Matthias Witt

Accelerated dereplication of crude extracts using HPLC–PDA–MS–SPE–NMR: Quinolinone alkaloids of Haplophyllum acutifolium

Direct hyphenation of analytical-scale high-performance liquid chromatography, photo-diode array detection, mass spectrometry, solid-phase extraction and nuclear magnetic resonance spectroscopy (HPLC-PDA-MS-SPE-NMR) has been used for accelerated dereplication of crude extract of Haplophyllum acutifolium (syn. Haplophyllum perforatum). This technique allowed fast on-line identification of six quinolinone alkaloids, named haplacutine A-F, as well as of acutine, haplamine, eudesmine, and 2-nonylquinolin-4(1H)-one. Acutine and haplacutine E, isolated by preparative-scale HPLC, showed moderate antiplasmodial activity with IC(50) values of 2.17+/-0.22 microM and 3.79+/-0.24 microM, respectively (chloroquine-sensitive Plasmodium falciparum 3D7 strain).

Solid-phase peptide synthesis and circular dichroism study of chiral β-peptoid homooligomers

Summary.N-alkyl-β-alanine oligomers (β-peptoids) with α-chiral side chains [(R)- or (S)-1-(phenylethyl)amino groups] were synthesized and analyzed by CD spectroscopy. These chiral β-peptoid homomers exhibited chain-length-dependent and solvent-dependent ellipticity, strongly indicating the presence of a secondary structure in solution. The CD behaviour was only slightly temperature-dependent upon heating, as also previously observed for stable α-peptoid helices containing the same type of side chains. Thus, the data presented here comprise the first evidence for a chain length-dependent secondary folding of compounds with this novel peptidomimetic backbone design. In addition, applicability of a novel hyphenated technique, HPLC-SPE-NMR/MS, for analysis of crude SPPS reaction products was demonstrated.

Piperidine and Tetrahydropyridine Alkaloids from Lobelia siphilitica and Hippobroma longiflora

The hyphenated technique HPLC-PDA-MS-SPE-NMR was used to assist targeted preparative-scale isolation of constituents of Lobelia siphilitica and Hippobroma longiflora (both Campanulaceae). This resulted in the isolation of two new alkaloids, (S)-2-[(2S,6R)-1-methyl-6-(2-oxo-2-phenylethyl)piperidin-2-yl]-1-phenylethyl acetate (3) and 6-[(E)-2-(3-methoxyphenyl)ethenyl]-2,3,4,5-tetrahydropyridine (4), the latter possessing a skeleton hitherto unseen among alkaloids of Lobelia and related genera. Lobeline (1), (1S,1S)-2,2-[(2R,6S)-1-methylpiperidine-2,6-diyl]bis(1-phenylethanol) (2), and lobetyolin (5) were also isolated. The structures of 1-5 were established using spectroscopic methods including homo- and heteronuclear two-dimensional NMR experiments and optical rotation data.

Microwave-Assisted Ring-Opening of Activated Aziridines with Resin-Bound Amines

This paper describes the first study of nucleophilic ring-opening of nosylamide-activated aziridines under microwave irradiation conditions in solid-phase synthesis (SPS). The effects of solvent, temperature, reaction time, and reagent ratio in SPS of partially protected triamines from aziridines and resin-bound diamines were investigated. The methodology was also optimized for the synthesis of novel amino acid derivatives.

Assessment of structurally diverse philanthotoxin analogues for inhibitory activity on ionotropic glutamate receptor subtypes: discovery of nanomolar, nonselective, and use-dependent antagonists.

An array of analogues of the wasp toxin philanthotoxin-433, in which the asymmetric polyamine moiety was exchanged for spermine and the headgroup replaced with a variety of structurally diverse moieties, was prepared using parallel solid-phase synthesis approaches. In three analogues, the spermine moiety was extended with an amino acid tail, six compounds contained an N-acylated cyclohexylalanine, and four analogues were based on a novel diamino acid design with systematically changed spacer length between N-cyclohexylcarbonyl and N-phenylacetyl substituents. The analogues were studied using two-electrode voltage-clamp electrophysiology employing Xenopus laevis oocytes expressing GluA1(i) AMPA or GluN1/2A NMDA receptors. Several of the analogues showed significantly increased inhibition of the GluN1/2A NMDA receptor. Thus, an analogue containing N-(1-naphtyl)acetyl group showed an IC(50) value of 47 nM. For the diamino acid-based analogues, the optimal spacer length between two N-acyl groups was determined, resulting in an analogue with an IC(50) value of 106 nM.

Selectively N-protected enantiopure 2,5-disubstituted piperazines: avoiding the pitfalls in solid-phase Fukuyama-Mitsunobu cyclizations.

An efficient solid-phase route to ring-substituted piperazines from O-linked resin-bound (S)-aziridine-2-methanol is described. Regioselective microwave-assisted aminolysis followed by intramolecular Fukuyama-Mitsunobu cyclization constitute the key features of the protocol. Simple piperazines and diazepanes were readily obtained without preceding N-protection of the acyclic intermediate, whereas attempts to extend this protocol to chiral 2,5-disubstituted piperazines failed. Modifications encompassing N-carbamoylation prior to ring-closure were therefore investigated. However, standard carbamoylating agents, for example, Fmoc-Cl and Alloc-Cl tended to give bis-protected by-products. Thus, novel microwave-assisted solid-phase N-protection procedures were developed for efficient introduction of Fmoc, Boc and Alloc groups. The subsequent cyclization proceeded in moderate to excellent yields depending on the bulk of the side chain and type of N-protecting group. This protocol readily provided novel cis- and trans-2,5-disubstituted piperazines displaying a variety of N-protecting group patterns after further on-resin manipulations. Also, unexpected by-products obtained during these optimization studies were identified and characterized. This includes nosylated ureas arising from an alternative cyclization pathway. Finally, post-cleavage oxidation gave access to the Fmoc/Boc-protected alpha-amino acid as well as the corresponding aldehyde. The chiral piperazines described in this work will enable construction of combinatorial libraries with a higher chemical diversity compared to those containing simple N,N-difunctionalized piperazines, often present in drug-like compounds.

Selagoline, A new Alkaloid from Huperzia Selago

A new natural product, named selagoline, and two known alkaloids, huperzine A and serratidine, were isolated from Huperzia selago (Lycopodiaceae) collected in Iceland. Their structures were determined using 600 and 800u2009MHz one- and two-dimensional NMR methods supported by Fourier-transform mass spectrometry. Possible role of selagoline as a precursor of 5,15-oxidolycopodane, a component of the classical alkaloids L28 and L31, is discussed.

Tuning Wasp Toxin Structure for Nicotinic Receptor Antagonism: Cyclohexylalanine‐Containing Analogues as Potent and Voltage‐Dependent Blockers

The wasp venom constituent philanthotoxin-433 (PhTX-433, 1) is a polyamine toxin that antagonizes ionotropic glutamate receptors (iGluRs) and nicotinic acetylcholine receptors (nAChRs) nonselectively (Figure 1). Thus, similar potencies have been reported for PhTX-433 and its synthetic sperminecontaining analogue PhTX-343 (2) toward a range of human and insect glutamateand ACh-gated ion channels. The broad selectivity observed for 1 is compatible with its natural role as a prey suppression tool. Attempts to improve selectivity toward human receptors, necessary for potential therapeutic applications, have been partly successful through manipulation of the parent polyamine structure, whereas no notable improvements in selectivity have yet been observed through structural modifications of the tyrosine head group. Thus, while PhTX-83 (3) has been shown to be a selective antagonist of the a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR), PhTX-12 (4) is a selective human muscletype nAChR antagonist, which is inactive toward AMPAR. Herein, we report analogues in which the tyrosine moiety present in 1–4 has been replaced by cyclohexylalanine (Cha). These novel head-group analogues (compounds 5–10) show pronounced selectivity toward human muscle-type nAChR, with one of them exhibiting unprecedented potency at nanomolar concentrations. The synthesis of polyamine toxins is greatly facilitated by the use of solid-phase synthesis (SPS) strategies. In particular, Fukuyama–Mitsunobu alkylation has been successfully applied as a means of stepwise construction of the polyamine moiety. However, a large excess of reagents in three repetitive couplings is required to obtain satisfactory yields. Accordingly, a method that involves SN2 amine alkylation was devised for the synthesis of 8 and 9 (Scheme 1). In preliminary alkylation experiments with various resin-bound sulfonates, mesylate proved superior, presumably owing to the absence of competing trans sulfonation, which becomes predominant in SPS with increasingly reactive sulfonates. Hence, the mesylate displacement strategy was chosen for the synthesis of 8 and 9. Argopore Wang resin 13 was derivatized with a mono-Teocprotected diamine (11 or 12 ; Scheme 1, Method A), and the resulting resins were treated successively with Boc2O and Bu4NF to give resin 15 or 16, respectively. Peptide and acyl couplings completed the syntheses, with isolation of the required analogues 8 and 9 in 8–9% yield. Moreover, 9 was re-synthesized on a polystyrene trityl resin 14 by using a modified procedure in an attempt to improve the overall yield; instead of the repetitive polyamine chain elongation, the monoprotected diamine 12 was employed at a concentration of 1m (Scheme 1, Method B), to give 9 in 24% yield. Compounds 5–7 were obtained by using Fukuyama–Mitsunobu alkylation in isolated yields of 30%. For the analogue 10, which contains the symmetrical spermine moiety, it was more rational to apply the original synthetic method in solution. Thus, tri-Boc spermine was coupled with Cha, protected with an Fmoc group and activated as a pentafluorophenyl (Pfp) ester, followed by deprotection and coupling with Pfp butanoate. All analogues 5–10 [a] Dr. C. A. Olsen, P. Wellendorph, Dr. H. Franzyk, Prof. J. W. Jaroszewski Department of Medicinal Chemistry The Danish University of Pharmaceutical Sciences 2100 Copenhagen (Denmark) Fax: (+45)35306040 E-mail : jj@dfuni.dk [b] Dr. I. R. Mellor, Prof. P. N. R. Usherwood School of Biology, University of Nottingham Nottingham NG72RD (UK)