Topoisomerase I
SW044248 targets human topoisomerase I (Topo I) (IC50 = 0.37 μM for recombinant Topo I enzymatic activity inhibition; IC50 > 100 μM for topoisomerase II (Topo II), indicating >270-fold selectivity for Topo I) [1]

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In nude mice bearing H23 (KRAS-mutant NSCLC) xenografts, intraperitoneal administration of SW044248 (20 mg/kg/day, 40 mg/kg/day) for 21 days dose-dependently inhibited tumor growth: high-dose treatment achieved a tumor growth inhibition (TGI) rate of 68% and reduced tumor weight from 1.25 ± 0.18 g (vehicle) to 0.39 ± 0.08 g [1]
- Immunohistochemical staining of H23 xenograft tissues showed that SW044248 (40 mg/kg) increased γ-H2AX-positive cells by ~2.8-fold, reduced Ki-67 (cell proliferation marker) positivity by ~62%, and increased TUNEL-positive apoptotic cells by ~4.5-fold compared to vehicle control [1]
- No significant changes in body weight (vehicle: 22.6 ± 1.3 g vs. high-dose: 21.8 ± 1.1 g) or histopathological abnormalities in major organs (liver, kidney, heart, lung) were observed in treated mice [1]

Topoisomerase I (Topo I) enzymatic activity inhibition assay: Recombinant human Topo I was incubated with supercoiled plasmid DNA substrate in reaction buffer containing MgCl2 and ATP. Serial dilutions of SW044248 (0.01-10 μM) were added to the reaction mixture, which was incubated at 37°C for 30 minutes. The reaction was terminated by adding SDS and proteinase K, followed by incubation at 50°C for 60 minutes. DNA products were separated by agarose gel electrophoresis and visualized by ethidium bromide staining. The percentage of relaxed DNA was quantified by densitometric analysis, and IC50 values were calculated from dose-response curves of Topo I inhibition [1]
- Topoisomerase II (Topo II) selectivity assay: Recombinant human Topo II was subjected to the same assay protocol as Topo I, using supercoiled plasmid DNA as substrate. SW044248 (0.01-100 μM) was tested to determine IC50 values for Topo II, confirming selective inhibition of Topo I [1]

In wells of 96-well plates, 100 μL of 50,000 cells/mL cell suspensions of distinct cell lines are added. The following day, 100 μL of cell medium was added to each well, substituting 2X concentration of either SW044248, camptothecin, or DMSO in triplicate. The wells' ATP concentration is determined using CelTiter-Glo after 96 and 120 hours. With the aid of a plate reader, luminescence is measured[1].

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NSCLC cell antiproliferation assay: H23, H460, A549, H1975, HCC827, and NHBE cells were seeded in 96-well plates at 5×10³ cells/well. After 24-hour attachment, serial dilutions of SW044248 (0.01-10 μM) were added, and cells were cultured for 72 hours. MTT reagent was added, and absorbance at 570 nm was measured to calculate cell viability and IC50 values [1]
- DNA damage detection assay: H23 cells were seeded on glass coverslips (2×10⁴ cells/coverslip) and treated with SW044248 (1 μM) for 24 hours. Cells were fixed with paraformaldehyde, permeabilized with Triton X-100, and stained with anti-γ-H2AX antibody and fluorescent secondary antibody. γ-H2AX foci were visualized under a confocal microscope and counted. For western blot, H23 cells were treated with SW044248 (0.5-2 μM) for 24 hours, lysed in RIPA buffer, and proteins were probed with anti-γ-H2AX and GAPDH antibodies [1]
- Cell cycle analysis: H23 cells were seeded in 6-well plates (2×10⁵ cells/well) and treated with SW044248 (1 μM) for 24 hours. Cells were harvested, fixed with ethanol, stained with propidium iodide (PI), and analyzed by flow cytometry to determine cell cycle distribution [1]
- Apoptosis assay: H23 cells were treated with SW044248 (0.5-2 μM) for 48 hours. Cells were stained with Annexin V-FITC and PI, then analyzed by flow cytometry to quantify apoptotic rate. Western blot was performed to detect cleaved caspase-3, cleaved PARP, and GAPDH [1]
- Clonogenic assay: H23 and H460 cells were seeded in 6-well plates (200 cells/well) and allowed to attach for 24 hours. Serial dilutions of SW044248 (0.1-0.5 μM) were added, and cells were cultured for 14 days. Colonies were fixed with methanol, stained with crystal violet, and counted. The colony-forming efficiency was calculated as the percentage of colonies formed relative to vehicle control [1]

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H23 KRAS-mutant NSCLC xenograft model: Female BALB/c nude mice (4-6 weeks old) were subcutaneously implanted with 5×10⁶ H23 cells. When tumors reached ~100 mm³, mice were randomly divided into vehicle control, SW044248 20 mg/kg, and 40 mg/kg groups (n=6 per group). The drug was dissolved in 10% DMSO + 90% cremophor EL (diluted with physiological saline to final cremophor EL concentration of 10%) and administered by intraperitoneal injection once daily for 21 days. Tumor volume was measured every 3 days using calipers, and tumor weight was recorded at the end of treatment. Tumor tissues were collected for immunohistochemical staining of γ-H2AX, Ki-67, and TUNEL [1]

In vitro cytotoxicity: SW044248 showed a CC50 > 10 μM in normal human bronchial epithelial cells (NHBE), indicating low toxicity to normal cells [1]
- Acute toxicity in mice: A single intraperitoneal injection of up to 100 mg/kg of SW044248 did not cause death or significant toxic reactions (drowsiness, weight loss, abnormal behavior) [1]
- Chronic toxicity in mice: Repeated intraperitoneal injections of SW044248 (40 mg/kg/day for 21 days) did not cause significant changes in hematological parameters (erythrocytes, leukocytes, platelets) or serum biochemical indicators (ALT, AST, creatinine, BUN) [1]

[1]. A Novel Inhibitor of Topoisomerase I Is Selectively Toxic for a Subset of Non-Small Cell Lung Cancer Cell Lines. Mol Cancer Ther. 2016 Jan;15(1):23-36.

[2]. Systematic identification of molecular subtype-selective vulnerabilities in non-small-cell lung cancer. Cell. 2013 Oct 24;155(3):552-66.

SW044248 is a novel selective topoisomerase I (Topo I) small molecule inhibitor that has been developed for the treatment of non-small cell lung cancer (NSCLC) [1]. The therapeutic mechanism of SW044248 includes selectively inhibiting Topo I enzyme activity, stabilizing the Topo I-DNA covalent complex, inducing DNA double-strand breaks, triggering S-phase cell cycle arrest, and ultimately promoting cancer cell apoptosis [1]. SW044248 exhibits subtype-selective antiproliferative activity in NSCLC, and its inhibitory efficacy against KRAS mutant NSCLC cell lines is significantly higher than that against EGFR mutant or wild-type EGFR/KRAS cell lines [1]. Preclinical data indicate that SW044248 has potent antiproliferative activity against a variety of NSCLC cell lines in vitro. SW044248 has significant in vivo antitumor efficacy against KRAS-mutant non-small cell lung cancer (NSCLC) cells, exhibits good antitumor activity in the H23 xenograft model, and has low toxicity to normal cells and organs [1]. Reference [2] identified SW044248 as a molecular subtype-selective targeted drug for KRAS-mutant NSCLC through systematic screening, supporting its potential as a targeted therapy for this clinically challenging NSCLC subtype [2].

C22H23N5O2S

421.52

421.157

C, 62.69; H, 5.50; N, 16.61; O, 7.59; S, 7.61

522650-83-5

3152990

Light yellow to yellow solid powder

1.3±0.1 g/cm3

1.675

4.48

1

6

7

30

582

0

S(C1N=NC2=C(N=1)N(C([H])([H])C([H])([H])[H])C1=C([H])C([H])=C([H])C([H])=C12)C([H])(C(N([H])C1=C([H])C([H])=C([H])C([H])=C1OC([H])([H])[H])=O)C([H])([H])C([H])([H])[H]

PEVRGVRHMMZNGI-UHFFFAOYSA-N

InChI=1S/C22H23N5O2S/c1-4-18(21(28)23-15-11-7-9-13-17(15)29-3)30-22-24-20-19(25-26-22)14-10-6-8-12-16(14)27(20)5-2/h6-13,18H,4-5H2,1-3H3,(H,23,28)

2-[(5-ethyl-[1,2,4]triazino[5,6-b]indol-3-yl)sulfanyl]-N-(2-methoxyphenyl)butanamide

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: ≥ 1.25 mg/mL (2.97 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 12.5 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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