The development of a convergent, chromatography-free synthesis of an allosteric Akt kinase inhibitor is described. The route comprised 17 total steps and was used to produce kilogram quantities of the target molecule. A key early transformation, for which both batch and flow protocols were developed, was formylation of a dianion derived by deprotonation and subsequent lithium-halogen exchange from a 2-bromo-3-aminopyridine precursor. Improved reaction yield and practicality were achieved in the continuous processing mode. Further significant process developments included the safe execution of a high temperature and pressure hydrazine displacement, separation of substituted cyclobutane diastereomers by means of chemoselective ester hydrolysis, and a late-stage Suzuki fragment coupling under mild conditions. ■ RESULTS AND DISCUSSIONSynthesis of Triflate 4. Pyridyl aldehyde 5 was previously prepared on gram scale in 5 steps from picoline derivative 9. 6 A

Compound (1) a poly(ADP-ribose)polymerase (PARP) inhibitor has been made by a fit-for-purpose large-scale synthesis using either a classical resolution or chiral chromatographic separation. The development and relative merits of each route are discussed, along with operational improvements and extensive safety evaluations of potentially hazardous reactions.

Synthetic diazeniumdiolate (DAZD)-based nitric oxide is utilized to modulate the nitric oxide (NO) concentration in cellular environments and to control physiological processes, yet chemists are still struggling to find efficient and scalable methodologies that will enable them to access sufficient quantities of the high-energy diazeniumdiolate intermediates for biological studies. Now, a general, scalable, safer, and high-yielding new methodology adaptable to the large-scale synthesis of DAZDs has been developed.

A six-step route starting from a readily available vinyl boronate was identified to produce an enantioenriched cyclopropanol in an overall 16% yield. Key steps involve the use of lithium acetylide-ethylene diamine complex 5 and an enzymatic resolution of a racemic cyclopropanol acetate. Process safety considerations surrounding the use of 5 were examined, and an improved procedure is described which was safely demonstrated at multikilogram scale.

The growth and function of primary rat hepatocytes was characterized on commercial, electrostatically treated nonwoven polyester carriers (Fibra-Cel) designed for use in a packedbed bioreactor. Such carriers are attractive in that they allow three-dimensional cellular interactions and afford shear protection. To initiate investigations of the potential of these carriers for hepatocyte support, novel hepatocyte media were evaluated along with a variety of carrier surface treatments in static cell culture. Different serum-free chemically defined media with recombinant growth factors known to be complete mitogens for hepatocytes were contrasted. Carrier treatments included unmodified surfaces contrasted with carriers coated with type I collagen or HepG2-derived extracellular matrix. Hepatocytes were also co-cultured with nonparenchymal cells. Cellular mitochondrial metabolism of 3-(4,5-dimethylthiozol-2-yellow)-2,5 diphenyl tetrazolium bromide (MTT) was used to ascertain continued viability. Cell proliferation was assessed by total DNA measurements. Cell morphology was monitored by phase microscopy. 7-Ethoxycoumarin deethylation was used to indicate cytochrome p450 function. MTT metabolism initially declined, but stabilized at 8 days, and then was maintained on the treated carriers for at least 30 days with a novel hepatocyte media supplemented with hepatocyte growth factor and epidermal growth factor. Carrier treatment also had significant effects on MTT metabolism with HepG2 matrices > collagen > untreated carriers. Co-culture with nonparenchymal cells also appeared to improve MTT conversion by hepatocytes substantially over those cultured on carriers treated with collagen. Evenly distributed throughout the carriers early in the culture period, hepatocyte clustering became apparent over time. Cytochrome p450 activity was sustained for at least 18 days in hepatocytes maintained in static culture on carriers treated with collagen. The combination of this commercial carrier system with specifically designed hepatocyte media and modified carrier substrates holds promise as a new avenue for bioartificial liver design.