Viktor Háda

Antioxidant activity-guided phytochemical investigation of Artemisia gmelinii Webb. ex Stechm.: Isolation and spectroscopic challenges of 3,5-O-dicaffeoyl (epi?) quinic acid and its ethyl ester

Although Artemisia gmelinii Webb. ex Stechm. has long been used in south and south-east Asia to treat many kinds of inflammatory diseases, up until now its bioactivity-coupled phytochemical characterization has not been reported. We identified one fraction of the methanolic extract of A. gmelinii as a hit in our antioxidant screening (DPPH) campaign. In order to identify the active radical scavenger components of the extract, a DPPH-HPLC spiking assay was carried out. Out of six detected known compounds caffeic acid and scopoletin had already been identified in the plant, but four of them, namely chlorogenic acid, 4-O-caffeoylquinic acid, luteolin-7-O-glucoside, and apigenin-7-O-glucoside are first described here. Moreover, the two most active compounds of the mixture, 3,5-O-dicaffeoylquinic acid (7) and its ethyl ester derivative (8) were isolated with preparative HPLC. The spectroscopic identification of 7 and 8 presented a surprising challenge due to literature ambiguities. These questions are discussed in detail.

Structure elucidation of indole–indoline type alkaloids: A retrospective account from the point of view of current NMR and MS technology

In this review our aim is to look back on how the structure elucidation of bisindoles, especially with focus placed on vinblastine and vincristine analogues, has evolved alongside with the development of MS and NMR over the last 60 years from the perspective of our present-day use of state-of-the-art MS and NMR instrumentation and on the basis of our own accumulated views and experience in the field.

Synthesis and in vitro antitumor effect of vinblastine derivative-oligoarginine conjugates.

Vinblastine is a widely used anticancer drug with undesired side effects. Its conjugation with carrier molecules could be an efficient strategy to reduce these side effects. Besides this, the conjugate could exhibit increased efficiency against resistant cells, e.g., due to the altered internalization pathway. Oligoarginines, as cell-penetrating peptides, can transport covalently attached compounds into different kinds of cells and enhance the efficiency of those compounds. We report here the coupling of vinblastine through its carboxyl group at position 16 with the N-terminal amino function of L-Trp methyl ester. After hydrolysis of the ester group, 17-desacetylvinblastineTrp was conjugated to the N-terminal amino group of oligoarginine via the C-terminal carboxyl group of the Trp moiety in solution. The antitumor effect of conjugates was studied on sensitive and resistant human leukemia (HL-60) cells in vitro. Our data suggest that all conjugates investigated possess an antiproliferative effect against the studied cells. However, the effect was dependent on the number of Arg residues in the conjugates: Arg₈ > Arg₆ ≫ Arg₄. The conjugate with Arg₈ exhibited similar efficicacy as compared with free 17-desacetylvinblastineTrp. The in vitro studies also showed that the tubulin binding ability of vinblastine was essentially preserved even in the octaarginine conjugate. We also observed that two isomers were formed during conjugation. These isomers showed different levels of activity against tubulin polymerization in vitro and in vivo. The 17-desacetylvinblastineTrp-Arg₈-1 isomer conjugate possessed high selectivity against the mitotic spindles. HRMS and NMR data suggest that 17-desacetylvinblastineTrp-Arg₈-1 and 17-desacetylvinblastineTrp-Arg₈-2 are epimers at the tryptophan α carbon atom.

New oxidative decomposition mechanism of estradiol through the structural characterization of a minute impurity and its degradants.

Herein we discuss the structure elucidation of a labile estradiol-related degradant, X1. X1 was detected at Gedeon Richter as an unknown trace impurity in a pharmaceutical formulation containing estradiol (1a) and norethisterone acetate (NA) as active ingredients. The structural identification of X1 proved to be an unusually complex task involving an initial structural hypothesis based on some limited analytical data (UV) obtained from the formulation, synthetic work targeting the proposed structure, chromatographic enrichment from the synthetic reaction mixture, (HPLC)-MS and MS-MS studies of the formulation and of samples from the synthesis using almost all available ionization modes, preparative LC enrichment, and the complementary use of off-line and on-line NMR techniques. Based on these results, X1 was finally characterized as a new oxidative product of estradiol, containing an epoxy function over the C9-C10 bond. During the structure determination of X1 its secondary and tertiary decomposition products were also identified as a new secoepoxy (6) and a known seco derivative (5a) of estradiol, respectively. On this basis a new oxidative decomposition mechanism of estradiol and its analogues could be proposed. A generalization of the mechanism of this pathway can more readily explain the formation of some oxidative secosteroid degradants than the mechanism proposed earlier in the literature.

Chapter 11 – The Adventurous Discovery of the Structure of a Novel Vincristine Impurity

In this chapter, we describe the NMR- and MS-based structure elucidation of a new impurity of the bisindole alkaloid vincristine (VCR). Owing to its size and internal conformational dynamics, VCR and its derivatives are highly “NMR-unfriendly” even if they are available in pure form and adequate quantity. For a VCR-related impurity, typically neither of these conditions holds, and therefore the structure determination of such an impurity can be a daunting task, requiring a “holistic” collaboration between NMR and MS. Here, we outline the subtleties and Mental Traps associated with such a collaboration in the case of an interesting impurity, which, after several apparently convincing but misleading deductions, turned out to be a novel iminium salt of VCR. Hitherto, this derivative was mentioned in the literature merely as a hypothetical intermediate in the peroxidase-catalyzed oxidative metabolism of VCR. The experiences described here illustrate, on the one hand, the reality and dangers of the don’t-look-any-further effect, professional chauvinism, and warped team dynamics; on the other hand, they also demonstrate the triumphs resulting from overcoming these hidden human factors.

Chapter 13 – The Case of an Emotion- and Emotycs-Laden Structure Determination of a Small Synthetic Molecule with an Unexpected Structure

Herein, we disclose the intriguing story of the structural elucidation of an organic molecule that gave a highly unexpected structure in an apparently well-predictable chemical reaction. Due to the interplay of a multitude of misleading and distracting factors, including the fact that the compound had broad NMR signals due to conformational exchange and had the same nominal mass as the expected molecule, in a first (routine) approximation, its structure seemed to correspond to the expected molecule. Only a deeper investigation, initiated by some further unexpected events in relation to this reaction scheme, revealed the true structure of the compound. This story demonstrates how easy it can be to misinterpret experimental data when one subconsciously seeks to confirm expectations (see Trap #29), especially in a time-pressed industrial research environment; it is also a story of the battle between emotycal and rational minds.

Recent advancements, challenges, and practical considerations in the mass spectrometry-based analytics of protein biotherapeutics: A viewpoint from the biosimilar industry

HighlightsAdvances of the past ten years in the MS‐based characterization of protein biotherapeutics.How to cope with industrial and regulatory requirements.How to obtain accurate and reliable analytical data in a time‐ and cost‐efficient way.New sample preparation approaches, measurement techniques and data evaluation strategies.Increasing role of mass spectrometry ABSTRACT The extensive analytical characterization of protein biotherapeutics, especially of biosimilars, is a critical part of the product development and registration. High‐resolution mass spectrometry became the primary analytical tool used for the structural characterization of biotherapeutics. Its high instrumental sensitivity and methodological versatility made it possible to use this technique to characterize both the primary and higher‐order structure of these proteins. However, even by using high‐end instrumentation, analysts face several challenges with regard to how to cope with industrial and regulatory requirements, that is, how to obtain accurate and reliable analytical data in a time‐ and cost‐efficient way. New sample preparation approaches, measurement techniques and data evaluation strategies are available to meet those requirements. The practical considerations of these methods are discussed in the present review article focusing on hot topics, such as reliable and efficient sequencing strategies, minimization of artefact formation during sample preparation, quantitative peptide mapping, the potential of multi‐attribute methodology, the increasing role of mass spectrometry in higher‐order structure characterization and the challenges of MS‐based identification of host cell proteins. On the basis of the opportunities in new instrumental techniques, methodological advancements and software‐driven data evaluation approaches, for the future one can envision an even wider application area for mass spectrometry in the biopharmaceutical industry.

The effect of conjugation on antitumor activity of vindoline derivatives with octaarginine, a cell-penetrating peptide

Some Vinca alkaloids (eg, vinblastine, vincristine) have been widely used as antitumor drugs for a long time. Unfortunately, vindoline, a main alkaloid component of Catharanthus roseus (L.) G. Don, itself, has no antitumor activity. In our novel research program, we have prepared and identified new vindoline derivatives with moderate cytostatic activity.

RATE AND STEREOSELECTIVITY CHANGES DURING HYDROGENATION OF N-HETEROCYCLES

High diastereoselectivities were obtained in the heterogeneous catalytic hydrogenation of chiral pyrrole and pyridine derivatives with complete conversion, in non-acidic medium. The products of the hydrogenations, secondary and tertiary amines act as catalyst modifiers, increasing the d.e. values in the last period of the reactions.