NZ209226A - Dimethylphosphoramidates - Google Patents

Description

New Zealand Paient Spedficaiion for Paient Number £09226 lender the provisions of Regii^ Iation 23 (I) the —,-f Specification has been ante-date^ to I9< 10922 ■"*wr *..lni«alt NEW ZEALAND PATENTS ACT, 1953 Divided frcm No. 198115 No.: Date: COMPLETE SPECIFICATION COMPOUNDS HAVING ANTITUMOUR ACTIVITY 3P?We, DEVELOPMENT FINANCE CORPORATION OF NEW ZEALAND, a body corporate established by the Development Finance Corporation Act 1964 and reconstituted by the Development Finance Corporation Act 1973, of Development Finance Centre, Corner Featherston and Grey Streets, Wellington 1, New Zealand, hereby declare the invention for which J.YJ we pray that a patent may be granted to rxue/us, and the method by which it is to be performed, to be particularly described in and by the following statement: - 209226 The present invention relates to intermediate compounds in the preparation of novel compounds having antibacterial and antitumour activity. More particularly, the present invention relates to novel diraethylphosphoramidates.

In earlier work with the 9-anilinoacridines the marked antitumour effect of the 1' -methanesulphonamide derivative (AMSA, compound 17_, Table II herein) was 10 revealed (G.J. Atwell, B.F. Cain and r.n. Seelye, J.Med. Chem. 15, 611-615 (1972) . A search for more dose-potent congeners culminated in the development of the clinical agent 4 ' - (9-acridinylamino) me thanesulphon-m-anisidide (m-AMSA, amsacrine, compound 18_ in Table II herein; B.F. 15 Cain and G.J. Atwell, Europ.J.Cancer 10, 539-549 (1974)) . The antitumour activity of a large range of AMSA and m-AMSA analogues containing variously substituted acridine nuclei has now been investigated, see for example B.F.

Cain, G.J. Atwell and W.A. Denny, J .Med .Chem. IB, 1110-20 1117 (1975) and B.F. Cain and G.J. Atwell, J.Med.Chem. 19, 1124-1129 and 1409-1415 (1976) . During this work a number of derivatives containing different oxygen-containing substituents were evaluated at the 1'-position (e.g.

NHCOCH^ r NHCOOCH^, COOH) but these compounds were less 25 active and/or less dose potent than those bearing the methanesulphonamide (B.C. Baguley, W.A. Denny, G.J. Atwell and B.F. Cain, J.Med.Chem. 24, 170-177 (1931). 2092 26 We have now unexpectedly found that, of a number of phosphorus derivatives examined, a 1'-dimethylphosphor-amidate group provides 9-anilinoacridine compounds of high potency and activity in in vivo antitumour tests.

The compounds are also bacteriostatic, and show toxicity towards mouse, hamster and human tumour cell lines in culture.

The novel class of compounds described and claimed in New Zealand Patent Specification No. 193115 is represented by the general formula (I): NH2, NHC0CH3 or NHC00CH3, and and R^ individually represent H, CH^, OCH^ or CONHCH3, and the acid addition salts thereof.

. - A preferred class; of compounds of the above formula (I) is that in which R^ represents H or OCH^, R^ represents H, CH^ or halogen, R^ represents H, CH^ or OCH^ and R^ represents H, CH^ or CONHCH^. ?~^77 6 In another class of compounds of the above formula (I), represents H or OCH^, R^ represents H, OCH^, CH^, Cl, Br, I, NHCOCH^ or NHCOOCH^, represents H, CH^ or OCH^ and R^ represents H.

The compounds of formula (I) form pharmaceutically acceptable salts with both organic and inorganic acids. Examples of suitable acids for salt formation are hydrochloric, hydrobromic, sulfuric, phosphoric, acetic, citric, oxalic, malonic, salicyclic, lactic, malic, fumaric, succinic, ascorbic, maleic, methanesulfonic, D-gluconic, 2-hydroxyethanesulphonic (i.e. isethionic) , and the like acids.

The formation of the organophosphorus anilino-acridines of formula (I) involves mild acid-catalysed coupling of an appropriate 9-substituted acridine derivative with either dimethyl N- (4-aminophenyl)phosphor-amidate or dimethyl N-(4-amino-3-methoxyphenyl)phosphor-amidate in anhydrous solvents.

Accordingly, the compounds of formula (I) , and acid addition salts thereof, are prepared by a process which comprises the coupling of a substituted acridine of the general formula (II): li_ 209226 wherein R , R^ and R^ are as defined above, and Z represents any suitable leaving group (e.g. methoxy, phenoxy, alkylthio or halogen but preferably chlorine),with a dimethylphosphor-amidate of the general formula (III): wherein is defined as above, and in an anhydrous solvent and in the presence of an acid, and, if desired, converting an acid addition salt of compound of formula (I) into a free base compound of formula (I) and/or ccxiverting a compound of formula (I) into an acid addition salt thereof.

The acid-catalysed coupling reaction of compound (II) with compound (III) is performed in an anhydrous solvent, for example methanol, ethanol, 2-ethoxyethanol or N-methyl-pyrrolidone, with methanol being the preferred solvent. The reaction is preferably performed at temperatures between 30°C and 100°C.

The acid addition salts are prepared by contacting the free base form with an equivalent amount of the desired acid in the conventional manner. The free base forms may be regenerated by treating the salt form with a base. For example, dilute aqueous base solutions may be utilized.

Dilute aqueous potassium carbonate, ammonia, and sodium bicarbonate solutions are suitable for'this purpose. The free base forms differ from-their respective salt forms somewhat in certain physical properties such as solubility in polar solvents, but the salts are otherwise equivalent R m o 209226 to their respective free base forms for purposes of the invention.

The intermediate compounds of formula (III) wherein represents H or OCH^, i.e. dimethyl N-(4-aminophenyl)-phosphoramidate and dimethyl N-(4-amino-3-methoxyphenyl)-phosphoramidate, are novel compounds and constitute the present invention.

The dimethylphosphoramidates of formula (III) may be prepared by the method of Scheme I in which Rn represents H and X represents -NO2 or PhC^OCONH- or R^ represents OCH^ 2 and X represents PhCH2OCONH-,and this general process also forms part of the present invention.

R» x NH^ *-<(5)- Cj I? ± 7 O ^ ' <> \H " Hi.

Scheme I For the preparation of dimethyl N-(4-aminophenyl) phosphoramidate (III; R^=H) , p-nitroaniline or benzyl N-(4-aminophenyl) carbamate is treated with PCCl^ to provide the dichloride (V; X=NO^ or PhCH^CCCNH,R-^=H) (A. Michaelis, Annalen 326 223 (1903)). Treatment with sodium methoxide followed by reduction (H^/Pd/C) yields 209226 the desired product, which can be isolated or used directly for coupling with a 9-. substituted acridine.

Dimethyl N-(4-amino-3-methoxyphenyl)phosphoramidate (III; R^OCH^) is elaborated from commercially available 5 2-methoxy-4-nitroaniline. Reaction with benzyl chloro- formate followed by reduction of the nitro group (Fe, HC1) gives benzyl N- (4-amino-2-methoxyphenyl) carbamate (IV; X=PhCH20C0NH, R^=0CH3) . Treatment with POCl^ followed by sodium methoxide yields (VI; X=PhCH20C0NH, R^OCH^). 10 Hydrogenolysis (H^/Pd/C) then provides the desired compound (III; R^=0CH^) which may be isolated or used directly for coupling with a 9-substituted acridine.

An alternative method for the preparation of dimethyl N- (4-amino-3-methoxyphenyl) phosphoramidate (III; R^OCH^) is 15 by direct reaction of benzyl N-(4-amino-2-rnethoxyphenyl) carbamate (IV; X=PhCH2OCONH, R^OCf^) with dimethyl phosphorobromidate or dimethylphosphorochloridate to give (VI; X=PhCH2OCONH, R^OCH^), which is treated as above-to provide the desired compound (III; R^OCH^) . 20 The 9-chloroacridines of formula (II; Z=C1) may be prepared using published methods (e.g. B.F. Cain, G.J.

Atwell and W.A. Denny, J.Med.Chem. 18, 1110-1117 (1975) to produce the appropriately substituted 9-chloroacridine derivatives. The following novel procedure (Scheme II) 25 in which Y represents halogen can be used for obtaining derivatives of general formula (II) where Z represents CI, R2 represents halogen, R^ represents H and R^ represents CONHCH3: 7 097- 2 Y CcoK YR vm CooV( IV \ "7 CONHch i JX cook Scheme II Reaction of a 2-chloro-4-halobenzoic acid with anthra-nilic acid in the presence of copper and an acid acceptor (preferably potassium carbonate) gives an essentially quantitative yield of the diacid (VII). Ring closure with mineral acid reagents gives an approximately 1:2 mixture of the carboxyacridanones (VIII) and (IX) , which can be conveniently separated by fractional crystallization of the potassium salts from aqueous EtOH. After removal of the less soluble l-halo-4-carboxyacridanone potassium salt, the more soluble 3-halo-5-carboxyacridanone potassium salt .is obtained by concentration of the mother liquors.

Crystallization of the liberated acid from DMF (in which the 3-halo-5-carboxyacridanone is the less soluble isomer) affords pure product. Activation-with S0C12 or POCl^ and reaction with aqueous CH^NH2 provides the desired 9-chloroacridine. The intermediate compounds of the general formula (IX) in Reaction Scheme II, are novel compounds.

The 9-bromoacridines of formula (II; Z=Br) can be prepared from either the appropriate diphenylamine-2-carboxylic acids by treatment with phosphoryl bromide, or from the acridone by reaction with thionyl bromide. 9-Phenoxy- and 9-methoxy-acridines can be prepared by the methods given in Albert, "The Acridines" Second Edition, Edward Arnold Ltd., London (1966). 9-Alkylthio heterocycles, and the precursor 9-acridanthiones can be prepared by the methods cited in E.F. Elslager et al, J.Med.Chem. 14, 782-788 (1971).

The coupling of the 9-chloroacridine of formula (II; Z-Cl) with the phosphoramidate of formula (III) may be performed according to the following procedure which is given by way of illustration of the process of the invention.

A methanolic solution of 1.1 equivalents of the phosphoramidate compound (preferably obtained in situ by hydrogenation of the nitro or benzylurethane precursor over palladium on charcoal) is combined with 1 equivalent of the appropriate 9-chloroacridine derivative dissolved or suspended in methanol, and one or two drops of concentrated hydrochloric acid are added to initiate the reaction (as evidenced by the appearance of a deep red colouration) . 209226 At the completion of the coupling reaction (5-10 minutes at room temperature in .the case of 9-chloroacridine and up to 15 minutes at reflux in the case of the much less soluble 4-methyl derivative) the solution is concentrated 5 to a small volume under vacuum, and allowed to stand as crystallization commences. After being diluted with ethyl acetate to ensure complete crystallization, the mixture is filtered and the dark-red hydrochloride salt washed with dry acetone. The product can be purified 10 by recrystallization from methanol-ethyl acetate.

Conversion of the hydrochloride salt to the free base can be achieved by the addition of 1.1 equivalents of aqueous KHCO^ to an aqueous methanolic solution of the salt. Removal of the methanol gives the free base 15 which can be recrystallized from either aqueous methanol or anhydrous solvents such as ethyl acetate or benzene. Alternatively, the free bases can be isolated by a procedure that eliminates the need to firstly isolate them as the hydrochloride salts. After completion of 20 the initial coupling reaction a slight excess of aqueous KHCO^ is added to the methanolic solution, and the solvent is removed under vacuum. After being extracted into ethyl acetate and washed with water, the product is extracted into aqueous methanesulphonic acid thereby 25 leaving any acridone impurities in the organic layer.

After neutralization of the aqueous layer with KHCO^ the product is again extracted into ethyl acetate, and d:: %j' r\ 2 -lithe solution dried over Na^SO^, and the solvent removed under vacuum to give the free base which can be recrystallized as described earlier.

Other acid addition salts (e.g. methanesulphonate) can be formed by treating the free base in methanol with an equivalent of the appropriate acid. Isolation of the product salt is achieved by dilution of the solution with ethyl acetate. 209226 The following Table I sets out physicochemical and biological data for 16 compounds within the general formula (I) and preparable by the above described process. In Table I the following terms and abbreviations are used: Anion = the anionic component of the acid addition salt of the compound of formula (I) . Compounds prepared as the free base form of formula (I) are shown as (base) . These compounds are converted to acid addition salts (normally by addition of the appropriate concentration of isethionic acid) before use in in vivo biological tests.

MW - molecular weight.

MP = melting point in °C-. Decomposition of the sample is shown by the abbreviation dec.

Rrn = a measure of the compound's lipophilic-hydrophilic balance from reversed-phase partition chromatography. Rm is linearly related to partition coefficients obtained in the 1-octanol/water system.

, R2, R^ and R^ refer to formula (I) . 209226 £ Pi 2 2 u 3 CU 2 <T3 M r-1 D <u £ rH u X* O fQ E-* c o •H c < "ST cs n Pi •w OJ (X en 1 o z /—1 C"t VJU t—1 r- r- . rr ro r- ao 00 VO ■^r TT , ! co VO in rH ro rH CN in n j • • • • • • • • • • • • • • • • o o O o O o o o o o o O o O o ° in XT in o O r- a\ TT cn <n cn • • • • • • • • • • • • • • • • a\ ro co TP CD 00 00 a\ ro vo vo r- r- rH r^ CO CN vo cn O ro CO in in ao rH o ro in in TT rr in in TT -*r in "CP rr in rr ^_L O o o o o o a O a o 0 o 0) <D 0 o <D a; d) <D 0) a) a) CD CD *o T3 in T3 T) TJ 'O ti >U "— -—' co *—* "—' -—- «w -w *»•—» "W o in o A in in CN <T> CN 03 in ro in ro in CO ro in o o rH rH ro CN CN 00 o CN i—( CN CN ro CN CN CN rH CN CN CN i—! CN CN 53 ro O rH i—i iH iH rH CO u CJ a O CJ ro i—I ffi EC w cn •—1 i—1 rH X rH »*r« a CJ CJ a a a • • • • : T» 53 • X . . a. 04 eu a. • • • ai • cn ro rr in Pu 0< 04 O o O o CU CU 04 04 04 04 04 O 04 ro CO n ro ro ro in •^r m in n ■^r CO O O o 2 2 2 Z O O O o O O O 2 O m m m r—I rH V~l S-l ro ro rs* ti* ro ro rH ro Z 2 2 u a C3 C3 2 2: 2 2: Z 2: 2 a 2 o CN CN CPl rH o rH cn CN •^r ro in CN •^r CN CN CN '—f CN »-1 CN rH CN CN CN CN CN CN CN CN 53 53 *r+ >—• 5n X 53 !*• 53 *T-» 53 >—• 53 rH CN IN r-H *CN t—I Tn i—1 cn To To rr Tn To ■^r co oj cn cn CN cn cn cn cn cn cn cn cn cn cn cn cn a O CJ U CJ a a O a a CJ a o a U a CO O OJ I 1 I i i <u 1 0} 1 i a) aj I i tf) cn rh i—I r—\ »-H rH cn rH cn rH rH tn cn rH rH ro a3 a a CJ U a K3 a <a a (J <0 to U CJ »-r« XI XI -O £ XI o -s~-' ' ' ro +r+ a ro n 53 hi_t *r* 2 a u o ro ro ro 53 CJ 53 ro cn 53 »!_• z ro ro ! a X r-4 1—1 u i-i O CJ o o o 53 X in 53 53 a O CJ ca cn % o o u CJ O CJ «» T3 i 1 1 i 1 1 rH 1 ro co m en rO ro **T tt •*3* u in 1 ro T CO ro ro ro ro ro ro X tr 53 »t* 53 53 CJ x k a SC U 53 K U t—1 M o a U X O o O o o o O o rH cn ro in vo 1—1 cn ro in vo r- 00 a\ rH rH rH i-H rH rH rH in o in rH - 14 - 9 « % The following Examples A, B and C illustrate the preparation of compounds of the general formula (III) and the preparation therefrom of compounds of the general formula (I).

Example A: Preparation of Compound 1 of Table I (via the method of 5 Scheme I ) .

N-(4-Nitrophenyl)phosphoramidoyl Dichloride (V; X=N02* Rj 75g (0.51 mol) of 4-nitroaniline and 150 ml distilled poci3 were mixed together in a 500 ml flatbottomed flask equipped with a reflux condenser and a calcium chloride 10 drying tube. The mixture was heated to reflux (oil bath) and maintained at that temperature until HC1 evolution ceased (2-3 hours) . The homogeneous solution was allowed cool and the product crystallized out on standing overnight at 5*C. After being filtered off the product was washed 15 with petroleum ether and dried _in vacuo to yield 130 g (9 of the dichloride.

Dimethyl N- ( 4-Ni trophenyl) phosphoramidate (VI; X=NC>2, R^ = 38.1 g (0.15 mol) of the crude N-(4-nitrophenyl)phosphor-amidoyl dichloride was added in portions with 20 stirring to a cooled solution of 11.4 g (0.5 mol, 3.3 equivalents) of sodium in 150 ml dry methanol.

After being stirred for a further 5 minutes at low temperature the mixture was diluted with 750 ml of ice cold water and filtered. Treatment of the filtrate with dilute 25.-. .hydrochloric acid gave a precipitate ;of-the diester which' was collected, washed several times with water, and dried in vacuo. Yield 33.4 g (90.5%) m.p. 163-164°C, after recrystallization from aqueous methanol. 209226 Dimethyl N-(4-aminophenyl)phosphoramidate (III; R^=H) The above nitrocompound (lGg, 0.04 mol) was hydrogenated over Pd/C in EtOH for 15 min. The solution was filtered, concentrated to small volume and the product precipitated with petroleum ether. Yield 8g(93%), m.p. 125°C after crystallization from ethyl acetate.

Alternative preparation of dimethyl N-(4-aminophenyl) phosphoramidate (III; R^=H) Benzyl N-(4-nitrophenyl)carbamate A mixture of 4-nitroaniline (20g,0.15 mol) and MgO (5g) in anhydrous acetone (150ml) was treated with benzyl chloro-formate (30ml) and the resulting slurry was stirred at 20°C for 12h. After this time DMF (25ml) was added, the mixture was heated to dissolve the product and filtered. Crystallization from aqueous acetone gave 34 .4g (87% yield) of the nitro compound, mp.155-156°C.

Benzyl N-(4-aminophenyl) carbamate (IV; X=PhCH20C0NH-, R-,=H) The above product (34g, 125 mmol) was dissolved in a mixture of DMF (100 ml), water (30 ml) and c.HCl (10 ml), and Fe powder (20g) was added to the hot solution at a rate sufficient to maintain gentle reflux.

After a further 30 min at reflux the mixture was treated with c. NH^OH (30 ml), filtered through celite and evaporated to dryness. The residue was extracted with 2N aqueous methanesulfonic acid, and the solution was clarified with charcoal and basified with c. NH^OH to give the amino compound (28g, 92% yield ) mp 102-103°C. - 16 - «- ^ & 2 N- (4-Benzyloxycarbonylaminopheny1) phosphoramidoyl dichloride (V; X=PhnH2OCONH, R =H) A solution of the above amino compound (20g, 83mmol) in CH2C12 (20ml) was added dropwise to a stirred icecold mixture of POCl^ (100ml) and pyridine (25ml). After stirring at 5°C for 2h, the product was precipitated by the addition of icecold petroleum ether (700 ml). The crude product was washed twice with petroleum ether by decantation.

Dimethyl N-(4-benzyloxycarbonylaminopheny1) phosphoramidate (VI; X=PhCH2 OCONH, Rj=H) The above crude product (2 8g)was added slowly with cooling to a solution of sodium (8g, 5 equivalents) in methanol.

After 10 min the solution was neutralized with acetic acid and all solvents removed under vacuum- The residue was extracted with ethyl acetate, washed with dilute acid and base, and the solvents removed under vacuum. Crystallization of the resulting solid from ethyl acetate-petroleum ether gave the desired product (55% yield) mp 135°C.

Hydrogenation of this compound in methanol over Eti/C provided dimethyl N-(4-aminophenyl) phosphoramidate (III; R1=H), m.p. 125°C. 2092^6 Compound 1 of Table 1 A solution of the above amine (l.Og, 4.7mM) in methanol was added to a suspension of 9-chloroacridine (0.90g, 4.2mM) in methanol. A drop of cHCl was added and the mixture was warmed until all solids dissolved. The solution was then concentrated to small volume (20mL) under vacuum, and ethyl acetate was added to complete crystallization of the hydrochloride salt.

EXAMPLE B : Preparation of Compound 14 of Table I (via the method of Scheme I) Benzyl N-(2-methoxy-4-nitrophenyl)carbamate 2-Methoxy-4-nitroaniline, 51g (0.3 mol), was dissolved in 135 ml of acetone containing 12 g of MgO, and 65 ml of commmercial benzylchloroformate was slowly added to the stirred mixture. After 4 hours the flask was warmed to redissolve the precipitate and the mixture was stirred overnight. DMF (10 0 ml) was added, and the mixture was heated to dissolve all of the product before being filtered through celiter- 209226 After dilution with 250 ml of ethanol the hot solution was further diluted with water until almost cloudy, and allowed to cool. The pale yellow crystals were collected, washed well with 50% aqueous ethanol, and dried- Yield 86.0 g, 94%, m.p. 130-131*C.

Benzyl N-(4-amino-2-methoxyphenyl)carbamate (IV; X=PhCH 2OCONH, =OCH3).

The benzyl N-(2-methoxy-4-nitrophenyl) carbamate, 50g (0.18mol), was dissolved in a hot mixture of 200 ml DMF, 100 ml H2O and 15 ml concentrated HCl, and the stirred solution was treated slowly with 75 g Fe powder at a rate so as to maintain gentle reflux. When the reaction was complete (15-30 minutes), concentrated NH3(aq) was added to precipitate Fe salts, the mixture was diluted with 250 ml EtOH and filtered through celite. After removal of the solvent under vacuum the residue was extracted with aqueous methanesulphonic acid and the solution was clarified with charcoal-celite. Neutralization with nh3 (aq) gave the aminocar-bamate which was collected, washed well with water, and dried. Yield 43.8 g, 97%, m.p. 77-78°C (toluene-petroleum ether) .

N— (4-Benzy loxycarbony lam i no- 3-methoxyphenvl) phosphoramidoyl Dichloride (V; X = PhCH2 COCONH, R} = och3) .

A solution of benzyl N-(4-amino-2-methoxyphenyl) carbamate (IV;X=PhCH2OCONH, RL =OCH3). (20g, 73.5 mM) in CH2C12 (25 mL) was added over 5 min to a stirred, icecold, mixture of dry . 2092^6 pyridine (25mL) and poci3 (lOOraL), and the resulting mixture was stirred below 5"C for a further 2h. Precipitation with petroleum ether (500mL) at -10"C for 15h gave crude solid product, which was isolated by decantation and washed 5 with two portions of petroleum ether.

Dimethyl N- (4-benzyloxycarbonylamino-3-methoxyphenyl) phosphoramidate (VI; X=PhCH20CONH, =OCH3) The above crude product (29g) was added slowly to a cooled solution of Na (8.5g, 5 equivalent) in CH3OH (250mL). After 10 5 mins the solution was neutralized with acetic acid, and the solvent removed under vacuum. The residue was extracted into ethyl acetate, washed successively with dilute HC1, water, khco3 solution, and brine, and dried over MgS04-Removal of the solvent gave an oil which was triturated with 15 a small amount of ethyl acetate to give 4.5g (16%) of dimethyl N- (4-benzyloxycarbonylamino—3-methoxyphenyl) phosphoramidate, m.p. 115*C (acetone-petroleum ether). Chromatography of the mother liquors yielded an additional 20% of crystalline compound.

Dimethyl N-(4-amino-3-methoxyphenyl) phosphoramidate (III; R1=OCH3) Hydrogenolysis of the above carbamate (5 g, 13mM) in EtOH over Pd/C for lh gave the aminocompound upon 5 filtration, concentration and precipitation with petroleum ether. Yield 3.Og (95%), m.p. 122-123°C after crystallization from ethyl acetate. 4-Methyl-9-chloroacridine (II; Z=C1, and R^=H, VCH3> A heterogeneous mixture of o-chlorobenzoic acid (3.12 g, 0.02 mol), o-toluidine (3.21 g, 0.03 mol), anhydrous (3.45 g, 0.025 mol), Cu powder (0.05 g), CuCl2 (0.05 g) and 2-ethoxyethanol (10 ml) was stirred and heated under reflux conditions in an oil bath at 105° for 3 15 hours. After cooling the mixture was acidified with excess concentrated HCl and diluted with water. The resulting solid was washed well with water, dissolved in an excess of aqueous Ha2C03 by boiling, stirred with a generous quantity of decolourizing charcoal and then 2o filtered through a Celite pad. Slow addition of dilute aqueous acetic acid to the hot stirred solution first initiated separation of a quantity of black impurity o which was filtered off and addition of excess aqueous HCl then precipitated the product, N-(2-methylphenyl) 25 anthranilic acid. One crystallization from acetone- methanol-water (by concentration of a boiling solution) provided material of acceptable purity (65% yield). 209226 The product may be recrystallized from benzene to provide TLC homogeneous product, m.p. 193-194°C (lit m.p. 190-191°C).

The N-(2-methylpheny1) anthranilic acid (5.0 g) and POCl^ (15 ml) were heated together under reflux conditions for 1 hour and then concentrated in vacuo on the steam bath to remove most of the excess POCl^. The resulting oily residue was cooled, dissolved in chloroform and then poured with stirring into an ice-excess NH^OH mix. The chloroform layer was washed with dilute aqueous NH^OH, water, dried boiling dry ligroine (bp 95-115°) or with a large volume of petroleum ether and the filtered solution was evaporated to dryness providing the product in 82% yield, sufficiently pure for use in the next stage (lit. m.p. 94°C).

Compound 14 of Table I A solution of the dimethyl N-(4-amino-3-methoxy-phenyl) phosphoramidate (1.5g, 6.2mM) in methanol was added to a suspension of 4-methyl-9-chloroacridine (1.5g, 6.06mM) in methanol and the mixture was gently refluxed with 1 drop of cHCl until all the solids had dissolved (about 15 min). The solution was then concentrated to small volume (20mL) and ethyl acetate was added to complete precipitation of the hydrochloride salt which was then converted to the free base. 2 09226 Example C: Preparation of Compound 15 of Table I Alternative Preparation of dimethyl N-(4-amino-3-methoxy-phenyl) phosphoramidate (III; R^OCH^) 5 A solution of 15.5ml Br^ (0.3mol) in 50 ml of petroleum ether was slowly added with stirring to a cooled solution of 30 ml trimethylphosphite (0.3 mol) in 100 ml of petroleum ether. At the completion of the addition (if necessary) trimethylphosphite was added 10 dropwise to decolorize any excess bromine, and the upper layer was decanted off. Dissolved methyl bromide was removed under vacuum at room temperature and the remaining dimethyl phosphorobromidate was used without further purification.

Benzyl N-(4-amino-2-methoxyphenyl) carbamate (IV; X=PhCH2OCONH, R^=OCH3) (10 g, 3.6 mmol), was dissolved in 50 ml of dry pyridine and an excess (1.5-2.0 equivalents) of freshly prepared dimethyl phosphorobromidate was slowly added at 0°C with stirring. The resulting 20 mixture was allowed to warm slowly to room temperature, and stirred overnight before being quenched with water and extracted with ethyl acetate. The organic layer was washed successively with dilute methanesulfonic acid, water, dilute KHCO^ solution, brine, and dried over 25 Na^SO^ and the solvent was removed under vacuum. The residue was chromatographed .on.; silica . (Cfr^Cl^MeOH , 25:1) to give dimethyl N-(4-benzyloxycarbonylamino-3-methoxy-phenyl)phosphoramidate (VI, X-PhCH^OCONH, R^=OCH^) as an oil, which crystallized on trituration with acetone.

Yield 5.68g (41%), mp 115°C (acetone-petroleum ether).

This was converted to the desired dimethyl N-(4-amino-3-methoxyphenyl)phosphoramidate (III; R^OCH^) by 5 the method given in Example B.

Preparation of methyl 3,9-dichloro-5-carboxamidoacridine (X,Y=C1) by the method of Scheme II 2-(2'-carboxy-N-phenyl)-4-chloroanthranilic acid (VII; Y=C1) Anthranilic acid (38g, 0.28M), 2,4-dichlorobenzoic acid (50g, 0.26M) and potassium carbonate (57g, 0^42M) were suspended in 2-ethoxyethanol (200mL) and heated at 50°C until gas evolution ceased- Copper/copper oxide (1:1 mixture, 0.4g) was added, and the mixture stirred 15 at 120°C for 45 min. The cooled mixture was diluted with water, filtered through celite and acidified with 2N HCl. The precipitate was collected and washed well with water to yield the desired diacid, suitable for the next step. Yield 73g, 96%. 3-Chloro-5-carboxyacridanone (IX; Y=C1).

The above crude diacid (50g) was dissolved in cH^SO^ (150mL) and kept at 100°C for 2h. The cooled mixture was poured slowly into hot water, and the mixture was boiled briefly to coagulate the precipitate. The 25 solid was collected and washed well with water to yield the mixed chlorocarboxyacridanones (VIII and IX; Y=C1) 45g, 96%.

This mixture (70g) was suspended in boiling EtOH - > (1200mL) and a hot solution of KOH (7Og) "in'water (1200mL) was added rapidly. All solids dissolved, followed rapidly by precipitation of the potassium salt 5 of l-chloro-4-carboxyacridanone (VIII; Y=C1). The suspension was allowed to cool to 30°c and the solid was collected (62g = 53g of free acid). The filtrate was concentrated to lOOOmL and kept at 20°C for 24h, when the K salt of the 3-chloro-5-carboxyacridanone (IX; Y=C1) 10 precipitated and was collected (12.5g =10.3g of free acid). Crystallization from DMF gave yellow micro-crystals, m.p. 360°C.

Methyl 3,9-dichloro-5-carboxamidoacridine 3-Chloro-5-carboxyacridinone (2.0g, 7-3mM) was 15 suspended in SOCl2 (25mL) and a drop of DMF, and refluxed gently for lh. Volatiles were evaporated under reduced pressure and the residue was azeotroped with dry benzene to remove all S0C12- The residue was dissolved in dry CH2C12 and poured into icecold aqueous CH^NH^ The 20 organic layer was washed with water and saturated NaCl and dried. Evaporation of solvent gave the desired chloroacridine (1.8g, 84%).

Compound 15 of Table I The above methyl 3,9-dichloro-5-carboxamidoacridine 25 (1.5g, 5.ImM) and 5.2mM of the dimethyl N-(4-amino-3- methoxyphenyl) phosphoramidate (III; R^OCH^) were coupled conventionally in methanol to give the red hydrochloride salt of compound 15.

The compounds of general formula (I) , and particularly the compounds"listed in Table I, have antitumour activity in both in vitro and in vivo test systems, as shown by the data of Table II. Many of them are more dose potent than the corresponding methanesulphonamido analogues (Table II). The compounds also show broad-spectrum antibacterial activity. Specifically, compound 1 is active against the bacterial aerobacter aerogenes, alcaligenes viscolactis, eschereichia coli, bacillus subtilis, sarcina lutea, micrococcus lysodeikticus, neisseria catarrhalis, staphylococcus aureus, xanthomonas phaseoli and streptococcus faecalis.

The following Table II shows the activity of compounds of general formula (I) towards L1210 tumour cells in culture and the P38 8 leukaemia in mice.

The P38 8 mouse leukaemia system has been shown by studies at the National Cancer Institute, U.S.A., to be a useful system for detecting agents with antitumour activity against clinical cancer (A. Goldin, J.M. Venditti, J.S. MacDonald, F.M. Muggia, J.E. Henney and V.T. De Vita, Europ.J.Cancer 17, 129-142 (1981).

The following terms and abbreviations are used in Table II:- Tumour P388 i.p. - P388 cells were obtained as frozen stocks from Mason Research Inc., U.S.A., and passaged introperitoneally according to standard methods (Cancer Chemother. Rep. 3_, Part 3, page 9 (1972) in DBA-2 mice of either sex. Groups of six F1 hybrid mice (DBA-2 male x C57 B1 female, g weight 20 i 1 g) were injected intraperitoneally with 10^ cells on day 0. : O.D. = optimal drug dose (in milligrams per kilogram), administered as a solution in 0.1ml of 30% v/v ethyl alcohol in water on days 1, 5 and 9 after tumour inoculation. The drug is administered as a soluble acid addition salt.

ILS = percentage increase in life span of treated animals over that of groups of control animals injected with tumour alone. The average survival of control mice 10 was 11 days. Values of ILS greater than 20% are considered statistically significant.

ID,-q = the nanomolar concentration of drug which when added to cultures of murine L1210 leukaemia cells over a period of 7 0 hours, reduces the resultant counted 15 number of leukaemia cells by 50% (B.C. Baguley and R.

Nash, Europ.J.Cancer 17, 671-679, 1981).

Y. implies a significant value of drug activity at the stated dose.

TABLE II BIOLOGICAL DATA FOR THE COMPOUNDS OF TABLE I No.

L1210 culture data P388 in vivo data ID 50 OD ILS Active 1 33 74 Y 2 70 8.9 67 Y 3 6.8 80 Y 4 16 54 Y 12 13. 3 78 Y 6 45 76 Y 7 4.9 13. 3 83 Y 8 74 8.9 22 Y 9 44 Y 12 8.9 76 Y 11 97 45 38 Y 12 18 118 Y 13 8.9 111 Y 14 18 8.9 106 Y 50 45 79 Y 16 17 45 109 Y 17 AMSA for comparison 150 93 Y 18 m-AMSA for Sompar ison 13. 3 78 Y NJ o ISJ Isi O* 2 0922 6 It is clear from the data of Table II that the dimethyl-phosphoramidates of general formula (I) are active antitumour agents, giving significant levels of life extension when tested against the P388 leukaemia system when given by intraperitoneal injection. The compounds also show antitumour activity when given by oral and intravenous routes. They also show high cytotoxicity towards cultured L1210 leukaemia cells (Table II), and are active in a number of cultured tumour cell lines, including those originating from human colon and breast tumours. 209226 - 29

Claims (5)

WHAT WE CLAIM IS:

1. A compound of the general formula (III): nh 2 NHP II 0 OCH och3 3 iii where represents H or OCH^.

2. Dimethyl N-(4-aminophenyl)-phosphoramidate.

3. Dimethyl N-(4-amino-3-methoxyphenyl)-phosphoramidate.

4. A process for the preparation of a compound of general formula (III) as defined in claim 1, which comprises reducing a compound of general formula (VI):

5. A process for the preparation of a compound of general formula (III) as defined in claim 1, substantially as hereinbefore described, with particular reference to any one of Examples A, B and C. X vi O where represents H or OCH^ and X represents NC>2 or C,HcCH_OCONH-. b d ^ :1r£Bi985 209226 - 30 - A compound represented by the general formula (III) as defined in claim 1, whenever prepared by the process according to either of claims 4 and 5. DATED THIS 1 DAY OF T ( % y A. J. PARK.A SON AGENTS FOR THE APftlCANTS