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

Imidazodiazepine Anticonvulsant, KRM-II-81, Produces Novel, Non-diazepam-like Antiseizure Effects

The need for improved medications for the treatment of epilepsy and chronic pain is essential. Epileptic patients typically take multiple antiseizure drugs without complete seizure freedom, and chronic pain is not fully managed with current medications. A positive allosteric modulator (PAM) of α2/3-containing GABAA receptors (5-(8-ethynyl-6-(pyridin-2-yl)-4H-benzo[f]imidazole[1,5-α][1,4]diazepin-3-yl) oxazole or KRM-II-81 (8) is a lead compound in a series of imidazodiazepines. We previously reported that KRM-II-81 produces broad-based anticonvulsant and antinociceptive efficacy in rodent models and provides a wider margin over motoric side effects than that of other GABAA receptor PAMs. The present series of experiments was designed to fill key missing gaps in prior preclinical studies assessing whether KRM-II-81 could be further differentiated from nonselective GABAA receptor PAMs using the anticonvulsant diazepam (DZP) as a comparator. In multiple chemical seizure provocation models in mice, KRM-II-81 was either equally or more efficacious than DZP. Most strikingly, KRM-II-81 but not DZP blocked the development of seizure sensitivity to the chemoconvulsants cocaine and pentylenetetrazol in seizure kindling models. These and predecessor data have placed KRM-II-81 into consideration for clinical development requiring the manufacture of kilogram amounts of good manufacturing practice material. We describe here a novel synthetic route amenable to kilogram quantity production. The new biological and chemical data provide key steps forward in the development of KRM-II-81 (8) as an improved treatment option for patients suffering from epilepsy

Design and Synthesis of Chiral and Achiral Benzodiazepines and Imidazodiazepines as Α–subtype Selective Gabaar Positive Modulators to Treat Schistosomiasis, Epilepsy, Asthma and Some Mental Disorders

Part I: Design, synthesis, and biological evaluation of non-sedating anti-schistosomal agents Schistosomiasis is a parasitic flatworm infection with only one drug therapy currently on the market, praziquantel. An old lead identified by Roche, meclonazepam (US Patent 4031078A) displayed antiparasitic efficacy but was not pursued due to dose-limiting sedation in human clinical trials (S. Afr. Med J. 1979 Apr 14;55(16):617-8, PMID: 380021). However, given the superior spectrum of activity meclonazepam displays relative to praziquantel (which is unable to cure immature parasites within the host) and advances in the understanding of benzodiazepine SAR at human GABAARs, the study of this benzodiazepine was resumed. Approximately 50 novel compounds were designed, synthesized, and tested for anti-schistosomal activity and 150 more from our BZR library. This resulted in many compounds that were active on the schistosome mobility assay. Of the various analogs ,which were synthesized MYM-III-10 (69), which contains a hydroxy group at the benzodiazepine C-3 position, was identified as the most active hit. MYM-III-10 (69) displayed anti-parasitic effects (causing parasite contractile paralysis in vitro and clearing the worm burden in vivo) comparable to meclonazepam, but was significantly less sedating in a mouse rotarod assay. It was also found that modification of the C3 position by replacing the ‘methyl’ in meclonazepam with a halogen resulted in the potent MYM-V-56 (84) that retains anti-parasitic effects (causing parasite contractile paralysis in vitro and clearing the worm burden in vivo) comparable to meclonazepam ,but was much less sedating in mice. This is a novel modification ,which eliminates the main side effects of this drug class, sedation and ataxia, which has so far prevented the clinical development of meclonazepam. When the enantiomers of MYM-V-56 (69) were compared, the S-MYM-V-56 was found to potently induce contractile paralysis on the worm muscle in vitro and clear the worms in vivo. A large-scale robust synthetic method was developed for both of these compounds (racemic) without employing any chromatographic purification in any step. Purification of compounds for each step simply required recrystallization by using different solvents such as ethyl acetate etc. Both MYM-III-10 and S-MYM-V-56 presented excellent preclinical data in different assays and further experiments will be performed to move these drugs toward the clinic for the treatment of this parasitic disease and other flatworm diseases (humans,cattle, horses,dogs, etc). Part II: Design, synthesis, and biological evaluation of chiral α5 subtype BZR/GABAAR modulators for the treatment of anxiety, major depressive disorder with agents that are procognitive and active against models of schizophrenia as well Gamma (γ)-amino butyric acid, a naturally occurring amino acid, is a proteinogenic amino acid that acts as the principal inhibitory neurotransmitter in the CNS, as well as the cerebrum of the mammalian brain. The benzodiazepines (BZDs) bind at the extracellular interface of the α+γ2-subunits of GABAAR. The binding of ligands at different subunits affects a wide variety of brain functions. The α1-subtypes are associated with the anxiolytic sedative, ataxic, amnesic, anticonvulsant and addictive effects, while the α2/3-subtypes are responsible for anxiolytic, anticonvulsant, and antinociceptive activities. The α5-containing GABAARs in the CNS are involved in cognition, as well as learning and memory processes; α5-subtypes in lungs are related with asthma. Their dysfunction in the CNS is also involved in other CNS disorders.The need for improved medications for the treatment of anxiety and depression is essential. Anxiety is comorbid with depression. Currently there are no drugs on the market for depression or schizophrenia that treat the cognition problems. This is an unmet medical need. Based on the privileged imidazodiazepine (IMZD) structure”, more than 150 novel α5 preferring ligands have been synthesized in Milwaukee for evaluation of pro-cognitive, anxiolytic, as well as anti-depressant activity. The lead, GL-II-73 (developed by Guanguan Li and later Daniel Knutson) was found to be anxiolytic, anti-depressant, as well as procognitive. A key finding. A large scale, industrial friendly synthetic strategy was developed for the lead, GL-II-73, without employing any chromatographic purification except for one-step and it was a flash column. Around 50 grams of the lead, GL-II-73 were synthesized and evaluated for additional biological activity. The ligand,GL-II-73, produced symptomatic, disease modifying, and neurotrophic effects in a mouse model of chronic stress. This ligand reversed the stress-induced or normal aging-related working memory deficits in old mice, as well as young mice. Further studies are ongoing by DAMONA PHARMACEUTICALS to move this ligand toward the clinic for depression and potentially Alzheimer\u27s disease. In addition, more than 30 novel α5 subtype selective ligands were synthesized based on the structure of GL-II-73 and evaluated for pro-cognitive, anxiolytic as well as anti-depressant activity. Of the synthesized derivatives, the amide analogs MYM-III-41 (97) and MYM-III-29(106) showed pro-cognitive effects on primary and secondary screening in the Y-maze assay. These ligands will be tested in the forced-swim test and elevated plus maze to determine their anti-depressive and anxiolytic behavioral profile. Also, the 1,2,4-oxadiazoles MYM-V-28 (129), MYM-III-43 (134), and MYM-IV-47 (128) showed strong pro-cognitive profiles in the Y-maze assay. These ligands may well be key backups for ligand GL-II-73, but require further testing such as the forced-swim test, UCMS, elevated plus maze, metabolism, etc. Part III: Design, synthesis and biological evaluation of α2/3-GABA(A)R subtype-selective imidazodiazepines to treat epilepsy, anxiety and neuropathic pain The novel imidazodiazepine, (5-(8-ethynyl-6-(pyridin-2-yl)-4H-benzo[f]-imidazole[1,5-α[1,4]diaze-pin-3-yl) oxazole ( KRM-II-81) is a positive allosteric modulator of GABAA receptors (a GABAkine) undergoing preparation for clinical development. 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 termed MYM-V-26 (193) was prepared. This salt is 14 times more water soluble than the free base. The oral administration of MYM-V-26 (193) was similar to that of the free base in the brain to plasma ratio in rats. High levels of both compounds were exposed in plasma and brain of rats with about 1 µM concentrations in plasma and 0.5 µM in brain at Cmax. Oral dosing in mice significantly elevated the latency to both clonic and tonic convulsions and decreased pentylenetetrazole-induced lethality. For clonus, the anticonvulsant effects lasted for at least 8 h. The newly synthesized MYM-V-26 (193) exhibited improved physicochemical properties such as high water solubility and a lower partition coefficient value as compared to those of the free base, KRM-II-81. Kinetic solubility experiments also indicated that the HCl salt allows KRM-II-81 to remain in the solution state over a longer period of time. One key to this study is the increased water solubility of the salt permits i.v. dosing in primates instead of just i.p. or i.m. dosing, which will permit better safety and efficacy studies. Part IV: Design, synthesis, analytical characterization, and biological evaluation of chiral α5 subtype GABAAR modulators for the treatment of asthma The novel α5-selective ligand MIDD0301, ((R)-8-bromo-6-(2-fluorophenyl)-4-methyl-4H-benzo[f]imidazo[1,5-a][1,4], diazepine-3-carboxylic acid), GL-II-93, is under development for the treatment of asthma by PANTHERICS. This lead compound, MIDD0301 exhibited relaxation ex vivo on human, guinea pig, and mouse airway smooth muscle (ASM), which express discrete GABAARs.A scalable and reproducible synthetic route for the lead asthma ligand MIDD0301 has been developed on multi-gram scale for large scale toxicity testing and for further biological evaluation. A number of impurities present in less than 2% are formed in each step during the large-scale synthesis. These impurities have been identified, synthesized and characterized by NMR, HPLC and LCMS. The carboxylic acid ligand MIDD0301 forms different metabolites in Phase I and Phase II metabolism studies. One of the phase II metabolites, MIDD0301-glucoronide (170) was synthesized to quantify this metabolite in vivo. In addition, more than 10 carboxylic acid analogs of MIDD0301 were synthesized and evaluated on an ex vivo ASM relaxation assay. Excitedly, MYM-III-100 (167) was the most potent compound in the ASM relaxation assay reported, to date . This ligand relaxed the ex vivo smooth muscle force in guinea pig tracheal rings more potently than the lead, MIDD0301. The 2’Cl version of MIDD0301, MYM-FR-II-88 (178) produced similar effects to MIDD0301 in the ASM relaxation assay. In addition, two other analogs MYM-III-20 (185) and MYM-V-71 (186) showed better ASM relaxation profiles than MIDD0301 in the ex vivo ASM relaxation assay. These newly synthesized ligands require further testing to establish the complete profile and to determine if they are better than MIDD0301 for the treatment of asthma

World Journal of Pharmaceutical Sciences Cytotoxic, thrombolytic, antioxidant and antimicrobial activities of Cocos nucifera linn. endocarp extracts

ABSTRACT Different partitionates of methanol extract of Cocos nucifera Linn. were subjected to screening for cytotoxic, thrombolytic, antioxidant and antimicrobial activities. Cytotoxicity was determined using brine shrimp nauplii in which vincristine sulfate was used as positive control and gave LC50 of 0.45±0.08 g/ml. Among the partitionates, carbon tetrachloride soluble fraction demonstrated the highest cytotoxic activity (LC50 value of 0.82±0.19 g/ml). While assaying for thrombolytic activity, the petroleum ether soluble fraction demonstrated highest thrombolytic activity (37.44±0.33%) compared to the standard streptokinase (66.76±0.03%). In total phenolic content assay, the highest amount of phenolic compounds was found in the crude methanol extract (113.94±2.01 mg of GAE/g of sample). In antioxidant assay, crude methanol extract (IC50 value of 4.39±0.69 μg/ml) showed maximum free radical scavenging activity whereas reference standards tert-butyl-1-hydroxytoluene and ascorbic acid gave IC50 values of 27.50±0.95 μg/ml and 5.80±1.03 μg/ml, respectively. In antimicrobial screening, the crude methanol extract and its carbon tetrachloride and chloroform soluble fractions exhibited mild zone of inhibition against the test organisms

Vasodilatory effects of a variety of positive allosteric modulators of GABAA receptors on rat thoracic aorta

Different subtypes of GABAA (gamma-aminobutyric acid A) receptors, through their specific regional and cellular localization, are involved in the manifestation of various functions, both at the central and peripheral levels. We hypothesized that various non-neuronal GABAA receptors are expressed on blood vessels, through which positive allosteric modulators of GABAA receptors exhibit vasodilatory effects. This study involved two parts: one to determine the presence of α1-6 subunit GABAA receptor mRNAs in the rat thoracic aorta, and the other to determine the vasoactivity of the various selective and non-selective positive GABAA receptor modulators: zolpidem (α1-selective), XHe–III–074 (α4-selective), MP–III–022 (α5-selective), DK-I-56-1 (α6-selective), SH-I-048A and diazepam (non-selective). Reverse transcription-polymerase chain reaction (RT-PCR) analysis data demonstrated for the first time the expression of α1, α2, α3, α4 and α5 subunits in the rat thoracic aorta tissue. Tissue bath assays on isolated rat aortic rings revealed significant vasodilatory effects of diazepam, SH-I-048A, XHe–III–074, MP–III–022 and DK-I-56-1, all in terms of achieved relaxations (over 50% of relative tension decrease), as well as in terms of preventive effects on phenylephrine (PE) contraction. Diazepam was the most efficient ligand in the present study, while zolpidem showed the weakest vascular effects. In addition, diazepam-induced relaxations in the presence of antagonists PK11195 or bicuculline were significantly reduced (P < 0.001 and P < 0.05, respectively) at lower concentrations of diazepam (10−7 M and 3 × 10−7 M). The present work suggests that the observed vasoactivity is due to modulation of “vascular” GABAA receptors, which after further detailed research may provide a therapeutic target

Synthesis and Receptor Binding Studies of α5 GABA_AR Selective Novel Imidazodiazepines Targeted for Psychiatric and Cognitive Disorders

GABA mediates inhibitory actions through various GABAA receptor subtypes, including 19 subunits in human GABAAR. Dysregulation of GABAergic neurotransmission is associated with several psychiatric disorders, including depression, anxiety, and schizophrenia. Selective targeting of α2/3 GABAARs can treat mood and anxiety, while α5 GABAA-Rs can treat anxiety, depression, and cognitive performance. GL-II-73 and MP-III-022, α5-positive allosteric modulators have shown promising results in animal models of chronic stress, aging, and cognitive disorders, including MDD, schizophrenia, autism, and Alzheimer’s disease. Described in this article is how small changes in the structure of imidazodiazepine substituents can greatly impact the subtype selectivity of benzodiazepine GABAAR. To investigate alternate and potentially more effective therapeutic compounds, modifications were made to the structure of imidazodiazepine 1 to synthesize different amide analogs. The novel ligands were screened at the NIMH PDSP against a panel of 47 receptors, ion channels, including hERG, and transporters to identify on- and off-target interactions. Any ligands with significant inhibition in primary binding were subjected to secondary binding assays to determine their Ki values. The newly synthesized imidazodiazepines were found to have variable affinities for the benzodiazepine site and negligible or no binding to any off-target profile receptors that could cause other physiological problems

Symptomatic and neurotrophic effects of GABAA receptor positive allosteric modulation in a mouse model of chronic stress

Chronic stress is a risk factor for Major Depressive Disorder (MDD), and in rodents, it recapitulates human behavioral, cellular and molecular changes. In MDD and after chronic stress, neuronal dysfunctions and deficits in GABAergic signaling are observed and responsible for symptom severity. GABA signals predominantly through GABAA receptors (GABAA-R) composed of various subunit types that relate to downstream outcomes. Activity at α2-GABAA-Rs contributes to anxiolytic properties, α5-GABAA-Rs to cognitive functions, and α1-GABAA-Rs to sedation. Therefore, a therapy aiming at increasing α2- and α5-GABAA-Rs activity, but devoid of α1-GABAA-R activity, has potential to address several symptomologies of depression while avoiding side-effects. This study investigated the activity profiles and behavioral efficacy of two enantiomers of each other (GL-II-73 and GL-I-54), separately and as a racemic mixture (GL-RM), and potential disease-modifying effects on neuronal morphology. Results confirm GL-I-54 and GL-II-73 exert positive allosteric modulation at the α2-, α3-, α5-GABAA-Rs and α5-containing GABAA-Rs, respectively, and separately reduces immobility in the forced swim test and improves stress-induced spatial working memory deficits. Using unpredictable chronic mild stress (UCMS), we show that acute and chronic administration of GL-RM provide pro-cognitive effects, with mild efficacy on mood symptoms, although at lower doses avoiding sedation. Morphology studies showed reversal of spine density loss caused by UCMS after chronic GL-RM treatment at apical and basal dendrites of the PFC and CA1. Together, these results support using a racemic mixture with combined α2-, α3-, α5-GABAA-R profile to reverse chronic stress-induced mood symptoms, cognitive deficits, and with anti-stress neurotrophic effects

A Structure-Activity Relationship Comparison of Imidazodiazepines Binding at Kappa, Mu, and Delta Opioid Receptors and the GABAA Receptor

Analgesic and anti-inflammatory properties mediated by the &kappa; opioid receptor (KOR) have been reported for oxadiazole imidazodiazepines. Affinities determined by radioligand competition assays of more than seventy imidazodiazepines using cell homogenates from HEK293 cells that overexpress KOR, &micro; opioid receptor (MOR), and &delta; opioid receptor (DOR) are presented. Affinities to synaptic, benzodiazepine-sensitive receptors (BZR) were determined with rat brain extract. The highest affinity for KOR was recorded for GL-I-30 (Ki of 27 nM) and G-protein recruitment was observed with an EC50 of 32 nM. Affinities for MOR and DOR were weak for all compounds. Ester and amide imidazodiazepines were among the most active KOR ligands but also competed with 3H-flunitrazepam for brain extract binding, which is mediated predominately by gamma aminobutyric acid type A receptors (GABAAR) of the &alpha;1-3&beta;2-3&gamma;1-2 subtypes. Imidazodiazepines with carboxylic acid and primary amide groups did not bind KOR but interacted strongly with GABAARs. Pyridine substitution reduced KOR affinity. Oxadiazole imidazodiazepines exhibited good KOR binding and interacted weakly with BZR, whereas oxazole imidazodiazepines were more selective towards BZR. Compounds that lack the imidazole moiety, the pendent phenyl, or pyridine substitutions exhibited insignificant KOR affinities. It can be concluded that a subset of imidazodiazepines represents novel KOR ligands with high selectivity among opioid receptors

Metabolism, pharmacokinetics, and anticonvulsant activity of a deuterated analog of the α2/3-selective GABAkine KRM-II-81

The 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 new α2/3‐selective GABAkine (gamma aminobutyric acid A receptor potentiator) with anticonvulsant, anxiolytic, and antinociceptive activity in preclinical models. Reducing metabolism was utilized as a means of potentially extending the half‐life of KRM‐II‐81. In vitro and in vivo studies were conducted to evaluate metabolic liabilities. Incubation of KRM‐II‐81 in hepatocytes revealed sites of potential metabolism on the oxazole and the diazepine rings. These sites were targeted in the design of a deuterated analog (D5‐KRM‐II‐ 81) that could be evaluated as a potentially longer‐acting analog. In contrast to computer predictions, peak plasma concentrations of D5‐KRM‐II‐81 in rats were not significantly greater than those produced by KRM‐II‐81 after oral administra- tion. Furthermore, brain disposition of KRM‐II‐81 was higher than that of D5‐KRM‐ II‐81. The half‐life of the two compounds in either plasma or brain did not statis- tically differ from one another but the tmax for D5‐KRM‐II‐81 occurred slightly earlier than for KRM‐II‐81. Non‐metabolic considerations might be relevant to the lack of increases in exposure by D5‐KRM‐II‐81. Alternative sites of metabolism on KRM‐II‐81, not targeted by the current deuteration process, are also possible. Despite its lack of augmented exposure, D5‐KRM‐II‐81, like KRM‐II‐81, significantly prevented seizures induced by pentylenetetrazol when given orally. The present findings introduce a new orally active anticonvulsant GABAkine, D5‐KRM‐II‐81