Note: Descriptions are shown in the official language in which they were submitted.
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SYNTHESIS AND ANTIVIRAL EVALUATION OF
NUCLEIC ACID BASED (NAB) LIBRARIES
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to the design and synthesis of focused nucleic acid
based
(NABT"') libraries with different diversity attributes. More specifically
these libraries are
useful for their antiviral activity and for rapidly identifying novel
molecular targets for
antiviral intervention.
2. Background
The discovery of safe and effective antiviral drugs presents a formidable
challenge
compared to bacterial and parasitic agents. Indeed, very few virus-specific
molecular targets
have been identified that can be specifically subjected to antiviral
intervention, because viral
metabolic processes closely resemble host cellular processes. Nevertheless,
three virus-
encoded enzymes have been the targets of most "small molecule-type" antiviral
drugs -
polymerases, proteases, and most recently, neuraminidase. However, the rapid
emergence of
resistance to antiviral drugs is a major problem and unwieldy "cocktail
regimens" often need
to be employed as a desperate measure. Clearly, there exists a substantial
unmet clinical need
for antiviral drugs with different structures and unique mechanisms of
aetion.z
Historically, antiviral drugs have been designed using both mechanism, and
structure-
based drug design approaches. The combinatorial methodology is emerging as a
powerful
contemporary drug discovery tool,3 and consists of two steps in which: (a)
biological
methods are used to select and validate molecular targets, and (b) structure-,
and mechanism-
guided drug design approaches are used, in which a library of compounds are
synthesized and
evaluated for their ability to interfere with the biosynthesis, structure
and/or function of the
target. When insufficient structure and/or function of a target is available,
combinatorial
methods such as "diversity oriented organic synthesis for therapeutic target
validation"4 or
alternatively "combinatorial target-guided ligand assembly" have been
employed.'
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2
The growing understanding of the molecular biology and biochemistry of viral
replication as well as of related protein structure has facilitated the
development of potent and
specific antiviral drugs. Many nucleoside analogs have been found to have
effective and
specific antiviral activity in the past two decades, and a number of compounds
in this class
have enjoyed considerable clinical and commercial success for the treatment of
viral diseases.
The primary mechanism for the antiviral drug action of the nucleoside analogs
was by acting
as the chain terminators or as competitive inhibitors of viral-related enzymes
or both; recently
an alternative strategy has been the development of agents that act in their
own right at an
allostatic site as noncompetitive inhibitors.
An approach that seems appropriate for antiviral drug discovery, is the use of
structurally diverse compounds to modulate biological pathway without regard
to specific
molecular target. This allows simultaneous functional validation of a target,
as well as, the
discovery of a lead structure that modulates the function of the target. We
describe here the
application of this concept for antiviral drug discovery.
SUMMARY OF THE INVENTION
The present invention provides for compositions and methods to generate a
library of
small molecules that mimic the repertoire of interactions that exist amongst
nucleic acids, and
proteins, as well as, that which exist between proteins and nucleic acids.
Indeed, a number of
proteins contain nucleotide-binding domains defined by the topology of protein
a-helices and
(3-sheets. More specifically, a nucleic acid-based (NABT"') scaffold is
described into which
such diversity attributes are engineered to potentially target "hot spots" in
protein-protein,
and protein-nucleic acid interaction surfaces. As an illustrative example of
an embodiment of
the invention, we report here the synthesis and antiviral evaluation ofNABT"'
libraries
against herpes simplex virus (HSV-1) using cell-based assays.
The invention provides for drug discovery in anti infectious agent
therapeutics, by
constructing libraries of nucleoside analogs in which a variety of functional
and structural
elements are attached to the nucleic acid-based (NABTM) scaffold through
proper linkers.
This pharmacore-based chemical class is intended to act as the competitive or
noncompetitive
viral replication inhibitor or serve as a prodrug of inhibitors.
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The advantages of the present system are many. For example, there are many
novel
diversity features associated with libraries bui_It around a NABTM scaffold:
(a) the scaffold
can be used to create variable spatial display of hydrogen-bonding,
hydrophobic, charge-
transfer, electrostatic and such other non-covalent interactions; (b) the
scaffold can be
conformationally rigid or flexible and by linking the individual scaffolds
together, one can
fashion diverse molecular topology into library members; indeed, such shape-
defining motifs
as circles, pseudoknots, bulges and stem loops can be incorporated into
libraries to target "hot
spots on the receptor; (c) From a synthetic perspective, the NABT"' libraries
can be assembled
using well established solid-phase or solution-phase methods in nucleic acids
field.'
The exquisite specificity of such interactions reside in topology-associated
molecular
recognition defined by local and global conformations of the ligand and
receptor, and a
network of hydrogen bonding, hydrophobic, ionic, and van der Waals
interactions that
facilitate the binding interactions.6
Examples of suitable compounds to generate a compound library are disclosed.
For
example, a compound library comprising two or more compounds of the following
Formula I
or I':
R~ R
B B
1 Ir
wherein L' and LZ are independently O or a linking group such as e.g. an
amide, ester,
diester or the like, or an optionally substituted alkylene (e.g. C~_zo
alkylene), optionally
substituted alkenylene (e.g., CZ_zo alkenylene) or alkynylene (e.g., C2_ZO
alkynylene) having
such groups either as a chain member of pendant to the chain, and which may be
optionally
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substituted with one or more substituents selected from a group consisting of
O, S, Se,
NR'NRz, CR'CR2, OR, SR and SeR, or an enzymatically reactive;
Q is carbon or a heteroatom such as O, S or N;
R', Rz, R3 are each independently a 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
I to 3 N, O or
S atoms;
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.
in another preferred embodiment, the compound library is comprised of two or
more
compounds of the following Formula 11 or LI':
HO HO O B
O OR3
II . ~ II~
wherein X and Y are each independently selected from a group consisting of O,
S, Se,
NR'NR2, CR'CRZ, OR, SR and SeR, or one or both of X and Y are an enzymatically
reactive
moiety;
R is hydrogen or an optionally substituted alkyl, optionally substituted
alkenyl,
optionally substituted alkynyl, optionally substituted aralkyl, optionally
substituted
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5 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~, RZ and R3 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;
and pharmaceutically acceptable salts thereof.
In another preferred embodiment, at least one of the compounds that comprise
the
compound library, has at least one furanose ring in the C-2' endo conformation
or has at
least one furanose ring in the C-3' endo conformation.
In another preferred embodiment, at least one of the compounds that comprise
the
compound library, has at least one furanose ring in the C-2' endo conformation
and at least
one furanose ring in the C-3' endo conformation.
In another preferred embodiment, the compound library is comprised of
compounds
having at least about I to 50 nucleoside residues, more preferably at least
about I to 10
nucleoside residues, most preferably at least about 1 to 5 nucleoside
residues.
In other preferred embodiments, the compound library is comprised of compounds
having at least about 4 nucleoside residues, or at least about 3 nucleoside
residues, or at least
about 2 nucleoside residues, or at least about 1 nucleoside residue.
In another preferred embodiment at least one compound of the following formula
III
or III' of the compound library is comprised of:
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6
HO HO
III R III'
wherein X and Y are each independently selected from a group consisting of O,
S, Se,
NR~NR2, CR~CRZ, OR, SR and SeR, or one or both ofX and Y are an enzymatically
reactive
moiety;
R is hydrogen 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 I to 3 N, O or S atoms;
R', Rz and R3 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 I 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; and pharmaceutically
acceptable salts
thereof.
In one preferred embodiment, the compound library is comprised of at least one
compound which has a sugar group in open chain form and wherein an
enantiomerically
enriched mixture of a compound is present.
A method is provided for construction of the compound library using solid-
phase
synthesis or solution-phase synthesis. In brief, one or more reagents are
added to a reaction
vessel capable of agitation and containing a resin reaction support material.
The reaction
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7
vessel is agitated during reaction of the reagents, and then centrifuging the
reaction vessel to
remove desired reaction materials therefrom. The library_may also be
constructed using an
automated solution-phase synthesis.
In one preferred embodiment the compound library is used to identify a
specific
interacting partner for a nucleic acid or a protein. The nucleic acid can be
for example, RNA
or DNA. Examples of proteins are antibodies, receptors or ligands.
Compositions and methods are also provided for the treatment of cells or
mammals
infected with viruses, for example, cells infected with a herpes virus, cells
infected with a
cytomegalovirus; or cells infected with bacteria.
In a preferred embodiments, the compositions ofthe present invention are
useful for
identifying specific inhibitors for viral kinases, viral polymerases,
identifying specific
compounds which cause disruption of the association between a helicase-primase
complex
and a viral nucleic acid to which it is bound. The compositions are useful in
treating a
mammal suffering from or susceptible to a fungal, viral, or a bacterial
infection.
Particularly preferred viral organisms causing human diseases according to the
present invention include (but not restricted to) Herpes viruses,
Hepatitisviruses,
Retroviruses, Orthomyxoviruses, Paramyxoviruses, Togaviruses, Picornaviruses,
Papovaviruses and Gastroenteritisviruses.
Particularly preferred bacteria causing serious human diseases are the Gram
positive
organisms: Staphylococcus az~reus, Staphylococcus epidermidis, Enterococcus
faecali.s and
E. faecium, Streptococcus pneumoniae and the Gram negative organisms:
Pseudoznonas
aeruginosa, Burkholdia cepacia, Xanthomonas maltophila, E.scherichia coli,
Enterobacter
.spp, Klebsiella pneumoniae and Salmonella .spp.
Particularly preferred protozoan organisms causing human diseases according to
the
present invention include (but not restricted to) Malaria e.g. Plasmodium
falciparum and M.
ovale, Trypanosomiasis (sleeping sickness) e.g. Trypanosoma cruzei,
Leischmaniasis e.g.
Leischmania donovani, Amebiasis e.g. Entamoeba histolytica.
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Particularly preferred fungi causing human diseases according to the present
invention
include (but not restricted to) Candida albicans, Histoplasma neoformans,
Coccidioides
immitis and Penicillium marneffei.
The methods of treatment comprise administering to a mammal susceptible to or
suffering from an infectious disease, a therapeutically effective amount of a
compound. The
compound may be administered alone or in combination with a pharmaceutically
acceptable
carrier.
Preferred compounds comprising compositions used for the treatment of cells or
mammals susceptible to or suffering from an infectious disease causing agent
such as, for
example, viruses such as herpes virus, or cytomegalovirus; bacteria, fungi and
the like are,
are preferably ofthe following Formula I or I':
25
R3 3
R
B
j 1'
wherein L' and LZ are independently O or a linking group such as e.g. an
amide, ester,
diester or the like, or an optionally substituted alkylene (e.g. C,_ZO
alkylene), optionally
substituted alkenylene (e.g., CZ_ZO alkenylene) or alkynylene (e.g., CZ_ZO
alkynylene) having
such groups either as a chain member of pendant to the chain, and which may be
optionally
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substituted with one or more substituents selected from a group consisting of
O, S, Se,
NR~NR2, CR~CR2, OR, SR and SeR, or an enzymatically reactive;
Q is carbon or a heteroatom such as O, S or N;
R~, R2, R3 are each independently a 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;
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.
The compound preferably is comprised of a sugar group, wherein the sugar group
is
in open chain form and an enantiomerically enriched mixture of a compound is
present.
Other preferred compounds, include compounds ofthe following Formula I1 or
II':
HO B HO
II ~ II'
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5 wherein X and Y are each independently selected from a group consisting of
O, S, Se,
NR~NR2, CR~CR2, OR, SR and SeR, or one or both of X and Y are an enzymatically
reactive
moiety;
R is hydrogen or an optionally substituted alkyl, optionally substituted
alkenyl,
optionally substituted alkynyl, optionally substituted aralkyl, optionally
substituted
10 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~, Rz and R3 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; and pharmaceutically
acceptable salts
thereof.
Other preferred compounds also include compounds of the following Formula IIT
or
III'
HO B HO
Y____I____X ____ ____
Y ~ X
III R III'
wherein X and Y are each independently selected from a group consisting of O,
S, Se,
NR~NR2, CR'CRZ, OR, SR and SeR, or one or both of X and Y are an enzymatically
reactive
moiety;
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11
R is hydrogen 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', RZ and R3 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; and pharmaceutically
acceptable salts
thereof.
The methods of the invention are preferably employed for treatment or
prophylaxis
against diseases caused by infectious agents, particularly for treatment of
infections as may
occur in tissues or organs of a subject. The methods of the invention also may
be employed
to treat systemic conditions such as viremia or septicemia. The methods of the
invention are
also preferably employed for treatment of diseases and disorders associated
with viral
infections or bacterial infections, as well as any other disorder caused by an
infectious agent.
The methods of the invention are also preferably employed for identifying
molecules that
interfere with the life cycle or biological pathways of an infectious disease
causing agent.
Other aspects of the invention are disclosed infra.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure I illustrates the general structure of the chemical library built
around the
NABTM scaffold. Two types of functional variants P2 and P4 were attached to
the furanose
scaffold Pl, providing divergent target recognition elements such as hydrogen
bonding,
lipophilicity, "shape-in-space", and electronic properties. The P3 elements
are served as
structural variants to restarin the furanose scaffold into certain
conformations, thereby
enabling overal changes in three dimensional projection for the functional
elements.
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Figure 2 shows the general structure of the libraries 1 and 2, with the
functional R
groups attached to the 5'- and 3'-ends of the nucleoside respectively. For
description of R, B,
and Z, see Figure 6 and Table 7.
Figure 3 shows the energy-favorable conformations of the furanose puckers
resulting
from the 2'-substitutions. The changes in the furanose puckers cause
dramatically different
projections in three-dimensional space for the functional groups attached to
the furanose
scaffold.
Figure 4 shows the chemical structures of the representative members of
library 2.
The 2'-deoxynucleotide analogs are listed in Panel A, and the 2'-O-
methylnucleotide analogs
are listed in Panel B.
Figure 5 shows the HPLC (C 18, at 260 nm, Panel A) and 3~P NMR (in DZO, Panel
B)
profiles of the representative members of library 2. The chemicals are
presented as
diastereomeric mixture of phosphorothioates. The purity of the products is in
general higher
than 95%.
Figure 6 is a schematic illustration of the generation of classes of libraries
with
varying diversities. (i) 1H-Tetrazole in CH3CH; (ii) 3H-1,2-benzodithiole-3-
one-1,1-dioxide
in CH3CN; (iii) partition between 2% NaHC03 and EtOAc; (iv) 28% NH40H; (v)
Dowex H';
(vi) partition between CHC13 and H20.
Figure 7 shows the general structure of a simple NAB library depicting
diversity
elements.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides for a library that possesses key diversity
attributes.
The libraries are comprised of di-, tri- and tetranucleotides that carry
modifications at the
backbone, sugar, and nucleobase as illustrated in figure I.
Important elements that contribute to diversity in the present invention
include, but
not limited to, backbone modifications such as for example, phosphorothioates
and
phosphoramidates which provide desirable metabolic stability to compounds when
used in
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13
cell-based assays - the former also could potentially participate in
electrostatic interactions,
while the latter could facilitate hydrophobic and hydrogen-bonding
interactions with the
target receptor; dominant furanose modifications, such as for example,
substitution of a 2'-
OMe group in place of a 2'-hydrogen in the deoxyribofuranoside ring, a 2'-
substituent which
acts as a "conformational switch", that transforms the furanose ring pucker
from the 2'-endo
to 3'-endo thereby affecting the global conformation of the individual library
members;
nucleobase modifications, which, for example can, include both the replacement
of the parent
heterocyclic moiety, as well as, substitution on the heterocycle, providing
additional
hydrophobicity to the library members; linkages between the different moieties
are, for
example, 3' to 5'. In this way a repertoire of diversity attributes can be
captured in a
representative NABT"' library with a molecular weight range of about 400 to
about 1200.
There are many novel diversity features associated with libraries built around
a
NABTM scaffold: (a) the scaffold can be used to create variable spatial
display of hydrogen-
bonding, hydrophobic, charge-transfer, electrostatic and such other non-
covalent interactions;
(b) the scaffold can be conformationally rigid or flexible and by linking the
individual
scaffolds together, one can fashion diverse molecular topology into library
members; indeed,
such shape-defining motifs as circles, pseudoknots, bulges and stem loops can
be
incorporated into libraries to target "hot spots on the receptor; (c) From a
synthetic
perspective, the NABT"' libraries can be assembled using well established
solid-phase or
solution-phase methods in nucleic acids field. An illustrative example of a
small molecule-
type NABTM library is shown in figure 7, and represents multiple elements of
diversity.
"Hot spots" as used herein refers to the high and low affinity binding sites
on a
receptor.
As used herein, "pseudoknots" refers to a three dimensional spatial
configuration in
the nucleic acid based scaffold that is formed by the inclusion of molecules
wherein the
molecular interactions, such as a network of hydrogen bonding, hydrophobic,
ionic, and van
der Waals interactions that facilitate the binding interactions between the
molecules, resulting
in a twist in the scaffold forming a "knot-like" structure.
Also described is a straightforward and facile solution-phase chemistry which
has
been developed to carry out the parallel combinatorial synthesis, from which
libraries of 3'-
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14
modified nucleoside analogs 2 were generated as individual compounds in high
purity. See
the Examples which follow.
As used herein, "nucleoside" includes the natural nucleosides, including 2'-
deoxy and
2'-hydroxyl forms, e.g., as described in Kornberg and Baker, DNA Replication,
2nd Ed.
(Freeman, San Francisco, 1992). For an excellent treatise on nucleic acids
chemistry, see:
Sanger, W. Principles of Nucleic Acids Structure. Springer- Verlag: New York,
1984.
"Analogs" in reference to nucleosides includes synthetic nucleosides having
modified
base moieties and/or modified sugar moieties, e.g., described generally by
Scheit, Nucleotide
Analogs, John Wiley, New York, 1980; Freier & Altmann, Nucl. Acid Res., 1997,
25(22),
4429-4443, Toulme, J.J., Nature Biotechnology 19:17-18 (2001); Manoharan M.,
Biochemica
et Biophysica Acta 1489:117-I 39( 1999); Freier S.,M., Nucleic Acid Research,
25:4429-4443
(1997), Uhlman, E., Drug Discovery & Development, 3: 203-213 (2000), Herdewin
P.,
Anti.sense & Nucleic Acid Drug Dev., 10:297-310 (2000), ); 2'-O, 3'-C-linked
[3.2.0]
bicycloarabinonucleosides (see e.g. N.K Christiensen., et al, J. Am. Chem.
Soc., 120: 5458-
5463 (1998). Such analogs include synthetic nucleosides designed to enhance
binding
properties, e.g., duplex or triplex stability, specificity, or the like.
The term "stability" in reference to duplex or triplex formation generally
designates
how tightly an antisense oligonucleotide binds to its intended target
sequence; more
particularly, "stability" designates the free energy of formation of the
duplex or triplex under
physiological conditions. Melting temperature under a standard set of
conditions, is a
convenient measure of duplex and/or triplex stability.
A compound library comprised of a nucleic acid based (NABTM) scaffold
typically
refers to a library of small molecules that mimic the repertoire of
interactions that exist
amongst nucleic acids and proteins, as well as that which exist between
proteins and nucleic
acids. For example, a number of proteins contain nucleotide-binding domains
defined by the
topology of protein a-helices and (3-sheets. For example, polymerases,
topoisomerases, p53
protein and the like.
An example of molecules which can mimic nucleic acid interactions are DNA
mimics
which are polymers composed of subunits capable of specific, Watson-Crick-like
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5 hybridization with DNA, or of specific hybridization with RNA. The nucleic
acids can be
modified at the base moiety, at the sugar moiety, or at the phosphate
backbone. Exemplary
DNA mimics include, e.g., phosphorothioates.
In accordance with the present invention, generation of compound libraries,
described
10 in detail in the material and methods and examples which follow, allow for
the identification
of interactions between nucleic acids and proteins, of an organism, for
example, infectious
disease causing agents and the biological pathways involved in replication of
an infectious
agent, inhibiting a virus-specific enzyme, such as viral thymidine kinase and
reverse
transcriptase, receptor-ligand interactions that allow an infectious agent to
infect a cell or
15 mammal; molecules that allow infection of mammals by disease causing
agents; and the like.
Based on the identification of the above-said interactions, drugs can be
designed which
inhibit the desired mechanism of pathogenesis of a disease causing agent.
As used herein, a biological pathway includes a collection of cellular
constituents that
influence one another through any biological mechanism, known or unknown, such
as by a
cell's synthetic, regulatory, homeostatic, or control networks. The influence
of one cellular
constituent on another can be, inter alia, by a synthetic transformation of
the one cellular
constituent into the other, by a direct physical interaction of the two
cellular constituents, by
an indirect interaction of the two cellular constituents mediated through
intermediate
biological events, or by other mechanisms. Further, certain pathways that are
of particular
interest in this invention can be said to originate at particular cellular
constituents, which
influence, but are not in turn influenced by, the other cellular constituents
in the pathway and
among such pathways, those without feedback loops are said to be hierarchical.
A feedback
loop in a biological pathway is a subset of cellular constituents of the
pathway, each
constituent of the feedback loop influences and also is influenced by other
constituents of the
feedback loop. Infectious disease causing agents may interfere with such
biological
pathways or make use of biological pathways for survival and replication in a
host organism.
For example, HIV stimulates the CD4+ T cell's production of certain cytokines.
Examples of biological pathways, as understood herein, are well known in the
art.
They depend on various biological mechanisms by which the cellular
constituents influence
one another. Biological pathways include well-known biochemical synthetic
pathways in
which, for example, molecules are broken down to provide cellular energy or
built up to
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16
provide cellular energy stores, or in which protein or nucleic acid precursors
are synthesized.
The cellular constituents of synthetic pathways include enzymes. and the
synthetic
intermediates, and the influence of a precursor molecule on a successor
molecule is by direct
enzyme-mediated conversion. Biological pathways also include signaling and
control
pathways, many examples of which are also well known. Cellular constituents of
these
pathways include, typically, primary or intermediate signaling molecules, as
well as the
proteins participating in the signal or control cascades usually
characterizing these pathways.
In signaling pathways, binding of a signal molecule to a receptor usually
directly influences
the abundances of intermediate signaling molecules and indirectly influences
on the degree of
phosphorylation (or other modification) of pathway proteins. Both of these
effects in turn
influence activities of cellular proteins that are key effectors of the
cellular processes initiated
by the signal, for example, by affecting the transcriptional state of the
cell. Control pathways,
such as those controlling the timing and occurrence of the cell cycle, are
similar. Here,
multiple, often ongoing, cellular events are temporally coordinated, often
with feedback
control, to achieve a consistent outcome, such as cell division with
chromosome segregation.
This coordination is a consequence of functioning of the pathway, often
mediated by mutual
influences of proteins on each other's degree of phosphorylation (or other
modification).
Also, well known control pathways seek to maintain optimal levels of cellular
metabolites in
the face of a fluctuating environment. Further examples of cellular pathways
operating
according to understood mechanisms will be known to those of skill in the art.
Infectious
disease causing agents may influence the biological pathways in any number of
ways, such as
for example, downregulation or upregulation of certain constituents of the
biological
pathway, etc.
As used herein, the term "infectious agent" or "infectious disease causing
agent",
refers to an organism wherein growth/multiplication leads to pathogenic events
in humans or
animals. Examples of such agents are: bacteria , fungi, protozoa and viruses.
The compound libraries are also useful in identifying interactions between
molecules
involved in autoimmune diseases such as diabetes or autoimmune diseases caused
by viruses
which mimic molecules of the immune system of a mammal, thereby developing
drugs which
inhibit autoimmune interactions.
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According to the invention, a preferred compound library comprises two or more
compounds of the following Formula I or I':
R3 3
R
B
wherein L' and L2 are independently O or a linking group such as e.g. an
amide, ester,
diester or the like, or an optionally substituted alkylene (e.g. C~_zo
alkylene), optionally
substituted alkenylene (e.g., CZ_ZO alkenylene) or alkynylene (e.g., Cz_ZO
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'NRz, CR~CR2, OR, SR and SeR, or an enzymatically reactive;
Q is carbon or a heteroatom such as O, S or N;
R~, R2, R3 are each independently a 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;
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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 I to 3 separate or fused rings and I to 3 N, O or S atoms, or a
heterocyclic
structure that is covalently linked to the sugar ring; n=I to 5; and
pharmaceutically acceptable
salts thereof.
Sugar groups of the nucleic acid base scaffold may be natural or modified
(e.g.
synthetic) and in an open chain or ring form. Sugar groups 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 a or (3 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 ~
4, 1 ~ 6 or
a mixture of 1 ~ 4 and 1 ~ 6 linkages. The sugar moiety may be attached to the
link group
through any of the hydroxyl or amino groups of the carbohydrate.
Preferred modifications to the sugar include modifications to the 2' position
of the
ribose moiety which include but are not limited to 2'-O-substituted with an -O-
lower alkyl
group containing 1-6 saturated or unsaturated carbon atoms, or with an -O-
aryl, or allyl group
having 2-6 carbon atoms wherein such -O-alkyl, aryl or allyl group may be
unsubstituted or
may be substituted (e.g., with halogen, hydroxy, trifluoromethyl, cyano,
vitro, acyl,~acyloxy,
alkoxy, carboxy, carbalkoxyl, or amino groups), or wherein the 2'-O-group is
substituted by
an amino, or halogen group. None of these substitutions are intended to
exclude the native
2'-hydroxyl group in case of ribose or 2'-H- in the case of deoxyribose.
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Heteroaliphatic moieties may be branched, unbranched or cycl(c and include
heterocycles such as morpholino, pyrrolidinyl, etc.
The term "heterocycle" as used herein refers to cyclic heteroaliphatic groups
and
preferably three to ten ring atoms total, includes, but is not limited to,
oxetane,
tetrahydrofuranyl, tetrahydropyranyl, aziridine, azetidine, pyrrolidine,
piperidine,
morpholine, piperazine and the like.
The terms "aryl" and "heteroaryl" as used herein refer to stable mono- or
polycyclic,
heterocyclic, polycyclic, and polyheterocyclic unsaturated moieties having 3-
14 carbon atom
which may be substituted or unsubstituted. Substituents include any of the
previously
mentioned substituents. Non-limiting examples of useful aryl ring groups
include phenyl,
halophenyl, alkoxyphenyl, dialkoxyphenyl, trialkoxyphenyl,
alkylenedioxyphenyl, naphthyl,
phenanthryl, anthryl, phenanthro and the like. Examples of typical heteroaryl
rings include 5-
membered monocyclic ring groups such as thienyl, pyrrolyl, imidazolyl,
pyrazolyl, furyl,
isothiazolyl, furazanyl, isoxazolyl, thiazolyl and the like; 6-membered
monocyclic groups
such as pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, triazinyl and the like;
and polycyclic
heterocyclic ring groups such as benzo[b]thienyl, naphtho[2,3-b]thienyl,
thianthrenyl,
isobenzofuranyl, chromenyl, xanthenyl, phenoxathienyl, indolizinyl,
isoindolyl, indolyl,
indazolyl, purinyl, isoquinolyl, quinolyl, phthalazinyl, naphthyridinyl,
quinoxalinyl,
quinazolinyl, benzothiazole, benzimidazole, tetrahydroquinoline cinnolinyl,
pteridinyl,
carbazolyl, beta-carbolinyl, phenanthridinyl, acridinyl, perimidinyl,
phenanthrolinyl,
phenazinyl, isothiazolyl, phenothiazinyl, phenoxazinyl, and the like (see e.g.
Katritzky,
Handbook of Heterocyclic Chemistry). The aryl or heteroaryl moieties may be
substituted
with one to five members selected from the group consisting of hydroxy, Cl-C8
alkoxy, C1-
C8 branched or straight-chain alkyl, acyloxy, carbamoyl, amino, N-acylamino,
nitro, halo,
trihalomethyl, cyano, and carboxyl. Aryl moieties thus include, e.g. phenyl;
substituted
phenyl bearing one or more substituents selected from groups including: halo
such as chloro
or fluoro, hydroxy, C1-C6 alkyl, acyl, acyloxy, C 1-C6 alkoxy (such as methoxy
or ethoxy,
including among others dialkoxyphenyl moieties such as 2,3-, 2,4-, 2,5-, 3,4-
or 3,5-
dimethoxy or diethoxy phenyl or such as methylenedioxyphenyl, or 3-methoxy-5-
ethoxyphenyl; or trisubstituted phenyl, such as trialkoxy (e.g., 3,4,5-
trimethoxy or
ethoxyphenyl), 3,5-dimethoxy-4-chloro-phenyl, etc.), amino, --SOZ NH2, --
SOZNH(aliphatic),
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5 --SOzN(aliphatic)z, --O-aliphatic--COON, and --O--aliphatic-NHZ (which may
contain one or
two N-aliphatic. or N-acyl substituents).
According to the present invention it is also preferred that the library is
comprised of
an enantiomerically enriched mixture of at least about one type of a compound
illustrated by
10 formula I or I' is present.
Other preferred compound libraries comprise two or more compounds of the
following Formula II or II':
15 Ho / B g
R3
,3
,~R II R II'
wherein X and Y are each independently selected from a group consisting of O,
S, Se,
NR'NR2, CR'CR2, OR, SR and SeR, or one or both of X and Y are an enzymatically
reactive
moiety;
R is hydrogen 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 I to 3 N, O or S atoms;
R', RZ and R3 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
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structure that is covalently linked to the sugar ring; and pharmaceutically
acceptable salts
thereof.
In accordance with the present invention, preferred compounds that comprise
the
compound library include at least about one compound of the following formula
III or III':
Ho HO O B
OR3
III ~ III'
wherein X and Y are each independently selected from a group consisting of O,
S, Se,
NR~NRz, CR~CRz, OR, SR and SeR, or one or both of X and Y are an enzymatically
reactive
moiety;
R is hydrogen 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 I to 3
separate or fused ring and 1 to 3 N, O or S atoms;
Rl, RZ and R3 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 I to 3 N, O or S atoms, or a
heterocyclic
structure that is covalently linked to the sugar ring; and pharmaceutically
acceptable salts
thereof.
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According to the present invention, the library is comprised of at least about
one
compound having at least about one furanose ring in the C-2' endo conformation
or at least
about one furanose ring in the C-3' endo conformation. Also preferred is a
library comprised
of compounds having at least one about furanose ring in the C-2' endo
conformation and at
least about one furanose ring in the C-3' endo conformation.
_
Preferably the compound library is comprised of compounds having a length of
at
least about 1 to about 50 nucleoside residues, more preferably the compounds
have a length
of at least about 1 to 10 nucleoside residues, most preferably the compounds
have a length of
at least about 1 to 5 nucleoside residues.
According to the present invention the library may be comprised of compounds
that
have at least about 1 nucleoside residue, or at least about 2 nucleoside
residues, or at least
about 3 nucleoside residues, or at least about 4 nucleoside residues.
The library can be constructed using techniques well-known to those of skill
in the
art. For example, the library is constructed using solid-phase synthesis or
solution-phase
synthesis. For a selected review of solid-phase combinatorial chemistry, see:
Houghten, R.
A.; Pinilla, C.; Appel, J. R.; Blondelle, S. E.; Dooley, C. T.; Eichler, J.;
Nefzi, A.; Ostresh, J.
M. J. Med Chem. 1999, 42, 3743-3778. For selected reviews of solution-phase
combinatorial
chemistry, see: (a) Baldino, C. M. J. Comb. Chem. 2000, 2, 89-103; (b) Boger,
D. L.;
Goldberg, J. Multiple solution-phase; combinatorial chemistry, In Combintorial
Chemistry: A
Practical Approach, Vol. 2; Fenniri, H., Ed.; Oxford University Press: Oxford,
1999. (c)
Gayo, L.M. Biotechnol. Bioeng. 1998, 61, 95-106.
According to the present invention, a facile parallel solution-phase chemistry
has been
developed to carry out the combinatorial synthesis of nucleoside analogs as a
source of
pharmacophore-based chemical diversity. The reactions adapted in the synthetic
cycle are
performed at room temperature in high yields in a one-pot reaction system,
except for the
final deprotection steps. An efficient and convenient liquid/liquid extraction
strategy
partitioning between organic solvents and water has been tailored to
successfully address the
challenge of intermediate/product purification often associated with the
application of
solution-phase combinatorial chemistry. The library members thus obtained are
present as
individual compounds with purity higher than 90%.
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As used herein, "pharmacophores-based diversity" are compounds representative
of
the same and/or closely related diversity based on physical and chemical
properties of
molecules with similar characteristics to a previously identified molecule or
pharmacophore.
The similarity principle requires that for any pair of molecules, differences
in activity are
related to differences in structure.
Various methods can be utilized to determine properties of compounds suitable
for
use in a compound library, for example US Patent No: 6,240,374, which is
incorporated
herein in its entirety. The reference makes use of the following measures: 2
dimensional and
3 dimensional measures. As used herein, "2 dimensional and 3 dimensional
measures" shall
mean a molecular representation which includes any terms which specifically
incorporate
information about the two dimensional and three dimensional features of the
molecule.
These measures take into account the geometric features of a molecule and also
reflect the
properties which are derivable from its topology; that is, the network of
atoms connected by
bonds.
As used herein, "2 dimensional fingerprints" means a 2D molecular measure in
which
a bit in a data string is set corresponding to the occurrence of a given 2-7
atom fragment in
that molecule. Typically, strings of roughly 900 to 2400 bits are used. A
particular bit may be
set by many different fragments.
As used herein, a "combinatorial screening library" means a subset of
molecules
selected from a combinatorial accessible universe of molecules to be used for
screening in an
assay.
As used herein, "molecular structural descriptor" or "descriptor" means a
quantitative
representation of the physical and chemical properties determinative of the
activity of a
molecule. The term "metric" is synonymous with molecular structural descriptor
and is used
interchangeably throughout this application.
"Patterson plots", as used herein, means two dimensional scatter plots in
which the
distance between molecules in some metric is plotted on the X axis and the
absolute
difference in some biological activity for the same molecules is plotted on
the Y axis.
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"Sigmoid plots", as used herein, means two dimensional plots for which the
proportion of molecular pairs in which the second molecule is also active is
plotted on the Y
axis and the pairwise Tanimoto similarity is plotted in intervals on the X
axis.
As used herein, "topomeric alignment", means conformer alignment based on a
set of
alignment rules.
Any method known to those of skill in the art may be used in designing a
compound
library with molecular diversity. Preferred methods include structure activity
relationships
(SAR), see for example, Clark-Lewis et al., "Structure-Activity Relationships
of Interleukin-8
Determined Using Chemically Synthesized Analogs", J. I3iol. Chem.
266(34):23128-23134
(1991); and the method described in US Patent No: 6,240,374. Briefly, the
latter method
requires that any valid molecular structural descriptor must have a
"neighborhood property".
That is: the descriptor must meet the similarity principle's constraint that
it measure the
chemical universe in such a way that similar structures (as defined by the
descriptor) have
substantially similar biological properties. Or stated slightly differently:
within some radius in
descriptor space of any given molecule possessing some biological property,
there should be
a high probability that other molecules found within that radius will also
have the same
biological property. If a descriptor does not have the neighborhood property,
it does not meet
the similarity principle, and can not be valid.
According to the present invention, to quantitatively analyze whether any
given
metric obeys the neighborhood principle is preferably used for determining
patterns for use in
identifying novel molecular targets for antiviral intervention. Absolute
values of biological
activity have also been considered the dependent variable with the structural
metric as the
independent variable. This is the case for traditional QSARs (quantitative
structure activity
relationships), which is also a preferred method for use in identifying
compounds in the
present invention.
It must be noted, although small differences in structure should be associated
with
small differences in activity, the converse is not necessarily true; large
differences in activity
are not necessarily associated with large differences in structure. Thus, it
is important to use
differences in both measures: biological differences and structural (metric)
differences. Thus,
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5 instead of looking at the values assigned by the metric to each molecule,
the absolute
differences in the metric values for each pair of molecules are the
independent variables and
the absolute differences in biological activity for each pair of molecules are
the dependent
variables. The absolute value is used since it is the difference, not its
sign, which is important.
Thus identified library members are used in constructing combinatorial
libraries
The library members are synthesized as individual compounds using the parallel
solution-phase chemistry (figure 6). The key precursor thiophosphotriesters 6
are synthesized
from the commercially available 3'-phosphoramidites 4 in a one-pot reaction
system at room
temperature in good yield by stepwise addition of reactants. Reaction of
alcohol 3 and the 3'-
phosphoramidite 4 in CH3CN in the presence of 1 H tetrazole provided
phosphosphite triester
5 in quantitative yield as indicated by TLC analysis. Without the necessity of
isolating 5, 3H
1, 2-benzodithiole-3-one (3H-BD)9 is added to the reaction mixture to
oxidatively sulfurize 5
to give thiophosphotriester 6. After a preliminary purification by
liquid/liquid extraction, the
crude triester 6 is converted to the final product phosphorothioate 2
quantitatively by
sequential treatment with NH40H (28%, 55 °C, 4 hrs) and Dowex resin to
remove the
corresponding protecting groups.
One of the challenges in applying the parallel solution-phase chemistry to the
synthesis of combinatorial libraries is to develop convenient and efficient
isolation/purification techniques to eliminate the otherwise time-consuming
purification
procedures (e.g. chromatography) often associated with solution-phase
chemistry. This
problem is overcome by designing a convenient two-stage liquid/liquid
extraction strategy
partitioning between organic solvents and water (figure 6). The extraction
strategy is based
on the uniqueness that the desired phosphorothioate product 2 is hydrophilic
(water soluble)
but its immediate precursor thiophosphotriester 6 is hydrophobic (organic
solvent soluble)
and also compound 6 could be converted to compound 2 quantitatively. The first
liquid/liquid extraction was applied after thiophosphotriester 6 was formed.
By partitioning
between ethyl acetate (EtOAc) and 2% aqueous NaHC03,,the organic,soluble 6 was
retained
in EtOAc, while the water-soluble byproducts (e.g. sulfurization byproducts 1-
benzothiole-2-
oxa-3-one sulfoxides, diisopropyl ammonium tetrazolides) and other excess
reagents from all
previous reactions (such as 1 H tetrazole) are removed by repetitive aqueous
washing. The
second extraction is applied after compound 2is formed. By partitioning
between CHC13 and
HZO, it is anticipated that the hydrophilic phosphorothioate 2 is retained in
H20, while the
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26
CHC13-soluble ingredients in the product mixtures - either generated from the
deprotection
reaction by ammonia or carried over with 6 from the previous reactions - are
removed by
repetitive CHC13 washing. After the second extraction, HPLC analysis'°
(C-18, reversed
phase, X260) shows that the desired product 2 is presented in the aqueous
layer in purity higher
than 90% (Figure 4).
Two approaches are used to remove the dimethoxytrityl group at the 5'-end of
compound 7. The conventional approach involves treatment in 80% acetic acid
for 1 to 2
hr"; a recent report showed that Dowex resin (strong acid form, Dowex 500 WX 8-
200,
Aldrich) could be used efficiently in the detritylation (10 min).'2 Although
both approaches
can be used, the preferred method is using the Dowex resin capture procedure.
The
advantage of using Dowex resin capture procedure in the parallel library
synthesis are: (a)
ease and convenience of operation; (b) reduced time of detritylation (10 min
of Dowex versus
1 - 2 hrs of acetic acid); (c) minimizing the depurization caused by prolonged
exposure of
nucleosides to acetic acid; (d) abolition of evaporation of the acrid acetic
acid. After the
ammonia treatment, the resulting mixture containing compound 7 is passed
through a short
column of Dowex H+ for the detritylation to give product 2.'3
The final desalting step (C-18 cartridges, Gilson) is used to remove the
inorganic
salts, and the product 2, thus obtained, is of purity higher than 95%.
Analysis by NMR and
MS confirmed the structures of the library members. Spectral analysis of
selected members
revealed that no detectable base-modifications had occurred during the
synthesis. The
chemical structures of the representative library members arc depicted in
Figure 5 and their
corresponding spectral analysis is displayed in Figure 4 and Table 6.
A preferred method for tethering of the 5'-end of a nucleoside with functional
hydrophobic groups is achieved through phosphorothioate linkers using the
parallel solid-
phase and solution-phase combinatorial chemistry. The "drug-like" attributes
of the 5'-
modified nucleotide analogs 1 (figure 2) are evaluated by screening the
library members
against hepatitis- and herpes-virus replication and a number of "hits" are
identified in cell-
based assays. Such cell-based assays are well-know in the art and include for
example,
plaque reduction assays, etc. Libraries of the general structure 2 (figure 2)
are constructed in
which the 3'-end of a nucleoside carries a variety of functional hydrophobic
groups.
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The library can also be constructed using an automated solution-ph:...~
synthesis.
The members of the library may be comprised of synthetic oligonucleotides.
Preferred synthetic oligonucleotides comprise at least one, and preferably
more than one,
modification. Modifications include, for example, modifications of the
internucleotide
linkage, the base or the sugar moiety, capped ends and chimeric or hybrid
oligonucleotides.
Synthetic oligonucleotides include chemically synthesized polymers of
deoxyribonucleotide and/or ribonucleotide monomers connected by
internucleotide linkages.
Oligonucleotides may be constructed entirely of deoxyribonucleotides, entirely
of
ribonucleotides or of a combination of deoxyribonucleotides and
ribonucleotides, including
hybrid and inverted hybrid oligonucleotides. Hybrid oligonucleotides contain a
core region
of deoxyribonucleotides interposed between flanking regions of
ribonucleotides. Inverted
hybrids contain a core region of ribonucleotides interposed between flanking
regions of
deoxyribonucleotides.
Synthetic oligonucleotides of the invention may be connected by standard
phosphodiester internucleotide linkages between the 5' group of one
mononucleotide pentose
ring and the 3' group of an adjacent mononucleotide. Such linkages could also
be established
using different sites of connection, including 5' to 5', 3' to 3', 2' to 5'
and 2' to 2', or any
combination thereof. In addition to phosphodiester linkages, the
mononucleotides may also
be connected by alkylphosphonate, phosphorothioate, phosphorodithioate,
alkylphosphonothioate, phosphoramidate, carbamate, carbonate, phosphate
triester,
acetamidate, or carboxymethyl ester linkages, or any combination thereof.
Preferably, an
oligonucleotide of the invention comprises at least one phosphorothioate
internucleotide
linkage, more preferably, all linkages in the oligonucleotide are
phosphorothioate
internucleotide linkages.
Oligonucleotides of the invention may be constructed such that all
mononucleotides
are connected by the same type of internucleotide linkages or by combinations
of different
internucleotide linkages, including chimeric or inverted chimeric
oligonucleotides. Chimeric
oligonucleotides have a phosphorothioate core region interposed between
methylphosphonate
or phosphoramidate flanking regions. Inverted chimeric oligonucleotides have a
nonionic
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core region (e.g. alkylphosphonate and/or phosphoramidate and/or
phosphotriester
internucleoside linkage) interposed between phosphorothioate flanking regions.
Synthetic oligonucleotides of the invention may be constructed of adenine,
cytosine,
guanine, inosine, thymidine or uracil mononucleotides. Preferred
oligonucleotides are
constructed from mononucleotides which contain modifications to the base
and/or sugar
moiety of the mononucleotide. Modifications to the base or sugar include
covalently attached
substituents of alkyl, carbocyclic aryl, heteroaromatic or heteroalicyclic
groups having from 1
to 3 separate or fused rings and 1 to 3 N, O or S atoms, or a heterocyclic
structure.
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.
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.
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.
Exemplary heteroaromatic and heteroalicyclic group include pyridyl, pyrazinyl,
pyrimidyl, furyl, pyrrolyl, thienyl, thiazolyl, oxazolyl, imidazolyl, indolyl,
benzothiazolyl,
tetrahydrofuranyl, tetrahydropyranyl, piperidinyl, morpholino and
pyrrolidinyl.
For good classification of various modifications to a scaffold, see:
Kaztritzky, A. R.;
Kiely, J. S.; Hebert, N.; Chassaing, C. Definition of templates within
combinatorial libraries.
J. Comb. Chem. 2000, 2, 2-5.
Other modifications include those which are internal or are at the ends) of
the
oligonucleotide molecule and include additions to the molecule at the
internucleoside
phosphate linkages, such as cholesteryl, cholesterol, or diamine compounds
with varying
CA 02415368 2003-O1-10
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29
numbers of carbon residues between the two amino groups, and terminal ribose,
deoxyribose
and phosphate modifications which cleave, or crosslink to the opposite chains
or to associated
enzymes or other proteins which bind to the viral genome. Additional linkers
including non-
nucleoside linkers include, but are not limited to, polyethylene glycol of
varying lengths, e.g.,
triethylene glycol, monoethylene glycol, hexaethylene glycol, (Ma et al.
(1993) Nucleic Acids
Res. 21: 2585-2589; Benseler et al. (1993) J. Am. Chem. Soc. 115: 8483-8484),
hexylamine,
and stilbene (Letsinger et al, (1995) J. Am. Chem. Soc. 117: 7323-7328) or any
other
commercially available linker including abasic linkers or commercially
available asymmetric
and symmetric linkers (CIoneTech, Palo Alto, California) (e.g., Glen Research
Product
Catalog, Sterling, VA).
Additionally oligonucleotides capped with ribose at the 3' end of the
oligonucleotide
may be subjected to NaI04 oxidation/reductive amination. Amination may include
but is not
limited to the following moieties, spermine, spermidine, Tris(2-
aminoethyl)'amine (TAEA),
DOPE, long chain alkyl amines, crownethers, coenzyme A, NAD, sugars, peptides,
dendrimers.
Oligonucleotides may also be capped with a bulky substituent at their 3'
and/or 5'
end(s), or have a substitution in one or both nonbridging oxygens per
nucleotide. Such
modifications can be at some or all of the internucleoside linkages, as well
as at either or both
ends of the oligonucleotide and/or in the interior of the molecule (reviewed
in Agrawal et al.
(1992) Trends Biotechnol. 10: 152-158). Some non-limited examples of capped
species
include 3'-O-methyl, 5'-O-methyl, 2'-O-methyl, and any combination thereof.
Synthetic oligonucleotides of the invention can be prepared by art recognized
methods. For example, nucleotides can be covalently linked using art-
recognized techniques
such as phosphoramidite, H-phosphonate chemistry, or methylphosphoramidite
chemistry
(see, e.g., Goodchild (1990) Bioconjugate Chem. 2: 165-187; Uhlmann et al.
(1990) Chem.
Rev. 90: 543-584; Caruthers et al. (1987) Meth. Enzymol. 154: 287-313; U.S.
Patent No.
5,149,798) which can be carried out manually or by an automated synthesizer
and then
processed (reviewed in Agrawal et al. (1992) Trends Biotechnol. 10: 152-158).
Oligonucleotides with phosphorothioate linkages can be prepared using methods
well known
in the field such as phosphoramidite (see, e.g., Agrawal et al. (1988) Proc.
Natl. Acad. Sci.
(USA) 85: 7079-7083) or H-phosphonate (see, e.g., Froehler (1986) Tetrahedron
Lett. 27:
CA 02415368 2003-O1-10
WO 02/08446 PCT/IBO1/02217
5 5575-5578) chemistry. The synthetic methods described in Bergot et al. (J.
Chromatog.
( 1992) 559: 35-42) can also be used. Oligonucleotides with other types of
modified
internucleotide linkages can be prepared according to known methods (see,
e.g., Goodchild
(1990) Bioconjugate Chem. 2: 165-187; Agrawal et al. (1988) Proc. Natl. Acad.
Sci. (USA)
85: 7079-7083; Uhlmann et al. (1990) Chem. Rev. 90: 534-583; and Agrawal et
al. (1992)
10 Trends Biotechnol. 10: 152-158).
In other aspects, the invention provides a pharmaceutical composition. The
pharmaceutical composition is a physical mixture of at least one, and
preferably two or more
classes of compound libraries.
As used herein, "classes of libraries" refers to those libraries generated
which fall into
categories of diversity based on their chemical or physical properties,
discussed supra. The
different compounds in the different classes of libraries may have different
specificities for
target molecules depending on the sequences of the scaffold, modification(s),
and/or lengths.
In some embodiments, this pharmaceutical formulation also includes a
physiologically or
pharmaceutically acceptable carrier. Specific embodiments include a
therapeutic amount of a
lipid carrier.
As used herein the terms, "an anti-fungal effective amount " or "an anti-viral
effective amount", or "an anti-bacterial viral effective amount", are used
interchangeably with
"therapeutically effective amount" or "effective amount" means an amount of a
drug or
pharmacologically active agent that is nontoxic but sufficient to provide the
desired local or
systemic effect and performance at a reasonable benefit/risk ratio attending
any medical
treatment.
As used herein, a "pharmaceutically acceptable" component is one that is
suitable for
use with humans and/or animals without undue adverse side effects (such as
toxicity,
irritation, and allergic response) commensurate with a reasonable benefit/risk
ratio.
The classes of libraries of the present invention are suitable for use as
therapeutically
active compounds, especially for use in the control or prevention of herpes
simplex virus
(HSV). In vitro assays (see example 5) indicate a significant inhibition of
HSV plaque
formation.
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31
In this aspect of the invention, a therapeutic amount of a pharmaceutical
composition
containing classes of libraries is administered to a cell to inhibit virus
replication. In a similar
aspect, the classes of libraries of the present invention can be used for
treating human or other
mammals infected with a virus comprising the step of administering to an
infected animal or
cell a therapeutic amount of a pharmaceutical composition containing at least
one class of
library, and in some embodiments, at Least two classes of libraries.
In all methods involving the administration of classes of libraries of the
invention, at
least one, and preferably two or more identical or different classes of
libraries may be
administered simultaneously or sequentially as a single treatment episode in
the form of
separate pharmaceutical compositions.
More specifically, the invention includes methods of treatment of a mammal
susceptible to (prophylactic treatment) or suffering from a disease associated
with viruses,
such as HSV. Methods in of the present invention comprise administration of a
therapeutical 1y effective amount of one or more compounds of the invention to
virally
infected cells, such as mammalian cells, particularly human cells.
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.
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
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32
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.
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.
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.
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).
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.
All documents mentioned herein are incorporated herein by reference.
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33
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.
EXAMPLES
MATERIALS AND METHODS
General Methods
'H,'3C, 3'P NMR spectra were collected on a 500 MHz NMR spectrometer (Bruker).
MS was performed by electrospray in negative mode. The HPLC analysis was run
on a
Waters 600 system equipped with a photodiode-array UV detector 996,
autosampler 717, and
Millenium, using a Radial-Pak liquid chromatography cartridge (8mm LD.,
8NVC18). The
C-18 cartridges (100 mg) were purchased from Gilson, and the desalting was
performed on
Gilson solid-phase extraction system (Gilson, ASPEC XL). The QIAshredder spin
column
was purchased from Qiagen. The N-protected nucleoside phosphoramidites 4a-h
were
purchased from CruaChem (Aston, PA). The Dowex resin (strong acid form, Dowex
500
WX 8-200) was cleaned before use. All other reagents were obtained from
commercial
sources and used without purification.
General procedures for library assembly (see schemel)
The general procedures for library assembly are illustrated in figure 6. Each
alcohol
3, at a concentration of 30 pmol, and each of the nucleoside amidites 4, at a
concentration of
20 pmol were added sequentially to a series of conical microtubes (2 mL,
Ultident Scientific)
containing 1 H-tetrazole solution in CH3CN (1 ml, 100 pmol) under argon. The
mixture was
shaken in a platform shaker at room temperature for 5 min. Then 3H-BD (40
~mol) in
CH3CN was evaporated in a Speed Vac. One ml of ethyl acetate (EtOAc) was then
added,
followed by 2% aqueous sodium bicarbonate (0.5 ml). Following thorough mixing
of the
phases, the organic layer containing the intermediate thiophosphate triester
6, was separated
and evaporated to dryness. Aqueous NH40H (28%, 1 ml) was then added to the
residue in
each microtube. The tightly capped tubes were heated at 55°C for 4 hrs.
The aqueous
ammoniacal solution was concentrated to dryness in a Speed Vac. The contents
were
dissolved in water (0.5 ml), then added to a spin column (QIAshredder)
containing Dowex H+
(50 mg). After shaking for 10 min, the column was placed in a receptacle vial
and
centrifuged.
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34
The flow-through was collected, diluted to 1 ml with water, and extracted with
chloroform (2 x 0.4m1). The aqueous layer was then passed through a C-18
cartridge. The
cartridge was then washed with water, and the product 2, was eluted with
CH3CN/ H20
(10/90 for 2 ml, 50/50 for 2 ml). The corresponding eluent was collected and
evaporated to
dryness in a Speed Vac to provide the product 2, as a white solid in high
purity (>95%, C-18
HPLC analysis at ~,zso). Quantitation was achieved on the basis of A26o units,
and the yields
of products 1, were found to range from 75 to 90% starting from 4.
"Drug-like"attribute.s and chemical diversity of the designed library
The pharmaceutical relevance and chemical diversity of the libraries building
around
the nucleic acid based (NABTM) scaffold (figure 1) could be recognized by a
number of
factors: (a) the nucleoside pharmacophores, including P1 and P2 elements,
could provide the
primary target (nucleic acids or proteins) recognition element by their unique
"shape-in-
space" and hydrogen-bonding characteristics; (b) the P4 elements (R groups,
figures 1 and 2
and Table 1 ) could serve as additional functional variants providing a handle
to incorporate
divergent hydrophobic elements to facilitate the specific "drug"-target
interaction; (c) the
negatively-charged element of phosphorothioate linker (figure 2) could provide
ionic
interaction of the nucleotide analog with the target, while imparting aqueous
solubility to the
compounds; (d) importantly, the 2'-substituents (P3 elements, figures 1 and 2)
could be used
as the structural variants to "lock" the furanose scaffold into significantly
different
conformational puckers (e.g. C2.-endo versus C3~-endo puckers, figure 3)
thereby enabling
overall changes in local and/or global projection of the functional groups
(e.g. P2 and P4)
attached to the furanose scaffold.
Alcohol building blocks
The alcohols shown in table 1 were used as the building blocks to incorporate
the
functional variants R groups (figure 2). The selection of alcohols for the
library construction
was based on the contribution of the alcohols to molecular diversity by
variable spatial
displays of shape-in-space, charge-transfer, and van der Waals interactions.
Such functional
divergency was intended to explore the specific interaction between a ligand
and its receptor:
(1) to provide hydrophobic interactions to facilitate high affinity binding
between the
nucleoside analogs and target receptors; and/or (2) to aid in the inter-
and/or intracellular
delivery of the nucleoside analogs;
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5
Assembly of the library using parallel solution phase chemistry ,
The strategy to generate the desired libraries of 3'-modified nucleoside
analogs, the
general structure of library 2 is illustrated in figure 2, was similar to the
method used to
assemble the libraries of the 5'-modified nucleoside analogs of library 1, the
general structure
10 illustrated in figure 2. The library members were synthesized as individual
compounds using
the parallel solution-phase chemistry depicted in figure 6. The key precursor
thiophosphotriesters 6, could be synthesized~from commercially available
3'phosphoramidites
4, in a one-pot reaction system at room temperature in good yield by step-wise
addition of
reactants. Reaction of alcohol 3, and the 3'phosphoramidite in CH3CN in the
presence of 1
15 H-tetrazole provided phosphosphite triester 5, in quantitative yields as
indicated by TLC
analysis. Without the necessity of isolating 5, 3H-1,2-benzodithiole-3-one (3H-
BD) was
added to the reaction mixture to oxidatively sulfurize 5, to produce
thiophosphotriesters 6.
After a preliminary purification by liquid/liquid extraction, the crude
triester 6, was then
converted to the final product phosphorothioate 2, quantitatively by
sequential treatment with
20 NH40H (28%, 55°C, 4 hrs) and Dowex resin to remove the corresponding
protecting groups.
To eliminate the other wise time-consuming purification procedures (e..g.
chromatography) often associated with solution-phase chemistry, a convenient
two-stage
liquid/liquid extraction strategy partitioning between organic solvents and
water (figure
25 6)was designed. The extraction strategy was based on the uniqueness that
the desired
phosphorothioate product 2, is hydrophilic but its immediate precursor
thiophosphotriesters 6,
is hydrophobic (organic solvent soluble) and also compound 6, could be
converted to
compound 2, quantitatively. The first liquid/liquid extraction was applied
after
thiophosphotriesters 6, was formed. By partitioning between EtOAc and 2%
aqueous
30 NaHC03, the organic soluble 6, was retained in EtOAc, while the water-
soluble byproducts,
for example, sulfurization byproducts such as 1-benzothiole-2-oxa-3-one;
sulfoxides such as
diisopropyl ammonium tetrazolides and other excess reagents from all previous
reactions
such as 1 H-tetrazole, were removed by repetitive aqueous washing.
35 The second extraction was applied after compound 2, was formed. By
partitioning
between CHC13 and H20, it is anticipated that the hydrophilic phosphorothioate
2, was
retained in HZO, while the CHC13-soluble ingredients in the product mixtures -
either
generated from the deprotection reaction by ammonia or carried over with 6,
from the
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36
previous reactions - were removed by repetitive CHC13 washing. After the
second
extraction, HPLC analysis (C-18, reversed phase, 7260) showed that the desired
product 2, was
presented in the aqueous layer in purity higher than 90% (figure 4).
Two approaches could be used to remove the dimethoxytrityl group at the 5'-end
of
compound 7. The conventional approach involves treatment in 80% acetic acid
for 1 to 2 hrs.
A recent report showed that Dowex resin (strong acid form, Dowex 500 WX 8-200,
Aldrich)
could be used efficiently in the detritylation (10 min).
Although both approaches can be used, the advantage of using Dowex resin
capture in
the parallel library synthesis are: (a) ease and convenience of operation; (b)
reduced time of
detritylation (10 min of Dowex versus 1-2 hrs of acetic acid); (c) minimizing
the depurization
caused by prolonged exposure of nucleosides to acetic acid; (d) abolition-of
evaporation of
the acrid acetic acid. After the ammonia treatment, the resulting mixture
containing
compound 7, was then passed through a short column of Dowex H+ for the
detritylation to
give product 2.
The final desalting step (C-18 cartridges, Gilson was used to remove the
inorganic
salts, and the product 2, thus obtained, was of purity higher than 95%.
Analysis by NMR and
MS confirmed the structures of the library members. Spectral analysis of
selected members
revealed that no detectable base-modifications had occurred during the
synthesis. The
chemical structures of the representative library members are depicted in
figure 5 and their
corresponding spectral analysis is displayed in figure 4 and Table 6.
ES-MS analysis (negative mode)
A scan spectrum was acquired first for each sample in order to verity the
presence of
the analyte in-the sample. Then a MS/MS spectrum was acquired for the analyte
to give
structural information on the samples. The samples were dissolved in
isopropanol/HZO (lit)
containing 20 mM of triethylamine.
Assembly of the Libraries 1-4
The library synthesis (5 to 15 pmol scale) was performed using standard
automated
DNA synthesis protocols (DMT-ofd. Oxidative sulfurization was effected using
3H 1,2-
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37
henzodithiole-3-one-1,1-dioxide.~3 After synthesis, the CPG was dried using
N2, and
transferred to 5-mL centrifuge tubes. Aqueous ammonium hydroxide (28%, 4 mL)
was
added and the mixture was heated at 55 °C for 3 - 6 h. The resulting
suspension was cooled
and centrifuged. The solution was evaporated in a Speed Vac. Each resulting
product was
dissolved in water (3 to 5 mL), and extracted with ethyl acetate (2 x 1 mL).
The aqueous
layer was evaporated to remove excess ethyl acetate and the residue taken up
in ultra pure
water and filtered through 0.2 pm filter. Lyophilization gave the products as
white foam.
Assembly of the library 5
The requisite dinucleotide H-phosphonates were assembled on solid support
using
standard 1-I-phosphonate chemistry. Following washing, the dry CPG was
transferred to 5
mL centrifuge tubes and a solution of amine in CC14 (10%, 3-4 mL).~4 The
mixture was
shaken for 20 to 30 min. Following washing, the CPG was treated with 28%
aqueous
NH40H (55 °C, 3-6 h). The suspension was cooled and centrifuged. The
solution was
evaporated to dryness in vacuo, the residue dissolved in H20 (5 mL), and
extracted with ethyl
acetate (2 mL). The aqueous layer was evaporated; residue taken up in ultra
pure water and
filtered.
Analysis of the library
Reversed-phase HPLC analysis of tile libraries was performed on a Waters 600
system equipped with a photodiode-array UV detector 996, autosampler 717, and
Millennium~ 2000 software, using a Radial-Pak~ liquid chromatography cartridge
[8 mm
LD., 8NVC18]. Mobile phase: Buffer A: 0.1 M NH40Ac; Buffer B: 20% A/80% CH3CN,
v/v: Gradient: 100% A, 0-3 min; 40% A, 40 min; 100% B, 49 min; 100% B. Product
purity
ranged from 85 to 95%. Yields were estimated to be 65 to 90% on the basis of
Az6o units.
Spectral characterization of representative library members
Before spectral acquisition, the representative library members (10% of the
library
size) were further purified by ion-exchange column (DEAE-5PW Resin, Buffer A:
H20,
Buffer B: 0.5 M NaCI) followed by desalting (C18 column, Buffer A: H20, Buffer
B: 20%
CH3CN in HZO) to give individual library members of 95-99% purity as
determined by
reversed-phase HPLC.
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38
3'P NMR analysis of selected library members revealed clear signals at S 58-59
ppm,
and 13-14 ppm, characteristic of phosphorothioate and phosphoramidate linkages
respectively. Other peaks in tile 3'P NMR spectra constituted less than 3% of
the total area
corresponding to the desired product peaks. Additionally, the ES-MS of
selected library
members were consistent with the expected molecular weights corresponding to
the assigned
structures
Antiviral assays against HSV I
Vero cells (African green monkey kidney cells) (ATCC) were infected with HSV-1
at
an MOI of 0.005 in 96 well plates in Dubelco's modified Eagle's (DMEM) medium.
The
plates were maintained at 37 °C for 3 h. The compounds were added at 25
micromolar
concentration, following which the plates were incubated at 37 °C: for
24 h. The media was
removed, and the cells were fixed with 10% formal saline for 10 minutes. The
cells were
stained with 0.1% crystal violet, incubated at room temperature for 30
minutes, and then
washed with distilled water. The viral plaques were counted, and the antiviral
effect
estimated as a percent reduction in the number of plaques compared with
untreated control.
Acyclovir was used as the positive control (EC9o, at 6 micromolar).
Example 1
Synthe.si.s of Libraries
Library I
A 64-member dinucleotide phosphorothioate library 1 (Table 1) was assembled on
solid support using commercially available deoxyribonucleoside and 2'-OMe
ribonucleoside
phosphoramidite building blocks in conjunction with the corresponding
controlled-pore-glass
(CPG)-linked nucleosides. The assembly was carried out in a parallel synthesis
mode (DMT-
off, 10 to 15 pmol scale). Following the assembly, each solid support was
treated with
aqueous ammonium hydroxide (28%, 55 °C) to remove nucleobase-, arid
phosphate-
protecting groups and to cleave the products off the support. The products
were purified and
evaluated as described in the experimental section.
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39
Tnble 1. 64-menlbcr dlnucleotlde phoephorothiuatc library
1 J 7 A M - .
M At. ' ' -
o
OA S'4C). y.~ A y~ -
. 7 .
,
1 7 ) 1 - - 1'T(i~
w . . a
)'AA!'AC!'AUTAT 'M -
- -
2V
)'GA7 ! 7 1 7~~ . )'GU
_ _ _-J';A -.
'
- 'UC TU 7 UA ~
~ '~ 7
A: C.. O. anU 1 ~trTClPcmu w uwwyrtotntuc.cvaww, ..nw,.a,, _r,. ..
and U correspond to 2'~OMc-ribonucleosidcs. All Internucleotidic
linkages ate phosphorothlontes.
Example 2
Library 2
Using eight of the commercially available monomers and nucleoside-bound-CPGs,
potentially 512 trinucleotide phosphorothioates could be assembled as a
mixture ofRp, Sp
diastereomers. We prepared a representative 64-member library (Table 2) using
phosphoramidite chemistry. Following work-up and extraction, the products were
obtained
85 to 95% pure as determined by reversed-phase HPLC.
Table L'J'rfnuc~cusidc phoaphorothioete Ilbrary
TAAA . ...
1'AT"~ . TACT ':!'Alt:
. -
.
7 . '7 JT
: LAC -
T 7'U . 7 J'UGC
:AA .A :
.
1' _ T
A ..
. , A 7 J'AAT.1'A
l A'ni .
7 3'
-.
~
~
a;(IAUJ'OCIT . a'ti(~J
~
n 3 11 .
. U
Example 3
Library 3
The parallel assembly of tetranucleotides posed a special challenge because a
4096-
member tetranucleotide library (representing an 8x8x8x8 array) could be
assembled. In order
to have a library amenable to parallel synthesis, three nucleotide positions
were fixed with the
fourth position being degenerate. Thus, each of the 64 trinucleotides were
first assembled on
CPG by parallel synthesis, and each CPG-bound trinucleotide was reacted with
an equimolar
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5 mixture of dA, dC, dG, and T nucleoside phosphoramidites (scheme 1). In this
way, 256-
member library was assembled (Table 3).
Scheme 1
~_.~.Ta . AeJ, Qie.. tae. T ohosOhorami6e~ ---
~-LTJ44 ~...~.T.p-0
-~C~T-A,C ~...~.TØt
Table 3: Deo><ynucleuaide phoephorothioaJe Ielranncleotide library
7'nnnYrncnv7'r,cnY7litnvJ'nnnr J racy
uv nJav
-
rcnnYrl:~nYracnvrcrnrrwrY~. ~ rcrcv
. ~tv
.Y
",( . n . 7 .1'111f.9
..
'J J' :~ 7.t 7 I !'AI'IJ
4A 'V A(: 'Y I
1 I
:1 :1 1 7 .11 . 1
. . . . Y .
Y1 . Iv 7
Y1
'1'UAIN'1' 7' ~ 11 . a 7'l!1'IV
:llY 1I IIVY lln nr -
Y
t"n ~ 7-1 1 V . w ~
~Y ICGY ~ t "Y
V
v: rcprrvcnt a mtxcurc or ara. w.., o~, ana J
10 Example 4
Library 4
A representative base-modified library of 24 members were assembled as di-,
tri-,
tetranucleoside phosphorothioates (Table 4) using the corresponding
commercially available
phosphoramidite monomers.
Table ~: 9~ae modified ohoanhorothinah lihrarv
7.w(:"'l.~C..w7.tk.swZ..t,~.w7.r.n.K..,..1.~C.,..
7.nn.lo~...7CA~""7T.",.ra..i.T.~..~-7.M.ryw.rJ
J'Atl~.vTr:u'sty.,. J..t.u.vi-CU..YC'4ltiUa
~ 7
7'nU''~ 1'(:U')'OU- 7'TUr TCV'C 7'CTGUr
G'M', 5-Mctt>,yl-dC; A''°°"', 7-Uelva-dA; l~'3°. SF-
dU; U''', 5-propync-
dU_ A. C, C, and T arc dcnxyrihonucleosidaa: tl C, Ci and U
rcproacnt 2'-OMe~ribonucleuaidcs.
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Example 5
Library 5
In the phosphoramidate dinucleotide library (Table 5), an additional diversity
element
was incorporated at the backbone. A 192-member dinucleotide phosphoramidate
library was
assembled by parallel synthesis using H-phosphonate chemistryt2 in conjunction
with a series
of amines. The requisite dinucleoside H-phosphonates (5'-DMT-off) were
assembled on
CPG using the corresponding commercially available H-phosphonates and the CPG-
bound
nucleosides in conjunction with 1-Adamantanecarbonyl chloride as the
activator. The CPG-
bond nucleoside H-phosphonates were converted to phosphoramidates by reported
procedure.t4
Table 5: Pboephoretnfdate Ltbruriee
a s k - J J'ACi.NyJ~~'1'.Ny
AI:Ny
Y
a - x x - 3 1'~4-J'_
,~ . ~I: Y ~'NY
V NY
7'UA.Hv)'U'.Na) 7'UTNw)'~ 3'f1:Ny_n.NYJ'ZST.Ny-.
~Na ~ -Ny_ Y
-a , n ~ - - ~
x y y Y ~
,r cnrrm, ~I Nv mon .
~.,rms ~T~ N. c:orrcs ,
L7 A hL. to N~..
A. C, G, T carrrspond to dcoxyribonucleosidea, ~ ~ C, ti correspond
to 2'-CIMe-rA, r(:, Ki, rll nucltosldes. All intemucla7tidic linkages me
phosphornn7idatcs.
/~ r
N~' ( r-NH
HN~~~ r
N : ~N~NFf N ~ ~Nw
Q~J ~,! CH3 ~ U
Ny --~ -N
i N":
J N
Example 5
Antiviral Evaluation
Different classes of NAB libraries, were prepared and evaluated. The results
demonstrate that the libraries represent biologically relevant chemical
diversity for drug
discovery.
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The libraries I-5 were evaluated in antiviral assays against HSV-1. A number
of
compounds, induced 30 to 60% inhibition of HSV-I-induced plaques at a dose of
25
micromolar when compared with acyclovir (EC9o of 6 micromolar).
Example 6
Characteristics of a possible new HSV 1 protein target for NABTM compounds
The results from preliminary screening of antiviral activity for NAB~~M
compounds by
SDS-PAGE and Western blotting have indicated that the compounds interact
either directly
or indirectly with one of the unique viral proteins. This protein has the
following
characteristics: A molecular weight of about 90kDa; it is an immediate early
(IE) or early (E)
protein as it appears during the early phases after infection around 6hr post
infection and is
most likely one of the important regulatory or structural proteins; the
protein is inhibited
completely by the NABTM compounds between 6 and 24 hrs post infection where
after it
reappears; the protein is stained by both Coomasie Blue and Silver Stains and
is most likely a
glycosylated protein as determined by tunicamycin treatment. The nature and
the kinetics of
the protein, its production and its role or function during the virus growth
cycle is determined
by pulse chase experiments, virus labeling with 35S-methionine, endo H/F
digestions.
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The following specific references, also incorporated herein by reference, are
indicated
in the above discussion and examples by the corresponding number generally
within
parenthesis or brackets.
Nucleotide Analogues as Antiviral Agents, Martin, J. C. (Ed). American
Chemical
Society, Washington, D. C. 1989.
2. Balfour, H. H. New Engl. J. Med. 1999, 340, 1255-1268.
3. (a) Geysen, H.; Meloen, R. H.; Barteling, S.J. Proc. Natl. Acad. Sci. USA,
1984, 81,
3998-4002; (b) Jung, G.; Beck-Sickinger, A. G. Angew. Chem., Intl. Ed Engl.
1992, 31,
367-383; (c) Pavia, M. R.; Sawyer, T. K.; Moos. W. H. Bioorg. Med. Chem. Lett.
1993,
3, 387-396; (d) Gordon, E. M.; Barrett, R. W.; Dower, W. J.; Fodor, S. P. A.;
Gallop,
M. A. J. Med Chem., 1994, 37, 1385-1401; (e) Thompson, L. A.; Ellman, J. A.
Chem.
Rev. 1996, 96, 555-600.
4. Schreiber, S. L. Science, 2000, 1964-1966.
Ellman, J. A. ACS Prospectives Symposium, Combinatorial Chemistry: 21 S'
Century
Chemical Synthesis, Tucson, Az., 2000
6. Saenger, W. Principles ofNucleicAcid Structure, Springer-Verlag, New York,
1984.
Oligonucleotide Synthesis, A Practical Approach; Gait, M. J. Ed.; IRL press,
New
York, 1990.
8. For related publications, see: (a) Zhou, W.; Roland, A.; Upendran, S.; Jin,
Y,; Iyer, R.
P. Tetrahedron Lett. 2000, 41, 445; (b) Zhou, W.; Roland, A.; Jin, Y.; Iyer;
R. P.
Bioorg. Mod. Chem Lett., 2000 (in press).
9. Cramer, R. D.; Patterson, D. E.; Clark, R. D.; Soltanshahi, F.; Lawless, M.
S. J. Chem.
Inf. Sci. 1998, 38, 1010-1023.
10. For examples see: (a) Jahnke, J.; Chao, Q.; and Nair, V. Nucleosides &
Nucleotides,
1997, 16, 1087-1090. (b) Hakimelahi, G. H.; Moosavi-Movahedi, A. A.; Sadeghi,
M.
M.; Tsay, S. C.; Hwu, J. R. J. Med. Chem. 1995, 38, 4648-4659. (c) Fathi, R.;
Rudolph,
M. J.; Gentles, R. G.; Patel . R.; MacMillan, E. W.; Reitman, M. S.; Pelham,
D.; Cook,
A. F. J. Org. Chem. 1996, 61, 5600-5609. (d) Davis, P. W.; Vickers, T. A.;
Wilson-
Lingardo, L.; Wyatt, J. R.; Guinosso, C. J.; Sanghvi, Y. S; DeBaets, E. A.;
Acevedo, O.
L.; Cook, P. D.; Ecker, D. J. J. Med Chem. 1995, 38, 4363-4366.
11. (a) Beaucage, S. L.; Caruthers, M. H. Tetrahedron Lett. 1981, 22, 1859-
1862; (b) For a
review of phosphoramidite chemistry, see: Iyer, R. P.; Beaucage, S. L.
Oligonucleotide
Synthesis In Comprehensive Natural Products Chemistry Vol. 7.: DNA and Aspects
of
Molecular Biology; Kool E. T., Ed. Elsevier Science: London, 1999; pp. 105-
152.
12. (a) Garegg, P. J.; Regberg, T.; Stawinski, J.; Stromberg, R. Chem. Scr.
1985, 25, 280
282. (b) Froehler, B. C.; Ng, P. G.; Matteucci, M. D. Nucl. Acids Res. 1986,
14, 5399
5407.
13. Iyer, R. P.; Egan, W.; Regan, J. B.; Beaucage, S. L. J. Am. Chem. Soc.
1990, 112, 1253-
1254.
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14. Froehler, B. C. Tetrahedron Lett. 1987, 5575-5578.
15. Saunders, J.; Cameron, J. M: Recent developments in the design of
antiviral agents.
Med Res. Rev. 1995, 15, 497-531.
16. (a) Young, S. D. Perspectives in Drug Discovery and Design, 1993, 1 , 181.
(b)
Declerq, F. Expert Opin. Invest. Drugs, 1994, 3, 253. (c) Nucleosides and
Nucleotides as
Antitumor and Antiviral Agents; Chu, C.K., Baker, D. C., Eds., Plenum Press:
New York,
1993.
17. (a) Jones, R. J.; Bischofberger, W. Antiviral Res. 1995, 27, t-17. (b)
Meier, C. Pro-
Nucleotides Recent Advances m the Design of Efficient Tools for the Delivery
of
Biologically Active Nucleosides Monophosphates. Synlett 1998, 233-242. (c)
Balzarini, J.;
Kruining, J.; Wedgwood, O.; Pannecouque, C.; Aquaro, S.; Perno, C.-F.;
Naesens, L.;
Witvrouw, M.; Heijtink, R.; De Clercq E.; McGuigan, C;. Conversion of 2' 3'-
Dideoxyadenosine (DDA) and 2', 3'-Didehydro-2', 3'-Dideoxyadenosine (d4A) to
their
corresponding aryloxyphosphoramidate derivatives markedly potentiates their
activity against
human immunodeficiency virus and hepatitis B virus. FEBSLett. 1997, 410, 324-
328.
18. (a) Zhou, W.; Roland, A.; Jin, Y.; Iyer, R. P. Tetrahedron Lett. 2000. 41,
441-445, (b)
Zhou, W.; Upendran, S.; Roland, A.; Jin, Y.; Iyer, R. P. Bioorg. Med Chem.
Lett. 2000 (in
press).
19. Gait, M. J. (ed.) Oligonucleotide Synthesis. A practical Approach. IRL
Press: Oxford
1984.
20. lyer, R. P.; Jiang, Z.; Yu, D.; Tan, W.; Agrawal, S. Improved procedure
for the
detritylation of DMT-oligonucleotides: use of Dowex. Syn. Comm. 1995, 25, 3611-
3623
