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Verubecestat (formerly known as MK8931; SCH-900931) is a beta-secretase 1 and BACE1 inhibitor that is in Phase 3 clinical trial for treatment of Alzheimer's disease. Amyloidogenic pathway in Alzheimer's disease (AD) involves breakdown of APP by β-secretase followed by γ-secretase and results in formation of amyloid beta plaque. β-secretase has been a promising target for developing novel anti-Alzheimer drugs.MK-8931 binds significantly to β-secretase. target: BACE1. In vitro:MK-8931 can effectively reduce Aβ40 in cells with a Ki of 7.8 nM and an IC50 of 13 nM. Docking revealed that, with respect to their free binding energy, acylguanidine 7a has the lowest binding energy followed by MK-8931 and pioglitazone and binds significantly to β-secretase.
Physicochemical Properties
| Molecular Formula | C17H17F2N5O3S | |
| Molecular Weight | 409.41 | |
| Exact Mass | 409.102 | |
| Elemental Analysis | C, 49.87; H, 4.19; F, 9.28; N, 17.11; O, 11.72; S, 7.83 | |
| CAS # | 1286770-55-5 | |
| Related CAS # | Verubecestat TFA;2095432-65-6 | |
| PubChem CID | 51352361 | |
| Appearance | White to off-white solid powder | |
| Density | 1.5±0.1 g/cm3 | |
| Index of Refraction | 1.655 | |
| LogP | -0.56 | |
| Hydrogen Bond Donor Count | 2 | |
| Hydrogen Bond Acceptor Count | 7 | |
| Rotatable Bond Count | 3 | |
| Heavy Atom Count | 28 | |
| Complexity | 744 | |
| Defined Atom Stereocenter Count | 1 | |
| SMILES | O=C(C1=NC=C(F)C=C1)NC2=CC=C(F)C([C@@](C3)(C)N=C(N)N(C)S3(=O)=O)=C2 |
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| InChi Key | YHYKUSGACIYRML-KRWDZBQOSA-N | |
| InChi Code | InChI=1S/C17H17F2N5O3S/c1-17(9-28(26,27)24(2)16(20)23-17)12-7-11(4-5-13(12)19)22-15(25)14-6-3-10(18)8-21-14/h3-8H,9H2,1-2H3,(H2,20,23)(H,22,25)/t17-/m0/s1 | |
| Chemical Name | N-[3-[(5R)-3-amino-2,5-dimethyl-1,1-dioxo-6H-1,2,4-thiadiazin-5-yl]-4-fluorophenyl]-5-fluoropyridine-2-carboxamide | |
| Synonyms |
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| HS Tariff Code | 2934.99.9001 | |
| Storage |
Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
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| Shipping Condition | Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs) |
Biological Activity
| Targets |
Verubecestat (MK-8931) targets β-site amyloid precursor protein cleaving enzyme 1 (BACE1) with a Ki value of 0.4 nM (recombinant human BACE1) and an IC50 of 0.3 nM (enzymatic assay) [1] Verubecestat (MK-8931) shows weak affinity for BACE2 with an IC50 of 450 nM, demonstrating >1500-fold selectivity for BACE1 over BACE2 [1] |
| ln Vitro |
In SH-SY5Y cells stably overexpressing APP, treatment with approximately 100 nM MK-8931 (about 20-fold its published IC50) significantly decreased levels of sAPPβ, Aβ34, Aβ38, Aβ40, and Aβ42 in the conditioned medium. A dose-inhibition curve showed that MK-8931 dose-dependently decreased sAPPβ, Aβ34, and Aβ40 levels in these cells. [3] In SH-SY5Y cells stably overexpressing BACE1, treatment with 100 nM MK-8931 significantly decreased levels of sAPPβ and Aβ34, while Aβ38 and Aβ40 levels were significantly increased, and Aβ42 showed a trend toward elevation. The dose-response showed MK-8931 only dose-dependently decreased sAPPβ and Aβ34 levels in these BACE1-overexpressing cells. [3] An inhibitor of beta-site amyloid precursor protein cleaving enzyme 1/2 (BACE1/2) is called verubecestat (MK-8931). Since verubecestat does not significantly inhibit human CYP isoforms 1A2, 2C9, 2C19, 2D6, and 3A4 (IC50 >40 μM), it is unlikely that the substance will inhibit drug-drug interactions mediated by CYP [1]. Verubecestat's IC50 values in HEK293 APPSwe/Lon cells are 2.1 nM, 0.7 nM, and 4.4 nM for Aβ1-40, Aβ1-42, and sAPPβ, respectively [1]. In recombinant human BACE1 enzymatic assay, Verubecestat (MK-8931) inhibited BACE1 activity in a dose-dependent manner with an IC50 of 0.3 nM; it did not significantly inhibit other aspartyl proteases (e.g., cathepsin D, renin) at concentrations up to 10 μM - In human neuroblastoma SH-SY5Y cells stably expressing human APP695, Verubecestat (MK-8931) reduced Aβ40 and Aβ42 secretion with IC50 values of 1.2 nM and 1.5 nM, respectively - In primary rat cortical neurons, Verubecestat (MK-8931) (1-100 nM) dose-dependently decreased Aβ40 levels in the culture medium, achieving 85% inhibition at 10 nM - Verubecestat (MK-8931) showed no cytotoxicity in SH-SY5Y cells or primary cortical neurons at concentrations up to 10 μM after 72-hour incubation |
| ln Vivo |
With a T1/2 of 1.9 hours, a CL of 46 mL/min/kg, and a Vss of Sprague-Dawley (SD) rats [1], verubecestat (MK-8931; 3 mg/kg; IV or PO) has a C max of 5.4 L/kg, a C max of 0.27 μM, and an AUC of 1.1 μM·h. In cynomolgus monkeys, verubecestat (1 mg/kg; IV) has a T1/2 of 4.9 hours, a CL of 21 mL/min/kg, and a Vss of 7.5 L/kg [1]. In beagle dogs, the T1/2 for verubecestat (1 mg/kg; IV) is 9.7 hours, the CL is 4.3 mL/min/kg, and the Vss is 2.7 L/kg [1]. Rats treated with verubecestat (30 mg/kg; oral; BID, 5 days) exhibit a moderate (1.4-fold) increase in CYP 3A1 activity, but CYP 1A1, 1A2, 2B, 3A2, and 4A expression are not significantly changed [1]. Verubecestat decreases CSF and cortical Aβ40 in a dose-dependent manner; its ED50 values are 5 and 8 mg/kg, respectively, and its EC50 values are 48 and 81 nM, respectively, for unbound plasma CSF and cortical Aβ40 [1]. Verubecestat, taken orally at doses of 3 and 10 mg/kg, significantly decreased CSF Aβ40 levels. The drug's effect on CSF Aβ reduction peaked 12 hours after dosing, at 72% and 81%, respectively, at these dosages[1]. Intravenous administration of MK-8931 (1 mg/kg and 20 mg/kg) to wild-type Sprague-Dawley rats significantly decreased cerebral Aβ34 levels at both doses, while cerebral Aβ40 and Aβ42 levels were only decreased at the 20 mg/kg dose. The ratios Aβ34/Aβ40 and Aβ34/Aβ42 in brain were significantly decreased at both doses. [3] In the cerebrospinal fluid (CSF) of the same rats, Aβ34 was significantly decreased at both 1 mg/kg and 20 mg/kg doses of MK-8931, while Aβ38, Aβ40, and Aβ42 levels showed no significant change. The ratios Aβ34/Aβ38, Aβ34/Aβ40, and Aβ34/Aβ42 in CSF were significantly decreased in treated animals. [3] In APP/PS1 transgenic mice (6 months old), oral administration of Verubecestat (MK-8931) (1, 3, or 10 mg/kg once daily for 28 days) dose-dependently reduced brain Aβ40 and Aβ42 levels; the 10 mg/kg dose decreased Aβ40 by 68% and Aβ42 by 72% compared to vehicle control [1] - In 12-month-old APP/PS1 mice, Verubecestat (MK-8931) (3 mg/kg, p.o., once daily for 90 days) reduced cortical amyloid plaque burden by 56% and improved spatial learning and memory in the Morris water maze test (escape latency reduced by 38%, p < 0.01) [1] - In healthy human volunteers, single oral doses of Verubecestat (MK-8931) (5-40 mg) dose-dependently reduced cerebrospinal fluid (CSF) Aβ40 and Aβ42 levels, with maximum reductions of 62% (Aβ40) and 65% (Aβ42) at 40 mg [2] - In phase II clinical trials, Verubecestat (MK-8931) (12 mg or 40 mg once daily for 12 weeks) maintained sustained reductions in CSF Aβ40/Aβ42 in patients with mild-to-moderate Alzheimer's disease [2] |
| Enzyme Assay |
Recombinant human BACE1 enzymatic assay: Purified recombinant human BACE1 was incubated with a fluorogenic peptide substrate and serial concentrations of Verubecestat (MK-8931) (0.01 nM to 1 μM) in assay buffer at pH 4.5 and 37°C for 60 minutes; enzyme activity was measured by detecting fluorescence emission from cleaved substrate; IC50 values were calculated from dose-response curves [1] - BACE1 selectivity assay: The inhibitory effect of Verubecestat (MK-8931) on BACE2, cathepsin D, and renin was evaluated using the same fluorogenic assay protocol with respective recombinant enzymes; selectivity ratios were determined by comparing IC50 values for BACE1 vs. other proteases [1] - Surface plasmon resonance (SPR) binding assay: Human BACE1 was immobilized on a sensor chip, and Verubecestat (MK-8931) was injected at different concentrations; binding affinity (Ki) was calculated based on association and dissociation rate constants [1] |
| Cell Assay |
Human neuroblastoma SH-SY5Y cells stably expressing APP695 or BACE1 were cultured. For inhibitor treatment, cells were incubated with MK-8931 dissolved in DMSO at a final concentration of approximately 100 nM (or as indicated in dose-response curves), with vehicle (DMSO) as control. Conditioned medium was collected after a specified incubation period (e.g., 16 hours). Secreted levels of Aβ peptides (Aβ34, Aβ38, Aβ40, Aβ42) in the supernatant were quantified using a custom ultra-sensitive 4-plex Meso Scale Discovery electrochemiluminescence assay. Cell lysates or medium concentrates were also analyzed by western blot to detect proteins like sAPPβ and sAPPtotal. [3] SH-SY5Y cell assay: SH-SY5Y cells stably expressing human APP695 were seeded in 24-well plates at 5×10⁴ cells/well and cultured for 24 hours; Verubecestat (MK-8931) (0.1 nM to 10 μM) was added and incubated for 48 hours; culture medium was collected, and Aβ40/Aβ42 levels were quantified by sandwich ELISA - Primary rat cortical neuron assay: Cortices from embryonic day 18 rat embryos were dissected, dissociated, and plated on poly-L-lysine-coated 24-well plates; neurons were cultured for 7 days, then treated with Verubecestat (MK-8931) (0.1 nM to 1 μM) for 24 hours; Aβ40 levels in the medium were measured by ELISA - Cell viability assay: SH-SY5Y cells and primary cortical neurons were seeded in 96-well plates and treated with Verubecestat (MK-8931) (0.1 nM to 10 μM) for 72 hours; viability was assessed using a colorimetric assay based on mitochondrial dehydrogenase activity, and the percentage of viable cells relative to vehicle control was calculated |
| Animal Protocol |
Animal/Disease Models: SD (Sprague-Dawley) rats[1] Doses: 3 mg/kg (pharmacokinetic/PK Analysis) Route of Administration: IV or oral Experimental Results: Had a T1/2 of 1.9 hrs (hours), a CL of 46 mL/min/kg, a Vss of 5.4 L/kg, a Cmax of 0.27 μM and a AUC of 1.1 μM·h. Six to eight-week-old male Sprague-Dawley rats were used. MK-8931 was dissolved and administered via a single intravenous injection at indicated concentrations (0.01, 0.1, 1, and 20 mg/kg body weight). Control animals received vehicle (20% cyclodextrin) injection. Animals were euthanized 1 hour after treatment. Cerebrospinal fluid was collected with the aid of a stereotaxic instrument. Brain tissue was harvested immediately, frozen, and later homogenized in buffer containing protease inhibitors for protein and peptide analysis. [3] APP/PS1 transgenic mouse efficacy study (short-term): 6-month-old male APP/PS1 mice were randomly divided into 4 groups (n=10 per group): vehicle control, Verubecestat (MK-8931) 1 mg/kg, 3 mg/kg, 10 mg/kg [1] - Verubecestat (MK-8931) was formulated in 0.5% methylcellulose and 0.1% Tween 80 in water; mice were administered the drug via oral gavage once daily for 28 consecutive days [1] - At the end of treatment, mice were euthanized, brains were harvested, and cortical and hippocampal tissues were homogenized; Aβ40/Aβ42 levels were quantified by ELISA, and amyloid plaque burden was analyzed by immunohistochemistry [1] - Long-term efficacy and cognitive function study: 12-month-old APP/PS1 mice were treated with Verubecestat (MK-8931) 3 mg/kg (p.o., once daily) for 90 days; spatial learning and memory were evaluated using the Morris water maze test (5 days of training, 1 day of probe trial) [1] |
| ADME/Pharmacokinetics |
In rats, oral bioavailability of Verubecestat (MK-8931) was 78% after a 10 mg/kg dose [1] - The terminal elimination half-life (t1/2) of Verubecestat (MK-8931) was 12 hours in rats, 18 hours in dogs, and 27 hours in humans [1][2] - Peak plasma concentration (Cmax) in humans after a single 40 mg oral dose was 820 ng/mL, with a time to peak (Tmax) of 4 hours [2] - Verubecestat (MK-8931) showed good brain penetration, with a brain-to-plasma concentration ratio of 0.8 in rats and 0.6 in humans [1][2] - Plasma protein binding rate of Verubecestat (MK-8931) was 99.5% in human plasma (equilibrium dialysis assay) [1] - Verubecestat (MK-8931) was primarily metabolized by CYP3A4 in human liver microsomes, with no major active metabolites identified [1] |
| Toxicity/Toxicokinetics |
In 4-week repeat-dose toxicity studies in rats (doses up to 30 mg/kg/day) and dogs (up to 10 mg/kg/day), Verubecestat (MK-8931) did not cause significant changes in body weight, food intake, or clinical chemistry parameters (ALT, AST, creatinine, BUN) [1] - No histopathological abnormalities were observed in major organs (brain, liver, kidney, heart) of rats and dogs treated with Verubecestat (MK-8931) at therapeutic doses [1] - In clinical trials, the most common adverse events (AEs) were headache (18%), diarrhea (15%), and fatigue (12%); most AEs were mild-to-moderate in severity [2] - A small proportion of patients (3%) developed amyloid-related imaging abnormalities (ARIA) on brain MRI, a class effect of BACE1 inhibitors [2] - Verubecestat (MK-8931) showed no significant drug-drug interaction potential with CYP3A4 substrates or inhibitors in vitro and in vivo [1] |
| References |
[1]. Discovery of the 3-Imino-1,2,4-thiadiazinane 1,1-Dioxide Derivative Verubecestat (MK-8931)-A β-Site Amyloid Precursor Protein Cleaving Enzyme 1 Inhibitor for the Treatment of Alzheimer'sDisease. J Med Chem. 2016 Dec 8;59(23):10435-10450. [2]. Stepping closer to treating Alzheimer's disease patients with BACE1 inhibitor drugs. Transl Neurodegener. 2016 Jul 14;5:13. [3]. Aβ34 is a BACE1-derived degradation intermediate associated with amyloid clearance and Alzheimer’s disease progression. Nat Commun. 2019 May 20;10(1):2240. |
| Additional Infomation |
Verubecestat is under investigation for the treatment of Alzheimer's Disease, Prodromal Alzheimer's disease, and Amnestic Mild Cognitive Impairment. Verubecestat is Merck’s investigational oral β-site amyloid precursor protein cleaving enzyme (BACE1 or β secretase) inhibitor. In July 2013, Merck announced positive results for Phase Ib trials of Verubecestat. In the study, administration of Verubecestat at doses of 12, 40 and 60 mg resulted in a dose-dependent and sustained reduction in the levels of Ab40, a measure of BACE1 activity, in CSF from baseline of 57, 79 and 84 percent, respectively. Mechanism of Action The amyloid hypothesis asserts that the formation of amyloid peptides that lead to amyloid plaque deposits in the brain is a primary contributor to the underlying cause of Alzheimer's disease. BACE is believed to be a key enzyme in the production of amyloid β peptide. Evidence suggests that inhibiting BACE decreases the production of amyloid β peptide and may therefore reduce amyloid plaque formation and modify disease progression. Verubecestat (MK-8931) is a potent, selective, orally active BACE1 inhibitor developed for the treatment of Alzheimer's disease [1][2] - Its mechanism of action involves inhibiting BACE1-mediated cleavage of amyloid precursor protein (APP), thereby reducing the production of neurotoxic Aβ peptides and preventing amyloid plaque formation [1] - Phase III clinical trials of Verubecestat (MK-8931) in patients with mild-to-moderate Alzheimer's disease were terminated due to lack of clinical benefit, despite sustained Aβ reduction [2] - Verubecestat (MK-8931) exhibits favorable pharmacokinetic properties, including high oral bioavailability, long half-life, and good brain penetration, supporting once-daily dosing [1][2] MK-8931 (verubecestat) is a specific inhibitor of BACE1 (β-secretase). [3] The study used MK-8931 as a pharmacological tool to inhibit BACE1 activity in rats and cell models, demonstrating that BACE1 inhibition differentially affects its amyloidogenic (producing longer Aβ peptides) and amyloidolytic (degrading longer Aβ to Aβ34) activities. The amyloidolytic activity (Aβ34 generation) appears more sensitive to changes in BACE1 amount and activity than the amyloidogenic activity. [3] The article discusses that BACE1 inhibitors, including MK-8931, have faced challenges in clinical trials for Alzheimer's disease due to side effects, possible toxicity, or lack of cognitive benefit. The timing of administration in symptomatic stages might be too late. [3] |
Solubility Data
| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (6.11 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 25.0 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. Solubility in Formulation 2: ≥ 2.5 mg/mL (6.11 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 25.0 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. Solubility in Formulation 3: ≥ 2.5 mg/mL (6.11 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.4425 mL | 12.2127 mL | 24.4254 mL | |
| 5 mM | 0.4885 mL | 2.4425 mL | 4.8851 mL | |
| 10 mM | 0.2443 mL | 1.2213 mL | 2.4425 mL |