U.S. patent application number 10/663155 was filed with the patent office on 2004-03-11 for oligonucleotides containing 2'-0-modified purines.
This patent application is currently assigned to ISIS Pharmaceuticals, Inc.. Invention is credited to Cook, Phillip Dan, Guinosso, Charles John, McGee, Daniel Peter Claude.
Application Number | 20040048826 10/663155 |
Document ID | / |
Family ID | 31999583 |
Filed Date | 2004-03-11 |
United States Patent
Application |
20040048826 |
Kind Code |
A1 |
Cook, Phillip Dan ; et
al. |
March 11, 2004 |
Oligonucleotides containing 2'-0-modified purines
Abstract
Novel 2'-O-alkyl guanosine compounds are provided. In accordance
with preferred embodiments compounds having the structure: 1
wherein X is R.sub.1--(R.sub.2).sub.n; R.sub.1 is C.sub.3-C.sub.20
alkyl, C.sub.4-C.sub.20 alkenyl or C.sub.2-C.sub.20 alkynyl;
R.sub.2 is halogen, hydroxyl, thiol, keto, carboxyl, nitro,
nitroso, nitrile, trifluoromethyl, trifluoromethoxy, O-alkyl,
S-alkyl, NH-alkyl, N-dialkyl, O-aryl, S-aryl, NH-aryl, O-aralkyl,
S-aralkyl, NH-aralkyl, amino, N-phthalimido, imidazole, azido,
hydrazino, hydroxylamino, isocyanato, sulfoxide, sulfone, sulfide,
disulfide, silyl, aryl, heterocycle, carbocycle, intercalator,
reporter molecule, conjugate, polyamine, polyamide, polyalkylene
glycol, polyether, a group that enhances the pharmacodynamic
properties of oligonucleotides, or a group that enhances the
pharmacokinetic properties of oligonucleotides; and n is an integer
from 0 to about 6, are provided.
Inventors: |
Cook, Phillip Dan;
(Carlsbad, CA) ; McGee, Daniel Peter Claude;
(Carlsbad, CA) ; Guinosso, Charles John; (Vista,
CA) |
Correspondence
Address: |
WOODCOCK WASHBURN LLP
ONE LIBERTY PLACE - 46TH FLOOR
PHILADELPHIA
PA
19103
US
|
Assignee: |
ISIS Pharmaceuticals, Inc.
|
Family ID: |
31999583 |
Appl. No.: |
10/663155 |
Filed: |
September 15, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10663155 |
Sep 15, 2003 |
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07967267 |
Oct 27, 1992 |
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07967267 |
Oct 27, 1992 |
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07918362 |
Jul 23, 1992 |
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5506351 |
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07918362 |
Jul 23, 1992 |
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07463358 |
Jan 11, 1990 |
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07918362 |
Jul 23, 1992 |
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07566977 |
Aug 13, 1990 |
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Current U.S.
Class: |
514/45 ;
536/27.23 |
Current CPC
Class: |
C12N 15/1137 20130101;
A61K 48/00 20130101; C07H 19/04 20130101; C12N 2310/32 20130101;
C12N 2310/3523 20130101; C12N 2310/3531 20130101; C07H 23/00
20130101; C12N 15/113 20130101; C12N 2310/321 20130101; C12N
2310/3533 20130101; C12N 2310/333 20130101; C12N 2310/3527
20130101; A61K 38/00 20130101; C07H 19/06 20130101; C12N 2310/321
20130101; C12Q 1/68 20130101; C07H 19/16 20130101; C12Y 113/11012
20130101; C12N 9/0069 20130101; C12N 2310/3521 20130101; C12N
2310/3125 20130101; C12N 2310/336 20130101; C12N 2310/33 20130101;
C12N 2310/315 20130101; C12N 2310/322 20130101; C07H 19/10
20130101; C07H 21/00 20130101; C07H 19/20 20130101; C12N 2310/321
20130101; C12N 2310/3523 20130101; C07J 43/003 20130101; C12N
2310/3527 20130101 |
Class at
Publication: |
514/045 ;
536/027.23 |
International
Class: |
A61K 031/7076; C07H
019/16 |
Claims
What is claimed is:
1. A compound having the structure: 14wherein X is
R.sub.1--(R.sub.2).sub.n; R.sub.1 is C.sub.3-C.sub.20 alkyl,
C.sub.4-C.sub.20 alkenyl or C.sub.2-C.sub.20 alkynyl; R.sub.2 is
halogen, hydroxyl, thiol, keto, carboxyl, nitro, nitroso, nitrile,
trifluoromethyl, trifluoromethoxy, O-alkyl, S-alkyl, NH-alkyl,
N-dialkyl, O-aryl, S-aryl, NH-aryl, O-aralkyl, S-aralkyl,
NH-aralkyl, amino, N-phthalimido, imidazole, azido, hydrazino,
hydroxylamino, isocyanato, sulfoxide, sulfone, sulfide, disulfide,
silyl, aryl, heterocycle, carbocycle, intercalator, reporter
molecule, conjugate, polyamine, polyamide, polyalkylene glycol,
polyether, a group that enhances the pharmacodynamic properties of
oligonucleotides, or a group that enhances the pharmacokinetiic
properties of oligonucleotides; and n is an integer from 0 to about
6.
2. The compound of claim 1 wherein R.sub.1 is C.sub.4-C.sub.20
alkyl.
3. The compound of claim 1 wherein R.sub.1 is C.sub.5-C.sub.20
alkyl.
4. A compound having the structure: 15wherein X is
R.sub.1--(R.sub.2).sub.n; R.sub.1 is C.sub.3-C.sub.20 alkyl;
R.sub.2 is NH.sub.2, imidazole, or N-phthalimido; Y is a hydroxyl
blocking group; Z is phosphate or an activated phosphate group;
Q.sub.1 and Q.sub.2 independently are H or a guanosine blocking
group; and n is an integer from 0 to about 6.
5. The compound of claim 4 wherein: Y is trityl, methoxytrityl,
dimethoxytrityl or trimethoxytrityl.
6. The compound of claim 4 wherein: Z is
.beta.-cyanoethyl-N,N-isopropylph- osphoramidate.
7. A compound having the structure: 16wherein X is
R.sub.1--(R.sub.2).sub.n; R.sub.1 is C.sub.3-C.sub.20 alkyl,
C.sub.4-C.sub.20 alkenyl or C.sub.2-C.sub.20 alkynyl; R.sub.2 is
halogen, hydroxyl, thiol, keto, carboxyl, nitro, nitroso, nitrile,
trifluoromethyl, trifluoromethoxy, O-alkyl, S-alkyl, NH-alkyl,
N-dialkyl, O-aryl, S-aryl, NH-aryl, O-aralkyl, S-aralkyl,
NH-aralkyl, amino, imidazole, N-phthalimido, azido, hydrazino,
hydroxylamino, isocyanato, sulfoxide, sulfone, sulfide, disulfide,
silyl, aryl, heterocycle, carbocycle, intercalator, reporter
molecule, conjugate, polyamine, polyamide, polyalkylene glycol,
polyether, a group that enhances the pharmacodynamic properties of
oligonucleotides, or a group that enhances the pharmacokinetic
properties of oligonucleotides; and n is an integer from 0 to about
6.
8. The compound 2'-O-propylguanosine, 2'-O-pentylguanosine,
2'-O-nonylguanosine, 2'-O-octadecylguanosine,
2'-O-(N-phthalimido)-pentyl- guanosine, or
2'-O-(imidazol-1-yl)butylguanosine.
9. An oligomer comprising at least one subunit having the
structure: 17wherein X is R.sub.1--(R.sub.2).sub.n; R.sub.1 is
C.sub.3-C.sub.20 alkyl, C.sub.4-C.sub.20 alkenyl, or
C.sub.2-C.sub.20 alkynyl; R.sub.2 is halogen, hydroxyl, thiol,
keto, carboxyl, nitro, nitroso, nitrile, trifluoromethyl,
trifluoromethoxy, O-alkyl, S-alkyl, NH-alkyl, N-dialkyl, O-aryl,
S-aryl, NH-aryl, O-aralkyl, S-aralkyl, NH-aralkyl, amino,
imidazole, N-phthalimido, azido, hydrazino, hydroxylamino,
isocyanato, sulfoxide, sulfone, sulfide, disulfide, silyl, aryl,
heterocycle, carbocycle, intercalator, reporter molecule,
conjugate, polyamine, polyamide, polyalkylene glycol, polyether, a
group that enhances the pharmacodynamic properties of
oligonucleotides, or a group that enhances the pharmacokinetic
properties of oligonucleotides; T.sub.3 and T.sub.5 independently
are OH or a further subunit of said oligomer that is joined to said
structure; and n is an integer from 0 to about 6.
10. An oligomer comprising at least one subunit having the
structure: 18wherein X is R.sub.1--(R.sub.2).sub.n; R.sub.1 is
C.sub.1-C.sub.20 alkyl, C.sub.2-C.sub.20 alkenyl, or
C.sub.2-C.sub.20 alkynyl; R.sub.2 is halogen, hydroxyl, thiol,
keto, carboxyl, nitro, nitroso, nitrile, trifluoromethyl,
trifluoromethoxy, O-alkyl, S-alkyl, NH-alkyl, N-dialkyl, O-aryl,
S-aryl, NH-aryl, O-aralkyl, S-aralkyl, NH-aralkyl, amino,
imidazole, N-phthalimido, azido, hydrazino, hydroxylamino,
isocyanato, sulfoxide, sulfone, sulfide, disulfide, silyl, aryl,
heterocycle, carbocycle, intercalator, reporter molecule,
conjugate, polyamine, polyamide, polyalkylene glycol, polyether, a
group that enhances the pharmacodynamic properties of
oligonucleotides, or a group that enhances the pharmacokinetic
properties of oligonucleotides; T.sub.3 and T.sub.5 independently
are OH or a further subunit of said oligomer that is joined to said
structure; and n is an integer from 0 to about 6.
11. A method of modulating the synthesis of a protein comprising
specifically hybridizing with mRNA coding for said protein an
oligomer comprising at least one subunit having the structure:
19wherein X is R.sub.1--(R.sub.2).sub.n; R.sub.1 is
C.sub.3-C.sub.20 alkyl, C.sub.4-C.sub.20 alkenyl, or
C.sub.2-C.sub.20 alkynyl; R2 is halogen, hydroxyl, thiol, keto,
carboxyl, nitro, nitroso, nitrile, trifluoromethyl,
trifluoromethoxy, O-alkyl, S-alkyl, NH-alkyl, N-dialkyl, O-aryl,
S-aryl, NH-aryl, O-aralkyl, S-aralkyl, NH-aralkyl, amino,
imidazole, N-phthalimido, azido, hydrazino, hydroxylamino,
isocyanato, sulfoxide, sulfone, sulfide, disulfide, silyl, aryl,
heterocycle, carbocycle, intercalator, reporter molecule,
conjugate, polyamine, polyamide, polyalkylene glycol, polyether, a
group that enhances the pharmacodynamic properties of
oligonucleotides, and a group that enhances the pharmacokinetic
properties of oligonucleotides; T.sub.3 and T.sub.5 independently
are OH or a further nucleotide or nucleoside of said
oligonucleotide or oligonucleoside that is joined to said
structure; and n is an integer from 0 to about 6.
12. The method of claim 11 wherein said oligonucleotide is in a
pharmaceutically acceptable carrier.
13. A method of modulating the synthesis of a protein comprising
specifically hybridizing with mgNA coding for said protein an
oligomer comprising at least one subunit having the structure:
20wherein X is R.sub.1--(R.sub.2).sub.n; R.sub.1 is
C.sub.1-C.sub.20 alkyl, C.sub.2-C.sub.20 alkenyl, or
C.sub.2-C.sub.20 alkynyl; R.sub.2 is halogen, hydroxyl, thiol,
keto, carboxyl, nitro, nitroso, nitrile, trifluoromethyl,
trifluoromethoxy, O-alkyl, S-alkyl, NH-alkyl, N-dialkyl, O-aryl,
S-aryl, NH-aryl, O-aralkyl, S-aralkyl, NH-aralkyl, amino,
imidazole, N-phthalimido, azido, hydrazino, hydroxylamino,
isocyanato, sulfoxide, sulfone, sulfide, disulfide, silyl, aryl,
heterocycle, carbocycle, intercalator, reporter molecule,
conjugate, polyamine, polyamide, polyalkylene glycol, polyether, a
group that enhances the pharmacodynamic properties of
oligonucleotides, and a group that enhances the pharmacokinetic
properties of oligonucleotides; T.sub.3 and T.sub.5 independently
are OH or a further nucleotide or nucleoside of said
oligonucleotide or oligonucleoside that is joined to said
structure; and n is an integer from 0 to about 6.
14. The method of claim 15 wherein said oligonucleotide is in a
pharmaceutically acceptable carrier.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of application
Ser. No. 918,362, filed on Jul. 23, 1992 and application Ser. No.
U.S. Pat. No. 91/00243 filed on Jan. 11, 1991 which is a
continuation-in-part of application Ser. No. 463,358 filed on Jan.
11, 1990 and application Ser. No. 566,977 filed on Aug. 13, 1990.
This application is related to application Ser. Number 566,977,
filed on Aug. 13, 1990. These applications are assigned to the
assignee of the present application and are incorporated by
reference herein in their entirety.
BACKGROUND OF THE INVENTION
[0002] This invention is directed to novel 2'-O-alkyl guanosine and
guanosine analogs and methods of use thereof.
[0003] A limited number of oligonucleotide analogs have been made.
One class of oligonucleotides that have been synthesized are the
2'-O-substituted oligonucleotides. Such oligonucleotides have
certain useful properties. In U.S. patent application Ser. No.
814,961, filed Dec. 24, 1991, entitled Gapped 2' Modified
Phosphorothioate Oligonucleotides, assigned to the same assignee as
this application, the entire contents of which are herein
incorporated by reference, 2' substituted nucleotides are
introduced within an oligonucleotide to induce increased binding of
the oligonucleotide to a complementary target strand while allowing
expression of RNase H activity to destroy the targeted strand. In a
recent article, Sproat, B. S., Beijer, B. and Iribarren, A.,
Nucleic Acids Research, 1990, 18, 41 the authors noted further use
of 2'-O-methyl substituted oligonucleotides as "valuable antisense
probes for studying pre-mRNA splicing and the structure of
spliceosomes". 2'-O-methyl and ethyl nucleotides have been reported
by a number of authors. Robins, et al., J. Org. Chem., 1974, 39,
1891; Cotten, et al., Nucleic Acids Research, 1991, 19, 2629;
Singer, et al., Biochemistry 1976, 15, 5052; Robins, Can. J. Chem.
1981, 59, 3360; Inoue, et al., Nucleic Acids Research, 1987, 15,
6131; and Wagner, et al., Nucleic Acids Research, 1991, 19,
5965.
[0004] A number of groups have taught the preparation of other
2'-O-alkyl guanosine. Gladkaya, et al., Khim. Prir. Soedin., 1989,
4, 568 discloses N.sub.1-methyl-2'-O-(tetrahydropyran-2-yl) and
2'-O-methyl guanosine and Hansske, et al., Tetrahedron, 1984, 40,
125 discloses a 2'-O-methylthio-methylguanosine. It was produced as
a minor by-product of an oxidization step during the conversion of
guanosine to 9-.beta.-D-arabinofuranosylguanine, i.e. the arabino
analogue of guanosine. The addition of the 2'-O-methylthiomethyl
moiety is an artifact from the DMSO solvent utilized during the
oxidization procedure. The 2'-O-methylthiomethyl derivative of
2,6-diaminopurine riboside was also reported in the Hansske et al.
publication. It was also obtained as an artifact from the DMSO
solvent.
[0005] Sproat, et al., Nucleic Acids Research, 1991, 19, 733
teaches the preparation of 2'-O-allyl-guanosine. Allylation of
guanosine required a further synthetic pathway. Iribarren, et al.,
Proc. Natl. Acad. Sci., 1990, 87, 7747 also studied 2'-O-allyl
oligoribonucleotides. Iribarren, et al. incorporated 2'-O-methyl-,
2'-O-allyl-, and 2'-O-dimethylallyl-substituted nucleotides into
oligoribonucleotides to study the effect of these RNA analogues on
antisense analysis. Iribarren found that 2'-O-allyl containing
oligoribonucleotides are resistant to digestion by either RNA or
DNA specific nucleases and slightly more resistant to nucleases
with dual RNA/DNA specificity, than 2'-O-methyl
oligoribonucleotides. However, Iribarren found that
2'-O-dimethylallyl containing oligoribonucleotides exhibited
reduced hybridization to complementary RNA sequences as compared to
2'-O-methyl oligoribonucleotides. Thus, Iribarren suggested that
further attempts to prepare alkylated RNA probes, especially those
superior to 2'-allyl cytidine containing oligoribonucleotides
should be limited to 2'-O-alkyl groups containing less than five
carbon atoms.
[0006] In some cases it is desireable to provide 2'-O-alkyl groups
having long chain alkyl groups (i.e. four or more carbon atoms).
For example, long chain alkyl groups may accomodate functional
groups in appropriate orientation with the opposing strand upon
strand hybridization. Thus, 2'-O-long chain alkyl nucleotides such
as 2'-O-long chain alkyl guanosine nucleotides are highly
desireable in some cases. Novel 2'-O-alkylated guanosine compounds
are greatly desired. The present invention provides such
compounds.
BRIEF DESCRIPTION OF THE INVENTION
[0007] Compounds having the structure: 2
[0008] wherein X is R.sub.1--(R.sub.2).sub.n;
[0009] R.sub.1 is C.sub.3-C.sub.20 alkyl, C.sub.4-C.sub.20 alkenyl
or C.sub.2-C.sub.20 alkynyl;
[0010] R.sub.2 is halogen, hydroxyl, thiol, keto, carboxyl, nitro,
nitroso, nitrile, trifluoromethyl, trifluoromethoxy, O-alkyl,
S-alkyl, NH-alkyl, N-dialkyl, O-aryl, S-aryl, NH-aryl, O-aralkyl,
S-aralkyl, NH-aralkyl, amino, N-phthalimido, imidazole, azido,
hydrazino, hydroxylamino, isocyanato, sulfoxide, sulfone, sulfide,
disulfide, silyl, aryl, heterocycle, carbocycle, intercalator,
reporter molecule, conjugate, polyamine, polyamide, polyalkylene
glycol, polyether, a group that enhances the pharmacodynamic
properties of oligonucleotides, or a group that enhances the
pharmacokinetic properties of oligonucleotides; and n is an integer
from 0 to about 6; are provided in some embodiments of the
invention. In more preferred embodiments of the present invention n
is from 1 to about 3. In still more preferred embodiments of the
present invention n is 1.
[0011] Preferred compounds of the invention include
2'-O-propylguanosine, 2'-O-pentylguanosine, 2'-O-nonylguanosine,
2'-O-octadecylguanosine, 2'-O-(N-phthalimido)-pentylguanosine, and
2'-O-(imidazol-l-yl)butylguanos- ine.
[0012] In other embodiments of the present invention compounds
having the structure: 3
[0013] wherein X is R.sub.1--(R.sub.2).sub.n;
[0014] R.sub.1 is C.sub.3-C.sub.20 alkyl;
[0015] R.sub.2 is NH.sub.2, H-imidazole or N-phthalimido;
[0016] Y is a hydroxyl blocking group;
[0017] Z is phosphate or an activated phosphate group;
[0018] Q.sub.1 and Q.sub.2 independently are H or a guanosine
blocking group; and
[0019] n is an integer from 0 to about 6, are provided.
[0020] In other aspects of the invention compounds are provided
having the structure: 4
[0021] wherein X is R.sub.1--(R.sub.2).sub.n;
[0022] R.sub.1 is C.sub.3-C.sub.20 alkyl, C.sub.4-C.sub.20 alkenyl
or C.sub.2-C.sub.20 alkynyl;
[0023] R.sub.2 is halogen, hydroxyl, thiol, keto, carboxyl, nitro,
nitroso, nitrile, trifluoromethyl, trifluoromethoxy, O-alkyl,
S-alkyl, NH-alkyl, N-dialkyl, O-aryl, S-aryl, NH-aryl, O-aralkyl,
S-aralkyl, NH-aralkyl, amino, imidazole, N-phthalimido, azido,
hydrazino, hydroxylamino, isocyanato, sulfoxide, sulfone, sulfide,
disulfide, silyl, aryl, heterocycle, carbocycle, intercalator,
reporter molecule, conjugate, polyamine, polyamide, polyethylene
glycol, polyether, a group that enhances the pharmacodynamic
properties of oligonucleotides, or a group that enhances the
pharmacokinetic properties of oligonucleotides; and n is an iteger
from 0 to about 6.
[0024] Compounds of the present invention may be incorporated into
oligomers. Thus, in some aspects of the present invention are
provided oligomers containing at least one subunit having the
structure: 5
[0025] wherein X is R.sub.1--(R.sub.2).sub.n;
[0026] R.sub.1 is C.sub.3-C.sub.20 alkyl, C.sub.4-C.sub.20 alkenyl
or C.sub.2-C.sub.20 alkynyl;
[0027] R.sub.2 is halogen, hydroxyl, thiol, keto, carboxyl, nitro,
nitroso, nitrile, trifluoromethyl, trifluoromethoxy, O-alkyl,
S-alkyl, NH-alkyl, N-dialkyl, O-aryl, S-aryl, NH-aryl, O-aralkyl,
S-aralkyl, NH-aralkyl, amino, imidazole, N-phthalimido, azido,
hydrazino, hydroxylamino, isocyanato, sulfoxide, sulfone, sulfide,
disulfide, silyl, aryl, heterocycle, carbocycle, intercalator,
reporter molecule, conjugate, polyamine, polyamide, polyalkylene
glycol, polyether, a group that enhances the pharmacodynamic
properties of oligonucleotides, and a group that enhances the
pharmacokinetic properties of oligonucleotides;
[0028] T.sub.3 and T.sub.5 independently are OH or a further
subunit of said oligomer that is joined to said structure; and
[0029] n is an integer from 0 to about 6.
[0030] In other aspects of the invention, are provided oligomers
containing at least one subunit having the structure: 6
[0031] wherein X is R.sub.1--(R.sub.2).sub.n;
[0032] R.sub.1 is C.sub.1-C.sub.20 alkyl, C.sub.2-C.sub.20 alkenyl
or C.sub.2-C.sub.20 alkynyl;
[0033] R.sub.2 is halogen, hydroxyl, thiol, keto, carboxyl, nitro,
nitroso, nitrile, trifluoromethyl, trifluoromethoxy, O-alkyl,
S-alkyl, NH-alkyl, N-dialkyl, O-aryl, S-aryl, NH-aryl, O-aralkyl,
S-aralkyl, NH-aralkyl, amino, imidazole, N-phthalimido, azido,
hydrazino, hydroxylamino, isocyanato, sulfoxide, sulfone, sulfide,
disulfide, silyl, aryl, heterocycle, carbocycle, intercalator,
reporter molecule, conjugate, polyamine, polyamide, polyalkylene
glycol, polyether, a group that enhances the pharmacodynamic
properties of oligonucleotides, and a group that enhances the
pharmacokinetic properties of oligonucleotides; T.sub.3 and T5
independently are OH or a further subunit of said oligomer that is
joined to said structure; and n is an integer from 0 to about
6.
[0034] Methods of modulating the synthesis of a protein are also
provided by the present invention comprising specifically
hybridizing with mRNA coding for said protein an oligomer
containing at least one subunit having the structure having the
structure: 7
[0035] wherein X is R.sub.1--(R.sub.2).sub.n;
[0036] R.sub.1 is C.sub.3-C.sub.20 alkyl, C.sub.4-C.sub.20 alkenyl
or C.sub.2-C.sub.20 alkynyl;
[0037] R.sub.2 is halogen, hydroxyl, thiol, keto, carboxyl, nitro,
nitroso, nitrile, trifluoromethyl, trifluoromethoxy, O-alkyl,
S-alkyl, NH-alkyl, N-dialkyl, O-aryl, S-aryl, NH-aryl, O-aralkyl,
S-aralkyl, NH-aralkyl, amino, imidazole, N-phthalimido, azido,
hydrazino, hydroxylamino, isocyanato, sulfoxide, sulfone, sulfide,
disulfide, silyl, aryl, heterocycle, carbocycle, intercalator,
reporter molecule, conjugate, polyamine, polyamide, polyalkylene
glycol, polyether, a group that enhances the pharmacodynamic
properties of oligonucleotides, or a group that enhances the
pharmacokinetic properties of oligonucleotides; T.sub.3 and T.sub.5
independently are OH or a further subunit of said oligomer that is
joined to said structure; and n is an integer from 0 to about
6.
[0038] In still other aspects of the invention methods of
modulating the synthesis of a protein are provided comprising
specifically hybridizing with mRNA coding for said protein an
oligomer containing at least one subunit having the structure:
8
[0039] wherein X is R.sub.1--(R.sub.2).sub.n;
[0040] R.sub.1 is C.sub.1-C.sub.20 alkyl, C.sub.2-C.sub.20 alkenyl
or C.sub.2-C.sub.20 alkynyl;
[0041] R.sub.2 is halogen, hydroxyl, thiol, keto, carboxyl, nitro,
nitroso, nitrile, trifluoromethyl, trifluoromethoxy, O-alkyl,
S-alkyl, NH-alkyl, N-dialkyl, O-aryl, S-aryl, NH-aryl, O-aralkyl,
S-aralkyl, NH-aralkyl, amino, imidazole, N-phthalimido, azido,
hydrazino, hydroxylamino, isocyanato, sulfoxide, sulfone, sulfide,
disulfide, silyl, aryl, heterocycle, carbocycle, intercalator,
reporter molecule, conjugate, polyamine, polyamide, polyalkylene
glycol, polyether, a group that enhances the pharmacodynamic
properties of oligonucleotides, or a group that enhances the
pharmacokinetic properties of oligonucleotides; T.sub.3 and T.sub.5
independently are OH or a further subunit of said oligomer that is
joined to said structure; and n is an integer from 0 to about
6.
DETAILED DESCRIPTION OF THE INVENTION
[0042] This invention includes compounds having the structure:
9
[0043] wherein X is R.sub.1--(R.sub.2).sub.n;
[0044] R.sub.1 is C.sub.3-C.sub.20 alkyl, C.sub.4-C.sub.20 alkenyl
or C.sub.2-C.sub.20 alkynyl;
[0045] R.sub.2 is halogen, hydroxyl, thiol, keto, carboxyl, nitro,
nitroso, nitrile, trifluoromethyl, trifluoromethoxy, O-alkyl,
S-alkyl, NH-alkyl, N-dialkyl, O-aryl, S-aryl, NH-aryl, O-aralkyl,
S-aralkyl, NH-aralkyl, amino, N-phthalimido, imidazole, azido,
hydrazino, hydroxylamino, isocyanato, sulfoxide, sulfone, sulfide,
disulfide, silyl, aryl, heterocycle, carbocycle, intercalator,
reporter molecule, conjugate, polyamine, polyamide, polyalkylene
glycol, polyether, a group that enhances the pharmacodynamic
properties of oligonucleotides, or a group that enhances the
pharmacokinetic properties of oligonucleotides; and n is an integer
from 0 to about 6.
[0046] In other embodiments of the present invention compounds
having the structure: 10
[0047] wherein X is R.sub.1--(R.sub.2).sub.n;
[0048] R.sub.1 is C.sub.3-C.sub.20 alkyl;
[0049] R.sub.2 is NH.sub.2, H-imidazole, N-phthalimido;
[0050] Y is a hydroxyl blocking group;
[0051] Z is phosphate or an activated phosphate group;
[0052] Q.sub.1 and Q.sub.2 independently are H or a guanosine
blocking group; and n is an integer from 0 to about 6, are also
provided.
[0053] In still other embodiments of the present invention
compounds having the structure: 11
[0054] wherein X is R.sub.1--(R.sub.2).sub.n;
[0055] R.sub.1 is C.sub.3-C.sub.20 alkyl, C.sub.4-C.sub.20 alkenyl
or C.sub.2-C.sub.20 alkynyl;
[0056] R.sub.2 is halogen, hydroxyl, thiol, keto, carboxyl, nitro,
nitroso, nitrile, trifluoromethyl, trifluoromethoxy, O-alkyl,
S-alkyl, NH-alkyl, N-dialkyl, O-aryl, S-aryl, NH-aryl, O-aralkyl,
S-aralkyl, NH-aralkyl, amino, imidazole, N-phthalimido, azido,
hydrazino, hydroxylamino, isocyanato, sulfoxide, sulfone, sulfide,
disulfide, silyl, aryl, heterocycle, carbocycle, intercalator,
reporter molecule, conjugate, polyamine, polyamide, polyalkylene
glycol, polyether, a group that enhances the pharmacodynamic
properties of oligonucleotides, and a group that enhances the
pharmacokinetic properties of oligonucleotides; and n is an integer
from 0 to about 6, are provided.
[0057] Compounds of Formulas I, II and III may be prepared by
alkylation effected directly on
2,6-diamino-9-(.beta.-D-ribofuranosyl)purine with an appropriate
compound having the formula R.sub.1-L.sub.1 wherein R.sub.1 is
C.sub.3-C.sub.20 alkyl, C.sub.4-C.sub.20 alkenyl or
C.sub.2-C.sub.20 alkynyl and L is a leaving group, in the presence
of a base of sufficient strength to effect removal of the proton
from the 2' or 3' (or both 2' and 3') hydroxyl of the ribofuranosyl
sugar moiety of 2,6-diamino-9-(.beta.-D-ribofuranosyl)purine. When
used in the general sense, the term "alkyl" or "alkylation" is
meant to refer to herein to alkyl, alkenyl and alkynyl groups.
Alkyl, alkenyl and alkynyl groups of the present invention may be
straight chain, branched or cyclic groups.
[0058] In more preferred embodiments of the present invention
R.sub.1 is C.sub.4-C.sub.20 alkyl and in still more preferred
embodiments of the present invention R.sub.1 is C.sub.5 to C.sub.20
alkyl. Alkylation can be limited to mono alkylation by limiting the
amount of either the R.sub.1-L group or the base to a
stoichiometric (or equivalent) amount. Alternately dialkylation (on
both the 2' and 3' positions) can be practiced by use of an excess
R.sub.1-L group and base to concurrently alkylate both the 2' and
the 3' positions.
[0059] It has been observed that alkylation predominates at the 2'
position compared to the 3' position. Generally a ratio of from
about 7:3 to about 8:2 of 2' to 3' alkylation products are obtained
(as determined by TLC). For both TLC as well as preparative scale
chromatography, the 2' product generally has a faster Rf than the
3' product. Advantage can be taken of this Rf difference to
separate the 2'-O- and 3'-O-products from each other or from
2'-O-,3'-O-dialkylated products. Thus the 2' and 3' alkylation
products can be separated by procedures such as silica gel
chromatography if desired.
[0060] For alkyl groups that are generally larger than propyl,
further advantage can be taken of the rate of deamination of the 2'
product versus the 3' product for separation of the 2'-O and 3'-O
products. Thus mixtures of 2'-O and 3'-O alkylated
2,6-diamino-9-(.beta.-D-ribofuranosyl- )-purine are subjected to
deamination with adenosine deaminase. The enzymatic deamination of
the 2'-O product is more facile than deamination of the 3'-O
product. This difference in the rate of deamination allows for
separation of the deaminated 2' product, i.e. the 2'-O-alkylated
guanosine, from the slower or non-deaminated 3' product, i.e. the
2,6-diamino-9-(3'-O-alkylated-.beta.-D-ribofuranosyl)purine.
Additionally procedures such as crystallization has been utilized
to further separate a 2' product from the corresponding 3' product
by separating the 2'-O-alkylated diaminopurine riboside product
from the corresponding 3'-O-alkylated diaminopurine riboside
product.
[0061] A preferred base utilized for alkylation is sodium hydride.
Other suitable bases may also be utilized, however such bases must
have sufficient base strength to remove the proton from the 2' (or
3') hydroxyl moiety of the 2,6-diamino-purine riboside starting
material. While not wishing to be bound by theory, generally any
base having a pK.sub.a about 10 pk.sub.a units greater than the
pK.sub.a of the proton of the 2' hydroxyl moiety of the
2,6-diaminopurine riboside starting material may be used. More
specifically, bases having a pK.sub.b greater than the pK.sub.b of
sodium hydride may conveniently be selected. Such bases can be
selected from compilations of base such as those given in Table 1,
page 220 of March, J. Advanced Organic Chemistry,
Wiley-Interscience, John Wiley & Sons, New York, 1985.
[0062] The alkylation reactions useful to prepare compounds of the
invention typically are conducted in DMF as the solvent. Other
suitable solvents include DMSO, N-methyl pyrolidone and
sulfolone.
[0063] Preferably, deamination is effected by use of deaminase
enzymes. Particularly preferred is adenosine deaminase.
Particularly suitable for use is Adenosine Deaminase Type II
available from Sigma Chemical Company, St. Louis, Mo. Other
deamination reagents may also be employed. The deamination
reactions of the invention typically are conducted in a mixture
solvent containing an organic solvent and an aqueous buffer.
Suitable for use as the organic solvent are DMSO, N-methyl
pyrolidone and sulfolone. In preferred embodiments of the present
invention deamination is achieved using DMSO as the organic
solvent. Suitable for use as the aqueous buffer are buffers having
a pH compatible to the pH range of use of the deaminse enzyme.
Preferred are phosphate buffers such as sodium phosphate and tris
buffers.
[0064] In order to enrich the 2' product verse 3' product by
elimination of any 3' product, a TIPDS (tetraisopropylsiloxane)
protecting group is utilized to protect the 3' and 5' hydroxyl
moieties of the sugar portions of the 2,6-diaminopurine riboside.
In the same manner, exclusive 3' product would be obtainable by use
of a base stable, non-migratory 2'-O-protecting group. Such base
stable, non-migratory protecting groups include but are not limited
to tetrahydropyranyl (THP), 4-methoxytetrahydropyran4-yl (Mthp),
1-(2-chloro-4-methyl)phenyl-4-methox- ypiperidin4-yl (Ctmp),
triphenylmethyl (trityl), mono-, di- and tri-methoxytrityl and
other similar protecting groups.
[0065] Suitable leaving groups of the present invention include
halides such as chloride, bromide, and iodide, sulfonates such as
tosyl, brosyl, nosyl, mesyl and trifyl and oxonium ions. In
preferred embodiments of the present invention the leaving group is
a halide. Still other suitable leaving groups are well known to
those skilled in the art.
[0066] The 3'-O-phosphoramidite of 2'-O-alkyl guanosine and
2,6-diamino-9-(2'-O-alkyl-.beta.-D-ribofuranosyl) purine are
provided in the present invention by reaction of 2NH.sub.2, 5'-OH
protected 2'-O-alkyl guanosine or 2NH.sub.2, 6NH.sub.2, and 5'-OH
protected2,6-diamino-9-(2'-O-alkyl-.beta.-D-ribofuranosyl) purine
with a reagent such as 2-cyanoethyl
N,N-diisopropylamino-chlorophosphine.
[0067] 2'-O-alkyl guanosine and 2'-o-alkyl-2,6-diaminopurine
riboside are phosphitylated at the 3'-OH to provide
phosphoramidites. In conducting such phosphitylation the NH.sub.2
moieties (2NH.sub.2 or 2NH.sub.2 and 6NH.sub.2, respectively) are
protected. Next the 5'-OH moiety is protected followed by reaction
with cyanoethyl N,N-diisopropyl aminochlorophosphine.
[0068] Compounds of the present invention can be incorporated into
oligomers by procedures known to those skilled in the art.
Oligomers of the present invention may contain at least one subunit
having the structure: 12
[0069] wherein X is R.sub.1--(R.sub.2).sub.n;
[0070] R.sub.1 is C.sub.3-C.sub.20 alkyl, C.sub.4-C.sub.20 alkenyl
C.sub.2-C.sub.20 alkynyl;
[0071] R.sub.2 is halogen, hydroxyl, thiol, keto, carboxyl, nitro,
nitroso, nitrile, trifluoromethyl, trifluoromethoxy, O-alkyl,
S-alkyl, NH-alkyl, N-dialkyl, O-aryl, S-aryl, NH-aryl, O-aralkyl,
S-aralkyl, NH-aralkyl, amino, imidazole, N-phthalimido, azido,
hydrazino, hydroxylamino, isocyanato, sulfoxide, sulfone, sulfide,
disulfide, silyl, aryl, heterocycle, carbocycle, intercalator,
reporter molecule, conjugate, polyamine, polyamide, polyalkylene
glycol, polyether, a group that enhances the pharmacodynamic
properties of oligonucleotides, or a group that enhances the
pharmacokinetic properties of oligonucleotides;
[0072] T.sub.3 and T.sub.5 independently are OH or a further
nucleotide or nucleoside of said oligonucleotide or oligonucleoside
that is joined to said structure; and
[0073] n is an integer from 0 to about 6.
[0074] In still other embodiments of the present invention
oligomers may contain at least one subunit having the structure:
13
[0075] wherein X is R.sub.1--(R.sub.2).sub.n;
[0076] R.sub.1 is C.sub.1-C.sub.20 alkyl, C.sub.2-C.sub.20 alkenyl
C.sub.2-C.sub.20 alkynyl;
[0077] R.sub.2 is halogen, hydroxyl, thiol, keto, carboxyl, nitro,
nitroso, nitrile, trifluoromethyl, trifluoromethoxy, O-alkyl,
S-alkyl, NH-alkyl, N-dialkyl, O-aryl, S-aryl, NH-aryl, O-aralkyl,
S-aralkyl, NH-aralkyl, amino, imidazole, N-phthalimido, azido,
hydrazino, hydroxylamino, isocyanato, sulfoxide, sulfone, sulfide,
disulfide, silyl, aryl, heterocycle, carbocycle, intercalator,
reporter molecule, conjugate, polyamine, polyamide, polyalkylene
glycol, polyether, a group that enhances the pharmacodynamic
properties of oligonucleotides, or a group that enhances the
pharmacokinetic properties of oligonucleotides;
[0078] T.sub.3 and T.sub.5 independently are OH or a further
nucleotide or nucleoside of said oligonucleotide or oligonucleoside
that is joined to said structure; and
[0079] n is an integer from 0 to about 6.
[0080] Such oligomers or oligonucleotides may be prepared by solid
state synthesis or by other means known to those skilled in the
art. For example, 2'-O-alkyl guanosine phosphoramidites and
derivatives thereof may be incorporated into oligonucleotides using
standard phosphoramidite chemistry. Incorporation of 2'-O-alkyl
guanosine nucleotides will confer desireable characteristics to an
oligonucleotide such as enhanced resistance to nuclease.
[0081] In the context of this invention, the term "oligonucleotide"
or "oligomer" refers to a polynucleotide formed from naturally
occuring bases and furanosyl groups joined by native phosphodiester
bonds. Oligonucleotides of the present invention will, of course,
comprise at least one 2'-O-alkyl guanosine or derivative thereof.
Thus, this term effectively refers to naturally occurring species
or synthetic species formed from naturally occurring subunits or
their close homologs. The term "oligonucleotide" or "oligomer" may
also refer to moieties which have portions similar to naturally
occurring oligonucleotides but which have non-naturally occurring
portions. Thus, oligonucleotides may have altered sugars, altered
base moieties, or altered inter-sugar linkages. Exemplary among
these are the phosphorothioate and other sulfur-containing species
which are known for use in the art. In accordance with some
preferred embodiments, at least some of the phosphodiester bonds of
the oligonucleotide have been substituted with a structure which
functions to enhance the stability of the oligonucleotide or the
ability of the oligonucleotide to penetrate into the region of
cells where the messenger RNA is located. It is preferred that such
substitutions comprise phosphorothioate bonds, phosphotriesters,
methyl phosphonate bonds, short chain alkyl or cycloalkyl
structures or short chain heteroatomic or heterocyclic structures.
Other preferred substitutions are CH.sub.2--NH--O--CH.sub.2,
CH.sub.2--N(CH.sub.3)--O--CH- .sub.2,
CH.sub.2--O--N(CH.sub.3)--CH.sub.2, CH.sub.2--N(CH.sub.3)--N(CH.su-
b.3)--CH.sub.2 and O--N(CH.sub.3)--CH.sub.2--CH.sub.2 structures
where phosphodiester intersugar linkage is replaced by the
substitutions. Also preferred are morpholino structures. Summerton,
J. E. and Weller, D. D., U.S. Pat. No. 5,034,506 issued Jul. 23,
1991. In other preferred embodiments, such as the protein-nucleic
acid (PNA) backbone, the phosphodiester backbone of the
oligonucleotide may be replace with a polyamide backbone, the bases
being bound directly or indirectly to the aza nitrogen atoms of the
polyamide backbone. P. E. Nielsen, et al., Science 1991 254 1497.
In accordance with other preferred embodiments, the phosphodiester
bonds are substituted with other structures which are, at once,
substantially non-ionic and non-chiral, or with structures which
are chiral and enantiomerically specific. Persons of ordinary skill
in the art will be able to select other linkages for use in
practice of the invention.
[0082] Oligonucleotides may also include species which include at
least some modified base forms. Thus, purines and pyrimidines other
than those normally found in nature may be so employed. Suitable
bases include, but are not limited to those described in U.S. Pat.
No. 3,687,808. Similarly, modifications on the furanosyl portion of
the nucleotide subunits, in addition to 2'-O-alkyl modifications of
the present invention, may also be effected, as long as the
essential tenets of this invention are adhered to. Examples of such
modifications are 2'-halogen-substituted nucleotides. Some specific
examples of modifications at the 2' position of sugar moieties
which are useful in the present invention are OH, SH, SCH.sub.3, F,
OCN, O(CH.sub.2).sub.nNH.sub.2, Cl, Br, CN, CF.sub.3, OCF.sub.3, S-
or N-alkyl; S- or N-alkenyl; SOCH.sub.3, SO.sub.2CH.sub.3;
ONO.sub.2; NO.sub.2; N.sub.3; NH.sub.2; heterocycloalkyl;
heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted
silyl; an RNA cleaving group; a conjugate; a reporter group; an
intercalator; a group for improving the pharmacokinetic properties
of an oligonucleotide; or a group for improving the pharmacodynamic
properties of an oligonucleotide and other substituents having
similar properties. Sugar mimetics such as cyclobutyls may also be
used in place of the pentofuranosyl group. Oligonucleotides may
also comprise other modifications consistent with the spirit of
this invention. Such oligonucleotides are best described as being
functionally interchangeable with yet structurally distinct from
natural oligonucleotides. All such oligonucleotides are
comprehended by this invention so long as they effectively function
as subunits in the oligonucleotide.
[0083] Preferably oligonucleotides of the present invention are
from about 6 to about 50 nucleotides in length. In still more
preferred embodiments of the present invention oligonucleotides are
from about 12 to about 20 nucleotides in length.
[0084] Intercalators are molecules which insert themselves between
neighboring bases of an oligonucleotide. A well known intercalator
is acridine. Other intercalators will be apparent to one skilled in
the art. Reporter molecules are molecules which may aid in the
identification of a molecule, either visually or otherwise. For
example, biotin and various fluorophores are effective reporter
groups. Conjugates, or bifunctional linkers effectively join two
groups. Some conjugates are commercially available such as biotin
or 3'maleimidobenzoyl-N-hydroxy-succinimide available from
Boehringer Mannheim (Indianapolis, Ind.). Pharmacodymanic property
improvement means, in this context, improved oligonucleotide
uptake, enhanced oligonucleotide resistance to degradation, and/or
strengthened sequence-specific hybridization with RNA. Such groups
do not initiate chemical reactions. Groups that enhance the
pharmacodynamic properties of an oligonucleotide preferrably
include alkyl chains, polyamines, ethylene glycols, polyamides,
alkyl chains, aminoalkyl chains and amphipathic moieties.
Pharmacokinetic property improvement means improved oligonucleotide
uptake, distribution, metabolism or excretion.
[0085] Antisense therapy involves the use of oligonucleotides which
are specifically hybridizable to target RNA or DNA.
Oligonucleotides of the present invention are preferably
specifically hydridizable with a target region. By "specifically
hybridizable" herein is meant capable of forming a stable duplex
with a target DNA or RNA. Upon binding to, or forming a stable
duplex with, the target RNA or DNA, the antisense oligonucleotide
can selectively inhibit the genetic expression of these nucleic
acids or can induce some other events such as destruction of a
targeted RNA or DNA or activation of gene expression. Destruction
of targeted RNA can be effected by RNase H activation or by linking
strand cleavers to the oligonucleotide. Antisense therapy is known
in the art. See for example, PCT/US91/05720 filed Dec. 3, 1991
entitled "Antisense Oligonucleotide Inhibitors of Papillomavirus"
and PCT/US91/01327 filed Feb, 25, 1991 entitled "Oligonucleotide
Therapies for Modulating the Effects of Herpesvirus".
[0086] In some embodiments of the present invention the
oligonucleotide portions of compounds of the present invention are
at least 60% complementary to a target sequence. In preferred
embodiments of the present invention the oligonucleotide portions
of compounds of the present invention are at least 80%
complementary to a target sequence. 100% complementarity of the
oligonucleotide portions of compounds of the present invention to a
target sequence is most preferred. In preferred embodiments of the
present invention, the oligonucleotide portions may be specifically
hybridizable with DNA or RNA from Candida, papilloma virus, Epstein
Barr virus, rhinovirus, hepatitis, human immunodeficiency virus,
herpes simplex virus, influenza virus and cytomegalovirus.
[0087] 2-O-alkyl guanosine containing oligonucleotides of the
present invention may be used to modulate the production of protein
by contacting a selected sequence of RNA or DNA coding for a
selected protein with an 2'-O-alkyl guanosine containing
oligonucleotide of the present invention having a sequence of
nucleotide bases specifically hybridizable with said selected
sequence of RNA or DNA coding for said protein.
[0088] The oligonucleotides of the present invention can be used in
diagnostics, therapeutics and as research reagents. For therapeutic
use, an animal having a disease characterized by the undesired
production of a protein is contacted with an oligonucleotide of the
present invention having a sequence of nucleotide bases
specifically hybridizable with a selected sequence of RNA or DNA
coding for said protein.
EXAMPLES
[0089] The following examples illustrate the invention, however,
they are not intended as being limiting.
Example 1
[0090] 2,6-Diamino-9-(.beta.-D-ribofuranosyl)purine
[0091] In accordance with modifications of the procedures described
in Robins, M. J., Hanske, F. and Beriner, S. E., Can. J. Chem.,
59:3360 (1981), guanosine hydrate (49 g, Aldrich Chemical Co.),
toluene (200 ml), hexamethyldisilazane (160 ml, 4.3 eq) and
trifluoromethanesulfonic acid (3.7 ml) were loaded in a stainless
steel Parr bomb. The bomb was sealed and heated approximately 1/3
submerged in an oil bath at 170.degree. C. for 5 days. The bomb was
cooled in a dry ice acetone bath and opened. The contents were
transferred to a 2 liter round bottom flask using methanol (MeOH)
and the solvent evaporated on a Buchii evaporator. 1:1
H.sub.2O/MeOH (600 ml) was added to the residue and the resulting
brown suspension was refluxed 4-5 hr. The resulting suspension was
evaporated on the Buchii evaporator to remove the methanol
(.apprxeq.1/2 volume). Additional H.sub.2O (.apprxeq.300 ml) was
added and the mixture was heated, treated with charcoal and
filtered through a Celite filter pad. Upon cooling, a crystalline
solid formed. The solid was isolated by filtration, washed with
H.sub.2O and dried under high vacuum at 90.degree. C. to yield the
product (43.7 g, 89% yield) as a tan solid. UV and NMR spectra of
this compound compared to literature values.
[0092] This variation of the procedures of Robins, et al. supra,
eliminated the need to utilize liquid ammonia in the reaction
mixture since the ammonia molecule is generation in situ from the
silazane reagent and the water of hydration of the guanosine
hydrate starting material. Further, the use of
chlorotrimethylsilane was not necessary nor was it necessary to
conduct the reaction under anhydrous conditions, do a preliminary
evaportaion, or open and re-seal the Parr bomb under a dry nitrogen
atmosphere.
Example 2
[0093] 2,6-Diamino-9-(2-O-propyl-.beta.-D-ribofuranosyl)purine
& 2,6-Diamino-9-(3-O-propyl-.beta.-D-ribofuranosyl)purine
[0094] Sodium hydride (NaH) (2.1 g) was added to
2,6-diamino-9-(.beta.-D-r- ibofuranosyl) purine (10.5 g) in dry
dimethylformamide (DMF) (150 ml). After stirring for 10 min,
iodo-propane (6 ml) was added. The solution was stirred for 45 min
at room temperature followed by the addition of a further aliquot
of NaH (600 mg). The reaction mixture was stirred overnight and
then quenched by the addition of ethanol (EtOH) (5 ml). The
reaction mixture was evaporated in vacuo, the residue suspended in
10% MeOH/CH.sub.2CL.sub.2 and purified by silica gel chromatography
(300 g) using 5.fwdarw.10% MeOH/CH.sub.2Cl.sub.2 as the eluent. The
2',3'-di-O-propyl product eluted first followed by the 2'-O-propyl
product and then the 3'-O-propyl product. The 2'-O-propyl product
containing fractions were pooled and the solvent stripped to yield
a crude foam. The foam was crystallized from H.sub.2O (40 ml),
washed with cold H.sub.2O and dried to yield 2.9 g of the
2'-O-propyl compound. The mother liquor was evaporated,
re-chromatographed and crystallized to yield an additional 2.4 g of
the 2'-O-propyl compound. The second mother liquor was evaporated
to yield 4 g of a mixture of 2'and 3'-O-propyl compounds as an oil.
Fractions containing the 3'-O-propyl product as the major product
were evaporated and residue foam crystallized from water. (See
Example 17 below for isolation and characterization of the
2',3'-di-O-propyl compound).
[0095] 2,6-Diamino-9-(2-O-propyl-.beta.-D-ribofuranosyl)purine
[0096] .sup.1H NMR (DMSO-d.sub.6) .delta.0.76 (t, 3, CH.sub.3), 1.4
(tq, 2, CH.sub.2), 3.3 (m, 1, H-5"+HDO), 3.65-3.45 (m, 3, H-5',
O--CH.sub.2), 3.9 (m, 1), 4.25 (br m, 1), 4.38 (dd, 1), 5.1 (br d,
1 3'-OH), 5.45 (br t, 1, 5'-OH), 5.75 (br s, 2, 6-NH.sub.2), 5.83
(d, 1, H-1'), 6.77 (br s, 2, 2-NH.sub.2) and 7.95 (s, 1, H-8).
Anal. Calcd. for C.sub.13H.sub.20N.sub.6O.sub.4{fraction
(1/2)}H.sub.2O: C, 46.91; H, 6.2; N,25.25. Found: C, 47.09; H,
6.37; N, 25.33.
[0097] 2,6-Diamino-9-(3-O-propyl-.beta.-D-ribofuranosyl)purine
[0098] .sup.1H NMR (DMSO-d.sub.6) .delta.0.75 (t, 3, CH.sub.3), 1.4
(tq, 2, CH.sub.2), 3.27-3.5 (ABX 2, O--CH.sub.2--), 3.5 and 3.6
(ABX, 2, H-5'), 3.9 (m,1), 4.22 (m, 1), 4.35 (m, 1), 5.1 (br d, 1,
2'-OH), 5.45 (br t, 1, 5'-OH), 5.75 (br s, 2, 6-NH.sub.2), 5.8 (d,
1, H-1'), 6.8 (br s, 2, 2-H.sub.2) and 7.95 (s, 1, H-8).
Example 3
[0099] 2'-O-Propylguanosine
[0100] A mixture of
2,6-Diamino-9-(2'-O-propyl-.beta.-D-ribofuranosyl) purine and
2,6-Diamino-9-(3,-O-propyl-.beta.-D-ribofuranosyl) purine (4.6 gm)
and adenosine deaminase (200 mg, Sigma Chemicals Type II) were
stirred at room temperature overnight in 0.1 M tris buffer (150 ml,
pH 7.4), DMSO (100 ml) and 0.1 M sodium phosphate buffer (10 ml). A
further aliquot of adenosine deaminase (140 mg) in 0.1 M phosphate
buffer (30 ml) and DMSO (20 ml) was added and the reaction stirred
an addition 24 hrs. The solvent was evaporated in vacuo and the
residue flash chromatographed on silica gel utilizing 5.fwdarw.20%
MeOH/CH.sub.2Cl.sub.2. Product-containing fractions were evaporated
in vacuo and the residue crystallized from H.sub.2O to yield 2.6 gm
of product. m.p. dec>270.degree. C. .sup.1H NMR (DMSO-d.sub.6)
.delta.0.75 (t, 3, CH.sub.3), 1.42 (tq, 2, CH.sub.2), 3.3-3.6 (m,
4, H-5', O--CH.sub.2), 3,85 (m, 1), 4.2 (m, 1), 4.23 (m, 1), 5.10
(t, 1, 5'-OH), 5.13 (d, 1, 3'-OH), 5.75 (d, 1, H-1'), 6.45 (br s,
2, NH.sub.2), 7.95 (s, 1, H-8) and 10.67 (br s, 1, NH). Anal.
Calcd. for C.sub.13H.sub.19N.sub.5O.sub.5: C, 47.99; H, 5.89; N,
21.53. Found: C, 47.90, H, 5.85; N, 21.44.
Example 4
[0101] N2-Isobutyryl-2'-O-propylguanosine
[0102] 2'-O-Propylguanosine (3.6 gm) in pyridine (50 ml) was cooled
in an ice bath and trimethylsilyl chloride (8.4 ml, 6 eq.) was
added. The reaction mixture was stirred for 30 min and isobutyryl
chloride (5.8 ml, 5 eq.) was added. The solution was stirred for 4
hours during which it was allowed to warm to room temperature. The
solution was cooled, H.sub.2O added (10 ml) and the solution was
stirred for an additional 30 mins. Concentrated NH.sub.4OH (10 ml)
was added and the solution evaporated in vacuo. The residue was
purified by silica gel chromatography using 10%
MeOH/CH.sub.2CL.sub.2 to elute the product. Product-containing
fractions were evaporated to yield 2.5 g of product as a foam. An
analytical sample was re-chromatographed on silica and eluted with
CH.sub.2Cl.sub.2.fwdarw.6% MeOH/CH.sub.2Cl.sub.2. .sup.1H NMR
(DMSO-d.sub.6) .delta.0.75 (t, 3, CH.sub.3), 1.13 [d, 6,
CH(CH.sub.3).sub.2], 1.4 (m, 2, CH.sub.2), 2.75 [m, 1,
CH(CH.sub.3).sub.2], 3.52 (m, 6, OCH.sub.2), 3.36 and 3.6 (ABX, 2,
H-5'), 3.95 (m, 1), 4.26 (m, 1), 4.33 (m, 1), 5.07 (t, 1, 5'-OH),
5.18 (d, 1, 3'-OH), 5.9 (d, 1, H-1'), 8.25 (s, 1, H-8), 11.65 (br
8, 1, NH) and 12.1 (br s, 1, NH). Anal. Calcd. for
C.sub.17H.sub.25N.sub.5O.sub.6.1/2H.sub.2- O: C, 50.49; H, 6.48; N,
17.32. Found: C, 50.81; H, 6.62; N, 17.04.
Example 5
[0103] N2-Isobutyryl-5'-dimethoxytrityl-2'-O-propylguanosine
[0104] N2-Isobutyryl-2'-O-propylguanosine (2.64 g) was
co-evaporated with pyridine and then solubilized in pyridine (180
ml). Dimethoxytrityl chloride (2.4 g, 1.1 eq) and
dimethyl-aminopyridine (50 mg) was added with stirring at room
temperature. The reaction mixture was stirred overnight and
evaporated in vacuo. The residue was partitioned between
CH.sub.2Cl.sub.2/2.times. dil Na.sub.2CO.sub.3. The organic phase
was dried (MgSO.sub.4) and evaporated. The residue was purified by
silica gel chromatography (1:1 EtOAc/Hex.fwdarw.5% MeOH/EtOAc, 1%
TEA) to yield 4.1 g of product. .sup.1H NMR (DMSO-d.sub.6)
.delta.0.78 (t, 3, CH.sub.3), 1.12 [d, 6, CH(CH.sub.3).sub.2], 1.46
(m, 2, CH.sub.2), 2.75 [m, 1, CH(CH.sub.3).sub.2], 3.35 and 3.55
(ABX, 2, H-5'), 3.73 (s, 6, OCH.sub.2), 4.0 (m, 1), 4.3 (m, 1), 4.4
(m, 1), 5.18 (d, 1, 3'-OH), 5.93 (d, 1, H-1'), 6.8, 7.2, 7.36 (m,
13, DMTr), 8.13 (s, 1, H-8), 11.63 (br s, 1, NH) and 12.1 (br s, 1,
NH). Anal. Calcd. for C.sub.38H.sub.42N.sub.5O.sub.8.H.sub.2O: C,
63.83; H, 6.20; N, 9.80. Found: C, 64.22; H, 6.35; N, 9.55.
Example 6
[0105] N2-Isobutyryl-5'-dimethoxytrityl-2'-O-propylguanosine
3'-.beta.-cyanoethyl-N,N-diisopropylphosphoramidate
[0106] A CH.sub.2Cl.sub.2 solution of
N2-Isobutyryl-5'-dimethoxy-trityl-2'- -O-propylguanosine (4.1 g),
bis-(N,N-diisopropyl-amino)-2-cyanoethylphosph- ite (3.7 ml, 2 eq)
and N,N-diiso-propylammonium tetrazolide (0.5 g, 0.5 eq) was
stirred at room temperature overnight. The solution was partitioned
against dil. Na.sub.2CO.sub.3 and then dil. Na.sub.2CO.sub.3/NaCl
and dried over MgSO.sub.4. The solvent was evaporated in vacuo and
the residue was purified by silica gel chromatograph (120 g, 1% TEA
in EtOAc) to yield 5.2 g of product as a foam. .sup.31P NMR
(CDCl.sub.3) .delta.150.5, 150.8.
Example 7
[0107] 2,6-Diamino-9-(2-O-pentyl-.beta.-D-ribofuranosyl)purine
& 2,6-Diamino-9-(3-O-pentyl-.beta.-D-ribofuranosyl)purine
[0108] 2,6-Diamino-9-(.beta.-D-ribofuranosyl)purine (10 g) was
treated with sodium hydride (1.7 g, 1.2 eq) and bromopentane (5.3
ml, 1.2 eq) in DMF (90 ml) as per the procedure of Example 2.
Silica gel chromatography yielded three components. The first
eluted component (not characterized but believed to be the
2,3-di-(O-pentyl) compound was isolated as an oil (700 mg). The
next component isolated as a foam (3.3 g) was crystallized from
MeOH to yield of 2.8 g of
2,6-diamino-9-(2-O-pentyl-.beta.-D-ribofur- anosyl)purine. The
third component isolated as a solid (200 mg) was crystallized from
MeOH to yield 80 mg of 2,6-diamino-9-(3-O-pentyl-.beta.-
-D-ribofuranosyl)purine. Fractions containing mixtures of the first
and second components were evaporated and the residue crystallized
from MeOH to yield a further 900 mg of the 2-O-pentyl compound.
Further fraction yielded 1.2 g of a mixture of the 2'-O-pentyl and
3'-O-pentyl compounds.
[0109] 2,6-Diamino-9-(2-O-pentyl-.beta.-D-ribofuranosyl)purine
[0110] .sup.1H NMR (DMSO-d.sub.6) .delta.0.75 (t, 3, CH.sub.3),
1.16 (m, 4, CH.sub.2), 1.39 (m, 2, CH.sub.2), 3.53 (m, 2,
CH.sub.2), 3.3 and 3.6 (ABX, 2, H-5'), 3.93 (br s, 1), 4.23 (m, 1),
4.38 (m, 1), 5.1 (d, 1 3'-OH), 5.5 (t, 1, 5'-OH), 5.75 (br s, 2,
6-NH.sub.2), 5.82 (d, 1, H-1'), 6.8 (br s, 2, 2-NH.sub.2) and 7.93
(s, 1, H-8).
[0111] 2,6-Diamino-9-(3-O-pentyl-.beta.-D-ribofuranosyl)purine
[0112] .sup.1H NMR (DMSO-d.sub.6) .delta.0.87 (t, 3, CH.sub.3), 1.3
(m, 4, CH.sub.2), 1.55 (m, 2, CH.sub.2), 3.5 (m, 2, O--CH.sub.2--),
3.6 (m, 2, H-5'), 3.86 (m, 1), 3.95 (m, 1), 4.6 (m, 1), 5.32 (br d,
1 2'-OH), 5.46 (br t, 1, 5'-OH), 5.70 (d, 1, H-1'), 5.75 (br s, 2,
6-NH.sub.2), 6.76 (br s, 2, 2-NH.sub.2) and 7.93 (s, 1, H-8).
Example 8
[0113] 2'-O-Pentylguanosine
[0114] 2,6-diamino-9-(2-O-pentyl-.beta.-D-ribofuranosyl)purine (1.9
g) in 0.1 M sodium phosphate buffer (50 ml, pH 6.0) and DMSO (25
ml) was treated with adenosine deaminase (added in two
aliquots--first aliquot 50 mg, second aliquot 80 mg) at 35.degree.
C. as per the procedure of Example 3 to yield 1.4 g of product.
.sup.1H NMR (DMSO-d.sub.6) .delta.0.8 (t, 3, CH.sub.3), 1.16 (m, 4,
2.times.CH.sub.2), 1.4 (m, 2, CH.sub.2), 3.38, 3.6 (m, 4,
OCH.sub.2, H1-5'), 3.93 (s, 1, H-4'), 4.28 (m, 2, H-2', H-3'), 5.17
(br, 2, 5', 3'-OH), 5.8 (d, 1, H1-1'), 6.53 (br s, 2, NH.sub.2),
8.0 (s, 1, H-8) and 10.68 (br, 1, NH).
Example 9
[0115] N2-Isobutyryl-2'-O-pentylguanosine
[0116] 2'-O-pentylguanosine (2.3 g) in pyridine (35 ml) was treated
with trimethylsilyl chloride (4.15 ml, 5 eq) and isobutyryl
chloride (3.4 ml, 5 eq) as per the procedure of Example 4 to yield
the product as a foam (2.3 g). An analytical sample was
crystallized from EtOAc/Hex. m.p. 178-180.degree. C. .sup.1H NMR
(DMSO-d.sub.6) .delta.0.75 (t, 3, CH.sub.3), 1.1 [m, 10,
2.times.CHhd 2, CH(CH.sub.3).sub.2], 1.4 (m, 2, CH.sub.2), 2.74 [m,
1, CH(CH.sub.3).sub.2], 3.56 (m, 4, OCH.sub.2, H-5'), 3.93 (m, 1,
H-4'), 4.25 (m, 1), 4.34 (m, 1), 5.05 (t, 1, 5'-OH), 5.17 (d, 1,
3'-OH), 5.88 (d, 1, H-1'), 8.27 (s, 1, H-8), 11.65 (br s, 1, NH)
and 12.05 (br s, 1, NH). Anal. Calcd. for
C.sub.19H.sub.29N.sub.5O.sub.6: C, 53.89; H, 6.90; N, 16.54. Found:
53.75; H, 6.92; N, 16.40.
Example 10
[0117] N2-Isobutyryl-5'-dimethoxytrityl-2'-O-pentylguanosine
[0118] N2-Isobutyryl-2'-O-pentylguanosine (2.3 g) was treated with
dimethoxytrityl chloride (1.7 g, 1.1 eq), and
dimethyl-aminopyridine (100 mg as a catalyst) in pyridine (50 ml)
as per the procedure of Example 5 to yield the product as a foam
(2.9 g). 'H NMR (DMSO-d.sub.6) .delta.0.83 (t, 3, CH.sub.3), 1.2
[m, 10, 2.times.CH.sub.2, CH(CH.sub.3).sub.2], 1.48 (m, 2,
CH.sub.2), 2.78 [m, 1, CH(CH.sub.3).sub.2], 3.4, 3.6 (m, 4,
OCH.sub.2, H-5'), 3.75 (s, 6, OCH.sub.3), 4.07 (m, 1), 4.27 (m, 1),
4.42 (m, 1), 5.2 (br d, 1, 3'-OH), 5.95 (d, 1, H-1'), 6.85, 7.25,
7.38 (m, 13, DMTr), 8.15 (s, 1, H-8), 11.67 (br s, 1, NH) and 12.1
(br s, 1, NH) . Anal. Calcd. for Anal. Calcd. for
C.sub.40H.sub.47N.sub.5O.sub.8.1/2H.sub- .2O: C, 65.38; H, 6.58; N,
9.53. Found: C, 65.37; H, 6.59; N, 9.39.
Example 11
[0119] N2-Isobutyryl-5'-dimethoxytrityl-2'-O-pentylguanosine
3'-.beta.-cyanoethyl-N,N-diisopropylphosphoramidate
[0120] N2-Isobutyryl-5'-dimethoxytrityl-2'-O-pentylguanosine (1.7
g) was treated with
bis-(N,N-diisopropylamino)-2-cyanoethyl-phosphite (1.48 g) and
N,N-diisopropylammonium tetrazolide (200 mg) as per the procedure
of Example 6 to yield the product (1.4 g). 31P NMR (CDCl.sub.3)
.delta.150.5, 150.85.
Example 12
[0121] 2,6-Diamino-9-(2-O-nonyl-.beta.-D-ribofuranosyl)purine
[0122] 2,6-Diamino-9-(.beta.-D-ribofuranosyl)purine (50 g, 180
mmol) was treated with sodium hydride (8.8 g, 220 mmol) and
bromononane (59 g, 54.4 ml, 285 mmol) in DMF (700 ml) as per the
procedure of Example 2 (the diamino compound in DMF was cooled in
an ice bath during the addition of NaH) to yield 83 g of crude
product. 50 g of crude product was purified by silica gel
chromatography. Fraction containing 2'-O-nonyl and 3'-O-nonyl
product were combined to give a 77:23 mixture (29 g) of the 2' and
3' product. Pure 2'-O-nonyl product is obtained by chromatography.
.sup.1H NMR (DMSO-d.sub.6) .delta.0.95 (t, 3, CH.sub.3); 1.17 [m,
12, O--CH.sub.2--CH.sub.2--(CH.sub.2).sub.6]; 1.42 [m, 2,
O--CH.sub.2CH.sub.2(CH.sub.2).sub.6]; 3.27-3.70 (m, 2, H-5');
3.50-3.70 ]m, 2, O--CH.sub.2(CH.sub.2).sub.7]; 3.95 (m, 1, H-4'),
4.24 (m, 1, H-3'); 4.40 (m, 1, H-2'); 5.10 (d, 1, 3'-OH, J=5 Hz);
5.50 (t, 1, 5'-OH, J=6 Hz); 5.76 (s, 2, 2-NH.sub.2); 5.83 (d, 1,
H-1', J=6.0 Hz); 6.81 (s, 2, 6-NH.sub.2); and 7.96 (s, 1, 8-H).
Example 13
[0123] 2'-O-Nonylguanosine
[0124] A mixture of
2,6-diamino-9-(2-O-nonyl-.beta.-D-ribofuranosyl)purine and
2,6-diamino-9-(3-O-nonyl-.beta.-D-ribofuranosyl)purine
(.apprxeq.80:20 mixture, 29 g) in 0.1 M sodium phosphate buffer (50
ml, pH 7.4), 0.1 M tris buffer (1800 ml, pH 7.4) and DMSO (1080 ml)
was treated with adenosine deaminase (1.6 g) as per the procedure
of Example 3 to yield 60 g of product as an oil. An analytical
product was purified by silica gel chromatography and
recrystallized from EtOAc. m.p. 258-259.degree. C. .sup.1H NMR
(DMSO-d.sub.6) .delta.0.96 (t, 3, CH.sub.3, J=7 Hz); 1.17 [m, 12,
O--CH.sub.2--CH.sub.2--(CH.sub.2).sub.6]; 1.42 [m, 2,
O--CH.sub.2CH.sub.2(CH.sub.2).sub.6]; 3.27-3.61 (m, 4, H-5',
O--CH.sub.2(CH.sub.2).sub.7]; 3.95 (m, 1, H-4'), 4.10-4.13 (m, 2,
H-2', H-3'); 5.13-6.06 (m, 2, 3'-OH 5'-OH); 5.80 (d, 1, H-1', J=6.4
Hz); 6.47 (s, 2, 2-NH.sub.2); 7.98 (s, 1, 8-H) and 10.64 (s, 1,
N.sub.1 amide) . Anal. Calcd. for C.sub.19H.sub.31N.sub.5O.sub.5:
C, 55.73; H, 7.63; N, 17.10. Found: C, 55.67; H, 7.66; N,
17.02.
Example 14
[0125] N2-Isobutyryl-2'-O-nonylguanosine
[0126] 2'-O-nonylguanosine (14.7 g) in pyridine (360 ml) was
treated with trimethylsilyl chloride (23.4 ml) and isobutyryl
chloride (30.6 ml) as per the procedure of Example 4 to yield crude
product (37 g). The crude material was purified by silica gel
chromatography (eluted with 90/10 CHCl.sub.3/MeOH) to yield 14.6 g
of product re-crystallized from EtOAc. m.p. 168-169.degree. C.
.sup.1H NMR (DMSO-d.sub.6) .delta.0.85 [t, 3, CH.sub.3(nonyl)],
1.14 [m, 18, O--CH.sub.2CH.sub.2(CH.sub.2).sub.6,
CH(CH.sub.3).sub.2], 1.40 [m, 2,
O--CH.sub.2CH.sub.2(CH.sub.2).sub.6], 2.79 [m, 1,
CH(CH.sub.3).sub.2], 3.31-3.63 (m, 4, H-5',
O--CH.sub.2(CH.sub.2).sub.7]; 3.96 (m, 1, H-4'), 4.27-4.37 (m, 2,
H-2', H-3'); 5.10 (t, 1, 5'-OH, J=5 Hz), 5.18 (d, 1, 3'-OH, J=4
Hz), 5.91 (d, 1, H-1', J=6.6 Hz), 8.31 (s, 1, 8-H), 11.73 (s, 1,
C.sub.2 amide) and 12.11 (s, 1, N, amide). Anal. Calcd. for
C.sub.23H.sub.37N.sub.5O.sub.6: C, 57.60; H, 7.78; N, 14.60. Found:
C, 57.63; H, 7.92; N, 14.62.
Example 15
[0127] N2-Isobutyryl-5'-dimethoxytrityl-2'-O-nonylguanosine
[0128] N2-Isobutyryl-2'-O-nonylguanosine (14.6 g, 30.4 mmol) was
treated with dimethoxytrityl chloride (12.1 g, 34 mmol) in pyridine
(200 ml) as per the procedure of Example 5 to yield 16 g of purple
foam prior to chromatography and 11.5 g after chromatography
purification. 'H NMR (DMSO-d.sub.6) .delta.0.84 [t, 3,
CH.sub.3(nonyl), J=7 Hz], 1.16 [m, 18,
O--CH.sub.2CH.sub.2(CH.sub.2).sub.6, CH(CH.sub.3).sub.2], 1.43 [m,
2, O--CH.sub.2CH.sub.2(CH.sub.2).sub.6], 2.77 [m, 1,
CH(CH.sub.3).sub.2], 3.18-3.63 (m, 4, H-5',
O--CH.sub.2(CH.sub.2).sub.7]; 3.74 (s, 6, DMTr --CH.sub.3) 4.06 (m,
1, H-4'), 4.27 (m, 1, H-3'); 4.42 (m, 1, H-2'); 5.19 (d, 1, 3'-OH,
J=5 Hz), 5.94 (d, 1, H-1', J=5.7 Hz), 6.83-7.38 (m, 13, DMTr
aromatic), 8.14 (s, 1, 8-H), 11.65 (s, 1, C.sub.2 amide) and 12.11
(s, 1, N, amide). Anal. Calcd. for C.sub.44H.sub.55N.sub.5O.sub.8:
C, 67.59; H, 7.27; N, 8.96. Found: C, 67.59; H, 7.11; N, 8.80.
Example 16
[0129] N2-Isobutyryl-5'-dimethoxytrityl-2'-O-nonylguanosine
3'-.beta.-cyanoethyl-N,N-diisopropylphosphoramidate
[0130] N2-Isobutyryl-5'-dimethoxytrityl-2'-O-nonylguanosine (2.1 g)
was treated with bis-(N,N-diisopropylamino)-2cyanoethyl-phosphine
(1.5 g) and N,N-diisopropylammonium tetrazolide (0.2 g) as per the
procedure of Example 6 to yield the product (2.0 g) .sup.31P NMR
(CDCl.sub.2) .delta.150.7 and 150.4 (diastereomers).
Example 17
[0131]
2,6-Diamino-9-(2,3-di-O-propyl-.beta.-D-ribofuranosyl]purine
[0132] The procedure of Example 2 was repeated utilizing
2,6-diamino-9-(.beta.-D-ribofuranosyl)purine (10 g), NaH (3 g) and
1-bromo-propane (10 ml) in DMF. After evaporation of the reaction
solvent, the reaction products were purified by silica gel
chromatography. The slower moving component yielded 4.3 g of the
2'-O-propyl product as a foam. This foam was crystallized from
water to yield 3.6 g of product. The faster moving component
isolated as an oil formed crystals upon standing. EtOH was added to
the crystals, they were filtered and wash 1.times.EtOH to yield 1.1
grams of 2',3'-di-O-propyl product. m.p. 165-167.degree. C. .sup.1H
NMR (DMSO-d.sub.6) .delta.0.80 and 0.92 (t, 6, CH.sub.3), 1.6 and
1.45 (m, 4, CH.sub.2), 3.7-3.45 (br m, 6), 4.07 (m, 2), 4.5 (dd,
1), 5.55 (br t, 1, 5'-OH), 5.8 (br s, 2, 6-NH.sub.2), 5.85 (d, 1,
H-1'), 6.84 (br s, 2, 2-NH.sub.2) and 8.0 (s, 1, H-8).
[0133] Anal. Calcd. for C.sub.16H.sub.26N.sub.6O.sub.4: C, 52.45;
H, 7.15; N, 22.94. Found: C, 52.18; H, 7.19; N, 22.75.
Example 18
[0134]
N2,N6-Diisobutyryl-2,6-diamino-9-(2-O-propyl-.beta.-D-ribofuranosyl-
)purine
[0135] 2,6-diamino-9-(2-O-propyl-.beta.-D-ribofuranosyl)purine (2.0
g) in pyridine (35 ml) was treated with trimethylsilyl chloride
(3.9 ml, 5 eq) and isobutyryl chloride (3.2 ml, 5 eq) as per the
procedure of Example 4 to yield a foam after silica gel
chromatography. The foam was crystallized from EtOAc/Hex to yield
2.2 g of product. m.p. 140-142.degree. C. .sup.1H NMR
(DMSO-d.sub.6) .delta.0.77 (t, 3, CH.sub.3), 1.07, 1.16 [d, 12,
2.times.CH(CH.sub.3).sub.2], 1.5 (m, 2, CH.sub.2), 2.9, 3.03 [m, 2,
2.times.CH(CH.sub.3).sub.2], 3.4 (m, 1, H-5"), 3.58 (m, 3,
OCH.sub.2, H-5'), 3.95 (m, 1, H-4'), 4.3 (m, 1), 4.5 (m, 1), 5.02
(t, 1, 5'-OH), 5.2 (d, 1, 3'-OH), 6.03 (d, 1, H-1'), 8.58 (s, 1,
H-8), 10.39 (br s, 1, NH), and 10.57 (br s, 1, NH).
Example 19
[0136]
N2,N6-Diisobutyryl-2,6-diamino-9-(5-O-dimethoxytrityl-2-O-propyl-.b-
eta.-D-ribofuranosyl)purine
[0137]
N2,N6-Diisobutyryl-2,6-diamino-9-(2-O-propyl-.beta.-D-ribofuranosyl-
)purine (1.9 g) was treated with dimethoxytrityl. chloride (1.5 g,
1.1 eq), and dimethylaminopyridine (20 mg as a catalyst) in
pyridine (50 ml) as per the procedure of Example 5 to yield the
product as a foam (2.8 g). .sup.1H NMR (DMSO-d.sub.6) .delta.0.79
(t, 3, CH.sub.3), 1.07, 1.16 [d, 12, 2.times.CH(CH.sub.3).sub.2],
1.5 (m, 2, CH.sub.2), 2.9, 3.03 [m, 2, 2.times.CH(CH.sub.3).sub.2],
3.58 (m, 3, OCH.sub.2, H-5'), 4.15 (m, 1, H-4'), 4.4 (m, 1), 4.6
(m, 1), 5.15 (d, 1, 3'-OH), 6.15 (d, 1, H-1'), 6.
[0138] 8-7.35 (m, 13, DMTr), 8.5 (s, 1, H-8), 10.3 (br s, 1, NH),
and 10.57 (br s, 1, NH).
Example 20
[0139]
N2,N6-Diisobutyryl-2,6-diamino-9-(5-O-dimethoxytrityl-2-O-propyl-.b-
eta.-D-ribofuranosyl)purine
3'-9-cyanoethyl-N,N-diisopropylphosphoramidate
[0140]
N2,N6-Diisobutyryl-2,6-diamino-9-(5-O-dimethoxy-trityl-2-O-propyl-.-
beta.-D-ribofuranosyl)purine (2.6g) was treated with
bis-(N,N-diisopropylamino)-2-cyanoethylphosphite (1.7 g) and
N,N-diisopropylammonium tetrazolide (300 mg) overnight at room
temperature. The reaction mixture was partitioned against dil.
Na.sub.2CO.sub.3/CHCl.sub.2 and then Na.sub.2CO.sub.3/NaCl and
dried over MgSO.sub.4. The organic layer was evaporated to a foam.
The foam was dissolved in CH.sub.2Cl2 (.apprxeq.8 ml) and slowly
added to Hexanes (500 ml). The solid was filtered and dried to
yield the product as a powder (3.1 g). .sup.31P NMR (CDCl.sub.3)
.delta.150.8 and 151.3.
Example 21
[0141]
2,6-Diamino-9-[2-O-(N-phthalimido)propyl-.beta.-D-ribofuranosyl]pur-
ine &
2,6-Diamino-9-[3-O-(N-phthalimido)propyl-.beta.-D-ribofuranosyl]puri-
ne
[0142] 2,6-Diamino-9-(.beta.-D-ribofuranosyl)purine (14.2 g) was
treated with sodium hydride (3 g, 1.5 eq) and N-(3-bromo-propyl)
phthalimide (5.3 ml, 1.5 eq) in DMF (20 g) at 70.degree. C.
overnight. The reaction mixture was proportioned between H.sub.2O
and Hexanes (1.times.), then extracted 4.times.CH.sub.2Cl.sub.2.
The organic layer was dried over MgSO.sub.4 and evaporated to a
residue. The residue was purified by silica gel chromatography
eluted with MeOH/CH.sub.2Cl.sub.2. The 2'-O-(N-phthalimido)propyl
product eluted first followed by mixed fractions and then the
3'-O-(N-phthalimido) product. Evaporations of the fractions gave
3.4 g of the 2'-O-(N-phthalimido)propyl product, 3.0 g of mixed 2'
and 3' products and 1.4 g of the 3'-O-(N-phthalimido)propyl product
all as foams. The 3'-O-(N-phthalimido)propyl product was
crystallized from EtOAc/MeOH to give 270 mg of solid.
[0143]
2,6-Diamino-9-[2-O-(N-phthalimido)propyl-.beta.-D-ribofuranosyl]pur-
ine
[0144] .sup.1H NMR (DMSO-d.sub.6) .delta.1.8 (tq, 2, --CH.sub.2--),
3.4-3.58 (m, 6, 2.times.CH.sub.2, H-5'), 3.9 (m, 1), 4.26 (m, 1),
4.37 (m, 1), 5.05 (br d, 1, 3'-OH), 5.4 (br t, 1, 5'-OH), 5.72 (br
s, 2, NH.sub.2), 5.8 (br d, 1, H-1'), 6.75 (br s, 2, NH.sub.2), 7.8
(br s, 4, Ar) and 8.93 (s, 1, H-8).
[0145]
2,6-Diamino-9-[3-O-(N-phthalimido)propyl-.beta.-D-ribofuranosyl]pur-
ine
[0146] m.p. 220-222.degree. C., .sup.1H NMR (DMSO-d.sub.6)
.delta.1.85 (tq, 2, -CH--N), 3.6-3.67 (m, 4, -O--CH.sub.2, H-5'),
3.85 (m, 1), 3.92 (m, 1), 4.6 (m, 1), 5.33 (d, 1, 2'-OH), 5.45 (br
t, 1, 5'-OH), 5.65 (d, 1, H-1'), 5.73 (br s, 2, NH.sub.2), 6.75 (br
d, 2, NH.sub.2), 7.8-7.85 (m, 4, Ar) and 7.85 (s, 1, H-8). Anal.
Calcd. for C.sub.21H.sub.23N.sub.7- O.sub.6: C, 53.73; H, 4.94;
N,.20.88. Found: C, 53.59; H, 4.89; N, 20.63.
Example 22
[0147] 2'-O-(N-Phthalimido)propylguanosine
[0148] 2,
6-diamino-9-[2-O-(N-phthalimido)propyl-.beta.-D-ribofuranosyl]
purine (3.1 g) in 0.1 M sodium phosphate buffer (3 ml, pH 7.4),
0.05 M tris buffer (65 ml, pH 7.4) and DMSO (45 ml) was treated
with adenosine deaminase (200 mg) at room temperature for 5 days as
per the procedure of Example 3. The product containing fractions
from the silica gel chromatography were evaporated and upon
concentration formed white crystals. The crystals were filtered and
washed with MeOH to yield 1.1 g of product. An analytical sample
was recrystallized from MeOH. m.p. 192-194.degree. C. 'H NMR
(DMSO-d.sub.6) .delta.1.82 (m, 2, CH.sub.2), 3.45-3.67 (m, 6, H-5',
OCH.sub.2, NCH.sub.2), 3.9 (m, 1), 4.3 (m, 2, H-2', H-3'), 5.1 (m,
2, 5' and 3'-OH), 5.8 (d, 1, H-1'), 6.5 (br s, 2, NH.sub.2), 7.83
(s, 4, phthal), 7.98 (s, 1, H-8) and 10.5 (br s, 1, NH). Anal.
Calcd. for C.sub.21H.sub.22N.sub.6O.sub.7.1/2H.sub.2O: C, 52.61; H,
4.83; N, 17.53. Found: C, 52.52; H, 4.78; N, 17.38.
Example 23
[0149] N2-Isobutyryl-2'-O-(N-phthalimido)propylguanosine
[0150] 2'-O-(N-phthalimido)propylguanosine (7.2 g, crude) in
pyridine (35 ml) was treated with trimethylsilyl chloride (11.6 ml,
5 eq) and isobutyryl chloride (8 ml, 5 eq) as per the procedure of
Example 4 to yield the product as a crude foam (6.5 g). An
analytical sample was obtained by crystallization from EtOAc. m.p.
166-1680 C. .sup.1H NMR (DMSO-d.sub.6) .delta.1.15 [d, 6,
--CH(CH.sub.3).sub.2], 1.85 (m, 2, CH.sub.2), 2.8 [m, 1,
CH(CH.sub.3).sub.2), 3.45-3.7 (m, 6, H-5', OCH.sub.2, NCH.sub.2),
3.95 (m, 1), 4.34 (m, 1), 4.4 (m, 1), 5.12 (t, 1, 5'-OH), 5.18 (d,
1, 3'-OH), 5.9 (d, 1, H-1'), 7.83 (s, 4, phthal), 8.3 (s, 1, H-8),
11.65 (br s, 1, NH) and 12.1 (br s, 1, NH). Anal. Calcd. for
C.sub.25H.sub.28N.sub.6O.sub.8.1/2H.sub.2O: C, 54.64; H, 5.32; N,
15.29. Found: C, 54.46; H, 5.39; N, 14.98.
Example 24
[0151]
N2-Isobutyryl-5'-dimethoxytrityl-2'-P-(N-phthalimido)propylguanosin-
e
[0152] N2-Isobutyryl-2'-O-(N-phthalimido)propylguanosine (1.2 g)
was treated with dimethoxytrityl chloride (820 mg, 1.1 eq), and
dimethylaminopyridine (20 mg as a catalyst) in pyridine (50 ml) as
per the procedure of Example 5 utilizing 1:1 Hex/EtOAc, then EtOAc
then 5% MeOH/EtOAc with 1% TEA as eluent. The product containing
fraction were evaporated to yield the product as a foam (1.7 g).
.sup.1H NMR (DMSO-d.sub.6) .delta.1.1 [d, 6, --CH(CH.sub.3).sub.2],
1.85 (m, 2, CH.sub.2), 2.75 [m, 1, CH(CH.sub.3).sub.2], 3.45-3.7
(m, 6, H-5', OCH.sub.2, NCH.sub.2), 3.75 (s, 6, OCH.sub.3), 4.0 (m,
1), 4.32 (m, 1), 4.4 (m, 1), 5.2 (d, 1, 3'-OH), 5.93 (d, 1, H-1'),
6.83, 7.2, 7.35 (m, 13, DMTr), 7.78 (s, 4, phthal), 8.15 (s, 1,
H-8), 11.6 (br s, 1, NH) and 12.05 (br s, 1, NH). Anal. Calcd. for
C.sub.46H.sub.46N.sub.6O.sub.10.H.s- ub.2O: C, 64.18; H, 5.62; N,
9.76. Found: C, 64.42; H, 5.78; N, 9.53.
Example 25
[0153]
N2-Isobutyryl-5'-dimethoxytrityl-2'-O-(N-phthalimido)propylguanosin-
e 3'-.beta.-cyanoethyl-N,N-diisopropylphosphoramidate
[0154] N2-Isobutyryl-5'-dimethoxytrityl-2'-O-(N-phthalimido)
propylguanosine (1.6 g) was treated with
bis-(N,N-diisopropylamino)-2-cya- noethylphosphite (1.48 g) and
N,N-diisopropylammonium tetrazolide (200 mg) as per the procedure
of Example 6 to yield the product (2.0 g). .sup.31P NMR
(CDCl.sub.3) .delta.150.9.
Example 26
[0155]
N2-Dimethylaminomethylidene-5'-dimethoxytrityl-2'-O-(N-phthalimido)-
propylguanosine
[0156] 2'-O-(N-phthalimido)propylguanosine (900 mg) in DMF (20 ml)
was treated with N,N-dimethylformamide dimethyl acetal (2 ml). The
reaction mixture was stirred for 2 hr and evaporated under high vac
at 52.degree. C. The residue was co-evaporated 1.times. with
pyridine and taken up in solution in pyridine. Dimethoxytrityl
chloride (713 mg, 1.1 eq) and dimethyl-aminopyridine (20 mg as a
catalyst) were added. The reaction mixture was stirred overnight,
partitioned between Na.sub.2CO.sub.3/CH.sub.2Cl.sub.2, dried over
MgSO.sub.4 and purified by silica gel chromatography as per the
procedure of Example 5 to yield 1.7 g of product as an off white
solid. .sup.1H NMR (DMSO-d.sub.6) .delta.1.88 (m, 2, CH.sub.2), 3.1
[d, 6, N.dbd.CHN(CH.sub.3).sub.2], 3.3 (m, 2, H-5'), 3.67 (m, 4,
OCH.sub.2, NC.sub.2), 3.78 (s, 6, 2.times.OCH.sub.3), 4.0 (m, 1,
H-4'), 4.35 (m, 2, H-2', H-3'), 5.2 (d, 1, 3'-OH), 5.95 (d, 1,
H-1'), 6.85, 7.25, 7.39 (m, 13, DMTr), 7.85 (s, 4, phthal), 7.95
[s, 1, H-8), 8.5 (s, 1, N.dbd.CHN(CH.sub.3).sub.2] and 11.39 (s, 1,
NH.sub.2). Anal. Calcd. for C.sub.45H.sub.45N.sub.7O.sub.9.1-
/2H.sub.2O: C, 64.58; H, 5.54; N, 11.71. Found: C, 64.10; H, 5.65;
N, 11.47.
Example 27
[0157]
N2-Dimethylaminomethylidene-5'-dimethoxytrityl-2'-O-(N-phthalimido)-
propylguanosine
3-.beta.-cyanoethyl-N,N-diisopropylphosphoramidate
[0158] N2-Isobutyryl-5'-dimethoxytrityl-2'-O-(N-phthalimido)
propylguanosine (1.7 g),
bis-(N,N-diisopropylamino)-2cyanoethylphosphite (1.4 ml) and
N,N-diisopropylammonium tetrazolide (170 mg) were stirred overnight
at room temperature. The reaction mixture was partitioned between
CH.sub.2Cl.sub.2 and Na.sub.2CO.sub.3 2.times.. The organic phase
was dried over MgSO.sub.4 and evaporated to an oil. The oil was
dissolved in a minimum of CH.sub.2Cl.sub.2 and added dropwise to
.apprxeq.900 ml Hexanes to precipitate the product. The solid was
isolated and dried to yield 2.1 g of product. .sup.1P NMR
(CDCl.sub.3) .delta.150.4, 150.6.
Example 28
[0159] 2,
6-Diamino-9-[2-O-(N-phthalimido)pentyl-.beta.-D-ribofuranosyl]pu-
rine
[0160] 2,6-Diamino-(9-.beta.-D-ribofuranosyl)purine (6.7 g) was
treated with sodium hydride (1.3 g) and N-(3-bromopentyl)
phthalimide (7.8 g, 1.1 eq) in DMF (60 ml) at room temperature for
three days. The reaction mixture was proportioned between H.sub.2O
and CH.sub.2Cl.sub.2 and extracted 4.times.CH.sub.2Cl.sub.2. The
combined organic layers were dried over MgSO.sub.4 and evaporated
to a residue. The residue was purified by silica gel chromatography
eluted with 5.fwdarw.10% MeOH/CH.sub.2Cl.sub.2. The
2'-O-(N-phthalimido)pentyl containing fractions were collected and
evaporated to a yellow foam to give 2.2 g of product. An analytical
sample was crystallized from EtOH. m.p. 173-175.degree. C. .sup.1H
NMR (DMSO-d.sub.6) .delta.1.2 (m, 2, --CH.sub.2--), 1.47 (m, 4,
2.times.CH.sub.2), 3.55, 3.65 (m, 6, O--CH.sub.2, H-5', NCH.sub.2),
3.95 (m, 1), 4.28 (m, 1), 4.4 (m, 1), 5.13 (d, 1, 3'-OH), 5.5 (t,
1, 5'-OH), 5.77 (br s, 2, 6-NH.sub.2), 5.84 (br d, 1, H-1'), 6.8
(br s, 2, 2-NH.sub.2), 7.86 (M, 4, phthal) and 7.95 (s, 1, H-8) .
Anal. Calcd. for C.sub.23H.sub.27N.sub.7O.sub.6: C, 55.50; H, 5.47;
N, 19.71. Found: C, 55.44; H, 5.51; N, 19.30.
Example 29
[0161] 2'-O-(N-Phthalimido)pentylguanosine
[0162] A mixture of the 2,6-diamino-9-[2-O-(N-phthalimido)
pentyl-.beta.-D-ribofuranosyl]purine and
2,6-diamino-9-[3-O-(N-phthalimid- o)
pentyl-.beta.-D-ribofuranosyl]purine isomers (2.2 g) in 0.1 M tris
buffer (60 ml, pH 7.4), 0.1 M NaPO.sub.4 buffer (2 ml, pH 7.4) and
DMSO (40 ml) was treated with adenosine deaminase (60 mg) at room
temperature for 5 days as per the procedure of Example 3. The
product containing fractions from the silica gel chromatography
were evaporated to give the product (1.0 g) as a crude white solid.
An analytical sample was prepared by the addition of MeOH to form
crystals. m.p. 178-180.degree. C. .sup.1H NMR (DMSO-d.sub.6)
.delta.1.24 (m, 2, CH.sub.2), 1.5 (m, 4, 2.times.CH.sub.2), 3.5-3.6
(m, 6, H-5', OCH.sub.2, NCH.sub.2), 3.87 (m, 1, H-4'), 4.25 (m, 2,
H-2', H-3'), 5.1 (m, 2, 5' and 3'-OH), 5.78 (d, 1, H-1'), 6.5 (br
s, 2, NH.sub.2), 7.84 (M, 4, phthal), 7.98 (s, 1, H-8) and 10.67
(br s, 1, NH). Anal. Calcd. for
C.sub.23H.sub.26N.sub.6O.sub.7.1/2H- .sub.2O: C, 54.43; H, 5.36; N,
16.56. Found: C, 54.79; H, 5.24; N, 16.61.
Example 30
[0163] N2-Isobutyryl-2'-O-(N-phthalimido)pentylguanosine
[0164] 2'-O-(N-phthalimido)pentylguanosine (1.6 g, crude) in
pyridine (35 ml) was treated with trimethylsilyl chloride (2.0 ml,
5 eq) and isobutyryl chloride (1.68 ml, 5 eq) as per the procedure
of Example 4 to yield the product as a foam. This foam was
co-evaporated 2.times. with EtOAc followed by the addition of EtOAc
and heating to yield white crystals (950 mg)., m.p. 202-204.degree.
C. .sup.1H NMR (DMSO-d.sub.6) .delta.1.1 [d, 6,
--CH(CH.sub.3).sub.2], 1.17 (m, 2, CH.sub.2), 1.43 (m, 4,
2.times.CH.sub.2), 2.74 [m, 1, CH(CH.sub.3).sub.2], 3.45-3.55 (m,
6, H-5', OCH.sub.2, NCH.sub.2), 3.9 (m, 1), 4.25 (m, 1), 4.3 (m,
1), 5.07 (t, 1, 5'-OH), 5.15 (d, 1, 3'-OH), 5.87 (d, 1, H-1'), 7.8
(s, 4, phthal), 8.27 (s, 1, H-8), 11.67 (br s, 1, NH) and 12.06 (br
s, 1, NH). Anal. Calcd. for C.sub.27H.sub.32N.sub.6O.sub.8.1/2H2O:
C, 56.14; H, 5.76; N, 14.55. Found: C, 56.45; H, 5.74; N,
14.41.
Example 31
[0165]
N2-Isobutyryl-5'-dimethoxytrityl-2'-O-(N-phthalimido)pentylguanosin-
e
[0166] N2-Isobutyryl-2'-O-(N-phthalimido) pentylguanosine (0.95 g)
was treated with dimethoxytrityl chloride (620 mg, 1.1 eq), and
dimethylaminopyridine (20 mg as a catalyst) in pyridine (50 ml) as
per the procedure of Example 5 utilizing EtOAc 1% TEA and then 5%
MeOH EtOAc/CH.sub.2Cl.sub.2 with 1% TEA as eluent. The product
containing fractions were evaporated to yield the product as a foam
(1.4 g). .sup.1H NMR (DMSO-d.sub.6) .delta.1.14 (d, 6,
--CH(CH.sub.3).sub.2], 1.25 (m, 2, CH.sub.2), 1.53 (m, 4,
2.times.CH.sub.2), 2.77 [m, 1, CH(CH.sub.3).sub.2], 3.3-3.6 (m, 6,
H-5', OCH.sub.2, NCH.sub.2), 3.75 (s, 6, OCH.sub.3), 4.07 (m, 1),
4.33 (m, 1), 4.4 (m, 1), 5.18 (d, 1, 3'-OH), 5.94 (d, 1, H-1'),
6.83, 7.2, 7.53 (m, 13, DMTr), 7.8 (s, 4, phthal), 8.15 (s, 1,
H-8), 11.6 (br s, 1, NH) and 12.1 (br s, 1, NH). Anal. Calcd. for
C.sub.48H.sub.50N.sub.6O.sub.10.1/2H.sub.2O: C, 65.52; H, 5.84; N,
9.55. Found: C, 65.55; H, 5.94; N, 9.20.
Example 32
[0167]
2,6-Diamino-9-[3,5-O-(tetraisopropyldisiloxane-1,3-diyl)-.beta.-D-r-
ibofuranosyl]purine
[0168] To a suspension of
2,6-diamino-9-(.beta.-D-ribofuranosyl)purine (10.5 g) in pyridine
(100 ml) was added 1,3-dichlorotetraisopropyldisilox- ane (TIPDS,
12.6 g). The reaction was stirred at room temperature for 4 hours
and an additional 1.3 g of 1,3-dichlorotetraisopropyldisiloxane was
added followed by stirring overnight. The reaction mixture was
poured into ice water and the insoluble product (11.6 g) collected
by filtration. An analytical sample was recrystallized from
EtOAc/Hexanes. m.p. 170-172.degree. C. Anal. Calcd. for
C.sub.22H.sub.40N.sub.6O.sub.5Si- .sub.2.1/2H.sub.2O: C, 49.5; H,
7.74; N, 15.7. Found: 49.57; H, 7.82; N, 15.59.
Example 33
[0169]
2,6-Diamino-9-[3,5-O-(tetraisopropyldisiloxane-1,3-diyl)-2-O-methyl-
-.beta.-D-ribofuranosyl]purine
[0170] A mixture of
2,6-Diamino-9-[3,5-O-(tetraisopropyl-disiloxane-1,3-di-
yl)-.beta.-D-ribofuranosyl]purine (8.8 g) in DMF (120 ml) and
methyl iodide (3 ml, 3 eq) was cooled in an ice bath and NaH (60%
in oil, 1.0 g, 1.5 eq) added. After 20 min the reaction was
quenched with MeOH and partitioned between sat. NH.sub.4Cl and
CH.sub.2Cl.sub.2. The organic phase was washed with
1.times.NH.sub.4Cl, dried over MgSO.sub.4 and evaporated. The
residue was crystallized from hot EtOH/H.sub.2O to yield the
product (8.5 g) as crystals. m.p. 87-89.degree. C. .sup.1H NMR
(DMSO-d.sub.6) .delta.1.05 (m, 28, TIPDS), 3.57 (s, 3, OCH.sub.3),
3.98 (m, 1, H-4'), 3.92 and 4.07 (ABX, 2, H-5'), 4.13 (d, 1), 4.6
(dd, 1, H-3'), 5.76 (br s, 2, NH.sub.2), 5.8 (s, 1, H-'), 6.77 (br
s, 2, NH.sub.2) AND 7.77 (s, 1 H-8).
Example 34
[0171] 2,6-Diamino-9-(2-O-methyl-.beta.-D-ribofuranosyl)purine
[0172] To a solution of
2,6-Diamino-9-[3,5-O-(tetraisopropyldisiloxane-1,3-
-diyl)-2-O-methyl-.delta.-D-ribofuranosyl]purine (8.5 g) in THF (50
ml) was added 1M tetrabutylammonium fluoride in THF (Aldrich, 20
ml). The reaction mixture was stirred for 2 hrs and filtered. The
filter cake was washed with 2.times.EtOAc and air dried to give 4.0
g of crude product. An analytical sample was crystallized from hot
MeOH. m.p. 133-135.degree. C. .sup.1H NMR (DMSO-d.sub.6) .delta.3.3
(s, 3, OCH.sub.3), 3.58 (m, 2, H-5'), 3.98 (m, 1, H-4'), 4.28 (m,
2, H-2', H-3'), 5.23.(br s, 1, 3'-OH), 5.48 (br t, 1, 5'-OH), 5.77
(br s, 2, NH.sub.2), 5.82 (d, 1, H-1'), 6.83 (br s, 2, NH.sub.2)
and 7.95 (s, 1, H-8). Anal. Calcd. for
C.sub.11H.sub.16N.sub.6O.sub.4.1/2H.sub.2O: C, 43.28; H, 5.61; N,
27.52. Found: C, 43.51; H, 5.62; N, 27.26.
Example 35
[0173] 2'-O-Methylguanosine
[0174] 2,6-Diamino-9-(2-O-methyl-.beta.-D-ribofuranosyl)purine (9.5
g) in 0.1M sodium phosphate buffer (200 ml, pH 7.4) and DMSO (25
ml) was treated with adenosine deaminase (Type II Sigma) at RT for
4 days. The resulting suspension was cooled and filtered and the
resulting filter cake washed with H.sub.2O and dried to a white
solid (4.0 g). The solid was recrystallized from hot H.sub.2O to
yield 2.9 g of product. m.p. 236-238.degree. C. .sup.1H NMR
(DMSO-d.sub.6) .delta.3.3 (s, 3, OCH.sub.3), 3.53 and 3.6 (ABX, 2,
H-5'), 3.87 (m, 1, H-4'), 4.15 (m, 1, H-2'), 4.25 (m, 1, H-3'),
5.13 (t, 1, 5'-OH), 5.23 (d, 1, 3'-OH), 5.8 (d, 1, H-1'), 6.48 (br
s, 2, NH-2), 7.96 (s, 1, H-8) and 10.68 (br s, 1, NH). Anal. Calcd.
for C.sub.11H.sub.15N.sub.5O.sub.5.1/2H.sub.2O: C, 43.14; H, 5.26;
N, 22.86. Found: C, 43.59; H, 5.34; N, 23.04.
Example 36
[0175] N2-Isobutyryl-2'-O-methylguanosine
[0176] 2'-O-methylguanosine (3.5 g) in pyridine (100 ml) was
treated with trimethylsilyl chloride (9 ml, 6 eq) and isobutyryl
chloride (6.2 ml) at RT for 4 hr. The reaction mixture was cooled
in an ice bath, H.sub.2O (20) was added and stirring continued for
an additional 20 min. NH.sub.4OH (20 ml) was added and after
stirring for 30 min the reaction mixture was evaporated. The
residue was triturated with H.sub.2O, filtered and the filtrate
evaporated and purified by silica gel chromatography as per the
procedure of Example 4 to yield the product as an off white solid
(1.5 g). .sup.1H NMR (DMSO-d.sub.6) .delta.1.1 [d, 6,
CH(CH.sub.3).sub.2], 2.77 [m, 1, CH(CH.sub.3).sub.2], 3.33-3.6 (m,
5, OCH.sub.3, H-5'), 3.93 (m, 1, H-4'), 4.22 (m, 1), 4.3 (m, 1),
5.1 (t, 1, 5'-OH), 5.28 (d, 1, 3'-OH), 5.9 (d, 1, H-1'), 8.28 (s,
1, H-8) and 11.9 (br s, 1, NH).
Example 37
[0177] N2-Isobutyryl-5'-dimethoxytrityl-2'-O-methylguanosine
[0178] N2-Isobutyryl-2'-O-methylguanosine (1.5 g) was treated with
dimethoxytrityl chloride (1.5 g, 1.1 eq), and dimethylaminopyridine
(100 mg as a catalyst) in pyridine (50 ml) as per the procedure of
Example 5 to yield the product as a foam (2.6 g) .sup.1H NMR
(DMSO-d.sub.6) .delta.1.14 (d, 6, CH(CH.sub.3).sub.2], 2.75 [m, 1,
CH(CH.sub.3).sub.2], 3.5 (m, 2, H-5'), 3.74 (s, 6, OCH.sub.3), 4.05
(m, 1), 4.33 (m, 1), 5.26 (d, 1, 3'-OH), 5.95 (d, 1, H-1'), 6.83,
7.2, 7.35 (m, 13, DMTr), 8.15 (s, 1, H-8), 11.6 (br s, 1, NH) and
12.1 (br s, 1, NH).
Example 38
[0179] N2-Isobutyryl-5'-dimethoxytrityl-2'-O-methylguanosine
3'-.beta.-cyanoethyl-N,N-diisopropylphosphoramidate
[0180] N2-Isobutyryl-5'-dimethoxytrityl-2'-O-methylguanosine (20 g)
was treated with bis-(N,N-diisopropylamino) 2-cyanoethylphosphite
(10.8 g) and N,N-diisopropylammonium tetrazolide (1.6 g) as per the
procedure of Example 6 to yield the product (15.7 g). .sup.31P NMR
(CDCl.sub.3) .delta.148.97 and 147.96.
Example 39
[0181]
N2,N6-Diisobutyryl-2,6-diamino-9-(2-O-methyl-.beta.-D-ribofuranosyl-
) purine
[0182] 2,6-diamino-9-(2-O-methyl-.beta.-D-ribofuranosyl)purine (700
mg) in pyridine (20 ml) was treated with trimethylsilyl chloride
(2.1 ml, 7 eq) and isobutyryl chloride (1.25 ml, 5 eq) as per the
procedure of Example 4 to yield the product as a foam (900 mg)
after silica gel chromatography.
Example 40
[0183]
N2,N6-Diisobutyryl-2,6-diamino-9-(5-O-dimethoxytrityl-2-O-methyl-.b-
eta.-D-ribofuranosyl)purine
[0184]
N2,N6-Diisobutyryl-2,6-diamino-9-(2-O-methyl-.beta.-D-ribofuranosyl-
)purine (900 mg) was treated with dimethoxytrityl chloride (1.0 g)
and dimethylaminopyridine (20 mg as a catalyst) in pyridine (30 m)
as per the procedure of Example 5 to yield the product as a foam
(700 mg). .sup.1H NMR (DMSO-d .sub.6) .delta.0.96-1.16 [m, 12,
2.times.CH(CH.sub.3).sub.2], 2.9 and 3.05 [M, 2,
2.times.CH(CH.sub.3).sub.2], 3.18 and 3.37 (ABX, 2, H-5'), 3.38 (s,
3, OCH.sub.3), 3.7 (s, 6, OCH.sub.3), 4.05 (m, 1, H-4' ), 4.44 (m,
2, H-2', H-3'), 5.24 (d, 1, 3'-OH), 6.06 (d, 1, H-1'), 6.78, 7.2,
7.33 (m, 13, Ar), 8.22 (s, 1, H-8), 10.3 (br s, 1, NH) and 10.57
(br s, 1, NH).
Example 41
[0185]
N2,N6-Diisobutyryl-2,6-diamino-9-(5-O-dimethoxytrityl-2-O-methyl-.b-
eta.-D-ribofuranosyl)purine
3'-9-cyanoethyl-N,N-diisopropylphosphoramidate
[0186]
N2,N6-Diisobutyryl-2,6-diamino-9-(5-O-dimethoxytrityl-2-O-methyl-.b-
eta.-D-ribofuranosyl)purine (600 mg) was treated with
bis-(N,N-diisopropylamino)-2-cyanoethylphosphite (500 .mu.l) and
N,N-diisopropylammonium tetrazolide (80 mg) overnight at RT. The
reaction mixture was partitioned against dil.
Na.sub.2CO.sub.3/CHC'.sub.2 and then Na.sub.2CO.sub.3/NaCl and
dried over MgSO.sub.4. The organic layer was evaporated to a foam
(500 mg). .sup.31P NMR (CDCl.sub.3) .delta.151.1 (doublet).
Example 42
[0187]
2,6-Diamino-9-(2-O-octadecyl-.beta.-D-ribofuranosyl)purine
[0188] 2,6-Diamino-9-(.beta.-D-ribofuranosyl)purine (50 g, 180
mmol) and sodium hydride (7 g) in DMF (1 l) were heated to boiling
for 2 hr. Iodooctadecane (100 g) was added at 150.degree. C. and
the reaction mixture allowed to cool to RT. The reaction mixture
was stirred for 11 days at RT. The solvent was evaporated and the
residue purified by silica gel chromatography. The product was
eluted with 5% MeOH/CH.sub.2Cl.sub.2. The product containing
fraction were evaporated to yield the product (11 g). .sup.1H NMR
(DMSO-d.sub.6) .delta.0.84 (t, 3, CH.sub.2) ; 1.22 [m, 32,
O--CH.sub.2--CH.sub.2--(CH.sub.2).sub.16--]; 1.86 (m, 2,
O--CH.sub.2CH.sub.2--); 3.25 (m, 2, O--CH.sub.2--); ; 3.93 (d, 1,
4'H), 4.25 (m, 1, 3'H) ; 4.38 (t, 1, 2'H); 5.08 (d, 1, 3'-OH); 5.48
(t, 1, 5'-OH) ; 5.75 (s, 2, 6-NH.sub.2); 5.84 (d, 1, 1'-H); 6.8 (s,
2, 2-NH.sub.2); and 7.95 (s, 1, 8-H).
Example 43
[0189] 2'-O-Octadecylguanosine
[0190] 2,6-Diamino-9-(2-O-octadecyl-.beta.-D-ribofuranosyl) purine
(10 g) in 0.1 M sodium phosphate buffer (50 ml, pH 7.4), 0.1 M tris
buffer (1000 ml, pH 7.4) and DMSO (1000 ml) was treated with
adenosine deaminase (1.5 g) as per the procedure of Example 3. At
day 3, day 5 and day 7 an additional aliquot (500 mg, 880 mg and
200 mg, respectively) of adenosine deaminase was added. The
reaction was stirred for a total of 9 day and after purification by
silica gel chromatography yielded the product (2 g). An analytical
sample was recrystallized from MeOH .sup.1H NMR (DMSO-d.sub.6)
.delta.0.84 (t, 3, CH.sub.3), 1.22 [s, 32,
O--CH.sub.2--CH.sub.2--(CH.sub.2).sub.16], 5.07 (m, 2, 3'-OH
5'-OH); 5.78 (d, 1, 1'-H); 6.43 (s, 2, NH.sub.2), 7.97 (s, 1, 8-H)
and 10.64 (s, 1, NH.sub.2) Anal. Calcd. for
C.sub.28H.sub.49N.sub.5O.sub.5: C, 62.80; H, 9.16; N, 12.95. Found:
C, 62.54; H, 9.18; N, 12.95.
Example 44
[0191] N2-Isobutyryl-2'-O-octadecylguanosine
[0192] 2'-O-Octadecylguanosine (1.9 g) in pyridine (150 ml) was
treated with trimethylsilyl chloride (2 g, 5 eq) and isobutyryl
chloride (2 g, 5 eq) as per the procedure of Example 4. The product
was purified by silica gel chromatography (eluted with 3%
MeOH/EtOAc) to yield 1.2 g of product. .sup.1H NMR (DMSO-d.sub.6)
.delta.0.85 [t, 3, CH.sub.3], 1.15 [m, 38,
O--CH.sub.2CH.sub.2(CH.sub.2).sub.16, CH(CH.sub.3).sub.2], 2.77 [m,
1, CH(CH.sub.3).sub.2], 4.25 (m, 2, 2'H, 3'H); 5.08 (t, 1, 5'-OH),
5.12 (d, 1, 3'-OH), 5.87 (d, 1, 1'-H), 8.27 (s, 1, 8-H), 11.68 (s,
1, NH.sub.2) and 12.08 (s, 1, NH.sub.2). Anal. Calcd. for
C.sub.32H.sub.55N.sub.5O.sub- .6: C, 63.47; H, 9.09; N, 11.57.
Found: C, 63.53; H, 9.20; N, 11.52.
Example 45
[0193]
2,6-Diamino-9-[2-O-(imidazol-1-yl)butyl-.beta.-D-ribofuranosyl]puri-
ne
[0194] 2,6-Diamino-(9-.beta.-D-ribofuranosyl)purine (5.0 g) in DMF
(400 ml) was treated with sodium hydride (0.78 g). After stirring
an additional 30 min a further portion of sodium hydride (2.6 g)
was added immediately followed by bromobutyl-imidazole (9.9 g) in
DMF (25 ml). The reaction mixture was stirred overnight and
quenched with H.sub.2O. The reaction mixture was filtered through
celite and evaporated to yield an oily product. TLC showed a
mixture of isomers.
Example 46
[0195] 2'-O-(Imidazol-1-yl)butylguanosine
[0196] A mixture of the
2,6-diamino-9-[2-O-(imidazol-1-yl)butyl-.beta.-D-r-
ibofuranosyl]purine and
2,6-diamino-9-[3-O-(imidazol-1-yl)butyl-.beta.-D-r-
ibofuranosyl]purine isomers in 0.1 M tris buffer (pH 7.4), 0.1 M
NaSO.sub.4 buffer (pH 7.4) and DMSO will treated with adenosine
deaminase at RT for 5 days as per the procedure of Example 3. The
product containing fractions will be purified by silica gel
chromatography and the product containing fraction evaporated to
give the product.
Example 47
[0197] N2-Isobutyryl-2'-O-(imidazol-1-yl)butylguanosine
[0198] 2'-O-(imidazol-1-yl) butylguanosine in pyridine will be
treated with trimethylsilyl chloride (5 eq) and isobutyryl chloride
(5 eq) as per the procedure of Example 4 to yield the product.
Example 48
[0199]
N2-Isobutyryl-5'-dimethoxytrityl-2'-O-(imidazol-1-yl)butylguanosine
[0200] N2-Isobutyryl-2'-O-(imidazol-1-yl)butylguanosine will be
treated with dimethoxytrityl chloride (1.1 eq), and
dimethylaminopyridine (as a catalyst) in pyridine as per the
procedure of Example 5. After chromatography purification, the
product containing fractions will be evaporated to yield the
product).
Example 49
[0201] A. Evaluation of the Thermodynamics of Hybridization of
2'-Modified Oligonucleotides.
[0202] The ability of the 2'-modified oligonucleotides to hybridize
to their complementary RNA or DNA sequences is determined by
thermal melting analysis. The RNA complement is synthesized from T7
RNA polymerase and a template-promoter of DNA synthesized with an
Applied Biosystems, Inc. 380B RNA species was purified by ion
exchange using FPLC (LKB Pharmacia, Inc.). Natural antisense
oligonucleotides or those containing 2'-O-alkyl guanosine at
specific locations are added to either the RNA or DNA complement at
stoichiometric concentrations and the absorbance (260 nm)
hyperchromicity upon duplex to random coil transition was monitored
using a Gilford Response II spectrophotometer. These measurements
are performed in a buffer of 10 mM Na-phosphate, pH 7.4, 0.1 mM
EDTA, and NaCl to yield an ionic strength of 10 either 0.1 M or 1.0
M. Data is analyzed by a graphic representation of 1/T.sub.m vs 1
n[Ct], where [Ct] was the total oligonucleotide concentration. From
this analysis the thermodynamic parameters is determined. Based
upon the information gained concerning the stability of the duplex
of heteroduplex formed, the placement of 2'-O-alkyl guanosine into
oligonucleotides are assessed for their effects on helix stability.
Modifications that drastically alter the stability of the hybrid
exhibit reductions in the free energy (delta G) and decisions
concerning their usefulness as antisense oligonucleotides are
made.
[0203] B. Fidelity of Hybridization of 2'-modified
Oligonucleotides
[0204] The ability of the 2'-O-alkyl guanosine modified antisense
oligo-nucleotides to hybridize with absolute specificity to the
targeted mRNA is shown by Northern blot analysis of purified target
mRNA in the presence of total cellular RNA. Target mRNA is
synthesized from a vector containing the cDNA for the target mRNA
located downstream from a T7 RNA polymerase promoter. Synthesized
mRNA was electrophoresed in an agarose gel and transferred to a
suitable support membrane (ie. nitrocellulose). The support
membrane was blocked and probed using [.sup.32P]-labeled antisense
oligonucleotides. The stringency will be determined by replicate
blots and washing in either elevated temperatures or decreased
ionic strength of the wash buffer. Autoradiography was performed to
assess the presence of heteroduplex formation and the autoradiogram
quantitated by laser densitometry (LKB Pharmacia, Inc.). The
specificity of hybrid formation was determined by isolation of
total cellular RNA by standard techniques and its analysis by
agarose electrophoresis, membrane transfer and probing with the
labeled 2'-modified oligonucleotides. Stringency was predetermined
for the unmodified antisense oligonucleotides and the conditions
used such that only the specifically targeted mRNA was capable of
forming a heteroduplex with the 2'-modified oligonucleotide.
Example 50
[0205] Nuclease Resistance
[0206] A. Evaluation of the Resistance of 2'-Modified
Oligonucleotides to Serum and Cytoplasmic Nucleases.
[0207] Natural phosphorothioate, and 2-modified oligonucleotides
were assessed for their resistance to serum nucleases by incubation
of the oligonucleotides in media containing various concentrations
of fetal calf serum or adult human serum. Labeled oligonucleotides
were incubated for various times, treated with protease K and then
analyzed by gel electrophoresis on 20% polyacrylamine-urea
denaturing gels and subsequent autoradiography. Autoradiograms were
quantitated by laser densitometry. Based upon the location of the
modifications and the known length of the oligonucleotide it was
possible to determine the effect on nuclease degradation by the
particular 2'-modification. For the cytoplasmic nucleases, a HL60
cell line was used. A post-mitochondrial supernatant was prepared
by differential centrifugation and the labeled oligonucleotides
were incubated in this supernatant for various times. Following the
incubation, oligo-nucleotides were assessed for degradation as
outlined above for serum nucleolytic degradation. Autoradiography
results were quantitated for comparison of the unmodified, the
phosphorothioates, and the 2'-modified oligonucleotides.
[0208] B. Evaluation of the Resistance of 2'-Modified
Oligonucleotides to Specific Endo- and Exo-Nucleases.
[0209] Evaluation of the resistance of natural and 2'-modified
oligonucleotides to specific nucleases (ie, endonucleases,
3',5'-exo-, and 5',3'-exonucleases) was done to determine the exact
effect of the modifications on degradation. Modified
oligonucleotides were incubated in defined reaction buffers
specific for various selected nucleases. Following treatment of the
products with proteinase K, urea was added and analysis on 20%
polyacrylamide gels containing urea was done. Gel products were
visualized by staining using Stains All (Sigma Chemical Co.). Laser
densitometry was used to quantitate the extend of degradation. The
effects of the 2'-modifications were determined for specific
nucleases and compared with the results obtained from the serum and
cytoplasmic systems.
* * * * *