DK161147B - METHOD OF ANALOGY FOR THE PREPARATION OF OE3HAA-IMIDAZO-OE5,1-DAA-1,2,3,5-TETRAZIN-4-UNDIVERIVES - Google Patents

Description

DK 161147 B

The present invention relates to an analogous process for the preparation of novel [3

The compounds prepared by the analogous process 5 of the present invention are [3 H] -imidazo- [5,1-d] -1,2,3,5-tetrazine-4-undivatives of the general formula (I) R 2 )

s \ I

T

. . Wherein R 1 represents a hydrogen atom or a straight or branched alkyl, alkenyl or alkynyl group containing up to 6 carbon atoms, any such group being unsubstituted or substituted with from 1 to 3 substituents selected from halogen atoms, straight chain or branched alk -20 oxy, alkylthio, alkylsulfinyl and alkylsulfonyl groups containing up to 4 carbon atoms, or R1 represents a cycloalkyl group containing from 3 to 8 carbon atoms or a phenyl group which is unsubstituted or substituted by one or more groups selected from straight chain 25 and branched alkoxy and alkyl groups each containing up to 4 carbon atoms and nitro groups, and R 2 represents a carbamoyl group which can carry on the nitrogen atom 1 or 2 groups selected from straight or branched alkyl and alkenyl groups each containing up to 4 carbon atoms, and from cycloalkyl groups containing from 3 to 8 carbon atoms, e.g. a methyl carbamoyl or dimethyl carbamoyl group, or are alkali metal salts thereof when R 1 is hydrogen.

If the symbol R1 represents an alkyl, alkenyl or alkynyl group substituted by 2 or 3 halogen atoms, the above halogen atoms may be the same or different.

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If the symbol R1 represents an alkyl, alkenyl or alkynyl; ! group substituted with 1, 2 or 3 optionally substituted; phenyl groups, the optional substituents on the phenyl group (s) may be selected from e.g. alkoxy and alkyl groups 5 containing up to 4 carbon atoms, e.g. methoxy and / or methyl groups, and from a nitro group; the symbol R · may e.g. represent a benzyl or p-methoxybenzyl group. Cycloalkyl groups within the definitions of the symbols R · and R₂ contain 3-8, preferably 6, carbon atoms.

Preferred tetrazine derivatives of the general formula (I) are those in which R 1 represents a straight or branched alkyl group containing from 1-6 carbon atoms optionally substituted with 1 or 2 halogen atoms, preferably chlorine, fluoro or bromine atoms or with an alkoxy group 15 containing 1-4 carbon atoms, preferably methoxy, or with a phenyl group optionally substituted with 1 or 2 alkoxy groups containing from 1-4 carbon atoms, preferably methoxy; or wherein R · represents an alkenyl group containing 2-6 carbon atoms, preferably allyl, or for a cyclohexyl group.

Particularly preferred tetrazine derivatives are those of the general formula (I) wherein R 1 represents a straight or branched alkyl group containing from 1-6 carbon atoms, and especially from 1-3 carbon atoms, unsubstituted or substituted with a halogen atom, preferably chlorine or fluorine atom. . IN

in particular, R1 represents a methyl or 2-haloalkyl group, e.g. a 2-fluoroethyl group or preferably a 2-chloroethyl group.

Preferably R2 represents a carbamoyl group or a monoalkylcarbamoyl group, e.g. methylcarbamoyl, or a monoalkenylcarbamoyl group.

The present invention also encompasses the preparation of alkali metal salts of the compounds of general formula (I) wherein R 1 represents a hydrogen atom and R 2 is as defined above, e.g. a sodium salt, and whenever the context allows, references to the compounds must

DK 161147 B

3 of the general formula (I) in this specification include reference to said salts.

The analogous process according to the invention is characterized in that 5 (A), when R

compound of the general formula II

n? © N

10 (II)

R2 N2 W

wherein R 2 is as defined above with an isocyanate of the general formula (III) R 3 NCO (III) wherein R 3 is as defined above for R 1 with the exception of hydrogen, or (B) when R 1 is different from hydrogen, a compound -20 with the general formula (IV) H-fr ”! (IV) o

wherein R 2 is as defined above or an alkali metal salt thereof having a compound of the general formula (V

R 3 X (V) wherein R 3 is as defined above and X represents an acid residue of a reactive ester, or (C) when R 1 in the derivative means hydrogen or an alkali metal salt thereof, react a compound of the general formula (II) )

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Wherein R 2 is as defined above with a compound of the general formula (VI) R 4 NCO (VI) wherein R 4 represents an alkali metal atom or a protecting group, preferably a benzyl or p methoxybenzyl group followed, if R4 represents a protecting group, by substituting the protecting group with a hydrogen atom in the compound thus obtained with the general formula (VII) 15 N ^ nvs ^ 'Nn ^ (VII) 20 II R5 in 0 wherein R2 is as defined above and R 5 represents a protecting group, preferably a benzyl or p-methoxybenzyl group, by known methods.

The reaction referred to above (A) may be carried out in the absence or presence of an anhydrous organic solvent, e.g. a chlorinated alkane, e.g. dichloromethane, or ethyl acetate, acetonitrile, N-methylpyrrolid-2-one or "preferably hexamethylphosphoramide, at a temperature between 30 ° C and 70 ° C, e.g. at room temperature. Turnover can be continued for up to 30 days. Light should preferably be excluded from the reaction mixture.

If, in a compound of general formula (V) in the reaction referred to above (B), R 3 represents a halo-alkyl, halo-alkenyl or halo-alkyl-nyl group, the acid residue of a reactive ester represented by X is selected from them.

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5 which is known to be less reactive than the halogen atom substituent in R 3. If X in a compound of general formula (V) represents a halogen atom, preferably an alkali metal salt of the compound of general formula 5 (IV) is used, and if X in a compound of general formula (V) represents a halogen atom, and R 3 is a haloalkyl, halogeno-alkenyl or haloalkynyl group in which the halogen atom is the same as that represented by X, preferably an excess amount of the dihalogen compound 10 of the general formula (V) is used. The reaction of a compound of general formula (IV) or an alkali metal salt thereof with a compound of general formula (V) wherein R 3 and X are as defined above can be carried out in a suitable anhydrous, inert organic solvent, e.g. dichloromethane, acetonitrile or N-methylpyrrolid-2-one or mixtures thereof, at a temperature of 0 to 120 ° C and, if a compound of general formula (IV) is used, in the presence of an acid-binding agent, e.g. . an alkali metal carbonate or bicarbonate such as sodium or potassium carbonate or bicarbonate.

The reaction referred to above (C) of a compound of the general formula (II) with a compound of the general formula (VI) wherein R 4 represents a protecting group can be carried out as mentioned above for the reaction of a compound with the general formula (II) having a compound of the general formula (III). Reaction of a compound of general formula (II) with a compound of general formula (VI) wherein R 4 represents an alkali metal atom can be carried out in a suitable inert organic solvent, e.g.

30 ethanol, acetonitrile or N-methylpyrrolidinone, optionally in the presence of an acid, at a temperature of 0 to 120 ° C. The group R5 in compounds of general formula (VII) wherein R5 is as defined above can be exchanged with a hydrogen atom by known methods to obtain a compound 35 of general formula (IV).

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6 j

Compounds of general formula (II) can be prepared by using or adapting methods known per se, e.g. methods described by Y.F. Shealy, R.F.

Struck, L.B. Holum & J.A. Montgomery: J. Org. Chem. (1961), 5 26, 2396.

Compounds of general formulas (III), (V) and (VI) can be prepared by using or adapting methods known per se.

By the term "methods known per se" as used in the present specification is meant methods which have been used previously or described in the literature.

The novel tetrazine derivatives of the general formula (I) possess valuable antineoplastic activity against e.g. carcinomas, melanomas, sarcomas, 15 lymphomas and leukemias. They have been found to be particularly active in mice against TLX5 (S) lymphoma at daily doses of between 0.5 and 16 mg / kg body weight, administered intraperitoneally, by the method of Gescher et al .: Biochem. Pharmacol.

(1981), 89, and ADJ / PC6A and M5076 (reticulum cell sarcoma).

20 against Leukemia L1210, transplanted intraperitoneally, 'intracerebrally and intravenously, and P388, according to the method described in "Methods of Development of New Anticancer Drugs" (NCI Monograph 45, March 1977, pages 147-149, National Cancer Institute, Bethesda, (USA) ), the compounds are active both intraperitoneally and orally at doses between 2.5 and 10 mg / kg body weight. Inhibition of both primary tumor and metastasis is achieved against Lewis lung carcinoma at similar doses.

Against B16 melanoma and C38 tumor in mice (NCI Monograph 45, op. Cit.), The compounds are active intraperitoneally at 30 doses of between 6.25 and 25 mg / kg body weight.

The tetrazine derivatives also possess valuable immunomodulatory activity and are useful in the treatment of organ transplantation and skin transplantation and in the treatment of immunological diseases.

Important individual compounds of general formula (I) include the following:

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A, 8-Carbamoyl-3-methyl- [3H] -imidazo [5,1-d] -1,2,3,5-tetrazin-4-one, B, 8-Carbamoyl-3-n-propyl [3H] -imidazo [5,1-d] -1,2,3,5-tetrazin-4-one, 5 C, 8-carbamoyl-3- (2-chloroethyl) - [3H] -imidazo [5 1,1-d] -1,2,3,5-tetrazin-4-one, D, 3- (2-chloroethyl) -8-methylcarbamoyl- [3H] -imidazo- [5,1-d] -1 , 2,3,5-Tetrazine> -4-Qn, E, 8-Carbamoyl-3- (3-chloropropyl) - [3H] -imidazo [5,1-d] -10,2,3,5 -tetrazin-4-one, F, 8-carbamoyl-3- (2,3-dichloropropyl) - [3H] -imidazo [5,1-d] - 1,2,3,5-tetrazin-4-one , G, 3-Allyl-8-carbamoyl- [3H] -imidazo [5,1-d] -1,2,3,5-tetrazin-4-one, 15H, 3- (2-chloroethyl) - 8-Dimethylcarbamoyl- [3H] -imidazo- [5,1-d] -1,2,3,5-tetrazin-4-one ,. 1,3- (2-bromethyl) -8-carbamoyl- [3H] -imidazo [5,1-d] -1,2,3,5-tetrazin-4-one, 1,3-benzyl-8-carbamoyl-4-one [3H] -imidazo [5,1-d] -1,2,3,5-20 -tetrazin-4-one, K, 8-carbamoyl-3- (2-methoxyethyl) - [3H] -imidazo [5 , 1-d] - 1,2,3,5-tetrazin-4-one, L, 8-carbamoyl-3-cyclohexyl- [3H] -imidazo [5,1-d] -1,2,3, 5-tetrazin-4-one, 25 m, 8-carbamoyl-3- (p-methoxybenzyl) - [3 H] -imidazo [5,1-d] -1,2,3,5-tetrazin-4-one.

Compounds A and D and especially C are of particular importance.

The letters A to M are assigned to the compounds for ease of reference later in the description.

The following examples illustrate the preparation of the compounds of general formula (I) using the process of the present invention, and the reference examples then illustrate the preparation of intermediates.

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DK 161147B

Example 1 500 mg of 4 [5] -diazoimidazole-5 [4] carboxamide is suspended in 3.0 ml of methyl isocyanate and stirred in the dark at room temperature for 21 days. Then, the reaction mixture is diluted with anhydrous diethyl ether and filtered. The residue is washed rapidly with anhydrous methanol, then with anhydrous diethyl ether and air dried in the dark at room temperature to give 198 mg of 8-carbamoy1-3-methyl- [3H] -imidazo [5,1-d] -1,2,3,5 -tetra-zin-4-one in the form of a light brown microcrystalline solid, m.p. at 210 ° C (foam up and darken at 160-210 ° C). Elemental analysis for C

Calculated: C: 37.1%, H: 3.09%, N: 43.3%.

Found: C: 36.8%, H: 3.10%, N: 44.2%.

15

Example 2

300 mg of 4 [5] diazoimidazole-5 [4] carboxamide is suspended in 10 ml of anhydrous dichloromethane and treated with an excess of 20; the same amount of n-propyl isocyanate. Then, the reaction mixture is stirred in the dark at room temperature for 30 days. The reaction mixture is then filtered and the residue is washed rapidly with anhydrous diethyl ether and air dried in the dark at room temperature to give 102 mg of 8-carbamoyl-3-n-propyl- [3H] -25-imidazo [5,1d] -1,3,3 , 5-tetrazin-4-one in the form of a pink powder, m.p. at 167 ° C (foaming).

Elemental analysis for C 8 H 10 N 6 ° 2;

Calculated: C: 43.2%: * H: 4.53%, N: 37.8%.

Found: C: 43.4%, H: 4.57%, N: 38.0%.

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Example 3 Compound_C

300 mg of 4 [5] -diazoimidazole-5 [4] carboxamide is suspended in 10 ml of anhydrous dichloromethane and 1.0 ml of 2-chloroethyl isocyanate is added. Then, the reaction mixture is stirred in the dark at room temperature for 30 days. The cream-colored suspension thus obtained is filtered and the residue is washed rapidly with anhydrous diethyl ether and air dried in the dark to give 483 mg of 8-carbamoyl-3- (2-chloroethyl) - [3H] -imide-10-azo [5,1d] -1 , 2,3,5-tetrazin-4-one in the form of a cream powder with m.p. 158 ° C (heavy decomposition).

Elemental analysis for C ^H ^ClNgC ^:

Calculated: C: 34.7%, H: 2.91%, N: 34.7%.

Found: C: 34.7%, H: 3.01%, N: 34.9%.

15

Repetition of the above process has also yielded 8-carbamoyl-3- (2-chloroethyl) - [3H] -imidazo [5,1-d] -1,2,3,5-tetrazin-4-one in another polymorph mold, mp: 164-165 ° C (dec.).

20

Example 4 Forb ± n ^ eXse_A.

A suspension of 1.37 g of 4 [5] -diazoimidazole-5 [4] -carboxamide in 20 ml of ethyl acetate is treated with 7.0 g of methyl isocyanate and stirred in a closed vessel in the dark at room temperature for 3 weeks. The resulting solid is filtered off and washed with diethyl ether to give 1.9 g of 8-carbamoyl-3-methyl- [3H] -imidazo [5,1-d] -1,2,3,5-tetrazin-4-one in the form of a cream colored solid, mp: 212 ° C (foam).

30.

This material is recrystallized from 3 different solvent systems to obtain 3 different products, each having a slightly different IR spectrum.

The 3 products are probably all polymorphic forms of 8-carbamoy1-3-methyl- [3H] -imidazo [5,1-d] -1,2,3,5-tetrazin-35 -4-one.

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(i) Colorless needles are obtained from a 3: 1 volura / vo lumen mixture of acetone and water max 3410/3205, 1758, 1730 and 1678 cm / mp: 212 ° C (foam).

(ii) White microcrystals are obtained from a 1: 3 volume / 5 volume mixture of acetone and water, V 3430.3200, 1740 and 1675 cm, mp: 210 ° C (foam).

(iii) A granular solid is obtained from hot water, max 3450 '3380, 3200, 1742, 1688 and 1640 cm -1, mp: 215 ° C

(foaming) (darkens from 200 ° C).

10

Example 5

A suspension of 1.37 g of 4 [5] -diazoimidazole-5 [4] carboxamide in 20 ml of acetonitrile is treated with 6.5 g of n-propyl isocyanate.

15 and stirred in a closed container in the dark at room temperature for 3 weeks. The resulting pink solid is filtered off, washed with diethyl ether and recrystallized from a mixture of water and acetone (1: 4 v / v) to give 1.6 g of 8-carbamoyl-3-n-propyl- [3H] -imidazo [5 , ld] -1,2,3,5-20 -tetrazin-4-one, mp: 170r-172 ° C (foam). Concentrating the mature liquor from the recrystallization yields an additional amount of 0.2 g of the same product. ;

Example 6 ConnectXse__C

A suspension of 1.0 g of 4 [5] -diazoimidazole-5 [4] carboxamide in 30 ml of ethyl acetate is treated with 3.3 ml of 2-chloroethyl isocyanate and the mixture is stirred in the dark at room temperature for 6 days. The reaction mixture is then diluted with diethyl ether} and the resulting solid is filtered off to give 1.6 g of 8-carbamoyl-3- (2-chloroethyl) - [3 H] -imidazo [5,1-d] -1,2,3 5-Tetrazin-4-one in the form of a colorless solid, mp: 164-165 ° C (dec.).

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11

Elemental analysis for C ^H ^ClNgC ^:

Calculated: C: 34.65%, H: 2.91%, N: 34.65%, Cl: 14.61%. Found: C: 34.5%, H: 2.88%, N: 34.5%, Cl: 14.6%.

Example 7

A suspension of 5.0 g of 4 [5] diazoimidazole-5 [4] carboxamide in a mixture of 158 ml of dichloromethane and 8.3 ml of N-methylpyrrolid-2-one is treated with 16.7 ml of 2-chloroethyl The 10 isocyanate yolk mixture is stirred in the dark at room temperature for 14 days. Then, the reaction mixture is diluted with anhydrous diethyl ether and the resulting solid is filtered off and washed with diethyl ether to give 6.3 g of 8-carbamoyl ether.

-3- (2-chloroethyl) - [3H] -imidazo [5,1-d] -1,2,3,5-tetrazine-4-

-one in the form of a purple solid, mp: 164-165 ° C

(Decomposition).

Elemental analysis for C

Calculated: C: 34.65%, H: 2.91%, N: 34.65%, Cl: 14.61%.

20 Found? C: 34.7%, H: 2.95%, N: 34.5%, Cl: 14.4%.

Example 8 Foxbind.el.se_C

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

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Example 9

A stirred suspension of 2.2 g of 4 [5] diazoimidazole-5 [4] carboxamide in a mixture of 70 ml of dichloromethane and 5 3.5 ml of N-methylpyrrolid-2-one is treated with 7.0 ml of methyl isocyanate. and stirred at room temperature for 4 weeks. The mixture is diluted with diethyl ether and the resulting solid is filtered off to give 2.38 g of 8-carbamoyl-3-methyl- [3H] -imidazo [5,1-d] -1,2,3,5-tetrazine-4 -one in the form of a pale purple solid, mp: 202-203 ° C (dec.). Elemental Analysis for CgHgNgO2:

Calculated: C: 37.11%, H: 3.14%, N: 43.3%.

Found: C: 36.8%, H: 2.94%, N: 43.1%.

15

A polymorphic form of 8-carbamoyl-3-methyl- [3H] -imidazo- [5,1-d] -1,2,3,5-tetrazin-4-one is obtained by dissolving in acetonitrile, filtering, concentrating the filtrate for dryness and trituration of the resulting residue with diethyl ether.

This material is in the form of an orange solid, 20 m.p .: ca. 200 C (decomposition).

Elemental Analysis C: 37.4%, H: 3.26%, N: 43.5%.

The NMR spectrum of dimethylsulfoxide-Dg is identical to the aforementioned slightly purple solid, but the IR spectrum 25 (KBr disk) shows some differences.

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Example 10 E2E ^ iS ^ ®ise_D

A stirred solution of 0.64 g of sodium nitrite in 4.6 ml of water is cooled to 5-10 ° C and treated dropwise at this temperature with a solution of 1.00 g of 5-amino-4-methylcarbamoylimidazole in 14.3 ml (1 M) aqueous acetic acid for 5 minutes. Stirring is continued at 5-10 ° C for 5 minutes. The dark red solution is then extracted 4 times with ethyl acetate, each time with 35 ml and the combined extracts are dried over magnesium sulfate. The resulting solution contains crude 4 [5] -diazo-5 [4] methylcarbamoylimidazole, which is unstable and is used immediately for the next step without further purification.

The solution of 4 [5] -diazo-5 [4] methylcarbamoylimide-azole in ethyl acetate, prepared as described above, is treated with 4.3 ml of 2-chloroethyl isocyanate and allowed to stand in the dark for 1 day. The solution is then evaporated at 40 ° C / 10 mm Hg and the residue triturated with petroleum ether (bp 40-60 ° C) to give 4.23 g of an orange 20 gum. This gum is treated with 50 ml of ethyl acetate and filtered and the filtrate is evaporated at 40 ° C / 10 mm Hg to give 2.94 g of an orange gum. This gum is purified by medium pressure column chromatography on silica gel, eluted with a mixture of ethyl acetate and acetonitrile (4: 1 v / v) to give 0.81 g of 3- (2-chloroethyl) -8-methylcarbamoyl - [3 H] -imidazo [5,1d] -1,2,3,5-tetrazin-4-one in the form of a purple solid, mp: 120-122 ° C (dec.).

Elemental Analysis for CgHgClNgO2:

Calculated: C: 37.4%, H: 3.53%, N: 32.7%.

30

Found: C: 37.3%, H: 3.58%, N: 31.9%.

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Example 11

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A suspension of 1.0 g of 4 [5] diazoimidazole-5 [4] carboxamide in 50 ml of ethyl acetate dried over anhydrous potassium carbonate is treated with 4.86 g of 3-chloropropyl isocyanate and the mixture is stirred at room temperature for 3 hours. days. Then, the reaction mixture is diluted with anhydrous diethyl ether and the resulting solid is filtered off and washed with anhydrous diethyl ether to give 1.05 g of 8-carbamoyl-3- (3-chloropropyl) -10 - [3 H] -imidazo [5,1-d] -1,2 , 3,5-tetrazin-4-one in the form of a pink solid, mp: 153-154 ° C (dec.). Elemental analysis for CgH ^ClNgC ^:

Calculated: C: 37.4%, H: 3.53%, N: 32.8%, Cl: 13.8%.

Found: C: 37.1%, H: 3.42%, N: 32.7%, Cl: 13.8%.

15

Example 12

By proceeding in the same manner as described above in Example 11, but by replacing the 3-chloropropyl isocyanate 20 used as starting material with the correct amount of 2,3-dichloropropyl isocyanate, 8-carbamoyl-3- (2.3 (dichloropropyl) - [3 H] -imidazo [5,1-d] -1,2,3,5-tetrazin-4-one in the form of a whitish solid, mp: 153-155 ° C (dec.). Elemental analysis for C

Calculated: C: 33.0%, H: 2.77%, N: 28.9%, Cl: 24.4%.

Found: C: 32.7%, H: 2.51%, N: 28.7%, Cl: 24.1%.

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Example 13 4.5 ml of stirred allylisocyanate redistilled immediately before use is treated with 1.0 g of 4 [5] -diazo-5-imidazole-5 [4] carboxamide and then with 20 ml of hexamethylphosphoramide. The mixture is stirred at room temperature in the dark for 18 hours and then diluted with anhydrous diethyl ether and filtered. The resulting colorless solid is washed with anhydrous diethyl ether to give 1.6 g of 3-allyl-8-10 -carbamoyl- [3H] -imidazo [5,1-d] -1,2,3,5-tetrazine-4 in the form of a colorless solid, mp: 149-150 ° C.

Ymax (KBr disk): 1730, 1675 cm- · * -.

NMR in DMSO-d6: singlets at 8.75, 7.67 and 7.606; double double triplet at 6.026 (J = 5.5, 8, 10 Hz), 15 double duplicate at 5.335 (J = 1.5, 8 Hz) and 5.206 (J = 1.5, 10 Hz), and double at 4.866 (J = 5.5).

Example 14 Compound H

20

A solution of 1.59 g of 4 [5] diazo-5 [4] dimethylcarbamoylimidazole, prepared as described in the following Reference Example 1, in 57 ml of dry ethyl acetate is treated with 6.36 g of 2-chloroethyl isocyanate and stirred at room temperature. in the dark for 24 hours. Then the solution is evaporated in vacuo at 35 ° C, finally at 0.1 mm Hg to remove the excess 2-chloroethyl isocyanate. The residual liquid is purified by medium pressure column chromatography on silica gel, eluting with a 4: 1 v / v mixture of ethyl acetate and acetonitrile to give 0.82 g of 3- (2-chloro-ethyl) -8-dimethylcarbamoyl- [3H] -imidazo [5,1-d] -1,2,3,5-te-trazin-4-one in the form of colorless crystals, mp: 114-116 ° C.

Elemental analysis for C ^H ^ClNgC ^:

Calculated: C: 39.9%, H: 4.10%, N: 31.0%.

Found: C: 39.7%, H: 3.95%, N: 30.8%.

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Example 15 E2

A stirred suspension of 1.0 g of 4 [5] -diazoimidazole-5 [4] carboxamide in 4 ml of hexamethylphosphoramide is treated with 4.5 ml of 2-bromomethyl isocyanate and the mixture is stirred in the dark at room temperature for 2 days. Then the reaction mixture is diluted with anhydrous diethyl ether and the resulting solid is filtered off, washed with anhydrous diethyl ether to give 1.17 g of 3- (2-bromethyl) -8-carbamoyl- [3H] -imidazo-10 [5,1-d] -1 2,3,5-tetrazin-4-one in the form of a colorless solid, mp: 156-157 ° C (dec.).

Elemental analysis for C

Calculated: C: 29.3%, H: 2.46%, N: 29.3%, Br: 27.8%.

Found: C: 29.5%, H: 2.36%, N: 29.1%, Br: 27.3%.

15

Example 16

By proceeding in the same manner as described above in Example 15, but replacing the 2-bromomethyl isocyanate used as the starting material with the correct amount of benzyl isocyanate, 0.83 g of 3-benzyl-8-carbamoyl- [3H] is prepared. - imidazo [5,1-d] -1,2,3,5-tetrazin-4-one in the form of a brown-yellow solid, mp: 176-177 ° C (dec.).

Elemental analysis for C2.2H10N6 ° 2:

Calculated: C: 53.3%, H: 3.73%, N: 31.1%.

Found: C: 53.6%, H: 3.66%, N: 31.0%.

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Example 17 Compound_K

A suspension of 0.3 g of 4 [5] -diazoimidazole-5 [4] -carboxamide in 5 ml of acetonitrile is treated with 0.5 g of 2-methoxy

5 ethyl isocyanate and the mixture is stirred at between 45 and 47 ° C

in the dark for 24 hours. The resulting solid is filtered off and washed with diethyl ether to give 0.45 g of crude 8-carbamoyl-3- (2-methoxyethyl) - [3 H] -imidazo [5,1-d] -1,2,3,5- tetrazin-4-one, mp: 145-147 ° C (dec.).

The product is purified by recrystallization from aqueous acetone to give pink rosettes or from aqueous dimethyl sulfoxide to give colorless needles, mp: 164-165 ° C (dec.).

Elemental analysis for C8H10N6 ° 3: 15

Calculated: C: 40.34%, H: 4.20%, N: 35.2%.

Found: C: 40.4%, H: 4.20%, N: 35.2%.

Example 18

Compound L 20 -------------

A suspension of 0.30 g of 4 [5] -diazoimidazole-5 [4] carboxamide in 10 ml of acetonitrile is treated with 1.0 g of cyclohexyl isocyanate and the mixture is stirred at 60 ° C in the dark for 3 days.

The resulting solid is filtered off and washed with a mixture of ethanol and 0.880 aqueous ammonia (100: 0.5 v / v, 20 ml) for 1 minute to give 0.015 g of 8-carbamoyl-3-cyclohexyl- [3H] -imidazo [5,1-d] -1,2,3,5-tetrazin-4-one, mp: 196 ° C (foam).

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Example 19

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5L2 £ ^ iSå§i§t-.i

A suspension of 0.4 g of 4 [5] -diazoimidazole-5 [4] carboxamide in 10 ml of acetonitrile is treated with 0.6 g of benzyl isocyanate, and the mixture is stirred at 60 ° C in the dark overnight. The reaction mixture is then cooled and filtered to give 0.75 g of 3-benzyl-8-carbamoyl- [3H] -imidazo [5,1-d] -1,2,3,5-tetrazin-4-one in the form of a pale pink solid, mp: 187-188 ° C (foam).

10

Example 20 E2 £ kiilS | else_M

A suspension of 0.1 g of 4 [5] -diazoimidazole-5 [4] carboxamide and 0.4 g of p-methoxybenzyl isocyanate in 5 ml of acetonitrile is stirred at 60 ° C in the dark for 4 hours. The resulting pale pink solid is filtered off and washed separately with cold diethyl ether to give 0.23 g of 8-carbamoyl-3- (p-methoxy-benzyl) - [3 H] -imidazo [5,1-d] -1,2,3, 5-tetrazin-4-one, mp: 180-182 ° C (foam).

20

Example 21

By proceeding in the same manner as in the previous examples, 8- (N-allylcarbamoyl) -3- (2-chloroethyl) - [3H] -imidazo [5,1-d] -25 -1,2,3 is prepared. 5-tetrazin-4-one (IR: 1750 cm-1; NMR (in DMSO-d6): multiples 3.96, 5.06 and 5.84 ppm; triplets 4.60 and 6.2 ppm; singlet 8, 78 ppm) from 5-amino-4-allylcarbamoyl) imidazole over 5-diazo-4-allylcarbamoylimidazole. 5-Amino-4-allylcarbamoylimidazole is prepared from 5-nitro-4-allylcarbamoylimidazole (mp: 218-220 ° C) by reduction by titanochloride.

35 19

ISLAND

DK 161147 B

Reference Examples (i) An inner mixture of 2.0 g of 5-nitroimidazole-4-carboxylic acid and 2.67 g of phosphorus pentachloride is stirred and heated in an oil bath at 120 ° C for 1 hour. The resulting yellow slurry is evaporated at 60 ° C / 0.1 mm Hg for 30 minutes to give 1.90 g, 6-dinitro-5H, 10H-diimidazo [1,5- a: 1 ', 5'-d ] pyrazine-5,10-dione in the form of a yellow solid, mp: 249-251 ° C (dec.).

10 (KBr disk): 1750 cm-1? m / e: 278 (M +).

Iug12 £

Windaus, Ber., 1923, 56, 684 ^ and Gireva ^ Chem. Abs. 59, 16222, using the same method, call their products "5-nitroimidazole-4-carbonyl chloride".

(Ii) An aqueous dimethylamine solution (25% w / v; 60 ml) is cooled to between 0 and 5 ° C and treated portionwise with stirring with 6.0 g of 1,6-dinitro-5H, 10H-diimidazo- [1, 5-a: 1 ', 5'-d] pyrazine-5,10-dione within this temperature-2Q range. The resulting deep purple solution is stirred for 2 hours. The solution is evaporated at 50 ° C / 10 mm Hg and then acidified by treatment with concentrated hydrochloric acid to give an orange solution. This solution is extracted 7 times with ethyl acetate, each time with 200 ml, and the combined 25 extracts are dried over magnesium sulfate and evaporated to give 6.6 g of a yellow solid. This solid is triturated with 50 ml of toluene and then recrystallized from ethyl acetate to give 2.53 g of 5 [4] -nitro-4 [5] -dimethylcarbamoylimidazole in the form of yellow crystals, mp: 193-195 ° C.

Elemental Analysis for CgHgN3:

Calculated: C: 39.1%, H: 4.38%, N: 30.4%.

Found: C: 38.9%, H: 4.23%, N: 30.4%.

35

DK 161147 B

20 j (iii) A solution of 1.62 g of 5 [4] -nitro-4 [5] dimethyl-1-ylcarbamoylimidazole in 32 ml of dry dimethylformamide is treated with 0.32 g of platinum oxide and shaken under hydrogen at atmospheric pressure and room temperature. After 3 hours, hydrogen ab-; The sorption is complete (710 ml). Then the mixture is treated with activated charcoal and filtered through diatomaceous earth. The dark brown filtrate is evaporated at 50 ° C / 0.1 mm Hg and the resulting residue is triturated with diethyl ether to give 1.75 g of crude 5 [4] -amino-4 [5] dimethylcarbamoylimidazole in the form of a dark brown crystalline solid, mp: 179--181 ° C.

-rmax (KBr disk): 1595 cm -1; NMR in DMSO-dg: singlets at 3.2 and 7.0i, still contaminated with colloidal platinum, in and used at the next step without further purification.

(Iv) A stirred solution of 0.79 g of sodium nitrite in 5.7 ml of water is cooled to between 5 and 10 ° C and treated dropwise within this temperature range with a solution of 1.75 g of 5 [4] amino-4 [ 5] -dimethylcarbamoylimidazole in 17.6 ml of 20 (1 M) aqueous acetic acid for 5 minutes. The resulting solution is extracted 4 times with ethyl acetate, each time with 40 ml, the combined extracts are dried over magnesium sulfate and evaporated at 30 ° C / 10 mm Hg to give 1.59 g of 4 [5] -diazo-5 [4] -dimethylcarbamoylimidazole in the form of orange-colored crystals, mp: 101-103 ° C (dec.).

in

Elemental Analysis for C5H7N5O:

Calculated: C: 43.6%, H: 4.27%, N: 42.4%. in

Found: C: 42.6%, H: 4.17%, N: 41.4%.

The compounds prepared by the process of the present invention can be used in pharmaceutical compositions containing as active ingredient at least one tetrazine derivative of the general formula (I) together with a pharmaceutical carrier or coating. In clinical practice, the compounds of the general formula (I) are usually administered orally,

DK 161147 B

21 rectally, vaginally or parenterally, e.g. intravenously or intraperitoneally.

Methods for designing pharmaceutically active compounds are well known in the art and a suitable matrix may be determined by the physician or pharmacist depending on such factors as the desired effect, size of patient, age, sex and condition and properties of active compound. The compositions may also contain such materials as solid or liquid diluents, wetting agents, preservatives, flavoring and coloring agents, which is common in the art.

Solid compositions for oral administration include compressed tablets, pills, dispersible powders and granules. In such solid compositions, one or more active compounds are mixed with at least one inert diluent such as calcium carbonate, potato starch, alginic acid or lactose. The compositions may also contain additional substances different from inert diluents, e.g. lubricants such as magnesium stearate, which is common in practice. Liquid compositions for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs containing inert diluents commonly used in the art such as water and liquid paraffin. In addition to inert diluents, such compositions may also contain adjuvants such as wetting agents and suspending agents, e.g. polyvinylpyrrolidone, and sweeteners, flavors, flavors and preservatives. Compositions for oral administration also include capsules of absorbable material, such as gelatin, containing one or more of the active substances with or without the addition of diluents or extenders.

Solid vaginal administration compositions comprise vaginal suppositories which are shaped in a manner known per se and contain one or more of the active compounds.

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22

Solid rectal administration compositions comprise suppositories formed in a manner known per se and containing one or more of the active compounds.

Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions or emulsions. Examples of non-aqueous solvents or suspending agents are polyethylene glycol, dimethyl sulfoxide, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifiers and dispersants. They can be sterilized, e.g. by filtration through a bacterial retaining filter, by incorporating sterilizing agents into the compositions or by irradiation. They may also be prepared in the form of sterile solid preparations which may be dissolved in sterile water or another sterile injectable medium immediately prior to use.

The percentage amount of the active ingredient in the composition may be varied, it being necessary that it be of a size large enough to achieve a suitable dose for the desired therapeutic effect. Obviously, several unit dosage forms can be administered at almost the same time. Generally, the compositions usually contain at least 0.025% by weight of the active substance to be administered by injection; For oral administration, the composition usually contains at least 0.1% by weight of active substance. The dose used depends on the desired therapeutic effect, route of administration and duration of treatment.

The following preparation examples illustrate pharmaceutical compositions containing compounds prepared by the process of the present invention.

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23

Preparation Example 1

A solution suitable for parenteral administration is prepared from the following ingredients: 5 8-Carbamoyl-3- (2-chloroethyl) - [3 H] - 1.0 g -imidazo [5,1-d] -1,2 , 3,5-tetrazin-4-one

Dimethylsulfoxide 10 ml

Arachis oil 90 ml 10 by dissolving the 8-carbamoyl-3- (2-chloroethyl) - [3H] -imidazo- [5,1d] -1,2,3,5-tetrazin-4-one in the dimethyl sulfoxide and adding the arachis oil . The resulting solution is divided into ampoules in an amount of 10 ml per ampoule under aseptic conditions. ampoule. The vials are sealed to obtain 10 vials, each containing 100 mg of 8-carbamoyl-3- (2-chloroethyl) - [3H] -imidazo- [5,1-d] -1,2,3,5- tetrazine-4-one.

Similar ampoules containing solutions suitable for parenteral administration may be prepared by proceeding in the same manner, but replacing 8-carbamoyl-3- (2-chloro-20-ethyl) - [3H] -imidazo- [5,1d] -1,2,3,5-tetrazin-4-one with another compound of the general formula (I).

Preparation Example 2

Capsules suitable for oral administration are prepared by applying 8-carbamoyl-3- (2-chloroethyl) - [3H] -imidazo- [5,1d] -1,2,3,5- tetrazin-4-one in size 2 gelatin shells in an amount of 10 mg per capsule.

Similar capsules may be prepared using another compound of the general formula (I) or any other capsule shell of appropriate size.

The tetrazine derivatives of general formula (I) are useful in the treatment of malignant neoplasms, e.g. carcinomas, melanomas, sarcomas, lymphomas and leukemias, in 35 doses, which are generally between 0.1 and 200, preferably between 1 and 20, mg / kg body weight per day. day.

DK 161147B

24 Table I below shows the results of standard experiments with compounds Ά, I, C, D and H prepared by the method of the invention against lymphoma TLX 5 implanted in mice.

5

Table I

Activity of compounds prepared according to DK patent application No. 3778/82 against TLX5 lymphoma implanted in mice.

10

Combine- Optimal dose mg / kg Survival- Survival submitted 3 days after treatment, referral time in 15 ter implantation led to days * controls A 160 157% 20 I 160 137%

All 5 treated mice 25 C 40 survived 60 60 days after implantation

All 5 treated mice D 40 survived 60 60 days after implantation 35 All 5 treated mice H 40 survived 60 60 days after implantation 40 * Mice that survived for 60 days remained alive and were not examined further. Table 45 below shows results of standard experiments with Compound C prepared by the method

DK 161147 B

25 according to the invention against leukemia tumors L1210 and P388 which are implanted in mice.

Table II 5

Activity of compound C prepared according to DK patent application 3778/82 against leukemia tumors implanted in mice.

10

Dose Number Survival time

Tumor type mg / kg-1 day "1 dose days per day * 15 L1210 40 1 60 20 5 60 10 5 60 25 9 60 12.5 9 60 20 P388 40 1 60 20 1 60 20 5 60 10 5 60 25 _ * Mice , who survived for 60 days, remained alive and were not investigated further.

Claims (13)

1. Analogous Process for Preparing [3H] -imidazo- [5,1-d] -1,2,3,5-tetrazine-4-undivatives of general formula (I) 5 2 TT »V? An analogous process according to claim 1, characterized in that X represents a halogen atom or a methoxysulfonyloxy, methanesulfonyloxy or p-toluenesulfonyloxy group. An analogous process according to claim 1 or 2, wherein the obtained product is a tetrazine derivative of the general formula (I) according to claim 1, characterized in that R 1 represents an alkyl, alkenyl or alkynyl group substituted by 1, 2, or 3 optionally substituted phenyl groups and wherein the optional substituents on the phenyl group (s) are selected from alkoxy and alkyl groups containing up to 4 carbon atoms and from a nitro group. An analogous process according to claim 1 or 2, wherein the product obtained is a tetrazine derivative of the general formula (I) according to claim 1, characterized in that R1 represents a straight or branched alkyl group containing from 1-6 carbon atoms optionally substituted with 1 or 2 halogen atoms or with an alkoxy group containing 1-4 carbon atoms or with a phenyl group optionally substituted with 1 or 2 alkoxy groups containing from 1 to 4 carbon atoms, or R1 represents an alkenyl group containing 2-6 carbon atoms or a cyclohexyl group. An analogous process according to claim 4, characterized in that the halogen atom (s) is chlorine, fluorine and / or bromine, the alkoxy group (s) is methoxy and the alkenyl group is allyl. An analogous process according to claim 1 or 2, wherein the product obtained is a tetrazine derivative of the general formula (I) according to claim 1, characterized in that R 1 represents a straight or branched alkyl group containing from 1-6 carbon atoms, which group is unsubstituted or substituted by a halogen atom. An analogous process according to claim 1 or 2, wherein the obtained product is a tetrazine derivative of the general formula (I) according to claim 1, characterized in that R 1 represents an alkyl group containing 1-3 carbon atoms which is unsubstituted or substituted by a halogen atom. An analogous process according to claim 1 or 2, wherein the obtained product is a tetrazine derivative of the general formula (I) according to claim 1, characterized in that R1 represents a methyl or 2-haloalkyl group. An analogous process according to claim 8, characterized in that the halogen atom of the alkyl group is chlorine or fluorine. An analogous process according to claim 1 or 2, wherein the obtained product is a tetrazine derivative of the general formula (I) according to claim 1, characterized in that R1 represents a 2-fluoroethyl or 2-chloroethyl group. R3NCO (III) wherein R3 is as defined above for R1 with the exception of hydrogen, or (B) when R1 is different from hydrogen, a compound of general formula (IV) converts R2 V '; Wherein R 2 is as defined above or an alkali metal salt thereof having a compound of the general formula (V 25 R 3 X (V) wherein R 3 is as defined above and X represents an acid residue of a reactive ester) or (C) when R 1 in the derivative means hydrogen or an alkali metal salt thereof, reacting a compound of the general formula (II) N.pV N λ n2 ® 35 as defined above with a compound of the general formula formula (VI) R4NCO (VI) wherein R4 represents an alkali metal atom or a protecting group, preferably a benzyl or p-methoxybenzyl group followed by R4 representing a protecting group, by substituting the protecting group with a hydrogen atom in the thus obtained compound of the general formula (VII) V-N. II (VII) Y x 5 wherein R 1 is as defined above and R 5 represents a protecting group, preferably a benzyl or p-methoxybenzyl group, by using known methods. Wherein R 1 represents a hydrogen atom or a straight or branched alkyl, alkenyl or alkynyl group containing up to 15 6 carbon atoms, any such group being unsubstituted or substituted with from 1 to 3 substituents selected from halogen atoms, straight chain or branched alkoxy, alkylthio, alkylsulfinyl and alkylsulfonyl groups containing up to 4 carbon atoms, or R 1 represents a cycloalkyl group containing from 3 to 8 carbon atoms or a phenyl group which is unsubstituted or substituted by one or more groups selected from straight chain and branched alkoxy and alkyl groups each containing up to 4 carbon atoms and nitro groups, and 25 R2 represents a carbamoyl group which can carry on the nitrogen atom 1 or 2 groups selected from straight chain or branched alkyl and alkenyl groups each containing up to 4 carbon atoms , and from cycloalkyl groups containing from 3 to 8 carbon atoms, and alkali metal salts thereof f, when R1 is hydrogen, characterized in that (A) when R1 is different from hydrogen, a compound of the general formula II N, q! Wherein R 2 is as defined above with an isocyanate of general formula (III) An analogous process according to claim 1 or 2, wherein the obtained product is a tetrazine derivative of the general formula (I) according to claim 1, characterized in that R1 represents a benzyl or p-methoxybenzyl group. DK 161147B Analogous process according to any one of claims 1 to 11, characterized in that R 4 carbon atoms in the alkenyl group. Process according to claim 1 or 2, wherein the obtained product is a tetrazine derivative of the general formula 10 (I) according to claim 1, characterized in that R 1 is a straight or branched alkyl, alkenyl or alkynyl group containing from 1 -6 carbon atoms, any such group being unsubstituted or substituted with from 1 to 3 halogen atoms, and R 2 represents a carbamoyl group.