CA1336073C - Treatment of malignant tumors with 8-aminoadenosine 3', 5'-cyclic phosphate and preparation thereof - Google Patents

Abstract

The compound 8-aminoadenosine 3',5'-cyclic phosphate is used to treat malignant tumors in warm blood animals. Two novel single step syntheses of 8-chloroadenosine 3',5'-cyclic phosphate and other related adenine and adenosine compounds from corresponding adenosine 3',5'-cyclic phosphate and other respective related adenosine compounds are also disclosed.

Description

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~

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 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 ~N~

wherein R is H or R2-0-- ~0 < H ~
'~ /'1 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.

N~ N~ NH2 [~ N~

HO--~ 0 ~ Cl HO--~O~

Adenosine 2 NtH2 7~2 ~N~ N~h\>' > G
HO- '-O--~O~ HO- -O--~O~

Na+ \~ ~/ Na+ \`l H OH OH OH

~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 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~).

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 (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.

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 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 ~.

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.

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.

~ 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.

_ 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 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.

-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.
-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 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.

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 -----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 control 334 549 control 204 463 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

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 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 control 29i 376 -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 Dosage Initial Mean ~ First-Last Change in Mean (mg/kg/day) Weight (mg)Tumor Weight2 Tumor Weight3 ~T/~C

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 control 361 726 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 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 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 control 332 SlS

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

62 350 - 1 - o.33 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

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.

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

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

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 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

1 33~73 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

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

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

13 SlS 119 S8 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.

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 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.

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 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.

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

~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.

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 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

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~

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-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.

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.
-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.

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 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~

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 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.

Claims (9)

1. An antitumor composition for the treatment of tumors in vivo containing as its active ingredient an effective amount of the compound 8-aminoadenosine 3',5'-cyclic phosphate or a pharmaceutically acceptable salt thereof in association with a pharmaceutically acceptable diluent or carrier.

2. A pharmaceutical composition according to claim 1 in a form suitable for administration to a warm blooded animal by injection.

3. A pharmaceutical composition according to claim 1 in a form suitable for adminstration to a warm blooded animal by infusion of said composition into said warm blooded animal.

4. A pharmaceutical composition in a dosage form suitable for administration in an amount so as to deliver an amount of from about 13 mg/kg/day to about 104 mg/kg/day of said 8-aminoadenosine 3',5'-cyclic phosphate to said warm blooded animal.

5. A commercial package comprising a pharmaceutically effective amount of 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.

6. Use of a pharmaceutically effective amount of a pharmaceutical composition comprising a pharmaceutically acceptable diluent or carrier and containing as the active component at least about 0.1 percent by weight, based on the total weight of the composition, of 8-aminoadenosine 3',5'-cyclic phosphate or a pharmaceutically acceptable salt thereof to treat a tumor in a warm blooded animal.

7. The use of claim 6 wherein: said pharmaceutical composition is in a form suitable for administration to said warm blooded animal by injection.

8. The use of claim 6 wherein: said pharmaceutical composition is in a form suitable for administration to said warm blooded animal by infusion of said composition into said warm blooded animal.

9. The use of claim 6 wherein: said pharmaceutical composition is in a dosage form suitable for administration in an amount so as to deliver an amount of from about 13 mg/kg/day to about 104 mg/kg/day of said 8-aminoadenosine 3',5'-cyclic phosphate to said warm blooded animal.