Discovery of E2730, a novel selective uncompetitive GAT1 inhibitor, as a candidate for anti-seizure medication.

OBJECTIVE: As of 2022, 36 anti-seizure medications (ASMs) have been licensed for the treatment of epilepsy, however, adverse effects (AEs) are commonly reported. Therefore, ASMs with a wide margin between therapeutic effects and AEs are preferred over ASMs that are associated with a narrow margin between efficacy and risk of AEs. E2730 was discovered using in vivo phenotypic screening and characterized as an uncompetitive, yet selective, inhibitor of γ-aminobutyric acid (GABA) transporter 1 (GAT1). Here, we describe the preclinical characteristics of E2730. METHODS: Anti-seizure effects of E2730 were evaluated in several animal models of epilepsy: corneal kindling, 6 Hz-44 mA psychomotor seizure, amygdala kindling, Fragile X syndrome, and Dravet syndrome models. Effects of E2730 on motor coordination were assessed in accelerating rotarod tests. The mechanism of action of E2730 was explored by [3 H]E2730 binding assay. The GAT1-selectivity over other GABA transporters was examined by GABA uptake assay of GAT1, GAT2, GAT3, or betaine/GABA transporter 1 (BGT-1) stably expressing HEK293 cells. To further investigate the mechanism for E2730-mediated inhibition of GAT1, in vivo microdialysis and in vitro GABA uptake assays were conducted under conditions of different GABA concentrations. RESULTS: E2730 showed anti-seizure effects in the assessed animal models with an approximately >20-|fold margin between efficacy and motor incoordination. [3 H]E2730 binding on brain synaptosomal membrane was abolished in GAT1-deficient mice, and E2730 selectively inhibited GAT1-mediated GABA uptake over other GABA transporters. In addition, results of GABA uptake assays showed that E2730-mediated inhibition of GAT1 positively correlated to the level of ambient GABA in vitro. E2730 also increased extracellular GABA concentration in hyperactivated conditions but not under basal levels in vivo. SIGNIFICANCE: E2730 is a novel, selective, uncompetitive GAT1 inhibitor, which acts selectively under the condition of increasing synaptic activity, contributing to a wide margin between therapeutic effect and motor incoordination.

Silinanyl Rhodamines and Silinanyl Fluoresceins for Super-Resolution Microscopy.

Single-molecule localization microscopy (SMLM) enables the visualization of biomolecules at unprecedented resolution and requires control of the fluorescent blinking (ON/OFF) states of fluorophores to detect single-molecule fluorescence without overlapping of the signals. Although SMLM probes based on the intramolecular spirocyclization of Si-xanthene fluorophores have been developed, fluorophores with lower ON/OFF ratios are required for SMLM visualization of high-density structures. Here, we describe a silinane structure that lowers the ON/OFF ratio of Si-xanthene fluorophores. On the basis of Mulliken population analysis, we replaced the dimethylsilane moiety in Si-rhodamine with a silinane moiety to increase the partial charge at the 9-position of the carbon atom in the Si-xanthene ring and to promote the ring-closure reaction. Evaluation of fluorescence properties in a solution and in single-molecule imaging indicated that introducing the silinane sufficiently stabilized the nonfluorescent spirocyclic forms, thus decreasing the fluorescence ON/OFF ratio. This novel substitution was applied to Si-rhodamines with various amine structures and to an Si-fluorescein to expand the color palette. We demonstrated SMLM observation of microtubules in fixed HeLa cells using the developed fluorophores in two color channels. The results demonstrated the feasibility of extending the design strategies of SMLM probes based on Si-xanthenes through modification of the substituents on the Si atom.

Design, synthesis, and structure-activity relationships of a series of novel N-aryl-2-phenylcyclopropanecarboxamide that are potent and orally active orexin receptor antagonists.

Herein we describe the design, synthesis, and structure-activity relationships (SARs) of a novel phenylcyclopropane series represented by 7 and 33 b as antagonists of orexin 1 and orexin 2 receptors. With 4 serving as the initial lead for the development of orexin antagonists, exploration of SAR resulted in improved binding affinity for orexin 1 and orexin 2 receptors. Among the synthesized compounds, 33 b ((-)-N-(5-cyanopyridin-2-yl)-2-[(3,4-dimethoxyphenyl)oxymethyl]-2-phenylcyclopropanecarboxamide) exhibited potent in vitro activity and oral efficacy in animal sleep measurement experiments. The results of our study suggest that compound 33 b may serve as a valuable template for the development of new orexin receptor antagonists.

Synthesis of 1-arylpiperazyl-2-phenylcyclopropanes designed as antidopaminergic agents: cyclopropane-based conformationally restricted analogs of haloperidol.

A series of the cyclopropane-based conformationally restricted analogs of haloperidol were designed as potential antidopaminergic agents and were effectively synthesized using highly stereoselective Grignard reaction with tert-butanesulfinyl imines as the key step. Pharmacological evaluation of the compounds showed that the conformational restriction method can effectively work for improving the pharmacological selectivity of a parent compound and also for investigating the bioactive conformation.

Stereochemical diversity-oriented conformational restriction strategy. Development of potent histamine H3 and/or H4 receptor antagonists with an imidazolylcyclopropane structure.

The stereochemical diversity-oriented conformational restriction strategy can be an efficient method for developing specific ligands for drug target proteins, especially in cases where neither the bioactive conformation nor the pharmacophore is known. To develop potent H3 and H4 receptor antagonists, a series of conformationally restricted analogues of histamine with a chiral cis- or trans-cyclopropane structure were designed on the basis of this strategy. These target compounds with stereochemical diversity were synthesized from the versatile chiral cyclopropane units (1S,2R)- and (1R,2R)-2-(tert-butyldiphenylsilyloxy)methyl-1-formylcyclopropane (6 and 7, respectively) or their enantiomers ent-6 and ent-7. Pharmacological profiles of these conformationally restricted analogues were shown to be different depending on the cyclopropane backbones. Among the analogues, (1R,2S)-2-[2-(4-chlorobenzylamino)ethyl]-1-(1H-imidazol-4-yl)cyclopropane (11a) with the (1R)-trans-cyclopropane structure has remarkable antagonistic activity to both the H3 (Ki = 8.4 nM) and H4 (Ki = 7.6 nM) receptors. The enantiomer of 11a, i.e., ent-11a, with the (1S)-trans-cyclopropane structure turned out to be a highly potent and selective H3 receptor antagonist with a Ki of 3.6 nM. Conversely, (1R,2R)-2-[(4-chlorobenzylamino)methyl]-1-(1H-imidazol-4-yl)cyclopropane (10a) with the (1R)-trans structure was selective for the H4 receptor (Ki = 118 nM) compared to the H3 receptor (Ki > 10(3) nM). Thus, a variety of compounds with different pharmacological profiles have been developed. These results show that when the structure of the target protein is unknown, the stereochemical diversity-oriented approach can be a powerful strategy in medicinal chemical studies.

Highly stereoselective grignard addition to cis-substituted C-cyclopropylaldonitrones. The bisected s-trans transition state can be stabilized effectively by the Lewis acid-coordination.

We previously found that Grignard addition to a C-cyclopropylaldonitrone, C-[cis-2-(N,N-diethylcarbamoyl)-trans-2-phenylcyclopropyl]-N-benzylaldonitrone (1), stereoselectively gave the anti-product 3, in which the stereoselectivity was particularly high when MgBr(2) was the additive. In this study, the reaction pathway was investigated in detail. The stereoselective addition was initially thought to occur via either a 1,5-chelation-controlled or a bisected s-trans conformation-controlled pathway. However, Grignard addition to a nonchelating silyl ether-type substrate, C-[cis-2-(tert-butyldiphenylsilyloxymethyl)-trans-2-phenylcyclopropyl]-N-benzylaldonitrone (7), also gave the anti-product 9 with high stereoselectivity suggesting that chelation is not important in the reaction. Theoretical calculations of C-cyclopropylaldonitrones showed that the coordination of Mg(2+) at the nitrone oxygen significantly stabilizes the bisected s-trans conformer due to the effective hyperconjugation between the pi* of the nitrone C=N bond and the electron-donating cyclopropane orbitals. This kind of orbital interaction is able to stabilize the transition state of the nucleophilic addition and is maximized in the bisected conformation, in which the orbitals of the forming bond and the cyclopropane C-C bond are in an almost planar arrangement. Thus, the high stereoselectivity can be explained by nucleophilic attack on the less hindered side of the C=N bond of the substrates in the Mg(2+)-coordinated bisected s-trans conformation.

Construction of a cis-cyclopropane via reductive radical decarboxylation. Enantioselective synthesis of cis- and trans-1-arylpiperazyl-2-phenylcyclopropanes designed as antidopaminergic agents.

(1S,2S)-, (1S,2R)-, and (1R,2S)-1-(2,4-Dimethylphenyl)piperazyl-2-phenylcyclopropane (2a, 3, and ent-3, respectively), which were designed as conformationally restricted analogues of haloperidol (1), a clinically effective antipsychotic agent, were synthesized from chiral epichlorohydrins using the Barton reductive radical decarboxylation as the key step. (1S,2R)-1-(tert-Butyldiphenylsilyloxy)methyl-2-carboxy-2-phenylcyclopropane (5), which was prepared from (S)-epichlorohydrin ((S)-7), was converted into its N-hydroxypyridine-2-thione ester 12, the substrate for the reductive radical decarboxylation. When 12 was treated with TMS3SiH in the presence of Et3B or AIBN, the decarboxylation and subsequent hydride attack on the cyclopropyl radical intermediate from the side opposite to the bulky silyloxymethyl moiety occurred, resulting in selective formation of the corresponding reductive decarboxylation product 4-cis with the cis-cyclopropane structure. From 4-cis, the cis-cyclopropane-type target compound 3 was readily synthesized. Starting from (R)-epichlorohydrin ((R)-7), ent-3 was similarly synthesized. Epimerization of the cyclopropanecarboxamide ent-16-cis, a synthetic intermediate for ent-3, on treatment with a base prepared from Bu2Mg and i-Pr2NH in THF occurred effectively to give the corresponding trans isomer 16-trans, which was converted into 2a with the trans-cyclopropane structure.

Cyclopropane-based conformational restriction of histamine. (1S,2S)-2-(2-aminoethyl)-1-(1H-imidazol-4-yl)cyclopropane, a highly selective agonist for the histamine H3 receptor, having a cis-cyclopropane structure.

A series of cyclopropane-based conformationally restricted analogues of histamine, the "folded" cis-analogues, i.e., (1S,2R)-2-(aminomethyl)-1-(1H-imidazol-4-yl)cyclopropane (11), (1S,2S)-2-(2-aminoethyl)-1-(1H-imidazol-4-yl)cyclopropane (13), and their enantiomers ent-11 and ent-13, and the "extended" trans-analogues, i.e., (1R,2R)-2-(aminomethyl)-1-(1H-imidazol-4-yl)cyclopropane (12) and its enantiomer ent-12, were designed as histamine H(3) receptor agonists. These target compounds were synthesized from the versatile chiral cyclopropane units, (1S,2R)- and (1R,2R)-2-(tert-butyldiphenylsilyloxy)methyl-1-formylcyclopropane (14 and 15, respectively) or their enantiomers ent-14 and ent-15. Among the conformationally restricted analogues, the "folded" analogue 13 (AEIC) having the cis-cyclopropane structure was identified as a potent H(3) receptor agonist, which showed a significant binding affinity (K(i) = 1.31 +/- 0.16 nM) and had an agonist effect (EC(50) value of 10 +/- 3 nM) on the receptor. This compound owes its importance to being the first highly selective H(3) receptor agonist to have virtually no effect on the H(4) subtype receptor. These studies showed that the cis-cyclopropane structure is very effective in the conformational restriction of histamine to improve the specific binding to the histamine H(3) receptor.

A systematic study of the hydride reduction of cyclopropyl ketones with structurally simplified substrates. highly stereoselective reductions of trans-substituted cyclopropyl ketones via the bisected s-cis conformation.

The stereoselective hydride reduction of the cis- and trans-substituted cyclopropyl ketones was systematically investigated using a series of structurally simplified substrates, trans-[tert-butyldiphenylsilyloxymethyl]cyclopropyl ketones 1a-e and trans-(benzyloxymethyl)cyclopropyl methyl ketone (2), and the corresponding cis congeners 3a,b,e and 4. The results showed that, not only in the reduction of the cis-substituted cyclopropyl ketones but also in that of the trans-substituted ketones, high stereoselectivity can be realized when the substrate has a bulky substituent on the cyclopropane ring, even though it is attached to the position trans to the acyl moiety. Ab initio calculations based on the density functional theory (DFT) of cyclopropyl ketones showed that (1) the bisected s-cis and s-trans conformers were the only two minimum energy conformers, while the s-cis conformer was more stable than the s-trans and (2) a bulky alkyl group in the acyl moiety and a cis substituent on the cyclopropane ring made the bisected s-cis conformer much more stable. On the basis of these calculations and experimental results, it is likely that the more stable the bisected s-cis conformer of the substrate, the more stereoselective the hydride reduction. Thus, the stereochemistry can be explained by hydride attack on the bisected s-cis conformation of the substrate from the less-hindered face. The predictability of the stereochemical results is predicated on the bisected s-cis transition-state model, which is very important from the viewpoint of synthetic organic chemistry.

Synthesis of derivatives of (1S,2R)-1-phenyl-2-[(S)-1-aminopropyl]-N,N-diethylcyclopropanecarboxamide (PPDC) modified at the 1-aromatic moiety as novel NMDA receptor antagonists: the aromatic group is essential for the activity.

(1S,2R)-1-Phenyl-2-[(S)-1-aminopropyl]-N,N-diethylcyclopropanecarboxamide (PPDC, 4a), which is a conformationally restricted analogue of antidepressant milnacipran [(+/-)-1], is a new class of potent noncompetitive NMDA receptor antagonists. A series of PPDC analogues modified at the 1-phenyl moiety, that is, the analogue 6 lacking 1-phenyl group, the 1-(fluorophenyl) analogues 4b,c,d, the 1-(methylphenyl) analogues 4e-g and the 1-(naphthyl) analogues 4h,i were synthesized. Analogue 6, lacking the 1-phenyl group, was completely inactive showing that the aromatic moiety is essential for the NMDA receptor binding. Among the analogues synthesized, the 1-o-fluorophenyl and 1-m-fluorophenyl analogues 4b and 4c showed potent affinities for the NMDA receptor [IC(50)=0.16+/-0.001 microM (4b), 0.15+/-0.02 microM (4c)], which were improved to some extent compared to those of the parent compound PPDC (IC(50)=0.20+/-0.02 microM). On the other hand, compounds 4b and 4c showed none of the 5-HT-uptake inhibitory effect, while PPDC turned out to be a weak 5-HT-uptake inhibitor.

Conformational analysis of the NMDA receptor antagonist (1S,2R)-1-phenyl-2-[(S)-1-aminopropyl]-N,N-diethylcyclopropanecarboxamide (PPDC) designed by a novel conformational restriction method based on the structural feature of cyclopropane ring.

(1S,2R)-1-phenyl-2-[(S)-1-aminopropyl]-N,N-diethylcyclopropanecarboxamide (2b, PPDC), a new class of potent N-methyl-D-aspartic acid (NMDA) receptor antagonist, was designed based on a new method for restricting the conformation of compounds having a cyclopropane ring. The three-dimensional structures of PPDC obtained by the three different methods of X-ray crystallographic analysis, usual MM2-calculations in vacuum, and MM2 calculations based on the nuclear Overhauser effect (NOE) data in D2O are similar, which are in accord with that hypothesized. These results suggest that this conformational restriction method is particularly effective in designing novel biologically active molecules.

Synthesis of (1S,2R)-1-phenyl-2-[(S)-1-aminopropyl]-N,N-diethylcyclopropanecarboxamide (PPDC) derivatives modified at the carbamoyl moiety as a new class of NMDA receptor antagonists.

(1S,2R)-1-Phenyl-2-[(S)-1-aminopropyl]-N,N-diethylcyclopropanecarboxamide (PPDC, ), which is a conformationally restricted analogue of the antidepressant milnacipran [(+/-)-1], represents a new class of potent NMDA receptor antagonists. A series of PPDC analogues modified at the carbamoyl moiety were synthesized. Among these, (1S,2R)-1-phenyl-2-[(S)-1-aminopropyl]-N,N-dipropylcyclopropanecarboxamide (4d) was identified as the most potent NMDA receptor antagonist in this series and clearly reduced the MMDA receptor mediated potentiation of rat hippocampal slices, a model of long-term potentiation (LTP). The three-dimensional structure of 4d was also analyzed in detail to clarify the receptor-binding conformation.

Development of versatile cis- and trans-dicarbon-substituted chiral cyclopropane units: synthesis of (1S,2R)- and (1R,2R)-2-aminomethyl-1-(1H-imidazol-4-yl)cyclopropanes and their enantiomers as conformationally restricted analogues of histamine.

The cyclopropane ring can be used effectively in restricting the conformation of biologically active compounds to improve activity and also to investigate bioactive conformations. We designed (1S,2R)- and (1R,2R)-2-aminomethyl-1-(1H-imidazol-4-yl)cyclopropanes (1 and 2, respectively) and their enantiomers (ent-1 and ent-2) as conformationally restricted analogues of histamine. The four types of chiral cyclopropanes bearing two differentially functionalized carbon substituents in a cis or trans relationship on a cyclopropane ring, (1S,2R)-2-(tert-butyldiphenylsilyloxy)methyl-1-formylcyclopropane (7) and (1R,2R)-2-(tert-butyldiphenylsilyloxy)methyl-1-formylcyclopropane (8) and their enantiomers (ent-7 and ent-8), were developed as the key intermediates for synthesizing 1, 2, ent-1, and ent-2. The reaction between (R)-epichlorohydrin [(R)-12] and phenylsulfonylacetonitrile (13a) in the presence of NaOEt in EtOH followed by treatment with acid gave the chiral cyclopropane lactone 11a with 98% ee in 82% yield. Compound 11a was converted into both the cis- and trans-chiral cyclopropane units 7 and 8, respectively, via reductive desulfonylation with Mg/MeOH as the key step. The corresponding enantiomers, the cis-substituted ent-7 and the trans-substituted ent-8, were also prepared starting from (S)-epichlorohydrin [(S)-12]. The four conformationally restricted target histamine analogues 1, 2, ent-1, and ent-2 were successfully synthesized from 7, 8, ent-7, and ent-8, respectively. The chiral cyclopropane units 7, 8, ent-7, and ent-8 should be useful as versatile intermediates for synthesizing various compounds having an asymmetric cyclopropane structure.