Anti-hepatic fibrosis drug 3 (compound 9a) can effectively inhibit the activity of the COL1A1 promoter in LX-2 cells, with an IC50 value of 53.18 μM and a selectivity index of 3.60[1]. Anti-hepatic fibrosis drug 3 (20-80 μM; 24 hours) can inhibit the expression of fibrosis marker proteins (COL1A1, fibronectin, α-SMA, p-Smad2) in TGFβ1-induced LX-2 cells, and inhibit the mRNA levels of fibrosis-related genes (COL1A1, fibronectin, TGFB1, MMP2, ACTA2, TIMP1), and reduce the expression of fibrosis proteins in mouse hepatic stellate cells (mHSC) in a concentration-dependent manner[1]. Anti-fibrotic drug 3 (20-80 μM; 24 hours) dose-dependently reduced the protein levels of precursor cathepsin D and cathepsin D in LX-2 and mHSC cells, while having no effect on cathepsin B and cathepsin L, and reduced the expression of inflammatory proteins (IL-1β, caspase-1) in lipopolysaccharide-stimulated LX-2 and mHSC cells [1]. Anti-fibrotic drug 3 (80 μM; 6-48 hours) accelerated the degradation of precursor cathepsin D and cathepsin D in LX-2 cells pretreated with cyclohexylimide [1].

Anti-hepatic fibrosis drug 3 (100-200 mg/kg; orally; once daily; for 14 days) dose-dependently reduced liver fibrosis and inflammation induced by bile duct ligation (BDL) in male Sprague-Dawley rats, significantly decreasing serum liver injury markers, collagen deposition, fibrosis and inflammation biomarkers, and cathepsin D levels at doses of 100 mg/kg and 200 mg/kg [1]. Anti-hepatic fibrosis drug 3 (100-200 mg/kg; orally; once daily; for 4 weeks) dose-dependently improved metabolic liver fibrosis and inflammation induced by congenital high-fat diet (CDAHFD) in C57BL/6 mice, significantly decreasing serum liver injury markers, collagen deposition, fibrosis and inflammation biomarkers, and cathepsin D levels at doses of 100 mg/kg and 200 mg/kg [1].

RT-PCR[1]
Cell Types: LX-2 cells
Tested Concentrations: 20 μM, 40 μM, 80 μM
Incubation Duration: 24 hours
Experimental Results: Dose-dependent downregulation of mRNA levels of fibrosis-related genes (COL1A1, fibronectin, TGFB1, MMP2, ACTA2, TIMP1).

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Western Blot Analysis [1]
Cell Types: LX-2 cells pretreated with cyclohexylimide (CHX)
Tested Concentrations: 80 μM
Incubation Duration: 6 h, 12 h, 24 h, 36 h, 48 h
Experimental Results: Significantly accelerated the degradation of pro-Cathepsin D and Cathepsin D.

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Western Blot analysis [1]
Cell Types: LPS-stimulated LX-2 cells, LPS-stimulated mHSCs
Tested Concentrations: 20 μM, 40 μM, 80 μM
Incubation Duration: 24 hours
Experimental Results: Significantly reduced the protein levels of inflammatory factors IL-1β and caspase-1.

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Western Blot Analysis [1]
Cell Types: LX-2 cells, mouse hepatic stellate cells (mHSC)
Tested Concentrations: 20 μM, 40 μM, 80 μM
Incubation Duration: 24 hours
Experimental Results: The protein levels of cathepsin D, procathepsin D, COL1A1, fibronectin, α-SMA and p-Smad2 were downregulated in a dose-dependent manner, but there was no significant effect on cathepsin B and cathepsin L.

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Western Blot Analysis [1]
Cell Types: LX-2 cells stimulated by TGFβ1
Tested Concentrations: Anti-fibrotic agent 3 (20 μM, 40 μM, 80 μM), L-theanine (20 μM, 40 μM, 80 μM)
Incubation Duration: 24 hours
Experimental Results: Dose-dependent downregulation of fibrosis-related proteins (COL1A1, fibronectin, α-SMA, p-Smad2, MMP2, TGFβ1, CTGF, TIMP1); showed stronger inhibitory activity than L-theanine and most of the tested analogues.

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Animal/Disease Models:Sprague-Dawley mice (male, 180-200 g, bile duct ligation-induced liver fibrosis) [1]
Doses: 100 mg/kg; 200 mg/kg
Route of Administration: Oral; once daily; 14 days
Experimental Results: The dose-dependent reduction in serum aspartate aminotransferase (AST), lactate dehydrogenase (LDH), and total bilirubin (TBIL) levels was significant. Compared with the bile duct ligation model group, liver elasticity and surface smoothness were improved. Histological analysis showed that the dose-dependent reduction in liver injury and collagen deposition was significant; the 200 mg/kg dose group significantly reduced liver necrosis. The 200 mg/kg dose group significantly reduced the liver hydroxyproline content. The protein levels of fibrosis biomarkers (fibronectin, COL1A1, α-SMA, TGFβ1, CTGF, TIMP1) in liver tissue were downregulated in a dose-dependent manner. The mRNA levels of Col1a1 and Acta2 were downregulated in the 200 mg/kg dose group. The protein levels of precursor cathepsin D and cathepsin D in liver tissue were decreased in a dose-dependent manner. The protein and mRNA expression of pro-inflammatory factors (IL-6, IL-1β) were both inhibited, and the inflammation score in pathological sections also decreased in a dose-dependent manner.

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Animal/Disease Models:C57BL/6 (8 weeks old, CDAHFD feeding-induced liver fibrosis) [1]
Doses: 100 mg/kg; 200 mg/kg
Route of Administration: Oral; once daily; for 4 weeks
Experimental Results: Serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and total bilirubin (TBIL) improved in a dose-dependent manner, and were superior to the positive control drug obeticholic acid (OCA) in reducing ALT and ALP. Compared with the CDAHFD model group, liver luster and elasticity were partially restored. Histological analysis showed a reduction in hepatic ballooning degeneration and collagen deposition; semi-quantitative scoring showed a dose-dependent decrease in liver fibrosis score. The liver hydroxyproline content was significantly reduced in both dosage groups. The protein levels of fibrosis biomarkers (COL1A1, fibronectin, α-SMA) and the mRNA levels of Col1a1, Mmp2, Timp1, and Acta2 in liver tissue were downregulated. The protein levels of precursor cathepsin D and cathepsin D in liver tissue decreased in a dose-dependent manner. The expression of pro-inflammatory cytokine (IL-6) protein and mRNA was inhibited in a dose-dependent manner.

[1]. Design, Synthesis, and Mechanistic Evaluation of l-Theanine Derivatives Targeting Cathepsin D for Anti-Hepatic Fibrosis. J Med Chem. 2026;69(4):4493-4511.

C22H28N2O3

368.47

Typically exists as solids at room temperature

O=C(OC)[C@H](CCC(NCC)=O)N(CC1=CC=CC=C1)CC2=CC=CC=C2

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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