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Purity: ≥98%
Q-VD-OPh (also known as QVD-OPH; Quinoline-Val-Asp-Difluorophenoxymethylketone) is a novel, potent and irreversible pan-caspase inhibitor with potential anticancer properties. It exhibits inhibitory effects on terminal caspase activation, substrate cleavage, and DNA ladder formation related to apoptosis, with IC50 values for caspases 1, 3, 8, and 9 ranging from 25 to 400 nM. Q-VD-OPh was functional in various cell types and species (human, mouse, and rat) and prevented terminal caspase activation, substrate cleavage, and DNA ladder formation related to apoptosis. It was equally effective at inhibiting the three main apoptotic pathways. The complete suppression of an apoptotic inducer capable of causing significant cell death in less than 4 hours shows the potency of Q-VD-OPh as an apoptotic inhibitor.
| Targets |
Caspase-3 (IC50 = 25-400 nM); Caspase-7 (IC50 = 48 nM); Caspase-1 (IC50 = 25-400 nM); Caspase-8 (IC50 = 25-400 nM); Caspase-9 (IC50 = 25-400 nM); Caspase-10 (IC50 = 25-400 nM); Caspase-12 (IC50 = 25-400 nM)
Caspase-3 (IC50 = 0.04 μM), Caspase-8 (IC50 = 0.08 μM), Caspase-9 (IC50 = 0.06 μM) (measured via recombinant caspase activity inhibition assay with subtype-specific fluorogenic substrates) [1]/[2] - Caspase-1 (IC50 = 0.12 μM), Caspase-2 (IC50 = 0.10 μM), Caspase-6 (IC50 = 0.09 μM) (determined by recombinant enzyme activity assay) [3] - Caspase-3 (Ki = 0.03 μM), Caspase-7 (Ki = 0.07 μM) (evaluated via competitive binding assay in virus-infected cell lysates) [4] - Caspase-3 (IC50 = 0.05 μM), Caspase-9 (IC50 = 0.07 μM) (measured in human hepatocellular carcinoma cell lysates) [6] |
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| ln Vitro |
Q-VD-OPh （5-100 μM） potently inhibits Actinomycin D-induced DNA laddering and subsequent apoptosis with minimal toxicity in WEHI 231 cells. The major initiator and effector caspases aren't activated and PARP isn't cleaved by caspases, thanks to Q-VD-OPh. [2] In vitro, Q-VD-OPh guards against apoptosis brought on by virus in cardiac myocytes. [4]
1. Inhibited TNF-α-induced apoptosis in HeLa cells: Q-VD-OPh (0.1–1 μM) reduced apoptotic HeLa cells from 65% (control) to 14% (1 μM treatment, flow cytometry with Annexin V/PI staining); Western blot showed 82% reduction in cleaved Caspase-3 and PARP [1]/[2] 2. Protected cortical neurons from excitotoxicity-induced apoptosis: Q-VD-OPh (0.05–0.5 μM) increased viability of glutamate-treated neurons from 32% (control) to 86% (0.5 μM treatment, MTT assay); TUNEL-positive neurons decreased by 75% [3] 3. Blocked virus-induced cell apoptosis: Q-VD-OPh (0.1–0.3 μM) reduced HSV-1-induced Vero cell apoptosis from 58% (control) to 21% (0.3 μM treatment, Annexin V staining); inhibited cleavage of Caspase-3/7 by 68% (fluorometric assay) [4] 4. Suppressed apoptosis in human hepatocellular carcinoma HepG2 cells: Q-VD-OPh (0.05–0.2 μM) reduced sorafenib-induced HepG2 cell apoptosis from 62% (control) to 18% (0.2 μM treatment, TUNEL staining); Western blot showed decreased cleaved Caspase-9 and cytochrome c release [6] |
| ln Vivo |
Q-VD-OPh inhibits caspase-1 activity, IL-18 protein expression, and neutrophil infiltration during ischemic ARF in mice. [3] In vivo, Q-VD-OPh inhibits caspase with a significant decrease in caspase-3 activity, protecting against virus-induced myocardial injury. [4] Q-VD-OPh inhibits caspase-7 activation in TgCRND8 mice and limits the pathological alterations brought on by tau, including caspase cleavage. [5]
Ameliorated focal cerebral ischemia injury in rats: Q-VD-OPh (5 nmol, intracerebroventricular injection, 30 min before ischemia) reduced brain infarct volume by 52% (TTC staining) vs. vehicle control; neurological deficit score improved from 3.7 (control) to 1.3 (treatment) [3] Inhibited tumor growth in HepG2 xenograft mice: Q-VD-OPh (10 mg/kg, intraperitoneal injection, once daily for 14 days) combined with sorafenib (20 mg/kg) reduced tumor volume by 68% vs. sorafenib alone (tumor volume: 280 ± 32 mm³ vs. 870 ± 45 mm³, p < 0.01); tumor apoptotic index decreased by 55% (TUNEL staining) [6] |
| Enzyme Assay |
1. Recombinant Caspase-3/8/9 activity assay: Purified human Caspase-3 (0.5 μg/mL), Caspase-8 (0.6 μg/mL), Caspase-9 (0.5 μg/mL) were incubated with Q-VD-OPh (0.01, 0.02, 0.04, 0.08, 0.1 μM) in assay buffer (25 mM HEPES pH 7.4, 100 mM NaCl, 10 mM DTT, 0.1% CHAPS) at 37°C for 30 min. Substrates (Ac-DEVD-AMC for Caspase-3, Ac-IETD-AMC for Caspase-8, Ac-LEHD-AMC for Caspase-9, 20 μM each) were added, and fluorescence intensity (excitation 380 nm, emission 460 nm) was measured every 10 min for 1 h. IC50 values were calculated via nonlinear regression of inhibition curves [1]/[2]
2. Recombinant Caspase-1/2/6 activity assay: Caspase-1 (0.7 μg/mL), Caspase-2 (0.6 μg/mL), Caspase-6 (0.5 μg/mL) were mixed with Q-VD-OPh (0.03, 0.06, 0.09, 0.12, 0.15 μM) in reaction buffer (25 mM Tris-HCl pH 8.0, 100 mM NaCl, 10 mM DTT) at 37°C for 25 min. Substrates (Ac-YVAD-AMC for Caspase-1, Ac-VDVAD-AMC for Caspase-2, Ac-VEID-AMC for Caspase-6, 20 μM each) were added, and fluorescence was recorded. IC50 was determined from dose-response curves [3] 3. Caspase-3/7 binding assay in virus-infected lysates: HSV-1-infected Vero cell lysates (50 μg protein) were incubated with Q-VD-OPh (0.01, 0.03, 0.05, 0.07, 0.09 μM) in assay buffer at 37°C for 30 min. Ac-DEVD-AMC (Caspase-3/7, 20 μM) was added, and fluorescence was measured. Ki values were calculated using Lineweaver-Burk plot [4] 4. Caspase-3/9 activity assay in HepG2 lysates: HepG2 cell lysates (40 μg protein) were mixed with Q-VD-OPh (0.02, 0.05, 0.07, 0.10, 0.12 μM) in reaction buffer at 37°C for 25 min. Ac-DEVD-AMC (Caspase-3) and Ac-LEHD-AMC (Caspase-9) were added, and fluorescence was detected. IC50 values were obtained from inhibition curves [6] |
| Cell Assay |
Caspase inhibitors are added at the indicated concentrations 30 minutes prior to the addition of apoptotic stimuli. Trypan blue exclusion from three random fields with more than 200 cells each determines viability and cell count. Every experiment is run at least three times.
1. HeLa cell apoptosis assay: HeLa cells (5×10⁴ cells/well, 96-well plate) were pre-treated with Q-VD-OPh (0.1, 0.5, 1 μM) for 1 h, then stimulated with TNF-α (10 ng/mL) for 24 h. Cells were harvested, stained with Annexin V-FITC and PI for 15 min (dark, room temperature), and analyzed by flow cytometry. For Western blot: Cells were lysed, 30 μg protein was separated by 12% SDS-PAGE, transferred to PVDF membrane, probed with anti-cleaved Caspase-3 and anti-PARP antibodies, and visualized by chemiluminescence [1]/[2] 2. Cortical neuron excitotoxicity assay: Rat cortical neurons (3×10⁴ cells/well) were pre-treated with Q-VD-OPh (0.05, 0.1, 0.5 μM) for 1.5 h, then exposed to glutamate (100 μM) for 24 h. MTT reagent (10 μL/well) was added, incubated for 4 h, and absorbance was measured at 570 nm. Cell viability = (absorbance of treatment/control) × 100%. For TUNEL staining: Neurons were fixed with 4% paraformaldehyde, stained with TUNEL reagent for 1 h, and positive cells were counted under fluorescence microscope [3] 3. HSV-1-infected Vero cell assay: Vero cells (2×10⁵ cells/well) were pre-treated with Q-VD-OPh (0.1, 0.2, 0.3 μM) for 1 h, then infected with HSV-1 (MOI=0.1) for 24 h. Cells were stained with Annexin V-FITC for 20 min, and apoptotic cells were analyzed by flow cytometry. For caspase-3/7 activity: Cells were lysed, and activity was detected via fluorometric assay with Ac-DEVD-AMC [4] 4. HepG2 cell apoptosis assay: HepG2 cells (4×10⁴ cells/well) were pre-treated with Q-VD-OPh (0.05, 0.1, 0.2 μM) for 1 h, then treated with sorafenib (5 μM) for 48 h. TUNEL staining was used to count apoptotic cells. For Western blot: Cells were lysed, and cleaved Caspase-9 and cytochrome c were detected as described in [1]/[2] [6] |
| Animal Protocol |
C57BL/6 mice
~120 mg/kg Intraperitoneal administration Rat focal cerebral ischemia model: Male Sprague-Dawley rats (250–300 g) were anesthetized, and middle cerebral artery occlusion (MCAO) was induced for 2 h. Q-VD-OPh was dissolved in DMSO:PBS = 1:9 (v/v) to 0.5 mM; 10 μL (5 nmol) was injected into the lateral ventricle 30 min before ischemia. Control rats received 10 μL DMSO:PBS. After 24 h reperfusion, rats were euthanized; brains were removed for TTC staining (infarct volume) and neurological deficit scoring (0–5 scale) [3] HepG2 xenograft model in nude mice: Female nude mice (6–8 weeks old) were subcutaneously injected with HepG2 cells (5×10⁶ cells/mouse, resuspended in PBS:Matrigel=1:1) into the right flank. When tumors reached ~100 mm³, mice were randomized into 2 groups (n=6/group): - Treatment group: Q-VD-OPh dissolved in 0.5% carboxymethyl cellulose sodium to 2 mg/mL (intraperitoneal injection, 10 mg/kg, daily) + sorafenib (20 mg/kg, oral gavage, daily) for 14 days. - Control group: Sorafenib (20 mg/kg, oral gavage, daily) + equal volume of 0.5% carboxymethyl cellulose sodium. At endpoint, mice were euthanized; tumors were collected for TUNEL staining and weight measurement [6] |
| Toxicity/Toxicokinetics |
1. In vitro toxicity: Q-VD-OPh (concentration up to 1 μM, treatment for 24 hours) had no effect on the viability of normal human foreskin fibroblasts (NHFF) (viability > 92% vs. control group) [1]/[2]
2. In vivo toxicity: Intraventricular injection of Q-VD-OPh (5 nmol) in rats did not cause brain inflammation (immunohistochemistry showed no increase in microglial cell activation) or behavioral abnormalities [3] 3. In vitro toxicity: Q-VD-OPh (concentration up to 0.3 μM, treatment for 24 hours) had no effect on the viability of uninfected Vero cells (viability > 88% vs. control group) [4] 4. In vivo toxicity: Intraperitoneal injection of Q-VD-OPh (10 mg/kg) in nude mice (14 days) did not cause significant weight loss (-2.0%). vs. control group -1.8%; serum ALT (32 ± 3 U/L vs. 30 ± 2 U/L) and creatinine (0.40 ± 0.02 mg/dL vs. 0.39 ± 0.03 mg/dL) were both within the normal range [6] |
| References | |
| Additional Infomation |
1. Q-VD-OPh is a cell-permeable, irreversible pan-cysteine inhibitor that covalently binds to the active sites of various caspase subtypes (such as Caspase-3, 8, 9, etc.), blocking their proteolytic activity and inhibiting extrinsic and endogenous apoptosis pathways [1]/[2]. 2. In neuroscience research, Q-VD-OPh protects neurons from excitotoxicity and ischemic damage by inhibiting caspase-mediated apoptosis, thus becoming a tool for studying the role of apoptosis in neurodegenerative diseases and stroke [3]. 3. Q-VD-OPh can inhibit virus-induced host cell apoptosis (e.g., HSV-1), which helps maintain cell viability in virus replication studies and also highlights its potential to regulate virus-host interactions through the apoptosis pathway. [4]
4. In oncology research, Q-VD-OPh has been used to study the role of apoptosis in chemosensitivity; it can reduce sorafenib-induced apoptosis in HepG2 cells in vitro and enhance the in vivo tumor growth inhibition effect induced by sorafenib by regulating apoptosis balance[6] |
| Molecular Formula |
C26H25F2N3O6
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| Molecular Weight |
513.49
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| Exact Mass |
513.171
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| Elemental Analysis |
C, 60.82; H, 4.91; F, 7.40; N, 8.18; O, 18.69
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| CAS # |
1135695-98-5
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| Related CAS # |
(R)-Q-VD-OPh
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| PubChem CID |
24794416
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| Appearance |
White to off-white solid powder
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| Density |
1.346±0.06 g/cm3
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| Boiling Point |
808.9±65.0 °C at 760 mmHg
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| Flash Point |
443.0±34.3 °C
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| Vapour Pressure |
0.0±3.0 mmHg at 25°C
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| Index of Refraction |
1.591
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| LogP |
4.61
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
9
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| Rotatable Bond Count |
11
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| Heavy Atom Count |
37
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| Complexity |
818
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| Defined Atom Stereocenter Count |
2
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| SMILES |
O=C(O)C[C@H](NC([C@@H](NC(C1=NC2=CC=CC=C2C=C1)=O)C(C)C)=O)C(COC3=C(F)C=CC=C3F)=O
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| InChi Key |
OOBJCYKITXPCNS-REWPJTCUSA-N
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| InChi Code |
InChI=1S/C26H25F2N3O6/c1-14(2)23(31-25(35)19-11-10-15-6-3-4-9-18(15)29-19)26(36)30-20(12-22(33)34)21(32)13-37-24-16(27)7-5-8-17(24)28/h3-11,14,20,23H,12-13H2,1-2H3,(H,30,36)(H,31,35)(H,33,34)/t20-,23-/m0/s1
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| Chemical Name |
(3S)-5-(2,6-difluorophenoxy)-3-[[(2S)-3-methyl-2-(quinoline-2-carbonylamino)butanoyl]amino]-4-oxopentanoic acid
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| Synonyms |
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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 |
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| 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) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (4.05 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. Solubility in Formulation 2: ≥ 2.08 mg/mL (4.05 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 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. View More
Solubility in Formulation 3: ≥ 2.08 mg/mL (4.05 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. Solubility in Formulation 4: 5%DMSO+40%PEG300+5%Tween80+50%ddH2O: 100mg/ml |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.9475 mL | 9.7373 mL | 19.4746 mL | |
| 5 mM | 0.3895 mL | 1.9475 mL | 3.8949 mL | |
| 10 mM | 0.1947 mL | 0.9737 mL | 1.9475 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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