IITZ-01 is a novel and potent lysosomotropic autophagy inhibitor which has single-agent antitumor efficacy in triple-negative breast cancer in vitro and in vivo. Treatment with IITZ-01 resulted in the vacuolated appearance of cells due to their specific accumulation in lysosomes. In addition, these basic compounds also deacidify lysosomes as evidenced by the decrease in lysotracker red staining and inhibit maturation of lysosomal enzymes leading to lysosomal dysfunction. IITZ-01 and IITZ-02 enhance autophagosome accumulation but inhibit autophagosomal degradation by impairing lysosomal function, finally resulting in the inhibition of autophagy. Interestingly, compound IITZ-01 exhibited more than 10-fold potent autophagy inhibition along with 12- to 20-fold better cytotoxic action than CQ. IITZ-01 and IITZ-02 also abolished mitochondrial membrane potential and triggered apoptosis through the mitochondria-mediated pathway. Furthermore, IITZ-01 and IITZ-02 displayed potent antitumor action in vivo through autophagy inhibition and apoptosis induction in MDA-MB-231 breast cancer xenograft model with IITZ-01exhibiting superior anticancer efficacy. Overall, these data demonstrate that IITZ-01 is potent autophagy inhibitor with single-agent anticancer activity and awaits further preclinical development as potential anticancer therapeutic.

Physicochemical Properties

Molecular Formula	C26H23FN8O
Molecular Weight	482.512227296829
Exact Mass	482.197
CAS #	1807988-47-1
Related CAS #	1807988-47-1
PubChem CID	134817273
Appearance	White to off-white solid powder
LogP	5.2
Hydrogen Bond Donor Count	3
Hydrogen Bond Acceptor Count	9
Rotatable Bond Count	6
Heavy Atom Count	36
Complexity	683
Defined Atom Stereocenter Count	0
SMILES	FC1C=CC(=CC=1)NC1N=C(NC2C=CC(=CC=2)C2=NC3C=CC=CC=3N2)N=C(N=1)N1CCOCC1
InChi Key	XGEDDGLPNSLBFE-UHFFFAOYSA-N
InChi Code	InChI=1S/C26H23FN8O/c27-18-7-11-20(12-8-18)29-25-32-24(33-26(34-25)35-13-15-36-16-14-35)28-19-9-5-17(6-10-19)23-30-21-3-1-2-4-22(21)31-23/h1-12H,13-16H2,(H,30,31)(H2,28,29,32,33,34)
Chemical Name	N2-(4-(1H-benzo[d]imidazol-2-yl)phenyl)-N4-(4-fluorophenyl)-6-morpholino-1,3,5-triazine-2,4-diamine
Synonyms	IITZ 01;IITZ-01;IITZ01
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	IITZ-01 (0-2 μM, 24 h) promotes autophagosomes formation as shown by increased expression of LC3-II levels time- and dose-dependently in triple-negative breast cancer (TNBC) cell lines (MDA-MB-231 and MDA -MB-453). IITZ-01 also showed substantial autophagy inhibitory effect in various breast, lung, and colon cancer cells[1].
ln Vivo	In mice with triple-negative breast tumor models, IITZ-01 (45 mg/kg, ip every other day for four weeks) reduces the growth of average breast tumors starting on the third day of therapy as compared to the control group[1].
Cell Assay	Western Blot Analysis[1] Cell Types: Triple-negative breast cancer (TNBC) cell lines (MDA -MB-231 and MDA-MB-453). Tested Concentrations: 0-2 μM. Incubation Duration: 24 hrs (hours). Experimental Results: Enhanced autophagosomes formation as indicated by increased expression of LC3-II levels.
Animal Protocol	Animal/Disease Models: MDA-MB -231 (TNBC)/green fluorescent protein (GFP) orthotropic breast cancer xenografts were developed in CrTac:NCr-Foxnnu BALB/c female nude mice[1]. Doses: 45 mg/kg. Route of Administration: intraperitoneal (ip)every alternate day for 4 weeks . Experimental Results: Inhibited average breast tumor growth when compared with control from third day of treatment.
References	[1]. IITZ-01, a novel potent lysosomotropic autophagy inhibitor, has single-agent antitumor efficacy in triple-negative breast cancer in vitro and in vivo. Oncogene. 2018 Aug 30.

Solubility Data

Solubility (In Vitro)

DMSO: >10mM Water:N/A Ethanol:N/A

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.18 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 (5.18 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. Solubility in Formulation 3: ≥ 2.5 mg/mL (5.18 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.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	2.0725 mL	10.3625 mL	20.7250 mL
	5 mM	0.4145 mL	2.0725 mL	4.1450 mL
	10 mM	0.2072 mL	1.0362 mL	2.0725 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.