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.

Automated ion exchange chromatography screening combined with in silico multifactorial simulation for efficient method development and purification of biopharmaceutical targets.

Bioprocess development of increasingly challenging therapeutics and vaccines requires a commensurate level of analytical innovation to deliver critical assays across functional areas. Chromatography hyphenated to numerous choices of detection has undeniably been the preferred analytical tool in the pharmaceutical industry for decades to analyze and isolate targets (e.g., APIs, intermediates, and byproducts) from multicomponent mixtures. Among many techniques, ion exchange chromatography (IEX) is widely used for the analysis and purification of biopharmaceuticals due to its unique selectivity that delivers distinctive chromatographic profiles compared to other separation modes (e.g., RPLC, HILIC, and SFC) without denaturing protein targets upon isolation process. However, IEX method development is still considered one of the most challenging and laborious approaches due to the many variables involved such as elution mechanism (via salt, pH, or salt-mediated-pH gradients), stationary phase's properties (positively or negatively charged; strong or weak ion exchanger), buffer type and ionic strength as well as pH choices. Herein, we introduce a new framework consisting of a multicolumn IEX screening in conjunction with computer-assisted simulation for efficient method development and purification of biopharmaceuticals. The screening component integrates a total of 12 different columns and 24 mobile phases that are sequentially operated in a straightforward automated fashion for both cation and anion exchange modes (CEX and AEX, respectively). Optimal and robust operating conditions are achieved via computer-assisted simulation using readily available software (ACD Laboratories/LC Simulator), showcasing differences between experimental and simulated retention times of less than 0.5%. In addition, automated fraction collection is also incorporated into this framework, illustrating the practicality and ease of use in the context of separation, analysis, and purification of nucleotides, peptides, and proteins. Finally, we provide examples of the use of this IEX screening as a framework to identify efficient first dimension (1D) conditions that are combined with MS-friendly RPLC conditions in the second dimension (2D) for two-dimensional liquid chromatography experiments enabling purity analysis and identification of pharmaceutical targets.

A kinase-cGAS cascade to synthesize a therapeutic STING activator.

The introduction of molecular complexity in an atom- and step-efficient manner remains an outstanding goal in modern synthetic chemistry. Artificial biosynthetic pathways are uniquely able to address this challenge by using enzymes to carry out multiple synthetic steps simultaneously or in a one-pot sequence1-3. Conducting biosynthesis ex vivo further broadens its applicability by avoiding cross-talk with cellular metabolism and enabling the redesign of key biosynthetic pathways through the use of non-natural cofactors and synthetic reagents4,5. Here we describe the discovery and construction of an enzymatic cascade to MK-1454, a highly potent stimulator of interferon genes (STING) activator under study as an immuno-oncology therapeutic6,7 (ClinicalTrials.gov study NCT04220866 ). From two non-natural nucleotide monothiophosphates, MK-1454 is assembled diastereoselectively in a one-pot cascade, in which two thiotriphosphate nucleotides are simultaneously generated biocatalytically, followed by coupling and cyclization catalysed by an engineered animal cyclic guanosine-adenosine synthase (cGAS). For the thiotriphosphate synthesis, three kinase enzymes were engineered to develop a non-natural cofactor recycling system in which one thiotriphosphate serves as a cofactor in its own synthesis. This study demonstrates the substantial capacity that currently exists to use biosynthetic approaches to discover and manufacture complex, non-natural molecules.

Generic anion-exchange chromatography method for analytical and preparative separation of nucleotides in the development and manufacture of drug substances.

Nucleotides are among the most frequently used chemical building blocks in the research, development and manufacture of drug substances. They are composed of three highly polar subunit molecules (a nucleobase, a sugar, and at least one phosphate group), which makes their separation and analysis very challenging by conventional liquid chromatography techniques. Herein, we describe a simple, efficient, and cost-effective ion-exchange chromatography (IEC) method for the separation and purification of over 20 nucleotides. This method combines the use of a Tosoh TSKgel SuperQ-5P W resin in conjunction with a fully aqueous eluent profile (ammonium bicarbonate-based) that allows for a straightforward scale-up transition and convenient drying process with minimal environmental impact. This generic method was optimized using chromatography simulation software (ACD Labs/LC Simulator) and successfully applied to the preparative purification of multicomponent nucleotide mixtures using readily available Fast Protein Liquid Chromatography (FPLC) instrumentation. These IEC method conditions can be effectively applied as the starting point for method development and isolation of other highly polar nucleotide species beyond those investigated in this study.

Synthesis of a naphthyridone p38 MAP kinase inhibitor.

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.

Stereoselective syntheses of highly functionalized bicyclo[3.1.0]hexanes: a general methodology for the synthesis of potent and selective mGluR2/3 agonists.

[Chemical reaction: See text] A Et3Al mediated intramolecular epoxide opening, cyclopropanation reaction is described. The transformation provided highly functionalized bicyclo[3.1.0]hexane systems in high efficiency and with perfect H or F endo selectivity. Application of this reaction to the synthesis of mGluR2/3 agonist 1 (43% overall yield) and a few intermediates suitable for the synthesis of other bicyclo[3.1.0]hexane mGluR2/3 agonists is discussed.

An efficient synthesis of a dual PPAR alpha/gamma agonist and the formation of a sterically congested alpha-aryloxyisobutyric acid via a Bargellini reaction.

A practical synthesis of benzisoxazole 1 and its conversion to alpha-aryloxyisobutyric acid 2 using 1,1,1-trichloro-2-methyl-2-propanol (chloretone) was developed. Benzisoxazole 1 was formed in high yields by the action of either methanesulfonyl chloride/base upon intermediate oxime 8 or with thionyl chloride/base, which initially forms cyclic sulfite 10. A highly reactive, short-lived intermediate derived from chloretone was detected by ReacIR and its half-life determined to be approximately 5 min. Reaction conditions for the Bargellini reaction were developed that resulted in a 95% yield of 2 from the reaction of highly hindered phenol 1 with chloretone hemihydrate and powdered NaOH in acetone. Thus highly hindered alpha-aryloxyisobutyric acids can be made in a single step in high yield.

Practical synthesis of the new carbapenem antibiotic ertapenem sodium.

[reaction: see text] A practical synthesis for the large-scale production of the new carbapenem antibiotic, [4R,5S,6S]-3-[[(3S,5S)-5-[[(3-Carboxyphenyl)amino]carbonyl]-3-pyrrolidinyl]thio]-6-[(1R)-1-hydroxyethyl]-4-methyl-7-oxo-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid monosodium salt (ertapenem sodium, 1), has been developed. The synthesis features the novel use of 1,1,3,3-tetramethylguanidine as base for the low-temperature reaction of a thiol, derived from trans-4-hydroxy-L-proline, with the carbapenem nucleus activated as the enol phosphate. Hydrogenolysis of a p-nitrobenzyl ester is effected using a palladium on carbon catalyst to give an overall yield for the two steps of 90%. The use of bicarbonate in the hydrogenolysis was key in providing protection of the pyrrolidine amine as the sodium carbamate improving both the performance of the reaction and the stability of the product. This discovery made processing at manufacturing scale possible. Experimental evidence for the formation of the sodium carbamate is provided. A remarkably expedient process for the simultaneous purification and concentration of the aqueous product stream relies on ion-pairing extraction for the removal of the water-soluble 1,1,3,3-tetramethylguanidine. Crystallization then affords 59-64% overall yield of the monosodium salt form of the product.

Stereoselective synthesis of an anti-HIV drug candidate.

The asymmetric synthesis of a Merck anti-HIV drug candidate is described. The target molecule contains four stereogenic centers, three of which are located in a highly functionalized cyclopentane unit. The convergent synthesis involves the preparation of two key advanced intermediates: the cyclopentane unit and a substituted pyrazole unit. The cyclopentane unit was prepared via two different procedures; a highly diastereoselective Diels-Alder reaction with a chiral oxazolidinone auxiliary and a sequence that incorporated a molybdenum-catalyzed asymmetric allylic alkylation reaction to set the stereocenters. The other key step was a highly diastereoselective hydroxyl-directed reductive amination. The overall yield for the 16-step synthesis was 10%.

Synthesis of 3-aminopyrazinone mediated by 2-pyridylthioimidate-ZnCl2 complexes. Development of an efficient route to a thrombin inhibitor.

A six-step preparation of thrombin inhibitor drug candidate 1 from pyrazinone 7 in 47% overall yield is described. The problem of low reactivity between weak amine nucleophile 4 and poor electrophile 3-bromopyrazinone 17 was overcome with the use of pyridinylthioimidate 27 in the presence of ZnCl(2) to afford adduct 3 in high yield. Several zinc complexes were characterized by solution and solid-state NMR and X-ray crystallographic analyses, and provided insight into the reaction mechanism. Preparation of pyridine N-oxide amine 4 was accomplished via a selective oxidation of the corresponding pyridinylamine 6. Pyridinylthioimidate 27 was prepared from pyrazinone 7 via a two-step one-pot process in near quantitative yield. Chlorination of the pyrazinone ring in 3 followed by hydrolysis and amide coupling completed the synthesis of 1. This chromatography-free synthesis was used successfully to prepare multikilogram quantities of the drug with reproducibility and high purity.

An improved preparation of 3,5-bis(trifluoromethyl)acetophenone and safety considerations in the preparation of 3,5-bis(trifluoromethyl)phenyl Grignard reagent.

An improved and efficient bromination of 3,5-bis(trifluoromethyl)benzene was developed. A safe and reliable preparation of the potentially explosive 3,5-bis(trifluoromethyl)phenyl Grignard and 3-trifluoromethylphenyl Grignard reagents, from the precursor bromides, is described. Reaction System Screening Tool (RSST) and Differential Thermal Analysis (DTA) studies suggest these trifluoromethylphenyl Grignard reagents can detonate on loss of solvent contact or upon moderate heating. When prepared and handled according to the methods described herein, these Grignard reagents can be safely prepared and carried on to advanced intermediates.

Development of a new and practical route to chiral 3,4-disubstituted cyclopentanones: asymmetric alkylation and intramolecular cyclopropanation as key C-C bond-forming steps.

An efficient and practical asymmetric synthesis of (+)-trans-3-hydroxymethyl-4-(3-fluorophenyl)cyclopentanone (1) is described. An asymmetric Mo-catalyzed alkylation reaction was used to establish the first stereocenter and a Cu-catalyzed intramolecular diastereoselective cyclopropanation reaction was used to set the second stereocenter. The last step involved a one-pot ring-opening/deprotection/hydrolysis/decarboxylation sequence that furnished the desired product in good yield.

Highly Chemoselective Trichloroacetimidate-Mediated Alkylation of Ascomycin: A Convergent, Practical Synthesis of the Immunosuppressant L-733,725.

L-733,725, a new immunosuppressant drug candidate, was prepared by a highly chemoselective alkylation of the macrolide ascomycin at the C32 hydroxy position with the imidazolyl trichloroacetimidate 16. The trichloroacetimidate-activated side chain 16 was prepared by an efficient four-step sequence in 42% overall yield. The high chemoselectivity in the alkylation of the C32 hydroxy group of the unprotected ascomycin was the result of the synergetic effects of the electron-donating protecting group on the imidazole 16, the polar, moderately basic solvent, and the strong acid catalyst. N,N-Dimethylpivalamide mixed with acetonitrile was found to be the best solvent and trifluromethanesulfonic acid the best catalyst. This synthesis coupled with a resin column purification of L-733,725 followed by crystallization of its tartrate salt has been used to make multi-kilogram quantities of the bulk drug with consistent and high purity.