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.

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.

Optimization of brain-penetrant picolinamide derived leucine-rich repeat kinase 2 (LRRK2) inhibitors.

The discovery of potent, kinome selective, brain penetrant LRRK2 inhibitors is the focus of extensive research seeking new, disease-modifying treatments for Parkinson's disease (PD). Herein, we describe the discovery and evolution of a picolinamide-derived lead series. Our initial optimization efforts aimed at improving the potency and CLK2 off-target selectivity of compound 1 by modifying the heteroaryl C-H hinge and linker regions. This resulted in compound 12 which advanced deep into our research operating plan (ROP) before heteroaryl aniline metabolite 14 was characterized as Ames mutagenic, halting its progression. Strategic modifications to our ROP were made to enable early de-risking of putative aniline metabolites or hydrolysis products for mutagenicity in Ames. This led to the discovery of 3,5-diaminopyridine 15 and 4,6-diaminopyrimidine 16 as low risk for mutagenicity (defined by a 3-strain Ames negative result). Analysis of key matched molecular pairs 17 and 18 led to the prioritization of the 3,5-diaminopyridine sub-series for further optimization due to enhanced rodent brain penetration. These efforts culminated in the discovery of ethyl trifluoromethyl pyrazole 23 with excellent LRRK2 potency and expanded selectivity versus off-target CLK2.

Development and optimization of a high-throughput micro-computed tomography imaging method incorporating a novel analysis technique to evaluate bone mineral density of arthritic joints in a rodent model of collagen induced arthritis.

Rheumatoid arthritis (RA) is a chronic autoimmune disease resulting in joint inflammation, pain, and eventual bone loss. Bone loss and remodeling caused by symmetric polyarthritis, the hallmark of RA, is readily detectable by bone mineral density (BMD) measurement using micro-CT. Abnormalities in these measurements over time reflect the underlying pathophysiology of the bone. To evaluate the efficacy of anti-rheumatic agents in animal models of arthritis, we developed a high throughput knee and ankle joint imaging assay to measure BMD as a translational biomarker. A bone sample holder was custom designed for micro-CT scanning, which significantly increased assay throughput. Batch processing 3-dimensional image reconstruction, followed by automated image cropping, significantly reduced image processing time. In addition, we developed a novel, automated image analysis method to measure BMD and bone volume of knee and ankle joints. These improvements significantly increased the throughput of ex vivo bone sample analysis, reducing data turnaround from 5 days to 24 hours for a study with 200 rat hind limbs. Taken together, our data demonstrate that BMD, as quantified by micro-CT, is a robust efficacy biomarker with a high degree of sensitivity. Our innovative approach toward evaluation of BMD using optimized image acquisition and novel image processing techniques in preclinical models of RA enables high throughput assessment of anti-rheumatic agents offering a powerful tool for drug discovery.

Etanercept ameliorates inflammation and pain in a novel mono-arthritic multi-flare model of streptococcal cell wall induced arthritis.

BACKGROUND: The impact of anti-TNF, corticosteroid and analgesic therapy on inflammation and pain was evaluated in a novel mono-arthritic multi-flare rat Streptococcal Cell Wall (SCW) model using Etanercept, Dexamethasone and Buprenorphine. METHODS: Multiple flares of arthritis were induced with an intra-articular injection of SCW in the hind ankle on day 1, followed by intravenous challenges on days 21 and 42. Inflammation and pain were monitored in the hind paws. Cytokine profiling, cell phenotyping, bioluminescence imaging and histopathological evaluation were also performed. RESULTS: Local injection of SCW caused a rapid onset of inflammation and pain in the injected ankle which resolved within 4 days (Flare 1). Intravenous injection 20 days after sensitization resulted in an increase in ankle diameter and pain, which partially resolved in 8 days (Flare 2). The subsequent intra-venous injection in the same animals 14 days after resulted in a more chronic disease with inflammation and pain persisting over a period of 10 days (Flare 3). In Flare 2, therapeutic administration of Dexamethasone inhibited paw swelling (95%; P<0.001) and pain (55%; P<0.05). Therapeutic administration of Buprenorphine inhibited pain (80%; P<0.001) without affecting paw swelling (0%). Prophylactic administration of Etanercept in Flare 2 inhibited paw swelling (≥60%; P<0.001) and pain by ≥30%. Expression of IL-1ß, IL-6, MCP-1 and CINC was reduced by >50% (P<0.001). Treatment with Etanercept in Flare 3 inhibited paw swelling by 60% (P<0.001) and pain by 25%. Prior treatment with Etanercept in Flare 2 followed by re-administration in Flare 3 led to a complete loss in the efficacy of Etanercept. Systemic exposure of Etanercept corroborated with lack of efficacy. Dexamethasone inhibited inflammation and pain in both Flares 2 and 3 (P<0.001). CONCLUSIONS: We established a novel multi-flare SCW arthritis model enabling drug intervention in different stages of disease. We show for the first time the evaluation of inflammation and pain simultaneously in this model. Etanercept and Dexamethasone inhibited inflammation, pain and proinflammatory cytokines in this model. Taken together, this model facilitates the assessment of anti-rheumatic agents targeting inflammation and pain in the multiple flare paradigm and offers a powerful tool for drug discovery.