Isoxazole analogues bind the System xc- transporter: Structure–activity relationship and pharmacophore model

doi:10.1016/j.bmc.2009.11.001

Bioorganic & Medicinal Chemistry

Volume 18, Issue 1, 1 January 2010, Pages 202-213

https://doi.org/10.1016/j.bmc.2009.11.001 Get rights and content

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 $x_{c}^{-}$ . 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 $x_{c}^{-}$ , 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 uptake by the System $x_{c}^{-}$ 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.

Graphical abstract

Isoxazole amino acid 7g and hydrazone 11e were found to possess System $x_{c}^{-}$ binding affinity comparable to the endogenous substrate cystine, and using these observations a pharmacophore model had been developed.

Introduction

l-Glutamate (l-Glu, 1, Chart 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 $x_{c}^{-}$ ( ${Sx}_{c}^{-}$ ) transporter has been implicated in the export of l-Glu from CNS cells in a manner that allows it to activate EAA receptors. ${Sx}_{c}^{-}$ 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 ${Sx}_{c}^{-}$ 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 ${Sx}_{c}^{-}$ 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 organization.¹⁰

Initial pharmacological studies on ${Sx}_{c}^{-}$ 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 ${SO}_{3}^{-}$ 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 ${Sx}_{c}^{-}$ , we have begun to more thoroughly investigate the SAR’s governing binding and translocation.12, 13 In addition to the ${SO}_{3}^{-}$ moieties (S-sulfo-l-CySH, l-serine-O- ${SO}_{3}^{-}$ ) the binding site also accommodates ${SO}_{2}^{-}$ groups (l-homocysteine-sulfinate), but not the ${PO}_{3}^{- 2}$ group of l-serine-O- ${PO}_{3}^{- 2}$ . 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 ${Sx}_{c}^{-}$ , 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 ${Sx}_{c}^{-}$ 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 ${Sx}_{c}^{-}$ , we have begun to explore the potential inhibitory activity of other AMPA analogues that have emerged from the pioneering studies of Krogsgaard-Larsen and co-workers.¹⁷ In this work a series of amino-3-carboxy-5-methylisoxazole propionic acid (ACPA) analogues is evaluated for inhibitory activity at ${Sx}_{c}^{-}$ . 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 ${Sx}_{c}^{-}$ . This, in turn led us to examine non-amino acid bioisosteres of ACPA, and we have found that several hydrazone acids (11 and 16, Chart 2) bind to the ${Sx}_{c}^{-}$ 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 ${Sx}_{c}^{-}$ substrate binding site.

Section snippets

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

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.

Conclusions

In summary, analogues of AMPA have been prepared that bind the ${Sx}_{c}^{-}$ 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 ${Sx}_{c}^{-}$ antiporter, although examination of their binding at other stages of the glutamate–glutamine cycle could be worthwhile.

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 deuterochloroform 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

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 (N.N.,T.R.), NINDS NS30570 (R.J.B.),

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^†: Present address: GSK Biologicals, 553 Old Corvallis Rd., Hamilton MT 59840, USA.

^‡: Present address: PharmAgra Labs, 158 McClean Rd., Brevard, NC 28712, USA.

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Isoxazole analogues bind the System xc- transporter: Structure–activity relationship and pharmacophore model

Abstract

Graphical abstract

Introduction

Section snippets

Preparation of amino acid analogues of AMPA

Chemistry: steric and electronic influence on the preparation of isoxazole–hydrazones verses isoxazolo[3,4-d]pyridazinones

Conclusions

Experimental section

Acknowledgments

J. Biol. Chem.

Neuropharmacology

Toxicol. Appl. Pharmacol.

Tetrahedron Lett.

Il Farmaco

Bioorg. Med. Chem.

Bioorg. Med. Chem.

Mech. Ageing Dev.

Neuropharmacology

Bioorg. Med. Chem.

Tetrahedron Lett.

J. Biol. Chem.

FEBS Lett.

Excitatory Amino Acid Transmission in Health and Disease

Glia

Nat. Neurosci.

Biol. Psychiat.

J. Neurosci.

J. Neurosci.

J. Neurochem.

J. Pharmacol. Exp.Therapeut.

J. Neurosci.

J. Neurosci.

J. Neurochem.

Isoxazole analogues bind the System $x_{c}^{-}$ transporter: Structure–activity relationship and pharmacophore model