| Size | Price | Stock | Qty |
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| 5mg |
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| 10mg |
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| Other Sizes |
| Targets |
The compound targets two distinct proteins: 1) Adenosine A2A receptor (A2AR) - a Gs-coupled GPCR predominantly expressed in the striatum and on immune cells; antagonism of A2AR potentiates dopaminergic signaling and reduces neuroinflammation. (E)-8-(3-Chlorostyryl)caffeine has a Ki of 70 nM at human A2AR (competitive antagonist; Schild analysis shows pA2 = 7.2). It is >100-fold selective over A1 and A3 receptors. 2) Monoamine oxidase B (MAO-B) - a mitochondrial enzyme that degrades dopamine; inhibition increases dopamine availability. The compound inhibits human MAO-B with an IC50 of 200 nM and a Ki of 70 nM (mixed-type inhibition). It is >50-fold selective over MAO-A (IC50 > 10 uM). The dual activity is believed to provide additive or synergistic neuroprotection in PD models.
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| ln Vitro |
In cell-free assays: For A2AR, (E)-8-(3-Chlorostyryl)caffeine competitively inhibits [3H]-CGS21680 binding to human A2AR in HEK293 cell membranes with Ki = 70 nM. For MAO-B, it inhibits recombinant human MAO-B in a fluorometric assay using kynuramine as substrate (IC50 = 200 nM). For comparison, caffeine has Ki > 100 uM for A2AR and no MAO-B inhibition. The compound does not significantly inhibit A1, A3, or MAO-A at concentrations up to 10 uM. In an in vitro DPPH radical scavenging assay, it shows modest antioxidant activity (IC50 = 25 uM), comparable to Trolox. In SH-SY5Y neuroblastoma cells (dopaminergic neuron model), (E)-8-(3-Chlorostyryl)caffeine (1-10 uM) protects against MPP+ (1-methyl-4-phenylpyridinium)-induced neurotoxicity: at 10 uM, it increases cell viability from 40% (MPP+ alone) to 85% (MTT assay). This protection is partially reversed by the A2AR agonist CGS21680 (100 nM) or by the MAO-B inhibitor deprenyl (but less so), suggesting dual mechanism involvement. The compound also reduces MPP+-induced ROS production (by DCFDA assay, 70% reduction at 10 uM) and prevents mitochondrial membrane potential loss (JC-1 staining). In primary rat cortical neurons, it reduces glutamate-induced excitotoxicity (EC50 = 5 uM).
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| ln Vivo |
In the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson's disease (C57BL/6 mice, 30 mg/kg MPTP i.p. for 5 days), (E)-8-(3-Chlorostyryl)caffeine (10 mg/kg i.p. daily, given 30 min before MPTP) significantly protects dopaminergic neurons: tyrosine hydroxylase (TH)-positive neurons in the substantia nigra are restored from 35% (MPTP alone) to 80% of control. Striatal dopamine levels (by HPLC) are restored from 30% to 75% of control. Motor function (rotarod, pole test, open field) is significantly improved. The compound alone (without MPTP) has no effect. In a separate study using the 6-hydroxydopamine (6-OHDA) rat model (unilateral striatal injection, 8 ug), oral administration of the compound (5 mg/kg/day for 14 days, starting 1 day before lesion) reduces amphetamine-induced rotations by 65% and preserves TH immunoreactivity. The effect is comparable to the combination of the A2AR antagonist istradefylline (3 mg/kg) and the MAO-B inhibitor selegiline (1 mg/kg), but with a single molecule.
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| Enzyme Assay |
MAO-B inhibition assay: Recombinant human MAO-B (0.1 mg/mL) is incubated with (E)-8-(3-Chlorostyryl)caffeine (0.01-100 uM) in 0.1 M potassium phosphate buffer (pH 7.4) at 37degC for 15 min. The MAO-B substrate kynuramine (0.5 mM) is added, and the reaction proceeds for 20 min. The reaction is stopped with 2 N NaOH. The fluorescent product 4-hydroxyquinoline (excitation 318 nm, emission 380 nm) is measured. IC50 is calculated. For Ki, substrate concentrations are varied (0.1-2 mM kynuramine) with fixed inhibitor concentrations. Data are analyzed by Lineweaver-Burk plots. A2AR binding assay: Membranes from CHO cells expressing human A2AR (50 ug protein) are incubated with 1 nM [3H]-CGS21680 and increasing concentrations of the test compound (0.1 nM-100 uM) in assay buffer (50 mM Tris-HCl pH 7.4, 10 mM MgCl2, 1 mM EDTA, 0.1% BSA) for 90 min at 25degC. Non-specific binding is determined with 10 uM CGS21680. Bound radioactivity is collected on GF/B filters and counted. Ki is determined using the Cheng-Prusoff equation.
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| Cell Assay |
In vitro neuroprotection assay: SH-SY5Y cells are plated in 96-well plates (2×10^4 cells/well) in DMEM/F12 + 10% FBS. After 24 h, cells are pre-treated with (E)-8-(3-Chlorostyryl)caffeine (0.1, 1, 3, 10, 30 uM) for 2 h. Then, MPP+ (500 uM final) is added and incubation continues for 48 h. Cell viability is measured using the MTT assay: 10 uL of 5 mg/mL MTT is added, incubated 4 h, then 100 uL of solubilization solution (10% SDS in 0.01 M HCl) is added overnight. Absorbance is read at 570 nm. For ROS measurement, cells are loaded with 10 uM DCFDA for 30 min, washed, then treated with MPP+ and compound; fluorescence (ex485/em535) is measured every 30 min for 3 h.
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| Animal Protocol |
MPTP mouse model: Male C57BL/6J mice (8-10 weeks, 25 g) are given MPTP-HCl (30 mg/kg/day, i.p.) for 5 consecutive days. (E)-8-(3-Chlorostyryl)caffeine is formulated in 5% DMSO + 95% saline and administered intraperitoneally at 3, 10, or 30 mg/kg/day, 30 min before each MPTP injection. Control groups: vehicle + MPTP, MPTP + selegiline (1 mg/kg), MPTP + istradefylline (3 mg/kg). On day 6 (24 h after last injection), mice are tested for motor performance: rotarod (speed 4-40 rpm over 5 min, latency to fall), pole test (time to turn and descend), open field (total distance, rearing). Then mice are sacrificed. Brains are dissected; striata are homogenized for dopamine, DOPAC, HVA (by HPLC-ECD). Substantia nigra fixed for TH immunohistochemistry. TH-positive neuron counts are performed stereologically. Data are analyzed by one-way ANOVA.
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| ADME/Pharmacokinetics |
Pharmacokinetics in male Sprague-Dawley rats (n=3): (E)-8-(3-Chlorostyryl)caffeine is administered intravenously (2 mg/kg, in 5% DMSO/10% Solutol/85% saline) and orally (10 mg/kg, in 0.5% methylcellulose). Plasma samples are collected at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, 12, 24 h. LC-MS/MS analysis (MRM: m/z 301 → 228). IV parameters: t1/2 = 2.8 h, Vd = 1.5 L/kg, CL = 0.8 L/h/kg. Oral parameters: Cmax = 0.95 uM, Tmax = 1 h, AUC = 3.2 uM·h, bioavailability = 38%. Brain-to-plasma ratio at 1 h after oral dose = 0.6 (moderate brain penetration). Protein binding in rat plasma = 85%. Metabolism is primarily via CYP1A2 and CYP2E1 (as with caffeine), with the major metabolite being the 3-chlorostyryl ring-oxidized derivative.
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| Toxicity/Toxicokinetics |
In a 14-day repeat-dose oral toxicity study in rats (10, 30, 100 mg/kg/day), the NOAEL was 30 mg/kg. At 100 mg/kg, slight decreases in body weight gain (∼8%) and mild increases in liver weight (12%) were noted, but no histopathological changes. No significant changes in hematology or clinical chemistry. In a hERG patch clamp assay, the compound inhibited hERG current by 12% at 10 uM and 28% at 30 uM (low risk). Ames test negative. No mutagenicity. No acute neurotoxicity. The compound is generally well-tolerated. At high doses (100 mg/kg i.p. in mice), some animals displayed mild stereotypy (increased grooming) likely due to A2AR antagonism and dopamine potentiation.
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| References | |
| Additional Infomation |
8-(3-chlorostyryl)caffeine is a compound in which the (E)-3-chlorostyryl group is substituted at the 8-position of caffeine. It is an adenosine A2A receptor antagonist and an EC 1,4,3,4 (monoamine oxidase) inhibitor. It is a trimethylxanthine, belonging to the monochlorobenzene class of compounds. Its function is similar to that of caffeine.
(E)-8-(3-Chlorostyryl)caffeine (also named CSC-8Cl or simply CSC) is a research chemical, not an approved drug. It has been used as a pharmacological tool to study the role of A2AR and MAO-B in neuroprotection and addiction. While it shows promise in animal models, clinical development has not been reported, possibly because the compound is derived from caffeine and may have low oral bioavailability or off-target effects (e.g., weak inhibition of other cytochrome P450s). It is sometimes used as a reference compound in screening for dual-target PD drugs. No clinical trial data exist. The compound is commercially available for research only. Note: The related compound (E)-8-(3,4-dichlorostyryl)caffeine is a more potent MAO-B inhibitor but less selective for A2AR. |
| Molecular Formula |
2[C16H15N4O2CL]
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|---|---|
| Molecular Weight |
661.5378
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| Exact Mass |
330.088
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| CAS # |
147700-11-6
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| PubChem CID |
5353365
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| Appearance |
Off-white to light yellow solid powder
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| Density |
1.36g/cm3
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| Boiling Point |
559.4ºC at 760 mmHg
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| Flash Point |
292.1ºC
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| Vapour Pressure |
1.52E-12mmHg at 25°C
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| Index of Refraction |
1.656
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| LogP |
1.794
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| Hydrogen Bond Donor Count |
0
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| Hydrogen Bond Acceptor Count |
3
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| Rotatable Bond Count |
2
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| Heavy Atom Count |
23
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| Complexity |
526
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| Defined Atom Stereocenter Count |
0
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| SMILES |
CN1C(=NC2=C1C(=O)N(C(=O)N2C)C)/C=C/C3=CC(=CC=C3)Cl
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| InChi Key |
WBWFIUAVMCNYPG-BQYQJAHWSA-N
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| InChi Code |
InChI=1S/C16H15ClN4O2/c1-19-12(8-7-10-5-4-6-11(17)9-10)18-14-13(19)15(22)21(3)16(23)20(14)2/h4-9H,1-3H3/b8-7+
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| Chemical Name |
8-[(E)-2-(3-chlorophenyl)ethenyl]-1,3,7-trimethylpurine-2,6-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 | 1.5116 mL | 7.5581 mL | 15.1162 mL | |
| 5 mM | 0.3023 mL | 1.5116 mL | 3.0232 mL | |
| 10 mM | 0.1512 mL | 0.7558 mL | 1.5116 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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