Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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Methylation Inhibitor Compounds
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims benefit of U.S. patent application 60/547,902, filed
February 25, 2004, the contents of which are incorporated by reference in
their
entirety.
STATEMENT REGARDING FEDERALLY SPONSORED
RESEARCH OR DEVELOPMENT
Research supporting this application was carried out by the United States of
America as represented by the Secretary, Department of Health and Human
Services.
BACKGROUND OF THE INVENTION
Aberrant de novo methylation of promotor regions of regulatory genes is
commonly associated with cancer. Several studies have shown that de novo
methylation of CpG islands in regulatory sequences of tumor suppressor genes
can
result in their silencing. Thus, DNA methylation may lead to abnormal growth
of
cancer cells. Examples of regulatory genes that are commonly hypermethylated
in
cancer cells include RB1 gene in retinoblastomas, the VHL gene in sporadic
renal cell
carcinomas, the H19 gene in Wihns' tumors, the p15 gene in leukemias, and the
p16
gene in several human cancer cell lines. Thus, modulation of the
hypermethylation of
genes involved in the control of cell proliferation by use of methylation
inhibitors has
been one strategy invoked for cancer therapy, and more specifically, the use
of DNA
methylation inhibitors to reactivate antiproliferative, apoptotic, and
differentiation-
inducing genes in cancer cells.
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fluorodeoxycytidine) the compounds typically suffer from one or more
characteristics
that detract from their use as therapeutic agents, including chemical
instability
(including in neutral solution), weak potency, short half life, and generation
of toxic
metabolites. Thus, there still exists a need for effective, stable, and
minimally toxic
DNA methylation inhibitor compounds.
SUMMARY OF THE INVENTION
Compounds and compositions having those compounds are provided herein,
as well as methods of making and using them. The compounds and compositions
herein are useful in treatment or modulation of disease, disease symptoms or
conditions in a subject. The compounds are useful as DNA methylation inhibitor
compounds (and methods thereof). The compounds, compositions, and methods
thereof axe useful in treatment or modulation of cancer, cancer symptoms or
cancer
conditions in a subject.
One aspect is an isolated compound of Formula I:
HRH
O~Zs
Formula T
wherein,
each X is independently NR1R2, or NR1RZR3 +;
each Rl, R2 and R3 is independently H or alkyl;
each Y is independently H, OH,~or halogen; and
each Z is independently a bond or -P(O)(OH)-O-;
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Another aspect is the isolated compound of Formula II or pharmaceutically
acceptable salt or hydrate thereof:
O
N N
O
,:
O
Y
O~Z
X
Formula II
wherein X, Rl, RZ, R3, Y and Z are as defined above.
Another aspect is the isolated compound of Formula III or pharmaceutically
acceptable salt or hydrate thereof,
O
N N
O
O
O ~ \\ O\'\\ Y
~Z
O
X
Formula III
wherein X, Rl, RZ, R3, Y and Z are as defined above.
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formulae herein, wherein X is NHa;. X is (NMe3)+ ; and the compounds as
delineated
in Tables I and II herein.
Another aspect is the isolated compound of Formula IV or pharmaceutically
acceptable salt or hydrate thereof
Y
O
Z O
X
Formula IV
wherein X, Rl; RZ, R3 and Z are as defined above and Y is OH.
Another aspect is the isolated compound of Formula V:
X ~~ /
o z P o
N N
OH
O
Formula V
wherein,
each X is independently NR1R2, or NR1R2R3 +;
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each Z is independently a bond or P(O)(OH)-O-;
or pharmaceutically acceptable salt or hydrate thereof.
Another aspect is the isolated compound of Formula VI or pharmaceutically
acceptable salt or hydrate thereof:
O Z P O II
N N
OH
O
Formula VI
wherein X, Rl, RZ, R3, Y and Z are as defined above.
Another aspect is the isolated compound of Formula VII or pharmaceutically
acceptable salt or hydrate thereof:
O Z P O
N N
OH
O
Formula VII
wherein X, Rl, R2, R3, Y and Z are as defined above.
Another aspect is the isolated compound of Formula VIII or pharmaceutically
acceptable salt or hydrate thereof:
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O
X
Z P
O
O
OH
Formula VIII
wherein X, Rl; R2, R3 and Z are as defined above and Y is OH.
Other aspects are compounds of the formulae herein, wherein Y is halogen;
wherein Y is fluoro; wherein Y is hydrogen. Other aspects are compounds of the
formulae herein, wherein X is NH2;. X is (NMe3)+ .
In another aspect, the invention relates to a composition having a
therapeutically effective amount of a compound (or pharmaceutically acceptable
salt
or hydrate thereof) according to any of the formulae herein and a
pharmaceutically
acceptable carrier. The composition can further have an additional therapeutic
agent,
the additional agent can be an anticancer agent.
Another aspect is a method of treating a DNA methyl transferase (DNMT)
mediated disease, disease symptom or condition that includes administration to
a
subject in need of such treatment a compound (or pharmaceutically acceptable
salt or
hydrate thereof) according to any of the formulae herein, or composition
thereof. The
disease, disease symptom or condition can involve hypermethylation of DNA; the
administration can be by oral administration.
Another aspect is a method of assessing the effect of a test compound on
methylation of DNA in a cell including: (i) contacting a test compound with a
cell that
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methylation; and measuring the methylation of DNA in the cell; and (iii)
comparing
the results of step (i) with the results of step (ii). In these methods, the
cell can
include a hypermethylated nucleic acid molecule; a CpG dinucleotide; and can
be a
mammalian tumor cell.
Another aspect is a method of reversing DNA methylation in a cell, including
administering to a cell a therapeutically effective amount of a compound of
any of the
formulae herein. The cell can be in a subject or in vitro.
The invention also relates to a method of treating cancer in a subject
including
administering an effective amount of a compound according to any of the
formulae
herein. The cancer can be ovarian, breast, colon, rectal, lung, prostate,
pancreatic,
bladder, solid tumor, or any tumor having a silenced tumor suppresser gene.
The
cancer can be any associated with or exemplified by cancer cell lines,
including for
example, T24, HCT15, CFPAC-1, SW48, HT-29, PC3, or CALU-1. The method can
further include administration of an additional anticancer agent; an anti-
nausea or an
anti-anemia agent. The administration of the additional agents) can be
concurrently
or sequentially, and can be individually or in a combined formulation.
The invention also relates to a kit having a compound of any of the formulae
herein and instructions for in vivo or in vitro use of the compound. The in
vitro use
can be screening for demethylation of a hypermethylated DNA.
The invention also relates to a kit having a compound of any of the formulae
herein and instructions for administration to a subject. The subject can be in
need of
treatment for a hypermethylated DNA mediated disease, disease symptom or
condition; in need of treatment for a hyperproliferative disease, disease
symptom or
condition; in need of treatment for cancer. The subject can be a human; or a
rat or
mouse. The administration can be oral;or intravenous or intraperitoneal.
Another aspect is a method of making a compound of any of the formulae
herein, comprising converting a compound of Formula B, wherein Y is H, OH, O-
PG,
or halo; and PG is a protecting group:
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H
O N N
O
0~~. Y
O O
Formula B
to a compound of Formula I herein. The process can further include
converting the compound of Formula B to the corresponding diphosphate; and
further
include removal of an oxygen- or nitrogen-protecting group.
In one aspect, a method of making any of the compounds delineated herein
involves one or more reactions and/or reagents as delineated herein.
In other aspects, the invention relates to a composition comprising a
compound of any of the formulae herein, an additional therapeutic agent, and a
pharmaceutically acceptable carrier. The additional therapeutic agent can be
an
anticancer agent (e.g., arabinofuranosyl cytosine (ara-C), 5-fluorouracil (5-
FU) and
taxol). The additional therapeutic agent can also be a histone deacetylase
inhibitor:
See, e.g., Lemaire et al. Leukemia Lym~ahorna 2004, 45, 147-154; Leone et al.
Clin
Imrnunol. 2003, 109, 89-102; Shaker et al. Leukemia Res 2003, 27, 437-444;
Primeau
et al. Int. J. Cancer 2003, 103, 177-184.
Yet another aspect of this invention relates to a method of treating a subject
(e.g., mammal, human, horse, dog, cat) having a DNA-methylation-mediated
disease
. 20 or disease symptom (including, but not limited to cancer). The method
includes
administering to the subject (including a subject identified as in need of
such
treatment) an effective amount of a compound described herein, or a
composition
described herein to produce such effect. Identifying a subject in need of such
_g_
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The invention also relates to a method of making a compound described
herein. Alternatively, the method includes taking any one of the intermediate
compounds described herein and reacting it With one or more chemical reagents
in
one or more steps to produce a compound described herein.
In other aspects the invention relates to a compound made by a process that
includes any one, or more, of the reactions delineated herein. In particular,
the
reactions in the general schemes and examples herein. In other aspects, the
process
includes the reagent or reagents, or reaction conditions delineated herein.
Also within the scope of this invention is a packaged product. The packaged
product includes a container, one of the aforementioned compounds in the
container,
and a legend (e.g., a label or an insert) associated with the container and
indicating
administration of the compound for treating a disorder associated with DNA-
methylation modulation.
In other embodiments, the compounds, compositions, and methods delineated
herein are any of the compounds of Table I or Table II (or salts or solvates
thereof)
herein or methods including them.
_g_
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/~O wZ
X
O N N
O
/P~~ O~\''' Y
O
Compound X Y z
1 NH2 H bond
2 NH2 F bond
3 NH2 OH bond
4 NH2 OBz bond
N(CH3)3 + H bond
6 N(CH3)3 + F bond
'7 N(CH3)3 + OH bond
g N(CH3)3 + OBz bond
NH2 H _P(O)(OH)_O_
NHZ F -P(O)(OH)-O_
11 NH2 OH -P(O)(OH)-O_
12 NH2 OBz -P(O)(OH)-O-
13 N(CH3)3 + H -P(O)(OH)-O-
14 N(CH3)3 + F -P(O)(OH)-O-
N(CH3)3 + OH -P(O)(OH)-O_
16 N(CH3)3 + pBz -P(O)(OH)-O_
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X
O Z
Compound
17 NH2 H bond
18 NHZ F bond
19 NHa OH bond
20 NH2 OBz bond
21 N(CH3)3 + H bond
22 N(CH3)3 + F bond
23 N(CH3)3 + OH bond
24 N(CH3)3 + OBz bond
25 NH2 H -P(O)(OH)-O_
26 NH2 F -P(O)(OH)-O-
27 NH2 OH -P(O)(OH)-O_
28 NH2 OBz -P(O)(OH)-O_
29 N(CH3)3 + H -P(0)(OH)-0-
30 N(CH3)3 + F -P(0)(OH)-0-
31 N(CH3)3 + OH -P(O)(OH)-O_
32 N(CH3)3 + OBz -P(O)(OH)-O_
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accompanying drawings and the description below. Other features, objects, and
advantages of the invention will be apparent from the description and from the
claims.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 shows the structures of zebularine and cytidine.
FIG. 2 illustrates the effects of 5-azacytidine (5-azaC) and zebularine
exposure
to T24 cell proliferation
FIG. 3 illustrates radiolabeled metabolites of zebularine incubated with T-24
cells.
FIG. 4 illustrates the enzymatic characterization of zebularine metabolites.
FIG. 5 illustrates HPLC radiochromatograms of zebularine metabolites.
FIG. 6 illustrates formation of phosphorylated zebularine metabolites.
FIG. 7 profiles zebularine metabolite levels in T-24 cells.
cells.
FIG. 8 illustrates incorporation of zebularine into DNA and RNA of T-24
FIG. 9 illustrates the HPLC radiochromatograms of zebularine metabolites in
vivo in nude mice in tumor and in normal muscle.
FIG. 10 illustrates the effect of zebularine on human bladder carcinoma cell
proliferation.
FIG. 11 illustrates a metabolic activation pathway for zebularine.
DETAILED DESCRIPTION OF THE INVENTION
The terms "halo" and "halogen" refer to any radical of fluorine, chlorine,
bromine or iodine. The term "alkyl" refers to a hydrocarbon chain that may be
a
straight chain or branched chain, containing the indicated number of carbon
atoms.
For example, C1-C10 indicates that the group may have from 1 to 10 (inclusive)
carbon atoms in it. The term "lower alkyl" refers to a C1-C6 alkyl chain. The
term
"alkenyl" refers to an unsaturated hydrocarbon chain that may be a straight
chain or
branched chain, containing the indicated number of carbon atoms. The term
"alkynyl" refers to an unsaturated hydrocarbon chain that may be a straight
chain or
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"ester" refers to a C(O)O-alkyl or C(O)O-aryl group. An "amido" is an C(O)NHZ,
an
"N-alkyl-substitited amido" is of the formula C(O)N(H)(alkyl).
The term "cycloalkyl" refers to a 6-carbon monocyclic or 10-carbon bicyclic
nonaromatic ring system wherein 0, 1, 2, 3, or 4 atoms of each ring may be
substituted
by a substituent. Examples of cycloalkyl groups include cyclopentyl,
cyclohexyl,
cyclohexenyl, bicyclo[2.2.1]hept-2-enyl, dihydronaphthalenyl,
benzocyclopentyl, and
the like.
The term "aryl" refers to a 6-carbon monocyclic or 10-carbon bicyclic
aromatic ring system wherein 0, 1, 2, 3, or 4 atoms of each ring may be
substituted by
a substituent. Examples of aryl groups include phenyl, naphthyl and the like.
The
term "arylalkyl" or the term "aralkyl" refers to alkyl substituted with an
aryl. The
term "arylalkoxy" refers to an alkoxy substituted with aryl.
The term "heteroaryl" refers to an aromatic 5-8 membered monocyclic, 8-12
membered bicyclic, or 11-14 membered tricyclic ring system having 1-3
heteroatoms
if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic,
said
heteroatoms selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9
heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic,
respectively), wherein
0, l, 2, 3, or 4 atoms of each ring may be substituted by a substituent.
Examples of
heteroaryl groups include pyridyl, furyl or furanyl, imidazolyl,
benzimidazolyl,
thienyl, indolyl, thiazolyl, and the like. The term "heteroarylalkyl" or the
term
"heteroaralkyl" refers to an alkyl substituted with a heteroaryl. The term
"heteroarylalkoxy" refers to an alkoxy substituted with heteroaryl.
The term "heterocyclyl" refers to a nonaromatic 5-8 membered monocyclic, 8-
12 membered bicyclic, or 11-14 membered tricyclic ring system comprising 1-3
heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if
tricyclic,
said heteroatoms selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or
1-9
heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic,
respectively), wherein
0, 1, 2, 3, or 4 atoms of each ring may be substituted by a substituent.
Examples of
heterocyclyl groups include tetrahydrofuryl, piperidinyl, pyrrolidinyl,
morpholinyl,
dihydrothiophenyl, dihydrobenzothiophenyl, indolinyl, and the like.
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the group hydroxy, mercapto, amino, alkoxy, carboxylic acid, ester, amido, N-
alkyl-
substitited amido, halo, nitro, and nitrite; or 1 to 4 independent NRERE,
C(O)NRERE,
ORE, SRE, C(O)ORE, C(O)RE, S(O)"RE, N02, CN, halo, NREC(O)RE, or NRES(O)"RE;
wherein n is 1 or 2; and each RE is independently alkyl, alkenyl, aryl,
arylalkyl, or
heteroarylalkyl, each optionally substituted with 1-4 independent substituents
selected
from the group hydroxy, mercapto, amino, alkoxy, carboxylic acid, ester,
amido,
N-alkyl-substitited amido, halo, nitro, and nitrite.
Combinations of substituents and variables envisioned by this invention are
only those that result in the formation of stable compounds. The term
"stable", as used
herein, refers to compounds which possess stability sufficient to allow
manufacture
and which maintains the integrity of the compound for a sufficient period of
time to
be useful for the purposes detailed herein (e.g., therapeutic formulations,
reagents,
kits). The compounds produced by the methods herein can be incorporated into
compositions, including pills, capsules, gel caps, solutions, tablets, cremes,
or
ointments for administration to a subject (e.g., human, animal).
Acids and bases useful in the methods herein are known in the art. Acid
catalysts are any acidic chemical, which can be inorganic (e.g., hydrochloric,
sulfuric,
nitric acids) or organic (e.g., camphorsulfonic acid, p-toluenesulfonic acid,
acetic
acid) in nature. Acids are useful in either catalytic or stoichiometric
amounts to
facilitate chemical reactions. Bases are any basic chemical, which can be
inorganic
(e.g., sodium bicarbonate, potassium hydroxide) or organic (e.g.,
triethylamine,
pyridine) in nature. Bases are useful in either catalytic or stoichiometric
amounts to
facilitate chemical reactions.
Alkylating agents are any reagent that is capable of effecting the alkylation
of
the functional group at issue (e.g., oxygen atom of an alcohol, nitrogen atom
of an
amino group). Alkylating agents are known in the art, including in the
references
cited herein, and include alkyl halides (e.g., methyl iodide, benzyl bromide
or
chloride), alkyl sulfates (e.g., methyl sulfate), or other alkyl group-leaving
group
combinations known in the art. Leaving groups are any stable species that can
detach
from a molecule during a reaction (e.g., elimination reaction, substitution
reaction)
and are known in the art, including in the references cited herein, and
include halides
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carbamates (e.g., N(Me)C(O)Ot-Bu), phosphonates (e.g., -OP(O)(OEt)2), water or
alcohols (erotic conditions), and the like.
Nucleophilic agents are known in the art and axe described in the chemical
texts and treatises referred to herein. The chemicals used in the
aforementioned
methods may include, for example, solvents, reagents, catalysts, protecting
group and
deprotecting group reagents and the Like. The methods described above may also
additionally comprise steps, either before or after the steps described
specifically
herein, to add or remove suitable protecting groups in order to ultimately
allow
synthesis of the compound of the formulae described herein. The methods
delineated
herein contemplate converting compounds of one formula to compounds of another
formula. The process of converting refers to one or more chemical
transformations,
which can be performed i~c situ, or with isolation of intermediate compounds.
The
transformations can include reacting the starting compounds or intermediates
with
additional reagents using techniques and protocols known in the art, including
those
in the references cited herein. Intermediates can be used with or without
purification
(e.g., filtration, distillation, crystallization, chromatography).
The compounds delineated herein can be synthesized using conventional
methods, as illustrated generally in the schemes herein.
In the structures in Schemes I and II, Y, X and Z are as defined in the
formulae herein, and PG is a protecting group (e.g., an oxygen-protecting
group, a
nitrogen-protecting group).
The term "protecting group," as used herein, refers to a labile chemical
moiety
which is known in the art to protect a chemical group (e.g., oxygen- or
nitrogen-
containing moiety) against undesired reactions during synthetic procedures.
After
said synthetic procedures) the protecting group as described herein may be
selectively removed. Protecting groups as known in the art are described
generally in
T.H. Greene and P.G. M. Wuts, Protective Groups in Organic S,.ynthesis, 3rd
edition,
John Wiley & Sons, New York (1999).
The term "oxygen protecting group," as used herein, refers to a labile
chemical
moiety which is known in the art to protect a hydroxyl group against undesired
reactions during synthetic procedures. After said synthetic procedures) the
hydroxy
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Protective Groups in Or~~anic Synthesis, 3rd edition, John Wiley ~ Sons, New
York
(1999). Examples of hydroxyl protecting groups include acetyl (Ac or -
C(O)CH3),
benzoyl (Bz or -C(O)C6Hs), benzyl (Bn), t-butyldimethylsilyl, (TBDMS), and
trimethylsilyl (TMS or-Si(CH3)3).
The term "nitrogen protecting group," as used herein, refers to a labile
chemical moiety which is known in the art to protect a nitrogen-containing
group
against undesired reactions during synthetic procedures. After said synthetic
procedures) the nitrogen protecting group as described herein may be
selectively
removed. Nitrogen protecting groups as known in the are described generally in
T.H.
Greene and P.G. M. Wuts, Protective Groups in Organic Synthesis, 3rd edition,
John
Wiley & Sons, New York (1999). Examples of nitrogen protecting groups include
acetyl (Ac or -C(O)CH3), phthalimido, BOC (t-butoxycarbonyl), and the like.
Compounds of Formula A can be converted to compounds of Formula B using
standard cyclization conditions such as those essentially described by Geniser
et al.
(S'yv~thesis, 53-54 (1989)). In instances where Y in Formula B is hydroxy, the
hydroxy
group can be protected (e.g., benzoyl) to give compounds of Formula B wherein
Y is
a protected hydroxy group (OPG). Compounds of Formula B can then be converted
to the diphosphate compounds of Formula C under standard conditions (e.g.,
DCC, (t-
Bu3N0)3P0) and then coupled (DCC and choline chloride or N-protected
ethanolamine, such as phthalimido-protected ethanolamine) followed by
deprotection,
if appropriate (e.g., removal of protecting groups, hydrolysis, aqueous acid,
ammonia/MeOH, hydrazine), to give compounds of Formula E where Z is
-P(O)(OH)-O-. Compounds of Formula B where Y is H or halogen can also be
directly coupled (DCC and choline chloxide or N-protected ethanolamine, such
as
phthalimido-protected ethanolamine) followed by deprotection, if appropriate
(e.g.,
removal of protecting groups, hydrolysis, aqueous acid, ammonia/MeOH,
hydrazine),
to give compounds of Formula E where Z is a bond. For compounds of Formula B
where Y is hydroxy, the hydroxy group is protected to give a compound of
Formula
D, which is coupled and then further deprotected to give compounds of Formula
E
where Z is a bond.
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O (/ O N IN
O
HO ~ O
O P~~O~~~ ' Y
HO~~~ y HO~ O
Formula A Formula B
I
O
HO~
~~O'
HO~
Formula C
HO'
Formula D
O N"N
O~ I~IZ
X~ O
O
Formula E ~ ~~ 0~~~~~ Y
Z = P~OWHOo- ~/O ~Z
O
X
Formula E
Z = bond
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~HO)2~0)p-O
\. i O
HO~ OOH
Formula F
X O O
~) -~P-p
O_P_O
O N' /N
OH O ~H
O
Formula G GPO OpG
X
O-P-O-P-O
O N' /N
OH O ~H
O
HO OH
Formula H
Scheme II illustrates general synthesis of compounds delineated herein.
Compounds of Formula F can be protected with oxygen protecting groups then
coupled (DCC and phosphorylcholine chloride or N-protected
ethanolaminephosphoric acid ester derivative, such as phthalimido-protected
ethanolaminephosphoric acid ester) followed by deprotection, if appropriate
(e.g.,
removal of protecting groups, hydrolysis, aqueous acid, ammonialMeOH,
hydrazine),
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if appropriate (e.g., removal of protecting groups, hydrolysis, aqueous acid,
ammonia/MeOH, hydrazine), to give compounds of Formula H.
As can be appreciated by the skilled artisan, the synthetic schemes herein are
not intended to comprise a comprehensive list of all means by which the
compounds
described and claimed in this application may be synthesized. Further methods
will
be evident to those of ordinary skill in the art. Additionally, the various
synthetic
steps described above may be performed in an alternate sequence or order to
give the
desired compounds. Synthetic chemistry transformations and protecting group
methodologies (protection and deprotection) useful in synthesizing the
compounds
described herein are known in the art and include, for example, those such as
described in R. Laxock, Comprehensive Organic Transformations, VCH Publishers
(1989); T.W. Greene and P.GM. Wuts, Protective Groups in Organic Synthesis,
2d.
Ed., John Wiley and Sons (1991); L. Fieser and M. Fieser, Fieser and Fieser's
Reagents for Organic Synthesis, John Wiley and Sons (1994); and L. Paquette,
ed.,
Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995) and
subsequent editions thereof.
The term "treating" or "treated" refers to administering a compound described
herein to a subject with the purpose to cure, heal, alleviate, relieve, alter,
remedy,
ameliorate, improve, or affect a disease, the symptoms of the disease or the
predisposition towaxd the disease.
"An effective amount" refers to an amount of a compound, which confers a
therapeutic effect on the treated subject. The therapeutic effect may be
objective (i.e.,
measurable by some test or marker) or subjective (i.e., subject gives an
indication of
or feels an effect). An effective amount of the compound described above may
range
from about 50 mg/Kg to about 200 mg/Kg. Effective doses will also vary
depending
on route of administration, as well as the possibility of co-usage with other
agents.
DNA methylation-modulating compounds can be identified through both in
vitro (e.g., cell and non-cell based) and in vivo methods. Representative
examples of
these methods are described in detail in the Examples.
Combinations of substituents and variables envisioned by this invention are
only those that result in the formation of stable compounds. The term
"stable", as used
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be useful for the purposes detailed herein (e.g., therapeutic or prophylactic
administration to a subject).
The compounds of this invention may contain one or more asymmetric centers
and thus occur as racemates and racemic mixtures, single enantiomers,
individual
diastereomers and diastereomeric mixtures. All such isomeric forms of these
compounds are expressly included in the present invention. The compounds of
this
invention may also be represented in multiple tautomeric forms, in such
instances, the
invention expressly includes all tautomeric forms of the compounds described
herein
(e.g., alkylation of a ring system may result in alkylation at multiple sites,
the
invention expressly includes all such reaction products). All such isomeric
forms of
such compounds are expressly included in the present invention. All crystal
forms of
the compounds described herein are expressly included in the present
invention.
As used herein, the compounds of this invention, including the compounds of
formulae described herein, are defined to include pharmaceutically acceptable
derivatives or prodrugs thereof. A "pharmaceutically acceptable derivative or
prodrug" means any pharmaceutically acceptable salt, ester, salt of an ester,
or other
derivative of a compound of this invention which, upon administration to a
recipient,
is capable of providing (directly or indirectly) a compound of this invention.
Particularly favored derivatives and prodrugs are those that increase the
bioavailability of the compounds of this invention when such compounds are
administered to a mammal (e.g., by allowing an orally administered compound to
be
more readily absorbed into the blood) or which enhance delivery of the parent
compound to a biological compartment (e.g., the brain or lymphatic system)
relative
to the parent species. Preferred prodrugs include derivatives where a group
which
enhances aqueous solubility or active transport through the gut membrane is
appended
to the structure of formulae described herein.
The compounds of this invention may be modified by appending appropriate
fzmctionalities to enhance selective biological properties. Such modifications
are
known in the art and include those which increase biological penetration into
a given
biological compartment (e.g., blood, lymphatic system, central nervous
system),
increase oral availability, increase solubility to allow administration by
injection, alter
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those derived from pharmaceutically acceptable inorganic and organic acids and
bases. Examples of suitable acid salts include acetate, adipate, alginate,
aspartate,
benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate,
camphorsulfonate, digluconate, dodecylsulfate, ethanesulfonate, formate,
fumarate,
glucoheptanoate, glycolate, hemisulfate, heptanoate, hexanoate, hydrochloride,
hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate,
malonate,
methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, palmoate,
pectinate,
persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate,
salicylate,
succinate, sulfate, tartrate, thiocyanate, tosylate and undecanoate. Other
acids, such
as oxalic, while not in themselves pharmaceutically acceptable, may be
employed in
the preparation of salts useful as intermediates in obtaining the compounds of
the
invention and their pharmaceutically acceptable acid addition salts. Salts
derived
from appropriate bases include alkali metal (e.g., sodium), alkaline earth
metal (e.g.,
magnesium), ammonium and N-(alkyl)4+ salts. This invention also envisions the
quaternization of any basic nitrogen-containing groups of the compounds
disclosed
herein. Water or oil-soluble or dispersible products may be obtained by such
quaternization.
The compositions delineated herein include the compounds of the formulae
delineated herein, as well as additional therapeutic agents if present, in
amounts
effective for achieving a modulation of disease or disease symptoms, including
ion
channel-mediated disorders or symptoms thereof.
The term "pharmaceutically acceptable carrier or adjuvant" refers to a carrier
or adjuvant that may be administered to a patient, together with a compound of
this
invention, and which does not destroy the pharmacological activity thereof and
is
nontoxic when administered in doses sufficient to deliver a therapeutic amount
of the
compound.
Pharmaceutically acceptable carriers, adjuvants and vehicles that may be used
in the pharmaceutical compositions of this invention include, but are not
limited to,
ion exchangers, alumina, aluminum stearate, lecithin, self emulsifying drug
delivery
systems (SEDDS) such as d-oc-tocopherol polyethyleneglycol 1000 succinate,
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substances such as phosphates, glycine, sorbic acid, potassium sorbate,
partial
glyceride mixtures of saturated vegetable fatty acids, water, salts or
electrolytes, such
as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen
phosphate,
sodium chloride, zinc salts, colloidal silica, magnesium trisilicate,
polyvinyl
pyrrolidone, cellulose-based substances, polyethylene glycol, sodium
carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-
block
polymers, polyethylene glycol and wool fat. Cyclodextrins such as a-, (3-, and
y-
cyclodextrin, or chemically modified derivatives such as
hydroxyalkylcyclodextrins,
including 2- and 3-hydroxypropyl-(3-cyclodextrins, or other solubilized
derivatives
may also be advantageously used to enhance delivery of compounds of the
formulae
described herein.
The pharmaceutical compositions of this invention may be administered
orally, parenterally, by inhalation spray, topically, rectally, nasally,
buccally,
vaginally or via an implanted reservoir, preferably by oral administration or
administration by injection. The pharmaceutical compositions of this invention
may
contain any conventional non-toxic pharmaceutically-acceptable carriers,
adjuvants or
vehicles. In some cases, the pH of the formulation may be adjusted with
pharmaceutically acceptable acids, bases or buffers to enhance the stability
of the
formulated compound or its delivery form. The term parenteral as used herein
includes subcutaneous, intracutaneous, intravenous, intramuscular,
intraarticular,
intraarterial, intrasynovial, intrasternal, intrathecal, intralesional and
intracranial
injection or infusion techniques.
The pharmaceutical compositions may be in the form of a sterile injectable
preparation, for example, as a sterile injectable aqueous or oleaginous
suspension.
This suspension may be formulated according to techniques known in the art
using
suitable dispersing or wetting agents (such as, for example, Tween ~0) and
suspending agents. The sterile injectable preparation may also be a sterile
injectable
solution or suspension in a non-toxic parenterally acceptable diluent or
solvent, for
example, as a solution in l,3-butanediol. Among the acceptable vehicles and
solvents
that may be employed are mannitol, water, Ringer's solution and isotonic
sodium
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employed including synthetic mono- or diglycerides. Fatty acids, such as oleic
acid
and its glyceride derivatives are useful in the preparation of injectables, as
are natural
pharmaceutically-acceptable oils, such as olive oil or castor oil, especially
in their
polyoxyethylated versions. These oil solutions or suspensions may also contain
a
long-chain alcohol diluent or dispersant, or carboxymethyl cellulose or
similar
dispersing agents which are commonly used in the formulation of
pharmaceutically
acceptable dosage forms such as emulsions and or suspensions. Other commonly
used
surfactants such as Tweens or Spans and/or other similar emulsifying agents or
bioavailability enhancers which are commonly used in the manufacture of
pharmaceutically acceptable solid, liquid, or other dosage forms may also be
used for
the purposes of formulation.
The pharmaceutical compositions of this invention may be orally administered
in any orally acceptable dosage form including, but not limited to, capsules,
tablets,
emulsions and aqueous suspensions, dispersions and solutions. In the case of
tablets
for oral use, carriers which are commonly used include lactose and corn
starch.
Lubricating agents, such as magnesium stearate, are also typically added. For
oral
administration in a capsule form, useful diluents include lactose and dried
corn starch.
When aqueous suspensions and/or emulsions are administered orally, the active
ingredient may be suspended or dissolved in an oily phase is combined with
emulsifying and/or suspending agents. If desired, certain sweetening and/or
flavoring
and/or coloxing agents may be added.
The pharmaceutical compositions of this invention may also be administered
in the form of suppositories for rectal administration. These compositions can
be
prepared by mixing a compound of this invention with a suitable non-irritating
excipient which is solid at room temperature but liquid at the rectal
temperature and
therefore will melt in the xectum to release the active components. Such
materials
include, but are not limited to, cocoa butter, beeswax and polyethylene
glycols.
Topical administration of the pharmaceutical compositions of this invention is
useful when the desired treatment involves areas or organs readily accessible
by
topical application. For application topically to the skin, the pharmaceutical
composition should be formulated with a suitable ointment containing the
active
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petroleum, white petroleum, propylene glycol, polyoxyethylene polyoxypropylene
compound, emulsifying wax and water. Alternatively, the pharmaceutical
composition can be formulated with a suitable lotion or cream containing the
active
compound suspended or dissolved in a carrier with suitable emulsifying agents.
Suitable carriers include, but are not limited to, mineral oil, sorbitan
monostearate,
polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyhdodecanol, benzyl
alcohol
and water. The pharmaceutical compositions of this invention may also be
topically
applied to the lower intestinal tract by rectal suppository formulation or in
a suitable
enema formulation. Topically-transdermal patches are also included in this
invention.
The pharmaceutical compositions of this invention may be administered by
nasal aerosol or inhalation. Such compositions are prepared according to
techniques
well-known in the art of pharmaceutical formulation and may be prepared as
solutions
in saline, employing benzyh alcohol or other suitable preservatives,
absorption
promoters to enhance bioavailability, fluorocarbons, and/or other solubilizing
or
dispersing agents known in the art.
A composition having the compound of the formulae herein and an additional
agent (e.g., a therapeutic agent) can be administered using an implantable
device.
Implantable devices and related technology are known in the art and are useful
as
delivery systems where a continuous, or timed-release delivery of compounds or
compositions delineated herein is desired. Additionally, the implantable
device
delivery system is useful for targeting specific points of compound or
composition
delivery (e.g., localized sites, organs). Negrin et al., Biomaterials,
22(6):563 (2001).
Timed-release technology involving alternate delivery methods can also be used
in
this invention. For example, timed-release formulations based on polymer
technologies, sustained-release techniques and encapsulation techniques (e.g.,
polymeric, liposomal) can also be used for delivery of the compounds and
compositions delineated herein.
Also within the invention is a patch to deliver active chemotherapeutic
combinations herein. A patch includes a material layer (e.g., polymeric,
cloth, gauze,
bandage) and the compound of the formulae herein as delineated herein. One
side of
the material layer can have a protective layer adhered to it to resist passage
of the
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natural or synthetic origin, that when contacted with the skin of a subject,
temporarily
adheres to the skin. It can be water resistant. The adhesive can be placed on
the patch
to hold it in contact with the skin of the subject for an extended period of
time. The
adhesive can be made of a tackiness, or adhesive strength, such that it holds
the
device in pace subject to incidental contact, however, upon an affirmative act
(e.g.,
ripping, peeling, or other intentional removal) the adhesive gives way to the
external
pressure placed on the device or the adhesive itself, and allows for breaking
of the
adhesion contact. The adhesive can be pressure sensitive, that is, it can
allow for
positioning of the adhesive (and the device to be adhered to the skin) against
the skin
by the application of pressure (e.g., pushing, rubbing,) on the adhesive or
device.
Compounds herein are administered in a dosage ranging from about 10 to
about 500 mg/kg of body weight, preferably dosages between 50 mg and 200
1 S mg/dose, every 4 to 120 hours, or according to the requirements of the
particular drug.
The methods herein contemplate administration of an effective amount of
compound
or compound composition to achieve the desired or stated effect. Typically,
the
pharmaceutical compositions of this invention will be administered from about
1 to
about 6 times per day or alternatively, as a continuous infusion. Such
administration
can be used as a chronic or acute therapy. The amount of active ingredient
that may
be combined with the carrier materials to produce a single dosage form will
vary
depending upon the host treated and the particular mode of administration. A
typical
preparation will contain from about 5% to about 95% active compound (wlw).
Alternatively, such preparations contain from about 20% to about 80% active
compound.
Lower or higher doses than those recited above may be required. Specific
dosage and treatment regimens for any particular patient will depend upon a
variety of
factors, including the activity of the specific compound employed, the age,
body
weight, general health status, sex, diet, time of administration, rate of
excretion, drug
combination, the severity and course of the disease, condition or symptoms,
the
patient's disposition to the disease, condition or symptoms, and the judgment
of the
treating physician.
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necessary. Subsequently, the dosage or frequency of administration, or both,
may be
reduced, as a function of the symptoms, to a level at which the improved
condition is
retained when the symptoms have been alleviated to the desired level,
treatment
should cease. Patients may, however, require intermittent treatment on a long-
term
basis upon any recurrence of disease symptoms.
When the compositions of this invention comprise a combination of a
compound of the formulae described herein and one or more additional
therapeutic or
prophylactic agents, both the compound and the additional agent should be
present at
dosage levels of between about 1 to 100%, and more preferably between about 5
to
95% of the dosage normally administered in a monotherapy regimen. The
additional
agents may be administered separately, as part of a multiple dose regimen,
from the
compounds of this invention. Alternatively, those agents may be part of a
single
dosage form, mixed together with the compounds of this invention in a single
composition.
"Prodrug", as used herein means a compound which is convertible in vivo by
metabolic means (e.g. by hydrolysis) to a compound of any of the formulae
herein.
Various forms of prodrugs are known in the art, for example, as discussed in
Bundgaard, (ed.), Design of Prodrugs, Elsevier (195); and "Hydrolysis In Drug
And
Prodrug Metabolism: Chemistry, Biochemistry And Enzymology," John Wiley and
Sons, Ltd. (2002).
The invention will be further described in the following examples. It should
be
understood that these examples are for illustrative purposes only and are not
to be
construed as limiting this invention in any manner.
All references cited herein, whether in print, electronic, computer readable
storage media or other form, are expressly incorporated by reference in their
entirety,
including but not limited to, abstracts, articles, journals, publications,
texts, treatises,
technical data sheets, Internet web sites, databases, patents, patent
applications, and
patent publications.
Embodiments are further described in the following representative examples,
which do not limit the scope of the invention described in the claims.
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Abb~eviatio~s: Zeb, zebularine; Zeb-MP, zebularine-5'- monophosphate;
Zeb-DP, zebularine-5'-diphosphate; Zeb-TP; zebularine-5'-triphosphate; Zeb-DP-
EA, zebularine-5'-diphosphoethanolamine; Zeb-DP-Chol, zebularine- 5'-
diphosphocholine; CPEU, cyclopentenyl uridine; ICSO, drug concentration
resulting
in 50% inhibition of growth; PDE-1, snake venom phosphodiesterase-1; AP,
alkaline
phosphatase; UCI~, uridine/cytidine kinase; NCI, US National Cancer Institute.
1. Chemistry
1.1. Zebularine and corresponding 2'-deoxy and 2'-fluoro analogues are known
in.the
literature. See, Driscoll et al., J. Med. Chem. 34, 3280-3284 (1991); Barchi
et al.,
Nucleosides & Nucleotides, 11, 1781-1793 (1992).
1.2
HzN
o n I~_ _~~
(Ha)z(~)P-o~~~.~ o-i o i -o
O
O OH OH
BzO~ ~Ogz O
loo HO~~~ ~pH
101
Compound 100 is reacted with phosphoric acid mono-[2-(1,3-dioxo-1,3-
dihydro-isoindol-2-yl)-ethyl] ester in the presence of DCC in methylene
chloride to
provide the corresponding diphosphate. The resulting diphosphate is
deprotected
(hydrazine; methanolic ammonia) to provide compound 10I. The 2'-deoxy and 2'-
fluoro analogs of compound 101 can be made similarly from available 2'-deoxy
and
2'-fluoro analogs of compound 100.
1.3
I (Hs0)sN+
a o
a N N
~O-IP-O-IP-O
on
(HO)z(O)P-O ~ I
O OH OH
Bz0 Ogz O
10o HO~~ ''OH
102
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1,3-dicyclohexylcarbodiimide (DCC) in methylene chloride to provide the
corresponding diphosphate. The diphosphate is hydrolized under methanolic
ammonia conditions to provide compound 102. The 2'-deoxy and 2'-fluoro analogs
of compound 102 can be made similarly from available 2'-deoxy and 2'-fluoro
analogs of compound 102.
1.4
103 104
Y=F
Compound I03 (available via literature procedures) is reacted with 2-
phthalimidoethyl bromide in the presence of DCC in methylene chloride. The
resulting compound is treated with hydrazine to provide compound 104.
2. Materials and methods
~.1. Chemicals ahd ~eagevcts
Zebularine (1-((3-D-ribofuranosyl)1,2-dihydropyrimidin-2-one, 2(1H)-
pyxiidinone
riboside, NSC 309132) and [2-14C]Zeb (51 mCi/mmol) were obtained from the
Developmental Therapeutics Program, Division of Cancer Treatment and
Diagnosis,
NCI (Bethesda, MD). [Methyl-3H]choline chloride (80 Ci/mmol) and [1 3H]ethanol-
1-ol-2-amine hydrochloride (31 Ci/mmol) were purchased from American
Radiolabeled Chemicals, Tnc. (St. Louis, MO). Choline chloride, ethanolarnine
HCI,
Tri-reagent~ kits and selected nucleoside and nucleotide standards were
obtained
from Sigma Chemical Co. (St. Louis, MO). The enzymes deoxyribonuclease I
(DNase I, type II [bovine pancreas], EC 3.1.21.1), ribonuclease A (type I-A,
jbovine
pancreas], EC 3.1.27.5), PDE-1 (type VII [Crotalus atrox venom], EC 3.1.4.1),
and
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CA) from parent nucleoside and had an HPLC purity >98%. A Zeb-MY standard was
produced by complete enzymatic conversion of Zeb-TP by PDE-1. 2'-
Deoxyzebularine [G] and CPEU [F] were synthetic products that were available
from
previous studies in our laboratories. AlI other chemicals and reagents were of
the
highest quality commercially available.
2.2 Cell culture
Human T-24 bladder carcinoma and human Molt-4 lymphoid cells were obtained
from the American Type Culture Collection (Rockville, MD). The EJ6 cell line,
a
tumorigenic derivative of T-24 cells, was kindly provided by Dr. Eric J.
Stanbridge,
Department of , University of California at Irvine, and marine MC38 colon
cancer
cells were a gift from Dr. Steven A. Rosenberg, Surgery Branch, Center for
Cancer
Research, NCI. T-24, EJ6 and MC38 cells were cultured in DMED media while
Molt-4 cells were grown in RPMI 1640 media. All media were supplemented with
10% heat-inactivated fetal calf serum, 4 mM glutaxnine, 100 U/ml penicillin,
and 100
~,g/ml streptomycin. Cells were in logarithmic growth at the time of use and
were
maintained at 37 °C in a humidified atmosphere of 95% air and 5% C02.
2. 3. Effect of zebulay~i~ce oh T 24 cell growth
Exponentially growing T-24 cells were cultured in 24-well plates (105 cells
per
well) overnight. Cells were then washed with fresh medium and varying
concentrations (0-1000 ~.M) of zebularine were added. After incubation for 48
h, cells
were trypsinized, collected and counted in a ZB 1 Coulter Counter. The cell
growth
rate was expressed as a percentage of the increase in cell number of the
untreated
control cultures. The ICSO was calculated from the linear portion of the
growth
inhibition curve.
~. 4. Separation and measur~eme~ct of zebulariv~e aid its cellular metabolites
2.4.1. Preparation of cell exty~acts
After the appropriate incubations with [2-14C]Zeb, cells were washed three
times
with PBS, treated with trypsin and collected by centrifugation at 1500 x g for
10 min.
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removed and heated at 95°C for 2.5 min. The heated extract was
centriiiiged at
12,000 x g for 10 min and the clear supernatant fraction was removed and
evaporated
to dryness under NZ. This sample was reconstituted in 250 ~,1 HZO and aliquots
were
subjected to gradient anion-exchange chromatography as described below.
2.4.2. Gradient av~ion-exchange HPLC
Separations of Zeb and its phosphorylated metabolites were carried out using a
Hewlett-Packaxd 1100 HPLC system with a diode-array ultraviolet absorption
detector and controlled by ChemStation software (Version 6.01). A Whatman
Partisil-10 SAX column (250 X 4.6 mm) certified to have greater than 100,000
plates/m was used with the following elution program: 0-5 min, 100% buffer A
(0.01
M ammonium phosphate, native pH); 5-20 min, linear gradient to 25% buffer B
(0.7
M ammonium phosphate with 10% methanol); 20-30 min, linear gradient to 100%
buffer B; 30-40 min, isocratic buffer B; 40-55 min, linear gradient to 100%
buffer A
and equilibration. The flow rate was 2 ml/min throughout. One-minute fractions
were collected and radioactivity was determined by scintillation spectrometry.
Fractions containing radiolabeled Zeb-containing nucleotides were quantified
based
on the known specific activity of [2-14C]Zeb. To confirm that the zebularine
base was
present in these radiolabeled nucleotides, aliquots of the cell extract were
enzymatically treated to degrade all nucleotides to nucleosides and analyzed
by anion-
exchange and reverse phase HPLC as described more fully below.
2.4.3. Reverse phase HPLC
Reverse-phase HPLC was employed for the separation and identification of
zebularine nucleosides in cell extracts, for measuring [2-14C]Zeb after
enzymatic
degradation of its phosphorylated metabolites, and for the DNA and RNA
studies.
Separations were carried out on a 5-~,m Beckman Ultrasphere C1$ column (250 X
4.6
mm) using the following gradient elution program with a flow rate of 2 ml/min
throughout: 0-25 min, linear gradient from 1% to 25% methanol; 25-30 min,
isocratic
with 25% methanol; 30-40 min, linear gradient to 1% methanol, and
equilibration.
One-minute fractions were collected and radioactivity was determined by liquid
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2.5. Enzymatic characterization of zebularine metabolites
Characterization of zebularine metabolites was carried out by selective
enzymatic
degradation of cellular extracts as previously described [A]. Briefly, the
lyophilized
methanolic cell extract was dissolved in 100 p1 of 0.01 M Tris~HCI, pH 9.0
containing 1 mM MgCl2, and 1.5 U AP or 0.03 U PDE-1 was added to the
appropriate aliquots. Samples were incubated for 6 h at 37 °C, enzymes
were
inactivated by heating at 95 °C for 2.5 min, and aliquots were then
analyzed by anion-
exchange and C18 reverse phase HPLC as described above.
2.6. Biosynthesis of doubly labeled choline and ethanolamine adducts
T-24 cells were cultured in DMEM medium supplemented with 10% FCS and 4
mM L-glutamine but without endogenous choline or ethanolamine. The cells so
grown exhibited doubling times (24 h) identical to those of T-24 cells in
choline
replete medium. To determine whether choline and ethanolamine adducts are
formed
during zebularine metabolism, T-24 cells were incubated with 100 ~.M [2-
14C]Zeb (1
~,Ci/ml) alone and in combination with either 2~ wM [methyl-3H]choline (10
~.Ci/ml)
or 50 ~,M [1 3H]ethanolamine (10 ~,Ci/m1) for 24 h in separate parallel
experiments.
Thereafter, cells were harvested, washed and extracted with 60% methanol.
Zebularine metabolites were determined by gradient ion-exchange HPLC as
described
above.
2. 7. Dose-dependent formation of zebularine metabolites
Triplicate aliquots of logarithmically growing T-24 cells (1 X 106 cells) were
incubated for 6 h with concentrations of [2-14C]Zeb (1 ~,Ci/ml) ranging from 1-
500
~,M. At the end of the incubations, methanolic extracts were prepared and
amounts of
zebularine metabolites were determined by gradient anion-exchange HPLC as
described before.
2.8. Rates of accumulation and decay of zebularine metabolites
T-24 cells in logarithmic growth were incubated with 10 ~,M [2-14C]Zeb (1
~,Ci/ml
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metabolites as previously described. After 24 h of exposure to drug, the cells
were
washed three times with fresh medium and re-incubated in drug-free medium.
Aliquots of this incubation mixture were then sampled periodically for the
ensuing
24-h period and the same work-up and analysis carried out. Apparent
disappearance
half lives after removal of drug were determined for individual metabolites by
non-
linear Ieast squares analysis of their concentration versus time profiles
using a
monoexponential decay function.
2.9. Effect of cytidine, uridine and CPELI ou zebularihe phosphorylatioh
Logarithmically growing T-24 cells (1 X 106 cells ) were incubated for 6 h
with 10
p.M [2-14C]Zeb (1 ~,Ci/ml) alone and in combination with either 10 or 50 ~,M
of
cytidine, uridine or CPEU, respectively. At the end of incubation, duplicate
aliquots
of cells were harvested and methanolic extracts were prepared and analyzed as
previously described
2.10. Incorporation ofzebularihe into cellular DNA and RNA
DNA and RNA were isolated using the TRI-reagent~ procedure [B,C]. Briefly, S
X 107 T-24 cells were incubated for 24 h with 10 u.M [2-14C]Zeb (1 p,Ci/ml).
At the
end of the incubation, cells were washed 3 times with cold PBS, harvested by
trypsinization and collected by centrifugation. One ml TRI-reagent~ (guanidine
thiocyanate and phenol in a monophasic solution) was added to the cell pellet,
solubilizing the DNA, RNA and protein. Chloroform ( 0.2 ml) was then added and
the mixture was centrifuged at 12,000 X g for 15 min. RNA (in the aqueous
phase)
and DNA (in the interphase) were then separated according to the TRI-reagent~
protocol, and the radioactivity in each quantified by liquid scintillation
counting.
The isolated DNA and RNA were hydrolyzed overnight at 37 °C in 1
ml of pH
7.4, 0.1 M HEPES buffer containing 140 ~cg of either DNase or Rnase and 0.02 U
PDE-1 and 5 U AP. One hundred-microliter aliquots of this reaction mixture
representing approximately 25 ~.g of hydrolyzed DNA or RNA were analyzed by
reverse-phase HPLC as described above to determine the presence and amount of
either [2-14C]2'-dZeb (DNA) or [2-14C]Zeb (RNA).
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2. 1l. Metabolism of zebularihe ih EJ6-derived tumors
All animal care and experiments were performed in accordance with the
guidelines of the Animal Caxe and Use Committee of the Ben-Gurion University
of
the Negev. Male BABL/c nulr~u mice (Harlan, San Diego, CA)(n=6), 6-8 weeks of
age, were kept at 22 t 1 ° C in 40- 60% relative humidity with
alternating 12-h periods
of light and dark and maintained on a diet of commercial-pelleted mouse food
(Purina
Chow) with free access to food and water. Mice were inoculated s.c. with 2.5 X
106
EJ6 cells each into both the left and right flanks. After a period of 2-3
weeks during
which macroscopic tumors (50-200mm3) developed, the mice (mean weight = 25 ~ 4
gm) were treated i.p. with 500 mg/kg [2-14C]Zeb (500 ~.Ci/ml). EJ6 tumors and
straight muscle were removed from mice under ether anesthesia 24 h after drug
administration and immediately frozen in liquid NZ and stored at -70 °C
pending
sample work-up and analysis. Tumors and muscle tissue (100-200 mg) were
homogenized at 4 °C in 1.0 ml of 60% methanol using a Polytron
homogenizes. The
homogenate was heated at 95°C for 3 min and centrifuged at 12,000 X g
for 10 min at
4 °C. The supernatant was collected and evaporated under a nitrogen
stream. The
resulting residue was then dissolved in deionized water and appropriate
aliquots
subjected to anion-exchange HPLC analysis as described by Noy et al. [D].
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Zebularine exhibited a moderate cytostatic effect on logarithmically growing T-
24
cells in culture. As seen in Fig. 2 and Fig. 10, a 48-hr exposure to the drug
was able
to inhibit T-24 cell proliferation with an ICSO of 145 ~M or less. This
antiproliferative
effect appeared to reach a plateau at concentrations of 500 ~M and above for
this
exposure time.
3.2. Cellular metabolism of zebula~i~e
A requirement for the biological activation of nucleoside analogues is their
anabolic
conversion to nucleotides. In the case of zebularine, conversion to the
triphosphate
appears to be a prerequisite for eventual incorporation into DNA and the
ability to
function as an inhibitor of DNA methylation. Therefore, the ability of
zebularine to
be phosphorylated was assessed using one marine and two human cell lines. This
was
accomplished by exposing exponentially growing cells to 10 ~,M [2 3H]Zeb for 6
h,
then extracting the cells with 60% methanol, and subjecting the extract to
gradient-
elution, ion-exchange chromatography with radiochemical detection. Typical
radiochromatograms obtained after zebularine treatment of T-24 cells are shown
in
Fig. 3 and Fig. 4A. In addition to the parent drug, which elutes close to the
void
volume (2 min), five acidic metabolites were observed. Three of these
metabolites
with retention times of 9, 18 and 29 min, respectively, corresponded to the 5'-
mono-,
di- and triphosphates of zebularine. These assignments were made by comparison
with authentic standards and are supported by enzymatic digestion of the
extracts with
PDE-1 and AP (Fig. 4B and 4C). Treatment of the cellular extract with PDE-1
resulted in the disappearance of the two HPLC peaks corresponding to Zeb-DP
and
Zeb-TP and produced a concomitant increase in the peak corresponding to Zeb-MP
(Fig. 4B). AP digestion of another aliquot of this extract eliminated the HPLC
peaks
corresponding to Zeb-MP, Zeb-DP and Zeb-TP and generated an increase in parent
nucleoside (Fig. 4C).
The remaining two metabolites, eluting at 5 min and 11 min respectively, were
identified as phosphodiester conjugates of ethanolamine and choline based on
the
following evidence. The fact that both metabolites were quantitatively
decomposed
by PDE-l, with one of the products being Zeb-MP (Fig. 4B), strongly suggested
that
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was unlikely. To assess whether choline and ethanolaxnme could be directly
utiiizea
by T-24 cells to form Zeb-DP conjugates, T-24 cells were incubated with [2-
14C]Zeb
and [3H]choline or [3H]ethanolamine. HPLC and radiometric analysis of the
cellular
extracts resulting from these double-label experiments indicated that the two
metabolites eluting with retention times of 5 and 11 min, respectively, did
indeed
contain both 14C and tritium. When T-24 cells were incubated with [2-14C]Zeb
and
[3H]ethanolamine, the peak eluting at 5 min contained both labels (Fig. 5B).
When
[3H]choline was used with [2-14C]Zeb, the double label was associated with the
peak
with a retention time of 11 min (Fig. SC). Furthermore, no tritium was
associated
with either peak when [3H]ethanolamine or [3H]choline was incubated with T-24
cells
in the absence of zebularine. Thus the two metabolites eluting at 5 and 11 min
can be
identified as phosphodiesters of zebularine conjugated with ethanolamine and
choline,
respectively.
Zebularine phosphorylation was also evaluated in Molt-4 human lymphoblasts
and in MC38 marine colon carcinoma under conditions comparable to those
employed for T-24 bladder carcinoma. As can be seen in Table 1, the five
zebularine
metabolites were also observed in these two cell lines after a 6-h incubation
with 10
~M [2-14C]Zeb. For this particular time point, levels of each of the five
zebularine
metabolites were highest in the MC38 cells with the concentration of Zeb-TP
being 6-
fold greater than in T-24 or Molt-4 cells. Zeb-TP and the Zeb-DP-Chol adduct
were
the major phosphorylated metabolites in all the cell lines, with the former
being
highest in Molt-4 and MC38 and the latter greatest in T-24 bladder carcinoma.
Individual metabolites levels were comparable in the two human cells lines
except for
the Zeb-DP-Chol adduct which was 3-fold higher in T-24 cells.
3.3. Dose-dependent formation of zebulay~ine metabolites
The formation of the five zebularine metabolites was evaluated as a function
of
zebularine dose in T-24 cells. Cells were incubated with increasing
concentrations of
[2-14C]Zeb (1 - 500 ~.M) for 6 h, at which time the levels of zebularine
metabolites
were measured. As can be seen in Fig. 6, levels of all metabolites increased
with
increasing zebularine dose. The rates of this dose-dependent formation were
the
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concentrations of these two major metabolites were more than 4-fold greater
than the
other metabolites and approached or exceeded the nanomole per million cell
level.
Furthermore, the formation of Zeb-TP did not appear to be saturable in
contrast to the
other metabolites whose levels start to plateau at 250 ~M zebularine (Fig. 6).
Thus in
the case where the zebularine dose was increased to 500 ~,M, Zeb-TP levels
exceeded
those of the Zeb-DP-Chol adduct.
3.4. Rates of accumulatiov~ and decay of zebula~i~e metabolites
The 24-h intracellular accumulation of individual zebularine metabolites was
evaluated for T-24 cells using a dose of 10 ~,M [2-14C]Zeb. After incubation
with
radiolabeled drug for an initial 24-h period, cells were washed three times
and
reincubated in drug-free media so that the decay rate of these metabolites
could be
determined over the ensuing 24-h period. As described in Materials and
Methods,
levels of the various zebularine metabolites were determined at timed
intervals over
the entire 48-h period. Fig. 7 depicts the concentration versus time profiles
of the
intracellular accumulation and decay of the individual zebularine metabolites.
Zeb-
MP, Zeb-DP and Zeb-TP all exhibited an initial rapid rate of intracellular
accumulation over the initial 4 h before reaching a more or less constant
steady-state
level by 8-12 h. The rate of accumulation of the phosphodiester adducts was
more
gradual and sustained, since both the Zeb-DP-EA and Zeb-DP-Chol adducts
increased
over the entire 24-h period and did not reach a steady-state. Upon removal of
parent
drug, Zeb-DP and Zeb-TP decayed rapidly with roughly equivalent half lives of
1.5 h
and 1.7 h, respectively. The intracellular disappearance of the phosphodiester
adducts
was much more gradual with estimated half lives of 6.1 h for the Zeb-DP-EA
adduct
and 5.4 h for the Zeb-DP-Chol adduct. In contrast to the zebularine 5'-
phosphates,
these two metabolites could still be detected in the cellular extract 24 h
after removal
of drug. The intracellular elimination rate of Zeb-MP was intermediate to that
of the
other metabolites with a half life of 4.1 h.
3.5. Effect of cytidine, ur~idine and CPEU on zebularine phosphorylation
Since zebularine is an analogue of cytidine (Fig. 1), it was of interest to
ascertain
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determined in T-24 cells incubated with 10 ~,M [2-14C]Zeb for 6 h in the
pxesence of
the natural UCK substrates cytidine and uridine and the UCK inhibitor CPEU. As
shown in Table 2, 10 ~.M cytidine reduced the concentration of all zebularine
metabolites except Zeb-DP by more than 50%. Increasing the cytidine
concentration
to 50 ~,M led to the almost complete abrogation of zebularine anabolism; Zeb-
MP and
the two phosphodiester adducts could no longer be detected in the cellular
extract. In
contrast to cytidine, uridine at either 10 p,M or 50 ~,M had no significant
inhibitory
effect on zebularine metabolism. Cyclopentenyl uridine, a potent inhibitor of
UCK
with low cytotoxicity [F], was more effective than cytidine in restricting
zebularine
phosphorylation at the 10 ~,M level and at least equally effective at 50 ~,M.
These
data strongly suggest that the initial phosphorylation of zebularine in T-24
cells is
indeed mediated by UCK.
3.6. Incorpor~atiou ofaebula~ihe into cellula~° DNA and RNA
The ability of zebularine to be incorporated into host cell nucleic acids was
evaluated by examining DNA and RNA isolates from T-24 cells following 24 - 72
h
exposure to 10 ~,M [2-14C]Zeb. As illustrated in Fig. 8A, the vast majority of
incorporated radioactivity was found in the RNA of treated cells. DNA
incorporation
of xadiolabel, while observable and significant, was only on average about 15%
that
of RNA. Subsequent reverse-phase HPLC analysis of the free nucleosides
generated
from the complete enzymatic digestion of the isolated DNA and RNA revealed
that
the radiolabel coeluted with zebularine itself in RNA and with authentic 2'-
deoxyzebularine in DNA and that no other radioactive peaks were seen in either
case
(Fig. 8B). Measurement of radiolabeled zebularine and 2'-deoxyzebularine in
the
RNA and DNA isolated from T-24 cells treated 10 ~,M [2-14C]Zeb for 48 and 72 h
indicated little change from the levels observed at 24 h.
3.7. Metabolism ofzebulat~ine in EJ6-derived tumor's
The in vivo phosphorylation of zebularine was assessed following an i.p dose
of 500 mg/kg [2-14C]Zeb to nude mice bearing EJ6-derived T-24 tumors. Both
tumor
and normal muscle tissue were examined for the presence of zebularine
metabolites
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occurred (Fig. 9A). Most of the radioactivity was accounted for by 6 principal
peaks,
which were identified as parent compound and the five metabolites observed i~c
vitro
(Zeb-MP, Zed-DP, Zeb-TP and the Zeb-DP-EA and Zeb-DP-Chol adducts). The
relative amounts of the various phosphorylated metabolites of zebularine were
quite
similar to that observed for T-24 cells in culture after drug exposure (Fig. 3
and 4A).
In contrast, only a small amount of radioactivity corresponding to
phosphorylated
metabolites could be detected in a similar extract of normal, straight muscle
tissue
(Fig. 9B). In this latter case, only limited metabolic conversion was
observed, with
the major zebularine-related compound being parent drug itself (Table 3).
Table 1
Intracellular levels of zebularine metabolites in human and murine tumor cells
after
incubation with 10 p,M [2-'4C]Zeb for 6 h
Metabolite Concentrations (pmole/106 cells)
T-24 Molt-4 MC38
Zeb-MP 13.8'40.84 12.842.5 30.6'42.3
Zeb-DP 10.0 'd 0.69 5.2 d 1.8 26.9 d
3.2
Zeb-TP 25.8 d 2.2 22.2 d 4.1 152 d 11
Zeb-DP-EA adduct 1.5 d 2.0 1.1 b 0.2 1.8 d 0.4
Zeb-DP-Chol adduct 57.9 d 2.8 17.4 b' 3.0 103 'd
8.7
smean d SD from three experiments.
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Relative effect of cytidine, uridine and cyclopentenyl uridine on zebularine
metabolism in T-24 cells after incubation with 10 pM [2-'4C]Zeb for 6 h
Zeb-MP Zeb-DP Zeb-TP Zeb-DP-EA Zeb-DP-Chol
Adduct Adduct
(% of Control)a
I S Control (10 NM 100b 100 100 100 100
Zeb)
10 uM Cyd + 10 NM 34 71 49 44 16
Zeb
50 uM Cyd + 10 NM ND 22 11 ND ND
Zeb
10 uM Urd + 10 pM 134 110 113 103 91
Zeb
50 ~rM Urd + 10 NM 100 115 100 66 71
Zeb
10 pM CPEU + 10 38 21 29 26 22
NM Zeb
50 uM CPEU + 10 NM 4 3 6 6 ND
Zeb
35
a Control levels (pmol / 106 cells) were: Zeb-MP, 5.1; Zeb-DP, 2.2; Zeb-
TP,6.7; Zeb-DP-EA Adduct, 2.4;
Zeb-DP-Chol Adduct, 22.
b Average of duplicate measurements.
~ Not detectable.
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mice bearing the EJ6 variant of T-24 human bladder carcinoma after i.p.
treatment
with 500 mg/kg [2-'aC]Zeb.
Metabolite Concentrations (pmole/100 mg tissue
EJ6 Tumorb Striated Muscleb
Zeb 1103 482 2682 +_ 255
Zeb-MP 694 239 229 23
Zeb-DP 833 328 24 10
Zeb-TP 1068 187 214 39
Zeb-DP-EA adduct 671 392 22 +_ 2
Zeb-DP-Chol adduct 2123 191 286 ~ 51
amean d SD from three animals.
btissue samples obtained 24 h after drug treatment.
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[A] Ford H, Jr, Cooney DA, Ahluwalia GS, Hao Z, Rommel ME, Hicks L, Bobyns
KA, Tomaszewski JE, Johns JG, Cellular pharmacology of cyclopentenyl cytosine
in
Molt-4 lymphoblasts. Cancer Res 1991; 51; 3733-40.
[B] Chomczynski P, Sacchi N, Single-step method of RNA isolation by acid
guanidinium thiocyanate-phenol-chloroform extraction, DNA and proteins from
cell
and tissue samples. Anal Biochem 1987; 162; 156-9.
[C] Chomczynski P, A reagent for the single-step simultaneous isolation of
RNA,
DNA and proteins from cell and tissue samples. Biotechniques 1993; 15; 532-7.
[D] Noy R, Ben-Zvi Z, Manor E, Candotti F, Morris JC, Ford H, Jr, Marquez VE,
Johns DG, Agbaria R, Antitumor activity and metabolic activation of N-
methanocarbathymidine, a novel thymidine analogue with a pseudosugar rigidly
fixed
in the northern conformation, in marine colon cancer cells expressing herpes
simplex
thymidine kinase. Mol Cancer Ther 2002; 1; 585-93.
[E] Hao Z, Stowe EE, Ahluwalia G, Baker DC, Hebbler AK, Chisena C, Musser SM,
Kelley JA, Perno C-F, Johns DG, Cooney DA, Characterization of 2'3'-
dideoxycytidine diphosphocholine and 2'3'-dideoxycytidine
diphosphoethanolamine.
Prominent phosphoodiester metabolites of the anti-HIV nucleoside 2',3'-
dideoxycytidine. Drug Metab Disp 1993; 21; 738-44.
[F] Lim M-I, Moyer JD, Cysyk RL, Marquez VE, Cyclopentenyluridine and
cyclopentenyl-cytidine analogues as inhibitors of uridine-cytidine kinase. J
Med
Chem 1984; 27; 1536-38.
[G] Barchi JJ, Jr, Haces A, Marquez VE, McCormack JJ, Inhibition of cytidine
deaminase by derivatives of 1-~-D-ribofuranosyl)-dihydropyrimin-2-one.
Nucleosides
Nucleotides 1992; 11; 1781-93.
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CA 02557075 2006-08-22
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J Med Chem 1991; 34; 3280-4.
[I] Marquez VE, Eritja R, Kelley JA, Vanbemmel D, Christman JK, Potent
inhibition of Hhal DNA methylase by the aglycon of 2-(1H)-pyrimidinone
riboside
(zebularine) at the GCGC recognition domain. Ann NY Acad Sci 2003: 1002; 154-
64.
[J] Cheng JC, Matsen CB, Gonzalez FA, Ye W, Greer S, Marquez VE, Jones PA,
Selker EU, Inhibition of DNA methylation and reactivation of silenced genes by
zebularine. J Natl Cancer Inst 2003: 95; 399-09.
[K] Lauzon GJ, Paterson AR, Belch AW, Formation of 1-beta-D
arabinofuranosylcytosine diphosphate choline in neoplastic and normal cells.
Cancer
Res 1978: 38; 1730-3.
Figure Legends
Fig. 1. Chemical structures of zebularine, cytidine and 5-azacytidine. The
asterisk indicates the position of the ['4C]radiolabel in zebularine.
Fig. 2. Effect of 5-azacytidine ( ! ) and zebularine (") on human bladder
carcinoma cell proliferation after treatment for 48 h. Points represent the
mean ~ SD
(n = 6). T24 cell number averaged 4 X 105 for control at 48 h. Curves were fit
(r2 >
0.990) to a sigmoidal dose-response function allowing variable slope.
Calculated
ICSOs are indicated on the graph.
Fig. 3. HPLC radiochromatogram of ['4C]metabolites arising from incubation
of T24 cells with 10 pM [2 '4C]Zeb (5 pCilml) for 6 h. Methanolic extracts
were
subjected to ion exchange HPLC as described in Materials and Methods.
Fig. 4. Enzymatic characterization of zebularine metabolites isolated from
T24 cells incubated with 10 pM [2-'4C]Zeb (1 pCi/ml) for 6 h. Equivalent
aliquots of
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(C). The peak corresponding to the Zeb-DP-EA adduct is indicated by an
asterisk.
Fig. 5. Anion-exchange HPLC radiochromatograms of zebularine metabolites
in cells treated with [2-'4C]Zeb and with either [3H]choline or
[3H]ethanolamine. T24
cells (5 X 106) were incubated with 100 pM [2-'4C]Zeb (1 pCi/ml) for 24 h
alone (A) or
in combination with either 50 pM [3H]ethanolamine (10 pCi/ml) (B) or 28 pM
[3H]choline (10 ~rCi/ml) (C). Cells were extracted and analyzed for zebularine
metabolites as described in Materials and Methods.
Fig. 6. Dose-dependent formation of phosphorylated zebularine metabolites
in T24 cells. Cells were incubated with [2-'4C]Zeb for 6 h and then extracted
and
analyzed as described in Materials and Methods. Data are mean ~ SD (n = 3).
Fig. 7. Concentration versus time profile of zebularine metabolites in T24
cells. Cells were incubated with 10 pM [2-'4C]Zeb (1 pCi/ml) for the indicated
times
before being collected, extracted and analyzed as described in Material and
Methods. The vertical dotted line indicates removal of drug-containing medium
and
replacement with drug-free media. Data points are the average of duplicate
measurements. A) Zebularine-5'-phosphates. B) Zebularine-5'-diphosphosocholine
and ethanolamine conjugates. Symbols are the same as in Fig. 6.
Fig. 8. Incorporation of zebularine into the DNA and RNA of T24 cells after
treatment with 10 pM [2-'4C]Zeb ( 1 pCi/ml). A) DNA and RNA were isolated by
the
Tri-reagent~ procedure and incorporated radioactivity was determined as
described
in Materials and Methods. Values are the mean ~ SD (n = 4). B) Reverse-phase
radiochromatograms of DNA ( ! ) and RNA (") from zebularine-treated cells
digested
to constituent nucleosides. DNA and RNA isolated from cells treated with drug
for 24
h were digested and analyzed as described in Materials and Methods. Arrows
indicate the retention times of authentic Zeb and 2'-deoxyzebularine.
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mice were inoculated S.C. Wtn tJ6 tumor ceus, wnicn were auuweu w y uw m ~
weeks. Mice were then treated i.p. with 500 mg/kg [2-'4C]Zeb (500 pCi/kg).
Twenty-
four hours after treatment, mice were sacrificed and tumors (A) and striated
muscle
(B) were removed and extracted for analysis.
Fig. 10. Effect of zebularine on human bladder carcinoma cell proliferation
after treatment for 48 h. Points represent the mean b' SD (n = 6). T-24 cell
number
averaged 4 X 105 for control at 48 h. The dashed arrow indicates the ICSO for
these
conditions.
Fig. 11 Metabolic activation pathway of zebularine.
A number of embodiments of the invention have been described.
Nevertheless, it will be understood that various modifications may be made
without
departing from the spirit and scope of the invention. Accordingly, other
embodiments
are within the scope of the following claims.
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