TREATMENT OF MALIGNANT TUMORS WITH 8-CHLOROADENOSINE 3',5'-CYCLIC PHOSPHATE, 8-AMINOADENOSINE 3',5'-CYCLIC PHOSPHATE AND PREPARATION THEREOF

Abstract: The compounds 8-chloroadenosine 3',5'-cyclic phosphate and 8-aminoadenosine 3',5'-cyclic phosphate are used together or separately to treat malignant tumors in warm blooded 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 disclosed.

Description: TITLE: TREATMENT OF MALIGNANT TUMORS WITH 8-CHLOROADENOSINE 3',5'-cyclic PHOSPHATE, 8-AMINOADENOSINE 3 » ,5'-cyclic PHOSPHATE AND PREPARATION THEREOF BACKGROUND OF INVENTION This invention is directed to treating malignant tumors in ivo utilizing the compounds 8-chloroadenosine 3',5'-cyclic phosphate and/or 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 synthesis. 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 confirm 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 Institute of Health, National Cancer Institute. In contrast to this report, in other studies 8-bromoadenosine 3',5'-cyclic phosphate has been found to be inactive as an antitumor agent in cell culture. Contemporaneously with the above report, Y.S. Cho-Chung, -2.-. Cyclic Nuclgoti.de Res_j. j[: 163, 1980, reported certain investigative studies on an antagonistic interaction between estrogen and adenosine 3', 5'-cyclic onophosphate (hereinafter alternately referred to as cAMP) and what role this might have in the control of growth of hormone-dependent mammary tumors. In studing the effects of mediated control of tumor growth by adenosine 3',5'-cyclic phosphate, Cho-Chung has suggested that cAMP functions by binding to a cAMP receptor protein which has two different cAMP binding sites. The cAMP receptor protein is a regulatory subunit of a cAMP dependent protein kinase. There apparently is site 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 chemothera- peutics to successfully control all neoplastic diseases, it is evident that there exists a need for new and additional cancer che otherapeutic agents. Further there exist a need for new and better preparative procedures for the syntesis 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, JX J . Garbohydr. 3Su£lsosid£s 3-lJ-lcl------..id£S, 1, 55, 1974. 8-Aminoadenosine 3',5'-cyclic phosphate was first reported by inventer R. K. Robins of this invention and other co-authors K. Muneyame et al., Sio£fce_mis £y., 10, 2390, 1971. It has now been found that 8- chloroadenosine 3',5'-cyclic phosphate (hereinafter alternately also identified as 8-chloro cAMP), and 8-aminoadenosine 3',5'- cyclic phosphate (hereinafter alternately also identified as 8- amino cAMP) together or separtely 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 present invention relates to the use of 8-chloro- adenoεine 3',5'-cyclic phosphate (8-chloro cAMP) and 8- aminoadenosine 3',5'-cyclic phosphate (8-amino cAMP) together or separately in treating malignant tumors in warm blooded animals. According to this invention the antitumor properties of 8-chloroadenosine 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 θ.ι < ?b by weight based on the total weight of the composition. 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, 8-chloroadenosine 3',5'-cyclic phosphate and/or 8-aminoadenosine 3 ',5 '-cyclic phosphate of the invention could be administered intraveneously, intramus¬ cularly, 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 invention includes improved processes for the preparation of 8-chloro derivatives of adenine, adenosine, adenosine 5'- onophosphate and adenosine 3',5'-cyclic phosphate and related compounds. In these improved processes 8- chlor oadenine, 8-chloroadenosine, 8-chloroadenosine 5'- monophosphate and 8-chloroadenosine 3',5'-cyclic phosphate or other related adenosine compounds are prepared directly from corresponding respective adenosine precursor compounds by processes for preparing chloro compounds of the formula wherein R is H or OH i i • wherein R 1 and R 2 are H or 0=P- or together R χ and R 2 are 0=P-, OH OH and R 3 and R 4 are H or one of R 3 or R 4 is OH and the other is H, and pharmaceutically acceptable salts thereof which comprise the steps of treating a starting compound of the formula OH I wherein R j and R 2 are H or 0=P- or together R j and R 2 are 0=P-, OH OH and R 3 and R 4 are H or one of R 3 or R 4 is OH and the other is H: either, 1) with N-chlorosuccinimide and a weak acid in a suitable solvent or 2 ) with hydrogen chlor ide and an oxidiz ing agent in a suitable solvent. DETAILED DESCRIPTION OF THE INVENTION 8-Aminoadenosine 3 ',5 '-cyclic phosphate of the invention is prepared as is described in the above referenced paper, K. Mumeya a et al. Bioc£ιe.mist, £y_, 10, 2390, 1971. 8- Chloroadenosine 3',5'-cyclic phosphate of the invention can be prepared as is described in above referenced paper, K. Muneya a et al, J. Carbohydr. Nucleosides £cle.o t i d≤s , 1, 55 1974, via an 8-bromo intermediate compound, however, by utilizing new and improved processes as discussed herein, 8-chloroadenosine 3',5'-cyclic phosphate and related compounds are obtained directly from respective adenosine precursors avoiding having to synthesis intermediate compounds. As such, improvements in the economy of the synthesis are obtained. In the first of these improved processes adenosine 3 ',5'- cyclic phosphate is directly chlorinated to 8-chloroadenosine 3',5'-cyclic phosphate utilizing N-chlorosuccinimide and a suitable weak acid in a suitable solvent. Suitable as the weak acid is acetic acid. Alternatively formic acid or other weak organic acid might be used. In choosing the acid, consideration is given whereby the acid is of such a strength that it is sufficiently weak so as not to cleave the sugar - heterocycle glycosidic bond (the Cl'-N9 bond). Suitable as the solvent are dimethylacetamide, dimethylf orma ide or aqueous medium. In the second of these improved processes adenosine 3 ',5'- cyclic phosphate is also directly chlorinated to 8-chloro¬ adenosine 3 ',5 '-cyclic phosphate utilizing anhydrous hydrogen chloride and a suitable oxidizing agent such as m-chloroperoxy- benzoic acid. This reaction is conduc edin a suitable solvent as for example dimethylacetamide or dimethylf ormamide. As an alternative oxidizing agent, sodium hypochlorite can be mentioned. Aside from the preparation of 8-chloroadenosine 3', 5'- cyclic phosphate, both of these processes can be utilized to prepare 8-chloroadenosine from adenosine or 8-chloroadenosine 5'-phosphate from adenosine 5'-phosphate. Further, if the hydrogen chlor ide/m-chloroperoxybenzoic acid reaction is carried out in the presence of moisture, it can be utilized to prepare 8-chloroadenine from adenosine. Additionally, these two novel processes might also be utilized to convert suitable derivatives of adenosine, adenosine 5'-phosphate, adenosine 3 '-phosphate and adenosine 3',5'-cyclic phosphate, to their respective 8-chloro derivatives. Such derivatives would include the 2-deoxy-β-D- er.y_th.ro -pent of uranosyl sugar derivative and the 2-β-D- arabinof uranosyl sugar derivative and might also include substituents on the purine amino group, the 2 position of the purine ring, sugar hydroxyl groups or even other heterocyclic moieties such as a deaza purine or the like. The synthesis of 8-chloroinosine 3',5'-cyclic phosphate, 8-chloroinosine 5 '-phosphate and 8-chloroinosine is facilitated utilizing these processes in that the respective 8-chloro¬ adenosine compounds can then be directly dea inated utilizing nitrous acid to respective 8-chloroinosine derivatives. For this reaction, the nitrous acid can be generated in situ from sodium nitrite and acetic acid. Thus 8-chloroinosine and its 5'-phosphate and 3',5'-cyclic phosphate derivatives are synthesised in a straight forward two step reaction scheme. In the following examples of the novel processes of the invention as is illustrated in scheme I, an adenosine compound, e.g. adenosine, AMP or cAMP, is directly chlorinated in the 8 position of its purine ring to an appropriate 8-chloroadenosine or adenine compound. Thus adensoine yields compounds l and 2, AMP yields compound 1 and cAMP yields compound ±. This is achieved using one, the other or both of the two novel processes. One process uses hydrogen chloride and ni-ohloro- peroxybenzoic acid in an appropiate solvent such as dimethylacetamide or dimethylf oramide. The other process uses N-chlorosuccinamide and acetic acid in an appropriate solvent such as dimethylacetamide, dimethylf ormamide or an aqueous acetic acid. Any of the 8-chloro compounds thus formed can then be further converted in a further steo to their 8- chloroinosine analogs as for instance the conversion of 8- chloroadenosine 3',5'-cyclic phosphate, compound 4_, to 8- chloroinosine 3',5'-cyclic phosphate, compound 5.. In the preparative examples below, melting points were taken on a Thomas-Hoover capillary melting point apparatus or on a Haake-Buchler digital melting point apparatus and are uncorrected. Nuclear magnetic resonance ( H NMR) spectra were determined at 300.1 MHz with an IBM NR300AF spectrometer. The chemical shifts are expressed in δ values (parts per million) relative to tetramethylsilane as internal standard. Ultra violet spectra (UV: sh = shoulder) were recorded on a Beckman DU-50 spectrophotometer. Elemental analyses were performed by Robertson Laboratory, Madison, N.J. Evaporations were carried out under reduced pressure with the bath temperature below 40°C. Thin layer chromatography (TLC) was run on silica gel 60 F-254 plates (EM Reagents). E. Merck silica gel (230-400 mesh) was used for flash column chromatography. cAMP SCHEME I EXAMPLE 1 8-Chloroadenine (_L). To a solution of adenosine (2.67 g, 10 mmol) in DMA/HC1 (0.5 M, 45 mL) was added m-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, 5 mmol) was added and stirring continued for another 1 h. Toluene (50 mL) was added to the reaction mixture and the solvents evaporated at 60°C under vacuo to dryness. The residue was dissolved in water (50 mL) and extracted with ether (3 x 50 mL). The pH of the aqueous phase was adjusted to 5 with 2N NaOH and then diluted with EtOH (100 L). The solution was stored in the refrigerator overnight. The light yellow solid that separated was collected, washed with cold EtOH (2 x 25 mL) and dried to give 1.44 g (85.2%) of 1 : mp 305-310<C (dec.) [Lit. mp >300°C (dec.)]: IR (KBr): 630 (C-Cl), 3100-3300 (NH 2 ) cm -1 : UV: λ maχ (pH 1) 262 nm (ε 8,700): λ maχ (ph 7) 268 nm (ε 7,900): λ maχ (pH 11) 269 nm (ε 8,300): X H NMR (Me 2 S0-d g ) : δ 7.48 (br S, 2, NH 2 ), 8.10 (s, l, C 2 H) and 13.60 (br s, 1, N 9 H). EXAMPLE 2 8-Chloroadenosine (.2) . Metfaod A. To a solution of adenosine (2.67 g, 10 mmol, dried at 80 β C under vacuum) in DMA/HC1 (0.5 M, 25 mL) was added rapidly a solution of purified MCPBA (3.09 g, 18 mmol) in DMA (20 mL) (adenosine will precipitate out from the reaction mixture if the MCPBA solution is 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: AcOH:H 2 0 (18:1:81, v/v) to give 1.25 g (41%) Of 2: mp 188-189°C [Lit. mp 188-190°C]: IR (KBr): 795 (C-Cl) . 3150-3400 (NH 2 , OH) cm "1 : UV: λ maχ (pH 1) 261 nm (ε 17,100): λ max ( P H 7) 262 nrn (ε 17,800 ) : λ maχ ( pH 11 ) 263 nm ( ε 16,700 ) : -Ε. NMR (Me 2 SO-d 6 ): δ 5.83 (d, 1, J χ , 2 , = 7.0 Hz, C-^H), 7.59 (br s, 2, NH 2 ) and 8.14 (s, 1, C 2 H) . Method B. To a solution of adenosine (1.09 g, 4.1 mmol) in DMF (50 mL) and AcOH (10 mL) was added N-Chlorosuccinamide (NCS, 2.0 g, 15 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:H 2 0 (18:1:81, v/v) to give 0.8 g (65%) of 2, which was identical to the title compound prepared by M≤thod A. EXAMPLE 3 8-Chiproadenosine. 5 '-monophosphate (3.). M≤thod A. To a solution of adenosine 5 '-monophosphate (1.3 g, 3.5 mmol) in dry DMF (75 mL, distilled over CaH 2 ) was added dropwise a solution of DMF saturated with anhydrous HC1 (5 mL) , followed by a solution of purified MCPBA (l.l g, 6.4 mmol) in DMF (10 mL) . The reaction mixture was stirred at room temperature for 2 1/2 h, before the DMF was evaporated to dryness under vacuo at 45°C. 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 0.5% aqueous AcOH to furnish 0.64 g (45%) of 3.: mp 180°C. IR (KBr): 635 (C-Cl), 3100-3400 (NH 2 .OH) cm "1 : UV: λ maχ (pH 1) 260 nm (ε 10,800): λ maχ (pH 7) 262 nm (ε 10,400): λ maχ (pH 11) 261 nm (ε 10,000): *H NMR (Me 2 S0-d g ): δ 5.86 ( d, 1, lt 2 » = 6 -° Hz ' c ι»B ) » 7-50 ( br s, 2, NH 2 ) and 8.16 (s, 1. C 2 H). Method B. Adenosine 5'-monophosphate monohydrate (3.11 g, 8.5 mmol), NaCl (3.11 g, 53 mmol) and NCS (3.33 g, 25 mmol) were dissolved in 50% aqueous AcOH (100 mL) and the solution was stirred at room tempeature for 5 days. Evaporation of the reaction mixture and purification of the residue by HPLC on a C-18 reverse phase column using 0.5% aqueous AcOH gave 2.30 g (60%) of the title compound, which -&ks identical to i prepared by Method B. EXAMPLE 4 8-Chloroadenosine 3'.5'-cγclic 2liQsp.hate (.4) . Me-.------.--l £• A mixture of dry 8-bromo-cAMP (20 g, 49 mmol) and CaCl 2 (20 g, 180 mmol, dried at 75° under vacuum overnight) in anhydrous DMF (800 L, distilled over CaH 2 under vacuum) was heated at 80-85 °C under anhydrous conditions with stirring for 15 h. DMF was evaporated under reduced pressure at 50°C and the residue was dissolved in cold 2N NaOH (20 L). The aqueous solution was neutralized (to pH 7) with cold 2N HC1, filtered through a membrane filter and purified by preparative HPLC on a C-18 reverse phase column. Initial washings with 0.5% AcOH/H 2 0 gave 8-hydroxy-cAMP and cAMP. Further elution with 15% MeOH/H 2 0 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, p 232-234 « C: IR (KBr): 655 (C-Cl), 3280 (NH 2 » OH) cm "1 : UV: λ maχ (pH 1) 260 nm (ε 15,600): λ maχ (pH 7) 261 nm (ε 15,700): λ maχ (pH 11) 261 nm (ε 15,900): 1 E NMR (Me 2 SO-d 6 ) : δ 4.13 (m, 2, C 5 ,H 2 ), 4.56-4.39 (m, 1, C 4 ,H), 4.99-4.97 and 5.17-5.13 (m, 2, C 2 ,H and C 3 ,I), 5.85 (s, 1, C j H , 7.67 (br s, 2, NH 2 ) and 8.21 (s, 1, C 2 H). Anal. Calcd for C 10 H 11 C1N 5 0 6 P.H 2 0: C, 31.47: H, 3.43: N, 18.35: CI, 9.29. Found: C, 31.69: H, 3.19: N, 18.26: CI, 9.52. Method A. To a solution of adenosine 3',5'-cyclic phosphate (1.15 g, 3.5 mmol) in dry DMF (75 mL, distilled over CaH 2 ) is added dropwise a solution of DMF saturated with anhydrous HC1 (5 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 vacuo at 45°C. The residue is then dissolved in minimum amount of water (~lθ 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 0.5% aqueous AcOH to furnish 4, identical to £ prepared by Mgtfrpd C. Method B. To a solution of cAMP (30.3 g, 92 mmol) and NaCl (30.6 g, 52 mmol) in AcOH (250 mL) and H 2 0 (150 mL) was added NCS (30.3 g, 227 mmol). The reaction mixture was stirred at room temperature for 3 days. The solvents were evaporated to dryness under reduced pressure and the residue purified as described above in Method A to yield 13.0 g (39%) of ± , which was identical to prepared by Method C. EXAMPLE 5 IzCllloxo nssine 3'.5'-cyclic phosphate (5.). To a suspension of 8-chloroadenosine 3',5'-cyclic phosphate (4, 1.0 g, 2.7 mmol) in H 2 0 (3 L) was added 2N NaOH dropwise until a clear solution was obtained. NaN0 2 (1.05 g, 15 mmol) was added to the reaction solution, followed by dropwise addition of AcOH (2 mL). The reaction mixture was stirred at room temperature overnight and then evaporated to dryness under reduced pressure at 35°C. The residue was purified on a Dowex 50 x 2 - 200 (H + ) resin column (5 x 20 cm) eluting with H 2 0. The homogeneous fractions containing 8- chloro-cIMP were pooled and evaporated to dryness. Co- evaporation of the residue with EtOH (3 x 25 mL) gave a white solid, which after drying at 78°C under vacuum for 15 h afforded 0.56 g (56%) of 5: mp 216-218°C: IR (KBr): 785 (C-Cl), 1680 (C=0), 3200-3400 (OH) cm "1 : UV: λ maχ <pH 1) 251 n (ε 17,100): λ maχ (pH 7) 251 nm (ε 17,000): λ maχ (pH 11) 255 nm (ε 17,300): 1 H NMR (Me 2 SO-d g ): δ 5.85 (s. 1, C^H) , 8.17 (s, 1, C 2 H), and 12.71 (br s, 1, χ H . Anal- Calcd for C 10 H 10 ClN 4 O 7 P): C, 32.94: H, 2.76: N, 15.37. Found: C, 32.75: H, 2.69: N, 15.14. For use in pharmaceutical preparations of the invention normally a salt of the 3 ',5 '-cyclic phosphate moiety of either 8-chloroadenosine 3',5'-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- al lyammonium, dialkylammonium, alkylam onium, 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',5'-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. 8-Chloro- adenosine 3 ',5 '-cyclic phosphate and/or 8-aminoadenosine 3 ',5'- cyclic phosphate might be appropriately used in the presence of a suitable buffer or as a salt as discussed above. The compounds of the invention 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. Preferredly, 8-chloroadenosine 3',5'-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',5'-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',5'-cyclic phosphate of the invention could be used where indicated. The pharmaceutical carrier, in addition to naving a solubilizing or suspending agent therein, might also include suitable dilutants, buffers, surface active agents and other similar agents 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',5*-cyclic phosphate and/or 8- aminoadenosine 3',5'-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 10% to about 90% by weight of the total composition. Based on present studies, effective amounts of 8-chloro¬ adenosine 3',5'-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',5'-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 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', 5'- cyclic phosphate and 8-aminoadenosine 3',5'-cyclic phosphate 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',5'-cyclic phosphate and 8- aminoadenosine 3',5'-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',5'-cyclic phosphate and 8- a inoadenosine 3',5'-cyclic phosphate exhibit synergistic activity. Singularly 8-chloroadenosine 3', 5 '-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',5'-cyclic phosphate exhibits significant activity against colon carcinoma. The combination of the compounds 8-chloroadenosine 3 ',5 '-cyclic phosphate and 8-aminoadenosine 3',5'-cyclic phosphate also exhibits significant activity against colon carcinoma, however, the activity of the combination of 8-chloroadenosine 3',5'-cyclic phosphate and 8-aminoadenosine 3*,5'-cyclic phosphate is greater than the activity of 8-chloroadenosine 3',5'-cyclic phosphate when used alone. When the compounds 8-chloroadenosine 3',5'-cyclic phosphate and 8-aminoadenosine 3',5'-cyclic phosphate are utilized in the dose ranges above but are utilized together, the activity is enhanced compared to 8-chloroadenosine 3',5'- cyclic phosphate when used independently. Further this enhanced activity is maintained beyond a post treatment period. Additionally with the combination of the two compounds 8- chloroadenosine 3',5'-cyclic phosphate and 8-aminoadenosine 3',5'-cyclic phosphate there is decreased drug rebound after discontinuous of the drug. The 8-chloroadenosine 3',5'-cyclic phosphate and/or 8- aminoadenosine 3',5'-cyclic phosphate of the invention can be given as single doses or as multiple doses divided into sub- doses given daily or over a period of days. As will be evident from the examples below. 8-chloroadenosine 3',5'-cyclic phosphate and 8-aminoadenosine 3',5'-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', 5'- cyclic phosphate and the combination of 8-chloroadenosine 3',5'-cyclic phosphate plus 8-aminoadenosine 3',5'-cyclic phosphate of the invention as a therapeutic agents against neoplastic diseases. In these examples the efficacy of 8- chloroadenosine 3',5'-cyclic phosphate and 8-aminoadenosine 3 ',5 '-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 In itro Cancer Modgls, National Institute of Health Publications No 84-2635, 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 forth 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 utilized as follows: i.p. - intraperitoneal: qd - once a day: and mg/kg/day - milligrams per kilograms per day. In the example utilizing L1210 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 125% is considered as having statically meaningful activity. For 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 ΔT/ΔC. However, if there was a net negative change in tumor weight the results are expressed as ΔT/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',5'-cyclic phosphate as a sodium salt was tested utilizing non-tumor bearing BDF j 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 1. As is evident from Table 1 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 100 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 of drug before lethal toxicity was seen. Table l. Influence of 8-Cl-cAMP Na + on the life span of non- tumor DF.^ mice when delivered i.p. by bolus injection Dosage Route and schedule Toxic deaths mg/kg/inj of delivery No killed/No treated 480 5/5 288 5/5 173 2/5 104 0 62 0 37 0 22 0 1. When delivered qd, day 1 to non-tumor BDF j mice, the 480 and 288 mb/kg dosages of S-Cl-cAMP Na + were lethally toxic for all treated mice. The 173 mg/kg dosage killed 2 of 5 mice and lower dosages were not lethally toxic. EXAMPLE 7 The activity of 8-chloroadenσsine 3',5'-cyclic phosphate against L1210 inoculated BDF j 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',5'-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',5'-cyclic phosphate is an effective antitumor agent against L1210 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 125 indicates significant activity. Tabl e 2. In f luenc e of 8 -Cl -cAMP Na + on the pos t inoculat i on lifespan of L12 l 0-inoculated BDF j mice when infused oi del ivered by bolus inj ection Dosage Route and schedule Postinoculation (mg/kg/day) of delivery liffreessopaa]n (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 1-5 62 toxic .3 480 ip: 24-hr infusion, day 1-5 93 toxic3 288 ip: 24-hr infusion, day 1-5 128 toxic 3 173 ip: 24-hr infusion, day 1-5 131 104 ip: 24-hr infusion, day 1-5 137 62 ip: 24-hr infusion, day 1-5 137 37 ip: 24-hr infusion, day 1-5 126 22 ip: 24-hr infusion, day 1-5 126 13 ip: 24-hr infusion, day 1-5 113 48 ip: 24-hr infusion, day 1-5 100 17 ip: 24-hr infusion, day 1-5 100 0.6 ip: 24-hr infusion, day 1-5 100 1. Mice were inoculated i.p. with 1x10° cells of murine leukemia L1210 24-hr before first treatment. Each treatment group consisted of 5 mice. Twenty control mice that received a 0.9«Ϊ solution of NaCl lived 6.2 + days. 2. Significant activity indicated at T/C > 125. 3. When infused, the 800 and 480 mg/kg dosages of 8-Cl-cAMP Na + were lethally toxic for all treated mice while the 288 mg/kg dosage killed 2 of 5 mice. 8-Chloroadenosine 3',5'-cyclic phosphate was tested against a variety of solid human tumor cell lines. In the tests of examples 8 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 Nation Cancer Institute protocol procedures for respective solid tumors cell lines which were tested. EXAMPLE 8 In this example, 8-chloroadenosine 3',5'-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 ΔT/T. In the study shown in the lower portion of Table 3a, at lower dose levels, the Δ 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 ΔT/ΔC. Any value for ΔT/ΔC or ΔT/T which is below 25 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. Table 3a. Influence of intraperitoneally infused 8-Cl-cAMP Na " on the growth in athy iS miiccee oof human mammary carcinoma 37 194 16 22 201 76 29' 13 216 95 37 ! 0 204 259 1. Tumor fragments (~ 14 mg) were implanted subcutaneously in the thigh region of athymic CD-I 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 ΔT/ΔC or ΔT/T. 3. ΔT/ΔC utilized for positive First-Last tumor weights and ΔT/T utilized for negative First-Last tumor weights. 4. ΔT/T 5. ΔT/ΔC 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-1 tumors in athymic mice intraperitoneally infused with 8-Cl-cAMP Ka + Dosage Initial Mean (mg/kg/day) Weight (mg) Weight (mg) Day i Day 8 336 317 288 549 210 277 311 463 EXAMPLE 9 8-chloroadenosine 3',5'-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 ΔT/ΔC for the positive value or ΔT/T for the negative values as was discussed with respect to Example 8 above. As is evident at the. 3 / 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 LoVo 1 Dosage Initial Mean Δ First-Last Change in Mean (mg/kg/day) Weight (mg) Tumor Weight' Tumor Weight 3 (ΔT/ΔC or ΔT/T) 104 288 29 35' 62 293 -3 - 1.0 : 37 288 -4 - 1.4 ; 0 292 84 1. Tumor fragments (~ 14 mg) were implanted subcutaneously in the thigh region of athymic CD-I 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 ΔT/ΔC or ΔT/T. 3. ΔT/ΔC utilized for positive First-Last tumor weights and ΔT/T utilized for negative First-Last tumor weights. 4. ΔT/ΔC 5. ΔT/T 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 LoVo tumors in athymic mice intraperitoneally infused with 8-Cl-cAMP Na + Dosage Initial Mean Mean Tumor (mg/kg/day) Weight (mg) Weight (mg) Day l Day 8 317 290 284 376 EXAMPLE 10 8-chloroadenosine 3',5'-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 5a 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. Table 5a. Influence of intraperitoneally infused 8-Cl-cAMP Na + on the growth in athymic mice of human mammary carcinoma MDA-MB-231 1 Dosage Initial Mean Δ First-Last Change in Mean (mg/kg/day) Weight (mg) Tumor Weight Tumor Weight ΔT/ΔC 104 367 66 18 62 375 96 26 37 367 149 41 0 361 365 1. Tumor fragments (~ 14 mg) were implanted subcutaneously in the thigh region of athymic CD-I 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 ΔT/ΔC or ΔT/T. 3. ΔT/ΔC utilized for positive First-Last tumor weights. The mean tumor weight for each dose shown in Table 5a for all the test animals at days l and 8 are indicated in Table 5b. Table 5b. Tumor weights of human mammary carcinoma MDA-MB- 231L ttuurnmo.rs in athymic mice intraperitoneally infused with 8-C1- c MP Na ' + Dosage Initial Mean Mean Tumor (mg/kg/day) Weight (mg) Weight (mg) Day l Day 8 104 367 433 62 375 471 37 367 516 control 361 726 EXAMPLE 11 8-chlor oadenos ine 3 ' , 5 '-cyclic phosphate was further tested in athymic mice against human lung carcinoma LX-1. As per the results shown in Table 6a below, the compound exhibited sign ificant activity at a dose range of from 37 mg to 104 mg/kg /day. For this test infusion was only for 5 days as opposed to the 7 days used for the previous solid tumor tests of Examples 8 , 9 and 10. Further , as pe r Example 8 the positive mean tumor weights results are shown as ΔT /ΔC and negative mean tumor weights as ΔT/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 regress ion. Table 6a. Influence of intraperitoneally infused 8-Cl-cAMP Na + on the growth in athymic mice of human lung carcinoma LX-1 1 Dosage Initial Mean Δ First-Last Change in Mean (mg/kg/day) Weight (mg) Tumor Weight Tumorr WWeeiicght 3 ΔT/ΔC 104 324 -54 -16.7' 62 327 -50 -15.3' 37 330 11 0 332 183 1. Tumor fragments (~ 14 mg) were implanted subcutaneously in the thigh region of athymic CD-I female mice. Three weeks later the tumors were staged and treatment lasting 5 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 ΔT/ΔC or ΔT/T. 3. ΔT/ΔC utilized for positive First-Last tumor weights and ΔT/T utilized for negative First-Last tumor weights. 4 . ΔT/T 5 . ΔT /ΔC The mean tumor weight for each dose shown in Table 6a for all the test animals at davs l and 8 are indicated in Table 6b. Table 6b. Tumor weights of human lung carcinoma LX-1 tumors in athymic mice intraperitoneally infused with 8-Cl-cAMP Na Dosage Initial Mean Mean Tumor (mg/kg/day) Weight (mg) Weight (mg) Day 1 Day 8 104 324 270 62 327 277 37 330 341 control 332 515 EXAMPLE 12 8-Chloroadenosine 3',5'-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. Table 6a. Influence of intraperitoneally infused 8-Cl-cAMP Na + on the growth in athymic mice of colon carcinoma MeC-1 Dosage Initial Mean Δ First-Last Change in Mean (mg/kg/day) Weight (mg) Tumor Weight' Tumor Weight ΔT/ΔC 104 356 -47 -13 3 62 350 - 1 - 0.3 3 37 339 18 14 0 335 133 1. Tumor fragments (~ 14 mg) were implanted subcutaneously in the thigh region of athymic CD-I 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 ΔT/ΔC. In instances of net tumor loss, ΔT/ΔT was calculated in lieu of ΔT/ΔC. 3. ΔT/T EXAMPLE 13 8-Aminoadenosine 3',5'-cyclic phosphate as a sodium salt was tested utilizing nontumor bearing BDF j mice to establish 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 173 mg/kg and 288 mg/kg there was lethal toxicity for 1/5 and 5/5 mice respectively. When 8-aminoadenosine 3',5'-cyclic phosphate was delivered 24 hr/day for 5 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-NH 2 -cAMP Na ÷ on the life span of non- tumor BDF j mice when delivered i.p. by bolus injection Dosage Route and schedule Toxic deaths g/kg/inj of delivery No killed/No treated 288 ip: qd, day 1 5/5 173 ip: qd, day 1 1/5 104 ip: qd, day 1 0 The activity of 8-aminoadenosine 3',5'-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 In this example 8-aminoadenosine 3',5'-cyclic phosphate 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-1 Dosage Initial Mean Δ First-Last Change in Mean (mg/kg/day) Weight (mg) Tumor Weight' Tumor Weight ΔT/ΔC 22 383 -49 -13 3 13 337 -10 - 3 3 392 142 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 ΔT/ΔC. In instances of net tumor loss, ΔT/T was calculated in lieu of ΔT/ΔC. 3. ΔT/T EXAMPLE 15 8-Aminoadenosine 3',5'-cyclic phosphate was further tested in athymic mice against 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- AMI NO -c AMP Na ÷ on the growth in athymic mice of human colon carcinoma HT-29 1 Dosage Initial Mean Δ First-Last Change in Mean (mg/kg/day) Weight (mg) Tumor Weight Tumorr WWeeiicght ΔT/ΔC 37 326 -27 - 8.0 22 346 -18 - 5.0 : 0 356 78 1. Tumor fragments (~ 14 g) were implanted subcutaneously in the thigh region of athymic CD-I 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 measurements recorded on the initial day of treatment (staging day) and again on day 7 were used to calculate mean tumor weight changes and ΔT/ΔC. In instances of net tumor loss, ΔT/T was calculated in lieu of ΔT/ΔC. 3. ΔT/T EXAMPLE 16 8-Aminoadenosine 3',5'-cyclic phosphate was further tested in athymic mice against human colon carcinoma CX-1. 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 15 with the exception that the CX-1 tumor line was evolved in vivo while the HT-29 tumor line was evolved in ϊi £θ. The results of this test are shown in Table ll below. As is evident from Table 11 the compound showed greater activity at a lower dose level at 8 mg/kg/day. While we do not wish to be bound by theory at this time it is believed that host toxicity to 8-aminoadenosine 3',5'-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- a inoadenosine 3',5'-cyclic phosphate exhibited particularly significant activity against human colon carcinomas. Table 11 . Influence of intraper itoneally infused 8-AMINO-cAMP Na on th e g r o wth i n athym ic m i c e o f hum an c o l on carcinoma CX-1 1 Dosage Initial Mean Δ First-Last Change in Mean (mg/kg/day) Weight (mg) Tumor Weight' Tumor Weight ΔT/ΔC 22 398 31 24 13 396 30 23 8 397 -8 -2 0 398 130 1. Tumor fragments (~ 14 mg) were implanted subcutaneously in the thigh region of athymic CD-I 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 ΔT/ΔC. In instances of net tumor loss, ΔT/T was calculated in lieu of ΔT/ΔC. 3. ΔT/T EXAMPLE 17 8-Aminoadenosine 3 r ,5'-cyclic phosphate was further tested in athymic mice against human lung carcinoma LX-l. 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 below . Table 12. Influence of intraperitoneally infused 8-AM NO-cAMP Na + on the growth in athymic mice of human lung carcinoma LX-l 1 Dosage Initial Mean Δ First-Last Change in Mean (mg/kg/day) Weight (mg) Tumor Weight' Tumor Weight ΔT/ΔC 22 475 78 38 13 515 119 58 0 484 204 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 5 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 ΔT/ΔC. EXAMPLE 18 In a l ike manner to example 17 8-aminoadenosine 3 ' , 5 '- cyclic phosphate was further tested in athymic m ice aga inst human lung carc inoma LX-l with treatment lasting f or seven days. The results in this test are shown in Table 13 below. Table 13 . Influence of intraperitoneally infused 8-AMINO-cAMP Na + on the g r ow th in athym ic m ic e o f human l ung carcinoma LX-1 1 Dosage Initial Mean Δ First-Last Change in Mean (mg/kg/day) Weight (mg) Tumor Weight Tumorr WWeeiight ΔT/ΔC 37 564 199 50 22 560 139 35 13 565 208 53 0 577 396 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 ΔT/ΔC. 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 protocol are shown in Tables 14a and 14b below. Table 14a. Influence of intraperitoneally infused 8-AMINO-cAMP Na + t + on the growth in athymic mice of human mammary carcinoma MX-l1 Dosage Initial Mean Δ First-Last Change in Mean (mg/kg/day) Weight (mg) Tumor Weight' Tumor Weight ΔT/ΔC 22 253 156 79 13 256 167 85 8 253 194 98 0 252 197 1. Tumor fragments (~ 14 mg) were implanted subcutaneously in the thigh region of athymic CD-I 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 ΔT/ΔC. Table 14b. Influence of intraperitoneally infused 8-AMINO-cAMP Ma + on the growth in athymic mice of human mammary carcinoma MX-l 1 Dosage Initial Mean Δ First-Last Change in Mean (mg/kg/day) Weight (mg) Tumor Weight" Tumorr WWeeiicght ΔT/ΔC 37 305 93 45 22 309 97 47 13 311 171 83 0 326 207 1. Tumor fragments (~ 14 mg) were implanted subcutaneously in the thigh region of athymic CD-I 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 ΔT/ΔC. EXAMPLE 20 8-Aminoadenosine 3 ',5 '-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 15 below. Table 15. Influence of intraperitoneally infused 8-AMINO-cAMP Na + on the growth in athymic mice of human colon carcinoma LOVO 1 Dosage Initial Mean Δ First-Last Change in Mean (mg/kg/day) Weight (mg) Tumor Weight' Tumor Weight ΔT/ΔC 22 263 130 76 13 326 98 58 8 330 153 90 0 326 170 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 ΔT/ΔC. The combination of 8-chloroadenosine 3',5'-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. 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 therapy was compared to administration of 8-chloroadenosine 3', 5 '-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 of this test in Table 16 below the combination of 8-aminoadenosine 3',5'-cyclic phosphate and 8- chlor oadenosine 3',5'-cyclic phosphate showed increased activity compared to administration of 8-chloroadenosine 3 ',5'- cyclic phosphate as an independent drug. This is indicative that 8-aminoadenosine 3',5'-cyclic phosphate and 8- chloroadenosine 3 ',5 '-cyclic phosphate are not antagonists. It is further believed that 8-aminoadenosine 3',5'-cyclic phosphate and 8-chloroadenosine 3 ',5 '-cyclic phosphate might be synergistic in their action as antitumor agents. Table 16. Influence of intraperitoneally infused 8-Cl-cAMP Na + on the growth in athymic mice of human colon carcinoma HT-29 1 when given alone or with 8-AMINO-cAMP Na + Dosage Initial Mean Δ First-Last Change in Mean (mg/kg/day) Weight (mg) Tumor Weight 2 Tumorr WWeeiight ΔT/ΔC 8-C1 62 327 28 18 8-C1 62 + 340 -44 -13 8-Amino 13 347 152 1. Tumor fragments (~ 14 mg) were implanted subcutaneously in the thigh region of athymic CD-I 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 ΔT/ΔC.. 3. in instances of net tumor loss, ΔT/T was calculated in lieu of ΔT/ΔC. In the test of Example 21 treatment was effected up to and including day seven. The test animals how-^fer were followed to day 21. On day 12 in the animals treated with the combination of 8-aminoadenosine 3',5'-cyclic phosphate and 8- chloroadenosine 3',5'-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',5'-cyclic phosphate plus 8-chloroadenosine 3',5*-cyclic phosphate exhibited less rebound activity compared to 8-chloroadenosine 3 ',5 '-cyclic phosphate as an independent drug. As is evident from the above examples 8-chloroadenosine 3',5'-cyclic phosphate exhibits a broad spectrum of activity against many human tumor cells, 8-Aminoadenosine 3 ',5 '-cyclic phosphate exhibits good specificity against human colon tumors and the combination of 8-aminoadenosine 3 ',5 '-cyclic phosphate and 8-chloroadenosine 3',5'-cyclic phosphate is not antagonistic. For delivery to a host inflicted with a neoplastic disease 8-chloroadenosine 3',5'-cyclic phosphate and 8-aminoadenosine 3',5 r -cyclic phosphate of the invention can be formulated in various formulations to prepare pharmaceutical compositions containing 8-chloroadenosine 3',5'-cyclic phosphate, 8- Aminoadenosine 3',5'-cyclic phosphate or a combination of 8- chloroadenosine 3 ',5 '-cyclic phosphate and 8-aminoadenosine 3',5'-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 ',5 '-cyclic phosphate and 8- aminoadenosine 3',5'-cyclic phosphate. In these examples, Pharmaceutical Preparative Example 22 illustrates the use of 8-chloroadenosine 3 ',5 '-cyclic phosphate sodium salt and/or 8-aminoadenosine 3',5'-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 preparation, Pharmaceutical Preparative Example 24 to an oral capsule preparation and Pharmaceutical Preparative Example 25 to oral tablets. Pharmaceutical Preparative Example 26 is directed to use of 8-chloroadenosine 3',5'-cyclic phosphate sodium salt and/or 8-aminoadenosine 3',5'-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 INJECTABLES 8-Chloroadenosine 3',5'-cyclic phosphate sodium salt or 8-aminoadenosine 3',5'-cyclic phosphate sodium salt 250 mg - 1000 mg Water for Injection USP q.s. 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 passed through a 0.22μ filter. The filtered solution is added to ampoules or vials, sealed and sterilized. EXAMPLE 23 SYRUP 250 mg Active ingredient/5 ml syrup 8-Chloroadenosine 3 ',5'-cyclic phosphate sodium salt or S-aminoadenosine 3',5'-cyclic phosphate sodium salt 50.0 g Purified Water USP q.s. or 200 ml Cherry Syrup q.s. ad 1000 ml The 8-chloroadenosine 3 ',5 '-cyclic phosphate sodium salt and/or 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 250 mg or 500 mg 8-Chloroadenosine 3 ',5 '-cyclic phosphate sodium salt and/or 8-aminoadenosine 3 ',5 '-cyclic phosphate sodium salt 500 g Lactose USP, Anhydrous q.s. or 200 g Sterotex Powder HM 5 g Combine the 8-chloroadenosine 3',5'-cyclic phosphate sodium salt and/or 8-aminoadenosine 3',5'-cyclic phosphate sodium salt and the Lactose in a twin-shell blender equipped with an intensifier bar. Tumble blend for two minutes, followed by blending for one minute with the intensifier bar artd 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, 352.5 mg or 705 mg of the blend, respectively, for the 100 mg, 260 mg and 500 mg containing capsules. EXAMPLE 25 TABLETS 100 mg , 200mg or 500 mg 8-Chloroadenosine 3',5'-cyclic phosphate and/or 8-aminoadenosine 3',5'-cyclic phosphate 500 g Corn Starch NF 200.0 g Cellulose Microcrystalline 46.0 g Sterotex Powder HM 4.0 g Purified Water q.s. or 300.0 ml Combine the corn starch, the cellulose and the 8- chloroadenosine 3 ',5 '-cyclic phosphate and/or 8-aminoadenoεine 3',5'-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 is 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 150 mg, 375 mg and 750 mg respectively, of the total mix are formed with appropriate sized punches for the 100 mg, 250 mg or 500 mg containing tables. EXAMPLE 26 SUPPOSITORIES 250 mg, 500 mg or 1000 mg per 3 g S-Chloroadenosine 3',5'-cyclic phosphate sodium salt and/or 8-aminoadenosine 3',5'-cyclic phosphate 250 mg 500 mg 1000 mg Polyethylene Glycol 1925 mg 1750 mg 1400 mg 1540 Polyethylene Glycol 825 mg 750 mg 600 mg 8000 Melt the Polyethylene Glycol 1540 and the Polyethylene Glycol 8000 together at 60°C. and dissolve the 8- chloroadenosine 3',5'-cyclic phosphate sodium salt and/or 8- Aminoadenosine 3',5'-cyclic phosphate into the melt. Mold this total at 25° C. into appropriate suppositories.

Claims: rece ve y e n erna ona . . original claims 1-17 unchanged;claim 18 amended; new claims 19, 20,29-34 added;original claims 19-26 amended and renumbered as claims 21-28 (4 pages)] 18. A method of treating tumors in warm blooded animals comprising: administering to said warm blooded animals an effective amount of a pharmaceutical composition containing as the active component, a compound chosen from the group consisting of 8-chloroadenosine 3 1 , 5'-cyclic phosphate and 8- aminoadenosine 3 1 , 5'-cyclic phosphate and pharmaceutically acceptable salts thereof. 19. A method as defined in claim 18 wherein the active component is present in at least about 0.1% by weight based on the total weight of the composition. 20. A method of treating tumors in warm blooded animals comprising: administering to said warm blooded animals an effective amount of a pharmaceutical composition containing as the active component, a compound chosen from the group consisting of 8-aminoadenosine 3', 5'-cyclic phosphate and pharmaceutically acceptable salts thereof. 21. The method of claim 18 or 20 wherein: said pharmaceutical composition is administered to said warm blooded animal by injection. 22. The method of claim 18 or 20 wherein: said pharmaceutical composition is administered to said warm blooded animal by infusion of said composition into said warm blooded animal. 23. The method of claim 18 or 20 wherein: said pharmaceutical composition is administered in an amount so as to deliver an amount of from about 13 mg/kg/day to about 288 mg/kg/day of said 8-chloroadenosine 3 • , 5'-cyclic phosphate to said warm blooded animal. 24. The method of claim 18 or 20 wherein: said pharmaceutical composition is administered 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. 25. The method of the claim 18 or 20 wherein: said pharmaceutical composition includes both of said 8-chloroadenosine 3', 5'-cyclic phosphate and 8- a inoadenosine 3', 5'-cyclic phosphate. 26. An antitumor composition for the treatment of tumors in vivo containing as its active ingredient an effective amount of the compound 8-chloroadenosine 3 ' , 5'- cyclic phosphate and pharmaceutically acceptable salts thereof. 27. 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 and pharmaceutically acceptable salts thereof. 28. An antitumor composition of claim 27 further including: an effective amount of the compound 8- chloroadenosine 3', 5'-cyclic phosphate and pharmaceutically acceptable salts thereof. 29. A process of preparing 8-chloroadenosine 3',5'- cyclic phosphate comprising contacting 8-bromoadenosine 3',5'- cyclic phosphate in a solvent with CaCl 2 . 30. A process as defined in claim 29 wherein the 8- bromoadenosine 3* ,5'-cyclic phosphate is contacted in a solvent with CaCl 2 under substantially anhydrous conditions. 31. A process as defined in claim 30 wherein the 8- bromoadenosine 3' ,5*-cyclic phosphate is contacted in a solvent with CaCl 2 at an elevated temperature. 32. A process of preparing 8-chloroadenosine 3*,5'- cyclic phosphate comprising; a) contacting 8-bromoadenosine 3* ,5'-cyclic phosphate in a solvent with CaCl 2 , under substantially anhydrous conditions, and at an elevated temperature; b) removingjgthe solvent to yield a residue; c) dissolving the residue in a basic solution; d) neutralizing the basic solution with an acid; e) purifying the 8-chloroadenosine 3 ',5'-cyclic phosphate from the neutralized solution. 33. A process as defined in claim 32 wherein the 8- chloroadenosine 3 ',5'-cyclic phosphate is purified by chromatography. 34. A process as defined in claim 32 or 33 wherein the solvent is dimethylformamide.

Inventors: CHANG YU-AN, REVANKAR GANAPATHI R, ROBINS ROLAND K, CHANG, YU-AN, REVANKAR, GANAPATHI, R., ROBINS, ROLAND, K.
Applicants: NUCLEIC ACID RESEARCH INST, NUCLEIC ACID RESEARCH INSTITUTE
Application year: 1988
Main classification: A61K31
IPC codes: A61K31, A61P35, C07D473, C07H19