NO301423B1 - Analogous Process for Preparation of Purine Nucleoside Phosphorylase Inhibitors - Google Patents

Description

The present invention relates to an analogous method for the production of a compound which is a purine nucleoside phosphorylase inhibitor.

Purine nucleoside phosphorylase (PNP) catalyzes the phosphorolysis of purine nucleosides in a reversible reaction. Individuals lacking PNP exhibit impaired T-cell development resulting in lower cell-mediated immunity, but normal B-cell development resulting in normal humoral immunity. Specific inhibitors of PNP that selectively inhibit T-cell development without damaging humoral immunity may be potentially effective against disorders where activated T-cells are pathogenic.

The present invention therefore relates to an analogous process for the preparation of a compound which is a purine nucleoside phosphorylase inhibitor selected from the group consisting of 2-amino-7-(R)-3H,5H-pyrrblo[3,2-d]pyrimidin-4-one ( I) and a compound of formula II

where for compound I R is cyclohexyl, cyclohexenyl or

-CH2-R1, and where R^ is a lower alkyl or trifluoromethyl substituted or unsubstituted C^C8 cycloalkyl or cycloalkenyl group, a tetrahydrofuranyl group, an adamantyl group or a pyridyl group and wherein the compound of formula II is R;^ H, NH2 or SCH3 , R<2>is a phenyl group possibly substituted with one or more halogen atoms or is a

C4-C8 cycloalkyl group, m is 0-4, X is OH CN, carboxy lower alkoxycarbonyl or aminocarbonyl, characterized by the preparation of the compound I in which R is cyclohexenyl or -CZtø-Ri when R^ is pyridinyl or alicyclic group comprises the following steps 1) to 7): 1) reacting a 3-substituted propanenitrile with an alkyl formate and a base to form a 3-cyclo-2-formyl-propanenitrile; 2) reaction of 2-formyl-3-substituted propanenitrile with glycine methyl ester hydrochloride and sodium or ammonium acetate to form the corresponding enamine; 3) reaction of the enamine with an alkyl chloroformate and DBN or DBU to form the corresponding N-blocked pyrrole; 4) reaction of N-blocked pyrrole with a base to form corresponding deblocked pyrrole; 5) reaction of deblocked pyrrole with benzoyl isothiocyanate to form the corresponding thioureido compound; 6) reacting the thioureido compound with an alkyl halide to form the corresponding methylthio compound; and 7) reacting the methylthio compound with methanolic or ethanolic ammonia to form a mixture of the 2-amino-7-(R)-3E,5H-pyrrolo[3,2-d]pyrimidin-4-one compound (I) and 2 the -methylthio byproduct; and when compound I is prepared in which R is cyclohexenyl or -CH 2 -R 1 when R 2 is a cycloalkenyl group, steps 1) to 7) as defined above and optionally the following step 8: 8) hydrogenation of 2-amino-7-(R)-3H ,5H-pyrrolo[3,2-d]pyrimidin-4-one compound from step 7) to 2-amino-7-(R)-3H,5H-pyrrolo[3,2-d]pyrimidin-4-one; and optionally the following steps 1) to 9) when the compound I is prepared where R is CEto-Ri when R^ is tetrahydrofuranyl, 1) reaction of a 3-substituted or unsubstituted furanyl-propanenitrile with an alkyl formate and a base to form a 3- substituted or unsubstituted furanyl-2-formyl-propanenitrile; 2) reaction of 3-substituted or unsubstituted furanyl-2-formyl-propanenitrile with glycine methyl ester hydrochloride and sodium or ammonium acetate to form the corresponding enamine; 3) reaction of the enamine with an alkyl chloroformate and DBN or DBU to form the corresponding N-blocked pyrrole; 4) reaction of N-blocked pyrrole with a base to form corresponding deblocked pyrrole; 5) reaction of deblocked pyrrole with benzoyl isothioureanate to form the corresponding thioureido compound; 6) reacting the thioureido compound with an alkyl halide to form the corresponding methylthio compound; 7) reaction of the methylthio compound with methanolic or ethanolic ammonia to form a mixture of 2-amino-7-substituted or unsubstituted furanyl-3H,5E-pyrrole[3,2-d]pyrimidin-4-one compound (I) and the 2-methylthiobi product; 8) hydrogenation of the 2-amino-7-substituted or unsubstituted furanyl-3H,5H-pyrrolol[3,2-d]pyrimidin-4-one compound from step 7) to 2-amino-7-substituted or unsubstituted tetrahydrofuranyl-3H ,5B>pyrro[3,2-d]pyrimidin-4-one; and and wherein the preparation of compound of formula II comprises the following steps: a) reacting an optionally substituted cyclic aldehyde with cyanoacetic acid in the presence of ammonium acetate to form a 3-substituted pentanedinitrile; b) reacting 3-substituted-pentanedinitrile with an alkyl formate and a base to form a 2-formyl-3-substituted-pentanenitrile; c) reacting 2-formyl-3-substituted-pentanedinitrile with glycine methyl ester hydrochloride and sodium or ammonium acetate to form methyl N-[3-substituted-2,4-dicyano)-2-butenyl]glycine; d) reacting methyl N-[3-substituted-2,4-dicyano)-2-butenyl]glycine with an alkyl chloroformate and DBN or DBU to form l-ethyl-2-methyl 3-amino-4-substituted lH- pyrrole-1,2-dicarboxylate; and e) reacting 1-ethyl-2-methyl-3-amino-4-substituted-1E-pyrrole-1,2-dicarboxylate with a base to form methyl 3-amino-4-substituted-1H-pyrrole-2 -carboxylate (i) and the following steps f)-h) for the preparation of 3-substituted-3-[2-amino-4-oxo-3H-5H-pyrrolo[3,2-d]-pyrimidin-7-yl] -propanenitrile; f) reacting methyl 3-amino-4-substituted-1E-pyrrole-2-carboxylate with benzoyl isothiocyanate to form N-benzoyl-N'-(2-methoxycarbonyl-4-substituted-1E-pyrrole-3-benzoylisothiocyanate to forming N-benzoyl-N'-(2-methoxycarbonyl-4-substituted-1E-pyrrol-3-yl)thiourea; g) reaction of N-benzoyl-N'-[2-methoxycarbonyl-4-substituted-1E-pyrrole -3-yl]thiourea with an alkyl halide to

prepare N-benzoyl-N'-2-methoxycarbonyl-4-substituted-1E-pyrrol-3-yl]-S-alkylthiourea; and

h) reaction of N-benzyl-N'-[2-methoxycarbonyl-4-substituted-1E-pyrrol-3-yl]-S-alkylthiourea with methanolic or

ethanolic ammonia to form a mixture of 3-substituted 3-(2-amino-4-oxo-3E,5E-pyrrolo[3,2-d]pyrimidine-

7-yl)propanenitrile and 3-substituted-3-(2-methylthio-4-oxo-3H,5H-pyrrolo[3,2-d]pyrimidin-7-yl)propanenitrile; (ii) and the following steps f)-j) in the preparation of 3-substituted-3-[2-alkoxy-4-oxo-3E,5H-pyrrolo[3,2-d]pyrimidin-7-yl]propanenitrile; f) reacting methyl 3-amino-4-substituted-1E-pyrrole-2-carboxylate with benzoyl isothiocyanate to form N-benzoyl-N'-2-methoxycarbonyl-4-substituted-1H-pyrrol-3-yl)thiourea; g) reacting N-benzoyl-N'-(2-methoxycarbonyl-4-substituted-1H-pyrrol-3-yl)thiourea with an alkyl halide to form N-benzoyl-N'-(2-methoxycarbonyl-4-substituted -1E-pyrrol-3-yl]-S-alkylthiourea; and h) reaction of N-benzoyl-N'-(2-methoxycarbonyl-4-substituted-1H-pyrrol-3-yl(-S-alkylthiourea) with methanolic or ethanolic ammonia to form a mixture of 3-substituted-3-(2-amino-4-oxo-3E,5B-pyrrolo[3,2-d]pyrimidin-7-yl)propanenitrile and 3-substituted-3 -(2-alkylthio-4-oxo-3E,5E-pyrrolo[3,2-d]pyrimidin-7-yl)propanenitrile; i) conversion of 3-substituted-3-(2-methylthio-4-oxo-3E ,5E-pyrrolo[3,2-d]pyrimidin-7-yl)propanenitrile with an oxidizing agent to form 3-substituted-3-(2-methyl-sulfonyl-4-oxo-3H,5H-pyrrolo[3 ,2-d]pyrimidin-7-yl)propanenitrile; and j) reaction of 3-substituted-3-(2-methylsulfonyl-4-oxo-3H,5H-pyrrolo[3,2-d]pyrimidin-7-yl ]propanenitrile with sodium alkoxide to form 3-substituted-3-(2-alkoxy 4-oxo-3H,5H-pyrrolo[3,2-d]pyrimidin-7-yl)propanenitrile; (iii) and the following step f)-g) in the production of 3- subst in tert-3-(4-oxo-3E,5H-pyrrolo[3,2-d]pyrimidin-7-yl)propanenitrile;

k) reacting methyl 3-amino-4-substituted-1E-pyrrole-2-carboxylate with dimethylformamide dimethyl acetal to form methyl 4-substituted-3-[N-(dimethylaminomethylene)amino]-1E-pyrrole-2-carboxylate; and

1) reaction of methyl 4-substituted-3-[N-(dimethylamino-methylene)amino]-1E-pyrrole-2-carboxylate with methanolic ammonia to form 3-substituted-3-(4-oxo-3H,5H- pyrrolo[3,2-d]pyrimidin-7-yl)propanenitrile.

A pharmaceutical composition for selective suppression of mammalian T-cell function without reducing the effect on humoral immunity comprising an effective amount of the compound and a pharmaceutically acceptable diluent can therefore be prepared. These are suitable for enteral, such as oral or rectal, transdermal and parenteral administration to mammals including humans, and can inhibit purine nucleoside phosphorylase activity.

The pharmaceutical compositions can be tablets and gelatin capsules comprising the active ingredient together with a) diluent, e.g. lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and/or glycerin; b) lubricants, e.g. silica, talc, stearic acid, its magnesium or calcium salt and/or polyethylene glycol; for tablets also c) binders, e.g., magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidone; if desired d) solvents, e.g. starches, agar, alginic acid or sodium salts thereof, or effervescent mixtures; and/or e) adsorbing agents, coloring agents, flavoring agents and sweetening agents. Injectable compositions are preferably aqueous isotonic solutions or suspensions, and suppositories are preferably prepared from fatty emulsions or suspensions. Said compositions can be sterilized and/or contain auxiliaries, such as preservatives, stabilizers, wetting agents or emulsifiers, dissolution promoting agents, salts for regulating the osmotic pressure and/or buffers. In addition, they may also contain other therapeutically valuable compounds. Said compositions are prepared according to conventional mixing methods, granulation methods or coating methods and may contain about 0.1 to 75%, preferably about 1 to 50% of the active ingredient.

Suitable formulations for transdermal application comprise an effective amount of a compound of the invention with a carrier. Advantageous carriers include adsorbable pharmacologically acceptable solvents to aid passage through the skin of the host. Transdermal devices are usually in the form of a bandage comprising a support joint, a reservoir containing the compound optionally with carriers, optionally a rate-controlling barrier to deliver the compound to the skin of the host at a controlled and predetermined rate over an extended period of time, and means for securing the device to the skin.

The purine nucleoside phosphorylase activity in mammals can inhibit diseases and conditions that react to it, e.g. autoimmune disorders, transplant rejection or psoriasis can be treated by administering to a mammal in need thereof an effective amount of a compound of the invention.

Pharmaceutically acceptable effective dosages of the active compound produced according to the invention to be administered depend on the species of the warm-blooded animal (mammal), body weight, age and individual condition, and on the form of administration.

The pharmaceutical composition may be oral, parenteral, suppository, or in some other form that delivers the compound into the bloodstream of a mammal to be treated. An oral form has from about 1 to about 150 mg of the compound for an adult (50 to 70 kg) which is mixed with pharmaceutically acceptable diluents such as lactose. In a regular capsule, 25 mg of the compound is mixed together with 192 mg of lactose, 80 mg of modified starch and 3 mg of magnesium stearate. Injectable forms of the compound are also being considered for administration.

The compound prepared according to the present invention is also useful with other therapeutic agents. A daily dosage for a human weighing 50 to 70 kg of 1-50 mg/kg inhibits the metabolic breakdown of certain anti-cancer agents such as beta-2'-deoxy-6-thioguanosine and antiviral agents such as 2',3'- dideoxyinosine, which is an anti-AIDS drug. These types of agents are known to be susceptible to cleavage. When split, the agents lose their effectiveness. The compounds produced according to the present invention can reduce such cleavage. This protection therefore enhances the effectiveness of other chemotherapeutic agents.

A process for preparing compound (I) uses 3-substituted propionitrile as starting materials. Such starting materials can be obtained by various methods which are well documented in the literature. Compound (I) is then prepared from the starting material by an adaptation of synthetic methodology described in M.I.Lim, R.S. Klein and J.J. Fox, J. Org. Chem., 44, 3826 (1979); M.I.Lim, R.S. Klein, and J.J. Fox, Tetrahedron Lett., 21, 1013 (1980); M.I. Lim and R.S. Klein Tetrahedron Easy. 22, 25 (1981); M.I. Lim, W.Y. Ren, B.A. Otter, and R.S. Klein, J. Org. Chem., 48, 780 (1983).

A method for preparing compound (II) uses 3-(pyridinyl)propionitrile as starting material to prepare compound (II). The appropriate 3-(pyridinyl)propionitrile can be prepared by converting the corresponding 3-(pyridinyl)propionyl chloride to the corresponding amide by aminolysis with, for example, ammonium hydroxide, which is then dehydrated to the desired nitrile by distillation with a dehydrating agent, such as POCI3 or SOCI2. Alternatively, the starting material is prepared by condensation of the 3-aldehyde with cyanoacetic acid followed by decarboxylation to provide the corresponding substituted acrylonitrile, which is hydrogenated to provide the corresponding 3-(pyridinyl)propionitrile by either catalytic hydrogenation or magnesium metal dissolution in methanol at 0°C, as described in Profitt, J., et al., J.Org.Chem., 40, 127 (1975). The compound (II) is then prepared from the starting material by an adaptation of the synthetic methodology described in M.I. Lim, E.S. Klein and J.J. Fox, J. Org. Chem., 44, 3826 (1979); M.I. Lim, R.S. Klein, and J.J. Fox, Tetrahedron Lett., 21, 1013 (1980); M.I. Lim and R.S. Klein, Tetrahedron Lett., 22, 25 (1981); M.I. Lim, W.Y. Ren, B.A. Otter, and R.S. Klein, J. Org. Chem, 48, 780 (1983).

Another method for producing the compound uses a known compound, i.e. cyclohexenyl acetonitrile, as starting material. The compound (III) is prepared by reacting the starting material in an adaptation of the synthetic methodology described in M.I. Lim, R.S. Klein and J.J. Fox, J. Org. Chem. 44, 3826 (1979); M.I. Glue. R. S. Klein and J.J. Fox, Tetrahedron Lett., 21, 1013 (1980); M.I. Lim and R.S. Klein, Tetrahedron Lett., 22, 25 (1981); M.I. Lim, W.Y. Ren, B.A. Otter and R.S. Klein, J.Org.Chem., 48, 780 (1983). Catalytic hydrogenation of either (HIA), (UIB) or (IIIC) affords the compound (IIID).

Compounds with different values of "m" according to the above formula are obtained by either ascending or descending the series of the required number of carbon atoms according to known procedures. Reactions involving some intermediates require protection of the nitrogen or oxygen atoms of the intermediates using known procedures.

To describe the present invention more completely, the following examples are now given. In the examples, all parts and percentages are by weight unless otherwise stated. Proportions of solvent mixtures used as chromatographic eluents are by volume.

EXAMPLE 1

3-(2-pyridinyl)propionitrile is prepared using the method of V. Boekelheide, et al., J.Am.Chem.Soc. , 75, 3243 (1953). A solution of potassium cyanide (83.74 g) in water (160 ml) is added to a solution of freshly distilled 2-vinylpyridine (67.59 g) in acetic anhydride (131.30 g), adjusting the addition rate to maintain gentle reflux. When the addition is complete, the resulting dark red mixture is heated for about 70 hours at 105°C in an oil bath with extensive stirring. The cooled reaction mixture is adjusted to pH 8 with a saturated Na 2 CO 3 solution. The mixture is extracted with CHCl 3 (4 x 150 mL), washed with water, dried with Na 2 SO 4 and evaporated to a dark viscous oil. The oil is fractionally distilled in vacuum through a Vigreaux column. After a small run containing 2-vinylpyridine (0.35 g), the desired 3-(2-pyridinyl)propionitrile is collected as a clear, fluorescent yellow-green, viscous oil: yield 59.8 g; 70.4$; bp 86 °C at 1.0 mm Hg.

EXAMPLE 2

3-(2-furanyl)propionitrile is prepared in this example. In a three-necked flask equipped with a condenser and a drying tube, pieces of magnesium (20 g) are added to a solution of 3-(2-furanyl)acrylonitrile (67.2 g) in dry methanol

(2 1). Additional magnesium is added in portions as the reaction rate allows until a total of 145 g has been added. After about 5 hours, the solvent is evaporated until the contents form a solid paste which is adjusted to a pH of about 6.5 with 6N HCl on cooling. The mixture is extracted with several portions of CECI 3 , dried with Na 2 SO 4 and concentrated to a dark oil. Distillation in vacuo through a short Vigreaux column affords propionitrile as a colorless oil; yield 49.5 g ($72); bp 46.0-46.5°C at 0.5 mm.

EXAMPLE 3a

Glycine methyl ester hydrochloride (67.05 g) and anhydrous sodium acetate (43.79 g) are added to a solution of crude formyl compound (53.05 g) in MeOH/H 2 O (4:1, 1500 mL). After 24 hours, the MeOH is evaporated in vacuo and the mixture of water and oil is extracted with CHCl 3 . The CHCl 3 layer is dried (Na 2 SO 4 ) and evaporated to provide a yellow oil (59.1

g)<.>

EXAMPLE 3b

Under a nitrogen atmosphere, ethyl chloroformate (43.68 g) is added dropwise to a solution of the yellow oil according to Example 3a (59.1 g) and DBN (133 g) in dry CH 2 Cl 2 (400 ml) with external cooling in an ice bath. After stirring at 0°C for 1 hour, the solution is left at room temperature overnight. After monitoring the progress by TLC, additional ClCO 2 Et and DBN were added to complete the conversion and the solution was allowed to stand for 24 hours. The volatile material is evaporated in vacuo, the viscous residue purified on a silica gel column (where the main purpose is to remove less mobile DBN) to provide an N-blocked pyrrole, a corresponding pyrrole without the ethoxycarbonyl blocking group on the pyrrole nitrogen and starting propionitrile.

EXAMPLE 3c

To a solution of N-blocked pyrrole according to Example 3b (11.66 g) in MeOH (200 ml) is added solid Na2CO3 (4.23

g), and the reaction mixture is stirred at room temperature for 24 hours with separation of the resulting deblocked pyrrole. The mixture is evaporated to dryness and the residue triturated thoroughly with H 2 O (200 mL) to dissolve inorganics and extracted with CHCl 3 (3 x 200 mL). The extract is dried (Na 2 SO 4 ) and evaporated to provide a viscous gum which crystallized on drying in vacuo for use as an intermediate without further purification. However, more extensive purification can be achieved using either column chromatography using silica gel/CECl3 or recrystallization from toluene/cyclohexane (1:3).

EXAMPLE 3d

Benzoyl isothiocyanate (4.58 g) is added dropwise to a solution of unblocked pyrrole according to Example 3c (5.15 g) in dry CH 2 Cl 2 (75 ml). After 1 hour at room temperature, the solution is evaporated and the gummy residue is dissolved in Et 2 O (100 mL) with almost immediate separation of the crystalline solid, which is collected by filtration. The Et20 filtrate is heated to boiling and diluted with an equal volume of warm cyclohexane. On slow cooling, the solution provides additional thioureido product.

EXAMPLE 3e

Methyl iodide (8.70 g) is added to a solution of the thioureido product according to Example 3d (10.68 g) and DBN (4.15

g) in dry CE 2 Cl 2 (250 ml) at 0°C. The solution is stirred at 0°C for 15 min., at room temperature for 1 hour and then

evaporated in vacuo. A solution of the residue in CECl 3 is chromatographed on a silica gel column with CEC 13 as eluent to provide homogeneous fractions of the methylthio intermediate.

EXAMPLE 3f

A solution of the methylthio compound of Example 3e (0.80 g, 2.0 mmol) in 25 ml of MeOH which has been saturated with NE 3 at 0°C is heated at 90-95°C for 24 hours in a glass-lined stainless steel bomb. The contents of the cooled bomb are evaporated in vacuo to provide a mixture of the 2-amino compound, benzamide and a by-product which is a 2-methylthio derivative, as opposed to the 2-amino compound. The mixture was vigorously stirred for several minutes with 30 mL of 2:1 Et20/cyclohexane, and the insoluble white solid was filtered off and washed with Et2. The filtrate contained most of the benzamide and 2-methylthio components. A solution of Et 2 O/cyclohexane insoluble solid (0.425 g) in MeOH is evaporated with about 10 g of silica gel. The powdered residue is distributed evenly on top of a silica gel column which is then eluted with CHCl 3 /MeOH/HOAc (95:5:1) to provide the 2-methylthio by-product and the desired furanyl intermediate. The intermediate product is recrystallized by extraction into boiling isopropyl acetate in a Soxhlet apparatus. The white crystals are collected in three portions and dried in vacuum over P2°5 at 110°C for 7 hours.

A solution of the intermediate (116 g, 0.5 mmol) in methanol (50 mL) is hydrated with 30% palladium-on-charcoal (40 mg) at 28 kg/6.4 cm<2>H 2 pressure for about 36 hours. The catalyst was removed by filtration under N2 pressure and the filtrate is evaporated and re-evaporated with toluene. The solid residue is recrystallized by extraction into boiling isopropyl acetate in a Soxhlet apparatus. The 2-tetrahydrofuranyl compound (IE) is obtained as a white crystalline solid, which is dried in vacuo over P2O5 at 110°C for about 6 hours to give 73 mg (61.8$); m.p. 284-286°C dec.

EXAMPLE 4a

1-Cyclohexenylacetonitrile is treated in the synthesis according to the present invention. Under an atmosphere of dry N2, a solution of 1-cyclohexenylacetonitrile (9.2 g; 75.92 mmol) in anhydrous tetrahydrofuran (THF, 10 mL) is added to a stirred mixture of sodium hydride (3.18 g; 132.86 mmol) and ethyl formate (30.14 g; 406.93 mmol) in 50 mL of THF, and the resulting mixture is stirred at room temperature for about 18

hours. Volatile material is evaporated in vacuum at room temperature. Water (30 ml) is added to the residue at 0°C, and the solution adjusted to pH 6 by dropwise addition of 6N HCl. The resulting precipitate of heavy oil is extracted into CHCl 3 . The extract is washed with water and dried with Na 2 SO 4 , and the resulting organic layer is evaporated to provide a crude formyl compound as a red-brown oil (9.6 g).

EXAMPLE 4b

Glycine methyl ester hydrochloride (16.68 g, 132.85 mmol) and anhydrous sodium acetate (10.89 g, 132.85 mmol) are added to a solution of crude formyl compound (9.6 g) according to Example 4a without further purification in MeOH/H 2 O (4:1, 150 ml). After 24 hours, the MeOH is evaporated in vacuo and the mixture of water and oil is extracted with CHCl 3 . The CHCl 3 layer is dried (Na 2 SO 4 ) and evaporated to provide a yellow oil which is applied to a silica gel column. Elution with CHCl 3 gave the desired enamine: yield 4.5 g.

EXAMPLE 4c

Under a nitrogen atmosphere, ethyl chloroformate (3.04 g; 28.06 mmol) is added dropwise to a solution of the enamine according to Example 4b (4.12 g, 18-70 mmol) and 1,5-diazabicyclo[4.3.0]-non-5-ene (DBN , 6.96 g, 56.11 mmol) in dry CH 2 Cl 2 (100 ml) with external cooling in an ice bath. After stirring at 0°C for 1 hour, the solution is left at room temperature overnight. After monitoring the progression by TLC, additional ClCO 2 Et (0.5 mL) and DBN (3.0 mL) are added to complete the conversion and the solution is allowed to stand for 24 h. Volatiles are evaporated in vacuo, the viscous residue purified on a short silica gel column (where the main purpose is to remove less mobile DBN) to provide an N-blocked pyrrole, which is used in the next step without further purification.

EXAMPLE 4d

To a solution of N-blocked pyrrole according to Example 4c (3.0 g, 10.26 mmol) in MeOH (100 ml) is added solid Na 2 CO 3 (2.71 g, 25.65 mmol) and the reaction mixture is stirred at room temperature for 48 h by separation of the resulting deblocked the pyrrole. The mixture is evaporated to dryness and the residue triturated thoroughly with H 2 O (50 mL) to dissolve inorganics and extracted with CHCl 3 (3 x 50 mL). The extract is dried (Na 2 SO 4 ) and evaporated to provide a viscous gum, which is purified on a silica gel column using CHCl 3 as eluent; yield 2.04 g; m.p. 125°C.

EXAMPLE 5

Benzoyl isothiocyanate (0.76 g, 4.65 mmol) is added dropwise to a solution of unblocked pyrrole according to Example 4d (0.91 g, 4.13 mmol) in dry CH 2 Cl 2 (30 ml). After 1 hour at room temperature, the solution is evaporated and the gum residue is triturated with methanol to provide a thioureido product; yield 0.70 g; m.p. 170"C.

EXAMPLE 6

Methyl iodide (0.678 g, 4.78 mmol) is added to a solution of the thioureido product according to Example 5 (0.630 g, 1.64 mmol) and DBN (0.230 g, 1.85 mmol) in dry CH 2 Cl 2 (50 ml) at 0°C. The solution is stirred at 0°C for 15 min. at room temperature for 1 hour, and then evaporated in vacuo. A solution of the residue in CHCl3 is chromatographed on a silica gel column with CHCl3 as eluent to provide homogeneous fractions of the methylthio intermediate; yield 0.7 g.

EXAMPLE 7

A solution of the methylthio compound according to Example 6 (0.70 g, 1.76 mmol) in 50 ml of MeOH which has been saturated with NH3 at 0°C is heated at 90-95°C for 24 hours in a glass-lined stainless steel bomb. The contents of the cooled bomb are evaporated in vacuo to provide a mixture of the compound (HIA) benzamide and a by-product which is a 2-methylthio derivative, relative to the 2-amino compound (HIA). The mixture is thoroughly stirred for several minutes with about 50 mL Et 2 O, and insoluble solid is filtered off and washed with Et 2 O. The filtrate contained most of the benzamide and the 2-methylthio components. A solution of Et 2 O-insoluble solid (0.342 g) in MeOH is evaporated with about 10 g of silica gel. The powdered residue is uniformly applied to the top of a silica gel column, which is then eluted with CHCl3/MeOH/HOAc (95:5:1) to provide 2-methylthio bi-product and the desired 2-amino product (HIA). (HIA) is recrystallized by extraction into boiling isopropyl acetate in a Soxhlet apparatus. The white crystals are collected in three portions and dried in vacuo over P2O5 at 110°C for 7 hours; dividend 44$; m.p. 280°C dec., anal. calcd for C12<H>14N40-0.6H20: C, 59.78; H, 6.35; N, 23.23. Found: C, 59.98; H, 6.46; N, 23.15.

EXAMPLE 8

The compound of Example 7 is tested for enzyme inhibition activity. A purine nucleoside phosphorylase (PNP) enzyme assay is performed in which the PNP activity of the compound is determined radiochemically by measuring the formation of [<14>C]-hypoxanthine from [<14>C]-inosine (see Biomedicine, 33, 39 (1980) using calf spleen as enzyme source At 1 mM phosphate the IC50 is 1.9 µM, and at 50 mM phosphate the IC50 is 19 uM.

EXAMPLE 9

Compound (IIID) is prepared using 2-amino-7-(1-cyclohexenyl)-3H,5H-pyrrolo[3,2-d]pyrimidin-4-one as an intermediate. A solution of the intermediate (0.2 g; 0.86 mmol) in ethanol (50 mL) is hydrated with 10% Pd-C (50 mg) at 20.4 kg/6.4 cm<2> for 16 h and filtered hot through Celite. The filtrate is evaporated to dryness and the residue is crystallized from hot ethanol to provide the compound (IIID); yield 157 mg (78%), m.p. >300°C dec. anal. calculated for C12H16N40'0. EtOH: C, 61.80; H, 7.10; N, 23.51. Found: C, 61.95; H, 7.43; N, 23.56.

EXAMPLE 10

The compound (IIID) prepared in Example 95 is tested for enzyme inhibitory activity as in Example 8. At 1 mM phosphate, I<C> is 501.3 µM, and at 50 mM phosphate IC50 is 145 µM.

EXAMPLE 11

3-(2-adamantyl)propionitrile is prepared in this example using a modification of the method of M. Ohno, et al., J. Org. Chem. 53, 1285 (1988). A solution of 2-bromoadmantane (20 g; 92.96 mmol); BusSnH (32.46 g; 111.5 mmol), acrylonitrile (9.86 g; 185.92 mmol) and AIBN (740 mg) in toluene (280 mL) are stirred at reflux for 3 h. The reaction mixture is washed with aqueous ammonia (0.4 M, 500 ml), the organic layer is washed with H 2 O and dried over MgSO 4 and evaporated. The residue is distilled between 110 - 118°C (about 0.2 mmEg); the fractions are combined to provide a crude sample of contaminated 3-(2-adamantyl)propionitrile with tin complexes, which is purified on a silica gel column with hexanes; followed by hexanes/ethyl-

acetate 97:3 and hexanes/ethyl acetate 95:5, gave 9.4 g (53.456); m.p. semi-solid.

EXAMPLE 12a

Glycyl methyl ester hydrochloride (6.96 g, 55.46 mmol) and anhydrous sodium acetate (4.55 g, 55.46 mmol) are added to a solution of crude formyl compound (8.0 g) in MeOH/E 2 O (4:1, 500 mL). After 24 hours, the MeOH is evaporated in vacuo and the mixture of water and oil is extracted with CHCl 3 . The CHCl 3 layer is dried (NagSO 4 ) and evaporated to provide a yellow oil which is applied to a silica gel column. Elution with CHCl 3 gave two major bands; (1) 3-(2-adamantyl )-propionitrile (used as starting material in the previous step) and (2) desired enamine, yield 6 g.

EXAMPLE 12b

Under a nitrogen atmosphere, ethyl chloroformate (2.82 g, 26.0 mmol) is added dropwise to a solution of the enamine of Example 12a (5.0 g, 17.34 mmol) and 1,5-diazabicyclo[4.3.0]-non-5-ene ("DBN ," 6.46 g, 52.0 mmol) in dry CH 2 Cl 2 (50 mL) with external cooling in an ice bath. After stirring at 0"C for 1 hour, the solution is left at room temperature overnight. After monitoring the process by TLC, additional ClCO 2 Et (1.81 mL) and DBN (3.23 g) are added to complete the conversion, and the solution is left for 24 hours The volatile material is evaporated in vacuo, the viscous residue purified on a short silica gel column (where the main purpose is to remove less mobile DBN) to provide an N-blocked pyrrole, which is used in the next step without further purification.

EXAMPLE 12c

To a solution of crude N-"blocked pyrrole according to Example 12b (6.0 g, 16.59 mmol) in MeOH (100 mL) is added solid Na 2 CO 3 (4.39 g, 41.49 mmol), and the reaction mixture is stirred at room temperature for 48 h with separation of resulting deblocked pyrrole. The mixture is evaporated to dryness and the residue triturated thoroughly with H 2 O (50 mL) to dissolve the inorganic material and extracted with CHCl 3 (3 x 100 mL). The extract is dried (Na 2 SO 4 ) and evaporated to provide a viscous gum which crystallized on trituration with ether, yield 4 g, mp 162-163°C.

EXAMPLE 13

Benzoyl isothiocyanate (1.22 g, 7.47 mmol) is added dropwise to a solution of unblocked pyrrole according to Example 12c (1.91 g, 6.62 mmol) in dry CH 2 Cl 2 (50 ml). After 1 h at room temperature, the solution is evaporated and the gummy residue is dissolved in Et 2 O (100 mL) with almost instantaneous separation of the crystalline solid. The filtrate is heated to boiling and diluted with an equal volume of warm cyclohexane. On slow cooling, the solution provides additional thioureido products; yield 2.81 g (95#); m.p. 193 - 194°C.

EXAMPLE 14a

Methyl iodide (2.46 g, 17.39 mmol) is added to a solution of the thioureido product according to Example 13 (2.7 g, 5.98 mmol) and DBN (0.82 g, 6.57 mmol) in dry CH 2 Cl 2 (50 ml) at 0"C. The solution becomes stirred at 0°C for 15 min., at room temperature for 1 h, and then evaporated in vacuo to provide a crude sample of the intermediate methylthio compound, yield 2.78 g (crude).

EXAMPLE 14b

A solution of the methylthio compound according to Example 14a (2.78 g, 5.18 mmol) in 150 ml of MeOH which has been saturated with NH3 at 0°C is heated at 90 - 95°C for 24 hours in a glass-lined stainless steel bomb. The contents of the cooled bomb are evaporated in vacuo to give a mixture of the compound (IVA), benzamide and a by-product which is a 2-methylthio derivative, as opposed to the 2-amino compound (IVA). The mixture is stirred vigorously for several minutes with about 75 mL of Et 2 O, and the insoluble white solid is filtered off and washed with Et 2 O. The filtrate contained most of the benzamide and the 2-methylthio components. A solution of Et 2 O-insoluble solid (1.38 g) in MeOH is evaporated with about 25 g of silica gel. The powdery residue is distributed evenly on top of a silica gel column which is then eluted with CHCl 23 /MeOH/HOAc (95:5:1) to provide the 2-methylthio bi-product and the desired 2-amino product (IVA). (IVA) is recrystallized by extraction into boiling isopropyl acetate in a Soxhlet apparatus. The white crystals are collected in three portions and dried in vacuo over P 2 O 5 at 110°C for 7 hours; dividends 51,656; m.p. >350°C dec.; anal. calcd for C17<H>22<N>40-0.21MeOH-0.22H2O: C, 66.88; H, 7.59; N, 18.12. Found: C, 66.86; H, 7.59; N, 18.12.

EXAMPLE 15

The compound of Example 14b is tested for enzyme inhibition activity. A purine nucleoside phosphorylase (PNP) enzyme assay is performed in which the PNP activity (IC50) of the compound is found, determined radiochemically by measuring the formation of [14C]-hypoxanthine from [14C]-inosine (see Biomedicine, 33, 39 (1980)) by using calf spleen as an enzyme source. At 1 mM phosphate, the IC50 is 090 µM, and at 50 mM phosphate, the IC50 is 2.5 µM.

EXAMPLE 16

3-Cyclopentylpropionitrile is prepared in this example. 3-Cyclopentylpropionyl chloride (57.7 g, 0.36 mol) is added dropwise to a large excess of concentrated ammonium hydroxide (400 mL) cooled in an ice/salt bath. The heavy suspension of white solid is stirred overnight, collected by filtration, washed with cold water, and recrystallized from about 2 liters of boiling water. Shiny white plates of amides are dried in vacuo over P2O5; yield 31.6 g (62.3$; m.p. 122°C).

Protected from atmospheric pressure, a solution of the amide (23.5 g, 0.166 mol) in POCl 3 (150 mL) is heated at 120°C for 1 hour. The oil bath is cooled to approximately 70°C and excess POCI3 is distilled off under vacuum, and the cooled residue is completely on ice (approximately 300 g). The mixture is neutralized by careful addition of solid Na2CC"3 and extracted with several portions of Et2O. The dried (Na2SO4) extract is evaporated to give a clear, slightly yellow oil which is distilled in vacuo to give the desired nitrile; yield, 16.86 g ( 82%) kp 88.0 - 88.5 "C/8.7 mm). MS (EI): m/z 122 (M-H)<+>; IR (cap.film), 2245 cm_<1>(CN), complex multiplets centered around 1.11, 1.63, 1.86 (cyclopentyl protons).

EXAMPLE 17a

3-Cyclopentylpropiontrile according to the previous example is further processed in the synthesis according to the present invention. Under an atmosphere of dry N2, a mixture of 3-cyclopentyl-propionitrile (14.8 g, 0.12 mol) sodium hyd-

rid (5.8, 0.24 mol) and anhydrous tetrahydrofuran (300 mL) heated at 52°C in a water bath for 15 min, and a solution of ethyl formate (13.3 g, 0.18 mol) in THF (100 mL) is added over a period of 45 min. After two hours at 50 - 55°C, a second portion of NaH (1.9 g) and HC02Et (5.0 mL) is added, followed for 30 minutes by a third portion of HC02Et (unreacted nitrile is inert in the next step and can be isolated by first cleaning stage). The thick paste is stirred overnight and cooled to room temperature. The volatile material is evaporated under reduced pressure and the remaining faint yellow material is dissolved in the minimum volume of cold water (approximately 150 mL) at 0°C. The solution is adjusted to pH 6.0 by addition of 6N HCl and extracted with CHCl 3 (3 x 100 ml). The extract is washed with H 2 O, dried over Na 2 SO 4 and evaporated in vacuo to a thick yellow oil. This crude product (15.6 g) is used in the aforementioned reaction without further purification.

EXAMPLE 17b

Glycine methyl ester hydrochloride (19.31 g, 0.154 mol) and anhydrous sodium acetate (12.61 g, 0.154 mol) were added to a solution of crude formyl compound of previous example (15.6 g) in MeOH/H 2 O (4:1, 500 mL). After 24 hours, the MeOH is evaporated in vacuo and the mixture of water and oil is extracted with CHCl 3 . The CHCl 3 layer is dried (Na 2 SO 4 ) and evaporated to provide a yellow oil which is applied to a silica gel column. Elution with CHCl 3 gave two major bands: (1) 3-cyclopentylpropionitrile (8.22 g, 66.7 mmol or 55.656 nitrile used as starting material in the previous step) and (2) the desired enamine (3.45 g; 29.156 based on the theoretical yield corrected for the amount of nitrile present in the starting material; MS (FAB): 223 (M + H)<+>).

EXAMPLE 18a

Under a nitrogen atmosphere, ethyl chloroformate (2.53 g, 23.3 mmol) is added dropwise to a solution of the enamine of the previous example (3.45 g, 15.5 mmol) and DBN (5.78 g, 46.6 mmol) in dry CH 2 Cl 2 (50 mL) with external cooling in an ice bath. After stirring at 0°C for 1 hour, the solution is left at room temperature overnight. After monitoring the process by TLC, additional ClCO 2 Et (0.5 mL) and DBN (3.0 mL) are added to complete the conversion and the solution is allowed to stand for 24 hours. The volatile material is evaporated in vacuo, the viscous residue purified on a short silica gel column (where the main function is to remove less mobile DBN) to provide an N-blocked pyrrole (4.50 g; 98%), which is used in the next step without further purification.

EXAMPLE 18b

To a solution of N-blocked pyrrole according to previous example (4.50 g, 15.3 mmol) in MeOH (100 mL) is added solid Na 2 CO 3 (1.62 g, 15.3 mmol), and the reaction mixture is stirred at room temperature for 48 h with separation of the resulting deblocked pyrrole. The mixture is evaporated to dryness and the residue triturated thoroughly with H 2 O (50 mL) to dissolve inorganics and extracted with CHCl 3 (3 x 100 mL). The extract is dried (Na 2 SO 4 ) and evaporated to give a viscous gum which crystallized on drying in vacuo; gave 2.97 g (87,456) of the material suitable for use as an intermediate without further purification. However, more extensive purification can be achieved by using either column chromatography using silica gel/CHCl3 or recrystallization from toluene/cyclohexane (1:3).

EXAMPLE 18c

Benzoyl isothiocyanate (2.62 g, 16.03 mmol) is added dropwise to a solution of unblocked pyrrole according to Example 18b (2.97 g, 13.36 mmol) in dry CH 2 Cl 2 (100 ml). After 1 hour at room temperature, the solution is evaporated and the gum residue is dissolved in Et 2 O (100 mL) with almost instantaneous separation of the crystalline solid; yield 1.75 g. The Et20 filtrate is heated to boiling and diluted with an equal volume of hot cyclohexane. On slow cooling, the solution gave a further 1.58 g of thioureido product; total yield 3.33 g (64,696). A small amount of the thioureido product is recrystallized from hot Et 2 O/cyclohexane (15 mL each); m.p. 123 - 125°C. MS (FAB): 386 (M + H)<+>. Analysis calculated for C20<E>23<N>3O3S-O.45C6<H>12<:>C, 64.40; H, 6.76;

N, 9.93. Found: C, 64.51; E, 7.10; N, 9.93.

EXAMPLE 19a

Methyl iodide (2.60 g, 18.32 mmol) is added to a solution of the thioureido product according to Example 18c (3.21 g, 8.33 mmol) and DBN (1.24 g, 9.99 mmol) in dry CE 2 Cl 2 (80 ml) at 0°C. The solution is stirred at 0°C for 15 min., at room temperature for 1 hour and then evaporated in vacuo. A solution of the residue in CEC13 is chromatographed on a silica gel column with CECl3 as eluent to provide homogeneous fractions of methylthio intermediates; yield, 2.46 g (7456).

EXAMPLE 19b

A solution of the methylthio compound according to Example 19a (2.07 g, 5.18 mmol) in 150 ml of MeOH which has been saturated with NH3 at 0°C is heated at 90 - 95°C for 24 hours in a glass-lined stainless steel bomb. The contents of the cooled bomb are evaporated in vacuo to provide a mixture of the compound (IVC), benzamide and a by-product which is a 2-methylthio derivative, as opposed to the 2-amino compound (IVC). The mixture is stirred vigorously for several minutes with about 75 mL of Et 2 O, and the insoluble white solid is filtered off and washed with Et 2 O. The filtrate contained most of the benzamide and 2-methylthio components. A solution of Et 2 O-insoluble solid (1.13 g) in MeOH is evaporated with approximately 10 g of silica gel. The powdered residue is uniformly applied to the top of a silica gel column which is then eluted with CHCl 3 /MeOH/HOAc (95:5:1) to provide the 2-methylthio bi-product (252 mg; MS (FAB): 264 (M + H)<+>) and the desired 2-amino product (IVC) (679 mg, 56.4$). (IVC) is recrystallized by extraction into boiling isopropyl acetate in a Soxhlet apparatus. The white crystals are collected in three portions and dried in vacuum over P2°5 at 110°C for 7 hours; yield 540 mg ($44.9); m.p. 324 - 326<*>C dec.; MS (FAB): 233 (M + H)<+>; analysis calculated for C12<H>16N40: c»62.05; H, 6.94; N, 24.12. Found: C, 62.04; H, 7.11; N, 24.48.

EXAMPLE 20a

Glycine methyl ester hydrochloride (30.60 g, 0.24 mol) and anhydrous sodium acetate (19.99 g, 0.24 mol) are added to a solution of the crude formyl compound of the previous example (25.22 g) in MeOH/H 2 O (4:1, 500 mL). After 24 hours, the MeOH is evaporated in vacuo and the mixture of water and oil is extracted with CHCl 3 . The CHCl 3 layer is dried (Na 2 SO 4 ) and evaporated to provide a yellow oil which is applied to a silica gel column. Elution with CHCl 3 gave two major bands: (1) 3-cyclohexylpropionitrile (used as starting material in the previous step) and (2) the desired enamine; yield 16 g.

EXAMPLE 20b

Under a nitrogen atmosphere, ethyl chloroformate (1.38 g, 12.7 mmol) is added dropwise to a solution of the enamine according to Example 20a (2.0 g, 8.46 mmol) and DBN (2.1 g, 16.9 mmol) in dry CE 2 Cl 2 (50 ml) with external cooling in an ice bath. After stirring at 0°C for one hour, the solution is left at room temperature overnight. After examining the process by TLC, additional ClCO 2 Et (0.5 mL) and DBN (1.5 mL) are added to complete the conversion, and the solution is allowed to stand for 24 hours. Volatile material is evaporated in vacuo, the viscous residue purified on a short silica gel column (where the main purpose is to remove the less mobile DBN) to provide an N-blocked pyrrole, which is used in the next step without further purification.

EXAMPLE 20c

To a solution of N-blocked pyrrole according to Example 20b (2.6 g, 8.43 mmol) in MeOH (100 ml) is added solid Na2CO3 (2.23 g, 21.07 mmol) and the reaction mixture is stirred at room temperature for 48 hours with separation of the resulting deblocked the pyrrole. The mixture is evaporated to dryness and the residue triturated thoroughly with H 2 O (50 mL) to dissolve inorganic materials and extracted with CHCl 3 (3 x 100 mL). The extract is dried (Na 2 SO 4 ) and evaporated to provide a viscous gum, which is purified on a silica gel column using CHCl 3 as eluent; yield 1.67 g (8496); m.p. 73 - 74°C.

EXAMPLE 21

Benzoyl isothiocyanate (0.74 g, 4.02 mmol) is added dropwise to a solution of unblocked pyrrole according to Example 20c (0.95 g) in dry CH 2 Cl 2 (20 ml). After 1 hour at room temperature, the solution is evaporated and the gummy residue is dissolved in Et 2 O (100 ml) with almost immediate separation of the crystalline solid. The Et 2 O filtrate is heated to boiling and diluted with an equal volume of warm cyclohexane. On slow cooling, a further thioureido product is obtained from the solution; total yield 1.41 g (8855); m.p. 156 - 157°C.

EXAMPLE 22a

Methyl iodide (1.1 g, 7.6 mmol) is added to a solution of the thioureido product according to Example 21 (0.96 g, 2.61 mmol) and 1,5-diazabicyclo[4.3.0]non-5-ene (0.38 g, 3.01 mmol) in dry CE 2 Cl 2 (20 mL) at 0°C. The solution is stirred at 0°C for 15 min., at room temperature for 1 hour, then evaporated in vacuo. A solution of the residue in CHCl3 is chromatographed on a silica gel column with CHCl3 as eluent to provide homogeneous fractions of the methylthio intermediate compound; yield 0.92 g.

EXAMPLE 22b

A solution of the methylthio compound according to Example 22a (0.8 g, 1.93 mmol) in 50 ml of MeOH which has been saturated with NH3 at 0°C is heated at 90-95°C for 24 hours in a glass-lined stainless steel bomb. The contents of the cooled bomb are evaporated in vacuo to provide a mixture of the compound (IVD) benzamide and a by-product which is a 2-methylthio derivative, as opposed to the 2-amino compound (IVD). The mixture is thoroughly stirred for several minutes with about 75 mL of Et 2 O, and the insoluble white solid is filtered off and washed with Et 2 O. The filtrate contained most of the benzamide and 2-methylthio components. A solution of Et 2 O-insoluble solid (0.390 g) in MeOH is evaporated with about 10 g of silica gel. The powdered residue is uniformly applied to the top of a silica gel column, which is then eluted with CHCl3/MeOH/HOAc (95:5:1) to provide the 2-methylthio bi-product and the desired 2-amino product (IVD). (IVD) is recrystallized by extraction in boiling isopropyl acetate in a Soxhlet apparatus. The white crystals are collected in three portions and dried in vacuo over P2°5 at 110°C for 7 hours; yield 495é, m.p. >300°C; analysis calcd for C 2 H 2 H 2 O: C, 63.39; H, 7.36; N, 22.74. Found: C, 63.50; H, 7.74; N, 22.67.

EXAMPLE 23

The compound according to Example 22b is tested for enzyme inhibition activity as in Example 105. At 1 mM phosphate, I<C> is 500.037 µM, and at 50 mM phosphate IC is 502.2 µM.

EXAMPLE 24a

The compound of Example 22b is tested to determine its effectiveness in potentiating the toxicity of 2'-deoxy-guanosine (d-Guo) (see D.A. Schewach et al., Cancer ..., 46, 519 (1986), and J.C. Sircar et al., Agents and ..., 21, 253

(1987)). The CCRF-CEM cells are grown in RPMI-1640 medium. To a suspension culture of these cells, d-Guo at a certain concentration (5.62 µM) is added and the compound at different concentrations is also added and the number of cells determined in a Coulter counter 24, 48 and .72 hours thereafter. From these data, the IC 50 is calculated to be 2.0 µM as the concentration of the compound necessary to reduce the increase in cell number between 0 and 72 hours to 50% of that of the control cultures.

EXAMPLE 24b

The compound 2-amino-7-(3-methylcyclohexylmethyl)-3H,5H-pyrrolo[3,2-d]pyrimidin-4-one is prepared. First by applying the method used in examples 20a - 22b above, but with 3-(3-methylbenzyl)-propionitrile as starting material, the aryl derivative 2-amino-7-(3-methylbenzyl)-3H,5H-pyrrolo[3,2- d]-pyrimidin-4-one prepared. A solution of the aryl derivative (0.2 g, 0.78 mmol) in trifluoroacetic acid (TFA)

(20 mL) is hydrated with PtO 2 at 27.2 kg/6.4 cm<2> for 24 hrs. The catalyst is filtered out through a Celite layer, and the filtrate evaporated. The rest is triturated with methanol and left in the freezer overnight. The resulting crystallized trifluoroacetate salt precipitates from the solution and is collected by filtration. The TFA salt is suspended in 8 ml of H2O, adjusted to pH 8 with concentrated NE4OE and sonicated. The pure product is collected, washed with H2O and dried: yield 165 mg ($81); m.p. 282"C. Analyzed: Calculated for

<C>14<H>20N40: c'64.60; H, 7.74; N, 21.52. Found: C, 64.24; H, 7.96; N, 21.5156.

EXAMPLE 25

The procedure described in Example 24b is repeated to prepare 2-amino-7-(3-trifluoromethylcyclohexyl-methyl)-3H,5H-pyrrolo-[3,2-d]pyrimidin-4-one using 3-(3- trifluoromethylbenzyl)-propionitrile as starting compound: yield 6956; m.p. 165°C. Analyzed: Calculated for C14<H>17N4OF3.0-6H20: c»51.72; H, 5.64; N, 17.23. Found: C, 51.82; H, 5.71; N, 16.8156.

EXAMPLE 26

The compound prepared in Example 24b is tested for enzyme inhibition activity as in Example 15. At 1 mM phosphate, IC is 500,025 pM, and at 50 mM phosphate, IC is 500,820 µM.

EXAMPLE 27

The compound prepared in Example 25 is tested for enzyme inhibitory activity as in Example 15. At 1 mM phosphate the IC is 500,020 µM, and at 50 mM phosphate the IC is 500,740 µM.

EXAMPLE 28a

3-cycloheptylpropionitrile is prepared in this example according to the procedure in Example 11 using a solution of 2-bromocycloheptane (25.57 g; 144.38 mmol); Bu3SnH (50.42 g; 173.26 mmol), acrylonitrile (15.32 g; 288.77 mmol) and AIBN (1.13 g) in toluene (300 mL). Yield is 16 g; m.p. oil.

EXAMPLE 28b

3-cycloheptylpropionitrile according to Example 28a is further processed in the preparation according to the present invention. Under an atmosphere of dry N 2 , a mixture of 3-cycloheptylpropionitrile (8.5 g, 56.19 mmol), sodium hydride (2.6 g, 112.39 mmol), and anhydrous tetrahydrofuran (100 mL) is heated at 52°C in a water bath for 15 min, and a solution of ethyl formate (20.81 g, 280.99 mmol) in THF (100 mL) is added over a period of 45 min. After two hours at 50-55°C, another portion of NaH (1.35 g) and HCO 2 Et (10.4 g) is added, followed for 30 min. of a third portion of HC02Et (unreacted nitrile is inert in the next step and can be isolated in the first purification step). The thick paste is stirred overnight and allowed to cool to room temperature. The volatile material is evaporated under reduced pressure, and

the remaining pale yellow material is dissolved in a minimal volume of cold water (about 150 ml) at 0°C. The solution is adjusted to pH 6.0 by addition of 6N HCl and extracted with CHCl 3 (3 x 100 ml). The extract is washed with H 2 O, dried over Na 2 SO 4 , and evaporated in vacuo to a thick yellow oil. This crude product is used in the next step without further purification.

EXAMPLE 28c

Glycine methyl ester hydrochloride (9.35 g, 74.47 mmol) and anhydrous sodium acetate (6.10 g, 74.47 mmol) are added to a solution of crude formyl compound according to Example 28b (8.9 g; 49.65 mmol) in MeOH/H 2 O (4:1, 250 mL ). After 24 hours, the MeOH is evaporated in vacuo and the mixture of water and oil is extracted with CHCl 3 . The CHCl 3 layer is dried (Na 2 SO 4 ) and evaporated to provide a yellow oil which is applied to a silica gel column. Elution with CHCl 3 gave two major bands; (1) 3-cycloheptylpropionitrile (used as starting material in the previous step) and (2) the desired enamine, which is recrystallized from a CHCl3/Et2O mixture; yield 6.18 g; m.p. 57 - 58°C.

EXAMPLE 29a

Under a nitrogen atmosphere, ethyl chloroformate (4.01 g, 37.03 mmol) is added dropwise to a solution of the enamine of Example 28c (6.18 g, 24.69 mmol) and DBN (9.19 g, 74.04 mmol) in dry CH 2 Cl 2 (100 mL) with external cooling in an ice bath. After stirring at 0"C for 1 hour, the solution is left at room temperature overnight. After monitoring the progression by TLC, additional ClCO 2 Et (0.5 mL) and DBN (3.0 mL) are added to complete the conversion, and the solution is left in 24 hours The volatile material is evaporated in vacuo, the viscous residue is purified on a silica gel column (where the main function is to remove less mobile DBN) to provide an N-blocked pyrrole, which is used in the next step without further purification.

EXAMPLE 29b

To a solution of N-blocked pyrrole according to Example 29a (7.8 g, 24.19 mmol) in MeOH (100 ml) is added solid NagCOs (6.41 g, 60.48 mmol) and the reaction mixture is stirred at room temperature for 48 hours with separation of the resulting deblocked the pyrrole. The mixture is evaporated to dryness and the residue triturated thoroughly with H 2 O (50 mL) to dissolve inorganic materials and extracted with CHCl 3 (3 x 100 mL). The extract is dried (NagSO 4 ) and evaporated to provide a viscous gum, which is purified by column chromatography using silica gel/CHCl 3 ; yield 4 g; mp 88-89°C.

EXAMPLE 30

Benzoyl isothiocyanate (1.5 g, 8.96 mmol) is added dropwise to a solution of unblocked pyrrole according to Example 29b (1.99 g, 7.95 mmol) in dry CH 2 Cl 2 (50 ml). After 1 hour at room temperature, the solution is evaporated and the gummy residue is dissolved in Et 2 O (100 mL) with almost instantaneous separation of the crystalline solid. The filtrate is heated to boiling and diluted with an equal volume of warm cyclohexane. On slow cooling, the solution provides further thioureido product: yield 2.89 g (88%); m.p. 158 - 159°C.

EXAMPLE 31a

Methyl iodide (1.7 g, 11.96 mmol) is added to a solution of thioureido product according to Example 30 (1.7 g, 4.1 mmol) and DBN (0.56 g, 4.52 mmol) in dry CH 2 Cl 2 (80 ml) at 0°C. The solution is stirred at 0"C for 15 min., at room temperature for 1 hour and then evaporated in vacuo. A solution of the residue in CHCl3 was chromatographed on a silica gel column with CHCl3 as eluent to provide homogeneous fractions of the methylthio intermediate compound.

EXAMPLE 31b

A solution of the methylthio compound according to Example 31a (1.72 g, 4.02 mmol) in 50 ml of MeOH which has been saturated with NH3 at 0°C is heated at 90 - 95°C for 24 hours in a glass-lined stainless steel bomb. The contents of the cooled bomb are evaporated in vacuo to provide a mixture of the compound (IVE), benzamide and a by-product which is a 2-methylthio derivative, as opposed to the 2-amino compound (IVE). The mixture is thoroughly stirred for several minutes with about 75 mL of EtgO and the insoluble white solid is filtered off and washed with EtgO. The filtrate contained most of the benzamide and 2-methylthio components. A solution of EtgO-insoluble solid (0.850 g) in MeOH is evaporated with about 10 g of silica gel. Powdered residue is distributed evenly on top of a silica gel column which is then eluted with CHCl3/MeOH/HOAc (95:5:1) to provide the 2-methylthio bi-product and the desired 2-amino product (IVE). (IVE) is recrystallized by extraction in boiling isopropyl acetate in a Soxhlet apparatus. The white crystals are collected in three portions and dried in vacuo over P2O5 at 110°C for 7 hours; yield 5456, m.p. >300°C dec.; analysis: calculated for C14<H>20<N>4O: C, 64.60; H, 7.74; N, 21.52. Found: C, 64.78; H, 8.01; N, 21.61.

EXAMPLE 32

The compound according to Example 31b is tested for enzyme inhibition activity as in Example 15. At 1 mM phosphate, I<C> is 500,030 uM, and at 50 mM phosphate, IC is 500,840 uM.

EXAMPLE 33

The above intermediate is prepared in this example by a modification of the method of Schiemenz, G.P.; Engelhard, H. (Chem. Ber., 1962, 95, 195).

A mixture of cyanoacetic acid (25.38 g, 298.38 mmol), 2,3,5-trichlorobenzaldehyde (25.0 g, 119.35 mmol), ammonium acetate (500 mg), toluene (120 mL) and pyridine (65 mL) is heated at reflux for 16 hours in a bottle fitted with a Dean-Stark trap and condenser. The solvents are evaporated in vacuo, the residue extracted with CHCl 3 , which is washed with HgO, dried with (NagSO 4 ) and evaporated to give the crude product, which is purified by silica gel column chromatography using the hexane-EtOAc mixture as eluent. Yield 23.69 g (7356 ); mp 90 - 91° C.

EXAMPLE 34a

The above intermediate compound is produced in this example. To a stirred mixture of NaH (1.56 g, 65.05 mmol) and ethyl formate (14.78 g 199.51 mmol) in THF (100 ml) is added substituted pentanedinitrile according to Example 33 (10.17 g, 37.17 mmol) at room temperature under a nitrogen atmosphere, and the resulting reaction mixture is stirred for 24 hours. Volatile materials are evaporated in vacuum at room temperature. Water (50 ml) is added to the residue at 0-5°C and the solution is adjusted to pH 5-6 with 2056 cone. HCl (v/v). The heavy oil is extracted into ethyl acetate, washed with HgO (1 x 100 mL) and dried (MgSO4). The ethyl acetate layer is evaporated to provide a reddish brown oil (11.0 g) which is used in the next step without further purification.

EXAMPLE 34b

The above intermediate compound is produced in this example. Glycine methyl ester hydrochloride (8.17 g, 65.06 mmol) and sodium acetate (5.33 g, 65.06 mmol) are added to a solution of crude formyl compound according to Example 34a (11.0 g) in a mixture of MeOH (80 ml) and HgO (20 ml) , and the resulting solution is stirred at room temperature for 22 hours. After evaporation of the solvent at room temperature, the residue is extracted with ethyl acetate. The washed (HgO) and dried (MgSO^ organic layer is evaporated to provide an oil. Flame column chromatography (silica gel) using CHCl3 as eluent gave the pure desired enamine as a mixture of cis-trans isomers which is recrystallized from MeOH, yield 10.41 g (7556), mp 142 - 143° C. EXAMPLE 35

The above intermediate compound is produced in this example. A solution of the enamine according to Example 34b (10.0 g, 26-84 mmol) in dry CE 2 Cl 2 (100 ml) is cooled to 0°C and treated with 1,5-diazabicyclo[4.3.0]non-5-ene (10.53 g , 84.79 mmol) under a nitrogen atmosphere followed by ethyl chloroformate (6.90 g, 63.57 mmol). The solution is stirred at 0°C for 1 hour and then at room temperature for 48 hours. The volatiles are evaporated in vacuo to provide a viscous dark gum which is purified by flame column chromatography over silica gel using CHCl 3 as eluent. All the fractions containing the desired N-protected pyrrole are combined and evaporated to give a foamy slightly pale yellow material which is stirred in MeOH (100 mL) to give the crystalline material which is recrystallized from CHCl3-MeOH, yield 8.92 g (74 . 756), m.p. 160 - 161°C.

EXAMPLE 36

The above intermediate compound is produced in this example. A suspension of N-protected pyrrole according to Example 35 (8.6 g, 19.34 mmol) in MeOH (300 mL) is treated with Na 2 CO 3 (5.12 g, 48.34 mmol) and the reaction mixture is stirred at room temperature for 17 h with separation of deblocked pyrrole during the first hour. Solid sodium carbonate is removed by filtration and washed thoroughly with MeOH. The filtrate is reduced to a volume of 25 ml and kept in a refrigerator for 1 hour to provide 5.23 g of crystalline product. Further concentration of the mother liquor gave a further 0.14 g of pure product; total yield 6.45 g ($89.5), m.p. 178-181°C.

EXAMPLE 37

The above intermediate compound is produced in this example. To a suspension of pyrrole in Example 36 (5.83 g, 15.64 mmol) in dichloromethane (100 ml) is added benzoyl isothiocyanate (2.88 g, 17.64 mmol) at room temperature under nitrogen. The reaction mixture is stirred for 30 min. with separation of the desired thioureido compound. Further benzoyl isothiocyanate (0.5 ml) is added to this and again stirred for 30 min. The solvent is evaporated to dryness and the pale yellow residue is triturated with methanol. The white crystalline material is isolated by filtration and recrystallized from a chloroform-ether mixture to provide the required thioureido compound as an analytically pure sample, yield 7.71 g (92%), m.p. 210 - 211'C.

EXAMPLE 38

The above intermediate compound is produced in this example. A solution of the thioureido compound according to Example 37 (6.75 g, 12.6 mmol) and 1,5-diazabicyclo[4.3.0]non-5-ene (1.76 g, 14.20 mmol) in dry CH 2 Cl 2 (200 ml) is cooled to 0' C and treated with methyl iodide (5.20 g, 36.65 mmol). The reaction mixture is stirred at 0°C for 10 min. and then for 1 hour at room temperature. The solvent is evaporated at room temperature and the residue is extracted with CHCl 3 , washed with E 2 O (2 x 50 mL), dried (Na 2 SO 4 ) and evaporated to provide a glassy foam (6.95 g) which is used in the next step without purification.

EXAMPLE 39

The above compounds A and B are prepared in this example. Compound A is a compound according to the present invention and compound B is an intermediate. A solution of the methylthio intermediate of Example 38 (6.90 g, 12.54 mmol) in MeOH (200 ml) is saturated at 0°C with ammonia and heated at 100°C for 20 hours in a glass-lined stainless steel bomb. The reaction mixture is brought to room temperature and the solvent evaporated at room temperature. Purification of the crude mixture by flame column chromatography over silica gel using CHCl3 as eluent gave 8B (1.1 g, 2156), mp 290 - 291°C and then CHCl3-MeOH (95:5) gave pure 8A (2.76 g, 57.556), mp 284-285°C.

EXAMPLE 40

The compound according to the present invention according to Example 39 is tested for enzyme inhibition activity. A purine nucleoside phosphorylase (PNP) enzyme assay is performed in which the PNP activity (IC50) of compound (8A) is found, determined radiochemically by measuring the formation of [<14>C]-hypoxanthine from [<14>C]-inosine (see Biomedicine, 1980, 33, 39) using calf spleen as an enzyme source. At 1 mM phosphate the IC500 is 64 µM and at 50 mM phosphate the IC50 is 10 µM.

EXAMPLE 41

According to the procedure set forth in Examples 33-39, 3-(3-chlorophenyl)-3-(2-amino-4-oxo-3E,5H-pyrrolo[3,2-d]pyrimidin-7-yl)propanenitrile is prepared by use of 3-(3-chlorophenyl)-pentanedinitrile as starting material, yield 54,596, m.p. 157 - 158°C.

EXAMPLE 42

The above compounds 3-(2-amino-4-oxo-3EJ,5E-pyrrolo[3,2-d]-pyrimidin-7-yl)-3-phenylpropanenitrile are prepared in this example. A solution of the compound obtained in Example 39 (2.0 g, 5.22 mmol) in hot ethanol (250 ml) and dimethylformamide (DMF) (150 ml) is hydrated over 3096 Pd/C catalyst (1.0 g) in the presence of triethylamine (2.64 g, 5.0 equivalent) at atmospheric pressure. After 5 hours, the reaction is complete and the catalyst is filtered out under Ng pressure. The solid obtained by evaporation of the filtrate is triturated and sonicated with EgO and dried, yield 1.28 g

(8896), m.p. 168 - 170° C.

EXAMPLE 43

The compound prepared in Example 42 is tested for enzyme inhibition activity as in Example 188. At 1 mM phosphate, I<C>500,023 µM and at 50 mM phosphate, IC50<4.>7 µM.

EXAMPLE 44

The above compound 3-(2-amino-4-oxo-3H,5H-pyrrolo[3,2-d]pyrimidin-7-yl)-3-phenylpropanoic acid is prepared in this example. A solution of the compound obtained in Example 42 (0.200 g, 0.72 mmol) in 6N HCl (3.0 mL) is heated at reflux for 18 hours. The solvent is evaporated in vacuo and the residue is triturated with HgO (6 mL), adjusted to pH 10 with conc. ammonium hydroxide. Insoluble material is collected by filtration and the filtrate is again adjusted to pH 6.8. White material that precipitates out is collected, washed with water and dried, yield 0.19 g (8996), m.p. 290°C dec.

EXAMPLE 45

The compound prepared in Example 44 is tested for enzyme inhibition activity as in Example 40. At 1 mM phosphate the IC500 is 012 µM and at 50 mM phosphate the IC50 is 0.19 µM.

EXAMPLE 46

The above compound 3-(2-amino-4-oxo-3E,5E-pyrrolo[3,2-d]pyrimidin-7-yl)-3-phenylpropanamide is prepared in this example. A solution of the compound obtained in Example 42 (0.200 g, 0.72 mmol) in conc. H2S04 (0.5 ml) is stirred at room temperature for 20 hours and then poured into crushed ice (5.0 g) and adjusted to pH 6.8 with conc. NH40H. The precipitated solid is collected, washed with HgO and dried, yield 0.180 g, m.p. 199 - 201 °C dec.

EXAMPLE 47

The compound prepared in Example 46 is tested for enzyme inhibition activity as in Example 40. At 1 mM phosphate, I<C>500.20 µM and at 50 mM phosphate IC506.6 >µM.

EXAMPLE 48

3-(2-Amino-4-oxo-3H,5E-pyrrolo[3,2-d]pyrimidin-7-yl)-3-cyclohexylpropanoic acid is prepared in this example. A solution of the compound obtained in Example 44 (83 mg, 0.28 mmol) in trifluoroacetic acid (TFA) (15 mL) is hydrated with PtO 2 (83 mg) at 27.2 kg/6.4 cm<2> for 24 hours. The catalyst is filtered out through a Celite layer, and the filtrate is evaporated at 25° C. The residue is suspended in HgO (8 ml) and adjusted to pE 8.5 with conc. NE4OE and filtered through a Whatman filter paper to remove brown impurities. The colorless filtrate is adjusted to pE 6.8 and the precipitate

the tert compound is filtered, washed with HgO and dried, yield 65 mg (11%), m.p. >300°C.

EXAMPLE 49

The compound prepared in Example 48 is tested for enzyme inhibition activity as in Example 40. At 1 mM phosphate, IC is 500,097 µM and at 50 mM phosphate IC is 501.0 µM.

Claims (8)

1. Analogous process for the preparation of a compound which is a purine nucleoside phosphorylase inhibitor selected from the group consisting of 2-amino-7-(R)-3H,5H-pyrrolo[3,2-d]pyrimidin-4-one (I) and a compound with formula II where for compound I R is cyclohexyl, cyclohexenyl or -CHg-Ri» and where R^ is a lower alkyl or trifluoromethyl substituted or unsubstituted C4-Cg cycloalkyl or cycloalkenyl group, a tetrahydrofuranyl group, an adamantyl group or a pyridyl group and in which the compound with formula II is R^H, NHg or SCH3, R<2>is a phenyl group possibly substituted with one or more halogen atoms or is a C4~Cg cycloalkyl group, m is 0-4, X is OH CN, carboxy lower alkoxycarbonyl or aminocarbonyl , characterized by the preparation of the compound I in which R is cyclohexenyl or -CHg-Rinwhere R is pyridinyl or an alicyclic group comprises the following steps 1) to 7): 1) reacting a 3-substituted propanenitrile with an alkyl formate and a base to form a 3-cyclo-2-formyl-propanenitrile; 2) reaction of 2-formyl-3-substituted propanenitrile with glycine methyl ester hydrochloride and sodium or ammonium acetate to form the corresponding enamine; 3) reaction of the enamine with an alkyl chloroformate and DBN or DBU to form the corresponding N-blocked pyrrole; 4) reaction of N-blocked pyrrole with a base to form corresponding deblocked pyrrole; 5) reaction of deblocked pyrrole with benzoyl isothiocyanate to form the corresponding thioureido compound; 6) reacting the thioureido compound with an alkyl halide to form the corresponding methylthio compound; and 7) reaction of the methylthio compound with methanolic or ethanolic ammonia to form a mixture of the 2-amino-7-(R)-3E,5E-pyrrolo[3,2-d]pyrimidin-4-one compound (I) and 2- the methylthio byproduct; and when compound I is prepared in which R is cyclohexenyl or -CEg-Rinwhere R^ is a cycloalkenyl group, steps 1) to 7) as defined above and optionally the following step 8: 8) hydrogenation of the 2-amino-7-(R)-3H,5H-pyrrolo[3,2-d]pyrimidin-4-one compound from step 7) to 2-amino-7-(R)-3E,5H -pyrrolo[3,2-d]pyrimidin-4-one; and optionally the following steps 1) to 9) when the compound I is prepared where R is CEg-R^ when R^ is tetrahydrofuranyl, 1) reacting a 3-substituted or unsubstituted furanyl-propanenitrile with an alkyl formate and a base to form a 3-substituted or unsubstituted furanyl-2-formyl-propanenitrile; 2) reaction of 3-substituted or unsubstituted furanyl-2-formyl-propanenitrile with glycine methyl ester hydrochloride and sodium or ammonium acetate to form the corresponding enamine; 3) reaction of the enamine with an alkyl chloroformate and DBN or DBU to form the corresponding N-blocked pyrrole; 4) reaction of N-blocked pyrrole with a base to form corresponding deblocked pyrrole; 5) reaction of deblocked pyrrole with benzoyl isothioureanate to form the corresponding thioureido compound; 6) reacting the thioureido compound with an alkyl halide to form the corresponding methylthio compound; 7) reaction of the methylthio compound with methanolic or ethanolic ammonia to form a mixture of 2-amino-7-substituted or unsubstituted furanyl-3H,5H-pyrrole[3,2-d]pyrimidin-4-one compound (I) and the 2-methylthiobi product; 8) hydrogenation of the 2-amino-7-substituted or unsubstituted furanyl-3H,5H-pyrrolol[3,2-d]pyrimidin-4-one compound from step 7) to 2-amino-7-substituted or unsubstituted tetrahydrofuranyl-3H ,5H-pyrrole [3,2-d]pyrimidin-4-one; and and wherein the preparation of compound of formula II comprises the following steps: a) reacting an optionally substituted cyclic aldehyde with cyanoacetic acid in the presence of ammonium acetate to form a 3-substituted pentanedinitrile; b) reacting 3-substituted-pentanedinitrile with an alkyl formate and a base to form a 2-formyl-3-substituted-pentanenitrile; c) reacting 2-formyl-3-substituted-pentanedinitrile with glycine methyl ester hydrochloride and sodium or ammonium acetate to form methyl N-[3-substituted-2,4-dicyano)-2-butenyl]glycine; d) reacting methyl N-[3-substituted-2,4-dicyano)-2-butenyl]glycine with an alkyl chloroformate and DBN or DBU to form l-ethyl-2-methyl 3-amino-4-substituted lH- pyrrole-1,2-dicarboxylate; and e) reacting 1-ethyl-2-methyl-3-amino-4-substituted-1E-pyrrole-1,2-dicarboxylate with a base to form methyl 3-amino-4-substituted-1H-pyrrole-2 -carboxylate (i) and the following steps f)-h) for the preparation of 3-substituted-3-[2-amino-4-oxo-3H-5H-pyrrolo[3,2-d]-pyrimidin-7-yl] -propanenitrile; f) reacting methyl 3-amino-4-substituted-1H-pyrrole-2-carboxylate with benzoyl isothiocyanate to form N-benzoyl-N'-(2-methoxycarbonyl-4-substituted-1H-pyrrole-3-benzoyl isothiocyanate to form N-benzoyl-N'-(2-methoxycarbonyl-4-substituted-1H-pyrrol-3-yl)thiourea; g) reaction of N-benzoyl-N'-[2-methoxycarbonyl-4-substituted-1H-pyrrole -3-yl]thiourea with an alkyl halide to prepare N-benzoyl-N'-2-methoxycarbonyl-4-substituted-1E-pyrrol-3-yl]-S-alkylthiourea; and h) reacting N-benzyl-N'-[2-methoxycarbonyl-4-substituted-1H-pyrrol-3-yl]-S-alkylthiourea with methanolic or ethanolic ammonia to form a mixture of 3-substituted 3-( 2-amino-4-oxo-3E,5H-pyrrolo[3,2-d]pyrimidin-7-yl)propanenitrile and 3-substituted-3-(2-methylthio-4-oxo-3H,5H-pyrrolo[3 ,2-d]pyrimidin-7-yl)propanenitrile; (ii) and the following steps f)-j) in the preparation of 3-substituted-3-[2-alkoxy-4-oxo-3H,5H-pyrrolo[3,2-d]pyrimidin-7-yl]propanenitrile; f) reacting methyl 3-amino-4-substituted-1H-pyrrole-2-carboxylate with benzoyl isothiocyanate to form N-benzoyl-N'-2-methoxycarbonyl-4-substituted-1H-pyrrol-3-yl)thiourea; g) reacting N-benzoyl-N'-(2-methoxycarbonyl-4-substituted-1H-pyrrol-3-yl)thiourea with an alkyl halide to form N-benzoyl-N'-(2-methoxycarbonyl-4-substituted -1H-pyrrol-3-yl]-S-alkylthiourea; and h) reaction of N-benzoyl-N'-(2-methoxycarbonyl-4-substituted-1H-pyrrol-3-yl(-S-alkylthiourea) with methanolic or ethanol in so-called ammonia to form a mixture of 3-substituted-3-(2-amino-4-oxo-3H,5H-pyrrolo[3,2-d]pyrimidin-7-yl)propanenitrile and 3-substituted -3-(2-alkylthio-4-oxo-3H,5H-pyrrolo[3,2-d]pyrimidin-7-yl)propanenitrile; i) reaction of 3-substituted-3-(2-methylthio-4-oxo -3H,5H-pyrrolo[3,2-d]pyrimidin-7-yl)propanenitrile with an oxidizing agent to form 3-substituted-3-(2-methyl-sulfonyl-4-oxo-3H,5E- pyrrolo[3,2-d]pyrimidin-7-ylpropanenitrile; and j) reaction of 3-substituted-3-(2-methylsulfonyl-4-oxo-3H,5H-pyrrolo[3,2-d]pyrimidine- 7-yl]propanenitrile with sodium alkoxide to form 3-substituted-3-(2-alkoxy 4-oxo-3H,5H-pyrrolo[3,2-d]pyrimidin-7-yl)propanenitrile; (iii) and the following tr into f)-g) in the preparation of 3-substitute-3-(4-oxo-3H,5H-pyrrolo[3,2-d]pyrimidin-7-yl)propanenitrile; k) reacting methyl 3-amino-4-substituted-1H-pyrrole-2-carboxylate with dimethylformamide dimethyl acetal to form methyl 4-substituted-3-[N-(dimethylaminomethylene)amino]-1H-pyrrole-2-carboxylate; and 1) reaction of methyl 4-substituted-3-[N-(dimethylamino-methylene)amino]-1H-pyrrole-2-carboxylate with methanolic ammonia to form 3-substituted-3-(4-oxo-3H,5H- pyrrolo[3,2-d]pyrimidin-7-yl)propanenitrile. 2. Analogous method according to claim 1, for the production of a compound of formula I or II, in which 2-, 3- or 4-pyridinyl, 1- or 2-adamantyl, cyclopentyl, cyclohexyl or cycloheptyl, characterized by using correspondingly substituted starting materials. 3. Analogous method according to claim 1 for the production of compound (I) in which R is 3-pyridinylmethyl, characterized in that correspondingly substituted starting materials are used.