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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Bioorg Med Chem. Author manuscript; available in PMC 2011 January 1.
Published in final edited form as:
PMCID: PMC2967674
NIHMSID: NIHMS158080

Isoxazole analogues bind the System xc Transporter: Structure-activity Relationship and Pharmacophore Model

Abstract

Analogues of amino methylisoxazole propionic acid (AMPA), were prepared from a common intermediate 12, including lipophilic analogues using lateral metalation and electrophilic quenching, and were evaluated at System xc. Both the 5-naphthylethyl-(16) and 5-naphthylmethoxymethyl-(17) analogues adopt an E-conformation in the solid state, yet while the former has robust binding at System xc, the latter is virtually devoid of activity. The most potent analogues were amino acid naphthyl-ACPA 7g, and hydrazone carboxylic acid, 11e Y=Y′=3,5-(CF3)2, which both inhibited glutamate up-take by the System xc transporter with comparable potency to the endogenous substrate cystine, whereas in contrast the closed isoxazolo[3,4-d] pyridazinones 13 have significantly lower activity. A preliminary pharmacophore model has been constructed to provide insight into the analogue structure-activity relationships.

1. Introduction

L-Glutamate (L-Glu, 1) is the primary excitatory neurotransmitter in the mammalian CNS. Through its activation of a wide variety of ionotropic (iGluRs) and metabotropic (mGluRs) excitatory amino acid (EAA) receptors, L-glutamate-mediated signaling contributes to fast synaptic neurotransmission, higher order signal processing (e.g., synaptic plasticity, development, learning and memory), and even to neuropathology 1. Concentrations of L-Glu in the CNS are regulated by a family of excitatory amino acid transporters (EAATs) that rapidly sequester and concentrate this dicarboxylic amino acid in glia and neurons, and thereby limit its extracellular accumulation and access to EAA receptors. In contrast to the EAAT-mediated uptake of L-Glu, the system xc (SXc) transporter has been implicated in the export of L-Glu from CNS cells in a manner that allows it to activate EAA receptors. SXc is a member of the heteromeric amino acid transporter family (HATs; a.k.a. glycoprotein-associated amino acid exchangers) that functions as an obligate exchanger. Under physiological conditions SXc employs the L-Glu concentration gradient generated by the EAATs as the driving force for the import of L-cystine (L-Cys2, 2). Thus, the transporter mediates the uptake of a vital sulfur-containing amino acid needed for the synthesis of glutathione (GSH) and oxidative protection 2, while simultaneously producing an efflux of L-Glu that has the potential to contribute to either excitatory signaling or excitotoxic pathology. The significance of these actions is reflected in the range of CNS processes, to which SXc has been linked, including: drug addiction 3, 4, brain tumor growth 5, oxidative protection 6, viral pathology 7, the operation of the blood brain barrier 8, neurotransmitter release 9, and synaptic organization10.

Initial pharmacological studies on SXc established L-Cys2 and L-Glu as substrates, verified it operated as an obligate exchanger, and defined key features of its specificity, for example: i) L-aspartate is neither a substrate nor inhibitor, ii) L-homocysteate is an inhibitor, (i.e., a SO3 can replace a distal COO) and iii) L-α-aminoadipate and L-α-aminopimelate are inhibitors (i.e., longer chain lengths are tolerated) 11. Using CNS-derived tumor cell lines that express high levels of SXc, we have begun to more thoroughly investigate the SAR’s governing binding and translocation 12, 13. In addition to the SO3 moieties (S-sulfo-L-CySH, L-serine-O-SO3) the binding site also accommodates SO2 groups (L-homocysteine-sulfinate), but not the PO3−2 group of L-serine-O- PO3−2. The ability to bind higher homologues of L-Glu is substantiated by the actions of S-carboxymethyl- and S-carboxyethyl-L-cysteine. However, there is a limit to this trend, as L-homocystine and L-djenkolate exhibit reduced activities 12. Several conformationally constrained analogues of Glu also inhibit SXc, including quisqualate (QA, 4), 4-S-carboxy-phenylglycine (4-S-CPG), ibotenate (IBO), (RS)-4-Br-homoibotenate, and (RS)-5-Br-willardiine 12, 14, 15. Interestingly, several of these latter analogues are much better known for activities at other iGluRs and mGluRs, where the conformationally restricted positioning of their functional groups have presumably increased their specificity of action, as well as added to their value in delineating the respective binding site pharmacophores for the various receptors. Among these analogues, the actions of natural products QA and IBO as SXc inhibitors highlighted the potential use of isoxazoles as a scaffold for the development of additional blockers for this transporter. One of the best known of the isoxazoles is aminomethyl isoxazole propionic acid (6, AMPA) 16, which was the defining agonist for the GluR1-4 receptor subtypes (a.k.a. AMPA receptors) 1. While AMPA itself exhibits little or no activity at SXc, we have begun to explore the potential inhibitory activity of other AMPA analogues that have emerged from the pioneering studies of Krogsgaard-Larsen and colleagues 17. In this work a series of amino-3-carboxy-5-methylisoxazole propionic acid (ACPA) analogues is evaluated for inhibitory activity at SXc. Interestingly, we find that the introduction of lipophilic groups to the ACPA base structure, as exemplified by 7, provides a point of divergence that distinguishes the binding sites of GluR2 and SXc. This, in turn led us to examine non-amino acid bioisosteres of ACPA, and we have found that several hydrazone acids (11 and 16) bind to the SXc with affinities comparable to those of the endogenous substrates. In contrast, the isoxazolo[3,4-d] analogues 13 exhibit little or no binding to this transporter. These novel isoxazole-based analogues are used in combination with SAR data from other structurally diverse inhibitors (e.g., 4-S-CPG, sulfasalazine (5)) to construct a pharmacophore model of the SXc substrate binding site.

2. Results and Discussion

2.1 Preparation of Amino Acid Analogues of AMPA

The amino acid analogues of AMPA were prepared using synthetic methodology previously described by our laboratory, with the exception of MOM- and BOM-ACPA, 7b and 7e respectively, which were prepared from the known 5-hydroxymethylene acetal 18, via a straightforward Williamson ether synthesis, and carried forward to the amino acids using our previously described sequence 19.

2.2. Chemistry: Steric and electronic influence on the preparation of Isoxazole hydrazones verses Isoxazolo[3,4-d]pyridazinones

The synthesis of isoxazole hydrazones proceeds to the open form only if electron withdrawing groups are present on the ring 20. In our hands, no conditions could be discovered to inhibit the cyclization of aryl containing electron donating groups, and these proceed to the isoxazolo[3,4-d]pyridazinones 13 ([3,4-d]) 21. Variable temperature NMR studies indicate that the E- to Z-conversion begins at elevated temperature (ca. 60°C) and the ring closure appears to be irreversible. Therefore, to deliberately synthesize the closed [3,4-d] analogues 13 the reactions were conducted at reflux 2232. In those cases where the open forms were isolated, the best yields were obtained by conducting the reaction at room temperature, and the subsequent hydrolysis at the lowest temperature practical to effect conversion to the products 11, 16 and 17.

The lipophilic analogues listed are only representative, and prepared using lateral metalation and electrophilic quenching as previously described by our group 19. After deprotection and hydrazone formation E- and Z-isomers of the esters are almost always observed. We have isolated and crystallized both E-14a (Figure 4A) and Z-14b (Figure 4B), and examined their structures by x-ray crystallography. Complete Crystallographic Information Files (CIF) are contained in the Supplementary Material. The Z-form 14b exhibits an almost orthogonal relationship between the isoxazole mean plane and C=N hydrazone, and the NH is virtually perfectly poised for cyclization to the [3,4-d] 13. On hydrolysis, the purified Z-forms convert on standing in solution to the [3,4-d]. Of paramount importance in comparing the biology of open to closed forms is the caveat that it was necessary to make the solutions up freshly before study, and to verify after the assay that the open formed was indeed still open, which in some cases (i.e., 11a to 13a) occurs in a matter of a few hours. The E-forms of the hydrazone acids with electron withdrawing groups appear to be more conformationally stable.

Figure 4
Panel A, X-ray structure of E-14a. Panel B. X-ray structure of Z-14b.

2.3. Inhibition of Uptake of Glutamate at the System xc transporter

Inhibitory activity at SXc was assessed by quantifying the ability of the analogues to attenuate the uptake of L-[3H]-Glu into human SNB-19 glioblastoma cells under Cl-dependent (Na-free) conditions. These cells, as well as other glial tumor-derived lines, exhibit much higher levels of SXc activity than do primary astrocytes and are thus well suited for pharmacological assays 12, 33. Compounds were initially screened at a single concentration (e.g., 250 μM or 500 μM) with SXc activity reported as a % of control uptake: 573 ± 16 pmol/min/mg protein (mean ± SEM, n=22). In this respect, lower values denote greater levels of inhibition. As summarized in Table 1, the first series of isoxazoles examined, which included the well-known AMPA receptor ligands AMPA (6) and ACPA (7), exhibited no discernable activity at SXc. A similar lack of activity was also observed with phosphonate derivative of ACPA (8), as well as those isoxazoles to which small aliphatic groups had been added to C(5), (7a-7c). In contrast, however, the placement of aromatic functional groups, such as phenethyl (7d), benzyloxymethyl (7e), and 1- or 2- naphthylethyl (7f and 7g, respectively), at C(5) yielded amino acids with the capacity to markedly inhibit the transporter. The increased inhibitory activity of the compounds suggest that the presence of the larger aromatic moieties enhanced binding to the transporter either by influencing the relative configurations of the functional groups participating in substrate binding or through direct interaction with lipophilic domains adjacent to the substrate site, or both. These structure-activity relationships are also noteworthy in light of the observation that 7d, 7f and 7g exhibit essentially no ability to displace radiolabeled AMPA in brain slice experiments 34.

Table 1
Percent of control uptake of L-[3H]-Glu in the presence of ACPA derivatives

This initial group of isoxazoles screened as SXc inhibitors (Table 1) also included modifications to the free amino group of ACPA, including: semicarbazide hydrazone (9a and 9b), benzene-suphonylhydrazone (10a and 10b), phenylsemicarbazide hydrazone (9c), and arylhydrazone (11e) bioisosteres. While for the most part inactive, the latter two analogues (9c and 11e) exhibited a moderate amount of inhibitory activity and prompted the synthesis and characterization of the hydrazone derivatives listed in Table 2. These compounds also included the corresponding pyridazones that formed following intramolecular ring closure. In general these bicyclic [3,4-d] analogues (13a–j) exhibited little or no activity and in the instances where inhibition was observed, in every case the structurally open parent hydrazones (11a–f) were markedly more potent. For example: 11a produced 44% inhibition compared to 26% for 13a; 11b produced 78% inhibition compared to 15% for 13b; and 11e produced 86% inhibition compared to 18% for 13j. While the results indicate that electron withdrawing groups on the aryl moiety appear to correlate with higher levels of SXc inhibition, this may be the case only because electron donating groups favor select diazene tautomers (Scheme 2) and, thus, encourage cyclization to the less active closed [3,4-d] (13). As was observed with modifications at C(5) (Table 1), the presence of lipophilic moieties linked to the amino group also have the potential to increase the apparent affinity of the ligands. To discern if these lipophilic groups are interacting with distinct transporter domains a hybrid analogue (16a) with lipophilic moieties at both C4 and C5 of the isoxazole was prepared. Interestingly, 16a exhibited the same level of inhibition observed with 11d, suggesting that the two different aryl groups could be simultaneously accommodated by SXc. There does appear, however, to be some specificity with regard to these lipophilic domains, as the presence of an additional oxygen in the linkage significantly diminishes activity (i.e., compare 17 with 16a). More specifically, we previously demonstrated that the corresponding esters of these two acids (14a and 15, respectively) 35 adopt similar E-geometries about the hydrazone moiety, and NMR chemical shifts indicate that this geometry is maintained within the corresponding carboxylic acids 16 and 17. Worthy of note is that the open hydrazones, such as 8, 9b, 9c, 10b and 11d showed no detectable GluR2 binding in Gouaux’s S1S2 construct filter assay (36; Gouaux, personal communication).

Scheme 2
E- to Z- conversion of 11, 16, and 17 likely occurs via tautomerization to a diazene 18, and the Z-geometry 19 can close rapidly to the isoxazolo[3,4d] pyridazinone 13.
Table 2
Percent of control uptake of L-[3H]-Glu in the presence of isoxazole-hydrazone derivatives

Those compounds exhibiting significant levels of inhibition in the initial screening assays were characterized in greater detail by quantifying the concentration-dependence with which the analogues blocked SXc and calculating corresponding Ki values. These values, which are summarized in Table 3, were determined for most of the blockers using the Cheng-Prusoff equation (i.e., Ki = (1 + [S]/Km)/[IC50] and IC50 values generated from standard inhibition curves (see Figure 1) 37, 38. To confirm that the inhibition could be attributed to a competitive mechanism, a few of the analogues, including the most potent inhibitor (S-2-naphthyl-ethyl-ACPA, 7g), were also examined using a Michaelis-Menten analysis in which both the concentration of the inhibitor and substrate were systematically varied. A replot of the resulting Km,apparent values vs. [I] was then used to determine a Ki value (Figure 2). Consistent with competitive inhibition, these plots revealed an increase in the apparent Km without a significant change in Vmax. A comparison of the Ki values demonstrate that inhibitors 7g, 11b, and 11e each bind to the transporter with an affinity comparable to that of the endogenous substrate L-Cys2, about 50–60 μM. Although all of the inhibitors share an isoxazole structure, the diversity among these ligands also reveals a number of novel properties of the substrate binding site. Thus, 11b and 11e stand apart from almost all of the recognized inhibitors of SXc in that the hydrazone linkage essentially removed the commonly found free α-amino and α-carboxylate groups that typify the amino acids. The aryl groups on these inhibitors, as well as on 7g, also suggest the presence of lipophilic domains readily adjacent to the substrate binding site that facilitate the binding of the compounds compared to ACPA itself. Further, the fact that hybrid analogue 16a includes aryl additions at both positions and retains inhibitory activity, suggests that these lipophilic groups are interacting with distinct regions of the protein.

Figure 1
IC50 determination for selected hydrazone inhibitors
Figure 2
Representative Michaelis-Menten analysis and Ki determination for S-2-naphthyl-ethyl-ACPA
Table 3
Ki values for the competitive inhibition of SXc-mediated uptake of L-Glu.

2.4. Substrate activity

While the competition assays discussed above address the specificity of ligand binding, the results provide little insight into whether the analogues are acting as alternative substrates or as non-substrate inhibitors (i.e., compounds that bind but are not translocated) to competitively block uptake. To examine this we have developed an exchange assay in which the substrate-induced efflux of L-Glu is quantified by measuring the conversion of NADP+ to NADPH as the extracellular L-Glu leaves the cells and is rapidly metabolized by glutamate dehydrogenase (GDH) included in the assay mixture 12, 13. Thus, the addition of an SXc substrate induces an efflux of L-Glu that can be followed fluorometrically in real time. Representative traces (calibrated with a standard curve) for the SXc-mediated exchange of external L-Cys2 with internal L-Glu in SNB19 cells under Na-free conditions is shown in the Figure 3. In contrast to L-Cys2, the addition of S-2-naphthyl-ethyl-ACPA (7g) did not induce an efflux of L-Glu over background levels, indicating that it is not a substrate of the transporter. Consistent with the action of a competitive inhibitor, however, the inclusion of 7g did attenuate the ability of L-Cys2 to exchange with intracellular L-Glu. When each of the identified isoxazole inhibitors listed in Table 3 was tested in this manner, none produced an efflux of L-Glu that was significantly different from background. These finding suggest that while the substrate binding site can accommodate the variously modified isoxazoles, the compounds carrying these lipophilic additions cannot be translocated across the membrane.

Figure 3
Representative fluorescent traces of SXc-mediated exchange of extracellular substrates for intracellular L-glutamate in SNB-19 cells

2.5 Computational Modeling of the SXc Binding Domain: Preliminary Pharmacophore Model

As a suitable crystal structure is lacking for the development of an SXc protein homology model, our approach has involved the construction of a ligand-based, superposition, three-dimensional (3D) pharmacophore model. Such models account for structural and functional group similarities in addition to select low energy ligand conformations that have been positioned in 3D space and can be correlated to the SAR data generated from the SXc transport assays. A similar approach has been successfully employed for delineating potent and selective ligands for the EAATs 39, 40. The ligand training set (Figure 5, Panel A) included the structurally diverse endogenous substrates L-Glu and L-Cys2 and the potent inhibitors quisqualate (QA) and (S)-4-carboxyphenyl-glycine (4-S-CPG). The latter two provided elements of structural rigidity to the central pharmacophore core region 12. The model employs both SXc substrates and non-substrate inhibitors, thereby, allowing it to be consistent with the fundamental hypothesis that an obligate exchange transporter operating with an alternate access mechanism will employ a single substrate binding site that may be defined with discrete overlapping binding domains for the various ligands.

Figure 5
A ligand-based, superposition, three-dimensional (3D) pharmacophore model of the SXc binding site

Conformational space searching of the training set ligands afforded global energy minima for the substrates and inhibitors that were then aligned in 3D space to maximize overlap of the α–C, α-COOH, α-NH2, and distal COOH groups or related isosteric moieties. Alignment of the training set L-α-amino acid head groups affords the initial composite superposition pharmacophore model shown in Figure 5, Panel B. A key model aspect is the pseudo chair-like conformations of the substrate L-Cys2 (Panels B–D), which are thought to correlate to the central core ring structures of QA and 4-S-CPG. With the amino acid head groups aligned, the model suggests the likelihood that there may be two ways in which the binding site can accommodate the distal COOH group (or isosteric moieties) as represented in Panels B, C and D. Although the distal COOH of L-Cys2 could reach either of these positions, based upon rotation about the C6-7 bond (Figure 5, Panels C and D), we have utilized the L-Cys2 conformation per Panel C in the model, since it is consistent with a conformation in which intramolecular interactions between the 4-position sulfur atom and a proton of the C7 terminal amine group afford an intramolecular hydrogen bonding interaction (depicted in Panel A, ligand 2) that is supported by NMR spectroscopy 41, 42. The distances between the various COOH binding domains (e.g., Panels C or D as C-C distances between the respective COOH carbon atoms) are distinct from those of L-aspartate and L-α-aminoadipate, further rationalizing the inactivity of L-aspartate as an inhibitor and the activity of L-α-aminoadipate as a substrate 12. It is also notable that the conformational rigidity of the central rings of 4-S-CPG and QA may provide more optimal SXc binding as reflected by lower Ki values (e.g., ≈ 5 μM). The lack of flexibility due to the rings, however, may also account for decreased substrate activity 12. Thus, 4-S-CPG is essentially a non-substrate inhibitor and QA exchanges with L-Glu at a rate of only 35% that of L-Cys2.

Three compounds of interest were incorporated into the initial pharmacophore model, 7g and 11e (both of which exhibited Ki values comparable to L-Cys2), as well as 16a, to identify previously unrecognized regions of the transporter protein that are able to accommodate lipophilic groups and steric bulk. Identifying and optimizing such interactions has been key to the development of high-affinity ligands for the EAATs 43. The challenge in trying to understand the relative position these lipophilic substituents with respect to the Figure 5 Panels A–D model lies with deciding how to appropriately pair the two COOH groups of these newer compounds with the α- and distal COOH model moieties. Of the three lead inhibitors, only 7g possesses an L-α-amino acid head group. When the α–C, α-COOH and α-NH2 of 7g are aligned with the corresponding groups of the initial superposition model, the naphthyl ring moiety occupies the position as shown in Panel E of Figure 5. When the naphthyl moiety position of 7g is used as a common 3D descriptor for the same naphthyl moiety type of 16a, then the 3-COOH group extending from the isoxazole ring of 16a is correlated to the distal COOH group of L-Glu within the model, resulting in a second lipophilic model domain defined by the region occupied by the 2,4-dinitrophenyl moiety, as shown in Figure 5, Panel F. In a similar way, alignment of the 3,5-bis-trifluoromethyl-phenyl group of 11e with the 2,4-dinitrophenyl group of 16a aligns the 3-COOH group extending from the isoxazole ring of 16a to the distal COOH moiety of L-Glu (Figure 5, Panel G). Although the above lipophilic group alignments are consistent to each other, it is acknowledged that regions occupied by the respective lipophilic moieties could be reversed resulting in altered alignments of the COOH groups and related isoteric moieties.

Similar alignment ambiguity arises when the more recently identified inhibitor sulfasalazine 5 (5-[4-(2-pyridylsulfamoyl)phenylazo]salicylic acid) is incorporated into the model. This compound has attracted considerable attention because it provides a potential therapeutic link between SXc inhibition and the treatment of CNS glial tumors 5, 44. Thus, the pyridylsulfamoylphenyl portion of sulfasalazine can be correlated to either of the lipophilic regions defined by 7g, 11e, or 16a depending upon whether the COOH group of the salicyclic moiety of 5 is aligned with the α- or distal COOH group of L-Glu (Figure 5, Panel H). Since either model alignments are possible with sulfasalzine, then it is thought that the lipophilic domains extending beyond the core SXc substrate binding area imparts enhanced SXc binding. Thus, while the parent analogues (e.g., ACAP (7), S-ethyl-ACPA (7a)) exhibit little or no activity, the inclusion of lipophilic groups with sufficient π–electron character and steric bulk at the C5 of the isoxazole ring or via a hydrazone linkage to the free amino group of ACPA, or both, markedly enhances the ability of these ligands to bind to the SXc site.

The ligand alignment computational modeling and SAR insights have allowed us to generate an initial perspective of an SXc pharmacophore model. A composite cartoon representation of the model is shown in Figure 5, Panel I. The model central core ring area is consistent with the notion that an alternate access transporter can be defined with discrete overlapping binding domains for the various ligands. The model regions that surround the central core ring area include: a) the L-amino acid head group; b) an electron lone pair region or a hydrogen bonding group, equivalent to the distal COOH group of L-Glu or a heteroatom of the isoxazole ring of the analogues; c) a hydrogen bonding group, equivalent to the distal L-amino acid moiety of L-Cys2 or a second heteroatom of an isoxazole ring within an analogue, d) a distal carboxyl or heteroatom area, equivalent to the distal COOH group of L-Cys2 or 4-S-CPG; e) lone pair electron or π-electron density, associated with the sulfur atoms of L-Cys2 or the aromatic ring of 4-S-CPG; f) an aromatic ring group region #1 which is represented by analogue 7g; and g) an aromatic ring group region #2 which found in analogue 16a. Importantly, not all of the pharmacophore regions A–G need to be represented in each ligand to produce significant SXc binding. Additionally, higher affinity SXc ligands may possess different select combinations of the model A–G structural facets.

As previously mentioned, the increased inhibitory activity of EAAT ligands possessing lipophilic groups has been similarly used to infer the presence lipophilic domains adjacent to transporter substrate binding sites. Interestingly, for both the EAAT and SXc transporters, the interactions between these lipophilic domains and the ligands is also associated with a decreased ability of the appended analogues to act as alternative substrates. Taken together, our initial SAR-based pharmacophore model supports a single site hypothesis for SXc in which functional groups on identified substrates and inhibitors most likely interact with different subsets of available binding domain residues or the same residues in the binding site altered with different relative conformations. This is perhaps best illustrated by inhibitors such as 11e and sulfasalazine, which may be binding to SXc in a manner that does involve the L-α-amino acid head group area, and that also is defined by the endogenous L-Glu and L-Cys2 substrates for the transporter.

3. Conclusions

In summary, analogues of AMPA have been prepared that bind the SXc transporter, with the most efficacious analogues amino acid 7g and bioisostere 11e having comparable activity to the endogenous substrate cystine. Both classes of compounds appear to exhibit competitive binding. The isoxazolo[3,4-d] pyridazinones 13, in contrast exhibit only very weak binding at the SXc antiporter, although examination of their binding at other stages of the glutamate-glutamine cycle could be worthwhile. Experiments to address these and related model-derived questions are planned to significantly advance the discovery paradigm. Future targets of second generation refined SXc inhibitor analogues will further elucidate which combinations of regions of the pharmacophore model provide the greatest SXc binding potency. Of particular interest will be the further characterization of the lipophilic ligand domains discovered in the present study.

4. Experimental Section

Commercial reagents are routinely examined for purity by NMR and TLC, and recrystallized or distilled as appropriate. All reactions were monitored by TLC. NMR was performed on a Varian Unity Plus spectrometer at (400 MHz for 1H, 101 MHz for 13C) in deuteriochloroform unless otherwise noted. Chemical shifts (δ) are reported using CHCl3 (7.26 ppm for 1H), CDCl3 (77 ppm for 13C) as references. High resolution mass spectra (HRMS) were obtained using a Micromass electrospray ionization (ESI)/time-of-flight mass spectrometry (LC-TOF). Mass spectrometer samples were introduced using a Waters model 2690 separations module HPLC fitted with a C-18 reversed phase column (2.1 mm i.d., 5 cm). Elemental analyses for C, H, and N were performed by Midwest Microlab, Indianapolis, IN. Melting points were uncorrected.

4.1. Chemistry: Amino Acid analogues of AMPA

Amino acids, racemic ACPA ((±)-7) 45, (±)-8 (S)-ACPA, ((−)-7), 7d, 7g, 7f 19, 7a and (S,S)-7c 18 were prepared using methods previously described by our laboratories, and full experimental details have been published. (S)-MOM-ACPA (7b) and BOM-ACPA (7e) 46 were prepared from 12 via the known 5-hydroxyl-methyl acetal 18 using the amino acid synthesis sequence previously described 19.

4-[(2S)-2-amino-2-carboxyethyl]-5-[methoxymethyl]isoxazole-3- carboxylic acid (7b, (S)-MOM-ACPA)

Overall yield 12 % for 4 steps, 7b was obtained as 60 mg of a yellowish solid, m.p.=166–168°C (dec.). 1H NMR (D2O) δ 3.25 (dd, 1H), 3.38 (dd, 1H), 3.41 (s, 3H), 4.29 (t, 1H, J=6.8 Hz), 4.65 (s, 2H). 13C NMR δ 23.8, 53.6, 59.4, 63.8, 113.0, 157.3, 164.0, 169.8, 171.9 FAB+ (m-glycerol) m/z 245 [M+1]+ (38.8% rel. intensity—glycerol matrix gave base peak at m/z 74.79). Anal. Calculated C9H13N2O6 [M+1]+ mass: 245.077361. Accurate mass: FAB+ 245.0769. Optical rotation: [α]20D -5.3 (c 0.15, H2O). Anal. Calc’d for C9H13N2O6Cl: C, 38.51; H, 4.66; N, 9.98. Found: C, 38.51; H, 4.52; N, 9.73.

4-[(2R,S)-2-amino-2-carboxyethyl]-5-[benzyloxymethyl]isoxazole-3- carboxylic acid (7e, BOM-ACPA)

Overall yield 7 % for 4 steps, 7e was obtained as 40 mg of a yellowish solid, m.p.=166–168°C (dec.). 1H NMR (DMSO-d6) δ 2.97 (dd, 1H), 3.16 (dd, 1H), 3.92 (t, 1H, J=5.75 Hz), 4.55 (s, 2H), 4.65 (q, 2H, J=13.05 Hz), 7.37 (m, 5H). 13C NMR (126 MHz) δ 23.5, 30.5, 33.02, 52.0, 58.5, 60.8, 63.4, 71.8, 110.8, 127.5, 128.1, 161.3, 170.1 FAB+ (m-glycerol/DMSP) m/z 321 [M+1]+, 277, 219, 91.6 Anal. Calc’d for C15H16N2O6 (1.5 H2O): C, 51.87; H, 5.51; N, 8.06. Found: C, 51.48; H, 4.81; N, 8.80. HRMS Calculated C15H17N2O6 [M+1]+: 321.1087. Found: FAB+ 321.1073.

4.2. Chemistry Bioisosteres of AMPA

Common intermediate 12 was prepared as described by dal Piaz, 21. Isoxazolo[3,4-d]pyridazinone 13a is known, also from that group’s extensive work in the area 2232. Compounds 13b–d and 13f–g, and 13j are reported in Chemical Abstracts, and are commercially available from Ryan Scientific, Inc, but are not referenced to Patent or Publication literature, therefore, the CAS registry number and full characterization data is provided for each in the Supplementary Material. Open hydrazones 11a–e were prepared in analogous fashion as previously described by our laboratories 20.

4.3

5-Methyl-4-[1-(phenyl-hydrazono)-ethyl]-isoxazole-3-carboxylic acid (11a) light brown solid; yield = 60%; mp = 138°C; 1H NMR (CD3OD) δ 2.18 (s, 3H), 2.58 (s, 3H), 6.78 (t, J= 8Hz, 1H), 7.12 (d, J = 8.0 Hz, 2H), 7.19 (t, J= 8Hz, 2H); 13C NMR (CD3OD) δ 12.3, 16.6, 114.2 (3C), 120.8, 130.1 (3C), 134.9, 147.3, 163.5, 170.0; MS (ES+) 260 (M+1), HRMS calcd for C13H14N3O3 [M+H]+ 260.1035, found 260.1021.

4.4

5-Methyl-4-{1-[(4-nitro-phenyl)-hydrazono]-ethyl}-isoxazole-3-carboxylic acid (11b) yellow solid; yield = 72%; mp = 240 °C; 1H NMR (Acetone-d6) δ 2.36 (s, 3H), 2.61 (s, 3H), 7.93 (d, J= 8.0 Hz, 2H), 8.44 (d, J= 8.0 Hz, 2H); 13C NMR (Acetone-d6) δ 11.7, 16.1, 111.8, 112.2 (2C), 125.9 (2C), 138.8, 140.1, 151.2, 155.0, 161.4, 169.1; MS (ES+) 305 (M+1), HRMS calcd for C13H13N4O5 [M+H]+ 305.0886, found 305.0901.

4.5

5-Methyl-4-{1-[(2-nitro-phenyl)-hydrazono]-ethyl}-isoxazole-3-carboxylic acid (11c) orange solid; yield = 97%; mp = 168–169 °C; 1H NMR (Acetone-d6) δ 2.36 (s, 3H), 2.65 (s, 3H), 6.96 (m, 1H), 7.64 (m, 1H), 7.89 (d, J= 8.0 Hz, 1H), 8.18 (d, J= 8.0 Hz, 1H), 10.86 (bs, 1H); 13C NMR (Acetone-d6) δ 11.6, 16.0, 116.0, 118.8 (2C), 125.9 (2C), 136.6 (2C), 141.2, 154.9, 161.3, 169.6; MS (ES+) 305 (M+1), HRMS calcd for C13H13N4O5 [M+H]+ 305.0886, found 305.0903.

4.6

4-{1-[(2,4-Dinitro-phenyl)-hydrazono]-ethyl}-5-methyl-isoxazole-3-carboxylic acid (11d) red solid; yield = 66%; mp = 189–191°C; 1H NMR (CD3OD) δ 2.39 (s, 3H), 2.64 (s, 3H), 7.96 (d, J= 8.0 Hz, 1H), 8.38 (d, J= 8.0 Hz, 1H), 9.07 (s, 1H); 13C NMR (CD3OD) δ 12.4, 17.1, 70.6, 117.2, 117.6 (2C), 124.1 (2C), 131.2 (2C), 146.2, 146.9, 171.5; MS (ES+) 350 (M+1), HRMS calcd for C13H12N5O7 [M+H]+ 350.0737, found 350.0767. Anal. Calc’d for C13H12N5O7 (1.5 H2O): C, 41.49; H, 3.75; N, 18.61. Found: C, 41.46; H, 3.47; N, 18.01.

4.7

4-{1-[(3,5-Bis-trifluoromethyl-phenyl)-hydrazono]-ethyl}-5-(2-naphthalen-2-yl-ethyl)-isoxazole-3-carboxylic acid (11e) Off white solid; yield = 95%; mp = 137–139 °C; 1H NMR (Acetone-d6) δ 2.28 (s, 3H), 2.59 (s, 3H), 7.33 (s, 1H), 7.71 (s, 2H), 9.41 (bs, 1H); 13C NMR (Acetone-d6) δ 11.5, 16.4, 111.5, 112.6 (2C), 116.6, 131.9 (2C), 138.7 (2C), 147.5, 156.3, 162.3, 168.5, 185.5; MS (ES+) 396 (M+1), HRMS calcd for C15H12N3O3F6 [M+H]+ 396.0783, found 396.0801.

4.8

3,4-dimethyl-6-(4-(trifluoromethyl)phenyl)isoxazolo[3,4-d]pyridazin-7(6H)-one (13e) brown crystalline solid; yield = 28% mp= 161–163°C; 1H NMR δ 2.56 (s, 3H), 2.90 (s, 3H), 7.72 (d, J= 8Hz, 2H), 7.78 (d, J= 8Hz, 2H); 13C NMR δ 13.5, 19.8, 37.0, 112.6, 126.2, 129.7, 129.9, 141.1, 143.8, 152.5, 153.2, 170.6; MS (ES+) 310 (M+1), HRMS calcd for C14H11N3O2F3 [M+H]+ 310.0803, found 310.0771.

4.9

3,4-dimethyl-6-(3-nitrophenyl)isoxazolo[3,4-d]pyridazin-7(6H)-one (13h) white solid; yield = 96% mp= 187–188°C; 1H NMR δ 2.59 (s, 3H), 2.91 (s, 3H), 7.63 (m, 1H), 8.07 (m, 1H), 8.21 (m, 1H), 8.54 (s, 1H); 13C NMR δ 13.5, 19.8, 112.6, 121.1, 122.5, 129.6, 131.7, 141.6, 141.8, 152.4, 153.2, 170.8; MS (ES+) 287 (M+1), HRMS calcd for C13H11N4O4 [M+H]+ 287.0780, found 287.0785.

4.10

3,4-dimethyl-6-(3,5-dichlorophenyl)isoxazolo[3,4-d]pyridazin-7(6H)-one (13i) white solid; yield = 93% mp= 230–232°C; 1H NMR (400MHz, CDCl3) δ 2.56 (s, 3H), 2.89 (s, 3H), 7.34 (s, 1H), 7.59 (s, 2H); 13C NMR (100MHz, CDCl3) δ 13.5, 19.8, 112.5, 124.5(2C), 128.1(2C), 135.1, 141.2, 142.4, 152.4, 153.0, 170.7; MS (ES+) 310 (M+), 312 (M+2); HRMS calcd for C13H10N3O2Cl2 [M+H] 310.0150, found 310.0190.

4.11. 4-{1-[(2,4-Dinitro-phenyl)-hydrazono]-ethyl}-5-(2-naphthalen-2yl-ethyl)-isoxazole-3-carboxylic acid ethyl ester (14)

To a solution of ketone 19 (0.56g, 1.66 mmol) in THF (10 ml), 2,4-DNP reagent (10.9ml, 1.66 mmol) was added drop wise at 0°C. The mixture was stirred at room temperature for 6 hours. Precipitate formed was filtered and purified by column chromatography (30% ether in hexanes). The major product was obtained as orange colored solid (14a), yield = 81%, mp = 160°C; 1H NMR δ 1.43 (t, J = 7.1 Hz, 3H), 1.93 (s, 3H), 3.26 (t, J = 6.8 Hz, 2H), 3.40 (t, J = 6.2 Hz, 2H), 4.44 (q, J = 7.1 Hz, 2H), 7.17 (dd, J = 8.4 & 1.7 Hz, 1H), 7.44 (m, 2H), 7.52 (m, 1H), 7.65 (m, 2H), 7.72 (d, J = 8.4 Hz, 1H), 7.76 (m, 1H), 8.14 (dd, J = 9.5 & 2.6 Hz, 1H), 9.14 (d, J = 2.5 Hz, 1H), 10.98 (brs, 1H, NH). 13C NMR δ 14.1, 17.2, 27.9, 34.0, 62.6, 116.2, 116.7, 123.3, 125.9, 126.4, 126.6, 126.8, 127.2, 127.6, 128.3, 129.9, 130.1, 132.2, 133.4, 136.8, 138.6, 144.5, 144.9, 153.9, 159.9, 172.2; MS (FAB): m/z 518(33), 517 (100), 501 (12), 248 (11), 154 (34), 141 (61), 136 (23).

Anal. Calcd. for C26H23N5O7: C, 60.35; H, 4.48; N, 13.53. Found: C, 60.51; H 4.62; N 13.41. Minor product (14b), yellow solid, 1H NMR δ 1.30 (t, J = 8.0 Hz, 3H), 2.08 (s, 3H), 3.21 (m, 4H), 4.32 (q, J = 8.0 Hz, 2H), 7.16 (dd, J = 8.0 & 4.0 Hz, 1H), 7.30 (m, 2H), 7.44 (m, 1H), 7.58 (m, 3H), 7.76 (d, J = 8.0 Hz, 1H), 8.20 (dd, J = 8.0 & 4.0 Hz, 1H), 8.84 (d, J = 4.0 Hz, 1H), 10.37 (brs, 1H, NH).

4.12. 4-{1-[(2,4-Dinitro-phenyl)-hydrazono]-ethyl}-5-(2-naphthalen-2-yl-ethyl)-isoxazole-3-carboxylic acid (16a)

To a solution of 14a (0.65g) in THF, 2ml of 3M NaOH was added drop wise at 0°C. After 0.5h stirring the disappearance of ester was observed. The reaction mixture was acidified with 1M HCl to pH 2 and extracted with EtOAc. The organic extracts were then washed with saturated NaHCO3 followed by brine and finally dried over sodium sulfate. The organic layer was filtered and evaporated. The residue obtained was purified by column chromatography with 5% MeOH in CHCl3 as eluent. The pure product was obtained as yellow solid, yield = 67%, mp = 169–171°C; 1H NMR (Acetone-d6): δ 2.10 (s, 3H), 3.27 (t, J = 8.0 Hz, 2H), 3.50 (t, J = 8.0 Hz, 2H), 7.34 (dd, J = 8.0 & 4.0 Hz, 1H), 7.41 (m, 2H), 7.66 (m, 1H), 7.71 (m, 1H), 7.79 (m, 2H), 7.88 (d, J = 8.0 Hz, 1H), 8.29 (dd, J = 8.0 & 4.0 Hz, 1H), 9.00 (m, 1H), 10.97 (brs, 1H, NH). 13C NMR (Acetone-d6) δ 16.7, 30.4, 42.0, 94.6, 115.6 (2C), 123.0 (2C), 125.5 (2C), 126.2, 126.7 (2C), 127.5, 127.6, 127.8, 128.2 (2C), 130.5 (2C), 133.9, 134.0, 139.0, 172.7; MS (FAB): m/z 492 (9.0), 491 (33), 490 (100), 489 (32), 460 (27), 446 (51), 445 (25). Anal. Calcd. for (C24H19N5O7)2.H2O: C, 57.83; H, 4.04; N, 14.05. Found: C, 57.74; H, 3.58; N, 13.80.

4.13. 4-{1-[(2,4-Dinitro-phenyl)-hydrazono]-ethyl}-5-(naphthalen-2-ylmethoxymethyl)-isoxazole-3-carboxylic acid (17)

Yield: 60% The major isomer 1H NMR (DMSO) δ 2.34 (s, 3H), 4.74 (s, 2H), 4.89 (s, 2H), 7.65 (d, 1H, J=9.5 Hz), 7.80 (m, 4H), 7.91 (m, 3H), 8.15 (dd, 1H, J=2.4, 9.5 Hz), 8.79 (d, 1H, J=2.6 Hz), 10.87 (brs, 1H, NH). 13C NMR (500 MHz) δ 15.3, 72.3, 73.4, 102.0, 120.9, 125.6, 125.7, 125.8, 125.9, 127.3, 127.5, 127.6, 127.7, 128.2, 129.6, 132.3, 132.6, 134.7, 135.6, 144.6, 146.2, 149.8, 152.7, 190.8. Accurate mass Calculated for C24H20N5O8 [M+1]+ : 506.131187. Found: FAB+ 506.131188.

The minor isomer 1H NMR (DMSO) δ 2.29 (s, 3H), 4.51 (s, 2H), 4.78 (s, 2H), 7.25 (d, 1H, J=9.5 Hz), 7.42 (m, 4H), 7.54 (m, 3H), 8.40 (dd, 1H, J=2.4, 9.5 Hz), 8.89 (d, 1H, J=2.6 Hz), 10.55 (brs, 1H, NH). FAB+ m/z 506 [M+1]+, 490, 349, 306, 288, 272.

4.14. Cell Culture

SNB-19 glioma cells, purchased from American Type Culture Collection (Manassas, VA), were grown in Ham’s F-10 medium (pH 7.4) containing 1 mM pyruvate and 16 mM NaHCO3 and supplemented with 10% fetal calf serum. The cells were cultured in 150 cm2 flasks (Corning) and maintained at 37 °C in a humidified atmosphere of 5% CO2. For L-glutamate uptake experiments, cells were seeded in 12 well culture plates (Costar) at a density of 5×104 cells/well and for fluorometric assays cells were seeded on 9.5 × 22 mm coverslips at a density of 1 × 105 cells/coverslip and maintained for 5 to 8 days until 80 – 90 % confluent. Cells formed a confluent monolayer with an estimated density of 1 – 2.5×106 cells and a protein concentration of 100–350 mg/well or coverslip as determined by the bicinchoninic acid (BCA) method (Pierce). Cells were given fresh medium two hours prior to an assay.

4.15. Glutamate uptake assay

Uptake of L-glutamate into cultured cells was quantified using a modification of the procedure of Martin and Shane as previously described 12, 13, 47. Individual wells, after removal of culture media, were rinsed three times and pre-incubated in 1 ml Na+-free HEPES buffered (pH 7.4) Hank’s balanced salt solution (HBHS) at 30 °C for 5 min. The Na+-free buffer contained: 137.5 mM choline Cl, 5.36 mM KCl, 0.77 mM KH2PO4, 0.71 mM MgSO4.7H2O, 1.1 mM CaCl2, 10 mM D-glucose, and 10 mM HEPES. Uptake was initiated by aspiration of the preincubation buffer and the addition of a 500 μl aliquot of Na+-free transport buffer containing L-[3H]-glutamate (4 – 16 mCi/ml) mixed with L-glutamate (10–500 μM, final concentration). In those assays that evaluated inhibitor activity, the 500 μl aliquot of transport buffer contained both the L-[3H]-glutamate and potential inhibitors to ensure simultaneous addition. Following a 5 min incubation at 30 °C, the assays were terminated by three sequential 1 ml washes with ice cold buffer and then the cells were dissolved in 1 ml of 0.4 M NaOH for 24 hours. An aliquot (200 μl) was then transferred into a 5 ml glass scintillation vial and neutralized with the addition of 5 μl glacial acetic acid followed by 3.5 ml Liquiscint@ scintillation fluid (National Diagnostics) to each sample. Incorporation of radioactivity was quantified by liquid scintillation counting (LSC, Beckman LS 6500). Values are reported as mean ± S.E.M and are corrected for non-specific uptake (e.g., leakage and binding) by subtracting the amount of L-[3H]-glutamate accumulation at 4 °C. Inhibitory and Lineweaver-Burk plots and associated kinetic parameters for transport inhibitors were estimated using a nonlinear curve fitting analysis (KaleidaGraph 3.6.5). Ki values were estimated on the basis of a replot of Km,app values or using IC50 values and the Cheng-Prusoff equation 37.

4.16. Fluorometric assay

Fluorometric determination of L-glutamate efflux was quantified using a modification of the procedures described by Nicholls 48 and Vesce et al., 49. NADPH fluorescence was quantified using a Hitachi F-2000 fluorescence spectrophotometer fitted with a thermostatted cuvette holder and a 1 cm2 electronically driven magnetic stirrer platform. Confluent monolayers of SNB-19 glioma cells grown on coverslips were paired, placed back to back and rinsed in Na+-free HBHS buffer for 10 min. The coverslips were transferred and incubated in a stirred quartz cuvette containing 2 ml HBHS at 30 °C. Assays were initiated with the addition of NADP+ (1 mM) at 0 sec followed by GDH (33 U) at 60 sec. Subsequent additions of L-cystine or other potential substrates (50–500 μM) were made as detailed in the figure legends. Each experiment was terminated with the addition of Triton X-100 (0.5 %) to determine the total amount of L-Glu in the cells. The amount of L-Glu efflux was determined via a standard curve generated in an identical experiment but omitting the cells. Following the enzyme, seven sequential additions of 4 nmol L-Glu were made yielding a non-linear curve. Rates of L-Glu efflux are reported as mean ± S.E.M and are corrected for non-specific efflux (e.g. leakage) by subtracting the amount of L-Glu efflux in the absence of L-cystine.

4.17. Computational Modeling

Computational modeling followed methods we have employed previously for the production of a gas phase, steric strain, 3D superposition, ligand-based pharmacophore model 39, 40. Conformational searching and alignment studies were carried out at the University of Montana, Molecular Computational Core Facility. In essence, training set ligands, including substrates L-Glu and L-Cys2 and inhibitors quisqualate (QA) and (S)-4-carboxyphenyl-glycine (4-S-CPG) were built with SYBYL 7.0 software suite (Tripos, Inc.; St. Louis, MO) and subjected to energy minimization (10,000 iterations) by Powell minimization standard method. Initial Optimization and Termination parameters were set to None and Energy Change options, respectively. Default parameters and values within the minimization dialogue (Minimize Details) were used. Respective ligand conformers were stored as MOL2 files within a molecular spreadsheet and sorted based on energy profile. The low energy conformation was identified per ligand. Similar conformational searches were performed for 7g, 11e, 16a and other analogues. Each low energy analogue per ligand was imported with SYBYL and positioned in 3D space utilizing the following common atoms (centroids) for alignment, a) amino acid head group: α–carbon, carbon of the COOH group and the amino moiety (α-NH2), and b) the distal COOH group carbon. When necessary, isosteric groups were directly correlated to the above alignment moieties. The various composite alignments were inspected visually in 3D and checked for desired atom superposition. Composite MOL2 files were exported to MacPyMol software (Version 0.99; DeLano Scientific, LLC; South San Francisco, CA) to provide the Figure 5 molecular renderings.

Scheme 1
Analogs of AMPA: open 11, 16, 17 and closed isoxazolo[3,4-d]pyridazinones 13.
Chart 1
Ligand structures which bind the System XC- Transporter.
Chart 2
Non-amino acid analogs of AMPA/ACPA.

Supplementary Material

01

Acknowledgments

The Bruker (Siemens) SMART APEX diffraction facility was established at the University of Idaho with the assistance of the NSF-EPSCoR program and the M. J. Murdock Charitable Trust, Vancouver, WA, USA. The authors thank Drs. C. Sean Esslinger and Mariusz Gajewski for helpful discussions of analogue design. The molecular modeling studies were carried out in the U.M. Molecular Core Computational Facility. This work was supported in part by NIH NINDS NS038444 (NN,TR), NINDS NS30570 (RJB), NCRR P20RR015583 (RJB, NN, JG, SP), and the Malcolm and Carol Renfrew Scholarship (MIS, TR).

Footnotes

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