Development and Validation of a Reliable and Robust Method for the Analysis of Cannabinoids and Terpenes in Cannabis.

The requirements for an acceptable cannabis assay have changed dramatically over the years resulting in a large number of laboratories using a diverse array of analytical methodologies that have not been properly validated. Due to the lack of sufficiently validated methods, we conducted a single- laboratory validation study for the determination of cannabinoids and terpenes in a variety of commonly occurring cultivars. The procedure involves high- throughput homogenization to prepare sample extract, which is then profiled for cannabinoids and terpenes by HPLC-diode array detector and GC-flame ionization detector, respectively. Spike recovery studies for terpenes in the range of 0.03-1.5% were carried out with analytical standards, while recovery studies for Δ9-tetrahydrocannabinolic acid, cannabidiolic acid, Δ9-tetrahydrocannabivarinic acid, and cannabigerolic acid and their neutral counterparts in the range of 0.3-35% were carried out using cannabis extracts. In general, accuracy at all levels was within 5%, and RSDs were less than 3%. The interday and intraday repeatabilities of the procedure were evaluated with five different cultivars of varying chemotype, again resulting in acceptable RSDs. As an example of the application of this assay, it was used to illustrate the variability seen in cannabis coming from very advanced indoor cultivation operations.

Non-enzymatic cyclization of creatine ethyl ester to creatinine.

Creatine ethyl ester was incubated at 37 degrees C in both water and phosphate-buffered saline and the diagnostic methylene resonances in the (1)H NMR spectrum were used to identify the resultant products. It was found that mild aqueous conditions result in the cyclization of creatine ethyl ester to provide inactive creatinine as the exclusive product, and this transformation becomes nearly instantaneous as the pH approaches 7.4. This study demonstrates that mild non-enzymatic conditions are sufficient for the cyclization of creatine ethyl ester into creatinine, and together with previous results obtained under enzymatic conditions suggests that there are no physiological conditions that would result in the production of creatine. It is concluded that creatine ethyl ester is a pronutrient for creatinine rather than creatine under all physiological conditions encountered during transit through the various tissues, thus no ergogenic effect is to be expected from supplementation.

An intra/intermolecular suzuki sequence to benzopyridyloxepines containing geometrically pure exocyclic tetrasubstituted alkenes.

A route to enable the preparation of 5-benzylidenyl-benzopyridyloxepine analogues was developed to continue our research in the field of nuclear hormone receptor modulators. The key steps are 1) a syn-stereoselective diboration of a tethered aryl alkyne; 2) an intramolecular Suzuki cross-coupling reaction, which forms in a stereo- and regiocontrolled fashion, the 5-exoalkylidenyl 7-membered ring imbedded within the core of the scaffold and; 3) an intermolecular Suzuki to furnish the final tetra-substituted olefinic benzopyridyloxepines.

Stereoselective synthesis of the rocaglamide skeleton via a silyl vinylketene formation/[4 + 1] annulation sequence.

The tricyclic core of the cyclopentabenzofurans has been prepared in an efficient and stereoselective manner utilizing an intramolecular silyl vinylketene formation/[4 + 1] annulation sequence. This novel approach affords the ABC ring system where the adjacent phenyl and aryl substituents of the C ring have the required cis relationship.

Stereoselective [4 + 1] annulation reactions with silyl vinylketenes derived from Fischer carbene complexes.

Stable silyl vinylketenes were prepared via the thermal reaction of Fischer carbene complexes with triisopropylsilyl- or tert-butyldimethylsilyl-substituted alkynes. The ability of these silyl vinylketenes to participate with carbenoid reagents in [4 + 1] annulation reactions was investigated. The best results were obtained with diazomethane and substituted diazomethane reagents, which provided cyclopentenone products in excellent yields and essentially complete stereoselectivity.

Construction of the benzindenoazepine skeleton via cyclopentannulation of Fischer aminocarbene complexes: total synthesis of bulgaramine.

A total synthesis of bulgaramine has been accomplished with a longest linear sequence of eight steps and an overall yield of 23% from commercially available 3,4-dimethoxyphenethyl alcohol. An intramolecular cyclopentannulation reaction of a Fischer aminocarbene complex provided the key step and occurred under significantly milder conditions and in higher yields than those of other reported examples of this reaction type. The reaction solvent was a critical factor in the cyclopentannulation reaction, with measurable amounts of the desired product observed only when THF was utilized. The product yield could be further enhanced by the addition of two-electron donor ligands, demonstrating the first example of this effect on the thermal reaction of aminocarbene complexes with alkynes.