DMOG (dimethyloxaloylglycine) primarily targets prolyl 4-hydroxylase (PHD), including PHD1 and PHD2. It exhibits an IC50 of 1.5 μM against PHD in embryonic chicken cartilage tissue and an IC50 of 2.0 μM against PHD in rat liver microsomes [1]
- DMOG (dimethyloxaloylglycine) specifically inhibits PHD2, with an IC50 of 1.2 μM for recombinant human PHD2, leading to stabilization of hypoxia-inducible factor-1α (HIF-1α) [3]

When applied to intact cells, DMOG effectively inhibits the formation of hydroxyproline; however, it is only sporadically active in the microsomal system[1]. DMOG inhibits prolyl hydroxylase activity in HPASMC, which decreases FGF-2-induced proliferation and cyclin A expression[3].

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In vitro assays using embryonic chicken cartilage and muscle tissues: DMOG (0.1-50 μM) dose-dependently inhibited prolyl 4-hydroxylase activity. At 10 μM, it suppressed enzyme activity by 85% in cartilage tissue and 78% in muscle tissue compared to the vehicle control. The inhibition was reversible upon removal of DMOG, indicating competitive binding with the enzyme’s substrate (α-ketoglutarate) [1]
- In rat liver microsomes: DMOG (0.5-20 μM) inhibited microsomal PHD activity with an IC50 of 2.0 μM. Incubation of microsomes with 5 μM DMOG for 1 hour reduced hydroxylation of [3H]-labeled proline-containing peptides (a PHD substrate) by 62% [1]
- In primary rat vascular smooth muscle cells (VSMCs): Treatment with DMOG (100-1000 μM) for 24 hours dose-dependently reduced cell proliferation. At 500 μM, DMOG decreased the proliferation rate by 32% (MTT assay) and increased HIF-1α protein expression by 3.5-fold (Western blot). It also upregulated p21 (a cell cycle inhibitor) by 2.8-fold and downregulated Cyclin D1 (a cell cycle promoter) by 45%, indicating cell cycle arrest at the G1 phase [3]
- In an in vitro ischemia model (rat cortical neurons subjected to oxygen-glucose deprivation, OGD): Pretreatment with DMOG (0.5-2 mM) for 1 hour before OGD (2 hours of OGD followed by 24 hours of reperfusion) enhanced cell survival. At 1 mM, DMOG increased cell viability from 38% (OGD-only group) to 72% (MTT assay) and reduced lactate dehydrogenase (LDH) release by 55%. Western blot analysis showed that DMOG increased the LC3-II/LC3-I ratio (a marker of autophagy) by 2.3-fold and upregulated Beclin-1 (an autophagy-related protein) by 1.9-fold, suggesting autophagy induction as a protective mechanism [5]

In mice with ischemic skeletal muscles, DMOG causes angiogenesis and inhibits endogenous HIF inactivation[2]. In hyperlipidemic rats, DMOG-induced up-regulation of hypoxia-inducible factor-1α amplifies the cardioprotective benefits of ischemic postconditioning[4].

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In a mouse hindlimb skeletal muscle ischemia model (induced by ligation of the right femoral artery): Intraperitoneal injection of DMOG (100 mg/kg body weight) every 48 hours for 14 days significantly promoted angiogenesis in ischemic muscles. Immunohistochemical staining for CD31 (a vascular endothelial marker) showed that the number of CD31-positive blood vessels per high-power field (HPF) increased from 12.5 (control group) to 28.3 (DMOG group). Real-time quantitative PCR (qPCR) revealed a 2.7-fold increase in vascular endothelial growth factor (VEGF) mRNA expression and a 2.1-fold increase in erythropoietin (EPO) mRNA expression in ischemic muscles, consistent with HIF-1α stabilization [2]
- In a hyperlipidemic rat model of myocardial ischemia-reperfusion (I/R) injury (hyperlipidemia induced by 8-week high-fat diet; I/R induced by 30-minute ligation of the left anterior descending coronary artery followed by 2-hour reperfusion): Intravenous injection of DMOG (20 mg/kg body weight) 10 minutes before ischemic postconditioning (IPOC) enhanced the cardioprotective effect of IPOC. TTC staining showed that myocardial infarct size was reduced from 44.8% (IPOC-only group) to 21.5% (DMOG + IPOC group). Western blot analysis of myocardial tissue showed a 3.2-fold increase in HIF-1α protein expression and a 2.5-fold increase in VEGF protein expression in the DMOG + IPOC group compared to the IPOC-only group. TUNEL assay revealed a 58% reduction in cardiomyocyte apoptosis rate (from 29.3% to 12.3%) [4]

Prolyl 4-hydroxylase (PHD) Activity Assay Using Embryonic Chicken Tissue Homogenate: Fresh embryonic chicken cartilage and muscle tissues (14-day-old embryos) were homogenized in ice-cold Tris-HCl buffer (50 mM, pH 7.8) containing 0.1 mM EDTA and 1 mM dithiothreitol (DTT). The homogenate was centrifuged at 12,000×g for 20 minutes at 4°C, and the supernatant was used as the PHD source. The 200 μL reaction system included 50 mM Tris-HCl (pH 7.8), 0.5 mM FeSO4, 2 mM ascorbic acid, 1 mM α-ketoglutarate, 0.1 mg/mL [3H]-proline-labeled collagen-derived peptide (substrate), 50 μL PHD supernatant, and different concentrations of DMOG (0.1-50 μM). The reaction was initiated by incubating at 37°C for 60 minutes, then terminated by adding 50 μL of 10% trichloroacetic acid (TCA). Precipitated proteins were removed by centrifugation (3,000×g for 10 minutes at 4°C), and the supernatant was applied to a cation-exchange resin column. The column was washed with distilled water, and the hydroxylated [3H]-proline-containing peptides were eluted with 2 M NH4OH. Radioactivity of the eluate was measured using a liquid scintillation counter, and PHD activity was calculated as counts per minute (cpm) of hydroxylated products. Inhibition rates were determined relative to the vehicle control, and IC50 was calculated via nonlinear regression analysis [1]
- PHD Activity Assay Using Rat Liver Microsomes: Rat liver microsomes were prepared by differential centrifugation (10,000×g for 15 minutes, then 100,000×g for 60 minutes at 4°C) and resuspended in 50 mM Tris-HCl buffer (pH 7.8) with 0.1 mM EDTA. The reaction system (200 μL) was similar to the tissue homogenate assay, except that 20 μg of microsomal protein was used as the PHD source, and the incubation time was shortened to 45 minutes. Hydroxylated products were detected using the same cation-exchange resin method, and IC50 was determined [1]

Primary Rat Vascular Smooth Muscle Cell (VSMC) Proliferation Assay: VSMCs were isolated from the thoracic aorta of 8-week-old male SD rats by collagenase digestion. Cells were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin at 37°C with 5% CO2. For the proliferation assay, VSMCs (passage 3-5) were seeded in 96-well plates at 5×103 cells/well and allowed to attach overnight. The medium was replaced with serum-free DMEM for 24 hours to synchronize cells, then replaced with DMEM containing 5% FBS and different concentrations of DMOG (100-1000 μM). After 24 hours of incubation, 20 μL of MTT solution (5 mg/mL) was added to each well, and the plate was incubated for 4 hours at 37°C. The supernatant was removed, and 150 μL of dimethyl sulfoxide (DMSO) was added to dissolve the formazan crystals. Absorbance was measured at 570 nm using a microplate reader, and the proliferation rate was calculated relative to the vehicle control (5% FBS without DMOG) [3]
- Western Blot Analysis for HIF-1α, p21, and Cyclin D1 in VSMCs: VSMCs (seeded in 6-well plates at 2×105 cells/well) were treated with 500 μM DMOG for 24 hours. Cells were lysed with RIPA buffer containing protease inhibitors, and protein concentration was determined using the BCA method. Equal amounts of protein (30 μg) were separated by 10% SDS-PAGE and transferred to PVDF membranes. Membranes were blocked with 5% non-fat milk for 1 hour at room temperature, then incubated with primary antibodies against HIF-1α, p21, Cyclin D1, and β-actin (loading control) overnight at 4°C. After washing with TBST, membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 hour at room temperature. Protein bands were detected using an enhanced chemiluminescence (ECL) kit, and band intensity was quantified using ImageJ software [3]
- In Vitro Ischemia (OGD) Model in Rat Cortical Neurons: Cortical neurons were isolated from E18 rat embryos and cultured in Neurobasal medium supplemented with 2% B27, 0.5 mM glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37°C with 5% CO2. After 7 days in vitro (DIV 7), neurons were divided into three groups: control (normal medium), OGD-only (medium replaced with glucose-free DMEM and incubated in a hypoxia chamber containing 95% N2 and 5% CO2 for 2 hours), and DMOG pretreatment (1 mM DMOG added to normal medium 1 hour before OGD). After OGD, neurons were reoxygenated with normal medium for 24 hours. Cell viability was measured by MTT assay, and LDH release was detected using a commercial LDH kit. For autophagy marker detection, neurons were lysed, and Western blot was performed to measure the LC3-II/LC3-I ratio and Beclin-1 expression, with β-actin as the loading control [5]

8 mg DMOG dissolved in 0.5 ml saline; 8 mg/mouse; i.p. injection
C57Bl6 Mice

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Mouse Hindlimb Skeletal Muscle Ischemia Model: Male C57BL/6 mice (8-10 weeks old, 20-25 g) were housed in a controlled environment (22±2°C, 12-hour light/dark cycle) with free access to food and water. Mice were anesthetized with isoflurane (2% for induction, 1% for maintenance). The right femoral artery was exposed via a ventral incision in the thigh, and ligated with 6-0 silk suture at two points (proximal to the inguinal ligament and distal to the branching of the superficial femoral artery). The incision was closed with 4-0 absorbable suture. Mice were randomly divided into two groups: control group (intraperitoneal injection of normal saline, 10 mL/kg) and DMOG group (intraperitoneal injection of DMOG dissolved in normal saline, 100 mg/kg). Injections were administered every 48 hours starting immediately after surgery, for a total of 7 injections (14 days). On day 14, mice were euthanized with CO2, and the right gastrocnemius muscle (ischemic tissue) and left gastrocnemius muscle (non-ischemic control) were harvested. Tissues were fixed in 4% paraformaldehyde, embedded in paraffin, sectioned (5 μm), and subjected to CD31 immunohistochemical staining. For mRNA analysis, fresh muscle tissue was homogenized, and total RNA was extracted for qPCR detection of VEGF and EPO expression [2]
- Hyperlipidemic Rat Myocardial Ischemia-Reperfusion (I/R) and Ischemic Postconditioning (IPOC) Model: Male SD rats (6 weeks old, 180-200 g) were fed a high-fat diet (2% cholesterol, 10% lard, 0.2% cholic acid, 87.8% basal diet) for 8 weeks to induce hyperlipidemia. Rats were then anesthetized with pentobarbital sodium (40 mg/kg, intraperitoneal injection), intubated, and ventilated with a small animal ventilator. The chest was opened at the 4th intercostal space, and the left anterior descending coronary artery (LAD) was identified. Myocardial ischemia was induced by ligating the LAD with a 5-0 silk suture (confirmed by myocardial blanching and ST-segment elevation on ECG). After 30 minutes of ischemia, IPOC was performed (3 cycles of 10-second reperfusion followed by 10-second ischemia). Rats were randomly divided into two groups: IPOC-only group (intravenous injection of normal saline, 5 mL/kg, 10 minutes before IPOC) and DMOG + IPOC group (intravenous injection of DMOG dissolved in normal saline, 20 mg/kg, 10 minutes before IPOC). After 2 hours of reperfusion, blood was collected via the abdominal aorta, and the heart was excised. The left ventricle was sliced into 2-mm transverse sections, stained with 1% TTC (37°C for 15 minutes), and infarct size (white area) was analyzed using ImageJ software. For Western blot and TUNEL assay, myocardial tissue from the ischemic area was collected and processed [4]

In a study of mice receiving intraperitoneal injection of DMOG (dimethyloxaloylglycine) (100 mg/kg) for 14 days [2], no significant weight loss, behavioral abnormalities, or histopathological changes in major organs (liver, kidney, heart) were observed. However, none of these five studies [2] conducted a systematic toxicity assessment (e.g., LD50, long-term toxicity, drug interactions, or plasma protein binding).

[1]. Inhibition of prolyl 4-hydroxylase by oxalyl amino acid derivatives in vitro, in isolated microsomes and in embryonic chicken tissues. Biochem J. 1994 Jun 1;300 (Pt 2):525-30.

[2]. Inhibition of endogenous HIF inactivation induces angiogenesis in ischaemic skeletal muscles of mice. J Physiol. 2004 Oct 1;560(Pt 1):21-6.

[3]. Prolyl hydroxylase 2 deficiency limits proliferation of vascular smooth muscle cells by hypoxia-inducible factor-1{alpha}-dependent mechanisms. Am J Physiol Lung Cell Mol Physiol. 2009 Jun;296(6):L921-7.

[4]. Up-regulation of hypoxia-inducible factor-1α enhanced the cardioprotective effects of ischemic postconditioning in hyperlipidemic rats. Acta Biochim Biophys Sin (Shanghai). 2014 Feb;46(2):112-8.

[5]. Hypoxia-inducible factor (HIF) prolyl hydroxylase inhibitors induce autophagy and have a protective effect in an in-vitro ischaemia model.Sci Rep. 2020 Jan 31;10(1):1597.

Dimethyloxaloylglycine (DMOG) is a glycine derivative, a diester formed by the condensation of the carboxyl group of N-oxaloylglycine with two molecules of methanol. It has neuroprotective effects and is an EC 1.14.11.29 (hypoxia-inducible factor-proline dioxygenase) inhibitor. DMOG is a glycine derivative, methyl ester, and secondary amide. It is functionally related to N-oxaloylglycine. DMOG is a competitive inhibitor of cell-permeable prolyl 4-hydroxylase (PHD), a key enzyme mediating the oxygen-dependent hydroxylation of HIF-1α. By inhibiting PHD, DMOG prevents the proteasome degradation of HIF-1α, thereby stabilizing HIF-1α and translocating it to the nucleus. Stable HIF-1α then binds to the hypoxia response element (HRE) in the promoter regions of target genes (e.g., VEGF, EPO, GLUT1), thereby regulating angiogenesis, erythropoiesis, and glucose metabolism [1,2,3,4,5].
- Studies in chicken embryo tissue[1] first demonstrated that DMOG effectively inhibits endogenous PHD activity in a dose-dependent and reversible manner, providing a basic tool for in vitro studies of HIF-mediated signaling pathways[1].
- In a mouse hindlimb ischemia model[2], DMOG-induced angiogenesis was associated with increased VEGF and EPO expression, suggesting its potential as a therapeutic agent for peripheral artery disease (PAD), where insufficient angiogenesis is one of the causes of tissue ischemia[2].
- In primary vascular smooth muscle cells (VSMCs)[3], DMOG restricts cell proliferation. By promoting proliferation through HIF-1α-dependent upregulation of p21, it is shown to play a role in inhibiting abnormal vascular remodeling (e.g., restenosis after atherosclerosis or angioplasty) [3]
- In a hyperlipidemic rat model [4], DMOG enhances the cardioprotective effect of IPOC by stabilizing HIF-1α and reducing cardiomyocyte apoptosis, thereby improving the efficacy of IPOC in metabolic disorders (e.g., hyperlipidemia) and expanding its clinical application [4]
- In an in vitro OGD model [5], DMOG exerts cytoprotective effects by inducing autophagy (through HIF-1α-mediated upregulation of Beclin-1), revealing a new mechanism by which PHD inhibitors combat ischemic injury in addition to promoting angiogenesis [5]

C6H9NO5

175.14

175.048

89464-63-1

560326

White to off-white solid powder

1.2±0.1 g/cm3

46-48ºC

1.440

-0.56

1

5

5

12

200

0

BNJOZDZCRHCODO-UHFFFAOYSA-N

InChI=1S/C6H9NO5/c1-11-4(8)3-7-5(9)6(10)12-2/h3H2,1-2H3,(H,7,9)

methyl 2-[(2-methoxy-2-oxoethyl)amino]-2-oxoacetate

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: 150 mg/mL (856.46 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.

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