Phthalazinone derivatives

0001The present invention relates to phthalazinone derivatives, and their use as pharmaceuticals. In particular, the present invention relates to the use of these compounds to inhibit the activity of the enzyme poly (ADP-ribose)polymerase, also known as poly(ADP-ribose)synthase and poly ADP-ribosyltransferase, and commonly referred to as PARP.

0002The mammalian enzyme PARP (a 113-kDa multidomain protein) has been implicated in the signalling of DNA damage through its ability to recognize and rapidly bind to DNA single or double strand breaks (<nplcit id="ncit0001" npl-type="s"><text>D'Amours, et al., 1999, Biochem. J. 342: 249-268</text></nplcit>).

0003Several observations have led to the conclusion that PARP participates in a variety of DNA-related functions including gene amplification, cell division, differentiation, apoptosis, DNA base excision repair and also effects on telomere length and chromosome stability (<nplcit id="ncit0002" npl-type="s"><text>d'Adda di Fagagna, et al., 1999, Nature Gen., 23(1): 76-80</text></nplcit>).

0004Studies on the mechanism by which PARP modulates DNA repair and other processes has identified its importance in the formation of poly (ADP-ribose) chains within the cellular nucleus (<nplcit id="ncit0003" npl-type="b"><text>Althaus, F.R. and Richter, C., 1987, ADP-Ribosylation of Proteins: Enzymology and Biological Significance, Springer-Verlag, Berl</text></nplcit>in). The DNA-bound, activated PARP utilizes NAD to synthesize poly (ADP-ribose) on a variety of nuclear target proteins, including topoisomerase, histones and PARP itself (<nplcit id="ncit0004" npl-type="s"><text>Rhun, et al., 1998, Biochem. Biophys. Res. Commun., 245: 1-10</text></nplcit>) Poly (ADP-ribosyl)ation has also been associated with malignant transformation. For example, PARP activity is higher in the isolated nuclei of SV40-transformed fibroblasts, while both leukemic cells and colon cancer cells show higher enzyme activity than the equivalent normal leukocytes and colon mucosa (<nplcit id="ncit0005" npl-type="s"><text>Miwa, et al., 1977, Arch. Biochem. Biophys. 181: 313-321</text></nplcit>; <nplcit id="ncit0006" npl-type="s"><text>Burzio, et al., 1975, Proc. Soc. Exp. Bioi. Med. 149: 933-938</text></nplcit>; and <nplcit id="ncit0007" npl-type="s"><text>Hirai, et al., 1983, Cancer Res. 43: 3441-3446</text></nplcit>).

0005A number of low-molecular-weight inhibitors of PARP have been used to elucidate the functional role of poly (ADP-ribosyl)ation in DNA repair. In cells treated with alkylating agents, the inhibition of PARP leads to a marked increase in DNA-strand breakage and cell killing (<nplcit id="ncit0008" npl-type="s"><text>Durkacz, et al., 1980, Nature 283: 593-596</text></nplcit>; <nplcit id="ncit0009" npl-type="s"><text>Berger, N.A., 1985, Radiation Research, 101: 4-14</text></nplcit>).

0006Subsequently, such inhibitors have been shown to enhance the effects of radiation response by suppressing the repair of potentially lethal damage (<nplcit id="ncit0010" npl-type="s"><text>Ben-Hur, et al., 1984, British Journal of Cancer, 49 (Suppl. VI): 34-42</text></nplcit>; <nplcit id="ncit0011" npl-type="s"><text>Schlicker, et al., 1999, Int. J. Radiat. Bioi., 75: 91-100</text></nplcit>). PARP inhibitors have been reported to be effective in radio sensitising hypoxic tumour cells (<patcit id="pcit0001" dnum="US5032617A"><text>US 5,032,617</text></patcit>; <patcit id="pcit0002" dnum="US5215738A"><text>US 5,215,738</text></patcit> and <patcit id="pcit0003" dnum="US5041653A"><text>US 5,041,653</text></patcit>).

0007Furthermore, PARP knockout (PARP -/-) animals exhibit genomic instability in response to alkylating agents and γ-irradiation (<nplcit id="ncit0012" npl-type="s"><text>Wang, et al., 1995, Genes Dev., 9: 509-520</text></nplcit>; <nplcit id="ncit0013" npl-type="s"><text>Menissier de Murcia, et al., 1997, Proc. Natl. Acad. Sci. USA, 94: 7303-7307</text></nplcit>).

0008A role for PARP has also been demonstrated in certain vascular diseases, septic shock, ischaemic injury and neurotoxicity (<nplcit id="ncit0014" npl-type="s"><text>Cantoni, et al., 1989, Biochim. Biophys. Acta, 1014: 1-7</text></nplcit>; <nplcit id="ncit0015" npl-type="s"><text>Szabo, et al., 1997, J. Clin.lnvest., 100: 723-735</text></nplcit>). Oxygen radical DNA damage that leads to strand breaks in DNA, which are subsequently recognised by PARP, is a major contributing factor to such disease states as shown by PARP inhibitor studies (<nplcit id="ncit0016" npl-type="s"><text>Cosi, et al., 1994, J. Neurosci. Res., 39: 38-46</text></nplcit>; <nplcit id="ncit0017" npl-type="s"><text>Said, et al., 1996, Proc. Natl. Acad. Sci. U.S.A., 93: 4688-4692</text></nplcit>). More recently, PARP has been demonstrated to play a role in the pathogenesis of haemorrhagic shock (<nplcit id="ncit0018" npl-type="s"><text>Liaudet, et al., 2000, Proc. Natl. Acad. Sci. U.S.A., 97(3): 10203-10208</text></nplcit>). Many of these diseases arise from massive cell loss and tissue damage caused by PARP activation.

0009It has also been demonstrated that efficient retroviral infection of mammalian cells is blocked by the inhibition of PARP activity. Such inhibition of recombinant retroviral vector infections was shown to occur in various different cell types (<nplcit id="ncit0019" npl-type="s"><text>Gaken, et al., 1996, J. Virology, 70(6): 3992-4000</text></nplcit>). Inhibitors of PARP have thus been developed for the use in anti-viral therapies and in cancer treatment (<patcit id="pcit0004" dnum="WO9118591A"><text>WO91/18591</text></patcit>).

0010Moreover, PARP inhibition has been speculated to delay the onset of aging characteristics in human fibroblasts (<nplcit id="ncit0020" npl-type="s"><text>Rattan and Clark, 1994, Biochem. Biophys. Res. Comm., 201 (2): 665-672</text></nplcit>). This may be related to the role that PARP plays in controlling telomere function (<nplcit id="ncit0021" npl-type="s"><text>d'Adda di Fagagna, et al., 1999, Nature Gen., 23(1): 76-80</text></nplcit>).

0011<patcit id="pcit0005" dnum="US5874444A"><text>US 5,874,444</text></patcit> discloses a number of PARP inhibitors, amongst which is 1(2H)-phthalazinone (100): <chemistry id="chem0001" num="0001"><img file="EP1501822B1_D0001.tif" /></chemistry>

0012The research team which includes the present inventors have previously discovered that certain derivatives of 1(2H)-phthalazinone and related compounds exhibit inhibition of the activity of PARP, and these compounds are described in <patcit id="pcit0006" dnum="GB0104729W"><text>PCT/GB01/04729, filed 25 October 2001</text></patcit> and <patcit id="pcit0007" dnum="US02150601A" dnum-type="L"><text>US Patent Application Serial No. 10/021,506, filed on 30 October 2001</text></patcit>, which are hereby incorporated by reference.

0013Following further study, the present inventors have discovered that the following classes of derivatives of 1(2H)-phthalazinone and related compounds also exhibit inhibition of the activity of PARP.

0014The first aspect of the present invention provides a compound of formula: <chemistry id="chem0002" num="0002"><img file="EP1501822B1_D0002.tif" /></chemistry> or an isomer, salt or solvate, wherein: A and B together represent a fused benzene ring; R^L is a phenyl group substituted in the <i>meta</i> position by the group R², and optionally further substituted in the para position by a group selected from halo and C_1-4 alkoxy; wherein R² is selected from: <ul id="ul0001" list-style="none" compact="compact"><li>a) <chemistry id="chem0003" num="0003"><img file="EP1501822B1_D0003.tif" /></chemistry> wherein: <ul id="ul0002" list-style="none" compact="compact"><li>n is 0;</li><li>Y is CR^C1R^C2;</li><li>R^C1, R^C2, R^C3, R^C4, R^C5, R^C6, R^C7 and R^C8 are independently selected from H, R, SR, NHC(=O)OR, and C_1-4 alkyl substituted by C_5-7 aryl, where R is C_1-10 alkyl or C_5-6 aryl optionally substituted by C_1-4 alkyl;</li><li>R^C4 and R^C6, R^C6 and R^C8 or R^C8 and R^C2 may optionally together form a double bond;</li><li>R^C1 and R^C2, R^C5 and R^C6 or R^C7 and R^C8 together with the carbon atom to which they are attached may optionally form a spiro-fused C_5-7 carbocylic or heterocyclic ring; and</li><li>R^C5 and R^C7 or R^C7 and R^C1 together with the carbon atoms to which they are attached form a carbocyclic or heterocyclic ring system optionally substituted with nitro, C_1-4 alkyl or</li><li>C_5-6 aryl groups;</li></ul></li><li>b) <chemistry id="chem0004" num="0004"><img file="EP1501822B1_D0004.tif" /></chemistry> wherein m is 0 or 1; X is selected from NR^N2 and CR^C9R^C10; R^N2 is selected from H and C_1-4 alkyl optionally substituted by C_5-7 aryl, or C_3-7 heterocyclyl; R^C9, R^C10, R^C11, R^C12, R^C13 and R^C14 are independently selected from H, R, SR and NHC(=O)OR, and C_1-4 alkyl substituted by C_5-7 aryl,where R is as defined above; R^C12 and R^C10 or R^C10 and R^C14 may optionally together form a double bond; R^C11 and R^C12, R^C9 and R^C10 or R^C13 and R^C14 together with the carbon atom to which they are attached may optionally form a spiro-fused C_5-7 carbocylic or heterocyclic ring; and R^C11 and R^C9 or R^C9 and R^C13 together with the carbon atoms to which they are attached may form a carbocyclic or heterocyclic ring system optionally substituted with nitro, C_1-4 alkyl or C_5-6 aryl groups; wherein the alkyl groups may be partially or fully unsaturated.</li></ul>

0015The structure of R² under a) above when n is 0 and Y is CR^C1R^C2 is as follows: <chemistry id="chem0005" num="0005"><img file="EP1501822B1_D0005.tif" /></chemistry>

0016The options for the structure of R² under b) above when m is 0 or 1 and X is NR^N2 or CR^C9R^C10 are as follows: <tables id="tabl0001" num="0001"><table frame="all"><tgroup cols="3"><colspec colnum="1" colname="col1" colwidth="24mm" /><colspec colnum="2" colname="col2" colwidth="39mm" /><colspec colnum="3" colname="col3" colwidth="41mm" /><thead><row><entry align="center" valign="middle" /><entry align="center" valign="middle">m=0</entry><entry align="center" valign="middle">m=1</entry></row></thead><tbody><row><entry align="center" valign="middle">X= NR^N2</entry><entry align="center" valign="middle"><chemistry id="chem0006" num="0006"><img file="EP1501822B1_D0006.tif" /></chemistry></entry><entry align="center" valign="middle"><chemistry id="chem0007" num="0007"><img file="EP1501822B1_D0007.tif" /></chemistry></entry></row><row><entry align="center" valign="middle">X=CR^C9R^C10</entry><entry align="center" valign="middle"><chemistry id="chem0008" num="0008"><img file="EP1501822B1_D0008.tif" /></chemistry></entry><entry align="center" valign="middle"><chemistry id="chem0009" num="0009"><img file="EP1501822B1_D0009.tif" /></chemistry></entry></row></tbody></tgroup></table></tables>

0017A second aspect of the present invention relates to a pharmaceutical composition comprising a compound of the first aspect and a pharmaceutically acceptable carrier or diluent.

0018A third aspect of the present invention relates to the use of a compound of the first aspect in a method of treatment of the human or animal body.

0019A fourth aspect of the present invention relates to the use of a compound of the first aspect in the preparation of a medicament for: <ol id="ol0001" compact="compact"><li>(a) the treatment of: vascular disease; septic shock; haemorraghic shock; ischaemic injury, both cerebral and cardiovascular; reperfusion injury, both cerebral and cardiovascular neurotoxicity, including acute and chronic treatments for stroke and Parkinsons disease; inflammatory diseases, such as arthritis; multiple sclerosis; secondary effects of diabetes; as well as the acute treatment of cytotoxicity following cardiovascular surgery;</li><li>(b) use as an adjunct in cancer therapy or for potentiating tumour cells for treatment with ionizing radiation or chemotherapeutic agents.</li></ol>

0020In particular, compounds as defined in the first aspect of the invention can be used in anti-cancer combination therapies (or as adjuncts) along with alkylating agents, such as methyl methanesulfonate (MMS) , temozolomide and dacarbazine (DTIC), also with topoisomerase-1 inhibitors like Irinotecan, Rubitecan, Exatecan, Lurtotecan, Gimetecan, Diflomotecan (homocamptothecins); as well as 7-substituted non-silatecans; the 7-silyl camptothecins, BNP 1350; and non-camptothecin topoisomerase-I inhibitors such as indolocarbazoles also dual topoisomerase-I and II inhibitors like the benzophenazines, XR 11576/MLN 576 and benzopyridoindoles. Such combinations could be given, for example, as intravenous preparations or by oral administration as dependent on the preferred method of administration for the particular agent.

<i><u>Definitions</u></i>

0021The term "fused ring system" as used herein pertains either to a system comprising in addition to the ring already defined in the formula, one or more aromatic rings, or one or more aliphatic rings.

0022The term "aromatic ring" is used herein in the conventional sense to refer to a cyclic aromatic structure, that is, a cyclic structure having delocalised n-electron orbitals.

0023The aromatic ring fused to the core rings, i.e. that formed by -A-B-, R^C5 and R^C7, R^C7 and R^C1, R^C11 and R^C9 and R^C9 and R^C13 may bear further fused aromatic rings (resulting in, e.g. naphthyl or anthracenyl groups). The aromatic ring(s) may comprise solely carbon atoms, or may comprise carbon atoms and one or more heteroatoms, including but not limited to, nitrogen, oxygen, and sulfur atoms. The aromatic ring(s) preferably have five or six ring atoms.

0024The aromatic ring(s) may optionally be substituted. If a substituent itself comprises an aryl group, this aryl group is not considered to be a part of the aryl group to which it is attached. For example, the group biphenyl is considered herein to be a phenyl group (an aryl group comprising a single aromatic ring) substituted with a phenyl group. Similarly, the group benzylphenyl is considered to be a phenyl group (an aryl group comprising a single aromatic ring) substituted with a benzyl group.

0025In one group of preferred embodiments, the aromatic group comprises a single aromatic ring, which has five or six ring atoms, which ring atoms are selected from carbon, nitrogen, oxygen, and sulfur, and which ring is optionally substituted. Examples of these groups include benzene, pyrazine, pyrrole, thiazole, isoxazole, and oxazole. 2-pyrone can also be considered to be an aromatic ring, but is less preferred.

0026If the aromatic ring has six atoms, then preferably at least four, or even five or all, of the ring atoms are carbon. The other ring atoms are selected from nitrogen, oxygen and sulphur, with nitrogen and oxygen being preferred. Suitable groups include a ring with: no hetero atoms (benzene); one nitrogen ring atom (pyridine); two nitrogen ring atoms (pyrazine, pyrimidine and pyridazine); one oxygen ring atom (pyrone); and one oxygen and one nitrogen ring atom (oxazine).

0027If the aromatic ring has five ring atoms, then preferably at least three, or even four or all, of the ring atoms are carbon. The remaining ring atoms are selected from nitrogen, oxygen and sulphur. Suitable rings include a ring with: one nitrogen ring atom (pyrrole); two nitrogen ring atoms (imidazole, pyrazole); one oxygen ring atom (furan); one sulphur ring atom (thiophene); one nitrogen and one sulphur ring atom (isothiazole or thiazole); one nitrogen and one oxygen ring atom (isoxazole or oxazole); two nitrogen and one oxygen (oxadiazole); and four nitrogen (tetrazole).

0028The aromatic ring may bear one or more substituent groups at any available ring position. These substituents are selected from nitro, C_1-4 alkyl and C_5-6 aryl.

0029The term "aliphatic ring" is used herein in the conventional sense to refer to a cyclic aliphatic structure, that is, a cyclic structure which is not aromatic.

0030The aliphatic ring fused to the core ring, i.e. that formed by R^C5 and R^C7, R^C7 and R^C1, R^C11 and R^C9 and R^C9 and R^C13 may bear further fused rings.

0031The aliphatic ring(s) may comprise solely carbon atoms (a carbocyclic ring), or may comprise carbon atoms and one or more heteroatoms, including but not limited to, nitrogen, oxygen, and sulfur atoms. The aliphatic ring(s) preferably have five to seven ring atoms, but may have more or less ring atoms than this.

0032The aliphatic ring(s) may be optionally substituted, and the substituent groups are selected from halo, nitro, C_1-4 alkyl and C_5-6 aryl. In one group of preferred embodiments, the aliphatic group comprises a single aliphatic ring, which has five or six ring atoms, which ring atoms are selected from carbon, nitrogen, oxygen and sulphur, and which ring is optionally substituted. Examples of these groups include, cyclohexane, cyclohexene, cyclopentane. Further examples are described with reference to the groups from which C_3-7 heterocyclic groups are derived below.

0033Spiro-fused rings: The term "spiro-fused rings" as used herein pertains to a carbocyclic or heterocyclic ring which is fused to the remainder of the molecule at a single carbon atom. The ring itself may contain only carbon ring atoms, and hence be a carbocyclic ring, or may contain one or more heteroatoms and thus be a heterocyclic ring. Examples of C_5-7 carbocyclic and heterocyclic rings are given herein.

0034Alkyl: The term "alkyl" as used herein, pertains to a monovalent moiety obtained by removing a hydrogen atom from a hydrocarbon compound having a specified number of carbon atoms, which may be aliphatic or alicyclic, or a combination thereof, and which may be saturated, partially unsaturated, or fully unsaturated.

0035In the context of alkyl groups, the prefixes (e.g. C_1-4, C_1-7, C_1-20, C_2-7, C_3-7, etc.) denote the number of carbon atoms, or range of number of carbon atoms. For example, the term "C_1-4alkyl," as used herein, pertains to an alkyl group having from 1 to 4 carbon atoms. Examples of groups of alkyl groups include C_1-4alkyl ("lower alkyl"), C_1-7alkyl, and C_1-20alkyl. Note that the first prefix may vary according to other limitations; for example, for unsaturated alkyl groups, the first prefix must be at least 2; for cyclic alkyl groups, the first prefix must be at least 3; etc.

0036Examples of saturated linear C_1-7 alkyl groups include, but are not limited to, methyl, ethyl, <i>n</i>-propyl, <i>n</i>-butyl, and <i>n</i>-pentyl (amyl).

0037Examples of saturated branched C_1-7 alkyl groups include, but are not limited to, <i>iso</i>-propyl, <i>iso</i>-butyl, <i>sec</i>-butyl, <i>tert</i>-butyl, and neo-pentyl.

0038Examples of saturated alicyclic (carbocyclic) C_1-7 alkyl groups (also referred to as "C_3-7 cycloalkyl" groups) include, but are not limited to, unsubstituted groups such as cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl, as well as substituted groups (e.g., groups which comprise such groups), such as methylcyclopropyl, dimethylcyclopropyl, methylcyclobutyl, dimethylcyclobutyl, methylcyclopentyl, dimethylcyclopentyl, methylcyclohexyl, cyclopropylmethyl and cyclohexylmethyl.

0039Examples of unsaturated C_1-7 alkyl groups which have one or more carbon-carbon double bonds (also referred to as "C_2-7 alkenyl" groups) include, but are not limited to, ethenyl (vinyl, -CH=CH₂), 2-propenyl (allyl, -CH₂-CH=CH₂), isopropenyl (-C(CH₃)=CH₂), butenyl, pentenyl, and hexenyl.

0040Examples of unsaturated C_1-7 alkyl groups which have one or more carbon-carbon triple bonds (also referred to as "C_2-7 alkynyl" groups) include, but are not limited to, ethynyl (ethinyl) and 2-propynyl (propargyl).

0041Examples of unsaturated alicyclic (carbocyclic) C_1-7alkyl groups which have one or more carbon-carbon double bonds (also referred to as "C_3-7 cycloalkenyl" groups) include, but are not limited to, unsubstituted groups such as cyclopropenyl, cyclobutenyl, cyclopentenyl, and cyclohexenyl, as well as substituted groups (e.g., groups which comprise such groups) such as cyclopropenylmethyl and cyclohexenylmethyl.

0042C_3-20 heterocyclyl: The term "C_3-20 heterocyclyl" as used herein, pertains to a monovalent moiety obtained by removing a hydrogen atom from a ring atom of a non-aromatic C_3-20 heterocyclic compound, said compound having one ring, or two or more rings (e.g., spiro, fused, bridged), and having from 3 to 20 ring atoms, atoms, of which from 1 to 10 are ring heteroatoms, and wherein at least one of said ring(s) is a heterocyclic ring. Preferably, each ring has from 3 to 7 ring atoms, of which from 1 to 4 are ring heteroatoms. "C_3-20" denotes ring atoms, whether carbon atoms or heteroatoms.

0043Examples of C_3-20 heterocyclyl groups having one nitrogen ring atom include, but are not limited to, those derived from aziridine, azetidine, azetine, pyrrolidine, pyrroline, piperidine, dihydropyridine, tetrahydropyridine, and dihydropyrrole (azoline).

0044Examples of C_3-20 heterocyclyl groups having one oxygen ring atom include, but are not limited to, those derived from oxirane, oxetane, oxolane (tetrahydrofuran), oxole (dihydrofuran), oxane (tetrahydropyran), dihydropyran, and pyran. Examples of substituted C_3-20 heterocyclyl groups include sugars, in cyclic form, for example, furanoses and pyranoses, including, for example, ribose, lyxose, xylose, galactose, sucrose, fructose, and arabinose.

0045Examples of C_3-20 heterocyclyl groups having one sulfur ring atom include, but are not limited to, those derived from thiolane (tetrahydrothiophene, thiane) and tetrahydrothiopyran.

0046Examples of C_3-20 heterocyclyl groups having two oxygen ring atoms include, but are not limited to, those derived from dioxane, for example 1,3-dioxane and 1,4-dioxane.

0047Examples of C_3-20 heterocyclyl groups having two nitrogen ring atoms include, but are not limited to, those derived from diazolidine (pyrazolidine), pyrazoline, imidazolidine, imidazoline, and piperazine.

0048Examples of C_3-20 heterocyclyl groups having one nitrogen ring atom and one oxygen ring atom include, but are not limited to, those derived from tetrahydrooxazole, dihydrooxazole, tetrahydroisoxazole, dihydroiosoxazole, morpholine, tetrahydrooxazine, dihydrooxazine, and oxazine.

0049Examples of C_3-20 heterocyclyl groups having one oxygen ring atom and one sulfur ring atom include, but are not limited to, those derived from oxathiolane and oxathiane.

0050Examples of C_3-20 heterocyclyl groups having one nitrogen ring atom and one sulfur ring atom include, but are not limited to, those derived from thiazoline, thiazolidine, and thiomorpholine.

0051Other examples of C_3-20 heterocyclyl groups include, but are not limited to, oxadiazine.

0052If the C_3-20 heterocyclyl is substituted, the substituents are on carbon, or nitrogen (if present), atoms.

0053Nitrogen-containing C_3-20 heterocyclyl: The term "nitrogen-containing C_3-20 heterocyclyl" as used herein, pertains to a C_3-20 heterocyclyl group as defined above having at least one nitrogen ring atom.

0054C_5-20 aryl: The term "C_5-20 aryl" as used herein, pertains to a monovalent moiety obtained by removing a hydrogen atom from an aromatic ring atom of a C_5-20 aromatic compound, said compound having one ring, or two or more rings (e.g. fused), and having from 5 to 20 ring atoms, and wherein at least one of said ring(s) is an aromatic ring. Preferably, each ring has from 5 to 7 ring atoms.

0055The ring atoms may be all carbon atoms, as in "carboaryl groups" in which case the group may conveniently be referred to as a "C_5-20 carboaryl" group.

0056Examples of C_5-20 aryl groups which do not have ring heteroatoms (i.e. C_5-20 carboaryl groups) include, but are not limited to, those derived from benzene (i.e. phenyl) (C₆), naphthalene (C₁₀), anthracene (C₁₄), phenanthrene (C₁₄), and pyrene (C₁₆).

0057Alternatively, the ring atoms may include one or more heteroatoms, including but not limited to oxygen, nitrogen, and sulfur, as in "heteroaryl groups". In this case, the group may conveniently be referred to as a "C_5-20 heteroaryl" group, wherein "C_5-20" denotes ring atoms, whether carbon atoms or heteroatoms. Preferably, each ring has from 5 to 7 ring atoms, of which from 0 to 4 are ring heteroatoms. Examples of C_5-20 heteroaryl groups include, but are not limited to, C₅ heteroaryl groups derived from furan (oxole), thiophene (thiole), pyrrole (azole), imidazole (1,3-diazole), pyrazole (1,2-diazole), triazole, oxazole, isoxazole, thiazole, isothiazole, oxadiazole, oxatriazole and tetrazole; and C₆ heteroaryl groups derived from isoxazine, pyridine (azine), pyridazine (1,2-diazine), pyrimidine (1,3-diazine; e.g., cytosine, thymine, uracil), pyrazine (1,4-diazine) and triazine.

0058The heteroaryl group may be bonded via a carbon or nitrogen ring atom.

0059Examples of C_5-20 heteroaryl groups which comprise fused rings, include, but are not limited to, C₉ heteroaryl groups derived from benzofuran, isobenzofuran, benzothiophene, indole, isoindole; C₁₀ heteroaryl groups derived from quinoline, isoquinoline, benzodiazine, pyridopyridine; C₁₄ heteroaryl groups derived from acridine and xanthene.

0060C_5-7 aryl: The term "C_5-7 aryl" as used herein, pertains to a monovalent moiety obtained by removing a hydrogen atom from an aromatic ring atom of a single C_5-7 aromatic ring having from 5 to 7 ring atoms.

0061If the ring atoms are all carbon, then the C_5-7 aryl group is derived from benzene, i.e. phenyl. Alternatively, the ring atoms may include one or more heteroatoms, including but not limited to oxygen, nitrogen, and sulfur, as in "heteroaryl groups". In this case, the group may conveniently be referred to as a "C_5-7 heteroaryl" group, wherein "C_5-7" denotes ring atoms, whether carbon atoms or heteroatoms. Upto 4 ring atoms may be heteroatoms. Examples of C_5-7 heteroaryl groups include, but are not limited to, C₅ heteroaryl groups derived from furan (oxole), thiophene (thiole), pyrrole (azole), imidazole (1,3-diazole), pyrazole (1,2-diazole), triazole, oxazole, isoxazole, thiazole, isothiazole, oxadiazole, oxatriazole, and tetrazole; and C₆ heteroaryl groups derived from isoxazine, pyridine (azine), pyridazine (1,2-diazine), pyrimidine (1,3-diazine; e.g., cytosine, thymine, uracil), pyrazine (1,4-diazine) and triazine.

0062The heteroaryl group may be bonded via a carbon or nitrogen ring atom.

0063Substituted in the <i>meta</i> position: The term "substituted in the <i>meta</i> position" as used herein, pertains to the substitution of the C_5-7 aryl group in a position 2 atoms away from where the group is bonded to the central moiety by -CH₂-. The following groups, which are given by way of example only, illustrate this position by the use of an asterix: <chemistry id="chem0010" num="0010"><img file="EP1501822B1_D0010.tif" /></chemistry>

0064C_1-6 alkylene: The term "C_1-6 alkylene" as used herein, pertains to a bidentate moiety obtained by removing two hydrogen atoms from separate carbon atoms of an aliphatic (i.e. non-cyclic) hydrocarbon compound having from 1 to 6 carbon atoms and which may be saturated, partially unsaturated, or fully unsaturated. Thus, the term "alkylene" includes the sub-classes alkenylene and alkynylene. Examples of these groups can be derived from the examples of alkyl groups given above, and thus include: saturated alkylene groups (e.g. methylene (C₁), propylene (C₃)); saturated linear alkylene groups (e.g. methylene (C₁), n-propylene (C₃)); saturated branched alkylene groups (e.g. iso-propylene (C₃), tert-butylene (C₄)); unsaturated alkenylene groups (e.g. ethenylene (-CH=CH-), isopropenylene (-C(CH₃)=CH-); unsaturated alkynylene groups (e.g. ethynylene (-C≡C-), 2-propynylene (-CH₂-C≡C-)).

0065The above C_1-4 alkyl, C_1-6 alkylene, C_3-7 heterocyclyl, nitrogen-containing C_3-7 heterocycly17 and C_5-7 aryl groups, whether alone or part of another substituent, may themselves optionally be substituted with one or more monovalent groups selected from themselves (unless otherwise stated) and the additional substituents listed below.

0066Nitro: -NO₂.

Further Preferences

0067The following preferences can apply to each aspect of the present invention, where applicable.

0068In the present invention, the fused aromatic ring(s) represented by -A-B- is benzene.

0069R^L is a phenyl group, and preferably has up to one further substituent in addition to the substituent defined as R² above.

0070This substituent is selected from halo and C_1-4 alkoxy. Halo groups are more preferred, with fluoro being most preferred. This further substituent is in the para position, i.e. adjacent R², and in a position 3 atoms away from where the group is bonded to the central moiety by - CH₂-.

0071R² is preferably of formula b). <i>R</i>² = a) Y is preferably CR^C1R^C2_. n is preferably 0.

0072R^C1 and R^C2 are preferably independently selected from H and R (C_5-6 aryl optionally substituted by C1-4 alkyl or C_1-10 alkyl), more preferably H and C_1-4 alkyl and are most preferably H.

0073It is preferred that none of R^C2, R^C4, R^C6 and R^C8 form a double bond, and that there are no spiro-fused rings.

0074It is also preferred that R^C5 and R^C7 and R^C7 and R^C1 do not form an optionally substituted ring system. If there is an optionally substituted ring system, it is preferably non-aromatic and carbocylic.

0075R^C3, R^C4, R^C5, R^C6, R^C7 and R^C8 are preferably independently selected from H and R (C_5-6 aryl optionally substituted by C_1-4 alkyl, or C_1-10 alkyl), more preferably H and C_1-4 alkyl and are most preferably H. <i>R²</i> = <i>b</i>) X is preferably CR^C9R^C10_. m is preferably 0.

0076R^N2 is selected from H and C_1-4 alkyl optionally substituted by C_5-7 aryl, or C_3-7 heterocyclyl, more preferably from H and unsubstituted C_1-4 alkyl.

0077R^C9 and R^C10 are preferably independently selected from H and R (C_5-6 aryl optionally substituted by C_1-4 alkyl or C_1-10 alkyl), more preferably H and C_1-4 alkyl and are most preferably H.

0078It is preferred that none of R^C10, R^C12 and R^C14 form a double bond, and that there are no spiro-fused rings. If there is a double bond it is preferably formed by R^C10 and R^C14_. If there is a spiro fused ring it is preferably carbocyclic, and is preferably formed by R^C9 and R^C10.

0079It is also preferred that R^C11 and R⁹ and R^C9 and R^C13 do not form an optionally substituted ring system. If there is an optionally substituted ring system, it is preferably non-aromatic and carbocylic, and it is preferably formed by R^C9 and R^C13.

0080R^C11, R^C12, R^C13 and R^C14 are preferably independently selected from H and R (C_1-10 alkyl or C5-6 aryl optionally substituted by C_1-4 alkyl) and more preferably from H, C_1-4 alkyl and phenyl.

0081It is preferred that at least two of R^C11, R^C12, R^C13 and R^C14 are H, and it is more preferred that three or four of them are H.

Compounds

0082Preferred compounds include, but are not limited to: <chemistry id="chem0011" num="0011"><img file="EP1501822B1_D0011.tif" /></chemistry><chemistry id="chem0012" num="0012"><img file="EP1501822B1_D0012.tif" /></chemistry>

0083Further preferred compounds are exemplified below.

0084Where appropriate, the above preferences may be taken in combination with each other.

Includes Other Forms

0085Included in the above are the well known ionic, salt and solvate forms of these substituents. For example, a reference to carboxylic acid (-COOH) also includes the anionic (carboxylate) form (-COO^-), a salt or solvate thereof. Similarly, a reference to an amino group includes the protonated form (-N⁺HR¹R²), a salt or solvate of the amino group, for example, a hydrochloride salt. Similarly, a reference to a hydroxyl group also includes the anionic form (-O^-), a salt or solvate thereof.

Isomers, Salts and Solvates

0086Certain compounds may exist in one or more particular geometric, optical, enantiomeric, diasteriomeric, epimeric, stereoisomeric, tautomeric, conformational, or anomeric forms, including but not limited to, <i>cis-</i> and <i>trans</i>-forms; <i>E</i>- and <i>Z</i>-forms; <i>c-, t-,</i> and <i>r</i>-forms; <i>endo</i>- and <i>exo</i>-forms; <i>R-, S-,</i> and meso-forms; <i>D</i>- and <i>L</i>-forms; <i>d</i>- and <i>l</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").

0087If the compound is in crystalline form, it may exist in a number of different polymorphic forms.

0088Note 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, -OCH₃, is not to be construed as a reference to its structural isomer, a hydroxymethyl group, -CH₂OH. 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_1-7 alkyl includes <i>n</i>-propyl and <i>iso</i>-propyl; butyl includes <i>n-, iso-, sec</i>-, and <i>tert</i>-butyl; methoxyphenyl includes <i>ortho-, meta-,</i> and <i>para</i>-methoxyphenyl).

0089The 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, <i>N</i>-nitroso/hyroxyazo, and nitro/aci-nitro.

0090Particularly relevant to the present invention is the tautomeric pair that exists when R_N is H, illustrated below: <chemistry id="chem0013" num="0013"><img file="EP1501822B1_D0013.tif" /></chemistry> 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 ¹H, ²H (D), and ³H (T); C may be in any isotopic form, including ¹²C, ¹³C, and ¹⁴C; O may be in any isotopic form, including ¹⁶O and ¹⁸O; and the like.

0091Unless 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.

0092Unless otherwise specified, a reference to a particular compound also includes ionic, salt and solvate forms of thereof, for example, as discussed below, as well as its different polymorphic forms.

0093It 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 <nplcit id="ncit0022" npl-type="s"><text>Berge et al., 1977, "Pharmaceutically Acceptable Salts," J. Pharm. Sci., Vol. 66, pp. 1-19</text></nplcit>.

0094For 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⁺ and K⁺, alkaline earth cations such as Ca²⁺ and Mg²⁺, and other cations such as Al³⁺. Examples of suitable organic cations include, but are not limited to, ammonium ion (i.e., NH₄⁺) and substituted ammonium ions (e.g., NH₃R⁺, NH₂R₂⁺, NHR₃⁺, NR₄⁺).

0095Examples 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(CH₃)₄⁺.

0096If the compound is cationic, or has a functional group which may be cationic (e.g., -NH₂ may be -NH₃⁺), 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.

0097It 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.

Acronyms

0098For convenience, many chemical moieties are represented using well known abbreviations, including but not limited to, methyl (Me), ethyl (Et), <i>n</i>-propyl (nPr), <i>iso</i>-propyl (iPr), <i>n</i>-butyl (nBu), <i>tert</i>-butyl (tBu), <i>n</i>-hexyl (nHex), cyclohexyl (cHex), phenyl (Ph), biphenyl (biPh), benzyl (Bn), naphthyl (naph), methoxy (MeO), ethoxy (EtO), benzoyl (Bz), and acetyl (Ac).

0099For 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 (Et₂O), acetic acid (AcOH), dichloromethane (methylene chloride, DCM), trifluoroacetic acid (TFA), dimethylformamide (DMF), tetrahydrofuran (THF), and dimethylsulfoxide (DMSO).

Synthesis

0100Compounds of the present invention in which R_L is a phenyl group substituted in the <i>meta</i>-position by the group R², and optionally further substituted, wherein R² is the group (a), and Y is CR^C1R^C2 and which can therefore be represented by Formula 1: <chemistry id="chem0014" num="0014"><img file="EP1501822B1_D0014.tif" /></chemistry> in which R^C1, R^C2, R^C3, R^C4, R^C5, R^C6, R^C7, R^C8, A, B and n are as defined previously and R^CX is a group selected from halo and C_1-4 alkoxy, for example fluorine, may be synthesised by reaction of a compound of Formula 2: <chemistry id="chem0015" num="0015"><img file="EP1501822B1_D0015.tif" /></chemistry> in which R^C1, R^C2, R^C3, R^C4, R^C5, R^C6, R^C7, R^C8, R^CX, A, B and n are as previously defined and Q is a leaving group, for example a halogen such as chlorine, with a base, for example sodium ethoxide, in a solvent, for example ethanol, at a temperature in the range of 0°C to the boiling point of the solvent used.

0101Compounds of Formula 2 may be synthesised by reaction of a compound of Formula 3: <chemistry id="chem0016" num="0016"><img file="EP1501822B1_D0016.tif" /></chemistry> in which R^CX, A and B are as previously defined, with a commercially available or readily accessible compound of formula QCR^C3R^C4CR^C5R^C6(CR^C7R^C8)_nCR^C1R^C2COZ, in which R^C1, R^C2, R^C3, R^C4, R^C5, R^C6, R^C7, R^C8 and Q are as previously defined and Z is a leaving group, for example a halogen such as chlorine, optionally in the presence of a base, for example triethylamine, in the presence of a solvent, for example dichloromethane or dioxane, at a temperature in the range of 0°C to the boiling point of the solvent used.

0102Compounds of Formula 2 may also be synthesised by reaction of a compound of Formula 3 with a commercially available or readily accessible compound of formula QCR^C3R^C4CR^C5R^C6(CR^C7R^C8)_nCR^C1R^C2CO₂H in the presence of a coupling reagent system, for example (dimethylaminopropyl)ethylcarbodiimide hydrochloride/hydroxybenzotriazole or <i>O</i>-benzotriazol-1-yl-<i>N,N,N'N'-</i>tetramethyluronium tetrafluoroborate, in the presence of a solvent, for example dichloromethane, dimethylformamide or dimethylacetamide, in the presence of a base, for example diisopropylethylamine, at a temperature in the range of 0°C to the boiling point of the solvent used.

0103Compounds of the present invention in which R_L is a phenyl group substituted in the <i>meta</i>-position by the group R², and optionally further substituted in the para postion by a group selected from halo and C_1-4 alkoxy, wherein R² is the group (b), m is 0 and X is NR^N2, and which can therefore be represented by Formula 8: <chemistry id="chem0017" num="0017"><img file="EP1501822B1_D0017.tif" /></chemistry> in which R^C13, R^C14, R^CX, A and B are as previously defined and R^N2 is optionally substituted C_1-10 alkyl or optionally substituted C_5-7 aryl, may be synthesised by reaction of a compound of Formula 8 in which R^N2 is H with an alkylating agent of formula R^N2Q, in which Q is a leaving group, for example a halogen such as bromine, in the presence of a base, for example sodium hydride, in a solvent, for example tetrahydrofuran or dimethylformamide, at a temperature in the range of 0°C to the boiling point of the solvent used.

0104Compounds of Formula 8 in which R^N2 is H may be synthesised by reaction of a compound of Formula 3 with a commercially available or readily accessible compound of formula EtO₂C.NH.CR^C13R^C14.CO₂Et in which R^C13 and R^C14 are as previously defined, optionally in a solvent, for example xylene, at a temperature in the range of 0-200°C.

0105Compounds of Formula 8 in which R^N2 is H may also be synthesised by reaction of a compound of Formula 3 with a commercially available or readily accessible compound of formula OCN.CR^C13R^C14.CO₂Et in which R^C13 and R^C14 are as previously defined, optionally in a solvent, for example xylene, at a temperature in the range of 0-200°C.

0106Compounds of the present invention in which R_L is a phenyl group substituted in the <i>meta</i>-position by the group R², and optionally further substituted in the para position by a group selected from halo and C_1-4 alkoxy, wherein R² is the group (b), m is 1 and X is NR^N2, and which can therefore be represented by Formula 10: <chemistry id="chem0018" num="0018"><img file="EP1501822B1_D0018.tif" /></chemistry> in which R^CX, R^C11, R^C12, R^C13, R^C14, A and B are as previously defined and R^N2 is a C_1-4 alkyl optionally substituted by C_5-7 aryl or C_1-10 alkyl or optionally substituted C_5-7 aryl group may be synthesised by reaction of a compound of Formula 10 in which R^N2 is H with an alkylating agent of formula R^N2Q, in which Q is a leaving group, for example a halogen such as bromine, in the presence of a base, for example sodium hydride, in a solvent, for example tetrahydrofuran or dimethylformamide, at a temperature in the range of 0°C to the boiling point of the solvent used. In the case of certain C5-7 aryl groups, palladium catalysts (Buchwald chemistry) may be required to effect the transformation.

0107Compounds of Formula 10 in which R^CX, R^C11, R^C12, R^C13, R^C14, A and B are as previously defined and R^N2 is a C_1-4 alkyl optionally substituted by C_5-7 aryl of formula -CHR^C20R^C21 may also be synthesised by reductive alkylation of a compound of Formula 10 in which R^N2 is H with an aldehyde or ketone of formula R^C20R^C21CO, in which R^C20 and R^C21 are H, a C_1-4 alkyl or C_5-7 aryl, in the presence of a reducing agent, for example sodium cyanoborohydride or sodium triacetoxyborohydride, in a solvent, for example methanol or 1,2-dichloroethane, optionally in the presence of an acidic catalyst, for example acetic acid, at a temperature in the range of 0°C to the boiling point of the solvent used.

0108Compounds of Formula 10 in which R^C8 is H may be synthesised by deprotection of a compound of Formula 12: <chemistry id="chem0019" num="0019"><img file="EP1501822B1_D0019.tif" /></chemistry> in which R^C11, R^C12, R^C13, R^C14, R^CX, A and B are as previously defined and P is an amine protecting group, for example a benzyl or tert-butoxycarbonyl group, under reaction conditions appropriate for the removal of the protecting group, for example catalytic hydrogenolysis using gaseous hydrogen or an <i>in situ</i> source of hydrogen, for example ammonium formate, and a catalyst, for example palladium-on-carbon, or an acid such as trifluoroacetic acid.

0109Compounds of Formula 12 may be synthesised by reaction of a compound of Formula 13: <chemistry id="chem0020" num="0020"><img file="EP1501822B1_D0020.tif" /></chemistry> or a compound of Formula 14: <chemistry id="chem0021" num="0021"><img file="EP1501822B1_D0021.tif" /></chemistry> or mixtures thereof, in which R^C11, R^C12, R^C13, R^C14, R^CX, A and B are as previously defined, with a coupling reagent system, for example (dimethylaminopropyl)ethylcarbodiimide hydrochloride/hydroxybenzotriazole or <i>O</i>-benzotriazol-1-yl-<i>N,N,N'N'</i>-tetramethyluronium tetrafluoroborate, in the presence of a solvent, for example dichloromethane, dimethylformamide or dimethylacetamide, in the presence of a base, for example diisopropylethylamine, at a temperature in the range of 0°C to the boiling point of the solvent used.

0110Compounds of Formula 13, 14 or mixtures thereof, may be synthesised by reaction of a compound of Formula 3 with a commercially available or readily accessible compound of Formula 15: <chemistry id="chem0022" num="0022"><img file="EP1501822B1_D0022.tif" /></chemistry> in which R^C11, R^C12, R^C13, R^C14 and P are as previously defined, in the presence of a solvent, for example toluene or acetonitrile, at a temperature in the range of 0°C to the boiling point of the solvent used.

0111Compounds of Formula 12 may also be synthesised directly by reaction of a compound of Formula 3 with a compound of Formula 15 in the presence of a solvent, for example acetic acid, at a temperature in the range of 0°C to the boiling point of the solvent used.

0112Compounds of Formula 12 may also be synthesised directly by reaction of a compound of Formula 3 with a commercially available or readily accessible compound of formula HO₂C.CR^C11R^C12.NP.CR^C13R^C14.CO₂H, in which R^C11, R^C12, R^C13, R^C14 and P are as previously defined, optionally in the presence of a coupling reagent system, for example (dimethylaminopropyl)ethylcarbodiimide hydrochloride/hydroxybenzotriazole or <i>O</i>-benzotriazol-1-yl-<i>N,N,N'N'</i>-tetramethyluronium tetrafluoroborate, optionally in the presence of a solvent, for example dichloromethane, dimethylformamide or dimethylacetamide, optionally in the presence of a base, for example diisopropylethylamine, at a temperature in the range of 0°C to the boiling point of the solvent used or, in the absence of solvent, in the range of 0°C to 250°C.

0113Where the substituents are compatible with the chosen methodologies, compounds of Formula 10 and 11 may also be synthesised using the methodologies described above for the synthesis of compounds of Formula 12.

0114Compounds of the present invention in which R_L is a phenyl group substituted in the <i>meta</i>-position by the group R², and optionally further substituted in the para position by a group selected from halo and C_1-4 alkoxy, wherein R² is the group (b) and X is CR^C9R^C10, and which can therefore be represented by Formula 16: <chemistry id="chem0023" num="0023"><img file="EP1501822B1_D0023.tif" /></chemistry> in which R^C9, R^C10, R^C11, R^C12, R^C13, R^C14, R^CX, A, B and m are as previously defined, may be synthesised by reaction of a compound of Formula 17: <chemistry id="chem0024" num="0024"><img file="EP1501822B1_D0024.tif" /></chemistry> or a compound of Formula 18: <chemistry id="chem0025" num="0025"><img file="EP1501822B1_D0025.tif" /></chemistry> or mixtures thereof, in which R^C9, R^C10, R^C11, R^C12, R^C13, R^C14, R^CX, A, B and m are as previously defined, with a coupling reagent system, for example (dimethylaminopropyl)ethylcarbodiimide hydrochloride/hydroxybenzotriazole or <i>O</i>-benzotriazol-1-yl-<i>N,N,N'N'</i>-tetramethyluronium tetrafluoroborate, in the presence of a solvent, for example dichloromethane, dimethylformamide or dimethylacetamide, in the presence of a base, for example diisopropylethylamine, at a temperature in the range of 0°C to the boiling point of the solvent used.

0115Compounds of Formula 17, 18 or mixtures thereof may be synthesised by reaction of a compound of Formula 3 with a commercially available or readily accessible compound of Formula 19: <chemistry id="chem0026" num="0026"><img file="EP1501822B1_D0026.tif" /></chemistry> in which R^C9, R^C10, R^C11, R^C12, R^C13, R^C14, R^CX, A, B and m are as previously defined, in a solvent, for example toluene, at a temperature in the range of 0°C to the boiling point of the solvent used.

0116Compounds of Formula 16 may also be synthesised directly by reaction of a compound of Formula 3 with a compound of Formula 19 in the presence of a solvent, for example acetic acid, at a temperature in the range of 0°C to the boiling point of the solvent used.

0117Compounds of Formula 16 may also be synthesised directly by reaction of a compound of Formula 3 with a commercially available or readily accessible compound of Formula 20: <chemistry id="chem0027" num="0027"><img file="EP1501822B1_D0027.tif" /></chemistry> in which R^C9, R^C10, R^C11_, R^C12, R^C13, R^C14 and m are as previously defined, optionally in the presence of a coupling reagent system, for example (dimethylaminopropyl)ethylcarbodiimide hydrochloride/hydroxybenzotriazole or <i>O</i>-benzotriazol-1-yl-<i>N,N,N'N'</i>-tetramethyluronium tetrafluoroborate, optionally in the presence of a solvent, for example dichloromethane, dimethylformamide or dimethylacetamide, optionally in the presence of a base, for example diisopropylethylamine, at a temperature in the range of 0°C to the boiling point of the solvent used or, in the absence of solvent, in the range of 0°C to 250°C.

0118Compounds of Formula 17 or 18 may also be synthesised using the above methodology, but employing a monoprotected analogue of a compound of Formula 20, for example a monoester, then deprotecting the resulting intermediate amidoester.

0119Compounds of Formula 3 may be synthesised by reaction of a compound of Formula 21: <chemistry id="chem0028" num="0028"><img file="EP1501822B1_D0028.tif" /></chemistry> in which R^CX, A and B are as previously defined, with a reducing agent, for example stannous chloride, titanium trichloride, iron powder/ammonium chloride, or hydrogen in the presence of an appropriate hydrogenation catalyst, for example palladium-on-carbon, in the presence of a solvent, for example ethanol and/or water, at a temperature in the range of 0°C to the boiling point of the solvent used, optionally at a pressure above 1 atmosphere.

0120Where the nature of the substituent, R^CX, is compatible with the methodologies used, compounds of Formula 21 may be synthesised by reaction of a compound of Formula 22: <chemistry id="chem0029" num="0029"><img file="EP1501822B1_D0029.tif" /></chemistry> in which R^CX, A and B are as previously defined, with hydrazine hydrate, optionally in the presence of a solvent, for example ethanol, at a temperature in the range of 0°C to the boiling point of the solvent or reagent used.

0121Where the nature of the substituent, R^CX, is compatible with the methodologies used, compounds of Formula 3 may also be synthesised directly from a compound of Formula 22 by reaction with hydrazine hydrate, optionally in the presence of a solvent, for example ethanol, at a temperature in the range of 0°C to the boiling point of the solvent or reagent used.

0122Compounds of Formula 22 may be synthesised by reaction of a compound of Formula 23: <chemistry id="chem0030" num="0030"><img file="EP1501822B1_D0030.tif" /></chemistry> in which A and B are as previously defined, with a commercially available or readily accessible compound of Formula 24: <chemistry id="chem0031" num="0031"><img file="EP1501822B1_D0031.tif" /></chemistry> in the presence of a base, for example sodium methoxide, lithium hexamethyldisilazide or triethylamine, in the presence of a solvent, for example methanol or tetrahydrofuran, at a temperature in the range of -80°C to the boiling point of the solvent used.

0123Compounds of Formula 3 may also be synthesised by reaction of a compound of Formula 25: <chemistry id="chem0032" num="0032"><img file="EP1501822B1_D0032.tif" /></chemistry> in which R^CX, A and B are as previously defined, with hydrazine hydrate, optionally in the presence of a solvent, for example ethanol, at a temperature in the range of 0°C to the boiling point of the solvent or reagent used.

0124Compounds of Formula 25 may be synthesised by reaction of a compound of Formula 26: <chemistry id="chem0033" num="0033"><img file="EP1501822B1_D0033.tif" /></chemistry> in which R^CX, A and B are as previously defined, with a reducing agent, for example stannous chloride, titanium trichloride, iron powder/ammonium chloride, or hydrogen in the presence of an appropriate hydrogenation catalyst, for example palladium-on-carbon, in the presence of a solvent, for example ethanol and/or water, at a temperature in the range of 0°C to the boiling point of the solvent used, optionally at a pressure above 1 atmosphere.

0125Compounds of Formula 26 may be synthesised by reaction of a compound of Formula 27: <chemistry id="chem0034" num="0034"><img file="EP1501822B1_D0034.tif" /></chemistry> in which A and B are as previously defined, with a compound of Formula 24, in the presence of a base, for example sodium methoxide, lithium hexamethyldisilazide or triethylamine, in the presence of a solvent, for example methanol or tetrahydrofuran, at a temperature in the range of -80°C to the boiling point of the solvent used.

0126Compounds of Formula 27 may be synthesised by reaction of a commercially available or readily accessible compound of Formula 28: <chemistry id="chem0035" num="0035"><img file="EP1501822B1_D0035.tif" /></chemistry> in which A and B are as previously defined, with dimethyl phosphite and a base, for example sodium methoxide, in a solvent, for example methanol, at a temperature in the range of -10°C to the boiling point of the solvent used.

0127In addition, in the circumstance in which a compound of the present invention contains a functional group suitable for commonly employed "Functional Group Interconversion" chemistry, then the invention also claims the products of such chemistry. Relevant examples are shown below in Scheme 1: <chemistry id="chem0036" num="0036"><img file="EP1501822B1_D0036.tif" /></chemistry>

Use

0128The present invention provides active compounds, specifically, active in inhibiting the activity of PARP.

0129The 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.

0130One assay which may conveniently be used in order to assess the PARP inhibition offered by a particular compound is described in the examples below.

0131The 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 <i>in vitro</i> or <i>in vivo.</i>

0132For example, a sample of cells may be grown <i>in vitro</i> 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.

0133The 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.

0134The term "adjunct" as used herein relates to the use of active compounds in conjunction with known therapeutic means. Such means include cytotoxic regimes of drugs and/or ionising radiation as used in the treatment of different cancer types.

0135Active compounds may also be used as cell culture additives to inhibit PARP, for example, in order to radio-sensitize cells to known chemotherapeutic or ionising radiation treatments <i>in vitro.</i>

0136Active compounds may also be used as part of an <i>in vitro</i> assay, for example, in order to determine whether a candidate host is likely to benefit from treatment with the compound in question.

Administration

0137The 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.

0138The 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

0139While 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.

0140Thus, 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.

0141The term "pharmaceutically acceptable" as used herein pertains to compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, 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.

0142Suitable carriers, excipients, etc. can be found in standard pharmaceutical texts, for example, <nplcit id="ncit0023" npl-type="b"><text>Remington=s Pharmaceutical Sciences, 18th edition, Mack Publishing Company, Easton, Pa., 1990</text></nplcit>.

0143The 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.

0144Formulations 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.

0145Formulations 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.

0146A 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.

0147Tablets may optionally be provided with an enteric coating, to provide release in parts of the gut other than the stomach.

0148Formulations 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.

0149Formulations 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.

0150Formulations 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.

0151Formulations 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.

0152Formulations 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, dichoro-tetrafluoroethane, carbon dioxide, or other suitable gases.

0153Formulations 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.

0154When 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.

0155Suitable 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.

0156Formulations suitable for rectal administration may be presented as a suppository with a suitable base comprising, for example, cocoa butter or a salicylate.

0157Formulations 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.

0158Formulations 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

0159It 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.

0160Administration <i>in vivo</i> 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.

0161In 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.

Examples

0162¹H NMR spectra were recorded using a Bruker Avance 250 spectrometer. Chemical shifts are reported in parts per million (ppm) on the δ scale relative to tetramethylsilane internal standard. Analytical LC-MS was carried out on a Micromass Platform LC-MS using a Phenomenex Luna C18 5µm - 50 x 2.1 mm column, mobile phase - 10-90% acetonitrile/water (containing 0.4% formic acid) over 3 minutes, hold for 2 minutes, return to 10% acetonitrile over 1 minute and re-equilibrate over 4 minutes, diode array detection at 220-350nm, cone voltage set at 30V, scan range 100-750 Daltons over 1.5s - interscan delay 0.3s, detection - +/- switching capturing positive and negative spectra.

Example 1

0163<chemistry id="chem0037" num="0037"><img file="EP1501822B1_D0037.tif" /></chemistry>

0164Sodium methoxide solution (27% in methanol, 400 g, 2 mol) was added over 40 minutes at 20-30°C to a stirred mixture of phthalide (67 g, 0.5 mol), 3-nitrobenzaldehyde (75.5 g, 0.5 mol), ethyl propionate (250 ml) and methanol (150 ml). The mixture was stirred at ambient temperature for 15 minutes then it was heated under reflux for 2.5 hours, cooled to ambient temperature and poured into water (2300 ml). The aqueous mixture was washed with ether (5 x 500 ml) then acetic acid (60 ml) was added. The resulting solid was collected by filtration, washed with water (200 ml) and dried <i>in vacuo</i> to give 2-(3-nitrophenyl)indan-1,3-dione (87.92 g) as a dark red-brown solid, m.pt. 216-226°C, which was used without further purification.

0165A stirred mixture of 2-(3-nitrophenyl)indan-1,3-dione (85 g, 0.318 mol) and hydrazine hydrate (450 ml) was heated under reflux for 2 hours then cooled to 0°C. The resulting solid was collected by filtration, washed with water (500 ml), ground to a fine powder, mixed with sufficient cold ethanol to give a thick paste, collected by filtration and dried <i>in vacuo</i> at 55°C. The crude solid was then recrystallised from ethanol to give 4-(3-aminobenzyl)-2<i>H-</i>phthalazin-1-one (32.6 g) as a pale brown solid, m.pt. 175-177°C.

01664-Chlorobutyryl chloride (5.42 g, 38.4 mmol) was added dropwise at ambient temperature to a stirred mixture of 4-(3-aminobenzyl)-2<i>H</i>-phthalazin-1-one (8 g, 32 mmol; prepared in a manner similar to that described above), triethylamine (5.35 ml, 38.4 mmol) and 1,4-dioxane (40 ml), the mixture was stirred at ambient temperature for 1 hour, then it was poured into ice-water (100 ml). The resulting solid was collected by filtration, washed with water (30 ml) and dried <i>in vacuo</i> to give 4-chloro-<i>N</i>-[3-(4-oxo-3,4-dihydrophthalazin-1-ylmethyl)phenyl]butyramide (12.34 g) as an off-white solid, m.pt. 180-184°C.

01674-Chloro-<i>N</i>-[3-(4-oxo-3,4-dihydrophthalazin-1-ylmethyl)phenyl]butyramide (1 g, 2.8 mmol) was added in portions at 0°C to a stirred solution of sodium ethoxide [from sodium (0.15 g, 6.7 mmol)] in ethanol (10 ml), then the mixture was heated under reflux for 3 hours, cooled to ambient temperature and added to ice-water (50 ml). The resulting solid was collected by filtration, washed with water (10 ml) and dried <i>in vacuo</i> to give 4-[3-(2-oxopyrrolidin-1-yl)benzyl]-2<i>H</i>-phthalazin-1-one (0.77 g) as an off-white solid, m.pt. 206-207°C; 250 MHz ¹H-nmr (d₆-DMSO) δ (ppm): 2.00 (m, 2<i>H</i>) (-CH₂C<b><i>H</i></b>₂CH₂-), 2.55 (m - partially obscured by DMSO peak, 2<i>H</i>) (-NC<b><i>H</i></b>₂-), 3.75 (t, 2<i>H</i>) (-COC<b><i>H</i></b>₂-), 4.3 (s, 2<i>H</i>) (ArC<b><i>H</i></b>₂-), 7.05 (d, 1<i>H</i>) (Ar<b><i>H</i></b>), 7.25 (t, 1<i>H</i>) (Ar<b><i>H</i></b>), 7.5 (d, 1<i>H</i>) (Ar<b><i>H</i></b>), 7.7 (s, 1<i>H</i>) (Ar<b><i>H</i></b>), 7.75-8.0 (m, 3<i>H</i>) (3 x Ar<b><i>H</i></b>), 8.25 (d, 1H)(Ar<b><i>H</i></b>); m/z (M+H)^+. 320, 100% purity.

Example 2

0168<chemistry id="chem0038" num="0038"><img file="EP1501822B1_D0038.tif" /></chemistry>

0169A solution of 4-(3-aminobenzyl)-2<i>H</i>-phthalazin-1-one (2 g, 8 mmol; prepared in a manner similar to that described in Example 1) in acetic acid (15 ml) was added to a stirred solution of succinic anhydride (0.96 g, 9.6 mmol) in acetic acid (15 ml), the mixture was heated under reflux for 4 hours, then it was allowed to stand at ambient temperature for 65 hours. Lc-ms analysis of the reaction mixture indicated it contained a mixture of the required product and the uncyclised amidoacid. The stirred mixture was heated under reflux for a further 9.25 hours and allowed to stand at ambient temperature overnight. The resulting solid was collected by filtration, washed with water (60 ml) and hexane (20 ml), and dried <i>in vacuo</i> to give 1-[3-(4-oxo-3,4-dihydrophthalazin-1-ylmethyl)phenyl]pyrrolidine-2,5-dione (1.142 g) as an off-white solid, 250 MHz ¹H-nmr (d₆-DMSO) δ (ppm): 2.75 (s, 4<i>H</i>) (-C<b>H</b>₂C<b><i>H</i></b>₂-) , 4.4 (s, 2<i>H</i>) (ArC<b><i>H</i></b>₂-), 7.15 (t, 1<i>H</i>) (Ar<b><i>H</i></b>), 7.25 (s, 1<i>H</i>) (Ar<b><i>H</i></b>), 7.5 (d, 2<i>H</i>) (2 x Ar<b><i>H</i></b>), 7.8-8.05 (m, 3<i>H</i>) (3 x Ar<b><i>H</i></b>), 8.3 (d, 1<i>H</i>) (Ar<b><i>H</i></b>), 12.7 (s, 1<i>H</i>) (CON<b><i>H</i></b>); m/z (M+H)⁺. 334, 100% purity.

Example 3

0170<chemistry id="chem0039" num="0039"><img file="EP1501822B1_D0039.tif" /></chemistry>

0171A stirred mixture of 4-(3-aminobenzyl)-2<i>H</i>-phthalazin-1-one (0.1 g, 0.4 mmol; prepared in a manner similar to that described in Example 1), 3-methylfuran-2,5-dione (0.045 g, 0.4 mmol) and acetic acid (4 ml) was heated under reflux for 8.25 hours and allowed to stand at ambient temperature for 65 hours, then it was diluted with water (10 ml). The resulting solid was collected by filtration, washed with water (10 ml) and dried <i>in vacuo</i> to give 3-methyl-1-[3-(4-oxo-3,4-dihydrophthalazin-1-ylmethyl)phenyl]pyrrole-2,5-dione (0.068 g) as an off-white solid, 250 MHz ¹H-nmr (d₆-DMSO) δ (ppm): 1.95 (s, 3<i>H</i>) (C<b><i>H</i></b>₃), 4.3 (s, 2<i>H</i>) (ArC<b><i>H</i></b>₂-), 6. 7 (s, 1<i>H</i>) (-COC<b><i>H</i></b>=CMeCO-), 7.15 (d, 1H) (Ar<b><i>H</i></b>), 7.2 (s, 1<i>H</i>) (Ar<b><i>H</i></b>), 7.25-7.35 (m, 2<i>H</i>) (2 x Ar<b><i>H</i></b>), 7.7-7.9 (m, 3<i>H</i>) ( 3 x ArH), 8.2 (d, 1<i>H</i>) (Ar<b><i>H</i></b>), 12.55 (s, 1<i>H</i>) (CON<b><i>H</i></b>); m/z (M+H)^+. 346, 100% purity.

0172The following Examples 3-14 were synthesised in a manner analogous to that described in Example 4, using appropriate starting materials, and heating under reflux until tlc indicated the reaction had progressed through the open chain amidoacid stage to the desired cyclised product (2 to 60 hours required). Any substantial variations in methodology are noted below.

Example 4

0173<chemistry id="chem0040" num="0040"><img file="EP1501822B1_D0040.tif" /></chemistry> The solid product was triturated with dichloromethane (20 ml), collected by filtration and dried <i>in vacuo</i> to give 4-nitro-2-[3-(4-oxo-3,4-dihydrophthalazin-1-ylmethyl)phenyl]isoindole-1,3-dione (0.025 g) as a pale yellow solid, 250 MHz ¹H-nmr (d₆-DMSO) δ (ppm): 4.45 (s, 2<i>H</i>) (ArC<b><i>H</i></b>₂-), 7.35 (t, 1<i>H</i>) (Ar<b><i>H</i></b>), 7.45 (s, 1<i>H</i>) (Ar<b><i>H</i></b>), 7.55 (d, 2<i>H</i>) (2 x Ar<b><i>H</i></b>), 7.8-8.4 (m, 7<i>H</i>) (7 x Ar<b><i>H</i></b>), 12.7 (s, 1<i>H</i>) (CON<b><i>H</i></b>); m/z (M+H)⁺, 427, 100% purity.

Example 5

0174<chemistry id="chem0041" num="0041"><img file="EP1501822B1_D0041.tif" /></chemistry>

0175The product was 2-[3-(4-oxo-3,4-dihydrophthalazin-1-ylmethyl)phenyl]-3a,4,5,6,7,7a-hexahydroisoindole-1,3-dione (0.076 g), obtained as an off-white solid, 250 MHz ¹H-nmr (d₆-DMSO) δ (ppm) : 1.3-1.55 (m, 4<i>H</i>) (2 x ring C<b><i>H</i></b>₂), 1.65-1.9 (m, 4<i>H</i>) (2 x ring C<b><i>H</i></b>₂), 3.05-3.15 (m, 2<i>H</i>) (2 x ring C<b><i>H</i></b>), 4.4 (s, 2<i>H</i>) (ArC<b><i>H</i></b>₂-), 7. 2 (d, 1<i>H</i>) (Ar<b><i>H</i></b>), 7.3 (s, 1<i>H</i>) (Ar<b><i>H</i></b>), 7.4-7.5 (m, 2<i>H</i>) (2 x Ar<b><i>H</i></b>), 7.8-8.05 (m, 3<i>H</i>) (3 x Ar<b><i>H</i></b>), 8.3 (d, 1<i>H</i>) (Ar<b><i>H</i></b>), 12.7 (s, 1<i>H</i>) (CON<b><i>H</i></b>); m/z (M+H)^+. 388, 94.7% purity.

Example 6

0176<chemistry id="chem0042" num="0042"><img file="EP1501822B1_D0042.tif" /></chemistry>

0177The product was dissolved in dichloromethane (30 ml), the solution was washed with saturated aqueous sodium hydrogencarbonate solution (10 ml) and water (10 ml), then it was dried (MgSO₄) and the solvent was removed <i>in vacuo</i> to give 5-methyl-2-[3-(4-oxo-3,4-dihydrophthalazin-1-ylmethyl)phenyl]isoindole-1,3-dione (0.055 g) as an off-white solid, 250 MHz ¹H-nmr (d₆-DMSO) δ (ppm): 2.5 (s - partially obscured by DMSO peak, 3<i>H</i>) (C<b><i>H</i></b>₃), 4.4 (s, 2H) (ArC<b><i>H</i></b>₂-), 7.3 (d, 1<i>H</i>) (Ar<b><i>H</i></b>), 7.4-7.5 (m, 3<i>H</i>) (3 x Ar<b><i>H</i></b>), 7.7-8.05 (m, 6<i>H</i>) (6 x Ar<b><i>H</i></b>), 8.3 (d, 1<i>H</i>) (Ar<b><i>H</i></b>), 12.65 (s, 1<i>H</i>) (CON<b><i>H</i></b>); m/z (M+<b><i>H</i></b>)^+. 396, 97.1% purity.

Example 7

0178<chemistry id="chem0043" num="0043"><img file="EP1501822B1_D0043.tif" /></chemistry>

0179The product was 1-[3-(4-oxo-3,4-dihydrophthalazin-1-ylmethyl)phenyl]-3-phenylpyrrole-2,5-dione (0.074 g), obtained as a tan solid, 250 MHz ¹H-nmr (d₆-DMSO) δ (ppm): 4.4 (s, 2<i>H</i>) (ArC<b><i>H</i></b>₂-), 7.3 (d, 1<i>H</i>) (Ar<b><i>H</i></b>), 7.4-7.7 (m, 7<i>H</i>) (6 x Ar<b><i>H</i></b> + -COC<b><i>H</i></b>=CPhCO), 7.85-8.15 (m, 5<i>H</i>) (5 x Ar<b><i>H</i></b>), 8.35 (d, 1<i>H</i>) (Ar<b><i>H</i></b>), 12.7 (s, 1<i>H</i>) (CON<b><i>H</i></b>); m/z (M+H)^+. 408, 100% purity.

Example 8

0180<chemistry id="chem0044" num="0044"><img file="EP1501822B1_D0044.tif" /></chemistry>

0181The product was dissolved in dichloromethane (20 ml), the solution was washed with saturated aqueous sodium hydrogencarbonate solution (10 ml) and water (10 ml), then it was decanted from some insoluble material, dried (MgSO₄) and the solvent was removed <i>in vacuo</i> to give 2-[3-(4-oxo-3,4-dihydrophthalazin-1-ylmethyl)phenyl]isoindole-1,3-dione (0.03 g) as a yellow solid, 250 MHz ¹H-nmr (d₆-DMSO) δ (ppm) : 4.45 (s, 2<i>H</i>) (ArC<b><i>H</i></b>₂-), 7. 35 (m, 1<i>H</i>) (Ar<b><i>H</i></b>), 7.4-7. 55 (m, 3H) (3 x Ar<b><i>H</i></b>), 7.8-8.05 (m, 7<i>H</i>) (7 x Ar<b><i>H</i></b>), 8.3 (dd, 1<i>H</i>) (Ar<b><i>H</i></b>), 12.7 (s, 1<i>H</i>) (CON<b><i>H</i></b>); m/z (M+H)^+. 382, 94.1% purity.

Example 9

0182<chemistry id="chem0045" num="0045"><img file="EP1501822B1_D0045.tif" /></chemistry>

0183The product was purified by flash chromatography over silica using an 85:15 mixture of ethyl acetate and hexane as eluant. Appropriate fractions were combined and the solvents were removed <i>in vacuo</i> to give 8-methyl-4-[3-(4-oxo-3,4-dihydrophthalazin-1-ylmethyl)phenyl]-4-azatricyclo[5.2.1.0^2,6]dec-8-ene-3,5-dione (0.012 g) as a beige solid, m/z (M+H)^+. 412, 91.5% purity.

Example 10

0184<chemistry id="chem0046" num="0046"><img file="EP1501822B1_D0046.tif" /></chemistry>

0185The product was dissolved in dichloromethane (30 ml), the solution was washed with saturated aqueous sodium hydrogencarbonate solution (10 ml) and water (10 ml), dried (MgSO₄) and the solvent was removed <i>in vacuo</i> to give 6-[3-(4-oxo-3,4-dihydrophthalazin-1-ylmethyl)phenyl]-2,3-dihydro[1,4]dithiino[2,3-<i>c</i>]pyrrole-5,7-dione (0.054 g) as a yellow solid, 250 MHz ¹H-nmr (d₆-DMSO) δ (ppm): 3.6 (s, 4<i>H</i>) (-SCH₂CH₂S-). 4.5 (s, 2<i>H</i>) (ArC<b><i>H</i></b>₂-), 7.35 (m, 1<i>H</i>) (Ar<b><i>H</i></b>), 7.45-7.6 (m, 3<i>H</i>) (3 x Ar<b><i>H</i></b>), 7.9-8.15 (m, 3<i>H</i>) (3 x Ar<b><i>H</i></b>), 8.4 (d, 1<i>H</i>) (Ar<b><i>H</i></b>), 12.8 (s, 1<i>H</i>) (CON<b><i>H</i></b>): m/z (M+H)^+. 422, 100% purity.

Example 11

0186<chemistry id="chem0047" num="0047"><img file="EP1501822B1_D0047.tif" /></chemistry>

0187The product was 3,4-dimethyl-1-[3-(4-oxo-3,4-dihydrophthalazin-1-ylmethyl)phenyl]pyrrole-2,5-dione (0.045 g), obtained as a beige solid, 250 MHz ¹H-nmr (d₆-DMSO) δ (ppm): 2.0 (s, 6<i>H</i>) (2 x C<b><i>H</i></b>₃) 4.4 (s, 2<i>H</i>) (ArC<b><i>H</i></b>₂-), 7.25 (d, 1<i>H</i>) (Ar<b><i>H</i></b>), 7.35 (s, 1<i>H</i>) (Ar<b><i>H</i></b>), 7.4-7.55 (m, 2<i>H</i>) (2 x Ar<b><i>H</i></b>), 7.8-8.05 (m, 3<i>H</i>) (3 x Ar<b><i>H</i></b>), 8.3 (d, 1<i>H</i>) (Ar<b><i>H</i></b>), 12.7 (s, 1<i>H</i>) (CON<b><i>H</i></b>); m/z (M+H)^+. 360, 90.3% purity.

Example 12

0188<chemistry id="chem0048" num="0048"><img file="EP1501822B1_D0048.tif" /></chemistry>

0189The product was 1-[3-(4-oxo-3,4-dihydrophthalazin-1-ylmethyl)phenyl]pyrrole-2,5-dione (0.026 g), obtained as a brown solid, 250 MHz ¹H-nmr (d₆-DMSO) δ (ppm): 4.3 (s, 2<i>H</i>) (ArC<b><i>H</i></b>₂-), 7.05 (s, 2<i>H</i>) (-COC<b><i>H</i></b>=C<b><i>H</i></b>CO-), 7.1 (d, 1<i>H</i>) (Ar<b><i>H</i></b>)<i>,</i> 7.2 (s, 1<i>H</i>) (Ar<b><i>H</i></b>), 7.25-7.4 (m, 2<i>H</i>) (2 x Ar<b><i>H</i></b>), 7.75-7.9 (m, 3<i>H</i>) (3 x Ar<b><i>H</i></b>), 8.2 (d, 1<i>H</i>) (Ar<b><i>H</i></b>), 12.55 (s, 1<i>H</i>) (CON<b><i>H</i></b>); m/z (M+H)^+. 332, 88% purity.

Example 13

0190<chemistry id="chem0049" num="0049"><img file="EP1501822B1_D0049.tif" /></chemistry>

0191The product was 3-[3-(4-oxo-3,4-dihydrophthalazin-1-ylmethyl)phenyl]-3-azabicyclo[3.2.0]heptane-2,4-dione (0.071 g), obtained as a beige solid, 250 MHz ¹H-nmr (d₆-DMSO) δ (ppm): 2.2 (m, 2<i>H</i>) (2 x ring C<b><i>H</i></b>), 2.65 (m, 2<i>H</i>) (2 x ring C<b><i>H</i></b>), 3.4 (m - obscured by water peak, 2H)(2 x ring COC<b><i>H</i></b>), 4.4 (s, 2<i>H</i>) (ArC<b><i>H</i></b>₂-), 7.25 (d, 1<i>H</i>) (Ar<b><i>H</i></b>), 7.35 (s, 1<i>H</i>) (Ar<b><i>H</i></b>), 7.45-7.55 (m, 2<i>H</i>) (2 x Ar<b><i>H</i></b>), 7.85-8.05 (m, 3<i>H</i>) (3 x Ar<b><i>H</i></b>), 8.35 (d, 1<i>H</i>) (Ar<b><i>H</i></b>), 12.65 (s, 1<i>H</i>) (CON<b><i>H</i></b>); m/z (M+H)^+. 360, 96.3% purity.

Example 14

0192<chemistry id="chem0050" num="0050"><img file="EP1501822B1_D0050.tif" /></chemistry>

0193A stirred mixture of 4-(3-aminobenzyl)-2<i>H</i>-phthalazin-1-one (0.126 g, 0.5 mmol; prepared in a manner similar to that described in Example 1), 3-phenyldihydrofuran-2,5-dione (0.088 g, 0.5 mmol) and toluene (8 ml) was heated under reflux for 3 hours then allowed to cool to ambient temperature. The resulting solid was collected by filtration, washed with hexane (20 ml) and dried <i>in vacuo</i> to give a mixture of <i>N</i>-[3-(4-oxo-3,4-dihydrophthalazin-1-ylmethyl)phenyl]-2-phenylsuccinamic acid and <i>N</i>-[3-(4-oxo-3,4-dihydrophthalazin-1-ylmethyl)phenyl]-3-phenylsuccinamic acid (0.142 g) as a beige solid which was used without further purification.

0194<i>O</i>-Benzotriazol-1-yl-<i>N</i>,<i>N</i>,<i>N,N,</i>-tetramethyluronium tetrafluoroborate (0.139 g, 0.429 mmol) and diisopropylethylamine (0.095 g, 0.726 mmol) were added sequentially at ambient temperature to a stirred solution of the above mixture of <i>N</i>-[3-(4-oxo-3,4-dihydrophthalazin-1-ylmethyl)phenyl]-2-phenylsuccinamic acid and <i>N</i>-[3-(4-oxo-3,4-dihydrophthalazin-1-ylmethyl)phenyl]-3-phenylsuccinamic acid (0.142 g, 0.33 mmol) in dimethylformamide (2ml), the mixture was stirred at ambient temperature for 3.25 hours and allowed to stand at ambient temperature for 18 hours, then it was added dropwise to water (10 ml). The mixture was stirred at ambient temperature for 30 minutes, then the resulting solid was collected by filtration, washed with water (5 ml) and hexane (10 ml), and dried <i>in vacuo</i> to give 1-[3-(4-oxo-3,4-dihydrophthalazin-1-ylmethyl)phenyl]-3-phenylpyrrolidine-2,5-dione (0.097 g) as an off-white solid, 250 MHz ¹H-nmr (d₆-DMSO) δ (ppm): 3.0 (m, 1<i>H</i>) (ring C<b><i>H</i></b>), 3.4 (m partially obscured by water peak, 1<i>H</i>) (ring C<b><i>H</i></b>), 4.4-4.55 (m, 3<i>H</i>) (ArC<b><i>H</i></b>₂- + ring C<b><i>H</i></b>), 7.25-7.65 (m, 9<i>H</i>) (9 x Ar<b><i>H</i></b>), 7.9-8.15 (m, 3<i>H</i>) (3 x Ar<b><i>H</i></b>), 8.4 (d, 1<i>H</i>) (Ar<b><i>H</i></b>), 12.8 (s, 1<i>H</i>) (CON<b><i>H</i></b>); m/z (M+H)^+. 410, 94.2% purity.

0195The following Examples 15-19 were synthesised in a manner analogous to that described in Example 14, using appropriate starting materials, and following both reaction stages by tlc until starting materials were consumed. Any substantial variations in methodology are noted below.

Example 15

0196<chemistry id="chem0051" num="0051"><img file="EP1501822B1_D0051.tif" /></chemistry>

0197The product was 3,3-dimethyl-1-[3-(4-oxo-3,4-dihydrophthalazin-1-ylmethyl)phenyl]pyrrolidine-2,5-dione (0.066 g), obtained as an off-white solid, 250 MHz ¹H-nmr (d₆-DMSO) δ (ppm): 1.55 (s, 6<i>H</i>) (2 x C<b><i>H</i></b>₃), 3.45 (s, 2<i>H</i>) (ring C<b><i>H</i></b>₂), 4.4 (s, 2<i>H</i>) (ArC<b><i>H</i></b>₂-), 7. 15 (d, 1<i>H</i>) (Ar<b><i>H</i></b>), 7. 3 (s, 1<i>H</i>) (Ar<b><i>H</i></b>), 7. 5 (m, 2<i>H</i>) (2 x Ar<b><i>H</i></b>), 7.8-8.05 (m, 3<i>H</i>) (3 x Ar<b><i>H</i></b>), 8.3 (d, 1<i>H</i>) (Ar<b><i>H</i></b>), 12.65 (s, 1<i>H</i>) (CON<b><i>H</i></b>); m/z (M+H)^+. 362, 97.4% purity.

Example 16

0198<chemistry id="chem0052" num="0052"><img file="EP1501822B1_D0052.tif" /></chemistry>

0199The cyclisation reaction mixture was added to water (10 ml), stirred at ambient temperature for 6 hours and allowed to stand at ambient temperature for 18 hours. The resulting solid was collected by filtration, washed with water (5 ml) and hexane (10 ml), and dried <i>in vacuo</i> to give 3-oct-2-enyl-1-[3-(4-oxo-3,4-dihydrophthalazin-1-ylmethyl)phenyl]pyrrolidine-2,5-dione (0.056 g) as a brown solid, 250 MHz ¹H-nmr (d₆-DMSO) δ (ppm): 0.9 (m, 3<i>H</i>) (C<b><i>H</i></b>₃), 1.15-1.35 (m, 6<i>H</i>) (3 x C<b><i>H</i></b>₂), 1.9-2.1 (m, 2<i>H</i>) (C<b><i>H</i></b>₂), 2.25-2.6 (m partially obscured by DMSO peak, 3<i>H</i>) (C<b><i>H</i></b>₂ + ring C<b><i>H</i></b>), 2.8-3.2 (m, 2<i>H</i>) (2 ring C<b><i>H</i></b>), 4.45 (s, 2<i>H</i>) (ArC<b><i>H</i></b>₂-), 5.35-5.7 (m, 2<i>H</i>) (-C<b><i>H</i></b>=C<b><i>H</i></b>-), 7.15-7.35 (m, 2<i>H</i>) (2 x Ar<b><i>H</i></b>), 7.5 (m, 2<i>H</i>) (2 x Ar<b><i>H</i></b>), 7.8-8.05 (m, 3<i>H</i>) (3 x Ar<b><i>H</i></b>), 8.3 (d, 1<i>H</i>) (Ar<b><i>H</i></b>), 12. 7 (s, 1<i>H</i>) (CON<b><i>H</i></b>); m/z (M+H)^+. 444, 98.0% purity.

Example 17

0200<chemistry id="chem0053" num="0053"><img file="EP1501822B1_D0053.tif" /></chemistry> The product was 3-[3-(4-oxo-3,4-dihydrophthalazin-1-ylmethyl)phenyl]-3-azabicyclo[3.1.0]hexane-2,4-dione (0.053 g), obtained as a beige solid, 250 MHz ¹H-nmr (d₆-DMSO) δ (ppm): 1.65 (m, 1<i>H</i>) (ring C<b><i>H</i></b>), 1.85 (m, 1<i>H</i>) (ring C<b><i>H</i></b>), 2.75 (m, 2<i>H</i>) (2 x ring C<b><i>H</i></b>), 4.4 (s, 2<i>H</i>) (ArC<b><i>H</i></b>₂-), 7.15 (m, 1<i>H</i>) (Ar<b><i>H</i></b>), 7.25 (s, 1<i>H</i>) (Ar<b><i>H</i></b>), 7. 4 (m, 2<i>H</i>) (2 x Ar<b><i>H</i></b>), 7.85-8.05 (m, 3<i>H</i>) (3 x Ar<b><i>H</i></b>), 8.3 (d, 1<i>H</i>) (Ar<b><i>H</i></b>), 12.65 (s, 1<i>H</i>) (CON<b><i>H</i></b>) ; m/z (M+H)⁺. 346, 95.4% purity.

Example 18

0201<chemistry id="chem0054" num="0054"><img file="EP1501822B1_D0054.tif" /></chemistry>

0202The cyclisation product was dissolved in dichloromethane (10 ml) and filtered through a short column of silica using dichloromethane as eluant. Appropriate fractions were combined and the solvent was removed <i>in vacuo</i> to give 3-hex-2-enyl-1-[3-(4-oxo-3,4-dihydrophthalazin-1-ylmethyl)phenyl]pyrrolidine-2,5-dione (0.04 g) as a pale brown solid, 250 MHz ¹H-nmr (d₆-DMSO) δ (ppm): 0.9 (t, 3<i>H</i>) (C<b><i>H</i></b>₃), 1.3-1.45 (m, 2<i>H</i>) (C<b><i>H</i></b>₂), 1.95-2.05 (m, 2<i>H</i>) (C<b><i>H</i></b>₂), 2.35-2.6 (m partially obscured by DMSO peak, 2<i>H</i>) (C<b><i>H</i></b>₂), 2.85-3.2 (m, 3<i>H</i>) (3 x ring C<b><i>H</i></b>), 4.4 (s, 2<i>H</i>) (ArC<b><i>H</i></b>₂-), 5.35-5.7 (m, 2<i>H</i>) (-C<b><i>H</i></b>=C<b><i>H</i></b>-), 7.1-7.3 (m, 2<i>H</i>) (2 x Ar<b><i>H</i></b>), 7.5 (m, 2<i>H</i>) (2 x Ar<b><i>H</i></b>), 7.85-8.05 (m, 3H)(3 x Ar<b><i>H</i></b>), 8.35 (d, 1<i>H</i>) (Ar<b><i>H</i></b>), 12.7 (s, 1<i>H</i>) (CON<b><i>H</i></b>); m/z (M+H)^+. 416, 89.1% purity.

Example 19

0203<chemistry id="chem0055" num="0055"><img file="EP1501822B1_D0055.tif" /></chemistry>

0204The product was 3-methyl-1-[3-(4-oxo-3,4-dihydrophthalazin-1-ylmethyl)phenyl]pyrrolidine-2,5-dione (0.086 g), obtained as a brown solid, 250 MHz ¹H-nmr (d₆-DMSO) δ (ppm): 1.2 (d, 3<i>H</i>)(C<b><i>H</i></b>₃), 2.35-2.5 (m partially obscured by DMSO peak, 1<i>H</i>) (ring C<b><i>H</i></b>), 2.8-3.0 (m, 2<i>H</i>) (2 x ring C<b><i>H</i></b>), 4.3 (s, 2<i>H</i>) (ArC<b><i>H</i></b>₂-), 7.05 (m, 2<i>H</i>) (2 x Ar<b><i>H</i></b>), 7.15 (s, 1<i>H</i>) (Ar<b><i>H</i></b>), 7.35 (m, 2<i>H</i>) (2 x Ar<b><i>H</i></b>), 7.75-7.95 (m, 3<i>H</i>) (3 x Ar<b><i>H</i></b>), 8.2 (d, 1<i>H</i>) (Ar<b><i>H</i></b>), 12.6 (s, 1<i>H</i>) (CON<b><i>H</i></b>); m/z (M+H)^+. 348, 100% purity.

Example 20

0205<chemistry id="chem0056" num="0056"><img file="EP1501822B1_D0056.tif" /></chemistry>

0206A stirred mixture of 4-(3-aminobenzyl)-2<i>H</i>-phthalazin-1-one (0.1 g, 0.4 mmol; prepared in a manner similar to that described in Example 1) and 1-(carboxymethyl)cyclopentanecarboxylic acid (0.0685 g, 0.4 mmol) was heated at 200°C for 2.5 hours then it was allowed to cool to ambient temperature. Methanol (1 ml) was added and the mixture was heated under reflux for 5 minutes. No solid precipitated, so the mixture was concentrated <i>in vacuo</i> and the residue was triturated with water (5 ml). The resulting solid was collected by filtration and dried <i>in vacuo</i> to give 2-[3-(4-oxo-3,4-dihydrophthalazin-1-ylmethyl)phenyl]-2-azaspiro[4.4]nonane-1,3-dione (0.108 g) as a brown solid, m/z (M+H)⁺. 388, 89.1% purity.

Example 21

0207<chemistry id="chem0057" num="0057"><img file="EP1501822B1_D0057.tif" /></chemistry> A stirred mixture of 4-(3-aminobenzyl)-2<i>H</i>-phthalazin-1-one (0.1 g, 0.4 mmol; prepared in a manner similar to that described in Example 1) and 2-phenyl-2-propylsuccinic acid (0.094 g, 0.4 mmol) was heated at 200°C for 1.5 hours then it was allowed to stand at ambient temperature for 18 hours. Methanol (1 ml) was added, the mixture was heated under reflux for 5 minutes, then it was filtered and the collected solid was washed with hot methanol (3 ml). The combined filtrate and washings were concentrated <i>in vacuo</i> and the residue was triturated with water (5 ml). The resulting solid was collected by filtration and dried <i>in vacuo</i> to give 1-[3-(4-oxo-3,4-dihydrophthalazin-1-ylmethyl)phenyl]-3-phenyl-3-propylpyrrolidine-2,5-dione (0.061 g) as a beige solid, 250 MHz ¹H-nmr (d₆-DMSO) δ (ppm): 0.95 (t, 3<i>H</i>) (C<b><i>H</i></b>₃), 1.25 (m, 2<i>H</i>) (-C<i>H</i>₂C<b><i>H</i></b>₂CH₃), 2.1 (m, 2H) (-C<b><i>H</i></b>₂CH₂CH₃), 3.3 (d, 2<i>H</i>) (ring C<b><i>H</i></b>₂), 4.4 (s, 2<i>H</i>) (ArC<b><i>H</i></b>₂-), 7.2 (d, 1<i>H</i>) (Ar<b><i>H</i></b>), 7.3-7.65 (m, 8<i>H</i>) (8 x Ar<b><i>H</i></b>), 7.85-8.1 (m, 3<i>H</i>) (3 x Ar<b><i>H</i></b>), 8.35 (d, 1<i>H</i>) (Ar<b><i>H</i></b>), 12.7 (s, 1<i>H</i>) (CON<b><i>H</i></b>); m/z (M+H)^+. 452, 93.6% purity.

Example 22

0208<chemistry id="chem0058" num="0058"><img file="EP1501822B1_D0058.tif" /></chemistry> Dimethyl phosphite (100 g, 0.909 mol) was added dropwise at 0°C under nitrogen to a stirred solution of sodium methoxide [from sodium (20.9 g, 0.909 gatom)] in methanol (730 ml), the mixture was stirred for 5 minutes, and 2-carboxybenzaldehyde (95.45 g, 0.64 mol) was added in portions. The stirred mixture was allowed to warm to ambient temperature, then it was stirred for 30 minutes and cooled in ice. Methanesulfonic acid (96 g, 1 mol) was added in portions at 5-10°C, then the solvent was removed <i>in vacuo.</i> The residue was partitioned between dichloromethane (1800 ml) and water (450 ml), and the organic layer was separated, washed with water (2 x 450 ml) and dried (MgSO₄). The solvent was removed <i>in vacuo,</i> the residue was triturated with ether (150 ml), and the resulting solid was collected by filtration, washed with ether (30 ml) and dried <i>in vacuo</i> to give dimethyl 3-oxo-1,3-dihydroisobenzofuran-1-ylphosphonate (139.94 g) as a white crystalline solid, m.pt 95-96.5°C; 250 MHz ¹H-nmr (d₆-DMSO) δ (ppm): 3.65 (d, 3<i>H</i>) (-OC<b><i>H</i></b>₃), 3.85 (d, 3<i>H</i>) (-OC<b><i>H</i></b>₃), 6.4 (d, 1<i>H</i>) (-C<b><i>H</i></b>-P), 7.75 (m, 2<i>H</i>) (2 x Ar<b><i>H</i></b>), 7.85-8.05 (m, 2<i>H</i>) (2 x Ar<b><i>H</i></b>); m/z (M+H)^+. 243, 100% purity.

0209A stirred solution of dimethyl 3-oxo-1,3-dihydroisobenzofuran-1-ylphosphonate (20 g, 0.083 mol) and 4-fluoro-3-nitrobenzaldehyde (13.97 g, 0.083 mol) in tetrahydrofuran (120 ml) was cooled to 15°C and triethylamine (11.5 ml, 0.083 mol) was added dropwise at &lt;25°C. The mixture thickened at this point, so further tetrahydrofuran (50 ml) was added to aid stirring. The mixture was stirred at ambient temperature for 65 hours and the resulting solid was collected by filtration. The filtrate was concentrated <i>in vacuo,</i> the residue was triturated with water (30 ml) and the resulting solid was collected by filtration. The two crops of solid were combined and suspended in water (120 ml). The mixture was stirred at ambient temperature for 30 minutes and the resulting solid was collected by filtration and dried <i>in vacuo</i> for 24 hours to give crude 3-(4-fluoro-3-nitrobenzylidene)-3<i>H</i>-isobenzofuran-1-one, still slightly wet with water, which was used directly in the next stage.

0210A stirred mixture of the above crude 3-(4-fluoro-3-nitrobenzylidene)-3<i>H</i>-isobenzofuran-1-one (23.5 g), industrial methylated spirit (400 ml), water (300 ml) and ammonium chloride (8.81 g, 0.165 mol) was heated to 70°C, and iron powder (46.0 g, 0.824 gatom) was added in portions. When the addition was complete, the stirred mixture was heated at 70°C for a further 2 hours, then it was filtered while hot through Celite. The collected inorganic solids were washed with hot industrial methylated spirit (6 x 200 ml), then the filtrate and washings were combined and the solvent was removed <i>in vacuo.</i> The residue was triturated with water (300 ml) and the resulting solid was collected by filtration and dried <i>in vacuo</i> for 24 hours to give crude 3-(3-amino-4-fluorobenzylidene)-3<i>H-</i>isobenzofuran-1-one as a yellow solid, m/z (M+H)^+. 256, 100% purity, which was used directly in the next stage.

0211A stirred mixture of the above crude 3-(3-amino-4-fluorobenzylidene)-3<i>H</i>-isobenzofuran-1-one (21 g), industrial methylated spirit (250 ml) and hydrazine monohydrate (4 ml, 0.082 mol) was heated under reflux for 1 hour then cooled to 0°C. The resulting solid was collected by filtration, washed with water (2 x 50 ml) and industrial methylated spirit (30 ml), and dried <i>in vacuo</i> to give a pale brown solid. The filtrate was concentrated <i>in vacuo,</i> the residue was dissolved in the minimum volume of hot industrial methylated spirit, then water was added until a solid precipitated. The resulting solid was collected by filtration and dried <i>in vacuo.</i> The overall yield of 4-(3-amino-4-fluorobenzyl)-2<i>H</i>-phthalazin-1-one was 14.5g, m.pt. 189-191°C; 250 MHz ¹H-nmr (d₆-DMSO) δ (ppm): 4.2 (s, 2<i>H</i>) (ArC<b><i>H</i></b>₂-), 5.2 (s, 2<i>H</i>) (-N<b><i>H</i></b>₂), 6. 5-6. 6 (m, 1<i>H</i>) (Ar<b><i>H</i></b>), 6. 65-6.75 (m, 1<i>H</i>) (Ar<b><i>H</i></b>), 6.9-7.05 (m, 1<i>H</i>) (Ar<b><i>H</i></b>), 7.85-8.0 (m, 3H) (3 x Ar<b><i>H</i></b>), 8.3-8.4 (m, 1<i>H</i>) (Ar<b><i>H</i></b>), 12.7 (s, 1<i>H</i>) (CON<b><i>H</i></b>); m/z (M+H)^+. 270, 100% purity.

0212A solution of 4-(3-amino-4-fluorobenzyl)-2<i>H</i>-phthalazin-1-one (1.62 g, 6 mmol) in acetonitrile (40 ml) was filtered to remove a trace of insoluble material. A solution of succinic anhydride (0.7 g, 7 mmol) in acetonitrile (10 ml) was filtered to remove traces of succinic acid impurity, and the two filtered solutions were combined. The stirred mixture was heated under reflux for 4 hours, allowed to stand at ambient temperature for 18 hours and heated under reflux for a further 2 hours. The resulting solid was collected by filtration from the hot mixture, washed with acetonitrile (10 ml) and dried <i>in vacuo.</i> The solid was suspended in dichloromethane (60 ml), the mixture was heated under reflux for 2 hours, then the resulting solid was collected by filtration from the hot mixture, washed with dichloromethane (20 ml) and dried <i>in vacuo</i> to give <i>N-</i>[2-fluoro-5-(4-oxo-3,4-dihydrophalazin-1-ylmethyl)phenyl]succinamic acid (1.73 g) as an off-white solid, m.pt. 210-213°C; 250 MHz ¹H-nmr (d₆-DMSO) δ (ppm): 2.4-2.7 (m, 4<i>H</i>) (-C<b><i>H</i></b>₂C<b><i>H</i></b>₂-) 4.4 (s, 2<i>H</i>) (ArC<b><i>H</i></b>₂-), 7.0-7.25 (m, 2<i>H</i>) (2 x Ar<b><i>H</i></b>), 7.7-8.0 (m, 4<i>H</i>) (4 x Ar<b><i>H</i></b>), 8.2-8.3 (m, 1<i>H</i>) (Ar<b><i>H</i></b>), 9. 7 (s, 1<i>H</i>) (chain CON<b><i>H</i></b>), 12.15 (br s, 1<i>H</i>) (-CO₂<b><i>H</i></b>), 12.65 (s, 1<i>H</i>) (ring CON<b><i>H</i></b>); m/z (M+H)^+. 370, 96% purity.

0213<i>O</i>-Benzotriazol-1-yl-<i>N</i>,<i>N</i>,<i>N</i>'<i>N</i>'-tetramethyluronium tetrafluoroborate (0.834 g, 2.6 mmol) and diisopropylethylamine (0.569g, 4.4 mmol) were added sequentially at ambient temperature to a stirred solution of <i>N</i>-[2-fluoro-5-(4-oxo-3,4-dihydrophalazin-1-ylmethyl)phenyl]succinamic acid (0.703g, 2 mmol) in dimethylformamide (2 ml), and the mixture was stirred at ambient temperature for 100 hours. Tlc indicated that starting material remained, so further <i>O</i>-benzotriazol-1-yl-<i>N</i>,<i>N</i>,<i>N</i>'<i>N</i>'-tetramethyluronium tetrafluoroborate (0.3 g) was added and stirring was continued for 24 hours. The resulting suspension was added dropwise to water (40 ml), the mixture was stirred at ambient temperature for 1 hour, and the resulting solid was collected by filtration, washed with water (20 ml) and hexane (20 ml), and dried <i>in vacuo</i> to give 1-[2-fluoro-5-(4-oxo-3,4-dihydrophalazin-1-ylmethyl)phenyl]pyrrolidine-2,5-dione (0.476g) as an off-white solid, 258-262°C; 250 MHz ¹H-nmr (d₆-DMSO) δ (ppm): 2.75-2.9 (m, 4<i>H</i>) (-C<b><i>H</i></b>₂C<b><i>H</i></b>₂-) 4.25 (s, 2<i>H</i>) (ArC<b><i>H</i></b>₂-), 7.25 (m, 1<i>H</i>) (Ar<b><i>H</i></b>), 7.35 (t, 1<i>H</i>) (Ar<b><i>H</i></b>), 7.5-7.6 (m, 1<i>H</i>) (Ar<b><i>H</i></b>), 7.8-8.05 (m, 3<i>H</i>) (3 x Ar<b><i>H</i></b>), 8.25 (d, 1<i>H</i>) (Ar<b><i>H</i></b>), 12.65 (s, 1<i>H</i>) (CON<b><i>H</i></b>).

Example 23

0214<chemistry id="chem0059" num="0059"><img file="EP1501822B1_D0059.tif" /></chemistry>

0215A stirred mixture of 4-(3-amino-4-fluorobenzyl)-2<i>H-</i>phthalazin-1-one (0.1g, 0.37mmol; prepared in a manner similar to that described in Example 22), glutaric anhydride (0.042g, 0.37mmol) and toluene (10ml) was heated under reflux until the starting materials were consumed (the reaction was followed by tlc using a 3:1 mixture of ethyl acetate and ethanol as eluant), then it was allowed to cool to ambient temperature. The resulting solid was collected by filtration and recrystallised from ethanol to give 4-[2-fluoro-5-(4-oxo-3,4-dihydrophthalazin-1-ylmethyl)phenylcarbamoyl]butyric acid (0.08 g) as a white solid, mpt. 190-194°C; 250 MHz ¹H-nmr (d₆-DMSO) δ (ppm): 1.5-1.65 (m, 2<i>H</i>) (-C<i>H</i>₂C<b><i>H</i></b>₂C<i>H</i>₂-), 2.0-2.25 (m, 4<i>H</i>) (-C<b><i>H</i></b>₂C<i>H</i>₂C<b><i>H</i></b>₂-) 4.1 (s, 2<i>H</i>) (ArC<b><i>H</i></b>₂-), 6.8-7.0 (m, 2<i>H</i>) (2 x Ar<b><i>H</i></b>), 7.5-7.8 (m, 4<i>H</i>) (4 x Ar<b><i>H</i></b>), 8.05 (d, 1<i>H</i>) (Ar<b><i>H</i></b>), 9.45 (s, 1<i>H</i>) (chain CON<b><i>H</i></b>), 12.85 (br s, 1<i>H</i>) (-CO₂<b><i>H</i></b>), 12.4 (s, 1<i>H</i>) (ring CON<b><i>H</i></b>); m/z (M-H)⁺, 382, 100% purity.

0216<i>O</i>-Benzotriazol-1-yl-<i>N</i>,<i>N</i>,<i>N</i>'<i>N</i>'-tetramethyluronium tetrafluoroborate (0.087 g, 0.27 mmol) and diisopropylethylamine (0.079 ml, 0.46 mmol) were added sequentially at ambient temperature to a stirred solution of 4-[2-fluoro-5-(4-oxo-3,4-dihydrophthalazin-1-ylmethyl)phenylcarbamoyl]butyric acid (0.08 g, 0.21 mmol) in dimethylformamide (2 ml), the mixture was stirred at ambient temperature for 50 hours, then it was added dropwise to ice-cold water (10 ml). The resulting solid was collected by filtration and dried <i>in vacuo</i> to give 1-[2-Fluoro-5-(4-oxo-3,4-dihydrophthalazin-1-ylmethyl)phenyl]piperidine-2,6-dione (0.05 g) as a white solid, m.pt. 259-263°C; 250 MHz ¹H-nmr (d₆-DMSO) δ (ppm): 1.8-2.1 (m, 2H) (-C<i>H</i>₂C<b><i>H</i></b>₂C<i>H</i>₂-), 2.7-2.9 (m, 4<i>H</i>) (-C<b><i>H</i></b>₂C<i>H</i>₂C<b><i>H</i></b>₂-) 4.35 (s, 2<i>H</i>) (ArC<b><i>H</i></b>₂-), 7.1-7.35 (m, 2<i>H</i>) (2 x Ar<b><i>H</i></b>), 7.4-7.5 (m, 1<i>H</i>) (Ar<b><i>H</i></b>), 7.8-8.05 (m, 3<i>H</i>) (3 x Ar<b><i>H</i></b>), 8.3 (d, 1<i>H</i>) (Ar<b><i>H</i></b>), 12.65 (s, 1<i>H</i>) (CON<b><i>H</i></b>) ; m/z (M+H)⁺ 366, 100% purity.

Example 24

0217<chemistry id="chem0060" num="0060"><img file="EP1501822B1_D0060.tif" /></chemistry>

0218A stirred mixture of 4-(3-amino-4-fluorobenzyl)-2<i>H-</i>phthalazin-1-one (0.7 g, 2.6 mmol; prepared in a manner similar to that described in Example 22), 3-phenyldihydrofuran-2,5-dione (0.458 g, 2.6 mmol) and toluene (35 ml) was heated under reflux for 2 hours then allowed to cool to ambient temperature. The resulting solid was collected by filtration, washed with ethyl acetate (3 ml) and dried <i>in vacuo</i> to give <i>N</i>-[2-fluoro-5-(4-oxo-3,4-dihydrophthalazin-1-ylmethyl)phenyl]-2-phenylsuccinamic acid (0.775 g) as an off-white solid, m.pt. 180-183°C; 250 MHz ¹H-nmr (d₆-DMSO) δ (ppm): 2.7-2.9 (m, 1<i>H</i>) (-C<b><i>H</i></b>HCHPh-), 3.1-3.3 (m, 1<i>H</i>) (-CH<b><i>H</i></b>CHPh-), 4.0-4.1 (m, 1<i>H</i>) (-C<b><i>H</i></b>Ph-) 4.3 (s, 2<i>H</i>) (ArC<b><i>H</i></b>₂-), 7.1-7.3 (m, 2<i>H</i>) (2 x Ar<b><i>H</i></b>), 7.3-7.5 (m, 5<i>H</i>) (5 x Ar<b><i>H</i></b>), 7.8-8.0 (m, 4<i>H</i>) (4 x Ar<b><i>H</i></b>), 8.3 (d, 1<i>H</i>)(Ar<b><i>H</i></b>)<i>,</i> 9.75 (s, 1<i>H</i>) (chain CON<b><i>H</i></b>), 12.5 (br s, 1<i>H</i>) (-CO₂<b><i>H</i></b>), 12.65 (s, 1<i>H</i>) (ring CON<b><i>H</i></b>); m/z (M+H)^+. 446, 100% purity.

0219<i>O</i>-Benzotriazol-1-yl-<i>N</i>,<i>N</i>,<i>N</i>'<i>N</i>'-tetramethyluronium tetrafluoroborate (0.726 g, 2.26 mmol) and diisopropylethylamine (0.494 g, 3.8 mmol) were added sequentially at ambient temperature to a stirred solution of <i>N</i>-[2-fluoro-5-(4-oxo-3,4-dihydrophthalazin-1-ylmethyl)phenyl]-2-phenylsuccinamic acid (0.775 g, 1.7 mmol) in dimethylacetamide (4 ml), the mixture was stirred at ambient temperature for 65 hours, then it was added dropwise to ice-cold water (40 ml). The resulting solid was collected by filtration, washed with water (3ml), then dried <i>in vacuo.</i> The crude product was dissolved in hot methanol (8 ml), the solution was filtered through a small plug of cotton wool, then the filtrate was added to water (30 ml). The resulting solid was collected by filtration and dried <i>in vacuo</i> to give 1-[2-fluoro-5-(4-oxo-3,4-dihydrophthalazin-1-ylmethyl)phenyl]-3-phenylpyrrolidine-2,5-dione (0.555 g) as an off-white solid, m.pt. 114-120°C; 250 MHz ¹H-nmr (CDCl₃) δ (ppm): 2.8-3.0 (m, 1<i>H</i>) (-C<b><i>H</i></b>HCHPh), 3.2-3.4 (m, 1<i>H</i>) (-CH<b><i>H</i></b>CHPh), 4.1-4.3 (m, <i>1H</i> + 2<i>H</i>) (-C<b><i>H</i></b>Ph- and ArC<b><i>H</i></b>₂-), 7.0-7.4 (m, 8<i>H</i>) (8 x Ar<b><i>H</i></b>), 7.6-7.8 (m, 3<i>H</i>) (3 x Ar<b><i>H</i></b>), 8.4 (d, 1<i>H</i>) (Ar<b><i>H</i></b>), 10.65 (s, 1<i>H</i>) (CON<b><i>H</i></b>); m/z (M+H)^+. 428, 92.1% purity.

Example 25

0220<chemistry id="chem0061" num="0061"><img file="EP1501822B1_D0061.tif" /></chemistry>

0221A stirred mixture of 4-(3-amino-4-fluorobenzyl)-2<i>H-</i>phthalazin-1-one (0.808 g, 3 mmol; prepared in a manner similar to that described in Example 22), 3,3-dimethyldihydrofuran-2,5-dione (0.384 g, 3 mmol) and toluene (50 ml) was heated under reflux for 8 hours, then the resulting solid was collected by filtration from the hot mixture, washed with toluene (20 ml) and dried <i>in vacuo</i> to give <i>N</i>-[2-fluoro-5-(4-oxo-3,4-dihydrophalazin-1-ylmethyl)phenyl]-2,2-dimethylsuccinamic acid (0.893 g) as an off-white solid, m.pt. 154-159°C; 250 MHz ¹H-nmr (d₆-DMSO) δ (ppm): 1.25 (s, 6<i>H</i>) (2 x C<b><i>H</i></b>₃), 2.65 (s, 2<i>H</i>) (-CMe₂C<b><i>H</i></b>₂-) 4.3 (s, 2<i>H</i>) (ArC<b><i>H</i></b>₂-), 7.1-7.25 (m, 2<i>H</i>) (2 x Ar<b><i>H</i></b>), 7.8-8.0 (m, 4<i>H</i>) (4 x Ar<b><i>H</i></b>), 8.3 (m, 1<i>H</i>) (Ar<b><i>H</i></b>), 9.65 (s, 1<i>H</i>) (chain CON<b><i>H</i></b>), 12.1 (br s, 1<i>H</i>)(-CO₂<b><i>H</i></b>), 12. 65 (s, 1<i>H</i>) (ring CON<b><i>H</i></b>); m/z (M+H)^+. 398, 100% purity.

0222<i>O</i>-Benzotriazol-1-yl-<i>N</i>,<i>N</i>,<i>N</i>'<i>N</i>'-tetramethyluronium tetrafluoroborate (0.942 g, 2.9 mmol) and diisopropylethylamine (0.641 g, 5 mmol) were added sequentially at ambient temperature to a stirred solution of <i>N</i>-[2-fluoro-5-(4-oxo-3,4-dihydrophalazin-1-ylmethyl)phenyl]-2,2-dimethysuccinamic acid (0.896 g, 2.3 mmol) in dimethylacetamide (5 ml), the mixture was stirred at ambient temperature for 2 hours, then it was added dropwise to ice-cold water (50 ml). The resulting solid was collected by filtration, washed with water (3 ml) and dried <i>in vacuo.</i> The product was crystallised from industrial methylated spirit to give 1-[2-fluoro-5-(4-oxo-3,4-dihydrophalazin-1-ylmethyl)phenyl]-3,3-dimethylpyrrolidine-2,5-dione (0.4 g) as an off-white solid, m.pt. 228-231°C; 250 MHz ¹H-nmr (d₆-DMSO) δ (ppm): 1.2 (s, 6<i>H</i>) (2 x C<b><i>H</i></b>₃), 2.7 (s, 2<i>H</i>) (-CMe₂C<b><i>H</i></b>₂-) 4.3 (s, 2<i>H</i>) (ArC<b><i>H</i></b>₂-), 7.25-7.35 (m, 2<i>H</i>) (2 x Ar<b><i>H</i></b>), 7.4-7.7.5 (m, 1<i>H</i>) (Ar<b><i>H</i></b>), 7.7-7.95 (m, 3<i>H</i>) (3 x Ar<b><i>H</i></b>), 8.2 (m, 1<i>H</i>) (Ar<b><i>H</i></b>), 12.55 (s, 1<i>H</i>) (CON<b><i>H</i></b>); m/z (M+H)^+. 380, 100% purity.

Example 26

0223<chemistry id="chem0062" num="0062"><img file="EP1501822B1_D0062.tif" /></chemistry>

0224A stirred mixture of 4-(3-amino-4-fluorobenzyl)-2<i>H-</i>phthalazin-1-one (1 g, 3.7 mmol; prepared in a manner similar to that described in Example 22), 3-oxabicyclo[3.1.0]hexane-2,4-dione (0.416 g, 3.7 mmol) and toluene (50 ml) was heated under reflux for 1.5 hours, then the resulting solid was collected by filtration from the hot mixture, washed with ethyl acetate (3 ml) and dried <i>in vacuo</i> to give 2-[2-fluoro-5-(4-oxo-3,4-dihydrophalazin-1-ylmethyl)phenylcarbamoyl]cyclopropanecarboxylic acid (1.3 g) as an off-white solid, m.pt. 219-221°C; 250 MHz ¹H-nmr (d₆-DMSO) δ (ppm): 1.1-1.25 (m, 1<i>H</i>)(cyclopropane C<b><i>H</i></b>), 1.35-1.45 (m, 1<i>H</i>)(cyclopropane C<b><i>H</i></b>), 1.95-2.1 (m, 1<i>H</i>) (cyclopropane C<b><i>H</i></b>), 2.25-2.35 (m, 1<i>H</i>) (cyclopropane C<b><i>H</i></b>), 4.3 (s, 2<i>H</i>) (ArC<b><i>H</i></b>₂-), 7.05-7.2 (m, 2<i>H</i>) (2 x Ar<b><i>H</i></b>), 7.75-8.0 (m, 4<i>H</i>) (4 x Ar<b><i>H</i></b>), 8.25 (m, 1<i>H</i>) (Ar<b><i>H</i></b>), 9.95 (s, 1<i>H</i>) (chain CON<b><i>H</i></b>), 12.15 (br s, 1<i>H</i>) (-CO₂<b><i>H</i></b>), 12.65 (s, 1<i>H</i>) (ring CON<b><i>H</i></b>); m/z (M+H)^+. 382, 100% purity.

0225<i>O</i>-Benzotriazol-1-yl-<i>N</i>,<i>N</i>,<i>N</i>'<i>N</i>'-tetramethyluronium tetrafluoroborate (1.419 g, 4.4 mmol) and diisopropylethylamine (0.965 g, 7.5 mmol) were added sequentially at ambient temperature to a stirred solution of 2-[2-fluoro-5-(4-oxo-3,4-dihydrophalazin-1-ylmethyl)phenylcarbamoyl]cyclopropanecarboxylic acid (1.3 g, 3.4 mmol) in dimethylacetamide (7 ml), the mixture was stirred at ambient temperature for 2 hours, then it was added dropwise to ice-cold water (70 ml). The resulting solid was collected by filtration, washed with water (3 ml) and dried <i>in vacuo</i> to give 3-[2-fluoro-5-(4-oxo-3,4-dihydrophalazin-1-ylmethyl)phenyl]-3-azabicyclo[3.1.0]hexane-2,4-dione (1.209 g) as an off-white solid, m.pt. 228-231°C; 250 MHz ¹H-nmr (d₆-DMSO) δ (ppm): 1.4-1.55 + 1.9-2.05 (2 x br m, 1<i>H</i>)(cyclopropane C<b><i>H</i></b>), 1.6-1.75 (m, 1<i>H</i>) (cyclopropane C<b><i>H</i></b>), 2.7-2.85 (m, 2<i>H</i>) (cyclopropane C<b><i>H</i></b>), 4. 35 (s, 2<i>H</i>) (ArC<i>H</i>₂-), 7.25-7. 35 (m, 2<i>H</i>) (2 x Ar<b><i>H</i></b>), 7.4-7.5 (m, 1<i>H</i>) (Ar<b><i>H</i></b>), 7.75-8.0 (m, 3H)(3 x Ar<b><i>H</i></b>), 8.3 (m, 1<i>H</i>) (Ar<b><i>H</i></b>), 12.65 (s, 1<i>H</i>) (CON<b><i>H</i></b>); m/z (M+H)^+. 364, 100% purity.

Example 27

0226<chemistry id="chem0063" num="0063"><img file="EP1501822B1_D0063.tif" /></chemistry>

0227A stirred mixture of 4-(3-amino-4-fluorobenzyl)-2<i>H-</i>phthalazin-1-one (0.1 g, 0.37 mmol; prepared in a manner similar to that described in Example 22), 3-oct-2-enyldihydrofuran-2,5-dione (0.078 g, 0.37 mmol) and toluene (10 ml) was heated under reflux for 20 hours, then the solvent was removed <i>in vacuo</i> to give crude 2-{[2-fluoro-5-(4-oxo-3,4-dihydrophthalazin-1-ylmethyl)phenylcarbamoyl]methyl}dec-4-enoic acid (0.133 g) as an oil which was used without further purification.

0228<i>O</i>-Benzotriazol-1-yl-<i>N</i>,<i>N</i>,<i>N</i>'<i>N</i>'-tetramethyluronium tetrafluoroborate (0.107 g, 0.33 mmol) and diisopropylethylamine (0.098 ml, 0.56 mmol) were added sequentially at ambient temperature to a stirred solution of 2-{[2-fluoro-5-(4-oxo-3,4-dihydrophthalazin-1-ylmethyl)phenylcarbamoyl]methyl}-dec-4-enoic acid (0.123 g, 0.26 mmol) in dimethylformamide (2 ml), the mixture was stirred at ambient temperature for 48 hours, then it was added dropwise to ice-cold water (10 ml). The product was extracted into ethyl acetate (2 x 5 ml), the extracts were combined, dried (MgSO₄) and the solvent was removed <i>in vacuo</i>. The residue was triturated with hexane (3 ml) and the resulting solid was collected by filtration and dried <i>in vacuo</i> to give 1-[2-fluoro-5-(4-oxo-3,4-dihydrophthalazin-1-ylmethyl)phenyl]-3-oct-2-enylpyrrolidine-2,5-dione (0.055 g) as an off-white solid, m.pt 138-141°C; 250 MHz ¹H-nmr (d₆-DMSO) δ (ppm): 0.7-0.9 (m, 3<i>H</i>) (C<b><i>H</i></b>₃), 1.1-1.4 4 (m, 6<i>H</i>) (-C<b><i>H</i></b>₂C<b><i>H</i></b>₂C<b><i>H</i></b>₂C<b><i>H</i></b>₃), 1.9-2.1 (m, 2<i>H</i>) (-C<b><i>H</i></b>₂-ⁿBu), 2.3-2.6 (m, 1<i>H</i> + <i>2H</i>) (-C<b><i>H</i></b>C<b><i>H</i></b>₂CH=CH-), 2.8-3.2 (m, 2<i>H</i>) (ring C<b><i>H</i></b>₂), 4.4 (s, 2<i>H</i>) (ArC<b><i>H</i></b>₂-), 5.25-5.45 and 5.45-5.7 (2 x m, 2 x 1<i>H</i>) (-C<b><i>H</i></b>=C<b><i>H</i></b>-) 7.2-7.3 (m, 1<i>H</i>) (Ar<b><i>H</i></b>), 7.3-7.5 (m, 1<i>H</i>) (Ar<b><i>H</i></b>), 7.5-7.6 (m, 1<i>H</i>) (Ar<b><i>H</i></b>), 7.8-8.05 (m, 3<i>H</i>) (3 x Ar<b><i>H</i></b>), 8.3 (d, 1<i>H</i>) (Ar<b><i>H</i></b>), 12.65 (s, 1<i>H</i>) (CON<b><i>H</i></b>); m/z (M+H)^+. 462, 90% purity.

Example 28

0229<chemistry id="chem0064" num="0064"><img file="EP1501822B1_D0064.tif" /></chemistry> A stirred mixture of 4-(3-amino-4-fluorobenzyl)-2<i>H-</i>phthalazin-1-one (0.1 g, 0.37 mmol; prepared in a manner similar to that described in Example 22), 3-hex-2-enyldihydrofuran-2,5-dione (0.068 g, 0.37 mmol) and toluene (10 ml) was heated under reflux for 20 hours, then the resulting solid was collected by filtration from the hot mixture, washed with ethyl acetate (3 ml) and dried <i>in vacuo</i> to give 2-{[2-fluoro-5-(4-oxo-3,4-dihydrophthalazin-1-ylmethyl)phenylcarbamoyl]methyl}oct-4-enoic acid (0.061 g) as an off-white solid, m.pt. 179-181°C; 250 MHz ¹H-nmr (d₆-DMSO) δ (ppm): 0.7-0.9 (t, 3<i>H</i>) (C<b><i>H</i></b>₃), 1.1-1.35 (m, 2<i>H</i>) (-C<b><i>H</i></b>₂CH₃), 1.75-2.0 (m, 2<i>H</i>)(-C<b><i>H</i></b>₂CH₂CH₃), 2.0-2.3 (m, 2H) (-C<b><i>H</i></b>₂CH=CH-), 2.3-2.8 (2 x m obscured by DMSO signal, 2H + 1<i>H</i>) (-C<b><i>H</i></b>₂C<b><i>H</i></b>(CO₂H)CH₂-), 4.2 (s, 2<i>H</i>)(ArC<b><i>H</i></b>₂-), 5.15-5.5 (m, 2<i>H</i>) (-C<b><i>H</i></b>=C<b><i>H</i></b>-) 6.9-7.2 (m, 2<i>H</i>) (2 x Ar<b><i>H</i></b>), 7.65-8.0 (m, 4<i>H</i>) (4 x Ar<b><i>H</i></b>), 8.2 (d, 1<i>H</i>)(Ar<b><i>H</i></b>), 12.1 (br s, 1<i>H</i>) (CO₂<b><i>H</i></b>), 12.6 (s, 1<i>H</i>) (CON<b><i>H</i></b>); m/z (M+H)^+. 452, 93.7% purity.

0230<i>O</i>-Benzotriazol-1-yl-<i>N</i>,<i>N</i>,<i>N</i>'<i>N</i>'-tetramethyluronium tetrafluoroborate (0.047 g, 0.15 mmol) and diisopropylethylamine (0.043 g, 0.25 mmol) were added sequentially at ambient temperature to a stirred solution 2-{[2-fluoro-5-(4-oxo-3,4-dihydrophthalazin-1-ylmethyl)phenylcarbamoyl]methyl}oct-4-enoic acid (0.051 g, 0.11 mmol) in dimethylformamide (2 ml), the mixture was stirred at ambient temperature for 48 hours, then it was added dropwise to ice-cold water (10 ml). The resulting solid was collected by filtration and dried <i>in vacuo</i> to give 1-[2-fluoro-5-(4-oxo-3,4-dihydrophthalazin-1-ylmethyl)phenyl]-3-hex-2-enylpyrrolidine-2,5-dione (0.029 g) as an off-white solid, m.pt. 146-149°C; 250 MHz ¹H-nmr (d₆-DMSO) δ (ppm): 0.8-1.0 (m, 3<i>H</i>) (C<b><i>H</i></b>₃), 1.2-1.4 (m, 2<i>H</i>) (-C<b><i>H</i></b>₂CH₃), 1.9-2.1 (m, 2<i>H</i>) (-C<b><i>H</i></b>₂CH₂CH₃), 2.3-2.5 (m, 2<i>H</i>) (-C<b><i>H</i></b>₂CH=CH-), 2.5-2.6 (m, 1<i>H</i>)(ring C<b><i>H</i></b>), 2.8-3.0 and 3.0-3.2 (2 x m, 2<i>H</i>) (ring C<b><i>H</i></b>₂), 4.4 (s, 2<i>H</i>) (ArC<b><i>H</i></b>₂-), 5.25-5.45 and 5.45-5.7 (2 x m, 2 x 1<i>H</i>) (-C<b><i>H</i></b>=C<b><i>H</i></b>-) 7.2-7.3 (m, 1<i>H</i>) (Ar<b><i>H</i></b>), 7.3-7.5 (m, 1<i>H</i>) (Ar<b><i>H</i></b>), 7.5-7.6 (m, 1<i>H</i>) (Ar<b><i>H</i></b>), 7.8-8.05 (m, 3<i>H</i>) (3 x Ar<b><i>H</i></b>), 8.3 (d, 1<i>H</i>) (Ar<b><i>H</i></b>), 12.65 (s, 1<i>H</i>) (CON<b><i>H</i></b>); m/z (M+H)^+. 434, 95.8% purity.

Example 29

0231<chemistry id="chem0065" num="0065"><img file="EP1501822B1_D0065.tif" /></chemistry> 1-[2-Fluoro-5-(4-oxo-3,4-dihydrophthalazin-1-ylmethyl)phenyl]-3-non-2-enylpyrrolidine-2,5-dione (0.041 g) was synthesised in a manner similar to that described in Example 27. It was isolated as an off-white solid, m.pt. 146-149°C; 250 MHz ¹H-nmr (d₆-DMSO) δ (ppm): 0.7-0.9 (m, 3<i>H</i>) (C<b><i>H</i></b>₃), 1.1-1.4 (m, 8<i>H</i>) (C<b><i>H</i></b>₂C<b><i>H</i></b>₂C<b><i>H</i></b>₂C<b><i>H</i></b>₂CH₃), 1.9-2.1 (m, 2<i>H</i>) (-C<b><i>H</i></b>₂-ⁿPentyl), 2.3-2.5 (m, 2<i>H</i>) (-C<b><i>H</i></b>₂CH=CH-) , 2.5-2.6 (m, 1<i>H</i>) (ring C<b><i>H</i></b>), 2.8-3.0 and 3.0-3.2 (2 x m, 2<i>H</i>) (ring C<b><i>H</i></b>₂), 4.4 (s, 2<i>H</i>) (ArC<b><i>H</i></b>₂-), 5.25-5.45 and 5.45-5.7 (2 x m, 2 x 1<i>H</i>) (-C<b><i>H</i></b>=C<b><i>H</i></b>-) 7.2-7.3 (m, 1<i>H</i>) (Ar<b><i>H</i></b>), 7.3-7.5 (m, 1<i>H</i>) (Ar<b><i>H</i></b>), 7.5-7.6 (m, 1<i>H</i>) (Ar<b><i>H</i></b>), 7.8-8.05 (m, 3<i>H</i>) (3 x Ar<b><i>H</i></b>), 8.3 (d, 1<i>H</i>) (Ar<b><i>H</i></b>), 12.65 (s, 1<i>H</i>) (CON<b><i>H</i></b>).

Example 30

0232<chemistry id="chem0066" num="0066"><img file="EP1501822B1_D0066.tif" /></chemistry>

0233A stirred mixture of 4-(3-amino-4-fluorobenzyl)-2<i>H-</i>phthalazin-1-one (2.51 g, 9.3 mmol; prepared in a manner similar to that described in Example 22), 4-benzylmorpholin-2,6-dione (2.3 g, 11.2 mmol) and toluene (15 ml) was heated under reflux for 20 hours, then the solvent was removed <i>in vacuo.</i> The residue was dissolved in dimethylacetamide (15 ml) and <i>O</i>-benzotriazol-1-yl-<i>N</i>,<i>N</i>,<i>N</i>'<i>N</i>'-tetramethyluronium tetrafluoroborate (3 g, 9.3 mmol) and diisopropylethylamine (1.63 ml, 9.3 mmol) were added sequentially. The mixture was stirred at ambient temperature for 2 hours, then it was added dropwise to stirred, ice-cold water (300 ml). The resulting solid was collected by filtration and dried <i>in vacuo</i> to give 4-benzyl-1-[2-fluoro-5-(4-oxo-3,4-dihydrophthalazin-1-ylmethyl)phenyl]piperazine-2,6-dione (4.01 g) as an off-white solid, 250 MHz ¹H-nmr (d₆-DMSO) δ (ppm) : 3.6 (s, 4<i>H</i>) (2 x ring C<b><i>H</i></b>₂) 3.75 (s, 2<i>H</i>) (NC<b><i>H</i></b>₂Ph), 4. 35 (s, 2<i>H</i>) (ArC<b><i>H</i></b>₂-), 7.17-7.5 (m, 8<i>H</i>) (8 x Ar<b><i>H</i></b>), 7.75-8.0 (m, 3<i>H</i>) (3 x Ar<b><i>H</i></b>), 8.25 (d, 1<i>H</i>) (Ar<b><i>H</i></b>), 12.6 (s, 1<i>H</i>) (CON<b><i>H</i></b>); m/z (M+H)⁺ 457, 100% purity.

Example 32

0234<chemistry id="chem0067" num="0067"><img file="EP1501822B1_D0067.tif" /></chemistry>

0235A stirred mixture of 4-(3-amino-4-fluorobenzyl)-2<i>H-</i>phthalazin-1-one (0.1 g, 0.37 mmol; prepared in a manner similar to that described in Example 22) and 1-(carboxymethyl)cyclopentane-1-carboxylic acid (0.064 g, 0.37 mmol) was heated at 200°C in a sealed tube until the starting materials had been consumed (the reaction was followed by tlc using a 1:1 mixture of ethyl acetate and hexane as eluant). The warm mixture was poured into an ice/water mixture (10ml) and the resulting solid was collected by filtration. The crude product was recrystallised from ethyl acetate/hexane (the hot solution required filtration to remove traces of undissolved solids) to give 2-[2-fluoro-5-(4-oxo-3,4-dihydrophthalazin-1-ylmethyl)phenyl]-2-azaspiro[4.4]nonane-1,3-dione (0.03 g) as an off-white solid, m.pt. 215-220°C; 250 MHz ¹H-nmr (d₆-DMSO) δ (ppm): 1.7-2.2 (m, <i>8H</i>) (4 x cyclopentyl C<b><i>H</i></b>₂), 2.9 (s, 2<i>H</i>) (-COC<b><i>H</i></b>₂-), 4.35 (s, 2<i>H</i>) (ArC<b><i>H</i></b>₂-), 7.3-7.45 (m, 2<i>H</i>) (2 x Ar<b><i>H</i></b>), 7.5-7.6 (m, 1<i>H</i>) (Ar<b><i>H</i></b>), 7.8-8.1 (m, 3<i>H</i>) (3 x Ar<b><i>H</i></b>), 8.3 (d, 1<i>H</i>) (Ar<b><i>H</i></b>), 12.7 (s, 1<i>H</i>) (CON<b><i>H</i></b>); m/z (M+H)^+. 406, 91.4% purity.

Example 32

0236<chemistry id="chem0068" num="0068"><img file="EP1501822B1_D0068.tif" /></chemistry>

0237A stirred mixture of 4-(3-amino-4-fluorobenzyl)-2<i>H-</i>phthalazin-1-one (0.135 g, 0.5 mmol; prepared in a manner similar to that described in Example 22), 3-benzyldihydrofuran-2,5-dione (0.095 g, 0.5 mmol) and toluene (8 ml) was heated under reflux for 1 hour, allowed to stand at ambient overnight, then heated under reflux for a further 6.5 hours and allowed to cool to ambient temperature. The resulting solid was collected by filtration, washed with hexane (10 ml) and dried <i>in vacuo</i> to give 2-benzyl-<i>N</i>-[2-fluoro-5-(4-oxo-3,4-dihydrophthalazin-1-ylmethyl)phenyl]succinamic acid (0.156 g) as an off white solid, 250 MHz ¹H-nmr (d₆-DMSO) δ (ppm): 2.2-2.95 (m 5<i>H</i>) (-C<b><i>H</i></b>₂C<b><i>H</i></b>(CO₂H)C<b><i>H</i></b>₂Ph), 4.2 (s, 2<i>H</i>) (ArC<b><i>H</i></b>₂-), 6.9-7.25 (m, 7<i>H</i>) (7 x Ar<b><i>H</i></b>), 7.6-7.9 (m, 4<i>H</i>) (4 x Ar<b><i>H</i></b>), 8.15 (d, 1<i>H</i>) (Ar<b><i>H</i></b>), 9.65 (s, 1<i>H</i>) (chain CON<b><i>H</i></b>), 12.2 (s, 1<i>H</i>) (-CO₂<b><i>H</i></b>), 12.5 (s, 1<i>H</i>) (ring CON<b><i>H</i></b>).

0238<i>O</i>-Benzotriazol-1-yl-<i>N</i>,<i>N</i>,<i>N</i>'<i>N</i>'-tetramethyluronium tetrafluoroborate (0.14 g, 0.44 mmol) and diisopropylethylamine (0.097 g, 0.44 mmol) were added sequentially at ambient temperature to a stirred solution of 2-benzyl-<i>N</i>-[2-fluoro-5-(4-oxo-3,4-dihydrophthalazin-1-ylmethyl)phenyl]succinamic acid (0.156 g, 0.34 mmol) in dimethylformamide (2 ml), the mixture was stirred at ambient temperature for 3 hours, then it was added dropwise to ice-cold water (10 ml). The mixture was stirred for 30 minutes, then the resulting solid was collected by filtration, washed with water (5 ml) and dried <i>in vacuo</i> to give 3-benzyl-1-[2-fluoro-5-(4-oxo-3,4-dihydrophthalazin-1-ylmethyl)phenyl]pyrrolidine-2,5-dione (0.120 g) as a white solid, 250 MHz ¹H-nmr (d₆-DMSO) δ (ppm): 2.6-3.4 (m 5<i>H</i>) (-C<b><i>H</i></b>₂C<b><i>H</i></b>(CO₂H)C<b><i>H</i></b>₂Ph), 4.5 (s, 2<b><i>H</i></b>) (ArC<b><i>H</i></b>₂-), 7. 3-7. 6 (m, 7<i>H</i>) (7 x Ar<b><i>H</i></b>), 7.6-7.7 (m, 1<i>H</i>) (Ar<b><i>H</i></b>), 7.9-8.2 (m, 3<i>H</i>) (3 x Ar<b><i>H</i></b>), 8.4 (m, 1<i>H</i>) (Ar<b><i>H</i></b>), 12.8 (br s, 1<i>H</i>) (CON<b><i>H</i></b>); m/z (M+H)⁺ 442, 96.8% purity.

Example 33

0239<chemistry id="chem0069" num="0069"><img file="EP1501822B1_D0069.tif" /></chemistry>

0240A stirred mixture of 4-(3-amino-4-fluorobenzyl)-2<i>H-</i>phthalazin-1-one (2.02 g, 7.5 mmol; prepared in a manner similar to that described in Example 22), 3-methyldihydrofuran-2,5-dione (0.856 g, 7.5 mmol) and toluene (100 ml) was heated under reflux for 2.5 hours (for the first 30 minutes of this period, traces of water in the mixture were removed by azeotropic distillation). The resulting solid was collected by filtration from the hot mixture, washed with toluene (20 ml) and dried <i>in vacuo</i> to give <i>N</i>-[2-fluoro-5-(4-oxo-3,4-dihydrophthalazin-1-ylmethyl)phenyl]-2-methylsuccinamic acid (1.91 g) as an off-white solid, m.pt. 140-144°C; 250 MHz ¹H-nmr (d₆-DMSO) δ (ppm): 0.95 (d, 3<i>H</i>) (C<b><i>H</i></b>₃), 2.2-2.6 (m, 3<i>H</i>) (-C<b><i>H</i></b>₂C<b><i>H</i></b>Me-), 4.1 (s, 2<i>H</i>) (ArC<b><i>H</i></b>₂-), 6.9-7.2 (m, 2<i>H</i>) (2 x Ar<b><i>H</i></b>), 7.6-7.9 (m, 4<i>H</i>) (4 x Ar<b><i>H</i></b>), 8.1 (d, 1<i>H</i>) (Ar<b><i>H</i></b>), 9.55 (s, 1<i>H</i>) (chain CON<b><i>H</i></b>), 12.0 (br s, 1<i>H</i>) (CO₂<b><i>H</i></b>), 12.5 (s, 1<i>H</i>) (ring CON<b><i>H</i></b>); m/z (M+H)⁺ 384, 100% purity.

0241<i>O</i>-Benzotriazol-1-yl-<i>N</i>,<i>N</i>,<i>N</i>'<i>N</i>'-tetramethyluronium tetrafluoroborate (1.09 g, 3.4 mmol) and diisopropylethylamine (0.98 g, 5.7 mmol) were added sequentially to a stirred solution of <i>N</i>-[2-fluoro-5-(4-oxo-3,4-dihydrophthalazin-1-ylmethyl)phenyl]-2-methylsuccinamic acid (1 g, 2.6 mmol) in dimethylacetamide (5 ml), the mixture was stirred at ambient temperature for 15 minutes, then it was poured onto water (50 ml) and allowed to stand at ambient temperature for 20 hours. The resulting solid was collected by filtration, washed with hexane (20 ml) and dried <i>in vacuo</i> to give 1-[2-fluoro-5-(4-oxo-3,4-dihydrophthalazin-1-ylmethyl)phenyl]-3-methylpyrrolidine-2,5-dione (0.654 g) as a beige solid, m.pt. 171-172°C; 250 MHz ¹H-nmr (d₆-DMSO) δ (ppm): 1.4 (d, 3<i>H</i>) (C<b><i>H</i></b>₃), 2.6-2.7 (m, obscured by DMSO signal, 1<i>H</i>) (-C<b><i>H</i></b>Me-), 3.05-3.3 (m, 2<i>H</i>) (-C<b><i>H</i></b>₂CHMe-), 4.5 (s, 2<i>H</i>) (ArC<b><i>H</i></b>₂-), 7.3-7.55 (m, 2<i>H</i>) (2 x Ar<b><i>H</i></b>), 7.6-7.7 7 (m, 1<i>H</i>) (Ar<b><i>H</i></b>), 7.9-8.2 (m, 3<i>H</i>) (3 x Ar<b><i>H</i></b>), 8. 4 (d, 1<i>H</i>) (Ar<b><i>H</i></b>), 12.8 (s, 1<i>H</i>) (CON<b><i>H</i></b>); m/z (M+H)⁺ 366, 98% purity.

Example 34

0242<chemistry id="chem0070" num="0070"><img file="EP1501822B1_D0070.tif" /></chemistry><i>O</i>-Benzotriazol-1-yl-<i>N</i>,<i>N</i>,<i>N</i>'<i>N</i>'-tetramethyluronium tetrafluoroborate (0.417 g, 1.3 mmol), diisopropylethylamine (0.284 g, 2.2 mmol) and 4-(3-amino-4-fluorobenzyl)-2<i>H</i>-phthalazin-1-one (0.269 g, 1 mmol; prepared in a manner similar to that described in Example 22) were added sequentially to a stirred solution of <i>N</i>-α-CBZ-L-aspartic acid β-benzyl ester (0.357 g, 1 mmol) in dimethylformamide (2 ml), the mixture was stirred at ambient temperature for 50 hours, then it was poured into ice-cold water (20 ml). The resulting solid was collected by filtration and dried <i>in vacuo</i> to give (<i>S</i>)-3-benzyloxycarbonylamino-<i>N</i>-[2-fluoro-5-(4-oxo-3,4-dihydrophthalazin-1-ylmethyl)phenyl]succinamic acid benzyl ester (0.57 g) as a beige solid, m.pt. 76-80°C; m/z (M+H)⁺ 607, 82% purity, which was used without further purification.

0243A mixture of the above crude (<i>S</i>)-3-benzyloxycarbonylamino-<i>N</i>-[2-fluoro-5-(4-oxo-3,4-dihydrophthalazin-1-ylmethyl)phenyl]succinamic acid benzyl ester (0.57 g) and saturated aqueous sodium carbonate solution (pH 9; 10 ml) was stirred at ambient temperature for 72 hours. The pH of the mixture was adjusted to pH10 by the addition of further saturated aqueous sodium carbonate solution, then the mixture was stirred at 70°C for 4 hours until a clear solution was obtained. The cooled solution was washed with ethyl acetate (2 x 5 ml) and the aqueous layer was acidified by the addition of 10% hydrochloric acid. The resulting solid was collected by filtration and dried <i>in vacuo</i> to give (S)-3-benzyloxycarbonylamino-<i>N</i>-[2-fluoro-5-(4-oxo-3,4-dihydrophthalazin-1-ylmethyl)phenyl]succinamic acid (0.35 g) as an off-white solid, m.pt. 188-190°C, which was used without further purification.

0244<i>O</i>-Benzotriazol-1-yl-<i>N</i>,<i>N</i>,<i>N</i>'<i>N</i>'-tetramethyluronium tetrafluoroborate (0.289 g, 0.9 mmol) and diisopropylethylamine (0.193 g, 1.5 mmol) were added sequentially to a stirred solution of (<i>S</i>)-3-benzyloxycarbonylamino-<i>N</i>-[2-fluoro-5-(4-oxo-3,4-dihydrophthalazin-1-ylmethyl)phenyl]succinamic acid (0.35 g, 0.68 mmol) in dimethylacetamide (3 ml), the mixture was stirred at ambient temperature for 24 hours, then it was added dropwise to stirred, ice-cold water (30 ml). The mixture was stirred for 1 hour, then the resulting solid was collected by filtration and dried <i>in vacuo</i> to give (S)-3-benzyloxycarbonylamino-1-[2-fluoro-5-(4-oxo-3,4-dihydrophthalazin-l-ylmethyl)phenyl]pyrrolidine-2,5-dione (0.238 g) as an off white solid, m.pt. 121-127°C (softens 102°C); m/z (M+H)⁺. 501, 96% purity.

Example 35

0245<chemistry id="chem0071" num="0071"><img file="EP1501822B1_D0071.tif" /></chemistry>

0246A stirred mixture of <i>N</i>-methyliminodiacetic acid (0.06 g, 0.4 mmol) and acetic anhydride (1 ml) was heated under reflux under nitrogen for 20 minutes, until a clear solution was obtained. The excess of acetic anhydride and the acetic acid produced in the reaction were removed <i>in vacuo</i> and the residual 4-methylmorpholine-2,6-dione was dissolved in toluene (7 ml) and used without purification.

02474-(3-Amino-4-fluorobenzyl)-2<i>H</i>-phthalazin-1-one (0.108 g, 0.4 mmol; prepared in a manner similar to that described in Example 22) was added to the above toluene solution, the stirred mixture was heated under reflux for 2 hours and allowed to stand at ambient temperature for 20 hours, then the resulting solid was collected by filtration and dried <i>in vacuo</i> to give <i>N</i>-[2-fluoro-5-(4-oxo-3,4-dihydrophthalazin-1-ylmethyl)phenylcarbamoylmethyl]-N-methylglycine (0.125 g) as a beige solid, m.pt. 194-198°C; m/z (M+H)^+. 399, 92.6% purity, which was used without further purification.

0248<i>O</i>-Benzotriazol-1-yl-<i>N</i>,<i>N</i>,<i>N</i>'<i>N</i>'-tetramethyluronium tetrafluoroborate (0.108 g, 0.34 mmol) and diisopropylethylamine (0.074 g, 0.57 mmol) were added sequentially at ambient temperature to a stirred solution of <i>N</i>-[2-fluoro-5-(4-oxo-3,4-dihydrophthalazin-1-ylmethyl)phenylcarbamoylmethyl]-N-methylglycine (0.103 g, 0.26 mmol) in dimethylacetamide (1 ml), the mixture was stirred at ambient temperature for 1 hour, then it was diluted with water (10 ml). Sodium chloride (1 g) was added, the mixture was stirred at ambient temperature for 30 minutes, then the resulting solid was collected by filtration, washed with water (1 ml) and dried <i>in vacuo</i> to give 1-[2-fluoro-5-(4-oxo-3,4-dihydrophthalazin-1-ylmethyl)phenyl]-4-methylpiperazine-2,6-dione (0.071 g) as a beige solid, m.pt. 205-208°C; 250 MHz ¹H-nmr (CDCl₃) δ (ppm) : 2. 4 (s, 3<i>H</i>) (C<b><i>H</i></b>₃), 3. 5 (q, 4<i>H</i>) (2 x C<b><i>H</i></b>₂), 4.25 (s, 2<i>H</i>) (ArC<b><i>H</i></b>₂-), 7.1 (m, 2<i>H</i>) (2 x Ar<b><i>H</i></b>), 7.25 (m, 1<i>H</i>) (Ar<b><i>H</i></b>), 7.7-7. 85 (m, 3<i>H</i>) (3 x Ar<b><i>H</i></b>), 8.4 (m, 1<i>H</i>) (Ar<b><i>H</i></b>), 10. 35 (s, 1<i>H</i>) (CON<b><i>H</i></b>) ; m/z (M+H)^+. 381, 100% purity.

Example 36

0249<chemistry id="chem0072" num="0072"><img file="EP1501822B1_D0072.tif" /></chemistry>

0250A solution of ammonium formate (0.55 g, 8.8 mmol) in water (5 ml) was added to a stirred mixture of 4-benzyl-1-[2-fluoro-5-(4-oxo-3,4-dihydrophthalazin-1-ylmethyl)phenyl]piperazine-2,6-dione (1 g, 2.2 mmol; prepared in a manner similar to that described in Example 30), 10% palladium on carbon catalyst (0.33 g) and methanol (15 ml), the mixture was heated under reflux for 1 hour, then it was cooled to ambient temperature and filtered through a pad of Celite filter aid. The filter pad was washed with methanol (50 ml), then the combined filtrate and washings were concentrated <i>in vacuo.</i> The residue was diluted with water (20 ml), the product was extracted into ethyl acetate (4 x 20 ml), the combined extracts were dried (MgSO₄) and the solvent was removed <i>in vacuo</i> to give 1-[2-fluoro-5-(4-oxo-3,4-dihydrophthalazin-1-ylmethyl)phenyl]piperazine-2,6-dione (0.8 g) as a pale brown solid, 250 MHz ¹H-nmr (d₆-DMSO) δ (ppm): 3.3 (2 x overlapping d, 4<i>H</i>) (2 x ring C<b><i>H</i></b>₂), 4.25 (s, 2<i>H</i>) (ArC<b><i>H</i></b>₂-), 6.95-7.2 (m, 2<i>H</i>) (2 x Ar<b><i>H</i></b>), 7.7-7.9 (m, 3<i>H</i>) (3 x Ar<b><i>H</i></b>), 7.95 (d, 1<i>H</i>) (Ar<b><i>H</i></b>), 8.2 (d, 1<i>H</i>) (Ar<b><i>H</i></b>), 9.7 (s, 1<i>H</i>) (piperazine N<b><i>H</i></b>), 12.6 (s, 1<i>H</i>) (CON<b><i>H</i></b>).

Example 37

0251<chemistry id="chem0073" num="0073"><img file="EP1501822B1_D0073.tif" /></chemistry>

0252A stirred solution of dimethyl 3-oxo-1,3-dihydroisobenzofuran-1-ylphosphonate (4.84 g, 0.02 mol; prepared in a manner similar to that described in Example 22) and 4-chloro-3-nitrobenzaldehyde (3.71 g, 0.02 mol) in tetrahydrofuran (30 ml) was cooled to 15°C and a solution of triethylamine (2.02 g, 0.02 mol) in tetrahydrofuran (3 ml) was added dropwise at &lt;25°C. The mixture was stirred at ambient temperature for 1 hour, allowed to stand at this temperature for a further 16 hours and the resulting solid was collected by filtration. The filtrate was concentrated <i>in vacuo,</i> the residue was triturated with water (5 ml) and the resulting solid was collected by filtration. The two crops of solid were combined and suspended in water (30 ml). The mixture was stirred at ambient temperature for 30 minutes and the resulting solid was collected by filtration and dried <i>in vacuo</i> for 24 hours to give crude 3-(4-chloro-3-nitrobenzylidene)-3<i>H</i>-isobenzofuran-1-one as a pale yellow solid, m.pt. 198-204°C, still slightly wet with water, which was used directly in the next stage.

0253A stirred mixture of the above crude 3-(4-chloro-3-nitrobenzylidene)-3<i>H</i>-isobenzofuran-1-one (6.2 g), industrial methylated spirit (80 ml), water (60 ml) and ammonium chloride (2.14 g, 0.04 mol) was heated to 70°C, and iron powder (11.2 g, 0.2 gatom) was added in portions. When the addition was complete, the stirred mixture was heated under reflux for a further 2 hours, then it was filtered while hot through Celite. The collected inorganic solids were washed with hot industrial methylated spirit (3 x 150 ml), then the filtrate and washings were combined and the solvent was removed <i>in vacuo.</i> The residue was triturated with water (60 ml) and the resulting sticky solid was collected by filtration and triturated with industrial methylated spirit (80 ml). The resulting solid was collected by filtration, washed with industrial methylated spirit (2 x 1 ml) and dried <i>in vacuo</i> for 24 hours to give 3-(3-amino-4-chlorobenzylidene)-3<i>H-</i>isobenzofuran-1-one (3.58 g) as a yellow solid, m.pt. 148-153°C; m/z (M+H)⁺, 272/274, 100% purity, which was used directly in the next stage.

0254A stirred mixture of the above 3-(3-amino-4-chlorobenzylidene)-3<i>H</i>-isobenzofuran-1-one (0.815 g, 3 mmol), industrial methylated spirit (10 ml) and hydrazine monohydrate (0.15 g, 3 mmol) was heated under reflux for 1 hour then cooled to ambient temperature. The resulting solid was collected by filtration, washed with industrial methylated spirit (5 ml) and water (5 ml), and dried <i>in vacuo</i> to give a pale grey solid. The crude material was recrystallised from acetonitrile (140 ml) and the resulting solid was collected by filtration and dried <i>in vacuo</i> to give 4-(3-amino-4-chlorobenzyl)-2<i>H</i>-phthalazin-1-one (0.607 g) as an off-white solid, m.pt. 227-228°C; 250 MHz ¹H-nmr (d₆-DMSO) δ (ppm): 4.05 (s, 2<i>H</i>) (ArC<b><i>H</i></b>₂-), 5.2 (s, 2<i>H</i>) (-N<b><i>H</i></b>₂), 6.4-6.5 (m, 1<i>H</i>) (Ar<b><i>H</i></b>), 6.6-6.7 (m, 1<i>H</i>) (Ar<b><i>H</i></b>), 6.95-7.05 (m, 1<i>H</i>) (Ar<b><i>H</i></b>), 7.65-7.85 (m, 3<i>H</i>) (3 x Ar<b><i>H</i></b>), 8.1-8.2 (m, 1<i>H</i>) (Ar<b><i>H</i></b>), 12.5 (s, 1<i>H</i>) (CON<b><i>H</i></b>) ; m/z (M+H)^+. 286/288, 100% purity.

0255A stirred mixture of 4-(3-amino-4-chlorobenzyl)-2<i>H-</i>phthalazin-1-one (0.171 g, 0.6 mmol), succinic anhydride (0.06 g, 0.6 mmol) and toluene (60 ml) was heated under reflux for 2 hours, then cooled to ambient temperature. The precipitated solid was collected by filtration and recrystallised from industrial methylated spirit (20 ml). The resulting solid was collected by filtration and dried <i>in vacuo</i> to give <i>N</i>-[2-chloro-5-(4-oxo-3,4-dihydrophalazin-1-ylmethyl)phenyl]succinamic acid (0.149 g) as an off-white solid, m.pt. 215-217°C; m/z (M+H)^+. not detected, 100% purity. This material was used without further purification.

0256<i>O</i>-Benzotriazol-1-yl-<i>N</i>,<i>N</i>,<i>N</i>'<i>N</i>'-tetramethyluronium tetrafluoroborate (0.150 g, 0.47 mmol) and diisopropylethylamine (0.102 g, 0.8 mmol) were added sequentially at ambient temperature to a stirred solution of <i>N</i>-[2-chloro-5-(4-oxo-3,4-dihydrophalazin-1-ylmethyl)phenyl]succinamic acid (0.139 g, 0.36 mmol) in dimethylformamide (2 ml), and the mixture was stirred at ambient temperature for 20 hours. Tlc indicated that starting material remained, so the stirred mixture was heated to 100°C for 1 hour then allowed to cool to ambient temperature. The resulting mixture was added dropwise to water (20 ml), the mixture was stirred at ambient temperature for 1 hour, and the resulting solid was collected by filtration, triturated with a mixture of industrial methylated spirit (1 ml) and ethyl acetate (2 ml), collected by filtration and dried <i>in vacuo</i> to give 1-[2-chloro-5-(4-oxo-3,4-dihydrophalazin-1-ylmethyl)phenyl]pyrrolidine-2,5-dione (0.07 g) as an off-white solid, m.pt. 260-263°C; 250 MHz ¹H-nmr (d₆-DMSO) δ (ppm): 2.75-2.95 (m, 4<b><i>H</i></b>) (-C<b><i>H</i></b>₂C<b><i>H</i></b>₂-) 4.4 (s, 2<b><i>H</i></b>) (ArC<b><i>H</i></b>₂-), 7.3 (s, 1<i>H</i>) (Ar<b><i>H</i></b>), 7.5-7.65 (m, 2<i>H</i>) (2 x Ar<b><i>H</i></b>), 7.8-8.05 (m, 3<i>H</i>) (3 x Ar<b><i>H</i></b>), 8.3 (d, 1<i>H</i>) (Ar<b><i>H</i></b>), 12.65 (s, 1<i>H</i>) (CON<b><i>H</i></b>); m/z (M+H)⁺. 368/370, 93.1% purity.

0257The following Examples 38-41 were synthesised in a manner analogous to the final two stages described in Example 38, using appropriate starting materials, and following both reaction stages by tlc until starting materials were consumed. Any substantial variations in methodology are noted below.

Example 38

0258<chemistry id="chem0074" num="0074"><img file="EP1501822B1_D0074.tif" /></chemistry>

0259The open chain intermediate was a mixture of <i>N</i>-[2-chloro-5-(4-oxo-3,4-dihydrophalazin-1-ylmethyl)phenyl]-2-phenylsuccinamic acid and <i>N</i>-[2-chloro-5-(4-oxo-3,4-dihydrophalazin-1-ylmethyl)phenyl]-3-phenylsuccinamic acid, obtained as an off-white solid, m.pt. 195-197°C.

0260The cyclisation reaction mixture was stirred for 2 hours at ambient temperature, then allowed to stand for a further 16 hours before work-up, without further heating, to give 1-[2-chloro-5-(4-oxo-3,4-dihydrophalazin-1-ylmethyl)phenyl]-3-phenylpyrrolidine-2,5-dione (0.073 g) as a white powder, m.pt. 131-135°C; m/z (M+H)^+. 444/446, 91.3% purity.

Example 39

0261<chemistry id="chem0075" num="0075"><img file="EP1501822B1_D0075.tif" /></chemistry>

0262The open chain intermediate required purification by suspension in 0.75 M aqueous sodium hydrogencarbonate solution (20 ml) and washing with dichloromethane (2 x 10 ml). The aqueous layer was reacidified by the addition of 5 M hydrochloric acid and the resulting solid was collected by filtration and dried <i>in vacuo</i> to give a mixture of <i>N</i>-[2-chloro-5-(4-oxo-3,4-dihydrophalazin-1-ylmethyl)phenyl]-2-hexen-2-ylsuccinamic acid and <i>N</i>-[2-chloro-5-(4-oxo-3,4-dihydrophalazin-1-ylmethyl)phenyl]-3-hexen-2-ylsuccinamic acid (0.089 g) as a beige solid, m/z (M+H)^+. 468/470, 93.3% purity.

0263The cyclisation reaction mixture was stirred for 48 hours at ambient temperature before work-up, without further heating, to give a sticky solid. The solid was dissolved in hot toluene (3 ml), the clear solution was decanted from insoluble residues, then it was diluted with hexane (20 ml). The resulting solid was collected by filtration and dried <i>in vacuo</i> to give 1-[2-chloro-5-(4-oxo-3,4-dihydrophalazin-1-ylmethyl)phenyl]-3-hexen-2-ylpyrrolidine-2,5-dione (0.034g) as a beige solid, m/z (M+H)^+. 450/452, 96.2% purity.

Example 40

0264<chemistry id="chem0076" num="0076"><img file="EP1501822B1_D0076.tif" /></chemistry>

0265The open chain intermediate was 2-[2-chloro-5-(4-oxo-3,4-dihydrophthalazin-1-ylmethyl)phenylcarbamoyl]cyclopropanecarboxylic acid (0.125 g), obtained as a beige solid; m/z (M+H)^+. 398/400, 97.3% purity

0266The cyclisation product was 3-[2-chloro-5-(4-oxo-3,4-dihydrophthalazin-1-ylmethyl)phenyl]-3-azabicyclo[3.1.0]hexane-2,4-dione (0.088g), obtained as an off-white powder, m.pt. 229-230°C; m/z (M+H)^+. 380/382, 97.7% purity.

Example 41

0267<chemistry id="chem0077" num="0077"><img file="EP1501822B1_D0077.tif" /></chemistry>

0268The open chain intermediate was 4-[2-chloro-5-(4-oxo-3,4-dihydrophthalazin-l-ylmethyl)phenylcarbamoyl]butyric acid (0.115g), obtained as a white powder, m.pt. 238-241°C; 250 MHz ¹H-nmr (d₆-DMSO) δ (ppm): 1.7-1.9 (m, 2<i>H</i>)(-CH₂C<b><i>H</i>₂</b>CH₂-), 2.25-2.5 (m, 4<i>H</i>) (-C<b><i>H</i>₂</b>CH₂C<b><i>H</i>₂</b>-), 4.4 (s,1<i>H</i>) (ArC<b><i>H</i>₂</b>-), 7.15-7.2 (m, 1<i>H</i>)(Ar<b><i>H</i></b>), 7.4-7.50 (m, 1<i>H</i>)(Ar<b><i>H</i></b>), 7.65-7.7 (s, 1<i>H</i>)(Ar<b><i>H</i></b>), 7.8-8.05 (m, 3<i>H</i>)(3 x Ar<b><i>H</i></b>), 8.3 (d, 1<i>H</i>)(Ar<b><i>H</i></b>), 9.5 (s, 1<i>H</i>)(chain CON<b><i>H</i></b>), 12.2 (s, 1<i>H</i>)(-CO₂<b><i>H</i></b>), 12.7 (s, 1<i>H</i>)(ring CON<b><i>H</i></b>); m/z (M+H)^+. 400/402, 100% purity.

0269The cyclisation reaction mixture was stirred for 3 hours at ambient temperature before work-up, without further heating, to give 1-[2-chloro-5-(4-oxo-3,4-dihydrophalazin-1-ylmethyl)phenyllpiperidine-2,5-dione (0.017 g) as an off-white powder, m/z (M+H)^+. 382/384, 98.0% purity.

Example 42

0270<chemistry id="chem0078" num="0078"><img file="EP1501822B1_D0078.tif" /></chemistry>

0271A stirred solution of dimethyl 3-oxo-1,3-dihydroisobenzofuran-1-ylphosphonate (2.42 g, 0.01 mol; prepared in a manner similar to that described in Example 22) and 4-methoxy-3-nitrobenzaldehyde (1.81 g, 0.01 mol) in tetrahydrofuran (15 ml) was cooled to 15°C and a solution of triethylamine (1.01 g, 0.01 mol) in tetrahydrofuran (1.5 ml) was added dropwise at &lt;25°C. The mixture was stirred at ambient temperature for 4 hours, allowed to stand at this temperature for a further 16 hours and the resulting mixture was concentrated <i>in vacuo.</i> The residue was triturated with water (50 ml) and stirred at ambient temperature for 2 hours before the resulting solid was collected by filtration and dried <i>in vacuo</i> for 24 hours to give crude 3-(4-methoxy-3-nitrobenzylidene)-3<i>H-</i>isobenzofuran-1-one (2.53 g) as a yellow solid, m.pt. 173-181°C, which was used directly in the next stage.

0272A stirred mixture of the above crude 3-(4-methoxy-3-nitrobenzylidene)-3<i>H</i>-isobenzofuran-1-one (2.5 g, 0.0085 mol), industrial methylated spirit (40 ml), water (30 ml) and ammonium chloride (0.91 g, 0.017 mol) was heated to 70°C, and iron powder (4.76 g, 0.085 gatom) was added in portions. When the addition was complete, the stirred mixture was heated under reflux for a further 2 hours, then it was filtered while hot through Celite. The collected inorganic solids were washed with hot industrial methylated spirit (3 x 80 ml), then the filtrate and washings were combined and the solvent was removed <i>in vacuo.</i> The residue was triturated with water (50 ml) and the resulting solid was collected by filtration and dissolved in ethyl acetate (300 ml). The solution was filtered, the filtrate was concentrated <i>in vacuo</i> and the residue was triturated with industrial methylated spirit (10 ml). The resulting solid was collected by filtration and dried <i>in vacuo</i> for 24 hours to give 3-(3-amino-4-methoxybenzylidene)-3<i>H</i>-isobenzofuran-1-one (1.49 g) as a yellow solid, m.pt. 148-153°C; m/z (M+H)^+. 268, 100% purity.

0273A stirred mixture of the above 3-(3-amino-4-methoxybenzylidene)-3<i>H</i>-isobenzofuran-1-one (1.336 g, 0.005 mol), industrial methylated spirit (20 ml) and hydrazine monohydrate (0.25 g, 0.005 mol) was heated under reflux for 1.5 hours then cooled to ambient temperature. The resulting solid was collected by filtration, washed with water (5 ml), and dried <i>in vacuo</i> to give 4-(3-amino-4-methoxybenzyl)-2<i>H</i>-phthalazin-1-one (1.15 g) as a beige solid, m.pt. 211.5-214.5°C; 250MHz ¹H-nmr (d₆-DMSO) δ (ppm): 3.75 (s, 3<i>H</i>)(-OC<b><i>H</i>₃</b>), 4.15 (s, 2<i>H</i>)(ArC<b><i>H</i>₂</b>-), 4.75 (s, 2<i>H</i>) (-N<b><i>H</i>₂</b>) 6.5-6.6 (m, 2<i>H</i>)(2 x Ar<b><i>H</i></b>), 6.7-6.75 (m, 1<i>H</i>)(Ar<b><i>H</i></b>) 7.8-8.0 (m, 3<i>H</i>) (3 x Ar<b><i>H</i></b>), 8.3 (d, 1<i>H</i>)(Ar<b><i>H</i></b>), 12.65 (s, 1<i>H</i>)(CON<b><i>H</i></b>); m/z (M+H)^+. 282, 100% purity.

0274A stirred mixture of 4-(3-amino-4-methoxybenzyl)-2<i>H-</i>phthalazin-1-one (0.281 g, 1 mmol), succinic anhydride (0.1 g, 1 mmol) and toluene (20 ml) was heated under reflux for 10 hours, then cooled to ambient temperature. The solvent was removed <i>in vacuo,</i> the residue was diluted with water (30 ml), and the mixture was heated under reflux for 30 minutes. The resulting solid was collected by filtration from the hot mixture and crystallised from glacial acetic acid (20 ml) to give <i>N</i>-[2-methoxy-5-(4-oxo-3,4-dihydrophalazin-1-ylmethyl)phenyl]succinamic acid (0.244 g) as an off-white solid, m.pt. 247-251°C; 250MHz ¹H-nmr (d₆-DMSO) δ (ppm): 2.45-2.55 (m, 2<i>H</i>)(-CH₂C<b><i>H</i>₂</b>-) 2.6-2.7(m, 2<i>H</i>)(-C<b><i>H</i>₂</b>CH₂-), 3.8 (s, 3<i>H</i>) (-OC<b><i>H</i>₃</b>), 4.25 (s, 2<i>H</i>) ( ArC<b><i>H</i>₂</b>-), 6.9-7.1 (m, 2<i>H</i>)(2 x Ar<b><i>H</i></b>), 7.8-8.05 (m, 4<i>H</i>) (4 x Ar<b><i>H</i></b>), 8.3 (d, 1<i>H</i>)(Ar<b><i>H</i></b>), 9.1 (s, 1<i>H</i>)(chain CON<b><i>H</i></b>), 12.15 (br.s, 1<i>H</i>)(-COO<b><i>H</i></b>), 12.65 (s, 1<i>H</i>)(ring CON<b><i>H</i></b>): m/z (M+H)^+. 382, 100% purity.

0275<i>O</i>-Benzotriazol-1-yl-<i>N,N,N'N'</i>-tetramethyluronium tetrafluoroborate (0.217 g, 0.68 mmol) and diisopropylethylamine (0.148 g, 1.14 mmol) were added sequentially at ambient temperature to a stirred solution of <i>N</i>-[2-methoxy-5-(4-oxo-3,4-dihydrophalazin-1-ylmethyl)phenyl]succinamic acid (0.2 g, 0.52 mmol) in dimethylacetamide (1 ml), the mixture was stirred at ambient temperature for 3 hours, then it was allowed to stand at ambient temperature for 16 hours. The mixture was added dropwise to water (10 ml) and stirred at ambient temperature for 1 hour. The resulting solid was collected by filtration, washed with water (2 x 1 ml) and dried <i>in vacuo</i> to give 1-[2-methoxy-5-(4-oxo-3,4-dihydrophalazin-1-ylmethyl)phenyl]pyrrolidine-2,5-dione (0.15 g) as an off-white solid, m.pt. 224-228°C; 250MHz ¹H-nmr (d₆-DMSO) δ (ppm): 2.7-2.9 (m, 4<i>H</i>)(-C<b><i>H</i>₂</b>C<b><i>H</i>₂</b>-), 3.7 (s, 3<i>H</i>)(-OC<i>H</i>₃), 4.3 (s, 2<i>H</i>)(ArC<b><i>H</i>₂</b>-), 7.05-7.2 (m, 2<i>H</i>)(2 x Ar<b><i>H</i></b>), 7.4-7.5 (d, 1<i>H</i>)(Ar<b><i>H</i></b>), 7.8-8.05 (m, 3H) (3 x Ar<b><i>H</i></b>), 8.3 (d, 1<i>H</i>)(Ar<b><i>H</i></b>), 12.65 (s, 1<i>H</i>)(CON<b><i>H</i></b>); m/z (M+H)^+. 364, 100% purity.

Biological Testing

0276In order to assess the inhibitory action of the compounds, the following assay was used to determine IC₅₀ values.

0277Mammalian PARP, isolated from Hela cell nuclear extract, was incubated with Z-buffer (25mM Hepes (Sigma); 12.5 mM MgCl₂ (Sigma); 50mM 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.

0278After 10 minutes incubation at 30°C the reactions were initiated by the addition of a 10 µl reaction mixture, containing NAD (5µM), 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.

0279Following 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.

0280The 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.

0281The % enzyme activity for each compound is then calculated using the following equation: <maths id="math0001"><math display="block"><mo mathvariant="normal">%</mo><mspace width="1em" /><mi>Inhibition</mi><mo mathvariant="normal">=</mo><mn mathvariant="normal">100</mn><mo mathvariant="normal">-</mo><mfenced><mn mathvariant="normal">100</mn><mo>⁢</mo><mi mathvariant="normal">x</mi><mo>⁢</mo><mfrac><mfenced><mi>cpm of unknowns</mi><mo mathvariant="normal">-</mo><mi>mean negative cpm</mi></mfenced><mfenced><mi>mean positive cpm</mi><mo mathvariant="normal">-</mo><mi>mean negative cpm</mi></mfenced></mfrac></mfenced></math><img file="EP1501822B1_D0079.tif" /></maths>

0282IC₅₀ 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 0.01 µM. Such IC₅₀ values are used as comparative values to identify increased compound potencies.

0283For comparison, the IC₅₀ of 100 (1(2H)-phthalazinone) was determined using the above test to be 7.2 µM.

0284All the compounds of the examples have an IC₅₀ of less than 0.30 µM, and the following compounds have an IC₅₀ of less than 0.03µM: 2, 3, 5, 7, 11-13, 15, 18-20, 23-39 and 41-43.

0285The Potentiation Factor (PF₅₀) for compounds is calculated as a ratio of the IC₅₀ of control cell growth divided by the IC₅₀ 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 200 nM. The concentrations of MMS were over a range from 0 to 10 µg/ml.

0286Cell growth was assessed using the sulforhodamine B (SRB) assay (<nplcit id="ncit0024" npl-type="s"><text>Skehan, P., et al., (1990) New colorimetric cytotoxicity assay for anticancer-drug screening. J. Natal. Cancer Inst. 82, 1107-1112.</text></nplcit>). 2,000 HeLa cells were seeded into each well of a flat-bottomed 96-well microtiter plate in a volume of 100 µl 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 200 nM. 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.

0287Cells 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 medium was then removed and the cells fixed with 100µl 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 100µl of 0.4% (w/v) SRB in 1% acetic acid for 20 minutes before washing four times with 1% acetic acid. Plates were then dried for 2 hours at room temperature. The dye from the stained cells was solubilized by the addition of 100µl of 10mM Tris Base into each well. Plates were gently shaken and left at room temperature for 30 minutes before measuring the optical density at 564nM on a Microquant microtiter plate reader.

0288The PF₅₀s of the following compounds were determined, and found to be greater than, or equal to 1, when tested at 200nM: 2, 5, 11, 13 and 15-43.

0289Assessment of compound stability was made both <i>in vitro</i> microsomal and heptatocyte preparations) and <i>in vivo</i> animal models. Selected compounds were tested and were shown to exhibit beneficial pharmacokinetic profiles.