DE4217679A1 - New cyclic guanosine-3,5-phosphoro-thioate derivs. - useful as ligands in affinity chromatography or for treating e.g. thromboses,. asthma, hypertonia or arteriosclerosis - Google Patents

Abstract

Guanosine-3',5'-phosphorothioate derivs. of formula (I) are new. R1 and R2 are H and X is Br, I, CF3, NR3R4, SR4 or OR4, or R1 and R2 together form styrylene so that (I) has a conensed tricyclic ring system of formula (II) in which X is H, I, Cl, Br, I, CF3, NR3R4, SR4 or OR4, R3 is H and R4 is alkyl, cycloalkyl, aralkyl or mono- or poly-substd. Ph of formula C6H4Y, or R3 and R4 are both alkyl, opt. joined to each other, opt. with the inclusion of a heteroatom, Y is F, Cl, Br, I, OH, SH, NH2, NO2, OMe, Me or CF3, Kat(+) is a proton or a physiologically acceptable metal cation or a trialkylammonium ion, provided that X is not thiobenzyl. (I) can be prepd. by cyclothiophosphorylation of the corresponding modified nucleoside in trialkyl phosphate with PSCl3 by halogen exchange on pre-formed 8-halogenated guanosine-3',5'-phosphonothioates with nucleophiles or by reacting pre-formed guanosine-3',5'-phosphonothioates with 2-bromo-acetophenone. USE/ADVANTAGE - (I) are cGMPS which have high lipophilia and are thus of better cell membrane permeability. They are competitive inhibitors of cGMP dependent protein kineases and are of satisfactory chemical, physical and metabolic stability. (I) can be used as inhibitors (Rp-contg. derivs.) or activators (Sp-configuration derivs.) of cGMP dependent ion channels, cyclonucleotrol dependent phosphodiesterases or other cGMP dependent bonding proteins. (I) can be used in the treatment of thromboses, asthma, hypertonia or arteriosclerosis. (I) can also be used as hydrolysis resistant ligands in liquid or affinity chromatography, in the prepn. of the stationary phase by bonding (I) onto an insoluble polymeric matrix of e.g. silica gel, glass, silicone polymer etc.

Description

The invention relates to new S _P - and R _P -configured cyclic guanosine-3,5'-phosphorothioate derivatives and physiologically acceptable salts of these compounds.

The central role of cyclic adenosine-3 ', 5'-monophosphate (cAMP) as a cellular signal transmitter (second messenger) since its discovery by Sutherland (Ann. Rev. Biochem. 37, 149 (1968)) in countless biochemical processes been confirmed.

The corresponding guanosine-3 ′, 5′-monophosphate (cGMP) also regulates obviously many cell biological processes, such as B. the Relaxation of contracted aortic muscles and inhibition of Platelet function (U. Walter, Rev. Physiol. Biochem. Pharma col. 113: 42-88 (1989)). It can both parallel the cAMP-Si gnalweg support as well as an opposite effect solve and act as a counter-regulator.  

cAMP and cGMP are usually based on external hormonal sti mulation formed by intracellular cyclases from ATP or GTP and bind to them in the course of signal transmission specific protein kinases, which in turn are other proteins activate or deactivate by phosphorylation and thus ultimately trigger a biological effect.

Various cyclonucleotide-dependent phosphodiesterases provide for the dismantling of cAMP and cGMP (J.A. Beavo, Second Mes senger and Phosphoprotein Res. 22, 1-38 (1988)) and switch in this way a triggered signal again.

The fact that many human diseases with one abnormally high or low levels of cAMP or cGMP has led to numerous attempts, the intrazel lular signal transmission to affect this "second messenger" rivers.

Too low a level of cAMP or cGMP can be caused by inhibition of the cyclonucleotide-dependent phosphodiesterases (Nicholson et al., TIBS 12, 19-27 (1991)). To this This prevents the degradation of cAMP or cGMP, so that the Concentration of these substances in the cell through accumulation increases. By administering membrane-compatible cAMP and cGMP derivatives, such as B. 8-bromo-cAMP / cGMP or 8- (4-chlorophenylthio) -cAMP / cGMP, the respective protein kinases can be activated directly and thus if the cyclonucleotide level is too low. Such modified derivatives can also be used for characterization The specificity of kinases can be used (Wolfe et al. J. Biol. Chem. 264, 7734-7741 (1989), Geiger et al., Proc. Natl. Acad. Sci. USA 89: 1031-1035 (1992)). An increase in intra cellular cAMP or cGMP concentration can also be determined by Achieve stimulation of the corresponding cyclases (forskolin, Cholera toxin, nitroprusside and the like. a.).  

Both in basic research of cellular processes, as well in terms of potential pharmacological use, however also the possibility of a lowering or even a complete one blockade of the cAMP or cGMP signal path of great importance tung.

In addition to some non-specific isoquinoline derivatives (Chÿawa et al., J. Biol. Chem. 265, 5267 (1990)) and a protein-based inhibitor (Walsh et al., J. Biol. Chem. 246, 1977 (1971)) mainly R _P - configured derivatives of adenosine-3 ′, 5′-monophosphorothioate (R _P -cAMPS). It acts as a cAMP antagonist and inhibitor of the CAMP-dependent protein kinases type I and II (cAK I + II) (Botelho et al., Methods Enzymol. 159, 159 (1988)). Further diastereomeric cAMP derivatives with R _P configuration have already been presented (Dostmann et al., J. Biol. Chem. 265, 10 484 (1990)) and are currently being examined for their biological properties.

Since it has been shown in the case of the cAMPS derivatives that not all R _P diastereomers are inhibitors of the cyclonucleotide-dependent protein kinases, but can also have an agonistic effect (Sandberg et al., Biochem. J. 279, 521-527 ( 1991)), and since most of the 20 R _P isomers examined so far are only partial antagonists and more agonists of cAK, there is currently no structural criterion that allows a reliable prediction of whether an R _P -configured adenosine -3 ', 5'-phosphorothioate derivative which can completely inhibit cAK I or II or not.

In contrast to the cAMPS derivatives, only two sulfur-modified compounds of cGMP with inhibitory properties have been described so far: R _P -guanosine-3 ′, 5′-monophosphorothioate (R _P -cGMPS) and R _P -8-chloroguanosine-3 ′, 5 'Monophosphorothioate (R _P - 8-Cl-cGMPS) (Butt et al., FEBS Lett. 263, 47 (1990)).

However, the potential use of these substances as inhibitors of the cGK signaling pathway has serious disadvantages. So z. B. for R _P -cGMPS the required kinase selectivity is not given. Furthermore, the lipophilicity, which is crucial for the penetration of the cell membrane, is quite low in both cases, although the modification with sulfur on average leads to a doubling of the lipophilicity (Braumann et al., J. Chromatogr. 350, 105-118 (1985)).

The task was therefore to find new cGMPS derivatives that a higher lipophilicity and thus a better cell membrane per possess meability that selectively select the cGMP-dependent protein ki competitively inhibit noses and also have sufficient chemi have physical, physical and metabolic stability.

To solve this problem, the compounds described in claim 1 are proposed. Surprisingly, it was found that the above-mentioned requirements for inhibitors of the cGMP-dependent protein kinase, which are particularly advantageous, are met by the R _P -configured cGMPS derivatives according to the invention. It is particularly surprising that even modifications that can clearly stimulate the cAMP-dependent protein kinases in the form of the corresponding R _P -cAMPS derivatives inhibit the cGK kinase as R _P cGMPS analogs. The high selectivity of the compounds according to the invention is also particularly advantageous. Inhibitory or activating effects on the corresponding cAK's only become apparent at very much higher concentrations than are necessary on the cGK. The S _P - configured cGMPS derivatives according to the invention are suitable as activators of cGMP-dependent protein kinases.

The substances found combine a number of properties that make their intensive use in cellular signal transmission research possible and enable use as a therapeutic agent for hyperfunction of the cGMP system as well as a pharmacologically useful blockade of normal cGMP levels. In addition, the compounds according to the invention are suitable as modulators, ie as inhibitors (R _P -configured derivatives) or activators (S _P -configured derivatives), from cGMP-dependent ion channels, cyclonucleotide-dependent phosphodiesterases or other cGMP-dependent binding proteins. As is known, the sulfur modification ensures that all R _P -configured compounds have full stability against all known cyclonucleotide-degrading phosphodiesterases in mammals, including humans; in the S _P -configured compound, the sulfur modification leads to a strong delay in the enzymatic degradation.

Compared to known nucleoside cyclophosphates, the compounds of the formula I according to the invention have an increased lipophilicity and thus an improved membrane permeability. Substitution with sulfur on the phosphate residue in the axial position (S _P -dia stereomers) leads to kinase-activating compounds, whereas an equatorial position of the sulfur leads to cGK antagonists (R _P -diastereomers).

R ¹ and R ² can each represent a proton, where X is -Br, -J, -CF ₃ or a radical of the general formulas -NR ³ R ⁴ , -SR ⁴ or -OR ⁴ , where R ^{3 is} hydrogen and R ⁴ an alkyl, cycloalkyl, Aral kyl- or a single or multiple, identical or mixed substituted phenyl radical of the general structure

and Y is -F, -Cl, -Br, -J, -OH, -SH, -NH ₂ , -NO ₂ , -OCH ₃ , -CH ₃ or -CF ₃ . In addition, R ³ and R ^{4 can} both be alkyl radicals, which can be linked together to form a ring, including other heteroatoms.

Furthermore, compounds with R _P configuration on the phosphate radical, in which R ¹ and R ² together represent a styrenic radical and form a condensed, tricyclic ring system of the formula II, have antagonistic properties.

In this case, X is hydrogen, -F, -Cl, -Br, -J, -CF ₃ or an organic radical of the general formulas -NR ³ R ⁴ , -SR ⁴ or -OR ⁴ . R ³ and R ⁴ have the same meaning as above.

Compounds with the hydrophobic aromatic substituents in Position 8 are preferred in both cases, since this is a be have particularly high lipophilicity and thus the use of this Enable connections even on intact cells. Especially Compounds which are in position 8 are preferred Phenylthio residues, such as. B. a 4-chlorophenlythio or 4-Hy droxyphenylthio radical are substituted.  

Primary and secondary amine, thiol or aliphatic and aromatic thioether, hydroxy or aliphatic and aromatic ether substituents are also preferred. Here are ringför shaped secondary amines, such as. B. piperidine is particularly preferred (X = NR ³ R ⁴ , R ³ and R ⁴ together are a pentamethylene radical). In addition, such compounds occupy a preferred position, the non-sulfur-modified analogues are already used in biochemistry and medicine, such as. B. 8-bromguanosine-3 ', 5'-monophosphate (8-Br-cGMP), 8- (4-chlorophenylthio) guanosine-3', 5'-monophosphate (8-pCPT-cGMP) or β-phenyl-1 , N ² -ethenoguano sin-3 ′, 5′-monophosphate (PET-cGMP).

Kat⁺ can be a proton, a physiologically compatible metal cation or a trialkylammonium ion. Preferred metal cations are Na⁺, K⁺, Li⁺, Ca ²⁺ and Mg ²⁺ .

To the membrane passage of the compounds of the invention to further increase, it may be advantageous to the 2'-hydroxyl group of the pentose residue and / or the phosphate residue with a hydrophobic group to derivatize pen by intracellular enzymes, hydroly table or by irradiation with light of a certain Wavelength in the visible or ultraviolet range Penetration of the cell membrane can be split off.

Suitable enzymatically removable protective groups for the 2'-hydroxyl group are esters of linear or branched carboxylic acids of short or medium chain lengths (C ₁ -C ₈ ), for the phosphate radical there are esters with linear or branched C ₁ -C ₈ alcohols with primary , secondary or tertiary hydroxyl group, with benzyl alcohol or the derivatization with the acetoxymethyl group.

Silyl groups may be mentioned as an example of groups which can be split off hydrolytically from both the 2′-hydroxyl group and from the phosphate residue. Trialkylsilyl groups, in particular trialkylsilyl groups with straight-chain or branched C ₁ -C ₅ -alkyl radicals, are particularly suitable. The trimethylsilyl group is particularly suitable.

Suitable groups which can be removed from light are suitable for the phosphate residue e.g. B. the 1- (2-nitrophenyl) ethyl or 4,5-dimethoxy-2-ni trobenzyl group, suitable for the derivatization of the 2'-OH group especially the butyryl group.

The derivatization of the phosphate group can be done on oxygen as well as on the sulfur atom, those on the acid are preferred Substance derivatized compounds. In addition, Gemi ces from oxygen and sulfur derivatives can be used.

It may also be useful to use the ver To derivatize bonds with fluorescent groups to them to be able to localize in the cell or to sensitive quan To enable certifications in connection with phosphodiesterases chen (J. Grewal et al., Biochemistry International, 19, 1287 (1989)).

A derivative, for example, is suitable as a fluorescence marker sation of the 2'-hydroxyl group with the anthraniloyl or N-Me thylanthraniloyl residue.

The use of the compounds according to the invention as medicaments is preferably carried out in combination with the usual pharmacological ones Carriers and / or diluents and / or auxiliaries e.g. B. as ointments, powder, tablets, capsules, drops, supposito rien, infusion or injection solutions. Indications of such Medicines are e.g. B. thrombosis, asthma, hypertension, arteros clerosis.

Because of their specific binding capacity and their hydrolysis resistance, the compounds according to the invention are suitable for the production of stationary phases for liquid chromatography, in particular for affinity chromatography, for example cyclonucleotide-dependent binding proteins. Here the compounds are bonded to insoluble polymeric supports via terminal reactive groups, such as halogens, amino or hydroxyl groups. The reactive groups are either bound directly (e.g. X = halogen, NH ₂ , OH) or via molecular spacers to the heterocycle.

Spacers of the structure X = -NHR ⁵ are preferred, where R ^{5 is} a linear alkyl, polyethylene glycol, polyethyleneimine or polypeptide residue with a terminal reactive group. Spacers with a length of 2 to 12 atoms or 1 to 5 amino acids in the case of the polypeptides are particularly preferred. Amino groups and in particular primary amino groups are preferred as terminal, reactive groups.

Suitable insoluble polymeric matrix are silica gel, glass, Silicone polymers, polyacrylamide, polystyrene, agarose, such as. B. Sepharose®, cellulose or dextran, such as. B. Sephadex®. Prefers are carrier materials that already contain spacers and therefore particularly suitable for binding the derivatives without spacers nen. These include, in particular, commercially for coupling Affinity ligands offered ethylene glycol methacrylic acid copo polymers such. B. Fraktogel TSK®, CNBr or epoxy activated Sepharose® or Sephadex® or derivatized porous glass balls (CPG). Preferably, the stationary invention Ready-to-use phases, d. H. in the form of carrier material alien with ligands bound to it.

The synthesis of the new compounds takes place in a manner known per se either by cyclothiophosphorylation of the correspondingly modified nucleosides in trialkyl phosphates with PSCl ₃ (Genie ser et al., Tetrahedron Lett. 29, 2803 (1988); WO 89/07108; DE-OS 38 02 685) by exchanging halogen on already preformed, 8-halogenated guanosine-3 ′, 5′-phosphorothioates with the corresponding nucleophiles (Miller et al., Biochemistry 12, 5310-5319 (1973)) or by converting preformed guano sin -3 ′, 5′-phosphorothioates with 2-bromoacetophenone (Miller et al., Biochem. Pharmacol. 30, 509-515 (1981)).

The derivatization of the 2'-hydroxyl and the phosphate group Compounds according to the invention are carried out according to standard methods. The 2'-hydroxyl group can be, for example, by reaction with Carboxylic acid chlorides or anthraniloyl chlorides and pyridine esterify, the esterification of the phosphate residue takes place, for. B. by Reaction with diazo compounds (J. Engels, Bioorg. Chem., 8, 9-16 (1979)).

Silyl protecting groups are, for example, by implementing the Phosphorothioates with trialkylsilyl chlorides and pyridine (Gaffney et al., J. Am. Chem. Soc., 104, 1316-1319 (1982)).

A derivatization of the phosphate residue with photolabile groups (cage compounds) are produced by reaction of the cyclothiophosphates with corresponding diazo compounds (McCray et al., Annu. Rev. Biophys. Biophys. Chem., 18, 239 (1986)).

Immobilization of the derivatives for the affinity chromatogra phie on coupling-capable carrier materials with training hy Drolytically stable bonds are made in a known manner. In the The rule is the affinity according to the invention to be bound ligand with the carrier material suspended in buffer at room temperature or a higher temperature.

The lipophilicity of the compounds according to the invention is determined in a manner known per se with the aid of reversed-phase high-pressure liquid chromatography as a Log K _w value which is roughly comparable to the Log P value (Braumann et al., J. Chromatogr. , 350: 105-118 (1985)). The log K _w value for the parent connection cGMP is 0.85.

The isolation of cGMP-dependent protein kinase type I from Rinder lung and cAMP-dependent protein kinase type II from rinder heart is carried out in a manner known per se (Walter et al., J. Biol. Chem. 255, 3757-3762 (1980)) . The measurement of the Proteinkina se activity is carried out by the method of Roskoski (Methods Enzy mol. 99, 3 -6 (1983)). The in vitro measurements on human platelet membranes are carried out in a manner known per se (Halbrügge et al., Eur. J. Biochem. 185, 41-50, (1989)).

cGK antagonists inhibit cGMP-dependent, depending on the concentration phosphorylation of a 46/50 kDa band, but not the cAMP specific phosphorylation of the 22 kDa and 240 kDa bands. Particularly well membrane-permeable compounds are in vivo intact human platelets in a manner known per se (Hal Brugge et al., J. Biol. Chem. 265, 3088-3093 (1990)).

The invention is explained below using examples.

example 1 Cyclic 8-bromoquanosine-3 ′, 5′-monophosphorothioate, R _P - and S _P -diastereomer (R _P - / S _P -8-Br-cGMPS)

The compounds are synthesized in a manner known per se by cyclothiophosphorylation of 8-bromoguanosine in trialkyl phosphates with PSCl ₃ (Genieser et al., Tetrahedron Lett. 29, 2803 (1988); WO 89/07108; DE-OS 38 02 685) . In addition to the target compounds, the reaction mixture contains, inter alia, the R _P and S _P diastereomers chlorinated in position 8 (R _P - / S _P -8-Cl-cGMPS). Chromatography on R _P -18 reversed phase silica gel (eluent: 10% methanol / 100 mM triethylammonium formate buffer) and lyophilization of the product-containing fractions results in mixtures of R _P -8-Br / Cl-cGMPS and S _P -8- Br / Cl-cGMPS, which are separated by renewed, preparative chromatography on R _P -18 silica gel (eluent: 7% or 8% methanol / 100 mM triethylammonium formate buffer). The diastereomers of 8-Br-cGMPS obtained by combining and lyophilizing product-containing fractions are optionally further purified by repeated chromatography under the same conditions. R _P -8-Br-cGMPS contains no detectable traces of the kinase-active S _P -diastereomers S _P -8-Cl / Br-cGMPS and less than 0.05% of the normal cyclophosphate 8-Cl / Br-cGMP. The purity is <98%. Yield: 6% of theory, as the triethylammonium salt. S _P -8-Br-cGMPS contains no detectable traces of the corresponding R _P -diastereomers. The purity is <98%. Yield: 5% of theory, as the triethylammonium salt.
Molecular formula: C₁₀H₁₁O₆N₅SPBr (MW: 440.12; free acid),
UV (H₂O): λ _max = 260 nm (ε = 15,000 at pH 7)

FAB-MS (glycerin)

Biological measurements

R _P -8-Br-cGMPS proves to be an antagonist of the cGK with an inhibition constant of K _i = 4 µM.

On the cAMP-dependent protein kinase II, the compound shows partial agonistic properties only at high concentrations (K _a = 60 µM).

R _P -8-Br-cGMPS inhibits the phosphorylation of a 46/50 kDa band in human platelet membranes.

The cAMP-dependent phosphorylation of the 22 and 240 kDa bands is not affected.

S _P -8-Br-cGMPS is a slightly poorer activator of cGK compared to cGMP and can also activate cAK II at a similar concentration.

Example 2 Cyclic 8- (4-chlorophenylthio) quanosine-3 ′, 5′-monophosphorothioate, R _P -diastereomer (R _P -8-pCPT-cGMPS)

The synthesis of the compound is carried out either by substitution of the 8-position halogenated cGMPS derivative (R _P -8-chlorine / bromine-cGMPS) with 4-chlorothiophenol (Miller et al., Biochemistry 12, 5310-5319 (1973)) or by cyclothiophosphorylation of 8- (4-chlorophenylthio) guanosine obtained by substitution of 8-bromoguanosine with 4-chlorothiophenol (Genieser et al., Tetrahedron Lett. 29, 2803 (1988)). Preparative chromatography on R _P -18 reversed phase silica gel (mobile solvent: 30% methanol / 100 mM triethyl ammonium formate buffer) and lyophilization of the combined product-containing fractions gives the product, which is optionally further purified by renewed chromatography under the same conditions. Yield: 62% of theory (substitution) or 5% of theory (cyclothiophosphorylation), (in each case as triethylammonium salt; purity: <98% (HPLC)). The product contains no detectable traces of the kinase-active S _P -diastereomer S _P -8-pCPT-cGMPS and less than 0.05% 8-pCPT-cGMP.

Molecular formula: C₁₆H₁₅O₆N₅S₂PCl (MW: 503.9; free acid)
UV: (H₂O): λ _max = 276 nm (ε = 21 500 at pH 7).

FAB-MS (glycerin):

Negative mode
m / z = 292 (10%: base -H) ⁻
m / z = 502 (55%; M-H) ⁻
m / z = 594 (35%; M - H + Gly) ⁻

Positive fashion
m / z = 294 (30%: B + H) ⁺
m / z = 504 (15%: M + H)) ⁺
m / z = 526 (20%: M + Na) ⁺
m / z = 548 (20%: M + 2Na) ⁺

1 H-NMR (DMSO-d₆ / TMS): δ = 5.85 ppm (s: H1 ′)
31 P NMR (MeOH / PO₄): δ = 58.4 ppm

Biological properties

R _P -8-pCPT-cGMPS proves to be an antagonist of the cGK with an inhibition constant of K _i = 0.7 µM, on the cAMP-dependent kinase the compound shows weak agonistic properties (400 µM) only at high concentrations.

R _P -8-pCPT-cGMPS inhibits the phosphorylation of a 46/50 kDa band in human platelet membranes, the cAMP-dependent phosphorylation of the 22 and 240 kDa bands is not affected.

The compound is sufficiently membrane-permeable and inhibits cGK Phosphorylation in intact human platelets.

Example 3 Cyclic β-phenyl-1, N ² -ethenoguanosine-3,5′-monophosphorothioate R _P -diastereomer (R _P -PET-cGMPS)

The compound is synthesized starting from R _P -cGMPS, which is obtained in a manner known per se by cyclothiophosphorylation of guanosine in trialkyl phosphates with PSCl ₃ (Genieser et al., Te trahdron Lett. 29, 2803 (1988). 231 mg R _P cGMPS (500 mmol; triethylammonium salt) are suspended in 5 ml of dimethyl sulfoxide, after adding 100 µl of 1,8-diazabicyclo [5.4.0] undec-7-ene as auxiliary base and 100 mg of 2-bromoacetophenone, the mixture is stirred at room temperature (yellow color) After 4 hours, another 100 μl of the auxiliary base and a further 100 mg of 2-bromoacetophenone are added. The course of the reaction is monitored using high pressure liquid chromatography (anion exchanger based on silica gel, 400 mM phosphate buffer, 40% methanol) and the reaction after the disappearance of the Signals for R _P -cGMPS stopped by adding 10 ml of water (reaction time about 24 hours).

The solution is adjusted to pH 7.5, the turbidity is filtered off and discarded. The free acid of the R _P -PET-cGMPS is then precipitated by adjusting the pH to 1.5 with hydrochloric acid and the yellowish precipitate obtained is washed first with water and then with acetone. The crude product is recrystallized in 10 ml of water and dissolved with 1 M NaOH at pH 7. Another precipitation as free acid is carried out with hydrochloric acid at pH 1.5. The precipitate is filtered off and washed with water and acetone. Yield: 46 mg (20% of theory); Purity <90%.

Molecular formula: C₁₈H₁₆O₆N₅SP (MW: 461.4; free acid)
UV (H₂O): λ _max = 252 nm
Lipophilic determination: Log K _w = 2.81

Example 4 Immobilization of R _P -8-Br-cGMPS on a support material for affinity chromatography

200 ml Fraktogel TSK AF Amino 650® (Merck) are washed in a glass sinter frit with water and then suspended in a 250 ml 2-neck flask in 100 ml sodium acetate buffer (20 mM). 280 mg of R _P -8-Br-cGMPS (500 μmol triethylammonium salt) are added and the suspension is moderately stirred at 80 ° C. under reflux. The decrease in unreacted R _P -8-Br-cGMPS is checked with high pressure liquid chromatography (R _P -18, 10% methanol / 100 mM triethylammonium formate buffer). After 48 hours, the suspension is filtered, the residue was washed with water until the absorption was constant at 260 nm and protected against microbial attack with 1 mM sodium acid.

Claims (29)

1. Guanosine-3 ', 5'-phosphorothioates of the general formula I in which R ¹ and R ^{2 are} hydrogen and X is -Br, -J, -CF ₃ , or a radical of the general formulas -NR ³ R ⁴ , -SR ⁴ or -OR ⁴ , where R ^{3 is} hydrogen and R ^{4 is} a Alkyl, cycloalkyl, aralkyl or a mono- or polysubstituted, identically or mixedly substituted phenyl radical of the general structure and Y is -F, -Cl, -Br, -J, -OH, -SH, -NH2, -NO ₂ , -OCH ₃ , -CH ₃ or -CF ₃ , or
R ³ and R ^{4 are} both alkyl radicals, it being possible for the alkyl radicals to be linked to form a ring, including further heteroatoms, but X is not a thiobenzyl radical, or
R ¹ and R ² together represent a styrenic radical and form a condensed tricyclic ring system according to formula II, in which X is hydrogen, -F, -Cl, -Br, -J, -CF ₃ or a radical of the general formulas -NR ³ R ⁴ , -SR ⁴ or -OR ⁴ , where R ³ and R ^{4 have the} same meaning as have above, and Kat⁺ is in each case a proton or a physiologically compatible metal cation or a trialkylammonium ion. 2. Compounds according to claim 1, characterized in that the sulfur atom of the thiophosphate residue is in the eguatorial position (R _P isomers). 3. Compounds according to claim 1, characterized in that the sulfur atom of the thiophosphate residue is in the axial position (S _P isomers). 4. Compounds according to claims 1 to 3, characterized records that X is -Br. 5. Compounds according to claims 1 to 3, characterized records that X is a 4-chlorophenylthio radical. 6. Compounds according to claims 1 to 3, characterized records that X is a 4-hydroxyphenylthio radical. 7. Compounds according to claims 1 to 3, characterized in that Kat⁺, Na⁺, K⁺, Li⁺, 1/2 Ca ²⁺ or 1/2 Mg ²⁺ is. 8. Compounds according to claims 1 to 3, characterized in that R ³ and R ⁴ together are a pentamethylene radical. 9. Compounds according to claims 1 to 3, characterized in that R ¹ and R ² together represent a styrenic radical and X is hydrogen. 10. Compounds according to claims 1 to 3, characterized in that R ¹ and R ² together represent a styrenic radical and X is bromine. 11. Compounds according to claims 1 to 10, characterized records that the 2'-hydroxyl group and / or the phosphate rest with an enzymatic, hydrolytic or photolytic removable hydrophobic group is derivatized. 12. Compounds according to claim 11, characterized in that the 2'-hydroxyl group is esterified with linear or branched C ₁ -C ₈ - carboxylic acids. 13. Compounds according to claim 12, characterized in that the 2'-hydroxyl group is esterified with butyric acid. 14. Compounds according to claim 11, characterized in that the phosphate residue with linear or branched primary, secondary or tertiary C ₁ -C ₈ alcohols, benzyl alcohol, the acetoxymethyl group or a 1- (2-nitrophenyl) ethyl or a 4, 5-Dimethoxy-2-nitro-benzyl group is derivatized. 15. Compounds according to claims 1 to 10, characterized records that the 2'-hydroxyl group and / or the phospha with an enzymatic, hydrolytic or photolytic cleavable fluorescent group is derivatized.   16. Compounds according to claim 15, characterized in that the 2'-hydroxyl group with an anthraniloyl or an N- Methylanthraniloylgruppe is derivatized. 17. Compounds according to claim 11, characterized in that the 2'-hydroxyl group and / or the phosphate residue with one Trialkylsilylgruppe is derivatized. 18. Compounds according to claim 17, characterized in that the 2'-hydroxyl group and / or the phosphate residue with one Trimethylsilyl group is derivatized. 19. Compounds according to general formulas I or II, since characterized in that X is a molecular spacer is that has terminal, reactive groups. 20. Compounds according to claim 19, characterized in that X is an organic radical of the general formulas -NHR ⁵ , -SR ⁵ or -OR ⁵ , where R ^{5 is} an alkylene, polyethylene glycol, polyethyleneimine or polypeptide radical. 21. Compounds according to claim 20, characterized in that the chain lengths of the alkyl, polyethylene glycol, polyethylene enamine residues 1 to 12 atoms and those of the polypeptide residues 1 to 5 amino acids. 22. Compounds according to claims 19 to 21, characterized records that the terminal reactive group is a primary or secondary amino group. 23. Use of compounds according to Claim 2 or R _P -configured compounds according to Claims 4 to 18 as inhibitors of cGMP-dependent protein kinases. 24. Use of compounds according to claim 3 or S _P -konfi-configured connections to claims 4 to 18 as activators cGMP-dependent protein kinases according. 25. Use of the compounds according to claims 1 to 18 as modulators of cGMP-dependent ion channels, cyclonucleo tid-dependent phosphodiesterases or other cGMP-dependent binding proteins. 26. Medicament containing one or more compounds according to claims 1 to 18 as an active pharmaceutical ingredient in combination with pharmacologically usual carriers and / or diluents and / or auxiliaries. 27. Medicament according to claim 26 for the treatment of thrombosis, Asthma, hypertension or arteriosclerosis. 28. Use of compounds according to claims 1 to 10 or 19 to 22 as enzymatically hydrolysis-resistant ligands in liquid or affinity chromatography. 29. Use of compounds according to claims 1 to 10 or 19 to 22 for the production of stationary phases for liquid or affinity chromatography, the Connections to an insoluble polymeric matrix made of pebble gel, glass, silicone polymers, polyacrylamide, polystyrene, Ethylene glycol-methacrylic acid copolymer, agarose, cellulose or dextran are bound and the matrix in the case of Ver bindings according to claims 1 to 11 suitable spacers for Binding of ligands.