An object of the invention are new (1-adamantylamino)-pyrimidines, salts thereof with inorganic and organic acids and method of their preparation.

New (1-adamantylamino)-pyrimidines of formula 1 are substituted at heterocyclic ring by alkyl groups or by halogen atoms. Many derivatives of adamantane exhibit biological activity.

The most known compounds of this group are derivatives of 1- adamantane (amantidine, rimantidine and tromantidine) used as antiviral drugs or in therapy of Parkinson's disease [W. L. Davies et al., Science, 144, 862, (1964), R. S. Schwab et al., J. Am. Med. Assoc., 208, 1168 (1969)]. Selected adamantyl esters of phthalimide substituted by amino acids inhibit growth of various bacteria strains to an extent that is comparable to activity of some antibiotics [A. Orzeszko, J. Stefariska, B. Starosciak, Z. Kazimierczuk, Acta Biochim. Polon., paper in press]. Our studies on biological activity of (1- adamantylamino)-pyrimidine derivatives have shown, that the derivatives stimulate secretion of TNF-a. Tumour necrosis factor-TNF-a is a cytokine exhibiting antineoplastic and immunomodulating properties. From a structural point of view it is identical with cachectin secreted by macrophages in the course of neoplastic processes and infection [B. Beutler et al., Nature, 320, 584, 1986].

Factors that stimulate secretion of TNF-a by macrophages are bacterial endotoxins of lipopolysaccharides (LPS) type.

Recently, it has been found that also chemical compounds, such as e. g. 5, 6- dimethylxanthenone-4-acetic acid, can stimulate secretion of TNF-a within a tumour [W. R. Joseph et al., Cancer Res. 59, 633, (1999] and this can possibly constitute a new targeted method of treatment of some tumours.

(1-Adamantylamino) pyrimidine derivatives and salts thereof constituting an object of the present invention exhibit considerable activity as substances that cause an increase in production of tumour necrosis factor of alpha-type (TNF-a) in some cell lines.

Our studies on an effect of stimulation of production of TNF-a by (1- adamantylamino) pyrimidine derivatives were conducted on genetically modified B78 murine melanoma cells, subjected to transduction by a gene for human TNF-a (the line was referred to as B78/TNF) [W. Lasek, A. Mackiewicz, A.

Czajka, T. Switaj, J. Gofab, paper in press]. These cells constitutively secreted human TNF-a in the culture. Exponential growing B78/TNF cells (2 x 105 cells per 1 mL) were incubated for 24 hours at 37°C in Dulbecco's medium comprising 10% of bovine foetal serum and the respective concentration of tested compounds (100, 10, 1 and 0. 1 rM). TNF-a concentrations were measured with a use of ELISA test. More intense secretion of TNF-a, depending on a position and a number of methyl groups in tested compounds were observed.

In a Table below some results of TNF-a secretion for that line with an addition of compounds of formula 1 of the same concentration (10, uM) are given.

Table Amount of secreted TNF-a Derivative (Background with no (concentration : 10 p. M) addition of inducing compound = 100) 1 4- (l-adamantylamino)-2, 6-dimethylpyrimidine 187 2 2-1-adamant lamino-4, 6-dimeth I rimidine 235 3 2-(1-adamantylamino)-pyrimidine 205 4 2- (1-adamantylamino)-6-methylpyridine 266 (Compound for the purpose of comparison) The new compounds stimulating production of tumour necrosis factor TNF-a that we have synthesised can constitute a promising group of drugs for topical treatment of neoplastic solid tumours.

The most important aspect of the invention are new (1-amantylamino)- pyrimidines of formula 1, 1 wherein substituents Ri, R2, R3 or R4 are adamantylamino group, hydrogen atom, methyl group or halogen atom, the compound of formula 1 containing one adamantylamino group and a substituent or substituents being methyl groups or halogen atoms at a position different from an ortho-position to the amino group.

According to the invention, the new compounds are as follows : 2- (1-adamantylamino)-4, 6-dimethylpyrimidine ; 2- 4-chloro-6-methylpyrimidine ; 2- 4-methylpyrimidine ; 2- (1-adamantylo-4-amino)-5-bromopyrimidine ; 4- (1-adamantylamino)-2, 6-dimethylpyrimidine ; 2- 4-methylpyrimidine hydrochloride.

Another important aspect of the invention is a method of preparation of new (1-adamantylamino) pyrimidines of formula 1, 1 wherein Ri, R2, Ra or R4, are adamantylamino group, methyl group or halogen atom, the molecule of formula 1 containing one 1-adamantyl- amino group and a substituent or substituents being methyl groups or halogen atoms at a position different from an ortho-position to the amino group, consisting in that, a pyrimidine derivative of formula 2, 2 wherein Ri, R2, R3 or R4 are : amino group, methyl group or halogen atom, the compound of formula 2 containing one amino group and a substituent or substituents being methyl groups or halogen atoms at a position different from an ortho-position to the amino group, is reacted with 1-adamantanol at elevated temperature in an acid solution, to yield a compound of formula 1, the formula of which and the substituents Ri, R2, R3 or R4 are as defined above, the compound of formula 1 containing one adamantylamino group and a substituent or substituents being methyl groups or halogen atoms at a position different from an ortho-position to the amino group, and the product of formula 1 is isolated and converted by a known method into a salt, preferably hydrochloride.

In the method of the invention trifluoracetic acid is used to provide an acid solution. The condensation reaction is carried out at elevated temperature 80- 120°C, preferably at boiling temperature of trifluoroacetic acid.

The final new (1-adamantylamino) pyrimidines are isolated by the method of the invention from reaction mixture by diluting the reaction mixture with water and adjusting the mixture to pH of 7. 0-14. 0.

The resulting precipitate is isolated by crystallization or adsorption chromatography on silica gel.

In the process of the invention adamantylation of an exocyclic amino group in pyrimidine derivatives results from preparation of an adamantyl carbocation from 1-adamantanol in trifluoroacetic acid solution at elevated temperature. It should be stressed that the reaction does not take place e. g. in case of aniline, where the amino group in acid solution is protonated.

A succesful course of the reaction of aminopyrimidine with the adamantyl carbocation is conditioned by the fact that it is a nitrogen atom of the heterocyclic ring which is subjected to protonation and not the amino group, which captures carbocations, to yield (1-adamantylamino) pyrimidine derivatives as final products.

As already mentioned, the new compounds exhibit activity stimulating production of tumour necrosis factor-TNF oc and can constitute a promising group of drugs for topical treatment of neoplastic solid tumours.

Accordingly, the present invention covers pharmaceutical or veterinary compositions comprising at least one compound defined by formula 1 as a single active substance used or combined with a pharmaceutically acceptable and compatible carrier and an excipient.

Hereinafter and in the patents claims the term"pharmaceutical composition"is meant as a mixture of an active substance in combination with a pharmaceutical acceptable and compatible carrier and an excipient, to be used for treatment of humans and animals.

For the purpose of topical treatment, the compounds of formula 1 can be formulated as an ointment or a cream or in another pharmaceutical form comprising the compound of formula 1 suspended in a liquid or solid carrier, used for preparation of such pharmaceutical forms. An additive of particular carriers to provide protection of an active substance from its decomposition or oxidation is also possible.

For the purpose of systemic treatment, the compounds of formula 1 can be formulated as injections or infusion solutions into a tumour. In all these cases, the pharmaceutical form has to be sterile and a form of liquid should make it possible to use syringe.

Another possibility is a dry pharmaceutical composition to be diluted or suspended prior to administration in an injection solution including e. g. liquid polyethylene glycol, propylene glycol, glycerol or the like.

Preferably, prolonged absorption of injection composition can be achieved through a use of known compounds that delay absorption and/or sustain release, such as aluminium monostearate and gelatine.

A unit dosage can include e. g. the main active ingredient in an amount from about 0. 1 to about 500 mg, preferably from about 0. 1 to about 250 mg per day.

In the embodiment examples of the invention chemical reagents were from Aldrich (apart from solvents). Column chromatography was performed on silica gel 60H (Merck). Analytical thin-layer chromatography was performed on aluminium plates with silica gel 60F254 (Merck). Melting points were determined with a use of Gallenkamp-5 apparatus in glass capillaries.

UV spectra were measured on Kontron-Uvikon-940 spectrophotometer.

NMR spectra (ppm) were measured on Varian-Gemini-200 MHz spectrometer.

The following examples illustrate the invention while not limiting its scope : Example 1.

2- (1-Adamantylamino)-4, 6-dimethylpyrimidine.

To a solution of 2-amino-4, 6-dimethylpyrimidine (1. 35g) in 15 cm3 of trifluoroacetic acid, 1-adamantanol (1. 52g) was added while stirring. The resulting reaction mixture was heated at reflux for 5 hrs while stirring. Then, the mixture was left to reach a room temperature, poured into 60 ml of ice water, adjusted to pH 7 with an aqueous ammonia solution and left overnight at temperature 4°C. The resulting precipitate was filtered off and recrystallised from ethanol-water mixture (1 : 1, v/v) to yield 1. 85g of colourless crystals (the product), mp 164-166°C. Yield 72%.

TLC (CHCI3, silica gel) : Rf 0. 40. UV (MeOH/H20 1 : 1) sax 242 nm, 303 nm.'H- NMR (CDCI3) : 1. 69 (br. s, 6H, Ada) ; 2. 11 (br. s, 9H, Ada) ; 2. 43 ; (s, 6H, CH3) ; 6. 33 (s, H-C (5)).

Elemental analysis. Calcd for C16H23N3 (257. 38) : C 74. 66, H 9. 01, N 16. 33 ; Found : C 74. 60, H 9. 11, N 16. 25.

Example 2.

2- (l-Adamantylamino)-4-chloro-6-methylpyrimidine To a solution of 2-amino-4-chloro-6-methylpyrimidine (1. 57g) in 8 cm3 of trifluoroacetic acid, 1-adamantanol (1. 52g) was added while stirring. The resulting reaction mixture was heated at reflux for 1 hour while stirring. Then, the mixture was left to reach a room temperature, poured into 60 ml of ice water, adjusted to pH 7 with an aqueous ammonia solution and left overnight at 4°C.

The resulting precipitate was filtered off, absorbed on silica gel and subjected to adsorption chromatography on a column with silica gel (3 x 25 cm) using methylene chloride as an eluent. Fractions containing product were evaporated to dryness and the residue was recrystallised from ethanol-water mixture (1 : 1 v/v) to yield 415 mg of colourless crystals, mp 124-126°C. Yield 15%.

TLC (CHCI3, silica gel) : Rf 0. 60. UV (MeOH/H20 1 : 1) : kmaX 245 nm, 304 nm.'H- NMR (CDC13) : 1. 70 (br. s, 6H, Ada) ; 2. 11 (br. s, 9H, Ada) ; 2. 36 (s, 3H, CH3) ; 6. 42 (s, H-C (5)).

Elemental analysis. Calcd for C15H2oN3CI (277. 80) : C 64. 85, H 7. 26, N 15. 13.

Found : C 64. 85, H 7. 31, N 15. 23.

Example 3.

2- (1-Adamantytamino)-4-methyfpyrimidine.

To a solution of 2-amino-4-methylpyrimidine (1. 9g) in 15 cm3 of trifluoroacetic acid, 1-adamantanol (2. 4g) was added while stirring. The resulting reaction mixture was heated at reflux for 5 hrs while stirring. Then, the mixture was left to reach a room temperature, poured into 150m1 of ice water containing 20% methanol, adjusted to pH 8 with an aqueous ammonia solution and left overnight at 4°C. The resulting precipitate was filtered off and recrystallised from ethanol- water mixture (1 : 1, v/v) to yield 1. 8g colourless crystals (the product), mp 122- 1240C. Yield 72%.

TLC (CHCI3, silica gel) : Rf 0. 45, UV (MeOH/H2O 4 : 1) : Xmax : 242 nm, 303 nm.

1H-NMR (CDOs) : 1. 69 (br. s, 6H, Ada) ; 2. 11 (br. s, 9H, Ada) ; 2. 26 ; (s, 3H, CH3) ; 6. 45 d, 1H, H-C (5)), 8. 10 (d, 1H, H-C (6)).

Elemental analysis : Calcd for C15H21N3 (243. 35) : C 74. 03, H 8. 70, N 17. 27.

Found : C 74. 15, H 8. 78, N 17. 40.

Example 4.

2- (1-Adamantylamino)-5-bromopyrimidine To a solution of 2-amino-5-bromopyrimidine (0. 97g) in 5 cm3 of trifluoroacetic acid, 1-adamantanol (0. 61g) was added while stirring. The resulting reaction mixture was heated at reflux for 4 hrs while stirring. Then, the mixture was left to reach a room temperature, poured into 60 ml of ice water, adjusted to pH 7 with an aqueous ammonia solution and left overnight at 4°C. The resulting precipitate was filtered off and recrystallised from ethanol-water mixture (1 : 1, v/v) to yield 0. 90g of colourless crystals (the product), m. p. 175-177°C. Yield 73%.

TLC (CHCI3, silica gel) : Rf 0. 60. UV (MeOH/H2O 1 : 1) : Xmax : 250 nm, 322nm.'H- NMR (CDC13) : 1. 69 (br. s, 6H, Ada) ; 2. 07 (br. s, 9H, Ada) ; 8. 21 (s, 1 H, H-C (4, 6)).

Elemental analysis. Calcd for Ci4HisN3Br (308. 22) : C 54. 56, H 4. 74, N 13. 63 ; Found : C 54. 60, H 4. 87, N 13. 45.

Example 5.

4- (1-Adamantylamino)-2, 6-dimethylpyrimidine To a solution of 4-amino-2, 6-dimethylpyrimidine (1. 35g) in 10 cm3 of trifluoroacetic acid, 1-adamantanol (1. 52g) was added while stirring. The resulting reaction mixture was heated at reflux for 5 hrs while stirring. Then, the mixture was left to reach a room temperature, poured into 60 ml of ice water, adjusted to pH 7 with an aqueous ammonia solution and left overnight at 4°C.

The resulting precipitate was filtered off and recrystallised from ethanol-water mixture (1 : 1, v/v) to yield 1. 65g of colourless crystals (the product), mp 200- 201°C. Yield 64%.

TLC (CHCI3-CH30H, 9 : 1, silica gel) : Rf 0. 56, UV (MeOH/H2O 1 : 1) : sax 279 nm.

1H-NMR (CDCI3) : 1. 70 (br. s, 6H, Ada) ; 2. 10 (br. s, 9H, Ada) ; 2. 37 and 2. 57 (2s, 6H, 2 CH3) ; 6. 35 (s, 1 H, H-C (5)).

Elemental analysis : Calcd for C16H23N3 (257. 38) : C 74. 66, H 9. 01, N 16. 33 ; Found : C 74. 63, H 9. 13, N 16. 22.

Example 6.

2- (1-Adamantylamino)-4-methylpyrimidine hydrochloride.

1g of 2- (l-adamantylamino)-4-methylpyrimidine was dissolved at 37°C in 20 cm3 of 20% methanolic HCI solution. Then, the solution was evaporated to dryness, and the residue was treated with 20 cm3 of ethyl ether-acetone mixture (1 : 1, v/v). The precipitate was filtered off and dried under reduced pressure over potassium hydroxide to yield 0. 95g of crystals, mp 210-212°C ; Yield 83%.