTE S HE ET (RU LE 26) WO 96/27379 PCTrUS96/03227 -137- The benzylidene is then azeotroped with benzene (2X 100 mL) and then dried overnight under vacuum over P205. A mixture of benzylidene, dibutyl tin oxide (1.2 equiv.) and dry methanol (.25 M) are heated at reflux for 4 h until the solution became clear and homogeneous. (An automatic stirring apparatus may be necessary.) The solvent is next removed in vacuo to give a foamy white tin complex which was then azeotroped with benzene (2X) and dried (2 h to overnight) under vacuum over P205. Next, anhydrous DMF is added to redissolve the tin complex and then CsF (1.2 equiv.) and finally Benzyl bromide equiv.) are added and then heated (40 overnight. The clear solution is partially distilled under vacuum, (3.3 mm Hg, 75-100 to obtain 1/5 the original volume of solvent.
Reaction mixture was then diluted with ethyl acetate (2L) and washed with a small amount of water (2X) to remove cesium salts. Aqueous layer is back extracted with ethyl acetate (3X) and then recombined with the organic layer which was then dried over MgSO4 and evaporated. Purification by flash column chromatography yields the desired benzyl ether 2000. For related chemistry see Nagashima, Ohno, M. Chemistry Letters, Chem. Soc. of Japan 1987, 141.
SUBSTITUTE SHEET(RULE 26) WO 96/27379 PCT/US96/03227 -138- Preparation of compound 2010.
Procedure adopted from Johansson Samuelsson; B. J. Chem.
Soc., Chem. Commun., 1984, 201. To a solution of the benzylidene acetal (1 equiv) and sodium cyanoborohydride equiv.) in DMF (.125 M) containing powedered 3 angtrsom molecular sieves is added trifluoroacetic acid (10 equiv) and the reaction is allowed to stir at 0 °C until no starting material remains. Reaction mixture is then diluted with ethyl acetate (2L) and washed with a small amount of water (2X) and brine Aqueous layer is back extracted with ethyl acetate (3X) and then recombined with the organic layer which was then dried over MgSO4 and evaporated. Purification by flash column chromatography yields the desired benzyl ether 2010.
Preparation of compound 2020.
To a solution of 2010 (1.0 equiv) was added methylene chloride M) and benzaldehyde 1.1 equiv), and the solution was exposed to ZnCl (1.1 equiv) at 25 °C and allowed to stir for hour. The solution is then diluted with ether and then washed with a saturated solution of sodium bicarbonate water (2X), brine (IX) and then dried over MgSO4. The compound is purified by flash column chromatography to yield the desired benzylidene 2020.
SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCTIUS96/03227 -139- Preparation of compound 2030.
To a solution of alcohol 2020 (22.0 g, .1068 mol, 1.0 equiv.) in THF (0.5 M) at 0 0 C, is added NaH (1.0 equiv., 35% dispersion in mineral oil) over several portions. The reaction mixture is warmed to room temperature and stirred lh. Next, the reaction iss cooled to 0 °C and treated with benzyl bromide (1.0 equiv.) and stirred for 1.5 h. A saturated solution of ammonium chloride (50 mL) is added dropwise to quench the reaction mixture at 0 OC and the mixture was diluted with ethyl acetate, washed with water brine dried over MgSO4 and evaporated. Purification by flash column chromatography yields tribenzyl ether 2030.
Preparation of compound 2040.
Procedure as adopted from Hanessian Organic Syntheses 1987, 243. To a suspension containing 1.0 equivalent of benzylidene 2030 in one molar carbon tetrachloride and 1,1,2,2-tetrachloroethane (1.5 equivalent) is added 1.2 equivalents of N-bromosuccinimide and 0.5 equivalents of barium carbonate. The resulting suspension is heated at the reflux temperature of the mixture with mechanical stirring for a period of 2.5 hour and filtered while hot. The solution is washed with water then dried over anhydrous sodium sulfate and evaporated. Purification by flash column chromatography yields SUBSTITUTE SHEET(RULE 26) WO 96/27379 PCT/US96/03227 -140tribenzyl ether 2040.
Preparation of compound 2050.
To a solution of 2040 (1.0 equivalents) in methylene chloride (.10 is added diisopropylethylamine (4.0 equivalents) at The reaction is stirred for 5 minutes and then 2-cyanoethyl- N,N-diisopropyl-chlorophosphoramidite (1.5 equiv) is added, as prepared from the procedures of Sinha et al. Nucl. Acids Res.
1984, 12, 4539. After 15 minutes the reaction is SUBSTITUTE SHEET (RULE 26) WO 96/27379 PrU9I32 PCT/'US96/03227 -141- Synthesis of a 01-02-Phosophodiester oligomer using a solid support B nO -'CO O'P'N'(Pr) 2 BnO 0 Sldupjort BnO 0~ BnO- 'OH 0 BnO BnO 2050 2060 S1. tetrazole, CH 3
CN
2.MCPBA,
CH
2
CI
2 3.1% NaOH, MeOH, 200C BNO 0 Solid>NfSup ort nO 0 BnO I
C
BnO 0 BnO Y OBn
OH
S1. 2050. then tetrazole, CH 3
CN
2.MCPBA, 0H 2 C1 2 3. 1 NaOH, MeOH, 2000 BnO 0 N-Sjort BncY '0 BnO 0 :p-O 0
CN
BnO-- 0 Bn& 0- BnOI B nO Cul j BnO'O01n
OH
ITERATE
Scheme 2001 SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCT/US96/03227 -142brine (IX) and is then dried (MgSO 4 and concentrated.
Purification by flash column chromatography (silica, 30% ethyl acetate in petroleum ether) affords compound 2050 (as shown in scheme 2000).
Preparation of compound 2060 To a solution of alcohol 2040 (1.0 equiv.) in THF (0.5 M) at 0 0
C,
is added NaH (1.0 equiv., 35% dispersion in mineral oil) over several portions. The reaction mixture is warmed to room temperature and stirred lh. Next, the reaction is cooled to 0 °C and exposed to the solid support functionalized with a bromide linker or any reasonable leaving group attached (1.0 equiv.) and stirred for 2 hours. A saturated solution of ammonium chloride mL) is added dropwise to quench the reaction mixture at 0 °C and the support was washed with ethyl acetate, 1% NaOH in methanol (2X) to remove the benzoate and finally brine (IX) to give 2060. The solid support used is the standard N-(2- Aminoethyl)-3-amino-propyl glass support; amino-polystyrene resin; aminopropyl glass; isothiocyanato glass and others as purchased from Sigma company. All supports may be with or without a linker extending from the amino group on the support (eg. succinate linkage, amide, ether, alkyl chain with terminal carbon activated as free alcohol, bromide etc.).
SUBSTITUTE SHEET (RULE 26) WO 96/27379 WO 9627379PCr/US96/03227 -143- Synthesis of a C-3 differentiated sugar H 0 0HC
HO
76 1. PhCHO, ZnCI, CH 2 C1 2 B '--r-OH OHW' *OH 2. NaCNBH, (5 eq), DMF OH7 (3 A sieves, TFA 10 eq) 27 3. TBDPSCI, Et 3 N, CH 2
CI
2 4. Bu 2 SnO, MeOH, IBzBr, CsF, DMF BnO
OH
B n 2090 7.
CI
5. BnBr. NaH, THF 6. TBAF, THF BnO"*llr-rOTBDPS HO-'r'OH OBz 2080 ll N'(Pr) 2
CH
2
CI
2 'Pr) 2 EtN Bz benzoate Bn benzyl Scheme 2002a SUBSTITUTE SHEET (RULE 26) WO 96/27379 WO 9627379PC1'1US96/03227 -144- 2.Connection of the C-3 differentiated sugar to a solid support Bno H BnO e OBn BzQ 2090 1. NaH, THF 2. then add to BryN§Spp 3. 1 NaOH, MeOH Scheme 2002b Bno' Bn 0
HO
2110 SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCrIUS96/03227 -145- Preparation of compound 2070 To a solution of 76 (1.0 equiv) was added methylene chloride (.1 M) and benzaldehyde 1.1 equiv), and the solution was exposed to ZnC1 (1.1 equiv) at 25 °C and allowed to stir for 2.5 hour. The solution is then diluted with ether and then washed with a saturated solution of sodium bicarbonate water brine (1X) and then dried over MgSO4. The compound is purified by flash column chromatography to yield the desired benzylidene.
Procedure adopted from Johansson Samuelsson; B. J. Chem.
Soc., Chem. Commun., 1984, 201. To a solution of the benzylidene acetal (1 equiv) and sodium cyanoborohydride equiv.) in DMF (.125 M) containing powedered 3 angtrsom molecular sieves is added trifluoroacetic acid (10 equiv) and the reaction is allowed to stir at 0 oC until no starting material remains. Reaction mixture is then diluted with ethyl acetate (2L) and washed with a small amount of water (2X) and brine Aqueous layer is back extracted with ethyl acetate (3X) and then recombined with the organic layer which was then dried over MgSO4 and evaporated. Purification by flash column chromatography yields the desired benzyl ether 2070.
Preparation of compound 2080 To a solution of 2070 (1.0 equivalents) in methylene chloride Molar), is added triethylamine (1.1 equivalents) at 0 °C.
SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCTIUS96/03227 -146- Subsequent addition of tertbutyldiphenylsilylchloride (1.1 equivalents) is followed by stirring for 2 hours and then the reaction is diluted with diethylether and washed with ammonium chloride brine (lX) and then dried (MgSO 4 and concentrated. Purification by flash column chromatography affords the TBDPS ether which is subsequently carried on as follows: The TBDPS ether is then azeotroped with benzene (2X 100 mL) and then dried overnight under vacuum over P205. A mixture of benzylidene, dibutyl tin oxide (1.2 equiv.) and dry methanol M) are heated at reflux for 4 h until the solution became clear and homogeneous. (An automatic stirring apparatus may be necessary.) The solvent is next removed in vacuo to give a foamy white tin complex which was then azeotroped with benzene (2X) and dried (2 h to overnight) under vacuum over P205. Next, anhydrous DMF is added to redissolve the tin complex and then CsF (1.2 equiv.) and finally Benzoyl bromide for the benzoate formation, (1.5 equiv.) are added and then heated (40 overnight. The clear solution is partially distilled under vacuum, (3.3 mm Hg, 75-100 to obtain 1/5 the original volume of solvent. Reaction mixture was then diluted with ethyl acetate (2L) and washed with a small amount of water (2X) to remove cesium salts. Aqueous layer is back extracted with ethyl acetate (3X) and then recombined with the SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCrIUS96/03227 -147organic layer which was then dried over MgSO4 and evaporated.
Purification by flash column chromatography yields the desired benzyl ether 2080. For related chemistry see Nagashima,
N.;
Ohno, M. Chemistry Letters, Chem. Soc. of Japan 1987, 141.
Preparation of compound 2090 To a solution of alcohol 2080 (1.0 equiv.) in THF (0.5 M) at 0 °C, is added NaH (1.0 equiv., 35% dispersion in mineral oil) over several portions. The reaction mixture is warmed to room temperature and stirred lh. Next, the reaction is cooled to 0 °C and treated with benzyl bromide (1.0 equiv.) and stirred for h. The compound is then treated with tetrabutylammonium fluoride (2.0 equivalents) and allowed to stir for an additional 2 hours. A saturated solution of ammonium chloride (50 mL) is then added dropwise to quench the reaction mixture at 0 °C and the mixture was diluted with ethyl acetate, washed with water brine dried over MgSO4 and evaporated. Purification by flash column chromatography yields tribenzyl ether 2090.
Preparation of compound 2100 To a solution of 2090 (1.0 equivalents) in methylene chloride is added diisopropylethylamine (4.0 equivalents) at The reaction is stirred for 5 minutes and then 2-cyanoethyl- N,N-diisopropyl-chlorophosphoramidite (1.5 equiv) is added, as SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCT/US96/03227 -148prepared from the procedures of Sinha et al. Nucl. Acids Res.
1984, 12, 4539. After 15 minutes the reaction is complete and is next diluted with ether and next washed with brine (IX) and is then dried (MgSO 4 and concentrated. Purification by flash column chromatography (silica, 30% ethyl acetate in petroleum ether) affords compound 2100 (as shown in scheme 2002).
SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCrIUS96/03227 -149- Synthesis of a C1 -C3-Phosophodiester oligomer using a solid support BnO-,O,-D-P.N'(Pr) 2 BnOY 1 OBn BzO 2100 BnO N- r BnO- 'QBn 0
OH
2110 S1. tetrazole, CH 3
CN
2MCPBA,
CH
2
CI
2 31% NaOH, MeOH, 1. 2100, then tetrazole, CH 3
CN
2. MCPBA,
CH
2 Gi 2 3. 1 NaOH, MeOH, 20 0
C
BnO o N- dUjrt BnO a O Bn 0 0 :p_ 0
-',CN
n0 Bn H
ITERATE
Scheme 2003 SU BSTITUTE S H EET (RU LE 26) WO 96/27379 PCT/US96/03227 -150- Preparation of compound 2110 To a solution of alcohol 2090 (1.0 equiv.) in THF (0.5 M) at 0 oC, is added NaH (1.0 equiv., 35% dispersion in mineral oil) over several portions. The reaction mixture is warmed to room temperature and stirred lh. Next, the reaction is cooled to 0 °C and exposed to the solid support functionalized with a bromide linker or any reasonable leaving group attached (1.0 equiv.) and stirred for 2 hours. A saturated solution of ammonium chloride (50 mL) is added dropwise to quench the reaction mixture at 0 oC and the support was washed with ethyl acetate, 1% NaOH in methanol (2X) to remove the benzoate and finally brine (IX) to give 2110. The solid support used is the standard N-(2- Aminoethyl)-3-amino-propyl glass support; amino-polystyrene resin; aminopropyl glass; isothiocyanato glass and others as purchased from Sigma company. All supports may be with or without a linker extending from the amino group on the support (eg. succinate linkage, amide, ether, alkyl chain with terminal carbon activated as free alcohol, bromide etc.).
Preparation of compound 2120 To a solution of 76 (1.0 equiv) was added methylene chloride M) and benzaldehyde 1.1 equiv), and the solution was exposed to ZnCI (1.1 equiv) at 25 °C and allowed to stir for SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCrIUS96/03227 -151hour. The solution is then diluted with ether and then washed with a saturated solution of sodium bicarbonate water (2X), brine (IX) and then dried over MgSO4. The compound is purified by flash column chromatography to yield the desired SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCT/US96/03227 -152- 1. Synthesis of a C-4 differentiated sugar HO-O CO 2 Et H0 0-H
HO
76 1. PhCHO, ZnCI, CH 2
CI
2 >'OC0E Ph OBn Pnr Nk-J fLL'IOBn 1 1 2 2 2120 3. NaCNBH 3 (5 eq), DMF (3 A sieves, TFA 10 eq) BnO '0
OTBDPS
H0 OBn OBn 2130 BnO 0 OTBOPS 4. TBDPSCI, Et 3 N, CH 2
CJ
2 BzO- OBn OBn 5. BzBr, NaH, THF 2140 6. TBAF, TI BnO
OH
BzO- OBn OBn 2150 7. c 0 Pr) 0H 2
CI
2 ('Pr) 2 EtN BnO 0 P1 6z0- OBn- OBn 2160 Bz benzoate Bn benzyl Scheme 2004a SUBSTITUTE SHEET (RULE 26) WO 96127379 PCTIUS96I03227 -153- 2.Connection of the C-4 differentiated sugar to a solid support BlnO
OH
BzoO B n OBn 2150 1. NaH, THF 2. then ald to...
BnO 0 0 Nj Siup 4rt H0 O03rn OBn 2170 3. 1% NaOH, MeOH Scheme 2004b SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCIUS96/03227 -154benzylidene and carried on as follows: To a solution of benzylidene (1.0 equiv.) in THF (0.5 M) at 0 oC, is added NaH (1.0 equiv., 35% dispersion in mineral oil) over several portions. The reaction mixture is warmed to room temperature and stirred lh. Next, the reaction is cooled to 0 °C and treated with benzyl bromide (1.0 equiv.) and stirred for h. A saturated solution of ammonium chloride (50 mL) is then added dropwise to quench the reaction mixture at 0 °C and the mixture was diluted with ethyl acetate, washed with water (2X), brine dried over MgSO4 and evaporated. Purification by flash column chromatography yields tribenzyl ether 2120.
Preparation of compound 2130 Procedure adopted from Johansson Samuelsson; B. J. Chem.
Soc., Chem. Commun., 1984, 201. To a solution of the benzylidene acetal 2120 (1 equiv) and sodium cyanoborohydride (5 equiv.) in DMF (.125 M) containing powedered 3 angtrsom molecular sieves is added trifluoroacetic acid (10 equiv) and the reaction is allowed to stir at 0 OC until no starting material remains. Reaction mixture is then diluted with ethyl acetate (2L) and washed with a small amount of water (2X) and brine Aqueous layer is back extracted with ethyl acetate (3X) and then recombined with the organic layer which was then dried over MgSO4 and evaporated. Purification by SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCr/US96103227 -155flash column chromatography yields the desired benzyl ether 2130.
Preparation of compound 2140 To a solution of 2130 (1.0 equivalents) in methylene chloride Molar), is added triethylamine (1.1 equivalents) at 0 °C.
Subsequent addition of tertbutyldiphenylsilylchloride (1.1 equivalents) is followed by stirring for 2 hours and then the reaction is diluted with diethylether and washed with ammonium chloride brine (1X) and then dried (MgSO 4 and concentrated. Purification by flash column chromatography affords the TBDPS ether which is subsequently carried on as follows: To a solution of TBDPS ether (1.0 equiv.) in THF (0.5 M) at 0 °C, is added NaH (1.0 equiv., 35% dispersion in mineral oil) over several portions. The reaction mixture is warmed to room temperature and stirred lh. Next, the reaction is cooled to 0 °C and treated with benzoyl bromide to afford benzoate formation equiv.) and stirred for 1.5 h. A saturated solution of ammonium chloride (50 mL) is then added dropwise to quench the reaction mixture at 0 °C and the mixture was diluted with ethyl acetate, washed with water brine dried over M g S 0 4 and evaporated. Purification by flash column chromatography yields tribenzyl ether 2140.
SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCTIUS96/03227 -156- Preparation of compound 2150 The compound 2140 is then treated with tetrabutylammonium fluoride (2.0 equivalents) in THF Molar) and allowed to stir for an additional 2 hours at 25 A saturated solution of ammonium chloride (50 mL) is then added dropwise to quench the reaction mixture at 0 °C and the mixture was diluted with ethyl acetate, washed with water brine dried over MgS0 4 and evaporated. Purification by flash column chromatography yields tribenzyl ether 2150.
Preparation of compound 2160 To a solution of 2150 (1.0 equivalents) in methylene chloride is added diisopropylethylamine (4.0 equivalents) at OC. The reaction is stirred for 5 minutes and then 2-cyanoethyl- N,N-diisopropyl-chlorophosphoramidite (1.5 equiv) is added, as prepared from the procedures of Sinha et al. Nucl. Acids Res.
1984, 12, 4539. After 15 minutes the reaction is complete and is next diluted with ether and next washed with brine (IX) and is then dried (MgSO 4 and concentrated. Purification by flash column chromatography (silica, 30% ethyl acetate in petroleum ether) affords compound 2160 (as shown in scheme 2004).
SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCT/US96/03227 -157- Preparation of compound 2170 To a solution of alcohol 2150 (1.0 equiv.) in THF (0.5 M) at 0 °C, is added NaH (1.0 equiv., 35% dispersion in mineral oil) over several portions. The reaction mixture is warmed to room temperature and stirred lh. Next, the reaction is cooled to 0 °C and exposed to the solid support functionalized with a bromide linker or any reasonable leaving group attached (1.0 equiv.) SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCTIUS96/03227 Synthesis of a C1-C4-Phosophodiester oligomer using a solid support BnO 0 P. Ni(Pr) 2 BzO- -OBn BnO 2160 BnO NX-N Soli H0 ,O OBn 0 -i BnO 2170 I1. tetrazole, CH 3
CN
2. MCPBA,
CH
2
CI
2 3. 1% NaOH, MeOH, 20 0
C
BnO 0 0 N4 N- rF BnO 1. 2160, then tetrazole, CH 3
CN
2. MCPBA,
CH
2
CI
2 3. 1% NaOH, Me0H, 20 0
C
BnO 0 o-,-Nf P t BnO ITERA TE Scheme 2005 SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCTIUS96/03227 -159and stirred for 2 hours. A saturated solution of ammonium chloride (50 mL) is added dropwise to quench the reaction mixture at 0 °C and the support was washed with ethyl acetate, 1% NaOH in methanol (2X) to remove the benzoate and finally brine (IX) to give 2170. The solid support used is the standard
N-(
2 -Aminoethyl)-3-amino-propyl glass support; aminopolystyrene resin; aminopropyl glass; isothiocyanato glass and others as purchased from Sigma company. All supports may be with or without a linker extending from the amino group on the support (eg. succinate linkage, amide, ether, alkyl chain with terminal carbon activated as free alcohol, bromide etc.).
Prepartion of compound 3010 Procedure as described in Methods in Carbohydrate chemistry, Whistler, II, 1963, p. 327. A mixture of 80 g anhydrous Dglucosamine hydrochloride or D-galactosamine hydrochloride from Aldrich chemical company, in 200 mL. methanol and Dowex 50 acidic resin, is stirred at the boiling point in a round bottom flask. After 24-hr. reaction time, the resin is removed by filtration and ished three times with 20 ml. of methanol. The filrate and washings are combined and concentrated to about 125 ml by rotovap. The concentrate is allowed to cool to room temperature and the product crystallizes overnight and carried on as follows: SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCT/US96/03227 -160- The methyl glycoside is dissolved in chloroform M) and to it, is added phthalic anhydride (1.5 equiv.) and the reaction mixture is allowed to reflux at 70 "C for 4 h. The product SUBSTITUTE SHEET (RULE 26) WO 96/27379 WO 9627379PCI US96/03227 -161- 1. C-2 differentiated amnine derivative HO 0O OH 1. MeOH, H' HO -0 OMe HO NH 2 HCI 2. Phthalic anhydridj-1 0 -V NPhth HO CHC1 3
HO
Glucose amine hydrochloride 3010 3. BnBr, NaH Bn& BnC 4. TMSCN, SnCI 4
OCO
2 Et rVO *NH 2 BnO 3020 D. r-LUn, t6. FMOIC-C I, K 2
C.
THF, H 2 0, 0 0
C
7. NaOEt, MeOH BnO C0 2
H
BnO' -"NHFMOC BnO 3030 03, 2. Connection of a C-2 differentiate amine sugar to a solid support BnO "0 C0 2
H
BnOV ThHFMOC BnO 3030 1. DCC, HOBT, Et 3 N, DMF 0 2dur HO-~SP~rtBnO'Y-NH 2 3040 2. Piperidine, DMF Scheme 3000 S U BSTITUTE S H EET (RU LE 26) WO 96/27379 PCT/US96/03227 -162- 3010 is then crystallized and carried onto the next step.
Preparation of compound 3020 To a solution of alcohol 3010 (1.0 equiv.) in THF (0.5 M) at 0 °C, is added NaH (3.3equiv., 35% dispersion in mineral oil) over several portions. The reaction mixture is warmed to room temperature and stirred lh. Next, the reaction is cooled to 0 °C and treated with benzyl bromide (3.3 equiv.) and stirred for h. A saturated solution of ammonium chloride (50 mL) is then added dropwise to quench the reaction mixture at 0 OC and the mixture was diluted with ethyl acetate, washed with water (2X), brine dried over MgSO4 and evaporated. Purification by flash column chromatography yields tribenzyl ether and is carried on as follows: To a solution of tribenzyl ether in nitromethane is added trimethylsilyl cyanide (3.0 equivalents) and then SnC14 (.02 equivalents). The mixture is stirred for one hour and then an aqueous solution of sodium acetate was added to hydrolyze the remaining trimethylsilyl cyanide. The mixture is evaporated and the remaining oil is resuspended in dichloromethane and washed with sodium acetate solution water brine (IX) and then dried over magnesium sulphate and concentrated. The crude solid is then recrystallized from methanol is next dissolved in ethanol (0.15 M) and then concentrated H 2 S0 4 (0.01 SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCT/US96103227 -163equivalents-catalytic) is added. The reaction mixture is heated to 85 °C for eight hours. The solution is next concentrated in vacuo and purification by flash column chromatography affords compound 3020 scheme 3000.
Prepartion of compound 3030 To a solution of 3020 (1.0 equivalents) in methylene chloride Molar), is added potassium carbonate (2.0 equivalents) at 0 0 C. Subsequent addition of 9-fluorenylmethyl chloroformate (FMOC-C1, 1.2 equivalents) is followed by stirring for 2 hours and then the reaction is diluted with diethylether and washed with ammonium chloride brine (1X) and then dried (MgSO 4 and concentrated. Purification by flash column chromatography affords product which is carried on as follows: To a solution of ester in ethanol (.13 Molar), is added sodium ethoxide (0.3 equivalents) and the reaction mixture is stirred for two hours at room temperature. The solution is then concentrated in vacuo and purification by flash column chromatography affords compound 3030 scheme 3000.
Preparation of compound 3040 To a stirred solution of the acid 3030 (1.0 equivalents) and the (1.1 equivalents) in dimethylformamide (.10 Molar) at 25 OC, is added 1-hydroxybenzotriazole (HOBT; 1.1 equivalents). Next SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCIIUS96/03227 -164dicyclohexylcarbodiimide (1.2 equivalents) is added and the reaction is stirred for 2 hours. The mixture is then exposed to the solid support and mixed for 24 hours. (The solid support used is the standard N-(2-Aminoethyl)-3-amino-propyl glass support; amino-polystyrene resin; aminopropyl glass; isothiocyanato glass and others as purchased from Sigma company. All supports may be with or without a linker extending from the amino group on the support (eg. succinate linkage, amide, ether, alkyl chain with terminal carbon activated as free alcohol, bromide The mixture is then diluted with ether, washed with aqueous NaHCO 3 water and brine Next, the compound/support equivalents) in dimethyl-formamide (.10 Molar) at 25 is added piperidine (1.1 equivalents). The support is stirred or exposed for 1 hour and is then diluted with ether, and washed with aqueous CuSO 4 water and brine The final step affords compound 3040.
SUBSTITUTE SHEET (RULE 26) WO 96/27379 WO 9627379PCr/US96/03227 -165- BnO 0 C0 2
H
BnO' "NHFMOC BnO 3030 BnO 0 1 IEDuort BnOf -NH 2 BnO 3040 I1. DCC, HOBT, EI 3 N, DMF 2.piperidine, DMF -0 BnO 0 O BnO 3030 BnO 0 0 24d~fort BnO~ 0 '-Bn
H
2 N OBn HNOBn 3050 1. OC. HOBT, Et 3 N, DMF 2.pprDC n DMF BnO -0 0 1 d ort Bno 'N O~ BnO-yO 0'H n 0 -n BnO '-'NHFMOC N):)OBn BnO B3nO-,O- 0 OBn 3030 1n H BnO 3060 1DCC, HOBT, Et 3 N, DMF piperidine, DMF Iterate as many steps as needed Scheme 3001 SUBSTITUTE SHEET (RULE 26) WO 96127379 WO 9627379PCTIUS96/03227 -166- Physical Data for scheme 9.
Pho spho raidate 138 (2 diastereoners) IR, (neat) cm- 1 3089, 2964, 2927, 2856, 2253, 1497, 1455, 1396, 1363, 1253, 1184, 1156, 1094, 1028, 978, 876, 836, 779, 735, 1H-NI'R (4 00 IvflZ, C 6
D
6 7 7.3 4 (mn, 5 H, Ph) 7. 14 Cm, H, Ph) 4. 97 (in, 4 H, CH 2 Ph) 4. 78 (in, 2 H, CH 2 Ph) 4.07-3.24 (in, 13 H, OCH, OCH 2
CH
2 CN) 1. 81 2 H, CH (CH 3 2 1. 16 (mn, 12 H, CH 3 CH) 1. 03, 1. 02 (2 s, 9 H, lBuSi), 0.20, 0.18, 0.16, 0.15, (4 s, 6 H, M4e 2 Si) HRMS: C 4 3
H
6 3
O
7
N
2 PSi, Caic. :911.3197;. found: 911.3185.
Naphthoylester 136 IR, (neat) cm- 1 3494, 3062, 2919, 1716, 1630, 1600, 1454, 1355, 1284, 1228, 1197, 1091, 779, 736; 1H-NMR (250 M'flz, CDC1 3 5 8.58 1 H, Ar) 8 .0 0 Cm, 2 H, Ar) 7 .8 9 2 H, Ar) 7. 59 (in, 2 H, Ar), 7.32 Cm, 15 H, Ph), 4.95 Cm, 3 4.90 (d, Hz, 1 H) 4.69 (mn, 3 H) 4. 52 (dd, J 3.9, 12.0 Hz, 1 H) 3 .9 1 Cdd J 2. 6, 12. 0, 1 H) 3 83 Cd, J 8.3, 1 H) 3.70 (in, 4 H) 3.96 (mn, 1 H) 2.25 Cs, 1 H, OH) HRNS: C 3 9
H
3 8 0 7 Caic. 751.1672; found: 751. 1668.
Diner 142 1K, (neat) cm-1: 3397, 3030, 2923, 2254, 1718, 1653, 1629, 1497, 1453, 1355, 1284, 1227, 1197, 1094, 1029, 780. IH-NI'R (400 M~iz, C 6
D
6 5 8.82 Cs, 1 H, Ar) 8. 26 Cd, 1 H, Ar) 7. 72 Cm, 1 H, Ar) 7. 61 Cm, 1 H, Ar), 7.48 (mn, 1 H, Ar) 7.37-6.95 32 H, Ar, Ph), 4.89-4.18 21 H, CH- 2 P:h, CH 2 -Ar, -CHT 2
CH
2 CN, ChRCH 2 -Ar and CH2OH) 3. 95-3.45 (mn, 13 H, CH- and CH 2 -sugar), 1. 71 Cs, 1 H, OH) HPYIS: C-1 70
H
72 0 5 NP caic. 1198.4718; found: 1198.4715.
SU BSTITUTE S HE ET (RU LE 26) WO 96/27379 WO 9627379PCIUS96/03227 -167- Tetrainer 150 IR, (neat) cm- 1 3420, 3064, 2924, 2255, 1721, 1497, 1455, 1357, 1278, 1028, 737. 1H-NMR~ (400 MHz, CDC1 3 68. 41 1 H, Ar) 8. 00 (in, 2 H, Ar) 7.91 (mn, 2 H, Ar), 7.55 (mn, 2 H, Ar), 7.30 (mn, 60 H, Ph),'4.93-4.05 (mn, 39 H, CH 2 Ph, CH 2 -Ar, CH 2
CH
2 CN and
CH
2 OH) 3 .88-3.27 (mn, 23 H, CH- and CH 2 -sugar) 2.58 1 H, OH) HRMS: C 1 3 2 Hl 4 0 0 3 lN 3
P
3 Caic. (M+Cs+) 2488.7738; found: 2488.7758.
Tetramer 154 TR, (neat) cm- 1 3376, 2934, 1450, 1244, 1110, 1088. 1H-NMR~ (400 MHz, D 2 0) 8 8.41 Cs, 1 H, Ar), 8.00 (in, 2 H, Ar), 7.91 (in, 2 H, Ar), 7.55 (in, 2 H, Ar) 4.93-4.05 (mn, 4 H, CH 2 -Ar and CH 2 OH) 3. 88-3.27 (in, 32 H, CH- and CH 2 -sugar) HRMS: C 3 9
H
5 9
O
3 2 P 3 Caic.
1117.2331; found: 1117 .2350.
SUBSTITUTE SHEET (RULE 26)

Claims (23)

1. An oligomeric carbopeptoid compound comprising carbohydrate amino acid subunits (CA's) coupled to one another via an amide linkage having a carbonyl carbon and an amido nitrogen represented by the following formula: CA- (CO-NH)-CA 2 wherein: CA is a first carbohydrate amino acid subunit having an anomeric carbon bonded to the carbonyl carbon of said amide linkage for forming a C-glycosidic linkage therewith and CA 2 is a second carbohydrate amino acid subunit having a non-anomeric carbon bonded to the amido nitrogen of said amide linkage.
2. In a process for synthesizing an oligomeric carbopeptoid compound, a coupling step wherein two or more carbohydrate amino acid subunits (CA's) are coupled by means of an amide linkage having a carbonyl carbon and an amido nitrogen for synthesizing said oligomeric carbopeptoid compound, said amide linkage being represented by a formula as follows: CA- (CO-NH)-CA 2 wherein: CA 1 is a first carbohydrate amino acid subunit having an anomeric carbon bonded to the carbonyl carbon of said amide linkage for forming a C-glycosidic linkage therewith; and CA 2 is a second carbohydrate amino acid subunit having a non-anomeric carbon bonded to the amido nitrogen of said amide linkage. SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCT/US96/03227 -169-
3. A library of oligomeric carbopeptoid compounds employable for drug screening, each oligomeric carbopeptoid compound including at least two carbohydrate amino acid subunits (CA's) coupled to one another via an amide linkage having a carbonyl carbon and an amido nitrogen, said amide linkage being represented by the following formula: CAI- (CO-NH)-CA 2 wherein: CA 1 is a first carbohydrate amino acid subunit having an anomeric carbon bonded to the carbonyl carbon of said amide linkage for forming a C-glycosidic linkage therewith; and CA 2 is a second carbohydrate amino acid subunit having a non-anomeric carbon bonded to the amido nitrogen of said amide linkage.
4. An improved process for synthesizing a library of oligomers, the process employing an elongation step wherein subunits are coupled to one another to produce the oligomers, wherein the improvement comprises: in said elongation step the oligomer includes at least two carbohydrate amino acid subunits (CA's) coupled to one another via an amide linkage having a carbonyl carbon and an amido nitrogen represented by the following formula: CA- (CO-NH)-CA 2 wherein: CAi is a first carbohydrate amino acid subunit having an anomeric carbon bonded to the carbonyl carbon of said amide linkage for forming a C-glycosidic linkage therewith; and SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCT/US96/03227 -170- CA 2 is a second carbohydrate amino acid subunit having a non-anomeric carbon bonded to the amido nitrogen of said amide linkage.
5. A derived carbohydrate amino acid having an anomeric carbon and non-anomeric carbons, said anomeric carbon being substituted with a carboxyl radical, each of said non-anomeric carbons being substituted with a radical selected from the group consisting of blocked hydroxyl, blocked amino, differentially protected amino, and hydrogen, with the proviso that at least one radical is a differentially protected amino.
6. A derived carbohydrate amino acid having an anomeric carbon and non-anomeric carbons, said anomeric carbon being substituted with a carboxyl radical, each of said non-anomeric carbons being substituted with a radical selected from the group consisting of blocked hydroxyl, blocked amino, unprotected amino, and hydrogen, with the proviso that at least one radical is an unprotected amino and at least one radical is a blocked hydroxyl or amino.
7. An oligomeric carbonucleotoid molecule comprising carbohydrate C-glycoside subunits (CG's) coupled to one another via a phosphodiester linkage represented by the following structure: CGI-Ci'-(O-PO(OH)-O)-CG 2 wherein: (O-PO(OH)-O) is said phosphodiester linkage; SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCT/US96/03227 -171- CGI-C 1 is a first carbohydrate C-glycoside subunit having an anomeric carbon forming a C-glycosidic bond with a carbon CI', said carbon C 1 being bonded to said phosphodiester linkage; and CG 2 is a second carbohydrateC-glycoside subunit having a non-anomeric carbon bonded to said phosphodiester linkage.
8. In a process for synthesizing an oligomeric carbonucleotoid molecule, a coupling step wherein two or more carbohydrate C-glycoside subunits (CG's) are coupled by means of a phosphodiester linkage, said phosphodiester linkage being represented by a formula as follows: CGI-C 1 (O-PO(OH) -O)-CG 2 wherein: (O-PO(OH)-O) is said phosphodiester linkage; CGI-Ci' is a first carbohydrate C-glycoside subunit having an anomeric carbon forming a C-glycosidic bond with a carbon C 1 said carbon C being bonded to said phosphodiester linkage; and CG 2 is a second carbohydrate C-glycoside subunit having a non-anomeric carbon bonded to said phosphodiester linkage.
9. A library of oligomeric carbonucleotoid molecules employable for drug screening, each oligomeric carbonucleotoid molecule including at least two carbohydrate C-glycoside subunits (CG's) coupled to one another by means of a phosphodiester linkage, said phosphodiester linkage being represented by a formula as follows: CGI-Cl'-(O-PO(OH)-0)-CG2 SUBSTITUTE SHEET (RULE 26) WO 96/27379 PCT1US96/03227 -172- wherein: (O-PO(OH)-O) is said phosphodiester linkage; CGI-C 1 is a first carbohydrate C-glycoside subunit having an anomeric carbon forming a C-glycosidic bond with a carbon C 1 said carbon C 1 being bonded to said phosphodiester linkage; and CG 2 is a second carbohydrate C-glycoside subunit having a non-anomeric carbon bonded to said phosphodiester linkage. An improved process for synthesizing a library of oligomers, the process employing an elongation step wherein subunits are coupled to one another to produce the oligomers, wherein the improvement comprises: in said elongation step the oligomer is a carbonucleotoid including at least two carbohydrate C- glycoside subunits (CG's) are coupled by means of a phosphodiester linkage, said phosphodiester linkage being represented by a formula as follows: CGi-C 1 '-(O-PO(OH)-O)-CG 2 wherein: (O-PO(OH)-O) is said phosphodiester linkage; CGI-C 1 is a first carbohydrate C-glycoside subunit having an anomeric carbon forming a C-glycosidic bond with a carbon C 1 said carbon Cl' being bonded to said phosphodiester linkage; and CG 2 is a second carbohydrate C-glycoside subunit having a non-anomeric carbon bonded to said phosphodiester linkage.
11. A derived carbohydrate C-glycoside having an anomeric carbon and non-anomeric carbons, said anomeric carbon forming a C-glycosidic bond SUBSTITUTE SHEET (RULE 26) 173 with a carbon C 1 said carbon C1' being bonded to an activated phosphite, each of said non-anomeric carbons being substituted with a radical selected from the group consisting of blocked hydroxyl, differentially protected hydroxyl, and hydrogen, with the proviso that at least one radical is a differentially protected hydroxyl.
12. A derived carbohydrate C-glycoside having an g o* anomeric carbon and non-anomeric carbons, said anomeric carbon forming a C-glycosidic bond 60 with a carbon Ci', said carbon C 1 being bonded to S* an activated phosphite, 0 each of said non-anomeric carbons being substituted with a radical selected from the group consisting of blocked hydroxyl, unprotected hydroxyl, and hydrogen, with the proviso that at least one radical is an unprotected hydroxyl and at least one radical is a blocked hydroxyl. 0
13. A compound selected from the group of compounds consisting of an oligomeric carbopeptoid compound, a derived carbohydrate amino acid having an anomeric carbon and non-anomeric carbon, an oligomeric carbonucleotoid molecule and a derived carbohydrate C-glycoside having an 0* anomeric and non-anomeric carbons, the being substantially as described herein with reference to the accompanying Examples. Pp-L L o 1 ~o (4/ 174 0 S 20 00 00 S 0 0 0 2 0 3 S
14. An oligomeric carbopeptoid compound comprising carbohydrate amino acid subunits (CA's) coupled to one another via an amide linkage having a carbonyl carbon and an amido nitrogen represented by the following formula: CA- (CO-NH)-CA 2 wherein: CAI is a first carbohydrate amino acid subunit having an anomeric carbon bonded to the carbonyl carbon of said amide linkage for forming a C-glycosidic linkage therewith and CA 2 is a second carbohydrate amino acid subunit having a non-anomeric carbon bonded to the amido nitrogen of said amide linkage, the oligomeric carbopeptoid compound being substantially as described herein with reference to the accompanying Examples. In a process for synthesizing an oligomeric carbopeptoid compound, a coupling step wherein two or more carbohydrate amino acid subunits (CA s) are coupled by means of an amide linkage having a carbonyl carbon and an amido nitrogen for synthesizing said oligomeric carbopeptoid compound, said amide linkage being represented by a formula as follows: CA 1 (CO-NH) -CA 2 AL' c QI wherein: CA 1 is a first carbohydrate amino acid subunit having an anomeric carbon bonded to the carbonyl carbon of said amide linkage for forming a C-glycosidic linkage therewith; and CA 2 is a second carbohydrate amino acid subunit having a non-anomeric carbon bonded to the amido nitrogen of said amide linkage the step being substantially as described herein with reference to the accompanying Example$. 175
16. A library of oligomeric carbopeptoid compounds employable for drug screening, each oligomeric carbopeptoid compound including at least two carbohydrate amino acid subunits (CA's) coupled to one another via an amide linkage having a carbonyl carbon and an amido nitrogen, said amide linkage being represented by the following formula: CAi- (CO-NH) -CA 2 wherein: CAI is a first carbohydrate amino acid subunit having an anomeric carbon bonded to the carbonyl carbon of said amide linkage for forming a C-glycosidic S U 0* linkage therewith; and S 3 CA 2 is a second carbohydrate amino acid subunit having a non-anomeric carbon bonded to the amido nitrogen of said amide linkage the library of oligomeric carbopeptoid compouns being substantially as described herein with reference to the accompanying ExampleS.
17. An improved process for synthesizing a library of oligomers, the process employing an elongation step wherein subunits are coupled to one another to produce the oligomers, wherein the improvement comprises: eas in said elongation step the oligomer includes at least two carbohydrate amino acid subunits (CA's) coupled to one another via an amide linkage having a carbonyl carbon and an amido nitrogen represented by the following formula: CAI- (CO-NH) -CA 2 wherein: CA 1 is a first carbohydrate amino acid subunit having an anomeric carbon bonded to the carbonyl carbon of said amide linkage for forming a C-glycosidic linkage therewith; and U T NV 0, 176 0 0 S 0 0S 0 SSOSS 0050 0 OOSS CA 2 is a second carbohydrate amino acid subunit having a non- anomeric carbon bonded to the amido nitrogen of said amide linkage, the process being substantially as described herein with reference to the accompanying Example.
18. A derived carbohydrate amino acid having an anomeric carbon and non-anomeric carbons, said anomeric carbon being substituted with a carboxyl radical, each of said non-anomeric carbons being substituted with a radical selected from the group consisting of-blocked hydroxyl, blocked amino, differentially protected amino, and hydrogen, with the proviso that at least one radical is a differentially protected amino, the derived carbohydrate amino acid being substantially as described herein with reference to the accompanying Examples.
19. A derived carbohydrate amino acid having an anomeric carbon and non-anomeric carbons, said anomeric carbon being substituted with a carboxyl radical, each of said non-anomeric carbons being substituted with a radical selected from the group consisting of blocked hydroxyl, blocked amino, unprotected amino, and hydrogen, with the proviso that at least one radical is an unprotected amino and at least one radical is a blocked hydroxyl or amino, the derived carbohydrate amino acid being substantially as described herein with reference to the accompanying ExampleS. An oligomeric carbonucleotoid molecule comprising carbohydrate C-glycoside subunits (CG's) coupled to one another via a phosphodiester linkage represented by the following structure: CGi-C 1 -CG 2 wherein: (O-PO(OH)-O) is said phosphodiester linkage; 11 177 CGI-C1' is a first carbohydrate C-glycoside subunit having an anomeric carbon forming a C-glycosidic bond with a carbon Ci', said carbon C 1 being bonded to said phosphodiester linkage; and CG 2 is a second carbohydrate C-glycoside subunit having a non-anomeric carbon bonded to said phosphodiester linkage, the oligomeric carbonucleotoid molecule being substantially as described herein with reference to the accompanying Examples.
21. In a process for synthesizing an oligomeric carbonucleotoid molecule, a coupling step wherein two or more carbohydrate C-glycoside subunits (CG's) are coupled by means of a phosphodiester linkage, said* phosphodiester linkage being represented by a formula as follows: flCGi-Ci'- (0-PO (OH) -CG 2 wherein: (O-PO(OH)-O) is said phosphodiester linkage; CGi-Ci' is a first carbohydrate C-glycoside subunit having an anomeric carbon forming a C-glycosidic bond with a carbon Ci', said carbon C 1 being sees bonded to said phosphodiester linkage; and CG 2 is a second carbohydrate C-glycoside subunit having a non-anomeric carbon bonded to said phosphodiester linkage, the process being substantially as described herein with reference to the accompanying Examples.
22. A library of oligomeric carbonucleotoid molecules employable for drug screening, each oligomeric carbonucleotoid molecule including at least two carbohydrate C-glycoside subunits (CG's) coupled to one another by means of a phosphodiester linkage, said phosphodiester linkage being represented by a formula as follows: CGI-Ci'-(O-PO(OH)-0)-CG2 7 (E (4-ssSS 178 wherein: (O-PO(OH)-O) is said phosphodiester linkage; CGi-C 1 is a first carbohydrate C-glycoside subunit having an anomeric carbon forming a C-glycosidic bond with a carbon C 1 said carbon CI' being bonded to said phosphodiester linkage; and CG 2 is a second carbohydrate C-glycoside subunit having a non-anomeric carbon bonded to said phosphodiester linkage, the library of oligomeric carbonucleotoid molecules being substantially as described herein with reference to the accompanying ExampleS.
23. An improved process for synthesizing a library of oligomers, the process employing an elongation step wherein subunits are coupled to one another to produce the oligomers, wherein the improvement comprises: 15 in said elongation step the oligomer is a carbonucleotoid including at least two carbohydrate C- glycoside subunits (CG's) are coupled by means of a i.i phosphodiester linkage, said phosphodiester linkage being represented by a formula as follows: CGI-Cl'-(0-PO (OH) -0)-CG 2 wherein: (O-PO(OH)-O) is said phosphodiester linkage; SCGi-Ci' is a first carbohydrate C-glycoside subunit having an anomeric carbon forming aC-glycosidic bond with a carbon CI', said carbon C 1 being bonded to said phosphodiester linkage; and CG 2 is a second carbohydrate C-glycoside subunit having a non-anomeric carbon bonded to said phosphodiester linkage, the process being substantially as described herein with reference to the accompanying ExampleS.
24. A derived carbohydrate C-glycoside having an anomeric carbon and non-anomeric carbons, said anomeric carbon forming a C-glycosidic bond .4L,VN\ 179 with a carbon C 1 said carbon Ci' being bonded to an activated phosphite, each of said non-anomeric carbons being substituted with a radical selected from the group consisting of blocked hydroxyl, differentially protected hydroxyl, and hydrogen, with the proviso that at least one radical is a differentially protected hydroxyl, the derived carbohydrate C-glycoside being substantially as described herein with reference to the accompanying Examples.
25. A derived carbohydrate C-glycoside having an anomeric carbon and non-anomeric carbons, said anomeric carbon forming a C-glycosidic bond with a carbon C 1 said carbon Ci' being bonded to San activated phosphite, 0 each of said non-anomeric carbons being substituted with a radical selected from the group consisting of blocked hydroxyl, unprotected hydroxyl, and hydrogen, with the proviso that at least one radical is an unprotected hydroxyl and at least one radical is a blocked hydroxyl, the derived carbohydrate C-glycoside being substantially as described herein with reference to the accompanying oo. oExampleS, r e6O
26. A library of oligomeric carbopeptoid compounds or a library of oligomeric carbonucleotoid molecules, the OO '"library being substantially as described herein with reference to the accompanying Examples. .0 Dated this 13th day of August 1999 THE SCRIPPS RESEARCH INSTITUTE By their Patent Attorneys GRIFFITH HACK
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