Umpolung Flow Chemistry for the Synthesis of a 3-Oxo-3H-spiro[benzofuran-2,4'-piperidine] Building Block.

An efficient and scalable route to tert-butyl 3-oxo-3H-spiro[benzofuran-2,4'-piperidine]-1'-carboxylate, a central prochiral intermediate in the synthesis of SHP2 inhibitor GDC-1971 (migoprotafib), was achieved. Preparation of the title compound from readily available 2-fluorobenzaldehyde included formation of a modified Katritzky benzotriazole hemiaminal, which, upon deprotonation by n-butyllithium, participated in umpolung reactivity via 1,2-addition to tert-butyl 4-oxopiperidine-1-carboxylate (N-Boc-4-piperidone). Most notably, this reaction was developed as a robust plug-flow process that could be executed on multiple kilograms without the need for pilot-scale reaction vessels operating at low cryogenic temperatures. Treatment of the resulting tetrahedral intermediate with oxalic acid resulted in collapse to the corresponding 4-(2-fluorobenzoyl)-4-hydroxypiperidine, which was isolated as a solid via crystallization. The synthesis concluded with an optimized intramolecular SNAr reaction and final crystallization to generate tert-butyl 3-oxo-3H-spiro[benzofuran-2,4'-piperidine]-1'-carboxylate as a stable, high-quality intermediate suitable for further functionalization toward GDC-1971.

Stereoselective Synthesis of the IDO Inhibitor Navoximod.

A highly efficient asymmetric synthesis of the IDO inhibitor navoximod, featuring the stereoselective installation of two relative and two absolute stereocenters from an advanced racemic intermediate, is described. The stereocenters were set via a crystallization-induced dynamic resolution along with two selective ketone reductions: one via a biocatalytic ketoreductase transformation and one via substrate-controlled hydride delivery from LiAlH(Ot-Bu)3. Following this strategy, navoximod was synthesized in 10 steps from 2-fluorobenzaldehyde and isolated in 23% overall yield with 99.7% ee and high purity.

Identification of an acetonitrile addition impurity formed during peptide disulfide bond reduction using dithiothreitol and Tris(2-carboxyethyl)phosphine.

Identification and localization of modifications in peptides containing multiple disulfide bonds is challenging due to inefficient fragmentation in mass spectrometry (MS) analysis. In cases where MS fragmentation techniques such as electron capture dissociation (ECD), electron transfer dissociation (ETD), and ultraviolet photodissociation (UVPD) fail to achieve efficient fragmentation, off-line disulfide bond reduction techniques are typically employed prior to MS analysis. Some commonly used reducing agents include dithiothreitol (DTT) and tris(2-carboxyethyl)phosphine (TCEP). In this work, we describe the detection and identification of an unexpected impurity that formed during the reduction of Peptide A, containing multiple disulfide bonds, while using DTT or TCEP as reducing agents and acetonitrile as a co-solvent. The DTT reduced products were found to be a mixture of the expected linear Peptide A (fully reduced) and an unknown product (>50%) with a mass corresponding to linear Peptide A plus 41 Da ([reduced-M + 41]). A series of experiments were subsequently performed to investigate the identity and origin of this impurity. Disulfide bond reduction with DTT was performed in aqueous mixtures containing acetonitrile, methanol, and deuterated acetonitrile; and with TCEP in aqueous mixtures containing acetonitrile. Additionally, glycine amino acid was used as a surrogate to investigate the mechanism. The liquid chromatography-mass spectrometry (LCMSMS) results demonstrated that the [reduced-M + 41] impurity was an acetonitrile addition on the peptide's N-terminal glycine. The corresponding impurity [M + 41] was also found in the native Peptide A (non-reduced), suggesting that small amounts of this impurity may also be generated during the synthesis in the upstream process steps. By understanding the formation of this process-related impurity [M + 41], one could potentially reduce or eliminate its presence in Peptide A through chemical controls. Finally, this observation provides caution against using acetonitrile as a co-solvent during DTT- or TCEP-promoted reduction of peptides with an uncapped N-terminus primary amine.

Pyrimidoaminotropanes as potent, selective, and efficacious small molecule kinase inhibitors of the mammalian target of rapamycin (mTOR).

We have recently reported a series of tetrahydroquinazoline (THQ) mTOR inhibitors that produced a clinical candidate 1 (GDC-0349). Through insightful design, we hoped to discover and synthesize a new series of small molecule inhibitors that could attenuate CYP3A4 time-dependent inhibition commonly observed with the THQ scaffold, maintain or improve aqueous solubility and oral absorption, reduce free drug clearance, and selectively increase mTOR potency. Through key in vitro and in vivo studies, we demonstrate that a pyrimidoaminotropane based core was able to address each of these goals. This effort culminated in the discovery of 20 (GNE-555), a highly potent, selective, metabolically stable, and efficacious mTOR inhibitor.