U.S. patent application number 10/424594 was filed with the patent office on 2004-05-06 for combinatorial library synthesis and pharmaceutically active compounds produced thereby.
This patent application is currently assigned to Micrologix Biotech Inc.. Invention is credited to Iyer, Radhakrishnan Parameswaran, Jin, Yi, Roland, Arlene, Zhou, Wenqiang.
Application Number | 20040087540 10/424594 |
Document ID | / |
Family ID | 26860197 |
Filed Date | 2004-05-06 |
United States Patent
Application |
20040087540 |
Kind Code |
A1 |
Zhou, Wenqiang ; et
al. |
May 6, 2004 |
Combinatorial library synthesis and pharmaceutically active
compounds produced thereby
Abstract
The invention provides new methods for synthesis of
nucleotide-based compounds and new libraries of such compounds.
Compounds of the invention are useful for a variety of therapeutic
applications, including treatment of viral or bacterial infections
and associated diseases and disorders.
Inventors: |
Zhou, Wenqiang; (Bedford
Hills, NY) ; Jin, Yi; (Carlsbad, CA) ; Roland,
Arlene; (Castries, FR) ; Iyer, Radhakrishnan
Parameswaran; (Shrewsbury, MA) |
Correspondence
Address: |
SEED INTELLECTUAL PROPERTY LAW GROUP PLLC
701 FIFTH AVE
SUITE 6300
SEATTLE
WA
98104-7092
US
|
Assignee: |
Micrologix Biotech Inc.
Vancouver
CA
|
Family ID: |
26860197 |
Appl. No.: |
10/424594 |
Filed: |
April 28, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10424594 |
Apr 28, 2003 |
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09709246 |
Nov 8, 2000 |
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6620796 |
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60164036 |
Nov 8, 1999 |
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60172508 |
Dec 17, 1999 |
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Current U.S.
Class: |
514/46 ;
435/6.14; 435/7.1; 436/518; 514/263.23; 514/269; 514/50; 544/276;
544/277; 544/310 |
Current CPC
Class: |
B01J 2219/00596
20130101; C40B 50/14 20130101; C40B 60/14 20130101; B01J 2219/00599
20130101; C07H 19/20 20130101; B01J 2219/00288 20130101; C40B 40/06
20130101; C07H 19/10 20130101; B01J 2219/00421 20130101; B01J
2219/00585 20130101; C07H 19/04 20130101; B01J 2219/00722 20130101;
B01J 2219/00306 20130101; C40B 50/08 20130101 |
Class at
Publication: |
514/046 ;
435/007.1; 436/518; 514/050; 514/263.23; 514/269; 544/276; 544/277;
544/310; 435/006 |
International
Class: |
C12Q 001/68; G01N
033/53; A61K 031/7072; A61K 031/7076; A61K 031/522; A61K
031/513 |
Claims
What is claimed is:
1. A compound library comprising two or more compounds of the
following 38Formula I or I': wherein L is a linking group such as
e.g. an amide, ester, diester or the like, or an optionally
substituted alkylene (e.g. C.sub.1-20 alkylene), optionally
substituted alkenylene (e.g., C.sub.2-20 alkenylene) or alkynylene
(e.g., C.sub.2-20 alkynylene) having such groups either as a chain
member of pendant to the chain, and which may be optionally
substituted with one or more substituents selected from a group
consisting of O, S, Se, NR.sup.1NR.sup.2, CR.sup.1CR.sup.2, OR, SR
and SeR, or an enzymatically reactive; Q is carbon or a heteroatom
such as O, S or N; R is hydrogen or a hydroxyl group or an
optionally substituted alkyl, optionally substituted alkenyl,
optionally substituted alkynyl, optionally substituted aralkyl,
optionally substituted cycloalkyl, optionally substituted
cycloalkenyl, optionally substituted carbocyclic aryl, an
optionally substituted mononucleotide, an optionally substituted
polynucleotide, or an optionally substituted heteroaromatic or
heteroalicyclic group preferably having from 1 to 3 separate or
fused ring and 1 to 3 N, O or S atoms; R.sup.1, R.sup.2, R.sup.3
and R.sup.4 are each independently selected from a group as defined
by R; B is optionally substituted adenine, optionally substituted
thymidine, optionally substituted cytosine or an optionally
substituted guanine, preferably where the optional substituents are
alkyl, carbocyclic aryl, or heteroaromatic or heteroalicyclic group
preferably having from 1 to 3 separate or fused rings and 1 to 3 N,
O or S atoms, or a heterocyclic structure that is covalently linked
to the sugar ring; n=1 to 5; and pharmaceutically acceptable salts
thereof.
2. The library of claim 1 wherein at least one compound has a sugar
group is in open chain form.
3. The library of claim 1 wherein an enantiomerically enriched
mixture of a compound is present.
4. A compound library comprising two or more compounds of the
following Formula II or II': 39X and Y are each independently
selected from a group consisting of O, S, Se, NR.sup.1NR.sup.2,
CR.sup.1CR.sup.2, OR, SR and SeR, or one or both of X and Y are an
enzymatically reactive moiety; R is optionally substituted alkyl,
optionally substituted alkenyl, optionally substituted alkynyl,
optionally substituted aralkyl, optionally substituted cycloalkyl,
optionally substituted cycloalkenyl, optionally substituted
carbocyclic aryl, an optionally substituted mononucleotide, an
optionally substituted polynucleotide, or an optionally substituted
heteroaromatic or heteroalicyclic group preferably having from 1 to
3 separate or fused ring and 1 to 3 N, O or S atoms; R.sup.1,
R.sup.2, and R.sup.3 are each independently selected from a group
as defined by R; B is optionally substituted adenine, optionally
substituted thymidine, optionally substituted cytosine or an
optionally substituted guanine, preferably where the optional
substituents are alkyl, alkynyl, carbocyclic aryl, or
heteroaromatic or heteroalicyclic group preferably having from 1 to
3 separate or fused ring and 1 to 3 N, O or S atoms, or a
heterocyclic structure that is covalently linked to the sugar ring;
and pharmaceutically acceptable salts thereof.
5. The library of claim 1 wherein at least one compound is of the
following formula IIA or IIA': 40X and Y are each independently
selected from a group consisting of O, S, Se, NR.sup.1NR.sup.2,
CR.sup.1CR.sup.2, OR, SR and SeR, or one or both of X and Y are an
enzymatically reactive moiety; R is optionally substituted alkyl,
optionally substituted alkenyl, optionally substituted alkynyl,
optionally substituted aralkyl, optionally substituted cycloalkyl,
optionally substituted cycloalkenyl, optionally substituted
carbocyclic aryl, an optionally substituted mononucleotide, an
optionally substituted polynucleotide, or an optionally substituted
heteroaromatic or heteroalicyclic group preferably having from 1 to
3 separate or fused ring and 1 to 3 N, O or S atoms; R.sup.1,
R.sup.2 and R.sup.3 are each independently selected from a group as
defined by R; B is optionally substituted adenine, optionally
substituted thymidine, optionally substituted cytosine or an
optionally substituted guanine, preferably where the optional
substituents are alkyl, alkynyl, carbocyclic aryl, or
heteroaromatic or heteroalicyclic group preferably having from 1 to
3 separate or fused ring and 1 to 3 N, O or S atoms, or a
heterocyclic structure that is covalently linked to the sugar ring;
and pharmaceutically acceptable salts thereof.
6. The library of claim 1 wherein the library has been constructed
using solid-phase synthesis.
7. The library of claim 1 wherein the library has been constructed
using solution phase synthesis.
8. Use of the library of claim 1 to find a specific interacting
partner for a nucleic acid.
9. Use of the library of claim 1 to find a specific interacting
partner for a protein.
10. The use of claim 8 wherein the nucleic acid is RNA or DNA.
11. The use of claim 9 wherein the protein is an antibody, receptor
or ligand.
12. A compound of the following Formula I or I': 41wherein L is a
linking group such as an amide, ester, diester or the like which
may be optionally substituted with one or more substituents
selected from a group consisting of O, S, Se, NR.sup.1NR.sup.2,
CR.sup.1CR.sup.2, OR, SR and SeR, or an enzymatically reactive
moiety; Q is carbon or a heteroatom such as O, S or N; R is
hydrogen or a hydroxyl group or an optionally substituted alkyl,
optionally substituted alkenyl, optionally substituted alkynyl,
optionally substituted aralkyl, optionally substituted cycloalkyl,
optionally substituted cycloalkenyl, optionally substituted
carbocyclic aryl, an optionally substituted mononucleotide, an
optionally substituted polynucleotide, or an optionally substituted
heteroaromatic or heteroalicyclic group preferably having from 1 to
3 separate or fused ring and 1 to 3 N, O or S atoms; R.sup.1,
R.sup.2, R.sup.3 and R.sup.4 are each independently selected from a
group as defined by R; B is optionally substituted adenine,
optionally substituted thymidine, optionally substituted cytosine
or an optionally substituted guanine, preferably where the optional
substituents are alkyl, carbocyclic aryl, or heteroaromatic or
heteroalicyclic group preferably having from 1 to 3 separate or
fused rings and 1 to 3 N, O or S atoms, or or B is heteroaromatic
or heteroalicyclic group other than an adenine, thymidine, cytosine
or guanine; N is an integer of from 1 (where to form a 5-membered
ring as depicted or 5-membered acyclic group) to 5; and
pharmaceutically acceptable salts thereof.
13. A compound of the following Formula II or II': 42X and Y are
each independently selected from a group consisting of O, S, Se,
NR.sup.1NR.sup.2, CR.sup.1CR.sup.2, OR, SR and SeR, or one or both
of X and Y are an enymatically reactive moiety; R is optionally
substituted alkyl, optionally substituted alkenyl, optionally
substituted alkynyl, optionally substituted aralkyl, optionally
substituted cycloalkyl, optionally substituted cycloalkenyl,
optionally substituted carbocyclic aryl, an optionally substituted
mononucleotide, an optionally substituted polynucleotide, or an
optionally substituted heteroaromatic or heteroalicyclic group
preferably having from 1 to 3 separate or fused ring and 1 to 3 N,
O or S atoms; R.sup.1, R.sup.2 and R.sup.3 are each independently
selected from a group as defined by R; B is optionally substituted
adenine, optionally substituted thymidine, optionally substituted
cytosine or an optionally substituted guanine, preferably where the
optional substituents are alkyl, carbocyclic aryl, or
heteroaromatic or heteroalicyclic group preferably having from 1 to
3 separate or fused ring and 1 to 3 N, O or S atoms, or a
heterocyclic structure that is covalently linked to the sugar ring;
and pharmaceutically acceptable salts thereof.
14. A compound of claim 12 wherein the sugar group is in open chain
form.
15. A compound of claim 12 wherein an enantiomerically enriched
mixture of a compound is present.
16. A compound of the following Formula IIA or IIA': 43X and Y are
each independently selected from a group consisting of O, S, Se,
NR.sup.1NR.sup.2, CR.sup.1CR.sup.2, OR, SR and SeR, or one or both
of X and Y are an enzymatically reactive moiety; R is optionally
substituted alkyl, optionally substituted alkenyl, optionally
substituted alkynyl, optionally substituted arakyl, optionally
substituted cycloalkyl, optionally substituted cycloalkenyl,
optionally substituted carbocyclic aryl, an optionally substituted
mononucleotide, an optionally substituted polynucleotide, or an
optionally substituted heteroaromatic or heteroalicyclic group
preferably having from 1 to 3 separate or fused ring and 1 to 3 N,
O or S atoms; R.sup.1, R.sup.2 and R.sup.3 are each independently
selected from a group as defined by R; B is optionally substituted
adenine, optionally substituted thymidine, optionally substituted
cytosine or an optionally substituted guanine, preferably where the
optional substituents are alkyl, carbocyclic aryl, or
heteroaromatic or heteroalicyclic group preferably having from 1 to
3 separate or fused ring and 1 to 3 N, O or S atoms, or a
heterocyclic structure that is covalently linked to the sugar ring;
and pharmaceutically acceptable salts thereof.
17. A method for treating virally infected cells comprising
administering to the cells an anti-viral effective amount of a
compound of claim 12.
18. The method of claim 17 wherein the cells are infected with a
herpes virus.
19. The method of claim 17 wherein the cells are infected with a
cytomegalovirus.
20. The method of claim 17 wherein the cells are infected with a
hepatitis B virus.
21. A method for treating bacterially infected cells comprising
administering to the cells an anti-bacterial effective amount of a
compound of claim 12.
22. The method of claim 21 wherein the mammal is suffering from a
mycobacterium infection.
23. A method for treating a mammal suffering from or susceptible to
a viral infection, comprising administering to the mammal an
anti-viral effective amount of a compound of claim 12.
24. The method of claim 23 wherein the mammal is suffering from a
herpes infection.
25. The method of claim 23 wherein the mammal is suffering from a
cytomegalovirus infection.
26. The method of claim 23 wherein the mammal is suffering from a
hepatitis B virus infection.
27. A method for treating a mammal suffering from or susceptible to
a bacterial infection, comprising administering to the mammal an
anti-bacterial effective amount of a compound of claim 12.
28. A pharmaceutical composition comprising a compound of any one
of claim 12 and a pharmaceutically acceptable carrier.
29. A method for synthesis of a compound library, comprising:
adding one or more reagents to a reaction vessel capable of
agitation and containing a resin reaction support material;
agitating the reaction vessel during reaction of the reagents; and
centrifuging the reaction vessel and removing desired reaction
materials therefrom.
30. The library of claim 1 wherein the library comprises
phosphorothioate compounds.
31. The library of claim 1 wherein the library comprises
thiophosphoramidate compounds.
32. The library of claim 1 wherein the library comprises
phosphoramidothionate compounds.
33. The library of claim 1 wherein the library comprises carbamate
compounds.
34. The compound of claim 12 wherein the compound is a
phosphorothioate compound.
35. The compound of claim 12 wherein the compound is a
thiophosphoramidate compound.
36. The compound of claim 12 wherein the compound is a
phosphoramidothionate compound.
37. The compound of claim 12 wherein the compound is a carbamate
compound.
Description
[0001] This application claims the benefit of U.S. Provisional
Application Serial No. 60/164,036 filed Nov. 8, 1999, and U.S.
Provisional Application Serial No. 60/172,508 filed Dec. 17, 1999,
the teachings of which are incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The invention provides new methods for synthesis of
nucleotide-based compounds and libraries of such compounds.
Compounds of the invention are useful for a variety of therapeutic
applications, including treatment of viral or bacterial infections
and associated diseases and disorders.
[0004] 2. Background
[0005] The important initial step in the development of therapeutic
agents is the discovery of compounds that bind to a protein, enzyme
or receptor of interest. Through careful structure/activity work of
resulting active compounds, one arrives at a lead compound for
further development into a clinical candidate. This traditional
process of drug discovery is a long and arduous endeavor. Often it
takes 10 to 15 years before a new drug makes it into the
marketplace.
[0006] Recent advances in molecular biology and genomics have led
to identification of new molecular targets for drug discovery. As a
result of the limitation of traditional drug discovery, new
approaches to the discovery of therapeutics have been developed. In
the more modern approaches, large libraries of diverse compounds
are synthesized by a number of methods and subjected to high
throughput in vitro screening against a particular molecular target
implicated in a disease. The active compounds so identified are
then subjected to Structure-Activity Relationship (SAR) work to
eventually identify the lead compound.
[0007] Modern drug discovery approaches entail the synthesis and
screening of libraries of compounds. The design and synthesis of
such libraries is often based on a unique molecular skeleton or
scaffold. By incorporating a variety of structural elements into a
scaffold, local as well as global molecular diversity can be
achieved which facilitates specific interactions between a ligand
and its receptor. The structural elements contribute to molecular
diversity by variable spatial display of ionic, hydrogen-bonding,
charge-transfer and van der Waals interactions thus allowing for
the selection of the best `fit` between the ligand and its
receptor.
[0008] Traditionally, libraries have been constructed using
solid-support synthesis methods, such as synthesis of a library on
`beads`. Solid support methods are useful because reactive products
can be readily isolated in a relatively pure form by simply washing
away excess reagents and solvents from the support matrix,
something that is not possible with solution based methods.
[0009] One method for generating compound libraries utilizes a
discrete compound approach. In the discrete compound approach,
compounds are synthesized in parallel each in a separate reaction
vessel. The identity of each compound is known or can be
ascertained by analytical methods. Various methods for constructing
discrete compound libraries are known in the art. For example, the
Pin method (H. M. Geyson et al., PNAS, USA 81: 3998-4002 (1984))
utilizes polyethylene pins placed in a 96-well supporting block.
Each pin is coated with polymeric material that is derivatized for
anchoring functional groups. The reactions can be run on 100 nmol
to 50 micromol scale and the products subjected to multiple
biological assays. The Diversomer apparatus approach (S. H. Dewitt
et al., PNAS, USA 90: 6909-6913 (1993)) utilizes a series of porous
gas dispersion tubes which serve as containers for resin beads and
reagents and solvents are placed in vials mounted on a reservoir
block. The ends of the gas dispersion tubes are placed in the vials
and the reagents are allowed to diffuse through the porous membrane
and contact the resin support. The apparatus can be placed in a
manifold with an injectable gasket. The porous frit apparatus
utilizes each well of a deep well microtiter plate fitted with
porous frits. The plate is clamped on to a viton gasket. In between
synthetic steps in a sequence, the reaction solution can be drained
and the resin rinsed by removal of the viton gasket. The spatially
addressable, light directed parallel synthesis method utilizes a
photolithographic method to synthesize 100,000 separate compounds.
The synthesis is done on a silicon wafer (chip) that is
functionalized to attach to a leader molecule which carries a
photolabile protecting group at its reaction site. Once unmasked by
illumination, the reactive group is unmasked which can then enter
into a specific chemical reaction with a reactant. The library of
compounds remain tethered to the solid support. The structure of
the compound in each specific location is known.
[0010] Another method for generating compound libraries utilizes a
mix and pool synthesis approach. This approach allows large
libraries of compounds to be synthesized by pooling different sets
of support-bound intermediates. However, this method only works
when all of the reactants in a mixture have similar reactivities.
Reaction conditions need to be optimized before attempting a split
and pool strategy. This strategy has been used to synthesize
libraries of peptides and oligonucleotides. Various mix and pool
synthesis approaches are known in the art. For example, Houghten et
al. (C. Pinilla et al., Biopolymers, Pept. Sci., 37: 221-240
(1995), pioneered this approach by preparing pools of compounds
that each contain structurally defined building blocks at one or
two positions. Once the pool with the highest activity is
identified in an in vitro assay, the deconvolution process begins.
Iterative rounds of synthesis and biological assays are carried out
until a molecule with the highest activity is identified.
Modifications of this approach include the positional scanning
approach developed by Houghten et al. (C. Pinilla et al.,
Biopolymers, Pept. Sci., 37: 221-240 (1995) and the orthogonal
approach developed by Tartar et al. (B. Deprez, et al., J. Am.
Chem. Soc., 117: 5405-5406 (1995). Another mix and pool synthesis
approach utilizes beads encoded by oligonucleotides of known
sequence to trace compounds.
[0011] Biological assays used to test the activity of compound
libraries can be carried out with the compounds immobilized on a
solid support or in solution. For example, a resin-bound library
can be treated with a fluorescent-labeled receptor and the
compound-bound receptors isolated using a fluorescence activated
cell sorting instrument. Structure determination can be done, for
example, by sequencing or mass spectrometry analysis. When assays
are performed in solution, the compounds need to be released from
the solid support. A portion of the beads are released and
contacted with the receptor. The active compounds are then traced
back to the original bead. Structure determination can be performed
by analytical methods.
[0012] See also: C. Pinilla et al., Biopolymers, Pept. Sci 37:
221-240 (1995); S. H. DeWitt et al., PNAS, USA 90: 6909-6913
(1993); B. Deprez et al., J. Am. Chem. Soc. 117: 5405-5406 (1995);
H. M. Geyson, et al. PNAS, USA 81: 3998-4002 (1984); G. Jung et
al., Angew. Chem. Intl. Ed. Engl. 31: 367-383 (1992); M. R. Pavia,
et al. Bioorg. Med. Chem. Lett. 3: 387-396 (1993); E. M. Gordon et
al., J. Med. Chem. 37: 1385-1401 (1994); L. A. Thompson et al.,
Chem. Rev. 96: 555-600 (1996); S. Verma et al., Annu. Rev. Biochem.
67: 99-134 (1998); S. L. Beaueage et al., Tetrahedron Lett. 22:
1859-1862 (1981); R. P. Iyer et al., In Comprehensive Natural
Products, D. H. R. Barton and K. Nakanishi Eds., Elsevier Science.
Vol 7 (In press); A. D. Barone et al., Nucl. Acids Res. 12:
4051-4061 (1984); R. P. Iyer et al., J. Am. Chem. Soc. 112: 1253-54
(1990).
SUMMARY OF THE INVENTION
[0013] We have now found new nucleotide-based compounds that are
useful for a variety of therapeutic applications, including to
treat against viral or bacterial infections.
[0014] The invention also provides new methods for synthesis of
nucleotide-based compounds and new libraries of such compounds. In
particular, the invention provides new methods for construction of
compound libraries utilizing a nucleic acid-based (NAB) scaffold.
This approach enables incorporating structural elements that can
provide both "sequence-specific" interactions (e.g.,
hydrogen-bonding interactions between nucleobases) as well as
"shape-specific" motifs (e.g., bulges and stem-loop structures)
that can allow specific recognition of other nucleic acids and
proteins. Libraries based on NAB scaffold can potentially mimic the
molecular recognition that exists between cellular macromolecules
and biomolecules such as hormones, nucleotides and their
receptors.
[0015] The invention provides methods for constructing compound
libraries by solution-phase or solid-phase approaches. Preferred
library syntheses of the invention are carried out on a solid
support. Suitable solid supports include, for example, pins, beads,
resins, chips, etc.
[0016] Preferred library syntheses of the invention include use of
columns capable of agitation (e.g. spin or other rotation) and that
may suitably contain a resin support material. Reactants are placed
in the column, and the column preferably shaken or otherwise
agitated during reaction. Additional reactants can be added to
provide repeated reaction cycles. Reagents and reaction products
also can be conveniently separated and removed from the column,
e.g. by centrifuging a reaction column to facilitate removal (e.g.
by filtration) of desired material.
[0017] Other aspects of the invention are disclosed infra.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIG. 1 provides a flow diagram for library assembly.
DETAILED DESCRITPTION OF THE INVENTION
[0019] As discussed above, we have discovered new methods for
construction of a compound library. Preferred library members
include compounds of the following Formula I or I': 1
[0020] wherein L is a linking group such as e.g. an amide, ester,
diester or the like, or an optionally substituted alkylene (e.g.
C.sub.1-20 alkylene), optionally substituted alkenylene (e.g.,
C.sub.2-20 alkenylene) or alkynylene (e.g., C.sub.2-20 alkynylene)
having such groups either as a chain member of pendant to the
chain, and which may be optionally substituted with one or more
substituents selected from a group consisting of O, S, Se,
NR.sup.1NR.sup.2, CR.sup.1CR.sup.2, OR, SR and SeR (R, R.sup.1 and
R.sup.2 defined below), or an enzymatically reactive (particularly,
cleavable) moiety such as an amide, ester, and the like;
[0021] Q is carbon or a heteroatom such as O, S or N;
[0022] R is hydrogen or a hydroxyl group or a hydrophobic group,
e.g. a moiety having from 1 to about 18 carbon atoms, such as
optionally substituted alkyl, optionally substituted alkenyl,
optionally substituted alkynyl, optionally substituted aralkyl,
optionally substituted cycloalkyl, optionally substituted
cycloalkenyl, optionally substituted carbocyclic aryl, an
optionally substituted mononucleotide, an optionally substituted
polynucleotide, or an optionally substituted heteroaromatic or
heteroalicyclic group preferably having from 1 to 3 separate or
fused ring and 1 to 3 N, O or S atoms;
[0023] R.sup.1, R.sup.2, R.sup.3 and R.sup.4 are each independently
selected from a group as defined by R;
[0024] B is optionally substituted adenine, optionally substituted
thymidine, optionally substituted cytosine or an optionally
substituted guanine, preferably where the optional substituents are
alkyl, carbocyclic aryl, or heteroaromatic or heteroalicyclic group
preferably having from 1 to 3 separate or fused rings and 1 to 3 N,
O or S atoms, or B is heteroaromatic or heteroalicyclic group other
than an adenine, thymidine, cytosine or guanine and preferably has
from 1 to 3 separate or fused rings and 1 to 3 N, O or S atoms;
[0025] n is an integer of from 1 to 5 and where n is greater than 1
designates that corresponding additional carbon ring or acyclic
members are present (i.e. where n is 2 an additional carbon ring
member (to form a 6-membered ring) or acyclic carbon is present;
where n is 3, two additional carbon ring members (to form a
7-membered ring) or acyclic carbon is present, and so on);
[0026] and pharmaceutically acceptable salts thereof.
[0027] In the above Formulae I and I', it is understood that the
dashed line indicates the ring may be in the open or closed
configuration.
[0028] The depicted sugar group may be natural or modified (e.g.
synthetic) form, or in an open chain form (where one of the
depicted ring bonds would not be present).
[0029] Preferred R groups of compounds of formulae I and I' include
cyclic groups, particularly alicyclic groups that may comprise one
or more single or polycyclic rings, particularly a bridged or fused
ring structure, with 0, 1 or 2 endocyclic carbon-carbon double
bonds. Additional preferred R groups include heteroalicyclic
moieties, particularly heteroalicyclic groups having from 5 to
about 8 ring member, preferably with one or two O, N or S ring
members, particularly one or two oxygen ring members.
[0030] Preferred compounds of the invention include those of
formulae I and I' where the nucleoside is linked to the R group via
a phosphorous group at the 5' end. Other dephospholinkers such as
carbonates, carbamates, ureas, acetals, etc., may also be used.
Such linkages could also be established via the 2' or 3' sites of
the nucleoside. When R is a nucleoside, linkages can be via 5' to
3', 5' to 5', 3' to 3', 2' to 5' and 2' to 2', or any combination
thereof, of the participating nucleosides.
[0031] Preferred library members include compounds of the following
Formula II or II': 2
[0032] wherein X and Y are each independently selected from a group
consisting of O, S, Se, NR.sup.1NR.sup.2, CR.sup.1CR.sup.2, OR, SR
and SeR, or one or both of X and Y are an enzymatically reactive
(particularly, cleavable) moiety such as an amide, ester, and the
like;
[0033] R is a hydrophobic group, e.g. a moiety having from 1 to
about 18 carbon atoms, such as optionally substituted alky,
optionally substituted alkenyl, optionally substituted alknyl,
optionally substituted aralkyl, optionally substituted cycloalkyl,
optionally substituted cycloalkenyl, optionally substituted
carbocyclic aryl, an optionally substituted mononucleotide, an
optionally substituted polynucleotide, or an optionally substituted
heteroaromatic or heteroalicyclic group preferably having from 1 to
3 separate or fused ring and 1 to 3 N, O or S atoms;
[0034] R.sup.1, R.sup.2 and R.sup.3 are each independently selected
from a group as defined by R;
[0035] B is optionally substituted adenine, optionally substituted
thymidine, optionally substituted cytosine or an optionally
substituted guanine, preferably where the optional substituents are
alkyl, carbocyclic aryl, alknyl, or heteroaromatic or
heteroalicyclic group preferably having from 1 to 3 separate or
fused rings and 1 to 3 N, O or S atoms, or any heterocyclic
structure that is covalently linked to the sugar ring;
[0036] and pharmaceutically acceptable salts thereof.
[0037] In the above Formulae II and II', it is understood that the
dashed line extending to each of the substituents X and Y
designates that one, but not both, of X and Y may have an
additional chemical bond (i.e. a double bond).
[0038] The depicted sugar group may be natural or modified (e.g.
synthetic) form, or in an open chain form (where one of the
depicted ring bonds would not be present).
[0039] Preferred R groups of compounds of formulae II and II'
include cyclic groups, particularly alicyclic groups that may
comprise one or more single or polycyclic rings, particularly a
bridged or fused ring structure, with 0, 1 or 2 endocyclic
carbon-carbon double bonds. Additional preferred R groups include
heteroalicyclic moieties, particularly heteroalicyclic groups
having from 5 to about 8 ring member, preferably with one or two O,
N or S ring members, particularly one or two oxygen ring
members.
[0040] As mentioned above, either one or both of X and Y may be an
enzymatically reactive group, i.e. the group may be cleavable or
otherwise reactive in vivo upon administration to a mammal,
particularly a human. Preferred enzymatically reactive groups
include e.g. amides (which may be cleaved in vivo with an amidase),
esters (which may be cleaved in vivo with an esterase), and acetal
and ketal groups.
[0041] Preferred compounds of the invention include those of
formulae II and II' where the nucleoside is linked to the R group
via a phosphorous group at the 5' end. Other dephospholinkers such
as carbonates, carbamates, ureas, acetals, etc., may also be used.
Such linkages could also be established via the 2' or 3' sites of
the nucleoside. When R is a nucleoside, linkages can be via 5' to
3', 5' to 5', 3' to 3', 2' to 5' and 2' to 2', or any combination
thereof, of the participating nucleosides.
[0042] Preferably, compounds of the invention will be present in
enantiomerically enriched mixtures, i.e. where one enantiomer is
present in a greater amount than other stereoisomer(s) of the
compound, particularly where one enantiomer is present in amount of
at least about 60 mole percent, relative to all stereoisomers
present of the compound; preferably where one enantiomer is present
in amount of at least about 70 or 80 mole percent, relative to all
stereoisomers present of the compound; still more preferably where
one enantiomer is present in amount of at least about 85, 90, 92,
95, 96, 97, 98 or 99 mole percent, relative to all stereoisomers
present of the compound
[0043] Preferred compounds of the invention include those of the
following Formulae IIA and IIA', having the depicted
configurations: 3
[0044] X, Y, R, R.sup.1, R.sup.2, B, R.sup.3 and n are the same as
defined above for Formulas II and II'; and pharmaceutically
acceptable salts thereof.
[0045] In the above Formulae IIA and IIA', it is understood that
the dashed line extending to each of the substituents X and Y
designates that one, but not both, of X and Y may have an
additional chemical bond (i.e. a double bond).
[0046] In the above Formulae IIA and IIA', the depicted sugar group
may be natural or modified (e.g. synthetic) form, or in an open
chain form (where one of the depicted ring bonds would not be
present).
[0047] In the above Formulae I, I', II, II', IIA and IIA', alkyl
groups preferably contain from 1 to about 18 carbon atoms, more
preferably from 1 to about 12 carbon atoms and most preferably from
1 to about 6 carbon atoms. Specific examples of alkyl groups
include, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl,
t-butyl, isobutyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl
etc.
[0048] In the above Formulae I, I', II, II', IIA and IIA', aralkyl
groups include the above-listed alkyl groups substituted by a
carbocyclic aryl group having 6 or more carbons, for example,
phenyl, naphthyl, phenanthryl, anthracyl, etc.
[0049] In the above Formulae I, I', II, II', IIA and IIA',
cycloalkyl groups preferably have from 3 to about 8 ring carbon
atoms, e.g. cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl,
1,4-methylenecyclohexane, adamantyl, cyclopentylmethyl,
cyclohexylmethyl, 1- or 2-cyclohexylethyl and 1-, 2- or
3-cyclohexylpropyl, etc.
[0050] In the above Formulae I, I', II, II', IIA and IIA',
exemplary heteroaromatic and heteroalicyclic group include pyridyl,
pyrazinyl, pyrimidyl, furyl, pyrrolyl, thienyl, thiazolyl,
oxazolyl, imidazolyl, indolyl, benzothiazolyl, tetrahydrofuranyl,
tetrahydropyranyl, piperidinyl, morpholino and pyrrolidinyl.
[0051] Mononucleotides of compounds of the invention (compounds of
Formulae I, I', II, II', IIA and IIA') invention include adenine,
cytosine, guanosine and thymidine.
[0052] Polynucleotides of compounds of the invention (compounds of
Formulae I, I', II, II', IIA and IIA') preferably contain from
about 1 to about 20 mononuculeotides, more preferably from 1 to
about 10 mononuculeotides and still more preferably from 1 to about
5 mononuculeotides. The polynucleotides are suitably constructed
such that the 5' group of one mononucleotide pentose ring is
attached to the 3' group of its neighbor in one direction via, for
example, a phosphodiester or a phosphorthioate internucleotide
linkage.
[0053] Sugar groups of compounds of the invention may be comprised
of mono-, di-, oligo- or poly-saccharides wherein each
monosaccharide unit comprises from 3 to about 8 carbons, preferably
from 3 to about 6 carbons, containing polyhydroxy groups or
polyhydroxy and amino groups. Non-limiting examples include
glycerol, ribose, fructose, glucose, glucosamine, mannose,
galactose, maltose, cellobiose, sucrose, starch, amylose,
amylopectin, glycogen and cellulose. The hydroxyl and amino groups
are present as free or protected groups containing e.g. hydrogens
and/or halogens. Preferred protecting groups include acetonide,
t-butoxy carbonyl groups, etc. Monosaccharide sugar groups may be
of the L or D configuration and a cyclic monosaccharide unit may
contain a 5 or 6 membered ring of the .alpha. or .beta.
conformation. Disaccharides may be comprised of two identical or
two dissimilar monosaccharide units. Oligosaccharides may be
comprised of from 2 to 10 monosaccharides and may be homopolymers,
heteropolymers or cyclic polysugars. Polysaccharides may be
homoglycans or heteroglycans and may be branched or unbranched
polymeric chains. The di-, oligo- and poly-saccharides may be
comprised of 1.fwdarw.4, 1.fwdarw.6 or a mixture of 1.fwdarw.4 and
1.fwdarw.6 linkages. The sugar moiety may be attached to the link
group through any of the hydroxyl or amino groups of the
carbohydrate.
[0054] Preferred compounds of the invention comprise R groups
containing one of the hydrophobic structures represented in Table 1
below.
[0055] Preferred library syntheses of the invention are carried out
on a solid support. Suitable solid supports include, for example,
pins, beads, resins, chips, etc. Particularly preferred methods are
those carried out using beads as the solid support.
[0056] Phospholinked compounds of the invention can be prepared as
generally depicted in Scheme I. In addition to the synthetic route
depicted in Scheme 1, library compounds may also be assembled using
a phosphodiester approach, a phosphortriester approach and/or the
H-phosphonate methodology (R. P. Iyer et al. In Comprehensive
Natural Products, D. H. R. Barton and K. Nakanishi Eds., Elsevier
Science Vol 7, in press). In Scheme I below, preferred
stereoisomers and substituent groups are depicted, although it is
understood that other compounds of the invention can be produced by
the same or similar procedures. 4
[0057] Scheme I shows the preparation of library compounds using
phosphoramidite chemistry (S. L. Beaueage et al., Tetrahedron Lett.
22: 1859-1862 (1981); R. P. Iyer et al., In Comprehensive Natural
Products, D. H. R. Barton and K. Nakanishi Eds. Elsevier Science.
Vol. 7 (in press)). A key synthon is the solid-support-bound
phosphoramidite, shown as compound 2 of Scheme I. Compound 2 can be
prepared by 5'-phosphitylation of controlled-pore-glass (CPG)-bound
nucleoside, shown as compound 3 of Scheme I. .beta.-cyanoethyl
bis(N,N-diisopropylamino)pho- sphine (CNP) in the presence of
N,N-diisopropylammonium tetrazolide was used as the phosphitylating
reagent, which produced byproducts soluble in methylene chloride.
The reaction mixture was filtered and a quantitative yield of
compound 2 was obtained. Each CPG-bound nucleoside phosphoramidite
(compound 2) could be stored in vacuo for subsequent use in library
synthesis.
[0058] The library synthesis was performed in a parallel format
using QIAquick.RTM. spin columns (Quiagen) each of which was
equipped with a sepharose resin supported by a nitrocellulose
filter at the bottom (FIG. 1). The reactants were placed in the
spin columns and the contents shaken were shaken during the
reaction. The columns were then placed in a recepticle vial and
centrifuged allowing facile filtration of the mixture into the
recepticle. The next reactant was then added to the spin columns
and the process repeated until the library synthesis was
complete.
[0059] Typically for the synthesis of the library (Scheme I, FIG.
1), a measured amount of CPG-bound phosphoramidite (compound 2) was
transferred to a series of spin columns. The alcohols 4a-f were
added to the spin columns along with tetrazole in acetonitrile and
incubated for 5 minutes. The spin columns were then centrifuged to
separate out the unreacted materials and byproducts from the
support-bound coupled product 5a-x as a mixture of Rp and Sp
diastereomers. The oxidative sulfurization of 5a-x was performed
using either 3H-1,2-berizodithiole-3-one-1,1-dioxide (0.1 M in
acetonitrile) or I.sub.2 solution (0.02 M in
Pyridine/H.sub.2O/tetrahy- rofuran) to produce the support bound
phosphotriesters 6a-x or 7a-x, respectively. The support-bound
library was heated with NH.sub.4OH (28%, 55.degree. C., 5 hours) to
remove the crude diesters. Each of the crude products were passed
through a Sep-Pak cartridge.RTM. (Waters) to give products 1a-x. It
is to be noted that oxidative sulfurization and oxidation can also
be carried out using other sulfurizing and oxidizing agents.
[0060] Table 1 below shows the members of a representative
24-member library. The library members were analyzed using
reversed-phase HPLC and determined to be 90-95% pure.
1TABLE 1 Representative Library members (1a-x), and their HPLC
retention time (R.sub.t min). 5 6 7 8 9 10 11 B Pdt R.sub.t Pdt
R.sub.t Pdt R.sub.t Pdt R.sub.t Pdt R.sub.t Pdt R.sub.t A 1a 30.5
1e 34.3 1i 47.8 1m 45.0 1q 36.0 1u 43.3 C 1b 28.6 1f 33.3 1j 47.5
1n 44.1 1r 35.1 1v 42.7 G 1c 28.1 1g 31.7 1k 46.5 1o 42.9 1s 33.6
1w 41.0 T 1d 29.7 1h 37.8 1l 48.2 1p 47.4 1t 38.2 1x 46.5 R =
hydrophobic group, Pdt = product, B = nucleobase, A = Adenine, C =
cytosine, G = guanine T = Thymine.
[0061] When prepared as a mixture, compound libraries of the
invention preferably will contain at least about 2, 3, 4 or 5
distinct compounds, more preferably at least about 10 distinct
compounds, still more preferably at least about 20, 30, 40, 50 ,60,
70, 80, 90 or 100 compounds, and may contain 200, 300, 400 or 500
or more compounds.
[0062] Table 2 shows examples of additional representative
hydrophobic groups which may be linked to the 5' end of ribo- and
deoxyribonucleosides via linkages. Specific examples of hydrophobic
groups include saturated and unsaturated acyclic and cyclic
alcohols, aromatic and heterocyclic alcohols. Exemplary groups
demonstrated in table 2 include primary and secondary alcohols.
However, one of skill in the art will recognize that the methods
may be extended to other kinds of structural variants, specifically
those hydrophobic groups bearing multiple functionalities, such as
for example, ether, keto, amino, halo in addition to the hydroxy
groups. Further, one of skill in the art would also recognize that
the methods disclosed may be useful for linkage to additional
available sites such as the 3' and 2' sites.
2TABLE 2 open-chain cyclic primary cyclic secondary aromatic
alcohol alcohol alcohol alcohol 12 13 14 15 16 17 18 19 20 21 22 23
24 25 26 27 28 29 30 31 32 33 34 35 36
[0063] Compounds of the invention (compounds of Formulae I, I', II,
II', IIA and IIA') will be useful for a variety or therapeutic
applications, such as bacterial or viral infections. For example,
methods of the invention include treatment against infections and
diseases associated with viruses, which methods in general comprise
administration of a therapeutically effective amount of one or more
compounds of Formulae I, I', II, II', IIA or IIA' to virally
infected cells, such as mammalian cells, particularly human
cells.
[0064] More specifically, the invention includes methods of
treatment of a mammal susceptible to (prophylactic treatment) or
suffering from a disease associated with DNA and RNA viruses;
examples include viruses of the herpes family, e.g. herpes simplex
viruses (HSV) including herpes simplex 1 and 2 viruses (HSV 1, HSV
2), varicella zoster virus (VZV; shingles), human herpes virus 6,
cytomegalovirus (CMV), Epstein-Barr virus (EBV), and other herpes
virus infections such as feline herpes infections, and diseases
associated with hepatitis viruses including hepatitis B (HBV) and C
(HCV) viruses. Examples of clinical conditions which are caused by
such viruses include herpetic keratitis, herpetic encephalitis,
cold sores and genital infections (caused by herpes simplex),
chicken pox and shingles (caused by varicella zoster) and
CMV-pneumonia and retinitis, particularly in immunocompromised
patients including renal and bone marrow transplant and patients
with Acquired Immune Deficiency Syndrome (AIDS). Epstein-Barr virus
can cause infectious mononucleosis, and is also suggested as the
causative agent of nasopharyngeal, immunoblastic lymphoma and
Burkitt's lymphoma.
[0065] Compounds of the invention also will be useful for cancer
therapy, particularly to treat solid tumors, such as may be present
in the liver, lung, brain or other tissue.
[0066] Compounds of the invention also will be useful for treatment
against bacterial infections, including both Gram positive and Gram
negative bacteria, and mycobacteria.
[0067] Administration of compounds of the invention may be made by
a variety of suitable routes including oral, topical (including
transdermal, buccal or sublingal), nasal and parenteral (including
intraperitoneal, subcutaneous, intravenous, intradermal or
intramuscular injection) with oral or parenteral being generally
preferred. It also will be appreciated that the preferred method of
administration and dosage amount may vary with, for example, the
condition and age of the recipient.
[0068] Compounds of the invention may be used in therapy in
conjunction with other pharmaceutically active medicaments, such as
another anti-viral agent, or an anti-cancer agent. Additionally,
while one or more compounds of the invention may be administered
alone, they also may be present as part of a pharmaceutical
composition in mixture with conventional excipient, i.e.,
pharmaceutically acceptable organic or inorganic carrier substances
suitable for parenteral, oral or other desired administration and
which do not deleteriously react with the active compounds and are
not deleterious to the recipient thereof. Suitable pharmaceutically
acceptable carriers include but are not limited to water, salt
solutions, alcohol, vegetable oils, polyethylene glycols, gelatin,
lactose, amylose, magnesium stearate, talc, silicic acid, viscous
paraffin, perfume oil, fatty acid monoglycerides and diglycerides,
petroethral fatty acid esters, hydroxymethyl-cellulose,
polyvinylpyrrolidone, etc. The pharmaceutical preparations can be
sterilized and if desired mixed with auxiliary agents, e.g.,
lubricants, preservatives, stabilizers, wetting agents,
emulsifiers, salts for influencing osmotic pressure, buffers,
colorings, flavorings and/or aromatic substances and the like which
do not deleteriously react with the active compounds.
[0069] For parenteral application, particularly suitable are
solutions, preferably oily or aqueous solutions as well as
suspensions, emulsions, or implants, including suppositories.
Ampules are convenient unit dosages.
[0070] For enteral application, particularly suitable are tablets,
dragees or capsules having talc and/or carbohydrate carrier binder
or the like, the carrier preferably being lactose and/or corn
starch and/or potato starch. A syrup, elixir or the like can be
used wherein a sweetened vehicle is employed. Sustained release
compositions can be formulated including those wherein the active
component is protected with differentially degradable coatings,
e.g., by microencapsulation, multiple coatings, etc.
[0071] Therapeutic compounds of the invention also may be
incorporated into liposomes. The incorporation can be carried out
according to known liposome preparation procedures, e.g. sonication
and extrusion. Suitable conventional methods of liposome
preparation are also disclosed in e.g. A. D. Bangham et al., J.
Mol. Biol., 23:238-252 (1965); F. Olson et al., Biochim. Biophys.
Acta, 557:9-23(1979); F. Szoka et al., Proc. Nat. Acad. Sci.,
75:4194-4198 (1978); S. Kim et al., Biochim. Biophys. Acta,
728:339-348 (1983); and Mayer et al., Biochim. Biophys. Acta,
858:161-168 (1986).
[0072] It will be appreciated that the actual preferred amounts of
active compounds used in a given therapy will vary according to the
specific compound being utilized, the particular compositions
formulated, the mode of application, the particular site of
administration, etc. Optimal administration rates for a given
protocol of administration can be readily ascertained by those
skilled in the art using conventional dosage determination
tests.
[0073] All documents mentioned herein are incorporated herein by
reference.
[0074] The present invention is further illustrated by the
following examples. These examples are provided to aid in the
understanding of the invention and are not to be construed as
limitations thereof.
EXAMPLE 1
[0075] Preparation of the CPG-bound Phosphoramidite (Compound 2)
2a-d.
[0076] Each of the CPG-bound nucleosides 3a-d (3 g, 0.228 mmol),
along with bis-(N,N-diisopropylamino)-2-cyanoethylphosphine (0.36
mL, 1.14 mmol), bisdiisopropylammonium tetrazolide (850 mg) in
methylene chloride (50 mL) were placed in flasks and shaken at
30.degree. C. overnight. The CPG-bound phosphoramidite 2a-d was
collected by filtration and washed sequentially with
CH.sub.2Cl.sub.2 (200 mL), CH.sub.3CN (100 mL) and anhydrous ether
(50 mL) and dried in vacuo.
EXAMPLE 2
[0077] Synthesis of a 24 Member Library 1-6, a-d (Products 1a-x)
(See Scheme 1, Table 1).
[0078] Step 1: The CPG-bound phosphoramidites 2a-d (5 mmol) were
placed in spin columns. A solution of tetrazole (0.5 mL, 0.45 M in
CH.sub.3CN) was added. The appropriate alcohols 4a-f (25 mmol) were
added to the reaction mixture. The mixture was shaken for five
minutes and the solvent removed by centrifugation and decanted. The
CPG was then washed with CH.sub.3CN (2.times.0.5 mL).
[0079] Step 2: The CPG was then soaked in a solution of
3H-1,2-benzodithiole-3-one-1,1-dioxide in CH.sub.3CN (0.1 M, 0.5
mL). The mixture was shaken at room temperature for five minutes.
The solvent was removed and the CPG washed with acetonitrile
(2.times.0.5 mL) and dried under argon.
[0080] Step 3: The CPG was transferred to a conical screwcap tube
(1.5 mL, VWR) and ammonium hydroxide (28%, 1.5 mL) added. The
mixture was heated at 55.degree. C. for 4 hours. The suspension was
cooled and centrifuged. The supernatant containing the desired
products were collected in a speed vac to give 1-6, a-d (products
1a-x).
[0081] Typical spectral data are as follows:
[0082] Nucleoside 1a: .sup.1H NMR (D.sub.2O): .delta. 8.44 (d, 1H,
J=9.2 Hz), 8.21 (s, 1H), 6.44-6.47 (m, 1H), 4.24 (s, 1H), 3.96-4.06
(m, 2H), 3.55-3.64 (m, 2H), 2.81-2.88 (m, 1H), 2.57-2.62 (m, 1H),
1.30-1.35 (m, 2H), 1.04-1.09 (m, 2H), 0.67 (t, 3H, J.sub.1=7.5 Hz,
J.sub.2=7.3 Hz) ppm; .sup.31P NMR (D.sub.2O): .delta. 58.97, 58.64
ppm; MS (negative mode): calcd. for
C.sub.14H.sub.21N.sub.5O.sub.5PS, 402 (M); found m/z, 402.
[0083] Nucleoside 1b: .sup.1H NMR (D.sub.2O): .delta. 8.05 (d, 1H,
J=7.7 Hz), 6.24 (t, 1H, J=6.6 Hz), 6.10 (d, 1H, J=7.8 Hz),
4.50-4.53 (m, 1H), 4.19 (s, 1H), 4.03-4.12 (m, 2H), 3.84-3.88 (m,
2H), 2.40-2.45 (m, 1H), 2.24-2.29 (m, 1H), 1.51-1.57 (m, 2H),
1.25-1.33 (m, 2H), 0.83 (t, 3H, J.sub.1=7.5 Hz) ppm; .sup.31P NMR
(D.sub.2O):.delta. 58.96 ppm; MS (negative mode): calcd. for
C.sub.13H.sub.21N.sub.3O.sub.6PS, 378 (M); found m/z, 378.
[0084] Nucleoside 1c: .sup.1H NMR (D.sub.2O): .delta. 8.04 (d, 1H,
J=5.4 Hz), 6.24-6.28 (m, 1H), 4.19 (s, 1H), 3.96-4.05 (m, 2H),
3.63-3.68 (m, 2H), 2.78-2.85 (m, 1H), 2.47-2.52 (m, 1H), 1.34-1.41
(m, 2H), 1.07-1.13 (m, 2H), 0.71 (t, 3H, J.sub.1=7.5 Hz) ppm;
.sup.31P NMR (D.sub.2O): .delta. 58.91, 58.61 ppm; MS (negative
mode): calcd. for C.sub.14H.sub.21N.sub.5O.sub.6PS, 418 (M); found
m/z, 418.
[0085] Nucleoside 1d: .sup.1H NMR (D.sub.2O): .delta. 7.71 (s, 1H),
6.28 (t, 1H, J=7.0 Hz), 4.53-4.54 (m, 1H), 4.14-4.17 (t, 1H, J=2.5
Hz), 4.06-4.07 (m, 2H), 3.83-3.88 (m, 2H), 2.30-2.33 (m, 2H), 1.90
(d, 3H, J=4.4 Hz), 1.50-1.56 (m, 2H), 1.24-1.31 (m, 2H), 0.81 (t,
3H, J.sub.1=7.4 Hz) ppm; .sup.31P NMR (D.sub.2O): .delta. 58.32,
58.24 ppm; MS (negative mode): calcd. for
C.sub.14H.sub.22N.sub.2O.sub.7PS, 393 (M); found m/z, 393.
[0086] Nucleoside 1e: .sup.1H NMR (D.sub.2O): .delta. 8.44 (d, 1H,
J=10 Hz), 8.23 (s, 1H), 6.44-6.47 (m, 1H), 4.25 (s, 1H), 3.94-4.06
(m, 2H), 3.32-3.46 (m, 2H), 2.83-2.89 (m, 1H), 2.57-2.62 (m, 1H),
1.84-1.90 (m, 1H), 1.40-1.44 (m, 2H), 1.29-1.30 (m, 4H), 0.82-0.90
(m, 2H) ppm; .sup.31P NMR (D.sub.2O): .delta. 58.73, 58.28 ppm.
[0087] Nucleoside 1f: .sup.1H NMR (D.sub.2O): .delta. 8.7.94 (d,
1H, J=7.6 Hz), 6.23 (t, 1H, J=6.7 Hz), 6.03 (d, 1H, J=7.8 Hz), 4.51
(d, 1H, J=2.9 Hz), 4.17 (s, 1H), 4.03-4.08 (m, 2H), 3.67-3.70 (m,
2H), 2.38-2.42 (m, 1H), 2.20-2.25 (m, 1H), 2.06-2.12 (m, 4H), 1.61
(br, 1H), 1.45, 1.46 (2xS, 4H) 1.14-1.16 (m, 2H) ppm; .sup.31P NMR
(D.sub.2O): .delta..quadrature.58.79, 58.70 ppm.
[0088] Nucleoside 1g: .sup.1H NMR (D.sub.2O): .delta. 8.10 (d, 1H,
J=7.3 Hz), 6.24-6.27 (m, 1H), 4.19 (s, 1H), 3.97-4.04 (m, 2H),
3.38-3.52 (m, 2H), 2.79-2.86 (m, 1H), 2.48-2.53 (m, 1H), 1.89-1.94
(m, 1H), 1.45-1.50 (m, 2H), 1.34 (bs, 4H), 0.87-1.00 (m, 2H) ppm;
.sup.31P NMR (D.sub.2O): .delta. 58.64, 58.23 ppm.
[0089] Nucleoside 1h: .sup.1H NMR (D.sub.2O): .delta. 7.71 (s, 1H),
6.28 (t, 1H, J=7.0 Hz), 4.55-4.56 (m, 1H), 4.15 (d, 1H, J=2.2 Hz),
4.04-4.10 (m, 2H), 3.66-3.74 (m, 2H), 2.31-2.34 (m, 2H), 2.08-2.14
(m, 1H), 1.90 (d, 3H, J=5.5 Hz), 1.61-1.63 (m, 2H), 1.46-1.47 (m,
4H), 1.12-1.17 (m, 2H) ppm; .sup.31P NMR (D.sub.2O): .delta. 58.76,
58.61 ppm.
[0090] Nucleoside 1k: .sup.1H NMR (D.sub.2O): .delta. 8.06-8.08 (m,
1H), 6.24 (t, 1H, J=6.6 Hz), 6.11-6.13 (m, 1H), 5.26 (s, 1H),
4.50-4.53 (m, 1H), 4.18 (d, 1H, J=2.0 Hz), 4.05-4.12 (m, 2H),
3.78-3.88 (m, 2H), 2.41-2.45 (m, 1H), 2.22 -2.30 (m, 4H), 2.09-2.18
(m, 2H), 1.95-2.01 (m, 2H), 1.18 (s, 3H), 1.00 (d, 1H, 3.9 Hz),
0.71 (d, 3H, 2.0 Hz) ppm; .sup.31P NMR (D.sub.2O): .delta. 58.91,
58.67 ppm.
[0091] Nucleoside 1l: .sup.1H NMR (D.sub.2O): .delta. 7.74 (d, 1H,
J=6.7 Hz), 6.30 (t, 1H, J=7.0 Hz), 5.23 (s, 1H), 4.54 (d, 1H, J=2.1
Hz), 4.14 (s, 1H), 4.06-4.08 (m, 2H), 3.80-3.84 (m, 2H), 2.22-2.35
(m, 5H), 2.07 -2.17 (m, 2H), 2.00 (s, 1H), 1.86-1.92 (m, 4H), 1.24
(d, 1H, J=6.8 Hz), 1.16 (s, 3H), 1.09 (d, 1H, J=6.8 Hz), 0.97 (d,
1H, J=8.3 Hz), 0.69 (s, 3H) ppm; .sup.31P NMR (D.sub.2O): .delta.
58.91, 58.67 ppm.
[0092] Nucleoside 1n: .sup.1H NMR (D.sub.2O): .delta. 7.93 (d, 1H,
J=7.6 Hz), 6.22 (t, 1H, J=6.3 Hz), 6.01-6.03 (m, 1H), 5.70 (s, 1H),
4.48-4.50 (m, 1H), 4.22, 4.23 (2xs, 2H), 4.16 (d, 1H, J=1.9 Hz),
4.05-4.10 (m, 2H), 2.37-2.42 (m, 1H), 2.18-2.22 (m, 1H), 1.98 -2.06
(m, 4H), 1.81-1.87 (t, 1H, J=14.7 Hz), 1.66 (s, 3H), 1.30-1.35 (m,
1H) ppm; .sup.31P NMR (D.sub.2O): .delta. 59.69, 59.15 ppm.
[0093] Nucleoside 1p: .sup.1H NMR (D.sub.2O): .delta. 7.71 (d, 1H,
J=5.1 Hz), 6.27 (t, 1H, J=6.9 Hz), 5.71 (s, 1H), 4.54 (t, 1H, J=2.5
Hz), 4.25 (m, 2H), 4.15 (s, 1H), 4.08 (m, 2H), 2.29-2.33 (m, 2H),
2.00-2.07 (m, 4H), 1.86 -1.89 (m, 4H), 1.71-1.76 (m, 1H), 1.68 (s,
3H), 1.31-1.38 (m, 1H) ppm; .sup.31P NMR (D.sub.2O): .delta. 59.09,
59.00 ppm.
[0094] Nucleoside 1r: .sup.1H NMR (D.sub.2O): .delta. 7.95 (d, 1H,
J=7.6 Hz), 6.20-6.24 (q, 1H), 6.04-6.06 (dd, 1H, J=7.6 Hz),
5.96-5.99 (dd, 1H, J.sub.1=12.9 Hz, J.sub.2=3.5 Hz), 4.80-4.90 (dd,
1H), 4.48-4.50 (m, 1H), 4.32-4.46 (m, 2H), 4.14-4.18 (m, 2H),
4.04-4.10 (m, 2H), 3.95-3.98 (m, 1H), 2.40-2.46 (m, 1H), 2.17-2.25
(m, 1H), 1.89 (s, 4H), 1.45 (d, 3H, 9.2 Hz), 1.38 (d, 3H, 4.8 Hz),
1.28 (d, 3H, 2.5 Hz), 1.24 (d, 3H, 9.2 Hz) ppm; .sup.31P NMR
(D.sub.2O): .delta. 59.75, 59.24 ppm.
[0095] Nucleoside 1t: .sup.1H NMR (D.sub.2O ): .quadrature..delta.
7.70 (s, 1H), 6.28, 6.23 (2xt, 1H), 5.99, 5.96 (dd, 1H, J.sub.1=7.6
Hz, J.sub.2=2.6 Hz), 5.96-5.99 (dd, 1H, J.sub.1=12.9 Hz,
J.sub.2=3.5 Hz), 4.80-4.90 (2xd, 1H), 4.90, 4.81 (2xd, 1H),
4.52-4.54 (m, 1H), 4.33-4.47 (m, 2H), 4.04-4.17 (m, 4H), 3.96-3.99
(m, 1H), 2.24-2.42 (m, 2H), 1.91 (d, 3H, J=2.3 Hz), 1.86 (s, 3H),
1.44 (2xs, 3H, 9.2 Hz), 1.38 (2xs, 3H), 1.24-1.29 (m, 6H) ppm;
.sup.31P NMR (D.sub.2O): .delta. 59.75, 59.24 ppm.
[0096] Nucleoside 1v: .sup.1H NMR (D.sub.2O): .delta. 7.95 (d, 1H,
J=7.9 Hz), 6.25 (t, 1H, J=6.8 Hz), 6.04 (d, 1H, J=7.5 Hz),
4.52-4.55 (m, 1H), 4.42 (t, 1H, J=9.9 Hz), 4.18 (d, 1H, J=2.1 Hz),
2.38-2.43 (m, 1H), 2.12 -2.28 (m, 2H), 1.64-1.79 (m, 2H), 1.06-1.25
(m, 4H), 0.76-0.79 (m, 9H) ppm; .sup.31P NMR (D.sub.2O): .delta.
58.66, 58.61 ppm.
[0097] Nucleoside 1x: .sup.1H NMR (D.sub.2O): .delta. 7.73 (d, 1H,
J=5.6 Hz), 6.27 (t, 1H, J=6.8 Hz), 4.54-4.57 (m, 1H), 4.43 (t, 1H,
J=9.7 Hz), 4.14 (s, 1H), 4.06-4.09 (m, 2H), 2.31 -2.36 (m, 2H),
2.13-2.20 (m, 1H), 1.91 (s, 3H), 1.73-1.79 (m, 1H), 1.64-1.69 (m,
1H), 1.07-1.25 (m, 4H), 0.75-0.79 (m, 9H) ppm; .sup.31P NMR
(D.sub.2O): .delta. 58.52, 58.46 ppm.
EXAMPLE 3
[0098] Biological Testing of Inhibition of CMV
[0099] Selected compounds of the invention were tested against
human cytomeglovirus (HCMV). Briefly, a 96 well cell-based assay
was used with human foreskin infected with HCMV strain with an MOI
of 0.05 plaque forming units per ml. Each well was treated once
with a 25 micromolar dose of test compound. Five days following
treatment with the test compound, total cellular DNA was harvested
after cell lysis. Cell lysates were applied to a Nylon membrane on
a dot blot apparatus, the blots hybridized with a probe specific
for HCMV DNA, and the blots scanned and analyzed using Scan
analysis software. Tested compounds showed significant inhibition
of viral growth relative to control samples.
EXAMPLE 4
[0100] Biological Testing of Inhibition of HSV-1
[0101] Selected compounds of the invention were tested against
Herpes Simplex Virus Type 1 (HSV-1). Briefly, a 48 well cell-based
assay was used with vero cells infected with HSV-1 strain with an
MOI of 0.005 plaque forming units per ml. Each well was treated
once with a 25 micromolar dose of test compound at three hours post
infection. Two days following treatment with the test compound,
plaque reduction was determined to determine cytotoxic compounds.
Approximately 30% of the compounds tested were non-cytotoxic in
plaque reduction assays.
[0102] Cytotoxicity determinations of those compounds of the
invention which demonstrated cytotoxicity in plaque reduction
assays were then based on a 96 well cell-based assay MTT assays
(Sigma). Briefly, 24 h following cells seeded on plates, dilutions
of test compounds were added and then incubated for two days in 5%
CO2 at 37.degree. C. The MTT assay was then carried out as directed
by the manufacturer. Absorbance values were then read at 570 and
600 nm using a multiscan plate reader.
EXAMPLE 5
[0103] Biological Testing of Inhibition of HBV
[0104] Selected compounds of the invention (W198-10, W198-21,
W198-22, and W198-24) were tested against hepatits B virus (HBV):
37
[0105] Briefly, confluent 2.2.15 cell cultures infected with an HBV
strain were treated with 9 consecutive daily doses of four
concentrations of test compound. Extracellular virion HBV DNA
levels were followed 24 hours after the last treatment. The level
of HBV virion DNA in the 24 control (untreated) cultures in these
experiments was 114.+-.16 pg/ml culture medium.
[0106] Cells for toxicity analyses were cultured and treated with
four concentrations of the test compound. Uptake of neutral red dye
was used to determine the relative level of toxicity 24 hours
following the last treatment. The absorbance of internalized dye at
510 nm (A.sub.510) was used for the quantitative analysis. The
percentage of dye uptake in the control (untreated) cell cultures
was 100.+-.3.
[0107] Tested compounds showed significant inhibition of viral
growth relative to control samples, with no significant toxicity at
concentrations for antiviral activity used, as shown in Tables
3-5.
3TABLE 3 Selectivity Indexes of test compounds against HBV
replication. Selectivity Index Compound CC.sub.50 (.mu.M) EC.sub.50
(.mu.M) EC.sub.90 (.mu.M) (CC.sub.50/EC.sub.90) W198-10 >300 7.7
.+-. 0.9 24 .+-. 0.8 13 W198-21 >300 5.3 .+-. 0.6 19 .+-. 2.1 16
W198-22 >300 10 .+-. 1.1 32 .+-. 3.2 >9.4 W198-24 >300 12
.+-. 1.2 39 .+-. 4.1 >7.7
[0108]
4TABLE 4 Toxicity analysis of test compounds in 2.2.15 cells.
Neutral Red Dye Uptake at Indicated Drug Concentration (% of
control) Compound 300 .mu.M 100 .mu.M 30 .mu.M 10 .mu.M W198-10 98
.+-. 1 99 .+-. 1 102 .+-. 2 100 .+-. 1 W198-21 100 .+-. 2 100 .+-.
1 99 .+-. 1 99 .+-. 3 W198-22 103 .+-. 2 102 .+-. 2 101 .+-. 3 99
.+-. 2 W198-24 102 .+-. 1 103 .+-. 1 102 .+-. 2 100 .+-. 1
[0109]
5TABLE 5 Antiviral analysis of test compounds in 2.2.15 cells. HBV
Virion DNA Levels at indicated Drug Concentration (pg/ml culture)
Compound 10 .mu.M 1.0 .mu.M 0.1 .mu.M 0.01 .mu.M W198-10 46 .+-. 2
139 .+-. 10 121 .+-. 10 127 .+-. 10 W198-21 35 .+-. 4 117 .+-. 12
121 .+-. 13 137 .+-. 14 W198-22 59 .+-. 2 152 .+-. 11 122 .+-. 13
129 .+-. 13 W198-24 62 .+-. 3 149 .+-. 25 133 .+-. 12 140 .+-.
15
[0110] The invention has been described in detail with reference to
preferred embodiments thereof. However, it will be appreciated that
those skilled in the art, upon consideration of this disclosure,
may make modifications and improvements within the spirit and scope
of the invention as set forth in the following claims.
* * * * *