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WO 96/40164 PCTIUS96110026
NUCLE051nES WITH ANTI HEPATITIS B VIRUS ACT1YITY
Background of the Inrentiou
This invention is in the area of methods for the treatment of hepatitis B
virus (also referred to as "HBV") that includes admuustermg an effective
amount of one or more of the active compounds disclosed herein, or a
pharmaceutically acceptable derivative or prodrug of one of these
1o compounds.
HBV is second only to tobacco as a cause of human cancer. The
mechanism by which HBV induces cancer is unknown, although it is
postulated that it may directly trigger tumor development, or indirectly
trigger tumor development through chronic inflammation, cirrhosis, and
cell regeneration associated with the infection.
Hepatitis B virus has reached epidemic levels worldwide. Aftex a two
to six month incubation period in which the host is unaware of the
infection, HBV infection can lead to xute hepatitis and liver damage, that
causes abdominal pain, jaundice, and elevated blood levels of certain
Z 0 enrymes. HBV can cause fitlminant hepatitis, a rapidly progressive, often
fatal form of the disease in which massive sections of the liver are
destroyed. Patients typically recover from acute viral hepatitis. In some
patients, however, high levels of viral antigen persist in the blood for an
extended, or indefinite, period, causing a chronic infection. Chronic
infections can lead to chronic persistent hepatitis. Patients infected with
chronic persistent HBV are most common in developing countries. By
mid-1991, there were approumatety 225 million chronic carriers of HBV
in Asia alone, and worldwide, almost 300 million carriers. Chronic
persistent hepatitis can cause fatigue, cirrhosis of the liver, and
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WO 96!40164 PCT/U596/10026
hepatocellular carcinoma, a primary liver cancer. In western industrialized
countries, high risk groups for IiBV infection include those in contact with
HBV carriers or their blood samples. The epidemiology of HBV is in fact
very similar to that of acquired immunodeficiency syndrome, which
s accounts for why HBV infection is common among patients with AIDS or
HIV-associated infections. However, HBV is more contagious than H1V.
Daily treatments with a-interferon, a genetically engineered prntein,
has shown promise. A human serum-derived vaccine has also been
developed to immunize patients against hIBV. Vaccines have been
1o produced through genetic engineering. While the vaccine has been found
effective, production of the vaccine is troublesome because the supply of
human serum from chronic carriers is limited, and the purification
procedure is long and ezpensive_ Further, each batch of vaccine prepared
from different serum must be tested in chimpanzees to ensure safety. In
15 addition, the vaccine does not help the patienES already infected with the
virus.
European Patent Application Na. 92304530.6 discloses that a group of
1,2-ozathiolane nucleosides are useful in the treatment of hepatitis B
infections. It has been reported that the 2-hydrnzymethyl-5-(cytosin-1-y1)-
20 1,3-oxarhiolarte has anti-hepatitis B activity. Doong, et al., Proc. of
Natl.
Acad, Sci. USA, 88, 8495-8499 (1991); Chang, et al., J. of Biologic
Chem., VoI 267(20), 13938-13942. The and-hepatitis B activity of the (-)
and (+)-enantiomers of 2-hydrozymethyl-5-(5-fluorocytQSin-I-yl)-1;3-
oxathiolane has been published by Furman, et al., in Antimicrobial Agents
2 s and Chemotheraov, Dec. 1992, pages 268fr2692.
PCT/US92/03144 (International Publication No. WO 92/18517) filed
by Yale University discloses a number of 9-L-nucleosides for the treatment
of both I38V and HIV. Other drugs ezlored for the treatment of HBV .
include adenosine arabinoside, thymosin, acyclovir, phosphonoformate,
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WO 96140164 PCTNS9611002b
zldavudinc, (+y-cyanidanol, quinacrine, and 2'-fluoroarabinosyl-5-
iodan~dl.
M essential step in the mode of anion of purine and pyrimidine
nucleosides against viral dexases, and In particular, HBV and HIV, is their
r~abolic acdvation by cellular and viral bltases, to yield the mono-, dl-,
and ttiphosphate derivatives. The biologically active spacies of many
auclaoaides is the triphaspahte form, Which inhibits DNA polymerase or
reNerae transcriptase, or causes chain termination. The nucleo~de
derivatives that have beat developed far the treabaent of HBV and HIV to
1 o dace have been presatted for administration to the host in
nnghosphorylated
form, notwithstanding the fact that the nucleoside must be phosphorylatied
is the cell prior bo exhibiting its antiviral effort, because the triphasphate
form has typically either been dephospborylated prior to r~a~ching the cell
or is poorly absorbed by the xll. NucleoudGS in generat cross cell
15 membrarxs very inefficiently and era gatdslly not very not very potent ju
~. Attempts at modifytt~g nucleotides to increase the absorption and
pararcy of nucloolides have been described by R. Tones and N.
l3iscawtberger, MtivJrrr! Reseal, Z9 (199 1-1'~,
2 0 In light of the fact that hepatitis B virus has readied ~d~iCC levels
worldwide, and has aevae and often tragic effocfs on the infocted patient,
there remains a strong aeod to provide nevi effective pharmaceutical agents
to treat humans infocted with the virus that have loW toxidty W the host.
Therefore; it Is another object of the present invention to provide a
z 5 method and composition for the treatment of human patients or other hosts
infected with HBV.
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Summary of the Invention
A method for the treatment of a host, and in particular, a human,
infected with HBV is provided that includes administering an HBV-
treatment amount of a nucleoside of the formula:
NHz
Rt NHz
N/\ N N/ / F
\ ~ 1N
N O~N
NaN N O' \ N
HO HO HO
p O O
wherein:
R' is hydrogen, fluoxo, bromo, chloro, iodo, methyl or ethyl; and R2
is OH, Cl, NHs, or H; or a pharmaceutically acceptable salt of the
compound, optionally in a pharmaceutically acceptable carrier or diluent.
In an alternative embodiment, the $-L-enantiomer of a compound of
the formula:
Rs
NO
O
wherein Rs is adenine, xanthine, hypozanthine, or other purine, including
an alkylated or halogenated purine is administered to a host in an HBV-
treatment amount as described more fully herein.
In another alternative embodiment, the nucleoside is of the formula:
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Y~
l3ase
AO
YZ
O
wherein B is a purine or pyrimidine base;
Yl, Y2, Y', and Y' are independently H, OH, N3, NR'R2, N02,
NOR', -O-alkyl, -O-aryl, halo (including F, Cl, Br, or n, -CN, -
C(O}NH2, SH, -S-alkyl, or -S-aryl, and wherein typically three of Y', Y~,
Y3, and Y4 are either H or OH. The -OH substituent, when present, is
typically a Y~ or Y3 group. As illustrated in the structure, YZ and Y°
are in
the arabino (aythro} configuration, and Y' and Y' are in the threo (ribose)
configuration. R is H, monophosphate, diphosphate, triphosphate, alkyl,
aryl or a phosphate derivative, as described in more detail below. R', R2,
and R3 are independently alley! (and in particular lower alkyl), aryl,
arallryl, atkaryl, acyl, or hydrogen.
In a preferred embodiment, the nucleoside is provided as the indicated
2 o enantiomer and substantially in the absence of its corresponding
enantiomer
(l.c., in enantiomerically enriched form).
In another embodiment, the invention includes a method for the
treatment of humans infected with HBV that includes administering an
HBV treatment amount of a prodrug of the specifically disclosed
2 5 nucleosides. A prodrug, as used herein, refers to a pharmaceutically
acceptable derivative of the specifically disclosed nucleoside, that is
converted into the nucleoside on administration in vivo, or that has activity
~ in itself. Nonlimiting examples are the 5' and 1V~-pyrinudine or N6-purine
acylated or alkylated derivatives of the active compound.
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In a preferred embodiment of the invention, the nucleoside is provided
as the monophosphate, diphosphate or triphosphate in a fonnuiation that
protects the compound from dephosphorylation. Formulations include
tiposomes, lipospheres, microspheres or nanospheres (of which the latter '
three can be targeted to infected cells). In an alternative preferred
embodiment, the nucleoside is provided as a monophosphate, diphosphate
or triphosphate derivative (i.e., a nucleotide prndrug), for example an
ester, that stabilizes the phosphate in vivo. In an alternative embodiment
of this invention, a stabilized phosphate derivative, as described further
1o below, of FTC, 13CH-189, or 3TC is provided for the treatment of
hepatits.
The disclosed nucleosides, or their pharmaceutically acceptable
prodxugs or salts or pharmaceutically acceptable formulations containing
these compounds are useful in the prevention and treatment of H13V
infections and other related conditions such as anti-HBV antibody positive
and H$V-positive conditions, chronic liver inflammation caused by HBV,
cirrhosis, acute hepatitis, fulminant hepatitis, chronic persistent hepatitis,
and fatigue. These compounds or formulations can also be used
P~PhY~tically to prevent or retard the progression of clinical ikltless in
2 o individuals who are anti-HBV antibody or HBV-antigen positive or who
have been exposed to HBV.
In one embodiment of the invention, one or more of the active
compounds is administered in alternation or combination with one or more
other anti-HBV agents, to provide effective anti-HBV treatment. Examples
of anti-HBV agents that can be used in alternation ar coubination therapy
include but are not limited to the Z-hydroxymethyl-5-{5-fluorocytosin-1-yl)-
1,3-oxathiolane {"FTC", see WO 92/14743), its physiologically acceptable
derivative, or physiologically acceptable salt; the 2-hydroxymethyl-5- -
(cytosin-1-yl)-1,3-oaathiotane (including the racemic BCH-189 form, or
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WO 96I40Ib4 PCT/L1S96/10026
3TC (BCH-189 enriched with the (-)-enxntiomer)) its physiologit~llly
acceptable derivative, or physiologically acceptable salt; 2'-fluoro-5-ethyl-
arabinosyluracil (FEAL>); carbovir, or interferon.
- Any method of alternation can 1x used that provides treatment to the
patient. Nonlimiti;ng examples of alternation patterns include 1-6 weeks of
administration of an effective amount of one agent followed by 1-6 weeks
of administration of an ei~ective amount of a second anti-HBV agent. The
alternation schedule can include periods of no treatment. Combination
therapy generally includes the simultaneous administration of an effective
io ratio of dosages of two or more anti-HBV agents.
In light of the fact that HBV is often found in patients who are also
anti-HIV antibody or HIV-antigen positive or who have been exposed to
HIY, the active anti-HBV compounds disclosed herein or their derivatives
or prodrugs can be administered in the appropriate circumstance in
combination or alternation with anti-HIV medications, including but not
limited to 3'-azido-3'~eozythymidine (AZT~, 2',3'-dideozyinosine (DDn,
2',3'-dideoxycytidine {DDC), Z',3'-dideoxy-2',3'-didehydrothymidine
(D4T), 2-hydrozymethyl-5-(5-fiuorocyWsin-1-yl)-1,3-oxathiolane (FTC,
or Z-hydroxymethyl-5-(cytosin-1-yl)-1,3-ozathiolane (racemic BCH-189 or
Z o BCH-189 enriched with the (-)-enantiomer, 3TC). Non-nucleoside RT-
inhibitors such as the Tibo class of compounds, nevirapine, or
pyrimidinone can also be administered in combination with the claimed
compounds.
The active anti-HBV agents can also be administered in combination
2 5 with antibiotics, other antiviral compounds, antifungal agents, or other
pharmaceutical agents administered for the treatment of secondary
infections.
In one embodiment, the nucleoside is provided as a phosphate
derivative that is stabilized to decrease or eliminate dephosphorylation prior
CA 02538205 1996-06-07
WO 96140164 PCT/US96J10026
to uptake into the infected cell. A number of stablized phosphate
derivative groups in the 5'-position of the nucleoside are known and have
been published in the literature. In one embodiment, the nucleoside is
administered as a SATE derivative, as disclosed in more detail below. Any .
alternative stablized phosphate derivative can be placed in the 5'-position of
the nucleoside that does not materially adversely affect the activity of the
compound.
Brief Description of the Frgures
to
Figure 1 is an illustration of the chemical structures of B-L-2',3'-
dideoxycytidine (B-L-FddC), B-D-2',3'-dideoxycytidine (B-D-ddC), B-L-
2',3'-dideoxy-5-fluoroeytidine (B-L-ddC), (-)-B-L-2-hydroaymethyi-5-(5-
fluorocytosin-1-y1r1,3,oxathiolane (( )-B-L-FTC), (+)-&D-2-
hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3-dioxolane ((+)-&D-FDOG~,
and B-L-2-amino-6-(R~-9-[(4-hydroaymethyl)-tetrahydrofiuan-1-yl]purine.
Figure 2 is an illustration of the numbering scheme used in the
chemical nomenclature for nucleosides in this text.
2 0 Detailed lDescrlption of the Invention
As used herein, the term "enantiomerically pure" refers to a nucleoside
composition that includes at least approximately 9596, and preferably
approximately 97%, 9$%, 99%, or 10096 of a single enantiomer of that
nucleoside.
The term alkyl, as used herein, unless otherwise specified, refers to a
saturated straight, branched, or cyclic, primary, secondary, or tertiary
hydrocarbon of C~ to C,o, and specifically includes methyl, ethyl, gropyl,
isopropyl, butyl, isobutyl, t-butyl, pentyl, cyclopentyl, isopentyl,
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neopentyl, hexyl, isohexyl, cyclohexyl, cyclohexylmethyl, 3-methylpentyl,
' 2,2-dimethylbutyl, arid 2,3-dimethylbutyl. The alkyl group can be
optionally substituted with one or more moieties selected from the group
consisting of hydroxyl, amino, alkylamino, arylamitto, alkoxy, aryloxy,
vitro, cyano, sulfonic acid, sulfate, phosphoric acid, phosphate, or
phosphonate, either unprotected, or protected as necessary, as known Lo
those skilled in the art, for example, as taught in Greene, et al.,
"Protective
Groups in Organic Synthesis," John Wiley and Sons, Second Edition,
i99I. The term lower alkyl, as used herein, and unless otherwise
1o spocified, refers to a C, to C, ethyl, propyl, butyl, pentyl, hexyl,
isopropyl, isobutyl, sec-butyl, or t-butyl group.
As used herein, the term aryl specifically includes but is not limited to
acetyl, propionyl, butyryl, pentanoyl, 3-methylbutyryl, hydrogen
succinate, 3-chlorobenzoate, benzoyl, acetyl, Pivaloyl, mesylate,
propionyl, valeryl, raproic, caprylic, capric, lauric, myristic, palmitic,
stearic, and oleic.
The term aryl, as used herein, and unless otherwise specified, refers to
phenyl, biphenyl, or naphthyl, and preferably phenyl. The aryl group can
be optionally substituted with one or more moieties selected from the group
z o consisting of hydroxyl, amino, alkylamino, arylamino, alkoxy, aryloxy,
vitro, cyano, sulfonic acid, sulfate, phosphoric acid, phosphate, or
phosphonate, either unprotected, or protected as necessary, as known to
those skilled in the art, for example, as taught in Greene, et al.,
"Protective
Groups in Organic Synthesis," John Whey and Sons, Second F~ctition,
1991.
. The term purine or pyrimidine base includes, but is not limited to,
adenine, 1V6-alkylpurines, N6-acylpurines (wherein acyl is C(O)(alkyl, aryl,
' alkylaryl, or arylalkyl), N6-bcnzylpurine, ~-halopurine, N6-vinylpurinc,
N6-acetylenic purirte, 1Vb-acyl purine, N6-hydroxyalkyl purine, 1V~-thioalkyl
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purine, Nz-alkyIpurines, Ni-alkyl-trthiopurines, thymine, cytosine, 6-
a~apyrimidine, 2- and/or 4-metcaptopyrmidine, uracil, Cs_
~YIPY~dines, Cs-benzylpyrimidines, C3-halopyrimidines, CS-
vinylpyrimidine, Cs-acetylrnic pyrimidine, Cs-aryl pyrimidine, Cs-
hydroxyalkyl purine, Cs-amidopyrimidine, Cs.cyanopyrimidine, Cs-
nitropyrimidine, G"5-aminc~pYrimidine, N1 allrylpurines, Nz-alkyl-6-
thiopurines, 5-azacytidinyl, 5-azauracilyl, triazalopyridinyl,
imidazolopyridinyl, pyrrolopyzimidinyl, pyrawlopyrimidinyl. Functional
oxygen and nitrogen groups on the base can be protected as neo~ssar~r or
l0 desired. Suitable protecting groups are well known to those slQlled in the
art, and include trimethylsilyl, dimethylhexylsilyl, t-butyWimethylsilyl, and
t-butyldiphenylsilyl, trityl, alkyl groups, acyl groups such as acetyl and
propionyl, methylsulfonyi, and p-Loluyisulfonyl.
As usod herein, the term natural amino acid includes but is not limited
15 to alanyl, valinyl, leucinyl, isoleucinyl, prolinyl, phenylalaninyl,
~YP~1~Y1. oninyl, glycinyl, serinyl, rhreoninYl, cysteinyl,
tyrosinyl, aspataginyi, giutaminyl, aspartoyl, glutaoyl, iysinyl, argininyl,
and histidinyl.
The invention as disclosed herein is a method and composition for the
z o treatment of HBV infection and oilier viruses replicating in a like
manner,
in humans or other host animals, that includes administering an effective
amount of one or morn of the above-identified compounds, or a
physiologi~lly acceptable derivative, or a physiologically acceptable salt
thereof, optionally in a pharmaceutically acceptable carrier. The
z ~ compounds of this invention either possess anti-HBV activity, or are
metabolized to a compound or compounds that exhibit anti-I3BV activity.
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I. Structure and Preparation of Active Nucleosides
The compounds used in the methods disclosed herein are enantiomers
of 2',3'-didcoxycytidine, 2',3'-dideoxy-5-(halo or methylxytidine, 2-
s hydmxymethyl-5-(5-fiuorocytosin-1-yl)-1,3-dioxolane, or 2-amino-b-(OH,
Cl, NH2, or I~-9-[(4-hydtnxymethyl)-tettahydrofuran-1-yl]purine.
Since the 1' and 4' carbons of the sugar or dioxolanyl moiety (referred
to below generically as the sugar moiety) of the nucleosides are chiral,
their nonhydrogen substituents (CHZOR and the pyrimidine or purine base,
respectively) can be zither cis (on the same side) or traps (on opposite
sides) with respect to the sugar ring system. The four optical isomers
therefore are represented by the following configurations (when orienting
the sugar moiety in a horizontal plane such that the "primary" oxygen (that
between the Cl' and C4'-atoms; see Figure 2) is in back): cis (with both
groups "up", which corresponds to the configuration of naturally occurring
nucleosides), cis (with both groups "down", which is a nonnaturally
occurring configuration), traps (with the C2 substituent "up" and the CS
substituent "down"), and traps (with the C2 substituent "down" and the CS
substituent "up"). As indicated schematically in Figure 1, the "D-
z o nucleosides" are cis nucleosides in a natural configuration and the "L
nucleosides" are cis nucleosides in the nonnaturally occurring
configuration.
The nucleosides useful in the disclosed method to treat HBV infection
are B-L-enantiomers, with the exception of FDOC, which is used in its B-
D-enantiomeric form, because it has been discovered that the B-D-
enantiomer of FDOC is surprisingly less toxic than the H-L-enantiomer of
FDOC.
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The nucleosides disclosed herein can be administered as any derivative
that upon administration to the rodpterct, is capable of providing ditscfty or
indiroctly. the parrot active compound, or that exhibits activity in itself.
1n
oae embodiment, dye hydrogen of the s~-QH gt~oup is by a C,-Cm
alkyl, including Ci to Cs alkyl; aryl in which the noo-carbonyl moiety of
tha ester group is seladed from straight, b:aa~hed. or cyclic Ci-Cm alkyl
including Ci to Cs atkyl, phenyl, or benryl; a naturally occurring or
rwiinaturally occurring amino acid; allooxyalkyl including mahoxymethyl;
to aralkyt including benzyl; aryloxyalkyl such as phartoxymethyl; aryl
ineludirig phenyl optionally substituted with haloga~, C, b C4 alkyl or Cs
to C, alkoxy; a dicarboxylie acid such as ~cinic acid; sulfonate eaters
such as alkyl or aralkyl sulphonyl including metheneeulfonyl; or a mono, dl
or triphosphate ester.
is Une or both hydmgens of the amino groups on the purine or
pyxlmidiile base can be replaced by a Ci-Cm alkyl, inctud'mg C, to Cs
alkyl; aryl in which the rwn-carbonyl moiety of the esta~ group is aeiected
from straight, branched. or cyclic Ci-C~ alkyl, including C, to Cs alkyl,
phenyl, or berizyl; sllwxyalkyl includiug methaxymethyl; aratkyl including
2 o benzyl; arybxyalkyl such at phenoxyrndhyl; aryl including phenyl
optiartally aubstitutod with halogea, C, to C~ alkyl or C, to C, alkozy.
The active nucleoside can also be provided :s a S'-ether fipid, as
disclosed in the following references,:
iCuoera, L.S., N. iyer, B. Leaks, A. Rabea, Modest E.1., D.
z5 L.W., and C. Piantadosi. 1990. Novel mernbrati~intersdive ether lipid
analogs that inhibit infectious HIV-1 production and induce defective virus
formation. A1DS Res Hum Retrovituses. 6:491-501; Piantadosi, C., I.
Maraxo C.1., S.L. Morris-Natschke, K.L. Meyer, P. Gumus, J.R.
Surles, K.S. Ishaq, L.S. Kucera, N. Iyer, C.A. Wallen, S. 1?iaetadosi, and
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WO 96/40164 PCT/US961t0026
B.J. Modest. 1991. Synthesis and evaluation of novel ether lipid
nucleoside conjugates for anti-HIV activity. J Med Chem. 34:1408.1414;
Hostetler, K.Y., D.D. ltichman, D.A. Carson, L.M. Stuhtniller, G,M. T.
' van Wijk, and H. van den Hosch. 1992. Greatly enhanced inhibition of
S human immunodeficiency virus type 1 replication in CfiM and HT4-6C
cells by 3'-deoxythymidine diphosphate dimyristoylglycerol, a lipid
prodrug of 3,-deoxythymidine. ~icrob AeentY emother.
36:2025.2029; Hosbetler, K.Y., L.M. SWhmiIler, H.B. Lenting, H. van
den Bosch, and D.D. ltichman, 1990. Synthesis and antiretroviral activity
1o of phospholipid analogs of azidothymidine and other andviial nucleosides.
J~~ Chem. 265:6112.7.
Any of the nucleosides described herein, or any other nucleoside that
i5 has anti-hepatitis B activity, can be administered as a nucleotide prodrug
to
increase the activity. bioavalability, stability or otherwise alter the
properties of the nucleoside. A number of nucleotide prodrug ligands are
known. A nucleotide prodrug, as described herein, refers to a nucleoside
that has a phosphate derivative on the 5'-position that is more stable in vivo
2 o than the parent phosphate, and which does not materially adversely affect
the anti-hepatits B activity of the nucleoside. Phosphonates are included as
phosphate derivatives. In general, alkylation, acylation or other Iipophilic
modification of the mono, di or triphosphoate of the nucleoside will
increase the stability of the nucleotide. Examples of substituent groups that
2 s cart replace one or more hydrogens on the the phosphate moiety are alkyl,
aryl, steroids, carbohydrates, including sugars, 1,2-diacylglycerol and
alcohols. Many are described in R_ Jones and N. Bischofberger, Antiviral
Research, 27 (1995) I-I'7. Any of these can be used in combination with
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WO 96/40164 PCTIUS96110026
the disclosed nucleosides to achieve a desired effect. Nonlimiting examples
of nucleotide pmdrugs are described in the following references. .
Ho, D.H.W. (1973) Distribution of Kinase and deaminase of lp-D-
arabinofuranosylcytosine in tissues of man and muse. Cancer Ices. 33, '
2816-2820; Holy, A. (i993) Isapolar phosphorous-modified nucleotide
analogues. In: De Clercq (Ed.), Advances in Antiviral Drug Design, Vot.
I, JAI Press, pp. 179-231; Hong, C.L, Nechaev, A., and West, C.R.
(1979x) Synthesis and antitumor activity of lp-D-arabinofuranosylcytosine
conjugates of oortisoI and cortisone. Bioc>~em. Biophys. Rs. Comrnun. 88,
1223-1229; Hong, C.L, Nechaev, A., Kirisits, A.J. Buchheit, D.J. and
West, C.R. (1980} Nucleoside conjugates as potential antitumor agents. 3.
Synthesis and antitumor activity of 1-((i-D-arabinofuranosyl)cytosine
conjugates of corticosteriods and selected lipophilic atcohols. J. Med.
Chem. Z8, 171-1?7; Hostetler, K.Y., Stuhtniller, L.M., Lenting, H.B.M.
van den Hosch,
H. and Richman, D.D. (1990) Synthesis and antiretriovira! activity of
phospholipid analogs of azido#hymidine and other antiviral nucleosides. J.
Biol. C~rem. 265, 6112-6117; Hostetler, K.Y., Carson, D.A. and
Richman, D.D. (1991); Phosphatidylazidothymidine: mechanism of
2 o antiretroviral action in CEM cells. J. Biol. Chem. 266, 11714-11717;
Hostetler, K.Y., Korha, B. Sridhar, C., Gardener, M. (1994x) Antivital
activity of phosphatidyl-dideoaycytidine in hepatitis B-infected cells and
enhanced hepatic uptake in mice. Antivtral Re',s. 24, 59-67;
Hostetler, K.Y., Richman, D.D., Sridhar, C. N. Felgner, P.L, Felgner, J.,
Ricci, 1., Gardener, M.F. Selleseth, D.W. and Ellis, M.N. (1994b)
Phosphatidylazidothymidine and phosphatidyl-ddC: Assessment of uptake
in mouse lymphoid tissues and antiviral activities in human
immunodeficiency virus-infected cells and in rauscher leukemia virus-
infected mice. Antimicrobial Agerrtr Cytemother. 38, 2792-2797; Hunston,
-14-
CA 02538205 1996-06-07
WO 961401b4 PCT/US9611002b
R.N., Jones, A.A. McGuigan, C., Walker, R,T., Balzarini, J., and De
Clercq, E. (1984) Synthesis and biological properties of some cyclic
phosphotriesters derived from 2'~eoay 5-fluorouridine. J. Med. Chem.
27, 440-444;11, Y.H., Moog, C., Schmitt, G., Bischoff, P. and Luu, B.
(1990); Monophosphoric acid diesters of 7~i-hydroxychoIesterol and of
pyrimidine nucleosides as potential antitumor agents: synthesis and
preliminary evaluation of antitumor activity. J. Med. Chem. 33, 2264-
2270; Jones, A.S., McGuigan, C.,11V~, R.T., Balzarini, J. and
DeClercq, E. (1984) Synthesis, properties, and biological activity of some
l o nucleoside cyclic phosphorxmidates. J. Chem. Soc. Perkin Trans. I, 1471-
1474; Juodka, H.A. and Smrt, J. (1974) Synthesis of ditribonucleoside
phosph(P-~N) amino acid derivatives. Coll. Czech. Chem. Comm. 39,
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CA 02538205 1996-06-07
WO 96/x0164 >'CTN596/10026
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deoxythymidine (AZ'17 as anti-HIV compounds. Aruiviral Q~em.
Chemother. I, 107-113; McGuigan, C., O'Connor, T.J., Nieholls, S.R.
2o Nickson, C. and Kinchington, D. (1990b) Synthesis and anti-HIV activity
of Borne novel substituted dialky phosphate derivatives of AZT and ddCyd.
Antiviml Deem. Chernother. l, 355-360; McGuigan, C., Nicholls, S.R.,
O'Connor, T.J., and Kinchington, D. (1990c) Synthesis of some novel
diatkyl phosphate derivative of 3'-modified nucleosides as potential anti-
AIDS drugs. Aruiviral C~rrr. Ctother. 1, 25-33; McGuigan, C.,
Devine, K.G., O'Connor, T.J., and Kinchington, D.(199I) Synthesis and
anti-HIV activity of some haloalky phosphoramidate derivatives of 3'-
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CA 02538205 1996-06-07
WO 96/40164 PCTIUS96110026
Pathirana, R.N., Mahmood, N., Devine, K.G.. and Hay, A.J. (1992) Aryl
phosphate derivatives of AZT retain activity against HIV 1 in cell lines
which are resistant to the action of AZT. .lruivirnl Res. 17, 311-321;
McGuigan, C., Fathirana, R.N., Choi, S.M., Kinchington, D. and
O'Cannor, T.J. (1993x) Phosphoialnidate derivatives of AZT as inhibitors
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97-101; McGuigan, C., Parhirana, R.N., B~alzarini, J. and De Clercq, E.
(1993b) Intracellular delivery of bioactive AZT nucleotides by aryl
phosphate derivatives of AZT. J. Med. t:7sem. 3as, 1048-1052.
io Alky hydrogen phosphonate derivatives of the anti-HIV agent AZT
may be less tonic than the parent nucleoside analogue. Antiviral tfiem.
C~entothtr. 5, 271-217; Meyer, R. H., Jr., Shaman, D.A. and Robins,
R.K. (1973) Synthesis of purine nucleoside 3',5'-cyclic phosphoramidates.
Tetrahedron Left. 2b9-272; Nagyvary, J. Gohil, R.N., Kirchner, C.R, and
Stevens, J.D. (1973) Studies on neutral esters of cyclic AMP, Biochem.
Biophys. Res t.onsmun. 55, 1072-10'17; Namane, A. Gouyette, C., '
Fillion, M.1'., Fillion, G. and Huynh-Dinh, T. (199Z) Improved brain
delivery of AZT using a glycosyl phosphotriester prodrug. J. Med. Chem.
35, 3039-3044; Nargeot, !. Nezbonne, 1.M. Bngels, J. and I,eser, H.A.
no (1983) Natl. Acad. Sci. U.S.A. 80, 2395-2399; Nelson, K.A., Hentrude,
W.G., Stser, W.N. and Hutchinson, J.P. (I987) The question of chair-
twist equilibria for the phosphate rings of nucleoside cyclic
3',3'monopl~sphatec. ~I~TMR and a-ray crystallographic study of the
diasteromers of thymidine pheayi cyclic 3',5'-monophosphate. 1. Am.
z5 Chem. Soc. 109, 4058-4064; Nerbonne, J.M., Richard, S., Nargeot, J.
and Lester, H.A. (1984) New photoactivatable cyclic nueieotides produce
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Gouyetie, C., Dupraz, B. and Huynh-Dinh, T. (1989) Synthesis and
-17-
CA 02538205 1996-06-07
WO 96!40164 PCT/IJ596l10028
transmembrane transport studies by NMR of a glucosyl phospholipid of
thymidine. J. Am. Chem. Sec. 111, 4270-4277; Ohno, R., Tatsumi, N.,
Hirano, M., imai, K. Mixoguchi, H., Nakamura, T., Kosaka, M.,
Takatuski, K., Ysmaya, T., Toyama, K., Yoshida. T., Masaoka, T.,
Hashimoto, S., Ohshima, T., IClmura, L, Yamada, K. and Kimura, J.
(I99I) Treatment of myelodysplastic syndromes with orally administered
1-~i-D-rabinofuranosylcytosine -5'stearylphosphate. Oncology 48, 451-
455.
Palomino, E., Kcssie, D. and Horwitz, J.P. {1989) A dihydropyridine
l0 carrier system for sustained delivery of 2',3'dideaaynucleosides to the
brain. J. Med. Chem. 32, 622-625; Perkins, R.M., Barney, S., Wittroek,
R., Clark, P.H., Levin, R. Lambert, D.M., PeEteway, S.R.,
Serafinowsl~, H.T., Banley, S.M., Jackson, S., Hamden, M.R. Ashton,
R., Sutton, D., Harvey, J.J. and Brown, A.G. (1993) Activity of
>3RI~47923 and its oral prodrug, SB20365yA against a rauscher routine
leukemia virus infection in mice. Antiviral Res. 20 (Suppl. n. 84;
Piantadosi, C., Marasco, C.J., Ir., Morns-Natschke, S_L., Meyer, K.L.,
Gumus, F., Surles, J.R., Ishaq, K.S., Kuceia, L.S. Iyer, N., Wallen,
C.A., Piantadosi, S. snd Modest, E.J. (1991) Synthesis and evaluation of
2 o novel ether lipid nucleoside conjugates for anti-HIV-1 activity. J. Med.
Chem. 34, 1408-1414; Pompon, A., Isfebvre, L, lmba~ch, J.L., Kahn, S.
and Farquhar, D. (1994) Decomposition pathways of the mono- and
bis(pivaloyloaymetllyl) esters of azidothymidine-5'-monophosphate in cell
extract and in tissue culture medium; an application of the ' on-line 1SRP-
cleaning' HPLC technique. Antiviral Chem. Chemother. 5, 91-98;
Postemark, T. (1974) Cyclic AMP and cyclic GMP. Annu. Rev.
Pharmacol. 14, 23-33; Prisbe, E.J., Martin, J.C.M., McGee, D.P.C.,
Barker, M.F., Smce, D.F. Duke, A.E., Matthews, T.R. and Verheyden,
1.P.J. (1986) Synthesis and antiherpes virus activity of phosphate an
-18-
CA 02538205 1996-06-07
WO 96/4016A PC'flI3S96It0026
phosphonate derivatives of 9-[(I,3-dihydroxy-2-propoxy)methyl] guanine.
1. Med. Chem. 29, 671-675; Pucch, F., G~selin, G., lxfebvre, L,
Pompon, A., Aubertin, A.M. Dirn, A. and Imbach, J.L. (1993)
Intracellulaur delivery of nucleoside monophosphate through a reductase-
mediated activation process. Antiviral Res. 22, 155-174; Pugaeva, V.P.,
Klochkeva, S.L, Mashbits, F.D. and Eiaengart, R.S. (I969).
Toxicological assessment and health standard ratings for ethylene
sulfide in the industrial atmosphere. Gig. Trf. Pm~ ?abol. 13, 47-48
CChem. Abstr. 72, 212); Robins, R.K. (1984) The potential of nucleotide
1 o analogs as inhibitors of retroviruses and tumors. Pharm. Res. ! 1-18;
Rosowsky, A., Kim. 5.H., Ross and J. Wick, M.M. (1982) Lipophilic 5'-
(alkylphosphate) esters of 1-p-D-arabinofuranosylcytosine and its 1V~-aryl
and 2.2'-anhydro-3'0-aryl derivatives as potential prodrugs. J. Med.
Chem. 25, 171-178; Ross, W. (1961) Increased sensitivity of the walker
turnout towards aromatic nitrogen mustards carrying basic side chains
following glucose pretreatment. Biochem. Pharm. 8, 23S-240; Ryu, e.K.,
Ross, R.J. Matsushita, T., MacCoss, M., Hong, C.I. and West, C.R.
(1982). Phospholipid-nucleoside conjugates. 3. Synthesis and preliminary
biological evaluation of 1-~-D-arabinofuranosyleytosine S'diphasphate[-j,
Z~iaeylglycemls. J. Med. Chem. 25, 1322-1329; Saffhill, R. and Hume,
W.J. (1986) The degradation of 5-iododeoxyurindine and S-
bromoeoxyuridine by serum from different sources and its consequences
for the use of these compounds for incorporation into DNA. Chem. Bial.
Interact. 57, 347-355; Saneyoshi, M., Morozumi, M., Kodama, K.,
Machida, J., Kuninaka, A. and Yoshino, H. (I980) Synthetic nucleosides
and nucleotides. XVI. Synthesis and biologics! evaluations of a series of
1-p-D-arabinofuranosyleytosine 5'-alley or arylphosphatrs. Chem. Pharm.
Bull. 28, 2915-2923; Sastry, J.K., Nehete, P.N., Khan, S., Nowak, B.J.,
Plunkett, W., Arlinghaus, R.B. and Farquhar, D. (1992) Membrane-
-19-
CA 02538205 1996-06-07
WO 96/40164 PCTlUS96J10026
permeable dideoxyuridine 5'-monophosphate analogue inhibits human
ilnmunodeficieacy virus infection. Mol. 1'harnlacol. 41, 441-445; Shaw, '
J.P., Jones, R.J. Arirnilli, M.N., I:ouie, M.S., Lee, W.A. and Cundy,
K.C. (I994) Oral bioavaiIability of PMF.A from PMEA prodrugs in male
Sprague-Dawley rats. 9th Annual AAPS Moelirtg. San Diego, CA
(Abstract). Shuto, S., Ueda, S., Imarnura, S., Fukukawa, K. Matsuda, A.
and Ueda, T. (19$'~ A facile ono-step synthesis of
5'phosphatidylnucleosides by an enrymatic two-phase reaction.
Tetrahedron Left. 28, 199-202; Shuto, S., Itch, H., Ueda, S., Imamura,
1o S., Knkukawa, K., Tsu,~ielo, M., Matsuda, A, and Ueda, T. (1988) A
facile enzymatic synthesis of 5'-(3-sn-phosphatidyl)nucleosides and their
antileukemic activities. Chem. Pharm. Bull. 36, 209-217. A prefenced
phosphate prodrug group is the S-aryl-2-thioethyi group, also referred to as
"SATE".
hreu~ration of the Active Compounds
The nucleosides used in the disclosed method to treat HBV infections
in a host organism can be prepared a~ccoidittg to published methods. &L-
Nucloosides can be prepared from methods disclosed in, or standard
modifications of methods disclosed in, for example, the following
publications: Jeang, et a1.,1. of Med. Chem., ~, 182-195, 1993;
European Patent Application Publication No. 0 285 884; Gbnu-DeIlac, C.,
G. Gosselin, A.-M. Aubertin, G. Obert, A. Kirn, and 1.-L. Imbach, 3-
Substituted thymine a-L-n~xleoside derivatives as potential antiviral agents;
z 5 synthesis and biological evaluation, ,pi,~~tiviral Chem. Chemother. 2:83-
92
(I991); 3ohansson, K. N. G., B. G. Lindbor~g, and R. Noreen, European
Patent Application 352 248; Mansuri, M. M., V. Farina, J. E. 5tarrett, D.
A. Berzigni, V. Brankovan, and J. C. Martin, Preparation of the geometric
isomers of DDC, DDA, D4C and D4T as potential anti-HIV agents,
-20-
CA 02538205 1996-06-07
WO 96!40164 PCTIU596ft0026
1:65-b8 (1991); Fujimori, S., N_ Iwanami, Y,
Hashimoto, and K. Shudo, A convenient and stereoselective synthesis of
2'-deoxy-B-~L-ribonucieosides, Nucleosides do ucleotides 11:341-349
. (1992); GErtu-Dellac, C., G. Gosselin, A.-M. Aubertin, G. Obett, A.
Kirn, and J.-L. Imbach, 3-Substiwted thymine ac-L-nucleoside derivatives
as potential antivirai agents; synthesis and biological evaluation, Antiviral
Chem. Chemothe~ 2:83-92 (1991); Holy, A, Synthesis of 2'-deoxy-L-
uridine, ~~. 2:189-192 (1992); Holy, A., Nucleic acid
components and their analogs. CLI)Z. Preparation of 2'-deoxy-L-
1o riba~nucleosides of the pyrimidine series. Coll~C,~h Chem Commun.
3y:4072-4087 (1992); Holy, A, 2'~eoxy-L-uridine: Total synthesis of a
uracil 2'-deoxynucleoside from a sugar 2-aminooxazoline through a 2.2'-
anhydronucleoside intermediate. In: Townsend LB, Tipsan RS, od.
Nucleic Acid Chem. New York: Whey, 1992: 347-353. vol 1) (1992);
Okabe, M., R.-C. Sun, S. Tan, L. Todaro, and D. L. Coffee, Synthesis
of the dideoxynucleosides ddC and CNT fmm glutamic acid,
ribonolactone, and pyrinudine bases. J~~ 53:4780-4786 (1988):
Robins, M. J., T. A. Khwja, and R. K. Robins. Purine nucleosides.
XXpC. Synthesis of 21-deoxy-L-adenosirve and 21-deoxy-L-guanosine and
2 o their alpha anomers. ~,QC~ Chem. 33:363-b39 (1992); Gnu-Dellac, C.,
Gosselin G., Aubertin A-M, Obert G., Kirn A., and Imbach J-L, 3'-
SubstiWted thymine a-L-nucleoside derivatives as potential antiviral agents;
synthesis and biological evaluation. A.ntivi_raf Chem hemQther. 2(2):83-
92 (1991); GEnu-Dellac, C., Gosselin G., Imbach J-L; Synthesis of new
~ s 2'-deoxy-3'-substituted-a-L-threo-pentofuranonucleosides of thymine as a
potential antiviral agents. Tel Lett 32(1):79-82 (1991); Gnu-Dellac, C.,
Gosselin G., Imbach 1-L. Preparation of new acylatod derivatives of L-
arabino-furanose and 2-deaxy-1-erythro-penwfvranose as precursors for the
synthesis of 1-pentofiuanosyl nucleosides. 216:240-255 (1991); and G~nu-
-21-
CA 02538205 1996-06-07
Dellac, C., Gosselin, G., Puech, F, et al. Systematic synthesis and antiviral
evaluation of
a-L-arabinofuranosyl and 2'-deoxy-a-L-erythro-pento-furanosyl nucleosides of
the five
naturally occurring nuclei acid bases. 10(b):1345-1376 (1991).
2',3'-Dideoxycytidine (DDC) is a known compound. The D-enantiomer of DDC
is currently being marketed by Hoffman-LaRoche under the name Zalcitabine for
use in
the treatment of persons infected with HIV. See U.S. Patent Nos. 4,879,277 and
4,900,828.
Enantiomerically pure ~i-D-dioxolane-nucleosides such as ~3-D-FDOC can be
prepared such as disclosed in detail in WO 92/010497. The process involves the
intitiaI
preparation of (2R,4R)- and (2R,4S)-4-acetoxy-2-(protected-oxymethyl)-
dioxolane from
1,6-anhydromannose, a sugar that contains all of the necessary stereochemistry
for the
entantiomerically pure final product, including the correct diastereorneric
configuration
about the 1 position of the sugar (that becomes the 4'-position in the later
formed
nucleoside). The (2R,4R)- and (2R,4S)-4-acetoxy-2-(protected-oxymethyl)-
dioxolane is
condensed with a desired heterocyclic base in the presence of SnCl4, other
Lewis acid, or
trimethylsilyl triflate in an organic solvent such as dichloroethane,
acetonitrile, or
methylene chloride, to provide the stereochemically pure dioxolane-nucleoside.
Enzymatic methods for the separation of D and L enantiomers of cis-nucleosides
are disclosed in, for example, Nucleosides and Nucleotides, 12(2), 225-236
(1993);
European Patent Application Nos. 92304551.2 and 92304552.0 filed by Biochem
Pharma, Inc.; and PCT Publication Nos. WO 91/11186, WO 92/14729, and WO
92/14743 filed by Emory University.
Separation of the acylated and alkylated racemic mixture of D and L
enantiomers
of cis-nucleosides can be acconrzplished by high performance
- 22 -
CA 02538205 1996-06-07
WO 96140164 PCTIUS96110026
Liquid chromatography with chiral stationary phases, as disclosed in PCT
Publication No. WO 92114729.
Mono, di, and triphosphate derivative of the active nucleosides can
be prcpared as described according to published methods. The
monophosp6ate can be pttpa~d accordeng to the procedure of In~ai ct al.,
J. Ora. Chem., 34(6), 154? 1350 (June 1969). The diphosphate can be
prepared according to the proceduze of Davisson et al., J. Orgasm.,
52(9), 1794-1801 (1987). The triphosphate can be prepared according to
the procedure of Hoard et a1.,1. Am. Chem. Soc., 87(8), 1785-1788
to (1965).
I O
S,~O ~ ~,~0 OH n R.~ ~O p,~g'~x
--~SH '''CS-~ - 'S
2
2 o O Base
~ O ~t Yt Ya
O p..._0 Y,
2
2 O
8is (SAT>~ p-L-ddoMP
an Y', Y2, Y', and Y4 are independently H, OH, Ns, NR'Ri, NOi, NOR3, -
O-alkyl,
_23_
CA 02538205 1996-06-07
WO 96/40164 PCTIUS961100Z6
-O-aryl, halo (including ~, Cl, Br, or n, -CN, -C{O)NHx, SH, -S-allryl, or
-S-aryl, and wherein typically three of Y~, Y2, Y3, and Y4 are either H or
OH. The -OH substituent, when present, is typically a Y' or Y' group.
As illustrated in the structure, Yx and Y4 are in the arabino (erythro)
configuration, and Yt and Y3 are in the threo (ribose) configuration. The
base is a purine or pyrimidine. AltGrnatirvely, the psuedo-sugar moiety is a
1,3-oaathiolane (as in FTC and BCH-184 or 3TC or is a 1,3-dioxolane
derivative). (t) ICHZCH20H, DBU/C6HsCH~: (ii) CIxPN(iPr)x,
NEt3lTHF; (iii) S-L-dideoxynucteoside, IH-tetrazole/THF, then
Zo C1C~C03H/CH2C111H-Tetrazole (0.218, 3.0 mmol) was added to a
stirred solution of p-I; dideoxynucleoside (1.0 mmol) and the appropriate
phosphoramidite ~ (1.2 mmol) in tettahydrofuran (2mL) at room
temperature. After 30 minutes, the reaction mixture was cooled to -40°C
and a soluti~ of 3-chiomperoxybenzaic acid {0.23 g, 1.3 mmol) in
1s dichlommethane (2.5 mL) was added; the mixture was then allowed to
warm to room temperature over 1 h. Sodium sulfite (1096 solution, 1.3
mL) was added to the mixture to destiny the excess 3-chtoroperoxybenzoic
acid, after which the organic layer was separated and the aqueous layer
washed with dichloromethane {2 s 10 mL). The combined organic layers
20 were washed with saturated aqueous sodium hydrogen carbonato (5 mL),
then water (3 a S mL), dried over sodium sulfate, filtered and evaporated
to dryness under reduced pressure. Column chromatography of the residue
on silica gel afforded the tine Bis(SA"Tfi) ~-L-ddxm~.
-24-
CA 02538205 1996-06-07
WO 96/4U164 PC'F/I3S96l10U26
= p-L-2',3'-Dideozyadenosin-5'-yI bis (2-pivaloylthioethyl)
phosphate [Bis (SATE) (3-L-ddAMp].
~o
(CH~3C-C~ ~CHZ_p PN(iPr)z
S Cliz 2
~-L-ddA. 1 H-teuazolelI'EiF
then CIC~HyC03HICf3~Ch
then silica gcl column chromatography
O
(Ct"ia)aC-C~ C
S-Cli2
$is (SATE)/3-L_ddAMp
Following the above general procedure, pure l3is(SATE~~-L-ddAMP_ was
obtained as a colorless oil in 72 % yield after silica gel column
chromatogtaptty [eluent: stepwise gradient of methanol (0-3%) in
dichloromethaale]; ' NMR (DMSO - d6) 8 ppm: 8.26 and 8. i3 (2s, 2H
each, H-2 and H-8), 7.20 (br s, 2H, NHS, 5.24 (t, 1H, H-1'; T=6.0 Hz),
4.35 - 4.25 (m, 1H, H-4'), 4.25-4.00 (m, 2H, H-5', 5"), 3.96 (m, 4H, 2
SCHzCHiO), 3.04 (t, 4H, 2 SCH1CH20 ; J = 6.3 Hz), 2.5 - 2.4 (m, 2H,
H-2',2") 2.2-2.0 (m, 2H, H-3',3"), 1.15 [s, 18H, 2 (CH3)3C]; 31~,NMR
(DMSO-db) 8 ppm = -0.76 (s) ; W {EtOH) , 7l ~ = 259 nm {e 15400);
CA 02538205 1996-06-07
WO 96140164 PCTN596/10026
mass spectrum (performed in: glycerol, thioglycerol, 1:1, ulu), FAB > O
604 {M+H)+, 136 (BHP+. .
~~~gueral s~~~,me for tie ~ros~xLt'N;~y~th~~,~~f 3'-cnbstit~ted B-L-
OH
RO~~~
Y' 11~~YO
Compound 8
[see Fig. L/2 of
tht French patent
~e Appendiz 4
Bass OX
Ro~Base
~/' .~/0
0
2 ~ ~ ease
OH
O Ra Baae
r
0
RO-t 8iee
V~-O~X
O
3 0 ~ ~o~aaae
~'' ~r
«erythrOo»
conftgtuation
RO
HO~~
~OIy
<atueo»
4 0 contiguranon
IE
v = any ~cH,-c, c6Hs-c)
-26-
CA 02538205 1996-06-07
WO 96/40164 PGTICJS96/10026
X = Leaving group [CH3 S4i, CH3 CaH, SOi, CF3 SOa]
Y. Y' = F. N3. W R: LRi.Rs = H. alkyl, aryl].
NOi, NOR [R = H, alkyl, aryl], O-alkyl, O-aryl, etc
EX9~E = 1-(3-Arido-2-3-dideozy-~i-Irerythno-penwfuranosyl)
thytnine [~i-L-AZTj
CH3
'(C6FIs)3
D!~AD
PA p
20
O
11N ~ CIi3
O~N
NO
a
O
~-L-AZT
4096 yield from g
30
CA 02538205 1996-06-07
WD 96140164 PCT/US96I1Q026
A mixture of diethyl azodicarboxyiate (0.46 mL; 2.9 mmol) and
diphenyl phosphorazidate (0.62 ml; 2.9 mmol) in 'TI-iF (2.9 ml) was added
dropwise over 30 min. to a solution of 1-(2-decay-5-O-monomethoxytrityl-
(i-L-three-pentofuranosyl) thymine $ [0.5 g, 0.97 mmol] and
triphenylphosphine (0.76g, 2.9 rnmol) in THF l 1.6 ml) at 0°C. The
mixture was stirred for 3.5h at room temperature, and ethanol was added.
After concentration to dryness in vacuo, the residue was dissolved in a
mixture of acidic acid (240 ml) and water (60 ml) in order to remove the
mMTr protecting group. The mixture was stirred for 5 hours at room
1o temperature and was diluted with toluene. T'he sepazated aqueous phase
was concentrated to dryness in vacuo. T'he residue was purified over a
silica gel column eluted with ethyl acetate to afford ~-L-AZT' (105 mg,
40%, crystallized from ethyl acetate). The physicochemical data of p-L_
AZT were in accordance with literature data [J. Wengel, J-Lau, 1~.B.
Ledersen, C.N. Nielsen, J. Org. Chem. ~ (11), 3591-3594 (199I)].
-28-
CA 02538205 1996-06-07
WO 96140164 PCT/LJS96/10026
~p~,~ral Scheme for the Stereas~fc Sv~~lhesis tt,~ ~~'_~,'b~tituted 9-1~.
RO~~
HO
L?
CO(IlpOtlnd j~
[see Fig. 1I2 of
~hc FrenCh patent'
5cx Appendix 4
OV
Beae
RO RO
xo
0
0
~ OH
RO
r
O R Hase
O
1 ox
RO
o r
Ho 8°'s
0
«thrto>s
conti oration
RO
'~yO
WO~~
Y
O
«erythtn»
configuration
O O
v = acyl [CH3-C C6H5-C]
X .= Leaving group [CHs SO~,~ CHs Cs~~ H. ~3 Sue]
Y. 1" = F. Ns~ NRtRx fRt~Rz = H. ~Yl~ ~1'1]>
3 o NOs, NOR [R = H, alkyl, aryl], O-alkyl, O-aryl, etc.
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CA 02538205 1996-06-07
WO 96140164 PCTlU596110026
E$~ ~ I-(2-Fluoro-2,3-dideoxy-(3-L-wren-pentofuranosyl)-5_
fluorocytosine [2'-F-~-L ~-L-FddC]
0
NON F H~CO CHZ_ F
o~N CH,C6~ N
DBUICH=CV
HO y~0
96~ yield
DAST.
I O a CFIICh,
CsH~V
O o
~F (NH,y~Ce(NO~)e H~O~~YN
15 F N CH3 ~ O~N
P
Bz0
8z0
6096 yitid ~7'b Yield
O
O
f~wesson's
~xgendCHZCh
roflux
NHZ
20 ~ F
_CH~OHIf~(H3 ,~
10~C O~N
F
NO
0
2'-F-d-L-Fddc
2 5 6x96 yirld
Hitherto unknown 2'-F-~i-L-FddC was synthesized in five steps from
1-(5-O-benzoyl-3-decay-~-I~-crydun-pentofuranosyl}-5-fluomutacil L7
with an overall yield of 2896, m.p. 209-210°C (crystallized from
absolute
-30-
CA 02538205 1996-06-07
WU 96140164 PC'1'1US96110026
ethanol); W (Et OH) l~,x 276 ~" (s, 9000), 7~ 226 (e, 4000); "F_
NMR (DMSO-d6) 8 ppm : -179.7 (m, F2,) , -167.2 (dd, Fs; JF.s = 7.3 Hz,
JF.,. = l:SHz);'H-NMR (DMSO~ 800m : 8.30 (d , 1H, H-6: Ja.F = 7.3
. Hz), 7.8-7.5 (br s, 2H, NHS}, 5.80 (d, 1H, H-1' J,.,F = 17.4 Hz), 5.34 (t,
1H, OH-5'; J = 4.8 Hz), 5.10 (c~, 1H, H-2'; J=.,F = 51.2 Hz; J2.,3. _
3.4 Hz), 4.3 (m, 1H, H-4'), 3.8-3.6 (m, 2H, H-5',5"), 2.2-2.0 (m, 2H,
H-3', H-3"); mass spectra (performed in: glycerol-thioglycerpl, 1:1 v!u),
FAB > 0:248 (M+H)*, 130 {BHP+; FAB < 0:246 (M-H)' ; [a]Z°a = -
16.5-(-c 0.85, DMSO). Anal. Calc. far C9HmN30jF2 : C, 43.73; H,
1o 9.49; N. 17.00; F. 15.37 . Found: C, 43.56; H,
4.78; N, 16.75; F, 14.96.
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CA 02538205 1996-06-07
WO 96140164 PCTNS96I10026
G~Cylose
«~iCO. HjSO,. CuSO,.
That NH,OH
HCIIH=O ~ NaHCO~hfiO
O ~~~,,/O
BxO~ ~H~OCI O-. O OBz
1 l p~~ HO-~ I ~~C~ Bz0
OH ~ 1 C3H~~I-CHCh (CH;CO),O. ~ OAc
(S)COm)= ~ HZSO, O
l(CHiG~
glyoosidic
(CH,S)),SiFi, AIHN O~ / O ooodmsation
O
Bz0 / T~ ~z
ZO O C lm ~ ~ Bzo--~8ase
t) CH,cnpH ss~
HrSO, O
2) (CH300yj0,
Bz0 ~~ ~Z~a-HxO
OAc lPYridioe-CH,COOHI
OAe -,.
L 14 Bz0 ease
O
1) CaFf,OC(SxI
D~ / CH~CT~J
Bx0 Base 2) Hu3SnH, A~N
Ac0 ldioxaoe 08z
O Bz0-~~Base
CH,ONa/CH,OH ~ HO Base O
NH, ! Ctt,OH m NHr l CH,OH ~
,~ OH
H''OZ=mM'1'r) HO-~8ase
O «
~ F (R=THDPSI) O
ltC1 l ~y.;a;ue « Z
NH, l CH,OH (R o Hz « Ac) RC1 I pyrid~oe
RO Bax
HO I) CatIspC(Sxi OH
O DMAP/CH,Ct~O ~ ~~(Sxl RO--~~Ba~e
-rt
2) Bu,Saff- AIBN -
l Talneoe
Q 2) Hu~SoFI, AIBN O
3 d ! dioxane
Schema I: Bases = pwines or pytimidines, eventuellemmt conveuablemeat
pcotegeea; R
a Beazoyl (Bz) Acetyl (Ac), monomethoaytrityl (mMTr) or
tertiarybutyldiphenylsilyl
(-ranpsn
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CA 02538205 1996-06-07
WO 96140164 t'CTIUS96/10026
II. Anti-HBV Activity of Nucleosides
The ability of the active compounds to inhibit HBV can be measured
by various experimental techniques. The assay used herein to evaluate the
ability of the disclosed, compounds to inhibit the replication of HBV is
described in detail in Korba and Germ, Antiviral Res. 19: 55-'70 (1992).
For purposes of illustration only, and without limiting the invention, the
results of the evaluation of toxicity and anti-HBV activity are provided
below for B-Ir2',3'-dideozycytidine (B-L-lrddG7, B-L-2',3'-didemcy-S-
1o fluorocytidine (B-L-ddC), and (+rB-D-2-hydrozymethyl-S-(5-
fluorocytasin-1-yl)-1,3~iiozolane ((+}-$-D-FDOC). The toxicity and anti-
HBV activity of (-)-&L-2-hydroxymethyl-5-(5-fluorocyWsin-1-yt)-1,3-
oxathiohme ((-)-$-L-FTC) and B-D-2',3'-dideozycytidine (&D-ddC) are
included as controls. The other compounds disclosed .herein can be
evaluated similarly.
The samples of B-L-ddC and B-L-5-FddC used in the anti-HHV assays
were characterized as follows.
2'.3~'-D'i~i~s~t-B-~v~j~e _f$-L'DDC1. m.p. = 220-220°C; W
2 o (EtOH 95) mix 273 nm, 7~min 252 nm; NMR-~H (DMSO-ds) Sppm =
7.89 (d. 1H. H-6; J = 7.4 Hz). 7.15-6.95 (d large, 2H, NHS, 5.91 (dd.
1H, H-1'; J = 3.0 et 6.5 Hz), 5.66 (d, IH, H-5; J = 7.4 Hz), 4.99 [E.
1H, OH-5'; J - 5.2 Hz]. 4.05-3.95 (m, IH, H-4'), 3.64-3.70 (m, IH, H-
5'; after DiO exchange: dd, 3.64 ppm, J = 3.6 et I2.0 Hz). 3.60-3.50
(m. 1H, H-5"; after D20 exchange: dd, 3.50 ppm, J = 4,1 et 12.0 Hz),
2.30-2.15 (m. 1H, H-2'), 1.9-1.65 (m. 3H, H-2", 3' et 3"); [a]p~°-103.6
(c 0.8 MeOH); mass spectrum (performed in: glytxrol-ttuoglycerol, 50
50. vlv); FAB>0 423 [2M+H]*, 304 (M+glyoerol+H]+. 212
[M+H]*, 112 [BHzJ*, 101 [s]+; FAB<O 210 [M-H]'. Anal. Calc. for
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CA 02538205 1996-06-07
WO 9b/40~ 6A PC'T/US9b/t002b
C9H,3N303 (M = 211.21); C SI.18; H 6.20; N 19.89 found; C 51.34; H
6.25; N 20.12.
~ m.p. .- 1S8-
160°C; UV (EtOH 95) kmax 281 am (e, 8100) et 237 nm (e, 8500); min
260 nm (e, 5700) et 225 nm (e, 7800); NMR - ~H (DMSO-d6) Sppm 8.28
(d. 1H, H-6; J - 7.4 Hz), 7.7-7.4 (d large, 2H, NHS, 5.83 (dd poorly
resolved, 1H, H-1'), 5.16(t. 1H, OH-5';1 = 5.1 Hz), 4.05-3.95 (m, 1H,
H-4'), 3.8-3.70 (m,1H, H 5'; after D20 exchange: dd, 3.71 ppm_ J = 2.7
et ~.3 Hzl, 3.60-3.50 [m. 1H, H-5"; after D20 exchange: dd, 3.52 ~r ;
1o J = 3.3 et 12.3 Hz], 2.35-2.15 (m, 1H, H-2'). 1.95-1.75 (m, 3H, H-2",
3' et 3"): jala~°-80.0 (~ 1.0, DMSO); Mass spectrum (performed in: 3-
nitrobenzyl alcohol] FAB > 0 230 [M+H]+ et 101 [sl+; FAB C O 228 [M-
IIl-. Anal. Calculated for C9H,zN~F'03(M = 229.2(); C 47.1b; II 5.28; N
18.33, F 8.29, Found. C 16.90; H 5.28; N 18.07; F 8.17.
The antiviral evaluations were performed on two separate passages of
cells, two cultures per passage (4 cultures total). All wells, in all plates,
were seeded at the same density and at the same time.
Due to the inherent variations in the levels of both intracellular and
extracellular HBV DNA, only depressions greater titan 3.0-fold (for HBV
Z o virion DNA) or 2.5-fold (for H13V DNA replication intermediates) from
the average levels for these HBV DNA forms in unu~eated cells are
generally considered to be statistically significant [P<0.051 (Korba and
Germ, Antiviral Res. 19: 55-70, 1992). The leveEs of integrated HBV
DNA in each cellular DNA preparation (which remain constant on a per
2s cell basis in these experiments) were used to calculate the leveEs of
intracellular HBV DNA forms, thereby eliminating technical variations
inherent in the blot hybridization assays. .
Typical values for extracelIular HHV virion DNA in untreated cells
range from 50 to 150 pg/ml culture modium (average of approximately 76
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CA 02538205 1996-06-07
WO 96/40164 PCTlUS96lI0o26
pg/ml). Intracellular HBV DNA replication intermediates in untreated
cells range from 50 to 100 pg/ug cell DNA (average approximately 74
pglug cell DNA). In general, depressions in the levels of intracellular
HBV DNA due to treatment with antiviral compounds are less pronounced,
and occur mole slowly, than depressions in the levels of HHV virion DNA.
For reference, the manner in which the hybridization analyses were
performed for these experiments results in an equivalence of approximately
L0 pg intracellular HBV DNA/ug cellular DNA to 2-3 genomic copies per
cell and 1.0 pg of extracellutar HHV DNA/ml culture medium to 3 x IOs
1o viral particles/mi.
Toxicity analyses were performed in order to assess whether any
observed antiviral effects were due to a general effect on cell viability.
The method used was based on the uptake of neutral red dye, a standard
and widely used assay for cell viability in a variety of virus-host systems,
including HSV (herpes simples virus) and HIV.
The test compounds were used in the form of 40 mM stock solutions in
DMSO (frozen on dry ice). Daily aliquots of the test samples were made
and frozen at -20°C so that each individual aliquot would be subjected
to a
single freeze-thaw cycle. The daily test aliquots were thawed, suspended
2 o into culture medium at room temperature and immediately added to the cell
cultures. The compounds were tested at 0.01 to 10 ~cM for antiviral
activity. The compounds were tested far toxicity at concentrations from 1
to 300 ACM. The results are provided in Table 1.
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CA 02538205 1996-06-07
WO 96/401b4 PC'f/US96I10026
....N
y1 0~0n
o T
N
.C N
U
y
W
1
V~ ~ ~ N
N
' ~ .~.--,...,
F7
.~ N W f N
U7 N
1 I I I 1
1
$ ~ M oo.-.
d ~D O1M V1
.-i'
'
N d ~ N
N
~!
O N ~O~ O
fV C O O O
CV
I I I
I
U -~ ~ ~ 000om o0
~
N ", N
.~ 'a'd O
..v
..rt
v
v N ,
'
~t N N_ 00
t~ O
~D etO
C O O O O
H ~ A ~ 1 1
I t~00N Oz
.-n
N
oo ~ nig
M
0 0 0
w ~
r
c 0
cmncEO o
O O O C
N
m
,a N ..
'
~ Q o o L1
W
U
A
A n g o g
w N 0 0 0 0
A 00
O ~ , 1 1 I 1
~I M cnN o n
p ~ ~~,
"'
-. o ~ Q ..
- 4 .
GTr W Q a ~ b
m
W ,Q
z
A
. b U U .c
A ~ A A
, ~ ..
c?' a
~
~yv
0 q
V GrlC1~L
n a ca
-3s-
CA 02538205 1996-06-07
WO 96/40164 PCTIU596/t002fi
Example 2 Toxicity De Compounds
The ability of the active compounds to inhibit the growth of virus in
2.2.15 cell cultures (HepG2 cells transformed with hepatitis virion) was
- evaluated. As illustrated in Table 1, no significant toxicity (greater than
s 50% depression of the dye uptake levels observed in untreated cells) was
observed for any of the test compounds at the concentrations 100 pM. The
compounds were moderately toxic at 300 itM, however, all three
compounds exhibited less toxicity at this concentration than &D-ddC. It
appears that the iC~ of &L-ddC and &IrFddC is approximately twice that
of B-D-ddC.
Toxicity analyses were performed in 9Crwell flat bottomed tissue
culture plates. Cells far the toxicity analyses were cultured and treated
with test compounds with the same schedule as used for the antivirai
evaluations. Each compound was tested at 4 concentrations, each in
triplicate cultures. Uptake of neutral red dye was used to determine the
relative level of toxicity. The absorbance of interna1ixed dye at 510 nM
(As~o) was used for the quantitative analysis. Values are presented as a
percentage of the average As,o values (t standard deviations) in 9 separate
cultures of untreated cells maintained on the same 96-well plate as the test
compounds. The percentage of dye uptake in the 9 control cultures on
plate d0 was 100 t 3. At 150-190 ~eM B-D-ddC, a 2-fold reduction in dys
uptake (versus the levels observed in untreated cultures) is typically
observod in these assays (Rorba and Gerin, Antiviral Res. 19: 55-70,
1992).
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WO 96/40164 1'CT/U596/tOD26
Eacamtpk 3 Anti-Hepatitis B Vinrs Activity ,
The positive tr~nent control, &D-2',3'-dideoxycytosine [&D-ddC],
induced significant depressions of HBV DNA replication at the
concentration used. htevious studies have indicted that at 9-12 pM of B-
D-ddC, a 90% depression of HBY RI (relative to average levels in
untreated cells) is typically observed in this assay system (Korba and Germ,
Antivira! Res. 19: 55-70, 1992). This is consistent with the data presented
in Table 1.
The data presented in Table 1 indicates that all three test compounds
20 ((&L-FddC), (B-L.ddC), and B-D-FDQC)), wera potent inhibitors of HBV
replication, causing depression of HBY virion DNA and HSV RI to a
degroc comparable to, or greater than, that observed following treatment
with B-D-ddC.
Fxampfe 4
The effect of selected B-L-derivatives against Hepatitis B virus
replication in transfectod fiep G-2 cells is described in Table 4.
_38-
CA 02538205 1996-06-07
W0 9b/40164 PGTNS96110026
P~~ h x N
v'
a
z N ~
a
U
N
~
U
W
,nO
~1G ~ 00h
U ~
a
o ~"a ~nr:
g
o m. ~.~ z
U ~ ~ ~ ~ ~ A
V
C~~1C~1f~N
w .o
a
-39-
CA 02538205 1996-06-07
WO 9b140t64 PC'TIU59b/Z002b
Example 5
The Comparative inhibitory effect of selected triphospahtes on
woodchuck hepatitis virus DNA polymexase is set out in Table 5.
Table 2: Comparative inhibitory activities of L..nucleoside
triphosphates on woochuck hepatitis virus DNA polymerise and human
DNA polymerise a and p.
Inhibitor WHB DNA Pol DNA Pol DNA Pol (i
ICS (p,M) a Ki (pM)
Ki (p.M)
~3-L-AZTPP 0.2 > 100 > 100
~i-L-ddATP 2.1 > 100 > 100
3-TC-TP 1.0 > 100 > 100
[3-L-SFDDCTP2.0 > 100 > 100
l0 ).)(I. Preparation of Pharmaceutical Compositions
The compounds disclosed herein and their pharmaceutically acceptable
salts, prodrugs, and derivatives, are useful in the prevenEion and treatment
of HBV infections and other related conditions such as anti-HBV antibody
positive and HBV-positive conditions, chronic liver inflammation caused
by HBV, cirrhosis, acute hepatitis, fulminant hepatitis, chronic persistent
hepatitis, and fatigue. These compounds or formulations can also be used
prophylactically to prevent or retard the progression of clinical illness in
individuals who are anti-HBV antibody or HBV-antigen positive or who
have been exposed to HBV.
2 o Humans suffering from any of these conditions can be treated by
administering to the patient an effective HBV-treatment amount of one or a
mixture of the active compounds described herein or a pharmaceutically
acceptable derivative or salt thereof, optionally in a pharmaceutically
acceptable carrier or diluent. The active materials can be administered by
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CA 02538205 1996-06-07
WO 96/40164 PCT/US96I1002b
any appropriate route, for example, orally, parerltenxlly, intravenously,
intradermally, subcutaneously, or topically, in liquid or solid form.
The active compound is included in the pharmaceutically acceptable
carrier or diluent in an amount sufficient to deliver to a patitnt a
therapeutically effective amount without causing serious toxic effects in the
pati~t treated.
A preferred dose of the active compound for all of the above-
mentioned conditions will be in the range from about 1 to 60 mg/kg,
preferably 1 to 20 mglkg, of body weight per day, more generally 0.1 to
1o about 100 mg per lalogram body weight of the recipient gcr day. The
effective dosage range of the pharnlaceufically acceptable derivatives can
be calculated based on the weight of the parent nucleoside to be delivered.
If the derivative exhibits activity in itself, the effxtive dosage can be
estimated as above using the weight of the derivative, or by other means
i5 known to those skilled in the art. In one embodiment, the active compound
is administered as described in the product insert or Physician's Desk
Reference for 3'-azido-3'~leoxythymidine (AZT), 2',3'-dideoxyinosine
(DDn, 2',3'-dideoxycytidine (DISC), or 2',3'-dideoxy-2',3'-
didehydrothymidine (D4T) for HIV indication.
2 o The compound is conveniently administered in unit any suitable dosage
form, including but not limited to one containing 7 to 3000 mg, preferably
70 to 1400 mg of active ingredient per unit dosage form. A oral dosage of
50-1000 mg is usually convenient.
Ideally the active ingredient should be administered to achieve peak
2 5 plasma concentrations of the active compound of from about 0.2 to 70 ~M,
preferably about 1.0 to 10 NM. This may be achieved, for example, by the
intravenous injection of a 0.1 to 596 solution of the active ingredient,
optionally in saline, or administered as a bolus of the active ingredient.
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WO 96/40164 PCT/US9611002b
The active compound can be provided in the form of pharmaceutically
acceptabie salts. As used herein, the term pharmaceutically acceptable salts
or complexes refers to salts or complexes of the nucloosides that retain the
desired biological activity of the parent compound and exhibit minimal, if
any, undesired toxicological effects. Nonlimiting examples of such salts
are (a) acid addition salts formed with inorganic acids (for example,
hydrochloric acid, hydrabmmic acid, sulfuric acid, phosphoric acid, nitric
acid, and the like), and salts formed with organic acids such as acetic acid,
oaaHc acid, tartaric acid, succinic acid, malic acid, ascorbic acid, benzoic
1 o acid, tannic acid, pamoic acid, alginic acid, polyglutamic acid,
naphthalenesulfonic acids, naphthalenedisuIfonic acids, and
polygalacturonic acid; (b) base addition salts formed with rations such as
sodium, potassium, zinc, calcium, bismuth, barium, magnesium,
aluminum, copper, cobalt, nickel, cadmium, sodium, potassium, and the
like, or with an organic ration formed from N,N-
dibenzylethylene-diamine, ammonium, or ethylenediamine; or (c)
combinations of (a) and {b); e.g., a zinc tannate salt or the like.
Modifications of the active compound, specifically at the 1i6 or N' and
5'-O positions, can affect the bioavailability and late of metabolism of the
Z o active species, thus providing control over the delivery of the active
species.
The concentration of active compound in the drug composition will
depend on absorption, inactivation, and excretion rates of the drug as well
as other factors known to those of skill in the art. It is to be noted that
dosage values will also vary with the severity of the condition to be
alleviated. It is to be further understood that for any particular subject,
specific dosage regimens should be adjusted over time according to the
individual need and the professional judgment of the person administering
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CA 02538205 1996-06-07
WO 9b/4U164 PCTNS96/10026
or supervising the administration of the compositions, and that the
concentration ranges set forth herein are exemplary only and are not
intended to limit the scope or practice of the claimed composition. The
active ingredient may be administered at once, or may be divided into a
number of smaller doses to be administered at varying intervals of time.
A preferred mode of administration of the active compound is oral.
t~ral compositions will generally include an inert diluent or an edible
carrier. They may be eneiosed in gelatin capsules or compressed into
tablets. For the purpose of oral therapeutic administration, the active
1 o compound can be incorporated with excipients and used in the form of
tablets, troches, or capsules. Pharmaceutically compatible binding agents,
andlor adjuvant materials can be included as part of the composition.
The tablets, pills, capsules, troches and the like can contain any of the
following ingredients, or compounds of a similar nature: a binder such as
microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as
starch or lactose, a disintegrating agent such as alginic acid, iflrimogel, or
corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant
such as colloidal silicon dioxide; a sweetening agent such as sucrose or
saccharin; or a flavoring agent such as peppermint, methyl salicylate, or
orange flavoring. When the dosage unit form is a ~SUIe, it can contain,
in addition to material of the above type, a liquid carrier such as a fatty
oil.
1n addition, dosage unit forms can contain various other materials which
modify the physical form of the dosage unit, for example, coatings of
sugar, shellac, or other enteric agents.
2 5 The active compound or pharmaceutically acceptable salt or derivative
thereof can be administered as a component of an elixir, suspension, syrup,
wafer, chewing gum or the like. A syrug may contain, in addition to the
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CA 02538205 1996-06-07
WO 96/90164 PC'f/U596/10026
active compounds, sucrose as a sweetEning agent and certain preservatives,
dyes and colorings and flavors.
The active compound, or pharmaceutically acceptable derivative or salt
thereof can also be mixed with other active materials that do not impair the
s desired action, or with materials that supplement the desired action, such
as
antibiotics, antifungals, antiinflammatories, or other antivirals, including
anti-HBV, anti-cytomegalovirus, or anti-HIV agents.
Solutions or suspensions used for parenteral, intradermal,
subcutaneous, or topical application can include the following compon~ts:
to a sterile diIuent such as water for injection, saline solution, fixed oils,
polyethylene glycols, glycerine, propylene glycol or other synthetic
solvents; antibacterial agents such as benzyl alcohol or methyl parabens;
antioxidants such as ascorbic acid or sodium bisulfate; chelating agents such
as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or
15 phosphates and agents for the adjustment of tenacity such as sodium
chloride or dextrose. The parental preparation can be enclosed in
ampoules, disposable syringes or multiple dose vials made of.glass or
plastic.
If administered intravenously, preferred carriers are physiological
2 o saline or phosphate buffered saline (PBS). In a preferned embodiment, the
active compounds are prepared with canters that will protect the compound
against rapid elimination from the body, such as a controlled release
formulation, including implants and microencapsulated delivery systems.
Biodegradable, biocompatible polymers can be use, such as ethylene vinyl
2 5 acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Methods for preparation of such formulations will be
apparent to those skilled in the art. The materials can also be obtained
commercially from Alza Corporation and Nova Pharmaceuticals, Inc.
CA 02538205 1996-06-07
WQ 96140164 PCT/US96/100~b
Liposontal suspensions rurcluding liposomes targeted to infected cells
with monoclonal antibodies to viral antigens) are also preferred as
pharma~ticxtlY acceptable carriers. Titex may be preparod according to
m~Ods latawtt to those sldllod in the art, for exampk, as described in
U.S. Patent No. ~t,522,$I I.
For example, liposome formulations may be by
dissolving appropriate tipid(s) (such as straooyl phosphatidYl ahanolamine,
st~earoyl phoephatidyl choliee, arachadoyl phosphatidyl c~oline, and
chaksterotj in an inorganic solvatt that is then evaporat0d, kaving behind
1o a thin liLn of dried lipid on the surface of the container. M aqueous
solution of the active compound or its motwphosphate, diphosphate. andlOr
trlpltosphaec derivatives arc then introduced into the oantaina. 'fhe
container is then swirled by hand to free Lipid material from the sides of the
container and to disperse lipid aggregates, thereby forming the liposo<nal
is suspensio,t.
This invention has been described with reference to its preferred
embodimatts. Variations and modifications of the invention, will be
obvious to thox sfdlled in the art from the foregoing detailed description of
the invention. It is intended that ail of these variations and modifications
2 o be included within the soopo of the appendod claims.
-45-
