Lipid nanoparticle purification by spin centrifugation-dialysis (SCD): a facile and high-throughput approach for small scale preparation of siRNA-lipid complexes.

This paper describes the use of spin centrifugation-dialysis (SCD) for small-scale concentration/purification of siRNA-lipid complexes designed for use as therapeutic agents for gene silencing. SCD consists of a two-step method for concentration, filtration and buffer exchange of lipid nanoparticles (LNP) to provide a homogeneous preparation suitable for injection. Here, we compare SCD with the more traditionally used tangential flow filtration (TFF), and demonstrate the physicochemical and biological comparability of LNPs produced with both methods. TFF is a highly scalable method used in both developmental and production applications, but is limited in terms of miniaturization. In contrast to TFF, SCD is faster, less expensive, and requires less oversight for assembling LNPs for small-scale applications, such as target screening both in vitro and in vivo. The finding that SCD is a viable method for filtering LNPs in a manner similar to TFF, producing particles with comparable properties and biological activity, is significant given the complexity and sensitivity of LNPs to processing conditions.

Development of a pharmaceutical cocrystal of a monophosphate salt with phosphoric acid.

We report the first case of a pharmaceutical cocrystal formed between an inorganic acid and an active pharmaceutical ingredient (API), which enabled us to develop a stable crystalline and bioavailable solid dosage form for pharmaceutical development where otherwise only unstable amorphous free form or salts could have been used.

Effective use of preparative chiral HPLC in a preclinical drug synthesis.

Access to a key 3-aryl-delta-lactone intermediate in enantiopure form using preparative chiral chromatography allowed expedited preparation of an important drug discovery target. A preclinical drug discovery strategy that combines rapid route discovery with effective use of preparative chiral chromatography can result in significant savings of both time and labor.

Crystal structure and surface properties of an investigational drug--a case study.

In this study we investigate the correlations between the single crystal structure, the crystal habitat and morphology, and surface energetics of an investigational pharmaceutical compound. Crystal structure of this investigational pharmaceutical solid has been solved from single crystal X-ray analysis. Crystallographic data are as follows: triclinic, P1 (no. 1), a = 6.1511 (8) A, b = 13.5004 (18) A, c = 17.417 (2) A, alpha = 68.259 (2) degrees, beta = 80.188 (2) degrees, gamma = 82.472 (2) degrees, V = 1320.2 (3) A(3), Z = 2. The external morphology of this crystalline solid was predicted by molecular modelling using attachment energies to be thin-plate like with a dominant face (001). The predicted morphology was confirmed by scanning electron micrographs (SEM) and the Miller Index of the dominant face was complemented by X-ray powder diffraction (XRPD) method. The microscopic layering structures of crystals and surface stability of the dominant faces were investigated using atomic force microscopy (AFM). Contact angle measurement showed that the surface of the dominant face is hydrophilic as predicted from crystal structure.

An efficient asymmetric synthesis of an estrogen receptor modulator by sulfoxide-directed borane reduction.

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 alpha,beta-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.

Mechanistic studies of the molybdenum-catalyzed asymmetric alkylation reaction.

Enantiomerically enriched, deuterated branched carbonates (Z)-(S)-PhCH(OCO(2)Me)-CH = CHD (1-D), (Z)-(R)-PhCH(OCO(2)Me)CH = CHD (2-D), and linear carbonate (E)-(S)-PhCH = CHCHD(OCO(2)Me) (3-D) were used as probes in the Mo-catalyzed asymmetric allylic alkylation with sodium dimethyl malonate, catalyzed by ligand-complex 11 derived from the mixed benzamide/picolinamide of (S,S)-transdiaminocyclohexane and (norbornadiene)Mo(CO)(4). The results of these studies, along with x-ray crystallography and solution NMR structural analysis of the pi-allyl intermediate, conclusively established the reaction proceeded by a retention-retention pathway. This mechanism contrasts with that defined for Pd-catalyzed allylic alkylations, which proceed by an inversion-inversion pathway. A proposed rationale for the retention pathway for nucleophilic substitution involves CO-coordination to form a tri-CO intermediate, followed by complexation with the anion of dimethyl malonate to produce a seven-coordinate intermediate, which reductively eliminates to afford product with retention of configuration.

Highly diastereoselective heterogeneously catalyzed hydrogenation of enamines for the synthesis of chiral beta-amino acid derivatives.

Pure (Z)-enamines readily prepared from beta-ketoesters and amides using (S)-phenylglycine amide were hydrogenated with very high diastereoselectivities (up to 200:1) using heterogeneous catalysis. Hydrogenolytic cleavage of the (S)-phenylglycine amide afforded the corresponding chiral beta-aminoesters and amides. The high geometrical purity of the (Z)-enamine and a simple activation procedure for the PtO2 catalyst are essential in achieving high selectivity.

Nucleophilic displacement at benzhydryl centers: asymmetric synthesis of 1,1-diarylalkyl derivatives.

[reaction: see text] Activation of substituted 1,1-diarylmethanols as their corresponding toluenesulfonates and subsequent displacement with a range of carbon, nitrogen, oxygen, and sulfur nucleophiles proceeds in 81-96% yield. Enantiomerically enriched diarylmethanols 8a-c were activated and displaced with pyridine acetate enolate with complete stereochemical inversion at carbon to yield 1,1-diarylalkyl derivatives 10a-c without loss of optical purity.

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.

Efficient synthesis of NK(1) receptor antagonist aprepitant using a crystallization-induced diastereoselective transformation.

An efficient stereoselective synthesis of the orally active NK(1) receptor antagonist Aprepitant is described. A direct condensation of N-benzyl ethanolamine with glyoxylic acid yielded a 2-hydroxy-1,4-oxazin-3-one which was activated as the corresponding trifluoroacetate. A Lewis acid mediated coupling with enantiopure (R)-1-(3,5-bis(trifluoromethyl)phenyl)ethan-1-ol afforded a 1:1 mixture of acetal diastereomers which was converted into a single isomer via a novel crystallization-induced asymmetric transformation. The resulting 1,4-oxazin-3-one was converted via a unique and highly stereoselective one-pot process to the desired alpha-(fluorophenyl)morpholine derivative. Interesting and unexpected [1,2]-Wittig and [1,3]-sigmatropic rearrangements were identified during the optimization of these key steps. In the final step, a triazolinone side chain was appended to the morpholine core. The targeted clinical candidate was thus obtained in 55% overall yield over the longest linear sequence.

Studies on the racemization of a stereolabile 5-aryl-thiazolidinedione.

The enantiomers of the stereolabile peroxisome proliferator-activated receptor (PPAR) agonist, 1, were isolated by preparative chiral chromatography and their absolute configuration established using a combination of chromatographic and NMR methods. Enantiomer interconversion was investigated under a variety of conditions, with rapid racemization being observed in most solvents, including all aqueous systems studied, irrespective of pH. Rapid racemization in both dog and human plasma was confirmed by chiral HPLC with MS detection.

The unusual role of CO transfer in molybdenum-catalyzed asymmetric alkylations.

Spectroscopic and crystallographic studies were undertaken to gain insight into the mechanism of the highly regio- and enantioselective allylic aklylation reaction catalyzed by molybdenum. The chiral ligand (L*) consisting of the mixed benzamide/picolinamide of (S,S,)-trans-1,2-diaminocyclohexane reacts with a typical Mo precatalyst, (norbornadiene)Mo(CO)4, to give a neutral complex L*Mo(CO)4 in which the ligand binds to the metal in a bidentate fashion through the pyridine and adjacent amide group. Reaction of this complex with the methyl carbonate of cinnamyl alcohol gives the corresponding pi-allyl complex L*(CO)2Mo(eta3-CH2=CH-CHPh). NMR and X-ray crystallographic characterization of this complex reveal the ligand binds in a facially capping tridentate fashion via the pyridine nitrogen, the nitrogen of the adjacent amide group, which has now been deprotonated, and the carbonyl oxygen of the remote amide. Surprisingly, the face of the allyl group open to attack with nucleophiles is that which would lead to the sense of stereochemistry opposite to that which is observed in catalytic reactions. Furthermore, the allyl complex in its isolated form is unreactive toward sodium dimethyl malonate. However, in the presence of a source of carbon monoxide (either Mo(CO)6 or gaseous CO), the allyl complex reacts with malonate to give the typically observed branched alkylated product in high yield and enantiomeric excess. The metal-containing product of this reaction is the molybdate complex [L*Mo(CO)4]-Na+. Reaction of the molybdate complex with linear or branched allylic carbonates regenerates the allyl complex, thus closing the catalytic cycle. Both the allyl complex and the molybdate complex are the only metal-containing species observed by NMR in typical catalytic reactions and thus appear to be catalyst resting states. Turnover of the catalytic cycle therefore involves shuttling of carbon monoxide between the two catalyst resting states. Coordination of CO appears to be necessary to activate the allyl complex toward nucleophilic attack, in effect stabilizing the molybdenum fragment as a leaving group.

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.