Identification and Strategies to Mitigate High Total Clearance of Benzylamine-Substituted Biphenyl Ring Systems.

For most oral small-molecule projects within drug discovery, the extent and duration of the effect are influenced by the total clearance of the compound; hence, designing compounds with low clearance remains a key focus to help enable sufficient protein target engagement. Comprehensive understanding and accurate prediction of animal clearance and pharmacokinetics provides confidence that the same can be observed for human. During a MERTK inhibitor lead optimization project, a series containing a biphenyl ring system with benzylamine meta-substitution on one phenyl and nitrogen inclusion as the meta atom on the other ring demonstrated multiple routes of compound elimination in rats. Here, we describe the identification of a structural pharmacophore involving two key interactions observed for both the MERTK program and an additional internal project. Four strategies to mitigate these clearance liabilities were identified and systematically investigated. We provide evidence that disruption of at least one of the interactions led to a significant reduction in CL that was subsequently predicted from rat hepatocytes using in vitro/in vivo extrapolation and the well-stirred scaling method. These tactics will likely be of general utility to the medicinal chemistry and DMPK community during compound optimization when similar issues are encountered for biphenyl benzylamines.

Transition-Metal-Mediated Chemo- and Stereoselective Total Synthesis of (-)-Galanthamine.

An asymmetric synthetic route to (-)-galanthamine (1), a pharmacologically active Amaryllidaceae alkaloid used for the symptomatic treatment of early onset Alzheimer's disease, was successfully established with very high levels of stereocontrol. The key to achieving high chemo- and stereo-selectivity in this approach was the use of transition-metal-mediated reactions, namely, enyne ring-closing metathesis, Heck coupling, and titanium-based asymmetric allylation.

Optimization of an Imidazo[1,2-a]pyridine Series to Afford Highly Selective Type I1/2 Dual Mer/Axl Kinase Inhibitors with In Vivo Efficacy.

Inhibition of Mer and Axl kinases has been implicated as a potential way to improve the efficacy of current immuno-oncology therapeutics by restoring the innate immune response in the tumor microenvironment. Highly selective dual Mer/Axl kinase inhibitors are required to validate this hypothesis. Starting from hits from a DNA-encoded library screen, we optimized an imidazo[1,2-a]pyridine series using structure-based compound design to improve potency and reduce lipophilicity, resulting in a highly selective in vivo probe compound 32. We demonstrated dose-dependent in vivo efficacy and target engagement in Mer- and Axl-dependent efficacy models using two structurally differentiated and selective dual Mer/Axl inhibitors. Additionally, in vivo efficacy was observed in a preclinical MC38 immuno-oncology model in combination with anti-PD1 antibodies and ionizing radiation.

GDC-9545 (Giredestrant): A Potent and Orally Bioavailable Selective Estrogen Receptor Antagonist and Degrader with an Exceptional Preclinical Profile for ER+ Breast Cancer.

Breast cancer remains a leading cause of cancer death in women, representing a significant unmet medical need. Here, we disclose our discovery efforts culminating in a clinical candidate, 35 (GDC-9545 or giredestrant). 35 is an efficient and potent selective estrogen receptor degrader (SERD) and a full antagonist, which translates into better antiproliferation activity than known SERDs (1, 6, 7, and 9) across multiple cell lines. Fine-tuning the physiochemical properties enabled once daily oral dosing of 35 in preclinical species and humans. 35 exhibits low drug-drug interaction liability and demonstrates excellent in vitro and in vivo safety profiles. At low doses, 35 induces tumor regressions either as a single agent or in combination with a CDK4/6 inhibitor in an ESR1Y537S mutant PDX or a wild-type ERα tumor model. Currently, 35 is being evaluated in Phase III clinical trials.

Discovery of GNE-149 as a Full Antagonist and Efficient Degrader of Estrogen Receptor alpha for ER+ Breast Cancer.

Estrogen receptor alpha (ERα) is a well-validated drug target for ER-positive (ER+) breast cancer. Fulvestrant is FDA-approved to treat ER+ breast cancer and works through two mechanisms-as a full antagonist and selective estrogen receptor degrader (SERD)-but lacks oral bioavailability. Thus, we envisioned a "best-in-class" molecule with the same dual mechanisms as fulvestrant, but with significant oral exposure. Through lead optimization, we discovered a tool molecule 12 (GNE-149) with improved degradation and antiproliferative activity in both MCF7 and T47D cells. To illustrate the binding mode and key interactions of this scaffold with ERα, we obtained a cocrystal structure of 6 that showed ionic interaction of azetidine with Asp351 residue. Importantly, 12 showed favorable metabolic stability and good oral exposure. 12 exhibited antagonist effect in the uterus and demonstrated robust dose-dependent efficacy in xenograft models.

Discovery of a class of highly potent Janus Kinase 1/2 (JAK1/2) inhibitors demonstrating effective cell-based blockade of IL-13 signaling.

Disruption of interleukin-13 (IL-13) signaling with large molecule antibody therapies has shown promise in diseases of allergic inflammation. Given that IL-13 recruits several members of the Janus Kinase family (JAK1, JAK2, and TYK2) to its receptor complex, JAK inhibition may offer an alternate small molecule approach to disrupting IL-13 signaling. Herein we demonstrate that JAK1 is likely the isoform most important to IL-13 signaling. Structure-based design was then used to improve the JAK1 potency of a series of previously reported JAK2 inhibitors. The ability to impede IL-13 signaling was thereby significantly improved, with the best compounds exhibiting single digit nM IC50's in cell-based assays dependent upon IL-13 signaling. Appropriate substitution was further found to influence inhibition of a key off-target, LRRK2. Finally, the most potent compounds were found to be metabolically labile, which makes them ideal scaffolds for further development as topical agents for IL-13 mediated diseases of the lungs and skin (for example asthma and atopic dermatitis, respectively).

The Rational Design of Selective Benzoxazepin Inhibitors of the α-Isoform of Phosphoinositide 3-Kinase Culminating in the Identification of (S)-2-((2-(1-Isopropyl-1H-1,2,4-triazol-5-yl)-5,6-dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepin-9-yl)oxy)propanamide (GDC-0326).

Inhibitors of the class I phosphoinositide 3-kinase (PI3K) isoform PI3Kα have received substantial attention for their potential use in cancer therapy. Despite the particular attraction of targeting PI3Kα, achieving selectivity for the inhibition of this isoform has proved challenging. Herein we report the discovery of inhibitors of PI3Kα that have selectivity over the other class I isoforms and all other kinases tested. In GDC-0032 (3, taselisib), we previously minimized inhibition of PI3Kß relative to the other class I insoforms. Subsequently, we extended our efforts to identify PI3Kα-specific inhibitors using PI3Kα crystal structures to inform the design of benzoxazepin inhibitors with selectivity for PI3Kα through interactions with a nonconserved residue. Several molecules selective for PI3Kα relative to the other class I isoforms, as well as other kinases, were identified. Optimization of properties related to drug metabolism then culminated in the identification of the clinical candidate GDC-0326 (4).

Discovery of thiazolobenzoxepin PI3-kinase inhibitors that spare the PI3-kinase ß isoform.

A series of suitable five-membered heterocyclic alternatives to thiophenes within a thienobenzoxepin class of PI3-kinase (PI3K) inhibitors was discovered. Specific thiazolobenzoxepin 8-substitution was identified that increased selectivity over PI3Kß. PI3Kß-sparing compound 27 (PI3Kß Ki,app/PI3Kα Ki,app=57) demonstrated dose-dependent knockdown of pAKT, pPRAS40 and pS6RP in vivo as well as differential effects in an in vitro proliferation cell line screen compared to pan PI3K inhibitor GDC-0941. A new structure-based hypothesis for reducing inhibition of the PI3K ß isoform while maintaining activity against α, δ and γ isoforms is presented.

A large geometric distortion in the first photointermediate of rhodopsin, determined by double-quantum solid-state NMR.

Double-quantum magic-angle-spinning NMR experiments were performed on 11,12-(13)C(2)-retinylidene-rhodopsin under illumination at low temperature, in order to characterize torsional angle changes at the C11-C12 photoisomerization site. The sample was illuminated in the NMR rotor at low temperature (~120 K) in order to trap the primary photointermediate, bathorhodopsin. The NMR data are consistent with a strong torsional twist of the HCCH moiety at the isomerization site. Although the HCCH torsional twist was determined to be at least 40°, it was not possible to quantify it more closely. The presence of a strong twist is in agreement with previous Raman observations. The energetic implications of this geometric distortion are discussed.

Light penetration and photoisomerization in rhodopsin studied by numerical simulations and double-quantum solid-state NMR spectroscopy.

The penetration of light into optically thick samples containing the G-protein-coupled receptor rhodopsin is studied by numerical finite-element simulations and double-quantum solid-state NMR experiments. Illumination with white light leads to the generation of the active bathorhodopsin photostate in the outer layer of the sample but generates a large amount of the side product, isorhodopsin, in the sample interior. The overall yield of bathorhodopsin is improved by using monochromatic 420 nm illumination and by mixing the sample with transparent glass beads. The implications of these findings on the interpretation of previously published rhodopsin NMR data are discussed.

Towards an interpretation of 13C chemical shifts in bathorhodopsin, a functional intermediate of a G-protein coupled receptor.

Photoisomerization of the membrane-bound light receptor protein rhodopsin leads to an energy-rich photostate called bathorhodopsin, which may be trapped at temperatures of 120 K or lower. We recently studied bathorhodopsin by low-temperature solid-state NMR, using in situ illumination of the sample in a purpose-built NMR probe. In this way we acquired (13)C chemical shifts along the retinylidene chain of the chromophore. Here we compare these results with the chemical shifts of the dark state chromophore in rhodopsin, as well as with the chemical shifts of retinylidene model compounds in solution. An earlier solid-state NMR study of bathorhodopsin found only small changes in the (13)C chemical shifts upon isomerization, suggesting only minor perturbations of the electronic structure in the isomerized retinylidene chain. This is at variance with our recent measurements which show much larger perturbations of the (13)C chemical shifts. Here we present a tentative interpretation of our NMR results involving an increased charge delocalization inside the polyene chain of the bathorhodopsin chromophore. Our results suggest that the bathochromic shift of bathorhodopsin is due to modified electrostatic interactions between the chromophore and the binding pocket, whereas both electrostatic interactions and torsional strain are involved in the energy storage mechanism of bathorhodopsin.

Double-quantum 13C nuclear magnetic resonance of bathorhodopsin, the first photointermediate in mammalian vision.

The 13C chemical shifts of the primary visual photointermediate bathorhodopsin have been observed by performing double-quantum magic-angle-spinning NMR at low temperature in the presence of illumination. Strong isomerization shifts have been observed upon the conversion of rhodopsin into bathorhodopsin.

Stereocontrolled synthesis of (-)-galanthamine.

An enantioselective synthesis of (-)-galanthamine has been realized in 11 linear steps starting from isovanillin. A Mitsunobu aryl ether forming reaction was used to assemble the galanthamine backbone, which was stitched together using enyne ring-closing metathesis, Heck, and N-alkylation reactions affording the tetracyclic ring system. Control of relative and absolute stereochemistry was derived from an easily accessible enantiomerically enriched propargylic alcohol 13.

Analytical theory of gamma-encoded double-quantum recoupling sequences in solid-state nuclear magnetic resonance.

Many important double-quantum recoupling techniques in solid-state NMR are classified as being gamma-encoded. This means that the phase of the double-quantum effective Hamiltonian, but not its amplitude, depends on the third Euler angle defining the orientation of the molecular spin system in the frame of the magic-angle-spinning rotor. In this paper, we provide closed analytical solutions for the dependence of the powder-average double-quantum-filtered signal on the recoupling times, within the average Hamiltonian approximation for gamma-encoded pulse sequences. The validity of the analytical solutions is tested by numerical simulations. The internuclear distance in a (13)C(2)-labelled retinal is estimated by fitting the analytical curves to experimental double-quantum data.

Accurate measurements of 13C-13C J-couplings in the rhodopsin chromophore by double-quantum solid-state NMR spectroscopy.

A new double-quantum solid-state NMR pulse sequence is presented and used to measure one-bond 13C-13C J-couplings in a set of 13C2-labeled rhodopsin isotopomers. The measured J-couplings reveal a perturbation of the electronic structure at the terminus of the conjugated chain but show no evidence for protein-induced electronic perturbation near the C11-C12 isomerization site. This work establishes NMR methodology for measuring accurate 1JCC values in noncrystalline macromolecules and shows that the measured J-couplings may reveal local electronic perturbations of mechanistic significance.