Bt354 (0.1-100 μM; 24-72 h) showed concentration- and time-dependent antiproliferative activity against U87 MG, GBM8401 and T98G glioblastoma cells, with IC50 values of 9.96 μM, 15.18 μM and 3.39 μM after 24 h of incubation, respectively [1]. Bt354 (0.01-5 μM; 48 h) induced apoptosis in U87 MG glioblastoma cells in a concentration-dependent manner, and after 48 h of incubation, a significant increase in the proportion of apoptotic cells was observed at concentrations of 0.1 μM and above [1]. Bt354 (0.1-5 μM; 24 hours) induced apoptosis in U87 MG glioblastoma cells by upregulating the expression of cleaved caspase-3 and cleaved PARP and reducing the activity of pro-caspase-3; significant effects were observed after incubation at concentrations of 0.1 μM and above for 24 hours [1]. Bt354 (0.1-5 μM; 24 hours) inhibited epithelial-mesenchymal transition in U87 MG glioblastoma cells by upregulating the expression of E-cadherin and downregulating the expression of vimentin, matrix metalloproteinase-2 (MMP-2), ZEB1 and FOXM1; significant effects were observed after incubation at concentrations of 0.1 μM and above for 24 hours (effective concentrations of E-cadherin and ZEB1 were 1 μM and above) [1]. Bt354 (0.01–5 μM; 6–18 h) inhibited the migration of U87 MG glioblastoma cells in a concentration- and time-dependent manner. Significant migration inhibition was observed at concentrations of 0.01 μM and above, with the inhibition reaching its maximum after 18 hours of incubation [1]. Bt354 (0.1–5 μM; 24 h) altered the polymerization state of F-actin in U87 MG glioblastoma cells, converting filamentous F-actin into a globular form near the nucleus; at concentrations of 0.1 μM and above, cell migration was inhibited after 24 hours of incubation [1]. Bt354 (0.1–5 μM; 24 h) regulated the epithelial-mesenchymal transition of U87 MG glioblastoma cells by upregulating E-cadherin expression and downregulating vimentin expression, an effect that was observed after 24 hours of incubation at concentrations of 0.1 μM or higher [1]. Bt354 (0.1–5 μM; 24 hours) reduced the production of MMP-2 in U87 MG glioblastoma cells in vitro in a concentration-dependent manner, with a significant reduction observed after 24 hours of incubation at concentrations of 0.1 μM and higher [1]. Bt354 (24 hours) effectively inhibited STAT3-dependent luciferase activity in DU145, MDA-MB-435, and MDA-MB-231 cells, with IC50 values of 4.6 μM, 6.5 μM, and 7.2 μM, respectively [3]. Bt354 (24–72 hours) inhibited the proliferation of DU145, MDA-MB-435, and MDA-MB-231 cells in a time- and dose-dependent manner, with IC50 values ranging from 0.07 μM to 35 μM, depending on the cell line and incubation time [3]. Bt354 specifically inhibits STAT3 phosphorylation at Tyr705 (without affecting phosphorylation at Ser727 or the upstream JAK2/Src signaling pathway) and downregulates the expression of STAT3-dependent pro-proliferative and anti-apoptotic proteins in MDA-MB-435 and MDA-MB-231 cells [3]. Bt354 (0.1–5 μM; 12 h) inhibits nuclear translocation of STAT3 in MDA-MB-435 and MDA-MB-231 cells in a dose-dependent manner, as evidenced by decreased nuclear STAT3 levels after 12 hours of treatment with 1 μM or 5 μM Bt354 [3]. Bt354 (0.5 μM; 24 h) induces G2/M phase cell cycle arrest in MDA-MB-435 and MDA-MB-231 cells [3]. Bt354 (48 hours) induced late apoptosis in MDA-MB-435 and MDA-MB-231 cells in a dose-dependent manner, increasing the proportion of late apoptotic cells to 26.0% and 29.5%, respectively [3]. Bt354 (1 μM) inhibited the migration of MDA-MB-435 and MDA-MB-231 cells in a dose-dependent manner, with a particularly significant inhibitory effect on the migration of MDA-MB-435 cells at a concentration of 1 μM [3].

Bt354 (0.1-20 μg; single injection; 24 hours) and (0.25-1 μg/h; continuous infusion; 7-14 days) produced dose-dependent acute and sustained prophylactic analgesia in CCI-induced neuropathic pain in rats by inhibiting the neuronal pSTAT3 signaling pathway, reducing neuroinflammation, glial cell activation and angiogenesis, with an ED50 of 1.176 μg[2]. Bt354 (10-40 mg/kg; oral; every 3 days; 21 days) showed dose-dependent antitumor activity in MDA-MB-231 xenograft mice[3]. Bt354 (10-40 mg/kg; oral; every 3 days; 21 days) showed dose-dependent antitumor activity in MDA-MB-435 xenograft mice[3].

Apoptosis Analysis [1]
Cell Types: U87 MG glioblastoma cells
Tested Concentrations: 0.01, 0.1, 1 and 5 μM
Incubation Duration: 24 hours; 48 hours
Experimental Results: After treatment with concentrations ranging from 0.1 μM to 5 μM for 48 hours, the proportion of apoptotic U87 MG cells increased significantly in a concentration-dependent manner. No significant apoptotic effect was observed after treatment with the same concentration for 24 hours.

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Western Blot Analysis [1]
Cell Types: U87 MG glioblastoma cells
Tested Concentrations: 0.1, 1, and 5 μM
Incubation Duration: 24 hours
Experimental Results: At 1 μM, the expression levels of cleaved caspase-3 and cleaved PARP were significantly increased. At concentrations of 0.1 μM and above, the activity of pro-caspase-3 in U87 MG cells was significantly decreased. E-cadherin expression increased in a concentration-dependent manner, with a significant increase observed at concentrations of 1 μM and above. Vimentin, MMP-2, and FOXM1 expression decreased in a concentration-dependent manner, with a significant decrease observed at concentrations of 0.1 μM and above. ZEB1 expression decreased in a concentration-dependent manner, with a significant decrease observed at concentrations of 1 μM and above. SLUG expression did not change significantly.

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Cell migration assay [1]
Cell Types: U87 MG glioblastoma cells
Tested Concentrations: 0.01, 0.1, 1 and 5 μM
Incubation Duration: 6 h; 12 h; 18 h
Experimental Results: The migration of U87 MG cells was significantly inhibited in a concentration- and time-dependent manner, with significant inhibition observed at concentrations of 0.01 μM and above. The inhibitory effect increased over time, reaching its maximum at 18 h.

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Immunofluorescence [1]
Cell Types: U87 MG glioblastoma cells
Tested Concentrations: 0.1, 1 and 5 μM
Incubation Duration: 24 hours
Experimental Results: Altered the polymerization of F-actin in U87 MG glioblastoma cells, causing filamentous F-actin to transform into globular form near the nucleus. Inhibited cell migration after treatment. \nWith increasing Bt354 concentration, the expression of E-cadherin increased significantly. With increasing Bt354 concentration, the expression of vimentin decreased significantly.

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ELISA detection [1]
Cell Types: U87 MG glioblastoma cells
Tested Concentrations: 0.1, 1 and 5 μM
Incubation Duration: 24 hours
Experimental Results: The concentration of MMP-2 in the supernatant of U87 MG cells was reduced in a concentration-dependent manner. Significant effects were observed at concentrations of 0.1 μM and above.

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Immunofluorescence [3] Cell Types: MDA-MB-435 human triple-negative breast cancer cells, MDA-MB-231 human triple-negative breast cancer cells Test concentrations: 0.1, 1 and 5 μM
Incubation Duration: 12 hours
Experimental Results: The level of STAT3 in the nucleus was significantly reduced in a dose-dependent manner. After treatment with 1 μM or 5 μM Bt354 for 12 hours, STAT3 was mainly retained in the cytoplasm.

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Cell cycle analysis [3]
Cell Types: MDA-MB-435 human triple-negative breast cancer cells, MDA-MB-231 human triple-negative breast cancer cells
Tested Concentrations: 0.5 μM
Incubation Duration: 24 hours
Experimental Results: This resulted in the accumulation of G2/M phase cells, with the percentage of G2/M phase cells increasing to 28.68% (MDA-MB-435) and 27.50% (MDA-MB-231), respectively.

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Animal/Disease Models:Wistar mice (adult males, 250-285 g, with chronic compression injury of the right sciatic nerve, and an intrathecal catheter was implanted before induction of CCI) [2]
Doses: 0.1-20 μg (acute test); 0.25-1 μg/h (prophylactic test)
Route of Administration: Single injection (acute test); continuous infusion for 7-14 days (prophylactic test)
Experimental Results: Dose-dependent analgesia was produced for CCI-induced thermal hyperalgesia, with a duration of action of up to 24 hours. The calculated ED50 was 1.176 μg. From day 3 to day 14 post-surgery, the paw retraction threshold was significantly higher than that of the CCI+carrier control group. Compared with the CCI+carrier control group, from day 1 to day 14 post-surgery, the paw retraction latency was significantly prolonged and improved in a dose-dependent manner. It significantly attenuated CCI-induced upregulation of STAT3 and its phosphorylated forms in the ipsilateral dorsal horn of the spinal cord, including reduced nuclear translocation of neuronal pSTAT3. It mitigated CCI-induced activation and pro-inflammatory polarization of microglia and astrocytes. It inhibited neuronal NLRP3 inflammasome expression. It reduced neuronal LDHA and pTBK1 expression. It attenuated microglia expression of pCREB and pP38. It reduced the production of pro-inflammatory cytokines in neurons and microglia. It inhibited CCI-induced angiogenesis.

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Animal/Disease Models:BalB/c-nu/nu (female, 8-10 weeks old) [3]
Doses: 10 mg/kg; 20 mg/kg; 40 mg/kg
Route of Administration: Oral; every 3 days; for 21 days
Experimental Results: Compared with the control group of 2604.3 mm³, the mean tumor volume in each group was reduced to 1303.2 mm³ (10 mg/kg group), 976.0 mm³ (20 mg/kg group), and 811.0 mm³ (40 mg/kg group), respectively. The tumor growth inhibition rate (T/C) in the 10 mg/kg, 20 mg/kg, and 40 mg/kg groups was 50%, 37.4%, and 31.3%, respectively. In the 40 mg/kg group, the tumor weight was reduced by 74.6%. The level of p-STAT3 (Y705) in tumor tissue decreased in a dose-dependent manner. Ki-67 positive cell counts were reduced in all Bt354 treatment groups. Tumor cell apoptosis (as detected by TUNEL assay) increased in a dose-dependent manner in all Bt354 treatment groups. Compared with the control group's 1365.8 mm³, the mean tumor volume decreased to 393.2 mm³ (10 mg/kg), 351.1 mm³ (20 mg/kg), and 321.4 mm³ (40 mg/kg), respectively. The tumor growth inhibition rates (T/C) in the 10, 20, and 40 mg/kg groups were 28.7%, 25.7%, and 23.5%, respectively. In the 10, 20, and 40 mg/kg dose groups, tumor weight decreased by 56.2%, 63.8%, and 77.1%, respectively. The level of p-STAT3 (Y705) in tumor tissue decreased in a dose-dependent manner. Ki-67 positive cell counts were reduced in all Bt354 treatment groups. Tumor cell apoptosis (detected by TUNEL assay) increased in a dose-dependent manner in all Bt354 treatment groups.

[1]. STAT3 phosphorylation inhibitor Bt354 exhibits anti-neoplastic activity in glioblastoma multiforme cells. Environ Toxicol. 2024;39(6):3292-3303.

[2]. Intrathecal STAT3 inhibitor Bt354 ameliorates chronic constriction injury-induced nociceptive sensitization by modulating neuroinflammation. Neurotherapeutics. 2025;22(6):e00763.

[3]. Bt354 as a new STAT3 signaling pathway inhibitor against triple negative breast cancer. J Drug Target. 2018;26(10):920-930.

C21H21N3O3S

395.48

931305-29-2

Typically exists as solids at room temperature

O=C(NCC=1C=NC=CC1)C2=CC=C(C(=C2)NS(=O)(=O)C3=CC=C(C=C3)C)C

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.)