Discovery of TRPA1 Antagonist GDC-6599: Derisking Preclinical Toxicity and Aldehyde Oxidase Metabolism with a Potential First-in-Class Therapy for Respiratory Disease.

Transient receptor potential ankyrin 1 (TRPA1) is a nonselective calcium ion channel highly expressed in the primary sensory neurons, functioning as a polymodal sensor for exogenous and endogenous stimuli, and has been implicated in neuropathic pain and respiratory disease. Herein, we describe the optimization of potent, selective, and orally bioavailable TRPA1 small molecule antagonists with strong in vivo target engagement in rodent models. Several lead molecules in preclinical single- and short-term repeat-dose toxicity studies exhibited profound prolongation of coagulation parameters. Based on a thorough investigative toxicology and clinical pathology analysis, anticoagulation effects in vivo are hypothesized to be manifested by a metabolite─generated by aldehyde oxidase (AO)─possessing a similar pharmacophore to known anticoagulants (i.e., coumarins, indandiones). Further optimization to block AO-mediated metabolism yielded compounds that ameliorated coagulation effects in vivo, resulting in the discovery and advancement of clinical candidate GDC-6599, currently in Phase II clinical trials for respiratory indications.

Tetrahydrofuran-Based Transient Receptor Potential Ankyrin 1 (TRPA1) Antagonists: Ligand-Based Discovery, Activity in a Rodent Asthma Model, and Mechanism-of-Action via Cryogenic Electron Microscopy.

Transient receptor potential ankyrin 1 (TRPA1) is a nonselective calcium-permeable ion channel highly expressed in the primary sensory neurons functioning as a polymodal sensor for exogenous and endogenous stimuli and has generated widespread interest as a target for inhibition due to its implication in neuropathic pain and respiratory disease. Herein, we describe the optimization of a series of potent, selective, and orally bioavailable TRPA1 small molecule antagonists, leading to the discovery of a novel tetrahydrofuran-based linker. Given the balance of physicochemical properties and strong in vivo target engagement in a rat AITC-induced pain assay, compound 20 was progressed into a guinea pig ovalbumin asthma model where it exhibited significant dose-dependent reduction of inflammatory response. Furthermore, the structure of the TRPA1 channel bound to compound 21 was determined via cryogenic electron microscopy to a resolution of 3 Å, revealing the binding site and mechanism of action for this class of antagonists.

The PAR2 inhibitor I-287 selectively targets Gαq and Gα12/13 signaling and has anti-inflammatory effects.

Protease-activated receptor-2 (PAR2) is involved in inflammatory responses and pain, therefore representing a promising therapeutic target for the treatment of immune-mediated inflammatory diseases. However, as for other GPCRs, PAR2 can activate multiple signaling pathways and those involved in inflammatory responses remain poorly defined. Here, we describe a new selective and potent PAR2 inhibitor (I-287) that shows functional selectivity by acting as a negative allosteric regulator on Gαq and Gα12/13 activity and their downstream effectors, while having no effect on Gi/o signaling and ßarrestin2 engagement. Such selective inhibition of only a subset of the pathways engaged by PAR2 was found to be sufficient to block inflammation in vivo. In addition to unraveling the PAR2 signaling pathways involved in the pro-inflammatory response, our study opens the path toward the development of new functionally selective drugs with reduced liabilities that could arise from blocking all the signaling activities controlled by the receptor.

Design of a brain-penetrant CDK4/6 inhibitor for glioblastoma.

CDK4 and CDK6 are kinases with similar sequences that regulate cell cycle progression and are validated targets in the treatment of cancer. Glioblastoma is characterized by a high frequency of CDKN2A/CCND2/CDK4/CDK6 pathway dysregulation, making dual inhibition of CDK4 and CDK6 an attractive therapeutic approach for this disease. Abemaciclib, ribociclib, and palbociclib are approved CDK4/6 inhibitors for the treatment of HR+/HER2- breast cancer, but these drugs are not expected to show strong activity in brain tumors due to poor blood brain barrier penetration. Herein, we report the identification of a brain-penetrant CDK4/6 inhibitor derived from a literature molecule with low molecular weight and topological polar surface area (MW = 285 and TPSA = 66 Å2), but lacking the CDK2/1 selectivity profile due to the absence of a basic amine. Removal of a hydrogen bond donor via cyclization of the pyrazole allowed for the introduction of basic and semi-basic amines, while maintaining in many cases efflux ratios reasonable for a CNS program. Ultimately, a basic spiroazetidine (cpKa = 8.8) was identified that afforded acceptable selectivity over anti-target CDK1 while maintaining brain-penetration in vivo (mouse Kp,uu = 0.20-0.59). To probe the potency and selectivity, our lead compound was evaluated in a panel of glioblastoma cell lines. Potency comparable to abemaciclib was observed in Rb-wild type lines U87MG, DBTRG-05MG, A172, and T98G, while Rb-deficient cell lines SF539 and M059J exhibited a lack of sensitivity.

Discovery of Potent, Orally Bioavailable Inhibitors of Human Cytomegalovirus.

A high-throughput screen based on a viral replication assay was used to identify inhibitors of the human cytomegalovirus. Using this approach, hit compound 1 was identified as a 4 µM inhibitor of HCMV that was specific and selective over other herpes viruses. Time of addition studies indicated compound 1 exerted its antiviral effect early in the viral life cycle. Mechanism of action studies also revealed that this series inhibited infection of MRC-5 and ARPE19 cells by free virus and via direct cell-to-cell spread from infected to uninfected cells. Preliminary structure-activity relationships demonstrated that the potency of compound 1 could be improved to a low nanomolar level, but metabolic stability was a key optimization parameter for this series. A strategy focused on minimizing metabolic hydrolysis of the N1-amide led to an alternative scaffold in this series with improved metabolic stability and good pharmacokinetic parameters in rat.

Monitoring drug self-aggregation and potential for promiscuity in off-target in vitro pharmacology screens by a practical NMR strategy.

A simple NMR assay was applied to monitor the tendency of compounds to self-aggregate in aqueous media. The observation of unusual spectral trends as a function of compound concentration appears to be signatory of the formation of self-assemblies. (1)H NMR resonances of aggregating compounds were sensitive to the presence of a range of molecular assemblies in solution including large molecular-size entities, smaller multimers, and mixtures of assembled species. The direct observation of aggregates via unusual NMR spectra also correlated with promiscuous behavior of molecules in off-target in vitro pharmacology assays. This empirical assay can have utility for predicting compound promiscuity and should complement predictive methods that principally rely on the computing of descriptors such as lipophilicity (cLogP) and topological surface area (TPSA). This assay should serve as a practical tool for medicinal chemists to monitor compound attributes in aqueous solution and various pharmacologically relevant media, as demonstrated herein.

Identification of potent and orally bioavailable nucleotide competing reverse transcriptase inhibitors: in vitro and in vivo optimization of a series of benzofurano[3,2-d]pyrimidin-2-one derived inhibitors.

Recently, a new class of HIV reverse transcriptase (HIV-RT) inhibitors has been reported. The novel mechanism of inhibition by this class involves competitive binding to the active site of the RT enzyme and has been termed Nucleotide-Competing Reverse Transcriptase Inhibitors (NcRTIs). In this publication we describe the optimization of a novel benzofurano[3,2-d]pyrimidin-2-one series of NcRTIs. The starting point for the current study was inhibitor 2, which had high biochemical and antiviral potency but only moderate permeability in a Caco-2 assay and high B-to-A efflux, resulting in moderate rat bioavailability and low Cmax. We present herein the results and strategies we employed to optimize both the potency as well as the permeability, metabolic stability and pharmacokinetic profile of this series. One of the key observations of the present study was the importance of shielding polar functionality, at least in the context of the current chemotype, to enhance permeability. These studies led to the identification of inhibitors 39 and 45, which display sub-nanomolar antiviral potency in a p24 ELISA assay with significantly reduced efflux ratios (ratios <1.5). These inhibitors also display excellent rat pharmacokinetic profiles with high bioavailabilities and low clearance.

Nucleotide competing reverse transcriptase inhibitors: discovery of a series of non-basic benzofurano[3,2-d]pyrimidin-2-one derived inhibitors.

A HTS screen led to the identification of a benzofurano[3,2-d]pyrimidin-2-one core structure which upon further optimization resulted in 1 as a potent HIV-1 nucleotide competing reverse transcriptase inhibitor (NcRTI). Investigation of the SAR at N-1 allowed significant improvements in potency and when combined with the incorporation of heterocycles at C-8 resulted in potent analogues not requiring a basic amine to achieve antiviral activity. Additional modifications at N-1 resulted in 33 which demonstrated excellent antiviral potency and improved physicochemical properties.

Identification of benzofurano[3,2-d]pyrimidin-2-ones, a new series of HIV-1 nucleotide-competing reverse transcriptase inhibitors.

Screening of our sample collection led to the identification of a set of benzofurano[3,2-d]pyrimidine-2-one hits acting as nucleotide-competing HIV-1 reverse transcriptase inhibitiors (NcRTI). Significant improvement in antiviral potency was achieved when substituents were introduced at positions N1, C4, C7 and C8 on the benzofuranopyrimidone scaffold. The series was optimized from low micromolar enzymatic activity against HIV-1 RT and no antiviral activity to low nanomolar antiviral potency. Further profiling of inhibitor 30 showed promising overall in vitro properties and also demonstrated that its potency was maintained against viruses resistant to the other major classes of HIV-1 RT inhibitors.

Naphthalene/quinoline amides and sulfonylureas as potent and selective antagonists of the EP4 receptor.

Two new series of EP(4) antagonists based on naphthalene/quinoline scaffolds have been identified as part of our on-going efforts to develop treatments for inflammatory pain. One series contains an acidic sulfonylurea pharmacophore, whereas the other is a neutral amide. Both series show subnanomolar intrinsic binding potency towards the EP(4) receptor, and excellent selectivity towards other prostanoid receptors. While the amide series generally displays poor pharmacokinetic parameters, the sulfonylureas exhibit greatly improved profile. MF-592, the optimal compound from the sulfonylurea series, has a desirable overall preclinical profile that suggests it is suitable for further development.

Potent and selective 5-LO inhibitor bearing benzothiophene pharmacophore: discovery of MK-5286.

The strategy and SAR studies that led to the discovery of a novel potent and orally available 5-lipoxygenase (5-LO) inhibitor 3-(4-fluorophenyl)-6-({4-[(1S)-1-hydroxy-1-(trifluoromethyl)propyl]-1H-1,2,3-triazol-1-yl}methyl)-1-benzothiophene-2-carboxamide ((S)-2l or MK-5286) were described.

Comparison of MALDI to ESI on a triple quadrupole platform for pharmacokinetic analyses.

This present work describes the systematic experimental comparison of electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI) for pharmacokinetic (PK) analysis of two drug candidates from rat plasma using single reaction monitoring (SRM) on a triple quadrupole mass spectrometer. The electrospray assay is an established method using a fast liquid chromatography (LC) separation of the sample extracts prior to mass spectrometry analysis. The novel MALDI assays measured the concentration levels of the drug candidates directly from the spotted sample extracts. Importantly, for both LC-ESI and MALDI the same solid-phase sample extraction protocol, internal standards, triple quadrupole mass analyzer platform, and SRM conditions were used, thus effectively standardizing all experimental parameters of the two assays. Initially, analytical figures of merit such as linearity, limit of quantitation, precision, and accuracy were determined from the calibration curves, indicating very similar performance for both LC-ESI and MALDI. Moreover, the LC-ESI rat plasma concentration time profiles of the drug candidates after orally dosing the animals were accurately reproduced by the MALDI assay, giving virtually identical PK results. The direct MALDI assay, however, was able to generate the data at least 50 x faster than the LC-ESI assay. It is shown in this study that analyzing the entire PK curve for one animal took less than 2 min using MALDI (with five replicate analyses per sample), whereas the corresponding LC-ESI assay required 80 min, however, allowing only two replicate measurements in that time frame.

Metabolic activation of indole-containing prostaglandin D2 receptor 1 antagonists: impacts of glutathione trapping and glucuronide conjugation on covalent binding.

Some DP1 receptor antagonists from an indole-containing series were shown to cause in vitro covalent binding to protein in rat and human liver microsomes. Glutathione trapping experiments along with in vitro labeling assays confirmed that the presence of a strong electron withdrawing group was necessary to abrogate in vitro covalent binding, leading to the discovery of MK-0524. Hepatocyte incubations and in vivo studies showed that acyl-glucuronide formation did not translate into covalent binding.

Discovery of a potent and selective prostaglandin D2 receptor antagonist, [(3R)-4-(4-chloro-benzyl)-7-fluoro-5-(methylsulfonyl)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl]-acetic acid (MK-0524).

The discovery of the potent and selective prostaglandin D2 (PGD2) receptor (DP) antagonist [(3R)-4-(4-chlorobenzyl)-7-fluoro-5-(methylsulfonyl)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl]-acetic acid (13) is presented. Initial lead antagonists 6 and 7 were found to be potent and selective DP antagonists (DP Ki = 2.0 nM for each); however, they both suffered from poor pharmacokinetic profiles, short half-lives and high clearance rates in rats. Rat bile duct cannulation studies revealed that high concentrations of parent drug were present in the biliary fluid (Cmax = 1100 microM for 6 and 3900 microM for 7). This pharmacokinetic liability was circumvented by replacing the 7-methylsulfone substituent present in 6 and 7 with a fluorine atom resulting in antagonists with diminished propensity for biliary excretion and with superior pharmacokinetic profiles. Further optimization led to the discovery of the potent and selective DP antagonist 13.

In vitro biotransformations of the prostaglandin D2 (DP) antagonist MK-0524 and synthesis of metabolites.

Metabolites of the potent DP antagonist, MK-0524, were generated using in vitro systems including hepatic microsomes and hepatocytes. Four metabolites (two hydroxylated diastereomers, a ketone and an acyl glucuronide) were characterized by LC-MS/MS and 1H NMR. Larger quantities of these metabolites were prepared by either organic synthesis or biosynthetically to be used as standards in other studies. The propensity for covalent binding was assessed and was found to be acceptable (<50 pmol-equiv/mg protein).

Antagonism of the prostaglandin D2 receptor 1 suppresses nicotinic acid-induced vasodilation in mice and humans.

Nicotinic acid (NA) is commonly used to treat dyslipidemia, but it elicits an adverse effect, termed flushing, which consists of cutaneous vasodilation with associated discomfort. An animal model of NA-induced flushing has been established in mice. As in humans, NA stimulated vasodilation in a dose-dependent manner, was associated with an increase of the vasodilatory prostaglandin (PG) D2 in plasma and could be blocked by pretreatment with aspirin. Two PGD2 receptors have been identified: PGD2 receptor 1 (DP1, also called DP) and PGD2 receptor 2 (DP2, sometimes termed CRTH2). DP2 does not mediate NA-induced vasodilation; the DP2-specific agonist DK-PGD2 (13,14-dihydro-15-keto-PGD2) did not induce cutaneous vasodilation, and DP2-/- mice had a normal vasodilatory response to NA. By contrast, BW245C, a DP1-selective agonist, induced vasodilation in mice, and MK-0524, a DP1-selective antagonist, blocked both PGD2- and NA-induced vasodilation. NA-induced vasodilation was also studied in DP1+/+, DP1+/-, and DP1-/- mice; although NA-induced vasodilation depended almost completely on DP1 in female mice, it depended only partially on DP1 in male mice. The residual NA-induced vasodilation in male DP-/- mice was aspirin-sensitive. Thus, in the mouse, DP1 appears to be an important component involved in NA-induced vasodilation, but other cyclooxygenase-dependent mechanisms also may be involved. A clinical study in healthy men and women demonstrated that treatment with MK-0524 reduced the symptoms of flushing and the increase in skin perfusion after the administration of NA. These studies suggest that DP1 receptor antagonism may be an effective means to suppress NA-induced flushing in humans.

Identification of an indole series of prostaglandin D2 receptor antagonists.

A novel indole series of PGD2 receptor (DP receptor) antagonists is presented. Optimization of this series led to the identification of potent and selective DP receptor antagonists. In particular, antagonists 35 and 36 were identified with Ki values of 2.6 and 1.8 nM, respectively. These two antagonists are also potent in a DP functional assay where they inhibit the PGD2 induced cAMP production in platelet rich plasma with IC50 values of 7.9 and 8.6 nM, respectively. The structure-activity relationships of this indole series of DP receptor antagonists will also be discussed.