Note: Descriptions are shown in the official language in which they were submitted.
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SMALL MOLECULE COMPOSITIONS AND METHODS FOR INCREASING
DRUG EFFICIENCY USING COMPOSITIONS THEREOF
RELATED APPLICATIONS
Benefit of priority under 35 U.S.C. ~119(e) to U.S. provisional
application Serial No. 60/505,033, filed September 22, 2003, to Aspland e~
al.,
entitled "DRUG IMPROVEMENT BY NUCLEOSIDE KINASE SPECIFIC
TARGETING AND TRAPPING" and U.S. provisional application Serial No.
60/581,835, filed June 22, 2004, to Aspland et al., entitled " SMALL
MOLECULE COMPOSITIONS AND METHODS FOR INCREASING
DRUG EFFICIENCY USING COMPOSITIONS THEREOF" is claimed. The
subj ect matter of the above-referenced applications are incorporated by
reference in their entirety.
FIELD
Conjugates, compositions and methods for improving drug efficiency
are provided. The conjugates provided are for delivery of therapeutic agents
for treating a variety of disorders, such as, proliferative diseases,
autoimmune
diseases, infectious diseases and inflammatory diseases, are provided. The
conjugates contain drug moieties and substrates for kinases other than
hexokinases, protein kinases and lipid kinases non-releasably linked thereto,
optionally by a non-releasing linker.
BACKGROUND
Many potent, but relatively non-specific drugs have been developed for the
treatment of cancer. Examples of drugs for the treatment of cancer include
taxanes or
taxoids, vinca alkaloids, alkylating agents, camptothecins, and
anthracyclines.
Furthermore, much of modern drug discovery and development is focused on the
identification of small molecules which enter cells and inhibit proteins
responsible for
the genesis or maintenance of the condition being treated, either by down-
regulating
the expression of the protein or directly inhibiting its function. Another
approach
inhibits DNA synthesis in proliferating cells by using an anti-metabolite
vcrhich
includes nucleoside analogs and pyrimidine and purine bases.
One method for antimetabolite treatment of cancer uses a pyrimidine
containing compound whose action depends upon inhibition of de novo pyrimidine
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nucleotide biosynthesis. Resistance is common due to upregulation of thymidine
synthetase which overcomes inhibition. Another method for antimetabolite
treatment
of cancer uses a purine containing compound whose action depends upon HGRT,
and
the most common cause of resistance is the deficiency of this enzyme.
Targeting is
lacking with antimetabolites used for treatment of cancer as evidenced by the
poor
discrimination between normal cells and cancer cells. Furthermore, the anti-
metabolites used in cancer chemotherapy are prodrugs, since additional
metabolic
events are needed once cellular entry is gained for a therapeutic effect to be
realized.
For example, the anti-metabolite 5-FdU, a pyrimidine base, must be converted
to a
nucleotide for inhibition of thymidine synthetase, which is responsible for
the
therapeutic effect.
Viral anti-metabolites target the viral thymidine kinase (TIC), but trapping
or
accumulation is not responsible for their therapeutic effect. Viral anti-
metabolites
acted upon by viral TK include pyrimidine and purine containing compounds.
Anti-
metabolites used in anti-viral therapy are also prodrugs, and must be subject
to
downstream intracellular enzymes for conversion to the nucleoside
triphosphate, and
incorporation into the developing DNA strand to inhibit the DNA synthesizing
machinery of a viral infected cell, which is the actual event responsible for
the
therapeutic effect. Viral diseases treated by anti-metabolites include the DNA
viruses
HSV-l, HSV-1, VZV, EBV, CMV and the RNA viruses HTLV-1 and HIV. Although
HSV infections are well treated with acyclovir, HSV encephalitis is either
fatal or
results in serious neurological outcomes. RNA viral infections are especially
problematic and despite the advances in treating AIDS caused by HIV infection,
the
disease caused by this virus is invariably fatal.
Thus, the effectiveness of drugs used to treat cancer and viral infections is
frequently limited by side effects produced in cells not directly involved in
the genesis
or maintenance of the disease being treated. Drug effectiveness can also be
limited by
resistance to the drug which develops during treatment. This resistance is
exemplified
by the treatment of cancer wherein drug is actively removed from the treated
cell by a
P-glycoprotein transporter or wherein the effectiveness of the drug is
diminished by
over-expression of the enzyme upon which the drug acts.
Accordingly, considerable efforts have been directed to effect targeting of
anti-cancer to tumors since many anti-cancer compounds of chemical origin are
extremely potent and able to kill virtually any cell. Yet current protocols
generally
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require high concentrations and/or prolonged administration of agents, or only
result
in low and/or variable concentrations in the cytosol or nucleus of the cell
and
therefore give inefficient cytotoxicity andlor significant systemic toxicity.
Similarly,
a significant need to improve dxug efficiency of anti-viral drugs exists.
SUMMARY
Provided herein are compounds and methods for targeted delivery of
drugs. The compounds are conjugates that contain a drug moiety and a
substrate for a kinase other than a hexokinase, a protein kinase or a lipid
kinase non-releasably linked thereto. The drug moieties include therapeutic
agents, such as a cytotoxic agents, and diagnostic agents, such as labeled
moieties and imaging agents other than compounds containing a carboranyl,
hydroxyboryl and rare earth crypate moiety. The substrates are substrates for
a kinase other than a hexokinase, a protein kinase or a lipid kinase. In
certain
embodiments, the drug moiety is a therapeutic agent other than a compound
containing a carboranyl or hydroxyboryl moiety. In certain embodiments, the
drug moiety is a label other than a compound containing a rare earth crypate
moiety.
The conjugates contain one or more substrates for one or a plurality of
kinases other than a hexokinase, a protein kinase or a lipid kinase non-
releasably linked thereto, either directly or via a non-releasing linker to a
drug
moiety, such as a cytotoxic agent. The conjugates provided herein contain the
following components: (substrate)t, (linker)q, and (drug)d in which: at least
one substrate for a kinase other than a hexokinase, a protein kinase or a
lipid
kinase is non-releasably linked, optinally via a linker, to a drug moiety. t
is 1
to 6 and each substrate is the same or different, and is generally 1 or 2; q
is 0
to 6; 0 to 4; 0 or 1; d is 1 to 6, in certain embodiment 1 or 2 and each drug
moieties are the same or different; linker refers to any non-releasing linker;
and the drug is any therapeutic agent, such as a cytotoxic agent, including an
anti-cancer drug, a diagnostic agent, such as an imaging agent or labeled
moiety other than compounds containing a carboranyl, hydroxyboryl and rare
earth crypate moiety. The drug moiety of the drug conjugate may be derived
from a naturally occurring or synthetic compound that may be obtained from a
wide variety of sources, including libraries of synthetic or natural
compounds.
Exemplary drug moieties can be cytotoxic agents, including, but not limited
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to, anti-infective agents, antihelininthic, antiprotozoal agents, antimalarial
agents, antiamebic agents, antileiscmanial drugs, antitrichomonal agents,
antitrypanosomal agents, sulfonamides, antimycobacterial drugs, or antiviral
chemotherapeutics.
The conjugates for use in the compositions and methods provided
herein have formula (1):
(D)e-(L)q (S)c (1)
or a pharmaceutically acceptable derivative thereof, wherein D is a drug
moiety; d is 1-6, or is 1 or 2; L is a non- releasing linker; q is 0 to 6, or
is 0 or 1; S is a
substrate for a kinase other than a hexokinase, a protein kinase or a lipid
kinase; and t
is 1 to 6, or is 1 or 2, or is 1. In the conjugates, the drug moiety is
covalently attached,
optionally via a non-releasing linker, to the substrate. In the conjugates
provided
herein, the conjugation of the drug moiety(s) or non-releasing linker linked
thereto
can be at various positions of the substrate.
In the conjugates that contain two drug moieties, which are the same or
different, conjugation to the drug moiety(s) or non-releasing linker linked
thereto can
be at various positions of the substrate.
In certain embodiments, the kinase is overexpressed, overactive or exhibits
undesired activity in a target system. The action of the kinase on the
substrate results
in a negative charge on the conjugate. The action of the kinase on the
substrate may
result in improved drug efficiency.
The target system may be a cell, tissue or organ. In particular embodiments,
the cell is a tumor cell or a tumor-associated endothelial cell. The target
system may
also be associated with cancer, inflammation, angiogenesis, autoimmune
syndromes,
transplant rejection or osteoporosis.
The substrate, in certain embodiments, has a molecular weight of between
about 50 amu and 1000 amu. In other embodiments, the substrate has a molecular
weight of more than 1000 amu such as when the substrate exists as a dimer.
In certain embodiments, the conjugates have formula (2)
D - L - S' (2)
wherein D and L are as defined in formula (1); and
S' is a substrate for a phosphotransferase other than a hexokinase, a protein
kinase or a lipid kinase. In certain embodiments, contemplated
phosphotransferase are
designated by the Enzyme Commission under the general category number EC 2.7.1
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with the exceptions of the specific EC numbers 2.7.1.1 (hexokinase), 2.71.37
(protein
kinase), 2.7.1.91 (sphinganine kinae) and EC numbers designating other lipid
kinases.
In one embodiment the phospho group acceptor is a nucleoside. The substrate in
the
conjugates herein can be a substrate for a kinase including, but not limited
to,
thymidine kinase, viral thymidine kinase, TK-1, deoxycytidine kinase,
deoxyguanosine kinase.
The substrate, in certain embodiments, is phosphorylated upon action of a
kinase such as thymidine kinase, viral thymidine kinase, TK-l, deoxycytidine
kinase,
deoxyguanosine kinase. In certain embodiments, the substrate is nucleoside.
Examples of nucleosides for use as substrates in the conjugates provided
herein
include, but are not limited to, cytosine, uridine, thymidine, guanosine,
adenosine, or
derivatives thereof.
In the above formula 1, the drug moiety can be a hydrophobic drug. In certain
embodiments, D can be a detectable label. In certain embodiments, the drug is
an anti
cancer drug.
Pharmaceutical compositions containing a conjugate of formula 1 and a
pharmaceutically acceptable carrier are provided herein.
Also provided are methods for using the conjugates. The methods provided
are methods for treating conditions caused by undesirable chronic or aberrant
cellular
activation, migration, proliferation or survival (ACAMPS). Furthermore,
methods for
ameliorating a cell-proliferative disorder including, but not limited to,
cancer are also
provided. In one embodiment, the conjugates are for used in methods for
treating
cancer.
Also provided are methods of improving drug efficiency by administering a
therapeutically effective amount of a conjugate provided herein to a target
system or
organism, wherein the action of the kinase on the substrate results in
improved drug
efficiency.
In one embodiment, methods for identifying kinase substrates capable of
selectively accumulating in a target system are provided. The methods contain
the
steps of a) contacting one or more conjugates with a kinase that is
overexpressed,
overactive or that exhibits undesired activity in a target system; and b)
determining
kinase activity on one or more conjugates. In other embodiment, the method for
identifying kinase substrates capable of selectively accumulating in a target
system
further contains the steps of c) determining a first amount or a plurality of
first
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amounts of one or more conjugates in the target system; and d) determining a
second
amount or a plurality of second amounts of one or more conjugates in a non-
target
system.
In one example, one or more conjugates may contain a detectable label. For
example, the label may be radioactive or fluorescent. The radioactive Table is
a
radioactive compound other than a compound containing raze earth crypate
moiety.
The target system may be associated with cancer, inflammation, angiogenesis,
autoimmune syndromes, transplant rejection or osteoporosis. The target system
may
be a cell, tissue or organ. In one embodiment, the cell may be a tumor cell or
a tumor-
associated endothelial cell.
In one embodiment, methods for identifying conjugates capable of exhibiting
selective toxicity against a target system are provided. The methods contain
the steps
of: a) contacting one or more conjugates containing a drug moiety with a
target
system; and b) determining the cytotoxicity of the one or more conjugates
against the
target system.
DETAILED DESCRIPTION OF EMBODIMENTS
A. Definitions
Unless defined otherwise, all technical and scientific terms used herein have
the same meaning as is commonly understood by one of ordinary skill in the
art. All
patents, applications, published applications and other publications are
incorporated
by reference in their entirety. In the event that there are a plurality of
definitions for a
term herein, those in this section prevail unless stated otherwise.
The singular forms "a," "an," and "the" include plural references, unless the
context clearly dictates otherwise. Thus, for example, references to a
composition for
delivering "a drug" include reference to one, two or more drugs.
As used herein, "drug conjugate" or a "conjugate" refers to compounds having
one or more drug moieties non-releasably linked, optionally via a non-
releasable
linker, to a substrate for one or more kinase other hexokinase, a protein
kinase or a
lipid kinase. The drug-substrate conjugates provided herein retain a
significant
fraction of drug activity within the conjugate and the desired therapeutic
effect is
elicited by the drug-substrate conjugate without having the need to cleave the
drug
from the substrate. In certain embodiments, the drug moiety or the substrate
moiety
in the conjugate can be present in a form of a pharmaceutically acceptable
derivative
that renders the conjugate biologically inactive. The inactive drug-substrate
conjugate
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can be converted to the active drug-substrate conjugate under physiological
conditions
without having the need to cleave the drug-substrate conjugate.
As used herein, "substrate" is a molecule which is subject to phosphorylation
by an enzyme, other than a hexokinase, a protein kinase or a lipid kinase, and
encompasses species which can be converted by chemical and/or enzymatic
reactions) to a substrate upon or after introduction of the molecule (in
conjugate
form) to a target system or organism. The substrates for use herein include,
but are
not limited to substrates for nucleoside kinases such as thymidine kinase,
deoxycytidine kinase and deoxyguanosine kinase. The substrates for nuceoside
kinases include, but are not limited to, natural and non-natural nucleosides
and their
analogs, and natural and non-natural bases for nucleosides, such as purines
and
pyrimidines and their analogs.
As used herein, "drug" or "drug moiety" is any drug or other agent that is
intended for delivery to a targeted cell or tissue, such as cells or tissues
associated
with aberrant cellular activation, migration, proliferation or survival, other
than a
compound containing a carboranyl, hydroxyboryl or rare earth cryptate
containing
moiety. Drug moiety for use herein, include, but are not limited to, anti-
cancer
agents, anti-angiogenic agents, cytotoxic agents and labels other than
compounds
containing a carboranyl, hydroxyboryl or rare earth cryptate containing
moieties, as
described herein and known to those of skill in the art.
As used herein, an anti-cancer agent (used interchangeably with "anti-tumor or
anti-neoplasm agent") refers to any agents used in the treatment of cancer.
These
include any agents, when used alone or in combination with other compounds,
that
can alleviate, reduce, ameliorate, prevent, or place or maintain in a state of
remission
of clinical symptoms or diagnostic markers associated with neoplasm, tumor or
cancer, and can be used in methods, combinations and compositions provided
herein.
Non-limiting examples of anti-neoplasm agents include anti-angiogenic agents,
alkylating agents, antimetabolite, certain natural products that are anti-
neoplasm
agents, platinum coordination complexes, anthracenediones, substituted areas,
methylhydrazine derivatives, adrenocortical suppressants, certain hormones,
antagonists and anti-cancer polysaccharides.
As used herein, anti-angiogenic agent refers to any compound, that, when used
alone or in combination with other treatment or compounds, can alleviate,
reduce,
ameliorate, prevent, or place or maintain in a state of remission, one or more
clinical
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symptoms or diagnostic markers associated with undesired andlor uncontrolled
angiogenesis. Thus, for purposes herein an anti-angiogenic agent refers to an
agent
that inhibits the establishment or maintenance of vasculature. Such agents
include,
but are not limited to, anti-tumor agents, and agents for treatments of other
disorders
associated with undesirable angiogenesis, such as diabetic retinopathies,
hyperproliferative disorders and others.
As used herein, "drug-linker construct" refers to a chemical combination
wherein a drug moiety and a linker moiety are covalently attached. Similarly,
a
"drug-substrate construct" refers to a chemical combination wherein a drug
moiety
and a substrate moiety are covalently attached.
As used herein, "linker-substrate construct" refers to a chemical combination
wherein a linker moiety and a substrate moiety are covalently attached.
As used herein, the term "fraction of activity" refers to an amount of the
desired biological activity of a test compound, such as a drug-substrate
conjugate
provided herein, compared with the biological activity of the unconjugated
drug or
unconjugated substrate. The desired biological activity for the conjugates,
the parent
drugs or the substrates can be measured by any method known in the art,
including,
but not limited to, cytotoxicity assay, tubulin polymerisation assay and
thymidine
kinase activity assays described herein. In certain embodiments, the
biological
activity of the conjugates provided herein is greater than the activity of the
parent
drug moiety. As used herein a "significant fraction" referes to the activity
of from
about 5% up to about 100% of the biological activity, from about 5% up to
about
95%, from about 5% up to about 90%, from about 5% up to about 80%, up to about
70%, up to about 60%, or up to about 50% of the biological activity.
Significant
fraction is also mean to include biological activity of 100% or more.
As used herein "subject" is an animal, typically a mammal, including human,
such as a patient.
As used herein, "aberrant" refers to any biological process, cellular
activation,
migration, proliferation or survival, enzyme level or activity that is in
excess of that
associated with normal physiology.
As used herein, "chronic" refers to a biological process, cellular activation,
migration, proliferation or survival, enzyme level or activity that is
persistent or lasts
longer than that associated with normal physiology.
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As used herein, "undesirable" refers to normal physiological processes that
occur at an undesirable time, such as but not limited to, immune responses
associated
with transplant rejection and/or graft versus host disease.
As used herein, "ACAMPS" refers to aberrant cellular activation, migration,
proliferation or survival. ACAMPS conditions are characterized by undesirable
or
aberrant activation, migration, proliferation or survival of tumor cells,
endothelial
cells, B cells, T cells, macrophages, granulocytes including neutrophils,
eosinophils
and basophils, monocytes, platelets, fibroblasts, other connective tissue
cells,
osteoblasts, osteoclasts and progenitors of many of these cell types. Examples
of
ACAMPS-related conditions include, but are not limited to, cancer, coronary
restenosis, osteoporosis and syndromes characterized by chronic inflammation
and/or
autoimmunity.
As used herein, "hydrophobic drug" refers to any organic or inorganic
compound or substance having biological or pharmaceutical activity with water
solubility of less than 100 mg/ml, having a log P greater than 2, being lipid
soluble or
not adsorbing water.
As used herein, the term "effective amount of therapeutic response" refers to
an amount which is effective in prolonging the survivability of the patient
beyond the
survivability in the absence of such treatment. Prolonging survivability also
refers to
improving the clinical disposition or physical well-being of the patient. When
used in
reference to cancer treatment methods, the term "therapeutically effective
amount"
refers to an amount which is effective, upon single or multiple dose
administration to
the patient, in controlling tumor growth. As used herein, "controlling tumor
growth"
refers to slowing, interrupting, arresting or stopping the migration or
proliferation of
tumor or tumor-associated endothelial cells.
The cytotoxic selectivity of the conjugates provided herein is assessed by
comparing conjugate cytotoxicity against normal cells to the conjugate
cytotoxicity in
the tumor cells. Typically, the conjugates show highter cytotoxicity
selectivity for
tumor cells as compared to the normal cells. As used herein, the term
"cytotoxic
selectivity index" refers to the ratio of ECso of the conjugate in tumor cells
to the ECSo
of the conjugate in normal cell. In certain embodiments, the conjugates
provided
herein have higher cytotoxic selectivity for tumor cells than that of the
parent drug. Tn
certain embodiments, the conjugates provided herein show inproved cytotoxic
selectivity index as compared to the parent drug. The cytotoxic selectivity
index
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values for the conjugates provided herein are calculated by the methods
provided
herein.
As used herein, the term "improved drug efficiency" refers to a property of a
drug within the conjugate which is improved relative to the drug in free form.
Improved drug efficiency includes, but is not limited to, increased
solubility, altered
pharmacokinetics, including adsorption, distribution, metabolism and
excretion, an
increase in maximum tolerated dose, a reduction of side effects, an increase
in
cytotoxic selectivity index, an ability to surmount or avoid resistance
mechanisms, or
an ability to be administered chronically or more frequently. For example, a
more
efficient drug may have an improved cytotoxic selectivity index as compared to
a less
efficient drug. In certain embodiments, the improvement in the cytotoxic
selectivity
index is at least 1.5 fold greater is the conjugate.
As used herein, "non releasing linker moiety" or "non releasable linker
moiety" refers to a linker moiety that is attached to a drug moiety through a
covalent
bond or functionality which remains substantially intact under physiological
conditions during a period of time required for eliciting a pharmacological
response
such that the pharmacological response is not due to free drug. Typically, the
time is
sufficient for uptake of the conjugate by the target system. In certain
embodiments,
the linkage remains from about 10% up to about 100% intact under physiologic
conditions in a period of about 0.1 hours up to about 3 hours. In certain
embodiments, the linker is more than 50% intact, in another embodiment, more
than
60%, more than 70%, 80% or 90% intact. Evaluation of the stability of such
linkage
can be made by one of skill in the art using methods known in the art.
As used herein, "linker moiety" refers to the intervening atoms between the
drug moiety and substrate. A linker precursor, used interchangeably with
linker
precursor moity, is a compound that is used in the synthesis of a drug linker
construct
or a substrate linker construct. The terms "linker" and "linking moiety"
herein refer to
any moiety that non-releasably connects the substrate moiety and drug moiety
of the
conjugate to one another. The linking moiety can be a covalent bond or a
chemical
functional group that directly connects the drug moiety to the substrate. The
linking
moiety can contain a series of covalently bonded atoms and their substituents
which
are collectively referred to as a linking group. Linking moieties are
characterized by a
first covalent bond or a chemical functional group that connects the drug
moiety to a
first end of the linker group and a second covalent bond or chemical
functional group
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that connects the second end of the linker group to the substrate. The first
and second
functionality, which independently may or may not be present, and the linker
group
are collectively referred to as the linker moiety. The linker moiety is
defined by the
linking group, the first functionality if present and the second functionality
if present.
As used herein, the linker moiety contains atoms interposed between the drug
moiety
and substrate, independent of the source of these atoms and the reaction
sequence
used to synthesize the conjugate.
As used herein "non-releasably linked" refers to linkage of a drug moiety
through a covalent bond or functionality wherein the linkage remains
substantially
intact under physiological conditions during a period of time required for
eliciting a
pharmacological response such that the pharmacological response is not due to
free
drug. In certain embodiments, the linkage remains from about 10% up to about
100%
intact under physiologic conditions in a period of about 0.1 hours up to about
3 hours.
In certain embodiments, the linker is more than 50% intact, in another
embodiment,
more than 60%, more than 70%, 80% or 90% intact.
In the conjugates provided herein, in certain embodiments, L', L", etc. refers
to linker groups or covalent bonds that connect the first and the second
functionalities
of the linker or the linking moiety.
As used herein, "label" or "labeling agent"is a molecule that allows for the
manipulation and/or detection of the conjugate which contains the label.
Examples of
labels include spectroscopic probes such as chromophores, fluorophores, and
contrast
agents. Other spectroscopic probes have magnetic or paramagnetic properties.
The
label may also be a radioactive molecule or a molecule that is part of a
specific
binding pair well known in the art such as biotin and streptavidin. The
radioactive
lable for use herein is a radioactive compound other than a compound
containing a
rare earth crypate moiety.
The term "nucleoside" as used herein, refers to a molecule composed of a
heterocyclic base and a carbohydrate. Typically, a nucleoside is composed of a
heterocyclic nitrogenous base in N-glycosidic linkage with a sugar.
Nucleosides are
recognized in the art to include natural bases (standard), and non-natural
bases well
known in the art. The carbohydrates include the true sugars found in natural
nucleosides or a species replacing the ribofuranosyl moiety or acyclic sugars.
The
heterocyclic nitrogenous bases are generally located at the 1' position of a
nucleoside
sugar moiety. Nucleosides generally contain a base and sugar group. The
nucleosides
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can be unmodified or modified at the sugar, andlor base moiety, (also referred
to
interchangeably as nucleoside analogs, modified nucleosides, non-natural
nucleosides,
non-standard nucleosides; see for example, Eckstein et al., International PCT
Publication No. WO 92/07065 and Usman et al., International PCT Publication
No.
WO 93/15187). In natural nucleosides the heterocyclic base is typically
thymine,
uracil, cytosine, adenine or guanine. The carbohydrate shall be understood to
mean
the true sugar found in natural nucleosides or a species replacing the
ribofuranosyl
moiety or acyclic sugars. In certain embodiments, acyclic sugars contain 3-6
carbon
atoms and include, for example, the acyclic sugar moieties present in
acyclovir (-
CH2-O-CH2 CH2-OH), ganciclovir (-CH2-O-CH(CH2 OH)-CH2-OH), and the like.
Natural nucleosides have the (3-D-configuration. The term "nucleoside" shall
be
understood to encompass unnatural configurations and species replacing the
true
sugar that lack an anomeric carbon. In natural nucleosides the heteocyclic
base is
attached to the carbohydrate through a carbon-nitrogen bond. The term
"nucleoside"
shall be understood to encompass species wherein the heterocyclic base and
carbohydrate are attached through a caxbon-caxbon bond (C-nucleosides).
As used herein, "target system" is a cell, tissue or organ which is
responsible
for the genesis or maintenance of a disease state or is responsible for or
associated
with the condition being treated.
As used herein, biological activity refers to the in vivo activities of a
compound or physiological responses that result upon ih vivo administration of
a
compound, composition or other mixture. Biological activity, thus, encompasses
therapeutic effects and pharmacokinetic behaviour of such compounds,
compositions
and mixtures. Biological activities can be observed in in vitro systems
designed to
test for such activities.
As used herein, pharmaceutically acceptable derivatives of a compound
include salts, esters, enol ethers, enol esters, acetals, ketals, orthoesters,
hemiacetals,
hemiketals, acids, bases, solvates, hydrates or prodrugs thereof. Such
derivatives may
be readily prepared by those of skill in this art using known methods for such
derivatization. The compounds produced may be administered to animals or
humans
without substantial toxic effects and either are pharmaceutically active or
are
prodrugs. Pharmaceutically acceptable salts include, but are not limited to,
amine
salts, such as but not limited to N,N'-dibenzylethylenediamine,
chloroprocaine,
choline, ammonia, diethanolamine and other hydroxyalkylamines,
ethylenediamine,
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N-methylglucamine, procaine, N-benzylphenethylamine, 1-para-chlorobenzyl-2-
pyrrolidin-1'-ylmethylbenzimidazole, diethylamineand other alkylamines,
piperazine
and Iris(hydroxymethyl)aminomethane; alkali metal salts, such as but not
limited to
lithium, potassium and sodium; alkali earth metal salts, such as but not
limited to
barium, calcium and magnesium; transition metal salts, such as but not limited
to
zinc; and inorganic salts, such as but not limited to, sodium hydrogen
phosphate and
disodium phosphate; and also including, but not limited to, salts of mineral
acids, such
as but not limited to hydrochlorides and sulfates; and salts of organic acids,
such as
but not limited to acetates, lactates, malates, tartrates, citrates,
ascorbates, succinates,
butyrates, valerates, mesylates, and fumaxates. Pharmaceutically acceptable
esters
include, but are not limited to, alkyl, alkenyl, alkynyl, aryl, heteroaryl,
axalkyl,
heteroaralkyl, cycloalkyl and heterocyclyl esters of acidic groups, including,
but not
limited to, carboxylic acids, phosphoric acids, phosphinic acids, sulfonic
acids,
sulfinic acids and boronic acids. Pharmaceutically acceptable enol ethers
include, but
are not limited to, derivatives of formula C=C(OR) where R is hydrogen, alkyl,
allcenyl, alkynyl, aryl, heteroaryl, aralkyl, heteroaralkyl, cycloalkyl ar
heterocyclyl.
Pharmaceutically acceptable enol esters include, but are not limited to,
derivatives of
formula C=C(OC(O)R) where R is hydrogen, alkyl, alkenyl, alkynyl, aryl,
heteroaryl,
aralkyl, heteroaralkyl, cycloalkyl ax heterocyclyl. Pharmaceutically
acceptable
solvates and hydrates are complexes of a compound with one or more solvent or
water
molecules, or 1 to about 100, or 1 to about 10, or one to about 2, 3 or 4,
solvent or
water molecules.
As used herein, treatment means any manner in which one or more of the
symptoms of a disease or disorder are ameliorated or otherwise beneficially
altered.
Treatment also encompasses any pharmaceutical use of the compositions herein,
such
as use for treating a cancer.
As used herein, amelioration of the symptoms of a particular disorder by
administration of a particular compound or pharmaceutical composition refers
to any
lessening, whether permanent or temporary, lasting or transient that can be
attributed
to or associated with administration of the composition.
As used herein, ECSO refers to a dosage, concentration or amount of a
particular test compound that elicits a dose-dependent response at 50% of
maximal
expression of a particular response that is induced, provoked or potentiated
by the
particular test compound.
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It is to be understood that the compounds provided herein may contain chiral
centers. Such chiral centers may be of either the (R) or (S) configuration, or
may be a
mixture thereof. Thus, the compounds provided herein may be enantiomerically
pure,
or be stereoisomeric or diastereomeric mixtures. As such, one of skill in the
art will
recognize that administration of a compound in its (R) form is equivalent, for
compounds that undergo epimerization in vivo, to administration of the
compound in
its (S) form.
As used herein, substantially pure means sufficiently homogeneous to appear
free of readily detectable impurities as determined by standard methods of
analysis,
such as thin layer chromatography (TLC), gel electrophoresis, high performance
liquid chromatography (HPLC) and mass spectrometry (MS), used by those of
skill in
the art to assess such purity, or sufficiently pure such that further
purification would
not detestably alter the physical and chemical properties, such as enzymatic
and
biological activities, of the substance. Methods for purification of the
compounds to
produce substantially chemically pure compounds are known to those of skill in
the
art. A substantially chemically pure compound may, however, be a mixture of
stereoisomers. In such instances, further purification might increase the
specific
activity of the compound. The instant disclosure is meant to include all such
possible
isomers, as well as, their racemic and optically pure forms. Optically active
(+) and
(-), (R)- and (S)-, or (D)- and (L)-isomers may be prepared using chiral
synthons or
chiral reagents, or resolved using conventional techniques, such as reverse
phase
HPLC. When the compounds described herein contain olefinic double bonds or
other
centers of geometric asymmetry, and unless specified otherwise, it is intended
that the
compounds include both E and Z geometric isomers. Likewise, all tautomeric
forms
are also intended to be included.
As used herein, the nomenclature alkyl, alkoxy, carbonyl, etc. is used as is
generally understood by those of skill in this art.
As used herein, alkyl, alkenyl and alkynyl carbon chains, if not specified,
contain from 1 to 20 carbons, or 1 to 16 carbons, and are straight or
branched.
Alkenyl carbon chains of from 2 to 20 carbons, in certain embodiments, contain
1 to 8
double bonds, and the alkenyl carbon chains of 2 to 16 carbons, in certain
embodiments, contain 1 to 5 double bonds. Alkynyl caxbon chains of from 2 to
20
carbons, in certain embodiments, contain 1 to 8 triple bonds, and the alkynyl
carbon
chains of 2 to 16 carbons, in certain embodiments, contain 1 to 5 triple
bonds.
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Exemplary alkyl, alkenyl and alkynyl groups herein include, but are not
limited to,
methyl, ethyl, propyl, isopropyl, isobutyl, n-butyl, sec-butyl, tert-butyl,
isopentyl,
neopentyl, tert-pentyl, isohexyl, ethene, propene, butene, pentene, acetylene
and
hexyne. As used herein, lower alkyl, lower alkenyl, and lower alkynyl refer to
carbon
chains having from about 1 or about 2 carbons up to about 6 carbons. As used
herein,
"alk(en)(yn)yl" refers to an alkyl group containing at least one double bond
and at
least one triple bond.
As used herein, "cycloalkyl" refers to a saturated mono- or multicyclic ring
system, in certain embodiments of 3 to 10 carbon atoms, in other embodiments
of 3 to
6 carbon atoms; cycloalkenyl and cycloalkynyl refer to mono- or multicyclic
ring
systems that respectively include at least one double bond and at least one
triple bond.
Cycloalkenyl and cycloalkynyl groups may, in certain embodiments, contain 3 to
10
carbon atoms, with cycloalkenyl groups, in further embodiments, containing 4
to 7
carbon atoms and cycloalkynyl groups, in further embodiments, containing 8 to
10
carbon atoms. The ring systems of the cycloalkyl, cycloalkenyl and
cycloalkynyl
groups may be composed of one ring or two or more rings which may be joined
together in a fused, bridged or spiro-connected fashion. "Cycloalk(en)(yn)yl"
refers
to a cycloalkyl group containing at least one double bond and at least one
triple bond.
As used herein, "substituted alkyl," "substituted alkenyl," "substituted
alkynyl," "substituted cycloalkyl," "substituted cycloalkenyl," and
"substitued
cycloalkynyl" refer to alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl and
cycloalkynyl groups, respectively, that are substituted with one or more
substituents,
in certain embodiments one to three or four substituents, where the
substituents are as
defined herein, generally selected from Ql.
As used herein, "aryl" refers to aromatic monocyclic or multicyclic groups
containing from 6 to 19 carbon atoms. Aryl groups include, but are not limited
to
groups such as fluorenyl, substituted fluorenyl, phenyl, substituted phenyl,
naphthyl
and substituted naphthyl.
As used herein, "heteroaryl" refers to a monocyclic or multicyclic aromatic
ring system, in certain embodiments, of about 5 to about 15 members where one
or
more, in one embodiment 1 to 3, of the atoms in the ring system is a
heteroatom, that
is, an element other than carbon, including but not limited to, nitrogen,
oxygen or
sulfur. The heteroaryl group may be optionally fused to a benzene ring.
Heteroaryl
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groups include, but are not limited to, furyl, imidazolyl, pyrrolidinyl,
pyrimidinyl,
tetrazolyl, thienyl, pyridyl, pyrrolyl, N-methylpyrrolyl, quinolinyl and
isoquinolinyl.
As used herein, a "heteroarylium" group is a heteroaryl group that is
positively
charged on one or more of the heteroatoms.
As used herein, "heterocyclyl" refers to a monocyclic or multicyclic non-
aromatic ring system, in one embodiment of 3 to 10 members, in another
embodiment
of 4 to 7 members, in a further embodiment of S to 6 members, where one or
more, in
certain embodiments, 1 to 3, of the atoms in the ring system is a heteroatom,
that is,
an element other than carbon, including but not limited to, nitrogen, oxygen
or sulfur.
In embodiments where the heteroatom(s) is(are) nitrogen, the nitrogen is
optionally
substituted with alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl,
heteroaralkyl,
cycloalkyl, heterocyclyl, cycloalkylalkyl, heterocyclylalkyl, acyl, guanidino,
or the
nitrogen may be quaternized to form an ammonium group where the substituents
are
selected as above.
As used herein, "substituted aryl," "substituted heteroaryl" and "substituted
heterocyclyl" refer to aryl, heteroaryl and heterocyclyl groups, respectively,
that are
substituted with one or more substituents, in certain embodiments one to three
or four
substituents, where the substituents are as defined herein, generally selected
from Ql.
As used herein, "aralkyl" refers to an alkyl group in which one of the
hydrogen
atoms of the alkyl is replaced by an aryl group.
As used herein, "heteroaralkyl" refers to an alkyl group in which one of the
hydrogen atoms of the alkyl is replaced by a heteroaryl group.
As used herein, "halo", "halogen" or "halide" refers to F, Cl, Br or I.
As used herein, pseudohalides or pseudohalo groups are groups that behave
substantially similar to halides. Such compounds can be used in the same
manner and
treated in the same manner as halides. Pseudohalides include, but are not
limited to,
cyano, thiocyanate, selenocyanate, trifluoromethoxy, and azide.
As used herein, "haloalkyl" refers to an alkyl group in which one or more of
the hydrogen atoms are replaced by halogen. Such groups include, but are not
limited
to, chloromethyl, trifluoromethyl and 1-chloro-2-fluoroethyl.
As used herein, "haloalkoxy" refers to RO- in which R is a haloalkyl group.
As used herein, "sulfinyl" or "thionyl" refers to -S(O)-. As used herein,
"sulfonyl" or "sulfuryl" refers to -S(O)Z-. As used herein, "sulfo" refers to -
S(O)20-.
As used herein, "carboxy" refers to a divalent radical, -C(O)O-.
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As used herein, "aminocarbonyl" refers to -C(O)NHa.
As used herein, "alkylaminocarbonyl" refers to -C(O)NHR in which R is alkyl,
including lower alkyl. As used herein, "dialkylaminocarbonyl" refers to -
C(O)NRR
in which R and R are independently alkyl, including lower alkyl; "carboxamide"
refers to groups of formula -NRCOR in which R and R are independently alkyl,
including lower alkyl.
As used herein, "diarylaminocarbonyl" refers to -C(O)NRR' in which R and R'
are independently selected from aryl, including lower aryl, such as phenyl.
As used herein, "arylalkylaminocarbonyl" refers to -C(O)NRR' in which one
of R and R' is aryl, including lower aryl, such as phenyl, and the other of R
and R' is
alkyl, including lower alkyl.
As used herein, "arylaminocarbonyl" refers to -C(O)NHR in which R is aryl,
including lower aryl, such as phenyl.
As used herein, "hydroxycarbonyl" refers to -COOH.
As used herein, "alkoxycarbonyl" refers to -C(O)OR in which R is alkyl,
including lower alkyl.
As used herein, "aryloxycarbonyl" refers to -C(O)OR in which R is aryl,
including lower aryl, such as phenyl.
As used herein, "alkoxy" and "alkylthio" refer to RO- and RS-, in which R is
2 0 alkyl, including lower alkyl.
As used herein, "aryloxy" and "arylthio" refer to RO- and RS-, in which R is
aryl, including lower aryl, such as phenyl.
As used herein, "alkylene" refers to a straight, branched or cyclic, in
certain
embodiments straight or branched, divalent aliphatic hydrocarbon group, in one
embodiment having from 1 to about 20 carbon atoms, in another embodiment
having
from 1 to 12 carbons. In a further embodiment alkylene includes lower
alkylene.
There may be optionally inserted along the alkylene group one or more oxygen,
sulfur, including S(=O) and S(=O)a groups, or substituted or unsubstituted
nitrogen
atoms, including -NR- and -N~RR- groups, where the nitrogen substituent(s)
is(are)
allcyl, aryl, aralkyl, heteroaryl, heteroaralkyl or COR', where R' is alkyl,
aryl, aralkyl,
heteroaryl, heteroaralkyl, -OY or NYY', where Y and Y' are each independently
hydrogen, alkyl, aryl, heteroaryl, cycloalkyl or heterocyclyl. Alkylene groups
include, but are not limited to, methylene (-CHZ-), ethylene (-CHzCH2-),
propylene
(-(CHa)3-), methylenedioxy (-O-CH2-O-) and ethylenedioxy (-O-(CH2)2-O-). The
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term "lower alkylene" refers to alkylene groups having 1 to 6 carbons. In
certain
embodiments, alkylene groups are lower alkylene, including alkylene of 1 to 3
carbon
atoms.
As used herein, "azaalkylene" refers to -(CRR)n NR-(CRR)m , where n and m
are each independently an integer from 0 to 4. As used herein, "oxaalkylene"
refers to
-(CRR)n-O-(CRR)m , where n and m are each independently an integer from 0 to
4.
As used herein, "thiaalkylene" refers to -(CRR)"S-(CRR)m ,
-(CRR)n S(=O)-(CRR)m , and -(CRR)"S(=O)z-(CRR)m , where n and m are each
independently an integer from 0 to 4. In certain embodiments herein, the "R"
groups
in the definitions of azaalkylene, oxaalkylene and thiaalkylene are each
independently
selected from hydrogen and Ql, as defined herein.
As used herein, "alkenylene" refers to a straight, branched or cyclic, in one
embodiment straight or branched, divalent aliphatic hydrocarbon group, in
certain
embodiments having from 2 to about 20 carbon atoms and at least one double
bond, in
other embodiments 1 to 12 carbons. In further embodiments, alkenylene groups
include lower alkenylene. There may be optionally inserted along the
alkenylene
group one or more oxygen, sulfur or substituted or unsubstituted nitrogen
atoms,
where the nitrogen substituent is alkyl. Alkenylene groups include, but are
not
limited to, -CH=CH-CH=CH- and -CH=CH-CHa-. The term "lower
alkenylene" refers to alkenylene groups having 2 to 6 carbons. In certain
embodiments, alkenylene groups are lower alkenylene, including alkenylene of 3
to 4
carbon atoms.
As used herein, "alkynylene" refers to a straight, branched or cyclic, in
certain
embodiments straight or branched, divalent aliphatic hydrocarbon group, in one
embodiment having from 2 to about 20 carbon atoms and at least one triple
bond, in
another embodiment 1 to 12 carbons. In a further embodiment, alkynylene
includes
lower alkynylene. There may be optionally inserted along the alkynylene group
one
or more oxygen, sulfur or substituted or unsubstituted nitrogen atoms, where
the
nitrogen substituent is alkyl. Alkynylene groups include, but are not limited
to, -
C---C-C---C-, -C---C- and -C---C-CHa-. The term "lower alkynylene" refers to
alkynylene groups having 2 to 6 carbons. In certain embodiments, alkynylene
groups
are lower alkynylene, including allcynylene of 3 to 4 carbon atoms.
l~
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As used herein, "alk(en)(yn)ylene" refers to a straight, branched or cyclic,
in
certain embodiments straight or branched, divalent aliphatic hydrocarbon
group, in
one embodiment having from 2 to about 20 carbon atoms and at least one triple
bond,
and at least one double bond; in another embodiment 1 to 12 carbons. In
further
embodiments, alk(en)(yn)ylene includes lower alk(en)(yn)ylene. There may be
optionally inserted along the alkynylene group one or more oxygen, sulfur or
substituted or unsubstituted nitrogen atoms, where the nitrogen substituent is
alkyl.
Alk(en)(yn)ylene groups include, but are not limited to, -C=C-(CHa)"C---C-,
where n is 1 or 2. The term "lower alk(en)(yn)ylene" refers to
alk(en)(yn)ylene
groups having up to 6 carbons. In certain embodiments, alk(en)(yn)ylene groups
have
about 4 carbon atoms.
As used herein, "cycloalkylene" refers to a divalent saturated mono- or
multicyclic ring system, in certain embodiments of 3 to 10 carbon atoms, in
other
embodiments 3 to 6 carbon atoms; cycloalkenylene and cycloalkynylene refer to
divalent mono- or multicyclic ring systems that respectively include at least
one
double bond and at least one triple bond. Cycloalkenylene and cycloalkynylene
groups may, in certain embodiments, contain 3 to 10 carbon atoms, with
cycloalkenylene groups in certain embodiments containing 4 to 7 carbon atoms
and
cycloalkynylene groups in certain embodiments containing 8 to 10 carbon atoms.
The
ring systems of the cycloalkylene, cycloalkenylene and cycloalkynylene groups
may
be composed of one ring or two or more rings which may be joined together in a
fused, bridged or spiro-connected fashion. "Cycloalk(en)(yn)ylene" refers to a
cycloalkylene group containing at least one double bond and at least one
triple bond.
As used herein, "substituted alkylene," "substituted alkenylene," "substituted
alkynylene," "substituted cycloalkylene," "substituted cycloalkenylene," and
"substitued cycloalkynylene" refer to alkylene, alkenylene, alkynylene,
cycloalkylene,
cycloalkenylene and cycloalkynylene groups, respectively, that are substituted
with
one or more substituents, in certain embodiments one to three or four
substituents,
where the substituents are as defined herein, generally selected from Q1.
As used herein, "arylene" refers to a monocyclic or polycyclic, in certain
embodiments monocyclic, divalent aromatic group, in one embodiment having from
5
to about 20 carbon atoms and at least one aromatic ring, in another embodiment
5 to
12 carbons. In further embodiments, arylene includes lower arylene. Arylene
groups
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include, but are not limited to, 1,2-, 1,3- and 1,4-phenylene. The term "lower
arylene"
refers to arylene groups having 5 or 6 carbons.
As used herein, "heteroarylene" refers to a divalent monocyclic or multicyclic
aromatic ring system, in one embodiment of about 5 to about 15 members where
one
or more, in certain embodiments 1 to 3, of the atoms in the ring system is a
heteroatom, that is, an element other than carbon, including but not limited
to,
nitrogen, oxygen or sulfur.
As used herein, "heterocyclylene" refers to a divalent monocyclic or
multicyclic non-aromatic ring system, in certain embodiments of 3 to 10
members, in
one embodiment 4 to 7 members, in another embodiment 5 to 6 members, where one
or more, including 1 to 3, of the atoms in the ring system is a heteroatom,
that is, an
element other than carbon, including but not limited to, nitrogen, oxygen or
sulfur.
As used herein, "substituted arylene," "substituted heteroarylene" and
"substituted heterocyclylene" refer to arylene, heteroarylene and
heterocyclylene
groups, respectively, that are substituted with one or more substituents, in
certain
embodiments one to three of four substituents, where the substituents are as
defined
herein, generally selected from Ql.
As used herein, "alkylidene" refers to a divalent group, such as =CR'R", which
is attached to one atom of another group, forming a double bond. Alkylidene
groups
include, but are not limited to, methylidene (=CH2) and ethylidene (=CHCH3).
As
used herein, "arylalkylidene" refers to an alkylidene group in which either R'
or R" is
an aryl group. "Cycloalkylidene" groups are those where R' and R" are linked
to form
a carbocyclic ring. "Heterocyclylidene" groups are those where at least one of
R' and
R" contain a heteroatom in the chain, and R' and R" are linked to form a
heterocyclic
ring.
As used herein, "amido" refers to the divalent group -C(O)NH-. "Thioamido"
refers to the divalent group -C(S)NH-. "Oxyamido" refers to the divalent group
-
OC(O)NH-. "Thiaamido" refers to the divalent group -SC(O)NH-. "Dithiaamido"
refers to the divalent group -SC(S)NH-. "LTreido" refers to the divalent group
-
HNC(O)NH-. "Thioureido" refers to the divalent group -HNC(S)NH-. As used
herein, aminocarbonyl refers to - NHC(O) group. As used herein,
aminocarbonyloxy
refers to - NHC(O) O- group.
As used herein, "semicarbazide" refers to -NHC(O)NHNH, "thiosemicarbizide
refers to - NHC(S)NHNH,. "Carbazate" refers to the divalent group -OC(O)NHIVH-
.
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"Isothiocarbazate" refers to the divalent group -SC(O)NFINH-. "Thiocarbazate"
refers
to the divalent group -OC(S)NHNH-. "Sulfonylhydrazide" refers to the group -
S02NHNH-. "Hydrazide" refers to the divalent group -C(O)NHNH-. "Azo" refers to
the divalent group -N N-. "Hydrazinyl" refers to the divalent group -NH-NH-.
S Where the number of any given substituent is not specified (e.g.,
"haloallcyl"),
there may be one or more substituents present. For example, "haloalkyl" may
include
one or more of the same or different halogens. As another example,
"C1_3alkoxyphenyl" may include one or more of the same or different alkoxy
groups
containing one, two or three carbons.
As used herein, PEG linker represents a polyethylene glycol chain containing
the designated number of atoms in the chain between the drug moiety and the
substrate, conjugated to the drug moiety at the first end and to the substrate
at the
second end.
As used herein, alkane linker represents an alkylene group having the
designated number of atoms in the chain between the drug moiety and the
substrate,
conjugated to the drug moiety at the first end and to the substrate at the
second end.
The following naming conventions have been used to name the conjugates
provided herein:
The conjugates are named in four parts: "Drug"-"Point of Attachment and
functionality to the "Drug"-"Linker Type (Linker Length)"-"Enzyme Substrate".
In
an exemplary conjugate, thymidine as the enzyme substrate is attached to the
linker at
N3 of the nucleoside base.
The drug moieties in exemplary conjugates provided herein have been
abbreviated as follows:
Paclitaxel or O1° deacetyl-paclitaxel = PXL
Vinblastine or 04-deacetyl-= VBL
Doxorubicin = DOX
In naming the conjugates, the abbreviated name of the drug is followed by the
point of attachment and functionality linking the drug to the substrate,
optinally via
linking atoms interspaced inbetween. The point of attachment to the substrate
is
indicated as a prefix to the substrate abrreviation. For example, conjugate
PXL-7Ca-
ALK(6)-N3-THY is a paclitaxel thymidine conjugate, wherein N3 of thymidine is
conjugated to paclitaxel at C7 with a C6 alkane unit via a carbamate
functionality.
Table 1 provides examples of various drug moieties with possible points of
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attachments and linking functionalities. Table 2 herein provides examples of
various
linker groups and the names thereof.
As used herein, the following terms have their accepted meaning in the
chemical literature:
AcOH acetic acid
CHCl3 chloroform
conc concentrated
DBU 1,8-diazabicyclo[5.4.0]undec-7-ene
DIEA N-ethyl-N,N-di-isopropylamine
DCM dichloromethane
DME 1,2-dimethoxyethane
DMF N,N dimethylformamide
DMSO dimethylsulfoxide
EtOAc ethyl acetate
EtOH ethanol (100%)
Et20 diethyl ether
Hex hexanes
HaSO4 sulfuric acid
MeCN acetonitrile
MeOH methanol
PdIC palladium on activated carbon
TEA triethylamine
THF tetrahydrofuran
TFA trifluoroacetic acid
As used herein, the abbreviations for any protective groups, amino acids and
other compounds, are, unless indicated otherwise, in accord with their common
usage,
recognized abbreviations, or the ILTPAC-ICTB Commission on Biochemical
Nomenclature (see, (1972) Bioehem. 11:942-944).
B. CONJUGATES
Provided herein are drug-substrate conjugates for use in the methods and
compositions for increasing drug efficiency. The drug-substrate conjugates
provided
herein retain a significant fraction of parent drug activity within the
conjugate and the
desired therapeutic effect is elicited by the drug-substrate conjugate without
having
the need to cleave the drug from the substrate.
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The conjugates provided herein are not limited to specific drug, linker and
substrate moieties. Various combinations of the drug, linker and substrate
moieties
can be prepared using synthetic methodologies known in the art and described
herein.
As discussed above, the conjugates can contain a plurality of substrates, a
plurality of
linkers and a plurality of drug moieties.
In certain embodiments, the conjugates provided herein retain a significant
fraction of biological activity of parent drug within the conjugate. In
certain
embodiments, the conjugates retain from about 5 % up to about 100% of the
biological activity, from 5% up to about 95%, from about 5% up to about 90%,
from
about 5% up to about 80%, up to about 70%, up to about 60% or up to about 50%
of
the biological activity of parent drug. In certain embodiment the biological
activity of
the drug in the conjugate exceeds that of parent drug.
Without being bound to any theory, in certain embodiments, the drug-substrate
conjugates are selectively trapped or accumulated in target cells. In certain
embodiments, the conjugates are selectively trapped or accumulated in target
cells due
to phosphorylation of the substrate in the conjugates by a kinase whose
activity is
involved in the condition being treated. As a result, doses of the drug-
substrate
conjugate required to elicit the same effective amount of therapeutic response
as the
parent drug can be reduced thereby resulting in a reduction of undesirable
side
effects. This allows for an increase in the duration of therapy, which is
highly
desirable in chronic disease settings. In addition, the standard drug dose in
conjugate
form can be increased without exceeding the tolerability of undesirable side
effects to
allow for more aggressive treatment. Furthermore, molecules capable of
eliciting a
desired pharmacological response but which elicit unacceptable side effects at
doses
below that required for an effective amount of therapeutic response may be
transformed by conjugation into a molecule useful in the treatment of an
ACAMPS
condition. Finally, trapping or accumulation of drug conjugates by
phosphorylation
may prevent the efflux of cancer drugs such as vinca alkaloids,
epipodophyllotoxins,
taxanes/taxoids, and anthracyclines, by the membrane transporter P-
glycoprotein,
thus, preventing a major form of MDR.
In certain embodiments, the substrate moiety in the conjugate may be any
substrate for a kinase other than a hexokinase, a protein kinase or a lipid
kinase that is
overexpressed, overactive or that exhibits undesired activity in a target
system. The
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action of the kinase on the substrate results in a modified conjugate wherein
significant fraction of the activity of the drug moiety as well as the
substrate moiety is
retained. In a target system (e.g. cell, tissue or organ) containing cells the
drug-
substrate conjugate is less able to exit the cell in comparison to the
unmodified drug.
Accumulation of the drug-substratre conjugate into the target cells will occur
by
pushing the equilibrium of passive diffusion towards the target cells because
of
preferential trapping or accumulation due to the higher kinase activity in
these cells.
In certain embodiments, the drug-substrate conjugates exhibit improved
cytotoxic selectivity index over the parent drug. In certain embodiments, the
drug-
substrate conjugates exhibit improved solubility over the parent drug. In
certain
embodiments, the conjugates exhibit better serum stability than the parent
drug. In
certain embodiments, the conjugates exhibit better shelf life than the parent
drug.
In one exemplary embodiment, the conjugates for use in the methods and
compositions provided herein have the formula (1):
l'-')d-(L)q'(S)t (1)
or a pharmaceutically acceptable derivative thereof, wherein D is a drug
moiety; d is
1-6, or is 1 or 2; L is a non- releasing linker; q is 0 to 6, or is 0 or 1; S
is a substrate
for a kinase other than a hexokinase, a protein kinase or a lipid kinase; and
t is 1 to 6,
or is 1 or 2, or is 1. In the conjugates, the drug moiety is covalently
attached,
optionally via a non-releasing linker, to the substrate.
In conjugates that contain two drug moieties, which are the same or different,
conjugated to the substrate moiety(s) or non-releasing linked thereto can be
at various
positions of the substrate.
In certain embodiments, the conjugates have formula (2):
D-L-S, (2)
or a pharmaceutically acceptable derivative thereof, where the variables are
as
defined elsewhere herein.
Exemplary substrates, drug moieties, linkers and exemplary conjugates are
described in further detail below. It is intended herein that conjugates
resulting from
all combinations and/or permutations of the groups recited below for the
variables of
formulae (1) and (2) are encompassed within the instant disclosure.
1. Drug Moiety
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The conjugates provided herein are intended for modifying a variety of
biological responses. The drug moiety may be any molecule, as well as a
binding
portion, fragment or derivative thereof that is capable of modulating a
biological
process other than compounds containing a carboranyl, hydroxyboryl or rare
earth
cryptate containing moiety. Thus, the drug moiety encompasses any molecule
that
elicits a pharmacological response that may be used for the treatment or
prevention of
a disease. Accordingly, the drug moities are any moities, including proteins
and
polypeptides, small molecules and other molecules that possess or potentiate a
desired
biological activity. Such molecules include cytotoxic agents, such as, but are
not
limited to, a toxin such as abrin, ricin A, pseudomonas exotoxin, shiga toxin,
diphtheria toxin and other such toxins and toxic portions and/or subunits or
chains
thereof; proteins such as, but not limited to, tumor necrosis factor, a-
interferon, y-
interferon, nerve growth factor, platelet derived growth factor, tissue
plasminogen
activator; or, biological response modifiers such as, for example,
lymphokines,
interleukin- I (IL-1), interleukin-2 (IL-2), interleukin-6 (IL-6), granulocyte
macrophage colony stimulating factor (GMCSF), granulocyte colony stimulating
factor (G-CSF), erythropoietin (EPO), pro-coagulants such as tissue factor and
tissue
factor variants, pro-apoptotic agents such FAS-ligand, fibroblast growth
factors
(FGF), nerve growth factor and other growth factors.
The drug moiety of the drug conjugate may be derived from a naturally
occurring or synthetic compound that may be obtained from a wide variety of
sources,
including libraries of synthetic or natural compounds. For example, numerous
means
are available for random and directed synthesis of a wide variety of organic
compounds and biomolecules. Alternatively, libraries of natural compounds in
the
form of bacterial, fungal, plant and animal extracts are available or readily
produced.
Additionally, natural or synthetically produced libraries and compounds are
readily
modified through conventional chemical, physical and biochemical means, and
may
be used to produce combinatorial libraries. Known pharmacological agents may
be
subj ected to directed or random chemical modifications, such as acylation,
alkylation,
esterification, amidification, etc., to produce structural analogs.
As such, the drug moiety may be obtained from a library of naturally
occurring or synthetic molecules, including a library of compounds produced
through
combinatorial means (i.e., a compound diversity combinatorial libraxy). When
obtained from such libraries, the drug moiety employed will have demonstrated
some
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desirable activity in an appropriate screening assay for the activity.
Combinatorial
libraries, as well as methods for the production and screening, are known in
the art.
In particular embodiments, the drug moiety is a chemotherapeutic agent.
Examples of chemotherapeutic agents include but are not limited to anti-
infective
agents, antihelminthic, antiprotozoal agents, antimalarial agents, antiamebic
agents,
antileiscmanial drugs, antitrichomonal agents, antitrypanosomal agents,
sulfonamides,
antimycobacterial drugs, or antiviral chemotherapeutics. Chemotherapeutic
agents
may also be antineoplastic agents or cytotoxic drugs, such as alkylating
agents, plant
alkaloids, antimetabolites, antibiotics, tubulin or microtubule binding agents
and other
anticellular proliferative agents.
Other specific drugs of interest include but are not limited to central
nervous
system depressants and stimulants, respiratory tract drugs, pharmacodynamic
agents,
such as histamines and antihistamines, cardiovascular drugs, blood and
hemopoietic
system drugs, gastrointestinal tract drugs, and locally acting drugs including
chemotherapeutic agents. Drug compounds of interest from which drug moieties
may
be derived are also listed in: Goodman & Gilman's, The Pharmacological Basis
of
Therapeutics (9th Ed) (Goodman, et al., eds.) (McGraw-Hill) (1996); and 1999
Physician's Desk Reference (1990 and Chu, E.; DeVita, V.T. Physicians' Cancer
Chemotherapy Drug Manual 2003, Jones and Bartlett Publishers.
Classes of cytotoxic agents for use herein include, for example, the a)
anthracycline family of drugs, b) vinca alkaloid drugs, c) mitomycins, d)
bleomycins, e) cytotoxic nucleosides, f) pteridine family of drugs, g)
diynenes, h)
estramustine, i) cyclophosphamide, j) taxanes, k) podophyllotoxins, l)
maytansanoids, m) epothilones, and n) combretastatin and analogs.
In certain embodiments, the drug moiety is selected from a) doxorubicin, b)
carminomycin, c) daunorubicin, d) aminopterin, e) methotrexate, f)
methopterin, g)
dichloromethotrexate, h) mitomycin C, i) porfiromycin, j) 5-fluorouracil, k) 6-
mercaptopurine, l) cytosine arabinoside, m) podophyllotoxin, n) etoposide,
o) etoposide phosphate, p) melphalan, q) vinblastine, r) vincristine, s)
leurosidine,
t) vindesine, u) estramustine, v) cisplatin, w) cyclophosphamide, x)
paclitaxel
y) leurositte, z) 4-desacetylvinblastine, aa) epothilone B, bb) taxotere,
cc) maytansanol, dd) epothilone A, and ee) combretastatin and analogs. In
certain
embodiments, the drug is selected from Paclitaxel, Doxorubicin, Vinblastine,
Methotrexate and Cisplatin.
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Table 1 provides exemplary drug moieties used in the conjugates provided
herein_ Also indicated are points of attachment of the linker to the drug
moieties and
the functionality connecting the drug and the linker.
Table 1
Structure of Drug/Drug Functional Group Abbreviation
I HN
O~O
O OH
O NH O
O"' l OCa-PXL
/ OH . O
OH - H p
O O
O
\I
~='O
O O OH
O NH O
v
I / ~H lOEs-PXL
OH . H 0 O
O O
O
/ I
I ~ HN
O ~O
O O O
O NH O
7Ca-PXL
OH . O
OH . H
O O ~ .
O
I
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~O ~O
O O O
O NH O
~! 'O m
I / OH 7Es-PXL
OH . H ~ O
O O
O
~I
N
I~ I
C02Me
H ~ N 'H I 3Am-VBL
I OH ''~./
Me0 ~ i 3= 4 OH
O~NH
O OH O
14 OH
I I ~~'OH
oCH~o off O 3'Alk-DOX
o
NH3
HO
O OH O
I ~ I ~ . OH
~'OH
OCH~O OH O 3'Am-DOX
O
HO ~O
a) Arrows indicates site of attachment to
drug (or functionality to drug) from Linker
grou of Table 2
Furthermore, other drug moieties that may have been tested and considered to
have poor properties for treating cancer or proliferative disorders may also
be used.
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When used in the conjugates provided herein, such drug moieties can exhibit
enhanced biological activity compared to the unconjugated drug.
2. Linking Moiety
A linking moiety is used to attach the drug covalently to the substrate. The
terms "linker" and "linking moiety" herein refer to any moiety that non-
releasably
connects the substrate moiety and drug moiety of the conjugate to one another.
The
linking moiety can be a covalent bond or a chemical functional group that
directly
connects the drug moiety to the substrate. The linking moiety can contain a
series of
covalently bonded atoms and their substituents which are collectively referred
to as a
linking group. Linking moieties are characterized by a first covalent bond or
a
chemical functional group that connects the drug moiety to a first end of the
linker
group and a second covalent bond or chemical functional group that connects
the
second end of the linker group to the substrate. The first and second
functionality,
which independently may or may not be present, and the linker group are
collectively
referred to as the linker moiety. The linker moiety is defined by the linking
group, the
first functionality if present and the second functionality if present. As
used herein,
the linker moiety contains atoms interposed between the drug moiety and
substrate,
independent of the source of these atoms and the reaction sequence used to
synthesize
the conj ugate.
In one embodiment, the linker moiety is chosen to serve as a spacer between
the drug and the substrate, to remove or relieve steric hindrance that may
interfere
with substrate activity and/or the pharmacological effect of the drug. The
linker
moiety can also be chosen based on its effect on the hydrophobicity of the
drug-substrate conjugate, to improve passive diffusion into the target cells
or tissue or
to improve pharmacokinetic or pharmacodynamic properties. Thus, linking
moieties
of interest can vary widely depending on the nature of the drug and substrate
moieties.
In certain embodiments, the linking moiety is biologically inert. Precursors
for a
variety of linkers are known to those of skill in the art, which may be used
in the
synthesis of conjugates provided herein. Linker precursors are desirably
synthetically
accessible and provide shelf stable products; and do not add any intrinsic
biological
activity that interferes with the conjugates activity. When incorporated into
the
conjugates, they can add desirable properties such as increasing solubility or
stability
to the conjugate.
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Any bifunctional linker precursor, in certain embodiments, heterobifunctional
linking precursors that can form a non-releasable bond between the drug moiety
and
the substrate moiety, when incorporated into the conjugates, can be used in
the
synthesis of conjugates provided herein. In certain embodiments, a linker
precusor
can be homobifunctional. In certain embodiments, one or more of substrate
moieties
are linked to one or more drug moieties via a multifunctional linking moiety.
In one embodiment, a linker precursor has functional groups that are used to
interact with and form covalent bonds with functional groups in the components
(e.g.,
drug moiety and substrate moiety) of the conjugates described and used herein.
Examples of functional groups on the linker precursors (prior to interaction
with other
components) include -NHS, -NHNHZ, -ONH2, NHC=(O)NHNH2, -OH, -CHO,
halogen, -C02H, and -SH. Each of these functional groups can form a covalent
linkage to a suitable functional group on the substrate or the drug to get a
drug-linker
or a substrate-linker construct. For example, amino, hydroxy and hydrazino
groups
can each form a covalent bond with a reactive carboxyl group (e.g., a
carboxylic acid
chloride or activated ester such as an N-hydroxysuccinimide ester (NHS)).
Other
suitable bond forming groups are well-known in the art.
The linking moiety, L can include linear or acyclic portions, cyclic portions,
aromatic rings or combinations thereof. In certain embodiments, the linking
moiety
can have from 1 to 100 main chain atoms other than hydrogen atoms, selected
from C,
N, O, S, P and Si. In certain embodiments the linking moiety contains up to SO
main
chain atoms other than hydrogen, up to 40, up to 30, up to 20, up to 15, up to
10, up to
5, up to 2 main chain atoms other than hydrogen. In certain embodiments the
linking
moiety is acyclic.
In certain embodiments, the linking moieties contain oligomers of ethylene
glycol or alkylene chains or mixtures thereof. These linking moieties are, in
certain
embodiments, attached to the substrate via either an alkyl or amide
connection. In
certain embodiments, the drug moiety is attached to the first end of the
linker via an
amide, sulfonamide, or ether connection. Illustrative synthetic schemes for
forming
such conjugates are discussed elsewhere herein for exemplary linkers for the
conjugates provided herein.
In one embodiment, the linking moiety is a covalent bond between the drug
moiety and the substrate moiety. Typically, this attachment is accomplished
via
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coupling of a functional group on the drug with a compatible functional group
on the
substrate. In certain embodiments, the drug has an isocyanate, isothiocyanate
or
carboxylic acid functional group that is used to attach the drug to a hydroxy
or amino
group present on the substrate moiety to form a carbamate, thiocarbamate, urea
or
thiourea linkage between the components.
A variety of linking moieties depending on the nature of the drug and
substrate
moieties can be used in the conjugates provided herein. Suitable linking
moieties can
be selected by one of skill in the art based on the criteria set forth herein.
In one
embodiment, the linking moiety can be selected by the following procedure: A
first
end of a linker precursor is used in synthesizing a linker-nucleoside
construct
according to the procedures illustrated by Schemess 2, 3 and 6 and described
herein.
It is subjected to a first test which determines nucleoside kinase activity.
In one
embodiment, the method of the first test is by observing ADP formation which
is an
obligatory product of phospho group transfer from ATP using a coupled enzyme
assay. ADP, formed from substrate phosphorylation (in conjugate form), is used
by
pyruvate kinase to generate pyruvate from phospoenolpyruvate which in turn is
converted to lactate by lactate dehydrogenase. The lactate results in the
consumption
of NADH which is followed spectrophotometrically. The rate of substrate
phosphorylation (in conjugate form) is then directly related to the rate of
decrease in
the observed NADH signal. By the aforementioned methods, a linker of
appropriate
length and a nucleoside or nucleoside analog is found with an effective amount
of
kinase activity which may be expected to be retained in the drug conjugate.
The linker found in the first test is subjected to a second test in certain
embodiments, to determine suitability of the linker by connecting a second end
of the
linker precursor to a drug moiety. The site on the drug wherein the second end
of the
linker is attached is known to tolerate modification or may be shown to
tolerate
modification through a suitable functional group either pre-existing on the
drug or on
an analog thereof that is known to have an effective amount of the
pharmacological
activity of the parent drug. For example, paclitaxel modifications at C7, C10
and
C3'-N are known to be tolerated as described in Kingston, Fortschr. Chem. Org.
Naturst. 2002:84, 53-225, the disclosure of which is incorporated by
reference. In
another example, camptothecin analogs with suitable functionalities for linker
attachment are described in Wall, et. al., J. Med. Chem. 1993: 36, 2689-2700
whose
disclosure is incorporated by reference.
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In another embodiment, conjugates based upon Vinblastine are prepared by
use of the natural product 04-deacetyl Vinblastine or Vindesine prepared
according to
Barnett, et. al. J. Med. Chem. 1978: 21, 88-96, whose disclosure is
incorporated by
reference. Vindesine and 04-deacetyl Vinblastine are characterized by a free
hydroxyl group at C-4. Alternatively, vinblastine conjugates are prepared from
04-
deacetyl-3-de-(methoxycarbonyl)-vinblastin-3-yl carbonyl azide through
condensation with amines as described in Lavielle, et.al. J. Med. Chem. 1991:
34,
1998-2003, the disclosure of which is incorporated by reference. A second test
of a
drug-linker construct may then be determined by a functional assay which is
predictive of pharmacological activity. For example, microtubule stabilization
for
paclitaxel drug linker constructs or microtubule disruption by vinblastine
drug-linker
constructs is determined with a tubulin polymerization assay as described in
Barron
et. al. Anal. Biochem. 2003:315, 49-56 the disclosure of which is incorporated
by
reference.
Tubulin assembly or inhibition thereof can be monitored by fluorescence using
the CytoDYNAMIX ScreenTM 10 kit available from Cytoskeleton (1830 S. Acoma
St., Denver, CO). The kit is based upon an increase in quantum yield of
florescence
upon binding of a fluorophore to tubulin and microtubules and a l OX
difference in
affinity for microtubules compared to tubulin. Compounds such as paclitaxel
which
enhance tubulin assembly will therefore give an increase in emission whereas
compounds such as vinblastine which inhibit tubulin assembly will give a
decrease in
emission. Tubulin assembly or inhibition thereof can also be monitored by
light
scattering which is approximated by the apparent absorption at 350 nm.
In certain embodiments, doxorubicin-linker constructs can be screened by
monitoring alteration in the ability of Topoisomerase II to catalyze the
formation of
relaxed conformation DNA from a super-coiled plasmid.
In another embodiment, a functional assay for camptothecin drug-linker
constructs depends on Topoisomerase I binding to DNA an example of which is
given
in Demarquay, Anti-Cancer Drugs 2001:12, 9-19 the disclosure of which is
incorporated by reference. It should be appreciated that an appropriate linker
may
also be found by interchanging the order of the first and second tests.
In one embodiment, the linking moiety in the conjugates provided herein
contains an alkylene chain containing from 1 up to 50 main chain atoms other
than
hydrogen. In certain embodiments, the alkylene chain contains 2, 3, 4, 5, 6,
7, 8, 9, 10
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or 15 main chain atoms other than hydrogen. In other embodiments, the alkylene
chain contains 3, 4, 5, 6, 7, ~ or 9 main chain atoms other than hydrogen.
In other embodiment, the linking moiety in the conjugates provided herein
contains a polyethylene glycol (PEG) chain. The PEGS for use herein can
contain up
to 50 main chain atoms other than hydrogen. hi certain embodiments, the PEG
contains 5, 1 l, 13, 14, 22 or 29 main chain atoms other than hydrogen. In
certain
embodiments, the PEG contains 5, 11, 13 or 29 main chain atoms other than
hydrogen. In other embodiment, the linker moiety contains a combination of
alkylene, PEG and maleimide units in the chain. Some exemplary linking groups
incorporated into the conjuagates are provided in Table 2. As exemplified in
Table 2,
the linking groups are named based on the chemical units present and the
number of
main atoms, other than hydrogen are indicated in the parenthesis.
Table 2
Structure of Linker Groups Abbreviation
H2C~0~0~0~0~0
PEG(29)
H2C~0~0~0~0 ,
PEG(13)
H C~O~O~O~CH
PEG 11
( )
H2C~0~ ~OvCH2
PEG(5)
H2C~O~CH2
ALK(6)
H c~eH2
ALK(n)
CH2-(CH2)n-2-CH2
from drugl
from substrate ~ drug functional
O group fragment pEGa(14)
H2C~N~O~O~O~CH2
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from substrate
from drug/
,~ drug functional
H C N CH group fragment ALKa(9)
2 ~ 2
from substrate
from drug/
~ dru
functional
H ALKa(6)
g
H2C~N~CH group fragment
2
I '
from drug/ ~ O
drug functional ~N~
group fragment HzC H ~ [MALaPEGj(22)
S 0
C
H
2
O
u
from substrata
~
O
-
O
from drug/ ~ MAL(8)
~,
drug functional H
C N
~
S
group fragment 2 ~
from substrate
O
from drug/ ~, O
~ MAL
N 9
~
drug functiona (
C~ )
~
rou
fra
ment 2 ~
g
p
g
from substrate
a) Arrows indicates
site of attachment
to drug (or functionality
to drug)
and to substrate (or
functionality to
substrate). For unsymrnetrical
linker groups directionality
of attachment to
drug and substrate
is so
indicated
Several linker precursors useful in the conjugates provide herein are
described
in U.S. Pat. Nos. 5,512,667; 5,451,463; and 5,141,813. In addition, U.S. Pat.
Nos.
5,696,251; 5,585,422; and 6,031,091 describe certain tetrafunctional linking
groups
that can be used for the conjugates provided herein.
3. Substrates
The substrate moiety may be any substrate for a kinase that is overexpressed,
overactive or that exhibits undesired activity in a target system, wherein the
kinase is
other than a hexokinase, a protein kinase or a lipid kinase. In certain
embodiments,
the substrate has a molecular weight between about 50 amu and 1000 amu. The
kinase is present at a higher concentration or operates at a higher activity,
or the
activity is undesired or persistent in a cell type that contributes to the
genesis or
maintenance of the condition being treated in the target cell in comparison to
other
cells. Addition of a phosphate group by action of the kinase on the substrate
confers a
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negative charge to the conjugate, thus trapping or accumulating the conjugate
inside
the targeted cells at concentrations higher than will be achieved in other
cells not
involved with the condition being treated.
The action of the kinase on the substrate results in a modified conjugate in
the
target system (e.g. cell, tissue, organ), which is less able to exit the
target system in
comparison to the unmodified conjugate. In another embodiment, the kinase is
associated with an ACAMPS-related condition.
In certain embodiments, the substrate is a substrate for a kinase such as a
nucleoside kinase. In certain embodiments, the substrate is a substrate for a
kinase
such as thymidine kinase, viral thymidine kinase, human thymidine kinase TK-1,
deoxycytidine kinase, deoxyguanosine kinase. In other embodiments, the
substrate is
a substrate for viral thymidine kinase, human thymidine kinase TK-1.
In certain embodiments, the substrate is selected from nucleosides and their
natural and non-natural analogs. Examples of nucleosides for use as substrates
in the
conj ugates herein, but axe not limited to, cytidine, uridine, thyrnidine,
guanosine,
adenosine, or derivatives thereof. In one embodiment, the substrate is a
nucleoside or
nucleoside analog for thyrnidine kinase, viral thymidine kinase, TK-1,
deoxycytidine
kinase or deoxyguanosine kinase known or found to be activated in cells
associated
with ACAMPS-related conditions. Natural and non-natural nucleoside analogs are
contemplated herein. In another embodiment, the substrate is a nucleoside or
nucleoside base which is converted to a substrate of thymidine kinase, viral
thymidine
kinase, TK-1 or deoxycytidine kinase by the action of thymidine phosphorylase
or
cytidine deaminase.
Table 3 shows illustrative examples of known pyrimidine nucleoside analogs
which are substrates for thymidine kinase, and deoxycytidine kinase; and
purine
analogs which are substrates for deoxycytidine kinase and deoxyguanosine
kinase, for
use in the conjugates and methods provided herein. (Johansson et al., Acta
Biochim.
Polonica 43: 143-160 (1996); Eriksson et al., Biochem. Biophys. Res. Common.
176:
586-592 (1991); Wang et al., Biochemistry 38: 16993-16999 (1999)).
Deoxycytosine
kinase shows low enantioselectivity for 2'-deoxycytidine and analogs thereof
(Vern,
A. et al. Mol. Pharm. 51: 132-138 (1997)) and deoxyguanosine kinase and
deoxycytidine kinase show low enantioselectivities for 2'deoxyadenosine,
2'deoxyguanosine and analogs thereof (Gaubert, G., et al. Biochimie 81: 1041-
1047
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(1999)). Therefore, D- and L-pyrimidine and purine nucleoside analogs are
contemplated substrates.
Other contemplated substrates include pyrimidine analogs covalently linked to
a non-deoxy-ribose sugar having an anomeric carbon (a and (i anomers). Such
substrates include but are not limited to (3-L-2',3'-dideoxy-3'-thiacytidine
(3TC), [3-L-
1,3-dioxolane-cytidine (L-OddC), (North)-methanocarba-thymidine and analogs
thereof. Still other contemplated substrates are acyclic and carbocyclic
analogs of
guanosine which are known in the art as substrates for viral thymidine kinase.
(For a
review see De Clerq, D.E. et al., Nucleosides, Nucleotides & Nucleic Acids
20:271-
285 (2001)).
TABLE 3
Johansson et al., supra
O
HzC_
TK1 TK2 dCK
R4=H, R3=OH (Thy) 100 100 2
R4=H, R3=F (FLT) 30
R4=H, R3 N3 (AZT) 50 5
R4=H, R3=CH2N3 15 3
R4=H, R3=CCH <l <1
BBCR 1991, 176, 586
TK 1 TK2 dCK
R4=OH, R3=OH 2 3
(RiboThymidine)
R4=H, R3=H 40 4 <1
R4=H, R3=F (FLT) 30 <1 <1
R4=H, R3=N3 (AZT) 40 5 <1
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TKl TK2 dCK
R2=H, R4=H, R3=OH (due 100 100 6
R2=CH3, R4=H, R3=OH (Thy) 100 100 2
R2=H, R4=H, R3=H (ddl~ 10 2 <1
R2=F, R4=H, R3=OH 90 90
R2=Br, R4=H, R3=OH 80 10 <1
R2=NH2, R4=H, R3=OH 3 50 17
R2=Et, R4=H, R3=OH 80 10
R2= -CH2=CHaCH3, R4=H, R3=OH<1 40
TK1 TK2
R2=CH3, R5=F FMAU 45 100
R2=I, R5=F FIAU 42 90
R2=CH3, R5=OH araT 60
R2=H, R5=OH araU 20
R2=c Clo ro I, R5=OH 15
R2= H CHCH3 R5=OH 50
R2= -C=C-CH3a R5=OH~ 30
R2= 2-thien I, R5=OH 6
Erilcsson et al., supra
NHZ
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TK2 dCK
R4=H, RS=H, R3=OH (dC) 90 100
R4=H, RS=OH, R3=OH <1 120
(Cytosar, AraC)
R4=OH, RS=H, R3=OH <1 20
R4=H, RS=H, R3=H (ddCy, <1 30
Zalcitabine)
R4=F, RS=H, R3=OH 30 300
R4=H, RS=H, R3=F <1 60
HO C
O <1 4
H
HO-C-C=C=CH-Cy
H2 <1 20
(~)-Cytallene
Johansson et al., supra
TKl TK2 dCK
R4=H, RS=OH, R2=H <1 <l 120
(Cytosar, AraC)
R4=H, RS=F, R2=H 110
R4=H, RS=F, R2=F 110
R4=H, RS=F, R2=2-thienyl<1 100 10
R4=OH, RS=H, R2=H <1 <1 20
(Cyto sine)
R4=H, RS=H, R2=H (dC) <1 90 100
R4=N3, RS=H, R2=H 20
R4=F, RS=H, R2=H <1 30 300
R4=OCH3, RS=H, R2=H 80
38
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~2
R~_
TK2 dCK
R2=H, X=C (dC) 90 100
R2=CH3, X=C 40 60
R2=cyclopropyl, X=C <1 20
R2=Br, X=C 40 20
R2=F, X=C 90 20
~=N (5-aza) 2p
R2=2-thienyl, X=C 30 2
R2=3-thienyl, X=C 30 <1
R', =2-furyl, X=C 10 3
R''=3-furyl, X=C 50 <1
R2=2- yridyl, X=C 7 <l
R2=3-pyridyl, X=C <1 4
R2=4-pyridyl, X=C 2 5
Wang et al., supra
TK2 dCK
D-Th _ 100
~
_
-L-Th 60 0.8
a-D-Th 2.4
a -L-Th 1.5
-D-dC 93 100
-L-dC 70 40
a -D-dC 1.8 1.8
a -L-d C 0.4
-ddT 3'-deox Th 3.2
a -ddT 9.8
-ddC(2',3'-dideox 7.1
C
a -ddC 29
Johansson et al., supra
HO
X
R5
R3
39
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R dCK dGK
X=O, R3=OH, R5=H, B=Adenine dado 350 100
X=O, R3=OH, R5=H, B=Guanine dGuo 300 50
X=O, R3=OH, R5=H, B=Hypoxanthine 120 100
dlno
X=O, R3=OH, R5=H, B=7-deaza-Adenine 50 100
X=O, R3=OH, R5=H, B=2-CI-Adenine 260 180
X=O, R3=CH20H, R5=H, B=2-CI-Adenine 40
X=O, R3=OH, R5=OH, B=Adenine AraA 50 15
X=O, R3=OH, R5=OH, B=Guanine AraG 6 180
X=O, R3=OH, R5=OH, B=H poxanthine 290
X=CH2, R3=OH, R5=H, B=Guanine 70
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In certain embodiments, the substrate is a nucleoside or nucleoside analog
substrate for a thymidine kinase (TK). The TK is active within a cell type
that
contributes to the genesis or maintenance of a disease. Phosphorylation of the
nucleoside or nucleoside analog by the TK leads to trapping or accumulation of
the
conjugate within the targeted cell type due to the drug-conjugate acquiring a
negative
charge. Due to failure to introduce the foreign gene into every cancer cell,
previous
efforts with gene therapy to effect targeting of a nucleoside prodrug to
tumors by
introducing a foreign TK into the cancer cell has not lead to clinical success
(for
review see Fillat, et. al. Curr. Gene Ther. 2003: 3:13-26).
In another embodiment, the substrate is a nucleoside or nucleoside analog
substrate for human thyrnidine TK-1 or a viral TK. The drug- nucleoside or
drug-
nucleoside analog conjugate, in one embodiment, is effective in treating
cancer
through phosphorylation of the drug- nucleoside or drug-nucleoside analog
conjugate
by TK-1, leading in certain embodiment, to trapping or accumulation of the
conjugate
and hence the anti-cancer agent within the cancer cell. Therefore, trapping or
accumulation is responsible for the therapeutic effect of these conjugates in
the
treatment of cancer. The therapeutic effect is due to the accumulated anti-
cancer drug
which is active in the conjugate and is not due to the nucleoside or
nucleoside analog,
which simply serves as a substrate for the targeting enzyme. Furthermore, the
therapeutic effect of the drug conjugate is not dependent on release of free
drug. In
one embodiment, no further intervention of intracellular proteins is required
for
activation of the drug within the conjugate. Further action by thymidylate
kinase and
incorporation into DNA is not precluded as an additional enhancement of the
therapeutic effect of the drug conjugate in the treatment of cancer.
In certain embodiments, the drug moiety and/or the substrate moiety in the
conjugate can be present in a form of a pharmaceutically acceptable derivative
that
renders the conjugate biologically inactive. The inactive drug-substrate
conjugate can
be converted to the active drug-substrate conjugate under physiological
conditions or
by intracellular proteins without having the need to cleave the drug-substrate
conjugate_
The anti-cancer drug-nucleoside conjugates are effective in treating viral
infections, such as DNA or RNA viral infections, by using a viral TK which
results in
trapping or accumulation of a drug which is responsible for the therapeutic
effect of
these conj ugates. A cell infected with a RNA or DNA virus is distinguished by
a TK
41
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WO 2005/030258 PCT/US2004/031147
activity introduced by the virus into the cell. The viruses include, but are
not limited
to, HSV-1, HSV-2, VZV, EBV, CMV, HTLV-1 and HIV. A thymidine kinase that is
aberrant in a cell type involved in the genesis or maintenance of the
condition is used
to target the cell types with a conjugate of a drug to a nucleoside or
nucleoside analog
that is a substrate of the kinase. Addition of a phosphate group by action of
the
thymidine kinase on the nucleoside or nucleoside analog confers a negative
charge to
the conjugate thus trapping or accumulating the drug inside the targeted cells
at
concentrations higher than will be achieved in other cells not involved with
the
condition being treated. The therapeutic effect is due to the accumulated drug
which
is active in the conjugate and is not due to the nucleoside which simply
serves as a
substrate for the targeted enzyme or release of free drug. Therefore, as
discussed
above, no further intervention of intracellular proteins is required for
activation of the
drug within the conjugate though cleavage of the linker to give free drug. In
certain
embodiments, where the conjugate is present as a pharmaceutically acceptable
derivative of the conjugates provided herein, the intracellular proteins may
activate
the conjugate by converting the conjugate in the active form. The nucleoside
conferring an additional therapeutic effect is not precluded. Further action
by
thymidylate kinase and incorporation into DNA is not precluded as an
additional
enhancement of the therapeutic effect of the drug conjugate in the treatment
of viral
infections.
Thymidylate synthase (TS) and thymidine kinase (TK) are the key enzymes in
the synthesis of pyrimidine nucleotides required for cell division. In the de
novo
pathway TS catalyzes the reductive methylation of dUMP to dTMP. In the salvage
pathway TK directly catalyzes the phosphorylation of thymidine released from
cells
by DNA catabolism. Both enzymes are highly expressed in breast, gastric,
ovarian,
colorectal and bladder carcinomas to name a few. There are already a large
number
of anti-metabolite drugs that target TS, notably 5-fluorouracil (5-FU).
However,
patients treated with 5-FU rapidly develop resistance, resulting from
increased
expression of TS, TK or both. Of the two known human TK isozymes, TK1 and TK2,
TKl is preferentially up-regulated in carcinomas. Some virus encoded TKs have
been shown to differ in substrate specificity from the corresponding TK
isozymes in
the host cells (for review see Hannigan, et. al. Cancer Biother. 1993: 8, 189-
97).
In another embodiment, the substrate is a substrate for deoxycytidine kinase,
including, but not limited to, cytidine and uridine derivatives. Deoxycytidine
kinase
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(dCK) is known to phosphorylate cytostatic drugs (e.g., gemcitabine) for
activation.
It is contemplated that dCK may have greater tolerance towards uridine
derivatives, in
comparison to TK. For example, 5-amino-uridine which has been shown to have a
17
activity towards dCK may be used as a substrate, utilizing the amino group as
a
potential site for linkage to the conjugate. Furthermore, dCK is known to have
greater
tolerance towards carbohydrate modifications than thymidine kinase. For
example,
dCK shows only modest discrimination between the natural nucleoside (3-D-
cytidine
and its enantiomer (3-L-cytidine. Furthermore, stereochemistry at C-1 is not
critical
for recognition since oc-anomers are also accepted as substrates (see Wang, J.
et al.
Biochemistry 1999, 38: 16993 and Verri, A. et al. Mol. Pharm. 1997, 51: 132.)
Contemplated substrates include, but are not limited to, cytostatic
nucleosides known
in the art to be substrates for TK andlor dCK and anti-viral nucleosides known
in the
art to be substrates for viral thymidine kinase. Additional contemplated
substrates
include but are not limited to (3-D-, (3-L-, a-D-, and a-L-nucleoside analogs
and
acyclic carbohydrate analogs, which may also utilize the acyclic carbohydrate
as a
potential site for linkage to the conjugate.
4. Exemplary conjugates
In certain embodiments, the conjugates provided herein contain a substrate
that is a substrate for a nucleoside kinase and the conjugates have formula:
N-L-D
or a pharmaceutically acceptable derivative thereof, wherein N is a natural or
non-
natural nucleoside; L, which may or may not be present, is a non-releasing
linker and
D is a drug moiety. The drug is non-releasably linked to a carbohydrate or a
base
moiety of the nucleoside. In certain embodiments, the drug is linked to the
carbohydrate moiety of the nucleoside. In other embodiments, the drug is
linked to
the base of the nucleoside moiety of the nucleoside.
In certain embodiments, the conjugates have a formula:
S~ pl-L-D
or a pharmaceutically acceptable derivative thereof, wherein S~ is ribose,
deoxyribose or analog thereof; P1 is a purine, pyrimidine or analog thereof
and other
variables are as defined herein.
In certain embodiments, the conjugates have a formula:
Pi_S~_L_D~
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or a pharmaceutically acceptable derivative thereof, wherein S~ is ribose,
deoxyribose or analog thereof; P1 is a purine, pyrimidine or analog thereof
and other
variables are as defined herein.
In certain embodiments, the conjugates provided herein have formula:
O
R2 ~ R
' ~ ~N
N_ 'O
R~
W
R5
Rs R4
or a pharmaceutically acceptable derivative thereof,
wherein
Rl, R3, R4 and RS are each independently Y, H, hydroxy, halo, azido, C1-6
alkyl and optionally containing a heteroatom, C2-6 alkenyl or C2-6 alkynyl;
RZ is Y, H, C1-6 alkyl and optionally containing a heteroatom, C2-6 alkenyl,
C2-6 alkynyl, C3-6 cycloalkyl, aryl or heteroaryl;
R is Y, H or Cl-6 alkyl, C2-6 alkenyl or C2-6 alkynyl;
W is CReRf or O; Re and Rf are each independently H or Cl-6 alkyl;
Y is a linker-drug construct L-D, wherein L, which may or may not be present,
is a non-releasing linker and D a drug moiety;
R, Rl and R3 -RS are selected such that at least one of R, Rl and R3 -RS is Y
and at least one of R, Rl and R3 -RS is OH;
Rl-RS and R are unsubstituted or substituted with one or more substituents, in
one embodiment 1-4 substituents, in another embodiment 1 or 2 substituents,
each
independently selected from Ql.
In certain embodiments, Ql is halo, pseudohalo, hydroxy, oxo, thia, nitrite,
nitro, formyl, mercapto, hydroxycarbonyl, hydroxycarbonylalkyl, alkyl,
haloalkyl,
polyhaloalkyl, aminoalkyl, diaminoalkyl, alkenyl containing 1 to 2 double
bonds,
allcynyl containing 1 to 2 triple bonds, cycloalkyl, cycloalkylalkyl,
heterocyclyl,
heterocyclylall~yl, aryl, heteroaryl, aralkyl, arallcenyl, aralkynyl,
heteroarylalkyl,
trialkylsilyl, dialkylarylsilyl, alkyldiarylsilyl, triarylsilyl, alkylidene,
arylalkylidene,
alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, alkoxycarbonyl,
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alkoxycarbonylalkyl, aryloxycarbonyl, aryloxycarbonylalkyl, aralkoxycarbonyl,
aralkoxycarbonylalkyl, arylcarbonylalkyl, aminocarbonyl, alkylaminocarbonyl,
dialkylaminocarbonyl, arylaminocarbonyl, diarylaminocarbonyl,
arylalkylaminocarbonyl, alkoxy, aryloxy, heteroaryloxy, heteroaralkoxy,
heterocyclyloxy, cycloalkoxy, perfluoroalkoxy, alkenyloxy, alkynyloxy,
aralkoxy,
alkylcarbonyloxy, arylcarbonyloxy, aralkylcarbonyloxy, alkoxycarbonyloxy,
aryloxycarbonyloxy, aralkoxycarbonyloxy, aminocarbonyloxy,
alkylaminocarbonyloxy, dialkylaminocarbonyloxy, alkylarylaminocarbonyloxy,
diarylaminocarbonyloxy, guanidino, isothioureido, ureido, N-alkylureido, N-
arylureido, N'-alkylureido, N',N'-dialkylureido, N'-alkyl-N'-arylureido, N',N'-
diarylureido, N'-arylureido, N,N'-dialkylureido, N-alkyl-N'-arylureido, N-aryl-
N'-
alkylureido, N,N'-diarylureido, N,N',N'-trialkylureido, N,N'-dialkyl-N'-
arylureido, N-
alkyl-N',N'-diarylureido, N-aryl-N',N'-dialkylureido, N,N'-diaryl-N'-
alkylureido,
N,N',N'-triarylureido, amidino, alkylamidino, arylamidino, aminothiocarbonyl,
allcylaminothiocarbonyl, arylaminothiocarbonyl, amino, aminoalkyl,
alkylaminoalkyl,
dialkylaminoalkyl, arylaminoalkyl, diarylaminoalkyl, alkylarylaminoalkyl,
alkylamino, dialkylamino, haloalkylamino, arylamino, diarylamino,
alkylarylamino,
alkylcarbonylamino, alkoxycarbonylamino, aralkoxycarbonylamino,
arylcarbonylarnino, arylcarbonylaminoalkyl, aryloxycarbonylaminoallcyl,
aryloxyarylcarbonylamino, aryloxycarbonylamino, alkylsulfonylamino,
arylsulfonylamino, heteroarylsulfonylamino, heterocyclylsulfonylamino,
heteroarylthio, azido, -N'RS1R52R53~ P(Rso)Z~ P(=O)(RS°)Z,
OP(=O)(RS°)2, -
~60~(=O)R63a dialkylphosphonyl, alkylarylphosphonyl, diarylphosphonyl,
hydroxyphosphonyl, alkylthio, arylthio, perfluoroalkylthio,
hydroxycarbonylalkylthio,
thiocyano, isothiocyano, alkylsulfinyloxy, alkylsulfonyloxy, arylsulfinyloxy,
arylsulfonyloxy, hydroxysulfonyloxy, alkoxysulfonyloxy, aminosulfonyloxy,
alkylaminosulfonyloxy, dialkylaminosulfonyloxy, arylaminosulfonyloxy,
diarylaminosulfonyloxy, alkylarylaminosulfonyloxy, alkylsulfinyl,
alkylsulfonyl,
arylsulfinyl, arylsulfonyl, hydroxysulfonyl, alkoxysulfonyl, aminosulfonyl,
alkylaminosulfonyl, dialkylaminosulfonyl, arylaminosulfonyl,
diarylaminosulfonyl or
alkylarylaminosulfonyl; or two Q1 groups, which substitute atoms in a 1,2 or
1,3
arrangement, together form alkylenedioxy (i. e., -O-(CHZ)Y O-), thioalkylenoxy
(i. e., -
S-(CHZ)y O-)or alkylenedithioxy (i.e., -S-(CH~)y S-) where y is 1 or 2; or two
Ql
groups, which substitute the same atom, together form alkylene; and
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each Q1 is independently unsubstituted or substituted with one or more
substituents, in one embodiment one, two or three substituents, each
independently
selected from Q2;
each QZ is independently halo, pseudohalo, hydroxy, oxo, thia, nitrite, vitro,
formyl, mercapto, hydroxycarbonyl, hydroxycarbonylalkyl, alkyl, haloalkyl,
polyhaloalkyl, aminoalkyl, diaminoalkyl, allcenyl containing 1 to 2 double
bonds,
alkynyl containing 1 to 2 triple bonds, cycloalkyl, cycloalkylalkyl,
heterocyclyl,
heterocyclylalkyl, aryl, heteroaryl, aralkyl, aralkenyl, aralkynyl,
heteroarylalkyl,
trialkylsilyl, dialkylarylsilyl, alkyldiarylsilyl, triarylsilyl, alkylidene,
arylalkylidene,
alkylcarbonyl, arylcarbonyl, heteroarylcaxbonyl, alkoxycarbonyl,
alkoxycarbonylalkyl, aryloxycarbonyl, aryloxycarbonylalkyl, aralkoxycarbonyl,
aralkoxycarbonylalkyl, arylcarbonylalkyl, aminocarbonyl, alkylaminocarbonyl,
dialkylaminocarbonyl, arylaminocarbonyl, diarylaminocarbonyl,
arylalkylaminocarbonyl, alkoxy, aryloxy, heteroaryloxy, heteroaralkoxy,
heterocyclyloxy, cycloalkoxy, perfluoroalkoxy, alkenyloxy, alkynyloxy,
aralkoxy,
alkylcarbonyloxy, arylcarbonyloxy, aralkylcarbonyloxy, alkoxycarbonyloxy,
aryloxycarbonyloxy, aralkoxycarbonyloxy, aminocarbonyloxy,
alkylaminocarbonyloxy, dialkylaminocarbonyloxy, alkylarylaminocarbonyloxy,
diarylaminocarbonyloxy, guanidino, isothioureido, ureido, N-alkylureido, N-
arylureido, N'-allcylureido, N',N'-dialkylureido, N'-alkyl-N'-arylureido,
N',N'-
diarylureido, N'-arylureido, N,N'-dialkylureido, N-alkyl-N'-arylureido, N-aryl-
N'-
alkylureido, N,N'-diarylureido, N,N',N'-trialkylureido, N,N'-diallcyl-N'-
arylureido, N-
alkyl-N',N'-diarylureido, N-aryl-N',N'-diallcylureido, N,N'-diaryl-N'-
alkylureido,
N,N',N'-triarylureido, amidino, alkylamidino, arylamidino, aminothiocarbonyl,
alkylaminothiocarbonyl, arylaminothiocarbonyl, amino, aminoalkyl,
alkylaminoalkyl,
dialkylaminoalkyl, arylaminoalkyl, diarylaminoalkyl, alkylarylaminoallcyl,
alkylamino, dialkylamino, haloalkylamino, arylamino, diarylamino,
alkylarylamino,
alkylcarbonylamino, alkoxycarbonylamino, aralkoxycarbonylamino,
arylcarbonylarnino, arylcarbonylaminoalkyl, aryloxycarbonylaminoalkyl,
aryloxyarylcarbonylamino, aryloxycarbonylamino, alkylsulfonylamino,
arylsulfonylamino, heteroarylsulfonylamino, heterocyclylsulfonylamino,
heteroarylthio, azido, -N~RsIRszRs3~ P(Rso)~~ P(=O)(Rso)Z~
OP(=O)(Rs°)a, -
~60C(=O)R63~ dialkylphosphonyl, alkylarylphosphonyl, diarylphosphonyl,
hydroxyphosphonyl, alkylthio, arylthio, perfluoroalkylthio,
hydroxycarbonylalkylthio,
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thiocyano, isothiocyano, alkylsulfinyloxy, alkylsulfonyloxy, arylsulfinyloxy,
arylsulfonyloxy, hydroxysulfonyloxy, alkoxysulfonyloxy, aminosulfonyloxy,
alkylaminosulfonyloxy, dialkylaminosulfonyloxy, arylaminosulfonyloxy,
diarylaminosulfonyloxy, alkylarylaminosulfonyloxy, alkylsulfinyl,
alkylsulfonyl,
arylsulfmyl, arylsulfonyl, hydroxysulfonyl, alkoxysulfonyl, aminosulfonyl,
alkylaminosulfonyl, dialkylaminosulfonyl, arylaminosulfonyl,
diarylaminosulfonyl or
alkylarylaminosulfonyl; or two Qz groups, which substitute atoms in a 1,2 or
1,3
arrangement, together form alkylenedioxy (i. e., -O-(CHz)y-O-), thioalkylenoxy
(i. e., -
S-(CHz)~O-)or all~ylenedithioxy (i. e., -S-(CHz)y S-) where y is 1 or 2; or
two Qz
groups, which substitute the same atom, together form alkylene;
RS° is hydroxy, alkoxy, aralkoxy, alkyl, heteroaryl, heterocyclyl,
aryl or -
NR~°R~l, where R~° and R~l are each independently hydrogen,
alkyl, aralkyl, aryl,
heteroaryl, heteroaralkyl or heterocyclyl, or R'° and R'1 together form
alkylene,
azaalkylene, oxaalkylene or thiaalkylene;
R51, Rsz and R53 are each independently hydrogen, alkyl, aryl, aralkyl,
heteroaryl, heteroaralkyl, heterocyclyl or heterocyclylalkyl;
R6° is hydrogen, alkyl, aryl, aralkyl, heteroaryl, heteroaralkyl,
heterocyclyl or
heterocyclylalkyl; and
R63 is alkoxy, aralkoxy, alkyl, heteroaryl, heterocyclyl, aryl or -
NR'°R~l.
In certain embodiments, Rl is, H, hydroxy, halo, azido, C1-6 alkyl and
optionally containing a heteroatom, C2-6 alkenyl or C2-6 alkynyl. In one
embodiment, Rl is OH.
In certain embodiments, R3 is Y, H, hydroxy, halo, azido, C1-6 alkyl and
optionally containing a heteroatom, C2-6 alkenyl or C2-6 alkynyl. In other
embodiments, R3 is hydroxy. In other embodiments, R3 is Y.
In certain embodiments, R4 is Y, H, hydroxy, halo, azido, Cl-6 alkyl and
optionally containing a heteroatom, C2-6 alkenyl or C2-6 alkynyl. In other
embodiments, R4 1S hydroxy. In other embodiments, R4 is H. In other
embodiments,
R4 is Y.
In certain embodiments, RS is Y, H, hydroxy, halo, azido, Cl-6 allcyl and
optionally containing a heteroatom, C2-6 alkenyl or C2-6 alkynyl. In other
embodiments, RS is H. In other embodiments, RS is Y.
In certain embodiments, Rz is Y, H, Cl-6 alkyl and optionally containing a
heteroatom, C2-6 alkenyl, C2-6 alkynyl, C3-6 cycloalkyl, aryl or heteroaryl.
In other
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embodiments, R2 is H. In other embodiments, R2 is C1-6 alkyl. In other
embodiments, R~ is methyl. In other embodiments, R2 is Y.
In certain embodiments, R is Y, H or C1-6 alkyl. In one embodiment, R is H.
In another embodiment, R is C1-6 alkyl. In other embodiments, R is Y.
In certain embodiments, W is CReRf or O. In certain embodiments, W is O.
In certain embodiments, W is CReRf. In other embodiments, Re and Rf are each
H.
In certain embodiments, Y is -L-D, where L is a non-releasing linker and D is
a drug moiety. In other embodiments, Y is D. In certain embodiments, -L- is
-O-Ll-, where Ll is non-releasing linker. In other embodiments, -Ll- is
selected from a mono or bifunctional alkelene chain or mono or bifunctional
polyethylene glycol chain.
In certain embodiments, the conjugates have formula:
O D
Rz s L
'N
N' 'O
R~
W
R5
R3 Ra
or a pharmaceutically acceptable derivative thereof,
wherein, Rl and R3 are Hydroxy; R4 is H or F ; RS is H, OH or F; RZ is H,
C1-6 alkyl or halo ,W is O and other variables are as described herein.
In certain embodiments, the conjugates have formula:
O D
HsC i L/
'N
N_ 'O
HO
W
OH
or a pharmaceutically acceptable derivative thereof,
wherein, L is a non-releasing linker and D a drug moiety.
In another embodiment, the conjugates have formula:
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Ra Rb
~N~
R2a
~~ N
Rya N O
Wa
R5a
R3a R4a
or a pharmaceutically acceptable derivative thereof,
yvherein
Ria, R3a, Raa and Rsa are each independently Y; H, hydroxy, halo, azido, C1-6
alkyl and optionally containing a heteroatom, C2-6 alkenyl or C2-6 alkynyl;
R2a is Y, H, C1-6 alkyl and optionally containing a heteroatom, C2-6 alkenyl,
C2-6 alkynyl, C3-6 cycloalkyl, hydroxy, aryl or heteroaryl or halo ;
Y is a linker-drug construct L-D, wherein L, which may or may not be present,
is a non-releasing linker and D is a drug moiety;
Ra and Rb are each independently Y, H, or C1-6 alkyl;
Rd is H or C 1-6 alkyl;
Wa is CReRf or O; Re and Rf are each independently H or Cl-6 alkyl;
Rla-Rsa, Ra and Rb are selected such that at least one of Rla-Rsa, Ra and Rb
is
Y and at least one of Rla, R3a- Rsa is OH;
Ria-Rsa, Raa Rb and Rd are unsubstituted or substituted with one or more
substituents, in one embodiment 1-4 substituents, in another embodiment 1 or 2
substituents, selected from Q1.
In certain embodiments, Rl~, R3a, R4~ and Rsa are each independently Y, H,
hydroxy, halo, azido, C1-6 alkyl and optionally containing a heteroatom, C2-6
alkenyl
or C2-6 alkynyl. In certain embodiments, RIa is Y, H, hydroxy, halo, azido, C1-
6
alkyl and optionally containing a heteroatom, C2-6 alkenyl or C2-6 alkynyl. In
certain embodiments, Ria is Y or hydroxy. In certain embodiments, Rla is Y. In
certain embodiments, Rla is hydroxy. In certain embodiments, R3a is Y or
hydroxy,
In certain embodiments, R3a is Y. In certain embodiments, R3a is hydroxy. In
certain
embodiments, R4a is Y or hydroxy. In certain embodiments, R4a is Y. In certain
embodiments, R4a hydroxy. In certain embodiments, Rsa is Y or hydroxy. In
certain
embodiments, Rsa is Y. In certain embodiments, Rsa is H.
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In one embodiment, RZa is Y, H or C1-6 alkyl. In other embodiments, RZa is
Y. In other embodiments, RZa is H.
In other embodiment, Ra and Rb are each independently Y, H or C1-6 alkyl.
In other embodiment, Ra and Rb are each independently H.
In other embodiment, Wa is O.
In certain embodiments, Y is -L-D, where L is a non-releasing linker and D is
a drug moiety. In other embodiments, Y is D. In certain embodiments, -L- is
-O-(Ll)9 , where Ll is non-releasing linker and q is 0-2. In other
embodiments, -Le-
is selected from a mono or bifunctional allcelene chain or mono or
bifunctional
polyethylene glycol chain.
In another embodiment, the conjugates have formula:
Ra Rb
~N~
R2b
~~ N
HO N O
Wb
R3b' R4b
or a pharmaceutically acceptable derivative thereof, wherein
R3b and R4b are each independently Y, H, C1-6 alkyl and optionally containing
a heteroatom, C2-6 alkenyl or C2-6 alkynyl;
R2b is Y, H, C1-6 alkyl and optionally containing a heteroatom, C2-6 alkenyl,
C2-6 alkynyl, C3-6 cycloalkyl, aryl, heteroaryl or halo;
Y is a linker-drug construct L-D, wherein L, which may or may not be present,
is a non-releasing linker and D is a drug moiety;
Wb is CReRf or O; Re and Rf are each independently H or C1-6 alkyl;
Ra and Rb are independently Y, H, or C1-6 alkyl;
Rd i s H or C 1-6 alkyl;
Rib-R4b, Ra and Rb are selected such that at least one of Rlb-Rab, Ra and Rb
is
Y;
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Rlb-R4b~ Ra~ Rr ~d Ra are unsubstituted or substituted with one or more
substituents, in one embodiment 1-4 substituents, in another embodiment 1 or 2
substituents, selected from Ql.
In other embodiments, RZb is Y or H. In other embodiments, Rzb is Y.
In another embodiment, the conjugates have formula:
O
R2c
N~Ra
HO N O
We
Rgc R4c
or a pharmaceutically acceptable derivative thereof, wherein
R3° and R4° are each independently Y, H, C1-6 alkyl and
optionally containing
a heteroatom, C2-6 alkenyl or C2-6 alkynyl;
RZ° is Y, H, C1-6 alkyl and optionally containing a heteroatom, C2-6
alkenyl,
C2-6 alkynyl, C3-6 cycloalkyl, hydroxy, aryl, heteroaryl, or halo;
Rq is Y, H or C 1-6 alkyl;
Y is a linker-drug construct L-D, wherein L, which may or may not be present,
is a non-releasing linker and D is a drug moiety;
We is CReRf or O; Re and Rf are each independently H or C1-6 alkyl;
Ri°-R4° and R9 are selected such that at least one of
Rl°-R4° or R9 is Y;
Ri°-R4° and R9 are unsubstituted or substituted with one or more
substituents,
in one embodiment 1-4 substituents, in another embodiment 1 or 2 substituents,
selected from Q'.
In certain embodiments, Rl~ is Y. In certain embodiments, Rz° is Y. In
certain
embodiments, Rq is Y.
In another embodiment, the conjugates have formula:
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Rid
Z1
N ~ ~~Z2 _ R9d
Rad ~ n / Zs
R1c
Rsd
R3d R4d
or a pharmaceutically acceptable derivative thereof, wherein
Ria~ R3d~ R4a and Rsa are each independently Y, H, hydroxy, halo, azido, Cl-6
alkyl and optionally containing a heteroatom, C2-6 alkenyl or C2-6 alkynyl;
Rya is Y, H, hydroxy, halo, azido, Cl-6 alkyl and optionally containing a
heteroatom, C2-6 alkenyl, C2-6 alkynyl, thioalkyl or NRaRb;
Rga is Y, H, alkyl, halo, SRa or NRaRb;
R9a is Y, H, or C 1-6 alkyl;
Y is a linker-drug construct L-D, wherein L, which may or may not be present,
is a non-releasing linker and D is a drug moiety;
Ria-R9a a~.e selected such that at least one of Rla, R3a~ Raa~ Rsa or Rya is Y
and
at least one of Rla, R3a, Raa, Rsa or Rya is OH;
R_a and Rb are each independently Y, H, or C1-6 alkyl;
Rd is H or C 1-6 alkyl;
Wa is CReRf or O; Re and Rf are each independently H or C1-6 alkyl;
Z 1, Z~ and Z3 are each independently C or N;
Rla-R9a, Ra, Rb and Ra are unsubstituted or substituted with one or more
substituents, in one embodiment 1-4 substituents, in another embodiment 1 or 2
substituents, selected from Ql.
In another embodiment, the conjugates have formula:
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O
Z1a
N R2e
~'z2b . R9e
~ I
R8e_ _ ni Z3c
R1e
R5e
R3e R4e
or a pharmaceutically acceptable derivative thereof, wherein Rle, R3e, Rae and
Rse are each independently Y, H, hydroxy, halo, azido, C 1-6 alkyl and
optionally
containing a heteroatom, C2-6 alkenyl or C2-6 alkynyl;
Rye is Y, H, C1-6 alkyl and optionally containing a heteroatom, C2-6 alkenyl,
C2-6 alkynyl or C3-6 cycloalkyl,;
Rse is Y, H, alkyl, halo, SRd or NRaRb;
R9e is Y, H, or Cl-6 alkyl;
Y is a linker-drug construct L-D, wherein L, which may or may not be present,
is a non-releasing linker and D is a drug moiety;
Rie-Rse, Rse and R9e are selected such that at least one of Rle- Rse, Rae and
R9e
is Y and at least one of Rle, R3e, Rae and Rse is OH;
We is CReRf or O; Re and Rf are each independently H or C1-6 alkyl;
Zl, Za and Z3 are each independently C or N;
Rle- Rse, Rse and R9e axe unsubstituted or substituted with one or more
substituents, in one embodiment 1-4 substituents, in another embodiment 1 or 2
substituents, selected from Ql.
In another embodiment, the conjugates have formula:
R7f
Z1c
N ' ~~Z2c , Rsf
i
8f ~ / Z3c
R N
Rsf
R1 f
or a pharmaceutically acceptable derivative thereof, wherein, R6f is C1-10
alkyl and optionally containing a heteroatom, C2-10 alkenyl or C2-10 alkynyl;
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Rlf is Y or hydroxy;
Ref 1S Y, H, hydroxy, halo, azido, C1-6 alkyl and optionally containing a
heteroatom, C2-6 alkenyl, C2-6 alkynyl, SRa or NRaR~;
R8f is Y, H, alkyl, SRa, halo or NRaRb;
R9e is Y, H, or C1-6 alkyl;
Y is a linker-drug construct L-D, wherein L, which may or may not be present,
is a non-releasing linker and D is a drug moiety;
R~ ; Rsf and R9f are selected such that at least one of Rl ; Ref, R8f and R9f
is Y
and at least one of Ref and Rlf is OH;
Zl ; ZZf and Z3f are each independently C or N;
R' ; Rgf and R9f are unsubstituted or substituted with one or more
substituents,
in one embodiment 1-4 substituents, in another embodiment 1 or 2 substituents,
selected from Q1.
In certain embodiments, the conjugates provided herein have formula (3):
R2 O .~~D
'N
N'~O
R~ O
O
R5
R3 R4 (3)
or a pharmaceutically acceptable derivative thereof, wherein R3, R4 and RS are
each independently H, optionally substituted hydroxy, halo, azido, C1-6
optionally
substituted alkyl and optionally containing a heteroatom, C2-6 alkenyl or
alkynyl;
wherein RZ is H, C1-6 optionally substituted alkyl and optionally containing a
heteroatom, C2-6 alkenyl or alkynyl, C3-6 cycloalkyl, optionally substituted
hydroxy,
aryl or heteroaryl;
formula (4):
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O
D~L N.R
N ~O
RIO
O
R5
R3 R4 (4)
wherein R3, R4 and RS are independently H, optionally substituted hydroxy,
halo, azido, C1-6 optionally substituted alkyl and optionally containing a
heteroatom,
C2-6 alkenyl or alkynyl;
wherein R is H or C1-6 alkyl;
formula (5):
O
R2 N.R
N'~O
R~ O
O
R5
X R4
L
D (5)
wherein R4 is H, optionally substituted hydroxy, halo, azido, C1-6 optionally
substituted alkyl and optionally containing a heteroatom, C2-6 alkenyl or
alkynyl;
wherein R2 is H, Cl-6 optionally substituted alkyl and optionally containing a
heteroatom, C2-6 alkenyl or alkynyl, C3-6 cycloalkyl, optionally substituted
hydroxy,
aryl or heteroaryl;
wherein R i s H or C 1-6 alkyl;
wherein X is O or is absent;
formula (6):
R~ .R"
p,L N
'N
RIO N~O
5
R
R3 R4 (6)
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wherein R3, R4 and RS are independently H, optionally substituted hydroxy,
halo, azido, C1-6 optionally substituted alkyl and optionally containing a
heteroatom,
C2-6 alkenyl or alkynyl;
wherein each R' and R" are independently H or lower alkyl;
formula (7):
R,' N, R"
D
L 'N
N~O
R~ O
O~
R~R4 (~)
wherein R3 and R4 are independently H, optionally substituted, Cl-6
optionally substituted alkyl and optionally containing a heteroatom, C2-6
alkenyl or
alkynyl;
wherein R' and R" are independently H or lower alkyl;
formula (8):
R'' . R"
N
R2
~N
RIO N~O
-O
R5
R4
L
i
D (8)
wherein R4 and RS are independently H, optionally substituted hydroxy, halo,
azido, C1-6 optionally substituted alkyl and optionally containing a
heteroatom, C2-6
alkenyl or alkynyl;
wherein R2 is H, C1-6 optionally substituted alkyl and optionally containing a
heteroatorn, C2-6 alkenyl or alkynyl, C3-6 cycloalkyl, optionally substituted
hydroxy,
aryl or heteroaryl;
wherein R' and R" are independently H or lower alkyl;
wherein X is O or is absent;
formula (9):
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R~
Z1
~~Z Z2-L-D
R N
R1 O
W
R5
R3 R4 (9)
wherein R3, R4 and RS are independently H, optionally substituted hydroxy,
halo, azido, Cl-6 optionally substituted alkyl and optionally containing a
heteroatom,
C2-6 alkenyl or alkynyl;
wherein R~ is H, optionally substituted hydroxy, halo, azido, Cl-6 optionally
substituted alkyl and optionally containing a heteroatom, C2-6 alkenyl, C2-6
alkynyl,
NR'R"', wherein R' and R" are independently H or lower alkyl, or SR"", wherein
R"' is H or lower alkyl;
wherein Rg is H, halo, NR'R", wherein R' and R" are independently H or
lower alkyl, or SR"", wherein R"' is H or lower alkyl;
wherein W is C or O;
wherein Zl, ZZ and Z3 are independently C or N;
formula (10)
O
RN z1
'~Z2-L-D
R$ ~~N
R1 O
W
R5
R3 Ra. (10)
wherein R3, R4 and RS are independently H, optionally substituted hydroxy,
halo, azido, C1-6 optionally substituted alkyl and optionally containing a
heteroatom,
C2-6 alkenyl or alkynyl;
wherein R8 is H, halo, NR'R", wherein R' and R" are independently H or
lower alkyl, or SR"", wherein R"' is H or lower alkyl;
wherein R is H or lower alkyl;
wherein W is C or O;
wherein Zl, ZZ and Z3 are independently C or N;
or formula (11):
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R7
Z1
~~Z ZZ-L-D
R ~N
Rs
R1~ (11)
or a pharmaceutically acceptable derivative thereof, wherein R6 is an acyclic
C1-10 alkyl optionally substituted alkyl and optionally containing a
heteroatom, or
C2-10 alkenyl or alkynyl;
wherein R' is H, optionally substituted hydroxy, halo, azido, C1-6 optionally
substituted alkyl and optionally containing a heteroatom, C3-6 cycloalkyl, C2-
6
alkenyl, C2-6 alkynyl, NR'R"', wherein R' and R" are independently H or lower
alkyl, or SR"", wherein R"' is H or lower alkyl;
wherein R$ is H, halo or NR'R", wherein R' and R" are independently H or
lower alkyl; wherein W is C or O;
wherein Z1, Z2 and Z3 are independently C or N;
wherein for formulas 3-11 each L, which may or may not be present, is a non-
releasing linker moiety;
each D is a drug moiety; and
each Rl is H, or acyl.
In the above formulas 3-11, the linker-drug moiety may be attached at other
positions of the pyrimidine or purine nucleoside analog.
In certain embodiments, the conjugates are paclitaxel-thymidine conjugates.
In other embodiments, the paclitaxel-thymidine conjugates contain a non-
releasing
linker between paclitaxel and thymidine. In certain embodiments, the linker
contains
an alkylene chain or PEG chain. In certain embodiments, the linker is bonded
to
thymidine via a covalent bond. In certain embodiments, the linker is bonded to
paclitaxel via a first functionality. In certain embodiments, the first
functionality is a
carbamate. In other embodiments, the substrate is conjugated to paclitaxel at
C7
position. In one embodiment, the paclitaxel-thymidine conjugates have formula:
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or a pharmaceutically acceptable derivative thereof.
In other embodiments, the conjugates are paclitaxel-thymidine conjugates in
which the linker contains an alkylene chain or PEG chain and is bonded to
thymidine
via a covalent bond and to paclitaxel via a carbamate group at C10. In one
embodiment, the paclitaxel-thymidine conjugates have formula:
or a pharmaceutically acceptable derivative thereof.
In other embodiment, the paclitaxel-thymidine conjugates have formula:
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\ O~ H
O O
O
N 0~
H -
O ' O
OH . H p
O O
N~ \
O~N
O X
HO
or a pharmaceutically acceptable derivative thereof, where n is 1, 2, 3, 4, 5,
6,
7, ~, 9, 10 or more.
In certain embodiments, the conjugates are vinblastine-thymidine conjugates.
In certain embodiments, the conjugates are desacetyl vinblastine-thymidine
conjugates. In certain embodiments, the vinblastine-thymidine conjugates
contain a
non-releasing linker between vinblastine and thymidine. In certain
embodiments, the
linker contains an alkylene chain or PEG chain. In certain embodiments, the
linker is
bonded to thymidine via a covalent bond. In other embodiments, the linker is
bonded
to C3 of vinblastine via an amide group. In one embodiment, the vinblastine-
thymidine conjugates have formula:
OH
N '~/./
~C02Me
H
' N
OH s'///
MeO~N _
I O~
or a pharmaceutically acceptable derivative thereof,
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In certain embodiments, the conjugates are doxorubicin-thymidine conjugates.
In certain embodiments, the doxorubicin-thymidine conjugates contain a non-
releasing linker between doxorubicin and thymidine. In certain embodiments,
the
linker contains one or more groups containing maleimide, an alkylene chain and
PEG
chain. In certain embodiments, the linker is bonded to thymidine via a
covalent bond.
In other embodiments, the linker is bonded to doxorubicin via an amino group.
In one
embodiment, the doxorubicin-thymidine conjugates have formula:
O OH O
OH
~'OH
OCH30 OH O
O O
NH
HO
L N' LS_ ~ N,L
O~ v ~O
OH
where L' and L" are each independently selected from alkylene or PEG or a
pharmaceutically acceptable derivative thereof.
More examples of conjugates provided herein are provided in Table ?
C. Preparation of the conjugates
The conjugates provided herein can be prepared using any convenient
methodology. In one approach, the conjugates are produced using a rational
approach. In a rational approach, the conjugates are constructed from their
individual
components (e.g., drug, linker precursor and substrate). The components can be
covalently bonded to one another through functional groups known in the art.
Furthermore, the particular portion of the different components modified to
provide
for covalent linl~age will be chosen so as not to substantially adversely
interfere with
that component' s desired binding activity. For example, in a drug moiety, a
region
that does not affect the target binding activity will be modified, such that a
sufficient
amount of the desired drug activity is preserved.
The fiulctional groups can be present on the components or introduced onto
the components using one or more steps, such as oxidation, reduction, cleavage
reactions and the like. Examples of functional groups that can be used in
covalently
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bonding the components to produce the conjugate include but are not limited to
hydroxy, sulfliydryl, amino, carbonyl, and the like. Where desirable, certain
moieties
on the components may be protected using blocking groups, as is known in the
art,
see, e.g., Green & Wuts, Protective Groups in Organic Synthesis (John Wiley &
Sons) (1991).
In the following discussion nucleoside and nucleoside analog are
interchangeable terms. For the purpose of teaching the use of the conjugates,
Scheme
1 illustrates the general syntheses of linker nucleoside constructs with
linker
attachment at N-3 of a pyrimidine base of a nucleoside, followed by attachment
of a
drug to the linker-nucleoside construct.
Scheme 1 0
R O
Rx~ ~-~H
N O * Y~Z-PG )BASE ~ n
3
PGz-p' I n 2) Remove PG3 PGz-O N 0
l ~~r_----~''JO
X~PG~ W Drug X
~PG~
a
O H
, ' Z=NHR, O or S
Rz~N~Z Drug W=Halogen, OS(O)zR
'N~' ,O
PGz O_ I Z.NR, O or S
,I~YY~IO
W Drug
Deprotection X
~PG~
X~C02H or CHO,
W=NHR
R N~ ~ YY~[\\ Dr~~u ,,//1g
V
Deprotection N O
Drug~Linker-Nucleoside ~ PGz-p_ I Va0 or H,H
(R=CH3. H) 1r'~'~O
X~PG~
Examples of functional groups on the drug for attaching to the linker-
nucleoside
construct include, but are not limited to, COOH, CHO, halogen, NHR, or OH,
wherein m is a positive integer preferably between 1 and 20. The drug, linker
precuser, and nucleoside used in the illustrated methods of conjugation are
suitably
protected in a manner consistent with the conditions required to affect
conjugation,
and the appropriate choice of protecting groups are within the ordinary skill
of one in
the art of chemical synthesis.
Furthermore, the methylenes interposed between the two ends of the linker are
for illustrative purposes only, and should not be construed as a limitation to
the
conjugates provided herein. For example the methylene subunits may be switched
to
ethyleneoxy subunits to give a polyethylene glycol based linker. The linker
nucleoside
construct is prepared by the condensation of thymidine or a suitable
nucleoside analog
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with a linker precusor having a first end and a second end wherein the first
end
contains an appropriate leaving group and the second end contains a suitably
protected functional group. Condensation is effected with a base with either
the
hydroxyl groups of the nucleoside carbohydrate protected or in free form. The
intermediate with carbohydrate hydroxyl groups suitably protected is then
subjected
to deprotection to selectively remove a protecting group on the functional
group on
the second end of the linker.
With COOH as the functional group on the drug, and an amine as the free
functional group on the linker precursor, attachment of the linker-nucleoside
construct
to the drug uses amide bond coupling procedures well known in the art of
peptide
chemistry. Where the functional group on the drug is CHO, reductive amination
is
performed with NaBH4, NaCNBH4 NaB(OAc)3H or other suitable reducing groups to
provide the drug conjugate. Where the functional group on the drug is OH,
coupling
is affected by activation of the linker COOH group with DCC, or with any other
acid
activation agent well known in the art for ester bond formation. Where a
functional
group on the drug is halogen, alkylsulfonyloxy, arylsulfonyloxy, or any other
suitable
leaving group for nucleophilic displacement, conjugation is through
nucleophilic
displacement by the free amine of the linker-nucleoside construct in the
presence of
Et3N or any other appropriate acid scavenger.
When the free functional group on the linker of the linker-nucleoside
construct
is a thiol, condensation with the drug is effected by nucleophilic
displacement of a
leaving group on the drug, as given for the aforementioned nucleophilic
displacement
by free amine, after conversion of the thiol to a thiolate. The procedure for
drug
conjugation to a linker-nucleoside construct bearing a free thiol groups is
also
applicable to a free hydroxyl group on the linker to give a drug-linker-
nucleoside
conjugate with ether attachment of the linker to the drug.
The conjugates may also be constructed using the same aforementioned
chemical transformations for synthesis of drug-linker-nucleophile conjugates
by first
attaching the linker to the drug followed by attachment to N-3 of the
nucleoside base.
The purpose of the linker is to serve as a spacer between the drug and the
nucleoside
in order to remove or relieve steric interactions that may interfere with the
kinase
substrate activity of the nucleoside and/or the pharmacological effect of the
drug so
effective amounts of kinase and pharmacological activities remain. The linker
may
also be chosen based on its effect on the hydrophobicity of the drug conjugate
to
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improve passive diffusion into the target cells or tissue. It should also be
understood
that cleavage of a bond within a linker to generate free drug is not required
for the
pharmacological effect of the drug that is incorporated into a conjugate.
Example syntheses of nucleoside-linker constructs with linker attaclnnent to
the nucleoside C3'-O is generally illustrated in Scheme 2a.
Scheme 2a
0
Rz~N.PGz
Br~C02H 'N~O
O PG~.O
Rz N,pGz Oxidation
~ ', COpH, OH
PG~-O'
l'0 O O
Rz~N.PGz ~N NHzNHz
O' 1) RS(O)02CI, Bas ' U~1 ,e ~
L 2) Nucleophile (X) N- S0 X=
OH PG - O' I O PdIC, Hz or
N3
0 1) PPh3
O 2) NH40H
~X
O O
1) Mltsinobu rxn, Rz N.PGzz N~PGz
CH3C(O)SH
N~O
~O
2) Hydrolysis PG j0'
PGq O 1r'
0
~
O ~
SH '
O
'NHz
In one example, nucleoside constructs may be synthesized from known 3'-O-
(2-hydroxyethyl)-2'-deoxy-thymidine suitably protected at CS'-O and N3. In
another
example, nucleoside constructs may be synthesized from a suitably protected
CS'-O,
N3 uridine or a suitably protected CS'-O, C4-N cytidine wherein the
hydroxyethyl
group is introduced as reported for 2'-deoxy-thymidine. The hydroxyethyl group
is
transformed by Swern oxidation to provide an aldehyde functional group. A
carboxylic acid functional group may be introduced by reaction of a suitably
protected
2'-deoxy-ribonucleoside with C1CH2COONa (Edge, M.D., et al. J. Chem. Soc.
Perkin
Trans. 1, 290-4 (1973)).
The aldehyde or carboxylic acid functional groups may be further elaborated
to extend the linker, or may be used directly for attachment to a drug bearing
an
amino group. Additionally, the hydroxyethyl group may be activated for
nucleophilic
displacement, for example, by treatment with triflic anhydride. Nucleophilic
displacement may be from a drug nucleophile to give a drug-linker-nucleoside
construct, or with the anion derived from a protected amine including
phthalimido or
bis-(t-butyloxycarbonyl)amine, to introduce a protected amine. After selective
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removal of the amine protecting group or groups, the free amine may be used to
further extend the linker or may be used to attach the linker to the drug as
described
for Scheme 1. An alternative method to introduce the amino functional group
and a
method to introduce a thiol functional group are given in Teng, K., et al. US
Pat. No.
6,087,482 the disclosure of which is incorporated by reference.
Alternatively, drug-linker nucleoside constructs with linker attachments at
C3'-O may be synthesized by selectively condensing thymidine or 2'-
deoxyuridine
with suitable protection at CS'-OH, or 2'-deoxy-cytosine with suitable
protection at
CS'-OH and C4-NH2, using a linker precursor bearing an electrophile on a first
end
and a suitably protected functional group on a second end. In one example, the
electrophile is a chloromethyloxy group (Scheme 2b).
Scheme 2b
0
RZ N.PGZ
O Y-PG3
N O
PG~-O
O 2) Remove PG3 '=OH, NHS, C02H, CHO
OH
where n is 1-30.
Selective deprotection of the functional group on the linker in the linker-
nucleoside conjugate would provide a functional group for attachment to a drug
as
described for Scheme 1. Alternatively the second end is a functional group
such as a
selectively protected OH, N3, ester or a terminal alkene which may be
converted once
incorporated into the nucleoside-linker construct into an electrophile by for
example
selective deprotection to give a free OH which is transformed to an aldehyde
by
Swern oxidation or is sulfonated to provide a leaving group, Staudinger
reduction of
the azide to give an amine, selective hydrolysis of the ester to give a
carboxylic acid,
or ozonolysis of the terminal alkene to provide an aldehyde or hydroboration
to give a
terminal OH which is further manipulated as described.
Example syntheses of nucleoside-linker constructs with direct linker
attachment to the nucleoside C3' carbon is generally illustrated in Scheme 2c.
CA 02539914 2006-03-22
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Scheme 2c
0 0 0 0
N.PGZ Ri N.PG2 Rx N.PGZ RZ N.PGZ
O O ~) X Y-PGy O ~O
PG~-0 or PG~-O ~~ PGi-O or PG~-O
O O ~ O O
2) Removal of PG3
r
Y'n
Y=OH, NHR, CHO, COZH
The known C3'-alkynyl or C3-methyl alkynyl thymidine with suitable
protection is treated with an appropriate base such as KH to generate the
acetylide
which is condensed with a molecule with a third and a four end wherein the
third end
is an electrophile such as an arene- or alkane sulfonate or a halogen and the
fourth end
is an appropriately protected functional group thereby extending the linker
attached to
C3'. Selective deprotection would then give a functional group of the
nucleoside-
linker for fiu they extension of the linker or to attach the linker to the
drug as described
for Scheme 1. Prior to selective deprotection of the functional group, or
after
formation of the drug conjugate the alkyne may be reduced to the allcene or
alkane to
proved further dxug conjugates. Alternative methods to synthesize nucleosides
having
a C3'-C bond wherein the substituent to C3' contains a suitable functional
group for
drug attachment as described for Scheme 1 are given in Teng, K., et al. US
Pat. No.
6,087,482 the disclosure of which is incorporated by reference.
For conjugates with linker attachment at C-5 Scheme 3 teaches the synthesis
of linker-nucleoside constructs at C-5. In one route given in general in
Scheme 3a the
known 5-iodouracil with protected C3' and CS' hydroxyl groups or the hydroxyl
groups in free form is condensed with a alkyne on the first end of the linker
through a
Sinorogoshi coupling using an appropriate palladium catalyst well know in the
art of
Pd catalyzed cross coupling reactions to give a linker-nucleoside conjugate
with direct
attachment of a linker to a nucleoside through a carbon-carbon bond.
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Scheme 3a
~Y-PGs
n
~Y-PG~
An alternative route for linker attachment to CS through a carbon-carbon bond
employs the Heck reaction using an alkene on the first end of the linker. An
example
of an alkene based linker with an appropriate second end is ethyl acrylate. In
this
example hydrolysis with optional reduction of the double bond would provide a
COOH group for further linker elaboration or attachment to a drug a described
for
Scheme 1 to give a drug-linker-nucleoside conjugate either with alkene or
alkane
functionality within the linker. Reduction of the alkyne in either the
aforementioned
linker-nucleoside construct or in a final drug conjugate would also provide
linkers
having alkene and alkane functionality within the linker. Conjugates with
linker
attachment at C-5 of 2'deoxy-cytidine are made in similar fashion starting
from 5-
iodo-2'deoxycyrtidine.
An alternative to nucleoside drug conjugates with linker attachment to CS is
shown in general in Scheme 3b and begins with S-thin-2-deoxy uracil.
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Scheme 3b
O x Y PG~ O
HS n ~S~NH HMDSITMSCI S OTMS
---~ PG3 Y'~ /n- ~ PG3-Y
N O a n
H 1i - ~N'~OTMS
O
PG3-Y~S~N.PG2
1) Drug attachment
CS Drug~Linker~Nucleoside < N O ~ PG~ O
2) Deprotecticn PG~ 0' I O
1) ZnClz CI
2) N3 protection
PG~
O.PG~
JO~ O
X~NH Ci Hg NH ~) PG3 Y~S~Y-PGs
/n ~ ~n
N~O N~O
HO'
l /Y0\ I ~ HO 2) C5'-0, C3'-O protection
OH OH
Alkylation with a bi-functional molecule having an electrophile at a third end
such as halogen, alkyl or arenesulfonylchloride or a Michael acceptor and a
protected
nucleophile at a fourth end provides a S alkylated pyrimidine base wherein a
linker is
attached to C5. The nucleoside linker construct is then formed by silylation
of the
pyrimidine base with hexamethyldisilazane and a catalytic amount of
trimethylsilylchloride followed by condensation with an appropriately
protected oc-D-
ribosylchloride using ZnCl2 in CC14. Alternatively, 5-thio-2'deoxy-urudine may
be
S-alkylated to directly give the nucleoside-linker construct. For the
bifunctional
molecule the fourth end may be a functionality that may be converted to a
electrophile
or nucleophile once incorporated into a nucleoside-linker construct and
includes but is
not limited to a selectively protected OH, N3, ester or a terminal alkene .
Selective
deprotection or conversion of the functional group in the linker of the
nucleoside-
linker construct as described would provide a functional group for attachment
to a
drug as described for Scheme 1. An alternative route to the general structure
in
Scheme 3b is through halogen-metal exchange of a 5-halo-2'deoxyuridine
suitably
protected or in free form followed by cross-coupling with a symmetrical di-
sulfide
bearing a suitably protected functional group (see Bashkin, J.I~., et al. J.
Org. Chem.
55:5125-5132 (1990) and Bergstom, D. J. Amer. Chem. Soc. 111:374-375 (1989)).
Selective deprotection of the functional group incorporated in the nucleoside-
liker
construct provides a functional group which may be used to further extend the
linker
or may be used to attach the linker to the drug as described for Scheme 1.
68
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It should be appreciated that thymidine or uracil nucleoside constructs or
drug
conjugates so described may be converted to their corresponding 2'deoxy
cytidine
analogs by treatment with a chlorinating agent such as POCl3 to form a 4-Cl
pyrimidine intermediate which is condensed with ammonia or a substituted
amine.
Syntheses of exemplary drug-linker-nucleoside conjugates for paclitaxel are
given in
Schemes 4 and 5 to further teach the conjugates provided herein. In Scheme 4a
paclitaxel, appropriately protected at the more reactive C2' hydroxyl, is
condensed
with a linker-nucleoside construct wherein n is 0 or a positive integer,
preferably
where n is between O and 20, wherein the second end of the linker bears a
carboxylic
acid group.
o~o
PG~~CI O OH
O O
Ba~ ~NH 3_ pu~ 1~ ~ 7
O 0
PG~ OH _ Ii
O O O
O O
R~N~OH
n
N~0
PG3 O'
1r_~~'0
Paclitaxel~Linker~Nucieoside Deprotection X
(linker attachment to C7 of drug E ~PGy
and N3 to nucleoside)
Condensation of the second end to the protected taxane is by DCC or any
other appropriate coupling agent used for ester bond formation. Deprotection
then
gives the paclitaxel-linker-conjugate with linker attachment at C7of
paclitaxel.
In Scheme 4b, the known paclitaxel derivative having a free C10-OH and
C2'OH and C7-OH groups protected as silyl ethers is condensed with the linker-
nucleoside construct of Scheme 4a to form an ester bond at C10.
69
Scheme 4a
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Scheme 4b o
~N~OH ~o
n
~
N
O ~
PGq-p
OH ~ ' O,
O OPGz O ~
O 3~ 10 l/
7 X z
~NH . 0u \~
OPG~ O ~ O
OOH O H p Coupling
rD agent
A ~ 3
~NH = On
II~~II
Pacgtaxe4LinkerNucleoslde OPG~
Deprotectfon
(linker attachment
to C10 of drug E
and N3 to nucleoside)
where n is 0-30.
Following the general procedures as described for Scheme 4a the paclitaxel-
linker-nucleoside conjugate with linker attachment at C-10 is obtained.
In Scheme Sa, Baccatin III, appropriately protected at C7 is condensed with an
appropriately protected phenylisoserine using standard ester bond forming
conditions
to give an intermediate that is deprotected to give the free C3' amino group.
Scheme 5a
3
PG$ N . OH
PG~~CI H O.PG
_~ z
Coupling agent
O
O~ O O PG~
O
1) Remove PGa 3 10
PGa~HN O '
O OH aPGz OH . ti . 0
O O
PG -
q HN ~,n
Coupling agent
Condensation with a benzoic acid derivative containing a suitably protected
amine wherein rn is preferentially zero, one or two using standard amide bond
forming conditions then provides a paclitaxel derivative wherein a
functionality
amenable to conjugation is introduced into the C3'-N benzamido group.
Deprotection
of the amine followed by amide bond formation to a linker-nucleoside conjugate
wherein the second end of the linker bears a COOH group gives after final
CA 02539914 2006-03-22
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deprotection the desired paclitaxel-linker-nucleoside conjugate with
attachment at the
C3'-N benzamido group.
Scheme 5b
Remove PG4
Paclitaxel-Linker-Nucleoside
(linker attachment to C3'-N
benzamido group of drug
and N3 to nucleoside)
n.
Drug-linker-purine nucleoside conjugates corresponding to Formulas 9, 10
and 11 are prepared according to the procedures illustrated in Schemes 1, 4
and 5
using a purine nucleoside linker construct wherein the linker is attached to
C8 of the
purine. Appropriate starting materials are exemplified by known compounds 8-
bromo-2'deoxy-adenosine and 8-bromo-2'-deoxy-guanosine. For the syntheses of
purine nucleoside-linker constructs according to Scheme 1, Y is a nucleophile,
preferably a thiolate, which displaces the C8-Br and becomes incorporated into
the
purine nucleoside-linker construct, and Z is an appropriately protected
nucleophile or
electrophile which may be used in the reaction sequences exemplified by
Schemes 4
and 5 after selective deprotection.
Method for Preparation of Paclitxel C10 carbamates
Existing examples of paclitaxel C-10 carbamates prepared directly from
paclitaxel include some simple analogs derived from 10-O-deacetyl-7-0,10-O-bis-
[N-
(2,2.,2-trichloroethyloxy)-aminocarbonyl]-paclitaxel as reported in Bourzat,
J.Det al.;
EPO Application 524,093 (1993). This synthetic methodology, however, is not
versatile since selective reaction of the amine input at C-10 is possible only
in
dichloromethane. A more general approach for the synthesis of C-10 carbamates
starts
from 10-deacetyl-baccatin-III. However, subsequent steps to install the
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phenylisoserine side chain are problematic for amine inputs containing
additional
functional groups that require protection. Due to the chemical sensitivity of
the
taxane core, the protecting group strategy required for such amine inputs
would be
complex. Disclosed in the instant application is a method which permits the
use of
amine inputs containing additional functionality in free form. The disclosed
method
allows for the syntheses of C10 carbamates directly from paclitaxel that
otherwise
would be inaccessible or difficult to prepare.
A procedure for preparation of Paclitaxel C10 carbamates as provided herein
is illustrated in Schemes 7 and 8. Accordingly, compound 5a can be converted
in
nearly quantitative yield into its C10 carbonylimidazole 6a by reaction with
carbonyl-
diimidazole (CDT) in dichloromethane at room temperature. Compound 6a can be
reacted with amines in suitable solvents to yield the corresponding carbamate
8a,
which can be deprotected to give 9a. Typically, for primary and secondary
amines,
the reaction can be carried out in non-polar solvents, such as dichloromethane
or in
protic solvents such as IPA or t-BuOH at elevated temperatures.
Scheme 7:
CDI
6a
5a
X
O
where X is an amine.
In certain embodiments, the C10- carbonylimidazole 6a can be activated with
an alkylating agent such as an alkyl halide, alkyl sulfonate or di-alkyl-
sulfate to give a
Nl-alkyl-N3-acyl imidazolium species represented by 7a.of Scheme 8. In certain
embodiments the alkylating agent is selected from dimetylsulfate and methyl
iodide.
The imidazolium species can then be reacted with various amines either in free
or salt
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forms in protic solvents or aprotic solvents such as DMF, DMSO or dioxane. For
amine salts condensation with 7a is conducted in the presence of a hindered
base such
as DIEA. In certain embodiments, less reactive amines, such as arylamines or
heteroarylamines may be condensed with 7a to obtain paclitaxel C10 carbamates
with
N aryl or N heteroaryl linker attachment.
Various nucleophiles can be used in the reactions provided herein to prepare
C10 paclitaxel carbamates. Certain exemplary nucleophiles include, but are not
limited to, primary and secondary amines, amine containing acids, such as a-
amino
acids, amino-sugars, such as glucosamine, arylamines, heteroarylamines, and
a,a-
disubstituted alcohols.
Scheme 8:
Sa
X
~a
9a
The following reaction schemes illustrate general methods for the preparation
of conjugates provided herein.
a. Preparation of Thymidine-Linker constructs
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Cbz-CI, TEA
HO-L'-NHZ HO-L'-NHCbz
MeOH
1 2
TsCI, pyridine (R = OTs)
or
PPh3, CBr4 (R = Br)
R-L'-NHCbz
3
O
1) 3, KZC03 (K1) NHZ
N O acetone ! DMF
HO O
2) Pd / C, Hz
OH
4
where L' represents atoms between the first fixnctionality and the second
functionality
of the linker moiety.
i) Reaction of aminoalcohols with benzylchloroformate
To an aminoalcohol (1, 100 mol%) in MeOH is added benzylchloroformate
(150 mol%) and triethylamine (150 mol%). The reaction mixture is stirred for
16 h at
RT then concentrated to dryness to give a residue which is purified by silica
gel
chromatography resulting in a mono-Cbz-protected aminoalcohol of structure 2.
ii) Reaction of N Cbz-protected aminoalcohols with tosylchloride or
triphenylphosphine and carbon tetrabromide
For R = OTs, to a mono Cbz-protected aminoalcohol (2, 100 mol%) in pyridine is
added tosylchloride (100 mol%) at 0°C. The reaction mixture is stirred
for 16 h while
the solution is warmed up to RT then partitioned between ethyl acetate and
water.
The aqueous layer is extracted with ethyl acetate and the organic layer is
dried over
Na2S04 and concentrated to dryness to give a residue which is purified by
silica gel
chromatography. Alternatively, for R = Br, to the mono Cbz-protected
aminoalcohol
(2, 100 mol%) in DCM are added triphenylphosphine (100 mol%) and carbon
tetrabromide (100 mol%). The reaction mixture is stirred for 90 min at RT then
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concentrated to dryness to give a residue which is purified by silica gel
chromatography resulting in a N Cbz-protected amino linker of general
structure 3.
iii) Reaction of thymidine with N Cbz-protected amino bromides or tosylates
To thymidine (100 mol%) in acetone / DMF are added a N Cbz-protected amino
linker precursor (3, 100 mol%) and K2CO3 (200 mol%) (for R = Br, KI [0.15 mol
%]
is also added). The reaction mixture is stirred at 50°C for 48 h then
partitioned
between ethyl acetate and water. The aqueous layer is extracted with ethyl
acetate
and the organic layer is dried over NaaS04 and concentrated to dryness to give
a
residue which is purified by silica gel chromatography. The thymidine-linker
intermediate so obtained is subjected to catalytic hydrogenation using
methanol with
10 wt% palladium on carbon and stirring under an atmosphere of HZ for 16 h.
Filtration of the reaction mixture on Celite, removal of volatiles ih vacuo
and
lyophilization provides the thymidine-linker-amine intermediate of general
structure
4.
b. Preparation of Paclitaxel-Linker-Thymidine Conjugates with
Carbamate Linker Attachment at Paclitaxel C7
o ~ ~ No;
c1-
HO O ~ ~ OAc DMAP
~O
2) Pd I C, HZ
O'I JJO~~ ~'
Ac0 O p~NH-L' N' Y
I / O / O~ JN
O
I _ O H0~
H OH O", HO O H OA ~c
OH
/
i) Preparation of 7-O-(p-nitrophenyloxycarbonyl)-paclitaxel (6)
To 2'-(benzyloxycarbonyl)-paclitaxel prepared according to the procedure
described in. Chen, S.-H., et al., Tetralaed~on (1993) 49:2805-2828, dissolved
in
DCM are addedp-nitrophenylchloroformate and DMAP. The reaction mixture is
1)4 DIEA
CHzCIZ / DMF
CA 02539914 2006-03-22
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stirred for 1 h and concentrated to dryness. The resulting residue is purified
by silica
gel chromatography column eluting with 1:1 hexanes:ethyl acetate to give 6.
ii) Reaction of 7-O-(p-nitrophenyloxycarbonyl)-paclitaxel with
thymidine-linker-amines
To 2'-D-(benzyloxycarbonyl)-O-7-(p-nitrophenyloxycarbonyl)-paclitaxe1 (6,
100 mol%) is added a thymidine-linker-amine (4, 100 mol%) dissolved in DMF
followed by DIEA (1000 mol%) prepared as described above. The reaction mixture
is
stirred for 90 min then partitioned between ethyl acetate and water. The
aqueous
layer is extracted with ethyl acetate and the organic layer dried over NaZS04
and
concentrated to dryness to give a residue directly injected onto a preparative
RP-HPLC C-18 reversed phase column for purification (Method A). Fractions
containing the appropriate mass, as determined by analytical HPLC-MS (Method
B),
are pooled and the solvent is removed under reduced pressure. The 2'-O-
(benzyloxycarbonyl)-paclitaxel(C7-carbamoyl)-linker-thymidine so obtained is
subjected to catalytic hydrogenation using MeOH and HCl (200 mol%, introduced
as
a 1 M aqueous solution) with 10 wt% palladium on carbon and stirring under an
atmosphere of HZ for 16 h. Filtration of the reaction mixture on Celite,
removal of
volatiles ih vacuo and lyophilization provides the paclitaxel-
(C7-carbamoyl)-linker-thymidine conjugate of general structure 7.
c. Preparation of Deacetyl-Vinblastine-Linker-Thymidine Conjugates with
Amide Linker Attachment at C3 of Vinblastine
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1) NaHC03
2) HZN-NHZ
O~H-NHZ
Vinblastine
NaN02, HCI
OH
N '~o/
~ COaMe 4, DIEA
H , N E
i ~ ~ 'H I DMF / CHZCIp
~H "~i/
Me0 ~ i H = OH O
O~H-L' N~ O~ Ns
~
O~N
HO~
l~O-''-~~ IH
i) Synthesis of O4-3-de-(methoxycarbonyl)-vinblastin-3-yl-carbonyl azide (9)
04-3-de-(methoxycalrbonyl)-vinblastin-3-yl-carbonyl hydrazide (8), prepared
5 according to the procedure described in Bhushana, K.S.P Rao, et al., J. Med.
Chem.
(1985) 28:1079, is dissolved in a mixture of methanol and an aqueous 1 M HCl
solution. The solution is cooled to -10°C and then NaNOa is added at
once with
stirring. After 10 min the pH of the brownish-red solution is adjusted to 8.8
with a
saturated aqueous sodium bicarbonate solution and is extracted rapidly with
DCM and
10 washed with a saturated aqueous NaCI solution. The extracts are dried over
Na~S04
and concentrated. The solution of deacetylvinblastine acid azide (9) is used
directly
in the next step.
ii) Reaction of 04-3-de-(methoxycarbonyl)-vinblastin-3-yl-carbonyl azide
with thymidine-linker-amines
To a DCM solution of deacetylvinblastine acid azide 9, (100 mol%) is added a
DMF solution of a thymidine-linker-amine (4, 200 mol%) followed by DIEA (200
mol%). The reaction mixture is stirred for 3 h then partitioned between ethyl
acetate
and water. The aqueous layer is extracted with ethyl acetate and the organic
layer is
dried over NaaSO4 and concentrated to dryness to give a residue directly
injected onto
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a preparative RP-HPLC C-18 reversed phase column for purification (Method A).
Fractions containing the appropriate mass, as determined by analytical HPLC-MS
(Method B), are pooled and CH3CN is removed under reduced pressure. The
remaining aqueous mixture is lyophilized to give vinblastine-linker-thymidine
conjugate of general structure 10.
d. Preparation of Paclitaxel-Linker-Thymidine Conjugates with Carbamate
Linker Attachment at Paclitaxel C10
CDI
DCM
71 ~2
1) 4, IPA, 82 °C
2) HFIPyridine
OH
0
HO~
O
i) Preparation of 2'-O-(tent-butlyldimethylsilyl)-7-O-(tri-ethylsilyl)-10-O-
deacetyl, 10-O-(imidazoylcarbonyl)-paclitaxel (12)
To 2'-O-(tent-butyldimethylsilyl)-7-O-(triethylsilyl)-10-O-deacetyl paclitaxel
(11, 845 mg, 0.812 mmol), prepared according to the procedure described in
Datta,
A.; Hepperle, M. I. G. J.Org.Chem. (1995) 60:761, in anhydrous DCM (6 mL) is
added carbonyldiimidazole (53 0 mg, 400 mol%). The reaction mixture is allowed
to
stir for 16 hours at room temperature under nitrogen atmosphere then extracted
with
water (5 mL). The organic layer is dried over sodium sulfate, filtered and
concentrated to give 890 mg of the title compound 12 which is subsequently
used
without purification.
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ii) Reaction of 2'-O-(tent-butlyldimethylsilyl)-7-O-(tri-ethylsilyl)-10-O-
deacetyl,10-O-(imidazoylcarbonyl)-paclitaxel) with thymidine-linker-amines
To 2'-O-(tent-butlyldirnethylsilyl)-7-O-(tri-ethylsilyl)-10-O-deacetyl, 10-0-
(imidazoylcarbonyl)-paclitaxel (12, 100 mol%), dissolved in anhydrous
isopropyl
alcohol is added thymidine-liriker-amine 4 (500 mol%). The reaction mixture
was
stirred under reflux for 16 hours. The volatiles are then removed ira vacuo
and the
resulting residue is re-dissolved in DCM. The organic solution is then
extracted with
water and dried over sodium sulfate. After filtration and evaporation of the
volatiles
the residue is desilylated following the procedure in Ojima, I. et al. J. Med.
Chem.
(1997), 40:267. The residue so obtained is purified by preparative RP-HPLC
(Method
A). Fractions containing the appropriate mass, as determined by analytical
HPLC-MS
(Method B) are pooled and CH3CN removed under reduced pressure. The remaining
aqueous mixture is then lyophilized obtaining the desired paclitaxel-10-
deacety1,10-
oxycarbonylamino-linker-thyrnidine of general structure 13.
e. Preparation of a Doxorubicin-Linker-Thymidine with Alkyl Linker
Attachment at C3'-N on Doxorubicin and Linker Attachment at N3'-
Thymidine
O OH O
O OH O OH
OH O O
~~'OH
~~'OH
+ N " n H OCH30 OH O
OCH30 OH O
14. O O
O 15 NHS
NHZ 14a: n=1 HO L-N
HO
Doxorubicin O
~ HCI
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0 0
~~ L'-NHZ ~ N-L'-N'~'SH
O ~ -O
II N O O
HO O + HO~SH ---~ HO
O
OH
4 OH 16
O OH O
OH
~~'OH
O OH O OCHaO OH O
OH
~~'OH ~ O
15 NH
Linker HO ~"L N
OCH30 OH O
O O O
NH\ O
HO
L"-N~S~N-L' ~N
O O O-"N
17 HO O
OH
where L' and L" represent atoms between the first functionality and the second
functionality of the linker moiety.
i) Preparation of a Thymidine-linker-NHCOCHZCH2SH (16) suitable for
reaction with the alkyl anthracycline-maleimide intermediate
To a thymidine-linker-NH2 4 (100 mol%) in DMF prepared according to the
procedure described herein is added BOP (100 mol%), DIEA (400 mol%) and
HOOCCHZCH~SH (100 mol%). The reaction mixture is stirred for 30 min
whereupon DMF is removed ih vacuo. The crude is purified by silica gel P-TLC
eluted with DCM/CH30H (9:1) to yield a thiol containing thymidine of general
structure 16.
ii) Preparation of 3-(2,5-dioxo-2,5-dihydropyrrol-1-yl)-propionaldehyde (14)
To 1-(3-hydroxypropyl)-1H pyrrole-2,5-dione dissolved in 5 mL DCM. DMP
(15% wt in DCM) is added in one portion. After stirring the mixture for 2 h,
2-propanol is added followed by stirring for an additional 30 min. The
resulting
solution is filtered through a silica gel pad eluted with EtOAc, and the
filtrate is
concentrated. The crude product is purified by silica gel chromatography
eluting with
EtOAc/Hexane (2/1) to provide
~0
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3-(2,5-Dioxo-2,5-dihydro-pyrrol-1-yl)-propionaldehyde (14a) which is used
immediately.
iii) Preparation of an anthracycline-maleimide intermediate with N alkyl
attached to 3'-N of the anthracycline
To a stirred solution of doxorubicin hydrochloride, an aldehyde-maleimide
intermediate (14, 200-300 mol%) and glacial AcOH (195 mol%) in CH3CN/H20
(2:1) is added a 1 M solution of NaCNBH3 in THF (0.33 mol%). The mixture is
stirred under nitrogen atmosphere in the dark at RT for 1 h. The solution is
then
concentrated under vacuum to give a residue which is diluted with an aqueous
5%
NaHC03 solution and extracted with DCM. Concentration of the organic solution
and purification of the resulting residue by silica gel chromatography eluting
with
DCM/CH30H (20:1) provided the anthracycline-maleimide intermediate of general
structure 15.
iv) Preparation of a Doxorubicin-Linker-Thymidine with alkyl linker
attachment at C3'-N on Doxorubicin and linker attachment at N3'-
thymidine
To a DCM/CH30H (9:1) solution of 15 (100 mol%) is added a thiol containing
thymidine of general structure 16 (100 mol%). The mixture is stirred under
nitrogen
atmosphere in the darl~ for 30 min. The solvent is removed ih vacuo and the
resulting
crude residue is dissolved into by DMSO and purified on a preparative RP-HPLC
C-18 reversed phase column for purification (Method A). Fractions containing
the
appropriate mass, as determined by analytical HPLC-MS (Method B), are pooled
and
CH3CN is removed under reduced pressure or N2 stream followed by
lyophilization to
give the anthracycline-linker-thymidine conjugate of general structure 17.
Several conjugates have been prepared by following the procedures described
herein and slight modifications thereof. Tables 4-6 provide mass spectroscopy
(Electrospray) data for exemplary conjugates.
Table 4
_. Retention
Systematic Name Formula Moi Purity MS Tji
Weight ObservedL
n~
~X~ HP
eted G
Method
8
.
PXL-7Ca-ALKa 9 C66H79N50211278.371>95% 1279 1279 8.39'
-N3-THY M+H M+H
PXL-7Ca-ALK 6 C64H76N40201221.3192>95% 1222 1222 6.22
-N3-THY M+H M+H
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PXL-7Ca-ALK 6 -N3-5'-deoxC64H76N40191205.319893% 1206 1206 6.94
-THY M+H M+H
PXL-7Ca-ALK 6 -N3-aTHYC64H76N40201221.319298% 1222 1222 6.22
M+H M+H
PXL-7Ca-ALK 6 -N3-5,6-dihC64H78N40201223.33596% 1224 1224 6.24
dro-THY M+H M+H
PXL-7Ca-PEG 11 C66H80N40231297.37193% 1298 1298 6.18
-N3-THY M+H M+H
PXL-10Ca-ALK 6 C62H74N40191179.282>95%1179 1179 5.87
-N3-THY M+H M+H
PXL-10Ca-ALK 6 C62H76N40191181.297893% 1181 1181 5.73
-N3-5,6-dih dro-THY M+H M+H
PXL-lOCa-ALK 6 C62H74N4O191179.282>95%1179 1179 5.82
-N3-aTHY M+H M+H
PXL-lOCa-PEG 5 C60H70N4O201167.227899% 1167 1167 5.57
-N3-THY M+H M+H
PXL-1 OCa-PEG 5 C60H70N40201167.2278>95%1167 1167 5.55
-N3-aTHY M+H M+H
PXL-7Ca-PEG 5 -N3-THYC62H72N40211209.26597% 1209 1209 5.89
M+H M+H
PXL-lOCa-PEG 11 C64H78N40221255.333899% 1256 1256 5.57
-N3-THY M+H M+H
PXL-lOCa-PEG 11 C64H78N40221255.3338>95%1256 1256 5.58
-N3-aTHY M+H M+H
PXL-7Ca-ALK 6 -N3-5'-C64H77N4O23P1301.299194% 1302 1302 5.97
hos ho-THY M+H M+H
PXL-l OCa-ALK 3 C59H68N40191137.201695% 1137 1137 5.53
-N3-THY M+H M+H
PXL-7Ca-ALK 3 -N3-THYC61 1179.238894% 1179 1179 5.84
H70N4020 M+H M+H
PXL-7Ca-ALK 4 -N3-THYC62H72N4O201193.265699% 1193 1193 5.92
M+H M+H
PXL-7Ca-ALK 5 -N3-THYC63H74N40201207.292499% 1208 1208 6.07
M+H M+H
PXL-10Ca-ALK 5 C61 1165.255295% 1165 1165 5.75
-N3-THY H72N4019 M+H M+H
PXL-7Ca-ALK(8)-N3-THYC66H80N40201249.372899% 1250 1250 6.62
(M+H)(M+H)
Table 5
Retention
'f'~m~
Systemaflc vr~rruta [ Weight'uriiy~ ' S i7fiservadlam)::
Name ~~p~~~~d. .
i ,. (~-IPL~-y
Methc~ct
8
BL-3Am-ALK C59H79N7O121078.3128>95% 1079 1079 6.39*
6 -N3-THY M+H M+H
BL-3Am-ALK C59H79N70111062.31398% 1063 1063 4.32
6 -N3-DeTHY M+H M+H
BL-3Am-PEGa C63H86N80161211.416>95% 1212 1212 6.17
14 -N3-THY M+H M+H
BL-3Am-ALKa C58H76N80131093.284>95% 1094 1094 6.01
6 -N3-THY M+H M+H
BL-3Am-PEG C61 H83N7O151154.364694 1155 1155 3.88
11 -N3-THY M+H M+H
BL-3Am-PEG C57H75N7O131066.258699 1067 1067 3.81
-N3-THY M+H M+H
BL-3Am-ALK C59H80N7O15P1158.29295% 1159 1159 3.97
6 -N3-PhTHY M+H M+H
BL-3Am-ALK C56H73N70121036.23294% 1037 1037 3.83
3 -N3-THY M+H M+H
BL-3Am-ALK C57H75N7O121050.25996% 1051 1051 3.77
4 -N3-THY M+H M+H
Table 6
;~ert(i~n:
'
rs . Ti~~
~ystematia Name ~ormuia Mol$Ne3gh],i~urtty Ms (~,ts,>
-
Ei~p~Cfedobserved-(HPL~
. '-
_ _ , : , - ' ., n :: '. Method=
=
-
DOX-3'ALK-MALa 17 C53H67N5019S1110.1944N NA NA
-N3-THY
~DOX-3'Alk-[MALaPEG](22)-N3-THYC57H72F3N5024S1300.270194% NA NA
x TFA
D. Formulation of pharmaceutical compositions
5 The pharmaceutical compositions provided herein contain therapeutically
effective amounts of one or more of conjugates provided herein that are useful
in the
prevention, treatment, or amelioration of one or more of the symptoms of
ACAMPS
conditions. Such conditions include, but are not limited to, cancer, coronary
restenosis, osteoporosis and syndromes characterized by chronic inflammation
and/or
autoimmunity. Examples of chronic inflammation and/or autoimmune diseases
include but are not limited to rheumatoid arthritis and other forms of
arthritis, asthma,
psoriasis, inflammatory bowel disease, systemic lupus erythematosus, systemic
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dermatomyositis, inflammatory ophthalmic diseases, autoimmune hematologic
disorders, multiple sclerosis, vasculitis, idiopathic nephrotic syndrome,
transplant
rejection and graft versus host disease.
The compositions contain one or more conjugates provided herein. The
conjugates are preferably formulated into suitable pharmaceutical preparations
such
as solutions, suspensions, tablets, dispersible tablets, pills, capsules,
powders,
sustained release formulations or elixirs, for oral administration or in
sterile solutions
or suspensions for parenteral administration, as well as transdermal patch
preparation
and dry powder inhalers. Typically the conjugates described above are
formulated
into pharmaceutical compositions using techniques and procedures well known in
the
art (see, e.g., Ansel Introduction to Pharmaceutical Dosage Forms, Fourth
Edition
1985, 126).
In the compositions, effective concentrations of one or more conjugates or
pharmaceutically acceptable derivatives is (are) mixed with a suitable
pharmaceutical
Garner or vehicle. The conjugates may be derivatized as the corresponding
salts,
esters, enol ethers or esters, acids, bases, solvates, hydrates or prodrugs
prior to
formulation, as described above. The concentrations of the conjugates in the
compositions are effective for delivery of an amount, upon administration,
that treats,
prevents, or ameliorates one or more of the symptoms of conditions associated
with
ACAMPS. Such conditions include, but are not limited to, cancer, coronary
restenosis, osteoporosis and syndromes characterized by chronic inflammation
and/or
autoimmunity.
Typically, the compositions are formulated for single dosage administration.
To formulate a composition, the weight fraction of conjugate is dissolved,
suspended,
dispersed or otherwise mixed in a selected vehicle at an effective
concentration such
that the treated condition is relieved or ameliorated. Pharmaceutical carriers
or
vehicles suitable for administration of the conjugates provided herein include
any
such Garners known to those skilled in the art to be suitable for the
particular mode of
administration.
In addition, the conjugates may be formulated as the sole pharmaceutically
active ingredient in the composition or may be combined with other active
ingredients. Liposomal suspensions, including tissue-targeted liposomes, such
as
tumor-targeted liposornes, may also be suitable as pharmaceutically acceptable
carriers. These may be prepared according to methods known to those skilled in
the
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art. For example, liposome formulations may be prepared as described in U.S.
Patent
No. 4,522,811. Briefly, liposomes such as multilamellar vesicles (MLV's) may
be
formed by drying down egg phosphatidyl choline and brain phosphatidyl serine
(7:3
molar ratio) on the inside of a flask. A solution of a compound provided
herein in
phosphate buffered saline lacking divalent cations (PBS) is added and the
flask
shaken until the lipid film is dispersed. The resulting vesicles are washed to
remove
unencapsulated compound, pelleted by centrifugation, and then resuspended in
PBS.
The active conjugate is included in the pharmaceutically acceptable carrier in
an amount sufficient to exert a therapeutically useful effect in the absence
of
undesirable side effects on the patient treated. The therapeutically effective
concentration may be determined empirically by testing the conjugates in ih
vitro and
ih vivo systems described herein and then extrapolated therefrom for dosages
for
humans.
The concentration of active conjugate in the pharmaceutical composition will
depend on absorption, inactivation and excretion rates of the active
conjugate, the
physicochemical characteristics of the conjugate, the dosage schedule, and
amount
administered as well as other factors known to those of skill in the art. For
example,
the amount that is delivered is sufficient to ameliorate one or more of the
symptoms
of diseases or disorders associated with ACAMPS condition as described herein.
Typically a therapeutically effective dosage should produce a serum
concentration of active ingredient of from about 0.1 ng/ml to about 50-100
p,g/ml.
The pharmaceutical compositions typically should provide a dosage of from
about
0.001 mg to about 2000 mg of conjugate per kilogram of body weight per day.
Pharmaceutical dosage unit forms are prepared to provide from about 1 mg to
about
1000 mg and preferably from about 10 to about 500 mg of the essential active
ingredient or a combination of essential ingredients per dosage unit form.
The active ingredient may be administered at once, or may be divided into a
number of smaller doses to be administered at intervals of time. It is
understood that
the precise dosage anei duration of treatment is a function of the disease
being treated
and may be determined empirically using known testing protocols or by
extrapolation
from in vivo or ih vitf-o test data. It is to be noted that concentrations and
dosage
values may also vary with the severity of the condition to be alleviated. It
is to be
further understood that for any particular subject, specific dosage regimens
should be
adjusted over time according to the individual need and the professional
judgment of
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the person administering or supervising the administration of the
compositions, and
that the concentration ranges set forth herein are exemplary only and are not
intended
to limit the scope or practice of the claimed compositions.
Pharmaceutically acceptable derivatives include acids, bases, enol ethers and
esters, salts, esters, hydrates, solvates and prodrug forms. The derivative is
selected
such that its pharmacokinetic properties are superior to the corresponding
neutral
conjugate.
Thus, effective concentrations or amounts of one or more of the conjugates
described herein or pharmaceutically acceptable derivatives thereof are mixed
with a
suitable pharmaceutical carrier or vehicle for systemic, topical or local
administration
to form pharmaceutical compositions. Conjugates are included in an amount
effective
for ameliorating one or more symptoms of, or for treating or preventing
diseases or
disorders associated with ACAMPS condition as described herein. The
concentration
of active conjugate in the composition will depend on absorption,
inactivation,
excretion rates of the active conjugate, the dosage schedule, amount
administered,
particular formulation as well as other factors known to those of skill in the
art.
The compositions are intended to be administered by a suitable route,
including orally, parenterally, rectally, topically and locally. For oral
administration,
capsules and tablets are presently preferred. The compositions are in liquid,
semi-
liquid or solid form and are formulated in a manner suitable for each route of
administration. Preferred modes of administration include parenteral and oral
modes
of administration. Oral administration is presently most preferred.
Solutions or suspensions used for parenteral, intradernzal, subcutaneous, or
topical application can include any of the following components: a sterile
diluent,
such as water for inj ection, saline solution, fixed oil, polyethylene glycol,
glycerine,
propylene glycol, dimethyl acetamide or other synthetic solvent; antimicrobial
agents,
such as benzyl alcohol and methyl parabens; antioxidants, such as ascorbic
acid and
sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid
(EDTA);
buffers, such as acetates, citrates and phosphates; and agents for the
adjustment of
tonicity such as sodium chloride or dextrose. Parenteral preparations can be
enclosed
in ampules, disposable syringes or single or multiple dose vials made of
glass, plastic
or other suitable material.
In instances in which the conjugates exhibit insufficient solubility, methods
for solubilizing conjugates may be used. Such methods are known to those of
skill in
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this art, and include, but are not limited to, using cosolvents, such as
dimethylsulfoxide (DMSO), using surfactants, such as TWEEN~, or dissolution in
aqueous sodium bicarbonate.
Upon mixing or addition of the conjugate(s), the resulting mixture may be a
solution, suspension, emulsion or the like. The form of the resulting mixture
depends
upon a number of factors, including the intended mode of administration and
the
solubility of the conjugate in the selected carrier or vehicle. The effective
concentration is sufficient for ameliorating the symptoms of the disease,
disorder or
condition treated and may be empirically determined.
The pharmaceutical compositions are provided for administration to humans
and animals in unit dosage forms, such as tablets, capsules, pills, powders,
granules,
sterile parenteral solutions or suspensions, and oral solutions or
suspensions, and
oil-water emulsions containing suitable quantities of the conjugates or
pharmaceutically acceptable derivatives thereof. The pharmaceutically
therapeutically active conjugates and derivatives thereof are typically
formulated and
administered in unit-dosage forms or multiple-dosage forms. Unit-dose forms as
used
herein refers to physically discrete units suitable for human and animal
subjects and
packaged individually as is known in the art. Each unit-dose contains a
predetermined
quantity of the therapeutically active conjugate sufficient to produce the
desired
therapeutic effect, in association with the required pharmaceutical earner,
vehicle or
diluent. Examples of unit-dose forms include ampules and syringes and
individually
packaged tablets or cap sules. Unit-dose forms may be administered in
fractions or
multiples thereof. A multiple-dose form is a plurality of identical unit-
dosage forms
packaged in a single container to be administered in segregated unit-dose
form.
Examples of multiple-dose forms include vials, bottles of tablets or capsules
or bottles
of pints or gallons. Hence, multiple dose form is a multiple of unit-doses
which are
not segregated in packaging.
The composition can contain along with the active ingredient: a diluent such
as lactose, sucrose, dicalcium phosphate, or carboxymethylcellulose; a
lubricant, such
as magnesium stearate, calcium stearate and talc; and a binder such as starch,
natural
gums, such as gum acaciagelatin, glucose, molasses, polvinylpyrrolidine,
celluloses
and derivatives thereof, povidone, crospovidones and other such binders known
to
those of skill in the art. Liquid pharmaceutically administrable compositions
can, for
example, be prepared by dissolving, dispersing, or otherwise mixing an active
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conjugate as defined above and optional pharmaceutical adjuvants in a carrier,
such
as, for example, water, saline, aqueous dextrose, glycerol, glycols, ethanol,
and the
like, to thereby form a solution or suspension. If desired, the pharmaceutical
composition to be administered may also contain minor amounts of nontoxic
auxiliary
substances such as wetting agents, emulsifying agents, or solubilizing agents,
pH
buffering agents and the like, for example, acetate, sodium citrate,
cyclodextrine
derivatives, sorbitan monolaurate, triethanolaxnine sodium acetate,
triethanolamine
oleate, and other such agents. Actual methods of preparing such dosage forms
are
known, or will be apparent, to those skilled in this art; for example, see
Remington's
Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 15th Edition,
1975.
The composition or formulation to be administered will, in any event, contain
a
quantity of the active conjugate in an amount sufficient to alleviate the
symptoms of
the treated subject.
Dosage forms or compositions containing active ingredient in the range of
0.005% to 100% with the balance made up from non-toxic carrier may be
prepared.
For oral administration, a pharmaceutically acceptable non-toxic composition
is
formed by the incorporation of any of the normally employed excipients, such
as, for
example pharmaceutical grades of mannitol, lactose, starch, magnesium
stearate,
talcum, cellulose derivatives, sodium crosscarmellose, glucose, sucrose,
magnesium
carbonate or sodium saccharin. Such compositions include solutions,
suspensions,
tablets, capsules, powders and sustained release formulations, such as, but
not limited
to, implants and microencapsulated delivery systems, and biodegradable,
biocompatible polymers, such as collagen, ethylene vinyl acetate,
polyanhydrides,
polyglycolic acid, polyorthoesters, polylactic acid and others. Methods for
preparation of these compositions are known to those skilled in the art. The
contemplated compositions may contain 0.001%-100% active ingredient,
preferably
0.1-85%, typically 75-95%.
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The active conjugates or pharmaceutically acceptable derivatives may be
prepared with carriers that protect the conjugate against rapid elimination
from the
body, such as time release formulations or coatings.
The compositions may include other active conjugates to obtain desired
combinations of properties. The conjugates provided herein, or
pharmaceutically
acceptable derivatives thereof as described herein, may also be advantageously
administered for therapeutic or prophylactic purposes together with another
pharmacological agent known in the general art to be of value in treating one
or more
of the diseases or medical conditions referred to hereinabove, such as
diseases or
disorders associated with ACAMPS. It is to be understood that such combination
therapy constitutes a further aspect of the compositions and methods of
treatment
provided herein.
1. Compositions for oral administration
Oral pharmaceutical dosage forms axe either solid, gel or liquid. The solid
dosage forms are tablets, capsules, granules, and bulk powders. Types of oral
tablets
include compressed, chewable lozenges and tablets which may be enteric-coated,
sugar-coated or film-coated. Capsules may be hard or soft gelatin capsules,
while
granules and powders may be provided in non-effervescent or effervescent form
with
the combination of other ingredients known to those skilled in the art.
In certain embodiments, the formulations are solid dosage forms, preferably
capsules or tablets. The tablets, pills, capsules, troches and the like can
contain any of
the following ingredients, or conjugates of a similar nature: a binder; a
diluent; a
disintegrating agent; a lubricant; a glidant; a sweetening agent; and a
flavoring agent.
Examples of binders include microcrystalline cellulose, gum tragacanth,
glucose solution, acacia mucilage, gelatin solution, sucrose and starch paste.
Lubricants include talc, starch, magnesium or calcium stearate, lycopodium and
stearic acid. Diluents include, for example, lactose, sucrose, starch, kaolin,
salt,
mannitol and dicalcium phosphate. Glidants include, but are not limited to,
colloidal
silicon dioxide. Disintegrating agents include crosscarmellose sodium, sodium
starch
glycolate, alginic acid, corn starch, potato starch, bentonite,
methylcellulose, agar and
carboxymethylcellu.lose. Coloring agents include, for example, any of the
approved
certified water soluble FD and C dyes, mixtures thereof; and water insoluble
FD and
C dyes suspended on alumina hydrate. Sweetening agents include sucrose,
lactose,
mannitol and artificial sweetening agents such as saccharin, and any number of
spray
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dried flavors. Flavoring agents include natural flavors extracted from plants
such as
fruits and synthetic blends of compounds which produce a pleasant sensation,
such as,
but not limited to peppermint and methyl salicylate. Wetting agents include
propylene
glycol monostearate, sorbitan monooleate, diethylene glycol monolaurate and
polyoxyethylene laural ether. Emetic-coatings include fatty acids, fats,
waxes, shellac,
ammoniated shellac and cellulose acetate phthalates. Filin coatings include
hydroxyethylcellulose, sodium carboxymethylcellulose, polyethylene glycol 4000
and
cellulose acetate phthalate.
If oral administration is desired, the conjugate could be provided in a
composition that protects it from the acidic environment of the stomach. For
example, the composition can be formulated in an enteric coating that
maintains its
integrity in the stomach and releases the active conjugate in the intestine.
The
composition may also be formulated in combination with an antacid or other
such
ingredient.
When the dosage unit form is a capsule, it can contain, in addition to
material
of the above type, a liquid carrier such as a fatty oil. In addition, dosage
unit forms
can contain various other materials which modify the physical form of the
dosage
unit, for example, coatings of sugar and other enteric agents. The conjugates
can also
be administered as a component of an elixir, suspension, syrup, wafer,
sprinkle,
chewing gum or the like. A syrup may contain, in addition to the active
conjugates,
sucrose as a sweetening agent and certain preservatives, dyes and colorings
and
flavors.
The active materials can also be mixed with other active materials which do
not impair the desired action, or with materials that supplement the desired
action,
such as antacids, H2 blockers, and diuretics. The active ingredient is a
conjugate or
pharmaceutically acceptable derivative thereof as described herein. Higher
concentrations, up to about 98% by weight of the active ingredient may be
included.
Pharmaceutically acceptable carriers included in tablets are binders,
lubricants,
diluents, disintegrating agents, coloring agents, flavoring agents, and
wetting agents.
Enteric-coated tablets, because of the enteric-coating, resist the action of
stomach acid
and dissolve or disintegrate in the neutral or alkaline intestines. Sugar-
coated tablets
are compressed tablets to which different layers of pharmaceutically
acceptable
substances are applied. Film-coated tablets are compressed tablets which have
been
coated with a polymer or other suitable coating. Multiple compressed tablets
are
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compressed tablets made by more than one compression cycle utilizing the
pharmaceutically acceptable substances previously mentioned. Coloring agents
may
also be used in the above dosage forms. Flavoring and sweetening agents are
used in
compressed tablets, sugar-coated, multiple compressed and chewable tablets.
Flavoring and sweetening agents are especially useful in the formation of
chewable
tablets and lozenges.
Liquid oral dosage forms include aqueous solutions, emulsions, suspensions,
solutions and/or suspensions reconstituted from non-effervescent granules and
effervescent preparations reconstituted from effervescent granules. Aqueous
solutions
include, for example, elixirs and syrups. Emulsions are either oil-in-water or
water-in-oil.
Elixirs are clear, sweetened, hydroalcoholic preparations. Pharmaceutically
acceptable carriers used in elixirs include solvents. Syrups are concentrated
aqueous
solutions of a sugar, for example, sucrose, and may contain a preservative. An
emulsion is a two-phase system in which one liquid is dispersed in the form of
small
globules throughout another liquid. Pharmaceutically acceptable Garners used
in
emulsions are non-aqueous liquids, emulsifying agents and preservatives.
Suspensions
use pharmaceutically acceptable suspending agents and preservatives.
Pharmaceutically acceptable substances used in non-effervescent granules, to
be
reconstituted into a liquid oral dosage form, include diluents, sweeteners and
wetting
agents. Pharmaceutically acceptable substances used in effervescent granules,
to be
reconstituted into a liquid oral dosage form, include organic acids and a
source of
carbon dioxide. Coloring and flavoring agents are used in all of the above
dosage
forms.
Solvents include glycerin, sorbitol, ethyl alcohol and syrup. Examples of
preservatives include glycerin, methyl and propylparaben, benzoic add, sodium
benzoate and alcohol. Examples of non-aqueous liquids utilized in emulsions
include
mineral oil and cottonseed oil. Examples of emulsifying agents include
gelatin,
acacia, tragacanth, bentonite, and surfactants such as polyoxyethylene
sorbitan
monooleate. Suspending agents include sodium carboxymethylcellulose, pectin,
tragacanth, Veegum and acacia. Diluents include lactose and sucrose.
Sweetening
agents include sucrose, syrups, glycerin and artificial sweetening agents such
as
saccharin. Wetting agents include propylene glycol monostearate, sorbitan
monooleate, diethylene glycol monolaurate and polyoxyethylene lauryl ether.
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adds include citric and tartaric acid. Sources of carbon dioxide include
sodium
bicarbonate and sodium carbonate. Coloring agents include any of the approved
certified water soluble FD and C dyes, and mixtures thereof. Flavoring agents
include
natural flavors extracted from plants such fruits, and synthetic blends of
compounds
which produce a pleasant taste sensation.
For a solid dosage form, the solution or suspension, in for example propylene
carbonate, vegetable oils or triglycerides, is preferably encapsulated in a
gelatin
capsule. Such solutions, and the preparation and encapsulation thereof, are
disclosed
in U.S. Patent Nos 4,328,245; 4,409,239; and 4,410,545. For a liquid dosage
form,
the solution, e.g., for example, in a polyethylene glycol, may be diluted with
a
sufficient quantity of a pharmaceutically acceptable liquid Garner, e.g.,
water, to be
easily measured for administration.
Alternatively, liquid or semi-solid oral formulations may be prepared by
dissolving or dispersing the active conjugate or salt in vegetable oils,
glycols,
triglycerides, propylene glycol esters (e.g., propylene carbonate) and other
such
Garners, and encapsulating these solutions or suspensions in hard or soft
gelatin
capsule shells. Other useful formulations include those set forth in U.S.
Patent Nos.
Re 28,819 and 4,35 x,603. Briefly, such formulations include, but are not
limited to,
those containing a conjugate provided herein, a dialkylated mono- or poly-
alkylene
glycol, including, but not limited to, 1,2-dimethoxymethane, diglyme,
triglyme,
tetraglyme, polyethylene glycol-350-dimethyl ether, polyethylene glycol-550-
dimethyl ether, polyethylene glycol-750-dimethyl ether wherein 350, 550 and
750
refer to the approximate average molecular weight of the polyethylene glycol,
and one
or more antioxidants, such as butylated hydroxytoluene (BHT), butylated
hydroxyanisole (BI3A), propyl gallate, vitamin E, hydroquinone,
hydroxycoumarins,
ethanolamine, lecithin, cephalin, ascorbic acid, malic acid, sorbitol,
phosphoric acid,
thiodipropionic acid and its esters, and dithiocarbamates.
Other formulations include, but are not limited to, aqueous alcoholic
solutions
including a pharmaceutically acceptable acetal. Alcohols used in these
formulations
are any pharmaceutically acceptable water-miscible solvents having one or more
hydroxyl groups, including, but not limited to, propylene glycol and ethanol.
Acetals
include, but are not limited to, di(lower alkyl) acetals of lower alkyl
aldehydes such as
acetaldehyde diethyl acetal.
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In all embodiments, tablets and capsules formulations may be coated as known
by those of skill in the art in order to modify or sustain dissolution of the
active
ingredient. Thus, for example, they may be coated with a conventional
enterically
digestible coating, such as phenylsalicylate, waxes and cellulose acetate
phthalate.
2. Injectables, solutions and emulsions
Parenteral administration, generally characterized by injection, either
subcutaneously, intramuscularly or intravenously is also contemplated herein.
Injectables can be prepared in conventional forms, either as liquid solutions
or
suspensions, solid forms suitable for solution or suspension in liquid prior
to injection,
or as emulsions. Suitable excipients are, for example, water, saline,
dextrose, glycerol
or ethanol. In addition, if desired, the pharmaceutical compositions to be
administered
may also contain minor amounts of non-toxic auxiliary substances such as
wetting or
emulsifying agents, pH buffering agents, stabilizers, solubility enhancers,
and other
such agents, such as for example, sodium acetate, sorbitan monolaurate,
triethanolamine oleate and cyclodextrins. Implantation of a slow-release or
sustained-release system, such that a constant level of dosage is maintained
(see, e.g.,
U.S. Patent No. 3,710,795) is also contemplated herein. Briefly, a conjugate
provided
herein is dispersed in a solid inner matrix, e.g., polymethylmethacrylate,
polybutylmethacrylate, plasticized or unplasticized polyvinylchloride,
plasticized
nylon, plasticized polyethyleneterephthalate, natural rubber, polyisoprene,
polyisobutylene, polybutadiene, polyethylene, ethylene-vinylacetate
copolymers,
silicone rubbers, polydimethylsiloxanes, silicone carbonate copolymers,
hydrophilic
polymers such as hydrogels of esters of acrylic and methacrylic acid,
collagen, cross-
linked polyvinylalcohol and cross-linked partially hydrolyzed polyvinyl
acetate, that
is surrounded by an outer polymeric membrane, e.g., polyethylene,
polypropylene,
ethylene/propylene copolymers, ethylene/ethyl acrylate copolymers,
ethylene/vinylacetate copolymers, silicone rubbers, polydimethyl siloxanes,
neoprene
rubber, chlorinated polyethylene, polyvinylchloride, vinylchloride copolymers
with
vinyl acetate, vinylidene chloride, ethylene and propylene, ionomer
polyethylene
terephthalate, butyl rubber epichlorohydrin rubbers, ethylene/vinyl alcohol
copolymer, ethylene/vinyl acetate/vinyl alcohol terpolymer, and
ethylene/vinyloxyethanol copolymer, that is insoluble in body fluids. The
conjugate
diffuses through the outer polymeric membrane in a release rate controlling
step. The
percentage of active conjugate contained in such parenteral compositions is
highly
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dependent on the specific nature thereof, as well as the activity of the
conjugate and
the needs of the subj ect.
Parenteral administration of the compositions includes intravenous,
subcutaneous and intramuscular administrations. Preparations for parenteral
administration include sterile solutions ready for injection, sterile dry
soluble
products, such as lyophilized powders, ready to be combined with a solvent
just prior
to use, including hypodermic tablets, sterile suspensions ready for injection,
sterile
dry insoluble products ready to be combined with a vehicle just prior to use
and sterile
emulsions. The solutions may be either aqueous or nonaqueous.
If administered intravenously, suitable carriers include physiological saline
or
phosphate buffered saline (PBS), and solutions containing thickening and
solubilizing
agents, such as glucose, polyethylene glycol, and polypropylene glycol and
mixtures
thereof.
Pharmaceutically acceptable carriers used in parenteral preparations include
aqueous vehicles, nonaqueous vehicles, antimicrobial agents, isotonic agents,
buffers,
antioxidants, local anesthetics, suspending and dispersing agents, emulsifying
agents,
sequestering or chelating agents and other pharmaceutically acceptable
substances.
Examples of aqueous vehicles include Sodium Chloride Injection, Ringers
Injection, Isotonic Dextrose Injection, Sterile Water Injection, Dextrose and
Lactated
Ringers Injection. Nonaqueous parenteral vehicles include fixed oils of
vegetable
origin, cottonseed oil, corn oil, sesame oil and peanut oil. Antimicrobial
agents in
bacteriostatic or fungistatic concentrations must be added to parenteral
preparations
packaged in multiple-dose containers which include phenols or cresols,
mercurials,
benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters,
thimerosal, benzalkonium chloride and benzethonium chloride. Isotonic agents
include sodium chloride and dextrose. Buffers include phosphate and citrate.
Antioxidants include sodium bisulfate. Local anesthetics include procaine
hydrochloride. Suspending and dispersing agents include sodium
carboxymethylcelluose, hydroxypropyl methylcellulose and polyvinylpyrrolidone.
Emulsifying agents include Polysorbate 80 (TWEEN~ 80). A sequestering or
chelating agent of metal ions include EDTA. Pharmaceutical carriers also
include
ethyl alcohol, polyethylene glycol and propylene glycol for water miscible
vehicles
and sodium hydroxide, hydrochloric acid, citric acid or lactic acid for pH
adjustment.
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The concentration of the pharmaceutically active conjugate is adjusted so that
an injection provides an effective amount to produce the desired
pharmacological
effect. The exact dose depends on the age, weight and condition of the patient
or
animal as is known in the art.
The unit-dose parenteral preparations are packaged in an ampule, a vial or a
syringe with a needle. All preparations for parenteral administration must be
sterile,
as is known and practiced in the art.
Illustratively, intravenous or intraarterial infusion of a sterile aqueous
solution
containing an active conjugate is an effective mode of administration. Another
embodiment is a sterile aqueous or oily solution or suspension containing an
active
material inj ected as necessary to produce the desired pharmacological effect.
Injectables are designed for local and systemic administration. Typically a
therapeutically effective dosage is formulated to contain a concentration of
at least
about 0.1 % w/w up to about 90% w/w or more, preferably more than 1 % w/w of
the
active conjugate to the treated tissue(s). The active ingredient may be
administered at
once, or may be divided into a number of smaller doses to be administered at
intervals
of time. It is understood that the precise dosage and duration of treatment is
a
function of the tissue being treated and may be determined empirically using
known
testing protocols or by extrapolation from ih vivo or ih vitro test data. It
is to be noted
that concentrations and dosage values may also vary with the age of the
individual
treated. It is to be further understood that for any particular subject,
specific dosage
regimens should be adjusted over time according to the individual need and the
professional judgment of the person administering or supervising the
administration
of the formulations, and that the concentration ranges set forth herein are
exemplary
only and are not intended to limit the scope or practice of the claimed
formulations.
The conjugate may be suspended in micronized or other suitable form or may
be derivatized to produce a more soluble active product or to produce a
prodrug. The
form of the resulting mixture depends upon a number of factors, including the
intended mode of administration and the solubility of the conjugate in the
selected
carrier or vehicle. The effective concentration is sufficient for ameliorating
the
symptoms of the condition and may be empirically determined.
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3. Lyophilized powders
Of interest herein are also lyophilized powders, which can be reconstituted
for
administration as solutions, emulsions and other mixtures. They may also be
reconstituted and formulated as solids or gels.
The sterile, lyophilized powder is prepared by dissolving a conjugate provided
herein, or a pharmaceutically acceptable derivative thereof, in a suitable
solvent. The
solvent may contain an excipient which improves the stability or other
pharmacological component of the powder or reconstituted solution, prepared
from
the powder. Excipients that may be used include, but are not limited to,
dextrose,
sorbital, fructose, corn syrup, xylitol, glycerin, glucose, sucrose or other
suitable
agent. The solvent may also contain a buffer, such as citrate, sodium or
potassium
phosphate or other such buffer known to those of skill in the art at,
typically, about
neutral pH. Subsequent sterile filtration of the solution followed by
lyophilization
under standard conditions known to those of skill in the art provides the
desired
formulation. Generally, the resulting solution will be apportioned into vials
for
lyophilization. Each vial will contain a single dosage (10-1000 mg, preferably
100-
500 mg) or multiple dosages of the conjugate. The lyophilized powder can be
stored
under appropriate conditions, such as at about 4 °C to room
temperature.
Reconstitution of this lyophilized powder with water for injection provides a
formulation for use in parenteral administration. For reconstitution, about 1-
50 mg,
preferably 5-35 rng, more preferably about 9-30 mg of lyophilized powder, is
added
per mL of sterile water or other suitable carrier. The precise amount depends
upon
the selected conjugate. Such amount can be empirically determined.
4. Topical administration
Topical mixtures are prepared as described for the local and systemic
administration. The resulting mixture may be a solution, suspension, emulsions
or
the like and are formulated as creams, gels, ointments, emulsions, solutions,
elixirs,
lotions, suspensions, tinctures, pastes, foams, aerosols, irrigations, sprays,
suppositories, bandages, dermal patches or any other formulations suitable for
topical
administration.
The conjugates or pharmaceutically acceptable derivatives thereof may be
formulated as aerosols for topical application, such as by inhalation (see,
e.g., U.S.
Patent Nos. 4,044,126, 4,414,209, and 4,364,923, which describe aerosols for
delivery
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of a steroid useful for treatment of inflammatory diseases, particularly
asthma). These
formulations for administration to the respiratory tract can be in the form of
an aerosol
or solution for a nebulizer, or as a microfine powder for insufflation, alone
or in
combination with an inert Garner such as lactose. In such a case, the
particles of the
formulation will typically have diameters of less than 50 microns, preferably
less than
microns.
The conjugates may be formulated for local or topical application, such as for
topical application to the skin and mucous membranes, such as in the eye, in
the form
of gels, creams, and lotions and for application to the eye or for
intracisternal or
10 intraspinal application. Topical administration is contemplated for
transdermal
delivery and also for administration to the eyes or mucosa, or for inhalation
therapies.
Nasal solutions of the active conjugate alone or in combination with other
pharmaceutically acceptable excipients can also be administered.
These solutions, particularly those intended for ophthalmic use, may be
formulated as 0.01 % - 10°1o isotonic solutions, pH about 5-7, with
appropriate salts.
5. Compositions for other routes of administration
Other routes of administration, such as topical application, transdermal
patches, and rectal administration are also contemplated herein.
For example, pharmaceutical dosage forms for rectal administration are rectal
suppositories, capsules and tablets for systemic effect. Rectal suppositories
are used
herein mean solid bodies for insertion into the rectum which melt or soften at
body
temperature releasing one or more pharmacologically or therapeutically active
ingredients. Pharmaceutically acceptable substances utilized in rectal
suppositories
are bases or vehicles and agents to raise the melting point. Examples of bases
include
cocoa butter (theobroma oil), glycerin-gelatin, carbowax (polyoxyethylene
glycol)
and appropriate mixtures of mono-, di- and triglycerides of fatty acids.
Combinations
of the various bases may be used. Agents to raise the melting point of
suppositories
include spermaceti and wax. Rectal suppositories may be prepared either by the
compressed method or by molding. The typical weight of a rectal suppository is
about
2 to 3 gm.
Tablets and capsules for rectal administration are manufactured using the same
pharmaceutically acceptable substance and by the same methods as for
formulations
for oral administration_
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6. Articles of manufacture
The conjugates or pharmaceutically acceptable derivatives can be packaged as
articles of manufacture containing packaging material, a conjugate or
pharmaceutically acceptable derivative thereof provided herein, which is used
for
treatment, prevention or amelioration of one or more symptoms associated with
ACAMPS condition, and a label that indicates that the conjugate or
pharmaceutically
acceptable derivative thereof is used for treatment, prevention or
amelioration of one
or more symptoms associated with ACAMPS condition.
The articles of manufacture provided herein contain packaging materials.
Packaging materials for use in packaging pharmaceutical products are well
known to
those of skill in the art. See, e.g., U.S. Patent Nos. 5,323,907, 5,052,558
and
5,033,252. Examples of pharmaceutical packaging materials include, but are not
limited to, blister packs, bottles, tubes, inhalers, pumps, bags, vials,
containers,
syringes, bottles, and any packaging material suitable for a selected
formulation and
intended mode of administration and treatment. A wide array of formulations of
the
conjugates and compositions provided herein are contemplated as are a variety
of
treatments for any disorder associated with ACAMPS conditions.
E. Evaluation of the Activity of the Conjugates
Standard physiological, pharmacological and biochemical procedures are
available for testing the conjugates to identify those that possess biological
activity,
including kinase activity. In vitro and in vivo assays that can be used to
evaluate
biological activity, such as cytotoxicity, which will depend upon the
therapeutic agent
being used in the conjugate.
Exemplary assays are discussed briefly below with reference to cytotoxic
conjugates (see, also, Examples). It is understood that the particular
activity assayed
will depend upon the conjugated therapeutic agent.
1. Kinase activity
Thymidine kinase, viral thymidine kinase, TK-1 deoxycytidine kinase, and
deoxyguanosine kinase activities are determined by subjecting a first end of a
linker
used in synthesizing linker-substrate constructs to a first test. The first
test may
involve observing ADP formation, an obligatory co-product of phospho group
transfer from ATP which is catalyzed by the kinase to the CS' hydroxyl group
or its
equivalent in the nucleoside or nucleoside analog. Formation of ADP is
followed by
a coupled enzyme assay well known in the art. ADP, formed from kinase
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phosphorylation, is used by pyruvate kinase to generate pyruvate from
phosphoenolpyruvate which in turn is converted to lactate by lactate
dehydrogenase.
The lactate results in the consumption of NADH which is followed
spectrophotometrically. The rate of nucleoside phosphorylation is then
directly
related to the rate of decrease in the observed NADH signal.
Another test may involve monitoring the consumption of ATP. For example,
ATP concentrations at time 0 or after 4 hour incubation may be monitored by
luciferase reaction (PKLightTM kit obtained from Cambrex Corporation, One
Meadowlands Plaza, East Rutherford, NJ 07073), which generate a luminescence
readout in the presence of ATP. Assays are initiated by mixing a kinase and a
drug
conjugate in the presence of 40 pM ATP. After 4 hour of incubation at
30°C,
PKLightTM reagent is added and mixed well, and luminescence readout measured.
The rate of drug conjugate phosphorylation is then directly related to the
rate of
decrease in the observed luminescence. Based on the first test, linkers of
appropriate
lengths and substrate with an effective amount of kinase activity which may be
expected to be retained in the drug conjugate may be found. For paclitaxel
drug
conjugates BSA is employed in the first test to prevent drug conjugate
aggregation.
2. Tubulin polymerization assay
Drug-linker constructs may further be screened using functional assays
predictive of biological activity. In one example, microtubule stabilization
for
paclitaxel drug linker constructs or microtubule disruption by vinblastine
drug-linker
constructs is determined with a tubulin polymerization assay (Barron, et al.,
Anal.
Biochem. (2003) 315:49-56). Tubulin assembly or inhibition thereof may be
monitored by fluorescence using the CytoDYNAMIX ScreenTM 10 kit available from
Cytoskeleton (1830 S. Acoma St., Denver, CO). The kit is based upon an
increase in
quantum yield of florescence upon binding of a fluorophore to tubulin and
microtubules and a 1 OX difference in affinity for microtubules compared to
tubulin.
Emission is monitored at 405 nm with excitation at 360 nm. The compounds such
as
paclitaxel which enhance tubulin assembly will therefore give an increase in
emission
whereas compounds such as vinblastine which inhibit tubulin assembly will give
a
decrease in emission_ Tubulin assembly or inhibition may also be monitored by
light
scattering which is approximated by the apparent absorption at 350 nm. For
paclitaxel drug conjugates BSA is employed to prevent aggregation and
glycerol,
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which is a tubulin polymerization enhancer, is omitted from the kit to
increase the
signal to noise ratio.
In certain embodiments, activity of doxorubicin conjugates was assayed by
monitoring alteration in the ability of Topoisomerase II, by electrophoresis,
to
catalyze the formation of relaxed conformation DNA from a super-coiled
plasmid.
The more active a conjugate is at a particular concentration the less relaxed
conformation DNA is produced by the action of Topoisomerase II.
In another example, a functional assay for camptothecin drug-linker constructs
depends on inhibition of Topoisomerase I binding to DNA. In another example, a
functional assay for camptothecin drug-linker constructs depends on inhibition
of
Topoisomerase I binding to DNA (Demarquay, Auti-Cancer Drugs (2001) 12:9-19).
For each type of functional assay, the enzyme (kinase) and biochemical
microtubule polymerization results for all synthetic lots of each compound
were
combined and analyzed using GraphPad Prism~ software to generate the mean ~
SD.
For each specific cell-based assay, results from all assays carned out with
all
synthetic lots of each compound were combined and analyzed using Graph Pad
Prism
software~ to generate the mean ~ SD. Outliers (<7% of the total dataset) were
identified and removed prior to analysis using the method of Hoaglin et al.,
J. Amer.
Statistical Assoc., 81, 991-999, 1986. Compounds were tested between five and
twenty times (in triplicate) in each assay. The significance of differences
between the
cytotoxic ECsos of each compound against normal and tumor cell types
(cytotoxic
selectivity index) was determined with an unpaired t test (95% confidence
interval)
using GraphPad Prism~ software.
Tables 7-9 provide results for cytotoxicity, kinase activity and Topoisomerase
II assay for exemplary conjugates and their parent drugs provided herein.
Detailed
procedures for conducting the assays are provided in the Examples section. The
conjugates provided herein typically exhibit higher cytotoxic selectivity
index in
tumor cells as compared to their parent drugs. The conjugates are more
selective for
the tumor cells than the normal cells.
Average EC50 ("EC50-AVG") for is provided as follows: A < 0.1 ~,M, B =
0.02-0.1 ~M, C > 0.1-1.0 p.M and N/A = not available or inactive. Average TI~1
kinase activity is provided as follows: A <20, B = 20-40 C > 40 and N/A = not
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available or inactive. Average MPA activity is provided as follows: A <60, B =
60-
100 C > 100 and N/A = not available or inactive.
TABLE 7
MCF
7 HUVE
Ave. (ECSMCF7 HT29 HT29C HFF
TKlAve.0 (EC50(EC50(EC50(EC50(EC50
Systematic NaYl2~KinasMPA Ave)Ave) Ave) Ave)Ave)Ave)
a Act.ML SA ML SA ML ML
Act.
Paclitaxel (PXL)NlAC A A A A A A
PXL-7Ca-ALKa(9)-N3-
THY N/AB N/AN/A N/A N/A C C
PXL-7Ca-ALK A B A A B B B B
6 -N3-THY
PXL-7Ca-ALK(6)-N3-
DeTHY A A B A B A B B
PXL-7Ca-ALK(6)-N3-
aTHY NlAB C B C C C C
PXL-7Ca-ALK(6)-N3-
H2THY A B A A B A A A
PXL-7Ca-PEG(11)-N3-THYB A C A C A A A
PXL-l OCa-ALK(6)-N3-
THY A A B A B A B C
PXL-l OCa-Alk(6)-N3-
H2THY A B B A A A A A
PXL-l OCa-ALK(6)-N3-
aTHY A B B A B A B B
PXL-l OCa-PEG B C B N/A B A C C
-N3-THY
PXL-l OCa-PEG(5)-N3-
aTHY A B B A B A B C
PXL-7Ca-PEG(5)-N3-THYA A C A C A A A
PXL-1 OCa-PEG(
11 )-N3-
THY B C A N/A A B A C
PXL-l OCa-PEG(
11 )-N3-
aTHY A B C N/A C N/A C C
PXL-7Ca-ALK(6)-N3-
PhTHY N/AB C N/A C N/A C C
PXL-lOCa-Alk B C B N/A B N/A A B
3 -N3-THY
PXL-7Ca-ALK B B C N/A C N/A C C
3)-N3-THY
PXL-7Ca-ALK C B C N/A C N/A C C
4- -N3-THY
PXL-7Ca-ALK(5)-N3-THYA B C N/A C NlA C C
PXL-l OCa-ALK(5)-N3-
THY B B B N/A B NlA B B
PXL-7Ca-ALK(8)-N3-THYA B A N/A A N/A A B
Vinblastine A C A A A A A A
(VBL)
VBL-3Am-ALK(6)-N3-
THY A B B A A A A A
VBL-3Am-ALK(6)-N3-
DeTHY A B A A A A A A
VBL-3Am-ALK(6)-N3-
DeTHY A B A A A A A A
VBL-3Am-PEGa(14)-N3-
THY A C A A A A B B
VBL-3Am-ALKa(6)-N3-
THY A C B A C A B B
VBL-3Am-PEG(
11 )-N3-
THY B A A A A B A A
IVBL-3Am-PEG(5)-N3-THYA C A A A A A A
'VBL-3Am-ALK(6)-N3-
PhTHY N/AC A N/A A N/A A A
VBL-3Am-ALK(3)-N3-
THY A C B C B A A A
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MCF
HUVE
Ave. (ECSMCF7 HT29 HT29C HFF
TKlAve.0 (EC50(EC50(EC50(EC50(EC50
Systematic Name~nasMPA Ave)Ave) Ave) Ave)Ave)Ave)
a Act.ML SA ML SA ML ML
Act.
VBL-3Am-ALK(4)-N3
-
THY B C B A NlA A A B
Doxorubicin A C A NlA B N/A A A
(DOX)
DOX-3'ALK-MALa(17)-A A C N/A N/A N/A C C
N3-THY
DOX-3'Alk- A N/A N/AN/A N/A N/A C N/A
[MALaPEG](22)-N3-T'HY
~,Vinblastine A N/A N/AN/A NlA N/A C N/A
(VBL)
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TABLE 8. PACLITAXEL NON-TARGETED DERIVATIVES
Ave. MCF7MCF7 HT29HT29HT29HUVECHFF
TKl Ave.(EC50(EC50MCF7(EC50(EC50(EC50(EC50(EC50
KinaseMPAAve)Ave)(EC50)Ave)Ave)Ave)Ave) Ave)
stematic Name Act.Act.ML SA SSA ML SA SSAML ML
Paclitaxel XL A C A A A A A A A A
PXL-7Es-ALK 5 -NH2 - A C A A C A A A A
PXL-7Ca-ALK 6 -NHZ - A C A A A A A A a
PXL-7Ca-ALK(6)-Phos ho(OPh,- A B A A A A A A A
N-Ala)
PXL-7Ca-ALK 6 -di hen - A B A A A A A A A
1 hos horamidate
PXL-2'Alloc - A A A A A A A A A
PXL-lOEs-Alk 6 -NH Z - A A A A A A A A A
Deacet I Taxol - - B A A A A A A A
PXL-lOEs-ALK 5 -NH2 - B A A A A A A A A
PXL-lOCa-PECl(13)-NH(Z) - B A A A A A A A A
TABLE 9. VINBLASTINE NON-TARGETED DERIVATIVES
5
Ave. MCF7MCF7 HT29HT29HT29HUVECHFF
Tie2Ave.(EC50(EC50MCF7(EC50(EC50(EC50(EC50(EC50
KinaseMPAAve)Ave)(EC50)Ave)Ave)Ave)Ave) Ave)
stematic Name Act.Act.ML SA SSA ML SA SSAML ML
Vinblastine BL - C A A A A A A A A
VBL-3Am-ALK(8 -NH2 - B A A A A A A A A
VBL-3Am-ALK 6 -NH - A A A A A A A A A
B
VBL-3Am-ALK(6)-NH2 - C A A A A A A A A
VBL-3Am-ALK 12 -NH - A B A A C A A A A
B
VBL-3Am-ALK 12)-NH2 - B A A A A A A A A
VBL-3Am-PEG 11 -NH - B A A A A A A A A
B
VBL-3Am-PEG(11 -NH2 - B B A A B A A A A
Desacet lvinblastine- C A A A A A A A A
monoh drazine
Desacetyl vinblastine- C A A A A A A A A
~ ~ ~ ~ ~ ~
In certain embodiments, as demonstrated by a comparison of cytotoxic
selectivity index for an exemplary conjugate and parent drug in tumors and
normal
10 cells, the conjugates show increase in the cytotoxic selectivity index of
the conjugate
for tumor cells as compared to the cytotoxic selectivity index of the parent
drug:
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HFF MCF-7
Monolayer Soft Agar HT-29 Soft Agar
EC50
EC50 (nM) (nM) EC50 (nM)
9~5 6~3 152
Paclitaxel (n=20) (n=8) (n=6)
PXL-7Ca-
ALK(6)- 457 ~ 310 40 ~ 41 120 ~ 4
N3-Thy (n=16) (n=9) (n=5)
The improvement in the cytotoxic selectivity index of the
PXL-7Ca-ALK(6)-N3-Thy conjugate as compared to the cytotoxic selectivity index
of paclitaxel in exemplary cell lines, as illustrated by improved cytotoxic
selectivity
index index, is shown below:
Cytotoxic Selectivity Index
HFF/MCF7 HFF/HT29
Paclitaxel 1.4 0.6
PXL-7Ca-
ALK(6)-N3 -Thy 11.4 3.8
In certain embodiments, the conjugates show better serum stability as
compared to the parent drug as demonstrated by an exemplary conjugate below:
Compound Initial Conc. (~,M) Relative Percent Remaining at
T1/2 (hr)
0 hr 4 hr 8 hr 24 hr 72 hr
Paclitaxel 8.9 100 73 59 28 <3.0 11
PXL-7Ca- 10 100 84 90 80 38 55
ALK(6)-N3-Thy
One skilled in the art will appreciate that the assays described here may also
be used to screen for direct substrate-drug conjugates (i.e., conjugates which
contain
no linker).
ZS F. Methods of use of the conjugates and compositions
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Methods of use of the conjugates and compositions provided herein are also
provided. The methods involve both ih vitro and in vivo uses of the conjugates
and
compositions. The methods provided herein can be used for increasing drug
efficiency. In certain embodiments, methods for treating conditions caused by
undesirable chronic or aberrant cellular activation, migration, proliferation
or survival
(ACAMPS) are provided.
ACAMPS conditions are characterized by undesirable or aberrant activation,
migration, proliferation or survival of tumor cells, endothelial cells, B
cells, T cells,
macrophages, granulocytes including neutrophils, eosinophils and basophils,
monocytes, platelets, fibroblasts, other connective tissue cells, osteoblasts,
osteoclasts
and progenitors of many of these cell types. Examples of ACAMPS-related
conditions include, but are not limited to, cancer, coronary restenosis,
osteoporosis
and syndromes characterized by chronic inflammation and/or autoimmunity.
Examples of chronic inflammation and/or autoimmune diseases include but are
not
limited to rheumatoid arthritis and other forms of arthritis, asthma,
psoriasis,
inflammatory bowel disease, systemic lupus erythematosus, systemic
dermatomyositis, inflammatory ophthalmic diseases, autoimmune hematologic
disorders, multiple sclerosis, vasculitis, idiopathic nephrotic syndrome,
transplant
rejection and graft versus host disease.
Examples of cancers include, but are not limited to, non-small cell lung
cancer, small cell lung cancer, head and neck squamous cancers, colorectal
cancer,
prostate cancer, and breast cancer, acute lymphocytic leukemia, adult acute
myeloid
leukemia, adult non-Hodgkin's lymphoma, brain tumors, cervical cancers,
childhood
cancers, childhood sarcoma, chronic lymphocytic leukemia, chronic myeloid
leukemia, esophageal cancer, hairy cell leukemia, kidney cancer, liver cancer,
multiple myeloma, neuroblastoma, oral cancer, pancreatic cancer, primary
central
nervous system lymphoma, skin cancer, and small-cell lung cancer. Childhood
cancers amenable to treatment by the methods and with the compositions
provided
herein include, but are not limited to, brain stem glioma, cerebellar
astrocytoma,
cerebral astrocytoma, ependymoma, Ewing's sarcoma and family of tumors, germ
cell
tumor, Hodgkin's disease, ALL, AML, liver cancer, medulloblastoma,
neuroblastoma, non-Hodgkin's lymphoma, osteosarcoma, malignant fibrous
histiocytoma of bone, retinoblastoma, rhabdomyosarcoma, soft tissue sarcoma,
supratentorial primitive neuroectodermal and pineal tumors, unusual childhood
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cancers, visual pathway and hypothalamic glioma, Wilms' tumor, and other
childhood
kidney tumors.
The methods and compositions provided can also be used to treat cancers that
originated from or have metastasized to the bone, brain, breast, digestive and
gastrointestinal systems, endocrine system, blood, lung, respiratory system,
thorax,
musculoskeletal system, and skin. The methods are generally applicable to all
cancers
but have particularly significant therapeutic benefit in the treatment of
solid tumors.
In certain embodiments, the solid tumors are characterized by extensive
regions of
hypoxic tissue. In certain embodiments, the drug moieties provided in Table 7
are
used in the conjugates, which are used in treating particular types of cancer.
TABLE 7
Drug Selection
Paclitaxel (Taxane susceptible to MDR)
~ Breast, Lung, Prostate, Ovarian, Head & Neck, Esophageal, Bladder
Doxorubicin (Anthracycline / MDR)
Breast, Lung, Ovarian, Bladder, Hepatoma, Neuroblastoma, Lymphoma
Vinblastine (Vinca Alkaloid l MDR)
~ Breast, Lung, Prostate, Testicular, Renal, Lymphoma
Methotrexate (Antimetabolite)
~ Breast, Colorectal, Head & Neck, Leukemia, Lymphoma
Cisplatin (DNA Crosslinking Agent)
~ Lung, Ovarian, Head & Neck, Esophageal, Bladder, Lymphoma
G. Library and Screening Methods
In certain embodiments, the conjugates provided herein are produced using
combinatorial methods to produce large libraries of potential conjugates.
Methods for
producing and screening combinatorial libraries of molecules are known in the
art.
The libraries of potential conjugates may then be screened for identification
of a
conjugate with the desired characteristics. Any convenient screening assay may
be
employed, where the particular screening assay may be known to those of skill
in the
art or developed in view of the specific molecule and property being studied.
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For example, the libraries of potential conjugates may be screened for
selectivity by comparing the conjugate activity in the target cell or tissue
type to
conjugate activity in cells or tissues in which drug activity is not desired.
A selective
conjugate will affect the target in the desired cells (e.g., cells involved in
a disease
process), but affect the target in undesired cells to a lesser extent or not
at all. In
another example, the libraries of potential conjugates may be screened for
conjugates
that exhibit enhanced drug efficiency as compared to the pharmacological
activity of
the unconjugated drug. For example, a more efficient drug will result in a
desirable
pharmacological response at a lower effective dose than a less efficient drug.
In
another example, a more efficient drug will have an improved cytotoxic
selectivity
index compared to a less efficient drug. In one example, the screening assay
will
involve observing the accumulation of the conjugate in the target system, in
comparison to that of the unconjugated drug.
H. High throughput screening and target identification methods for kinase
substrate trapping using drug-linker- conjugate libraries
Provided herein is a broadly applicable method for specifically targeting and
trapping non-specific drugs in cancer. In one embodiment, the conjugates
provided
are distinguished by retention of drug activity or a significant fraction
thereof within
the conjugate and therefore do not rely on release of free drug or activation
of the
drug by an infra-cellular protein. In one embodiment, the drug moiety and/or
the
substrate moiety in the conjugate can be present in a form of a
pharmaceutically
acceptable derivative that renders the conjugate biologically inactive. The
inactive
drug-substrate conjugate can be converted to the active drug-substrate
conjugate
under physiological conditions or by intracellular proteins without having the
need to
cleave the drug-substrate conjugate. In other embodiments, the conjugates are
selectively targeted or trapped by cancer or viral infected cells due to
phosphorylation
of the substrate (e.g. , nucleoside or nucleoside analog by a TK) whose
activity is
involved in the condition being treated.
Accumulation of the drug conjugate into the cancer or viral infected cell
types
will occur by pushing the equilibrium of passive diffusion towards the cancer
or viral
infected cells as a result of preferential trapping due to the higher kinase
activity
within these cell types. As a result, standard doses of the drug (in conjugate
form)
will produce enhanced efficacy, without an increase in undesirable side
effects. In
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addition, the standard drug dose (in conjugate form) can be reduced, without
loss of
efficacy, but with a reduction in undesirable side effects. This allows for an
increase
in the duration of therapy, which is highly desirable in chronic disease
settings.
Finally, trapping or accumulation of drug conjugates by phosphorylation may
prevent
the efflux of cancer drugs, including vinca alkaloids, epipodophyllotoxins,
taxanes/taxoids, and anthracyclines by the membrane transporter P-
glycoprotein,
preventing a major form of MDR.
Drugs such as paclitaxel and vinblastine can be prepared with a biotin moiety
or fluorescent tag using procedures known in the art. (See, e.g., Guillemard
et al.,
Anticancer Res. 1999 Nov-Dec; 19(6B):5127-30; Dubois et al., Bioorg Med Chem.
1995 Oct; 3(10):1357-68; Chatterjee et al., Biochemistry. 2002 Nov 26;
41(47):14010-8; Baloglu et al., BioorgMed Chem Lett. 2001 Sep 3; 11(17):2249-
52;
Li et al., Biochemistry. 2000 Jan 25; 39(3):616-23; Rao et al., Bioorg Med
Chem.
1998 Nov; 6(11):2193-204; Bicamumpaka et al., Int JMoI Med. 1998 Aug; 2(2):161-
165; Sengupta et al., Biochemistry. 1997 Apr 29; 36(17):5179-84; Han et al.,
Biochenaistry. 1996 Nov 12; 35(45):14173-83; Sengupta et al., Biochemistry.
1995
Sep 19; 34(37) :11889-94). Substrate libraries can be conjugated to drugs
(such as
paclitaxel or vinblastine) which contain a biotin moiety or a fluorescent tag.
A
fluorescent drug (such as doxorubicin) can also be used. In the case of
biotinylated
conjugates, the libraries need not be purified. Large mixtures of conjugates
can be
incubated with various target cells (ACAMPS disease or normal), followed by
removal of the extracellular medium, cell washing and isolation of
phosphorylated
(trapped or accumilated) conjugates from cell lysates using streptavidin or
avidin
affinity chromatography. Determination of the trapped or accumulated substrate
by
ZS standard methods will identify a substrate of an overexpressed or activated
kinase
expressed in the diseased cell type (or disease-associated normal cell type).
This
provides a trapping or accumilation of the substrate candidate, which can then
be used
with the original drug or linked to other drugs and optimized.
Fluorescently tagged conjugates can be used with drug conjugate libraries that
are produced in a "one conjugate per well" format. The libraries are incubated
with
tumor cells, endothelial cells or cells derived from any (ACAMPS) disease
tissue, in a
mufti-well format, followed by washing and determination of well-associated
fluorescence. Fluorescent drug conjugates that are retained to a high extent
by
diseased or other target cells represent novel drug candidates. Additionally,
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specificity can be assessed by comparing fluorescence uptake in the target
cell to that
in a normal cell type or one not associated with the disease of interest. The
above
methods are not limited to biotinylated or fluorescently tagged conjugates,
but can be
earned out with any tag or inherent property that facilitates purification or
spectrophotometric visualization of conjugates specifically trapped or
accumulated in
target cells.
Since substrates are known for a large number of kinases, it is also possible
to
use these methods to identify new drug discovery (enzyme inhibition) targets
for any
ACAMPS disease_ In certain embodiments, the methods can be used to identify an
overexpressed or aberrantly activated kinase that has not previously been
associated
with a particular disease. In the instances where a biotinylated drug-
substrate
conjugate is employed, it could also be used to isolate the kinase in question
from cell
extracts via affinity chromatography. The kinase may be a previously
identified or
novel enzyme. The library and screening methods can be applied to small
molecule
or metabolic kinase substrates.
G. Combination Therapy
The conjugates provided herein may be administered as the sole active
ingredient or in combination with other active ingredients. Other active
ingredients
that may be used in combination with the conjugates provided herein include
but are
not limited to, compounds known to treat ACAMPS conditions, anti-angiogenesis
agents, anti-tumor agents, other cancer treatments and autoimmune agents. Such
compounds include, in~general, but are not limited to, alkylating agents,
toxins,
antiproliferative agents and tubulin binding agents. Classes of cytotoxic
agents for
use herein include, for example, the anthracycline family of drugs, the vinca
drugs,
the mitomycins, the bleomycins, the cytotoxic nucleosides, the pteridine
family of
drugs, diynenes, the maytansinoids, the epothilones, the taxanes and the
podophyllotoxins.
It is understood that the foregoing detailed description and accompanying
examples are merely illustrative, and are not to be taken as limitations upon
the scope
of the subject matter. Various changes and modifications to the disclosed
embodiments will be apparent to those skilled in the art. Such changes and
modifications, including without limitation those relating to the chemical
structures,
substituents, derivatives, intermediates, syntheses, formulations and/or
methods of use
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provided herein, may be made without departing from the spirit and scope
thereof.
U.S. patents and publications referenced herein are incorporated by reference.
EXAMPLES
Syntheses of representative paclitaxel drug-linker constructs with carbamate
linker to
paclitaxel C10 are given in Examples 1-3 and Example 4. Example 6 provides a
synthesis of a representative vinblastine drug-linker construct with amide
linker to C3.
Abbreviations used: Cbz, benzyloxycarbonyl; CDI, 1,1'-carbonyldiimidazole;
DCM, dichloromethane; DIEA, N,N diisopropylethylamine; DMAP, 4-
(dimethylamino)pyridine; DMF, N,N dimethylformamide; IPA, isopropyl alcohol;
MeOH, methanol; MS, mass spectroscopy; RP-HPLC, reversed phase high
performance liquid chromatography; RT, room temperature; TEA, triethylamine;
TFA, trifluoroacetic acid; Ts = Tosyl. Preparative RP-HPLC purification was
conducted on YMC-Pack ODS-A columns (S-S ~,M, 300 X 20 mm ID) with gradient
elution between 0% B to SO% B or 0% B to 100% B (A=0.105% TFA in H20;
B=0.1 OS% TFA in CH3CN) with gradient times of 10 min and a flow rate of 25
mL/min with UV 220nm detection (Method A). Analytical HPLC-MS was conducted
on a YMC Combi-Screen ODS-A column (S-S wM, 50 X 4.6 mm ID) with gradient
elution of %0 B to 100% B (A=0.105% TFA in H20;
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B=O.IOS% TFA in CH3CI~ with gradient times of 10 min and a flow rate of 3.S
mh/min with IN 220nrn and Electrospray MS detection (Method B).
EXAMPLE 1
Preparation of 2'-O-(tert-butyldimethylsilyl}-7-O-(triethylsilyl)-10-D-
deacetyl-10-O-
(carbonylimidazolyl)paclitaxel (12)
CDI
12
11
To 10-deacetyl-2'-O-(tert-butyldimethylsilyl)-7-O-(triethylsilyl)-10-O-
deacetyl--paclitaxel {11, 845 mg, 0.81 mmol), prepared according to the
procedure in
Datta, A.; Hepperle, M. I. G. J.Org.Chem. (1995) 60:761, in anhydrous DCM (6
mL)
was added carbonyldiimidazole (530 mg, 400 mol%). The reaction mixture was
allowed to stir for 16 hours at room temperature under nitrogen atmosphere
then
extracted with water (5 mL). The organic layer was dried over sodium sulfate,
filtered
and concentrated to give 890 mg (96% yield) of the title compound 12 which was
subsequently used without purification.1H NMR (CDC13, 300 MHz) ~ 8.26 (s, 1H),
8.18 {d, J--9 Hz, 2H), 7.77 (d, J--8 Hz, 2H), 7.53 (m, 11H), 7.14 (s, 1H),
7.09 (d, J--9
Hz, 1H), 6.59 (s, 1H), 6.32 (t, J--9 Hz, 1H), 5.78 (m, 2H), 5.02 (d, J--8 Hz,
1H), 4.72
(d, J--2 Hz, 1H), 4.56 (m, 1H), 4.38 (d, J--8 Hz, 1H), 4.25 (d, J--8 Hz, 1H),
3.88 (d,
.I--7 Hz, 1 H), 2.62 (s, 2 Vii), 2.45 (m, I H), 2.2 (m, 1 H), 2.18 (m, 1 H),
1. 98 (m, I H),
1.81 (s, 1H), 1.78 (s, 3H), 1.62 (s, 3H), 1.32 (m, 3H), 1.22 (s, 3H), 0.95 (m,
6H), 0.83
(s, 9H), 0.62(m, 9H), 0.1 (s, 3H), -0.2 (s, 3H); Electrospray (LCMS) m/z 1134
(M +
H+, C61H$oN30~4Si2 requires 1134); retention time = 9.92 min. (1 % to 99% B,
Method
B)
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EXAMPLE 2
Preparation of 2'-O-(tart-butyldimethylsilyl)-7-O-(triethylsilyl)-paclitaxel-
10-O-
(deacetyl)-10-O- f N [(3-[2-[2-[3-CBz-aminopropoxy]-ethoxy]-ethoxy]-propyl]-
aminocarbonyl}-paclitaxel) (19}
" f
CBz'N~ O ~~ O~~
O~O~O ,8 ~ N
l -- " o
-s~-
,z ,s
To 2'-O-(tart-butyldimethylsilyl)-7-O-(triethylsilyl)-10-deacetyl-10-D-
(carbonylimidazolyl)-paclitaxelpaclitaxel-2'-(tart-butlyldimethylsilyl)-7-
(triethylsilyl)-10-(deacetyl-carbonyl-imidazole) (12, 250 mg, 0.22 mrnol),
prepared
according to Example l, dissolved in anhydrous tart-butyl alcohol (5 mL) was
added
commercially available 3-[2-[2-[3-CBz-aminopropoxy]-ethoxy]-ethoxy]-
propylamine
mono-N-Cbz-amidoPEG-diamine (18, 398 mg, 510 mol%). The reaction mixture was
stirred at 80 °C for 16 hours. The volatiles were then removed in vacuo
and the
resulting residue was re-dissolved in DCM (15 mL). The organic solution was
then
extracted with water (10 mL), dried over sodium sulfate, filtered and
concentrated to
give 284 mg of the title carnpound 19 which was subsequently used without
purification. Electrospray (LCMS) m/z 1421 (M + H+, C~6Hto6N3419Si~ requires
1421); retention time 10.49 min. (1% to 99% B, Method B);
EXAMPLE 3
Preparation of paclitaxel-10-O-deoxy-10-O- f N [(3-[2-[2-[3-aminopropoxy]-
ethoxy]
ethoxy]-propyl]-aminocarbonyl}-paclitaxel) (20}
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1) HF/Pytidirte
2) Pd ! C, HZ
3) HPLC purif.
Compound 19 (284 mg, 0.2 mmol) prepared according to Example 2 was
desylilated following the procedure in Ojima, I. et al. J. Med. Chem. (I997),
40:267.
The residue so obtained (225 mg) was dissolved in methanol (20, mL) whereupon
10
wt% palladium on carbon (100 rng) was added. The resulting mixture was stirred
for
40 minutes under one atmosphere of H2. The reaction mixture was filtered
through
Celite and concentrated under reduced pressure. The residue so obtained was
purified
by preparative RP-HPLC (Method A). Fractions containing the appropriate mass,
as
deterniined by analytical HPLC-MS (Method B) were pooled and CH3CN removed
under reduced pressure. The remaining aqueous mixture was then lyophilized
obtaining 140 mg (55% overall yield) of the desired paclitaxel-linker
construct 20
with PEG carbamate linker at C10.1H NMR (CD30D, 300 MHz) b 8.38 (d, J 8 Hz,
1H), 8.I4 (d, .I--8 Hz, 2H~, 7.89 (d, J--8 Hz, 2H), 7.45 (m, 1 IH), 6.29 (s,
1H), 6.19 (t,
1H), 5.66 (m, 2H), 5.03 (d, .I--10 Hz, 2H), 4.76 (d, .T=5 Hz, 2H), 4.35 (m,
1H), 4.22 (s,
2H), 3.85 (d, 1H), 3.60 (m, 8H), 3.12 (m, 2H), 2.50 (m, 1H), 2.40 (s, 3H),
2.26 (rn,
1H), 2.I9 (s, 2H), 1.94 (m, 4H), 1.82 (m, 4H), 1.68 (s, 2H), 1.18 (s, 6H);
Electrospray
(LCMS) mlz 1058 (M + Hue, CSr~~2N301~ requires 1058); retention time 5.07 min.
(1% to 99% B, Method B).
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EXAMPLE 4
Preparation of -10-O-(deacetyl)-10-O-(N (4-(3-carboxylic acid)-prop-1-yl)-
phenyl)-
aminocarbonyl)-paclitaxel (22)
Mel
acetonit~ile, aT
21
HZN /
O 1) ~ ~ OH
HO
2) HFIPy O
N~H
O~O O OH
O
\ N _ O"' n Ii -
H ~H HO O OAc
22
To (20 mg, 0.018 mm~l) 2'-O-(text-butyldimethylsilyl)-T-O-(triethylsilyl)-10-
deacetyl-10-O-(imidazoylcarbonyl)-paclitaxel (12), prepared according to the
procedure of Example 1, dissolved in CH3CN (0.3 mL), is added MeI (0.2 mL) .
The
reaction mixture is stirred for 3 hours at 55 °C in a sealed tube. A
stream of N2 is then
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used to remove the volatiles and the residue is exposed to high-vacuum to
remove
volatiles giving intermediate 21. The imidazolium salt 21 is then dissolved in
DMSO
(0.5 mL) and 3-(4-amino-phenyl)-propionic acid (500 mol%) is added. The
reaction
mixture is stirred for 30 minutes at room temperature, diluted with pyridine
(0.5 mL).
The resulting mixture is cooled to 0°C and HF/Py (233 p1) is added.
Stirring is
continued for 3 hours at room temperature. The reaction mixture is then
diluted with
EtOAc (5 mL) and extracted with saturated aqueous solution of CuS04 (3 x 1 mL)
followed by water (2 x 2 mL). The organic phase is then dried over sodium
sulfate,
filtered and concentrated. The resulting residue is purified by preparative RP-
HPLC
C-18 column (Method A). Fractions containing the appropriate mass, as
determined
by analytical HPLC-MS (Method B) are pooled and CH3CN is removed under
reduced pressure. The remaining aqueous mixture is then lyophilized to give
the
desired paclitaxel-linker construct 22 with aryl carbamate linker at C10.
EXAMPLE 4a
Preparation of 10-O-deacetyl-10-O-(N-(2-deoxyglucosyl)-aminocarbonyl)-
paclitaxel (7a)
To the imidazolium salt 21 prepared according to the procedure of Example 4,
dissolved in DMSO (0.5 mL), was added D-glucosamine hydrochloride (500 mol%)
followed by DIEA (500 mol%). The reaction mixture was stirred for 30 minutes
at
SSoC and then diluted with pyridine (0.5 mL). After cooling to 0 oC, HF/Py
(233 ml)
was added and the resulting mixture was stirred for an additional 3 hours at
room
temperature. The reaction mixture was then diluted with EtOAc (5 mL) and
extracted
with saturated aqueous solution of CuS04 (3 x 1 mL) followed by water (2 x 2
mL).
The organic phase was then dried over sodium sulfate, filtered and
concentrated. The
resulting residue was purified by preparative RP-HPLC C-18 column (Method A).
Fractions containing the appropriate mass, as determined by analytical HPLC-MS
(Method B) were pooled and CH3CN was removed under reduced pressure. The
remaining aqueous mixture was then lyophilized to give paclitaxel-sugar-
conjugate of
formula 7a. Electrospray (LCMS) m/z 1017 (M+H+, C52H61N2O19 requires 10I7);
retention time 5.12 min. (1%-99% B, Method B)
EXAMPLE 5
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Preparation of mono-N Boc-2-[2-[2-[2
aminoethoxy]ethoxy]ethoxy]ethylaminediaminoPEG (24)
Boc20, Et~N
HiN~ ~O~O~./~NH= _ HiN~0~0~0~~
23 CHzC~2 24
To 2-[2-[2-[2-aminoethoxy]ethoxy]ethoxy]ethylamine (23 (, 0.5 g, 2.6 mmol),
dissolved in CH2C12 (50 mL), was added triethylamine (0.36 mL, 100 mol%) and
Bac20 (0.55 g, 100 mol%). The reaction mixture was stirred for 4 hours and
concentrated to dryness. The resulting residue was purified by silica gel
column
chromatography eluting with 9:1:0.1 chloroform:methanol:ammonium hydroxyde to
give 0.26 g (34% yield) of the title compound 24.1H NMR (CDC13, 300 MHz) ~
3.66
(m, 8H), 3.57 (m, 4H), 3.28 (m, 2H), 2.90 (t, 2H), 1.63 (bs, ZH), 1.47 (s,
9H).
EXAMPLE 6
Preparation of deacetylvinblastine-3-(amido-PEG-amine)Reaction between 4
deacetyl-3-de-(methoxycarbonyl)-vinblastin-3-yl-carbonyl azide (9) and N Boc-2-
[2
[2-[2-aminoethoxy]ethoxy]ethoxy]ethylamine (24)
OH
N '~~./
1) 9, DIEA, DCM I / N ~ , COZMe
H
N I
,~H
2) TFA, DCM
OH "'~/
Me ~ (4 H : OH
O~N~O~O
H
8 25 HzN~O
To a DCM solution of 4-deacetyl-3-de-(methoxycarbonyl)-vinblastin-3-yl-
carbonyl azide (9 deacetylvinblastine acid azide , 0.46 mmol)), prepared
according to
the procedure of K.S.P. Bhushana Rao et al., J. Med. Chem. (1985), 28:1079,
was
added N Boc-2-[2-[2-[2-aminoethoxy]ethoxy]ethoxy]ethylamine (the mono Boc-
protected diarnine 24, ( 0.2 g, 150 mol%), followed by DIEA (0.12 mL, 150
mol%).
The reaction mixture was stirred at room temperature for 3 hours then
concentrated in
vacuo to give a residue that was purified by silica gel column chromatography
eluting
with 95:5 chloroform:methanol. The intermediate, 4-deacetyl-3-de-
(methoxycarbonyl)-vinblastin-3-yl-N {N'-Boc-2-[2-[2-[2-
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ethoxy]ethoxy]ethoxy]ethylamino}-aminocarbonyl) was dissolved into 120 mL 1:1
of
DCM:TFA and *he mixture was stirred at room temperature for 10 minutes. The
mixture was concentrated with a flow of NZ and the resulting residue
lyophilized to
give 0.31 g (65% overall yield) of the desired vinblastine-linker construct 25
with
PEG amide linker at C3 which was used without further purification.
Electrospray
(LCMS) m/z 929.5 (M + H+, CslH~3NsOio requires 929.5); retention time 3.46
min.
(1% to 99% B, Method B).
EXAMPLE 7
Preparation of (6-hydroxy-hexyl)-carbamic acid benzyl ester (27)
HO~~NH2 Cbz~CI, TEA
HO~NHCbz
MeOH
26 27
To 6-aminohexan-1-of (26, 0.49 g, 100 mol%) in MeOH (25 mL) were added
benzylchloroformate (1.0 mL, 165 mol%) and triethylamine (1.0 mL, 165 mol%).
The
reaction mixture yeas stirred for S hours at room temperature then
concentrated to
dryness to give a residue which was purified by silica gel column
chromatography
using 1:1 hexanes:EtOAc to give 0.9 g (85% yield) of title compound 27. 1H NMR
(CDCl3, 300 MHz) 8 7.37 (m, SH), 5.13 (s, 2H), 3.67 (m, 2H), 3.24 (m, 2H),
1.56 (m,
4H), 1.35 (m, 2I3)
EXAMPLE 8
Preparation of (6-Bromo-hexyl)-carbarnic acid benzyl ester (28)
PPb~, CBr4
HO~NHCbx _,.~ Br~NHCbz
CHZCIz
27 28
To 27 (0.53 g, 100 mol%) dissolved in DCM (50 mL) were added
triphenylphosphine (0.66 g, I20 rnol%) and carbon tetrabromide (0.84 g, 120
mol%).
The reaction mixture was stirred for 90 minutes at room temperature then
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concentrated to dryness to give a residue which was purified by silica gel
column
chromatography eluting with 1:1 hexanes:EtOAc to give 0.32 g (48%) of title
compound 28. 1H NMR (CDCI3, 300 MHz) 8 7.39 (m, SH), 5.13 (s, 2H), 3.43 (t,
2H),
3.25 (m, 2H), 1.74 (m, 2H), 1.45 (m, 6H)
EXAMPLE 9
Reaction of thymidine with (6-Bromo-hexyl)-carbamic acid benzyl ester (28)
and deprotection to give 3-(6"-aminohexyl)-thymidine (29)
0
0
~NH N,~NH2
//~N"-O 1) 15, KzCO~, KI
HO O acetone I DMF N~O
HO O
2) Pd I C, H~
OH MeOH OH
29
To thymidine (0.2 g, 100 0.83 mmol%) dissolved in acetone (3 mL) and DMF
(3 mL) were added 28 (0.26 g, 100 mol%) and K~C03 (0.22 g, 200 mol%). The
reaction mixture was stirred at 50°C for 48 hours then partitioned
between EtOAc and
water. The aqueous layer was extracted with ethyl acetate and the organic
layer was
dried over Na2S04 and concentrated to dryness to give a residue which was
purified
by silica gel colunnn chromatography eluting with 95:5 chloroform:methanol.
The
CBz protected thyrnidine-N3-linker intermediate so obtained was dissolved in
methanol (10 mL) and 10 wt% palladium on carbon (33 mg) was added. The
reaction
mixture was stirred at room temperature under 1 atm of H2 for 16 hours then
filtered
through Celite. The filtrate was concentrated under reduced pressure to give
0.17 g
(60% overall yield) of the title compound 29. 'H NMR (CDC13, 300 MHz) 8 7.88
(s,
1H), 6,33 (t, J 7 Hz, 1H), 3.96 (m, 3H), 3.78 (m, 2H), 2.96 (m, 2H), 2.54 (m,
2H),
1.94 (s, 3IT), 1.65 (m, 4H), 1.45 (m, 4H)
EXAMPLE 10
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Preparation of 2'-O-{benzyloxycarbonyl)-7-O-(4-nitrophenyloxycarbonyl)-
paclitaxe1
(6)
o ~ ~ No,
c~--
0
DMAP
To 2'-O-(benzyloxycarbonyl)--paclitaxel (5, 0.52 g, 0.53 mmol), prepared
according to the procedure described in. Chen, S.-H., et al., Tetrahedron
(1993)
49:2805-2828, dissolved in DCM (150 mL) were addedp-nitrophenylchloroformate
(0.64 g, 600 mol%) and DMAP {0.6 g, 920 rnol%). The reaction mixture was
stirred
for 2 hours and concentrated to dryness. The resulting residue was purified by
silica
gel column chromatography eluting with 1:1 hexanes:EtOAc to give 0.48 g (79%
yield) of the title compound 6.: 1H NMR (CDCI3, 300 MHz) 8 8.31 (d, J 9 Hz,
2H),
8.17 (d, J 9 Hz, 2H), 7.77 (d, J--8 Hz, 2H), 7.32 (m, 18H), 6.98 (d, J 9 Hz,
1H), 6.43
(s, 1 H), 6.31 (t, J--9 Hz, 1 H), 6.01 (d, J--9 Hz, 1 H), S .18 (s, 2H), 5.03
(d, J--9 Hz, 1 H),
4.40 (d, J--8 Hz, 1H), 4.25 (d, J 8 Hz, 1H), 4.03 (d, J--8 Hz, 1H), 2.77 (m,
2H), 2.50
(s, 3H}, 2.33 (m, 2H), 2_24 (s, 3H), 2.08 (m, 4H), 1.91 (s, 3H), 1.80 (s, 3H),
1.28 (m,
6H); Electrospray (LCMS) m/z 1154 (M + H+, C6zH61NzOzo requires 1154);
retention
time = 8.48 min (1% to 99% B, Method B).
EXAMPLE 11
Reaction of 2'-O-(benzyloxycarbonyl)-7-O-(4-nitrophenyloxycarbonyl)-
paclitaxe17 with 3-(6"-aminohexyl)thymidine and deprotection to give O-(N (6-
hexyl-
[thymidin-3-yle) ]hexan-1-yl) aminocarbonyl)-paclitaxel (30)
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~--~~ ~f ~s, mea
WO", HO(\CHZCii I D~MF
O
O~O ~O 2) Pd / C, Hi
/ 6 __
To 2'-O-(benzyloxycarbonyl)-, 7-O-(p-nitrophenyloxycarbonyl)-paclitaxel (6,
50 mg, 0.043100 mrnol%), dissolved in DMF (2 mL} and CHZC12 (3 mL), was added
3-(6"-aminohexyl)-thymidine (29, 57 mg, 380 mol%) followed by DIEA (30 p.L,
380
mol%). The reaction mixture was stirred at room temperature for 3 hours then
partitioned between EtOAc and water. The aqueous layer was extracted with
EtOAc
and the organic layer was dried over NaZS04 and concentrated to dryness to
give a
residue directly injected onto a preparative RP-HPLC C-18 reversed phase
column for
purification (Method A). Fractions containing the appropriate mass, as
determined by
analytical HPLC-MS (Method B), were pooled and the solvent was removed under
reduced pressure. To the paclitaxel-thymidine intermediate so obtained,
dissolved in
methanol (5 mL), was added 10 wt% palladium on carbon (13 mg) and the reaction
mixture was stirred at room temperature under 1 atm of HZ for 16 hours. The
mixture
was filtered through Celite and concentrated under reduced pressure to give 37
mg
(71 % yield) of the title compound 30. Electrospray (LCMS) m/z 1221 (M + Hue,
C64H~~NøOzo requires 1221); retention time = 6.20 min (1% to 99% B, Method B).
E~~AMPLE 12
Preparation of 3-(2,5-dioxo-2,5-dihydropyrrol-1-yl)propionaldehyde (31a)
To 1-(3-hydroxypropyl)-1H pyrrole-2,5-dione (200 mg, 1.29 mmol) dissolved
in 5 mL DCM. DMP (1S% wt in DCM, 4 rnL, 1.93 mmol) was added in one portion.
After stirring the mixture for 2 h, 2-propanol (3 mL) was added followed by
stirring
for an additional 30 min. The resulting solution was filtered through a
silica.gel pad
eluted with EtOAc, and the filtrate was concentrated. The crude product was
purified
by silica gel chromatography eluting with EtOAc/Hexane (2/1) to provide
3-(2,5-Dioxo-2,5-dihydro-pyrrol-1-yl)-propionaldehyde (110.0 mg, 0.72 mmol,
56%
yield) which was used immediately. 1H NMR (CDCI~, 300 MHz) 8 9.74(t, 1H, J=
i.2
Hz), 6.69 (s, 2H), 3.84 (t, ZH, J= 6.9 Hz), 2.77 (td, 2H, J= 6.9, 1.2 Hz).
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EXAMPLE 13
Preparation of N3'-maleimidopropyl doxorubicin (31)
O OH O
~ off
~~'
~
I
OH
I
O H
O ~'''~ NaHCO~,AcOH
~
OCHSD OH ~N
+ O
O ~.
l
OH~ ~HCI ~~
O
H 31a
Doxorubicin
To a stirred solution of doxorubicin hydrochloride (100 rng, 0.172 mmol), 31a
(68.2 mg, 0.446 mmol) and glacial AcOH {20 ~,L, 195 mol%) in CH3CN/H20 (2:1, 5
mL) was added a 1 M solution of NaCNBH3 in THF (57 pL, 0.33 mol%). The
mixture was stirred under nitrogen atmosphere in the dark at RT for 1 h. The
solution
was then concentrated in vacuo to give a residue which was diluted with an
aqueous
5% NaHC03 solution and extracted with DCM. Concentration of the organic
solution
and purification of the resulting residue by silica gel chromatography eluting
with
DCMlCH30H {20:1) provided 26.0 mg of N-3-maleimidopropyl doxorubicin 31
(21.4% yield). 1H NMR (CDCl3, 300 MHz) 8 8.03 (d, 1H, J= 8.4 Hz), 7.79 (t, 1H,
J=
8.4 Hz), 7.41 (d, 1H, J= 8.4), 6.68 (s, 2H), 5.51 (m, 1H), 5.32 (m, 1H), 4.82-
4.76 (m,
2H), 4.09 (s, 3H), 3.36 (m, 1H), 3.58 (m, 3H), 3.32-2.98 (m, 2H), 2.76 (m,
1H), 2.54
(m, 2H), 2.37 (m, 1H), 2.15 (m, 1H), 1.85 -1.54 (m, 4H), 1.37 (d, 3H, J= 7.0
Hz).
Electrospray (LCMS) m/z 681.2 (M + Hue, C3.4H36N2O13 requires 681.2)
EXAMPLE 14
Preparation of (6-(thymidin-3-yl)-hexan-1-yl) 3-mercaptopropanamide (32)
0 0
N~NHZ N~N~SH
O ~~O [J _O
~ ~ BOP, DIEA
HO O '~ HO~SH >t~- HO
H OH 32
29
To a solution of 3-(6-aminohexyl) thymidine (29, 81.0 mg, 0.237 mmol) in
DMF prepared according to the procedure described herein were added BOP (192
mg,
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0.350 mmol), DIEA (123 mg, 0.948 mmol) and 3-mercaptopropionic acid (37.2 mg,
0.350 mmol). The reaction mixture was stirred for 30 min whereupon DMF was
removed in vacuo. The crude was purified by silica gel P-TLC eluting with
DCM/CH30H (9:1) to give 51.3 mg compound 32 (50.4% yield). 1H NMR (CDCl3,
300 MHz) S 7.40 {s, 1H), 6.16 (t, 1H, J= 6.6 Hz), 6.0 (m, IH), 4.60 (m, 1H),
4.06-
3.78 (m, 4H), 3.70 (m, 1 H), 3.29 - 3.11 (m, 3H), 2.81 (q, 2H, J= 7.5 Hz),
2.50 (t, 2H,
J= 6.6 Hz), 2.46 - 2.27 (m, 2H), 1.92 {s, 3H), 1.68 -1.17 (m, 8H).
Electrospray
(LCMS) m/z 430.2 (M + H+, C19H31N3O6S requires 430.2)
EXAMPLE 15
Preparation of 3-(1-(Doxorubicin-N3'-propyl)-2,5-dioxopyrrolidin-3-ylthio)-N
(6-(thymidin-3-yl)-hexylpropanamide (33)
~SH
O
O OH O
OH
_ ~''OH
O OCH30 OH O
O
'"OH OH 31 NH O
linkln moie HO~~,
9 ty ~N~
O
O H
S~N
~N
O
O
33
To a DCM/CH30H (9:1) solution of N-3-maleimidopropyl doxorubicin 31 (17.5
mg, 0.026 mmol) was added the thiol containing thymidine derivative 32 (11.2
mg,
0.026 mmol). The mixture was stirred under nitrogen atmosphere in the dark for
30
min. The solvent was removed in vacuo and the resulting crude residue was
dissolved
into by DMSO and purified on a preparative RP-HPLC C-18 reversed phase column
(Method A). Fractions containing the appropriate mass, as determined by
analytical
HPLC-MS (Method B), were pooled and CH3CN was removed under reduced
pressure or Nz stream followed by lyophilization to give 6.1 mg of the
anthracycline-
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linker-thymidine conjugate 33 (21% yield). Electrospray (LCMS) m/z 1110.5 (M +
H~, C53H6~N5O~9S requires 1110.4)
EXAMPLE I6
Cytotoxicity Assay
Cytotoxicity Assay (monolayer)
Monolayer assays with tumor cell lines (MCF-7 breast carcinoma and HT-29
colorectal carcinoma from ATCC) were carried out in triplicate in 96-well
plates with
RPMI1640 medium containing 5% fetal bovine serum, 100 U/ml penicillin and 100
p.g/mI streptomycin. Normal human foreskin fibroblasts (I~F #CC-2509) were
from
Cambrex and were cultured in FGM-2 medium. Exponentially growing cells (5,000
MCF-7 or HT-29; 1,500 HFF) were plated in 100 ~l medium and incubated
overnight
(5% COZ, 37°C). Compounds (20 pM to 20 p,M final concentration, 6-8
doses) and
vehicle (DMSO) controls were added and the incubation was continued for an
additional 72 hours. Final cell density was determined by incubating cultures
with 25
~1 AlamarBlue reagent (BioSource, Camarillo, CA) for 4 hours, followed by
determination of fluorescence at excitation of 544 nm and emission of 590 nm
with a
SpectroMax Gemini EM fluorescence plate reader (Molecular Devices, Sunnyvale,
CA). ECSO values were generated from dose-response curves by a 4-parameter
method using Softmax PRO software. Mean ECSOs (+ SD) represent the average of
all tests carried out for all Iots of a given compound. Outlier ECso values
(<7%) were
identified and removed prior to analysis using the method of Hoaglin et al.,
J. Amer.
Statistical Assoc., 81, 991-999 (1986).
Cytotoxicity Assay (soft agar)
Assays were carned out in 24-well plates with 0.5 ml bottom layers (0.8%
agar) and 0.5 ml top layers (0.38% agar) in RPMI1640 medium containing 5%
fetal
calf serum. Top layers were plated with 1,250 MCF-7 or 5,000 HT-29 cells per
well
and drugs, compounds or vehicle controls in triplicate as described above.
Plates
were incubated as above for 10-14 days and then colony formation was assessed
by
adding 50 ~,l AlarnarBIue to each well and determining EC50s as described
above for
monolayer assays. The ECSO values for exemplary conjugates and patent drugs
for
normal and tumor cells different cell lines are provided in Figure 1-3.
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EXAMPLE 17
Thymidine Kinase Cloning, Expression, Assay
Human thymidine kinase 1 (TKl) cDNA clone (cat. # OHS1166-7304119)
was obtained from Open Biosystems (Huntsville, AL). TKl was amplified using
PCR with forward primer S'-CAATCCATATGAGCTGCATTAACCTGC-3' and
reverse primer 5'-TATTAAGCTTCTAGTTGGCAGGGCTGCAT-3'. The PCR
product was digested with Nde I and Hind III. An N-terminal His-tagged TKl
construct, TKl/pET28b(+), was generated by subcloning TKl into the Nde I and
Hind
III sites of pFOkaryotic expression vector pET28b {+) (Novagen, San Diego,
CA).
TKl was expressed from TKIlpET28b(+) using E, coli strain BL21-codon plus
(Stratagene, San Diego, CA) and purified by Ni column chromatography.
A coupled (kinetic) ATP depletion assay was developed to measure thymidine
kinase activity. The reaction contained 100 mM Tris HCI, pH 7.5, 20 mM MgCl2,
100 mM KCl, 0.4 mM PEP, 0.2 mM NADH, Pyruvate Kinase (0.7 units) / Lactate
Dehydrogenase (1.0 unit) (Sigma #P0294), 2.5-10 ~.g Thymidine Kinase, 100 p,M
thymidine or thymidine-drug conjugate in a volume of 7S p1. ATP (5 mM in 25
p,1
H20) was added to initiate the reaction and the velocity of ATP depletion was
monitored at 340 nm continuously for 15 minutes. Results for conjugate
phosphorylation represent the initial velocity relative to thymidine and are
provided in
Tables 7-9 for exemplary conjugates.
EXAMPLE 18
Fluorescence-based assays for enhancement (paclitaxel) and inhibition
(vinblastine) of tubulin polymerization
The assay kit (#BKO11) was purchased from Cytoskeleton (Denver, CO). The
assays were carried out according to the manufacturer's instructions, except
that 1
mg/ml BSA (Sigma #A3059) was included in all assays. Paclitaxel assays were
carried out in the absence of glycerol and vinblastine assays were carried out
in the
presence of 20% glycerol. Parent drugs and conjugates were tested at 0.75,
1.5, 3 and
micromolar final concentration and results represent a comparison of conjugate
and parent drug curves obtained from the linear range of the dose responses.
Mean
percentages of paclitaxel or vinblastine activity (~ SD) represent the average
of all
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10
20
30
tests carried out for all lots of a given compound. Results for exemplary
conjugates
are provided in Tables 7-9.
E~~AMPLE 19
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Topoisomerase II Assay
Doxorubicin conjugates were assayed for their effect on Topoisomerase II
using the Topoisomerase II Drug Screening Kit (Catalog # 1009-1) produced by
TopoGEN Inc. (Columbus, Ohio). Specifically the kit was used to assay whether
Doxorubicin conjugates altered the ability of Topoisomerase II to catalyze the
formation of relaxed conformation DNA from a super-coiled plasmid. Doxorubicin
conjugates were compared directly to Doxorubicin at 10, 3, 1, 0.3, 0.1 and
0.03
micromolar concentrations. The quantity of relaxed conformation DNA was
quantified from an agarose gel on which is it is separated from the super-
coiled DNA
by standard electrophoresis. The more active a drug is at a particular
concentration
the less relaxed conformation DNA is produced by the action of Topoisomerase
II.
The results are presented in terms of percent activity of Doxorubicin. Results
for
exemplary conjugates are provided in Tables 7-9.
EXAMPLE 20
Serum Stability
The stability of conjugates was measured in RPMI1640 cell culture medium
containing 10% fetal bovine serum. The serum-containing medium was pre-warmed
at 37°C for 3 min prior to addition of test articles. Test articles,
prepared in DMSO as
5 mM stocks, were added to the cell culture media to a final concentration of
10 mM.
Aliquots ( 150 ml) were withdrawn in triplicate at 0, 4, 8, 24 and 72 hours
and
combined with the same volume of ice-cold acetonitrile to terminate the
reaction. The
mixture was centrifuged at 2,000 x g for 10 minutes. One part of the
supernatant was
mixed with four parts of deionized water to bring down the percentage of
organic
solvent. The diluted samples were then assayed by LC/MS for the test article.
The
natural log of the percent remaining was plotted versus time. A linear fit was
used to
determine the rate constant. The fit was truncated after the percent of
remaining test
article was less than 10%. The elimination half lives associated with the
disappearance of test articles were determined to compare their relative
stability. The
assays were carried out by Absorption Systems (Exton, PA).
EXAMPLE 21
Liver microsome metabolic stability
Human and mouse liver microsomes were obtained from Absorption Systems
(Exton, PA) and Xenotech (Lenexa, KS), respectively. The reaction mixture
contained microsomes (human or mouse) 1.0 mg/ml, potassium phosphate, pH 7.4
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100 mM, magnesium chloride 10 mM, test article 10 mM, and was equilibrated at
37°C for 3 min. The reaction was initiated by adding NADPH (1 mM
final), and the
system was then incubated in a shaking water bath at 37°C. Aliquots
(100 ml) were
withdrawn in triplicate at 0, 15, 30, and 60 minutes and combined with 900 ml
of ice-
s cold 50/50 acetonitrileldH20 to terminate the reaction. Two controls
(testosterone and
propranolol) were run simultaneously with the test articles in separate
reactions. The
samples were assayed by LC/MS for the test article. The natural log of the
percent
remaining was plotted versus time. A linear fit was used to determine the rate
constant. The fit was truncated when percent remaining of the test article was
less
than 10°fo. The elimination half lives associated with the
disappearance of test and
control articles were determined to compare their relative metabolic
stability. The
assays vcpere carried out at Absorption Systems (Eaton, PA).
EXAMPLE 22
Thymidine Kinase-mediated Drug Trapping Results
The pharmacological activity of a paclitaxel-thymidine conjugate and a
vinblastine-thymidine conjugate were compared to the corresponding
unconjugated
paclitaxel and vinblastine. The paclitaxel-thymidine conjugate shows a TTY
substrate activity corresponding to 25 % of thymidine, and a paclitaxel
activity
corresponding to 77 % of paclitaxel. The paclitaxel-thymidine conjugate
exhibits
cytotoxity against breast carcinoma, colon carcinoma and leukemia. ECSO values
for paclitaxel and paclitaxel-thymidine conjugate were determined to be 5-S nM
and 75-170 nM, respectively.
The vinblastine-thymidine conjugate shows a TK substrate activity
corresponding to 27 % of thymidine, and a vinblastine activity corresponding
to 76
% of vinblastine. The vinblastine-thymidine conjugate exhibits cytotoxicity
against breast carcinoma, colon carcinoma and leukemia. ECso values for
vinblastine and vinblastine-thymidine conjugate were determined to be 1-2 nM
and 11-43 nM, respectively.
EXAMPLE 23
A comparison of cytotoxic selectivity index for an exemplary conjugate
and paxent drug in tumors and normal cells shows the increase in the cytotoxic
selectivity index of the conjugate for tumor cells as compared to the
cytotoxic
selectivity index of the parent drug:
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HFF
Monolay MCF-7 MCF-7 Soft HT-29 HT-29 Soft
er Monolayer Agar Monolayer Agar
EC50
(nM) EC50 (nM) EC50 (nM) EC50 (nM) EC50 (nM)
95 52 63 53 152
Paclitaxel(n=20) (n=19) (n=8) (n=18) (n=6)
PXL-7Ca-457
ALK(6)- 310 120 73 40 41 258 129 120
4
N3-Thy (n=16) (n=16) (n=9) (n=16) (n=5)
127
