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AZ10606120 diHCL (AZ-10606120), the dihydrochloride salt of AZ10606120, is a novel and potent negative allosteric modulator of the P2X7 receptor with anti-depressant and anticancer effects.
AZ10606120 is a potent and highly selective antagonist of the P2X7 receptor (P2X7R), an ATP‑gated ion channel involved in inflammation, immune modulation, and cell proliferation. It exhibits sub‑micromolar affinity for human and rat P2X7Rs (IC₅₀ ~10 nM) and shows >1000‑fold selectivity over other P2X receptor subtypes. Preclinical studies have demonstrated that AZ10606120 effectively reverses diabetes‑induced retinal vascular leakage and reduces VEGF/IL‑6 expression in a rat model of type 1 diabetes, alleviates lipopolysaccharide (LPS)‑induced anhedonia in mice, and significantly suppresses the growth of patient‑derived glioblastoma cells in vitro. These findings highlight the therapeutic potential of AZ10606120 for conditions such as diabetic retinopathy, mood disorders, and glioblastoma.| Targets |
P2X7 receptor (P2X7R) [1]
P2X7 receptor (P2X7R) [2] |
|---|---|
| ln Vitro |
In Vitro: In Xenopus laevis oocytes expressing the human P2X7 receptor (modified P2X7-2N3 construct), AZ10606120 inhibited ATP-evoked currents with a pIC50 of approximately 7.9 (IC50 ~12.6 nM). The compound showed >1,000 fold selectivity for P2X7R over other P2X receptor subtypes, being ineffective at the P2X1 receptor at concentrations up to 10 μM [1].
In human U251 glioblastoma cells, treatment with 15 μM AZ10606120 for 72 hours significantly depleted tumour cell numbers (DAPI+ cells) compared to untreated cells (1309 ± 378.3 vs. 4744 ± 302.4 cells). This effect was more effective than treatment with 50 μM temozolomide (TMZ) (5921 ± 495.1 cells). No synergistic anti-tumour effect was observed when AZ10606120 was co-administered with TMZ [2]. In patient-derived primary glioblastoma cultures, treatment with 15 μM AZ10606120 for 72 hours significantly reduced the number of GFAP+/DAPI+ tumour cells compared to untreated cells (114.3 ± 35.9 vs. 569.1 ± 157 cells). A significant reduction in tumour cell number was observed with concentrations of 15 μM or greater. There was no statistically significant difference in cell count between the AZ-treated group and the TMZ-treated group [2]. Treatment of primary glioblastoma cultures with AZ10606120 at concentrations of 15 μM and above for 72 hours significantly increased lactate dehydrogenase (LDH) release into the culture supernatant, indicating plasma membrane damage and cellular cytotoxicity. TMZ treatment did not induce LDH release [2]. |
| ln Vivo |
In Vivo: In a streptozotocin (STZ)-induced type 1 diabetes rat model, intraperitoneal administration of AZ10606120 (100 μg/kg) every other day for 15 days (total of 7 injections) fully reversed the diabetes-induced increase in retinal microvasculature permeability, as measured by Evans blue leakage. The treatment also completely reversed the diabetes-induced accumulation of VEGF and IL-6 expression in the retina, with no effect on blood glucose levels [3].
In C57BL/6J mice, lipopolysaccharide (LPS)-induced anhedonia (measured by sucrose preference test) was significantly attenuated by acute intraperitoneal injection of AZ-10606120 (2 mg/kg) administered 30 minutes before the test. The anti-anhedonic effect of acute AZ-10606120 was completely absent in P2rx7 knockout mice [4]. Subacute treatment with AZ-10606120 (2 mg/kg i.p. once daily for 4 days) also alleviated LPS-induced anhedonia, and this effect was significantly attenuated in P2rx7 knockout mice compared to wild-type mice [4]. |
| Enzyme Assay |
Enzyme Assay: No classical enzyme assays were performed. However, molecular modelling and ligand docking studies (RosettaLigand) were conducted. An unbiased search for potential binding sites on the human P2X7 receptor homology model using FTsite predicted an orthosteric site and a putative allosteric site at the subunit interface at the apex of the receptor. Flexible ligand docking with RosettaLigand, sampling the full extracellular region, showed that 60% of the 1000 lowest-energy poses for AZ10606120 were located in this putative allosteric site, suggesting it as the most likely binding site. A second round of targeted docking focused on the allosteric site identified two main clusters of binding poses. The favoured docking solution showed the unique residue F88 and conserved residues D92 and M105 aligning the aromatic moiety of AZ10606120, while the adamantane group sat deep in the pocket near T94 [1].
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| Cell Assay |
Cell Assay: For the U251 human glioblastoma cell line, cells were cultured in DMEM supplemented with sodium pyruvate, penicillin-streptomycin, FBS, and non-essential amino acids. At 80% confluency, cells were treated with AZ10606120 (1, 5, 15, 25 μM) and/or temozolomide (50 μM) for 72 hours (or 24 and 48 hours in some experiments). Following treatment, cells were fixed in a 1:1 acetone-methanol solution, and cell nuclei were stained with DAPI for 1 hour at room temperature. The total number of DAPI+ cells was quantified over 16 random fields per sample using fluorescence microscopy and ImageJ software [2].
For patient-derived primary glioblastoma cultures, tumour samples were obtained from patients undergoing routine resection. Tissue was digested with papain, and cells were seeded onto poly-D-lysine-coated coverslips in MEM supplemented with D-glucose, L-glutamine, penicillin-streptomycin, FBS, and serum extender. At 80% confluency, cells were treated with AZ10606120 (1, 5, 15, 25, 50, 100 μM) and/or temozolomide (50 μM) for 72 hours. Cells were then fixed and incubated with primary rabbit anti-GFAP antibody overnight at 4°C, followed by secondary goat anti-rabbit antibody conjugated to Texas Red-X for 2 hours at room temperature. Cell nuclei were counterstained with DAPI for 1 hour at room temperature. The total number of GFAP+/DAPI+ cells was quantified across 16 random fields per sample using fluorescence microscopy and ImageJ software [2]. The lactate dehydrogenase (LDH) cytotoxicity assay was performed on primary glioblastoma cultures. Cells were treated with AZ10606120 (1, 5, 15, 25, 50, 100 μM) or temozolomide (50 μM) for 72 hours. Cell-free culture supernatants were then incubated with an LDH reaction mixture for 25 minutes at room temperature. Absorbance, which is directly proportional to LDH levels, was measured at 492 nm using a plate reader [2]. |
| Animal Protocol |
Animal Protocol: For the rat model of type 1 diabetes: Male Wistar rats (150–180 g) were rendered diabetic by a single intraperitoneal injection of streptozotocin (60 mg/kg). Fifteen days after STZ injection, rats received intraperitoneal injections of AZ10606120 at a dose of 100 μg/kg or vehicle every 2 days for an additional 15 days (total of 7 injections). The stock solution of AZ10606120 was prepared in sterile PBS and diluted in sterile PBS for administration [3].
For the mouse model of LPS-induced anhedonia: Male wild-type C57BL/6J mice (2–3 months old, approximately 30 g) were used. For acute treatment, AZ-10606120 (2 mg/kg) was injected intraperitoneally 30 minutes before the sucrose preference test. For subacute treatment, AZ-10606120 (2 mg/kg) was injected intraperitoneally once daily for 4 consecutive days. Lipopolysaccharide (LPS, 0.2 mg/kg or 1 mg/kg) was injected intraperitoneally to induce depressive-like behavior [4]. |
| Toxicity/Toxicokinetics |
In patient-derived primary glioblastoma cultures, treatment with AZ10606120 at concentrations of 15, 25, 50, and 100 μM for 72 hours resulted in a significant increase in lactate dehydrogenase (LDH) release compared to untreated cells, indicating cellular cytotoxicity and plasma membrane damage (p = 0.01, 0.03, 0.02, and 0.015, respectively). There was a trend for increasing LDH release with increasing AZ concentration. Treatment with temozolomide did not promote LDH release [2].
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| References |
[1]. Unique residues in the ATP gated human P2X7 receptor define a novel allosteric binding pocket for the selective antagonist AZ10606120. Sci Rep. 2017 Apr 7;7(1):725.
[2]. P2X7 receptor antagonism by AZ10606120 significantly reduced in vitro tumour growth in human glioblastoma. Sci Rep. 2023 May 24;13(1):8435. [3]. Pharmacological blockade of the P2X7 receptor reverses retinal damage in a rat model of type 1 diabetes. Acta Diabetol. 2019 Sep;56(9):1031-1036. [4]. Neurochemical Changes in the Mouse Hippocampus Underlying the Antidepressant Effect of Genetic Deletion of P2X7 Receptors. PLoS One. 2013 Jun 21;8(6):e66547. |
| Additional Infomation |
AZ10606120 (N-[2-[[2-[(2-hydroxyethyl)amino]ethyl]amino]-5-quinolinyl]-2-tricyclo[3.3.1.13,7]dec-1-ylacetamide dihydrochloride) is a selective, high-affinity antagonist at human and rat P2X7Rs, with little or no effect at other P2X receptor subtypes [1].
Molecular dynamics simulations suggested that the unique P2X7R features (the 73-79 loop insertion and threonine residues T90 and T94) regulate access of AZ10606120 to the allosteric site by shaping the pocket's dynamics and volume. The mean volume of the allosteric pocket in the wild-type P2X7R was 410 ± 38 ų, which was significantly reduced in the deletion mutant (301 ± 30 ų) and the T90V/T94V mutant (260 ± 33 ų) [1]. This study identifies a novel allosteric binding pocket for AZ10606120 at the subunit interface at the apex of the P2X7 receptor, providing a new target for rational drug development [1]. The authors propose that selectivity of AZ10606120 for P2X7R over other P2X subtypes does not come from direct interactions with unique amino acids in the binding pocket, but rather indirectly, by modulation of the dynamic structure and access to the allosteric pocket which is shaped by P2X7R-unique residues [1]. P2X7 receptor antagonism by AZ10606120 is suggested as a potential novel and effective alternative therapeutic approach for patients with glioblastoma, showing superior tumour cell killing compared to temozolomide in U251 cells [2]. AZ10606120 is a small molecule P2X7 receptor antagonist. In a rat model of type 1 diabetes, P2X7R blockade with AZ10606120 fully reversed retinal microvasculature permeability increase, VEGF accumulation, and IL-6 expression, suggesting that P2X7R antagonism may be a promising strategy for treating microvascular changes in early diabetic retinopathy [3]. AZ-10606120 is a potent P2X7 receptor antagonist. In the LPS-induced anhedonia model, acute AZ-10606120 treatment showed higher potency than the selective serotonin reuptake inhibitor citalopram. The antidepressant-like effect of AZ-10606120 was dependent on P2X7 receptor expression, as it was abolished or attenuated in P2rx7 knockout mice [4]. |
| Molecular Formula |
C25H34N4O2
|
|---|---|
| Molecular Weight |
422.573
|
| Exact Mass |
494.221
|
| Elemental Analysis |
C, 60.60; H, 7.32; Cl, 14.31; N, 11.31; O, 6.46
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| CAS # |
607378-18-7
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| Related CAS # |
607378-18-7 (2HCl); 777851-64-6
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| PubChem CID |
56972227
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| Appearance |
White to off-white solid powder
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| Hydrogen Bond Donor Count |
6
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| Hydrogen Bond Acceptor Count |
5
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| Rotatable Bond Count |
9
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| Heavy Atom Count |
33
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| Complexity |
585
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| Defined Atom Stereocenter Count |
0
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| InChi Key |
BVFONFUUWORSPO-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C25H34N4O2.2ClH/c30-9-8-26-6-7-27-23-5-4-20-21(28-23)2-1-3-22(20)29-24(31)16-25-13-17-10-18(14-25)12-19(11-17)15-25;;/h1-5,17-19,26,30H,6-16H2,(H,27,28)(H,29,31);2*1H
|
| Chemical Name |
2-(1-adamantyl)-N-[2-[2-(2-hydroxyethylamino)ethylamino]quinolin-5-yl]acetamide;dihydrochloride
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| Synonyms |
AZ10606120 2HCl; AZ 10606120 DIHYDROCHLORIDE; AZ-10606120 dihydrochloride; 2-(Adamantan-1-yl)-N-(2-((2-((2-hydroxyethyl)amino)ethyl)amino)quinolin-5-yl)acetamide dihydrochloride; az10606120 2hcl; AZ10606120 dihydrochloride
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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 Note: Please store this product in a sealed and protected environment, avoid exposure to moisture. |
| 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) |
DMSO : ~8.33 mg/mL (~16.81 mM)
H2O : ~2 mg/mL (~4.04 mM) |
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| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). View More
Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.3665 mL | 11.8324 mL | 23.6647 mL | |
| 5 mM | 0.4733 mL | 2.3665 mL | 4.7329 mL | |
| 10 mM | 0.2366 mL | 1.1832 mL | 2.3665 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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