HK1105960B - Quinazolinone derivatives as parp inhibitors - Google Patents

Description

Quinazolinone derivatives as PARP inhibitors

Technical Field

The present invention relates to PARP inhibitors and provides compounds and compositions containing the disclosed compounds. Furthermore, the present invention provides methods of using the disclosed PARP inhibitors, for example, as medicaments.

Background

The ribozyme poly (ADP-ribose) polymerase-1 (PARP-1) is a member of the PARP enzyme family. This evolving family of enzymes includes PARPs such as PARP-1, PARP-2, PARP-3 and Vault-PARP, and Tankyrase (TANKs); such as TANK-1, TANK-2 and TANK-3. PARP is also known as poly (adenosine 5' -diphospho ribose) polymerase or PARS (poly (ADP-ribose) synthetase).

PARP-1 is a major nuclear protein of 116kDa, consisting of three domains: an N-terminal DNA binding domain comprising two zinc fingers, a self-modifying domain and a C-terminal catalytic domain. It is present almost entirely in eukaryotic cells. The enzyme synthesizes poly (ADP-Ribose), a branched polymer that may consist of more than 200 ADP-ribose units. The protein acceptor of poly (ADP-ribose) is directly or indirectly involved in maintaining DNA integrity. They include histones, topoisomerases, DNA and RNA polymerases, DNA ligases and Ca²⁺-and Mg²⁺-dependent endonucleases. PARP proteins are expressed at high levels in many tissues, most notably in the immune system, heart, brain and microbial lineage cells. Under normal physiological conditions, there is minimal PARP activity. However, DNA damage leads to up to 500-fold immediate activation of PARP.

TANKs (TANKs) are identified as components of the human telomeric complex. They have also been thought to play a role in vesicle trafficking and may also serve as scaffolds for proteins involved in various other cellular processes. Telomeres, which are essential for chromosome maintenance and stability, are maintained by telomerase, a specific reverse transcriptase. TANKs are (ADP-ribose) transferases with some characteristics of signaling and cytoskeletal proteins. They contain a PARP domain (catalyzing poly-ADP-ribosylation of substrate proteins), a sterile alpha motif (shared by certain signaling molecules) and an ANK domain (containing 24 ankyrin repeats homologous to the cytoskeletal protein ankyrin). The ANK domain interacts with telomeric protein, telomere repeat binding factor-1 (TRF-1). These proteins are therefore called TRF 1-interacting, ankyrin-related ADP-ribose polymerases (TANKs).

One of the more specific functions of TANK is ADP-ribosylation of TRF-1. Human telomere function requires two telomere-specific DNA binding proteins TRF-1 and TRF-2. TRF-2 protects the ends of chromosomes and TRF-1 regulates telomere length. ADP-ribosylation inhibits the ability of TRF-1 to bind to telomeric DNA. This poly-ADP-ribosylation of TRF-1 releases TRF-1 from the telomeres, opens up the telomere complex, and allows access to telomerase. Thus, TANK functions as a positive regulator of telomere length, allowing telomere elongation by telomerase.

Among the many functions attributed to PARP, especially PARP-1, the promotion of DNA repair by ADP-ribosylation is its major roleThus regulating many DNA repair proteins. As a result of PARP activation, NAD⁺The level was significantly reduced. Extensive PARP activation leads to NAD in cells that suffer from extensive DNA damage⁺Is severely worn out. The short half-life of poly (ADP-ribose) results in a rapid turnover rate. Once poly (ADP-ribose) is formed, it is rapidly degraded by constitutively activated poly (ADP-ribose) active glycohydrolases (PARG), as well as phosphodiesterases and (ADP-ribose) proteolytic enzymes. PARP and PARG form a cycle, converting large amounts of NAD⁺To ADP-ribose. Over-stimulation of PARP can result in NAD in less than 1 hour⁺And ATP drops to less than 20% of normal levels. Such a situation is particularly harmful during ischemia, when the lack of oxygen has already significantly threatened cellular energy output. Subsequent generation of free radicals during reperfusion is believed to be the primary cause of tissue damage. A partial ATP drop, which is typically present in many organs during ischemia and reperfusion, is associated with NAD due to poly (ADP-ribose) turnover⁺Losses are associated. Thus, inhibition of PARP or PARG is expected to preserve cellular energy levels, thereby enhancing survival of ischemic tissue after injury.

Poly (ADP-ribose) synthesis also includes the inducible expression of many genes essential for inflammatory response. PARP inhibitors inhibit the production of Inducible Nitric Oxide Synthase (iNOS) in macrophages, and P-type selectin and intracellular adhesion molecule-1 (ICAM-1) in endothelial cells. Such activity is responsible for the strong anti-inflammatory effects exhibited by PARP inhibitors. PARP inhibition reduces necrosis by preventing migration and infiltration of neutrophils to the damaged tissue.

PARP is activated by damaged DNA fragments and, once activated, catalyzes the attachment of up to 100 ADP-ribose units to a variety of nuclear proteins, including histones and PARP itself. Extensive activation of PARP during major cellular stress can rapidly lead to cell damage or death by loss of energy storage. Since one NAD is produced per second⁺The molecule consumes four ATP molecules, NAD⁺Are activated and exhausted by a large amount of PARP, and efforts are being made to resynthesize NAD⁺ATP may also become depleted.

PARP activation has been reported to play an important role in both NMDA-and NO-induced neurotoxicity. This has been demonstrated in cortical cultures and hippocampal slices, where toxicity prevention is directly related to PARP inhibitory potency. The potential role of PARP inhibitors in the treatment of neurodegenerative diseases and head trauma is therefore recognized, however the exact mechanism of this effect has not yet been elucidated.

Similarly, a single injection of PARP inhibitor has been shown to reduce infarct size in rabbits resulting from ischemia and reperfusion of the heart or skeletal muscle. In these studies, a single injection of 3-amino-benzamide (10mg/kg) one minute prior to closure or one minute prior to reperfusion resulted in a similar reduction in infarct size in the heart (32-42%), 1, 5-dihydroxyisoquinoline (1mg/kg), another PARP inhibitor, reducing infarct size to a comparable extent (38-48%). From these results it is reasonable to speculate that PARP inhibitors could rescue cardiac ischemia or muscle tissue reperfusion injury in advance.

PARP activation can also be used to determine damage following neurotoxic insult caused by exposure to any of the following inducers, such as glutamate (via NMDA receptor stimulation), reactive oxygen intermediates, amyloid beta-protein, N-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) or its active metabolite N-methyl-4-phenylpyridine (MPP)⁺) Which are involved in pathological conditions such as stroke, alzheimer's disease and parkinson's disease. Other studies have continued to explore the role of PARP activation in cerebellar granule cells in vitro and in MPTP neurotoxicity. Excessive neural exposure to glutamate, most often as a result of stroke or other neurodegenerative processes, acts as the leading central nervous system neurotransmitter at the N-methyl D-aspartate (NMDA) receptor and other subtype receptors. During ischemic brain injury such as stroke or heart attack, oxygen deficient neurons release glutamate in large amounts. This excess glutamate release in turn leads to N-methyl-D-aspartate (NMDA),Hyperstimulation (excitotoxicity) of AMPA, kainate and MGR receptors, opening ion channels and allowing uncontrolled ion flow (e.g., Ca)²⁺And Na⁺Flow into cells and K⁺Efflux out of the cell) leading to over-stimulation of neurons. Over-stimulated neurons secrete more glutamate, producing a feedback loop or domino effect that ultimately leads to cell damage or death through the production of proteases, lipases, and free radicals. Excessive activation of glutamate receptors has been implicated in a variety of neurological diseases and disorders, including epilepsy, stroke, alzheimer's disease, parkinson's disease, Amyotrophic Lateral Sclerosis (ALS), huntington's chorea, schizophrenia, chronic pain, ischemia and tissue hypoxia, hypoglycemia, ischemia, trauma, and neuronal loss following nerve injury. Glutamate exposure and stimulation have also been implicated as a basis for obsessive-compulsive disorders, particularly drug dependence. Evidence includes findings in many animal species, as well as in cerebral cortical culture media treated with glutamate or NMDA, that glutamate receptor antagonists (i.e., compounds that block glutamate binding to or activating receptors) block nervous system damage following vascular stroke. Attempts to prevent excitotoxicity by blocking NMDA, AMPA, kainate and MGR receptors have proven difficult because each receptor has multiple sites to which glutamate can bind, and it has been difficult to find effective mixed or universal antagonists to prevent glutamate binding to all receptors and to test this theory. Moreover, many compositions that effectively block the receptor are toxic to animals. Therefore, there is currently no known effective treatment for glutamate abnormalities.

Stimulation of NMDA receptors by glutamate, for example, activates the enzyme neuronal nitric oxide synthase (nNOS), leading to the formation of Nitric Oxide (NO), which also modulates neurotoxicity. NMDA neurotoxicity can be prevented by use of Nitric Oxide Synthase (NOS) inhibitors or by targeting in vitro disruptions of the nNOS gene.

Another use of PARP inhibitors is in the treatment of peripheral nerve injury and the resulting pathological pain syndrome known as neuropathic pain, such as pain induced by Chronic Constriction Injury (CCI) of the common sciatic nerve, where transsynaptic alterations of the spinal cord dorsal horn are characterized by the occurrence of hyperpigmentation of the cytoplasm and nucleoplasm (so-called "black neurons").

Evidence also exists for the use of PARP inhibitors for the treatment of inflammatory bowel diseases, such as colitis. Specifically, colitis was induced in rats by intraluminal administration of the hapten trinitrobenzene sulfonic acid in 50% ethanol. The treated rats received 3-aminobenzamide, an inhibitor of the specific PARP activity. Inhibition of PARP activity reduces inflammatory responses, restoring morphology and the energy status of the distal colon.

Further evidence suggests that PARP inhibitors are useful in the treatment of arthritis. Further PARP inhibitors appear to be useful in the treatment of diabetes. PARP inhibitors have been shown to be useful in the treatment of endotoxic shock or septic shock.

PARP inhibitors have also been used to prolong lifespan and extend cellular proliferative capacity, including treatment of diseases such as skin aging, alzheimer's disease, atherosclerosis, osteoarthritis, osteoporosis, muscular dystrophy, degenerative diseases of skeletal muscle including repetitive aging, age-related muscle degeneration, immune aging, AIDS, and other immune aging diseases; and altering gene expression of aging cells.

PARP inhibitors, such as 3-aminobenzamide, are also known to affect overall DNA repair, e.g., in response to hydrogen peroxide or ionizing radiation.

The key role of PARP in DNA strand break repair is well established, especially when breaks are initiated directly by ionizing radiation, or indirectly after methylation agents, topoisomerase I inhibitors, and other chemotherapeutic agents such as cisplatin and bleomycin induce DNA damaging enzyme repair. Various studies using "knockout" mice, trans-dominant inhibition models (overexpression of DNA-binding domains), antisense, and small molecular weight inhibitors have shown a role for PARP in repair and cell survival following induction of DNA damage. Inhibition of PARP enzyme activity may lead to enhanced sensitivity of tumor cells to DNA damaging treatments.

PARP inhibitors have been reported to be effective in radiosensitizing (hypoxic) tumor cells and to prevent tumor cell recovery in potentially lethal and sublethal damage of DNA following radiation therapy, presumably due to their ability to prevent DNA strand break recombination and due to the influence on several DNA damage signaling pathways.

PARP inhibitors have been used to treat cancer. In addition, U.S. Pat. No. 5,177,075 discusses several isoquinolines for enhancing the lethal effects of ionizing radiation or chemotherapeutic agents on tumor cells. Weltin et al in "Effect of Poly (ADP-ribose) Polymerase inhibitor 6- (5-Phenanthridinone) on tumor cells in culture" ("Effect of 6(5-Phenanthridinone), anihibitor of Poly (ADP-ribose) Polymerase, on filtered TumorCells"), Oncol. Res., 6: inhibition of PARP activity, reduction of tumor cell proliferation, and significant synergy when tumor cells are co-treated with alkylating drugs are discussed in 9,399-403 (1994).

A review of the state of the art is given by Li and Zhang in IDrugs 2001, 4 (7): 804-812, Ame et al in Bioassays 2004, 26: 882-883 and Nguewa et al in Biophysic & Molecular Biology 2005, 88: 143, 172.

These continue to be a need for potent and potent PARP inhibitors, more particularly PARP-1 inhibitors, which produce minimal side effects. The present invention provides compounds, compositions and methods for inhibiting PARP activity for treating cancer and/or preventing cell, tissue and/or organ damage due to cell damage or death, for example, by necrosis or apoptosis. The compounds and compositions of the invention are particularly useful for enhancing the effects of chemotherapy and radiotherapy, where the primary effect of the treatment is to cause DNA damage to the target cells.

Prior Art

GB1062357, published on 3/22 in 1967, discloses quinazolinone derivatives having antihypertensive effects.

Published in 1973 at 20 th 6 th DE2258561 discloses substituted pyridone derivatives having antihypertensive effect.

EP13612, published 11.11.1983, discloses substituted piperidinylalkylquinazoline derivatives. The compounds described are 5-hydroxytryptamine-antagonists.

EP669919, published on 9/6/1994, discloses dimethylbenzofuran and dimethylbenzopyran as 5-HT3 antagonists. More particularly, compounds 8, 4, 5, 10, 11, 12, 13, 15, 16, 17 and 14 of the present application are disclosed. US5374637 published in 20 mesh, 12 months, 1994 discloses benzamide derivatives. The disclosed compounds have kinetic stimulatory properties of the gastrointestinal tract. Specifically disclosed are compounds 8, 6 and 9 of the present application. EP885190, published in 1998 at 23.12.1998, is a1, 4-disubstituted piperidine derivative having gastric motility properties. Specifically disclosed is compound 7 of the present application.

EP1036073, published 17.6.1999, discloses substituted quinazolinedione derivatives. The compounds described have basic relaxing properties. EP1355888, published on 6/20/2002, discloses quinazolinone derivatives as PARP inhibitors.

Description of the invention

The invention relates to compounds of formula (I)

The N-oxide forms, the pharmaceutically acceptable addition salts and the stereochemically isomeric forms thereof, wherein the dashed line represents an optional bond;

x is > N-or > CH-;

is-N-C (O) -or-N ═ CR⁴-, wherein R⁴Is a hydroxyl group;

l is a direct bond or is selected from the group consisting of-C (O) -, -C (O) -NH-, -C (O) -C_1-6Alkanediyl-, -C (O) -O-C_1-6alkanediyl-or-C_1-6Alkanediyl-divalent radicals;

R¹is hydrogen, halogen, C_1-6Alkoxy or C_1-6An alkyl group;

R²is hydrogen, hydroxy, C_1-6Alkoxy or aminocarbonyl;

when X is substituted by R²Substituted, R²Together with-L-Z, form a divalent radical of formula

-C(O)-NH-CH₂-NR¹⁰- (a-1)

Wherein R is¹⁰Is phenyl;

R³is hydrogen, or C_1-6An alkoxy group;

z is amino, cyano or a group selected from

Wherein each R⁵、R⁶、R⁷And R⁸Independently selected from hydrogen, halogen, amino, C_1-6Alkyl or C_1-6An alkoxy group; or R⁷And R⁸May together form a divalent group of the formula:

-CH₂-CR⁹ ₂-O- (c-1)、

-(CH₂)₃-O- (c-2)、

-O-(CH₂)₂-O- (c-3) or

-CH＝CH-CH＝CH- (c-4)

Wherein each R⁹Independently selected from hydrogen or C_1-6An alkyl group;

provided that

When X is > N-, then Z is not a group (b-2), and

when X is > CH-and L is-C (O) -NH-or-C (O) -O-C_1-6When alkanediyl-Z is a group (b-2), and R⁷And R⁸When taken together to form a divalent group of formula (c-1), (c-2) or (c-3), then R⁵Is not chlorine.

The compounds of formula (I) may also exist in their tautomeric form. Such forms, although not explicitly shown in the above formula, are included within the scope of the present invention.

Many of the terms defined above and used hereinafter are to be interpreted as follows. These terms are sometimes used as such or in combination terms.

Halogen as used in the foregoing definitions and hereinafter is typically fluorine, chlorine, bromine and iodine; c_1-6Alkyl is defined as straight and branched chain saturated hydrocarbon radicals containing from 1 to 6 carbon atoms such as methyl, ethyl, propyl, butyl, pentyl, hexyl, 1-methylethyl, 2-methylpropyl, 2-methyl-butyl, 2-methylpentyl and the like; c_1-6Alkanediyl is defined as a straight-chain and branched divalent saturated hydrocarbon radical containing from 1 to 6 carbon atoms, such as methylene, 1, 2-ethanediyl, 1, 3-propanediyl, 1, 4-butanediyl, 1, 5-pentanediyl, 1, 6-hexanediyl and branched isomers thereof, such as 2-methylpentanediyl, 3-methylpentanediyl, 2-dimethylbutanediyl, 2, 3-dimethylbutanediyl and the like.

The term "pharmaceutically acceptable salt" means a pharmaceutically acceptable acid or base addition salt. The pharmaceutically acceptable acid or base addition salts mentioned above are meant to be the forms which the compounds of formula (I) are able to form, comprising therapeutically active non-toxic acid and non-toxic base addition salts. The compounds of formula (I) having basic properties can be converted into their pharmaceutically acceptable acid addition salts by treatment with a suitable acid. Suitable acids include, for example, inorganic acids, such as hydrohalic acids, e.g., hydrochloric or hydrobromic acid; sulfuric acid; nitric acid; acids such as phosphoric acid; or organic acids such as acetic, propionic, glycolic, lactic, pyruvic, oxalic, malonic, succinic (i.e., butanedioic acid), maleic, fumaric, malic, tartaric, citric, methanesulfonic, ethanesulfonic, benzenesulfonic, p-toluenesulfonic, cyclohexylsulfamic, salicylic, p-aminosalicylic, pamoic and the like acids.

The compounds of formula (I) having acidic properties can be converted into their pharmaceutically acceptable base addition salts by treating the acid form with a suitable organic or inorganic base. Suitable base salt forms include, for example, ammonium salts, alkali and alkaline earth metal salts such as lithium, sodium, potassium, magnesium, calcium salts and the like, salts with organic bases such as benzathine, N-methyl-D-glucamine, hydrabamine, and salts with amino acids such as arginine, lysine and the like.

The term acid or base addition salt also encompasses hydrates and solvent addition forms which the compounds of formula (I) are able to form. Examples of such forms are hydrates, alcoholates and the like. The term stereochemically isomeric forms of a compound of formula (I), as used hereinbefore, defines all the possible compounds bonded by the same atoms in the same order but having different three-dimensional structures and not being interchangeable, which the compound of formula (I) may possess. Unless otherwise mentioned or indicated, the chemical designation of a compound encompasses the mixture of all possible stereochemically isomeric forms which said compound may possess. The mixture may contain all diastereomers and/or enantiomers of the basic structure of the compound. All stereochemically isomeric forms of the compounds of formula (I) both in pure form and in admixture with each other are intended to be embraced within the scope of the present invention.

The N-oxide forms of the compounds of formula (I) are meant to comprise those compounds of formula (I) wherein one or several nitrogen atoms are oxidized to the so-called N-oxides, in particular those N-oxides wherein one or more piperidine-or piperazine nitrogens are N-oxidized.

Whenever the term "compound of formula (I)" is used hereinafter, it is meant to also include the N-oxide forms, the pharmaceutically acceptable acid or base addition salts and all stereoisomeric forms.

GB1062357 discloses quinazolinone derivatives having antihypertensive effect. DE2258561 discloses substituted pyridone derivatives having antihypertensive effect. EP13612 discloses substituted piperidinylalkylquinazoline derivatives which are 5-hydroxytryptamine-antagonists. EP669919 discloses as 5-HT₃Antagonists of the class of dimethylbenzofurans and dimethylbenzopyrans. US5374637 discloses benzamide derivatives having gastrointestinal motility stimulating properties. EP885190 discloses 1, 4-disubstituted piperidine derivatives having gastric motility properties. EP1036073 discloses substituted quinazolinedione derivatives having basic relaxation properties.

Unexpectedly, it has been found that the compounds of the present invention exhibit PARP inhibitory activity.

A first group of interesting compounds consists of those compounds of formula (I) wherein one or more of the following restrictions apply:

a) each X is > N-;

b) l is selected from the group consisting of-C (O) -, -C (O) -NH-, -C (O) -C_1-6Alkanediyl-, -C (O) -O-C_1-6alkanediyl-or-C_1-6Alkanediyl-divalent radicals;

c)R¹is hydrogen;

d)R²is hydroxy, C_1-6Alkoxy or aminocarbonyl;

e) z is amino, cyano or a group selected from (b-1), (b-3), (b-4), (b-5), (b-6), (b-7), (b-8) or (b-9);

f) each R⁵And R⁶Independently selected from hydrogen or amino.

A second group of interesting compounds consists of those compounds of formula (I) wherein one or more of the following restrictions apply:

a) x is > CH-;

b) l is a direct bond or is selected from the group consisting of-C (O) -, -NH-, -C (O) -C_1-6alkanediyl-or-C_1-6Alkanediyl-divalent radicals;

c) z is amino, cyano or a group selected from (b-1), (b-3), (b-4), (b-5), (b-6), (b-7), (b-8) or (b-9);

d) each R⁵Independently selected from hydrogen, fluorine, iodine, bromine, amino, C_1-6Alkyl or C_1-6An alkoxy group;

e) each R⁶Independently selected from hydrogen, chlorine, iodine, bromine, amino, C_1-6Alkyl or C_1-6An alkoxy group.

A third group of interesting compounds consists of those compounds of formula (I) wherein one or more of the following restrictions apply:

a) l is a direct bond or a divalent group selected from-C (O) -or-C (O) -NH-;

b)R²is hydrogen, hydroxy or C_1-6An alkoxy group;

c) z is a group selected from (b-2), (b-3), (b-4), (b-5), (b-6), (b-7), (b-8) or (b-9);

d) each R⁵、R⁶、R⁷And R⁸Independently selected from hydrogen, halogen, C_1-6Alkyl or C_1-6An alkoxy group; or

e)R⁷And R⁸May together form a divalent group of formula (c-1) or (c-4).

A fourth group of compounds of interest consists of those of formula (I), wherein one or more of the following restrictions apply:

a) l is a direct bond or is selected from-C (O) -, -C (O) -NH-or-C (O) -O-C_1-6Alkanediyl-divalent radicals;

b)R²is hydrogen, hydroxy or C_1-6An alkoxy group;

c) z is a group selected from (b-2), (b-3), (b-4), (b-5), (b-6), (b-7), (b-8) or (b-9);

d) each R⁵、R⁶、R⁷And R⁸Independently selected from hydrogen, halogen, amino, C_1-6Alkyl or C_1-6An alkoxy group; or

e)R⁷And R⁸May together form a divalent group of formula (c-1), (c-2), (c-3) or (c-4).

A fifth group of compounds of interest consists of those of formula (I), wherein one or more of the following restrictions apply:

a) l is a direct bond;

b)R¹is hydrogen, halogen or C_1-6An alkyl group;

c)R²is hydrogen;

d)R³is hydrogen;

e) z is a group selected from (b-5) or (b-7);

f) each R⁵Independently selected from hydrogen or halogen.

A preferred group of compounds consists of those of formula (I) wherein L is a direct bond or is selected from the group consisting of-C (O) -, -C (O) -NH-or-C (O) -O-C_1-6Alkanediyl-divalent radicals; r²Is hydrogen, hydroxy or C_1-6Alkoxy, Z is a group selected from (b-2), (b-3), (b-4), (b-5), (b-6), (b-7), (b-8) or (b-9); each R⁵、R⁶、R⁷And R⁸Independently selected from hydrogen, halogen, amino, C_1-6Alkyl or C_1-6An alkoxy group; or R⁷And R⁸May together form a divalent group of formula (c-1), (c-2), (c-3) or (c-4).

A more preferred group of compounds consists of those compounds of formula (I) wherein L is a direct bond; r¹Is hydrogen, halogen orC_1-6An alkyl group; r²Is hydrogen; r³Is hydrogen; z is a group selected from (b-5) or (b-7); and each R⁵Independently selected from hydrogen or halogen.

The most preferred compounds are compounds No.35, No.36, No.39, No.1 and No. 43.

Compound 35 Compound 36

Compound 39 Compound 1

Compound 43

The compounds of formula (I) may be prepared according to the general methods described in EP1036073, EP885190, US5374637, EP669919 and EP 13612. The starting materials and some intermediates are known compounds, are commercially available or can be prepared according to conventional reaction methods generally known in the art.

Some of the preparation methods will be described in more detail below. Other methods of obtaining the final compound of formula (I) are described in the examples.

Compounds of formula (I) may be prepared by reacting an intermediate of formula (II) with an intermediate of formula (III) wherein W is an appropriate leaving group, for example halogen, such as fluorine, chlorine, bromine or iodine, or a sulfonyloxy group such as methylsulfonyloxy, 4-methylphenylsulfonyloxyAnd the like. The reaction may be carried out in a reaction-inert solvent such as an alcohol, e.g., methanol, ethanol, 2-methoxy-ethanol, propanol, butanol, etc.; ethers, e.g. 4, 4-bisAlkyl, 1' -oxybispropane, and the like; or ketones such as 4-methyl-2-pentanone, N-dimethylformamide, nitrobenzene, and the like. The addition of a suitable base, such as an alkali or alkaline earth metal carbonate or bicarbonate, e.g. triethylamine or sodium carbonate, may be used to capture the acid released during the reaction. A small amount of a suitable metal iodide such as sodium iodide or potassium iodide may be added to facilitate the reaction. Agitation can enhance the reaction rate. The reaction is conveniently carried out at from room temperature to the reflux temperature of the reaction mixture, and if desired, under increased pressure.

The compounds of formula (I) may also be interconverted by reactions or functional group transformations known in the art. Some such transformations have been described above. Other examples are the hydrolysis of carboxylic esters to the corresponding carboxylic acids or alcohols; hydrolysis of the amide to the corresponding carboxylic acid or amine; hydrolysis of the nitrile to the corresponding amide; the amino group on the imidazole or phenyl group may be replaced by hydrogen by a diazotisation reaction known in the art, and subsequent replacement of the diazo group by hydrogen; alcohols can be converted into esters and ethers; primary amines can be converted to secondary or tertiary amines; the double bonds may be hydrogenated to the corresponding single bonds; the iodine group on the phenyl group can be converted to an ester group by carbon monoxide insertion in the presence of a suitable palladium catalyst.

The invention also relates to a compound of formula (I) as defined above for use as a medicament.

The compounds of the present invention have PARP inhibitory properties as can be seen in the experimental section below.

The term "PARP" as used herein means a protein having poly-ADP-ribosylation activity. Within the meaning of this term, PARP includes all proteins encoded by the PARP gene, mutants thereof and optional fragment proteins thereof. Furthermore, as used herein, the term "PARP" includes PARP analogs, homologs, and other animal analogs.

The term "PARP" includes, but is not limited to, PARP-1. Within the meaning of this term, PARP-2, PARP-3, vacuum-PARP (PARP-4), PARP-7(TiPARP), PARP-8, PARP-9(Bal), PARP-10, PARP-11, PARP-12, PARP-13, PARP-14, PARP-15, PARP-16, TANK-1, TANK-2 and TANK-3 may be included.

Compounds that inhibit both PARP-1 and tankyrase can have advantageous properties in that they have enhanced growth inhibitory activity in cancer cells.

The invention also contemplates the use of a compound for the manufacture of a medicament for treating any disease or condition in an animal as described herein, wherein said compound is a compound of formula (I),

the N-oxide forms, the pharmaceutically acceptable addition salts and the stereo-chemical isomeric forms thereof, wherein the dotted line represents an optional bond;

x is > N-or > CH-;

is-N-C (O) -or-N ═ CR⁴-, wherein R⁴Is a hydroxyl group;

l is a direct bond or is selected from the group consisting of-C (O) -, -C (O) -NH-, -C (O) -C_1-6Alkanediyl-, -C (O) -O-C_1-6alkanediyl-or-C_1-6Alkanediyl-divalent radicals;

R¹is hydrogen or halogen、C_1-6Alkoxy or C_1-6An alkyl group;

R²is hydrogen, hydroxy, C_1-6Alkoxy or aminocarbonyl;

when X is substituted by R²Substituted, R²Together with-1-Z, form a divalent radical of formula

-C(O)-NH-CH₂-NR¹⁰- (a-1)

Wherein R is¹⁰Is phenyl;

R³is hydrogen, or C_1-6An alkoxy group;

z is amino, cyano or a group selected from the following formulae:

wherein each R⁵、R⁶、R⁷And R⁸Independently selected from hydrogen, halogen, amino, C_1-6Alkyl or C_1-6An alkoxy group; or

R⁷And R⁸May together form a divalent radical of the formula

-CH₂-CR⁹ ₂-O- (c-1)，

-(CH₂)₃-O- (c-2)，

-O-(CH₂)₂-O- (c-3) or

-CH＝CH-CH＝CH- (c-4)

Wherein each R⁹Is independently selected fromHydrogen or C_1-6An alkyl group.

Furthermore, the present invention relates to the use of a compound as described above for the preparation of a medicament for the treatment of a condition mediated by PARP.

In particular, the present invention relates to the use of a compound as described above for the preparation of a medicament for the treatment of a condition mediated by PARP.

Compounds that inhibit both PARP-1 and TANK-2 have advantageous properties in that they have enhanced growth inhibitory activity in cancer cells.

In view of their PARP binding properties, the compounds of the invention may be used as reference compounds or tracer compounds, wherein one atom of the molecule may be substituted, for example by a radioactive isotope.

To prepare the pharmaceutical compositions of this invention, an effective amount of the particular compound, in base or acid addition salt form, as the active ingredient is combined in intimate admixture with a pharmaceutically acceptable carrier, which carrier may take a wide variety of forms depending on the form of preparation desired for administration. These pharmaceutical compositions are desirably in unit dosage form, preferably suitable for administration orally, rectally, transdermally or by parenteral injection. For example, in the case of oral liquid preparations such as suspensions, syrups, elixirs and solutions in the preparation of oral dosage form compositions, any of the usual pharmaceutical media may be employed such as water, glycols, oils, alcohols and the like; or in the case of powders, pills, capsules and tablets, solid carriers such as starches, sugars, kaolin, lubricants, binders, disintegrating agents and the like. Because of their ease of administration, tablets and capsules represent the most advantageous oral dosage unit form, obviously employing solid pharmaceutical carriers. For parenteral compositions, the carrier will typically comprise at least a major proportion of sterile water, although other ingredients, such as those to aid solubility, may also be included. Injectable solutions, for example, may be prepared in which the carrier comprises saline solution, glucose solution or a mixture of saline and glucose solution. Injectable suspensions may also be prepared in which case appropriate liquid carriers, suspending agents and the like may be employed. In compositions suitable for transdermal administration, the carrier optionally comprises a penetration enhancer and/or a suitable moisturizer, optionally mixed with a minor proportion of a suitable additive of any nature, wherein the additive does not cause significant deleterious effects to the skin. The additives may facilitate application to the skin, and/or may aid in the preparation of the desired composition. These compositions can be administered in various ways, for example as transdermal patches, as spot-on, as an ointment. It is particularly advantageous to formulate the pharmaceutical compositions in the aforementioned dosage unit forms for ease of administration and uniformity of dosage. Dosage unit form as used in the specification and claims herein refers to physically discrete units suitable as unitary dosages, each unit containing a predetermined quantity of active ingredient calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. Examples of such dosage unit forms are tablets (including scored or coated tablets), capsules, pills, powder packets, wafers, injectable solutions or suspensions, teaspoonfuls, tablespoonfuls and the like, and segregated multiples thereof.

The compounds of the present invention are capable of treating or preventing tissue damage resulting from cell damage or death caused by necrosis or apoptosis; can improve nervous system or cardiovascular tissue damage, including following focal ischemia, myocardial infarction and reperfusion injury; can treat various diseases and conditions caused or exacerbated by PARP activity; can prolong or increase life span or cell proliferation ability; can alter gene expression in senescent cells; can radiosensitize and/or chemosensitize cells. In general, inhibition of PARP activity blocks cellular energy loss, preventing irreversible depolarization of neurons in nervous system cells, thus providing neuroprotection.

For the foregoing reasons, the present invention further relates to a method of administering a therapeutically effective amount of a compound as defined above in an amount sufficient to inhibit PARP activity to treat or prevent tissue damage due to cell damage or death resulting from necrosis or apoptosis to produce neuronal activity not mediated by NMDA toxicity to produce neuronal activity mediated by NMDA toxicity to treat tissue damage of the nervous system, neurological disorders and neurodegenerative diseases resulting from ischemia and reperfusion injury; to prevent or treat stroke; to treat or prevent cardiovascular disorders; to treat other conditions and/or diseases such as age-related muscle degeneration, AIDS and other immune aging diseases, inflammation, gout, arthritis, atherosclerosis, cachexia, cancer, degenerative diseases of skeletal muscle including replicative aging, diabetes, head trauma, inflammatory bowel disease (e.g., colitis and Crohn's disease), muscular dystrophy, osteoarthritis, osteoporosis, chronic and/or acute pain (e.g., neuropathic pain), renal failure, retinal ischemia, septic shock (e.g., endotoxic shock), and skin aging, prolonging life and cell proliferative capacity; altering gene expression in senescent cells; chemosensitize and/or radiosensitize (hypoxic) tumor cells. The invention also relates to the treatment of diseases and conditions in animals comprising administering to said animals a therapeutically effective amount of a compound identified above.

In particular, the invention relates to a method of treating, preventing or inhibiting a neurological disorder in an animal comprising administering to said animal a therapeutically effective amount of a compound identified above. The neurological condition is selected from the group consisting of peripheral neurological disease resulting from physical injury or disease state, traumatic brain injury, physical injury to the spinal cord, stroke associated with brain injury, focal ischemia, global ischemia, reperfusion injury, demyelinating disease and neurological conditions associated with neurodegeneration.

The present invention also contemplates the use of a compound of formula (I) to inhibit PARP activity for treating, preventing or inhibiting tissue damage resulting from cell damage or death due to necrosis or apoptosis, for treating, preventing or inhibiting a neurological disorder in an animal.

The term "preventing neurodegeneration" includes the ability to prevent neurodegeneration in a patient newly diagnosed with a neurodegenerative disease or at risk of developing a new degenerative disease, as well as preventing further neurodegeneration in a patient who has suffered from or has symptoms of a neurodegenerative disease.

The term "treatment" as used herein includes any treatment of a disease and/or condition in an animal, particularly a human, including: (i) preventing the occurrence of a disease and/or condition in a subject predisposed to having the disease and/or condition but not yet diagnosed as having the disease; (ii) inhibiting the disease and/or disorder, i.e., arresting its development; (iii) alleviating the disease and/or condition, i.e., causing regression of the disease and/or condition.

The term "radiosensitizer," as used herein, is defined as a molecule, preferably a low molecular weight molecule, administered to an animal in a therapeutically effective amount to increase the sensitivity of cells to ionizing radiation and/or to facilitate treatment of diseases treatable by ionizing radiation. Diseases that can be treated with ionizing radiation include neoplastic diseases, benign and malignant tumors, and cancer cells. Ionizing radiation treatment of other diseases not listed herein is also contemplated by the present invention.

The term "chemosensitizer", as used herein, is defined as a molecule, preferably a low molecular weight molecule, administered to an animal in a therapeutically effective amount to increase the sensitivity of cells to chemotherapy and/or to promote the treatment of diseases treatable with chemotherapy. Diseases treatable with chemotherapy include neoplastic diseases, benign and malignant tumors, and cancer cells. Other chemotherapeutic treatments of diseases not listed herein are contemplated by the present invention.

The compounds, compositions and methods of the invention are particularly useful for treating or preventing tissue damage resulting from cell damage or death due to necrosis or apoptosis.

The compounds of the invention may be "anti-cancer agents", which term also includes "anti-tumor cell growth agents" and "anti-neoplastic agents". For example, the methods of the invention are useful for treating cancer, and tumor cells that are chemosensitize and/or radiosensitize the cancer, e.g., ACTH-producing tumors, acute lymphoid leukemia, acute non-lymphoid leukemia, renal cortex cancer, bladder cancer, brain cancer, breast cancer, cervical cancer, chronic lymphoid leukemia, chronic myeloid leukemia, colorectal cancer, cutaneous T-cell lymphoma, endometrial cancer, esophageal cancer, Ewing's sarcoma, gallbladder cancer, hairy cell leukemia, head and neck cancer, Hodgkin's lymphoma, Kaposi's sarcoma, kidney cancer, liver cancer, lung cancer (small and/or non-small cells), malignant peritoneal effusion, malignant pleural effusion, melanoma, mesothelioma, multiple myeloma, neuroblastoma, non-Hodgkin's lymphoma, osteosarcoma, ovarian cancer, ovarian (germ cell) cancer, cancer of the head and neck, cancer cells, cancer, Prostate cancer, pancreatic cancer, penile cancer, retinoblastoma, skin cancer, soft tissue sarcoma, squamous cell carcinoma, gastric cancer, testicular cancer, thyroid cancer, embryotrophoblastic tumors, uterine cancer, vaginal cancer, vulvar cancer, and Wilm's tumor.

Thus, the compounds of the present invention may be used as "radiosensitizers" and/or "chemosensitizers".

Radiosensitizers are known to increase the sensitivity of cancer cells to the toxic effects of ionizing radiation. Several mechanisms of the mode of action of radiosensitizers have been proposed in the literature, including: hypoxic cell radiosensitizers (e.g., 2-nitroimidazole compounds and benzotriazine dioxide compounds) mimic oxygen, or act as bioreductive agents under tissue hypoxia; non-hypoxic cell radiosensitizers (e.g., halogenated pyrimidines) can be analogs of DNA bases and preferentially bind to the DNA of cancer cells, thus promoting radiation-induced DNA molecule fragmentation and/or preventing normal DNA repair mechanisms; various other possible mechanisms of action of radiosensitizers in the treatment of disease have been proposed.

Many current cancer treatment regimens employ radiosensitizers in combination with x-ray radiation. Examples of x-ray activated radiosensitizers include, but are not limited to, the following: metronidazole, misonidazole, desmethonidazole (demethyl isoniazole), pimonidazole, etanidazole, nimorazole, mitomycin C, RSU 1069, SR 4233, EO9, RB 6145, niacinamide, 5-bromodeoxyuridine (BUdR), 5-iododeoxyuridine (IUdR), bromodeoxycytidine, fluorodeoxyuridine (FudR), hydroxyurea, cisplatin and therapeutically effective analogs and derivatives of the drugs.

Photodynamic therapy (PDT) of cancer employs visible light as a radiation activator of a sensitizer. Examples of photodynamic radiosensitizers include, but are not limited to, the following: hematoporphyrin derivatives, photosensitizers, benzoporphyrin derivatives, tin protoporphyrin, Pheoborbide-a, bacteriochlorophyll-a, naphthalocyanine, phthalocyanines, zinc phthalocyanines, and therapeutically effective analogs and derivatives of said drugs.

The radiosensitizer can be administered in combination with one or more therapeutically effective amounts of other compounds including, but not limited to: a compound that promotes binding of the radiosensitizer to the target cell; compounds that control the flow of therapeutic agents, nutrients, and/or oxygen to target cells; chemotherapeutic agents that act on the tumor with or without the administration of additional radiation; or other therapeutically effective compounds for the treatment of cancer or other diseases. Examples of additional therapeutic agents that may be used in combination with radiosensitizers include, but are not limited to: 5-fluorouracil, folinic acid, 5 '-amino-5' deoxythymidine, oxygen, carbopol, erythrocyte infusions, perfluorocarbons (e.g., Fluosol10 DA), 2, 3-DPG, BW12C, calcium channel blockers, pentoxyfylline, anti-angiogenic compounds, hydralazine, and lbo. Examples of chemotherapeutic agents that may be used in combination with radiosensitizers include, but are not limited to: doxorubicin, camptothecin, carboplatin, cisplatin, daunomycin, docetaxel, doxorubicin, interferon (α, β, γ), interleukin 2, irinotecan, paclitaxel, topotecan, and therapeutically effective analogs and derivatives of said drugs.

Chemosensitizers may be administered in combination with one or more other compounds in a therapeutically effective amount, including but not limited to: a compound that promotes binding of the target cell to a chemosensitizer; compounds that control the flow of therapeutic agents, nutrients, and/or oxygen to target cells; chemotherapeutic agents that act on tumors or other therapeutically effective compounds for the treatment of cancer or other diseases. Examples of additional therapeutic agents that may be combined with chemosensitizers include, but are not limited to: methylating agents, topoisomerase I inhibitors and other chemotherapeutic agents such as cisplatin and bleomycin.

The compounds of formula (I) may also be used to detect or identify PARP, more particularly the PARP-1 receptor. For this purpose, the compounds of formula (I) may be labeled. The label may be selected from the group consisting of a radioisotope, a rotational label, an antigen label, an enzyme-labeled fluorophore or a chemiluminescent group.

Effective amounts can be readily determined by those skilled in the art from the experimental results set forth below. An effective amount of 0.001mmg/kg to 100mg/kg body weight, in particular 0.005mg/kg to 10mg/kg body weight, is generally contemplated. It is appropriate to administer the required dose in two, three, four or more sub-doses at appropriate time intervals throughout the day. The sub-doses may be formulated in unit dosage forms, for example containing from 0.05 to 500mg, especially from 0.1mg to 200mg, of active ingredient per unit dosage form.

Experimental part

Hereinafter, "DCM" is defined as dichloromethane, "DMF" is defined as N, N-dimethylformamide, "MeOH" is defined as methanol, "MIK" is defined as methyl isobutyl ketone, "MEK" is defined as methyl ethyl ketone, "TEA" is defined as triethylamine and "THF" is defined as tetrahydrofuran.

A. Preparation of intermediate compounds

Example A1

a) Preparation of intermediate 1

A mixture of 3- (1-piperazinyl) -1H-indazole (0.11mol), chloroacetonitrile (0.16mol) and TEA (13g) in toluene (200ml) and acetonitrile (200ml) was stirred and refluxed for 3 hours. The cooled reaction mixture was washed with water (250 ml). The organic layer was separated and dried (MgSO)₄) Filtering and evaporating the solvent. The residue was dissolved in chloroform and purified on silica gel on a glass filter (eluent: chloroform/MeOH 90/10). Collecting the purestIn part, the solvent was evaporated. The residue was crystallized from acetonitrile. The crystals were filtered and dried to yield 26g (99%) of intermediate 1, mp 136 ℃.

b) Preparation of intermediate 2

Intermediate 1(0.11mol) in NH₃The mixture in MeOH (600ml) was hydrogenated at 50 ℃ with Raney nickel (4g) as catalyst. In absorption of H₂After (2 eq), the catalyst was removed by filtration and the filtrate was evaporated. The residue was crystallized from acetonitrile. The crystals were filtered and dried to yield 21g (77.5%) of intermediate 2, mp 121 ℃.

Example A2

a) Preparation of intermediate 3

Phosphorus oxychloride (110.9ml) was added dropwise to DMF (81.5ml) at 5 ℃. The mixture was stirred until completely dissolved. 4- [ (1-oxobutyl) amino ] -benzoic acid ethyl ester (0.34mol) was added. The mixture was stirred at 100 ℃ for 15 hours, then cooled to room temperature and poured into ice water. The precipitate was removed by filtration, washed with water and dried to yield 42.35g (47%) of intermediate 3.

b) Preparation of intermediate 4

Combine intermediate 3(0.1606mol) in 30% sodium methoxide in MeOH (152.8ml) and MeOH (400ml)The mixture was stirred and refluxed for 15 hours, then cooled and poured into ice water. The precipitate was removed by filtration, washed with water and taken up in DCM. The organic layer was separated and dried (MgSO)₄) Filtration and evaporation of the solvent until dry gave 31.64g (85%) of intermediate 4.

c) Preparation of intermediate 5

At 0 ℃ N₂Lithium aluminium tetrahydrochloride (0.1288mol) was added portionwise to a solution of intermediate 4(0.1288mol) in THF (263ml) under reduced pressure. The mixture was stirred for 30min, poured into ice water and extracted with DCM. The organic layer was separated and dried (MgSO)₄) Filtration and evaporation of the solvent to dryness gave 27.4g (98%) of intermediate 5.

d) Preparation of intermediate 6

At 0 ℃ N₂Methanesulfonyl chloride (0.104mol) was added dropwise to a mixture of intermediate 5(0.069mol) and TEA (0.207mol) in DCM (120ml) under stream. The mixture was stirred at 0 ℃ for 4 hours. The solvent was evaporated to dryness (no heating). The product was used without further purification, yielding 20.4g of intermediate 6.

Example A3

a) Preparation of intermediate 7

Stirring at 180 ℃ for adding 4-Ethyl (2-aminoethyl) -1-piperazinecarboxylate (0.0586mol) and 2- (methylthio) -4(1H) -quinazolinone (0.0588mol) were heated for 2 hours, treated with bleached water and then taken up in DCM and MeOH. Evaporation of the solvent to dryness the residue was purified by column chromatography on silica gel (15-35 μm) (eluent: DCM/MeOH/NH)₄OH 94/6/0.5). The pure fractions were collected and the solvent was evaporated. The oily residue was crystallized from diethyl ether. The precipitate was filtered and dried to give intermediate 7, melting point: 138 ℃.

b) Preparation of intermediate 8

A mixture of intermediate 7(0.0223mol) and potassium hydroxide (0.223mol) in 2-propanol (100ml) was stirred and refluxed for 4 days. The solvent was evaporated to dryness. The residue was taken up in MeOH while stirring at 60 ℃. The salt is removed by filtration. Evaporation of the solvent gave 6.5g of intermediate 8.

B. Preparation of the Final Compounds

Example B1

Preparation of Compound 1

Intermediate 2(0.000815mol) and 6-chloro-2- (methylthio) -4(1H) -quinazolinone (0.00097mol) were heated at 160 ℃ for 1 hour, then taken up in 10% water and potassium carbonate and extracted with DCM/MeOH 90/10. The organic layer was separated and dried (MgSO)₄) Filtering and evaporating the solvent. Chromatography over silica gel (15-40 μm) column (eluent: DCM/MeOH/NH)₄OH 92/8/0.5) purification of the residue (0.3 g). The pure fractions were collected and the solvent was evaporated. The residue was crystallized from MEK and DIPE. Filtering the precipitateAnd dried to give 0.2g (58%) of Compound 1, m.p. 186 ℃.

Example B2

Preparation of Compound 2

A mixture of 1- (3-aminopropyl) -4- (4-chlorophenyl) -4-piperidinol (0.015mol) and 2-chloro-4 (1H) -quinazolinone (0.018mol) in dimethylacetamide (5ml) was stirred at 120 ℃ for 1 hour. The reaction mixture was cooled, dissolved in DCM and washed with aqueous ammonia solution. The organic layer was separated and dried (MgSO)₄) Filtering and evaporating the solvent. The residue was purified by column chromatography on silica gel (eluent: DCM/(MeOH/NH)₃)92/8). The pure fractions were collected and the solvent was evaporated. The residue was suspended in DIPE. The precipitate was filtered and dried (vacuum; 70 ℃ C.) to give 3.72g (60%) of Compound 2, m.p. 178.4 ℃.

Example B3

Preparation of Compound 3

A mixture of intermediate 6(0.0124mol), intermediate 8(0.0137mol) and potassium carbonate (0.0373mol) in DMF (80ml) was stirred at 60 ℃ for 1 hour, poured into ice water and stirred at room temperature for 30 min. The precipitate was filtered, washed with water and taken up in 2-propanone. The precipitate was filtered and dried to yield 1.5g (26%) of compound 3, m.p. 118 ℃.

Table F-1 lists compounds prepared according to one of the above examples.

Examples of pharmacology

In vitro Scintillation Proximity Assay (SPA) for PARP-1 inhibitory Activity

The compounds of the invention were analyzed in vitro based on SPA technology (assigned to Amersham Pharmacia Biotech).

In general, the assay relies on the well established SPA technique for the detection of poly (ADP-ribosylation) of biotinylated target proteins such as histones. This ribosylation is induced using a DNA-activated PARP-1 enzyme having a notch and is expressed by³H]-nicotinamide adenine dinucleotide ([ 2 ]³H]-NAD⁺) As ADP-ribosyl donor.

As an inducer of PARP-1 enzyme activity, a nicked DNA was prepared. For this purpose, 25mg of DNA (supplier: Sigma) were dissolved in 25ml of DNase buffer (10mM Tris-HCl, pH 7.4; 0.5mg/ml Bovine Serum Albumin (BSA); 5mM MgCl)₂.6H₂O and 1mM KCl), 50. mu.l of DNase solution (1mg/ml in 0.15M NaCl) was added. After incubation at 37 ℃ for 90min, the reaction was stopped by adding 1.45g NaCl and subsequently incubated further at 58 ℃ for 15 min. The reaction mixture was cooled on ice and treated with 1.510.2M KCl at 4 ℃ respectivelyThe dialysis was performed for 1.5 and 2 hours, and for 1.5 and 2 hours, respectively, on 1.510.01M KCl. The mixture was divided into equal portions and stored at-20 ℃. Histones (1mg/ml, type II-A, supplier: Sigma) were biotinylated using the biotinylation kit from Amersham and stored in aliquots at-20 ℃. A stock solution of 100mg/ml SPA poly (vinyl toluene) (PVT) beads (supplier: Amersham) was prepared in PBS. By adding to 6ml of a culture buffer (50mM Tris/HCl, pH 8; 0.2mM DTT; 4mM MgCl)₂) To which 120. mu.l of a [ sic ] solution was added³H]-NAD⁺(0.1mCi/ml, supplier: NEN) preparation [ 2 ]³H]-NAD⁺The ready-to-use solution of (1). Preparation of 4mM NAD in culture buffer (from 100mM stock solution stored in water at-20 ℃)⁺(supplier: Roche) solution. The PARP-1 enzyme is produced using techniques known in the art, i.e., cloning and expressing the protein starting from human liver cDNA. Information included in the reference regarding the protein sequence of the PARP-1 enzyme used may be found in the Swiss-Prot database, originally obtained under the number P09874. Biotinylated histones and PVT-SPA beads were mixed and pre-incubated at room temperature for 30 min. The PARP-1 enzyme (concentration depending on the batch) was mixed with the nicked DNA and the mixture was pre-incubated at 4 ℃ for 30 min. Equal parts of this histone/PVT-SPA bead solution and PARP-1 enzyme/DNA solution were mixed, and 75. mu.l of this mixture was taken with 1. mu.l of the compound in DMSO and 25. mu.l³H]-NAD⁺Add to each well of a 96-well microtiter plate the final concentration in the incubation mixture was 2. mu.g/ml biotinylated histone, 2mg/ml PVT-SPA beads, 2. mu.g/ml nicked DNA and 5-10. mu.g/ml PARP-1 enzyme. After incubation of the mixture at room temperature for 15min, by adding 100. mu.l of 4mM NAD in the incubation buffer (final concentration 2mM)⁺The reaction was terminated and the plates were mixed.

The beads were allowed to settle for at least 15min, and the plates were transferred to TopCount NXT^TM(Packard) scintillation counting was performed and values are expressed as counts per minute (cpm). For each assay, controls (containing PARP-1 enzyme and DMSO without compound), blank medium (containing DMSO but no PARP-1 enzyme or compound) and samples (containing PARP-1 enzyme and compound dissolved in DMSO) were run in parallel. Dissolving all tested compoundsLysed, and further diluted in DMSO. In the first case, the compound is at 10^-5Test at M concentration. When the compound is at 10^-5When M shows activity, a dose-response curve was prepared with compound concentration at 10^-5And 10^-8M is greater than or equal to the total weight of the composition. In each experiment, blank values were subtracted from control and sample values. Control samples represent maximal PARP-1 enzyme activity. For each sample, the cpm amount was expressed as a percentage of the control mean cpm value. When appropriate, the IC of the drug was calculated using linear interpolation between the upper and lower test points adjacent at the 50% level₅₀Value (drug concentration that reduces PARP-1 enzyme activity to 50% of control). The effect of the test compounds is expressed herein as pIC₅₀(IC₅₀Negative log value of the value). As a reference compound, 4-amino-1, 8-naphthalimide was included to validate the SPA assay. Test Compound at initial test concentration 10^-5M showed inhibitory activity (see Table-2).

In vitro filtration assay for PARP-1 inhibitory Activity

A method of evaluating PARP-1 activity (triggered by the presence of nicked DNA) by testing a compound of the present invention in an in vitro filtration assay, by its histone poly (ADP-ribosylation) activity, is used³²P]NAD as ADP-ribosyl donor. Radioactive ribosylation histone was precipitated in 96-well filter plates by trichloroacetic acid (TCA), and bound [ 2 ] was measured using a scintillation counter³²P]。

Preparation of Histone (stock solution: 5mg/ml in H)₂In O), NAD⁺(stock solution: 100mM in H₂In O) and [ 2 ]³²P]-NAD⁺In culture buffer (50mM Tris/HCl, pH 8; 0.2mM DTT; 4mM MgCl)₂) The mixture of (1). PARP-1 enzyme (5-10. mu.g/ml) and nicked DNA were also prepared. The nicked DNA was prepared as described in the in vitro SPA for PARP-1 inhibitory activity. Mu.l of PARP-1 enzyme/DNA mixture was mixed with 1. mu.l of compound in DMSO and 25. mu.l of histone-NAD⁺/[³²P]-NAD⁺The mixture was added to each well of a 96-well filter plate (0.45 μm, supplier Millipore). In cultureFinal concentration of histone 2. mu.g/ml, 0.1mM NAD in the nutrient mixture⁺、200μM(0.5μC)[³²P]-NAD⁺And 2. mu.g/ml of nicked DNA. Plates were incubated at room temperature for 15min by adding 10. mu.l of ice-cooled 100% TCA followed by 10. mu.l of ice-cooled BSA solution (1% in H)₂O) to terminate the reaction. The protein fraction was precipitated at 4 ℃ for 10min and the plates were vacuum filtered. Each well of the plate was then washed with 1ml 10% ice-cooled TCA, 1ml 5% ice-cooled TCA and 1ml 5% TCA at room temperature. Add a final 100. mu.l scintillation solution (Microscint 40, Packard) to each well and transfer the plates to TopCount NXT^TM(supplier: Packard) scintillation counting was performed and values are expressed as counts per minute (cpm). For each assay, controls (containing PARP-1 enzyme and DMSO without compound), blank medium (containing DMSO but no PARP-1 enzyme or compound) and samples (containing PARP-1 enzyme and compound dissolved in DMSO) were run in parallel. All test compounds were dissolved and finally further diluted in DMSO. In the first case, the compound is at 10^-5And (4) carrying out M concentration test. When the compound is at 10^-5When M shows activity, a dose-response curve was prepared with compound concentration at 10^-5And 10^-8M is greater than or equal to the total weight of the composition. In each experiment, blank values were subtracted from control and sample values. Control samples represent maximal PARP-1 enzyme activity. For each sample, the cpm amount was expressed as a percentage of the control mean cpm value. When appropriate, the IC of the drug was calculated using linear interpolation between the upper and lower test points adjacent at the 50% level₅₀Value (drug concentration that reduces PARP-1 enzyme activity to 50% of control). The effect of the test compounds is expressed herein as pIC₅₀(IC₅₀Negative log value of the value). As a reference compound, 4-amino-1, 8-naphthalimide was included to validate the filtration assay. The test compounds showed inhibitory activity at the initial test concentration of 10-5M (see Table-2).

In vitro Scintillation Proximity Assay (SPA) for TANK-2 inhibitory Activity

The compounds of the invention were subjected to in vitro assay based on SPA technology using Ni flash plates (96 or 384 wells).

Generally, the assay uses [ 2 ]³H]-nicotinamide adenine dinucleotide ([ 2 ]³H]-NAD⁺) As an ADP-ribosyl donor, the autopoly (ADP-ribosyl) ation of TANK-2 protein was detected by means of SPA technique.

Mu.l of assay buffer (60mM Tris/HCl, pH 7.4; 0.9mM MDTT; 6mM MgCl) was added to 1888.7. mu.l₂) To which 64.6. mu.l of a [ sic ] solution was added³H]-NAD⁺(0.1mCi/ml, supplier: Perkin Elmer) and 46.7. mu.l of NAD-stock solution (10.7mM, stored at-20 ℃ C., supplier Roche)³H]-NAD⁺NAD stock solution. The TANK-2 enzyme was produced as described in EP 123806. Mu.l of assay buffer was mixed with 1. mu.l of the compound in DMSO, 20. mu.l of³H]-NAD⁺NAD and 20. mu.l TANK-2 enzyme (final concentration 6. mu.g/ml) were added to each well of a 96-well Ni-coated flash plate (Perkin Elmer). After incubating the mixture at room temperature for 120min, stop solution (42.6mg NAD in 6ml H) was added by 60. mu.l₂O) to terminate the reaction. The plates were covered with a plate sealer and placed in a TopCount NXT^TMScintillation counting was performed in (feeder: Packard), and the values were expressed as counts per minute (cpm). For each assay, controls (containing TANK-2 enzyme and DMSO without compound), blank medium (containing DMSO but no TANK-2 enzyme or compound) and samples (containing TANK-2 enzyme and compound dissolved in DMSO) were run in parallel. All test compounds were dissolved and finally further diluted in DMSO. In the first case, the compound is at 10^-5And (4) carrying out M concentration test. When the compound is at 10^-5When M shows activity, a dose-response curve was prepared with compound concentration at 10^-5And 10^-8M is greater than or equal to the total weight of the composition. In each experiment, blank values were subtracted from control and sample values. Control samples represent maximal TANK-2 enzyme activity. For each sample, the cpm amount was expressed as a percentage of the control mean cpm value. When appropriate, the IC of the drug was calculated using linear interpolation between the upper and lower test points adjacent at the 50% level₅₀Value (drug concentration that reduces TANK-2 enzyme activity to 50% of control). The effect of the test compounds is expressed herein as pIC₅₀(IC₅₀-value ofNegative log value of). As reference compounds, 3-aminobenzamide and 4-amino-1, 8-naphthalimide were included to validate the SPA assay. The assay is described herein using 96-well plates. The same final concentration was used in this assay in 384-well plates and the volume was appropriate. If 96-well plate results are available, these results are combined in Table-2, otherwise the results of the 384-well plate assay are shown.

Compound numbering	In vitro filtration assay PARP-1pIC50	In vitro SPA analysis PARP-1pIC50	In vitro SPA analysis TANK-2pIC50
				1		8.11	＜5
2	6.012	6.876	＜5
				3		6.272	5.759
4	5.439	6.144	＜5
				5	5.579	6.195	＜5
6	5.563	6.412	＜5
				7	5.464	6.228	6.127
8	5.676	6.272	＜5
				11			＜5
12			＜5
				13			＜5
16			＜5
				17			＜5
18	5.345	6.072	＜5
				19	6.204	7.498	＜5
20	5.276	6.171	＜5
				21	6.284	7.593	＜5
22	6.331	7.334	5.073
				23	5.595	6.163	＜5
24	5.305	6.105	＜5
				25	5.635	6.721	＜5
26	5.789	6.372	＜5
				27	6.373	7.353	5.099
28	5.55	5.827	＜5
				29	5.333	6.105	＜5
30		7.491	6.1

Compound numbering	In vitro filtration assay PARP-1pIC50	In vitro SPA analysis PARP-1pIC50	In vitro SPA analysis TANK-2pIC50
				31		7.405	＜5
32		7.345
				33		7.458	6.028
34		7.664
				35		7.971
36		7.965	＜5
				37		7.816	＜5
38		6.373	6.27
				39		8.003	＜5
40	5.649	6.492	＜5
				41	6.263	7.352	＜5
42	5.048	6.075	＜5
				43		7.908	＜5
44		7.533	＜5
				45	5.516	6.647	＜5
45	5.729	6.637	＜5

The compounds may further be evaluated in cellular chemosensitization and/or radiosensitization assays that measure the inhibition of endogenous PARP-1 activity in cancer cell lines and ultimately in vivo radiosensitization assays.

Claims (16)

1. A compound of formula (I)

Pharmaceutically acceptable addition salts and stereochemically isomeric forms thereof, wherein

The dotted line represents an optional bond;

x is > N-or > CH-;

—N ^…… Y-is-NH-C (O)-or-N = CR⁴-, wherein R⁴Is a hydroxyl group;

l is a direct bond or is selected from the group consisting of-C (O) -, -C (O) -NH-, -C (O) -C_1-6Alkanediyl-, -C (O) -O-C_1-6alkanediyl-or-C_1-6Alkanediyl-divalent radicals;

R¹is hydrogen, halogen, C_1-6Alkoxy or C_1-6An alkyl group;

R²is hydrogen, hydroxy, C_1-6Alkoxy or aminocarbonyl;

or, when X is substituted by R²When substituted, R²Together with-L-Z, form a divalent radical of formula

-C(O)-NH-CH₂-NR¹⁰-　　　（a-1）

Wherein R is¹⁰Is phenyl;

R³is hydrogen, or C_1-6An alkoxy group;

z is amino, cyano or a group selected from

Wherein each R⁵And R⁶Independently selected from hydrogen, halogen, amino, C_1-6Alkyl or C_1-6An alkoxy group.

2. The compound of claim 1, wherein

L is a direct bond or is selected from-C (O) -, -C (O) -NH-, or-C (O) -O-C_1-6Alkanediyl-divalent radicals; r²Is hydrogen, hydroxy or C_1-6An alkoxy group; z is a group selected from (b-3), (b-4), (b-5), (b-6), (b-7), (b-8) or (b-9); each R⁵And R⁶Independently selected from hydrogen, halogen, amino, C_1-6Alkyl or C_1-6An alkoxy group.

3. The compound of claim 1, wherein

L is a direct bond; r¹Is hydrogen, halogen or C_1-6An alkyl group;

R²is hydrogen; r³Is hydrogen; z is a group selected from (b-5) or (b-7); and each R⁵Independently selected from hydrogen or halogen.

4. The compound of claim 1, wherein said compound is selected from the group consisting of compounds 35, 36, 39, 1 and 43

Compound 35 Compound 36

Compound 39 Compound 1

Compound 43.

5. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and, as active ingredient, a therapeutically effective amount of a compound according to any one of claims 1 to 4.

6. A process for preparing a pharmaceutical composition as claimed in claim 5, wherein a pharmaceutically acceptable carrier and a compound as claimed in any one of claims 1 to 4 are intimately mixed.

7. Use of a compound of any one of claims 1-4 in the manufacture of a medicament for treating a PARP mediated disorder.

8. Use of a compound of any one of claims 1-4 in the manufacture of a medicament for the treatment of a PARP-1 mediated disorder.

9. The use of claim 7, wherein the medicament is a chemosensitizer.

10. The use of claim 7, wherein the medicament is a radiosensitizer.

11. A combination of a compound of formula (I) as claimed in any one of claims 1 to 4 and a chemotherapeutic agent.

12. A compound of the formula and pharmaceutically acceptable addition salts

。

13. A compound of the formula and pharmaceutically acceptable addition salts

。

14. A compound of the formula and pharmaceutically acceptable addition salts

。

15. A compound of the formula and pharmaceutically acceptable addition salts

。

16. Use of a compound according to any one of claims 1-4 and 12-15 in the manufacture of a medicament for the treatment of cancer.