Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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r2~RRo~~ oIDs AND ~'ARRr~cLl iO ~ S
Specification
Field of thç 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.
Backaround:
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
(Scienti~ic American, January 1993, 82). The emerging
importance of glycobiology is further characterized by
Mekelburger et al. (Angew. Chem. Int. Ed. Engl. 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 dvsfunctional mediation of such process. The
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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 ~or the design of libraries of
oligosaccharide analogs include the following:
(a) A need to maximize the potential of the
designed oligosaccharides as ligand and drug
candidates;
(b) 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
(c) A need for ~lexibility 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 ~acilitated by the
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availability of ef~icient techniques and sophisticated
instrumentation for synthesizing peptide and phosphate
bonds with high yields. The synthesis of oligope~tides
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 ~or constructing oligosaccharides give
comparatively lower yields and are complicated by the two
isomer possibilities (~ and $) in glycoside bond
formation.
Techniques and chemical methods ~or simultaneously
synthesizing multiple oligopeptides, e.g. 100-150
completely differe~t peptides having lengths of up to 20
amino acid residues, are reviewed by Jung, G. et al.
(Angew. Chem, Int . Ed . ~ngl . 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 p~rm~ceutically useful binding
~1-
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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.
Summarv:
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 ph~rm~sological 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
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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 o~ new libraries o~ 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 CAl-(CO-NH)-CA2. The amide linkage (CO-NH)
includes a carbonyl carbon and an amido nitrogen. A
first carbohydrate amino acid subunit CAl or glycoside
amino acid subunit GAl has an anomeric carbon bonded to
the carbonyl carbon of the amide linkage. The anomeric
carbon of the ~irst carbohydrate amino acid subunit CAl
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 C~2 has a non-anomeric carbon bonded to the amido
nitrogen of the amide linkage. The second carbohydrate
amino acid subunit CA2, like the ~irst amino acid subunit
CAl, may include an anomeric carbon bonded to the
carbonyl carbon o~ a second amide linkage linking the
second carbohydrate amino acid subunit CA2 to a third
carbohydrate amino acid subunit CA3, etc. In this
instance, the anomeric carbon of the second
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carbohydrate amino acid subunit CA2 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 C~2 is a t~rm; n~ ~
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 (Q'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 ~or
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
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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 int~rm~iate is a
derived carbohydrate amino acid similar to the first
except that the non-anomeric carbons are substituted with
a radical selected ~rom 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
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practical 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: CG1-C1 -(O-PO(OH)-O)-CG2. The first
carbohydrate C-glycoside subunit (CG1-C1') has an anomeric
carbon forming a C-glycosidic bond wilh a carbon C1'. In
turn the carbon C1' is bonded to the phosphodiester
linkage. The second carbohydrate C-glycoside subunit CG2
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
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improved process for synthesizing a library of oligomers.
The process employs an elongation step wherein subunits
are 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. ~he ~n~iC carbon
forms a C-glycosidic bond with carbon C1 . In turn, the
carbon Cîl is bonded to an phosphoramidite. Each of the
non-anomeric carbons is substituted with a radical
selected from the group consisting of blocked hydroxyl,
differentizlly protected hydroxyl, and hydrogen, with the
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 hydrox~rl, and hydrogen, with the proviso that
at least one radical is an unprotected hydroxyl and at
least one radical is a blocked hydroxyl.
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H ~~, H ~~, H ~~ ' H ~~' H
... , NO-~ , N o_~ , N o_~ , N _o_ , N--
I carbopeptoid (CPD)
H2N ~COOH H2N O_ COOH H2N O_ COOH H2N O COOH
~0~ ~0~ ~0
1 1 l l 1 1 l l 1 1
~ o ~', P-' ~~0_/--~'' 'P-' ~~0_~--~''''P-' ~~o_~--~'
~f '0' ~ ~0' ~J ~0~ ~ ~
H _ H H
I l carbonucleotoid (CND)
HO OH HO o_ OH HO O_ OH HO o ~OH
Scheme 1. Designed carbopeptoids (CPD's) and carbonucleotoids (CND's)
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Detailed ~escri~tion:
Retrosynthetic sch~s 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 no~-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 phosphodies~er 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 (CG's). Preferred carbohydrate amino acid
subunits (CA's) include the ~ollowing:
D-glucose having an unprotected carboxyl at the
anomeric C(1) position, an unprotec~ed amino group
at the C(6) position, and blocked hydroxyls at the
C(2), C(3), and C(4) positions;
D-mannose having an unprotected carboxyl at the
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.
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anomeric C(l) position, an unprotected amino group
at the C(6) position, and blocked hydroxyls at the
C(2), C(3), and C(4) positions;
D-galactose having an unprotected carboxyl at the
anomeric C(l) position, an unprotected amino group
at the C(6) position, and blocked hydroxyls at the
C(2), C(3), and C(4) positions;
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sH2N ~ ~ COOH ~ O ~ COOH ~ ~ r COOH ~ O ~ COOH
TESO ~ 'OTES TESO ~ ~OTESTESO ~ "OTES TESO ~ NHAc
OTES OTES OTES OTES
D-glucose (a + 0 D-mannose (a + ~) D-galactose (a + ~) N-acetyl-D-glucosa
2 4 6 mine (a + p)
H2N ~ ~ ~COOH H2N ~ ~COOH~ ~'COOH HOOC r ~ ~ O-NH2
TESO ~ OTES TESO . OTES TES "OTES TESO ~ OTES
OTES OTES OTES OTES
a - D- idose a - D- altrosea - D- gulose D-glucose (a + ~)
12 14 16
HOOC~O~O-NH2 HOOC~O~O-NH2 HOOC~O~O-NH2
TESO' ~ OTES TESO ~ 'OTES TESO' ~ 'NHAc
OTES OTES OTES
D- mannose (~ + 0 D- galactose (o~ + o N-acetyl- D-gl~ c~minp(c~+~) 18 20 22
H2N ~ COOH H2N ~ COOH
TESO OlES TESO OTES
D- ribose D- arabinose
21 26
OCH2CH2CN OCH2CH2CN
~ ~ O NPr2 ~ ~ ~ NPr2
TESO' ~ 'OTES TESO ~ "OTES
OTES OTES
D-glucose (c~ + ~) D-galactose (a + ~)
Z8 OCH2CH2CN 32 OCH2CH2CN
HO ~ ~ O~ ~NiPr2 ~ ~ ~ NPr2
TESO ~ OTES TESO' ~ NHAc
OTES OTES
D-mannose (a + ,B) N-acetyl-D-glll- nc~min,~ + ~)
34
Scheme 2. StrLlctu}es of carbohydrate amirloacids (CA's) and C-glycosides (CG's)
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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;
~-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
C(2), C(3), and C(4) positions;
~-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
C(2), C(3), and C(4) positions;
~-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
C(2), C(3), and C(4) positions;
~-D-glucose 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 C(2), C(3), and C(4) positions;
D-mannose having an unprotected O-glycosidic amino
at the ~n~m~ric C(1) position, an unprotected
carboxyl as the C(6) position, and blocked hydroxyls
at the C(2), C(3), and C(4) positionsi
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 C(2), C(3), and C(4) positions;
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N-acetyl-D-glucosamine having an unprotected O-
glycosidic amino at the ~nom~ric 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 ~ollowing:
D-glucose having a C(1) C1 -glycosidic carbon
bonded to a phosphoramidite, an unprotected hydroxyl
at the C(6) position and blocked hydroxyls at the
C(2), C(3), and C(4) positions;
D-mannose having a C(1) C1 -glycosidic carbon
bonded to a phosphoramidite, an unprotected hydroxyl
at the C(6) position and blocked hydroxyls at the
C(2), C(3), and C(4) positions;
D-galactose having a C(1) C1 -glycosidic carbon
bonded to a phosphoramidite, an unprotected hydroxyl
at the C(6) position and blocked hydroxyls at the
C(2), C(3), and C(4) positions; and
N-acetyl-D-glucosamine having a C(1) C1 -glycosidic
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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 D-
glucose, i.e. compound 46.
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AcO~ ~ 1. TMSCN, SnCll ~OrCOOEt
AcO' 'OAc 2. EtOH, H2S04 (cat) HO" y 'OH
OAc OH 38
36
1. PivC1, DMAP, pyridine
y 2. Et3SiOTf, iPr2EtN, CH2C12
N3~ r 1 NaOEt, EtOH PivO--~OrCOOEt
TESO" ~ "OTES 2. TDHPFA, DIAD, Ph3P TESO' ~ 'OTES
OTES (41~2) OTES
42 40
1. H~, Pd on C
~I
H2N ~ COOEt FMOCN ~ COOH
~~ ~ 1. NaOH, p-dioxane l r
TESO' y 'OTES 2. FMOC-Cl, NaHCO3, H~O TESO ~ 'OTES
OTES OTES
44 46
Scheme 3. Svnthesis of glucose-derived carbohydrate amino acid (CA): 46
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Scheme 4 outlines the synthesis of suitably
protected carbohydrate amino acid subunits (CA's) ~rom N-
acetyl-D-glucosamine, i.e. compound 62.
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- 19 --
AcO--~ r L TMSCN, SnCl4 ~OrCOOEt
AcO' ~ 'NPhth 2. EtOH, H2SO~L (cat) HO' ~ 'NHAc
3. Ac2O, MeOH
OAc OH 54
48
1. PivCl, DMAP, pyridirle
~, 2. Et3SiOTf, 'Pr2EtN, CH2Ck
N3~ r 1 NaOEt EtOH PivO~OrCOOEt
TESO" I "NHAc 2. DPPA DIAD, Ph3P TESO" 'NHAc
OTES (57-58) OTES
58 56
l.H2,PdonC
H2N ~~ COOEt Fl\/lOCN~O~ COOH
~ 1. NaOH p-dioxane ¦ ~
TESO' ~ "NHAc 2. FMOC-Cl, NaHCO3, H20 ~ 'NHAc
OTES (61-62) OTES
62
Scheme 4. Synthesis of N-acetyl~ s~mine-derived amino acid (CA): 62
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Scheme 5 summarizes the synthesis of hexamer 74, i.e
glucose-glucosamine hetero carbopeptoid (CPD).
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o ~ z~ $
O =~ O t~ O Z~ O U . O =~ O
C/~ ~~ ~
O ~ ~ Z C
a ~
~ I 0=~ ~~
~ 0~ 0
,_ ~ \ /
-- LL ~ G
X
-
. _
..
C~
U)
SUBSTITUTE SHEET (RULE 26)
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0~0 ~~~ ~~~ ~~
~z Z r' =~Z G =~ ~=~
T ~ 0~0 ~ ~~ ~- =
~0 ~~~'~r ~ O,,
~ ~~ O = ~ o 0~ l o=~ O
~0 o~o 0~0 ~~0
T =~--0 ~--0 o~ T
0=~ 0 ~=~ ~
o \>_ O O '>--O
~/ ~ ~/ I
I I
5,
_G
~ SUBST~TUTE Sl IEET (RULE 26)
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Scheme 6 illustrates the constructio~ of suitably
protected and activated C-glycoside subunits (CG's~
corresponding to glucose.
-
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- 24 -
ACO ~ ~ ~ scN~sncl4 HO ~ O r COOEt
AcO~ ''OAc 2.EtOH,H2SO4(cat) HO~ 'OH
OAc OH 76
36
1.~im~th~xytritylrhl~n~,
DMAP, DMF
~ 2. Et3SiOTf, iP}2EtN, CH2C12
DMTO ~ O ~ OH iBU2A1H,CH2Cl2 DMTO ~ ~ r COOEt
TESO' ~ 'OTES TESO' 'OTES
OTES OTES
1. Et3SiCl, imid., DMF 78
OCH2CH2CN ~Cl3CCOOH, CH3NO2, THF
Cl N Pr2
\~
Pr2EtN, CH2Cl2 OCH2CH2CN
DMTO ~ ~ ~ ~P~Nipr2 DMTO ~ O ~ OTES
TESO~ y 'OTES TESO~' y 'OTES
OTES OTES
82 84
Scheme 6. Synthesis of glucose-derived C-glycoside (CG)
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Scheme 7 illustrates the construction of suitably
protected and activated C-glycoside subunits (CG's)
corresponding to glucosamine.
-
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AcO ~ ~ scN~sncl4 HO ~ O r COOEt
AcO' ~ 3 Ac20, MeOH HO' 'NHAC
OAc OH 86
48
m~thnxytrityl chloride,
DMAP, DMF
~ 2. Et3SiOTf, iPr2EtN, CH2Cl2
DMTO~ ~ OH iBU2AlH,CH2Cl2 DMTO ~ ~ r COOEt
TESO' ~ 'NHAc TESO' ~ 'NHAc
OTES OTES
~ 1. Et3SiCl, irnid., DMF 88
OCH2CH2CN ~CCOOH, CH3NO2, THF
Cl NiPr
Pr2EtN, CH2Cl2 OCH2CH2CN
DMTO ~O~l ~O N Pr2 DMTO ~ OTES
TESO' ~ 'NHAc TESO' ~J 'NHAc
OTES OTES
92 94
Scheme ~. Synthesis of N-acetylglllcos~mine-derived C-glycoside (CG)
Scheme 8 summarizes the synthesis of hexamer 116, i.e.
glucose-glucosamine hetero carbonucleotoid (CND).
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U ~ ~ ~ O ~ ~
o ~ ~ ~ C ~ ~ J ~ ~ ~ Z O llJ
1~ " O=~L--~ U ~ ~ ~--I --~ ~ U o=n--C O
1~ 0~0 ~ ~~~ -~ 0~0
O IO O ~1~ 0
0=~--O 0=~--O
0~
o I ~ llJ
O ~ O--~--O
O
- S ~~
0~ o
O
x
-
U~
. _
s~:
" oo
SUBSTITUTE SHEET (RULE 26)
CA 02214789 1997-09-05
W 096/27379 PcT/u~ 2~7
- 28 -
~_ 0~0 ~ ~0
"~ o=c---o o o
~o u~-u ~ - o~
O=l~O ~ ~ ~
~ o=~--o o=~--o
¢o=~L--o o o
~0 ~ ; O ~ ~Z~ O_
~ o I UJ I ~
r I 0=~1--00= 1 --O
O O
CJ Or~--O O \~--0
~ ¢~ ~ ~
SUBSTITUTE SHEET (RULE 26)
CA 02214789 1997-09-05
WO 96/27379 PCTnUS~v~v3227
- 29 -
The chemistries illustrated in Schemes 5 and 8 for
synthesizing heteroh~m~r CPD 74 and heterohexamer CND
116 can also be employed for synthesizing homohexamer
CPD's 118 (glucose) and 120 (glucosamine) and homohexamer
CND~s 122 (glucose) and 124 (glucosamine).
SUBSTITUTE SH EFT (RULE 26)
CA 02214789 1997-09-05
W 096r27379 PCTrUS96/03227
- 30 -
O O ~1:
~\ I ~\ I
0~0 0~0
o ~ I0=~--0 O=tL--O
C,) ~0 ~ Z 1 ~
0~ 0 0~0
"'IO ~"'I
~ \ I o=~l_o o=~l_o
0~ 0~ 0~0 0~0
o=~_o -- 0=~--0 "
\z -- 2 Z ~2 0 0 C
~_0 =~}O y ~_
= ~_o o = ~ _ O
O ~ ~
~I ~J''~~=g--~ g ~
~ ~
A
o~o o~o
O
SUBSTITUTE SH EET (RULE 26)
CA 022l4789 l997-09-0~
W 096/27379 PCTrUS9G)v~227
- 31 -
In analogy with the construction o~ oligopeptide and
oligonucleotide libraries, a oligosaccharide carbopeptoid
(CPD) library may be constructed by per~orming using a
split synthesis method o~ 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 o~ a several reaction vessels,
each reacrtion vessel cont~ini~g 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 o~ 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 ~or
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.
SU BSTITUTE SH EET (RU LE 26)
CA 02214789 1997-09-05
W096/27379 PCT/u~r~32~7
- 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 ~rom glucose pentaacetate The
reagents and conditions for synthesizing CG 82 are
provided as follows:
Steps (a)-(d): according to Fuchs (supra).
Step (e): (1) DMTCl, DMAP, Pyridine; room
temperature.
(2) TESTfl; 0~C.
Step (f): DIBAL-H,=CH2Cl2; -78~C; and
Step (g): (NCCH2CH2)(NiPr2)PCl, tetrazole,
CH2Cl2-
The reagents and conditions for synthesizing CA 46
from CA 45 are provided in Step M as follows:
Step (m): FMOC-Cl, K2CO3, THF, H2O; 0~C.
SUBSTITUTE SHEET (RULE 26)
CA 02214789 1997-09-05
W 096/27379 ~-lIU~_5.'~3227
AcO ~ o ~ OAc HO ~ ~ ~ COOMe DMTO ~ O ~ COOMe
AcO' ~ 'OAc HO' ~ "OH TESO' ~ 'OTES
OAcOH OTES
36~ 76 78
RNH~O CO2H ~o
TESO' ~OTESDMTO ~ O~ ~ ~O NiPr2 DMTO ~O~ 'OH
45OTEHTESO' ~ "OTES ~ TESO' ~ 'OTES
r r ~ OTES OTES
~146: R=FMOC ~ ~ 80
Scheme 20. Conventional route to CAs and a variation for the synthesis of
CG. Reagents and conditions: (a) - (d) corresp. Lit.: (e) (1) DMTCl, DMAP,
Py; RT. (2) TESTfl; oor (f) DIBAL-H, CH2Cl2; -78 ~C. (g)
(NCCH2CH2)(NzPr2)PCl, tetrazole CH~C12. (m~ FMOC-Cl, K2CO3, l~IF,
H20; 0 ~C.
SUBSTITUTE SHEET (RULE 26)
CA 02214789 1997-09-05
W 096/27379 PCTrUS96/03227
A synthetic route for producing C-glycosides (CG's)
with B-configuration at the former anomer 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 (a): Co2(CO)8, HSiEt2Me, CO.
Step (b): (1) AcOH, H20, THF;
(2) RuC13, NalO4, CH3CN, H20, CCL4, room
temperature;
Step (c): NaOMe, MeOH;
Step (d): (1) DMTCl, DMAP, Pyridine, room
temperature;
(2) TESOTf;
Step (e): BH3-THF;
Step (f): (NCCH2CH2)(NiPr2)PCl, tetrazole,
Ch2C12 i
Step (g): (1) 1 equiv TsCl. base
(2) TESOTf;
Step (h): NaN3;
Step (i): H2, Pd(OH)2-C;
Step (j): FMOC-Cl, base.
SUBSTITUTE SHEET (RULE 26)
CA 02214789 1997-09-05
W 096127379 ~CTSUS96S032~7
AeO--~O~OACAcO~ ~1--OSiMe3 b ACO ~O~CO2H
AeO' ~ 'OAeAcO~ ~ 'R AeO' ~ 'R
OAe OAe OAe
36 176
R = OAc or NPhth or NHAc
c
N ~ O
DMTO f ~~-- NiPr2 DMTO~O~--OH HO ~,O~CO2H
TESO' ~J 'R ~ TESO~ ~IJ 'R d,e HO' ~J 'R
OTES OTES OH
181 ~ 180 178
R = OTES or NPhth or NHAc R = OAc or NPhth or NHAc
FMOCNH 1~~ CO2H N3~O~CO2H TsO I~O~CO2H
TESO' ~R TESo' ~ 'R TESO' ~ 'R
OTES OTES OTES
183 182
185 R = OTES or NPhth or NHAc
Scheme 21. Synthesis of C-glycosides with ~-configuration at the former
,~nomPric center. Re~lgents and conditions: (a) Co2(CO)8, HSiEt2Me, CO. (b) (1)
AcOH, H20, THF; (2) Rua3, NaIO4, CH3CN, H20, CCl4, RT. (c) NaOMe,
MeOH. (d) (1) DMTCl, DMAP, Py, RT: (2) TESOTF. (e) BH3-THF. (fl
(NCCH2CH3)(NiPr~)PCl, tetrazole, CH2Cl2. (g) (1) 1 equiv TsCl, base: (2)
TESOTf. (H) NaN3. (i) H~, Pd(OH)2-C. (j) FMOC-Cl, base.
SUBSTITUTE SHEET (RULE 26~
CA 02214789 1997-09-05
W O 96/27379 PC~rrUS96/03227
BnO ~ .~ ~ a-b TsO ~ ~ ~ COzH
BnO' 'OBn 191-192 TESO' 'OTES TESO' 'OTES
OBn OTES OTES
190 192 194
a,~,h,i ef
FMOCNH ~ O ~CO2H
DMTO ~ ~ ' ~ OH HO2C ~ ~ ' ~ NHFMOCTESO' ~ 'OTES
TESO' ~ 'OTES 201-204 TESO' ~ 'OTES OTES
OTES OTES
200 ~
Scheme 22. Svnthesis of C-glycosides with a-configuration at the former
anomeric center. Reage~2ts nt2d CO?lditiO?25: (a) reductive debenzylation. (b) (1) 1
equiv TsC1, base; (2) TESOTf. (c) NaN3. (d) RuCl3, NaIO4, CH3CN, H20, CCl4.
(e) H2, Pd-C. (f) FMOC-Cl, base. (g) (1) DMTC1, DMAP, Py, RT; (2) TESOTf.
(h) (1) RuC13, NaIO4, CH3CN, H~O, CC14; (2) CH~N2. (i) DIBAL-H. (j) PPh3,
DIAD, diphenyl phosphoryl azide (DPPA), THF. (k) KMnO4, t-BuOH, buffer.
SUBSTITUTE SHEET (RULE 26)
CA 02214789 1997-09-05
W 096127379 P~-llu~ 3227
Synthetic routes for producing with C-glycosides
with a-con~igurations at the ~ormer 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 lea~ing to CG 196 and CG 204
are as follows:
Step (a): reductive debenzylation;
Step (b): (1) e~uiv TsCl. base;
(2) TESOTf.
Step (c): NaN3.
Step (d): RuCl3, NalO4, CH3CN, H20, CCl4.
Step (e): H2, Pd-C.
Step (f): FMOC-Cl, base.
Step (g): (1) DMTCl, DMAP, Pyridine, room
temperature;
(2) TESOTf.
Step (h): (1) RuCl3, NalO4, CH3CN, H20, CC14;
(~) CH2N2
Step (i): DI3AL-H.
Step (j): PPh3, DIAD, diphenyl phosphoryl azide
(DPPA),THF.
Step (k): KMnO4, t-BuOH, buf~er.
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)
CA 02214789 1997-09-0
W O 96127379 PC~rrUS96/03227
- 38 -
Petrus, L. et al. (Chem. zvesti. 1982, 3 6, 103) . The
reagents and conditions required for the synthesis of
compound 209, compound 2 14, compound 220, and compound
224 are indicated below:
Step (a): (1) 1.1 e~uivalent DMTCl, DMAP, Pyridine,
12 hour, 20~C;
(2) TesOTf, CH2, 0~C, 1 hour, 83%.
Step (b): (1) LAH, ether, reflux, 2 hour;
(2)FMOC-Cl, K2CO3, THF, H2O, 0~C, 1 hour,
55%;
Step (c): 10% HCOOH in CH2Cl2, 0~C, 2 minutes, 100%.
Step (d): RuCl3, NalO4, CH3CN, H2O, CCl4, 20~C, 10
minutes,
54%.
Step (e~: (1) 1 equiv. TsCl, base;
(2) TESOTf.
Step (f): NaN3.
Step (g): oxidative NEF.
Step (h): Pd-C, H2.
Step (i): FMOC-Cl, base.
Step (j): (1) 1 equiv. PivCl, base;
(2) TESOTf.
Step (k): (1) oxidative Nef; (2) CH2N2.
Step (l): DI~AL-H.
Step (m): DMTCl, DMAP, Pyridine.
Step (n): LAH.
Step (o): Nef reaction
Step (p): LAH.
SUBSTITUTE SH EET ~RULE 26)
CA 02214789 1997-09-05
WO 96/27379 PCI)US96/03227
6 6
HO2C~O~f N(H)FMOC a-d i FMOCNH l~orc02H
TESO~ 'OTES 206-209 ¦ ¦ 210-~14 TESO~ 'OTES
OTES O OTES
[~ HO~ ~ NO
HO~ 'OH
OH
138
DMTO~ ~--OH l l HO/~ ~ODMT
TESO~ 'OTES j-n TESO~ 'OTES
OTES a, o, p 215-2Z0 OTES
~ I
Scheme 23. Development of the galactose derived C-glycoside 138 to
protected CAs and diols. Reagents and conditions: (a) (1) 1.1 equiv Dl~TCl,
DMAP, Py, 12h, 20 ~C; (2) TesOTf, CH2Cl2, 0 ~C, lh, 83%. (b) (1) LAH, ether,
reflux, 2h; (2) FMOC-Cl, K2C03, THF, H20, 0 ~C, lh, 55%. (c) 10% HCOOH ~n
CH2Cl2 0 ~C, 2 min, 100%. (d) RuCl3, NaIO4, CH3CN, H2O, CCl4, 20 ~C, 10
min, 54%. (e) (1) 1 equiv TsCl, base; (2) TESOTf. (f) NaN3. (g) oxidative Nef. (h)
Pd-C, H2. (i) FMOC-Cl, base. (j) (1) 1 equiv PivCl, base; (2) TESOTf. (k) (1)
oxidative Nef; (2) CH2N2. (l) DIBAL-H. (m) DMTCl, DMAP, Py. (n) LAH. (o)
Nef reac~ion. (p) LAH
SUBSTITUTE SHEET (RULE 26)
CA 02214789 1997-09-05
W 096/27379 PCTrUS9GI'~32~7
- 40 -
An exemplary protocol ~or synthesizing a hexamer
carbopeptoid (CPD 234) starting from galactose derived CA
214, glucosamine derived CA 62, and glucose derived CA,
using standard methods ~or solid phase peptide synthesis
is illustrated in Scheme 24 The reagents and condition
~or these reactions are as ~ollows:
Step 1: DCC, HOBT, Et3, DMF;
Step 2: Piperidine, DMF
SUBSTITUTE SHEFT (RULE 26)
CA 022l4789 l997-09-05
W 096127379 PCTnUS9~03227
- 41 -
Q -~
O _ f O
~_ Cl
(~3 ~ G ~ Z G
0~
I I
,_ @ o G
~ C ~
0~0 ~,:, 0~0
O G
C ~, )
Z O ~
,.
SUBSTITUTE SHEFT (RULE 26)
CA 02214789 1997-09-05
W096/27379 P~lIU~,'/03227
- 42 -
Q ~ ~
O--/ ~ O--/ ~ 0=/ ~~ ~,~
c
~Z =~ ~ ~ 0~ ~
0~ ~0 0=~,~0 ==~
or~
cO~ o ~ 0~ < " ~ I ~;
~0
G
I
SUBSTITUTE SHEET (RULE 26)
,
CA 02214789 1997-09-05
W096~7379 ~lJU~J~3227
-43-
n~ lC D~ilnOL~S
Preparation of 37
A ~--1'~' ~C N
AcO' ~ OAc
AcO
To a solution of ,B-D-Glucose pentaacetate 36 i n
nitromethane from Aldrich company (.13 Molar), is added
trimethylsilyl cyanide (3.0 equivalents) and then SnCl4 (.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 (lX), water (lX), brine (lX)
and then dried over ma nesium sulphate and concentrated. The
crude solid is then recrystallized from methanol to yield 37 as a
white solid (47%). scheme 3 step 1; scheme 9, step a.
SUBSTITUTE SH EET (RU LE 26)
CA 02214789 1997-09-05
W096~7379 r~l/u~-5~o3227
Preparation of 38
HO--l--O~COOEt
HO' (~ 'OH
OH
38
The crude product 37 is next dissolved in ethanol (0.15 M)
and then concentrated H2S O4 (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
10flash column chromatography affords compound 38. scheme 3
step 2
Preparation of 39
PivO~o~cooE~
HO' ~ OH
OH
39
To a solution of 38 (1.0 equivalents) in pyridine (.10
Molar), is added trimethylacetyl chloride (pivaloyl chloride) (2.5
20equivalents) at 0 ~C. The reaction is stirred for 2 hours and
then diluted with diethylether and washed with ammonium
SUBSTITUTE StlEET tRULE Z6)
CA 02214789 1997-09-05
W 096~ m 9 ~-llU' ~CJD3227
-45-
chloride (2X), copper sulfate (2X), brine ( lX), dried over MgSO4
and concentrated. Purification by flash column chromatography
affords compound 39. scheme 3 step 1
Preparation of 40
PivO~T~O~COOEt
TESO' ~ OTES
OTES
To a solution of 39 (1.0 equivalents) in methylene chloride
(.10 Molar), is added diisopropylethylamine (3.3 equivalents) 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 (2X), brine
(lX) and then dried (MgSO4) and concentrated. Purification by
flash column chromatography affords compound 40. scheme 3
step 2
SUBSTITUTE SHEET (RULF 26)
CA 02214789 1997-09-05
W096~7379 ~ 3??7
-46-
Preparation of 41
HO--~O~COOEt
TESO' ~ 'OTES
OTES
41.
s
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
N3~0~COOEt
TESO' ~ 'OTES
OTES
42
A solution of 41 (l.0 equivalents) in tetrahydrofuran (.18
M) is treated with DPPA (diphenylphosphorylazide, 2.0
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 StlEET (RULE 26)
CA 02214789 1997-09-05
W 096/27379 ~l~U'_ÇJ~
-47-
washed with .5 M aqueous NaOH (2X). The organic layer is dried
over MgSO4 and evaporated. Purification by flash column
chromatography affords compound 42. scheme 3 step 2
Preparation of 44
H2N ~O~,~COOEt
TESO' ~ 'OTES
OTES
44
10A solution of 42 (1.0 equivalents) is dissolved in ethanol
(.01 M total) at 25 ~C. The mixture is next exposed to 10% Pd/C
(.1 equivalents) and is then subsequently capped with a
hydrogen balloon at l atmosphere. The reaction is stirred for 72
hours and is then filtered through celite. The crude mixture is
15subsequently diluted with ether and washed with NaHCO3 (3X),
brine (lX) and dried (MgSO4) and concentrated. Purification by
flash column chromatography affords compound 44. scheme 3
step 1
SUBSTITUTE SHE~T (RULE 26)
CA 02214789 1997-09-0~
wog6n7379 ~/u.~'~03227
-48-
Preparation of 45
2 ~~ r
TESO" ~ 'OTES
OTES
s
A solution of 4 4 ( 1.0 equivalents) is dissolved in p-
dioxanes (.1 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 NH4Cl (3X), brine (lX)
and dried (MgSO4) and concentrated. Purification by flash
column chromatography affords compound 45. scheme 3 step
Preparation of 46
To a solution of 45 (1.0 equivalents) in methylene chloride
(.10 Molar), is added sodium bicarbonate (2.0 equivalents) at 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 diethylether and washed
with ammonium chloride (2X), brine ( lX) and then dried
( M g S O 4 ) and concentrated. Purification by flash column
SUBSTITUTE SHEET (RULE 26)
CA 02214789 1997-09-0~
wo s6/2ms ~ V~ ?7
--49--
chromatography affords compound 46. scheme 3 step 2
Preparation of 48
r
ACo--~o ~OAc
AcO' ~ ' NPhth
OAc
Procedure as described in Methods in Carbohydrate
chemistry, Whistler, R., II, 19 6 3, p. 327. A mixture of 80g
anhydrous D-glucosamine hydrochloride or D-galactosamine
hydrochloride from Aldrich chemical company, in 200 mL.
methanol and 20g Dowex 50 (H+) 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 (.5 M), 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 (.5 M), is added
SUBSTITUTE SHEET (RULE 26)
CA 02214789 1997-09-05
wog6r27379 ~ 1u~,5~'03n7
-50-
acetic anhydride (3.5 equiv.) and triethyl amine (3.5 equiv.) and
the reaction mixture is allowed to stir at O ~C for 4 h. The
product 4 8, is then crystallized or purified by flash column
chromatography and carried onto the next step.
SUBSTITUTE StlEET (RULE 26)
CA 022l4789 l997-09-0~
W 096/27379 1~-~J~6JD3227
-51-
Preparation of 50
Aco--~~~C N
AcO' ~ ' NPhth
AcO
S To a solution of N-phthalamido-D-Glucosamine tetraacetae
48 in nitromethane (.13 Molar), is added trimethylsilyl cyanide
(3.0 equivalents) and then SnCl4 (.02 equivalents). The mixture
is stirred for one hour and then an aqueous solution of sodium
acetate was added to hydrolyze the rem~ining trimethylsilyl
cyanide. The mixture is evaporated and the rem~ining oil is
resuspended in dichloromethane and washed with sodium
acetate solution ( 1 X), water ( 1 X), brine ( 1 X) and then dried over
magnesium sulphate and concentrated. The crude solid is then
recrystallized from methanol to yield 51) as a white solid (47%).
scheme 4
Preparation of 52
The crude product 50 is next dissolved in ethanol (0.15 M)
and then concentrated H2S O4 (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)
CA 02214789 1997-09-05
W 096127379 r~ 6/03Z27
flash column chromatography affords compound 52. scheme 4
Preparation of 54
HO--~O~cooEt
HO' ~ NHAc
5OH
54
A solution of 52 (1.0 equivalents) is dissolved in methanol
(.1 M total). The reaction is then charged with acetic anhydride
( 1.1 equivalents) and is subsequently stirred for 2 hours at 30
~C. The reaction is next diluted with ether and washed with
NaHCO3 (3X), brine (lX) and dried (MgSO4) and concentrated.
Purification by flash column chromatography affords compound
54. scheme 4
Preparation of 55
Pivo - ~o~cooEt
HO' ~ NHAc
OH
SUBSTITUTE SHEET ~RULE 26)
CA 02214789 1997-09-05
W 0961273n ~ r J '03227
To a solution of 54 ( 1.0 equivalents) in pyridine (.10
Molar), is added trimethylacetylchloride (pivaloyl chloride) (2.5
equivalents) at 0 ~C. The reaction is stirred for 2 hours and
then diluted with diethylether and washed with ammonium
chloride (2X), copper sulfate (2X), brine ( 1 X), dried over MgSO4
and concentrated. Purification by flash column chromatography
affords compound 55. schenne 4
Preparation of 56
Pivo - ~o~cooEt
TESO' ~ 'NHAc
OTES
56
To a solution of 55 (l.0 equivalents) in methylene chloride
(. l0 Molar), is added diisopropylethylamine (2.2 equivalents) at
0 ~C. 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 (2X), brine
(lX) and then dried (MgSO4) and concentrated. Purification by
flash column chromatography affords compound 56. scheme 4
SUBSTITUTE SHEET tRULE 26)
CA 02214789 1997-09-05
W096127379 ~-l/U~961o3227
-54-
Preparation of 57
HO--~O~rcooEt
TESO' ~ 'NHAc
OTES
57
s
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
10chromatography affords compound 57. scheme 4
Preparation of 58
N3~0~COOEt
TESO' ~ NHAc
OTES
58
A solution of 57 (1.0 equivalents) in tetrahydrofuran (.18
M) is treated with DPPA (diphenylphosphorylazide, 2.0
equivalents), triphenylphosphine ( 1.3 equivalents) and DIAD
20(diisopropyl-azo-dicarboxylate, 1.3 equivalents). The reaction is
SUE3STITUTE StlEET (RULE 26)
CA 02214789 1997-09-05
W 096127379 ~-lIU~ 03227
-55-
heated to 80 ~C for 3 hours and then diluted with ether (2X) and
washed with .5 M aqueous NaOH (2X). The organic layer is dried
over MgS O4 and evaporated. Purification by flash column
chromatography affords compound 58. scheme 4
Preparation of 60
H2N ~OrCOOEt
TESO' ~ NHAc
OTES
A solution of 58 (1.0 equivalents) is dissolved in ethanol
(.01 M total) at 25 ~C. The mixture is next exposed to 10% Pd/C
(.1 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 NaHCO3 (3X),
brine (lX) and dried (MgSO4) and concentrated. Purification by
flash column chromatography affords compound 60. scheme 4
SUBSTITUTE SH EET (RU LE 26~
CA 02214789 1997-09-05
WO 96/27379 ~- -lIU~;~ "03227
--56--
Preparation of 61
H2N ~~~,C O O H
TESO' ~ NHAc
OTES
61
A solution of 6 0 ( 1.0 equivalents) is dissolved in p-
dioxanes (.1 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 NH4Cl (3X), brine ( 1 X)
and dried (MgSO4) and concentrated. Purification by flash
column chromatography affords compound 61. scheme 4
Preparaffon of 62
H
FMocN~~O~,~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)
CA 02214789 1997-09-05
W096/27379 ~ lU~61o3227
-57-
~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 diethylether and washed
with ammonium chloride (2~), brine ( lX) and then dried
( M g S O 4 ) and concentrated. Purification by flash column
chromatography affords compound 62. scheme 4
Preparation of 63
FMOCN--~~~ 11 N ro~1~COOEt
TESO"' ~/ 'OTESTESO ~/ 'NHAc
OTES OTES
63
To a stirred solution of the acid 46 ( 1.0 equivalents) and
the amine 6 0 ( 1 .1 equivalents) in dimethylformamide ( .10
Molar) at 25 ~C, 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 NaHCO3 (2X), water (2X), and brine (2X). The organic
phase is dried over MgSO4 and then concentrated. Purification
by flash column chromatography affords compound 6 3 .
SUBSTITUTE SHEET (RULE 26)
CA 02214789 1997-09-05
W096~7379 ~1/U~6~'~32>7
-58-
scheme 5 step 1
Preparation of 64
H2N~ ~ I N--~o~COOEt
TESO"'y 'OTESTESO ~ 'NHAc
OTES OTES
64
To a stirred solution of 63 ( 1.0 equivalents) in
dimethylformamide (.10 Molar) at 25 ~C, is added piperidine
( 1.1 equivalents) . The reaction is stirred for 1 hour and is then
diluted with ether, and washed with aqueous CuS O4 (2 X)7 water
(2X), and brine (2X). The organic phase is dried over MgSO4 and
then concentrated. Purification by flash column
chromatography affords compound 64. scheme 5 step 2
Preparation of 65
FMOCN~O~ N--~COOEt
TESO' ' NHAc 'OTESTESO ' NHAc
OTES OTES OTES
SUBSTITUTE SHEET (RULE 26)
CA 02214789 1997-09-05
wos~nn ~1J~ D3227
--59--
To a stirred solution of the acid 62 ( 1.0 equivalents) and
the amine 64 (1.1 equivalents) in dimethylformamide (.10
Molar) at 25 ~C, is added 1-hydroxybenzotriazole (HOBT; 1.1
equivalents). Note: numerous iterations can be performed using
S 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 ~ ) to obtai7l 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 NaHCO3 (2X), water (2X), and brine (2X).
The organic phase is dried over MgSO4 and then concentrated.
Purification by flash column chromatography affords compound
65. scheme 5 step 1
Preparation of 66
NH2 ~~ ll N/~O-- ll N l o~COOEt
ESO '~J OTESTESO"'~"""N
OTES OTES OTES
66
To a stirred solution of 65 ( 1.0 equivalents) in
SUBSTITUTE 5HEET (RULE 26)
CA 02214789 1997-09-05
W 096~7379 r~~ o3227
-60-
dimethylformamide (.10 Molar) at 25 ~C, is added piperidine
( 1.1 equivalents) . The reaction is stirred for 1 hour and is then ~,
diluted with ether, and washed with aqueous CuSO4 (2X), water
(2X), and brine (2X). The organic phase is dried over MgSO4 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 S). scheme ~ step 2
Preparation of 67
o o o
MOCN--~O-- ll N~O-- --N--~O~ ~ N~O~cOoEt
--~ TESO ~ 'NHAC TEso~--~J 'OTESTEso~ NHA
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 ( 10
Molar) at 25 ~C, 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
infinit~ (a hexamer is shown in scheme 5 ) to obtain large
carbopeptoid libraries. Next dicyclohexylcarbodiimide ( 1.2
SUBSTITUTE S~EET (RULE 26)
CA 02214789 1997-09-05
wa 96 m 379 ~-lJU~Jv3227
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 NaHCO3 (2X), water (2X), and brine (2X).
The organic phase is dried over MgSO4 and then concentrated.
Purification by flash column chromatography affords compound
67. scheme 5 step 1
SUBSTITUTE SHEET (RULE 26)
CA 02214789 1997-09-05
W096~7379 ~l/U~Ç~'03Z27
-62-
Preparation of 68
"
o o ,,
H2N--~ H ~ --N--~o~ rl N l O~COOEt
TESO ',J OTESTESO' ~ '''NHAcTESO'' 'lJ 'OTESTEso~"~ "'NHA
68
To a stirred solution of 67 ( 1.0 equivalents) in
dimethylformamide (.10 Molar) at 25 ~C, is added piperidine
( 1.1 equivalents) . The reaction is stirred for l hour and is then
diluted with ether, and washed with aqueous CuSO4 (2X), water
(2X), and brine (2X). The organic phase is dried over MgSO4 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 5). scheme ~ step 2
Preparation of 69
H ~ 1I N- ~ ~r ~ N ~ 1I N ~ COOEt
TESO' 'NHAc 'OTESTESO' 'NHAcTESO "OTESTESO' 'NHAc
OTES OTES OTES OTES OTES
69
A
To a stirred solution of the acid 62 ( 1.0 equivalents) and
SUBSTtTUTE SH EET (RULE 26)
CA 02214789 1997-09-OF,
WO 96127379 ~-llU' ~ 13~7
-63-
the amine 6 8 ( 1.1 equivalents) in dimethylformamide (.10
~, Molar) at 25 ~C, is added l-hydroxybenzotriazole (HOBT; 1.1
equivalents). No~e: 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 NaHCO3 (2X), water (2X), and brine (2X).
The organic phase is dried over MgSO4 and then concentrated.
Purification by flash column chromatography affords compound
69. scheme 5 step 1
.
Preparation of 70
O o o o
~2N~~~L1~ 1 H--~ ~N ~O~N--~O~COOEt
y TESff--~ 'OTESTESO" ~
To a stirred solution of 69 ( 1.0 equivalents) in
dimethylformamide (.10 Molar) at 25 ~C, is added piperidine
( 1.1 equivalents). The reaction is stirred for 1 hour and is then
SUBSTITUTE SHEET(RUI_E 26)
CA 02214789 1997-09-05
W 096~7379 ~ 5f'~ 7
-64-
diluted with ether, and washed with aqueous CuSO4 (2X), water
(2X), and brine (2X). The organic phase is dried over MgSO4 and
then concentrated. Purification by flash column
chromatography affords compound 70. Note: numerous
iterations can be performed using variable length oligomers of
70 to form peptoid oligomers where n=2 to infinity (a hexamer
is shown in scheme 5). scheme 5 step 2
Preparation of 71
FMOCN ~~~ ~ N ~ i--N--~r~~r~ O~COOEt
TESO~y ~OTE~SO 'NHAcTESO" ~OTESTESO~ 'NHAcESO (,~lt~>lt~7C/~ ~ 'NHAc
OTES OTES OTES OTES OTES OTES
To a stirred solution of the acid 46 ( 1.0 equivalents) and
the amine 70 (1.1 equivalents) in dimethylformamide (.10
Molar) at 25 ~C, is added l-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
infirzity (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 SH EET (RULE 26~
CA 02214789 1997-09-05
WO961273n ~ U~_.'/03227
-65-
washed with aqueous NaHC03 (2X), water (2X), and brine (2X).
The organic phase is dried over MgS04 and then concentrated.
Purification by flash column chromatography affords compound
71. scheme 5 step 1
SUBSTITUTE SHEET (RULE 26)
CA 02214789 1997-09-05
W096/27379 r~ ',5~03227
Preparation of 72
H2N~~N~--~N--~, r~ L N~ ~-- N ,~COOEt
TESO' ""~OT~SO 'NHAcTESO" OTES TESO' 'NHAcESO ~ t~ t~ ' NHAc
OTES OTES OTES OTES OTES OTES
s
To a stirred solution of 71 ( 1.0 equivalents) in
dimethylformamide (.10 Molar) at 25 ~C, is added piperidine
( 1.1 equivalents) . The reaction is stirred for 1 hour and is then
diluted with ether, and washed with aqueous CuSO4 (2X), water
(2X)~ and brine (2X). The organic phase is dried over MgSO4 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 ~=2 to infinity (a hexamer
is shown in scheme 5). scheme ~ step 2
Preparation of 74
H2N r~O~ ~LNH ~ ~ N--~O~ O~N ~O~COOH
,, H H "'NHA HO'" '~; HO' ~ 'OH ~ OH HO' ~ 'NHAC
74
To a stirred solution of 72 ( 1.0 equivalents) in acetonitrile
SUBSTITUTE SHEET (RULE 26)
CA 02214789 1997-09-05
W096~7379 r~l/u~5~o3227
-67-
(.50 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
NH4Cl (2X) and subsequently lyophilized. Purification by HPLC
chromatography affords compound 74. scheme
Preparation of 76
HO--I~OvcOOEt
HO' ~ 'OH
OH
76
To a solution of ,B-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 ,B-D-Mannose, ,B-D-Galactose pentaacetate and other
lewis acids such as BF30Et~ 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
SU85TITUTE SH EET (RULE 26)
CA 02214789 1997-09-0~
W096/27379 ~-llU~r~r~7
-68-
rem~ining trimethylsilylcyanide. The mixture is evaporated and
the remaining oil is resuspended in dichloromethane and
washed with sodium acetate solution (lX), water (lX), brine (lX)
and then dried over magnesium sulphate and concentrated. The
crude product is next dissolved in ethanol (or methanol if the O-
methyl glycoside is desired as in scheme 20), (0.15 M) and
then concentrated H2 S O 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
20 (as the O-methyl glycoside).
Preparation of 78
~MTO~ COOEt
TESO' ~ 'OTES
OTES
78
To Tetrol 7 6 ( 1.0 equivalents) in pyridine ( .10 Molar), is
added dimethyoxytritylchloride (DMT chloride) (2.5 equivalents)
at 0 ~C. The reaction is stirred for 2 hours and then diluted with
diethylether and washed with ammonium chloride (2X), copper
sulfate (2X), brine (lX) dried over MgSO4 and concentrated.
SUBSTITUTE SHEET (RULE 26)
CA 02214789 1997-09-0~
W096/~7379 ~ ,.S'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 (2X), brine (lX) and then dried (MgSO4) and
concentrated. Purification by flash column chromatography
affords compound 78, scheme 6; 78, scheme 20 (as the O-
methyl glycoside).
Preparation of 80
~MTO~ OH
TESO' ~ 'OTES
OTES
To a solution of 78 (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
~C. Subsequent stirring for 2 hours is followed by dilution with
diethylether and washing with sodium-potassium tartrate (2X)~
brine (lX) and then MgSO4. The solution is then concentrated
and purification by flash column chromatography affords
..
SU~STITUTE SHEET (RULF 26)
CA 02214789 1997-09-05
W096~7379 r~liU~3G/03227
-70-
compound 80. scheme 6
Preparation of 82
N(~Pr)2
~MTO--~O O'P'O~--C N
TESO' ~ 'OTES
OTES
82
To a solution of 80 (1.0 equivalents) in methylene chloride
(.10 M), is added diisopropylethylamine (4.0 equivalents) at 25
~C. 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 ( lX) and is then dried (MgSO4) and concentrated.
Purification by flash column chromatography (silica, 30% ethyl
acetate in petroleum ether) affords compound 82 (66~o yield).
scheme 6
SUBSTITUTE SHEET (RULE 26)
CA 02214789 1997-09-05
W096/27379 ~ 961n3227
Preparation of 84
,.
H O~,l' 1~ OTFS
TESO' ~ 'OTES
OTES
84
To 80 (1.0 equivalents) in methylene chloride (.10 Molar)
at 0 ~C, 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 (2X), brine (lX) and then dried (MgSO4) and
concentrated. The crude is then resuspended in nitromethane
and exposed to 10% Cl3COOH (1.1 equivalents) in THF (.10
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 8 4 .
scheme 6
SUBSTITUTE SHEET (RULE 26)
CA 02214789 1997-09-0~
W 096/27379 ~-1/U~_5J03227
Preparation of 86
~,
Ho--~OrCO~Et
HO' \~/ 'NHAc
OH
86
5 To a solution of N-phthalamido-D-Glucosamine tetraacetate
48 in nitromethane (.13 Molar), is added trimethylsilyl cyanide
(3.0 equivalents) and then SnCl4 (.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 rem~ining oil is
resuspended in dichloromethane and washed with sodium
acetate solution (lX), water (lX), brine (lX) and then dried over
magnesium sulphate and concentrated. The crude product is
next dissolved in ethanol (0.15 M) and then concentrated H~SO4
(0.04 equivalents) is added. The reaction mixture is heated to
85 ~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
SU BSTITUTE S H EET (RU LE 26)
CA 02214789 1997-09-0
W~96~7379 ~ ,56J0322
Preparation of 88
)MTO--I' ~COOEt
TESO' ~ NHAc
OTES
88
To Triol 86 (1.0 equivalents) in pyridine (.10 Molar),
is added dimethyoxytritylchloride (DMT chloride) (2.5
equivalents) at 0 ~C. The reaction is stirred for 2 hours and
then diluted with diethylether and washed with ammonium
chloride (2X), copper sulfate (2X), brine ( lX), 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 (2X), brine (lX) and then dried (MgSO4) and
concentrated. Purification by flash column chromatography
affords compound 88. scheme 7
SUBSTITUTE SHEET (RULE 26)
CA 02214789 1997-09-05
W 096/27379 ~1/US96103227
-74-
Preparation of 90
~MTO~ OH
TESO~ ~ 'NHAc
OTES
To a solution of 88 (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
~C. Subsequent stirring for 2 hours is followed by dilution with
diethylether and washing with sodium-potassium tartrate (2X),
brine (lX) and then MgSO4. The solution is then concentrated
and purification by flash column chromatography affords
compound 90. scheme 7
Preparation of 92
N(~Pr)2
~MTO~ ~~O' O~
TESO' ~ 'NHAc
OTES
92
To a solution of 90 ( 1.0 equivalents) in methylene chloride
(.10 M), is added diisopropylethylamine (4.0 equivalents) at 25
SUBSTITUTE SHEET tRULE 26)
CA 02214789 1997-09-0~
W096~7379 ~-lJU~,5Jo3227
-75-
~C. 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 ( lX) and is then dried (MgS04 ) and concentrated.
Purification by llash column chromatography (silica, 30% ethyl
acetate in petroleum ether) affords compound 92 (66% yield).
scheme 7
Preparation of 94
HO~ '~--OTES
TESO' ~ 'NHAc
OTES
94
To 90 (1.0 equivalents) in methylene chloride (.10 Molar)
at O ~C, 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 (2X), brine ( lX) and then dried (MgS04) and
concentrated. The crude is then resuspended in nitromethane
and exposed to 10% Cl3COOH (1.1 equivalents) in THF (.lO
Molar). The reaction is stirred at O ~C for 2 hours and is then
SUBSTITUTE SHEET (RUEE 26)
CA 02214789 1997-09-0~
W 0961~7379 ~-1/U'-.,"03227
-76-
diluted with ether and washed with sodium bicarbonate (2X),
brine (lX) and then dried (MgSO4) and concentrated. Purification
by flash column chromatography affords compound 9 4 .
scheme 7
Preparation of 98 (homodimer scheme 8)
To a solution of 94 (1.0 equivalents) in methylene chloride
(.10 M), is added 1-H-tetrazole from Aldrich company ( 10.0
equivalents) at 25 ~C. Next, a solution of 82 (3.0 equivalents) in
methylene chloride ( 1.0 M), is added dropwise with stirring at
25 ~C. After 25 minutes, the mixture is cooled to 0 ~C and I2 (4.0
e~uivalents), 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 ( 1 X) and
dried (MgSO4) and concentrated. Purification by flash column
chromatography and then the product is suspended in acetic
acid-tetrahydrofuran-water (3: 1:1), (.01 M) and stirred for 18
hours at 25 ~C. The reaction is then diluted with ether and
washed with NaHCO3 (3X), brine (lX) and dried (MgSO4) and
concentrated. Purification by flash column chromatography
affords compound 98 (scheme 8).
SUBSTITUTE SHEET ~RULE 263
CA 02214789 1997-09-0~
W096~7379 ~ ,Cl03227
Preparation of 102 (heterotrimer scheme 8)
To a solution of 9 8 ( 1 .0 equivalents) in methylene chloride
(.10 M), is added 1-H-tetrazole from Aldrich company ( 10.0
equivalents) at 25 ~C. Next, a solution of 92 (3.0 equivalents) in
methylene chloride ( 1.0 M), is added dropwise with stirring at
25 ~C. 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 (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 ( 1 X) and
dried (MgSO4) and concentrated. Purification by flash column
chromatography and then the product is suspended in acetic
acid-tetrahydrofuran-water (3: 1: 1 ), (.01 M) and stirred for 18
hours at 25 ~C. The reaction is then diluted with ether and
washed with NaHCO3 (3X), brine (lX) and dried (MgSO4) and
concentrated. Purification by flash column chromatography
affords compound 102 (scheme 8).
Preparation of 106 (heterotetrarner scheme 8)
To a solution of 102 (1.0 equivalents) in methylene
chloride (.10 M), is added 1-H-tetrazole from Aldrich company
( 10.0 equivalents) at 25 ~C. Next, a solution of 8 2 (3.0
equivalents) in methylene chloride ( 1.0 M), is added dropwise
SUBSTITUTE SHEET (RULE 26)
CA 02214789 1997-09-0~
W096~7379 ~1/U'~ 3227
with stirring at 25 ~C. 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 (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 (lX) and dried (MgSO4) and concentrated.
Purification by flash column chromatography and then the
product is suspended in acetic acid-tetrahydrofuran-water
(3:1:1), (.01 M) and stirred for 18 hours at 25 ~C. The reaction is
then diluted with ether and washed with NaHCO3 (3X), brine
(lX) and dried (MgSO4) 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 M), is added 1-H-tetrazole from Aldrich company
(10.0 equivalents) at 25 ~C. Next, a solution of 92 (3.0
equivalents) in methylene chloride ( 1.0 M), is added dropwise
with stirring at 25 ~C. 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 (4.5
equivalents) is added). The reaction is next stirred for an
SUBSTITUTE SHEET (RULE 26)
.
CA 02214789 1997-09-0~
WO 96/273'79 ~ u.,~OES03227
--79-
additional 5 minutes and is next diluted with ether and washed
, with brine ( 1 X) and dried (MgS04 ) and concentrated.
Purification by flash column chromatography and then the
product is suspended in acetic acid-tetrahydrofuran-water
(3:1:1), (.01 M) and stirred for 18 hours at 25 ~C. The reaction is
then diluted with ether and washed with NaHC03 (3X), brine
(lX) and dried (MgS04) and concentrated. Purification by flash
column chromatography affords compound 110 (scheme 8).
Preparation of 114 (heterohexarner scheme 8)
To a solution of 110 ( 1.0 equivalents) in methylene
chloride (.10 M), is added 1-H-tetrazole from Aldrich company
( 10.0 equivalents) at 25 ~C. Next, a solution of ~ 2 (3.0
equivalents) in methylene chloride ( 1.0 M), is added dropwise
with stirring at 25 ~C. 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 (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 (lX) and dried (MgS04) and concentrated.
Purification by flash column chromatography and then the
product is suspended in acetic acid-tetrahydrofuran-water
(3 :1:1), (.01 M) and stirred for 18 hours at 25 ~C. The reaction is
SUE~STITUTE S~tEET (RULE 26)
CA 02214789 1997-09-0~
W09612 m 9 ~-1/U~6/03227
-80-
then diluted with ether and washed with NaHCO3 (3X), brine
(1~) and dried (MgSO4) 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 M), is added a solution of HF-pyridine ( 1.0 M) at O
~C. The reaction is next stirred for an additional 30 minlltes and
is next diluted with ether and washed with a saturated solution
of sodium bicarbonate (3X), copper sulfate solution to remove
the pyridine (2X) brine (lX), dried (MgS04) and concentrated.
Purification by flash column chromatography and then the
product is resuspended in concentrated aqueous ammonium
hydroxide and acetonitrile ( 1:1), (.1 M total). The reaction is
then stirred for 2 hours at 50 ~C and is subsequently diluted
with ether and washed with NaHCO3 (3X), brine (lX) and dried
(MgSO4) and concentrated. Purification by flash column
chromatography affords compound 116 scheme 8.
SUBSTITUTE SHEET (RULE 26)
CA 02214789 1997-09-05
~ JU~.~~'03227
WO 96127379
--8~ -
ACo~r~r~AC a - d HO--rOrCOOMee g Bu~Me2SiO~rOl~OH
AcO"y 'OAc (126-130)HO' ~ 'OH (131-134) BnO''y 'OBn
OAc OH OBn
36 130 134
cl~P o~CN~ N(~Pr)2 1 0
Bu~Me2SiO~rO'I~OP O~CN HO ,O~
BnO' ~ 'OBnBnO'' y "OBn
OBn OBn
138 136
Ii
R10--rO'I--O-P-O--~O~--OR
R20' y 'OR2 R20 Y oR2
OR2 OR2
k 140: R1 = SitBuMe2; R2 = Bn o
C 142: R1 = H; R2 = Bn; R4 = N~3
~li
R o~~~o OpRo~o~ ~R3 ~o~
R20' ~ 'OR2 ~ R20' Y 'OR2 ~ RZO' Y ~oR2
OR2 OR2 OR2
k 144: R1 = SitBuMe2; R2 = Bn; R4 = Nphth
C146 R1 = H; R2 = Bn; R4 = Nphth
E~i
R10 ~ OR3,~,o~ 0 OpRo3 ~0~ 0 p 0~~~~l~OR4
R2~'0R2 ~ R20 y 'OR2 ~ R20' Y 'OR2 ~ R20' Y ~oR2
OR2 OR2 OR2 OR2
k 148: R1 = Si~BuMe2; R2 = Bn; R3 = CH2CH2CN; R4 = Nphth
150 R1 = H; R2 = Bn; R3 = CH2CH2CN; R4 = Nphth
_ 152: R1 = R3 = H; R2 = Bn; R4 = Nphth
mL- 154: R1 = R2 = R3 = H; R4 = Nphth
Scheme 9. Synthesis of a Carbonucleotoid
SUBSTITUTE SHEET (RULE 26)
CA 02214789 1997-09-0=.
W096/27379 ~ G~227
Preparation of 125
A O ~O~C N
AcO' ~ 'OAc
OAc
125
To a solution of ,~-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 ~-D-Mannose, ,B-D-Galactose
0 pentaacetate and other lewis acids such as SnCI4, 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 ( lX),
water ( lX), brine ( lX) 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 (47%). scheme
9 step a
SUBSTITUTE SHEET (RULE 26)
CA 02214789 1997-09-05
W096~7379 ~ 56J03227
~ -83-
Preparation of 126
Ho ~O~C N
H O' ~ O H
OH
126
5To a solution of 12 5 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
10as a crude oil for the next step. scheme 9 step b
Preparation of 127
HO--rO~COOH
HO' ~,J '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)
20and is then stirred. The supernatant is then decanted and the
SU~STITUTE SH EET (RULE 26)
CA 02214789 1997-09-05
W096127379 ~-~/U~,''03227
resin 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 (47%).
Preparation of 130
HO~l~O~cOoMe
HO' ~ 'OH
OH
130
The crude product 127 is next dissolved in methanol (0.15
M) and then concentrated HCl (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 - l~o~cooMe
H O' ~ 'O H
OH
131
SlJBSTITUTE SHEET (RULE 26)
CA 02214789 1997-09-05
W096~7379 ~1J~6JD3227
-85-
To a solution of 130 ( 1.0 equivalents) in
dimethylformamide (.23 Molar), is added imidazole (2.5
equivalents) at 0 ~C. Subsequent addition of tert-Butyl-
dimethylsilylchloride (2.5 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. 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 (dinzethoxytrityl), and TBDPS
tert-butyldiphenlysilyl, etc.
Preparation of 132
ButMe2s jo~r~~C~~Me
BnO' ~ 'OBn
OBn
132
To a solution of 13 1 (1.0 equivalents) in
dimethylformamide (.23 M), is added Ag2O (6.0 equivalents) at
25 ~C. Benzyl bromide (9.0 equivalents) is next added and the
SUBSTITUTE 5HEET (RULE 26)
CA 02214789 1997-09-0~
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-86-
reaction is allowed to stir for 20 hours. The reaction is diluted
with diethylether and washed with ammonium chloride (2X),
brine (lX) and then dried (MgSO4) and concentrated.
Purification by flash column chromatography (silica, 20% ethyl
acetate) affords compound 132 (83% yield). scheIne 9 step f
Note: the choice of the protecting group ~s relative and the
molecule can be protected with other protecting groups at C2,
C3, C4, such as PMB (paramethoxvbenzyl), TES (triethvlsilyl),
TBS (tertbutvldimethyls~lyl), etc.
Preparation of 134
ButMe2SiO~i ~--OH
BnO' ~ 'OBn
OBn
134
To a solution of 132 (l.0 equivalents) in tetrahydrofuran
(.08 M), is added diisobutylaluminumhydride (DIBALH) (3.0
equivalents) at 0 ~C. 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 (2X), brine ( lX) and is
then dried (MgS04) and concentrated. Purification by flash
column chromatography (silica, 20% ethyl acetate) affords
compound 134 (66% yield). scheme 9 step g
SUBSTITUTE SHEET (RULE 26)
CA 02214789 1997-09-05
w~ s6~2ms ~ u~g6/~3227
Preparation of 136
B~ 'fOB ~3
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 ~C. The reaction is
stirred for 45 minutes and then diluted with ether and worked-
up with a saturated solution of CuSO4 (2X), brine (lX) and is
then dried (MgSO4) and concentrated. The crude product is then
exposed to acetic acid/tetrahydrofuran/water (3 :1:1 ) at 25 ~C
and allowed to stir for l 5 hours. The reaction is then diluted
with ether and worked-up with brine (2X) and is then dried
( M g S O 4 ) 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 Cl position as a TES (triethyl silyl) group.
Subsequent mild acid hydrolysis of the DMT group leads ~o the
SIJ~STITUTE SHEET (RULE 26)
CA 02214789 1997-09-05
W096/27379 P~l/u~ 3227
-88-
above compound with the TES group at ~he C1 position and a
free hydroxvl at the C7 position. scheme 9 step h
Preparation of 138
N(~Pr)2
ButMe2SiO~I' ~~O O~~
BnO' ~'OBn
OBn
138
To a solution of 134 (1.0 equivalents) in methylene
chloride (. lO M), is added diisopropylethylamine (4.0
equivalents) at 25 ~C. 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 ( lX) 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 ~l oted that the oligomerization process as
shown below in scheme 9, uses the same C-glycoside
138 in an iterative fashior2. The process can be
SUBSTITUTE SHEET (RULE 26)
CA 02214789 1997-09-05
Wo 96/27379 ~-llU!,,C~3227
--89--
extended however to include a pool of random or
ordered C-glycosides as depicted i~ scheme 8.
Preparation of 140
~ CN O
rBDMSO~ ~--P--0~ ~1 o l~
BnO' ~ 'OBn BnO' ~ 'OBn ~J
OBn OBn
140
To a solution of 13 6 ( 1.0 equivalents) in methylene
chloride (.10 M), is added 1-H-tetrazole ~rom Aldrich company
(10.0 equivalents) at 25 ~C. Next, a solution of 138 (3.0
equivalents) in methylene chloride ( 1.0 M), is added dropwise
with stirring at 25 ~C. 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 S minutes and is next
diluted with ether and washed with brine ( 1 X) and dried
( M g S O 4 ) 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.
SlJBSTITUTE SH EET (RULE 26)
CA 02214789 1997-09-0=.
W096~7379 ~ 96103227
--90--
Preparation of 142
~ C N O
H ~--~~~1--~--P--~--1'~~-- ~
BnO' ~ 'OBn BnO' ~'OBn ~J
OBn OBn
142
A solution of 14 0 ( 1.0 equivalents) in acetic acid-
tetrahydrofuran-water (3 :1:1), (.01 M) is stirred for 18 hours at
25 ~C. The reaction is then diluted with ether and washed with
NaHCO3 (3X), brine (lX) and dried (MgSO4) 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
o~ CN o~,CN O
rB D M S O~ ~--P--O ~ ~--P--~ ~ ~ J~
BnO' ~ 'OBn BnO' ~ 'OBn BnO' ~ 'OBn ~J
OBn OBn OBn
144
To a solution of 13 8 ( 1.0 equivalents) in methylene
chloride (.10 M), is added 1-H-tetrazole from Aldrich company
SUBSTITUTE SH EET (RU LE 26)
CA 02214789 1997-09-05
wog6nn79 PCTnUS96)03227
_91 _
( 10.0 equivalents) at 25 ~C. Next, a solution of 14 2 (3 .0
equivalents) in methylene chloride ( 1.0 M), is added dropwise
with stirring at 25 ~C. After 25 minutes, the mixture is cooled to
O ~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
( M g S 0 4 ) and concentrated. Purification by flash column
chromatography (silica, 50% ethyl acetate in petroleum ether)
affords compound 144 (97% yield). scheme 9 step j Nole the
0 process can iterate as many times as possible to build large
carbonucleotide libraries.
SUBSTITUTE SHEET (RULE 26)
CA 02214789 1997-09-05
W 096/27379 ~ 103227
-92-
Preparation of 146
HO ~O--P--o ~~O--P--o~
Bn~ OBn BXJ'OBn BnO' \I~'OBn
OBn OBn OBn
146
s
A solution of 14 4 ( 1.0 equivalents) in acetic acid-
tetrahydrofuran-water (3:1:1), (.01 M total) is stirred for 18
hours at 25 ~C. The reaction is then diluted with ether and
washed with NaHCO3 (3X), brine ( lX) and dried (MgSO4 ) 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
~CN ~CN ~CN O
-BDMSO--~~~--O--P--0~~~~~0--P--O~~ ~~O--P--0~~~~~o~
BnO' I 'OBn BnO' I 'OBn BnO' ~ 'OBn BnO' ~ 'OBn b~J
OBn OBn OBn OBn
148
To a solution of 13 g ( 1.0 equivalents) in methylene
SUBSTITUTE SHFET (RULE 26)
CA 02214789 1997-09-05
W096~7379 1~ 961o3227
-g3-
chloride (.10 M), is added l-H-tetrazole from Aldrich company
(10.0 equivalents) at 25 ~C. Next, a solution of 146 (3.0
equivalents) in methylene chloride ( 1.0 M), is added dropwise
with stirring at 25 ~C. 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 ( 1 X) and dried
( M g S 0 4 ) 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 b~ild large
carbonucleotide libraries.
Preparation of 150
~C~ ~ O~CN o
~o~--r-o~~o_r_o~ 1 5 0
A solution of 14 8 ( 1.0 equivalents) in acetic acid-
tetrahydrofuran-water (3:1:1), (.01 M total) is stirred for 18
hours at 25 ~C. The reaction is then diluted with ether and
washed with NaHCO3 (3X), brine (lX) 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)
CA 02214789 1997-09-05
W096127379 ~1/U'~6'03227
-94-
libraries.
Preparation of 152
~--O ~ o OH ~_o ~
onxl ~o~ nO ~ oun 1 5 2
A solution of 15 0 ( 1.0 equivalents) is dissolved in
concentrated aqueous ammonium hydroxide and acetonitrile
( 1:1), (.1 M total). The reaction is then stirred for 2 hours at 50
~C and is subsequently diluted with ether and washed with
NaHCO3 (3X), brine (lX) and dried (MgSO4) 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 D "~ U
"~~ "0 ~0 o ~ ~ 1 5 4
A solution of 15 2 ( 1.0 equivalents) is dissolved in a
mixture of ethanol-tetrahydrofuran-acetic acid (2:1:1), (.01 M
total) at 25 ~C. The mixture is next exposed to l O~o Pd/C ( 1.0
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
SUBSTtTUTE Slt EET (RULE 26)
CA 02214789 1997-09-0~
W096/27379 ~ U~ 7
-95-
subsequently diluted with ether and washed with NaHCO3 (3X),
brine (lX) and dried (MgSO4) and concentrated. Purification by
flash colurnn 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 (.1 molar) is added
a 1.0 molar solution of Co2(CO)g (1.5 equivalents ) in methylene
chloride and diethylmethylsilane ( 1.5 equivalents) at 0 ~C. To
the stirring reaction mixture, a stream of carbon monoxide is
bubbled at l 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 (2x), brine (lx) 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 (l:l ratio,
.1 molar combined), is added RuCl3 (.03 equiv.) and NaIO4 (4.0
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 ( lX 5 mL). The
SUBSTITUTE SHEET (RULE 26)
CA 02214789 1997-09-0
WO96127379 ~ 32
--96--
aqueous layer is back extracted (2X), recombined, and the
organic layer was then dried MgS04 and evaporated.
Purification by flash column chromatography yields the desired
product 176.
s
Preparation of 178 (R group = OTES, NPhth or NHAc)
A solution of triacetate 17 6 ( 1.0 equiv.) in methanol (0.5 M), 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.~ equivalents) at O
~C. The reaction is stirred for 2 hours and then diluted with
diethylether and washed with ammonium chloride (2X), copper
sulfate (2X), brine (lX), 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 O ~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 (2X), brine (lX) and then dried (MgS04) and
SUBSTITUTE SHEET (RUI E 26)
CA 02214789 1997-09-0~
W~ 96~7379 ~-1r~56/03227
-97-
concentrated. Purification by flash column chromatography
affords the intermediate acid, which is then resuspended in THF
(1.0 M) and exposed to a 1.0 M solution of BH3-THF (1.5
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
(2X), brine ( lX) and then dried (MgSO4) 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 (.10
M), is added tetrazole (4.0 equivalents) at 25 ~C. 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. 198~, 12, 4539.
After 15 minutes the reaction is complete and is next diluted
with ether and next washed with brine ( lX) and is then dried
( M g S O 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 (.0 equiv.) in CH2Cl2 (.5 M) at 0 ~C, was
SUBSTITUTE SHEET (RULE 26)
CA 02214789 1997-09-0
W096~7379 r~l/
-98-
added triethylamine (1.2 equiv.), 4-DMAP (.10 equiv.) and then
TOSCl ( 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
(lX 5 mL), back-extracted (2X), recombined, dried (MgSO4) and
evaporated. The compound is purified by flash column
chromatography and then a solution of the crude intermediate
( 1.0 equivalents) is dissolved in methylene chloride (.10 Molar)
and diisopropylethylamine (2.2 equivalents) is added at 0 ~C.
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 (2X), brine (lX) and then dried (MgSO4) and
concentrated. Purification by flash column chromatography
affords the protected tosylate/acid 182.
Preparation of 183 (1~ group = OTES. NPhth or NHAc)
To a solution of triol 182 (.0 equiv.) in CH2C12 (.5 M) at 0 ~C, is
added sodium-azide ( 1.2 equiv.) from Aldrich chemical company
at 0 ~C. 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 (2X), recombined, dried (MgSO4) and evaporated.
The compound is purified by flash column chromatography and
SUBSTITUTE SHEET (RULE 26)
CA 02214789 1997-09-05
WO96~?379 ~ U~C~o3227
_99_
affords compound 183.
Preparation of 18~ (R group = OTES, NPhth or NHAc)
A solution of 183 (1.0 equivalents) in ethanol (.01 M total) at 25
~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 NaHCO3 (3X), brine (lX) and dried (MgSO4) and
concentrated. Purification by flash column chromatography
affords compound 185 scheme 21.
Preparation vf 191
H O ~ ~~
~ 'OH
OH
191
A solution of starting material 19 0 as disclosed by
Schmidt, R. R. et al. (Liebigs Ann. Chem. 1 9 8 7, 825), ( 1.0
equivalents) is dissolved in a mixture of ethanol-
tetrahydrofuran-acetic acid (2:1:1), (.01 M total) at 25 ~C. The
" mixture is next exposed to 10% Pd/C ( 1.0 equivalents) and is
SUBSTITUTE SHEET tRuLE 26)
CA 02214789 1997-09-05
W096127379 ~ 96/03227
--100--
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 NaHCO3 (3X), brine (lX) and
dried (MgSO4) and concentrated. Purification by flash column
chromatography (silica, 100% ethyl acetate in petroleum ether)
affords compound 191. scheme 22 step a
SUE~STITUTE SHEET (RULE 26)
CA 02214789 1997-09-0~
W096~7379 r~l~U~36,'~3~27
--101--
Preparation of 192
r TsO ~O
TES~ ~ OTES
OTES
192
To a solution of 191 ( 1.0 equivalents) in methylene
chloride (.10 Molar) is added tosylchloride ( 1.2 equivalents) at
0 ~C. 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
(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 (2X), brine (lX) and then dried (MgSO4) and
concentrated. Purification by flash column chromatography
affords compound 192. scheme 22 step b
SUI~STITUTE SHEET (RULE 26)
CA 022l4789 l997-09-0=,
W096~ m 9 ~ ,''03227
-102-
Preparation of 193
N3--~~
TESC~ ~ 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 O ~C. Subsequent stirring
for 2 hours is followed by dilution with diethylether and
washing with ammonium chloride (2X), brine ( lX) and then
M g S 0 4 . The solution is then concentrated and purification by
flash column chromatography affords compound 193. scheme
22 step c
Preparation of 194
N3--~o~ ~CO2H
TESb~ ~ OTES
OTES
194
To solution of 193 in CCl4 (.33 M), CH3CN (.33 M) and
water (.22 M) at O ~C is added RuCl3 (.03 equiv.) and NaI04 (4.0
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
SU B STITUTE S H EET (RU LE 26)
CA 02214789 1997-09-05
WO 9612m9 ~ 03227
--103--
water (2X 5 .0 mL) and brine ( lX 5 mL). The aqueous layer is
back extracted (2X), 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 ~O~ "CO2H
TESC~ 'jJ OTES
OTES
196
A solution of 194 (1.0 equivalents) is dissolved in ethanol
(.01 M total) at 25 ~C. The mixture is next exposed to 10% Pd/C
(.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 NaHCO3 (3X),
brine ( lX) and dried (MgSO4 ) 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
~C. Subsequent addition of 9-fluorenylmethyl chloroformate
(FMOC-C1, 1.2 equivalents) is followed by stirring for 2 hours
SIJBSTITUTE SHEET (RULE 26)
CA 022l4789 l997-09-05
W 096/27379 PCTnUS96/03227
-104-
and then the reaction is diluted with diethylether and washed
with ammonium chloride (2X), brine (lX) and then dried
( M g S 0 4 ) and concentrated. Purification by flash column
chromatography affords compound 196. scheme 22 steps e-f
s
SU~STITUTE SHEET (RULE 26)
CA 02214789 1997-09-0~
W096127379 ~ 96J03227
-105-
Preparation of 197
DMTo~O~.~
TESo'~f OTES
OTES
197
To Tetrol 191 (1.0 equivalents) in pyridine (.10 Molar), is
added dimethyoxytritylchloride (DMT chloride) (2.5 equivalents)
at 0 ~C. The reaction is stirred for 2 hours and then diluted with
diethylether and washed with ammonium chloride (2X), copper
sulfate (2X), brine (lX), 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 (2X), brine (lX) and then dried (MgSO4) and
concentrated. Purification by flash column chromatography
affords compound 197. scheme 22 step g
S~J~STITUTE SHEET (RULE 26)
CA 022l4789 l997-09-0=.
W096127379 ~ U~ 3227
-106-
Preparation of 198
DMTO ~o~ c02CH3
TESC~ ~J OTES
OTES
198
s
To solution of 197 in CCl4 (.33 M), CH3CN (.33 M) and
water (.22 M) at 0 ~C is added RuCl3 (.03 equiv.) and NaIO4 (4.0
equiv.) and the muddy black mixture is allowed to stir for l.5 h.
The mixture is then diluted with ether (25 mL), washed with
water (2X 5.0 mL) and brine ( lX 5 mL). The crude is then
resuspended in a mixture of methylene chloride/water ( l :1, . l
M total) and diazomethane ( l .2 equivalents) is gradually
dropped into the flask via an addition funnel at the rate of l
drop/10 seconds. After complete addition the mixture is diluted
lS 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 198. scheme 22 step
h
SUBSTITUTE SHEET (RULE 26)
CA 022l4789 l997-09-05
W096~7379 ~-~JU~ D3227
-107-
Preparation of 200
D MTO ~O~ ~C H20 H
TESC~ ~J OTES
OTES
200
To a solution of 198 ( 1.0 equivalents) in methylene
chloride (.10 Molar) is added a l.0 M solution of DIBALH in
methylene chloride from Aldrich chemical company ( 1.2
equivalents) at 0 ~C. Subsequent stirring for 2 hours is followed
by dilution with diethylether and washing with sodium-
potassium tartrate (2X), brine (lX) and then MgSO4. The
solution is then concentrated and purification by flash column
chromatography affords compound 200. scheme 22 step i
lS Preparation of 201
DMTO--~O~ N3
TES~ ~ OTES
OTES
201
A solution of 2 0 0 ( 1.0 equivalents) in tetrahydrofuran
(. l 8 M) is treated with DPPA (diphenylphosphorylazide, 2.0
SUBSTITUTE SHEET (RULE 26)
CA 02214789 1997-09-0~
W096127379 ~ 03227
-108-
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
washed with .5 M aqueous NaOH (2X). The organic layer is dried
over MgSO4 and evaporated. Purification by flash column
chromatography affords compound 201. scheme 22 step j
Preparaffon of 202
DMTO--~O~ N H FMOC
TESC~ ~IJ OTES
OTES
202
A solution of 201 (1.0 equivalents) is dissolved in ethanol
(.01 M total) at 25 ~C. The mixture is next exposed to 10% Pd/C
(.1 equivalents) and is then subsequently capped with a
hydrogen balloon at 1 atrnosphere. The reaction is stirred for 72
hours and is then filtered through celite. The crude mixture is
subsequently diluted with ether and washed with NaHCO3 (3X),
brine ( lX) and dried (MgSO4 ) 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
~C. Subsequent addition of 9-fluorenylmethyl chloroformate
(FMOC-CI, 1.2 equivalents) is followed by stirring for 2 hours
SUBSTITUTE SHEET (RULE 26)
CA 02214789 1997-09-05
W096~1379 r~Ju~5~D3227
--109--
and then the reaction is diluted with diethylether and washed
with ammonium chloride (2X), brine ( 1 X) and then dried
( M g S O 4 ) and concentrated. Purification by flash column
chromatography affords compound 202. scheme 22 step e
s
Preparation of 204
HO2C----~O~ NHFMOC
TES~ ~ OTES
OTES
204
To asolution of 202 ( 1.0 equivalents) in methylene
chloride (.10 Molar) is added 10% HCOOH from Aldrich chemical
company ( 1.2 equivalents) at 0 ~C. Subsequent stirring for 2
hours is followed by dilution with diethylether and washing
with sodium bicarbonate (2X), brine ( lX) and then MgSO4. The
solution is then resuspended in t-BuOH (.10 M) and pH 7 buffer
(.10 M) and is then exposed to KMnO4 ( 1.2 equivalents) for 2
hours at 0 ~C. The reaction mixture is next washed with sodium
bicarbonate (2X), brine (lX) and then MgSO4. The organic layer
is then concentrated and purified by flash column
chromatography to afford compound 204. scheme 22 step k
5UBSTlTUTE SHEET (RULE 25~
CA 02214789 1997-09-0~
W096127379 ~ 7
--1 10--
Preparation of 206
DMTO--~O~--NO2
TESO~ OTES
OTES
206
To Tetrol 205 (1.0 equivalents) (as disclosed by Petrus, L.
et ahChem. zvesti. 1982, 36, 103) in pyridine (.10 Molar), is
added dimethyoxytritylchloride (DMT chloride) (2.5 equivalents)
at 0 ~C. The reaction is stirred for 2 hours and then diluted with
diethylether and washed with ammonium chloride (2X), copper
sulfate (2X), brine (lX), dried over MgS04 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 (2X), brine (lX) and then dried (MgSO4) and
concentrated. Purification by flash column chromatography
affords compound 206. scheme 23 step a
SUBSTITUTE SHEET (RULE 26)
CA 02214789 1997-09-0~ -
W~9612~379 ~JU~,~103227
_111_
Preparation of 207
DMTO~O~--NHFMOC
TESO~J OTES
OTES
207
Toa solution of 206 (1.0 equivalents) in diethylether (.08
M), is added lithiumaluminumhydride (LAH) ( 1.5 equivalents) at
30 ~C. 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 (2X), brine (lX) and
is then dried (MgS04) and concentrated. The crude mixture is
resuspended in methylene chloride (.10 Molar) and to it is
added sodium bicarbonate (2.0 equivalents) at O ~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 diethylether and washed with
ammonium chloride (2X), brine (lX) and then dried (MgSO4) and
concentrated. Purification by flash column chromatography
affords compound 207. scheIne 23 step b
5U13STITUTE S~tE~T tRULE 26)
CA 022l4789 l997-09-05
W096127379 P~ ,''03227
-112-
Preparation of 208
HO--I'O~--NHFMOC
TESO~ OTES
OTES
208
To a solution of 2 0 7 ( 1.0 equivalents) in methylene
chloride (.10 Molar) is addedlO% 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 (2X), brine ( 1 X) and
then dried (MgSO4) and concentrated. Purification by flash
column chromatography affords compound 208. scheme 23
step c
Preparation of 209
HOOC~O~ NHFMOC
TESO~ 'OTES
OTES
209
To solution of 208 in CC14 (.33 M), CH3CN (.33 M) and
water (.22 M) at 20 ~C is added RuC13 (.03 equiv.) and NaIO4
(4.0 equiv.) and the muddy black mixture is allowed to stir for
10 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
SUBSTITUTE SHEET (RULE 26)
CA 022l4789 l997-09-0~
W096~73~9 ~ 6103227
-113-
is back extracted (2X), recombined, and the organic layer iss
then dried MgSO4 and evaporated. Purification by flash column
chromatography affords the compound 209. schelme 23 step
d
Preparation of 210
TOSO--~O~--NO2
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 ~C. Subsequent addition of triethylamine ( 1.5 equivalents) is
followed by stirrin~; 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
( 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
SUI~STITUTE SHEET (RULE 26)
CA 022l4789 l997-09-0~
W096~7379 ~ ''03227
-114-
ammonium chloride (2X), brine (lX) and then dried (MgSO4) and
concentrated. Purification by flash column chromatography
affords compound 210. scheme 23 step e
Preparation of 211
N3~ ~ NO2
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 ~C. Subsequent stirring
for 2 hours is followed by dilution with diethylether and
washing with ammonium chloride (2X), brine (lX) and then
M g S O 4 . The solution is then concentrated and purification by
flash column chromatography affords compound 211. sche~ne
23 step f
SUBSTITUTE SHEET (RULE 26)
CA 022l4789 l997-09-05
W 096127379 l~ '3~103227
-115-
Preparation of 212
N3,~0rCO2H
TESO~ OTES
OTES
212
To solution of 211 in CCl4 (.33 M), CH3CN (.33 M)
and water (.22 M) at 20 ~C is added RuCl3 (.03 equiv.) and NaIO4
(4.0 equiv.) and the muddy black mixture is allowed to stir for
10 min. The mixture is then diluted with ether (25 mL), washed
with water (2X 5 .0 mL) and brine ( 1 X S mL) . The aqueous layer
is back extracted (2X), 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
H2N ~O~I~C02H
TESO~ OTES
OTES
213
A solution of 212 (l.0 equivalents) in ethanol (.01 M total)
at 25 ~C is exposed to 10% Pd/C (0. l equivalents) and is then
SUBSTITUTE SHEET (RULE 26)
CA 022l4789 l997-09-0~
WO961Zn79 ~l/U~,''03227
-116-
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 NaHCO3 (3X), brine (lX) and dried (MgSO4) and
concentrated. Purification by flash column chromatography
affords compound 213. scheme 23 step h
Preparation of 214
FMOCH N ~O~CO2H
TESO~ 'OTES
OTES
214
Compound 213 is suspended in methylene chloride (.10
Molar) and to it is added sodium bicarbonate (2.0 equivalents)
at 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 diethylether
and washed with ammonium chloride (2X), brine ( l X) and then
dried (MgSO4) and concentrated. Purification by flash column
chromatography affords compound 214. scheme 23 step i
SUBSTITUTE S~EET (RULE 26)
CA 022l4789 l997-09-0~
W096~7379 ~ ~9~D3227
-117-
Preparation of 215
P IVO--~O~--NO2
TESO~ OTES
OTES
215
To a solutionof 205 ( 1.0 equivalents) in pyridine (.10
Molar), is added trimethylacetyl chloride (pivaloyl chloride) (2.5
e~uivalents) at 0 ~C. The reaction is stirred for 2 hours and
then diluted with diethylether and washed with ammonium
chloride (2X), copper sulfate (2X), brine (lX), 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 (2X), brine (lX) and then dried (MgSO4) and
concentrated. Purification by flash column chromatography
affords compound 215. scheme 23 step j
SlJBSTITUTE SHEET (RULE 26)
CA 022l4789 l997-09-0~
W 096~7379 ~ '3~103227
-118-
Preparation of 216
PlVO--~O~C02M e
TESO~ OTES
OTES
216
To solution of 215 in CCl4 (.33 M), CH3CN (.33 M) and
water (.22 M) at 20 ~C is added RuCl3 (.03 equiv.) and NaIO4
(4.0 equiv.) and the muddy black mixture is allowed to stir for
10 min. The mixture is then diluted with ether (25 mL), washed
with water (2X 5 .0 mL) and brine ( 1 X 5 mL) . The aqueous layer
is back extracted (2X), recombined, and the organic layer is then
dried MgSO4 and evaporated. The crude is then resuspended in
a mixture of methylene chloride/water ( 1: l, .1 M total) and
diazomethane ( 1.2 equivalents) is gradually dropped into the
flask via an addition funnel at the rate of l drop/ l 0 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 StlEET (RULE 26)
CA 02214789 1997-09-05
W096~7379 ~-lJU'~ 3227
_1 19--
Preparation of 217
.
Plvo - ~o~cH2oH
TESO~J OTES
OTES
217
To a solution of 216 ( 1.0 equivalents) in methylene
chloride (. l 0 Molar) is added a 1.0 M solution of DIBALH in
methylene chloride from Aldrich chemical company ( l .2
equivalents) at 0 ~C. Subsequent stirring for 2 hours is followed
by dilution with diethylether and washing with sodium-
potassium tartrate (2X), brine (lX) and then MgSO4. The
solution is then concentrated and purification by flash column
chromatography affords compound 217. scheme 23 step l
Preparation of 218
PIVO--~CH20DMT
TESO OTES
OTES
218
To 217 (l.0 equivalents) in pyridine (.10 Molar), is added
dimethyoxytritylchloride (DMT chloride) ( l .1 equivalents) at 0
~C. The reaction is stirred for 2 hours and then diluted with
SUBSTITUTE SHEET (RULE 26)
CA 02214789 1997-09-0~
W096~ m g ~ 6tO3227
-120-
diethylether and washed with ammonium chloride (2X), copper
sulfate (2X), brine (lX), dried over MgSO4 and concentrated.
Purification by flash column chromatography affords compound
218. scheme 23 step m
Preparation of 220
Ho - l~o~l - cH2oDMT
TESO~J 'OTES
OTES
220
To a solution of 218 (1.0 equivalents) in diethylether (.08
M), is added lithiumaluminumhydride (LAH) ( 1.5 equivalents) at
30 ~C. 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 (2X), brine ( 1 X) and
is then dried (MgSO4) and concentrated. Purification by flash
column chromatography affords compound 220. scheme 23
step n
SUBSTITUTE S~l EET (RU LE 26)
CA 02214789 1997-09-05
WO 96t2mg ~I_lJ~Y6J1~3227
--121--
Preparation of 221
D MT~X~~ N ~2
TESO 'OTES
OTES
221
To Tetrol 20~ (1.0 equivalents) in pyridine (.10
Molar), is added dimethyoxytritylchloride (DMT chloride) (2.5
equivalents) at 0 ~C. The reaction is stirred for 2 hours and
then diluted with diethylether and washed with ammonium
chloride (2X), copper sulfate (2X), brine (lX), 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 (2X), brine (lX) and then dried (MgSO4) and
concentrated. Purification by flash column chromatography
affords compound 221. scheme 23 step a
SUBSTITUTE SHEET (RULE 26)
CA 022l4789 l997-09-05
W096127379 ~-l/U~ 03227
-122-
Preparation of 222
DMTO~O~C02H
TESO~J OTES
OTES
222
To solution of 221 in CC14 (.33 M), CH3CN (.33 M) and
water (.22 M) at 20 ~C is added RuCl3 (.03 equiv.) and NaIO4
(4.0 equiv.) and the muddy black mixture is allowed to stir for
10 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 (2X)~ recombined, and the organic layer iss
then dried MgSO4 and evaporated. Purification by flash column
chromatography affords the compound 222. scheme 23 step
o.
Preparation of 224
DMTO O CH2OH
TESOX~'OTES
OTES
224
To a solution of 222 ( 1.0 equivalents) in diethylether (.08
SUBSTITUTE SHEET (RULE 26)
CA 022l4789 l997-09-0~
wo96~73n ~ U~6103227
-123-
M), is added lithiumaluminumhydride (LAH) ( 1.5 equivalents) at
30 ~C. 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 (2X), brine ( 1 X) and
Sis then dried (MgSO4) 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 (.10Molar) at 25 ~C, is added 1-
hydroxybenzotriazole(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
15substituted solid support (N-(2-Aminoethyl)-3-amino-propyl
glass; aminopolystyrene resin; aminopropyl glass; isothiocyanato
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 NaHCO3 (2X), water (2X), and brine (2X). The organic
phase is dried over MgSO4 and then concentrated.
Preparation of 226; 228; 230 or 232
To a stirred solution of the acid 214; 62; 215 or 62 ( 1 . 0
SUI~STITUTE SHEET (RULE 26)
CA 02214789 1997-09-0~
WO9612?379 PCTrUS96103227
-124-
equivalents) and the amine 216; 226; 228 or 230 ( 1.1
equivalents) in dimethylformamide (.10 Molar) at 25 ~C, 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 NaHCO3
(2X), water (2X), and brine (2X). The organic phase is dried over
M g S 0 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 ~C, is added piperidine ( 1.1 equivalents). The reaction is
stirred for 1 hour and is then diluted with ether, and washed
with aqueous CuSO4 (2X), water (2X), and brine (2X). The
organic phase is dried over MgSO4 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 (.50
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 3.0
Molar solution of NaOH (3.0 equivalents). The reaction is stirred
SUBSTITUTE SHEET (RUEE 26)
CA 02214789 1997-09-05
WO 96127'379 ~ U~56~03227
-125-
for 1 hour at SO ~C and is then quenched with aqueous NH4C l
(2X) and subsequently Iyophilized. Purification by HPLC
chromatography affords compound 234. scheme 24
SUBSTITUTE SHEET (RUI_E 26)
CA 02214789 1997-09-0~
WO 96127379 ~ l/U~ '??7
--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 itsel~ 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. ~ll 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.).
SUI~STITUTE SHEET (RULE 26)
CA 02214789 1997-09-05
wa 96~7379 ~-11u-_~D3227
--1 27--
cq
~:~~ 2i~ ~--~~ '~ \
~ ~o c~~~~ 1' 0~0
~Z 0 , ~ ~O
O ~ ", ~ I ~
~a
O ~ Q 5 ~ ~ ~
~ ~ ~ ~' ~
O ~, ~L I ~
~Q ~
O ~ I O
SUBSmU~E SHEET (RULE 26)
CA 022l4789 l997-09-05
W096127379 ~ 96103227
-128-
o ~ Ga I ~
o~@ ~ d~ ' ~ ~
O C/~ o ~0 X
C~-O~ ~C~"_
E ~ E
E_ ~" 'Z ~ G;;l I ~
~ Z
C~-~ C~ ~ I . X x
UJ n
O C~ o
C~O ~ ' X X 1~
_I O ~ ~ t7 - ' -- 0
LL
SUBSTITUTE SHEET (RULE 26)
.
CA 02214789 1997-09-05
WO 96/27379 ~ iu~56JD3227
-129-
C ~
X X 1l \ Z
T O ~ U~ O=~U~
O~u z ~ I c Q \ ~ ~~
~ b ", ~ r n ~ ~ ~
I . X
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x m
a ~ Q . a ~
rD ~ X '
N~
Q ~ _ c~ _
O L' D C~l I 11, C~ o
~ ---- u~
X
_~ aJ /
C., "_ ~I ~ /
l-- O ~
O ~ XX m
m ~,. m
Z
O ~ c
~n
C~ . X
N X
SUBSmUTE SHEET (RULE 26)
CA 02214789 1997-09-05
W 096127379 ~ 6/03227
-130-
Z Z
~ ~Z @ ~ . 8 ~
~~ ~ ~
o ~- 9 ~ ~ o ~1
~o ~ 0 ~ ~~
d ~ o ~~ O ~ ~ o ~
~z ~ /
~0 ~ o
~u~ E ~ ~ O~O m
a ~ o@ ~
o~ o
~0 I UJ
SUBSTITUTE SHEET (RULE 26)
CA 022l4789 l997-09-05
W096~7379 ~-1J~3~D3227
-131-
~ llJ ~ ~ ~ X _ I
O O 0 t
~~ t ~ ~ ~l \
cn O O ~ O. ~ ~ ~ o
~ ~L~ t Z i~/ ~ ~ ~ G~ I t
ID Z ~0~-o ~ ~ = Z 0
0
C~ ~ ~ ,c ~
UJ~ N ~ C E tn
~~C~
,N5 1-- N N
~ O ~ ~, . ~q /
o-~L cn o ~ ~ ~ g N X I /
0~0
O ~J~
SUBSTITUTE SHEET (RULE 26)
CA 022l4789 l997-09-05
W 096/27379 l~l/u~,G1~227
-132-
N
N N N j~ I CL ~~
~W ~0 ~ ~ ~ X ~ ~
o d oO~ l 0 ~~~
~ ~ e~ o
~ ~ IL 0~
~ ~ Q E ~
UJ - 'S ~ _ _ \
~0 ~ ~ID XX ~,
_- ~ ~ o~ Z O
' z 0 c~ o m t , x x a~ E
- c' o x u c
~0 _ _, ~ x, - ",'~ a~
DE r
~ Q G
~Z0~ - .~ t x xN 11 /
O 0
Q ~ ~ = ~ E-~: m
O ~ --~ ~
~ Z 0 C) ~ E~ . X x ~
~--O ~ ~
~ 0
SUBSTITUTE SHEET (RULE 26)
CA 022l4789 l997-09-0~
W096~7379 ~ 6'C~?7
-133-
Preparation of Nucleotoid ~ombinatorial libraries
(Scheme 550)
A depiction of the generation of a combinatorial library for
S 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 7 6 ( 1.0
equiv) was added methylene chloride (.1 M) and benzaldehyde
1.1 equiv), and the solution was exposed to ZnCl ( 1.1 equiv) at
25 ~C and allowed to stir for 2.5 hour. The solution is then
SUBSTITUTE SHEET tRULE 26)
CA 022l4789 l997-09-05
W096/2 m 9 ~ ,Cl03227
-134-
diluted with ether and then washed with a saturated solution of
sodium bicarbonate (2X), water (2X), brine ( 1 X) and then dried
over MgS04. The compound is purified by flash column
chromatography to yield the desired benzylidene.
SUBSTITUTE SHEET tRULE 26)
-
CA 02214789 1997-09-OS
W096~7379 ~-1lu~Y6JD3227
-135-
1 Synthesis of a C-2 differentiated sugar
HO~O C02Et 1. PhCHO, ZnCI, CH2C~Orco2Et
HO J~OH ~ Ph O'~'OH
HO 2. Bu2SnO, MeOH OBn
76 then BnBr, CsF, DMF 2C00
3. NaCNBH3 (~ eq), DMF
(3 A sieves, TFA 10 eq)
BnO~O o 4. PhCHO,ZnCI, CH2C12 BnO~l~O~I~oH
HO'~O Ph OBn
2C 20 2010
5. BnBr, NaH, THF
BnO~O~I - o 6. NBS, BaCO3 BnO~Ol~OH
OBn CCI4-C2H2Cl4~ H2O OBn
2030 7 o~CN
Cl' Ni(Pr)2
CH2CI2, (iPr)2EtN
o~CN
BnO~O~~O~ Ni(Pr)2 Bz = Benzoate
BnO ~'OBz Bn = Benzyl ether
OBn
20~0
Scheme 2000a
SUBSTITUTE SHEET (RULE 26)
CA 022l4789 l997-09-05
WO 96127379 r~~ 9~03227
-136-
2.Connection of the C-2 differentiated sugar to a solid support
BnO ~ OH 1. NaH, THF BnO~o ~N~ Solid Support ¦
Bn~'OBz BnO 'OH ~
OBn 2. then add to OBn
2040 Br ~N~ Solid Support ¦ 2060
3. 1% NaOH, MeOH
Scheme 2000b
SUBSTITUTE SHEET (RlJLE 26)
CA 022l4789 l997-09-0~
W096/Z7379 ~llU~,~6103227
-137-
" The benzylidene is then azeotroped with benzene (2X 100 mL)and then dried overnight under vacuum over P2 O 5 . 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
P2Os. Next, anhydrous DMF (.2M) is added to redissolve the tin
complex and then CsF ( 1.2 equiv.) and finally Benzyl bromide
(1.5 equiv.) are added and then heated (40 ~C) overnight. The
clear solution is partially distilled under vacuum, (3.3 mm Hg,
75-100 ~C) 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, N.; Ohno, M. Chemistry Letters,
Chem. Soc. of Japan 1987, 141.
S~JBSTITUTE SH EET (RULE 26)
CA 022l4789 l997-09-0~
W 096~7379 ~ 61o3227
-138-
Preparation of compound 2010.
Procedure adopted from Johansson R.; Samuelsson; B. J. Chem.
Soc., Chem. Commun., 19 8 4, 201. To a solution of the
benzylidene acetal ( 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 ~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
(2X). 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
(.1 M) and benzaldehyde ( 1.1 equiv), and the solution was
exposed to ZnCl ( 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 (2X), water (2X),
brine (lX) and then dried over MgSO4. The compound is
purified by flash column chromatography to yield the desired
benzylidene 2020.
SlJBSTITUTE SH EET (RULE 26)
CA 02214789 1997-09-0~
wa s6t2ms ~-h~u:3~6Jr~7
--139--
Preparation of compound ~030.
To a solution of alcohol 2020 (22.0 g, .1068 mol, 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
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 ~C and the mixture was diluted with ethyl acetate,
washed with water (2X), brine (lX), dried over MgSO4 and
evaporated. Purification by flash column chromatography yields
tribenzyl ether 2030.
Preparation of compound 2040.
Procedure as adopted from Hanessian S.; Organic Synt~eses
19 8 7, 243. To a suspension containing 1.0 equivalent of
benzylidene 2 0 3 0 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 (3X), then dried over anhydrous sodium sulfate and
evaporated. Purification by flash column chromatography yields
SUBSTITUTE SH EET tRULE 26)
CA 022l4789 l997-09-05
W096~7379 r~l/U~96/03227
-140-
tribenzyl ether 2040.
Preparation of compound 2050.
To a solution of 2040 (1.0 equivalents) in methylene chloride
(.10 M), is added diisopropylethylamine (4.0 equivalents) at 25
~C. 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 StlEET (RULE 26)
CA 022l4789 l997-09-05
W096~7379 ~ /03227
-14t-
Synthesis of a C1-C2-Phosophodiester oligomer using a solid support
o~CN
Bno~ol - o~pNi(pr)2 BnO~O~--o~~lrN--¦ Solid Sup )ort
BnO'~'OBz BnO
2050 2060
1. tetrazole, CH3CN
2. MCPBA, CH2Clz
~ 3. 1% NaOH, MeOH, 20OC
BnO~o ~N--¦ Solid Sup )ort
BnO ~ O
o p_o~CN
Bno~~l~d
BnO' i 'OBn
OH
1. 2050. then tetrazole, CH3CN
2. MCPBA, CH2C12
~ 3. 1% NaOH, MeOH, 20~C
BnO~O~_o ~N--¦ Solid Sup ~ort
BnO' ~3 ~
O FI_o~CN
Bno~~l~d
BnO'~3
BnO
O-F ~_o~CN
Bno~~~~d
,~BnO'~'OBn
OH
I TERA TE
.. .
Scheme 2001
SUBSTITUTE SHEET (RULE 26)
CA 022l4789 l997-09-0~
W 096~7379 ~ U~ 3227
-142-
brine (lX) and is then dried (MgSO4) and concentrated.
Purification by flash column chromatography (silica, 30% ethyl
acetate in petroleum ether) affords compound 2050 (as shown
in scheme 2000).
s
Preparation of compound 2060
To a solution of alcohol 2040 (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 . ) and
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 ( 1 X) to
give 2 0 6 0 . 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 amzno group on the support
(eg. succinate linkage, amide, ether, alkyl chain with terminal
carbon activated as free alcohol, bromide etc.).
SUBSTITUTE SHEET (RULE 26
CA 022l4789 l997-09-05
W096~7379 ~ 0~ 7
-143-
1. Synthesis of a C-3 ~lir~ , ILidLed sugar
1~
HO~CO2Et 1. PhCHO. ZnCI. CH2C12 ~'oH
HO 2 NaCNBH3(5 eq), DMF 2070
76 (3 A sieves, TFA 10 eq)
3. TBDPSCI, Et3N, CH2C12
4. Bu2SnO, MeOH,
BzBr, CsF, DMF
'1
BnO~OH 5. BnBr, NaH. THF BnO~OTBDPS
BzO 6. TBAF. THF OBz
2~90 2080
o~CN
7. ,p .
Cl N'(Pr)2
y CH2CI2, (iPr)2EtN
o~CN
BnO~rOrO~ N (Pr)2 Bz = benzoate
BnO ~~'OBn Bn = benzyl
BzO
2100
Scheme 2002a
SUBSTITUTE SHEET (RULE 26)
_ _ ,
CA 02214789 1997-09-05
WO 96127379 ~ 961Q3227
Z.Connection of the C-3 .li~el ~l ILid~d sugar to a solid support
BnO~OH 1. NaH, THF BnO~~o ~N--l SoGd Suppor
BzO 2. then add to HO
2090 Br~,N--l Solid Suppo~ 2110
o
3. 1% NaOH, MeOH
S~heme 2002b
SUBSTITUTE 5HEET (RUL~ 26)
CA 022l4789 l997-09-0~
W096~7379 ~ 03227
-145-
Preparatfon 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 ZnCl (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 (2X), water (2X), brine
(lX) and then dried over MgSO4. The compound is purified by
flash column chromatography to yield the desired benzylidene.
Procedure adopted from Johansson R.; Samuelsson; B. J. Chem.
Soc., Chem. Commun., 19 8 4, 201. To a solution of the
benzylidene acetal ( 1 equiv) and sodium cyanoborohydride (5
equiv . ) in DMF ( .125 M) containin~, 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
(2X). 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
(.10 Molar), is added triethylamine ( 1.1 equivalents) at 0 ~C.
SU~STITUTE SHEET (RULE 26)
,
CA 022l4789 l997-09-0~
W 096r27379 PCTÇUS96/03227
-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 (2X), brine ( lX) and then dried (MgSO4) 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 P2 O 5 . 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
P2O5. Next, anhydrous DMF (.2M) 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 ~C) overnight. The clear solution is partially distilled
under vacuum, (3.3 mm Hg, 75-100 ~C) 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)
CA 022l4789 l997-09-05
W 096~7379 r~-l/U~6/03227
-147-
organic 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 l etters, Chem. Soc. of Japan 1987, 141.
s
lPreparation 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
severaI portions. The reaction mixture is warmed to room
temperature and stirred 1 h. Next, the reaction is cooled to 0 ~C
and treated with benzyl bromide ( 1.0 equiv.) and stirred for 1.5
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
(2X), brine ( lX), 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
(.10 M), is added diisopropylethylamine (4.0 equivalents) at 25
~C. The reaction is stirred for 5 minutes and then 2-cyanoethyl-
N, N-diisopropyl-chlorophosphoramidite ( 1.5 equiv) is added, as
SUE~STITUTE StlEET (RULE 26)
CA 022l4789 l997-09-05
W096127379 PCT/u~ C~7
-148-
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 (lX) and
is then dried (MgSO4) and concentrated. Purification by flash
S column chromatography (silica, 30% ethyl acetate in petroleum
ether) affords compound 2100 (as shown in scheme 2002).
SUE~STITUTE SHE~:T (RUL~ 26)
CA 02214789 1997-09-05
W096127379 ~ /U~961o3227
-149-
Synthesis of a C1-C3-Phosophodiester oligomer using a solid support
o~CN
BnO~--o~PNi(Pr)2BnO'~-O~ o~~N--¦ Solid Supp )rt
BnO 'OBnBnO'~'OBn ~
J 'BZO OH
2100 211 0
1. tetrazole, CH3CN
2. MCPBA. CH2CI2
~ 3. 1% NaOH, MeOH, 20~C
BnO~O~ - O - ~N-- Solid Supp )rt
BnO'~'OBn ~
O=P--o~CN
BnO~O~
BnO'~f 'OBn
OH
1. 2100, then tetrazole, CH3CN
2. MCPBA, CH2C12
~ 3. 1% NaOH, MeOH, 20~C
BnO~~o ~N--¦ Solid Supp ~rt
o
O=P--o~CN
Bno~~~d
BnO~ OBn
O=P--o~CN
Bno~~l~d
BnO'~'OBn
OH /TERATE
Scheme 2003
SUBSTITUTE SHEET (RULE 26)
CA 022l4789 l997-09-0~
W096/27379 ~-1J~3G/03227
-150-
Preparation of compound 2110
To a solution of alcohol 2090 (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 1 h. 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
~C and the support was washed with ethyl acetate, 1% NaOH in
methanol (2X) to remove the benzoate and finally brine ( 1 X) 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
f eg. succinate linkage, amide, ether, alkyl chain with terminal
carbon activated as free alcohol, bromide etc.).
Preparation of compound 2120
To a solution of 7 6 ( 1 .0 equiv) was added methylene chloride
(.1 M) and benzaldehyde ( 1.1 equiv), and the solution was
exposed to ZnCl ( 1.1 equiv) at 25 ~C and allowed to stir for 2.5
SUBSTITUTE SHEET (RULF 26)
CA 022l4789 l997-09-05
W096~7379 ~-lIV~96/03227
-151-
hour. The solution is then diluted with ether and then washed
with a saturated solution of sodium bicarbonate (2X), water (2X),
brine (lX) and then dried over MgS04. The compound is
J purified by flash column chromatography to yield the desired
SUBSTITUTE SHEET (RULE 26)
CA 022l4789 l997-09-05
W096/27379 PCT/u~ tt7
-152-
1. Synthesis of a C-4 differentiated sugar
HO~CO2Et 1. PhCHO, ZnCI, CH2CI2 phl~'OBn
7H6O 2. BnBr, NaH. CH2C12 2120
3. NaCNBH3 (5 eq), DMF
(3 A sieves, TFA 10 eq)
BnO~OTBDPS 4 TBDPSCI, Et3N, CH2CI2 gno~OTBDPS
BzO 'OBn HO 'OBn
OBn 5. BzBr, NaH, THF OBn
2140 21 30
6. TBAF, THF
BnO~OH 7- Cl'P ~ (P" BnO~ o~CN
OBn CH2CI~ Pr)2EtN OBn Bn = benzyl
2150 21 60
Scheme 2004a
SU~STITUTE SHEET (RULE 26)
CA 022l4789 l997-09-05
wog6n7379 ~ U~C~3227
-153-
'~ 2.Connection of the C-4 differentiated sugar to a solid support
BnO~rO~~OH 1. NaH, THF BnO~O~~o~~N--l Solid Supp )rt
BzO y' OBn HO I 'OBn ~
OBn 2. then ~ 1 lo OBn
2150 Br ~N-- Solid Supp )rt 2170
3.1% NaOH, MeOH
Scheme 2004b
SUSSTITUTE SHEET (RUL~ 26)
CA 022l4789 l997-09-0~
W096/2n79 ~-l/U~/03227
-154-
benzylidene and carried on as follows:
To a solution of benzylidene (1.0 equiv.) in ~HF (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 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 (2X),
brine ( lX), dried over MgSO4 and evaporated. Purification by
flash column chromatography yields tribenzyl ether 2120.
Preparation of compound 2130
Procedure adopted from Johansson R., Samuelsson; B. J. Chem.
Soc., Chem. Commun., 19 8 4, 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 ~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 (2X). 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~
CA 022l4789 l997-09-0~
W096~7379 ~ Y6103227
-155-
flash column chromatography yields the desired benzyl ether
2130.
Preparation of compound 2140
To a solution of 2130 ( 1.0 equivalents) in methylene chloride
(.10 Molar), is added triethylamine ( 1.1 equivalents) at 0 ~C.
Subsequent addition of tertbutyldiphenylsilylchloride ( 1. l
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. 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 1 h. Next, the reaction is cooled to 0 ~C
and treated with benzoyl bromide to afford benzoate formation
( 1.0 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 (2X), brine (lX), dried over
M g S O 4 and evaporated. Purification by flash column
chromato~raphy yields tribenzyl ether 2140.
,.
SUBSTiTUTE StlEET ~RULE 26)
CA 022l4789 l997-09-0~
W096/27379 r~l/U~6/03227
-156-
Preparation of compound 2150
The compound 2140 is then treated with tetrabutylammonium
fluoride (2.0 equivalents) in THF (.1 Molar) and allowed to stir
for an additional 2 hours at 25 ~C. 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 (lX), dried over
M g S O 4 and evaporated. Purification by flash column
chromatography yields tribenzyl ether 2150.
Preparation of compound 2160
To a solution of 215 0 ( 1.0 equivalents) in methylene chloride
(.10 M), is added diisopropylethylamine (4.0 equivalents) at 25
~C. 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 ( l X) and
is then dried (MgSO4) and concentrated. Purification by flash
column chromatography (silica, 30~o ethyl acetate in petroleum
ether) affords compound 2160 (as shown in scheme 2004).
SUBSTITUTE SHEET (RULE 26)
-
CA 022l4789 1997-09-OF.
WO 96t2mg PCr/~S96~03227
--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.)
SU B STITI JTE SH EET (RU LE 26)
CA 022l4789 l997-09-05
WO9612 m 9 ~tLllU~,''03n7
-158-
Synthesis of a C1-C4-Phosophodiester oligomer using a solid support
o~CN
BnO~O~o' Ni(Pr)2BnO~~O~l o ~N--~ Solid Supp ~rt
BzO' ~ 'OBnHO'~f'OBn ~
BnO BnO
2160 2170
I
1. tetrazole, CH3CN
2. MCPBA. CH2CI2
~, 3. 1% NaOH, MeOH, 20~C
BnO ~O ~fN--¦ Solid Supp 7rt
~' 'OBn O
¦ BnO CN
BnO~O~I J
HO'~'OBn
BnO
1. 2160, then tetrazole, CH3CN
2. MCPBA. CH2C12
~, 3.1% NaOH, MeOH, 20~C
BnO~o~~N--¦ Solid Supp )rt
~' 'OBn ~
¦ BnO CN
BnO~O~--J
~;)'~'OBn
I BnO
O=P--o~CN
BnO~O~--J
HO'~'OBn
OBn tTERATE
Scheme 2005
SUBSTITUTE SHEET (RULE 26)
CA 022l4789 l997-09-05
W096~7379 ~ 7~61o3227
-159-
and stirred for 2 hours. A saturated solution of ammonium
Y 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 (lX) to give 2170. 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 pllrchased from Sigma company. All supports may be
with or without a linker extending from the amino group on the
0 support (eg. succinate linkage, amide, ether, alkyl chain with
terminal carbon activated as free alcohol, bromide elc.).
Prepartion of compound 3010
Procedure as described in Methods in Carbohydrate chemistry,
Whistler, R., II, 1963, p. 327. A mixture of 80 g anhydrous D-
glucosamine hydrochloride or D-galactosamine hydrochloride
from Aldrich chemical company, in 200 mL. methanol and 20g
Dowex 50 (H+) 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 Sl IEET (RULE 26)
CA 022l4789 l997-09-05
W096127379 PCTnUS96/03227
-160-
The methyl glycoside is dissolved in chloroform (.S 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 (RUI_E 26)
CA 02214789 l997-09-05
W096~7379 PCTnUS96103227
-161-
1. C-2 differentiated amine derivative
Ho'~,~r~H 1. MeOH, H+ HO~OrOMe 3- BnBr, NaH BnO~OrCO2Et
HO ~ Y HO 4. TMSCN, SnC4 BnO
CHCI3 3020
Glucose amine hydrochloride 3010 5. EtOH, H2SO4 (cat)
6. FMOC-CI, K2CO3,
THF, H2O, 0 ~C.
~ 7. NaOEt, MeOH
BnO~O C02H
BnO'~XNHFMOC
BnO 3030
2. Connection of a C-2 dirr~ liaL~ amine sugar to a solid support
BnO~O CO2H 1. DCC, HOBT, Et3N, DMF ~
r ~ BnO'~O~o_ Solid Su~ ~ort
BnO' ~ NHFMOC
BnO I BnO' '~'NHz
3030 HO--lSolid Supp )rtBn3oo4o
2. Piperidine, DMF
Scheme 3000
SUE~STITUTE SH EET (RULE 26)
CA 02214789 1997-09-0~
W 096~7379 ~ 1U~g6/03227
-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 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 (2X),
brine ( lX), 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 SnCl4 (.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 rem~ining oil is resuspended in dichloromethane and
washed with sodium acetate solution (lX), water (lX), brine (lX)
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 H2SO4 (0.01
SUBSTITUTE SHEET (RULE 26)
CA 022l4789 l997-09-0~
W0961~7379 ~ C~03227
-163-
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 3020 scheme 3000.
Prepartion of compound 3030
To a solution of 3020 ( 1.0 equivalents) in methylene chloride
(.10 Molar), is added potassium carbonate (2.0 equivalents) at 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 diethylether and washed
with ammonium chloride (2X), brine ( lX) and then dried
( M g S 0 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 ~C~ is
added l-hydroxybenzotriazole (HOBT; 1.1 equivalents). Next
SUBSTITUTE SHEET (RULE 26)
CA 022l4789 l997-09-05
W 096r27379 PCTIU~ ?7
-164-
dicyclohexylcarbodiimide ( 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, alkvl chain with terminal carbon activated
0 as free alcohol, bromide etc.)J. The mixture is then
diluted with ether, washed with aqueous NaHCO3 (2X), water
(2X), and brine (2X) . Next, the compound/support ( 1.0
equivalents) in dimethyl-formamide (.10 Molar) at 25 ~C, 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 CuSO4 (2X), water (2X), and brine (2X). The final
step affords compound 3040.
SUBSTITUTE SHEET (RULE 26)
CA 02214789 1997-09-05
WO 96127379 ~ 3C~03227
_165-
BnO O CO2H BnO O~'o{ Soiid Sui~ort
Bn;~XNHFMOC Bn~NH2
BnO BnO
3030 3040
1. DCC, HOBT, Et3N, DMF
2. piperidine, DMF
o
BnO~O~o{ Solid Sui~ort
~BnO'~'N
BnO~O~OHBnO O~ ~ ~OBn
BnO'~'NHFMOC+H2N~OBn
BnO OBn
3030 3050 1
1. DCC, HOBT, Et3N, DMF
~,~ 2. piperidine, DMF
BnO~O~ Solid Sui-4~ort
OBnO- 'N
BnO,~,O~ OH BnO o~rO~--OBn
BnO'JyL'NHFMOC N~. OBn
BnOBnO~~O~OOBn
3030BnO~L NH2
BnO
3060
1. DCC, HOBT, Et3N, DMF
~ 2. piperidine, DMF
/terate as many steps as needed
Scheme 3001
SUBSTITUTE SHEET (RULE 26)
CA 022l4789 l997-09-0~
W096~7379 r~ 96/03227
-166-
Physical Data for scheme 9.
Phosphoramidate 138 (2 diastereomers): IR, (neat) cm-l:
3089, 2964, 2927, 2856, 2253, 1497, 1455, 1396, 1363,
1253, 1184, 1156, 1094, 1028, 978, 876, 836, 779, 735,
lH-NMR (400 MHz, C6D6): ~ 7.34 (m, 5 H, Ph), 7.14 (m, 10
H, Ph), 4.97 (m, 4 H, CH2Ph), 4.78 (m, 2 H, CH2Ph),
4.07-3.24 (m, 13 H, OCH, OCH2, CH2CN), 1.81 (m, 2 H,
CH(CH3)2), 1.16 (m, 12 H, CH3CH), 1.03, 1.02 (2 s, 9 H,
lBuSi), 0.20, 0.18, 0.16, 0.15, (4 s, 6 H, Me2Si);
HRMS: C43H6307N2PSi, Calc. (M+Cs+): 911.3197; ~ound:
911.3185.
Naphthoylester 135 IR, (neat) cm-l: 3494, 3062, 2919,
1716, 1630, 1600, 1454, 1355, 1284, 1228, 1197, 1091,
779, 736i lH-NMR (250 MHz, CDCI3): ~ 8.58 (s, 1 H,
Ar), 8.00 (m, 2 H, Ar), 7. 89 (m, 2 H, Ar), 7.59 (m, 2
H, Ar), 7.32 (m, 15 H, Ph), 4.95 (m, 3 H), 4.90 (d,
,;r=4 5 Hz, 1 H), 4.69 (m, 3 H), 4.52 (dd, J = 3.9, 12.0
Hz, 1 H), 3.91 (dd J = 2.6, 12.0, 1 H), 3.83 (d, ~J --
8.3, 1 H), 3.70 (m, 4 H), 3.96 (m, 1 H), 2.25 (s, 1 H,
OH). HRMS: C3gH3807 Calc. (M+Cs+): 751.1672i found:
751.1668.
Dimer 142 IR, (neat) cm-l: 3397, 3030, 2923, 2254, 1718,
1653, 1629, 1497, 1453, 1355, 1284, 1227, 1197, 1094,
1029, 780. lH-NMR (400 MHz, C6D6): ~ 8.82 (s, 1 H,
Ar), 8.26 (d, 1 H, Ar), 7.72 (m, 1 H, Ar), 7.61 (m,
H, Ar), 7.48 (m, 1 H, Ar), 7.37-6.95 (m, 32 H, Ar, Ph),
4.89-4.18 (m, 21 H, CH2Ph, CH2-Ar, -CH2CH2CN, CHCH2-Ar
and CH20H), 3.95-3.45 (m, 13 H, CH- and CH2-sugar),
1.71 (s, 1 H, OH); HRMS: Cl70H7205NP calc. (M+H+):
1198.4718; found: 1198.4715.
SUBSTtTUTE SHEET (RULE 26)
CA 022l4789 l997-09-05
W096127379 ~JU~ 3227
-167-
Te~ramer 150 IR, (neat) cm-l: 3420, 3064, 2924, 2255,
1721, 1497, 1455, 1357, 1278, 1028, 737. lH-NMR (400
MHz, CDCI3): ~ 8.41 (s, 1 H, Ar), 8.00 (m, 2 H, Ar),
7.91 (m, 2 H, Ar), 7.55 (m, 2 H, Ar), 7.30 (m, 60 H,
Ph), 4.93-4.05 (m, 39 H, CH2Ph, CH2-Ar, CH2CH2CN and
CH2OH), 3.88-3.27 (m, 23 H, CH- and CH2-sugar), 2.58
(s, 1 H, OH). HRMS: Cl32Hl40O3lN3P3 Calc. (M+Cs+):
2488.7738; found: 2488.7758.
Tetramer 154 IR, (neat) cm-l: 3376, 2934, 1450, 1244,
1110, 1088. lH-NMR (400 MHz, D20): ~ 8.41 (s, 1 H,
Ar), 8.00 (m, 2 H, Ar), 7.91 (m, 2 H, Ar), 7.55 (m, 2
H, Ar), 4.93-4.05 (m, 4 H, CH2-Ar and CX2OH), 3.88-3.27
(m, 32 H, CH- and CH2-sugar); HRMS: C39H59O3lP3 Calc.
(M+H+): 1117.2331; ~ound: 1117.2350.
SUBSTITUTE S~tEET (RULE 26)
