Chemical strategies for development of ATR inhibitors

Chemical strategies for development of

ATR inhibitors

Sabin Llona-Minguez*, Andreas Höglund, Sylvain A. Jacques,Tobias Koolmeister and Thomas Helleday*

ATR protein kinase is one of the key players in maintaining genome integrity andcoordinating of the DNA damage response and repair signalling pathways.Inhibition of ATR prevents signalling from stalled replication forks andenhances the formation of DNA damage, particularly under conditions ofreplication stress present in cancers. For this reason ATR/CHK1 checkpointinhibitors can potentiate the effect of DNA cross-linking agents, as evidencedby ATR inhibitors recently entering human clinical trials. This review aims tocompile the existing literature on small molecule inhibitors of ATR, both fromacademia and the pharmaceutical industry, and will provide the reader with acomprehensive summary of this promising oncology target.

IntroductionA replicating mammalian cell is estimated tosuffer as many as 105 DNA lesions daily (Ref. 1).In order to protect the genomic integrity of thecell, complex biochemical pathways have beenpreserved to ensure the propagation of genomicmaterial with a minimum of sustained damage.The DNA damage response and repair pathwaysare dependent on the signal transducers ofthe phosphoinositide 3-kinase (PI3 K)-relatedprotein kinases (PIKKs) family, including ataxia-telangiectasia-mutated (ATM), ATM and RAD3-related (ATR) and DNA-dependent proteinkinase (DNA-PK) (Ref. 2).Single-stranded DNA (ssDNA) is present

during transcription and replication or isgenerated during the repair of DNA double-strand breaks (DSBs), stalled replication forks ordamage triggering nucleotide excision repair

(Refs 1, 3, 4). The signal emanating from ssDNAis relayed through a complex biochemicalpathway that centres on the activity of ATR(Fig. 1). Activation and regulation of ATRensures faithful replication as well as contributesto DNA damage signalling after DNA damageto enforce cell cycle arrest, maintenance ofreplication fork integrity and recruitment ofDNA repair factors to lesions. As DNA lesionsare repaired, the signal emanating from suchsites dissipates and the cell is free to resumereplication and cell division. ATR recruitment tosites of DNA lesions is dependent on the ATR-interacting protein (ATRIP) and its bindingof replication protein A (RPA) coated ssDNA(Refs 5, 6). The activity of the ATR–ATRIPcomplex is in turn regulated by the evolutionaryconserved Rad9–Rad1–Hus1 (9–1–1) complex(Refs 7, 8), which upon being phosphorylated

Science for Life Laboratory, Division of TranslationalMedicine and Chemical Biology, Department ofMedical Biochemistry and Biophysics, Karolinska Institutet, S-171 21 Stockholm, Sweden

*Corresponding authors: Thomas Helleday and Sabin Llona-Minguez, Science for Life Laboratory,Division of Translational Medicine and Chemical Biology, Department of Medical Biochemistry andBiophysics, Karolinska Institutet, S-171 21 Stockholm, Sweden. E-mail: [email protected]; [email protected]

expert reviewshttp://www.expertreviews.org/ in molecular medicine

1Accession information: doi:10.1017/erm.2014.10; Vol. 16; e10; May 2014

© Cambridge University Press 2014

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mailto:[email protected]



binds topoisomerase-binding protein 1 (TOPBP1)to activate the ATR-signalling cascade (Ref. 9).Recruitment of the 9–1–1 and ATR–ATRIPcomplexes occurs independently, possiblycreating a built-in regulatory element of thepathway ensuring that activation will only occurwhen both complexes are present, minimisingthe chance of illicit firing of this regulatorypathway (Refs 10, 11). The recruitment andactivation of ATR triggers the phosphorylationof a large number of target proteins. In fact,activation of ATR and ATM – responsible forsignalling downstream of double-stranded DNAdamage – regulates approximately 700 targetproteins, spanning functional hubs, such as cell-cycle regulation, DNA replication and DNArepair (Ref. 12). Arguably the most importantphosphorylation target of ATR is the p53tumour suppressor. Ultraviolet (UV) irradiationinduces ATR-dependent phosphorylation ofp53Ser15, leading to p53 activation that,depending on cellular context, will either inducecell-cycle arrest, repair or apoptosis (Refs 13, 14).The rationale for targeting ATR in cancertherapy comes from the evident engagementof this signalling hub following both radiation-and chemically induced DNA damage incancer therapy, as well as the consistentand constitutive activation of ATR in fastproliferating cancers with aberrant replicationstress (Ref. 15). The interest in ATR inhibitors(ATRi) is also linked with the vast interestin inhibitors of the CHK1 kinase, which isregulated downstream of ATR. Blocking of thedownstream activation of repair and signallingfactors by ATR inhibition would induce a more

severe DNA damage phenotype of currentlyapplied therapy. This in turn would cause amore efficient killing of cancer cells, enablingbetter disease control in the clinic. Indeed, byeither genetically or chemically inhibiting ATR,several pre-clinical studies have shown thepotential of applying this combination therapyto various cancer models (Ref. 16). The increasedefficacy of ATR ablation in combination withchemotherapy has also been shown in cells withfaulty checkpoint regulation, such as lack of p53,which is important for proper G2/M and G1arrest following DNA damage. The inhibition ofATR causes a p53-independent death due topremature chromatin condensation and mitoticentry (Ref. 17). Several inhibitors of ATR’s maindownstream effector, CHK1, have beenpublished and reviewed elsewhere (Ref. 18). Amajority of clinical trials using CHK1 inhibitorsare combination studies with different cytotoxicdrugs, e.g. gemcitabine, showing positiveclinical outcome (Ref. 19). This data uniformlysupport the use of ATRi’s combined with DNA-damaging agents. Targeting ATR, a more centralhub in the cell-cycle machinery and the directupstream regulator of CHK1, will likely furtherincrease the potential of these combinationtherapies.

The potential of using ATR inhibition, or bytargeting its main downstream signal transducerCHK1, in a mono-therapy approach has also beenevaluated in cancers with a high degree ofreplication stress or in those that carry an inherentdefect in DNA repair, with positive results(Refs 20, 21, 22, 23). Even though the efficacy ofthis approach have been questioned due to the

ATR is recruited to sites of ssDNA damage

Expert Reviews in Molecular Medicine © 2014 Cambridge University Press

RPA RPA RPADNArepair

DNArepair

Chk 1

Cell-cycleregulation

Cell-cycleregulation

ReplicationorigincontrolATRIP

ATRIP ATRIPATR

ATR ATRTOPBP1P P9-1-1

9-1-1 9-1-1

Figure 1. ATR is recruited to sites of ssDNA damage. This in turn enables the recruitment of ATR–ATRIP andthe 9–1–1 complex. Subsequent phosphorylation of 9–1–1 then enables TOPBP1 binding, starting the ATR-mediated phosphorylation cascade. ATR phosphorylates numerous proteins, including CHK1 and p53, toinitiate DNA repair, cell-cycle regulation and control of replication origin firing.




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lack of positive clinical outcome, the recent pre-clinical studies with encouraging data usingmodel systems with inherent high degree ofreplication stress (N-MYC or C-MYC transformedcells) highlight the importance of patientstratification (Refs 21, 22, 23). Selecting disease orpatients with, e.g. amplified MYC expressioncould potentially find a use for CHK1 and ATRias monotherapeutic agents. Interestingly, the firstclinical trials using ATRi’s have been initiated byAstraZeneca, preferably in ATM-mutated chroniclymphocytic leukaemia (CLL), prolymphocyticleukaemia (PLL) or B-cell lymphoma patients(Ref. 24) and by Vertex Pharmaceuticals inadvanced solid tumours (Ref. 25).

ATRi’s published by academic groupsResearch on ATRi’s has been on-going for morethan a decade and although the first small-molecule inhibitors were identified in the late1990′s, most of the novel patented chemicalmatter appeared in 2010 and onwards. LY2940021 (Fig. 2) was originally developed by Eli Lillyas a PI3 K inhibitor (Ref. 26) but was lateracknowledged as a pan-inhibitor of the PIKKfamily (Refs 27, 28). Although no enzymaticinhibition data were available from the initialpublications, the ATR IC50 of 1 was determinedto be >100 μM in a later publication focused oninvestigating the pharmacological roles of thePI3 K family (Ref. 29). In this study, Knight et al.screened a chemically diverse set of PI3 Kinhibitors in pre-clinical development againstseveral lipid kinases and identified compounds2, 3, 4, 5, 6 and 7 as moderate ATRi’s in anenzymatic assay (ATR IC50≥ 0.85 μM but ≤21 μM,Fig. 2), with no selectivity over ATM or DNA-PK.The fungalmetabolitewortmannin 8 (Fig. 2) has

been characterised as an irreversible pan-inhibitorof the PIKK family (ATR IC50= 1.8 μM, DNA-PKIC50= 16 nM, ATM IC50= 150 nM), suggestingthat the radio-sensitising effect of 8 is probablydue to the inhibition of DNA-PK kinase activityrather than ATR inhibition (Ref. 30). Caffeine 9(Fig. 2) has also been identified as a weak pan-PIKK inhibitor (ATR IC50= 1.1 mM, DNA-PKIC50= 10 mM, ATM IC50= 0.2 mM), but in thiscase the radio-sensitising effect of 9 wasattributed to both ATM and ATR kinases (Ref. 31).The first patent on the therapeutic use of ATR

inhibition was filed in 2003 by the Universities ofRochester and Utah (Ref. 32). This filing claimedthe use of ATR or Rad17 inhibitors to inhibit HIV

replication but did not exemplify any novelchemical matter. Interestingly, in 2005 researchersat the Massachusetts General Hospital filed a USpatent on the use of ATRi’s to reduce UVB-induced skin damage (Ref. 33). The ATR-mediated checkpoint-signalling cascade isactivated upon UV-induced DNA damage inreplicating cells. Working under this hypothesis,topical application of an ATRi (1.2% solution of9 in acetone) reduced UV-induced wrinkles in amurine model. No additional biological datawere provided.

Nishida et al. filed in 2007 a patent on the firstATR-selective small-molecule inhibitors (Ref. 34),the dibenzocyclooctadiene lignans schisandrinsand gomisins and published a subsequent paperwith detailed information on the mechanismof action of one of the compounds of interest,schisandrin B 10 (Fig. 2) (Ref. 35). 10 (30 μM)blocked the G2/M and S-phase checkpointand reduced viability of UV-exposed A549adenocarcinoma cells. In addition, 10 inhibitedp53 and CHK1 ATR-dependent phosphorylationbut did not prevent ATR-activating associationto the ATRIP. 10 selectively inhibited ATR kinaseactivity in vitro with an IC50 of 7.25 μM (ATMIC50= 1.74 mM, no inhibition detected forCHK1, PI3 K, DNA-PK and Mammalian targetof rapamycin (mTOR). 10 (30 μM) blocked p53,CHK1, SMC1 and BRCA1 phosphorylation inATM-deficient UV-exposed AT2KY fibroblasts andp53 and CHK1 phosphorylation in ATM-siRNA-depleted UV-exposed A549 cells, confirming theATR-specificity of 10. Furthermore, 10 did notaffect cell viability of UV-exposed ATR-deficientSeckel syndrome fibroblasts, in agreement withthe other findings presented in this work.

Liu et al. at the Harvard Medical Schoolcharacterised Torin 2 11 (Fig. 2) as a potent ATP-competitive inhibitor of mTOR, ATM and ATR(mTOR IC50=<0.01 μM, ATM IC50=<0.01 μM,ATR IC50=<0.01 μM) using a KiNativ chemicalproteomics assay and in cellular assays lookingat phosphorylation status of downstreamsubstrates (mTOR EC50= 0.25 nM, ATM EC50=28 nM, ATR EC50= 35 nM) (Ref. 36). In the caseof ATR, 11 inhibited the cellular activity of thekinase as assessed by phosphorylation status ofCHK1S317 following exposure of HCT116 coloncancer cells to UV-induced DNA damage. 11showed significant inhibition of tumour volumein a KRAS-driven murine model of lung cancerwhen administered in combination with the




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mitogen-activated protein/extracellular signal-regulated kinase (MEK) inhibitor AZD6244.A group led by Dr Fernandez-Capetillo at the

Spanish National Cancer Research Centre alsoscreened a collection of PI3 K inhibitors with theaim of finding hits against the PIKK family, inparticular ATR (Ref. 37). This phenotypic screenis very interesting as it exploit the fact that ATRis activated by binding the TOPBP1 protein(Ref. 38). The team expressed the ATR-bindingdomain of TOPBP1 fused with ER which canenter the nucleus and activate ATR uponaddition of 4-hydroxytamoxifen (4-OHT) in theabsence of DNA damage (Ref. 39). Using this

system the team carried out a high-throughputmicroscopy screen that quantified nuclearγH2AX, an early and reliable marker of double-stranded DNA damage, identified compound 12(Fig. 2) as a promising hit. Compound 12 notonly inhibited γH2AX, but also CHK1phosphorylation, abolished the G2/M checkpointand induced nuclei damage after ionisingradiation (IR) exposure. 12 potently inhibitedATR in vitro and although it showed someselectivity over ATM (enzymatic ATR/ATMIC50’s= 14 nM/545 nM) and 26 additional kinases,it strongly inhibited mTOR and DNA-PK(enzymatic mTOR/DNA-PK IC50’s= 0.6 nM/36

Chemical structures, reported enzymatic IC50 values and assaytype for ATRi’s published by academic institutions

Expert Reviews in Molecular Medicine © 2014 Cambridge University Press

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0.021 µMd

N

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NH2N

11Torin 2

IC50 10 nMc

SS

Figure 2. Chemical structures, reported enzymatic IC50 values and assay type for ATRi’s published byacademic institutions. aRadiolabelled assay; Rad17-derived peptide substrate; bRadiolabelled assay;PHAS-I substrate; cChemical proteomics, KiNativ ActivX Biosciences; dflag-IP/Radiolabelled assay, GST-p53 substrate.




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nM). Given the complex network of ATR-binding/activating partners, using a flag-IP assay allowedthe group to efficiently quantify ATR inhibition.12 displayed outstanding ATR activity in thecellular assay quantifying γH2AX intensity in 4-OHT-treated U2OS cells (ATR IC50= 25 nM).Upon co-treatment of U2OS cells with 12 andthe DNA-damaging agent hydroxyurea, therewas an increase in p53-binding protein 1 (53BP1)and RPA foci, together with a rise of ATM andChk2 phosphorylation, indicating chromosomaldamage. Cells treated with 12 stalled in G2 phase16 h after removal of hydroxyurea, following acommon pattern seen in selective ATR deficiency.12 also showed synergy with the CHK1 inhibitor7-hydroxystaurosporine. It is worth notingthat staurosporine derivatives are well-knownpromiscuous compounds that inhibit a widevariety of protein kinases and this could blurthe interpretation of these results (Ref. 40).Compound 13 (NVP-BEZ235), a dual PI3 K/mTOR inhibitor developed by Novartis (Ref. 41),was also identified as a potent pan-PIKKinhibitor (ATR, ATM DNA-PK IC50’s <21 nM)using the cell-free kinase assay and the ATR cell-based assay (ATR IC50= 25 nM). 13 inhibitedionising radiation-induced ATM, CHK1, Chk2and DNA-PK phosphorylation and affectedionising radiation-induced γH2AX formation,whereas 12 did not, in agreement with the lessselective profile of 13. Compound 12 suffers frompoor pharmacological properties in mice and hasnot been further developed, whereas compound13 is currently undergoing clinical trials for solidtumours (Refs 27, 42). The authors suggestedthat mTOR inhibition of 12 and 13 could bedetrimental for the generation of replicative stressand such selectivity issue should be addressedin future generations of ATRi’s. Furthermore,dual inhibition of PI3 K/mTOR did not showsynthetic lethal interaction with cyclin Eoverexpression and/or p53 deficiency. Thisevidence, together with other data recentlypublished (Refs 43, 44), indicates that clinicaleffect of 13 could be due to potent inhibition ofATR rather than PIK3 or mTOR.Prof Curtin’s team at Newcastle University and

colleagues at the Mayo Clinic identified the CDK2inhibitor NU6027 14 (Fig. 2) as amoderate ATRi ina cell-free enzymatic assay (ATR IC50’s= 0.1 μM;CDK2 IC50= 2.2 μM) and using pCHK1S345 asa specific marker of ATR activity in GM847KDand human breast cancer MCF7 cells (IC50’s=

2.8 and 6.7 μM, respectively), with goodselectivity over related PIKK’s, ATM and DNA-PK (Ref. 45). 14 was mildly cytotoxic inclonogenic assays when administered alone at10 μM and sensitised GM847KD, MCF7 andA2780 cancer cell lines to the major classes ofDNA-damaging agents by around 2-fold, butdid not potentiate the cytotoxic effect of themitotic inhibitor paclitaxel, indicating a specificchemo-sensitisation mechanism. 14 decreasedDNA damage-induced G2/M arrest andinhibited RAD51 foci formation in cells treatedwith the poly(ADP-ribose) polymerase (PARP)inhibitor rucaparib. 14 was synergistic withrucaparib in MCF7 cells and synthetically lethalin XRCC1-defective EM9 cells, confirming thatimpaired DNA single-strand break repair issynthetically lethal with ATR inhibition.

D’Andrea et al. at the Dana Farber CancerInstitute filed a patent on inhibitors of theFanconi anaemia pathway and their use asinhibitors of DNA damage repair (Ref. 46).During the screening campaign H-9 15,alsterpaullone 16 and curcumin 17 (Fig. 3) werefound to inhibit mono-ubiquitination of theFANCD2 polypeptide. Further investigationindicated the compounds did not affect ATM-dependent phosphorylation of FANCD2 but didinhibit ATR-dependent phosphorylation ofCHK1Ser345 in a concentration-dependentmanner, suggesting that the compounds inhibitATR either directly or indirectly, although nospecific data were provided. This is inagreement with previous evidence suggestingthat ATR, together with ATM and RPA, areupstream regulators of FANCD2 mono-ubiquitination (Refs 47, 48). Additionally, 15 and16 sensitised the 2008 ovarian tumour cell line(deficient in FANCF protein) to cisplatin.

ATRi’s published by pharmaceuticalcompanies

In 2007 CGK Co. Ltd. filed a patent on a series ofurea derivatives as ATM/ATR inhibitors (Fig. 4)(Ref. 49). The compounds claimed blocked ATMand ATR-mediated phosphorylation of p53Ser15

in a concentration-dependent manner, both inan enzymatic assay and in RKO and GM847cells, although no IC50 values were provided inthis filing. The compounds inhibited the ATMand ATR-mediated phosphorylation selectivelyover a panel of known p53-targeting kinases(Table 1). Compound 18 showed synergistic




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effectwith the cytotoxic agents cisplatin, etoposideand/or doxorubicin in Va-13, HeLa and/orMCF-7cells. Additionally, co-treatment of RKO cells with19 and doxorubicin-inhibited cell cycle at the G2/M phase. When BJ cells in replicative senescencewere treated with compound 20, the celldivision was increased and the process showedto be reversible. Compounds 19 and 20 werewell tolerated in mice acute toxicity tests. It isworth noting that the same research group

published data on compound 19 elsewhere(Ref. 50) but subsequently retracted the paperafter an investigation (Ref. 51).

The first patents on potent and selective small-molecule inhibitors of ATR were filed in 2010 byAstraZeneca and Vertex Pharmaceuticals. Thesecompounds were claimed for the treatment ofcancer, as single agents or in combination withother anti-cancer drugs (Refs 52, 53, 54, 55, 56,57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,

Chemical structures of ATRi’sExpert Reviews in Molecular Medicine © 2014 Cambridge University Press

N

SO

HN

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NH2

HN

O

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NO2

HO

O

O O

OH

O

15H-9

16alsterpaullone

17curcumin

Figure 3. Chemical structures of ATRi’s published by Dana Farber Cancer Institute.

Chemical structures of ATRi’sExpert Reviews in Molecular Medicine © 2014Cambridge University Press

18 R1 = diphenylmethyl, R2 = 3-cyanophenyl19 R1 = diphenylmethyl, R2 = 4-fluoro-3-nitrophenyl20 R1 = diphenylmethyl, R2 = 3-nitrophenyl

R1 NH

O

NH

CCl3

NH

SR2

Figure 4. Chemical structures of ATRi’s published by CGK Co.

Table 1. Biological data of ATRi’s published by CGK Co.

Example ID Inhibition intensityof p53Ser15 phosphorylation at 10 μM (%)

18 28 30

19 33 22

20 1 28




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71, 72, 73). AstraZeneca presented compoundswith ATR and/or mTOR IC50 values and IC50’sfor HT29 colon cancer cell growth, alone and incombination with the DNA-damaging agentcarboplatin (IC50= 3.97 μg/ml in HT29 cells)(Table 2) (Refs 52, 53). The synergism of thecompounds was assessed by the potentiationfactor 50 (PF50) which is calculated as a ratio ofthe IC50 of control cell growth in the presence ofcarboplatin, divided by the IC50 of cell growth inthe presence of this agent and the ATRi.Compounds were compared using the PF50value at concentrations of ATRi that showedminimal growth inhibition on their own in asulforhodamine B (SRB) assay after 5 days ofculture. In accordance with Toledo et al.(Ref. 37), the AstraZeneca group suggested thatcompounds with reduced mTOR inhibition arepreferred since they may ameliorate off-target effects. A follow-up publication by thesame group reported on the development ofcompound 21 (Fig. 5) from the 2010 patent(Ref. 74). Initial hit 22 (Fig. 5) was identified as a

potent ATRi from an mTOR screening campaign(enzymatic/cellular ATR IC50’s= 30 nM/1.1 nM;enzymatic/cellular mTOR IC50’s= 0.33 μM/5.9 μM) with high selectivity against other PIKKand PI3 K kinases. The group determined cell-free ATR inhibition using an ELISA-based assayfor the detection of p53Ser15 phosphorylationwhilst cellular ATR inhibition was measured inHT29 cells using pCHK1Ser345 phosphorylationas a marker. Medicinal chemistry optimisation of22 lead to the discovery of 21 (enzymatic/cellular ATR IC50’s= 5 nM/50 nM). 21 showedweak potency in a mTOR cell-based pAKT assayand displayed good selectivity against PI3Kα,ATM and DNA-PK, amongst a panel of 442kinases, and no inhibition of the hERG channel,but suffered from time-dependent inhibitionof CYP450 3A4. Compound 21 inhibited cellgrowth in LoVo colorectal adenocarcinoma cells(GI50= 0.2 μM), as measured by an MTS assayafter 72 h. Despite poor aqueous solubility(10 μM), 21 showed high Caco-2 permeabilityand good stability in rat hepatocytes, which

Table 2. Biological data of ATRi’s published by AstraZeneca.

Patentapplication

ExampleID

EnzymaticATRinhibitionIC50 (nM)a

EnzymaticmTORinhibitionIC50 (nM)

HT29 IC50 –carboplatin+0.3 μM ATRi(μg/ml)

Potentiationfactor(PF50)

WO2010073034A1 21 3.01 4 26 0.39 10.08

23 2.02 4 – 2.99 4.21

WO2011154737A1 24 2.03 6 – 0.63 18.72

aELISA-based assay, p53 substrate.

Chemical structures of ATRi’sExpert Reviews in Molecular Medicine © 2014 Cambridge University Press

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Figure 5. Chemical structures of ATRi’s published by AstraZeneca.




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led to respectable bioavailability and plasmaexposure in a rat pharmacokinetic (PK) study.Female nude mice grafted with LoVo cells andtreated with 21 alone (25 mg/kg twice a day or50 mg/kg once a day) for 13 days presented asignificant decrease in tumour growth andthe treatment was generally well tolerated by theanimals (Ref. 74). No mention was made in thepaper to the fact that 21 also exhibitedgood synergy with carboplatin in HT29 colonadenocarcinoma cell growth inhibition (Table 2)(Ref. 74). Sulfonimides 23 and 24 (Fig. 5) from asubsequent patent were also potent ATRi’s andcompound 24 strongly increased the cytostaticeffect of carboplatin (Table 2) (Ref. 53). Thecompany has recently announced a clinical trialin patients with 11q-deleted relapsed/refractoryCLL, PLL or B-cell lymphomas to determine thesafety, tolerability, PKs and biological activityafter oral administration of the ATRi AZD6738alone (undisclosed structure). The study willexamine biomarkers of ATR inhibition/statussuch as and γH2AX, pCHK, pATR and willidentify patients with ATM-deficient status,determine ATM mutation load and correlate thiswith clinical efficacy (Ref. 24).In 2010, researchers at Vertex Pharmaceuticals

filed two patents covering a broad numberof pyrazines as ATRi’s (Fig. 6) (Refs 54, 56).The initial filings were followed-up by a numberof patents providing detailed information onselected compounds, including enzymatic/cellular inhibition of ATR kinase and viabilityvalues for HCT116 cell growth, as measuredby an MTS assay after 96 h, alone and incombination with the DNA-damaging agentcisplatin (Table 3). Many of the examples listedin these filings inhibited the full-length ATRwith Ki <100 nM. Vertex expressed the synergylevel of the inhibitors using the ‘CP3 shift’,which is the concentration of compoundrequired to reduce the IC50 of cisplatin alone byat least 3-fold. This metric does not take intoaccount the cytotoxicity displayed by theinhibitors alone in contrast to AstraZeneca’smeasure of synergism.The compounds claimed generally feature

a central 2-aminopyrazine core, but 2-aminopyridine (33) or pyrrolopyrazine (43, 46,47, 48) are also described (Fig. 7) (Refs 55, 59,61, 63, 66, 70, 71, 73). Examples lacking the2-amino group such as 39, 40 and 41 are lessfrequent and result in higher enzymatic ATR

Ki ranges (Ref. 61). The pyrazine ring isoften flanked at the 3-position by an amide (29)or a 5-membered heterocyclic linker, mostcommonly isoxazole (30) but also oxadiazole(26) (Refs 56, 57), pyrazole (59) (Ref. 67), oxazole(60) (Ref. 67), triazole (58) (Ref. 62) anddihydroisoxazole (61) amongst others (Ref. 64).Iodinated isoxazole rings (63, 64) maintainacceptable enzymatic activity and present anattractive option to access triterated isotopes ofATRi, useful as tool compounds (Ref. 64). Otherlinkers such as ether (34) (Ref. 63), ketone (37)(Ref. 63), alkane (36) (Ref. 63), alkene (35)(Ref. 63), alkyne (31) (Ref. 59) or bicyclic systems(25, 32) (Refs 54, 58) are generally mentionedwith higher Ki ranges and to a lesser extent.Structural motifs that allow for intramolecularhydrogen bonding with the exocyclic aminogroup at the 2-position of the pyrazine ringusually display good enzymatic potency. Theabove-mentioned linker usually leads to anaromatic ring, mainly benzene (29) but alsothiophene (55) (Ref. 57), often containing analiphatic amine substituent (30).

The central heterocyclic core is usuallysubstituted on the 5-position with an appropriatespacer leading to a hydrogen bond acceptorplaced within a ‘sphere’ located 6 Å away fromthe pyrazine’s C5 on a line defined by the C5–C2axis (Fig. 8) (Ref. 54). This hydrogen bond acceptoris usually presented as an aliphatic sulfone, butoccasionally a pyridone or a cyanopyridine canalso assume this role, as seen with examples 42and 44 (Refs 55, 65, 68). It is worth noting that3-piperidine sulfones (54, 55, 56, 57) confer goodPK properties when compared with similar4-piperidine compounds (Table 3) (Ref. 57).

During 2013 additional patents providingbiological data on very potent ATRi’s werepublished. Compounds 49 (Ref. 69) and 50(Ref. 62) are sub-nanomolar inhibitors withencouraging synergy in combination withcisplatin and good PK profiles (Tables 3 and 4).Compound 51 was characterised as varioussolid forms and several deuterated analoguessuch as 52 were described (Ref. 57). Deuteriumbioisostere 52 showed the same biological efficacyas the parent compound 51. The tetrahydropyranring in 51 and 52 led to an improvement incomparison with the tetrahydrofuran derivative28 included in a 2010 filing (Ref. 56), not only interms of cytotoxic effect but also in the PK profile(Tables 3 and 4). Compound 51 was tested in two




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mouse models: colon adenocarcinoma Colo205, 51(40 mg/kg), in combination with irinotecan(20 mg/kg) and primary non-small cell lungcancer (NSCLC), 51 (30 mg/kg), in combination

with cisplatin (3 mg/kg). In both cases there wasa marked tumour regression in comparison withthe control groups (<98% reduction in tumoursize in the case of cisplatin combination).

N O N

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44 R = 3-(4-((methylamino)methyl)phenyl)isoxazol-5-yl45 R = 3-(4-(((tetrahydro-2H-pyran-4-yl)amino)methyl)phenyl)isoxazol-5-yl

R1

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SOO

R2

26 R1 = 5-(4-((methylamino)methyl)phenyl)-1,3,4-oxadiazol-2-yl, R2 = iPr27 R1 = 3-(4-(((2-hydroxypropyl)amino)methyl)phenyl)isoxazol-5-yl, R2 = iPr28 R1 = (R)-(3-(4-(((tetrahydrofuran-3-yl)amino)methyl)phenyl)isoxazol-5-yl, R2 = iPr31 R1= 2-(3-hydroxyphenyl)ethyn-1-yl, R2 = tetrahydro-2H-pyran-4-yl34 R1= benzyloxy, R2 = Me35 R1= 2-phenylethen-1-yl, R2 = Me36 R1= 2-phenylethan-1-yl, R2 = iPr37 R1= oxo(phenyl)methyl, R2 = iPr38 R1= (Z)-1-fluoro-2-phenylethen-1-yl, R2 = Me49 R1 = (R)-(3-(4-(morpholin-3-yl)phenyl)isoxazol-5-yl, R2 = iPr50 R1= 3-(4-(((oxetan-3-ylmethyl)amino)methyl)phenyl)isoxazol-5-yl, R2 = iPr51 R1 = 3-(4-((((tetrahydro-2H-pyran-4-yl)methyl)amino)methyl)phenyl)isoxazol-5-yl, R2 = iPr52 R1 = 3-(4-((((tetrahydro-2H-pyran-4-yl)methyl)amino)methyl)phenyl)isoxazol-5-yl, R2 = heptadeutero-iPr53 R1 = 3-(4-((trideuteromethylamino)methyl)phenyl)isoxazol-5-yl, R2 = iPr54 R1 = 3-phenylisoxazol-5-yl, R2 = piperidin-3-yl55 R1 = 3-(thiophen-2-yl)isoxazol-5-yl, R2 = piperidin-3-yl56 R1 = 5-(3-methylthiophen-2-yl)-1,3,4-oxadiazol-2-yl, R2 = piperidin-3-yl57 R1 = 5-(2-methoxyphenyl)-1,3,4-oxadiazol-2-yl, R2 = piperidin-3-yl58 R1 = 1-phenyl-1H-1,2,3-triazol-4-yl, R2 = iPr59 R1 = 3-phenyl-1H-pyrazol-5-yl, R2 = iPr60 R1 = 5-phenyloxazol-2-yl, R2 = iPr61 R1 = 3-(4-((methylamino)methyl)phenyl)-4,5-dihydroisoxazol-5-yl, R2 = iPr62 R1 = 3-phenyl-4,5-dihydroisoxazol-5-yl, R2 = iPr63 R1 = 4-iodo-3-(4-((methylamino)methyl)phenyl)isoxazol-5-yl, R2 = iPr64 R1 = 4-iodo-3-(4-((((tetrahydro-2H-pyran-4-yl)methyl)amino)methyl)phenyl)isoxazol-5-yl, R2 = iPr

N

N

SOO

29 VE-821

N

N

SOO

N O

NH

30 VE-822

NH

O

Chemical structures of pyrazine ATRi’s published by Vertex PharmaceuticalsExpert Reviews in Molecular Medicine © 2014 Cambridge University Press

Figure 6. Chemical structures of pyrazine ATRi’s published by Vertex Pharmaceuticals.




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Table 3. Biological data of ATRi’s published by Vertex Pharmaceuticals.

Patentapplication

Example ID EnzymaticATR inhibitionKi (nM)a

Cellular ATRinhibition Ki(nM)b

HTC116IC50 (nM)

CP3shift(nM)

WO2010054398A1 25 I-27 ≤100 <1000 – –

WO2010071837A1 26 IA-159 ≤100 ≤100 >100 but≤1000

≤100

27 IIA-11 ≤100 ≤100 >100 but≤1000

≤100

28 P110 0.29 54 205 78

29 I-82 orVE-821

≤100 >1000 but≤20000

>1000but≤20000

>100but≤1000

30 IIA-7 orVE-822

≤100 – – –

WO2011143399A1 31 I-34 ≤100 – – 80

WO2011143419A1 32 II-56 ≤50 – – –

WO2011143422A1 33 III-10 ≤100 – – 80

WO2011143423A2 34 V-9 ≤100 – – –

35 V-10 ≤100 – – –

36 V-11 ≤1000 – – –

37 V-12 ≤100 – – –

38 V-16 ≤100 – – –

WO2011143425A2 39 IV-1 ≥250 but <500 – – –

40 IV-2 ≥100 but <250 – – –

41 IV-3 <100 – – –

WO2011143426A1 42 I-94 ≤5 – – 7

WO2011163527A1 43 I-70 ≤10 – – –

WO2012138938A1 44 I-1 ≤1 – – –

45 I-53 ≤1 – – –

WO2012178123A1 46 I-1 ≤10 – – –

WO2012178124A1 47 I-1 ≤100 – – –

WO2012178125A1 48 I-1 ≤50 – – –

WO2013049719A1 49 1 0.5 – 365 117

WO2013049720A1 50 1 0.2 16 62 39

WO2013049722A1 51 I-1 0.14 13 83 39

(continued on next page)




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Equivalent doses of a compoundwith a favourablein vitroprofile such as 28, did not show sensitisationin similar in vivo experiments. The greatereffectiveness of 51 versus 28 in the animal modelscan be explained by the improved in vitro and PKproperties of 51.The Vertex group expanded on their findings

with two follow-up publications. The firstcommunication presented extensive biologicalcharacterisation of the synthetic lethal interactionbetween ATR and the ATM-p53 tumoursuppressor pathway in cells responding to DNAdamage (Ref. 75). Compound 29 is a potentATP-competitive ATRi (enzymatic Ki= 13 nM)with good selectivity over a panel of kinasesincluding ATM, DNA-PK, mTOR and PI3Kγ(Ki of 16 μM, 2.2 μM, >1 μM and 3.9 μM,respectively), although Rsk3 was highlyinhibited at 2 μM. 29 showed synergy with arange of genotoxic agents in HCT116 cells, asmeasured by an MTS assay, in particular withthe DNA cross-linking drugs cisplatin andcarboplatin, nevertheless this effect was not

observed in normal cells or ATR-defective celllines. HCT116 cells express normal levels of p53but are ATM-deficient, indicating that these cellsare more reliant on ATR for survival from DNAdamage. This hypothesis was confirmed withATM-null cells from an individual with ataxiatelangiectasia (AT1BR cells) and with cellsfrom a healthy donor (161BR cells). In bothcases 29 showed clear synergism with cisplatin.Apoptosis levels were studied in a p53-mutantcancer line (H23); 29 in combination withcisplatin did not induce cell death after 24 h andapoptosis only became apparent at the 96 h timepoint. When administered alone, 29 reversiblylimited cell cycle progression in 161BR normalcells without inducing significant cell death orlong-term detrimental effects but inducedsubstantial cell death in AT1BR cancer cells. Thegroup claimed that ATR inhibition promotesDSB formation at sites of DNA replicationstress which in normal conditions activates anATM-dependant checkpoint that stops DNAreplication, decreasing further DNA damage

Table 3. Biological data of ATRi’s published by Vertex Pharmaceuticals. (continued)

Patentapplication

Example ID EnzymaticATR inhibitionKi (nM)a

Cellular ATRinhibition Ki(nM)b

HTC116IC50 (nM)

CP3shift(nM)

52 II-2 0.10 – 59 39

WO2013049726A2 53 II-3 0.15 10 66 39

WO2013071085A1 54 I-1 ≤5 – – –

55 I-3 ≤5 – – –

56 I-10 ≤5 – – –

57 I-14 ≤5 – – –

WO2013071088A1 58 I-1 6 – – –

WO2013071090A1 59 I-1 2.86 – – –

60 I-2 4.03 – – –

WO2013071093A1 61 I-1 16 – – –

62 I-2 90 – – –

WO2013071094A1 63 I-1 16 – – –

64 I-2 90 – – –

aRadiolabelled assay, Rad17-derived peptide substrate.bImmunofluorescence microscopy assay, γH2AX substrate, after hydroxyurea treatment.




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and promoting cell survival. In ATM pathway-deficient cancer cells, S phase arrest does notoccur, leading to cytotoxic levels of DNAdamage accumulation. The second paper

expanded on the structure–activity relationships(SAR) of some pyrazine ATRi’s and introducedan ATR homology model based on the structureof the related kinase PI3Kγ (Ref. 76). Althoughthe sequence identity between the kinase domainof the two enzymes is low, the group identifiedkey amino acid residues and discussed thepredicted interactions. Compound 65 washighlighted as a potent ATP-competitive inhibitorof ATR (enzymatic Ki= 12 nM, cellular ATR IC50=

Chemical structures of non-pyrazine ATRi’sExpert Reviews in Molecular Medicine © 2014 Cambridge University Press

43

S

OOH

N

N

NH

SO

O

O

Cl

N

N

SO

O

H2N

O

N N

HN

33

HN

N

N

NH

SO

O

46

O

47

N

N

NH

S OO

NH

O

N

N

NH

SO

O

48

N

O

Figure 7. Chemical structures of non-pyrazine ATRi’s published by Vertex Pharmaceuticals.

N

5

N

3

2

6 Å

4 Å

R

NH2

Schematic representation of theposition of the hydrogenbond acceptor present in pyrazine ATRi’sExpert Reviews in Molecular Medicine © 2014Cambridge University Press

Figure 8. Schematic representation of theposition of the hydrogen bond acceptor.

Table 4. PK data of ATRi’s published byVertex Pharmaceuticals.

ExampleID

Clearance(ml/min/kg)

Volume ofdistribution(litre/kg)

28 P110 12.7 2.7

49 1 3.5 1.1

50 1 6 0.9

51 I-1 7.1 4.7

54 I-1 0.9 0.9

55 I-3 3.3 1

56 I-10 9.3 3

57 I-14 16 2




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420nM)withexcellent selectivityoverapanelofmorethan 50 kinases, includingATMandDNA-PK (IC50’s>8 μM), good aqueous solubility as well as cellpermeability and minimal efflux in a Caco-2 assay.65 affected ATM-defective HCT116 cancer cells,both alone (IC50= 1.1 μM) and in combinationwith cisplatin or ionising radiation (IR), butshowed no effect in HFL1 non-cancerous cells,in agreement with the results observed whencancer cells are treated with other ATR-selectiveinhibitors in combination with DNA cross-linking agents.Compounds 29 and 30 have also been

specifically claimed for the treatment ofpancreatic cancer and NSCLC (Ref. 72). Bothcompounds sensitised pancreatic cancer cells,but not normal cells, to radiation therapy. 30synergised well with cisplatin and gemcitabinein pancreatic cancer cells and enhanced notonly their anti-tumour effect, but also theeffect of IR in pancreatic cancer xenograftmouse models. Additionally 30 synergisedwith cisplatin, oxaliplatin, etoposide,gemcitabine and irinotecan in several lungcancer cell lines. Compound 30 has beencharacterised as various solid forms anddeuterated analogues such as 53 have also beendescribed (Ref. 68). Compound 30 (also knownas VX-970) has entered a clinical trial to evaluatethe safety, tolerability and response afterintravenous administration, alone and indifferent combinations with gemcitabine,cisplatin or etoposide in subjects with advancedsolid tumours, squamous NSCLC), relapsed orrefractory small cell lung cancer (SCLC) andmetastatic oestrogen receptor, progesteronereceptor, and HER2 negative (triple negative)breast cancer (TNBC) with and without BRCA1/BRCA2 mutation(s) (Ref. 25).

Summary/OutlookThe efforts towards the development of ATRi’shave undergone a remarkable upswing inthe past years. From classical phenotypicapproaches based on complex natural products(Refs 30, 31, 35) or simple small molecules(Ref. 45), through rational discoveries associatedwith investigations of the PIKK family and/orrelated lipid kinases (Refs 29, 37) to industrialhigh-throughput screening campaigns (Refs 74,76), the ATR field has benefited from the workof both academic groups and pharmaceuticalcompanies. Although the first ATR-selective

inhibitor schizandrin B 10 was published in 2007(Ref. 34), the weak enzymatic potency and lowmicrosomal stability in mice has limited itspreclinical development (Ref. 77). NVP-BEZ23513 entered clinical trials as a dual PI3 K/mTORinhibitor but recent data published by Toledoet al. questions the mechanism of action ofthis clinical candidate and starts a debate onthe true molecular target behind the observedtherapeutic effect (Ref. 37). There is a growingbody of opinion suggesting that in the oncologytherapy, and especially within the kinase field, a‘selective polypharmacology’ approach might berequired to overcome cancer resistance (Refs 78,79, 80). The early clinical success of the Novartispan-PIKK inhibitor NVP-BEZ235 13 in mono-therapy could be attributed to a favourable‘non-selective’ profile as seen with othermarketed kinase inhibitors such as imatinibor sorafenib (Ref. 78). On the other hand,AstraZeneca and Vertex Pharmaceuticalsentered the ATR arena with selective small-molecule inhibitors which have been progressedinto clinical evaluation. The AstraZeneca clinicaltrial will help to establish the safety andtolerability of AZD6738, as well as givinginsight on ATR inhibition and ATM functionalstatus, which will aid to determine whatpatients will benefit the most from thetreatment. The AstraZeneca inhibitors slowcancer cell growth when used alone, as seenwith 21, indicating a potential use in mono-therapy. However, the compounds show aremarkable synergistic effect when combinedwith carboplatin, in accordance with theliterature: ATRi’s kill cancer cells more effectivelywhen combined with DNA-damaging agentssuch as antimetabolites, alkylating agents,topoisomerase inhibitors or platinatingcompounds, being the later most commonlyused, translating to marked tumour regressionin cancer mouse models (Ref. 81). In contrastto the mono-therapy trial sponsored byAstraZeneca, Vertex Pharmaceuticals hasapplied the pre-clinical information available onsynergistic combinations with ATRi’s for therational design of the on-going clinical trial withVX-970 30. The scientific community will followwith interest the outcome of the NVP-BEZ235,AZD6738 and VX-970 clinical trials, whichwill provide invaluable information on thetherapeutic validity of DNA-damage enhancingagents.




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AcknowledgementsThe authors would like to thank OliverMortusewicz, Martin Scobie and UlrikaWarpman-Berglund for insightful comments.

Financial supportWewould like to acknowledge the Knut and AliceWallenberg Foundation, Vinnova, the SwedishResearch Council, Swedish Cancer Society, theSwedish Pain Relief Foundation and the Torstenand Ragnar Söderberg Foundation for funding.

Conflicts of interestNone.

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Features associated with this article

FiguresFigure 1. ATR is recruited to sites of ssDNA damage.Figure 2. Chemical structures, reported enzymatic IC50 values and assay type for ATRi’s published by academic

institutions.Figure 3. Chemical structures of ATRi’s published by Dana Farber Cancer Institute.Figure 4. Chemical structures of ATRi’s published by CGK Co.Figure 5. Chemical structures of ATRi’s published by AstraZeneca.Figure 6. Chemical structures of pyrazine ATRi’s published by Vertex Pharmaceuticals.Figure 7. Chemical structures of non-pyrazine ATRi’s published by Vertex Pharmaceuticals.Figure 8. Schematic representation of the position of the hydrogen bond acceptor.

TablesTable 1. Biological data of ATRi’s published by CGK Co.Table 2. Biological data of ATRi’s published by AstraZeneca.Table 3. Biological data of ATRi’s published by Vertex Pharmaceuticals.Table 4. PK data of ATRi’s published by Vertex Pharmaceuticals.




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Citation details for this article

Sabin Llona-Minguez, Andreas Höglund, Sylvain A. Jacques,Tobias Koolmeister and Thomas Helleday (2014) Chemical strategies for development of ATR inhibitors.Expert Rev. Mol. Med. Vol. 16, e10, May 2014, doi:10.1017/erm.2014.10




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Chemical strategies for development of ATR inhibitors

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