Metabolism-driven in vitro/in vivo disconnect of an oral ERɑ VHL-PROTAC.

Targeting the estrogen receptor alpha (ERα) pathway is validated in the clinic as an effective means to treat ER+ breast cancers. Here we present the development of a VHL-targeting and orally bioavailable proteolysis-targeting chimera (PROTAC) degrader of ERα. In vitro studies with this PROTAC demonstrate excellent ERα degradation and ER antagonism in ER+ breast cancer cell lines. However, upon dosing the compound in vivo we observe an in vitro-in vivo disconnect. ERα degradation is lower in vivo than expected based on the in vitro data. Investigation into potential causes for the reduced maximal degradation reveals that metabolic instability of the PROTAC linker generates metabolites that compete for binding to ERα with the full PROTAC, limiting degradation. This observation highlights the requirement for metabolically stable PROTACs to ensure maximal efficacy and thus optimisation of the linker should be a key consideration when designing PROTACs.

Vinylpyridine as a Tunable Covalent Warhead Targeting C797 in EGFR.

To further facilitate the discovery of cysteine reactive covalent inhibitors, there is a need to develop new reactive groups beyond the traditional acrylamide-type warheads. Herein we describe the design and synthesis of covalent EGFR inhibitors that use vinylpyridine as the reactive group. Structure-based design identified the quinazoline-containing vinylpyridine 6 as a starting point. Further modifications focused on reducing reactivity resulted in substituted vinyl compound 12, which shows high EGFR potency and good kinase selectivity, as well as significantly reduced reactivity compared to the starting compound 6, confirming that vinylpyridines can be applied as an alternative cysteine reactive warhead with tunable reactivity.

Discovery of the Potent and Selective Inhaled Janus Kinase 1 Inhibitor AZD4604 and Its Preclinical Characterization.

JAK-STAT cytokines are critical in regulating immunity. Persistent activation of JAK-STAT signaling pathways by cytokines drives chronic inflammatory diseases such as asthma. Herein, we report on the discovery of a highly JAK1-selective, ATP-competitive series of inhibitors having a 1000-fold selectivity over other JAK family members and the approach used to identify compounds suitable for inhaled administration. Ultimately, compound 16 was selected as the clinical candidate, and upon dry powder inhalation, we could demonstrate a high local concentration in the lung as well as low plasma concentrations, suggesting no systemic JAK1 target engagement. Compound 16 has progressed into clinical trials. Using 16, we found JAK1 inhibition to be more efficacious than JAK3 inhibition in IL-4-driven Th2 asthma.

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.

AZD4625 is a Potent and Selective Inhibitor of KRASG12C.

AZD4625 is a potent, selective, and orally bioavailable inhibitor of oncogenic KRASG12C as demonstrated in cellular assays and in vivo in preclinical cell line-derived and patient-derived xenograft models. In vitro and cellular assays have shown selective binding and inhibition of the KRASG12C mutant isoform, which carries a glycine to cysteine mutation at residue 12, with no binding and inhibition of wild-type RAS or isoforms carrying non-KRASG12C mutations. The pharmacology of AZD4625 shows that it has the potential to provide therapeutic benefit to patients with KRASG12C mutant cancer as either a monotherapy treatment or in combination with other targeted drug agents.

Discovery of AZD4625, a Covalent Allosteric Inhibitor of the Mutant GTPase KRASG12C.

KRAS is an archetypal high-value intractable oncology drug target. The glycine to cysteine mutation at codon 12 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 21, AZD4625, a clinical development candidate for the treatment of KRASG12C positive tumors. Highlights include a quinazoline tethering strategy to lock out a bio-relevant binding conformation and an optimization strategy focused on the reduction of extrahepatic clearance mechanisms seen in preclinical species. Crystallographic analysis was also key in helping to rationalize unusual structure-activity relationship in terms of ring size and enantio-preference. AZD4625 is a highly potent and selective inhibitor of KRASG12C with an anticipated low clearance and high oral bioavailability profile in humans.

Identification and optimization of a novel series of selective PIP5K inhibitors.

Phosphatidyl inositol (4,5)-bisphosphate (PI(4,5)P2) plays several key roles in human biology and the lipid kinase that produces PI(4,5)P2, PIP5K, has been hypothesized to provide a potential therapeutic target of interest in the treatment of cancers. To better understand and explore the role of PIP5K in human cancers there remains an urgent need for potent and specific PIP5K inhibitor molecules. Following a high throughput screen of the AstraZeneca collection, a novel, moderately potent and selective inhibitor of PIP5K, 1, was discovered. Detailed exploration of the SAR for this novel scaffold resulted in the considerable optimization of both potency for PIP5K, and selectivity over the closely related kinase PI3Kα, as well as identifying several opportunities for the continued optimization of drug-like properties. As a result, several high quality in vitro tool compounds were identified (8, 20 and 25) that demonstrate the desired biochemical and cellular profiles required to aid better understanding of this complex area of biology.

Drugging the undruggable: a computational chemist's view of KRASG12C.

In recent years, the emergence of targeted covalent inhibitors which bind to the G12C mutant of KRAS have offered a solution to this previously intractable target. Inhibitors of KRASG12C tend to be structurally complex, displaying features such as atropisomerism, chiral centres and a reactive covalent warhead. Such molecules result in lengthy and challenging syntheses, and as a consequence critical decisions need to be made at the design level to maximise the chances of success. Here we take a retrospective look into how computational chemistry can help guide and prioritise medicinal chemistry efforts in the context of a series of conformationally restricted tetracyclic quinolines.

Alkynyl Benzoxazines and Dihydroquinazolines as Cysteine Targeting Covalent Warheads and Their Application in Identification of Selective Irreversible Kinase Inhibitors.

With a resurgence in interest in covalent drugs, there is a need to identify new moieties capable of cysteine bond formation that are differentiated from commonly employed systems such as acrylamide. Herein, we report on the discovery of new alkynyl benzoxazine and dihydroquinazoline moieties capable of covalent reaction with cysteine. Their utility as alternative electrophilic warheads for chemical biological probes and drug molecules is demonstrated through site-selective protein modification and incorporation into kinase drug scaffolds. A potent covalent inhibitor of JAK3 kinase was identified with superior selectivity across the kinome and improvements in in vitro pharmacokinetic profile relative to the related acrylamide-based inhibitor. In addition, the use of a novel heterocycle as a cysteine reactive warhead is employed to target Cys788 in c-KIT, where acrylamide has previously failed to form covalent interactions. These new reactive and selective heterocyclic warheads supplement the current repertoire for cysteine covalent modification while avoiding some of the limitations generally associated with established moieties.

Discovery and pharmacological characterization of AZD3229, a potent KIT/PDGFRα inhibitor for treatment of gastrointestinal stromal tumors.

Gastrointestinal stromal tumor (GIST) is the most common human sarcoma driven by mutations in KIT or platelet-derived growth factor α (PDGFRα). Although first-line treatment, imatinib, has revolutionized GIST treatment, drug resistance due to acquisition of secondary KIT/PDGFRα mutations develops in a majority of patients. Second- and third-line treatments, sunitinib and regorafenib, lack activity against a plethora of mutations in KIT/PDGFRα in GIST, with median time to disease progression of 4 to 6 months and inhibition of vascular endothelial growth factor receptor 2 (VEGFR2) causing high-grade hypertension. Patients with GIST have an unmet need for a well-tolerated drug that robustly inhibits a range of KIT/PDGFRα mutations. Here, we report the discovery and pharmacological characterization of AZD3229, a potent and selective small-molecule inhibitor of KIT and PDGFRα designed to inhibit a broad range of primary and imatinib-resistant secondary mutations seen in GIST. In engineered and GIST-derived cell lines, AZD3229 is 15 to 60 times more potent than imatinib in inhibiting KIT primary mutations and has low nanomolar activity against a wide spectrum of secondary mutations. AZD3229 causes durable inhibition of KIT signaling in patient-derived xenograft (PDX) models of GIST, leading to tumor regressions at doses that showed no changes in arterial blood pressure (BP) in rat telemetry studies. AZD3229 has a superior potency and selectivity profile to standard of care (SoC) agents-imatinib, sunitinib, and regorafenib, as well as investigational agents, avapritinib (BLU-285) and ripretinib (DCC-2618). AZD3229 has the potential to be a best-in-class inhibitor for clinically relevant KIT/PDGFRα mutations in GIST.

Discovery of (2R)-N-[3-[2-[(3-Methoxy-1-methyl-pyrazol-4-yl)amino]pyrimidin-4-yl]-1H-indol-7-yl]-2-(4-methylpiperazin-1-yl)propenamide (AZD4205) as a Potent and Selective Janus Kinase 1 Inhibitor.

JAK1, JAK2, JAK3, and TYK2 belong to the JAK (Janus kinase) family. They play critical roles in cytokine signaling. Constitutive activation of JAK/STAT pathways is associated with a wide variety of diseases. Particularly, pSTAT3 is observed in response to the treatment with inhibitors of oncogenic signaling pathways such as EGFR, MAPK, and AKT and is associated with resistance or poorer response to agents targeting these pathways. Among the JAK family kinases, JAK1 has been shown to be the primary driver of STAT3 phosphorylation and signaling; therefore, selective JAK1 inhibition can be a viable means to overcome such treatment resistances. Herein, an account of the medicinal chemistry optimization from the promiscuous kinase screening hit 3 to the candidate drug 21 (AZD4205), a highly selective JAK1 kinase inhibitor, is reported. Compound 21 has good preclinical pharmacokinetics. Compound 21 displayed an enhanced antitumor activity in combination with an approved EGFR inhibitor, osimertinib, in a preclinical non-small-cell lung cancer (NSCLC) xenograft NCI-H1975 model.

The Pharmacokinetic-Pharmacodynamic (PKPD) Relationships of AZD3229, a Novel and Selective Inhibitor of KIT, in a Range of Mouse Xenograft Models of GIST.

PURPOSE: The emergence of secondary mutations is a cause of resistance to current KIT inhibitors used in the treatment of patients with gastrointestinal stromal tumors (GIST). AZD3229 is a selective inhibitor of wild-type KIT and a wide spectrum of primary and secondary mutations seen in patients with GIST. The objective of this analysis is to establish the pharmacokinetic-pharmacodynamic (PKPD) relationship of AZD3229 in a range of mouse GIST tumor models harboring primary and secondary KIT mutations, and to benchmark AZD3229 against other KIT inhibitors. EXPERIMENTAL DESIGN: A PKPD model was developed for AZD3229 linking plasma concentrations to inhibition of phosphorylated KIT using data generated from several in vivo preclinical tumor models, and in vitro data generated in a panel of Ba/F3 cell lines. RESULTS: AZD3229 drives inhibition of phosphorylated KIT in an exposure-dependent manner, and optimal efficacy is observed when >90% inhibition of KIT phosphorylation is sustained over the dosing interval. Integrating the predicted human pharmacokinetics into the mouse PKPD model predicts that an oral twice daily human dose greater than 34 mg is required to ensure adequate coverage across the mutations investigated. Benchmarking shows that compared with standard-of-care KIT inhibitors, AZD3229 has the potential to deliver the required target coverage across a wider spectrum of primary or secondary mutations. CONCLUSIONS: We demonstrate that AZD3229 warrants clinical investigation as a new treatment for patients with GIST based on its ability to inhibit both ATP-binding and A-loop mutations of KIT at clinically relevant exposures.

Structure-Based Design and Pharmacokinetic Optimization of Covalent Allosteric Inhibitors of the Mutant GTPase KRASG12C.

Attempts to directly drug the important oncogene KRAS have met with limited success despite numerous efforts across industry and academia. The KRASG12C mutant represents an "Achilles heel" and has recently yielded to covalent targeting with small molecules that bind the mutant cysteine and create an allosteric pocket on GDP-bound RAS, locking it in an inactive state. A weak inhibitor at this site was optimized through conformational locking of a piperazine-quinazoline motif and linker modification. Subsequent introduction of a key methyl group to the piperazine resulted in enhancements in potency, permeability, clearance, and reactivity, leading to identification of a potent KRASG12C inhibitor with high selectivity and excellent cross-species pharmacokinetic parameters and in vivo efficacy.

Covalent inhibitors of the GTPase KRASG12C: a review of the patent literature.

Introduction: KRAS is one of the most important oncology drug targets, playing a pivotal role in the initiation and progression of many human tumors. It has long been held undruggable due to many previously failed attempts to both directly and indirectly target this challenging GTPase protein family.Areas covered: This review covers patent applications claiming inhibitors of the mutant GTPase KRASG12C that act via covalent modification of cysteine at codon 12 in the period of 2014 to the present. A total of 37 PCT applications from 9 applicants are evaluated, with the discussion organized alphabetically by assignee name.Expert opinion: The last 5 years have seen an explosion in interest around this important target with many companies aiming to capitalize on the breakthrough discovery of covalent allosteric inhibitors of the glycine to cysteine mutant form of the enzyme. The first agents from this effort have now entered clinical trials and preliminary data are encouraging with responses seen in both lung adenocarcinoma and colorectal cancer patients.

Discovery of Mcl-1-specific inhibitor AZD5991 and preclinical activity in multiple myeloma and acute myeloid leukemia.

Mcl-1 is a member of the Bcl-2 family of proteins that promotes cell survival by preventing induction of apoptosis in many cancers. High expression of Mcl-1 causes tumorigenesis and resistance to anticancer therapies highlighting the potential of Mcl-1 inhibitors as anticancer drugs. Here, we describe AZD5991, a rationally designed macrocyclic molecule with high selectivity and affinity for Mcl-1 currently in clinical development. Our studies demonstrate that AZD5991 binds directly to Mcl-1 and induces rapid apoptosis in cancer cells, most notably myeloma and acute myeloid leukemia, by activating the Bak-dependent mitochondrial apoptotic pathway. AZD5991 shows potent antitumor activity in vivo with complete tumor regression in several models of multiple myeloma and acute myeloid leukemia after a single tolerated dose as monotherapy or in combination with bortezomib or venetoclax. Based on these promising data, a Phase I clinical trial has been launched for evaluation of AZD5991 in patients with hematological malignancies (NCT03218683).

Discovery of N-(4-{[5-Fluoro-7-(2-methoxyethoxy)quinazolin-4-yl]amino}phenyl)-2-[4-(propan-2-yl)-1 H-1,2,3-triazol-1-yl]acetamide (AZD3229), a Potent Pan-KIT Mutant Inhibitor for the Treatment of Gastrointestinal Stromal Tumors.

While the treatment of gastrointestinal stromal tumors (GISTs) has been revolutionized by the application of targeted tyrosine kinase inhibitors capable of inhibiting KIT-driven proliferation, diverse mutations to this kinase drive resistance to established therapies. Here we describe the identification of potent pan-KIT mutant kinase inhibitors that can be dosed without being limited by the tolerability issues seen with multitargeted agents. This effort focused on identification and optimization of an existing kinase scaffold through the use of structure-based design. Starting from a series of previously reported phenoxyquinazoline and quinoline based inhibitors of the tyrosine kinase PDGFRα, potency against a diverse panel of mutant KIT driven Ba/F3 cell lines was optimized, with a particular focus on reducing activity against a KDR driven cell model in order to limit the potential for hypertension commonly seen in second and third line GIST therapies. AZD3229 demonstrates potent single digit nM growth inhibition across a broad cell panel, with good margin to KDR-driven effects. Selectivity over KDR can be rationalized predominantly by the interaction of water molecules with the protein and ligand in the active site, and its kinome selectivity is similar to the best of the approved GIST agents. This compound demonstrates excellent cross-species pharmacokinetics, shows strong pharmacodynamic inhibition of target, and is active in several in vivo models of GIST.

Discovery and Optimization of a Novel Series of Highly Selective JAK1 Kinase Inhibitors.

Janus kinases (JAKs) have been demonstrated to be critical in cytokine signaling and have thus been implicated in both cancer and inflammatory diseases. The JAK family consists of four highly homologous members: JAK1-3 and TYK2. The development of small-molecule inhibitors that are selective for a specific family member would represent highly desirable tools for deconvoluting the intricacies of JAK family biology. Herein, we report the discovery of a potent JAK1 inhibitor, 24, which displays ∼1000-fold selectivity over the other highly homologous JAK family members (determined by biochemical assays), while also possessing good selectivity over other kinases (determined by panel screening). Moreover, this compound was demonstrated to be orally bioavailable and possesses acceptable pharmacokinetic parameters. In an in vivo study, the compound was observed to dose dependently modulate the phosphorylation of STAT3 (a downstream marker of JAK1 inhibition).

Inhibitors of JAK-family kinases: an update on the patent literature 2013-2015, part 2.

INTRODUCTION: Janus kinases (JAKs) are a family of four enzymes; JAK1, JAK2, JAK3 and tyrosine kinase 2 (TYK2) that are critical in cytokine signalling and are strongly linked to both cancer and inflammatory diseases. There are currently two launched JAK inhibitors for the treatment of human conditions: tofacitinib for Rheumatoid arthritis (RA) and ruxolitinib for myeloproliferative neoplasms including intermediate or high risk myelofibrosis and polycythemia vera. Areas covered: This review covers patents claiming activity against one or more JAK family members in the period 2013-2015 inclusive, and covers 95 patents from 42 applicants, split over two parts. The authors have ordered recent patents according to the primary applicant's name, with part 2 covering J through Z. Expert opinion: Inhibition of JAK-family kinases is an area of growing interest, catalysed by the maturity of data on marketed inhibitors ruxolitinib and tofacitinib in late stage clinical trials. Many applicants are pursuing traditional fast-follower strategies around these inhibitors, with a range of chemical strategies adopted. The challenge will be to show sufficient differentiation to the originator compounds, since dose limiting toxicities with such agents appear to be on target and mechanism-related and also considering that such agents may be available as generic compounds by the time follower agents reach market.