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Enzymes are capable of unique and selective transformations that can enable sustainable chemical production. While many industrial processes have been developed using free enzymes in aqueous solutions, immobilizing enzymes on a solid support can offer considerable advantages, including improved reaction efficiency, enzyme stability, the ability to perform reactions in non-aqueous media, and simplified separation of product from enzyme. Herein, we describe the development of a biocatalytic transaminase reaction of CyreneTM (2) utilizing an immobilized, evolved transaminase enzyme in an organic solvent to provide amine intermediate 3a en route to the Brutons tyrosine kinase (BTK) inhibitor nemtabrutinib. Enzyme immobilization is critical to facile isolation of the water-soluble product. Improved reaction kinetics and diastereoselectivity were achieved by bridging directed enzyme evolution with the selection of an optimal reaction solvent and solid support for immobilization, enabling a unified solvent system and direct isolation of 3a as a crystalline salt with dr > 50:1.

The development of a protecting group-free, 2-step synthesis of 5-amino-2-hydroxymethyltetrahydropyran 1a from biorenewable Cyrene™ is described which renders access to BTK-inhibitor nemtabrutinib (2) more efficient and sustainable.

A low-cost compact mass spectrometer was effectively utilized for identifying and tracking process impurities in the early stage development of a multistep synthetic protocol used to prepare the verubecestat drug substance. Process optimizations were influenced by the type, quantity, and final fate of impurities generated during synthesis. Employing a compact mass spectrometer for impurity identification and tracking served as a guide to redesign synthetic strategies which enabled the minimization or avoidance of undesired side products.

Verubecestat is a β-site amyloid precursor protein cleaving enzyme 1 (BACE1) inhibitor which was previously evaluated for the treatment of Alzheimer’s disease. The synthesis of verubecestat relies ...

A hemiaminal derivative identified during preparation of the penultimate of ceftolozane 1, mandated a thorough investigation of the source of the impurity. N,N-Dimethylacetamide (DMAc) subjected to oxidation was found to be the culprit generating oxidized products that serve as sources of the hemiaminal derivative. Identification of DMAc as a source of formaldehyde derivatives and the mechanism of DMAc oxidation are elaborated.

Single-stranded silencing RNAs (ss siRNA), while not as potent as duplex RNAs, have the potential to become a novel platform technology in RNA interference based gene silencing by virtue of their simplicity and plausibly favorable characteristics in pharmacokinetics and biodistribution. Like other therapeutic pharmaceutical agents, ss siRNA can be optimized to achieve higher potency through a structure-activity based approach. Systematic chemical modification at each position of a 21-mer oligonucleotide identified 2',5'-linked 3'-deoxythymidine (3dT) at position 1 and locked nucleic acids (LNAs) at the seed region as key components to afford significant enhancement in knockdown activity both in vitro and in vivo. Further optimization by additional chemical modifications should enable ss siRNA as an alternative gene silencing modality.

An efficient multicomponent orthogonal protocol was developed for post-synthetic oligonucleotide modification using commercially available 2'- O-methyl ester and 2'- O-propargyl nucleoside scaffolds. Amidation of methyl esters with primary amines was achieved in the presence of 2'-propargyl groups which were utilized for subsequent copper catalyzed cycloaddition with GalNAc-azide. The methodology was applied to generate siRNA composed of multiple amide and triazole conjugates. Computational methods were used to illustrate the impact of substitution at the 2'-position. This a powerful post-oligomerization technique for rapidly introducing diversity to oligonucleotide design.

Volatile amines are among the most frequently used chemicals in organic and pharmaceutical chemistry. Synthetic route optimization often involves the evaluation of several different amines requiring the development and validation of analytical methods for quantitation of residual amine levels. Herein, a simple and fast generic GC-FID method on an Agilent J&W CP-Volamine capillary column (using either He or H2 as the carrier gas) capable of separating over 25 volatile amines and other basic polar species commonly used in pharmaceutical chemistry workflows is described. This 16min method is successfully applied to the analysis and quantitation of volatile amines in a variety of pharmaceutically-related drugs and synthetic intermediates. Method validation experiments showed excellent analytical performance in linearity, recovery, repeatability, and limit of quantitation and detection. In addition, diverse examples for the application of this method to the simultaneous determination of other amine-related chemicals in reaction mixtures are illustrated, thereby indicating that these GC-FID method conditions can be effectively used as starting point during method development for the analysis of other basic polar species beyond the validated list of amines described in this study.

A practical synthesis of a macrocyclic HCV protease inhibitor, MK-1220, is described. The key features are a new synthesis of the trisubstituted isoquinoline, Sonogashira fragment coupling, and a high-yielding, 18-membered macrolactam formation.

Development of a practical synthesis of MK-7009, a 22-membered macrocycle, is described. A variety of ring-closing strategies were evaluated, including ring-closing metathesis, intermolecular palladium-catalyzed cross-couplings, and macrolactamization. Ring closure via macrolactamization was found to give the highest yields under relatively high reaction concentrations. Optimization of the ring formation step and the synthesis of key intermediates en route to MK-7009 are reported.

Taranabant (1) is a cannabinoid-1 receptor (CB1R) inverse agonist that was recently in late-stage clinical development for the treatment of obesity. The previously employed synthesis exhibited a number of shortcomings for continuing development, and in this paper we report an improved synthesis of the target molecule that is suitable for large-scale implementation. Palladium-catalyzed amidation of an enol tosylate afforded a stereodefined tetrasubstituted enamide, and asymmetric hydrogenation thereof provided the target molecule.

A highly stereoselective aldol reaction was observed on chromane carboxylate ester 1 via the corresponding diisopinocampheyl boron-enolate using commercially available (−)-DIP-Cl reagent. The aldol product 2c was obtained in 89% yield with 48 dr and 92% ee. Further studies indicate that stereoselective formation of the enolate and proper chiral ligand on boron are responsible for the exceptional diastereo- and enantioselectivity in the aldol reaction.

Chemical modification of siRNA is achieved in a high-throughput manner (96-well plate format) by copper catalyzed azide–alkyne cycloadditions. This transformation can be performed in one synthetic operation at up to four positions with complete specificity, good yield, and acceptable purity. As demonstrated here, this approach extends the current synthetic options for oligonucleotide modifications and simultaneously facilitates the systematic, rapid biological evaluation of modified siRNA.

An efficient synthesis of a structurally unique, novel M(3) antagonist 1 is described. Compound 1 is conveniently disconnected retrosynthetically at the amide bond to reveal the acid portion 2 and the amine fragment 3. The synthesis of key intermediate 2 is highlighted by a ZnCl(2)-MAEP complex 19 catalyzed diastereoselective Michael reaction of dioxolane 7 with 2-cyclopenten-1-one (5) to establish the contiguous quaternary-tertiary chiral centers and a subsequent geminal difluorination of ketone 17 using Deoxofluor in the presence of catalytic BF(3).OEt(2). The synthesis of the amine moiety 3 is highlighted by the discovery of a novel n-Bu(3)MgLi magnesium-halogen exchange reaction for selective functionalization of 2,6-dibromopyridine. This new and practical metalation protocol obviated cryogenic conditions and upon quenching with DMF gave 6-bromo-2-formylpyridine (26) in excellent yield. Further transformations afforded the amine fragment 3 via reductive amination with 35, Pd-catalyzed aromatic amination, and deprotection. Finally, the highly convergent synthesis of 1 was accomplished by coupling of the two fragments. This synthesis has been used to prepare multi-kilogram quantities of the bulk drug.

Bromopyridine 4 was converted into aminopyridine 5 under Cu2O catalysis with an ethylene glycol solution of ammonia in excellent yield (90%). The amination reaction features low (0.5 mol%) catalyst loading, mild reaction temperature (80°C) and low reaction pressure (50 psi). This protocol is further studied in the amination of a variety of aryl halides.

We report a practical global deprotection of RNA 2'-O-tert-butyldimethylsilyl (TBS) ethers using commercially available aqueous NH(4)F. The procedure is applicable to both 96-well plate format and large-scale production of RNA. This improved procedure provides a safe, mild, and cost-effective alternative to highly hazardous Et(3)N x 3 HF, a reagent commonly used in the routine synthesis of RNA.

The title racemate, C13H19NO2, is a pre-dehydration inter­mediate isolated from the base-catalysed Robinson annulation of (+/−)-2-(2-cyano­ethyl)cyclo­hexa­none. Centrosymmetric dimers are formed by reciprocal hydroxyl-to-ketone hydrogen bonding [O⋯O = 2.7902 (13) A and O—H⋯O = 176°]. One inter­molecular C—H⋯O close contact is found to the hydroxyl group.

[reaction: see text] A practical, ligand-free cyanation of aryl bromides that utilizes as little as 0.1 mol % Pd(OAc)(2) in combination with a nontoxic cyanide source, M(4)[Fe(CN)(6)] (M = K, Na), is described. The reactions are performed in DMAC at 120 degrees C and provide the corresponding aryl nitrile in 83-96% yield, typically in less than 5 h. TON values of up to 7100 were attained.

New mixed-mode columns consisting of reversed-phase and ion-exchange separation modes were evaluated for the analysis of short RNA oligonucleotides (∼20mers). Conventional analysis for these samples typically involves using two complementary methods: strong anion-exchange liquid chromatography (SAX-LC) for separation based on charge, and ion-pair reversed-phase liquid chromatography (IP-RPLC) for separation based on hydrophobicity. Recently introduced mixed-mode high performance liquid chromatography (HPLC) columns combine both reversed-phase and ion-exchange modes, potentially offering a simpler analysis by combining the benefits of both separation modes into a single method. Analysis of a variety of RNA oligonucleotide samples using three different mixed-mode stationary phases showed some distinct benefits for oligonucleotide separation and analysis. When using these mixed-mode columns with typical IP-RPLC mobile phase conditions, such as ammonium acetate or triethylammonium acetate as the primary ion-pair reagent, the separation was mainly based on the IP-RPLC mode. However, when changing the mobile phase conditions to those more typical for SAX-LC, such as salt gradients with NaCl or NaBr, very different separation patterns were observed due to mixed-mode interactions. In addition, the Scherzo SW-C18 and SM-C18 columns with sodium chloride or sodium bromide salt gradients also showed significant improvements in peak shape.

This unit describes two protocols for the deprotection of 2'-O-TBS groups in oligoribonucleotides under mild conditions. Desilylation using ammonium fluoride is applicable to fully protected "RNA only" substrates and desilylation using potassium fluoride is applicable to "mixed RNA/non-RNA" substrates. Characterization of products is accomplished using LC/MS, RP HPLC and SAX HPLC.

Development of a practical synthesis of MK-7009, a 20-membered [corrected] macrocycle, is described. A variety of ring-closing strategies were evaluated, including ring-closing metathesis, intermolecular palladium-catalyzed cross-couplings, and macrolactamization. Ring closure via macrolactamization was found to give the highest yields under relatively high reaction concentrations. Optimization of the ring formation step and the synthesis of key intermediates en route to MK-7009 are reported.

We report a practical global deprotection of RNA 2′-O-tert-butyldimethylsilyl (TBS) ethers using commercially available aqueous NH4F. The procedure is applicable to both 96-well plate format and large-scale production of RNA. This improved procedure provides a safe, mild, and cost-effective alternative to highly hazardous Et3N·3HF, a reagent commonly used in the routine synthesis of RNA.

Bromopyridine 4 was converted into aminopyridine 5 under Cu2O catalysis with an ethylene glycol solution of ammonia in excellent yield (90%). The amination reaction features low (0.5 mol%) catalyst loading, mild reaction temperature (80°C) and low reaction pressure (50 psi). This protocol is further studied in the amination of a variety of aryl halides.

A short and practical synthesis of glucokinase activator 1 was achieved utilizing a convergent strategy involving S(N)Ar coupling of activated aryl fluoride 11 with hydroxypyridine 9. The key to the success of the synthesis was the development of a novel method for enantioselective formation of alpha-arylpyrrolidines during the course of the project. In this method, (-)-sparteine-mediated enantioselective lithiation of N-Boc-pyrrolidine was followed by in situ transmetalation to zinc and Pd-catalyzed coupling with aryl bromide 3, proceeding in 92% ee. This transformation allowed the preparation of compound 1 in a 31% overall yield over six steps.

The cycloacylation of aniline derivatives to 4-quinolones in the presence of Eaton's reagent is described. This high-yielding methodology is applicable to a wide variety of functionalized anilines and requires milder conditions than those traditionally employed. This cyclization protocol is used to prepare a host of heterocycles and bis-quinolones and is characterized by relatively low reaction temperature and ease of product isolation.

Compound 1 is a p38 MAP kinase inhibitor potentially useful for the treatment of rheumatoid arthritis and psoriasis. A novel six-step synthesis suitable for large-scale preparation was developed in support of a drug development program at Merck Research Laboratories. The key steps include a tandem Heck-lactamization, N-oxidation, and a highly chemoselective Grignard addition of 4-(N-tert-butylpiperidinyl)magnesium chloride to a naphthyridone N-oxide. The N-oxide exerted complete chemoselectivity via chelation in directing the Grignard addition to the alpha position as opposed to 1,4-addition on the ene-lactam. The dihydropyridyl adduct was in situ aromatized with isobutylchloroformate followed by heating in pyridine. Syntheses of Grignard precursor, N-tert-butyl-4-chloro-piperidine, were accomplished via transamination with a quaternary ammonium piperidone or via addition of methylmagnesium chloride to an iminium ion. Utilizing this chemistry, multi-kilogram preparation of compound 1 was successfully demonstrated.

The crystal structure of the title compound, (±)-cis-2-oxoperhydronaphthalene-4a-acetic acid, C12H18O3, involves hydrogen-bonding catemers. Hydrogen bonds progress from the carboxyl group of one molecule to the ketone group of a translationally related neighbor [O⋯O 2.715 (3) A]. Eight parallel hydrogen-bonding chains proceed in the a direction through the chosen cell. Two C=O⋯H—C close contacts were found, involving the acid carbonyl group.

The preparation of 3-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)propan-1-amine 2a and 3-[(7R)-7-methyl-5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl]propan-1-amine 2b, key intermediates in the synthesis of alpha(V)beta(3) antagonists, is described. The syntheses rely on the efficient double Sonogashira reactions of 2,5-dibromopyridine 3 with acetylenic alcohols 4a/4b and protected propargylamines 10a-e followed by Chichibabin cyclizations of 3,3'-pyridine-2,5-diyldipropan-1-amines 9a/9b.

Dibenzyloxydihydrobenzoxathiin 1 , which resisted debenzylation with SiCl 4 /LiI, was effectively debenzylated with SiCl 4 /LiI in the presence of a catalytic amount of BF 3 . The HCl salt of the bis-debenzylated product 2 was isolated in 90% yield and 99% purity. This enhanced dealkylation activity has also been observed with other substrates.

An efficient asymmetric synthesis of a selective estrogen receptor modulator (SERM) that has a dihydrobenzoxathiin core structure bearing two stereogenic centers is reported. The stereogenic centers were established by an unprecedented chiral sulfoxide-directed stereospecific reduction of an α,β-unsaturated sulfoxide to the saturated sulfide in one step. Studies to elucidate the mechanism for this reduction are reported. Highly efficient Cu(I)-mediated ether formation was used to install the ether side chain, and selective debenzylation conditions were developed to remove the benzyl protecting groups on the phenols.

A practical and convenient synthesis of azetidine-3-carboxylic acid (1) that proceeded in 55% overall yield from commercially available diethylbis(hydroxymethyl)malonate (3) is reported. Azetidine ring-formation was achieved in high yield by cyclization of bistrifiate of the diol (3) and benzylamine. Decarboxylation under carefully pH-controlled conditions gave the mono acid azetidine that was hydrogenated to give the title compound.

The title racemate, C13H19NO2, was isolated from the Michael condensation of methyl vinyl ketone with 3,4,5,6,7,8-hexa­hydro-2-quinolone under acidic conditions. The compound aggregates as dimers by centrosymmetric hydrogen-bonded pairing of the carboxamide groups [N⋯O = 2.9321 (16) A and N—H⋯O = 174 (2)°]. The packing includes three close inter­molecular dipolar contacts, involving both O atoms.

The title racemate, C17H18O2, is a tetra­cylic diketone isolated from the base-catalysed Robinson annulation of 2-(2-carbethoxy­ethyl)cyclo­hexa­none. It represents a previously unreported four-ring carbocyclic system. In the crystal structure, the packing includes three close inter­molecular C—H⋯O dipolar contacts, involving both ketones.