NO751925L - - Google Patents

Description

"Biologically active rlbofuranosylpurln phosphates"

It is known that 3<1>,5'-cyclic purine ribonucleotides are produced in vivo in living animals, including humans, and that cell levels of some of these, such as cyclic guanosine monophosphate (C-GMP) and cyclic adenosine monophosphate (C-AMP), is regulated by specific phosphodiesterases. The biological activity of these cyclic nucleotides follows naturally from such in vivo production and regulation. In fact, as stated in Sutherland et al., "Cyclic Amp", Am. Fox. Biochem. 37, 149 (1968), cyclic AMP has now been established as an intracellular "second messenger" that mediates many of the effects of a variety of hormones.

According to "second messenger" theories, first hormone messengers influence adenyl cyclase contained on or in cell walls to intracellularly form C-AMP from adenosine triphosphate after receiving an extracellular hormone signal.

The formed C-AMP in turn stimulates intracellular functions, especially on the disc cells of the hormone. C-AMP has been shown to "activate" protein kinases which in turn cause physiological effects such as muscle contraction, glycogenolysis, steroidogenesis and lipolysis. As a specific example of mediating steroidogenesis with C-AMP, cellular biosynthesis and excretion of corticosteroids can be mentioned as caused by CAMP formed by adenyl cyclase within the cell walls of the adrenal cortex after receiving an extracellular signal carried by the peptide hormone ACTH.

In addition to the foregoing and as suitable examples of the various roles played by C-AMP in biological processes, mention can be made of the involvement of C-AMP as a participant or mediator of the following metabolic reactions or pharmacological agents: glucagon, vasopressin, luteinizing hormone, thyroid -stimulating hormone, insulin, UDPG-α-trach-glucosylase, phosphofructokinase, tryptophan-pyrrolase, ketogenesis, amino acid uptake in liver proteins. acetate incorporation into fatty acids and cholesterol in the liver, conversion of lactate into glucose (gluconeogenesis), release of amylase, water and ion permeability, sugar transport, acid secretion in the gastric mucus, inhibition of lamellar aggregation, suppression of catabolite , strengthening of antiviral activity in interferon, inhibition of HeLa and lineage L cells in cultures and stimulation of antibody production (immunological mechanism).

The so-called adrenergic effects of many hormones and drugs have now been attributed to the intracellular effects of cyclic AMP, whose concentration is regulated by adenyl cyclase and cyclic nucleotide phosphodiesterase. Recent investigations have shown that the physiological effect of cyclic AMP is at least partly a result of the activation of specific protein kinases by cyclic AMP, such as, for example, in small nerve tubes isolated from the central nervous system.

A logical consequence of the growing recognition of the role played by this cyclic pyrin nucleotide is the suggestion that it be administered to assist subsequent cellular processes. As an example, the report that asthma can be caused by a genetic deficiency of adenyl cyclase can be mentioned. A consequence of such a deficiency is of course a reduced capacity for intracellular conversion of ATP to cyclic aderiosin monophosphate.

Phosphodiesterase enzymes degrade purine nucleotides, such as C-GMP and C-AMP. In the latter case, the enzyme catalyzes hydrolysis of the 3<*>,5'-cyclic adenosine monophosphate to 5'-adenosine monophosphate with accompanying loss of function. There has been a need for cyclic purine nucleotide analogs which, while maintaining the biological activity of the naturally occurring nucleotides, are resistant to phosphodiesterase degradation. The availability of such C-AMP analogues could, for example, maintain the desired level of cyclic nucleotide monophosphate at doses lower than those required with C-AMP itself.

Notwithstanding the foregoing, in certain cases adenyl cyclase will be found to produce harmfully high intracellular amounts of cyclic AMP. For example shows

L.C. Chen et al. in The Lnacet, p. 939 (May 8, 1971) that excess cyclic AMP produced by adenyl cyclase underpins the debilitating dehydration that accompanies cholera. Furthermore, there is substantial evidence that the positive inotropic and chronotropic effects of catecholamines on the heart are mediated by adenyl cyclase stimulation (S.E. Epstein et al., Annals Int. Med., 72:561-568, (1970) . Accordingly, compounds that inhibit adenylate cyclases may act to slow heart rate and may be valuable in preventing arrhythmia. The discovery that the nitrogen, phytohemagglutinin, stimulates adenylate cyclase in human peripheral blood lymphocytes (J. W. Smith et al.,

J. Clin. Invest. 50:432-441 (1971) suggests that adenylate cyclase inhibitors may also be useful as immunosuppressive or anti-inflammatory agents.

According to this invention, compounds with the structure (a) are provided.

where X is -NR^, -Cl, -Br or SR, Y is -X, -OH or -NH2, Z is hydrogen or"NH2, and R' is hydrogen or C^C^-acyl, R is hydrogen, phenyl, p-chlorophenyl, benzyl, or lower alkyl (e.g., C 1 -C 4 alkyl), and R 1 and R 4 are independently selected from hydrogen, benzyl, and lower alkyl groups (e.g., C 1 - C 1 -alkyl) alone or together form a 5- or 6-membered heterocyclic ring, with the proviso that when one of R 1 and R 1 is hydrogen, the other is not hydrogen. Substantially all compounds are better than cyclic AMP itself with respect to resistance to phosphodiesterase degradation.The compounds according to the invention are themselves bioactive or useful as intermediates in the formation of bioactive compounds, and they have varying anti-inflammatory,

anti-hypertensive, adenyl cyclase inhibitory, phosphodiesterase inhibitory and positive inotropic activity.

A first series of the compounds according to the invention is prepared by selective nucleophilic substitution of S/S-dichloro-g<->B-D-ribofuranosylpyrine-S<*>,5'-cyclic-phosphate, as in reaction series I (examples 1 -7). Compound 8 is outside the scope of the invention and was simply prepared to confirm the structure of compound 5.

REACTION SERIES I

Selective nucleophilic substitution of 6,8-dichloro-9-3-D-ribofuranosylpyrine-3',5•-cyclic-phosphate:

Example 1 2'-O-acetyl-6,8-dichloro-9-&-D-ribofuranosylpyrine-3',5'-cyclic-phosphate (2a)

a. 2'-O-acetyl-8-brominosine-3'^'-cyclic-phosphate-triethyl-ammonium salt ( la)

A solution of 8-brominosine-3',5'-cyclic-phosphate (29 g, 71 mmol) in 10 mL of triethylamine, 200 mL of dry pyridine, and 150 mL of acetic anhydride was stirred overnight at room temperature. The solvent was removed in vacuo and the gum that remained crystallized by scraping. The crude crystals were filtered, washed with EtOAc and dissolved in a small volume of MeOH. EtOAc was added to MeOH until solids began to crystallize out. The crystals were filtered off and dried to

give 27 g (69%) with.la.

Analysis:

Calculated for C18<H>27<N5>0gBrP: C 39.14, H 4.95, N 12.68, Br 14.46 Found: C 38.85, H 4.65 N 12.55, Br 14, 66

B. 2'-O-acetyl-6,8-dichloro-9-&-D-ribofuranosylpyrine-3',5•-cyclic- phosphate- sodium salt ( 2a).

A stirred mixture of 21-0-acetyl-8-brominosine-3', 5'-cyclic-phosphate-triethylammonium salt (1a, 2g, 3.6 mmol) in 150 ml POCl3 was placed in an oil bath at 160° and refluxed. treated for 10 minutes. The resulting solution was cooled and the liquid evaporated until a light oil remained. The oil was added dropwise to a mechanically stirred ice-water mixture. The resulting fine suspension was filtered and the solid was washed with an ice-water mixture. The moist solid was dissolved in 25 mL of 0.5N NaOAc pH 5 and extracted three times with 25 mL of Et₂O. The aqueous solution was evaporated to dryness and the residue co-distilled with EtOH to dryness. The final dry residue was stirred with CHCl-j and filtered. The filter cake was washed several times with CHCl 2 to ensure complete removal of the product from residual salts. The filtrate and washings were evaporated to a small volume and applied to a column of 30 mg silica gel (packed in CHCl 2 ). The column was washed with 1 liter of CHCl^ and then the product was eluted with MeOH-CHCl^ (20:80). The fractions containing the product were collected and evaporated. The residue was dissolved in a small volume of MeOH and 20 volumes of Et 2 O were added. This suspension was evaporated, Et 2 O was added and the suspension was again evaporated to dryness to give 300 mg (18%) of 2a.

Analysis:

Calculated for C12<H1>0N4<0>7<C>l2NaP'1 1/4H20:

C 30.68, H 2.68, N 11.93, Cl 15.09, P 6.59.

Found: C 30.48, H 2.27, N 11.64, Cl 15.16, P 6.57.

Example 2

2•-o-butyryl-6,8-dichloro-9-3-D-ribofuranosylpyrine-3',5'-cyclic-phosphate- sodium salt ( 2b)

A. 8-Bromo-2'-O-butyrylinosine-3',5'-cyclic-phosphate-triethylammonium salt ( lb)

A solution of 8-brominosine-3',5'-cyclic-phosphate

(5 g, 9.8 mmol) in MeOH containing 3 mL of triethylamine was evaporated to dryness. EtOH was added to the residue and the resulting crystals were filtered, washed with ether and dried. A solution of the dry triethylammonium salt and 4-dimethylaminopyridine (0.2 g, 1.6 mmol) in 50 mL DMF and 30 mL butyric anhydride was stirred for 3 hours. The solvent was evaporated and the residue

was co-distilled 2 times with EtOH. The residue was dissolved in

EtOH and EtOAc were added until crystals formed. The crystals were filtered, washed with EtOAc, Et 2 O and dried

to give 4.6 g (81%) with lb.

B. 2<1->O-butyryl-6,8-dichloro-9-3-D-ribofuranosylpyrine-3■,5'-cyclic-phosphate-sodium salt ( 2b).

A solution of 8-bromo-2'-O-butyrylinosine-3',5'-cyclic-phosphate-triethylammonium salt (1b, 4.5 g, 9.4 mmol) was refluxed for 4 min in 30 mL POCl 3 and was worked up as for compound 2a to give 2.65 g (56%) of 2b.

Analysis:

Calculated for C^H^N^C^NaP^O: C 34.04, H 3.30, N 11.10, Cl 14.45. Found: C 34.09, H 3.27, N 11.36, Cl 14.37.

Example 3

8- amino- 6- chloro- 9- B- D- ribofuranosylpyrine- 3', 5'- cyclic- phosphate ( 4)

A solution of 2'-O-acetyl-6,8-dichloro-9-O-D-ribofuranosylpyrine-3',5'-cyclic-phosphate sodium salt (2a, 1 g, 2.1 mmol) in 60 mL of MeOH saturated at 0°C with NH3, was stirred at room temperature overnight in a bomb. The solvent was evaporated and the residue was dissolved in H 2 O and applied to a Dowex 50 column

(H+, 100-200 mesh, 5 x 30 cm). The column was eluted with H 2 O and appropriate fractions were collected and evaporated. MeOH was added to the residue. The solid that formed was filtered off and dried to give 350 mg (43%) of 4. An analytical sample was obtained by recrystallization from H 2 O.

Analysis:

Calculated for C^<H>^Ng<OgC>IP^O: C 31.46, H 3.43, N 18.35, Cl 9.28 Found: C 31.24, H 3.58, N 18, 30, Cl 9,31.

Example 4 8- amlno- 9- B- D- ribofuranosyl- 6- thiopurine- 3', 5'- cyclic- phosphate ( 6)

A solution of 8-amino-6-chloro-9-B-D-ribofuranosylpurine-3*,5'-cyclic-phosphate (4,600 mg, 1.6 mmol), thiourea (300 mg,

4.7 mmol) and 5 drops of formic acid in 20 ml H 2 O were refluxed for 1 hour. The solution was cooled to room temperature and the precipitated solid was filtered, washed with H 2 O and dried to give 447 mg (78%) of impure 6. An analytical sample was obtained by acidifying a basic solution of the impure

product with n HCl to a pH of 2. The solid was filtered, washed with H 2 O and dried.

Analysis:

Calculated for C10H12N5OPS: C 33.24, H 3.34, N 19.38, S 8.87.

Found: C 33.26, H 3.36, N 19.13, S 8.78.

Example 5

6-chloro-8-hydroxy-9-B-D-ribofuranosylpurine-3',5'-cyclic-phosphate-sodium salt ( 5 )

A solution of 2'-O-acetyl-6,8-dichloro-9-p-D-ribo-furanosylpurine-3',5'-cyclic-phosphate sodium salt (2a, 1 g, 2.1 mmol)

in 20 ml ln NaOH was stirred at room temperature overnight. A small amount of the solid was filtered off and the filtrate was passed through a Dowex 50 column (H + , 100-200 mesh, 4 x 10 cm). The column was washed with H 2 O and the appropriate fractions were evaporated to dryness. The residue was dissolved in H 2 O and passed through

a Dowex 50 column (Na+, 100-200 mesh, 4x5 cm). The column

was washed with H 2 O and the appropriate fractions were evaporated to dryness. The residue was co-distilled with absolute EtOH. The final dry residue was dissolved in MeOH and 4 g of silica gel was added. The solvent was evaporated and the dry powder was added to a 20 g column of silica gel (packed in CHCl 2 ). The column was eluted with MeOH-CHCl 3 (20:80). Appropriate fractions were pooled,

evaporated and the resulting residue was dissolved in MeOH. Addition of ether to MeOH precipitated 319 mg (33%) with 5. Analysis: Calculated for C10HgN407ClNap»3 1/2 H20: C 26.70, H 3.58, N 12.46 Found: C 26.69, H 3.26, N 12.40.

Example 6

6,8-bis(diethylamino)-9-B-D-ribofuranosylpurine-3* ^'-cyclic-phosphate (11) and 8-chloro-6-(diethylamino)-9-3-D-ribofuranosylpyrine-3', 5' - cyclic phosphate ( 9). '

A solution of 2'-O-acetyl-6,8-dichloro-9-p-D-ribo-furanosylpurine-3',5'-cyclic-phosphate sodium salt (2a, 2 g, 4.2 mmol) in 20 mL of diethylamine and 30 mL DMF was stirred at room temperature for 4 days. The emollient was evaporated. The residue was taken up

in H 2 O and after adjusting to a pH value of 7 with n HCl, the solution was placed on a Dowex 1x8 column (formate, 100-200 mesh, 70 ml). The column was washed with H 2 O and then eluted with a

1.4 liter gradient of 0-4n formic acid to elute 11 and then 9.

The appropriate fractions were collected, evaporated to dryness and co-distilled twice with MeOH, and the dry residue was taken up

in MeOH and 20 volumes of Et2O were added. The solids formed were collected on a filter, washed with Et 2 O and dried to give 0.538 g (28%) of 11 and 0.661 g (35%) of 9 .

Analysis:

Calculated for C18<H2>gN6<0gP:>C 47.36, H 6.40, N 18.41,

Found: C 47.17, H 6.32, N 18.21.

Analysis:

Calculated for C 14 Hig N 5 OgCIP: C 40.05, H 4.56, N 16.68, Cl 8.44 Found: C 39.77, H 4.51, N 16.46, Cl 8.52.

Example 7

6-chloro-8-(diethylamino)-9-3-D-ribofuranosulpurine-3<1>,5'-cyclic-phosphate ( 10 ).

A solution of 2<1->0-butyryl-6,8-dichloro-9-B-D-ribo-furanosylpurine-3',5'-cyclic-phosphate sodium salt (2b, 0.5 g,

1.05 mmol) in 10 ml diethylamine and 20 ml DMF was stirred at room temperature for 1 hour. The solvent was evaporated and the residue was dissolved in 50 mL of 2N NH 4 OH. After stirring for 1 hour at room temperature, the solvent was evaporated and the residue was chromatographed on a Dowex 1 column (35 mL), as for compound 9, to give 11 (<3% at n.f.) 173 mg of 9 (41%) and 176 mg out of 10 (40%).

Analysis:

Calculated for C14H19N506CIP: C 40.05, H 4.56, N 16.68.

Found: C 40.25, H 4.63, N 16.42.

Although position 2 of the aglycon is unsubstituted in the preferred compounds according to the invention, the methodology of the invention is equally suitable for the production of 6,8-disubstituted analogues of guanosine-3<*>,5'-cyclic-phosphate. Reaction series Ia includes such a preparation (that of compound 37), where the methodology of reaction series I is applied, and this is more fully shown in the following examples 8 and 9. Reaction series Ia illustrates, for similar reasons, preparation of a 6,8-substituted cyclic GMP analogue (compound 42) via nucleophilic attack on the 6-chloro substituent in the preformed 2-amino-8-substituted 3',5'-cyclic phosphates (see the following examples 9a and 9b and compare with reaction series II, below).

REACTION SERIES IA

Preparation of 6,8-disubstituted guanosine-3',.5'-cyclic-phosphate analogues:

Example 8 2-amino-2•-O-butyryl-6,8-dichloro-9-B-D-ribofuranosylpurine-3<1>,5'-cyclic-phosphate (37) A. 2'-O-butyrylguanosine-3' , 5'- cyclic phosphate sodium salt ( 35 ) cGMP sodium salt (2.0 g, 5.4 mmol) and 4-dimethylaminopyridine (0.15 g, 1.23 mmol) were dissolved in 50 mL DMF and 5 mL butyric anhydride. After stirring at room temperature for 3 hours, the mixture was filtered and the filtrate was evaporated to dryness. EtOH was added to the residue and the resulting solid was filtered, washed with EtOH and dried to give 0.870 g (37%) of 35: X<P>"kg 257 nm, 282 sh (e 1,800, 8,300), X j ^ks253nm'277 sh

(e 13,300, 8,700), X j^g<1>260nm (e 11,800).

Analysis:

Calculated for C14H14<N>5<O>g<N>aP: C 38.45, H 3.91, N 16.01

Found: C 38.35, H 4.07, N 15.93.

B. 8-bromo-2'-O-butyrylguanosine-3',5'-cyclic-phosphate-triethylammonium salt ( 36 )

150 ml of bromine water (saturated at room temperature) was added dropwise over the course of 2 hours to a solution of 2'-0-butyryl-

cGMP Na<+>, (35, 11.5 g, 26 mmol) in 500 ml HjP. The solution was stirred for a further 1/2 hour and then N2 was bubbled through until the color changed from orange to pale yellow. The solution was evaporated to 300 mL and diluted with 300 mL of MeOH. It was passed through a Dowex 50 column (H + , 100-200 mesh, 5 x 24 cm, prewashed with MeOH- 2 O [1:1]). The eluate was evaporated to dryness and the residue co-distilled with EtOH until a dry foam. Acetone was added to the foam and the resulting solid was filtered and dried to give 10.5 g of crude free acid of 4b. The free acid was dissolved in MeOH containing 5 ml of triethylamine. The solution was evaporated to dryness and the residue was dissolved in CHCl3 and placed on a column with 30 g of silica gel (packed in CHCl3). The column was washed with CHCl 2 and then the product was eluted away with MeOH-CHCl 2 (10:90). The appropriate fractions were pooled and evaporated to dryness. CHCl₂ was added to the residue and the resulting crystals were filtered off and dried to give 5 g (32%) of 36:1 j^k* 260 nm, 275 sh (e 15,500, 12,800), Xj^g1 270 nm (e 13,600).

Analysis:

Calculated for C20<H3>2N6<0>8<B>rP: C 40.34, H 5.41, N 14.11, Br 13.42 Found: , C 40.18, H 5.40, N 14 ,06, Br 13,45. C. 2-amino-2'-0-butyryl-6,8-dichloro-9-3-D-ribofuranosypurin-3', 5'- cyclic- phosphate (37).

To 8-bromo-2'-0-butyrylguanosine-3'^'-cyclic-phosphate-triethylammonium salt (36, 0.7 g, 1.18 mmol), moistened with 2,6-lutidine (0.17 g, 1 .6 mmol), 50 ml of phosphoryl chloride was added.

The mixture was refluxed for 3 minutes in an oil bath at 160°. The resulting solution was evaporated to a light oil. The oil was added dropwise to a mechanically stirred ice-water mixture. The resulting fine suspension was filtered and the solid was washed with an ice-water mixture. The moist solid was dissolved in 20 mL of 1.0 M NaOAc at pH 5.3 and extracted with Et 2 O three times. The solution was evaporated and the residue was triturated with EtOH to dryness. The dry residue was extracted with CHCl 3 . The CHCl 3 was evaporated to a small volume and 20 volumes of Et 2 O were added to precipitate 320 mg (55%). with impure (contains 1/2 mol NaOAc by elemental analysis and nmr) sodium salt of 37. An analytical sample was obtained by

passing a sample of the sodium salt through a Dowex 50 column (H+, 100-200 mesh): X jj*^ and 11 252, 311 nm (e 9,700, 8,200). Analysis: Calculated for C14H16N507C12P: C35.91, H, 3.44, N 14.95.

Found: C 36.04, H 3.37, N 14.80.

Example 9

2-amino-8-chloro-6-dimethylamino-9-8-D-ribofuranosylpurine-3',5'-cyclic-phosphate (38).

A solution of 2-amino-2'-O-butyryl-T6,8-dichloro-9-G-D-ribofuranosylpurine-3',5'-cyclic-phosphate sodium salt (37, 0.6 g, 1.2 mmol) in 30 ml of EtOH and 2 ml of dimethylamine was stirred under reflux for 1 hour and then at room temperature overnight. The solvent was evaporated and the residue was co-distilled twice with EtOH. The final residue in H,0 was placed on a Dowex 50 column; (H , 100-200 mesh, 2x6 cm). The column was eluted with H2O and the appropriate fractions were collected and evaporated to dryness. After co-distillation 2 times with EtOH, the residue was suspended

in EtOH, was filtered, washed with Et 2 O and dried to give 95 mg

(20%) with 38. X 262, 304 nm (e 14,800, 13,800), Xm"ks^230, 285 nm, 272 sh (e 18,200, 16,000, 13,700).

Analysis:

Calculated for C12<H>16<N60>6<C>1P: C 35.43, H 3.96, N 20.66, Cl 8.71. Found: C 35.20, H 4.06, N 20.44, Cl 8.79.

Example 9a 2'-0-acetyl-2-amino-6-chloro-8-(p-chlorophenylthio)-9-B-D-ribofuranosyl-purine-31,5'-cyclic-phosphate-sodium salt (41)

A. 8-(p-Chlorophenylthio)guanosine-3',5'-cyclic-phosphate- sodium salt ( 39)

A H 2 O (25 mL) - MeOH (500 mL) suspension of 8-bromo-cGMP (10 g, 23.6 mmol), p-chlorophenylthiol (3.7 g, 25.7 mmol) and NaOAc (4 g) was refluxed for 24 hours. The cloudy solution was filtered and evaporated to a solid. The solid was triturated with 3 x 100 mL of hot EtOH and then dissolved in a minimal amount of hot H 2 O. The aqueous solution was cooled and filtered to give 8.3 g (66%) of 39: X m^kg# 295 nm (e 21,500), X P?,,11 296 nm (e 21,500). Analysis:

Calculated for C^H^ClNgPO.jSNa.1.25 H2<0:>

C 36.18, H 2.94, N 13.32, S 6.30.

Found: C 36.17, fl 2.94, N 13.18, S 6.03.

B. 2'-0-acetyl-2-amino-8-(p-chlorophenylthio)guanosine-3',5'-cyclic-phosphate ( 40 )

H 2 O was added dropwise to 8-(p-chlorophenylthio)guanosine-3',5•-cyclic-phosphate sodium salt (39.3 g, 5.6 mmol) in hot (90°C) DMF until dissolution occurred. The solution was cooled to room temperature and 4-dimethylaminopyridine (150 mg, 1.2 mmol) and 10 mL of acetic anhydride were added and the resulting solution was stirred for 45 minutes. The solvent was evaporated and the residue in MeOH-H 2 O (1:1) was passed through a Dowex 50 column (H+, 100-200 mesh, 4x9 cm). The column was eluted with MeOH-H 2 O. (1:1), the eluate was evaporated and the residue was co-distilled with EtOH to dryness to give 2.1 g at 40. C. 2'-O-acetyl-2-amino- 6--chloro-8-(p-chlorophen<y>ltio^<g>-p-D-ribo-furanosylpurln- S' , 5°- cyclic- phosphate- sodium salt ( 41). A suspension of 2'-0-acety1 -8-(p-chlorophenylthio)guanosine-3,5'-cyclic-phosphate (40.2 g, 3.7 mmol) and 2,6-lutidine (400 mg, 3.7 mmol) in 30 ml of phosphorus oxychloride were placed in a preheated oil bath at 160°C and refluxed for 4 minutes. The solution was evaporated to a small volume and added dropwise to a mechanically stirred ice-water mixture. The resulting fine suspension was filtered and the solid was washed with an ice-water mixture.The solid was dissolved in 25 ml of 5n NaOAc

with a pH of 5.0 and was extracted three times with ether. The aqueous phase was evaporated and the residue was co-distilled with EtOH to dryness. The dry residue was extracted with MeOH and 4 g of silica gel was added. This was then evaporated and the residue was added to a column with 20 g of silica gel (packed in CHCl^)• The column was washed with CHCl^ and the product was eluted with MeOH-CHCl^

(10-90). The fractions were slowly evaporated. The crystals that formed after approximately 1/2 volume was evaporated were filtered off and dried to give 40 mg of 41. The filtrate was evaporated to give an additional 262 mg of 41. Total yield was 302 mg (13%): X max!<11>324nm'262 sh <e 16,700, 14,200).

Analysis:

Calculated for C18H15N507ClNaPS*4H20: C 35.62, H 3.81, N 11.54. Found: C 35.71, H 3.19, N 11.48.

Example 9b

2-amino-8-(p-chlorophenylthio)-6-piperidino-9-B-D-ribofuranosylpurine-3', 5'-cyclic- phosphate (42)

A solution of 2'-0-acetyl-2-amino-8-(p-chlorophenylthio)-6-chloro-9-B-D-ribofuranosylpurine-3',5'-cyclic-phosphate sodium salt (41, 220 mg) in 10 ml of H 2 O and 4 ml of piperidine were stirred overnight at room temperature. The solvent was evaporated and the residue was dissolved in water and acidified to a pH of 1 with 1N HCl. The residue was filtered and recrystallized from 1:1 water:EtOH to give 120 mg.

Analysis:

Calculated for C21H24<N>6<O>g<C>1PS: C 45.44, H 4/35, N 15.14. Found: C 45.20, H 4.44, N 14.95. >

Reaction series II illustrates a route to further 6,8-disubstituted compounds according to the invention, which comprises nucleophilic substitution of 6-chloro-8-substituted-9-B-D-ribofuranosylpurine-3',5'-cyclic-phosphate.

(See the following examples 10-21).

REACTION SERIES II

Nucleophilic substitution of 6-chloro-8-substituted-9-3-D-ribo-furanosylpurine-3',5'-cyclic-phosphates obtained by chlorination of 8-substituted inosine-3',5'-cyclic-phosphates:

Example 10

2'-0-acetyl-8-(benzylthio)-6-chloro-9-p-D-ribofuranosylpurine-3', 5' -

. cyclic phosphate sodium salt ( 13)

A. A solution of 8-(benzylthio)inosine-<3>\5'-cyclic-phosphate (15.5 g, 11.1 mmol) in 10% aqueous triethylamine was evaporated to dryness. The residue was co-distilled twice with pyridine. The final residue was stirred in 250 ml of pyridine together with 150 ml of acetic anhydride overnight. The resulting solution was evaporated. to dryness and the residue co-distilled with ethanol until all traces of pyridine were removed. 50 ml POCl3 was added to the residue and after refluxing for 10 minutes the reaction mixture was worked up as for compound 2a to give 3.2 g (51%) of 13.

B. To a solution of 2'-O-acetyl-6,8-dichloro-9-p-D-ribofuranosylpurine-3',5'-cyclic-phosphate sodium salt (2a, 1 g, 2.1 mmol) in 25 mL 0.1 M NaOAc with a pH of 8.7, 20 drops of benzyl mercaptan were added to 20 ml of EtOH. The solution was stirred at room temperature overnight. The solution was evaporated and the residue was dissolved in 10 ml CHCl 4 . This was added to a column (2.5 cm) of 25 g silica gel (packed in CHClg). The column was washed with CHCl 2 to remove excess benzyl mercaptan. The product was then eluted with MeOH-CHCl 3 (20:80). The solvent was evaporated, the residue was dissolved in EtOH and 20 volumes of Et 2 O were added. The residue was filtered off, washed with ether and dried to give 612 mg (51%) with 13.

Analysis:

Calculated for C^<H>^N^<C>iNaPS^^O: C 39.97, H 3.70, N 9.81. Found: C 39.94, H 3.36, N 9.92.

Example 11

6,8-bis-(benzylthio)-9-B-D-ribofuranosylpurine-3',5'-cyclic-phosphate-sodium salt ( 14 )

A solution of 2'-0-acetyl-8-benzylthio-6-chloro-9-fS-D-ribofuranosylpurine-3',5'-cyclic-phosphate sodium salt (13, 750 mg, 1.3 mmol) and 2 ml of benzyl mercaptan in 30 ml of 0.25M NaOAc was refluxed overnight. The solvent was evaporated and the residue was co-distilled with EtOH to dryness. The dry residue was dissolved in MeOH and 4 g of silica gel was added. The MeOH was evaporated and the residue was added to a column (2.5 cm) of 15 g silica gel (packed in CHCl 3 ). The column was eluted with CHCl 3 to remove the contaminating benzyl mercaptan, followed by MeOH-CHCl 3 , 500 mL of each (5:95 and 10:90) and then (15:85) until the product was eluted. The fractions containing the product were pooled and the solvent was evaporated. A portion of the residue was taken up in EtOH and 20 volumes of Et 2 O were added and the solid was filtered and dried to give 80 mg of crude 2'-O-acetyl-6,8-bis-(benzylthio)-9- 3-D-ribofuranosyl-purine-3',5'-cyclic-phosphate sodium salt. The rest of the residue was taken up in 20 ml with 2n NH 2 OH and left at room temperature in

2 hours. The solvent was evaporated and the residue co-distilled once with H 2 O to ensure complete removal of NH 2 . The final residue was taken up in 50 ml of EtOH-H 2 O (1:1) and 50 ml of a saturated NaCl solution was added. The resulting gel was filtered off, washed well with H 2 O and then with ethanol. The final gel was dried under vacuum

for 12 h at 100° to give 290 mg (38%) with 14.

Analysis:

Calculated for C24H22N406NaPS2 «1/2^0:0 48.84, H 3.90, N 9.50, S 10.87. Found: C 48.71, H 4.03, N 9.53, S 10.79.

Example 12

8-(Benzylthio)-6-(diethylamino)-9-3-D-ribofuranosylpurine-3',5'-cyclic-phosphate ( 12 )

A solution of 2'-0-acetyl-8-(benzylthio)-6-chloro-9-3~D-ribofuranosylpurine-3',5'-cyclic-phosphate sodium salt (13, 3.5 g, 6.1 mmol) in 90 ml of 20% aqueous diethylamine was stirred overnight at room temperature. The solvent was evaporated and the residue triturated twice with ether. The final residue was dissolved in . H 2 O and was placed on a Dowex 50 column [H+, 100-200 mesh,

2.5 x 10 cm, prewashed with MeOH-H 2 O (1:1)]. The column was washed with H 2 O followed by 100 ml MeOH-H 2 O (20:80), 100 ml MeOH-H 2 O (1:1) and finally MeOH-H 2 O (75:25) until all product was eluted. The appropriate fractions were combined and evaporated to dryness. The residue was dissolved in boiling EtOH and 5 volumes of H 2 O were added. The solution was boiled down to 1/5 volume. The crystals which settled on cooling were filtered off and dried to give 2.4 g (75%) with 120

Analysis:

Calculated for C^H^^OgPS^O: C 47.99, H 5.37, N 13.32, S 6.10. Found: C 47.71, H 5.52, N 13.12, S 6.08.

Example 13

8-(Benzylthio)-6-piperidino-9-3-D-ribofuranosylpurine-3',5'-cyclic-phosphate ( 21 )

A solution of <2>'-O-acet<y>l-8-(benz<y>ltio)-6-chloro-9-B-D-ribofuranosylpurine-3<*>,5'-cyclic-phosphate sodium salt ( 13, 1.3 g, 2.3 mmol) in .20 mL H 2 O and 10 mL piperidine was treated as for compound 12„ The residue from the column was crystallized from H 2 O to give 700 mg (59%) of 21.

Analysis:

Calculated for C22<H2>6N506<P>S: C 50.86, H 5.04, N 13.48, S 6.17. Found: C 51.01, H.5.11, N 13.52, S 6.05.

Example 14 8-(Benzylthio)-6-(n-butylamino)-9~G-D-ribofuranosylpurine-3',5'-cyclic phase (22)

A solution of 2'-O-acetyl-8-(benzylthio)-6-chloro-9-B-D-ribofuranosylpurine-3',5'-cyclic-phosphate sodium salt (13, .3.5 g, 5.25 mmol ) in 50 ml H 2 O and 10 ml n-butylamine was stirred overnight at room temperature. The solvent was evaporated and the residue triturated twice with ether, dissolved in H 2 O and acidified to a pH of 1 with n HCl. The crude solid was filtered and then dissolved in EtOH. To this EtOH was added 5 volumes of ether and the precipitated solid was collected. Recrystallization from EtOH-H 2 O (1:1) gave 1.68 g (61%) of 22.

Analysis:

Calculated for C21H26N5OPS»H20: C 47.99, H 5.32, N 13.32, S 6.10. Found: C 47.77, H 5.48, N 13.24, S 6.30. Example 15 8-(benzylthio)-6-pyrrolidino-9-3-D-ribofuranosylpurine-3',5'-cyclic-phosphate (23). A solution of 2'-O-acetyl-8-(benzylthio)-6-chloro-9-B-D-ribofuranosylpurine-3',5'-cyclic-phosphate (13.2g, 3.5 mmol) in 20 mL H 2 O containing 5 mL of pyrrolidine was treated as for compound 12. H 2 O was added to the residue from the column and the resulting crystals were filtered off and dried to give 947 mg (51%) of 23.

Analysis:

Calculated for C21H24N506PS»1 1/2 H2O: C 47.36, H 5.11, N 13.15 Found: C 47.05, H 5.26, N 12.94.

Example 16

8-(Benzylthio)-6^(ethylamino)-9-3-D-ribofuranosylpurine-3',5'-cyclic-phosphate ( 24 )

A solution of 2'-O-acetyl-8-(benzylthio)-6-chloro-9-3~D-ribofuranosylpurine-3'*5'-cyclic-phosphate sodium salt (13.5 g, 8.8 mmol) in 60 ml of 35% aqueous ethylamine was stirred for 2 days at room temperature. The solvent was evaporated and the residue was dissolved in H 2 O and acidified to a pH of 2 with 1N HCl. The liquid was decanted from the gum that formed. After rubbing with warm water, the rubber became solid. The solid was filtered off, washed with water and boiling EtOH and was dried to give 2.4 g (56%) of 24. A sample was recrystallized from EtOH-H 2 O (1:1) to analyze.

Analysis:

Calculated for C^H^NgOgPS* 3/4H 2 O: C 46.29, H 4.76, N 14.20, S 6.30. Found: C 46.23, H 4.73, N 14.08, S 6.63.

Example 17

2'- O- acetyl- 8-( p- chlorophenylthio) lnosln- 3', 5'- cyclic- phosphate ( 19)

8-(p-chlorophenylthio)inosine-3',5'-cyclic-phosphate [J.P. Miller et al., Biochemistry, 12, 5310 (1973)] (17, 6.0 g, 12.2 mmol) was dissolved in MeOH containing 3 mL of triethylamine. The solution was evaporated to dryness and the residue was dissolved in a mixture of DMF (100 mL) and acetic anhydride (50 mL) containing 4-dimethylaminopyridine (305 mg, 2.5 mmol). The solution was stirred for 2 hours at room temperature, the solvent was evaporated and the residue was dissolved in a minimal volume of MeOH-H 2 O (1:1). This was then passed through a Dowex 50 column [H + , 100-200 mesh, 4.5 x 15 cm, pre-washed with MeOH-H 2 O (1:1)]. The eluent was evaporated to a small volume and the formed crystals were filtered off and washed with H 2 O. Recrystallization from H 2 O gave 5.43 g (86%) in two portions. Analysis:

Calculated for C18<H1>6N408C1PS: C 41.99, H 3.13, N 10.88.

Found: C 41.79, H 3.36, N 10.75.

Example 18

2'-O-acetyl-6-chloro-8-(p-chlorophenylthio)-9-3-D-ribofuranosylpurine-3°, 5'- cyclic- phosphate- sodium salt ( 20).

To 2'-O-acetyl-8-(p-chlorophenylthio)-inosine-3',5'-cyclic-phosphate (19, 1.9 g, 3.7 mmol) and 2,6-lutidine (394 mg, 3.7 mmol) 30 ml of POCl 3 were added. The mixture was placed in an oil bath at 160° and after refluxing for 4 minutes, the reaction mixture was worked up as for compound 2a to give 822 mg (40%) with 20

Analyzed. Calculated for C18H14N40._Cl2NaPSs C 38.93,- H 2.54, N 10.09.

Found? C 38.72, H 2.52, N 10.10.

Example 19 8-(p-Chlorophenylthio)-6-piperidino-9-3~D-ribofuranosylpurine-3',5'-cyclic-phosphate (25)

A solution of 2'-O-acetyl-6-chloro-8-(p-chlorophenylthio)-9-3-D-ribofuranosylpurine-3',5'-cyclic-phosphate sodium salt (20, 1.0 g, 1 .8 mmol) in 20 ml H 2 O containing 3 ml piperidine was stirred overnight at room temperature. The solvent was evaporated and the residue triturated twice with ether. The final residue

was dissolved in H 2 O and the pH was adjusted to 1 with n HCl., The liquid was decanted from the gum that formed. The gum was rubbed with H 2 O and then dissolved in hot H 2 O. After cooling, a semi-solid substance was separated from the solution, and after standing overnight it became solid. The solid was filtered off and dried to give 670 mg (70%) with 25.

Analysis:

Calculated for C21<H>23<N>5<0>6C1PS: C 46.71, H 4.29, N 12.97

Found: C 46.55, H 4.28, N 12.80.

Example 20

8-(p-Chlorophenylthio)-6-(diethylamino)-9-3-D-ribofuranosulpurine-3',5'-cyclic- phosphate (26)

A. A solution of 2'-O-acetyl-6-chloro-8-(p-chlorophenylthio)-9-3-D-ribofuranosylpurine-3',5'-cyclic-phosphate sodium salt (20,

0.8 g, 1.4 mmol) in 30 mL of 20% aqueous diethylamine was treated as for compound 12. The residue from the column was dried to give a yield of 395 mg (51%) of 26.

Analysis:

Calculated for C2o<H2>3N5<0>6<C>lPS: C 45.15, H 5.29, N 13.16

Found: C 45.29, H 5.07, N 13.19.

B. A solution of 8-chloro-6-(diethylamino)-9-3-D-ribofuranosylpurine-3',5'-cyclic-phosphate (9, 270 mg, 0.65 mmol), sodium methoxide (200 mg, 3 .7 mmol) and p-chlorobenzenethiol (300 mg,

21 mmol) in 50 mL MeOH was refluxed overnight. The solvent was evaporated and the residue triturated twice with ether. The final residue was dissolved in H 2 O and acidified to a pH of 1 with n HCl. The resulting solid was collected and dissolved in H 2 O by addition of 2N NaOH. The solvent was evaporated and the residue was co-distilled twice with EtOH. The dry residue was dissolved in MeOH and 2 g of silica gel was added. After evaporation of the solvent, the residue was placed on a column with 10 g of silica gel (packed in CHCl-j). The column was washed with CHCl3 to remove excess p-chlorobenzenethiol and then the product was eluted away with MeOH-CHCl3 (30.70). The fractions containing the product were collected and evaporated

to dryness to give 35 mg (9%) with 26.

Analysis

Calculated for C 20 H 22 N 5 O 6 ClNaPS' 2 H 2 O: C 41.06, 'H 4.44, N 11.97. Found: C 40.82, H 4.27, N 12.38.

Example 21

6,8-bis-(methylthio)-9-3-D-ribofuranosylpurine-3',5'-cyclic-phosphate-sodium salt ( 27) ..

8-methylthioinosine-3',5'-cyclic-phosphate [J.P. Miller et al., Biochemistry, 12, 5310 (1973)] (16.2 g, 5.3 mmol) was treated as for compound 13 up to and including the addition of the oil to the ice-water mixture. The resulting solid that formed was filtered, washed with ice water and then dissolved in 50 ml of 1M NaOAc pH 7. The pH of the solution was adjusted to 9 with 2N NaOH and 10 ml of methylmercaptan was added. The reaction mixture was stirred for 3 days at 50°C under a condenser.

After cooling to room temperature, the volume was evaporated to half. The solid which precipitated was filtered and dissolved in 30 ml of hot water. 30 ml EtOH was added. On cooling M>e 27 separated out. 27 was filtered, washed with EtOH and dried to give 300 mg (12%).

Analysis:

Calculated for C12H14N4O6NaPH2'^O: C 32.28, H 3.58, N 12.55. Found: C 32.07, H 3.98, N 12.21.

Reaction Series III illustrates yet another synthetic route to preferred 6,8-disubstituted bioactive compounds (see the following Examples 22-25).

REACTION SERIES III

N^-Alkylation of 8-Substituted Adenosine-3',5<1->cyclic-Phosphates Followed by Dimroth Rearrangement

Example 22 6-(Benzylamino)-8-bromo-9-3-D-ribofuranosylpurine-3<1>,5'-cyclic-phosphate ( 31 ) A solution of 8-bromoadenosine-3',5!-cyclic-phosphate [Muneyama et al., Biochemistry, 10, 2390 (1971)] (1/22 g, 3 mmol), 1,5-diazabicyclo[5„4oO]-undecene-5 (1.0 ml, 6.0 mmol) and α-bromotoluene (0.4 mL) in 8 mL of DMSO was stirred for 18 h. A further aliquot of α-bromotoluene was added and stirring was continued for 36 hours. The solution was added to 150 ml of H 2 O containing NaHCO 3 (1.3 g) and Na 2 CO 3 (1.0 g), and was heated on a steam bath for 2 hours. After adjusting the pH to 1.5 with concentrated HCl, the solution was absorbed on a charcoal column (30 ml, Barnebey-Cheney, UU 50-200 mesh) and was washed well with H 2 O. The nucleotide was eluted with EtOH-NH 4 OH-H 2 O (4:1:5). The solvent was evaporated and the residue Me in a small volume of H 2 O was added to a Dowex 1=2 column (formate, 100-200 mesh, 2.5 x 3 cm). The column was washed with water and then eluted with a 1 liter gradient of

0 to 4n formic acid. The appropriate fractions were pooled and evaporated. The residue was suspended in a small amount of H 2 O, filtered and air-dried to give 142 mg (9.0% with 31.

Analysis:

Calculated for C17<H1>7N506<B>rP'13/4H20: C 38.53, H 3.95, N 13.21. Found: C 38.79, H 3.74, N 12.92.

Example 23

6-(Benzylamino)-8-(methylthio)-9-B-D-ribofuranosylpurine-3',5 cyclic-phosphate ( 32 )

Aliquots of α-bromotoluene (0.25 mL) were left at times

0, 20 min., 40 min., 80 min. and 5 hours added to a solution of 8-(methylthio)adenosine-3",5'-cyclic-phosphate [K. Muneyama et al., Biochemistry,,10, 2390 (1971)] (1.0 g, 249 mmol ) and 1,5-diaza-bicyclo[5.4.0]undecene-5 (812 mg, 3.2 mmol) in 5 mL of DMSO at 60°. After cooling to room temperature overnight, the reaction mixture was added to 150 mL of water containing NaHCO 3 (1.2 g) and Na 2 CO 3 (0.9 g). The solution was heated on a steam bath for 2 1/2 hours. After filtration, the volume was diluted to 1 liter,

10 g of NaCl were added and the solution was extracted twice with 50 ml of CHCl 3 . The aqueous phase was added to an amberlite XAD-4 column (130 mL, 2.5 x 25 cm). The column was washed well with water and the nucleotide was eluted with a 5 liter gradient of H 2 O vs MeOH. The solvent was evaporated and the residue was suspended

in MeOH, filtered and dried at room temperature to give 340 mg (29%) of 32. Analysis: Calcd for C18H20N50gPS: C 46.44, H 4.33, N 15.04. Found: C 46.20, H 4.19, N 14.77.

Example 24

6-(Benzylamino)-8-(benzylthio)-9-3-D-ribofuranosylpurine-3',5'-cyclic-phosphate ( 33 )

Aliquots of α-bromotoluene (0.2 ml) were added to a solution of 8-(benzylthio)adenosine sodium salt [K. Menyama et al., Biochemistry, 10, 2390 (1971)] (442 mg, 1 mmol) and 1,5-diaza-bicyclo[S.4.0]unde.cen-5 (0.2 ml, 1.2 mmol) in 1 ml DMSO at times 0, 1 hour and 24 hours. After stirring for a further 18 hours, 50 ml of H 2 O were added and the resulting suspension was extracted with Et 2 O and then with CHCT-j. NaHCO 3 (0.4 g) and Na 2 CO 3

(0.3 g) was added to the aqueous phase and the resulting solution was heated on a steam bath for 2 hours. After cooling, the pH value was adjusted to 1.5 with HCl. The eluate was filtered and reprecipitated from H 2 O with conc.HCl to give 326 mg (66%) of 33. An analytical sample was obtained by chromatography on

Dowex 1, as for compound 31.

Analysis: Calculated for C24<H>24<N>5<0>5PS: C 53.23, H 4.46, N 12.93. Found: C 53.14, H 4.46, N 13.05.

Example 25

8-(Benzylthio)-6-(methylamino)-9-3-D-ribofuranosylpurine-3*,5'-cyclic-phosphate ( 34 )

A solution of 8-benzylthioadenosine-3°,5'-cyclic-phosphate-sodium salt [K. Menyama et al., Biochemistry, 10, 2390 (1971)]

(1.32 g, 2.7 mmol), 1,5-diazabicyclo[5.4.0]undecene-5 (0.67 mL,

4.05 mmol) and methyl iodide (0.5 mL) in 4 mL of DMSO was stirred at room temperature overnight. The reaction mixture was added to 150 ml of H 2 O containing NaHCO 3 (1.3 g) and Na 2 CO 3 (1.0 g) and heated on a steam bath for 3 hours. After adjusting the pH value to 1.5 with conc. HCl, the reaction mixture was placed under an aspirator vacuum for 1 hour. The pH was readjusted to 8.5 with 2N NaOH

and the solution was chromatographed on Dowex 1 as for compound 31. The residue from the column was suspended in MeOH and was filtered to give 375 mg (28%) of 34. An analytical sample was obtained by dissolving a sample in a large vol. with MeOH-H2O, filter and

evaporate to dryness. The residue was suspended in H 2 O, filtered and dried.

Analyzes Calculated for C18H20N506PS•1 1/2H20s C 43.90, H 4.70, N 14.22. Found C 43.72, H 4.36, N 14.36.

In the previous examples of preferred embodiments of the invention, evaporations were carried out in vacuum at < 40°. The UV spectra were determined on a Cary 15 spectrometer. The silica gel for column chromatography was E.M. Reagent Silica Gel 60 (particle size 0.063 - 0.200 mm). The eluates from the column chromatography were examined at 254 nm to detect the presence of u.f. absorbent compounds. Unless otherwise stated, the analytical samples were dried at 80-100°C at 0.01 mm for 12 hours. Tic for the NH4 salt of the compounds was determined on E. Merck Silica Gel F-254 plates and was developed with solvent system A CH^CN: 0.1M NH4Cl (4:1) or § CH^OH: CHCl-j (35:65) o The elemental analyzes were at Galbraith Laboratories, Inc., Knoxville, Tenn.

Compounds 22, 25 and 26 show positive inotropic effects in in vitro tests, and this suggests that they are active as cardiac stimulants. Many of the compounds according to the invention (e.g. compounds 4, 6, 9, 10, 11, 12, 13,

14, 21, 22, 23, 24, 25, 26, 31, 32, 33 and 38) clearly inhibit phosphodiesterase. Compounds 2a, 2b, 4, 9, 12, 21-26 and 31

is superior to cyclic AMP with regard to protein kinase activation, while compounds 2a, 2b, 11 and 21 inhibit adenyl cyclase function. Compounds 11 and 20 have exhibited anti-hypertensive properties in animal tests in vivo, and in such

tests, compounds 20 and 21 exhibited anti-inflammatory activity.

The compounds according to the invention can be provided in 2?-O-acyl form by reaction with e.g. C_~C-^3

(preferably C^-C^) acid anhydrides or acid halides in base.

Sutherland et al., Biochem. a Biophys. Acta, 148, 106 (1967)

have shown that acylation of cyclic AMP increases the cellular transport of the purine nucleotide.

Claims (21)

1. 9-3-D-ribofuranosylpurine->3',5'-cyclic-phosphate with the structure where X is -NP^R-j, -Cl, -Br or -SR, Y is -X, -OH or -NH2 , Z is hydrogen or -NH2 and R' is hydrogen or c_" C^ g acyl, R is hydrogen, phenyl, p-chlorophenyl, benzyl or lower alkyl, and R₂ and R₂ are independently selected from the group consisting of hydrogen, benzyl, lower alkyl groups alone or united to form a 5- or 6-membered heterocyclic ring, with the proviso that when one of R₂ and R₂ is hydrogen, the other is not. 2. 9-3-D-ribofuranosylpurine-3 <1> ,5 <*-> cyclic-phosphate with the structure where Z is hydrogen or -NH2 and X and Y are independently selected from the group consisting of -NR^ R2 , -Cl- -Br and -SR, R is hydrogen, phenyl, p-chlorophenyl, benzyl or lower alkyl and R, and R 2 is independently selected from the group consisting of hydrogen, benzyl, lower alkyl groups alone or joined to form a 5- or 6-membered heterocyclic ring, with the proviso that when one of R 1 and 2 is hydrogen, the other it not. 3. Compound according to claim 2 where X is -N^Rg or -SR. 4. Compound according to claim 2 where X is -NR1<R>2 or -SR, R is hydrogen, phenyl, p-chlorophenyl or lower alkyl and Y is -S-CH2. 5. Compound according to claim 3 where Z is hydrogen. 6. Compound according to claim 4 where Z is hydrogen. , 7. Compound according to claim 3, where R-^ and R^ are independently selected from the group consisting of hydrogen, methyl, ethyl, benzyl and butyl. 8. Compound according to claim 4 where R^ and R- are independently selected from the group consisting of hydrogen, methyl, ethyl, benzyl and butyl. 9. Compound according to claim 5 where R 1 and R 2 are independently selected from the group consisting of hydrogen, methyl, ethyl, benzyl and butyl. 10. Compound according to claim 6 where R 1 and R 1 are independently selected from the group consisting of hydrogen, methyl, ethyl and benzyl. 11. Connection with the structure where Z is hydrogen or amino and R" is hydrogen or C 1 -C 4 acyl. 12. Compound according to claim 11 where R' is acetyl. 13. Compound according to claim 11 where R' is hydrogen. 14. Compound according to claim 11 where Z is hydrogen. 15. 8-(Benzylthio)-6-(diethylamino)-9-B-D-ribofuranosylpurine-3',5'-cyclic-phosphate. 16. 8-(Benzylthio)-6-piperidino-9-3-D-ribofuranosylpurine-3',5'-cyclic-phosphate. 17. 8-(Benzylthio)-6-(butylamino)-9-|J-D-ribofuranosylpurine-3',5'-cyclic-phosphate. 18. 8-(p-Chlorophenylthio)-6-piperidino-9-B-D-ribofuranosyl-purine-3',5'-cyclic-phosphate. 19. 8-(p-Chlorophenylthio)-6-(diethylamino)-9-p-D-ribofuranosyl-purine-3',5'-cyclic-phosphate. 20. 8-Chloro-6-(diethylamino)-9-B-D-ribofuranosylpurine-3',5'-cyclic-phosphate. 21. 6-(Benzylamino)-8-(methylthio)-9-3-D-ribofuranosylpurine-3',5'-cyclic-phosphate.