HK1105631B - Substituted 2-alkyl quinazolinone derivatives as parp inhibitors - Google Patents

Description

Substituted 2-alkyl quinazolinone derivatives as PARP inhibitors

Technical Field

The present invention relates to PARP inhibitors and provides compounds and pharmaceutical compositions containing the disclosed compounds. In addition, the present invention provides methods of using the disclosed PARP inhibitors, for example as pharmaceuticals.

Background

The ribozyme poly (ADP-ribose) polymerase-1 (PARP-1) is one member of the PARP enzyme family. This growing enzyme family consists of PARP classes such as PARP-1, PARP-2, PARP-3 and Vault-PARP with Tankyrases (TANKyrases, TANKs) such as TANK-1, TANK-2 and TANK-3. PARP also refers to poly (adenosine 5' -diphosphate-ribose) polymerase or PARS (poly (ADP-ribose) synthetase).

PARP-1 is a major nuclear protein of 116kDa, which consists of three domains: an N-terminal DNA binding domain comprising two zinc finger domains, an auto-modifying domain and a C-terminal catalytic domain. It is present in almost all eukaryotic cells. The enzyme synthesizes poly (ADP-ribose), a branched polymer that may contain more than 200 ADP-ribose units. The protein receptors for poly (ADP-ribose) are directly or indirectly involved in maintaining DNA integrity. They include histones, topoisomerases, DNA and RNA polymerases, DAN ligases and Ca-dependent²⁺And Mg²⁺The endonuclease of (4). PARP protein is highly expressed in many tissues, most notably in the immune system, heart, brain and germ cells (germ-lines). Under normal physiological conditions, PARP is in the least active state. However, when DAN is damaged it immediately causes up to 500-fold PARP activation.

TANKs (TANKs) are recognized as components of the human telomere complex. They are also proposed to play a role in vesicle trafficking and are likely to be platforms for proteins involved in various cellular processes. Telomeres, which are essential for maintaining and stabilizing chromosomes, are maintained primarily by a specific reverse transcriptase, telomerase. Tankyrase is an (ADP-ribose) transcriptase that functions as both a signaling and cytoskeletal protein. They contain a PARP domain that catalyzes the poly-ADP-ribosylation of the base protein, a sterile alpha motif shared with certain signaling molecules, and an ANK domain containing 24 ankyrin repeat units homologous to the cytoskeletal protein ankyrin. The ANK domain interacts with the telomeric protein, telomere repeat binding factor-1 (TRF-1). These proteins were therefore designated TRF 1-interacting, ankyrin-associated 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, while TRF-1 regulates the length of telomeres, and ADP-ribosylation inhibits the ability of TRF-1 to bind to telomere DNA. This ADP-ribosylation of TRF-1 releases TRF-1 from telomeres, opening the telomere complex and bringing it into proximity with telomerase. Thus, TANK plays a positive role in regulating telomere length, allowing telomeres to be lengthened by telomerase.

Among the many functions provided by PARP (especially PARP-1), promotion of DNA repair by ADP-ribosylation is its primary function and thus collectively constitutes a number of DNA repair proteins. After PARP activation, NAD⁺The level is significantly reduced. Activation of large amounts of PARP leads to NAD in DNA-damaged cells on a large scale⁺Horizontal, substantial slippage. The short half-life of poly (ADP-ribose) results in a rapid rate of reversal. Once poly (ADP-ribose) is formed, it is rapidly degraded by the action of structurally active poly (ADP-ribose) glycohydrolases (PARG), phosphodiesterases, and poly (ADP-ribose) protein lyases. PARP forms a cycle with PARG, and large amounts of NAD⁺Converted to ADP-ribose. Over-stimulation of PARP results in NAD in less than 1 hour⁺Slip off and drop ATP to less than 20% of normal levels. Such a situation is extremely disadvantageous when, during ischemia, oxygen deprivation already jeopardizes cellular energy output. The free radicals produced during subsequent reperfusion are believed to be the main cause of tissue damage. Partial drop of ATP during ischemia and reperfusion is a typical response in many organs and can be thought of as NAD due to reversal of poly (ADP-ribose)⁺The wear and tear of (2). Thus, it is desirable to preserve the energy level of cells by inhibiting PARP or PARG, thereby enhancing the viability of ischemic tissue after damage.

The synthesis of poly (ADP-ribose) is also involved in the inducible expression of a number of essential genes for the inflammatory response. PARP inhibitors suppress the production of nitric oxide (iNOS) induced in macrophages, P-type selectins and intercellular adhesion molecule-1 (ICAM-1) in endothelial cells. This activity makes PARP inhibitors exhibit potent anti-inflammatory effects. Inhibition of PARP can reduce tissue necrosis by preventing neutrophil translocation and penetration into damaged tissues.

PARP is activated by damaged DNA fragments and once activated, catalyzes up to 100 adhesionsADP-ribose to various nuclear proteins, including tissue proteins and PARP itself. Under greater cellular stress, extensive activation of PARP can rapidly lead to cell damage or death through depletion of stored energy. Since each NAD⁺Regeneration of the molecule requires consumption of 4 ATP molecules, while NAD⁺Is depleted by massive activation of PARP, in order to resynthesize NAD⁺ATP is also depleted.

Activation of PARP has been reported to play a key role in NMDA and NO-induced neurotoxicity. This has been demonstrated in cortical culture and hippocampal neuronal fragments, where prevention of toxicity is directly related to the potential for PARP inhibition. The potential role of PARP inhibitors in the treatment of neurodegenerative diseases and head trauma is therefore recognized, even though the true mechanism of action has not yet been elucidated.

Similarly, it has been demonstrated that simple injection of PARP inhibitors can reduce the size of emboli in rabbit heart or skeletal muscle due to ischemia and reperfusion. In these studies, a simple injection of 3-amino-benzamide (10mg/kg), either 1 minute prior to embolization or 1 minute prior to reperfusion, resulted in a similar reduction in cardiac embolization size (32-42%), while the use of another PARP inhibitor, 1, 5-dihydroxyisoquinoline (1mg/kg), reduced embolization size to an extent comparable to the former (38-48%). From these results it is reasonable to assume that PARP inhibitors can be used to rescue reperfusion injury of ischemic heart or skeletal muscle tissue in advance.

Activation of PARP can also be used to measure neurotoxic damage due to exposure to the following inducers: glutamic acid (stimulation via NMDA receptor), intermediate having reactive oxygen species, amyloid beta-protein, N-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPP), or N-methyl-4-phenylpyridine (MPP), an active metabolite thereof⁺) It is 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 granulocytes in vitro and in MPTP neurotoxicity. The most frequent post-stroke or other neurodegenerative processesThe effect is excessive exposure of the nerve to glutamate, which acts to control central nervous system neurotransmitters and acts on the N-methyl D-aspartate (NMDA) receptor and other subtype receptors. During ischemic brain injury such as stroke or heart disease, oxygen deprived neurons release considerable amounts of glutamate. These excessive releases of glutamate in turn lead to overstimulation of N-methyl D-aspartate (NMDA), AMPA, kainate and MGR receptors (excitotoxicity), opening ion channels and allowing unregulated ion flux (e.g., Ca)²⁺And Na⁺Enter 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, eventually leading to cell damage or death by producing proteases, lipases and free radicals. Over-activation of glutamate has been implicated in a variety of neurological diseases and conditions, including epilepsy, stroke, alzheimer's disease, parkinson's disease, Amyotrophic Lateral Sclerosis (ALS), huntington's disease, personality schizophrenia, chronic pain, post-hypoxic ischemia and nerve loss, hypoglycemia, ischemia, trauma, and nerve injury. Glutamate exposure and irritation have been implicated as a cause of obsessive compulsive disorder, particularly drug dependence. Evidence includes the finding that glutamate receptor antagonists (i.e., compounds that block the binding of glutamate to its receptor or activate its receptor) block nerve damage following vascular impingement in a wide variety of animal species and brain cortical cultures treated with glutamate or NMDA. Attempts to prevent excitotoxicity by blocking NMDA, AMPA, kainate and MGR receptors have proven to be difficult because each receptor has multiple sites to which glutamate can bind, and it has therefore been difficult to find effective antagonist cocktails or universal antagonists to prevent glutamate from binding to all receptors and to test this theory. In addition, many compositions that effectively block the receptor are toxic to animals. Thus, no effective treatment for glutamate abnormalities has been known. Activation of the NMDA receptor, stimulated by glutamate, e.g. the enzyme neuronal nitric oxide synthase (nNOS), leads to the formation of Nitric Oxide (NO), which in turn triggersNeurotoxicity. NMDA neurotoxicity can be prevented by treatment with Nitric Oxide Synthase (NOS) inhibitors or by targeted gene disruption of nNOS in vitro.

Another use of PARP inhibitors is in the treatment of peripheral nerve injury, and the resultant pathological pain syndrome known as neuropathic pain, such as that caused by chronic stenotic lesions (CCI) of the common sciatic nerve and in which transsynaptic changes in the spinal cord's posterior horn are characterized by the appearance of hyperpigmentation of the cytoplasm and nucleoplasm (so-called "dark" neurons).

Evidence also shows that PARP inhibitors are useful in the treatment of inflammatory bowel disorders such as colitis.

Specifically, rat lumen is administered heptenyltrinitrobenzenesulfonic acid dissolved in 50% ethanol to cause colitis, and then treated rats are subjected to 3-aminobenzamide, which is a specific PARP activity inhibitor. Inhibition of PARP activity reduces the inflammatory response and restores the morphology and energy status of the distal colon.

Further evidence suggests that PARP inhibitors may be useful in arthritis. In addition, PARP inhibitors appear to be useful in diabetes. Evidence has shown that PARP inhibitors are useful in the treatment of endotoxic and septic shock.

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

PARP inhibitors, such as 3-aminobenzamide, are also known to affect overall DNA repair, for example, by hydrogen peroxide or ionizing radiation.

The critical role played by PARP in the repair of DNA strand breaks is well established, particularly in the enzymatic repair of DNA damage caused by direct strand breaks caused by ionizing radiation, or indirectly by the action of methylating agents, topoisomerase I inhibitors, and other chemotherapeutic agents (e.g., cisplatin and bleomycin). Many studies using "knockout" mice, transdominant inhibition patterns (over-expression of DNA-binding domains), antisense, and small molecular weight inhibitors have demonstrated a role for PARP in repair and in the induction of cell survival following DAN injury. Inhibition of PARP enzyme activity will lead to an increased sensitivity of tumor cells to DNA damage treatment.

PARP inhibitors have been reported to have an effect on tumor cell radiosensitization (hypoxia) and at the same time prevent tumor cells from recovering from potentially lethal and sublethal DNA damage following radiation therapy, presumably by the mechanism that PARP inhibitors have the ability to prevent DNA strand breaks from rejoining, while at the same time destroying some DAN damage signaling pathways.

PARP inhibitors have been used to treat cancer. In addition, U.S. Pat. No.5,177,075 discusses some isoquinolines which enhance the lethal effect of ionizing radiation or chemotherapeutic agents on tumor cells. "Effect of 6(5-Phenanthridione), an Inhibitor of Poly (ADP-ribose) Polymerase, on Current Tumor Cells", Oncol. Res., 6: 9,399-.

Li and Zhang in Idrugs 2001, 4 (7): 804-: 882-883 and Nguewa et al in Progress in Biophysic & molecular biology 2005, 88: 143- "172 discloses a review of the state of the art.

There is a continuing need for potent and potent PARP inhibitors, and in particular for PARP-1 inhibitors that produce minimal side effects. The present invention provides compounds, compositions and methods for inhibiting PARP activity for treating cancer and/or preventing damage to cells, tissues and/or organs due to cell damage or death caused by, for example, necrosis or apoptosis. The compounds and compositions of the present invention are particularly advantageous for enhancing the efficacy of chemotherapy and radiation therapy, the primary therapeutic effect of which is to cause DNA damage to targeted cells.

GB 1062357 published 3/22 in 1967 discloses quinazolinone derivatives having antihypertensive effects.

DE 20,1973 published in 1973 on 6/20 discloses substituted pyridone derivatives having antihypertensive effect.

EP 13612 published 11.11.1983 discloses substituted piperidinylalkylquinazoline derivatives. The compounds are serotonin antagonists. In particular, the compound No. 63 disclosed in the present invention.

EP 669919 published on 9/6 in 1994 is disclosed as 5-HT₃The antagonists dimethylbenzofuran and dimethylbenzopyran.

US5374637 published on 20.12.1994 discloses benzamide derivatives. The disclosed compounds have the property of stimulating gastrointestinal motility.

EP 885190 published on 23.12.1998 discloses 1, 4-disubstituted piperidine derivatives having gastric motility enhancing properties.

EP 1036073 published 12.6.1999 discloses substituted quinazolinedione derivatives. The compounds have fundic relief properties.

EP 1355888 published on 6/20/2002 discloses quinazolinone derivatives as PARP inhibitors.

Disclosure of 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-, > CH-or > CR²-, wherein R²Is aminocarbonyl; or

When X is > CR²When is, then R²Together with-L-Z there may be formed a divalent radical of the formula (a-1)

-C(O)-NH-CH₂-NR¹⁰- (a-1)

Wherein R is¹⁰Is phenyl;

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

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

R³is hydrogen or C_1-6An alkoxy group;

z is 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 R⁸Together may 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 R is⁹Independently selected from hydrogen or C_1-6An alkyl group; and is

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

When X is > CR²-or when Z is a group of formula (b-11), L may be a direct bond; the limiting conditions are as follows: when X is > CH-, -N...When Y-is-NH-C (O) -, the second dotted line is not a bond, Z is a group of formula (b-2) and L is-C (O) -, then-C_1-6alkanediyl-not-CH₂-CH₂-。

The compounds of formula (I) may also exist in their tautomeric form. These types are not explicitly shown in the above formula, but are within the scope of the present invention.

A number of terms used in the definitions herein before and hereinafter will be explained in the following. These terms are sometimes used individually, and sometimes in combination.

As defined hereinbefore and hereinafter, halogen generally refers to fluorine, chlorine, bromine and iodine; c_1-6Alkyl means straight and branched chain saturated hydrocarbon groups containing 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 means divalent straight-chain and branched-chain saturated hydrocarbon radicals having from 1 to 6 carbon atoms, e.g. methylene, 1, 2-ethanediyl, 1, 3-propanediyl, 1, 4-butanediyl, 1, 5-pentanediyl, 1, 6-hexanediyl and branched-chain isomers thereof, e.g. 2-methylpentanediyl, 3-methylpentanediyl, 2-dimethylbutanediyl,2, 3-dimethylbutylene and the like.

"pharmaceutically acceptable salt" means a pharmaceutically acceptable acid or base addition salt. The pharmaceutically acceptable acid or base addition salts referred to above are non-toxic acid and non-toxic base addition salt forms which can be formed from the compounds of formula (I) and which are therapeutically active. The compounds of formula (I) having basic properties may be converted into 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 acid 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. succinic), maleic, fumaric, malic, tartaric, citric, methanesulfonic, ethanesulfonic, benzenesulfonic, p-toluenesulfonic, cyclamic (cyclohexylsulfamic), salicylic, p-aminosalicylic, pamoic and the like acids.

The compounds of formula (I) having acidic properties may be converted into pharmaceutically acceptable base addition salts by treatment with a suitable organic or inorganic base. Suitable base salts include, for example, ammonium salts, alkali metal 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-glucosamine, hydrabamine salts and salts with amino acids such as arginine, lysine and the like.

The term "acid or base addition salts" also includes addition salts which may be formed from the compounds of formula (I) in the form of hydrates and solvates. Examples of such forms are hydrates, alcoholates and the like.

The term "stereochemically isomeric forms of a compound of formula (I)" as used hereinbefore denotes all the possible compounds possessed by the compounds of formula (I) which are linked by the same linking atoms in the same order, but which have a non-interchangeable three-dimensional structure. 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 contains all diastereomers and/or enantiomers of the basic molecular 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 those in which one or several of the nitrogen atoms on the compound of formula (I) are oxidized to the so-called N-oxide, in particular those in which one or more of the nitrogens on the piperidine or piperazine are oxidized.

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

GB 1062357 discloses quinazolinone derivatives having antihypertensive effects. DE2258561 discloses substituted pyridone derivatives having antihypertensive effect. EP 13612 discloses substituted piperidinylalkylquinazoline derivatives as serotonin antagonists. EP 669919 discloses a 5-HT₃The antagonists dimethylbenzofuran and dimethylbenzopyran. US5374637 discloses benzamide derivatives having gastrointestinal motility stimulating properties. EP 885190 discloses 1, 4-disubstituted piperidine derivatives having gastric motility enhancing properties. EP 1036073 discloses substituted quinazolinedione derivatives having fundic relief properties. EP 1355888 discloses quinazolinone derivatives as PRAP inhibitors.

Surprisingly, the compounds of the present invention also have the ability to inhibit PRAP.

A first group of interesting compounds comprises those compounds of formula (I) which satisfy one or more of the following constraints:

a) x is > N-or > CR²-, wherein R²Is aminocarbonyl;

b) when X is > CR²When is, then R²Together with-L-Z, may form a divalent group of formula (a-1);

c)-N...Y-is-NH-C (O) -or-N ═ CR⁴-, wherein R⁴Is hydroxy and the second dotted line isA key;

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

e)R³is C_1-6An alkoxy group;

f) z is a group selected from (b-1), (b-3), (b-4), (b-5), (b-6), (b-7), (b-8), (b-9), (b-11), and (b-11);

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

h) l is a group selected from-C (O) -NH-, -C (O) -C_1-6alkanediyl-or-C (O) -O-C_1-6Alkanediyl-divalent radicals;

a second group of interesting compounds comprises those compounds of formula (I) which satisfy one or more of the following constraints:

a) x is > CH-or > CR²-, wherein R²Is aminocarbonyl;

b) when X is > CR²When is, R²Then together with-L-Z a divalent group of formula (a-1) may be formed;

c)R¹is hydrogen;

d)R³is hydrogen;

e) z is a group of formula (b-1), (b-2) or (b-11);

f) each R⁵、R⁶、R⁷And R⁸Independently selected from hydrogen or halogen;

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

and the number of the first and second electrodes,

h) when X is > CR²Or L may be a direct bond when Z is a group of formula (b-11).

A third group of interesting compounds comprises those compounds of formula (I) which satisfy one or more of the following constraints:

a) x is > CH-or > CR²-, wherein R²Is aminocarbonyl; (ii) a

b)R¹Is hydrogen;

c)R³is hydrogen;

d) z is a group of formula (b-1) or (b-2);

e) each R⁵、R⁶、R⁷And R⁸Independently selected from hydrogen or halogen;

f) l is a divalent group selected from-C (O) -or-C (O) -NH-; and the number of the first and second electrodes,

g) when X is > CR²When-is, L may be a direct bond.

Preferred compounds of the formula (I) are those in which X is > N-or > CR²-, wherein R²Is aminocarbonyl; when X is > CR²When is, then R²Together with-L-Z, may form a divalent group of formula (a-1); r¹Is hydrogen; r³Is hydrogen; z is a group of formula (b-1), (b-2) or (b-11); each R⁵、R⁶、R⁷And R⁸Independently selected from hydrogen or halogen; l is a group selected from-C (O) -, -C (O) -NH-or-C (O) -C_1-6Alkanediyl-divalent radicals; and when X is > CR²When-or Z is a group of formula (b-11), L may be a direct bond.

Preferred compounds of the formula (I) are those in which X is > N-or > CR²-, wherein R²Is aminocarbonyl; r¹Is hydrogen; r³Is hydrogen; z is a group of formula (b-1) or (b-2); each R⁵、R⁶、R⁷And R⁸Independently selected from hydrogen or halogen; l is a divalent group selected from-C (O) -, or-C (O) -NH-; and when X is > CR²-, L may be a direct bond.

The most preferred compounds are compounds No.4, No.5, No.11, No.12 and No. 14.

Compound 4 Compound 5

Compound 11 Compound 12

Compound 14

The compounds of formula (I) may be prepared according to the general method described in EP 13612. The starting materials and some intermediates are known compounds and are either commercially available or prepared according to conventional reaction procedures well known in the art.

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

wherein-N...Compounds of formula (I) wherein Y-is-NH-C (O) -and the second dotted line is a bond, here formula (I-a), may be prepared by cyclisation of an appropriate substituted 2-aminocarbonylbenzamide of formula (II) according to art-known cyclisation procedures.

Or, wherein C_1-6alkanediyl-is-CH₂-CH₂-and-N...Compounds of formula (I) wherein Y-is-NH-C (O) -and the second dotted line is a bond, here compounds of formula (I-b), may be prepared by cyclisation of the appropriate substituted 2-aminocarbonylbenzamide of formula (IV) using an intermediate of formula (III) according to art-known cyclisation procedures.

wherein-N...Compounds of formula (I) wherein Y-is-NH-C (O) -and the second dotted line is not a bond, here compounds of formula (I-c), may be prepared by cyclisation of a suitable substituted 2-aminobenzamide of formula (V) using a suitable substituted intermediate of formula (VI) according to art-known cyclisation procedures. The cyclization reaction is conveniently carried out by stirring the various reactants together in the presence of a suitable solvent such as an alcohol (methanol, ethanol, propanol, etc.). The reaction rate can be increased by slightly increasing the temperature and adding a suitable catalytic amount of a strong acid such as hydrochloric acid or the like.

The compounds of formula (I) may also be converted into each other by reactions or functional group transfer known in the art. Some of these transformations have been described previously. 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; nitrile hydrolysis to the corresponding amide; the amino group on the imidazole or phenyl group can be replaced with hydrogen by a diazotization reaction known in the art and subsequent replacement of the diazo group with hydrogen; alcohols can be converted into esters and ethers; primary amines can be converted to secondary or tertiary amines; double bonds may be hydrogenated to the corresponding single bonds; the iodo group on the phenyl group can be converted to an ester group by insertion of carbon monoxide in the presence of a suitable palladium catalyst.

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

From the following examples, it can be seen that the compounds of the present invention have PARP inhibitory effects.

The term "PARP" as used herein means a protein having poly-ADP-ribosylation activity. Within the meaning of this term, PARP covers all proteins encoded by the PARP gene, variants thereof and fragments thereof substituted. In addition, the term "PARP" as used herein includes homologs, homologs and other animal PARP homologs of PARP.

The term "PARP" includes, but is not limited to, PARP-1. In the definition of this term, PARP-2, PARP-3, Vault-PARP (PARP-4), PARP-7(TiPARP), PARP-8, PARP-9(Bal), PARP-10, PARP-11, PARP-12, PARP-13, PARP-14, PARP-15, TANK-1, TANK-2 and TANK-3 are also encompassed.

Compounds that inhibit both PARP-1 and tankyrase-2 are more beneficial in enhancing the inhibition of cancer cell growth activity.

The invention also contemplates the use of such compounds for the manufacture of a medicament for the treatment of diseases and disorders of the animal body wherein such compounds are of formula (I).

Its N-oxide form, its pharmaceutically acceptable addition salts and its stereochemically isomeric forms,

wherein the content of the first and second substances,

the dotted line represents an optional bond;

x is > N-, > CH-or > CR²-, wherein R²Is aminocarbonyl; or

When X is > CR²When is, then R²Together with-L-Z there may be formed a divalent radical of the formula (a-1)

-C(O)-NH-CH₂-NR¹⁰- (a-1)

Wherein R is¹⁰Is phenyl;

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

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

R³is hydrogen or C_1-6An alkoxy group;

z is a group selected from

Wherein each R⁵、R⁶、R⁷And R⁸Independently selected from hydrogen, halo, amino, C_1-6Alkyl or

C_1-6An alkoxy group; or the like, or, alternatively,

R⁷and R⁸Together, divalent groups of the formula may be formed.

-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; and

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

When X is > CR²Or L may be a direct bond when Z is a group of formula (b-11).

In view of the PARP binding properties of the compounds of the invention, they may be used as reference compounds or tracer compounds, in which case one atom of the molecule may be substituted, for example by a radioisotope.

To prepare the pharmaceutical compositions of this invention, an effective amount of a particular compound, in base or acid addition salt form, as the active ingredient is intimately admixed with a pharmaceutically acceptable carrier, which may take a wide variety of forms depending on the intended mode of administration and the form of preparation. The pharmaceutical compositions are formulated in unit dosage forms acceptable to the individual, preferably in a form suitable for oral, rectal, subcutaneous or parenteral injection. For example, in preparing the composition for oral administration, any of the usual pharmaceutical vehicles may be used, such as water, glycols, oils, alcohols, etc. in the case of preparing suspensions, syrups, medicated liquors and solutions; or in the preparation of powder, pill, capsule and tablet, starch, sugar, laponite, lubricant, binder, and dispersant can be used as solid carrier. Capsules and tablets are the best oral dosage form because of their ease of use, in which case solid pharmaceutical carriers are obviously preferred. For compositions for parenteral administration, the carrier will usually comprise sterile water, the majority of which is sterile water, although other ingredients, such as co-solvents, may also be added. Injectable solutions can be prepared in which case the carrier can comprise, for example, a saline solution, a dextrose solution, or a mixed saline and dextrose solution. Injectable suspensions may also be prepared by choice of appropriate liquid carriers, suspensions and the like. In compositions suitable for subcutaneous administration, the carrier optionally includes a penetration enhancer and/or a suitable wetting agent, optionally in admixture with minor amounts of suitable natural additives that do not cause serious damage to the skin. The additives facilitate dermal administration and/or facilitate formulation into a desired composition. These compositions can be administered in various ways, for example, as a plaster, spray, ointment. It is particularly advantageous to formulate the aforementioned pharmaceutical compositions in dosage unit form for convenient administration and dosage form for uniform administration. Dosage unit form as used in the specification and claims herein refers to physically discrete units of medicament suitable for separate use, each unit containing a predetermined quantity of active ingredient calculated to achieve the desired therapeutic effect in association with the required pharmaceutical carrier. Examples of unit dosage forms are tablets (scored or sugar-coated tablets), capsules, pills, powder packets, wafers, injectable solutions or suspensions, teaspoonfuls, tablespoonfuls and the like, and their individual dosage forms

Several parts of (a).

The compounds of the present invention may treat or prevent tissue damage caused by cell damage or death resulting from necrosis or apoptosis; can improve neurological or cardiovascular tissue damage, including damage caused by focal ischemia, myocardial infarction and reperfusion; can treat a variety of diseases and conditions caused or exacerbated by PARP activity; can prolong or increase the life span or reproductive capacity of cells; can alter gene expression in senescent cells; can improve sensitivity of cell radiotherapy and/or chemotherapy; in general, inhibition of PARP activity reduces energy loss from the cell, and in the case of nerve cells, prevents irreversible depolarization of neurons, thereby providing protection to the nerve.

In view of the foregoing, the present invention further relates to the use of a compound as defined above in a therapeutically effective amount sufficient to inhibit PARP activity for the treatment or prevention of tissue damage resulting from cell damage or death due to necrosis and apoptosis, without affecting neuronal activity mediated by NMDA toxicity, for the treatment of neuronal tissue damage, neurological disorders and neurodegenerative diseases resulting from ischemia and reperfusion injury; treating or preventing vascular embolism; to treat or prevent cardiovascular disease; treating other conditions and/or disorders, for example, age-related muscle degeneration, AIDS and other immune aging diseases, inflammation, rheumatism, arthritis, arteriosclerosis, cachexia, cancer, skeletal muscle degeneration involving replicative senescence, diabetes, brain injury, inflammatory bowel disease (e.g., colitis and crohn's disease), muscle atrophy, osteoarthritis, osteoporosis, chronic and/or acute pain (e.g., neuralgia), renal failure, retinal ischemia, septic shock (e.g., endotoxic shock), and skin aging; prolonging the life span of the cell or increasing its reproductive capacity; changes the gene expression of the senescent cells and increases the sensitivity of the tumor cells to chemotherapy and/or radiotherapy (hypoxia). The invention also relates to the treatment of diseases and conditions of the animal body comprising administering to said body a therapeutically effective amount of a compound as defined above.

In particular, the present invention relates to a method of treatment, prevention or inhibition of neurological diseases of the animal body which comprises administering to said body a therapeutically effective amount of a compound as defined above. The neurological disorder is selected from the group consisting of peripheral neuropathy caused by physical injury or disease symptoms, traumatic brain injury, physical injury to the spinal cord, stroke associated with brain injury, focal ischemia, global ischemia, reperfusion injury, demyelinating disease, and neurological disorder due to neurodegeneration.

The present invention also contemplates the use of compounds of formula (I) to inhibit PARP activity, treat, prevent or inhibit tissue damage resulting from cell damage or death due to necrosis and apoptosis, treat, prevent or inhibit neurological diseases in animals.

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

The term "treatment" as used herein encompasses any treatment of a disease and/or condition in an animal, particularly a human, and includes: (i) preventing a disease and/or condition in a subject who has not yet been diagnosed with the disease, but who is predisposed to developing the disease and/or condition; (ii) inhibiting the disease and/or condition, i.e., arresting its development; (iii) alleviating, i.e., recovering from, the disease and/or condition.

The term "radiosensitizer" as used herein refers to a molecule, preferably a low molecular weight molecule, which is administered to an animal in a therapeutically effective amount to increase the sensitivity of cells to ionizing radiation and/or to promote a therapeutic effect on a disease treatable with ionizing radiation. Diseases that can be treated with ionizing radiation include neoplastic diseases, benign and malignant tumors, and cancer cells. Other diseases that may be treated with ionizing radiation not listed herein are also within the scope of the present invention.

The term "chemosensitizer" as used herein refers to a molecule, preferably a low molecular weight molecule, which is administered to an animal in a therapeutically effective amount to increase the sensitivity of cells to chemotherapy and/or to promote the therapeutic effect on a disease treatable with chemotherapy. Diseases treatable with chemotherapy include neoplastic diseases, benign and malignant tumors, and cancer cells. Other diseases that may be treated with ionizing radiation not listed herein are also within the scope of the present invention.

The compounds, compositions and methods of the invention are particularly useful for treating or preventing tissue damage caused by cell damage or death due to gangrene or self-apoptosis.

The compounds of the invention may be "anti-cancer agents", which term also encompasses "anti-tumor cell growth agents" and "anti-neoplastic agents". For example, the methods of the invention can be used to treat cancer and tumor cells in cancer that are sensitive to radiation and/or chemotherapy, e.g., ACTH-induced tumors, acute lymphoid blood cancer, acute non-lymphoid blood cancer, adrenocortical cancer, bladder cancer, brain cancer, breast cancer, cervical cancer, chronic lymphoid cancer, chronic medulloblastoma blood cancer, rectal cancer, subcutaneous T-cell lymphoid cancer, endometrial cancer, esophageal cancer, Ewing's sarcoma gallbladder cancer, hairy cell blood cancer, head & neck cancer, Hodgkin's lymphoma, Kaposi's sarcoma, kidney cancer, liver cancer, lung cancer (small and/or non-small cells), malignant ascites, malignant pleural ascites, melanoma, mesodermoma, myeloma, neuroblastoma, non-Hodgkin's lymphoma, bone cancer, ovarian cancer, ovo (seminal) cell cancer, prostate cancer, pancreatic cancer, penile cancer, pancreatic cancer, cervical cancer, retinoblastoma, skin cancer, soft tissue sarcoma, squamous cell carcinoma, gastric cancer, testicular cancer, thyroid cancer, trophoblastic cancer, uterine cancer, vaginal cancer, perineal cancer, and Wilm's tumor.

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

As is known, radiosensitizers can increase the sensitivity of cancer cells to the toxic effects of ion irradiation. Some mechanisms concerning the mode of action of radiosensitizers have been mentioned in the following documents, including: hypoxic cell radiotherapy sensitizers (such as 2-nitroimidazole compounds and benzotriazine dioxide compounds) can mimic oxygen or behave like a bioreductive agent in a hypoxic environment; non-hypoxic cell radiosensitizers (e.g., halopyrimidines) can act as analogs of DNA bases and can preferentially bind to the DNA of cancer cells, thereby promoting radiation-induced fragmentation of DNA molecules and/or inhibiting normal DNA repair mechanisms; and other various hypotheses regarding the potential mechanisms of radiosensitizers to treat disease.

Currently, many cancer treatment protocols combine radiosensitizers with X-ray radiation. Examples of radiosensitizers capable of activating X-rays include, but are not limited to, the following: metronidazole, misonidazole (misonidazole), desmethyisonidazole (desmodazole), pimozide (pimodazole), etanidazole (etanidazole), nimorazole (nimorazole), mitomycin C, RUS 1069, SR 4233, EO9, RB 6145, nicotinamide, 5-bromodeoxyuridine (BUdR), 5-iododeoxyuridine (IUdR), bromodeoxycytidine, fluorodeoxyuridine (FudR), hydroxyurea, cisplatin, and analogs and derivatives thereof having a therapeutic effect.

Photodynamic therapy (PDT) of cancer uses visible light as a radiation activator of sensitizers. Examples of photodynamic radiosensitizers include, but are not limited to, the following: hematoporphyrin derivatives, Photofrin (Photofrin), benzoporphyrin derivatives, protoporphyrin tin salts (tinotoporphyrin), phenobiode-a, bacteriochlorophyll-a, naphthalocyanine, phthalocyanine, zinc phthalocyanine, and analogs and derivatives thereof having therapeutic effects.

Radiosensitizers can be used with a therapeutically effective amount of one or more other compounds, including but not limited to the following: a compound that promotes binding of a radiosensitizer to target cells; a compound capable of controlling the flow of therapeutic agents, nutrients and/or oxygen towards the target cells; chemotherapeutic agents that act on tumors with or without additional irradiation; or other compounds effective in treating cancer or other diseases. Examples of other therapeutic agents that may be used with radiosensitizers include, but are not limited to, the following: 5-fluorouracil, folinic acid, 5 '-amino-5' deoxythymidine, oxygen, carbon-oxygen mixtures, blood transfusions, perfluorocarbons (e.g. Fluosol 10 DA), 2, 3-DPG, BW12C, calcium channel blockers, pentoxifylline (pentoxyfylline), anti-angiogenic compounds, hydralazine and LBSO. Examples of chemotherapeutic agents that may be used with radiosensitizers include, but are not limited to, the following: doxorubicin, camptothecin, carboplatin, cisplatin, daunomycin, docetaxel, adriamycin, interferon (α, β, γ), interleukin 2, irinotecan, paclitaxel, rocarb, and therapeutically effective analogs and derivatives thereof.

Chemotherapeutic sensitizers may be used with a therapeutically effective amount of one or more other compounds, including but not limited to the following: a compound that promotes binding of the chemotherapeutic sensitiser to the target cell; a compound capable of controlling the flow of therapeutic agents, nutrients and/or oxygen towards the target cells; chemotherapeutic agents that act on tumors or other compounds that are effective in treating cancer or other diseases. Examples of other therapeutic agents that may be used with chemotherapeutic sensitizers include, but are not limited to, the following: 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 and recognize PARP, in particular the PARP-1 receptor. In use for this purpose, the compounds of formula (I) may be labelled. The label may be selected from the group consisting of a radioisotope, a spin label, an antigenic label, an enzymatic label, a fluorescent group or a chemiluminescent group.

The effective amount can be readily determined by one skilled in the art from the experimental results set forth below. Generally, an effective amount of 0.001-100 mg/kg body weight, preferably 0.005-10 mg/kg body weight, is recommended. The dosage required for one day may be suitably divided into two, three, four or more sub-portions for use at appropriate intervals. The aliquots may be prepared in unit dosage forms, e.g. containing from 0.05 to 500 mg, preferably from 0.1 to 200 mg of active ingredient per unit dose.

Experimental part

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

A. Preparation of intermediate compounds

Example A1

Preparation of intermediate 1

A mixture of 2- [ (4-chloro-1-oxobutyl) amino ] -benzamide (0.03mol), 4-phenyl-4-piperidinecarboxamide (0.03mol) and TEA (0.1mol) dissolved in acetonitrile (150ml) was stirred and refluxed overnight, and after evaporation of the solvent, water was added to the residue. The oil layer was extracted with chloroform, dried, filtered and the solvent removed by evaporation. The residue was purified over silica gel on a glass filter (eluent: chloroform/MeOH 95/5). The product fractions were collected and the solvent was evaporated to yield 5g (40.8%) of intermediate 1.

Example A2

Preparation of intermediate 6

A mixture of 4-chloro-1, 1-diethoxy-butane (0.05mol), 4-phenyl-4-piperidinecarboxamide (0.03mol), sodium carbonate (0.05mol) and potassium iodide (q.s.) in 4-methyl-2-pentanone (250ml) was stirred and refluxed overnight, the reaction mixture was cooled, water was added and the layers were separated. The organic layer was dried, filtered and the solvent evaporated to give 9g (86%) of intermediate 6.

B. Preparation of the Final Compounds

Example B1

Preparation of Compound 3

A mixture of 2- [ (1-oxo-2-propenyl) amino ] -benzamide (0.02mol), 1- (4-fluorophenyl) -2- (4-piperidinyl) -ethanone (0.02mol) and sodium bicarbonate (4g) in 2-propanol (150ml) was stirred and refluxed overnight, the reaction mixture was filtered while hot and the solvent was evaporated off. The residue was purified twice by column chromatography over silica gel (eluent: chloroform/MeOH 95/5), the product fractions were collected and the solvent was evaporated off. The residue was recrystallized from 2-propanol and the resulting precipitate was collected to give 2g (25%) of compound 3, melting point 159.1 ℃.

Example B2

Preparation of Compound 4

Intermediate 1(0.012mol) was dissolved in sodium hydroxide (60ml) and ethanol (100ml), and the resulting mixture was stirred and refluxed for 1.5 hours, then the solvent was removed by evaporation and water was added to the residue. The oil layer was extracted with chloroform, dried, filtered and the solvent removed by evaporation. The residue was recrystallized from MIK/2-propanol and the desired product was collected and dried to yield 2g (41.7%) of compound 4 having a melting point of 138.9 ℃.

Example B3

Preparation of Compound 5

A mixture of 2-amino-benzamide (0.027mol) and intermediate 6(0.027mol) dissolved in ethanol (100ml) was heated and the mixture was acidified with chemically pure hydrochloric acid (3 ml). The reaction mixture was stirred and refluxed overnight, then the solvent was evaporated off, and water and concentrated NH were added to the residue₄OH solutionAnd (4) liquid. Filtering to obtain precipitate, dissolving in chloroform, drying, and filtering. After evaporation of the solvent, the residue was recrystallized from MeOH, the resulting precipitate was collected and dried to yield 3g (28%) of compound 5, melting point 216.1 ℃.

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

Pharmaceutical embodiment

In vitro PARP-1 inhibitory Activity Using the proximity scintillation assay (SPA)

The compounds of the invention were tested in vitro according to SPA technology (owned by Amersham Pharmacia Biotech).

In principle, the present assay relies on the well established SPA technique to detect poly (ADP-ribosyl) ation on biotinylated target proteins (i.e., histones). Using PARP-1 enzyme and³H]-nicotinamide adenosine dinucleotide (,)³H]-NAD⁺) The activated nicked DNA acts as an ADP-ribosyl donor to induce the ribosylation reaction.

A nicked DNA was prepared as an inducer for inducing the activity of PARP-1 enzyme. For this, 25mg of DNA (supplier: Sigma) was 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 a DNAse solution (1mg/ml in 0.15M NaCl) was added. After incubation at 37 ℃ for 90 minutes, the reaction was stopped by adding 1.45g of NaCl and followed by an additional incubation at 58 ℃ for 15 minutes. The reaction mixture was cooled on an ice bath and dialyzed against 0.2M KCl 1.5L for 1.5 hours and 2 hours, respectively, at 4 ℃ and then against 0.01M KCl 1.5L for 1.5 hours and 2 hours, respectively, twice to portionwise and store at-20 ℃. Biotinylation reaction set with AmershamTissue protein (1mg/ml, type II-A, supplier: Sigma) was biotinylated and aliquoted, stored at-20 ℃. A stock of 100mg/ml SPA poly (vinyl toluene) (PVT) beads (supplier: Amersham) was prepared in PBS. 6ml of incubation buffer (50mM Tris/HCl, pH 8; 0.2mM DTT; 4mM MgCl)₂) To which 120. mu.l of a solution of [ mu ] l of a copolymer of [³H]-NAD⁺(0.1mCi/ml, supplier: NEN) made of³H]-NAD⁺And (4) storing the liquid. Preparation of 4mM NAD in incubation buffer (taken from 100mM stock stored at-20 ℃ in water)⁺(provider: Roche). The PARP-1 enzyme is prepared by techniques known in the art, i.e.asexual propagation and protein expression starting from human liver cDNA. Information concerning the protein sequence of the PARP-1 enzyme used, including references, can be found in the database of Swiss-Prot under the main accession number P09874. Biotinylated histones and PVT-SPA beads were mixed and pre-incubated for 30 minutes at room temperature. The PARP-1 enzyme (concentration batch-wise) was mixed with the nicked DNA and the mixture was pre-incubated at 4 ℃ for 30 minutes. An aliquot of this histon/PVT-SPA bead solution was mixed with a PARP-1 enzyme/DNA solution, and 75. mu.l of this mixture was mixed with 1. mu.l of the compound of the present invention dissolved in DMSO and 25. mu.l of³H]-NAD⁺Added together to each well on a 96 well microtiter plate. The final concentrations of the various components in the incubation mixture were: the concentration of biotinylated histones was 2. mu.g/ml, the concentration of PVT-SPA beads was 2mg/ml, the concentration of nicked DNA was 2. mu.g/ml and the concentration of PARP-1 enzyme was 5-10. mu.g/ml. After incubating the mixture at room temperature for 15 minutes, 100. mu.l of 4mM NAD was added to the incubation buffer (final concentration of 2mM)⁺To stop the reaction and mix the plates.

After allowing the beads to settle for at least 15 minutes, the plate was moved to ToppCountNXT^TM(Packard) scintillation counting was performed and the resulting value is expressed as the number of flashes per minute (cpm). For each experiment, a control group (containing PARP-1 enzyme and DMSO without compound), a blank culture solution (containing DMSO without PARP-1 enzyme and compound), and a sample group (containing PARP-1 enzyme and compound dissolved in DMSO) were tested in parallel. All compounds tested were dissolvedSolubilized in DMSO, and finally further diluted with DMSO. In a first embodiment, the compound is at 10^-5The test was performed at the concentration of M. When the compound is at 10^-5M shows activity at the tested concentration range of 10 for the compound^-5-10^-8Dose-response curves are plotted between M. At each test, the test value of the blank was subtracted from the test values of the control and sample groups. Control samples represent maximal PARP-1 enzyme activity. For each sample, the cpm amount was expressed as a percentage of the average cpm value of the control. Suitably, the IC is calculated in computer using linear interpolation at 50% above and below the experimental point₅₀Value (drug concentration, used to reduce the activity of PARP-1 enzyme to 50% of control). Here, pIC was used to test the effect of the compounds₅₀(IC₅₀Negative logarithm of value). As a reference compound, 4-amino-1, 8-naphthalimide was also used to validate the SPA experiments. The compounds tested were at an initial experimental concentration of 10^-5M, the inhibitory activity was exhibited (see Table-2).

In vitro PARP-1 inhibitory Activity assays Using filtration assays

Use 2³²P]NAD as ADP-ribosyl donor, tested in vitro in a filtration experiment by its histon poly (ADP-ribosyl) activity, to evaluate the activity of PARP-1 (triggered in the presence of nicked DNA). The ribosylated tissue protein having radioactivity was precipitated with trichloroacetic acid (TCA) on a 9696-well microtiter plate, and the amount of the added [ alpha ], [ beta ] -was measured with a scintillation counter³²P]。

Tissue protein (stock solution: H)₂Concentration in O5 mg/ml), NAD⁺(stock solution: H)₂O concentration 100mM) and incubation buffer (50mM tris/HCl, pH 8; 0.2mM DTT; 4mM MgCl₂) The term³²P]-NAD⁺A mixture is made. A mixture of PARP-1 enzyme (5-10. mu.g/ml) and nicked DNA was prepared simultaneously. Nicked DNA was prepared as described in the SPA method for determining in vitro PARP-1 inhibitory activity. Mu.l of the PARP-1 enzyme/DNA mixture was mixed with 1. mu.l of the present solution in DMSOInventive Compounds and 25. mu.l of cathepsin-NAD⁺/[³²P]-NAD⁺The mixture was added together to each well of a 96-well filter plate (0.45 μm, supplier: Millipore). The final concentration of tissue protein in the incubation mixture was 2. mu.g/ml, NAD⁺Is 0.1mM, and³²P]-NAD⁺the final concentration of (2) was 200. mu.M (0.5. mu.C) and the final concentration of nicked DNA was 2. mu.g/ml. The plates were incubated at room temperature for 15 minutes, quenched by the addition of 10. mu.l of ice-cold 100% TCA, followed by the addition of 10. mu.l of ice-cold BSA solution (1% strength in water). The protein fraction was allowed to settle at 4 ℃ for 10 minutes and the plate was then vacuum filtered. Each well of the plate was washed sequentially with 1ml of 10% ice-cold TCA, 1ml of 5% ice-cold TCA and 1ml of 5% TCA at room temperature. Finally, 100. mu.l of scintillation solution (Microscint 40, Packard) was added to each well and the plates were transferred to TopCount NXT^TM(Packard) scintillation counting was performed and the resulting value is expressed as the number of flashes per minute (cpm). For each experiment, a control group (containing PARP-1 enzyme and DMSO without compound), a blank culture solution (containing DMSO without PARP-1 enzyme and compound), and a sample group (containing PARP-1 enzyme and compound dissolved in DMSO) were tested in parallel. All compounds tested were dissolved in DMSO and finally further diluted with DMSO. In a first embodiment, the compound is at 10^-5The test was performed at the concentration of M. When the compound is at 10^-5M shows activity at the tested concentration range of 10 for the compound^-5-10^-8Dose-response curves are plotted between M. At each test, the test value of the blank was subtracted from the test values of the control and sample groups. Control samples represent maximal PARP-1 enzyme activity. For each sample, the cpm amount was expressed as a percentage of the average cpm value of the control. Suitably, the IC is calculated in computer using linear interpolation at 50% above and below the experimental point₅₀Value (drug concentration, used to reduce the activity of PARP-1 enzyme to 50% of control). Here, pIC was used to test the effect of the compounds₅₀(IC₅₀Negative logarithm of value). As a reference compound, 4-amino-1, 8-naphthalimide was also used to validate the SPA experiments. The compounds tested were at the initial experimental concentration of10^-5M, the inhibitory activity was exhibited (see Table-2).

In vitro TANK-2 inhibitory Activity determination by proximity scintillation assay (SPA)

The compounds of the invention were tested according to SPA technology (owned by Amersham pharmacia Biotech) using Ni Flash plates (96 or 384 wells).

In principle, the test relies on the SPA technique, with [ 2 ]³H]-nicotinamide adenosine dinucleotide (,)³H]-NAD⁺) As ADP-ribosyl donors, self-polymerization (ADP-ribosyl) of TANK-2 protein was examined.

-NAD⁺Preparation of NAD stock: the [ 2 ] solution of 64.6. mu.l³H]-NAD⁺(0.1mCi/ml, supplier: Perkin Elmer) and 46.7. mu.l of NAD-stock (10.7mM, stock at-20 ℃ C., supplier: Roche) were added to 1888.7. mu.l of assay buffer (60mM Tris-HCl, pH 7.4; 0.9mM DTT, 6mM MgCl)₂). The TANK-2 enzyme was produced according to the method of EP 1238063. To each well of a 96-well nickel-coated scintillation plate (Perkinelmer), 60. mu.l of an assay buffer, 1. mu.l of a compound dissolved in DMSO, and 20. mu.l of³H]-NAD⁺NAD and 20. mu.l of TANK-2 enzyme (final concentration 6 mg/ml). After 120 min incubation at room temperature, 60. mu.l of stop solution (42.6 mg of NAD in 6ml of water) was added, the scintillation plate was covered with a sealing plate, and the plate was placed in TopCount NXT^TMScintillation counting was performed in (Packard) and the resulting value is expressed as number of flashes per minute (cpm). For each experiment, a control group (containing TANK-2 enzyme and DMSO without compound), a blank medium (containing DMSO without TANK-2 enzyme and compound), and a sample group (containing TANK-2 enzyme and compound dissolved in DMSO) were tested in parallel. All compounds tested were dissolved in DMSO and finally further diluted with DMSO. In a first embodiment, the compound is at 10^-5The test was performed at the concentration of M. When the compound is at 10^-5M shows activity at the tested concentration range of 10 for the compound^-5-10^-8Dose-response curves are plotted between M. At each test, the null was subtracted from the test values for the control and sample groupsTest values for white samples. Control samples represent maximal TANK-2 enzyme activity. For each sample, the cpm amount was expressed as a percentage of the average cpm value of the control. Suitably, the IC is calculated in computer using linear interpolation at 50% above and below the experimental point₅₀Value (drug concentration, used to reduce the activity of TANK-2 enzyme to 50% of control). As reference compounds, 3-aminobenzamide and 4-amino-1, 8-naphthalimide were also used to validate the SPA experiments. The assays described herein were performed in 96-well plates. In the analysis using 384-well plates, the same final concentration and volume were used. If the results of the 96-well plate experiment are valid, these results are incorporated in Table-2, otherwise, the results listed are obtained from the 384-well plate experiment.

TABLE-2

Compound numbering	In vitro filtration analysis of PARP-1 pIC50	In vitro SPA analysis PARP-1 pIC50	In vitro SPA analysis TANK-2 pIC50
				1			＜5
2	5.303	6.414	＜5
				3	5.371	6.252	5.605
4	7.115	7.699	＜5
				5	6.523	7.64	＜5
6	5.669	6.75	5.129
				7	5.381	6.233	＞5
8	5.804	6.627	＜5
				9	5.243	6.214	＜5
10	5	6.376	5.674
				11	5.942	6.725	＜5
12	6.172	6.931	＜5
				13	5.332	6.497	6.028
14	6.263	7.232	＜5

These compounds can be further evaluated by assaying for chemo-and/or radiosensitivity in cells, assaying for inhibition of endogenous PARP-1 activity in cancer cell lines, and finally performing radiosensitization experiments in vivo.

Claims (13)

1. A compound of formula (I)

Or a pharmaceutically acceptable addition salt thereof, wherein,

the dotted line represents an optional bond;

x is > CH-or > CR²-, wherein R²Is aminocarbonyl;

-N...Y-is-NH-C (O) -;

R¹is hydrogen;

R³is hydrogen;

z is a group selected from

Wherein each R⁵、R⁶、R⁷And R⁸Independently selected from hydrogen or halogen; and is

L is selected from-C (O) -NH-or-C (O) -C_1-6A divalent radical of alkanediyl-; or

When X is > CR²Or L may be a direct bond when Z is a group of formula (b-11).

2. The compound of claim 1, wherein Z is a group of formula (b-1), (b-2), or (b-3).

3. A compound according to claim 1 or 2, wherein Z is a group of formula (b-1) or (b-2); and L is a divalent group-C (O) -NH-or when X is > CR²When-is, L may be a direct bond.

4. The compound of claim 3, wherein said compound is selected from the group consisting of compounds numbered 4, 5, 11, and 12:

compound 4 Compound 5

Compound 11 compound 12.

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 is intimately mixed with a compound as claimed in any one of claims 1 to 4.

7. Use of a compound of any one of claims 1 to 4 in the manufacture of a medicament for the treatment of a PARP mediated disease.

8. The use of claim 7, wherein said PARP mediated disease is a PARP-1 mediated disease.

9. Use according to claim 7 or 8, wherein the treatment involves chemosensitization.

10. Use according to claim 7 or 8, wherein the treatment involves radiosensitisation.

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 process for preparing a compound according to claim 1, characterized in that

a) Cyclizing an appropriate substituted 2-aminocarbonylbenzamide of formula (II) to give a compound of formula (I-a),

b) cyclisation of an appropriate intermediate of formula (IV) with an intermediate of formula (III) gives a compound of formula (I-b), wherein-C_1-6alkanediyl-is-CH₂-CH₂-, or

c) Cyclisation of the appropriate substituted 2-aminobenzamide of formula (V) using an intermediate of formula (VI) gives a compound of formula (I-c),

13. use of a compound according to any one of claims 1 to 4 in the manufacture of a medicament for the treatment of cancer.