Note: Descriptions are shown in the official language in which they were submitted.
CA 02203674 1997-04-24
WO96/12728 PCT~S95/13717
L-E~, - ~1 Nucl~
Field of the Invontion
This invention relates to L-pyranosyl nucleosides, processes for
their preparation, pharmaceutical compositions containing such and
methods of using such compounds as anticancer, antiviral,
antifungal, antiparasitic and/or antibacterial agents in mP~mm;315.
Bac}.~ of th~ Invention
Perigaud, C. et al., Nucleosides and Nucleotides, 11(2-4), 903-945,
(1992) provide a useful overview of the current state of the art
relating to the use of nucleosides and/or nucleotides as
chemotherapeutic agents (including use as anticancer, antiviral and
antibacterial agents). As described in this review article, the
term "nucleoside(s)" relates to naturally-occurring nucleosides
which are distinguished dep~n~;ng on the base, for example, adenine
and guanine (A and G, respectively) have a purine base, whereas
cytosine, uracil, thymine and hypoxanthine (C, U, T, and H,
respectively) have a pyrimidine base.
Nagasawa, N., et al., J. Ora. Chem., 32, 251-252, (1967) describe
the production of certain D-ribopyranosyl nucleosides (particularly
9-(2'-Deoxy-~-D-ribopyranosyl)adenosine. Fucik, V., et al., Nucleic
Acids Research, Vol. 1, No. 4, (1974), 639-644, describe structural
effects of chemical modification upon the affinity of purine
nucleosides to cytidine-transport system in Bacil l us subtil is using
a series of modified derivatives including certain ribopyranosyl
nucleosides.
As is well known, sugars found in natural nucleic acids are D-ribose
and D-deoxyribose in almost all cases. Much research has been done
to investigate the chemical and biological activities of the D-
isomers of ribonucleotides and ribonucleosides, however, far less
work has been done with the L-isomers. This is primarily due to the
fact that the synthesis of the L-isomers is much more difficult,
often involving the optical resolution of the D,L-isomers of
nucleosides with the aid of microorg~n;.C~c and enzymes. (See
generally, Asai, M., et al., t~h~m. ph~rm. Bull., 15(12), 1863-1870,
(1967)). The known activity of D-nucleoside compounds, and the
successful commercialization of many of such D-sugar nucleosides
(see Perigaud, C., et al., supra, for a discussion of D-nucleoside
analogs which have gained commercial acceptance) lead in part to the
CA 02203674 1997-04-24
W096/12728 PCT~S95/13717
present work relating to the L-isomers of certain nucleoside
analogs.
Perhaps the best known commercial nucleobase analog is 5-
fluorouracil (5-FU) the structure of which is shown below:
~ N~
o
(5-Fln
5-FU is an antimetabolite compound commercially available from Roche
and is one of the most commonly used drugs for treating certain
types of cancer. The high acceptance of this drug is due in part to
its extreme cytotoxic effects. However, this highly toxic compound
has a narrow margin of safety and it has many side effects
including, for example, GI side effects like nausea, vomiting and
diarrhea, leukopenia, thrombocytopenia, alopecia, etc.
Additionally, 5-FU is primarily used as an intravenous formulation.
Therefore, there is a need for a nucleoside analog which is perhaps
as cytotoxic as 5-FU or which is less cytotoxic but more specific
than 5-FU, and which preferably can be ~m;n; stered orally.
5-FU is currently dosed at short intervals due to the damage it does
to normal cells. The patient is taken off chemotherapy for a time
to allow recovery from the cytotoxic effects of the treatment. It
is contemplated that if a drug is developed that is less cytotoxic
to healthy cells it would no longer be necessary to treat the
patient in periodic intervals, which may be associated with the
development of multiple drug resistance often exhibited in treated
cancer cells. Specifically, as a tumor is being killed the cells
that are most resistant to the drug die slower and, therefore, when
the treatment is stopped (often because of the toxicity to normal
cells), the more resistant tumor cells are left to multiply.
A significant commercial nucleoside analog is azidothymidine (AZT),
commercially available as Retrovir from Burroughs Wellcome. AZT, a
~-D-deoxy-ribofuranosyl derivative of the formula:
CA 02203674 1997-04-24
W O 96/12728 PCTrUS95/13717
o ~ ~ C~3
N
HO ~
(A~
is useful as an antiviral agent, particularly against the virus
responsible for the Acquired Immune Deficiency Syndrome (AIDS).
This compound, like 5-FU, is associated with a number of undesirable
side effects including hematologic toxicity such as granulocytopenia
and/or severe anemia.
Without inten~;ng to be limited, applicants believe that the L-
nucleoside compounds, as claimed in the present invention, may be
beneficial over compounds such as 5-FU and AZT since it is believed
that L-nucleosides (as claimed) exhibit selective pPrme~hility to
compromised cells. By compromised cells we mean cells such as
cancer cells or other infected cells whether the infection is
bacterial, fungal, viral or parasitic. It is believed that the L-
nucleosides of the present invention may be transported into or
permeate these compromised cells, whereas in normal cells the L-
nucleosides would not permeate. (See, for example, Lin, T.S., et
al., Abstract entitled n Synthesis and Biological Evaluation of
2',3'-Dideoxy-L-Pyrimidine Nucleosides as Potential Antiviral Agents
against HIV and HBV:' published J. Med. Ch~m., (1994), 37, 798-803;
and Spadari, S., et al., J. Med. Chem., (1992), ~ 4214-4220.)
Therefore, to the extent these L-nucleosides are selective for
compromised cells, they are less harmful to normal cells than
compounds like 5-FU.
In addition to this concept of selective per~hility, in viral-
infected cells where therapeutic compounds often have an inhibitory
mechanism related to the RNA of the cell, it is contemplated that
the enzymes of such viral-infected cells may be less specific than
CA 02203674 1997-04-24
W O96/12728 PCTrUS95/13717
in a normal cell and, therefore, if you can permeate the cell with
an L-nucleoside, a more primitive enzyme (such as an organic
phosphorylases, kinase or thymidylate synthase) may recognize the
compound in such a way as to cause inhibition.
The present invention, therefore, relates to a novel group of such
L-pyranosyl nucleosides which have interesting activity as
anticancer, antiviral, antiparasitic, antifungal and/or
antimicrobial agents. These compounds are generally water soluble,
which suggests that oral deliver may be achieved. This would be
specifically advantageous versus anticancer compounds such as 5-FU.
And the activity of these compounds may be more selective for
compromised cells as compared to normal cells, suggesting that
compounds of this invention will cause fewer side effects than
similar compounds such as 5-FU.
Detailed DescriDtion of the ~n~ention
There is provided by this invention pyranosyl nucleoside compounds
having the formula (I):
R ~ R3
~ R,
or a p~rm~ceutically acceptable salt thereof wherein:
B is a naturally-occurring nucleobase tA,G,C,U,T or
hypoxanthine) or a substituted nucleobase comprising one or
more substitutions selected from the group consisting of H,
halogen, C1-C6 alkyl, C2-C6 alkenyl, C1-C6 alkoxy, C3-C6
cycloalkyl-C1-C6 alkoxy, C3-C8 cycloalkyloxy, C3-C8
cycloalkylthio, C1-C6 alkylthio, a substituted amino group, an
aryl, aralkyl, aryloxy, aralkoxy, arylthio, aralkylthio, a
heterocyclic ring and an amino group, provided that when the
base is a pyrimidine, the atom at position 4 in the base can
be sulfur and that when the base is a purine, the atom at
position 6 in the base may be sulfur;
R is OR5 (wherein R5 is H, COR6, or P(O)nR7Ra (wherein R6 is
substituted or unsubstituted alkyl of 1-5 carbon atoms or a
CA 02203674 1997-04-24
W O 96/12728 PCTrUS9S/13717
substituted or unsubstituted aromatic ring structure, R7 and R~
are each H or alkyl of 1-5 carbon atoms and n is 2 or 3));
Rl and R2 are independently H, mono- or di-halogen, ORg, or B
(wherein Rg is H, CORlo, P(O)~RllRl2 (wherein Rlo is substituted
or unsubstituted alkyl of 1-5 carbon atoms or a substituted or
unsubstituted aromatic ring structure and Rll and Rl2 are each H
or alkyl of 1-5 carbon atoms and m is 2 or 3)); and
R3 and R4 are independently B, H or ORl3 (wherein Rl3 is H, CORl4,
P(O)pRl5Rl6 (wherein Rl4 is substituted or unsubstituted alkyl of
1-5 carbon atoms or a substituted or unsubstituted aromatic
ring structure and Rls and Rl6 each are H or alkyl of 1-5 carbon
atoms and p is 2 or 3)), provided that only one of Rl-R4 can be
B; and further provided that when R and Rl are each OH, R2 is
H, and R3 is B, then B can not be thymine; and when R and R
are each OH, R2 is H, and R4 is B, then B cannot be thymine.
Preferred compounds of the present invention include those compounds
of formula (I) wherein:
one of R3 or R4 is B and the other is H, such that when R3 is B
the series is a and when R4 is B the series is ~;
B is C, T, U, G, A or 5-fluorouracil; and
R-R2 are each OH.
Specifically preferred compounds of the present invention are the
following:
~-L-ribopyranosylcytosine; ~-L-ribopy~-anosylguanine; ~-L-
ribopyranosyladenosine; ~-L-ribopyranosyluracil; ~-L-ribopyranosyl-
5-fluorouracil; and ~-L-ribopyranosyl-5-fluorouracil and
pharmaceutically acceptable salts thereof.
Also provided by this invention are processes for the preparation of
the compounds of formula (I), phArm~ceutical compositions contA;n;ng
the compounds of formula (I), and methods of using the compounds of
formula (I) for the treatment of cancer in a m~mmAl (and
particularly a solid tumor in a mAmmAl), as well as methods of using
CA 02203674 1997-04-24
W O96/12728 PCTrUS95/13717
the compounds of formula (I) as antiviral, antifungal, antiparasitic
and/or antibacterial agents in a mammal.
SYnthesis
The present invention describes a series of L-pyranosyl nucleosides
use~ul for treating various diseases (including cancer~. Compounds
of this invention may be orally~active based on their water
solubility.
The compounds of this invention, wherein the nucleoside has a
pyrimidine base (U,T,C or substituted pyrimidine base) which is
linked to the pyranosyl sugar via ~ linkage (B is R4 in a compound
of Formula (I)), can be made by the general Scheme A.
SCHEME A
General procedure to make ~-L-Ribopyranosyl pyrimidines:
+ RQ~C 1~ S
2.NE~McOH
o~
N~OMk~McOH B~
To a mixture of tetraacetyl-L-ribopyranoside (1 mol) and pyrimidine
base (1 mol) in anhydrous MeCN are successively added HMDS (1 mol),
ClSiMe3 (0.8 mol) and SnCl4 (1.2 mol). The resulting clear solution
is refluxed for 1 hour when TLC indicates completion of the
reaction. The solvent is evaporated and the residue dissolved in
EtOAc, washea with NaHCO3 and H20. The EtOAc layer is dried,
filtered and evaporated to give the crude product, which is either
crystallized or purified on a silica column to obtain the pure
2,3,5-tri-0-acetyl-L-ribopyranosyl pyrimidine compounds. These
compounds are either stirred with NH3tMeOH or NaOMe in MeOH to give
the pure ~-L-ribopyranosyl pyrimidines after purification and
crystallization.
A more detailed scheme for the synthesis of specific ~-linked
pyrimidine compounds is provided as Scheme A-1.
SUBSTITUTE SHEET (RULE 26)
CA 02203674 1997-04-24
W O 96/12728
PCTrUS95/13717
SCHEME A- 1
AC201PY ~ ~OAc
1l
0 ~ H~DSS~TMS ~ N~lOMe/MeOH ~
a~N~ l~N\
O O 3
HN~ o~
J~ J TMS ~ ~ NaOMe,/MeOH~
H O~/ ~ Ql~
H~p HN~
O O 5
NHSiMe3
~ TMSOTf ~~\ NaOMe/MeOH
Me3S10 N N~J N~l
NH2 7 NH2 8
SUBSTITUTE SHEET (RULE 26)
CA 02203674 1997-04-24
W096/12728 PCT~S95/13717
The compounds of this invention wherein the nucleoside has a purine
base (A or G or substituted purine base) which is linked to the
pyranosyl sugar via ~ link (B is R4 in a compound of Formula (I))can
be made by the general scheme B below.
SCHEME B
General procedure to make ~-L-ribopyranosyl purines:
TG--o ~ OAc + Silylated 1. TMSOTf/ClC~CH2Cl
Base 2. N~/MeOH
B~
A mixture of purine base (2 mol) and (NH4)2S04 (catalytic amount) in
HMDS is refluxed until the solution becomes clear. The resulting
clear solution is concentrated to yield silylated base to which
anhydrous dichloroethane is added, and the solution is cooled to
0C. Under nitrogen atmosphere a solution of tetraacetyl-L-
ribopyranoside in dichloroethane (l mol) and TMSOTf (2.1 mol) are
added to the above solution and stirred at room temperature for 16
hours. The reaction is quenched with saturated NaHCO3 solution and
the solvent is evaporated. The residue is dissolved in EtOAc,
washed with water and brine. After drying and evaporating the
solvent, the residue obtained is purified on a silica gel column to
give pure 2,3,5-tri-0-benzyl-~-L-ribopyranosyl purines and which,
after stirring with NH3/MeOH and usual purification, give pure ~-L-
ribopyranosyl purines.
A more detailed schematic for the synthesis of specific ~-linked
purine compounds is provided as Scheme B~
SUBSTITUTE SHEET (RULE 26)
CA 02203674 1997-04-24
W O96/12728 PCTrUS95/13717
SCHEME B-l
IOSiMe3
N~h
N~N TMSOTf/ , ~ MeOHlNH3 . f~
1~ J CICH2CH2CI ~ \
SiMe3 N~ N~N
1 1 NH2
1 + ~ f~ MeO~NH3 , ~ ~
SiM~ ~ Me ~N ~ N ~ NHAc ~N ~ N ~ NH2
OSiMe3 N ~ NH N ~ NH
12
13 14
The compounds of this invention wherein the nucleoside has a
pyrimidine base (U,T,C,H or substituted pyrimidine base) which is
linked to the pyranosyl sugar via ~ linkage (B is R3 in a compound
of Formula (I)) can be made by the general scheme C below.
SCHEME C
General procedure to make ~-L-Ribopyranosyl pyrimidines:
B æ
+ Silylatcd . NBS/MS.4A/C~CI~
Base 2. ~/PdlC/EtOH
A mixture of pyrimidine base ~2 mol) in HMDS and ammonium sulfate
(catalytic amount) is refluxed until the solution becomes clear.
SUBSTITUTE SHEET (RULE 26)
CA 02203674 1997-04-24
W O96/12728 PCTrUS95/13717
The resulting clear solution is concentrated in vacuo to yield
silylated base. To this silylated base in anhydrous CH2Cl2 under
nitrogen atmosphere, 1-thio-2,3,5-tri-0-benzyl-L-ribopyranoside (2
mol), 4A molecular sieves and NBS (1.1 mol) are added. The reaction
mixture is stirred at room temperature overnight and ~uenched with
addition of Na2S203 solution. The organic layer is washed with
water, brine and dried over Na2SO4. Evaporation of the solvent gave
the crude product which is purified on a silica gel column to obtain
pure 2,3,5-tri-0-benzyl-a-L-ribopyranosyl pyrimidine compounds.
These compounds are subjected to H2/Pd/C reduction followed by
purification and crystallization to give pure a-L-ribopyranosyl
pyrimidines.
A more detailed schematic for the synthesis of specific a-linked
pyrimidine compounds is provided in Scheme C-1.
SCHEME C-l
PhSH/SnCl~ ~ ~S~h l.NaOMe/MeOH, ~--SPn
CH2Ck 2.NaH/BnBr/DMF
16
O, SiMe~ ,O, O
Me3510 ~ ~/ Os~
I Bn O / BC~/C~zCl~ . 1~}
NBS/M.S.4A/CE~ 78C
17 18
Compounds of this invention wherein the nucleoside has a purine base
which is linked to the pyranosyl via a linkage can be made by the
general Scheme D below.
SCHEME D
General procedure to make a-L-ribopyranosyl purines:
SUBSTITUTE SHEET (RllLE 26
CA 02203674 1997-04-24
W096/12728 PCT~S95/13717
~"~ E~O~ BllOj~
B~
B~
l.S~ OH o
2.H~n~C~OH ~ OH o~
A mixture of purine base (2 mol) and (~H4)2SO4 (catalytic amount) in
HMDS is refluxed until the solution becomes clear. The resulting
clear solution is concentrated to yield silylated base to which
anhydrous dichloroethane is added and the solution is cooled to 0C.
Under nitrogen atmosphere a solution of 1-0-acetyl-2,3,5-tri-0-
benzyl-L-ribopyranose and TMSOTf (2.1 mol) are added to the above
solution and stirred at room temperature for 16 hours. The reaction
is quenched with saturated NaHCO3 solution and the solvent is
evaporated. The residue is dissolved in EtOAc, washed with water
and brine. After drying and evaporating the solvent, the residue
obtained is separated on a silica gel column to give pure a and ~-
2,3,5-tri-0-benzyl-L-ribopyranosyl purines and which after reduction
with hydrogen in presence of Pd/C catalyst (H2/Pd/C) and usual
purification give pure -L-ribopyranosyl purines.
A more detailed schematic for a-linked purine compounds within the
scope of the present invention is provided below as Scheme D-1.
SUBSTITUTE SHEET (RULE 26)
CA 02203674 1997-04-24
WO 96/12728 PCT/US95113717
SCHEME D- 1
f
+ ~ o(~o~o~
~ 19 20
lDME~
2E~VP~C~OH ~ 21
l~U I~R~4
2E~C~OH '
22
~ ' ~
A~ ~ ~ TMSOTq ~ ~
~ ~ F~U~ ~ ~ ,~ 2 ~ C~OH
L OSNo
2~ burJ ~ '1 23 ~
22~ hJr OH~_
2S
In addition to the teachings provided herein, the skilled artisan
will readily understand how to make compounds within the scope of
the present invention by applying well known techniques such as
those described in Nucleic Acid Chemistr~, Im~roved and New
Svnthetic Procedures, Methods and Techniaues, Edited by Leroy B.
Townsend and R. Stuart Tipson, John Wiley ~ Sons, New York (1978);
and Chemistrv of Nucleosides and Nucleotides, Edited by Leroy B.
Townsend, New York, Plenum Press (1988-1991). Suitable methods for
making various substitutions on purine nucleosides are provided in
WO90/08147. Suitable methods for making substitutions on pyrimidine
nucleosides are provided in W088/04662. The disclosure of both such
applications and their US equivalent applications/patents being
readily available to those skilled in ~he art and incorporated
herein. Suitable methods for making substitutions within the sugar
12
SUBSTITUTE SHEET (RULE 26)
CA 02203674 1997-04-24
WO96tl2728 PCT~S95/13717
moiety of the presently claimed compounds are known to those skilled
in the art and are described in various publications including: US
Patent 4,880,782; W088/00050; EPO 199451 A2; US Patent 3,817,982;
Lange, P., et al., Pro~ress in Antimicrobial and Anticancer
Chemotherapv, Proceedings of the 6th International Congress of
Chemotherapy, Univ. Park Press, England, 1970, Vol. II, p. 394-397;
and Townsend, et al., supra, all of which are incorporated herein by
reference.
This invention can be further understood by referring to the
following Examples and Tables below:
ment~l
Exam~le 1
~-L-ribopyranosvluracil and
~-L-ribo~Yranosvl-5-fluorouracil
Part A
Synthesis of 1,2,3,4-tetraacetyl-L-ribopyranoside (1)
The mixture of L-ribose (commercially available from Sigma Chemical
Co.) (6.0 g), acetic anhydride (80 ml) and pyridine (12 ml) was
heated to 100C with stirring until the L-ribose dissolved. After
cooling acetic anhydride was evaporated in vacuo. The residue was
dissolved in 100 ml of dichloromethane and washed 3 X 50 ml of
saturated NaHCO3 solution and 50 ml satd. NaCl solution. Organic
phase was dried over sodium sulfate and the solvent was evaporated
on a rotovapor. Finally, the oily residue was kept overnight at
high vacuum to give (1) as a white crystalline solid (12.3 g,
96.7%). TLC:Rf=0.63 in ethyl acetate-petroleum ether (7:3J.
pArt B
Synthesis of 0-L-ribopyranosyluracil triacetate (~) and 0-L-
ribopyranosyl-S-fluorouracil triacetate (~)
To 1,2,3,4-tetraacetyl-L-ribopyranoside (1, 3.5 g, 11 mmol) and
uracil (commercially available from Sigma Chemical Co.) (1.23 g, 11
mmol) or 5-fluorouracil (commercially available from Sigma Chemical
Co.) (1.43 g, 11 mmol) in 80 ml of dry acetonitrile were added
hexamethyldisilazane (2.3 ml, 8.8 mmol), trimethyl-chlorosilane (1.1
ml, 8.8 mmol) and trimethylsilyl triflate (3.6 ml, 18 mmol). The
reaction mixture was stirred for about 16 h at room temperature,
then refluxed for 1.5 h to complete the reaction. After cooling the
reaction mixture was diluted with 100 ml of dichloromethane and
13
CA 02203674 1997-04-24
WO96/12728 PCT~S95/13717
washed with 3 X 50 ml of satd. sodium hydrocarbonate solution. The
combined aqueous phases were re-extracted with 50 ml of
dichloromethane. The combined organic phases were washed with 50 ml
satd. NaCl solution. The organic phase was dried over sodium
sulfate and the solvent was evaporated on a rotovapor to give pale
yellow crystals. Yields were 3.1 g (76%J for-~-L-
ribopyranosyluracil triacetate (~) and 2.9 g (69.9%) for ~-L-
ribopyranosyl-5-fluorouracil (4) derivate. Crude products were
purified by flash chromatography on a silica gel column (150 g)
using ethylacetate-petroleum ether (7:3) eluent. Yields: 2.0 g
(52%) and 2.5 g(56%), respectively. TLC:Rf=0.18 in ethylacetate-
petroleum ether (7:3) for both compounds.
Part C
Synthesis of ~-L-ribopyranosyluracil (~)and ~-L-ribopyranosyl-5-
fluorouracil (5)
Triacetates from Part B were deprotected by treating with 2.0 M
methanolic ~mmo~;a (80 ml for 10 mmol nucleoside) for 24 h at room
temperature. (The methanolic ammonia was evaporated in vacuum and
the residue was purified by flash chromatography on silica gel (150
g) with chloroform-methanol-water (65:35:4) eluent to give 1.4 g
(36% overall yield) L-ribopyranosyluracil (3), and 1.8 g (40%
overall yield) L-ribopyranosyl-5-fluorouracil (5) white crystals.
TLC:Rf=0.4 for both compounds in chloroform-methanol-water (65:35:4)
solvent.
'H-NMR For (3)
(DMSO-d6) ~
3.52-3.75 (m,4H,H-3',4' & 5') 3.95 (m,lH,H2') 4.84 (d,lH,OH)
5.06 (d,lH,OH) 5.10 (d,lH,OH) 5.58 (d,lH,H-2') 5.60 (d,lH,H-l')
7.65 (d,lH,H-6)
'H-NMR For (5)
(DNSO-d6) ~
3.45-3.80 (m,4H,H-3',4' & 5') 4.0 (m,lH,H2') 4.8-5.4(br s,3H,2',3' & 5'
OH)
5.60 (d,lH,H-l') 8.10 (d,lH,H-6)
WO96/12728 PCT~S95/13717
~nle 2
CA 02203674 1997-04-24
W 096/12728 PCTrUS95/13717
~m~le 2
B-L-~;hOPVranOSV1CYtOSine
Part A
Synthesis of 2-trimethylsilyloxy-4-trimethylsilyl amino pyrimidine
(6)
The mixture of cytosine (1.7 g, 15.3 mmol), hexamethyldisilazane
(HMDS) (15 ml), trimethyl-chlorosilane (TCS) (0.1 ml) and pyridine
(10 ml) was refluxed at 130C bath temperature until the complete
dissolving of cytosine (about 1.5 h). The excess HMDS and pyridine
was removed by co-distillation with 2 X 50 ml of dry toluene. White
solid was dried in high vacuum. Yield: 3.8 g (6) (97.3%).
TLC:Rf=0.42 ethyl acetate-petroleum ether (7:3).
Part B
Synthesis of 0-L-ribopyranosylcytosine triacetate (7)
To 1,2,3,4-tetraacetyl-L-ribopyranoside (1, 4.1 g, 12.9 mmol) and
silylated cytosine (6) (3.3 g, 12.9 mmol) dissolved in 80 ml of dry
acetonitrile was slowly added trimethylsilyltriflate (2.8 ml, 14
mmol). The mixture was stirred 72 h at room temperature. The
reaction was completed by refluxing the mixture for 1 h. The dark
brown solution was diluted with 150 ml of dichloromethane and washed
3 X 50 ml of satd. sodium bicarbonate solution. The aqueous layer
was re-extracted with 50 ml of dichloromethane. The combined
organic phases were dried over sodium sulfate and the solvent
evaporated on a rotovapor. The residue was dried in vacuo to give
3.8 g (80~) brownish foam. The product was purified by flash
chromatography on a silica gel column (150 g) with ethyl acetate-
methanol (9:1) eluent. Yield: 1.5 g (7) (31.5%) orange crystalline
material. TLC:Rf=0.51 ethyl acetate-methanol (9:1).
Part C
Synthesis of 0-L-ribopyranosylcytosine (8)
~-L-ribopyranosylcytosine triacetate (7) was deprotected by treating
with 2.0 M methanolic ~mm~;a (80 ml for 10 mmol nucleoside) for 24
h at room temperature. The methanolic ~mmo~;a was evaporated in
vacuo and the residue was purified by flash chromatography on silica
gel (150 g) with chloroform-methanol-water (65:35:4) eluent to give
(~) 0.8 g (17% overall yield) of white needles. TLC:Rf=0.17 in
chloroform-methanol-water (65:35:4) as a mobile phase.
CA 02203674 1997-04-24
W O 96/12728 PCTrUS95/13717
'H-NMR For (8)
(DMSO-d6) ~
3.42-3.65 (m,4H,H-3',4' & 5') 3.95 (m,lH,H-2') 4.79 (d,lH,OH)
4.84 (d,lH,OH) 5.01 (d,lH,OH) 5.67 (d,lH,H-5') 5.71 (d,lH,H-5)
7.13 (br d, lH,NH2) 7.55 (d,lH,H-6)
~xam~le 3
~-L-ribo~vranosYladenosine (11)
N6-benzoyl-adenine (2.392 g, 10 mmol) was silylated by heating at
reflux temperature for 7 hrs with hexamethyldisilazane (35 ml),
trimethyl-chlorosilane (0.5 ml) in the presence of dry pyridine (10
ml). The solvents were removed in vacuo . The traces of silylating
agents were removed by co-distillation with dry toluene (2 X 20 ml)
and the resulting off-white solid (9) was used for nucleoside
synthesis.
Silylated N6-benzoyladenine (9) (10 mmol) was reacted with 1,2,3,4-
tetraacetyl-L-ribopyranoside (1, 3.18 g, 10 mmol) in dry
acetonitrile (50 ml) using trimethylsilyl triflate catalyst (2.2 ml,
11 mmol). The clear solution after the addition of catalyst was
refluxed for 14 h. TLC indicated no starting sugar and silylated
benzoyladenine. The reaction mixture was diluted with 100 ml of
dichloromethane and washed with 3 X 50 ml of cold satd. sodium
bicarbonate solution. The organic phase was dried over sodium
sulfate and evaporated to give brown glassy product. Purification
by flash chromatography (chloroform-methanol 95:5) yielded 3.5 g of
greenish solid. After dissolving in methanol (50 ml) the product
was treated with active carbon (1.2 g). Filtration of this solution
followed evaporation on a rotovapor afforded 2.5 g (10) of pale
yellow amorphous crystalline material. After deprotection with 100
ml of methanolic ammonia yielded (11) 1.0 g (39~) as off-white
crystals. Rf=0.36 in n-butanol-acetic acid-water (12:3:5).
'H-NMR For (11)
(DNSO-d6) ~
3.55-3.78 (m,3H,H-4' & 5') 4.04 (m,lH,H-3') 4.23 (t,lH,H-2')
4.91 (d,lH,OH) 5.08 (d,lH,OH) 5.15 (d,lH,OH) 5.63 (d,lH,H-l')
7.25 (s,2H,NH2) 8.14 & 8.30 (s,2H,H-2 & 8)
CA 02203674 l997-04-24
WO96/12728 PCT~S95/13717
~mnle 4
~ -L-ribo2vranosyl~n~ne (14)
Part A
N2-acetyl-guanine (2.13 g, 11 mmol) was silylated by heating at
reflux temperature for 7 h with hexamethyldisilazane (35 ml),
trimethyl-chlorosilane (0.5 ml) in the presence of dry pyridine (10
ml). The solvents were removed in vacuo . The traces of silylating
agents were removed by co-distillation with dry toluene (2 X 20 ml).
The resulting off-white solid (12) was used for nucleoside
synthesis.
Part B
Silylated N2-acetyl-guanine (12) (10 mmol) was reacted with 1,2,3,4-
tetraacetyl-L-ribopyranoside (1, 3.5 g, 11 mmol) in dry acetonitrile
(50 ml) using trimethylsilyl triflate catalyst (2.42 ml, 12.1 mmol).
The clear solution after the addition of catalyst was refluxed for
1.5 h and stirred at room temperature for 12 h. TLC showed no
parent base of silylated base or sugar. The reaction mixture was
diluted with 100 ml of dichloromethane and washed with 3 X 50 ml of
cold satd. sodium bicarbonate solution. Organic phase was dried
over sodium sulfate and evaporated to give 3.4 g yellow amorphous
crystalline material. Purification by flash chromatography
(chloroform-methanol 95:5) yielded 1.9 g (13) of off-white crystals.
After deprotection with 100 ml of methanolic ~mmon;a yielded (14)
1.0 g (45~) as off-white crystals. Rf=0.22 in n-butanol-acetic
acid-water (12:3:5).
'H-NMR For (14)
(DMSO-d6) ~
3.50-3.65 (m,3H,H-4' & 5') 3.95-4.15 (m,2H,H-2' & 3')
5.0 (br s,3H,2',3' & 5'-OH) 5.50 (d,lH,H-l') 6.60 (br s,2H,NH2) 7.83 (s,lH,H-8)
Ex~mnle 5
a-L-ribo~vranos~1-5-fluorouracil (18)
Part A
1,2,3,4-Tetraacetyl -L-ribopyranoside (1)
To a solution of L-ribose (2.15 g, 14.32 mmol) in pyridine (10 ml),
acetic anhydride (10 ml) was added and the reaction mixture was
stirred at room temperature overnight. Solvents were evaporated and
the residue was dissolved in EtOAc, washed with water, CuS04
solution, NaHCO3 solution and brine. After drying and evaporating
CA 02203674 1997-04-24
W O 96/12728 ~C~rrUS95/13717
the solvent, an oil was obtained and it was used without further
purification in the next step.
Part B
l-Thio-2,3,4-tri-0-acetyl-L-ribopyranoside (~J
To a solution of (1) (4.54 g, 14.28 mmol), in CH2Cl2 (80 ml),
thiophenol (1.6 ml, 15.71 mmol) was added and stirred at room
temperature for 15 min. Then the reaction mixture was cooled in an
ice bath and SnCl4 (1 ml, 8.56 mmol) was added dropwise and stirred
at room temperature overnight. The reaction mixture was w-che~ with
2N HCl (2 X 100 ml), water (150 ml), NaHCO3 solution (100 ml) and
then with brine. After drying over Na2SO~, the solvent was
evaporated and the residue was purified on a silica gel column using
20-30% EtOAc/petroleum ether as solvent to give pure compound 15
(2.61 g, 49.7%) as an oil.
Part C
l-Thio-2,3,4-tri-0-benzyl-L-ribopyranoside (16)
To a solution of (~) (2.61 g, 7.08 mmol) in MeOH (50 ml), NaOMe
(0.3 ml, 1.4 mmol) was added and stirred for 18 h. The reaction
mixture was neutralized by Dowex 50 ion exchange resin, filtered and
evaporated. To this residue, DMF (50 ml) was added and cooled in an
ice bath. To this cooled solution NaH (2.67 g, 66.85 mmol) was
added in portion and stirred for 15 min. Benzyl bromide (8 ml, 6.85
mmol) was added dropwise and stirred at 0C for 2-3 h. The reaction
was quenched with MeOH and water after diluting with EtOAc. EtOAc
layer was washed with water (2 X 100 ml) and brine. After drying
and evaporation of the solvent, the crude product obtained was
purified on a silica gel column using 5-10% EtOAc/petroleum ether as
solvent to yield pure (16)(3.29 g, 93.5%) as an oil.
Part D
1-(2,3,4-Tri-O-benzyl-~-L-ribopyranosyl)-S-fluorouracil (17)
A mixture of 5-fluorouracil (1.62 g, 12.48 mmol) in
hexamethyldisilazane (30 ml) and Am~on;um sulfate (catalytic amount)
was refluxed for 4 h. The resulting clear solution was concentrated
in vacuo to yield silylated 5-fluorouracil as colorless oil. To a
solution of silylated 5-fluorouracil in CH2Cl2 (20 ml) under nitrogen
atmosphere were added NBS (1.22 g, 6.86 mmol), 4A molecular sieves
(2.4 g) and compound (16)(3.2 g, 6.24 mmol) in CH2Cl2 (20 ml). The
reaction mixture was stirred at room temperature overnight and
18
CA 02203674 1997-04-24
W O96/12728 PCTrUS95/13717
quenched with the addition of Na2S2O3 solution. The organic layer
was washed with water and brine and then dried over Na2SO4.
Evaporation of the solvent gave the crude product and it was
purified on a silica gel column using 50% EtOAc/petroleum ether as
solvent to give the pure ~ isomer (17) (2.42 g, 73%) as white solid.
Part E
a-L-ribopyranosyl-5-fluorouracil (18)
To a solution of ~ (from Part D) (2.42 g, 4.54 mmol) in CH2Cl2 (100
ml), at -78C under nitrogen atmosphere, 1 M solution of BCl3 (50 ml,
49.94 mmol) was added dropwise. The reaction mixture was stirred at
-78C for 4 h and 1:1 mixture of CH2Cl2/MeOH (100 ml) was added and
the reaction mixture was brought to room temperature and the
solvents were evaporated to dryness. The residue was coevaporated
with MeOH (50 ml) 5 times. The residue obtained was dissolved in
water and washed with CHCl3 (50 X 2) and CC14 (50 ml). The water
layer was evaporated to give a white solid and which was
crystallized from EtOH/ether to give the pure 18 (0.94 g, 79.4%) as
white crystals: mp 231C dec.
'H-NMR For (18)
(DMSO-d6) ~
3.62-3.80 (m,3H,H-3~,4~ ~ 5') 3.95 (d,lH,H-2') 5.0-5.2 (2 br s,2H,OH)
5.30 (d,lH,OH) 5.50 (s,lH,H-l~) 7.95 (d,lH,H-6) 11.90 (brd,lH,NH)
Ut~ l~tY
In vitro activity against certain human tumor cell lines.
CELL LINES: Eight different established human cell lines (CALU
(lung), COLO320 (colon), H578St (breast), HT-29 (colon), MCF-7
(breast), OM-l (colon), SKLU (lung), and SKMES (lung) and two
control cell lines (bone marrow cell lines and/or fibroblasts) were
utilized. All cell lines were obtained from the Tumor Cloning
Laboratory, Institute for Drug Development, Cancer Therapy and
Research Center, San Antonio, Texas. All cell lines grew as
monolayers in the appropriate culture medium supplemented with heat-
inactivated calf serum. All reagents were obtained from Grand
Island Biological Co., Grand Island, New York.
IN VITRO EXPOSURE OF TUMOR CELLS TO COMPOUNDS: Stock solutions of
intravenous (iv) formulations of certain of the compounds of the
present invention (as shown in Table I below), as well as
CA 02203674 1997-04-24
W O96/12728 PCT~US95/13717
intravenous formulations of 5-FU (control) were used. The iv
formulations of the compounds of the present invention were prepared
with sterile buffered saline and stored at -70C until required for
testing. The 5-FU control formulation was prepared as suggested in
the commercial product literature.
Following trypsinization, tumor cells were suspended in tissue
culture medium and exposed to the antitumor agents continuously at
three different concentrations: 10, 1 and 0.1 ~ug/ml.
RADIOMETRIC MEASU~EMENT OF GROWTH INHIBITION: Growth inhibition was
assessed with the BACTEC System 460 (Johnston Laboratories, Towson,
MD) after addition of the antitumor agent to the cells in the
respective growth medium cont~;n;ng 14C-glucose at a final
concentration of 2 ,uCi/ml. (See generally, C. Arteaga, et al., A
Radiometric Method for Evaluation of Chemothera~v SensitivitY:
Results of Screenina a Panel of Human Breast Cancer Cell Lines,
Cancer Research, 47, 6248-6253, (1987).)
Two mls of the tumor cell suspension cont~;n;ng radioactive glucose
were seeded into sterile, disposable 15 ml vials by injection
through self-sealing rubber-aluminum caps. For each cell line, the
optimal number of tumor cells needed per vial in order to show
significantly measurable growth in this radiometric system varied.
The seeded vials were then incubated at 37C. Measurement of the
release of l4co2 resulting from the metabolism of 14C-glucose were
performed on days 6, 9, 12, and 15 in the BACTEC instrument. This
instrument flushes the 14co2 cont~;ning air out of the vials into an
ionization chamber that converts dpm to growth index values.
Chemotherapy sensitivity was calculated by comparing the growth
index values of drug-treated vials to that observed in control
vials. Each data point represents triplicate values.
Results are shown in Table I below. All compounds were compared on
an equimilimolar level.
CA 02203674 1997-04-24
W O 96/12728 PCTAUS95/13717
TABLE I
COMPOUND % SURVIVAL BONE % SURVIVAL IC 50 (~g/ml~
MARROW TUMOR
5-FU (control) 38.6 CALU - 9.9 c0.6
6 ~g/ml SKMES29.1 <0.6
SKLU 24.7 1.05
COLO3201.0 <0.6
HT-29 5.7 0.61
OM-1 20.1 1.47
HS578T12.5 <0.6
MCF-7 4.2 <0.6
(8) 10 ,ug/ml 93.1 OM-1 78.9 >10
HS578T89.2 >10
MCF-7 72.2 >10
(14) 10 ~g/ml 105.9 HT-29 94.8 >10
(11) 10 ~g/ml 94.1 OM-1 41.6 8.3
HS578T43.6 7.8
(3) 10 ug/ml 96.0 HS578T86.7 >10
(5) 10 ~g/ml 115.8 SKMES68.2 >10
SKLU 90.1 >10
MCF-7 76.2 >10
(18) 10 ~g/ml 165.7 SKLU 78.4 57.1
HT-29 83.1 119
OM-1 82.5 65.1
HS578T83.6 50.8
The data presented in Table I are compared to results achieved with
5-FU as the control. All compounds were dosed on an equimilimolar
basis. Inhibitory concentration (IC 50) is defined as the
concentration required to kill 50% of the untreated cancer cells.
Although the IC 50 of certain of the compounds listed in Table I may
be higher than that for 5-FU (the control), the compounds of the
present invention are generally less toxic to normal cells such as
bone marrow or fibroblasts. This implies that the compounds of the
present invention may have advantages over known cancer therapies as
the claimed compounds may be less toxic and/or more selective for
the tumor cells, thereby causing less serious side effects.
Additionally, because of their lower toxicity to normal cells, it is
anticipated that the present compounds may be dosed at a higher rate
to selectively increase toxicity to the cancer cells. In this
regard, a therapeutic ratio for a given compound is typically
determined by the following calculation.
CA 02203674 1997-04-24
WO96/12728 PCT~S95/13717
% sllrvival bone mArrow
% survival tumor
A therapeutic ratio of <80% is considered active.
In ~ivo Evaluation
Representative compounds of the present invention have been and/or
are being tested in a variety of preclinical tests of anti-cancer
activity which are indicative of clinical utility. For example,
certain compounds were tested in vivo against human tumors
xenografted into nude mice, specifically B16, MX-1 and P388 Leukemia
tumor lines were used.
B16 Melanoma
B6D2F1 mice receive i.p. inocula of B16 murine melanoma brei
prepared from B16 tumors growing s.c. in mice (day 0). On day 1,
tumored mice are treated with drugs or vehicle controli the drugs,
route of drug administration, and sche~]le are selected as
appropriate for the study in question. If dosing information for
agents is not available, the maximum tolerated dose (MTD) is
determined in initial dose f;n~lng experiments in non-tumored mice.
In a typical experiment, drugs are given at their MTD and 1/2 MTD
doses i.p. on a daily x 5 sche~ule.
The mean survival times of all groups are calculated, and results
are expressed as mean survival of treated mice/mean survival of
control mice (T/C) x 100. A T/C value of 150 means that the treated
group lived 50% longer than the control group; this is sometimes
referred to as the increase in life span, or ILS value.
Mice that survive for 60 days are considered long term survivors, or
cures, in the B16 model. The universally accepted cut-off for
activity in this model, which has been used for years by the NCI, is
T/C=125. Conventional use of B16 over the years has set the
following levels of activity: T/C<125, no activity; T/C=125-150,
weak activity; T/C=150-200, modest activity; T/C=200-300, high
activity; T/C>300, with long term survivors excellent, curative
activity.
Statistics are performed on the data using primarily the log rank p-
value test.
CA 02203674 1997-04-24
W O 96/12728 PCTrUS95113717
P388 T.eukemia
This test is conducted in exactly the same way as the B16 test. The
tumor inoculum is prepared by removing ascites fluid contA;nlng P388
cells from tumored DBA/2 mice, centrifuging the cells, and then
resuspending the leukemia cells in saline. Mice receive 1 x 105
P388 cells i.p. on day 0.
MX-l Human Breast Tumor Xenoaraft
Nude mice are implanted s.c. by trocar with fragments of MX-l
mAm~Ary carcinomas harvested from s.c. growing MX-l tumors in nude
mice hosts. When tumors are approximately 5 mm x 5 mm in size
(usually about ten days after inoculation), the An;m~l5 are pair-
matched into treatment and control groups. Each group contains 10
tumored mice, each of which is ear-tagged and followed individually
throughout the experiment. The administration of drugs or vehicle
begins the day the Anim~l5 are pair-matched (day 1). The doses,
route of drug administration and schedule are selected as
appropriate for the study in question. If the MTD dose of an agent
is not known, it is determined in an initial dosing experiment in
non-tumored mice. In a typical experiment, drugs are given at their
MTD and 1/2 MTD doses i.p. on a daily x 5 sche~nle.
The experiment is usually terminated when control tumors reach a
size of 2-3 g. Mice are weighed twice weekly, and tumor
measurements are taken by calipers twice weekly, starting on day 1.
These tumor measurements are converted to mg tumor weight by a well-
known formula, and from these calculated tumor weights the
term;nAtion date can be determined. Upon term;nAtion, all mice are
weighed, sacrificed, and their tumors excised. Tumors are weighed,
and the mean tumor weight per group is calculated. In this model,
the mean control tumor weight/mean treated tum~or weight x 100% (C/T)
is subtracted from 100% to give the tumor growth inhibition (TGI)
for each group.
Some drugs cause tumor shrinkage in the MX-l model. With these
agents, the final weight of a given tumor is subtracted from its own
weight at the start of treatment on day 1. This difference divided
by the initial tumor weight is the % shrinkage. A mean % tumor
shrinkage can be calculated from data from the mice in a group that
experienced MX-l regressions. If the tumor completely disappears in
a mouse, this is considered a complete regression or complete tumor
23
CA 02203674 1997-04-24
WO96/12728 PCT~S95/13717
shrinkage. If desired, mice with partial or total tumor regressions
can be kept alive past the term;n~tion date to see whether they live
to become long term, tumor-free survivors.
Statistics are performed on the data using primarily the log rank p-
value test.
Protocols for ~rV-l Inacti~ation ~tudi-s
General protocols for the testing of compounds in in vitro antiviral
screens are disclosed in the following references:
1) Perez, V.L., Rowe, T., Justement, J.S., Butera, S.T., June,
C.H., and Folks, T.M., An HIV-1-infected T cell clone
defective in IL-2 production and Ca" mobilization after CD3
stimulation. J. Immunol . 147:3145-3148, 1991.
2) Folks, T.M., Justement, J., Kinter, A., Dinarello, C., and
Fauci, A.S., Cytokine-induced expression of HIV-1 in a
chronically infected promonocyte cell line. Science 238:800-
802, 1987.
3) Folks, T.M., Clouse, K.A., Justement, J., Rabson, A., Duh, E.,
Kehrl, J.H., and Fauci, A.S., Tumor necrosis factor a induces
expression of human immunodeficiency virus in a chronically
infected T-cell clone. Proc . Natl . Acad. sci . USA 86:2365-
2368, 1989.
4) Clouse, K.A., Powell, D., Washington, I., Poli, G., Strebel,
K., Farrar, W., Barstad, P., Kovacs, J., Fauci, A.S., and
Folks, T.M., Monokine regulation of human immunodeficiency
virus-1 expression in a chronically infected human T cell
clone. J. Immunol . 142:431-438, 1989.
1. ~n~ctivation of cell-free ~rv-l. .
Cell-free HIV-1 stocks are derived from culture supernatants of H-9
human T cells chronically infected with the HTLV-IIIB strain of HIV-
1. Other HIV-1 strains including the MN and some African strains
may be used later for confirmatory purposes.
a) Cell-free HTLV-ITIB:
Cell-free HIV-1 (5 x 105 to 1 x 106 TCID50/ml, or median tissue
culture infectious dose) is either left untreated, or treated
24
CA 02203674 1997-04-24
W O96/12728 PCTrUS95/13717
with RPMI 1640 culture medium, or with different
concentrations of antivirals for various time intervals at
37C, or at a temperature to be deter~;neA. After incubation,
the treated and untreated are added to 5 x 105 washed and
pelleted target MT-4 cells. After 1 h incubation at 37C, the
MT-4 cells are washed three times with RPMI 1604, resuspended
in RPMI 1640 supplemented with 15% fetal bovine serum (FBS),
and cultured in a 5% CO2 humidified incubator at 37C. Cell
viability is determined on day 7 of culture by the addition of
the 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyitetrazolium
bromide (MTT) dye, which changes in color in the presence of
live mito~ho~ria. All determ;n~tions are done in
triplicates.
b) Cell-free JR-CSF:
In addition to assessing the effects of antivirals on a lab
strain of HIV-1 (HTLV-IIIB), it is also important to determine
antiviral effects on a primary isolate of HIV-1 (JR-CSF),
which only infects primary human peripheral mononuclear cells
(PBMCs). Human PBMCs activated with phytohemagglutinin A
(PHA, Sigma Chemical Co.) are prepared by culturing PBMCs in
RPMI 1640 culture medium supplemented with 10% FBS (complete
medium) and 2.0~ug of PHA/ml for 1 day before used in
infectivity studies. HIV-1 (JR-CSF) untreated or treated as
above are added to PHA-activated human PBMCs, and incubated
for 1 h at 37C. After incubation, 1.0 ml of complete RPMI
1640 culture medium is added to the cells. Culture
supernatants are collected on days 3, 6 and 9 of culture, and
the amounts of HIV-1 p24 core protein are determined in
triplicate by the HIV-1 p24 antigen capture assay (Coulter
Immunology, FL, or NEN-Du Pont, Wilmington, DE).
2. Inact~rat~on of cell --~oc~rt~
HIV-1-infected human cells to be used include the chronically
infected H-9 cells (HTLV-IIIB or MN strains), and human PBMCs
infected with HTLV-IIIB or with JR-CSF, HTLV-IIIB and MN infected H-
9 cell lines are available in our laboratory. For infected human
PBMCs, fresh human PBMCs are obtained from normal volunteers and
stimulated with PHA, and infected with HTLV-IIIB or JR-CSF, as
described above. On day 7 after in vitro infection, infectivity is
checked by testing for the presence of HIV-1 p24 in the culture
CA 02203674 1997-04-24
WO96/12728 PCT~S95113717
supernatants. Infected cultures are divided in equal aliquots. One
set is then treated with antivirals at different concentrations for
various time intervals, whereas one set is left untreated. Culture
supernatants collected on days 3, 6 and 9 of culture will be
assessed for HIV-l p24 levels by the p24 antigen capture assay kit.
Cells from these cultures can also be used in immunofluorescence
(IF) studies to determine the percentage of cells expressing HIV-l
antigen(s).
3. Inacti~ation of HIV-l lat~ntly infect~d cells.
These assays are designed to study the effects of antivirals on HIV-
l-latently infected cells. One or more of the following HIV-l
latently infected human cell lines can be used (Jl-l, Ul/HIV, and
ACH-2 obtained from the NIH AIDS Research and Reagent Reference
Program, Rockville, MD). These cells are characterized by HIV-l
infection without significant HIV-l viral replication unless they
are stimulated with different cytokines which results in a 10-100
fold increase in HIV-l replication. Jl-l, or Ul/HIV, or ACH-2 cells
are seeded in 96-well round-bottom tissue culture plates to give 5 x
105/well in RPMI 1640 supplemented with 15% fetal bovine serum
(FBS). The cells are either left untreated or treated with
different concentrations of antivirals for various time intervals.
Subsequent to treatment, treated and untreated cells are washed
three times in RPMI 1640 and are stimulated as follows.
The Jl-l cells are stimulated with 1000 U of tumor necrosis factor
(~-TNF, Genzyme) for 48 h at 37C, as previously described (ref.l).
The Ul/HIV-l cells are stimulated with 20~-40% PHA-culture
supernatant (Electronucleonics) for 48 h at 37C (ref.2). The PHA-
sup will either be purchased from Electronucleonics or will be
prepared by us. The prepared PHA-sup, normal human PBMC will be
cultured at a cell density of 106 cells/ml in RPMI 1640 supplemented
with 15~ FBS and 10 ,ug/ml of phytohemagglutinin A (PHA, Sigma
Chemical Co.). The culture supernatant will be harvested, filtered
through a 2 ~m filter and used to stimulate the Ul/HIV cells as
described above.
The ACH-2 cells will be stimulated by addition of 1.0 ~uM of phorbal
12-myristate 13 acetate (PMA, Sigma Chemical Co.) for 48 h at 37C
as described (3,4). At the end of the stimulation period, culture
26
CA 02203674 1997-04-24
W 096/12728 PCTrUS95113717
supernatants are collected and HIV-l expression is assessed by the
HIV-l p24 antigen capture ELISA (Du Pont) and by the reverse
transcriptase (RT).
In inactivation of cell-associated HIV-l experiments, the treated
and untreated cells could also be submitted to PCR analysis.
4. T~ it~on of ~IIV~ ct~ L~um for~ t~on.
HIV-l-infected H-9 cells are left untreated or treated with
antivirals as described above. Treated and untreated cells (5 x 104
cells/well) are added to 96-well flat-bottom microtiter tissue
culture plates contA;n;ng 1 x 105 indicator SupTl human T cells/well
in complete RPMI 1640 culture medium. Following overnight
incubation at 37C, syncytium formation is scored by two independent
people using an inverted microscope scope.
5. Cytotoxicity studie~.
The cytotoxicity of the antivirals can be tested on a variety of
cell types. All of the cell lines used above and normal human PBMCs
are incubated with different antiviral concentrations for various
time intervals as described above. Cytotoxicity is determined by
the MTT dye method (see above) and by [3H]thymidine uptake and
scintillation counting.
Dosa~e and ~ t~on
The antitumor compounds (active ingredients) of this invention can
be A~m;n;stered to inhibit tumors 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 A~m; n; stered 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 ~m; n; stered alone, but
are generally a~m;n;stered with a phAr~ceutical carrier selected on
the basis of the chosen route of A~m;n;stration and st~n~Ard
pharmaceutical practice.
The dosage A~m;nistered will be a tumor-inhibiting amount of active
ingredient and will, of course, vary depending upon known factors
such as the pharmacodymanic characteristics of the particular active
ingredient, and its mode and route of ~m; n; stration; age, health,
and weight of the recipient; nature and extent of symptoms; kind of
CA 02203674 1997-04-24
W O96/12728 PCT~US95/13717
concurrent treatment, frequency of treatment, and the effect
desired. Usually a daily dosage (therapeutic effective amount or
cancer-inhibiting amount) of active ingredient can be about 5 to 400
milligrams per kilogram of body weight. Ordinarily, 10 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 milligram to about 500 milligrams of active
ingredient per unit. In these ph~rm~ceutical 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 A~m;n; stered orally in solid dosage
forms, such as capsules, tablets, and powders, or in liquid dosage
forms, such as elixirs, syrups, and suspensions. It can also be
administered parentally, in sterile liquid dosage forms.
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 sust~; ne~ 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.
Liquid dosage forms for oral ~m;nistration 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 glycols 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 stabilizing
CA 02203674 1997-04-24
WO96/12728 PCT~S95/13717
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, Mack Publ;sh;ng Company, a stAn~Ard
reference text in this field.
Useful pharmaceutical dosage forms for administration of the
compounds of this invention can be illustrated as follows.
Capsules: Capsules are prepared by filling stAn~rd two-piece hard
gelatin capsulates each with lO0 milligrams 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 soft gelatin capsules cont~;n;ng lO0
milligrams of the active ingredient. The capsules are washed and
dried.
Tablets: Tablets are prepared by conventional procedures so that
the dosage unit is lO0 milligrams of active ingredient, 0.2
milligrams of colloidal silicon dioxide, 5 milligrams of magnesium
stearate, 275 milligrams of microcrystalline cellulose, ll
milligrams of cornstarch and 98.8 milligrams of lactose.
Appropriate coatings may be applied to increase palatability or
delay absorption.
Injectable: A parenteral composition suitable for A~min;stration by
injection is prepared by stirring l.5% by weight of active
ingredients in lO~ by volume propylene glycol and water. The
solution is made isotonic with sodium chloride and sterilized.
Suspension: An aqueous suspension is prepared for oral
A~m;n;stration so that each 5 millimeters contain lO0 milligrams of
finely divided active ingredient, 200 milligrams of sodium
carboxymethyl cellulose, 5 milligr_ms of sodium benzoate, l.0 grams
of sorbitol solution, U.S.P. and 0.025 millimeters of vanillin.
CA 02203674 1997-04-24
W096/12728 PCT~S95/13717
In the present disclosure it should be understood that the specified
materials and conditions are important in practicing the invention
but that unspecified materials and conditions are not excluded so
long as they do not prevent the benefits of the invention from being
realized.
