NL192739C - 3H-imidazo-5,1-d-1,2,3,5-tetrazine-4-one derivatives and pharmaceutical compositions containing these derivatives. - Google Patents

Description

1 192739 [3H] -imidazo- {5,1-d] -1,2,3,5-tetrazine-4-one derivatives and pharmaceutical compositions containing these derivatives

The present invention relates to new [3 H] imidazo [5,1-d] -1,2,3,5-4-tetrazine-4-one derivatives with antineoplastic activity and to pharmaceutical compositions containing these derivatives.

DE-A-2,932,305 and Tetrahedron Letters, 1979, pages 4253-4256 disclose the preparation of azole [5,1 -dj-1,2,3,5-tetrazine-4-one derivatives by the cycload edition of diazoazoles and isocyanates. The compounds thus obtained are suitable as diuretics, sedatives, tranquilizers and anti-inflammatory agents.

The compounds of the present invention are the [3H] -imidazo [5,1-d] -1,2,3,5-tetrazine-4-one derivatives of the general formula 1, wherein R 1 is a methyl, 2- chloroethyl or 2-bromoethyl group and R2 represents a carbamoyl, methylcarbamoyl or dimethylcarbamoyl group.

More particularly preferred tetrazine derivatives are those of the general formula 1 wherein R 1 represents a methyl or 2-chloroethyl group.

Preferably R2 represents a carbamoyl group or a methyl carbamoyl group.

The compounds of the general formula I according to the invention can be prepared by reacting a compound of the general formula 2, wherein R 2 is as defined above, with an isocyanate of the general formula 3, wherein R 1 is as defined above.

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. Preferably, light should be closed outside the reaction mixture.

The compounds of the general formula I according to the invention may also be prepared by the reaction of a compound (within the general formula 1) of the general formula 4, wherein R 2 is as defined above, or an alkali metal salt, for example sodium salt thereof, with a compound of the general formula 5, wherein R 1 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 star residue, for example a methoxisulfonyloxy, methanesulfonyloxy or toluene -p-sulfonyloxi group. When R 1 in a compound of the general formula 5 represents a 2-chloroethyl or 2-bromoethyl 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 substituent in R 1. 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 2-chloroethyl- or 2-bromoethyl group, in which the halogen atom is the same as that represented by X, an excess of the dihalogen compound of the 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 are as defined above, can be carried out in a suitable anhydrous inert organic solvent, for example dichloromethane, acetonitrile or N -methylpyrrolide-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.

Compounds of general formula 4 or their alkali metal salts prepared by the reaction of a compound of general formula 2 with a compound of general formula 6, wherein R4 represents an alkali metal atom (e.g. sodium) or a protecting group such as a benzyl or p -methoxy-benzyl group, followed, when R 4 represents a protecting group, by replacing the protecting group with a hydrogen atom in the compound of general formula 7 thus obtained, wherein R 2 is as defined above and R 5 represents a protecting group, such as a 50 benzyl or p-methoxybenzyl group, according to methods known per se. Reaction of a compound of the general formula 2 with a compound of the general formula 6, wherein R 4 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, wherein R 4 represents an alkali metal atom 55, may be carried out in a suitable inert organic solvent, for example ethanol, acetonitrile or N-methylpyrrolidone, optionally in the presence of a acidic, at a temperature of 0 ° to 120 ° C. The group R5 of compounds of the general formula 7, wherein R5 is as defined above, 192739 2 can be replaced by a hydrogen atom by 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 5 L.B. and Montgomery J.A., J. Org. Chem. 26, 2396-2401 (1961).

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

The term "per se known methods" as used herein is to be understood to mean methods 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, lymphones and leukemias. They have been shown to be particularly effective in mice at daily doses between 0.5 and 16 mg / kg body weight administered intraperitoneally against TLX5 (S) lymphoma according to the method of Gescher et al., Biochem. Pharmacol. 30, (1981) 89-93, and ADJ / PC6A (a tumor cell line) and M5076 (reticulum cell sarcoma). Against leukemia L1210, grafted intraperitoneally, intracerebral, and intravenously, and P388 by the method described in "Methods of Development of New Anticancer Drugs" (NCI Monograph 45, March 1977), pages 147-149, National Cancer Institute, Bethesda, United States ), the compounds were active both intraperitionally and orally at doses between 2.5 and 10 mg / kg body weight. Inhibition of both primary tumor and metastasis was obtained against the Lewis lung carcinoma by similar dosing regimens. Against the 20 B16 melanomas and C38 tumor in mice (NCI Monograph 45, op cit.), the compounds were active intraperitoneally 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- [3Hj-imidazo [5,1-dj- 1,2,3,5-tetrazine-4-one A, 8 -carbamoyl-3- (2-chloroethyl) - [3H] imidazo [5,1-d] -1,2,3,5-tetrazine-4-one B, 30 3- (2-chloroethyl) -8 -methylcarbamoyl- [3H] imidazo [5,1-d] -1,2,3,5-tetrazine-4-one C, 3- (2-chloroethyl) -8-dimethylcarbamoyl- [3H] imidazo [5 1,1-d] -1,2,3,5-tetrazine-4-one D, 3- (2-bromoethyl) -8-carbamoyl- [3 H] imidazo [5,1-d] -1, 2,3,5-tetrazine-4-one E,

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

Example 1 Compound A

4 [5] -Diazoimidazole-5 [4] -carboxamide (500 mg) was suspended in 3.0 ml of methyl isocyanate and stirred at ambient temperature for 21 days in the dark. Then the reaction mixture was diluted with anhydrous diethyl ether and filtered. The residue was quickly washed with anhydrous methanol, then with anhydrous diethyl ether, and dried in the air at ambient temperature in the dark to give 8-carbamoyl-3-methyl- [3 H] imidazo [5.1-d] -1.2. 3,5-Tetrazine-4-one was obtained in the form of a 45 light brown microcrystalline solid (198 mg), mp 210 ° C (with effusion and darkening from 160 ° to 210 ° C).

[Elemental analysis: found: C 36.8; H 3.10; N 44.2%; C6 H6 N6 O2 calculated: C 37.1; H 3.09; N 43.3%].

50

Example II Compound B

4 [5] -Diazoimidazole-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 in the dark 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) - [3H] -imidazo [5.1- -1,2,3,5-tetrazine-4-one (483 mg) 3 192739 was obtained in the form of a cream-colored powder, mp 158 ° C (under vigorous decomposition). [Elemental analysis: found: C 34.7; H 3.01; N 34.9%; C7H7ClN602 calculated: C 34.7; H 2.91; N 34.8%] 5 Repeating the above method also has 8-carbamoyl-3- (2-chloroethyl) - [3 H} imidazo [5,1-d] -1,2,3,5-etrazine -4-one in another polymorphic form, m.p. 164-165 ° C (with decomposition).

Example III 10 Compound A

A suspension of 4 [5] -diazoimidazole-5 [4] -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 in the dark for 3 weeks . The resulting solid was filtered off and washed with diethyl ether to give 8-carbamoyl-3-methyl- [3 H] imidazo [5,1-d] -1,2,3,5-tetrazine-4-one (1.9 g) was obtained in the form of a cream-colored solid, mp 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-methyl- [3 H] imidazo [5,1-d] -1,2,3,5-4-tetrazine-4-one.

(I) Colorless needles were obtained from a 3: 1 mixture of volumes of acetone and water, 3410, 3205, 1758, 1730 and 1678 cm @ s, mp 212 ° C (with foaming.) (Ii) White microcrystals were obtained from a 1 : 3 mixture of parts by volume of acetone and water, 3430, 3200, 1740 and 1675, melting point 210 ° C (with foaming).

(iii) A granular solid was obtained from hot water, vmax> 3450, 3380, 3200, 1742.1688, and 1640 cm -1, mp 215 ° C (with foaming) (dark color from 200 ° C).

Example IV Compound C

A suspension of 4 [5] -diazoimidazole-5 [4] -carboxamide (1.0 g) in ethyl acetate (30 ml) was treated with 3.3 30 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] imadazo [5,1-d] -1,2,3,5-tetrazine -4-one (1.6 g) was obtained in the form of a colorless solid, mp 164-165 ° C (with decomposition).

[Elemental analysis: found: C 34.5; H 2.88; N 34.5; C1 14.6%; C7H7ClN602 calculated: V 34.65; N 34.65; C1, 14.61%].

Example V 40 Compound 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 and the mixture was stirred at ambient temperature in the dark for 14 days. Then, the reaction mixture was diluted with anhydrous diethyl ether, the solid obtained was filtered and washed with 45 diethyl ether, to give 8-carbamoyl-3- (2-chloroethyl) - [3H] -imidazo [5,1-d] -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 Analyzes: Found: C 34.7; H 2.95; N 34.5; C1 14.4%; C7 H7 ClN6 O2 50 calculated: C 34.65; H 2.91; N 34.65; C1, 14.61%].

Example V!

Connection C

A suspension of 4 [5] -diazoimidazole-5 [4] -carboxamide (145 g) in ethyl acetate (2175 ml) was treated with 55 478.5 ml of 2-chloroethyl isocyanate and stirred at 30 ° C under light for 2 days. . The mixture was then filtered to give 8-carbamoyl-3- (2-chloroethyl) - [3 H] imidazo [5,1-d] -1,2,3,5-tetrazine-4-one (250 g). in the form of a peach-colored solid, 192739 mp 166 ° C.

Example VII Compound A

A stirred suspension of 4 [5] -diazoimidazole-5 [4] -carboxamide (2.2 g) in a mixture of dichloromethane (70 ml) and N-methyl-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- [3 H] imidazo [5,1-d] -1,2,3,5-tetrazine-4-one ( 2.38 g), were obtained in the form of a light purple solid, mp 202-203 ° C (with decomposition).

[Elemental analysis: found: C 36.8; H 2.94; N 43.1%; C6 H6 N6 O2 calculated: C 37.11; H 3.14; N 43.3%].

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

[Elemental analysis: C37.4; H 3.26; N 43.5%].

Its NMR spectrum in dimethylsulfoxide-D6 was identical to that of the aforementioned light-20 purple solid, but the IR spectrum (KBr disc) showed some differences.

Example VIII Compound 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 of 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-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 DEG-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 was purified by medium pressure column chromatography over silica gel, eluting with a mixture of ethyl acetate and acetonitrile in the volume ratio of 4: 1 to 3- (2-chloroethyl) -6-methylcarbamoyl- [3 H] imidazo [5.1- d] -1,2,3,5-tetrazine-4-one (0.81 g) was obtained as a purple solid, mp 120-122 ° C (with decomposition).

40 [Elemental analysis: found: C 37.3; H 3.58; N 31.9%; CeH9ClN602 calculated: C 37.4; H 3.53; IM 32.7%].

Example IX 45 Compound D

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 left in the dark for Stirred at room temperature for 24 hours. Then, the solution was 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 column chromatography on medium over silica gel, eluting with a mixture of ethyl acetate and acetonitrile in the volume ratio 4: 1, whereby 3- (2-chloroethyl) -8-dimethylcarbamoyl- [3Hj-imidazo [5 1,1-d] -1,2,3,5-tetrazine-4-one (0.82 g) was obtained in the form of colorless crystals with a melting point of 114-116 ° C.

[Elemental analysis: 55 found: C 39.7; H 3.95; N 30.8%; CgH 2 CINgO 3, calculated: C 39.9; H 4.10; N 31.0%].

5 192739

Example X Connection E

A stirred suspension of 4 [5] -diazoimidazole-5 [4] -carboxamide (1.0 g) in hexamethylphosphoramide (4 ml) was treated with 4.5 ml of 2-bromoethyl isocyanate and the mixture was treated at ambient temperature for 2 days 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 to give 3- (2-bromoethyl) -8-carbamoyl- [3 H] imidazo [5.1] -1.2.3 5-tetrazine-4-one (1.17 g) was obtained as a colorless solid, mp 156-157 ° C (decomposition). [Elemental analysis: 10 found: C 29.5; N 2.36; N 29.2; Br 27.3%; C7H7BrN602 calculated: C 29.3; H 2.46; N 29.3; Br 27.8%].

Reference Example (i) An intimate mixture of 5-nitroimidazole carboxylic acid (2.0 g) and phosphorus pentachloride (2.67 g) was stirred and heated in an oil bath for 1 hour at 120 ° C. The resulting yellow suspension was evaporated at 60 ° C / 0.13 kPa for 30 minutes, yielding 1,6-dinitro-5H, 10H-dimidazole [1,5-a: 15'-d] pyrazine-5, 10-dione (1.90 g) was obtained in the form of a yellow solid with a melting point of 249-152 ° C (with decomposition). [υ ^ (ΚΒΓ disc) 1750 cm -1; mIe 278 ([*)].

Windaus et al., Ber., 56 (1923), 684 (683-686) and Gireva, et al., Chem. Abs. 59,1622e (1963), using the same method, describe their products as "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 in portions with stirring with 1,6-dinitro-5H, 10H-dimidazo [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 50 ° C / 1.3 kPa and then acidified by treatment with concentrated hydrochloric acid to yield 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. 30 [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 - Shook for 35 hours. After 3 hours, the hydrogen absorption was complete (710 ml). The mixture was treated with charcoal and filtered through diatomaceous earth. The dark brown Altrate was evaporated at 50 ° C / 0.013 kPa and the residue obtained was triturated with diethyl ether to give impure 5 [4] amino-4 [5] dimethylcarbamoylimidazole (1.75 g) as a dark brown, crystalline solid, mp 179-181 ° C [µmax (KBr disk) 1595cm -1; NMR in DMSO-d6: singlets at 3.2 and 7.05], which was still 40 contaminated with colloidal platinum, obtained and used in the next step without further purification.

(iv) A stirred solution of 0.79 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 45 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] dimethylcarbamoylimidazole (1.59 g ) in the form of orange crystals, mp 101-103 ° C (with decomposition).

[Elemental analysis: 50 found: C 42.6; H 4.17; N 41.4%; C6H7N7N50 calculated: C 43.6; H 4.27; N 42.4%].

Claims (4)

192739 6 1. [3H] -imidazo- [5,1-d] -1,2,3,5-tetrazine-4-one derivatives with antineoplastic activity, characterized in that the derivatives have the general formula 1, wherein R1 represents a methyl, 2-chloroethyl or 2-bromoethyl group and R2 represents a carbamoyl, methylcarbamoyl or dimethylcarbamoyl group. Tetrazine derivatives according to claim 1, characterized in that R 1 represents a methyl or 2-chloroethyl group. Tetrazine derivatives according to claim 1 or 2, characterized in that R 2 represents a carbamoyl group or a methyl carbamoyl group. 4. 8-Carbamoyl-3-methyl- [3 H] imidazo [5,1-d] -1,2,3,5-tetrazine-4-one. 5. 8-Carbamoyl-3- (2-chloroethyl) - [3 H] imidazo [5,1-d] -1,2,3,5-tetrazine-4-one. 6. 3- (2-Chloroethyl) -8-methylcarbamoyl- [3H] irididazo [5,1-d] -1,2,3,5-tetrazine-4-one. 7. 3- (2-Chloroethyl) -8-dimethylcarbarnoyl- [3 H] imidazo [5,1-dJ-1,2,3,5-tetrazino 4 one. 8. 3- (2-Bromoethyl) -8-carbamoyl- [3Hi] irinidazo [511-d] -1,2,3,5-tetrazine-4-one. Pharmaceutical composition, which contains as active ingredient at least one tetrazine derivative according to one or more of claims 1 to 8 together with a pharmaceutical carrier or a coating. Hereby 1 sheet drawing