David A. Hunt

Development of Small Molecular Proteasome Inhibitors Using a Caenorhabditis elegans Screen

We have developed a screening protocol to identify compounds with characteristics of small molecule proteasome inhibitors using the real-time analysis of the Caenorhabditis elegans germ line. This screen is able to identify compounds that induce germ line phenotypes characteristic of a reduction in proteasome function such as changes in polarity, aberrant nuclear morphology, and stimulation of apoptosis. This basic protocol is amenable to a high throughput (96-well) format and has been used successfully to identify multiple compounds for further analysis based on structural elements from the naturally occurring compounds lactacystin and the β-lactone homologs omuralide and salinosporamide A. The further development of this assay system should allow for the generation of novel small molecule proteasome inhibitors in a genetically tractable whole animal amenable to biochemical analysis.

Expedient Preparation of 4,6-Dihalo-3-arylisobenzofuran-1(3H)-ones from 3,5-Dihalo-N-ethylbenzamides

3,5-Dihalobenzoic acid derivatives have long been known as scaffolds for compounds exhibiting a variety of biological activities. For example, compounds incorporating this moiety exhibit phytotoxicity,1 selective inhibition of 11β-HSD1 for the treatment of metabolic syndrome,2 MCH 1 receptor antagonism,3 and serve as intermediates in the preparation of liquid crystals, fungicides, organo-tin antitumor compounds, insecticides, and quinolones possessing antibiotic activity.4 While our previous studies have shown that 3,5-dichloro tertiary benzamides are metalated para to the carboxamide group,5 we have found that use of secondary amides derived from 3,5-dihalobenzoic acids (1, X = Cl, F) result in exclusive ortho-metalation. In light of the paucity of methods available to prepare highly ring-functionalized derivatives of 3,5-dihalobenzoic acids, coupled with recent studies documenting the unique antitumor properties of benzolactones,6 we now describe the application of the chelation-controlled directed ortho-metalation reaction in this series as a useful method for the preparation of 4,6-dihalo-3-arylisobenzofuran-1(3H)-ones. Metalation of 3,5-dihalo secondary benzamides (1) with sec-butyllithium resulted in exclusive ortho-directed metalation. Use of an aromatic aldehyde as the electrophile results in a lithioalkoxy intermediate (3) which undergoes nucleophilic attack upon the adjacent amide carbonyl during work-up with a concomitant loss of ethylamine, resulting in the formation of an aryl substituted dihalobenzofuranone (4, Table 1). This methodology should be applicable to a wide variety of both aromatic and aliphatic aldehydes based on reported reactions of aldehydes with aryllithium reagents generated by the directed orthometalation strategy.7 Studies of these systems as well as the scope and limitations of this process will be the focus of future investigations.

1-Methyl-4H-3,1-benzoxazine-2,4(1H)dione

In its crystal structure, the title compound, C9H7NO3, forms π-stacked dimers, with a centroid–centroid distance of 3.475 (5) Å between the benzenoid and the 2,4 dicarbonyl oxazine rings. These dimers then form staircase-like linear chains through further π-stacking between the benzenoid rings [centroid–centroid distance of 3.761 (2) Å]. The methyl-H atoms are disordered due to rotation about the C—N bond and were modeled with equal occupancy.