Poly(ADP-ribose) polymerase inhibition in cancer therapy: are we close to maturity?

Review

10.1517/13543770903215883 © 2009 Informa UK Ltd ISSN 1354-3776 1377All rights reserved: reproduction in whole or in part not permitted

Poly(ADP-ribose)polymeraseinhibitionincancertherapy:areweclosetomaturity?Gianluca Papeo, Barbara Forte, Paolo Orsini, Claudia Perrera, Helena Posteri, Alessandra Scolaro & Alessia Montagnoli††Nerviano Medical Sciences, Viale Pasteur 10, 20014 Nerviano (MI), Italy

Background: During the last few years an increasing number of poly(ADP-ribose) polymerase (PARP) inhibitors have been appearing in the context of cancer therapy. This is mainly due to a better knowledge of the best-characterized member of the PARP family of enzymes, PARP-1, further reinforced by the recognition of the clinical benefits arising from its inhibition. Objective/method: The aim of this review is to give the reader an update on PARP inhibition in cancer therapy, by covering both the scientific (SciFinder® search) and the patent literature (Chemical Abstract®/Derwent® search) published recently (2005 – 2008). Conclusions: More patient-compliant orally available PARP-1 inhibitor clinical candidates, along with their possible use as single agents in specific, molecularly defined cancer indications, increase the expectations for this therapeutic approach. The growing understanding of the biological role of other PARPs, such as Tankyrase 1, may be of interest as new potential targets. Besides the classical NAD-mimicking pharmacophore, additional compounds, which either do not resemble nicotinamide or exploit different binding sites, are emerging.

Keywords: BRCA, cancer therapy, PARP inhibitors, poly(ADP-ribose) polymerase, Tankyrase

Expert Opin. Ther. Patents (2009) 19(10):1377-1400

1. Introduction

1.1 Thepoly(ADP-ribose)polymerasefamilyPoly(ADP-ribose) polymerases (PARPs) belong to a family of 17 members (Figure 1) [1] that catalyze the addition of ADP-ribose units to DNA or different acceptor proteins, which affect cellular processes as diverse as replication, transcription, differentiation, gene regulation, protein degradation and mitotic spindle maintenance [2,3].

Poly(ADP-ribose) polymerase-1 and PARP-2 are the only enzymes among the PARPs that are activated by DNA damage and are involved in the repair of DNA single strand breaks (SSBs) [4].

Poly(ADP-ribose) polymerase-1 is the most abundant isoform of the PARP enzyme family [5]. It is a nuclear protein consisting of three domains: the N-terminal DNA binding domain (DBD) containing two zinc fingers, the auto modification domain and the C-terminal catalytic domain. The crystal structure of PARP-1 catalytic domain has been solved [6].

Poly(ADP-ribose) polymerase-1 acts by binding through the zinc-finger domain to DNA SSBs [7] followed by cleavage of NAD+ and resulting in the attachment of multiple ADP-ribose units to several proteins such as histones and various DNA repair enzymes. This results in a highly negatively charged target, which in turn leads to the unwinding and repair of the damaged DNA through the base excision repair pathway. In knockout mouse models, deletion of PARP-1 impairs DNA repair but it is not embryonic lethal [8,9]. These mice were hypersensitive

1. Introduction

2. Poly(ADP-ribose) polymerase

inhibitors

3. Expert opinion

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Poly(ADP-ribose)polymeraseinhibitionincancertherapy:areweclosetomaturity?

1378 ExpertOpin.Ther.Patents(2009) 19(10)

PARP-1

PARP-2

PARP-3

BRCT

BRCT

DNA binding

DNA binding

Znf Znf

Auto modificationdomain

PARP catalytic VWA

SAM2

SAM2 PARP catalytic

PARP catalytic

PARP catalytic

PARP catalyticPARP regulatory

PARP regulatory

PARP regulatory

PARP catalytic

PARP catalytic

PARP catalytic

PARP catalytic

PARP catalytic

Myc binding

MACRO1MACRO1

MACRO1

MACRO1

MACRO2

MACRO2

MACRO3

PARP catalytic

PARP catalytic

PARP catalytic

PARP catalytic

PARP catalytic

PARP catalytic

PARP catalytic

WGR

WGR

WGR

Ankyrin repeats

Ankyrin repeats

PARP-6

PARP-8

PARP-10 RRM

PARP-11

PARP-12

PARP-13

PARP-14

PARP-15

PARP-16

vParp(PARP-4)

Tankinase 1(PARP-5a)

Tankinase 2(PARP-5b)

PARPT(PARP-7)

PARP-9(BAL)

Zn finger

BRCT

WGR

VIT

VWA

SAM2

RRM

PARP regulatory,PARP catalytic

Ankyrin repeatsZnf

Znf Znf Znf

ZnfZnf

Znf

Znf

Ubiquitin-interactingmotif

MACRO

WWE

WWE

WWE

WWE1 WWE2

WWE

WWE

VIT

PADR1

PADR1

Figure1. Schematicdomainarchitectureof the17membersofPARP superfamily. Protein domains are drawn to scale with respect to full-length protein sequence; low complexity regions have been omitted. The architecture and boundaries of the domains are derived from Pfam 23.0 database [142]. A brief description of the most significant domains/motifs follows: The Znfingers can be either DNA binding or RNA binding (putative) such as those present in Parpt, Parp12 and Parp13. PADR1 domain, present in Parp1, is of unknown function. BRCT (BRCA1 C terminus) domain is a protein–protein interaction domain and is found predominantly in proteins involved in checkpoint function following DNA damage. WGR domain is present in polyA polymerases and it might be involved in DNA binding. VWA (von Willebrand factor A) domain seems to be a metal ion binding site, involved in protein binding. VIT (Vault protein Inter-α-Trypsin) is associated with the vault particles, large ribonucleoproteins whose function is still unknown. Ankyrin repeats mediate protein–protein interaction. SAM2 (sterile-α-motif) is a putative protein–protein interaction domain. WWE is predicted to mediate specific protein–protein interactions in ubiquitin and ADP ribose conjugation systems. MACRO domains are high-affinity ADP-ribose binding modules. UIM (ubiquitin-interacting motif) is known to interact with ubiquitin. RRM is probably an RNA binding motif.PARP: Poly(ADP-ribose) polymerase.

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Papeo,Forte,Orsini,Perrera,Posteri,Scolaro&Montagnoli

ExpertOpin.Ther.Patents(2009) 19(10) 1379

to ionizing radiation and alkylating agents while showing protection against various inflammatory processes such as ischemia [10].

Poly(ADP-ribose) polymerase-1 is responsible for the majority of the DNA damage dependent PARP activity (> 90%). The residual activity in PARP-1-deficient cells is due to PARP-2 [11,12], which is a nuclear protein that shares 68% homology with PARP-1 in its catalytic domain. The crystal structure of murine PARP-2 is very similar to that of PARP-1, with the exception of differences in a region close to the acceptor site [13]. Poly(ADP-ribose) polymerase-2 knockout mice are viable and show peculiar phenotypes that include infertility and defects in T-lymphocyte devel-opment and in fat storage capacity, while mice deficient in both PARP-1 and PARP-2 die during early embryogenesis, suggesting that the two enzymes do not display completely overlapping functions [4,14-16]. In particular, PARP-2, and not PARP-1, is an important mediator of T-cell survival during thymopoiesis by preventing the activation of DNA damage dependent Noxa mediated apoptotic response, during the multiple rounds of rearrangements preceding a positively selected T-cell receptor [17]. In addition, an exacerbation of cell death is observed in PARP-2 knockout mice and in hippocam-pal slices treated with selective PARP-2 inhibitors indicating a PARP-2-specific role in post-ischemic brain damage [18,15].

In contrast to PARP-1 and PARP-2, PARP-3 activity is not linked to DNA damage but could instead have a role in the regulation of gene expression, being part of the Polycomb group proteins responsible for epigenetic modifications leading to gene silencing [19].

Tankyrase 1 (PARP5a) was identified by its ability to bind telomere repeat-binding factor 1 (TRF1), a positive regulator of telomere length, through the ankyrin (ANK) repeats C-terminal domain. Tankyrase 1 poly ADP-ribosylation of TRF1 causes its release from telomeres and its proteolytic degradation. In agreement with this observation, Tankyrase 1 overexpression results in telomere elongation and its inhibi-tion induces telomere shortening, indicating that Tankyrase 1 may be a possible therapeutic target in cancer [20,21]. The function of Tankyrase 1 is partially redundant with that of Tankyrase 2 (PARP5b) [22].

Among the other less well-characterized PARPs, of special interest are the macro-PARPs (PARP-9, PARP-14 and PARP-15), also designated as B aggressive lymphoma (BAL) proteins, that are overexpressed in lymphoma and that may play a role in malignant B-cell migration [23].

1.2 Poly(ADP-ribose)polymeraseinhibitionEarly discovered PARP inhibitors included nicotinamide (NA) and 3-aminobenzamide (3-AB) that showed activity on PARP in the micromolar range. The next generation of inhibitors shows a higher potency and specificity and generally serve as competitors for NAD+ binding.

The first clinical application for PARP inhibitors in cancer therapy was in combination with DNA damaging

agents (e.g., temozolomide [TMZ], platinums, topoisomerase inhibitors and radiation). In fact, these cytotoxic agents induce PARP-1 activity for the repair of DNA damage that they generate, allowing tumor cells to withstand genotoxic stress. As a consequence, inhibition of PARP-1 through small molecules sensitizes tumor cells to DNA damaging agents [24-29]. Whereas the therapeutic use of PARP inhibitors in combina-tion with DNA damaging agents is not novel, the use of these agents as monotherapy in tumor genetic backgrounds deficient in specific DNA repair pathways represents a new approach. In 2005, two papers demonstrating the preferential sensitivity of breast cancer-1 (BRCA-1), early onset and BRCA-2 defi-cient cells to different PARP inhibitors given as single agents were published [30-32]. BRCA proteins are necessary for the accurate repair of DNA double strand breaks through the homologous recombination repair (HR) pathway [33-36]. It is known that on PARP-1 inhibition, base excision repair is reduced and single strand breaks that are generated during the normal cell cycle persist. In normal cells, replication forks that encounter single breaks can form double strand breaks that are repaired by HR without introducing errors [37].

Individuals with heterozygous germ line mutations in either the BRCA-1 or BRCA-2 genes show high risks of developing breast and other cancers throughout their lifetime [38-40]. Tumors arising in mutation carriers have generally lost the wild-type allele and do not express functional BRCA-1 and BRCA-2 proteins. Tumor cells derived from these patients are highly sensitive to PARP inhibition compared with wild-type or heterozygous cells because double strand breaks can-not be properly repaired. This is in line with the concept of synthetic lethality, in which defects within two independent pathways have no effect but become lethal when combined [41]: PARP inhibitors may be more effective in patients with tumors with specific DNA repair defects without affecting normal heterozygous tissues. Besides BRCA mutants that represent the majority of hereditary breast and ovarian cancer, a substantial fraction of sporadic cancers with defects in homologous recombination repair might also extend the putative patient population [42,43]. This phenomenon, termed ‘BRCAness’, includes, for example, methylation of the pro-moters of the BRCA-1 [44-46] or FANCF genes [47] and ampli-fication of the EMSY gene [48-50], which encodes a BRCA-2 interacting protein. By extending the rationale of synthetic lethality of PARP and BRCA-1 and BRCA-2, it is likely that deficiencies in any gene that is not redundant in double strand break repair should be sensitive to PARP inhibition. For example, ATM deficiency, found in patients with T-cell pro-lymphocytic leukemia, B-cell chronic lymphocytic leukemia and breast cancer [51,52] or CHK2 germ line mutations iden-tified in sarcoma, breast cancer, ovarian cancer and brain tumors [53] have also been shown to be synthetically lethal in combination with PARP deficiency as well as deficiencies in other known HR pathway proteins (including RAD51, DSS1, RAD54, RPA1, NBS1, ATR, CHK1, FANCD2, FANCA and FANCC) [54,55].

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There has been increasing evidence that PARP-1 is also involved in the inflammatory response. Pro-inflammatory stimuli trigger the release of pro-inflammatory mediators that induce the production of peroxynitrate and hydroxyl radicals, which in turn yield DNA SSBs with consequent over-activation of PARP-1. This results in depletion of NAD+ and energy stores, culminating in cell dysfunction and necrosis or apoptosis through the release of a mitochondrial pro-apoptotic protein called apoptosis-inducing factor (AIF) [56]. The choice between the activation of the PARP dependent DNA repair or cell death induction is guided by the extent of the DNA damage. This cellular suicide mechanism has been implicated in the pathomechanism of stroke, myocardial ischemia, diabetes, diabetes-associated cardiovascular dysfunc-tion, shock, traumatic CNS injury, arthritis, colitis, allergic encephalomyelitis and various other forms of inflammation where PARP inhibitors can offer therapeutic benefit.

Poly(ADP-ribose) polymerase-1 also regulates the expression of different proteins at the transcriptional level, loosening up the chromatin structure and making it more accessible to the transcriptional machinery. In particular, PARP-1 activates NF-κB-mediated transcription, which plays a central role in the expression of inflammatory cytokines, chemokines [57] and inflammatory mediators confirming an application for PARP inhibitors also in the cardiovascular and inflammatory disease area.

1.3 Poly(ADP-ribose)polymeraseinhibitorsinclinicaltrialsThe first clinical evidence that BRCA-mutated cancer may be sensitive to PARP inhibitor monotherapy comes from the preliminary data for the Phase I trial of the oral, small molecule PARP inhibitor AZD2281 [58,59]. This compound, developed by KuDOS/AstraZeneca, is a potent inhibitor of both PARP-1 and PARP-2 activities in vitro (PARP-1 Ki = 5 nM; PARP-2 Ki = 1 nM; PARP5a IC50 = 1.5 μM) with preclinical antitumor activity as a single agent only in HR deficient models [60,61]. The Phase I trials consisted of a patient population enriched for hereditary BRCA mutation carriers. Dose levels rose from 10 to 600 mg/day for 2 out of 3 weeks. Substantial PARP inhibition was seen in surro-gate PBMCs and tumor tissue as a confirmation of the mechanism of action at doses > 60 mg b.i.d. At these doses and schedules, antitumor activity was seen in ovarian, breast and prostate cancer patients with 46% of ovarian cancer patients showing a response (combined RECIST and GCIG CA-125 criteria) with a median duration of 6 months. Dose-limiting toxicities were reversible neurocognitive disturbance and myelosuppression. AZD2281 is currently in Phase II trials as monotherapy in patients with advanced BRCA associated breast (NCT00494234) and ovarian cancer (NCT00494442) as well as in combination with cytotoxic agents (i.e., irinotecan, dacarbazine, gemcitabine, carboplatin, topotecan, cisplatin, paclitaxel, doxorubicin) in different clinical indications. The Phase II data presented at

the American Society of Clinical Oncology (ASCO) meeting this year confirmed that AZD2281 is well tolerated and highly active in BRCA deficient ovarian and breast cancers after continuous treatment at 400 mg b.i.d. in 28-day cycles (objective response rates: 33 and 38%, respectively) [62,63].

AG-014699 (PARP-1 and PARP-2 Ki = 1.4 and 0.17 nM, respectively) from Pfizer was the first PARP inhibitor to be evaluated in cancer patients [64]. A Phase II combination trial with TMZ has been completed in patients with meta-static malignant melanoma with 18% partial responses seen, which corresponds to doubling compared to regression rates of TMZ [65]. Toxicities observed were an exacerbation of TMZ related toxicities, mainly thrombocytopenia and neutropenia. A Phase II study in breast and ovarian cancer in patients carrying the BRCA mutations is currently continuing (NCT00664781).

BSI-201 (BiPar/Sanofi-Aventis) is currently in Phase II clinical trials both in combination with gemcitabine and carboplatin in triple negative breast cancer and as a single agent in BRCA deficient ovarian, fallopian tube and perito-neal cancer (NCT00677079) using an intravenous adminis-tration. Preliminary results from Phase I studies show low toxicity and a wide therapeutic window with gastrointestinal toxicity as the main adverse effects. Of 24 patients, 6 had stable disease for > 2 months with 1 subject showing stable disease for > 9 months.

INO-1001 (Inotek) has been shown to be effective in Phase II in acute heart attack patients undergoing primary percutaneous coronary intervention [66-68]. It is also being studied in Phase I in combination therapy in metastatic melanoma [69].

ABT-888 (Abbott) is an oral PARP inhibitor (PARP-1 and PARP-2 Ki = 5 nM) studied in Phase 0 to determine the dose range at which PARP is effectively inhibited both in peripheral white blood cells and tumor cells and to deter-mine the pharmacokinetic parameters [70]. Phase I studies are conducted in combination with DNA damaging agents (i.e., TMZ, carboplatin, topotecan radiation, irinotecan, cyclophosphamide, paclitaxel) in solid tumors and hemato-logic cancer or as single agent in BRCA mutation carriers. The combination with TMZ is well tolerated and a Phase II trial evaluating the efficacy of this combination versus TMZ alone is currently continuing in subjects with metastatic melanoma [71-73]. Other companies have entered PARP inhibi-tors in Phase I trials or are in different stages of preclinical development (Table 1).

Preclinical evidence and preliminary clinical data suggest that PARP inhibitors can have an application in different therapeutic areas in combination or as single agents. In par-ticular, the concept of the synthetic lethality, to be able to specifically kill cancer cells defective in one DNA repair pathway by inhibiting another, represents a major potential breakthrough in treating cancer cells specifically, thus reducing undesirable toxic effects. We await the results of continuing and future studies to see if these promising preliminary clinical findings are confirmed.

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Table1.PARPinhibitorsinclinicaltrials.

Compound/company Route Application Clinicalstatus

AG-014699/Pfizer

i.v. Melanoma, in combination with temozolomide (completed)Breast and ovarian cancer BRCA mutated, single agent

Phase II

INO-1001/Inotek

i.v. Patients undergoing heart surgeryMelanoma, glioblastoma, in combination with temozolomide (Phase I completed)

Phase II

AZD2281AstraZeneca/Kudos

Oral Breast and ovarian cancer BRCA mutated, single agentSolid tumors in combination with different cytotoxic

Phase II

BSI-201/BiPar(Sanofi-Aventis)

i.v. Ovarian, fallopian, peritoneal cancer BRCA mutated, single agentTriple negative breast cancer in combination with gemcitabine/carboplatinUterine carcinosarcoma in combination with carboplatin/paclitaxel carboplatin/paclitaxel

Phase II

ABT-888/Abbott

Oral Melanoma, in combination with temozolomideSolid tumors and lymphoid malignancies in combination or as single agent

Phase II

MK-4827/Merck

Oral Solid tumors + ovarian cancer BRCA mutated, single agent Phase I

BRCA: Breast cancer genes; PARP: Poly(ADP-ribose) polymerase.

1.4 Updateonstructuraldataregardingpoly(ADP-ribose)polymeraseenzymesA large number of PARP inhibitors have been developed so far and were designed to mimic, to some degree, the NA moiety of NAD+ and bind to the donor site of the catalytic domain of PARP enzyme (Figure 2).

The majority of this kind of inhibitors does not discriminate between PARP-1 and PARP-2 isoforms. The high degree of similarity of the catalytic domain between PARP-1 and PARP-2 can explain the extremely scarce number of compounds claimed to be PARP-1 or PARP-2 selective. The two catalytic domains share 68% homology and > 90% identity in proximity of the NAD+ binding site; the residues in the NA-ribose binding site (NI) of PARP-1, which are responsible for hydrogen bonding interactions with these inhibitors, are completely conserved in PARP-2. The first published selective inhibitors of PARP-1 and PARP-2 were discovered by Fujisawa by using a combination of X-ray analysis and homology modeling (human PARP-2 crystal structure is still missing) [74]. Fujisawa compounds belonging to the quinazolinone chemical class proved to be ≤ 38-fold selective for PARP-1, as exemplified by compound 1 (Figure 3), whereas quinoxaline derivatives (e.g., compound 3; Figure 3) was 10-fold more potent against PARP-2 than PARP-1. Both scaffolds bind in a canonical way, establishing hydrogen bonds between the amide/lactam moieties of the ligands and the Gly863 and Ser904 residues (PARP-1 numbering) of NI site of PARP [75]. Selectivity can be essentially ascribed to the different ability of the two scaffolds to bind to the enzyme regions, in the catalytic pocket, close to the donor site. In particular, the difference between the adenosine hydrophobic site in PARP-1 and in PARP-2 is responsible for selectivity. X-ray analysis of the catalytic domain of human PARP (hPARP) in complex with

compound 2 (Figure 3) showed that the side chain of Arg878 at the bottom of the AD site moves on inhibitor binding to avoid steric clashes of the fluorophenyl moiety of compound 2. This way a structural change is induced, which generates a new hydrophobic subsite in PARP-1. Replacement of Leu769 in hPARP-1 with Gly314 in hPARP-2 causes a loss of hydrophobicity thus leading to a lack of potency against PARP-2 [74].

On the other hand, quinoxaline derivative 3 was 10-fold more potent against PARP-2 than against PARP-1. Differences in the environment of the binding site of the phenyl group of the quinoxaline scaffold in PARP-1 and in PARP-2 are responsible for the observed selectivity. X-ray analysis high-lighted that the side chain of Glu763 in hPARP-1 does not freely move because of hydrogen bonds with neighboring residues Gln759 and Ala760, differently from the corre-sponding Gln308 in hPARP-2 that can accommodate the p-substituted phenyl moiety [74]. A structurally different PARP-2 selective inhibitor (compound 4, 60-fold more selective versus PARP-1; Figure 3) was also disclosed by Pellicciari et al. [76]. Possible reasons for the selectivity observed arise from docking studies of compound 4 on a model of catalytic domain of bovine PARP-1 and of the catalytic domain of mouse PARP-2. Authors ascribed the selectivity to certain differences found between these two catalytic domains (e.g., Glu764/Lys308 substitution, PARP-1/PARP-2 numbering). However, due to some differences within the amino-acid sequence of PARP catalytic domains among different species, it would be prefer-able, in a drug design process, to consider the non-conserved residues of the PARP catalytic domains in the context of the human species.

The first structure of the PARP catalytic domain of Tankyrase 1 (PARP5a) has been obtained for the human apo

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Tankyrase 1 (TANKS-1) [77]. The TANKS-1 PARP domain (residues 1,091 – 1,313) is similar to the previously reported crystal structure of PARP-enzymes. Significant variability, however, is found in the region surrounding the catalytic core: N-terminal α-helical domain of PARP-1 is missing in TANKS-1 and in the backside of the catalytic domain a 32-amino-acid loop in PARP-1 is replaced by a small loop in TANKS-1. Moreover, TANKS-1 catalytic domain has a short zinc binding motif that is present only in Tankyrase 1 and not in other members of the PARP family. The AD subsite of the donor site of TANKS-1 compared to the AD

subsite in PARP-1 is different. In particular the D-loop, which adopts an open conformation in PARP-1, resulted in a closed conformation in TANKS-1 (residue 1,197 – 1,211). Consequently inhibitors like 1 (Figure 2) would sterically clash with TANKS-1 D-loop when overlaid [77]. Interaction with this non-conserved D-loop could provide a route for the development of selective inhibitors, keeping in mind that the reported differences are probably amplified by the absence of a ligand in the apo form of Tankyrase 1.

Recently the PARP domain (residue 178 – 532) crystal structure of human PARP-3, in complex with some small

NO

H

OH

Ser-904 (hPARP-1)Ser-446 (hPARP-2)

Gly-863 (hPARP-1)Gly-405 (hPARP-2)

OH

Tyr-907 (hPARP-1)Tyr-449 (hPARP-2)

O

OH

Glu-988 (hPARP-1)Glu-534 (hPARP-2)

NAD+

DONOR site

ACCEPTOR site

P O

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OP

O

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OH OH

N

N

N+

NHO

H

O

OHOH

O

N

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H2N

NI site

PH siteAD site

Figure2.ActivesiteofPARP:thecatalyticdomaincanbegenerallydividedintotwosites,theDONORsite,whereNAD+isaccommodated,andtheACCEPTORsite,occupiedbytheADPmoietyofthepoly-(ADP-ribose)chain. The DONOR site is further divided into three subsites, the nicotinamide-ribose binding site (NI site), the phosphate binding site (PH site) and the adenosine binding site (AD site) [75].PARP: Poly(ADP-ribose) polymerase.

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molecules inhibitors, was solved. Although the structures of the PARP domains of PARP-3 and PARP-1 are overall similar, they differ in the length and position of the donor site loops (D-loop). The PARP-3 D-loop is four residues shorter than those in PARP-1 giving again the possibility of a rationale design of selective inhibitors [78].

2. Poly(ADP-ribose)polymeraseinhibitors

2.1 Theoldandthenew:classicpharmacophoremotifsandbeyondA plethora of PARP inhibitors can be found in both scientific and patent literature. As mentioned in the previous section, the vast majority of them tend to replace NAD+ NA interac-tions at the donor site of the C-terminal catalytic domain of the protein. Competition with NA implies the inhibitors exploiting the classical interactions with Ser904 side chain and Gly863 (PARP-1 numbering) backbone through hydrogen bonds (Figure 2). Further π–π hydrophobic interactions of the aromatic portion of the inhibitor with Tyr896 and Tyr907 (PARP-1 numbering) reinforce the potency of the compound. Historically, the minimal pharmacophore suitable for playing

this game was represented by aromatic primary amides such as 3-AB or NA [79]. Recognition of the conformational flexi-bility of the carboxamide moiety, whose anty-syn equilibrium is thermodynamically driven towards the most stable biologically silent syn conformation (≅ 0.5 Kcal/mol) [80], resulted in the development of both lactams (bi- up to tetracyclic) and intramolecularly hydrogen-bonded locked primary amides. These approaches also succeeded in increasing the potency of the newly generated inhibitors by picking up additional interactions in the active site. This is facilitated by the accessible co-crystal structures with non-human [81] and, more recently, human PARP-1 catalytic domains [75]. Polycyclic lactams and specific substituents on known scaf-folds were claimed to get patentable structures in light of the extremely crowded field. More innovative approaches deal with the generation of aromatic N-oxide pro-drugs (see Section 2.7, Sentinel Oncology). The reductive environment notoriously present in hypoxic cancer cells restores the aromatic nitrogen lone pair that is responsible for locking the adjacent primary amide into the PARP-inhibitory active anti confor-mation. Scaffolds that do not present classical NA-like features started to appear on the PARP inhibitors’ arena quite

1

N

NH

O

N

F

Cl

2

IC50(PARP-1) = 9000 nMIC50(PARP-2) = 150 nM

4

NH

O

O O

N

NH

O

N

F



3

N

N

NH2

O

N


Figure3.PARP-1andPARP-2selectiveinhibitors.PARP: Poly(ADP-ribose) polymerase.

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5 6 7AZD2281

8 9 Ki = 0.6 nM

10 Ki = 39.6 nM 11 Ki = 1.2 nM 12 Ki = 191 nM

N

N

N

NH

O

F

O

ON

NH

O

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NH

O

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O

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4

N

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NN

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HN

O

HN

O

N

NH

O

N

O

N

NH

O

N

F

O

N

NH

O

F

N

O

Figure4.Evolutionofphthalazinoneinhibitors.

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recently, such as substituted coumarines that were identified using computer-assisted design (see Section 2.4, BiPar). Finally, new binding sites were envisaged in the DBD of the protein, namely the F1 zinc finger. Damage on F1 zinc finger resulted in the complete loss of enzymatic activity independently of the nature of the DNA breaks [82]. By establishing a cation-π interaction with the guanidinium moiety of the Arg34 present in this PARP-1 zinc finger, certain electron-rich aromatic molecules could act as inhibitors at this stage (see Section 2.4, BiPar). Alternatively, oxidation of the first zinc finger by some C-nitroso derivatives results in zinc ion ejection with the corresponding inactivation of PARP-1 activity [83].

The following sections give an overview of the efforts in the development of new PARP inhibitors, in which both companies and academies are active players. However, as the main focus of this review is the patent literature, we deliberately concentrate our attention on this field, notoriously more accessed by industrial institutions.

Sections are divided according to the companies whose patents were assigned. We have also added the synthetic schemes for those compounds that successfully reached the clinical stage.

2.1.1 KuDOS/AstraZenecaThe phthalazinone core structure 5 (IC50 = 12 μM; Figure 4) was elaborated for the first time in 2000 by Ono Pharmaceuticals [84] with the introduction of an aromatic

substitution in position 4, which led to a 300-fold increased potency for compound 6 (IC50 = 42 nM; Figure 4). Other 4-substituted phthalazinones were then disclosed by Icos, but it was KuDOS, at first in collaboration with Maybridge [85,86], who intensively worked on the series, introducing a methylene bridge between the scaffold and the aromatic moiety [87], which in the end resulted in the development of the potent inhibitor KU59436 (7; Figure 4) (then AZD2281 after acquisition of KuDOS by AstraZeneca) [59].

AZD2281 (7), disclosed explicitly in a 2004 patent [85], was then the subject of two more recent patents [88,89] where X-ray characterization of its crystalline form and improved methods of preparation are claimed.

This compound was chosen to be developed among some hundreds analogues, where 4-benzyl moiety was also het-eroaromatic, not only because of its higher potency but also for its great level of absorption and oral exposure, especially in dogs. The 4-fluorine substitution seems to enhance permeability (improved cellular potency over the corresponding 4-unsubstituted derivative but similar in terms of biochemical activity). Repulsive interactions between the fluorine atom and the adjacent carbonyl- group may be responsible, through the consequent restriction of the conformational flexibility, for the observed result.

The synthesis [59] is short and high-yielding. Key step is the formation of the phthalazinone core from the isobenzofura-nylidenemethylbenzonitrile intermediate and hydrazine hydrate (Scheme 1).

OH

O

O

H

O

O

PO

O

O

O

O

CN

F75:25 E:Z

i ii iii

iv

7

N

NH

O

F

OH

O

N

N

N

NH

O

F

O

O

Scheme1.SynthesisofAZD2281. i) Dimethylphosphite, NaOMe, MeOH, 0°C then CH3SO3H, 20°C (89%); ii) 2-fluoro-5-formylbenzonitrile, TEA, THF, 20°C (96%); iii) NaOH 13N, water, 90°C, 1 h, then hydrazine hydrate, 70°C, 18 h (77%); iv) 1-cyclopropylpiperazine- 1-ylmethanone, DIPEA, HBTU, DMA, room temperature, 16 h (67%).

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In 2008, improved potency and better solubility and stability were claimed [90] by KuDOS/straZeneca for compound 8 (PARP-1 IC50 = 3.8 nM; Figure 4). Racemic compound was claimed as epimerization of the enantiomers in solution was observed.

Recently, Abbott reported 491 tetrahydrophthalazinone derivatives, such as compounds depicted in Figure 4 (9 – 12), which inhibit PARP with a Ki value ranging from 0.6 to 9.2 μM [91]. Benzyl-substituted compounds (e.g., 9 – 11; Figure 4) substantially resemble the phthalazinones devel-oped by KuDOS. The presence of a fluorine atom onto the benzene ring and the methylene bridge are pivotal to improve potency in vitro against PARP (compare Ki of compounds 10 – 12).

In 2006, KuDOS, capitalizing on the phthalazinones chemical class, disclosed another group of PARP inhibitors, the benzyloxy amides series (Figure 5) [92,93]. The dissection of the heterocyclic ring in phthalazinones resulted initially in a detrimental effect for potency, as the intramolecular hydrogen bond between the aryl C-2 oxygen and benzamide NH is not strong enough to balance the corresponding loss in entropy. After medicinal chemistry investigation on the series, however, potent compounds were finally secured. 5-Fluorine substitution confers an enhancement of the in vitro potency (e.g., compound 13; Figure 5), whereas 4′-fluorine sub-stitution also potentiates cellular response (e.g., compounds 14 and 15; Figure 5). Best substitution in position 3′- is a piper-azinocarbonyl moiety as for AZD2281 (7) (e.g., compounds 14 and 15; Figure 5), without, however, reaching comparable levels of potency.

No information was provided about the activity displayed by these compounds towards other PARP isoforms.

To a subclass of benzyloxy amides, thiophene derivatives of formula 16 (Figure 5), was dedicated another recent patent [94]. Only some 30 compounds are claimed (comprising synthetic intermediates) with only generic biological data reported for some representatives of the series (IC50 < 2 μM and PF50 < 1.1 μM).

In a recent patent [95], AstraZeneca claimed the use of a Chk (Checkpoint kinase 1 – 2) inhibitor (specifically, thio-phenes patented by AstraZeneca itself, and among them AZD7762) in combination with a PARP 1 – 2 inhibitor for the treatment of cancer (specifically AZD2281, 7). In vitro and in vivo studies reported were designed to assess the antipro-liferative action against cancer cells. The in vivo results suggest significant tumor growth inhibition (TGI) when the two compounds are administered in combination (TGI 64% with AZD2281@50 mg/kg + AZD7762@25 mg/kg), while no statistically significant TGI was observed using either inhibitor as a single agent.

In the combination experiment AZD2281 (7) was admin-istered 5 times a week and AZD7762 was administered 2 h later twice a week. The mice (female nude with SW620 tumors) were treated for 3 weeks and the treatment was generally well tolerated.

2.1.2 Pfizer/Cancer Research TechnologyIn recent years, Pfizer has collaborated with Cancer Research Technology and these efforts resulted in the clinical candidate compound 17, which displays low nanomolar activity values (PARP-1 Ki = 1.4 nM; PARP-2 Ki = 0.17 nM) [96]. A series of patents reported the convergent synthesis (Scheme 2) of lactam 17 as free base [97] or salt derivative (to increase solubility) [98].

Key steps are i) Sonogashira coupling between a terminal alkyne already bearing the benzylamino-methyl moiety and the properly functionalized O-triflate benzoic acid methyl ester; and ii) copper mediated cyclization to form the indole ring. Compound 17 is reported to be very potent against the VC8 cell line that lacks BRCA-2, having an LC50 value of 12 nM.

2.1.3 BiParIn 2006, BiPar patented [99] electron-rich coumarin and indole derivatives (see compounds 18 – 23; Figure 6) as PARP-1 inhibitors. These molecules can bind to the F1 zinc finger of the DBD of the protein through a cation-π interaction with the guanidinium moiety of the Arg34.

This amino acid was demonstrated to be crucial for the interaction of PARP-1 with ATP, whose ability in inhibiting the protein at physiological concentration has been well-established. Authors claimed this new binding site will potentially avoid side effects observed with unselective NAD-competitive PARP-1 inhibitors. BiPar also claimed in 2007 [100] the use of the PARP inhibitor 4-iodo-3-nitrobenzamide (BA, 24; Figure 6) alone or in combination with another PARP inhibitor, 6-amino-5-iodobenzopyrone (25; Figure 6), or with buthionine sulfoximine (an inhibitor of glutanylcysteine synthetase, a key enzyme in the biosynthesis of glutathione). Compound 24 was reported to be able to inhibit, alone or in combination, the growth of several cancer cell lines. Complete inhibition of cellular proliferation was achieved, after 120-h exposure at a concentration ranging from 4 to 200 μM. Compound 24 was also demonstrated to possess antitumor efficacy in vivo as single agent on a human ovarian adenocarcinoma (OVCAR3) xenograft model in nude mice at 50 mg/kg i.p. b.i.d. (5 days of treatment with 2 days of rest before the start of the next cycle). More recently, BiPar also patented a computer-assisted method for designing PARP inhibitors [101]. Superimposition of well-documented PARP inhibitors whose co-crystal structures with the catalytic domain of the protein are known with 5-iodo-6-nitrocou-marin allowed for the design of new coumarin derivatives. The replacement of the iodine atom with proper substitu-ents generates compounds able to pick up more interactions within the NAD binding domain of the protein, thus delivering more efficient inhibitors. Preferred substituents (e.g., 26 – 28; Figure 6) somehow capitalized on PARP-1 inhibi-tors previously published by other companies (e.g., Fujisawa PARP-1 selective inhibitors; Figure 3). No biological data are reported in these patents.

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IC50 = 770 nM

Cellular activity enhancement

Enzimatic, notcellular, activityenhancement

14 IC50 = 14 nMPF50 = 1.5

15 IC50 = 12 nMPF50 = 1.12

16

13

O

N

NH NH2

O

O

1

2

5

3′

4′

NH2

O

O

F

N

O

O

HN

R

best R1 =

NH2

O

O

F

N

O

N

O

R1

F

O

NH2

O

O

F

N

O

N

O

R

S

Figure5.KuDOS/AstraZenecabenzyloxyamides.

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17 PfizerAG-014699

N

CO2Me

F

OMeO

NO2

N

CO2Me

ab

NH

HN

O

FN

F

OTf

OMeO

NO2

NHF N

MeO2C

CO2Me

H

Scheme2.SynthesisofAG-014699.

18 19 R = I; R1 = OH; R2 = H20 R = OH; R1 = I; R2 = H21 R = H; R1 = OH; R2 = I

22 R = I; R1 = OH; R2 = H23 R = H; R1 = OH; R2 = I

26 R =

27 R =

28 R =25

24

O O

I

HONH

R

R1

R2 NH

R

R1

R2

NH2

O

O

NO2

R

N

N(CH2)4

N N(CH2)4

O O

I

H2N

I

NO2

NH2

O

Figure6.BiParinhibitors.

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2.1.4 AbbottSeveral potent PARP inhibitors have been described in recent years containing a benzimidazole carboxamide scaffold [102-106]. The high intrinsic potency of this relatively small scaffold is due to an intramolecular hydrogen-bond that locks the carbox-amide moiety into the biologically active conformation thus mimicking the NA binding interactions in the PARP-1 active site. Contributing to the potency of this scaffold is also a π-stacking interaction with Tyr-907 [107] and a water-mediated hydrogen bond of 1-NH of the benzimidazole ring system with Glu-988 [108]. This scaffold was previously disclosed by the University of Newcastle (compound 29, NU-1085;

Figure 7) [79,109]. Incorporation of a basic amine at the 2-position of the benzimidazole ring system (compound 30; Figure 7) is reported to increase water solubility also giving, in many cases, an improvement in pharmacokinetic properties and in cellular potency, maintaining good enzyme affinity [108].

In agreement with the presence of a large pocket in the PARP-1 active site [110,111], alkylation on the nitrogen sub-stituent (e.g., compound 32; Figure 7), also with a sterically demanding group (e.g., compound 33; Figure 7), beneficially influenced the cellular potency of the compounds.

A quaternary carbon substitution at the 2-position of the benzimidazole ring resulted in a further increase of affinity

NU-1085Ki = 6 nM

Ki = 5 nMEC50 = 31 nM

Ki = 5 nMEC50 = >1000 nM

Ki = 3 nMEC50 = 21 nM

3332 A-620223

Ki = 8 nMEC50 = 3 nM

35 ABT-888

Ki = 5 nMEC50 = 2 nM

34

Ki = 30 nMEC50 = 16 nM

29 30 31

NH

N

NOH

H

OH

NH

N

NOH

H

NHNH

N

NOH

H

NH

NH

N

NOH

H

N N

NH

N

NOH

H

N

NH

NH

N

NOH

H

NH

N

NOH

H

NH

Figure7.Benzimidazolecarboxamideevolution.

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a b c

d e f

ABT-888

N

Cbz

O

MeO

NH2O

NH2

NH

ON

Cbz

NH

N

NH2O

N

Cbz

NH

N

NH2O

N

Cbz

N

Cbz

O

MeON

Cbz

O

HO

NH

N

NH2O

NH

Scheme3.SynthesisofABT-888. a) MeI, base, THF; b) LiOH, H2O, THF; c) 2,3-diaminobenzamide-HCl, CDI, DMF, pyridine; d) HOAc, heat; e) resolution by chiral HPLC (Chiralcel OD, 80/10/10 hexane/EtOH/MeOH), 99.4% ee; f) H2, 10% Pd/C.

for PARP (as exemplified by comparison of compound 34 and compound 35; Figure 7) as well as single-digit nanomolar cellular potency (C41 cells), oral bioavailability > 50% and good exposure [104,72]. The lead compound ABT-888 (35) is currently in Phase II clinical trials in combination with TMZ (NCT00804908). (R)-enantiomer was preferred to the (S)-enantiomer because of its superior oral bioavailability and exposure. Generally no significant differences in biochemical inhibitory activity against PARP-1 were observed between enantiomers, in agreement with X-ray data showing the absence of any specific interactions involving that portion of the inhibitor with the enzyme [72]. It showed moderate to high oral bioavailability, high plasma clearance and moderate volume of distribution in all species (mouse, rat, dog and monkey), T1/2 in the 1.2 – 2.7 h range and high water solu-bility (> 5 mg/ml) at physiological pH. In vitro data suggest that ABT-888 (35) displays excellent potency against PARP-1 and PARP-2 with a Ki value of ∼ 5 nM and was also potent in a C41 whole-cell assay with an EC50 value of 2 nM (Figure 7) [72]. In vivo ABT-888 (35) enhanced TMZ antitumor activity with no observed toxicity [72]. The synthesis of 35 is depicted in Scheme 3 [72]. The (R)-enantiomer was separated through preparative chiral HPLC.

Abbott is currently developing back-up series of the benzimidazole class where an aryl [105] or a heteroaryl spacer [106] is present between the scaffold and the hetero-cycle (e.g., compounds 36 – 38; Figure 8). The 2-fluoro substitution on the phenyl spacer together with a 3-piperidinyl or 2-pyrrolidinyl at the 4-position of the same phenyl ring

provided compounds with excellent activity (Ki < 10 nM; Figure 8) [105].

Among them, (S)-enantiomers were generally more potent than the corresponding (R)-enantiomers. Compound 38 (Figure 8) was highlighted for its potency, acceptable pharma-cokinetic properties in rats, dogs and monkeys, oral bioavail-ability (34% in monkeys, 71% in dogs) and improved brain penetration compared to ABT-888 (35) [112,113]. Abbott more recently disclosed tricyclic lactam scaffolds as potent PARP inhibitors (Figure 8) [114,115].

As far as the general bicyclic scaffolds A and B are concerned (Figure 8), both ring size and ring electron density proved to be crucial for potency [115]. Compounds with a quinox-alinone core (e.g., compound 39; Figure 8) were shown to be more potent than the ones with a benzodiazepinone core (e.g., compound 43; Figure 8). Furthermore, pyrroloquinox-alinone derivatives (e.g., compound 39; Figure 8) demon-strated higher affinity for PARP-1 than imidazole quinoxalinones (e.g., compound 41; Figure 8), tetrahydropyrrolo- (e.g., com-pound 40; Figure 8) and tetrahydropyrido-quinoxalinones (e.g., compound 42; Figure 8) [115].

Also derivatives belonging to the pyrazoloquinolone class [114] showed excellent inhibitory activity against PARP. A set of 249 compounds (e.g., compound 44; Figure 8) has been reported. Many of them were tested both in biochemical and cellular assays. Several derivatives proved to have Ki values in the single-digit range and also demonstrated penetration of cell membranes inhibiting PARP in intact cells with EC50 values, calculated on a C41 whole-cells assay, < 1 nM.

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Ki = 2 nM

36 37

38

39Ki = 6 nMEC50 = 5.4 nM

Ki = 280 nM

Ki = 1120 nM

Ki = 97 nM

Ki = 114 nM

40 41

42 43

A B

NH

NH

N

NOH

H

F

NH

N

NOH

H

F

N NH

N

NOH

H

F

N

HN

N

ON

HN

NN

ON

N

HNO

A1

A2

R1

R2

R3

R4

HN

N

O

A1 A2

R1

R2

R3

R4

N

N

O

NHN

N

NO

HN

NO

N

H H

H

Figure8.AbbottrecentlydisclosedPARPinhibitors.PARP: Poly(ADP-ribose) polymerase.

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Abbott also came up with novel pyrazoloquinazolinones (e.g., compound 45; Figure 8) as PARP inhibitors [116]. A total of 100 derivatives were prepared with Ki values ranging from 31 nM to values > 9.5 μM. Among them, 7 compounds have been tested in cellular assay (C41 cells) giving EC50 values between 1.1 nM and 1 μM.

2.1.5 Sanofi-AventisSanofi-Aventis reported a series of substituted pyridones as new inhibitors of PARP.

In two patent applications [117,118] they disclosed few hundred compounds with several derivatives with IC50 values < 100 nM such as compounds 46 and 47 (Figure 9).

Preferred substituents in position 3 and 6 of the pyridone ring seem to be ethyl and methyl while most of the derivatives contain aryl, heteroaryl and arylamino moieties in position 5. These substituents are installed by Suzuki-Miyaura or Buch-wald couplings and are then further functionalized with solubilizing groups. Cell based (HL60) fluorescence assay reveals for compound 46 an EC50 value of 20 nM. Experi-ments on male Sprague-Dawley and Fisher rats demonstrated the efficacy of compound 46 in treating patients suffering from myocardial ischemia and stroke.

2.1.6 Sentinel OncologySentinel Oncology has recently designed a series of quinoxa-lines N-oxide and benzo[1,2,4]triazines N-oxide as potential PARP inhibitors. Their invention [119] relates to compounds that inhibit or modulate the activity of PARP and that are activated under hypoxic conditions. A number of neoplasms

have hypoxic regions that may permit reduction of the N-oxides to the corresponding tertiary amines thus leading to compounds demonstrating preferential toxicity to malig-nant cells. Quinoxaline N-oxides can be considered tumor targeting agents that in areas of very low oxygenation are reduced to quinoxalines having already disclosed PARP-inhibiting activity [120,121]. The synthesis of a series of quinoxalines and benzotriazines is reported but just a few compounds are evaluated from a biological point of view. The best IC50 value was found for compound 48 (Figure 9), which shows low nanomolar potency in the biochemical assay.

2.1.7 Cylene PharmaceuticalsCylene Pharmaceuticals recently came up with a series of tricyclic lactam compounds active as PARP modulators that, in certain cases, show low nanomolar potency in biochemical assays. Two classes emerged from their patent [122] as the most representative: phenanthridin-6(5H)-ones (e.g., compound 49; Figure 9) and thieno[3,2-c]quinolin-4(5H)-ones (core scaffold of compound 50; Figure 9).

In both series, the presence of a polar group or a carboxylate bioisostere, a moiety expected to be negatively charged at physiological pH, in the south-east portion of the molecule is reported. Cylene Pharmaceuticals refers the use of these compounds as modulators in the treatment of diseases such as cancer and inflammatory disorders, in combination with known therapeutic agents that capitalize on a synergistic effect and, for this reason, can be administered at a lower dosage than when used as single agents.

Figure8.AbbottrecentlydisclosedPARPinhibitors(continued).PARP: Poly(ADP-ribose) polymerase.

X = NR4 44Pyrazoloquinolones

45

HN

N

XO

R1

R2

R3

HN

N

NO

N

NH

N

N

O

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46 IC50 = 80 nM 47 IC50 = 60 nM

48 IC50 = 13 nM

49 IC50 = 30 nM 50 IC50 = 20 nM

NH

O

S

SO2

N

N

3 5

6 NH

O

S

SO2

N

NH

N

N

NH2O Cl

NH

O

N N

N

HN

NH

O

S

N

O

NH2

Q2

Q1

Z1

Z2

Z3

Z4

α

H H

Figure9.RecentlydisclosedPARPinhibitors.PARP: Poly(ADP-ribose) polymerase.

2.1.8 MGI Guilford PharmaceuticalsFollowing previous Guilford expertise in discovering PARP inhibitors, MGI Guilford Pharmaceuticals (GP) disclosed new potent tri- and tetracyclic lactams [123]. Tetracyclic derivatives are chromeno[4,3,2-de]phthalazin-3(2H)-one com-pounds bearing a solubilizing group in position 10 of the scaffold. Several compounds show potency in the double-digit nanomolar range (such as for compound 51; Figure 10) and are also characterized for their in vivo efficacy activity. In mice bearing B16 melanoma, a 5-day treatment with compound 51 (two doses: 10 and 40 mg/kg p.o.) in

combination with TMZ (100 mg/kg i.p.) indicated that the mean survival time of the group was significantly higher than that observed in animals receiving TMZ as single agent. Good results were also obtained against B16 melanoma growing subcutaneously in B6D2F1 mice. The combination treatment of compound 51 + TMZ significantly reduced the growth of the melanoma versus TMZ alone. Tricyclic deriva-tives [124] are 2,9-dihydro-3H-pyridazino[3,4,5-de]quinazo-lin-3-one (as for compounds of general formula 52; Figure 10) with a substituted methylamino residue in position 8. Several compounds are reported to have low nanomolar activity

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51 IC50 = 40 nM 52

53 R =

54 R =

55 IC50 = 6 nM

56 57

59PARP-1 (SPA assay) = 158 nMTANK-2 (SPA assay) = 7.1 µM

58PARP-1 (SPA assay) = 79 nMTANK-2 (SPA assay) = 501 nM

NH

N

O

O

N

OH

NH

N

O

N NH

N

R2

R1

NH

O

N

R

*NH

N

O

N* N

NF

N

O

N N

2

8

N

HN O

N

HNO

1

3

7

6NH

O

N

O

O 1

36

7

NH

O

N NH

O

Br

H

N

Figure10.RecentPARPinhibitors.PARP: Poly(ADP-ribose) polymerase.

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(< 100 nM) both in enzymatic and in cell based assays. Recently, MGI GP came out with a patent [125] in which new benzo[c][1,5]naphthyridin-6(5H)-one structures are dis-closed as, for instance, compounds 53 and 54 (Figure 10). As reported for compound 51, compounds 53 and 54 were also used to chemosensitize cancers to the cytotoxic effects of TMZ. Oral administration for 5 days of both compounds 53 (two doses: 10 and 40 mg/kg) or 54 (three doses: 10, 40 and 100 mg/kg) + TMZ (100 mg/kg i.p.) enhances survival of mice bearing intracerebral malignant melanoma with respect to the treatment of TMZ as single agent.

2.1.9 Altana Pharma4,5-Dihydro-6H-imidazo[4,5,1-ij]quinolin-6-one is the core scaffold optimized from Altana to obtain a very potent PARP inhibitor with a non-classical mode of binding [126]. The potency has been increased by varying the substituents in position 2 and 8 of the scaffold: best activity has been reported for compound 55 (Figure 10), bearing a polar group para to the phenyl ring in position 2 of the scaffold, such as an N-methylpiperazino-, and with a fluorine atom in position 8, responsible for a dramatic enhancement of the potency. Compound 55 shows single-digit nanomolar biochemical activity (IC50 = 6 nM).

2.1.10 Janssen PharmaceuticaRecently, Janssen Pharmaceutica filed a series of patent applications on 2-quinoxalinone and 2-quinolinone scaf-folds. They thoroughly explored positions 6 and 7 of each bicyclic system, with the ethyl group being the preferred substituent for position 3 of both scaffolds. In two patent applications [127,128] compounds 56 and 57 (Figure 10) were claimed to inhibit PARP-1 activity in vitro with IC50 values of 7 and 8 nM, respectively. In 2008, they disclosed several hundreds of 2-quinolinone-based com-pounds as PARP-1 and TANKS inhibitors [129]. Most active members were characterized by a nitrile-bearing benzylic stereocenter. Representative derivatives were 58 and 59 (Figure 10), which showed IC50 values of 39 and 50 nM, respectively, in an antiproliferative assay on HCT116 cell line.

2.1.11 InotekOver the past 4 years, Inotek has still been engaged in developing indenoisoquinolinones as PARP inhibitors (Figure 11). In two recent patent applications [130,131] they disclosed a large number of compounds, most having an EC50 value < 100 nM. Analysis of the most active compounds revealed the structures possessing common features. Ring A (Figure 11) better tolerates small substituents, such as H or F, X is usually CH2 and ring B contains an aliphatic amine. In an antiproliferative assay (murine RAW macrophages) both compounds 60 and 61 (Figure 11) resulted to be extremely potent, with an EC50 value ∼ 3 nM each.

2.1.12 IRBMIn 2007, IRBM claimed intramolecularly hydrogen-bonded indazole/benzotriazole carboxamides (62; Figure 11) as efficient PARP inhibitors [132]. The wide general formula described was associated with some generic statements of biochemical (IC50 < 5 μM) and cellular (CC50 < 10 μM in BRCA-1 silenced HeLa cell line, tenfold selectivity over BRCA-proficient cells; CC50 < 5 μM in either BRCA-1 or BRCA–2 naturally deficient cell lines) potency. A subsequent restriction of the general formula claimed a limited number of com-pounds (e.g., 63; Figure 11) and their corresponding sepa-rated enantiomers that generally show increased biochemical (IC50 < 50 nM) and cellular (CC50 < 5 μM in BRCA-1 silenced HeLa cell line) activity [133].

The pyrazoloquinazolinones chemical class was the subject of a pair of patents disclosed, respectively, in 2007 [134] and 2008 [135] by IRBM. While the 2007 publication mainly claimed preferred (furtherly substituted) phenyl, 2-furyl, 2-thienyl and aminocarbonylphenyl R3 substituents (64; Figure 11), the more recent one focused on aminocarbonyl substituents directly linked to the pyrazole ring, again in position R3 of the general formula (65; Figure 11). However, neither SAR information nor indication of the most potent compounds are given in those patents, as the more active derivatives are generically claimed to possess IC50 < 10 μM in biochemical assays.

A number of patents were also published by IRBM in which general structures of mono- up to tri-cyclic lactams were repetitively claimed to possess, for selected representatives, IC50 < 5 μM in biochemical assay (pyridones and pyridazi-nones [136]; pyrrolopyrazinones and pyrrolotriazinones [137]; benzonaphthyridinones [138]; fused indoles and indazoles [139]; oxo-dihydropyrrolequinoxalines [140]; imidazolopyrimidines and imidazolotriazines [141]).

3. Expertopinion

During the last few years, a number of PARP inhibitor candi-dates reached the clinical stage. As the inhibition of PARP is supposed to show beneficial effects in selected cancer patients after a prolonged time of treatment, more patient-compliant orally available candidates (e.g., ABT-888, 35; AZD2281, 7) represent a significant improvement. Another advancement could be envisaged in the recent discovery that PARP inhib-itors, as single agent, may be successfully used wherever specific cancer phenotypes are present. One of the current unanswered questions is if selective PARP-1 inhibitors are really a plus. So far, not one of the observed side effects in clinics is strictly linked to the inhibition of any specific PARP isoform, thus rendering the positive evaluation of any kind of selectivities premature. Moreover, other PARP isoforms seem to be involved in cancer maintenance (e.g., Tankyrase 1, BAL proteins), and then poly-PARP inhibition may be useful. Considering the number of companies actively involved in finding PARP inhibitors and the NAD-mimicking scaffolds claimed (both

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as lactams and as intramolecularly hydrogen-bonded benz-amides), one could argue that this still remains the most easy and successful way to rapidly produce PARP inhibitors. In this respect, the newborn development of non-NAD-mimicking small molecules (e.g., DBD-targeting compounds), as well as compounds that do not relapse into the classical NA-like pharmacophore paradigm, may represent the future pathways for the creation of novel PARP inhibitors. These approaches could lead to the generation of selective inhibitors,

thus answering the question of whether they are really advantageous and, moreover, create new room for patentable chemical classes in the growing scenario of PARP inhibition.

Declarationofinterest

The authors are full-time employees of Nerviano Medical Sciences, which has an interest in the target subject of the present review.

60 EC50 = 3 nM 61 EC50 = 3 nM

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Figure11.Recentlydisclosedhydrogen-bondedcarboxamidesandlactamsinhibitors.

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AffiliationGianluca Papeo1, Barbara Forte1, Paolo Orsini1, Claudia Perrera2, Helena Posteri1, Alessandra Scolaro1 & Alessia Montagnoli†3

†Author for correspondence1Nerviano Medical Sciences, Department of Medicinal Chemistry, Italy2Nerviano Medical Sciences, Department of Biotechnology, Italy3Nerviano Medical Sciences, Department of Cell Biology, Viale Pasteur 10, 20014 Nerviano (MI), Italy Tel: +039 033 158 1071; Fax: +039 033 158 1233; E-mail: [email protected]

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Documents

Poly(ADP-ribose) polymerase inhibition in cancer therapy: are we close to maturity?