CA-5f (CA5f) is a novel and potent late-stage macroautophagy/autophagy inhibitor via inhibiting autophagosome-lysosome fusion with anti-tumor activity. It has potent anti-tumor effect against non-small cell lung cancer. CA-5f neither impaired the hydrolytic function nor the quantity of lysosomes. Use of an isobaric tag for relative and absolute quantitation (iTRAQ)-based proteomic screen in combination with bioinformatics analysis suggested that treatment of human umbilical vein endothelial cells (HUVECs) with CA-5f for 1 h suppressed the levels of cytoskeletal proteins and membrane traffic proteins. Subsequent studies showed that CA-5f exhibited strong cytotoxicity against A549 non-small cell lung cancer (NSCLC) cells, but low cytotoxicity to normal human umbilical vein endothelial cells (HUVECs), by increasing mitochondrial-derived reactive oxygen species (ROS) production.

Physicochemical Properties

Molecular Formula	C24H24N2O3
Molecular Weight	388.46
Exact Mass	388.178
CAS #	1370032-19-1
Related CAS #	1370032-19-1
PubChem CID	57341148
Appearance	Light yellow to yellow solid powder
LogP	3.7
Hydrogen Bond Donor Count	1
Hydrogen Bond Acceptor Count	4
Rotatable Bond Count	4
Heavy Atom Count	29
Complexity	659
Defined Atom Stereocenter Count	0
SMILES	C(/C1=CNC2=CC=CC=C12)=C1\C(/C(/CN(C)C\1)=C/C1C=CC(OC)=C(OC)C=1)=O
InChi Key	JYOLPDWVAMBMQN-UBIAKTOFSA-N
InChi Code	InChI=1S/C24H24N2O3/c1-26-14-18(10-16-8-9-22(28-2)23(11-16)29-3)24(27)19(15-26)12-17-13-25-21-7-5-4-6-20(17)21/h4-13,25H,14-15H2,1-3H3/b18-10+,19-12+
Chemical Name	(3E,5E)-3-(3,4-dimethoxybenzylidene)-5-[(1H-indol-3-yl)methylene]-1-methylpiperidin-4-one
Synonyms	CA-5f;CA5f;CA 5f;
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	Macroautophagy/autophagy[1]
ln Vitro	In A549 cells and HUVEC, CA-5f (0-40 μM, 6 hours) elevates the levels of SQSTM1 protein and LC3B-II (an autophagy monitoring marker) in a concentration- and time-dependent manner [1]. A549 cells and HUVECs treated with CA-5f (20 μM, 6 hours) or in combination with Bafilomycin A1 (100 nM) or Chloroquine (30 μM) do not exhibit autophagosome degradation [1]. Neither the number of lysosomes nor the hydrolytic function are impacted by CA-5f (20 μM) [1]. CA-5f (20 μM, 96 hours) is less harmful to normal HUVECs and suppresses the development of A549 cells [1].
ln Vivo	In nude mice containing A549 lung cancer cells, CA-5f (40 mg/kg, intraperitoneal injection, once every two days, up to 30 days) is well tolerated and can successfully stop tumor growth [1]. Using A549 lung cancer cells, CA-5f (40 mg/kg, ip) suppresses autophagy flux and causes apoptosis in nude mice [1].
Cell Assay	Cell Viability Assay[1] Cell Types: A549, HUVECs Tested Concentrations: 20 μM Incubation Duration: 96 hrs (hours) Experimental Results: demonstrated more cytotoxicity against A549 cells compared with normal HUVECs. Western Blot Analysis[1] Cell Types: A549 cells and HUVECs Tested Concentrations: 0 -40 μM Incubation Duration: 6 hrs (hours) Experimental Results: Elevated LC3B-II (a marker to monitor autophagy) and SQSTM1 protein levels in a concentration- and time-dependent manner.
Animal Protocol	Animal/Disease Models: Nude mice bearing A549 lung cancer cells[1] Doses: 40 mg/kg Route of Administration: Injected via caudal vein, every 2 days for up to 30 days Experimental Results: Dramatically suppressed tumor volume and weight in mice, increased the number of apoptotic cells in mice.
References	[1]. Identification of compound CA-5f as a novel late-stage autophagy inhibitor with potent anti-tumor effect against non-small cell lung cancer. Autophagy. 2019 Mar;15(3):391-406.

Solubility Data

Solubility (In Vitro)

DMSO:≥ 10 mM Water:N/A Ethanol:N/A

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.08 mg/mL (5.35 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 20.8 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: 2.08 mg/mL (5.35 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 20.8 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: ≥ 2.08 mg/mL (5.35 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 20.8 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	2.5743 mL	12.8713 mL	25.7427 mL
	5 mM	0.5149 mL	2.5743 mL	5.1485 mL
	10 mM	0.2574 mL	1.2871 mL	2.5743 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.