Linker Architectures as Steric Auxiliaries for Altering Enzyme-Mediated Payload Release from Bioconjugates.

Protease-activated prodrugs leverage the increased activity of proteases in the tumor microenvironment and the tight regulation in healthy tissues to provide selective activation of cytotoxins in the tumor while minimizing toxicity to normal tissues. One of the largest classes of protease-activated prodrugs are composed of therapeutic agents conjugated to macromolecular carriers via peptide motifs that are substrates for cathepsin B, and antibody-drug conjugates are one of the most successful designs within this class. However, many of these peptide motifs are also cleaved by extracellular enzymes such as elastase and carboxylesterase 1C. Additionally, some peptide sequences have little selectivity for other lysosomal cathepsins, which have also been found to have extracellular activity in normal physiological processes. A lack of selectivity or oversensitivity to other extracellular enzymes can lead to off-target release of the cytotoxic payload and subsequent toxicities. In this report, we describe an approach for modulating cathepsin-mediated release of the cytotoxic payload through steric shielding provided by the synergistic effects of appropriately designed hydrophilic linkers and the conjugated carrier. We prepared a fluorogenic model payload with a Val-Cit cleavable trigger and attached the trigger-payload to a variety of PEG-based linker architectures with different numbers of PEG arms (y), different numbers of ethylene oxide units in each arm (n), and different distances between the cleavable trigger and PEG branch point (D'). These linker-payloads were then used to prepare DAR2 conjugates with the cleavable triggers at three different distances (D) from the antibody, and cathepsin-mediated payload release was monitored with in vitro assays. The results show that structural variables of the linker architectures can be manipulated to effectively shield enzymatically labile trigger-payloads from enzymes with readily accessible binding sites, and may offer an additional strategy for balancing off-target and tumor-targeted payload release.

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.

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.

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 [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.