General Synthesis of Conformationally Constrained Noncanonical Amino Acids with C(sp3)-Rich Benzene Bioisosteres.

Recent years have seen novel modalities emerge for the treatment of human diseases resulting in an increase in beyond rule of 5 (bRo5) chemical matter. As a result, synthetic innovations aiming to enable rapid access to complex bRo5 molecular entities have become increasingly valuable for medicinal chemists' toolkits. Herein, we report the general synthesis of a new class of noncanonical amino acids (ncAA) with a cyclopropyl backbone to achieve conformational constraint and bearing C(sp3)-rich benzene bioisosteres. We also demonstrate preliminary studies toward utilities of these ncAA as building blocks for medicinal chemistry research.

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.

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.

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.

Comprehensive Strategies to Bicyclic Prolines: Applications in the Synthesis of Potent Arginase Inhibitors.

Comprehensive synthetic strategies afforded a diverse set of structurally unique bicyclic proline-containing arginase inhibitors with a high degree of three-dimensionality. The analogs that favored the Cγ-exo conformation of the proline improved the arginase potency over the initial lead. The novel synthetic strategies reported here not only enable access to previously unknown stereochemically complex proline derivatives but also provide a foundation for the future synthesis of bicyclic proline analogs, which incorporate inherent three-dimensional character into building blocks, medicine, and catalysts and could have a profound impact on the conformation of proline-containing peptides and macrocycles.

Synthesis and evaluation of azabicyclo[3.2.1]octane derivatives as potent mixed vasopressin antagonists.

A series of biaryl amides containing an azabicyclooctane amine headpiece were synthesized and evaluated as mixed arginine vasopressin (AVP) receptor antagonists. Several analogues, including 8g, 12g, 13d, and 13g, were shown to have excellent V(1a)- and good V(2)-receptor binding affinities. Compound 13d was further profiled for drug-like properties and for an in vitro comparison with conivaptan, the program's mixed V(1a)/V(2)-receptor antagonist standard.

Desmethoxymajusculamide C, a cyanobacterial depsipeptide with potent cytotoxicity in both cyclic and ring-opened forms.

Cytotoxicity-guided fractionation of the organic extract from a Fijian Lyngbya majuscula led to the discovery of desmethoxymajusculamide C (DMMC) as the active metabolite. Spectroscopic analysis including 1D and 2D NMR, MS/MS, and chemical degradation and derivatization protocols were used to assign the planar structure and stereoconfiguration of this new cyclic depsipeptide. DMMC demonstrated potent and selective anti-solid tumor activity with an IC(50) = 20 nM against the HCT-116 human colon carcinoma cell line via disruption of cellular microfilament networks. A linear form of DMMC was generated by base hydrolysis, and the amino acid sequence was confirmed by mass spectrometry. Linearized DMMC was also evaluated in the biological assays and found to maintain potent actin depolymerization characteristics while displaying solid tumor selectivity equivalent to DMMC in the disk diffusion assay. A clonogenic assay assessing cytotoxicity to HCT-116 cells as a function of exposure duration showed that greater than 24 h of constant drug treatment was required to yield significant cell killing. Therapeutic studies with HCT-116 bearing SCID mice demonstrated efficacy at the highest dose used (%T/C = 60% at 0.62 mg/kg daily for 5 days).

Unexpected rearrangement of enantiomerically pure 3-aminoquinuclidine as a simple way of preparing diastereomeric octahydropyrrolo[2,3-c]pyridine derivatives.

Reaction of (S)- or (R)-3-aminoquinuclidine with 2-chloropyrimidine or 2-bromopyrimidine led to an unexpected formation of both cis- and trans-octahydropyrrolo [2,3]pyridine derivatives. A single-step synthesis of two of the four stereoisomers of these octahydropyrrolo[2,3]pyridine derivatives provides a convenient way of generating stereochemically defined isomers. Optimization of reaction conditions was carried out by (1)H NMR monitoring. The relative and absolute stereochemistry of all four stereoisomers was determined by a combination of (1)H, (13)C, and (15)N NMR spectroscopy and vibrational circular dichroism spectroscopy.

Development of a Platform To Enable Efficient Permeability Evaluation of Novel Organo-Peptide Macrocycles.

As more macrocycle structures are utilized to drug intracellular targets, new platforms are needed to facilitate the discovery of cell permeable compounds in this unique chemical space. Herein, a method is disclosed that allows for the efficient synthesis and permeability evaluation of novel organo-peptide macrocycle libraries. Thoughtful library design allows for the collection of crude permeability data using supercritical fluid chromatography mass spectrometry (SFC-MS) (EPSA) by mass-encoding the stereochemistry, ring size, and organic linker of the desired macrocycles. Library synthesis was aided via the development of a new on-resin N-arylation reaction. Further insights on the permeation of these organo-peptide macrocycles will be discussed, such as the permeability enhancement when utilizing a 2-substituted phenethyl linker versus a 3-substituted phenethyl linker. Lastly, selected macrocycles were scaled up and tested in the MDCK-II permeability assay, and the results of this assay reiterated the permeability trends from the crude SFC-MS data.

Carboxylic acid based quinolines as liver X receptor modulators that have LXRbeta receptor binding selectivity.

A series of potent and binding selective LXRbeta agonists was developed using the previously reported non-selective LXR ligand WAY-254011 as a structural template. With the aid of molecular modeling, it was found that 2,3-diMe-Ph, 2,5-diMe-Ph, and naphthalene substituted quinoline acetic acids (such as quinoline 33, 37, and 38) showed selectivity for LXRbeta over LXRalpha in binding assays.

Synthesis and activity of 1-(3-amino-1-phenylpropyl)indolin-2-ones: a new class of selective norepinephrine reuptake inhibitors.

Norepinephrine and serotonin play an important role in a wide variety of biological processes and are implicated in a number of neurological disorders. A novel class of 1-(3-amino-1-phenylpropyl)indolin-2-ones was designed and synthesized that displays potent norepinephrine reuptake inhibition while maintaining high selectivity (>100-fold) against the human serotonin and dopamine transporters.

Sub-2 microm HPLC coupled with sub-ppm mass accuracy for analysis of pharmaceutical compound libraries.

Recent advances in accurate mass analysis are poised to allow the high-throughput production of accurate mass data on many more compounds than was previously available. It is shown that sub-ppm mass accuracy (producing elemental compositions) can be obtained on a simple TOF mass spectrometer operating in the manufacturer's standard mode. Concomitantly, there have been important technological advances in LC with respect to speed of analysis using sub-2 microm particle columns. Much of the sub-2 microm work in the literature has been under the label ultra performance LC (UPLC), however, we show that very high-speed results can be obtained using other manufacturer's pumps by using elevated column temperatures. Using elevated temperatures, HPLC peak widths on the order of 1 s can be obtained. We report the coupling of these two technologies (sub-ppm mass accuracy MS with high-speed HPLC) for the rapid analysis of compounds entering pharmaceutical libraries.

Regioselective synthesis of substituted pyrroles: efficient palladium-catalyzed cyclization of internal alkynes and 2-amino-3-iodoacrylate derivatives.

The first efficient and regioselective palladium-catalyzed cyclization of internal alkynes and 2-amino-3-iodoacrylates to give moderate to excellent yields of highly functionalized pyrroles has been developed. This approach is applicable to a range of alkynes and affords the deacylated pyrrole under reaction conditions for most substrates. [reaction: see text]

Preparative separation and identification of derivatized beta-methylphenylalanine enantiomers by chiral SFC, HPLC and NMR for development of new peptide ligand mimetics in drug discovery.

A direct preparative purification of all four isomers of the unnatural amino acid beta-methylphenylalanine was achieved using supercritical fluid chromatography (SFC) with stacked-injection. Final purification of the Cbz-methyl ester derived isomers was performed on a Daicel Chiralpak AD-H column (20 mm x 250 mm), using 50:50 methanol/ethanol as the organic modifier and resulted in purification of over 3.4 g of material in 6.25 h with >90% total recovery. The absolute stereochemical assignment of the purified amino acids was determined through a combination of chiral HPLC, NMR and optical rotation studies. To our knowledge, this is the first reported preparative approach that has yielded all four compounds in a single chromatographic run.

Integrity profiling of high throughput screening hits using LC-MS and related techniques.

Integrity profiling of HTS hits is valuable for verification of the hit identity and purity. This provides early discovery researchers with more confident SAR theories. Methodology for integrity profiling of HTS hits must be high throughput, consume little material, and selectively provide structure-based data. Analytical techniques that can be utilized for integrity profiling methods are reviewed for their appropriateness in sample preparation, component separation, detection, purity quantitation, identity confirmation, and follow-up.

Structure and biosynthesis of the jamaicamides, new mixed polyketide-peptide neurotoxins from the marine cyanobacterium Lyngbya majuscula.

A screening program for bioactive compounds from marine cyanobacteria led to the isolation of jamaicamides A-C. Jamaicamide A is a novel and highly functionalized lipopeptide containing an alkynyl bromide, vinyl chloride, beta-methoxy eneone system, and pyrrolinone ring. The jamaicamides show sodium channelblocking activity and fish toxicity. Precursor feeding to jamaicamide-producing cultures mapped out the series of acetate and amino acid residues and helped develop an effective cloning strategy for the biosynthetic gene cluster. The 58 kbp gene cluster is composed of 17 open reading frames that show an exact colinearity with their expected utilization. A novel cassette of genes appears to form a pendent carbon atom possessing the vinyl chloride functionality; at its core this contains an HMG-CoA synthase-like motif, giving insight into the mechanism by which this functional group is created.

Somocystinamide A, a novel cytotoxic disulfide dimer from a Fijian marine cyanobacterial mixed assemblage.

[reaction: see text] Bioassay-guided investigation of the extract from a Lyngbya majuscula/Schizothrix sp. assemblage of marine cyanobacteria led to the discovery of somocystinamide A (1), an extraordinary disulfide dimer of mixed PKS/NRPS biosynthetic origin. Somocystinamide A (1) was highly acid-sensitive, rapidly and completely converting to a characterizable derivative (2). Compound 1 exhibits significant cytotoxicity against mouse neuro-2a neuroblastoma cells (IC50 = 1.4 microg/mL), whereas 2 has no activity.

Wewakazole, a novel cyclic dodecapeptide from a Papua New Guinea Lyngbya majuscula.

Phytochemical examination of a Papua New Guinea collection of Lyngbya majuscula resulted in the discovery of wewakazole (1), a novel cyclic dodecapeptide containing an unprecedented six five-membered heterocycles. Multiple NMR experiments and MS/MS data were required to assemble its planar structure because of its extensively signal-overlapped NMR spectra. In particular, a 1D HMBC was utilized to orient a three amino acid fragment that could not be placed by standard spectroscopic methods. [structure--see text]

Rapid method development for chiral separation in drug discovery using multi-column parallel screening and circular dichroism signal pooling.

A novel strategy for rapid chiral method development has been developed using multi-column parallel screening and circular dichroism (CD) signal pooling. Described is the first use of a customized HPLC system that integrates an HPLC auto-sampler, one pump and five divided channels with five columns and five UV detectors to screen five chiral stationary phases (CSPs) simultaneously in parallel. A high-pressure semi-prep on-line pre-filter, a six-port manifold and five individually adjusted backpressure restrictors were installed in the system which allowed the sample and mobile phase to be evenly distributed over the five columns and UV detectors. The five CSPs, namely Chiralpak AD and AS, Chiralcel OJ and OD and Whelk-O1, were screened. The system guarantees a five-fold increase in speed for chiral column scouting compared with the widely used automated sequential column switching approach, and does not have the limitations of the coupled column screening approach for enantiomers whose elution order could be reversed on CSPs. Furthermore, the five channels after the UV detectors were recombined using a reversed flow splitter into a CD detector. The pooled CD signal from the five channels was recorded to track the elution order of the resolved enantiomers and to determine their sign, positive or negative. The signal pooling allows for the effective use of a single CD detector for multiple columns since unresolved racemate has little CD signal, and observing the sign of CD signal for one of the two enantiomer UV peaks is sufficient for tracking the enantiomeric elution order.

Evaluation of non-conventional polar modifiers on immobilized chiral stationary phases for improved resolution of enantiomers by supercritical fluid chromatography.

An evaluation of the use of non-conventional polar modifiers for the supercritical fluid chromatographic separation of enantiomers on immobilized chiral stationary phases is presented. The resolution of a group of nine commercially available racemates is studied on the Chiralpak IA, IB, IC, ID, IE, and IF chiral stationary phases using CO2-based eluents containing non-conventional polar modifiers such as dichloromethane, chloroform, tetrahydrofuran, 2-methyl tetrahydrofuran, methyl tert-butyl ether, cyclopentyl methyl ether, acetone, ethyl acetate, toluene, 2,2,2-trifluoroethanol, and N,N-dimethylformamide. Screening experiments and method development for the commercial racemates on the immobilized columns with the non-conventional solvents demonstrated an ability to adjust the retention and improve resolution. From these results we were able to assign a general eluotropic relationship between the non-conventional solvents and methanol. A general ability to selectively adjust chromatographic retention while improving analyte solubility can lead to improved preparative chromatographic performance.