ZLDI-8 is an inhibitor (blocker/antagonist) of Notch activating/cleaving enzyme ADAM-17, which inhibits the cleavage of Notch protein and reduces the expression of pro-survival/anti-apoptotic and epithelial-to-mesenchymal transition-related proteins. ZLDI-8 is also a competitive and irreversible inhibitor of tyrosine phosphatase (Lyp) with IC50 of 31.6 μM and Ki of 26.22 μM. ZLDI-8 suppresses the growth of MHCC97-H cells with IC50 of 5.32 μM.

Physicochemical Properties

Molecular Formula	C₂₄H₂₃N₃O₃S
Molecular Weight	433.52
Exact Mass	433.146
Elemental Analysis	C, 66.49; H, 5.35; N, 9.69; O, 11.07; S, 7.40
CAS #	667880-38-8
PubChem CID	989566
Appearance	Yellow to orange solid powder
Density	1.3±0.1 g/cm3
Index of Refraction	1.662
LogP	5.26
Hydrogen Bond Donor Count	2
Hydrogen Bond Acceptor Count	4
Rotatable Bond Count	5
Heavy Atom Count	31
Complexity	732
Defined Atom Stereocenter Count	0
SMILES	S=C1N([H])C(/C(/C(N1[H])=O)=C(/[H])\C1C2=C([H])C([H])=C([H])C([H])=C2N(C([H])([H])C([H])([H])OC2C([H])=C([H])C(C([H])([H])[H])=C([H])C=2C([H])([H])[H])C=1C([H])([H])[H])=O
InChi Key	GNBZGGYWSRGVBG-UHFFFAOYSA-N
InChi Code	InChI=1S/C24H23N3O3S/c1-14-8-9-21(15(2)12-14)30-11-10-27-16(3)18(17-6-4-5-7-20(17)27)13-19-22(28)25-24(31)26-23(19)29/h4-9,12-13H,10-11H2,1-3H3,(H2,25,26,28,29,31)
Chemical Name	5-[[1-[2-(2,4-dimethylphenoxy)ethyl]-2-methylindol-3-yl]methylidene]-2-sulfanylidene-1,3-diazinane-4,6-dione
Synonyms	ZLDI8; ZLDI 8
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	ZLDI-8 (0.03-30 μM; 6-72 hours; MHCC97-H cells) treatment decreases cell viability in a dose- and time-dependent manner [1]. ZLDI-8 (1-10 μM; 6-72 hours; MHCC97-H cells) dramatically decreased the amount of NICDs present and their accumulation in the nucleus. ZLDI-8 also inhibits the expression of cIAP1/2 and Survivin, two pro-survival/anti-apoptotic regulators. Moreover, it decreases the expression of the mesenchymal markers Vimentin and N-Cadherin and increases the expression of the epithelial marker E-Cadherin [1]. By inhibiting the Notch pathway and preventing chemoresistance, ZLDI-8 increases the impact of chemotherapy on tumor cell proliferation, induction of apoptosis, and cell cycle arrest [1].
ln Vivo	In tumor-bearing nude animal models, ZLDI-8 (0.2-2 mg/kg; intraperitoneal injection; every two days; 20 consecutive days; nude mice) treatment improved sorafenib's inhibitory effect on tumor growth [1].
Cell Assay	Cell viability assay[1] Cell Types: MHCC97-H Cell Tested Concentrations: 0.03 μM, 0.1 μM, 0.3 μM, 1 μM, 3 μM, 10 μM, 30 μM Incubation Duration: 6 hrs (hours), 12 hrs (hours), 24 hrs (hours), 48 hrs (hours), 72-hour Experimental Results: Cytotoxic effects on MHCC97-H cells in a time- and dose-dependent manner. Western Blot Analysis[1] Cell Types: MHCC97-H Cell Tested Concentrations: 1 μM, 3 μM, 10 μM Incubation Duration: 6 hrs (hours), 12 hrs (hours), 24 hrs (hours), 48 hrs (hours), 72 hrs (hours) Experimental Results: NICD levels were Dramatically diminished, NICD was accumulate in the cell nucleus. Also diminished the expression of pro-survival/anti-apoptotic regulators Survivin and cIAP1/2
Animal Protocol	Animal/Disease Models: MHCC-97H cell nude mice [1] Doses: 2 mg/kg, 1 mg/kg, 500 μg/kg, 200 μg/kg Route of Administration: intraperitoneal (ip) injection; every two days; 20 days Experimental Results: Inhibition Tumor growth in a nude mouse model of HCC.
References	[1]. Novel ADAM-17 inhibitor ZLDI-8 enhances the in vitro and in vivo chemotherapeutic effects of Sorafenib on hepatocellular carcinoma cells. Cell Death Dis. 2018 Jul 3;9(7):743. [2]. Fast identification of novel lymphoid tyrosine phosphatase inhibitors using target-ligand interaction-based virtual screening. J Med Chem. 2014 Nov 26;57(22):9309-22.

Solubility Data

Solubility (In Vitro)

DMSO : ~62.5 mg/mL (~144.17 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	2.3067 mL	11.5335 mL	23.0670 mL
	5 mM	0.4613 mL	2.3067 mL	4.6134 mL
	10 mM	0.2307 mL	1.1533 mL	2.3067 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.