 Chemical Structure.png)
Rupintrivir (AG-7088) is a novel, selective and irreversible rhinovirus 3C protease inhibitor potentially for the treatment of human rhinovirus-HRV infection. Rupintrivirvr inhibits the replication of a panel of 48 different HRV serotypes in H1-HeLA and MRC-5 cell protection assays with a mean EC50 of 0.023 μM.
Physicochemical Properties
| Molecular Formula | C31H39FN4O7 |
| Molecular Weight | 598.66 |
| Exact Mass | 598.28 |
| Elemental Analysis | C, 62.19; H, 6.57; F, 3.17; N, 9.36; O, 18.71 |
| CAS # | 223537-30-2 |
| PubChem CID | 6440352 |
| Appearance | White to off-white solid powder |
| Density | 1.213g/cm3 |
| Boiling Point | 866.7ºC at 760mmHg |
| Melting Point | 170-171°C |
| Flash Point | 477.9ºC |
| Vapour Pressure | 1.87E-30mmHg at 25°C |
| Index of Refraction | 1.537 |
| LogP | 3.935 |
| Hydrogen Bond Donor Count | 3 |
| Hydrogen Bond Acceptor Count | 9 |
| Rotatable Bond Count | 16 |
| Heavy Atom Count | 43 |
| Complexity | 1010 |
| Defined Atom Stereocenter Count | 4 |
| SMILES | CCOC(=O)/C=C/[C@H](C[C@@H]1CCNC1=O)NC(=O)[C@H](CC2=CC=C(C=C2)F)CC(=O)[C@H](C(C)C)NC(=O)C3=NOC(=C3)C |
| InChi Key | CAYJBRBGZBCZKO-BHGBQCOSSA-N |
| InChi Code | InChI=1S/C31H39FN4O7/c1-5-42-27(38)11-10-24(16-21-12-13-33-29(21)39)34-30(40)22(15-20-6-8-23(32)9-7-20)17-26(37)28(18(2)3)35-31(41)25-14-19(4)43-36-25/h6-11,14,18,21-22,24,28H,5,12-13,15-17H2,1-4H3,(H,33,39)(H,34,40)(H,35,41)/b11-10+/t21-,22+,24+,28-/m0/s1 |
| Chemical Name | ethyl (S,E)-4-((2R,5S)-2-(4-fluorobenzyl)-6-methyl-5-(5-methylisoxazole-3-carboxamido)-4-oxoheptanamido)-5-((S)-2-oxopyrrolidin-3-yl)pent-2-enoate |
| Synonyms | AG-7088; AG7088; AG 7088; 223537-30-2; ag7088; Ruprintrivir; Rupintrivir-d4; RGE5K1Q5QW; |
| 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 | Antiviral; HRV (human rhinovirus) 3C protease |
| ln Vitro | Rupintrivirvr (AG7088), in H1-HeLa and MRC-5 cell protection assays, inhibited the replication of all HRV serotypes (48 of 48) tested, as well as that of related picornaviruses, such as coxsackieviruses A21 and B3, enterovirus 70, and echovirus 11. The mean 50% effective concentration (EC50) was 0.023 μM (range, 0.003 to 0.081 μM) and the mean EC90 was 0.082 μM (range, 0.018 to 0.261 μM). |
| ln Vivo | Rupintrivirvr (AG7088) decreases the TH-2 cytokine IL-4 that is induced by RV in ex vivo HDM-sensitized mouse lung slices (PCLS)[2]. |
| Enzyme Assay |
Enzyme assays.[1] The proteolytic activity of HRV 14 3C protease was measured by a continuous fluorescence resonance energy transfer assay as described previously. In brief, cleavage of the substrate peptide was monitored by the appearance of fluorescent emission at 490 nm (following excitation at 336 nm) in a Perkin-Elmer LS50-B spectrophotometer. Data were analyzed with the nonlinear regression analysis program ENZFITTER, which calculates a first-order rate constant for the inactivation of HRV 14 3C protease. Protease selectivity assays were performed with commercially available proteases (at approximately 10 nM concentrations) essentially as described by the supplier. Analysis of proteolytic processing. [1] The ability of Rupintrivirvr (AG7088) to inhibit HRV 14 3C-mediated proteolytic processing was assessed by polyacrylamide gel electrophoresis (PAGE) of radiolabeled sodium dodecyl sulfate (SDS)-solubilized lysates of HRV 14-infected cells. Initially, H1-HeLa cells were infected with HRV 14 at an MOI of 10. Eight and one-half hours after infection, the cells were washed with PBS and the medium was replaced with methionine- and cysteine-deficient medium. At 9 h after infection, appropriate concentrations of compounds were added. After a 30-min exposure to compounds, 50 μCi of [35S]Met-[35S]Cys (Expre35S 35S protein label) was added. One hour later, the monolayers were washed twice with cold PBS and lysed in 250 μl of radioimmunoprecipitation assay buffer (50 mM Tris-Cl [pH 8.0], 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS), sonicated, and stored at −70°C for subsequent analysis. Proteins present in the solubilized cell lysates were resolved by 12% PAGE. Following electrophoresis, gels were stained with Coomassie brilliant blue, destained, and treated with Amplify. Gels were air dried overnight in cellulose sheets and exposed to film at −80°C. |
| Cell Assay |
Time-of-addition assay. [1] Subconfluent monolayers of H1-HeLa cells in six-well plates were infected with HRV 14 at an MOI of 15. After 1 h of adsorption, cell monolayers were washed three times with phosphate-buffered saline (PBS) and replenished with medium. AG7088 (0.5 μM) or WIN 51711 (3.0 μM) was added at concentrations 20-fold above the EC50 (as determined by the cell protection assay) at the time of infection and at various times thereafter. Eight hours after infection, samples were processed by three freeze-thaw cycles followed by sonication for 15 s and clarification by centrifugation (5 min at 15,000 × g at 4°C). Clarified cell and supernatant lysates were stored at −70°C for subsequent analysis for infectious virus. HCMV antiviral assay. [1] The antiviral activity of AG7088 against HCMV AD169 replication in MRC-5 cells was determined by an enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibody MAb directed against the HCMV major immediate-early gene product. Briefly, following a 2-h virus adsorption, the inoculum was removed and medium containing the appropriate concentrations of compound was added. Five days after infection, the MRC-5 monolayers were incubated with MAb 810, followed by goat anti-mouse antibody conjugated with horseradish peroxidase, and viral antigen was then detected spectrophotometrically at 650 nm with the tetramethylbenzidine liquid substrate system. The EC50 was calculated as the concentration of compound that reduced the optical density to 50% of that of the virus control. The CC50 was measured by the XTT reduction method as described above. RV infection of PCLS[2] Two PCLS per well were infected with 1 × 105 IU/mL of RV for 48 h in culture medium at cell culture conditions (33 °C, 5% CO2, and 100% humidity). To determine the replication dependent immune response, UV-inactivated virus (260 nm for 1 h) from the same batch was used under the same conditions. Pharmacological intervention was performed by infection with RV (1 × 10~5 IU/mL) in the presence of 100 nM rupintrivir. |
| Animal Protocol | Mice were sensitized with HDM. Precision-cut lung slices (PCLS) were prepared from HDM-sensitized or non-sensitized mice. Lung slices were infected ex vivo with RV or RV together with rupintrivir. Modulation of immune responses was evaluated by cytokine secretion 48 h post infection. Results: In vivo HDM sensitization resulted in a TH-2/TH-17-dominated cytokine response that persisted in PCLS ex vivo. RV infection of PCLS from non-sensitized mice resulted in the induction of an antiviral and pro-inflammatory immune response, as indicated by the secretion of IFN-α, IFN-β, IFN-γ, TNF-α, MCP-1, IP-10, IL-10, and IL-17A. In contrast, PCLS from HDM-sensitized mice showed an attenuated antiviral response, but exaggerated IL-4, IL-6, and IL-10 secretion upon infection. Rupintrivir inhibited exaggerated pro-inflammatory cytokine IL-6 and TH-2 cytokine IL-4 in HDM-sensitized mice.[2] |
| References |
[1]. In vitro antiviral activity of AG7088, a potent inhibitor of human rhinovirus 3C protease. Antimicrob Agents Chemother. 1999 Oct;43(10):2444-50. [2]. Rupintrivir reduces RV-induced TH-2 cytokine IL-4 in precision-cut lung slices (PCLS) of HDM-sensitized mice ex vivo. Respir Res. 2019 Oct 22;20(1):228. [3]. Structure-based design, synthesis, and biological evaluation of irreversible human rhinovirus 3C protease inhibitors. 3. Structure-activity studies of ketomethylene-containing peptidomimetics. J Med Chem. 1999 Apr 8;42(7):1203-12. |
| Additional Infomation |
Rupintrivir is a rhinovirus 3C protease inhibitor in development for use against human rhinoviral (HRV) infections. Rupintrivir was active against all 48 HRV serotypes tested in a cell protection assay in H1-HeLa cells. It is designed to combat colds caused by rhinovirus. Rupintrivir is a peptidomimetic inhibitor with activity against human rhinoviruses. Rupintrivir is an irreversible 3C protease inhibitor which blocks processing of viral proteins, thus blocking viral replication. Drug Indication Investigated for use/treatment in viral infection. Mechanism of Action The 3C protease (3CP) enzyme is central to rhinovirus replication. By binding to and inhibiting this enzyme, AG7088 prevents rhinovirus replication in cells of the respiratory tract and stops cold symptoms developing. |
Solubility Data
| Solubility (In Vitro) | DMSO : ~50 mg/mL (~83.52 mM) |
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 1.67 mg/mL (2.79 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 16.7 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. Solubility in Formulation 2: 10% DMSO+90% Corn Oil: ≥ 1.67 mg/mL (2.79 mM) (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.6704 mL | 8.3520 mL | 16.7040 mL | |
| 5 mM | 0.3341 mL | 1.6704 mL | 3.3408 mL | |
| 10 mM | 0.1670 mL | 0.8352 mL | 1.6704 mL |