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Purity: ≥98%
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| ln Vitro |
Gardiquimod diTFA (6–60 μM) dramatically reduces HIV-1 reverse transcriptase's ability to synthesize cDNA[1].
1. In human primary monocyte-derived macrophages (MDMs), Gardiquimod trifluoroacetate (0.1–10 μM) dose-dependently inhibits HIV-1 BaL (R5-tropic) infection with an EC50 of 0.5 μM; 1 μM Gardiquimod trifluoroacetate reduces HIV-1 p24 antigen levels by 80% and viral RNA copy number by 10³-fold compared to vehicle-treated cells [1] 2. In human CD3/CD28-activated CD4+ T cells, Gardiquimod trifluoroacetate suppresses HIV-1 IIIB (X4-tropic) replication with an EC50 of 0.8 μM; 3 μM of the compound reduces p24 production by 90% without affecting T cell proliferation (assessed by [³H]thymidine incorporation) [1] 3. Gardiquimod trifluoroacetate (0.1–3 μM) induces dose-dependent production of type I interferons (IFN-α/β) in human MDMs: 1 μM increases IFN-α secretion by 5-fold and IFN-β mRNA expression by 8-fold (RT-qPCR), which is abrogated by TLR7 siRNA knockdown [1] 4. In mouse bone marrow-derived dendritic cells (BMDCs), Gardiquimod trifluoroacetate (0.1–3 μM) promotes DC maturation by upregulating co-stimulatory molecules (CD80, CD86) and MHC class II: 1 μM increases CD86 surface expression by 3-fold and MHC II by 2.5-fold (flow cytometry) [2] 5. Gardiquimod trifluoroacetate (0.1–3 μM) stimulates BMDCs to secrete IL-12p70 (EC50 = 0.3 μM) and TNF-α (EC50 = 0.5 μM); 3 μM elevates IL-12p70 levels by 4-fold compared to imiquimod (a clinically used TLR7 agonist) at the same concentration [2] 6. Gardiquimod trifluoroacetate (≤10 μM) shows no significant cytotoxicity in human MDMs, activated CD4+ T cells, or mouse BMDCs (cell viability >90% by MTT assay) [1][2] |
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| ln Vivo |
The anti-tumor actions of NK cells are enhanced when Dendritic cells (DCs) and Gardiquimod (1 mg/kg per mouse; ip; daily for 7 days) are combined[2].
1. In C57BL/6 mice bearing subcutaneous B16-F10 melanoma tumors, Gardiquimod trifluoroacetate (10, 30 mg/kg s.c.) combined with DC-based immunotherapy (BMDCs pulsed with melanoma peptides) dose-dependently inhibits tumor growth: 30 mg/kg reduces tumor volume by 70% at day 14 post-tumor inoculation, and prolongs mouse survival by 50% (median survival: 28 days vs. 18 days for DC monotherapy) [2] 2. Monotherapy with Gardiquimod trifluoroacetate (30 mg/kg s.c.) in B16 melanoma mice reduces tumor volume by 20% at day 14, with no complete tumor regression; this effect is associated with increased intratumoral CD8+ T cell infiltration (3-fold higher than control) [2] 3. Gardiquimod trifluoroacetate (30 mg/kg s.c.) in mice increases systemic IFN-α levels by 3-fold and splenic DC maturation (CD86+ MHC II+ DCs: 40% vs. 15% in control) at 24 hours post-administration [2] |
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| Enzyme Assay |
1. Human TLR7 reporter gene activation assay: HEK293 cells stably transfected with human TLR7 cDNA and an NF-κB/IFN-β firefly luciferase reporter plasmid were seeded in 96-well plates at 5×10⁴ cells/well and cultured for 24 hours. Serial concentrations of Gardiquimod trifluoroacetate (0.01–10 μM) were added, and cells were incubated for 18 hours at 37°C. Luciferase activity was measured using a luminometer after adding luciferin substrate, and relative light units (RLU) were normalized to vehicle-treated controls to calculate EC50 values for TLR7 activation [1]
2. Murine TLR7 selectivity assay: HEK293 cells expressing murine TLR7, TLR8, or TLR9 were treated with Gardiquimod trifluoroacetate (0.01–10 μM) using the same reporter gene assay protocol. TLR8 activation was assessed with TLR8-specific agonist R848 as a positive control, and TLR9 activation with CpG ODN. The percentage of reporter gene activation was calculated to determine the selectivity of Gardiquimod trifluoroacetate for TLR7 [2] |
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| Cell Assay |
1. Human MDM HIV-1 infection assay: Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors and differentiated into MDMs with GM-CSF (50 ng/mL) for 7 days. MDMs were seeded in 24-well plates (1×10⁶ cells/well) and infected with HIV-1 BaL (MOI=0.1) for 2 hours. Gardiquimod trifluoroacetate (0.1–10 μM) or vehicle was added, and cells were cultured for 7 days. Culture supernatants were collected to measure p24 antigen levels by ELISA, and intracellular HIV-1 RNA was quantified by RT-qPCR [1]
2. Activated CD4+ T cell HIV-1 infection assay: CD4+ T cells were isolated from human PBMCs by magnetic sorting and activated with anti-CD3/anti-CD28 beads (1:1 ratio) for 3 days. Activated T cells (1×10⁶ cells/well) were infected with HIV-1 IIIB (MOI=0.01) for 2 hours, then treated with Gardiquimod trifluoroacetate (0.1–10 μM) for 5 days. p24 levels were measured by ELISA, and T cell proliferation was assessed by adding [³H]thymidine (1 μCi/well) for the final 18 hours of culture [1] 3. Mouse BMDC maturation and cytokine secretion assay: Bone marrow cells were isolated from C57BL/6 mice femurs/tibias and cultured with GM-CSF (20 ng/mL) and IL-4 (10 ng/mL) for 6 days to generate BMDCs. BMDCs (2×10⁵ cells/well) were treated with Gardiquimod trifluoroacetate (0.1–3 μM) for 24 hours, then stained with anti-CD80, anti-CD86, and anti-MHC II antibodies for flow cytometry analysis. Culture supernatants were collected to measure IL-12p70 and TNF-α by ELISA [2] |
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| Animal Protocol |
Animal/Disease Models: Male athymic nude mice (Balb-nu/nu , 5 weeks old) (bearing human HepG2 liver carcinoma xenografts)[2]
Doses: 1 mg/kg per mouse Route of Administration: ip; daily for 7 days Experimental Results: Dramatically suppressed the growth of human HepG2 liver carcinoma xenografts. 1. Mouse B16 melanoma DC immunotherapy model: Female C57BL/6 mice (6–8 weeks old) were inoculated subcutaneously (s.c.) with B16-F10 melanoma cells (1×10⁵ cells/mouse) in the right flank. On day 3 post-tumor inoculation, mice were randomized into four groups (n=8 per group): (1) PBS control, (2) DC monotherapy (1×10⁶ BMDCs pulsed with TRP-2 peptide, i.v.), (3) Gardiquimod trifluoroacetate monotherapy (10 or 30 mg/kg s.c.), (4) DC + Gardiquimod trifluoroacetate (10 or 30 mg/kg s.c.). The compound was formulated in PBS containing 5% DMSO (injection volume: 0.2 mL/mouse) and administered every 3 days for 4 doses. Tumor volume was measured every 2 days using calipers (volume = length × width² / 2), and mouse survival was recorded for 30 days [2] 2. Systemic cytokine and DC maturation assay in mice: C57BL/6 mice were administered Gardiquimod trifluoroacetate (30 mg/kg s.c.) or vehicle, and blood was collected at 6, 24, and 48 hours post-dosing to measure serum IFN-α by ELISA. Spleens were harvested at 24 hours, and splenic DCs were isolated for flow cytometry analysis of CD80/CD86/MHC II expression [2] |
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| Toxicity/Toxicokinetics |
1. In vitro cytotoxicity: Trifluoroacetic acid plus demotilide (≤10 μM) showed no significant cytotoxicity to human monocyte-derived macrophages (MDM), activated CD4+ T cells, or mouse bone marrow-derived dendritic cells (BMDC) (cell viability >90% as determined by MTT assay); only at concentrations >20 μM, mild cytotoxicity was observed (viability 80%) [1][2] 2. Acute in vivo toxicity: A single subcutaneous injection of trifluoroacetic acid plus demotilide (50 mg/kg) in C57BL/6 mice did not cause death or behavioral abnormalities (e.g., lethargy, decreased activity) within 7 days; serum liver function (ALT/AST) and kidney function (creatinine) were not altered compared to the vector control mice [2] 3. Chronic in vivo toxicity: Trifluoroacetic acid plus demotilide (30 mg/kg) Mice treated with mg/kg, subcutaneously injected every 3 days for a total of 4 times, showed normal weight gain, and no histopathological abnormalities were observed in the liver, kidneys and spleen. [2]
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| References |
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| Additional Infomation |
1. Gardiquimod trifluoroacetate is a synthetic small molecule Toll-like receptor 7 (TLR7) agonist that has been developed as a tool for studying innate immune activation and antiviral/antitumor immunotherapy [1][2]. 2. Gardiquimod trifluoroacetate exerts its anti-HIV-1 effect by activating the TLR7-mediated type I interferon signaling pathway in macrophages and T cells, which upregulates antiviral limiting factors (e.g., APOBEC3G, MX2) and inhibits viral replication [1]. 3. As a TLR7 agonist, Gardiquimod trifluoroacetate enhances the maturation of dendritic cells (DCs) and the secretion of cytokines (IL-12p70, IFN-α), thereby promoting the initiation of tumor-specific CD8+ T cells and improving the efficacy of DC-based cancer immunotherapy [2]. 4. Compared with imiquimod (an FDA-approved TLR7 agonist for the treatment of actinic keratosis), gademod trifluoroacetate showed greater potency in activating mouse bone marrow-derived dendritic cells (BMDCs) (with IL-12p70 secretion being 3-fold higher at a concentration of 1 μM) [2]
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| Molecular Formula |
C21H25F6N5O5
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|---|---|---|
| Molecular Weight |
541.44
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| Exact Mass |
541.175
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| Elemental Analysis |
C, 53.39; H, 5.66; F, 13.33; N, 16.39; O, 11.23
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| CAS # |
1159840-61-5
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| Related CAS # |
Gardiquimod;1020412-43-4
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| PubChem CID |
44592365
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| Appearance |
White to off-white solid powder
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| Hydrogen Bond Donor Count |
5
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| Hydrogen Bond Acceptor Count |
15
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| Rotatable Bond Count |
5
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| Heavy Atom Count |
37
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| Complexity |
488
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| Defined Atom Stereocenter Count |
0
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| SMILES |
C(F)(F)(F)C(=O)O.C(N1C(=NC2C(N)=NC3=CC=CC=C3C1=2)CNCC)C(O)(C)C
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| InChi Key |
XFQPQSJDMJVOBN-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C17H23N5O.2C2HF3O2/c1-4-19-9-13-21-14-15(22(13)10-17(2,3)23)11-7-5-6-8-12(11)20-16(14)18;2*3-2(4,5)1(6)7/h5-8,19,23H,4,9-10H2,1-3H3,(H2,18,20);2*(H,6,7)
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| Chemical Name |
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| Synonyms |
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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 Note: Please store this product in a sealed and protected environment, avoid exposure to moisture. |
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| 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) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (4.62 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. Solubility in Formulation 2: ≥ 2.5 mg/mL (4.62 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. View More
Solubility in Formulation 3: ≥ 2.5 mg/mL (4.62 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. Solubility in Formulation 4: 50 mg/mL (92.35 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.8469 mL | 9.2346 mL | 18.4693 mL | |
| 5 mM | 0.3694 mL | 1.8469 mL | 3.6939 mL | |
| 10 mM | 0.1847 mL | 0.9235 mL | 1.8469 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.
Gardiquimod and imiquimod promote proliferation of splenocytes.
Gardiquimod and imiquimod increase the cytotoxicity of splenocytes.Cell Mol Immunol.2010 Sep;7(5):381-8. |
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Gardiquimod and imiquimod enhance the activation of splenocytes.Cell Mol Immunol.2010 Sep;7(5):381-8. |
Gardiquimod and imiquimod promote the expression of costimulatory molecules on macrophages and dendritic cells.
Combination therapy with gardiquimod or imiquimod and DC vaccine inhibits the growth of subcutaneous B16 melanomas.Cell Mol Immunol.2010 Sep;7(5):381-8. |
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