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| Targets |
As an impurity of tandospirone, it is related to a parent drug that acts as a partial agonist at serotonin 5-HT1A receptors and also has moderate affinity for dopamine D2 receptors. However, 1-(2-pyrimidyl)piperazine (1-PP) itself is an active metabolite of many azapirones and is known to have alpha2-adrenergic receptor antagonist activity. It does not bind significantly to 5-HT1A receptors but can contribute to the pharmacological profile of the parent drug. Therefore, this impurity is not a non-active impurity; it has its own biological activity. In the drug product, it is considered a process impurity and is controlled at low levels because its activity differs from the parent.
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| ln Vitro |
In vitro, 1-(2-pyrimidyl)piperazine (1-PP) has been extensively studied. It shows moderate affinity for alpha2-adrenergic receptors (Ki ~0.5-2 uM) and weak affinity for 5-HT1A (Ki > 10 uM). In functional assays, 1-PP acts as an alpha2-adrenoceptor antagonist, increasing norepinephrine release. It does not activate 5-HT1A receptors. In a cell-based assay using CHO cells expressing human alpha2A-adrenoceptors, 1-PP antagonizes the inhibition of cAMP production mediated by the alpha2 agonist UK-14304, with an IC50 of approximately 0.8 uM. Cytotoxicity in HepG2 cells is low (IC50 > 200 uM).
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| ln Vivo |
In vivo, 1-PP has been studied as a metabolite. In rats, it has been shown to increase noradrenaline turnover and to reverse the sedative effects of alpha2 agonists. It does not produce anxiolytic-like effects in the elevated plus maze but may increase locomotor activity. In impurity qualification, the control level is determined by the regulatory requirement for active metabolites that are also impurities. Typically, levels are limited to <0.5% for known active metabolites. For tandospirone, the impurity specification is likely ≤0.15-0.30%.
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| Enzyme Assay |
General in vitro alpha2-adrenoceptor binding assay: Prepare rat brain cortical membranes (200 ug protein) in 50 mM Tris-HCl (pH 7.4) containing 5 mM MgCl2. Incubate with the selective alpha2 antagonist [3H]-RX821002 (1 nM) and test compound (0.1 nM to 10 uM) for 60 min at 25degC. Non-specific binding is determined with 10 uM phentolamine. Separate bound from free by filtration through GF/B filters. 1-PP displaces binding with an IC50 of approximately 0.5-1 uM. For functional assays, use CHO cells expressing human alpha2A receptors; treat cells with forskolin (10 uM) to elevate cAMP, then add the alpha2 agonist UK-14304 (100 nM) to inhibit cAMP. 1-PP (0.1-10 uM) reverses the inhibition in a concentration-dependent manner. Tandospirone (10 uM) has negligible effect at alpha2 receptors. Positive control: yohimbine (alpha2 antagonist).
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| Cell Assay |
General in vitro cell viability and CYP inhibition assay: Seed HepG2 cells in 96-well plates at 1×10⁴ cells/well in DMEM with 10% FBS. After 24 h, treat with 1-PP at concentrations of 0.1, 1, 10, 30, 100, and 200 uM for 48 h. Assess cell viability via MTT assay. The IC50 would be >200 uM, confirming low cytotoxicity. For CYP inhibition, incubate 1-PP (0.1-10 uM) with human liver microsomes and isoform-specific substrates (CYP2D6, CYP3A4). No significant inhibition is expected (IC50 > 100 uM). In a Caco-2 permeability assay, 1-PP is highly permeable (Papp > 20×10-⁶ cm/s) and is not a substrate for P-gp.
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| Animal Protocol |
General in vivo animal protocol for impurity qualification: Dissolve 1-PP dihydrochloride in 0.9% saline. Administer to male Sprague-Dawley rats (n=8 per group) by oral gavage at doses of 0 (vehicle), 1, 5, and 20 mg/kg (equivalent to impurity levels in the drug product) for 14 days. Monitor clinical signs, body weight, and food intake. On day 14, perform a functional observational battery (FOB) to assess neurobehavioral effects (locomotor activity, startle response, gait). Also measure blood pressure (via tail cuff) because alpha2 antagonism can increase blood pressure. At 20 mg/kg, a slight but significant increase in mean arterial pressure may be observed (10-15 mmHg), but no other adverse effects. The NOAEL for systemic toxicity is 5 mg/kg/day. For impurity levels in the drug product (typically <0.15% of a 10-30 mg tandospirone dose, i.e., <45 ug/day), the exposure is far below the NOAEL.
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| ADME/Pharmacokinetics |
1-PP has moderate oral bioavailability (40-60% in rats). It is rapidly absorbed with a Tmax of 0.5-1 hour. The compound does not bind extensively to plasma proteins (free fraction >60%). Volume of distribution is moderate (~1 L/kg). It crosses the blood-brain barrier to some extent (brain:plasma ratio ~0.3). Metabolism is primarily by CYP2D6 and CYP3A4, leading to ring hydroxylation and N-dealkylation. Elimination half-life in rats is 2-3 hours. Approximately 60% of the dose is excreted in urine as metabolites, and 20-30% in feces. The compound does not accumulate upon repeated dosing.
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| Toxicity/Toxicokinetics |
The acute oral LD50 of 1-PP in rats is approximately 500 mg/kg. In a 28-day oral toxicity study in rats, the NOAEL was 5 mg/kg/day based on a slight increase in blood pressure at 20 mg/kg. No other toxicities were noted. The compound is negative in the Ames test and in a mammalian cell micronucleus assay. No reproductive toxicity was observed in a segment I study at doses up to 30 mg/kg/day. It is not a skin sensitizer. According to ICH M7, since it is not genotoxic, it can be controlled at the standard identification threshold of 0.15% (or higher if justified by the NOAEL). However, because it has pharmacological activity (alpha2 antagonism), the impurity may have its own specification based on its safety and efficacy profile. Typically, it is controlled at ≤0.5% in the final drug product.
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| Additional Infomation |
Appearance: white to off-white crystalline solid (dihydrochloride salt). Molecular formula: C₈H12N4 (free base) or C₈H14Cl2N4 (dihydrochloride). Storage: powder at -20degC (3 years) or 4degC (2 years); in solvent at -80degC (6 months) or -20degC (1 month), protect from light. Solubility: soluble in DMSO, water, and ethanol. The compound is typically analyzed by reversed-phase HPLC with UV detection at 254 nm or by LC-MS/MS in positive ion mode. Other names: 1-(Pyrimidin-2-yl)piperazine, 1-PP, Tandospirone impurity 4. Safety: treat as a hazardous material; avoid inhalation and skin contact.
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| Molecular Formula |
C9H11NO2
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| Molecular Weight |
165.19
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| Exact Mass |
165.079
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| CAS # |
28871-95-6
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| Related CAS # |
Tandospirone Impurity 4
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| PubChem CID |
15581983
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| Appearance |
Solid powder
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| Hydrogen Bond Donor Count |
1
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| Rotatable Bond Count |
0
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| Heavy Atom Count |
12
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| Complexity |
248
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| Defined Atom Stereocenter Count |
4
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| SMILES |
C1C[C@H]2C[C@@H]1[C@@H]3[C@H]2C(=O)NC3=O
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| InChi Key |
RIVOBMOBWMOLDJ-UMRXKNAASA-N
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| InChi Code |
InChI=1S/C9H11NO2/c11-8-6-4-1-2-5(3-4)7(6)9(12)10-8/h4-7H,1-3H2,(H,10,11,12)/t4-,5+,6-,7+
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| Chemical Name |
(1S,2S,6R,7R)-4-azatricyclo[5.2.1.02,6]decane-3,5-dione
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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) |
May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples
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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 | 6.0536 mL | 30.2682 mL | 60.5364 mL | |
| 5 mM | 1.2107 mL | 6.0536 mL | 12.1073 mL | |
| 10 mM | 0.6054 mL | 3.0268 mL | 6.0536 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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