Topo I/COX-2-IN-2 (Compound W10) is a potent dual-target inhibitor of Topo I and COX-2 with IC50s of 0.90 μM and 2.31 μM respectively. Topo I/COX-2-IN-2 causes apoptosis in cancer cells through the mitochondrial pathway.

Physicochemical Properties

Molecular Formula	C24H25CLN4O
Molecular Weight	420.93
CAS #	2841455-91-0
Appearance	Typically exists as solid at room temperature
HS Tariff Code	2934.99.9001
Storage	Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month
Shipping Condition	Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Biological Activity

Targets	Topoisomerase I 0.90 μM (IC50) COX-2 2.31 μM (IC50)
ln Vitro	Compound W10 (Topo I/COX-2-IN-2) (0–30 μM) exhibits good toxicity against cancer cells[1]. To enhance Topo I inhibition, Topo I/COX-2-IN-2 forms an ionic bonding interaction with DNA's DA13[1]. Topo I/COX-2-IN-2 (0-9 μM, 24 h) inhibits aberrant activation of the NF-κB/IκB pathway, induces apoptosis through the mitochondrial pathway, and stops the cell cycle of HT29 and RKO cells at the G1/G0 phase. [1].
ln Vivo	Topo I/COX-2-IN-2 (Compound W10) suppresses the growth of tumors and causes clear necrosis on tumor tissue (15 and 30 mg/kg; ip; bid for 2 weeks)[1]. The pharmacokinetic characteristics of Topo I/COX-2-IN-2 are suitable for oral and intraperitoneal administration[1].
Cell Assay	Cell Proliferation Assay[1] Cell Types: HT29, RKO, HCT116, LoVo and SW480 Tested Concentrations: 0-30 μM Incubation Duration: 72 h Experimental Results: demonstrated good toxicity with IC50 values of 1.48 ± 0.08 μM, 2.06 ± 0.01 μM, 4.89 ± 0.36 μM, 8.42 ± 0.22 μM and 7.36 ± 0.64 μM for HT29, RKO, HCT116, LoVo and SW480 cells, respectively. Cell Cycle Analysis[1] Cell Types: HT29 and RKO Tested Concentrations: 2, 4 and 8 μM for HT29; 3, 6 and 9 μM for RKO Incubation Duration: 24 h Experimental Results: Blocked the cell cycle in G1/G0 phase in both HT29 and RKO. In HT29 cells, the arresting activity was not obvious for the ratio of G1/G0 phase in high-concentration treatment group increased from 55.20% to 65.17% slightly, while in RKO cells, the ratio of G1/G0 phase obviously increased from 37.57% to 76.99 %. Apoptosis Analysis[1] Cell Types: HT29 and RKO Tested Concentrations: 2, 4 and 8 μM for HT29; 3, 6 and 9 μM for RKO Incubation Duration: 24 h Experimental Results: Mainly induced the late apoptosis in HT29 cells and demonstrated dual Induction of late and early apoptosis
Animal Protocol	Animal/Disease Models: Male BALB/c nude mice weighing 20–25 g, HT29 xenograft model[1] Doses: 15 and 30 mg/kg Route of Administration: intraperitoneal (ip) injection , twice (two times) daily for 2 weeks Experimental Results: 30 mg/kg group immediately slowed down the tumor growth rate after administration, and almost completely prevented tumor growth in the later stage, and its tumor growth inhibition (TGI) was 57.86%. 15 mg/kg group demonstrated 40.67% TGI. demonstrated obvious necrosis on tumor tissue. Animal/Disease Models: 250–280 g male SD rats[1] Doses: 100 mg/kg and 30 mg/kg Route of Administration: intragastric (po) administration (100 mg/kg) or intraperitoneal (ip) injection (30 mg/kg) (pharmacokinetic/PKs Study) Experimental Results: pharmacokinetic/PK data of Topo I/COX-2-IN-2 (W10) in vivoa[1] Comp. Dose (mg/kg) Route T1/2(h) Tmax( h) Cmax (μg/mL) AUC0-t (μg⋅h/mL) Topo I/COX-2-IN-2 100 po 8.87 ± 1.92 3.67 ± 0.58 2.00 ± 0.41 24.81 ± 5.76 30 ip 4.27 ± 0.22 0.28 ± 0.05 1.60 ± 0.34 5.41 ± 0.20 a Topo I/COX-2-IN-2 was administered to 6 SD rats with different administration methods a
References	[1]. Discovery of dual inhibitors of topoisomerase I and Cyclooxygenase-2 for colon cancer therapy. Eur J Med Chem. 2022 Jun 23;240:114560.

Solubility Data

Solubility (In Vitro)

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

Solubility (In Vivo)

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. Injection Formulations (e.g. IP/IV/IM/SC) 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). 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) Oral Formulations 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). 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 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.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	2.3757 mL	11.8785 mL	23.7569 mL
	5 mM	0.4751 mL	2.3757 mL	4.7514 mL
	10 mM	0.2376 mL	1.1878 mL	2.3757 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.