TW-37 (TW37 or TW 37) is a novel and potent small-molecule inhibitor of Bcl-2, Bcl-xL and Mcl-1 with Ki of 0.29 μM, 1.11 μM and 0.26 μM in cell-free assays, respectively. It may have anticancer properties. TW-37 inhibits several BCL-2 family members, including BCL-xL (Ki = 1,110 nmol/L) and MCL-1 (Ki = 260 nmol/L), and attenuates BCL-2 activation. It binds to the BCL-2 homology domain 3 (BH3) groove, which stops proapoptotic proteins (like Bid, Bim, and Bad) from heterodimerizing with BCL-2 and subsequently allows them to trigger apoptosis. TW-37 induces S-phase cell cycle arrest with regulation of several significant cell cycle related genes, including p27, p57, cyclin D1 and cyclin E, according to recent studies, which suggests it can inhibit the growth of a wide variety of cancer cells.

Physicochemical Properties

Molecular Formula	C33H35NO6S
Molecular Weight	573.7
Exact Mass	573.218
Elemental Analysis	C, 69.09; H, 6.15; N, 2.44; O, 16.73; S, 5.59
CAS #	877877-35-5
Related CAS #	877877-35-5
PubChem CID	11455910
Appearance	White to off-white solid powder
Density	1.3±0.1 g/cm3
Boiling Point	723.7±60.0 °C at 760 mmHg
Flash Point	391.5±32.9 °C
Vapour Pressure	0.0±2.4 mmHg at 25°C
Index of Refraction	1.631
LogP	8.74
Hydrogen Bond Donor Count	4
Hydrogen Bond Acceptor Count	6
Rotatable Bond Count	8
Heavy Atom Count	41
Complexity	963
Defined Atom Stereocenter Count	0
SMILES	O=C(C1C(O)=C(O)C(O)=C(CC2C(C(C)C)=CC=CC=2)C=1)NC1C=CC(S(C2C(C(C)(C)C)=CC=CC=2)(=O)=O)=CC=1
InChi Key	PQAPVTKIEGUPRN-UHFFFAOYSA-N
InChi Code	InChI=1S/C33H35NO6S/c1-20(2)25-11-7-6-10-21(25)18-22-19-26(30(36)31(37)29(22)35)32(38)34-23-14-16-24(17-15-23)41(39,40)28-13-9-8-12-27(28)33(3,4)5/h6-17,19-20,35-37H,18H2,1-5H3,(H,34,38)
Chemical Name	5-(2-isopropylbenzyl)-N-(4-(2-tert-butylphenylsulfonyl)phenyl)-2,3,4-trihydroxybenzamide
Synonyms	TW-37; TW37; TW 37
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	Mcl-1 (Ki=260 nM); Bcl-2 (Ki=290 nM); Bcl-xL (Ki=1110 nM)
ln Vitro	TW-37 (TW37) is a novel nonpeptide small-molecule inhibitor designed using a structure-based design strategy. TW-37 targets the proapoptotic Bcl-2 proteins Bak, Bax, and Bid in the BH3-binding groove of Bcl-2. TW-37 binds to recombinant Bcl-2 and Bcl-xL proteins with Ki values of 290 and 1110 nM, respectively, in fluorescence polarization-based binding assays. TW-37 does not have any cytotoxic effects on fibroblasts at concentrations up to 50 M, but it does have an IC50 of 1.8 M for endothelial cells. Apoptosis, a process mediated by mitochondrial depolarization and activation of caspase-9 and caspase-3, is the mechanism of TW-37-induced endothelial cell death. Coexposure to the growth factor milieu secreted by tumor cells does not counteract the effect of TW-37 on the apoptosis of endothelial cells. At subapoptotic TW-37 concentrations (0.005–0.05 M), the angiogenic chemokines CXCL1 and CXCL8 are expressed and the angiogenic potential of endothelial cells is inhibited (i.e., migration and capillary sprouting assays)[1]. TW-37 is a potent Bcl-2 and Mcl-1 inhibitor. TW-37 binds to recombinant Bcl-2, Bcl-xL, and Mcl-1 proteins with Ki values of 290, 1,110, and 260 nM, respectively, in fluorescence polarization-based binding assays[2].
ln Vivo	We examine the biological impact of TW-37 (TW37) on human microvascular endothelial cells in vivo using a murine model of humanized vasculature. According to this model, both 3 and 30 mg/kg TW-37 are significantly less effective than the vehicle control in terms of the total number of blood vessels (P0.05). Along with a decrease in the overall number of blood vessels, the treated groups are also experiencing an unusually high number of occluded vessels. By counting completely blocked vessels and calculating their percentage of all vessels, the levels of vessel occlusion are evaluated. When compared to control, TW-37 concentration significantly increases the number of occluded vessels[1].
Enzyme Assay	For this assay, the 21-residue BH3 peptide QEDIIRNIARHLAQVGDSMDR derived from Bid labeled with 6-carboxyfluorescein succinimidyl ester (FAM-Bid) and recombinant proteins derived from human Bcl-2,Bcl-X L,and Mcl-1 are employed. It is determined that FAM-Bid has a Ki of 11 nM to Bcl-2 protein,25 nM to Bcl-XL protein,and 5.7 nM to Mcl-1 protein. The competitive binding assay for Bcl-XL is same as that for Bcl-2 with the following exceptions: 30 nM Bcl-XL protein and 2.5 nM FAM-Bid peptide in the following assay buffer [50 mM Tris-Bis (pH 7.4) and 0.01% bovine gamma-globulin].
Cell Assay	The sulforhodamine B (SRB) cytotoxicity assay is used as described. Briefly, optimal cell density for cytotoxicity assay is determined by growth curve analysis. HDMECs are seeded in a 96-well plate and allowed to adhere overnight. Drug or control is diluted in EGM2-MV and layered onto cells, which are allowed to incubate for times as indicated in the figures. Alternatively, HDMECs are coincubated with TW37 and 0 to 100 ng/mL recombinant human VEGF (rhVEGF)165 or 0 to 100 ng/mL recombinant human CXCL8. Cells are fixed on the plates by addition of cold trichloroacetic acid (10% final concentration) and incubation for 1 hour at 4 °C. Cellular protein is stained by addition of 0.4% SRB in 1% acetic acid and incubation at room temperature for 30 minutes. Unbound SRB is removed by washing with 1% acetic acid and the plates are air dried. Bound SRB is resolubilized in 10 mM unbuffered Tris-base and absorbance is determined on a microplate reader at 560 nm. Test results are normalized against initial plating density and drug-free controls. Data are obtained from triplicate wells per condition and are representative of at least three independent experiments
Animal Protocol	~40 mg/kg TW-37 is resuspended in Tween 80/ethanol (1:1 ratio, diluted 10-fold with 0.9% saline before use). Athymic NCr-nu/nu mice bearing SK-Mel-147 melanoma xenografts
References	[1]. Antiangiogenic effect of TW37, a small-molecule inhibitor of Bcl-2. Cancer Res. 2006 Sep 1;66(17):8698-706. [2]. Preclinical studies of TW-37, a new nonpeptidic small-molecule inhibitor of Bcl-2, in diffuse large cell lymphoma xenograft model reveal drug action on both Bcl-2 and Mcl-1. Clin Cancer Res. 2007 Apr 1;13(7):2226-35.
Additional Infomation	N-[4-(2-tert-butylphenyl)sulfonylphenyl]-2,3,4-trihydroxy-5-[(2-propan-2-ylphenyl)methyl]benzamide is a member of benzamides. TW-37 is a small-molecule inhibitor of Bcl-2 investigated for its anti-cancer properties.

Solubility Data

Solubility (In Vitro)

DMSO: ~115mg/mL (~200.5 mM) Water: <1 mg/mL(slightly soluble or insoluble) Ethanol: ~4 mg/mL (~7.0 mM)

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.36 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: 2.08 mg/mL (3.63 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 (3.63 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.. Solubility in Formulation 4: 30% Propylene glycol , 5% Tween 80 , 65% D5W: 30 mg/mL  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	1.7431 mL	8.7154 mL	17.4307 mL
	5 mM	0.3486 mL	1.7431 mL	3.4861 mL
	10 mM	0.1743 mL	0.8715 mL	1.7431 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.