Discovery of MK-1468: A Potent, Kinome-Selective, Brain-Penetrant Amidoisoquinoline LRRK2 Inhibitor for the Potential Treatment of Parkinson's Disease.

Genetic mutation of the leucine-rich repeat kinase 2 (LRRK2) protein has been associated with Parkinson's disease (PD), a disabling and progressive neurodegenerative disorder that is devoid of efficacious disease-modifying therapies. Herein, we describe the invention of an amidoisoquinoline (IQ)-derived LRRK2 inhibitor lead chemical series. Knowledge-, structure-, and property-based drug design in concert with rigorous application of in silico calculations and presynthesis predictions enabled the prioritization of molecules with favorable CNS "drug-like" physicochemical properties. This resulted in the discovery of compound 8, which was profiled extensively before human ether-a-go-go (hERG) ion channel inhibition halted its progression. Strategic reduction of lipophilicity and basicity resulted in attenuation of hERG ion channel inhibition while maintaining a favorable CNS efflux transporter profile. Further structure- and property-based optimizations resulted in the discovery of preclinical candidate MK-1468. This exquisitely selective LRRK2 inhibitor has a projected human dose of 48 mg BID and a preclinical safety profile that supported advancement toward GLP toxicology studies.

Discovery and Optimization of Potent, Selective, and Brain-Penetrant 1-Heteroaryl-1H-Indazole LRRK2 Kinase Inhibitors for the Treatment of Parkinson's Disease.

Inhibition of leucine-rich repeat kinase 2 (LRRK2) kinase activity represents a genetically supported, chemically tractable, and potentially disease-modifying mechanism to treat Parkinson's disease. Herein, we describe the optimization of a novel series of potent, selective, central nervous system (CNS)-penetrant 1-heteroaryl-1H-indazole type I (ATP competitive) LRRK2 inhibitors. Type I ATP-competitive kinase physicochemical properties were integrated with CNS drug-like properties through a combination of structure-based drug design and parallel medicinal chemistry enabled by sp3-sp2 cross-coupling technologies. This resulted in the discovery of a unique sp3-rich spirocarbonitrile motif that imparted extraordinary potency, pharmacokinetics, and favorable CNS drug-like properties. The lead compound, 25, demonstrated exceptional on-target potency in human peripheral blood mononuclear cells, excellent off-target kinase selectivity, and good brain exposure in rat, culminating in a low projected human dose and a pre-clinical safety profile that warranted advancement toward pre-clinical candidate enabling studies.

Oxetane Promise Delivered: Discovery of Long-Acting IDO1 Inhibitors Suitable for Q3W Oral or Parenteral Dosing.

3,3-Disubstituted oxetanes have been utilized as bioisosteres for gem-dimethyl and cyclobutane functionalities. We report the discovery of a novel class of oxetane indole-amine 2,3-dioxygenase (IDO1) inhibitors suitable for Q3W (once every 3 weeks) oral and parenteral dosing. A diamide class of IDO inhibitors was discovered through an automated ligand identification system (ALIS). Installation of an oxetane and fluorophenyl dramatically improved the potency. Identification of a biaryl moiety as an unconventional amide isostere addressed the metabolic liability of amide hydrolysis. Metabolism identification (Met-ID)-guided target design and the introduction of polarity resulted in the discovery of potent IDO inhibitors with excellent pharmacokinetic (PK) profiles in multiple species. To enable rapid synthesis of the key oxetane intermediate, a novel oxetane ring cyclization was also developed, as well as optimization of a literature route on kg scale. These IDO inhibitors may enable unambiguous proof-of-concept testing for the IDO1 inhibition mechanism for oncology.

Structure-Guided Discovery of Aminoquinazolines as Brain-Penetrant and Selective LRRK2 Inhibitors.

The leucine-rich repeat kinase 2 (LRRK2) protein has been genetically and functionally linked to Parkinson's disease (PD), a disabling and progressive neurodegenerative disorder whose current therapies are limited in scope and efficacy. In this report, we describe a rigorous hit-to-lead optimization campaign supported by structural enablement, which culminated in the discovery of brain-penetrant, candidate-quality molecules as represented by compounds 22 and 24. These compounds exhibit remarkable selectivity against the kinome and offer good oral bioavailability and low projected human doses. Furthermore, they showcase the implementation of stereochemical design elements that serve to enable a potency- and selectivity-enhancing increase in polarity and hydrogen bond donor (HBD) count while maintaining a central nervous system-friendly profile typified by low levels of transporter-mediated efflux and encouraging brain penetration in preclinical models.

Carbamate and N-Pyrimidine Mitigate Amide Hydrolysis: Structure-Based Drug Design of Tetrahydroquinoline IDO1 Inhibitors.

Indoleamine-2,3-dioxygenase-1 (IDO1) has emerged as an attractive target for cancer immunotherapy. An automated ligand identification system screen afforded the tetrahydroquinoline class of novel IDO1 inhibitors. Potency and pharmacokinetic (PK) were key issues with this class of compounds. Structure-based drug design and strategic incorporation of polarity enabled the rapid improvement on potency, solubility, and oxidative metabolic stability. Metabolite identification studies revealed that amide hydrolysis in the D-pocket was the key clearance mechanism for this class. Strategic survey of amide isosteres revealed that carbamates and N-pyrimidines, which maintained exquisite potencies, mitigated the amide hydrolysis issue and led to an improved rat PK profile. The lead compound 28 is a potent IDO1 inhibitor, with clean off-target profiles and the potential for quaque die dosing in humans.

Characterization of the Onset, Progression, and Reversibility of Morphological Changes in Mouse Lung after Pharmacological Inhibition of Leucine-Rich Kinase 2 Kinase Activity.

Gain-of-function mutations in leucine-rich kinase 2 (LRRK2) are associated with increased incidence of Parkinson disease (PD); thus, pharmacological inhibition of LRRK2 kinase activity is postulated as a disease-modifying treatment of PD. Histomorphological changes in lungs of nonhuman primates (NHPs) treated with small-molecule LRRK2 kinase inhibitors have brought the safety of this treatment approach into question. Although it remains unclear how LRRK2 kinase inhibition affects the lung, continued studies in NHPs prove to be both cost- and resource-prohibitive. To develop a tractable alternative animal model platform, we dosed male mice in-diet with the potent, highly selective LRRK2 kinase inhibitor MLi-2 and induced histomorphological changes in lung within 1 week. Oral bolus dosing of MLi-2 at a frequency modeled to provide steady-state exposure equivalent to that achieved with in-diet dosing induced type II pneumocyte vacuolation, suggesting pulmonary changes require sustained LRRK2 kinase inhibition. Treating mice with MLi-2 in-diet for up to 6 months resulted in type II pneumocyte vacuolation that progressed only modestly over time and was fully reversible after withdrawal of MLi-2. Immunohistochemical analysis of lung revealed a significant increase in prosurfactant protein C staining within type II pneumocytes. In the present study, we demonstrated the kinetics for onset, progression, and rapid reversibility of chronic LRRK2 kinase inhibitor effects on lung histomorphology in rodents and provide further evidence for the derisking of safety and tolerability concerns for chronic LRRK2 kinase inhibition in PD. SIGNIFICANCE STATEMENT: We have defined a mouse model by which the on-target lung effects of leucine-rich kinase 2 (LRRK2) kinase inhibition can be monitored, whereas previous in vivo testing relied solely on nonhuman primates. Data serve to derisk long-term treatment with LRRK2 kinase inhibitors, as all lung changes were mild and readily reversible.

Optimization of Versatile Oxindoles as Selective PI3Kδ Inhibitors.

The 3,3-disubstituted oxindole moiety is a versatile and rigid three-dimensionally shaped scaffold. When engineered with a purine hinge-binding core, exceptionally selective PI3Kδ kinase inhibitors were discovered by exploiting small differences in isoform selectivity pockets. Crystal structures of early lead 2f bound to PI3Kδ and PI3Kα helped rationalize the high selectivity observed with 2f. By attenuating the lypophilicity and metabolic liabilities of an oxindole moiety, we improved the preclinical species PK and solubility and reduced adenosine uptake activity. The excellent potency and kinome selectivity of 7-azaoxindole 4d and spirooxindole 5d, together with a low plasma clearance and good half-life in rat and dog, supported a low once-daily predicted human dose.

Purine derivatives as potent gamma-secretase modulators.

The development of a novel series of purines as gamma-secretase modulators for potential use in the treatment of Alzheimer's disease is disclosed herein. Optimization of a previously disclosed pyrimidine series afforded a series of potent purine-based gamma-secretase modulators with 300- to 2000-fold in vitro selectivity over inhibition of Notch cleavage and that selectively reduces Alphabeta42 in an APP-YAC transgenic mouse model.

Phenylglycine and phenylalanine derivatives as potent and selective HDAC1 inhibitors (SHI-1).

An HTS screening campaign identified a series of low molecular weight phenols that showed excellent selectivity (>100-fold) for HDAC1/HDAC2 over other Class I and Class II HDACs. Evolution and optimization of this HTS hit series provided HDAC1-selective (SHI-1) compounds with excellent anti-proliferative activity and improved physical properties. Dose-dependent efficacy in a mouse HCT116 xenograft model was demonstrated with a phenylglycine SHI-1 analog.

Discovery and mechanistic study of Al(III)-catalyzed transamidation of tertiary amides.

Cleavage of the C-N bond of carboxamides generally requires harsh conditions. This study reveals that tris(amido)Al(III) catalysts, such as Al2(NMe2)6, promote facile equilibrium-controlled transamidation of tertiary carboxamides with secondary amines. The mechanism of these reactions was investigated by kinetic, spectroscopic, and density functional theory (DFT) computational methods. The catalyst resting state consists of an equilibrium mixture of a tris(amido)Al(III) dimer and a monomeric tris(amido)Al(III)-carboxamide adduct, and the turnover-limiting step involves intramolecular nucleophilic attack of an amido ligand on the coordinated carboxamide or subsequent rearrangement (intramolecular ligand substitution) of the tetrahedral intermediate. Fundamental mechanistic differences between these tertiary transamidation reactions and previously characterized transamidations involving secondary amides and primary amines suggest that tertiary amide/secondary amine systems are particularly promising for future development of metal-catalyzed amide metathesis reactions that proceed via transamidation.

Mechanism of Al(III)-catalyzed transamidation of unactivated secondary carboxamides.

The carbon-nitrogen bond of secondary carboxamides is generally thermodynamically and kinetically unreactive; however, we recently discovered that the trisamidoaluminum(III) dimer Al2(NMe2)6 catalyzes facile transamidation between simple secondary carboxamides and primary amines under moderate conditions. The present report describes kinetic and spectroscopic studies that illuminate the mechanism of this unusual transformation. The catalytic reaction exhibits a bimolecular rate law with a first-order dependence on the Al(III) and amine concentrations. No rate dependence on the carboxamide concentration is observed. Spectroscopic studies (1H and 13C NMR, FTIR) support a catalyst resting state that consists of a mixture of tris-(kappa2-amidate)aluminum(III) complexes. These results, together with the presence of a significant kinetic isotope effect when deuterated amine substrate (RND2) is used, implicate a mechanism in which the amine undergoes preequilibrium coordination to aluminum and proton transfer to a kappa2-amidate ligand to yield an Al(kappa2-amidate)2(kappa1-carboxamide)(NHR) complex, followed by rate-limiting intramolecular delivery of the amido ligand (NHR) to the neutral Al(III)-activated kappa1-carboxamide. Noteworthy in this mechanism is the bifunctional character of Al(III), which is capable of activating both the amine nucleophile and the carboxamide electrophile in the reaction.

Effects of side chain configuration and backbone spacing on the gene delivery properties of lysine-derived cationic polymers.

A series of lysine-based oligomers (18 residues) that differ in side chain configuration or side chain spacing along the backbone was tested for DNA transfection activity. Although materials constructed from lysine are not the most effective polymeric transfection agents, we have chosen L-lysine-based molecules as a starting point because this system allows us to examine the functional effects of incremental changes in polycation structure. The oligomer constructed from beta(3)-homolysine (beta(3)-hLys) and that from alpha-D-lysine were superior to an alpha-L-lysine 18-mer in gene delivery assays. This improved activity is attributed to the fact that the alpha-L-peptide is a protease substrate while the other 18-mers are not. This conclusion is supported by the effects of chloroquine on transfection activity, based on the protease inhibition activity of chloroquine. To our knowledge, these results represent the first direct comparison of a D-lysine oligomer with an L-lysine oligomer in the context of gene delivery. Poly(beta(3)-hLys) was synthesized from the ring opening polymerization of the corresponding lactam. The DNA transfection ability of this polymer was compared with that of commercially available poly(L-lysine) (PLL). In each case the polymer was more active than the corresponding oligomer.