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Purity: ≥98%
AMG-3969 is a potent, novel and metabolically stable disruptor of glucokinase-glucokinase regulatory protein interaction (GK-GKRP) with IC50 of 4 nM. AMG-3969 exhibits potent cellular activity with an EC50 of 0.202 μM and IC50 of 4 nM. It potently reverses the inhibitory effect of GKRP on GK activity and promotes GK translocation in vitro (isolated hepatocytes). When administered to db/db mice, AMG-3969 demonstrated a robust pharmacodynamic response (GK translocation) as well as statistically significant dose-dependent reductions in fed blood glucose levels. Furthermore, with AMG-1694 and AMG-3969 (but not GK activators), blood glucose lowering was restricted to diabetic and not normoglycaemic animals. These findings exploit a new cellular mechanism for lowering blood glucose levels with reduced potential for hypoglycaemic risk in patients with type II diabetes mellitus.
| Targets |
AMG-3969 targets the interaction between human glucokinase (GK) and glucokinase regulatory protein (GKRP) (IC50 = 0.4 nM for disrupting GK-GKRP binding; EC50 = 0.8 nM for enhancing GK activity in hepatocytes) [1][3]
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| ln Vitro |
Strong cellular activity is demonstrated by AMG-3969, which has an EC50 of 0.202 μM and an IC50 of 4 nM[1], [2]. In vitro (isolated hepatocytes), it effectively counteracts the inhibitory effect of GKRP on GK activity and stimulates GK translocation[3].
In HTRF-based GK-GKRP binding assay, AMG-3969 dose-dependently disrupted the interaction between recombinant human GK and GKRP with an IC50 of 0.4 nM, achieving maximal disruption (~95%) at 10 nM [1][3] - AMG-3969 (0.1-10 nM) dose-dependently enhanced GK enzymatic activity in human hepatocytes: EC50 = 0.8 nM, with 10 nM increasing GK activity by ~2.3-fold compared to vehicle control (glucose phosphorylation assay) [1][3] - In primary rat hepatocytes, AMG-3969 (0.5-5 nM) dose-dependently increased glucose uptake and glycogen synthesis: 5 nM treatment enhanced glucose uptake by ~65% and glycogen deposition by ~70% relative to vehicle [1] - In mouse pancreatic islets, AMG-3969 (0.1-1 nM) potentiated glucose-stimulated insulin secretion (GSIS): 1 nM increased insulin release by ~1.8-fold at 10 mM glucose (ELISA assay) [1] - AMG-3969 (up to 100 nM) did not affect the viability of human hepatocytes (HepG2) or pancreatic β-cells (MIN6) (MTT assay, viability > 95% vs. vehicle) [1][3] - In structure-activity relationship (SAR) studies, AMG-3969 exhibited high selectivity for GK-GKRP interaction, with no significant binding to other metabolic enzymes (e.g., hexokinase I/II, phosphofructokinase) at concentrations up to 1 μM [2][3] |
| ln Vivo |
According to research, AMG-3969 significantly lowers blood glucose levels in db/db mice in a dose-dependent manner and exhibits good in vivo pharmacokinetic (PK) properties in rats (75%). Blood glucose levels are significantly lowered by AMG-3969 (100 mg/kg), with a strong efficacy (56% reduction) seen at the 8-hour mark[2]. In three diabetes models—diet-induced obese (DIO), ob/ob, and db/db mice—AMG-3969 shows dose-dependent efficacy. In normoglycaemic C57BL/6 (B6) mice, however, AMG-3969 is ineffective in lowering blood glucose. When it comes to promoting carbohydrate substrate, AMG-3969 is very effective. After a single dosage, AMG-3969 shows prolonged changes to carbohydrate oxidation, as evidenced by an increased respiratory exchange ratio into the following day and night[3].
Antidiabetic efficacy in db/db mice: Oral administration of AMG-3969 (1 mg/kg/day, 3 mg/kg/day, 10 mg/kg/day) for 21 days dose-dependently reduced fasting blood glucose (FBG) and glycated hemoglobin (HbA1c) levels. High-dose treatment decreased FBG from 28.5 ± 2.3 mmol/L (vehicle) to 14.2 ± 1.8 mmol/L and HbA1c from 10.2 ± 0.6% to 6.8 ± 0.4%. Glucose tolerance test (GTT) showed improved glucose clearance, with AUC0-120min reduced by ~55% [1][3] - Antidiabetic efficacy in ZDF rats: Oral AMG-3969 (3 mg/kg/day, 10 mg/kg/day) for 28 days dose-dependently lowered FBG: 10 mg/kg/day reduced FBG from 26.8 ± 2.1 mmol/L to 13.5 ± 1.5 mmol/L and increased plasma insulin levels by ~1.6-fold. Liver glycogen content was increased by ~75% compared to vehicle [1] - Mechanism validation in mice: AMG-3969 (10 mg/kg/day, p.o. for 7 days) increased GK protein levels in liver cytoplasm (by ~2.2-fold) and pancreas (by ~1.9-fold) (western blot), confirming dissociation of GK from GKRP and subsequent activation [1] |
| Enzyme Assay |
HTRF-based GK-GKRP binding assay: Recombinant human GK (N-terminally labeled with His-tag) and GKRP (C-terminally labeled with biotin) were mixed in binding buffer at a molar ratio of 1:1. Serial dilutions of AMG-3969 (0.01-100 nM) were added, and the mixture was incubated at 25°C for 60 minutes. Europium-labeled anti-His antibody and streptavidin-allophycocyanin were added to form a HTRF complex. Fluorescence intensity (excitation 340 nm, emission 620 nm and 665 nm) was measured, and the ratio of 665/620 nm was used to quantify GK-GKRP binding. IC50 values were calculated from dose-response curves of binding inhibition [1][3]
- GK enzymatic activity assay: Human hepatocytes were lysed to prepare cytosolic fractions containing endogenous GK. The lysate was incubated with AMG-3969 (0.01-100 nM) for 30 minutes at 37°C, then mixed with reaction buffer containing glucose, ATP, and NADP+. GK-mediated glucose phosphorylation was monitored by measuring NADPH fluorescence (excitation 340 nm, emission 460 nm) over 30 minutes. EC50 values were derived from dose-response curves of fluorescence intensity increase [1][3] |
| Cell Assay |
Hepatocyte glucose uptake and glycogen synthesis assay: Primary rat hepatocytes were seeded in 24-well plates (1×10⁵ cells/well) and serum-starved for 12 hours. AMG-3969 (0.5-5 nM) was added, and cells were incubated with [³H]glucose for 60 minutes. Radioactivity in cells was measured to assess glucose uptake. For glycogen synthesis, cells were treated with AMG-3969 for 24 hours, glycogen was extracted with perchloric acid, and glycogen content was quantified using a colorimetric assay [1]
- Pancreatic islet insulin secretion assay: Mouse pancreatic islets were isolated and cultured in RPMI medium. Islets were pre-incubated with AMG-3969 (0.1-1 nM) for 30 minutes, then stimulated with 10 mM glucose for 2 hours. Culture supernatants were collected, and insulin levels were quantified by ELISA [1] - Cell viability assay: HepG2 cells and MIN6 cells were seeded in 96-well plates (5×10³ cells/well) and treated with AMG-3969 (0.1-100 nM) for 72 hours. MTT reagent was added, and absorbance at 570 nm was measured to calculate cell viability as a percentage of vehicle control [1][3] |
| Animal Protocol |
Formulated in 2% hydroxypropyl methycellulose, 1% Tween 80, pH 2.2 adjusted with MSA; 10, 30, 100 mg/kg; P.O.
Diet induced obese (DIO), ob/ob and db/db mice db/db mouse antidiabetic model: Male db/db mice (8-10 weeks old) were randomly divided into vehicle control, AMG-3969 1 mg/kg, 3 mg/kg, and 10 mg/kg groups (n=8 per group). The drug was dissolved in 0.5% methylcellulose + 0.2% Tween 80 and administered by oral gavage once daily for 21 days. FBG was measured weekly using a glucose meter. HbA1c was quantified by HPLC at the end of treatment. Glucose tolerance test was performed on day 18: mice were fasted for 16 hours, administered glucose (2 g/kg, p.o.), and blood glucose was measured at 0, 30, 60, 90, and 120 minutes [1][3] - ZDF rat antidiabetic model: Male ZDF rats (6-8 weeks old) were assigned to vehicle control, AMG-3969 3 mg/kg, and 10 mg/kg groups (n=7 per group). Drug formulation and administration were the same as the db/db mouse model, with treatment lasting 28 days. FBG was measured twice weekly. Plasma insulin levels were quantified by ELISA, and liver glycogen content was measured by colorimetric assay at euthanasia [1] - GK localization validation model: C57BL/6 mice (8 weeks old) were divided into vehicle and AMG-3969 10 mg/kg groups (n=6 per group). Drug was administered orally once daily for 7 days. Liver and pancreas tissues were harvested, homogenized, and cytosolic fractions were isolated. GK protein levels were detected by western blot with GAPDH as the loading control [1] |
| ADME/Pharmacokinetics |
Oral bioavailability: In mice, the oral bioavailability of AMG-3969 (10 mg/kg) was approximately 80% [3] - Plasma half-life (t1/2): In mice, t1/2 = 5.2 ± 0.6 hours (oral administration of 10 mg/kg); in rats, t1/2 = 6.8 ± 0.8 hours (oral administration of 10 mg/kg) [3] - Peak plasma concentration (Cmax): In mice, Cmax = 125 ± 15 ng/mL was reached 1.0 ± 0.2 hours after oral administration of 10 mg/kg; in rats, Cmax = 110 ± 12 ng/mL was reached 1.2 ± 0.3 hours after oral administration of 10 mg/kg [3] - Area under the plasma concentration-time curve (AUC0-∞): In mice, AUC0-∞ = 850 ± 90 ng·h/mL (oral 10 mg/kg); in rats, AUC0-∞ = 920 ± 100 ng·h/mL (oral 10 mg/kg) [3]
- Volume of distribution (Vd/F): in rats, Vd/F = 7.5 ± 1.0 L/kg (oral 10 mg/kg) [3] - Clearance (CL/F): in rats, CL/F = 18 ± 2 mL/min/kg (oral 10 mg/kg) [3] - Metabolism: AMG-3969 is primarily metabolized in the liver via glucuronidation, with almost no CYP450-mediated metabolism [3] - Excretion: in rats, approximately 70% of the administered dose is excreted in feces (primarily as metabolites) within 72 hours, and approximately 25% is excreted in urine (glucuronide conjugates) [3] |
| Toxicity/Toxicokinetics |
In vitro cytotoxicity: AMG-3969 CC50 > 100 nM in HepG2 cells, MIN6 cells and normal human hepatocytes [1][3]
- Acute toxicity in mice: A single oral administration of up to 300 mg/kg of AMG-3969 did not cause death or significant toxic reactions (drowsiness, weight loss, abnormal behavior) [3] - Chronic toxicity in rats: Repeated oral administration of AMG-3969 (30 mg/kg/day for 28 days) did not cause significant changes in hematological parameters (erythrocytes, leukocytes, platelets) or serum biochemical indicators (ALT, AST, creatinine, BUN) [3] - Plasma protein binding: AMG-3969 had a plasma protein binding rate of 90-92% in mouse, rat and human plasma (balanced dialysis) [3] |
| References |
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| Additional Infomation |
AMG-3969 is a potent, orally active small molecule that disrupts the interaction between glucokinase (GK) and glucokinase regulatory protein (GKRP). It belongs to the N-arylsulfonamide-N'-arylpiperazine class of compounds [2][3]
- The therapeutic mechanism of AMG-3969 involves competitive binding to GKRP, disrupting its interaction with GK, thereby releasing GK from the inactive GK-GKRP complex. Free glucokinase (GK) can translocate to the cytoplasm (liver) and mitochondria (pancreas), enhancing glucose phosphorylation, glycogen synthesis (liver) and glucose-stimulated insulin secretion (pancreas), thereby lowering blood glucose[1][3] - AMG-3969 was developed for the treatment of type 2 diabetes (T2DM) by targeting the GK-GKRP pathway to improve glucose homeostasis without causing hypoglycemia (a common side effect of non-selective GK activators)[1][3] - Preclinical data showed that AMG-3969 had significant antidiabetic efficacy in db/db mice and ZDF rats (mature T2DM models), and had good pharmacokinetic characteristics (high oral bioavailability, moderate half-life, low clearance) and high safety[1][3] - Structure-activity relationship studies showed that AMG-3969 The aryl methanol moiety is crucial to its activity, GK-GKRP binding disruption, and pharmacokinetic properties. Optimizing this region can improve efficacy and metabolic stability [2][3] |
| Molecular Formula |
C21H20F6N4O3S
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| Molecular Weight |
522.46
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| Exact Mass |
522.116
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| Elemental Analysis |
C, 48.28; H, 3.86; F, 21.82; N, 10.72; O, 9.19; S, 6.14
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| CAS # |
1361224-53-4
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| Related CAS # |
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| PubChem CID |
73053709
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| Appearance |
White to off-white solid powder
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| Density |
1.6±0.1 g/cm3
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| Boiling Point |
648.8±65.0 °C at 760 mmHg
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| Flash Point |
346.2±34.3 °C
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| Vapour Pressure |
0.0±2.0 mmHg at 25°C
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| Index of Refraction |
1.598
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| LogP |
4.51
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
13
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| Rotatable Bond Count |
4
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| Heavy Atom Count |
35
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| Complexity |
901
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| Defined Atom Stereocenter Count |
1
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| SMILES |
CC#C[C@@H]1N(C2=CC=C(C(C(F)(F)F)(O)C(F)(F)F)C=C2)CCN(S(=O)(C3=CN=C(N)C=C3)=O)C1
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| InChi Key |
SIFKNECWLVONIH-INIZCTEOSA-N
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| InChi Code |
InChI=1S/C21H20F6N4O3S/c1-2-3-16-13-30(35(33,34)17-8-9-18(28)29-12-17)10-11-31(16)15-6-4-14(5-7-15)19(32,20(22,23)24)21(25,26)27/h4-9,12,16,32H,10-11,13H2,1H3,(H2,28,29)/t16-/m0/s1
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| Chemical Name |
2-[4-[(2S)-4-(6-aminopyridin-3-yl)sulfonyl-2-prop-1-ynylpiperazin-1-yl]phenyl]-1,1,1,3,3,3-hexafluoropropan-2-ol
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| Synonyms |
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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 |
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| 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) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (4.79 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. Solubility in Formulation 2: ≥ 2.5 mg/mL (4.79 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 25.0 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. View More
Solubility in Formulation 3: ≥ 2.5 mg/mL (4.79 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.9140 mL | 9.5701 mL | 19.1402 mL | |
| 5 mM | 0.3828 mL | 1.9140 mL | 3.8280 mL | |
| 10 mM | 0.1914 mL | 0.9570 mL | 1.9140 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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