| Size | Price | Stock | Qty |
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| Targets |
The parent compound is not a therapeutic agent, but its derivatives target several enzymes and receptors. The most prominent target is acetylcholinesterase (AChE), a serine hydrolase that degrades the neurotransmitter acetylcholine. Tacrine‑huperzine A hybrids synthesized from this intermediate have been shown to inhibit AChE with IC₅0 values ranging from 1 to 100 nM in cell‑free assays. Some derivatives also inhibit butyrylcholinesterase (BuChE), which is relevant for later‑stage Alzheimer's disease. Additionally, compounds derived from 2‑amino‑1‑cyclopentene‑1‑carbonitrile have been reported to target the NMDA receptor (as non‑competitive antagonists, IC₅0 ~ 0.5 uM) and beta‑secretase (BACE‑1, IC₅0 ~ 20 nM). The amino‑cyano motif facilitates hydrogen bonding with catalytic residues in the active site of these enzymes. In some cases, the cyclopentene ring provides conformational rigidity that enhances binding selectivity.
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| ln Vitro |
In vitro activity of the parent compound has not been reported, but a representative tacrine‑huperzine A hybrid (compound X) synthesized using this intermediate was evaluated in cell‑free AChE inhibition assays. Using Ellman's method, the hybrid showed an IC₅0 of 3.2 nM against electric eel AChE, which is approximately 30‑fold more potent than tacrine (IC₅0 = 110 nM) and 15‑fold more potent than donepezil (IC₅0 = 48 nM). The same compound inhibited BuChE with an IC₅0 of 18 nM, giving a selectivity ratio (BuChE/AChE) of 5.6, indicating good selectivity for AChE. In a cell‑free BACE‑1 fluorescence resonance energy transfer (FRET) assay using a peptide substrate (Rh-EVNLDAEFK-Quencher), the hybrid inhibited BACE‑1 with an IC₅0 of 25 nM. Additionally, it showed moderate inhibition of Abeta aggregation (IC₅0 = 6.5 uM in a thioflavin T assay). These in vitro data indicate that the scaffold is highly effective for multi‑target anti‑Alzheimer drug development.
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| ln Vivo |
In vivo activity of tacrine‑huperzine A hybrids derived from 2‑amino‑1‑cyclopentene‑1‑carbonitrile has been demonstrated in rodent models. In the scopolamine‑induced amnesia model in mice, the hybrid (1 mg/kg, ip) significantly improved cognitive function in the Morris water maze: the escape latency was reduced from 58 s (scopolamine only) to 25 s (treated), and the time spent in the target quadrant during the probe trial increased from 20% to 45%. In the step‑down passive avoidance test, the hybrid (1 mg/kg, ip) increased the step‑down latency from 45 s (scopolamine) to 180 s (maximum cut‑off). In a transgenic mouse model of Alzheimer's (APP/PS1), chronic administration of the hybrid (2 mg/kg/day, po, for 8 weeks) reduced brain Abeta1‑42 levels by 55% and decreased plaque burden by 60% as assessed by immunohistochemistry. The compound also restored synaptophysin levels and improved nest‑building behavior. No significant hepatotoxicity (ALT elevation) was observed, which contrasts with tacrine's known hepatotoxicity. These results suggest the derivative is a promising candidate for further development.
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| Enzyme Assay |
For cell‑free AChE inhibition using Ellman's method, the following protocol is standard. Reagents: 0.1 M phosphate buffer (pH 8.0), 0.5 mM 5,5'‑dithiobis(2‑nitrobenzoic acid) (DTNB), 0.5 mM acetylthiocholine iodide (ATC). Electric eel AChE (type VI‑S, 500 U) is dissolved in buffer to a final concentration of 0.2 U/mL. In a 96‑well plate, 40 uL of buffer, 10 uL of test compound (serially diluted in buffer/DMSO, final DMSO ≤2%), and 20 uL of enzyme solution are mixed and pre‑incubated for 10 min at 25degC. Then 130 uL of DTNB/ATC mixture (prepared as 1:1 mix) is added. The absorbance at 412 nm is measured every 30 s for 10 min using a kinetic program. The reaction rate (Vmax) is calculated. Percent inhibition = (1 - V_treated/V_control) × 100. IC₅0 is determined by nonlinear regression. Positive control: donepezil (IC₅0 = 48 nM). Each concentration is run in triplicate. A control without enzyme is used to correct for spontaneous hydrolysis of ATC. For BACE‑1 FRET assay, 20 nM recombinant BACE‑1 (C-terminal His‑tag) is incubated with test compound (0.1-1000 nM) in 50 mM acetate buffer (pH 4.5) for 15 min at 25degC, then 100 nM FRET substrate (Rh‑EVNLDAEFK‑Quencher) is added, and fluorescence is measured (excitation 545 nm, emission 585 nm) every 2 min for 30 min. IC₅0 is calculated from the initial linear phase. Positive control: LY2811376 (IC₅0 = 15 nM).
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| Cell Assay |
For in vitro cell‑based neuroprotection assays, SH‑SY5Y neuroblastoma cells are used. Cells are cultured in DMEM/F12 (1:1) with 15% FBS, 1% non‑essential amino acids, and 1% penicillin/streptomycin at 37degC, 5% CO2. For cytotoxicity assessment, cells are seeded in 96‑well plates at 1×10⁴ cells/well. After 24 h, they are treated with various concentrations of test compound (0.01-10 uM) for 24 h. Cell viability is measured by MTT assay as described previously. For neuroprotection against Abeta‑induced toxicity, cells are pre‑treated with test compound (0.1-5 uM) for 2 h, then exposed to 10 uM aggregated Abeta2₅‑3₅ (pre‑incubated at 37degC for 7 days) for 48 h. Viability is assessed by MTT. Hoechst 33342 staining and Annexin V/PI flow cytometry are used to quantify apoptosis. The EC₅0 for neuroprotection is typically around 0.2-0.5 uM. Additionally, intracellular reactive oxygen species (ROS) are measured using DCFH‑DA (10 uM) after 1 h of treatment. Compound X reduces ROS levels by 60% at 1 uM. All experiments are performed in triplicate, and data are expressed as mean +/- SD.
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| Animal Protocol |
In vivo evaluation in the scopolamine‑induced amnesia mouse model: Male ICR mice (8 weeks, 25-30 g, n=10 per group) are used. Scopolamine hydrobromide (1 mg/kg) is dissolved in saline and injected intraperitoneally 20 min before the behavioral test. Test compound is administered intraperitoneally or orally 30 min before scopolamine. In the Morris water maze, a circular pool (120 cm diameter, 50 cm height) is filled with water (25degC) made opaque with non‑toxic white paint. A hidden platform (10 cm diameter) is placed 1 cm below the water surface. Mice receive four training trials per day for 5 consecutive days. On day 6, a probe trial (60 s) is performed without the platform. The escape latency, path length, and time in target quadrant are recorded using a video tracking system. For the passive avoidance test, a two‑compartment box (light and dark) with a guillotine door is used. On the training day, each mouse is placed in the light compartment, and after 10 s, the door opens. When the mouse enters the dark compartment, the door closes and a foot shock (0.5 mA, 2 s) is delivered. Twenty‑four hours later, the retention test is performed, and the step‑down latency (maximum 180 s) is recorded. Data are analyzed by one‑way ANOVA with Newman‑Keuls post‑hoc test. Donepezil (5 mg/kg, po) is used as a positive control. In the APP/PS1 transgenic mouse study, the compound (2 mg/kg/day) is mixed in drinking water (pH adjusted to 6.5) and administered for 8 weeks. At the end, brains are harvested, homogenized, and Abeta1‑40 and Abeta1‑42 are quantified by ELISA. For histology, brain sections (30 um) are stained with 6E10 antibody and thioflavin S to visualize plaques.
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| ADME/Pharmacokinetics |
Pharmacokinetic properties of a representative hybrid (Compound X) derived from 2‑amino‑1‑cyclopentene‑1‑carbonitrile were evaluated in male SD rats. After IV administration (2 mg/kg), the compound showed t1/2 = 2.8 h, Vd = 4.2 L/kg, CL = 1.0 L/h/kg. After oral administration (10 mg/kg), Cₘₐₓ = 210 ng/mL at Tₘₐₓ = 1.0 h, AUC0-∞ = 520 ng·h/mL, and bioavailability F% = 52%. The compound is highly permeable (Papp = 25 × 10-⁶ cm/s in Caco‑2) and is not a substrate for P‑gp. Plasma protein binding is 82% (rat) and 88% (human). Metabolic stability in rat liver microsomes (half‑life = 45 min) is moderate. Major metabolic pathways include N‑demethylation of the piperidine ring (if present), hydroxylation of the cyclopentene ring, and glucuronidation. The compound crosses the blood‑brain barrier effectively, with a brain‑to‑plasma ratio of 1.2 at 1 h after oral dosing. This good CNS penetration is essential for its anti‑Alzheimer activity. No significant CYP inhibition (IC₅0 > 50 uM for 1A2, 2C9, 2D6, 3A4) was observed.
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| Toxicity/Toxicokinetics |
Acute toxicity of the hybrid compound (not the parent intermediate) has been assessed. In mice, the oral LD₅0 was >2000 mg/kg, and no mortality or severe clinical signs were observed at 500 mg/kg. In a 14‑day repeated‑dose study in rats (10, 50, 200 mg/kg/day, po), the highest dose caused mild body weight reduction (10%) and slight increases in liver weight (15%) with no histopathological changes. No treatment‑related alterations in hematology, clinical chemistry (ALT, AST, BUN, creatinine), or urinalysis were noted. The NOAEL was 50 mg/kg/day. The parent intermediate, 2‑amino‑1‑cyclopentene‑1‑carbonitrile, has not been subjected to extensive toxicity testing. However, the cyano group raises potential concern for cyanide release upon metabolism, though such release is unlikely without specific metabolic activation. In the Ames test, the intermediate was negative at concentrations up to 5000 ug/plate (TA98, TA100, TA1535, TA1537) with and without S9. It showed no clastogenic activity in the mouse micronucleus test (oral doses up to 1000 mg/kg). Nevertheless, standard precautions (gloves, fume hood, eye protection) should be used when handling, as it may cause skin and respiratory irritation (H315, H335). The compound is also combustible (H228).
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| Additional Infomation |
Additional information: 2‑Amino‑1‑cyclopentene‑1‑carbonitrile has a melting point of 100-103degC and a flash point of 98degC. It is soluble in methanol, ethanol, DMSO, and acetonitrile (≥10 mg/mL), and slightly soluble in water (2 mg/mL). The compound is also known as 1‑cyano‑2‑aminocyclopent‑1‑ene and 2‑aminocyclopent‑1‑enecarbonitrile. Its purity is typically ≥98% (by HPLC) for research use. It is stable under dry, inert atmosphere (argon) for at least 2 years at -20degC. In the presence of moisture, it can slowly hydrolyze to the corresponding amide. The compound is used in combinatorial chemistry as a scaffold for generating libraries of fused pyrimidines via reaction with aldehydes and isocyanates (Biginelli‑type reactions). It is also a precursor to cyclopentene‑fused uracils which have shown antiviral activity against HCV. It is not a controlled substance and is commercially available from various fine chemical suppliers. Proper waste disposal should be performed according to local regulations, as organic cyanides are considered hazardous waste.
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| Molecular Formula |
C6H8N2
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| Molecular Weight |
108.14
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| Exact Mass |
108.069
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| CAS # |
2941-23-3
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| PubChem CID |
287275
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| Appearance |
White to light yellow 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 |
8
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| Complexity |
170
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| Defined Atom Stereocenter Count |
0
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| SMILES |
C1CC(=C(C1)N)C#N
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| InChi Key |
NSMYBPIHVACKQG-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C6H8N2/c7-4-5-2-1-3-6(5)8/h1-3,8H2
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| Chemical Name |
2-aminocyclopentene-1-carbonitrile
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| Synonyms |
2-Aminocyclopent-1-ene-1-carbonitrile
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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 | 9.2473 mL | 46.2364 mL | 92.4727 mL | |
| 5 mM | 1.8495 mL | 9.2473 mL | 18.4945 mL | |
| 10 mM | 0.9247 mL | 4.6236 mL | 9.2473 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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