Note: Descriptions are shown in the official language in which they were submitted.
1 336073
1 60724-1808D
This application i5 a divisional application of Canadian
Patent Application 564,584 filed on April 20th, 1988.
The invention of the parent application is directed to
treating malignant tumors _ vivo utilizing the compound 8-
chloroadenosine 3',5'-cyclic phosphate. The invention of this
divisional application relates to such u~es of 8-aminoadenosine
3',5'-cyclic phosphate. The compound 8-chloroadenosine 3',5'-
cyclic phosphate and other related adenine and adenosine compounds
are prepared by two novel syntheses utilizing hydrogen chloride
and m-chloroperoxybenzoic acid in a suitable solvent in the first
synthesis and N-chlorosuccinamide and acetic acid in a suitable
solvent in the second synthesi~.
While the arsenal of chemotherapeutic agents for
treating neoplastic diseases includes a number of clinically
useful agents, control of malignant tumors in warm blooded animals
still remains a much sought after goal.
In a study reported from the People's Republic of China
but not confirmed elsewhere, 8-bromoadenosine 3',5'-cyclic
phosphate was noted as inhibiting the solid form of uterine tumor
14 Ehrlich carcinoma, sarcoma-180 and reticulum-cell sarcoma in
mice. An abstract of this study appeared in a Cancergram of the
International Cancer Research Data Bank, Series CB14 Number 80/03,
March 1980, published by the United States Department Health,
Education and Welfare National In~titute of Health, National
Cancer Institute. In contrast to this report, in other studies 8-
bromoadenosine 3',5'-cyclic pho~phate has been found to be
inactive as an antitumor agent in cell culture.
~'~ ,
~ 3~7~
2 60724-1808
Contemporaneously wlth the above report, Y.S. Cho-Chung,
J. CYclic Nucleotide Res. 6: 163, 1980, reported certain
investigative studies on an antagonistic interaction between
estrogen and adenosine 3',5'-cyclic monophosphate (hereinafter
alternately referred to as cAMP) and what role this might have
in the control of growth of hormone-dependent mammary tumors.
In studying the effects of mediated control of tumor
growth by adenosine 3',5'-cyclic phosphate, Cho-Chung has
suggested that cAMP functlons by binding to a cAMP receptor
protein which ha~ two different _ AMP binding sites. The cAMP
receptor protein is a regulatory subunit of a _AMP dependent
protein kinase. There apparently is ~ite selectivity in binding
to one or the other of two sites. This activity can thus be
described as site l-selectivity and site 2-selectivity.
In view of the inability of current cancer chemo-
therapeutics to successfully control all neoplastic disease~, it
is evident that there exists a need for new and additional cancer
chemotherapeutic agents. Further there exist a need for new and
better preparative procedures for the synthesis of such new and
additional cancer chemotherapeutic agents.
8-Chloroadenosine 3',5'-cyclic phosphate was first
reported by inventor R.K. Robins of this invention and other
co-authors in K. Muneyama et al. J. Carbohydr. Nucleosides
Nucleotides, 1, 55, 1974. 8-Aminoadenosine 3',5'-cyclic phosphate
was first reported by inventor R.K. Robins of this invention and
other co-authors K. Muneyame et al., Biochemistry, 10, 2390, 1971.
It has now been found that 8-chloroadenosine 3',5'-cyclic
phosphate (hereinafter alternately also identified as 8-chloro
2a 1 3 3 6 0 7 3 60724-1808D
cAMP) and 8-aminoadenosine 3',5'-cyclic phosphate (hereinafter
al~ernately also identified as 8-amino cAMP) together or
separately exhibit such significant antitumor activity so as to be
useful as antitumor agents in vivo. Further, two novel
preparative syntheses yield 8-chloroadenosine compounds such as 8-
chloroadenosine 3',5'-cyclic phosphate directly from respective
adenosine precursors.
BRIEF DESCRIPTION OF THE INVENTION
The invention of the parent application relates to the
use of 8-chloroadenosine 3',5'-cy~lic phosphate (8-chloro cAMP)
optionally with 8-aminoadenosine 3',5'-cyclic phosphate (8-amino
AMP) in treating malignant tumors in warm blooded
3 1 336073 60724-1808D
animals. According to the invention of the parent application and
this divisional application the antitumor properties of 8-chloro-
adenosine 3',5'-cyclic phosphate and 8-aminoadenosine 3',5'-cyclic
phosphate are achieved by administering to a warm blooded animal
an effective amount of a pharmaceutical composition containing 8-
chloroadenosine 3',5'-cyclic phosphate or 8-aminoadenosine 3',5'-
cyclic phosphate, together or separately, or pharmaceutically
acceptable salts thereof as active compounds in at least 0.1% by
weight based on the total weight of the composition.
The inventions also relate to a commercial package
comprising a pharmaceutically effective amount of 8-
chloroadenosine 3',5'-cyclic phosphate or 8-aminoadenosine 3',5'-
cyclic phosphate or a pharmaceutically acceptable salt thereof
together with instructions for use thereof to treat a tumor in a
warm blooded animal.
For use in pharmaceutical compositions of the invention
a pharmaceutical carrier would be utilized. Preferredly the
carrier would be chosen to allow for administration of a suitable
concentration of 8-chloroadenosine 3',5'-cyclic phosphate and/or
8-aminoadenosine 3',5'-cyclic phosphate either by oral
administration, ophthalmic administration, topical administration,
suppository administration or by suitable injection as a solution
or suspension into the affected warm blooded animal. The dose and
choice of administration of 8-chloroadenosine 3',5'-cyclic
phosphate and/or 8-aminoadenosine 3',5'-cyclic phosphate of the
invention would be dependent upon the host harboring the malignant
tumor, the type of tumor and the tumor site. For injection,
3a 1 336~73 60724-1808
8-chloroadenosine 3',5'-cyclic phosphate and/or 8-aminoadenosine
3',5'-cyclic phosphate of the invention could be administered
intravenously, intramuscularly, intracerebrally, subcutaneously
or intraperitoneally. Further, for facilitating the use of
8-chloroadenosine 3',5'-cyclic phosphate and 8-aminoadenosine
3',5'-cyclic phosphate, a physiologically accepted salt, as for
instance the sodium, potassium or ammonium salt, could be used.
Presently it is preferred to administer the compounds by infusion.
Further, the inventions include improved processes
for the preparation of 8-chloro derivatives of adenine, adenosine,
adenosine 5'-monophosphate and adenosine 3',5'-cyclic phosphate
and related compounds. In these improved processes 8-chloro-
adenine, 8-chloroadenosine, 8-chloroadenosine 5'-monophosphate
and 8-chloroadenosine 3',5'-cyclic phosphate or other related
adenosine compounds are prepared directly from
~ ' 4 1 336073
corresponding respective adenosine precursor compounds by
processes for preparing chloro compounds of the formula
NH2
~N~
wherein R is H or
R2-0-- ~0
< H ~
'~ /'1
OR1 R3
OH
wherein R1 and R2 are H or O~P- or together R1 and R2 are O~P-,
OH OH
and R3 and R4 are H or one of R3 or R4 is OH and the other is
H, and pharmaceutically acceptable salts thereof which comprise
the steps of:
t~eating a starting compound of the formula
~ 5 l 33~73 60724-1808
~H2
~ Y~
R2-0-- ~0
H
ORl R3
OH
wherein ~1 and R2 are H or 0~'- or together Rl and R2 are 0~
OH OH
and R3 and R4 are H or one of R3 or R4 i9 OH and the other is
H:
either.
1) with N-chlorosuccinimide and a weak acid in a suitable
solvent or
2) with hydrogen chlorlde and an oxidizing agent in a
suitable solvent.
In preferred embodiments R3 is OH and R4 is H,
OH
Rt and R2 are 05P- or together R1 and R2 are O=P-.
OH OH
6 1 33~73 6~724-1~0~
DET~ILED D~SCRIPTION OF TtlE I~vENTIO~
8-Aminoadenosine 3',5'-cyclic phosphate
ls prepared as i~ described in the above reerenced paper, ~.
~lumeyama et al. ~1_ h ~ m 1 s ~ L ~, 1 0, 2 3 9 ~, 1 9 7 1 ~ -
Chloroadenosine 3~s~-cyclic phosphate can be
prepared as is described in above referenced paper, K. ~uneyama
et al, J, C~L~Qhy~r. N-l--s~ o~ , 1, ss 1974, vic~
an ~-bromo intermediate compound, however, by utilizing ll~W antl
improved processes as discussed herein, 8-chloroadenosine
3',5'-cyclic phosphate and related compounds are obtained
dlrectly Erom respective adenosine precursors avoiding havitlg
to synthesis intermediate compounc~s. ~6 Such, improvemellts in
the economy of the synthesis are obtained.
In the first of these improved proce~ses adenosine 3',5'-
cyclic phosphate is directly chlorinated to 8-chloroadenosine
3',5'-cyclic phosphate utilizing N-chlorosuccinimide and a
suitable weak acld in a suitable solvent. Suitable as the weak
acid ls acetic acid. Alternatively ~ormic acid or other wea~
organic acld mlght ~e used. Jn chooslng the acid,
consideratlon is given whereby ~he acid is o~ such a streng~l
that it is sufflciently weak 50 as not to cleave the sugar -
heterocycle glycosidic bond (the C1'-N9 bond). Suitable as the
solvent are dlmethylacetamide, dimethylformamide or aqueous
medi~m.
In the 6econd o~ these lmproved processes adenosine 3',5'-
cyclic phosphate i5 also directly chlorinated to 8-chloro-
adenosine 3',S'-cyclic phosphate utilizing anhydrous hydrogen
chloride and a sultable oxidi71ng agent such as m-chloroperoxy-
ben%olc acid. This reaction isconducted1n a sultable 501Vellt
as or example dimethylacetamide or dimetllylormamide. ~s an
alternative oxidlzing aqent, sodium hypochlorite can be
mentioned.
~ side from the preparation o~ 8-chloroadenosine 3',5'-
cyclic phosphate, both o these processes can be utiliied to
prepare 8-chloroadenosine from adeno~ine or 8-chloroadeno~ine
S'-phosphate from adenosine S'-phosphate. Further, i~ the
1336073 ~ 724-1~n~
hydrogen chlorLde/~-chloroperoxyben7,01c acid reaction is
carried out ln the presence o~ molsture. it can be utilized to
prepare 8-chloroadenlne from adenosine.
~ dditionally, these two novel processes might a]so be
utili~ed to convert su1table derivatives o~ adenosii-l?~
adenosine 5'-phosphate, adenosine 3'-phosphate and adenosine
3',5'-cyclic phosphate, to their respec~ive ~-chloro
derivative~. Such derivatives would include the 2-dcoxy-~-D
_LY~hLQ-pentofurano9yl sugar derivative and the 2-~
arab1nofuranosyl sugar derivative and might also include
substituents on the purine amino group, the 2 posltion of the
purlne ring, sugar hydroxyl groups or even other hetecocyclic
moieties such as a deaza purlne or the like.
The synthesis of 8-chloroinos1ne 3',S'-cyclic phospllate.
8-chloroinoslne S'-phosphate and 8-chloroinos1ne is ~acilitatecl
utilizing these processes in that the respective 8-chloro-
adenosLne compounds can then be directly deaminated utilizing
nitrous acid to respective 8-chloroinoslne derivatives. For
this reactlon, the nitrou6 acid can be generated in _it~ ~rom
sodium nltrite and acetic acid. Thus 8-chloroinosine and its
S~-phosphate and 3',S'-cyclic phosphate derivatives are
synthesised in a straight forward two step reaction scheme.
In the ~ollowing examples of the novel processes
as is illustrated in sctleme 1, an adenosine compound.
e.g. adenosine, AMP or _~MP, is directly chlorinated in t~le 8
position o~ its purine ring to anappropriate 8-chloroadenosine
or adenine compound. Thu~ adenosine yields compounds 1 and 2,
~MP yields compound 3 and c~MP yields compound 4. Tilis is
achieved uslng one, the other or both of the two novel
processes.
One process uses hydrogen chloride and ~-c~]oro-
peroxybenzoic acid in an appropiate solvent such as
dimethylacetamide or dLmethyl~oramide. The other process uses
N-chlorosucclnamlde and acetic acid in an appropriate solvent
such as dimethylacetamide, dimethylformamide or an aqueous
acetic acid. ~ny of the 8-chloro compounds thus formed can
then be further converted in a further step to their 8-
8 ~ 7 3
chloroinosine analogs as for instance the conversion o$ 8-
chloroadenosine 3',S'-cyclic phosphate, compound 4, to 8-
chloroinosine 3',S'-cyclic phosphate, compound S.
In the preparative examples below, melting points were
taken on a Thomas-Hoover~capillary melting point apparatus or
on a Haake-Buchle~ digital melting point apparatus and are
uncorrected. Muclear magnetic resonance (1H NMR) spectra were
determined at 300.1 MHz with an IBM NR300AF spectrometer. The
chemical shifts are expressed in ~ values (parts per million)
relative to tetramethylsilane as internal standard. Ultra
violet spectra (UV: sh = shoulder) were recorded on a Beckman
DU-S0 spectrophotometer. Elemental analyses were performed by
Robertson Laboratory, Madison, N.J. Evaporations were carried
out under reduced pressure with the bath temperature below
40C. Thin layer chromatography (TLC) was run on silica gel 60
F-2S4~plates (EM Reagents). E. Merck silica gel (230-400 mesh)
was used for flash column chromatography.
1 336073
NH2 INH2
N~ N~ NH2 [~ N~
HO--~ 0 ~ Cl HO--~O~
HO OH 1 HO OH
Adenosine 2
NtH2 7~2
~N~ N~h\>'
> G
HO- '-O--~O~ HO- -O--~O~
Na+ \~ ~/ Na+ \`l
H OH OH OH
AMP 3
NH2 NH2 0
~N N~N\>_Cl HN~ Cl
O--~0~ 0-- 0~ 0-- 0
O=P- \~ OH O~P- ~ OH O=P--O OH
O ~ Na + o~ Na O~ Na +
~AMP 4 5
SCHEME I
lO 1 336073
EXAMPLE 1
8-Chloroadenine (1).
To a solution of adenosine (2.67 g, 10 mmol) in DMA/HCl
(o.s M, 45 mL) was added ~-chloroperoxybenzoic acid (MCPBA,
3.22 g, 16 mmol, 87~) and stirred at room temperature for 2.5
h. An additional portion of MCPBA (0.9 g, S mmol) was added
and stirring continued for another 1 h. Toluene (So mL) was
added to the reaction mixture and the solvents evaporated at
600C under vAcuo to dryness. The residue was dissolved in
water (S0 mL) and extracted with ether (3 x 50 mL). The pH of
the aqueous phase was adjusted to S with 2N NaOH and then
diluted with EtOH (100 mL). The solution was stored in the
refrigerator overnight. The light yellow solid that separated
was collected, washed with cold EtOH (2 x 2S mL) and dried to
give 1.44 g (8S.2~) of 1: mp 30S-310C (dec.) lLit. mp >300'C
(dec.j~: IR (KBr): 630 (C-CI). 3100-3300 (NH2) cm 1 UV: AmaX
(pH 1) 262 nm (~ 8,700): AmaX (ph 7) 268 nm (e 7,900): AmaX (pH
11) 269 nm (~ 8,300): 1H NMR (Me2SO-~6): ~ 7.48 (br s, 2, N~2),
8.10 (s, 1, C2~) and 13.60 (br s, 1, N9~).
EXAMPLE 2
8-ChloroAdenosine (2).
Method A.
To a solution of adenosine (2.67 g, 10 mmol. dried at 80-C
under vacuum) in DMA/HCl (o.S M, 2S mL) was added rapidly a
soluti~n of purified MCP8A (3.09 g, 18 mmol) in DMA (20 mL)
(adenosine will precipitate out from the reaction mixture if
the MCPBA solution i~ not added quickly). After stirring at
room temperature for 2 h, additional MCPBA (0.7 g, 4 mmol) was
added and the mixture was stirred for another 1/2 h until all
the adenosine disappeared. The DMA was evaporated in vacuo
and the residue was purified by HPLC on a C-18 reverse phase
column using MeOH: AcO~:H2O (18:1:81, v/v) to give 1.2S g (41~)
of 2: mp 188-189C rLit. mp 188-190Cl: IR (RBr): 79S (C-C1),
31S0-3400 (NHz, OH) cm 1 UV: AmaX (pH 1) 261 nm (~ 17,100):
~max (pH 7) 262 nm (~ 17,800): Amax (pH 11) 263 nm (~ 16,700):
H NMR (Me2SO-d6): ~ S.83 (d, 1, J1~ 2~ s 7.0 Hz, C1.H), 7.Ss
11 1 336073
(br s, 2, NH2) and 8.14 ts, 1, C2~),
Method B.
To a solution of adenosine (1.09 g, 4.1 mmol) in D~F (S0
mL) and AcOH (10 mL) was added N-Chlorosuccinamide (NCS, 2.0 g,
lS mmol). The reaction mixture was stirred at room temperature
for 6 days. The solvents were evaporated to dryness and the
residue was purified by HPLC on a C-18 reverse phase column
using MeOH:AcOH:H2O (18:1:81, v/v) to give 0.8 g (6S~) of 2,
whlch was identical to the title compound prepared by Method A.
EXAMPLE 3
8-Chloroadenosine S'-~onophosph~te (3).
Method A.
To a solution of adenosine S'-monophosphate (1.3 g, 3.S
mmol) in dry DMF (7S mL, distilled over CaR2) was added
dropwise a solution of DMF saturated with anhydrous HCl (S mL),
followed by a solution of purified MCP8A (1.1 g, 6.4 mmol) in
~MF (10 mL). The reaction mixture was stirred at room
temperature for 2 1/2 h, before the DMF was evaporated to
dryness under v~cuo at 4SC. The residue was dissolved in
minimum amount of water (~10 mL) and the product was
precipitated from the solution by the dropwise addition of
MeOH. The solid was collected and purified by HPLC on a C-18
reverse phase column using 0.S~ aqueous AcOH to furnish 0.64 g
(~S~) of 3: mp 180C. IR (KBr~: 63S (C-Cl), 3100-3400 (NH2,OH)
cm 1 UV: AmaX (pH 1) 260 nm (s 10,800): AmaX (pH 7) 262 nm (~
10,400): ~max (pH 11) 261 nm (~ 10,000): lH NMR (Me2SO-~6): ~
S.86 (d, 1, Jl' 2~ = 6.0 Hz, C1,~), 7.S0 (br s, 2, N~2) and
8.16 (s, 1, C2H).
Method ~.
Adenosine S'-monophosphate monohydrate (3.11 g, 8.S mmol),
NaCl (3.11 g, S3 mmol) and NCS (3.33 g, 2S mmol) were dissolved
in 50~ aqueous AcOH (100 mL) and the solution was stirred at
room tempeature for S days. Evaporation of the reaction
mixture and purification of the residue by HPLC on a C-18
12
1 336~73
reverse phase column using O.S~ aqueous AcOH gave 2.30 g (60~
of the title compound, which was identical to 3 prepared by
Method ~.
EXAMPLE 4
8-Chloroadenosine 3',S'-cyçlic phos~ha~e (4).
Method ~.
A mixture of dry 8-bromo-~AMP (20 g. 49 mmol) and CaCl2
(20 g, 180 mmol, dried at 7S under vacuum overnight) in
anhydrous ~MF (800 mL, distilled over CaH2 under vacuum) was
heated at 80-85C under anhydrous conditions with stirring for
lS h. DMF was evaporated under reduced pressure at SOC and
the residue was dissolved in cold 2N NaOH (20 mL). The aqueous
solution was neutralized (to pH 7) with cold 2N HCl, filtered
through a membrane filter and purified by preparative HPLC on a
C-18 reverse phase column. Initial washings with O.S* AcOH/H20
gave 8-hydroxy-~AMP and ~AMP. Further elution with lS~
MeOH/~20 gave pure title compound. Evaporation of the solvent
gave white solid, which was collected, washed with cold water
followed by EtOH and dried to furnish 14.0 g (79~) of 4, mp
232-2340C: IR (Rsr): Css (C-Cl), 3280 (NH2, OH) cm 1 UV: AmaX
(pH 1) 260 nm (8 15,600): AmaX (pH 7) 261 nm (~ lS,700): ~max
(pH 11) 261 nm (~ 15,900): lH NMR (Me2SO-d6): ~ 4.13 (m, 2,
Cs,~2), 4.S6-4.39 (m, 1, C~,~), 4.99-4.97 and S.17-S.13 (m, 2,
C2,~ and C3,~), S.8S (8, 1, C1~), 7.67 (br s, 2, N~2) and 8.21
(s 1 ~2~) ~n~l. Calcd for C1oHllClN506P.H20: C,
3.43: N, 18.3S: Cl, 9.29. Found: C, 31.69: H, 3.19: N, 18.26:
C1, 9.S2.
Method A.
To a solution of adenosine 3',S'-cyclic phosphate (l.lS g,
3.S mmol) in dry DMF (7S mL, distilled over CaH2) is added
dropwise a solution of DMF saturated with anhydrous HCl (S mL),
followed by a solution of purified MCPBA (1.1 g, 6.4 mmol) in
DMF (10 mL). The reaction mixture is then stirred at room
temperature for 2 1/2 h, before the DMF is evaporated to
dryness under Y~c_Q at 45C. The residue is then dissolved in
13 1 33~073
minimum amount of water (~lo mL) and the product precipitated
from the solution by the dropwise addition of MeOH. The solid
is then collected and purified by HPLC on a C-18 reverse phase
column using 0.S~ aqueous AcOH to furnish 4, identical to 4
prepared by Method C.
Method ~.
To a solution of cAMP (30.3 g, 92 mmol) and NaCl t30.6 g,
S2 mmol) in AcOH (2So mL) and H2O (ls0 mL) was added NCS (30.3
g, 227 mmol). The reaction mixture was stirred at room
temperature for 3 days. The ~olvents were evaporated to
dryness under reduced pressure and the residue purified as
described above in ~ethod A to yield 13.0 g (39~) of 4. which
was identical to 4 prepared by Method Ç.
EXAMPLE 5
8-Chloroinosine 3'.S'-cyclic ~hos~hAte (S).
To a suspension of 8-chloroadenosine 3',S'-cyclic
phosphate (4, 1.0 q, 2.7 mmol) in H2O (3 mL~ was added 2N NaOH
dropwise until a clear solution was obtained. NaN02 (l.OS g,
lS mmol) was added to the reaçtion solution, followed by
dropwise addition of AcOH t2 mL). The reaction mixture was
stirred at room temperature overnight and then evaporated to
dryness under reduced pressure at 3S'C. The residue was
purifi~d on a Dowex~So x 2 - 200 (H+) resin column (S x 20 cm)
eluting with H2O. The homogeneous fractions containing 8-
chloro-~IMP were pooled and evaporated to dryness. Co-
evaporation of the residue with EtOH (3 x 2S mL) gave a white
solid, which after drying at 78OC under vacuum for lS h
afforded o.S6 g (S6~J) of S: mp 216-il8-C: IR (RBr): 78s (C-C1),
1680 (C=O), 3200-3400 (OH) cm 1 UV: AmaX ~pH 1) 2Sl nm (~
17,100): AmaX (pH 7) 2Sl nm (8 17,000): AmaX (pH 11) 2SS nm (~
17,300): lH NMR (Me2SO-~6): ~ s.8S (s, 1, C1,~), 8.17 (s, 1,
C2H), and 12.71 (br ~, 1, Nl~ al- Calcd for
C1oHloClN4O7P): C, 32.94: H, 2.76: N, lS.37. Found: C, 32.7S:
H, 2.69: N, 15.14.
14
1 336073
~ or use in pharmaceutical preparations of the invention
normally a salt of the 3',5'-cyclic phosphate moiety of either
8-chloroadenosine 3',S'-cyclic phosphate or 8-aminoadenosine
3',5'-cyclic phosphate would be utilized and would be suitably
given to a host as a solution in a suitable carrier.
Alternately, the free acid form of the compounds could be
utilized.
Acceptable salts of the phosphate moiety can be selected
from, but not necessarily limited to the group consisting of
alkali and alkaline earths. e.g. sodium, potassium, calcium,
magnesium, lithium, or ammonium and substituted ammonium, tri-
alklyammonium, diallcylammonium, alkylammonium, e.g. triethyl-
ammonium, trimethylammonium, diethylammonium, octylammonium,
cetyltrimethylammonium and cetylpridium. Such a salt would
preferredly be chosen from the group consisting of alkali metal
salt, as for instance, a sodium or a potassium salt or an
ammonium salt.
In performing the invention, 8-chloroadenosine 3',5'-
cyclic phosphate and/or 8-aminoadenosine 3',S'-cyclic
phosphate, as free acids or as salts, are appropriately mixed
with a suitable pharmaceutical carrier which, since the
compounds of the invention are water soluble, may be as simple
as sterilized water or could be a complex carrier having
appropriate agents to suitably mimic certain biological
environmental, i.e., pH or salt adjusted for solution suitable
for intravenous, intramuscular or other injections.
In selecting a suitable pharmaceutical carrier,
consideration of the type of tumor, the site of the tumor and
the health and age of the host would be given. ~-Chloro-
adenosine 3',S'-cyclic phosphate and/or 8-aminoadenosine 3',s'-
cyclic phosphate might be approp~iately used in the presence of
a suitable buffer or as a salt as discussed above. The
compounds of the inventlon are especially useful in treating
carcinoma. Included in such a class are mammary, colon,
bladder, lung, prostate, stomach and pancreas carcinoma. The
treatment method is effective in bringing about regression,
palliation, inhibition of growth, and remission of tumors.
_
60724-1808D
lS ~ 33~073
Preferably, 8-chloroadenosine 3',S'-cyclic phosphate
and/or 8-aminoadenosine 3',5'-cyclic phosphate of the invention
or salts thereof would be mixed with an appropriate
pharmaceutical carrier such that 8-chloroadenosine 3',S'-cyclic
phosphate and/or 8-aminoadenosine 3',5'-cyclic phosphate would
be suitably soluble in the carrier. Alternately, however,
suspensions, emulsions and other formulations of 8-
chloroadenosine 3',5'-cyclic phosphate and 8-aminoadenosine
3~,S'-cyclic phosphate of the invention could be used where
indicated. The pharmaceutical carrler, in addition to having a
solubilizing or suspending agent therein, might also include
suitable dilutants, buffers, surface active agents and other
similar a~ents as are typically used in pharmaceutical
carriers. The total composition of the pharmaceutical carrier
would, however, be chosen to be compatible with the site of
delivery, the concentration of the active ingredient and other
parameters as are standard in pharmaceutical industry.
8-Chloroadenosine 3~,S~-cyclic phosphate and/or 8-
aminoadenosine 3',S'-cyclic phosphate of the invention would be
suitably admixed with the pharmaceutical carrier such that they
would be present in a concentration of at least 0.1 percent by
weight of the total composition. Preferredly, they would be
present in the pharmaceutical carrier at a concentration of
about lo~ to about 90~ by weight of the total composition.
Based on presen~ studies, effective amounts of 8-chloro-
adenosine 3',S'-cyclic phosphate typically would range from
about 13 milligrams per kilogram per day (mg/kg/day) of the
total body weight of the treated warm blooded animal to about
288 mg/kg/day. Preferredly, this range would be from 22 mg/kg
to about 173 mg/kg/day, Based on present studies, effective
amounts of 8-aminoadenosine 3~,S~-cyclic phosphate typically
would range from about 13 milligrams per kilogram per day
(mg/kg/day) of ~he total body weight of the treated warm
blooded animal to about 104 mg/kg/day. Preferredly, this range
would be from 13 mg/kg to about 37 mg/kg/day. As with other
factors noted above, the amounts of 8-chloroadenosine 3~,S~-
cyclic phosphate and 8-aminoadenosine 3~,S~-cyclic phosphate
16 ~ ~3~
utilized in treating an afflicted animal would take into
account parameters such as the type of tumor, the tumor site,
the form of administering and the physical size and condition
of the host. In any event, the actual amount should be
sufficient to provide a chemotherapeutically effective amount
of the agent in the host in a convenient volume. which will be
readily within the ability of those skilled in the art to
determine given the disclosure herein.
8-Chloroadenosine 3',S'-cyclic phosphate and 8-
aminoadenosine 3',S'-cyclic phosphate can be administered
together admixed with a suitable pharmaceutical carrier. As is
evident from the examples and tables below both of these
compounds when administered independently to a host exhibit
certain antitumor activity. When both of the compounds are
administered together to a host the antitumor activity is
maintained and the compounds are not antagonistic to one
another. Further, while we do not wish to be bound by theory,
at this time it is believed that when administered together the
compounds 8-chloroadenosine 3',S'-cyclic phosphate and 8-
aminoadenosine 3',S'-cyclic phosphate exhibit synergistic
activity.
Singularly 8-chloroadenosine 3',S'-cyclic phosphate, as is
evident from the experiments and tables below, exhibits a very
broad spectrum of activity against many different tumors.
Singularly 8-aminoadenosine 3',S'-cyclic phosphate exhibits
significant activity against colon carcinoma. The com~ination
of the compounds 8-chloroadenosine 3',S'-cyclic phosphate and
8-aminoadenosine 3',S'-cyclic phosphate also exhibits
significant activity against colon carcinoma, however, the
activity of the combination of 8-chloroadenosine 3',S'-cyclic
phosphate and 8-aminoadenosine 3 ', S '-cyclic phosphate is
greater than the activity of 8-chloroadenosine 3',S'-cyclic
phosphate when used alone.
When the compounds 8-chloroadenosine 3',S'-cyclic
phosphate and 8-aminoadenosine 3',S'-cyclic phosphate are
utilized in the dose ranges above but are utilized together,
the activity is enhanced compared to 8-chloroadenosine 3~,S~-
17 l 3 3 6 0 7 3
cyclic phosphate when used independently. Further thisenhanced activi~y is maintained beyond a post treatment period.
Additionally with the combination of the two compounds 8-
chloroadenosine 3',S'-cyclic phosphate and 8-aminoadenosine
3 ~, S ~-cyclic phosphate there is decreased drug rebound after
discontinuous of the drug.
The 8-chloroadenosine 3',S'-cyclic phosphate and/or 8-
aminoadenosine 3',S'-cyclic phosphate of the invention can be
given as single doses or as multiple doses divided into sub-
doses qiven daily or over a period of days. As will be evident
from the examples below, 8-chloroadenosine 3 ~,S '-cyclic
phosphate and 8-aminoadenosine 3',S'-cyclic phosphate of the
invention exhibits certain inhanced responses when administered
as an infusion and, as such, this will be taken in to account
in the optimization of a dosage schedule as is well within the
skill of the Art given the disclosure herein.
The following examples are given for use of 8-
chloroadenosine 3',5'-cyclic phosphate, 8-aminoadenosine 3~,S~-
cyclic phosphate and the combination of 8-chloroadenosine
3',S'-cyclic phosphate plus 8-aminoadenosine 3',S'-cyclic
phosphate of the invention as a therapeutic agents against
neoplastic diseases. In these examples the efficacy of 8-
chloroadenosine 3',S'-cyclic phosphate and 8-aminoadenosine
3',S'-cyclic phosphate as antitumor agents is demonstrated by
using standard tests against certain malignant tumors.
These standard tests utilize protocols developed under the
auspices of the Developmental Therapeutic Program, Division of
Cancer Treatment, United States National Cancer Institute,
Bethesda, Maryland, United States of America, as set forth in
I~ Yit~Q C~nce~ MQd~l~, National Institute of Health
Publications No 84-263S, February 1984, United States
Department of Health and Human Services, Public Health Service,
National Institute of Health.
Staging, growth and testing of tumors was done as is set
orth in the publication of the preceeding paragraph. The mode
of administration and delivery of 8-chloroadenosine 3',5'-
cyclic phosphate and~or 8-aminoadenosine 3',5'-cyclic
18 1 336073
phosphate. however, deviated slightly from these protocols and
is as is set forth in each individual example. Evaluation
protocols with respect to activity of 8-chloroadenosine 3',5'-
cyclic phosphate and 8-aminoadenosine 3',5'-cyclic phosphate as
anti-tumor agents. however, follow the criteria as is defined
in the above referenced publication.
For the purposes of these examples certain standard
abbreviations are utili~ed as follows: i.p. - intraperitoneal:
qd - once a day: and mglkg1day - milligrams per kilograms per
day.
In the example utilizing Ll210 as the test tumor cell
line. the test results are indicated as ~T/C. According to
protocols of the United States National Cancer Institute for
the L1210 tumor line, a value greater than 12S~ is considered
as having statically meaningful activity. ~or test against
solid human tumor cell lines, test results are given as change
in mean tumor weight. This is expressed in two ways. If there
was an increase in tumor weight the results are expressed as
~T/~C. However, if there was a net negative change in tumor
weight the results are expressed as ~T/T. These two ways of
data expressions again follow the protocols and criteria set
forth as noted above by the National Cancer Institute.
EXAMPLE 6
-
8-chloroadenosine 3~,S'-cyclic phosphate as a sodium salt
was tested utilizing non-tumor bearing BDFl mice to establish a
lethal toxicity for this drug. For this test the drug was
delivered i.p. by a bolus injection given as a single dose on
day l. As is evident from Table l below, at 104 milligrams per
kilograms per injection there were no toxic deaths. At a level
of 173 milligrams per kilograms per injection there was a 40
percent toxic death and at 288 milligrams per kilograms per
injection the compound exhibited lO0 percent lethal toxicity.
As will be evident in example 6 below, when the compound is
delivered by infusion, the test animals tolerated a higher dose
19 1 336~73
of drug before lethal toxicity was seen.
Table 1. Influence o 8-Cl-cAMP Na+ on the life span of non-
tumor BDFl mice when delivered i.p. by bolus injection
Dosage Route and scheduleToxic deaths1
mg/kg/inj of deliveryNo killed/Mo treated
480 ip: qd, day 1 S/S
288 ip: qd, day ~ s/s
173 ip: qd, day 1 2/S
104 ip: qd, day 1 0
6 2 ip: qd, day 1 0
37 ip: qd, day 1 o
22 ip: qd, day 1 0
1. When delivered qd, day 1 to non-tumor BDFl mice, the 480 and
288 mb/kg dosages of 8-Cl-~AMP Na+ were lethally toxic for all
treated mice. The 173 mg/kg dosage killed 2 of S mice and lower
dosages were not lethally toxic.
-
1 336073
EXAMPLE 7
The activity of 8-chloroadenosine 3',5'-cyclic phosphate
against L1210 inoculated BDF1 mice was determined by both bolus
injection and by infusion. As shown in Table 2 below, when
delivered by bolus injection there was insignificant activity,
however, when infused into a test animal in a dose range of
from 22 mg to 173 mg/kg/day, 8-chloroadenosine 3',S'-cyclic
phosphate exhibited significant antitumor activity. Further,
the toxicity was determined for the infusion test animals. As
is also evident from Table 2 when infused at 173 mg/kg/day the
compound was not toxic, however, when infused at the 288
mg/kg/day level both activity and toxicity are noted and at
higher levels, at 480 and 800 mg/kg/day, the compound is
lethally toxic.
The results of Table 2 indicate that 8-chloroadenosine
3',S'-cyclic phosphate is an effective antitumor agent against
Ll210 inoculated mice when infused into the test animals.
Further the compound demonstrated a dose response for this
activity. As was indicated above, a T/C of greater than 12S
indicates significant activity.
~ 21 1 336073
Table 2. Influence of 8-Cl-_AMP Na on the postinoculation
lifespan of L1210-inoculated BDF1 micel when infused or
delivered by bolus injection
Dosage Route and schedule Postinoculation
(mg/kg/day) of delivery lifespan2
(T/C)
104 ip: qd, day 1-7 98
62 ip: qd, day 1-7 103
37 ip: qd, day 1-7 93
22 ip: qd, day 1-7 103
800 ip: 24-hr infusion, day 1-s62 toxic3
480 ip: 24-hr infusion, day 1-593 toxic3
288 ip: 24-hr infusion, day 1-S128 toxic3
173 ip: 24-hr infusion, day l-S131
04 ip: 24-hr infusion, day 1-S137
62 ip: 24-hr infusion, day 1-S137
37 ip: 24-hr infusion, day l-S126
22 ip: 24-hr infusion, day l-S126
13 ip: 24-hr infusion, day l-S113
48 ip: 24-hr infusion, day 1-S100
17 ip: 24-hr infusion, day l-SloO
Q.6 ip: 24-hr infusion, day 1-S100
1. Mice were inoculated i.p. with 1x106 cells of murine leukemia
L1210 24-hr before first treatment. Each treatment group
consisted of S mice. Twenty control mice that received a 0.9
solution of NaCl lived 6.2 + days.
2. Significant activity indicated at T/C > 12S.
3. When infused, the 800 and 480 mg/kg dosages of 8-Cl-~AMP Na~
were lethally toxic for all treated mice while the 288 mg/kg
dosage killed 2 of s mice.
~ 1 336~73
22
8-Chloroadenosine 3',S'-cyclic phosphate was tested
against a variety of solid human tumor cell lines. In the
tests of examples B through 11 shown in Tables 3 through 6,
what is being measured is tumor regression size and not
increase in life span of the test animal. This expression
follows the accepted ~Jation Cancer Institute protocol
procedures for respective solid tumors cell lines which were
tested.
EXAMPLE 8
In this example, 8-chloroadenosine 3',S'-cyclic phosphate
given by infusion was tested against human mammary carcinoma in
athymic mice. In a first study shown in the upper portion of
Table 3a at the dose levels given there was a mean reduction in
tumor weight. As such the change in this mean reduction in
tumor weight, following the Nation Cancer Institute protocols
as noted above, is expressed as ~T/T. In the study shown in
the lower portion of Table 3a, at lower dose levels, the
weight between the starting tumor weight and the final tumor
weight was greater than unity and as such again following the
established protocols the results are shown as ~T/AC.
Any value for ~T/~C or ~T/T which is below 2~ is
considered by the National Cancer Institute protocols as
indicative of significant activity. As is evident from Table
3a significant activity was indicated over a large dosage range
of from 22 mg/kg up to and including 173 mg/kg.
23 l 336073
Table 3a. Tnfluence of intraperitoneally infused 8-Cl-A~P Na+
on the growth in athymic mice of human mammary carcinoma
MX_ll
Dosage Initial Mean ~ First-Last Change in Mean
(mglkg/day) Weight (mg) Tumor Weight2Tumor Weight3
(~T/~C
or
~T/T)
173 37S -39 -10.44
104 346 -23 - 6.74
62 337 -49 -14.S4
0 334 2~S -----
37 194 16 6S
22 201 76 29S
13 216 9S 37S
o 204 259 --
1. Tumor fragments (- 14 mg) were implanted subcutaneously in
the thigh region of athymic CD-l female mice. Three weeks later
the tumors were staged and treatment lasting 7 days was started,
each treatment group consisted of 7 mice.
2. Tumor measurements recorded on the initial day of treatment
(staging day) and again on day 8 were used to calculate mean
tumor weight changes and ~T/~C or ~T/T.
3. ~T/~C utilized for positive First-Last tumor ~weights and
~T/T utilized for negative First-Last tumor weights.
4. ~T/T
5. ~TtAC
24 ~ 336073
The mean tumor weight at each dose shown in Table 3a for
all the test animals at days 1 and 8 are indicated in Table 3b.
Table 3b.Tumor weights of human mammary carcinoma MX-l1
tumors in athymic mice intraperitoneally infused with 8-Cl-cAMP
Na+
Dosage Initial MeanMean Tumor
(mg/kg/day) Weight (mg)Weight (mg)
Day 1 Day 8
173 37S 336
104 346 317
62 337 288
control 334 549
37 194 210
22 201 277
13 216 311
control 204 463
EXAMPLE 9
8-chloroadenosine 3~,S~-cyclic phosphate was further
tested in athymic mice against human colon carcinoma LoVo. As
per example 8, administration was also by infusion. The
results of this test are shown in Table 4a below. The mean
change in tumor weight is indicated either as ~T/~C for the
positive value or ~T/T for the negative values as was discussed
2~ 1 336073
with respect to Example 8 above. As is evident at th~ 37 and
62 kg/mg dosage the compound exhibited significant activity.
Table 4a. Influence of intraperitoneally infused 8-Cl-cAMP Na
on the growth in athymic mice of human colon carcinoma
LoVol
Dosage Initial Mean ~ First-LastChange in Mean
(mg/kg/day~ Weight tmg) Tumor Weight2Tumor Weight3
(~T/~C
or
~T/T)
104 288 29 3s4
62 293 -3 - l.oS
37 28~ -4 _1.4S
0 292 84 ------
1. Tumor fragments (~ 14 mg) were implanted subcutaneously in the
thigh region of athymic CD-1 female mice. Three weeks later the
tumors-were staged and treatment lasting 7 days was started, each
treatment group consisted of 7 mice.
2. Tumor measurements recorded on the initial day of treatment
(staging day) and again on day 8 were used to calculate mean
tumor weight changes and ~T/~C or AT/T.
3. ~T/~C utilized for positive First-Last tumor weights and ~T/T
utilized for negative First-Last tumor weights.
4. ~T/~C
S. ~T/T
~ 1 336073
26
The mean tumor weight for each dose shown in Table 4a for
all the test animals at days 1 and 8 are indicated in Table 4b.
Table 4b. Tumor weights of human colon carcinoma LoVol
tumors in athymic mice intraperitoneally infused with 8-Cl-AMP
Na
Dosage Initial Mean Mean Tumor
(mg/kg~day~ Weight (mg) Weight (mg~
Day 1 Day 8
104 288 317
62 293 290
37 288 284
control 29i 376
EXAMPLE 10
-
8-chloroadenosine 3',S'-cyclic phosphate was further
tested in athymic mice against human mammary carcinoma MDA-MB-
231. The test was also done using infusion as the route of
administration. The results of this test are shown in Table Sa
below. The compound exhibited significant activity at the dose
levels of 62 and 104 mg/kg~day. Further while it did not
exhibit significant activity at all dose levels it did show a
linearity of response throughout the tested dosage range
against this tumor line.
27 ~ ~6073
Table Sa. Influence of intraperitoneally infused 8-Cl-AMP Na+
on the growth in athymic mice of human mammary carcinoma
MDA-MB-2311
Dosage Initial Mean ~ First-Last Change in Mean
(mg/kg/day) Weight (mg)Tumor Weight2 Tumor Weight3
~T/~C
104 367 66 18
62 37S 96 26
37 367 149 41
0 361 36S ------
1. Tumor fragments (~ 14 mg) were implanted subcutaneously in the
thigh region of athymic CD-1 female mice. Three weeks later the
tumors were staged and treatment lasting 7 days was started, each
treatment group consisted of 7 mice.
2. Tumor measurements recorded on the initial day of treatment
(staging day) and again on day 8 were used to calculate mean
tumor weight changes and ~T/~C or ~T/T.
3. AT/~C utilized for positive First-Last tumor weights.
28 l 336073
The mean tumor weight for each dose shown in Table Sa for
all the test animals at days 1 and 8 are indicated in Table Sb.
Table Sb. Tumor weights of human mammary carcinoma MDA-MB-
231 tumors in athymic mice intraperitoneally infused with 8-Cl-
c~lP ~a+
Dosage Initial Mean Mean Tumor
(mg/kg/day) Weight (mg) Weight (mg)
Day 1 Day 8
104 367 433
62 37S 471
37 367 S16
control 361 726
EXAMPLE 11
8-chloroadenosine 3 ', S ~-cyclic phosphate was further
tested in athymic mice against human lung carcinoma LX-l. As
per the results shown in Table 6a below, the compound exhibited
significant activity at a dose range of from 37 mg to 104
mg~kg/day. For this test infusion was only for S days as
opposed to the 7 days used for the previous solid tumor tests
of Examples 8, 9 and 10. Further, as per Example 8 the
positive mean tumor weights results are shown as AT/~C and
negative mean tumor weights as ~T/T. It is significant to note
at 62 and 104 mg dosage range there was a high degree of tumor
weight loss as opposed to only inhibition of tumor growth, i.e.
at the 62 and 104 mg dose levels there was tumor regression.
~ 29
1 336073
Table 6a. Influence of intraperitoneally infused 8-Cl-cAMP Na
on the growth in athymic mice of human lung carcinoma
LX_ll
Dosage Initial Mean~ First-Last Change in Mean
(mg/kg/day) Weight (mg)Tumor Weight2Tumor ~eight3
~TI~C
104 324 -S4 -16.74
62 327 -S0 -lS.34
37 330 11 65
o 332 183 ------
1. Tumor fragments (~ 1~ mg) were implanted subcutaneously in the
thigh region of athymic CD-1 female mice. Three weeks later the
tumors were staged and treatment lasting S days was started, each
treatment group consisted of 7 mice.
2. Tumor measurements recorded on the initial day of treatment
(staging day) and again on day 8 were used to calculate mean
tumor weight changes and ~T/~C or ~T/T.
3. ~T/~C utilized for positive First-Last tumor weights and ~T/T
utilized for negative First-Last tumor weights.
4. ~T/T
S. ~T/~C
30 1 33607~
The mean tumor weight for each dose shown in Table 6a for
all the test animals at days 1 and 8 are indicated in Table 6b.
Table 6b. Tumor weights of human lung carcinoma LX-l tumors
in athymic mice intraperitoneally infused with 8-Cl-cAMP Na+
Dosaqe Initial Mean Mean Tumor
(mg/kg/day) Weight (mg) Weight (mg)
Day 1 Day 8
1~4 324 270
62 327 277
37 330 341
control 332 SlS
EXAMPLE 12
8-Chloroadenosine 3',S'-cyclic phosphate was further
tested in athymic mice against human colon carcinoma MeC-1. As
per the results shown in Table 7a below the compound exhibited
significant activity in a dose range of 37 mg/kg/day to 104
mg/kg/day. The drug was administered by infusion as the route
of administration. As per the result of Table 7a significant
activity was exhibited throughout the tested dose range against
this tumor line.
31 l 336073
Table 6a. Influence of intraperitoneally infused 8-Cl-AMP Na+
on the growth in athymic mice of colon carcinoma MeC-l1
Dosage Initial Mean ~ First-Last Change in Mean
(mg/kg~day) Weight (mg)Tumor Weight2 Tumor Weight
~T/~C
104 3S6 -47 -13 3
62 350 - 1 - o.33
37 339 18 14
33S ~33 ______
1. Tumor fragments (- 14 mg) were implanted subcutaneously in the
thigh region of athymic CD-1 female mice. Three weeks later the
tumors were staged and treatment lasting 7 days was started, each
treatment group consisted of 7 mice.
2. Tumor measurements recorded on the initial day of treatment
(staging day) and again on day 8 were used to calculate mean
tumor weight changes and ~T/~C. In instances of net tumor
loss, ~T/~T was calculated in lieu of ~T/~C.
3. ~T/T
EXAMPLE 13
8-Aminoadenosine 3',S'-cyclic phosphate as a sodium salt
was tested utilizing nontumor bearing BDF1 mice to estabiish a
lethal toxicity for the drug. For this test the drug was
delivered i.p. by a bolus injection given as a single dose on
day one. As is evident from Table 8 below at 104 mg/kg per
injection there were no toxic deaths. At the higher levels of
32 ~ 336073
173 mg/kg and 288 mg/kg there was lethal toxicity for l/S and
S/S mice respectively. When 8-aminoadenosine 3',5'-cyclic
phosphate was delivered 24 hr/day for S consecutive days by
i.p. infusion, 62 mg/kg was the maximum tolerated dose and at a
dose of 104 mg/kg or more the compound was lethally toxic for
13/13 mice.
Table 8. Influence of 8-NH2-~AMP Na on the life span of non-
tumor BDFl mice when delivered i.p. by bolus injection
Dosage Rou~e and scheduleToxic deaths
mg/kg/inj of deliveryNo killed/No treated
288 ip: qd, day 1 S/S
ip: qd, day 1 l/S
104 ip: qd, day 1 0
The activity o~ 8-aminoadenosine 3',S'-cyclic phosphate
against various solid human tumors was tested utilizing athymic
mice treated by infusion with the active compound. The
protocols discussed in Example 8 above were utilized for these
tests.
EXAMPLE 14
Tn this example 8-aminoadenosine 3',S'-cyclic phosphate
33 l 33607~
was given by infusion against human colon carcinoma MeC-1. As
is evident from Table 9 significant activity was indicated over
the dose range tested.
Table 9. Influence of intraperitoneally infused 8-AMINo-cAMp
Na+ on the growth in athymic mice of human colon
carcinoma MeC-l1
Dosage Initial Mean ~ First-LastChange in Mean
(mg/kg/day) Weight (mg) Tumor Weight2Tumor Weight
~T/~C
22 383 -49 -13 3
13 337 -10 - 3 3
0 3g2 142 ------
l. Tumor fragments (- 14 mg) were implanted subcutaneously in the
thigh region of athymic CD-l female mice. Three weeks later the
tumors-were staged and treatment lasting 7 days was started, each
treatment group consisted of 7 mice.
2. Tumor measurements recorded on the initial day of treatment
(staging day) and again on day 8 were used to calculate mean
tumor weight changes and ~T/~C. In instances of net tumor
loss. ~T/T was calculated in lieu of ~T/~C.
3. ~T/T
EXAMPLE 1S
8-Aminoadenosine 3~,S~-cyclic phosphate was further tested
~ 336~7~
in athymic mice aqainst human colon carcinoma HT-29. As per
Example 14 administration was also done by infusion. As is
evident from Table 10 the compound exhibited significant
activity over the totality of the range of doses tested.
Table 10. Influence of intraperitoneally infused 8-AMINO-~AMP
Na+ on the growth in athymic mice of human colon
carcinoma HT-2 91
Dosage Initial Mean ~ First-Last Change in Mean
(mq/ks/day) Weight (mg)Tumor Weight2 Tumor Weight
~T/~C
37 326 -27 - 8.o3
22 346 -18 - s.o3
0 3S6 78 ------
1. Tumor fragments (~ 1~ mg) were implanted subcutaneously in the
thigh Eegion of athymic CD-l female mice. Three weeks later the
tumors were staged and treatment lasting 6 days was started. each
treatment group consisted of 6 mice.
2. Tumor measurement~ recorded on the initial day of treatment
(staging day~ and again on day 7 were used to calculate mean
tumor weight changes and ~T/~C. In instances of net tumor
loss, ~T/T was calculated in lieu of AT/~C.
3 . /'T /T
35
1 33~73
EXAMPLE 16
8-Aminoadenosine 3',S'-cyclic phosphate was further tested
in athymic mice against human colon carcinoma CX-l. This test
was also done utilizing infusion as the route of
administration. This particular human tumor line has common
origins with the above referred to HT-29 tumor line of Example
lS with the exception that the CX-1 tumor line was evolved i~
ViyQ while the HT-29 tumor line was evolved in vitro. The
results of this test are shown in Table 11 below. As is
evident from Table 11 the compound showed greater activity at a
lower dose level at 8 mg/kgtday. While we do not wish to be
bound by theory at this time it is believed that host toxicity
to 8-aminoadenosine 3',S'-cyclic phosphate appears to be
influenced by the tumor line which is being treated. Further.
as per the results shown in Tables 9, 10 and 11 the compound 8-
aminoadenosine 3',S'-cyclic phosphate exhibited particularly
significant activity against human colon carcinomas.
36 1 336~73
Table 11. Influence of intraperitoneally infused 8-AMINO-cAMP
Na+ on the growth in athymic mice of human colon
carcinoma CX-11
Dosage Initial Mean ~ First-Last Change in Mean
(mg/kg/day~ Weight (mg)Tumor Weight2 Tumor Weight
AT/~C
22 398 31 24
13 396 30 23
8 397 -8 -2 3
0 398 130 ----
1. Tumor f~agments (~ 14 mg~ were implanted subcutaneously in the
thigh region of athymic CD-1 female mice. Three weeks later the
tumors were staged and treatment lasting 7 days was started. each
treatment group consisted of 6 mice.
2. Tumor measurements recorded on the initial day of treatment
(staging day) and again on day 8 were used to calculate mean
tumor ~eight changes and aT/~c. In instances of net tumor
loss, ~T/T was calculated in lieu of ~T/~C.
3. AT/T
EXAMPLE 17
8-Aminoadenosine 3',S'-cyclic phosphate was further tested
in athymic mice against human lung carcinoma LX-1. This test
was further done by infusion and with treatment over a period
of five days. The results of this test are shown in Table 12
37 l 336073
below.
Table 12. Influence of intraperitoneally infused 8-AMIN0-_AMP
Na+ on the growth in athymic mice of human lung
carcinoma LX-ll
Dosage Initial Mean ~ First-Last Change in Mean
(mg/kglday) Weight (mg)Tumor Weight2 Tumor Weight
~T/~C
22 47S 78 38
13 SlS 119 S8
0 484 204 ----
1. Tumor fragments (~ 14 mg) were implanted subcutaneously in the
thigh region of athymic CD-1 female mice. Three weeks later the
tumors were staged and treatment lasting S days was started, each
treatment group consisted of 6 mice.
2. Tumor measurements recorded on the initial day of treatment
(staging day) and again on day 6 were used to calculate mean
tumor weight changes and ~T/~C.
EXAMPLE 18
In a like manner to example 17 8-aminoadenosine 3',S'-
cyclic phosphate was further tested in athymic mice against
human lung carcinoma LX-l with treatment lasting for seven
days. The results in this test are shown in Table 13 below.
38 l 336073
Table 13. Influence of intraperitoneally infused 8-AMIN0-AMP
Na+ on the growth in athymic mice of human lung
carcinoma LX-l1
Dosage Initial Mean ~ First-LastChange in Mean
(mg/kg/day) Weight (mg) Tumor Weight2Tumor Weight
~T/~C
37 S6~ 1g9 S0
22 S60 139 3S
13 S6S 208 S3
0 S77 396 ----
1. Tumor fragments (^ 14 mg) were implanted subcutaneously in the
thigh region of athymic CD-l female mice. Three weeks later the
tumors were staged and treatment lasting 7 days was started, each
treatment group consisted of 6 mice.
2. Tumor measurements recorded on the initial day of treatment
(staging day) and again on day 8 were used to calculate mean
tumor weight changes and ~T/~C.
EXAMPLE 19
8-Aminoadenosine 3~,5'-cyclic phosphate was further tested
in athymic mice against human mammary carcinoma MX-l. This
test was also done by infusion and treatment was conducted for
seven days. The results of two separate studies utilizing this
39 l 336073
protocol are shown in Tables 14a and 14b below.
Table 14a. Influence of intraperitoneally infused 8-AMINo-_AMP
Na+ on the growth in athymic mice of human mammary
carcinoma MX-ll
Dosage Initial Mean A First-LastChange in Mean
(mg/kg/day) Weight (mg) Tumor Weight2Tumor Weight
~T/~C
22 2S3 lS6 79
13 2S6 167 8S
8 2S3 194 98
o 2s2 197 ----
1. Tumor fragments (- 14 mg) were implanted subcutaneously in the
thigh ~egion of athymic CD-l female mice. Three weeks later the
tumors were staged and treatment lasting 7 days was started, each
treatment group consisted of 7 mice.
2. Tumor measurements recorded on the initial day of treatment
(staging day) and again on day 8 were used to calculate mean
tumor weight changes and ~T/~C.
1 336073
Table 14b. Influence of intraperitoneally infused 8-AMINO-AMP
Na+ on the growth in athymic mice of human mammary
carcinoma MX-11
Dosage Initial Mean ~ First-Last Change in Mean
(mg/kg/day) Weight (mg)Tumor Weight~ Tumor Weight
~T/~C
37 305 93 4S
22 309 97 47
~3 311 171 83
o 326 207 ----
1. Tumor fragments (- 14 mg) were implanted subcutaneously in the
thigh region of athymic CD-l female mice. Three weeks later the
tumors were staged and treatment lasting 7 days was started, each
treatment group consisted of 6 mice.
2. Tumor measurements recorded on the initial day of treatment
(staging day) and again on day 8 were used to calculate mean
tumor weight changes and ~T/~C.
EXAMPLE 20
8-Aminoadenosine 3~,S~-cyclic phosphate was further tested
in athymic mice against human colon carcinoma LOVO. This test
was also done utilizing infusion as the route of
administration. The results of this test against this
41 1 3 3 6 0 1 3
particular human colon carcinoma are shown in Table lS below.
Table lS. Influence of intraperitoneally infused 8-AMINO-cAMP
Na+ on the growth in athymic mice of human colon
carcinoma LOW 1
Dosage Initial Mean ~ First-Last Cnange in Mean
(mg/kg/day) Weight (mg~Tumor Weight2 Tumor Weight
~T/~C
22 263 130 76
13 326 98 S8
8 330 lS3 go
o 326 170 ----
1. Tumor ~ragments (~ 14 mg) were implanted subcutaneously in the
thigh region of athymic CD-1 female mice. Three weeks later the
tumors were staged and treatment lasting 7 days was started. each
treatment group consisted of 6 mice.
2. Tumor measurements recorded on the initial day of treatment
(staging day) and again on day 8 were used to calculate mean
tumor weight changes and T/~C.
The combination of 8-chloroadenosine 3',S'-cyclic
phosphate and 8-aminoadenosine 3',5'-cyclic phosphate when
administered together was further tested. Each compound was
given in amount within its active dose range as illustrated in
the above examples.
42 1 336~7~
EXAMPLE 21
In this test 8-chloroadenosine 3',5'-cyclic phosphate and
8-aminoadenosine 3',5'-cyclic phosphate are administered
together in athymic mice against human colon carcinoma HT-29.
This combination drug ~herapy was compared to administration of
8-chloroadenosine 3',S'-cyclic phosphate as an independent drug
at the same dose level as utilized in the combination therapy.
Both the combination drug and the single drug therapy were
given by infusion with treatment lasting for seven days. As
per the results o~ this test in Table 16 below the combination
of 8-aminoadenosine 3',S'-cyclic phosphate and 8-
chloroadenosine 3',S'-cyclic phosphate showed increased
activity compared to administration of 8-chloroadenosine 3 ', S '-
cyclic phosphate as an independent drug. This is indicative
that 8-aminoadenosine 3',5'-cyclic phosphate and 8-
chloroadenosine 3',S'-cyclic phosphate are not antagonists. It
is further believed that 8-aminoadenosine 3',S'-cyclic
phosphate and 8-chloroadenosine 3',S'-cyclic phosphate might be
synergistic in their action as antitumor agents.
43 l 336073
Table 16. Influence of intraperitoneally infused 8-Cl-AMP
Na+ on the growth in athymic mice of human colon
carcinoma HT-291 when given alone or with 8-AMINO-AMP
Na
Dosage Initial Mean A First-Last Change in Mean
(mglkg/day) Weight (mg) Tumor Weight2 Tumor Weight
~T/~C
8-C1 62 327 28 18
8-Cl 62
~ 340 -44 -133
8-Amino 13
347 152 ____
1. Tumor fragments (~ 14 mg) were implanted subcutaneously in the
thigh region of athymic CD-l female mice. Three weeks later the
tumors were staged and treatment lasting 7 days was started, each
treatment group consisted of 7 mice.
2. Tumor measurements recorded on the initial day of treatment
(staging day) and again on day 8 were used to calculate mean
tumor weight changes and ~T/~C.
3. In instances of net tumor loss, ~T/T was calculated in lieu
of ~T/~C.
44 l 336073
In the test of Example 21 treatment was effected ue to and
including day seven. The test animals however were followed to
day 21. On day 12 in the animals treated with the combination
of 8-aminoadenosine 3',S'-cyclic phosphate and 8-
chloroadenosine 3',S'-cyclic phosphate tumor weight was still
less than that of the controlled animals on day 1, that is
tumor weight at day 12 for the combination of the two compounds
was still less than the initial tumor weight at day one. At
day 21 the combination of 8-aminoadenosine 3',S'-cyclic
phosphate plus 8-chloroadenosine 3',S'-cyclic phosphate
exhibited less rebound activity compared to 8-chloroadenosine
3',S'-cyclic phosphate as an independent drug.
As is evident from the above examples 8-chloroadenosine
3',S'-cyclic phosphate exhibits a broad spectrum of activity
against many human tumor cells, 8-Aminoadenosine 3',S'-cyclic
phosphate exhibits good specificity against human colon tumors
and the combination of 8-aminoadenosine 3',S'-cyclic phosphate
and 8-chloroadenosine 3',S'-cyclic phosphate is not
antagonistic.
For delivery to a host inflicted with a neoplastic disease
8-chloroadenosine 3~,S~-cyclic phosphate and 8-aminoadenosine
3',S'-cyclic phosphate of the invention can be formulated in
various formulations to prepare pharmaceutical compositions
containing 8-chloroadenosine 3',S~-cyclic phosphate, 8-
Aminoadenosine 3',S'-cyclic phosphate or a combination of 8-
chloroadenosine 3',5'-cyclic phosphate and 8-aminoadenosine
3',S'-cyclic phosphate the invention as active ingredients.
The following illustrative examples are given for the
formulations of such pharmaceutical compositions utilizing the
sodium salt of 8-chloroadenosine 3',S'-cyclic phosphate and 8-
aminoadenosine 3',S'-cyclic phosphate.
In these examples, Pharmaceutical Preparative Example 22
illustrates the use of 8-chloroadenosine 3',S'-cyclic phosphate
sodium salt and/or 8-aminoadenosine 3',S'-cyclic phosphate
sodium salt in injectables suitable for intravenous or other
types of injection into the host animal. Pharmaceutical
Preparative Example 23 is directed to an oral syrup
4~ 1 336073
preparation, Pharmaceutical Preparative Example 24 to an oral
capsule preparation and Pharmaceutical Preparative Example 2S
to oral tablets. Pharmaceutical Preparative Example 26 is
directed to use of 8-chloroadenosine 3',S'-cyclic phosphate
sodium salt and/or 8-aminoadenosine 3',S'-cyclic phosphate
sodium salt in suitable suppositories. For Pharmaceutical
Preparative Examples 22 through 26, the ingredients are listed
followed by the methods of preparing the composition.
EXAMPLE 22
I NJECTABLES
8-Chloroadenosine 3',S'-cyclic phosphate
sodium salt or 8-aminoadenosine 3',5'-cyclic
phosphate sodium salt 2S0 mg - 1000 mg
~ater for Injection USP q.s.
The 8-chloroadenosine 3',S'-cyclic phosphate sodium salt
and/or 8-aminoadenosine 3',S'-cyclic phosphate sodium salt is
dissolved in the water and passed through a 0.22~ filter. The
filtered solution i$ added to ampoules or vials, sealed and
sterilized.
-
EXAMPLE 23
SYRUP
2S0 mg Active ingredient/S ml syrup
8-Chloroadenosine 3',S'-cyclic phosphate sodium salt or
8-aminoadenosine 3~,S'-cyclic phosphate sodium salt 50.0 g
Purified l~ater USP q.s. or 200 ml
Cherry Syrup q.s. ad looO ml
46 l 336073
The 8-chloroadenosine 3',5'-cyclic phosphate sodium salt
and/or 8-aminoadenosine 3',5'-cyclic phosphate sodium salt is
dissolved in the water and to this solution the syrup is added
with mild stirring.
EXAMPLE 24
CAPSULES
100 mg 2S0 mg or S00 mg
8-Chloroadenosine 3',~'-cyclic phosphate sodium salt and/or
8-aminoadenosine 3',S'-cyclic phosphate sodium salt
sOo g
Lactose USP, Anhydrous q.s. or 200 g
Sterotex~Powder HM S g
Combine the 8-chloroadenosine 3',S'-cyclic phosphate
sodium salt and/or 8-aminoadenosine 3',S'-cyclic phosphate
sodium salt and the Lactose in a twin-shell blender equipped
with an intensifier bar. Tumble blend for two mînutes,
follow~d by blending for one minute with the intensifier bar
and then tumble blend again for one minute. A portion of the
blend is then mixed with the Sterotex Powder, passed through a
#30 screen and added back to the remainder of the blend. The
mixed ingredients are then blended for one minute, blended for
the intensifier bar for thirty seconds and tumble blended for
an additional minute. Appropriate sized capsules are filled
with 141 mg, 3s2.S mg or 705 mg of the blend, respectively, for
the loo mg, 260 mg and soo mg containing capsules.
~ t r
47 1336073
EX~MPLE 2 S
TABLETS
100 mg, 200mg or 500 mg
8-Chloroadenosine 3',5'-cyclic phosphate
and~or 8-aminoadenosine 3',S'-cyclic phosphate Soo g
Co{n Starch NF 200.0 g
Cellulose Microcrystalline 46 .0 g
Sterotex Powder HM 4 ~ O g
Purified Water q.s. or 300.0 ml
Combine the corn starch. the cellulose and the 8-
chloroadenosine 3',S'-cyclic phosphate and/or 8-aminoadenosine
3~,S'-cyclic phosphate together in a planetary mixer and mix
for two minutes. Add the water to this combination and mix for
one minute. The resulting mix is spread on trays and dried in
a hot air oven at 50 C. until a moisture level of 1 to 2
percent is obtained. The dried mix i~ then milled with a
Fitzmill through a #RH2B screen at medium speed. The Sterotex
Powder is added to a portion of the mix and passed thrugh a #30
screen~ and added back to the milled mixture and the total
blended for five minutes by drum rolling. Compressed tables of
lS0 mg, 37S mg and 7S0 mg respectively. of the total mix are
formed with appropriate sized punches for the loO mg, 2so mg or
S00 mg containing tables.
.
4~
1 336073
ExAMPLE z6
SUPPOSITORIES
2S0 mg, S00 mg or 1000 mg per 3 g
8-Chloroadenosine 3',S'-cyclic phosphate
sodium salt and/or 8-aminoadenosine
3',S'-cyclic phosphate2So mg S00 mg 1000 mg
Polyethylene Glycol192S mg 17S0 mg 1400 mg
lS40
Polyethylene Glycol82S mg 1S0 mg 600 mg
8000
Melt the Polyethylene Glycol lS40 and the Polyçthylene
Glycol 8000 together at 60C. and dissolve the 8-
chloroadenosine 3',S'-cyclic phosphate sodium salt and/or 8-
Aminoadenosine 3',S' cyclic phosphate into the melt. Mold this
total at 2S C. into appropriate suppositories.
