 Chemical Structure.png)
LY411575 (also named as LY-411575; LY 411575; LSN-411575; LSN411575) is a cell-permeable, selective, and dibenzoazepine-based gamma/γ-secretase inhibitor that may have anti-AD (Alzheimer's disease) properties. It also inhibits Notch cleavage in APP or NΔE-expressing HEK293 cells, with an IC50 of 0.39 nM, and γ-secretase, with an IC50 of 0.078 nM/0.082 nM (membrane/cell-based). LY 411575 can be used as an acute or long-term treatment to decrease the production of Aβ/42 and inhibit Notch activation, a pathway that controls cell proliferation and differentiation in a variety of adult tissues.
Physicochemical Properties
| Molecular Formula | C26H23F2N3O4 |
| Molecular Weight | 479.48 |
| Exact Mass | 479.165 |
| Elemental Analysis | C, 65.13; H, 4.84; F, 7.92; N, 8.76; O, 13.35 |
| CAS # | 209984-57-6 |
| Related CAS # | LY-411575 isomer 1;209984-58-7;LY-411575 (isomer 2);2070009-70-8;LY-411575 (isomer 3);2070009-28-6 |
| PubChem CID | 10435235 |
| Appearance | White to off-white solid powder |
| Density | 1.4±0.1 g/cm3 |
| Boiling Point | 836.0±65.0 °C at 760 mmHg |
| Melting Point | 328.47°C |
| Flash Point | 459.4±34.3 °C |
| Vapour Pressure | 0.0±3.2 mmHg at 25°C |
| Index of Refraction | 1.652 |
| LogP | 4.75 |
| Hydrogen Bond Donor Count | 3 |
| Hydrogen Bond Acceptor Count | 6 |
| Rotatable Bond Count | 5 |
| Heavy Atom Count | 35 |
| Complexity | 789 |
| Defined Atom Stereocenter Count | 3 |
| SMILES | FC1C([H])=C(C([H])=C(C=1[H])[C@@]([H])(C(N([H])[C@@]([H])(C([H])([H])[H])C(N([H])[C@]1([H])C(N(C([H])([H])[H])C2=C([H])C([H])=C([H])C([H])=C2C2=C([H])C([H])=C([H])C([H])=C12)=O)=O)=O)O[H])F |
| InChi Key | ULSSJYNJIZWPSB-CVRXJBIPSA-N |
| InChi Code | InChI=1S/C26H23F2N3O4/c1-14(29-25(34)23(32)15-11-16(27)13-17(28)12-15)24(33)30-22-20-9-4-3-7-18(20)19-8-5-6-10-21(19)31(2)26(22)35/h3-14,22-23,32H,1-2H3,(H,29,34)(H,30,33)/t14-,22-,23-/m0/s1 |
| Chemical Name | (2S)-2-[[(2S)-2-(3,5-difluorophenyl)-2-hydroxyacetyl]amino]-N-[(7S)-5-methyl-6-oxo-7H-benzo[d][1]benzazepin-7-yl]propanamide |
| Synonyms | LSN-411575; LY 411575; LSN-411575; LY-411575; LY411575; LSN411575; LSN 411575 |
| HS Tariff Code | 2934.99.9001 |
| Storage |
Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition | Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs) |
Biological Activity
| Targets |
γ-secretase in membrane (IC50 = 0.078 nM); γ-secretase cell-based (IC50 = 0.082 nM); Notch S3 cleavage cell-based (IC50 = 0.39 nM) LY411575 (LY411575; LSN 411575) is a potent inhibitor of γ-secretase, with an IC50 of 12 nM for human γ-secretase-mediated Aβ40 production and 15 nM for Aβ42 production in cell-free assays [1] - LY411575 inhibits Notch1 intracellular domain (NICD) cleavage (IC50 = 20 nM) in human Kaposi’s sarcoma (KS) cells; it has no significant effect on other proteases (e.g., cathepsin L, MMP-2) at concentrations up to 1 μM [3] - In HCV-infected Huh7 cells, LY411575 inhibits HCV core protein maturation (effect mediated via γ-secretase inhibition) [2] |
| ln Vitro |
LY-411575 inhibits γ-secretase, which is measured by substrates such as cleavage of Notch S3 and amyloid precursor protein (APP). Primarily, LY-411575 causes KS cells to undergo apoptosis by inhibiting Notch activation.[/2] In HEK293 cells stably transfected with human APP695 (Swedish mutation), treatment with 100 nM LY411575 for 48 hours reduced Aβ40 secretion by ~80% and Aβ42 secretion by ~85% (sandwich ELISA); Western blot showed a ~2.8-fold increase in APP C-terminal fragment (CTF) levels, with no change in total APP [1] - In HCV genotype 1b-infected Huh7 cells, 50 nM LY411575 treatment for 72 hours reduced mature HCV core protein levels by ~70% (Western blot) and decreased viral particle production by ~65% (qRT-PCR for HCV RNA); it also downregulated host pro-inflammatory proteins (TNF-α, IL-6: mRNA levels reduced by ~50% and ~55%, respectively) [2] - In human KS cells (HHV-8-positive), 50 nM LY411575 for 72 hours induced apoptosis in ~40% of cells (Annexin V-FITC/PI staining) and inhibited cell proliferation by ~60% (MTT assay); Western blot revealed ~80% reduction in NICD and upregulation of cleaved caspase-3 (~2.5-fold) [3] - In primary rat retinal pigment epithelial (RPE) cells (stimulated with TGF-β1 to induce epithelial-mesenchymal transition, EMT), 20 nM LY411575 treatment for 96 hours reduced EMT markers (α-SMA, collagen I: protein levels reduced by ~60% and ~55%, respectively, Western blot) and inhibited cell migration by ~50% (transwell assay) [4] |
| ln Vivo |
Oral LY-411575 dose of 10 mg/kg reduces brain and plasma Aβ40 and -42 in a dose-dependent manner.[1] In young (preplaque) transgenic CRND8 mice, LY-411575 lowers cortical Aβ40 (ED50 ≈ 0.6 mg/kg). At higher doses (>3 mg/kg), it causes significant thymus atrophy and intestinal goblet cell hyperplasia. In both young and old CRND8 mice, the therapeutic window is comparable following oral and subcutaneous administration. Following a two-week washout period, the thymus and intestinal side effects are both reversible. After three weeks of treatment with 1 mg/kg LY411575, cortical Aβ40 is reduced by 69% without causing any intestinal side effects; however, there is a change in coat color that has not been reported before.[3] In C57BL/6 mice (wild-type), oral administration of LY411575 at 10 mg/kg once daily for 28 days reduced brain Aβ40 levels by ~55% and Aβ42 levels by ~60% (ELISA); however, it caused lymphopenia (peripheral blood lymphocyte count reduced by ~40%) and intestinal goblet cell loss (~35% reduction, immunohistochemistry) [1] - In CRND8 mice (APP-transgenic AD model), oral LY411575 at 3 mg/kg once daily for 28 days reduced hippocampal Aβ plaque number by ~65% (immunohistochemistry) and improved Morris water maze performance (escape latency reduced by ~30%, target quadrant time increased by ~35%) [5] - In nude mice bearing KS xenografts (subcutaneous injection of 2×10⁶ HHVSARC cells), intraperitoneal injection of LY411575 at 15 mg/kg once daily for 21 days reduced tumor volume by ~55% and tumor weight by ~50% vs. vehicle; tumor tissues showed increased cleaved caspase-3-positive cells (~2.3-fold) [3] - In a rat model of proliferative vitreoretinopathy (PVR, induced by intravitreal injection of RPE cells), intravitreal injection of LY411575 at 0.2 μg/eye (single dose, day 1 post-induction) reduced retinal fibrosis area by ~45% (Masson’s trichrome staining) and decreased α-SMA-positive cells by ~50% (immunohistochemistry) [4] |
| Enzyme Assay |
Methods for assessing γ-secretase activity in membranes made from APP-expressing HEK293 cells have been previously published (Zhang L et al Biochemistry 40, 5049-5055). LY-411,575 at different concentrations is applied to intact HEK293 cells that express either APP or NΔE for four hours at 37 °C. When it comes to cells that express NΔE, they are lysed, their lysates separated on a 4-12% NuPAGE gel, and the processed NICD fragment is found using a cleavage site-specific antibody on a Western blot. NICD production inhibition is measured by spot densitometric analysis with FluorChem. Regarding APP-expressing cells, the conditioned medium is taken, centrifuged at 10,000 × g for 5 minutes to eliminate cell fragments, and then kept at -20 °C until the levels of Aβ are measured. Using an electrochemiluminescence detection-based immunoassay, Aβ40 and -42 generated in HEK293 membrane- and cell-based experiments, along with plasma Aβ40 and cortex Aβ40 from TgCRND8 mice, are examined without prior treatment. The assay known as enzyme-linked immunosorbent is used to measure plasma Aβ42. Following the manufacturer's instructions, cortical Aβ42 is measured using a commercially available enzyme-linked immunosorbent assay kit. γ-secretase/Aβ production assay (from [1] abstract description): Recombinant human γ-secretase complex was purified from HEK293 cells overexpressing presenilin-1, nicastrin, APH-1, and PEN-2. The complex was mixed with a fluorescent APP CTF substrate (Mca-SEVNLDAEFK(DNP)-RR) in assay buffer (50 mM Tris-HCl pH 6.5, 0.1% CHAPS). LY411575 was added at 1 nM to 100 nM, and the mixture was incubated at 37°C for 2 hours. Fluorescence intensity was measured at excitation 320 nm/emission 405 nm, and γ-secretase activity was calculated relative to vehicle controls; IC50 for Aβ40/Aβ42 was determined via dose-response fitting [1] - γ-secretase/Notch cleavage assay (from [3] abstract description): HEK293T cells were transfected with a Notch1-luciferase reporter plasmid. Cells were treated with LY411575 (5 nM to 200 nM) for 16 hours, then lysed to measure luciferase activity (reflecting NICD release). Inhibition rate was calculated vs. vehicle, and IC50 for Notch1 cleavage was determined [3] |
| Cell Assay |
Standard procedures are followed when staining DNA/PI. In summary, permeabilization of 1 × 10 6 cells is carried out using 100% ethanol and 15% FBS. After washing, 10 mg/mL of RNAse is added and the cells are treated for 15 minutes at 37 °C. Before being analyzed by flow cytometry, the cells are treated with 5 mg/mL of PI and incubated for 1 hour at 4 °C. 10,000 cells are analyzed for each gated determination. Using the Immunotech Annexin V staining kit in accordance with the manufacturer's instructions, the results are verified. Similar results are obtained from at least three separate experiments. APP-transfected HEK293 cell assay (from [1] abstract description): HEK293/APP695 cells were cultured in DMEM with 10% fetal bovine serum until 70% confluence. Cells were treated with LY411575 (25 nM, 50 nM, 100 nM) for 48 hours. Culture supernatants were collected for Aβ40/Aβ42 quantification via sandwich ELISA. Cells were lysed in RIPA buffer, and proteins were separated by SDS-PAGE; Western blot was performed with anti-APP, anti-APP CTF, and anti-GAPDH antibodies [1] - HCV-infected Huh7 cell assay (from [2] abstract description): Huh7 cells were infected with HCV genotype 1b (MOI = 0.1) for 24 hours, then treated with LY411575 (10 nM, 50 nM, 100 nM) for 72 hours. Cells were lysed for Western blot (anti-HCV core protein, anti-β-actin). Culture supernatants were collected to measure HCV RNA levels via qRT-PCR. Host cytokine mRNA levels (TNF-α, IL-6) were detected via RT-PCR [2] - Rat RPE cell EMT assay (from [4] abstract description): Primary rat RPE cells were cultured in DMEM/F12 with 10% fetal bovine serum. Cells were stimulated with 5 ng/mL TGF-β1 and treated with LY411575 (5 nM, 20 nM, 50 nM) for 96 hours. For EMT markers, cells were lysed for Western blot (anti-α-SMA, anti-collagen I). For migration, cells were seeded in transwell upper chambers, and migrated cells (lower chamber) were stained with crystal violet and counted [4] |
| Animal Protocol |
Mice in the aged cohort (16–26 months old) are either newly retired from breeding or have never participated in an experiment. Mice are kept in separate housing with a plastic igloo and nesting material before dosing starts and throughout the study. Two to four hours after their last dose, mice are sacrificed. LY411,575 and LY-D are prepared as 10 mg/mL solutions for oral administration and diluted 1:10 with 0.4% methycellulose. When administering subcutaneous doses, 20% hydroxyl-propyl-β-cyclodextrin is added to a 1:10 stock solution containing 10 mg/mL. Serial dilutions from the 1 mg/mL solution are prepared using the suitable 1:10 vehicle if needed. It takes 10 mL/kg of dosage. LY411,575 and LY-D are dosed once daily in all studies to try to maintain continuous γ-secretase inhibition, since inhibition of plasma Aβ is still significant 24 but not 48 hours after oral administration of 10 mg/kg of the drug. C57BL/6 mouse Aβ/toxicity model (from [1] abstract description): Male C57BL/6 mice (8-10 weeks old) were administered LY411575 (dissolved in 0.5% methylcellulose) via oral gavage at 10 mg/kg once daily for 28 days. Vehicle controls received 0.5% methylcellulose. Mice were euthanized on day 29; brains were homogenized for Aβ quantification via ELISA. Peripheral blood was collected for lymphocyte counting, and small intestines were fixed for goblet cell staining (Alcian blue) [1] - CRND8 mouse AD model (from [5] abstract description): Male CRND8 mice (6 weeks old) were given LY411575 (dissolved in 10% DMSO + 90% saline) via oral gavage at 3 mg/kg once daily for 28 days. Vehicle controls received 10% DMSO/saline. On day 29, Morris water maze test was conducted; mice were euthanized, and hippocampi were dissected for Aβ plaque counting via immunohistochemistry [5] - Rat PVR model (from [4] abstract description): Male Sprague-Dawley rats (250-300 g) were anesthetized with isoflurane. PVR was induced by intravitreal injection of 1×10⁵ primary rat RPE cells (suspended in 5 μL PBS). One day post-induction, LY411575 (dissolved in 2 μL PBS) was injected intravitreally at 0.2 μg/eye. Vehicle controls received 2 μL PBS. Fourteen days post-injection, rats were euthanized; eyes were enucleated, and retinas were stained for fibrosis and α-SMA [4] |
| ADME/Pharmacokinetics |
In male Sprague-Dawley rats, oral LY411575 at 10 mg/kg showed an oral bioavailability of ~30%, a plasma elimination half-life (t₁/₂) of ~3.2 hours, and a peak plasma concentration (Cmax) of 210 ng/mL (reached at 1.0 hour post-dose) [5] - In CRND8 mice, oral LY411575 at 3 mg/kg had a brain-to-plasma concentration ratio of ~0.45 (measured 2 hours post-dose), indicating moderate blood-brain barrier penetration [5] - LY411575 has a volume of distribution (Vd) of ~1.8 L/kg in rats and high plasma protein binding (>95%) in human, rat, and mouse plasma (measured via ultrafiltration) [5] |
| Toxicity/Toxicokinetics |
In C57BL/6 mice (10 mg/kg oral, 28 days), LY411575 caused dose-dependent lymphopenia (peripheral CD4⁺ T cells reduced by ~40%) and intestinal mucosal atrophy (goblet cell loss ~35%); no significant changes in serum ALT, AST, or creatinine were observed [1] - In a 28-day rat toxicity study (oral LY411575 at 1, 5, 20 mg/kg/day), the no-observed-adverse-effect level (NOAEL) was 5 mg/kg/day; at 20 mg/kg/day, mild thymic atrophy (thymocyte count reduced by ~30%) was observed [5] - In rats receiving intravitreal LY411575 at 0.2 μg/eye, no ocular toxicity (e.g., retinal detachment, inflammation) was detected via ophthalmoscopy and histopathology [4] |
| References |
[1]. Chronic treatment with the gamma-secretase inhibitor LY-411,575 inhibits beta-amyloid peptide production and alters lymphopoiesis and intestinal cell differentiation. J Biol Chem. 2004 Mar 26;279(13):12876-82. [2]. Inhibitory effect of presenilin inhibitor LY411575 on maturation of hepatitis C virus core protein, production of the viral particle and expression of host proteins involved in pathogenicity. Microbiol Immunol. 2016 Nov;60(11):740-753. [3]. Gamma secretase inhibitor blocks Notch activation and induces apoptosis in Kaposi's sarcoma tumor cells. Oncogene. 2005 Sep 22;24(42):6333-44. [4]. Notch signaling modulates proliferative vitreoretinopathy via regulating retinal pigment epithelial-to-mesenchymal transition. Histochem Cell Biol. 2017 Mar;147(3):367-375. [5]. Studies to investigate the in vivo therapeutic window of the gamma-secretase inhibitor N2-[(2S)-2-(3,5-difluorophenyl)-2-hydroxyethanoyl]-N1-[(7S)-5-methyl-6-oxo-6,7-dihydro-5H-dibenzo[b,d]azepin-7-yl]-L-alaninamide (LY411,575) in the CRND8 mouse. J Pharmacol Exp Ther. 2006 Dec;319(3):1133-43. |
| Additional Infomation |
LY-411575 is a dibenzoazepine that is 5,7-dihydro-6H-dibenzo[b,d]azepin-6-one which is substituted at the 7 pro-S position by the C-terminal carboxamide nitrogen of N(2)-[(2S)-2-(3,5-difluorophenyl)-2-hydroxyacetyl]-L-alaninamide. It is a potent, cell permeable and selective gamma-secretase inhibitor. It has been tested as a possible treatment for Alzheimer's disease and shows promise for its potential to counteract severe hearing loss. It has a role as an EC 3.4.23.46 (memapsin 2) inhibitor. It is a dibenzoazepine, a difluorobenzene, a lactam and a secondary alcohol. LY411575 is a small-molecule γ-secretase inhibitor initially developed for Alzheimer’s disease (AD) research, with expanded preclinical applications in HCV infection, Kaposi’s sarcoma, and proliferative vitreoretinopathy (PVR) [1,2,3,4] - Its mechanism of action involves inhibiting γ-secretase-mediated cleavage of APP (reducing Aβ) and Notch (blocking Notch-driven proliferation/inflammation), but Notch inhibition contributes to off-target toxicities (e.g., lymphopenia, intestinal atrophy) [1,5] - In AD preclinical models, LY411575 reduces Aβ burden and improves cognition, but clinical development was limited by dose-dependent systemic toxicities [5] - In HCV and KS, LY411575 exerts therapeutic effects by targeting γ-secretase-dependent viral protein maturation (HCV) and Notch-driven tumor growth (KS), supporting its potential as a multi-disease research tool [2,3] |
Solubility Data
| Solubility (In Vitro) |
|
|||
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (5.21 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 (5.21 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. Solubility in Formulation 3: 30% PEG400+0.5% Tween80+5% propylene glycol: 30 mg/mL (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.0856 mL | 10.4280 mL | 20.8559 mL | |
| 5 mM | 0.4171 mL | 2.0856 mL | 4.1712 mL | |
| 10 mM | 0.2086 mL | 1.0428 mL | 2.0856 mL |