Non-clinical characterization of the disposition of EMA401, a novel small molecule angiotensin II type 2 receptor (AT2R) antagonist.

EMA401, (the S-enantiomer of 5-(benzyloxy)-2-(2,2-diphenylacetyl)-6-methoxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid), also known as Olodanrigan, is an orally active selective angiotensin II type 2 receptor (AT2 R) antagonist that is in Phase IIb clinical development as a novel analgesic for the relief of chronic pain. The main purpose of the present work was to investigate the disposition of a single 14 C- labeled EMA401 in non-clinical studies. The in vitro metabolism studies of EMA401 were undertaken to understand the hepatic biotransformation pathways in animal species used in toxicology studies and how they compare to human. Furthermore, investigation of EMA401's PK was carried out in vivo in rats. The study demonstrates the rapid absorption and distribution of drug-related material mainly to the tissues associated with absorption and elimination (GI tract, liver, and kidney). EMA401was then readily eliminated metabolically via the bile (95% of dose) predominantly in the form of the direct acylglucuronide (40% of dose), which was further hydrolysed by the intestinal flora to the active parent drug. Other metabolic pathways such as dealkylations and hydroxylation were also involved in the elimination of EMA401 to a lesser extent. EMA401 was metabolically unstable in hepatocytes of all species investigated and the key metabolites produced in the in vitro model were also detected in vivo. Independent of the dosing route, the S-enantiomer EMA401 showed a good in vivo chiral stability. Overall, the present study provides the first full characterization of the disposition of EMA401 in preclinical species.

Cladosporin Derivatives Obtained by Biotransformation Provide Guidance for the Focused Derivatization of this Antimalarial Lead Compound.

Cladosporin, a natural product known for decades, has recently been discovered to display potent and selective antiplasmodial activity by inhibition of lysyl-tRNA synthetase. It was subjected to a panel of oxidative biotransformations with one fungal and two actinomycetes strains, as well as a triple mutant bacterial CYP102A1, yielding eight, mostly hydroxylated, derivatives. These new compounds covered a wide chemical space and contained two pairs of epimers in the tetrahydropyran ring. Although less potent than the parent compound, all analogues showed activity in a cell-based synthetase assay, thus demonstrating uptake and on-target activity in living cells with varying degrees of selectivity for the enzyme lysyl-tRNA synthetase from Plasmodium falciparum and highlighting sites suitable for synthesis of future cladosporin analogues. Compounds with adjacent hydroxy functions showed different MS/MS fragmentation that can be explained in terms of an, in some cases, regioselective loss of water followed by a retro-Diels-Alder reaction.

Gas-Phase Rearrangement of the O-Glucuronide of Vildagliptin Forms Product-Ion Fragments Suggesting Wrongly an N-Glucuronide.

The O-glucuronide of vildagliptin, a dipeptidyl peptidase 4 inhibitor, is a major metabolite in monkeys and a minor metabolite in humans, rats, and dogs. Its product ion spectrum shows fragments that can be explained only by an N-glucuronide. Biotransformation using rat liver yielded milligram amounts of the O-glucuronide, and its structure was assigned unambiguously by nuclear magnetic resonance. The tandem mass spectra (MS/MS) of this compound was investigated in detail using MSn and accurate mass spectrometers and was identical to the animal metabolite. Thus, the MS/MS fragments suggesting an N-glucuronide had to be formed by gas-phase rearrangement. This gas-phase rearrangement can be observed on quadrupole time-of-flight and ion-trap mass instruments. The literature on gas-phase rearrangements is reviewed.

KAE609 (Cipargamin), a New Spiroindolone Agent for the Treatment of Malaria: Evaluation of the Absorption, Distribution, Metabolism, and Excretion of a Single Oral 300-mg Dose of [14C]KAE609 in Healthy Male Subjects.

KAE609 [(1'R,3'S)-5,7'-dichloro-6'-fluoro-3'-methyl-2',3',4',9'-tetrahydrospiro[indoline-3,1'-pyridol[3,4-b]indol]-2-one] is a potent, fast-acting, schizonticidal agent in clinical development for the treatment of malaria. This study investigated the absorption, distribution, metabolism, and excretion of KAE609 after oral administration of [(14)C]KAE609 in healthy subjects. After oral administration to human subjects, KAE609 was the major radioactive component (approximately 76% of the total radioactivity in plasma); M23 was the major circulating oxidative metabolite (approximately 12% of the total radioactivity in plasma). Several minor oxidative metabolites (M14, M16, M18, and M23.5B) were also identified, each accounting for approximately 3%-8% of the total radioactivity in plasma. KAE609 was well absorbed and extensively metabolized, such that KAE609 accounted for approximately 32% of the dose in feces. The elimination of KAE609 and metabolites was primarily mediated via biliary pathways. M23 was the major metabolite in feces. Subjects reported semen discoloration after dosing in prior studies; therefore, semen samples were collected once from each subject to further evaluate this clinical observation. Radioactivity excreted in semen was negligible, but the major component in semen was M23, supporting the rationale that this yellow-colored metabolite was the main source of semen discoloration. In this study, a new metabolite, M16, was identified in all biologic matrices albeit at low levels. All 19 recombinant human cytochrome P450 enzymes were capable of catalyzing the hydroxylation of M23 to form M16 even though the extent of turnover was very low. Thus, electrochemistry was used to generate a sufficient quantity of M16 for structural elucidation. Metabolic pathways of KAE609 in humans are summarized herein and M23 is the major metabolite in plasma and excreta.

Evaluation of the optimization space for atmospheric pressure photoionization (APPI) in comparison with APCI.

The usefulness of atmospheric pressure photoionization (APPI) is difficult to evaluate for unknowns due to the fragmented literature. Specifically, the variation of dopants with a wide set of compounds or the use of APPI in the negative mode have rarely been explored. Thirty compounds were selected that were not suitable for ESI with a wide variety of functional groups and investigated with atmospheric pressure chemical ionization (APCI) and APPI in the positive and negative ion modes. The influence of the mobile phase (eluents containing acetonitrile or methanol) and--for APPI--four different dopants (acetone, chlorobenzene, toluene, and toluene/anisole) were explored. Stepwise variation of the organic mobile phase allowed to elucidate the ionization mechanism. Atmospheric pressure photoionization was especially useful for compounds, where the M(●+) and not the [M + H](+) was formed. The dopants chlorobenzene and anisole promoted the formation of molecular ions M(●+) for about half of the compounds, and its formation was also positively influenced by the use of mobile phases containing methanol. In the negative ion mode, APPI offered no advantage toward APCI. Best results were generally achieved with the dopant chlorobenzene, establishing that this dopant is suitable for a wide set of compounds. For one quarter of the compounds, significantly better results were achieved with mobile phases containing methanol for both APPI and APCI than those with acetonitrile, but only in the positive mode. With either of the methods--APPI or APCI--about 10% of the compounds were not detected. Strategies to get results quickly with difficult unknowns will be discussed.

Biocatalytic synthesis and structure elucidation of cyclized metabolites of the deacetylase inhibitor panobinostat (LBH589).

Panobinostat (LBH589) is a novel pan-deacetylase inhibitor that is currently being evaluated in phase III clinical trials for treatment of Hodgkin's lymphoma and multiple myeloma. Under catalysis of recombinant human CYP3A4 and CYP2D6 coexpressed with human cytochrome P450 reductase in Escherichia coli JM109, five metabolites of panobinostat were produced via whole-cell biotransformation. The structures of the metabolites were elucidated with the spectroscopic methods mass spectrometry (MS) and NMR and revealed an oxidative cyclization of the ethyl-amino group to the methylindole moiety. The MS(2) spectrum of the cyclized metabolite showed a base peak, where the closed ring is reopened and that, taken as sole base for structure proposals, would have lead to wrong conclusions. The metabolites were substantially less potent deacetylase inhibitors than the parent compound.

Structure determination of neoefrapeptins A to N: peptides with insecticidal activity produced by the fungus Geotrichum candidum.

The structures of neoefrapeptins A to N, peptides with insecticidal activity, were elucidated. They showed a close similarity to efrapeptin. However, all neoefrapeptins contained the very rare amino acid 1-amino-cyclopropane-carboxylic acid and some of them also contained (2S,3S)-3-methylproline. The neoefrapeptins are the first case, in which these amino acids are found as building blocks for linear peptides. They were identified by comparison of the silylated hydrolyzate to reference material by GC/MS (EI-mode). The sequence was elucidated using mass spectrometry (ESI+ mode). Full scan spectra showed two fragments in high yield, even under mild ionization conditions. MS/MS spectra of these two fragments yielded fragment rich spectra from which the sequence of the compounds was determined almost completely. The proteolytic cleavage with the proteinase papain yielded products that allowed to prove the rest of the sequence and the identity of the C-terminus to efrapeptin. The proteolytic cleavage products allowed furthermore to determine the position of the isobaric amino acids, pipecolic acid and 3-methylproline in neoefrapeptin F, as well as the location of R-isovaline and S-isovaline. Papain digestion was such established as a tool for structure elucidation of peptides rich in alpha,alpha-dialkylated amino acids. CD spectra suggested a 3(10) helical structure for neoefrapeptins A and F.

An MS/MS library on an ion-trap instrument for efficient dereplication of natural products. Different fragmentation patterns for [M + H]+ and [M + Na]+ ions.

The structural novelty of lead compounds is very important in the agrochemical and pharmaceutical industries, and as such, natural products can be an important source. Taking into account that the isolation of lead compounds is very time-consuming, the efficient and safe identification of compounds in microorganism and plant extracts isolated previously is essential. A suitable procedure for this task based on an HPLC system interfaced with an electrospray (ESI) source and a Thermo Finnigan LCQ deca XP plus ion-trap mass spectrometer was developed, and an extensive MS/MS spectral library of characterized natural products was built up. This report summarizes the parameters used for acquiring the library spectra and discusses current limitations of the NIST library and search algorithm. The advantages of the newly introduced Mass Frontier 4.0 for the search of MS/MS product-ion spectra are discussed. Different mechanisms for fragmentation of some [M + H](+) and [M + Na](+) ions that were found are proposed. Oligomycin A, a macrolide antibiotic, exhibits different fragmentation mechanisms in positive and negative ion modes. The cleavage of the ester bond is the preferred mechanism in the positive ion mode, whereas two different pathways-one showing a rare retro-Michael-addition-are observed in the negative ion mode.