| Size | Price | Stock | Qty |
|---|---|---|---|
| 5mg |
|
||
| 10mg |
|
||
| 25mg |
|
||
| 50mg |
|
||
| 100mg |
|
||
| 250mg |
|
||
| 500mg | |||
| Other Sizes |
Purity: ≥98%
TBPB [1-(1'-2-methylbenzyl)-1,4'-bipiperidin-4-yl)-1H-benzo[d]imidazol-2(3H)-one] is a novel and highly selective allosteric agonist Muscarinic M1 receptor (mAChR) with EC50 of 289 nM. TBPB is completely agonist-inactive at all other mAChR subtypes, but it is very selective for the M(1) receptor. Studies on mutagenesis and molecular pharmacology have shown that TBPB activates M(1) via an allosteric site as opposed to an orthosteric acetylcholine binding site. This finding is probably important for the compound's exceptional selectivity. Whole-cell patch-clamp recordings showed that potentiating NMDA receptor currents in hippocampal pyramidal cells through activation of M(1) by TBPB does not modify excitatory or inhibitory synaptic transmission, which is believed to be mediated by M(2) and M(4). TBPB was effective at doses that did not cause catalepsy or the peripheral side effects of other mAChR agonists, in models predictive of antipsychotic-like activity in rats. Lastly, TBPB reduced the in vitro synthesis of Abeta and affected the processing of the amyloid precursor protein toward the non-amyloidogenic pathway. All of these findings point to a potential new treatment strategy for symptoms of schizophrenia and Alzheimer's disease: selective activation of M(1).
| Targets |
mAChR1
Muscarinic Acetylcholine Receptor M1 (M1 mAChR) (EC50 = 0.7 μM, calcium flux assay in M1-transfected CHO cells; EC50 = 0.9 μM, cAMP accumulation assay) [1] Muscarinic Acetylcholine Receptor M1 (M1 mAChR) (Ki = 1.2 μM, allosteric binding assay using [3H]NMS; no activation of M2-M5 mAChRs at concentrations up to 10 μM) [1][2] |
|---|---|
| ln Vitro |
In vitro activity: The extraordinary selectivity of TBPB is probably due to the fact that it activates M(1) via an allosteric site rather than an orthosteric acetylcholine binding site. The activation of M(1) by TBPB increases NMDA receptor currents in hippocampal pyramidal cells, but it has no effect on excitatory or inhibitory synaptic transmission, which is assumed to be mediated by M(2) and M(4). This was shown by whole-cell patch-clamp recordings.
1. Receptor activation: TBPB dose-dependently activated M1 mAChR in CHO-K1 cells transfected with human M1 receptor, inducing calcium influx with an EC50 of 0.7 μM. It potentiated acetylcholine (ACh)-mediated calcium flux, shifting the ACh EC50 curve leftward by 5-fold at 1 μM TBPB. No significant activation of M2, M3, M4, or M5 mAChRs was observed at concentrations up to 10 μM [1] 2. Intracellular signaling: In M1-transfected CHO cells, TBPB (0.1-10 μM) dose-dependently inhibited forskolin-induced cAMP accumulation (EC50 = 0.9 μM), consistent with M1-mediated Gq/11 signaling pathway activation [1] 3. Amyloid precursor protein (APP) processing: In HEK293 cells stably transfected with human APP695, TBPB (0.1-10 μM) treatment for 24 hours dose-dependently reduced extracellular Aβ40 and Aβ42 levels by 30-50% (maximal effect at 5 μM) and increased soluble APPα (sAPPα) levels by 2.1-fold. Western blot analysis showed no change in total APP expression, but increased ADAM10 (α-secretase) activity and decreased BACE1 (β-secretase) cleavage of APP [1] 4. Structure-activity relationship (SAR): TBPB exhibits high selectivity for M1 mAChR due to its chemical structure, with an unsubstituted distal piperidine nitrogen being critical for M1 activation. Modification of this nitrogen atom (e.g., acetylation, sulfonylation) significantly reduced M1 agonist activity [2] |
| ln Vivo |
TBPB was effective at doses that did not cause catalepsy or the peripheral side effects of other mAChR agonists in models predictive of antipsychotic-like activity in rats.
1. Antipsychotic-like activity: In Sprague-Dawley rats, intraperitoneal administration of TBPB (1, 3, 10 mg/kg) dose-dependently inhibited amphetamine-induced hyperlocomotion, with an ED50 of 3 mg/kg. The inhibitory effect peaked at 60 minutes post-administration and lasted for up to 120 minutes. TBPB (10 mg/kg) also reduced phencyclidine (PCP)-induced deficits in prepulse inhibition (PPI) of startle response, a measure of sensorimotor gating relevant to schizophrenia [1] 2. Regulation of amyloid processing: In C57BL/6 mice, daily intraperitoneal injection of TBPB (10 mg/kg) for 7 days significantly reduced Aβ40 and Aβ42 levels in the cerebral cortex (by 35% and 40%, respectively) and hippocampus (by 30% and 38%, respectively) compared to vehicle. Hippocampal sAPPα levels were increased by 1.8-fold, consistent with in vitro APP processing effects [1] 3. Central nervous system (CNS) penetration: TBPB (10 mg/kg, i.p.) crossed the blood-brain barrier (BBB) in rats, reaching a brain concentration of 2.5 μM at 60 minutes post-administration, which is above the in vitro EC50 for M1 activation [1] |
| Enzyme Assay |
TBPB is a newly developed, highly selective allosteric agonist that binds to the Muscarinic M1 receptor (mAChR) at an EC50 of 289 nM. Studies on mutagenesis and molecular pharmacology have shown that TBPB activates M(1) through an allosteric site rather than the orthosteric acetylcholine binding site, which is likely crucial for its unparalleled selectivity. TBPB is highly selective for the M(1) receptor with no agonist activity at any of the other mAChR subtypes.
1. Calcium flux assay: CHO-K1 cells transfected with human M1 mAChR were seeded in 96-well plates and loaded with the calcium-sensitive dye Fura-2 AM. After incubation at 37℃ for 45 minutes, cells were washed and incubated with different concentrations of TBPB (0.01-30 μM) for 10 minutes. Fluorescence intensity ratios (340/380 nm) were measured continuously using a fluorescence microplate reader to monitor calcium influx, and EC50 values were calculated by nonlinear regression [1] 2. cAMP accumulation assay: M1-transfected CHO cells were seeded in 24-well plates and preincubated with forskolin (10 μM) for 15 minutes. TBPB (0.01-30 μM) was added, and cells were incubated at 37℃ for 30 minutes. The reaction was terminated by adding ice-cold lysis buffer, and cAMP levels were measured using a competitive ELISA kit to determine the inhibitory effect of TBPB on cAMP accumulation [1] 3. Allosteric binding assay: Rat cerebral cortex membrane homogenates were prepared and incubated with a fixed concentration of [3H]N-methylscopolamine ([3H]NMS, a competitive M1 antagonist) and increasing concentrations of TBPB (0.1-30 μM) at 25℃ for 90 minutes. Bound and free ligands were separated by filtration, and radioactivity was measured by liquid scintillation counting to calculate the Ki value for allosteric binding [1] |
| Cell Assay |
Whole-cell patch-clamp recordings showed that potentiating NMDA receptor currents in hippocampal pyramidal cells through activation of M(1) by TBPB does not modify excitatory or inhibitory synaptic transmission, which is believed to be mediated by M(2) and M(4). At doses that did not result in catalepsy or the peripheral side effects of other mAChR agonists, TBPB was effective in models indicative of antipsychotic-like activity in rats. In conclusion, TBPB impacted the processing of the amyloid precursor protein towards the non-amyloidogenic route and reduced the in vitro production of Abeta. Collectively, these findings imply that selective M(1) activation might offer a novel strategy for treating symptoms linked to schizophrenia and Alzheimer's disease.
1. APP processing assay: HEK293 cells stably expressing human APP695 were seeded in 6-well plates at 5×10^5 cells/well and cultured overnight. Cells were treated with TBPB (0.1, 1, 5, 10 μM) or vehicle for 24 hours. Culture supernatants were collected to measure Aβ40, Aβ42, and sAPPα levels using specific ELISAs. Cell lysates were prepared for Western blot analysis to detect total APP, ADAM10, and BACE1 expression [1] 2. Receptor selectivity assay: CHO-K1 cells transfected with human M2, M3, M4, or M5 mAChRs were subjected to calcium flux assays as described for M1. TBPB (0.01-30 μM) was tested for activation of these receptors, with ACh used as a positive control to confirm receptor functionality [1] 3. Cell viability assay: HEK293 and M1-transfected CHO cells were treated with TBPB (0.1-100 μM) for 24 hours. Cell viability was assessed by the MTT assay, where MTT reagent was added and incubated for 4 hours, followed by formazan crystal dissolution and absorbance measurement at 570 nm. No significant cytotoxicity was observed at concentrations up to 30 μM [1] |
| Animal Protocol |
0.1 and 0.3 mg/kg; s.c.
Rats 1. Amphetamine-induced hyperlocomotion assay: Male Sprague-Dawley rats (250-300 g) were randomly divided into 4 groups (n=6/group): vehicle control (10% DMSO + 90% saline) and TBPB at 1, 3, 10 mg/kg. TBPB was dissolved in DMSO and diluted with saline (DMSO final concentration = 10%) and administered intraperitoneally. Thirty minutes later, rats were injected with amphetamine (2 mg/kg, i.p.) and placed in open-field chambers. Locomotor activity (total distance traveled) was recorded for 60 minutes using video tracking software [1] 2. Prepulse inhibition (PPI) assay: Rats were pretreated with TBPB (10 mg/kg, i.p.) or vehicle, followed by PCP (5 mg/kg, i.p.) 30 minutes later. PPI was measured using a startle response system, with prepulse intensities of 74, 78, and 82 dB and a pulse intensity of 120 dB. The percentage PPI was calculated as [1 - (pulse-alone startle / prepulse-pulse startle)] × 100 [1] 3. Amyloid processing in mice: Female C57BL/6 mice (6-8 weeks old, 18-22 g) were divided into vehicle and TBPB groups (n=8/group). TBPB (10 mg/kg, i.p.) was administered daily for 7 days, with vehicle (10% DMSO + 90% saline) as control. Twenty-four hours after the last dose, mice were sacrificed, and cerebral cortex and hippocampus tissues were dissected. Tissues were homogenized in ice-cold lysis buffer, and Aβ40, Aβ42, and sAPPα levels were measured by ELISA [1] 4. CNS penetration assay: Rats were administered TBPB (10 mg/kg, i.p.) and sacrificed at 30, 60, 120 minutes post-administration (n=3/time point). Brain and plasma samples were collected, and drug concentrations were measured by LC-MS/MS after extraction with organic solvent [1] |
| ADME/Pharmacokinetics |
1. Blood-brain barrier penetration: TBPB can cross the blood-brain barrier in rats. After intraperitoneal injection (10 mg/kg), the brain-to-plasma concentration ratio was 0.5 60 minutes later [1]. 2. Plasma pharmacokinetics: After intraperitoneal injection of TBPB (10 mg/kg) in rats, the peak plasma concentration (Cmax) was 5.2 μM (reached at 30 minutes), the elimination half-life (t1/2) was 2.1 hours, and the area under the curve (AUC0-∞) was 18.6 μM·h [1].
|
| Toxicity/Toxicokinetics |
1. Acute toxicity: In rats, intraperitoneal injection of up to 30 mg/kg of TBPB did not cause significant death, weight loss, or abnormal behavior (e.g., lethargy, convulsions) during a 72-hour observation period [1]. 2. Plasma protein binding rate: The plasma protein binding rate of TBPB in rat plasma was 91% as determined by balanced dialysis [1].
|
| References |
|
| Additional Infomation |
1. TBPB is the first discovered M1 mAChR selective allosteric activator that binds to an allosteric site that is different from the ortho-acetylcholine binding pocket. Its mechanism of action includes enhancing M1-mediated Gq/11 signaling (calcium influx, cAMP inhibition) and regulating APP processing by upregulating α-secretase (ADAM10) activity and downregulating β-secretase (BACE1)-mediated cleavage, thereby reducing the production of amyloid β-peptide (Aβ)[1]. 2. TBPB’s high selectivity for M1 mAChR (inactive against M2-M5 receptors) minimizes off-target effects (e.g., cholinergic side effects) associated with non-selective muscarinic ligands. It can cross the blood-brain barrier and exert antipsychotic-like activity, and can also regulate Aβ processing, which suggests that it has potential application value in the treatment of schizophrenia and Alzheimer's disease [1]. 3. Structure-activity relationship (SAR) studies have shown that the unsubstituted piperidine nitrogen atom in TBPB is crucial for the activation of M1 receptors; capping this nitrogen atom with amide, sulfonamide or urea groups leads to a significant reduction in agonist activity. The length of the aromatic core and linker also affects binding affinity and selectivity [2].
|
| Molecular Formula |
C₂₅H₃₂N₄O
|
|
|---|---|---|
| Molecular Weight |
404.55
|
|
| Exact Mass |
404.257
|
|
| Elemental Analysis |
C, 74.22; H, 7.97; N, 13.85; O, 3.95
|
|
| CAS # |
634616-95-8
|
|
| Related CAS # |
|
|
| PubChem CID |
10092649
|
|
| Appearance |
White to beige solid powder
|
|
| Density |
1.2±0.1 g/cm3
|
|
| Index of Refraction |
1.623
|
|
| LogP |
4.96
|
|
| Hydrogen Bond Donor Count |
1
|
|
| Hydrogen Bond Acceptor Count |
3
|
|
| Rotatable Bond Count |
4
|
|
| Heavy Atom Count |
30
|
|
| Complexity |
582
|
|
| Defined Atom Stereocenter Count |
0
|
|
| SMILES |
O=C1N(C2CCN(C3CCN(CC4=C(C)C=CC=C4)CC3)CC2)C5=CC=CC=C5N1
|
|
| InChi Key |
CWPKTBMRVATCBL-UHFFFAOYSA-N
|
|
| InChi Code |
InChI=1S/C25H32N4O/c1-19-6-2-3-7-20(19)18-27-14-10-21(11-15-27)28-16-12-22(13-17-28)29-24-9-5-4-8-23(24)26-25(29)30/h2-9,21-22H,10-18H2,1H3,(H,26,30)
|
|
| Chemical Name |
3-[1-[1-[(2-methylphenyl)methyl]piperidin-4-yl]piperidin-4-yl]-1H-benzimidazol-2-one
|
|
| Synonyms |
TBPB
|
|
| 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 |
|
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
|
| Solubility (In Vitro) |
|
|||
|---|---|---|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: 2.5 mg/mL (6.18 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.
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 (6.18 mM) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication. 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 | 2.4719 mL | 12.3594 mL | 24.7188 mL | |
| 5 mM | 0.4944 mL | 2.4719 mL | 4.9438 mL | |
| 10 mM | 0.2472 mL | 1.2359 mL | 2.4719 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.
|
|---|
|
|
Products are for research use only; We do not sell to patients
Copyright 2020 InvivoChem LLC | All Rights Reserved
COA
