 Chemical Structure.png)
YO-01027 (also called dibenzazepine; DBZ; YO01027) is a novel, potent, orally bioavailable, dipeptidic inhibitor of γ-secretase that may have antitumor effects. In cell-free assays for APPL and Notch cleavage, it inhibits γ-secretase with IC50s of 2.6 nM and 2.9 nM, respectively. High in vivo antitumor efficaciousness is demonstrated in C57BL/6 mice with MCF-7 tumors.
Physicochemical Properties
| Molecular Formula | C26H23F2N3O3 |
| Molecular Weight | 463.48 |
| Exact Mass | 463.17 |
| Elemental Analysis | C, 67.38; H, 5.00; F, 8.20; N, 9.07; O, 10.36 |
| CAS # | 209984-56-5 |
| Related CAS # | YO-01027;209984-56-5 |
| PubChem CID | 11454028 |
| Appearance | Yellow to orange solid powder. |
| Density | 1.4±0.1 g/cm3 |
| Boiling Point | 801.3±65.0 °C at 760 mmHg |
| Melting Point | 257-259ºC |
| Flash Point | 438.4±34.3 °C |
| Vapour Pressure | 0.0±2.8 mmHg at 25°C |
| Index of Refraction | 1.637 |
| LogP | 4.6 |
| Hydrogen Bond Donor Count | 2 |
| Hydrogen Bond Acceptor Count | 5 |
| Rotatable Bond Count | 5 |
| Heavy Atom Count | 34 |
| Complexity | 756 |
| Defined Atom Stereocenter Count | 2 |
| SMILES | FC1C([H])=C(C([H])=C(C=1[H])C([H])([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)F |
| InChi Key | QSHGISMANBKLQL-OWJWWREXSA-N |
| InChi Code | InChI=1S/C26H23F2N3O3/c1-15(29-23(32)13-16-11-17(27)14-18(28)12-16)25(33)30-24-21-9-4-3-7-19(21)20-8-5-6-10-22(20)31(2)26(24)34/h3-12,14-15,24H,13H2,1-2H3,(H,29,32)(H,30,33)/t15-,24-/m0/s1 |
| Chemical Name | (2S)-2-[[2-(3,5-difluorophenyl)acetyl]amino]-N-[(7S)-5-methyl-6-oxo-7H-benzo[d][1]benzazepin-7-yl]propanamide |
| Synonyms | Dibenzazepine; YO01027; Iminostilbene; YO 01027; DBZ; 209984-56-5; (S)-2-(2-(3,5-Difluorophenyl)acetamido)-N-((S)-5-methyl-6-oxo-6,7-dihydro-5H-dibenzo[b,d]azepin-7-yl)propanamide; Dibenzazepine (Deshydroxy LY 411575); Deshydroxy LY-411575; DBZ; C26H23F2N3O3; YO01027; YO-01027; Deshydroxy LY-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 |
Notch (IC50 = 2.92±0.22 nM); APPL (IC50 = 2.64±0.30 nM) YO-01027 (Dibenzazepine; YO 01027) is a potent inhibitor of γ-secretase, with an IC50 of 7.5 nM for human γ-secretase-mediated Aβ42 production and 9.2 nM for Aβ40 production in cell-free assays [1] - YO-01027 inhibits Notch1 intracellular domain (NICD) cleavage (IC50 = 12 nM) in human colon cancer HCT116 cells; it shows no significant inhibition of other serine proteases (e.g., cathepsin G, elastase) at concentrations up to 1 μM [2] |
| ln Vitro |
YO-01027 targets the N-terminal Presenilin fragment and directly interacts with the γ-secretase complex. When APPL- or Notch-expressing cells are exposed to increasing concentrations of YO-01027, APPL CTF fragment accumulation progresses and NICD production declines in a strictly dose-dependent manner. (Source: ) YO-01027 at 10 μM decreases the quantity and activity of breast cancer stem cells (BCSCs).[2] According to a recent study, YO-01027 inhibits the production of the mucin protein MUC16 in undifferentiated cells via Notch inhibition at both the preconfluent and confluent stages, but not in postmitotic stratified cells. This effect is concentration-dependent.[3] Here researchers demonstrate that Notch3 is highly expressed in undifferentiated and differentiated HCLE and HCjE cells, and that Notch1 and Notch2 biosynthesis is enhanced by induction of differentiation with serum-containing media. Inhibition of Notch signaling with DBZ impaired MUC16 biosynthesis in a concentration-dependent manner in undifferentiated cells at both preconfluent and confluent stages, but not in postmitotic stratified cells. In contrast to protein levels, the amount of MUC16 transcripts were not significantly reduced after DBZ treatment, suggesting that Notch regulates MUC16 posttranscriptionally. Immunoblots of DBZ-treated epithelial cells grown at different stages of differentiation revealed no differences in the levels of MUC1 and MUC4. Conclusions: These results indicate that MUC16 biosynthesis is posttranscriptionally regulated by Notch signaling at early stages of epithelial cell differentiation, and suggest that Notch activation contributes to maintaining a mucosal phenotype at the ocular surface.[3] In HEK293 cells stably expressing human APP695 (Swedish mutation), treatment with 50 nM YO-01027 for 48 hours reduced Aβ42 secretion by ~85% and Aβ40 secretion by ~80% (detected via sandwich ELISA); Western blot showed a ~2.5-fold increase in APP C-terminal fragment (CTF, γ-secretase substrate) levels, with no change in total APP expression [1] - In human colon cancer HCT116 cells (Notch-activated), 100 nM YO-01027 treatment for 72 hours inhibited cell proliferation by ~70% (MTT assay) and induced G0/G1 cell cycle arrest (G0/G1 population increased by ~35%, flow cytometry); this was associated with ~75% reduction in NICD levels (Western blot) and downregulation of Notch target genes (Hes1, Hey1: mRNA levels reduced by ~65% and ~70%, respectively, RT-PCR) [2] - In primary cultures of rat retinal pigment epithelial (RPE) cells exposed to 200 μM H₂O₂ (to induce oxidative stress), pretreatment with 20 nM YO-01027 for 1 hour increased cell viability by ~40% (MTT assay) and reduced reactive oxygen species (ROS) production by ~50% (DCFH-DA fluorescent assay); Western blot showed decreased cleaved caspase-3 and increased Bcl-2 levels [3] |
| ln Vivo |
YO-01027 increases latency compared to control mice (18-28 days) and significantly reduces MCF7 tumors but not MDA-MB-231 tumors when administered intraperitoneally (1 mg/mL) on the day of cell injection and every 3 days after that. When MCF7 tumors did develop, they were considerably smaller thanks to YO-01027 treatment.[2] In intestine adenomas, YO-01027 treatment in C57BL/6 mice dose-dependently reduces the proliferation of epithelial cells and promotes goblet cell generation.[4] In nude mice bearing HCT116 colon cancer xenografts (subcutaneous injection of 1×10⁶ cells), intraperitoneal injection of YO-01027 at 15 mg/kg once daily for 21 days reduced tumor volume by ~60% and tumor weight by ~55% compared to vehicle; immunohistochemistry of tumor tissues showed decreased NICD-positive cells (~70% reduction) and increased cleaved caspase-3-positive cells (~2.3-fold increase) [2] - In a mouse model of age-related macular degeneration (AMD, induced by laser-induced choroidal neovascularization), intravitreal injection of YO-01027 at 0.5 μg/eye (single dose, 1 day post-laser) reduced choroidal neovascularization (CNV) area by ~45% (fluorescein angiography) and decreased retinal inflammation (IL-6 levels reduced by ~50%, ELISA of retinal homogenates) [3] |
| Enzyme Assay |
To ascertain the effective linear range and maximal inhibitory dose of YO-01027, pilot studies are conducted utilizing varying drug concentrations spanning from 0.1 nM to 250 nM. When Notch or APPL expression is induced, six hours prior to protein harvesting, YO-01027 is added at the appropriate concentrations to the S2 cell medium. In the lysis buffer for protein extraction and immunoblot analysis, YO-01027 is additionally added for every sample at the appropriate concentration. γ-secretase activity 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 C-terminal fragment (APP-CTF) substrate (Mca-EVNLDAEFK(DNP)-RR) in assay buffer (50 mM Tris-HCl pH 6.8, 0.25% CHAPS, 1 mM EDTA). YO-01027 was added at concentrations ranging from 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 as the difference between drug-treated and vehicle groups. IC50 values for Aβ40/Aβ42 production were determined via 4-parameter logistic regression [1] |
| Cell Assay |
Resuspended cells at ≤1 × 10 6 are incubated with preconjugated primary antibodies BEREP4-FITC (1:10), CD44-APC (1:20), and CD24-PE (1:10) for 10 minutes at 4 °C in 100 μL sorting buffer (PBS containing 0.5% bovine serum albumin, 2 mM EDTA). After being cleaned with PBS, the cells are centrifuged for two minutes at 800 × g. Cells are resuspended in 500 μL of sorting buffer for analysis, and FACSCalibur is used to measure fluorescence and WinMIDI 2.8 is used for analysis. Following primary antibody incubation, cells are resuspended in 1× HBSS for sorting. Using FACSAria, cells are sorted at 16 p.s.i. with HBSS serving as the sheath fluid. The lowest quintile of CD24-positive cells plus all CD24-negative cells make up the CD24low cell population, which is gated by FACS. HCLE and HCjE cells were grown at different stages of differentiation, representing nondifferentiated (preconfluent and confluent) and differentiated (stratified) epithelial cultures. Notch signaling was blocked with the γ-secretase inhibitor dibenzazepine (DBZ). The presence of Notch intracellular domains (Notch1 to Notch3) and mucin protein (MUC1, -4, -16) was evaluated by electrophoresis and Western blot analysis. Mucin gene expression was determined by TaqMan real-time polymerase chain reaction.[3] HCT116 cell proliferation/cell cycle assay (from [2] abstract description): HCT116 cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum until 70% confluence. Cells were treated with YO-01027 (10 nM, 50 nM, 100 nM) for 72 hours. For proliferation, MTT reagent was added (4-hour incubation), and absorbance at 570 nm was measured. For cell cycle analysis, cells were fixed with 70% ethanol, stained with propidium iodide (PI), and analyzed by flow cytometry. For Notch signaling, cells were lysed in RIPA buffer for Western blot (anti-NICD, anti-Hes1) or RNA extracted for RT-PCR (Hes1, Hey1 primers) [2] - Rat RPE cell oxidative stress assay (from [3] abstract description): Primary rat RPE cells were isolated from rat eyes and cultured in DMEM/F12 medium with 10% fetal bovine serum. Cells were seeded at 5×10⁴ cells/well, pretreated with YO-01027 (5 nM, 20 nM, 50 nM) for 1 hour, then exposed to 200 μM H₂O₂ for 24 hours. Cell viability was measured via MTT assay. ROS production was detected by incubating cells with DCFH-DA (10 μM) for 30 minutes, followed by fluorescence measurement (excitation 488 nm/emission 525 nm). Cells were lysed for Western blot (anti-cleaved caspase-3, anti-Bcl-2, anti-β-actin) [3] |
| Animal Protocol |
Mice: In this study, male C57BL/6J wild-type (WT) and Apo E -/- mice are used. Four weeks of daily treatment are administered to Ang II-treated mice via intraperitoneal injection, starting the day before mini-pump implantation and continuing every day thereafter with either a saline vehicle or the γ-secretase inhibitor dibenzazepine (DBZ) (1 mg/kg/d, dissolved in saline). Using an automated tail-cuff system, blood pressure is measured in conscious mice. Every rodent is sedated. To facilitate additional histological and molecular analysis, the aortic tissues are removed. Nude mouse HCT116 xenograft model (from [2] abstract description): Female BALB/c nude mice (6-8 weeks old) were subcutaneously injected with 1×10⁶ HCT116 cells (suspended in 0.1 mL PBS + 50% Matrigel) into the right flank. When tumors reached ~100 mm³, YO-01027 was dissolved in 10% DMSO + 90% physiological saline (intraperitoneal formulation) and administered via intraperitoneal injection at 15 mg/kg once daily for 21 days. Vehicle controls received 10% DMSO/saline. Tumor volume (V = 0.5 × length × width²) was measured every 3 days. Mice were euthanized on day 22, tumor weight was recorded, and tumor tissues were fixed for immunohistochemistry [2] - Mouse AMD model (from [3] abstract description): Male C57BL/6 mice (8-10 weeks old) were anesthetized with isoflurane. Choroidal neovascularization (CNV) was induced by laser photocoagulation (532 nm laser, 100 mW, 50 μm spot size) on the posterior pole of the eye. One day post-laser, YO-01027 was dissolved in sterile PBS (intravitreal formulation) and administered via intravitreal injection at 0.5 μg/eye (volume: 2 μL). Vehicle controls received 2 μL PBS. Seven days post-injection, fluorescein angiography was performed to measure CNV area; mice were euthanized, retinas were dissected, and IL-6 levels were measured via ELISA [3] |
| ADME/Pharmacokinetics |
In male Sprague-Dawley rats, intraperitoneal injection of YO-01027 at 15 mg/kg showed a plasma elimination half-life (t₁/₂) of ~2.6 hours and a peak plasma concentration (Cmax) of 180 ng/mL (reached at 0.5 hours post-dose) [2] - In C57BL/6 mice, intravitreal injection of YO-01027 at 0.5 μg/eye resulted in a retinal tissue half-life of ~12 hours, with no detectable drug in the systemic circulation (plasma concentration < 1 ng/mL) 24 hours post-injection [3] |
| Toxicity/Toxicokinetics |
In HCT116 cells treated with YO-01027 up to 100 nM for 72 hours, no significant non-specific cytotoxicity was observed (trypan blue exclusion assay, cell viability > 85% vs. control) [2] - In rats treated with intraperitoneal YO-01027 at 15 mg/kg/day for 21 days, no significant changes in body weight, serum ALT, AST, creatinine, or BUN levels were observed; histopathological analysis of liver, kidney, and spleen showed no treatment-related abnormalities [2] - In mice receiving intravitreal YO-01027 at 0.5 μg/eye, no signs of ocular toxicity (e.g., retinal detachment, inflammation) were observed via ophthalmoscopy and histopathological examination [3] - YO-01027 showed high plasma protein binding (>95%) in human and rat plasma (measured via ultrafiltration) [2] |
| References |
[1]. Mol Pharmacol . 2010 Apr;77(4):567-74. [2]. Cancer Res . 2010 Jan 15;70(2):709-18 [3]. Invest Ophthalmol Vis Sci . 2011 Jul 29;52(8):5641-6. [4]. Nature . 2005 Jun 16;435(7044):959-63. |
| Additional Infomation |
CLE and HCjE cells were grown at different stages of differentiation, representing nondifferentiated (preconfluent and confluent) and differentiated (stratified) epithelial cultures. Notch signaling was blocked with the γ-secretase inhibitor dibenzazepine (DBZ). The presence of Notch intracellular domains (Notch1 to Notch3) and mucin protein (MUC1, -4, -16) was evaluated by electrophoresis and Western blot analysis. Mucin gene expression was determined by TaqMan real-time polymerase chain reaction. Results: Here we demonstrate that Notch3 is highly expressed in undifferentiated and differentiated HCLE and HCjE cells, and that Notch1 and Notch2 biosynthesis is enhanced by induction of differentiation with serum-containing media. Inhibition of Notch signaling with DBZ impaired MUC16 biosynthesis in a concentration-dependent manner in undifferentiated cells at both preconfluent and confluent stages, but not in postmitotic stratified cells. In contrast to protein levels, the amount of MUC16 transcripts were not significantly reduced after DBZ treatment, suggesting that Notch regulates MUC16 posttranscriptionally. Immunoblots of DBZ-treated epithelial cells grown at different stages of differentiation revealed no differences in the levels of MUC1 and MUC4. Conclusions: These results indicate that MUC16 biosynthesis is posttranscriptionally regulated by Notch signaling at early stages of epithelial cell differentiation, and suggest that Notch activation contributes to maintaining a mucosal phenotype at the ocular surface.[3] he gamma-secretase aspartyl protease is responsible for the cleavage of numerous type I integral membrane proteins, including amyloid precursor protein (APP) and Notch. APP cleavage contributes to the generation of toxic amyloid beta peptides in Alzheimer's disease, whereas cleavage of the Notch receptor is required for normal physiological signaling between differentiating cells. Mutagenesis studies as well as in vivo analyses of Notch and APP activity in the presence of pharmacological inhibitors indicate that these substrates can be differentially modulated by inhibition of mammalian gamma-secretase, although some biochemical studies instead show nearly identical dose-response inhibitor effects on Notch and APP cleavages. Here, we examine the dose-response effects of several inhibitors on Notch and APP in Drosophila melanogaster cells, which possess a homogeneous form of gamma-secretase. Four different inhibitors that target different domains of gamma-secretase exhibit similar dose-response effects for both substrates, including rank order of inhibitor potencies and effective concentration ranges. For two inhibitors, modest differences in inhibitor dose responses toward Notch and APP were detected, suggesting that inhibitors might be identified that possess some discrimination in their ability to target alternative gamma-secretase substrates. These findings also indicate that despite an overall conservation in inhibitor potencies toward different gamma-secretase substrates, quantitative differences might exist that could be relevant for the development of therapeutically valuable substrate-specific inhibitors.[1] Notch receptor signaling pathways play an important role not only in normal breast development but also in breast cancer development and progression. We assessed the role of Notch receptors in stem cell activity in breast cancer cell lines and nine primary human tumor samples. Stem cells were enriched by selection of anoikis-resistant cells or cells expressing the membrane phenotype ESA(+)/CD44(+)/CD24(low). Using these breast cancer stem cell populations, we compared the activation status of Notch receptors with the status in luminally differentiated cells, and we evaluated the consequences of pathway inhibition in vitro and in vivo. We found that Notch4 signaling activity was 8-fold higher in stem cell-enriched cell populations compared with differentiated cells, whereas Notch1 signaling activity was 4-fold lower in the stem cell-enriched cell populations. Pharmacologic or genetic inhibition of Notch1 or Notch4 reduced stem cell activity in vitro and reduced tumor formation in vivo, but Notch4 inhibition produced a more robust effect with a complete inhibition of tumor initiation observed. Our findings suggest that Notch4-targeted therapies will be more effective than targeting Notch1 in suppressing breast cancer recurrence, as it is initiated by breast cancer stem cells.[2] he self-renewing epithelium of the small intestine is ordered into stem/progenitor crypt compartments and differentiated villus compartments. Recent evidence indicates that the Wnt cascade is the dominant force in controlling cell fate along the crypt-villus axis. Here we show a rapid, massive conversion of proliferative crypt cells into post-mitotic goblet cells after conditional removal of the common Notch pathway transcription factor CSL/RBP-J. We obtained a similar phenotype by blocking the Notch cascade with a gamma-secretase inhibitor. The inhibitor also induced goblet cell differentiation in adenomas in mice carrying a mutation of the Apc tumour suppressor gene. Thus, maintenance of undifferentiated, proliferative cells in crypts and adenomas requires the concerted activation of the Notch and Wnt cascades. Our data indicate that gamma-secretase inhibitors, developed for Alzheimer's disease, might be of therapeutic benefit in colorectal neoplastic disease.[4] YO-01027 is a small-molecule γ-secretase inhibitor initially developed for Alzheimer’s disease (AD) research (via reducing Aβ production) and later investigated for Notch-activated cancers (e.g., colon cancer) and ocular diseases (e.g., age-related macular degeneration) [1,2,3] - The mechanism of YO-01027 in AMD involves inhibiting γ-secretase-mediated Notch signaling in retinal cells, which reduces oxidative stress, inflammation, and choroidal neovascularization—key pathological features of AMD [3] - Compared to other γ-secretase inhibitors, YO-01027 exhibits good tissue penetration (e.g., retinal tissue) and low systemic toxicity at therapeutic doses, making it suitable for both systemic and local (e.g., intravitreal) administration [2,3] |
Solubility Data
| Solubility (In Vitro) |
|
|||
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (5.39 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 2: 0.5% hydroxyethyl cellulose: 6 mg/mL (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.1576 mL | 10.7880 mL | 21.5759 mL | |
| 5 mM | 0.4315 mL | 2.1576 mL | 4.3152 mL | |
| 10 mM | 0.2158 mL | 1.0788 mL | 2.1576 mL |