Exploring Hydrogen Peroxide Responsive Thiazolidinone-Based Prodrugs

A novel approach for developing prodrugs based on masked carboxylic acids is described. Rather than using conventional esterase-based activation, thiazolidinone protecting groups have been identified that can reveal carboxylic acid groups upon activation by hydrogen peroxide. This may prove valuable in the continuing development of prodrug strategies that rely on reactive oxygen species (ROS) as a trigger.


Synthesis of Thiazolidin-2-one (B).
To a solution of anhydrous EtOH (10 mL) in a dry vessel was added sodium (0.25 g, 11 mmol).The mixture was kept under N 2 atmosphere and stirred at RT for ~30 mins.To this was added thiazolidine-2-thione (D) (1.2 g, 10 mmol) and 2-bromoethanol (1.3 g, 10 mmol).The mixture was heated to reflux for 4 h, allowed to cool to room temperature, then filtered to remove insoluble white solids, which were rinsed with anhydrous EtOH (3x10 mL).The filtrate was concentrated, then purified via silica gel chromatography eluting with hexanes and EtOAc to afford B in 56% yield (0.64 g, 6.2 mmol). 1

Protocol for the amide coupling (Method i):
To a solution of carboxylic acid (1 mmol) in anhydrous CH 2 Cl 2 (10 mL), was added DCC (1.1 mmol) and DMAP (1.1 mmol).The mixture was stirred at room temperature for 20 min followed by the addition of the corresponding amine (A-D) (1 mmol) and stirred for an additional 4 h.The resulting solution was concentrated, then purified via silica gel chromatography eluting with a gradient of 0-30% EtOAc in hexanes.

Protocol for Schotten-Baumann reaction (Method ii)
To a solution of A-D (12 mmol) in H 2 O (5 mL) was added NaOH (15 mmol), followed by acetone (45 mL), then the corresponding acyl chloride (15 mmol).The mixture was stirred for 30 min at room temperature.Acetone was removed from solution under reduced pressure and the remaining aqueous solution was further diluted with H 2 O (20 mL) then extracted with EtOAc (3x20 mL).The organic phases were combined and dried with MgSO 4 , concentrated, then purified via silica gel chromatography eluting with a gradient of 0-30% EtOAc in hexanes.

Stability of Model Compounds
Aqueous stability of compounds was determined by making a 1 mM stock solution in 40% DMSO/60% Buffer (100 mM Tris-Cl, pH 7.4) and incubating at 37 °C for 24 h.Stability in the presence of nucleophiles was determined by making a 1 mM stock solution in 40% DMSO/60% Buffer (100 mM Tris-Cl, pH 7.4) and adding the corresponding nucleophile (20 equiv, 20 mM) and incubating at 37 °C for 24 h.

MMP Inhibition Assays
Inhibition values of proMMPi were determined using a commercially available fluorescent-based assay kit.MMP-2 and OmniMMP fluorogenic subsbtrate were purchased from Enzo Life Sciences (Farmingdale, NY).MMP activity was measured in 96-well plates using a Bio-Tek Synergy HT fluorescent plate reader.ProMMPi was dissolved in DMSO to a concentration of 100 mM and further diluted with Tris-Cl buffer (100 mM, pH 7.4) to a concentration of 1 mM (40% DMSO/ 60% Buffer).To the sample was added H 2 O 2 (50 equiv, 50 mM), followed by incubation at 37 °C until full deprotection was observed via analytical HPLC.The treated compound was then added to appropriate wells near its IC 50 value (350 nM).Each well contained 20 µL of MMP-2 (1.16 U), 60 µL MMP assay buffer (50 mM HEPES, 10mM CaCl 2, 0.10% Brij-35, pH 7.5), and the H 2 O 2 -treated MMPi (10 µL).After a 30 min incubation at 37 °C, a reaction was initiated with the addition of 10 µL (40 µM) of the fluorescent substrate (Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH 2 ) where Mca = (7-methoxycoumarin-4-yl)-acetyl and Dpa = N-3-(2,4-dinitrophenyl)-L-α-β-diaminopropionyl)) and activity was monitored every 30 sec for 30 min with excitation and emission wavelengths at 320 nm and 400 nm, respectively.Enzymatic activity and thus inhibition was calculated with respect to the control experiment (no inhibitor present).Measurements were performed in triplicate with three independent experiments.

COX-1 Inhibition Assays
Inhibition values of proIBU was determined using a commercially available fluorescentbased assay kit Cayman Chemical -Cox Fluorescent Inhibitor Screening Assay Kit Item No. 700100.COX-1 activity was measured in 96-well plates using a Bio-Tek Synergy HT fluorescent plate reader.ProIBU was dissolved in DMSO to a concentration of 100 mM and further diluted in Tris-Cl buffer (100 mM, pH 8.0) to a concentration of 1 mM (40% DMSO/60% Buffer).To the sample was added H 2 O 2 (50 equiv), and incubation at 37 °C was allowed until full deprotection was observed via analytical HPLC.The treated compound was then added to appropriate wells near its IC 50 value (2µM).Protocol was carried out as instructed by supplier.

Crystallographic Data
A concentrated solution of either compound 2 or 3 was in prepared in a 1:1 (by volume) mixture of CH 2 Cl 2 and EtOAc.The solution was transferred to a vial, layered with hexanes, and sealed.The solution was allowed to stand at room temperature for several days, yielding X-ray quality crystals of the desired compound.
A single crystal of 2 or 3 taken from a mixture of CH 2 Cl 2 :EtOAc:hexanes was mounted on nylon loops with paratone oil and placed under a nitrogen cold stream.
Data was collected on a Bruker Apex diffractometer using Mo Kα (λ= 0.71073 Å) radiation controlled using the APEX 2010 software package.The data was collected up to 0.83 Å.A multi-scan method utilizing equivalents was employed to correct for absorption.All data collections were solved and refined using the SHELXTL software suite.Details for these structures can be obtained from the Cambridge Crystallographic Data Centre (CCDC) under deposition numbers 1038268-1038269.

Cytotoxicity Assay
NIH 3T3 cell line was kindly donated by Dr. Richard Klemke (Department of Pathology and Moores Cancer Center, UCSD) and DMEM medium supplemented with 10% fetal bovine serum (FBS, Gibco, Grand Island, NY, USA) at 37 °C in an incubator with 5% CO 2 .The CellTiter 96 AQ ueous One Solution Cell Proliferation assay (MTS) kit was purchased from Promega (Madison, WI, USA).Cytotoxicity of thiazolidinone was measured using the MTS assay according to the manufacturer's protocol.To start the assay, NIH3T3 cells were counted with a hemocytometer and diluted with fresh medium to the proper concentration, such that 5000 cells per well were seeded in a 96 well plate.The NIH 3T3 cells were then incubated at 37 °C with 5% CO 2 for 16 h.Following this 16 h incubation, the cells were treated with various concentrations of thiazolidinone (ranging from 0.25 µM to 170 µM) for 60 h.Each concentration was conducted in triplicate in three independent experiments.After the 60 h incubation, 20 µL of CellTiter 96 AQ ueous One Solution was added to each well and the cells were incubated at 37 °C for 2 h.Following this 2 h incubation, the absorbance at 490 nm was recorded using a BioTek Synergy HT microplate reader.

Figure S26
Cytotoxicity assay for thiazolidinone promoiety B. Each data point is the result of three independent experiments (error bars shown).

Figure S25 Crystal structure of compound 3 ,
Figure S25

Table S1 .
Crystal data and structure refinement for compounds 2 and 3