EC50: 3 nM (GABAA α5)[1] Ki: 0.83 nM (Human GABAA α1β3γ2), 0.85 nM (Human GABAA α2β3γ2), 0.77 nM (Human GABAA α3β3γ2), 1.4 nM (Human GABAA α5β3γ2)[1]

With an EC50 of 3 nM for GABAA α5, and Kis of 0.83, 0.85, 0.77, and 1.4 nM for human GABAA α1β3η2, GABAA α2β3η2, GABAA α3β3η2, and GABAA α5β3γ2, respectively, MRK-016 is a selective, orally bioavailable inverse agonist of GABAA α5 receptor. As a complete inverse agonist at the α5-subtype, MRK-016 exhibits very low affinity for the GABAA α4β3γ2-subtype (Ki 395 ± 173 nM), and at the GABAA α6β3γ2 receptor (Ki > 4000 nM), it is basically inactive[1]. At 400 nM, MRK-016 had a negligible impact on GABAA α4β3γ2. In mouse hippocampus slices, MRK-016 (100 nM) alao enhances long-term potentiation[2].

For 20 days in mice, MRK-016 did not generate seizures at 30 mg/kg by po or worsen convulsions induced by pentylenetetrazole at 10 mg/kg via ip. In rats, MRK-016 does not exhibit overt anxiogenic-like effects at dosages that occupy more than 95% of the binding sites for benzodiazepines (BZs). In rats, MRK-016 (0.3, 1, and 3 mg/kg, po) dose-dependently enhances hippocampal-dependent memory task performance[1]. In rats, MRK-016 (0.3–30 mg/kg, po) results in excellent receptor occupancy. The delayed matching-to-position version of the Morris water maze exhibits cognitive-enhancing activity when administered at doses of 0.3, 1, or 3 mg/kg po. Mice treated with MRK -016 (1, 3, or 10 mg/kg ip) do not produce kindling[2]. In mice, MRK-016 (3 mg/kg, ip) prevents learning and memory deficits brought on by LPS[3].

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The oral bioavailability of 13 (MRK-016) in rats and dogs is 52% and 8%, respectively. The proconvulsant potential of 13 was determined in mice.36 At doses up to 10 mg/kg ip, where 99% of GABAA receptor subtypes are occupied, 13 did not potentiate pentylenetetrazole-induced convulsions in mice. This contrasts with the significant convulsant effects reported in mice for the nonselective BZ full inverse agonist 212 and the proconvulsant effects reported for the nonselective BZ partial inverse agonist 15 (FG 7142).13 While nonselective BZ partial inverse agonists are without direct convulsant effects in mice when given acutely, they can induce seizures if given daily over a number of days, a process known as ‘kindling'.13 Repeated dosing of 13 (30 mg/kg po for 20 days) did not induce seizures, although seizures were induced with 15 (40 mg/kg ip for 20 days) which occupied fewer receptors than 13 and had a shorter duration. The rat elevated plus maze test37 is sensitive to the anxiogenic effects of nonselective BZ inverse agonists such as 15.11 In this test, 13 was without significant anxiogenic-like effects in rats at doses that occupy >95% of BZ binding sites. In contrast, 15 produced a robust anxiogenic effect.[1]
To investigate the effect that 13 has on learning and memory we assessed the performance of rats dosed orally with 13 in the delayed ‘matching-to-place' variant of the Morris water maze, a hippocampal-dependent cognitive test.38,39 In this assay, the difference in time taken to find a hidden platform (the position of which varies day-to-day but is fixed on any given day) on the first and second trial is used as an index of how well the rat has remembered the position of the platform. Spatial cues around the pool assist the rat in navigating its way to the platform.39 As can be seen in Figure 2, 13 showed a dose-dependent (0.3−3 mg/kg po) improvement in performance between trial 1 and trial 2 compared with vehicle-treated animals (p < 0.05). The occupancy of BZ sites on GABAA receptors in a satellite group of rats dosed at 0.3, 1, and 3 mg/kg po was 61%, 72%, and 85%, respectively. Since 13 was without effect on swim speed, these data indicates that the functionally selective GABAA α5 inverse agonist 13 significantly enhanced performance in a dose-dependent manner in this hippocampal-dependent memory task. These data complements the findings obtained with the structurally diverse thiophene 427 and triazolophthalazine 528 and supports the hypothesis that a selective α5-subtype inverse agonist may have therapeutic utility for the treatment of cognitive disorders.[1]

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The inhibition of in vivo binding of [3H]Ro 15-1788 by 13 was used to determine the occupancy of this compound at the BZ site of rat brain GABAA receptors. [3H]Ro 15-1788 binds with equal affinity to the BZ site of GABAA receptors containing α1-, α2-, α3-, and α5-subunits, and, therefore, in vivo binding of [3H]Ro 15-1788 reflects binding to this combined receptor population. However, since 13 also binds with equal affinity to GABAA receptors containing α1-, α2-, α3-, and α5-subtypes, the dose of 13 required to occupy the α5-subtype is the same as that required to occupy the combined α1-, α2-, α3-, and α5-subtypes. As shown in Figure 1, the occupancy of 13 in rats was dose-dependent after oral dosing, with the mean dose of 13 required to occupy 50% of GABAA receptor subtypes (0.75 h postdose) being 0.35 mg/kg.[1]

Compound 13 (MRK-016) has high in vitro binding affinity 32 at all four BZ-sensitive GABAA receptor subtypes, ranging from 0.8 to 1.4 nM. It has very weak affinity at the GABAA α4β3γ2-subtype (Ki 395 ± 173 nM) and is essentially inactive at the GABAA α6β3γ2 receptor (Ki > 4000 nM). Furthermore, when examined in 147 radioligand binding and enzyme assays, 3313 showed no significant off-target activity (IC50 values > 10 μM). The efficacy values shown in Table 1 were determined using whole cell patch clamp recordings 34 from mouse fibroblast cells stably expressing the human GABAA receptor subtypes.32 The in vitro efficacy is measured as the percentage maximum modulation of the GABA-evoked current using a submaximal (EC20) GABA concentration. Positive values represent a potentiation of the GABA-induced current (agonist) whereas negative values represent an attenuation (inverse agonist). The values for 13 are compared to the clinical compound 5, the nonselective full inverse agonist 2,9 and the nonselective full agonist chlordiazepoxide (CDZ; 14). 13 is a full inverse agonist at the α5-subtype and is functionally selective over the α1-, α2-, and α3-subtypes. In mouse fibroblast L(tk-) cells expressing the α5β3γ2-subtype, 13 attenuates the GABA-evoked current by 55% (efficacy = −55%), which is essentially identical to that of the full inverse agonist 2 (efficacy = −57%) and greater than that produced by 5 (efficacy = −40%). In contrast, efficacy at the α1-, α2-, and α3-subtypes is much lower, with respective efficacy values of −16%, +6%, and −9% being in the range of weak partial inverse agonists or antagonists. The efficacy of 13 at the α1- and α3-subtypes is comparable with the recordings obtained for 5; whereas at the α2-subtype 13 is essentially an antagonist (efficacy = +6%), 5 exhibits partial agonism (efficacy = +16%). Indeed, in our quest for a compound with full inverse agonism at the α5-subtype and little functional activity at the other GABAA receptor subtypes, pyrazolotriazine 13 has a more impressive efficacy profile compared to the clinical compound 5. The EC50 value of 13 at the α5-subtype is 3.0 nM, which complements its in vitro binding affinity of 1.4 nM[1].

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3-tert-Butyl-7-(5-methylisoxazol-3-yl)-2-(1-methyl-1H-1,2,4-triazol-5-ylmethoxy)-pyrazolo[1,5-d][1,2,4]triazine (MRK-016) is a pyrazolotriazine with an affinity of between 0.8 and 1.5 nM for the benzodiazepine binding site of native rat brain and recombinant human α1-, α2-, α3-, and α5-containing GABAA receptors. It has inverse agonist efficacy selective for the α5 subtype, and this α5 inverse agonism is greater than that of the prototypic α5-selective compound 3-(5-methylisoxazol-3-yl)-6-[(1-methyl-1,2,3-triazol-4-hdyl)methyloxy]-1,2,4-triazolo[3,4-a]phthalazine (α5IA). Consistent with its greater α5 inverse agonism, MRK-016 increased long-term potentiation in mouse hippocampal slices to a greater extent than α5IA[2].

Consistent with its greater α5 inverse agonism, MRK-016 increased long-term potentiation in mouse hippocampal slices to a greater extent than α5IA. MRK-016 gave good receptor occupancy after oral dosing in rats, with the dose required to produce 50% occupancy being 0.39 mg/kg and a corresponding rat plasma EC50 value of 15 ng/ml that was similar to the rhesus monkey plasma EC50 value of 21 ng/ml obtained using [11C]flumazenil positron emission tomography. In normal rats, MRK-016 enhanced cognitive performance in the delayed matching-to-position version of the Morris water maze but was not anxiogenic, and in mice it was not proconvulsant and did not produce kindling. MRK-016 had a short half-life in rat, dog, and rhesus monkey (0.3–0.5 h) but had a much lower rate of turnover in human compared with rat, dog, or rhesus monkey hepatocytes. Accordingly, in human, MRK-016 had a longer half-life than in preclinical species (∼3.5 h). Although it was well tolerated in young males, with a maximal tolerated single dose of 5 mg corresponding to an estimated occupancy in the region of 75%, MRK-016 was poorly tolerated in elderly subjects, even at a dose of 0.5 mg, which, along with its variable human pharmacokinetics, precluded its further development.[2]

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In this study, researchers tested the ability of the inverse benzodiazepine agonist, MRK-016 (MRK) to protect against LPS-induced deficits in memory acquisition and consolidation, using a contextual fear conditioning (CFC) paradigm. In Experiment One, mice received lipopolysaccharide (LPS) and/or MRK injections prior to CFC training, and were then tested 24h after training. In Experiment Two, animals received similar treatment injections immediately after training, and were tested 24h later. Additionally, hippocampal samples were collected 4h after LPS injections and immediately after testing, to evaluate brain-derived neurotrophic factor (BDNF) and insulin-like growth factor 1 (IGF-1) mRNA expression. Results indicate that MRK can protect against LPS-induced learning/memory decrements in both paradigms. We also found, in both paradigms, that animals treated with LPS/Saline expressed significantly less BDNF mRNA when compared to Saline/Saline-treated animals 4h after LPS administration, but that MRK did not restore BDNF expression levels. Further, treatment administrations had no effect on IGF-1 mRNA expression at any collection time-point. In summary, MRK-016 can protect against LPS-induced deficits in memory acquisition and consolidation, in this hippocampus-dependent paradigm, though this protection occurs independently of recovery of BDNF expression.[3]

[1]. An orally bioavailable, functionally selective inverse agonist at the benzodiazepine site of GABAA alpha5 receptors with cognition enhancing properties. J Med Chem. 2004 Nov 18;47(24):5829-32.

[2]. In vitro and in vivo properties of 3-tert-butyl-7-(5-methylisoxazol-3-yl)-2-(1-methyl-1H-1,2,4-triazol-5-ylmethoxy)-pyrazolo[1,5-d]-[1,2,4]triazine (MRK-016), a GABAA receptor alpha5 subtype-selective inverse agonist. J Pharmacol Exp Ther. 2009 Nov;331(2):470-84.

[3]. Administration of the inverse benzodiazepine agonist MRK-016 rescues acquisition and memory consolidation following peripheral administration of bacterial endotoxin. Behav Brain Res. 2015 Jul 15;288:50-3.

(3-tert-Butyl-7-(5-methylisoxazol-3-yl)-2-(1-methyl-1H-1,2,4-triazol-5-ylmethoxy)pyrazolo[1,5-d][1,2,4]triazine (13) has been identified as a functionally selective, inverse agonist at the benzodiazepine site of GABA(A) alpha5 receptors. 13 is orally bioavailable, readily penetrates the CNS, and enhances performance in animal models of cognition. It does not exhibit the convulsant, proconvulsant, or anxiogenic activity associated with nonselective GABA(A) inverse agonists.[1]

C17H20N8O2

368.3931016922

368.17

C, 55.43; H, 5.47; N, 30.42; O, 8.69

342652-67-9

6918583

White to off-white solid powder

1.5

0

8

5

27

518

0

QYSYOGCIDRANAR-UHFFFAOYSA-N

InChI=1S/C17H20N8O2/c1-10-6-11(23-27-10)15-21-19-7-12-14(17(2,3)4)16(22-25(12)15)26-8-13-18-9-20-24(13)5/h6-7,9H,8H2,1-5H3

3-[3-tert-butyl-2-[(2-methyl-1,2,4-triazol-3-yl)methoxy]pyrazolo[1,5-d][1,2,4]triazin-7-yl]-5-methyl-1,2-oxazole

TXZ4DVJ9MF; 3-[3-tert-butyl-2-[(2-methyl-1,2,4-triazol-3-yl)methoxy]pyrazolo[1,5-d][1,2,4]triazin-7-yl]-5-methyl-1,2-oxazole; CHEMBL363211;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: 50 mg/mL (135.73 mM)

Solubility in Formulation 1: ≥ 2.75 mg/mL (7.46 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 27.5 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.75 mg/mL (7.46 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 27.5 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.75 mg/mL (7.46 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 27.5 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)