Journal of Medicinal Chemistry egg incubator (Lyon Electric, Chula Vista, CA) ﬁtted with an automatic egg turner before being transferred to a Roll-X static incubator for the rest of the incubation period. Eggs were kept at 37 °C in a 60% relative humidity atmosphere for the entire incubation period. By use of a hobby drill (Dremel, Racine, WI), a hole was drilled on the side of the egg, and negative pressure was applied to create a new air sac. A window was opened in this new air sac and was covered with transparent adhesive tape to prevent contamination. A freshly prepared HT-1080 cell suspension (40 μL, 3.5 × 105 cells/egg) was applied directly on the freshly exposed CAM tissue. On day 11, drugs dissolved in DMSO (40 μM) were extemporaneously diluted at the required concentrations in the excipient (Cremophor EL/99% ethanol/PBS, 1/1/14 v/v). The drug solution (100 μL) was injected into a vein under the CAM. Each experimental group contained 10− 12 eggs that were incubated until day 17. Embryos were euthanized by cooling at 4 °C for at least 4 h. Tumors were collected, and tumor wet weight was recorded. The number of dead embryos and signs of toxicity from the diﬀerent groups were also recorded. Chemical Procedures. General. Proton NMR spectra were recorded on a Bruker AM-300 spectrometer (Bruker, Germany). Chemical shifts (δ) are reported in parts per million. Reactions using microwave heating were performed with an Initiator system (Biotage, Charlottesville, VA). IR spectra were recorded with a Magna FT-IR spectrometer (Nicolet Instrument Inc., Madison, WI). Uncorrected melting points were determined on an electrothermal melting point apparatus. HPLC analyses of compounds 4−8 and 10 were performed using an Acquity UPLC sample with binary solvent manager equipped with a Quattro Premier XE tandem quadrupole mass spectrometer (Waters, Milford, MA). Compounds were analyzed with a Waters BECH C18 reversed-phase column (1.7 μm, 2.1 mm × 50 mm, 50 °C) and eluted within 7 min with a MeOH/H2O linear gradient containing 0.1% TFA at 0.6 mL/min. HPLC analysis of other end compounds was performed using a Prominence LCMS-2020 system with binary solvent equipped with a UV/vis photodiode array (Shimadzu, Columbia, MD). Compounds were eluted in 30 min on an Alltech Alltima C18 reversed-phase column (5 μm, 250 mm × 4.6 mm) equipped with an Alltech Alltima C18 precolumn (5 μm, 7.5 mm × 4.6 mm) with a MeOH/H2O linear gradient at 1.0 mL/min. Purity of the ﬁnal compounds was >95%. All reactions were performed under a dried Ar atmosphere. All chemicals were supplied by Aldrich Chemicals (Milwaukee, WI) or VWR International (Mont-Royal, Quebec, Canada) and used as received unless speciﬁed otherwise. Liquid ﬂash chromatography was performed on silica gel F60, 60A, 40−63 μm supplied by Silicycle (Queb́ec, Canada) using a FPX ﬂash puriﬁcation system (Biotage, Charlottesville, VA) and using solvent mixtures expressed as v/v ratios. Solvents and reagents were used without puriﬁcation unless speciﬁed otherwise. The progress of all reactions was monitored by TLC on precoated silica gel 60 F254 TLC plates (VWR). The chromatograms were viewed under UV light at 254 and/or 265 nm. General Procedure for the Synthesis of Compounds 4−55. Method A. The appropriate isocyanate (1.2 mmol) was added dropwise to the appropriate aniline (1.0 mmol) in dry methylene chloride or dry tetrahydrofuran (10 mL) under an Ar atmosphere. The reaction mixture was stirred at room temperature for 7 days. The solvent was evaporated under reduced pressure, and the compound was puriﬁed by ﬂash chromatography. Method B. 2-Chloroethyl isocyanate (1.2 mmol) and 4- dimethylaminopyridine were added dropwise to a solution of the appropriate aniline (1.0 mmol) in dry tetrahydrofuran (10 mL) under an Ar atmosphere. The reaction mixture was heated to reﬂux and stirred for 7 days. After the mixture was cooled to room temperature, the solvent was evaporated under reduced pressure and the crude compound was puriﬁed by ﬂash chromatography. Method C. The appropriate isocyanate (2.0 mmol) was added dropwise to appropriate aniline (1.0 mmol) in dry acetonitrile or dry tetrahydrofuran (10 mL). The reaction was performed either in the absence or presence of pyridine (1 mmol). The reaction mixture was stirred from 60 to 130 °C under microwave heating (100 W) for 15− 50 min. The solvent was evaporated and the residue dissolved in ethyl Article acetate. The solution was washed with hydrochloric acid (1 N) and brine, dried over anhydrous Na2SO4, ﬁltered, and evaporated to dryness. Method D. The appropriate isocyanate (1.2 mmol) was added dropwise to the appropriate aniline (1.0 mmol) in dry acetonitrile (10 mL) under an Ar atmosphere. Pyridine (1.0 mmol) was added to the solution. The reaction mixture was stirred at room temperature for 7 days. The solvent was evaporated under reduced pressure, and the compound was puriﬁed by ﬂash chromatography. Method E. The appropriate compound (9, 14, 27, 32, 43, or 48 (0.1 mmol)) was dissolved in dry tetrahydrofuran (5 mL). Tetrabuty- lammonium ﬂuoride (1M) in dry THF was added dropwise. The mixture was stirred at room temperature for 24 h. The solvent was evaporated and the residue dissolved with ethyl acetate (40 mL). The solution was washed with 40 mL of HCL (1 N), brine, dried over Na2SO4, ﬁltered, and evaporated to dryness. The crude product was puriﬁed by ﬂash chromatography. 2-Tolyl 4-[3-(2-Chloroethyl)ureido]benzenesulfonate (4). Method A in dry DCM was used. The crude product was puriﬁed by ﬂash chromatography (silica gel, methylene chloride to methylene chloride/ethyl acetate (90:10)). Yield, 57%; yellow solid; mp, 101 °C. IR ν: 3369 (NH), 1592 (CO) cm−1. 1H NMR (CDCl3): δ 8.18 (s, 1H, NH), 7.69−7.67 (m, 2H, Ar), 7.53−7.51 (m, 2H, Ar), 7.12−7.04 (m, 3H, Ar), 6.98−6.95 (m, 1H, Ar), 6.12 (t, 1H, J = 4.8 Hz, NH), 3.58 (brs, 4H, 2 × CH2), 2.05 (s, 3H, CH3). 13C NMR (CDCl3): δ 155.1, 148.1, 145.3, 131.8, 131.5, 129.8, 127.9, 127.3, 127.1, 122.2, 118.1, 44.2, 41.9, 16.3. MS (ESI+) m/z found 368.9; C16H18ClN2O4S (M+ + H) requires 369.1. 3-Tolyl 4-[3-(2-Chloroethyl)ureido]benzenesulfonate (5). Method C in dry THF under microwave at 100 °C for 15 min without washing with HCl (1 N) was used. The crude product was puriﬁed by ﬂash chromatography (silica gel, methylene chloride to methylene chloride/ethyl acetate (90:10)). Yield, 29%; orange oil. IR ν: 3348 (NH), 1594 (CO) cm−1. 1H NMR (CDCl3): δ 8.05 (s, 1H, NH), 7.67−7.64 (m, 2H, Ar), 7.54−7.51 (m, 2H, Ar), 7.13−7.00 (m, 2H, Ar), 6.84 (s, 1H, Ar), 6.70−6.67 (m, 1H, Ar), 6.06 (t, 1H, J = 5.4 Hz, NH), 3.62−3.56 (m, 4H, 2 × CH2), 2.26 (s, 3H, CH3). 13C NMR (CDCl3): δ 155.0, 149.4, 145.2, 140.2, 129.9, 129.3, 128.1, 127.3, 122.9, 119.0, 117.9, 44.3, 41.9, 21.2. MS (ESI+) m/z found 368.9; C16H18ClN2O4S (M+ + H) requires 369.1. 4-Tolyl 4-[3-(2-Chloroethyl)ureido]benzenesulfonate (6). Method A in dry DCM was used. The crude product was puriﬁed by ﬂash chromatography (silica gel, hexanes/ethyl acetate (80:20) to hexanes/ethyl acetate (60:40)). Yield, 33%; colorless oil. IR ν: 3369 (NH), 1539 (CO) cm−1. 1H NMR (CDCl3): δ 8.17 (s, 1H, NH), 7.66−7.63 (m, 2H, Ar), 7.53−7.50 (m, 2H, Ar), 7.02 (d, 2H, J = 8.4 Hz, Ar), 6.81 (d, 2H, J = 8.4 Hz, Ar), 6.13 (brs, 1H, NH), 3.58 (brs, 4H, 2 × CH2), 2.25 (s, 3H, CH3). 13C NMR (CDCl3): δ 155.2, 147.2, 145.2, 137.3, 130.3, 129.9, 127.1, 122.0, 118.0, 44.2, 41.9, 20.9. MS (ESI+) m/z found 368.9; C16H18ClN2O4S (M+ + H) requires 369.1. 4 - M e t h o x y p h e n y l 4 - [ 3 - ( 2 - C h l o r o e t h y l ) u r e i d o ] - benzenesulfonate (7). Method A in THF was used. The crude product was puriﬁed by ﬂash chromatography (silica gel, methylene chloride to methylene chloride/ethyl acetate (80:20)). Yield, 46%; colorless oil. IR ν: 1500 (CO) cm−1. 1H NMR (CDCl3): δ 7.92 (s, 1H, NH), 7.64−7.62 (m, 2H, Ar), 7.50−7.48 (m, 2H, Ar), 6.86−6.83 (m, 2H, Ar), 6.75−6.72 (m, 2H, Ar), 5.97 (t, 1H, J = 5.2 Hz, NH), 3.72 (s, 3H, CH3), 3.62−3.57 (m, 4H, 2 × CH2). 13C NMR (CDCl3): δ 158.4, 154.9, 145.1, 142.8, 130.0, 127.1, 123.3, 118.0, 114.6, 55.6, 44.3, 42.0. MS (ESI+) m/z found 385.0; C16H18ClN2O5S (M+ + H) requires 385.1. 4-(Dimethylamino)phenyl 4-[3-(2-Chloroethyl)ureido]- benzenesulfonate (8). Method C in dry THF under microwave at 60 °C for 15 min without washing with HCl (1 N) was used. The crude product was puriﬁed by ﬂash chromatography (silica gel, methylene chloride to methylene chloride/ethyl acetate (95:5)). Yield, 22%; white sticky solid. IR ν: 3355 (NH), 1569 (CO) cm−1. 1H NMR (CDCl3): δ 7.95 (s, 1H, NH), 7.65−7.63 (m, 2H, Ar), 7.51− 7.49 (m, 2H, Ar), 6.78−6.76 (m, 2H, Ar), 6.50−6.48 (m, 2H, Ar), 5.98 (t, 1H, J = 5.3 Hz, NH), 3.63−3.57 (m, 4H, 2 × CH2), 2.87 (s, 6H, 2 6200 dx.doi.org/10.1021/jm3006492 | J. Med. Chem. 2012, 55, 6194−6208