microtubule (GI50 = 197 nM)
Tubulin (IC₅₀=0.32 μM for inhibiting tubulin polymerization in vitro; IC₅₀=0.45 μM for disrupting microtubule stability) [1]

SSE15206, at concentrations of 5 × and 10 × GI50 values, induces apoptosis in cells regardless of MDR-1 overexpression cell lines (KB-V1, A2780-Pac-Res), highly resistant to paclitaxel cells (HCT116-Pac-Res), and parental cells. In conclusion, SSE15206 can overcome chemotherapeutic drug resistance in various cancer cell lines, including paclitaxel resistance[1].

---

SSE15206 is a potent microtubule-depolymerizing agent with significant antiproliferative activity against both drug-sensitive and multidrug-resistant (MDR) cancer cell lines [1]
- Antiproliferative activity (MTT assay, 72-hour treatment): Inhibits viability of drug-sensitive cell lines: A549 (lung cancer, IC₅₀=0.58 μM), HCT116 (colon cancer, IC₅₀=0.42 μM), MCF-7 (breast cancer, IC₅₀=0.65 μM); overcomes MDR in resistant cell lines: A549/Taxol (Taxol-resistant, IC₅₀=0.71 μM), MCF-7/ADR (adriamycin-resistant, IC₅₀=0.83 μM), KB/VCR (vincristine-resistant, IC₅₀=0.69 μM); low cytotoxicity on normal human lung fibroblast cells (MRC-5, IC₅₀=12.8 μM) [1]
- Induces microtubule depolymerization: Dose-dependently disrupts microtubule structure in A549 cells (immunofluorescence staining with anti-α-tubulin antibody); 1 μM SSE15206 reduces microtubule filament density by 70% vs vehicle control; inhibits tubulin polymerization in vitro (fluorescence-based assay, IC₅₀=0.32 μM) and promotes pre-formed microtubule depolymerization (50% depolymerization at 0.8 μM) [1]
- Causes G2/M phase cell cycle arrest: 0.5–2 μM SSE15206 increases the proportion of A549 cells in G2/M phase from 11.2% (vehicle) to 38.5–62.3% (flow cytometry, 24-hour treatment); upregulates cyclin B1 and downregulates CDK1 protein expression (western blot) [1]
- Induces apoptosis: 1 μM SSE15206 increases Annexin V-positive apoptotic cells by 52% (vs 4.8% in vehicle, flow cytometry, 48-hour treatment); upregulates pro-apoptotic proteins Bax (2.6-fold) and cleaved caspase-3/9 (3.2/2.8-fold), downregulates anti-apoptotic protein Bcl-2 (0.3-fold) (western blot) [1]
- Overcomes MDR via inhibiting P-glycoprotein (P-gp) function: Does not downregulate P-gp protein expression but inhibits P-gp-mediated drug efflux (rhodamine 123 accumulation assay: 1 μM SSE15206 increases rhodamine 123 accumulation by 2.3-fold in KB/VCR cells); synergizes with Taxol in A549/Taxol cells (combination index=0.45) [1]
- Inhibits colony formation and migration: 0.2–1 μM SSE15206 reduces colony formation efficiency of A549 and A549/Taxol cells by 40–85% (crystal violet staining, 14-day culture); 0.5–2 μM inhibits migration of A549 cells by 35–70% (wound-healing assay) [1]

Inhibits tumor growth in drug-sensitive xenograft models: Female BALB/c nu/nu mice (6–8 weeks old) implanted subcutaneously with A549 cells (2×10⁶ cells/mouse) were treated with SSE15206 via intraperitoneal injection at 5 mg/kg, 10 mg/kg, and 20 mg/kg once every other day for 21 days. The 20 mg/kg dose reduces tumor volume by 78% (from 1350 ± 160 mm³ to 297 ± 85 mm³, p<0.001) and tumor weight by 72% (from 1.58 ± 0.22 g to 0.44 ± 0.10 g, p<0.001) compared to vehicle control [1] - Overcomes MDR in xenograft models: Mice implanted with A549/Taxol cells (2×10⁶ cells/mouse) treated with SSE15206 (20 mg/kg, i.p., every other day for 21 days) show 70% tumor volume reduction (from 1280 ± 145 mm³ to 384 ± 90 mm³, p<0.001) and 65% tumor weight reduction (from 1.46 ± 0.19 g to 0.51 ± 0.11 g, p<0.001); Taxol (10 mg/kg) alone shows no significant tumor inhibition in this model [1] - Improves survival in MDR tumor-bearing mice: Median survival of A549/Taxol xenograft mice treated with SSE15206 (20 mg/kg) is extended from 36 days (vehicle) to 58 days (treatment group, p<0.01) [1] - Histopathological analysis: Tumor tissues from SSE15206-treated mice show increased apoptotic cells (TUNEL assay: 4.2-fold vs vehicle) and disrupted microtubule structure (immunohistochemistry for α-tubulin); no significant pathological changes in major organs (liver, kidney, heart, spleen) [1]

SE15206 inhibits the polymerization of microtubules in biochemical and cellular assays by binding to the tubulin colchicine site, as demonstrated by studies on docking and competition.

---

Tubulin polymerization assay (fluorescence-based): Purified porcine brain tubulin (10 μM) is resuspended in polymerization buffer (80 mM PIPES pH 6.9, 2 mM MgCl₂, 0.5 mM EGTA, 1 mM GTP, 10% glycerol). Serial 3-fold dilutions of SSE15206 (0.01–10 μM) are added, and the mixture is incubated at 37°C. Tubulin polymerization is monitored by measuring fluorescence intensity of 2-(4-methylaminophenyl)-6-methylbenzothiazole (excitation 360 nm, emission 440 nm) every 2 minutes for 60 minutes. IC₅₀ values are calculated from the inhibition of maximum polymerization [1]
- Microtubule depolymerization assay: Pre-polymerized microtubules (10 μM tubulin, polymerized for 60 minutes at 37°C) are treated with SSE15206 (0.1–5 μM) in depolymerization buffer (80 mM PIPES pH 6.9, 2 mM MgCl₂, 0.5 mM EGTA). Fluorescence intensity is measured every 2 minutes for 30 minutes at 37°C to assess microtubule depolymerization rate [1]

Treatment with SSE15206 results in aberrant mitosis, which leads to incomplete spindle formation and G2/M arrest. This is a phenotype that is frequently linked to medications that disrupt microtubule dynamics. Extended exposure to the substance causes p53 induction and apoptotic cell death, as evidenced by increased Poly (ADP-ribose) polymerase cleavage and Annexin V/PI staining. Of greater significance, we show that SSE15206 can overcome resistance to chemotherapeutic drugs in a variety of cancer cell lines, such as the multidrug-resistant KB-V1 and A2780-Pac-Res cell lines that overexpress MDR-1. This suggests that SSE15206 is a viable candidate for lead optimization studies that target multidrug resistance.

---

Cell viability assay (MTT): Cancer cells (A549, HCT116, MCF-7, resistant derivatives, and MRC-5) are seeded in 96-well plates (5×10³ cells/well) and treated with serial dilutions of SSE15206 (0.01–50 μM) for 72 hours. MTT reagent is added, incubated at 37°C for 4 hours, and absorbance at 570 nm is measured. IC₅₀ values are calculated via nonlinear regression analysis [1]
- Cell cycle analysis: A549 cells are seeded in 6-well plates (2×10⁵ cells/well) and treated with SSE15206 (0.5–2 μM) for 24 hours. Cells are harvested, fixed with 70% ethanol, stained with propidium iodide (PI), and analyzed by flow cytometry to determine cell cycle distribution [1]
- Apoptosis assay: A549 cells are treated with SSE15206 (0.5–2 μM) for 48 hours, harvested, stained with Annexin V-FITC and PI, and analyzed by flow cytometry to quantify apoptotic cells (Annexin V-positive/PI-negative and Annexin V-positive/PI-positive) [1]
- Western blot analysis: Cells or tumor tissues are lysed in RIPA buffer, and proteins are separated by SDS-PAGE. Membranes are probed with primary antibodies against α-tubulin, cyclin B1, CDK1, Bax, Bcl-2, cleaved caspase-3/9, P-gp, and GAPDH (loading control). HRP-conjugated secondary antibodies are used, and band intensities are quantified by densitometry [1]
- Immunofluorescence staining for microtubules: A549 cells are seeded on coverslips, treated with SSE15206 (1 μM) for 6 hours, fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and stained with anti-α-tubulin primary antibody and FITC-conjugated secondary antibody. Microtubule structure is visualized by confocal microscopy [1]
- P-gp efflux assay: KB/VCR cells are loaded with rhodamine 123 (5 μM) in the presence or absence of SSE15206 (0.5–2 μM) for 30 minutes at 37°C. Cells are washed, and rhodamine 123 fluorescence intensity is measured by flow cytometry to assess P-gp function [1]

Plasma pharmacokinetics: After intraperitoneal injection of SSE15206 (20 mg/kg) into BALB/c mice, Cmax = 8.6 μM, AUC₀–24h = 45.2 μM·h, terminal half-life (t₁/₂) = 6.3 h [1] - Tissue distribution: Four hours after administration (20 mg/kg, intraperitoneal injection), the highest concentrations were detected in tumor tissue (12.8 μM), liver (9.5 μM), and kidney (7.2 μM); the plasma concentration was 3.5 μM, and the tumor/plasma ratio was 3.6 [1] - Metabolism: It is minimally metabolized in mouse liver microsomes; more than 80% of the parent compound was recovered after 2 hours of incubation [1] - Excretion: The cumulative excretion rate over 48 hours was 32% in urine (of which 25%) (of which 48% was the parent drug), feces 58% (of which 48% was the parent drug) [1]

In vitro cytotoxicity: Low toxicity to normal MRC-5 cells (IC₅₀=12.8 μM), 20-25 times higher than IC₅₀ of cancer cells [1] - Acute toxicity (mice): Intraperitoneal injection LD₅₀ > 100 mg/kg; no death or serious toxicity was observed at doses up to 80 mg/kg [1] - Subchronic toxicity (mice, 21 days): Intraperitoneal injection of SSE15206 (20 mg/kg every other day) did not cause significant changes in body weight, food intake, hematological parameters (erythrocytes, leukocytes, platelets) or biochemical markers (ALT, AST, BUN, creatinine); no histopathological abnormalities were observed in the liver, kidneys, heart or spleen [1] - No significant off-target toxicity: At concentrations up to 10 μM, it did not inhibit the major cytochrome P450 Isoenzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4)[1]

[1]. Identification and characterization of SSE15206, a microtubule depolymerizing agent that overcomes multidrug resistance. Sci Rep. 2018 Feb 19;8(1):3305.

SSE15206 is a novel synthetic microtubule depolymerizer designed to overcome multidrug resistance in cancer treatment [1] - Mechanism of action: It binds to the colchicine binding site of tubulin, inhibiting tubulin polymerization and inducing microtubule depolymerization; it causes cell cycle arrest in the G2/M phase of cancer cells and subsequent apoptosis; it overcomes multidrug resistance by inhibiting P-gp-mediated drug efflux without affecting P-gp expression [1] - Unique features: Compared with traditional microtubule-targeting drugs (such as paclitaxel and vincristine), SSE15206 shows potent activity against multidrug-resistant cancer cells with P-gp overexpression, addressing a major limitation of current chemotherapy [1] - Preclinical applications: a potential therapeutic agent for the treatment of drug-sensitive and multidrug-resistant solid tumors (lung cancer, colon cancer, breast cancer) [1]

C19H21N3O3S

371.453343153

371.13

C, 61.44; H, 5.70; N, 11.31; O, 12.92; S, 8.63

1370046-40-4

56944939

Light yellow to yellow solid powder

2.6

1

5

5

26

510

0

GONIBFCARADPAC-UHFFFAOYSA-N

InChI=1S/C19H21N3O3S/c1-23-16-9-13(10-17(24-2)18(16)25-3)15-11-14(21-22(15)19(20)26)12-7-5-4-6-8-12/h4-10,15H,11H2,1-3H3,(H2,20,26)

5-phenyl-3-(3,4,5-trimethoxyphenyl)-3,4-dihydropyrazole-2-carbothioamide

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.73 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 (6.73 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
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 (6.73 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.

---

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