Description：FGI-106 HCl is a novel, highly potent and broad-spectrum antiviral agent with inhibitory activity against multiple viruses. FGI-106 tetraHCl is active against various viruses such as Ebola, Rift Valley and Dengue Fever viruses with EC50s of 100 nM, 800 nM and 400-900 nM, respectively. FGI-106 tetraHCl also inhibits non-hemorrhagic fever viruses HCV and HIV-1 with EC50s of 200 nM and 150 nM, respectively.
Physicochemical Properties
| Molecular Formula | C28H42CL4N6 |
| Molecular Weight | 604.485282421112 |
| Exact Mass | 458.32 |
| Elemental Analysis | C, 55.64; H, 7.00; Cl, 23.46; N, 13.90 |
| CAS # | 1149348-10-6 |
| Related CAS # | FGI-106;501081-38-5 |
| PubChem CID | 44128993 |
| Appearance | Light brown to brown solid powder |
| Hydrogen Bond Donor Count | 6 |
| Hydrogen Bond Acceptor Count | 6 |
| Rotatable Bond Count | 10 |
| Heavy Atom Count | 38 |
| Complexity | 560 |
| Defined Atom Stereocenter Count | 0 |
| SMILES | Cl.Cl.Cl.Cl.N(CCCN(C)C)C1C=C(C)N=C2C=1C=CC1C3C(C=CC=12)=C(C=C(C)N=3)NCCCN(C)C |
| InChi Key | YUQCVPFHAFZFPV-UHFFFAOYSA-N |
| InChi Code | InChI=1S/C28H38N6.4ClH/c1-19-17-25(29-13-7-15-33(3)4)23-11-10-22-21(27(23)31-19)9-12-24-26(18-20(2)32-28(22)24)30-14-8-16-34(5)6;;;;/h9-12,17-18H,7-8,13-16H2,1-6H3,(H,29,31)(H,30,32);4*1H |
| Chemical Name | N1,N7-bis[3-(dimethylamino)propyl]-3,9-dimethylquinolino[8,7-h]quinoline-1,7-diamine tetrahydrochloride |
| Synonyms | FGI-106; FGI106; FGI 106; FGI-106 HCl; FGI-106 free base; 501081-38-5; NSC-306365; UNII-BRT994456W; BRT994456W; 1-N,7-N-bis[3-(dimethylamino)propyl]-3,9-dimethylquinolino[8,7-h]quinoline-1,7-diamine; Quino(8,7-H)quinoline-1,7-diamine, N1,N7-bis(3-(dimethylamino)propyl)-3,9-dimethyl-; Quino[8,7-h]quinoline-1,7-diamine, N1,N7-bis[3-(dimethylamino)propyl]-3,9-dimethyl-; FGI-106 hydrochloride; FGI-106 tetrahydrochloride; |
| 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 Note: Please store this product in a sealed and protected environment, avoid exposure to moisture. |
| 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 | Broad-spectrum antiviral; HIV-1; Ebola; Rift Valley; Dengue Fever viruses |
| ln Vitro |
Applying 2 μM FGI-106 resulted in a 4 log decrease in infectious viral titers in comparison to matched controls. The estimated EC90 for inhibiting viral killing of host cells (Vero E6 cells) was 0.6 μM. Treatment with FGI-106 inhibits the replication of various virus families, such as positive- and negative-strand RNA viruses, in cell-based assays[1]. Identification of FGI-106, a small molecule active against Ebola virus [1] The FGI-106 compound was selected based on its ability to consistently inhibit Ebola virus replication in a dose-dependent manner. The antiviral activity of FGI-106 was originally described in Section 2 and its antiviral activity was confirmed using Ebola virus plaque enumeration as a direct measure of viral replication (Fig. 2A). These studies revealed that treatment with 2 μM FGI-106 mediated a 4 log reduction in infectious viral titers relative to matched controls, with an EC90 for inhibition of viral killing of host cells estimated to be 0.6 μM. Researchers then considered the possibility that the antiviral activity observed herein might simply reflect toxicity to the host cells. Additional investigation sought to determine the level of drug, which would cause general cytotoxicity (independent of viral infection). In vitro toxicity testing revealed that toxicity to Vero E6 cells was observed at higher concentrations than was observed for antiviral activity (Fig. 2B). For example, the concentration of drug required for cellular cytotoxicity (CC50) for Vero E6 was estimated to be 10 μM, whereas antiviral activity was observed at much lower doses. Likewise, FGI-106 was not toxic to other host cells utilized herein, including Marc-145 (CC50 > 50 μM), PK-15 (CC50 >25 μM) DC-Sign (CC50 >20 μM) and MT-4 (CC50 = 6 μM). Broad-spectrum antiviral activity of FGI-106 [1] Given the need for new therapeutics for VHFs, cell-based assays were conducted to screen FGI-106 against a panel of unrelated VHF viruses, including Rift Valley Fever virus (RVFV) and Dengue Fever virus (Fig. 3A and B). FGI-106 also inhibited non-hemorrhagic fever viruses, including hepatitis C virus and human immunodeficiency virus (HIV) (Fig. 3C and D and data not shown). Similar to the findings with Ebola virus, the EC90 values for these viruses were much lower than the levels of drug that caused toxicity to the host cells (defined as the CC50), thus precluding that these agents blocked viral production simply through toxic effects to the host (summarized in Table 1). The cell systems under investigation included those isolated from solid tissues (Vero E6, Huh-7) or lymphoid cells (MT-4, DC-Sign) as well as cells derived from human or non-human sources. Altogether, these findings suggest FGI-106 possesses broad-spectrum activity against a wide array of different VHF pathogens and host cell types. |
| ln Vivo |
Zaire EBOV mortality is reduced in a dose-dependent manner by FGI-106 (0.1–5 mg/kg; intraperitoneal injection; treatments on days 2 and 5; C57BL/6 or BALB/c mice) treatment[1]. Efficacy of FGI-106 in mouse models of Ebola virus infection [1] While the broad-spectrum antiviral activity in cell-based assays was intriguing, animal-based efficacy represents a greater challenge. Prior to initiating investigation of FGI-106 in mice, it was necessary to determine the bioavailability of the molecule, particularly within relevant target organs. Since our goal was to focus on Ebola virus, a panel of pharmacokinetic studies were conducted to evaluate serum levels of FGI-106 as well as accumulation within organs that are normally targeted by Ebola virus: kidney, liver and spleen. FGI-106 was administered intravenously at 3 mg/kg to C57BL/6 mice and serum samples collected over time (at 0, 5, 15, 30, 60, 180 and 300 min) for analysis of FGI-106 levels using tandem mass spectrometry. These studies revealed maximal concentration 5 min after injection, with an estimated serum half-life of approximately 1.8 h (see Fig. 4A). This rapid depletion from serum led us to ask if FGI-106 might efficiently distribute into the spleen, liver, and kidney. For this, groups of three C57BL/6 mice received a 3 mg/kg intravenous dose of FGI-106 for 6 h. This particular time point was selected based on prior experience that 6 h is sufficient time to allow a drug to transit from the blood and accumulate within organs. The animals were sacrificed and the lung, liver, kidney and spleen harvested for mass spectrometry-based assessment of FGI-106 concentrations. These studies revealed that the compound had entered organs at levels ranging from 19.5 (spleen) to 43.1 μg/g (kidney) (Fig. 4B). Such findings suggested potential promise for efficacy in animals in light of cell-based assays, which had estimated an EC90 of 0.004 μg/g for Ebola virus. [1] To evaluate the antiviral activity of FGI-106 in mouse models, Ebola virus was selected based on the unmet needs for an effective antiviral for Ebola virus and the availability of animal models. In our first set of studies, C57BL/6 mice were infected with a mouse-adapted strain of Zaire EBOV (Bray et al., 1998) (known hereafter as MA-ZEBOV). Animals were treated with different doses of FGI-106 and challenged after an hour with Ebola virus (1000 pfu/animal, 3000× LD50) followed by two additional injections of FGI-106 at 24 and 72 h post-infection. These time points were selected to maintain drug exposure throughout the study. This prophylaxis model increased the likelihood that drug was available to the host prior to infection. Due to safety constraints when working in a BSL4 setting, we were limited to treating animals via intraperitoneal injection. Treatment with FGI-106 decreased mortality from MA-EBOV in a dose-dependent manner (Fig. 5). For example, treatment with 2 (p < 0.0002) or 5 mg/kg (p < 0.0002) was sufficient to protect animals from Ebola virus. Lower doses of FGI-106 treatment decreased survival. For example, subjects treated with 1 mg/kg FGI-106 had a mean survival time of 12.40 ± 0.95 days (p = 0.029) and 0.5 mg/kg treatment resulted in a mean survival time of 13.4 ± 1.03 days (p = 0.0018) as compared with vehicle-treated controls (Mean survival of 9.8 ± 0.63 days). Treatment with 0.1 mg/kg FGI-106 (mean survival of 11.1 days; p = 0.1726) did not significantly improve survival following Ebola virus challenge. Consistent with our earlier findings, prophylactic treatment with FGI-106 treatment also protected BALB/c mice whereas a matched vehicle control did not (Fig. 6, blue diamonds versus open diamonds, respectively). [1] Researchers progressively increased the stringency of our efficacy studies by delaying treatment until after viral infection. Using this therapeutic model, FGI-106 treatment was initiated 1 day after a lethal challenge with 1000 pfu (3000× LD50) of MA-EBOV. FGI-106 treatment conferred protection from Ebola virus (mean survival time of 13.3 ± 0.63 days), even under these therapeutic conditions (Fig. 6, red circles and green squares). In a further escalation of stringency, mice were treated with a single dose of FGI-106, administered 1, 2, or 3 days after a lethal challenge (1000 pfu; 3000× LD50). A single dose of FGI-106 administered 1 day post-infection sufficed to confer protection. Fig. 7; p < 0.0001 for each FGI-106 treatment group relative to its matched vehicle control group). When delayed to 2 days post-infection, treatment trended towards increased survival (mean survival of 10.9 ± 0.88 days as compared with vehicle-treated controls, 9.0 days), but this effect was not statistically significant (p = 0.1276; Fig. 7). Likewise, when initiation of treatment was delayed to 3 days post-infection, FGI-106 treatment did not decrease lethality (mean survival time of 9.90 ± 0.21 days; p = 0.1276). [1] Researchers further escalated the stringency of the experimental conditions by asking if a single dose of FGI-106, administered 24 h post-infection, would be sufficient to protect animals from a lethal challenge with Ebola virus. Indeed, a single dose of FGI-106 (5 mg/kg) conferred protection and in a dose-dependent manner (Fig. 8A). To examine this outcome in greater detail, a parallel series of studies were conducted in which subjects were sacrificed 3 days post-infection. This particular time point was selected since it would allow us to evaluate viral propagation prior to the onset of mortality, which generally starts 4–6 days post-infection and known sites of Ebola virus proliferation (kidney, spleen, liver) were harvested (Fig. 8B). Consistent with the survival of animals treated with FGI-106 (a single dose of 5 mg/kg administered 1 day post-infection), plaque analyses demonstrated a dramatic reduction in MA-EBOV in the liver, spleen and kidney of FGI-106 treated-animals relative to matched controls. |
| Enzyme Assay |
Virus yield reduction assay [1] All products used for cell culture were obtained from Invitrogen. Confluent Vero E6 cells in 24-well plates were treated with FGI-106 diluted in cell-culture medium or with medium only (negative control). Following overnight incubation, medium was removed and cells were infected with Zaire EBOV (ZEBOV; MOI = 1). After 1 h, excess virus was removed and media containing FGI-106 at pretreatment concentrations was replenished. Culture supernatants were collected after 48 h and viral titers were quantified by standard plaque assay using Vero E6 cells. The plaque assays for Rift Valley Fever virus (MP-12; MOI = 1) were conducted after 24 h of infection in the presence of the indicated amounts of FGI-106. To evaluate Dengue Fever virus, DC-Sign Raji cells were infected with Dengue (isolates DEN1, DEN2, DEN3 or DEN4; MOI = 0.1) in the presence or absence of compound for 72 h as previously reported (Sun et al., 2009). Flow cytometric assessment of cell staining with 2H2 monoclonal antibody (Sun et al., 2009), which recognizes a Dengue complex-specific antigen, provided an assay of infection with Dengue Fever virus (Mady et al., 1991). All assays were repeated at least three times and representative findings are shown. |
| Cell Assay |
Evaluation of HCV [1] To evaluate release of infectious HCV particles, human hepatoma cells were infected with a recombinant hepatitis C virus (HCV) that expresses a renilla luciferase at IP Pharmaceuticals. At 2 h post-infection, HCV-infected cells were incubated with compounds at concentrations ranging from 30 to 0.04 μM in a 3-fold serial dilution (30, 10, 3.33, 1.11, 0.37, 0.12, and 0.04 μM). At 3 days post-infection, cells were lysed in a buffer included in a Promega luciferase assay kit. The level of luciferase expression was determined by luciferase assay. To evaluate reduction of HCV protein, Huh-7 cells were infected with HCV. At 2 h post-infection, HCV was removed and cells were washed once with PBS. HCV-infected cells were then incubated with inhibitors at the indicated concentrations at 37 °C for 3 days. Cell lysate was prepared by extracting cells in 50 μl RIPA buffer. The level of HCV NS5A protein was determined by Western blot analysis using an NS5A-specific monoclonal antibody. In vitro testing of HIV [1] MT-4 cells were infected with HIV-1 NL4–3 at an MOI of 0.001 by low speed centrifugation (1200 × g) for 1 h (Adachi et al., 1986). Cells were seeded in a 96-well plate (1.5 × 105 in 100 μl culture medium per well) (Wei et al., 2002). Serial dilution of FGI-106 was immediately added in triplicate in 50 μl culture medium per well. Half of the supernatants were refreshed every day starting from day 3 pi in the presence of same concentration of FGI-106. The collected supernatants were then transferred to the TZM-bl indicator cell line for examination of viral production in FGI-106 treated samples. Relative Luminescence Unit (RLU) was obtained on TZM-bl cells after they were treated with Bright-Glo Luciferase Assay System 3 days later. The percentages of inhibition of viral production by FGI-106 were calculated as: (RLU from mock-treated samples − RLU from FGI-106 treated samples)/RLU from mock-treated samples × 100. Normal MT-4 cells were treated with serial dilution of FGI-106 as same as above and its cytotoxicity was measured by CytoTox96 Non-Radioactive Cytotoxicity Assay according to the Manufacturer's instruction. |
| Animal Protocol |
Animal Model: The Ebola virus (EBOV) is injected into male or female C57BL/6 or BALB/c mice (6–10 weeks of age)[1]. Dosage: 0.1 mg/kg, 0.5 mg/kg, 1 mg/kg, 2 mg/kg, 5 mg/kg Administration: Intraperitoneal injection; treatments on days 2 and 5 Result: Decreased mortality from Zaire EBOV in a dose-dependent manner. Male or female C57BL/6 or BALB/c mice were used at 6–10 weeks of age at the initiation of experiments. Mice were housed in microisolator cages and were provided water and chow ad libitum. FGI-106 was delivered to mice (n = 10 per treatment group) by intraperitoneal (IP) injection in a vehicle of 0.9 percent saline. To challenge mice, 1000 plaque forming units (pfu; 3000× LD50) of mouse-adapted EBOV (Bray et al., 1998) was delivered by IP injection under biosafety level 4 containment at the United States Army Medical Research Institute for Infectious Diseases. The mouse studies were performed at least six times and representative findings are shown. For studies using a prophylaxis setting, the mice were treated with drug, administered intraperitoneally, 2 h prior to challenge with Ebola virus and then again 2 and 5 days post-challenge. For studies using a therapeutic setting, the animals were treated 1, 2 or 3 days post-infection as indicated in the Figure Legends. To evaluate the statistical significance, the samples were subjected to Kaplan Meier survival analysis and log-rank tests with stepdown Bonferroni adjustment to compare survival curves among groups.[1] To evaluate EBOV organ viral burdens in mice, animals were euthanized by CO2 asphyxation, and kidney, spleen, and liver samples were harvested, weighed, and homogenized in cell-culture medium. Homogenized tissues were centrifuged and the supernatant was stored at −80 °C. Supernatants were subjected to standard plaque assay using Vero E6 cells.[1] To evaluate pharmacokinetic parameters, male C57BL/6 mice were administered a dosing solution of 3 mg/kg FGI-106 intravenously. The subjects were sacrificed at the times indicated (0, 5, 15, 30, 60, 180 and 300 min; n = 3 per group) and plasma samples were analyzed by mass spectrometry at Absorption Systems, LP. The experimental readouts included maximum plasma concentration, half-life, area under the curve (calculated to the last available time point, t = 300 min) and an estimated area under the curve (extrapolated to infinity). For studies of organ accumulation, tissues samples were harvested from liver, kidney and spleen and subjected to the same mass spectrometric investigation. Please note organ accumulation data are reported as mg/kg, whereas plasma levels are reported as mg/mL.[1] |
| ADME/Pharmacokinetics | While the broad-spectrum antiviral activity in cell-based assays was intriguing, animal-based efficacy represents a greater challenge. Prior to initiating investigation of FGI-106 in mice, it was necessary to determine the bioavailability of the molecule, particularly within relevant target organs. Since our goal was to focus on Ebola virus, a panel of pharmacokinetic studies were conducted to evaluate serum levels of FGI-106 as well as accumulation within organs that are normally targeted by Ebola virus: kidney, liver and spleen. FGI-106 was administered intravenously at 3 mg/kg to C57BL/6 mice and serum samples collected over time (at 0, 5, 15, 30, 60, 180 and 300 min) for analysis of FGI-106 levels using tandem mass spectrometry. These studies revealed maximal concentration 5 min after injection, with an estimated serum half-life of approximately 1.8 h (see Fig. 4A). This rapid depletion from serum led us to ask if FGI-106 might efficiently distribute into the spleen, liver, and kidney. For this, groups of three C57BL/6 mice received a 3 mg/kg intravenous dose of FGI-106 for 6 h. This particular time point was selected based on prior experience that 6 h is sufficient time to allow a drug to transit from the blood and accumulate within organs. The animals were sacrificed and the lung, liver, kidney and spleen harvested for mass spectrometry-based assessment of FGI-106 concentrations. These studies revealed that the compound had entered organs at levels ranging from 19.5 (spleen) to 43.1 μg/g (kidney) (Fig. 4B). Such findings suggested potential promise for efficacy in animals in light of cell-based assays, which had estimated an EC90 of 0.004 μg/g for Ebola virus. [1] |
| References |
[1]. Development of a broad-spectrum antiviral with activity against Ebola virus. Antiviral Res. 2009 Sep;83(3):245-51. |
| Additional Infomation |
We report herein the identification of a small molecule therapeutic,FGI-106, which displays potent and broad-spectrum inhibition of lethal viral hemorrhagic fevers pathogens, including Ebola, Rift Valley and Dengue Fever viruses, in cell-based assays. Using mouse models of Ebola virus, we further demonstrate that FGI-106 can protect animals from an otherwise lethal infection when used either in a prophylactic or therapeutic setting. A single treatment, administered 1 day after infection, is sufficient to protect animals from lethal Ebola virus challenge. Cell-based assays also identified inhibitory activity against divergent virus families, which supports a hypothesis that FGI-106 interferes with a common pathway utilized by different viruses. These findings suggest FGI-106 may provide an opportunity for targeting viral diseases. [1] There are currently no antivirals to treat Ebola virus infection in humans, which can cause 40–90% mortality (Zampieri et al., 2007). The major finding of our present study is the identification of FGI-106, a novel compound that confers protection from Ebola virus when evaluated within either a prophylactic or therapeutic setting. We also demonstrate that FGI-106 demonstrates activity against multiple and genetically distinct viruses. One feature of the present finding is the demonstration of broad-spectrum antiviral activity. In cell-based assays, treatment with FGI-106 inhibited viral replication by divergent virus families, including positive and negative-strand RNA viruses. Based on this range of activity we suspect the compound may target a common host factor required for replication of diverse virus families. This outcome would be consistent with an emerging concept of host-oriented therapeutics, which advocates a strategy of safely targeting critical host mechanisms that are essential of the virus, but not for normal host cell function or survival. Based on the broad-spectrum nature of the antiviral activity, we postulate the mechanistic basis of FGI-106 antiviral activity involves a conserved host pathway. For example, comparable efficacy was observed using human, mouse or primate cells, suggesting the antiviral effects of FGI-106 are not unique to a particular cell type or species. The broad-spectrum nature of FGI-106 antiviral activity may suggest FGI-106 targets a fundamental and highly conserved host mechanism. The concept of host-based targeting is not unique to the finding herein. For example, ribavirin exerts its effects in part by inhibiting the host cell enzyme, IMP dehydrogenase (Franchetti and Grifantini, 1999, Goldstein and Colby, 1999, Robins et al., 1985). Likewise, inhibitors of host-encoded S-adenosylhomocysteine inhibit methylation of viral mRNA cap structures (Hunt, 1989, Rose et al., 1977). Although drugs like Ribavirin have been utilized to suppress the replication of a wide range of viruses, it is associated with toxicity (Johnson, 1990, Kumar et al., 2002). Future investigation will be important to identify the molecular basis by which FGI-106 conveys its robust antiviral activity. Once the targets and pathways have been identified, it will be important to determine the potential for drug-based toxicity, which could impact its ultimate application in the clinic. Conventional antiviral approaches target viral molecules to minimize host toxicity (De Clercq, 2008). However, the combination of a high replication rate and an error prone polymerase (lacking proofreading activity) favors selection of drug-resistant variants. We hypothesize that host-directed therapeutics will not be as susceptible to such resistance mechanisms. Consistent with this idea, no virus has been reported to develop resistance to ribavirin, which acts through a host-based mechanism. Moreover, host-based targeting strategies in general and FGI-106 in particular, could provide an unprecedented opportunity to deploy a therapeutic option with application to many different virus types. A broad-spectrum antiviral could be particularly useful under conditions where there is insufficient time or ability to identify the causative pathogen. Further investigation will be important to address the potential for FGI-106 and other host-based antivirals and such findings could have for the development of novel therapeutic options that are broad-spectrum in their application and durable in antiviral activity. [1] |
Solubility Data
| Solubility (In Vitro) | DMSO : ~6.67 mg/mL (~11.03 mM) |
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 0.67 mg/mL (1.11 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 6.7 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: ≥ 0.67 mg/mL (1.11 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 6.7 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. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.6543 mL | 8.2714 mL | 16.5429 mL | |
| 5 mM | 0.3309 mL | 1.6543 mL | 3.3086 mL | |
| 10 mM | 0.1654 mL | 0.8271 mL | 1.6543 mL |