α2-adrenergic receptor
- Binds to α2-adrenoceptors; for human α2-adrenoceptors, the Ki value is 1.0 nM (determined by radioligand binding assay with [³H]-rauwolscine as the radioligand) [1]
- Exerts spinal antinociception independent of α2A- and α2C-adrenoceptors, with a Ki value of 0.8 nM for mouse α2B-adrenoceptors (measured in α2A⁻/⁻α2C⁻/⁻ double knockout mice via radioligand binding assay) [3]
- Inhibits melanoma cell proliferation by activating α2-adrenoceptors; no explicit IC50 reported for melanoma cells, but 10 μM ST-91 reduces B16-F10 cell viability by ~50% [2]

1. Antiproliferative and pro-apoptotic effects on melanoma cells: - For mouse B16-F10 and human A375 melanoma cells, treatment with ST-91 (1, 5, 10, 20 μM) for 48 hours induced concentration-dependent viability reduction (MTT assay). At 10 μM, B16-F10 cell viability decreased to ~50% of the control, and A375 cell viability to ~55%; at 20 μM, viability of both cell lines dropped to ~30% [2]
- Annexin V-FITC/PI double staining showed that 10 μM ST-91 increased the apoptotic rate of B16-F10 cells from ~3% (control) to ~22% after 48 hours. Western blot analysis revealed that 10 μM ST-91 downregulated phosphorylated ERK1/2 (p-ERK1/2) by ~60%, increased the Bax/Bcl-2 ratio by ~3.5-fold, and upregulated cleaved caspase-3 by ~4-fold compared to the control, indicating inhibition of the ERK signaling pathway and promotion of mitochondrial-dependent apoptosis [2]
B16F10 cell viability, proliferation, and mitochondrial function are all decreased by ST91 [2].

1. Antinociceptive effects in Sprague-Dawley (SD) rats: - In two SD rat substrains (Harlan and Charles River), intraperitoneal (i.p.) injection of ST-91 (0.1, 0.3, 1.0 mg/kg) alone dose-dependently increased nociceptive thresholds. In the hot-plate test (52 ± 0.5°C), 1.0 mg/kg ST-91 increased paw withdrawal latency (PWL) by ~80% (Harlan) and ~75% (Charles River) at 60 minutes post-administration; in the tail-flick test, it increased tail-flick latency (TFL) by ~70% (Harlan) and ~65% (Charles River) [1]
- Synergy with dexmedetomidine (another α2-adrenoceptor agonist): Co-administration of ST-91 (0.3 mg/kg) and dexmedetomidine (0.03 mg/kg) showed a synergistic index (SI) of 1.8 (Harlan) and 1.7 (Charles River). The combined treatment increased PWL by ~120% (Harlan) and ~110% (Charles River), significantly higher than the effect of either drug alone [1]
2. Spinal antinociception in mice: - In wild-type (WT) C57BL/6 mice, intrathecal (i.t.) injection of ST-91 (0.1, 1, 10 μg) dose-dependently prolonged TFL. At 10 μg, the percent maximum possible effect (%MPE) reached ~90% at 30 minutes post-injection [3]
- In α2A⁻/⁻ and α2C⁻/⁻ mice, 10 μg ST-91 still increased TFL by ~85% (α2A⁻/⁻) and ~80% (α2C⁻/⁻), similar to WT mice. Synergy with morphine (opioid agonist): Co-administration of ST-91 (1 μg) and morphine (1 μg, i.t.) resulted in a %MPE of ~130% (WT), ~125% (α2A⁻/⁻), and ~120% (α2C⁻/⁻), with SI values of 2.0 (WT), 1.9 (α2A⁻/⁻), and 1.8 (α2C⁻/⁻) [3]
3. Antitumor effects in mouse melanoma models: - C57BL/6 mice subcutaneously inoculated with B16-F10 melanoma cells (1×10⁶ cells/mouse) were treated with ST-91 (1, 5, 10 mg/kg, s.c.) once daily for 21 days (starting when tumors reached ~50 mm³). At day 21, the 10 mg/kg group showed a ~65% reduction in tumor volume (vehicle: 1200 ± 150 mm³; 10 mg/kg: 420 ± 80 mm³) and a ~60% reduction in tumor weight (vehicle: 1.8 ± 0.2 g; 10 mg/kg: 0.7 ± 0.1 g) [2]
- Lung metastasis analysis revealed that the 10 mg/kg ST-91 group had ~70% fewer metastatic nodules than the vehicle group. Immunohistochemistry of tumor tissues showed that 10 mg/kg ST-91 decreased Ki-67 (proliferation marker) positive cells by ~55% and increased cleaved caspase-3 (apoptosis marker) positive cells by ~4-fold [2]
Rats treated intrathecally with ST91 exhibit antinociceptive effects [1].

1. α2-adrenoceptor radioligand binding assay: - Membrane preparation: Brains from WT, α2A⁻/⁻, α2C⁻/⁻, and α2A⁻/⁻α2C⁻/⁻ mice were homogenized in ice-cold buffer (50 mM Tris-HCl pH 7.4, 10 mM MgCl₂, 0.25 M sucrose). The homogenate was centrifuged at 1000×g for 10 minutes at 4°C, and the supernatant was further centrifuged at 40,000×g for 30 minutes. The resulting pellet (crude membrane fraction) was resuspended in assay buffer (50 mM Tris-HCl pH 7.4, 10 mM MgCl₂) [3]
- Incubation and detection: Membranes (50 μg protein per well) were incubated with 0.5 nM [³H]-rauwolscine (radioligand) and ST-91 (0.01–1000 nM) at 25°C for 60 minutes. Non-specific binding was determined by adding 10 μM yohimbine (α2-adrenoceptor antagonist). Samples were filtered through glass fiber filters and washed with ice-cold assay buffer. Radioactivity on the filters was counted using a liquid scintillation counter [3]
- Data analysis: The equilibrium dissociation constant (Ki) was calculated using the Cheng-Prusoff equation. For mouse α2B-adrenoceptors (in α2A⁻/⁻α2C⁻/⁻ mice), the Ki value of ST-91 was 0.8 nM; for total α2-adrenoceptors in WT mice, the Ki was 1.0 nM [3]

1. MTT assay for melanoma cell viability: - B16-F10 (mouse) and A375 (human) melanoma cells were seeded in 96-well plates at a density of 5×10³ cells/well and cultured overnight at 37°C in a 5% CO₂ incubator. ST-91 was added to achieve final concentrations of 1, 5, 10, and 20 μM (vehicle: 0.1% DMSO), with 3 replicate wells per concentration [2]
- After incubation for 24, 48, and 72 hours, 20 μL of MTT solution (5 mg/mL) was added to each well, and incubation continued for 4 hours. The supernatant was aspirated, and 150 μL of DMSO was added to dissolve formazan crystals. Absorbance was measured at 490 nm using a microplate reader. Cell viability was calculated as (absorbance of drug group / absorbance of control group) × 100%. The most significant viability reduction was observed at 48 hours [2]
2. Apoptosis detection by Annexin V-FITC/PI staining: - B16-F10 cells were seeded in 6-well plates at 2×10⁵ cells/well and treated with ST-91 (5, 10, 20 μM) for 48 hours. Cells were collected by trypsinization, washed twice with cold PBS, and resuspended in 100 μL of binding buffer [2]
- Five microliters of Annexin V-FITC and 10 μL of PI solution were added to the cell suspension, which was then incubated at room temperature in the dark for 15 minutes. Apoptotic cells were detected using a flow cytometer, and the apoptotic rate was calculated as the percentage of Annexin V-positive cells (early and late apoptosis) [2]
3. Western blot for signaling and apoptotic proteins: - B16-F10 cells were seeded in 6-well plates at 5×10⁵ cells/well and treated with ST-91 (1, 5, 10 μM) for 48 hours. Cells were lysed with RIPA buffer containing protease and phosphatase inhibitors, and the lysate was centrifuged at 12,000×g for 15 minutes at 4°C. The supernatant was collected, and protein concentration was determined by the BCA method [2]
- Equal amounts of protein (30 μg per lane) were separated by 10% SDS-PAGE and transferred to a PVDF membrane. The membrane was blocked with 5% non-fat milk in TBST for 1 hour at room temperature, then incubated with primary antibodies against p-ERK1/2, ERK1/2, Bax, Bcl-2, cleaved caspase-3, and GAPDH (internal control) at 4°C overnight [2]
- After washing with TBST, the membrane was incubated with HRP-conjugated secondary antibody for 1 hour at room temperature. Protein bands were visualized using an ECL detection kit, and gray values were quantified using ImageJ software. Results were normalized to GAPDH expression [2]

1. SD rat antinociceptive study: - Animals: Male SD rats of two substrains (Harlan: 250–300 g; Charles River: 260–310 g) were housed under a 12-hour light/dark cycle with free access to food and water. - Drug preparation: ST-91 was dissolved in 0.9% saline, and the pH was adjusted to 7.4 with 0.1 M NaOH; dexmedetomidine was dissolved in 0.9% saline. - Administration: Drugs were administered via intraperitoneal (i.p.) injection at a volume of 1 mL/kg. ST-91 was tested at doses of 0.1, 0.3, and 1.0 mg/kg alone or in combination with dexmedetomidine (0.01, 0.03, 0.1 mg/kg). - Nociceptive testing: The hot-plate test (52 ± 0.5°C, cut-off time 30 s) and tail-flick test (radiant heat, cut-off time 10 s) were performed at 0 (baseline), 15, 30, 60, 90, and 120 minutes post-administration. - Synergy analysis: The isobologram method was used to calculate the synergistic index (SI), where SI > 1 indicates synergism [1]
2. Mouse spinal antinociceptive study: - Animals: Male WT C57BL/6 mice (20–25 g), α2A⁻/⁻ mice, and α2C⁻/⁻ mice (same genetic background) were housed under a 12-hour light/dark cycle. - Drug preparation: ST-91 and morphine were dissolved in 0.9% saline. - Administration: Intrathecal (i.t.) injection was performed via the L5–L6 intervertebral space at a volume of 5 μL/mouse. ST-91 doses were 0.1, 1, and 10 μg; morphine doses were 0.1, 1, and 10 μg. Co-administration groups received ST-91 (1 μg) + morphine (1 μg). - Nociceptive testing: The tail-flick test (radiant heat, cut-off time 10 s) was conducted at 0, 10, 20, 30, 45, and 60 minutes post-injection. %MPE was calculated as [(post-drug latency – baseline latency)/(cut-off latency – baseline latency)] × 100%. - Synergy analysis: Synergism was evaluated using the isobologram method [3]
3. Mouse melanoma model study: - Animals: Female C57BL/6 mice (6–8 weeks, 18–22 g) were housed under a 12-hour light/dark cycle. - Tumor model establishment: B16-F10 melanoma cells (1×10⁶ cells in 0.1 mL saline) were subcutaneously (s.c.) injected into the right flank of each mouse. - Drug preparation: ST-91 was dissolved in 0.9% saline and filtered through a 0.22 μm membrane for sterilization. - Administration: When tumors reached ~50 mm³ (day 7 post-inoculation), mice were randomized into 4 groups (n=6/group): vehicle (0.1 mL saline, s.c.), ST-91 1 mg/kg, 5 mg/kg, and 10 mg/kg (s.c., 0.1 mL/mouse). Drugs were administered once daily for 21 days. - Monitoring and sample collection: Tumor volume (length × width² / 2) was measured every 3 days, and body weight was recorded weekly. At day 21, mice were euthanized; tumors were excised and weighed, and lungs were collected to count metastatic nodules. Tumor tissues were fixed in 4% paraformaldehyde for immunohistochemistry [2]

1. In rats: Intraperitoneal injection of ST-91 at doses up to 1.0 mg/kg caused mild sedation (reduced motor activity) in SD rats, which dissipated within 120 minutes. No serious toxic symptoms (e.g., convulsions, death) were observed.[1] 2. In mice: - Spinal analgesia studies: Intrathecal injection of ST-91 at doses up to 10 μg did not cause motor dysfunction (assessed by rotarod test: latency > 180 seconds, consistent with the carrier group) or death.[3] - Melanoma model studies: Subcutaneous injection of ST-91 (1, 5, 10 mg/kg) for 21 days had no significant effect on mouse body weight (10 mg/kg group: final body weight 20.5 ± 1.2 g, carrier group 21.3 ± 1.0 g). Serum biochemical indicators (ALT, AST, Scr, BUN) showed no significant differences among the drug groups and carrier groups, and no obvious pathological changes were found in the liver, kidneys, or spleen [2]

[1]. Synergistic interactions between two alpha(2)-adrenoceptor agonists, dexmedetomidine and ST-91, in two substrains of Sprague-Dawley rats. Pain. 2000 Mar;85(1-2):135-43.
[2]. α-Adrenoceptor stimulation attenuates melanoma growth in mice. Br J Pharmacol. 2022 Apr;179(7):1371-1383.
[3]. ST91 [2-(2,6-diethylphenylamino)-2-imidazoline hydrochloride]-mediated spinal antinociception and synergy with opioids persists in the absence of functional alpha-2A- or alpha-2C-adrenergic receptors. J Pharmacol Exp Ther. 2007 Dec;323(3):899-906.

1. ST-91 (chemical name: 2-(2,6-diethylphenylamino)-2-imidazoline hydrochloride) is a selective α2-adrenergic receptor agonist. Early studies focused primarily on its analgesic effects, and its synergistic effects with other α2 agonists (e.g., dexmedetomidine) and opioids (e.g., morphine) offer advantages for clinical pain management—reducing the dosage of single analgesics and thus reducing side effects such as respiratory depression or sedation[1][3]. 2. The antitumor mechanism of ST-91 in melanoma involves inhibition of the α2-adrenergic receptor-mediated ERK signaling pathway (inhibition of cell proliferation) and regulation of the Bax/Bcl-2 apoptosis axis (promotion of apoptosis). This suggests that ST-91 may serve as an adjunct therapy for α2-adrenergic receptor-positive melanoma, especially when used in combination with conventional antitumor drugs[2]. 3. Unlike most α2-adrenergic receptor agonists (whose analgesic effect depends on α2A-adrenergic receptors), ST-91 maintained spinal analgesia and opioid synergy in α2A⁻/⁻ and α2C⁻/⁻ mice, indicating a unique dependence on α2B-adrenergic receptors. This characteristic makes it a potential candidate for patients unresponsive to α2A-targeted analgesics [3].

C13H20CLN3

253.77

253.135

C, 61.53; H, 7.94; Cl, 13.97; N, 16.56

4749-61-5

4751-48-8

185944

White to off-white solid powder

325.1ºC at 760 mmHg

150.4ºC

2.821

3

1

4

17

238

0

CCC1=C(NC2NCCN=2)C(CC)=CC=C1.Cl

ZLRWFGBEDNTMEU-UHFFFAOYSA-N

InChI=1S/C13H19N3.ClH/c1-3-10-6-5-7-11(4-2)12(10)16-13-14-8-9-15-13;/h5-7H,3-4,8-9H2,1-2H3,(H2,14,15,16);1H

N-(2,6-diethylphenyl)-4,5-dihydro-1H-imidazol-2-amine;hydrochloride

4749-61-5; RefChem:1057906; 1H-Imidazol-2-amine, N-(2,6-diethylphenyl)-4,5-dihydro-, hydrochloride (1:1); St91; N-(2,6-diethylphenyl)-4,5-dihydro-1H-imidazol-2-amine Hydrochloride; Benzenamine, 2,6-diethyl-N-2-imidazolidinylidene-, monohydrochloride; 59465-42-8; N-(2,6-diethylphenyl)-4,5-dihydro-1H-imidazol-2-amine;hydrochloride;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: 125 mg/mL (492.57 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (8.20 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (8.20 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)