Stable and Storable N(CF3 )2 Transfer Reagents.

Fluorinated groups are essential for drug design, agrochemicals, and materials science. The bis(trifluoromethyl)amino group is an example of a stable group that has a high potential. While the number of molecules containing perfluoroalkyl, perfluoroalkoxy, and other fluorinated groups is steadily increasing, examples with the N(CF3 )2 group are rare. One reason is that transfer reagents are scarce and metal-based storable reagents are unknown. Herein, a set of CuI and AgI bis(trifluoromethyl)amido complexes stabilized by N- and P-donor ligands with unprecedented stability are presented. The complexes are stable solids that can even be manipulated in air for a short time. They are bis(trifluoromethyl)amination reagents as shown by nucleophilic substitution and Sandmeyer reactions. In addition to a series of benzylbis(trifluoromethyl)amines, 2-bis(trifluoromethyl)amino acetate was obtained, which, upon hydrolysis, gives the fluorinated amino acid N,N-bis(trifluoromethyl)glycine.

Crystalline Sponges as a Sensitive and Fast Method for Metabolite Identification: Application to Gemfibrozil and its Phase I and II Metabolites.

Understanding the metabolism of new drug candidates is important during drug discovery and development, as circulating metabolites may contribute to efficacy or cause safety issues. In the early phase of drug discovery, human in vitro systems are used to investigate human relevant metabolism. Though conventional techniques are limited in their ability to provide complete molecular structures of metabolites (liquid chromatography mass spectrometry) or require a larger amount of material not available from in vitro incubation (nuclear magnetic resonance), we here report for the first time the use of the crystalline sponge method to identify phase I and phase II metabolites generated from in vitro liver microsomes or S9 fractions. Gemfibrozil was used as a test compound. Metabolites generated from incubation with microsomes or S9 fractions, were fractionated using online fraction collection. After chromatographic purification and fractionation of the generated metabolites, single crystal X-ray diffraction of crystalline sponges was used to identify the structure of gemfibrozil metabolites. This technique allowed for complete structure elucidation of 5'-CH2OH gemfibrozil (M1), 4'-OH gemfibrozil (M2), 5'-COOH gemfibrozil (M3), and the acyl glucuronide of gemfibrozil, 1-O-ß-glucuronide (M4), the first acyl glucuronide available in the Cambridge Crystallographic Data Centre. Our study shows that when optimal soaking is possible, crystalline sponges technology is a sensitive (nanogram amount) and fast (few days) method that can be applied early in drug discovery to identify the structure of pure metabolites from in vitro incubations. SIGNIFICANCE STATEMENT: Complete structure elucidation of human metabolites plays a critical role in early drug discovery. Low amounts of material (nanogram) are only available at this stage and insufficient for nuclear magnetic resonance analysis. The crystalline sponge method has the potential to close this gap, as demonstrated in this study.

Small molecules-Giant leaps for immuno-oncology.

Immuno-oncology therapies are revolutionizing the oncology landscape with checkpoint blockade becoming the treatment backbone for many indications. While inspiring, much work remains to increase the number of cancer patients that can benefit from these treatments. Thus, a new era of immuno-oncology research has begun which is focused on identifying novel combination regimes that lead to improved response rates. This review highlights the significance of small molecules in this approach and illustrates the huge progress that has been made to date.

Small Molecules Drive Big Improvements in Immuno-Oncology Therapies.

Immuno-oncology therapies have the potential to revolutionize the armamentarium of available cancer treatments. To further improve clinical response rates, researchers are looking for novel combination regimens, with checkpoint blockade being used as a backbone of the treatment. This Review highlights the significance of small molecules in this approach, which holds promise to provide increased benefit to cancer patients.

Discovery of potent and selective CDK8 inhibitors from an HSP90 pharmacophore.

Here we describe the discovery and optimization of 3-benzylindazoles as potent and selective inhibitors of CDK8, also modulating CDK19, discovered from a high-throughput screening (HTS) campaign sampling the Merck compound collection. The primary hits with strong HSP90 affinity were subsequently optimized to potent and selective CDK8 inhibitors which demonstrate inhibition of WNT pathway activity in cell-based assays. X-ray crystallographic data demonstrated that 3-benzylindazoles occupy the ATP binding site of CDK8 and adopt a Type I binding mode. Medicinal chemistry optimization successfully led to improved potency, physicochemical properties and oral pharmacokinetics. Modulation of phospho-STAT1, a pharmacodynamic biomarker of CDK8, was demonstrated in an APC-mutant SW620 human colorectal carcinoma xenograft model following oral administration.

Tetrahydro-naphthols as orally available TRPV1 inhibitors.

Starting from a naphthol-based lead series with low oral bioavailability, we have identified potent TRPV1 antagonists with oral bioavailability in rats. These compounds emerged from SAR studies aimed at replacing the lead's phenol structure whilst maintaining potency. Compound rac-6a is an orally available TRPV1 antagonist with single-digit nanomolar activity. The enantiomers show a low eudismic ratio at the receptor level.

The expanding role for small molecules in immuno-oncology.

The advent of immune checkpoint inhibition (ICI) using antibodies against PD1 and its ligand PDL1 has prompted substantial efforts to develop complementary drugs. Although many of these are antibodies directed against additional checkpoint proteins, there is an increasing interest in small-molecule immuno-oncology drugs that address intracellular pathways, some of which have recently entered clinical trials. In parallel, small molecules that target pro-tumorigenic pathways in cancer cells and the tumour microenvironment have been found to have immunostimulatory effects that synergize with the action of ICI antibodies, leading to the approval of an increasing number of regimens that combine such drugs. Combinations with small molecules targeting cancer metabolism, cytokine/chemokine and innate immune pathways, and T cell checkpoints are now under investigation. This Review discusses the recent milestones and hurdles encountered in this area of drug development, as well as our views on the best path forward.

Naphthol derivatives as TRPV1 inhibitors for the treatment of urinary incontinence.

We have identified naphthol derivatives as inhibitors of the vanilloid receptor TRPV1 by high throughput screening. The initial lead showed high clearance in rats and has been optimized by enhancing the acidity of the phenol group. Compound 6b has reduced clearance, improved potency and is active in rat cystometry models of urinary incontinence after intravenous administration.

Glycine amides as PPARalpha agonists.

The design, synthesis and pharmacological properties of a novel class of PPARalpha agonists is described. Compound 2 is a novel, potent and specific glycine amide with oral bioavailability in rodents. The compound is active in vivo and alters plasma lipids in hAPO-A1 transgenic mice after oral administration.

Targeted Covalent Inhibitors for the Treatment of Malaria?

Malaria is a vector-borne disease caused by protozoan parasites of the genus Plasmodium. According to the World Health Organization, it is one of the most serious infectious diseases threatening more than 3 billion people worldwide. In recent years, targeted covalent inhibitors (TCIs) have gained a lot of attention and several TCI-based drugs have been approved across different therapeutic areas. For malaria, surprisingly, this approach has not been explored in depth even though lot of advancements have been made in understanding the biology of the parasite. Herein, we present our views on exploring TCIs as a new class of antimalarial agents.

Discovery of Evobrutinib: An Oral, Potent, and Highly Selective, Covalent Bruton's Tyrosine Kinase (BTK) Inhibitor for the Treatment of Immunological Diseases.

Bruton's tyrosine kinase (BTK) inhibitors such as ibrutinib hold a prominent role in the treatment of B cell malignancies. However, further refinement is needed to this class of agents, particularly in terms of adverse events (potentially driven by kinase promiscuity), which preclude their evaluation in nononcology indications. Here, we report the discovery and preclinical characterization of evobrutinib, a potent, obligate covalent inhibitor with high kinase selectivity. Evobrutinib displayed sufficient preclinical pharmacokinetic and pharmacodynamic characteristics which allowed for in vivo evaluation in efficacy models. Moreover, the high selectivity of evobrutinib for BTK over epidermal growth factor receptor and other Tec family kinases suggested a low potential for off-target related adverse effects. Clinical investigation of evobrutinib is ongoing in several autoimmune diseases, including multiple sclerosis, rheumatoid arthritis, and systemic lupus erythematosus.

Overcoming undesirable HERG potency of chemokine receptor antagonists using baseline lipophilicity relationships.

The inhibition of the hERG channel by noncardiovascular drugs is a side effect that severely impedes the development of new medications. To increase hERG selectivity of preclinical compounds, we recommend the study of nondesolvation related interactions with the intended target and hERG using a baseline lipophilicity relationship approach. While this approach is conventionally used in studies of potency, we demonstrate here that it can help in selectivity issues. Studies of hERG selectivity in four in-house classes of chemokine receptor (CCR) antagonists suggest that the selectivity is improved most effectively by structural alterations that increase the lipophilicity-adjusted primary potency, pIC 50 (CCR) - Log D. Fragment-based QSAR analysis is performed using the lipophilicity-adjusted hERG potency, pIC 50 (hERG) - Log D, to identify moieties that form nonhydrophobic interactions with the hERG channel. These moieties, which erode hERG selectivity, can then be avoided. A novel two-dimensional fragment-based QSAR analysis helps visualizing the lipophilicity-adjusted hERG and CCR potencies within chemical series.

2,8-Disubstituted-1,6-Naphthyridines and 4,6-Disubstituted-Isoquinolines with Potent, Selective Affinity for CDK8/19.

We demonstrate a designed scaffold-hop approach to the discovery of 2,8-disubstituted-1,6-naphthyridine- and 4,6-disubstituted-isoquinoline-based dual CDK8/19 ligands. Optimized compounds in both series exhibited rapid aldehyde oxidase-mediated metabolism, which could be abrogated by introduction of an amino substituent at C5 of the 1,6-naphthyridine scaffold or at C1 of the isoquinoline scaffold. Compounds 51 and 59 were progressed to in vivo pharmacokinetic studies, and 51 also demonstrated sustained inhibition of STAT1(SER727) phosphorylation, a biomarker of CDK8 inhibition, in an SW620 colorectal carcinoma human tumor xenograft model following oral dosing.

Structure-Based Optimization of Potent, Selective, and Orally Bioavailable CDK8 Inhibitors Discovered by High-Throughput Screening.

The mediator complex-associated cyclin dependent kinase CDK8 regulates ß-catenin-dependent transcription following activation of WNT signaling. Multiple lines of evidence suggest CDK8 may act as an oncogene in the development of colorectal cancer. Here we describe the successful optimization of an imidazo-thiadiazole series of CDK8 inhibitors that was identified in a high-throughput screening campaign and further progressed by structure-based design. In several optimization cycles, we improved the microsomal stability, potency, and kinase selectivity. The initial imidazo-thiadiazole scaffold was replaced by a 3-methyl-1H-pyrazolo[3,4-b]-pyridine which resulted in compound 25 (MSC2530818) that displayed excellent kinase selectivity, biochemical and cellular potency, microsomal stability, and is orally bioavailable. Furthermore, we demonstrated modulation of phospho-STAT1, a pharmacodynamic biomarker of CDK8 activity, and tumor growth inhibition in an APC mutant SW620 human colorectal carcinoma xenograft model after oral administration. Compound 25 demonstrated suitable potency and selectivity to progress into preclinical in vivo efficacy and safety studies.

Assessing the mechanism and therapeutic potential of modulators of the human Mediator complex-associated protein kinases.

Mediator-associated kinases CDK8/19 are context-dependent drivers or suppressors of tumorigenesis. Their inhibition is predicted to have pleiotropic effects, but it is unclear whether this will impact on the clinical utility of CDK8/19 inhibitors. We discovered two series of potent chemical probes with high selectivity for CDK8/19. Despite pharmacodynamic evidence for robust on-target activity, the compounds exhibited modest, though significant, efficacy against human tumor lines and patient-derived xenografts. Altered gene expression was consistent with CDK8/19 inhibition, including profiles associated with super-enhancers, immune and inflammatory responses and stem cell function. In a mouse model expressing oncogenic beta-catenin, treatment shifted cells within hyperplastic intestinal crypts from a stem cell to a transit amplifying phenotype. In two species, neither probe was tolerated at therapeutically-relevant exposures. The complex nature of the toxicity observed with two structurally-differentiated chemical series is consistent with on-target effects posing significant challenges to the clinical development of CDK8/19 inhibitors.

Increasing selectivity of CC chemokine receptor 8 antagonists by engineering nondesolvation related interactions with the intended and off-target binding sites.

The metabolic stability and selectivity of a series of CCR8 antagonists against binding to the hERG ion channel and cytochrome Cyp2D6 are studied by principal component analysis. It is demonstrated that an efficient way of increasing metabolic stability and selectivity of this series is to decrease compound lipophilicity by engineering nondesolvation related attractive interactions with CCR8, as rationalized by three-dimensional receptor models. Although such polar interactions led to increased compound selectivity, such a strategy could also jeopardize the DMPK profile of compounds. However, once increased potency is found, the lipophilicity can be readjusted by engineering hydrophobic substituents that fit to CCR8 but do not fit to hERG. Several such lipophilic fragments are identified by two-dimensional fragment-based QSAR analysis. Electrophysiological measurements and site-directed mutagenesis studies indicated that the repulsive interactions of these fragments with hERG are caused by steric hindrances with residue F656.

Biphenyls as potent vitronectin receptor antagonists. Part 3: Squaric acid amides.

Vitronectin receptor (alpha(V)beta(3)) antagonists have been implicated as a possible new treatment of restenosis following balloon angioplasty. In this work we investigate a series of novel arginine mimetic scaffolds leading to new insight of the alpha(V)beta(3)/ligand interaction. Squaric acid amide 10 is a subnanomolar alpha(V)beta(3) antagonist with improved potency on human smooth muscle cell migration.

IKCa-channel blockers. Part 2: discovery of cyclohexadienes.

Novel cyclohexadienes have been identified as potent and specific IK(Ca)-channel blockers. In this communication we describe their synthesis as well as their chemical and biological properties. A selected derivative is being enriched in rat brain and reduces the infarct volume, intracranial pressure as well as the water content in a rat subdural hematoma model of traumatic brain injury after iv administration.

Benzimidazoles as non-peptide luteinizing hormone-releasing hormone (LHRH) antagonists. Part 3: Discovery of 1-(1H-benzimidazol-5-yl)-3-tert-butylurea derivatives.

1-(1H-Benzimidazol-5-yl)-3-tert-butylurea derivatives have been identified as a novel class of non-peptide luteinizing hormone-releasing hormone (LHRH) antagonists. Herein, we disclose the synthesis and structure-activity relationships (SAR) of this class resulting in the identification of compound 12c, with dual functional activity on human and rat receptors (rat LHRH: IC50=120 nM; human LHRH: IC50=18 nM). These SAR studies suggest that 1-(1H-benzimidazol-5-yl)-3-tert-butylurea is a new pharmacophore for small molecule LHRH antagonists.

Benzimidazole derivatives as novel nonpeptide luteinizing hormone-releasing hormone (LHRH) antagonists. Part 1: Benzimidazole-5-sulfonamides.

A new class of benzimidazole-5-sulfonamides has been identified as nonpeptide luteinizing hormone-releasing hormone (LHRH) antagonists. Initial structure-activity relationships are presented resulting in compounds 19 and 28 with submicromolar dual functional activity on human and rat receptors.