RSV604 targets the nucleocapsid (N) protein of respiratory syncytial virus (RSV) [1].
The N protein is a highly conserved major structural protein that encapsulates the RNA genome and is essential for viral replication and transcription [1].

Four laboratory strains of RSV (RSS, Long, A2, and B) with EC50 ranging from 0.5 to 0.9 μM in plaque reduction experiments are inhibited in growth by RSV604 (5 days) [1]. RSV604 (6 days) has an EC50 of 0.86 μM and prevents HEp-2 cell death caused by RSV[1]. In RSV-infected HEp-2 cells, RSV604 (3 days) decreases viral antigen synthesis with an EC50 of 1.7 μM [1]. Human airway epithelial (HAE) cells are dose-dependently inhibited from RSV infection by RSV604 (1–20 μM; 7 days) without exhibiting any cytotoxicity, basolateral fluid leaking to the apical surface, or ciliary beating frequency alterations [1].

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In XTT assays using HEp-2 cells, RSV604 demonstrated an EC₅₀ (50% effective concentration) of 0.5-0.9 μM against RSV strain RSS. The CC₅₀ (50% cytotoxic concentration) was >50 μM, giving a therapeutic index >58 [1].
In cell ELISA measuring viral antigen synthesis, RSV604 was approximately 2.5 times more active than ribavirin [1].
In plaque reduction assays, RSV604 showed EC₅₀ values of 0.5 ± 0.1 μM against RSV strain RSS, 0.6 ± 0.2 μM against RSV Long strain, 0.5 ± 0.1 μM against RSV A2 strain, and 0.6 ± 0.2 μM against RSV B strain [1].
RSV604 was active against bovine RSV with an EC₅₀ of 0.3 μM in plaque reduction assay, but showed no activity against pneumonia virus of mice, parainfluenza virus type 1 (PIV1), PIV3, or human metapneumovirus (all EC₅₀ >50 μM) [1].
When tested against 40 clinical isolates of RSV (both A and B subtypes) from various geographic regions spanning 15 years, RSV604 showed equipotent activity with an average EC₅₀ of 0.8 ± 0.2 μM by plaque reduction assay. No clinical isolates showed resistance to RSV604 [1].
Increasing the multiplicity of infection (MOI) from 0.02 to 1.5 had only a slight effect on the EC₅₀ of RSV604 (1.4 μM and 2.55 μM, respectively) [1].
RSV604 was able to inhibit virus replication at similar levels when given prior to infection and at up to 6 hours post-infection, suggesting it acts at a step subsequent to adsorption and viral penetration [1].
Resistant mutants generated after multiple passages showed mutations in the conserved N-terminal region of the nucleocapsid protein (amino acids 105-139). Amino acid substitutions identified included N105D, K107N, I129L, and L139I [1].
Reverse genetics experiments confirmed that mutations I129L, L139I, and N105D in the N protein conferred resistance to RSV604. Single mutants showed 5-7 fold resistance, while the I129L-plus-L139I double mutant showed >20-fold resistance [1].

For XTT assays, HEp-2 cells were seeded at 4 × 10³ cells per well in 96-well plates in DMEM containing 10% FBS. Cells were infected the next day with sufficient RSV (RSS strain) to produce approximately 80% cytopathic effect after 6 days. Cells were incubated in the presence or absence of serial dilutions of RSV604. Cell viability was assessed after 6 days using XTT [2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide, disodium salt], which is reduced by living cells to colored formazan products. Results were expressed as EC₅₀ or CC₅₀ values. A cell control (no virus) assay was performed in parallel to measure growth inhibitory effects [1].
For cell ELISA, HEp-2 cells were seeded in 96-well plates (5 × 10³ cells per well in DMEM containing 10% FBS) for 24 hours. Cells were infected at an MOI of approximately 0.02 and incubated in the presence or absence of serial dilutions of RSV604 for 3 days. Cells were fixed and permeabilized with 75% methanol-25% acetone, blocked with 2% nonfat milk-0.05% Tween, incubated with mouse anti-RSV monoclonal antibody followed by rabbit anti-mouse horseradish peroxidase-labeled secondary antibody, and developed with O-phenylene diamine in the presence of hydrogen peroxide. Absorbance was measured at 650 nm [1].
For plaque reduction assays, HEp-2 cell monolayers in 6-well plates were infected with 0.2 ml of 500 PFU/ml RSV (RSS strain). After 2 hours, cells were overlaid with DMEM containing 2% FBS and 0.6% agarose, and RSV604 was added from a dilution series. Plates were incubated for 5 days, fixed with 10% formaldehyde, and plaques were visualized by methylene blue staining or immunostaining. Plaques were counted and results expressed as percentages of virus replication compared to wells infected in the absence of inhibitor [1].
For generation of resistant mutants, HEp-2 cells were infected with RSV strain RSS (MOI 0.01) and grown in the presence of RSV604 until cytopathic effect reached 80-90%. Virus was harvested and used to reinfect cells, with compound concentration either kept constant or doubled at each passage until resistant mutants were isolated. Resistant mutants were plaque picked three times under agarose [1].
For human airway epithelial (HAE) cell model studies, differentiated HAE cells cultured on permeable membrane supports at air-liquid interface for 4-6 weeks were inoculated via the luminal surface with recombinant RSV expressing GFP (rgRSV; 10⁶ PFU). RSV604 at various concentrations was added to the basolateral medium concomitantly with virus. Infection was monitored by visualization of GFP with fluorescence microscopy [1].

A preclinical safety program was performed to assess the safety profile of RSV604 before commencing human studies. RSV604 was well tolerated by all species tested, and evaluation of major organ systems in vivo demonstrated no effects attributable to treatment [1].

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A preclinical safety program was performed to assess the safety profile of RSV604 before commencing human studies. RSV604 was well tolerated by all species tested, and evaluation of major organ systems in vivo demonstrated no effects attributable to treatment [1].

RSV604 has a molecular formula of C₂₂H₁₇FN₄O₂ and a molar mass of 388.4 g/mol. It is a white powder stable at room temperature with a melting point of 243-245°C (decomposition) [1].
In human airway epithelial cell model studies, RSV604 administered to the basolateral medium was able to penetrate the multilayered epithelium to efficaciously inhibit viral replication and spread after mucosal inoculation, indicating that systemic delivery of this compound will be useful against luminal RSV infection [1].
Phase I trials in humans showed that RSV604 is well absorbed, and human pharmacokinetics support once-daily dosing to achieve antiviral EC₉₀ levels [1].

In XTT assays using HEp-2 cells, RSV604 showed a CC₅₀ (50% cytotoxic concentration) of >50 μM, with a therapeutic index >58 [1].
In the HAE cell model, no gross cytotoxicity, leakage of basolateral fluid to the apical surface, or alteration of cilium beat frequency was observed when HAE cultures were exposed to 20 μM RSV604 for 7 days [1].
A preclinical safety program demonstrated that RSV604 was well tolerated by all species tested, with no effects attributable to treatment on major organ systems [1].

[1]. RSV604, a novel inhibitor of respiratory syncytial virus replication. Antimicrob Agents Chemother. 2007 Sep;51(9):3346-53.

[2]. Mechanism of action for respiratory syncytial virus inhibitor RSV604. Antimicrob Agents Chemother. 2015 Feb;59(2):1080-7.

RSV-604 is being investigated in the clinical trial NCT00416442 (Safety, tolerability and pharmacokinetics of single and multiple intravenous injections of RSV604 in healthy subjects).

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RSV604 is a novel benzodiazepine with the systematic name (S)-1-(2-fluorophenyl)-3-(2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazepin-3-yl)-urea [1].
Respiratory syncytial virus (RSV) is the most common cause of lower respiratory tract infections worldwide, yet no effective vaccine or antiviral treatment is available. RSV604 represents the first in a new class of RSV inhibitors targeting the nucleocapsid protein [1].
RSV604 exhibits submicromolar activity against numerous clinical isolates of both A and B subtypes of RSV, indicating a low probability of preexisting resistance mutations in the population [1].
The nucleocapsid protein target is highly conserved and essential for viral replication and transcription, making it an excellent antiviral target. Mutations conferring resistance to RSV604 map to the conserved N-terminal region of the N protein (amino acids 105-139) [1].
Unlike fusion inhibitors that must be given prophylactically, RSV604 is active when given post-infection and can inhibit viral spread even when administered 24 hours post-infection in the HAE cell model [1].
RSV604 is currently in phase II clinical trials, representing the most promising candidate to date for the treatment of RSV disease in humans [1].

388.134

676128-63-5

RSV604 racemate;676128-62-4;RSV604 (R enantiomer);932108-20-8

5279172

White to off-white solid powder

3.8

3

4

3

29

634

1

FC1=C([H])C([H])=C([H])C([H])=C1N([H])C(N([H])[C@]1([H])C(N([H])C2=C([H])C([H])=C([H])C([H])=C2C(C2C([H])=C([H])C([H])=C([H])C=2[H])=N1)=O)=O

MTPVBMVUENFFLL-HXUWFJFHSA-N

InChI=1S/C22H17FN4O2/c23-16-11-5-7-13-18(16)25-22(29)27-20-21(28)24-17-12-6-4-10-15(17)19(26-20)14-8-2-1-3-9-14/h1-13,20H,(H,24,28)(H2,25,27,29)/t20-/m1/s1

(S)-1-(2-fluorophenyl)-3-(2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazepin-3-yl)urea

RSV-604 A-60444 RSV604 A60444 RSV 604 A 60444

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~100 mg/mL (~257.47 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.44 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.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (6.44 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 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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (6.44 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.

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 (Please use freshly prepared in vivo formulations for optimal results.)