PHTHALAZINONE DERIVATIVES

Abstract

Compounds of the formula (I):

##STR00001##

wherein A and B together represent an optionally substituted, fused aromatic ring; X can be NR.sup.X or CR.sup.XR.sup.Y; if X=NR.sup.X then n is 1 or 2 and if X=CR.sup.XR.sup.Y then n is 1; R.sup.X is selected from the group consisting of H, optionally substituted C.sub.1-20 alkyl, C.sub.5-20 aryl, C.sub.3-20 heterocyclyl, amido, thioamido, ester, acyl, and sulfonyl groups; R.sup.Y is selected from H, hydroxy, amino; or R.sup.X and R.sup.Y may together form a spiro-C.sub.3-7 cycloalkyl or heterocyclyl group; R.sup.C1 and R.sup.C2 are both hydrogen, or when X is CR.sup.XR.sup.Y, R.sup.C1, R.sup.C2, R.sup.X and R.sup.Y, together with the carbon atoms to which they are attached, may form an optionally substituted fused aromatic ring; and
R.sup.1 is selected from H and halo.

Claims

1.-12. (canceled)

13. A method of synthesising a compound of formula III: ##STR00446## from 2-carboxybenzaldehyde, wherein R.sup.1 is selected from H and halo.

14. A method according to claim 13 comprising the steps of: (a) synthesizing (3-oxo-1,3-dihydro-isobenzofuran-1-yl)phosphonic acid dimethyl ester from 2-carboxybenzaldehyde; (b) synthesizing a compound of formula II: ##STR00447## from (3-oxo-1,3-dihydro-isobenzofuran-1-yl)phosphonic acid dimethyl ester; (c) reacting a compound of formula II with sodium hydroxide, followed by reaction with hydrazine hydrate, followed by acidification with an acid.

15. A method according to claim 14, where in step (a), 2-carboxybenzaldehyde is reacted with the sodium salt of dimethyl phosphite, which is formed in situ by adding dimethyl phosphite to a cooled solution of sodium methoxide in methanol.

16. A method according to claim 15, where in step (a), the reaction with the sodium salt of dimethyl phosphite is followed by reaction with methanesulphonic acid.

17. A method according to claim 14, wherein step (b) is carried out in tetrahydrofuran, with the addition of triethylamine.

18. A method according to claim 14, where in step (c), the acid is hydrochloric acid.

19. A method according to claim 13, wherein R.sup.1 is fluoro.

20. A method of synthesizing a compound of formula I: ##STR00448## from a compound of formula II ##STR00449## wherein R.sup.1 is selected from H and halo; R.sup.2 is selected from optionally substituted C.sub.1-20 alkyl, C.sub.3-20 cycloalkyl, C.sub.3-20 heterocyclyl, and C.sub.5-20 aryl; and n is 1 or 2.

21. A method according to claim 20, comprising the steps of: (a) synthesizing a compound of formula III: ##STR00450## from a compound of formula II by reaction with sodium hydroxide, followed by reaction with hydrazine hydrate; (b) synthesizing a compound of formula IV: ##STR00451## from a compound of formula III; (c) synthesizing a compound of formula V: ##STR00452## from a compound of formula IV; (d) reacting a compound of formula V with an appropriate acid chloride in the presence of a base.

22. A method of synthesizing a compound of formula I: ##STR00453## from 2-carboxybenzaldehyde, wherein R.sup.1 is selected from H and halo; R.sup.2 is selected from optionally substituted C.sub.1-20 alkyl, C.sub.3-20 cycloalkyl, C.sub.3-20 heterocyclyl, and C.sub.5-20 aryl; and n is 1 or 2.

23. A method according to claim 22, comprising the steps of: (a) synthesizing (3-oxo-1,3-dihydro-isobenzofuran-1-yl)phosphonic acid dimethyl ester from 2-carboxybenzaldehyde; (b) synthesizing a compound of formula II: ##STR00454## from (3-oxo-1,3-dihydro-isobenzofuran-1-yl)phosphonic acid dimethyl ester; (c) synthesizing a compound of formula III: ##STR00455## from a compound of formula II by reaction with sodium hydroxide, followed by reaction with hydrazine hydrate; (d) synthesizing a compound of formula IV: ##STR00456## from a compound of formula III; (e) synthesizing a compound of formula ##STR00457## from a compound of formula IV; (f) reacting a compound of formula V with an appropriate acid chloride in the presence of a base.

Description

BRIEF DESCRIPTION OF FIGURES

[0089] FIGS. 1A-B show clonogenic survival curves of cells proficient and deficient in Braca 1 exposed to compound (4) of the present invention.

[0090] FIG. 1A shows Brca1 wild type (11CO:.square-solid.), heterozygous (Cre6: .box-tangle-solidup.) and deficient (Cre10:.circle-solid.) embryonic fibroblastic stem (ES) cells under continuous exposure to compound 4. Error bars represent standard errors of the mean.

[0091] FIG. 1B shows Brca2 wild type (D3:.square-solid.), heterozygous (Cre6: .box-tangle-solidup.) and deficient (Cre24:.circle-solid.) ES cells under continuous exposure to compound 4. Error bars represent standard errors of the mean.

[0092] FIG. 2 shows an analysis of the effects of a compound of the invention (4) in another cell line lacking BRCA2 function in comparison to a BRCA2 complemented line. Data shown is clonogenic survival of Brca2 deficient (V-C8:.square-solid.) and complemented (V-C8 BAC+:.box-tangle-solidup.) cells under continuous exposure to compound 4 at varying concentrations.

FURTHER PREFERENCES

[0093] The following preferences can apply to each aspect of the present invention, where applicable.

[0094] In the present invention, the fused aromatic ring(s) represented by -A-B- preferably consist of solely carbon ring atoms, and thus may be benzene, naphthalene, and is more preferably benzene. As described above, these rings may be substituted, but in some embodiments are preferably unsubstituted.

[0095] If the fused aromatic ring represented by -A-B- bears a substituent group, it is preferably attached to the atom which itself is attached to the central ring meta- to the carbonyl group. Thus, if the fused aromatic ring is a benzene ring, the preferred place of substitution is shown in the formula below by *:

##STR00009##

which is usually termed the 5-position of the phthalazinone moiety.

[0096] R.sup.1 is preferably selected from H, Cl and F, and is more preferably F.

[0097] It is preferred that R.sup.C1 and R.sup.C2 are independently selected from hydrogen and C.sub.1-4 alkyl, and more preferably H and methyl. It is more preferred that at least one of R.sup.C1 and R.sup.C2 are hydrogen, with the most preferred option being that both are hydrogen.

[0098] When n is 2, X is NR.sup.X. In these embodiments, R.sup.X is preferably selected from the group consisting of: H; optionally substituted C.sub.1-20 alkyl (for example, optionally substituted C.sub.5-20 arylmethyl); optionally substituted C.sub.5-20 aryl; optionally substituted ester groups, wherein the ester substituent is preferably C.sub.1-20 alkyl; optionally substituted acyl groups; optionally substituted amido groups; optionally substituted thioamido groups; and optionally substituted sulfonyl groups. R.sup.X is more preferably selected from the group consisting of: H; optionally substituted C.sub.1-20 alkyl; optionally substituted C.sub.5-20 aryl; and optionally substituted ester groups, wherein the ester substituent is preferably C.sub.1-20 alkyl.

[0099] When n is 1, X may be NR.sup.X or CR.sup.XCR.sup.Y.

[0100] In embodiments where X is NR.sup.X, R.sup.X is preferably selected from the group consisting of: H; optionally substituted C.sub.1-20 alkyl (for example, optionally substituted C.sub.5-20 arylmethyl); optionally substituted C.sub.5-20 aryl; optionally substituted acyl; optionally substituted sulfonyl; optionally substituted amido; and optionally substituted thioamido groups.

[0101] In embodiments where X is CR.sup.XR.sup.Y, R.sup.Y is preferably H. R.sup.X is preferably selected from the group consisting of: H; optionally substituted C.sub.1-20 alkyl (for example, optionally substituted C.sub.5-20 arylmethyl); optionally substituted C.sub.5-20 aryl; optionally substituted C.sub.3-20 heterocyclyl; optionally substituted acyl, wherein the acyl substituent is preferably selected from C.sub.5-20 aryl and C.sub.3-20 heterocyclyl (e.g. piperazinyl); optionally substituted amido, wherein the amino groups are preferably selected from H and C.sub.1-20 alkyl or together with the nitrogen atom, form a C.sub.5-20 heterocyclic group; and optionally substituted ester groups, wherein the ester substituent is preferably selected from C.sub.1-20 alkyl groups.

[0102] Particularly preferred compounds include: 1, 2, 3, 4, 10, 20, 59, 80, 135, 146, 192, 194, 195, 211 and 212.

[0103] Where appropriate, the above preferences may be taken in combination with each other.

Includes Other Forms

[0104] Included in the above are the well known ionic, salt, solvate, and protected forms of these substituents. For example, a reference to carboxylic acid (—COOH) also includes the anionic (carboxylate) form (—COO—), a salt or solvate thereof, as well as conventional protected forms. Similarly, a reference to an amino group includes the protonated form (—N.sup.+HR.sup.1R.sup.2), a salt or solvate of the amino group, for example, a hydrochloride salt, as well as conventional protected forms of an amino group. Similarly, a reference to a hydroxyl group also includes the anionic form (—O.sup.−), a salt or solvate thereof, as well as conventional protected forms of a hydroxyl group.

Isomers, Salts, Solvates, Protected Forms, and Prodrugs

[0105] Certain compounds may exist in one or more particular geometric, optical, enantiomeric, diasterioisomeric, epimeric, stereoisomeric, tautomeric, conformational, or anomeric forms, including but not limited to, cis- and trans-forms; E- and Z-forms; c-, t-, and r-forms; endo- and exo-forms; R-, S-, and meso-forms; D- and L-forms; d- and i-forms; (+) and (−) forms; keto-, enol-, and enolate-forms; syn- and anti-forms; synclinal- and anticlinal-forms; α- and β-forms; axial and equatorial forms; boat-, chair-, twist-, envelope-, and halfchair-forms; and combinations thereof, hereinafter collectively referred to as “isomers” (or “isomeric forms”).

[0106] If the compound is in crystalline form, it may exist in a number of different polymorphic forms.

[0107] Note that, except as discussed below for tautomeric forms, specifically excluded from the term “isomers”, as used herein, are structural (or constitutional) isomers (i.e. isomers which differ in the connections between atoms rather than merely by the position of atoms in space). For example, a reference to a methoxy group, —OCH.sub.3, is not to be construed as a reference to its structural isomer, a hydroxymethyl group, —CH.sub.2OH. Similarly, a reference to ortho-chlorophenyl is not to be construed as a reference to its structural isomer, meta-chlorophenyl. However, a reference to a class of structures may well include structurally isomeric forms falling within that class (e.g., C.sub.1-7 alkyl includes n-propyl and iso-propyl; butyl includes n-, iso-, sec-, and tert-butyl; methoxyphenyl includes ortho-, meta-, and para-methoxyphenyl).

[0108] The above exclusion does not pertain to tautomeric forms, for example, keto-, enol-, and enolate-forms, as in, for example, the following tautomeric pairs: keto/enol, imine/enamine, amide/imino alcohol, amidine/amidine, nitroso/oxime, thioketone/enethiol, N-nitroso/hyroxyazo, and nitro/aci-nitro.

[0109] Particularly relevant to the present invention is the tautomeric pair illustrated below:

##STR00010##

[0110] Note that specifically included in the term “isomer” are compounds with one or more isotopic substitutions. For example, H may be in any isotopic form, including .sup.1H, .sup.2H (D), and .sup.3H (T); C may be in any isotopic form, including .sup.12C, .sup.13C, and .sup.14C; O may be in any isotopic form, including .sup.16O and .sup.18O; and the like.

[0111] Unless otherwise specified, a reference to a particular compound includes all such isomeric forms, including (wholly or partially) racemic and other mixtures thereof. Methods for the preparation (e.g. asymmetric synthesis) and separation (e.g. fractional crystallisation and chromatographic means) of such isomeric forms are either known in the art or are readily obtained by adapting the methods taught herein, or known methods, in a known manner.

[0112] Unless otherwise specified, a reference to a particular compound also includes ionic, salt, solvate, and protected forms of thereof, for example, as discussed below, as well as its different polymorphic forms.

[0113] It may be convenient or desirable to prepare, purify, and/or handle a corresponding salt of the active compound, for example, a pharmaceutically-acceptable salt. Examples of pharmaceutically acceptable salts are discussed in Berge, et al., “Pharmaceutically Acceptable Salts”, J. Pharm. Sci., 66, 1-19 (1977).

[0114] For example, if the compound is anionic, or has a functional group which may be anionic (e.g., —COOH may be —COO—), then a salt may be formed with a suitable cation. Examples of suitable inorganic cations include, but are not limited to, alkali metal ions such as Na.sup.+ and K.sup.+, alkaline earth cations such as Ca.sup.2+ and Mg.sup.2+, and other cations such as Al.sup.3+. Examples of suitable organic cations include, but are not limited to, ammonium ion (i.e., NH.sub.4.sup.+) and substituted ammonium ions (e.g., NH.sub.3R.sup.+, NH.sub.2R.sub.2.sup.+, NHR.sub.3.sup.+, NR.sub.4.sup.+). Examples of some suitable substituted ammonium ions are those derived from: ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as amino acids, such as lysine and arginine. An example of a common quaternary ammonium ion is N(CH.sub.3).sub.4.sup.+.

[0115] If the compound is cationic, or has a functional group which may be cationic (e.g., —NH.sub.2 may be —NH.sub.3.sup.+), then a salt may be formed with a suitable anion. Examples of suitable inorganic anions include, but are not limited to, those derived from the following inorganic acids: hydrochloric, hydrobromic, hydroiodic, sulfuric, sulfurous, nitric, nitrous, phosphoric, and phosphorous. Examples of suitable organic anions include, but are not limited to, those derived from the following organic acids: acetic, propionic, succinic, gycolic, stearic, palmitic, lactic, malic, pamoic, tartaric, citric, gluconic, ascorbic, maleic, hydroxymaleic, phenylacetic, glutamic, aspartic, benzoic, cinnamic, pyruvic, salicyclic, sulfanilic, 2-acetyoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethanesulfonic, ethane disulfonic, oxalic, isethionic, valeric, and gluconic. Examples of suitable polymeric anions include, but are not limited to, those derived from the following polymeric acids: tannic acid, carboxymethyl cellulose.

[0116] It may be convenient or desirable to prepare, purify, and/or handle a corresponding solvate of the active compound. The term “solvate” is used herein in the conventional sense to refer to a complex of solute (e.g. active compound, salt of active compound) and solvent. If the solvent is water, the solvate may be conveniently referred to as a hydrate, for example, a mono-hydrate, a di-hydrate, a tri-hydrate, etc.

[0117] It may be convenient or desirable to prepare, purify, and/or handle the active compound in a chemically protected form. The term “chemically protected form,” as used herein, pertains to a compound in which one or more reactive functional groups are protected from undesirable chemical reactions, that is, are in the form of a protected or protecting group (also known as a masked or masking group or a blocked or blocking group). By protecting a reactive functional group, reactions involving other unprotected reactive functional groups can be performed, without affecting the protected group; the protecting group may be removed, usually in a subsequent step, without substantially affecting the remainder of the molecule. See, for example, “Protective Groups in Organic Synthesis” (T. Green and P. Wuts; 3rd Edition; John Wiley and Sons, 1999).

[0118] For example, a hydroxy group may be protected as an ether (—OR) or an ester (—OC(═O)R), for example, as: a t-butyl ether; a benzyl, benzhydryl (diphenylmethyl), or trityl (triphenylmethyl) ether; a trimethylsilyl or t-butyldimethylsilyl ether; or an acetyl ester (—OC(═O)CH.sub.3, —OAc).

[0119] For example, an aldehyde or ketone group may be protected as an acetal or ketal, respectively, in which the carbonyl group (>C═O) is converted to a diether (>C(OR).sub.2), by reaction with, for example, a primary alcohol. The aldehyde or ketone group is readily regenerated by hydrolysis using a large excess of water in the presence of acid.

[0120] For example, an amine group may be protected, for example, as an amide or a urethane, for example, as: a methyl amide (—NHCO—CH.sub.3); a benzyloxy amide (—NHCO—OCH.sub.2C.sub.6H.sub.5, —NH-Cbz); as a t-butoxy amide (—NHCO—OC(CH.sub.3).sub.3, —NH-Boc); a 2-biphenyl-2-propoxy amide (—NHCO—OC(CH.sub.3).sub.2C.sub.6H.sub.4C.sub.6H.sub.5, —NH-Bpoc), as a 9-fluorenylmethoxy amide (—NH-Fmoc), as a 6-nitroveratryloxy amide (—NH-Nvoc), as a 2-trimethylsilylethyloxy amide (—NH-Teoc), as a 2,2,2-trichloroethyloxy amide (—NH-Troc), as an allyloxy amide (—NH-Alloc), as a 2(-phenylsulphonyl)ethyloxy amide (—NH-Psec); or, in suitable cases, as an N-oxide (>NO.).

[0121] For example, a carboxylic acid group may be protected as an ester for example, as: a C.sub.1-7 alkyl ester (e.g. a methyl ester; a t-butyl ester); a C.sub.1-7haloalkyl ester (e.g. a C.sub.1-7 trihaloalkyl ester); a triC.sub.1-7 alkylsilyl-C.sub.1-7 alkyl ester; or a C.sub.5-20 aryl-C.sub.1-7 alkyl ester (e.g. a benzyl ester; a nitrobenzyl ester); or as an amide, for example, as a methyl amide.

[0122] For example, a thiol group may be protected as a thioether (—SR), for example, as: a benzyl thioether; an acetamidomethyl ether (—S—CH.sub.2NHC(═O)CH.sub.3).

[0123] It may be convenient or desirable to prepare, purify, and/or handle the active compound in the form of a prodrug. The term “prodrug”, as used herein, pertains to a compound which, when metabolised (e.g. in vivo), yields the desired active compound. Typically, the prodrug is inactive, or less active than the active compound, but may provide advantageous handling, administration, or metabolic properties.

[0124] For example, some prodrugs are esters of the active compound (e.g. a physiologically acceptable metabolically labile ester). During metabolism, the ester group (—C(═O)OR) is cleaved to yield the active drug. Such esters may be formed by esterification, for example, of any of the carboxylic acid groups (—C(═O)OH) in the parent compound, with, where appropriate, prior protection of any other reactive groups present in the parent compound, followed by deprotection if required. Examples of such metabolically labile esters include, but are not limited to, those wherein R is C.sub.1-20 alkyl (e.g. -Me, -Et); C.sub.1-7 aminoalkyl (e.g. aminoethyl; 2-(N,N-diethylamino)ethyl; 2-(4-morpholino)ethyl); and acyloxy-C.sub.1-7 alkyl (e.g. acyloxymethyl; acyloxyethyl; e.g. pivaloyloxymethyl; acetoxymethyl; 1-acetoxyethyl; 1-(1-methoxy-1-methyl)ethyl-carbonxyloxyethyl; 1-(benzoyloxy)ethyl; isopropoxy-carbonyloxymethyl; 1-isopropoxy-carbonyloxyethyl; cyclohexyl-carbonyloxymethyl; 1-cyclohexyl-carbonyloxyethyl; cyclohexyloxy-carbonyloxymethyl; 1-cyclohexyloxy-carbonyloxyethyl; (4-tetrahydropyranyloxy) carbonyloxymethyl; 1-(4-tetrahydropyranyloxy)carbonyloxyethyl; (4-tetrahydropyranyl)carbonyloxymethyl; and 1-(4-tetrahydropyranyl)carbonyloxyethyl).

[0125] Further suitable prodrug forms include phosphonate and glycolate salts. In particular, hydroxy groups (—OH), can be made into phosphonate prodrugs by reaction with chlorodibenzylphosphite, followed by hydrogenation, to form a phosphonate group —O—P(═O)(OH).sub.2. Such a group can be cleaved by phosphatase enzymes during metabolism to yield the active drug with the hydroxy group. Also, some prodrugs are activated enzymatically to yield the active compound, or a compound which, upon further chemical reaction, yields the active compound. For example, the prodrug may be a sugar derivative or other glycoside conjugate, or may be an amino acid ester derivative.

Acronyms

[0126] For convenience, many chemical moieties are represented using well known abbreviations, including but not limited to, methyl (Me), ethyl (Et), n-propyl (nPr), iso-propyl (iPr), n-butyl (nBu), tert-butyl (tBu), n-hexyl (nHex), cyclohexyl (cHex), phenyl (Ph), biphenyl (biPh), benzyl (Bn), naphthyl (naph), methoxy (MeO), ethoxy (EtO), benzoyl (Bz), and acetyl (Ac).

[0127] For convenience, many chemical compounds are represented using well known abbreviations, including but not limited to, methanol (MeOH), ethanol (EtOH), iso-propanol (i-PrOH), methyl ethyl ketone (MEK), ether or diethyl ether (Et.sub.2O), acetic acid (AcOH), dichloromethane (methylene chloride, DCM), trifluoroacetic acid (TFA), dimethylformamide (DMF), tetrahydrofuran (THF), and dimethylsulfoxide (DMSO).

Synthesis

[0128] In the synthesis routes given below, the A-B fused ring is shown as a fused benzene ring for convenience. Compounds in which the A-B ring is other than benzene may be synthesised using methodologies analogous to those described below by the use of appropriate alternative starting materials.

[0129] Compounds of the present invention may be synthesised by reaction of a compound of Formula 1:

##STR00011##

in which R.sup.1 is as previously defined, with a compound of Formula 2:

##STR00012##

in which n, R.sup.C1, R.sup.C2 and X are as previously defined, in the presence of a coupling reagent system, for example 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate, 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate or (dimethylaminopropyl)ethylcarbodiimide hydrochloride/hydroxybenzotriazole, in the presence of a base, for example diisopropylethylamine, in a solvent, for example dimethylacetamide or dichloromethane, at a temperature in the range of 0° C. to the boiling point of the solvent used.

[0130] Alternatively, compounds of the present invention may be synthesised by conversion of a compound of Formula 1 into an activated species, for example an acid chloride or an activated ester such as an N-hydroxysuccinimide ester, using well-known methodologies, and reaction of the activated species with a compound of Formula 2.

[0131] Compounds of Formula 1 may be synthesised by reaction of a compound of Formula 3:

##STR00013##

in which R.sup.1 is as previously defined, or a compound of Formula 4:

##STR00014##

in which R.sup.1 is as previously defined, or a mixture of a compound of Formula 3 and a compound of Formula 4, with a source of hydrazine, for example hydrazine hydrate, optionally in the presence of a base, for example triethylamine, optionally in the presence of a solvent, for example industrial methylated spirit, at a temperature in the range of 0° C. to the boiling point of the solvent used.

[0132] Compounds of Formula 3 or Formula 4, or mixtures thereof, may be synthesised by reaction of a compound of Formula 5:

##STR00015##

in which R.sup.1 is as previously defined, with a reagent capable of hydrolysing a nitrile moiety, for example sodium hydroxide, in the presence of a solvent, for example water, at a temperature in the range of 0° C. to the boiling point of the solvent used.

[0133] Compounds of Formula 5 may be synthesised by reaction of a compound of Formula 6:

##STR00016##

in which R.sup.1 is as previously defined, with a compound of Formula 7:

##STR00017##

in the presence of a base, for example sodium methoxide, in a solvent, for example methanol, optionally in the presence of a water scavenger, for example ethyl propionate, at a temperature in the range of 0° C. to the boiling point of the solvent used.

[0134] Compounds of Formula 1 may also be synthesised by reaction of a compound of Formula 8:

##STR00018##

in which R.sup.1 is as previously defined, with a reagent capable of hydrolysing a nitrile moiety, for example sodium hydroxide, in the presence of a solvent, for example water, at a temperature in the range of 0° C. to the boiling point of the solvent used, followed by reaction of the resulting intermediate with a source of hydrazine, for example hydrazine hydrate, at a temperature in the range of 0° C. to the boiling point of the solvent used.

[0135] Compounds of Formula 8 may be synthesised by reaction of a compound of Formula 9:

##STR00019##

in which R.sub.a is a C.sub.1-4 alkyl group, with a compound of Formula 6, in the presence of a base, for example triethylamine or lithium hexamethyldisilazide, in the presence of a solvent, for example tetrahydrofuran, at a temperature in the range of −80° C. to the boiling point of the solvent used.

[0136] Compounds of Formula 9 may be synthesised by methods analogous to those described in WO 02/26576.

[0137] Compounds of Formula 1 may also be synthesised by methods analogous to those described above in which the nitrile moiety in all Formulae is replaced by other moieties capable of generating a carboxylic acid, for example ester or carboxamide moieties.

[0138] Compounds of Formula 2 are commercially available or may be synthesised by methods reported in the chemical literature.

[0139] Compounds of the present invention in which X is CR.sup.XR.sup.Y, in which one of R.sup.X or R.sup.Y is an amido moiety, and which may therefore be represented by Formula 10:

##STR00020##

in which n, R.sup.C1, R.sup.C2, R.sup.1 and R.sup.X are as previously defined and R.sup.N1 and R.sup.N2 are each individually selected from the group consisting of H, optionally substituted C.sub.1-20 alkyl, C.sub.5-20 aryl, C.sub.3-20 heterocyclyl, or may together form an optionally substituted C.sub.3-7 cycloalkyl or heterocyclyl group, may be synthesised by reaction of a compound of Formula 11:

##STR00021##

in which n, R.sup.C1, R.sup.C2, R.sup.1 and R.sup.X are as previously defined, with a compound of Formula HNR.sup.N1R.sup.N2 in which R.sup.N1 and R.sup.N2 are as previously defined, in the presence of a coupling reagent system, for example 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate, 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate or (dimethylaminopropyl)ethylcarbodiimide hydrochloride/hydroxybenzotriazole, in the presence of a base, for example diisopropylethylamine, in a solvent, for example dimethylacetamide or dichloromethane, at a temperature in the range of 0° C. to the boiling point of the solvent used.

[0140] Alternatively, compounds of Formula 10 may be synthesised by conversion of a compound of Formula 11 into an activated species, for example an acid chloride or an activated ester such as an N-hydroxysuccinimide ester, using well-known methodologies, and reaction of the activated species with a compound of Formula HNR.sup.N1R.sup.N2.

[0141] Compounds of Formula 11 may be synthesised by deprotection of a protected form of a compound of Formula 11, for example a compound of Formula 12:

##STR00022##

in which n, R.sup.C1, R.sup.C2, R.sup.1 and R.sup.X are as previously defined and R.sup.01 is a C.sub.1-4 alkyl group, using well known methodologies, for example base-catalysed hydrolysis in the presence of a source of hydroxide, for example sodium or lithium hydroxide, in the presence of a solvent, for example water and/or tetrahydrofuran, at a temperature in the range of 0° C. to the boiling point of the solvent used.

[0142] Compounds of Formula 12 may be synthesised from compounds of Formula 1 by the previously described methods.

[0143] Compounds of Formula HNR.sup.N1R.sup.N2 are commercially available or may be synthesised by methods reported in the chemical literature.

[0144] Compounds of the present invention in which X is NH and which may therefore be represented by Formula 13:

##STR00023##

in which n, R.sup.C1, R.sup.C2 and R.sup.1 are as previously defined, may be synthesised by deprotection of a protected form of a compound of Formula 13, for example a compound of Formula 14:

##STR00024##

in which n, R.sup.C1, R.sup.C2 and R.sup.1 are as previously defined, using well known methodologies, for example acid-catalysed cleavage, in the presence of an acid, for example trifluoroacetic acid or hydrochloric acid, in the presence of a solvent, for example dichloromethane or ethanol and/or water, at a temperature in the range of 0° C. to the boiling point of the solvent used.

[0145] Compounds of Formula 14 may be synthesised from compounds of Formula 1 by the previously described methods.

[0146] Compounds of the present invention in which X is NR.sup.X, in which R.sup.X is an acyl moiety, and which may therefore be represented by Formula 15:

##STR00025##

in which n, R.sup.C1, R.sup.C2 and R.sup.1 are as previously defined and R.sup.C3 is selected from the group consisting of optionally substituted C.sub.1-20 alkyl, C.sub.5-20 aryl and C.sub.3-20 heterocyclyl, may be synthesised by reaction of a compound of Formula 13 with a compound of Formula R.sup.C3COX, in which R.sup.C3 is as previously defined and X is a suitable leaving group, for example a halogen such as chloro, optionally in the presence of a base, for example pyridine, triethylamine or diisopropylethylamine, optionally in the presence of a solvent, for example dichloromethane, at a temperature in the range of 0° C. to the boiling point of the solvent used.

[0147] Compounds of Formula R.sup.C3COX are commercially available or may be synthesised by methods reported in the chemical literature.

[0148] Compounds of Formula 15 may also be synthesised by reaction of a compound of Formula 13 with a compound of Formula R.sup.C3CO.sub.2H, in which R.sup.C3 is as previously defined, in the presence of a coupling reagent system, for example 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate, 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate or (dimethylaminopropyl)ethylcarbodiimide hydrochloride/hydroxybenzotriazole, in the presence of a base, for example diisopropylethylamine, in a solvent, for example dimethylacetamide or dichloromethane, at a temperature in the range of 0° C. to the boiling point of the solvent used.

[0149] Compounds of Formula R.sup.C3CO.sub.2H are commercially available or may be synthesised by methods reported in the chemical literature.

[0150] Compounds of the present invention in which X is NR.sup.X, in which R.sup.X is an amido or thioamido moiety, and which may therefore be represented by Formula 16:

##STR00026##

in which n, R.sup.C1, R.sup.C2 and R.sup.1 are as previously defined, Y is O or S and R.sup.N3 is selected from the group consisting of optionally substituted C.sub.1-20 alkyl, C.sub.5-20 aryl and C.sub.3-20 heterocyclyl, may be synthesised by reaction of a compound of Formula 13 with a compound of Formula R.sup.N3NCY, in which Y and R.sup.N3 are as previously defined, in the presence of a solvent, for example dichloromethane, at a temperature in the range of 0° C. to the boiling point of the solvent used.

[0151] Compounds of Formula R.sup.N3NCY are commercially available or may be synthesised by methods reported in the chemical literature.

[0152] Compounds of the present invention in which X is NR.sup.X, in which R.sup.X is a sulfonyl moiety, and which may therefore be represented by Formula 17:

##STR00027##

in which n, R.sup.C1, R.sup.C2 and R.sup.1 are as previously defined and R.sup.S1 is selected from the group consisting of optionally substituted C.sub.1-20 alkyl, C.sub.5-20 aryl and C.sub.3-20 heterocyclyl, may be synthesised by reaction of a compound of Formula 13 with a compound of Formula R.sup.S1SO.sub.2Cl, in which R.sup.S1 is as previously defined, optionally in the presence of a base, for example pyridine, triethylamine or diisopropylethylamine, in the presence of a solvent, for example dichloromethane, at a temperature in the range of 0° C. to the boiling point of the solvent used.

[0153] Compounds of Formula R.sup.S1SO.sub.2Cl are commercially available or may be synthesised by methods reported in the chemical literature.

[0154] Compounds of the present invention in which X is NR.sup.X, in which R.sup.X is selected from the group consisting of optionally substituted C.sub.1-20 alkyl or C.sub.3-20 heterocyclyl, and which may therefore be represented by Formula 18:

##STR00028##

in which n, R.sup.C1, R.sup.C2 and R.sup.1 are as previously defined and R.sup.C4 and R.sup.C5 are each individually selected from the group consisting of H, optionally substituted C.sub.1-20 alkyl, C.sub.5-20 aryl, C.sub.3-20 heterocyclyl, or may together form an optionally substituted C.sub.3-7 cycloalkyl or heterocyclyl group, may be synthesised by reaction of a compound of Formula 13 with a compound of Formula R.sup.C4COR.sup.C5, in which R.sup.C4 and R.sup.C5 are as previously defined, in the presence of a reducing agent, for example sodium cyanoborohydride or sodium triacetoxyborohydride, in the presence of a solvent, for example methanol, optionally in the presence of an acid catalyst, for example acetic acid, at a temperature in the range of 0° C. to the boiling point of the solvent used.

[0155] Compounds of Formula R.sup.C4COR.sup.C5 are commercially available or may be synthesised by methods reported in the chemical literature.

Use

[0156] The present invention provides active compounds, specifically, active in inhibiting the activity of PARP.

[0157] The term “active” as used herein, pertains to compounds which are capable of inhibiting PARP activity, and specifically includes both compounds with intrinsic activity (drugs) as well as prodrugs of such compounds, which prodrugs may themselves exhibit little or no intrinsic activity.

[0158] One assay which may conveniently be used in order to assess the PARP inhibition offered by a particular compound is described in the examples below.

[0159] The present invention further provides a method of inhibiting the activity of PARP in a cell, comprising contacting said cell with an effective amount of an active compound, preferably in the form of a pharmaceutically acceptable composition. Such a method may be practised in vitro or in vivo.

[0160] For example, a sample of cells may be grown in vitro and an active compound brought into contact with said cells, and the effect of the compound on those cells observed. As examples of “effect”, the amount of DNA repair effected in a certain time may be determined. Where the active compound is found to exert an influence on the cells, this may be used as a prognostic or diagnostic marker of the efficacy of the compound in methods of treating a patient carrying cells of the same cellular type.

[0161] The term “treatment”, as used herein in the context of treating a condition, pertains generally to treatment and therapy, whether of a human or an animal (e.g. in veterinary applications), in which some desired therapeutic effect is achieved, for example, the inhibition of the progress of the condition, and includes a reduction in the rate of progress, a halt in the rate of progress, amelioration of the condition, and cure of the condition. Treatment as a prophylactic measure (i.e. prophylaxis) is also included.

[0162] The term “adjunct” as used herein relates to the use of active compounds in conjunction with known therapeutic means. Such means include cytotoxic regimens of drugs and/or ionising radiation as used in the treatment of different cancer types. In particular, the active compounds are known to potentiate the actions of a number of cancer chemotherapy treatments, which include the topoisomerase class of poisons and most of the known alkylating agents used in treating cancer.

[0163] Active compounds may also be used as cell culture additives to inhibit PARP, for example, in order to sensitize cells to known chemotherapeutic agents or ionising radiation treatments in vitro.

[0164] Active compounds may also be used as part of an in vitro assay, for example, in order to determine whether a candidate host is likely to benefit from treatment with the compound in question.

Administration

[0165] The active compound or pharmaceutical composition comprising the active compound may be administered to a subject by any convenient route of administration, whether systemically/peripherally or at the site of desired action, including but not limited to, oral (e.g. by ingestion); topical (including e.g. transdermal, intranasal, ocular, buccal, and sublingual); pulmonary (e.g. by inhalation or insufflation therapy using, e.g. an aerosol, e.g. through mouth or nose); rectal; vaginal; parenteral, for example, by injection, including subcutaneous, intradermal, intramuscular, intravenous, intraarterial, intracardiac, intrathecal, intraspinal, intracapsular, subcapsular, intraorbital, intraperitoneal, intratracheal, subcuticular, intraarticular, subarachnoid, and intrasternal; by implant of a depot, for example, subcutaneously or intramuscularly.

[0166] The subject may be a eukaryote, an animal, a vertebrate animal, a mammal, a rodent (e.g. a guinea pig, a hamster, a rat, a mouse), murine (e.g. a mouse), canine (e.g. a dog), feline (e.g. a cat), equine (e.g. a horse), a primate, simian (e.g. a monkey or ape), a monkey (e.g. marmoset, baboon), an ape (e.g. gorilla, chimpanzee, orangutang, gibbon), or a human.

Formulations

[0167] While it is possible for the active compound to be administered alone, it is preferable to present it as a pharmaceutical composition (e.g., formulation) comprising at least one active compound, as defined above, together with one or more pharmaceutically acceptable carriers, adjuvants, excipients, diluents, fillers, buffers, stabilisers, preservatives, lubricants, or other materials well known to those skilled in the art and optionally other therapeutic or prophylactic agents.

[0168] Thus, the present invention further provides pharmaceutical compositions, as defined above, and methods of making a pharmaceutical composition comprising admixing at least one active compound, as defined above, together with one or more pharmaceutically acceptable carriers, excipients, buffers, adjuvants, stabilisers, or other materials, as described herein.

[0169] The term “pharmaceutically acceptable” as used herein pertains to compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgement, suitable for use in contact with the tissues of a subject (e.g. human) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. Each carrier, excipient, etc. must also be “acceptable” in the sense of being compatible with the other ingredients of the formulation.

[0170] Suitable carriers, diluents, excipients, etc. can be found in standard pharmaceutical texts. See, for example, “Handbook of Pharmaceutical Additives”, 2nd Edition (eds. M. Ash and I. Ash), 2001 (Synapse Information Resources, Inc., Endicott, N.Y., USA), “Remington's Pharmaceutical Sciences”, 20th edition, pub. Lippincott, Williams & Wilkins, 2000; and “Handbook of Pharmaceutical Excipients”, 2nd edition, 1994.

[0171] The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. Such methods include the step of bringing into association the active compound with the carrier which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the active compound with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product.

[0172] Formulations may be in the form of liquids, solutions, suspensions, emulsions, elixirs, syrups, tablets, losenges, granules, powders, capsules, cachets, pills, ampoules, suppositories, pessaries, ointments, gels, pastes, creams, sprays, mists, foams, lotions, oils, boluses, electuaries, or aerosols.

[0173] Formulations suitable for oral administration (e.g., by ingestion) may be presented as discrete units such as capsules, cachets or tablets, each containing a predetermined amount of the active compound; as a powder or granules; as a solution or suspension in an aqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion; as a bolus; as an electuary; or as a paste.

[0174] A tablet may be made by conventional means, e.g. compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active compound in a free-flowing form such as a powder or granules, optionally mixed with one or more binders (e.g. povidone, gelatin, acacia, sorbitol, tragacanth, hydroxypropylmethyl cellulose); fillers or diluents (e.g. lactose, microcrystalline cellulose, calcium hydrogen phosphate); lubricants (e.g. magnesium stearate, talc, silica); disintegrants (e.g. sodium starch glycolate, cross-linked povidone, cross-linked sodium carboxymethyl cellulose); surface-active or dispersing or wetting agents (e.g., sodium lauryl sulfate); and preservatives (e.g., methyl p-hydroxybenzoate, propyl p-hydroxybenzoate, sorbic acid). Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active compound therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile. Tablets may optionally be provided with an enteric coating, to provide release in parts of the gut other than the stomach.

[0175] Formulations suitable for topical administration (e.g. transdermal, intranasal, ocular, buccal, and sublingual) may be formulated as an ointment, cream, suspension, lotion, powder, solution, past, gel, spray, aerosol, or oil. Alternatively, a formulation may comprise a patch or a dressing such as a bandage or adhesive plaster impregnated with active compounds and optionally one or more excipients or diluents.

[0176] Formulations suitable for topical administration in the mouth include losenges comprising the active compound in a flavored basis, usually sucrose and acacia or tragacanth; pastilles comprising the active compound in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising the active compound in a suitable liquid carrier.

[0177] Formulations suitable for topical administration to the eye also include eye drops wherein the active compound is dissolved or suspended in a suitable carrier, especially an aqueous solvent for the active compound.

[0178] Formulations suitable for nasal administration, wherein the carrier is a solid, include a coarse powder having a particle size, for example, in the range of about 20 to about 500 microns which is administered in the manner in which snuff is taken, i.e. by rapid inhalation through the nasal passage from a container of the powder held close up to the nose. Suitable formulations wherein the carrier is a liquid for administration as, for example, nasal spray, nasal drops, or by aerosol administration by nebuliser, include aqueous or oily solutions of the active compound.

[0179] Formulations suitable for administration by inhalation include those presented as an aerosol spray from a pressurised pack, with the use of a suitable propellant, such as dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane, carbon dioxide, or other suitable gases.

[0180] Formulations suitable for topical administration via the skin include ointments, creams, and emulsions. When formulated in an ointment, the active compound may optionally be employed with either a paraffinic or a water-miscible ointment base. Alternatively, the active compounds may be formulated in a cream with an oil-in-water cream base. If desired, the aqueous phase of the cream base may include, for example, at least about 30% w/w of a polyhydric alcohol, i.e., an alcohol having two or more hydroxyl groups such as propylene glycol, butane-1,3-diol, mannitol, sorbitol, glycerol and polyethylene glycol and mixtures thereof. The topical formulations may desirably include a compound which enhances absorption or penetration of the active compound through the skin or other affected areas. Examples of such dermal penetration enhancers include dimethylsulfoxide and related analogues.

[0181] When formulated as a topical emulsion, the oily phase may optionally comprise merely an emulsifier (otherwise known as an emulgent), or it may comprises a mixture of at least one emulsifier with a fat or an oil or with both a fat and an oil. Preferably, a hydrophilic emulsifier is included together with a lipophilic emulsifier which acts as a stabiliser. It is also preferred to include both an oil and a fat. Together, the emulsifier(s) with or without stabiliser(s) make up the so-called emulsifying wax, and the wax together with the oil and/or fat make up the so-called emulsifying ointment base which forms the oily dispersed phase of the cream formulations.

[0182] Suitable emulgents and emulsion stabilisers include Tween 60, Span 80, cetostearyl alcohol, myristyl alcohol, glyceryl monostearate and sodium lauryl sulphate. The choice of suitable oils or fats for the formulation is based on achieving the desired cosmetic properties, since the solubility of the active compound in most oils likely to be used in pharmaceutical emulsion formulations may be very low. Thus the cream should preferably be a non-greasy, non-staining and washable product with suitable consistency to avoid leakage from tubes or other containers. Straight or branched chain, mono- or dibasic alkyl esters such as di-isoadipate, isocetyl stearate, propylene glycol diester of coconut fatty acids, isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate, 2-ethylhexyl palmitate or a blend of branched chain esters known as Crodamol CAP may be used, the last three being preferred esters. These may be used alone or in combination depending on the properties required. Alternatively, high melting point lipids such as white soft paraffin and/or liquid paraffin or other mineral oils can be used.

[0183] Formulations suitable for rectal administration may be presented as a suppository with a suitable base comprising, for example, cocoa butter or a salicylate.

[0184] Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations containing in addition to the active compound, such carriers as are known in the art to be appropriate.

[0185] Formulations suitable for parenteral administration (e.g., by injection, including cutaneous, subcutaneous, intramuscular, intravenous and intradermal), include aqueous and non-aqueous isotonic, pyrogen-free, sterile injection solutions which may contain anti-oxidants, buffers, preservatives, stabilisers, bacteriostats, and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents, and liposomes or other microparticulate systems which are designed to target the compound to blood components or one or more organs. Examples of suitable isotonic vehicles for use in such formulations include Sodium Chloride Injection, Ringer's Solution, or Lactated Ringer's Injection. Typically, the concentration of the active compound in the solution is from about 1 ng/ml to about 10 μg/ml, for example from about 10 ng/ml to about 1 μg/ml. The formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets. Formulations may be in the form of liposomes or other microparticulate systems which are designed to target the active compound to blood components or one or more organs.

Dosage

[0186] It will be appreciated that appropriate dosages of the active compounds, and compositions comprising the active compounds, can vary from patient to patient. Determining the optimal dosage will generally involve the balancing of the level of therapeutic benefit against any risk or deleterious side effects of the treatments of the present invention. The selected dosage level will depend on a variety of factors including, but not limited to, the activity of the particular compound, the route of administration, the time of administration, the rate of excretion of the compound, the duration of the treatment, other drugs, compounds, and/or materials used in combination, and the age, sex, weight, condition, general health, and prior medical history of the patient. The amount of compound and route of administration will ultimately be at the discretion of the physician, although generally the dosage will be to achieve local concentrations at the site of action which achieve the desired effect without causing substantial harmful or deleterious side-effects.

[0187] Administration in vivo can be effected in one dose, continuously or intermittently (e.g., in divided doses at appropriate intervals) throughout the course of treatment. Methods of determining the most effective means and dosage of administration are well known to those of skill in the art and will vary with the formulation used for therapy, the purpose of the therapy, the target cell being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician.

[0188] In general, a suitable dose of the active compound is in the range of about 100 μg to about 250 mg per kilogram body weight of the subject per day. Where the active compound is a salt, an ester, prodrug, or the like, the amount administered is calculated on the basis of the parent compound and so the actual weight to be used is increased proportionately.

Synthesis Data

General Experimental Methods

Preparative HPLC

[0189] Samples were purified with a Waters mass-directed purification system utilising a Waters 600 LC pump, Waters Xterra C18 column (5 μm 19 mm×50 mm) and Micromass ZQ mass spectrometer, operating in positive ion electrospray ionisation mode. Mobile phases A (0.1% formic acid in water) and B (0.1% formic acid in acetonitrile) were used in a gradient; 5% B to 100% over 7 min, held for 3 min, at a flow rate of 20 ml/min.

Analytical HPLC-MS

[0190] Analytical HPLC was typically carried out with a Spectra System P4000 pump and Jones Genesis C18 column (4 μm, 50 mm×4.6 mm). Mobile phases A (0.1% formic acid in water) and B (acetonitrile) were used in a gradient of 5% B for 1 min rising to 98% B after 5 min, held for 3 min at a flow rate of 2 ml/min. Detection was by a TSP UV 6000LP detector at 254 nm UV and range 210-600 nm PDA. The Mass spectrometer was a Finnigan LCQ operating in positive ion electrospray mode.

NMR

[0191] .sup.1H NMR and .sup.13C NMR were typically recorded using Bruker DPX 300 spectrometer at 300 MHz and 75 MHz respectively. Chemical shifts were reported in parts per million (ppm) on the δ scale relative to tetramethylsilane internal standard. Unless stated otherwise all samples were dissolved in DMSO-d.sub.6.

Synthesis of Key Intermediates

a. 3-(4-Oxo-3,4-dihydrophthalazin-1-ylmethyl)benzoic Acid (A)

[0192] ##STR00029##

[0193] A mixture of 27% sodium methoxide solution in methanol (400 g, 2 mol) and methanol (150 ml) was added dropwise between ambient temperature and 30° C. over 15 minutes to a stirred mixture of phthalide (67 g, 0.5 mol), 3-formylbenzonitrile (65.5 g, 0.5 mol) and ethyl propionate (250 ml), the mixture was stirred at ambient temperature for 40 minutes and at reflux temperature for 1 hour, then it was allowed to cool to ambient temperature. The resulting red solid was collected by filtration, washed with ethyl acetate (2×50 ml) and dissolved in water (1800 ml). The solution was acidified by the addition of acetic acid (60 ml) and the resulting red solid was collected by filtration, washed with water (2×200 ml) and dried in vacuo to give 3-(1,3-dioxoindan-2-yl)benzonitrile (83.2 g) as a dark red solid, m.pt. 179-182° C., m/z (M+H).sup.+ 248, which was used without further purification.

[0194] 3-(1,3-Dioxoindan-2-yl)benzonitrile (74.18 g, 0.3 mol) was added in portions to a solution of sodium hydroxide (36 g, 0.9 mol) in water (580 ml), the resulting dark red suspension was stirred at reflux temperature for 5 hours, then it was cooled to ambient temperature and washed with ethyl acetate (3×300 ml). The aqueous solution was acidified by the dropwise addition of concentrated hydrochloric acid (110 ml), the mixture was stirred at ambient temperature for 1 hour, then the resulting solid was collected by filtration, washed with water (2×200 ml) and dried in vacuo to give a 1:1 mixture of 3-(1,3-dioxoindan-2-yl)benzoic acid, (M+H).sup.+ 267, and 2-[2-(3-carboxyphenyl)acetyl]benzoic acid, (M+H).sup.+ 285, (69.32 g), which was used without further purification.

[0195] The mixture obtained in the previous step (52.8 g) was added to a solution of triethylamine (37.55 g, 0.372 mol) in industrial methylated spirit (500 ml) and the resulting cloudy solution was filtered through a pad of filter-aid to give a clear solution. Hydrazine monohydrate (9.3 g, 0.186 mol) was added in one portion at ambient temperature, the stirred mixture was heated under reflux for 1 hour, then it was concentrated in vacuo to approximately 250 ml and added to a solution of sodium acetate (41 g, 0.5 mol) in water (500 ml). The mixture was brought to pH 7 by the dropwise addition of concentrated hydrochloric acid, then it was stirred at ambient temperature for 3 hours. The resulting solid was collected by filtration, washed with water (50 ml) and dried in vacuo to give a white solid (15.62 g). The combined filtrate and washings were acidified to pH 6 by the addition of hydrochloric acid, then the mixture was stirred at ambient temperature for 3 hours. The resulting solid was collected by filtration, washed with water (50 ml) and dried in vacuo to give a second crop of off-white solid (17.57 g). The combined filtrate and washings from the second crop were readjusted to pH 6 and treated as before to give a third crop of pale orange solid (6.66 g). The three crops were combined to give essentially pure 3-(4-oxo-3,4-dihydrophthalazin-1-ylmethyl)benzoic acid (A), (M+H).sup.+ 281, δ.sub.H 4.4 (2H, s), 7.2-7.4 (1H, m), 7.5-7.6 (1H, m), 7.7-8.0 (5H, m), 8.1-8.2 (1H, m), 12.6 (1H, s)

b. 2-Fluoro-5-(4-oxo-3,4-dihydro-phthalazin-1-ylmethyl)benzoic Acid (B)

[0196] ##STR00030##

[0197] Dimethyl phosphite (22.0 g, 0.2 mol) was added drop-wise to a solution of sodium methoxide (43.0 g) in methanol (100 ml) at 0° C. 2-Carboxybenzaldehyde (21.0 g, 0.1 mol) was then added portion-wise to the reaction mixture as a slurry in methanol (40 ml), with the temperature kept below 5° C. The resulting pale yellow solution was warmed to 20° C. over 1 hour. Methanesulphonic acid (21.2 g, 0.22 mol) was added to the reaction drop-wise and the resulting white suspension was evaporated in vacuo. The white residue was quenched with water and extracted into chloroform (3×100 ml). The combined organic extracts were washed with water (2×100 ml), dried over MgSO.sub.4, and evaporated in vacuo to yield (3-oxo-1,3-dihydro-isobenzofuran-1-yl)phosphonic acid dimethyl ester as a white solid (32.0 g, 95%, 95% purity). This was then used without further purification in the next stage.

[0198] To a mixture of (3-oxo-1,3-dihydro-isobenzofuran-1-yl)phosphonic acid dimethyl ester (35.0 g, 0.14 mol) in tetrahydrofuran (200 ml) and 2-fluoro-5-formylbenzonitrile (20.9 g, 0.14 mol) in tetrahydrofuran (130 ml) was added triethylamine (14 ml, 0.14 mol) drop-wise over 25 min, with the temperature kept below 15° C. The reaction mixture was warmed slowly to 20° C. over 1 hour and concentrated in vacuo. The white residue was slurried in water (250 ml) for 30 minutes, filtered, washed with water, hexane and ether, and dried to yield 2-fluoro-5-(3-oxo-3H-isobenzofuran-1-ylidenemethyl)benzonitrile as a 50:50 mixture of E and Z isomers (37.2 g, 96%); m/z [M+1].sup.+ 266 (98% purity)

[0199] To a suspension of 2-fluoro-5-(3-oxo-3H-isobenzofuran-1-ylidenemethyl)benzonitrile in water (200 ml) was added aqueous sodium hydroxide (26.1 g in 50 ml water) solution and the reaction mixture was heated under nitrogen to 90° C. for 30 minutes. The reaction mixture was partially cooled to 70° C., and hydrazine hydrate (100 ml) was added and stirred for 18 hours at 70° C. The reaction was cooled to room temperature and acidified with 2M HCl to pH 4. The mixture was stirred for 10 min and filtered. The resulting solid was washed with water, hexane, ether, ethyl acetate and dried to yield 2-fluoro-5-(4-oxo-3,4-dihydrophthalazin-1-ylmethyl)benzoic acid as a pale pink powder (30.0 g, 77%). m/z [M+1].sup.+ 299 (96% purity), δ.sub.H 4.4 (2H, s), 7.2-7.3 (1H, m), 7.5-7.6 (1H, m), 7.8-8.0 (4H, m), 8.2-8.3 (1H, m), 12.6 (1H, s).

c. 1-[3-(4-Oxo-3,4-dihydrophthalazin-1-ylmethyl)benzoyl]piperidine-4-carboxylic Acid (C)

[0200] ##STR00031##

[0201] 3-(4-Oxo-3,4-dihydrophthalazin-1-ylmethyl)benzoic acid (A)(7.0 g, 0.25 mol), ethyl isonipecotate (5 ml, 0.32 mol), 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) (12.3 g, 0.32 mol) and N,N,-diisopropylethylamine (10.0 ml, 0.55 mol) were added to dimethylacetamide (40 ml) and stirred for 18 h. Water (100 ml) was added to the reaction mixture and the product was extracted into dichloromethane (4×50 ml). The combined organic layers were washed with water (3×100 ml), dried over MgSO.sub.4, filtered and evaporated in vacuo to yield an oil. To a solution of the oil in tetrahydrofuran (100 ml) was added 10% aqueous sodium hydroxide solution (20 ml) and the reaction was stirred for 18 hours. The reaction was concentrated, washed with ethyl acetate (2×30 ml) and acidified with 2M HCl to pH 2. The aqueous layer was extracted with dichloromethane (2×100 ml), then the extracts were dried over MgSO.sub.4, filtered and evaporated to yield 1-[3-(4-oxo-3,4-dihydrophthalazin-1-ylmethyl)benzoyl]piperidine-4-carboxylic acid (C) as a yellow solid (7.0 g, 65%), m/z [M+1].sup.+ 392 (96% purity), δ.sub.H 1.3-1.8 (5H, m), 2.8-3.1 (4H, m), 4.4 (2H, s), 7.2-7.3 (1H, m), 7.3-7.4 (1H, m), 7.7-8.0 (5H, m), 8.2-8.3 (1H, m), 12.6 (1H, s).

d. 1-[2-Fluoro-5-(4-oxo-3,4-dihydrophthalazin-1-ylmethyl)benzoyl]piperidine-4-carboxylic Acid (D)

[0202] ##STR00032##

[0203] 2-Fluoro-5-(4-oxo-3,4-dihydrophthalazin-1-ylmethyl)benzoic acid (B)(3.1 g, 0.14 mol), ethyl isonipecotate (1.7 ml, 0.11 mol), 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) (5.1 g, 0.13 mol) and N,N,-diisopropylethylamine (10.0 ml, 0.55 mol) were added to dimethylacetamide (15 ml) and stirred for 18 hours. Water (100 ml) was added to the reaction mixture and the product was extracted into dichloromethane (4×50 ml). The combined organic layers were, filtered, washed with water (3×100 ml), dried over MgSO.sub.4, filtered and evaporated in vacuo to yield an orange oil. The oil was purified by flash chromatography (ethyl acetate) to yield 1-[2-fluoro-5-(4-oxo-3,4-dihydrophthalazin-1-ylmethyl)benzoyl]piperidine-4-carboxylic acid as the methyl ester (1.5 g, 33%, 96% purity). To a solution of the methyl ester in tetrahydrofuran: water (2:1, 40 ml) was added sodium hydroxide (0.3 g, 0.075 mol) and the reaction was stirred for 18 h. The reaction was concentrated, washed with ethyl acetate (2×20 ml) and acidified with 2M HCl to pH 2. The aqueous layer was extracted with dichloromethane (2×20 ml), and the combined extracts were dried over MgSO.sub.4 and evaporated to yield 1-[3-(4-oxo-3,4-dihydrophthalazin-1-ylmethyl)benzoyl]piperidine-4-carboxylic acid (D) as a yellow solid (0.6 g, 65%), m/z [M+1].sup.+ 392 (96% purity)

Example 1

a. Synthesis of 4-[3-(piperazine-1-carbonyl)benzyl]-2H-phthalazin-1-one (1)

[0204] ##STR00033##

[0205] 3-(4-Oxo-3,4-dihydrophthalazin-1-ylmethyl)benzoic acid (A)(5.0 g, 0.17 mol), tert-butyl 1-piperazinecarboxylate (3.9 g, 0.21 mol), 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) (8.6 g, 0.22 mol) and N,N,-diisopropylethylamine (6.7 ml, 0.38 mol) were added to dimethylacetamide (40 ml) and stirred for 18 hours. Water (100 ml) was added and the reaction mixture was heated to 100° C. for 1 hour. The suspension was cooled to room temperature, filtered and dried to yield a white solid. The solid was dissolved in a solution of 6M HCl and ethanol (2:1, 50 ml) and stirred for 1 hour. The reaction was concentrated, basified with ammonia to pH 9, and the product was extracted into dichloromethane (2×50 ml). The combined organic layers were washed with water (2×50 ml), dried over MgSO.sub.4, and evaporated in vacuo to yield 4-[3-(piperazine-1-carbonyl)benzyl]-2H-phthalazin-1-one (1) as a yellow crystalline solid (4.0 g, 77%); m/z [M+1].sup.+ 349 (97% purity), δ.sub.H 2.6-3.8 (8H, m), 4.4 (2H, s), 7.2-7.5 (4H, m), 7.7-8.0 (3H, m), 8.2-8.3 (1H, m), 12.6 (1H, s)

b. Synthesis of 4-[4-Fluoro-3-(piperazine-1-carbonyl)benzyl]-2H-phthalazin-1-one (2)

[0206] ##STR00034##

[0207] The synthesis was carried out according to the method described in (a) above using 2-fluoro-5-(4-oxo-3,4-dihydrophthalazin-1-ylmethyl)benzoic acid (B) to yield 4-[4-fluoro-3-(piperazine-1-carbonyl)benzyl]-2H-phthalazin-1-one (2) as a white crystalline solid (4.8 g, 76%); m/z [M+1].sup.+ 367 (97% purity), δ.sub.H 2.6-3.8 (8H, m), 4.4 (2H, s), 7.2-7.5 (3H, m), 7.7-8.0 (3H, m), 8.2-8.3 (1H, m), 12.6 (1H, s).

c. Synthesis of 4-[3-([1,4]diazepane-1-carbonyl)benzyl]-2H-phthalazin-1-one (3)

[0208] ##STR00035##

[0209] The synthesis was carried out according to the method described in (a) above using 3-(4-oxo-3,4-dihydrophthalazin-1-ylmethyl)benzoic acid (A) and tert-butyl 1-homopiperazine carboxylate to yield 4-[3-([1,4]diazepane-1-carbonyl)benzyl]-2H-phthalazin-1-one (3) as a grey crystalline solid (5.3 g, 97%); m/z [M+1].sup.+ 363 (97% purity); δ.sub.H 2.6-3.8 (10H, m), 4.4 (2H, s), 7.2-7.5 (4H, m), 7.7-8.0 (3H, m), 8.2-8.3 (1H, m), 12.6 (1H, s).

d. Synthesis of 4-[3-([1,4]diazepane-1-carbonyl)-4-fluorobenzyl]-2H-phthalazin-1-one (4)

[0210] ##STR00036##

[0211] The synthesis was carried out according to the method described in (a) above using 2-fluoro-5-(4-oxo-3,4-dihydrophthalazin-1-ylmethyl)benzoic acid (B) and tert-butyl 1-homopiperazinecarboxylate to yield 4-[3-([1,4]diazepane-1-carbonyl)benzyl]-2H-phthalazin-1-one (4) as a yellow crystalline solid (5.3 g, 68%); m/z [M+1].sup.+ 381 (97% purity); δ.sub.H 2.6-3.8 (10H, m), 4.4 (2H, s), 7.2-7.5 (3H, m), 7.7-8.0 (3H, m), 8.2-8.3 (1H, m), 12.6 (1H, s).

Example 2

a. 4-{3-[4-(6-Chlorobenzothiazol-2-yl)-1,4-diazepan-1-ylcarbonyl]benzyl}-1(2H)-phthalazinone

[0212] ##STR00037##

[0213] 2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (150 mg, 0.47 mmol), diisopropylethylamine (102 mg, 0.8 mmol) and 6-chloro-2-(1,4-diazepan-1-yl)-1,3-benzothiazole (115 mg, 0.43 mmol) were added sequentially at ambient temperature to a stirred solution of 3-(4-oxo-3,4-dihydrophthalazin-1-ylmethyl)benzoic acid (A)(100 mg, 0.36 mmol) in dry dimethylacetamide (1 ml), the mixture was stirred at ambient temperature for 1 hour and allowed to stand at ambient temperature for 16 hours, then it was added dropwise to stirred cold water (10 ml). After 30 minutes, the resulting solid was collected by filtration, washed with water (2×1 ml) and hexane (1 ml), dried in vacuo and purified using preparative HPLC to give the desired compound (5)(166 mg) as a grey solid; HPLC purity 90%; HPLC Retention time 4.21 minutes; m/z (M+H).sup.+ 530.

[0214] b. The following compounds were synthesised in a manner analogous to that described in (a) above, but using appropriate alternative amine starting materials.

##STR00038##

TABLE-US-00001 Com- LC RT LC Purity pound R (minutes) M + 1 (%) 6 [00039] 4.18 508 90 7 [00040] 3.22 551 90 8 [00041] 4.13 508 90 9 [00042] 3.95 483 90 10 [00043] 3.79 465 90 11 [00044] 3.76 406 90 219 [00045] 2.80 407 90

##STR00046##

TABLE-US-00002 LC Com- LC RT Purity pound R (minutes) M + 1 (%) 12 (Note 1) [00047] 3.56 494 100 13 [00048] 3.71 451 90 14 [00049] 4.39 538 90 15 [00050] 3.66 498 90 16 [00051] 4.33 533 90 Note 1: 12 did not require purification via preparative scale HPLC- the product from the reaction was essentially pure.

##STR00052##

TABLE-US-00003 Com- LC RT LC Purity pound R (minutes) M + 1 (%) 17 [00053] 4.64 526 90 18 [00054] 3.99 482 90 19 [00055] 4.00 452 90 20 [00056] 4.15 466 90

Example 3

a. 4-{3-[4-(4-fluorophenyl)piperazin-1-ylcarbonyl]benzyl}-1(2H)-phthalazinone (21)

[0215] ##STR00057##

[0216] 2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (150 mg, 0.47 mmol), diisopropylethylamine (102 mg, 0.8 mmol) and 1-(4-fluorophenyl)piperazine (65 mg, 0.47 mmol) were added sequentially at ambient temperature to a stirred solution of 3-(4-oxo-3,4-dihydrophthalazin-1-ylmethyl)benzoic acid (A)(100 mg, 0.36 mmol) in dry dimethylacetamide (1 ml), the mixture was stirred at ambient temperature for 4 hours and allowed to stand at ambient temperature for 16 hours, then it was added dropwise to stirred cold water (10 ml). After 30 minutes, the resulting solid was collected by filtration, washed with water (2×1 ml) and hexane (1 ml), dried in vacuo and purified using preparative HPLC to give 4-{3-[4-(4-fluorophenyl)piperazin-1-ylcarbonyl]benzyl}-1(2H)-phthalazinone (21)(76 mg) as a cream solid; m/z (M+H).sup.+ 443; HPLC Purity 90%; HPLC Retention time 4.00 minutes.

[0217] b. The following compounds were synthesised in a manner analogous to that described in (a) above, but using appropriate alternative amine starting materials.

##STR00058##

TABLE-US-00004 Com- LC RT LC Purity pound R (minutes) M + 1 (%) 22 [00059] 4.00 470 90 23 [00060] 4.26 486 90 24 [00061] 3.18 504 85 25 [00062] 3.78 473 90 26 [00063] 4.46 583 90 27 [00064] 4.96 509 90 28 [00065] 3.73 511 90 29 [00066] 3.78 553 90 30 [00067] 3.71 459 90 31 [00068] 3.94 546 90 32 [00069] 3.84 485 90 33 [00070] 4.37 552 90 34 [00071] 3.77 485 90 220 (Note 2) [00072] 2.89 440 100 Note 2: 220 did not require purification via preparative scale HPLC- the product from the reaction was essentially pure.

Example 4

[0218] 1-[3-(4-Oxo-3,4-dihydro-phthalazin-1-ylmethyl)-benzoyl]-piperidine-4-carboxylic acid (C) (0.24 mmol) was added to a solution of the appropriate amine (0.2 mmol) in dimethylacetamide (2 ml). 2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (0.3 mmol) and Hunigs base (0.4 mmol) were then added and the reaction was stirred at room temperature for 16 hours. The reaction mixtures were then purified by preparative HPLC.

[0219] The compounds synthesised are set out below.

##STR00073##

TABLE-US-00005 LC RT LC Purity Compound R (minutes) M + 1 (%) 35 [00074] 4.39 572 90 36 [00075] 3.71 496 90 37 [00076] 3.63 474 80 38 [00077] 3.76 474 90 39 [00078] 3.56 502 90 40 [00079] 3.58 568 90 41 [00080] 3.81 508 90 42 [00081] 4.39 531 90 43 [00082] 3.52 460 85 44 [00083] 3.77 508 90 45 [00084] 3.59 488 90 46 [00085] 3.83 488 90 47 [00086] 3.85 488 90 48 [00087] 3.47 448 90 49 [00088] 3.36 446 90 50 [00089] 3.77 488 90 51 [00090] 3.74 472 90 52 [00091] 3.82 498 90 53 [00092] 3.52 460 90 54 [00093] 3.86 510 90 55 [00094] 3.75 546 90 56 [00095] 3.01 565 90 57 [00096] 3.93 549 90 373 [00097] 4.17 663 90 374 [00098] 3.08 595 90 375 [00099] 4.16 551 90 376 [00100] 4.3 565 90 377 [00101] 4.1 571 90 378 [00102] 3.64 567 90 379 [00103] 3.62 579 90 380 [00104] 4.27 605 90 381 [00105] 3.89 555 90 382 [00106] 3.84 565 90 383 [00107] 2.92 565 90 384 [00108] 3.02 543 90

Example 5

[0220] The appropriate sulphonyl chloride (0.24 mmol) was added to a solution of 4-[3-(piperazine-1-carbonyl)benzyl]-2H-phthalazin-1-one (1) (0.2 mmol) in dichloromethane (2 ml). Hunigs base (0.4 mmol) was then added and the reaction was stirred at room temperature for 16 hours. The reaction mixtures were then purified by preparative HPLC.

[0221] The compounds synthesised are set out below.

##STR00109##

TABLE-US-00006 LC RT LC Purity Compound R (minutes) M + 1 (%) 58 [00110] 3.85 504 90 59 [00111] 3.44 442 90 60 [00112] 4.09 558 90 61 [00113] 3.93 525 90 62 [00114] 3.73 543 90 63 [00115] 4.38 532 90 64 [00116] 3.76 509 90 65 [00117] 3.82 470 90

Example 6

[0222] The appropriate acid chloride (0.24 mmol) was added to a solution of 4-[3-(piperazine-1-carbonyl)benzyl]-2H-phthalazin-1-one (1) (0.2 mmol) in dichloromethane (2 ml). Hunigs base (0.4 mmol) was then added and the reaction was stirred at room temperature for 16 hours. The reaction mixtures were then purified by preparative HPLC.

[0223] The compounds synthesised are set out below.

##STR00118##

TABLE-US-00007 LC RT Compound R (minutes) M + 1 LC Purity (%) 66 [00119] 4.04 530 85 67 [00120] 3.71 460 90 68 [00121] 3.69 506 90 69 [00122] 3.19 450 85 70 [00123] 3.44 478 90 71 [00124] 3.94 538 90 72 [00125] 3.68 526 90 73 [00126] 3.6 464 90 74 [00127] 3.63 472 90 75 [00128] 3.73 535 80 76 [00129] 3.49 530 90 77 [00130] 3.5 512 85 78 [00131] 3.52 459 90 79 [00132] 4.01 588 90 80 [00133] 4.05 406 90 81 [00134] 3.84 513 90 82 [00135] 4.07 520 90 83 [00136] 4.41 671 90 84 [00137] 3.62 582 90 85 [00138] 3.82 508 90 86 [00139] 3.81 507 90 87 [00140] 3.33 445 90 88 [00141] 4.08 571 90 89 [00142] 3.67 480 90 90 [00143] 3.54 577 90 91 [00144] 3.49 498 90 92 [00145] 4.04 510 90 93 [00146] 3.75 512 90 94 [00147] 3.67 482 90 95 [00148] 3.54 474 90 96 [00149] 3.52 537 90 97 [00150] 4.13 475 90 98 [00151] 3.8 512 85 99 [00152] 4.09 544 90 100 [00153] 3.63 486 90 101 [00154] 3.91 502 90 102 [00155] 3.61 511 90 103 [00156] 3.57 474 90 104 [00157] 3.67 504 90 105 [00158] 4.02 508 90 106 [00159] 3.81 500 90 107 [00160] 4.11 540 90 108 [00161] 4.19 560 90 109 [00162] 3.61 468 90 110 [00163] 3.69 582 90 111 [00164] 3.85 549 85 112 [00165] 4.37 573 90 113 [00166] 3.84 496 90 114 [00167] 3.62 573 90 115 [00168] 3.72 500 90 116 [00169] 3.8 500 85 117 [00170] 3.9 496 90 118 [00171] 4.03 560 90 119 [00172] 4.16 560 90 120 [00173] 4.71 472 80 121 [00174] 3.47 526 90 122 [00175] 3.78 632 90 123 [00176] 3.27 506 90 124 [00177] 3.92 590 90 125 [00178] 4.76 706 90 126 [00179] 4.27 605 90 127 [00180] 3.71 557 90 128 [00181] 3.98 551 90 129 [00182] 3.9 496 90 130 [00183] 3.57 454 90 131 [00184] 3.21 450 90 132 [00185] 3.61 446 90 133 [00186] 3.39 418 85 134 [00187] 3.81 494 90 135 [00188] 3.31 418 90 136 [00189] 3.38 444 90 137 [00190] 3.56 506 85 138 [00191] 3.48 484 90 139 [00192] 3.84 510 90 140 [00193] 3.56 472 90 141 [00194] 3.34 476 90 142 [00195] 3.56 490 90 143 [00196] 3.53 488 90 144 [00197] 3.99 523 90 145 [00198] 3.7 468 90 192 [00199] 3.11 392 90 350 [00200] 3.16 462 90 351 [00201] 2.77 474 90

Example 7

[0224] The appropriate isocyanate (0.24 mmol) was added to a solution of 4-[3-(piperazine-1-carbonyl)benzyl]-2H-phthalazin-1-one (1) (0.2 mmol) in dichloromethane (2 ml). The reaction was stirred at room temperature for 16 hours. The reaction mixtures were then purified by preparative HPLC.

[0225] The compounds synthesised are set out below.

##STR00202##

TABLE-US-00008 LC RT LC Purity Compound R (minutes) M+ 1 (%) 146 [00203] 3.34 435 85 147 [00204] 3.76 483 90 148 [00205] 3.49 449 90 149 [00206] 3.57 487 90 150 [00207] 4.03 537 90 151 [00208] 3.77 519 85 152 [00209] 3.72 487 90 153 [00210] 3.57 505 90 154 [00211] 4.08 537 80 155 [00212] 3.68 497 85 156 [00213] 4.03 553 90 157 [00214] 3.93 497 90 158 [00215] 3.62 494 90 159 [00216] 3.68 497 90 160 [00217] 3.73 475 90 161 [00218] 3.9 513 90 162 [00219] 4.11 511 90 163 [00220] 3.58 483 90 164 [00221] 3.71 517 90 165 [00222] 3.34 435 85 166 [00223] 3.71 497 90 167 [00224] 3.56 513 90 168 [00225] 4.04 552 90 169 [00226] 4 547 90 170 [00227] 3.54 507 90 171 [00228] 3.42 497 90 172 [00229] 3.95 552 90 173 [00230] 3.79 541 85 174 [00231] 3.66 529 90 175 [00232] 3.92 527 85 176 [00233] 3.62 527 90 177 [00234] 4.28 569 90 178 [00235] 3.81 610 90 352 [00236] 3.73 555 90 353 [00237] 3.79 541 90

Example 8

[0226] The appropriate isothiocyanate (0.24 mmol) was added to a solution of 4-[3-(piperazine-1-carbonyl)benzyl]-2H-phthalazin-1-one (1) (0.2 mmol) in dichloromethane (2 ml). The reaction was stirred at room temperature for 16 hours. The reaction mixtures were then purified by preparative HPLC.

[0227] The compounds synthesised are set out below.

##STR00238##

TABLE-US-00009 LC RT Compound R (minutes) M + 1 LC Purity (%) 179 [00239] 3.68 515 90 180 [00240] 4.05 513 90 181 [00241] 3.94 465 90 182 [00242] 3.55 449 90 183 [00243] 4.21 575 90 184 [00244] 3.79 543 90 185 [00245] 4.28 557 85 186 [00246] 3.63 527 90 187 [00247] 3.18 528 90 188 [00248] 3.32 423 90 189 [00249] 3.69 485 80 190 [00250] 3.68 515 90 191 [00251] 3.72 503 90 354 [00252] 4.67 542 90 355 [00253] 3.96 543 90

Example 9

[0228] 3-(4-Oxo-3,4-dihydrophthalazin-1-ylmethyl)benzoic acid (A) (0.24 mmol) was added to a solution of the appropriate amine (0.2 mmol) in dimethylacetamide (2 ml). 2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (0.3 mmol) and Hunigs base (0.4 mmol) were then added and the reaction was stirred at room temperature for 16 hours. The reaction mixtures were then purified by preparative HPLC.

[0229] The compounds synthesised are set out below.

##STR00254##

TABLE-US-00010 LC RT LC Purity Compound R (minutes) M + 1 (%) 193 [00255] 2.72 378 90 221 [00256] 2.77 407 90

##STR00257##

TABLE-US-00011 Compound R LC RT (minutes) M + 1 LC Purity (%) 194 [00258] 2.96 427 90 195 [00259] 4.08 444 90 196 [00260] 3.9 456 95 197 [00261] 3.83 450 95 198 [00262] 2.98 432 90 199 [00263] 4.17 440 90 200 [00264] 2.9 449 90 201 [00265] 4.31 460 90 202 [00266] 3.63 468 90 203 [00267] 3.78 456 90 204 [00268] 3.08 378 90

##STR00269##

TABLE-US-00012 LC RT Compound R (minutes) M + 1 LC Purity (%) 205 [00270] 2.88 432 90 206 [00271] 3.61 421 95 207 [00272] 3.96 397 90 356 [00273] 4.34 564 90 357 [00274] 3.84 554 90

Example 10

[0230] 2-Fluoro-5-(4-oxo-3,4-dihydrophthalazin-1-ylmethyl)benzoic acid (B) (0.24 mmol) was added to a solution of the appropriate amine (0.2 mmol) in dimethylacetamide (2 ml). 2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (0.3 mmol) and Hunigs base (0.4 mmol) were then added and the reaction was stirred at room temperature for 16 hours. The reaction mixtures were then purified by preparative HPLC.

[0231] The compounds synthesised are set out below.

##STR00275##

TABLE-US-00013 LC RT LC Purity Compound R (minutes) M + 1 (%) 208 [00276] 2.8 396 90 359 [00277] 2.78 425 85

##STR00278##

TABLE-US-00014 Com- LC RT LC Purity pound R (minutes) M + 1 (%) 209 [00279] 3.9  456 95 210 [00280] 3.97 467 90 211 [00281] 2.84 426 90 212 [00282] 3.46 368 90 222 [00283] 5.07 461 90 223 [00284] 4.46 511 90 224 [00285] 2.96 499 90 225 [00286] 3.55 445 85 226 [00287] 4.19 457 90 227 [00288] 4.34 471 90 228 [00289] 2.88 466 90 229 [00290] 4.23 477 90 230 [00291] 4.03 473 90 231 [00292] 3.07 500 90 232 [00293] 3.92 473 90 233 [00294] 4.55 471 90 234 [00295] 4.32 477 90 235 [00296] 3.94 443 90 236 [00297] 4.03 461 90 237 [00298] 2.69 411 90 238 [00299] 2.76 438 90 239 [00300] 2.77 494 90 240 [00301] 2.91 444 90 358 [00302] 4.59 472 90

##STR00303##

TABLE-US-00015 LC Com- LC RT Purity pound R (minutes) M + 1 (%) 213 [00304] 3.81 439 90 214 [00305] 3.95 415 90

##STR00306##

TABLE-US-00016 LC RT LC Purity Compound R (minutes) M + 1 (%) 360 [00307] 4.08 472 90

Example 11

[0232] An appropriate aldehyde (0.2 mmol) and 4-[3-(piperazine-1-carbonyl)benzyl]-2H-phthalazin-1-one (1)(0.24 mmol) were dissolved in dichloromethane (2 ml). Sodium triacetoxyborohydride (0.28 mmol) and glacial acetic acid (6.0 mmol) were then added and stirred at room temperature for 16 hours. The reaction mixtures were then purified by preparative HPLC.

[0233] The compounds synthesised are set out below.

##STR00308##

TABLE-US-00017 Com- LC RT LC Purity pound R (minutes) M + 1 (%) 215 [00309] 2.89 406 90 216 [00310] 2.91 406 90 217 [00311] 3.16 493 90 218 [00312] 3.09 479 90 361 [00313] 2.98 419 90 362 [00314] 2.74 377 90 363 [00315] 3.04 419 90 364 [00316] 2.82 391 85 365 [00317] 2.84 403 90 366 [00318] 2.96 419 90 367 [00319] 3.15 445 90 368 [00320] 3.04 419 90 369 [00321] 2.75 391 85

Example 12

[0234] An appropriate aldehyde (0.2 mmol) and 4-[3-([1,4]diazepane-1-carbonyl)benzyl]-2H-phthalazin-1-one (4)(0.24 mmol) were dissolved in dichloromethane (2 ml). Sodium triacetoxyborohydride (0.28 mmol) and glacial acetic acid (6.0 mmol) were then added and stirred at room temperature for 16 hours. The reaction mixtures were then purified by preparative HPLC.

[0235] The compounds synthesised are set out below.

##STR00322##

TABLE-US-00018 LC RT LC Purity Compound R (minutes) M + 1 (%) 241 [00323] 2.9  437 90 242 [00324] 3.05 451 90 243 [00325] 2.84 409 90 244 [00326] 3.12 465 90 245 [00327] 3.16 451 90 246 [00328] 2.86 423 90 247 [00329] 2.89 435 90 248 [00330] 3.04 451 90 249 [00331] 3.23 477 90 250 [00332] 3.09 451 90 370 [00333] 2.80 423 90

Example 13

[0236] An appropriate aldehyde (0.2 mmol) and 4-[4-fluoro-3-(piperazine-1-carbonyl)benzyl]-2H-phthalazin-1-one (2)(0.24 mmol) were dissolved in dichloromethane (2 ml). Sodium triacetoxyborohydride (0.28 mmol) and glacial acetic acid (6.0 mmol) were then added and stirred at room temperature for 16 hours. The reaction mixtures were then purified by preparative HPLC.

[0237] The compounds synthesised are set out below.

##STR00334##

TABLE-US-00019 LC RT LC Purity Compound R (minutes) M + 1 (%) 251 [00335] 2.97 423 90 252 [00336] 3.06 437 90 253 [00337] 2.8  395 90 254 [00338] 3.11 451 90 255 [00339] 3.09 437 90 256 [00340] 2.87 409 90 257 [00341] 2.89 421 90 258 [00342] 3.01 437 85 259 [00343] 3.14 463 90 260 [00344] 3.08 437 90 261 [00345] 2.83 458 90 262 [00346] 3.04 464 90 263 [00347] 2.79 409 90

Example 14

[0238] The appropriate acid chloride (0.24 mmol) was added to a solution of 4-[4-fluoro-3-(piperazine-1-carbonyl)benzyl]-2H-phthalazin-1-one (2) (0.2 mmol) in dichloromethane (2 ml). Hunigs base (0.4 mmol) was then added and the reaction was stirred at room temperature for 16 hours. The reaction mixtures were then purified by preparative HPLC.

[0239] The compounds synthesised are set out below.

##STR00348##

TABLE-US-00020 LC Com- LC RT Purity pound R (minutes) M + 1 (%) 264 [00349] 3.89 409 90 265 [00350] 4.17 462 85 266 [00351] 4.04 423 90 267 [00352] 4.03 417 90 268 [00353] 3.19 480 90 269 [00354] 2.84 492 90 270 [00355] 2.71 521 90 271 [00356] 2.83 508 90 272 [00357] 2.86 478 90 273 [00358] 2.63 521 90

Example 15

[0240] An appropriate aldehyde (0.2 mmol) and 4-[3-([1,4]diazepane-1-carbonyl)benzyl]-2H-phthalazin-1-one (3)(0.24 mmol) were dissolved in dichloromethane (2 ml). Sodium triacetoxyborohydride (0.28 mmol) and glacial acetic acid (6.0 mmol) were then added and stirred at room temperature for 16 hours. The reaction mixtures were then purified by preparative HPLC.

[0241] The compounds synthesised are set out below.

##STR00359##

TABLE-US-00021 LC RT LC Com- (min- Purity pound R utes) M + 1 (%) 274 [00360] 2.92 419 90 275 [00361] 3.04 433 90 276 [00362] 2.78 391 90 277 [00363] 3.09 433 90 278 [00364] 2.86 405 90 279 [00365] 2.88 417 90 280 [00366] 2.99 433 90 281 [00367] 3.11 459 90 282 [00368] 3.06 433 90 283 [00369] 2.93 443 90 284 [00370] 2.92 451 90 285 [00371] 2.99 459 90 286 [00372] 2.94 441 90 287 [00373] 3.36 589 85 288 [00374] 2.72 487 85 289 [00375] 3.24 499 90 290 [00376] 3.14 497 90 291 [00377] 3.9  483 85 292 [00378] 3.22 493 90 293 [00379] 2.91 419 90 294 [00380] 3.08 511 90 295 [00381] 2.92 443 90 296 [00382] 3.03 478 90 297 [00383] 3.48 501 90 298 [00384] 3.39 545 90 299 [00385] 3.06 456 90 300 [00386] 3.8  483 90 301 [00387] 3.08 472 90 302 [00388] 3.22 506 90 303 [00389] 3.13 522 90 304 [00390] 3.29 533 90 305 [00391] 3.39 589 90 306 [00392] 3.07 492 90 307 [00393] 3.41 589 90 308 [00394] 2.87 460 85 309 [00395] 3.06 496 90 310 [00396] 3.13 513 90 311 [00397] 2.96 459 90 312 [00398] 2.98 453 90 313 [00399] 3.09 501 90 314 [00400] 2.76 405 90

Example 16

[0242] The appropriate sulphonyl chloride (0.24 mmol) was added to a solution of 4-[3-([1,4]diazepane-1-carbonyl)benzyl]-2H-phthalazin-1-one (3) (0.2 mmol) in dichloromethane (2 ml). Hunigs base (0.4 mmol) was then added and the reaction was stirred at room temperature for 16 hours. The reaction mixtures were then purified by preparative HPLC.

[0243] The compounds synthesised are set out below.

##STR00401##

TABLE-US-00022 Com- LC RT LC Purity pound R (minutes) M + 1 (%) 315 [00402] 4.15 571 90 316 [00403] 4.36 579 90 317 [00404] 3.68 483 90 371 [00405] 3.71 523 90

Example 17

[0244] The appropriate acid chloride (0.24 mmol) was added to a solution of 4-[3-([1,4]diazepane-1-carbonyl)benzyl]-2H-phthalazin-1-one (3) in dichloromethane (2 ml). Hunigs base (0.4 mmol) was then added and the reaction was stirred at room temperature for 16 hours. The reaction mixtures were then purified by preparative HPLC.

[0245] The compounds synthesised are set out below.

##STR00406##

TABLE-US-00023 LC Com- LC RT Purity pound R (minutes) M + 1 (%) 318 [00407] 3.84 533 90 319 [00408] 3.23 458 90 320 [00409] 3.63 532 90 321 [00410] 3.61 525 90 322 [00411] 3.63 495 90 323 [00412] 3.62 513 90 324 [00413] 3.16 415 90 325 [00414] 3.52 459 90 326 [00415] 3.95 489 90 327 [00416] 3.89 549 90 328 [00417] 4.00 557 90

Example 18

[0246] The appropriate isocyanate (0.24 mmol) was added to a solution of 4-[3-([1,4]diazepane-1-carbonyl)benzyl]-2H-phthalazin-1-one (3) (0.2 mmol) in dichloromethane (2 ml). The reaction was stirred at room temperature for 16 hours. The reaction mixtures were then purified by preparative HPLC.

[0247] The compounds synthesised are set out below.

##STR00418##

TABLE-US-00024 LC RT LC Purity Compound R (minutes) M + 1 (%) 329 [00419] 3.4  512 90 330 [00420] 3.95 550 90 331 [00421] 3.76 510 90 332 [00422] 3.58 526 90 333 [00423] 4.04 582 90

Example 19

[0248] The appropriate isothiocyanate (0.24 mmol) was added to a solution of 4-[3-([1,4]diazepane-1-carbonyl)benzyl]-2H-phthalazin-1-one (3) (0.2 mmol) in dichloromethane (2 ml). The reaction was stirred at room temperature for 16 hours. The reaction mixtures were then purified by preparative HPLC.

[0249] The compounds synthesised are set out below.

##STR00424##

TABLE-US-00025 Com- LC RT LC Purity pound R (minutes) M + 1 (%) 334 [00425] 3.86 512 90 335 [00426] 3.75 516 90 336 [00427] 3.66 556 90 337 [00428] 3.57 540 90 338 [00429] 4.17 569 90 339 [00430] 3.56 540 90 340 [00431] 3.1  541 85 341 [00432] 3.84 498 90 342 [00433] 3.64 556 90 343 [00434] 3.53 528 90 344 [00435] 3.92 556 90 345 [00436] 4.09 566 90 346 [00437] 3.64 516 90 347 [00438] 3.57 528 90 348 [00439] 3.78 512 90 349 [00440] 3.62 512 90

Example 20

[0250] The appropriate acid chloride (0.24 mmol) was added to a solution of 4-[3-([1,4]diazepane-1-carbonyl)-4-fluorobenzyl]-2H-phthalazin-1-one (4) in dichloromethane (2 ml). Hunigs base (0.4 mmol) was then added and the reaction was stirred at room temperature for 16 hours. The reaction mixtures were then purified by preparative HPLC.

[0251] The compounds synthesised are set out below.

##STR00441##

TABLE-US-00026 LC RT LC Purity Compound R (minutes) M + 1 (%) 372 [00442] 2.81 466 80 385 [00443] 3.19 495 90

Example 21

[0252] 1-[2-Fluoro-5-(4-Oxo-3,4-dihydrophthalazin-1-ylmethyl)benzoyl]-piperidine-4-carboxylic acid (D) (0.24 mmol) was added to a solution of the appropriate amine (0.2 mmol) in dimethylacetamide (2 ml). 2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (0.3 mmol) and Hunigs base (0.4 mmol) were then added and the reaction was stirred at room temperature for 16 hours. The reaction mixtures were then purified by preparative HPLC.

[0253] The compounds synthesised are set out below.

##STR00444##

TABLE-US-00027 LC RT LC Purity Compound R (minutes) M + 1 (%) 386 [00445] 3.21 480 90

Example 22

[0254] In order to assess the inhibitory action of the compounds, the following assay was used to determine IC.sub.50 values (Dillon, et al., JBS., 8(3), 347-352 (2003)).

[0255] Mammalian PARP, isolated from Hela cell nuclear extract, was incubated with Z-buffer (25 mM Hepes (Sigma); 12.5 mM MgCl.sub.2 (Sigma); 50 mM KCl (Sigma); 1 mM DTT (Sigma); 10% Glycerol (Sigma) 0.001% NP-40 (Sigma); pH 7.4) in 96 well FlashPlates (TRADE MARK) (NEN, UK) and varying concentrations of said inhibitors added. All compounds were diluted in DMSO and gave final assay concentrations of between 10 and 0.01 μM, with the DMSO being at a final concentration of 1% per well. The total assay volume per well was 40 μl.

[0256] After 10 minutes incubation at 30□C the reactions were initiated by the addition of a 10 μl reaction mixture, containing NAD (5 μM), .sup.3H-NAD and 30mer double stranded DNA-oligos. Designated positive and negative reaction wells were done in combination with compound wells (unknowns) in order to calculate % enzyme activities. The plates were then shaken for 2 minutes and incubated at 30□C for 45 minutes.

[0257] Following the incubation, the reactions were quenched by the addition of 50 μl 30% acetic acid to each well. The plates were then shaken for 1 hour at room temperature.

[0258] The plates were transferred to a TopCount NXT (TRADE MARK) (Packard, UK) for scintillation counting. Values recorded are counts per minute (cpm) following a 30 second counting of each well.

[0259] The % enzyme activity for each compound is then calculated using the following equation:

[00001]$\%\mathrm{Inhibition}=100-\left(100⨯\frac{\left(\mathrm{cpm}\mathrm{of}\mathrm{unknowns}-\mathrm{mean}\mathrm{negative}\mathrm{cpm}\right)}{\left(\mathrm{mean}\mathrm{positive}\mathrm{cpm}-\mathrm{mean}\mathrm{negative}\mathrm{cpm}\right)}\right)$

[0260] IC.sub.50 values (the concentration at which 50% of the enzyme activity is inhibited) were calculated, which are determined over a range of different concentrations, normally from 10 μM down to 0.001 μM. Such IC.sub.50 values are used as comparative values to identify increased compound potencies.

[0261] All compounds tested had a IC.sub.50 of less than 0.1 μM.

[0262] The following compounds have an IC.sub.50 of less than 0.01 μM: 2-5, 9, 10, 12-20, 24, 26-28, 30, 32-35, 38, 39, 42, 44-47, 49, 51, 58-68, 80-82, 84, 86, 88-90, 94-97, 100-105, 113-116, 121-123, 126, 132, 136, 138-140, 142-144, 147, 149, 151, 152, 156, 158, 159, 161-164, 166-175, 177-180, 182-184, 186-190, 194, 199, 202, 205, 207, 208, 213, 221-223, 225, 233-236, 239-274, 277, 279, 287, 288, 292, 293, 301-303, 306, 315, 316, 318-323, 325, 327-336, 338-350, 352-357, 359, 361, 363, 365 and 367-370.

[0263] The following compounds, as well as those above, have an IC.sub.50 of less than 0.02 μM: 1,6-8, 11, 21-23, 25, 31, 36, 37, 40, 41, 43, 48, 52-54, 56, 57, 69-79, 83, 87, 91-93, 98, 99, 106, 109, 110, 111, 117, 118, 120, 124, 128-130, 133-135, 137, 141, 145, 146, 148, 150, 153-155, 157, 160, 165, 176, 181, 185, 191, 192, 195, 196, 197, 201, 203, 204, 206, 211, 212, 215-217, 219, 220, 224, 226, 227, 229-232, 237, 238, 275, 276, 278, 281-286, 289-291, 294, 295, 297-299, 304, 305, 307-309, 311, 314, 317, 324, 326, 337, 351, 358, 360, 362, 364, 366, 371 and 372.

[0264] The Potentiation Factor (PF.sub.50) for compounds is calculated as a ratio of the IC.sub.50 of control cell growth divided by the IC.sub.50 of cell growth+PARP inhibitor. Growth inhibition curves for both control and compound treated cells are in the presence of the alkylating agent methyl methanesulfonate (MMS). The test compounds were used at a fixed concentration of 0.2 micromolar. The concentrations of MMS were over a range from 0 to 10 μg/ml.

[0265] Cell growth was assessed using the sulforhodamine B (SRB) assay (Skehan, P., et al., (1990) New colorimetric cytotoxicity assay for anticancer-drug screening. J. Natl. Cancer Inst. 82, 1107-1112.). 2,000 HeLa cells were seeded into each well of a flat-bottomed 96-well microtiter plate in a volume of 100 □l and incubated for 6 hours at 37° C. Cells were either replaced with media alone or with media containing PARP inhibitor at a final concentration of 0.5, 1 or 5 μM. Cells were allowed to grow for a further 1 hour before the addition of MMS at a range of concentrations (typically 0, 1, 2, 3, 5, 7 and 10 μg/ml) to either untreated cells or PARP inhibitor treated cells. Cells treated with PARP inhibitor alone were used to assess the growth inhibition by the PARP inhibitor.

[0266] Cells were left for a further 16 hours before replacing the media and allowing the cells to grow for a further 72 hours at 37° C. The media was then removed and the cells fixed with 100 μl of ice cold 10% (w/v) trichloroacetic acid. The plates were incubated at 4° C. for 20 minutes and then washed four times with water. Each well of cells was then stained with 100 μl of 0.4% (w/v) SRB in 1% acetic acid for 20 minutes before washing four times with 10% acetic acid. Plates were then dried for 2 hours at room temperature. The dye from the stained cells was solubilized by the addition of 100 μl of 10 mM Tris Base into each well. Plates were gently shaken and left at room temperature for 30 minutes before measuring the optical density at 564 nM on a Microquant microtiter plate reader.

[0267] All the compounds tested had a PF.sub.50 at 200 nM of at least 2.0.

Example 23

[0268] To assess the stand alone activity of a PARP inhibitor on Braca 1 and 2 deficient cells the following protocols were used.

Small Molecule Inhibitors of PARP:

[0269] Compound (4) was dissolved in DMSO at 10 mM and stored at −20° C. in the dark.

Cell Lines

[0270] VC8 cells and the mouse Brca2 BAC complemented derivatives were as described in M. Kraakman-van der Zwet, et al., Mol Cell Biol 22, 669-79 (2002)). ES cells defective in Brca2 function have been described previously (Tutt, et al., EMBO Rep 3, 255-60 (2002)). The construction of ES cells defective in Brca1 will be described elsewhere but have previously been validated (Foray, et al., Embo J, 22, 2860-71 (2003)).

Clonogenic Assays

[0271] For measurement of cellular sensitivity to a PARP inhibitor (compound 4), cell cultures in exponential growth were trypsinised and seeded at various densities in 6-well plates onto Mitomycin C inactivated mouse embryonic fibroblasts and where appropriate treated with the test compound after 18 hours. For continuous exposure, cells were re-fed every 4 days with fresh medium and inhibitor. After 10-14 days, cells were washed with PBS, fixed in methanol and stained with crystal violet. Colonies containing greater than approximately 50 cells were counted. Experiments were performed at least three times in triplicate.

Results

Reduction in the Viability of BRCA1 and BRCA2 Deficient Cells

[0272] Compound 4 was used to probe the sensitivity of cells deficient in Brca1 or Brca2 to the inhibition of PARP activity. Clonogenic assays showed that both Brca1 and Brca2 deficient cell lines were extremely sensitive to compound 4 compared to otherwise isogenic cells (FIG. 1A, 1B). The SF.sub.50 (dosage at which 50% of cells survived) for Compound 4 was 1.5×10.sup.−8M for cells deficient in Brca1, whilst the SF.sub.50 for matched wild type cells was 7×10.sup.−6M (FIG. 1A). This represents a factor of 467 fold enhanced sensitivity of Brca1 mutant cells compared to wild type cells.

[0273] The SF.sub.50 for Compound 4 was 1.2×10-8M for cells deficient in Brca2 whilst the SF.sub.50 for matched wild type cells was 1.8×10.sup.−5M (FIG. 1B). This represents a factor of 1,500 fold enhanced sensitivity of Brca2 mutant cells compared to wild type cells. Similar results were obtained with Chinese hamster ovary cells deficient in Brca2 (VC8) compared to a Brca2-complemented derivative (VC8-BAC)(FIG. 2). The SF.sub.50 for Compound 4 was 5×10.sup.−8M for the Brca2 deficient VC8 line whilst the SF.sub.50 for matched control, VC8-BAC, was 3×10.sup.−5M (FIG. 2). This represents a factor of 600 fold enhanced sensitivity of Brca2 mutant cells compared to wild type cells.