Search

Supplementary figues and tables: Figure S1. In vitro AZD1152-hQPA reduces proliferation and induces polyploidy across a panel of human AML cancer cell lines. Figure S2. AZD1152 has a modest effect on the growth of the HL-60 xenograft models in SCID mice Figure S3. AZD1152 in combination with Ara-C has a modest effect on the growth of the HL-60 xenograft models in SCID mice Figure S4. AZD2811 affect the neutrophil count in a dose-dependent manner Table S1: Cytogenetics of the different cell lines Table S2: Summary table of the pIC50 and nuclear area.

Supplementary Information

U87/Centrin2-EGFP/H2B-mCherry/puro cells were seeded on dishes with cover slip bottoms, treated with AZ32 (3 uM), and irradiated (5 Gy). Time-lapse videos were recorded intermittently (every 7 min) over 16 hrs. Treatment results in 11% aberrant mitoses (see Fig. 3F).

U87/shp53/Centrin2-EGFP/H2B-mCherry/shp53 cells were seeded on dishes with cover slip bottoms and irradiated (5 Gy). Time-lapse videos were recorded intermittently (every 7 min) over 16 hrs. Treatment results in 12% aberrant mitoses (see Fig. 3F).

U87/Centrin2-EGFP/H2B-mCherry/puro cells were seeded on dishes with cover slip bottoms and irradiated (5 Gy). Time-lapse videos were recorded intermittently (every 7 min) over 16 hrs. Treatment results in 2.7% aberrant mitoses (see Fig. 3F).

Inhibition of ataxia-telangiectasia mutated (ATM) during radiotherapy of glioblastoma multiforme (GBM) may improve tumor control by short-circuiting the response to radiation-induced DNA damage. A major impediment for clinical implementation is that current inhibitors have limited central nervous system (CNS) bioavailability; thus, the goal was to identify ATM inhibitors (ATMi) with improved CNS penetration. Drug screens and refinement of lead compounds identified AZ31 and AZ32. The compounds were then tested in vivo for efficacy and impact on tumor and healthy brain. Both AZ31 and AZ32 blocked the DNA damage response and radiosensitized GBM cells in vitro. AZ32, with enhanced blood–brain barrier (BBB) penetration, was highly efficient in vivo as radiosensitizer in syngeneic and human, orthotopic mouse glioma model compared with AZ31. Furthermore, human glioma cell lines expressing mutant p53 or having checkpoint-defective mutations were particularly sensitive to ATMi radiosensitization. The mechanism for this p53 effect involves a propensity to undergo mitotic catastrophe relative to cells with wild-type p53. In vivo, apoptosis was >6-fold higher in tumor relative to healthy brain after exposure to AZ32 and low-dose radiation. AZ32 is the first ATMi with oral bioavailability shown to radiosensitize glioma and improve survival in orthotopic mouse models. These findings support the development of a clinical-grade, BBB-penetrating ATMi for the treatment of GBM. Importantly, because many GBMs have defective p53 signaling, the use of an ATMi concurrent with standard radiotherapy is expected to be cancer-specific, increase the therapeutic ratio, and maintain full therapeutic effect at lower radiation doses. Mol Cancer Ther; 17(8); 1637–47. ©2018 AACR.

The PARP inhibitor AZD2461 was developed as a next-generation agent following olaparib, the first PARP inhibitor approved for cancer therapy. In BRCA1-deficient mouse models, olaparib resistance predominantly involves overexpression of P-glycoprotein, so AZD2461 was developed as a poor substrate for drug transporters. Here we demonstrate the efficacy of this compound against olaparib-resistant tumors that overexpress P-glycoprotein. In addition, AZD2461 was better tolerated in combination with chemotherapy than olaparib in mice, which suggests that AZD2461 could have significant advantages over olaparib in the clinic. However, this superior toxicity profile did not extend to rats. Investigations of this difference revealed a differential PARP3 inhibitory activity for each compound and a higher level of PARP3 expression in bone marrow cells from mice as compared with rats and humans. Our findings have implications for the use of mouse models to assess bone marrow toxicity for DNA-damaging agents and inhibitors of the DNA damage response. Finally, structural modeling of the PARP3-active site with different PARP inhibitors also highlights the potential to develop compounds with different PARP family member specificity profiles for optimal antitumor activity and tolerability. Cancer Res; 76(20); 6084–94. ©2016 AACR.

Supplementary materials and methods. Suppl Table 1. MMS combination PF50 cell line assays. Concentration-dependent potentiation of MMS (PF50 ratios). Suppl Table 2. Permeability of AZD2461 and olaparib were assessed. Suppl Table 3. Activity of olaparib and AZD2461 alone or in combination with the P-gp inhibitor verapamil in the colorectal cancer cell line HCT-15, which expresses high levels of P-gp. Suppl Fig. 1. Immunofluorescence for poly(ADP-ribose) in human A549 cells. Suppl Fig. 2. Analysis of both in vivo pharmacokinetics (PK) and pharmacodynamics (PD) in tumors taken from SW620 (human colon carcinoma) xenograft-bearing mice. Suppl Fig. 3. Colony formation (clonogenic) assays in the intrinsically high P-gp-expressing MRE11-deficient HCT-15 human colorectal cell line. Suppl. Fig. 4. Combination efficacy studies. Suppl Fig. 5. Relative body weight changes in mice following treatment with 10 mg/kg AZD2461 and olaparib in combination with temozolomide. Suppl. Fig. 6. A serial dilution of RNA concentration was used to establish a standard curve for determining the reaction efficiency of PARP1, PARP2, PARP3, and housekeeper gene (PPIA: cyclophilin A) TaqMan RT-PCR assays in both rat and mouse species; Determination of optimal reference gene for rat-mouse data normalization. Suppl Fig. 7.A. A serial dilution of reference RNA (Qiagen) concentration was used to establish a standard curve for determining the reaction efficiency of PARP3 and housekeeper genes (PPIA: cyclophilin and YWHAZ: tyrosine 3-monooxygenase/tryptohpan 5-monooxygenase activation protein, zeta polypeptide) SybrGreen RT-PCR assays in both rat and human species; Determination of optimal reference gene for rat-human data normalization. Suppl Fig. 8. Relative body weight changes in rat following treatment with 10 mg/kg AZD2461 and olaparib in combination with temozolomide; Relative body weight changes in rat following treatment with 20 mg/kg AZD2461 and olaparib in combination with temozolomide.

DNA damage checkpoint kinases ATR and WEE1 are among key regulators of DNA damage response pathways protecting cells from replication stress, a hallmark of cancer that has potential to be exploited for therapeutic use. ATR and WEE1 inhibitors are in early clinical trials and success will require greater understanding of both their mechanism of action and biomarkers for patient selection. Here, we report selective antitumor activity of ATR and WEE1 inhibitors in a subset of non-germinal center B-cell (GCB) diffuse large B-cell lymphoma (DLBCL) cell lines, characterized by high MYC protein expression and CDKN2A/B deletion. Activity correlated with the induction of replication stress, indicated by increased origin firing and retardation of replication fork progression. However, ATR and WEE1 inhibitors caused different amounts of DNA damage and cell death in distinct phases of the cell cycle, underlying the increased potency observed with WEE1 inhibition. ATR inhibition caused DNA damage to manifest as 53BP1 nuclear bodies in daughter G1 cells leading to G1 arrest, whereas WEE1 inhibition caused DNA damage and arrest in S phase, leading to earlier onset apoptosis. In vivo xenograft DLBCL models confirmed differences in single-agent antitumor activity, but also showed potential for effective ATR inhibitor combinations. Importantly, insights into the different inhibitor mechanisms may guide differentiated clinical development strategies aimed at exploiting specific vulnerabilities of tumor cells while maximizing therapeutic index. Our data therefore highlight clinical development opportunities for both ATR and WEE1 inhibitors in non-GCB DLBCL subtypes that represent an area of unmet clinical need.Significance:ATR and WEE1 inhibitors demonstrate effective antitumor activity in preclinical models of DLBCL associated with replication stress, but new mechanistic insights and biomarkers of response support a differentiated clinical development strategy.

Methods Table: Antibodies

This file contains all supplementary figures. Figure 1-7 provide additional analysis of genetic and cell features associated with AZD6738 and AZD1775 activity in DLBCL cell lines, including immunochemistry, immunoblots, immunofluorescence and flow cytometry data to further support the conclusions made from data presented in the main manuscript. Fig 8 provides tumour volume and body weight measurements for individual animals used in the in vivo studies.

Supplementary Data from ATR Inhibitor AZD6738 (Ceralasertib) Exerts Antitumor Activity as a Monotherapy and in Combination with Chemotherapy and the PARP Inhibitor Olaparib

This file contains Supplementary Tables 1-6. Table S1 lists the growth inhibitory values of AZD6738 and cell doubling rates in DLBCL cell lines used in the study. Table S2 details the median replication fork velocities of DLBCL cell lines treated with DMSO and AZD6738 in Figure 3. Table S3 lists the statistical significance for DNA fibre analysis in Figure 3. Tables S4-S6 summarise the in vivo experiment groups used in the manuscript, including animal numbers in study, efficacy and body weight loss.

Supplementary Figures 1-3, Table 1 from AZD1152 Rapidly and Negatively Affects the Growth and Survival of Human Acute Myeloid Leukemia Cells In vitro and In vivo

Supplementary Table 4 from CHR-2797: An Antiproliferative Aminopeptidase Inhibitor that Leads to Amino Acid Deprivation in Human Leukemic Cells

CHR-2797 is a novel metalloenzyme inhibitor that is converted into a pharmacologically active acid product (CHR-79888) inside cells. CHR-79888 is a potent inhibitor of a number of intracellular aminopeptidases, including leucine aminopeptidase. CHR-2797 exerts antiproliferative effects against a range of tumor cell lines in vitro and in vivo and shows selectivity for transformed over nontransformed cells. Its antiproliferative effects are at least 300 times more potent than the prototypical aminopeptidase inhibitor, bestatin. However, the mechanism by which inhibition of these enzymes leads to proliferative changes is not understood. Gene expression microarrays were used to profile changes in mRNA expression levels in the human promyelocytic leukemia cell line HL-60 treated with CHR-2797. This analysis showed that CHR-2797 treatment induced a transcriptional response indicative of amino acid depletion, the amino acid deprivation response, which involves up-regulation of amino acid synthetic genes, transporters, and tRNA synthetases. These changes were confirmed in other leukemic cell lines sensitive to the antiproliferative effects of CHR-2797. Furthermore, CHR-2797 treatment inhibited phosphorylation of mTOR substrates and reduced protein synthesis in HL-60 cells, both also indicative of amino acid depletion. Treatment with CHR-2797 led to an increase in the concentration of intracellular small peptides, the substrates of aminopeptidases. It is suggested that aminopeptidase inhibitors, such as CHR-2797 and bestatin, deplete sensitive tumor cells of amino acids by blocking protein recycling, and this generates an antiproliferative effect. CHR-2797 is orally bioavailable and currently undergoing phase II clinical investigation in the treatment of myeloid leukemia. [Cancer Res 2008;68(16):6669–79]

Supplementary Methods and Materials from CHR-2797: An Antiproliferative Aminopeptidase Inhibitor that Leads to Amino Acid Deprivation in Human Leukemic Cells

AZD6738 (ceralasertib) is a potent and selective orally bioavailable inhibitor of ataxia telangiectasia and Rad3-related (ATR) kinase. ATR is activated in response to stalled DNA replication forks to promote G2–M cell-cycle checkpoints and fork restart. Here, we found AZD6738 modulated CHK1 phosphorylation and induced ATM-dependent signaling (pRAD50) and the DNA damage marker γH2AX. AZD6738 inhibited break-induced replication and homologous recombination repair. In vitro sensitivity to AZD6738 was elevated in, but not exclusive to, cells with defects in the ATM pathway or that harbor putative drivers of replication stress such as CCNE1 amplification. This translated to in vivo antitumor activity, with tumor control requiring continuous dosing and free plasma exposures, which correlated with induction of pCHK1, pRAD50, and γH2AX. AZD6738 showed combinatorial efficacy with agents associated with replication fork stalling and collapse such as carboplatin and irinotecan and the PARP inhibitor olaparib. These combinations required optimization of dose and schedules in vivo and showed superior antitumor activity at lower doses compared with that required for monotherapy. Tumor regressions required at least 2 days of daily dosing of AZD6738 concurrent with carboplatin, while twice daily dosing was required following irinotecan. In a BRCA2-mutant patient-derived triple-negative breast cancer (TNBC) xenograft model, complete tumor regression was achieved with 3 to5 days of daily AZD6738 per week concurrent with olaparib. Increasing olaparib dosage or AZD6738 dosing to twice daily allowed complete tumor regression even in a BRCA wild-type TNBC xenograft model. These preclinical data provide rationale for clinical evaluation of AZD6738 as a monotherapy or combinatorial agent. Significance: This detailed preclinical investigation, including pharmacokinetics/pharmacodynamics and dose–schedule optimizations, of AZD6738/ceralasertib alone and in combination with chemotherapy or PARP inhibitors can inform ongoing clinical efforts to treat cancer with ATR inhibitors.

This study describes a novel series of UDP-N-acetylglucosamine acyltransferase (LpxA) inhibitors that was identified through affinity-mediated selection from a DNA-encoded compound library. The original hit was a selective inhibitor of Pseudomonas aeruginosa LpxA with no activity against Escherichia coli LpxA. The biochemical potency of the series was optimized through an X-ray crystallography-supported medicinal chemistry program, resulting in compounds with nanomolar activity against P. aeruginosa LpxA (best half-maximal inhibitory concentration (IC50) 20 μM and MIC > 128 μg/mL). The mode of action of analogues was confirmed through genetic analyses. As expected, compounds were active against multidrug-resistant isolates. Further optimization of pharmacokinetics is needed before efficacy studies in mouse infection models can be attempted. To our knowledge, this is the first reported LpxA inhibitor series with selective activity against P. aeruginosa.

This study describes a novel series of UDP

Background AZD0156 and AZD6738 are potent and selective inhibitors of ataxia-telangiectasia-kinase (ATM) and ataxia-telangiectasia-mutated and Rad3-related (ATR), respectively, important sensors/signallers of DNA damage. Methods We used multiplexed targeted-mass-spectrometry to select pRAD50(Ser635) as a pharmacodynamic biomarker for AZD0156-mediated ATM inhibition from a panel of 45 peptides, then developed and tested a clinically applicable immunohistochemistry assay for pRAD50(Ser635) detection in FFPE tissue. Results We found moderate pRAD50 baseline levels across cancer indications. pRAD50 was detectable in 100% gastric cancers (n = 23), 99% colorectal cancers (n = 102), 95% triple-negative-breast cancers (TNBC) (n = 40) and 87.5% glioblastoma-multiformes (n = 16). We demonstrated AZD0156 target inhibition in TNBC patient-derived xenograft models; where AZD0156 monotherapy or post olaparib treatment, resulted in a 34–72% reduction in pRAD50. Similar inhibition of pRAD50 (68%) was observed following ATM inhibitor treatment post irinotecan in a colorectal cancer xenograft model. ATR inhibition, using AZD6738, increased pRAD50 in the ATM-proficient models whilst in ATM-deficient models the opposite was observed, suggesting pRAD50 pharmacodynamics post ATR inhibition may be ATM-dependent and could be useful to determine ATM functionality in patients treated with ATR inhibitors. Conclusion Together these data support clinical utilisation of pRAD50 as a biomarker of AZD0156 and AZD6738 pharmacology to elucidate clinical pharmacokinetic/pharmacodynamic relationships, thereby informing recommended Phase 2 dose/schedule.

Third-generation cephalosporin (3GC)-resistant Enterobacteriaceae are classified as critical priority pathogens, with extended-spectrum β-lactamases (ESBLs) as principal resistance determinants. Enmetazobactam (formerly AAI101) is a novel ESBL inhibitor developed in combination with cefepime for empirical treatment of serious Gram-negative infections in settings where ESBLs are prevalent. Cefepime-enmetazobactam has been investigated in a phase 3 trial in patients with complicated urinary tract infections or acute pyelonephritis. This study examined pharmacokinetic-pharmacodynamic (PK-PD) relationships of enmetazobactam, in combination with cefepime, for ESBL-producing isolates of Klebsiella pneumoniae in 26-h murine neutropenic thigh infection models. Enmetazobactam dose fractionation identified the time above a free threshold concentration (fT > C(T)) as the PK-PD index predictive of efficacy. Nine ESBL-producing isolates of K. pneumoniae, resistant to cefepime and piperacillin-tazobactam, were included in enmetazobactam dose-ranging studies. The isolates encoded CTX-M-type, SHV-12, DHA-1, and OXA-48 β-lactamases and covered a cefepime-enmetazobactam MIC range from 0.06 to 2 μg/ml. Enmetazobactam restored the efficacy of cefepime against all isolates tested. Sigmoid curve fitting across the combined set of isolates identified enmetazobactam PK-PD targets for stasis and for a 1-log(10) bioburden reduction of 8% and 44% fT > 2 μg/ml, respectively, with a concomitant cefepime PK-PD target of 40 to 60% fT > cefepime-enmetazobactam MIC. These findings support clinical dose selection and breakpoint setting for cefepime-enmetazobactam.

The kinase ataxia telangiectasia mutated and rad3 related (ATR) is a key regulator of the DNA-damage response and the apical kinase which orchestrates the cellular processes that repair stalled replication forks (replication stress) and associated DNA double-strand breaks. Inhibition of repair pathways mediated by ATR in a context where alternative pathways are less active is expected to aid clinical response by increasing replication stress. Here we describe the development of the clinical candidate 2 (AZD6738), a potent and selective sulfoximine morpholinopyrimidine ATR inhibitor with excellent preclinical physicochemical and pharmacokinetic (PK) characteristics. Compound 2 was developed improving aqueous solubility and eliminating CYP3A4 time-dependent inhibition starting from the earlier described inhibitor 1 (AZ20). The clinical candidate 2 has favorable human PK suitable for once or twice daily dosing and achieves biologically effective exposure at moderate doses. Compound 2 is currently being tested in multiple phase I/II trials as an anticancer agent.

Barasertib (AZD1152), a highly potent and selective aurora kinase B inhibitor, gave promising clinical activity in elderly acute myeloid leukemia (AML) patients. However, clinical utility was limited by the requirement for a 7-day infusion. Here we assessed the potential of a nanoparticle formulation of the selective Aurora kinase B inhibitor AZD2811 (formerly known as AZD1152-hQPA) in preclinical models of AML. When administered to HL-60 tumor xenografts at a single dose between 25 and 98.7 mg/kg, AZD2811 nanoparticle treatment delivered profound inhibition of tumor growth, exceeding the activity of AZD1152. The improved antitumor activity was associated with increased phospho-histone H3 inhibition, polyploidy, and tumor cell apoptosis. Moreover, AZD2811 nanoparticles increased antitumor activity when combined with cytosine arabinoside. By modifying dose of AZD2811 nanoparticle, therapeutic benefit in a range of preclinical models was further optimized. At high-dose, antitumor activity was seen in a range of models including the MOLM-13 disseminated model. At these higher doses, a transient reduction in bone marrow cellularity was observed demonstrating the potential for the formulation to target residual disease in the bone marrow, a key consideration when treating AML. Collectively, these data establish that AZD2811 nanoparticles have activity in preclinical models of AML. Targeting Aurora B kinase with AZD2811 nanoparticles is a novel approach to deliver a cell-cycle inhibitor in AML, and have potential to improve on the clinical activity seen with cell-cycle agents in this disease. Mol Cancer Ther; 16(6); 1031–40. ©2017 AACR.

The PARP inhibitor AZD2461 was developed as a next-generation agent following olaparib, the first PARP inhibitor approved for cancer therapy. In BRCA1-deficient mouse models, olaparib resistance predominantly involves overexpression of P-glycoprotein, so AZD2461 was developed as a poor substrate for drug transporters. Here we demonstrate the efficacy of this compound against olaparib-resistant tumors that overexpress P-glycoprotein. In addition, AZD2461 was better tolerated in combination with chemotherapy than olaparib in mice, which suggests that AZD2461 could have significant advantages over olaparib in the clinic. However, this superior toxicity profile did not extend to rats. Investigations of this difference revealed a differential PARP3 inhibitory activity for each compound and a higher level of PARP3 expression in bone marrow cells from mice as compared with rats and humans. Our findings have implications for the use of mouse models to assess bone marrow toxicity for DNA-damaging agents and inhibitors of the DNA damage response. Finally, structural modeling of the PARP3-active site with different PARP inhibitors also highlights the potential to develop compounds with different PARP family member specificity profiles for optimal antitumor activity and tolerability. Cancer Res; 76(20); 6084–94. ©2016 AACR.

Inhibition of ataxia-telangiectasia mutated (ATM) during radiotherapy of glioblastoma multiforme (GBM) may improve tumor control by short-circuiting the response to radiation-induced DNA damage. A major impediment for clinical implementation is that current inhibitors have limited central nervous system (CNS) bioavailability; thus, the goal was to identify ATM inhibitors (ATMi) with improved CNS penetration. Drug screens and refinement of lead compounds identified AZ31 and AZ32. The compounds were then tested in vivo for efficacy and impact on tumor and healthy brain. Both AZ31 and AZ32 blocked the DNA damage response and radiosensitized GBM cells in vitro. AZ32, with enhanced blood–brain barrier (BBB) penetration, was highly efficient in vivo as radiosensitizer in syngeneic and human, orthotopic mouse glioma model compared with AZ31. Furthermore, human glioma cell lines expressing mutant p53 or having checkpoint-defective mutations were particularly sensitive to ATMi radiosensitization. The mechanism for this p53 effect involves a propensity to undergo mitotic catastrophe relative to cells with wild-type p53. In vivo, apoptosis was >6-fold higher in tumor relative to healthy brain after exposure to AZ32 and low-dose radiation. AZ32 is the first ATMi with oral bioavailability shown to radiosensitize glioma and improve survival in orthotopic mouse models. These findings support the development of a clinical-grade, BBB-penetrating ATMi for the treatment of GBM. Importantly, because many GBMs have defective p53 signaling, the use of an ATMi concurrent with standard radiotherapy is expected to be cancer-specific, increase the therapeutic ratio, and maintain full therapeutic effect at lower radiation doses. Mol Cancer Ther; 17(8); 1637–47. ©2018 AACR.

The mTOR kinase inhibitor AZD8055 inhibits both mTORC1 and mTORC2 leading to disruption of glucose metabolism and proliferation pathways. This study assessed the impact of single and multiple doses of AZD8055 on the uptake of the glucose metabolism marker 2-deoxy-2-[(18) F]fluoro-D-glucose ([(18) F]FDG) and the proliferation marker 3'-deoxy-3'-[(18) F]fluorothymidine ([(18) F]FLT) in U87-MG glioma xenografts.Mice bearing U87-MG tumours received either vehicle or AZD8055 (20 mg/kg) once daily p.o. Mice were imaged with either [(18) F]FDG or [(18) F]FLT PET to assess treatment response. Comparisons were made between in vivo imaging and ex vivo histopathology data.Tumour uptake of [(18) F]FDG was reduced by 33 % 1 h after a single dose of AZD8055 and by 49 % following 4 days of dosing. These changes coincided with suppression of the mTOR pathway biomarkers pS6 and pAKT. In contrast, the effect of AZD8055 on [(18) F]FLT uptake was inconsistent.The very rapid change in [(18) F]FDG uptake following acute AZD8055 treatment suggests that this could be used as an early mechanistic biomarker of metabolic changes resulting from mTOR inhibition. The utility of [(18) F]FLT for measuring the anti-proliferative effect of AZD8055 remains unclear.

Reversed-phase gradient LC-MS was used to perform untargeted metabonomic analysis on extracts of human colorectal cancer (CRC) cell lines (COLO 205, HT-29, HCT 116 and SW620) subcutaneously implanted into age-matched athymic nude male mice to study small molecule metabolic profiles and examine possible correlations with human cancer biopsies. Following high mass accuracy data analysis using MS and MS/MS, metabolites were identified by searching against major metabolite databases including METLIN, MASSBANK, The Human Metabolome Database, PubChem, Biospider, LipidMaps and KEGG. HT-29 and COLO 205 tumor xenografts showed a distribution of metabolites that differed from SW620 and HCT 116 xenografts (predominantly on the basis of relative differences in the amounts of amino acids and lipids detected). This finding is consistent with NMR-based analysis of human colorectal tissue, where the metabolite profiles of HT-29 tumors exhibit the greatest similarity to human rectal cancer tissue with respect to changes in the relative amounts of lipids and choline-containing compounds. As the metabolic signatures of cancer cells result from oncogene-directed metabolic reprogramming, the HT-29 xenografts in mice may prove to be a useful model to further study the tumor microenvironment and cancer biology.

ATR is an attractive new anticancer drug target whose inhibitors have potential as chemo- or radiation sensitizers or as monotherapy in tumors addicted to particular DNA-repair pathways. We describe the discovery and synthesis of a series of sulfonylmorpholinopyrimidines that show potent and selective ATR inhibition. Optimization from a high quality screening hit within tight SAR space led to compound 6 (AZ20) which inhibits ATR immunoprecipitated from HeLa nuclear extracts with an IC50 of 5 nM and ATR mediated phosphorylation of Chk1 in HT29 colorectal adenocarcinoma tumor cells with an IC50 of 50 nM. Compound 6 potently inhibits the growth of LoVo colorectal adenocarcinoma tumor cells in vitro and has high free exposure in mouse following moderate oral doses. At well tolerated doses 6 leads to significant growth inhibition of LoVo xenografts grown in nude mice. Compound 6 is a useful compound to explore ATR pharmacology in vivo.

Cediranib (AZD2171) is a highly potent inhibitor of all three vascular endothelial growth factor receptors. The aim of this preclinical study was to examine the effect of combining cediranib with mechanistically distinct anti-tumour therapies.Cediranib (1.5 or 3 mg/kg/day) was evaluated alone and in combination with either gefitinib, imatinib, ZD6126, saracatinib, selumetinib, bevacizumab, 5-fluorouracil (5-FU), docetaxel, oxaliplatin, gemcitabine, pemetrexed, irinotecan or cisplatin in human tumour xenograft models. Anti-tumour activity was measured by assessing the change in tumour volume following treatment compared with vehicle-treated time-matched controls.In all cases, the combination regimens, at tolerated doses and schedules, inhibited tumour growth to a greater extent than the corresponding monotherapy treatments. Compared with cediranib alone, statistically significant enhancements in anti-tumour activity were observed with all combination regimens. Notably, after 14 days of treatment, the combination of cediranib with ZD6126 induced substantial tumour regression (60 % compared with pre-treatment volume), whilst treatment with each agent alone led only to partial growth inhibition. A combination of cediranib with gefitinib also induced tumour regressions, and cediranib combined with either gemcitabine or irinotecan was found to inhibit tumour growth profoundly (by 99 and 98 %, respectively).Combining cediranib with selected cytotoxic or targeted agents proved efficacious in a range of human tumour xenograft models.

Background: The Ras/RAF/MEK/ERK pathway is frequently deregulated in cancer and a number of inhibitors that target this pathway are currently in clinical development. It is likely that clinical testing of these agents will be in combination with standard therapies to harness the apoptotic potential of both the agents. To support this strategy, it has been widely observed that a number of chemotherapeutics stimulate the activation of several intracellular signalling cascades including Ras/RAF/MEK/ERK. The MEK1/2 inhibitor selumetinib has been shown to have anti-tumour activity and induce apoptotic cell death as a monotherapy. Methods: The aim of this study was to identify agents, which would be likely to offer clinical benefit when combined with selumetinib. Here, we used human tumour xenograft models and assessed the effects combining standard chemotherapeutic agents with selumetinib on tumour growth. In addition, we analysed tumour tissue to determine the mechanistic effects of these combinations. Results: Combining selumetinib with the DNA-alkylating agent, temozolomide (TMZ), resulted in enhanced tumour growth inhibition compared with monotherapies. Biomarker studies highlighted an increase in γH2A.X suggesting that selumetinib is able to enhance the DNA damage induced by TMZ alone. In several models we observed that continuous exposure to selumetinib in combination with docetaxel results in tumour regression. Scheduling of docetaxel before selumetinib was more beneficial than when selumetinib was dosed before docetaxel and demonstrated a pro-apoptotic phenotype. Similar results were seen when selumetinib was combined with the Aurora B inhibitor barasertib. Conclusion: The data presented suggests that MEK inhibition in combination with several standard chemotherapeutics or an Aurora B kinase inhibitor is a promising clinical strategy.

Gadolinium nitride films have been deposited on Si(100) using a plasma-enhanced ALD (PEALD) based process. The deposition was carried out using tris(methylcyclopentadienyl)gadolinium {Gd(MeCp) 3 } and remote nitrogen plasma, separated by argon pulses. Films were deposited at temperatures between 150 and 300 °C and capped with tantalum nitride to prevent post-deposition oxidation. Film composition was initially assessed using EDX and selected samples were subsequently depth profiled using medium energy ion scattering (MEIS) or AES. X-ray diffraction appears to show that the films are effectively amorphous. Films deposited at 200 °C were found to have a Gd:N ratio close to 1:1 and a low oxygen incorporation (∼5%). Although the growth was affected by partial thermal decomposition of the Gd(MeCp) 3 , it was still possible to obtain smooth (Ra.=∼0.7 nm) films with good thickness uniformity (97%). Less successful attempts to deposit gadolinium nitride using thermal ALD with ammonia or mono-methyl-hydrazine are also reported.

TaN x thin films were grown at temperatures ranging from 200 to 375 °C using atomic layer deposition (ALD). Pentakis(dimethylamino)tantalum (PDMAT) was used as a tantalum source with either ammonia or monomethylhydrazine (MMH) as a nitrogen co-reactant. Self-limiting behaviour was observed for both ammonia and MMH processes, with growth rates of 0.6 and 0.4 A/cycle, respectively at 300 °C. Films deposited using ammonia were found to have a mono-nitride stoichiometry with resistivities as low as 70 mΩ cm. In contrast, films deposited using MMH were found to be nitrogen rich Ta 3 N 5 with high resistivities. A Quartz Crystal Microbalance (QCM) was used to measure mass gain and loss during the cyclic ALD processes and the data was used in combination with medium energy ion scattering (MEIS) to elucidate the PDMAT absorption mechanisms.

Vertically aligned zinc oxide (ZnO) nanowires (NWs) have been grown by liquid injection Metal Organic Chemical Vapour Deposition, using oxygen donor adducts of Me2Zn. The growth and characterisation of the nanowires grown using [Me2Zn(L)] where L = monodentate ethers, tetrahydrofuran (C4H8O) (1), tetrahydropyran (C5H10O) (2), furan (C4H4O) (3) and the bidentate ethers, 1,2-dimethoxyethane (C4H12O2,) (4) 1,4-dioxane (C4H8O2) (5) and 1,4-thioxane (C4H8SO) (6) is discussed. Single crystal X-ray structures of (4), (5), (6) have been established and are included here. The ZnO NWs were deposited in the absence of a seed catalyst on Si(111) and F-doped SnO2/glass substrates over the temperature range 350-600 degrees C. X-ray diffraction (XRD) data shows that the nanowires grown from all adduct precursors were deposited in the wurtzitic phase.

The dimethylzinc-bidentate ether adducts [Me2Zn(1,4-dioxane)] (1), [Me2Zn(1,2-dimethoxyethane)] (2) and [Me2Zn(1,4-thioxane)] (3) are used as precursors for the growth of vertically aligned zinc oxide (ZnO) nanowires (NWs) by liquid-injection, metal-organic (MO)CVD. The ZnO NWs are deposited on Si(111) and F-doped SnO2/glass substrates in the absence of a seed catalyst. The precursors (1) and (2) are used to deposit ZnO NWs at substrate temperatures of 450 °C and 500 °C, whilst higher deposition temperatures of 550–600 °C are necessary to obtain ZnO NWs using (3). X-ray diffraction (XRD) data show that the NWs grown from all three adduct precursors are deposited in the wurtzitic ZnO phase. Room-temperature photoluminescence (PL) data for the ZnO NWs grown using (1) and (2) show an intense peak at 3.28 eV due to near band-edge emission with a very low intensity of defect-related green luminescence at 2.42 eV. In contrast, room-temperature PL data for the ZnO NWs deposited using (3) is dominated by deep centre, defect-related emission at 2.42 eV. Auger electron spectroscopy (AES) shows that the ZnO films deposited from (1) and (2) are high purity with no detectable carbon, but the ZnO films grown from (3) are contaminated with sulfur (1 at.-%). Single-crystal X-ray structures show that (1) is polymeric containing bridging 1,4-dioxane ligands while (2) is a monomeric complex featuring a chelating 1,2-dimethoxyethane ligand. The wide [Me-Zn-Me] bond angles in (1) and (2) (152.0(3)° and 154.5(2)°, respectively) suggest moderately weak [Me2Zn]-ligand interactions. Complex (3) is polymeric and exhibits nearly linear [Me-Zn-Me] bond angles, indicative of much weaker [Me2Zn]-ligand interactions.

The dimethylzinc adduct [Me 2 Zn(1,4-dioxane)] has been used for the growth of vertically aligned ZnO nanowires by liquid injection MOCVD. The ZnO nanowires were deposited at temperatures of 450 and 500 °C on Si(1 1 1) and F-doped SnO 2 /glass substrates and XRD data showed that the ZnO nanowires were deposited in the wurtzitic phase. Room temperature PL data for the nanowires showed that they are of high crystalline quality with intense near band-edge emission at 3.28 eV and a very low intensity of defect-related green luminescence at 2.42 eV. Single crystal XRD data showed that the [Me 2 Zn (1,4-dioxane)] adduct is polymeric with repeating [Me 2 Zn] units bridged by monodentate 1,4-dioxane ligands.

The deposition of ruthenium thin films is investigated using a newly synthesized precursor (cyclopentadienyl ethylruthenium dicarbonyl, Ru(Cp)(CO)2 Et) and O2 gas as reactants. The conditions to achieve self-terminated surface reactions (sample temperature, precursor pulse length and precursor gas pressure) are investigated and the resulting composition, conductivity, and surface morphology are determined during/after deposition on hydrogen-terminated silicon (111) surfaces using in situ FTIR, and ex situ Rutherford back scattering, X-ray photoelectron spectroscopy, and atomic force microscopy. Higher growth rates (∼1.5-3 A˚ ) are obtained compared to those typical of ALD of metals (∼0.5-1 A˚ ), under conditions of saturation, i.e., through self-terminated surface reactions. Infrared absorption measurements reveal that bridged CO formed by the self-reaction of Ru(Cp)(CO)2 Et leads to surface passivation, thus terminating the precursor self-reaction. They also show that, under these “saturation” growth conditions, metallic Ru develops during the early stage of deposition (1-5 cycles), and RuO2 is observed later in the growth. The deposition rate is linear with cycles after an initially slow nucleation stage and the film becomes metallic after∼22 cycles. Thick films (∼45 nm) grown with short pulses produce metallic polycrystalline ruthenium with hcp structure.

Aurora kinases play a critical role in regulating mitosis and cell division, and their overexpression has been implicated in the survival and proliferation of human cancer. In this study, we report the in vitro and in vivo activities of AZD1152, a compound that has selectivity for aurora B kinase, in acute myeloid leukemia (AML) cell lines, primary AML samples, and cord blood cells. AZD1152 exerted antiproliferative or cytotoxic effects in all cell lines studied, inhibited the phosphorylation of histone H3 (pHis H3) on Ser10 in a dose-dependent manner, and resulted in cells with >4N DNA content. THP-1 cells treated with AZD1152 accumulated in a state of polyploidy and showed a senescent response to the drug, in contrast to the apoptotic response seen in other cell lines. Accordingly, AZD1152 profoundly affected the growth of AML cell lines and primary AML in an in vivo xenotransplantation model. However, concentration-dependent effects on cell growth, apoptosis, and cell cycle progression were also observed when human cord blood and primary lineage–negative stem and progenitor cells were analyzed in vitro and in vivo. These data suggest that the inhibition of aurora B kinase may be a useful therapeutic strategy in the treatment of AML and that further exploration of dosing and treatment schedules is warranted in clinical trials. [Cancer Res 2009;69(10):4150–8]

Atomic Layer Deposition (ALD) has been identified as one of the primary technologies to fabricate devices with novel architecture. This requires design and development of advanced ALD precursors that can withstand the rigors of various integration steps while delivering the high quality layers essential to the new device design. The design criteria require these precursors to have suitable thermal stability, volatility, purity, reactivity, scalability, and economic viability. This brief review describes various elements of a precursor screening/development cycle from the materials company perspective. Specific examples will be presented for precursors of selected metals (such as ruthenium and cobalt) and high-k dielectrics (Hf, Zr, Ln) oxide materials.

Thin films of HfO2 are deposited by liquid injection metal-organic (MO) CVD and atomic layer deposition (ALD) using the new cyclopentadienyl precursors [(MeCp)2HfMe(OiPr)] and [(MeCp)2HfMe(mmp)] (mmp = OCMe2CH2OMe). Both precursors evaporate at moderate temperatures, making them very suitable for MOCVD and ALD applications. Thin films of HfO2 are deposited by MOCVD in the temperature range 400–650 °C, and by ALD in the temperature range 300–450 °C, using both [(MeCp)2HfMe(OiPr)] and [(MeCp)2HfMe(mmp)] . Analysis by X-ray diffraction (XRD) shows that the films are deposited with an essentially amorphous microstructure, but subsequent annealing above 700 °C in dry air crystallizes the films into the α- or monoclinic phase. Auger electron spectroscopy (AES) shows that residual carbon (0–5 at.- %) is present in the oxide films and that the HfO2 films grown by MOCVD generally contain more carbon than those grown by ALD. The dielectric properties of the as-deposited HfO2 films are evaluated using [Al/HfO2/SiO2/n-Si] MOS capacitor structures with various HfO2 thicknesses ranging from 1.7 nm to 102 nm. The calculated permittivity (ϵox), assuming the presence of a 1 nm native SiO2 interlayer, is found to be ϵox ∼ 14 for a HfO2 film grown by ALD using [(MeCp)2HfMe(OiPr)], whilst for a film grown by ALD using [(MeCp)2HfMe(mmp)], ϵox is found to be ∼10. Equivalent oxide thicknesses of 2.5 and 0.78 nm are measured for HfO2 films deposited by ALD using [(MeCp)2HfMe(OiPr)] and [(MeCp)2HfMe(mmp)], respectively. The leakage current density for a film grown by ALD using [(MeCp)2HfMe(OiPr)] remains very low (

The development of novel precursors for advanced semiconductor applications requires molecular engineering and chemical tailoring to obtain specific physical properties and performance capabilities. Having identified promising avenues in ligand design, the first priority when isolating a new compound is to ensure that when used it is safe and will not cause detrimental effects in deposition equipment due to thermal decomposition. We have employed different techniques to study the physical properties and stabilities of highly promising Hafnium and Zirconium compounds particularly suited to the Atomic Layer Deposition technique for fabrication of state-of-the-art metal oxide layers for both logic and memory semiconductor devices to ensure that they may be employed safely. In particular mixed alkyl/alkoxide and alkylcyclopentadienyl substituted compounds [(MeCp) 2 M(OMe) x Me 2 − x M = Hf and Zr x = 0 or 1] were compared to conventional alkylamine sources (M(NEtMe) 4 M = Hf and Zr). The use of thermogravimetric analysis (TGA) to determine evaporation rates under a variety of conditions is employed to determine initial temperature regimes where decomposition may occur. More detailed studies using Nuclear Magnetic Resonance (NMR) are then conducted at fixed temperatures over a prolonged time period with regular monitoring to establish degradation effects more accurately. Our studies show similar trends between both Hf and Zr materials with the Zr derivatives less stable in all cases. Operation temperatures required to achieve suitable vapour transport in the case of MeCp 2 ZrMe 2 have been found to lead to catastrophic decomposition after a relatively short time thus negating the usefulness of this precursor in a production environment. All other sources were deemed safe to employ under standard process conditions.

Thin films of ZrO 2 have been deposited by liquid injection MOCVD and ALD using the cyclopentadienyl-based Zr precursors [(MeCp) 2 ZrMe(OMe)] and [(MeCp) 2 ZrMe(OBu t )]. Analysis by X-ray diffraction showed that films grown at low temperature (300 °C) by ALD were amorphous, whist films deposited at higher temperature (600 °C) by MOCVD exist in the tetragonal phase. Auger electron spectroscopy showed that residual carbon (1.4–7.0 at.%) was present in the oxide films and that the MOCVD-grown films contained more carbon contamination than those grown by ALD.

Atomic layer deposition (ALD) of HfO2 thin films was studied using four novel cyclopentadienyl precursors, namely, (CpMe)2HfMe2, Cp2Hf(OMe)2, (CpMe)2Hf(OMe)Me, and (CpMe)2Hf(OMe)2. Ozone was used a...

The transport of trimethylindium (TMI) must be highly reliable throughout the lifetime of a bubbler. This study reports stability data and transport rates obtained for a number of alternative bubbler designs when tested under different conditions of use. The best results, with exceptionally stable flux output across a wide range of operating parameters throughout the bubbler lifetime, were obtained using a novel perforated disc design. This design is an extension of the cross dipleg concept which provides multiple gas pathways to minimise channelling effects and allow uniform and efficient depletion of the bubbler contents.

Precursors for use in metal organic vapour phase epitaxy (MOVPE) of compound semiconductor films are supplied in high integrity stainless steel containers to ensure the highest levels of purity and safety are maintained. However, this means that an accurate level determination is difficult to obtain in situ and growers must rely on usage calculations based on source vapour pressure values quoted in the literature. A number of level sensing systems have been investigated with some degree of success to address this problem, but all have suffered from excessive complexity, being limited to a fixed level indication, fragility and other issues. The most suited technology has been identified as capacitance related and data is reported here obtained from a simple, compact, robust probe fitted to a standard MOVPE bubbler. Results from standard source materials trimethylaluminium (TMA), trimethylgallium (TMG), dimethylzinc (DMZ) and diethylzinc (DEZ) under static and simulated chemical vapour deposition (CVD) operating conditions reported here clearly demonstrate that an accurate continuous level can be measured. Information on the repeatability, material compatibility and temperature dependence of the probe is also presented.

Topoisomerase I inhibitors are used as standard-of-care chemotherapy in many types of cancer but are associated with significant toxicities. There is potential to improve their efficacy further by combining with inhibitors of the DNA damage response, such as the poly ADP ribose polymerase (PARP) inhibitor olaparib. However, while preclinical data highlight the improved efficacy of this combination, subsequent clinical trials have struggled due to dose limiting myelotoxicity. CRLX101 is an investigational nanoparticle-drug conjugate (NDC) containing the payload camptothecin, a potent topoisomerase I inhibitor. This agent is preferentially targeted to tumours and demonstrated a generally favorable toxicity profile in the clinic. Here, we explored the molecular mechanism and therapeutic potential of combining CRLX101 with either olaparib or the WEE1 G2 checkpoint kinase inhibitor AZD1775, by testing both efficacy and safety in preclinical models. In vitro studies using NCI-H417a small cell lung cancer (SCLC) cells demonstrated that combination with both olaparib and AZD1775 potentiated the efficacy of CRLX101 although by different mechanisms. Cellular analyses revealed that CRLX101 treatment alone predominantly activated ATM-mediated DNA damage response and resulted in late S/G2 cell cycle arrest. Combination with a PARP inhibitor further enhanced the CRLX101-induced DNA damage response and prolonged cell cycle arrest in late S/G2 phase. In contrast, WEE1 inhibition abrogated late S/G2 cell cycle arrest induced by CRLX101, resulting in aberrant mitotic entry and enhanced cell death. Our in vivo studies using wild type Wistar rat model showed that CRLX101, olaparib and AZD1775, are well tolerated as single agents. However, concurrent combination of CRLX101 with either olaparib or AZD1775 resulted in a dose-dependent decrease in hematological parameters. We investigated sequenced schedules and demonstrated that at a 24h delay between the CRLX101 and olaparib mitigates much of the combined bone marrow toxicity, while improving the efficacy above CRLX101 alone in xenograft tumors from NCI-H417a cells. Collectively, these preclinical data demonstrate increased anti-tumor efficacy of CRLX101 when combined with DDR inhibitors. The combination schedule for CRLX101 and olaparib identified in our preclinical models as providing an increased therapeutic index has been used to develop protocols to test this combination in a relapsed (2nd line) SCLC human clinical trial (in collaboration with NCI). Citation Format: Lenka Oplustil O'Connor, Anderson T. Wang, David Jones, Rajesh Odedra, Michael Spreadborough, Joanne Wilson, Aaron Smith, Peter Cotton, Jaimini Reens, Jen Barnes, Victoria Sheridan, Andres Tellez, Alan Lau, Claire Sadler, Mark J. O'Connor, Scott Eliasof. CRLX101, an investigational camptothecin-containing nanoparticle-drug conjugate, combined with DDR agents provides a novel approach to increasing therapeutic index [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr B32.

The synthesis of dimethylzinc (Me2Zn) and diethylzinc (Et2Zn) by Frankland in the mid-nineteenth century marks a cornerstone in the history of chemistry.1 Not only were they among the first organometallic compounds, but studies on their chemical reactions and vapor densities led to the first clear exposition of valency theory.2 Since then both compounds have found widespread applications: They are important reagents in organic synthesis,3 for example in the enantioselective alkylation of carbonyls4 and imines5 and in cyclopropanation reactions.6 Their high vapor pressures have led to extensive uses in metalorganic chemical vapor deposition (MOCVD) for the preparation of wide band gap II–VI semiconducting films (e.g. ZnS, ZnSe, ZnTe),7 ZnO nanostructures, and as p-dopant precursors for III–V semiconductors (e.g. GaAs, InP, AlxGa1−xAs), which have numerous electronic and photonic applications.8

Bis(cyclopentadienyl)magnesium (Cp2Mg) is a common source for p-type doping in GaN and AlInGaP materials. It is a white crystalline solid with very low vapor pressure, leading to transport problems similar to solid trimethyindium (TMI). Some of these problems can be alleviated by a newly developed source-solution magnesocene, Cp2Mg, dissolved in a solvent that is essentially nonvolatile. In this paper, we report the growth and comparative results of Mg-doped GaN grown by OMVPE using solid and solution Cp2Mg. Using both sources, we optimized parameters to obtain high-quality GaN growth with hole concentrations up to 1 1018/cm3.