NCT-58 is a potent inhibitor of the C-terminus of HSP90. NCT-58 does not induce heat shock response (HSR) as it targets the C-terminal region and elicits anti-tumor activity by simultaneously downregulating HER family members and inhibiting Akt phosphorylation. NCT-58 kills trastuzumab-resistant breast cancer stem-like cells. NCT-58 causes apoptosis in HER2-positive breast cancer cells. NCT-58 inhibits growth and angiogenesis in a trastuzumab-resistant xenograft model.

Physicochemical Properties

Molecular Formula	C27H34N2O5
Molecular Weight	466.57
Exact Mass	466.25
Elemental Analysis	C, 69.51; H, 7.35; N, 6.00; O, 17.15
CAS #	2411429-33-7
PubChem CID	149033485
Appearance	White to off-white solid powder
LogP	4.4
Hydrogen Bond Donor Count	2
Hydrogen Bond Acceptor Count	6
Rotatable Bond Count	8
Heavy Atom Count	34
Complexity	699
Defined Atom Stereocenter Count	0
SMILES	C1(C)(C)OC2=CC=C(C(NC3=CC=C(OCCC4CCCCN4)C(OC)=C3)=O)C(OC)=C2C=C1
InChi Key	QGEKYFVMAGLOKW-UHFFFAOYSA-N
InChi Code	InChI=1S/C27H34N2O5/c1-27(2)14-12-20-22(34-27)11-9-21(25(20)32-4)26(30)29-19-8-10-23(24(17-19)31-3)33-16-13-18-7-5-6-15-28-18/h8-12,14,17-18,28H,5-7,13,15-16H2,1-4H3,(H,29,30)
Chemical Name	5-methoxy-N-(3-methoxy-4-(2-(piperidin-2-yl)ethoxy)phenyl)-2,2-dimethyl-2H-chromene-6-carboxamide
Synonyms	NCT-58; NCT 58; NCT58;
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	HSP90 Apoptosis
ln Vitro	In HER2-positive BT474 and SKBR3 cells, NCT-58 treatment (0.1–20 μM; 72 hours) dose-dependently lowers cell viability[1]. HER2-positive BT474 and SKBR3 cells exhibit an increase in early and late apoptotic cells in response to NCT-58 treatment (0.1–10 μM; 72 hours)[1]. In JIMT-1 and MDA-MB-453 cells, NCT-58 treatment (2–10 μM; 72 hours) significantly decreased the amounts of truncated p95HER2 and its phosphorylated version, as well as downregulated the protein contents of Akt and phospho-Akt (Ser473)[1].
ln Vivo	NCT-58 (30 mg/kg; intraperitoneally; every other day for 47 days) inhibits the growth of tumors resistant to trastuzumab[1]. NCT-58 (30 mg/kg; ip; every other day for 47 days) significantly inhibits the growth of tumors and significantly reduces their weight[1].
Cell Assay	Cell Viability Assay[1] Cell Types: BT474 and SKBR3 cells Tested Concentrations: 0, 0.1, 0.5, 1, 5, 10, 15, 20 μM Incubation Duration: 72 hrs (hours) Experimental Results: Dramatically decreased cell growth. Apoptosis Analysis[1] Cell Types: BT474 and SKBR3 cells Tested Concentrations: 0, 2, 10 μM Incubation Duration: 72 hrs (hours) Experimental Results: Increased the number of early and late apoptotic cells. Western Blot Analysis[1] Cell Types: Trastuzumab-resistant JIMT-1 and MDA-MB-453 cells Tested Concentrations: 0, 2, 10 μM Incubation Duration: 72 hrs (hours) Experimental Results: Effectively decreased the levels of truncated p95HER2 and its phosphorylated form, as well as downregulation of Akt and phospho-Akt (Ser473) protein contents in JIMT-1 and MDA-MB-453 cells.
Animal Protocol	Animal/Disease Models: Trastuzumab-resistant xenograft model (female nude mice; 6 weeks; BALB/c )[1] Doses: 30 mg/kg Route of Administration: ip; every other day for 47 days Experimental Results: Dramatically diminished tumor growth.
References	[1]. The C-terminal HSP90 inhibitor NCT-58 kills trastuzumab-resistant breast cancer stem-like cells. Cell Death Discov. 2021;7(1):354.

Solubility Data

Solubility (In Vitro)

DMSO : 50 mg/mL (107.17 mM)

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	2.1433 mL	10.7165 mL	21.4330 mL
	5 mM	0.4287 mL	2.1433 mL	4.2866 mL
	10 mM	0.2143 mL	1.0717 mL	2.1433 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.