Discovery of BLU-945, a Reversible, Potent, and Wild-Type-Sparing Next-Generation EGFR Mutant Inhibitor for Treatment-Resistant Non-Small-Cell Lung Cancer.

While epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have changed the treatment landscape for EGFR mutant (L858R and ex19del)-driven non-small-cell lung cancer (NSCLC), most patients will eventually develop resistance to TKIs. In the case of first- and second-generation TKIs, up to 60% of patients will develop an EGFR T790M mutation, while third-generation irreversible TKIs, like osimertinib, lead to C797S as the primary on-target resistance mutation. The development of reversible inhibitors of these resistance mutants is often hampered by poor selectivity against wild-type EGFR, resulting in potentially dose-limiting toxicities and a sub-optimal profile for use in combinations. BLU-945 (compound 30) is a potent, reversible, wild-type-sparing inhibitor of EGFR+/T790M and EGFR+/T790M/C797S resistance mutants that maintains activity against the sensitizing mutations, especially L858R. Pre-clinical efficacy and safety studies supported progression of BLU-945 into clinical studies, and it is currently in phase 1/2 clinical trials for treatment-resistant EGFR-driven NSCLC.

Mechanistic Insights into a CDK9 Inhibitor Via Orthogonal Proteomics Methods.

Cyclin-dependent-kinases (CDKs) are members of the serine/threonine kinase family and are highly regulated by cyclins, a family of regulatory subunits that bind to CDKs. CDK9 represents one of the most studied examples of these transcriptional CDKs. CDK9 forms a heterodimeric complex with its regulatory subunit cyclins T1, T2 and K to form the positive transcription elongation factor b (P-TEFb). This complex regulates transcription via the phosphorylation of RNA polymerase II (RNAPolII) on Ser-2, facilitating promoter clearance and transcription elongation and thus remains an attractive therapeutic target. Herein, we have utilized classical affinity purification chemical proteomics, kinobeads assay, compressed CEllular Thermal Shift Assay (CETSA)-MS and Limited Proteolysis (LiP) to study the selectivity, target engagement and downstream mechanistic insights of a CDK9 tool compound. The above experiments highlight the value of quantitative mass spectrometry approaches to drug discovery, specifically proteome wide target identification and selectivity profiling. The approaches utilized in this study unanimously indicated that the CDK family of kinases are the main target of the compound of interest, with CDK9, showing the highest target affinity with remarkable consistency across approaches. We aim to provide guidance to the scientific community on the available chemical biology/proteomic tools to study advanced lead molecules and to highlight pros and cons of each technology while describing our findings in the context of the CDKs biology.

Discovery of a Series of 7-Azaindoles as Potent and Highly Selective CDK9 Inhibitors for Transient Target Engagement.

Optimization of a series of azabenzimidazoles identified from screening hit 2 and the information gained from a co-crystal structure of the azabenzimidazole-based lead 6 bound to CDK9 led to the discovery of azaindoles as highly potent and selective CDK9 inhibitors. With the goal of discovering a highly selective and potent CDK9 inhibitor administrated intravenously that would enable transient target engagement of CDK9 for the treatment of hematological malignancies, further optimization focusing on physicochemical and pharmacokinetic properties led to azaindoles 38 and 39. These compounds are highly potent and selective CDK9 inhibitors having short half-lives in rodents, suitable physical properties for intravenous administration, and the potential to achieve profound but transient inhibition of CDK9 in vivo.

Discovery of AZD4573, a Potent and Selective Inhibitor of CDK9 That Enables Short Duration of Target Engagement for the Treatment of Hematological Malignancies.

A CDK9 inhibitor having short target engagement would enable a reduction of Mcl-1 activity, resulting in apoptosis in cancer cells dependent on Mcl-1 for survival. We report the optimization of a series of amidopyridines (from compound 2), focusing on properties suitable for achieving short target engagement after intravenous administration. By increasing potency and human metabolic clearance, we identified compound 24, a potent and selective CDK9 inhibitor with suitable predicted human pharmacokinetic properties to deliver transient inhibition of CDK9. Furthermore, the solubility of 24 was considered adequate to allow i.v. formulation at the anticipated effective dose. Short-term treatment with compound 24 led to a rapid dose- and time-dependent decrease of pSer2-RNAP2 and Mcl-1, resulting in cell apoptosis in multiple hematological cancer cell lines. Intermittent dosing of compound 24 demonstrated efficacy in xenograft models derived from multiple hematological tumors. Compound 24 is currently in clinical trials for the treatment of hematological malignancies.

Quest for a COVID-19 Cure by Repurposing Small-Molecule Drugs: Mechanism of Action, Clinical Development, Synthesis at Scale, and Outlook for Supply.

The outbreak of the COVID-19 pandemic has spurred an intense global effort to repurpose existing approved drugs for its treatment. In this review, we highlight the development of seven small-molecule drugs that are currently being assessed in clinical trials for the treatment of COVID-19. Three sections are presented for each drug: (1) history, mechanism of action, and status of clinical trials; (2) scalable synthetic routes and final forms; and (3) outlook for supply should clinical trials show treatment efficacy. A brief overview of diagnostic testing and vaccine development is also presented.

AZD9496: An Oral Estrogen Receptor Inhibitor That Blocks the Growth of ER-Positive and ESR1-Mutant Breast Tumors in Preclinical Models.

Fulvestrant is an estrogen receptor (ER) antagonist administered to breast cancer patients by monthly intramuscular injection. Given its present limitations of dosing and route of administration, a more flexible orally available compound has been sought to pursue the potential benefits of this drug in patients with advanced metastatic disease. Here we report the identification and characterization of AZD9496, a nonsteroidal small-molecule inhibitor of ERα, which is a potent and selective antagonist and downregulator of ERα in vitro and in vivo in ER-positive models of breast cancer. Significant tumor growth inhibition was observed as low as 0.5 mg/kg dose in the estrogen-dependent MCF-7 xenograft model, where this effect was accompanied by a dose-dependent decrease in PR protein levels, demonstrating potent antagonist activity. Combining AZD9496 with PI3K pathway and CDK4/6 inhibitors led to further growth-inhibitory effects compared with monotherapy alone. Tumor regressions were also seen in a long-term estrogen-deprived breast model, where significant downregulation of ERα protein was observed. AZD9496 bound and downregulated clinically relevant ESR1 mutants in vitro and inhibited tumor growth in an ESR1-mutant patient-derived xenograft model that included a D538G mutation. Collectively, the pharmacologic evidence showed that AZD9496 is an oral, nonsteroidal, selective estrogen receptor antagonist and downregulator in ER(+) breast cells that could provide meaningful benefit to ER(+) breast cancer patients. AZD9496 is currently being evaluated in a phase I clinical trial. Cancer Res; 76(11); 3307-18. ©2016 AACR.

Optimization of a Novel Binding Motif to (E)-3-(3,5-Difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic Acid (AZD9496), a Potent and Orally Bioavailable Selective Estrogen Receptor Downregulator and Antagonist.

The discovery of an orally bioavailable selective estrogen receptor downregulator (SERD) with equivalent potency and preclinical pharmacology to the intramuscular SERD fulvestrant is described. A directed screen identified the 1-aryl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole motif as a novel, druglike ER ligand. Aided by crystal structures of novel ligands bound to an ER construct, medicinal chemistry iterations led to (E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid (30b, AZD9496), a clinical candidate with high oral bioavailability across preclinical species that is currently being evaluated in phase I clinical trials for the treatment of advanced estrogen receptor (ER) positive breast cancer.

Investigation of (E)-3-[4-(2-Oxo-3-aryl-chromen-4-yl)oxyphenyl]acrylic Acids as Oral Selective Estrogen Receptor Down-Regulators.

A novel estrogen receptor down-regulator, 7-hydroxycoumarin (5, SS5020), has been reported with antitumor effects against chemically induced mammary tumors. Here, we report on our own investigation of 7-hydroxycoumarins as potential selective estrogen receptor down-regulators, which led us to the discovery of potent down-regulating antagonists, such as 33. Subsequent optimization and removal of the 7-hydroxy group led to coumarin 59, which had increased potency and improved rat bioavailability relative to SS5020.

Efficacious inhaled PDE4 inhibitors with low emetic potential and long duration of action for the treatment of COPD.

Oral phosphodiesterase 4 (PDE4) inhibitors, such as cilomilast and roflumilast, have been shown to be efficacious against chronic obstructive pulmonary disease (COPD). However, these drugs have been hampered by mechanism-related side effects such as nausea and emesis at high doses. Compounds administered by inhalation are delivered directly to the site of action and may improve the therapeutic index required to overcome side effects. This paper describes systematic and rational lead optimization to deliver highly potent, long-acting, and efficacious preclinical inhaled PDE4 inhibitors with low emetic potential.

Hydantoin based inhibitors of MMP13--discovery of AZD6605.

Piperidine ether and aryl piperazine hydantoins are reported as potent inhibitors of MMP13. A medicinal chemistry campaign focused on replacing the reverse hydroxamate zinc binding group associated with historical inhibitors with a hydantoin zinc binding group then optimising MMP13 potency, solubility and DMPK properties whilst maintaining good selectivity over MMP14. A number of high quality candidates were progressed and following rat and dog safety evaluation, AZD6605 (3m) was identified as a candidate drug.

Lead optimisation of selective non-zinc binding inhibitors of MMP13. Part 2.

Directed screening has identified a novel series of non-zinc binding MMP13 inhibitors that possess good levels of activity whilst demonstrating excellent selectivity over related MMPs. A lead optimisation campaign has delivered compounds with enhanced MMP13 potency, good selectivity and acceptable bioavailability profiles leading to a predicted twice-a-day dosing regimen in man.

Selective non zinc binding inhibitors of MMP13.

Directed screening has identified a novel series of MMP13 inhibitors that possess good levels of activity whilst possessing excellent selectivity over related MMPs. The binding mode of the series has been solved by co-crystallisation and demonstrates an interesting mode of inhibition without interaction with the catalytic zinc atom.

Orally active achiral N-hydroxyformamide inhibitors of ADAM-TS4 (aggrecanase-1) and ADAM-TS5 (aggrecanase-2) for the treatment of osteoarthritis.

A new achiral class of N-hydroxyformamide inhibitor of both ADAM-TS4 and ADAM-TS5, 2 has been discovered through modification of the complex P1 group present in historical inhibitors 1. This structural change improved the DMPK properties and greatly simplified the synthesis whilst maintaining excellent cross-MMP selectivity profiles. Investigation of structure-activity and structure-property relationships in the P1 group resulted in both ADAM-TS4 selective and mixed ADAM-TS4/5 inhibitors. This led to the identification of a pre-clinical candidate with excellent bioavailability across three species and predicting once daily dosing kinetics.

The design and synthesis of novel N-hydroxyformamide inhibitors of ADAM-TS4 for the treatment of osteoarthritis.

Two series of N-hydroxyformamide inhibitors of ADAM-TS4 were identified from screening compounds previously synthesised as inhibitors of matrix metalloproteinase-13 (collagenase-3). Understanding of the binding mode of this class of compound using ADAM-TS1 as a structural surrogate has led to the discovery of potent and very selective inhibitors with favourable DMPK properties. Synthesis, structure-activity relationships, and strategies to improve selectivity and lower in vivo metabolic clearance are described.

Construction of perhydro indol-2-ones by a methoxide catalyzed deacetylation-Michael-aldol cascade.

An efficient, stereoselective Michael-aldol cascade for the one-pot construction of the perhydro indol-2-one bicyclic ring system using an acetate protected doubly-activated pyrrole-2-one pro-nucleophile and alpha,beta-unsaturated carbonyl compounds has been developed. Initiated by a methoxide deacetylation in methanol at room temperature, the cascade is easy to perform, stereoselective, efficient and broad in scope to this synthetically relevant structure.

Discovery, synthesis, and in vivo activity of a new class of pyrazoloquinazolines as selective inhibitors of aurora B kinase.

The Aurora kinases have been the subject of considerable interest as targets for the development of new anticancer agents. While evidence suggests inhibition of Aurora B kinase gives rise to the more pronounced antiproliferative phenotype, the most clinically advanced agents reported to date typically inhibit both Aurora A and B. We have discovered a series of pyrazoloquinazolines, some of which show greater than 1000-fold selectivity for Aurora B over Aurora A kinase activity, in recombinant enzyme assays. These compounds have been designed for parenteral administration and achieve high levels of solubility by virtue of their ability to be delivered as readily activated phosphate derivatives. The prodrugs are comprehensively converted to the des-phosphate form in vivo, and the active species have advantageous pharmacokinetic properties and safety pharmacology profiles. The compounds display striking in vivo activity, and compound 5 (AZD1152) has been selected for clinical evaluation and is currently in phase 1 clinical trials.

Discovery of novel and potent thiazoloquinazolines as selective Aurora A and B kinase inhibitors.

The synthesis of a novel series of quinazolines substituted at C4 by five-membered ring aminoheterocycles is reported. Their in vitro structure-activity relationships versus Aurora A and B serine-threonine kinases is discussed. Our results demonstrate that quinazolines with a substituted aminothiazole at C4 possess potent Aurora A and B inhibitory activity and excellent selectivity against a panel of various serine-threonine and tyrosine kinases, as exemplified by compound 46. We found also that the position and nature of the substituent on the thiazole play key roles in cellular potency. Compounds with an acetanilide substituent at C5' have the greatest cellular activity. The importance of the C5' position for substitution has been rationalized by ab initio molecular orbital calculations. Results show that the planar conformation with the sulfur of the thiazole next to the quinazoline N-3 is strongly favored over the other possible planar conformation. Compound 46 is a potent suppressor of the expression of phospho-histone H3 in tumor cells in vitro as well as in vivo, where 46, administered as its phosphate prodrug 54, suppresses the expression of phospho-histone H3 in subcutaneously implanted tumors in nude mice.