Development of a Series of Pyrrolopyridone MAT2A Inhibitors.

The optimization of an allosteric fragment, discovered by differential scanning fluorimetry, to an in vivo MAT2a tool inhibitor is discussed. The structure-based drug discovery approach, aided by relative binding free energy calculations, resulted in AZ'9567 (21), a potent inhibitor in vitro with excellent preclinical pharmacokinetic properties. This tool showed a selective antiproliferative effect on methylthioadenosine phosphorylase (MTAP) KO cells, both in vitro and in vivo, providing further evidence to support the utility of MAT2a inhibitors as potential anticancer therapies for MTAP-deficient tumors.

Discovery of AZD4747, a Potent and Selective Inhibitor of Mutant GTPase KRASG12C with Demonstrable CNS Penetration.

The glycine to cysteine mutation at codon 12 of Kirsten rat sarcoma (KRAS) represents an Achilles heel that has now rendered this important GTPase druggable. Herein, we report our structure-based drug design approach that led to the identification of 14, AZD4747, a clinical development candidate for the treatment of KRASG12C-positive tumors, including the treatment of central nervous system (CNS) metastases. Building on our earlier discovery of C5-tethered quinazoline AZD4625, excision of a usually critical pyrimidine ring yielded a weak but brain-penetrant start point which was optimized for potency and DMPK. Key design principles and measured parameters that give high confidence in CNS exposure are discussed. During optimization, divergence between rodent and non-rodent species was observed in CNS exposure, with primate PET studies ultimately giving high confidence in the expected translation to patients. AZD4747 is a highly potent and selective inhibitor of KRASG12C with an anticipated low clearance and high oral bioavailability profile in humans.

Chemical Biology Approaches Confirm MCT4 as the Therapeutic Target of a Cellular Optimized Hit.

Lactic acid transport is a key process maintaining glycolytic flux in tumors. Inhibition of this process will result in glycolytic shutdown, impacting on cell growth and survival and thus has been pursued as a therapeutic approach for cancers. Using a cell-based screen in a MCT4-dependent cell line, we identified and optimized compounds for their ability to inhibit the efflux of intracellular lactic acid with good physical and pharmacokinetic properties. To deconvolute the mechanism of lactic acid efflux inhibition, we have developed three assays to measure cellular target engagement. Specifically, we synthesized a biologically active photoaffinity probe (IC50 < 10 nM), and using this probe, we demonstrated selective engagement of MCT4 of our parent molecule through a combination of confocal microscopy and in-cell chemoproteomics. As an orthogonal assay, the cellular thermal shift assay (CETSA) confirmed binding to MCT4 in the cellular system. Comparisons of lactic acid efflux potencies in cells with differential expression of MCT family members further confirmed that the optimized compounds inhibit the efflux of lactic acid through the inhibition of MCT4. Taken together, these data demonstrate the power of orthogonal chemical biology methods to determine cellular target engagement, particularly for proteins not readily amenable to traditional biophysical methods.

Discovery of Clinical Candidate AZD0095, a Selective Inhibitor of Monocarboxylate Transporter 4 (MCT4) for Oncology.

Due to increased reliance on glycolysis, which produces lactate, monocarboxylate transporters (MCTs) are often upregulated in cancer. MCT4 is associated with the export of lactic acid from cancer cells under hypoxia, so inhibition of MCT4 may lead to cytotoxic levels of intracellular lactate. In addition, tumor-derived lactate is known to be immunosuppressive, so MCT4 inhibition may be of interest for immuno-oncology. At the outset, no potent and selective MCT4 inhibitors had been reported, but a screen identified a triazolopyrimidine hit, with no close structural analogues. Minor modifications to the triazolopyrimidine were made, alongside design of a constrained linker and broad SAR exploration of the biaryl tail to improve potency, physical properties, PK, and hERG. The resulting clinical candidate 15 (AZD0095) has excellent potency (1.3 nM), MCT1 selectivity (>1000×), secondary pharmacology, clean mechanism of action, suitable properties for oral administration in the clinic, and good preclinical efficacy in combination with cediranib.

Optimization of hERG and Pharmacokinetic Properties for Basic Dihydro-8H-purin-8-one Inhibitors of DNA-PK.

The DNA-PK complex is activated by double-strand DNA breaks and regulates the non-homologous end-joining repair pathway; thus, targeting DNA-PK by inhibiting the DNA-PK catalytic subunit (DNA-PKcs) is potentially a useful therapeutic approach for oncology. A previously reported series of neutral DNA-PKcs inhibitors were modified to incorporate a basic group, with the rationale that increasing the volume of distribution while maintaining good metabolic stability should increase the half-life. However, adding a basic group introduced hERG activity, and basic compounds with modest hERG activity (IC50 = 10-15 µM) prolonged QTc (time from the start of the Q wave to the end of the T wave, corrected by heart rate) in an anaesthetized guinea pig cardiovascular model. Further optimization was necessary, including modulation of pK a, to identify compound 18, which combines low hERG activity (IC50 = 75 µM) with excellent kinome selectivity and favorable pharmacokinetic properties.

The Discovery of 7-Methyl-2-[(7-methyl[1,2,4]triazolo[1,5-a]pyridin-6-yl)amino]-9-(tetrahydro-2H-pyran-4-yl)-7,9-dihydro-8H-purin-8-one (AZD7648), a Potent and Selective DNA-Dependent Protein Kinase (DNA-PK) Inhibitor.

DNA-PK is a key component within the DNA damage response, as it is responsible for recognizing and repairing double-strand DNA breaks (DSBs) via non-homologous end joining. Historically it has been challenging to identify inhibitors of the DNA-PK catalytic subunit (DNA-PKcs) with good selectivity versus the structurally related PI3 (lipid) and PI3K-related protein kinases. We screened our corporate collection for DNA-PKcs inhibitors with good PI3 kinase selectivity, identifying compound 1. Optimization focused on further improving selectivity while improving physical and pharmacokinetic properties, notably co-optimization of permeability and metabolic stability, to identify compound 16 (AZD7648). Compound 16 had no significant off-target activity in the protein kinome and only weak activity versus PI3Kα/γ lipid kinases. Monotherapy activity in murine xenograft models was observed, and regressions were observed when combined with inducers of DSBs (doxorubicin or irradiation) or PARP inhibition (olaparib). These data support progression into clinical studies (NCT03907969).

AZD7648 is a potent and selective DNA-PK inhibitor that enhances radiation, chemotherapy and olaparib activity.

DNA-dependent protein kinase (DNA-PK) is a critical player in the DNA damage response (DDR) and instrumental in the non-homologous end-joining pathway (NHEJ) used to detect and repair DNA double-strand breaks (DSBs). We demonstrate that the potent and highly selective DNA-PK inhibitor, AZD7648, is an efficient sensitizer of radiation- and doxorubicin-induced DNA damage, with combinations in xenograft and patient-derived xenograft (PDX) models inducing sustained regressions. Using ATM-deficient cells, we demonstrate that AZD7648, in combination with the PARP inhibitor olaparib, increases genomic instability, resulting in cell growth inhibition and apoptosis. AZD7648 enhanced olaparib efficacy across a range of doses and schedules in xenograft and PDX models, enabling sustained tumour regression and providing a clear rationale for its clinical investigation. Through its differentiated mechanism of action as an NHEJ inhibitor, AZD7648 complements the current armamentarium of DDR-targeted agents and has potential in combination with these agents to achieve deeper responses to current therapies.

Synthesis of 3-(hetero)aryl tetrahydropyrazolo[3,4-c]pyridines by Suzuki-Miyaura cross-coupling methodology.

A new synthetic route to 3-(heteroaryl) tetrahydropyrazolo[3,4-c]pyridines has been developed that uses the Suzuki-Miyaura cross-coupling of a triflate 6 with (hetero)aryl boronic acids or esters. Using Pd(OAc)2 and XPhos or an XPhos precatalyst, a diverse range of substituents at the C3 position of the tetrahydropyrazolo[3,4-c]pyridine skeleton were prepared. The use of pivaloyloxymethyl and benzyl protection also offers the potential to differentially functionalize the pyrazole and tetrahydropyridine nitrogens.

Sulfonyl-morpholino-pyrimidines: SAR and development of a novel class of selective mTOR kinase inhibitor.

High throughput screening to identify inhibitors of the mTOR kinase revealed sulfonyl-morpholino-pyrimidine 1 as an attractive start point. The compound displayed good physicochemical properties and selectivity over related kinases such as PI3Kα. Library preparation of related analogs allowed the establishment of additional SAR understanding and in particular the requirement for a key hydrogen bond donor motif at the 4-position of the phenyl ring in compounds such as indole 19. Isosteric replacement of the indole functionality led to the identification of urea compounds such as 32 that show good levels of mTOR inhibition in both enzyme and cellular assays.