M1 mAChR ( IC50 = 477 nM ); M1 mAChR ( Ki = 9.91 μM )
Muscarinic acetylcholine receptor M1 (M₁ mAChR): EC₅₀ = 198 ± 13.2 nM (calcium mobilization assay in CHO cells expressing rat M₁), AChₘₐₓ = 80.52% ± 7.67% max[2]
- Muscarinic acetylcholine receptor M1 (M₁ mAChR): EC₅₀ = 379 ± 912 nM (calcium mobilization assay in rM₁ Y381A cells with orthosteric mutation)[2]
- Muscarinic acetylcholine receptor M1 (M₁ mAChR): No agonism at M₂-M₅ mAChR subtypes up to 30 μM[2]
- Muscarinic acetylcholine receptor M1 (M₁ mAChR): IC₅₀ > 30 μM for antagonism of ACh-induced responses at M₂-M₅ mAChR subtypes[2]
- Muscarinic acetylcholine receptor M1 (M₁ mAChR): Does not compete with [³H]-NMS for binding to M₁-M₅ mAChRs (IC₅₀ > 30 μM)[2]
- Muscarinic acetylcholine receptor M1 (M₁ mAChR): Shows agonist activity in calcium release assay (EC₅₀ not specified in this context) and ERK phosphorylation assay, but no effect on β-arrestin recruitment in hM₁ CHO cells[3]
- Muscarinic acetylcholine receptor M1 (M₁ mAChR): At high concentrations, completely displaces [³H]-NMS binding to the orthosteric site of M₁-M₅ receptors (no specific IC₅₀ value provided)[1]

In vitroactivity: VU0357017 is an M1-selective agonist that acts at an allosteric site to appear to activate M1. Based on competition binding experiment, the Ki values of VU0357017 are 9.91 (rM1), 21.4 (rM2), 55.3 (rM3), 35 (rM4), and 50 (rM5), in that order. Being a highly selective M1 agonist, VU0357017 indicates that these substances are more likely to function as allosteric agonists than at the highly conserved orthosteric site on M1.

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1. In intracellular calcium mobilization assays using CHO cells expressing rat M₁ mAChR, VU0357017 HCI exhibited potent and efficacious agonism with an EC₅₀ of 198 ± 13.2 nM and AChₘₐₓ of 80.52% ± 7.67% max; no agonistic activity was detected at M₂-M₅ mAChR subtypes up to 30 μM[2]
2. In rM₁ Y381A cells (bearing an orthosteric mutation that ablates orthosteric agonist binding), VU0357017 HCI still elicited a functional response with an EC₅₀ of 379 ± 912 nM, confirming its allosteric agonism mechanism[2]
3. In antagonism assays evaluating the ability to attenuate EC₈₀ responses of ACh at M₂-M₅ mAChR subtypes, VU0357017 HCI showed IC₅₀ > 30 μM for all M₂-M₅ subtypes, indicating high selectivity for M₁[2]
4. In [³H]-NMS competition binding assays for M₁-M₅ mAChRs, VU0357017 HCI had IC₅₀ > 30 μM and did not compete with the orthosteric ligand [³H]-NMS, further supporting its allosteric binding mode[2]
5. In CHO-K1 cells transiently transfected with M₁ receptor mutants (E401H single-point mutant), VU0357017 HCI showed substantial loss of potency (no specific EC₅₀ provided), while mutations at K392D, E397V, or E397D had little effect on its potency, indicating E401 in the third extracellular loop is critical for its binding[2]
6. At high concentrations, VU0357017 HCI completely displaced [³H]-NMS binding to the orthosteric site of M₁-M₅ receptors in membrane preparations from CHO cells expressing these receptors; Furchgott analysis using a cell line with inducible M₁ expression indicated it acts as a weak orthosteric partial agonist of M₁, and it slowed the rate of [³H]-NMS dissociation from CHO-rM₁ membranes, suggesting a bitopic binding mode (both orthosteric and allosteric interactions)[1]
7. In CHO-K1 cells stably expressing human M₁ mAChRs, VU0357017 HCI induced calcium release (normalized to CCh maximum response) and ERK1/2 phosphorylation (fold change over basal ERK levels), but had no effect on β-arrestin recruitment in β-arrestin recruitment assays using PathHunter detection kit (normalized to % CCh max)[3]
8. In hM₁ TREx CHO cells treated with varying concentrations of tetracycline (to modulate M₁ receptor density), VU0357017 HCI induced calcium release in a concentration-dependent manner (normalized to % ionomycin); induction of M₁ expression with 50 ng/ml or 1 μg/ml tetracycline had little effect on its maximal response in ERK phosphorylation assays[3]
9. In hM₁ TREx CHO cells expressing β-arrestin2-YFP, treatment with 100 μM VU0357017 HCI did not induce β-arrestin2 recruitment even in cells treated with 50 ng/ml or 1 μg/ml tetracycline overnight[3]
10. In rat hippocampal slices, bath application of 500 nM VU0357017 HCI for 10 min before threshold theta-burst stimulation (TBS) significantly potentiated field excitatory postsynaptic potential (fEPSP) slope at the Schaffer collateral-CA1 synapse, enhancing threshold theta-burst long-term potentiation (LTP); bath application of 30 μM VU0357017 HCI for 10 min had no effect on long-term depression (LTD) of fEPSP slope at the same synapse[3]
11. In medium spiny neurons (MSNs) of rats, application of VU0357017 HCI induced a small but significant increase in evoked action potential firing frequency (p ≤ 0.001), though the effect was weaker than that of CCh (10 μM)[3]
12. In mouse medial prefrontal cortical (mPFC) pyramidal cells, VU0357017 HCI had no measurable agonist activity and no effect on spontaneous excitatory postsynaptic currents (sEPSCs)[3]

VU0357017 significantly enhances hippocampal-dependent learning in rats. In rats, VU0357017 improves contextual fear conditioning and Morris water maze performance.

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1. In a rodent model of contextual fear conditioning, VU0357017 HCI (10 mg/kg, intraperitoneal injection) significantly reversed scopolamine (0.2 mg/kg, subcutaneous injection)-induced disruption of the acquisition of contextual fear conditioning response, as shown by an increase in the percent of time spent freezing in the same context environment as the training session (p < 0.05 vs. vehicle/scopolamine group)[2]
2. In rat Morris Water Maze (MWM) assays, treatment with VU0357017 HCI enhanced spatial memory performance (swim distance across 5 days of testing was collapsed across four daily trials); however, no significant effects were observed on spatial memory on days 4 and 5 of testing (p > 0.445), on memory retention between day 4 Trial 4 and day 5 Trial 1 (p > 0.755), or on platform crossings during the first 30 s of the probe trial (p > 0.981)[3]
3. In rat contextual fear conditioning (CFC) assays, treatment with VU0357017 HCI enhanced the acquisition of contextual fear (p < 0.05 vs. vehicle group)[3]
4. In rats, VU0357017 HCI (administered intraperitoneally 30 min before amphetamine injection at a dose of 4 mg/kg, subcutaneous) failed to reverse amphetamine-induced hyperlocomotion, suggesting it does not have an antipsychotic-like profile[3]

1. [³H]-NMS competition binding assay for M₁-M₅ mAChRs: Membranes were prepared from CHO cells expressing rat M₁-M₅ mAChRs and incubated with [³H]-NMS (0.3 nM final concentration in 100 mM NaCl and 20 mM HEPES, pH = 7.4) for 3 h at room temperature in the presence of varying concentrations of VU0357017 HCI or atropine (positive control). Equilibrium binding was terminated by rapid filtration, and data were plotted as a percentage of specific [³H]-NMS binding. The assay confirmed that VU0357017 HCI did not compete with [³H]-NMS for binding to M₁-M₅ mAChRs (IC₅₀ > 30 μM)[2]
2. [³H]-NMS dissociation assay for M₁ mAChRs: Membrane preparations from CHO cells stably expressing rat M₁ receptors were incubated with [³H]-NMS to reach equilibrium binding. Then, atropine was added to induce dissociation of [³H]-NMS, and the effects of 10 μM or 1 mM VU0357017 HCI on the dissociation rate (k_off) were measured. VU0357017 HCI slowed the rate of [³H]-NMS dissociation (k_off for 10 μM: 0.030 ± 0.006/min; k_off for 1 mM: 0.025 ± 0.006/min) compared with atropine alone (k_off: 0.041 ± 0.001/min), indicating negative cooperativity with orthosteric ligands via an allosteric site[1]

1. Calcium mobilization assay for M₁ mAChR agonism: CHO cells expressing rat M₁-M₅ mAChRs were seeded in appropriate plates and loaded with calcium-sensitive fluorescent dye. After incubation, VU0357017 HCI was added at varying concentrations, and changes in intracellular calcium concentration were measured using a fluorescence plate reader. Concentration-response curves were generated, and EC₅₀ and maximal efficacy (AChₘₐₓ) values were calculated. The assay showed VU0357017 HCI was a potent M₁ agonist with no activity at M₂-M₅ up to 30 μM[2]
2. Calcium mobilization assay in M₁ receptor mutant cells: CHO-K1 cells transiently transfected with M₁ receptor mutants (E401H, K392D, E397V, etc.) were subjected to calcium mobilization assays as described above. VU0357017 HCI was added at varying concentrations, and EC₅₀ values were determined to assess the impact of mutations on its potency[2]
3. ERK1/2 phosphorylation assay: hM₁ CHO cells or hM₁ TREx CHO cells (treated with varying concentrations of tetracycline to modulate M₁ expression) were treated with VU0357017 HCI at different concentrations. Cells were lysed, and ERK1/2 phosphorylation levels were assessed using the SureFire ERK phosphorylation assay. Data were expressed as fold change over basal ERK levels and normalized to the maximum response elicited by CCh[3]
4. β-arrestin recruitment assay: hM₁ CHO cells were used in β-arrestin recruitment assays with PathHunter detection kit. VU0357017 HCI was added at varying concentrations, and β-arrestin recruitment was measured and normalized to % CCh max; additionally, hM₁ TREx CHO cells expressing β-arrestin2-YFP were treated with 100 μM VU0357017 HCI, and β-arrestin2 recruitment was visualized via confocal microscopy and quantified by counting puncta[3]
5. Furchgott analysis in inducible M₁ cell line: hM₁ TREx CHO cells were treated with 1 μg/mL or 25 ng/mL tetracycline for 24 h to modulate M₁ receptor density. Concentration-response curves for VU0357017 HCI were generated in calcium release assays, and double reciprocal plots of equiactive concentrations were used to calculate equilibrium dissociation constants (pK_d and K_d), confirming its weak orthosteric partial agonist activity at M₁[1]

Male Sprague−Dawley rats (380-420 g) were pretreated with scopolamine
1, 3, 10 mg/kg
A single i.p.

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1. Contextual fear conditioning assay in rodents: Rodents were pretreated with scopolamine (0.2 mg/kg, subcutaneous injection) to induce cognitive deficits. VU0357017 HCI was dissolved in an appropriate vehicle (not specified) and administered at 10 mg/kg via intraperitoneal injection. The animals were then subjected to contextual fear conditioning training (exposure to a context paired with a mild foot shock). After a retention interval, the percentage of time spent freezing in the same context was measured to assess cognitive function; VU0357017 HCI reversed scopolamine-induced deficits (n = 3−4 rats per treatment group)[2]
2. Morris Water Maze (MWM) assay in rats: VU0357017 HCI was dissolved in a suitable vehicle (not specified) and administered to rats (n = 8 per group) via an unspecified route (frequency not specified). Rats were trained in the MWM for 5 days (four daily trials per day) to locate a hidden platform; swim distance was recorded daily. On day 5, a probe trial was conducted (platform removed), and platform crossings in the first 30 s were counted to assess spatial memory retention[3]
3. Contextual fear conditioning (CFC) assay in rats: VU0357017 HCI was dissolved in a vehicle (not specified) and administered to rats (n = 4–6 per group) via an unspecified route. Rats were trained in CFC (exposure to a context and a foot shock), and the percentage of freezing behavior was measured post-training to assess acquisition of contextual fear[3]
4. Amphetamine-induced hyperlocomotion assay in rats: VU0357017 HCI was dissolved in a vehicle (not specified) and administered intraperitoneally 30 min before subcutaneous injection of amphetamine (4 mg/kg). Rats were placed in open-field chambers, and locomotor activity (distance traveled) was measured over a specified period to evaluate the ability of VU0357017 HCI to reverse amphetamine-induced hyperlocomotion[3]
5. Electrophysiological studies in rat hippocampal slices: Rat hippocampal slices were prepared and maintained in artificial cerebrospinal fluid (ACSF) at 32°C. VU0357017 HCI was dissolved in ACSF and bath-applied at 500 nM (for LTP studies) or 30 μM (for LTD studies) for 10 min before/stimulus application. Field excitatory postsynaptic potentials (fEPSPs) were recorded at the Schaffer collateral-CA1 synapse to assess LTP and LTD[3]
6. Electrophysiological recordings in rat striatal medium spiny neurons (MSNs): Rat striatal slices were prepared, and whole-cell patch-clamp recordings were performed on MSNs. VU0357017 HCI was applied via bath perfusion, and action potential firing frequency in response to current steps was measured to assess its effect on neuronal excitability[3]
7. Electrophysiological recordings in mouse medial prefrontal cortical (mPFC) pyramidal cells: Mouse mPFC slices were prepared, and whole-cell patch-clamp recordings were conducted on pyramidal cells. VU0357017 HCI was bath-applied, and spontaneous excitatory postsynaptic currents (sEPSCs) were recorded to evaluate its agonist activity[3]

VU0357017 HCI provides good brain exposure after systemic administration (specific pharmacokinetic parameters, such as half-life, oral bioavailability, or clearance, are not provided) [2]

[1]. Chemical modification of the M(1) agonist VU0364572 reveals molecular switches in pharmacology and a bitopic binding mode. ACS Chem Neurosci. 2012 Dec 19;3(12):1025-36.

[2]. Discovery and characterization of novel subtype-selective allosteric agonists for the investigation of M(1) receptor function in the central nervous system. ACS Chem Neurosci. 2010;1(2):104-121.

[3]. Novel allosteric agonists of M1 muscarinic acetylcholine receptors induce brain region-specific responses that correspond with behavioral effects in animal models. J Neurosci. 2012 Jun 20;32(25):8532-44.

1. VU0357017 HCI is a highly selective M₁ muscarinic acetylcholine receptor allosteric agonist, discovered through functional high-throughput screening (HTS) and diversity-directed synthesis; it has unprecedented purity in auxiliary pharmacology, showing no significant activity against a large number of targets at a concentration of 10 μM [2]
2. Targeted mutagenesis studies have shown that the third extracellular loop (especially E401) and the seventh helical loop in the transmembrane region of the M₁ receptor are crucial for the binding of VU0357017 HCI; flexible ligand docking experiments have shown that VU0357017 HCI can form up to four hydrogen bonds with E401, E397, Y381 and Q181 of the M₁ receptor [2]
3. VU0357017 HCI as M₁ mAChR The dual-site ligands of VU0357017 HCI have weak ortho-and-parallel agonist activity and allosteric interactions (slowing the dissociation of [³H]-NMS), which distinguishes it from pure allosteric agonists [1]. 4. VU0357017 HCI differentially activates the coupling of M₁ with different signaling pathways (inducing calcium release and ERK phosphorylation, but not recruiting β-arrestin), thereby having a selective effect on brain circuits: it has a significant effect in the hippocampus (enhancing LTP and cognitive function), a weaker effect in the striatum, and no effect in the medial prefrontal cortex [3]. 5. VU0357017 HCI is a valuable research tool for studying M₁-mediated central nervous system (CNS) effects and a potential lead compound for new therapies for Alzheimer's disease and schizophrenia [2].

C18H28CLN3O3

369.89

369.182

C, 58.45; H, 7.63; Cl, 9.58; N, 11.36; O, 12.98

1135242-13-5

25010775

White to off-white solid powder

3.64

3

4

7

25

408

0

Cl[H].O(C([H])([H])C([H])([H])[H])C(N1C([H])([H])C([H])([H])C([H])(C([H])([H])C1([H])[H])N([H])C([H])([H])C([H])([H])N([H])C(C1=C([H])C([H])=C([H])C([H])=C1C([H])([H])[H])=O)=O

XKJQVUIXSBOCPP-UHFFFAOYSA-N

InChI=1S/C18H27N3O3.ClH/c1-3-24-18(23)21-12-8-15(9-13-21)19-10-11-20-17(22)16-7-5-4-6-14(16)2;/h4-7,15,19H,3,8-13H2,1-2H3,(H,20,22);1H

ethyl 4-[2-[(2-methylbenzoyl)amino]ethylamino]piperidine-1-carboxylate;hydrochloride

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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

DMSO: 25~38 mg/mL (67.6~102.7 mM)
Water: ~10 mg/mL
Ethanol: ~2 mg/mL

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.76 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.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (6.76 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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (6.76 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.

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Solubility in Formulation 4: 33.33 mg/mL (90.11 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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