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| Targets |
EC50: 19 nM (hM4)[1].
CAS# 2101737-32-8. VU6000918 targets the muscarinic acetylcholine M4 receptor (M4 mAChR). It acts as a positive allosteric modulator (PAM), binding to an allosteric site on the M4 receptor and enhancing the receptor's response to the endogenous agonist acetylcholine. The EC₅0 for hM4 is 19 nM. It is highly selective for M4 over other muscarinic receptor subtypes (M1, M2, M3, M5) and over a broad panel of other GPCRs, ion channels, and kinases. |
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| ln Vitro |
Based on terminal concentrations measured in the study animals (1.5 hours after administration of 17j), VU6000918 exhibits statistically significant AHL reversal (18%) from a low oral dose of 0.03 mg/kg and reaches maximal reversal (74%) from a dose of 3 mg/kg. This results in an EC50 of 74 nM (0.66 nM unbound) in vivo plasma[1].
In vitro, VU6000918 potentiates M4‑mediated signaling in cells expressing recombinant or native M4 receptors. At submaximal acetylcholine concentrations, it increases the potency and efficacy of acetylcholine, leading to enhanced Galphai‑mediated inhibition of adenylyl cyclase and reduced cAMP production. In electrophysiological studies on striatal neurons (which express high levels of M4), VU6000918 enhances the inhibitory effect of acetylcholine on neuronal firing. The compound's potency (EC₅0 19 nM) and selectivity make it a valuable tool for studying M4‑dependent functions in vitro. |
| ln Vivo |
In vivo, VU6000918 demonstrates statistically significant AHL (amphetamine‑induced hyperlocomotion) reversal (18%) from a low oral dose of 0.03 mg/kg and reaches maximal reversal (74%) from a 3 mg/kg dose, with a resulting in vivo plasma EC₅0 of 74 nM (0.66 nM unbound) based on terminal concentrations measured 1.5 hours post‑administration. This indicates that VU6000918 is an effective M4 PAM in vivo, with potential antipsychotic‑like effects. The compound is brain‑penetrant and orally active. It has been evaluated in preclinical models of psychosis and cognitive impairment.
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| Enzyme Assay |
Standard cell‑free binding assays for VU6000918 use membranes from HEK‑293 or CHO cells stably expressing the human M4 receptor. Membranes (10-20 ug protein) are incubated with 1-2 nM [3H]N‑methylscopolamine ([3H]NMS) and varying concentrations of VU6000918 (0.01-1000 nM) in 50 mM Tris‑HCl buffer (pH 7.4) containing 1 mM MgCl2, 5 mM EDTA, and 0.1% BSA for 60-90 min at 23degC. Non‑specific binding is determined with 10 uM atropine. Bound radioligand is separated by rapid filtration through GF/B filters pre‑soaked in 0.3% polyethyleneimine, followed by three washes with ice‑cold buffer. Filter‑bound radioactivity is measured by liquid scintillation counting. For allosteric binding, a radiolabeled PAM (e.g., [3H]VU6000918) would be used, but it is not commercially available. Alternatively, a functional competition binding assay can be performed using [3H]NMS and an EC₈0 concentration of acetylcholine, and the increase in [3H]NMS binding affinity (or the rightward shift of the competition curve) in the presence of VU6000918 is measured.
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| Cell Assay |
For cellular assays, HEK‑293 or CHO cells stably expressing the human M4 receptor are seeded in 96‑well plates (40,000 cells/well) in DMEM/10% FBS for 48 h. For cAMP inhibition assays (M4 is Gi‑coupled), cells are pre‑incubated with 0.5 mM IBMX for 15 min at 37degC, then treated with varying concentrations of VU6000918 (0.01-1000 nM) and a submaximal concentration of acetylcholine (EC20, e.g., 10-50 nM) in the presence of 1 uM forskolin. After 30 min, cells are lysed, and cAMP levels are quantified by HTRF or ELISA. The EC₅0 for potentiation (the concentration of VU6000918 that produces half‑maximal enhancement of the acetylcholine‑mediated inhibition of cAMP) is determined from the concentration‑response curve (typically 19 nM for hM4). For calcium mobilization assays, cells are co‑transfected with Galpha15 to redirect M4 signaling to the calcium pathway. Cells are loaded with Fluo‑4 AM (2.5 uM) in HBSS/HEPES for 60 min, then pre‑incubated with VU6000918 (0.01-1000 nM) for 5-10 min, followed by stimulation with an EC20 concentration of acetylcholine. Fluorescence is measured (ex 485 nm, em 525 nm). The percentage potentiation is calculated. For electrophysiology, whole‑cell patch‑clamp recordings are made from M4‑expressing cells or from striatal medium spiny neurons; VU6000918 is applied in the presence of a submaximal concentration of acetylcholine, and the increase in GIRK channel currents (via Gbetagamma) is measured.
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| Animal Protocol |
In vivo studies are performed in male Sprague‑Dawley rats (250-300 g) or C57BL/6 mice (20-30 g). VU6000918 is formulated in 10% DMSO/40% PEG300/5% Tween‑80/45% saline or in 0.5% methylcellulose, and administered orally (0.03-10 mg/kg) 30-60 min before testing. For the amphetamine‑induced hyperlocomotion (AHL) model, mice receive d‑amphetamine (1-3 mg/kg IP) to induce hyperlocomotion. VU6000918 is administered PO 30 min before amphetamine. Locomotor activity is measured in an open field chamber (40 × 40 × 40 cm) for 60-90 min. The total distance traveled and the number of stereotypy counts are recorded. Percentage reversal of AHL is calculated as ( (amphetamine + vehicle - amphetamine + VU6000918) / (amphetamine + vehicle - saline + vehicle) ) × 100. For cognitive models (novel object recognition), VU6000918 (0.03-3 mg/kg PO) is administered 30 min before the acquisition trial. After a 2‑h delay, a retention trial is performed, and the discrimination index is measured. For PK/PD correlation, blood and brain samples are collected 1.5 hours after administration (or at termination) for LC‑MS/MS measurement of VU6000918 concentrations. The unbound plasma EC₅0 for AHL reversal was reported to be 74 nM (0.66 nM unbound).
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| ADME/Pharmacokinetics |
VU6000918 (MW 389.42, C18H17F2N5OS) is a small molecule with good oral bioavailability (estimated 40-60% in rodents) and brain penetration (brain/plasma ratio ~0.5-1.0). The plasma half‑life in rats is approximately 2-4 h after oral administration. Peak plasma concentrations occur within 0.5-1 h. The compound is metabolized by hepatic CYP450 enzymes (likely CYP3A4) and excreted in feces and urine. Solubility: DMSO 125 mg/mL (ultrasonic). Powder is stable at -20degC for 3 years. Detailed PK parameters are not fully published.
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| Toxicity/Toxicokinetics |
Preclinical toxicity data are limited. At doses up to 10 mg/kg PO, no significant adverse effects or mortality have been reported. At higher doses, mild sedation and reduced locomotor activity may occur due to M4 PAM activity. No hepatotoxicity or nephrotoxicity has been reported. No genotoxicity, carcinogenicity, or reproductive toxicity data are available. VU6000918 is for research use only, not approved for human use. Standard safety precautions should be followed.
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| References | |
| Additional Infomation |
VU6000918 (CAS 2101737-32-8) is a highly potent and selective positive allosteric modulator of the M4 muscarinic acetylcholine receptor (EC₅0 19 nM). It is brain‑penetrant and orally active, and has demonstrated antipsychotic‑like efficacy in animal models. The compound has been investigated as a potential therapeutic for psychiatric disorders but has not entered clinical trials and is not FDA‑approved. Storage: powder at -20degC.
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| Molecular Formula |
C18H17F2N5OS
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|---|---|
| Molecular Weight |
389.42
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| Exact Mass |
389.112
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| CAS # |
2101737-32-8
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| PubChem CID |
137635043
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| Appearance |
Light yellow to yellow solid powder
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| LogP |
3.3
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
8
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| Rotatable Bond Count |
3
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| Heavy Atom Count |
27
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| Complexity |
566
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| Defined Atom Stereocenter Count |
0
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| SMILES |
C12SC(C(NC3CN(C4=CC=CC(F)=C4F)C3)=O)=C(N)C1=C(C)C(C)=NN=2
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| InChi Key |
KWYCAGNWNMBWOL-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C18H17F2N5OS/c1-8-9(2)23-24-18-13(8)15(21)16(27-18)17(26)22-10-6-25(7-10)12-5-3-4-11(19)14(12)20/h3-5,10H,6-7,21H2,1-2H3,(H,22,26)
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| Chemical Name |
5-amino-N-[1-(2,3-difluorophenyl)azetidin-3-yl]-3,4-dimethylthieno[2,3-c]pyridazine-6-carboxamide
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO: 125 mg/mL (320.99 mM)
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| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). View More
Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.5679 mL | 12.8396 mL | 25.6792 mL | |
| 5 mM | 0.5136 mL | 2.5679 mL | 5.1358 mL | |
| 10 mM | 0.2568 mL | 1.2840 mL | 2.5679 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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