CYP1A1-Mediated Intramolecular Rearrangement of Aminoazepane in GDC-0339.

GDC-0339 is a novel small molecule pan-Pim kinase inhibitor that was discovered as a potential treatment of multiple myeloma. During the in vitro and in vivo metabolite profiling of GDC-0339, a metabolite was detected that had the same elemental composition as the parent but was distinct with respect to its chromatographic separation and mass spectrometric fragmentation pattern. High resolution tandem mass spectrometry data indicated the metabolite was modified at the aminoazepane moiety. The structure was solved by nuclear magnetic resonance analysis of the isolated metabolite and further confirmed by comparing it to a synthetic standard. These results indicated that the metabolite was formed by an intramolecular amine replacement reaction with the primary amine forming a new attachment to pyrazole without any change in stereochemistry. In vitro experiments showed cytochrome P450s catalyzed the reaction and demonstrated high isoform selectivity by CYP1A1. Results from kinetic experiments showed that the CYP1A1-mediated rearrangement of GDC-0339 was an efficient reaction with apparent turnover number (kcat) and Michaelis constant (Km) of 8.4 minutes-1 and 0.6 µM, respectively. The binding of GDC-0339 to the cytochrome P450 active site was examined by characterizing the direct inhibition of CYP1A1-mediated phenacetin O-deethylation, and GDC-0339 was a potent competitive inhibitor with Ki of 0.9 µM. This high affinity binding was unexpected given a narrow active site for CYP1A1 and GDC-0339 does not conform structurally to known CYP1A1 substrates, which are mostly polyaromatic planar molecules. Further, we explored some of the structural requirements for the rearrangement reaction and identified several analogs to GDC-0339 that undergo this biotransformation.

Targeted drug delivery through the traceless release of tertiary and heteroaryl amines from antibody-drug conjugates.

The reversible attachment of a small-molecule drug to a carrier for targeted delivery can improve pharmacokinetics and the therapeutic index. Previous studies have reported the delivery of molecules that contain primary and secondary amines via an amide or carbamate bond; however, the ability to employ tertiary-amine-containing bioactive molecules has been elusive. Here we describe a bioreversible linkage based on a quaternary ammonium that can be used to connect a broad array of tertiary and heteroaryl amines to a carrier protein. Using a concise, protecting-group-free synthesis we demonstrate the chemoselective modification of 12 complex molecules that contain a range of reactive functional groups. We also show the utility of this connection with both protease-cleavable and reductively cleavable antibody-drug conjugates that were effective and stable in vitro and in vivo. Studies with a tertiary-amine-containing antibiotic show that the resulting antibody-antibiotic conjugate provided appropriate stability and release characteristics and led to an unexpected improvement in activity over the conjugates previously connected via a carbamate.

Degradation of a pharmaceutical in HPLC grade methanol containing trace level formaldehyde.

An anomalous peak was observed in the HPLC/UV analysis of a developmental drug product. High resolution LC/MS revealed that the mass of this degradant was 12 Da greater than the drug substance, corresponding to a net gain of a single carbon atom. The degradant was reproduced by incubating the drug substance with formaldehyde, followed by isolation using normal phase chromatography and structure elucidation by NMR. It was determined to be an analytical artifact caused by the nucleophilic reaction of the drug substance with trace levels of formaldehyde in the methanol diluent. Typical formaldehyde levels in various grades of methanol were determined, leading to the adoption of spectrophotometric purity solvent to mitigate the recurrence of this artifact. This work demonstrates that even ppm levels of impurities in solvents can cause significant degradation of drug product and the HPLC grade solvents are not always suitable for HPLC analysis in drug product development.

Formation of a quinoneimine intermediate of 4-fluoro-N-methylaniline by FMO1: carbon oxidation plus defluorination.

Here, we report on the mechanism by which flavin-containing monooxygenase 1 (FMO1) mediates the formation of a reactive intermediate of 4-fluoro-N-methylaniline. FMO1 catalyzed a carbon oxidation reaction coupled with defluorination that led to the formation of 4-N-methylaminophenol, which was a reaction first reported by Boersma et al. (Boersma et al. (1993) Drug Metab. Dispos. 21 , 218 - 230). We propose that a labile 1-fluoro-4-(methylimino)cyclohexa-2,5-dienol intermediate was formed leading to an electrophilic quinoneimine intermediate. The identification of N-acetylcysteine adducts by LC-MS/MS and NMR further supports the formation of a quinoneimine intermediate. Incubations containing stable labeled oxygen (H(2)(18)O or (18)O(2)) and ab initio calculations were performed to support the proposed reaction mechanism.

Investigation of brominated tryptophan alkaloids from two thorectidae sponges: Thorectandra and Smenospongia.

Chemical investigation of an NCI-DTP collection of Thorectandra sp. and a UCSC collection of Smenospongia sp. yielded six new brominated tryptophan derivatives: 6-bromo-1'-hydroxy-1',8-dihydroaplysinopsin (4), 6-bromo-1'-methoxy-1',8-dihydroaplysinopsin (5), 6-bromo-1'-ethoxy-1',8-dihydroaplysinopsin (6), (-)-5-bromo-N,N-dimethyltryptophan (7), (+)-5-bromohypaphorine (8), and 6-bromo-1H-indole-3-carboxylic acid methyl ester (11). Additionally, the known compounds aplysinopsin (1), 1',8-dihydroaplysinopsin (2), 6-bromo-1',8-dihydroaplysinopsin (3), (1H-indole-3-yl)acetic acid (9), and (6-bromo-1H-indol-3-yl)acetic acid methyl ester (10) were also encountered. The structures of 4-8 and 11 were confirmed on the basis of analysis of (1)H and (13)C (1D and 2D) NMR data as well as comparison to known compounds. Compounds 1, 3-8, 10, and 11 were found to inhibit the growth of Staphylococcus epidermidis with either weak or moderate MICs.

Probing the activity differences of simple and complex brominated aryl compounds against 15-soybean, 15-human, and 12-human lipoxygenase.

Lipoxygenases (LO) have been implicated in asthma, immune disorders, and various cancers. As a consequence of these broad biological implications, there is great interest in understanding the effects of naturally occurring and environmental contaminants against its activity. On the basis of our earlier studies indicating that polybrominated diphenol ethers are potent inhibitors to mammalian 15-LO, we expanded our structure-activity study to include marine-derived brominated phenol ethers (including a newly discovered tribrominated diphenyl ether), dioxins, and bastadins, as well as the synthetic brominated fire retardants, brominated bisphenol A (BBPA), and polybrominated diphenyl ethers (PBDEs). We report herein the effects of 21 simple and complex organobromine compounds against human platelet 12-LO, human reticulocyte 15-LO, and soybean 15-LO-1.

Comparison of fascaplysin and related alkaloids: a study of structures, cytotoxicities, and sources.

The fascaplysin class of compounds have been further investigated from six organisms consisting of four sponge collections (Fascaplysinopsis reticulata) and two tunicate collections (Didemnum sp.). This work is an extension of an earlier communication and reports the isolation of 12 new fascaplysin derivatives: 10-bromofascaplysin (7), 3,10-dibromofascaplysin (8), homofascaplysate A (9), homofascaplysin B-1 (11), 3-bromohomofascaplysins B (12), B-1 (13), and C (15), 7,14-dibromoreticulatine (17), reticulatol (20), 14-bromoreticulatol (21), and 3-bromosecofascaplysins A (22) and B (23), along with known compounds: fascaplysin (1), reticulatine (4), 3-bromofascaplysin (6), and homofascaplysin C (14). Selected compounds were screened in a cell-based cytotoxicity assay with compounds 1, 6, and fascaplysin A (24) also screened in the NCI 60 cell line panel. A biogenetic pathway for the brominated fascaplysins and brominated related alkaloids is proposed and discussed.