Highly Efficient Synthesis of a Staphylococcus aureus Targeting Payload to Enable the First Antibody-Antibiotic Conjugate.

A practical synthesis of the complex payload for an anti-Staphylococcus aureus THIOMABTM antibody-antibiotic conjugate (TAC) is described. The route takes advantage of a delicate oxidative condensation, achieved using a semi-continuous flow procedure. It allows for the generation of kilogram quantities of a key intermediate to enable a mild nucleophilic aromatic substitution to the tertiary amine free drug. The linker component is introduced as a benzylic chloride, which allows formation of the quaternary ammonium salt linker-drug. This chemical process surmounts numerous synthetic challenges and navigates deeply colored and unstable compounds to support clinical studies to counter S. aureus bacterial infections.

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.

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.

An isolable acyclic hemiacetal of ansamitocin P-3.

During impurity analysis of maytansinol (2), produced from the reduction of ansamitocin P-3 (AP-3, 1), a surprisingly stable acyclic hemiacetal (4) was isolated. A combination of 1D and 2D NMR experiments, along with liquid chromatography-mass spectrometry data was used to confirm the structure. Comparison of NMR data to the previously reported bridged acetal (3), a by-product of AP-3 reduction, supports reassignment of the latter to the former. Additionally, ROESY data, in conjunction with minimum energy calculations, support intramolecular hydrogen bonding that is involved in stabilizing the hemiacetal. This report adds another example to the very short list of isolable acyclic hemiacetals.

Challenges in the structure determination of a dimeric impurity found during development of GDC-0326.

While developing a synthetic route for GDC-0326, a PI3Kα selective inhibitor, a side product was identified which was adversely impacting process chemistry development. To aid in optimization of a viable synthetic pathway for the drug, it was decided to characterize this impurity. Initial efforts using typical high-resolution mass spectrometry data coupled with NMR analysis were unable to unambiguously identify the structure. The NMR analysis was hampered by a severe lack of protons in the core of the structure. While efforts were being made to produce suitable crystals for definitive x-ray analysis, Raman analysis was undertaken. The vibrational data were compared to DFT calculations for the two most likely structures. This data, along with chemical reasoning, eventually led to successful prediction of structure 2, which was ultimately confirmed by single crystal x-ray diffractometry data.

Assessing pressurized liquid extraction for the high-throughput extraction of marine-sponge-derived natural products.

In order to compare the utility of standard solvent partitioning (SSP) versus accelerated solvent extraction (ASE), a series of experiments were performed and evaluated. Overall yields, solvent consumption, processing time, and chemical stability of the fractions obtained by both methods were compared. Five marine sponges were selected for processing and analysis containing 12 structurally distinct, bioactive natural products. Extracts generated using SSP and ASE were assessed for chemical degradation using comparative LC MS-ELSD. The extraction efficiency (EE) of the ASE apparatus was 3 times greater than the SSP method on average, while the total extraction yields (TEY) were roughly equivalent. Furthermore, the ASE methodology required only 2 h to process each sample versus 80 h for SSP, and the LC MS-ELSD from extracts of both methods appeared comparable. These results demonstrate that ASE can serve as an effective high-throughput methodology for extracting marine organisms to streamline the discovery of novel and bioactive natural products.

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.

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.

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.

A Madagascar Sponge Batzella sp. as a source of alkylated iminosugars.

Three new C-alkylated iminosugars, batzellasides A (3), B (4), and C (5), along with the known halitoxin (2) polymer were isolated from a Batzella sp. sponge, collected off the west coast of Madagascar. Although this class of azasugars is well known from terrestrial sources, our report represents the first examples of iminosugars from a marine organism. Comparison with the properties of known natural and synthetic iminosugars assisted in the structure determinations. Compounds 3-5 inhibited the growth of Staphylococcus epidermidis with MICs of < or =6.3 microg/mL.

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.