This invention relates to novel 2,4,6-triamino- 1,3,5-triazines, compositions containing them, processes for making them and their use in the treatment of carcinomas, particularly ovarian carcinomas. Trimelamol (1) [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 di ers during formulation.

It has been established that the half-life of trimelamol in humans is short and this may limit its clinical efficacy (reference 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 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.

Our earlier filed European Patent Application 92302491.3 (EP-A-505,220) describes novel 2,4,6-triamino- 1,3,5-triazines having the following general formula:

1 CK,0H

N

I I

wherein R is hydrogen, alkyl or hydroxymethyl, and R ¹ and R ² are hydrogen, alkyl or an electron-withdrawing organic group containing at least. two carbon atoms, at least one of R ¹ and R ³ being an electron withdrawing group, and with the proviso that: when R ³ is hydroxymethyl, R ¹ and R ² are electron-withdrawing organic groups. The electron-withdrawing organic groups are -CH ₂ CF ₃ and -CH ₂ CsCH.

The present invention provides 2,4,6-triamino- 1,3,5-triazines of the general formula:

where R° is hydrogen, alkyl or hydroxymethyl, and R* and R ^b are hydrogen, alkyl or an electron withdrawing group; at least one of R* and R ^b being an electron withdrawing group which is cyanomethyl or cyanoethyl. We have found that the compounds of formula IA, whilst retaining the valuable anti-cancer properties of the compounds of our above-mentioned EP-A-505,220, have improved water solubility that facilitates the formulation of the compounds for clinical use. In the compounds of the present invention, at least one of the R* and R ^b groups is cyanomethyl or cyanoethyl. It is preferred that all the electron withdrawing groups present are cyanomethyl or cyanoethyl but the present invention also includes compounds where one or two of the electron withdrawing groups may be electron withdrawing groups of the type disclosed in our earlier Patent Application EP-A-505,220 e.g. trifluoroethyl and/or propargyl. Our present preference is for compounds where R" and R ^b groups are cyanomethyl. The compounds of the present invention are prepared via intermediate compounds of the general formula:

R ^a H \ /

P ¹ R ^c2

wherein R ^cl and R ^c2 are hydrogen, alkyl or hydroxymethyl, but not both hydroxymethyl, and R* and R ^b are as defined above.

The intermediates II can be prepared by reacting a cyanuric halide of general formula:

wherein X is fluoro or chloro with an amine of the formula R*-NH ₂ or R ^b -NH ₂ , wherein R* or

R ^b are as defined in formula (I) , optionally in the presence of caesium fluoride. The amine is preferably used in free base form rather than as the hydrochloride or other salt and can be used in excess. The excess amine R*-NH ₂ or R ^b -NH ₂ can be used to neutralise the acid HF or HCl liberated in the reaction. Alternatively, another base, e.g. NaHC0 ₃ can be used for this purpose.

Fewer than three of the halogen substituents on the 1,3,5-triazine ring may be displaced by reaction with the amine, 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 (IA) . The

reaction proceeds satisfactorily without the need to adjust the pH of the formaldehyde solution which is about pH4. Unlike the procedure described in our above-mentioned EP-A-505,220, the product does not precipitate from the reaction medium. This means that a bis-hydroxymethyl product can be isolated from the reaction medium by column chromatography after a reduced reaction time, while the tris-hydroxymethyl product can be isolated as the sole product after a further reaction time. The compounds IA of this invention are clinically active and are of use against ovarian carcinomas, particularly against cisplatin-resistant ovarian carcinomas (see Table 1 below) .

Also included within the scope of the present invention are pharmaceutical compositions which comprise, as active ingredient, at least one compound of general formula IA, 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 administration 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 (a) Generation of aminoacetonitrile from its hydrochloride

A suspension of aminoacetonitrile hydrochloride (9.25 g, 0.1 mole) and NaHC0 ₃ (42 g, 0.5 mole) in dichloromethane (150 ml) was vigorously stirred for 16 h. Solid was removed by filtration and the filtrate was concentrated at 50oC on a rotary evaporator to give aminoacetonitrile as a mobile, colourless oil (4.62 g, 82%) which contained, on the basis of ^X H-NMR spectroscopy, only 1% w/w of residual dichloromethane: δ _E (DMSO-d ₆ )<5 2.05 (br s, 2, NH ₂ ) , 3.48 (s, 2, CH ₂ ) , 5.73 (s, integration 0.7% of signal at δ 3.48, residual CH ₂ C1 ₂ ) .

(b) 2,4,6-Tris [ (Cyanomethyl)amino] -1,3,5-triazine (1)

To a stirred solution of cyanuric fluoride (2.025 g, 14 mmol) in dry DMF (15 ml) in a flask surrounded by an ice bath was added dropwise through a septum by syringe aminoacetonitrile (5.89 g, 105 mmol, 7 mol equiv) , during 5 min. The cooling bath was removed and the

reaction mixture stirred at ambient temperature for 3 h, then treated with ice-cold water (150 ml) . The resulting white solid was recovered by filtration, washed with water and the still moist solid recrystallised from boiling water (200 ml) , a small insoluble residue being removed by filtration of the hot solution, to yield (1) as colourless needles 2,778 g (76%): mp 232-234oC, (Schδnenberger et al, Arzneim. Forsch. 15, 30-36 (1965) give mp 216-228oC for analytically impure compound) . δ _H (CDC1 ₃ ) 4.21-4.33 (m, 6, CH ₂ ) , 7.59-7.87 (m, 3, NH) ; m/z 243 (M ⁺ ; 79%), 189

( [M-NCH ₂ CN] ⁺ , 71%). Anal. C ₉ H ₉ N ₉ requires, C, 44.44; H, 3.73; N, 51.83: found C, 44.41; H, 3.70; N, 51.85%. (c) 2,4,6-Tris [ (Cyanomethyl) (hydroxymethyl)amino] -1,3,5- triazine (2) (CB7669) 2,4,6-Tris [ (Cyanomethyl)amino] -1,3,5-triazine

(1,2.432 g, 10 mmol) was added to 40% aqueous formaldehyde (50 ml) and the suspension was stirred at ambient temperature for 3 days. The now clear solution was extracted with ethyl acetate (150 ml, then 100 ml) and the combined extracts dried over Na ₂ S0 ₄ , concentrated, and the residual clear syrup was triturated with water (50 ml) to give a white crystalline solid which was recovered by filtration, washed with water and dried in vacuo over CaCl ₂ . The yield of (2), obtained as the hemihydrate was 1.804 g (53%): 6 _S (Me ₂ S0-d ₆ ) 4.61 (s, 6, CH ₂ CN) , 5.12 (br d, 6, CH ₂ OH, J = 6.2 Hz), 5.95 ( br s, OH). Anal.

C ₁₂ H ₁₅ N ₉ 0 ₃ . 0 . 5H ₂ 0 requires C , 42 . 10 ; H, 4 . 71 ; N, 36 . 83 ; Found 41 . 97 ; H, 4 . 75 ; N, 36 . 68% .

EXAMPLE 2

2,4-Bis [ (Cyanomethyl) (hydroxymethyl)amino] -6- [ (cyanomethyl)amino] -1,3.5-triazine (3) (CB7682) and 2,4,6- tris [ (cyanomethyl) (hydroxymethyl)amino] 1.3.5-triazine (2) (CB7669)

The procedure described in Example 1(c) was carried out on the same scale for 18 h. only. The white solid similarly obtained by treating the concentrated ethyl acetate extract with water was a mixture of (3) and (2) . This was dissolved in a minimum volume of acetone and applied to a column of silica gel (Merck, Art. No. 7734) which was eluted with ethyl acetate to give successively the bis- (hydroxymethyl) derivative (3) and the tris-

(hydroxymethyl) derivative (2) . The products were isolated as white solids after addition of water. The yield of (2) was 0.971 g (28%) and of (3) (also as hemihydrate) 0.952 g

(30%) . Analytical data for (3); δ _H (Me ₂ S0-d ₆ ) 4.37 (s, 2, HNCH-CN) , 4.64 (m, 4, H ₂ CNCH ₂ CN) , 5.14 (m, 4, CH-OH) , 6.03 (br s, 2, OH), 7.98 (br d, 1, NH) . Anal. C^H^N _J O _J .0.5H ₂ 0 requires C, 42.31; H, 4.52; N, 40.37: Found 42.63; H, 4.60; N, 39.64%.

EXAMPLE 3 2,4, 6-Tris [ (2-Cyanoethyl) amino] -1,3, 5-triazine (4)

To a stirred solution of cyanuric fluoride (1.35 g, 10 mol) in dry DMF (25 ml) containing sodium bicarbonate (6.72 g, 80 mmol) and in a flask surrounded by an ice bath was added dropwise through a septum 3-aminopropionitrile (2.804 g, 40 mmol) . The mixture was allowed to attain ambient temperature then stirred for a further 18 hours, then added to ice-cold water (500 ml) and the product extracted with ethyl acetate (2 x 200 ml) . The dried

(MgS0 ₄ ) extracts were concentrated to an oil which later solidified. Recrystallisation from aqueous ethanol afforded (4) as white crystals, 2.23 g (78%); m.p. 91-93oc. δ _H (Me ₂ S0-d ₆ ) 2.76 (t, 6, HNCH ₂ ) , 3.45 (t, 6, CH ₂ CN) , 6.96- 7.22 ( , 3, NH) ; m/z 285 (M ^* ; 27%), 245 ([M-CH ₂ CN] ⁺ , 45%. Anal. C ₁₂ H ₁₅ N ₉ H ₂ 0 requires C, 47.52; H, 5.65; N, 41.56: Found C, 47.37; H, 5.28; N, 42.02%. (4) can be converted to 2,4,6-tris [ (2-cyanoethyl) (hydroxymethyl)amino] -1,3, 5- triazine by reaction with formaldehyde following the general procedure described in Example 1(c) .

EXAMPLE 4

2- (2-Cyanoethyl)amino-4, 6-bis (cyanomethyl)amino-1,3, 5- triazine (5)

To a stirred solution of 2-chloro-4, 6- bis (cyanomethyl)amino-1,3,5-triazine (Schδnenberger et al, Arzneim. Forsch. L5, 30-36 (1965)) (2.234 g, 10 mmol) in dry DMF was added dropwise 3-aminopropionitrile (1.402 g, 20 mmol) during 5 minutes. The reaction mixture was stirred at ambient temperature for 16 hours then water (100 ml) was added and the mixture left for a further 16 hours and the resulting white solid recrystallised from boiling water to give (5), 2.054 g, (80%); m.p. 183-185oC. δ _H (Me ₂ SO-d ₆ ) 2.81 (t, 2, NCH ₂ CH ₂ CN) , 3.47 (t, 2, NCH ₂ CH ₂ CN) , 4.20 (s, 4, NCHjCN) , 7.39-7.54 (m, 3, NH) ; m/z 257 (M*; 67%), 217 ([M-CH ₂ CN] ⁺ , 55%, 190 ( [M-NCH ₂ CH ₂ CN] *, 45%, Anal. CioHuNj requires C, 46.66; H, 4.32; N, 49.02: Found C, 46.32; H, 4.46; N, 48.23%. (5) can be converted to 2- [ (2-cyanoethyl) (hydroxymethyl)amino] -4, 6- bis [ (cyanomethyl) (hydroxymethyl)amino] -1,3,5-triazine by reaction with formaldehyde following the general procedure described in Example 1 (c) .

Biological Results

The anti-tumour activities of the compounds of the invention against ADJ/PC6 plasmacytoma from mice,

Walker 256 carcinoma from rats, L1210 leukaemia from mice and against a human small cell lung cancer line H69 and human ovarian tumour cell lines CHI and 41M were compared with that of trimelamol and the results are set out in Table 3 below. The results show that Compound (2) (CB

7669) is comparable to trimelamol in all the test systems.

Aqueous Stabilities of the Hydroxymethyl Drivatives

The hydroxymethyl derivatives (2, CB7669) and (3, CB7682) were both compared with trimelamol in respect of their stabilities in aqueous solutions at physiological temperature and various pH values (Table 1) and their stabilities at ambient temperature in media which might be suitable for formulation (Table 2) . CB7669 was more stable than trimelamol at all pH values at 37°C. In particular its stability at pH 2.0 should be sufficient to allow oral administration in patients. Likewise its much greater stability at ambient temperature should facilitate its administration by continuous infusion. This could be advantageous since clinical studies on trimelamol (Judson et al , Cancer Research), 49., 5475-5479, (1989) suggest that prolonged exposure would result in an improved therapeutic index.

TABLE 1

STABILITY OF CYANOMETHYL SUBSTITUTED TRIMELAMOL ANALOGUES AT 37oC IN DEIONISED WATER AT VARYING pH

Notes:

Trimelamol data is shown for comparison.

T ¹² measurements were performed using HPLC analysis. This involved an isocratic elution using a mobile phase of 10% acetonitrile: 90% water with a 15cm column packed with C8 octyl Spherisorb material. T ¹² measurements were made by measurement of the disappearance of compound by decreasing peak area with time.

TABLE 2

STABILITY OF CYANOMETHYL SUBSTITUTED TRIMELAMOL ANALOGUES AT ROOM TEMPERATURE IN VARIOUS MEDIA

Note:

Room temperature 21-24oC. Other notes as for Table 1.

TABLE 3

CYTOTOXICITY OF CYANOMETHYL SUBSTITUTED ANALOGUES OF TRIMELAMOL. RESULTS EXPRESSED AS IC ₅₀ VALUES in μM (± standard deviation)

Notes:

Results obtained using the MTT assay with continual drug exposure.

IC ₅₀ value is the drug concentration giving 50% inhibition of cell growth as compared to control values. ND - not done. Cell lines used : ADJ/PC6 - murine plasmacytoma L1210 - murine leukaemia Walker 256 - rat mammary carcinoma H69 - human small cell lung cancer CHI, 41M - human epithelial ovarian cancer

Antitumour Tests on CB 7669 (2)

Table 4 shows that CB 7669 was not active against the ADJ/PC6 tumour in mice in vivo . It was however active against the human ovarian xenograft, though less so than trimelamol (1) . Table 5 compares the activity of CB 7669 in terms of growth delay at doses of 60 mg/kg against the PXN65 cell line as xenograft compared with trimelamol against a number of ovarian xenografts (including PXN65) . Figure 1 shows the growth delay at 3 doses of CB 7669 compared with untreated animals. In view of these results, further experiments were carried out (see below) which indicate the appropriate route of administration could be continuous infusion, comparable with the aforementioned preference based on stability data.

TABLE 4

N.A. not assessable

16

TABLE 5

SUMMARY OF DATA OBTAINED IN HUMAN XENOGRAFTS TRANSPLANTED INTO MICE

CB 7669 initiates a respectable growth delay in the PXN 65 xenograft, although it is substantially less effective than Trimelamol. Specimen growth curves for the PXN 65 xenograft are included.(Fig.1 )

Time dependency of drug exposure for stable analogues of trimelamol (TM) .

The method for cytotoxicity testing was the MTT assay, as previously used. Cells in 96 well plates were plated in the usual manner and left for 24 hours. Drug treatments were carried out for the designated time periods after which drug-containing medium was removed and replaced by fresh, drug-free medium. The assay was then terminated at 96 hours in the usual fashion.

Experiments (Figure 2) involving varied drug exposure time using the human cancer cell line CHI showed a delay onset of cytotoxic activity for the stable analogue CB 7669. Substantial cell killing (>70%) due to exposure with TM for 1 hour was seen at the 200 μM dose, whereas the corresponding data for CB 7669 showed <10% cell kill.

At 6 hours the levels of cell killing increased with values of 56% and 85% for CB 7669 at 100 and 200 μM doses, compared with the corresponding data for TM of 88% and 90%. We found that at least 12 hours exposure was required in order for the stable analogue CB 7669 to exert a similar level of cytotoxicity to that seen for TM.

Plasma pharmacokinetics of CB 7669 in mice

CB 7669 was administered to Balb C female mice at a dose of 120 mg/kg I.P.. Three animals were treated per time point with freeze-dried drug in 5% DMSO/Dextrose.

Animals were anaesthetised with a mixture of halothane and oxygen at 5, 10, 20, 30 and 60 minutes post drug administration, and exsanguinated by direct cardiac puncture.

Blood was centrifuged rapidly at 1800 g for 10 minutes and the plasma removed and mixed with 2 volumes of

ice-cold methanol to precipitate plasma proteins. Following a further centrifugation at 1800 g and 4oC for 10 minutes, the supernatant was removed for analysis by HPLC (as previously described) .

The plasma decay curve of CB 7669 was monophasic and the half-life (t ¹² ) determined using the formula t ¹² = 0 .693/β (β = the first order rate constant). For CB 7669 t ¹² was found to be 9.0 minutes. Previous work carried out using TM in similar experiments gave a t ¹² of 11.8 minutes (i.e. IP dose in female balb C mice at a dose of 150 mg/kg) .

Given these two sets of data, it would appear necessary to administer CB 7669 as a continual infusion. This is based on the observation that due to its stability, CB 7669 requires several hours to exert its cytotoxic effects. In addition, a single IP dose of this compound appears to be cleared from plasma at a similar rate as TM. These factors will inevitably compromise any in vivo activity if administered in the conventional manner. Therefore, administration of CB 7669 by continual infusion using osmotic mini-pumps in our in vivo model systems is of interest.