ROC-325 is a novel, potent and orally bioactive autophagy inhibitor with potent anticancer activity. ROC-325 exhibited superior in vitro anticancer effects compared with the existing autophagy inhibitor hydroxychloroquine (HCQ) in 12 different cancer cell lines with diverse genetic backgrounds. ROC-325 antagonized RCC growth and survival in an ATG5/7-dependent manner, induced apoptosis, and exhibited favorable selectivity.

Physicochemical Properties

Molecular Formula	C28H27CLN4OS
Molecular Weight	503.0582
Exact Mass	502.159
CAS #	1859141-26-6
Related CAS #	1859141-26-6
PubChem CID	129626603
Appearance	Light yellow to orange solid powder
LogP	7
Hydrogen Bond Donor Count	2
Hydrogen Bond Acceptor Count	6
Rotatable Bond Count	8
Heavy Atom Count	35
Complexity	712
Defined Atom Stereocenter Count	0
SMILES	ClC1C=CC2C(C=1)=NC=CC=2NCCN(C)CCNC1C=CC(C)=C2C=1C(C1=CC=CC=C1S2)=O
InChi Key	HXUYKEGAEIYPKY-UHFFFAOYSA-N
InChi Code	InChI=1S/C28H27ClN4OS/c1-18-7-10-23(26-27(34)21-5-3-4-6-25(21)35-28(18)26)32-14-16-33(2)15-13-31-22-11-12-30-24-17-19(29)8-9-20(22)24/h3-12,17,32H,13-16H2,1-2H3,(H,30,31)
Chemical Name	1-((2-((2-((7-chloroquinolin-4-yl)amino)ethyl)(methyl)amino)ethyl)amino)-4-methyl-9H-thioxanthen-9-one
Synonyms	ROC-325 ROC 325 ROC325.
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	Renal cell carcinoma (RCC) development and activation are inhibited by ROC-325 in an antagonistic way towards ATG5/7, resulting in cell apoptosis and excellent selectivity. A498, A549, CFPAC-1, COLO -205, DLD-1, IGROV-1, MCF-7, MiaPaCa-2, NCI-H69, PC-3, RL, and UACC-62 cells are all inhibited by ROC-325; the IC50 values for these cells are 4.9 μM, 11 μM, 4.6 μM, 5.4 μM, 7.4 μM, 11 μM, 8.2 μM, 5.8 μM, 11 μM, 8.4 μM, and 6.0 μM. Autophagy labeling is induced by ROC-325, which also counteracts autophagy staining [1]. Tissue stain D (CTSD) levels were increased in response to ROC-325. After subjecting A498 and 786-0 RCC cells to a 24-hour treatment with 5 μM ROC-325, LC3B solution was formed and LC3B levels dramatically increased. ROC-325 facilitated dose-increasing LC3B expression in A498 and 786-0 cells, according to immunoblot analysis, which was correlated with increases in p62 and histone D levels [1].
ln Vivo	suggested that ROC-325 (25 mg/kg, 40 mg/kg, and 50 mg/kg) applied to the anatomical surface of 786-0 RCC xenografts was well tolerated, inhibited autophagy in the body, and was more effective at halting tumor growth in vitro than chemotherapy[1].
References	[1]. Disruption of Autophagic Degradation with ROC-325 Antagonizes Renal Cell Carcinoma Pathogenesis. Clin Cancer Res. 2017 Jun 1;23(11):2869-2879.

Solubility Data

Solubility (In Vitro)

DMSO : ~4.55 mg/mL (~9.04 mM) H2O : ~1 mg/mL (~1.99 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	1.9878 mL	9.9392 mL	19.8783 mL
	5 mM	0.3976 mL	1.9878 mL	3.9757 mL
	10 mM	0.1988 mL	0.9939 mL	1.9878 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.