The Effects of Conformational Constraints in the Polyamine Moiety of Philanthotoxins on AMPAR Inhibition

Philanthotoxin‐433 (PhTX‐433) is a known potent inhibitor of ionotropic glutamate receptors, and analogues have been synthesised to identify more potent and selective antagonists. Herein we report the synthesis of four PhTXs with a cyclopropane moiety introduced into their polyamine chain, and their inhibition of an α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) receptor subtype by using two‐electrode voltage‐clamp assays on Xenopus oocytes expressing the GluA1flop subunit. All analogues were found to be more potent than PhTX‐343, with trans‐cyclopropyl‐PhTX‐343 being the most potent (∼28‐fold) and cis‐cyclopropyl‐PhTX‐343 least potent (∼4‐fold). Both cis‐ and trans‐cyclopropyl‐PhTX‐444 had intermediate potency (both ∼12‐fold). Molecular modelling indicates that a cyclopropane moiety confers a favourable steric constraint to the polyamine part, but this is compromised by a cis conformation due to enhanced intramolecular folding. Elongated PhTX‐444 analogues alleviate this to some extent, but optimal positioning of the amines is not permitted.


Introduction
Philanthotoxin-433 (PhTX-433, 1; the numerals indicate the number of methylene groups between the nitrogen atoms in the polyamine moiety; Figure 1) is a toxin found naturally in the venom of the solitary wasp, Philanthus triangulum. [1,2] PhTX-433 and many of its synthetic analogues have been shown to have noncompetitive inhibitory effects at both ionotropic glutamate receptors and nicotinic acetylcholine receptors. [3][4][5][6] In that respect, PhTXs are attractive molecules for further investigation given that both of these receptor types are accepted as valid drug targets for a variety of neurodegenerative and other disorders of the central nervous system (CNS). [7] The modular butyryl-tyrosyl-thermospermine composition of 1 has allowed the efficient generation of many synthetic analogues, demonstrating the importance of all of these structure segments. [4,6,[8][9][10][11] PhTXs 1, 2, and an array of other analogues have been shown to produce powerful voltage-dependent inhibition of a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) currents, suggesting a binding mode with the polyamine inserted deeply within the pore region of the ion channel. [3] This hypothesis is supported by the observation that AMPARs containing the GluA2 subunit with arginine at the "Q/R" site in the selectivity filter of the pore exhibit dras-tically decreased inhibition by PhTX-343 and other polyaminecontaining molecules. [12] In the last two decades advanced methodologies for solidphase synthesis (SPS) of polyamines have been developed; [13] however, no examples of SPS of cyclopropane-containing polyamine derivatives have been reported. The commonly used solution-phase method for obtaining polyamines displaying a cyclopropane moiety is alkylation of mesitylenesulfonamides with mesitylenesulfonates of cyclopropane diols, but this is not readily transferred into an SPS protocol due to the harsh conditions required for deprotonation of the sulfonamide and the risk of cross-linking the resin due to the bifunctional building block. [14] Cyclopropane-trans-1,2-dicarboxylic acid [15] may be Philanthotoxin-433 (PhTX-433) is a known potent inhibitor of ionotropic glutamate receptors, and analogues have been synthesised to identify more potent and selective antagonists. Herein we report the synthesis of four PhTXs with a cyclopropane moiety introduced into their polyamine chain, and their inhibition of an a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subtype by using two-electrode voltage-clamp assays on Xenopus oocytes expressing the GluA1flop subunit. All analogues were found to be more potent than PhTX-343, with trans-cyclopropyl-PhTX-343 being the most potent (~28-fold) and cis-cyclopropyl-PhTX-343 least potent (~4-fold). Both cis-and trans-cyclopropyl-PhTX-444 had intermediate potency (both~12-fold). Molecular modelling indicates that a cyclopropane moiety confers a favourable steric constraint to the polyamine part, but this is compromised by a cis conformation due to enhanced intramolecular folding. Elongated PhTX-444 analogues alleviate this to some extent, but optimal positioning of the amines is not permitted. readily obtained from the corresponding ethyl diester, and the cis-anhydride 3-oxabicyclo[3.1.0]hexane-2,4-dione was commercially available. Therefore, we chose an approach involving on-resin reduction of the diamide corresponding to the desired 4,4'-dimethoxytrityl-protected polyamine. [16] In the present work we focus on incorporating unprecedented structural variations of the polyamine moiety present in both 1 and its well-studied close structural analogue, PhTX-343 (2), and examine how these influence the inhibitory effects of the resulting PhTX analogues on a specific subunit, GluA1flop, present in members of the AMPAR subdivision of the ionotropic glutamate receptor family. This subunit is characteristic of a calcium-permeable and polyamine-sensitive subtype of AMPARs, with the flop splice variant (a 38-residue region upstream of the fourth transmembrane region) being up-regulated in place of the flip splice variant during early development to become dominating in adult CNS. Constraints were introduced in the central region of the polyamine moiety in an attempt to assess the importance of rigidity in this region. This was achieved by solid-phase synthesis incorporating cyclopropane moieties into the polyamine chain to give trans and cis analogues with 3-4-3 or 4-4-4 (i.e., PhTX-343-or PhTX-444-like) spacing between the nitrogen atoms (3-6: trans-343, trans-444, cis-343, cis-444, respectively; Figure 1). Analogues 2-6 were tested for inhibitory potency against homomeric rat GluA1flop AMPARs expressed in Xenopus oocytes by measurement of their reduction of currents evoked by stimulation with kainic acid (KA; 100 mm) by using a two-electrode voltageclamp at a holding potential of À80 mV. Although KA is a weaker agonist of AMPARs, it is often used instead of AMPA or l-Glu in these assays to avoid the problem of rapid receptor desensitisation.

Results and Discussion
Solid-phase synthesis of philanthotoxin analogues First, cyclopropane-trans-1,2-dicarboxylic acid diethyl ester (7) was converted into the corresponding pentafluorophenyl diester 8 (Scheme 1) via alkaline hydrolysis and subsequent transesterification by using pentafluorophenyl trifluoroacetate/pyridine (1:1) in DMF. [15,17] Building blocks 9 a and 9 b were obtained upon condensation of pentafluorophenyl diester 8 with one equivalent of the appropriate 2-(trimethylsilyl)ethoxycarbonyl (Teoc)-monoprotected diamine. [16b] To minimise cross-linking of the 2-chlorotrityl chloride resin, it was loaded with the appropriate Teoc-monoprotected diamine (Scheme 2). A prolonged reaction time of two days enabled a cost-efficient use of these selectively protected diamines, as only 10 % excess was required. The resulting resinbound diamines 10 a/10 b were then coupled with building blocks 9 a/9 b to give Teoc-protected diamide intermediates 11 a/11 b that were subjected to exchange of the N-protecting group followed by borane reduction to yield the 4,4'-dimethoxytrityl (Dmt)-protected polyamine intermediates 12 a/12 b. The thus formed secondary amino functionalities were Bocprotected, and then the Dmt group on the terminal primary amine was removed under weakly acidic conditions to give selectively protected resin-bound polyamines 13 a/13 b. Successive acylation with the activated ester, Fmoc-Tyr(tBu)-OPfp, Fmoc deprotection, acylation with pentafluorophenyl butanoate, [18] and cleavage from the linker afforded the crude trans-cyclopropane-containing target philanthotoxins that were readily purified by reversed-phase preparative HPLC to give PhTXs 3 and 4 as the tris(TFA) salts.
SPS of the corresponding cis-cyclopropane-containing PhTXs also started from resin-bound diamines 10 a/10 b, but in this case these were acylated with the cis-anhydride 3-oxabicyclo-  Elongation with the respective Teoc-monoprotected diamines under DIC/HOBt amide coupling conditions followed by removal of the Teoc group furnished the unprotected diamides 15 a/15 b. Introduction of the Dmt-protecting group and subsequent borane reduction of the amide functionalities gave rise to resins 16 a/16 b, which upon Boc-protection and Dmt removal, afforded primary amines 17 a/17 b that were acylated and cleaved from the resin as described for the trans analogues to yield PhTXs 5 and 6 as the corresponding tris(TFA) salts upon purification (Scheme 3).

Receptor inhibition by philanthotoxins
PhTX analogues 2-6 were all able to cause potent inhibition of currents evoked by exposure of oocytes to 100 mm KA ( Figure 2), with their IC 50 values listed in Table 1. The present study is the first to report on PhTX-343 inhibition of homomeric rat GluA1flop channels, whereas previous reports have focused on rat GluA1flip or AMPARs expressed from mRNA extracted from rat brain. We found that the IC 50 for GluA1flop inhibition is similar to that for GluA1flip, [12] but is~10-fold higher than that observed for KA-activated channels expressed from rat brain mRNA. [4,10] The subunit combination of the latter is not known, but presumably it contains other AMPAR subunits that may be more sensitive to PhTX 2. All of the cyclopropane-containing PhTX analogues (3-6) were significantly more potent inhibitors than 2, with the trans-configured 3 being the most potent, nearly 30-fold more so than the corresponding straight-chain PhTX 2.

Modelling studies on philanthotoxins
It is well known that the potency of a philanthotoxin analogue is influenced by changes in the length of the polyamine moiety as well as by the number and distribution of the posi-   Table 1. tively charged amino groups, as these factors determine the overall shape and electronic properties of the resulting molecule via intramolecular hydrogen bonding. [3,4,19,20] PhTX analogues can adopt three general low-energy structures: "head and tail" (HT; extended), "semi-folded" (SF), and "folded" (F) (as depicted in Figure 3), depending on whether hydrogen bonding involves the first, second, or third amino group, respectively. The HT structure is believed to be the most active at AMPARs due to previous observations showing that the monocationic analogues are virtually inactive at AMPARs. [4,14] In terms of general shape, monocationic analogues are "folded" because they do not possess a "tail" at all. Our modelling studies show that for all compounds the vast majority (99.8 %) of low-energy conformations possessed at least one intramolecular hydrogen bond. Ensembles of lowenergy conformations obtained for the PhTX analogues stud-ied contained all three types of structures (HT, SF, and F) within 3 kcal mol À1 from the apparent energy minimum (0.5-1.0 kcal mol À1 ). To reveal the conformational preferences of the compounds we compared the relative numbers of conformations belonging to the different types within these ensembles. Compound 2 was found to be distributed as 85 % HT (Figure 3 a), 13 % SF, and 2 % F (Figure 3 b). This was unchanged for 3, so the additional hydrocarbon bulk, which in fact becomes part of the head group and thus relocating the tyrosyl group (Figure 3 c), appears to be responsible for the increased potency of 3. This is reminiscent of analogues in which short hydrocarbon chains were introduced to the central section of the polyamine moiety resulting in more potent analogues at insect quisqualate-sensitive ionotropic glutamate receptors. [9] The cis analogue of 3 (i.e., compound 5) was less potent, as the cis arrangement caused the molecule to adopt a greater proportion of SF structure (44 %; Figure 3 d) at the expense of HT (54 %). The cause of this effect is that in the cis form, both amino groups occur at the same side of the bulky and rigid cyclopropane ring. While the trans configuration dictates a rather large distance between amino groups (6.2 in the lowest-energy structure), the cis configuration results in a significantly smaller distance (4.6 ). As a result, the probability that the second amino group is involved in intramolecular hydrogen bonding (and thus the structure is semi-folded) is larger for the cis than for the trans configuration (Figure 3 g).
PhTX analogues 4 and 6 showed intermediate potency between 3 and 5 because the additional carbon in the chain between the head group and the first amine functionality allowed for greater flexibility and hence a higher proportion of SF structures (Figure 3 e,f). The SF structures for these analogues are likely to be more active because of the longer chain between the second and terminal amine groups (giving it a more HT-like structure).

Conclusions
Introduction of a cyclopropane moiety between the first and second amino groups of PhTX-343 proved beneficial for AMPAR antagonism, but less so for the cis analogue due to its greater tendency to become semi-folded as inferred by modelling studies. The positive (for trans analogues) and negative (for cis analogues) effects of this structural alteration are, to some extent, obscured by the increased spacing of the amide and the first amine functionality as well as of the second and third amines, such that both PhTX-444 analogues display similar potency.

Experimental Section
General procedures: Unless otherwise stated, starting materials and solvents were purchased from commercial suppliers (H-Tyr-(tBu)-OH from Novabiochem, reagents and solvents from Sigma-Aldrich, Fluka, or Lancaster) and used as received. CH 2 Cl 2 was distilled from P 2 O 5 and stored over 4 molecular sieves. THF was distilled from Na/benzophenone immediately before use. 1 H and 13 C NMR spectra were recorded at 400.14 and 100.62 MHz, respec- Figure 3. Structures of philanthotoxin analogues 2-6, showing the three general conformations that can be adopted: "head and tail" for 2 (a) and 3 (c), "semi-folded" for 4, 5, and 6 (d-f), and "folded" for 2 (b). A clearer view of hydrogen bonding (g, pink lines) between amide oxygen atoms and amine hydrogens in 3 for an HT conformation and in 5 for an SF conformation.
SPS of trans-cyclopropane PhTX analogues: Resin-bound diamines (10 a/10 b, 0.44 mmol) were swelled in dry DMF for 30 min. The solvent was removed by suction, and the resin was treated with a cyclopropane building block (9 a or 9 b, 2 equiv), HODhBt (1 equiv), and DIPEA (2 equiv) in dry DMF (10 mL (mmol resin) À1 ). The mixture was shaken for 16 h at room temperature. The resin was drained and then washed with DMF, MeOH, CH 2 Cl 2 , and DMF (3 12 mL (mmol resin) À1 with each solvent). The Teoc group was removed by treatment with TBAF (5 equiv) in DMF (7.5 mL (mmol resin) À1 ) at 558C for 15 min. Upon draining and wash with DMF, this deprotection step was repeated. The resin was drained and then washed with DMF, MeOH, and CH 2 Cl 2 (3 ). Dmt-Cl (6 equiv) in dry CH 2 Cl 2 (9 mL (mmol resin) À1 ) and DIPEA (6 equiv) were added to the resin. After 3.5 h, the resin was drained and washed with CH 2 Cl 2 , CH 2 Cl 2 /MeOH/DIPEA (80:15:5), DMF, CH 2 Cl 2 , and THF (each 3 ). The resin was transferred to a 25 mL flask with a minimum of THF, and then 1 m BH 3 ·THF in THF (20 equiv) was added. The flask was heated at reflux under N 2 in an oil bath (70 8C) for 16 h. The resin was transferred back to a Teflon reactor using THF. Upon draining, the resin was washed with MeOH (3 ). Then the resin was treated with piperidine (9 mL (mmol resin) À1 ) at 608C for 1.5 h. After washing with MeOH (12 mL (mmol resin) À1 ), this decomplexa-tive energy minimisations did not decrease the energy of the apparent global minimum. For statistical analysis, the 1000 lowest energy conformations were selected.