Marc Geoffery Hummersone

AZD8055 Is a Potent, Selective, and Orally Bioavailable ATP-Competitive Mammalian Target of Rapamycin Kinase Inhibitor with In vitro and In vivo Antitumor Activity

The mammalian target of rapamycin (mTOR) kinase forms two multiprotein complexes, mTORC1 and mTORC2, which regulate cell growth, cell survival, and autophagy. Allosteric inhibitors of mTORC1, such as rapamycin, have been extensively used to study tumor cell growth, proliferation, and autophagy but have shown only limited clinical utility. Here, we describe AZD8055, a novel ATP-competitive inhibitor of mTOR kinase activity, with an IC50 of 0.8 nmol/L. AZD8055 showed excellent selectivity (approximately 1,000-fold) against all class I phosphatidylinositol 3-kinase (PI3K) isoforms and other members of the PI3K-like kinase family. Furthermore, there was no significant activity against a panel of 260 kinases at concentrations up to 10 micromol/L. AZD8055 inhibits the phosphorylation of mTORC1 substrates p70S6K and 4E-BP1 as well as phosphorylation of the mTORC2 substrate AKT and downstream proteins. The rapamycin-resistant T37/46 phosphorylation sites on 4E-BP1 were fully inhibited by AZD8055, resulting in significant inhibition of cap-dependent translation. In vitro, AZD8055 potently inhibits proliferation and induces autophagy in H838 and A549 cells. In vivo, AZD8055 induces a dose-dependent pharmacodynamic effect on phosphorylated S6 and phosphorylated AKT at plasma concentrations leading to tumor growth inhibition. Notably, AZD8055 results in significant growth inhibition and/or regression in xenografts, representing a broad range of human tumor types. AZD8055 is currently in phase I clinical trials.

Improved ATM kinase inhibitor KU-60019 radiosensitizes glioma cells, compromises insulin, AKT and ERK prosurvival signaling, and inhibits migration and invasion

Ataxia telangiectasia (A-T) mutated (ATM) is critical for cell cycle checkpoints and DNA repair. Thus, specific small molecule inhibitors targeting ATM could perhaps be developed into efficient radiosensitizers. Recently, a specific inhibitor of the ATM kinase, KU-55933, was shown to radiosensitize human cancer cells. Herein, we report on an improved analogue of KU-55933 (KU-60019) with Ki and IC50 values half of those of KU-55933. KU-60019 is 10-fold more effective than KU-55933 at blocking radiation-induced phosphorylation of key ATM targets in human glioma cells. As expected, KU-60019 is a highly effective radiosensitizer of human glioma cells. A-T fibroblasts were not radiosensitized by KU-60019, strongly suggesting that the ATM kinase is specifically targeted. Furthermore, KU-60019 reduced basal S473 AKT phosphorylation, suggesting that the ATM kinase might regulate a protein phosphatase acting on AKT. In line with this finding, the effect of KU-60019 on AKT phosphorylation was countered by low levels of okadaic acid, a phosphatase inhibitor, and A-T cells were impaired in S473 AKT phosphorylation in response to radiation and insulin and unresponsive to KU-60019. We also show that KU-60019 inhibits glioma cell migration and invasion in vitro, suggesting that glioma growth and motility might be controlled by ATM via AKT. Inhibitors of MEK and AKT did not further radiosensitize cells treated with KU-60019, supporting the idea that KU-60019 interferes with prosurvival signaling separate from its radiosensitizing properties. Altogether, KU-60019 inhibits the DNA damage response, reduces AKT phosphorylation and prosurvival signaling, inhibits migration and invasion, and effectively radiosensitizes human glioma cells. [Mol Cancer Ther 2009;8(10):2894–902]

Optimization of potent and selective dual mTORC1 and mTORC2 inhibitors: The discovery of AZD8055 and AZD2014

The optimization of a potent and highly selective series of dual mTORC1 and mTORC2 inhibitors is described. An initial focus on improving cellular potency whilst maintaining or improving other key parameters, such as aqueous solubility and margins over hERG IC(50), led to the discovery of the clinical candidate AZD8055 (14). Further optimization, particularly aimed at reducing the rate of metabolism in human hepatocyte incubations, resulted in the discovery of the clinical candidate AZD2014 (21).

The discovery and optimisation of pyrido[2,3-d]pyrimidine-2,4-diamines as potent and selective inhibitors of mTOR kinase.

We describe a novel series of potent inhibitors of the kinase activity of mTOR. The compounds display good selectivity relative to other PI3K-related kinase family members and, in cellular assays, inhibit both mTORC1 and mTORC2 complexes and exhibit good antiproliferative activity.

DNA-Dependent Protein Kinase (DNA-PK) Inhibitors. Synthesis and Biological Activity of Quinolin-4-one and Pyridopyrimidin-4-one Surrogates for the Chromen-4-one Chemotype

Following the discovery of dibenzo[b,d]thiophen-4-yl)-2-morpholino-4H-chromen-4-one (NU7441) ( Leahy , J. J. J. ; Golding , B. T. ; Griffin , R. J. ; Hardcastle , I. R. ; Richardson , C. ; Rigoreau , L. ; Smith , G. C. M. Bioorg. Med. Chem. Lett. 2004 , 14 , 6083 - 6087) as a potent inhibitor (IC₅₀ = 30 nM) of DNA-dependent protein kinase (DNA-PK), we have investigated analogues in which the chromen-4-one core template has been replaced by aza-heterocyclic systems: 9-substituted 2-morpholin-4-ylpyrido[1,2-a]pyrimidin-4-ones and 8-substituted 2-morpholin-4-yl-1H-quinolin-4-ones. The 8- and 9-substituents were either dibenzothiophen-4-yl or dibenzofuran-4-yl, which were each further substituted at the 1-position with water-solubilizing groups [NHCO(CH₂)(n)NR¹R², where n = 1 or 2 and the moiety R¹R²N was derived from a library of primary and secondary amines (e.g., morpholine)]. The inhibitors were synthesized by employing a multiple-parallel approach in which the two heterocyclic components were assembled by Suzuki-Miyaura cross-coupling. Potent DNA-PK inhibitory activity was generally observed across the compound series, with structure-activity studies indicating that optimal potency resided in pyridopyrimidin-4-ones bearing a substituted dibenzothiophen-4-yl group. Several of the newly synthesized compounds (e.g., 2-morpholin-4-yl-N-[4-(2-morpholin-4-yl-4-oxo-4H-pyrido[1,2-a]pyrimidin-9-yl)dibenzothiophen-1-yl]acetamide) combined high potency against the target enzyme (DNA-PK IC₅₀ = 8 nM) with promising activity as potentiators of ionizing radiation-induced cytotoxicity in vitro.

1-substituted (Dibenzo[b,d]thiophen-4-yl)-2-morpholino-4H-chromen-4-ones endowed with dual DNA-PK/PI3-K inhibitory activity.

Analogues of (dibenzo[b,d]thiophen-4-yl)-2-morpholino-4H-chromen-4-one (NU7441), a potent inhibitor of DNA-dependent protein kinase (DNA-PK; IC50 = 42 ± 2 nM), have been synthesized in which water-solubilizing groups [NHCO(CH₂)nNR¹R², where n = 1 or 2 and the moiety R¹R²N was derived from a library of primary and secondary amines, e.g., morpholine] were placed at the 1-position. Several of the newly synthesized compounds exhibited high potency against DNA-PK and potentiated the cytotoxicity of ionizing radiation (IR) in vitro 10-fold or more (e.g., 2-(4-ethylpiperazin-1-yl)-N-(4-(2-morpholino-4-oxo-4H-chromen-8-yl)dibenzo[b,d]thio-phen-1-yl)acetamide, 39; DNA-PK IC₅₀ = 5.0 ± 1 nM, IR dose modification ratio = 13). Furthermore, 39 was shown to potentiate not only IR in vitro but also DNA-inducing cytotoxic anticancer agents, both in vitro and in vivo. Counter-screening against other members of the phosphatidylinositol 3-kinase (PI-3K) related kinase (PIKK) family unexpectedly revealed that some of the compounds were potent mixed DNA-PK and PI-3K inhibitors.

Certain Imidazotetrazines Escape O6-Methylguanine-DNA Methyltransferase and Mismatch Repair

Resistance to temozolomide (TMZ), conferred by O6-methylguanine-DNA methyltransferase (MGMT) or mismatch repair (MMR) deficiency, presents obstacles to successful glioblastoma multiforme (GBM) treatment. Activities of novel TMZ analogs, designed to overcome resistance, were tested against isogenic SNB19 and U373 GBM cell lines (V = vector control, low MGMT; M = MGMT overexpression). TMZ and triazene MTIC demonstrated >9-fold resistance in SNB19M cells (cf SNB19V). N-3 methyl ester analog 11 and corresponding triazene 12 inhibited growth of TMZ-sensitive (V) and TMZ-resistant (M) cells (GI50 <50 µM). Ethyl ester 13 and triazene 14 gave similar profiles. MMR-deficient colorectal carcinoma cells, resistant to TMZ (GI50 >500 µM), responded to analog 11 and 13 treatment. Cross-resistance to these agents was not observed in cell lines possessing acquired TMZ resistance (SNB19VR; U373VR). Methyl ester 11 blocked SNB19V, SNB19M and SNB19VR cells in S and G2/M, causing dose- and time-dependent apoptosis. DNA damage, recruiting excision repair was detected by alkaline comet assay; H2AX phosphorylation indicated a lethal DNA double-strand break formation following analog 11 exposure. Compounds 11 and 13 demonstrated 3.7- and 5.1-fold enhanced activity in base excision repair-deficient Chinese hamster ovary cells; furthermore, poly (ADP-ribose) polymerase-1 inhibition potentiated HCT-116 cells’ sensitivity to analog 11. In conclusion, analogs 11 and 13 exert anticancer activity irrespective of MGMT and MMR.

On and off-target effects of telomere uncapping G-quadruplex selective ligands based on pentacyclic acridinium salts

Quadruplexes DNA are present in telomeric DNA as well as in several cancer-related gene promoters and hence affect gene expression and subsequent biological processes. The conformations of G4 provide selective recognition sites for small molecules and thus these structures have become important drug-design targets for cancer treatment.The DNA G-quadruplex binding pentacyclic acridinium salt RHPS4 (1) has many pharmacological attributes of an ideal telomere-targeting agent but has undesirable off-target liabilities. Notably a cardiovascular effect was evident in a guinea pig model, manifested by a marked and sustained increase in QTcB interval. In accordance with this, significant interaction with the human recombinant β2 adrenergic receptor, and M1, M2 and M3 muscarinic receptors was observed, together with a high inhibition of the hERG tail current tested in a patch clamp assay.Two related pentacyclic structures, the acetylamines (2) and (3), both show a modest interaction with β2 adrenergic receptor, and do not significatively inhibit the hERG tail current while demonstrating potent telomere on-target properties comparing closely with 1. Of the two isomers, the 2-acetyl-aminopentacycle (2) more closely mimics the overall biological profile of 1 and this information will be used to guide further synthetic efforts to identify novel variants of this chemotype, to maximize on-target and minimize off-target activities.Consequently, the improvement of toxicological profile of these compounds could therefore lead to the obtainment of suitable molecules for clinical development offering new pharmacological strategies in cancer treatment.

N3-Substituted Temozolomide Analogs Overcome Methylguanine-DNA Methyltransferase and Mismatch Repair Precipitating Apoptotic and Autophagic Cancer Cell Death

Glioblastoma multiforme (GBM) treatment includes temozolomide (TMZ) chemotherapy. O6-Methylguanine lesions are repaired by methylguanine-DNA methyltransferase (MGMT). Response to TMZ requires low MGMT and functional mismatch repair (MMR); resistance, conferred by MGMT or MMR deficiency, represents a barrier to successful treatment. TMZ analogs were synthesized, substituting N3-methyl with propargyl (1) or sulfoxide (2). MTT assays were conducted in SNB19 and U373 isogenic glioma cell lines (V = vector control; M = MGMT-transfected). TMZ potency was reduced >5-fold in SNB19M and U373M cells; in contrast, MGMT-expressing cells were equisensitive as vector controls to analogs 1 and 2. GI50 values <50 VM of analogs 1 or 2 were detected in V cells possessing acquired TMZ resistance: SNB19VR (hMSH6 loss) and U373VR (MGMT upregulation). Analogs 1 and 2 inhibited MMR-deficient colorectal carcinoma cell growth (irrespective of p53); G2/M cell cycle arrest preceded apoptosis. GH2AX foci inferred the generation of DNA double-strand breaks by analogs 1 and 2. Acridine orange-stained vesicles, intracellular punctate GFP-LC3 protein and double-membraned autophagosomes indicate that TMZ, 1 and 2 induce autophagy in apoptotis-resistant GBM cells. Analogs 1 and 2 elicit in vitro antitumor activity irrespective of MGMT, MMR and p53. Such imidazotetrazines may treat MGMT+ GBM and possess broader spectrum activity causing apoptosis and autophagy in malignancies which evade apoptosis. i 2014 S. Karger AG, Basel

Antitumour imidazotetrazines. Synthesis and chemistry of 4-oxo-3,4-dihydroimidazo[5,1-d][1,2,3,5]tetrazine-8-carboxamide (nor-temozolomide): an intermediate for the preparation of the antitumour drug temozolomide and analogues, avoiding the use of isocyanates

An efficient synthesis of 4-oxo-3,4-dihydroimidazo[5,1-d][1,2,3,5]tetrazine-8-carboxamide (nor-temozolomide) and in situ generation of the anion of nor-temozolomide from an N-3-hydroxymethyl derivative of temozolomide are reported. Alkylation of the anion of nor-temozolomide with methyl iodide gave a new route to temozolomide avoiding the use of methyl isocyanate. A series of new 3-substituted analogues of temozolomide were also prepared by electrophilic substitution of the nor-temozolomide anion but this approach is so far restricted to certain electrophiles.