GB2195636A - Isoquinoline derivatives - Google Patents

Description

g 1 GB2195636A 1

SPECIFICATION

Isoquinoline derivatives This invention relates to new isoquinoline derivatives, to processes for their preparation, to 5 pharmaceutical compositions containing them, and to their use in medicine.

Isoquinoline compounds have been reported in, for example, EP-A-108620 and EP-A-161102 as having anti-cancer activity. We have now found a novel group of isoquinoline compounds which have good anti-cancer activity. The novel compounds of the invention also exhibit particu larly advantageous physico-chemical properties.

The invention thus provides compounds of the general formula (1) H 3 R 1 1 N\ 13 11 1 R 0 R 2 wherein R' and R2, which may be same or different, each represents a hydrogen atom, a hydroxyl group or a group -OP(O)(OH)(OR4) [where R4 represents a hydrogen atom, or an alkyl (optionally substituted by a hydroxyl, alkoxy, cyclic ether or cycloalkyl group), alkeny], cycloalky], aryi, aralkyl or aroylalkyl group], with the proviso that at least one of R' and R2 represents a group -OP(O)(OH)(OR4); and R3 is a hydrogen or halogen atom or a methyl group; and salts, especially 25 physiologically acceptable salts, thereof.

Compounds of formula (1) may exist as stereoisomers and/or tautomers and the invention. is to be understood to include all such isomers of compounds of formula (1), including mixtures thereof.

Compounds of formula (1) may form salts with bases. It will be appreciated that, for pharma- 30 ceutical use, these salts should be physiologically acceptable, but other salts may find use, for - example in the preparation of compounds of formula (1) as well as physiologically acceptable salts thereof. Examples of salts of compounds of general formula (1) include alkali metal salts - such as lithium, sodium and potassium salts, amine salts. for example ammonium and mono-, di- or tri-substituted ammonium, e.g. triethylammonium salts and amino acid salts, for example arginine salts. Sodium salts are particularly useful.

References hereinafter to compounds of formula (1) and their use and preparation should, unless the context dictates otherwise, be taken to be references to the compounds and their salts, eg the physiologically acceptable salts.

In the compounds of formula (1), when the group R4 is an alkyl group it may be for example a 40 straight or branched C,, alkyl group, or more especially a straight or branched Cl-, alkyl group such as a methyl, ethyl, n-propyi, i-propyl, n-butyl or n-pentyl group. Such alkyl groups may be substituted by for example, a hydroxyl, C1-4 alkoxy, eg methoxy or ethoxy, 3 to 7 membered cyclic ether eg tetrahydrofuranyl, or C3-7 cycloalkyl eg cyclohexyl, groups.

When R4 in the compounds of formula (1) represents an alkenyl group it may be for example a 45 C3-8 alkenyl group, eg allyl.

When R4 in the compounds of formula (1) represents a cycloalkyl group it may be for example a C3-7 cycloalkyl group, eg cyclohexyl.

When R4 in compounds of formula (1) represents an aryl group it may be for example a phenyl group optionally substituted by a group R5 where R5 is a hydroxy, C1-4 alkoxy, eg methoxy, or nitro group.

When R4 is an aralkyl group it may be for example a phenCl-3 alkyl group in which the phenyl portion is optionally substituted by a group R5 as just defined, and may be in particular a benzy] group.

When R4 in the compounds of formula (1) represents an aroylalkyl group it may be for example a benzoyl C1-3 alkyl group in which the benzoyl portion is optionally substituted in the phenyl ring by a group R5 as defined above, and may be in particular a benzoyimethyl group.

When the group R3 in the compounds of formula (1) is a halogen atom it may be a fluorine, chlorine, bromine or iodine atom.

According to one embodiment the invention provides compounds according to formula (1) in 60 which; R' and R2, which may be the same or different, each represents a hydrogen atom, a hydroxyl group or a group -OP(O)(OH)(OR4) [where R4 represents a hydrogen atom or an alkyl (optionally substituted by a hydroxyl, alkoxy or cycloalkyl group) aryl or aralkyl group], with the proviso that at least one of R' and R2 represents a group -OP(O)(Offi(OR4), and R3 is a hydrogen or halogen 65 1 2 GB2195636A 2 atom or a methyl group, and salts thereof.

In the compounds of formula (1), R3 is preferably a methyl group or, more particularly, a hydrogen atom.

Particular examples of the group R4 include a hydrogen atom, a straight or branched C,-, alkyl group, a C,-, alkoxyethyl group (such as 2-methoxyethyl), a tetrahydrofuranyimethyl group (eg 2- 5 tetrahydrofuranyimethyi), a C,-, cycloalkylmethyl group (eg cyclohexyImethyl), a C,-, alkenyl group (eg ally[), a C,-, cycloalkyl group (eg cyclohexyl), phenyl, phenyl C,3 alkyl (eg benzyl) in which the phenyl portion is optionally substituted by a nitro (eg p-nitro) group or a benzoyl Cl3 alkyl group (eg benzoyimethyi).

In the compounds of formula (1), R4 iS preferably a straight Cr, alkyl group (eg methyl or n- 10 propyl), a tetrahydrofuranyl methyl group, a phenyl C,-, alkyl group (eg benzyi) or a benzoyl C,-, alkyl group (eg benzoyimethyi).

Compounds of formula (1) in which R' is a hydrogen atom and R 2 is a group -OP(O)(OH)(OR4) (where R4 is as defined above, especially benzyl or benzoyImethyl) and salts thereof are pre- ferred.

A preferred group of compounds of formula (1) are those in which R' is a hydrogen atom, R2 is a group -OP(O)(OH)(OR4) and R3 is a hydrogen atom and salts thereof. Such compounds in which R4 is a straight C,-5 alkyl group (eg methyl or n propyl), a tetrahydrofuranyimethyl group, a phenyl C,-, alkyl group (eg benzyi) or a benzoyl C,,-alkyl group (eg benzoyImethyl) are particu- larly preferred.

A particularly important compound according to the invention is phenyimethyi, [5,8,13,14 tetrahydro-14-methyi-8,13-dioxobenz[5,61-isoindolo[2,1-blisoquinolin-9yllph osphoric acid and the salts thereof, particularly the sodium salt.

Another important compound according to the invention is 2-oxo-2phenylethyl, [5,8,13,14 tetra hydro- 1 4-methy]-8,1 3 -dioxobenz[5,6]-isoindolo[2, 1 - blisoquinolin-9-yi]phosphoric acid and the 25 salts thereof, particularly the sodium salt.

The compounds of formula (1) possess anticancer activity, particularly against tumours such as carcinomas, sarcomas and hepatomas.

Thus, when a compound of formula (1) is administered intra peritonea] ly or intravenously to mice with a subcutaneous tumour arising from an implant of S180 cells, subsequent examination 30 has shown that tumour growth has been significantly reduced and in some cases total regres sion of the tumour has occurred. Activities against HT29 Human Colon Xenograft in Nude Mice and Mouse Adenocarcinoma MAC30T have also been shown.

Compounds according to the invention also have especially favourable physico-chemical proper- ties. Thus, for example, compounds of the invention have improved water solubility at near to physiological pH [e.g. pH 6.0 to pH 8.0] over the isoquinoline compounds described in EP-A 108620 and EP-A-161102 which makes them particularly suitable for formulation for parenteral administration.

According to a further aspect of the present invention we therefore provide a compound of formula-(1) or a physiologically acceptable salt thereof for use in the treatment of the human or 40 non-human animal body to combat cancer, particularly tumours, therein.

According to a yet further aspect of the present invention we provide the use of a compound of formula (1) or a physiologically acceptable salt thereof for the manufacture of a therapeutic agent for the treatment of the human or non-human animal body to combat cancer, particularly tumours, therein.

According to a still further aspect of the present invention we provide a method of treatment of the human or non-human animal body to combat cancers, particularly tumours, therein, which method comprises administering to the said body an effective amount of a compound of formula (1) or a physiologically acceptable salt thereof.

It will be appreciated that the compounds according to the invention may advantageously be 50 used in conjunction with one or more other therapeutic agents, such as for example anti-emetic agents, immunosuppressive agents or different anti-cancer agents. It is to be understood that the present invention covers the use of a compound of formula (1) or a physiologically acceptable salt thereof in combination with one or more other therapeutic agents.

In a further aspect of the present invention we provide a pharmaceutical composition compris- 55 ing as an active ingredient a compound of formula (1) or a physiologically acceptable salt thereof together with one or more pharmaceutical carriers or excipients.

The compounds according to the invention may be formulated for administration in any convenient way, and the invention therefore also includes within its scope pharmaceutical corn- positions comprising a compound of formula (1) or a physiologically acceptable salt thereof adapted for use in human or veterinary medicine. Such compositions may be presented for use in conventional manner in admixture with one or more physiologically acceptable carriers or excipients. The compositions may optionally further contain one or more other therapeutic agents such as a different anti-cancer agent or an anti-emetic agent or an immunosuppressive agent.

Thus, the compounds according to the invention may be formulated for oral, buccal, topical, 65 3 GB2195636A 3 rectal or, preferably, parenteral administration (eg by bolus injection or intravenous infusion).

Injections are sterile products and may be presented in unit dose form, for instance as ampoules, vials, small volume infusions or pre-filled syringes, or in multi-dose containers, for example, with an added preservative. The compositions may take such forms as solutions, suspensions or emulsions in aqueous or non-aqueous vehicles, and may contain formulatory agents such as anti-oxidants, buffers, bulking agents, chelating agents, antimicrobial agents, solubilising agents, surfactants and/or tonicity adjusting agents. Alternatively, the active ingredient (with our without added substances) may be in a dry form for constitution with a suitable vehicle, eg sterile pyrogen-free water or dextrose, before use. The dry solid presentation may be prepared by filling a sterile powder aseptically into individual sterile containers or by filling a 10 sterile solution aseptically into each container and freeze-drying.

The compounds according to the invention may also be formulated as compositions for oral administration. As tablets or capsules, they may contain conventional excipients such as binding agents, fillers, lubricants, disintegrants, or wetting agents. The tablets may be coated according to methods well known in the art. Oral liquid preparations may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid prepara tions may contain conventional additives such as suspending agents, surfactants, non-aqueous vehicles, preservatives, sugars, sweetening agents, buffers, colours, antioxidants and/or flavours.

The compounds may also be formulated as suppositories, eg containing conventional suppository 20 bases such as cocoa butter or other glycerides.

For buccal administration the compositions may take the form of tablets or lozenges formu lated in conventional manner.

For topical administration the compounds according to the invention may be formulated as ointments, creams, lotions, powders, pessaries, sprays, aerosols or drops (eg eye or nose drops). Ointments and creams may, for example, be formulated with an aqueous or oily base with the addition of suitable thickening and/or gelling agents. Such bases may thus, for example, include water and/or an oil such as liquid paraffin or-a vegetable oil such as arachis oil or castor oil. Lotions may be formulated with an aqueous or oily base and will in general also contain one or more emulsifying agents, stabilising agents, dispersing agents, suspending agents, thickening 30 agents and/or colouring agents. Powders may be formed with the aid of any suitable powder base, for example talc, lactose or starch. Drops may be formulated with an aqueous or non aqueous base also comprising one or more dispersing agents, solubilising agents or suspending agents. Aerosol sprays are conveniently delivered from pressurised packs, with the use of a suitable propellant, eg dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, 35 carbon dioxide or other suitable gas.

The compositions may contain 0.1-100% of the active material.

It may be possible to target a compound of the invention to a tumour by including in the composition specialised drug carrier systems such as liposomes, albumen microspheres or mo noclonal antibodies.

For systemic administration the daily dosage as employed for adult human treatment will generally be within the range of from 5 mg to 5000 mg, preferably 50 mg to 200Orng, which may be administered in 1 to 5 divided daily doses, for example, depending on the route of administration and the condition of the patient. When the compositions comprise dosage units, each unit will preferably contain 10 mg to 2000 mg of active ingredient, for example 50 mg to 45 1 00Orng.

For topical administration the daily dosage as employed for adult human treatment will gener ally range from 0.1 mg to 100Orng, depending on the condition of the patient.

The compounds useful according to the invention may be prepared by a number of processes described in the following, wherein the various groups and symbols are as defined for formula 50 (1) unless otherwise specified.

Thus, according to one process (A) a compound of formula (1) may be prepared by reacting a compound of formula (2) H 3 '1 13 11 1 0 R 7 (2) [in which one of R6 and R7 represents a group -OP(O)(Xl)(X2) (where X' represents an atom or group convenable to a hydroxyl group and X2 represents a hydroxyl group or an X' moiety) and the other represents a hydrogen atom, a hydroxyl group or a group -OP(O)(Xl)(X2) as just defined] to convert at least one X' moiety to hydroxy to produce a compound of formula (1), for 65 4 GB2195636A 4 example by hydrolysis or ether cleavage.

Examples of X1 as atoms capable of being converted to hydroxy include halogen atoms such as chlorine or bromine atoms.

Examples of X1 as groups capable of being converted to hydroxy include the group OX3 [where X3 may be, for example, an alkyl group (optionally substituted by a hydroxyl, alkoxy, cyclic ether or cycloalkyl group) or an alkenyl, cycloalkyl, aryl, aralkyl or aroylalkyl group).

The conversion of X1 to hydroxy may be carried out using conventional procedures. Thus, for example, a halogen atom may be displaced by a hydroxyl group by hydrolysis, eg basic hydrolysis using a suitable base such as an alkali metal hydroxide in a suitable solvent eg aqueous tetrahydrofuran.

The conversion of OX3 to hydroxy may be carried out using a nucleophile eg a halide ion such as an iodide ion (obtained, for example, from an alkali metal halide such as sodium iodide) or a quaternary ammonium halide such as triethylamine hydroiodide in an inert solvent such as a ketone (eg acetone or butan-2-one) or a substituted amide (eg dimethylformamide), or by reaction with a trialkylsilyl iodide such as trimethylsilyl iodide in a solvent such as a halogenated hydrocarbon (eg carbon tetrachloride) followed by hydrolysis, eg basic hydrolysis using a suitable base such as an alkali metal hydroxide.

The reaction may be carried out at any suitable temperature, for example in the range -10' to + 1 OOOC.

It will be appreciated that in accordance with conventional practice the reaction may be 20 managed such that when -OP(O)(X')(X2) represents -OP(O)(OX3)(OX3) only one of the two OX3 groups is converted to a hydroxyl group. In these circumstances secondary phosphates of formula (1) [ie in which R4 is an alkyl group (optionally substituted by a hydroxyl, alkoxy, cyclic ether or cycloalkyl group) or an alkenyl, cycloalkyl, aryl, aralkyl or aroylalkyl group] are prepared.

Alternatively, both OX3 groups may be converted to hydroxyl groups using trimethylsilyl iodide.

In these circumstances primary phosphates of formula (1) [i.e. in which R4 is a hydrogen atom] are prepared.

It will be further appreciated that the reaction may be managed such that when an alkali metal halide (such as lithium, sodium or potassium iodide) is used the appropriate alkali metal salt of a compound of formula (1) is prepared.

According to another process (B) a compound of formula (1) may be prepared by reacting a compound of formula (3) H3 0 R6 / N / 101 9 (3) (in which one of W' and R9 represents a hydroxyl group and the other represents a hydrogen atom or a hydroxyl group) to convert a hydroxyl group at the 9 and/or 12 position to a group -OP(O)(OH)(OR4) where R4 is as defined in formula (1) above.

The conversion may be effected by conventional means, for example by reaction of a corn pound of formula (3) with a halophosphate HaIP(O)(OH)(OR4) or HaIP(O) (Xl)(X2) (where Hal is a 45 halogen atom eg chlorine and X' and X2 are as previously defined) or a pyrophosphate (R40)( H0)(0)P-O-P(O)(01-1)(OR4) or (XI)(X2)(0)P-0-P(O)(X2) (where X' and X2 are as previously defined), followed, where necessary, by conversion of one or more X' moieties to hydroxy according to the procedure described in process (A) above. The reaction with the phosphorylating agent may conveniently be carried out in the presence of a base, for example an alkali metal hydride such 50 as sodium hydride, or an alkali or alkaline earth metal carbonate eg potassium carbonate in an inert solvent such as an ether, e.g. tetra hyd rofuran, an aromatic hydrocarbon eg toluene or a ketone eg acetone or a mixture of such solvents. The reaction may conveniently be carried out at a temperature in the range WC to 1OWC eg 2WC. It will be appreciated that reaction with a halophosphate HaIP(O)(Xl)(X2) or a pyrophosphate (X1))(X2)(0)P-0- P(O)(X1)(X2) leads initially to 55 the formation of a compound of formula (2) which may, if desired, be isolated.

According to another process (C) a compound of formula (1) in which R3 is a hydrogen atom or a methyl group may be prepared by condensing a quinone of formula (4) GB2195636A 5 0 R10 0 0' (4) [in which one of RIO and W' represents a group -OP(O)(OH)(OR4) or - OP(O)(Xl)(X2) (where X' and X2 are as defined above) and the other represents a hydrogen atom, a hydroxyl group, a 10 group -OP(O)(OH)(OR4) or a group -OP(O)(Xl)(X2) (where X' and X2 are as definedd above)] with a compound of formula (5) H3 COOH 15 (5) R17 (in which R12 represents a hydrogen atom or a group -CHO or -COCH,) and subsequently, if 20 required, converting one or more X' moieties to hydroxy to form a compound of formula (1).

The reaction between the compounds of formulae (4) and (5) may be carried out in the presence of an alkanoic acid anhydride, such as acetic anhydride, at an elevated temperature, eg 1OWC. The conversion of one or more X' moieties to hydroxy may be effected using conditions described in process (A) above.

The compounds of formulae (1), (2) and (3)) in which R3 is a halogen atom may be prepared by halogenating a corresponding compound in which R3 is a hydrogen atom. Standard halogena tion procedures may be used, for example, by reaction with a N-chloro-, N- bromo- or N iodoimide, eg N-chloro, N-bromo- or Modosuccinimide, or perchloryl fluoride in an inert solvent such as dichloromethane at ambient temperature.

The compounds of formula (2) may be prepared from compounds of formula (3) as described above in Process (B). Alternatively, the compounds of formula (2) in which R3 is hydrogen or methyl may be prepared by condensing a compound of formula (4) in which one of RIO and W' is a group -OP(O)(Xl)(X2) and the other is a hydrogen atom, a hydroxyl group or a group -OP(O)(Xl)(X2) with a compound of formula (5) accordinng to the method of process (C) above. 35 The compounds of formula (3) in which R 3 is hydrogen or methyl may be prepared by condensing a quinone of formula (6) 0 R13 11 1 -+V / \ /\\ 0 R14 (6) (in which one of R 13 and R14 is a hydroxyl group or a protected hydroxyl group such as an acyloxy group eg acetoxy or halomethylcarbonyloxy and the other is a hydrogen atom, a hydroxyl group or a protected hydroxy group as just defined) with a compound of formula (5) according to the method of process (C) above, followed, where necessary, by removal of the protecting group, eg under acidic conditions using, for example, hydrochloric acid in a solvent 50 such as tetra hydrof u ran.

The compounds of formula (4) may be prepared by phosphorylation of a compound of formula (7) 0 R'S r' ri6 0 R (7) (in which one of R's and R16 is a hydroxyl group and the other is a hydrogen atom or a hydroxyl group) according to the method described in process (B) above.

The compounds of formulae (5), (6) and (7) are known compounds described in EP-A-161102 65 and GB-A-2175587.

6 GB2195636A 6 The intermediates of formula (2) are novel compounds and form a further aspect of the invention. Key intermediates of formula (2) are those in which R3 is a hydrogen atom, R6 is a hydrogen atom and R7 is a group - OP(O)(Xl)(X2) where X' and X2 both represent OX3. Such intermediates in which X3 is benzoyimethyl or, more particularly, benzy] are of special interest.

Where a salt of a compound of formula (1) is formed, the corresponding acid may be obtained by conventional means by addition of a suitable acid e.g. a mineral acid such as hydrochloric acid.

Where a compound of formula (1) is produced in the acid form, a corresponding salt may be obtained by conventional means, e.g. by addition of a base providing the required cation, for example sodium hydroxide or an alkali metal carboxylate such as sodium carboxylate (eg sodium 10 2-ethyihexanoate). Thus, a salt of a compound of formula (1) may be prepared by treating a compound of formula (1) in the acid form with a suitable base in a solvent such as water, an ether eg tetrahydrofuran or an alcohol eg isopropyl alcohol or a mixture thereof. The reaction may conveniently be carrieed out at ambient temperature. Alternatively, a salt of a compound of formula (1) may be obtained by treating a suitable compound of formula (2) with an alkali metal halide (eg lithium iodide, sodium iodide or potassium iodide) in a suitable solvent such as water or a ketone (eg acetone or butan-2-one) or a mixture thereof, conveniently at ambient tempera ture. An amino acid salt of a compound of formula (1) may be obtained by treating a compound of formula (1) in the acid form with an amino acid, eg L-arginine monohydrate in water at ambient temperature.

A salt of a compound of formula (1) may be converted to a different salt, eg a physiologically acceptable salt, by exchange of ion using conventional means.

When a specific enantiomer of formula (1) is required, this may be prepared by conventional methods of resolution known per se. Thus, for example, racemic compounds of the invention may be resolved as salts with optically active bases, eg (+)-dehydroa bietyla mine or (R)-(+)- or 25 (S)-(-)-a-methyibenzylamine. Alternatively, optically active compounds of formula (1) may be prepared from optically active intermediates, eg optically active intermediates of formula (5).

Racemic compounds of formula (5) in which R12 is hydrogen may be resolved, for example, as salts with optically active bases as above or with optically active acids, eg (L)-(+)- or (D)-(-)- tartaric acid. Compounds of formula (5) in which R12 is CHO or COCH, may be similarly resolved 30 as salts with optically active bases.

The following non-limiting Examples illustrate the invention. All temperatures are in 'C. Unless otherwise stated all UV spectral data relate to solutions in ethanol of the compounds concerned.

Intermediate 1 Juglone 5-bis(phenylmethyl)phosphate A solution of sulphuryl chloride (3.489) in toluene (25mi) was added to a stirred solution of dibenzyl phosphite (6.08mi) in toluene (75m1) under nitrogen. The mixture was stirred for 75 min. under nitrogen, washed with 8% sodium bicarbonate solution (75m]) and the organic layer separated and dried over anhydrous sodium sulphate. After filtration the filtrate was evaporated 40 and dried to give dibenzyl chlorophosphate (7.759) as a colourless oil. Potassium carbonate (1.819) was added to a solution of 5-hydroxy-1, 4-naphthalenedione (2. 189) in acetonitrile (300m1) with stirring at romm temperature. Freshly prepared dibenzyl chlorophosphate (7.759, see above) in acetonitrile (50mi) was added and the mixture stirred for 20h. Potassium carbo- nate (907m9) was then added and a further quantity (907rng) of potassium carbonate added after another 24h. The mixture was evaporated to dryness and dried in vacuo to give an oil. The oil was subjected to column chromatography (silica, 500g, 70-230 mesh) eluting with dich loromethane: acetone (96:4). Fractions (250mi) were collected and fractions 4-12 combined and the solvent evaporated. After drying in vacuo the title compound (3,18g) was obtained as an orange solid; (5 (CDCI,) 5.28 (OCH,), 7.38(Ph).

Intermediate 2 Bis(phenylmethyl),[5,8,13,14-tetrahydro- 14-methyl-B, 13-dioxobenz[5,61- isoindolo[2, 1-blisoquibolin 9-yllphosphate Acetic anhydride (12mi) was added to a mixture of N-formyi-1,2,3,4tetrahydroisoquinoline-4- methyl-3-carboxylic acid (625mg) and intermediate 1 (3.1659). The reaction mixture was heated at 100' for 30 min with stirring, then cooled and evaporated to dryness under reduced pressure.

The resulting oil was dissolved in dichloromethane (5mi) and ether was added and the solution cooled in an acetone-drikold bath. The resulting yellow solid was collected by filtration and dried at room temperature to give the title compound (1. 16g); 6(CDC13) 5.2-5.4 (OCH2), 5.23 +5.13 60 (51-1) 4.95 (14H), 1.55 (14CH,).

Intermediate 3 5,6,13,14- Tetrahydro-9-hydroxy14-methylbenz[5,6]isoindolo[2, 1 - blisoquinolin-8, 13-dione Water (312mi) and concentrated hydrochloric acid (169mi) were added to a suspension of 65 7 GB2195636A 7 5,8,13,14-tetrahydro-9-[iodoacetoxy]- 1 4-methyibenz[5,6]isoindolo[2, 1 - blisoquinolin-8,13-dione (80.0 9, Intermediate 9 in GB-A-2175587) in tetrahydrofuran (1.3351---). This mixture was stirred and heated to reflux under nitrogen for 18 h and part (550mi) of the solvent was removed by distillation at atmospheric pressure. The resulting suspension was stirred and cooled to ca. 5' for 4.5 h, and the solid was collected by filtration, washed with cold tetrahydrofuran and dried 5 in vacuo to give the title compound (48.8g); A,,,, 243nrn (MeOH, E] 1022; (5 (MCIj 12.98 (OH,s,1H), 5.27 (5-H,d,16Hz,1H), 5.16 (5-H,d,16Hz,1H), 4.98 (14-H,9,7Hz, 1H),1.55 (14 CH,d,71-1z,31-1).

Intermediate 4 Dimethyl,[5,8,13,14-tetrahydro- 14-methyl-8, 13-dioxobenz[5,6]isoindolo[2, 1-blisoquinolin-9-yllphos phate (a) A 60%-dispersion of sodium hydride in oil (20Orng) was washed free of oil with 40-60' petroleum spirit (2 X 1 Omi) under a nitrogen atmosphere. Dry tetrahydrofuran (25mi) was added to the sodium hydride, followed by addition of Intermediate 3 (1.59) in dry tetrahydrofuran (150mi). The resulting solution was stirred at 20' under nitrogen for 15 min and then treated with a solution of freshly distilled dimethylchloro phosphate (723rng) in dry tetrahydrofuran (5mi).

After 1h the reaction mixture was diluted with water (500mi) and extracted with dichlorometh ane (3x250mi). The combined organic extracts were dried over anhydrous sodium sulphate and evaporated to a green foam which was purified by column chromatography on silica (100g) eluting with dichloromethane-methanol (29:1) to give the title compound as a green foam (1.539g); Ania, 241.6m E] 1052, 369.4nm, E] 158; (5(CDC13) 5.26 (5-H,d, 17Hz,1H), 5.14 (5 H,d,17Hz,1H), 4.96 (14-H,q,7Hz,1H), 4.02 (OCH,m, 6H), 1.57 (14-CH,,d,7Hz, 3H). The following compounds were prepared in a similar manner using

Intermediate 3 and the appropriate halophosphate:

(b) Diethyl,[5,8,13,14-tetrahydro- 14-methyl-8, 13-dioxobenz[5,61isoindolo[2, 1-blisoquinolin-9- yllphosphate, (5,609g) from Intermediate 3 (4.0g) and diethyl chlorophosphate (2.305g); A,,a,, 243.5nm, E] 1007, 372nm, E] 155; J (CDC13) 5.23 (5-H,d,16Hz,1H), 5.13 (5-H,d, 16Hz,1H), 4.93 (14-H,q,7Hz,1H), 4.37 (OCH2CH3,M,4H), 1.55 (14-CH3,d,7Hz,3H), 1.38 (OCH2CH,,m,6H).

(c) Dipropyl,[5,8,13,14-tetrahydro-14-methyl-8,13-dioxobenz[5, 6]isoindolo[2,1-bl isoquinolin-9yllphosphate, (2.068g) from Intermediate 3 (2.0g) and dipropyl chlorophosphate (1.339g);- A,. 242.4nnm, E] 940, 369.6nm, E] 132; J (MCIj 5.25 (5-H,d,16Hz,1H), 5.15 (5-H,d, 16Hz,1H), 4.95 (14-H,q,7Hz,1H), 4.27 (OCHCH,CH,,m,4H), 1.76 (OCH2CH2CH,,m,4H), 1.56 (14CH,d,7Hz,31-1), 0.97 (OCH2CH,CH,,m,6H).

(d) Bis(l-methylethyl),[5,8,13,14-tetrahydro-14-methyl-8,13-dioxobenz[5, 6]isoind olo[2,1-blisoquinolin-9-yllphosphate, (1.563g) from Intermediate 3 (1.5g) and diisopropyl chlorophosphate (1.003g); A... 241.8nm, E] 880, 369.6nm, E] 132; 6(CDCI,) 5.25 (5-H,d, 16Hz,1H), 5.15 (5H,d,161-1z,11-1), 4.85-5.10 (14-H+OCH(CH,),,m,3H), 1.56 (14-CH,,d,7Hz,3H), 1.30-1.45 (OCH(CH,)2,M,12H).

(e) Dibutyl,[5,8,13,14-tetrahydro-14-methyl-8,13-dioxobenz[5,61isoindolo[2,1-bl isoquinolin-9- yllphosphate, (1.628g) from Intermediate 3 (1.5g) and dibutyl chlorophosphate (1.143g); A 241.8nm, E] 956, 369.8nm, E] 129; (5(CDCI,) 5. 25 (5-H,d,16Hz,1H), 5.15 (5-H,d,16Hz,1H), 4.96 (14-H,q,7Hz,1H), 4.31 (OCH2CH,CH2CH3,m,4H), 1.62-1.80 (OCH,CH2CH2CH3,M,4H), 1.56 (14CH,d,71-1z, 31-1), 1.32-1.52 (OCH2CH2CH2CH,,m,4H), 0.92 (OCH2CH2CH2CH,,m,6H).

(f) Bis(tetrahydro-2-furanylmethyl),[5,8,13,14-tetrahydro-14-methyl-8,13dioxobe nz[5,6]isoin- 50 dolo[2, 1-blisoquinolin-9-yljphosphate, (1.00g) from Intermediate 3 (2. 0g) and bis(tetrahydro-2 furanyimethyi) chlorophosphate (6.78g, freshly prepared from phosphorus trichloride and tetrahy drofurfuryl alcohol) except that purification was initially carried out by column chromatography on silica (6509) eluting with dichloromethane-acetone (4:1) and the resulting yellow foam further purified by column chromatography on silica (70g) eluting with dichloromethane-methanol (96:4); 55 A ax 242.2nm, E] 809, 369.8nm, E] 117; 6(CDCI,), 5.24(5-H, d, 17Hz, 11-1); 5.16(5-H, 17Hz, 11-1); 4.96(14-H, q, 7Hz, 11-1); 4.30(POCH2, m, 4H), 4.21(OCH2CHO, m, 21-1); 1. 56(14-CH, d, 7Hz, 31-1) (9) Cyclohexyl methyl, [5,8,13,14-tetrahydro- 14-methyl-8, 13-dioxobenz[5, 61)isoindolo[2, 1-bli soquinolin-9-yljphosphate, (1.4099) from Intermediate 3 (3.09) and cyclohexyl methyl chlorophos- 60 phate (2.9g, freshly prepared from methyi dichlorophosphate and cyclohexanol); A,,,,x 242.4 nm, E] 899, 369.6nm, E] 150; (5(CDCl,) 5.23 (5-1-1, cl, 16Hz, 1H), 5.13(5-1-1, d, 161-1, 11-1), 4.95(14-1-1, q,7Hz, 1H), 4.64(OCH, m, 11-1), 4.01(0CH, m, 31-1), 1.54(14-CH, d, 71-1, 31-1).

(h) Bis (2-propenyl),[5,8,13,14-tetrahydro-14-methyl-8,13-dioxobenz[5, 6]isoindoto[2, 1-blisoqui- 65 8 GB2195636A 8 nolin-9-yllphosphate, (3.5549) from Intermediate 3 (3.0g) and diallyl chlorophosphate (2.689) except that purification was carried out by column chromatography on silica (3009) eluting with dichloromethane- acetone (94:4); Anzx 242.2rim, E] 973, 371.8nm, E] 148; d(CDCI,), 5.90-6. 10(CH, m, 2H), 5.20-545 (CH, m, 4H), 5.25(5-H, d, 17Hz, 1H), 5.15(5-H, d, 17Hz, 11-1), 4.94 (14-1-1, q, 7Hz, 11-1) 4.75-4.90(OCH2, m, 4H), 1.58(14- CH, d, 71-13, 31-1).

(i) Dipentyl,[5,8,13,14-tetrahydro-14-methyl-8,13-dioxobenz[5,61isoindolo[2,1-b lisoquinolin-9yllphosphate, (2.079) from Intermediate 3 (2.0g) and dipentyl chiorophosphate (1.715g, freshly prepared from phosphorus trichloride and pentan-l-ol) except that after evaporation the oil was dissolved in diethyl ether (50mi), stirred at 20' for 30 min. and the resulting yellow solid filtered 10 and washed with diethyl ether; A,,, 242.4 rim, E] 913, 369.4rim, E] 137; 8(CDCi,) 5.24 (5-H, d, 17Hz, 1H), 5.14(5-H, d, 17Hz, 1H), 4.96 (14-1-1, q, 7Hz, 11-1), 4.20-4.40 (POCHCH,CH,CH,CH,, m, 41-1), 1.65-1.85 (POCH,CH2CH, Cl-1, m, 41-1), 1.56(14-CH3, d_ 7Hz, 3H), 1.20-1.45 (POCH2CH2CH,CH2CH, m, 8H), 0.88(POCH2CH2CH2CH2CH3, m, SH).

0) Bis(2-methoxyethyl),[5,8,13,14-tetrahydro- 14-methyl-B, 13-dioxobenz[5, 6]isoindo[2, 1-blisoquinolin-9-yllphosphate (2.07g) from Intermediate 3 (2.09) and bis (2-methoxyethyl) chlorophosphate (1.55g. freshly prepared from 2-methoxyethanol and phosphorus oxychloride) except that the column was eluted with dichloromethane acetone (4: 1); A,,,. 242.2rim, E] 920, 371,6nm, E] 142; (5(CDC13), 5.23(5-H, d_ 17Hz, 1H), 5.13(5-H, d, 17Hz, 1H), 4.95 (14- H, q, 7Hz, 11-1), 4.48(POCH2. m, 4H), 3.68(POCH2CH, m, 4H), 3.39(0CH, s, 31-1), 3.40(0CH, s, 31-1), 1.57(14CH3, d, 7Hz, 3H).

(k) Bis(cyclohexylmethyl),[5,8,13,14-tetrahydro- 14-methyl-8,13dioxobenz[5,6]isoindolo[2, 1-bli- 25. soquinolin-9yllphosphate (5.3029) from intermediate 3 (4.0g) and bis(cyclohexyimethyi) chloro phosphate (8.229, freshly prepared from phosphorus trichloride and cyciohexyimethanol) except that after evaporation the solid was triturated with ether (100mi) and crystallised from ethyl acetate/chloroform; 242.4rim, E] 778, 369.6rim, E] 119; (5(CDCI,) 5.25(5- H, d, 16Hz, 1H), 5.15(5-H, d, 16Hz, 1H), 4.96(14-H, q, 7Hz, 1H), 4.09(OCH2, m, 4H), 1. 54(14-CH, d, 7Hz, 31-1).

Intermediate 5 (a) Bis(phenylmethyl),[5,8,13,14-tetrahydro- 14-methyl-B, 13-dioxobenz[5, 6]isoindoolo[2, 1-bliso quinolin-9-yllphosphate N-chlorosuccinimide (1.017g) was added to a stirred solution of dibenzyl phosphite (3.3m1) in dry benzene (30mi) and the resulting solution was stirred at 20', the succinimide was removed 35 by filtration and the filtrate was concentrated to a low volume (about 5- 10mf). This solution was then used in the following preparation.

A 60%-dispersion of sodium hydride in oil (134mg) was washed free of oil with 40-600 petroleum spirit (2 x 15mi) under a nitrogen atmosphere. Dry tetrahydrofuran (25mi) was added to the sodium hydride followed by addition of Intermediate 3 (1.0g) in dry tetrahydrofuran (100mi). The resulting red solution was cooled to 10' in an ice-bath and treated with freshly prepared dibenzyi chlorophosphate in benzene (about 5-10mi, see above). After 2h at 10', the reaction mixture was allowed to warm to 20' and maintained at this temperature for 3h. The reaction mixture was diluted with water (500mi) and extracted with dichloromethane (3x250mi).

The combined organic extracts were dried over anhydrous sodium sulphate and evaporated to a 45 green oil which was dissolved in dichloromethane and applied to a column of silica (3009) prepared using dichloromethane-acetone (96:4). The column was eluted with dichioromethane acetone (96:4) and 25mi fractions were collected. The product-containing fractions were com bined and evaporated to yield the title compound as a green foam (1.2119); 244rim, E] 743, 374rim, E] 112; (5(CDCI,) 5.20-5.40 (OCH2C,Hs,m,4H), 5.23 (5-H,d, 16Hz,1H), 5,13 (5- 50 H,d,16Hz,1H), 4.95 (14-H,q,7Hz,1H), 1.55 (14-CH,,d,7Hz,3H).

The following compound was prepared in a similar manner:

(b) Bis[4-nitrophenyl)methyll,[5,8,13,14-tetrahydro- 14-methyl-B, 13dioxobenz[5,6]isoindolo[2, 1 blisoquinolin-9-yllphosphate, (3.6629) from intermediate 3 (2.09) and di(p-nitrobenzyi) chloro- 55 phosphate (2.5829); Am,, 244rim, E] 330; (5(d,-DMSO) 5.48 (OOCH,,d,8Hz, 4H), 5.42 (5 H,d,18Hz,1H), 5.32 (5-H,d,18Hz,1H), 4.79 (14-H,q,7Hz,1H), 1.48 (14-CH,,d, 7Hz,3H).

In termediate 6 5,8,13,14- Tetrahydro- 12-hydroxy14-methylbenz[5,61isoindolo[2, I - b]isoquinolin-8,13-dione 60 Acetic anhydride (160ml) was added to a mixture of N-formyl-1,2,3,4- tetra hydroisoqu inoline 4-methyl-3-carboxylic acid (20g) and 5-hydroxy-1,4-naphthoquinone (31. 78g). The reaction mix ture was heated at 100' for 30 min and then left to cool (4') overnight. A precipitate formed which was filtered, washed with ether, dried in vacuo and purified by column chromatography on silica gel eluting with dichloromethane to yield the title compound; Am., ), 243nm, E,1 1103, 397nm,65 Z 9 GB2195636A 9 E] 381.

Intermediate 7 Bis(phenylmethyl),[7-bromo-5,8,13,14-tetrahydro14-methyl-B, 13- dioxobenz[5,6]isoindolo[2, 1-bli soquinolin-9-yllphosphate To a solution of Intermediate 5a) (1.0g) in dichloromethane (50mi) was added N-bromosuccin imide (0.604g) in dichloromethane (50mi). After 1h at 20', the reaction mixture was evaporated and the residue was purified by column chromatography, firstly on silica (150g) eluting with chloroform-acetone (96/4) followed by silica (40g) eluting with dichloromethane-acetone (96/4) to give the title compound as a brown foam (0.81 g); Am 242.Onm, E] 760, 378.Onm, E] 108; J 10 (c16-DMS0) 5.42 (5-H, d,17Hz,1H), 5.34-5.17 (5-H+OCH2,M,51-1), 4.89 (14-H, q,7Hz,1H), 1.49 (1 4-CH,,d,7Hz,3H).

Intermediate 8 Bis(phenylmethyl),[7-chforo-5,8_,13,14-tetrahydro-14-methyl-8,13dioxobenz[5,6lisoindolo[2,1-bliso-15 quinolin-9-yllphosphate 1 To a solution of Intermediate (5a) (1.0g) in chloroform (200mi) was added N-chlorosuccinimide (0.2269) followed by a saturated solution of hydrogen chloride in chloroform (0.5mi). After 30 min at 20', the reaction mixture was evaporated to a yellow foam which was purified by column chromatography on silica (100g) eluting with dichloromethane-acetone (96/4) to give a yellow 20 foam (0.9439). Crystallisation of this foam from acetone gave the title compound as a yellow solid (0.6979); Am,,,, 241.6nm, E] 820, 377.2nm, E] 125; J (d6-DMSO) 5.43 (5-H,d,17Hz,1H), 5.32-5.16 (5-H+OCH2,m,5H), 4.87 (14-H,q,7Hz,1H),1.49 (14-CH3,d,7Hz,3H).

Intermediate 9 Bis(2-oxo-2-phenylethyl),[5,8,13,14-tetrahydro14-methyl-B, 13- dioxobenz[5,6]isoindolo[2, 1-bliso quinotin-9-yllphosphate A solution of dicyclohexylcarbodiimide (3.039) in dichloromethane (125mi) was added dropwise over 3h to a stirred solution of phosphoric acid bis(2-oxo-2- phenylethyl) ester (8.939) in dichloromethane (500mi) at 2WC. The reaction mixture was evaporated to low volume (ca. 30 100mi) and the precipitated solid was removed by filtration. The filtrate was stirred at 20' for 1 h and then evaporated to give the crude phosphorylating reagent as a colourless oil.

A 60% dispersion of sodium hydride in oil (534m9) was washed free of oil with 40-600 petroleum ether (2x25mi) under a nitrogen atmosphere. Dry tetrahydrofuran (100mi) was added to the sodium hydride followed by a solution of Intermediate 3 (4g) in dry tetrahydrofuran (400mi). The resulting solution was stirred at 20' for 30 min, and then treated with the crude phosphorylating reagent. After 15 min. at 20' the reaction mixture was diluted with water (2.51---) and extracted with dichloromethane (3x60Ornl). The organic extracts were dried over anhydrous sodium sulphate and evaporated to a yellow solid (8.96g). To this solid (7.96g) was added acetone (200mi) and insoluble impurities were removed by filtration. The filtrate was evaporated 40 and the residue was subjected to column chromatography on silica (2709) eluting with dichloro methane-methanol (96/4) to give a yellow foam. This foam was purified by further column chromatography on silica (1009) eluting with dichforomethane-acetone (96/4) to give the title compound as a yellow foam (1.14g); I.R. (CHBr,) 1710cm--', 1659cm-1; 5(CDCI,) 5.68-7.81(OCH2, m, H), 5.22(5-H, cl, 16Hz, 1H), 5.15(5-H, d, 16Hz, 11-1), 4.95(14-H, q, 7Hz, 45 1H), 1.55(114-CH, cl, 7Hz, 3H).

Intermediate 10 (a) Diethyl,[5,8,13,14-tetrahydro-14-methyl-8,13-dioxobenz[5, 6]isoindolo[2,l-bli soquinolin-12- yllphosphate A 50% dispersion of sodium hydride in oil (158m9) was washed free of oil with 40-600 petroleum spirit (2 X 1 Orril) under a nitrogen atmosphere. Dry tetrahydrofuran (25mi) was added to the sodium hydride followed by addition of Intermediate 6 (1.09) in dry tetrahydrofuran (100mi). The resulting solution was stirred at 20' under nitrogen for 15 min and then treated with a solution of freshly distilled diethyl chlorophosphate (569mg) in dry tetrahydrofuran (5mi). 55 After 1.5h, the reaction mixture was diluted with water (500mi) and extracted with dichloromethane (3x25Ornl). The combined organic extracts were dried over anhydrous sodium sulphate and evaporated to a green solid (1.4849) which was purified by column chromatography on silica (1509) eluting with chloroform-ethanol (49/1) to give a green solid (1.2549). Crystallisation of this solid from a mixture of chloroform and ethyl acetate gave the title compound as green 60 feathery crystals (904m9); Am.,, 243nm, E] 1005, 372nm, E] 146; 5(CDCI,) 5.25 (5-H,d,16Hz,1H), 5.15 (5-H,d,16Hz,1H), 5.01 (14-H,q,7Hz,1H) 4.39 (OCHCH,,m,4H), 1.54 (14CH,,d,7Hz,3H), 1.42 (OCH,CHm,6H).

The following compound was prepared in a similar manner:

GB2195636A 10 (b) Bis(phenylmethyl),[5,8,13,14-tetrahydro- 14-methyl-B, 13-dioxobenz[5, 6]isoindolo[2, 1-bliso quinotin-12-yllphosphate (1.59) from Intermediate 6 (3.09) and dibenzyl chlorophosphate (freshly prepared from dibenzyi phosphate (9.9mi) and N-chlorosuccinimide) in benzene (ca. 20mi) except that elution was carried out using dichioromethane-acetone (96:4) to give an oil which crystal lised on standing and was recrystallised from ethyl acetate/chloroform; A 242.6 nm, E] 733, 5 371.4 nm, E] 109; 5(CDCI,) 5.20-5.45(OCH,+5-H, m, 5H), 5.15 (5-H, d, 16Hz, 1H), 4.97(14-H, q, 7Hz, 1H), 1.53(14-CH3, cl, 7Hz, 3H).

Intermediate 11 Dipropyl,[1,4-dioxonaphthalene5,8-diyllphosphate 5,8-Dihydroxy-1,4-naphthalenedione (19) was suspended in acetone (100mi) and potassium carbonate (4g) and dipropyl chlorophosphate (5mi) were added. The reaction mixture was stirred at 40-50' for 90 min., filtered to remove excess potassium carbonate and evaporated to dryness, The residue was dissolved in dichloromethan and purified by column chromatography on silica using dichloromethane and dichioromethane-methanol (99:1) as eluant to give the title 15 compound as a yellow oil (2.3 1 g); (5(CDCI,9) 4.23(CH2), 1.78(CH,), 1.0 1 (CH,).

Intermediate 12 Dipropyl,[5,8,13,14-tetrahydro- 14-methyl-B, 13dioxobenz[5,6]isoindolo[2, 1-blisoquinotin-9,12-diyll- phosphate Acetic anhydride (10mi) was added to a mixture of N-formyi-1,2,3,4,tetrahydroisoquinoline-4methyl-3-carboxylic acid (440rng) and Intermediate 11 (2.169). The reaction mixture was kept at 10' for 30 min. and then evaporated to dryness to give a brown oil. Chromatography on silica using petrol (60-80')-ethyl acetate (1A, 1:2 and 1:3) as eluant gave the title compound (984mg); Am,,, 237.6nm, E] 609, 371nm, E] 94; J(CDC]3) 5.13+5.23 (5H), 4. 93(14H), 4,20-4.35 (CH2), 1.60-1.90(CH,), 0.8-1.05(CH3).

Example 1 [5,8,13,14- Tetrahydro14-methyl-8,13-dioxobenz[5,6]isoindolo[2, 1 bJisoquinofin-9-y1]dZhydrogen phosphate, disodium salt A cooled solution (0 to 5') of Intermediate 4b) (465mg) in carbon tetrachloride (3ml) was treated under a nitrogen atmosphere with trimethylsilyl iodide (0.32ml). After 5 min, the reaction mixture was evaporated to dryness and the residual oil was treated with methanol (5ml). The mixture was stirred at 20' and after about 5 min a clear brown solution was obtained. After 20 min the solution was evaporated to a dark foam which was suspended in water (20ml) and the 35 suspension adjusted to pH 7.5 with 1M-sodium hydroxide solution (4.00ml). The resulting mixture was extracted with chloroform (25ml) and was filtered through a prewashed bed of kieselguhr using water (50ml) to wash the product through. The resulting layers were separated and the aqueous phase was washed with more chloroform (10ml). The combined aqueous extracts were filtered through a millipore filter, degassed and freeze- dried. The freeze-dried solid 40 was redissolved in water (100ml) filtered through a millipore filter and freeze-dried to give the title compound as a bright yellow solid (0.398g); Am,,,, 246nm, Ell 667, 388nm, Ell 165.

Example 2 (a) Phenylmethyl,[5,8,13,14-tetrahydro- 14-methyl-B, 13-dioxobenz[5, 6]isoindolo[2, 1-blisoqui- 45 nolin-9-yllphosphoric acid, sodium salt Sodium iodide (1.551g) was added to a solution of Intermediate (5a) (5. 5469) in acetone (100m1) and the resulting solution was heated under reflux for 1.5h. The reaction mixture was evaporated to dryness and the residue was triturated with ether (100mi) to give a yellow solid.

This solid was dissolved in water (200mi) and the pH of the solution was adjusted to pH 1 to 2 50 by the addition of 2M-hydrochloric acid. The resulting mixture was diluted with water (200mi) and extracted with dichloromethane (2x400mi). Salt solution was added during the extraction procedure to disperse the emulsion which was formed. The combined organic extracts were dried over anhydrous sodium sulphate and evaporated to a green solid. This solid was sus pended in water (300mi) and the suspension was adjusted to pH 7.0 by the dropwise addition 55 of 110-sodium hydroxide solution. The resulting solution was filtered and the filtrate was freeze dried to a solid which was treated with acetone (400mi) and evaporated to dryness to give a solid (4.49g). Trituration of this solid with ether (200mi) gave the title compound as a yellow solid (4.232g); Arna., 243.8nm, E] 679, 367.2nm, E] 132; 8(d,-DMSO) 5.41 (5-H,d,16Hz,1H), 5.31 (5-H,d,16Hz,1H), 493 (OCH,,d,6Hz,2H), 4.82 (14-H,q,7Hz,1H), 1.47 (14-CH3, d,7Hz,3H).

The following compounds were prepared in a similar manner:

(b) Propyl,[5,8,13,14-tetrahydro-14-methyl-8,13-dioxobenzyl[5, 6]isoindolo[2,1-bl isoquinolin-9yllphosphoric acid, sodium salt, (1.2569) from Intermediate 4c) (1.59) and sodium iodide (1.3679) using butan-2-one (35mi) in place of acetone; Am 243.6nm, E] 963, 360.Onm, E] 265; 65 1 11 GB2195636A 11 5(d,-DMSO) 5.42 (5-H,d,16Hz,1H), 5.32 (5-H,d,16Hz,1H), 4.81 (14-H,mlH), 3. 78 (OCH,CH,CH,,m,2H), 1.40-1.60 (14-CH3+ OCH,CHCHn3H), 0.82 (OCH2CH, CH3^3H).

(c) (1-Methylethyl),[5,8,13,14-tetrahydro-14-methyl-8,13-dioxobenz[5, 6]isoindol o[2,1-blisoqui- nolin-9-yllphosphoric acid, sodium salt, (91Orng) from Intermediate 4d) (1.29) and sodium iodide 5 (1.094g) using butan2-one (25mi) in place of acetone; 243.4nm, E] 943, 366.8nm, E] 199; 6 (dc,-DMSO) 5.40 (5-H,d,16Hz,1H), 5.30 (5-H,d,16Hz,1H), 4.81 (14-H,q,7HZ, 1H), 4.43 (OCH(CH,)2,M,1H), 1.48 (14-CH3,d,7Hz,3H), 1.11 (OCH(CH3)2,d,5Hz,6H).

(d) Phenyimethyl,[7-bromo-5,8,13,14-tetrahydro-14-methyl-8,13-dioxobenz[5, 6lisoi ndolo[2,1- 10 blisoquinotin-9-yllphosphoric acid, sodium salt, (338mg) from Intermediate 7 (705rng) and so dium iodide (174mg); An 243.6nm, E] 601, 367.8nm, E] 120; J (d,-DMSO) 5. 40 (5 H,d,171-1z,1H), 5.21 (5-H,d,17Hz,1H), 4.97-4.82 (14-H+OCH2,m,3H), 148 (14- CH3,d,7Hz,3H).

(e) Phenylmethyl,[7-chloro-5,8,13,14-tetrahydro-14-methy]-8,13dioxobenz[5,6]iso indolo[2,1- 15 blisoquinolin-9-yllphosphoric acid, sodium salt, (674m9) from Intermediate 8 (1.0689) and so dium iodide (282mg); A,,,, 243.Onm, E] 688, 374.Onm, E] 98; 6 (d6-DMSO) 5. 40 (5 H,d,17Hz,1H), 5.23 (5-H,d,17Hz,1H), 5.00-4.79 (14H+OCH,,m,3H), 1.50 (14CH3,d,7Hz,3H).

(f) Pentyl,[5,8,13,14-tetrahydro- 14-methyl-8,13-dioxobenz[5, 6]isoindolo[2, 1blisoquinotin-9-yll- 20 phosphoric acid, sodium salt (0. 8369) from Intermediate 4i) (1.2079) and sodium iodide (724m9) using butan-2-one (30mi) in place of acetone; A,, 244.Onm, E] 899, 366.8nm, E] 190; 5(d,DMSO) 5.40(5-H, d, 17Hz, 1H), 5.32(5-H, d, 17Hz, 1H), 4.81(14-H, q, 7Hz, 1H), 3.80(POCH2CH2CH2CH2CH3, 40, 7Hz, 2H), 1.35-1.55(POCH,CH2CH, CH2CH3-1-14-CH3, m, 5H), 25 1.10-1.30 (POCH2CH2CH2CHCH,, m, 4H), 0. 79(POCH,CH2CH2CH2CH3, t, 7Hz, 3H).

(9) Cyclohexylmethyl,[5,8,13,14-tetrahydro-14-methyl-8,13-dioxobenz[5, 6]isoindol o[2,1-blisoquinolin-9-yllphosphoric acid, sodium salt (1.0179) from Intermediate 4k) (1.5g) and sodium iodide (411 mg) using butan-2-one (40m1) in place of acetone; A... 244.Onm, E] 849, 366.8nm, E] 198; 3(d,-DIVISO) 5.41(5-H, cl, 16Hz, 11-1), 5.31(5-H, d, 16Hz, 1H), 4. 81(14-H, q, 7Hz, 11-1), 30 3.62(0CH, t, 6Hz, 2H), 1.48(14-CH3, d, 7Hz, 3H).

(h) Phenylmethyl,[5,8,13.14-fetrahydro-14-methyl-8,13-dioxobenz[5, 6]isoindolo[2, 1-blisoqui- nolin-12-yllphosphoric acid, sodium salt (781rng) from Intermediate 10b) (1.2g) and sodium iodide (336rng) except that after the pH was adjusted with hydrochloric acid the resulting mixture was extracted with dichloromethane-methanol (3:2, 40OmI and 2X200mi); 244.Onm, E] 842, 265.8nm, E1295, 356.Onm, E] 97; d(d6-DMSO) 5.41(5-H, d, 16Hz, 1H), 5.31(5-H, d, 16Hz, 1H), 4.93(OCH2, d, 7Hz, 2H), 4.88(14-H, q, 7Hz, 1H), 1.47(14-CH, d, 7Hz, 3H).

Example 3 (a) Methyl,[5,8,13,14-tetrahydro-14-methyl-8,13-dioxobenz[5,6]isoindolo[2, 1-blis oquinolin-9-yll- phosphoric acid, sodium salt A mixture of Intermediate (4a) (1.0g) and sodium iodide (377m9) in acetone (35mi) was heated under reflux for 5h. After this time, a second portion of sodium iodide (189m9) was added, and the reaction mixture was maintained under reflux for 1h. The reaction mixture was 45 cooled to 20' and the precipitated product was collected by filtration, washed with acetone and ether, and dried to give a green solid which was dissolved in water (50mi) the pH of the resulting solution being adjusted to pH 1 to 2 by the addition of 2M- hydrochloric acid. The resulting mixture was diluted with water (50mi) and extracted with dichloromethane (2 x 1 00mi).

Salt solution was added during the extraction procedure to disperse the emulsion which was formed. The combined organic extracts were dried over anhydrous sodium sulphate and evapo rated to a green solid which was suspended in water (70m1) the resulting suspension being adjusted to pH 7.0 by the dropwise addition of 1M-sodium hydroxide solution. The resulting solution was filtered and the filtrate was freeze-dried to a solid which was suspended in acetone (100mi) and evaporated to dryness to give a solid. Trituration of this solid with ether (100mi) gave the title compound as a yellow solid (858mg); A... 243.4nm, E], 991, 365.4nm, E] 199; 6(cl,-DMS0) 5.41 (5-H,d, 16Hz,1H), 5.31 (5-H,d,16Hz,1H), 4.82 (14-H,q,7Hz,1H) 3.50 (OCH,,d,11Hz, 3H) 1.47(14-CH,,d,7Hz,3H).

The following compounds were prepared in a similar manner:

(b) Butyl,[5,8,13,14-tetrahydro- 14-methyl-B, 13-dioxobenz[5, 6]isoindolo[2, 1-blisoquinolin-9-yllphosphoric acid, sodium salt, (1. 0419) from Intermediate 4e) (1.255g) and sodium iodide (1.0829); A... 243. 2nm, E] 984, 366.6nm, E] 189; J(d,,-DMSO) 5.41 (5-H,d,16Hz,1H), 5.31 (5H, d,16Hz,11-1), 4.82 (14-H,q,7Hz,1H), 3.81 (OCH2CH2CH2CH,,q,6Hz,2H), 1.15-1. 60 (OCH,CHCH2CH,+14-CH,,m,7H), 0.83 (OCH,CH2CH2CH3,t,6Hz,3H).

12 GB2195636A 12 (c) Cyclohexyl,[5,8,13,14-tetrahydro- 14-methyl-B, 13-dioxobenz[5, 6]isoindolo[2, 1-blisoquinolin9-yllphosphoric acid, sodium salt, (686m9) from Intermediate 4g) (1.0g) except that the final trituration step was not necessary; A x 244.0 nm, Ell 850, 367.0 nm, E] 182; (5(d,-DMSO) 5.42(5-H, d, 16Hz, 11-1), 5.32(5-H, d, 16Hz, 11-1), 5.48(14-H, q, 7Hz, 1H), 4.13(OCH, m, 1H), 1.46(14-CH, d, 7Hz, 1H).

Example 4

Ethyl,[5,8,13,14-tetrahydro- 14-methyl-B, 13-dioxobenz[5,6]isoindolo[2, 1blisoquinolin-9-yllphos- phoric acid, sodium salt Sodium iodide (3.09) was added to a solution of Intermediate 4b) (3.09) in acetone (150mi) and the mixture was heated under reflux for 4h. After this time, a second portion of sodium iodide (3.0g) was added, and the reaction mixture was maintained under reflux for 16h. The reaction mixture was cooled to 20' and the precipitated product was collected by filtration, washed with acetone (3 x 30m1) and dried to give the title compound as a green solid (2.379); 15 Zm,,,, 244.5nm, Ell 1050, 368,5nrn Ell 222; 8(d,-DMSO) 5.41 (5-H,d,16Hz, 1H), 5.30 (5H,d,16Hz,1H), 4.82 (14-H,q,7Hz,1H), 3.90 (OCHCH,,m,2H), 1.49 (14-CH,,d,7Hz,3H), 1.14 (OCH2CH3,t,8Hz,3H).

Example 5

Phenyl,[5,8,13,14-tetrahydro- 14-methyl-B, 13-dioxobenz[5,6]isoindolo[2, 1-blisoquinolin-9-yllphosphoric acid, sodium salt A 60%-dispersion of sodoum hydride in oil (67mg) was washed free of oil with 40-600 petroleum spirit (2x 10mi) under a nitrogen atmosphere. Dry tetrahydrofuran (10mi) was added to the sodium hydride, followed by addition of Intermediate 3 (50Orng) in dry tetrahydrofuran (50mi). The resulting solution was stirred at 20' under nitrogen for 15min and then treated with phenyl dichforophosphate (381mg). After 1h, the reaction mixture was diluted with water (250mi) and extracted with dichforomethane (3 x 1 00mi). Salt solution was added, during the extraction procedure, to disperse the emulsion which was formed. The combined organic ex tracts were dried over anhydrous sodium sulphate and evaporated to an oily foam (769m9). This 30 foam was dissolved in a mixture of tetrahydrofuran (15mi) and water (5mi) and basified with 1M-sodium hydroxide solution. After 2h at pH 9 and 2h at pH 12, the reaction mixture was warmed to 50-55' and diluted with water (5mi) and tetrahydrofuran (bmi). After 20 min, the reaction mixture was concentrated in vacuo, diluted with water (150m1) and acidified to pH 1 to 2 with 2M-hydrochloric acid, The resulting mixture was extracted with dichloromethane (3 x 1 00mi). Salt solution was added during the extraction procedure to disperse the emulsion which was formed. The combined organic extracts were dried over anhydrous sodium sulphate and evaporated to a dark green foam (705m9). This foam was suspended in water (100mi) and the suspension was adjusted to pH 7.1 by the dropwise addition of 1M-sodiurn hydroxide solution. The resulting mixture was filtered and the filtrate was freeze- dried to a foam which was 40 treated with acetone (100mi) and evaporated to dryness to give a solid (440rng). Trituration of this solid with ether (100mi) gave the title compound as a dark green solid (341mg); 4ax 243. 8nm, E] 918, 369.2nm, Ell 174; (5(d,-DMSO) 5.42 (5-H,d,16Hz,1H), 5.32 (5- H,d,16Hz,1H), 4.82 (14-H,q,7Hz,1H), 1.48 (14-CH,,d,7Hz,3H).

Example 6 (4-Nitrophenyl)methyl,[5,8,13,14-tetrahydro- 14methyl-8, 13-dioxobenz[5, 6]isoindolo[2, 1-blisoqui nolin-9-yllphosphoric acid, sodium salt Sodium iodide (330m9) was added to a suspension of Intermediate 5b) (1. 3599) in butan-2 one (100mf) and the mixture was heated under reflux for 1.5h. The reaction mixture was 50 evaporated to dryness and the residue was triturated with ether (3 X 1 00mi) to give a green soid (1.055g). The solid was recovered by filtration to give the title compound as a green solid (91 '1 mg); A,x 243.8nm, E] 837, 265.2nm,. Ell 438, 368.8nm, E] 142; J(d,- DMSO) 5.39 (5 H,d,16Hz,1H), 5.29 (5-H,d,16HzlH), 5.09 (OCH,,d,7Hz,2H) 4.80 (14-H,q,7Hz, 1H), 1.46 (14 CH,d,71-1z,31-1).

Example 7 [5,8,13,14- Tetrahydro-8, 13-dioxo14-methylbenz[5,6]isoindolo[2, 1- blisoquinolin-9-ylldihydrogen phosphate, disodium salt Intermediate 5a) (1.69) was dissolved in dry carbon tetrachloride (5mi) and the resulting 60 solution was cooled in an ice-bath at 0 to W. The mixture was allowed to warm,to 20' and dry carbon tetrachloride (8m]) was added, followed by dichloromethane (5mi). The resulting solution was cooled at 0 to 5' under nitrogen and treated with trimethylsilyl iodide (0.54mi). After 5 min at 0 to W, the reaction mixture was evaporated to dryness and the residual oil was treated with methanol (10mi). The mixture was stirred at 20' and after about 5 min, a clear brown solution 65 t 13 GB2195636A 13 was obtained. After 10 min, the solution was evaporated to an oil which was then triturated with diethyl ether (4x25ml) to give a dark yellow solid which was suspended in sterile water (70ml). The pH was adjusted to pH 8.0 by the addition of OAM-sodium hydroxide solution to give a solution which was filtered through a glass-fibre filter to remove a small amount of coarse solid and then twice through a large (5cm) millipore filter. The filtrate was freeze-dried to yield 5 the title compound as a dark yellow solid (0.738g); Am,, 246nm, Ell 667, 388nm, Ell 165.

Example 8

2-Oxo-2-phenylethyl,[5,8,13,14-tetrahydro- 14-methyl-B, 13-dioxobenz[5, 6]isoindolo[2, 1-blisoqui nolin-9-yllphosphoric acid, sodium salt Sodium iodide (243m9) was added to a solution of Intermediate 9 (1.129) in acetone (25mi).

The resulting solution was refluxed for 10 min. and then evaporated. The residue was triturated with ether (3 X 30mi) to give a solid. Water (140mi) was added to this solid and insoluble impurities removed byfiltration. The resulting solution was acidified to pH 1.5 by the addition of 2M hydrochloric acid and extracted with dichloromethane (3x300mi). The organic extracts were dried over.anhydrous sodium sulphate and evaporated to a solid. The solid was suspended in water (40mi) and 0.1M sodium hydroxide solution was added to pH 7.0. The resulting solution was filtered and freeze-dried to a foam. Treatment of this foam with acetone (100mi) gave a solution which was filtered and evaporated. The residue was triturated with ether (50mi) to give the title compound as a yellow solid (50Orng); 243.6 nm, Ell 955, 366.4nm, E] 147; 6(d6- 20 DIVISO) 5.39(5-H, d, 17Hz, 1H), 5.30(5-H, d, 17Hz, 1H), 5.23(OCH2, d, 8Hz, 2H), 4.82(14-H, q, 7Hz, 11-1), 1.48(14-CH, d, 7Hz, 3H).

1-5 Example 9

Tetrahydro-2-furanylmethyl,[5,8,13,14tetrahydro- 14-methyl-B, 13dioxobenz[5,6]isoindolo[2, 1bli- 25 soquinolin-9-yllphosphoric acid, sodium salt Sodium iodide (272mg) was added to a solution of Intermediate 4f) (952rng) in butan-2-one (50mi). The resulting solution was refluxed for 75min, further sodium iodide (272rng) was added and refluxing continued for 24h. The reaction mixture was evaporated to low volume (ca. 25mi) and refluxing continued for -24h. The precipitated solid was filtered and washed with ether (2x40m1). This solid was dissolved in water (75m1) and the solution acidified to pH 1.5 by the addition of 2M hydrochloric acid and extracted with dichloromethane (5x750mi). The organic extracts were dried over anhydrous sodium sulphate and evaporated to a foam. This foam was suspended in water (50mi) and 0.1M sodium hydroxide solution was added to pH 7.0. The resulting solution was filtered and freeze-dried to a foam. This foam was treated with acetone (50m1) and the resulting suspension was evaporated. The residue was triturated with ether (50mi) to give the title compound as a yellow solid (588mg); Amax 244.Onm, E] 867, 366.2nm, E] 178; J(d6- DMSO) 5.40(5-H, d, 17Hz, 1H), 530(5-H, d, 17Hz, 1H), 4.80 (14-H, q, 7Hz, 1H), 3.50-4. 00 (POC H2 + OCH,C HO + CH2CH20 (ob scu red), m, 5H), 1. 48(14-CH3, d, 7Hz, 3H).

Example 10 (2-Propenyl),[5,8,13,14-tetrahydro- 14-methyl-B, 13-dioxobenz[5, 6]isoindolo[2, 1-blisoquinolin-9-yll phosphoric acid, sodium salt Sodium iodide (697rng) was added to a solution of Intermediate 4h) (1.59) in acetone (30mi) and the mixture was heated under reflux. After 3h, the reaction mixture was cooled to 20' and 45 the precipitated product was collected by filtration, washed with acetone (10mi) and ether (10mf) and dried to a yellow solid. This solid was dissolved in water (300mi) and the pH of the solution was adjusted to pH 1.5 by the addition of 2114-hydrochloric acid. The resulting mixture was extracted with dichloromethane-ethanol (4A, 300mi) and then dichloromethane (300mi). Salt solution was added during the extraction procedure to disperse the emulsion which was formed. 50 The combined organic extracts were dried over anhydrous sodium sulphate and evaporated to a green foam. This foam was suspended in water (150m1) and the suspension was adjusted to pH 7.0 by addition of 110-sodium hydroxide solution. The resulting solution was filtered and the filtrate was freeze-dried to a yellow foam. Trituration of this foam with ether (100mi) gave the title compound as a yellow solid (1.214g); A,,,, 244.Onm, Ell 883, 367Anm, E] 183; J(d6-DMSO), 55 5.85-6.00(CH, m, 1H), 4.95-5.30(CH, m, 2H), 5.39(5-H, d, 17Hz, 1H), 5. 30(5-H, cl, 17Hz, 1H), 4.82(14-H, q, 7Hz, 1H), 4.30-4.50(OCH2, m, 2H), 1.48(14-CH, d, 7Hz, 3H).

Example 11

Ethyl,[5,8,13,14-tetrahydro- 14-methyl-B, 13-dioxobenz[5,6]isoindolo-[2, 1-blisoquinolin- 12-yllphos- 60 phoric acid, sodium salt Sodium iodide (2.09) was added to a solution of Intermediate 10a) (2.09) in acetone (100mi) and the mixture was heated under reflux for 5h. After this time, a second portion of sodium iodide (2.09) was added and the reaction mixture was maintained under reflux for 16h. The reaction mixture was cooled to 20' and the precipitated product was collected by filtration, 14 GB2195636A 14 washed with acetone (2 X 20mi) and dried to give a green solid (1.759). Crystallisation of this solid (1.759) from a mixture of water (20m1) and acetone (800mi) gave the title compound as a green solid (1.1259); Am,, 243.6nm, E, 973, 367.2m E] 173; & (d6-DMSO) 5.41 (5H,d,16Hz,1H), 5.31 (5- H,d,16Hz,1H), 4.87 (14-H, q, 7Hz, 1H), 3.88 (OCH2CH, m, 2H), 1.45 (145 CH, ,d,71-1z,31-1), 1.10 (OCH,CH,,t,6Hz,3H).

Example 12 2-Methoxyethyl,[5, 8, 13,14-tetrahydro- 14-methyl-8, 13dioxobenz[5,6]isoindolo[2, 1 -blisoquinolin-9yllphosphoric acid, sodium salt Sodium iodide (0.637g) was added to a solution of Intermediate 4j) (2. 039) in acetone (40mi). 10 The resulting solution was refluxed for 3h and more sodium iodide (1. 2749) was added. Refluxing was continued for 20h. The reaction mixture was evaporated and the residue was redissolved in 2-butanone (40mi). The resulting solution was refluxed for 2h and then evaporated. The residue was triturated with ether (3x25mi) to give a solid. Water (200mi) was added and the resulting suspension was acidified to pH 1.5 with 2M hydrochloric acid and extracted with 15 dichloromethane (3x300mi). Salt solution was added during the extraction to disperse an emulsion. The organic extracts were dried over anhydrous sodium sulphate and evaporated to a solid. This solid was suspended in water (200mi) and 0.1M sodium hydroxide solution was added to pH 7.0. The mixture was filtered and the filtrate was freeze-dried to a foam. Treatment of the foam with acetone (120mi) gave a solution which was filtered and evaporated. The residue was triturated with ether (40mi) to give the title compound as a yellow solid (1.34g); Ar,, 244.Onm, E] 1034, 366.8nm, E] 208;.5(d,-DMSO) 5.40(5-H, d, 17Hz, 11-1), 5.30(5-H, cl, 17Hz, 11-1), 4. 80(14-H, q, 7Hz, 11-1), 3.96(POCH, m, 2H), 3.44(POCH2CH2, q, 5Hz, 2H), 3. 22(0CH, s, 31-1), 1.47(14-CH, cl, 7Hz, 3H).

Example 13 Propyl,[5,8,13,14-tetrahydro- 14-methyl-B, 13-dioxobenz[5, 6]isoindolo[2, 1-blisoquinolin-9,12-diyllphosphoric acid, sodium salt Sodi - urn iodide (2.5g) was added to as solution of Intermediate 12 in butan-2-one (50mi) and the reaction mixture was stirred under reflux for 4h and evaporated to dryness. The residue was 30 dissolved in methanol and chromatographed on Sephadex LH 20 in methanol twice to give title compound as a dark yellow solid (595m9), (water) 244.8nm, E] 637, 356.4nm, E] 140; 9 (DIVISO) 5.28+5.38 (5H), 4.82 (14H), 3.65-3.85 (CH2), 1.35-1.6(14CH3+CH2), 0.70-0.90CH, Example 14

4-Nitrophenylmethyl,[5,8,13,14-tetrahydro- 14-methyl-8, 13-dioxobenz[5, 6]isoindolo[2, 1-blisoqui nolin-9-yllphosphoric acid, triethylamine salt Triethylamine hydroiodide (278rng) was added to a solution of Intermediate 5b) (750rng) in acetone (75mi). The resulting solution was refluxed for 4h. Further triethylamine hydroiodide (278rng) was added and refluxing continued for a further 17h. The reaction mixture was evapo- 40 rated and the residual oil was washed with ether. The oil was dissolved in water (100mi) and extracted with dichloromethane (2 x 1 00m1) then with dichforomethane- methanol (1: 1, 1 00mi) then with dichloromethane (100mi). The combined organic extracts were dried over anhydrous sodium sulphate and evaporated. Water (250mi) was added to the residue and the resulting solution was filtered and freeze-dried to a foam. This foam was dissolved in water (50mi) and 45 the solution filtered. The filtrate was acidified to pH 1.5 with 2M hydrochloric acid and extracted with dichloromethane-methanol (2A, 450m1) then with dichloromethane (100mi). The combined organic extracts were washed with water and dried over anhydrous sodium sulphate. Triethylam ine (0.1mi, 0.79) was added and the solution was evaporated. The residual oil was dissolved in water- (30mi), the solution was filtered and the filtrate was freeze- dried to give the title corn pound as a yellow foam (256mg); A,,,, 244.2nrn, E] 739, 365.8nm, E] 129; 05(d,-DMSO) 5.39(5 H, d, 17Hz, 1H), 5.29(5-H, d, 17Hz, 11-1), 5.10(POCH, cl, 8Hz, 2H), 4. 80(14-H, q, 7Hz, 11-1), 2.98-3.13(N±CH,CH3, m, 6H), 1.47(14-CH, d, 7Hz, 3H), 1.18(N-I-CH,CH3, t, 7Hz, 9H).

Example 15

Phenylmethyj,[5,8.13,14-tetrahydro- 14-methyl-B,.13-dioxobenz[5, 6]isoindolo[2, 1blisoquinolin-9-yll phosphoric acid Sodium iodide (285m9) was added to a solution of Intermediate 5a) (1. 019g) in acetone (25mi) and the mixture was heated under reflux. After 1.5h, the reaction mixture was evaporated to dryness and the residue was diluted with water (100mi), acidified to pH 2 to 3 with 2M- 60 hydrochloric acid and extracted with dichloromethane (3 x 1 00mi). Salt solution was added during the extraction procedure to disperse the emulsion which was formed. The combined organic extracts were dried over anhydrous sodium sulphate and evaporated to a green oil which - solidified. The solid was stirred in ether (50m1) for 10 min. and then collected by filtration to give a green solid. Crystallisation of the solid from a mixture of chloroform (containing a trace of 65 1 1 Z1 GB2195636A 15 ethanol) and ether gave the title compound as green crystals (427mg); A,,, ,,,245.Onm, Ell 979, 369.Onm, Ell 193; J(dCIDIVISO) 5.44 (5-H, d, 16Hz, 1H), 5.36(5-H, d, 16Hz, 1H), 5.23(OCH, d, 8Hz, 2H), 4.86 (14-H, q, 7Hz, 1H), 1.51(14-CH3, d, 7Hz, 3H).

1 r Example 16 Phenylmethyl,[5,8,13,14-tetrahydro- 14-methyl-S, 13dioxobenz[5,6]isoindo1o[2, 1 -b]isoquino1in-9-y1Jphosphoric acid, sodium salt A solution of sodium 2-ethylhexanoate (1.55g) in tetrahydrofuran (14ml) was added over 2 min to a stirred suspension of the product of Example 15 (4g) under nitrogen. Water (0.4ml) was added to the resulting clear solution which was then diluted with propan-2-ol (215ml) over 10 min. The resulting suspension was stirred for 20 min and the solid was collected by filtration, washed with propan-2-ol (3 x 30ml), sucked dry under nitrogen and dried in vacuo to give the title compound as a yellowgreen amorphous solid (3.19g); Am 244nm (MeOH), Ell 843, having a similar 1H N.m.r. spectrum to the sample obtained in Example 2a).

Example 17

Phenylmethyl,[5,8,13,14-tetrahydro- 14-methyl-8,13-dioxobenz[5, 6]isoindolo[2, 1-blisoquinolin-9-yl jphosphoric acid, potassium salt Potassium iodide (1.83g) was added to a suspension of Intermediate 5a) (5. 909) in a mixture of acetone (118mi) and water (2.93m]). This mixture was stirred and heated to reflux under 20 nitrogen for 2.5 h, a crystalline precipitate forming after 40 min. The suspension was cooled to 21' and stirred for 1 h and the solid was collected by filtration, washed with acetone (2x25mi) and dried in vacuo to give the title compound (4.329); Amz,>, 244nrn (MeOH), Ell 813; (5 (d,-DMSO) 5.36 (5-H,d,16Hhz,1H), 5.24 (5-H,d,16Hz,1H), 4.96 (OCH2,d,6Hz,2H), 4.80 (14-H,q,7Hz,1H), 1.48 (14-CH3,d,7Hz,3H).

Example 18

Phenylmethyl,[5,8,13,14-tetrahydro- 14-methyl-B, 13-dioxobenz[5, 6]isoindolo[2, 1-blisoquinolin-9-yll phosphoric acid, lithium salt Lithium iodide trihydrate (1.88g) was added to a suspension of Intermediate 5a) (5.909) in a 30 mixture of acetone (1 18mi) and water (2.3mi). This mixture was stirred and heated to reflux under nitrogen for 2.7 h, a crystalline precipitate forming after 20 min. The suspension was cooled to ca. 20' and stirred for 2 h and the solid was collected by filtration, washed with acetone (3x50mi) and dried in vacuo at 21' to give the title compounnd (4. 729); Am,,, 244nrn (MeOH), E] 847; 3 (d,-DMSO) 5.43 (5-H,d, 1 6Hz, 1 H), 5.31 (5-H,d, 1 6Hz, 1 H), 5.04 (OCH,d,6Hz,2H), 4.84 (14-H,q,7Hz,1 H), 1.49 (14-CH,,d,7Hz,3H) Example 19 Phenylmethyl,[5,8,13,14-tetrahydro- 14-methyl-B, 13dioxobenz[5,61-isoindolo[2, 1-blisoquinolin-9-yl- 40]phosphoric acid, L-arginine salt A suspension of the product of Example 15 (29) in water (80mi) was stirred under nitrogen and a solution of L-arginine monohydrate (0.70g) in water (10m1) was added dropwise over 15 min to adjust the pH to 6.1. The pH was raised to 7.0 by the addition of part (2m1) of a solution of L-arginine (0.1g) in water (5mi), and the resulting clear solution was filtered. The filtrate was freeze-dried to give the title compound as a yellow-green solid (2.61g); Am 244 nm 45 (MeOH), E] 648; (5 (d,-DMSO) 5.41 (5-H,d,16Hz,1H), 5.29 (5-H,d,16Hz,1H), 5.02 (OCH,,d,6Hz,2H), 4.83 (14-H,q,7Hz,1H), 3.14 (NCH,,t,6Hz,2H), 1.50 (14-CH,, d,7Hz,3H).

Example 20

Phenylmethyl,[5,8,13,14-tetrahydro- 14-methyl-B, 13-dioxobenz[5, 6]isoindolo[2, 1-blisoquinolin-9-yll- 50 phosphoric acid, sodium salt (a) Sodium iodide (5.94g) was added to a suspension of Intermediate (5a) (21.2g) in acetone (420mi). This mixture was stirred and heated to reflux under nitrogen for 1.5h, and the resulting solution was cooled to ca. 35' and filtered. The filtrate was concentrated in vacuo to ca. 50 mi, and the residue was stirred while diisopropyl ether (350mi) was added. The resulting suspension 55 was stirred for 45 min, and the green solid was collected by filtration, washed with diisopropyl ether (2 x 1 00mi) and dried in vacuo at 30'. Part (2.09) of this solid was suspended in ref luxing acetone (209) and water (2.Omi) was added. The resulting solution was seeded and stirred under reflux for 5 min while crystallisation occurred. The mixture was cooled to ca 20 over 30 min and refrigerated overnight, and the solid was collected by filtration, washed with chilled acetone (2 x 5m1) and dried in vacuo to give the title compound as crystals (1.779); Am.X 244nrn (MeOH), Ell 857; v,,,,., (Nujol mull) 1662 (C=O), 1250 and 111 Ocm-1 (phosphate); (5(d6-DMSO) 5.41 (5-Hd, 1 6Hz, 1 H), 5.29 (5-H,d, 1 6Hz, 1 H), 4.96 (OCH2,d,6Hz,2H), 4.82 (14-H,q,7Hz, 1 H), 1.49 -(14-CH3,d,7Hz,3H).

(b) Sodium iodide (1.659) was added to a suspension of Intermediate (5a) (5.909) in a 65 16 GB2195636A 16 mixture of acetone (1 18mi) and water (2.93mi). This mixture was stirred and heated to reflux under nitrogen for 2 h, a pale yellow crystalline precipitate forming after 0.8 h. The suspension was cooled to 21' and stirred for 1 h, and the solid was collected by filtration, washed with acetone (2 x 25mi) and dried in vacuo to give the title compound as crystals (4.96g); A a 244nrn 5 (MeOH) Ell 842, having infrared and 'H N.m. r. spectra similar to the sample obtained in Example 20a).

The following are examples of pharmaceutical compositions according to the invention. The term 'Active Ingredient' as used hereinafter means a compound of the invention and may be for example the compounds of Examples 2a) or 8.

Example A-Dry Powder for Injection Active ingredient Trisodium citrate 15 Citric acid mglvial equivalent to 10Orng acid 8.8 0.2 Method 1 Blend the sterile ingredients until homogeneous. Fill aseptically into glass vials. Purge the headspace with nitrogen and close the vials using rubber closures and metal overseals.

Method 2 Dissolve the ingredients in water for injections 13.P. Sterilise the solution by membrane filtration. Aseptically fill into freeze-drying vials and place suitable rubber freeze-drying closures in position. Freeze-dry, filling the vials with nitrogen at the end of the cycle. Fully insert the closures and apply metal overseals.

Constitution Constitute with a suitable sterile vehicle, e.g. using water for injections or using a 5% w/v dextrose solution, as an injection (e.g. in a 10mf volume) or an infusion (e.g. in a 100mi volume).

Example B-Oral Tablet Active Ingredient 35 Microcrystalline cellulose Sodium starch glycolate Magnesium stearate mgItablet equivalent to 250rng acid 231 6 2 Sieve the ingredients and blend until homogeneous. Compress with appropriate punches. The tablets may be covered with a thin polymer coat applied by the usual film coating techniques. A 40 pigment may be included in the film coat.

Claims (19)

CLAIMS -

1. Compounds of the general formula (1) H 3 R 1 1 # T T2 R3 R (I) (wherein R' and R2, which may be the same or different, each represents a hydrogen atom, a hydroxyl group or a group -OP(O)(OH)(OR4) [where R4 represents a hydrogen atom or an alkyl (optionally substituted by a hydroxyl, alkoxy, cyclic ether or cycloalkyl group), alkenyl, cycloalky], 55 - aryl, aralkyl or aroylaikyl group], with the proviso that at least one of R' and R2 represents a group -OP(O)(OH)(OR4); and R3 represents a hydrogen or halogen atom or a methyl group), and salts thereof.

2. Compounds of formula (1) as claimed in claim 1 wherein R4 represents a hydrogen atom or a straight-chain or branched C,-, alkyl (optionally substituted by a hydroxyl, C,-, alkoxy, 3-7 60 membered cyclic ether, or C,_7 cycloalkyl group), C,-, alkenyl, C3-7 cycloalkyl, phenyl, phen C1-3 aikyl or benzoyl Cl-3 alkyl group, wherein any phenyl moiety may optionally be substituted by a hydroxyl, C1-4 alkoxy or nitro group, and salts thereof.

3. Compounds of formula (1) as claimed in either of claims 1 and 2 wherein R4 represents a straight chained C,-, alkyl group, a tetra hyd rofuranyl methyl group, a phen C,3 alkyl group or a 65 Z k 1 1 17 n 15 GB2195636A 17 benzoyl C,3 alkyl group, and salts thereof.

4. Compounds of formula (1) as claimed in any one of claims 1 to 3 wherein R3 represents a hydrogen atom, and salts thereof.

5. Compounds of formula (1) as claimed in any one of claims 1 to 4 wherein R' represents a 5 hydrogen atom, and salts thereof.

6. Compounds of formula (1) as claimed in claim 1 wherein R' and R 2, which may the same or different, each represents a hydrogen atom, a hydroxyl group or a group -OP(O)(OH)(OR4)[where R4 represents a hydrogenn atom or an alkyl (optionally substituted by a hydroxy], alkoxy or cycloalkyl group), aryl or aralkyl group], and R3 is a hydrogen or halogen atom or a methyl group, and salts thereof

7. A compound as claimed in claim 1 being phenyimethyi, [5,8,13,14- tetrahydro-14-methyl8,13-dioxobenz[5,6]isoindolo[2,1-blisoquinolin-9-yilphosphoric acid, or 2oxo-2-phenyle-.

thyi,[5,8,13,14-tetrahydro-14-methyi-8,13-dioxobenz[5,6]isoindolo[2,1b]isoq uinolin-9-yi]phos- phoric acid, or a salt thereof.

8. A compound as claimed in claim 1 being the sodium salt of phenyimethy], [5,8,13,14 tetrahydro-14-methy]-8,13-dioxobenz[5,6]-isoindolo[2,1-b]isoquinolin-9yl]ph osphoric acid.

9. A compound as claimed in claim lbeing the sodium salt of 2-oxo-2phenyle thyl,[5,8,13,14-tetrahydro-14-methyi-8,13-dioxobenz[5,6]isoindolo[2,1blisoq uinolin-9-yllphos- phoric acid.

10. A compound of formula (1) as claimed in any one of the preceding claims, or a physiologically acceptable salt thereof, for use in the treatment of the human or non-human animal body to combat cancer therein.

11. lsoquinoline compounds substantially as herein disclosed in any one of Examples 1 to 20.

12. A pharmaceutical composition comprising as an active ingredient a compound of formula 25 (1) (as defined in claim 1), or a physiologically acceptable salt thereof, together with one or more pharmaceutical carriers or excipients.

13. Pharmaceutical compositions as claimed in claim 12 adapted for parenteral administration.

14. A process for the preparation of compounds of formula (1) (as defined in claim 1) and salts thereof, which process comprises at least one of the following steps:

(A) reacting a compound of formula (2) H 3 IN\ A 1 3 1117 R (] R (2) [wherein R3 is as defined in claim 1; and one of R6 and R7 represents a group -OP(O)(Xl)(X2) 40 (where X' represents an atom or group convenable to a hydroxyl group and X2 represents a hydroxyl group or an X' moiety) and the other represents a hydrogen atom, a hydroxyl group or a group -OP(O)(Xl)(X2)l to convert at least one X' moiety to hydroxyl to produce a compound of formula (1); (B) reacting a compound of formula (3) H 3 0 R8 % N \ / 0 9 (3) [wherein R3 is as defined in claim 1; and one of W' and R9 represents a hydroxyl group and the other represents a hydrogen atom or a hydroxyl group] to convert a hydroxyl group at the 9 55 and/or 12 position to a -OP(O)(OH)(OR4) group (wherein R4 is as defined in claim 1); (C) condensing a quinone of formula (4) 0 R1O 0 R" (4) 60.

18 GB2195636A 18 [wherein one of R10 and W' represents a group -OP(O)(OH)(OR4) or - OP(O)(X1)(X2) (where X' and X2 are as herelinbefore defined) and the other represents a hydrogen atom, a hydroxyl group or a group - OP(O)(OH)(OR4) or -0p(o)(X1)(X2)l with a compounnd of formula (5) H3 COOH N (5) - R12 [wherein R12 represents a hydrogen atom or a -CHO or -COCH3 group] and subsequently, if required, converting one or more X' moieties to hydroxyl; (D) halogenating a compound of formula (1) in which R3 represents a hydrogen atom; and (E) converting a free acid of formula (1) into a salt thereof or a salt of a compound of formula (1) into the free acid.

15. A process as claimed in claim 14 wherein process step (A) is effected by hydrolysis or ether cleavage.

16. A process as claimed in claim 14 wherein process step (B) is effected by reaction of the compound of formula (3) with a halophosphate or pyrophosphate of formula HaW(O)(OH)(OR4), HaIP(O)(X1) (X2), O(P(O)(OH)(OR4))2 or O(P(O)(X1)(X2))2 (where R4 is as defined in claim 1 and X' and X2 are as defined in claim 14), followed where required by conversion of one or more X' moieties to hydroxy].

17. A process as claimed in claim 14 wherein process step (C) is effected in the presence of an alkanoic acid anhydride and at elevated temperature.

18. The use of a compound of formula (1) as defined in claim 1 or a physiologically acceptable salt thereof for the manufacture of a therapeutic agent for the treatment of the human or non-human animal body to combat cancer therein.

19. Compounds of formula (2) (as defined in claim 14) and salts thereof.

Ir Published 1988 at The Patent Office, State House, 66/71 High Holborn, London WC 1 R 4TP. Further copies may be obtained from The Patent Office, Sales Branch, St Mary Cray, Orpington, Kent BR5 3RD. Printed by Burgess & Son (Abingdon) Ltd. Con. 1/87.

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