Journal of Medicinal Chemistry Figure 3. Eﬀect of PUB-SOs 4, 10, 16, 17, 30, 45−47, CA-4, and cDDP on the growth of HT-1080 tumors and their toxicity on chick embryos in the CAM assay. Gray bars represent the percentage of wet weight of tumors treated with or without excipient. Black bars represent the percentage of chick embryo mortality. death rate, respectively), in a manner similar to that of cDDP and CA-4. ■ CONCLUSION We have identiﬁed and characterized PUB-SOs as a novel class of anticancer agents that block cell cycle progression in S-phase. Structure−activity relationships of PUB-SOs indicate that modiﬁcation of their sulfonyl group by a bioisosteric sulfonamide moiety, yielding PUB-SAs, abolishes both their antiproliferative and cell cycle blocking activities. The pharmacophoric EU and CEU moieties with a substitution at C4 on aromatic ring A are required to achieve optimal antiproliferative activity and S-phase arrest, whereas substitu- tions with alkyl groups at C2 or a hydroxyl group at C4 on the B-ring do not signiﬁcantly aﬀect cytocidal activity. In the series of PUB-SOs herein synthesized, we have identiﬁed compounds with an antiproliferative activity and ability to cause S-phase arrest comparable to those of cDDP. Moreover, compounds 4, 16, 17, 29−31, 33, 40, 41, 45−47, and 49 induce H2AX phosphorylation, in support for a mechanism of action that involves DNA double-strand breaks, although the molecular details have yet to be identiﬁed. Finally, compounds 4 and 46 are at least as active as cDDP and CA-4 in the CAM assay while displaying little or no toxic eﬀect on chick embryos, suggesting that these compounds might represent a promising new class of anticancer agents. ■ EXPERIMENTAL SECTION Biological Methods. Antiproliferative Activity. HT-29 colon carcinoma cells, M21 skin melanoma cells, and MCF-7 breast carcinoma cells (all of human origin) were purchased from the American Type Culture Collection (Manassas, VA). Cells were cultured in high-glucose Dulbecco’s minimal essential medium (DMEM) supplemented with 5% (v/v) fetal bovine serum (Hyclone, Logan, UT). The cell lines were maintained at 37 °C in a water- saturated atmosphere containing 5% CO2. The growth inhibition potency of all compounds was assessed using the procedure recommended by the National Cancer Institute for its drug screening program.17 Brieﬂy, 96-well microtiter plates were seeded with 75 μL of a suspension of HT-29 (4 × 103), M21 (3.5 × 103), or MCF-7 (3 × 103) cells per well in DMEM. Plates were incubated at 37 °C and 5% CO2 for 24 h. Drugs freshly solubilized in DMSO (40 mM) were Article diluted in fresh DMEM, and 75 μL aliquots containing serially diluted concentrations of the drug were added. Final drug concentrations ranged from 200 μM to 780 nM. DMSO was maintained at a concentration of <0.5% (v/v) to avoid any related toxicity. Plates were incubated for 48 h, after which growth was stopped by the addition of cold trichloroacetic acid to the wells (10% w/v, ﬁnal concentration), followed by a 1 h incubation at 4 °C. Plates were then washed 5 times with water. An amount of 75 μL of a sulforhodamine B solution (0.1% w/v) in 1% acetic acid was added to each well, and the plates were incubated for 15 min at room temperature. After staining, unbound dye was removed by washing 5 times with 1% acetic acid. Bound dye was solubilized in 20 mM Tris base, and the absorbance was read using an optimal bandwidth (530−568 nm) with a μQuant Universal microplate spectrophotometer (Biotek, Winooski, VT). Readings obtained from treated cells were compared with measurements from control cell plates ﬁxed on treatment day, and the percentage of cell growth inhibition was thus calculated for each drug. The experiments were performed at least twice in triplicate. The assays were considered valid when the coeﬃcient of variation for a given set of conditions and within the same experiment was <10%. Cell Cycle Analysis. Jurkat E6 human leukemic T-cell lymphoblasts were purchased from the American Type Culture Collection. Cells were cultured in RPMI 1640 medium supplemented with 10% (v/v) fetal bovine serum. Cells were maintained at 37 °C in a water-saturated 5% CO2 atmosphere. PUB-SOs, PUB-SAs, cDDP, and DMSO were serially diluted in culture medium in a 12-well plate, starting at a concentration 50% above their respective IC50 toward M21 cells. Next, 4.0 × 105 Jurkat cells suspended in culture medium were added to each well and incubated with the drugs for 24 h. Cells were then harvested, washed with PBS, and resuspended in 250 μL of PBS containing 3.0 × 105 chicken red blood cells as an internal standard. Cells were ﬁxed by the addition of 750 μL of ice-cold EtOH and stored at −20 °C until analysis. Prior to ﬂuorometry, cells were washed with PBS and resuspended in 1 mL of PBS containing 1 μg/ mL DAPI. Fixed cell suspensions were incubated on ice for 1 h, and cell cycle distribution was then analyzed using an LSR II ﬂow cytometer (BD Biosciences, Franklin Lakes, NJ). Immunoﬂuorescence. Cover slides (22 mm × 22 mm) sterilized with 70% (v/v) EtOH were placed in six-well plates. To promote cell adhesion, cover slides were treated with 1.5 mL of a ﬁbronectin solution in PBS (10 μg/mL) for 1 h at 37 °C. Slides were then rinsed twice with PBS. M21 cells (1 × 105) were seeded onto the plates and incubated for 24 h. Cells were then incubated with the test compound at its IC50 for 24 h at 37 °C. The control solution consisted of DMSO dissolved in culture medium (0.5%, v/v). Cells were ﬁxed using 1 mL of formaldehyde at 3.7% and permeabilized by addition of a saponin solution (0.1% in PBS) containing 3% (w/v) BSA (saponin−BSA). Cells were incubated with mouse anti-H2AX pS139 antibody (Millipore, Billerica, MA). Cover slides were next incubated for 3 h at room temperature and then washed twice with PBS supplemented with 0.05% (v/v) Tween 20 (PBS-T). Saponin−BSA containing goat anti-mouse IgG conjugated to AlexaFluor 594 (Invitrogen, Burlington, Ontario, Canada), and DAPI (Sigma, Oakville, Ontario, Canada) (0.3 μg/mL) was then added. The cover slides were incubated for 2 h at room temperature and then washed twice with PBS-T and twice with PBS. The cover slides were mounted with polyvinyl alcohol−1,4,- diazobicyclo[2.2.2]octane (10−2.5%, v/v) in buﬀer (5% (v/v) glycerol and 25 mM Tris buﬀer, pH 8.7) (Sigma, Oakville, Ontario, Canada). Cells were visualized using an epiﬂuorescence microscope (Olympus BX51, Center Valley, PA) with a Qimaging RETIGA EXi camera (Qimaging, Surrey, British Columbia, Canada). CAM Assay. Human HT-1080 ﬁbrosarcoma cells were cultured in Dulbecco’s minimal essential medium containing 58 mM NaHCO3 25 mM D-glucose, 4 mM L-glutamine, and 0.11 mM sodium pyruvate supplemented with 5% (v/v) fetal bovine serum. Cells were maintained at 37 °C in a water-saturated, 5% CO2 atmosphere. HT- 1080 cells were used to assess the antitumoral activity of candidate drugs in the CAM assay. Brieﬂy, on day 0, freshly fertilized chicken eggs were purchased from Couvoirs Victoriaville (Victoriaville, Quebec, Canada). The eggs were incubated for 10 days in a Pro-FI 6199 dx.doi.org/10.1021/jm3006492 | J. Med. Chem. 2012, 55, 6194−6208