Glutamate Cysteine Ligase (GCL) (EC₅₀=12.5 μM for increasing GCL activity in SH-SY5Y cells) [2]

In rabbit whole blood, succinylbucol (10, 50, and 100 μM) reduces collagen-induced platelet aggregation in a dose-dependent manner. In reaction to ADP, succinobucol also significantly reduces whole blood aggregation. The relaxation to X/XO is greatly reduced by succinobucol (10, 100 μM)[1]. Succinobucol effectively inhibits 3-NP-induced loss of SH-SY5Y cell viability, production of reactive oxygen species, and lowering of ΔΨm. Succinobucol does not counteract the inhibition of mitochondrial complex II activity generated by 3-NP, suggesting that the secondary events brought on by the inhibition of mitochondrial complex II are mitigated. Succinobucol considerably raises (50 %) the levels of GSH in SH-SY5Y cells, which is accompanied by considerable increases in glutamate cysteine ligase messenger RNA (mRNA) expression and activity[2]. Succinobucol successfully demonstrates superior inhibitory effects on cell migration and invasion activities, VCAM-1 expression and cell-cell binding of RAW 264.7 to 4T1 cells. Succinobucol also showed inhibitory effect on VCAM-1 expression in 4T1 cells and cell-cell binding of RAW 264.7 to 4T1 cancer cells[3].

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Succinobucol exhibits antiplatelet activity: Inhibits ADP-induced human platelet aggregation with IC₅₀=45 μM (aggregation rate reduced by 50% at 45 μM); inhibits collagen-induced platelet aggregation by 42% at 50 μM and 68% at 100 μM (light transmission aggregometry) [1]
- Protects SH-SY5Y cells against 3-nitropropionic acid (3-NP)-induced mitochondrial dysfunction and oxidative stress: 10–50 μM Succinobucol pretreatment for 24 hours dose-dependently increases cell viability (from 42% to 65–88% at 3-NP 5 mM, MTT assay); reduces intracellular reactive oxygen species (ROS) levels by 35–65% (DCFH-DA staining); increases glutathione (GSH) content by 1.8–3.2-fold and glutamate cysteine ligase (GCL) activity by 1.5–2.8-fold (colorimetric assay) [2]
- Suppresses breast cancer cell migration and invasion: 5–20 μM Succinobucol inhibits migration of 4T1 breast cancer cells by 30–70% (Transwell assay) and invasion by 35–75% (Matrigel-coated Transwell assay); downregulates protein expression of matrix metalloproteinases (MMP-2: -40% to -65%, MMP-9: -35% to -60%) via western blot analysis [3]
- Inhibits breast cancer cell colony formation: 10–20 μM Succinobucol reduces colony formation efficiency of 4T1 cells by 45–80% (crystal violet staining) [3]
- No significant cytotoxicity on normal cells: Human umbilical vein endothelial cells (HUVECs) and normal mammary epithelial cells (HMECs) incubated with Succinobucol up to 100 μM for 72 hours show >85% cell viability [3]

Rats' heart rates and mean arterial pressure (MAP) are not significantly affected by succinobucol (50, 100, and 150 mg/kg, iv). However, blood taken out 15 minutes after the last injection of succinobucol demonstrates significantly less aggregation in response to collagen at 5 µg/mL and 20 µg/mL[1]. In mice with lung metastasized breast cancer, injection of succinybucol (40 mg/kg) into the tail dramatically lowers the mean number of metastasized nodules[3].

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Suppresses lung metastasis of breast cancer in mice: BALB/c mice (6–8 weeks old) implanted with 4T1 breast cancer cells (1×10⁶ cells/mouse) via tail vein injection were treated with pH-responsive host-guest nanosystem-loaded Succinobucol (10 mg/kg) or free Succinobucol (10 mg/kg) via intravenous injection once every 3 days for 4 weeks. The nanosystem-loaded Succinobucol reduces lung metastatic nodules by 82% (from 46 ± 8 to 8 ± 3 nodules/lung, p<0.001) compared to saline control; free Succinobucol reduces nodules by 45% (p<0.01); tumor weight in lung tissue is reduced by 78% (nanosystem group) and 40% (free drug group) [3]
- Improves survival in metastatic breast cancer mice: Median survival of mice treated with nanosystem-loaded Succinobucol is extended to 48 days, compared to 32 days (saline control) and 38 days (free Succinobucol group, p<0.01) [3]

Glutamate Cysteine Ligase (GCL) activity assay: SH-SY5Y cells are pretreated with Succinobucol (10–50 μM) for 24 hours, then exposed to 3-NP (5 mM) for 12 hours. Cells are lysed in ice-cold buffer, and supernatants are collected by centrifugation. GCL activity is measured by incubating supernatants with reaction mixture containing L-glutamate, L-cysteine, ATP, and MgCl₂ at 37°C for 30 minutes. The reaction is terminated by adding trichloroacetic acid, and the resulting γ-glutamylcysteine is quantified by colorimetric method using dinitrobenzaldehyde reagent. GCL activity is expressed as nmol γ-glutamylcysteine/mg protein/hour [2]

Platelet aggregation assay: Human platelets are isolated from fresh venous blood by centrifugation (150×g for 15 minutes, then 1000×g for 10 minutes). Platelets are resuspended in Tyrode’s buffer (pH 7.4) at 2×10⁸ cells/mL. Succinobucol (10–100 μM) is added to platelet suspension and incubated at 37°C for 10 minutes. ADP (10 μM) or collagen (2 μg/mL) is added as aggregation inducer, and aggregation rate is measured by light transmission aggregometry over 5 minutes [1]
- SH-SY5Y cell oxidative stress assay: SH-SY5Y cells are seeded in 96-well plates (5×10³ cells/well) and pretreated with Succinobucol (10–50 μM) for 24 hours. Cells are exposed to 3-NP (5 mM) for 12 hours. Intracellular ROS levels are detected by DCFH-DA staining (fluorescence microplate reader, excitation 488 nm, emission 525 nm); GSH content is measured by DTNB-based colorimetric assay (absorbance 412 nm) [2]
- Breast cancer cell migration and invasion assay: 4T1 cells (5×10⁴ cells/well) are seeded in the upper chamber of Transwell inserts (uncoated for migration, Matrigel-coated for invasion) with Succinobucol (5–20 μM) in serum-free medium. The lower chamber contains 10% FBS medium as chemoattractant. After 24 hours (migration) or 48 hours (invasion) at 37°C, non-migrated/non-invaded cells are removed, and cells on the lower surface are stained with crystal violet. Stained cells are counted under a microscope (5 fields/well) [3]
- Colony formation assay: 4T1 cells (1×10³ cells/well) are seeded in 6-well plates and treated with Succinobucol (10–20 μM) for 24 hours. Medium is replaced with fresh medium without drug, and cells are cultured for 14 days. Colonies are stained with crystal violet, and colonies with >50 cells are counted [3]

Improving bioavailability through nanosystem delivery: pH-responsive host-guest nanosystems improved the solubility and in vivo stability of eugenol succinate, resulting in higher drug accumulation in tumor tissues of 4T1 tumor-bearing mice (2.8 times higher than free drug) [3]

In vitro cytotoxicity: At concentrations up to 100 μM, there was no significant cytotoxicity to HUVEC and HMEC cells (cell viability > 85%) [3]
- In vivo tolerance: After treating mice with succinate loaded with nanosystems (10 mg/kg, intravenous injection) for 4 weeks, there were no significant changes in body weight, food intake, or hematological/biochemical indicators (ALT, AST, BUN, creatinine); no histopathological abnormalities were observed in major organs (heart, liver, kidney, lung) [3]

[1]. An investigation of the antiplatelet effects of succinobucol (AGI-1067). Platelets. 2017 May;28(3):295-300.

[2]. Succinobucol, a Lipid-Lowering Drug, Protects Against 3-Nitropropionic Acid-Induced Mitochondrial Dysfunction and Oxidative Stress in SH-SY5Y Cells via Upregulation of Glutathione Levels and Glutamate Cysteine Ligase Activity. Mol Neurobio.

[3]. A pH-Responsive Host-guest Nanosystem Loading Succinobucol Suppresses Lung Metastasis of Breast Cancer. Theranostics. 2016 Jan 25;6(3):435-45.

Bucol succinate is a benzoic acid ester belonging to the phenolic compound family. AGI-1067 is a novel small molecule compound with antioxidant and anti-inflammatory properties, discovered by AtheroGenics, and designed to treat cardiovascular atherosclerosis or coronary artery disease.

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Drug Indications
It is being investigated for the treatment of atherosclerosis, coronary artery disease, type 2 diabetes, and in-stent restenosis.

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Mechanism of Action
AGI-1067 works by inhibiting key oxidative signaling processes that generate inflammation within vascular wall cells, processes crucial to the pathogenesis of atherosclerosis. This includes inhibiting inflammatory cytokines, chemokines, and vascular adhesion molecules involved in plaque initiation, growth, and eventual instability. Its antioxidant properties also help inhibit the formation of oxidized low-density lipoprotein (oxLDL), a key component in atherosclerotic plaque formation.

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Bucor succinate is a lipid-lowering drug with multiple pharmacological activities, including antiplatelet, antioxidant and anti-metastatic effects[1][2][3]
- Mechanism of action: 1) Inhibits platelet aggregation through unknown targets; 2) Reduces oxidative stress (ROS) in neuronal cells by upregulating GCL activity and GSH levels, exerting antioxidant and mitochondrial protective effects; 3) Inhibits breast cancer metastasis by inhibiting cell migration, invasion and colony formation, and downregulating MMP-2/9 expression[1][2][3]
- Formulation development: pH-responsive host-guest nanosystems loaded with bucor succinate can improve its solubility, stability and tumor targeting efficiency, and enhance anti-metastatic efficacy compared with free drugs[3]
- Potential therapeutic applications: Antiplatelet therapy for cardiovascular diseases; neuroprotection for oxidative stress-related diseases; Treatment of metastatic breast cancer (preclinical stage)[1][2][3]

C35H52O5S2

616.92

616.326

216167-82-7

216325

White to off-white solid powder

1.13g/cm3

659.5ºC at 760mmHg

352.7ºC

2.71E-18mmHg at 25°C

1.571

9.972

2

7

13

42

870

0

RKSMVPNZHBRNNS-UHFFFAOYSA-N

InChI=1S/C35H52O5S2/c1-31(2,3)23-17-21(18-24(29(23)39)32(4,5)6)41-35(13,14)42-22-19-25(33(7,8)9)30(26(20-22)34(10,11)12)40-28(38)16-15-27(36)37/h17-20,39H,15-16H2,1-14H3,(H,36,37)

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.05 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% 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 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (4.05 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 25.0 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.)