Orally Bioavailable Macrocyclic Peptide That Inhibits Binding of PCSK9 to the Low Density Lipoprotein Receptor.

BACKGROUND: Inhibition of PCSK9 (proprotein convertase subtilisin/kexin type 9)-low density lipoprotein receptor interaction with injectable monoclonal antibodies or small interfering RNA lowers plasma low density lipoprotein-cholesterol, but despite nearly 2 decades of effort, an oral inhibitor of PCSK9 is not available. Macrocyclic peptides represent a novel approach to target proteins traditionally considered intractable to small-molecule drug design. METHODS: Novel mRNA display screening technology was used to identify lead chemical matter, which was then optimized by applying structure-based drug design enabled by novel synthetic chemistry to identify macrocyclic peptide (MK-0616) with exquisite potency and selectivity for PCSK9. Following completion of nonclinical safety studies, MK-0616 was administered to healthy adult participants in a single rising-dose Phase 1 clinical trial designed to evaluate its safety, pharmacokinetics, and pharmacodynamics. In a multiple-dose trial in participants taking statins, MK-0616 was administered once daily for 14 days to characterize the safety, pharmacokinetics, and pharmacodynamics (change in low density lipoprotein cholesterol). RESULTS: MK-0616 displayed high affinity (Ki = 5pM) for PCSK9 in vitro and sufficient safety and oral bioavailability preclinically to enable advancement into the clinic. In Phase 1 clinical studies in healthy adults, single oral doses of MK-0616 were associated with >93% geometric mean reduction (95% CI, 84-103) of free, unbound plasma PCSK9; in participants on statin therapy, multiple-oral-dose regimens provided a maximum 61% geometric mean reduction (95% CI, 43-85) in low density lipoprotein cholesterol from baseline after 14 days of once-daily dosing of 20 mg MK-0616. CONCLUSIONS: This work validates the use of mRNA display technology for identification of novel oral therapeutic agents, exemplified by the identification of an oral PCSK9 inhibitor, which has the potential to be a highly effective cholesterol lowering therapy for patients in need.

Total Synthesis of Aflastatin A.

The total syntheses of aflastatin A and its C3-C48 degradation fragment (6a, R = H) have been accomplished. The syntheses feature several complex diastereoselective fragment couplings, including a Felkin-selective trityl-catalyzed Mukaiyama aldol reaction, a chelate-controlled aldol reaction involving soft enolization with magnesium, and an anti-Felkin-selective boron-mediated oxygenated aldol reaction. Careful comparison of the spectroscopic data for the synthetic C3-C48 degradation fragment to that reported by the isolation group revealed a structural misassignment in the lactol region of the naturally derived degradation product. Ultimately, the data reported for the naturally derived aflastatin A C3-C48 degradation lactol (6a, R = H) were attributed to its derivative lactol trideuteriomethyl ether (6c, R = CD3). Additionally, the revised absolute configurations of six stereogenic centers (C8, C9, and C28-C31) were confirmed.

Ultrahigh performance liquid chromatography methods facilitate the development of glucose-responsive insulin therapeutics.

Insulin oligosaccharide conjugates hold promise as potential glucose-responsive insulins (GRIs), which can improve the therapeutic index of insulins and mitigate the risk of hypoglycemia. A key challenge for the analytical development of such molecules is finding an efficient method to characterize the purity and impurities of conjugated insulins. Using the S-Matrix Fusion QbD-ultrahigh performance liquid chromatography (UHPLC) integrated system, we were able to quickly screen and develop two short UHPLC methods. These methods were used to support process development, clinical batch drug substance (DS) release, and stability studies of MK-2640, an insulin oligosaccharide conjugate. Both methods used a Waters CSH C18 column, with a shallow gradient of acetonitrile to aqueous mobile phase containing 25 mM sodium perchlorate and 0.05% perchloric acid. The 10-min run time method was well suited for process development and monitoring as it was able to separate the main product, MK-2640, six oligosaccharide-substituted recombinant human insulin (RHI) impurities, A21 deamidated MK-2640, and the starting material RHI. The 13-min run time method provided improved separation of the major impurities and demonstrated good chromatographic reproducibility on different instruments or using columns from different lots of stationary phase, which made it ideal for the final DS release. Validation of the 13-min method demonstrated great linearity for both the MK-2640 main peak and its related impurities, low limit of detection (0.02%), and limit of quantitation (0.05%). The high specificity of the method allowed the separation of the degradation products from main peak, thus makes it suitable for stability monitoring. The major impurities in the DS were characterized by two-dimensional liquid chromatography-mass spectrometry (2D-LC-MS).

Highly Diastereoselective Synthesis of a HCV NS5B Nucleoside Polymerase Inhibitor.

An asymmetric synthesis of HCV NS5B nucleoside polymerase inhibitor (1) is described. This novel route features several remarkably diastereoselective and high-yielding transformations, including construction of the all-carbon quaternary stereogenic center at C-2 via a thermodynamic aldol reaction. A subsequent glycosylation reaction with activated uracil via C-1 phosphate and installation of the cyclic phosphate group using an achiral phosphorus(III) reagent followed by oxidation provides 1.

A new class of non-C2-symmetric ligands for oxidative and redox-neutral palladium-catalyzed asymmetric allylic alkylations of 1,3-diketones.

We report the discovery, synthesis, and application of a new class of non-C2-symmetric phosphoramidite ligands derived from pyroglutamic acid for use in both oxidative and redox-neutral palladium-catalyzed asymmetric allylic alkylations of 1,3-diketones. The resulting chiral products are typically obtained in high yield with good to excellent levels of enantioselectivity.

Synthesis of a Complex and Highly Potent PCSK9 Inhibitor.

The solution-based gram-scale synthesis of complex and highly potent proprotein convertase subtilisin-like/kexin type 9 (PCSK9) inhibitor 1 is presented. Construction of Northern fragment 2, followed by stepwise installation of Eastern 3, Southern 4, and Western 5 fragments, provided macrocyclic precursor 19. This intermediate was cross-linked via an intramolecular azide-alkyne click reaction, which preceded macrolactamization to afford the core framework of compound 1. Finally, coupling with poly(ethylene glycol) side-chain-based 6 gave the PCSK9 inhibitor 1.

Palladium-catalyzed alkylation of 1,4-dienes by C-H activation.

Activated: the title reaction proceeds with a broad range of nucleophiles and variously substituted 1,4-dienes under mild conditions, and provides direct access to the corresponding 1,3-diene-containing products with high regio- and stereocontrol (see scheme; 2,6-DMBQ=2,6-dimethylbenzoquinone, EWG=electron-withdrawing group). This is the first catalytic allylic C-H alkylation that proceeds in the absence of sulfoxide ligands.

Palladium-catalyzed asymmetric allylic alkylations of polynitrogen-containing aromatic heterocycles.

We report the palladium-catalyzed asymmetric allylic alkylation (AAA) reaction of a variety of nitrogen-containing aromatic heterocycles, including pyrazine, pyrimidine, pyridazine, quinoxaline, and benzoimidazole derivatives. The mesityl ester, whose steric bulk prevents competitive deacylation of the electrophile from "hard" nucleophiles, is introduced as a new leaving group in allylic alkylation chemistry. In contrast to our previous studies of AAA reactions with pyridine-based substrates, no precomplexation with a Lewis acid is required before deprotonation with LiHMDS, underscoring the relative acidity of these electron-deficient nucleophiles.

Palladium-catalyzed regio-, diastereo-, and enantioselective benzylic allylation of 2-substituted pyridines.

We report a new method for the highly regio-, diastereo-, and enantioselective palladium-catalyzed allylic alkylation of 2-substituted pyridines that allows for the formation of homoallylic stereocenters containing alkyl, aryl, heteroaryl, and nitrogen substituents. When the reaction is conducted with asymmetric acyclic electrophiles, both linear and branched products may be obtained exclusively by selecting the appropriate regioisomeric starting material and ligand, an example of the "memory effect." Deuterium-labeling studies reveal that though no such phenomenon occurs with racemic cyclic electrophiles, the chiral ligand employed reacts kinetically faster with the enantiomer of the substrate for which it is "matched" and yet eventually converts all "mismatched" substrate to product.

Strategy for employing unstabilized nucleophiles in palladium-catalyzed asymmetric allylic alkylations.

We report a strategy for the employment of highly unstabilized anions in palladium-catalyzed asymmetric allylic alkylations (AAA). The "hard" 2-methylpyridyl nucleophiles studied are first reacted in situ with BF3.OEt2; subsequent deprotonation of the resulting complexes with LiHMDS affords "soft" anions that are competent nucleophiles in AAA reactions. The reaction is selective for the 2-position of methylpyridines and tolerates bulky aryl and alkyl substitution at the 3-, 4-, and 5-positions. Investigations into the reaction mechanism demonstrate that the configuration of the allylic stereocenter is retained, consistent with the canonical outer sphere mechanism invoked for palladium-catalyzed allylic substitution processes of stabilized anions.

A Next Generation Synthesis of BACE1 Inhibitor Verubecestat (MK-8931).

The development of a commercial manufacturing route to verubecestat (MK-8931) is described, highlights of which include the application of a continuous processing step to outcompete fast proton transfer in a Mannich-type ketimine addition, a copper-catalyzed amidation reaction, and an optimized guanidinylation procedure to form the key iminothiadiazine dioxide core.

Synthesis of Verubecestat, a BACE1 Inhibitor for the Treatment of Alzheimer's Disease.

Verubecestat is an inhibitor of ß-secretase being evaluated for the treatment of Alzheimer's disease. The first-generation route relies on an amide coupling with a functionalized aniline, the preparation of which introduces synthetic inefficiencies. The second-generation route replaces this with a copper-catalyzed C-N coupling, allowing for more direct access to the target. Other features of the new route include a diastereoselective Mannich-type addition into an Ellman sulfinyl ketimine and a late-stage guanidinylation.