HI5 is a potent inhibitor of tubulin and IDO with IC50 of 70 nM in HeLa cells. HI5 inhibits the expression of IDO and reduces the production of kynurenine, thereby stimulating T cell activation and proliferation. HI5 can inhibit tubulin polymerization and cell migration in HeLa cells, causing G2/M phase arrest, while inducing apoptosis through the mitochondria-dependent apoptotic pathway and causing reactive oxidative stress. HI5 could be used in anti-cancer research.

Physicochemical Properties

Molecular Formula	C42H43N5O8
Molecular Weight	745.819530725479
Exact Mass	745.311
CAS #	2411548-90-6
PubChem CID	171360936
Appearance	Typically exists as solid at room temperature
LogP	6.2
Hydrogen Bond Donor Count	1
Hydrogen Bond Acceptor Count	11
Rotatable Bond Count	18
Heavy Atom Count	55
Complexity	1230
Defined Atom Stereocenter Count	0
SMILES	CN1C=C(C2=CC=CC=C21)CC(C(=O)OCCC3=CN(N=N3)CC4=CC(=CC=C4)C(=O)OC5=C(C=CC(=C5)/C=C\C6=CC(=C(C(=C6)OC)OC)OC)OC)N
InChi Key	ZXDJTIHUHXGWOK-YPKPFQOOSA-N
InChi Code	InChI=1S/C42H43N5O8/c1-46-25-31(33-11-6-7-12-35(33)46)23-34(43)42(49)54-18-17-32-26-47(45-44-32)24-29-9-8-10-30(19-29)41(48)55-37-20-27(15-16-36(37)50-2)13-14-28-21-38(51-3)40(53-5)39(22-28)52-4/h6-16,19-22,25-26,34H,17-18,23-24,43H2,1-5H3/b14-13-
Chemical Name	[2-methoxy-5-[(Z)-2-(3,4,5-trimethoxyphenyl)ethenyl]phenyl] 3-[[4-[2-[2-amino-3-(1-methylindol-3-yl)propanoyl]oxyethyl]triazol-1-yl]methyl]benzoate
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

ln Vitro	In HeLa, PC-3, A549, and HUVEC, HI5 (0–10 μM; 72 hours) demonstrates strong antiproliferative action [1]. Microtubule organization is dramatically disrupted in a concentration-dependent manner by HI5 (0.1 and 0.5 μM; 24 h) [1]. HI5 (0.5 μM; 24 hours) considerably increases the expression of the Bax protein, significantly decreases the expression of the Bcl-2 protein, and significantly increases the expression of the PARP and caspase-3 proteins [1].
ln Vivo	In a dose-dependent manner, HI5 (15 and 30 mg/kg; intravenously administered daily for 21 days) significantly inhibits the growth of tumors [1].
Cell Assay	Cell proliferation assay Cell Types: HeLa, PC-3, A549 and HUVEC[1] Tested Concentrations: 0-10 μM Incubation Duration: 72 hrs (hours) Experimental Results: demonstrated effective anti-proliferative activity in HeLa, PC-3, A549 and HUVEC, The IC50 is 0.07 ± 0.005 μM, 0.46 ± 0.06 μM, 0.22 ± 0.10 μM, and 1.52 ± 0.09 μM, respectively. Immunofluorescence Cell Types: HeLa[1] Tested Concentrations: 0.1 and 0.5 μM Incubation Duration: 24 hrs (hours) Experimental Results: Significant disruption of microtubule organization in a concentration-dependent manner. Western Blot Analysis Cell Types: HeLa[1] Tested Concentrations: 0.5 μM Incubation Duration: 24 hrs (hours) Experimental Results: Bax protein expression level was Dramatically up-regulated, Bcl-2 protein expression level was Dramatically up-regulated, and Bcl-2 protein expression level was Dramatically up-regulated. Protein expression of caspase-3 and PARP.
Animal Protocol	Animal/Disease Models: BALB/c nude mice (18-22 g, 5 weeks old, injected with HeLa cells) [1] Doses: 15 and 30 mg/kg Route of Administration: intravenous (iv) (iv)injection; one time/day for 21 days Experimental Results: in dose It Dramatically inhibited tumor growth in a dependent manner, with inhibition rates of 50.73% and 65.76% at doses of 15 and 30 mg/kg, respectively.
References	[1]. Dual-functional conjugates improving cancer immunochemotherapy by inhibiting tubulin polymerization and indoleamine-2,3-dioxygenase. Eur J Med Chem. 2020;189:112041.

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	1.3408 mL	6.7040 mL	13.4081 mL
	5 mM	0.2682 mL	1.3408 mL	2.6816 mL
	10 mM	0.1341 mL	0.6704 mL	1.3408 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.