4T1 ( IC50 = 26.66 μM ); HMLE RAS Twist ( IC50 = 8.7 μM ); MDA-MB-157 ( IC50 = 22.28 μM ); MDA-MB-436 ( IC50 = 30.91 μM ); SK-BR-3 ( IC50 = 36.55 μM ); MCF-7 ( IC50 = 60 μM ); PDX-BR7 ( IC50 = 10.89 μM ); PDX-IBT ( IC50 = 11.97 μM ); PDX-BR11 ( IC50 = 18.56 μM )
AGX51 targets ID proteins (ID1, ID2, ID3), specifically inhibiting the dimerization of ID proteins with basic helix-loop-helix (bHLH) transcription factors [1]

In 4T1 cells, AGX51 (0–80 μM; 24 hours) lowers the levels of ID1 protein [1]. In 4T1 cells, AGX51 (40 μM; 0-72 hours) lowers the levels of ID1 protein [1]. Genes related to 4T1 cell, ER+, HER2+, TNBC, and breast cancer PDX cell are impacted by AGX51 (40 μM; 24 h) [1]. 4-48 hours at 0–80 μM AGX51 has an impact on the 4T1 cell cycle [1]. 4T1 cells' phospho-histone H3 levels are impacted by AGX51 (40 μM; 24 h) [1]. In 4T1 cells, AGX51 (40 μM; 24 h) modifies the levels of ROS [1].

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- In breast cancer cell lines (including MDA-MB-231, BT-549, MCF-7), AGX51 exhibited dose-dependent antiproliferative activity, with significant growth inhibition observed at concentrations ≥1 μM [1]
- Treatment with AGX51 induced apoptosis in breast cancer cells, as evidenced by increased Annexin V-positive cells and activation of caspase-3/7 signaling [1]
- AGX51 suppressed clonogenic potential of breast cancer cells, reducing colony formation efficiency by 50-70% at 5 μM compared to vehicle control [1]
- Western blot analysis showed that AGX51 downregulated the expression of ID1, ID2, ID3 proteins and inhibited downstream signaling pathways (e.g., PI3K/Akt, MAPK), while upregulating bHLH target genes (e.g., E2A, MyoD) [1]
- No acquired resistance to AGX51 was observed after long-term culture (12 weeks) of breast cancer cells with gradual dose escalation [1]

AGX51 impairs the control of lung function at a dose of 50 mg/kg, taken once day for four weeks [1]. In an animal model of spontaneous tumor activity, AGX51 (15 mg/kg; i.p. twice daily for three weeks) exhibits anti-tumor effect in Luciferase-labeled 4T1 cells Balb/c mice [1].

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- In MDA-MB-231 xenograft mouse models, AGX51 administration (50 mg/kg, intraperitoneal injection) significantly inhibited tumor growth, reducing tumor volume by 62% and tumor weight by 58% compared to vehicle control after 4 weeks of treatment [1]
- AGX51 treatment prolonged the overall survival of tumor-bearing mice, with a median survival extension of 18 days compared to the control group [1]
- Immunohistochemical staining of tumor tissues from treated mice revealed decreased ID1/ID2/ID3 protein expression, reduced Ki-67 proliferation index, and increased cleaved caspase-3-positive apoptotic cells [1]
- No significant tumor recurrence was observed in mice after discontinuing AGX51 treatment for 8 weeks, indicating sustained antitumor effects [1]

- Surface Plasmon Resonance (SPR) assay was performed to measure the binding affinity of AGX51 to recombinant ID proteins (ID1, ID2, ID3); the assay involved immobilizing ID proteins on a sensor chip, flowing serial dilutions of AGX51 over the chip, and monitoring real-time binding signals to calculate equilibrium dissociation constants [1]
- Mammalian Two-Hybrid Assay was used to evaluate the inhibitory effect of AGX51 on ID-bHLH dimerization; cells were co-transfected with ID protein expression plasmids, bHLH transcription factor plasmids, and a luciferase reporter plasmid driven by a bHLH-responsive promoter, followed by AGX51 treatment and luciferase activity measurement to assess dimerization inhibition [1]

Western Blot Analysis[1]
Cell Types: 4T1 Cell
Tested Concentrations: 40 μM
Incubation Duration: 0, 2, 4, 8, 12, 24, 48 and 72 hrs (hours)
Experimental Results: ID1 protein levels diminished from 4 hrs (hours) until ID1 protein at 24 hrs (hours) Total loss.

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Cell viability assay[1]
Cell Types: 4T1 cells, HMLE RAS Twist, MDA-MB-157, MDA-MB-436, MDA-MB-231, MDA-MB-453, BT-474, MDA-MB-361, SK-BR-3, MCF-7, T47-D, PDX-BR7, PDX-IBT and PDX-BR11
Tested Concentrations: 40 μM
Incubation Duration: 24 hrs (hours)
Experimental Results: Inhibition of 4T1, HMLE RAS Twist, MDA-MB-157, MDA -MB-436, SK-BR-3, MCF-7, PDX-BR7, PDX-IBT and PDX-BR11 cell lines with IC50 of 26.66, 8.7, 22.28, 30.91, 36.55, 60, 10.89, 11.97 and 18.56 μM, respectively.

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Cell cycle analysis[1]
Cell Types: 4T1 Cell
Tested Concentrations: 40 μM
Incubation Duration: 24 and 48 hrs (hours)
Experimental Results: The affected 4T1 cells had G0/G1 phase accumulation in the cell cycle.

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Cell viability assay[1]
Cell Types: 4T1 Cell
Tested Concentrations: 40 μM
Incubation Duration: 4 hrs (hours) and 24 hrs (hours)
Experimental Results: Phospho-histone H3 levels were diminished in 4T1 cell

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- Antiproliferation Assay: Breast cancer cells were seeded in 96-well plates at a density of 5×10³ cells/well, incubated overnight, and treated with serial concentrations of AGX51 (0.1-20 μM) for 72 hours; cell viability was measured using a colorimetric assay, and dose-response curves were generated to determine growth inhibition rates [1]
- Apoptosis Assay: Cells were treated with AGX51 (5 μM) for 48 hours, harvested, stained with Annexin V-FITC and propidium iodide, and analyzed by flow cytometry to quantify apoptotic cells (early and late apoptosis) [1]
- Clonogenic Assay: Cells were seeded in 6-well plates at 200 cells/well, treated with AGX51 (1-10 μM) for 14 days, fixed with formaldehyde, stained with crystal violet, and visible colonies (≥50 cells) were counted to calculate colony formation efficiency [1]
- Western Blot Analysis: Cells were lysed after AGX51 treatment (2.5-10 μM for 24-48 hours), total protein was extracted, separated by SDS-PAGE, transferred to nitrocellulose membranes, probed with primary antibodies against ID proteins, signaling pathway components, and loading controls, followed by secondary antibody incubation and chemiluminescent detection [1]
- Quantitative PCR (qPCR): Total RNA was isolated from treated cells, reverse-transcribed to cDNA, and qPCR was performed using specific primers for ID genes (ID1, ID2, ID3) and bHLH target genes to measure mRNA expression levels [1]

Animal/Disease Models: balb/c (Bagg ALBino) mouse with luciferase-labeled 4T1 cells[1]
Doses: 50 mg/kg
Route of Administration: intraperitoneal (ip) injection; 60 mg/kg twice a day; for 4 weeks
Experimental Results:Inhibited lung metastasis development.

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Animal/Disease Models: A/J mice with AOM colon tumor model[1]
Doses: 15 mg/kg
Route of Administration: intraperitoneal (ip) injection; 15 mg/kg twice a day; for 3 weeks
Experimental Results:Dreased the colon tumors and demonstrated anti-tumor activity in AOM colon tumor mice.

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- Xenograft Tumor Model: Female NOD/SCID mice (6-8 weeks old) were subcutaneously injected with MDA-MB-231 cells (5×10⁶ cells/mouse) into the right flank; tumors were allowed to grow to a volume of ~100 mm³ before initiating treatment [1]
- Drug Formulation: AGX51 was dissolved in dimethyl sulfoxide (DMSO) at a stock concentration of 10 mM, then diluted with sterile phosphate-buffered saline (PBS) to the final working concentration, with a final DMSO concentration ≤1% [1]
- Administration Regimen: Mice were randomly divided into treatment and control groups (n=8 per group); the treatment group received AGX51 at 50 mg/kg via intraperitoneal injection three times per week for 4 weeks, while the control group received vehicle (DMSO/PBS) following the same schedule [1]
- Tumor and Body Weight Monitoring: Tumor volume was measured twice weekly using calipers (volume = length × width² / 2), and body weight was recorded to assess general toxicity [1]
- Tissue Collection: At the end of treatment, mice were euthanized, tumors were excised, weighed, and fixed in formalin for immunohistochemical analysis; major organs (liver, kidney, spleen) were also collected for histopathological examination [1]

AGX51 did not show significant acute toxicity in tumor-bearing mice; throughout the treatment period, the weight of the mice remained stable, with a weight loss of no more than 10% compared to the control group [1]
- Histopathological examination of the liver, kidneys and spleen of AGX51-treated mice revealed no significant tissue damage or abnormal lesions [1]
- No hematological toxicity (e.g., changes in white blood cell count and red blood cell count) was observed in AGX51-treated mice [1]

[1]. Anti-tumor effects of an ID antagonist with no observed acquired resistance. NPJ Breast Cancer. 2021 May 24;7(1):58.

AGX51 is a small molecule ID antagonist designed to target ID-bHLH protein interactions, which are key pathways driving breast cancer proliferation and survival[1]. Unlike conventional chemotherapy drugs and targeted therapies, AGX51 does not induce acquired resistance in breast cancer cells even with long-term exposure, which is attributed to its unique mechanism of targeting transcriptional regulatory pathways rather than a single oncogenic driver[1]. AGX51 exhibits selective antitumor activity against breast cancer cells with high expression of ID protein, while having minimal effect on normal breast epithelial cells[1]. The antitumor mechanisms of AGX51 include disrupting ID-bHLH dimerization, restoring bHLH transcription factor activity, inducing cell cycle arrest in the G1 phase, and promoting cancer cell apoptosis[1].

C27H29NO4

431.52346777916

429.23

C, 78.29; H, 7.27; N, 3.26; O, 11.17

330834-54-3

2920302

Light yellow to yellow solid powder

5.2

0

4

9

32

578

0

CCC(=O)N(CCC(C1=CC2=C(C=C1)OCO2)C3=CC=CC=C3OC)CC4=CC=CC=C4

SRADCMOCDMFMPS-UHFFFAOYSA-N

InChI=1S/C27H29NO4/c1-3-27(29)28(18-20-9-5-4-6-10-20)16-15-22(23-11-7-8-12-24(23)30-2)21-13-14-25-26(17-21)32-19-31-25/h4-14,17,22H,3,15-16,18-19H2,1-2H3

N-[3-(1,3-benzodioxol-5-yl)-3-(2-methoxyphenyl)propyl]-N-benzylpropanamide

AGX-51; AGX51; AGX 51

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)

DMSO: ~100 mg/mL (~231.7 mM)
Ethanol: ~100 mg/mL (~231.7 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.82 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 20.8 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: 2.08 mg/mL (4.82 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 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.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (4.82 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly..

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Solubility in Formulation 4: 5%DMSO+ 40%PEG300+ 5%Tween 80: 5.0mg/ml (11.59mM)

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