PAC-1 (VO 100) is a potent small-molecule activator of procaspase-3 that catalyzes the maturation of procaspase-3 into the active caspase-3 by inducing the time-dependent cleavage of procaspase-3. Studies have shown that PAC-1 can cause cell death in both primary cancerous cells and nearby normal tissues, with 50% inhibition concentration (IC50) values ranging from 0.003 to 1.41 μM and 5.02 to 9.98 μM, respectively.

Physicochemical Properties

Molecular Formula	C23H28N4O2
Molecular Weight	392.49
Exact Mass	392.221
Elemental Analysis	C, 70.38; H, 7.19; N, 14.27; O, 8.15
CAS #	315183-21-2
Related CAS #	315183-21-2
PubChem CID	135421197
Appearance	White to yellow solid powder
Density	1.1±0.1 g/cm3
Index of Refraction	1.597
LogP	4.26
Hydrogen Bond Donor Count	2
Hydrogen Bond Acceptor Count	5
Rotatable Bond Count	8
Heavy Atom Count	29
Complexity	539
Defined Atom Stereocenter Count	0
SMILES	O=C(N/N=C/C1=CC=CC(CC=C)=C1O)CN2CCN(CC3=CC=CC=C3)CC2
InChi Key	YQNRVGJCPCNMKT-LFVJCYFKSA-N
InChi Code	InChI=1S/C23H28N4O2/c1-2-7-20-10-6-11-21(23(20)29)16-24-25-22(28)18-27-14-12-26(13-15-27)17-19-8-4-3-5-9-19/h2-6,8-11,16,29H,1,7,12-15,17-18H2,(H,25,28)/b24-16+
Chemical Name	2-(4-benzylpiperazin-1-yl)-N-[(E)-(2-hydroxy-3-prop-2-enylphenyl)methylideneamino]acetamide
Synonyms	PAC-1; PAC1; PAC 1; VO-100; VO100; VO 100; procaspase activating compound-1
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	Procaspase-3 (EC50 = 0.22 μM)
ln Vitro	PAC-1 activates procaspase-7 in a less efficient manner with an EC50 value of 4.5 μM. With an IC50 ranging from 0.35 M for NCI-H226 cells to 3.5 M for UACC-62 cells, elevated caspase 3 levels in cancer cell lines enable PAC-1 to selectively induce apoptosis. PAC-1 more potently induces apoptosis in primary cancerous cells with IC50 values of 3 nM to 1.41 μM than in neighboring noncancerous cells with IC50 values of 5.02 μM to 9.98 μM, which is also closely related to the distinct procaspase-3 concentration. [1]
ln Vivo	The growth of an ACHN renal cancer xenograft in mice is significantly inhibited by the administration of PAC-1 at a dose of 5 mg with low and steady release. In a dose-dependent manner, oral administration of PAC-1 (50 or 100 mg/kg) significantly slows the growth of the lung cancer xenograft NCI-H226 and significantly reduces the cancer cells' ability to invade lung tissue. Procaspase-3 activation and subsequent induction of apoptosis, both of which are consistent with the activity in vitro, are thought to be the causes of PAC-1's anti-tumor effects in vivo. [1]
Enzyme Assay	Procaspase-3 is expressed and purified in Escherichia coli. In a 96-well plate, 90 μL of procaspase-3 solution containing 50 ng/mL is added. The solution is then mixed with various concentrations of PAC-1. The plate is then incubated for 12 hours at 37 °C. The caspase-3 peptidic substrate acetyl Asp-Glu-Val-Asp-p-nitroanilide (Ac-DEVD-pNa) in a 10 μL volume of caspase assay buffer is then added to each well. A Spectra Max Plus 384 well plate reader reads the plate every two minutes at a wavelength of 405 nm for two hours. Calculating the relative increase in activation from untreated control wells requires determining the slope of the linear portion for each well.
Cell Assay	For 72 hours, cells are exposed to various PAC-1 concentrations. The MTS/PMS CellTiter 96 Cell Proliferation Assay reagent is used to measure cell death. The plates are incubated at 37 °C for roughly an hour (until the colored product formed), and the absorbance is measured at 490 n.
Animal Protocol	0, 50 or 100 mg/kg; Mixed with cholesterol and pelleted into a 3-mm-diameter 20-mg (total weight) pellet, or dissolved in a mixture of 24:1 vegetable oil/DMSO Mice s.c. injected with NCI-H226 (lung cancer) cells
References	[1]. Nat Chem Biol . 2006 Oct;2(10):543-50. [2]. J Mol Biol . 2009 Apr 24;388(1):144-58. [3]. Cell Death Dis . 2011 Sep 8;2(9):e207.
Additional Infomation	PAC-1 has been used in trials studying the treatment of Lymphoma, Melanoma, Solid Tumors, Breast Cancer, and Thoracic Cancers, among others. Procaspase Activating Compound-1 VO-100 is an orally bioavailable procaspase activating compound-1 (PAC-1), with potential proapoptotic and antineoplastic activities. Upon administration, VO-100 binds to and forms a chelating complex with zinc (Zn) ions inside cells, which prevents the binding of Zn ions to procaspase-3 (PC3) and abrogates the Zn-mediated inhibition of PC3. This allows for the proteolytic autoactivation of PC3 into the active form caspase-3. This results in the selective caspase-3-mediated induction of apoptosis and cell death in cancer cells. In addition, VO-100 is able to cross the blood-brain-barrier (BBB). PC3, a Zn-inhibited proenzyme, is upregulated in a variety of cancer cell types, while its expression is minimal in normal healthy cells.

Solubility Data

Solubility (In Vitro)

DMSO: ~78 mg/mL (~198.7 mM) Water: <1 mg/mL (slightly soluble or insoluble) Ethanol: ~16 mg/mL (~40.8 mM)

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.37 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 (6.37 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 (6.37 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.. Solubility in Formulation 4: 30% PEG400+0.5% Tween80+5% Propylene glycol : 30mg/mL  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	2.5478 mL	12.7392 mL	25.4784 mL
	5 mM	0.5096 mL	2.5478 mL	5.0957 mL
	10 mM	0.2548 mL	1.2739 mL	2.5478 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.