Note: Descriptions are shown in the official language in which they were submitted.
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~ -Ribofur~no-yl Nucl~osid-s
Field of the In~ention
This invention relates to L-ribofuranosyl nucleosides and
intermediate or derivatives thereof useful in the synthesis of such
nucleosides, processes for their preparation, pharmaceutical
compositions containing such, and methods of using such compounds to
treat various diseases, particularly cancer,in mammals.
B~c~u.~r,d of the In~ention
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 depending 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. Or~. 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
Bacillus subtilis using a series of modified derivatives including
certain ribopyranosyl nucleosides.
Baud, M.V., et al., Tetrahedron Letters, Vol. 31, No. 31, pp. 4437-
4440 (1990), describes the synthesis of certain 2'-
deoxyribonucleoside compounds starting from available sugars (2-
deoxyribofuranosyl or pyranosyl). The compounds described in this
paper are all D-isomers.
Spadari, S., et al, J. Med. Chem., 35, pp. 4214-4220 (1992),
describes certain L-~-nucleosides useful for treating viral
infections including Herpes Simples Virus Type I.
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Holy, A., Nucleic Acid Chemistr~, Vol. 1, 347-353 (1978), describes
the synthesis of 2'-deoxy-L-uridine.
WO 92/08727 describes certain 2'-L-desoxyuridines and their use for
treating viruses.
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 microorganisms and enzymes. (See
generally, Asai, M., et al., Chem. Pharm. Bull., 15(12), 1863-1870
(1967).) The known activity of D-nucleoside compounds, and the
successful commercialization of several of such D-sugar-nucleoside
compounds, (see Perigaud, C., et al., supra, for a discussion of D-
nucleoside analogs which have gained commercial acceptance) led in-
part to the present work relating to the L-isomers of certain
nucleoside analogs.
Perha~c the best known commercial nucleobase analog is 5-
fluorc~~a- l (5-FU) the structure of which is shown below:
,~D,\N~H
o
(s~
5-FU is 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, it also has a narrow margin of safety and is,
therefore, associated with many serious side effects including, for
example, nausea, vomiting, diarrhea, alopecia, leukopenia,
thrombocytopenia, etc. Additionally, 5-FU is primarily used in an
intravenous formulation.
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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:
~ ~ CH3
N3
(AZT
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 intending 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 permeability to
compromised cells. By compromised cells we mean cells such as
cancer cells or other infected cells, whether the infection is
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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 "Synthesis and Biological Evaluation of
2l,3'-Dideoxy-L-Pyrimidine Nucleosides as Potential Antiviral Agents
against HIV and HBV~ published J. Med. Chem., 37 (1994) 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 permeability, 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
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, kinases or thymidilate synthase may recognize the
compound in such a way as to cause inhibition.
Therefore, although certain nucleoside analogs and/or nucleobase
analogs have been commercialized for indications such as cancer
and/or AIDs treatment, there is a need for a nucleoside analog which
is perhaps as cytotoxic as 5-FU or is less cytotoxic but more
specific than 5-FU for cancer therapy and/or a compound which is
more effective and/or better tolerated than AZT for treatment of
viruses.
The present invention relates to a novel group of such L-
ribofuranosyl nucleosides which have interesting activity as
anticancer, antiviral, antiparasitic, antifungal, antibacterial
and/or antimicrobial agents. These compounds are generally water
soluble, which suggests that oral deliver may be achieved, and the
activity of these compounds may be more selective for compromised
cells as compared to normal cells.
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Det~ilod De~criDtion of the Inv-nt$on
There is provided by this invention ribofuranosyl nucleoside
compounds having the formula (I):
B
O ~ R3
RO ~l ~
or a pharmaceutically acceptable salt thereof, wherein:
B is a naturally-occurring nucleobase (A, G, C, U,
hypoxanthine or T) or a modified base comprising one or more
substitutior.s selected from the group consisting of H,
hzlogen, C1-C6 alky_, C2-C6 alkenyl, C1-C6 alkoxy, C3-C6
cycloalkv'-C1-C6 alko~.~, C3-C8 cycloalkyloxy, C3-C8
cycloalkylthio, C1-C6 alkylthio, a substituted amino group, an
aryl, aralkyl, aryloxy, aralkoxy, arylthio, aralkylthio, a
heterocyciic 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
posil c~ 6 in the base may be sulfur;
R is H, CGR-, P(5) R~R or 503H (wherein R5 is alkyl of 1-5
carbon a~sms cr an aromatic ring structure, R6 and R, are each
H or alkyl of 1-5 carbon atoms and n is 2 or 3);
R. and R~ are independe..lly H, halogen, mono- or di-difluro, ORE
or B (wherein R~ is H, CORg, P(O)mRioR;l (wherein Rg is H2,
substituted or unsubstituted alkyl of 1-5 carbon atoms or a
substituted or unsubstituted aromatic ring structure, R1Q and
R are each H or alkyl of 1-5 carbon atoms and m is 2 or 3)),
provided that when R~ is OH, R2 and B can combine to form a 5-
membered cyclic ring structure;
R3 and R~ are independently B, H or OR~2 (where R;2 is H, COR 3,
P(O)FR;~R:~ (wherein R-. is substituted or unsubstituted alkyl of
1-5 carbor. atoms or a substituted or unsubstituted aromatic
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ring structure, R;~ and Rls are each H or alkyl of C1-C5 carbon
atoms and p is 2 or 3)), provided that:
only one of R,-R4 can be B;
when R=H, R~ =OH, R2=H, R3=H and R4=B, then B cannot be ~,
C, T, 5-FU, hypoxanthine, A, or G;
when R=H, Rl=OH, R2=OH, R3=B and R~=H, then B cannot be C;
when R=H, R~=OH, R2=OH, R3=H and R4=B, then B cannot be 5-
FU, C, U, A or hypoxanthine;
when R=H, R;=OH, R,=H, R3=B and R4=H, then B cannot be 5-
FU, A, C, G, T, U or hypoxanthine;
when R=H, R~=H, R2=H, R.=B and R~=H, then B cannot be A, C,
G, ~, U, 5-FU, or hypoxanthine; and
wher, R=H, R =H, R2=H, R3=H and R~=B, then B cannot be A, C,
G, T, U, 5-FU or hypoxanthine.
Preferred compounds of the present invention include those compounds
of formula (~) wherein:
R~or P.; is ~ an~i the other is H, such that when R3 is B the
series is afid when R~ is B, the series is ~;
B is C, T, U, G, I, A, 5-fluorouracil, 6-thioguanine or 4-
thiouracil;
R is H; and
R-R~ are each H or OH, or when R2 is OH, R2 and B combine to
form a five-membered cyclic ring.
Specifically preferred compounds of the present invention are the
followina: ~-L-ribofuranosyluracil; 1-(2,3,5-tri-0-benzoyl-~-L-
ribofuranosyl)-4-thiouracil; ~-L-ribofuranosyl-4-thiouracil; 1-(3,5-
di-O-benzoyl-2-deoxy-~-L-~ibofuranosyl)-4-thiouracil; 2'-~-L-
deox~rib^furanosyl-4-thiouracil; ~-L-ribofuranosyl-5-fluorouracil;
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~-L-ribofuranosyl guanine; ~-L-ribofuranosyl-6-thioguanine and
pharmaceutically acceptable salts thereof.
Also proYided by this invention are processes for the preparation of
the compounds of formula (I), r~r~Aceutical compositions contAi~ing
the compounds of formuls (I) _nd methods of using the compounds of
formula (I) for the treatment of c ncer in a r~mmAl, ~s well as
methods of using the compounds of formula (I) as antivirAls,
antiparasitics, antibacterials, _ntifungals and ~ntimicrobial agents
in a m~mmal.
The present invention describes a series of L-ribofuranosyl
nucleosides usefut for treating various dise~ses (including cAncer
and certain viruses). Compounds of this invention may be orally
a~tive 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 ribofuranosyl sugar via ~ linkage (B is R, in a
compound cf Formula (I)) can be made by the general Scheme I.
SC~EME I
General procedure to make ~-L-ribofuranosyl pyrimidines:
1. HMDS ClSiM~/ ~ \
BzO ~ ~ Ac + Base Snc ~ CH3CN ~ H
To a mixture of l-O-acetyl-2,3,5-tri-0-benzoyl-~-L-ribofur~nose ~1
mol) and pyrimidine base (1 mol) in ~nh~drous MeCN are successively
added HMDS (1 mol), ClSiMe3 (O.8 mol) nd SnC14 (1.2 mol). The
resulting clear solution is refluxed for 1 hour when TLC indie~tes
completion of the reaction. The ~olvent is ev~porated ~nd the
residue dissolved in EtOAc, washed with NaXC03 and H20. The EtOAc
layer is dried, filtered and evaporated to give the crude product,
which is either crystallized or purified on a silica gel column to
obtain the pure 2,3,5-tri-0-benzoyl-~-L-ribofuranosyl pyrimidine
compounds These oompounds are stirred with NH3/~eOH to give pure
SUBSTITUTE SHEET (RULE 26~
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PCT~S95/13716
~-L-ribofuranosyl pyrimidines after purification and
crystallization.
A more detailed schematic ior ~-linked pyrimidine compounds within
the scope of the present invention is shown in SchPme I-A.
SCHEME I-A
L,R~x~e l.MeOH~2SO~ ~ ~ Mc 1.HE~HOA~n~O
2.BzCVPz ~ ~ 2.HOA~AQO ~ o~
1 2
OSiMc
2+ N ~ TMSOTf . ~ ~ NH~eOH ~ ~ ~
Mc3SiO 1 N ~ C1CH2CH2C1 ~ ~ \ HO ~ ~ \
0~_~ O~
o 4
H~ H~S/CISiM~ ~\ NH~lMeOH >
o~H SnC~CH3CN \ HO
5 ~P 6
NH~
2+ ~ H~DS/CIS~ f~ N]H~ OH r
~ SllC~CH3CN
0~ 0~
7 8
The compounds of this invention wherein the nucleoside has a purine
base (A, I, G or su~stituted purine base such as thio-G) which is
SU~STITUTE SHEET (RULE 26)
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linked to the ribofuranosyl sugar via ~ linkage (B is R, in a
compound of Formula (I)) can be m~de by the general scheme II below.
SCHEME ~I
General procedure to make ~-L-ri~ofuranosyl purines:
Silylated l.TMSOTf/C~C~CH2Cl ~ \
B ~ ~ Ac + Base 2. ~eOH HB~
A mixture of purine base (2 mol) and (NH,)2SO, (cat~lytic amount) in
~MDS is refluxed until the solution becomes cle~r. The resulting
clear solution is concentrated to yield silylated base to which
anhydrous dichloroethane is added and the solution is cooled to 0C.
~nder nitrogen atmosphere a solution of 1-0-acetyl-2,3,5-tri-0-
benzoyl-~-L-ribofuranose in dichloroethane (1 mol) nd TMSOTf (2.1
mol) are added to the above solution and stirred at room temperature
for 16 hours. The rea~tion 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 2,3,5-tri-0-benzoyl-~-L-ribofur nosyl purines,
which after stirring hith NH /Me~H and usual purification give pure
~-L-ribofuranosyl purines.
A more detailed schematic for the synthesis of ~-linked purine
compounds within the scope of the present invention is provided in
Scheme II-A.
SUBSTITUTE SHEET (RULE 26)
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SCHEME II-A
~ ~ l.DM~JN~/80C
Nl ~ - Nl TMSOTft ~ 2.MeOH/NH3
N / ~ ~ ClCH2CH2CL ~ \ HO ~ ~ \
N ~ N N ~ N
9 10
t NH~
HSCH2CH20H/
NaOMeJ
MeOH
e~?\
~ N ~ N ~
11 ~ NH
C~
N~N TMSOTf/ ~ / \
~NJ~NJ\NH~C
3 ~ N~NHAc
12 --~N
NaOMe /\ l.l~ol~an~OH
MeOH CH OH / ~ .NH3~eOH
HO ~ \ HO J ~
~N~l~N~NH2 ~N~N~NH2
N ` NH N~NH
SUBSTITUTE SHEET (RULE 26)
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The compounds of this invention wherein the nucleoside has a
pyrimidine base (U, T, C or substituted pyrimidine base ~uch as 5-F~
or thio-U) which is linked to the ribofuranosyl ~ugar ~ia ~ linkage
(B is R3 in a compound of Formula (I)) can be made by the general
Scheme III below.
SC~ME III
General procedure to make c-L-ribofuranosyl pyrimidines:
B~
~SPh Silylated 1. ~BS/M.S.4AIC~C12 ,~ \
~ ~ + B~ 2.H~/P~C~OH HO
A mixture of pyrimidine base (2 mol) in HMDS and ~mmonium sulfate
(catalytic amount) is refluxed until the solution becomes clear.
The resulting clear solution is concentrated in vacuo to yield
silylated base. To this silylated base in anhydrous CH2Cl2 under
nitrogen atmosphere, l-thio-2,3,5-tri-0-benzyl-L-ribofuranoside (2
mol) 4A molecular sieves and NBS (l.l mol) are added. The reaction
mixture is stirred at room temperature overnight and quenched with
addition of Na,S;Oj solution. The organic layer is washed with water
brine a~nd dried over Na250~. ~vaporation of the solvent gives the
crude product which is pu-ified on a silica gel column to obtain
pure 2,3,5-tri-0-benzyl-c-L-ribofuranosyl pyrimidines. These
compounds are subjected to H2/Pd~C reduction, followed by
purification and crystallization to give pure c-L-ribofuranosyl
pyrimidines.
A more detailed schematic for the synthesis of ~-linked pyrimidines
within the scope of the present invention is provided in c~h~me III-
A.
SUBSTITUTE SHEET (RULE 26)
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SCHEME I 11 -A
NHk ~NHC ~ --C- OCH3~ ~ H~ot
CN 50% E~OH ~ ~ 0.2 ~ Ha
o ~ C~H5CN .
CH~CN~3N ~ ~ P2S5ÇPy /
H~ E~\~ ~/
17 18 19
NH~eOH / \ NE~cOH
100C / \ r.~
NHl ~ 5
H~ H~J
21
~` PhSFVSnC~4 . ~ ~ s~ 2 N~H~h~31DMF~ ~ s~
2 22 23
~' ~ Ba3/CB2a2 ~/
NBSI~LS.4A~ ~ 2C12 H
24 25
The compounds of this invention wherein the nucleoside has a purine
base whi~h is linked to the ribofurAnosyl sugar ~ia ~ linkage (B is
R3 in a compound of formula (I)) and only one of Rl or R2 is OH, or
where R2 is O~ and com~ines with B to form a cyclic ring structure,
can be made by the general Scheme IV below.
SUBSTITUTE SHEET (RULE 26J
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SCHE~E IV
General procedure to make ~-L-2'-Deoxyribofuranosyl purines:
Cl
\ Silylated 1. NaH,/C~}CN ~/ \
~Tol ~ 2. ~eOH HO
B~
A mixture of purine ~ase (2 mol) ~nd NaH ~2.2 mol) is ~tirred in
anhydrous C~3CN under nitrogen atmosphere at room temperature for 30
min. 1-Chloro-2-deoxy-3,~-di-0-p-toluoyl-c-L-pentofuranose is added
to the reaction mixture and stirred for 2 hours. The reaction
mixture is diluted with CHCl3 and filtered through Celite~. The
filtrate is conce~trated, redissolved in EtOAc ~nd washed with water
and brine. After drying and evaporatiny the solvent, the residue
obtained is purified on a silica gel column to give pure 2'-deoxy-
3,5-di-0-p-toluoyl-~-L-ribofuranosyl purines. These compounds Are
treated with NH,/MeOH and then purified and crystallized to give
pure ~-L-2'-deoxyribofuranosyl purines.
A detailed schematic for similar ~-linked deoxy-ribofuranosyl having
pyrimidine bases is sho~ in Scheme IV-A.
SUBSTITLITE SHEET (RULE 26
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SCHEME IV-A
NH2CN ~----~N HC=C~--~CH~
,h;-~e
N~
26
HO~ BzCN, ~ HCI/DM~'.
DMI' N
27 o 28
9 AIBN ~ ~ NaOMe/MeOH. H~
~ 3 Bu3 Sn~VBenz~e ~ 31
29 30
P~Ss~o~ane 2.H2,P~C
~ ~ NH3/MeOH ~ H~
34 ~ 32 - ~ 35
NH3/MeOH
lOoC
o\
H~/~/
~N~ 33
NX2
SUBSTITUTE SHEET (RULE 26)
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The compounds of this invention comprising a-L-2'deoxy ribofur nosyl ~pyrimidines and purines (including 5-FU analogs of such) are shown
in Scheme V, whereas Scheme V-A ~hows methods for making ~-linked L-
2~-deoxy-ribofuranosyl compounds.
SC~ V
General procedure to make -L-2'-doexyribofuranosyl pyrimidines and
purlnes:
B~
/(~)2
~ ~ 1,3~ ~ O~}1~,3,3-t~la- o / ~
H ~ ~ ~opn~l di~ n~ PhCXC(S)
~H ~ ~ ~ D~LAP~
6~ )2 ~H
36a
~ 2 ~)2
0/ ~<o\~jCc B~Sn~VAIBN,
~ i~\~/ Toluene ' i~o W ~ H
'.-~)2 (~ OPn ~ )2 39a
38a
37a
- ~ 3 l ~ 5 ~ -o- ( l, l, 3 ~ 3 -~-tr~ G~ 3-~llyl)-~-L-
~r~blnofur~no~yll-~ur~n- or ~yr~ (36~)
To a stirred suspension of (~ 1 mol) in pyridine is added 1,3-
di~hloro-1,1,3,3-tetraisopropyldisiloxane (1.2 mol). This is
stirred at room temperature until the completion of the reaction
(five hours), the solvent is evaporated ~nd the residue is dissolved
in ~tOAc and washed with water, 5~ HCl, water, ~aturated ~gueous
NaHCO3 and brine. After drying over ~nhydrous Na2SO, it was filtered
and evaporated to give the crude produot (~6~) which is used in the
next step without further purifi~ation.
1-12'-O-P~ h~ocar~onyl-3',S'-0-(1,1,3,3-t-tr~
~1-llox n~ 3-~lyl)-t-L-~rab~nofur~nol~yl~-~urln- or ~y~
(~7~)
To a solution of (~g) (1 mol) in anhydrous CH3CN is added 4-
dimethly amino pyridine (DMAP) (1.9 mol) and phenyl
lS
SUBSTITUTE SHEET (RULE 26)
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chlorothionoformate (1.1 mol). The solution is stirred at room
temperature for 24 hours. Then the solvent is evaporated and the
residue dissolved in EtOAc and washed with water, 5~ HCl, water,
saturated aqueous NaHCO, and brine. The EtOAc layer is dried
(Na2SO~), filtered and evaporated. The residue is purified on a
silica gel column to give pure (37c).
3~,5~-0~ ,3,3-T--traioovro~yldi-iloxane-1,3-diyl)-~-~-~urine or
~yrimudine (~g)
To a mixture of (~1~) (1 mol), AIBN (0.2 mol) in dry toluene is
added Bu~SnH (5 mcl). The solution is deoxygenated with oxygen-free
Ar then heated at 75C for four hours. The solvent is then
evaporated and the residue is purified on a silica gel column to
yield pure (38~!.
2~-Deoxy-~-L-~rine or pyrimudine (39~)
A mixture of (~) (1 mo ) and TBAF (2 mol) in THF is stirred at
room temperature. After completion of the reaction the solvent is
evaporated anc the residue is dissolved in water and washed with
ether. The water is evaporated and the residue purified on a silica
gel colu~. to give pure 2'-Deoxy-c-L-purine or pyrimidine.
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SC~EME V-A
O \ ~ h
~ 1,3~i~hln~}1~1,3,3-teba- ~ \
HOJ ~o~u~ si~n~ ~ , O ~ ~ PhOC(S)CU
O ~,~ N ' ~OJ ~ DMAp/py
6 l ~ 36
h /(~
~ Toluene ~ f ~ TBAF~HF
37 P~ , ~ ~p 38 P~
R =--C-OPh
A general schematic for the synthesis of e and ~-L-2'-3'-
dideoxyribofuranosyl pyrimidines and purines is pro~ided below in
Scheme VI. Detailed schematics for ~-linXed 2'3' dideoxy
pyrimidines and ~-linked 2'deoxyinosine are shown in Sch~mes VI-A
and VII.
SC~EME VI
General procedure to make e and ~-L-2',3'-dideoxyribofuranosyl
pyrimidines and purines:
UTE S~ET`~R~2~)
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OAc Silyl~tcd EtAlC~ r 0~ c~
~i + Basc CH2C~2
1. Scparation
2.TBAFr~HF and H
A mixture of purine or pyrimidine base (1 mol) in HMDS and G onium
sulfate (catalytic amount) is refluxed until the solution becomes
clear. The resulting clear solution is concentrated in vacuo to
yield silylated base. To a solution of this silylated base in
nhydrous CHaCl2 under nitrogen atmosphere, a solution of l-~-acetyl-
5-O-(tert-butyldiphenylsilyl)-2,3-dideoxy-L-ribofurnose is ~dded
followed by the addition of EtAlCl2. The reaction mixture is
stirred at room temperature for an hour and then poured into an ice
cold mixture of CH2Cl, and saturated NaHCO3 solution. The mixture is
stirred for lO m.n and filtered through Celite0. The organic l~yer
is washed ~ith saturated NaHCO3 solution and brine. After
evaporating the solvent, the c,0 crude product is separated on a
silica gel column to give pure ~ and 0-5'-O-(tert-
butyldiphenylsilyl)-2',3'-dideoxy purines and pyrimidines. These
compounds are treated with TBAF to remove the ~ilyl protection, and
then purified on a silica gel column to give pure ~ ~nd ~-dideoxy
purines and pyrimidines.
The compounds of this invention wherein the nucleoside ~s ~
pyrimidine base (U, T, C or substituted pyrimidine base) which is
linked to the ribofuranosyl sugar via ~ linka~e (B is R, in a
compound of formula (I)) and are 2',3~-dideoxy compounds, can be
made by the yeneral Scheme VI-A below.
SUBSTITUTE SHEET (RULE 26~
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SCHEME VI-A
H~/ MM~ ~1 P~OC(S)
DMUP~
31 ~ 40 ~ ~ 4
B~SnHyAlHN B0~ HOA~ , H~ lJ~O~ ~ H~
~1 ~ r.L 2~4CPhOPOC~h ',~
~ ~ ~ 4.N ~ sOH N~
42 O 43 O 44 NE~
H~ T~DMSC~ , TRn~ ~ NaOHyC~/B~CH~CN ~ r~
~0~ DMSO
6 ~ 45 ~ 46
B~SnH ~DM~ ~ l.~C H~
AIHN2.TBAF~rHF R=~--C~-so~o~cN
~ U~p
o 48
Compounds of this in~ention wherein the nu~leoside is inosine
(hypoxanthine) linked to the sugar via ~-linkage ~re shown in the
general scheme VII below.
SUBSTITUTE SHEET (RULE 26)
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WO 96/13512 PCT/US95/13716
~ - L - 2 ' -Deoxyino s ine C~O
L~ binn~:e BnOH~ICl ~ ~0~
~ C~Ph p-TsOH~DMF
HO
0~0 0~0
I~\o \~ NaHlCS2/MeI l~\o ~ BU3SnH
~1 ~ OC~h THF ~1 ~ OC~Ph Toluene
OH O-C-SC~
51 52 S
O ~ O HO
oc~2Ph 2Ø5M HCl 2.p-TolCVPY
53 54
D~_OC~ HcvEther
~Tol ~Tol
56
~ N Na~CN
O~Tol / + ~ O~Tol /
~TolO~ ~ / ~ N ~ ~ ~TolO~ ~ /
56 ~ ~ N
HSCH2CH20H/H ~ 57 Cl
NaOMe/MeOH
58
SUBSTITUTE SHEET (RULE 26
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other compounds within the scope of the invention can be made based
on the teachings of the schematics provided herein, as well as the
specific examples incorporated herein and in view with what is known
to the skilled artisan.
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 Chemistrv. Imnroved ~nd 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, Ne~ 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 being readily available to those skilled in the art and
incorpGrate~ herein. Suitable methods for making substitutions
within the sugar moiety of the presently claimed compounds are known
to those skilied in the art and are described in various
publica~ions including: US Patent 4,880,782; W088/00050; EP 199,451
A2; ~S Patent 3,817,982; Lange, P., et al, Progress in Antimicrobial
and ~-~icancer Chemotherapy, Proceedinqs of the 6th International
Cc~ ess -f C~ .hera~v, Univ. Park Press, England, 1970, Vol. II,
p. 3~ ?; and To~send, et al., supra, all of which are
inc_-p--ated herein by reference.
This in~JentiOn can be further understood by referring to the
fo'lo~ins Examples and Tables below:
~xDeriment~l
Fx~mnle 1
L-RibofuranosYll~racil
Ste~ A
l-O-Acetyl-2, 3, 5-tri-0-benzoyl-~-L-ribose (~)
To a solution of L-ribose (5.0 g, 33.31 mmol) in MeOH (150 ml),
concentrated H25O~ (0.5 ml) was added and refluxed for 2 hours.
After cooling the reaction mixture, pyridine (30 ml) was added and
the solven~s were evaporated. To the residue another 30 ml of
pyridine was added and evaporated to dryness. The residue was
dissolved in pyridine (5G ml) and CH2Cl2 (25 ml) then cooled to 0C
~i
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and benzoyl chloride (19 ml, 166.55 mmol) was added dropwise and
stirred at room temperature overnight. The solvents were evaporated
and the residue dissolved in CHCl~ and washed with H~O and NaHCO3 and
dried over anhydrous Na2SO4. After evaporating the CHCl3, the
residue was coevaporated with toluene to give a brown residue. The
brown residue was dissolved in 30% HBr/OHAc (67 ml) and the solution
was stirred at room temperature for 30 minutes, after which time
glacial acetic acid (47 ml) was added and the mixture was cooled to
8~C (internal temperature) in an ice-salt bath. Stirring and
cooling were continued while H2O (34 ml) was added dropwise. The
mixture was removed from the cooling bath and stirred another 30
minutes. Then the solvents were evaporated and the residue was
dissolved in CHCl3 and washed with H2O and NaCHO3. The CHC13 was
evaporated do~ to 50 ml, pyridine (50 ml) and acetic anhydride (9.5
ml) were added and stirred overnight at room temperature. Then the
solvents we-e evaporated and the residue dissolved in CHCl;, washed
with H O and NaHCO.. After evaporation of the solvent a ~rown
residue was obtained and it was coevaporated with toluene. The
brown residue was triturated with EtOH to give light brown crystals.
This was recrystallized from EtOH/EtOAc (5:2) to give 2 (8.15 g,
g8.5~) as whi~e crystals: m.p. 126 - 127CC.
Ste~ B
1- (2, 3, ~-Tr ~ - ^-be~,zcy 7-~ -ribcfura..osyl ) uraci1 (~)
A mixture of uracil (Q.4~ g, 3.96 mmol) and (NH~)2SO4 (catalytic
amour.t) ir. HMDS (25 ml) was refluxed for five hours. The resulting
solution was concer.trated under anhydrous conditions to yield
silylated uracil. To a cooled (0C) and stirred solution of
silylated uracil and 2 (1.0 g, 1.98 mmol) in dry dichloroethane (50
ml~, TMSCmf (0.77 ml, 3.96 mmol) was added. The reaction mixture
was warmed to room temperature and stirred for 16 hours. The
reaction was quenched with saturated NaCHO3 solution (5 ml) and
evaporated to dryness. The residue was dissolved in EtOAc (100 ml),
washed with water, brine, dried, filtered and evaporated to give a
solid residue and it was purified on a silica gel column using
EtOAc/CHCl~ (30 - 40~) to give a white solid which was crystallized
from EtOH/petroleum ether to give pure 3 (0.914 g, 82.7~) as white
crystals: m.p. 143 - 144C.
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Ste~ C
~ L-Ribofuranosyluracil (4)
Compound 3 (0.87 g, 1.56 mmol) in NH3/MeOH (100 ml) was stirred at
room temperature overnight. The solvent was evaporated and the
residue was dissolved in H2O (50 ml), washed with ether (3 X 25 ml)
and evaporated to give a white solid and it was crystallized from
95% EtOH to give pure compound 4 (0.335 g, 87.2~) as white crystal:
m.p. 165 - 166C.
~xamDle 2
~ L-Ribofuranosvl-5-fluorouracil
Ste~ A
1-(2,3,5-Tri-O-benzoyl-~-L-ribofuranosyl)-5-fluorouracil (~)
To a mixture of 5-fluorouracil (0.85 g, 6.54 mmol) and compound
(3.0 g, 5.94 mmol) in anhydrous MeCN (100 ml) were successively
adaed HMDS (1.25 ml, 5.95 mmsl), ClSiMe~ (0.6 ml, 4.76 mmol) and
SnC1; (0.83 ml, 7.13 mmol). The resulting clear solution was
refluxed for 1 hcur when TLC indicated completion of the reaction.
The solvent w2~ evaporated and the residue dissolved in EtOAc t250
ml), washed with NaHCO, and H~O. The EtOAc layer was dried, filtered
and evaporated to give a white solid and it was crystallized from
CHCl !MeOH (1 - 2~) to give compound 5 (2.11 g, 61.9~) as white
crystals: m.p. 20& - 209'C.
Ste~ B
l-~-L-~ibofura.._syl-5-fluorouracil (6)
Compound 5 (0.75 g, 1.30 mmol) in NH./MeOH (100 ml) was stirred at
room temperature overnight and worked up as ln Example 1, Step C to
give pure 6 (0.33 g, 96~) as white crystals: m.p. 147 - 148C.
F~mnle 3
1-~-L-Ribofuranosvlcvtosine
Stec A
1-(2,3,~-~ri-0-benzoyl-~-L-ribofuranosyl)-~-acetylcytosine r7)
To a mixture of N~-acetylcytosine (0.18 g, 1.19 mmol) and compound 2
(0.50 g, 0.99 mmol) in anhydrous MeCN (30 ml) were successively
added HMDS (O.17 ml, 0.79 mmol), ClSiMe3 (0.10 ml, 0.79 mmol) and
SnCl~ (0.14 ml, 1.19 mmol). The resulting clear solution was
refluxed for one hour. Then the solvent was evaporated and the
residue dissolved in EtOAc (100 ml), washed with NaHCO, and H2O.
After evaporaticn of the solvent the residue was purified on a
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silica gel colu~n using EtOAc/petroleum ether (70~) to give pure 7
(0.41 g, 70~) as a white foam.
Ste~ E
~ L-Ribofuranosylcytosine (8)
Compound 7 (0.85 g, 1.42 mmol) in NH3/MeOH (100 ml) was stirred at
room temperature overnight and worked up as in Example 1, Step C to
give pure 8 (0.18 ~, 52~) as white crystals: m.p. 210C.
~mnl e 4
9-~-L-Ribofuranosvladenine
SteD A
9-(2,3,5-Tri-O-benzoyl-~-L-ribofuranosyl)-6-chloropurine (9)
A mixture of 6-chloropurine (1.22 g, 7.93 mmol) and (NH4)2SO4
(catalytic amount) in HMDS (25 ml) was refluxed for eight hours.
The resulting solution was concentrated under anhydrous conditions
to yield silylated 6-chloropurine. To a cooled (0C) and stirred
solution of si'ylated 6-chloropurine and 2 (2.0 g, 3.97 mmol) in dry
dichloroethane (25 ml), TMSOTf (1.87 g, 1.6 ml, 7.93 mmol) was
added. The reaction mixture was warmed to room temperature and
stirred for 16 hours. The reaction was quenched with saturated
NaHCO. solution (10 ml) and the solvent was evaporated. The residue
was dissc ved in EtOAc (150 ml), washed with water, brine, dried,
fiitered and evap^rated to give a solid residue and it was purified
on a silica gel columLn using CHCl.:MeOH (5~) to give pure ~ (2.35 g,
99~) as foa-..
Ste~ B
g-~-T,-Ribofuranosyl~denine (10)
A solution of 9 (1.00 g) in DME/NH; (50 ml) was heated at 80C in a
steel bomb for 24 hours. After cooling, the solvent was evaporated
and the solid obtained was stirred in NH,/MeOH (100 ml) overnight.
After the evaporation of the solvent, the residue was dissolved in
water (50 ml), washed with CHC13 (2 x 25 ml) and ether (2 x 25 ml).
The water layer was evaporated and the residue crystallized from
water to give pure 10 (0.30 g, 67~) as white crystals: m.p. 225C
(dec).
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Fx~mnle 5
9-~-L-Ribofuranosvlh~Doxanthine (~1)
A mixture of 2 (1.05 g, 1.70 mmol), mercaptoethanol (0.48 ml, 6.9
mmol), and NaOMe in MeOH (100 ml) was refluxed for four hours. The
reaction mixture was cooled, neutralized with glacial acetic acid
and evaporated to dryness. The solid obtained was washed with CHCl3
and the residue was crystallized from EtOH to give pure 11 (6.198 g,
47~) as white crystals: m.p. 212 - 213C (dec).
F~xamnle 6
9-~-T,-RibofuranosvlcuAnine
Ste~ A
9- (2, 3, 5-Tri-O-benzoyl-~-L-ribofuranosyl) -2-acetarnido-6-chloropurine
(12 )
A mixture of 2-acetamido-6-chloropurine (1.68 g, 7.93 mmol) and
(NH;) 253~ (catalytic amour,t) in HMDS (75 ml) was refluxed for 16
hours. The res~llting clear solution was concentrated under
anhydrous cor,d_tions to yield silylated 2-acetamido-6-chloropurine.
To a cooled (0 C) and stirred solution of silylated 6-chloropurine
and 2 (2.0 g, 3.96 mmol) in dry dichloroethane (100 ml), TMSOTf tl.6
ml, 7.93 mmol) was added. The reaction mixture was quenched with
saturated NaHCO. solution (10 ml) and the solvent was evaporated.
The residue was dissolved in EtOAc (150 ml), washed with water,
brine, dried, filtered and evaporated to give a solid residue and it
was pu-ified on a silica gel colu~ using EtOAc/petroleum ether (40
- 50C) to give pure 12 (1.38 g, 53%) as white foam.
Ste~ E
9-~-L-Ri~of..ranosylgua.~ine (~)
A mixture of 12 (0.58 g, 0.88 mmol), mercaptoethanol (0.25 ml, 3.53
mmol) and NaOMe (0.76 ml, 3.53 mmol, 25~ weight solution in MeOH (10
ml)) was refluxed for six hours. The reaction mixture was cooled,
neutralized with glacial acetic acid and evaporated to dryness. The
solid obtained was washed with CHC13 and the residue was
crystallized from water to yield pure 13 (0.215 g, 86~) as white
crystal: m.p. 248C (dec).
F~ATr~1e 7
9-~-L-Ribofuranosvl-6-thio~uanine (14)
To a solution of 12 (0.68 g, 1.03 mmol) in anhydrous EtOH was added
thio~rea (3.15 g, 2.06 mmol). The reaction mixture was refluxed for
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an hour and then the solvent was evaporated. The residue was
dissolved in EtOAc and washed with water and dried. After
evaporation of the solvent the crude product was purified on a
silica gel column (5~ MeOH/CHCl3) to yield benzoylated thioguanine.
The product was debenzoylated by stirring with NH3/~eOH (100 ml) at
room temperature overnight. After evaporating the solvent the solid
obtained was dissolved in water and washed with CHCl3 (3 x 50 ml).
Then the water was concentrated and crystallized from water to give
pure 14 (0.15 g, 58~) as yellow crystals: m.p. 227C (dec).
~Amnle 8
2-Amino-~-L-ribofurano~1'.2':4.5loxazoline (1~)
A mixture of L-ribose (7.0 g, 46.63 mmol), cyanamide (3.92 g, 93.27
m~mcl) and lN NH~OH (20 ml) was heated in a 30 - 35~C water bath until
the solids were dissolved. The reaction mixture was kept at room
temperature for 30 minutes and heated again at 60C for an hour,
during which time a white solid started to precipitate. After
adding MeO~. (35 ml), this was kept in the refrigerator overnight,
filtered and washed with MeOH and ether to give compound 15 (7.46 g,
92%) as white crystals: m.p. 195 - 196C (dec).
ExamDle 9
O ~--Anhvdrc-1-c-L-ribofuranosvluracil (~6)
A mixture of compound 15 (7.86 g, 45.13 mmol) and methyl propiolate
(14.04 mi, 15,.95 mmol) in 50% EtOH (100 ml) was refluxed for six
hours. After cooling, the solvent was evaporated to dryness and
coevaporated Iwice with EtOH. Then the solid obtained was boiled ir.
EtOH, cooled a~G filtered to give compound 16 (5.88 g, 57.6%) as
white crystals: m.p. 215C (dec).
F:xAmnle 10
1-~-L-Ribofuranosvluracil tl7)
A solution of compound 1~ (4.50 g, 19.89 mmol) in 30 ml of 0.2 N HCl
was refluxed for two hours. After cooling, it was neutralized with
Dowex (2 x 8-100) ion exchange resin. The resin was filtered and
washed with water and the combined filtrates were evaporated and
coevaporated with ethanol to give a hygroscopic foam, to which 1:1
mixture of acetone-ether (100 ml) was added and kept at room
temperature fo~ two days. This was filtered to yield compound 17
(4.80 g, 86.6%) as white solid: m.p. 137~C.
26
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~am~le 11
1-(2.3,5-Tri-O-benzovl-~-L-ribofuranosyl)-4-thiouracil (19)
Ste~ A
1-(2,3,5-Tri-O-Benzoyl-~-L-ri~ofuranosyl)uracil rls)
A solution of benzoyl cyanide (4.29 g, 32.76 mmol) in CH3CN (25 ml)
was added dropwise to a suspension of compound 17 (2.0 g, 8.19 mmol)
in CH3CN (50 ml) followed by Et3N. The mixture was stirred at room
temperature for three hours and the solvent was evaporated to
dryness. The crude material obtained was purified on a silica
column using 50~ EtOAc/hexane as solvent to yield compound ~ (4.55
g, quantitative) as pale yellow foam.
Ste~ B
1-(2,3~5-Tri-O-benzoyl-~-L-ri~ofuranosyl)-4-thiouracil (12)
Phosphorus pentasulfide (5.99 g, 26.95 mmol) was added to a solution
of compound 18 (3.75 g, 6.73 mmol) in pyridine (70 ml) and was
refluxed for four hours. After cooling, the solvent was evaporated
anc the residue dissolved in CHCl~, washed with water and brine.
After drying over anhydrous Na2SO~, evaporation of the solvent gave
the crude product which was purified on a silica gel column using
30% EtOAc/pelroleum ether as solvent to give compound ~2 (3.68 g,
95~) 2S golde~ yello~ foam.
Exæm~'e 12
~ L-RibofuranosYlcYtosine (20~
CompGund 19 (3.6& g, 6.42 mmol) was treated with 200 ml of
methanolic ammonia in a bomb at 100~C for 18 hours. After cooling
to room tempera'ure, the solvent was evaporated to dryness and the
residue dissolved in water (200 ml). The aqueous solution was
extracted successively with CHCl3 and CCl4 (3 x 100 ml) to remove
benzamide and methyl benzoate. The aqueous layer was treated with
active charcoal, filtered through Celite~, evaporated to dryness and
coevaporated with EtOH. The solid obtained was recrystallized from
MeOH to give pu~e compound 20 (1.36 g, 81~) as white solid: m.p. 206
- 207^C (dec).
n le 13
~ L-Ribofuranosvl-4-thiouracil (~1~
Compo~nd 19 (0.61 g, 1.06 mmol) in NH3/MeOH (40 ml) was stirred at
room temperature overnight. The solvent was evaporated and the
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residue was purified on preparative plates using MeOH/CHCl3 (20~) to
give pure compound ~ (0.185 g, 66.5~) as yellow foam.
~ m~le 14
l-~-L-ribofuranosyl-5-fluorouracil
Ste~ A
1-Thio-2, 3, 5-tri-0-benzoyl-L-ribofuranoside (~)
To a solution of 2 (0.50 g, 0.99 mmol) in CH2Cl2 (50 ml), thiophenol
(0.11 ml, 1.09 mmol) was added and stirred at room temperature for
15 minutes. Then the reaction mixture was cooled in an ice bath and
SnC1~ (O.07 ml, 0.59 mmol) was added dropwise and stirred at room
temperature overnight. The reaction mixture was washed with 2N HCl
(2 x 20 ml), water (25 ml), NaHCO3 solution (25 ml) and then with
brine. After drying over Na2SO~, the solvent was evaporated and the
residue was purified on a silica gel column using 15 - 20~
EtOA~/pet-Gle~. ether as solvent to give pure compound 22 (0.45 g,
82~) as an oil.
Ste~ B
1-Thio-2,3,~-tri-O-benzyl-L-ribofuranoside (~)
To a sclution of 22 (0.45 g, 0.81 mmol) in MeOH (20 ml), NaOMe (0.03
ml, 0.16 mmcl) was added and stirred for 18 hours. The reaction
mixture was ne-_.ralized by Dowex 50 ion exchange resin, filtered and
evapcrated. ,_ this residue, DMF (20 ml) was added and cooled in an
ice bath. To this cooled solution NaH (0.32 g, B.11 mmol) was added
in portion and s.irred for 15 minutes. Benzyl bromide (0.96 ml,
8.11 mmol) was adde dropwise and stirred at 0~C for 2 - 3 hours.
The reaction was quenched with water after diluting with EtOAc. The
EtOAc layer was washed with water (2 X 25 ml) and brine. After .
dryina and evaporation of the sol~ent, the crude product obtained
was purified on a silica gel column using 5 - 10% EtOAc/petroleum
ether as solver.t to yield pure Z3 (0.30 g, 73.5%) as an oil.
Ste~ C
1- (2, 3, ~-Tri-O-benzyl-~-L-ribOfUranOsyl) -5-fluorouracil (~)
A mixture of 5-fluorouracil (0.15 g, 1.17 mmol) in
hexamethyldisilazane (30 ml) and ammonium sulfate (catalytic amount)
was refluxed for four hours. The resulting clear solution was
concentrated in vacuo to yield silylated 5-fluorouracil as colorless
oil. To a solution of silylated 5-fluorouracil in CH2C12 (20 ml)
unde- nitrogen atmosphere were added NBS (0.11 g, 0.64 mmol), 4A
28
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W O 96/13512 PCT~US95/13716
molecular sieves (0.21 g) and compound ~ (0.30 g, 0.58 mmol) in
CH2Cl, (20 ml). The reaction mixture was stirred at room temperature
overnight and quenched with the addition of Na2S203 solution. The
organic layer was washed with water, brine and dried over Na2SO4.
Evaporation of the solvent gave the crude product and it was
purified on a silica gel column using 5% MeOH/CH2Cl2 as solvent to
give the pure c isomer ~ (0.23 g, 74.5%) as yellow oil.
Stez D
l-c-L-ribofuranosyl-5-fluorouracil (2~)
To a solution of ~ (1.0 g, 1.87 mmolJ in CH2C12 (50 ml), at -78C
under nitrogen atmosphere, 1 M solution of BCl3 (20 ml, 20.57 mmol)
was added dropwise. The reaction mixture was stirred at -78C for
four hours, a 1:1 mixture of CH2Cl2/MeOH (50 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
(25 ml) 5 times. The residue obtained was dissolved in water and
washed with CH~_- (2 x 50 ml). The water layer was evaporated to
give a white sclid which was crystallized from EtOH/ether to give
the pure 2S (0.41 g, 83%) as white crystals: m.p. 150C.
Exam~le 15
2-~mino-~-L-arabinofurano~1' 2':4 5loxazoline (~6)
A mixture of L-ara~inose (10.0 g, 66.60 mmol), cyanamide (5.60 g,
133.20 mmol), methanol (30 ml) and NH~OH (3.3 ml) was stirred at
room temperature for three days and then kept at -10C overnight
The product was ccllec~ed with suction, washed with methanol and
ether to give compound 26 (9.60 g, 82.7%) as white crystals: m.p.
175C.
~mnle 16
C~,O--Anh~dro-~-L-arabinofuranos~luracil (27)
A solution of compound 26 (15.0 g, 85.15 mmol) and methyl propiolate
(23.0 ml, 261.75 mmol), in 50% aqueous ethanol (250 ml) was refluxed
for five hours. After cooling, the solvent was evaporated to
dryness and the solid obtained was coevaporated with EtOH twice.
Then the residue was dissolved in hot EtOH, cooled and filtered tG
give 27 (12.52 g, 65~) as white solid: m.p. 236C.
29
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F.x~mnle 17
2'-Deoxv-~-T,-uridine (31)
Ste~ A
3 ~, 5 ~, -Di-O-benzoyl -02, O' -anhydro-~-L-uridine (~8)
A suspension of compound 27 (12.52 g, 55.31 mmol) and benzyl cyanide
(15.96 g, 121.77 mmol), in DMF (100 ml) was treated dropwise with
triethylamine (1.9 ml). The mixture dissolved rapidly and
spontaneously deposited the product. The mixture was diluted with
DMF (50 ml), stirred for three hours and finally diluted with
ethanol (15 ml). This was poured into ether (250 ml), the
precipitate collected with suction, washed with ether and dried to
give 28 (21.92 g, 88%) as white solid: m.p. 260C.
Ste~ B
3 ~, 5 ~, -Di - O-benzoyl -2 ' -chl oro -2 ' -deoxy-~-L-uridine (~2)
A mixture of compound 28 (21.72 g, 50.46 mmol), DMF (200 ml) and 6 M
HCl in DMF (4C.5 ml) was stirred at 100C for 90 minutes under
exclusion cf a~mospheric moisture, cooled down and poured under
stirring intc 1.5 L of water. The precipitate was collected with
su~tio,., washed with 1 L of water and recrystallized from ethanol
(60G m') ~c give pure 29 (19.9 g, 83.7%) as white crystals: m.p. 166
Ste~ '
3 ,~ De.._-yl-2~-deoxy-~1-T-uridine (30)
A m x~_-e -~ co.mpour.d 29 (18.89 g, 39.93 mmol), tri-a-butyltin
hyd~_d~ .l. 159.7 mmol), benzene (400 ml), and
azob_s~sob~tyronitrile (0.160 g) was refluxed under stirring for one
hour. ~fter cooling, the solid was filtered and washed with
benzen~. This solid was portion-wise recrystallized from ethanol
(3.2 L~ t- yield pure 30 (15.9 g, 91.3~) as white crystals: m.p. 223
- 224-C.
Ste~ D
2'-Deoxy-~-L-uridine (31)
To a solution of compound 30 (5.75 g, 13.17 mmol) in MeOH (75 ml)
was added 4.62 M NaOMe (3.17 ml) and the reaction mixture was
stirred at room temperature overnight. Then the solvent was
evaporated and the residue was dissolved in water (250 ml) and
washed with ether (3 x 100 ml). The aqueous layer was neutralized
with Dowex 50(H ) ion exchange resin, filtered and evaporated. The
CA 02203672 1997-04-24
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.
crude product obtained was coevaporated with ethanol and
crystallized from ethanol to give ~ (2.53 g, 84.3%) as white
crystals: m.p. 162 - 163C.
~mnle 18
3~.5~.-Di-O-benzoYl-2'~eoxv-4-thio B-T,-uri~ine (32)
The boiling solution of compound ~ (5.0 g, 11.45 mmol) in anhydrous
dioxane was treated with phosphorus pentasulfide (2.58 g, 12.83
mmol) and the mixture refluxed under nitrogen atmosphere for 30
minutes. The mixture was then treated with additional phosphorus
pentasulfide (2.85 g), refluxed for another 30 minutes, filtered
while hot, and the solid washed with dioxane. The filtrate was
evaporated to dryness and the crude product obtained was purified on
a silica gel column using 20 - 30% EtOAc/petroleum ether as solvent
to give an oil, which was coevaporated with ethanol and then
crysta'iizea from ethanol-petroleum ether to give pure 32 (2.62 g,
50.5~) as yellow crystals: m.p. 136 - 137C.
~xam~le 19
2~-Deoxv-B-L-cvtidine (33)
Comp~un 32 (3.0 g, 6.63 mmol) was treated with 200 ml of methanolic
ammonia in a bomb at 10CC for 10 hours. After cooling to room
tempC-2~ure, the solven~ was evaporated to dryness and the residue
dis~-:.e_ ir. water (2GC ml). The aqueous layer was washed with
ethe- ~ x 100 r, ) and treated with active charcoal, filtered
thrc_~ e'ite~, and evaporated to dryness and coevaporated with
ethan-:. T;r.e solid obtained was recrystallized from ethanol-
aceto~ ile mixture (1:10) to give 33 (1.13 g, 75%) as white
crystalc: m.~. 210~C.
~xamrle 20
2~-Deoxv-6-L-4-thiouridine (34)
Compound 32 (0.25 g, 0.55 mmol) in NH3/MeOH (30 ml) was stirred at
room temperature overnight. The solvent was evaporated and the
residue was purified on preparative plates using MeOH/CHCl3 (20%) to
give pure 34 (0.12 g, 88%) as yellow oil.
~x~mnle 21
2'-Deoxv-~-L-thvmidine (35~
Compound 31 (0.5 g, 2.19 mmol) was heated at 60 - 70C for six days
in a mixture of 1.2 mi 37~ aqueous formaldehyde ana 1.2 ml lM KO~.
CA 02203672 1997-04-24
WO96/13512 PCT~S95/13716
~very 24 hours the reaction mixture was treated with an additional
0.55 ml of lM KOH and 0.55 ml of aqueous formaldehyde. The solution
was then diluted with water, adjusted to pH4 with Dowex 50(H~),
filtered, the filtrate evaporated in vacuo and the residue
coevaporated several times with ethanol. The final residue was
dissolved in ethanol and made alkaline (pH10) by the addition of
triethylamine, evaporated and coevaporated several times with
toluene. The residue was dissolved in 22 ml ethanol and 50 ,ul
concentrated hydrochloric acid was added. The solution was refluxed
for five hours. Then the reaction mixture was made alkaline with
triethylamine, evaporated and the residue was purified by flash-
chromatography (20~ methanol-chloroform). 240 mg pure product and
280 mg mixture (containing starting material) was obtained. The
product was dissolved in 20 ml ethanol, acidified with 27 ~ul
concentrated hydrochloric acid and hydrogenated over 55 mg of 10%
palladium on cha~coal catalyst overnight under atmospheric pressure.
The mixture wac filtered, the filtrate made alkaline with
triethylamine a~d evaporated to dryness. Flash-chromatography of
the residue (20~ ethanol-chloroform) gave 35 (0.18 g, 34% overall
yield) as a white powder.
Exam~le 22
2'-Deoxv-~-L-5-fluorouridine (39)
Ste~ ~
1-[3',5'-0-(l,i,',3-Tetraisopropyldlsiloxane-1,3-diyl)-~-L-
ribGfuranos~l]-~-fluorouracil (36)
To a stirred suspension of (1.0 g, 3.90 mmol) in pyridine (40 ml)
was addea 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane tl.65 ml,
4.68 mmol). This was stirred at room temperature until the
completion o, the reaction tfive hours), the solvent was evaporated
and the residue was dissolved in EtOAc and washed with water, 5%
HCl, water, saturated aqueous NaHCO3 and brine. After drying over
anhydrous Na2SO4 it was filtered and evaporated to give the crude
product 36 and it was used in the next step without further
purification.
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WO96/13512 PCT~S95/13716
Ste~c B
1-~2~-0-Phenoxythiocarbonyl-3',5'-0-(1,1,3,3-
tetraisopropyldisiloxane-1,3-diyl)-~-L-ribofuranolsyl]-5-
fluorouracil r37)
To a solution of 36 (3.90 mmol) in anhydrous CH3CN (50 ml) was added
4-(dimethyl amino) pyridine (DMAP) (0.92 g, 7.56 mmol) and phenyl
chlorothionoformate (0.6 ml, 4.29 mmol). The solution was stirred
at room temperature for 24 hours. Then the solvent was evaporated
and the residue was dissolved in EtOAc and washed with water, 5%
HCl, water, saturated agueous NaHCO3 and brine. The EtOAc layer was
dried (Na2SO~), filtered and evaporated. The resultant oil was
purified on a silica gel column using MeOH/CHCl3 (5~) to give pure
37 (0.47 g) as white foam.
~te~ C
3~,51-G-(1,1,3,3-~e t'-2i sopropyldisiloxane-1,3-diyl)-~-L-5-
flucrouridlne (38'
To a mixture c r 37 (0.46 g, C.72 mmol), AIBN (0.02 g, 0.14 mmol) in
dry toluene (3C ml) was added Bu3SnH (2.0 ml, 5.05 mmol). The
solution was deoxygenated with oxygen-free air and then heated at
75CC for four hours. Then the solvent was evaporated and the
residue was purified on a silica gel column using EtOAc/petroleum
ethe~ (30~) to yield pure 38 (0.34 g, 97~) as white foam.
Ste~ D
2~-Deoxy~ - fluorou~idine (39)
A mixture of 38 (0.32 g, 0.66 mmol) and TBAF (1.4 ml, l M solution)
in THF (15 ml) was stirred at room temperature. After completion of
the reaction the solvent was evaporated and the residue was
dissolved in water and washed with ether. The water was evaporated
and the residue was purified on a silica gel column using MeOH/CHCl.
(l0~) to yield pure 30 (1.30 g, 80~) as an oil.
~xample 23
2~-~eoxv-~-L-5-fluorollri~ine (49~
Following the method of Example 22, this compound was made starting
from l-~-L-ribcfuranosyl-5-flUorouraCil (25): m.p. 150C.
NMR: (DMSOd~) ~ l.90 (m,lH, H-2'a)
2.55 (m, lH, H-2'b), 3.33 (m, 2H, H-5'), 4.l9 (m, 2H, H-3' ~ 4'),
4.85 (br s, lH, OH), 5.43 (br s, lH, OH), 6.10 (dd, lH, H-l~), 8.15
(d, l:-, H-6), ll.78 (br s, lH, N-H)
CA 02203672 1997-04-24
W O96/13512 PCTrUS95/13716
~x~mn le 24
2l.3l-DideoxY-~-L-uridine (43)
Ste~ A
2'-Deoxy-5'-0-(4-monomet~oxytrityl)-~-L-uridine (~)
To a solution of compound ~ (2.0 g, 9.16 mmol) in pyridine (100
ml), 4-monomethoxytrityl chloride (3.11 g, 10.08 mmol) was added and
stirred at room temperature for 24 hours. Then the solvent was
evaporated and the residue was dissolved in EtOAc, washed with
water, NaHCO3 and brine. After drying over anhydrous Na2SO~, the
solvent was evaporated to give the crude product and it was purified
on a silica gel column (5~ MeOH/CHCl3) to yield 40 (4.13 g, 94~) as
white foam.
Ste~ B
2'-Deoxy-~'-0-(4-monomethoxytrityl)-3'-0-phenoxythiocarbonyl-~-L-
uridine (41~
To a solution cf 40 (4.13 g, 8.25 mmol) in anhydrous CH3CN (100 ml)
wzs added D1~ (0.46 g, 3.76 mmol) and phenyl chlorothiono formate
(1.26 ml, 9.07 mmol). The solution was stirred at room temperature
for 24 hours. Then the solvent was evaporated and the residue was
dissolved in EtOAc and washed with water, 5% HCl, water, saturated
aqueous Na~C5 and brine. Evaporation of the EtOAc layer gave the
crude product and it was purified on a silica gel column (3.5%
MeOH/CHCl.) to yield pure 41 (3.95 g, 75~) as foam.
Ste C
2',3'-Dideoxy-~1-0-(4-monomethoxytrityl)-~-L-uridine (42)
To a mixture Gf 41 (3.95 g, 6.20 mmol), AIBN (0.20 g, 1.24 mmol) in
dry toluene (150 ml) was added Bu3SnH (16.7 ml, 62.0 mmol). The
solution was deo~genated with oxygen-free air and then heated at
75C for five hours. The solvent was evaporated and the residue was
chromatographed on a silica gel column (50 - 60~ EtOAc/petroleum
ether) to yield pure 42 (2.36 g, 78.8~) as white foam.
Ste~ D
2',3'-Dideoxy-~-L-uridine (~3)
Compound 42 (0.37 g, 0.76 mmol) in 80% acetic acid (5.0 ml) was
stirred at room temperature for two hours, the solvent was
evapcrated and the residue was coevaporated with toluene. The
residue was pur~fied on a silica gel column (15% MeOH!CHCl,) to give
- 34
CA 02203672 1997-04-24
W O 96/13512 PCTrUS95/13716
pure 43 and it was crystallized from MeOH/ether to give ~ (0.118 g,
73~) as white crystals: m.p. 122C.
F~mnle 25
2'-Deoxv-~-T,-inosine (58
Ste~ A
9-(2-Deoxy-3,5-di-0-~-toluoyl-~-L-ribofuranosyl)-6-chloropurine (57)
A mixture of 6-chloropurine (0.40 g, 2.62 mmol) and sodium hydride
(60% in oil, 0.11 g, 2.88 mmol) in anhydrous CH3CN (50 ml) was
stirred under a nitrogen atmosphere for 30 minutes at room
temperature. Crystalline compound 56, made according to the
procedure described in Tetrahedron 43, 2355-2368, 1987, for the D-
isomer (0.85 g, 2.18 mmol), was added and stirring was continued for
2 hours. After addition of CHCl3 (50 ml), the mixture was filtered
through Celite~. The filtrate was evaporated and the crude was
purified on a silica gel column using 50~ EtOAc/petroleum ether to
give pure compound 57 (0.55 g, 50~) as white solid.
Ste~ B
2'-Deoxy-~-~-inosine (45)
A mixture of 57 (0.20 g, 0.39 mmol), mercaptoethanol (0.11 ml, 1.55
mmoll, and NaOMe (0.34 ml, 1.55 mmol) in MeOH was refluxed for four
hours. The reaction mixture was cooled, neutralized with glacial
acet_c acid an_ evaporated to dryness. The solid obtained was
washed with CH_'- and the residue was crystallized from H2OtMeOH to
give pure 58 (C.070 g, 71~) as white crystals: m.p. 219 - 220C.
~tilitY
In ~itro 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 and/or fibroblast cells) 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 Riological Co.,
Grand Island, Ne~ York.
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WO96/13512 PCT~S95/13716
IN V~T~O 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
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-F~ control formulation was prepared as suggested in
the product literature.
Following trypsinization, tumor cells were suspended in tissue
culture medium and exposed to the antitumor agents continuously at
three different concentrations: l0, l and 0.l ~g/ml.
RADIOMETRIC MEAS'JREMENT OF GROWTH INHIBITION: Growth inhibition was
assessed with the BACTEC System 460 (Johnston Laboratories, Towson,
MD) after addi.ion of the antitumor agent to the cell in the
respective growth medium containing '6C-glucose at a final
concentrat.on of 2 uCi/ml. (See generally, C. Arteaga, et al-, k
Radiometric Method for Evaluation of Chemothera~v Sensitivitv:
Results of Screenina a Panel of Hl~m~n Breast C~ncer Cell Tlines,
Cancer Research, 47, 6248-6253 (l987).
Two mis of the tumor cell suspension containing radioactive glucose
were seeded int~ sterile, disposable 15 ml vials by injection
through self-sealing rubber-aluminum caps. For each cell line, the
optimal number c r 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 1~CO2 resulting from the metabolism of 1~C-glucose were
performed on days 6, 9, 12 and 15 in the BACTEC instrument. This
instrument flushes the ~'CO2 containing 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.
36
CA 02203672 1997-04-24
W O96/13512 PCT/US95/13716
TART.~ I
COMPOUND~ SURVIVAL BONE~ SURVIVAL IC 50
MARRO~ TUMOR
5-FU 1.9 CALU 2.2 <0.6
COLO3201.0 <0.6
HS578T43.5 <0.6
HT29 1.2 0.613
MCF-7 0.8 <0.6
OM-1 1.7 1.47
SKLU 5.0 1.05
SKMES11.2 <0.6
~-l- 96.6 CALU 89.1 >10
ribofuranosyl-
uracil
2-amino--L- 114.6 OM-l 2.6 0.026
ribofurano
[1',2':4,5]
oxazoline
2-amino-c-L- 109.5 CALU 89 44.6
arabinofurans MCF-7 88.8 187
[1',2':4,5' OM-1 41.3 5
oxazoline
Oi,Oi-anhy-rc_~_ 70.9 OM-1 46.4 2.97
c-L-
ribofuranosyl
uracll
~-L- 120.1 COLO32082.3 34.9
ribcfuranosyl SKLU 88.8 240
cytosine
1-(2,3,5-tri-G- 85.9 HT29 75 >10
be.,zcyl-~
ribofuranosyl~-
4-thiourac~_
1-(3,5-di-C- 102.1 MCF-7 84.5 >10
benzoyl-2- OM-1 35.7 0.9&
deoxy-~-L- SKLU 77.7 88.7
ribofuranosyl)-
4-thiouracii
2'-~-L-deo~ 98.2 OM-1 82.3 34.3
ribofuranosyl- SKLU 79.4 >10
4-thiouracil
~-L- 94.6 OM-1 0.4 0.87
ribofuranosy,- SKLU 71 43.4
5-fluorouracil
~-L- 129.9 COLO32081.9 188
ribofuranosyl- HT29 64.7 51
5-fluorouracil OM-1 71.9 37.8
SKLU 69.7 32.2
~-L- 72.6 OM-1 39.2 5.9
ribofuranosyl
cytosine
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WO 96/13512 PCTrUS95/13716
COMPOUND~ SURVIVAL BONE% SURVIVAL IC 50
MARROW TUMOR
~-L- 169.8 HT29 80.9 >10
ribofuranosyl OM-1 44.7 0.097
guanine
2~-~-L-deoxy 104 HT29 81.7 85.2
ribofuranosyl- MCF-7 66.9 16.6
5-fluorouracil OM-1 66.7 62.9
SKLU 83.9 32.5
2'-~-L-deoxy 158.9 COLO32067.0 57.4
ribofuranosyl HT29 72.2 30.4
thymine OM-1 37.6 6.5
SKLU 72.4 38.2
~-L- 112.5 HT29 78.7 >10
ribofuranosyl OM-1 30.8 0.094
uracil
2',3'-~-L- 79.4 CALU 0.4 0.623
dideo~y
ribofuranos~'
ura-il
~-L- 91.2 OM-1 50.9 4.23
ribofuranos~_
adenine
~-L- 118.4 COLO32068.2 17.9
ribofuranosv' ~S578T93.4 >10
hypoxanthine MCF-7 96.8 38.1
OM-1 87.3 82.7
SKLU 82.5 48.1
~-L- 97.4 HS578T66.6 17.1
ribcFuranosv_- OM-1 83.1 42.7
6-~hioguanine SKLU 45.1 8.7
2'-~-L-deoxy 108.8 COLO32019.6 6.56
ribofuranosyl- HT29 42.5 8.46
5-fiuorourac l MCF-7 86.9 60.7
OM-1 85.2 56.1
SKLU 86.2 59.9
2'-~-L-deoxy 64.9 CALU 28.9 0.696
ribofuranosyl
adenine
2'-~-L-deoxy 109.6 OM-1 83.1 >10
ribofuranosyl
hypoxanthine
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 recl~ired to kill 50~ of the untreated cancer cells.
CA 02203672 1997-04-24
WO96/13512 PCT~S95/13716
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.
survival bone marrow
~ survival tumor
A therapeutic ratio of <80~ is considered ac'ive.
~n vi~o ~v~uation
Representative compounds of the present invention are being tested
in a variety of preclinical tests of anti-cancer activity which are
indicative of clinical utility. For example, certain compounds will
be tested i.~ vivo agai-.st human tumors xenografted into nude mice,
specifically B15, MX-l and P388 Leukemia tumor lines were used.
Bl6 Melanoma
B6D2Fl mice receive i.p. inocula of Bl6 murine melanoma brei
prepared from B'6 tumors growing s.c. in mice (day 0). On day l,
tumored mice are treated with drugs or vehicle controli the drugs,
route of drug administration and schedule 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-finding experiments in non-tumored mice.
In a typical experiment, drugs are given at their MTD and l/2 MTD
doses i.p. on a daily x 5 schedule.
39
CA 02203672 1997-04-24
W O96113~12 PCTAUS95/13716
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~s 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=150-20C, modest activity; T/C=200-300, high
activity; T/C>300, with long term survivors~ excellent, curative
activity.
Statistics are performed o-. the data using primarily the log rank p-
value test.
P388 Leu~.emia
This tes~ is conducted in exactly the same way as the B16 test. The
tumor inoculu~, is prepared by removing ascites fluid containing P388
cells fr_.. tumored DB~'2 mice, centrifuging the cells, and then
resuspe~ ng the leukemi2 cells in saline. Mice receive 1 x 105
P3~ c~::c i.~. or day C.
M~ a.. Ereact Tumor Xeno~raft
Nude m~ re a-e implanted s.c. by trocar with fragments of MX-1
mammary carcinomas harvested from s.c. growing MX-1 tumors in nude
mice hosts. When tumors are approximately 5 mm x 5 mm in size
(usually about ten days after inoculation), the animals 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 animals are pair-matched (day 1). The doses,
route of dru~ administration and schedule are selected as
appropriate for the study in question. If the MTD dose of an agent
is not kno~., it is determined in an initial dosing experiment in
non-tumored mice. In a typical experiment, drugs are given at their
MTD and i'2 MTD doses i.p. on a daily x 5 schedule.
CA 02203672 1997-04-24
W O96/13512 PCT/US95/13716
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
termination date can be determined. Upon termination, 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 tumor 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-1 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 M~-1 regressions. If the tumor completely disappears in
a mouse, this is considered a complete regression or complete tumor
shrinkage. If desired, mice with partial or total tumor regressions
can be kept alive past the termination date to see whether they live
to become iong term, tumor-free survivors.
S~ali~tics are pe-formed on the data using primarily the log rank p-
value tes'.
Protocols for ~Iv-l In~cti~tion 8tudi~c
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. Immuncl . 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.
CA 02203672 1997-04-24
W O96/13S12 PCT~US95/13716
3) Folks, T.M., Clouse, K.A., Justemer.t, 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. I~unol . 142:431-438, 1989.
1. Inactivation of c~ fr~- ~IV-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 HI~- strains including the MN and some African strains
ma~ be used iate- for confirmatory purposes.
a) Ce'l-f-ee HTLV-III~:
Cell-free HIV-1 (5 x 10 to 1 x 10~ TCIDs0/ml, or median tissue
culture infectious dose~ is either left untreated, or treated
with RPr'_ 1640 culture medium, or with different
concen~ra.ions of antivirals for various time intervals at
_7'C, or at a temperature to be determined. After incubation,
the trea~ed and untreated stocks are added to 5 x 105 washed
ar,d pelle'ed target MT-4 cells. After 1 h incubation at 37C,
the M.-4 ce~is are washed three times with RPMI 1604,
resuspenae- in RPMI 1640 supplemented with 15~ fetal bovine
serum (FB'`, 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-
d.phenyitetrazolium bromide ~MTT) dye, which changes in color
in the presence of live mitochondria. All determinations are
aone in triplicates.
b) Cell-free J~-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),
wr.ich only infects primary human peripheral mononuclear cells
CA 02203672 1997-04-24
W O96/13512 PCTrUS95/13716
(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~g of PHA/ml for 1 day before used in
infectivity studies. HIV-l (JR-CSF) untreated or treated as
above are added to PHA-activated human PBMCs and incubated for
1 h at 37~C. 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-DuPont, Wilmington, DE).
2. Inact~vat~on of cell~ oc~at-d xrv-l.
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 from various laboratories including
those listed in the references cited above. For infected human
PBMCs, fresh human PBMCs are obtained from normal volunteers and
stimulated with PHA, then infected with HTLV-IIIB or JR-CSF, as
described above. On day 7 after in ~itro infection, infectivity is
checked by testing for the presence of HIV-1 p24 in the culture
supernatants. Infected cultures are divided in equal aliquots. One
set is then treated with antivirals at different concentrations for
va-ious time intervals, whereas one set is left untreated. Culture
supernatants collected on days 3, 6 and 9 of culture will be
assessed for HIV-1 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-1
antigen(s).
3. Inact~vation of ~IV-l lat--ntly ~nf--ct--~ c~ll~.
These assays are designed to study the effects of antivirals on HIV-
1 latently infected cells. One or more of the following HIV-1
latently infected human cell lines can be used (J1-1, U1/HIV and
ACH-2 obtained from the NIH AIDS Research and Reagent Reference
Program, Rockville, MD). These cells are characterized by HIV-1
infe_ticr. without significant HIV-1 viral replication unless they
43
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W O96/13512 PCT/US95/13716
are stimulated with different cytokines which results in a 10 - 100
fold increase in HIV-1 replication. J1-1, or U1/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 J1-1 cells are stimulated with 1000 U of ~ tumor necrosis factor
(-TNF, Genzyme) for 48 h at 37C as previously described (Reference
1) .
The U1/HIV-1 cells are stimulated with 20~5 - 40~5 PHA-culture
supernatant (Electronucleonics) for 48 h at 37C (Reference 2). The
PHA-superna_z..t will either be purchased from Electronucleonics or
will be prepare~ in our laboratory. To prepare PHA-supernatant,
normal human PBM_ will be cultured at a cell density of 10~ cells/ml
in RPMI 1640 supplemented with 15~ FBS and 10 ,ug/ml of
phytohemagglu inin A (PHA, Sigma Chemical Co.). The culture
supernatant will be harvested, filtered through a 2 ~m filter and
used tc stimulate the U1/HIV cells as described above.
The ACH-2 cells will be stimulated by addition of 1.0 ~M of phorbal
12-myristate _^~ acetate (PMA, Sigma Chemical Co.) for 48 h at 37~C
as described (References 3 and 4 above). At the end of the
stimulation period, culture supernatants are collected and HIV-1
expression is assessed by the HIV-1 p24 antigen capture ELISA
(DuPont) and by the reverse transcriptase (RT).
In ina~'ivation of cell-associated HIV-1 experiments, the treated
and un.reated cells could also be submitted to PCR analysis.
4. Inhibition of ~IV~ c-~ syncytiu~ form~t~on.
HIV-1-infected H-9 cells are left untreated or treated with
antiviral as described above. Treated and untreated cells (5 x 10
cells/well) are added to 96-well flat-bottom microtiter tissue
culture plates containing 1 x 105 indicator SupT1 human T cells/well
in comple.e RPMI 1640 culture medium. Following overnight
CA 02203672 1997-04-24
W O96/13512 PCTrUS9S/13716
incubation at 37C, syncytium formation is scored by two independent
people using an inverted microscope scope.
5. Cytotoxicity studl--s.
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.
Dosaae ~nd Formulation
The antitumor compounds (active ingredients) of this invention can
be administered 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 administered by any conventional means
available fGr use in conjunction with pharmaceuticals, either as
individual therapeutic active ingredients or in a combination of
therapeutic a- ive ingredients. They can be administered alone, but
are generally administered with a pharmaceutical carrier selected on
the basis of the chosen route of administration and standard
pharmaceutical practice.
The dosage ad.-,-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 administration; age, health and
weight of the recipient; nature and extent of symptoms; kind of
concurrent treatment, frequency of treatment and the effect desired.
Usually a dai'y 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 milligram to about 500 milligrams of active
ingredient per unit. In these pharmaceutical compositions the
CA 02203672 1997-04-24
W O96/13S12 PCTrUS9~tl3716
active ingredient will ordinarily ~e present in an amount of about
0.05 - 95~ by weight, based on the total weight of the composition.
The active ingredient can be administered orally in solid dosage
forms such as capsules, tablets and powders, or in liquid dosage
forms such as elixirs, syrups and suspensions. It can also be
administered parenterally, 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 sustained release products to provide for
continuous release of medication over a period of hours. Compressed
tablets can be sugar coated or film coated to mask any unpleasant
taste and protect the tablet from the atmosphere, or enteric coated
for selective dis~ntegration in the gastrointestinal tract.
Liquid dosage forms for oral administration can contain coloring and
flavoring to increase patient acceptance.
In general, water, a su1table oil, salinej aqueous dextrose
(glucose), and related sugar solutions and glycols such as propylene
glycol or polyethylene glycols are suitable carriers for parenteral
solu.ions. So'utions 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
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-para~en and chlorobutanol.
Suitable pharmaceutical carriers are described in Pemington's
Pharmaceutical Sciences, Mack Publishing Company, a standard
reference text in this field.
Useful pharmaceutical dosage forms for administration of the
compounds of this invention can be illustrated as follows.
46
CA 02203672 1997-04-24
WO96/13512 PCT~S95/13716
Capsules: Capsules are prepared by filling standard 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 containing lO0
milligrams of the active ingredient. The capsules are then washed
and dried.
Tablets: Tablets are prepared by conventional procedures so that
the dosage unit is 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 to
delay absorp.icr..
Injectable: A parenteral composition suitable for administration 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.
Suspensicn: An ac,uecus suspension is prepared for oral
administraticn so that each 5 millimeters contain lO0 milligrams of
finely divided active ingredient, 200 milligrams of sodium
carboxymethyl cellulose, 5 milligrams of sodium benzoate, l.0 grams
of sorbitol sclution U.S.P. and 0.025 millimeters of vanillin.
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 cor.ditions are not excluded so
long as they do not prevent the benefits of the invention from being
realized.
