Publications by authors named "Teresa Klinowska"

32 Publications

Discovery of AZD9833, a Potent and Orally Bioavailable Selective Estrogen Receptor Degrader and Antagonist.

J Med Chem 2020 12 29;63(23):14530-14559. Epub 2020 Sep 29.

Advanced Drug Delivery, Pharmaceutical Sciences, R&D, Boston, Massachusetts, United States.

Herein we report the optimization of a series of tricyclic indazoles as selective estrogen receptor degraders (SERD) and antagonists for the treatment of ER breast cancer. Structure based design together with systematic investigation of each region of the molecular architecture led to the identification of -[1-(3-fluoropropyl)azetidin-3-yl]-6-[(6,8)-8-methyl-7-(2,2,2-trifluoroethyl)-6,7,8,9-tetrahydro-3-pyrazolo[4,3-]isoquinolin-6-yl]pyridin-3-amine (). This compound was demonstrated to be a highly potent SERD that showed a pharmacological profile comparable to fulvestrant in its ability to degrade ERα in both MCF-7 and CAMA-1 cell lines. A stringent control of lipophilicity ensured that had favorable physicochemical and preclinical pharmacokinetic properties for oral administration. This, combined with demonstration of potent in vivo activity in mouse xenograft models, resulted in progression of this compound, also known as AZD9833, into clinical trials.
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http://dx.doi.org/10.1021/acs.jmedchem.0c01163DOI Listing
December 2020

A Randomized, Open-label, Presurgical, Window-of-Opportunity Study Comparing the Pharmacodynamic Effects of the Novel Oral SERD AZD9496 with Fulvestrant in Patients with Newly Diagnosed ER HER2 Primary Breast Cancer.

Clin Cancer Res 2020 Aug 31;26(16):4242-4249. Epub 2020 Mar 31.

Breast Center, Department of Obstetrics and Gynecology and Comprehensive Cancer Center, University of Munich (LMU), Munich, Germany.

Purpose: Fulvestrant, the first-in-class selective estrogen receptor (ER) degrader (SERD), is clinically effective in patients with ER breast cancer, but it has administration and pharmacokinetic limitations. Pharmacodynamic data suggest complete ER degradation is not achieved at fulvestrant's clinically feasible dose. This presurgical study (NCT03236974) compared the pharmacodynamic effects of fulvestrant with AZD9496, a novel, orally bioavailable, nonsteroidal, potent SERD, in treatment-naïve patients with ER HER2 primary breast cancer awaiting curative intent surgery.

Patients And Methods: Patients were randomized 1:1 to receive AZD9496 250 mg twice daily from day 1 for 5-14 days, or fulvestrant 500 mg on day 1. On-treatment imaging-guided core tumor biopsies were taken between day 5 and 14 and compared with pretreatment diagnostic biopsies. The primary objective was to compare the effects of AZD9496 and fulvestrant on ER expression. Secondary objectives included changes in progesterone receptor (PR) and Ki-67 pharmacokinetic/pharmacodynamic relationships and safety.

Results: Forty-six women received treatment (AZD9496 = 22; fulvestrant = 24); 35 paired biopsies were evaluable (AZD9496 = 15; fulvestrant = 20). The least square mean estimate for ER H-score reduction was 24% after AZD9496 versus 36% after fulvestrant treatment ( = 0.86). AZD9496 also reduced PR H-scores (-33.3%) and Ki-67 levels (-39.9%) from baseline, but was also not superior to fulvestrant (PR: -68.7%, = 0.97; Ki-67: -75.4%, = 0.98). No new safety findings were identified.

Conclusions: This was the first presurgical study to demonstrate that an oral SERD affects its key biological targets. However, AZD9496 was not superior to fulvestrant at the dose tested.
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http://dx.doi.org/10.1158/1078-0432.CCR-19-3387DOI Listing
August 2020

HO-1 drives autophagy as a mechanism of resistance against HER2-targeted therapies.

Breast Cancer Res Treat 2020 Feb 8;179(3):543-555. Epub 2019 Nov 8.

Edinburgh Cancer Research UK Centre, Institute of Genetics & Molecular Medicine, University of Edinburgh, Crewe Road South, Edinburgh, EH4 2XR, UK.

Purpose: Targeted therapies have resulted in major advances in the treatment of HER2-positive breast cancers. Despite this, up to 70% of patients will develop resistance to treatment within 2 years and new strategies for targeting resistant disease are needed.

Methods: To identify potential resistance mechanisms, we used the mouse MMTV-NIC-PTEN spontaneous model of HER2-positive breast cancer and the pan-HER family kinase inhibitor sapatinib. Vehicle and sapatinib-treated tumors were evaluated by immunohistochemistry and proteomic analysis. In vitro studies were carried out to define the role of heme oxygenase 1 (HO-1) and autophagy in resistance to sapatinib and lapatinib, another pan-HER family kinase inhibitor.

Results: Treatment of tumor-bearing MMTV-NIC-PTEN mice with sapatinib resulted in delayed tumor progression and increased survival. However, tumors eventually progressed on treatment. Proteomic analysis identified proteins associated with cellular iron homeostasis as being upregulated in the sapatinib-treated tumors. This included HO-1 whose overexpression was confirmed by immunohistochemistry. Overexpression of HO-1 in HER2-expressing SKBR3 breast cancer cells resulted in reduced sensitivity to both pan-HER family kinase inhibitors sapatinib and lapatinib. This was associated with increased autophagy in the HO-1 over-expressing cells. Furthermore, increased autophagy was also seen in the sapatinib-treated tumors. Treatment with autophagy inhibitors was able to increase the sensitivity of the HO-1 over-expressing cells to both lapatinib and sapatinib.

Conclusion: Together these data indicate a role for HO-1-induced autophagy in resistance to pan-HER family kinase inhibitors.
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http://dx.doi.org/10.1007/s10549-019-05489-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6997276PMC
February 2020

Building Bridges in a Series of Estrogen Receptor Degraders: An Application of Metathesis in Medicinal Chemistry.

ACS Med Chem Lett 2019 Oct 23;10(10):1492-1497. Epub 2019 Sep 23.

Oncology R&D, AstraZeneca, R&D Boston, 35 Gatehouse Drive, Waltham, Massachusetts 02451, United States.

Herein we report the use of metathesis to construct a novel tetracyclic core in a series of estrogen receptor degraders. This improved the chemical stability, as assessed using an NMR-MS based assay, and gave a molecule with excellent physicochemical properties and pharmacokinetics in rat. X-ray crystallography established minimal perturbation of the bridged compounds relative to the unbridged analogues in the receptor binding pocket. Unfortunately, despite retaining excellent binding to ERα, this adversely affected the ability of the compounds to degrade the receptor.
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http://dx.doi.org/10.1021/acsmedchemlett.9b00370DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6792171PMC
October 2019

Combined Inhibition of PI3Kβ and mTOR Inhibits Growth of PTEN-null Tumors.

Mol Cancer Ther 2018 11 10;17(11):2309-2319. Epub 2018 Aug 10.

Bioscience, Oncology, IMED Biotech Unit, AstraZeneca, Cambridge, United Kingdom.

Loss of the tumor suppressor PTEN confers a tumor cell dependency on the PI3Kβ isoform. Achieving maximal inhibition of tumor growth through PI3K pathway inhibition requires sustained inhibition of PI3K signaling; however, efficacy is often limited by suboptimal inhibition or reactivation of the pathway. To select combinations that deliver comprehensive suppression of PI3K signaling in PTEN-null tumors, the PI3Kβ inhibitor AZD8186 was combined with inhibitors of kinases implicated in pathway reactivation in an extended cell proliferation assay. Inhibiting PI3Kβ and mTOR gave the most effective antiproliferative effects across a panel of PTEN-null tumor cell lines. The combination of AZD8186 and the mTOR inhibitor vistusertib was also effective controlling growth of PTEN-null tumor models of TNBC, prostate, and renal cancers. , the combination resulted in increased suppression of pNDRG1, p4EBP1, as well as HMGCS1 with reduced pNDRG1 and p4EBP1 more closely associated with effective suppression of proliferation. biomarker analysis revealed that the monotherapy and combination treatment consistently reduced similar biomarkers, while combination increased nuclear translocation of the transcription factor FOXO3 and reduction in glucose uptake. These data suggest that combining the PI3Kβ inhibitor AZD8186 and vistusertib has potential to be an effective combination treatment for PTEN-null tumors. .
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http://dx.doi.org/10.1158/1535-7163.MCT-18-0183DOI Listing
November 2018

Circulating Biomarkers and Resistance to Endocrine Therapy in Metastatic Breast Cancers: Correlative Results from AZD9496 Oral SERD Phase I Trial.

Clin Cancer Res 2018 12 6;24(23):5860-5872. Epub 2018 Aug 6.

University of Michigan Rogel Cancer Center and the Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan.

Purpose: Common resistance mechanisms to endocrine therapy (ET) in estrogen receptor (ER)-positive metastatic breast cancers include, among others, ER loss and acquired activating mutations in the ligand-binding domain of the ER gene (). mutational mediated resistance may be overcome by selective ER degraders (SERD). During the first-in-human study of oral SERD AZD9496, early changes in circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) were explored as potential noninvasive tools, alongside paired tumor biopsies, to assess pharmacodynamics and early efficacy.

Experimental Design: CTC were enumerated/phenotyped for ER and Ki67 using CellSearch in serial blood draws. ctDNA was assessed for the most common by droplet digital PCR (BioRad).

Results: Before starting AZD9496, 11 of 43 (25%) patients had ≥5 CTC/7.5 mL whole blood (WB), none of whom underwent reduction to <5 CTC/7.5 mL WB on C1D15. Five of 11 patients had baseline CTC-ER, two of whom had CTC-ER reduction. CTC-Ki67 status did not change appreciably. Patients with ≥5 CTC/7.5 mL WB before treatment had worse progression-free survival (PFS) than patients with <5 CTC ( = 0.0003). Fourteen of 45 (31%) patients had ctDNA at baseline, five of whom had ≥2 unique mutations. Baseline status was not prognostic. Patients with persistently elevated CTC and/or ctDNA at C1D15 had worse PFS than patients who did not ( = 0.0007).

Conclusions: Elevated CTC at baseline was a strong prognostic factor in this cohort. Early on-treatment changes were observed in CTC-ER and ctDNA, but not in overall CTC number. Integrating multiple biomarkers in prospective trials may improve outcome prediction and ET resistance mechanisms' identification over a single biomarker.
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http://dx.doi.org/10.1158/1078-0432.CCR-18-1569DOI Listing
December 2018

A First-in-Human Study of the New Oral Selective Estrogen Receptor Degrader AZD9496 for ER/HER2 Advanced Breast Cancer.

Clin Cancer Res 2018 08 13;24(15):3510-3518. Epub 2018 Feb 13.

Department of Internal Medicine, Seoul National University Hospital, Cancer Research Institute, Seoul National University College of Medicine, Seoul, South Korea.

AZD9496 is an oral nonsteroidal, small-molecule inhibitor of estrogen receptor alpha (ERα) and a potent and selective antagonist and degrader of ERα. This first-in-human phase I study determined the safety and tolerability of ascending doses of oral AZD9496 in women with estrogen receptor (ER)/HER2 advanced breast cancer, characterized its pharmacokinetic (PK) profile, and made preliminary assessment of antitumor activity. Forty-five patients received AZD9496 [20 mg once daily (QD) to 600 mg twice daily (BID)] in a dose-escalation, dose-expansion "rolling 6" design. Safety, tolerability, and PK activity in each cohort were reviewed before escalating to the next dose. PK was determined by mass spectrometry. Adverse events (AEs) were graded according to the Common Terminology Criteria for Adverse Events (CTCAE) v4.0. Objective tumor response was evaluated by Response Evaluation Criteria in Solid Tumors (RECIST) v1.1. Most common causally related AEs were diarrhea (35.6%), fatigue (31.1%), and nausea (22.2%), and seven patients had grade ≥3 AEs. Three patients experienced a dose-limiting toxicity: one each at 150 mg BID (abnormal hepatic function), 400 mg BID (diarrhea and elevated liver function tests), and 600 mg BID (diarrhea), and all were reversible. The maximum tolerated dose was not reached. Partial response was confirmed in one patient, who also had decreased tumor marker Ca15.3. Four patients had stable disease at 12 months' follow-up. AZD9496 is well tolerated with an acceptable safety profile, showing evidence of prolonged disease stabilization in heavily pretreated patients with ER/HER2 advanced breast cancer. .
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http://dx.doi.org/10.1158/1078-0432.CCR-17-3102DOI Listing
August 2018

Overcoming mTOR resistance mutations with a new-generation mTOR inhibitor.

Nature 2016 06 18;534(7606):272-6. Epub 2016 May 18.

Howard Hughes Medical Institute and Department of Cellular and Molecular Pharmacology, University of California San Francisco, San Francisco, California 94158, USA.

Precision medicines exert selective pressure on tumour cells that leads to the preferential growth of resistant subpopulations, necessitating the development of next-generation therapies to treat the evolving cancer. The PIK3CA-AKT-mTOR pathway is one of the most commonly activated pathways in human cancers, which has led to the development of small-molecule inhibitors that target various nodes in the pathway. Among these agents, first-generation mTOR inhibitors (rapalogs) have caused responses in 'N-of-1' cases, and second-generation mTOR kinase inhibitors (TORKi) are currently in clinical trials. Here we sought to delineate the likely resistance mechanisms to existing mTOR inhibitors in human cell lines, as a guide for next-generation therapies. The mechanism of resistance to the TORKi was unusual in that intrinsic kinase activity of mTOR was increased, rather than a direct active-site mutation interfering with drug binding. Indeed, identical drug-resistant mutations have been also identified in drug-naive patients, suggesting that tumours with activating MTOR mutations will be intrinsically resistant to second-generation mTOR inhibitors. We report the development of a new class of mTOR inhibitors that overcomes resistance to existing first- and second-generation inhibitors. The third-generation mTOR inhibitor exploits the unique juxtaposition of two drug-binding pockets to create a bivalent interaction that allows inhibition of these resistant mutants.
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http://dx.doi.org/10.1038/nature17963DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4902179PMC
June 2016

Utilization of Structure-Based Design to Identify Novel, Irreversible Inhibitors of EGFR Harboring the T790M Mutation.

ACS Med Chem Lett 2016 May 21;7(5):514-9. Epub 2016 Mar 21.

Oncology iMed, Innovative Medicines & Early Development, AstraZeneca R&D Boston, 35 Gatehouse Drive, Waltham, Massachusetts 02451, United States.

A novel series of covalent inhibitors of EGFR (epidermal growth factor receptor) kinase was discovered through a combination of subset screening and structure-based design. These compounds preferentially inhibit mutant forms of EGFR (activating mutant and T790M mutant) over wild-type EGFR in cellular assays measuring EGFR autophosphorylation and proliferation, suggesting an improved therapeutic index in non-small cell lung cancer patients would be achievable relative to established EGFR inhibitors. We describe our design approaches, resulting in the identification of the lead compound 5, and our efforts to develop an understanding of the structure-activity relationships within this series. In addition, strategies to overcome challenges around metabolic stability and aqueous solubility are discussed. Despite limitations in its physical properties, 5 is orally bioavailable in mice and demonstrates pronounced antitumor activity in in vivo models of mutant EGFR-driven cancers.
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http://dx.doi.org/10.1021/acsmedchemlett.6b00058DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4867489PMC
May 2016

Identification of novel pathways linking epithelial-to-mesenchymal transition with resistance to HER2-targeted therapy.

Oncotarget 2016 Mar;7(10):11539-52

Edinburgh Cancer Research Centre, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh EH4 2XR, UK.

Resistance to human epidermal growth factor receptor 2 (HER2)-targeted therapies in the treatment of HER2-positive breast cancer is a major clinical problem. To identify pathways linked to resistance, we generated HER2-positive breast cancer cell lines which are resistant to either lapatinib or AZD8931, two pan-HER family kinase inhibitors. Resistance was HER2 independent and was associated with epithelial-to-mesenchymal transition (EMT), resulting in increased proliferation and migration of the resistant cells. Using a global proteomics approach, we identified a novel set of EMT-associated proteins linked to HER2-independent resistance. We demonstrate that a subset of these EMT-associated genes is predictive of prognosis within the ERBB2 subtype of human breast cancers. Furthermore, targeting the EMT-associated kinases Src and Axl potently inhibited proliferation of the resistant cells, and inhibitors to these kinases may provide additional options for the treatment of HER2-independent resistance in tumors.
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http://dx.doi.org/10.18632/oncotarget.7317DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4905492PMC
March 2016

Use of a genetically engineered mouse model as a preclinical tool for HER2 breast cancer.

Dis Model Mech 2016 Feb 31;9(2):131-40. Epub 2015 Dec 31.

Edinburgh Cancer Research UK Centre, University of Edinburgh, Crewe Road South, Edinburgh EH4 2XR, UK

Resistance to human epidermal growth factor receptor 2 (HER2)-targeted therapies presents a major clinical problem. Although preclinical studies have identified a number of possible mechanisms, clinical validation has been difficult. This is most likely to reflect the reliance on cell-line models that do not recapitulate the complexity and heterogeneity seen in human tumours. Here, we show the utility of a genetically engineered mouse model of HER2-driven breast cancer (MMTV-NIC) to define mechanisms of resistance to the pan-HER family inhibitor AZD8931. Genetic manipulation of MMTV-NIC mice demonstrated that loss of phosphatase and tensin homologue (PTEN) conferred de novo resistance to AZD8931, and a tumour fragment transplantation model was established to assess mechanisms of acquired resistance. Using this approach, 50% of tumours developed resistance to AZD8931. Analysis of the resistant tumours showed two distinct patterns of resistance: tumours in which reduced membranous HER2 expression was associated with an epithelial-to-mesenchymal transition (EMT) and resistant tumours that retained HER2 expression and an epithelial morphology. The plasticity of the EMT phenotype was demonstrated upon re-implantation of resistant tumours that then showed a mixed epithelial and mesenchymal phenotype. Further AZD8931 treatment resulted in the generation of secondary resistant tumours that again had either undergone EMT or retained their original epithelial morphology. The data provide a strong rationale for basing therapeutic decisions on the biology of the individual resistant tumour, which can be very different from that of the primary tumour and will be specific to individual patients.
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http://dx.doi.org/10.1242/dmm.023143DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4770148PMC
February 2016

AZD2014, an Inhibitor of mTORC1 and mTORC2, Is Highly Effective in ER+ Breast Cancer When Administered Using Intermittent or Continuous Schedules.

Mol Cancer Ther 2015 Nov 10;14(11):2508-18. Epub 2015 Sep 10.

AstraZeneca Oncology, CRUK Cambridge Institute, Cambridge, United Kingdom.

mTOR is an atypical serine threonine kinase involved in regulating major cellular functions, such as nutrients sensing, growth, and proliferation. mTOR is part of the multiprotein complexes mTORC1 and mTORC2, which have been shown to play critical yet functionally distinct roles in the regulation of cellular processes. Current clinical mTOR inhibitors only inhibit the mTORC1 complex and are derivatives of the macrolide rapamycin (rapalogs). Encouraging effects have been observed with rapalogs in estrogen receptor-positive (ER(+)) breast cancer patients in combination with endocrine therapy, such as aromatase inhibitors. AZD2014 is a small-molecule ATP competitive inhibitor of mTOR that inhibits both mTORC1 and mTORC2 complexes and has a greater inhibitory function against mTORC1 than the clinically approved rapalogs. Here, we demonstrate that AZD2014 has broad antiproliferative effects across multiple cell lines, including ER(+) breast models with acquired resistance to hormonal therapy and cell lines with acquired resistance to rapalogs. In vivo, AZD2014 induces dose-dependent tumor growth inhibition in several xenograft and primary explant models. The antitumor activity of AZD2014 is associated with modulation of both mTORC1 and mTORC2 substrates, consistent with its mechanism of action. In combination with fulvestrant, AZD2014 induces tumor regressions when dosed continuously or using intermittent dosing schedules. The ability to dose AZD2014 intermittently, together with its ability to block signaling from both mTORC1 and mTORC2 complexes, makes this compound an ideal candidate for combining with endocrine therapies in the clinic. AZD2014 is currently in phase II clinical trials.
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http://dx.doi.org/10.1158/1535-7163.MCT-15-0365DOI Listing
November 2015

Combining AZD8931, a novel EGFR/HER2/HER3 signalling inhibitor, with AZD5363 limits AKT inhibitor induced feedback and enhances antitumour efficacy in HER2-amplified breast cancer models.

Int J Oncol 2015 Aug 22;47(2):446-54. Epub 2015 Jun 22.

Oncology iMED, AstraZeneca, Alderley Park, Macclesfield SK10 4TG, UK.

The phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signalling network is frequently de-regulated in breast cancer and has been shown to mediate resistance to anti-HER2 agents. Whilst constitutive activation of this pathway is emerging as a marker of sensitivity to various PI3K pathway inhibitors, activity of these agents in the clinic may be limited by the presence of feedback loops, leading to reactivation of receptor tyrosine kinases, such as HER2/HER3. To determine whether inhibition of HER2 could increase the efficacy of AZD5363, a novel AKT inhibitor, a panel of breast cancer cells was dosed with AZD5363 in combination with AZD8931, an inhibitor of EGFR/HER2/HER3 signalling. We show that the combined treatment resulted in synergistic growth inhibition and enhanced cell death, specifically in the HER2-amplified cell lines. Investigation of the mechanism by western blot analysis revealed that the addition of AZD8931 prevented the induction of HER2/HER3 phosphorylation induced by AZD5363 and resulted in concomitant inhibition of both the PI3K/AKT/mTOR and ERK signalling pathways and induction of apoptosis. Using the HCC1954 xenograft model, which is resistant to trastuzumab, we show that the combination of AZD5363 and AZD8931 is more efficacious than either agent alone, resulting in profound tumour regressions. We conclude that the activity of AZD5363 in HER2-amplified breast cancer cells is enhanced by the addition of AZD8931 and that dual targeting of AKT and EGFR/HER2/HER3 signalling is an attractive treatment option to be explored in the clinic.
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http://dx.doi.org/10.3892/ijo.2015.3062DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4501645PMC
August 2015

Discovery of a potent and selective EGFR inhibitor (AZD9291) of both sensitizing and T790M resistance mutations that spares the wild type form of the receptor.

J Med Chem 2014 Oct 1;57(20):8249-67. Epub 2014 Oct 1.

Oncology Innovative Medicines, ‡Drug Safety and Metabolism, §Global Medicines Development, and ∥Discovery Sciences, AstraZeneca , Mereside, Alderley Park, Macclesfield, Cheshire SK10 4TG, U.K.

Epidermal growth factor receptor (EGFR) inhibitors have been used clinically in the treatment of non-small-cell lung cancer (NSCLC) patients harboring sensitizing (or activating) mutations for a number of years. Despite encouraging clinical efficacy with these agents, in many patients resistance develops leading to disease progression. In most cases, this resistance is in the form of the T790M mutation. In addition, EGFR wild type receptor inhibition inherent with these agents can lead to dose limiting toxicities of rash and diarrhea. We describe herein the evolution of an early, mutant selective lead to the clinical candidate AZD9291, an irreversible inhibitor of both EGFR sensitizing (EGFRm+) and T790M resistance mutations with selectivity over the wild type form of the receptor. Following observations of significant tumor inhibition in preclinical models, the clinical candidate was administered clinically to patients with T790M positive EGFR-TKI resistant NSCLC and early efficacy has been observed, accompanied by an encouraging safety profile.
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http://dx.doi.org/10.1021/jm500973aDOI Listing
October 2014

Overcoming endocrine resistance due to reduced PTEN levels in estrogen receptor-positive breast cancer by co-targeting mammalian target of rapamycin, protein kinase B, or mitogen-activated protein kinase kinase.

Breast Cancer Res 2014 Sep 11;16(5):430. Epub 2014 Sep 11.

Introduction: Activation of the phosphatidylinositol 3-kinase (PI3K) pathway in estrogen receptor α (ER)-positive breast cancer is associated with reduced ER expression and activity, luminal B subtype, and poor outcome. Phosphatase and tensin homolog (PTEN), a negative regulator of this pathway, is typically lost in ER-negative breast cancer. We set out to clarify the role of reduced PTEN levels in endocrine resistance, and to explore the combination of newly developed PI3K downstream kinase inhibitors to overcome this resistance.

Methods: Altered cellular signaling, gene expression, and endocrine sensitivity were determined in inducible PTEN-knockdown ER-positive/human epidermal growth factor receptor 2 (HER2)-negative breast cancer cell and/or xenograft models. Single or two-agent combinations of kinase inhibitors were examined to improve endocrine therapy.

Results: Moderate PTEN reduction was sufficient to enhance PI3K signaling, generate a gene signature associated with the luminal B subtype of breast cancer, and cause endocrine resistance in vitro and in vivo. The mammalian target of rapamycin (mTOR), protein kinase B (AKT), or mitogen-activated protein kinase kinase (MEK) inhibitors, alone or in combination, improved endocrine therapy, but the efficacy varied by PTEN levels, type of endocrine therapy, and the specific inhibitor(s). A single-agent AKT inhibitor combined with fulvestrant conferred superior efficacy in overcoming resistance, inducing apoptosis and tumor regression.

Conclusions: Moderate reduction in PTEN, without complete loss, can activate the PI3K pathway to cause endocrine resistance in ER-positive breast cancer, which can be overcome by combining endocrine therapy with inhibitors of the PI3K pathway. Our data suggests that the ER degrader fulvestrant, to block both ligand-dependent and -independent ER signaling, combined with an AKT inhibitor is an effective strategy to test in patients.
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http://dx.doi.org/10.1186/s13058-014-0430-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4303114PMC
September 2014

Exploring mechanisms of acquired resistance to HER2 (human epidermal growth factor receptor 2)-targeted therapies in breast cancer.

Biochem Soc Trans 2014 Aug;42(4):822-30

*Edinburgh Cancer Research Centre, Institute of Genetics and Molecular Medicine, University of Edinburgh, Crewe Road South, Edinburgh EH4 2XR.

HER2 (human epidermal growth factor receptor 2)-targeted therapy in breast cancer is one of the earliest and arguably most successful examples of the modern class of targeted drugs. Initially identified in the 1980s, the observation that HER2 acts as an independent predictor of poor prognosis in the 20% of breast cancer cases carrying a gene amplification or protein overexpression cemented its place at the forefront of research in this field. The outlook for patients with HER2-positive breast cancer has been revolutionized by the introduction of HER2-targeted agents, such as trastuzumab and lapatinib, yet resistance is frequently encountered and multiple different resistance mechanisms have been identified. We have explored resistance to a novel pan-HER inhibitor, AZD8931, and we examine mechanisms of resistance common to trastuzumab, lapatinib and AZD8931, and discuss the current problems associated with translating the wealth of pre-clinical data into clinical benefit.
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http://dx.doi.org/10.1042/BST20140109DOI Listing
August 2014

Discovery of AZD8931, an Equipotent, Reversible Inhibitor of Signaling by EGFR, HER2, and HER3 Receptors.

ACS Med Chem Lett 2013 Aug 31;4(8):742-6. Epub 2013 May 31.

Oncology iMed, AstraZeneca , Alderley Park, Macclesfield, Cheshire SK10 4TG, United Kingdom.

Deregulation of HER family signaling promotes proliferation and tumor cell survival and has been described in many human cancers. Simultaneous, equipotent inhibition of EGFR-, HER2-, and HER3-mediated signaling may be of clinical utility in cancer settings where the selective EGFR or HER2 therapeutic agents are ineffective or only modestly active. We describe the discovery of AZD8931 (2), an equipotent, reversible inhibitor of EGFR-, HER2-, and HER3-mediated signaling and the structure-activity relationships within this series. Docking studies based on a model of the HER2 kinase domain helped rationalize the increased HER2 activity seen with the methyl acetamide side chain present in AZD8931. AZD8931 exhibited good pharmacokinetics in preclinical species and showed superior activity in the LoVo tumor growth efficacy model compared to close analogues. AZD8931 is currently being evaluated in human clinical trials for the treatment of cancer.
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http://dx.doi.org/10.1021/ml400146cDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4027407PMC
August 2013

AZD9291, an irreversible EGFR TKI, overcomes T790M-mediated resistance to EGFR inhibitors in lung cancer.

Cancer Discov 2014 Sep 3;4(9):1046-61. Epub 2014 Jun 3.

Department of Medicine and Vanderbilt-Ingram Cancer Center, Vanderbilt University, Nashville, Tennessee; and

Unlabelled: First-generation EGFR tyrosine kinase inhibitors (EGFR TKI) provide significant clinical benefit in patients with advanced EGFR-mutant (EGFRm(+)) non-small cell lung cancer (NSCLC). Patients ultimately develop disease progression, often driven by acquisition of a second T790M EGFR TKI resistance mutation. AZD9291 is a novel oral, potent, and selective third-generation irreversible inhibitor of both EGFRm(+) sensitizing and T790M resistance mutants that spares wild-type EGFR. This mono-anilino-pyrimidine compound is structurally distinct from other third-generation EGFR TKIs and offers a pharmacologically differentiated profile from earlier generation EGFR TKIs. Preclinically, the drug potently inhibits signaling pathways and cellular growth in both EGFRm(+) and EGFRm(+)/T790M(+) mutant cell lines in vitro, with lower activity against wild-type EGFR lines, translating into profound and sustained tumor regression in EGFR-mutant tumor xenograft and transgenic models. The treatment of 2 patients with advanced EGFRm(+) T790M(+) NSCLC is described as proof of principle.

Significance: We report the development of a novel structurally distinct third-generation EGFR TKI, AZD9291, that irreversibly and selectively targets both sensitizing and resistant T790M(+) mutant EGFR while harboring less activity toward wild-type EGFR. AZD9291 is showing promising responses in a phase I trial even at the first-dose level, with first published clinical proof-of-principle validation being presented.
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http://dx.doi.org/10.1158/2159-8290.CD-14-0337DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4315625PMC
September 2014

AZD8931, an equipotent, reversible inhibitor of signaling by epidermal growth factor receptor (EGFR), HER2, and HER3: preclinical activity in HER2 non-amplified inflammatory breast cancer models.

J Exp Clin Cancer Res 2014 May 30;33:47. Epub 2014 May 30.

Department of Medical Oncology, Thomas Jefferson University; and Kimmel Cancer Center, Philadelphia, PA 19107, USA.

Introduction: Epidermal growth factor receptor (EGFR) overexpression has been associated with prognostic and predictive value in inflammatory breast cancer (IBC). Epidermal growth factor receptor 2 (HER2) overexpression is observed at a higher rate in IBC compared with noninflammatory breast cancer. Current clinically available anti-HER2 therapies are effective only in patients with HER2 amplified breast cancer, including IBC. AZD8931 is a novel small-molecule equipotent inhibitor of EGFR, HER2, and HER3 signaling. In this study, we investigated the antitumor activity of AZD8931 alone or in combination with paclitaxel using preclinical models of EGFR-overexpressed and HER2 non-amplified IBC cells.

Methods: Two IBC cell lines SUM149 and FC-IBC-02 derived from pleural effusion of an IBC patient were used in this study. Cell growth and apoptotic cell death were examined in vitro. For the in vivo tumor growth studies, IBC cells were orthotopically transplanted into the mammary fat pads of immunodeficient mice. AZD8931 was given by daily oral gavage at doses of 25 mg/kg, 5 days/week for 4 weeks. Paclitaxel was subcutaneously injected twice weekly.

Results: AZD8931 significantly suppressed cell growth of IBC cells and induced apoptosis of human IBC cells in vitro. Significantly, we showed that AZD8931 monotherapy inhibited xenograft growth and the combination of paclitaxel + AZD8931 was demonstrably more effective than paclitaxel or AZD8931 alone treatment at delaying tumor growth in vivo in orthotopic IBC models.

Conclusion: AZD8931 single agent and in combination with paclitaxel demonstrated signal inhibition and antitumor activity in EGFR-overexpressed and HER2 non-amplified IBC models. These results suggest that AZD8931 may provide a novel therapeutic strategy for the treatment of IBC patients with HER2 non-amplified tumors.
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http://dx.doi.org/10.1186/1756-9966-33-47DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4061513PMC
May 2014

Therapeutic potential of the dual EGFR/HER2 inhibitor AZD8931 in circumventing endocrine resistance.

Breast Cancer Res Treat 2014 Apr 20;144(2):263-72. Epub 2014 Feb 20.

Lester and Sue Smith Breast Center and Dan L Duncan Cancer Center, Baylor College of Medicine, One Baylor Plaza, BCM:600, Houston, TX, 77030, USA.

Modest up-regulation of either HER-ligands or receptors has been implicated in acquired endocrine resistance. AZD8931, a dual tyrosine kinase inhibitor (TKI) of epithelial growth factor receptor (EGFR)/HER2, has been shown to more effectively block ligand-dependent HER signaling than the HER TKIs lapatinib or gefitinib. We therefore examined the effect of AZD8931 in ER-positive/HER2-negative breast cancer cells with acquired resistance to tamoxifen, where there is ligand up-regulation associated with HER pathway activation. RNA-seq ligand profiling and levels of HER receptors and signaling by western blotting were conducted in ER+ MCF7 and T47D parental cells and their Tam-resistant derivatives (TamRes). In vitro cell growth and apoptosis and HER ligand-stimulated signaling were measured in response to endocrine and HER TKIs. For studies in vivo, transplantable MCF7/TamRes xenografts were treated with tamoxifen or fulvestrant, either alone or in combination with AZD8931. AZD8931 only minimally enhanced endocrine sensitivity in MCF7 parental cells, but showed a greater effect in the T47D parental model. AZD8931 combined with either tamoxifen or fulvestrant inhibited cell growth more than lapatinib in T47D TamRes cells, and was also significantly, though modestly, more potent in MCF7 TamRes cells. In both TamRes models, AZD8931 significantly inhibited cell proliferation and induced apoptosis. Under ligand-stimulated conditions, AZD8931 more potently inhibited HER signaling than lapatinib or gefitinib. AZD8931 also significantly delayed the growth of MCF7 TamRes xenografts in the presence of tamoxifen or fulvestrant. The strongest inhibition was achieved with a fulvestrant and AZD8931 combination, though no tumor regression was observed. This study provides evidence that AZD8931 has greater inhibitory efficacy in tamoxifen-resistant settings than in an endocrine therapy naïve setting. The absence of tumor regression, however, suggests that additional escape pathways contribute to resistant growth and will need to be targeted to fully circumvent tamoxifen resistance.
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http://dx.doi.org/10.1007/s10549-014-2878-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4030601PMC
April 2014

Structure- and reactivity-based development of covalent inhibitors of the activating and gatekeeper mutant forms of the epidermal growth factor receptor (EGFR).

J Med Chem 2013 Sep 30;56(17):7025-48. Epub 2013 Aug 30.

AstraZeneca , Oncology & Discovery Sciences Innovative Medicines, Mereside, Alderley Park, Macclesfield, Cheshire SK10 4TG, United Kingdom.

A novel series of small-molecule inhibitors has been developed to target the double mutant form of the epidermal growth factor receptor (EGFR) tyrosine kinase, which is resistant to treatment with gefitinib and erlotinib. Our reported compounds also show selectivity over wild-type EGFR. Guided by molecular modeling, this series was evolved to target a cysteine residue in the ATP binding site via covalent bond formation and demonstrates high levels of activity in cellular models of the double mutant form of EGFR. In addition, these compounds show significant activity against the activating mutations, which gefitinib and erlotinib target and inhibition of which gives rise to their observed clinical efficacy. A glutathione (GSH)-based assay was used to measure thiol reactivity toward the electrophilic functionality of the inhibitor series, enabling both the identification of a suitable reactivity window for their potency and the development of a reactivity quantitative structure-property relationship (QSPR) to support design.
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http://dx.doi.org/10.1021/jm400822zDOI Listing
September 2013

AZD4547: an orally bioavailable, potent, and selective inhibitor of the fibroblast growth factor receptor tyrosine kinase family.

Cancer Res 2012 Apr 27;72(8):2045-56. Epub 2012 Feb 27.

AstraZeneca Innovation Center China, Building 7, 898 Halei Road, Zhangjiang Hi-Tech Park, Shanghai 201203, PR China.

The fibroblast growth factor (FGF) signaling axis is increasingly implicated in tumorigenesis and chemoresistance. Several small-molecule FGF receptor (FGFR) kinase inhibitors are currently in clinical development; however, the predominant activity of the most advanced of these agents is against the kinase insert domain receptor (KDR), which compromises the FGFR selectivity. Here, we report the pharmacologic profile of AZD4547, a novel and selective inhibitor of the FGFR1, 2, and 3 tyrosine kinases. AZD4547 inhibited recombinant FGFR kinase activity in vitro and suppressed FGFR signaling and growth in tumor cell lines with deregulated FGFR expression. In a representative FGFR-driven human tumor xenograft model, oral administration of AZD4547 was well tolerated and resulted in potent dose-dependent antitumor activity, consistent with plasma exposure and pharmacodynamic modulation of tumor FGFR. Importantly, at efficacious doses, no evidence of anti-KDR-related effects were observed, confirming the in vivo FGFR selectivity of AZD4547. Taken together, our findings show that AZD4547 is a novel selective small-molecule inhibitor of FGFR with potent antitumor activity against FGFR-deregulated tumors in preclinical models. AZD4547 is under clinical investigation for the treatment of FGFR-dependent tumors.
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http://dx.doi.org/10.1158/0008-5472.CAN-11-3034DOI Listing
April 2012

AZD8931, an equipotent, reversible inhibitor of signaling by epidermal growth factor receptor, ERBB2 (HER2), and ERBB3: a unique agent for simultaneous ERBB receptor blockade in cancer.

Clin Cancer Res 2010 Feb 9;16(4):1159-69. Epub 2010 Feb 9.

AstraZeneca, Macclesfield, United Kingdom; AstraZeneca, Reims, France.

Purpose: To test the hypothesis that simultaneous, equipotent inhibition of epidermal growth factor receptor (EGFR; erbB1), erbB2 (human epidermal growth factor receptor 2), and erbB3 receptor signaling, using the novel small-molecule inhibitor AZD8931, will deliver broad antitumor activity in vitro and in vivo.

Experimental Design: A range of assays was used to model erbB family receptor signaling in homodimers and heterodimers, including in vitro evaluation of erbB kinase activity, erbB receptor phosphorylation, proliferation in cells, and in vivo testing in a human tumor xenograft panel, with ex vivo evaluation of erbB phosphorylation and downstream biomarkers. Gefitinib and lapatinib were used to compare the pharmacological profile of AZD8931 with other erbB family inhibitors.

Results: In vitro, AZD8931 showed equipotent, reversible inhibition of EGFR (IC(50), 4 nmol/L), erbB2 (IC(50), 3 nmol/L), and erbB3 (IC(50), 4 nmol/L) phosphorylation in cells. In proliferation assays, AZD8931 was significantly more potent than gefitinib or lapatinib in specific squamous cell carcinoma of the head and neck and non-small cell lung carcinoma cell lines. In vivo, AZD8931 inhibited xenograft growth in a range of models while significantly affecting EGFR, erbB2, and erbB3 phosphorylation and downstream signaling pathways, apoptosis, and proliferation.

Conclusions: AZD8931 has a unique pharmacologic profile providing equipotent inhibition of EGFR, erbB2, and erbB3 signaling and showing greater antitumor activity than agents with a narrower spectrum of erbB receptor inhibition in specific preclinical models. AZD8931 provides the opportunity to investigate whether simultaneous inhibition of erbB receptor signaling could be of utility in the clinic, particularly in the majority of solid tumors that do not overexpress erbB2.
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http://dx.doi.org/10.1158/1078-0432.CCR-09-2353DOI Listing
February 2010

Analyzing how cell adhesion controls mammary gland function by transplantation of embryonic mammary tissue from knockout mice.

Methods Mol Biol 2009 ;522:331-45

AstraZeneca Plc, Chesire, SK10 4TG, UK.

Understanding how cell adhesion to the extracellular matrix controls mammalian development has been explored extensively using gene knockout technology. However, in some knockout mice, animals die during late embryogenesis or shortly after birth. In such cases, it is possible to analyze embryonic developmental phenotypes, but it is less easy to determine the in vivo role of cell-matrix interactions in adult tissues. Although this problem has been partially solved by the development of tissue-specific knockouts, the approach relies on appropriate tissue-specific promoters. In many cases, genes that uniquely characterize specific cell types within complex tissues have not been identified. Thus, knockout technology can be restrictive when analyzing cell-matrix interactions in specific cases of tissue development and/or homeostasis. Here we describe how transplantation of mammary tissue into recipient hosts can be used to extend the understanding of cell adhesion functions in developmental processes.
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http://dx.doi.org/10.1007/978-1-59745-413-1_22DOI Listing
April 2009

Urinary retention and cystitis associated with subcutaneous estradiol pellets in female nude mice.

Toxicol Pathol 2009 Feb 29;37(2):227-34. Epub 2009 Jan 29.

GSK Research and Development, The Frythe, Welwyn, Hertfordshire, UK.

Unexpected deaths occurred in studies involving a nude mouse model of mammary cancer that required subcutaneous implantation of 0.5 mg twenty-one-day release estrogen pellets for growth of the estrogen-dependent mammary tumor xenograft BT474c. Early deaths occurred in female nude mice and were associated with urinary retention, frequently with cystitis. Drug treatment had no effect on the incidence or severity of cystitis. Histological findings did not alter significantly over various time points following pellet implantation. Changes were not seen in males or in females receiving lower doses of estradiol even when the duration of administration was prolonged, suggesting that a threshold level was required for the onset of urinary retention. Because of the influence of estrogen on micturition, immunohistochemistry for estrogen receptor alpha (ERalpha) in the urinary bladder was carried out, which did not demonstrate any differences between females implanted with 0.5 mg twenty-one-day release estrogen pellets and nonimplanted females. Although previous publications have concentrated on possible mechanisms of action, this paper describes the histopathological changes seen in the urinary bladder of female nude mice resulting from exposure to high levels of estradiol.
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http://dx.doi.org/10.1177/0192623308329281DOI Listing
February 2009

Neutral 5-substituted 4-indazolylaminoquinazolines as potent, orally active inhibitors of erbB2 receptor tyrosine kinase.

Bioorg Med Chem Lett 2008 Mar 16;18(6):1799-803. Epub 2008 Feb 16.

AstraZeneca, Centre de Recherches, Z.I.S.E. La Pompelle, B.P. 1050, 51689 Reims Cedex 2, France.

We have identified a new series of C-5 substituted indazolylaminoquinazolines as potent erbB2 kinase inhibitors. The lead compound 22 showed excellent in vitro potency, good physical properties, acceptable oral pharmacokinetics in rat and dog, and low human in vitro clearance. It showed at least equivalent activity dose for dose compared to lapatinib in various erbB2- or EGFR-driven xenograft models after chronic oral administration.
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http://dx.doi.org/10.1016/j.bmcl.2008.02.035DOI Listing
March 2008

A new series of neutral 5-substituted 4-anilinoquinazolines as potent, orally active inhibitors of erbB2 receptor tyrosine kinase.

Bioorg Med Chem Lett 2008 Jan 21;18(2):674-8. Epub 2007 Nov 21.

AstraZeneca, Centre de Recherches, Z.I.S.E. La Pompelle B.P. 1050, 51689 Reims Cedex 2, France.

Starting from initial lead 1 containing a basic 5-substituent, optimisation of the glycolamide-derived neutral 5-substituent led to potent inhibitors of erbB2 with good pharmacokinetics. Representative compounds 19 and 21 inhibited phosphorylation of erbB2 in a mouse BT474C xenograft model after oral administration.
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http://dx.doi.org/10.1016/j.bmcl.2007.11.052DOI Listing
January 2008

Neutral 5-substituted 4-anilinoquinazolines as potent, orally active inhibitors of erbB2 receptor tyrosine kinase.

Bioorg Med Chem Lett 2007 Nov 2;17(22):6326-9. Epub 2007 Sep 2.

Cancer and Infection Research, AstraZeneca, Mereside, Alderley Park, Macclesfield, UK.

Neutral 5-substituted 4-anilinoquinazolines addressed high in vivo clearance and phospholipidosis associated with previous basic compounds. A representative compound 8a inhibited tumor growth in a mouse xenograft model when co-administered with the cytochrome P450 inhibitor 1-aminobenzotriazole (ABT), and data are consistent with pharmacology primarily reflecting inhibition of erbB2 receptor tyrosine kinase.
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http://dx.doi.org/10.1016/j.bmcl.2007.08.073DOI Listing
November 2007

Inhibitors of epidermal growth factor receptor tyrosine kinase: optimisation of potency and in vivo pharmacokinetics.

Bioorg Med Chem Lett 2006 Sep 27;16(18):4908-12. Epub 2006 Jun 27.

AstraZeneca, Mereside, Alderley Park, Macclesfield, Cheshire SK10 4TG, UK.

The structure-activity and structure-property relationships of anilinoquinazoline inhibitors of EGFR were investigated. Strategies to lower volume of distribution and shorten half-life through structure and pKa modulation are discussed.
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http://dx.doi.org/10.1016/j.bmcl.2006.06.054DOI Listing
September 2006

Inhibitors of epidermal growth factor receptor tyrosine kinase: Novel C-5 substituted anilinoquinazolines designed to target the ribose pocket.

Bioorg Med Chem Lett 2006 Mar 27;16(6):1633-7. Epub 2005 Dec 27.

AstraZeneca, Mereside, Alderley Park, Macclesfield, Cheshire SK10 4TG, UK.

A series of novel C-5 substituted anilinoquinazolines, selected on the basis of docking experiments and overlays with ATP in the active site of EGFR tyrosine kinase, have been prepared and found to be potent inhibitors. In vivo pharmacokinetics and disease model activity are discussed.
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http://dx.doi.org/10.1016/j.bmcl.2005.12.028DOI Listing
March 2006