Microscale Purification with Direct Charged Aerosol Detector Quantitation Using Selective Online One- or Two-Dimensional Liquid Chromatography.

The pharmaceutical industry is increasingly faced with challenging separations of complex crude reaction mixtures at the microscale that require the adoption of new platforms for rapid target isolation, impurity determination, and quantitation. In this study, we describe an online microscale one- or two-dimensional liquid chromatography (1D/2D-LC) system with heart-cutting and multi (triple) detector triggering in either dimension to address this need. The advantages of charged aerosol detection (CAD) are discussed for the direct quantitation of limited quantity samples, without utilizing a second analytical instrument or gradient compensation pump. In addition to the significant time and cost savings, there is no minimum recovery requirement that exists when compared to gravimetric methods for accurate microscale quantitation. This platform has been successfully used to purify 0.5-5.0 mg scale reactions in 96- or 384-well reaction plates with a gradient time of 4 min per sample. Separations performed in both dimensions are complete in less than 12 min, including trapping and column equilibration time. The isolated arrays of small-quantity investigational compounds at a high purity enable rapid exploration of chemical reaction parameters and synthetic route scouting for biological target validation.

Potent, selective and orally bioavailable leucine-rich repeat kinase 2 (LRRK2) inhibitors.

Familial Parkinson's disease cases have recently been associated with the leucine rich repeat kinase 2 (LRRK2) gene. It has been hypothesized that inhibition of the LRRK2 protein may have the potential to alter disease pathogenesis. A dihydrobenzothiophene series of potent, selective, orally bioavailable LRRK2 inhibitors were identified from a high-throughput screen of the internal Merck sample collection. Initial SAR studies around the core established the series as a tractable small molecule lead series of LRRK2 inhibitors for potential treatment of Parkinson's disease. It was also found that incorporation of a lactam into the core drastically improved the CNS and DMPK properties of these small molecules.

Synthesis of Complex Druglike Molecules by the Use of Highly Functionalized Bench-Stable Organozinc Reagents.

The reactivity of a representative set of 17 organozinc pivalates with 18 polyfunctional druglike electrophiles (informers) in Negishi cross-coupling reactions was evaluated by high-throughput experimentation protocols. The high-fidelity scaleup of successful reactions in parallel enabled the isolation of sufficient material for biological testing, thus demonstrating the high value of these new solid zinc reagents in a drug-discovery setting and potentially for many other applications in chemistry. Principal component analysis (PCA) clearly defined the independent roles of the zincates and the informers toward druggable-space coverage.

Predictions of Chromatography Methods by Chemical Structure Similarity to Accelerate High-Throughput Medicinal Chemistry.

We introduce a new workflow that relies heavily on chemical quantitative structure-retention relationship (QSRR) models to accelerate method development for micro/mini-scale high-throughput purification (HTP). This provides faster access to new active pharmaceutical ingredients (APIs) through high-throughput experimentation (HTE). By comparing fingerprint structural similarity (e.g., Tanimoto index) with small training data sets containing a few hundred diverse small molecule antagonists of a lipid metabolizing enzyme, we can predict retention time (RT) of new compounds. Machine learning (ML) helps to identify optimal separation conditions for purification without performing the traditional crude QC step involving ultrahigh performance liquid chromatography (UHPLC) analyses of each compound. This green-chemistry approach with the use of predictive tools reduces cost and significantly shortens the design-make-test (DMT) cycle of new drugs by way of HTE.

Rapid Affinity and Microsomal Stability Ranking of Crude Mixture Libraries of Histone Deacetylase Inhibitors.

The science of drug discovery involves multiparameter optimization of molecular structures through iterative design-make-test cycles. For medicinal chemistry library synthesis, traditional workflows involve the isolation of each individual compound, gravimetric quantitation, and preparation of a standard concentration solution for biological assays. In this work, we explore ways to expedite this process by testing unpurified library mixtures using a combination of mass spectrometry-based assays for affinity selection and microsomal metabolic stability. Utilizing this approach, microgram quantities of crude library mixtures can be used to identify high affinity, metabolically stable library members for isolation and full characterization. This streamlined approach was demonstrated for the synthesis and evaluation of two libraries of histone deacetylase inhibitors and was shown to generate decision-making data in line with traditional workflows. The advantages of this paradigm include greatly reduced cycle time, reduced material requirements, and concentration of resources on the most promising compounds.

Ultra-fast microscale purification of chiral racemates and achiral pharmaceuticals with analytical supercritical fluid chromatography.

Improving efficiency while maintaining high quality separations is a central theme for specialized analytical/purification groups supporting discovery chemistry programs. Supercritical fluid chromatography (SFC) has become the prevalent technique for chiral separation and a complementary technique to reverse phase high-pressure liquid chromatography (RP-HPLC). In this manuscript we demonstrate the successful micro-isolation of chiral racemates, small molecules, and peptides using a sub-minute method on an analytical SFC system. The addition of a custom gas liquid separator (GLS) and alterations to the fluidic pathways allow the fractionation of desired products on a micro-scale SFC platform, providing analytical method development, purifications, and purity confirmation on a single SFC system. This enables micro-purification of pharmaceuticals including chiral racemates at high speed and reduced cost of materials. The resulting small-quantity, high-purity products enable follow-up enantioselective isolations from racemic products of parallel synthesis libraries. The processes established here will be beneficial for the isolations of other desired products in complex crude mixtures.

Association of respiratory failure with inhibition of NaV1.6 in the phrenic nerve.

As part of a drug discovery effort to identify potent inhibitors of NaV1.7 for the treatment of pain, we observed that inhibitors produced unexpected cardiovascular and respiratory effects in vivo. Specifically, inhibitors administered to rodents produced changes in cardiovascular parameters and respiratory cessation. We sought to determine the mechanism of the in vivo adverse effects by studying the selectivity of the compounds on NaV1.5, NaV1.4, and NaV1.6 in in vitro and ex vivo assays. Inhibitors lacking sufficient NaV1.7 selectivity over NaV1.6 were associated with respiratory cessation after in vivo administration to rodents. Effects on respiratory rate in rats were consistent with effects in an ex vivo hemisected rat diaphragm model and in vitro NaV1.6 potency. Furthermore, direct blockade of the phrenic nerve signaling was observed at exposures known to cause respiratory cessation in rats. Collectively, these results support a significant role for NaV1.6 in phrenic nerve signaling and respiratory function.

Optimization and Mechanistic Investigations of Novel Allosteric Activators of PKG1α.

Activation of PKG1α is a compelling strategy for the treatment of cardiovascular diseases. As the main effector of cyclic guanosine monophosphate (cGMP), activation of PKG1α induces smooth muscle relaxation in blood vessels, lowers pulmonary blood pressure, prevents platelet aggregation, and protects against cardiac stress. The development of activators has been mostly limited to cGMP mimetics and synthetic peptides. Described herein is the optimization of a piperidine series of small molecules to yield activators that demonstrate in vitro phosphorylation of vasodilator-stimulated phosphoprotein as well as antiproliferative effects in human pulmonary arterial smooth muscle cells. Hydrogen/deuterium exchange mass spectrometry experiments with the small molecule activators revealed a mechanism of action consistent with cGMP-induced activation, and an X-ray co-crystal structure with a construct encompassing the regulatory domains illustrated a binding mode in an allosteric pocket proximal to the low-affinity cyclic nucleotide-binding domain.

Notoamide E: Biosynthetic incorporation into notoamides C and D in cultures of Aspergillus versicolor NRRL 35600.

Notoamide E, a short-lived secondary metabolite, has been proposed as a biosynthetic intermediate to several advanced metabolites isolated from Aspergillus versicolor. In order to verify the role of this indole alkaloid along the biosynthetic pathway, synthetic doubly (13)C-labeled notoamide E was fed to Aspergillus versicolor. Analysis of the metabolites showed significant incorporation of notoamide E into the natural products notoamides C and D.

Discovery of Arylsulfonamide Nav1.7 Inhibitors: IVIVC, MPO Methods, and Optimization of Selectivity Profile.

The voltage-gated sodium channel Nav1.7 continues to be a high-profile target for the treatment of various pain afflictions due to its strong human genetic validation. While isoform selective molecules have been discovered and advanced into the clinic, to date, this target has yet to bear fruit in the form of marketed therapeutics for the treatment of pain. Lead optimization efforts over the past decade have focused on selectivity over Nav1.5 due to its link to cardiac side effects as well as the translation of preclinical efficacy to man. Inhibition of Nav1.6 was recently reported to yield potential respiratory side effects preclinically, and this finding necessitated a modified target selectivity profile. Herein, we report the continued optimization of a novel series of arylsulfonamide Nav1.7 inhibitors to afford improved selectivity over Nav1.6 while maintaining rodent oral bioavailability through the use of a novel multiparameter optimization (MPO) paradigm. We also report in vitro-in vivo correlations from Nav1.7 electrophysiology protocols to preclinical models of efficacy to assist in projecting clinical doses. These efforts produced inhibitors such as compound 19 with potency against Nav1.7, selectivity over Nav1.5 and Nav1.6, and efficacy in behavioral models of pain in rodents as well as inhibition of rhesus olfactory response indicative of target modulation.

Nav1.7 target modulation and efficacy can be measured in nonhuman primate assays.

Humans with loss-of-function mutations in the Nav1.7 channel gene (SCN9A) show profound insensitivity to pain, whereas those with gain-of-function mutations can have inherited pain syndromes. Therefore, inhibition of the Nav1.7 channel with a small molecule has been considered a promising approach for the treatment of various human pain conditions. To date, clinical studies conducted using selective Nav1.7 inhibitors have not provided analgesic efficacy sufficient to warrant further investment. Clinical studies to date used multiples of in vitro IC50 values derived from electrophysiological studies to calculate anticipated human doses. To increase the chance of clinical success, we developed rhesus macaque models of action potential propagation, nociception, and olfaction, to measure Nav1.7 target modulation in vivo. The potent and selective Nav1.7 inhibitors SSCI-1 and SSCI-2 dose-dependently blocked C-fiber nociceptor conduction in microneurography studies and inhibited withdrawal responses to noxious heat in rhesus monkeys. Pharmacological Nav1.7 inhibition also reduced odor-induced activation of the olfactory bulb (OB), measured by functional magnetic resonance imaging (fMRI) studies consistent with the anosmia reported in Nav1.7 loss-of-function patients. These data demonstrate that it is possible to measure Nav1.7 target modulation in rhesus macaques and determine the plasma concentration required to produce a predetermined level of inhibition. The calculated plasma concentration for preclinical efficacy could be used to guide human efficacious exposure estimates. Given the translatable nature of the assays used, it is anticipated that they can be also used in phase 1 clinical studies to measure target modulation and aid in the interpretation of phase 1 clinical data.

Discovery of a new class of integrin antibodies for fibrosis.

Lung fibrosis, or the scarring of the lung, is a devastating disease with huge unmet medical need. There are limited treatment options and its prognosis is worse than most types of cancer. We previously discovered that MK-0429 is an equipotent pan-inhibitor of αv integrins that reduces proteinuria and kidney fibrosis in a preclinical model. In the present study, we further demonstrated that MK-0429 significantly inhibits fibrosis progression in a bleomycin-induced lung injury model. In search of newer integrin inhibitors for fibrosis, we characterized monoclonal antibodies discovered using Adimab's yeast display platform. We identified several potent neutralizing integrin antibodies with unique human and mouse cross-reactivity. Among these, Ab-31 blocked the binding of multiple αv integrins to their ligands with IC50s comparable to those of MK-0429. Furthermore, both MK-0429 and Ab-31 suppressed integrin-mediated cell adhesion and latent TGFß activation. In IPF patient lung fibroblasts, TGFß treatment induced profound αSMA expression in phenotypic imaging assays and Ab-31 demonstrated potent in vitro activity at inhibiting αSMA expression, suggesting that the integrin antibody is able to modulate TGFß action though mechanisms beyond the inhibition of latent TGFß activation. Together, our results highlight the potential to develop newer integrin therapeutics for the treatment of fibrotic lung diseases.

Genome-based characterization of two prenylation steps in the assembly of the stephacidin and notoamide anticancer agents in a marine-derived Aspergillus sp.

Stephacidin and notoamide natural products belong to a group of prenylated indole alkaloids containing a core bicyclo[2.2.2]diazaoctane ring system. These bioactive fungal secondary metabolites have a range of unusual structural and stereochemical features but their biosynthesis has remained uncharacterized. Herein, we report the first biosynthetic gene cluster for this class of fungal alkaloids based on whole genome sequencing of a marine-derived Aspergillus sp. Two central pathway enzymes catalyzing both normal and reverse prenyltransfer reactions were characterized in detail. Our results establish the early steps for creation of the prenylated indole alkaloid structure and suggest a scheme for the biosynthesis of stephacidin and notoamide metabolites. The work provides the first genetic and biochemical insights for understanding the structural diversity of this important family of fungal alkaloids.

Synthetic Studies on Palau'amine. Construction of the Cyclopentane Core via an Asymmetric 1,3-Dipolar Cycloaddition.

The cyclopentane core of palau'amine has been constructed in optically pure form through the use of an asymmetric azomethine ylid [1,3]-dipolar cycloaddition reaction.

Isolation of notoamide E, a key precursor in the biosynthesis of prenylated indole alkaloids in a marine-derived fungus, Aspergillus sp.

Notoamide E was identified to be a short-lived precursor in the biosynthesis of prenylated indole alkaloids in the mussel-derived Aspergillus sp. In addition, the feeding experiment of the (13)C-labeled notoamide E afforded structurally novel metabolites.

Synthesis and conformation-activity relationships of the peptide isosteres of FK228 and largazole.

The peptide isosteres (10 and 11) of the naturally occurring and potent histone deacetylase (HDAC) inhibitors FK228 and largazole have been synthesized and evaluated side-by-side with FK228, largazole, and SAHA for inhibition of the class I HDACs 1, 2, 3, and 6.

Subminute micro-isolation of pharmaceuticals with ultra-high pressure liquid chromatography.

The drive for faster separations while maintaining quality and yield remains an important consideration for enhanced productivity as well as cost reduction for drug discovery laboratories in the pharmaceutical industry. High-throughput experimentation (HTE) and high-throughput screening (HTS) techniques can benefit from rapid and efficient isolation of product at high purity and recovery from microgram-scale crude reaction mixtures. In this study we describe the isolation of small molecule and biomolecule crude mixtures at the microgram-scale (100-2500 µg) in single or library format with methods as fast as 1.0 min and system pressures averaging 10,000 psi with an ultra-high pressure liquid chromatography (UHPLC) setup. UHPLC technology provides several advantages for rapid (<1.0 min) separations with small-particle (1.8-3.5 µm) size 4.6 × 50 mm C18 columns such as minimal extra column and delay volume, fast detector response time, and higher linear velocities for improved speed and resolution. We typically see a 5-10 fold improvement in purification time and overall sample processing time with low fraction volumes and same-day drying when compared with traditional semi-preparative techniques. There is a significant 50-fold reduction in solvent usage per run, resulting in a much lower cost of solvent and waste handling. Fluidic pathways have been optimized for collection into tared high-density 96 or 384 well 2D barcoded storage tubes in a microtiter plate (MTP) layout. Coupling the system to robotics has enabled us to implement a fully integrated automation platform with additional capabilities for small-scale purification at high speed and reduced cost of materials. The resulting arrays of small-quantity, high-purity compounds enable synthetic route scouting for HTE and HTS for biological target validation.

Improved total synthesis of the potent HDAC inhibitor FK228 (FR-901228).

A scaleable synthesis of the potent histone deacetylase (HDAC) inhibitor FK228 is described. A reliable strategy for preparing the key beta-hydroxy mercapto heptenoic acid partner was accomplished in nine steps and 13% overall yield. A Noyori asymmetric hydrogen-transfer reaction established the hydroxyl stereochemistry in >99:1 er via the reduction of a propargylic ketone.

Calmodulin inhibitory activity of the malbrancheamides and various analogs.

The preparation and biological activity of various structural analogs of the malbrancheamides are disclosed. The impact of indole chlorination, C-12a relative stereochemistry, and bicyclo[2.2.2]diazaoctane core oxidation state on the ability of these analogs to inhibit calmodulin dependent phosphodiesterase (PDE1) was studied, and a number of potent compounds were identified.

Detection of VM55599 and preparaherquamide from Aspergillus japonicus and Penicillium fellutanum: biosynthetic implications.

The secondary metabolites VM55599 (4) and preparaherquamide (5) have been identified by LC-MS(n) analysis as natural metabolites in cultures of Penicillium fellutanum, whereas preparaherquamide has been identified only in cultures of Aspergillus japonicus. In accord with a previous proposal, the identification of both metabolites, which have a diastereomeric relationship, provides indirect support for a unified biosynthetic scheme.