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Purity: ≥98%
Azoramide is a small-molecule modulator of the unfolded protein response (UPR) and an ER stress alleviator that is reported to have antidiabetic activity. It can improve ER protein-folding ability and activates ER chaperone capacity to protect cells against ER stress. Azoramide also displayed potent antidiabetic efficacy in two mouse models with obesity by improving by improving peripheral insulin sensitivity and pancreatic β-cell function. Therefore, azoramide has the potential to be developed as a drug for type 2 diabete. In addition, a recent study showed that azoramide may serve as an antagonist against GLP-1R in MSC (mesenchymal stem cells) lineage determination, and azoramide favors adipogenesis against osteogenesis through inhibiting the GLP-1 receptor-PKA-β-catenin pathway.
| Targets |
UPR
Unfolded protein response (UPR) pathway; endoplasmic reticulum (ER) function; CREB signaling pathway [1] Unfolded protein response (UPR) pathway; endoplasmic reticulum (ER) function; CREB signaling pathway [2] |
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| ln Vitro |
Azoramide may be protective because it increases chaperone expression while decreasing protein synthesis and does not cause cytotoxicity or apoptosis. To fully increase chaperone capacity, azoramide may need the IRE1 and PERK branches of the UPR to be present and functional. Azoramide has been discovered to be a particular type of compound with the dual ability to promote ER homeostasis by activating ER chaperone capacity both acutely and chronically. Its treatment effectively shields cells from ER stress conditions brought on by chemicals. During ER stress brought on by metabolic stress, azoramide maintains beta cell survival and function. Azoramide pretreatment's initial action does not compromise ER function. Treatment with azoramide increases SERCA expression, which improves Ca+2 retention in the ER. Azoramide interacts with UPR pathways to support ER stress resolution and enhance ER functionality[1]. Azoramide modulated the unfolded protein response (UPR) in ER-stressed cells. Treatment of mouse embryonic fibroblasts (MEFs) with 10 μM Azoramide for 6 hours reduced ER stress-induced Xbp1 splicing and downregulated the expression of UPR target genes (BiP, CHOP, Atf4) at the mRNA and protein levels. It enhanced insulin sensitivity in 3T3-L1 adipocytes: pre-treatment with 1-10 μM Azoramide for 24 hours increased insulin-stimulated glucose uptake (2-NBDG assay) and Akt phosphorylation (Ser473) in a dose-dependent manner, reversing palmitate-induced insulin resistance. In MIN6 β-cells, 10 μM Azoramide protected against ER stress-induced cell death (thapsigargin or tunicamycin treatment) by reducing caspase-3/7 activation and CHOP expression [1] Azoramide protected iPSC-derived dopaminergic (DA) neurons carrying the PLA2G6 D331Y mutation. Treatment with 1-10 μM Azoramide for 72 hours increased the viability of mutant DA neurons (MTT assay) and reduced apoptotic cell death (Annexin V/PI staining), with maximal effects at 5 μM. It restored ER function by reducing ER stress markers (GRP78, p-eIF2α, ATF4, CHOP) at the protein level and decreasing ER dilation (transmission electron microscopy). Azoramide also reactivated CREB signaling: it increased p-CREB (Ser133) expression and upregulated CREB target genes (BDNF, Bcl-2) at the mRNA and protein levels, which was abrogated by the CREB inhibitor 666-15. Additionally, it reduced reactive oxygen species (ROS) production (DCFH-DA assay) and lipid peroxidation (MDA assay) in mutant DA neurons [2] |
| ln Vivo |
In mice with genetic obesity and diet-induced obesity, azoramide improves glucose homeostasis. Surprisingly, azoramide treatment significantly increases beta cell function, glucose tolerance, and insulin sensitivity in obese mice in several preclinical models[1].
Azoramide exhibited antidiabetic activity in mouse models of type 2 diabetes. In db/db mice (10 weeks old, male), oral administration of 30 mg/kg Azoramide once daily for 4 weeks significantly reduced fasting blood glucose (FBG) levels (from ~350 mg/dl to ~250 mg/dl) and improved glucose tolerance (IPGTT assay: AUC₀₋₁₂₀ min reduced by ~30% compared to vehicle). It also increased insulin sensitivity (ITT assay: glucose disappearance rate increased by ~25%) and reduced hepatic steatosis, as evidenced by decreased liver triglyceride content and Oil Red O staining. In high-fat diet (HFD)-fed mice, 30 mg/kg Azoramide PO once daily for 8 weeks lowered FBG, improved glucose tolerance, and increased insulin-stimulated Akt phosphorylation in liver and adipose tissues. Azoramide did not cause hypoglycemia in non-diabetic C57BL/6 mice [1] Azoramide rescued motor deficits in PLA2G6 D331Y knock-in (KI) mice. Intraperitoneal injection of 10 mg/kg Azoramide three times weekly for 8 weeks (starting at 8 weeks of age) improved motor function in KI mice, as demonstrated by increased latency to fall in the rotarod test (from ~100 s to ~180 s) and reduced hindlimb clasping score (from ~3 to ~1). Histological analysis showed that Azoramide preserved DA neuron number in the substantia nigra pars compacta (SNpc) of KI mice (from ~40% loss to ~15% loss compared to wild-type mice) and reduced microgliosis (Iba1 staining) and astrogliosis (GFAP staining) in the SNpc and striatum. It also restored ER function (reduced GRP78 and CHOP expression) and CREB signaling (increased p-CREB and BDNF expression) in the brain tissues of KI mice [2] |
| Cell Assay |
In either the absence or presence of 20 mM azoramide, INS1 cells are treated for 60 hours with 25 mM glucose and 500 mM palmitate (G/P). Using the CellTiter-Glo cell viability assay system, viability is evaluated after incubation.
ER stress and UPR modulation assay: MEFs or MIN6 β-cells were pre-treated with 1-10 μM Azoramide for 24 hours, then exposed to ER stressors (thapsigargin, tunicamycin) for 6-24 hours. Xbp1 splicing was detected by RT-PCR and agarose gel electrophoresis. mRNA expression of UPR target genes (BiP, CHOP, Atf4) was quantified by qPCR. Protein levels of BiP, CHOP, and p-eIF2α were measured by Western blot [1] Insulin sensitivity assay: 3T3-L1 adipocytes were differentiated, pre-treated with palmitate (to induce insulin resistance) and 1-10 μM Azoramide for 24 hours, then stimulated with insulin (100 nM) for 30 minutes. Glucose uptake was assessed by incubating cells with fluorescent glucose analog 2-NBDG for 1 hour, followed by flow cytometry or fluorescence microscopy. Akt phosphorylation (Ser473) was detected by Western blot [1] DA neuron viability and apoptosis assay: iPSC-derived DA neurons (wild-type or PLA2G6 D331Y mutant) were treated with 1-10 μM Azoramide for 72 hours. Cell viability was measured by MTT assay (absorbance at 570 nm). Apoptosis was analyzed by Annexin V-FITC/PI staining and flow cytometry. ER morphology was observed by transmission electron microscopy after fixing cells with glutaraldehyde and osmium tetroxide [2] ROS and lipid peroxidation assay: Mutant DA neurons were treated with 5 μM Azoramide for 72 hours. ROS production was detected by incubating cells with DCFH-DA for 30 minutes, followed by fluorescence measurement. Lipid peroxidation was assessed by measuring malondialdehyde (MDA) levels using a colorimetric assay kit [2] CREB signaling assay: Mutant DA neurons were treated with 5 μM Azoramide (with/without 666-15 pre-treatment) for 72 hours. Protein levels of p-CREB (Ser133) and CREB target genes (BDNF, Bcl-2) were detected by Western blot. mRNA expression of BDNF and Bcl-2 was quantified by qPCR [2] |
| Animal Protocol |
i.p. injection; azoramide compound (20 mg/ml, 30 μl per injection) was administered via intraperitoneal injection once a day for 14 consecutive days.
C57BL/6 mice Type 2 diabetes mouse models: 10-week-old male db/db mice were randomly divided into vehicle and Azoramide groups (n=8/group). Azoramide was dissolved in a suitable vehicle and administered orally at 30 mg/kg once daily for 4 weeks. Fasting blood glucose was measured weekly using a glucose meter. Intraperitoneal glucose tolerance test (IPGTT) and insulin tolerance test (ITT) were performed at the end of treatment. Mice were euthanized, and liver, adipose, and pancreatic tissues were collected for triglyceride measurement, Oil Red O staining, and Western blot (Akt phosphorylation) [1] High-fat diet (HFD)-fed mice: C57BL/6 mice were fed HFD for 8 weeks to induce insulin resistance, then treated with 30 mg/kg Azoramide PO once daily for another 8 weeks (n=8/group). Glucose and insulin tolerance tests were performed, and tissue samples were collected for molecular analysis [1] PLA2G6 D331Y KI mouse model: 8-week-old KI mice (n=10/group) were treated with 10 mg/kg Azoramide via intraperitoneal injection three times weekly for 8 weeks. Wild-type (WT) and KI vehicle groups were included as controls. Motor function was evaluated by rotarod test (latency to fall at 10 rpm) and hindlimb clasping scoring (0-4 scale) every 2 weeks. After euthanasia, brain tissues (substantia nigra, striatum) were collected for immunohistochemistry (TH, Iba1, GFAP staining), Western blot (GRP78, CHOP, p-CREB, BDNF), and stereological counting of DA neurons [2] |
| Toxicity/Toxicokinetics |
Azolamed showed low toxicity in vivo. Oral administration of 30 mg/kg azolamed to db/db mice and mice fed a high-fat diet for 4–8 weeks did not cause weight loss, death, or significant organ damage (histological analysis of the liver, kidneys, and pancreas was normal). Intraperitoneal injection of 10 mg/kg azolamed to KI mice for 8 weeks did not cause significant toxicity or behavioral changes [1][2]
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| References |
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| Additional Infomation |
Azoramix is a small molecule unfolded protein response (UPR) modulator that was discovered through phenotypic screening of antidiabetic drugs [1]. Its antidiabetic mechanism includes reducing endoplasmic reticulum stress, enhancing insulin sensitivity, and improving glucose metabolism in peripheral tissues (adipose tissue, liver, pancreas) [1]. Azoramix exerts a neuroprotective effect in PLA2G6-related neurodegenerative diseases (PLAN) by restoring endoplasmic reticulum homeostasis, reactivating the CREB signaling pathway, reducing oxidative stress, and inhibiting neuroinflammation [2]. The PLA2G6 D331Y mutation leads to endoplasmic reticulum stress, impaired CREB signaling pathway, and loss of dopaminergic neurons, which are key pathological features of PLAN; Azoramix targets these pathways to rescue neuronal dysfunction [2].
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| Molecular Formula |
C15H17CLN2OS
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| Molecular Weight |
308.83
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| Exact Mass |
308.075
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| Elemental Analysis |
C, 58.34; H, 5.55; Cl, 11.48; N, 9.07; O, 5.18; S, 10.38
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| CAS # |
932986-18-0
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| Related CAS # |
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| PubChem CID |
7518316
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| Appearance |
White to off-white solid powder
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| Density |
1.2±0.1 g/cm3
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| Index of Refraction |
1.574
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| LogP |
3.97
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
3
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| Rotatable Bond Count |
6
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| Heavy Atom Count |
20
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| Complexity |
308
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| Defined Atom Stereocenter Count |
0
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| SMILES |
O=C(CCC)NCCC1=CSC(C2C=CC(Cl)=CC=2)=N1
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| InChi Key |
VYBFWKKCWTXCQX-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C15H17ClN2OS/c1-2-3-14(19)17-9-8-13-10-20-15(18-13)11-4-6-12(16)7-5-11/h4-7,10H,2-3,8-9H2,1H3,(H,17,19)
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| Chemical Name |
N-[2-[2-(4-chlorophenyl)-1,3-thiazol-4-yl]ethyl]butanamide
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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 (8.10 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 (8.10 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 (8.10 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 | 3.2380 mL | 16.1901 mL | 32.3803 mL | |
| 5 mM | 0.6476 mL | 3.2380 mL | 6.4761 mL | |
| 10 mM | 0.3238 mL | 1.6190 mL | 3.2380 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.
Reduced BMP2-induced bone formation in azoramide-treated mice.Stem Cell Res Ther. 2018; 9: 57. |
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Inhibited osteogenic differentiation potential of C3H10T1/2 cells with azoramide treatment in vitro.Stem Cell Res Ther. 2018; 9: 57. |
Enhanced adipogenic differentiation potential of C3H10T1/2 cells and mouse-derived MSCs with azoramide treatment in vitro.Stem Cell Res Ther. 2018; 9: 57. |
Ex-4 treatment attenuated azoramide effects on suppressing C3H10T1/2 cell osteoblast differentiation and promoting their differentiation into adipocytes.Stem Cell Res Ther. 2018; 9: 57. |
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GLP-1R silencing abolished the azoramide (Azo) regulatory effects of suppressing C3H10T1/2 cell osteoblast differentiation and promoting their differentiation into adipocytes.Stem Cell Res Ther. 2018; 9: 57. |
Decreased expression levels of protein kinase A (PKA) with azoramide (Azo) treatment.Stem Cell Res Ther. 2018; 9: 57. |
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Image depicting the main facts observed of azoramide pre-treatment on ER homeostasis.Ann Transl Med. 2016 Oct; 4(Suppl 1): S45 |
Azoramideregulates ER folding and secretion capacity without inducing ER stress.Sci Transl Med.2015 Jun 17;7(292):292ra98. |
Azoramideprotects against chemically-induced ER stress in vitro.Sci Transl Med.2015 Jun 17;7(292):292ra98. |
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Azoramideinduces weight loss, changes in energy expenditure and improved metabolic profile in mice with diet-induced obesity.Sci Transl Med.2015 Jun 17;7(292):292ra98. |
Azoramideimproves ER function, insulin secretion and survival in beta cells.Sci Transl Med.2015 Jun 17;7(292):292ra98. |
Azoramidetreatment alters ER calcium homeostasis.Sci Transl Med.2015 Jun 17;7(292):292ra98. |
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Azoramidereduces ER stress and improves metabolism inob/obmice.Sci Transl Med.2015 Jun 17;7(292):292ra98. |
Reporter systems monitor ER chaperone availability and activity.Sci Transl Med.2015 Jun 17;7(292):292ra98. |
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