Note: Descriptions are shown in the official language in which they were submitted.
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WO 99/45935 PCT/US99/05360
NOVEL NUCLEOSIDE ANALOGS AND USES 1N TREATING DISEASE
This Application is a continuation-in-part from U.S. Pat. Appl. Ser. No.
081531,875, filed on September 21, 1995.
BACKGROUND OF THE INVENTION
Field of Invention
This invention relates to novel nucleosides and dinucleoside dimers and
derivatives of these compounds, including, L-deoxyribofuranosyl nucleoside
phosphodiester dimers in which the sugar moiety of at feast one of the
nucleosides
has an L-configuration. These compounds are highly effective in the treatment
of
various diseases. They may be used to treat parasitic infections such as the
one
caused by Plasmodium falciparum, the etiologic agent responsible for the most
fatal
form of malaria. They may also be used to treat bacterial, viral, and fungal
infections, and may also be used to treat cancer.
Prior Art
Modified nucleoside analogs are an important class of antineoplastic and
antiviral drugs. The present application discloses novel compounds for of this
type
for use in the treatment of P. falcfparum infection and other parasitic
infections.
Plasmodium falciparum is the etiologic agent responsible for the most fatal
form of
malaria, a disease which afflicts between 200 and 300 million people per year
(all
forms), including over one million childhood deaths. Additionally, greater
than 40%
of the world's population lives in areas in which malaria is at epidemic
levels. Due
to the extraordinary morbidity and mortality associated with malaria and other
parasitic infections, related research has intensified during the past decade
in a
desperate search for effective treatments. Safe and effective vaccines still
do not
exist. Instead, many victims must depend upon chemotherapy.
These modified nucleoside analogs may also be used to treat various other
parasitic infections, bacterial infections, fungal infections, viral
infections, and
cancer.
These chemotherapeutic agents can be classified into two groups: those that
act post-translationaliy, and those that act by interfering with nucleic acid
synthesis.
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Most drugs are in the first group, which means that they exert their
therapeutic effect by interfering with a cell's protein synthesis, and hence
its
metabolism (rather than its nucleic acid synthesis). Examples of drugs in this
group
include: the antifolate compounds (which inhibit dihyrdofolate reductase), and
sulfonamide drugs (which inhibit dihydropteroate synthetase. Yet these drugs
have
serious drawbacks. For example, the protozoan responsible for malaria very
quickly
develops resistance to these drugs. The reason is that, since resistance
occurs
through adaptive mutations in successive generations of the parasite, a one or
two
point mutation is often sufficient to confer resistance. Bacterial, viral, and
fungal
infections are frequently also susceptible to these types of resistance
mutations.
The second group of compounds includes the nucleic acid intercalators such
as acridines, phenanthrenes and quinolines. These intercalators partially
mimic the
biochemical activity of nucleic acids, and therefore are incorporated into the
protozoan's, or a cell's, nucleic acid (DNA and RNA), though once
incorporated, do
not allow further nucleic acid synthesis, hence their effectiveness. At the
same time,
these intercalators interfere with host nucleic acid synthesis as well, and
thus give
rise to toxic side effects. Because of the potential for toxic side effects,
these drugs
can quite often be given only in very small doses. Once again, a resistance
pattern
may develop. For example, a number of protozoans are known to develop
"cross-resistance," which means that the parasites develop resistance to other
classes of drugs even though they were exposed to a different class of drug.
Indeed, all of the currently known drugs or drug candidates utilizing the
delivery of cytotoxic pyrimidine or purine biosynthesis inhibitors to invading
cells are
extremely toxic. Therefore, while drugs of this type-i.e., those that
interfere with
nucleic acid synthesis-are effective, they lack selectivity. It is this latter
parameter
that must be maximized in the development of a safe and effective drug. In
other
words, such a drug would target host tissues that are infected, or cancerous,
yet
leave the host tissue unchanged.
Recent advances in our understanding of the biochemistry of parasite cells
serves as a valuable example regarding the design of effective therapies. One
investigator (H. Ginsburg, Biochem. Pharmacol. 48, 1847-185fi (1994)) observed
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that normal and parasite-infected erythrocytes exhibit significant differences
with
respect to purine and pyrimidine metabolism in single enzymes, as well as in
whole
branches of related pathways. The parasite satisfies all of its purine
requirements
through scavenger pathways; meanwhile, the host cell lacks the enzymes
necessary
to exploit this pathway, and so therefore must meet its pyrimidine
requirements
largely through de novo synthesis. Put another way, the parasite is more
efficient
than normal or host cells since it can synthesize the nucleic acid building
blocks.
Other investigators (G. Beaton, D. Pellinqer, W.S. Marshall & M. H.
Caruthers, In: Oligonucleofides and Analogues: A Practical Approach, F.
Eckstein
Ed., IRL Press, Oxford, 909-136 (1991)) have established that a malaria-
infected
erythrocyte is capable of effectively transporting the non-naturally occurring
"L-nucleosides" (in contrast to the "D-nucleosides" which are the naturally
occurring
form) for use in nucleic acid synthesis. Yet, normal mammalian cells are
nonpermeable to this class of compounds, which suggests that the L-nucleosides
are non-toxic to normal mammalian or host cells. Thus, derivatives of these
compounds may be used as highly selective drugs against parasite infection, or
against any other type of cell or organism utilizing the L-nucleosides. The
chemical
modification of the L-nucleosides consists generally of modifying the
nucleosides so
that they are still recognized by the invading cell or organism's nucleic acid
synthetic machinery, and therefore incorporated into a nucleic acid chain, but
yet
once this incorporation occurs, no further synthesis will take place.
Currently, there are no therapeutic compounds in use that are based on
_._ _ dimers of these nucleoside analogs. While dimers of the naturally
occurring
D-deoxyribofuranosyl nucleosides are well known, dimers in which one or both
nucleosides are of the unnatural L-configuration are much less known, and
their use
in therapy of neoplastic and viral diseases is unknown.
In the synthesis of DNA-related oligomers, types of nucleoside dimers are
synthesized as part of the overall process. These dimers usually include bases
from naturally occurring DNA or RNA sequences. There is much known in the art
about nucleoside monophosphate dimers. Many of these compounds have been
synthesized and are available commercially. However, these dimers are made
from
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nucleosides containing a sugar moiety in D-configuration.
Reese, C.B., Tetrahedron 34 (1978) 3143 describes the synthesis of
fully-protected dinucleoside monophosphates by means of the phosphotriester
approach.
Littauer, U.Z., and Soreg, H. (1982) in The Enzymes, Vol. XV, Academic
Press, NY, p. 517 is a standard reference which describes the enzymatic
synthesis
of dinucleotides.
Heikkilb, J., Stridh, S., C~berg, B. and Chattopodhyaya, J., Acta Chem. Stand.
B 39 (1985) 657-669, provides an example of the methodology used in the
synthesis
of a variety of ApG nucleoside phosphate dimers. Included are references and
methods for synthesis of 3'~5' phosphates and 2'~5' phosphates by solution
phase
chemistry.
Gait, M., "Oligonucleotide Synthesis", IRL Press, Ltd., Oxford, England, 1984,
is a general reference and a useful overview for oligonucleotide synthesis.
The
methods are applicable to synthesis of dimers, both by solution phase and
solid
phase methods. Both phosphitetriester and phosphotriester methods of coupling
nucleosides are described. The solid phase method is useful for synthesizing
dimers.
Gulyawa, V. and Holy, A., Coll. Czec. Chem. Commun. 44 613 (1979),
describe the enzymatic synthesis of a series of dimers by reaction of 2',-3'
cyclic
phosphate donors with ribonucleoside acceptors. The reaction was catalyzed by
non-specific RNases. The donors are phosphorylated in the 5'-position,
yielding the
_--..--.. .-following compounds: donor nucleoside-(3'~5') acceptor nucleoside:
Dimers were
made with acceptors, ~i-L-cytidine, ~i-L-adenosine, and 9(a-L-lyxofuranosyl)
adenine. Also, a large number of dimers with D-nucleosides in the acceptor 5'-
position were made.
Holy, A., Sorm, F., Collect. Czech. Chem. Commun., 34, 3383 (1969),
describe an enzymatic synthesis of ~i-D-guanylyl-(3'~5')-~3-L-adenosine and
~i-D-guanylyl-(3'-.5')-~i-L-cytidine.
Schirmeister, H. and Pffeiderer, W., Helv. Chim. Acta 77, 10 (1994), describe
trimer synthesis and intermediate dimers, all from ~i-D-nucleosides. They used
the
4
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phosphoramidite method which gave good yields.
Thus, dimers with L-deoxyribofuranosyl moieties in any position are new, as
are dimers with L-ribofuranosyi moieties bonded to the 3'-position of the
phosphate
intemucleotide bond.
Modified nucleoside analogues represent an important class of compounds in
the available arsenal of antineoplastic and antiviral drugs. The anticancer
agents
5-fluorodeoxyuridine (floxuridine), cytarabine and deoxycoformycin and the
antiviral
drugs 3'azidodeoxythymidine (AZT), dideoxycytidine (ddC), dideoxyinosine
(ddl),
acyclovir, 5-iododeoxyuridine (idoxuridine) fludarabine phosphate and
vidarabine
(adenine arabinosidelara A) are representative of this class of monomeric
nucleoside-derived compounds which are used therapeutically.
More recently, "antisense" oligonucleotide analogues with modified bases
andlor phosphodiester backbones have been actively pursued as antiviral and
antitumor agents. While no clinically approved drug has yet emerged from this
class of compounds, it remains a very active field of research. Recently,
antipodal
L-sugar-based nucleosides also have found application as potent antiviral
agents
because they can inhibit viral enzymes without affecting mammalian enzymes,
resulting in agents that have selective antiviral activity without concomitant
mammalian cytotoxicity.
Most naturally occurring nucleosides have the D-configuration in the sugar
moiety. While the chemical properties of L-nucleosides are similar to those of
their
~i-D-enantiomers, they exhibit very different biological profiles in mammalian
cells
. . .. . . ._ ... .. .. ._ a~d..do .not interferewith the transport of normal
D-nucleosides. For.example, .. . __.
(3-L-uridine is not phosphorylated at the 5'-position by human prostate
phosphotransferase, which readily phosphorylates the enantiomeric ~i-D-
uridine.
Apparently, L-nucleosides are not substrates for normal human cell kinases,
but
they may be phosphorylated by viral and cancer cell enzymes, allowing their
use for
the design of selective antiviral and anticancer drugs.
Oligonucleotides based on L-nucleosides have been studied previously.
Octamers derived from a- and (3-L-thymidine were found resistant to fungal
nucleases and calf spleen phosphodiesterase, which readily degrades the
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corresponding (3-D-oligonucleotide. Fujimory, et al., S. Fujimory, K Shudo, Y.
Hashimoto, J. Am. Chem. Soc., 112, 7436, have shown that enantiomeric
poly-a-DNA recognizes complementary RNA but not complementary DNA. This
principle has been used in the design of nuclease-resistant antisense
oligonucleotides for potential therapeutic applications.
Thus, L-nucleoside-based compounds have potential as drugs against
neoplastic, fungal, and viral diseases, as well as against parasitic
infections. While
L-sugar-derived nucleosides and their oligonucleotides have been widely
evaluated
for such activities, little is known regarding the biological activities of
shorter
oligomers such as dimers obtained by L-nucleoside substitution.
This invention comprises novel L-nucleoside-derived antitumor, antiviral,
antibacterial, antifungal, and antiparasitic agents. Novel L-nucleoside-
derived
dinucleoside monophosphates, based on L-a-5-fluoro-2'-deoxyuridine showed a
remarkably high potency activity profile in in vitro assays, with indications
of unique
mechanisms of action, including inhibition of telomerase. Therefore, the
L-nucleosides can serve as building blocks for new drugs with the special
advantage of low toxicity.
SUMMARY OF THE INVENTION
A further embodiment of the present invention is the administration of a
therapeutically effective amount of the compounds of the present invention for
the
treatment of cancer, viral infections, parasitic infections, fungal
infections, and
bacterial infect~onsr - _. . . _ _ . _ _ __ __ _.~...___ _ . _.. .._ _ _ .
_.._ _.... _. . __ ._ .. _. _..
Other and further objects, features and advantages will be apparent from the
following description of the present preferred embodiments of the invention
given for
the purposes of disclosure when taken in conjunction with the accompanying
drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a schematic representation of examples of the dinucleotide dimers
of the present invention.
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WO 99/45935 PCT/US99105360
Figures 2-14 are schematic representations of examples of the synthesis
schemes followed in the present invention.
Figures 15A and 15B are schematic representations of examples of
dinucleoside phosphate dimers containing alternate backbones.
Figures 16A-16D are schematic representations of dinucleoside phosphate
dimers used in the examples.
Certain features of the invention may be exaggerated in scale or shown in
schematic form in accordance with the customary practices in the biochemical
arts.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
It is readily apparent to one skilled in the art that various substitutions
and
modifications may be made to the invention disclosed in this Application
without
departing from the scope and spirit of the invention.
The term "dimers" as used herein is defined by the structures shown in
Figure 1. These compounds are L-nucleoside-derived dinucleoside
monophosphates. The B, and B2 units will consist of either a (3-D, a (3-L or
an a-L
nucleoside and at least one of B, or B2 will be ~3-L or a-L. R, and R2 will be
the
pyrimidine bases cytosine, thymine, uracil, or 5 fluorouridine (5-FUdR) other
5-halo
compounds, or the purine bases, adenosine, guanosine or inosine. As can be
seen
in Figure 1, the dimers can be bound by various linkages. Permissible linkages
include 5'-.3', 3'-.5', 3'-.3', 5'-.5', 2'-.3', 3'-.2', 2'~2', 2'-.5', 5'~2',
or any other
stereochemicaiiy permissible linkage. The sugar part of the nucleoside may be
fully
.., . . _ . . .. _. oXy9enated;. or may be +n the deoxy or dideoxy form. _ . .
. ,. . . .~ .._ _ .
Specific antidisease compounds which are useful in the present invention
include 3'-0-(a-L-5 fluoro-2'-deoxyuridinyl)-~i-D-5-fluoro-2'-deoxyuridine,(L-
102),
3'-0-((3-D-5-fluoro-2'-deoxyuridinyl)-a-L-5-fluoro-2'-deoxyuridine, (L-103),
3'-O-((3-D-5-fluoro-2'-deoxyuridinyl)-a-L-2'-deoxyuridine, (L-107),
3'-O-(a-L-5-fluoro-2'-deoxyuridinyl)-a-L-5-fluoro-2'-deoxyuridine, (L-108),
3'-O-(~i-L-5-fluoro-2'-deoxyuridinyl)-~i-L-5 fluoro-2'-deoxyuridine, (L-109),
3'-O-(~i-D-5-fluoro-2'-deoxyuridinyl)-~i-L-5-fluoro-2'-deoxyuridine, (L-110),
3'-0-(~i-D-5 fluoro-2'-deoxyuridinyl)-a-L-2'-deoxycytidine, (L-111 ),
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3'-O-((3-D-5 fluoro-2'-deoxyuridinyl)-2'-deoxy-~i-L-cytidine (L-113),
3'-O-(2'-deoxy-[3-L-cytidinyl)-~-D-5 fluoro-2'-deoxyuridine (L-114),
3'-O-(2'-deoxy-a-L-cytidinyl)-~3-D-5 fluoro-2'-deoxyuridine (L-115),
3'-O-(~i-D-5-fluoro-2'-deoxyuridinyl)-~3-L-2'-deoxyuridine (L-117),
3'-O-((3-L-5-fluoro-2'-deoxyuridinyl)-a-L-5 fluoro-2'-deoxyuridine (L-119),
3'-O-((3-D-5-fluoro-2'-deoxyuridinyl)-a-L-5-fluoro-2'-deoxyuridine (3', 3') (L-
122),
3'-0-(3'-deoxy-(3-D-adenosinyl)-~-L-2'-deoxyuridine (L-150),
3'-O-(3'-deoxy-~i-D-adenosinyl)-(3-L-2'-deoxyadenosine {L-151 ),
3'-O-(3'-deoxy-~3-D-adenosinyl)-a-L-2'-deoxyuridine (L-152),
3'-O-(3'-deoxy-~3-D-adenosinyl)-(3-L-2'-deoxycytidine (L-153),
3'-O-(3'-deoxy-(3-D-adenosinyl)-cc-L-2'-deoxycytidine (L-154),
3'-O-(3'-deoxy-(3-D-adenosinyl)-~3-L-2'-deoxyadenosine (L-155),
3'-O-(2'-deoxy-~i-D-adenosinyl)-a-L-2'-deoxyadenosine (L-210), or a
therapeutically
acceptable salts of these foregoing compounds. In the currently preferred
embodiment, 3'-O-(~3-D-5-fluoro-2'-deoxyuridinyl)-a-L-5-fluoro-2'-
deoxyuridine,
(L-103) is used.
The term "intemucleotide binding agent" or "IBA" means the backbone
binding which links the nucleosides together. Although one skilled in the art
will
readily recognize a variety of other backbones are available and useful in the
present invention. For example, see Figure 6, where methoxy phosphotriesters,
methylphosphonates, phosphorodithioates, phosphorothioates, silyl ethers,
sulphonates and ethylenedioxy ethers are shown. Although shown schematically
_.._ ..._._ _ ~. . _3~.T-5' ~e.tBp,~s.can-be.~sed to link the
sugars~5_'.~3',_3'-..5~, 3'~3',. 5'-.5', 2'-.3', . _ __ . _ . .. .. ._
3'~2', 2'--2', 2'~5', 5'~2', or any other stereochemicalfy permissible
linkages. The
sugars may be fully oxygenated, or may be in the deoxy or dideoxy form as
permitted. In the preferred embodiment, the IBA of the compounds is either
phosphodiester or phosphorothioate. The term "antidisease" as used herein
refers
to any of the activities of the compounds of the present invention to affect a
disease
state, including antitumor, antineoplastic, anticancer, antiparasitic and
antiviral
activity.
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A compound or composition is said to be "pharmacologically acceptable" if its
administration can be tolerated by a recipient mammal. Such an agent is said
to be
administered in a "therapeutically effective amount" if the amount
administered is
physiofogicaliy significant. An agent is physiologically significant if its
presence
results in technical change in the physiology of a recipient mammal. For
example,
in the treatment of cancer or neoplastic disease, a compound which inhibits
the
tumor growth or decreases the size of the tumor would be therapeutically
effective;
whereas in the treatment of a viral disease, an agent which slows the
progression of
the disease or completely treats the disease, would be considered
therapeutically
effective.
Dosage and Formulation
The antidisease compounds (active ingredients) of this invention can be
formulated and administered to inhibit a variety of disease states (including
tumors,
neoplasty, cancer, bacterial, fungal, parasitic and viral diseases) by any
means that
produces contact of the active ingredient with the agent's site of action in
the body
of a mammal. They can be administered by any conventional means available for
use in conjunction with pharmaceuticals, either as individual therapeutic
active
ingredients or in a combination of therapeutic active ingredients. They can be
administered alone, but are generally administered with a pharmaceutical
carrier
selected on the basis of the chosen route of administration and standard
pharmaceutical practice.
. . . .. _ __.. .. .T.~_.dosages given as examples. herein:~8 the dosag~s
uswaaly used in , ... ... ... _. .T
treating tumors, neopfasty and cancer. Lower doses may also be used. Dosages
for
antiparasitic and antiviral applications will, in general; be 10-50% of the
dosages for
anticancer applications.
The dosage administered will be a therapeutically effective amount of active
ingredient and will, of course, vary depending upon known factors such as the
pharmacodynamic characteristics of the particular active ingredient and its
mode
and route of administration; age, sex, health and weight of the recipient;
nature and
extent of symptoms; kind of concurrent treatment, frequency of treatment and
the
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effect desired. Usually a daily dosage (therapeutic effective amount) of
active
ingredient can be about 5 to 400 milligrams per kilogram of body weight.
Ordinarily,
to 200, and preferably 10 to 50, milligrams per kilogram per day given in
divided
doses 2 to 4 times a day or in sustained release form is effective to obtain
desired
results.
Dosage forms (compositions) suitable for internal administration contain from
about 1.0 to about 500 milligrams of active ingredient per unit. In these
pharmaceutical compositions, the active ingredient will ordinarily be present
in an
amount of about 0.05-95% by weight based on the total weight of the
composition.
The active ingredient can be administered orally in solid dosage forms such
as capsules, tablets and powders, or in liquid dosage forms such as elixirs,
syrups,
emulsions and suspensions. The active ingredient can also be formulated for
administration parenterally by injection, rapid infusion, nasopharyngeal
absorption
or dermoabsorption. The agent may be administered intramuscularly,
intravenously,
or as a suppository.
Gelatin capsules contain the active ingredient and powdered carriers such as
lactose, sucrose, mannitol, starch, cellulose derivatives, magnesium stearate,
stearic acid, and the like. Similar diluents can be used to make compressed
tablets.
Both tablets and capsules can be manufactured as sustained release products to
provide for continuous release of medication over a period of hours.
Compressed
tablets can be sugar coated or film coated to mask any unpleasant taste and
protect
the tablet from the atmosphere, or enteric coated for selective disintegration
in the
gastrointestinal tract. ..._J.._.. .. _ . . _ _ ..
Liquid dosage forms for oral administration can contain coloring and flavoring
to increase patient acceptance.
In general, water, a suitable oil, saline, aqueous dextrose (glucose), and
related sugar solutions and glycols such as propylene glycol or polyethylene
glycois
are suitable carriers for parenteral solutions. Solutions for parenteral
administration
contain preferably a water soluble salt of the active ingredient, suitable
stabilizing
agents and, if necessary, buffer substances. Antioxidizing agents such as
sodium
bisulfate, sodium sulfite or ascorbic acid, either alone or combined, are
suitable
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WO 99/45935 PCT/US99/05360
stabilizing agents. Also used are citric acid and its salts and sodium EDTA.
In
addition, parenteral solutions can contain preservatives such as benzalkonium
chloride, methyl- or propyl-paraben and chlorobutanol. Suitable pharmaceutical
carriers are described in Remington's Pharmaceutical Sciences, a standard
reference text in this field.
Additionally, standard pharmaceutical methods can be employed to control
the duration of action. These are well known in the art and include control
release
preparations and can include appropriate macromolecules, for example polymers,
polyesters, polyaminoacids, polyvinyl, pyrolidone, ethylenevinylacetate,
methyl
cellulose, carboxymethyl cellulose or protamine sulfate. The concentration of
macromolecules as well as the methods of incorporation can be adjusted in
order to
control release. Additionally, the agent can be incorporated into particles of
polymeric materials such as polyesters, polyaminoacids, hydrogels, poly
(lactic acid)
or ethylenevinylacetate copolymers. In addition to being incorporated, these
agents
can also be used to trap the compound in microcapsules.
Useful pharmaceutical dosage forms for administration of the compounds of
this invention can be illustrated as follows.
Capsules: Capsules are prepared by filling standard two-piece hard gelatin
capsulates each with 100 milligram of powdered active ingredient, 175
milligrams of
lactose, 24 milligrams of talc and 6 milligrams magnesium stearate.
Soft Gelatin Capsules: A mixture of active ingredient in soybean oil is
prepared and injected by means of a positive displacement pump into gelatin to
form
. . . . _.. _ _ _ . s~oft_.gelatin .capsules containing 100 milligrams of
..the.active. ingredient. The .... .. ,
capsules are then washed and dried.
Tablets: Tablets are prepared by conventional procedures so that the
dosage unit is 100 milligrams of active ingredient. 0.2 milligrams of
colloidal silicon
dioxide, 5 milligrams of magnesium stearate, 275 milligrams of
microcrystaliine
cellulose, 11 milligrams of cornstarch and 98.8 milligrams of lactose.
Appropriate
coatings may be applied to increase palatability or to delay absorption.
Injectable: A parenteral composition suitable for administration by injection
is
prepared by stirring 1.5°~ by weight of active ingredients in 10% by
volume
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propylene glycol and water. The solution is made isotonic with sodium chloride
and
steri I ized.
Suspension: An aqueous suspension is prepared for oral administration so
that each 5 millimeters contain 100 milligrams of finely divided active
ingredient, 200
milligrams of sodium carboxymethyl cellulose, 5 milligrams of sodium benzoate,
1.0
grams of sorbitol solution U.S.P. and 0.025 millimeters of vanillin.
Summary of Compounds Synthesized
The following examples are offered by way of illustration and are not intended
to limit the invention in any manner. The nucleosides and dimers may
incorporate
any stereochemcially permissible linkage and may include various oxygenated,
deoxy, and dideoxy forms of the sugar rings. The synthetic nucleosides and
dimers
described in the examples can include any of the substitutions discussed
earlier.
The backbone and base modifying groups can be added. Various substitutions
will
enhance the affinity, the chemical stability and the cellular uptake
properties of the
specific dimers treatments.
Example 1
S~rnthesis of 2'-deoxyr-a-L-5-flourouridine
While ~i-D-5-fluoro-deoxyuridine is commercially available, the a-L-isomer
2'-deoxy-a-L-5-fluorouridine is not, and this component of the dimers was
synthesized from L-arabinose.
_. . .. .. .. __. _ _. .1__i~2.~~ .3.~;_5~,art-O-benzo~rl-a-L-
arabinofuranosyll-5-fluorouracil-(3)r-..___._.. . _ ...._ _. __. ._ .. . . . .
. . . __
To a mixture of 5-fluorouracil (4.01 g, 30.87 mmol) and compound 2 (15.57 g,
30.87 mmol) in anhydrous MeCN were successively added HMDS (5.20 ml, 24.69
mmol), CISiMe3 (3.10 ml, 24.69 mmol), and SnCl4 (4.30 ml, 37.04 mmol). The
resulting clear solution was refluxed for one hour. Then the solvent was
evaporated
and the residue was dissolved in EtOAc (750 ml), washed with HZO, and
saturated
NaHC03 solution. The EtOAc layer was dried over sodium sulfate, filtered and
evaporated to give the crude product. This crude product was purified on a
silica
gel column using 40-50°~ EtOAclpetroleum ether to give pure 3 (11.7 g,
66.0°~
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WO 99145935 PCTIUS99I05360
yield) as a white foam.
NMR: (CDCI3) S = 4.65 (dd, 1 H), 4.78 (dd, 1 H), 4.97 (dd, 1 H, 5.75-5.88 (2
t, 2H),
6.27 (d, 1 H), 7.36-7.62 and 8.00-8.10 (m, 5H), 8.94 (d, 1 H).
1-oc-L-arabinofuranosyrl-5-fluorouracil (4)
To a solution of compound 3 (11.7 g, 20.37 mmol) in MeOH (300 ml), NaOMe
(4.2 ml of a methanolic 25°~ wlv solution) was added and the solution
was stirred
until the reaction was complete. The solvent was then evaporated and the
residue
was dissolved in H20 (200 ml), washed with ether and neutralized with Dowex 50
ion exchange resin. After filtration of the resin, the aqueous solution was
evaporated to give compound 4 (4.92 g, 92°~ yield) as a white foam.
NMR: (DMSO-dg) S = 3.48 (m, 2H), 3.93-4.00 (2 t, 2H), 4.16 (q, 1 H), 5.69 (dd,
1 H),
8.03 (d, 1 H).
1-L3'.5'-O-(1.1.3.3-tetraisoprop~rldisiloxane-1.3-diy I)-a-L-arabinofuranosyl]-
5-fluorou
racil 5
To a stirred suspension of 4 (6.43 g, 24.52 mmol) in pyridine (200 ml) was
added 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane (10.3 ml, 29.43 mmol).
This was
stirred at room temperature until the reaction was complete (5 hours). The
solvent
was evaporated to a residue which was dissolved in EtOAC and washed
successively with H20, 5% HCI, H20, saturated NaHC03, and brine. After drying
the
EtOAc portion over Na2S04, the solution was filtered and evaporated to give
the
crude product 5 which was used in the next step without further purification.
1-(2'-O phenoxythiocarbonyl-3'.5'-0-(1.1.3.3-tetraisopropyldisifoxane-1.3-
divl)-a-L-
_ arabinofuraos~l-5-fluorouracil (61 . . ._.. ... . . .. .... . . _.. . . _ _
.
To a solution of 5 (24.52 mmol) in anhydrous MeCN (300 ml) were added
4-dimethylaminopyridine (DMAP) (5.80 g, 47.58 mmol), and
phenylchlorothionoformate (3.85 ml, 26.98 mmol). The solution was stirred at
room
temperature for 24 hours. Then, the solvent was evaporated to a residue which
was
dissolved in EtOAc and washed successively with H20, 5°~ HCI, H20,
saturated
NaHC03, and brine. After drying the EtOAc portion over Na2S04, the solution
was
filtered and evaporated to an oil. The oil was purified on a silica gel column
using
30% EtOAclpetroleum ether to produce pure 6 (8.9 g, 56.7°~ yield) as a
yellow
13
CA 02322494 2000-09-07
WO 99/45935 PCT/US99/05360
foam.
NMR: (CDC13) s = 4.02 (m, 2H), 4.32 (m, 1 H), 4:76 (dd, 1 H), 6.10 (dd, 1 H),
6.18 (dd,
1 H}, 7.07-7.48 (m, 6H}, 8.41 (br s, 1 H).
3'.5'-O-l1.1.3.3-tetraisoprop)rldisiloxane-1.3-divly-a-L-2'-deoxv-5
fluorouridine ~(7)
To a solution of 6 (8.92 g, 13.91 mmol), in dry toluene (300 ml) was added
AIBN (0.46 g, 2.78 rnmol) followed by Bu3SnH (20.0 ml, 69.35 mmof). The
solution
was deoxygenated with argon and heated at 75°C for four hours. The
solvent was
then evaporated and the residue was purified on a silica gel column using
30°r6
EtOAclpetroleum ether to give pure 7 (5.44 g, 80% yield) as a white foam.
NMR: (CDCI3) S = 2.16 (m, 1 H), 2.84 (m, 1 H), 3.8 Cm, 1 H), 4.07 (m, 1 H),
4.60 (m,
1 H}, 6.19 (ddd, 1 H), 7.92 (m, 1 H}.
2'-deox~r-a-L-5-fluorouridine l8)
A solution of compound 7 (5.44 g, 11.13 mmol) and NH4F (4.12 g, 111.3
mmol) in MeOH was stirred in an oif bath at 60°C for 3 hours. Silica
gel (3 g) was
added and the mixture was evaporated to a dry powder. This powder was added to
a silica column and eluted with 10-15°~ MeOHlCHCI3 to produce pure 8
(2.4 g,
87.6°~ yield) as a white foam.
NMR: (DMSO-de) S = 1.90 (m,1 H), 2.55 (m, 1 H), 3.33 (m,2H), 4.19 (m, 2H),
4.86 (br
s, 1 H), 5.43 (br s, 1 H), 6.10 (dd, 1 H), 8.15 (d, 1 H), 11.78 (br s, 1 H).
Example 2
Syrnthesis of 2'-deoxy-a-L-uridine
1_(2w.3,: 5,:tri-O=benzoyl-a-L-arabinofuranosyl) ur~c~I~fH) - _ _. _. . . _ .
. __..
To a mixture of uracil (1.17g, 10.49 mmol) and compound 2 (5 g) in
anhydrous MeCN (100 ml) were successively added HMDS (1.77 ml, 8.39 mmol),
CISiMe3 (1.06 ml, 8.39 mmol), and SnCI, (1.47 ml, 12.58 mmol). The resulting
clear
solution was refluxed for one hour. Then the solvent was evaporated and the
residue was dissolved in EtOAc (200 ml), washed with H20, and saturated NaHC03
solution. The EtOAc layer was dried over sodium sulfate, filtered and
evaporated to
give the crude product, which was purified on a silica gel column using 40-
50°~
EtOAclpetroleum ether to give pure 9 (3.66 g, 62.7% yield) as a white foam.
14
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WO 99/45935 PCT/US99105360
NMR: (CDCI3) 8 = 4.70 (m, 1 H), 5.77 (5, 1 H), 5.80 (dd, 1 H), 5.94 (t, 1 H),
6.20 (d,
1 H), 7.40-8.10 (m, 16H), 8.58 (br s, 1 H).
1-oc-L-arabinofuranosyl-uracil (10)
To a solution of compound 8 (17.83 g, 32.03 mmol) in MeOH (400 ml),
NaOMe (5.0 ml of a methanolic 25°~ wlv solution) was added and the
solution was
stirred until the reaction was complete. The solvent was then evaporated and
the
residue was dissolved in H20 (250 ml), washed with ether and neutralized with
Dowex 50 ion exchange resin. After filtration of the resin, the aqueous
solution was
evaporated to give compound 10 (7.4 g, 94.6% yield) as a white foam. This was
used in the next step without further purification.
1-[3'.5'-0-11,1.3.3-tetraisoprolaYldisiloxane-1.3-did,)-a-L-arabinofuranosyrl)-
uracil (11)
To a stir-ed suspension of 10 (7.4 g, 30.3 mmol) in pyridine was added
1.3-dichloro-1,1,3,3-tetraisopropyldisiloxane (12.74 ml, 36.36 mmol). This was
stirred at room temperature until the reaction was complete (5 hours). The
solvent
was evaporated to a residue which was dissolved in EtOAC (500 ml) and washed
successively with HZO, 5% HCI, H20, saturated NaHC03, and brine. After drying
the
EtOAc portion over Na2S04, the solution was filtered and evaporated to give
the
crude product 11 which was used in the next step without further purification.
1-[2'-o-phenoxythiocarbonyl-3'.5'-0-( 1.1.3.3-tetraiso~ro~~rldisiloxane-1.3-
divl)-a-L~ar
abinofuranos~,rlLuracil j12)
To a solution of 11 (30.3 mmoi) in anhydrous MeCN were added
4-dimethylaminopyridine (DMAP) (7.2 g, 58.78 mmol), and
pfienylchlorothionoformatew(4:7-ml, 33.33 mmol). The solution was stirred at
room
temperature for 24 hours. Then, the solvent was evaporated to a residue which
was
dissolved in EtOAc (750 ml) and washed successively with HzO, 5°~ HCI,
H20,
saturated NaHC03, and brine. After drying the EtOAc portion over Na2S04, the
solution was filtered and evaporated to an oil. The oil was purified on a
silica gel
column using 30% EtOAclpetroleum ether to produce pure 12 (13.14 g, 74.5%
yield)
as a white foam.
NMR: (CDC13)a = 4.04 (m, 2H), 4.38 (m, 1 H), 4.73 (dd, 1 H), 5.79 (dd, 1 H),
5.93 (d,
1 H), 6.31 (dd, 1 H), 7.08-7.33 (m, 6H), 9.2 (br s, 1 H).
CA 02322494 2000-09-07
WO 99145935 PCfNS99105360
3',5'-0-(1.1.3.3 tetraisopropyldisiloxane-1.3-diyly-a-L-2'-deoxyuridine l13)
To a mixture of 12 (13.14 g, 21.09 mmol), in dry toluene (300 ml) was added
AIBN (0.69 g, 4.2 mmol) followed by Bu3SnH (28.4 ml, 105.4 mmol). The solution
was deoxygenated with argon and heated at 75°C for four hours. The
solvent was
then evaporated and the residue was purified on a silica gei column using
30°~
EtOAclpetroleum ether to give pure 13 (9.29 g, 88.4°~ yield) as a white
foam.
NMR: (CDC13) 5 = 2.15 (2 t, 1 H), 2.81 (m, 1 H), 3.82 (dd,1 H), 4.05 (m, 2H),
4.56 (q,
1 H), 5.75 (dd, 1 H), 6.16 (t, 1 H), 7.69 (d, 1 H), 9.38 (br s, 1 H).
2'-deoxy-a-L-uridine (14)
A mixture of compound 13 (9.2 g, 18.63 mmol) and NH4F (6.9 g, 186.3 mmol)
in MeOH (200 ml) was stirred in an oil bath at 60°C for 3 hours. Silica
gel (5 g.) was
added and the mixture was evaporated to a dry powder. This powder was added to
a silica column and eluted with 10-15% MeOHICHCl3 to produce pure 14 (3.70 g,
83% yield) as a white foam.
NMR: (DMSO-de) b = 1.87 (m, 1 H), 2.56 (m, 1 H), 3.41 (m, 2H), 4.15 (m, 1 H),
4.22
(m, 1 H), 4.44 (t, 1 H), 4.92 (t, 1 H), 5.38 (d, 1 H), 5.62 (d, 1 H), 6.09
(dd, 1 H).
Example 3
Synthesis of 2'-deox~r-a-L-clrtidine
3'.5'-di-O-benzoyl-2'-deoxy-a-L-uridine (15)
A solution of BzCN (0.61 g, 4.67 mmol) in MeCN (10 ml) was added dropwise
to a suspension of compound 14 (0.43 g, 1.87 mmol) in MeCN (10 ml) followed by
~~ Et3N (0:1 ml).-The reaction"was stirred at room~empe~atia~e fog t~iree
hours after ~ ~~ ~ -~~
which time the solvent was evaporated to dryness. The crude material was
purified
on a silica gel column using 50% EtOAcJpetroleum ether to give pure 15 (0.57
g,
70°~ yield} as yellow foam.
NMR: (CDC13) 5 = 2.55 (d, 1 H), 2.96 (dt, 1 H), 4.56 (m, 2H), 4.86 (t, 1 H),
5.61 (d,
1 H), 5.73 (dd, 1 H), 6.31 (dd, 1 H), 7.40-7.63 (m, 7H), 7.87-8.06 (m, 4H),
8.82 (br s,
1 H).
3'.5'-di-O-benzo~rl-2'-deox~r-4-thin-a-L-uridine (16)
A boiling solution of compound 15 (0.54 g, 1.25 mmol) in anhydrous dioxane
16
CA 02322494 2000-09-07
WO 99/45935 PCTNS99/05360
was treated with PzSs (0.61 g, 2.75 mmol) and the mixture was refluxed under a
nitrogen atmosphere for one hour. Remaining solids were filtered from the hot
solution and washed on the filter with additional dioxane. The filtrate was
evaporated to dryness and the crude product was purified on a silica gel
column
using 30°~ EtOAclpetroleum ether to give pure 16 (0.42 g, 74°~
yield) as a yellow
oil.
NMR: (CDCI3) b = 2.59 (d, 1 H), 2.93 (dt, 1 H), 4.58 (m, 2H), 4.89 (t, 1 H),
5.63 (d,
1 H), 6.26 (dd, 1 H), 6.41 (dd, 1 H), 7.40-8.10 (m, 11 H), 9.54 (br s, 1 H).
2'-deoxy-a-L-cytidine i(17~
Compound 16 (0.42 g, 9.28 mural) was treated with NH~IMeOH (50 ml) in a
steel bomb at '100°C for 10 hours. After cooling, the solvent was
evaporated to
dryness, the residue was dissolved in water (50 ml) and washed with ether (3 x
50
m1). The water layer was treated with charcoal, filtered through Celite and
evaporated to dryness by coevaporation with EtOH. The semi-solid obtained was
crystallized from EtOHlether to give compound 17 (0.18 g, 85.7°~
yield).
NMR: (DMSO-de) b = 1.86 (Cd,H), 2.50 (m, 1 H), 3.40 (m, 1 H), 4.12 (m, 1 H),
4.15
(m, 1 H), 4.86 (t, 1 H), 5.21 (d, 1 H), 5.69 (d, 1 H), 6.03 (dd, 1 H), 7.02
(br d, 1 H), 7.74
(d, 1 H).
N4-benzo~rl-2'-deox~r-a-L-c idine (18y
CISiMe3 (2.3 ml, 18.05 mmol) was added dropwise over 30 minutes to a
stirring suspension of compound 17 (0.82 g, 3.61 mmol) in pyridine (50 ml)
chilled in
an ice bath. BzCI (2.1 ml, 18.05 mmol) was then added dropwise and the
reaction
- --~ - - - - mixture was eoofed at roomtemperature for two~ours. The reaction
mixture ~nras --
again cooled in an ice bath and cold water (10 ml) was added dropwise. Fifteen
minutes later, concentrated NH40H (10 ml) was added to produce a solution of
ammonia of a concentration of about 2M. Thirty minutes after the addition of
the
ammonia solution, a solvent was evaporated, dissolved in water and washed with
ether. Evaporation of this aqueous solution provided the crude product (18)
which
was used in the next step without further purification.
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F~cample 4
Synthesis of Dimers
The dimers were prepared from the monomeric materials by the general
scheme shown in Scheme 2.
A. a-L. [3-D 5 FUdR Dimer
5'-O-dimethoxytrityl-a-L-5-fluoro-2'-deox~uridine (20a)
a-L-5-fluoro-2'-deoxyuridine (8) (500 mg, 2.0 mmol) was dissolved in 10 ml of
dry, distilled pyridine. To this solution was added 4,4'-dimethoxytrityl
chloride (813
mg, 2.4 mmol) and 4-dimethylaminopyridine (DMAP) (50 mg, 0.4 mmof). The
mixture was stirred under an argon atmosphere for 16 hours. After this time,
the
pyridine was stripped off in vacuo. The residue was dissolved in EtOAc (50
ml).
The organic layer was washed with saturated NaHC03, water and with brine. The
organic layer was dried over NaaS04, filtered and evaporated in vacuo to a
residue
which was purified on a silica gel column using 10% MeOHICHCl3. Pure fractions
were pooled and evaporated to give the pure product as an off-white foam (679
mg,
86°~ yield). Rf = 0.48 in 10% MeOHICHCl3.
NMR: (DMSO-de) b = 2.3 (dd, 1 H), 2.72-2.81 (m, 1 H), 3.15-3.26 (m, 2H), 3.75
(s,
6H), 4.45 (m, 2H), 6.23 (dd, 1 H), 6.92 (d, 1 H), 7.2-7.3 (m, 13 H), 7.94 (d,
1 H).
5'-O-dimetho rityri-a-L-5 fluoro-2'-deoxyuridine-3'-N.N-diisopro~pylmethoxy
phos~horamidite (21a)
The 5'-O-dimethoxytrityl-a-L-5-fluoro-2'-deoxyuridine (20a, 548 mg, 1 mmol)
~-~- - w ~~ - w - vas ~dissotved-irr ~anhydrous-dichloromethane (20wm1). ~ N,N-
diisopropylethylamine
(700 NI, 4 mmol) was added through a septum, followed by
chloro-N, N-diisopropylmethoxyphosphine (290 pl, 1.5 mmoi), under an argon
atmosphere. The reaction was stirred for 30 minutes. The solvent was
evaporated
and the residue was partitioned between an 80% EtOAcltriethylamine mixture and
brine. The organic layer was washed with saturated NaHC03 solution and brine.
The organic residue was evaporated to dryness and the residue was purified on
a
silica gel column using a mixture of dichloromethane, EtOAc and triethylamine
(45:45:10;Rf = 0.69). The product (390 mg) was isolated as a yellow foam and
it
18
CA 02322494 2000-09-07
WO 99/45935 PGT/US99/05360
was used in the next step without further purification.
3' AcetoxY (3-D-5-fluoro-2'-deox~,ruridine (24a)
(3-D-5 fluoro-2'-deoxyuridine (500 mg, 2.2 mmol) was dissolved in 10 ml of
dry, distilled pyridine. To this solution was added 4,4'-dimethoxytrityl
chloride (813
mg, 2.4 mmole) and 4-dimethylaminopyridine (DMAP) (50 mg, 0.4 mmole). The
mixture was stirred at room temperature for 16 hours. The pyridine was
stripped off
in vacuo. The residue was dissolved in dichloromethane (50 ml). The organic
layer
was washed with 0.3 N HCI, brine, saturated NaHC03, and again with brine. The
organic layer was dried over Na2S04, filtered and evaporated in vacuo to a
residue
which was purified on a silica gel column, eluting with 10°~
MeOH/CHCl3. Pure
fractions were pooled and evaporated to give the pure product as an off-white
foam
(685 mg, 86°r6 yield). This material was dissolved in pyridine (12 ml)
and treated
with acetic anhydride (2.5 ml) for 3 hours at room temperature. The solvent
was
evaporated, and the residue was dissolved in ethyl acetate. The ethyl acetate
was
washed as described above, dried over sodium sulfate and evaporated. The
residue was then treated with 80°~ acetic acid (10 ml) for 2.5 hours at
room
temperature. The solvent was evaporated in vacuo and the residue was
chromatographed on silica gel, eluting with 10°~ MeOHICHCl3 to give
pure 24a as a
white foam, yield 422 mg.
NMR: (DMSO-dg) a = 1.95 (s, 3H), 2.08-2.24 (m, 2H), 3.65-3.9 (m, 2H), 4.45 (m,
1 H), 4.72 (m, 1 H), 6.24 (dd, 1 H), 8.24 (d, 1 H).
5'-O-dimethoxvtrit5rl-3'-I~O-~3'-O-acetyl)-~3-D-5 fluoro-2'-deoxyuridinyl]-a-L-
5-fluoro-2
_r~ ....~"d~~y~idine 25a) _. . . _ .... __.._-_..,r _.._._ .... __. ..._ ..
._... _ .. ._.._ _ ._ .___.._..~ _~
The 3'-O-acetyl-(3-D-5-fluoro-2'-deoxyuridine (188 mg, 0.65 mmol) was
dissolved in dry acetonitrile (5 ml). Sublimed 1 H-tetrazole (80 mg) was added
and
the mixture was stirred under an argon atmosphere for 15 minutes. The solution
of
21a (380 mg, 0.54 mmol), dissolved in 5 ml of dry acetonitrile was added via
syringe
to the reaction solution over 5 minutes. The mixture was allowed to stir at
room
temperature for three hours. The acetonitrile was evaporated in vacuo to a
residue.
This residue was triturated with a 70°~ EtOAclether mixture. The
undissolved
tetrazole was filtered off and the filtrate was evaporated to give a dry
yellow foam
19
CA 02322494 2000-09-07
WO 99/45935 PCT/US99/05360
(468 mg). This foam was used in the next step without further purification.
(3'-acetoxy-J3-D-5 fluoro-2'-deoxyuridinyl)-a-L-5 fluoro-2'-deoxyuridine
methyl
phosphonate ester (,26a)
The dimer, 25a (504 mg), was dissolved in 8 ml of THF and 2 ml of pyridine
containing 0.2 ml of water. Iodine crystals (26 mg) were added and the
contents of
the loosely stoppered flask were allowed to stir for 2.1 hours. Excess iodine
was
discharged by the addition of a few drops of saturated sodium thiosulfate. The
reaction mixture was then evaporated to dryness. The crude product was
dissolved
in EtOAc washed with saturated NaHC03 solution and brine. The organic layer
was
dried over Na2S04, filtered and evaporated in vacuo. The residue (530 mg) was
dissolved in 10 ml of 80% acetic acidlwater solution and was stirs-ed until
the
reaction was completed. The solvent was evaporated and the residue was
purified
on a silica gel column, using 20°~ MeOH/CHCl3. Fractions containing one
spot by
TLC (10% MeOHICHCl3 Rf=0.35} were pooled and evaporated to give the pure
product (316 mg}.
NMR: (CD30D) i5 = 2.08 (s, 3H), 2.25-2.45 (m, 3H), 2.65-2.72 (m, 1 H), 3.60
(m, 2H),
3.80 (2d, 3H), 4.18 (m, 1 H), 4.28 (m, 1 H}, 4.35 (dd, 1 H), 4.62 (dd, 1 H),
5.05 (dd,
1 H), 5.23 {m, 1 H), 6.13 (m, 1 H), 6.18 (m, 1 H), 7.85 (m, 2H).
P3' NMR: (CD30D) 5 = 0.77 (s), 1.16 (s).
3'-O-((3-D-5-fluoro-2'-deoxyuridinyiJi-a-L-5 fluoro-2'-deoxyuridine {27a)
The O-protected dimer, 26a (280 mg) was treated with 20 ml of saturated
methanolic ammonia at room temperature until the reaction was completed at
room
-- -~- - -- - .-#emperatur~: The-sowsnt-was-stripped-off in-vacuoand the-
residue was purified on - -----
DEAE cellulose ion exchange column using gradient of NH4C03 buffer from 0.02-
0.2
M. Pure fractions were evaporated at 40°C in high vacuo to dryness to
give the
pure product (162 mg).
NMR: (D20) a = 2.2-2.4 (m, 3H), 2.65-2.71 (m, 1 H), 3.65 (m, 2H), 4.01 (m, 1
H), 4.11
(t, 1 H), 4.45 (m, 1 H), 4.65 (t, 1 H), 6.14 (d, 1 H), 6.24 (td, 1 H), 8.06
(d, 1 H), 8.02 (d,
1 H).
3'P NMI~: (D20) 6 = 0.04 (s).
CA 02322494 2000-09-07
WO 99/45935 PCT/US99/05360
B. ~i-D. a-L SFUdR Dimer
5'-O-dimethoxlrtrit)rl-3'-[~3'-O-acetvl)~-L-5-fluoro-2'-deoxvuridinyl]-~3-D-5-
fluoro-2
'-deoxyuridine i(25b)
The 3'-0-acetyl-a-L-5-fluoro-2'-deoxyuridine, 24b (188 mg, 0.65 mmol) was
dissolved in dry acetonitrile (5 ml). Sublimed 1 H-tetrazole (80 mg) was added
and
the mixture was stirred under an argon atmosphere for 15 minutes. The solution
of
29b (380 mg, 0.54 mmol), dissolved in 5 ml of dry acetonitrile was added via
syringe
to the reaction solution over 5 minutes. The mixture was allowed to stir at
room
temperature for three hours. The acetonitrile was evaporated in vacuo to a
residue.
This residue was triturated with a 70% EtOAGether mixture. The undissolved
tetrazole was filtered off and the filtrate was evaporated to give a dry
yellow foam
(484 mg). This foam was used in the next step without further purification.
(3'-acetox~a-L-5-fluoro-2'-deoxyuridinyl~-~i-D-5-fluoro-2'-deoxyuridine meth
phosphate ester (26b)
The dimer, 25b (526 mg), was dissolved in 8 ml of THF and 2 ml of pyridine
containing 0.2 ml of water. Iodine crystals (26 mg) were added and the
contents of
the loosely stoppered flask were allowed to stir for 1 hour. Excess iodine was
discharged by the addition of a few drops of saturated sodium thiosulfate. The
reaction mixture was then evaporated to dryness. The crude product was
dissolved
in EtOAc washed with saturated NaHC03 solution and brine. The organic layer
was
dried over Na2S04, filtered and evaporated in vacuo. The residue (578 mg) was
dissolved in 10 ml of 80°~ acetic acidlwater solution and was stirred
for three hours.
__ . __ ._. _ .. . ... __.. Tie ~ivent was-evaporated and the residue
waswpuri#ied-on a silica gel column; ._ . ... ._
using 10-15% MeOHlCHCI3. Fractions containing one spot by TLC (10°~
MeOHICHCl3 Rf - 0.35) were pooled and evaporated to give the pure product (342
mg).
NMR: (DMSO-de) i5 = 1.98 (s, 3H), 2.2-2.4 (m, 3H), 2.62-2.71 (m, 1 H), 3.5-3.8
(m,
4H), 4.02 (m, 1 H), 4.42 (m, 2H), 6.10 (dd, 1 H), 6.26 (dt, 1 H), 8.00 (d, 1
H), 8.04 (d,
1 H).
3'-O- a-L-5 fluoro-2'-deo uridinyl)-[3-D-5 fluoro-2'-deoxyuridine y27b)
The O-protected dimer, 26b (170 mg) was treated with 20 ml of saturated
21
CA 02322494 2000-09-07
WO 99145935 PCT/US99/05360
methanolic ammonia at room temperature until the reaction was completed. The
solvent was stripped off in vacuo and the residue was purified on DEAF
cellulose
ion exchange column using gradient of NH4C03 buffer from 0.02-0.2 M. Pure
fractions were evaporated at 40°C in high vacuo to dryness to give the
pure product
(89 mg).
NMR: (D20) a = 2.2-2.4 (m, 3H), 2.65-2.71 (m, 1 H), 3.54-3.85 (m, 5H), 4.05
(t, 1H),
4.42 (m, 2H), 6.06 (dd, 1 H), 6.23 (dt, 1 H), 8.00 (d, 1 H), 8.04 (d, 1 H).
C. a-L uridine. ~i-D 5 F~IdR dlmer
5'-O-(di-p-methoxytritvl)-2'-deoxy-a-L-uridine (20c~~
a-L-2'-deoxyuridine (1.5 g, 6.57 mmol) was dissolved in 25 ml of dry,
distilled
pyridine. To this solution was added 4,4'-dimethoxytrityl chloride (2.9 g,
7.89 mmol)
and 4-dimethylamino pyridine (DMAP) (160 mg, 1.31 mmol). The mixture was .
stirred under an argon atmosphere for 16 hours. After this time, the pyridine
was
stripped off in vacuo. The residue was dissolved in EtOAc (150 ml). The
organic
layer was washed with saturated NaHC03, water and again with brine. The
organic
layer was dried over NaZSOa, ~Itered and evaporated in vacuo to a residue
which
was purified on a silica gel column using 5°~ MeOHJCHCl3. Pure
fractions were
pooled and evaporated to give the pure product as an off white foam (2.84 g,
81 °~
yield).
NMR: (CDC13-de) 8 = 2.29 (d, 1 H), 2.70 (m, 2H), 3.17 (m, 2H), 3.78 (s, 6H),
4.44 (m,
2H), 5.63 (d, 1 H), 6.19 (d, 1 H), 6.83 (d, 4H), 7.28 (m, 9 H), 7.68 (d, 1 H),
9.30 (br s,
1 H), _ . . .. . ._ ... _ . __. . .. _. .. _ . ... . . .... . ... _. . _.... .
.. ._..~
5'-O-(dimethoxyrtrityl_)-a-L-2'-deoxyuridine-3'-N. N-diisopro~p)rlmethoxy
phosehoramidite (21c)
The 5'-0-dimethoxytrityl-a-L-2'-deoxyuridine (2.35 g, 4.43 mmol) was
dissolved in anhydrous dichloromethane (50 ml). N,N-diisopropylethylarnine
(3.1
ml, 17.72 mmol) was added through a septum, followed by
chioro-N,N-diisopropylmethoxyphosphine (1.3 ml, 6.64 mmol), under an argon
atmosphere. The reaction was stirred for 30 minutes. The solvent was
evaporated
and the residue was partitioned between an 80% EtOAGtriethylamine mixture and
22
CA 02322494 2000-09-07
WO 99/45935 PCT/US99I053b0
brine. The organic layer was washed with saturated NaHC03 solution and brine.
The organic residue was evaporated to dryness and the residue was purified on
a
silica gel column using a mixture of dichloromethane, EtOAc and triethylamine
(40:50:10; Rf = 0.69). The product was isolated quantitatively as a yellow
foam and
it was used in the next step without further purification.
5'-0-dimethoxytrityl-3'-[0-(3'-0-acetyl~i-D-5 fluoro-2'-deo ux r~yll-2'-deoxv-
a-L-
uridine 125c)
The 3'-O-acetyl-(3-D-5 fluoro-2'-deoxyuridine (0.95 g, 3.29 mmol) was
dissolved in dry acetonitrile (125 ml). Sublimed 1 H-tetrazole (350 mg, 4.91 )
was
added and the mixture was stirred under an argon atmosphere for 15 minutes.
The
solution of 21 c (4.91 mmol), dissolved in 5 ml of dry acetonitrile was added
via
syringe to the reaction solution over 5 minutes. The mixture was allowed to
stir at
room temperature for three hours. The acetonitriie was evaporated in vacuo to
a
residue. This residue was triturated with a 70% EtOAclether mixture. The
undissolved tetrazole was filtered off and the ~Itrate was evaporated to give
a dry
yellow foam. This compound was further purified on a silica gel column using
5%
MeOHICHCl3 to give the pure product (2.81 g, 97°r6 yield).
3'-acetox~~i-D-5' fluoro-2'-deoxvuridinvl)-cx-L-2'-deoxvuridine meth~rl
phosphate
ester 26c)
The dimer, 25c (2.81 g, 3.2 mmol), was dissolved in a mixture of
THF:pyridine:water (25:6:0.6). Iodine crystals (150 mg) were added and the
contents of the loosely stoppered flask were allowed to stir for 1 hour.
Excess
-~ - -- iodine was-discharged by the addition of a few drops of saturated
sodium
thiosuifate. The reaction mixture was then evaporated to dryness. The crude
product was dissolved in EtOAc washed with saturated NaHCO$ solution and
brine.
The organic layer was dried over NazSO,,, filtered and evaporated in vacuo.
The
residue (1.48 g) was dissolved in 25 ml of 80% acetic acidlwater solution and
was
stirced until the reaction was completed. The solvent was evaporated and the
residue was purified on a silica gel column, using 10°~ MeOH/CHCl3.
Fractions
containing one spot by TLC (10% MeOHICHCl3 Rf = 0.4) were pooled and
evaporated to give the pure product (0.465 g, 25°~ yield).
23
CA 02322494 2000-09-07
WO 99145935 PC'TIUS99/05360
NMR: (CD30D) 8 = 2.09 (d, 3H), 2.40 (m, 3H), 2.80 (m, 1 H), 3.78 (dd, 3H),
4.30 (m,
3H), 4.63 (m, 1 H), 5.05 (m, 1 H), 5.23 (m, 1 H), 5.70 (d, 1 H), 6.13 (m, 1
H), 6.20 {m,
1 H), 7.73 (d, 1 H), 7.82 (d, 1 H).
P3' NMR: (CD30D) b = 0.56 (s), 0.84 (s).
3'-O-(f3-D-5 fluoro-2'-deoxyuridinyl)-a-L-2'-deoxyuridine (27c)i
The O-protected dimer, 26c (465 mg, 0.78 rnmol) was treated with 50 ml of
saturated methanolic ammonia at room temperature until the reaction was
completed. The solvent was stripped off in vacuo and the residue was purified
on
DEAE cellulose ion exchange column using gradient of NH4C03 buffer from 0.02-
0.2
M. Pure fractions were evaporated at 40°C in high vacuo to dryness to
give the
pure product (370 mg, 87.7°~ yield).
NMR: (CD30D) 8 = 2.23 (m, 2H), 2.29 (d, 1 H), 2.73(m, 1 H), 4.0 {d, 2H), 4.42
(m,
1 H), 4.56 (m, 1 H), 4.81 (m, 1 H), 5.69 (d, 1 H), 6.24 (m, 2H), 7.85 (d, 1
H), 8.02 (d,
1 H).
P3' NMR: (CD30D) S = 1.25 (s).
D. j3-Ls j3-L 5 FUdR dimer
5'-O-dimethoxvtrityl-3L-L-5 fluoro-2'-deoxyuridine (20d1
j3-L-5 fluoro-2'-deoxyuridine (19d, 1.42 g, 5.77 mmol) was dissolved in 25 ml
of dry, distilled pyridine. To this solution was added 4,4'-dimethoxytrityl
chloride
{2.34 g, 6.92 mmol) and 4-dimethylamino pyridine (DMAP) (140 mg, 1.15 mmol).
The mixture was stirred under an argon atmosphere for 16 hours. After this
time,
.._ _ _ __.___._... _ the.pyridy was.stripped off~in vacuo: The residue was
dissolved in ~tOAc (100 . __ ._. .... ....... .. _ ..
ml). The organic layer was washed with saturated NaHC03, and with brine. The
organic layer was dried over Na2S04, filtered and evaporated in vacuo to a
residue
which was purified on a silica gel column using 5% MeOH/CHCl3. Pure fractions
were pooled and evaporated to give the pure product as an off-white foam (2.88
g,
88.7°~ yield).
NMR: (CDCI3) b = 2.25 (m, 1 H), 2.50 (m, 1 H), 3.50 (m, 2H), 3.80 (s, 6H),
4.08 (m,
1 H), 4.58 (m, 1 H), 6.30 (t, 1 H), 6.84 {d, 4H), 7.28 (m, 9 H), 7.82 (d, 1
H), 8.58 (br s,
1 H).
24
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WO 99/45935 PC"f/US99/05360
5'-O-dimethoxvtrityl~i-L-5-fluoro-2'-deoxyuridine-3'-N N-diisopropylmethoxy
iphOSphoramidite 121d)
The 5'-O-dimethoxytrityl-~i-L-5-fluoro-2'-deoxyuridine (20d, 840 mg, 1.53
mmol) was dissolved in anhydrous dichloromethane (50 ml).
N,N-diisopropylethylamine (1.1 ml, 6.13 mmol) was added through a septum,
followed by chloro-N,N-diisopropylmethoxyphosphine (0.42 ml, 2.3 mmol), under
an
argon atmosphere. The reaction was stin-ed for 30 minutes. The solvent was
evaporated and the residue was partitioned between an 80°~
EtOAcltriethylamine
mixture and brine. The organic layer was washed with saturated NaHC03 solution
and brine. The organic residue was evaporated to dryness and the residue was
purified on a silica gel column using a mixture of dichloromethane, EtOAc and
triethylamine (45:45:10; Rf = 0.69). The product (700 mg, 65%) was isolated as
a
yellow foam and it was used in the next step without further purification.
5'-O-dimethoxytriyl-3'-[O-{3'-O-acetyl)-a-L-5 fluoro-2'-deoxvuridin~l-Q-L-5
fluoro-2
'-deoxyuridine {25d)
The 3'-acetyl-~i-L-5-deoxyuridine, 24d (330 mg, 1.15 mmol) was dissolved in
dry acetonitrife (50 ml). Sublimed 1 H-tetrazole (120 mg, 1.77 mmol) was added
and
the mixture was stirred under an argon atmosphere for 15 minutes. The solution
of
21d (950 mg, 1.36 mmol), dissolved in 5 ml of dry acetonitrile was added via
syringe
to the reaction solution over 5 minutes. The mixture was allowed to stir at
room
temperature for three hours. The acetonitrile was evaporated in vacuo to a
residue.
This residue was triturated with a 70% EtOAclether mixture. The undissolved
. _ _.._ .. . w. _ ... .. ._ .tetrazole was-Tittered-off- and-the filtrat$-was-
evaporated to~-give-a tlry Yellow-foam. ___ _ _ . ._. ._.._. _. ... _
This foam was purified on a silica gel column using 5°r6 MeOHICHCl3 to
give the
pure product (960 mg, 93% yield).
NMR: (CDCI3) 8 = 2.10 (d, 3H), 2.28 (m, 2H), 2.49 (m, 2H), 3.42 (m, 3H), 3.51
(dd,
3H), 3.76 (s, 6H), 4.07 (m, 1 H), 4.55 (m, 1 H), 4.87 (m, 1 H), 5.23 (m, 1 H),
6.30 (m,
2H), 6.84 (d, 4H), 7.30 (m, 9H), 7.82 (m, 2H).
~3'-acetox~(3-L-5-fluoro-2'-deoxyuridin~rl~J3-L-5-fluoro-2'-deoxyuridine
methyl
phosphate ester~26d~
The dimer, 25d (960 mg, 1.07 mmol), was dissolved in a mixture containing
CA 02322494 2000-09-07
WO 99145935 PCT/US99/05360
THF:pyridine:water (12:3:0.3). Iodine crystals (50 mg) were added and the
contents
of the loosely stoppered flask were allowed to stir for 1 hour. Excess iodine
was
discharged by the addition of a few drops of saturated sodium thiosulfate. The
reaction mixture was then evaporated to dryness. The crude product was
dissolved
in EtOAc washed with saturated NaHC03 solution and brine. The organic layer
was
dried over Na2S04, filtered and evaporated in vacuo. The residue (530 mg) was
dissolved in 20 ml of 80°~ acetic acidlwater solution and was stirred
until the
reaction was completed. The solvent was evaporated and the residue was purred
on a silica gel column, using 10°r6 MeOHICHCl3. Fractions containing
one spot by
TLC (10°~ MeOHlCHCI3 Rf = 0.35) were pooled and evaporated to give the
pure
product (310 mg, 46% yield).
NI-x-11 R: (CD30D) a = 2.08 (s, 3H), 2.35-2.54 (m, 4H), 3.79 (m, 2H), 3.83
(dd, 3H),
4.18 (m, 2H), 5.08 (m, 1 H), 5.29 (m, 1 H), 6.24 (m, 1 H), 7.86 (dd, 1 H),
8.19 (dd, 1 H).
P3' NMR: (CD30D) S = 0.82 (s), 1.03 (s).
3'-O-y3-L-5-fluoro-2'-deoxvuridinyly-~i-L-5-fluoro-2'-deoxyuridine (27d)
The O-protected dimer, 26d (300 mg, 0.49 mmol) was treated with 50 ml of
saturated methanolic ammonia at room temperature until the reaction was
completed. The solvent was stripped off in vacuo and the residue was purified
on
DEAE cellulose ion exchange column using gradient of NH4C03 buffer from 0.02-
0.2
M. Pure fractions were evaporated at 40°C in high vacuo to dryness to
give the
pure product (240 mg, 85°~ yield).
NMR: (CD30D) i5 = 2.25 (m, 3H), 2.50 (m, 1 H), 3.79 (d, 2H), 4.03 (m, 1 H),
4.08 (m,
.. _. . . _2H),..4,1-8.(m,.1.H~,.4.44 (m,..1H), 4.90 (m, lH), 6.25.(x*
1H),.8..01. (d,..l.H), 8.24 (d, _ . . H.__._.
1 H).
P3' NMR: (CD30D) i3 = 0.18 (s).
E. Q-L. I3-D 5 FUdR dimer
5'-O-dimethoxlrtritvl-3'-f0-(3'-O-acetyl)-(3-D-5 fluoro-2'-deo ux~r ridinvl]-a-
L-5-fluoro-2
'-deoxyuridine (25e)
The 3'-O-acetyl-(3-D-5 fluoro-2'-deoxyuridine (250 mg, 0.97 mmol) was
dissolved in dry acetonitrile (50 ml). Sublimed 1 H tetrazole (100 mg, 1.46
mmol)
26
CA 02322494 2000-09-07
WO 99/45935 PC'T/US99/05360
was added and the mixture was stirred under an argon atmosphere for 15
minutes.
The solution of 21e (1.02 g, 1.46 mmol), dissolved in 5 ml of dry acetonitrile
was
added via syringe to the reaction solution over 5 minutes. The mixture was
allowed
to stir at room temperature for three hours. The acetonitrile was evaporated
in
vacuo to a residue. This residue was triturated with a 70% EtOAclether
mixture.
The undissolved tetrazole was filtered off and the filtrate was evaporated to
give a
dry yellow foam. This foam was purified on a silica gel column using
5°~
MeOHICHCl3 to give the pure product quantitatively. .
(3'-acetoxv~'i-D-5-fluoro-2'-deo u~yl)-~-L-5 fluoro-2'-deoxyuridine methK
phosphonate ester (26e)
The dimer in reduced form, 25 (700 mg, 0.78 mmol), was dissolved in a
mixture containing THF:pyridine:water (25:6:0.6). Iodine crystals (100 mg)
were
added and the contents of the loosely stoppered flask were allowed to stir for
2.5
hours. Excess iodine was discharged by the addition of a few drops of
saturated
sodium thiosulfate. The reaction mixture was then evaporated to dryness. The
crude product was dissolved in EtOAc washed with saturated NaHC03 solution and
brine. The organic layer was dried over Na2S04, ~Itered and evaporated in
vacuo.
The residue was dissolved in 25 ml of 80% acetic acidlwater solution and was
stirred until the reaction was completed. The solvent was evaporated and the
residue was purified on a silica gel column, using 10°~ MeOH/CHCl3.
Fractions
containing one spot by TLC (10°~ MeOHICHCl3 Rf = 0.35) were pooled and
evaporated to give the pure product (340 mg, 71.4°~ yield).
. _._ __ ~. ~MR: - (DMSO-ds) b -_.2.06 (s; 3H), 2.37 {m; 4H); 3.45 (m; .2H.);
.3:fi5 (d; 3H), 4.20 (m;- . __ ..
3H), 4.95 (m, 1 H), 5.30 (m, 1 H), 5.96 (m, 1 H), 6.15 (t, 2H), 7.99 (d, 1 H),
8.16 (d,
1 H), 11.90(br s, 2H).
P3' NMR: (DMSO-dg) 8 = 1.93 (s), 2.01 (s).
3'-O-~(3-D-5-fluoro-2'-deoxyuridinyl~(3-L-5-fluoro-2'-deoxYuridine i(27e)
The 0-protected dimer, 26e (34.0 mg, 0.57 mmol) was treated with 100 ml of
saturated methanolic ammonia at room temperature until the reaction was
completed. The solvent was stripped off in vacuo and the residue was purified
on
DEAE cellulose ion exchange column using gradient of NH4C03 buffer from 0.02-
0.2
27
CA 02322494 2000-09-07
WO 99/45935 PCTNS99/05360
M. Pure fractions were evaporated at 40°C in high vacuo to dryness to
give the
pure product (200 mg, fi6.9% yield).
NMR: (CD30D) a = 2.20 (m, 3H), 2.53 (m, 1 H), 3.79 (d, 2H), 4.05 (m, 3H), 4.16
(m,
1 H), 4.45 (m, 1 H), 6.27 (t, 2H), 8.01 (d, 1 H), 8.04 (d, 1 H), 8.26 (d, 1
H).
5'-O-~dimethoxytrityl)-a-L-5-fluoro-2'-deoxvuridine-3'-N.N-
diisopropylcvanoethyl
phosohoramidite (21f1
The 5'-o-dimethoxytrityl-a-L-5-fluoro-2'-deoxyuridine (1.48 g, 2.71 mmol) was
dissolved in anhydrous dichloromethane (50 ml). N,N-diisopropylethylamine
(1.9 ml, 10.84 mmol) was added through a septum, followed by
2'-cyanoethyl-N,N-diisopropylchlorophosphoramidite (0.78 ml, 3.52 mmol), under
an
argon atmosphere. The reaction was stirred for 30 minutes. The solvent was
evaporated and the residue was partitioned between an 80% EtOAcltriethylamine
mixture and brine. The organic layer was washed with saturated NaHC03 solution
and brine. The organic residue was evaporated to dryness and the residue was
purified on a silica gel column using a mixture of dichloromethane, EtOAc and
triethylamine (45:45:10:Rf=0,7). The product was isolated quantitatively as a
yellow
foam and it was used in the next step without further purification.
5'-O-dimethox~rtrityl-3'-(~5'-0-dimethox~rtritvl)-~-D-5-fluoro-2'-deo
ux~ridinyl]i-a-L-5
-fluoro-2'-deoxyuridine~25f~
The 5'-O-dimethoxytrityl-~i-D-5-fluoro-2'-deoxyuridine (0.44 g, 0.81 mmol)
was dissolved in dry acetonitrile (20 ml). Sublimed 1 H-tetrazole (90 mg) was
added
and the mixture was stirred under an argon atmosphere for 15 minutes. The
_. . _. - ._. . - - olution of-21f-(0..51- mg; -0:67 mmol), dissolved in 10 ml-
of dry-.acetonitrile was _ .. .._ _..
added via syringe to the reaction solution over 5 minutes. The mixture was
allowed
to stir at room temperature for three hours. The acetonitrile was evaporated
in
vacuo to a residue. This residue was triturated with a 70°~ EtOAclether
mixture.
The undissolved tetrazole was filtered off and the filtrate was evaporated to
give a
dry yellow foam (970 mg). This foam was used in the next step without further
purification.
28
CA 02322494 2000-09-07
WO 99/45935 PCT/US99I05360
-D-5 fiuoro-2'-deoxvuridinvl)-a-L-5-ffuoro-2'-deoxvuridine cvanoeth
phosphonate ester (26f)
The dimer, 25f (970 mg), was dissolved in 16 ml of THF and 4 ml of pyridine
containing 0.4 ml of water. Iodine crystals (50 mg) were added and the
contents of
the loosely stoppered flask were allowed to stir for 1 hour. Excess iodine was
discharged by the addition of few drops of saturated sodium thiosulfate. The
reaction mixture was then evaporated to dryness. The crude product was EtOAc
washed with saturated NaHC03 solution and brine. The organic layer was dried
over Na2S0,,, filtered and evaporated in vacuo. The residue was dissolved in
20 ml
of 80% acetic acidlwater solution and was stirred until the reaction was
completed.
The solvent was evaporated and the residue was purified on a silica gel
column,
using 10-15% MeOHICHCL3. Fractions containing one spot by TLC (10°~
MeOHICHCL3 Rf=0.35) were pooled and evaporated to give the pure product (330
mg).
3'-O-y(3-D-5 fluoro-2'-deoxvuridinyly-a-L-5-fluoro-2'-deoxvuridine i(27f)
The O-protected dimer, 26f (200 mg) was treated with 20 ml of concentrated
ammonia solution until the reaction is completed. The solvent was stripped off
in
vacuo and the residue was purified on DEAE cellulose ion exchange column using
gradient of NH4C03 buffer from 0.02-0.2 M. Pure fractions were evaporated at
40°C
in high vacuo to dryness to give the pure product (79 mg).
NMR: (CD30D) i5 = 2.45 (m, 3H), 2.69 (m, 1 H), 3.67 (m, 2H), 3.76 (m, 2H),
4.13 (t,
1 H), 4.65 (m, 2H), 6.19 (m, 2H), 7.98 (td, 2H).
p3? NMR:. (DZO) 8 = _1:0(s) . ... . . __ . ... ..._. _ _ .. . . __.....
5'-0-dimethoxytrityl-3'-(O- 3'-O-acet~~3-L-5 fluoro-2'-deoxvuridinvll-a-L-5-
fluoro-2
'-deoxyuridine (254)
The 3'-O-acetyl-~i-D-5-fluoro-2'-deoxyuridine (0.19 g, 0.67 mmol) was
dissolved in dry acetonitrile (20 ml). Sublimed 1 H-tetrazole (70 mg) was
added and
the mixture was stirred under an argon atmosphere for 15 minutes. The solution
of
21f (0.51 mg, 0.67 mmol), dissolved in 10 ml of dry acetonitrile was added via
syringe to the reaction solution over 5 minutes. The mixture was allowed to
stir at
room temperature for three hours. The acetonitrile was evaporated in vacuo to
a
29
CA 02322494 2000-09-07
WO 99/45935 PCTNS99/05360
residue. This residue was triturated with a 70°~ EtOAcJether mixture.
The
undissolved tetrazole was filtered off and the filtrate was evaporated to give
a dry
yellow foam (611 mg). This foam was used in the next step without further
purification.
(3'-acetox~ti-L-5-fluoro-2'-deoxyuridinyl)-a-L-5-fluoro-2'-deox~dine
cyanoethyl
phosnhonate ester (26a)
The dimer, 25g (611 mg), was dissolved in 8 ml of THF and 2 ml of pyridine
containing 0.2 ml of water. Iodine crystals (30 mg) were added and the
contents of
the loosely stoppered flask were allowed to stir for 1 hour. Excess iodine was
discharged by the addition of few drops of saturated sodium thiosulfate. The
reaction mixture was then evaporated to dryness. The crude product was EtOAc
washed with saturated NaHC03 solution and brine. The organic layer was dried
over Na2S04, filtered and evaporated in vacuo. The residue was dissolved in 20
ml
of 80% acetic acidlwater solution and was stirred until the reaction was
completed.
The solvent was evaporated and the residue was purified on a silica gel
column,
using 10-15°~ MeOH/CHCL3. Fractions containing one spot by TLC (10%
MeOHICHCL3 Rf=0.35) were pooled and evaporated to give the pure product
(200 mg).
3'-O-(~3-L-5 fluoro-2'-deoxyruridin~,)-ar-L-5 fluoro-2'-deoxyuridine 127x) (a-
L p3-L
5FUdR Dimerl
The o-protected dimer, 26g (200 mg) was treated with 20 ml of concentrated
ammonia solution until the reaction is completed. The solvent was stripped off
in
--. -- - .., ... . .v~uo.and the.c8sidue was purifed on DEAF Eellulose-ion-
exchange column using -~ -
gradient of NH4C03 buffer from 0.02-0.2 M. Pure fractions were evaporated at
40°C
in high vacuo to dryness to give the pure product (134 mg).
NMR: (DZO) 8 = 2.30 (m, 3H), 2.71 (m, 1 H), 3.fi5 (m, 2H), 4.03 (m, 2H), 4.08
(t, 1 H),
4.47 (m, 1 H), 4.68 (m, 2H), 6.13 (d, 1 H), 6.24 (td, 1 H), 7.89 (d, 1 H),
7.95 (d, 1 H).
P3'NMR: (DZO) b = 0.32(s)
55'-O-(dimethoxvtritvl)-Q-L-2'-deoxyuridine-3'-N. N-diisopropylmethoxY
phosphoramidite l2lh)
The 5'-O-dimethoxytrityl-a-L-2'-deoxyuridine (1.0 g, 1.88 mmol) was
CA 02322494 2000-09-07
WO 99/45935 PCT/US99105360
dissolved in anhydrous dichloromethane (50 ml). N,N-diisopropylethylamine
(1.31 ml, 7.55 mmol) was added through a septum, followed by
chloro-N,N-diisopropylmethoxyphosphine (0.55 ml, 2.83 mmol), under an argon
atmosphere. The reaction was stirred for 30 minutes. The solvent was
evaporated
and the residue was partitioned between an 80°~ EtOAcltriethylamine
mixture and
brine. The organic layer was washed with saturated NaHC03 solution and brine.
The organic residue was evaporated to dryness and the residue was purified on
a
silica gel column using a mixture of dichloromethane, EtOAc and triethylamine
(50:40:10; Rf=0.8). The product was isolated quantitatively as a yellow foam
and it
was used in the next step without further purification.
5'-O-dimethoxytrityl-3'-[O-(3'-O-acet~)-~3-D-5 fluoro-2'-deox,~ruridin~l-Q-L-
2'-deoxyu
ridine (25h)
The 3'-O-acetyl-~i-D-5-fiuoro-2'-deoxyuridine (0.54 g, 1.88 mmol) was
dissolved in dry acetonitrile (50 ml). Sublimed 1 H-tetrazole (200 mg) was
added
and the mixture was stirred under an argon atmosphere for 15 minutes. The
solution of 21 h (1.88 mmol), dissolved in 15 ml of dry acetonitrile was added
via
syringe to the reaction solution over 5 minutes. The mixture was allowed to
stir at
room temperature for three hours. The acetonitrile was evaporated in vacuo to
a
residue. This residue was triturated with a 70% EtOAclether mixture. The
undissolved tetrazole was filtered off and the filtrate was evaporated to give
a dry
yellow foam (1.08 g). This foam was used in the next step without further
purification.
. . 3'- t -fl r -2'-d uri in ' r o at .._ ._. .
ester 126h~
The dimer, 25h (1.08 g), was dissolved in 15 ml of THF and 3 ml of pyridine
containing 0.3 ml of water. Iodine crystals (100 mg) were added and the
contents of
the loosely stoppered flask were allowed to stir for 1 hour. Excess iodine was
discharged by the addition of few drops of saturated sodium thiosulfate. The
reaction mixture was then evaporated to dryness. The crude product was EtOAc
washed with saturated NaHC03 solution and brine. The organic layer was dried
over NaZS04, filtered and evaporated in vacuo. The residue was dissolved in 25
ml
31
CA 02322494 2000-09-07
WO 99/45935 PCT/US99105360
of 80°~ acetic acidlwater solution and was stirred until the reaction
was completed.
The solvent was evaporated and the residue was purified on a silica gel
column,
using 10-15% MeOHICHCL3. Fractions containing one spot by TLC (10%
MeOHICHCL3 Rf=0.4) were pooled and evaporated to give the pure product
(400 mg).
3'-0-(j3-D-5-fluoro-2'-deoxvuridinylJi 3~-L-2'-deoxyuridine (27hJi
The 0-protected dimer, 26h (400 mg) was treated with 100 ml of methnolic
ammonia solution until the reaction is completed. The solvent was stripped off
in
vacuo and the residue was purified on DEAE cellulose ion exchange column using
gradient of NH4C03 buffer from 0.02-0.2 M. Pure fractions were evaporated at
40°C
in high vacuo to dryness to give the pure product (175 mg).
NMR: (D20} a = 2.40 (m, 3H), 2.61 (m, 1 H), 3.80 (m,2H), 4.10 (m, 2H), 4.18
(m,2H),
4.55 (m, 1 H), 4.80 {m, 1 H), 5.85 (d, 1 H), 6.30 (q, 2H), 7.85 (d, 1 H), 8.06
(d, 1 H).
P3'NMR: (D20) b = 0.20(s)
5'-O-dimethoxytri-tyl-N°-benzoyl-2'-deoxy-Q-L-cytidine (20i)
N4-benzoyl-2'-deoxy-~i-L-cytidine (0.8 g, 2.42) was dissolved in 50 ml of dry,
distilled pyridine. To this solution was added 4,4'-dimethoxytrityl chloride
(3.0 g,
8.85 mmol) and 4-dimethyiamino pyridine (DMAP) (60 mg, 0.48 mmol). The mixture
was stirred under an argon atmosphere for 16 hours. After this time, the
pyridine
was stripped off in vacuo. The residue was dissolved in EtOAc (100 ml). The
organic layer was washed with water, saturated NaHC03, and brine. The organic
layer was dried over Na2S04, filtered and evaporated in vacuo to a residue
which
.._ . . ._. . _ _.~_._. was.purified on a silica gel-column using
10°/fl-MeOHICHCl3. Pure fractions were
pooled and evaporated to give the pure product as an off-white foam (1.49 g,
(97°~
yield). Rf=0.48 in 10% MeOH/CHCl3.
NMR: (CDCI3-de) S = 2.3 (m, 1 H), 2.75 (m, 2H), 3.42 (ddd, 2H), 3.80 (s, 6H),
4.15
(q, 2H}, 4.52 (m, 1 H), 6.30 (t, 1 H), 6.82 (dd, 4H), 7.2-7.6 (m, Ar), 7.85
{d, 2H), 8.32
(d, 1 H), 8.76 (br s, 1 H).
5'-O-{dimethoxytrityl)-N"-benzoyl-2'-deoxy ~3-L-cvtidine-3'-N.N-
diisohropylmeth~
phosehoramidite {21i)
The 5'-O-dimethoxytrityl-N°-benzoyl-2'-deoxy-~i-L-cytidine (0.6 g, 0.95
mmol)
32
CA 02322494 2000-09-07
WO 99/45935 PCT/US991053b0
was dissolved in anhydrous dichloromethane (50 ml). N,N-diisopropylethylamine
(0.66 ml, 3.79 mmol) was added through a septum, followed by
chloro-N,N-diisopropylmethoxyphosphine (0.28 ml, 1.42 mmol), under an argon
atmosphere. The reaction was stirred for 30 minutes. The solvent was
evaporated
and the residue was partitioned between an 80% EtOAcltriethylamine mixture and
brine. The organic layer was washed with saturated NaHC03 solution and brine.
The organic residue was evaporated to dryness and the residue was purified on
a
silica gel column using a mixture of dichloromethane, EtOAc and triethylamine
(60:30:10;Rf=0.8). The product was isolated quantitatively as a yellow foam
and it
was used in the next step without further purification.
5'-O-dimethoxytritvl-3' ~~3'-O-acet5rl)i-~i-D-5-fluoro-2'-deoxyuridinylt-
N°-benzo~rl-2'
-deoxy-~3-L-cytidine (25i)
The 3'-O-acetyl-~i-D-5 fluoro-2'-deoxyuridine (0.23 g, 0.78 mmol) was
dissolved in dry acetonitrile (30 ml). Sublimed 1 H-tetrazote (110 mg) was
added
and the mixture was stirred under an argon atmosphere for 15 minutes. The
solution of 21i (0.94 rnmol), dissolved in 15 ml of dry acetonitrile was added
via
syringe to the reaction solution over 5 minutes. The mixture was allowed to
stir at
room temperature for three hours. The acetonitriie was evaporated in vacuo to
a
residue. This residue was triturated with a 70% EtOAclether mixture. The
undissolved tetrazole was filtered off and the filtrate was evaporated to give
a dry
yellow foam (0.73 g). This foam was used in the next step without further
purification.
... . _ . _ .__. _. __(3..'._aceto~rt~3.=.p.-5.-~uoro-2'.-deo ux~i_nyrl rN4-
benzovl:~'.-de~y~:L-cytidine. methyl ~ .. . ____ _...
phosphonate ester (26i)
The dimer, 25i (0.73 g), was dissolved in 20 ml of THF and 4 ml of pyridine
containing 0.4 ml of water. Iodine crystals (100 mg) were added and the
contents of
the loosely stoppered flask were allowed to stir for 1 hour. Excess iodine was
discharged by the addition of few drops of saturated sodium thiosulfate. The
reaction mixture was then evaporated to dryness. The crude product was EtOAc
washed with saturated NaHC03 solution and brine. The organic layer was dried
over Na2S04, filtered and evaporated in vacuo. The residue was dissolved in 25
ml
33
CA 02322494 2000-09-07
WO 99145935 PCT/US99/05360
of 80°~ acetic acidlwater solution and was stirred until the reaction
was completed.
The solvent was evaporated and the residue was purified on a silica gel
column,
using 10-15°~ MeOHICHCl3. Fractions containing one spot by TLC
(10°~
MeOHICHCl3 Rf=0.4) were pooled and evaporated to give the pure product
(108 mg).
3'-0-lt3-D-5 fluoro-2'-deoxyuridinyl)-2'-deoxy-(3-L-cytidine~,27i)
The 0-protected dimer, 26i (108 mg) was treated with 100 ml of methnolic
ammonia solution until the reaction is completed. The solvent was stripped off
in
vacuo and the residue was purified on DEAE cellulose ion exchange column using
gradient of NH4C03 buffer from 0.02-0.2 M. Pure fractions were evaporated at
40°C
in high vacuo to dryness to give the pure product (56 mg).
NMR: (D20) 5 = 2.30 (m, 3H), 2.55 (m, 1 H), 3.80 (m, 2H), 4.05 (m, 2H), 4.18
(m,
2H), 4.52 (m, 1 H), 4.78 (m, 1 H), 6.02 (d, 1 H), 6.25 (m, 2H), 7.80 (d, 1 H),
8.04 (d,
1 H).
P3'NMR: (DZO) 6 = 0.05(s)
5'-0-dimethoxvtritvl-3'-[~3'-O-acet~)-N'-benzovl-2'-deoxy-(3-L-cvtidinyl~(3-D-
5 flu
oro-2'deoxvuridine (25j)
The 3'-O-acetyl-[i-D-5 fluoro-2'-deoxyuridine (0.25 g, 0.67 mmol) was
dissolved in dry acetonitrile (30 ml). Sublimed 1 H-tetrazole (94 mg) was
added and
the mixture was stirred under an argon atmosphere for 15 minutes. The solution
of
21 j (0.51 g, 0.67 mmol), dissolved in 15 ml of dry acetonitriie was added via
syringe
to the reaction solution over 5 minutes. The mixture was allowed to stir at
room
.__-.-.. .--_.. ._,..._t~perature-for-.three.hours.. -ThE
acetonitrilewas.~vaporatedla.vacuoao.aresidue: _.
This residue was triturated with a 70% EtOAclether mixture. The undissolved
tetrazole was filtered off and the filtrate was evaporated to give a dry
yellow foam
(0.49 g). This foam was used in the next step without further purification.
~3'-acetoxy-N4-benzoyl-2'-deoxy-(3-L-cytidinyl)-(3-D-5-fluoro-2'-deoxyuridinyl
cyanoethvl phosphonate ester (26j)
The dimer, 25j (0.49 g), was dissolved in 8 ml of THF and 2 ml of pyridine
containing 0.2 ml of water. Iodine crystals (30 mg) were added and the
contents of
the loosely stoppered flask were allowed to stir for 1 hour. Excess iodine was
34
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WO 99/45935 f'CTIUS99/05360
discharged by the addition of few drops of saturated sodium thiosulfate. The
reaction mixture was then evaporated to dryness. The crude product was EtOAc
washed with saturated NaHC03 solution and brine. The organic layer was dried
over Na2S04, filtered and evaporated in vacuo. The residue was dissolved in 20
mi
of 80°~ acetic acidlwater solution and was stirred until the reaction
was completed.
The solvent was evaporated and the residue was purified on a silica gel
column,
using 10-15°~ MeOHICHCl3. Fractions containing one spot by TLC
(10°~
MeOHICHCl3 Rf=0.4) were pooled and evaporated to give the pure product
(1B8 mg).
3'-O-(2'-deoxy-[i-L-c idinyl)-[i-D-5-fluoro-2'-deoxvuridine (27i)
The O-protected dimer, 26j (188 mg) was treated with 100 ml of concentrated
ammonia solution until the reaction is completed. The solvent was stripped off
in
vacuo and the residue was purified on DEAE cellulose ion exchange column using
gradient of NH4C03 buffer from 0.02-0.2 M. Pure fractions were evaporated at
40°C
in high vacuo to dryness to give the pure product (105 mg).
NMR: (DZO) S = 2.30 (m, 3H), 2.50 (m, 1 H), 3.80 (m, 2H), 4.05 (m, 2H), 4.10
(m,
2H), 4.20 (m, 1 H), 4.52 (m, 1 H), 4.75 (m, 1 H), 6.05 (d, 1 H), 6.29 (q, 2H),
7.89 (d,
1 H), 9.03 (d, 1 H).
P3'NMR: (D20) b = 0.05(s)
5'-O-dimethox)rtrityl-3'-[0-(3'-O-acetyl)-N''-benzoyl-2'-deoxy-a-L-cytidin~)-
(3-D-5-flu
oro-2'-deoxyuridine (25k)
The 3'-O-acetyl-(3-D-5-fluoro-2'-deoxyuridine (0.19 g, 0.51 mmol) was
..._._........ .. ..., _. . dissolued.in_dry~acetonitrile.(30.m1)...
Sublimed..lH-.tetrazole. (80. mg). was added and.... ._ ... _ ...._. . _..~
the mixture was stirred under an argon atmosphere for 15 minutes. The solution
of
21 k (0.45 g, 0.61 mmol), dissolved in 15 m1 of dry acetonitrile was added via
syringe
to the reaction solution over 5 minutes. The mixture was allowed to stir at
room
temperature for three hours. The acetonitrile was evaporated in vacuo to a
residue.
This residue was triturated with a 70°r6 EtOAclether mixture. The
undissolved
tetrazole was filtered off and the filtrate was evaporated to give a dry
yellow foam
(0.42 g). This foam was used in the next step without further purification.
CA 02322494 2000-09-07
WO 99/45935 PCT/US99105360
~3'-acetoxY N°-benzovl-2'-deoxy-a-L-cytidinylZ-~-D-5 fluoro-2'-
deoxyuridinvl
c~anoethvJahosphonate ester (26k)
The dimer, 25k (0.42 g), was dissolved in 10 ml of THF and 2 ml of pyridine
containing 0.2 ml of water. Iodine crystals (45 mg) were added and the
contents of
the loosely stoppered flask were allowed to stir for 1 hour. Excess iodine was
discharged by the addition of few drops of saturated sodium thiosulfate. The
reaction mixture was then evaporated to dryness. The crude product was EtOAc
washed with saturated NaHC03 solution and brine. The organic layer was dried
over Na2S0,, filtered and evaporated in vacuo. The residue was dissolved in 25
ml
of 80% acetic acidlwater solution and was stirred until the reaction was
completed.
The solvent was evaporated and the residue was purified on a silica gel
column,
using 10-15% MeOHICHCl3. Fractions containing one spot by TLC (10°~
MeOHICHCl3 Rf=0.4) were pooled and evaporated to give the pure product
(125 mg).
3'-O-(2'-deoxy-a-L-cytidin~rly3-D-5-fluoro-2'-deoxyuridin~27k)
The O-protected dimer, 26k (125 mg) was treated with 100 ml of
concentrated ammonia solution until the reaction is completed. The solvent was
stripped off in vacuo and the residue was purified on DEAF cellulose ion
exchange
column using gradient of NH4C03 buffer from 0.02-0.2 M). Pure fractions were
evaporated at 40°C in high vacuo to dryness to give the pure product
(40 mg).
NMR: (D20) S = 2.15 (m, 1 H), 2.35 (m, 1 H), 2.60 (m, 1 H), 2.71 (m, 1 H),
3.81 (m,
2H), 3.97 (m, 2H), 4.22 (m, 1 H), 4.52 (m, 2H), 6.02 (d, 1 H), 6.15 (dd, 1 H),
6.28 (t,
1.H)~:.7:87 (d;-1H), 8.03 (d; lhl). ...... . . _ . N. _ ._.._...... ..... _.__
...~. . ___.___..,..... .. _.
P3'NMR: (D20) 5 = 0.12(s)
Example 5
Dimers Testing In Vitro in B16 Melanoma and
P388 Leukemia and in Inhibition Assays Against
293 Processive Tefomerase
The biological effects of the dimers were compared with those of the
monomeric 5-FUdR on P388 leukemia and B16 melanoma cell lines and in
inhibition
36
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WO 99145935 PCT/US99/05360
assays against 293 processive telomerase. Telomerase is a DNA-processive
enzyme that is not expressed in normal somatic cells but generally only in
germ-line
cells and fetal cells. In many types of cancer cells, enzyme activity is
reactivated,
and others, telomerase inhibitors can therefore serve as a valuable new class
of
antineoplastic agents. The results are shown in Table 2.
Table 1 - Inhibition of Tumor Cell Growth and Telomerase Activity by 5-FUdR
Dinucleoside Monophosphates
[Compound Growth Telomerase
Inhibition Inhibition
IC nM
P388 B16 (Mean+SEM)
~at1 mN
[i-D FUDR 2.8 28 0
a-L, ~i-D Dimer 0.41 2.45 84+11
a-L FUDR NT 389 500 NT
NT = Not Tested
The results indicate that the prototype dimers inhibit the growth of
murine-cultured leukemic L1210 and melanoma B16 cells with great potency (some
ICS values of less than 1 nM.) The ICS values are several times more potent
than
FUdR: These results are unexpected and thus these compounds aye truly unique. -
--
The preliminary results of the telomerase inhibition are also intriguing. The
a-L, [i-D dimer inhibited the enzyme by 84% compared to control.
These data indicate that dimers containing an L-sugar have extremely
interesting biological proi:lles and represent a novel class of potent
antineoplastic
agents. The activity profile of the L-dimers is different from that of the
parent
monomeric drug (3-D-5FUdR.
The biological effects of the dimers were compared with those of the
monomeric 5-FUdR on P388 leukemia and B16 melanoma cell lines. The results
37
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WO 99/45935 PCTNS99I05360
are shown in Table 2.
Table 2 - In Vitro Testing Data
IC50
CODE # I COMPOUND ~ P388 I B16
L-102 ~-D-FUdR, a-L-FUdR 0.71 3.0
L-103 a-L-FUdR, ~-D-FUdR 0.57 2.45
L-107 a-L-dU, ~i-D-FUdR 7.0 219
L-108 a-L-FUdR, a-L-FUdR 22,200 52,200
L-109 ~3-L-FUDR, (3-L-FUdR5860 45,900
L-110 S-L-FUdR, p-D-FUdR 2.0 6.3
L-111 a-L-dC, ~i-D-FUdR 0.7 5.0
-- [3-D-FUdR 2.8 28]
These data indicate that the dinucleoside monophosphate compounds
containing (3-D-SFUdR in conjunction with a-L or ~i-L-nucleosides show
superior in
vitro activities against murine P388 leukemia and B16 melanoma cell lines, as
is
evidenced by lower ICS values. This indicates that such nucleoside dimers may
indeed be acting by mechanisms different from those of 5-FUdR or are
metabolized
andlor transported differently from 5-FUdR.
Example 6
_ _._ . _. . _...... _ ._. . _ _. .. _. ...__ _ . ,.. _ . _ . _ petermi nation
of Thyrmid~,rl ate-8vnthase Activit~r
And its Inhibition In Intact X1210 Leukemia Cells !n Vitro
Thymidylate synthase is one suspected site of action of the compounds.
Hence, the activity of selected compounds of the present invention, measured
on
thymidylate synthase activity measured in vitro, is a reliable indicium of the
behavior
of these compounds in in vivo systems.
Mouse leukemia L1210 cells are harvested from the cell culture flasks and
the cell concentration is determined. The cells are then resuspended in the
desired
amount of the medium to give a stock concentration 5x10' ceIIsImL. Series of
the
38
CA 02322494 2000-09-07
WO 99/45935 PCT/US99/05360
dilution of the stock solution of the compounds to be tested are prepared
(concentrations are ranged from 10g M to 10'~ M). The solution of the compound
to
be tested in the desired concentration is pipetted into a microcentrifuge tube
and
incubated at 37°C using a shaking water bath. The reaction is started
by addition of
[5 3H]-2'-deoxycytidine (10 NL, concentration of the stock solution -10~ M)
after a
30 or 60 min. preincubation with 80 NL of the cell suspension and allowed to
proceed for 30 min. in a shaking water bath at 37°C. The reaction is
terminated by
adding 100 NL of the 10°~ charcoal in 4°~ HC104. The tubes are
vigorously stirred
by vortexing and then centrifuged for 10 min. in a Beckman Microfuge. The
radioactivity of a 100 NL of supernatant fraction from each tube is counted in
a
Packard Tri-Carb (model 2450 or 3255) liquid scintillation spectrometer using
a
toluene based scintillation mixture. The release of tritium is expressed as a
percentage of the total amount of radioactivity added. ICS values determined
from
dose response curves represent the concentration of inhibitors required for
50%
inhibition of the release of tritium. Table 3 below shows the results of the
analysis of
tritium release and determination of the ICS.
TABLE 3
Inhibition
Sample of Tritium
Code Release,
.. . . .~... . . IC . M . . _ . ,_.. _....
. ... ..
5FUdR 0.035
L-102 0.035
L-103 0.035
L-107 > 100
L-108 > 100
L-109 > 100
L-110 6
L-111 NIT
39
CA 02322494 2000-09-07
WO 99145935 PCT/US99/05360
Example 7
in Vivo Testing of Dimers in
P388 Leukemia B96 Melanoma
A. EXPERIMENTAL
1. P388 Leukemia
B6D2F1 mice received i.p. inocula of P388 murine leukemia cells prepared
by removing ascites fluid containing P388 cells from tumored B6D2F1 mice,
centrifuging the cells, and then resuspending the leukemia cells in saline.
Mice
received 1 x 108 P388 cells i.p. on day 0. On day 1, tumored mice were treated
with
the dimers or vehicle control. The route of drug administration was i.p. and
the
schedule selected was daily x 5. The maximum tolerated doses (MTD) was 200
mglkg for each dimer and was determined in initial dose experiments in
non-tumored mice. In the actual experiments, L-103 was given at 100 mglkg and
50
mglkg.
2. B16 Melanoma
B6D2F1 mice received i.p. inocula of B16 murine melanoma brei prepared
from B16 tumors growing s.c. in mice (day 0). On day 1, tumored mice were
treated
with the dimers or vehicle control. The route of drug administration was i.p.
and the
schedule selected was daily x 5. The maximum tolerated doses (MTD) was 200
mglkg for each dimer and was determined in initial dose experiments in
-- w w-- -- - - won-tumored mice: In the-actual experiments, L-103 was given
at 100 mg/kg and 50
mglkg.
3. Survival Standard .
The mean survival times of all groups were calculated, and results are
expressed as mean survival of treated mice I mean survival of control mice
(TIC) x
100°~. A TIC value of 150 means that the mice in the treated group
lived 50%
longer than those of the control group; this is sometimes referred to as the
increase
in life span, or ILS value.
In the P388 studies, mice that survive for 30 days are considered long term
CA 02322494 2000-09-07
WO 99145935 PCTIUS99/05360
survivors or cures while in B16; mice that survive for 60 days are considered
long
term survivors or cures. The universally accepted cut-off for activity in both
models,
which has been used for years by the NCI, is TIC = 125. Conventional use of
B16
and P388 over the years has set the following levels of activity: TlC < 125,
no
activity; TIC = 125-150, weak activity; TlC = 150-200, modest activity; TIC =
200-300, high activity; TIC > 300, with long term survivors; excellent,
curative
activity.
B. RESULTS
1. P388 Leukemia
L-103 demonstrated modest activity in the P388 leukemia in mice at all doses
tested (Table 5). L-103 gave i.p. daily x 5 at doses of 100 mglkg and 50 mg/kg
resulted in TIC values of 149 and 144 respectively. Fluorodeoxyuridine (FUdR)
was
used as the positive drug control in this study; FUdR produced a TIC =164 in
the
P388 test (Table 4). All agents were well-tolerated in this experiment; there
was
little or no body weight loss and no toxic deaths were recorded.
TABLE 4
L-103 vs. Murine P388 Leukemia
Weight
Change
I Group ~ pose .. _ . ~.._~~Dav ~7) . T/C _... . . . _. _. ._ . . _ ._. ..
Control (10) 0.9~ Saline +9.1 ~ 100
L-103 (10) 100 mg/kg +1.5% 149
L-103 (10) 50 mg/kg -1.6% 144
FUdR (10) 100 mg/kg -2.7% 164
2. B16 Melanoma
L-103 demonstrated modest activity against B16 melanoma implanted in mice
(Table 6). L-103 (i.p:; daily x 5) gave TIC values of 139 and 134
respectively. The
41
CA 02322494 2000-09-07
WO 99/45935 PCTNS99/05360
positive control drug FUdR resulted in modest efficacy in the B16 test; a TIC
value
of 135 was obtained (Table 5). All agents were well-tolerated, with little or
no weight
loss; no drug-related deaths occurred.
i;
TABLE 5
L-1 D3 vs. Murine B16 Leukemia)
Weight
Change
n Dose (Day 7) TIC
Control (10) 0.9~ Saline +4.9~ 100
L-103 (10) 100 mglkg +2.3~ 139
L-103 (10) 50 mglkg +6.3% 134
FUdR ( 10) 100 mg/kg -0.5% 1
L-103 demonstrated modest activity against both the P388 and B16
experimental murine tumors at the two doses tested. L-103 was approximately as
active as the positive control drug FUdR in the B16 test, and was somewhat
less
active than FUdR in the P388 test.
From the foregoing, the significance of L-sugar-based a- and (3-enantiomeric
nucleosides, nucleotides and their analogues as versatile, highly effective
chemotherapeutic agents is apparent. Our results on derivatives of 5-FUdR show
w w ~ ww y -~-that-L=5-FlidR-containing isomers are less toxic than those
containing (3-D-SFUdR,
perhaps because they are not phosphorylated or transported as the latter. We
have
found that dimeric derivatives designed and prepared from a-L-SFUdR show very
potent activity against P388 leukemia cells and B16 melanoma cell fines,
exceeding
that of ~i-D-SFUdR. They appear to have unusual mechanisms of action,
including
inhibition of telomerase.
Example 8
!n Vivo Activity of Nucleoside Analogs
42
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WO 99/45935 PCT/US99/05360
A. In Vitro Culture of the Malarial Parasite
P. falciparum, FCQ27, was maintained in culture using the techniques
described by Trager & Jensen (W. Traqer and J.B. Jensen, Science, 193 673-675
(1976)). Cultures containing 2% hematocrit suspensions of parasitized human
type
O+ erythrocytes in RPMI 1640 medium, supplemented with 25 mM HEPES-KOH, pH
7.2, 25 mM NaHC03 and 10% human type 0+ serum (vlv) are maintained in
modular incubator chambers at 37°C in a gas mixture of 5°~ 02,
5°~ C02 and 90°~
N2. The isolate of P. falciparum used in these experiments was FCQ27,
routinely
maintained in synchronized or asynchronous in vitro cultures at low
hematocrit.
B. In Vitro Toxicity against P. falciparum
The potential toxicity of nucleoside analogues against P. faiciparum in
culture
was tested in microtitre plates over the range of drug concentrations for 24
hours.
The procedures for monitoring parasite viability is well established (A.M.
Gero, H.V.
Scott, W.J. O'Sullivan and R.I. Christopherson, Mol. Biochem. Parasitol. 34,
87-89
(1989)) and is based on radiolabelled hypoxanthine or isoleucine
incorporation. The
incorporation of [G-3H]hypoxanthine into the nucleic acids of P. falciparum
was
used to assess the viability of the parasite in vitro. Microculture plates
were
prepared with each well containing 225NI of a 2% hematocrit culture of
asynchronous parasited erythrocytes (1 % parasitized cells). Each plate,
containing
varying concentrations of the drug to be studied (up to 200 NM anal
concentration
for initial screen), was incubated for 24 h at 37°C in a gas mixture of
5% 02, 5%
_ _ C02 .a._nd. 90%. N2,. at which point [G-3H] hypoxa_nthine.was added ao
aa_c~_well and _ . ~__._.
the incubation continued under identical conditions for a further 18-20 h. The
control
infected cells (i.e. without drug), routinely reached a parasitemia of 6-
8°~ before
harvesting. Expediency was aided by 96-well plate counter using lactate
dehydrogenase for the drug susceptibility assay (L. K. Basco, F. Marguet, M.T.
Makler and J. Lebbras, Exp. Parasitol. 80, 260-271 (1995); M.T. Makler and
D.J.
Hinrichs; Am. J. Trop. Med. Hyg. 48, 205-210 (1993)). This assay gave the
identical
results to the hypoxanthine technique. In addition, for each experiment,
microscopic
counting of Giemsa stained thin slides was used as a control.
43
CA 02322494 2000-09-07
WO 99/45935 PCT/US99/05360
C. Transport and Metabolism in P. falciparum infected erythrocytes
The metabolism of the L-nucleoside conjugates was studied by HPLC
analysis. The primary aim was to determine their ability to be catabolized by
parasite purine salvage enzymes. Some effect on the purine metabolic pools was
also observed.
For each HPLC determination 200 NL of packed cells of 80-90% trophozoite
infected cells were used. These were isolated from in vitro cultures by
synchronization of the parasites in in vitro cultures using sterile D-sorbitol
(L.
Lambros and J.P. Vanderberg, Parasitol. 65, 418-420 (1979)) followed by
separation of the trophozoites from non infected erythrocytes by Percoll
gradients as
described previously (A.M. Gero, H.V. Scott, W.J. O'Sullivan and R.I.
Christopherson, Mol. Biochem. Parasitol. 34, 87-89 (1989)). Trophozoites were
incubated at 37°C for 2 hours with each compound to be tested.
Compounds were
incubated with both whole infected cells as whole and lyzed uninfected normal
erythrocytes to determine:
a) entry to the cell (whether they were transported);
b) the metabolic effect within the cell (was the compound metabolized);
c) the capacity of broken or lyzed cells to catabolize the compound which may
not
be able to enter the unbroken cell (i.e. if the compound was transported into
the cell,
would it be metabolized to the active form).
Drug incubation was terminated by centrifugation through silicon oil using the
method of Upston and Gero (J.M. Upston and A.M. Gero, Biochem. Biophys. Acta.
1236, 249-258 (1995)). This procedure separated intact trophozoites from
extracellular non-transported drug solution.
The metabolism of nucleosides with potential chemotherapeutic activity was
assessed by the analysis of cytoplasmic samples by reverse phase ion-pair high
performance liquid chromatography (R.S. Toguzov, Y.V. Tikhonov, A.M. Pimenov,
V.
Prokudin, Journal of Chromatography, 1. Biomedical Appl. 434, 447-453 (1988)).
Nucleotides, nucleosides and bases were separated by this HPLC method.
The transport and metabolism of purine nucleosides differ considerably
between the normal human erythrocyte and human erythrocytes which have been
44
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infected with Plasmodium falciparum. The malaria parasite is unable to
synthesize
purines de novo and so therefore must rely on salvage pathways to obtain
purines it
requires for growth and division (L.W. Scheibel & T.W. Shennan, In: Malaria:
Principles and practice of Malariology (W.H. Wemsdorfer & I. McGregor, Eds.)
V. 1,
234-242 (1988)). Additionally, normal human erythrocytes do not contain
significant
levels of pyrimidine nucleotides (E. Szabodos & R.I. Christopherson, Biochem.
Edu.
19, 90-94 (1991 )), and the parasite is unable to obtain pyrimidine bases by
salvage
pathways and again has to rely on de novo synthesis (L.W. Scheibel & T.W.
Shennan, In: Malaria: Principles and practice of Malariology (W.H. Wernsdorter
& I.
McGregor, Eds.) V. 1, 234-242 (1988)). These modifications to the metabolic
pathways of the infected erythrocytes, along with modifications of their
transport
system, represent significant variations from normal erythrocytes and may
present
an opportunity for the use of selectively toxic compounds against the
parasites.
Nucleosides have attracted researchers as potential therapeutic agents.
Naturally occurring nucleosides are usually in the ~i-D configuration.
Therefore most
of nucleoside analogues designed for the treatment of cancer, viral and
parasitic
diseases have been synthesized in this stereochemical configuration. Recent
discoveries in our laboratories at the University of Georgia, the University
of Iowa
and at Yale University, as well as at universities in France and Italy, have
confirmed
that most L-nucleosides exhibit low toxicity because normal cells do not
utilize them
for building RNA or DNA and don't metabolize them.
Recently, Dr. Gero and her coworkers discovered that the nonphysiological
_ .. _ . _ . _ ~i-L-adenosine_.can_be. selectively transported into an
erythrocyte infected with P.
falciparum (A.M. Gero & J.M. Upston, In: Pyrine & Pyrimidine Metabolism in Man
VIII
(A. Sahota & M. Taylor, Eds) Plenum Press, NY, 495-498 (1995). Normal
erythrocytes and other cell types are completely impermeable to this compound.
During the Phase I study, we used this unique ability of the non-natural
nucleoside analogues for selective transport to the malaria infected cells to
create a
novel synthetic L-nucleoside based class of non-toxic antimalarial agents. Our
working hypothesis was based on the design and biological evaluation of novel
chemical entities which would consist of both 5-fluorodeoxyuridine (FUdR), a
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CA 02322494 2000-09-07
WO 99/45935 PCT/US99I05360
inhibitor of thymidylate synthase, and an L-nucleoside or its derivatives. The
number
of "dimers" consisting of a-or ~i-L isomeric modification of physiological
nucleosides
or their derivatives was conjugated with FUdR by phosphate or pro-phosphate
linkage. Along with anticancer activity, FUdR has a potential as an
antimalarial
agent (S.A. Queen, D.L. Vander Jagt & P. Reyer, Antimicrobial Agents &
Chemotherapy. 34, 1393-1398 (1990). Unfortunately, FUdR's toxicity limits its
use.
In theory, combining FUdR with an L-nucleoside unit would result in an entity
that
could selectively transport an active component to infected cells while having
no
effect on normal cells.
During Phase I research a total of 42 L-nucleoside analogs were screened in
an in vitro assay against P. falciparum. These compounds are shown in Figure
7.
From these forty-two compounds, 31 were available for screening from Lipitek
Intemationai's library and 11 were specifically synthesized for the purpose of
this
project. The detailed synthesis of 11 L-nucleoside conjugates is described in
the
Methods and Procedures. They were prepared in 100 mg scale and were fully
characterized by analytical methods (NMR, HPLC, mass spectra, TLC). The
forty-two compounds tested were representative of L-nucleoside monomers or 4
different types of L-nucleosides conjugates. The conjugates tested were: a)
dinucleoside phosphates, b) dinucleoside phosphorothioates, c) SATE
derivatives of
L-nucleosides, and d) L-nucleoside conjugates of nitrobenzylthionosine
(NBMPR). It
should be emphasized that even more diversification resulted from utilizing
characteristic to nucleosides 3' to 5' versus 5' to 3' phosphodiester linkages
as well
as variations of purines and pyrimidines in both.parts.of_the dimers, _ _ ~_.
_ , __ _ , . _ ,
The biological screen involved evaluation of the compounds against the
protozoan P. falciparum in in vitro culture. The range of drug concentrations
was
used independently by two assays. One, radiolabelled hypoxanthine
incorporation
into the nucleic acid of P. falciparum, and the other, more expedient assay, a
96-well plate susceptibility assay using lactate dehydrogenase. Both assays
gave
identical results. In addition, microscopic counting of Giemsa stained thin
slides was
used as a control. The results of the biological assays are presented in Table
1.
Examples of experimental curves are attached as Appendix 2. The biological
tests
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WO 99/45935 PCTlUS99105360
were done at several concentrations. The highest concentration was 200 pM, the
compounds were considered active at concentrations less than 40pM.
Discussions
In vitro activity
A careful analysis of the data presented in Table 6 (below) indicates that
nine
(9) analogs from 42 screened had IC50 less than 40NM (for the structure of the
tested compounds see Figure 14). The most active representative of
dinucleoside
phosphates were L-101, L-103, L-110, L-111, L-113, L-133 and L-138.
Table 6. Results of the in vitro testing.
. N CODE COMPOUND IC , M
1. L-101 -D -FUdR 15
2. L-103 a-L, D FUdR 20
3. L-103 thio a-L, -D -FUdR, S=P-O' >200
4. L-108 L -FUdR >200
5. L-110 -L, 0 -FUdR 20
8. L-111 a-L-dC, -D-FUdR 38
7. L-113 L-dC, D-FUdR 17
8. L-117 L-dU, -D-FUdR 35
8. L-117 thio -L-dU, D-FUdR S=P-O- >200
10. L-125 a-L-dA, D-FUdR 60
11. L-128 D, -D-FUdR S=P-O- 1.5
12. L-133 -L-dG, -D-FUdR 14
13. L-138 L-dA -D-FUdR 5
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WO 99/45935 PCT/US99/05360
14. L-138-thio L-dA D-FUdR S=P-O' 100
15. L-144 -D-FUdR, L-A 140
16. L-145 L-dU, NBMPR >200
17. L-14g a-L dT, NBMPR >200
18. L-147 NBMPR, MP Solubility
roblem
19. NBMPR NBMPR 100
20. GCI 1007 a-L-a hrofuranos i-5-fluorouracil>200
21. GCI 1018 a-L-arabinofuranos ~I adenine>200
22. GC11027 a-L-FUdR >200
23. GCI1030 L-G >200
24. GCI1032 L-A >200
25. GCI1033 L-I >200
2g. GCI1034 L-mercy o-G >200
27. GCI1038 -L-Da >200
28. GCI1037 -L-dl >200
29. GCI108g a-L-A >200
30. GCI10g9 a-L-dA >200
31. GCI1070 -L-dG >200
32. GCI1077 L-ddA >200
2 0 33. GCI 1079 a-L-ddA >200
34. GCI 1085 N-meth I- -L-A >200
35. GCI 107___ g_thio- =L urine -._ .. .. _.. _. >200
_ .. __ . . . .
3g. B01 D-FUdR 3'SATE 100
37. B02 D-FUdR 5'SATE g0
2 5 38. B03 D-FUdR 3'-5'SATE g
39. B04 -L-FUdR 5'SATE 200
40. B05 Ei-L-FUdR 3'-5' SATE Solubility
roblem
41. 80g a-L-FUdR 5'SATE 150
Q8
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WO 99145935 PC'fIUS99/05360
42. B07 a-L-FUdR 3'-5'SATE Solubility
roblem
The 14 L-nucleoside monomers in a- and Vii- forms and even
a-L-FUdR showed no activity against P. falciparum. Because of that,
further research on monomers was halted (see Table 6).
s The dimer containing only the "non-natural" isomeric form of
nucleoside (L-109) did not exhibit any activity.
Careful analysis of the data in Table 6 indicates that ~3-D-isomer of
FUdR is the active component of the dimer molecules. The position of the
active component in the dimer is important. The ~i-D-FUdR needs to be
~o connected to the 3'-OH end of the L-nucleoside through a phosphodiester
linkage to its 5'-OH. Compounds which are linked through 3'-OH of FUdR
are much less active (see Table 7). This indicates that the substitution
pattern of ~i-D-FUdR is critical for the activity of the dimers and most
probably the mechanism involves thymidylate synthase inhibition. It is well
i5 known that TS inhibitors of FUdR have very rigid structural requirements
and do not allow for any substitution at the 3' end.
49
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Table 7. The activity of the L-nucleoside containing dimers
versus position of FUdR linkage
5' 3'
Compound ICS, Compound ICS,
M M
L-101 ([[3-D]2-FUdR) 15 L-144 (~-D-FUdR, 140
13-L-
A
L-103 a-L-FJdR, -D-FUdR 20
L-110 L-FUdR -D-FUdR 20
L-111 a-L-dC, D-FUdR 38
L-113 L-dC, D-FUdR 17
L-125 a-L-dA D-FUdR 60
L-133 L-dC D-FUdR 14
L-138 (~-L-dA, (3-D-FUdR)5
Table 8. The activity of the dimers versus chemical configuration
of the L-nucleoside
a-L -L
Compound ICS, Compound ICS,
M M
L-103 -L-FUdR ~ 20 L-110 -L-FUdR 20
L-111 a-L-dC 38 L-113 -L-dC 17
L-125 a-L-dA 60 L-138 -L-dA 5
2 o In the case of purine nucleoside, the attachment of the a-L
nucleoside to ~i-D-FUdR monomer reduces the dimer activity in
comparison with dimers containing the ~i-L unit (see Table 8, L-125 and
CA 02322494 2000-09-07
WO 99!45935 PCT/US99/05360
L-138). In the case of pyrimidine nucleosides there is no obvious
difference in the activity (L-103 & L-110, L-111 & L-113).
Table 9. The activity of the dimers versus nature of the linkage
between two nucleosides analogs
Phosphate "bridge" ICS,, Phosphorothioate "bridge"ICS,
O=P-O M S=P-O M
L-101 D -FUdR 15 L-128 -D FUdR 1.5
L-103 a-L, -D -FUdR 20 L-103 a-L, D -FUdR >200
L-117 L-dU, -D-FUdR 35 L-117 -L-dU, D-FUdR >200
L-138 -L-da, D-FUdR 5 L-138 L-dA, D-FUdR 100
The different activity of the dimers is dependent on the structure of the
second nucleoside.
The following plausible pathways for metabolic activation andlor
1 s mode of action of the dimer. molecules tested are that:
(1 ) Dimer may act as a new chemical entity without hydrolysis of the
phosphate or pro-phosphate bond between the two monomeric
units;
(2) Hydrolysis to L-nucleoside and FUdR nucleotide may occur, in
2 0 ~ which case the dimer is a prodrug: The L-nucleoside is used for
protection and to increase the bioavailability of (3-D-FUdR
monophosphate.
It is also important to note that hydrolysis can take place intracellularly as
well as outside the cell.
2 s In the last decade, monumental efforts have been directed toward
the synthesis of oligonucleotide analogs with altered phosphodiester
linkage. The goal was to improve the stability of duplex and triplex
51
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formation, to improve the cellular uptake and to decrease the rate of
degradation of oligonucleotides by endo and exo nucleases which cleave
the phosphodiester linkage. We selected one such chemical modification
for our study. As a consequence, several dimers with phosphorothioate
linkage between two nucleosides were synthesized and tested. The
phosphorothioate comprises a sulfur-for-oxygen substitution at
phosphorus of the phosphodiester linkage (for the structure of the
corresponding dimers see Appendix 1 ). It has been shown (M.
Matsukura, K. Shinozuka, G. Zon, H. Mitsuya, M. Reitz, J.S. Cohen, L.M.
to Neckers, Proc. Natl. Acad. Sci. USA. 84, 7706 (1987)) that the S
homologues are more resistant to cellular nucleases and are readily taken
up by cells. Several oligonucleotides of this type are currently in clinical
studies (ISIS Pharmaceuticals and others).
Table 10. The activity of (3-D-FUdR and some possible products of
its metabolism
COMPOUND IC , M
-D-FUdR 34
-D-5'-FUdRmP 50
SFUracil 6
2 o The replacement of the phosphate linkage by the phosphorothioate bond
in the dimer, containing two (3-D-FUdR units, increases the activity of the
compounds by a factor of 10 (see Table 9, data for L-101 & L-128). The
only active phosphorothioate analog appears to be compound L-128. The
activity of L-128 is greater in comparison with all possible products of
2 5 hydrolysis (see Table 10). Moreover, L-128 was the most active
52
CA 02322494 2000-09-07
WO 99!45935 PCTNS99105360
compound tested.
Table 11. The activity of the SATE derivatives of FUdR
Compound ID , M
-D-FUdR 34
s -D-FUdR mono hos hate 50
B01 -D-FUdR 3' SATE 100
B02 -D-FUdR 5' SATE 60
B03 -D-FUdR 3',5' SATE 6
-L-FUdR >200~
-L-FUdR mono hos hate NIA
B04 -L-FUdR 5' SATE 200
B05 -L-FUdR 3',5' SATE Solubili roblem
a-L-FUdR >200
a-L-FUdR mono hos hate NIA
B06 a-L-FUdR 5' SATE 150
B07 a-L-FUdR 3',5' SATE Solubili roblem
The introduction of the phosphorothioate bond into molecules of dimers
containing "non-natural" nucleoside isomer was not successful: the
activity of the compounds was reduced dramatically (see Table 9, data for
2 o L-103, L-117 & L-138). As was discussed before, one of the possible
mechanisms of dimer action is the participation in the metabolic pathways
of the whole non-hydrolyzed molecule. In this case the increasing of the
dimer stability by the introduction of the phosphorothioate linkage results
in the increasing of the activity of L-109 . For the dimers containing the
53
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WO 99/45935 PCTNS99/05360
"non-natural" isomeric modification of the nucleoside the metabolism of
whole non-hydrolyzed molecule is probably impossible.
It is well established that most of the nucleoside analogs are
dependent in kinase-mediated activation to generate the bioactive
nucleotide and ultimately, the nucleoside triphosphate (C. Periqaud, G.
Gosselin, J.L. Imbach, Nucleoside Nucleotides. 11, 903 (1992)).
Activation takes place in the cytosol after nucleoside uptake and involves
three successive viral andlor cellar kinases, the first one being highly
specific (M.C. Statues, Y.C. Cheng, J. Biol. Chem. 262, 988 (1987)). One
io possibility to improve the efficiency of the nucleoside analog is a
therapeutic agent could be to bypass the phosphorylation step.
Unfortunately, nucleoside monophosphates themselves, due to their polar
nature, are not able to cross the cell membrane efficiently (K.C. Leibman,
C.J. Heidelberg, J. Biol. Chem. 216, 823 (1995)). Hence the idea of
i5 temporarily masking or reducing the phosphate negative charges with
neutral substituents, thereby forming more lipophilic derivatives which
would be expected to revert back to the nucleoside mono-phosphate once
inside the cell.
One of the possible structural modifications for the kinase bypass
2 0 is the use of the bis-S-acetylthioethyl (SATE) derivatives pioneered by
J.-L. Imbach (I. Lefebvre, C. Perigaud, A. Pompon, A.M. Auberth, J.L.
Grardet, A. Kirn, G. Gosselin and J.L. Imbach, J. Med. Chem. 38,
3941-3950 (1995); C. Perigaud, G. Gosselin, I. Lefebvre, J.L. Girardet, S.
Benzaria, I. Barber and J.L. Imbach, Bio. Org. Med. Chem. Lett. 3,
2s 2521-2526 (1993)). Several SATE derivatives of FUdR isomers were
synthesized and tested their in vitro activity against P. falciparum. The
obtained results are listed in Table 11.
It should be emphasized that all of the SATE derivitization was
pertormed on monomers of FUdR varying the conformation. Thus
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derivatives of a- and ~i D and L-FUdR were prepared. Three types of
SATE analogs were produced, a) decorated at 5' of the nucleoside, b)
decorated at 3' of the nucleoside, and c) decorated at both 3' and 5' of the
nucleoside resulting in disubstitution. Fifteen L-nucleoside dimers (L-101,
L-103, L-103A, L-107, L-110, L-111, L-112, L-114, L-117, L-120, L-122,
L-124, L-125, L-133 & L-138) from Lipitek's library were submitted for in
vitro screen to the U.S. Army Antimalarial Test Program (for the structures
of the compounds, see Figure 7). The compounds have been tested for
their activity against two P. falciparum strains: D6 (chioroquin
1 o non-resistant) and W2 (chloroquin resistant). Seven (7) of the tested
compounds exhibited activities below 40 NM against both strains of P.
falciparum. The most active dimers were L-101, L-110, L-112, L-117,
L-133 & L-138.
The transport and metabolism study
i5 It has been established that transport and uptake in parasite
invaded cells is different from that of normal blood erythrocytes (A.M.
Gero & A.M. Wood, In: Pyrine & Pyrimidine Methabolism in Man VII, Part
A. (R.A. Harkness et al., Eds) Plenum Press, NY, 169-172 (1991 )).
Invasion by the malaria parasite comprises the cell membrane, allowing
2 o penetration of unnatural substances of various size and shape, whereas
normal cells are very selective in uptake. It was shown that L-nucleosides
and their derivatives easily penetrate invaded cells, while they have a
very slow rate of uptake into normal cells, if they enter at all. In order to
obtain preliminary data on transport, uptake and metabolism, the HPLC
2 s method was used to analyze the following 10 Lipitek compounds: L-101,
L-103, L-109, L-111, L-117, L-133, L-138, GCI 1007, GCI 1027, GCI
1069.
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HPLC retention times for standard compounds purchased from Sigma is
presented in Table 12.
Table 12. HPLC Retention Time for the Standard Compounds
Com ound R.t., min Com ound R.t., min
Nucleobase Nucleotide
Adenine 4.93 5'AMP 16.56
Guanine 5.38 3'AMP 19.46
Uracil 4.37 5'ADP 23.89
H oxanthine 5.59 5' /ATP 30.09
s o Nucleoside 5'GMP 15.24
Adenosine 12.47 5'-GDP 23.13
Guanosine 9.96 5'GTP 29.01
Inosine 8.88 5'-UMP 14.85
Th midine 12.36 FUdRMP 16.40
2'-Deo adenosine 12.29
~
2'-Deo uanosine 11.03
2'-Deo uridine 7.41
2'-Deo c idine 8.25
FUdR 10.21
Table 13. HPLC Retention Time of Lipitek's Compounds
(Trophozoite Incubations)
Compound Neat InjectionUnmetabolized Metabolic
Peak Product
L-138 18.17 18.89 18.26 16.93 15.73
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L-133 16.72 17.03 15.65
L-101 19.05 19.01 10.62
L-103 18. 31 18.25 -
L-117 17.41 17.61 -
L-111 15.69 15.69 -
L-109 16.36 - -
GC I 1027 10.21 10.61 -
GCI 1007 12.61 12.42 -
GC1 1069 12.29 - 9.65
z o These compounds were chosen for the identification of possible
metabolites. In this experiment the compounds were incubated with both
whole infected cells, and with whole and lysed uninfected cells, followed
by the separation of unreacted compound and HPLC analysis.
The results are presented in Table 13:
Column 1 shows the retention times of the original compound (not
incubated with any cells).
Column 2 shows the retention times of the original compound remaining
after incubation with whole parasite infected cell.
Column 3 shows the metabolic products i.e. new peaks due to conversion
w -- °-~ ow-w- -- of the original compound or alteration in the natural
purine or pyrimidine
profile of the infected cell.
All nucleosides monophosphate dimers containing (3-D-FUdR unit
in combination with any L-nucleosides (L-101, L-103, L-111, L-117, L-133,
and L-138) as welt as tested L-nucleoside monomer analogs (GCI 1007,
GCI 1027 & GCI 1069) entered the infected cells. All these compounds
were toxic against P. falciparum. The L-109, combination of two L-dimers,
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WO 99/45935 PCT/US99/05360
could not enter the infected cell, and was also not toxic.
Compounds L-101, L-133 & L-138 appear to be metabolized by the
infected cells, each producing at least one new peak (see Table 13). It is
possible that L-138 and L-133 may be cleaved to a nucleotide and
nucleoside.
None of the above 10 compounds were found to enter normal
erythrocyte. Metabolism of any of the above compounds did not occur in
lysates of human erythrocytes or lymphocytes. So even if the compounds
were able to get into the normal cells, the normal cells cannot metabolize
1 o them into active ingredients. This underscores again the low toxicity and
the selectivity of the L-nucleoside conjugates disclosed and claimed in
this Application.
Example 9
Syrntheeis of N°-Benzoyrl-3'-deoxyr-~-D-adenosine
15 To a stirring solution of 3'-deoxyadenosine (2.0 g, 7.96 mmol) in
pyridine (80 ml) chilled in an ice bath, CISiMe3 (5.0 ml, 39.8 mmol) was
added dropwise and stirred for 30 minutes. Benzoyl chloride (3.7 ml,
31.84 mmol) was then added dropwise and the reaction mixture was
stirred at room temperature for two hours. This was cooled in an ice bath
2 o and water (16 ml) was added dropwise. 15 minutes later concentrated
NH40H (16 ml) was added to give a solution approximately 2M in
ammonia. After 30 minutes the solvent was evaporated and the residue
was dissolved in water and washed with ether. The water layer was
concentrated and the compound was crystallized from water as white solid
2 5 (2.32 g. 82%).
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Example 1 Q
Syrnthesis of Na-Benzo~rl-5'-O-i(di
p-methoxvtrityrll-3'-deoxyr- ~~i-D-adenosine
To a solution of compound N°-Benzoyl-3'-deoxy-(3-D-adenosine
s 1 (2.32 g, 6.53 mmol) in pyridine (100 ml) was added 4,4'-dimethoxytrityl
chloride (3.328, 9.79 mmol) and DMAP (0.24 g, 1.96 mmol) and stirred at
room temperature for 2 hours under argon. To complete the reaction,
additional DMTCI (0.5 g) was added and stirred for another 2 hours. The
reaction was quenched with the addition of MeOH (5 ml) and the solvent
to was evaporated. The residue was dissolved in EtOAc, washed with water,
NaHC03 and brine. After drying over Na2S04, the EtOAc layer was
evaporated and the crude compound was purified on a silica gel column
using 80°~ EtOAGCHCl3 as solvent to give pure compound 2 (4.338,
83°~)
as a white foam.
Example 11
Syrnthesis of N°-Benzoyi-2'-O-acetoxyr-~3-D-3'-deox~radenosine
To a solution of Ng-Benzoyl-5'-0-(di
p-methoxytrityl)-3'-deoxy-(3-D-adenosine (4.3 g, 6.58 mmol) in pyridine
(100 ml) acetic anhydride (1 ml, 9.87 mmol), and DMAP (0.08 g, 0.65
2 o mmol) was added and stirred at room temperature for 15 minutes. Then
the solvent was evaporated and the residue was dissolved in EtOAc,
washed with water, NaHC03, brine and dried over NAZS04. After the
evaporation of EtOAc, then the crude material was dissolved in 80°~
AcOH (50 ml) and stirred at room temperature for one hour. Then the
2 s solvent was evaporated and coevaporated with tolune and purified on a
silica gel column using 3-5% MeOHICHCl3 to give pure
Ng-Benzoyl-2'-O-acetoxy-~i-D-3-deoxyadenosine (2.11 g, 81 %) as a foam.
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Example 12
Synthesis of (3-L-dU. Cordyrcepin dimer yL-150
(3-L-dU (1.0 g, 4.38 mmol) was dissolved in dry pyridine (50 ml), to
this solution was added 4,4'-dimethoxytrityl chloride (1.78 g, 5.25 mmol)
and DMAP (0.1 g, 0.87 mmol). This was stirred under argon at room
temperature for 2 hours and quenched with MeOH (5 ml). The solvent
was evaporated, the residue was dissolved in EtOAc, washed with water,
NaHC03 and brine. After drying and evaporation of the solvent, the crude
material was purified on a silica gel column using 60-80% EtOAcICHCl3
io as solvent to give pure 5'-O Dimethoxytrityl-(3-L-2'-deoxyuridine (2.2 g,
94.8°~) as white foam.
Dimethoxytrityl-(3-L-2'-deoxyuridine (1.5 g, 2.83 mmol) was
dissolved in anhydrous dichloromethane (50 ml). N,
N-diisopropylethylamine (2.0 ml, 11.3 mmol) was added uner argon
i5 followed by 2'-cyanoethyl-N,N-diisopropylchlorophosphoramidite (0.82 ml,
3.68 mmol). The reaction was stirred for 30 minutes and the solvent was
evaporated. The residue was dissolved in 80°~ EtOAc/Et3N (75 ml) and
washed with water, NaHC03 and brine. The organic layer was
evaporated and purified on a short silica gel column using a mixture of
2 o EtOAc, CHZCIZ and ET3N (40:50:10) to give
5'-O-Dimethoxytrityl-(3-L-2'-deoxyuridine-3'-N, N-diisopropylcyanoethyl
phosphoramidite in quantitative yield.
5'-O-D i methoxytrityl-3'-[0-(2'-0-acetyl )-Ne-benzoyl-~i-D-3'-deoxy
adenosinyl]-2'-deoxy-~3-L-uridine cyanoethyl phosphite ester (7).
2 s To a solution of compound
5'-0-Dimethoxytrityl-(3-L-2'-deoxyuridine-3'-N, N-diisopropylcyanoethyl
phosphoramidite (2.83 mmol) in anhydrous acetonitrile (60 ml),
Ne-BenzoYl-2'-O-acetoxy-(3-D-3-deoxyadenosine (1.12 g, 2.83 mmol) in
acetonitrile (40 ml) was added and stirred for 10 minutes under argon. To
so
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this solution, sublimed 1 H-tetrazole (0.6 g, 8.5 mmol) was added and
stirred over night. The solvent was evaporated and the residue was
triturated with 70°~ EtOAclether and filtered. The filtrate was
evaporated
to give 5'-O-Dimethoxytrityl-3'-[O-(2'-0-acetyl)-Ne-benzoyl-~i-D-3'-deoxy
s adenosinyl]-2'-deoxy-~i-L-uridine cyanoethyl phosphite ester as a foam
and this was used in the next step without further purification.
The
5'-O-Dimethoxytrityl-3'-[O-(2'-O-acetyl)-Ne-benzoyl-~i-D-3'-deoxy
adenosinyl]-2'-deoxy-~i-L-uridine cyanoethyl phosphite ester was
io dissolved in THF (24 ml), pyridine (6 ml) and water (0.6 ml). Iodine
crystals (1.0 g) were added portion wise until the iodine color persists.
The reaction mixture was stirred for another 15 minutes and the excess
iodine was removed by the addition of saturated sodium thiosulfate. The
solvent was evaporated and the residue was dissolved in EtOAc and
15 washed with water, NaHC03 and brine. EtOAc layer was evaporated and
the residue was dissolved in 80°~ acetic acid/water solution (40 ml)
and
stirred for 1 hour. Then the solvent was evaporated and the crude
product was purified on a silica gel column using 8-15°r6 MeOHICHCl3 as
solvent to give pure
2 0 (2'-Acetoxy-NB-benzoyl-3'-deoxy-(3-D-adenosinyl)-~i-L-2'-deoxyuridinyl
cyanoethyl phosphate ester (0.75 g) as a foam.
The dimer
(2'-Acetoxy-Ne-benzoyl-3'-deoxy-~-D-adenosinyl)-~i-L-2'-deoxyuridinyl
cyanoethyl phosphate ester (0.75 g) was treated with ammoniun
25 hydroxide solution (100 ml) over night. The solvent was evaporated and
the residue was purified on DEAE Cellulose ion exchange column using
gradient of NH4HC03 buffer (0.05-0.2M). The pure fractions were
collected and lyophillized to give pure
3'-O-{3'-deoxy-(3-D-adenosinyl)-~i-L-2'-deoxyuridine (L-150){0.486 g) as
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white sOlld.
Example 13
Synthesis of (3-L-dA. Cordyrcepin dimer jL-151
s To a stirring solution of 2'-deoxy-(3-L-adenosine (2.05 g, 8.16 mmol)
in pyridine (75 ml) chilled in an ice bath, CISiMe3 (5.17 ml, 40.8 mmol)
was added dropwise and stirred for 30 minutes. Benzoyl chloride {4.7 ml,
40.8 mmol) was then added dropwise and the reaction mixture was stirred
at room temperature for two hours. This was cooled in an ice bath and
io water {15 ml) was added dropwise. 15 minutes later concentrated NH40H
(15 ml) was added to give a solution approximately 2 M in ammonia. After
30 minutes the solvent was evaporated and the residue was dissolved in
water and washed with ether. The water layer was concentrated and the
Ng-benzoyl-2'-deoxy-[3-L-adenosine was crystallized from water as white
15 solid (2.48 g, 85.8%).
To a solution of Ne-benzoyl-2'-deoxy-(3-L-adenosine (2.488, 6.98
mmol) in pyridine (100 ml) was added 4,4'-dimethoxy trityl chloride (3.55
g, 10.47 mmol) and DMAP (0.25 g, 2.09 mmol) and stirred at room
temperature for 2 hours under argon. To complete the reaction, additional
2 o DMTCI (1.3 g) was added and stirred for another 2 hours. The reaction
was quenched with the addition of MeOH (5 ml) and the solvent was
evaporated. The residue was dissolved in EtOAc, washed with water,
NaHC03 and brine. After drying over Na2S04, the EtOAc layer was
evaporated and the crude compound was purified on a silica gel column
25 using 3-5°~ MeOHICHCI, as solvent to give pure
Ng-benzoyl-5'-O-(di-p-methoxy trityl)-2'-deoxy-~i-L-adenosine (3.42 g,
74.5°~) as pale yellow foam.
Ng-benzoyl-5'-O-(di-p-methoxy trityl)-2'-deoxy-(3-L-adenosine (1.64
g, 2.5 mmol) was dissolved in anhydrous dichloromethane (50 ml).
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N,N-diisopropylethylamine (1.75 ml, 10.0 mmol) was added under argon
followed by 2'-cyanoethyl-N,N-diisopropylchlorophosphoramidite (0.8 ml,
3.25 mmol). The reaction was stirred for 30 minutes and the solvent was
evaporated. The residue was dissolved in 80% EtOAc/Et3N (75 ml) and
s washed with water, NaHC03 and brine. The organic layer was
evaporated and purified on a short silica gel column using a mixture of
EtOAc, CH2CL2 and Et3N (40:50:10) to give
NB-Benzoyl-5'-0-(d i methoxytrityl )-[3-L-2'-deoxyadenosine-3'-N, N-d i
isoprop
ylcyanoethyl phosphoramidite in quantitative yield.
i o To a solution of
Ng-Benzoyl-5'-O-(dimethoxytrityl)-~i-L-2'-deoxyadenosine-3'-N,N-diisoprop
ylcyanoethyl phosphoramidite (2.5 mmol) in anhydrous acetonitriie (60
ml), Ne-Benzoyl-2'-O-acetoxy-~i-D-3'-deoxyadenosine (0.94 g, 2.36 mmol)
in acetonitrile (40 ml) was added and stirred for 10 minutes under argon.
i5 To this solution, sublimed 1 H-tetrazole (0.5 g, 7.2 mmol) was added and
stirred over night. The solvent was evaporated and the residue was
triturated with 70°~ EtOAclether and filtered. The filtrate was
evaporated
to give
Ne-Benzoyl-5'-O-(dimethoxytrityl)-3'-[0-(2'-O-acetyl)-Ne-benzoyl-(3-D-3-de
20 oxy adenosinylj-2'-deoxy-[3-L-adenosine cyanoethyi phosphite ester as a
foam and this was used in the next step without further purification.
The dimer
Ng-Benzoyl-5'-O-(d i methoxytrityl )-3'-[0-(2'-0-acetyl )-Ng-benzoyl-[i-D-3-de
oxy adenosinyl]-2'-deoxy-[3-L-adenosine cyanoethyl phosphite was
25 dissolved in THF (24 ml), pyridine (6 ml) and water (0.6 ml). Iodine
crystals (0.63 g) were added portion wise until the iodine color persists.
The reaction mixture was stirred for another 15 minutes and the excess
iodine was removed by the addition of saturated sodium thiosulfate. The
solvent was evaporated and the residue was dissolved in EtOAc and
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washed with water, NaHC03 and brine. EtOAc layer was evaporated and
the residue was dissolved in 80°r6 acetic acidlwater solution (50 ml)
and
stirred for 1 hour. Then the solvent was evaporated and the crude
product was purified on a silica gel column using 5-10% MeOH/CHCl3 as
solvent to give pure
(2'-Acetoxy-Ng-benzoyl-3'-deoxy-~i-D-adenosinyl )-Ne-benzoyl-~i-L-2'-deoxy
adenosinyl cyanoethyl phosphate ester (0.97 g) as a foam.
The dimer
(2'-Acetoxy-Ne-benzoyl-3'-deoxy-~3-D-adenosinyl)-Ng-benzoyl-~i-L-2'-deoxy
io adenosinyl cyanoethyl phosphate ester (0.97 g) was treated with
ammonium hydroxide solution (100 ml) over night. The solvent was
evaporated and the residue was purified on DEAE Cellulose ion exchange
column using gradient of NH4HC03 buffer (0.05 - 0.2 M). The pure
fractions were collected and lyophillized to give pure compound
3'-O-(3'-deoxy-~3-D-adenosinyl)-~i-L-2'-deoxyadenosine (L-151 ) (0.55 g)
as white solid.
Example 14
Syrnthesis of a-L-dU. Cordyrcepin dimer (L-1521
a-L-dU (1.04 g, 4.5 mmol) was dissolved in dry pyridine (50 ml), to
2 o this solution was added 4, 4'-dimethoxytrityl chloride (2.4 g, 6.86 rnmol)
and DMAP (0.11 g, 0.91 mmol). This was stirred under argon at room
temperature for 2 hours and quenched with MeOH (5ml). The solvent was
evaporated, the residue was dissolved in EtOAc, washed with water,
NaHC03 and brine. After drying and evaporation of the solvent, the crude
2s material was purified on a silica gel column using 3-5°r6 MeOHICHCl3
as
solvent to give pure 5'-O-Dimethoxytrityl-a-L-2'-deoxyuridine (2.4 g.,
99°~)
as white foam.
5'-O-Dimethoxytrityl-a-L-2'-deoxyuridine (1.73 g, 3.26 mmol) was
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dissolved in anhydrous dichloromethane (30 ml), N,
N-diisopropylethylamine (2.3 ml, 13.04 mmol) was added under argon
followed by 2'-cyanoethyl-N,N-diisopropylchlorophosphoramidite (0.95 ml,
4.23 mmol). The reaction was stirred for 30 minutes and the solvent was
s evaporated. The residue was dissolved in 80°r6 EtOAc/Et3N (75 ml) and
washed with water, NaHC03 and brine. The organic layer was
evaporated and purified on a short silica gel column using a mixture of
EtOAc, CHZCL2 and Et3N (40:50:10) to give 5'-O-Dimethoxytrityl-
a-L-2'-deoxyuridine-3'-N, N-diisopropylcyanoethyl phosphoramidite (2.26
io g, 95%) as a foam.
To a solution of 5'-O-Dimethoxytrityl- a-L-2'-deoxyuridine-3'-N,
N-diisopropylcyanoethyl phosphoramidite (2.26 g, 3.09 mmoi) in
anhydrous acetonitrile (60 ml),
Ng-Benzoyl-2'-O-acetoxy-[3-D-3'-deoxyadenosine (1.35 g, 3.4 mmol) in
i5 acetonitrile (40 ml) was added and stirred for 10 minutes under argon. To
this solution, sublimed 1 H-tetrazole (0.65 g, 8.5 mmol) was added and
stirred over night. The solvent was evaporated and the residue was
triturated with 70% EtOAclether and filtered. The filtrate was evaporated
to give
20 5'-O-Dimethoxytrityl-3'-(O-(2'-O-acetyl)-Ng-benzoyl-[i-D-3'-deoxyadenosin
yl]-2'-deoxy-a-L-uridine cyanoethyl phosphite ester as a foam and this
was used in the next step without further purification.
The dimer
5'-O-Dimethoxytrityl-3'-[O-(2'-0-acetyl)-NB-benzoyl-[i-D-3'-deoxyadenosin
25 yl]-2'-deoxy-a-L-uridine cyanoethyl phosphite ester was dissolved in THF
(24 ml), pyridine (6 ml) and water (0.6 ml). Iodine crystals (0.7 g) were
added portion wise until the iodine color persists. The reaction mixture
was stirred for another 15 minutes and the excess iodine was removed by
the addition of saturated sodium thiosulfate. The solvent was evaporated
CA 02322494 2000-09-07
wo 99i4s~s PcTiusmos~o
and the residue was dissolved in EtOAc and washed with water, NaHC03
and brine. EtOAc layer was evaporated and the residue was dissolved in
80°~ acetic acidlwater solution (50 ml) and stirred for 1 hour. Then
the
solvent was evaporated and the crude product was purified on a silica gel
s column using 8-15% MeOHICHCl3 as solvent to give pure
{2'-Acetoxy-Ne-Benzoyl-3'-deooy-(3-D-adenosinyl}-a-L-2'-deoxyuridinyl
cyanoethyl phosphate ester (1.29 g) as a foam.
[3'0-(3'-deoxy-(3-D-adenosinyl)-a-L-2'-deoxyuridine (6) (L-152)]
The dimer
i o {2' Acetoxy-Ne-Benzoyl-3'-deoxy-(3-D-adenosinyl}-a-L-2'-deoxyuridinyl
cyanoethyl phosphate ester (1.29 g) was treated with ammonium
hydroxide solution (100 ml) over night, The solvent was evaporated and
the residue was purified on DEAE Cellulose ion exchange column using
gradient of NH4HC03 buffer (0.05-0.2M). The pure fractions were
15 collected and lyophillized to give pure
[3'-O]-{3'-deoxy-[3-D-adenosinyl)-a-L-2'-deoxyuridine (L-152) (0.856 g} as
white solid.
Example 15
S~~thesis of ~i-L-dC. Cordvcepin dimer i[L-153)
2o To a solution of [i-L-dCBz (1.7 g, 5.22 mmol) in pyridine (100
ml) was added 4, 4'-dimethoxy trityl chloride {2.65 g, 7.83 mmol) and
DMAP (0.13 g, 1.04 mmol) and stirred at room temperature for 2 hours
under argon. To complete the reaction, additional DMTCI (0.9 g) was
added and stirred for another 2 hours. The reaction was quenched with
25 the addition of MeOH (5 ml) and the solvent was evaporated. The residue
was dissolved in EtOAc, washed with water, NaHC03 and brine. After
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drying over Na2S04, the EtOAc layer was evaporated and the crude
compound was purified on a silica gel column using 3-5°~ MeOHICHCl3 as
solvent to give pure N4-Benzoyl-5'-O-(di-p-methoxy
trityl)-2'-deoxy-[i-L-cytidine (2.98 g, 90°~) as pale yellow foam.
[N''-Benzoyl-5'-O-(dimethoxytrityl)-(3-L-2'-deoxycytidine-3'-N,
N-diisopropylcyanoethyl phosphoramidite (3)]
N4-Benzoyl-5'-O-(di-p-methoxy trityl)-2'-deoxy-~i-L-cytidine (1.7 g,
2.68 mmol) was dissolved in anhydrous dichloromethane (50 ml), N,
N-diisopropylethylamine (1.9 ml, 10.72 mmol) was added under argon
io followed by 2'-cyanoethyl-N, N-diisopropyichlorophosphoramidite (0.85
ml, 3.5 mmol). The reaction was stirred for 30 minutes and the solvent
was evaporated. The residue was dissolved in 80% EtOAcIEt3N (75 ml)
and washed with water, NaHC03 and brine. The organic layer was
evaporated and purified on a short silica gel column using a mixture of
EtOAc, CH2C12 and Et3N (30:60:10) to give
N4-Benzoyl-5'-O-(dimethoxytrityl)-~i-L-2'-deoxycytidine-3'-N,
N-diisopropylcyanoethyl phosphoramidite (2.06 g, 92°~) as a foam.
To a solution of
N4-Benzoy!-5'-0-(dimethoxytrityl)-~i-L-2'-deoxycytidine-3'-N,
2 o N-diisopropylcyanoethyl phosphoramidite (2.06 g, 2.47 mmol) in
anhydrous acetonitrile (100
ml),N8-benzoyl-2'-O-acetoxy-(3-D-3'-deoxyadenosine (1.08 g, 2.72 mmol)
in acetonitrile (40 ml) was added and stirred for 10 minutes under argon.
To this solution, sublimed 1 H-tetrazole (0.52 g, 7.4 mmol) was added and
2 5 stirred over night. The solvent was evaporated and the residue was
triturated with 70°~ EtOAclether and filtered. The filtrate was
evaporated
to give
N4-Benzoyl-5'-0-dimethoxytrityl-3'-[0-(2'-O-acetyl)-Ng-benzoyl-(3-D-3'-deo
xy adenosinyl]-2'-deoxy-(3-L-cytidine cyanoethyl phosphite ester as a foam
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and this was used in the next step without further purification.
The dimer
N"-Benzoyl-5'-0-dimethoxytrityl-3'-[O-(2'-O-acetyl)-Ne-benzoyl-(3-D-3'-{deo
xyadenosinyl-2'-deoxy-[3-L-cytidine cyanoethyl phosphite ester was
dissolved in THF {24 ml), pyridine (6 ml) and water (0.6 ml). Iodine
crystals (0.55 g) were added portion wise until the iodine color persists.
The reaction mixture was stirred for another 15 minutes and the excess
iodine was removed by the addition of saturated sodium thiosulfate. The
solvent was evaporated and the residue was dissolved in EtOAc and
io washed with wafer, NaHC03 and brine. EtOAc layer was evaporated and
the residue was dissolved in 80°~ acetic acidlwater solution (50 ml)
and
stirred for 1 hour. Then the solvent was evaporated and the crude
product was purified on a silica gel column using 5-10°~ MeOH/CHCl3 as
solvent to give pure compound
i5 (2'-Acetoxy-Ng-benzoyl-3'-deoxy-(3-D-adenosinyl)-N"-Benzoyl-[3-L-2'-deoxy
citydinyl cyanoethyl phosphate ester (1.46 g) as a foam.
3'-O-(3'-deoxy-[i-D-adenosinyl)-[3-L-2'-deoxycytidine (6) (L-153)
The dimer
(2'-Acetoxy-Ng-benzoyl-3'-deoxy-(3-D-adenosinyl)-N"-Benzoyl-[3-L-2'-deoxy
2o citydinyl cyanoethyl phosphate ester (1.46 g) was treated with ammoniun
hydroxide solution {100 ml) over night. The solvent was evaporated and
the residue was purified on DEAE Cellulose ion exchange column using
gradient of NH4HC03 buffer (0.05 - 0.2 M). The pure fractions were
collected and lyophillized to give pure
25 3'-O-(3'-deoxy-(3-D-adenosinyl)-[3-L-2'-deoxycytidine (L-153) (0.81 g) as
white solid.
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Example16
Synthesis of a-L-dC. Cord~rcepin dimer (L-154
To a solution of a-L-dC (1.6 g, 4.88 mmol) in pyridine (100 ml) was
added 4, 4'-dimethoxy trityl chloride (2.43 g, 7.2 mmol) and DMAP (0.13 g,
1.04 mmol) and stirred at room temperature for 2 hours under argon. To
complete the reaction, additional DMTCI (1.0 g} was added and stin-ed for
another 2 hours. The reaction was quenched with the addition of MeOH
(5 ml) and the solvent was evaporated. The residue was dissolved in
EtOAc, washed with water NaHC03 and brine. After drying over Na2S04,
s o the EtOAc layer was evaporated and the crude compound was purified on
a silica gel column using 3-5°~ MeOHICHCl3 as solvent to give pure
N4-Benzoyl-5'-O-(di-p-methoxy trityl)-2'-deoxy-a-L-cytidine (2.34 g,
76°~)
as pale yellow foam.
N4-Benzoyl-5'-0-(di-p-methoxy trityl)-2'-deoxy-a-L-cytidine (1.84 g,
2.9 mmol) was dissolved in anhydrous dichloromethane (50 ml). N,
N-diisopropylethylamine (2.0 ml, 11.6 mmol) was added under argon
followed by 2'-cyanoethyl-N, N-diisopropylchlorophosphoramidite (0.85
ml, 3.5 mmol). The reaction was stirred for 30 minutes and the solvent
was evaporated. The residue was dissolved in 80°~ EtOAcIEt3N (75 ml)
2 o and washed with water, NaHC03 and brine. The organic layer was
evaporated and purified on a short silica gel column using a mixture of
EtOAc, hexane and Et3N (50:40:10) to give
N4-Benzoyl-5'-O-(dimethoxytrityl}-a-L-2'-deoxycytidine-3'-N,
N-diisopropylcyanoethyl phosphoramidite (2.01 g, 83°~) as a foam.
To a solution of compound
N"-Benzoyl-5'-O-(dimethoxytrityl)-a-L-2'-deoxycytidine-3'-N,
N-diisopropylcyanoethyl phosphoramidite (2.01 g, 2.4 mmol) in anhydrous
acetonitrile (100 ml),Ng-Benzoyl-2'-0-acetoxy-(3-D-3'-deoxyadenosine
(1.05 g, 2.65 mmol) in acetonitrile (40 ml) was added and stirred for 10
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minutes under argon. To this solution, sublimed 1-H-tetrazole (0.5 g, 7.2
mmol) was added and stirred over night. The solvent was evaporated and
the residue was triturated with 70% EtOAclether and filtered. The filtrate
was evaporated to give
N4-Benzoyl-5'-O-(dimethoxytrityl)-3'-[O-(2'-acetyl)-Ne-Benzoyl-[i-D-3'-deox
y adenosinyl]-2'-deoxy-a-L-cytidine cyanoethyl phosphite ester as a foam
and this was used in the next step without purification.
The dimer
N4-Benzoyl-5'-O-(dimethoxytrityl)-3'-[O-(2'-acetyl)-Ne-Benzoyl-(3-D-3'-deox
1 o y adenosinyl]-2'-deoxy-a-L-cytidine cyanoethyl phosphite ester was
dissolved in THF (24 ml), pyridine (6 ml) and water (0.6 mi). Iodine
crystals (0.5 g) were added portion wise until the iodine color persists.
The reaction mixture was stirred for another 15 minutes and the excess
iodine was removed by the addition of saturated sodium thiosulfate. The
z5 sulfate was evaporated and the residue was dissolved in EtOAc and
washed with water, NaHC03 and brine. EtOAc layer was evaporated and
the residue was dissolved in 80% acetic acid/water solution (50 ml) and
stirred for 1 hour. Then the solvent was evaporated and the crude
product was purified on a silica gel column using 5-10% MeOHICHCl3 as
2 o solvent to give pure
(2'-Acetoxy-Ng-Benzoyl-3'-deoxy-(3-D-adenosinyl )-N4-Benzoyl-a-L-2-deoxy
citydinyl cyanoethyl phosphate ester (1.8 g) as a foam.
The dimer
(2' Acetoxy-Ne-Benzoyl-3'-deoxy-(3-D-adenosinyl)-N~-Benzoyl-a-L-2-deoxy
2s citydinyl cyanoethyl phosphate ester (1.8 g) was treated with ammonium
hydroxide solution (100 ml) over night, The solvent was evaporated and
the residue was purified on DEAE Cellulose ion exchange column using
gradient of NH4HC0s buffer (0.05 - 0.2M). The pure fractions were
collected and lyophillized to give pure
CA 02322494 2000-09-07
WO 99/45935 PCT/US99/053b0
3'-O-(3'-deoxy-~i-D-adenosinyl)-a-L-2'-deoxycytidine (L-154) (1.08 g) as
white solid.
Example 17
Synthesis of a-L-dA. Cordyrcepin dimer (L-155
To a stirring solution of 2'-deoxy-a-L-adenosine (2.05 g, 8.16 mmol)
in pyridine (75 ml) chilled in an ice bath, CISiMe3 (5.17 ml, 40.8 mmol)
was added dropwise and stirred for 30 minutes. Benzoyl chloride (4.7 ml,
40.8 mmol) was then added dropwise and the reaction mixture was stirred
at room temperature for two hours. This was cooled in an ice bath and
to water (15 ml) was added dropwise, 15 minutes later concentrated NH40H
(15 ml) was added to give a solution approximately 2M in ammonia. After
30 minutes the solvent was evaporated and the residue was dissolved in
water and washed with ether. The water layer was concentrated and the
Ne-Benzoyl-2'-deoxy-a-L-adenosine crystallized from water as white solid
(2.48 g, 85.8°~).
To a solution of compound Ne-Benzoyl-2'-deoxy-a-L-adenosine
(2.48 g, 6.98 mmol) in pyridine (100 ml) was added 4, 4'-dimethoxy trityl
chloride (3.55 g, 10.47 mmol) and DMAP (0.25 g, 2.09 mmol) and stirred
at room temperature for 2 hours under argon. To complete the reaction,
2o additional DMTCI (1.3 g) was added and stirred for another 2 hours. The
reaction was quenched with the addition of MeOH (5 ml) and the solvent
was evaporated. The residue was dissolved in EtOAc, washed with water,
NaHC03 and brine. After drying over NaZSO,, the EtOAc layer was
evaporated and the crude compound was purred on a silica gel column
25 using 3-5% MeOHICHCl3 as solvent to give pure
Ne-Benzoyl-5'-O-(di-p-methoxy trityl)-2'-deoxy-a-L-adenosine (3.42 g,
74.5°~) as pale yellow foam.
N'~-Benzoyl-5'-O-(di-p-methoxy trityl)-2'-deoxy-a-L-adenosine (1.64
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g, 2.5 mmol) was dissolved in anhydrous dichloromethane (50 ml). N,
N-diisopropylethylamine (1.75 ml, 10.0 mmol) was added under argon
followed by 2'-cyanoethyl-N, N-diisopropylchlorophosphoramidite (0.8 ml,
3.25 mmol). The reaction was stin-ed for 30 minutes and the solvent was
s evaporated. The residue was dissolved in 80°~ EtOAcIEt3N (75 ml) and
washed with water, NaHC03 and brine. The organic layer was
evaporated and purified on a short silica gel column using a mixture of
EtOAc, CH2CI2 and Et3N (40:50:10) to give
Ng-Benzoyl-5'-O-(dimethoxytrityl)-a-L-2'-deoxyadenosine-3'-N,
io N-diisopropylcyanoethyl phosphoramidite in quantitative yield.
To a solution of
NB-Benzoyl-5'-O-( di methoxytrityl )-a-L-2'-deoxyadenosi ne-3'-N,
N-diisopropylcyanoethyl phosphoramidite(2.5 mmol) in anhydrous
acetonitrile {60 ml), Ne-Benzoyl-3'-0-acetoxy-[i-D-2'-deoxyadenosine
m (0.94 g, 2.36 mmol) in acetonitrile (40 ml) was added and stirred for 10
minutes under argon. To this solution, sublimed 1 H-tetrazole (0.5 g,
7.2 mmol) was added and stirred overnight. The solvent was evaporated
and the residue was triturated with 70% EtOAcI ether and filtered. The
filtrate was evaporated to give
2 o Ne-Benzoyl-5'-0-dimethoxytrityl-3'-[O-(2'-)-acetyl)-Ne-benzoyl-~i-D-3'-
deoxy
adenosinyl]-2'-deoxy-a-L-adenosine cyanoethyl phosphite ester as a foam
and this was used in the next step without further purification.
The dimer
Ne-Benzoyl-5'-0-dimethoxytrityl-3'-[O-(2'-)-acetyl )-Ne-benzoyl-[i-D-3'-deoxy
2 s adenosinyl]-2'-deoxy-a-L-adenosine cyanoethyl phosphite ester was
dissolved in THF (24 ml), pyridine (6 ml) and water (0.6 ml). Iodine
crystals (0.63 g) were added portion wise until the iodine color persists.
The reaction mixture was stirred for another 15 minutes and the excess
iodine was removed by the addition of saturated sodium thiosulfate. The
72
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solvent was evaporated the residue was dissolved in EtOAc and washed
with water, NaHC03 and brine. EtOAc layer was evaporated and the
residue was dissolved in 80% acetic acid I water solution (50 ml) and
stirred for 1 hour. Then the solvent was evaporated and the crude
s product was purified on a silica gel column using 5-10°~ MeOH I CHC13
as
solvent to give pure compound
(2'-Acetoxy-Ne-benzoyl-3'-deoxy-[i-D-adenosi nyl )-Ns-benzoyl-a-L-2'-deoxy
adenosinyl cyanoethyl phosphate ester (0.97 g) as a foam.
The dimer
to (2'-Acetoxy-Ne-benzoyl-3'-deoxy-(3-D-adenosinyl)-Ne-benzoyl-a-L-2'-deoxy
adenosinyl cyanoethyl phosphate ester(0.97 g) was treated with
ammonium hydroxide solution (100 ml) overnight. The solvent was
evaporated and the residue was purified on DEAE Cellulose ion exchange
column using gradient of NH4HC03 buffer (0.05-0.2 M). The pure
i5 fractions were collected and iyophillized to give pure ~
[3']-O-(3'-deoxy-~i-D-adenosinyl)-~i-L-2'-deoxyadenosine (0.55 g)(L-155)
as white solid.
Example 18
Syrnthesis of ~i -L-dA. ~3 -D-dA ~(L-210)
2 o To a stirring solution of 2'-deoxy- [i -L-adenosine (2.05 g,
8.16 mmol) in pyridine (75 ml) chilled in an ice bath, CISiMe3 (5.17 ml,
40.8 mmol) was added dropwise and stirred for 30 minutes. Benzoyl
chloride (4.7 m1, 40.8 mmol) was then added dropwise and the reaction
mixture was stirred at room temperature for two hours. This was cooled in
25 an ice bath, and water (15 ml} was added dropwise. 15 minutes later
concentrated NH40H (15 ml} was added to give a solution approximately
2 M in ammonia. After 30 minutes, the solvent was evaporated and the
residue was dissolved in water and washed with ether. The water layer
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was concentrated, and the Ne-Benzoyl-2'-deoxy-(3-L-adenosine was
crystallized from water as white solid (2.48 g, 85.8°rb).
To a solution of Ng-Benzoyl-2'-deoxy-~-L-adenosine(2.48 g,
6.98 mmol) in pyridine (100 ml) was added 4,4'-dimethoxy trityl chloride
(3.55 g, 10.47 mmol) and DMPA (0.25 g, 2.09 mmol) and stirred at room
temperature for 2 hours under argon. To complete the reaction, additional
DMTC1 (1.3 g) was added and stirred for another 2 hours. The reaction
was quenched with the addition of MeOH (5 ml), and the solvent was
evaporated. The residue was dissolved in EtOAc, washed with water,
io NaHC03 and brine. After drying over Na2S04, the EtOAc layer was
evaporated and the crude compound was purified on a silica gel column
using 3l5°~ MeOHICHC1~ as solvent to give pure
Ng-Benzoyl-5'-O-(di-p-methoxy trityl)-2'-deoxy-(3-L-adenosine (3.42 g,
74.5°~) as pale yellow foam.
i 5 Ne-Benzoyl-5'-0-(di-p-methoxy
trityl)-2'-deoxy-~i-L-adenosine(1.71 g, 2.61 mmol) was dissolved in
anhydrous dichloromethane (50 ml). N,N-diisopropylethylamine (1.8 ml,
10.34 mmol) was added under argon followed by
2'-cyanoethyl-N,N-diisopropylchlorophosphoramidite (1.0 ml, 4.47 mmol).
2 o The reaction was stirred for 30 minutes, and the solvent was evaporated.
The residue was dissolved in 80°~ EtOAcIEt3N (75 ml) and washed
with
water, NaHC03 and brine. The organic layer was evaporated and purified
on a short silica gel column using a mixture of EtOAc, hexane and Et3N
(50:40:10) to give
a5 Ng-Benzoyl-5'-O-(dimethoxytrityl)-(3-L-2'-deoxyadenosine-3'-N,N-diisoprop
yicyanoethyl phosphoramidite(1.87 g, 84°~) as a foam.
To a solution of
Ng-Benzoyl-5'-0-(dimethoxytrityl)-(3-L-2'-deoxyadenosine-3'-N,N-diisoprop
ylcyanoethyl phosphoramidite(1.87 g, 2.18 mmol) in anhydrous
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WO 99/45935 PCTNS99I05360
acetonitrile (60 ml), N8-Benzoyl-3'-O-acetoxy-(3-D-2'-deoxyadenosine
(0.95 g, 2.4 mmol) in acetonitrile (40 rnl) was added and stirred for
minutes under argon. To this solution, sublimed 1 H-tetrazole (0.46 g,
6.6 mmol) was added and stirred overnight. The solvent was evaporated,
5 and the residue was triturated with 70°~ EtOAclether and filtered.
The
filtrate was evaporated to give
Ne-Benzoyl-5'-O-dimethoxytrityl-3'-[O-(3'-0'acetyl)-Ng-benzoyl-[3-D-2'-deox
y adenosinyl]-2'-deoxy-[i-L-adenosine cyanoethyl phosphite ester as a
foam, and this was used in the next step without further purification.
io The dimer
Ne-Benzoyl-5'-O-dimethoxytrityl-3'-[O-(3'-O'acetyl}-Ne-benzoyl-(3-D-2'-deox
y adenosinyl]-2'-deoxy-[i-L-adenosine cyanoethyl phosphite ester was
dissolved in THF (24 ml), pyridine (6 ml) and water (0.6 ml). Iodine
crystals (0.5 g) were added portion wise until the iodine color persists.
The reaction mixture was stirred for another 15 minutes, and the excess
iodine was removed by the addition of saturated sodium thiosulfate. The
solvent was evaporated, and the residue was dissolved in EtOAc and
washed with water, NaHCO$ and brine. EtOAc layer was evaporated, and
the residue was dissolved in 80% acetic acidlwater solution (50 ml) and
2 o stin-ed for 1 hour. Then the solvent was evaporated, and the crude
product was purified on a silica gel column using 5-10°~ MeOHlCHC13 as
solvent to give pure compound
(3'-Acetoxy-Ne-benzoyl-2'-deoxy-[3-D-adenosinyl)-Ng-benzoyl-[i-L-2'-deoxy
adenosinyl cyanoethyl phosphate ester (0.13 g} as a foam.
2 s The dimer
(3'-Acetoxy-N'~-benzoyl-2'-deoxy-[i-D-adenosinyl}-Ne-benzoyl-[3-L-2'-deoxy
adenosinyl cyanoethyl phosphate ester(1.3 g) was treated with ammonium
hydroxide solution (100 ml) overnight. The solvent was evaporated, and
the residue was purified on DEAF Cellulose ion exchange column using
CA 02322494 2000-09-07
WO 99145935 PCT/US99/05360
gradient of NH4HC03 buffer (0.05-0.2 M). The pure fractions were
collected and lyophillized to give pure
3'-O-(2'-deoxy-~3-D-adenosinyl)-~i-L-2'-deoxyadenosine (L-210) (0.640 g)
as white solid.
s All patents and publications mentioned in the specifications are
indicative of the levels of those skilled in the art to which the invention
pertains. All patents and publications are herein incorporated by
reference to the same extent as if each individual publication was
specifically and individually indicated to be incorporated by reference.
~ o One skilled in the art readily appreciates that the patent invention is
well adapted to carry out the objectives and obtain the ends and
advantages mentioned as well as those inherent therein. Dimers,
pharmaceutical compositions, treatments, methods, procedures and
techniques described herein are presently representative of the preferred
i5 embodiments and are intended to be exemplary and are not intended as
limitations of the scope. Changes therein and other uses will occur those
skilled in the art which are encompassed within the spirit of the invention
or defined by the scope of the pending claims.
76
