pubs.acs.org/jmc Published on Web 09/27/2010 r 2010 American Chemical Society J. Med. Chem. 2010, 53, 7441–7451 7441 DOI: 10.1021/jm100886h Assessment of Structurally Diverse Philanthotoxin Analogues for Inhibitory Activity on Ionotropic Glutamate Receptor Subtypes: Discovery of Nanomolar, Nonselective, and Use-Dependent Antagonists Sidsel Frølund, † Angelo Bella, † Anders S. Kristensen, † Hanne L. Ziegler, † Matthias Witt, ‡ Christian A. Olsen, †,§ Kristian Strømgaard, † Henrik Franzyk,* ,† and Jerzy W. Jaroszewski* ,† † Department of Medicinal Chemistry, Faculty of Pharmaceutical Sciences, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark, and ‡ Bruker Daltonik GmbH, Fahrenheitstrasse 4, D-28359 Bremen, Germany. § Present address: Department of Chemistry, Technical University of Denmark, Kemitorvet 207, DK-2800 Kgs. Lyngby, Denmark. Received July 15, 2010 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. Introduction Because of the complexity of their physiological and bio- chemical manifestations, disorders of the central nervous sys- tem represent a major challenge for modern medicine. At the same time, the burden of age-related neurodegenerative diseases increases worldwide in consequence of aging populations. While the etiology of neurodegenerative diseases is often un- known and the underlying mechanisms are exceedingly intri- cate, degeneration of isolated neurons is known to be associated with an imbalance in their neurotransmitter receptor systems. The most common cause of dementia is Alzheimer’s disease, 1-3 and two types of drugs for its relief are currently in use: acetylcholine esterase (AChE a ) inhibitors, used mainly to slow down progress during early stages of the disease, and meman- tine, which is prescribed in moderate to severe stages. 4 The use of AChE inhibitors, originating from the cholinergic deficit hypothesis of Alzeimer’s disease, counteracts low levels of cholinergic neurotransmission by increasing the availability of acetylcholine, 5,6 whereas memantine acts on the glutamatergic system by blocking N-methyl-D-aspartate (NMDA) receptors. Memantine 7-10 is believed to protect neurons from glutamate excitotoxicity without affecting physiological glutamate recep- tor activation required for normal function of the brain. Novel modulators of ionotropic glutamate receptors are therefore of interest as potential leads for neuroactive drug development. Analogues of philanthotoxin-433 (PhTX-433, 1, Figure 1) constitute a group of compounds that modulate the function of pore-forming ligand-gated receptors, 11 including ionotropic glu- tamate receptors (iGluR). PhTX-433 (1) was originally isolated from the venom of a solitary wasp, Philanthus triangulum. 12 PhTX-433 and its analogues inhibit iGluR by an uncompetitive mechanism, i.e., they require preactivation of the receptor, after which the toxin apparently enters and binds to the ion channel region of the receptor. This mechanism of action is believed to be similar to that of memantine. However, PhTX-433 (1) and its more readily available and extensively studied synthetic ana- logue, PhTX-343 (2), display potent activity on both acetylcholine- gated and glutamate-gated ion channels, and their selectivity on various subtypes of iGluR still remains largely unexplored. iGluR are classified into three main subgroups according to their preferential activation by (RS)-2-amino-3-(3-hydroxy-5- methylisoxazol-4-yl)propionate (AMPA), NMDA, or kainic acid, respectively. AMPA and NMDA receptors are the most abundant iGluR and are involved in more than 80% of all ex- citatory transmission in the central nervous system. The recep- tors are composed of four subunits, where the membrane- embedded domains of the four subunits form an ion channel that controls flux of cations such as Na þ ,K þ , and Ca 2þ in res- ponse to binding of glutamate. 13-15 AMPA receptors are homo- meric or heteromeric assemblies of four different subunits, *To whom correspondence should be addressed. Phone: þ45 35336372. Fax: þ45 35336041. E-mail: jj@farma.ku.dk. a Abbreviations: AChE, acetylcholine esterase; Adm, adamantyl; AMPA, (RS)-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionate; BAL, back- bone amide linker; Boc, tert-butyloxycarbonyl; Cha, cyclohexylalanine; Dab, (S)-2,4-diaminobutyric acid; Dap, (S)-2,3-diaminopropionic acid; DIC, diisopropylcarbodiimide; Fmoc, 9-fluorenylmethoxycarbonyl; gDap, (S)-2- amino-4-guanidinylpropionic acid; hArg, homoarginine; HOBt, N-hydroxy- benzotriazole; iGluR, ionotropic glutamate receptor; nAChR, nicotinic acetylcholine receptor; NMDA, N-methyl-D-aspartate; Pfp, pentafluorophe- nyl; Pyrazol(Boc) 2 , N, N 0 -bis(tert-butyloxycarbonyl)-1-guanylpyrazol; SPS, solid-phase synthesis; Teoc, 2-(trimethylsilyl)ethyloxycarbonyl; Teoc-ONp, 2-(trimethylsilyl)ethyl para-nitrophenyl carbonate; TFA, trifluoroacetic acid or trifluoroacetate; TFFH, tetramethylfluoroformamidinium hexafluoro- phosphate; VLC, vacuum liquid chromatography.