NL8203286A - 3H-IMIDAZO-5,1-D-1,2,3,5-TETRAZINE-4-ON DERIVATIVES, METHODS FOR PREPARING THEM AND PHARMACEUTICAL COMPOSITIONS, CONTAINING THESE DERIVATIVES. - Google Patents

Description

m i.

v N.O. 31302 1 [3H] -imidazo [5,1-d] -1,2,3,5-tetrazine-4-one derivatives, methods of preparation thereof and pharmaceutical compositions containing these derivatives.

The present invention relates to new [3H] imidazo [5,1-d] 1,2,3-tetrazine-4-one derivatives, methods of preparation thereof and pharmaceutical compositions containing these derivatives.

The compounds of the present invention are the [3 H] -imidazo [5, 1d] -1,2,3,5-tetrazine-4-one derivatives of the general formula 1, wherein R 1 is a hydrogen atom or a straight or branched alkyl, alkenyl or alkynyl group containing from 1 to 6 carbon atoms, any such group may be unsubstituted or substituted with ββη to three substituents selected from halogen (ie bromine, iodine or preferably chlorine or fluorine) atoms, straight or branched chain alkoxy, (for example, methoxy), alkylthio, alkylsulfinyl and alkylsulfonyl groups having up to 4 carbon atoms, and optionally substituted phenyl groups or a cycloalkyl group, and R 1 represents a carbamoyl group which has one or two on the nitrogen atom groups can be selected from straight and branched chain alkyl and alkenyl groups, each of which contains at most 4 carbon atoms and cycloalkyl groups, for example a methyl carbamoyl or dimethyl carbamoy group.

When the symbol R 1 represents an alkyl, alkenyl or alkynyl group substituted with two or three halogen atoms, the aforementioned halogen atoms may be the same or different. When the symbol represents an alkyl, alkenyl or alkynyl group substituted with §, two or three optionally substituted phenyl groups, the optional substituents on the phenyl group (s) may be selected from, for example, alkoxy and alkyl groups having up to 4 carbon atoms ( for example methoxy and / or methyl group (s)) and the nitro group; the symbol R1 may represent, for example, a benzyl or p-methoxybenzyl group. Cycloalkyl groups within the definitions of the symbols R 3 and contain 3 to 8, preferably 6, carbon atoms.

Preferred tetrazine derivatives of the general formula 1 are those in which R * * represents a straight or branched chain alkyl group of 1 to 6 carbon atoms, optionally substituted with S§n or two halogen (preferably chlorine, fluorine or bromine) atoms or with an alkoxy group containing 1 to 4 carbon atoms (preferably 8203286 2 methoxy) or with a phenyl group (optionally substituted with SSn or two alkoxy groups containing 1 to 4 carbon atoms), preferably methoxy), or wherein an alkenyl group containing 2 to 6 carbon atoms (preferably allyl) or represents a cyclohexyl group.

More particularly preferred tetrazine derivatives are those derivatives of the general formula 1, wherein R 1 represents a straight or branched chain alkyl group having 1 to 6 carbon atoms and more particularly 1 to 3 carbon atoms, unsubstituted or substituted with a halogen, preferably chlorine or fluorine, atom. More particularly, represents a methyl or 2-haloalkyl, for example 2-fluoroethyl or, preferably, 2-chloroethyl group.

Preferably R 1 represents a carbamoyl group or a monoalkyl carbamoyl, for example, methyl carbamoyl or monoalkenyl carbamoyl group.

The present invention also relates to salts of the compounds of the general formula 1, wherein R 1 represents a hydrogen atom and R 1 as defined above, more in particular alkali metal salts, eg sodium salts, and where the context permits reference to the compounds of general formula 1 in the present specification also refers to the salts. The salts are particularly suitable as intermediates.

According to a feature of the present invention, the compounds of the general formula 1, wherein R 2 as defined above and R 1 is other than hydrogen, are prepared by the reaction of a compound of the general formula 2, wherein R 2 as defined above is, with an isocyanate of the general formula 3, which represents an alkyl, alkenyl or alkynyl group optionally substituted with one to three substituents selected from halogen atoms, alkoxy, alkylthio, alkylsulfinyl and alkylsulfonyl and optionally substituted phenyl groups or a cycloalkyl group within the definition of the aforementioned symbol R * *. The reaction can be carried out in the absence or presence of an anhydrous organic solvent, for example a chlorinated alkane, for example dichloromethane or ethyl acetate, acetonitrile, N-methylpyrrolide-2-one or preferably hexamethylphosphoramide, at a temperature between 0 ° and 70 ° C, for example at ambient temperature. The reaction can be continued for up to 30 days. Light should preferably be closed outside of the reaction mixture.

In accordance with another feature of the present invention, 40 compounds of the general formula 1, wherein R ^ is as defined above 8203285 t (3 and R ^ is other than hydrogen, are prepared by the reaction of a compound (within the general formula 1) of the general formula 4, wherein goals are defined above, or an alkali metal salt, for example sodium salt thereof, with a compound of the general formula 5, wherein R 2 is as defined above and X represents the acid residue of a reactive ester for example a halogen (for example chlorine) atom or a sulfuric or sulfonic acid ester residue, for example a methoxysulfonyloxy, methanesulfonyloxy or toluene p-sulfonyloxy group When R 1 in a compound of the general formula 5 is a halo-10alkyl or haloalkenyl or haloalkynyl group, the acid moiety of a reactive ester represented by X will be selected from those known to be no less reactive than the halogen a substituent in R ^. For example, when X in a compound of the general formula 5 represents a halogen atom, an alkali metal salt of the compound of the general formula 4 is used and when X in a compound of the general formula 5 represents a halogen atom and R 1 is a haloalkyl -, haloalkenyl or haloalkynyl group, in which the halogen atom is the same as that represented by X, an excess of the dihalo compound of general formula 5 is preferably used. The reaction of a compound of the general formula 4 or an alkali metal salt thereof with a compound of the general formula 5, wherein R 1 and X as defined above, can be carried out in a suitable anhydrous inert organic solvent, for example dichloromethane, acetonitrile or N-methylpyrroli-de-2-one or mixtures thereof, at a temperature of 0 ° C to 120 ° C, when a compound of the general formula 4 is used, in the presence of an acid-binding agent, for example an alkali metal -, for example, sodium or potassium carbonate or hydrogen carbonate.

As a further feature of the invention, compounds of general formula 4 (ie compounds of general formula 1, wherein R 2 represents a hydrogen atom and R 2 as defined above) or its alkali metal salts are prepared by the reaction of a compound with the general formula 2 with a compound of the general formula 6, wherein R 1 represents an alkali metal atom (eg sodium) or a protecting group such as a benzyl or p-methoxy-benzyl group, followed when R 1 represents a protecting group, by the replacement of the protecting group by a hydrogen atom in the compound of the general formula (7) thus obtained, wherein R 4 is as defined above and R 4 represents a protecting group, such as a benzyl or p-methoxybenzyl group, according to itself - 820 3 286 «» 4 known working methods. Reaction of a compound of the general formula 2 with a compound of the general formula 6, which represents a protecting group, can be carried out as described above for the reaction of a compound of the general formula 2 with a compound of the general formula 3. Reaction of a compound of the general formula 2 with a compound of the general formula 6, which represents an alkali metal atom, can be carried out in a suitable inert organic solvent, for example ethanol, acetonitrile or N-methylpyrrolidone, if desired in the presence of an acid, at a temperature of 0 ° to 120 ° C. The group R ^ of compounds of the general formula 7, wherein R ^ as defined above, can be replaced by a hydrogen atom according to methods known per se to obtain a compound of the general formula 4.

Compounds of general formula 2 can be prepared by the application or adaptation of methods known per se, for example, methods described by Shealy Y.F., Struck R.F., Houlk L.B and Montgomery J.A., J. Org. Chem. 26, 2396 (1961).

Compounds of general formulas 3, 5 and 6 can be prepared by application or adaptation by methods known per se.

The term "in known per se methods" as used in the description is understood to mean processes which have been used heretofore or have been described in the literature.

The new tetrazine derivatives of the general formula 1 have valuable antineoplastic activity, for example against carcinomas, melanomas, sarcomas, lymphomas and leukemias. They have been found to be particularly effective in mice at daily doses between 0.5 and 16 mg / kg body weight administered intraperitoneally, against TLX5 (S) 30 lymphoma by the method of Gescher et al., Biochem. Pharmacol. 30, (1981) 89, and ADJ / PC6A and M5076 (reticulum cell sarcoma). Against leukemia L1210, intraperitoneal, intracerebral and intravenous, and P388 by the method described in "Methods of Development of New Anticancer Drugs" (NCI Monograph 45, March 1977, pages 147-149, 35 National Cancer Institute, Bethesda, United States), the compounds were active both intraperitoneally and orally at doses between 2.5 and 10 mg / kg body weight. Remitting of both primary tumor and metastasis was obtained against Lewis lung carcinoma by similar dosing regimens. Against the B16 melanomas and C38 tumor in mice 40 (NCI Monograph 45, cit.), The compounds were active intraperitoneally at 8203288 4% at doses between 6.25 and 25 mg / kg body weight.

The tetrazine derivatives also have valuable immunomodulatory activity and can be used in the treatment of organ grafts and skin grafts and in the treatment of immunological diseases.

Important individual compounds of the general formula 1 include the following: 8-carbamoyl-3-methyl- [3 H] imidazo [5,1-d] -1,2,3,5-tetrazine-4-one A, 10 8-carbamoyl- 3-n-propyl- [3H] -imidazo [5.1d] - 1.2.3.5-tetrazine-4-one B, 8-carbamoyl-3 ~ (2-chlorophenyl) - [3H] -imidazo [5.1 -83-1,2,3,5-ίθίΓηζ1ηθ-4-οη C, 3- (2-chloroethyl) -8-methylcarbamoyl- [3H] - 15 imidazo [5.1d] -1,2,5.5- tetrazine-4-one D, 8-carbamoyl ~ 3- (3-chloropropyl) - [3H] imidazo [5, 1d] -1,2,3,5-tetrazine-4-one E, 8-carbamoyl- 3- (2,3-dichloropropyl) -1H] -imidazo [5, 1d] -1,2,3,5-tetrazine-4-one F, 20 3-allyl-8-carbamoyl- [3H] -imidazo [ 5.1) 1.2.3.5-Tetrazine-4-one G, 3- (2-chloroethyl) -8-dimethylcarbamoyl- [3 H] imidazo [5,1d] -1,2,3,5-tetrazine-4 -one H, 3- (2-bromoethyl) -8-carbamoyl- [3 H] imidazo-25 [5, 1d] -1,2,3,5-tetrazine-4-one 1,3-benzyl-8- carbamoyl- [3H] imidazo [5.1]] 1.2.3.5-tetrazine-4-one J, 8-carbamoyl-3- (2-methoxyethyl) - [3H] imidazo [5.1] -1.2 3,5-tetrazine-4-one K, 30 8-carbamoyl-3-cyclohexyl ~ [3H] imidazo [5.1d] - 1.2.3.5-tetr azine-4-one L, and 8-carbamoyl-3- (p-methoxybenzyl) - [3H] -

imidazo [5,1-dj-1,2,3,5-tetrazine-4-one. M

The compounds A and D and in particular C are of particular interest.

The letters A to M are assigned to the compounds for later reference.

EXAMPLE I

40 Connection A

8203286 * 6 4 [5] -DiazoImidazole-5 [4] -carboxamide (500 mg) was suspended in 3.0 ml of methyl isocyanate and stirred at ambient temperature in the dark for 21 days. The reaction mixture was then diluted with anhydrous diethyl ether and filtered. The residue was quickly washed with anhydrous methanol, then with anhydrous diethyl ether, and dried in air in the dark at ambient temperature to give 8-carbamoyl-3-methyl- [3 H] imidazo [5.1] -1.2 3,5-tetrazine-4-one was obtained in the form of a light brown microcrystalline solid (198 mg), mp 210 ° C (with effusion and darkening from 160 ° 10 to 210 ° C).

[Elemental analysis: found: C 36.8; H 3.10; N 44.2%; C6 H6 N5 O2 calculated: C 37.1; H 3.09; N 43.3%].

EXAMPLE II

15 Connection B

4 [5] -Diazoimidazole-5 [4] -carboxamide (300 mg) was suspended in 10 ml of anhydrous dichloromethane and treated with an excess of n-propyl isocyanate. The reaction mixture was then stirred at ambient temperature in the dark for 30 days. Then the reaction mixture was filtered and the residue was washed quickly with anhydrous diethyl ether and dried in air in the dark at ambient temperature to give 8-carbamoyl-3-n-propyl- [3 H] imidazo [5.1 -d] -1,2,3,5-tetrazine-4-one (102 mg) was obtained in the form of a light pink powder, m.p. 167 ° C (with effusion).

[Elemental analysis: found: G 43.4; H 4.57; N 38.0%; C8H10N6O2 calculated: C 43.2; H 4.53; N 37.8%].

EXAMPLE III

Connection C

4 [5] -Diazolmidazole-5 [4] -carboxamide (300 mg) was suspended in 10 ml of anhydrous dichloromethane and 2-chloroethyl isocyanate (1.0 ml) was added. Then the reaction mixture was stirred at ambient temperature for 30 days. The cream-colored suspension thus obtained was filtered and the residue was quickly washed with anhydrous diethyl ether and dried in the air in the dark to give 8-carbamoyl-3- (2-chloroethyl) - [3 H] imidazo [5 -1-1,3,3,3-tetrazine-4-one (483 mg) was obtained in the form of a cream colored powder, mp 158 ° C (under vigorous decomposition).

[Elemental analysis: 40 found: C 34.7 H 3.01; N 34.9%; C7H7CIN5O2 8203236 4 → 7 Calculated: C 34.7; H 2.91; N 34.8%].

Repeating the above method also has 8-carbamoyl-3- (2-chloroethyl) - [3 H] imidazo [5,1-d] -1,2,3,5-tetrazine-4-one in another polymorphic shape, mp 164-165 ° C (with decomposition).

EXAMPLE IV

Connection A

A suspension of 4 [5] -diazoimidazole-5 [4J-carboxamide (1.37 g) in ethyl acetate (20 ml) was treated with 7.0 g of methyl isocyanate and stirred in a closed kettle at room temperature for 3 weeks. . The solid obtained was filtered off and washed with diethyl ether to give 8-carbamoyl-3-methyl- [3 H] imidazo [5.1] -1,2,3,5-tetrazine-4-one (1.9 g) was obtained in the form of a cream-colored solid, melting point 212 ° C (under foaming).

This material was recrystallized from three different solvent systems to yield three different products, each of which had a slightly different IR spectrum. The three products were probably all polymorphs of 8-carbamoyl-3- [3 H] imidazo [5,1-d] -1,2,3,5-tetrazine-4-one.

(I) Colorless needles were obtained from a 3: 1 mixture of volume parts of acetone and water, 1? 3410, 3205, 1758, 1730 and max 1678 cm, melting point 212 ° C (with foam).

(ii) White microcrystals were obtained from a 1: 3 mixture of parts by volume of acetone and water, 3430, 3200, 1740 and

IH3X

25 1675, melting point 210 ° C (with foaming).

(iii) A granular solid was obtained from bite water, 343450, 3380, 3200, 1742, 1688 and 1640 c, melting point max 215 ° C (with foaming) (darkening from 200 ° C).

EXAMPLE V

30 Connection B

A suspension of 4 [5] -diazolmidazole-5 [4] -carboxamide (1.37 g) in acetonitrile (20 ml) was treated with 6.5 g of n-propyl isocyanate and was placed in a closed kettle in the dark at room temperature for Stirred for 3 weeks. The resulting pink solid was filtered off, washed with 35 diethyl ether and recrystallized from a mixture of water and acetone (volume ratio 1: 4) to give 8-carbamoyl-3-n-propyl- [3 H] imidazo [5 1,1d-1,2,3,5-tetrazine-4-one (1.6 g) mp 170-172 ° C (with foaming) was obtained. By concentrating the mother liquor from the recrystallization, a further amount (0.2 g) of the same product was obtained.

8203286 • ψ 8

EXAMPLE VI

Connection C

A suspension of 4 [5] -diazoImidazole-5 [4] -carboxamide (1.0 g) in ethyl acetate (30 ml) was treated with 3.3 ml of 2-chloroethyl isocyanate and the mixture was left in the dark at ambient temperature for 6 stirred for days. Then the reaction mixture was diluted with diethyl ether and the solid obtained was filtered off, whereby 8-carbamoyl-3- (2-chloroethyl) - [3 H] imidazo [5, 1d] -1,2,3,5-tetrazine-4 -one (1.6 g) was obtained as a colorless solid, mp 164-165 ° C (with decomposition).

[Elemental analysis: found: C 34.5; H 2.88; N 34.5; Cl 14.6%; C7H7CIN6O2 calculated: V 34.65; N 34.65; Cl, 14.61%].

EXAMPLE VII

15 Connection C

A suspension of 4 [5] -diazoImidazole-5 [4] -carboxamide (5.0 g) in a mixture of dichloromethane (158 ml) and N-methylpyrrolide-2-one (8.3 ml) was mixed with 16.7 ml of 2-chloroethyl isocyanate were treated and the mixture was stirred at ambient temperature in the dark for 14 days. The reaction mixture was then diluted with anhydrous diethyl ether and the solid obtained was filtered off and washed with diethyl ether, yielding 8-carbamoyl-3- (2-chloroethyl) - [3H] -imidazo [5.1] 2.3. 5-tetrazine-4-one (6.3 g) was obtained in the form of a purplish solid, mp 164-165 ° C (with decomposition).

[Elemental analysis: found: C 34.7; H 2.95; N 34.5; Cl 14.4%; C7 H7 ClN6 O2 calculated: C 34.65; H 2.91; N 34.65; Cl, 14.61%].

EXAMPLE VIII

Connection C

A suspension of 4 [5] -diazolmidazole-5 [4] -carboxamide (145 g) in ethyl acetate (2175 ml) was treated with 478.5 ml of 2-chloroethyl isocyanate and at 30 ° C under the exclusion of light for 2 stirred for days. The mixture was then filtered to give 8-carbamoyl-3- (2-chloroethyl) - [3 H] imidazo [5.1] -1,2,3,5-tetrazine-4-one (250 g). gene was in the form of a peach solid, mp 166 ° C.

EXAMPLE IX

Connection A

A stirred suspension of 4 [5] -diazolmidazole-5 [4] -carboxamide 40 (2.2 g) in a mixture of dichloromethane (70 ml) and N-methyl-8203286 9% pyrrolidine-2-one (3, 5 ml) was treated with 7.0 ml of methyl isocyanate and stirred at ambient temperature for 4 weeks. The mixture was diluted with diethyl ether and the resulting solid filtered off to give 8-carbamoyl-3-methyl-[3H] imidazo [5, 1d] -1,2,3,5-tetrazine-4-one 5 ( 2.38 g), were obtained in the form of a light purple solid, mp 202-203 ° G (with decomposition).

[Elemental analysis: found: C 36.8; H 2.94; N 43.1%; CgH6NgO2 calculated: C 37.11; H 3.14; N 43.3%].

A polymorphic form of 8-carbamoyl-3-methyl-[3 H] imidazo [5.1] -1,2,3,5-tetrazine-4-one was obtained by dissolving it in acetonitrile, filtering, concentrate the filtrate to dryness and triturate the resulting residue with diethyl ether. This product was in the form of an orange-colored solid, melting point about 200 ° C (under decomposition).

[Elemental analysis: C 37.4; H 3.26; N 43.5%].

Its NMR spectrum in dimethylsulfoxide-Dg was identical to that of the aforementioned light purple solid, but the IR spectrum (KBr disc) showed some differences.

20 EXAMPLE X

Connection D

A stirred solution of 0.64 g of sodium nitrite in 4.6 ml of water was cooled to 5-10 ° C and treated dropwise at that temperature with a solution of 1.00 g of 5-amino-4-methylcarbamoylimidazole in 14.3 ml. 1 molar aqueous acetic acid for 5 minutes. Stirring was continued at 5 to 10 ° C for 5 minutes. The dark red solution was then extracted with 4 x 35 ml ethyl acetate and the combined extracts were dried over magnesium sulfate. The resulting solution contained crude 4 [5] -diazo-5 [4] -methylcarbamoyl-30 imidazole, which was unstable and was immediately used for the next step without further purification.

The solution of 4 [5] -diazo-5 [4] -methylcarbamoylimidazole in ethyl acetate, prepared as described above, was treated with 4.3 ml of 2-chloroethyl isocyanate and stored in the dark for 1 day.

Then the solution was evaporated at 40 ° C / 1.3 kPa and the residue was triturated with petroleum ether (boiling point 40-60 ° C) to obtain 4.23 g of an orange gum. This gum was treated with 50 ml of ethyl acetate and filtered and the filtrate was evaporated at 40 ° C / 1.3 kPa to obtain 2.94 g of an orange gum. This gum 40 was purified by medium pressure column chromatography on silica gel, eluting with a 4: 1 by volume mixture of ethyl acetate and acetonitrile, to give 3- (2-chloroethyl) -8-methylcarbamoyl- [3H] -imidazo. [5.1d] -1,2,3,5-tetrazine-4-one (0.81 g) was obtained in the form of a purple solid, mp 120-122 ° C (decomposition).

[Elemental analysis: found: C 37.3; H 3.58; N 31.9%; CgHgClN2 calculated: C 37.4; H 3.53; N 32.7%].

EXAMPLE XI

10 Connection B

A suspension of 4 [5] -diazolmidazole-4 [4] -carboxamide (1.0 g) in ethyl acetate (50 ml dried over anhydrous potassium carbonate) was treated with 4.86 g of 3-chloropropyl isocyanate and the mixture was treated for 3 days at ambient temperature stirred. Then, the reaction mixture was diluted with anhydrous diethyl ether and the solid obtained was filtered and washed with anhydrous diethyl ether to give 8-carbamoyl-3- (3-chloropropyl) - [3H] -imidazo [5.1 -d] -1,2,3,5-tetrazine-4-one (1.05 g) was obtained as a pink solid, mp 153-154 ° C (with decomposition).

[Elemental analysis: found: C 37.1; H 3.42; N 32.7; Cl 13.8% C8H8ClN602 calculated: C 37.4; H 3.53; N 32.8; Cl 13.8%].

EXAMPLE XII

Connection F

Proceeding in a similar manner as described above in Example XI, but replacing the 3-chloropropyl isocyanate used as starting product with the appropriate amount of 2,3-dichloropropyl isocyanate, 8-carbamoyl-3- (2 , 3-dichloro-propyl) - [3H] imidazo [5, 1d] -1,2,3,5-tetrazine-4-one prepared in the form of an off-white solid, mp 153-155 ° C (with decomposition).

[Elemental analysis: found: C 32.7; H 2.51; N 28.7; Cl 24.1% C8H8C12N602 calculated: C 33.0; H 2m77; N 28.9; Cl, 24.4%].

EXAMPLE XIII

Connection G

Stirred 4.5 ml immediately before use, stirred allyl isocyanate with 1.0 g of 4 [5] -diazoimidazole-5 [4] -carboxamide and then with 20 ml of hexamethylphosphoramide. The mixture was stirred in the dark at ambient temperature for 18 hours and then 8203286 Π Π then diluted with anhydrous diethyl ether and filtered.

The resulting colorless solid was washed with anhydrous diethyl ether to obtain 3-allyl-8-carbamoyl- [3 H] imidazo [5,1-d] -1,2,3,5-tetrazine-4-one (1.6 g) was obtained in the form of a colorless solid 5, mp 149-150 ° C.

[l) (KBr disc): 1730, 1675 cm -1, NMR in MMSO-d *: singlets at 8.75, 7.67 and 7.60 (7; double triplet at 6.02 a (Js5.5) , 8, 10Hz), double doublet at 5.35 / (J-1.5, 8 Hz) and 5.20 (f (J = 1.5, 10 Hz) and doublet at 4.88 (/ (J = 5.5)].

VQQR IMAGE XIV

Connection H

A solution of 4 [5] -diazo-5 [4] -dimethylcarbamoylimidazole (1.59 g as described in Reference Example I below) in 57 ml of dry ethyl acetate was treated with 6.36 g of 2-chloroethyl isocyanate and 15 in the dark for 24 stirred at room temperature for hours. The solution was then evaporated under vacuum at 35 ° C and finally at 0.013 kPa to remove the excess 2-chloroethyl isocyanate. The residual liquid was purified by silica gel column chromatography over silica gel, slurried with a mixture of ethyl acetate and acetonitrile in the volume ratio 4: 1, whereby 3- (2-chloroethyl) -8-dimethylcarbamoyl- [3H] - imidazo [5, 1d] -1,2,3,5-tetrazine-4-one (0.82 g) was obtained in the form of colorless crystals, m.p. 114-116 ° C.

[Elemental analysis: 25 found: C 39.7j H 3.95; N 30.8%; C9HHCIN6O2 calculated: C39.9; H 4.10; N 31.0%].

EXAMPLE XV

Connection I

A stirred suspension of 4 [5] -diazoImidazole-5 [4] -carboxamide 30 (1.0 g) in hexamethylphosphoramide (4 ml) was treated with 4.5 ml of 2-bromoethyl isocyanate and the mixture was stirred for 2 days -fing temperature stirred in the dark. Then the reaction mixture was diluted with anhydrous diethyl ether and the residual solid was filtered off and washed with anhydrous diethyl ether, whereby 3- (2-bromoethyl) -8-carbamoyl- [3H] -imidazo [5,1-d] - 1,2,3,5-Tetrazine-4-one (1.17 g) was obtained as a colorless solid, m.p. 156-157 ° C (with decomposition). [Elemental analysis: found: C 29.5; N 2.36; N 29.2; Br 27.3%; C7H7BrN602 40 calculated: C 29.3; H 2.46; N 29.3; Br 27.8%].

6203286 ψ »12

EXAMPLE XVI

Blinding J

Proceeding in a similar manner to that described above in Example XV, however, replacing the 2-broth-5-isocyanate, which was used as starting material, by the appropriate amount of benzyl-isocyanate, 3-benzyl-8-carbamoyl- [3H] imidazo [5, X-d] -1,2,3,5-tetrazine-4-one (0.83 g) prepared in the form of a strong yellowish-colored solid, m.p. 176- 177 ° C (with decomposition).

[Elemental analysis: found: C 53.6; H 3.66; N 31.0%; 0 ^ 2 ^ 10 ^ 6 ^ 2 calculated: C 53.3; H 3.73; N 31.1%].

EXAMPLE XVII

Connection K.

A suspension of 4 (5] -diazoImidazole-5 [4] -carboxamide (0.3 g) in acetonitrile (5 ml) was treated with 0.5 g of 2-methoxyethyl isocyanate and the mixture was left in the dark for 24 hours Stirred at 45 ° and 47 ° C. The solid obtained was filtered off and washed with diethyl ether to give crude 8-carbamoyl-3 ~ (2-methoxyethyl) - [3 H] ~ 20 imidazo [5.1] 2.3. 5-tetrazine-4-one (0.45 g), mp 145-147 ° C (with decomposition) was obtained.

The product was purified by recrystallization from aqueous acetone to give pink rosettes, or from aqueous dimethyl sulfoxide to obtain colorless needles, melting point 164-165 ° C (with decomposition).

[Elemental analysis: C 40.4; H 4.20; N 35.2%; GgHioN6 ° 3 calculated: C 40.34; H 4.20; N 35.2%].

EXAMPLE XVIII

Connection L.

A suspension of 4 [5] -diazolmidazole-5 [4] -carboxamide (0.30 g) in acetonitrile (10 ml) was treated with 1.0 g of cyclohexyl isocyanate and the mixture was left in the dark at 60 ° for 3 days C stirred. The resulting solid was filtered off and washed with a mixture of ethanol and 0.880 aqueous ammonia (volume ratio 100: 0.5, 20 ml) for 35 minutes, using 8-carbamoyl-3-cyclohexyl- [3 H] imidazo [5 1,1d-1,2,3,5-tetrazine-4-one (0.015 g), mp 196 ° C (with effusion) was obtained.

EXAMPLE XIX

Connection J

40 A suspension of 4 [5] -diazoimidazole-5 [4] -carboxamide (0.4 g) in 8203236 * 13 acetonitrile (10 ml) was treated with 0.6 g of benzyl cyanate and the mixture was washed overnight in the stirred dark at 60 ° C. Then the reaction mixture was cooled and filtered to give 3-benzylcarbamoyl- [3 H] imidazo [5.11] -1,2,3,5-tetrazine-4-one (0.75 g) in the form of a light pink solid with a melting point of 187-188 ° C (with effusion).

EXAMPLE XX

Connection M

A suspension of 4 [5] -diazoimidazole-5 [4] -carboxamide (0.1 g) and 10 p-methoxyb etc ylisocyanate (0.4 g) in acetonitrile (5 ml) was left in the dark for 4 hours stirred at 60 ° C. The resulting pale pink solid was filtered off and washed repeatedly with cold diethyl ether, 8-carbamoyl-3- (p-methoxybenzyl) - [3 H] imidazo [5,1-d] -1,2,3, 5-Tetrazine-4-one (0.23 g), mp 180-182 ° C (with effusion) was obtained.

EXAMPLE XXI

By proceeding in a similar manner to the previous examples, 8- (n-allylcarbamoyl) -3- (2'-chloroethyl) - [3 H] imidazo [5.1] -1.2,3 5-tetrazine-4-one (IR 1750 NMR (in 20 DMSO-Dg): multiplets 3.96, 5.06 and 5.84 ppm; triplets 4.60 and 6.2 ppm: singlet 8.78 ppm, prepared from 5-amino "-4-allylcarbamoylimidazole via 5-diazo-4-allylcarbamoylimidazole. The 5-amino-4-allylcarbamoyl imidazole was prepared from 5-nitro'-4- <allylcarbamoylimidazole (melting point 218-220 ° C) by reduction with titanium chloride.

25 REFERENCE EXAMPLE

(i) An intimate mixture of 5-nitroImidazole-4-carboxylic acid (2.0 g) and phosphorus pentachloride (2.67 g) was stirred and heated in an oil bath at 120 ° C for 1 hour. The resulting yellow slurry was evaporated at 60 ° C / 0.13 kPa for 30 minutes to give 1,6-dinitro-5H, 10H-diimidazol-1H-1'JS'-pypyrazine-SiO-dione (1.90 g). in the form of a yellow solid with a melting point of 249-152 ° C (with decomposition). [\) (KBr disc) 1750 cm m / e 278 ([+)]. mflx

Windaus, Ber., 56, 68 (1923) 684 and Givera, Chem. Abs. 59, 1622e, using the same method, describe their products as 35 "5-nitroImidazole-4-carbonyl chloride".

(ii) An aqueous dimethylamine solution (25 w / v%, 60 ml) was cooled to a temperature between 0 ° and 5 ° C and treated with 1,6-dinitro-5H, 10H in portions with stirring. -dilmidazo [1,5-a: 1,5'-d] -pyrazine-5,10-dione (6.0 g) within that temperature range. The resulting dark purple solution was stirred for 2 hours. The solution was evaporated at 8203286-150 ° C / 1.3 kPa and then acidified by treatment with concentrated hydrochloric acid to obtain an orange solution. This solution was extracted with 7 x 200 ml ethyl acetate and the combined extracts were dried over magnesium sulfate and evaporated to yield 6.6 g of a yellow solid. This solid was triturated with 50 ml of toluene and then recrystallized from ethyl acetate to give 5 [4] nitro-4 [5] dimethylcarbamoylimidazole (2.53 g) as yellow crystals, mp 193-195 ° C. became.

[Elemental analysis: found: C 38.9; H 4.23; N 30.4%; C6 H8 N4 O3 calculated: C 39.1; H 4.38; N 30.4%].

(iii) A solution of 5 [4] -nitro-4 [5] -dimethylcarbamoylimidazole (1.62 g) in dry dimethylformamide (32 ml) was treated with 0.32 g of platinum oxide and under hydrogen at atmospheric pressure and room temperature shaken. After 3 hours, the hydrogen absorption was complete (710 ml). The mixture was treated with charcoal and filtered through diatomaceous earth. The dark brown filtrate was evaporated at 50 ° C / 0.013 kPa and the resulting residue was triturated with diethyl ether, whereby 5 [4] -amino-4 [5] -dimethylcarbamoylimidazole (1.75 g), in an impurity, was shape of a dark brown, crystalline solid with a melting point of 179-181 ° C [ί) (KBr disk) 1595 cm -1; NMR in max DMSO-dg: singlets at 3.2 and 7.0 µl, which was still contaminated with colloidal platinum, was obtained and used in the next step without further purification.

(iv) A stirred solution of 0.79 g of sodium nitrite in 5.7 ml of water was cooled to a temperature between 5 ° and 10 ° C and treated dropwise within this temperature range with a solution of 5 [4] -amino-4 [5] dimethylcarbamoylimidazole (1.75 g) in aqueous acetic acid (1 molar, 17.6 ml) for 5 minutes. The resulting solution was extracted with 4 x 40 ml ethyl acetate, the combined extracts were dried over magnesium sulfate and evaporated at 30 ° C / 1.3 kPa to give 4 [5] diazo-5 [4] dimethyl carbamoylimidazole (1.59 g ) in the form of orange crystals, mp 101-103 ° C (with decomposition).

[Elemental analysis: found: C 42.6; H 4.17; N 41.4; C6 H7 N5 O calculated: C 43.6; H 4.27; N 42.4%].

The present invention also relates to pharmaceutical compositions which contain as active ingredient at least one trazyl derivative of the general formula 1, together with a pharmaceutical carrier or coating. In clinical practice, the compounds of the general formula 1 will be administered orally, rectally, vaginally or parenterally, for example intravenously or intraperitoneally.

Methods of providing pharmaceutically active compounds are known and a suitable carrier can be determined by the physician or pharmacist depending on factors such as the intended effect, size, age, sex and condition of the patient and of the properties of the active compound. The compositions may also, as usual, contain such materials as solid or liquid diluents, wetting agents, preservatives, flavors and dyes and the like.

Solid compositions for oral administration include compressed tablets, pills, dispersible powders and granules. In such solid compositions, one or more active compounds is or are mixed with at least one inert diluent such as calcium carbonate, potato starch, alginic acid or lactose. The compositions may also, as in normal practice, contain additional substances other than inert diluents, for example lubricants such as magnesium stearate. Liquid compositions for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs containing inert diluents as commonly used, for example, water and liquid paraffin.

In addition to inert diluents, such compositions may also contain additives, such as wetting agents and suspending agents, for example, polyvinylpyrrolidone, and sweeteners, flavors, flavors, and preservatives. The compositions of the invention for oral administration also include capsules of absorbable material, such as gelatin, containing or measuring active ingredients with or without addition of diluents or extenders.

Solid compositions for vaginal administration include pessaries, formulated in a manner known per se and containing Sen 35 or more active compounds.

Solid compositions for rectal administration include suppositories which are formulated in known manner and contain one or more active compounds.

Compositions of the invention for parenteral administration 40 include sterile aqueous or non-aqueous solutions, suspensions or 8203286 φ 16 emulsions. Examples of non-aqueous solvents or suspending media are polyethylene glycol, dimethyl sulfoxide, vegetable oils such as olive oil and injectable organic esters such as ethyl oleate. These compositions may also contain additives such as preservatives, wetting agents, emulsifying agents and dispersing agents. They can be sterilized, for example, by filtration through a bacteria retaining filter, by incorporation of sterilizing agents into the compositions or by irradiation. They can also be prepared in the form of sterile solid compositions, which are dissolved in sterile water or some sterile injectable medium immediately before use.

The percentage of active ingredient in the compositions of the invention may be varied, it being necessary that it will form such a portion that an appropriate dosage for the desired therapeutic effect will be obtained. Obviously, different unit dosage forms can be administered at about the same time. In general, the compositions will usually contain at least 0.025% by weight of the active ingredient when intended for administration by injection; for oral administration, the composition will usually contain at least 0.1% by weight of active ingredient. The dosage used depends on the desired therapeutic effect, the route of administration and the duration of the treatment.

The tetrazine derivatives of general formula 1 are suitable for the treatment of malignant neoplasms, for example carcinomas, melanomas, sarcomas, lymphomas and leukemias at doses generally between 0.1 and 200, preferably between 1 and 20 mg / kg body weight per day.

The following composition examples illustrate pharmaceutical compositions of the present invention.

30 COMPOSITION TYPE 1

A solution suitable for parenteral administration was prepared from the following ingredients: 8-Carbamoyl-3- (2-chloroethyl) - [3H] - imidazo [5, 1d] -1,2,3,5-tetrazine- 4-on 1.0 g 35 Dimethyl sulfoxide 10 ml

Peanut oil 90 ml by dissolving the 8-carbamoyl-3- (2-chloroethyl) - [3 H] imidazo [5, 1 d] -1,2,3,5-tetrazine-4-one in the dimethyl sulfoxide and add the peanut oil. The resulting solution was divided into ampoules under aseptic conditions in an amount of 10 ml per amp 8203286 17 s # pul. The ampoules were sealed to yield 10 ampoules, each containing 100 mg of 8-carbamoyl-3- (2-chloroethyl) - [3 H] imidazo [5.1] 1,2,3,5-tetrazine-4-one contain.

Similar ampoules containing solutions suitable for parenteral administration can be prepared by proceeding in a similar manner, but the 8-carbamoyl-3- (2-chloroethyl) - [3 H] imidazo [54, 1d] -1,2,3,5-tetrazine-4-one by replacing with another compound of the general formula I COMPOSITION EXAMPLE 2 Capsules suitable for oral administration were prepared by 8-carbamoyl-3- (2 chloroethyl) - [3 H] imidazo [5, 1d] -1,2,3,5-tetrazine-4-one in gelatin coat size No. 2 in an amount of 10 mg per capsule.

Similar capsules can be prepared using another compound of the general formula 1 or any other appropriately sized capsule shell.

8203286

Claims (22)

1. [3H] -imidazo [5, 1d] -1,2,3,5-tetrazine-4-one derivatives of the general formula 1, wherein R 1 is a hydrogen atom or a straight or branched alkyl , alkenyl or alkynyl group containing up to 6 carbon atoms, each such group being unsubstituted or substituted with §Sn up to three substituents selected from halogen, straight or branched chain alkoxy, alkylthio, alkylsulfinyl and alkylsulfonyl groups containing up to 4 carbon atoms, optionally representing substituted phenyl groups, or wherein a cycloalkyl group represents 10 containing 3 to 8 carbon atoms, and R 1 represents a carbamoyl group, which on the nitrogen atom may contain βδη or two groups selected from straight or branched chain alkyl and alkenyl groups, each of which contains at most 4 carbon atoms and represents cycloalkyl groups, which contain 3 to 8 carbon atoms and, when R 1 represents hydrogen, alkali metal salts thereof. Tetrazine derivatives according to claim 1, characterized in that r! represents an alkyl, alkenyl or alkynyl group substituted with §, two or three optionally substituted phenyl groups and the optional substituents on the phenyl group (s) are selected from alkoxy and alkyl groups containing up to 4 carbon atoms and the nitro group. Tetrazine derivatives according to claim 1, characterized in that R - * - represents a straight or branched alkyl group with 1 to 6 carbon atoms, optionally substituted with one or two halogen atoms or with an alkoxy group containing 1 to 4 carbon atoms or with a phenyl group, which is optionally substituted with one or two alkoxy groups containing 1 to 4 carbon atoms, or wherein R 1 represents an alkenyl group containing 2 to 6 carbon atoms or represents a cyclohexyl group. Tetrazine derivatives according to claim 3, characterized in that the halogen atom (s) is (are) chlorine, fluorine and / or bromine, the alkoxy group (s) is (are) methoxy and the alkenyl group is allyl. Tetrazine derivatives according to claim 1, characterized in that R 1 represents a straight or branched chain alkyl group having 1 to 6 carbon atoms, which may or may not be substituted with a halogen atom. Tetrazine derivatives according to claim 5, characterized in that r! represents an alkyl group containing 1 to 3 carbon atoms unsubstituted or substituted with a halogen atom. Tetrazine derivatives according to claim 1 or 5, characterized in that R * represents a methyl or 2-haloalkyl group. Tetrazine derivatives according to claims 5 to 7, characterized in that 8203286 is that the halogen atom on the alkyl group is chlorine or fluorine. Tetrazine derivatives according to claim 1, characterized in that R 1 represents a 2-fluoroethyl or 2-chloroethyl group. Tetrazine derivatives according to claim 1, characterized in that 5 R1 represents a benzyl or p-methoxybenzyl group. 11. Tetrazine derivatives according to one or more of claims 1 to IQ, characterized in that R 1 is a carbamoyl group, a monoalkyl carbamoyl group, which contains at most 4 carbon atoms in the alkyl group, or a monoalkenyl carbamoyl group, which contains at most 4 carbon atoms in the alkenyl group. Tetrazine derivatives according to claim 1, characterized in that r! represents a straight or branched chain alkyl, alkenyl or alkynyl group containing 1 to 6 carbon atoms, such group being unsubstituted or substituted by δέη to three halogen atoms and the carbamoyl group. 13. 8-Carbamoyl-3-methyl- [3 H] imidazo [5,1-d] -1,2,3,5-tetrazine-4-one. 14. 8-Carbamoyl-3-n-propyl- [3 H] imidazo [5,1-d] -1,2,3,5-tetrazine-4-one. 15. 8-Carbamoyl-3- (2-chloroethyl) - [3 H] imidazo [5.1-d] ~ 1.2.3.5-tetrazine-4-one. 16. 3- (2-Chloroethyl) -8-methylcarbamoyl- [3 H] imidazo [5.1-d] -1.2.3.5-tetrazine-4-one. 17. 8-Carbamoyl-3- (3-chloropropyl) - [3 H] imidazo [5,1-d] -25 1,2,3,5-tetrazine-4-one. 18. 8-Carbamoyl-3- (2,3-dichloropropyl) - [3 H] imidazo [5.1-d] -1.2.3.5-tetrazine-4-one. 19. 3-Allyl-8-carbamoyl- [3 H] imidazo [5,1-d] -1,2,3,5-tetrazine-4-one. 30 20. 3- (2-Chloroethyl) -8-dimethylcarbamoyl ~ [3 H] imidazo [5,1-d] - 1.2.3.5-tetrazine-4-one · 21. 3- (2-Bromoethyl) -8- carbamoyl-3H] imidazo [5, 1d] -1,2,3,5-tetrazine-4-one. 22. 3-Benzyl-8-carbimoyl- [3 H] imidazo [5,1-d] -1,2,3,5-35 tetrazine-4-one. 23. 8-Carbamoyl-3- (2-methoxyethyl) - [3 H] imidazo [5,1-d] -1,2,3,5-tetrazine-4-one. 24. 8-Garbamoyl-3-cyclohexyl- [3 H] imidazo [5,1-d] -1,2,3,5-tetrazine-4-one. 40 25. 8-Carbanoyl-3- (p-methoxybenzyl) - [3 H] imidazo [5.1, d] - 8203286 1,2,3,5-tetrazine-4-one. 26. 8- (n-Allylcarbamoyl) -3- (2-chloroethyl) - [3 H] imidazo [5, 1-d] -1,2,3,5-tetrazine-4-one. 27. A process for the preparation of tetrazine derivatives, characterized in that compounds of formula 1, in which the symbols have the meanings stated in claim 1, are prepared, but with the exclusion of hydrogen for the definition of R * *, in that a compound of the general formula 2, wherein R 2 defined as in claim 1, is reacted with an isocyanate of the general formula 3, wherein R 2 represents an alkyl, alkenyl or alkynyl group containing at most 6 carbon atoms, wherein any such group is unsubstituted or substituted with δδη to three substituents selected from halogen atoms, alkoxy, alkylthio, alkylsulfinyl and alkylsulfonyl groups containing up to 4 carbon atoms and optionally substituted phenyl groups, or R 1 represents a cycloalkyl group, which Contains 3 to 8 carbon atoms. 28. Process for the preparation of tetrazine derivatives, characterized in that compounds of the general formula 1, in which the symbols have the meanings stated in claim 1, are prepared, however, with the exclusion of hydrogen for the definition of R *, in that a compound of the general formula 4, wherein R 2 is defined as in claim 1, or an alkali metal salt thereof is reacted with a compound of the general formula 5, wherein R 2 is defined as in claim 26 and X the acid residue of a reactive ester proposes. 29. Process according to claim 27, characterized in that compounds of formula 5 are used, in which X represents a halogen atom, a methoxysulfonyloxy, methanesulfonyloxy or toluene p-sulfonyl oxy group. 30. A process for preparing tetrazine derivatives, characterized in that compounds of the general formula 1 according to claim 1, wherein R 1 represents a hydrogen atom and R 1 is defined as in claim 1, or an alkali metal salt thereof are prepared, by reacting a compound of the general formula II with a compound of the general formula 6, wherein R 1 represents an alkali metal atom or a protecting group such as a benzyl or p-methoxybenzyl group, followed by when R 1 represents a protective group, the replacement of the protecting group by a hydrogen atom in the compound of general formula 7 thus obtained, wherein R 1 is defined as in claim 1 and R 1 represents a protecting group, such as 40 a benzyl or p- methoxybenzyl group, according to methods known per se 8203286: 21. A process for preparing a tetrazine derivative of the general formula as defined in claim 1, wherein R * and r2 are defined as in claim 1, or when R * represents a hydrogen atom, an alkali metal salt thereof substantially as before described with reference to each of Examples 1 to 20. 32. A pharmaceutical composition containing as active ingredient at least one Sen tetrazine derivative according to S§n or more of claims 1 to 25 together with a pharmaceutical carrier or a coating. The pharmaceutical composition according to claim 31, substantially as described above with particular reference to the composition example 1 or 2. 34. Tetrazine derivatives of the general formula according to claim 1, wherein R 1 and r 2 are defined as in claim 1, for use in the treatment of malignant neoplasms such as carcinomas, melanomas, sarcomas, lymphomas and leukemia Sn. 35. Tetrazine derivatives of the general formula according to claim 1, wherein R 1 and R 1 are defined as in claim 1, for use in the treatment of organ transplants and skin grafts and for the treatment of immunological diseases. A method of preparing pharmaceutical compositions, characterized in that 6 and more compounds according to claim 1, wherein the symbols have the meanings stated in claim 1, are brought into a form suitable for such use. 25 ™ 8203286