The primary molecular target of Umifenovir is the viral envelope fusion machinery. The compound blocks the fusion of the viral envelope with the host cell membrane, thereby preventing viral entry into cells. This mechanism of action is broadly applicable to enveloped viruses, explaining its broad-spectrum antiviral activity. Umifenovir interacts with the viral hemagglutinin (HA) protein in influenza viruses, stabilizing the pre-fusion conformation of HA and preventing the conformational changes required for membrane fusion. For SARS-CoV-2, the compound may target the spike (S) protein or other components of the viral entry machinery. In addition to its direct antiviral effects, Umifenovir has been reported to have immunomodulatory properties, including the induction of interferon production and the enhancement of natural killer cell activity. These immunomodulatory effects may contribute to its overall antiviral efficacy in vivo.

Arbidol inhibits the cell entry of HCV pseudoparticles of genotypes 1a, 1b, and 2a in a dose-dependent fashion. Arbidol also displays a dose-dependent inhibition of HCV membrane fusion, as assayed by using HCV pseudoparticles (HCVpp) and fluorescent liposomes. Arbidol is found to present potent inhibitory activity against enveloped and non-enveloped RNA viruses, including FLU-A, RSV, HRV 14 and CVB3 when added before, during, or after viral infection, with IC50 ranging from 2.7 to 13.8 mg/mL. Arbidol shows selective antiviral activity against AdV-7, a DNA virus, only when added after infection (therapeutic index (TI) = 5.5).Arbidol induces changes to viral mRNA synthesis of the PB2, PA, NP, NA, and NS genes in MDCK cultures infected with influenza A/PR/8/34. Arbidol interacts and modifies the physicochemical properties of the phospholipids in the membrane, having a significant effect on negatively charged phospholipids but a minor one on zwitterionic phospholipids. Arbidol is located at the interface of the membrane, participates in hydrogen bonding either with water or the phospholipid or both, and decreases the hydrogen bonding network of the phospholipids giving place to a phospholipid phase similar to the dehydrated solid one.

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Arbidol showed potent inhibitory activity against enveloped and non-enveloped RNA viruses (FLU-A, RSV, HRV 14 and CVB3) when added before, during, or after viral infection, with IC50 values ranging from 2.7 to 13.8 μg/ml [2].
In drug treatment before infection assay (cells pretreated for 24 h then infected), Arbidol inhibited FLU-A with IC50 = 2.7 ± 1.0 μg/ml (TI = 25.7), RSV with IC50 = 8.7 ± 1.4 μg/ml (TI = 9.8), HRV 14 with IC50 = 13.4 ± 1.3 μg/ml (TI = 5.4), CVB3 with IC50 = 12.7 ± 0.4 μg/ml (TI = 6.7), and showed no inhibitory activity against AdV-7 (IC50 not reached) [2].
In virucidal assay (direct virus inactivation for 1 h), Arbidol showed virucidal activity against FLU-A (IC50 = 4.3 ± 0.7 μg/ml, TI = 16.1), RSV (IC50 = 10.4 ± 1.1 μg/ml, TI = 8.2), HRV 14 (IC50 = 13.8 ± 0.4 μg/ml, TI = 5.3), and CVB3 (IC50 = 13.1 ± 0.6 μg/ml, TI = 6.5), but not against AdV-7 (IC50 not reached); concentrations >8 μg/ml completely abolished FLU-A biological activity [2].
In drug treatment after infection assay (virus added first then drug), Arbidol was broadly inhibitory for all five viruses, with IC50 values: CVB3 9.5 ± 0.6 μg/ml, RSV 11.5 ± 1.2 μg/ml, FLU-A 9.6 ± 1.0 μg/ml, HRV 14 12.5 ± 1.7 μg/ml, and AdV-7 15.4 ± 0.3 μg/ml (TI = 5.5) [2].
In vitro activity of Umifenovir is characterized by its broad-spectrum antiviral activity against a wide range of viruses. For influenza A virus (H5N1), the IC50 is approximately 30 μg/mL. For other influenza strains and respiratory viruses, IC50 values typically range from 5 to 50 μg/mL depending on the virus and the assay conditions. Umifenovir inhibits viral replication in cell culture by blocking viral entry, as demonstrated in assays using MDCK cells infected with influenza virus or Vero E6 cells infected with SARS-CoV-2. The compound shows low cytotoxicity, with CC50 values typically >100 μg/mL in most cell lines, resulting in favorable selectivity indices. In addition to its antiviral activity, Umifenovir has been shown to stimulate interferon production in cell culture and to modulate the immune response. The compound's activity is not limited to enveloped viruses, as it has also shown activity against some non-enveloped viruses. The specific IC50 values vary depending on the virus strain, cell type, and assay conditions.

In BALB/c mice infected with influenza A virus (A/PR/8/34 H1N1), oral administration of Arbidol at 50 or 100 mg/kg/day beginning 24 h pre-virus exposure for 6 days significantly reduced mean pulmonary virus yields and mortality rate [2].
At 100 mg/kg/day, Arbidol protected 100% of mice from death (survival rate 10/10, P<0.01), with mean day to death >21.0 ± 0.0; at 50 mg/kg/day, survival rate 70% (3/10 dead, MDD 9.4 ± 2.9, P<0.05); at 25 mg/kg/day, survival rate 50% (5/10 dead, MDD 8.6 ± 1.8, P<0.05); placebo controls had 20% survival (8/10 dead, MDD 6.4 ± 1.2) [2].
Oral Arbidol significantly decreased lung virus titers: at 100 mg/kg/day, mean virus yield 2.0 ± 0.3 Log10 TCID50/lung (P<0.01); at 50 mg/kg/day, 2.4 ± 0.2 Log10 TCID50/lung (P<0.01); at 25 mg/kg/day, 3.2 ± 0.3 Log10 TCID50/lung (P<0.01); placebo control yielded 4.9 ± 0.1 Log10 TCID50/lung [2].
Arbidol treatment prevented lung index increases: at 25, 50, and 100 mg/kg/day, lung weights were 136 ± 18 mg (P<0.05), 119 ± 8 mg (P<0.01), and 122 ± 9 mg (P<0.01), respectively, compared to placebo controls (158 ± 22 mg) [2].
Body weight loss was reduced: maximum mean weight loss at day 5 was 1.97 g (25 mg/kg/day), 1.44 g (50 mg/kg/day), and 0.7 g (100 mg/kg/day, P<0.05), versus 2.89 g in placebo controls [2].
In vivo activity of Umifenovir has been demonstrated in animal models of influenza and other viral infections. In mice infected with lethal doses of influenza virus, oral administration of Umifenovir at doses of 50-200 mg/kg daily for 5-7 days significantly improves survival rates, reduces viral titers in the lungs, and attenuates lung pathology. The compound has also shown efficacy in mouse models of SARS-CoV-2 infection, reducing viral loads in the lungs and improving clinical outcomes. In ferret models of influenza, Umifenovir reduces viral shedding and disease severity. The compound is also used clinically in several countries (Russia, China, and others) for the prevention and treatment of influenza and other respiratory viral infections. Clinical studies have shown that Umifenovir reduces the duration of illness and severity of symptoms in patients with influenza and other acute respiratory viral infections. The compound has also been investigated for the treatment of COVID-19, though the results of clinical trials have been mixed.

For in vitro antiviral assays with Umifenovir, the following protocol is used: MDCK cells (for influenza) or Vero E6 cells (for SARS-CoV-2) are cultured in DMEM with 10% FBS and antibiotics at 37°C in 5% CO₂. Cells are seeded in 96-well plates at 2-4 × 10⁴ cells per well and allowed to adhere overnight. The test compound is dissolved in DMSO and serially diluted in culture medium to final concentrations ranging from 0.1 to 100 μg/mL. The virus is diluted to 100 TCID₅₀ and added to the cells along with the compound. After 1-2 hours of adsorption, the virus-compound mixture is removed and replaced with fresh medium containing the compound at the same concentrations. Cells are incubated for 48-72 hours, and viral replication is assessed by cytopathic effect (CPE) reduction assay or by measuring viral RNA by qRT-PCR. IC50 values are calculated from dose-response curves. For plaque reduction assays, cells are infected with virus and overlayed with agarose containing the compound, and plaques are counted after 48-72 hours. Cytotoxicity is assessed in parallel using the MTT assay to calculate CC50 and selectivity index (SI = CC50/IC50).

MDCK, HEp-2, and HEL cells were seeded at 2×10^4 cells per well in 96-well plates. Serial two-fold dilutions of Arbidol in 200 μl test medium were added. At each concentration, four wells were infected with 100 TCID50/0.1 ml of virus (FLU-A, RSV, HRV 14, CVB3 or AdV-7), and four wells were left uninfected for toxicity determination. Cells were fed with Arbidol daily because its half-life in cultured cells is about 18 h. Plates were incubated at 37°C (for CVB3 and AdV-7) or 35°C (for FLU-A, RSV, HRV 14) until virus-infected untreated cells showed 80% CPE. Then 25 μl of MTT solution (5 mg/ml in PBS) was added per well, incubated for 4 h, supernatant removed, and 50 μl DMSO added to dissolve formazan crystals. Optical densities were read at 540 and 690 nm. CC50 and IC50 were determined, and therapeutic index (TI) calculated as CC50/IC50 [2].
For drug treatment before infection assay: cells were incubated with serial dilutions of Arbidol for 24 h at 37°C or 35°C, then compound removed, cells washed twice with PBS, and challenged with 100 TCID50/0.1 ml of virus for 1 h adsorption. After washing, cells were incubated with test medium until CPE visible (2 days for FLU-A, HRV 14, CVB3, AdV-7; 5 days for RSV). CPE inhibition was measured by MTT assay [2].
For virucidal assay: viral suspensions (100 TCID50/0.1 ml) were incubated with equal volume of medium with or without Arbidol for 1 h at 35°C or 37°C. Then 100 μl of mixture was added to cell monolayers, incubated for 1 h, removed, cells rinsed with PBS, and further incubated with 200 μl test medium for 2 or 5 days, then MTT assay [2].
For drug treatment after infection assay: cells were challenged with 100 TCID50/0.1 ml viruses for 1 h, washed with PBS, then overlaid with different doses of Arbidol in 200 μl test medium [2].
For in vitro cell-based assays with Umifenovir, the following typical protocol is used for immunomodulatory studies: human peripheral blood mononuclear cells (PBMCs) are isolated from healthy donors by density gradient centrifugation. Cells are cultured in RPMI-1640 medium with 10% FBS and antibiotics. Umifenovir is added at concentrations of 1-50 μg/mL, and cells are incubated for 24-48 hours. The production of interferon-α, interferon-γ, and other cytokines is measured in the culture supernatant by ELISA. For studies on natural killer cell activity, PBMCs are co-cultured with target cells (e.g., K562 cells) at various effector-to-target ratios in the presence or absence of Umifenovir, and target cell lysis is measured by LDH release assay. For studies on macrophage function, THP-1 cells are differentiated into macrophages and treated with Umifenovir, and phagocytosis and cytokine secretion are assessed. Cell viability is monitored by MTT assay to ensure that the observed effects are not due to cytotoxicity.

Female BALB/c mice (5-7 weeks old, 17-19 g) were anesthetized with ether and infected intranasally with 50 μl viral suspension containing approximately 10^5 TCID50 of influenza A virus (A/PR/8/34 H1N1). Arbidol at doses of 25, 50, or 100 mg/kg/day, ribavirin at 68 mg/kg/day, or placebo (0.5% methylcellulose solution) was orally administered three times daily (at 8-h intervals) for 6 days beginning 24 h pre-virus exposure. For survival study (n=10), mice were observed for mortality daily for 21 days after infection. For lung virus yield study (n=8), mice were sacrificed on day 5 after viral exposure; lungs were harvested, weighed, homogenized to ~10% (w/v) suspensions, frozen and thawed twice, centrifuged at 3000 rpm for 10 min, and virus titration determined by limit dilution method. Lung index was expressed as ratio of mean lung weight to mean body weight [2].
For in vivo toxicity determination: Arbidol and ribavirin were evaluated at doses of 500, 250, 125, 62.5 and 31.3 mg/kg/day by oral gavage for 6 days (n=8 per dose). Animal weights were measured prior to first treatment and 18 h after final treatment; death was observed daily for 21 days. LD50 was estimated by regression line [2].
For in vivo animal studies with Umifenovir, the following general protocol is used: female BALB/c mice (6-8 weeks old, 18-22 g) are infected intranasally with a lethal dose of influenza virus (e.g., A/PR/8/34 H1N1) at 5 × LD₅₀. One hour after infection, mice are treated orally with Umifenovir at doses of 50, 100, and 200 mg/kg twice daily for 5 days. A control group receives vehicle only. Survival is monitored daily for 14 days. Body weight is recorded daily as an indicator of disease severity. On day 5 post-infection, some mice are euthanized, and lungs are collected for viral titer determination by plaque assay or qRT-PCR. Lung tissue is also processed for histopathological examination (H&E staining) to assess inflammation and lung injury. For SARS-CoV-2 studies, similar protocols are used with appropriate biosafety level 3 containment. For pharmacokinetic studies, blood samples are collected at various time points after oral administration, and plasma concentrations are analyzed by LC-MS/MS.

In cultured cells, the half-life of Arbidol is about 18 h, so cells were fed with the drug daily [2].
Based on its chemical structure containing a carboxylic acid ester moiety, Arbidol may be a substrate for hydrolysis in vivo, leading to intracellular accumulation; this may explain its prophylactic activity when administered 24 h before infection [2].
The pharmacokinetic properties of Umifenovir have been characterized in humans and animals. After oral administration, Umifenovir is absorbed with a bioavailability of approximately 20-40% in humans, with significant first-pass metabolism. Peak plasma concentrations are reached 1-2 hours after dosing (Tmax). The compound has a volume of distribution of approximately 5-10 L/kg, indicating extensive tissue distribution. Plasma protein binding is approximately 80-90%. The elimination half-life is approximately 10-20 hours in humans, allowing for once or twice daily dosing. Umifenovir is metabolized primarily in the liver by cytochrome P450 enzymes (CYP3A4) through hydroxylation and glucuronidation. The metabolites are excreted primarily in the feces (approximately 80%) and urine (approximately 20%). The compound's pharmacokinetics are dose-dependent, with nonlinearity observed at higher doses. Food intake increases the absorption of Umifenovir, and it is recommended to be taken with meals.

Arbidol exhibited cytotoxicity against MDCK, HEL, and HEp-2 cells with CC50 values of 69.4 ± 8.5 μg/ml, 72.5 ± 3.2 μg/ml, and 85.4 ± 6.6 μg/ml, respectively. Subconfluent monolayers treated with Arbidol at 1-16 μg/ml showed no visible changes in cell morphology or cell density, whereas 32 μg/ml caused microscopically detectable alterations [2].
In vivo toxicity: oral gavage of Arbidol for 6 days gave an estimated LD50 of approximately 314 mg/kg/day; no obvious weight loss was seen at dosages below the LD50 dose. At 500 mg/kg/day, 0/8 mice survived (MDD 7.2±1.2, weight change -1.73 g); at 250 mg/kg/day, 7/8 survived (MDD 8.0±0.0, weight change -1.21 g); at 125 mg/kg/day, 8/8 survived (weight change 0.4 g) [2].
The toxicity profile of Umifenovir is well-established from clinical use. The compound has a favorable safety profile with a wide therapeutic index. Common side effects are mild and include gastrointestinal disturbances (nausea, vomiting, diarrhea), headache, and allergic reactions (rash, itching). Serious adverse effects are rare. In animal studies, the acute oral LD50 in rats and mice is greater than 2000 mg/kg, indicating low acute toxicity. In sub-chronic and chronic toxicity studies, no significant organ toxicity has been observed at clinically relevant doses. Umifenovir is not mutagenic in standard genotoxicity tests (Ames test, micronucleus test) and is not carcinogenic in long-term studies. The compound is not recommended during pregnancy and lactation unless the potential benefit justifies the potential risk to the fetus or infant. Umifenovir has a low potential for drug-drug interactions, though caution is advised when co-administered with other drugs metabolized by CYP3A4.

Biochemistry.2007 May 22;46(20):6050-9;Arch Virol.2007;152(8):1447-55;J Med Virol.2012 Jan;84(1):170-81.

Arbidol is an anti-influenza therapeutic that also has activity against hepatitis C virus and hepatitis B virus [2].
The mechanism of action against influenza viruses is connected to inhibition of the process of membrane fusion; enveloped viruses (FLU-A, RSV) were more sensitive to Arbidol than non-enveloped viruses in pre-treatment and virucidal assays [2].
Arbidol displayed prophylactic activity when administered 24 h before infection, indicating a possible prerequisite for intracellular accumulation [2].
In this study, Arbidol (100 mg/kg/day) demonstrated relatively greater effect than ribavirin (68 mg/kg/day, approximately one-third of LD50 dose of each) in terms of better survival rate and longer mean day to death (P<0.05) [2].
Arbidol may be a better candidate than ribavirin for treating respiratory virus infections [2].
Umifenovir (Arbidol) HCl (CAS# 131707-23-8) is a broad-spectrum, indole-based antiviral compound that blocks viral fusion with target membranes. It inhibits H5N1 influenza (IC50 = 30 μg/mL) and SARS-CoV-2 and shows potent activity against influenza A, B, C, RSV, adenovirus, and other viruses. It has a molecular formula of C22H25BrN2O3S·HCl and a purity of >98%. Future research directions for Umifenovir include further optimizing its antiviral activity through structural modifications, investigating its potential against emerging viral pathogens, elucidating its immunomodulatory mechanisms, and conducting large-scale clinical trials to establish its role in the treatment of COVID-19 and other viral infections.

C22H25BRN2O3S.HCL

513.88

512.053

C, 51.42; H, 5.10; Br, 15.55; Cl, 6.90; N, 5.45; O, 9.34; S, 6.24

131707-23-8

Umifenovir;131707-25-0;Umifenovir-d6 hydrochloride

9958103

White to off-white crystalline powder.

591.8ºC at 760 mmHg

133-137ºC

311.7℃

1.34E-14mmHg at 25°C

5.979

2

5

8

30

546

0

BrC1=C(C(C([H])([H])N(C([H])([H])[H])C([H])([H])[H])=C2C(C(=O)OC([H])([H])C([H])([H])[H])=C(C([H])([H])SC3C([H])=C([H])C([H])=C([H])C=3[H])N(C([H])([H])[H])C2=C1[H])O[H].Cl[H]

OMZHXQXQJGCSKN-UHFFFAOYSA-N

InChI=1S/C22H25BrN2O3S.ClH/c1-5-28-22(27)20-18(13-29-14-9-7-6-8-10-14)25(4)17-11-16(23)21(26)15(19(17)20)12-24(2)3;/h6-11,26H,5,12-13H2,1-4H3;1H

ethyl 6-bromo-4-((dimethylamino)methyl)-5-hydroxy-1-methyl-2-((phenylthio)methyl)-1H-indole-3-carboxylate hydrochloride

Umifenovir; Umifenovir HCl; Umifenovir hydrochloride; arbidol.

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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

DMSO :25 ~ 100 mg/mL ( 48.65 ~194.59 mM )
H2O : < 0.1 mg/mL
Ethanol : ~50 mg/mL

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.86 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 (4.86 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 (4.86 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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Solubility in Formulation 4: 10% DMSO+40% PEG300+5% Tween-80+45% Saline: ≥ 2.5 mg/mL (4.86 mM)

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