MMP-2 (Ki = 13.9 nM); MMP-9 (Ki = 600 nM)

(R)-MG-132, the stereoisomer of MG-132, is being investigated as a possible inhibitor of the proteasome's ability to hydrolyze peptidylglutamyl peptide, trypsin, and chymotrypsin-like activities[1]. The effects of MG-132 and (R)-MG-132 on the inhibition of trypsin-like (TL), peptidylglutamyl peptide hydrolyzing (PGPH), and ChTL of purified 20S proteasomes isolated from human erythrocytes are being studied. MG-132 has IC₅₀ values of 0.89 μM, 104.43 μM, and 5.7 μM for ChTL, TL, and PGPH, in that order. The IC₅₀ values for ChTL, TL, and PGPH of (R)-MG-132 are 0.22 μM, 34.4 μM, and 2.95 μM, respectively[1].

SB-3CT (i.p.; 50 mg/kg; every other day; five weeks) prevents the intraosseous growth of human PC3 cells in the marrow of human fetal femur fragments that have been implanted in SCID mice[3].

The fluorescence quenched substrate MOCAcPLGLA2pr(Dnp)-AR-NH2 is used to measure the enzymatic activity of MMP-2, MMP-9, and MMP-7. Using a PTI spectrofluorometer, fluorescence is measured. The temperature of the cuvette compartment is set to 25 °C.

Cell proliferation assay[3]
PC3 cells were seeded in 35-mm dishes (5 × 104 cells/dish) in complete culture medium. The next day, the medium was replaced with complete medium supplemented with 1% DMSO alone (vehicle) or SB-3CT (final concentrations 0.1–50 μM) in 1% DMSO. At various times, the cells were harvested with trypsin and counted.

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Effect of SB-3CT on BMEC-1 cell viability[3]
BMEC-1 cells were seeded in 96-well culture plates (104 cells/well) in complete culture medium. Twenty-four h later, the medium was replaced with serum-free, phenolred-free media supplemented with either vehicle (1% DMSO) or SB-3CT (1 nM–50 μM final concentrations). After 72 h, 10 μL of WST-1 were added to each well, and the optical density was measured at 450 nm, according to the manufacturer's instructions.

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Capillary-like tubule formation assay[3]
Twenty-four-well plates were coated with 300 μL of an ice-cold Matrigel solution (10 mg/mL). The plates were then incubated for 30 min at 37°C to allow Matrigel polymerization, and then 5 × 104 BMEC-1 cells were placed onto the Matrigel-coated wells in the presence of complete medium supplemented with various amounts of SB-3CT (0.1–1 μM) or vehicle (1% DMSO). After overnight incubation at 37°C, digital photographs of three randomly selected areas from each well were taken at 10× magnification, using an Olympus® DP12 Microscope Camera. The area occupied by the capillary-like structures was calculated using Adobe Photoshop 7.0.

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Endothelial cell invasion assay[3]
BMEC-1 cells suspended in Medium-199 with 0.1 % bovine serum albumin supplemented with either SB-3CT (0.1–1 μM) or 1% DMSO (vehicle) were seeded (2 × 105 cells per insert) onto Transwell inserts (8-μm pore size) coated with 25 μg/filter Matrigel. Culture medium supplemented with 5% FBS was placed in the lower chamber as a chemoattractant. After 24 h incubation at 37°C, the cells that migrated to the lower side of the filter were stained with Diff-Quik® and counted under 200× magnification.

Five-week-old male C.B.-17.SCID mice[3]
50 mg/kg
IP; every other day; five weeks

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In situ gelatin zymography[3]
Frozen tissue sections were obtained from HT1080 tumors grown subcutaneously in SCID mice, which were intraperitonially (i.p.) treated for two consecutive days before sacrifice either with 1 mL vehicle (10% DMSO in PBS) or 1 ml containing 1.25 mg SB-3CT in 10% DMSO (equivalent to 50 mg/kg of mouse weight). In situ gelatin zymography was performed in 8-μm thick unfixed cryostat tumor sections incubated for 1 h with 100 μg/ml DQ™-gelatin and 1 μg/mL DAPI (Molecular Probes), as described previously. Establishment of PC3 human bone tumors and experimental treatment[3]
One fourth human fetal femur fragments were implanted subcutaneously in SCID mice as described previously.29 Four weeks later, 1 × 105 PC3 cells were injected through the mouse skin directly into the marrow of the previously implanted bone, as described.29 Twenty-four h after tumor cell inoculation, the mice were injected i.p. with either vehicle (10% DMSO) or SB-3CT in 10% DMSO (50 mg/kg of mouse weight) every other day. Each experimental group contained 9 animals.
Five weeks after tumor cell inoculation, the mice were killed and bone implants harvested, weighed, fixed overnight in 10% buffered formalin, and then X-ray imaged using a Lo-Rad M-IV mammography unit with a magnified specimen technique. Images were developed using a Kodak 2000 screen and radiography film. For histomorphometrical and histological analyses, bone tumors were decalcified with 10% ethylenediaminetetraacetic acid (EDTA) (pH 6.5) in PBS, dehydrated, infiltrated and paraffin-embedded.

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SB-3CT, a discovery from the Mobashery laboratory, was synthesized for this study by reported methodology. Mice were divided into four groups: vehicle-treated group and SB-3CT-treated one with treatment for either one day or seven days after embolic MCA occlusion. SB-3CT (12.5 mg/mL) was freshly dissolved in 25% DMSO/65% PEG-200/10% water and filtered through an Acrodisc syringe filter with a 0.2 μm, 13-mm diameter sterile hydrophobic PTFE membrane. Mice were ip injected with 2 μL/gram body weight of this solution (equivalent to 25 mg/kg) 2 hours after embolic ischemia, followed by an additional dose at 4 hours. In repeated-dose treatment conditions, the same dose of SB-3CT was ip administered 2 and 4 hours after embolic ischemia, followed by once daily from post-ischemia day 1 to 6. Earlier work indicated that ip administration of SB-3CT does not alter mean arterial blood pressure, pH, PCO2, and PO2[4].

[1]. Water-Soluble MMP-9 Inhibitor Reduces Lesion Volume after Severe Traumatic Brain Injury. ACS Chem Neurosci. 2015 Oct 21;6(10):1658-64.

[2]. Potent and Selective Mechanism-Based Inhibition of GelatinasesJ. Am. Chem. Soc.2000122286799-6800

[3]. Inhibition of human prostate cancer growth, osteolysis and angiogenesis in a bone metastasis model by a novel mechanism-based selective gelatinase inhibitor. Int J Cancer. 2006, 118(11), 2721-2726.

[4]. Inhibition of MMP-9 by a selective gelatinase inhibitor protects neurovasculature from embolic focal cerebral ischemia. Mol Neurodegener. 2012, 15, 7-21.

2-[(4-phenoxyphenyl)sulfonylmethyl]thiirane is an aromatic ether.

C15H14O3S2

306.40

306.038

C, 58.80; H, 4.61; O, 15.67; S, 20.93

292605-14-2

9883002

White to pink solid powder

1.3±0.1 g/cm3

501.4±46.0 °C at 760 mmHg

101 °C

257.1±29.0 °C

0.0±1.2 mmHg at 25°C

1.628

3.36

0

4

5

20

401

0

S1C([H])([H])C1([H])C([H])([H])S(C1C([H])=C([H])C(=C([H])C=1[H])OC1C([H])=C([H])C([H])=C([H])C=1[H])(=O)=O

LSONWRHLFZYHIN-UHFFFAOYSA-N

InChI=1S/C15H14O3S2/c16-20(17,11-14-10-19-14)15-8-6-13(7-9-15)18-12-4-2-1-3-5-12/h1-9,14H,10-11H2

2-[(4-phenoxyphenyl)sulfonylmethyl]thiirane

SB3CT; SB3-CT; 2-[(4-phenoxyphenyl)sulfonylmethyl]thiirane; 2-((4-phenoxyphenylsulfonyl)methyl)thiirane; 2-(((4-Phenoxyphenyl)sulfonyl)methyl)thiirane; (4-phenoxyphenylsulfonyl)methylthiirane; CHEMBL483857; Thiirane, 2-[[(4-phenoxyphenyl)sulfonyl]methyl]-; SB-3CT

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: 5 mg/mL (16.32 mM) in 10% DMSO 20% Cremophor EL + 70% ddH2O (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.

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Solubility in Formulation 2: 2.5 mg/mL (8.16 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% 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 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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (8.16 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 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.

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Solubility in Formulation 4: ≥ 2.5 mg/mL (8.16 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 corn oil and mix evenly.

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Solubility in Formulation 5: 4% DMSO+corn oil: 10mg/mL

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 (Please use freshly prepared in vivo formulations for optimal results.)