Hydrochloride Salt of the GABAkine KRM-II-81

Imidazodiazepine (5-(8-ethynyl-6-(pyridin-2-yl)-4H-benzo[f]imidazole[1,5-α][1,4]diazepin-3-yl) oxazole or KRM-II-81) is a potentiator of GABAA receptors (a GABAkine) undergoing preparation for clinical development. KRM-II-81 is active against many seizure and pain models in rodents, where it exhibits improved pharmacological properties over standard-of-care agents. Since salts can be utilized to create opportunities for increased solubility, enhanced absorption, and distribution, as well as for efficient methods of bulk synthesis, a hydrochloride salt of KRM-II-81 was prepared. KRM-II-81·HCl was produced from the free base with anhydrous hydrochloric acid. The formation of the monohydrochloride salt was confirmed by X-ray crystallography, as well as 1H NMR and 13C NMR analyses. High water solubility and a lower partition coefficient (octanol/water) were exhibited by KRM-II-81·HCl as compared to the free base. Oral administration of either KRM-II-81·HCl or the free base resulted in high concentrations in the brain and plasma of rats. Oral dosing in mice significantly increased the latency to both clonic and tonic convulsions and decreased pentylenetetrazol-induced lethality. The increased water solubility of the HCl salt enables intravenous dosing and the potential for higher concentration formulations compared with the free base without impacting anticonvulsant potency. Thus, KRM-II-81·HCl adds an important new compound to facilitate the development of these imidazodiazepines for clinical evaluation

Stereospecific Total Synthesis of the Indole Alkaloid Ervincidine. Establishment of the C‑6 Hydroxyl Stereochemistry

The total synthesis of the indole alkaloid ervincidine (<b>3</b>) is reported. This research provides a general entry into C-6 hydroxy-substituted indole alkaloids with either an α or a β configuration. This study corrects the errors in Glasby’s book (Glasby, J. S. Encyclopedia of the Alkaloids; Plenum Press: New York, 1975) and Lounasmaa et al.’s review (Lounasmaa, M.; Hanhinen, P.; Westersund, M. In The Alkaloids; Cordell, G. A., Ed.; Academic Press: San Diego, CA, 1999; Vol. 52, pp 103–195) as well as clarifies the work of Yunusov et al. (Malikov, V. M.; Sharipov, M. R.; Yunusov, S. Yu. Khim. Prir. Soedin. 1972, 8, 760−761. Rakhimov, D. A.; Sharipov, M. R.; Aripov, Kh. N.; Malikov, V. M.; Shakirov, T. T.; Yunusov, S. Yu. Khim. Prir. Soedin. 1970, 6, 724–725). It establishes the correct absolute configuration of the C-6 hydroxyl function in ervincidine. This serves as a structure proof and corrects the misassigned structure reported in the literature

Vascular effects of midazolam, flumazenil, and a novel imidazobenzodiazepine MP-III-058 on isolated rat aorta

Hypotensive influences of benzodiazepines and other GABAA receptor ligands, recognized in clinical practice, seem to stem from the existence of “vascular” GABAA receptors in peripheral blood vessels, besides any mechanisms in the central and peripheral nervous systems. We aimed to further elucidate the vasodilatatory effects of ligands acting through GABAA receptors. Using immunohistochemistry, the rat aortic smooth muscle layer was found to express GABAA γ 2 and α1-5 subunit proteins. To confirm the role of “vascular” GABAA receptors, we investigated the vascular effects of standard benzodiazepines, mida-zolam, and flumazenil, as well as the novel compound MP-III-058. Using two-electrode voltage clamp electrophysiology and radioligand binding assays, MP-III-058 was found to have modest binding but substantial functional selectivity for α5β3γ 2 over other αxβ3γ 2 GABAA receptors. Tissue bath assays revealed comparable vasodilatory effects of MP-III-058 and midazo-lam, both of which at 100 μmol/L concentrations had efficacy similar to prazosin. Flumazenil exhibited weak vasoactivity per se, but significantly prevented the relaxant effects of midazolam and MP-III-058. These studies indicate the existence of functional GABAA receptors in the rat aorta, where ligands exert vasodilatory effects by positive modulation of the benzodiazepine binding site, suggesting the potential for further quest for leads with optimized pharmacokinetic properties as prospective adjuvant vasodilators

Evidence that sedative effects of benzodiazepines involve unexpected GABAA receptor subtypes: Quantitative observation studies in rhesus monkeys

In nonhuman primates we tested a new set of behavioral categories for observable sedative effects using pediatric anesthesiology classifications as a basis. Using quantitative behavioral observation techniques in rhesus monkeys, we examined the effects of alprazolam and diazepam (nonselective benzodiazepines), zolpidem (preferential binding to α1 subunit-containing GABAA receptors), HZ-166 (8-ethynyl-6-(2′-pyridine)-4H-2,5,10b-triaza-benzo[e]azulene-3-carboxylic acid ethyl ester; functionally selective with relatively high intrinsic efficacy for α2 and α3 subunit-containing GABAA receptors), MRK-696 [7-cyclobutyl-6-(2-methyl-2H-1,2,4-triazol-2-ylmethoxy)-3-(2-flurophenyl)-1,2,4-triazolo(4,3-b)pyridazine; no selectivity but partial intrinsic activity], and TPA023B 6,2′-diflouro-5′-[3-(1-hydroxy-1-methylethyl)imidazo[1,2-b][1,2,4]triazin-7-yl]biphenyl-2-carbonitrile; partial intrinsic efficacy and selectivity for α2, α3, α5 subunit-containing GABAA receptors]. We further examined the role of α1 subunit-containing GABAA receptors in benzodiazepine-induced sedative effects by pretreating animals with the α1 subunit-preferring antagonist β-carboline-3-carboxylate-t-butyl ester (βCCT). Increasing doses of alprazolam and diazepam resulted in the emergence of observable ataxia, rest/sleep posture, and moderate and deep sedation. In contrast, zolpidem engendered dose-dependent observable ataxia and deep sedation but not rest/sleep posture or moderate sedation, and HZ-166 and TPA023 induced primarily rest/sleep posture. MRK-696 induced rest/sleep posture and observable ataxia. Zolpidem, but no other compounds, significantly increased tactile/oral exploration. The sedative effects engendered by alprazolam, diazepam, and zolpidem generally were attenuated by βCCT pretreatments, whereas rest/sleep posture and suppression of tactile/oral exploration were insensitive to βCCT administration. These data suggest that α2/3-containing GABAA receptor subtypes unexpectedly may mediate a mild form of sedation (rest/sleep posture), whereas α1-containing GABAA receptors may play a role in moderate/deep sedation

Base-Mediated Stereospecific Synthesis of Aryloxy and Amino Substituted Ethyl Acrylates

The stereospecific synthesis of aryloxy and amino substituted <i>E</i>- and <i>Z</i>-ethyl-3-acrylates is of interest because of their potential in the polymer industry and in medicinal chemistry. During work on a copper-catalyzed cross-coupling reaction of ethyl (<i>E</i>)- and (<i>Z</i>)-3-iodoacrylates with phenols and <i>N</i>-heterocycles, we discovered a very simple (nonmetallic) method for the stereospecific synthesis of aryloxy and amino substituted acrylates. To study this long-standing problem on the stereoselectivity of aryloxy and amino substituted acrylates, a series of <i>O-</i> and <i>N-</i>substituted nucleophiles was allowed to react with ethyl (<i>E</i>)- and (<i>Z</i>)-3-iodoacrylates. Screening of different bases indicated that DABCO (1,4-diazabicyclo[2.2.2]­octane) afforded successful conversion of ethyl (<i>E</i>)- and (<i>Z</i>)-3-iodoacrylates into aryloxy and amino substituted ethyl acrylates in a stereospecific manner. Herein are the details of this DABCO-mediated stereospecific synthesis of aryloxy and amino substituted <i>E-</i> or <i>Z-</i>acrylates

Structural Analogs of the GABAkine KRM-II-81 Are Orally Bioavailable Anticonvulsants without Sedation

To provide back-up compounds to support the development of the GABAA receptor (GABAAR) potentiator KRM-II-81, three novel analogs were designed: replacing the pyridinyl with 2'-Cl-phenyl (FR-II-60), changing the positions of the N and O atoms in the oxazole ring with addition of an ethyl group (KPPIII-34 and KPP-III-51), or substituting a Br atom for the ethynyl of KRM-II-81 (KPP-III-34). The compounds bound to brain GABAARs. Intraperitoneal administration of FR-II-60 and KPP-III-34 produced anticonvulsant activity in mice [maximal electroshock (MES)-induced seizures or 6 Hz-induced seizures], whereas KPPIII-51 did not. Although all compounds were orally bioavailable, structural changes reduced the plasma and brain (FR-II-60 and KPP-III-51) exposures relative to KRM-II-81. Oral administration of each compound produced dose-dependent increases in the latency for both clonic and tonic seizures and the lethality induced by pentylenetetrazol (PTZ) in mice. Since KPP-III-34 produced the highest brain area under the curve (AUC) exposures, it was selected for further profiling. Oral administration of KPP-III-34 suppressed seizures in corneal-kindled mice, hippocampal paroxysmal discharges in mesial temporal lobe epileptic mice, and PTZ-induced convulsions in rats. Only transient sensorimotor impairment was observed in mice, and doses of KPP-III-34 up to 500 mg/kg did not produce impairment in rats. Molecular docking studies demonstrated that all compounds displayed a reduced propensity for binding to a1His102 compared with the sedating compound alprazolam; the bromine-substituted KPP-III-34 achieved the least interaction. Overall, these findings document the oral bioavailability and anticonvulsant efficacy of three novel analogs of KRM-II-81 with reduced sedative effects. SIGNIFICANCE STATEMENT A new non-sedating compound, KRM-II-81, with reduced propensity for tolerance is moving into clinical development. Three new analogs were orally bioavailable, produced anticonvulsant effects in rodents, and displayed low sensorimotor impairment. KPP-III-34 demonstrated efficacy in models of pharmacoresistant epilepsy. Docking studies demonstrated a low propensity for compound binding to the a1His102 residue implicated in sedation. Thus, three additional structures have been added to the list of non-sedating imidazodiazepine anticonvulsants that could serve as backups in the clinical development of KRM-II-81. Copyright © 2023 by The American Society for Pharmacology and Experimental Therapeutics