This invention relates to novel 2,4,6-triamino-l,3,5-triazines, compositions containing them, processes for making them and their use in the treatment of carcinomas, particularly ovarian carcinomas.

Trimelamol [2,4,6-tris{(hydroxymethyl) (methyl) amino)-l,3,5-triazine] is clinically active, particularly against ovarian carcinomas, but its clinical development has been halted due to difficulties with formulation due to instability with respect to the formation of dimers during formulation. It has been established that the half-life of trimelamol activity in humans is short and that may limit its clinical efficacy (I.R. Judson, et al Cancer Res. 49, 5475-5479, 1989). We believe that this is, in part, due to the chemical instability of the N- hydroxymethyl functions resulting in the release of formaldehyde. We have investigated reducing the number of N-hydroxymethyl functions and stabilizing these functions using electron-withdrawing organic groups (defined in the present context as electron-withdrawing relative to methyl), with a view to lengthening the half-life and also improving amenability to formulation, for example in aqueous solutions.

Accordingly this invention provides novel 2,4, 6-triammo-l,3,5-triazines having the following general formula:

wherein each R ¹ which may be the same or different, is hydrogen, alkyl or an electron- withdrawing group and R ² is hydrogen, alkyl or an electron-withdrawing organic group. Preferably, all three groups R ¹ are not hydrogen. The alkyl group R ¹ and/or R ² is preferably

a C _r C ₄ alkyl group, particularly methyl and it is preferred that all three R ¹ groups, when alkyl, are all methyl.

Preferred electron-withdrawing organic groups are -CH ₂ CF ₃ and -CH ₂ C≡CH. Because of the greater stability conferred on such compounds by the presence of such electron withdrawing substituents, which may constitute in lengthening the half-life and also in improving amenability to formulation, they may be prepared by allowing tris-hydroxymethyl compounds or precursors thereof to decompose in aqueous organic media and separating from the mixture of products (see Fig. 1) thus generated the appropriate compounds of the present invention, for example by chromatography on silica gel.

We have found that these new analogues of trimelamol have a similar level of activity against carcinomas, particularly ovarian carcinomas, as trimelamol, but are more stable and do not form dimers and polymers and thus are more amenable to formulation.

The compounds of the present invention are also prepared via novel intermediate compounds of the general formula:

R ¹ H

R ¹ R ¹

wherein R ¹ and R ² are as defined above for the formula I

The intermediates are prepared by reacting a cyanuric halide of general formula:

wherein X is fiuoro or chloro with an amine of the formula R'-NH ₂ or R ¹ R ² NH ₂ , wherein R ¹ and R ² are as defined in formula (I), optionally in the presence of caesium fluoride.

In the absence of caesium fluoride, less than three of the substituents on the 1,3,5-triazine ring may be displaced, which allows for the preparation of asymmetrical compounds.

Treatment of the intermediates II with aqueous formaldehyde, optionally in the presence of potassium carbonate, gives the compounds of formula (I). In order to provide compounds of the formula I in which R ¹ is methyl and R ² is hydrogen, starting from compounds of the formula II in which R ¹ and R ² are also methyl and hydrogen respectively, we prefer to use a concentration of formaldehyde of from about 2 to 5% (w/v), for example about 3% (w/v). This provides a final product which contains as the major product the compound of the formula I. A small amount of the corresponding trimelamol (i.e. R-=methyl, R ² =CH ₂ OH) and 'monomelamol' (i.e. three methyls but only one hydroxymethyl group) compounds will be produced. The presence of these compounds does not significantly affect the activity of the preparation of the compound of the invention in biological assays. However, if desired, the purity of the preparation may be increased by recrystallisation. For example, the material may be dissolved in methanol-water (eg at a ratio of 9:1), and recrystallised.

The compounds of this invention are biologically active and are of use against ovarian carcinomas, particularly against cisplatin-resistant ovarian carcinomas.

Also included within the scope of the present invention are pharmaceutical compositions which comprise, as active ingredient, at least one compound of general formula I, in association with a pharmaceutically acceptable carrier or diluent.

The compounds of the invention will normally be administered orally or by injection.

Compositions for parenteral adinimstration will normally be solutions in aqueous saline, which is pyrogen free for human use. Such compositions can be administered intravenously or intraperitoneally.

Compositions for oral administration will mostly be in solid or liquid form, mostly as tablets, capsules, lozenges, etc. Liquid compositions can be solutions or dispersions in aqueous or non-aqueous media. Ideal solutions are of neutral or alkaline pH and of low ionic strength e.g. 5% dextrose.

Suitable daily doses of the compounds of the invention in therapeutic treatment of the human or animal body range from about lOOmg to 3g/m ² body-surface.

The following Examples illustrate the preparation of the compounds of the present invention.

Example 1

2.4-Bisr(nv roxymethyl) (methyl) amino1-6-methylamino-1.3.5-triazine

To a 3 % w/v aqueous solution of formaldehyde (15 ml) was added potassium carbonate (691 mg, 5 mmol) then (841 mg, 5 mmol). The reaction mixture was stirred at room temperature until the initially clear solution (pH 11.5) became cloudy (2-3 h) then set aside overnight (16 h). The white granular solid which separated was recovered by filtration, washed with water (4 x 5 ml) and the product dried in vacuo over anhydrous CaCl ₂ . Yield 593 mg (52%); Η-NMR spectrum δ _H 2.75 (app d, 3, HNCJfc), 4.99 (br s, 4, HOCHj), 5.36 (br s, 2, OH) 6.61 (br s, 1, NH); mass spectrum (FAB; glycerol/thioglycerol matrix) m/z 229 ([M+H] ⁺ , 70%), 211 (229-H ₂ O, 100%), 199 (229- CH ₂ O, 35%), 181 (199-H ₂ O, 50%), 169 (199-CH ₂ O, 30%). Anal. C ₈ H ₁₆ N ₆ O ₂ requires C, 42.10; H, 7.07; N, 36.82: found C, 41.87; H, 7.01; N, 36.55%.

In the Examples which follow, this compound is referred to as CB7646.

Example 2

Further purification of title compound of Example 1.

Using the procedures described in Example 1 above, but with 10 times the amount of starting materials, 6.325 g of product was obtained. HPLC analysis revealed the preparation to have the following composition: title compound: 65%, trimelamol 22%, monohydroxymethyl derivative 12%.

This material (3 g) was dissolved in methanol-water, 9:1 (100 ml) at 37°C and cooled at - 20°C for 24 h. The white crystalline solid was recovered by rapid filtration and dried in vacuo over anhydrous CaCl ₂ to give 1.37 g of material having the following composition: title compound 87% trimelamol 4%, monohydroxymethyl derivative 9%. Signals in the Η-NMR spectrum (D ₂ O, determined at 37°C) were: title compound δ 3.08 (HNCH ₃ ), 3.30

5.29 (HOCH-,): trimelamol 3.33 and 5.32; monohydroxymethyl derivative 3.05, 3.27 and 5.26.

Example 3

2.4-Bfcrflιvdroχymethviy2.2.2-ιrtfluor^^ triazine

A solution of 2-[([hydroxymethoxy]methyl)(2,2,2-trifIuoroethyl)amino]-4,6- bis (hydroxymethyl) (2,2,2-trifluoroethyl) amino-l,3,5-triazine(500 mg, 1.02 mmol) in a mixture of acetone (3 ml) and water (2 ml) was set aside at room temperature for 18 h. Acetone was removed under vacuum and the organic materials were extracted with diethyl ether.

The organic phase was concentrated and applied to a column (50 g, 3 cm dia.) of silica gel (Merck, Art. No. 9385) which was eluted with diethyl ether. There was successively eluted 2-[0ιydroxymemyI)(2,2,2-trifluoroeΛyl)aπιino]-4,6-bis [2,2,2-trifiuoroethyl)amino]-l,3,5- triazine (23 mg), the title compound (144 mg, 33% yield) and 2,4,6-tris [(hydroxymethyl) (2,2,2-trifluoroethyl) amino] 1,3,5-triazine (111 mg). The title compound is obtained as a white solid by trituration of the appropriate fractions with ice-cold water, recovery by filtration and desiccation in vacuo over calcium chloride. NMR spectrum: δ _H (Me ₂ SO-dβ) 4.09 (brq, 2, F ₃ CCI NH), 4.41 (brq, 4, F^d^NCH.OH) 5.06 (d, 4, J=7.1Hz, CKflR), 5.78 (brs, 2, OH), 7.80 (brs, 1, NH). δ _r 70.23, -70.03 (2s, 3, F ₃ CCH ₂ NH) -68.3 (s, 6, F ₃ CCH ₂ NCH ₂ OH).

In the Examples which follow, this compound is referred to as CB7683.

Example 4

Stability of Compounds of the invention.

(ϊ) Stability in solution.

Compounds were dissolved in DMSO to a concentration of 50mM. Aliquots were then dispersed into the appropriate medium to give a final concentration of 100μM in a volume of about 10ml. The diluted preparations of trimelamol and CB7646 (see Example 1) for

_ _

HPLC analysis were stored in a water batii at 21°-24°C (to simulate room temperamre) or at 37°C in water, 0.9% NaCl or 5% dextrose. Aliquots were removed from each preparation at intervals to assess their stability (i.e. half-life, T ^1/2 ) which was measured using HPLC analysis. This entailed an isocratic elution using a mobile phase comprising 10% acetonitrile, 90% 0.05M ammonium bicarbonate. The 15 cm column was packed with C8 octyl Spherisorb material. The column was encased in a cooling cabinet which was maintained at 14-17°C. Standards of freshly prepared solutions were run throughout the analysis period by way of controls.

T ^{1 2} measurements were made by measurement of the disappearance of compound by decreasing peak area with time, using a Data System 450MT2 data acquisition system (Kontron Instruments, Watford, UK) linked directly to the detector on the HPLC system (set at 225 nM). T ^{, 2} measurements were read from a semi-logarithmic plot of peak area (y) versus time (x).

The results are shown in table 1 and indicate that CB7646 has superior stability.

TABLE 1

(if) Dimer/polvmer formation in solution.

An aqueous solution of CB7646 and trimelamol in 4ml aliquots at a concentration of 4-5 mg ml was left to stand overnight (14-16 hours) at room temperature. By the end of this period, the trimelamol solution had formed a heavy precipitate, indicative of dimer and polymer formation. Similar polymerisation of trimelamol over a period of time proved problematic during its Phase I and II clinical trials (Judson et al, 1989, Cancer Res. 49;5475- 5479; Judson et al, 1991, Br. J. Cancer 63; 311-313). In contrast, preparations of CB7646 prepared in Examples 1 and 2 did not form a precipitate, indicating the monomeric form is more stable that trimelamol.

Example 5

Cvtotoxicitv of Compounds of the Invention

The cytotoxicity of CB7646 and CB7683 was compared with trimelamol against mammalian tumour cell lines using the MTT assay. This assay is based upon the selective ability of

living but not dead cells to reduce the tetrazolium salt MTT (3-[4,5-dimethylthiazol-2-yl]-25- diphenyl tetrazolium bromide) to purple formazan (Mosmann et al, 1983, J. Immun. Methods 65; 55-63; Carmichael et al (1987) Cancer Res. £7; 936-942). Cell lines were grown in culture with continual drug exposure. The ICs, values (in μm) of the compounds (i.e. concentration giving 50% inhibition of cell growth as compared with untreated control) were determined, and are shown in Table 2. Figures in parenthesis refer to standard deviation or +/- values for 2 or more measurements.

TABLE 2

Cell lines used:

PC6- murine plasmacytoma

Walker 256- rat mammary carcinoma

H69 - human small cell lung cancer

CHI, 41M- human epithelial ovarian cancer

The tests on Walker 256 and H69 cells were repeated using a preparation of CB7646 prepared by the recrystallisation method of Example 2. The results were:

Walker 256 - 10.5

H69 - 16.5

Example 6

Antitumour Activity Towards the ADJ/PC6 Tumour in Mice

The anti-tumour activity of CB 7646 prepared in accordance with Example 1 against ADJ/PC6 tumour in mice were compared with that of trimelamol. An implant of 1mm ³ of tumour was made on day 1. On day 20, animals bearing tumours of comparative size were placed into groups of 4 and treated with drug on 5 consecutive days, and then left until day 30. Tumours from the σeated and controls were dissected and weighed as a measure of tumour growth. Compounds were given in 5% DMSO/dextrose.

TABLE 3

% Inhibition at various Doses fTumour wt as % of Control Value)

For CB7646 (dimelamol) the results give LD^ > lOOmg/kg, EDgo 74mg/kg Therapeutic Index (IT) > 1.4

Example 7

Example 6 was repeated to obtain more precise LD ₅₀ values. The LD ₅₀ , EDo _n and T.I. values were calculated and shown in Table 4.

TABLE 4

Example 8

CB7646 was tested in vivo against ovarian cancer xenografts of the PXN65 cell line transplanted into mice, substantially in accordance with Harrap et al, Annals of Oncology, 1990, l;65-76. PXN65 is a cisplatin-sensitive line. Mice treated with either trimelamol or CB7646 showed tumour regression within 28 days whereas in untreated controls tumour growth was uncontrolled, leading to death. The results are summarised in Table 5.

TABLE 5

Activity in vivo against PXN65 Xenografts

GD = Growth delay.

The data show that CB7646 has a comparable efficacy to trimelamol.