Synthesis and Characterization of New V1A Antagonist Compounds: The Separation of Four Atropisomeric Stereoisomers.

A new class of selective vasopressin receptor 1A (V1A) antagonists was identified, where "methyl-scan" was performed around the benzene ring of the 5-hydroxy-triazolobenzazepine core. This led to the synthesis of two 10-methyl derivatives, each possessing a chiral axis and a stereogenic center. The four atropisomeric stereoisomers (involving two enantiomer pairs and atropisomeric diastereomers) could be successfully isolated and spectroscopically characterized. According to the in vitro pharmacological profiles of the compounds, the human V1A receptor has a strong preference toward the isomers having an aR axial chirality, the most active isomer being the aR,5S isomer. Furthermore, the structure-activity relationships obtained for the isomers and for the newly synthesized analogues could be tentatively explained by an in silico study.

On the utility of nonuniformly sampled two-dimensional NMR spectra in the pharmaceutical industry.

In this work, we discuss representative examples of the application of nonuniform sampling (NUS) in small-molecule structure determination in a pharmaceutical research and development and quality control setting. We demonstrate the advantages of NUS over traditional sampling in various industrial applications of nuclear magnetic resonance (NMR). We propose an optimal trade-off between the quality and the time efficiency of 'routine' measurements, as demonstrated via a test sample of vinpocetine analyzed on a 'work horse' NMR spectrometer. In addition, we present case studies where the application of NUS contributed significantly to the successful completion of some challenging structure determination task at hand. To that end, in some cases, we combined NUS with a method of decoupling in the indirect dimension that gives 'constant time' nuclear Overhauser effect spectroscopy (NOESY) and 'constant time' heteronuclear multiple bond correlation (HMBC) spectra. NUS proved to be superior over traditional sampling even for the analysis of trace impurities, where the need to cope with a thousand-fold or even larger dynamic range and low signal-to-noise ratio for the impurity signals is quite a challenge.

Synthesis and Cytotoxic Activity of New Vindoline Derivatives Coupled to Natural and Synthetic Pharmacophores.

New Vinca alkaloid derivatives were synthesized to improve the biological activity of the natural alkaloid vindoline. To this end, experiments were performed to link vindoline with various structural units, such as amino acids, a 1,2,3-triazole derivative, morpholine, piperazine and N-methylpiperazine. The structure of the new compounds was characterized by NMR spectroscopy and mass spectrometry (MS). Several compounds exhibited in vitro antiproliferative activity against human gynecological cancer cell lines with IC50 values in the low micromolar concentration range.

Synthesis and In Vitro Anticancer Evaluation of Novel Chrysin and 7-Aminochrysin Derivatives.

Chrysin is a naturally occurring flavonoid with mild anticancer activity. In this paper we report the synthesis of new chrysin derivatives alkylated with N-phenylchloroacetamides in position 7. A novel method was developed for the preparation of 7-aminochrysin derivatives via the Smiles rearrangement, resulting in diphenylamine-type compounds. In silico studies of the Smiles rearrangement were performed. We also present the in vitro antiproliferative activity of the synthesized compounds against 60 human tumor cell lines (NCI60). The most potent derivative exhibited nanomolar antitumor activity on the MCF7 cell line of breast cancer (GI50 = 30 nM) and on the HCT-15 cell line of colon cancer (GI50 = 60 nM).

Attempted Synthesis of Vinca Alkaloids Condensed with Three-Membered Rings.

Our successful work for the synthesis of cyclopropanated vinblastine and its derivatives by the Simmons⁻Smith reaction was followed to build up further three-membered rings into the 14,15-position of the vindoline part of the dimer alkaloid. Halogenated 14,15-cyclopropanovindoline was prepared by reactions with iodoform and bromoform, respectively, in the presence of diethylzinc. Reactions of dichlorocarbene with vindoline resulted in the 10-formyl derivative. Unexpectedly, in the case of the dimer alkaloids vinblastine and vincristine, the rearranged products containing an oxirane ring in the catharanthine part were isolated from the reactions. The attempted epoxidation of vindoline and catharanthine also led to anomalous rearranged products. In the epoxidation reaction of vindoline, an o-quinonoid derivative was obtained, in the course of the epoxidation of catharanthine, a hydroxyindolenine type product and a spiro derivative formed by ring contraction reaction, were isolated. The coupling reaction of vindoline and the spiro derivative obtained in the epoxidation of catharanthine did not result in a bisindole alkaloid. Instead, two surprising vindoline trimers were discovered and characterized by NMR spectroscopy and mass spectrometry.

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

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. Here, we describe the effect of conjugation of vindoline derivative with oligoarginine (tetra-, hexa-, or octapeptides) cell-penetrating peptides on the cytostatic activity in vitro and in vivo. Br-Vindoline-(l)-Trp-OH attached to the N-terminus of octaarginine was the most effective compound in vitro on HL-60 cell line. Analysis of the in vitro activity of two isomer conjugates (Br-vindoline-(l)-Trp-Arg8 and Br-vindoline-(d)-Trp-Arg8 suggests the covalent attachment of the vindoline derivatives to octaarginine increased the antitumor activity significantly against P388 and C26 tumour cells in vitro. The cytostatic effect was dependent on the presence and configuration of Trp in the conjugate as well as on the cell line studied. The configuration of Trp notably influenced the activity on C26 and P388 cells: conjugate with (l)-Trp was more active than conjugate with the (d)-isomer. In contrast, conjugates had very similar effect on both the HL-60 and MDA-MB-231 cells. In preliminary experiments, conjugate Br-vindoline-(l)-Trp-Arg8 exhibited some inhibitory effect on the tumor growth in P388 mouse leukemia tumor-bearing mice. Our results indicate that the conjugation of modified vindoline could result in an effective compound even with in vivo antitumor activity.

What NMR can do in the biopharmaceutical industry.

Nuclear magnetic resonance (NMR) spectroscopy has a unique capability to probe the primary and higher order molecular structure and the structural dynamics of biomolecules at an atomic resolution, and this capability has been greatly fortified over the last five decades by an astonishing NMR instrumental and methodological development. Because of these factors, NMR has become a primary tool for the structure investigation of biomolecules, spawning a whole scientific subfield dedicated to the subject. This role of NMR is by now well established and broadly appreciated, especially in the context of academic research dealing with proteins that are purified and isotope-labeled in order to facilitate the necessary sophisticated multidimensional NMR measurements. However, the more recent industrial development, manufacturing, and quality control of biopharmaceuticals provide a different framework for NMR. For example, protein drug substances are not isotope-labeled and are present in a medium of excipients, which make structural NMR measurements much more difficult. On the other hand, biotechnology involves many other analytical requirements that can be efficiently addressed by NMR. In this respect the scope and limitations of NMR are less well understood. Having the non-expert reader in mind, herein we wish to highlight the ways in which modern NMR can effectively support biotechnological developments. Our focus will be on biosimilar proteins, pointing out certain cases where its use is probably essential. Based partly on literature data, and partly on our own hands-on experience, this paper is intended to be a guide for choosing the proper NMR approach for analytical questions concerning the structural comparability of therapeutic proteins, monitoring technology-related impurities, protein quantification, analysis of spent media, identification of extractable and leachable components, etc. Also, we focus on critical considerations, particularly those coming from drug authority guidelines, which limit the use of the well-established NMR tools in everyday practice.

The Chemistry of Galanthamine. Classical Synthetic Methods and Comprehensive Study on its Analogues.

Galanthamine as an Amaryllidaceae alkaloid has an important role in the treatment of Alzheimer's disease. Some efforts were made to elaborate the total synthesis, and hundreds of its derivatives were prepared to find a more effective molecule with advantageous properties. Moreover, almost every part of the tetracycle was changed; in members of the rings, in the nature and position of the heteroatoms, and ring-opened analogues were also synthesized. In this review the basic synthetic works and the most important derivatives and analogues are overviewed.

The influence of 5-HT(2A) activity on a 5-HT(2C) specific in vivo assay used for early identification of multiple acting SERT and 5-HT(2C) receptor ligands.

As a result of our exploratory programme aimed at elaborating dually acting compounds towards the serotonin (5-HT) transporter (SERT) and the 5-HT2C receptor a novel series of 3-amino-1-phenylpropoxy substituted diphenylureas was identified. From that collection two promising compounds (2 and 3) exhibiting highest 5-HT2C receptor affinity strongly inhibited the 5-HT2C receptor agonist 1-(3-chlorophenyl)piperazine (mCPP) induced hypomotility in mice. In further pursuance of that objective (2-aminoethyl)(benzyl)sulfamoyl diphenylureas and diphenylpiperazines have also been elaborated. Herein we report the synthesis of potent multiple-acting compounds from this new class. However, when two optimized representatives (6 and 14) possessing the desired in vitro profile were tested neither reduced the motor activity of mCPP treated animals. Comparative albeit limited in vitro structure-activity relationship (SAR) analysis and detailed in vivo studies are discussed and explanation for their intricate behaviour is proposed.

Biomimetic synthesis and HPLC-ECD analysis of the isomers of dracocephins A and B.

Starting from racemic naringenin ((±)-1), a mixture of dracocephin A stereoisomers 6-(2"-pyrrolidinone-5"-yl)naringenin (±)-2a-d and its regioisomer, dracocephin B 8-(2"-pyrrolidinone-5"-yl)naringenin (±)-3a-d originally isolated from Dracocephalum rupestre, have been synthesized in a one-pot reaction. The separation of 2a-d and 3a-d was achieved by preparative HPLC. The four stereoisomers of each natural product were separated by analytical chiral HPLC and their absolute configuration was studied by the combination of HPLC-ECD measurements and TDDFT-ECD calculations. The synthesized flavonoid alkaloids were further characterized by physicochemical and in vitro pharmacological studies.

Detection by HPLC and structural characterization by NMR and MS of a natural deuterium isotopologue of ulipristal acetate.

Herein we discuss the structure elucidation of an unknown peak detected by HPLC in the active pharmaceutical ingredient ulipristal acetate during analytical method development. An extensive chromatographic, MS and NMR spectroscopic study gave the surprising result that the detected component is the natural-abundance mono-deuterium isotopologue of the API. To the best of our knowledge this is the first example where such a mono-deuterium isotopologue could be resolved from its mother component by HPLC and structurally fully characterized by NMR and MS. The reason for this separation could be rationalized in terms of some special structural features of the molecule.

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.

NMR and mass spectrometric characterization of vinblastine, vincristine and some new related impurities - part I.

In the course of exploring the possibilities of developing a new, improved process at Gedeon Richter for the production of the "bisindole" alkaloids vinblastine (VLB) and vincristine (VCR), some novel VLB/VCR-related trace impurities were detected by analytical HPLC. Following isolation by preparative HPLC, a combination of 1D and 2D ultra high-field NMR and high-resolution (HR) (LC-)MS/MS studies allowed the structural identification and complete spectral characterization of several hitherto unpublished VLB/VCR-analogue impurities. Since the impurities could not be isolated in entirely pure forms and were available only in minute, mass-limited quantities, accessing the spectral information needed for their ab initio structure determination was met with various practical difficulties. Successful structure determination therefore relied heavily on the availability and use of detailed and definitive spectral data for both VLB and VCR. In particular, the utilization of detailed (1)H, (13)C, and (15)N NMR assignments as well as (1)H-(1)H, (1)H-(13)C and (1)H-(15)N spin-spin connectivities pertaining to different solvents for VLB/VCR base and sulphate salt was required. Although NMR studies on VLB base and other bisindoles were reported earlier in the literature, an NMR characterization of VLB and VCR under the above-mentioned circumstances and using ultra-high field instrumentation is either scarcely available or entirely lacking, therefore the necessary data had to be obtained in-house. Likewise, a modern tandem HR-ESI-MS/MS(n) fragmentation study of VLB and VCR has not been published yet. In the present paper we therefore give a thorough NMR and MS characterization of VLB and VCR specifically with a view to filling this void and to provide sufficiently extensive and solid reference data for the structural investigation of the aforementioned VLB/VCR impurities. Besides being scientifically relevant in its own right, the disclosed data should be useful for anyone interested in VLB/VCR-related molecules at a structural level.

NMR and mass spectrometric characterization of vinblastine, vincristine and some new related impurities--part II.

In the course of developing a new, improved process at Gedeon Richter for the production of the "bisindole" alkaloids vinblastine (VLB) and vincristine (VCR), some novel VLB/VCR-related trace impurities were detected by analytical HPLC at the production site. Repeated attempts to isolate and purify these unknown impurities by preparative liquid chromatography yielded small amounts of materials whose main components were the unknown impurities, but were still contaminated with other VLB/VCR-related compounds. In spite of these difficulties, by using a combination of high-resolution (LC-)MS/MS and off-line 1D and 2D ultra high-field NMR techniques and leaning on the relevant spectroscopic data for VLB and VCR as discussed in Part 1 [1], we could unambiguously solve the structures of, and could give a complete spectral characterization for, the trace impurities. Among these, although "cyclo-VCR" (impurity-2), "[VCR]-C(16)-COOEt" (impurity-4) and "[VLB]-C(16)-COOEt" (impurity-5) are known synthetic VLB/VCR-derivatives, and "[VLB]-C(14')-OH(α)" is a known natural alkaloid (leurocolombine), they are new VLB/VCR impurities, and "[VCR]-N(4')-C(21')-iminium-salt" (impurity-3) is also a new chemical structure which provides direct proof of a hypothetic metabolic pathway of VLB/VCR. The structure determination of impurity-4 and impurity-5, and the rationalization of their origin was a particularly challenging task: since VCR is produced by the oxidation of VLB, it may be assumed that [VCR]-C(16)-COOEt (impurity-4) originates from the oxidization of [VLB]-C(16)-COOEt (impurity-5). This is consistent with the finding that [VLB]-C(16)-COOEt (impurity-5) could be detected by LC-MS/MS in the raw VLB samples in similar amounts as [VCR]-C(16)-COOEt (impurity-4) in the final VCR product. Our investigations indicate that [VLB]-C(16)-COOEt (impurity-5) does not form directly from VLB during extraction or chromatographic separation, suggesting that it may be a new natural product.

Modifications on the basic skeletons of vinblastine and vincristine.

The synthetic investigation of biologically active natural compounds serves two main purposes: (i) the total synthesis of alkaloids and their analogues; (ii) modification of the structures for producing more selective, more effective, or less toxic derivatives. In the chemistry of dimeric Vinca alkaloids enormous efforts have been directed towards synthesizing new derivatives of the antitumor agents vinblastine and vincristine so as to obtain novel compounds with improved therapeutic properties.

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.

Structure of the major degradant of ezetimibe.

In a recent contribution to this Journal Gajjar and Shah described the isolation and structure elucidation of the major alkaline degradant of ezetimibe, a lipid lowering agent [A.K. Gajjar, V.D. Shah, J. Pharm. Biomed. Anal. 55 (2011) 225-229]. Based on (1)H NMR, (13)C NMR and mass spectrometric studies the authors concluded that the structure of the degradant is 5-(4-fluorophenyl)-2-[(4-fluorophenylamino)-(4-hydroxyphenyl) methyl]-pent-4-enoic acid. In a subsequent "Letter to the Editor" submitted to the Journal, Barhate and Mohanra pointed out that the aforementioned structure is inconsistent with the spectroscopic data reported by Gajjar and Shah, consequently it must be wrong [Ch.R. Barhate, K. Mohanra, J. Pharm. Biomed. Anal. 55 (2011) 1237-1238]. However, Barhate and Mohanra did not offer a correct structure in their critical letter. Based on the cited NMR data we realised that previously we had had the same degradant in hand and had unambiguously determined its structure from detailed (1)H, (13)C, COSY, H-C HSQC, H-C HMBC and 1D-NOESY NMR investigations. Herein we report the correct structure to be (2R,3R,6S)-N,6-bis(4-fluorophenyl)-2-(4-hydroxyphenyl)-3,4,5,6-tetrahydro-2H-pyran-3-carboxamide. However, the structure is not new and was described earlier [G.Y.S.K. Swamy et al., Acta Cryst. E 61 (2005) o3608-o3610; K. Filip et al., J. Mol. Struct. 991 (2011) 162-170]. The aim of our present communication is to bring together the various threads of analytical effort involving this degradant into a compact and hopefully instructive conclusion on the pages of this Journal. For the sake of completeness and clarity we also list the correct NMR spectral assignments for ezetimibe which was also given partly erroneously in the earlier literature, and we propose a mechanism for the formation of the degradant.

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.

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.