EP0147443A1

EP0147443A1 - Pyrimidine derivatives

Info

Publication number: EP0147443A1
Application number: EP19840902351
Authority: EP
Inventors: Edward A. Bliss; Roger John Griffin; Malcolm Francis Graham Stevens
Original assignee: Aston Molecules Ltd; University of Birmingham; Wellcome Foundation Ltd
Current assignee: Aston Molecules Ltd; Wellcome Foundation Ltd
Priority date: 1983-05-26
Filing date: 1984-05-29
Publication date: 1985-07-10
Also published as: GB2152508A; JPS60502255A; AU3016884A; WO1984004746A1; GB8501742D0; GB2152508B; GB8314643D0

Abstract

Des 2,4-diamino-5-phénylepyrimidines sous la forme de la base libre ou d'un de leurs sels d'addition d'acide présentent la formule générale (I), où R1 est de l'hydrogène, un halogène ou un groupe alcoxy possédant une chaîne de carbone comportant de 1 à 4 atomes de carbone tandis que R2 est de l'hydrogène ou un groupe alkyle comportant de 1 à 4 atomes de carbone. Les composés sont utiles dans le traitement thérapeutique, par exemple en tant qu'agents antitumoraux, antipsoriatiques et antimalariens, et des préparations pharmaceutiques en permettant l'administration à des mammifères sont décrites. Des composés particulièrement utiles, notamment dans la thérapie antitumorale, possèdent le groupe azido en position 3' ou 4' et R1 en position 4' ou 3' du groupe phényle.2,4-diamino-5-phenylepyrimidines in the form of the free base or one of their acid addition salts have the general formula (I), where R1 is hydrogen, halogen or a alkoxy group having a carbon chain having from 1 to 4 carbon atoms while R2 is hydrogen or an alkyl group having from 1 to 4 carbon atoms. The compounds are useful in therapeutic treatment, for example as anti-tumor, anti-psoriatic and anti-malarial agents, and pharmaceutical preparations allowing administration to mammals are described. Particularly useful compounds, in particular in the antitumor therapy, have the azido group in position 3 'or 4' and R1 in position 4 'or 3' of the phenyl group.

Description

PYRIMIDINE DERIVATIVES

TECHNICAL FIELD

The present invention relates to azido-substituted pyrixaidine derivatives. More specifically it concerns azido derivat ives of 2 ,4diamino-5-phenylpyrimidines , the preparat ion thereof , pharmaceutical formulations thereof and the use thereof in therapy.

BACKGROUND ART

It is known that the azido group may act as a "pseudohalide" group (see A. Treinin, in "The Chemist ry of the Azido Group" , Editor S . Patai; publisher John Wiley & Sons , 1971 , pp 1-55) and in its lipophilic and electronic characteristics bears affinities with halogen substituents which have found wide use as biologically acceptable groups in drug molecules . It has also been reported that aromat ic azides can act as pro-drug modif icat ions for the corresponding aromatic amines (E .A. Bliss , T .B . Brown, M.F.G. Stevens, and C.K. Wong , J. Pharm. Pharmacol . , 1979 , 315 , 66P; E.A. Bliss , Ph.D Thesis , University of Aston in Birmingham, Birmingham, England , 1980) . It is also known that some aromatic azides can potentially degrade in vivo to short-lived reactive chemical intermediates in the manner in which they degrade thermally and photochemically ("The Chemistry of the Azido Group," Editor S . Patai, John Wiley & Sons , 1971 , pp. 221330 and pp. 441-502) and this property might perhaps suggest exploitation of the use of such aromat ic azides as radiosensitizing agents in the treatment of cancer or in photochemotherapy of overproliferative skin disorders ( skin cancer or psoriasis) .

However, so far as is known no aromatic azido compounds have ever been used therapeutically for treatment in connection with human or animal disease and no aromatic azido compounds known to be useful or effeective for this purpose have hitherto been available. DISCLOSURE OF THE INVENTION

The present invention provides, in a first aspect, compounds of the general formula (I):

wherein the or each R¹ radical is selected from hydrogen, halogen, or alkoxy having a carbon chain of 1-4 carbon atoms, while R² is hydrogen or an alkyl group of 1-4 carbon atoms, and the invention further includes acid addition salts of compounds of the said general formula (I).

In a first class of preferred compounds, the azido group is in the 3'-position of the phenyl group, a said radical R¹ consisting of a chlorine, bromine or iodine atom or an alkoxy group of 1-4 carbon atoms is in the 4'-position of the phenyl group, and the said radical R² is an alkyl group of 1-4 carbon atoms. Particularly preferred compounds within this first class comprise azido-substituted pyrimidine derivatives of formulas (la - Id) below and acid addition salts thereof:

H

In a second class of preferred compounds, the azido group is in the 4'-posit ion of the phenyl group, a said radical R¹ consisting of a chlorine, bromine or iodine atom or an alkoxy group of

1-4 carbon atoms is in the 3 '-position of the phenyl group, and the said radical R² is an alkyl group of 1-4 carbon atoms. Particularly preferred compounds within this second class comprise azido-substituted pyrimidine derivatives of formulas (Ie - If) below and acid addition salts thereof :

Acid addition salts of compounds of formula (I) [including compounds of formulas ( la - If) ] are preferably pharmaceutically acceptable although other acid addition salts are within the scope of the invention. Suitable salts are those derived from, for example, the following acids : hydrochloric, hydrobromic, sulphuric, nitric, isethionic, phosphoric, maleic, salicylic, p-toluenesulphonic, tartaric , citric , lactobionic , formic , malonic , pantothenic , succinic , naphthalene-2-sulphonic, benzenesulphonic, methanesulphonic and ethanesulphonic. The preferred salt s in terms of pharmaceutical acceptability are the ethanesulphonic acid salts. The compounds of formula (I) may be prepared by adapting or using any method or synthetic procedure known ih the art for the preparation of compounds of analogous structure. For example, compounds of formula (I), at least wherein the azido group is in the 3'- or 4'- position of the phenyl group, may be prepared by using either of the following procedures:

(1) Diazotisation of the corresponding amino compound, for example the 3'- or 4'-amino compound, of formula (II) below, wherein R¹ and R² are as hereinbefore defined in relation to the compounds of formula (I), followed by treatment with azide. The compounds of formula (II) may themselves be obtained by reduction of the corresponding nitro compounds.

(2) React ion with guanidine of an azido compound of formula (III) ,

wherein R¹ and R² are as hereinbefore defined, and Y is a residue or group such as alkoxy (e.g. methoxy, ethoxy), aralkoxy (e.g. benzyloxy), alkylthio (e.g. methylthio), aralkylthio (e.g. benzylthio) or a secondary or tertiary amino group (e.g. dimethylamino, diethyl amino, or other dialkylamino group; anilino; morpholino).

There is therefore provided, as a further aspect of the invention, a method for the preparation of a compound of formula (I) comprising adapting or using any method known for the preparation of analogous compounds, and in particular those methods defined in (1) to

(2) hereinabove.

The compounds of the invention have been found to inhibit dihydrofolate reductase (DHFR) in mammals and other biological systems and to have antitumour activity; they also appear to have some antipsoriatic and antimalarial activity. Such antitumour activity is evidenced for example by reduction of tumour cell number in mammals bearing ascitic tumours and their consequent increase in survival as compared to a control group which is untreated. Antitumour activity is further evidenced by measurable reduction in the size of solid tumours following treatment with the compounds of this invention compared to the tumours of untreated control animals. The murine tumour lines against which the compounds of formula (I) are active include, but are not limited to, lymphocytic leukemia P388, lymphocytic leukemia L1210, melanotic melanoma B16 , Colon 38, TLX5 lymphoma, W3129 myeloma, Walker 256 and M5 recticulura cell sarcoma.

As has been described above, the compounds of the present invention are useful for the treatment of tumours. The invention thus further provides a method for the treatment of tumours in animals, including mammals, which comprises the administration of an effective, non-toxic amount of the compound of formula (I), or an acid addition salt thereof, orally, parenterally (including subcutaneous, intramuscular and intravenous), or applied topically. The administration will generally be carried out repetitively at intervals, for example once or several times a day. There is further provided as a further, or alternative, aspect of the invention, a compound of formula (I) or an acid addition salt thereof for use in therapy, for example as an antitumour agent. The amount of compounds of formula (I) required to be effective as an antitumour agent for treating mammals will, of course, vary and is ultimately at the discretion of the medical or veterinary practitioner treating the mammal in each particular case. The factors to be considered by such a practitioner, e.g. a physician, include the route of administration and pharmaceutical formulation; the mammal's body weight, surface area, age and general condition; and the particular salt or ester to be administered. However, a suitable effective antitumour dose is in the range of about 1.0 to about 75mg/kg bodyweight, preferably in the range of about 5 to 40mg/kg with most suitable doses being for example in the range 10 to 30mg/kg. In daily treatment for example, the total daily dose may be given as a single dose, multiple doses, e.g. two to six times per day, or by intravenous infusion for any selected duration. For example, for a 75kg mammal, the dose range would be about 75 to 500mg per day, and a typical dose would cqmraonly be about 100mg per day. If discrete multiple doses are indicated, treatment might typically be 50mg of a compound, of formula (I) given 4 times per day in the form of a tablet, capsule, liquid (e.g. syrup) or injection.

The antitumour activity of the compounds of formula (I) resides in the free base and thus the nature of the acid participating in the acid addition salts is of minor importance. However, when used in medicine, the salts of the compound of formula (I) should be both pharmacologically and pharmaceutically acceptable, but nonpharmaceutically and non-pharmacologically acceptable salts may conveniently be used to prepare the free active compound or pharmaceutically acceptable salts thereof and are not excluded from the scope of this Invention.

While it is possible for the active compound [defined herein as compound of formula (I)] to be administered alone as the raw chemical, it Is preferable to present the active compound as a pharmaceutical formulation. Formulations of the present invention, for medical use, comprise the active compound together with one or more pharmaceutically acceptable carriers thereof and, optionally, any other therapeutic ingredients. The carrier(s) must be pharmaceutically acceptable in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof .

The present invention therefore further provides a pharmaceutical formulation comprising a compound of formula (I) (in the form of the free base or a pharmaceutically acceptable acid addition salt) together with a pharmaceutically acceptable carrier therefor.

The formulations include those suitable for oral, rectal or parenteral (including subcutaneous, intramuscular and intravenous) administration.

The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include generally the step of bringing the active compound into association with a carrier which constitutes one or more accessory Ingredients. Usually, the formulations are prepared by uniformly and intimately bringing the active compound into association with a liquid carrier or with a finely divided solid carrier or with both and then, if necessary, shaping the product into desired formulations.

Formulations of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets, tablets or lozenges, each containing a predetermined amount of the active compound; as a powder or granules; or a suspension In an aqueous liquid or non-aqueous liquid such as a syrup, an elixir, an emulsion or a draught. The active compound may also be presented as a bolus, electuary or paste.

A tablet may be made by compression or moulding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing, in a suitable machine, the active compound in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant, Inert diluent, surface active or dispersing agent. Moulded tablets may be made by moulding, In a suitable machine, a mixture of the powdered active compound with any suitable carrier.

A syrup may be made by adding the active compound to a concentrated, aqueous solution of a sugar, for example sucrose, to which may be added any accessory ingredient. Such accessory ingredient(s) may include flavourings, an agent to retard crystallisation of the sugar or an agent to increase the solubility of any other ingredient, such as a polyhydric alcohol for example glycerol or sorbitol.

Formulations for rectal administration may be presented as a suppository with a usual carrier such as cocoa butter.

Formulations suitable for parental administration conveniently comprise a sterile aqueous preparation of the active compound which is preferably isotonic with the blood of the recipient.

In addition to the aforementioned Ingredients, the formulations of this Invention may further include one or more accessory ingredient(s) selected from diluents, buffers, flavouring agents, binders, surface active agents, thickeners, lubricants, preservatives (including antioxidants) and the like.

Overall, the invention comprises each and any novel feature or combination of features disclosed herein, but different aspects of the invention principally but not exclusively comprise broadly the following:-

(i) A compound of formula (I) as defined herein;

(ii) Processes for the preparation of compounds of formula (I) as defined herein; (iii) A pharmaceutical formulation comprising a compound of formula (I) as defined herein together with a pharmaceutically acceptable carrier therein; (iv) Processes for the preparation of a pharmaceutical formulation as defined in (iii) above by methods described herein; (v) Compounds of formula (I) for therapy or for use in medicine, for example as antitumour agents, antipsoriatic agents and antimalarial agents; (vi) A method for the therapy of a mammal having a malignant tumour comprising administration of a compound of formula (I).

DESCRIPTION OF EXAMPLES OF PREFERRED EMBODIMENTS

The following examples and descriptions of stages in synthetic routes of preparation of preferred compounds of formula (I) illustrate the invention but should not be construed in any way as a limitation thereof.

EXAMPLE 1 2,4-Diamino-5-(3-azido-4-chlorophenyl)-6-ethylpyrimidine - Compound (la)

Stages of Synthetic route of preparation

(i) 2,4-Diamino-5-(3-nitro-4-chlorophenyl)-6-ethylρyrimidine

To a stirred mixture of concentrated sulphuric acid (15 ml) and nitric acid (15 ml: D1.42) maintained below 15°C was added 2,4diamino-5-(4-chlorophenyl)-6-ethylpyrimidine ('pyrimethamine') (5g) over 1 hour. The mixture was heated to 50°C for 2 hours, cooled, poured onto ice (approx. 200g) and basified by the slow addition of concentrated aqueous ammonia. The cream nitropyrimidine (4.1g) was collected and crystallised from aqueous ethanol to afford bright yellow rosettes, m.p. 203-205°C; ν _max 1340, 1560 (NO₂), 1630, 3120, 3300 an add 3450 (NH ) cm^-1; δ 1.00(3H,t,CH₃), 2.15(2H,q,CH₂), 5.93(2H,s,NH₂), 6.03(2H,s,NH₂), 7.51(lH,dd,6'-H), 7.81(2H,s,2'-H and 5'-H).

Found C, 49.3; H, 4.0; N, 23.6%

C₁₂H₁₂C11N₅O₂ requires C, 49.1; H, 4.1; N, 23.85%

This compound has been referred to twice in the literature but not characterised (J. Medicinal Chem. , 1969, 12, 662 and J. Chromatography, 1975, 106, 41).

(ii) 2, 4-Diamino-5-(3-amino-4-chlorophenyl)-6-ethylpyrimidine

2,4-diamino-5-(3-nitro-4-chlorophenyl)-6-ethylpyrimidine ( 16g) was added in small portions with stirring over 15 minutes to a solution of tin (II) chloride dihydrate (38g) in 10N-hydro- chloric acid (160ml) that was maintained at 5-10°C. The mixture was stirred thoroughly for 1.5 hours (using a mortar) and allowed to stand overnight. The white stannicomplex that was produced was collected, dissolved in hot water and basified to pH 14 with 10N sodium hydroxide. The resulting white precipitate was collected and crystallised from 100% ethanol to yield small amber prisms of the anhydrous base (9.5g), m.p 215-217°C; ν max 1622 br, 3180 br, 3320 and 3480 (NH₂) cm^-1; δ 1.00(3H,t,CH₃), 2.18(2H,t,CH₂), 5.35(2H,s,3'NH₂), 5.69(2H,s,NH₂), 5.90(2H,s,NH₂), 6.65(lH,d, benzene 2'-H),

6.38 (1H,q, benzene 6'-H, J₂₆1.7Hz, J₆₅8Hz) 7.25(1H,d, benzene 5'-H).

Found: C, 54.4; H, 5.1; N, 26.4%

C₁₂H₁₄N₅Cl requires C, 54.6; H, 5.3; N, 26.7%

This compound has been referred to (J. Medicinal Chem. 1969, 12, 662) but not characterised.

A sample of the crude white precipitate was also crystallised from 50% aqueous ethanol to afford colourless needles of the amine monohydrate, m.p. 215-217°C, m/e 265(31%), 263(96%; M⁺) , 247(100%);

1602, 3180 br, 3330, 3480 (NH₉) cm^-1. ν max

Found C, 50.0; H, 5.4; N, 24.9% C₁₂H₁₄N₅C1,1¼H₂O requires C, 50.4; H, 5.8; N, 24.5%

(iii) 2, 4-Diamino-5-( 3-azido-4-chlorophenyl)-6-ethy Ipyrimidine

A fine suspension of the 3-amino compound from (i) above (1.84g) in 5N-hydrochloride acid (60ml) was diazotised at 0°C by the addition of sodium nitrite (0.6g) dissolved in water (2ml), with stirring, over 15 min. When the resulting suspension had been stirred for 1 hour, sodium azide (1.8g) was added in small portions over 2 hours, the agitation being maintained.

Following basification to pH 9 with concentrated aqueous ammonia, and stirring for 0.5 hours, the solid product was collected (1.82g; 90%) and crystallised from ethanol to yield photosensitive cream microprisms of the azide base, m.p. 185-188°C(eff.); m/e 291(15%), 289(41%;M⁺),

263(21%), 262(39%), 226(66%), 65(100%); ν 1450, 1564 br, 1639 br, 2150 (N₃), 3140 br, max

3300 and 3460 (NH.) cm ; δ 1.00(3H,t,CH₃), 2.18(2H,q,CH₂), 5.84(2H,s,NH₂), 5.96(2H,s,NH₂), 7.00(lH,q, benzene 6'-H, J₆₂2Hz, J₆₅8.4Hz), 7.19(lH,d, benzene 2'-H), 7.55( lH,d, benzene 5'-H).

Found: C, 49.9; H, 4.2; N, 33.3%

C_l2H₁₂N₇Cl requires C, 49.7; H, 4.2; N, 33.8% EXAMPLE 2 2, 4-Diamino-5-(3-azido-4-chloroρhenyl)-6-ethyIpyrimidine (la) salts

(I) The azide base (from Example 1) (1.0g) was dissolved in a minimal quantity of acetic acid (about 8ml). Following the addition of 10M-hydrochloric acid (2 ml) and standing for 48 hours ac 4°C in the dark, white microprisms of the azide monohydrochloride (0.5g), m.p.220-225°C, were deposited;

1542, 1575, 1600, 1642, 1660 infl., ν max

2180 (N₃) cm ^-1 δ 1.08(3H,t,CH₃), 2.30(q,CH₂), 7.13(1H,q,6'-H), 7.39(1H,d,2'-H), 7.48(exchangeable,s), 7.68(1H,d,5'-H).

Found C, 44.3; H, 4.0; N, 30.3%

C₁₂H₁₂N₇Cl,HCl requires C, 44.2; H, 4.0; N, 30.0%

(ii) Upon crystallisation in the dark of the azide base from acetic acid (8 days at 0°C) the azide diacetate was formed as colourless precipitate. This was dried at 100°C/750 torr for 3 hours to give a white powder, m.p. 190-195°C; ν 1320, 1542 infl., 1575, 1645, 1660 br, max

2180 (N₃); no band 1700-1720 cm^-1 (therefore acetic acid of crystallisation absent); & 1.02(3H,t,CH₃), 1.93(1.5H,s,CH₃COO), 2.20(q,CH₂),

6.21(2H,br s,NH₂), 6.50(2H,br s,NH₂), 7.15 (H,q, benzene 6'-H), 7.22(lH,d,2'-H), 7.58 (1H,5'-H), 9.40 (br s,⁺NH).

Found: C, 46.5; H, 4.6% C₁₂H₁₄N₇Cl,2C₂H₃O₂ requires C, 46.9; H, 4.9%

(iii) The azide free base was treated with ethanesulphonic acid (1.1 mol. equiv.) to afford the azide ethanes ulp ho nate as a white precipitate. The azide ethanes ulpho nate was crystallised from water to give white crystals, m.p. 190-191°C (decomp); ν 2130 (N max ₃) cm^-1;

Found: C, 41.90; H, 4.3; N, 24.1%

C₁₂H₁₂C 1lN₇.C₂H₆SO₃ requires C, 42.05; H, 4.5; N, 24.5%

(iv) The azide free base (2.0g) was treated with raethanesulphonic acid (1.1 mol. equiv.) to afford the azide methanesulphonate salt (cream solid). The salt crystallised from water to give cream prisms, m.p. 201-202°C (decomp.) (0.9 g); v 2150 (N cm^-1; max ₃)

Found: C, 40.1; H, 4.3; N, 25.2% C₁₂H₁₂ClN₇.CH₃SO₃H requires C, 40.2; H, 4.15; N, 25.4%

EXAMPLE 3 2, 4-Diamino-5-(3-azido-4-chloroρhenyl)-6-methy Ipyrimidine - Compound ( lb)

(i) 2, 4- Diamino-5-(3-amino-4-chlorophenyl)-6-me thy Ipyrimidine

To a suspension of 2,4-diamino-5-(3-nitro-4-chlorophenyl)-6- methylpyrimidine (10.0g) in ethanol (100ml) was added Raney nickel (10.0g) and the mixture was stirred at 60-65°C. Hydrazine hydrate (33 ml) was added dropwise over an hour with vigorous effervescence. The solution was filtered hot, through celite, and the ethanol was removed by vacuum evaporation. The cream solid (9.7g) crystallised from aqueous ethanol to afford microcrystals, m.p. 242-244°C.

(ii) 2 ,4-Diamino-5-(3-azido-4-chlorophenyl)-6-methy Ipyrimidine

A solution of the 2,4-diamino-5-(3-amino-4-chlorophenyl)-6methy Ipyrimidine (prepared above) (8.0g) in 5M-hydrochloric acid (130 ml) was stirred at 0°C and sodium nitrite (2.4g) dissolved in water (10 ml) was added over 30 minutes. Sodium azide (5.4g) was added in portions over one hour. After stirring for a further one hour concentrated aqueous ammonia was added (to pH 9.0) and the cream solid (95% yield) was collected.

The ethanesulphonate salt was prepared as follows: the azide (3.0g) was suspended in water (20ml) and ethanesulphonic acid (1.2g) was added. The mixture was heated to boiling and the solid dissolved. On cooling the salt crystallised out and was collected; it was then recrystallised from water to afford cream microcrystals (2.2g), m.p. 202-203°C (with decomposition); v 3350, 3175, 2900 (NH,) and max

2125 (N₃) cm^-1; δ 1.14(3H,t,CH₃), 2.02(3H,s,CH₃), 2.58(2H,q,CH₂), 3.38(1H,s,NH), 7.06, 7.82, 8.18(4H, NH₂), 7.17(1H,dd,6'-H), 7.45(1H,d,2'-H), 7.70(1H,d,5'-H).

Found: C, 40.0; H, 4.3; N, 25.2% C _{1 1}H₁₀CIN₇.C₂H₆SO₃ requires C, 40.2 ; H, 4.15 ; N, 25 .4%

EXAMPLE 4 2 ,4-Diamino-5-(4-azido-3-chlorophenyl)-6-ethyIpyrimidine

- Compound ( le)

Synthetic route

( i) 2 ,4-Diamino-5-(3-chlorophenyl)-6-ethyIpyrimidine

α -Propionyl-3-chlorophenylacetonitrile ( 10g) was dissolved in ether (200ml) and diazomethane (4g) in ether (500ml) was added. After standing for 12 hours excess diazomethane was destroyed with acet ic acid. Removal of ether left an oil which was dissolved in ethanol (50ml) and a solution of guanidine (prepared from guanidine hydrochloride , 5 .8g) in ethanol (50ml) was added. The solution was refluxed for 4 hours and the chloropyrimidine (8.1g) was collected from the cooled solution, m.p. 211-213°C.

(ii) 2, 4-Diamino-5-(3-chloro-4-nitroplιenyl)-6-ethy Ipyrimidine

2, 4-diamino-5-(3-chlorophenyl)-6-ethy Ipyrimidine (19.0g) was added over 1 hour to a stirred mixture of nitric acid (60 ml, D 1.42) and sulphuric acid (60 ml) at 25°C. The yellow solution was stirred for a further 4 hours at 30°C and then quenched with water and basified with ammonia. The yellow nit ropy rimidine (23.0g) had a m.p. over 300°C (decomp).

Found: C, 49.3; H, 4.0; N, 23.6%

C.₂H₁₂ClN₅O₂ requires C, 49.1; H, 4.1; N, 23.85%

(iii) 2, 4-Diamino-5-(4-azido-3-chloroρhenyl)-6-ethy Ipyrimidine

2 ,4-diamino-5-(3-chloro-4-nitrophenyl)-6-ethylpyrimidine (16g) was reduced with a solution of tin (II) chloride dihydrate (38g) in ION-hydrochloric acid (160 ml) that was maintained at 5-10°C. The mixture was stirred overnight and the white stannicomplex collected. The stannicomplex was basified to pH 14 with ION- sodium hydroxide and the white residue of 2,4-diamino-5-(4- amino-3-chlorophenyl)-6-ethylpy rimidine was collected and washed with water. The crude aminopyrimidine (1.84g) was converted to 2, 4-diamino-5-(4-azido-3-chlorophenyl)-6-ethy Ipyrimidine by dissolving in 5N-hydrochloric acid (60 ml) at 0°C and treating the mixture, successively with sodium nitrite (0.6g) followed by sodium azide (1.8g) in the manner described for the synthesis of compound (la). The ethanesulphonate salt was prepared from the azide base and ethanesulphonic acid and crystallised from water as cream crystals, m.p. 196-197°C (decomp.) Found : C, 42.2; H, -4.4; N, 24.2%

C.₂H₁₂ClN₇.C₂H₆SO₃ requires C, 42.05; H, 4.5; N, 24.5%

EXAMPLE 5 2, 4-Diamino-5-(4-azido-3-chlorophenyl)-6-methyIpyrimidine

- Compound (If)

This azidopyrimidine was prepared from 2,4-diamino-5-(4-amino-3chlorophenyl)-6-methylpyrimidine (m.p. 242-244ºC) by the diazotisation and azidation procedure described in detail for the synthesis of compound (la). The ethanesulphonate salt was prepared from the azide base and ethanesulphonic acid in the manner described previously and crystallised from water as cream crystals, m.p. 202-203°C; ν _max 2140 (N₃) cm^-1.

Found; C, 40.3; H, 4.4; N, 25.2% C₁₁H₁₀CLN₇.C₂H₆SO₃ requires C, 40.2; H, 4.15; N, 25.4%

EXAMPLE 6 2, 4-Diamino-5-(3-azido-4-methoxyphenyl)-6-ethyIpyrimidine

- Compound (Ic)

(I) 2 ,4-Diamino-5-(4-methoxy-3-nitrophenyl)-6-ethyIpyrimidine

To a solution of sodium metal (1.0g) in dry methanol (50 ml) was added 2,4-diamino-5-(4-chloro-3-nitrophenyl)-6-ethylpyrimidine (2.0g). The mixture was boiled (18 hours), cooled, and concentrated to 25 mis. Addition of water (50 ml) precipitated a yellow solid (1.9g) which crystallised from dimethylformamide as yellow crystals, m.p. 277-278°C (decomp.); & 0.90(3H,t,CH₃), 2.10(2H,q,CH₂), 3.75(3H,s,CH₃), 7.1(1H,d,H'-2), 7.2(1H,dd,H'-6), 7.7(1H,d,H'-5).

Found C, 53.8; H, 4.9; N, 24.1%

C₁₃H₁₅N₅O₃ requires C, 54.0; H, 5.2; N, 24.22% (ii) 2,4-Diamino-5-(3-azido-4-methoxyphenyl)-6-ethyIpyrimidine

Reduction of 2,4-diamino-5-(4-methoxy-3-nitrophenyl)-6-ethylpyrimidine (10g) in ethanol (150mls) at 65°C with hydrazine hydrate (50 mLs) and Raney nickel catalyst gave a grey precipitate of crude 2 ,4-diamino-5-(3-amino-4-methoxyphenyl)-6ethyIpyrimidine (6.9g) when the catalyst was removed and the filtered solution was evaporated to dryness. The crude aminopyrimidine was used without further purification.

Synthesis of the azidopyrimidine was accomplished by diazotisation and azidation of 2,4-diamino-5-(3-amino-4-methoxyρhenyl)-6-ethyIpyrimidine in the manner described in detail for the synthesis of compound (la). The azide base was converted to the ethanesulphonate salt by treatment with ethanesulphonic acid in the manner already described and the salt was crystallised from water to furnish cream crystals, m.p. 272-274°C (decomp.); ν _max 2130 (N₃) cm^-1;

Found C , 45 .8; H , 5.1 ; N, 24.6% C_{1 3}H₁₅N₇O .C₂H₆SO₃ requires C, 45.6 ; H, 5 .3 ; N, 24 .8%

EXAMPLE 7 2 , 4-Diamino-5-( 3-azido-4-ethoxyρhenyl)-6-ethylpyrimidine

- Compound ( Id)

This azidopyrimidine was likewise prepared from 2 ,4-diamino-5(3-amino-4-ethoxyphenyl)-6-ethylpyrimidine (m.p. 176-177 °C) by t reatment with sodium nitrite and sodium azide using the procedure used successfully for the synthesis of compound ( la ) . The azide base was converted to the ethanesulphonate salt by treatment with ethanesulphonic acid in the manner previous ly described . The cream salt crystallised from water as microcrystals , m.p. 173-174 °C ( decomp .) ; ν 2140 (N₃) cm^-1 m_ax Found C, 47.1; H, 5.8; N, 23.8%

C₁₄H₁₇N₇O.C₂H₆SO₃ requires C, 46.95; H, 5.6; N, 24.0%

EXAMPLE 8 Activity of compounds of formula ( I) against dihydrofolate reductase

The inhibitory activity of 2 ,4-diamino-5-(3-azido-4-chlorophenyl)-6-ethylpyrimidine (compound la) against mouse lymphoma L1210 dihydrofolate reductase at various concentrations was determined and compared to that of pyrimethamine. The results are shown in Table I.

Compounds (Ia-If) were tested against dihydrofolate reductase obtained from rat. All the compounds tested had IC ₅₀ concentrations , i.e. the concentrations required to inhibit the euzyme by 50%, of less than 10 ^-6 molar.

EXAMPLE 9 In vivo Antitumour Activity of Compound (la)

Methods for evaluating the antitumour activity of this compound are essentially those used In the Tumor Panel by the Developmental Therapeutics Program, Division of Cancer Treatment, National Cancer Institute, A. Goldin, et al., Methods in Cancer Research, Vol. XVI, p.165, Academic Press (1979). (i) Lymphocytic Leukemia P388

CD2-F mice, of the same sex, weighing within a 3 gram range surrounding 20g, are used for this test. Control and test animals are injected intraperitoneally with a suspension of 10⁶ viable P388/0 tumour cells on day 0. In each test several dose levels which bracket the LD ₂₀ for the compound are evaluated; each dose level group contains 6 animals. The test compounds are prepared either in physiologic saline containing 0.05% Tween 80 or distilled water containing 5% dextrose and are administered intraperitoneally on days 1 to 9 relative to tumour implant. Doses are on a mg/kg basis according to individual animals' body weights. The day of death for each animal is recorded, the median identified for each group and the ratios of median survival time for treated (T)/control (C) groups are calculated. The criterion for activity is T/C x 100 120%. The results are summarised in Table II balow. No toxicity was observed with 9 daily doses of up to 200 mg/kg and increases in survival time of up to 57% were observed.

(ii) TLX5 Lymphoma

Five female CBA/LAC mice received an inoculum (s.c.) of 2 x

10 cells and were then treated with 5 daily i.p. injections of compound (la) in 10% DMSO/arachis oil starting on day 2. Survival time was determined and compared with controls receiving solvent only.

The results obtained are shown in Table (III) .

(iii) W3129 Myeloma

Groups of 5 female BALB/C mice were inoculated s.c. with 5 x

10⁵ W3129 myeloma cells. A single dose of 100mg/kg of compound (la) in 25% DMSO/arachis oil was administered i.p. on day 2 and mean tumour diameter was determined on days 8, 10, 11 and 14 and compared to untreated controls. The results are shown in Table (IV).

a significantly untreated p 0.005 b significantly untreated p 0.01 d tumours having diameters 13mm were not accurately measurable

Claims

1. A compound comprising an azido-substituted pyrimidine derivative, or an acid addition salt thereof, having the general formula (I):

wherein the or each R¹ is selected from hydrogen, halogen or alkoxy having a carbon chain of 1-4 carbon atoms, while R² is hydrogen or an alkyl group of 1-4 carbon atoms.

2. A compound as claimed in Claim 1 wherein the azido group is in the 3'-position of the phenyl group, a said radical R¹ consisting of a chlorine, bromine or iodine atom or an alkoxy group of 1-4 carbon atoms is in the 4'-position of the phenyl group, and the said radical R² is an alkyl group of 1-4 carbon atoms.

3. A compound as claimed in Claim 2 wherein the formula is one of the following (la - Id):

H

or an acid addition salt thereof.

4. A compound as claimed in Claim 1 wherein the azido group is in the 4'-position of the phenyl group, a said radical R¹ consisting of a chlorine, bromine or iodine atom or an alkoxy group of 1-4 carbon atoms is in the 3'-position of the phenyl group, and the said radical R² is an alkyl group of 1-4 carbon atoms.

5. A compound as claimed in Claim 4 wherein the formula is one of the following (Ie - If):

or an acid addition salt thereof.

6. A compound as claimed in any one of Claims 1 to 5 further characterised in that It is an acid addition salt derived from an acid selected from the group comprising: hydrochloric, hydrobromic, sulphuric, nitric, isethionic, phosphoric, maleic, salicylic, ptoluenesulphonic, tartaric, citric, lactobionic, formic, malonlc, pantothenic, succinic, naphthalene-2-sulphonic, benzenesulphonic, methanesulphonic and ethanesulphonic.

7 . A process for preparing a compound as claimed in any one of

Claims 1 to 6 wherein the azido group is in the 3 ' - or 4 '-position of the phenyl group, characterised in that it includes the step of diazotisation of the corresponding amino corαpound of formula ( II) :

wherein R¹ and R² are as hereinbefore defined in Claim 1, followed by the step of treatment with azide.

8. A process as claimed in Claim 7 wherein the compound of formula II is first prepared by reduction of the corresponding nitro compound.

9. A process for preparing a compound as claimed in any one of Claims 1 to 6 wherein the azido group is in the 3'- or 4'-position of the phenyl group, characterised in that it includes the step of reacting with guanidine a compound of formula (III):

wherein R¹ and R² are as hereinbefore defined in Claim 1 , and Y is a residue or group selected from alkoxy, aralkoxy, alkylthio, aralkylthio, dialkylamino, anilino and morpholino.

10. A compound as claimed in any one of Claims 1 to 6 for therapeutic use as an antitumour, antipsoviatic or antimalarial agent in treating mammals.

11. A compound as claimed in Claim 10 in the form of a pharmacologically and pharmaceutically acceptable acid addition salt.

12. A compound as claimed in Claim 10 or 11 in unit dosage form made up for administration to the mammal.

13. A compound as claimed in Claim 10 or 11 in combination with a pharmaceutically acceptable carrier or vehicle.

14. A pharmaceutical formulation for medical use comprising, as the active compound, a compound as claimed in any one of Claims 1 to 6 in the form of the free base or a pharmaceutically acceptable acid addition salt together with a pharmaceutically acceptable carrier therefor.

15. A method of preparing a pharmaceutical formulation as specif ied in Claim 14, said method comprising the step of bringing the said act ive compound into associat ion with said carrier which const itutes one or more accessory ingredients.

16. An azido-substituted pyrimidine derivat ive having the general formula ( I) :

wherein R¹ is a chlorine, bromine or iodine atom or an alkoxy group of 1-4 carbon atoms in the 4'- or 3 '-position of the phenyl group, the azido group (N₃) is in the 3'- or 4'-position of the phenyl group, and R² is an alkyl group of 1-4 carbon atoms, provided in the form of the free base or an acid addition salt thereof, for use as an active therapeutic substance.

17. A medical preparation containing an azido-substituted pyrimidine derivative having the general formula (I) as claimed in Claim 16, or an acid addition salt thereof, and a pharmaceutical excipient.

18. 2,4-Diamino-5-(3-azido-4-chlorophenyl)-6-ethyIpyrimidine, or an acid addition salt thereof, for use as an active therapeutic substance.

19. 2,4-Diamino-5-(3-azido-4-chlorophenyl)-6-methyIpyrimidine, or an acid addition salt thereof, for use as an active therapeutic substance.

20. 2,4-Diamino-5-(3-azido-4-methoxyphenyl)-6-ethyIpyrimidine, or an acid addition salt thereof, for use as an active therapeutic substance.

21. 2,4-Diamino-5-(3-azido-4-ethoxyphenyl)-6-ethyIpyrimidine, or an acid addition salt thereof, for use as an active therapeutic substance.

22. 2,4-Diamino-5-(4-azido-3-chlorophenyl)-6-ethyIpyrimidine, or an acid addition salt thereof, for use as an active therapeutic substance.

23. 2,4-Diamino-5-(4-azido-3-chlorophenyl)-6-methyIpyrimidine, or an acid addition salt thereof, for use as an active therapeutic substance.

24. A medical preparation containing an azido-substituted 2 , 4diamino-5-pheny Ipyrimidine as claimed in any one of Claims 18 to 23 , or an acid addition salt thereof , and a pharmaceutical excipient .

25. Use in antitumour, antipsoriatic or antimalarial therapy of the free base form, or an acid addition salt , of an azido-substituted pyrimidine derivative having the general formula ( I) :

wherein R ¹ is a chlorine, bromine or iodine atom or an alkoxy group of 1-4 carbon atoms in the 4 '- or 3 '-posit ion of the phenyl group, the azido group (N₃) is in the 3 '- or 4 '-position of the phenyl group, and R² is an alkyl group of 1-4 carbon atoms.