In the mammalian central nervous system, (S)-glutamate (Glu) is released from the presynaptic neuron where it activates a plethora of pre- and postsynaptic Glu receptors. The fast acting ionotropic ...Glu receptors (iGluRs) are ligand gated ion channels and are believed to be involved in a vast number of neurological functions such as memory and learning, synaptic plasticity, and motor function. The synthesis of 14 enantiopure 2,4-syn-Glu analogues 2b–p is accessed by a short and efficient chemoenzymatic approach starting from readily available cyclohexanone 3. Pharmacological characterization at the iGluRs and EAAT1–3 subtypes revealed analogue 2i as a selective GluK1 ligand with low nanomolar affinity. Two X-ray crystal structures of the key analogue 2i in the ligand-binding domain (LBD) of GluA2 and GluK3 were determined. Partial domain closure was seen in the GluA2-LBD complex with 2i comparable to that induced by kainate. In contrast, full domain closure was observed in the GluK3-LBD complex with 2i, similar to that of GluK3-LBD with glutamate bound.
The excitatory amino acid transporters (EAATs) play essential roles in regulating the synaptic concentration of the neurotransmitter glutamate in the mammalian central nervous system. To date, five ...subtypes have been identified, named EAAT1–5 in humans, and GLAST, GLT-1, EAAC1, EAAT4, and EAAT5 in rodents, respectively. In this paper, we present the design, synthesis, and pharmacological evaluation of seven 7-N-substituted analogues of UCPH-101/102. Analogue 9 inhibited EAAT1 in the micromolar range (IC50 value 20 μM), whereas analogues 8 and 10 were inactive (IC50 values >100 μM). The diastereomeric pairs 11a/11b and 12a/12b were separated by HPLC and the absolute configuration assigned by VCD technique in combination with ab initio Hartree–Fock calculations. Analogues 11a (RS-isomer) and 12b (RR-isomer) inhibited EAAT1 (IC50 values 5.5 and 3.8 μM, respectively), whereas analogues 11b (SS-isomer) and 12a (SR-isomer) failed to inhibit EAAT1 uptake (IC50 values >300 μM).
Uptake of the major excitatory neurotransmitter in the CNS, (
S
)-glutamate, is mediated by a family of excitatory amino acid transporters (EAAT). Previously we have explored the structure–activity ...relationship (SAR) of a series of EAAT1 selective inhibitors, leading to the development of the potent inhibitors UCPH-101 and UCPH-102. In the present study, we set out to improve the solubility properties of these EAAT1 inhibitors with the objective to develop analogs more suited as pharmacological tools for in vivo studies of EAAT1 in terms of their bioavailability. A total of 23 novel UCPH-101/102 analogs were designed, synthesized and characterized pharmacologically at EAAT1-3 in a
3
H-
d
-aspartate uptake assay. Most notably, the potent EAAT1 inhibition displayed of UCPH-101 and UCPH-102 was retained in analog
1d
in which the napht-1-yl group in the 7-position of UCPH-102 has been replaced by an
o
-biphenyl moiety. In contrast, EAAT1 activity was dramatically compromised in analogs
1e
and
1f
comprising
m
- and
p
-biphenyl groups as 7-substituents, respectively. Analog
1d
displayed low bioavailability after oral administration in rats, and this problem was addressed by the synthesis of a series of analogs with different chloro, fluoro, methoxy, triflouromethyl and carboxy substitution patterns at the
o
-biphenyl group of
1d
(
1h
–
1s)
and
m
- and
p
-pyridine analogs of
1d
(
1t
and
1v
). Unfortunately, all of the modifications resulted in substantial decreased EAAT1 inhibitory activity, which supports the notion of a very lipophilic binding pocket in EAAT1 for the aromatic 7-substituent in these ligands. In conclusion, while we have not succeeded in developing UCPH-101/102 analogs possessing improved bioavailability properties, this study does offer interesting SAR information about this inhibitor class, and analog
1d
seems to be an interesting lead for future SAR studies with focus on the development of more potent EAAT1 inhibitors.
Graphical Abstract
Neuregulin-1 has a key role in mediating signaling between axons and Schwann cells during development. A limitation to studying its role in adulthood is the embryonic lethality of global Nrg1 gene ...deletion. We used the Cre-loxP system to generate transgenic mice in which neuregulin-1 is conditionally ablated in the majority of small-diameter and a proportion of large-diameter sensory neurons that have axons conducting in the C- and Adelta-fiber range, respectively. Sensory neuron-specific neuregulin-1 ablation resulted in abnormally large Remak bundles with axons clustered in "polyaxonal" pockets. The total number of axons in the sural nerve was unchanged, but a greater proportion was unmyelinated. In addition, we observed large-diameter axons that were in a 1:1 relationship with Schwann cells, surrounded by a basal lamina but not myelinated. There was no evidence of DRG or Schwann cell death; the markers of different DRG cell populations and cutaneous innervation were unchanged. These anatomical changes were reflected in a slowing of conduction velocity at the lower end of the A-fiber conduction velocity range and a new population of more rapidly conducting C-fibers that are likely to represent large-diameter axons that have failed to myelinate. Conditional neuregulin-1 ablation resulted in a reduced sensitivity to noxious mechanical stimuli. These findings emphasize the importance of neuregulin-1 in mediating the signaling between axons and both myelinating and nonmyelinating Schwann cells required for normal sensory function. Sensory neuronal survival and axonal maintenance, however, are not dependent on axon-derived neuregulin-1 signaling in adulthood.
Although the selective excitatory amino acid transporter subtype 1 (EAAT1) inhibitor UCPH‐101 has become a standard pharmacological tool compound for in vitro and ex vivo studies in the EAAT research ...field, its inability to penetrate the blood–brain barrier makes it unsuitable for in vivo studies. In the present study, per os (p.o.) administration (40 mg kg−1) of the closely related analogue UCPH‐102 in rats yielded respective plasma and brain concentrations of 10.5 and 6.67 μm after 1 h. Three analogue series were designed and synthesized to improve the bioavailability profile of UCPH‐102, but none displayed substantially improved properties in this respect. In vitro profiling of UCPH‐102 (10 μm) at 51 central nervous system targets in radioligand binding assays strongly suggests that the compound is completely selective for EAAT1. Finally, in a rodent locomotor model, p.o. administration of UCPH‐102 (20 mg kg−1) did not induce acute effects or any visible changes in behavior.
EAAT1 inhibition beyond the BBB: In the present study, oral administration (40 mg kg−1) of the selective excitatory amino acid transporter subtype 1 (EAAT1) inhibitor UCPH‐102 in rats yielded respective plasma and brain concentrations of 10.5 and 6.67 μm after 1 h. In vitro profiling of UCPH‐102 (10 μm) at 51 central nervous system targets in radioligand binding assays strongly suggests that the compound is fully selective for EAAT1.
Although the selective excitatory amino acid transporter subtype1 (EAAT1) inhibitor UCPH-101 has become a standard pharmacological tool compound for invitro and exvivo studies in the EAAT research ...field, its inability to penetrate the blood-brain barrier makes it unsuitable for invivo studies. In the present study, per os (p.o.) administration (40mgkg-1) of the closely related analogue UCPH-102 in rats yielded respective plasma and brain concentrations of 10.5 and 6.67µm after 1h. Three analogue series were designed and synthesized to improve the bioavailability profile of UCPH-102, but none displayed substantially improved properties in this respect. Invitro profiling of UCPH-102 (10µm) at 51 central nervous system targets in radioligand binding assays strongly suggests that the compound is completely selective for EAAT1. Finally, in a rodent locomotor model, p.o. administration of UCPH-102 (20mgkg-1) did not induce acute effects or any visible changes in behavior.
In the present study, we made further investigations on the structure-activity requirements of the selective excitatory amino acid transporter1 (EAAT1) inhibitor, ...2-amino-4-(4-methoxyphenyl)-7-(naphthalen-1-yl)-5-oxo-5,6,7,8-tetrahydro-4H-chromene-3-carbonitrile (UCPH-101), by exploring 15 different substituents (R1) at the 7-position in combination with eight different substituents (R2) at the 4-position. Among the 63 new analogues synthesized, we identified a number of compounds that unexpectedly displayed inhibitory activities at EAAT1 in light of understanding the structure-activity relationship (SAR) of this inhibitor class extracted from previous studies. Moreover, the nature of the R1 and R2 substituents were observed to contribute to the functional properties of the various analogues in additive and non-additive ways. Finally, separation of the four stereoisomers of analogue 14g (2-amino-4-(1,1'-biphenyl-4-yl)-3-cyano-7-isopropyl-5-oxo-5,6,7,8-tetrahydro-4H-chromene) was carried out, and in agreement with a study of a related scaffold, the R configuration at C4 was found to be mandatory for inhibitory activity, while both the C7 diastereomers were found to be active as EAAT1 inhibitors. A study of the stereochemical stability of the four pure stereoisomers 14g-A-D showed that epimerization takes places at C7 via a ring-opening, C-C bond rotation, ring-closing mechanism.
In the present study, we made further investigations on the structure–activity requirements of the selective excitatory amino acid transporter 1 (EAAT1) inhibitor, ...2‐amino‐4‐(4‐methoxyphenyl)‐7‐(naphthalen‐1‐yl)‐5‐oxo‐5,6,7,8‐tetrahydro‐4H‐chromene‐3‐carbonitrile (UCPH‐101), by exploring 15 different substituents (R1) at the 7‐position in combination with eight different substituents (R2) at the 4‐position. Among the 63 new analogues synthesized, we identified a number of compounds that unexpectedly displayed inhibitory activities at EAAT1 in light of understanding the structure–activity relationship (SAR) of this inhibitor class extracted from previous studies. Moreover, the nature of the R1 and R2 substituents were observed to contribute to the functional properties of the various analogues in additive and non‐additive ways. Finally, separation of the four stereoisomers of analogue 14 g (2‐amino‐4‐(1,1′‐biphenyl‐4‐yl)‐3‐cyano‐7‐isopropyl‐5‐oxo‐5,6,7,8‐tetrahydro‐4H‐chromene) was carried out, and in agreement with a study of a related scaffold, the R configuration at C4 was found to be mandatory for inhibitory activity, while both the C7 diastereomers were found to be active as EAAT1 inhibitors. A study of the stereochemical stability of the four pure stereoisomers 14 g‐A–D showed that epimerization takes places at C7 via a ring‐opening, C−C bond rotation, ring‐closing mechanism.
Inhibition of glutamate uptake: 63 new analogues of the selective EAAT1 inhibitor UCPH‐101 were synthesized and characterized pharmacologically. Analogues that unexpectedly displayed inhibitory activities at EAAT1 were identified. Furthermore, separation of the four stereoisomers of analogue 14 g revealed that, in agreement with a study of a related scaffold, the R configuration at C4 is mandatory for inhibitory activity, whereas both C7 diastereomers are active as EAAT1 inhibitors.
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