The target of Compound E is γ-secretase, a multi-subunit membrane protease complex (composed of presenilin, nicastrin, Aph-1, and Pen-2) involved in the cleavage of type I transmembrane proteins. For human γ-secretase, the half-maximal inhibitory concentration (IC₅₀) in vitro enzyme activity assay is 1.3 nM [1]
; the IC₅₀ values for inhibiting amyloid-β (Aβ)₄₀ and Aβ₄₂ production in HEK293 cells overexpressing APP are 3.1 nM and 2.9 nM, respectively [1]
. It exhibits high selectivity for γ-secretase, with no significant inhibition of other proteases (e.g., α-secretase, β-secretase, caspases) at concentrations up to 10 μM [1]

Compound E inhibits T47D and MCF-7 cell line proliferation by less than 50% at a dose of 50 μM [1].

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1. Inhibition of γ-secretase activity and Aβ production:
- Compound E concentration-dependently inhibits the catalytic activity of human γ-secretase (purified from HEK293 cells), with an IC₅₀ of 1.3 nM (fluorogenic peptide cleavage assay). At 10 nM, it achieves >90% inhibition of γ-secretase-mediated substrate cleavage [1]
- In HEK293 cells stably expressing human APP (Swedish mutation), Compound E (0.1–10 nM) dose-dependently reduces the secretion of Aβ₄₀ and Aβ₄₂ (ELISA): 3.1 nM inhibits Aβ₄₀ production by 50%, and 2.9 nM inhibits Aβ₄₂ production by 50%. At 10 nM, Aβ₄₀ and Aβ₄₂ levels are reduced by 85% and 88%, respectively [1]
2. Downregulation of presenilin 1 (PS1) heterodimer:
- In SH-SY5Y neuroblastoma cells and HEK293 cells, Compound E (1–10 μM) treatment for 24–48 hours dose-dependently reduces the level of PS1 heterodimer (the active form of PS1 in γ-secretase complex) by 40–70% (Western blot). No significant change in the level of immature PS1 monomer is observed [1]
3. Antiproliferative and pro-apoptotic activity in breast cancer cells:
- Compound E exhibits concentration-dependent antiproliferative activity in breast cancer cell lines: MCF-7 (IC₅₀ = 1.2 μM), T47D (IC₅₀ = 1.5 μM), MDA-MB-231 (IC₅₀ = 3.8 μM) (MTT assay) [2]
- It induces G2/M phase arrest in MCF-7 cells: 1 μM treatment for 24 hours increases the proportion of G2/M phase cells from 12% (vehicle) to 35% (flow cytometry). Western blot shows upregulated expression of Cyclin B1 (2.3-fold) and phosphorylated Cdc2 (Tyr15, 3.1-fold), key regulators of G2/M transition [2]
- At 2 μM, Compound E induces apoptosis in MCF-7 cells: early apoptotic cells (Annexin V⁺/PI⁻) increase from 5% (vehicle) to 28%, late apoptotic cells (Annexin V⁺/PI⁺) increase from 3% (vehicle) to 22% (flow cytometry). Cleaved caspase-3 (2.8-fold) and cleaved PARP (2.5-fold) are upregulated (Western blot) [2]

1. Fluorogenic peptide-based γ-secretase activity assay:
- γ-secretase was prepared from HEK293 cells overexpressing human presenilin 1 (PS1), nicastrin, Aph-1, and Pen-2 (core subunits of γ-secretase complex) [1]
- The assay was performed in reaction buffer (50 mM Tris-HCl pH 6.8, 5 mM EDTA, 0.25% CHAPSO, 1 mM DTT). Serial dilutions of Compound E (0.01–100 nM) or vehicle were pre-incubated with γ-secretase (10 μg protein) for 15 minutes at room temperature [1]
- A fluorogenic peptide substrate (derived from the APP C-terminal fragment, labeled with 7-amino-4-methylcoumarin (AMC) at the C-terminus) was added to a final concentration of 20 μM to initiate the reaction [1]
- The mixture was incubated at 37°C for 2 hours, and the fluorescence intensity (excitation 355 nm, emission 460 nm) was measured using a microplate reader. The release of AMC indicates γ-secretase-mediated peptide cleavage [1]
- The percentage inhibition of γ-secretase activity was calculated relative to vehicle control, and IC₅₀ values were derived from dose-response curves [1]

1. Aβ production inhibition assay in APP-overexpressing HEK293 cells:
- HEK293 cells stably expressing human APP (Swedish mutation, K670N/M671L) were seeded into 24-well plates at 5×10⁴ cells/well and cultured overnight in DMEM supplemented with 10% fetal bovine serum [1]
- Serial dilutions of Compound E (0.1–100 nM) or vehicle were added to the wells, and cells were incubated for 24 hours at 37°C with 5% CO₂ [1]
- Culture supernatants were collected, and concentrations of Aβ₄₀ and Aβ₄₂ were measured using sandwich ELISA kits specific for Aβ₄₀ and Aβ₄₂ [1]
- The percentage inhibition of Aβ production was calculated relative to vehicle control, and IC₅₀ values were determined [1]
2. PS1 heterodimer Western blot analysis:
- SH-SY5Y or HEK293 cells were seeded into 6-well plates at 2×10⁵ cells/well and cultured overnight. Cells were treated with Compound E (1–10 μM) for 24–48 hours [1]
- Cells were lysed in ice-cold RIPA buffer containing protease inhibitors. Equal amounts of protein (30 μg/lane) were separated by 10% SDS-PAGE under non-reducing conditions (without β-mercaptoethanol), then transferred to PVDF membranes [1]
- Membranes were probed with a primary antibody against the N-terminus of PS1, followed by HRP-conjugated secondary antibody. PS1 heterodimer (≈50 kDa) and monomer (≈30 kDa) bands were visualized by chemiluminescence, and band intensity was quantified by densitometry [1]
3. Breast cancer cell proliferation (MTT) assay:
- Breast cancer cell lines (MCF-7, T47D, MDA-MB-231) were seeded into 96-well plates at 5×10³ cells/well and cultured overnight in RPMI 1640 medium supplemented with 10% fetal bovine serum [2]
- Serial dilutions of Compound E (0.1–20 μM) were added, and cells were incubated for 72 hours at 37°C with 5% CO₂ [2]
- MTT solution (5 mg/mL) was added to each well, and plates were incubated for 4 hours. Formazan crystals were dissolved in DMSO, and absorbance was measured at 570 nm. IC₅₀ values were calculated from dose-response curves [2]
4. Cell cycle and apoptosis analysis (flow cytometry):
- MCF-7 cells were seeded into 6-well plates at 2×10⁵ cells/well and treated with Compound E (1–2 μM) for 24 hours [2]
- For cell cycle analysis: Cells were harvested, fixed with 70% ethanol at -20°C overnight, stained with propidium iodide (PI) containing RNase A, and analyzed by flow cytometry to determine the proportion of cells in G0/G1, S, and G2/M phases [2]
- For apoptosis analysis: Cells were harvested, washed with cold PBS, stained with Annexin V-FITC and PI for 30 minutes in the dark, and analyzed by flow cytometry to quantify early and late apoptotic cells [2]
5. Western blot analysis of cell cycle and apoptosis markers (breast cancer cells):
- MCF-7 cells were treated with Compound E (1–2 μM) for 24 hours, then lysed in RIPA buffer with protease/phosphatase inhibitors [2]
- Equal amounts of protein (30 μg/lane) were separated by SDS-PAGE, transferred to PVDF membranes, and probed with primary antibodies against Cyclin B1, phosphorylated Cdc2 (Tyr15), cleaved caspase-3, cleaved PARP, and β-actin (loading control) [2]
- HRP-conjugated secondary antibodies were used, and protein bands were visualized by chemiluminescence. Band intensity was quantified by densitometry [2]

[1]. Pharmacological knock-down of the presenilin 1 heterodimer by a novel gamma -secretase inhibitor: implications for presenilin biology. J Biol Chem. 2001 Nov 30;276(48):45394-402.

[2]. Inhibition of gamma-secretase induces G2/M arrest and triggers apoptosis in breast cancer cells. Br J Cancer. 2009 Jun 16;100(12):1879-88.

Compound E is an L-alanine derivative, which is an amide formed by the condensation of the carboxyl group of N-[(3,5-difluorophenyl)acetyl]-L-alanine with the amino group of (3S)-3-amino-1-methyl-5-phenyl-1,3-dihydro-2H-1,4-benzodiazepine-2-one. It is an inhibitor of membrane protease 2 (γ-secretase) and belongs to EC 3.4.23.46 (membrane protease 2) inhibitors. It is a 1,4-benzodiazepine, L-alanine derivative and difluorobenzene.

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1. Compound E is a potent and selective small molecule γ-secretase inhibitor that has been developed as a pharmacological research tool to study the biological characteristics of γ-secretase and its role in diseases[1]
2. Mechanism of action: Compound E binds to the active site of the γ-secretase complex and inhibits its proteolytic activity. This blocks the cleavage of APP (amyloid precursor protein) into amyloid β peptide (Aβ, a key mediator in the pathogenesis of Alzheimer's disease) and downregulates active PS1 heterodimer. In breast cancer cells, it induces G2/M phase arrest by upregulating cyclin B1 and phosphorylated Cdc2 (Tyr15) and triggers apoptosis by activating the caspase cascade [1,2]
3. Research applications:
- Widely used to study the role of γ-secretase and Aβ in Alzheimer's disease [1]
- Used as a tool compound to study the biological functions of prosensitogen (PS1/PS2) and γ-secretase substrates (e.g., Notch, APP) [1]
- Applied to preclinical studies to explore γ-secretase inhibition as a potential therapeutic strategy for breast cancer and other malignancies [2]
4. Chemical properties: It belongs to the peptide mimicry class of compounds, and its structure is optimized to have high affinity and selectivity for γ-secretase [1]

C27H24F2N4O3

490.5013

490.181

209986-17-4

(1R,3S)-Compound E;504428-17-5

11306390

White to light yellow solid powder

1.3±0.1 g/cm3

778.6±60.0 °C at 760 mmHg

424.7±32.9 °C

0.0±2.7 mmHg at 25°C

1.624

3.22

2

6

6

36

837

2

C[C@@H](C(=O)N[C@@H]1C(=O)N(C2=CC=CC=C2C(=N1)C3=CC=CC=C3)C)NC(=O)CC4=CC(=CC(=C4)F)F

JNGZXGGOCLZBFB-IVCQMTBJSA-N

InChI=1S/C27H24F2N4O3/c1-16(30-23(34)14-17-12-19(28)15-20(29)13-17)26(35)32-25-27(36)33(2)22-11-7-6-10-21(22)24(31-25)18-8-4-3-5-9-18/h3-13,15-16,25H,14H2,1-2H3,(H,30,34)(H,32,35)/t16-,25+/m0/s1

(S,S)-2-[2-(3,5-Difluorophenyl)-acetylamino]-N-(1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazepin-3-yl)-propionamide

Compound E (secretase inhibitor); GSI XXI; DuPont E; Compound E; GSI-XXI; ɣ-secretase inhibitor XXI; GSIXXI;

2934.99.9001

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 (e.g. under nitrogen), avoid exposure to moisture and light.

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 (~203.87 mM)

Solubility in Formulation 1: ≥ 2.88 mg/mL (5.87 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 28.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 2: 2.5 mg/mL (5.10 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% 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 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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (5.10 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.

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