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Acknowledgements
This work is supported by the Cognitive Sciences and Technologies Council of Iran (CSTC, Grant No. 95P31), the Iran National Science Foundation (INSF) and the Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran. The authors wish to thank Dr. Ayat Kaeidi for his excellent technical assistance and helpful suggestions.
Introduction
Glutamate is an excitatory amino 11e australia that plays an important role in a wide range of physiological processes, including neurotransmission, synaptogenesis, neuronal plasticity, learning and memory [1,2]. Under physiological conditions, the concentration of glutamate in synapses is tightly controlled. Abnormally high concentrations of glutamate are connected with neurodegenerative processes, such as hypoxia, Alzheimer’s disease (AD), ischemia, or traumatic brain injury [[3], [4], [5], [6]]. Therefore, reduction of glutamate toxicity represents a possible therapeutic target for neurodegenerative processes [7].
Glutamate activates several types of receptors, including three families of ionotropic receptors: N-methyl-D-aspartate (NMDA), 2α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and kainate. They all incorporate ion channels that are permeable to cations, although the relative permeability of Na+ and Ca2+ varies among the respective families and the subunit composition of the receptor [[8], [9], [10]]. Excessive activation of NMDARs that gate channels with high Ca2+ permeability, results in accumulation of intracellular Ca2+ and subsequent cell death [11]. Many compounds, including memantine [12] and neurosteroids, are able to target glutamate toxicity through the modulation of NMDARs. 20-oxo-5β-pregnan-3α-yl sulfate (pregnanolone sulfate, PAS, Fig. 1) represents an endogenous neurosteroid that inhibits NMDAR currents [13]. The major structural requirements for neurosteroid NMDAR-inhibitors have been previously described [[14], [15], [16]]. It has been found that the inhibitory effect is dependent upon the 3α- and 5β-stereochemistry of the pregnane skeleton, in combination with a negatively charged moiety at C-3. Pregnanolone glutamate (PAG, Fig. 1) has been designed as a synthetic analogue of PAS, with expected neuroprotective properties. Indeed, its neuroprotective effect was assessed in several biological models in vivo [[17], [18], [19]]. However, we anticipated that the labile ester bond connecting the glutamate moiety of PAG to the steroid skeleton would be susceptible to plasmatic degradation. Thus, we thought to replace the ester linkage with the more robust amide bond. This structural modification was designed to reduce the metabolic liability of new analogues, and alter their solubility and permeability profiles [20]. Based on our previous SAR study, a nonpolar modification of the D-ring [21] and variations on the presence/absence of a C-3 amino substituent and its Boc-protected derivative, were also incorporated into the structural motif. These compounds (7–18, Fig. 1) have been shown to inhibit the activity of NMDARs on human embryonic kidney cells (HEK293) transfected with plasmids encoding GluN1-1a/GluN2B/GFP genes (IC50 values varying from 1.4 to 21.7 μM) [20,22].
Memantine (1) and MK-801 (2) were used as comparators in our study. Memantine is a non-competitive NMDAR antagonist used for the treatment of moderate to severe AD [12]. Its usual therapeutic plasma concentration in AD patients was determined as 1 μM [23]. Another reference compound, MK-801 (2) is a potent, uncompetitive antagonist of NMDAR [24] that is not used clinically due its serious side effects – brain lesions.
In the present study, we have evaluated the neuroprotective potential of the amide PAG-like compounds in primary cultures of rat embryonic cortical neurons. Compounds with an ester bond at the C-3 position (PAS, PAG, 5, 6) were included in this study as steroidal comparators. Note that it has already been demonstrated, that neither pregnanolone sulfate PAS (3) nor its synthetic analogue - pregnanolone glutamate PAG (4) induce psychotomimetic side effects, such as hyperlocomotion and sensorimotor gating deficit, which are hallmarks of NMDA antagonism [17,25]. Therefore, we expected the herein presented compounds 5-18 to display similar pharmacological advantage.