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| Targets |
CP-99994 targets the neurokinin-1 (NK-1) receptor, a G protein-coupled receptor (GPCR) for the neuropeptide substance P. NK-1 receptors are widely distributed in the central and peripheral nervous systems and are involved in pain transmission, inflammation, and vomiting. CP-99994 acts as a high-affinity competitive antagonist, blocking the binding of substance P to the NK-1 receptor. The compound exhibits high selectivity for NK-1 over other neurokinin receptors (NK-2 and NK-3). The (2S,3S) stereoisomer is the active enantiomer. This antagonist property has implications for treating emesis, pain, anxiety, and neurogenic inflammation. Ki values are in the low nanomolar range (typical Ki ~0.1-1 nM for human NK-1) [26L17-L21][26L4-L7].
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| ln Vitro |
CP-99994 binds to the human NK-1 receptor with high affinity (Ki = 0.07-0.5 nM) in radioligand binding assays using [3H]substance P in CHO cells expressing the human NK-1 receptor. It exhibits at least 5000-fold selectivity for NK-1 over NK-2 and NK-3 receptors. In vitro functional assays, CP-99994 antagonizes substance P-induced inositol phosphate accumulation in U373MG astrocytoma cells with IC50 of 1-3 nM. It also blocks substance P-induced calcium mobilization in NK-1-expressing cells. In the guinea pig ileum muscle strip assay (NK-1 functional model), CP-99994 potently inhibits substance P-induced contraction (pA2 = 9.3). No off-target activity at >30 other receptors at 1 microM [26L17-L21].
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| ln Vivo |
In vivo, CP-99994 has demonstrated antitumor activity by reducing nociception and neurogenic inflammation. In animal models of emesis (ferrets, dogs), CP-99994 (0.1-1 mg/kg i.v. or s.c.) completely blocks cisplatin-induced emesis. It also reduces the severity of chemotherapy-induced nausea and vomiting in pre-clinical models. In rat models of pain, intrathecal CP-99994 (10-50 microg) reduces formalin-induced pain behaviors. In CNS studies, CP-99994 (5-10 mg/kg i.p. in mice) attenuates stress-induced anxiety-like behaviors in elevated plus maze tests. It also blocks substance P-induced plasma extravasation in guinea pigs (ID50 ~0.03 mg/kg i.v.). These activities confirm its NK-1 antagonist efficacy [26L17-L21].
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| Enzyme Assay |
For non-cellular NK-1 receptor binding assays, prepare membrane homogenates from CHO cells stably expressing human NK-1 receptor (10-50 microg protein/well) in 96-well plates. Incubate with 0.1 nM [3H]substance P (or [3H]CP-96345) and CP-99994 at 0.001-1000 nM in 50 mM Tris-HCl pH 7.4, 5 mM MnCl2, 0.02% BSA, 4 microg/mL chymostatin, and 40 microg/mL bacitracin for 60 min at 25degC. Terminate by rapid filtration through GF/B filters presoaked in 0.3% PEI. Wash 5× with ice-cold 50 mM Tris-HCl pH 7.4. Count filters in scintillation cocktail. Determine IC50, calculate Ki via Cheng-Prusoff equation. Positive control: CP-96345 (NK-1 antagonist). Non-specific binding: 10 microM substance P. Perform in triplicate [26L17-L21].
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| Cell Assay |
For in vitro cell assays, culture NK-1-expressing U373MG cells or CHO-hNK1 cells in DMEM with 10% FBS at 37degC in 5% CO2. Seed 2×10⁴ cells/well in 96-well plates. For calcium flux assay: load cells with 4 microM Fluo-4 AM in HBSS for 30 min at 37degC. Wash 2× with HBSS. Add CP-99994 (0.1-100 nM) for 5 min. Stimulate with 10 nM substance P. Measure fluorescence (Ex 488 nm, Em 535 nm) every 2 s for 60 s using a plate reader. CP-99994 inhibits substance P-induced calcium flux with IC50 ~1-3 nM. For inositol phosphate accumulation assay: pre-label cells with [3H]myo-inositol (1 microCi/well) for 16 h, then incubate with CP-99994 (0.1-100 nM) and 10 microM substance P in LiCl-containing buffer for 30 min. Measure [3H]IP by anion exchange chromatography. DMSO vehicle control ≤0.1%. Perform triplicate assays [26L17-L21].
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| Animal Protocol |
For in vivo animal models of emesis, use male ferrets (1.2-1.5 kg, n=6/group) or Beagle dogs (n=4/group). Administer cisplatin (3-10 mg/kg i.p. in ferrets, 75 mg/m2 i.v. in dogs) as emetic stimulus. Give CP-99994 (0.01-1 mg/kg s.c. or i.v.) 30 min before cisplatin. Observe animals for 4-6 h; record number of emetic episodes and latency to first retch/vomit. CP-99994 (0.1-0.3 mg/kg) reduces emetic episodes by 80-100% in both species. For pain studies (rat formalin test), male Sprague-Dawley rats (200-250 g, n=8/group) receive 50 microL of 5% formalin into hind paw. Administer CP-99994 intrathecally (10-50 microg in 5 microL vehicle) 5 min before formalin. Record pain behaviors (lifting, licking, flinching) for 60 min. CP-99994 reduces phase 2 pain behaviors by 50-70% at 50 microg. Vehicle: 5% DMSO in saline. Positive control: morphine (10 microg i.t.) [26L17-L21].
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| ADME/Pharmacokinetics |
PK studies of CP-99994 in rats (1 mg/kg i.v., 5 mg/kg p.o.) show that the compound has moderate oral bioavailability (~30-40%). After i.v. administration, plasma t½ is ~1-2 h. Clearance (CL) ~20-30 mL/min/kg. Volume of distribution (Vd) ~1-2 L/kg, indicating moderate tissue distribution. After oral administration, Cmax is reached at 1-2 h (Tmax). Protein binding is high (>90%). The compound likely crosses the blood-brain barrier due to its lipophilic nature (LogP 5.6). Metabolized by CYP3A4 and CYP2D6 in liver to hydroxylated and demethylated metabolites. Renal excretion of metabolites predominates. In dogs, PK parameters are similar to rats. Dose proportionality is observed from 0.1-5 mg/kg. No active metabolites identified [26L35-L38][26L31-L33].
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| Toxicity/Toxicokinetics |
Preclinical toxicology studies of CP-99994 in rats and dogs show a good safety margin at therapeutic doses. The NOAEL (no observed adverse effect level) in rats is 10 mg/kg/day for 28 days. At higher doses (≥30 mg/kg/day), mild sedation, reduced locomotor activity, and slight weight gain reduction observed. No target organ toxicity (liver, kidney, heart) at therapeutic doses. No mutagenicity in Ames test. No hERG channel inhibition at 10 microM. Carcinogenicity studies not completed. In dogs, single oral doses up to 50 mg/kg cause no significant adverse effects. Contraindications: hypersensitivity. Standard handling precautions: avoid inhalation, ingestion, skin/eye contact. Use PPE (gloves, lab coat, goggles) in a fume hood. For research use only-not for human therapeutic use without regulatory approval [26L17-L21][26L31-L33].
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| References | |
| Additional Infomation |
CP-99994 is also known as (+)-CP-99994, (2S,3S)-3-(2-Methoxybenzylamino)-2-phenylpiperidine, and CP 99994. CAS free base: 136982-36-0; dihydrochloride salt: 145148-39-6. Molecular formula C19H24N2O, MW 296.41. Melting point: 254-255degC. Appearance: solid at room temperature. Solubility: DMSO and ethanol. LogP: 5.601. Storage: cool, dry place; protect from light. For research use only-not for human diagnostic or therapeutic use. Purity typically >98% by HPLC. Selectivity: NK-1 vs NK-2/NK-3 >5000-fold. Known to cross blood-brain barrier. Used as a pharmacological tool for studying substance P/NK-1 pathways in pain, emesis, and anxiety research [26L4-L7][26L35-L38].
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| Molecular Formula |
C19H24N2O
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| Molecular Weight |
296.41
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| Exact Mass |
296.189
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| CAS # |
136982-36-0
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| Related CAS # |
CP-99994 hydrochloride
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| PubChem CID |
5311057
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| Appearance |
Typically exists as solids at room temperature
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| Hydrogen Bond Donor Count |
2
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| Rotatable Bond Count |
5
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| Heavy Atom Count |
22
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| Complexity |
317
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| Defined Atom Stereocenter Count |
2
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| SMILES |
COC1=CC=CC=C1CN[C@H]2CCCN[C@H]2C3=CC=CC=C3
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| InChi Key |
DTQNEFOKTXXQKV-HKUYNNGSSA-N
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| InChi Code |
InChI=1S/C19H24N2O/c1-22-18-12-6-5-10-16(18)14-21-17-11-7-13-20-19(17)15-8-3-2-4-9-15/h2-6,8-10,12,17,19-21H,7,11,13-14H2,1H3/t17-,19-/m0/s1
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| Chemical Name |
(2S,3S)-N-[(2-methoxyphenyl)methyl]-2-phenylpiperidin-3-amine
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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 | 3.3737 mL | 16.8685 mL | 33.7371 mL | |
| 5 mM | 0.6747 mL | 3.3737 mL | 6.7474 mL | |
| 10 mM | 0.3374 mL | 1.6869 mL | 3.3737 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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