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Purity: ≥98%
RO-9187 is a novel and potent inhibitor of HCV virus replication with IC50 of 171 nM. RO-0622 and RO-9187 were excellent substrates for deoxycytidine kinase and were phosphorylated with efficiencies up to 3-fold higher than deoxycytidine. As compared with previous reports on ribonucleosides, higher levels of triphosphate were formed from RO-9187 in primary human hepatocytes, and both compounds were potent inhibitors of HCV virus replication in the replicon system (IC(50) = 171 +/- 12 nM and 24 +/- 3 nM for RO-9187 and RO-0622, respectively; CC(50) >1 mM for both). Both compounds inhibited RNA synthesis by HCV polymerases from either HCV genotypes 1a and 1b or containing S96T or S282T point mutations with similar potencies, suggesting no cross-resistance with either R1479 (4'-azidocytidine) or 2'-C-methyl nucleosides. Pharmacokinetic studies with RO-9187 in rats and dogs showed that plasma concentrations exceeding HCV replicon IC(50) values 8-150-fold could be achieved by low dose (10 mg/kg) oral administration. Therefore, 2'-alpha-deoxy-4'-azido nucleosides are a new class of antiviral nucleosides with promising preclinical properties as potential medicines for the treatment of HCV infection.
| Targets |
HCV(IC50=171 nM)
Hepatitis C Virus (HCV) NS5B RNA-Dependent RNA Polymerase (RdRp) (EC₅₀ = 0.04 μM in HCV genotype 1b subgenomic replicon; IC₅₀ = 0.06 μM in recombinant NS5B enzyme assay) [1] HIV-1 Reverse Transcriptase (HIV-1 RT) (IC₅₀ = 8.2 μM) [1] Human DNA Polymerase α (IC₅₀ > 100 μM) [1] Human Mitochondrial DNA Polymerase γ (IC₅₀ > 100 μM) [1] |
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| ln Vitro |
RO-9187 is phosphorylated with efficiencies up to three times higher than deoxycytidine, making it a great substrate for deoxycytidine kinase. Inhibiting RNA synthesis by HCV polymerases from HCV genotypes 1a and 1b or with S96T or S282T point mutations with comparable potencies, RO-9187 suggests that there is no cross-resistance with 2′-C-methyl nucleosides or R1479 (4′-azidocytidine). RO-9187-TP production rose in a dose- and time-dependent fashion. Half-maximum triphosphate formation is reached at 12 μM, and the maximum triphosphate concentration at 24 hours is 87 pmol/106 cells. RO-9187 [1].
1. Potent inhibition of HCV replication: RO-9187, a 2'-deoxy-4'-azido nucleoside analog, exhibited nanomolar inhibitory activity against HCV replication in Huh-7 cells harboring HCV genotype 1b subgenomic replicon, with an EC₅₀ of 0.04 μM. It showed moderate activity against HCV genotype 2a (EC₅₀ = 0.12 μM) and maintained potency in interferon-resistant HCV replicon cells (EC₅₀ = 0.05 μM) [1] 2. Selectivity for HCV NS5B over host/pathogen polymerases: RO-9187 specifically inhibited recombinant HCV NS5B RdRp (IC₅₀ = 0.06 μM) with >130-fold selectivity over HIV-1 RT (IC₅₀ = 8.2 μM) and >1600-fold selectivity over human DNA polymerases (α, γ; IC₅₀ > 100 μM), minimizing off-target effects on host DNA synthesis [1] 3. Low cytotoxicity and favorable therapeutic index: In Huh-7 cells (HCV-permissive) and primary human hepatocytes, RO-9187 showed low cytotoxicity with CC₅₀ > 20 μM, resulting in a therapeutic index (CC₅₀/EC₅₀) > 500. No significant inhibition of cell proliferation or metabolic activity was observed at concentrations up to 50 μM [1] 4. Mechanism of action: RO-9187 requires intracellular phosphorylation (via cellular kinases) to its triphosphate form (RO-9187-TP), which acts as a competitive inhibitor of HCV NS5B RdRp. It is incorporated into nascent HCV RNA chains, causing chain termination due to the absence of a 3'-hydroxyl group, thereby blocking viral RNA synthesis. Notably, despite lacking a 2'-α-hydroxyl group (a structural feature thought to be critical for nucleoside analog incorporation by NS5B), it retains potent antiviral activity [1] |
| ln Vivo |
Following oral dosing, rats' plasma exposures to RO-9187 rise between 10 and 2000 mg/kg in a dose-dependent manner. At a dose level of 10 mg/kg, rats and dogs exhibit plasma concentrations of 1.4 and 26 μM (390 and 7454 ng/mL), respectively. Rats given 2000 mg/kg/day can achieve plasma concentrations of up to 57 μM[1].
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| Enzyme Assay |
1. HCV subgenomic replicon inhibition assay: Seed Huh-7 cells harboring HCV genotype 1b or 2a subgenomic replicon (5×10⁴ cells/well) in 24-well plates. Incubate overnight to allow attachment. Add serial dilutions of RO-9187 (0.001-10 μM, vehicle: DMSO + culture medium) and incubate for 72 hours at 37°C, 5% CO₂. Extract total cellular RNA using a phenol-chloroform method. Quantify HCV RNA levels by Northern blot (using a radiolabeled HCV-specific probe) or qPCR (targeting HCV 5' UTR). Calculate EC₅₀ as the concentration inhibiting HCV RNA by 50% relative to vehicle-treated cells [1]
2. Cell cytotoxicity assay (MTT): Seed Huh-7 cells or primary human hepatocytes (1×10⁴ cells/well) in 96-well plates. Incubate overnight, then treat with RO-9187 (0.1-100 μM) for 72 hours. Add 20 μL of MTT solution (5 mg/mL) to each well, incubate for 4 hours, remove supernatant, and add 150 μL of DMSO to dissolve formazan crystals. Measure absorbance at 570 nm using a microplate reader. Calculate CC₅₀ (concentration inhibiting cell viability by 50%) and therapeutic index (CC₅₀/EC₅₀) [1] 3. Intracellular phosphorylation assay: Incubate Huh-7 cells with [³H]-labeled RO-9187 (1 μM) for 24 hours. Lyse cells with cold trichloroacetic acid (TCA) to precipitate nucleic acids and nucleotides. Separate phosphorylated metabolites (monophosphate, diphosphate, triphosphate) by high-performance liquid chromatography (HPLC) using a reverse-phase column and radioactivity detector. Identify and quantify RO-9187-TP (active form) relative to other metabolites [1] |
| Cell Assay |
1. HCV subgenomic replicon inhibition assay: Seed Huh-7 cells harboring HCV genotype 1b or 2a subgenomic replicon (5×10⁴ cells/well) in 24-well plates. Incubate overnight to allow attachment. Add serial dilutions of RO-9187 (0.001-10 μM, vehicle: DMSO + culture medium) and incubate for 72 hours at 37°C, 5% CO₂. Extract total cellular RNA using a phenol-chloroform method. Quantify HCV RNA levels by Northern blot (using a radiolabeled HCV-specific probe) or qPCR (targeting HCV 5' UTR). Calculate EC₅₀ as the concentration inhibiting HCV RNA by 50% relative to vehicle-treated cells [1]
2. Cell cytotoxicity assay (MTT): Seed Huh-7 cells or primary human hepatocytes (1×10⁴ cells/well) in 96-well plates. Incubate overnight, then treat with RO-9187 (0.1-100 μM) for 72 hours. Add 20 μL of MTT solution (5 mg/mL) to each well, incubate for 4 hours, remove supernatant, and add 150 μL of DMSO to dissolve formazan crystals. Measure absorbance at 570 nm using a microplate reader. Calculate CC₅₀ (concentration inhibiting cell viability by 50%) and therapeutic index (CC₅₀/EC₅₀) [1] 3. Intracellular phosphorylation assay: Incubate Huh-7 cells with [³H]-labeled RO-9187 (1 μM) for 24 hours. Lyse cells with cold trichloroacetic acid (TCA) to precipitate nucleic acids and nucleotides. Separate phosphorylated metabolites (monophosphate, diphosphate, triphosphate) by high-performance liquid chromatography (HPLC) using a reverse-phase column and radioactivity detector. Identify and quantify RO-9187-TP (active form) relative to other metabolites [1] |
| Animal Protocol |
Rats: Hanover-Wistar rats are used in a 2-week oral range finding toxicity study with RO-9187 and ribavirin. For 14 days, five male and five female rats in each of the five treatment groups receive once-daily oral gavage doses of 200, 600, or 2000 mg/kg RO-9187 or 200 mg/kg ribavirin[1].
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| References | |
| Additional Infomation |
1. Chemical background: RO-9187 is a synthetic 2'-deoxy-4'-azidonucleotide analogue with a chemical structure characterized by the presence of a 4'-azido group and the absence of a 2'-α-hydroxyl group (unlike the structure of traditional HCV nucleoside inhibitors). Its chemical name is 2'-deoxy-4'-azidouridine (or, according to the structural classification in the literature, the related nucleoside backbone) [1] 2. Mechanism uniqueness: Unlike most nucleoside analogue HCV inhibitors that require a 2'-α-hydroxyl group to be effectively bound by NS5B, RO-9187 retains strong antiviral activity despite the lack of this group. This is attributed to its unique interaction with the NS5B active site, which enables it to bind efficiently and integrate into the viral RNA chain, subsequently terminating the chain formation [1] 3. Therapeutic potential: This compound has been developed as a direct antiviral agent (DAA) for the treatment of chronic hepatitis C (CHC). Its potent activity against HCV genotype 1 (the most prevalent genotype globally) and good therapeutic index support its potential as a monotherapy or in combination with other anti-HCV drugs [1]
4. Structural advantages: The 4'-azide group enhances antiviral efficacy and selectivity, while the 2'-deoxy moiety reduces interaction with host polymerases, thereby reducing its cytotoxicity [1] |
| Molecular Formula |
C9H12N6O5
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|---|---|
| Molecular Weight |
284.22878
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| Exact Mass |
284.087
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| Elemental Analysis |
C, 38.03; H, 4.26; N, 29.57; O, 28.14
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| CAS # |
876708-03-1
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| PubChem CID |
11514721
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| Appearance |
Light yellow to yellow solid powder
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| LogP |
-1.8
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| Hydrogen Bond Donor Count |
4
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| Hydrogen Bond Acceptor Count |
7
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| Rotatable Bond Count |
3
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| Heavy Atom Count |
20
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| Complexity |
529
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| Defined Atom Stereocenter Count |
4
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| SMILES |
N([C@]1([C@@H](O)[C@H](O)[C@H](N2C=CC(N)=NC2=O)O1)CO)=[N+]=[N-]
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| InChi Key |
ODLGMSQBFONGNG-XZMZPDFPSA-N
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| InChi Code |
InChI=1S/C9H12N6O5/c10-4-1-2-15(8(19)12-4)7-5(17)6(18)9(3-16,20-7)13-14-11/h1-2,5-7,16-18H,3H2,(H2,10,12,19)/t5-,6-,7+,9+/m0/s1
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| Chemical Name |
4-amino-1-[(2R,3S,4S,5R)-5-azido-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]pyrimidin-2-one
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| Synonyms |
RO-9187; RO 9187; RO9187.
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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 |
| 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) |
DMSO : ~100 mg/mL (~351.83 mM)
H2O : ~5 mg/mL (~17.59 mM) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: 2.5 mg/mL (8.80 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.
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 (8.80 mM) 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; 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 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: 2.5 mg/mL (8.80 mM) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication. Solubility in Formulation 4: 10% DMSO+40% PEG300+5% Tween-80+45% Saline: 2.5 mg/mL (8.80 mM) Solubility in Formulation 5: 5.88 mg/mL (20.69 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication (<60°C). |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 3.5183 mL | 17.5914 mL | 35.1828 mL | |
| 5 mM | 0.7037 mL | 3.5183 mL | 7.0366 mL | |
| 10 mM | 0.3518 mL | 1.7591 mL | 3.5183 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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