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Purity: =100%
Danoprevir (formerly ITMN-191, R-7227; RO-5190591; RG 7227; RG-7227; trade name Ganovo) is an orally bioavailable and 15-membered macrocyclic peptidomimetic inhibitor of the NS3/4A protease of hepatitis C virus (HCV) with IC50s in the range of 0.2-3.5 nM. Due to its role in viral replication and ability to suppress the host's response to viral infection, HCV NS3/4A protease is a prime candidate for anti-HCV medication targeting. Ascletis was the company that developed and sold danoprevir in China.
| Targets |
NS3/4A protease (IC50 = 0.29 nM); HCV genotypes (IC50 = 0.2-3.5 nM)
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| ln Vitro |
Danoprevir (0.29 nM) inhibits the reference genotype 1 NS3/4A protease half-maximally; however, a panel of 79 proteases, ion channels, transporters, and cell surface receptors does not exhibit any discernible inhibition at a high dose of Danoprevir (10 μM). After its initial association, danoprevir binds to and inhibits NS3/4A for over five hours. A patient-derived HCV genotype 1b replicon is eliminated by danoprevir (45 nM) from Huh7 cells derived from hepatocytes with an EC50 of 1.8 nM.[1] The R155K substitution confers a high level (62-fold increase) of resistance to Danoprevir in HCV subgenomic replicon cell lines containing the individual mutations, whereas the V36M, R109K, and V170A substitutions confer little or no resistance to the drug.[2] Danoprevir exhibits antiviral inhibition effects against HCV genotypes 1, 4, and 6 in Huh7.5 cells transfected with chimeric recombinant virus (IC50 of 2-3 nM), which is >100-fold lower than genotypes 2/3/5 (280-750 nM).[3]
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| ln Vivo |
Danoprevir (30 mg/kg) administered to rats or monkeys demonstrates that its liver concentrations 12 hours post-dosage surpass the concentration of Danoprevir needed to eradicate replicon RNA from cells.[1]
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| Enzyme Assay |
The assay buffer comprises the following: 0.5 μM fluorescein/QXL520-labeled FRET substrate {Ac-DE-Dap(QXL520)-EE-Abu-ψ-[COO]-AS-Cys(5-FAMsp)-NH2}; 25 μM NS4A peptide; 50 mM Tris-HCl, pH 7.5; 15% (vol/vol) glycerol; 0.6 mM lauryldimethylamine N-oxide; 10 mM dithiothreitol). The reaction is started by adding 50 pM of the K2040 enzyme. Fluorescence data is gathered and reactions are set up in black 96-well plates. Included are control reactions that lack both inhibitors and enzymes. Initial rates, which are used to determine IC50, are computed from the reaction's linear phase (up to one hour).The evaluation of the activity recovered from the preformed Danoprevir-NS3/4A complex involves a 15-minute preincubation of 10 nM NS3/4A with a two-fold excess of Danoprevir in 1× assay buffer. Subsequently, the preformed complex is rapidly diluted 200 times into assay buffer that contains substrate. A control reaction is started by adding an enzyme to an otherwise complete reaction mixture, resulting in the same final conditions but without the preincubation of NS3/4A and Danoprevir. Either NS3 or Danoprevir are absent from additional control reactions. Over the course of five hours, the reactions are monitored.
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| Cell Assay |
One day after cell plating, Huh7 cells containing the K2040 replicon are treated with serially diluted Danoprevir. After a 48-hour incubation period, intracellular RNA is extracted for antiviral assays. Using an ABI Prism 7900 sequence detection system, the amount of HCV replicon RNA is measured by reverse transcription (RT)-PCR assay using the primers (5-CACTCCCCTGTGAGGAACTACTG-3 and 5-AGGCTGCACGACACTCATACT-3) and a probe (5-6-FAM-CTTCACGCAGAAAGCGTCTAGCCATGG-MGBNFQ-3). Here, MGBNFQ is a molecular groove binding non-fluorescence quencher that is specific to the HCV 5 untranslated region, and FAM is 6-carboxyfluorescin. The TaqMan Gold RT-PCR kit is used to conduct single-tube reactions. 5 μL of intracellular RNA (50 ng) is used in triplicate reactions for both the RNA standards and samples, in 50 μL. The RT process lasts 30 minutes at 48 °C and then 10 minutes at 95 °C. This is how the PCR is conducted: 40 cycles of 15 s at 95 °C and 1 s at 60 °C. Three duplicate measurements of each RNA concentration are made. A standard curve created by known concentrations of an in vitro-transcribed RNA corresponding to an untranslated region of genotype 1b 5 is used to calculate the absolute concentration of replicon RNA. The EC50 is obtained by fitting the replican levels in the presence of Danoprevir to a logistic function with four parameters.
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| Animal Protocol |
In monkeys and rats, pharmacokinetic characteristics are assessed. An oral gavage treatment of 30-mg/kg of body weight (a 6-mg/mL solution in water) is given to three Sprague-Dawley rats per time point. ITMN-191 is given orally via gavage to two cynomolgus monkeys per time point at a dose of 30 mg/kg (three milligrams per milliliter in water). After the dose is administered, 1, 4, 8, 12, and 24 hours later, terminal blood samples and the whole perfused liver are taken for every species. Before being analyzed, blood samples are centrifuged at 5°C to extract plasma, then they are kept at -20°C. The samples are collected in EDTA. Liver samples are kept at −70°C after being snap-frozen, pending analysis. Acidified acetonitrile is used to treat blank, standard, and unknown plasma samples as well as homogenized liver containing an internal standard (ITMN-191 analog). Precipitated proteins are then removed by centrifugation. In order to express concentrations in both compartments as weight per unit volume, the density of liver tissue is taken into consideration. A 4000 Q-trap liquid chromatography tandem mass spectrometer equipped with a Turbo-Ionspray source operating in negative-ion mode is used to analyze the cleared supernatants after they have been diluted 1:1 into high-performance liquid chromatography grade water. ABI Analyst software, version 1.4.2, is used to calibrate the analytes and internal standards, and multiple-reaction monitoring scans are used for monitoring. For the quantification of plasma samples and liver homogenates, the calibration standards range from 7.47 ng/mL to 5,440 ng/mL and from 0.0169 ng/mL to 37.0 ng/mL, respectively. In situations where both matrices have an R2 value of > 0.999, quadratic fitting with 1/x weighting is applied.
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| References | |
| Additional Infomation |
Danoprevir is a keratan 6'-sulfate and an azamacrocycle.
Danoprevir has been used in trials studying the treatment of Hepatitis C, Chronic. Danoprevir is an orally bioavailable, peptidomimetic inhibitor of hepatitis C virus (HCV) NS3/4A protease, with antiviral activity against HCV and potential antiviral activity against SARS-CoV-2. Upon oral administration, danoprevir binds to and blocks the activity of HCV NS3/4A protease. This prevents the cleavage and processing of HCV viral proteins leading to the inhibition of HCV replication. Danoprevir may also bind to and block of the activity of SARS-CoV-2 protease. This prevents the cleavage and processing of SARS-CoV-2 viral proteins leading to the inhibition of SARS-CoV-2 replication. NS3/4A, a chymotrypsin-like serine protease, is responsible for cleavage at four sites of the HCV polyprotein to form the viral proteins required for HCV replication. It plays a key role in the HCV viral replication process. HCV infection is associated with the development of hepatocellular carcinoma (HCC). A chymotrypsin-like protease is responsible for cleavage of the SARS-CoV-2 viral polyprotein to form the RNA replicase-transcriptase complex, which plays a key role in the SARS-CoV-2 viral transcription and replication process. Mechanism of Action Danoprevir is a NS3/4A protease inhibitor. The HCV NS3/4A protease is an attractive drug target because of its potential involvement in viral replication and suppressive effects on host response to viral infection. Inhibitors of the HCV protease, such as Danoprevir, represent a promising new class of drugs for HCV. |
| Molecular Formula |
C35H46FN5O9S
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| Molecular Weight |
731.831251621246
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| Exact Mass |
731.3
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| Elemental Analysis |
C, 57.44; H, 6.34; F, 2.60; N, 9.57; O, 19.68; S, 4.38
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| CAS # |
850876-88-9
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| Related CAS # |
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| PubChem CID |
11285588
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| Appearance |
White to off-white solid powder
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| Density |
1.4±0.1 g/cm3
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| Index of Refraction |
1.622
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| LogP |
1.12
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
10
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| Rotatable Bond Count |
8
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| Heavy Atom Count |
51
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| Complexity |
1530
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| Defined Atom Stereocenter Count |
5
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| SMILES |
CC(C)(C)OC(=O)N[C@H]1CCCCC/C=C\[C@@H]2C[C@]2(NC(=O)[C@@H]3C[C@H](CN3C1=O)OC(=O)N4CC5=C(C4)C(=CC=C5)F)C(=O)NS(=O)(=O)C6CC6
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| InChi Key |
ZVTDLPBHTSMEJZ-JSZLBQEHSA-N
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| InChi Code |
InChI=1S/C35H46FN5O9S/c1-34(2,3)50-32(45)37-27-13-8-6-4-5-7-11-22-17-35(22,31(44)39-51(47,48)24-14-15-24)38-29(42)28-16-23(19-41(28)30(27)43)49-33(46)40-18-21-10-9-12-26(36)25(21)20-40/h7,9-12,22-24,27-28H,4-6,8,13-20H2,1-3H3,(H,37,45)(H,38,42)(H,39,44)/b11-7-/t22-,23-,27+,28+,35-/m1/s1
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| Chemical Name |
[(1S,4R,6S,7Z,14S,18R)-4-(cyclopropylsulfonylcarbamoyl)-14-[(2-methylpropan-2-yl)oxycarbonylamino]-2,15-dioxo-3,16-diazatricyclo[14.3.0.04,6]nonadec-7-en-18-yl] 4-fluoro-1,3-dihydroisoindole-2-carboxylate
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| Synonyms |
Danoprevir; RG-7227; RG7227; RG 7227; ITMN-191; ITMN 191; ITMN191; RO-5190591; RO5190591; RO 5190591; Danoprevir (ITMN-191); Ganovo; Intermune ITMN-191;
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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: ≥ 2.5 mg/mL (3.42 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 (3.42 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. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.3664 mL | 6.8322 mL | 13.6644 mL | |
| 5 mM | 0.2733 mL | 1.3664 mL | 2.7329 mL | |
| 10 mM | 0.1366 mL | 0.6832 mL | 1.3664 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.
| NCT Number | Recruitment | interventions | Conditions | Sponsor/Collaborators | Start Date | Phases |
| NCT03019991 | Completed | Drug: Danoprevir Drug: Ritonavir |
Healthy | Ascletis Pharmaceuticals Co., Ltd. |
October 2015 | Phase 1 |
| NCT03020082 | Completed | Drug: Danoprevir Drug: Ritonavir |
Chronic Hepatitis C | Ascletis Pharmaceuticals Co., Ltd. |
June 2016 | Phase 3 |
| NCT03020004 | Completed | Drug: Danoprevir Drug: Ritonavir |
Chronic Hepatitis C | Ascletis Pharmaceuticals Co., Ltd. |
January 2016 | Phase 2 |
| NCT03020095 | Completed | Drug: Danoprevir Drug: Ritonavir |
Chronic Hepatitis C | Ascletis Pharmaceuticals Co., Ltd. |
November 28, 2011 | August 2015 |
| NCT01531647 | Completed | Drug: danoprevir Drug: raltegravir |
Healthy Volunteer | Hoffmann-La Roche | January 2012 | Phase 1 |
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