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Small Molecule Antagonists of Cell-Surface Heparan Sulfate and Heparin–Protein Interactions

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Small Molecule Antagonists of Cell-Surface Heparan Sulfate and Heparin–Protein Interactions Electronic Supplementary Material (ESI) for Chemical Science. This journal is © The Royal Society of Chemistry 2015 Small Molecule Antagonists of Cell-Surface Heparan Sulfate and Heparin–Protein Interactions Ryan J. Weiss,a Philip L.S.M. Gordts,b Dzung Le,c,d Ding Xu,e Jeffrey D. Esko,b,d and Yitzhak Tor*a,d aDepartment of Chemistry and Biochemistry, bDepartment of Cellular and Molecular Medicine, cDepartment of Medicine, dGlycobiology Research and Training Center, University of California, San Diego, 9500 Gilman Dr., La Jolla, California 92093, United States. eDepartment of Oral Biology, University at Buffalo, 12 Capen Hall, Buffalo, New York 14260. Table of Contents S.1 – Synthesis and characterization of surfen analogs ............................................. 2 S.2 – Synthesis and characterization of building blocks .........................................11 S.3 – General procedure for precipitation of HCl salts ...........................................11 S.4 – X-ray crystal structures ...................................................................................14 S.5 – FGF2 binding inhibition curves ......................................................................21 S.6 – sRAGE Binding Inhibition Data .....................................................................22 S.7 – Biotinylation of FGF2 .....................................................................................22 S.8 – Biotinylation of sRAGE .................................................................................23 S.9 – Example of histograms for FACS binding inhibition experiments ................23 S.10 – Neutralization of heparinoids in vitro ...........................................................24 S.11 – References .....................................................................................................24 S.1 – Synthesis and characterization of surfen analogs The synthesis for certain analogs (3, 7–12) was adapted from previously published procedures.1 (4-amino-2-methyl-6-quinolyl)-urea (Hemisurfen) (2). 4,6-diamino-2-methylquinoline (14) (40 mg, 0.23 mmol) was suspended in water (1 ml). Potassium cyanate (22.5 mg, 0.28 mmol), dissolved in 0.8 ml of 10% acetic acid, was added dropwise to the suspension at room temperature and allowed to react overnight. Once the reaction was complete, 37% NaOH was added dropwise to the reaction mixture until basic. A precipitate formed overnight. The crude solid was filtered, washed with water, and dried overnight. The solid was recrystallized from 1 water. Product: tan solid (15 mg, 0.069 mmol, 30%). H NMR (400 MHz, DMSO-d6): δ 8.47 (s, 1H), 7.82 (d, J = 2 Hz, 1H), 7.60 (d, J = 2.4 Hz, 1H), 7.53 (d, J = 9.2 Hz, 1H), 6.38 (s, 1H), 6.29 13 (brd, 2H), 5.90 (brd, 2H), 2.36 (s, 3H). C NMR (125 MHz, DMSO-d6): δ 156.13, 155.97, 150.67, 144.48, 135.53, 128.35, 122.85, 117.48, 108.81, 102.29, 24.60. HR-ESI-MS calculated + for C11H13N4O [M+H] 217.1084, found 217.1083. 6-Acetamido-4-aminoquinaldine (Acetyl-hemisurfen) (3). 4,6-diamino-2-methyl-quinoline (14) (40 mg, 0.23 mmol) was dissolved in glacial acetic acid (0.5 ml) under argon. Acetyl S2 chloride (16.4 μl, 0.23 mmol) was slowly added. The mixture was stirred for 3 hours at room temperature and a heavy precipitate formed. Diethyl ether (3 ml) was added to the reaction mixture, and the solid was filtered and washed with diethyl ether. The hydrochloride salt was dissolved in 4 ml of warm water, cooled to room temperature, and made basic with 37% NaOH. The precipitated solid was filtered, washed with water, and dried under high vacuum overnight. The crude product was then recrystallized from water. Product: tan solid (25 mg, 0.12 mmol, 1 52%). H NMR (400 MHz, DMSO-d6): δ 10.00 (s, 1H), 8.16 (s, 1H), 7.65-7.47 (m, 2H), 6.41 (s, 13 1H), 6.35 (brd, 2H), 2.37 (s, 3H), 2.07 (s, 3H). C NMR (125 MHz, DMSO-d6): δ 168.19, 156.95, 151.07, 145.50, 134.03, 128.59, 123.46, 117.29, 111.25, 102.50, 24.77, 23.89. HR-ESI- + MS calculated for C12 H14 N3O [M+H] 216.1131, found 216.1132. N,N’-bis-(4-amino-2-methyl-6-quinolyl)-thiourea (Thio Surfen) (4). 4,6-diamino-2-methyl- quinoline (14) (100 mg, 0.58 mmol) was dissolved in dry DMF (0.6 ml) under argon. The solution was placed on ice and thiophosgene (TOXIC! 22.1 μl, 0.29 mmol) was slowly added. The mixture was stirred overnight and allowed to return to room temperature. A precipitate formed overnight with stirring. Methanol (2 ml) was added to quench any remaining thiophosgene. The reaction mixture was evaporated to dryness and dried under high vacuum overnight. The crude product was dissolved in 7.5 ml of warm water. The solution was cooled to room temperature and made basic with 37% NaOH. The precipitated solid was filtered, washed with water, and dried overnight under high vacuum. Recrystallization was accomplished by dissolving the solid in a minimum amount of hot DMF, filtering, and adding diethyl ether until S3 the solution became turbid. The crystals were collected by filtration, washed with ether, and dried under high vacuum. Product: dark yellow solid (43 mg, 0.11 mmol, 38%). IR spectrum: –1 1 1631 cm (C=S). H NMR (400 MHz, DMSO-d6): δ 9.79 (s, 2H), 7.93 (s, 2H), 7.68–7.53 (m, 13 4H), 6.57 (brd, 4H), 6.41 (s, 2H), 2.39 (s, 6H). C NMR (125 MHz, DMSO-d6): δ 180.74, 157.57, 151.24, 146.30, 134.07, 128.31, 127.85, 117.72, 116.92, 102.02, 24.55. HR-ESI-MS + calculated for C21H21N6S [M+H] 389.1543, found 389.1542. 1,3-bis(4-methoxy-2-methyl-6-quinolyl)-urea (Methoxy Surfen) (5). 6-amino-4- methoxyquinaldine (20) (105 mg, 0.56 mmol) was dissolved in glacial acetic acid (3 mL) under argon. Triphosgene (TOXIC! 28 mg, 0.093 mmol) was dissolved in dioxane (150 µL) and slowly added to the reaction mixture. A heavy precipitate formed, and the reaction was stirred at room temperature for 2 hrs. Methanol (8 mL) was added to the solution to quench any unreacted triphosgene. The solution was then evaporated and the residue co-evaporated with toluene to azeotropically remove remaining acetic acid. The product was then dried under high vacuum. The hydrochloride salt was dissolved in warm water (2 ml). The solution was cooled to room temperature and was made basic with 37% NaOH. The precipitated solid was filtered, washed with water, and dried on high vacuum overnight. Recrystallization was accomplished by dissolving the solid in a minimum amount of hot DMF, filtering, and adding diethyl ether until the solution became turbid. The crystals were collected by filtration, washed with ether, and dried under high vacuum. Product: tan solid (22 mg, 0.055 mmol, 59%). 1H NMR (400 MHz, DMSO-d6): δ 9.03 (s, 2H), 8.41 (d, J = 2.3 Hz, 2H), 7.78 (d, J = 9.0 Hz, 2H), 7.58 (dd, J = 9.0, S4 13 2.4 Hz, 2H), 6.89 (s, 2H), 4.04 (s, 6H), 2.58 (s, 6H). C NMR (125 MHz, DMSO-d6): δ 160.95, 157.61, 152.56, 144.32, 136.28, 128.41, 122.70, 119.55, 107.37. 101.36, 55.80, 25.09. HR-ESI- + MS calculated for C23H23N4O3 [M+H] 403.1765, found 403.1766. 1,3-bis(2-methyl-6-quinolyl)-urea (Deaminated Surfen) (6). 6-Amino-2-methylquinoline (106 mg, 0.67 mmol) was dissolved in glacial acetic acid (2 mL) under argon. Triphosgene (TOXIC! 33 mg, 0.11 mmol) was dissolved in 150 µL of dioxane and slowly added dropwise to the reaction mixture. A heavy precipitate formed, and the reaction was stirred at room temperature for 2 hours. Next, 8 mL of methanol was added to the solution to quench any unreacted triphosgene. Solution was then rotoevaporated completely. Toluene was added to the reaction flask and rotoevaporated to aziotrope remaining acetic acid. The crude product was dried on high vacuum overnight. The hydrochloride salt was dissolved in 2 ml of warm water, the solution was cooled to room temperature, and it was made basic with 37% NaOH. The precipitated solid was filtered, washed with water, and dried on high vacuum overnight. Recrystallization was accomplished by dissolving the solid in a minimum amount of hot DMF, filtering, and adding diethyl ether until the solution became turbid. The crystals were collected by filtration, washed with diethyl ether, and dried under high vacuum. Product: tan solid (35 mg, 0.10 mmol, 91%). 1H NMR (400 MHz, DMSO-d6): δ 9.09 (s, 2H), 8.17 (d, J = 4.9 Hz, 2H), 8.14 (s, 2H), 7.85 (d, J = 8.9 Hz, 2H), 7.67 (d, J = 8.9 Hz, 2H), 7.36 (d, J = 8.4 Hz, 2H), 2.62 (s, 6H). 13C NMR (125 MHz, DMSO-d6): δ 156.62, 152.78, 143.82, 136.93, 135.46, 128.86, 126.82, 122.90, 122.50, + 113.39, 24.71. HR-ESI-MS calculated for C21H19N4O [M+H] 343.1553, found 343.1551. S5 Bis(4-amino-2-methyl-6-quinolyl)-oxalamide (Oxalyl Surfen) (7). 4-6-diamino-2-methyl- quinoline (14) (100 mg, 0.58 mmol) was dissolved in glacial acetic acid (0.6 ml) under argon. Oxalyl chloride (25 μl, 0.29 mmol) was slowly added to the solution. A heavy precipitate formed, and the mixture was stirred for 1 hour at room temperature. After addition of diethyl ether (2.5 ml), the solid was filtered, washed with diethyl ether and dried under vacuum overnight. The hydrochloride salt was dissolved in warm water (8 ml), the solution was cooled to room temperature, and it was made basic with 37% NaOH. The precipitated solid was filtered, washed with water, and dried on high vacuum. Recrystallization was accomplished by dissolving the solid in a minimum amount of hot DMF, filtering, and adding diethyl ether until the solution became turbid.
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