Design, synthesis and evaluation of ph-dependent hydrolysable emetine analogs as treatment for prostate cancer.

Transcription

1 Design, synthesis and evaluation of p-dependent hydrolysable emetine analogs as treatment for prostate cancer. Emmanuel S. Akinboye, 1 Marc D. Rosen, 2 Samuel R. Denmeade, 2 Bernard Kwabi-Addo 3 and ladapo Bakare 1* 1 Department of Chemistry, oward University, 525 College Street W, Washington DC, 20059, USA 2 The Sidney Kimmel Comprehensive Cancer Center at Johns opkins, The Johns opkins University School of Medicine, Baltimore, MD, 21239, USA 3 Department of Biochemistry and Cancer Center, oward University, Washington DC * Address correspondence to this author at the Department of Chemistry, oward University, 525 College Street W, Washington DC, 20059, USA; Tel: ; Fax: ; E- mail: obakare@howard.edu Preparation and characterization of emetine analogs General Information: All solvents and reagents used were bought from commercial sources and used without further purification. Melting points were determined in open capillary tubes on a Mel-Temp melting point apparatus and are uncorrected. The IR spectra were recorded on a Perkin Elmer PE 100 spectrometer with an Atenuated Total Reflectance (ATR) window. The 1 - and 13 C-MR spectra were obtained on a Bruker Avance 400 Mz spectrometer at 400 Mz and 100 Mz, respectively in deuterated chloroform (CDCl 3 ) or deuterated methanol (CD 3 D). Chemical shifts are in δ units (ppm) with TMS (0.00 ppm), CCl 3 (7.27 ppm), or C 3 (3.34 ppm) as the internal standard for 1 -MR, and CDCl 3 (77.00 ppm) or CD 3 D (49.90 ppm) for 13 C-MR. Accurate mass determination was carried out on Agilent 6224 Accurate-Mass TF LC/MS system and also with Voyager-DE-STR (Applied Biosystems) Matrix-assisted laser S1

2 desorption/ionization (MALDI) time-of-flight mass spectrometry. Analytical thin-layer chromatography was performed on EMD precoated silica gel 60 F254 plates. Purity of the compounds was determined with reverse phase-plc. The purity of all the compounds was determined to be 95%. PLC Methods In this study, the reverse phase PLC studies were carried out using the following methods. PLC Method 1 This method was used for analytical reversed-phase PLC of synthetic emetine analogues: PLC Unit: Waters Delta 600 Controller equipped with a variable wavelength UV-vis detector (Waters 2487 Dual λ Absorbance Detector) set to detect at 215 nm and 285 nm and a Luna 5µ C18 reversed phase analytical column (150 X 4.60 mm, Waters). Software: Laura TM Lite 3 PLC Conditions: Flow rate: 1 ml/min; mobile phase A: 50% p M phosphate buffer, 50%. Mobile phase B: 100%. Gradient: 0-2 min 56% mobile phase A and 44% mobile phase B; 2-9 min gradual change to 10% mobile phase A and 90% mobile phase B; 9-26 min 10% mobile phase A and 90% mobile phase B; min gradual change to 56% mobile phase A and 44% mobile phase B; min 56% mobile phase A and 44% mobile phase B. Injection volume 100 µl. S2

3 PLC Method 2 This method was used for analytical reversed-phase PLC of p responsive studies on emetine analogues: PLC Unit: Waters Delta 600 Controller equipped with a variable wavelength UV-vis detector (Waters 2487 Dual λ Absorbance Detector) set to detect at 215 nm and 285 nm and a Waters XBridge 5µ C18 reversed phase analytical column (150 X 4.60 mm, Waters). Software: Empower 2 PLC Conditions: Flow rate: 1 ml/min; mobile phase A: 30% p M phosphate buffer, 70%. Mobile phase B: 100%. Gradient: 0-20 min 100% mobile phase A; min gradual change to 100% mobile phase B; min 100% mobile phase B; Injection volume: 100 µl. General procedure for the synthesis of thiourea analogs 4-6 Synthesis of intermediate isothiocyanate Procedure adapted from report by Wong & Colman 1. To a solution of appropriate amine (16.0 mmol) in TF (15 ml) at 0 o C was added triethylamine (10.0 ml). This was set to moderate stirring and CS 2 (34.0 mmol) was added in drops over about 30 min at 0 o C. The mixture was allowed to stir at this temperature for 15 minutes after which it was stirred at room temperature for 1 h. The reaction temperature was then cooled to 0 o C again while stirring continued and a solution of tosyl chloride (20.8 mmol) in TF was added gently. The reaction mixture was allowed to warm up to room temperature, stirred for 1 h and then 20 ml 1 Cl was added while stirring continued. This was followed by 25 ml diethyl ether and the reaction stirred for S3

4 another 5 minutes. The aqueous layer was separated and extracted with diethyl ether (2 X 20 ml). The combined organic layers were dried over anhydrous a 2 S 4, solvent evaporated invacuo, and the crude product was purified by column chromatography over silica gel with hexanes as eluent. Reaction of isothiocyanate with emetine to produce the thiourea analogs To a stirred solution of pyridine (0.5 ml) in C 2 Cl 2 (15 ml) at room temperature was added emetine dihydrochloride (200 mg, 0.36 mmol). After all the emetine was completely dissolved, the appropriate isothiocyanate (0.72 mmol) was added. The reaction mixture was stirred at room temperature for 8 to 16 h. C 2 Cl 2 (15 ml) was then added to the mixture and the resulting mixture washed with water (2 x 25 ml) and brine (1 x 25 ml). The organic layer was dried over anhydrous MgS 4 followed by solvent removal in-vacuo. The crude product was purified either by precipitation in a diethyl ether/hexane mixture or by column chromatography using C 2 Cl 2 / mixture in appropriate ratio as eluent. Compound 4 was purified by column chromatography over silica gel using gradient elution. 100% C 2 Cl 2 was employed to elute nonpolar impurities followed by elution with 10% in C 2 Cl 2 to obtain the desired product 4. Compounds 5 and 6 were purified by dissolving the crude product in a small amount of ethyl acetate to dissolve it and to this solution was added a 3:7 mixture of diethyl ether and hexanes to precipitate 5 and 6 in pure form. C S S4

5 Isothiocyanatomethyl-benzene Yield: 73%, yellow oil, IR (cm -1 ) 3053, 3033, 2906, 2855, 2167, 2077, 1593, 1496, 1454, 1439: 1 MR (400Mz, CDCl 3 ) δ 4.74 (2, s), (2, m), (3, m); 13 C MR (100 Mz, CDCl 3 ) δ 48.7, 126.0, 126.9, 127.2, 128.4, 129.0, 129.4, S 4 1-(3-Ethyl-9,10-dimethoxy-1,3,4,6,7,11b-hexahydro-2-pyrido[2,1 α]isoquinol-2-ylmethyl)- 6,7-dimethoxy-3,4-dihydro-1-isoquinoline-2-carbothioic acid benzylamide (4) Yield: 67%, mp = o C. 1 MR (400 Mz, CDCl 3 ) δ 0.89 (3, t, J = 7.4 z), (1, m), (1, m), (3, m), (3, m), 2.09 (1,br s), 2.38 (1, t, J = 12.5 z), 2.51 (1, br s), 2.63 (1, br d, J = 15.6 z), 2.76 (1, dt, J = 3.9, 15.8 z), (2, m), (4, m), (1, m), 3.83 (3, s), 3.84 (3, s), 3.86 (3, s), 3.87 (3, s), 4.92 (2, d, J = 4.7 z), 5.84 (1, t, J = 4.7 z), 6.56 (1, s), 6.58 (1, s), 6.63 (1, s), 6.92 (1, s), (2, m), (3, m); 13 C MR (100 Mz, CDCl 3 ) δ 11.2, 23.8, 27.2, 29.1, 37.8, 38.4, 40.0, 41.3, 50.5, 52.4, 55.9, 56.0, 56.1 (2C), 56.3 (2C), 57.8, 61.4, 108.9, 110.2, 111.1, 111.3, 124.7, 125.9, 127.1, 127.9, 128.0, 128.7, 128.9, 129.2, 130.9, 138.1, 147.3, 147.4, 147.7, 147.8, 182.3; ESI-TF-MS m/z , ([C S + ] + calcd ). t R is 8.39 min (100% purity). S5

6 C S 1-Isothiocyanatomethyl-4-methoxy-benzene Yield: 93%, pale yellow oil, IR (cm -1 ) 3007, 2955, 2837, 2163, 2077, 1611, 1587, 1440, MR (400 Mz, CDCl 3 ) δ 3.83 (3, s), 4.65 (2, s), (2, m), (2, m). 13 C MR (100 Mz, CDCl 3 ) δ 48.3, 55.4, 114.0, 114.4, 128.1, 128.2, 130.3, 131.9, S 5 1-(3-Ethyl-9,10-dimethoxy-1,3,4,6,7,11b-hexahydro-2-pyrido[2,1 α]isoquinol-2-ylmethyl)- 6,7-dimethoxy-3,4-dihydro-1-isoquinoline-2-carbothioic acid 4-methoxybenzylamide (5) Yield: 48%, mp = o C; 1 MR (400 Mz, CDCl 3 ) δ 0.88 (3, t, J = 7.4 z), (2, m), (3, m), (2, m), (1, m), 2.37 (1, t, J = 12.4 z), (1, m), 2.62 (1, br d, J = 15.6 z), 2.75 (1, dt, J = 4.0, 16.4 z), (6, m), (1, m), 3.76 (3, s), 3.81 (1, s), 3.82 (3, s), 3.83 (3, s), 3.84 (3, s), 3.89 (3, s), (2, m), 5.77 (1, t, J = 4.8 z), 6.55 (1, s), 6.57 (1, s), 6.63 (1, s), 6.85 (2, d, J = 9.6 z), 6.93 (1, s), 7.27 (2, d, J = 9.6 z); 13 C MR (100 Mz, CDCl 3 ) δ 11.2, 23.8, 27.1, 29.1, 37.8, 38.4, 40.0, 50.0, 52.4, 55.4, 55.9, 56.0 (2C), 56.1 (2C), 56.3, 57.8, 61.5, 108.9, 110.2, 111.2, 111.3, 114.2, 114.3, 124.8, 126.3, 128.4, 129.0, 129.2, 129.4, 130.0, S6

7 (2C), (2C), 159.3, ESI-TF-MS m/z , ([C S + ] + calcd ). t R is 8.11 min (100% purity). Cl C S S Cl 6 1-Chloro-4-Isothiocyanatomethyl-benzene Yield: 85%, yellow oil, IR (cm -1 ) 3027, 2953, 2850, 2173, 2069, 1597, 1579, 1491, MR (400 Mz, CDCl 3 ) δ 4.69 (2, s), (2, m), (2, m). 13 C MR (100 Mz, CDCl 3 ) δ 48.7, 128.8, 129.0, 129.5, 129.7, 133.3, (3-Ethyl-9,10-dimethoxy-1,3,4,6,7,11b-hexahydro-2-pyrido[2,1 α]isoquinol-2-ylmethyl)- 6,7-dimethoxy-3,4-dihydro-1-isoquinoline-2-carbothioic acid 4-chloro-benzylamide (6) Yield: 51%, mp = o C; 1 MR (400 Mz, CDCl 3 ) δ 0.88 (3, t, J = 7.4 z), (5, m), (2, m), (1, m), (2, m), 2.61 (1, br d, J = 15.5 z), 2.75 (1, dt, J = 4.0, 16.0 z), (6, m), (1, m), 3.83 (3, s), 3.84 (3, s), 3.86 (3, s), 3.87 (4, s), 4.87 (1, dd, J = 4.9, 14.8 z), 4.95 (1, dd, J = 5.0, 14.7 z), 5.91 (1, t, J = 4.8 z), 6.56 (1, s), 6.58 (1, s), 6.64 (1, s), 6.90 (1, s), 7.28 (4, s). 1 MR (400 Mz, CD 3 D) δ 0.93 (3, t, J = 7.4 z), (2, m), (2, m), (2, S7

8 m), (1, m), 2.12 (1, t, J = 11.2 z), 2.38 (1, t, J = 12.4 z), (1, m), (2, m), (6, m), (1, m), 3.73 (3, s), 3.81 (6, s), 3.82 (3, s), 4.28 (1, br s), (2, m), 6.69 (2, s), 6.74 (2, s), 6.96 (1, br s), 7.17 (2, d, J = 8.4 z), 7.30 (2, d, J = 8.4 z). 13 C MR (100 Mz, CDCl 3 ) δ 11.2, 23.9, 27.2, 29.2, 37.9, 38.5, 39.9, 40.3, 49.5, 52.4, 55.9, 56.0, 56.1 (2C), 56.3 (2C), 58.0, 61.6, 108.9, 110.2, 111.1, 111.3, (2C), (2C), 129.2, 129.3, 130.1, 130.9, 133.5, 136.8, 147.2, 147.4, 147.8, 147.9, ESI-TF-MS m/z , ([C Cl 3 4 S + ] + calcd ). t R is min (100% purity). General procedure for the synthesis of urea analogs 7-9 Synthesis of the isocyanate was carried out as reported by Sigurdsson 2 et al with slight modifications. A solution of Trichloromethyl chloroformate (5.741 mmol) in C 2 Cl 2 (15 ml) was stirred at 0 o C. A solution of an appropriate amine, (3.827 mmol) and 1,8- bis(dimethylamino)-naphthalene or DMAP (7.654 mmol) in C 2 Cl 2 was then added dropwise over a period of 5 to 10 min. Thereafter, the ice bath was removed and the reaction mixture was allowed to warm up to room temperature and then stirred for another 45 min. Solvent and all volatiles were evaporated in-vacuo and fresh C 2 Cl 2 (30 ml) was added followed by 1 Cl solution (20 ml); this was stirred for about 3-5 min. The organic layer was separated and then washed with 1 Cl (3 x 15 ml) and 1 a (1 x 15 ml). It was dried over anhydrous a 2 S 4 and solvent was evaporated in vacuo to give the respective isocyanates that were used without further purification. S8

9 To a solution of a given isocyanate, (0.72 mmol) in C 2 Cl 2 (10 ml), was added a solution of emetine dihydrochloride (200 mg, 0.36 mmol) and DMAP (1.44 mmol) in C 2 Cl 2 (15 ml) at room temperature. The reaction mixture was stirred overnight at room temperature. Additional C 2 Cl 2 (10 ml) was added to the mixture and the resulting mixture washed with water (2 x 25 ml) and brine (1 x 25 ml). The organic layer was dried over anhydrous MgS 4 and the solvent removed in-vacuo. All the products were purified by flash column chromatography using gradient elution beginning with 100 % EtAc and followed with a gradual addition of from 5% to 10 % in EtAc to afford 7-9 in pure form. 7 1-(3-Ethyl-9,10-dimethoxy-1,3,4,6,7,11b-hexahydro-2-pyrido[2,1 α]isoquinol-2-ylmethyl)- 6,7-dimethoxy-3,4-dihydro-1-isoquinoline-2-carboxylic acid benzylamide (7) Yield: 82%, mp = o C; 1 MR (400 Mz, CDCl 3 ) δ 0.89 (3, t, J = 7.5 z), (2, m), (3, m), (2, m), 1.75 (2, m), (1, m), 2.25 (1, 11.6 z), (1, m), 2.67 (2, br t, J = 15.0 z), (2, m), (1, m), (1, m), 3.83 (3, s), 3.85 (6, s), 3.86 (3, s), 4.42 (2, dd, J = 5.6, 16.8 z), 4.55 (1, dd, J = 5.8, 14.6 z), 4.93 (1, t, J = 5.4 z), 5.60 (1, d, J = 10.0 z), 6.58 (1, s), 6.59 (2, s), 6.91 (1, s), (5, m); 13 C MR (100 Mz, CDCl 3 ) δ 11.4, 23.9, 27.8, 29.4, 36.6, 37.2, 38.1, 38.7, 45.2, 45.4, 51.3 (2C), 52.8, 56.2, 56.3, 56.4 (2C), 61.8, 109.2, 110.5, 111.5, 111.9, 125.6, S9

10 TF-MS m/z , ([C ] + calcd ). t R is 8.48 min (97 % purity) , 127.8, 128.1, 128.9, (2C), (2C), 140.1, 147.6, 147.7, (2C), ESI- 1-(3-Ethyl-9,10-dimethoxy-1,3,4,6,7,11b-hexahydro-2-pyrido[2,1 α]isoquinol-2-ylmethyl)- 6,7-dimethoxy-3,4-dihydro-1-isoquinoline-2-carboxylic acid 4-methoxy-benzylamide (8) Yield: 75%, mp = o C; 1 MR (400 Mz, CDCl 3 ) δ 0.89 (3, t, J = 7.4 z), (2, m), (2, m), (3, m), (1, m), 2.19 (1, t, J = 12.4 z), 2.67 (1, d, J = 15.2 z), (1, m), (2, m), (3, m), 3.65 (2, d, J = 15.6 z), 3.72 (3, s), 3.81 (3, s), 3.85 (3, s), 3.86 (3, s), 3.92 (3, s), (3, m), 5.05 (1, s), 5.61 (1, d, J = 8.8 z), 6.57 (2, s), 6.59 (1, s), 6.76 (2, d, J = 8.2 z), 6.98 (1, br s), 7.21 (2, d, J = 8.2 z); 13 C MR (100 Mz, CDCl 3 ) δ 11.2, 22.7, 27.5, 30.1, 36.2 (2C), 38.3 (2C), 44.5 (2C), 50.4 (2C), 55.2, 55.3, 55.9, 56.1 (2C), 56.2 (2C), 108.8, 109.9, 111.0, 111.4, (2C), (2C), 129.1, (2C), (2C), (4C), 157.3, 158.9; ESI- TF-MS m/z , ([C ] + calcd ). t R is 9.17 min (98.8 % purity). 8 S10

11 Cl 9 1-(3-Ethyl-9,10-dimethoxy-1,3,4,6,7,11b-hexahydro-2-pyrido[2,1 α]isoquinol-2-ylmethyl)- 6,7-dimethoxy-3,4-dihydro-1-isoquinoline-2-carboxylic acid 4-chloro-benzylamide (9) Yield: 85%, mp = o C; 1 MR (400 Mz, CDCl 3 ) δ 0.87 (3, t, J = 7.6 z), (2, m), (3, m), (4, m), (1, m), 2.20 (1, t, J = 12.4 z), (1, m), 2.65 (2, t, J = 14.6 z), (2, m), (1, m), 3.62 (1, d, J = 14.0 z), 3.82 (3, s), 3.83 (6, s), 3.84 (3, s), 4.36 (2, dd, J = 5.6, 14.8 z), 4.45 (1, dd, J = 5.8, 15.0 z), 4.93 (1, s), 5.55 (1, d, J = 9.2 z), 6.56 (3, s), 6.87 (1, s), 7.20 (2, s), 7.25 (2, s); 13 C MR (100 Mz, CDCl 3 ) δ 11.0, 23.5, 27.5, 36.3, 37.8 (2C), 38.4, 39.5, 44.3, 51.0 (2C), 52.5 (2C), 55.9 (2C), 56.0, 56.1, 61.4, 108.8, 110.1, 111.2, 111.5, (2C), (2C), 129.0, (2C), (2C), 138.4, 147.4, (3C), 157.3; ESI-TF-MS m/z , ([C Cl ] + calcd ). t R is min (100% purity). General procedure for the preparation of sulfonamide analogs To a stirred solution of dimethyl amino pyridine (DMAP) ( mg, 1.45 mmol) in C 2 Cl 2 (20 ml) was added emetine dihydrochloride (200 mg, 0.36 mmol) at room temperature. After minutes, the appropriate sulfonyl chloride (0.9 mmol) solution in C 2 Cl 2 (5 ml) was added. The reaction mixture was then stirred at room temperature for h. The solvent was removed in- S11

12 vacuo and water (25 ml) was added to the residue to dissolve all the water soluble impurities. The crude product was isolated by extraction into C 2 Cl 2 (3 x 25 ml). The combined organic phase was washed with brine (2 x 20 ml), dried over anhydrous MgS 4 and the solvent evaporated in-vacuo. The crude product was purified by flash column chromatography over silica gel using gradient elution starting with 100% C 2 Cl 2 to remove the least polar impurities and gradually varying this to C 2 Cl 2 : eluent mixture. ptimum components vary from 5 to 20% depending on the specific analog to afford the desired product. S 10 2-(2-Benzenesulfonyl-6,7-dimethoxy-1,2,3,4-tetrahydro-isoquinolin-1-ylmethyl)-3-ethyl- 9,10-dimethoxy-1,3,4,6,7,11b-hexahydro-2-pyridol[2,1-a]isoquinoline (10) The product was purified using flash column chromatography with of 5% in C 2 Cl 2. Yield: 75%, mp = o C; 1 MR (400 Mz, CDCl 3 ) δ 0.87 (3, t, 7.4 z), (2, m), 1.33 (1, t, J = 11.6 z), (1, m), (3, m), (1, m), 2.22 (1, t, J = 12.1 z), (2, m), (1, m), 2.64 (1, d, J = 16.2 z), (1, m), (4, m), 3.73 (3, s), 3.83 (3, s), 3.85 (3, s), 3.89 (3, s), 4.01 (1, dt, J = 3.9, 14.5 z), 5.16 (1, dd, J = 3.6, 11.8 z), 6.21 (1, s), 6.43 (1, s), 6.58 (1, s), 7.07 (1, s), 7.25 (2, t, J = 7.8 z), (1, m), 7.67 (2, dd, J = 1.2, 8.4 z); 13 C MR (100 Mz, CDCl 3 ) δ S12

13 147.8, 147.6, 147.4, 147.3, 140.8, (2C), (2C), 128.6, (2C), (2C), 111.4, 111.1, 109.2, 108.9, 56.2, 56.0, 55.9, 55.8, 53.4, 52.3 (2C), 41.6, 40.6, 40.0, 37.9, 36.7, 36.2, 29.3, 24.6, 23.6, 11.1; ESI-TF-MS m/z , ([C S + ] + calcd ). t R is 9.29 min (99.8% purity). S Cl 11 2-[2-(4-Chloro-benzenesulfonyl)-6,7-dimethoxy-1,2,3,4-tetrahydro-isoquinolin-1-ylmethyl)- 3-ethyl-9,10-dimethoxy-1,3,4,6,7,11b-hexahydro-2-pyridol[2,1-a]isoquinoline (11). The product was purified using flash column chromatography with of 7% in C 2 Cl 2. Yield: 86%, mp = o C; 1 MR (400 Mz, CDCl 3 ) δ 0.87 (3, t, 7.4 z), (3, m), (1, m), (1, m), (2, m), (1, m), (1, m), (2, m), (1, m), 2.63 (1, d, J = 15.4 z), (1, m), (3, m), (1, m), 3.74 (3, s), 3.83, (3, s), 3.84 (3, s), 3.87 (3, s), 3.99 (1, dd, J = 6.4, 15.3 z), 5.15 (1, dd, J = 3.5, 11.8 z), 6.22 (1, s), 6.44 (1, s), 6.56 (1, s), 7.04 (1, s), 7.21 (2, d, J = 8.4 z), 7.58 (2, d, J = 8.4 z); 13 C MR (100 Mz, CDCl 3 ) δ 148.0, 147.7, 147.4, 147.3, 139.4, 138.8, (2C), (2C), (2C), (2C), 111.4, 111.2, 109.2, 108.9, 56.2 (2C), 56.1, 55.9, 55.8, 53.6, 52.3, 41.6, 40.4, 37.9, 36.8, 36.1, 30.9, 29.2, 24.5, 23.6, 11.1; ESI-TF-MS m/z , ([C Cl 2 6 S + ] + calcd ). t R is min (100% purity). S13

14 S 12 2-[6,7-Dimethoxy-2-(4-methoxy-benzenesulfonyl)-1,2,3,4-tetrahydro-isoquinolin-1- ylmethyl]-3-ethyl-9,10-dimethoxy-1,3,4,6,7,11b-hexahydro-2-pyridol[2,1-a]isoquinoline (12) The product was purified using flash column chromatography with of 5% in C 2 Cl 2. Yield: 81%, mp = o C; 1 MR (400 Mz, CDCl 3 ) δ 0.87 (3, t, J = 7.4 z), (2, m), (1, m), (1, m), (2, m), (1, m), (1, m), 2.21 (1, t, J = 12.2 z), (2, m), (1, m), 2.63 (1, d, J = 15.7 z), (1, m), (3, m), (1, m), 3.74 (3, s), 3.75 (3, s), 3.84 (3, s), 3.85 (3, s), 3.88 (3, s), (1, m), 5.14 (1, dd, J = 3.5, 11.7 z), 6.24 (1, s), 6.44 (1, s), 6.58 (1, s), 6.71 (2, d, J = 8.9 z), 7.07 (1, s), 7.59 (2, d, J = 8.9 z) 13 C MR (100 Mz, CDCl 3 ) δ 162.6, 147.7, 147.6, 147.5, 147.4, (2C), (2C), 129.2, (2C), 114.2, 114.1, 111.4, 111.3, 111.1, 110.1, 56.2 (2C), 56.0, 55.9, 55.8, 55.5, 53.2, 52.3, 48.9, 42.9, 39.7, 38.2, 36.7, 36.2, 29.2, 24.6, 23.7, ESI-TF-MS m/z , ([C S + ] + calcd ). t R is 9.55 min (100% purity). S14

15 a +- S S 13 Sodium 1-(3-Ethyl-9,10-dimethoxy-1,3,4,6,7,11b-hexahydro-2-pyrido[2,1-a]isoquinolin-2- ylmethyl)-6,7-dimethoxy-3,4-dihydro-1-isoquinoline-2-carbodithioate (13). A solution of a (240 mg, 6.00 mmol) in water (1.00 ml) and ethanol (20.00 ml) was added to a solution of emetine dihydrochloride hydrate (1.11 g, 2.00 mmol) in ethanol (10.0 ml) at -8 o C. This was stirred at this temperature for 15min after which CS 2 (0.30 ml, 4.97 mmol) was added. The resulting mixture was stirred at -5 to 1 o C for 2 h and at room temperature for 30 min. The solvent was removed in-vacuo and the residue triturated with acetonitrile and then filtered. The filtrate was evaporated to dryness and the residue dissolved in ethyl acetate (3 ml). To this was added diethyl ether to precipitate 13 as a white solid (950 mg, 82%). Mp: o C; 1 MR (400 Mz, CD 3 D) δ 0.93 (3, t, J = 7.4 z), (1, m), (2, m), (2, m), (1, m), 2.10 (1, t, J = 11.0 z), 2.38 (1, t, J = 12.8 z), 2.50 (1, tb, J = 4.1, 11.6 z ), (2, m), (5, m), 3.35 (1, s), (1, m) 3.80 (3, s), 3.81 (6, br s), 3.84 (3, s), 5.95 (1, dd, J = 5.9, 12.8 z), 6.66 (1, s), 6.69 (1, s), 6.72 (1,s), 7.20 (1,s), 7.25 (1, dd, J = 3.6, 11.8 z). 13 C MR (100 Mz, CD 3 D) δ 216.5, 147.4, 147.3, 147.2, 147.1, 133.0, 131.1, 127.0, 126.5, 112.6, 112.0, 111.0, 110.3, 65.4, 63.3, 61.6, 58.0, 56.5, 56.2, 56.1, 56.0, 55.9, 52.5, 41.7, 41.3, 37.6, 29.4, 27.3, 23.7, MALDI- MS m/z ([C a 4 S 2 + a] + calcd ). S15

16 S S 14 1-(3-Ethyl-9,10-dimethoxy-1,3,4,6,7,11b-hexahydro-2-pyrido[2,1-a]isoquinolin-2- ylmethyl)-6,7-dimethoxy-3,4-dihydro-1-isoquinoline-2-carbodithioic acid benzyl ester (14): Compound 13 (200 mg, 0.35 mmol) was dissolved in acetonitrile (15 ml) and to this was added a solution of benzyl bromide (0.27 mmol.) in acetonitrile (5 ml). The reaction mixture was stirred for 24 h at room temperature. Solvent was evaporated in vacuo and the residue obtained triturated with water (20 ml), and filtered under suction. The crude product was airdried and then purified by flash chromatography on silica gel using EtAc: (10:1) as eluent. Yield: 45%, mp = o C; 1 MR (400 Mz, CDCl 3 ) δ 0.89 (3, t, J = 7.4 z), (1, m), (3, m), (1, m), (1, m), (1, m), 2.05 (1, t, J = 11.0 z), (2, m), 2.62 (1, d, J = 14.9 z), (1, m), (1, m), (4, m), 3.35 (1, s), (1, m), 3.80 (3, s), 3.84 (3, s), 3.91 (3, s), 3.95 (3, s), 4.54 (1, d, J = 13.1 z), 4.63 (1, d, J = 13.1 z), 6.56 (1, s), 6.58 (2, s), 6.86 (1, dd, J = 3.8, 11.3 z), 6.93 (1, s), (3, m), 7.38 (2, d, J = 6.7 z); 13 C MR (100 Mz, CDCl 3 ) δ 197.1, 148.3, 148.0, 147.8, 147.4, 147.3, 135.8, 135.3, 130.0, 129.5, 128.3, 127.8, 127.6, 126.2, 124.6, 111.5, 111.3, 111.0, 108.8, 62.8, 62.3, 61.4, 56.3, 56.1, 56.0, 55.9, S16

17 52.4, 52.3, 45.2, 43.4, 42.0, 38.9, 37.7, 29.7, 28.0, 23.8, 11.2; ESI-TF-MS m/z ([C S 2 + ] + calcd ). t R is min (98% purity) (3-Ethyl-9,10-dimethoxy-1,3,4,6,7,11b-hexahydro-2-pyrido[2,1-a]isoquinolin-2- ylmethyl)-6,7-dimethoxy-3,4-dihydro-1-isoquinoline-2-carboxylic acid benzyl ester (15): Yield: 46%, mp = o C; 1 MR (400 Mz, CDCl 3 ) δ 0.88 (3, t, J = 7.6 z), (2, m), (7, m), (1, m), (1, m), (1, m), (1, m), (1, m), (1, m), (1, m), (3, m), 3.84 (3, s), 3.89 (6, s), 3.96 (3, s), (1, m), 5.14 (1, d, J = 12.6 z), 5.24 (1, d, J = 12.6 z), 6.56 (1, s), 6.58 (1, s), 6.62 (1, s), 6.98 (1, s), (2, m), (3, m); 13 C MR (100 Mz, CDCl 3 ) δ 9.3, 22.3, 27.0, 29.3, 31.8, 33.1, 37.3, 38.7, 51.1, 52.2, 53.4, 53.8, 55.9, 56.0, 56.1 (2C), 59.7, 67.1, 108.3, 109.4, 111.1, 111.5, 127.7, 127.8, 127.9, 128.5, 128.8, (2C), (2C), 139.2, 147.0, 147.8, 148.4, 148.7, 155.5; MALDI-MS m/z , ([C ] + calcd ). t R is min (100 % purity). S17

18 General Reaction procedure for all the amide analogues of emetine A solution of emetine dihydrochloride (500 mg, 0.89 mmol) in chloroform (10 ml) and triethylamine (2 ml) was stirred for about 5 minutes. To this was added a solution of the appropriate anhydride (3.55 mmol) in chloroform. The reaction mixture was stirred at room temperature for 12 to 20 h. All volatiles and solvent was evaporated in-vacuo from the reaction mixture to obtain a crude solid. Purification methods vary slightly from one analog to another and these are specified below. For compounds 20 and 21 the crude product was dissolved in chloroform (40 ml). This was washed with water (2 x 15 ml) with gentle shaking, brine (1 x 15 ml), dried over anhydrous MgS 4 and the solvent evaporated in vacuo. The residue was dissolved in ethyl acetate (2-4 ml) and pure product was obtained by precipitation with 20% diethyl ether in hexanes at 0 to 10 o C. Product was filtered and washed with cold (0 to 5 o C) 20% ether in hexanes. The solid isolated was further dried in a vacuum oven at 60 o C for 4 hour. 20 S18

19 ylmethyl)-6,7-dimethoxy-3,4-dihydro-1-isoquinolin-2-yl]-4-oxo-butyric acid (20): Yield. 90%, white solid, mp = o C; 1 MR (400 Mz, CD 3 D) δ 0.81 (3, t, J = 7.5 z), (3, m), (2, m), (1, m), 2.14 (1, t, J = 12.4 z), 2.29 (1, t, J = 11.6 z), (2, m), (2, m), (3, m), (1, m), (1, m), (1, m), (1, m), (2, m), (2, m), 3.66 (3, s), 3.68 (3, s), 3.69 (3, s), 3.77 (3, s), 3.98 (1, dd, J = 5.0, 13.5 z), 5.66 (1, dd, J = 3.8, 11.6 z), 6.58 (1, s), 6.59 (1, s), 6.60 (1, s), 6.84 (1, s); 13 C MR (100 Mz, CD 3 D) δ 11.2, 24.3, 28.7, 29.6, 30.6, 30.9, 33.6, 37.0, 38.3, 40.0, 49.7 (2C), 52.8, 55.8, 56.5, 56.6, 56.8, 57.0, 64.2, 110.6, 111.9, 113.0, 113.5, 126.6, 127.1, 129.5, 131.7, 149.3, 149.4, 149.5, 149.6, 174.3, ESI-TF-MS m/z , ([C ] + calcd ). t R is 3.05 min (97.5% purity) [1-(3-Ethyl-9,10-dimethoxy-1,3,4,6,7,11b-hexahydro-2-pyrido[2,1-a]isoquinolin-2-4-[1-(3-Ethyl-9,10-dimethoxy-1,3,4,6,7,11b-hexahydro-2-pyrido[2,1-a]isoquinolin-2- ylmethyl)-6,7-dimethoxy-3,4-dihydro-1-isoquinolin-2-yl]-4-oxo-but-2-enoic acid (21): Yield. 90%, white solid, mp = o C 1 MR (400 Mz, CD 3 D) δ 0.86 (3, t, J = 7.1 z), (3, m), (4, m), (1, m), 2.23 (1, t, J = 12.8 z), 2.67 S19

20 (1, br d, J = 12.9 z), (5, m), (1, m), (3, m), 3.82 (4, s), 3.83 (4, s), 3.86 (4, s), 4.07 (1, dd, J = 2.4, 7.1 z), 5.70 (1, br d, J = 8,6 z), 6.10 (1, dd, J = 3.0, 11.9 z), 6.31 (1, dd, J = 3.0, 11.9 z), 6.49 (1, s), 6.51 (1, s), 6.55 (1, s), 6.87 (1, s). 13 C MR (100 Mz, CDCl 3 ) δ 10.3, 22.7, 26.3, 28.4, 34.7 (2C), 35.4, 38.4, 39.4, 45.3, 48.5 (2C), 55.8, 55.9, 56.1, 56.2, 60.4, 109.3, 109.8, 110.8, 111.6, (2C), (2C), 132.5, 135.7, 147.7, 147.9, 148.2, 148.4, 167.2, 169.5; MALDI-MS m/z , ([C ] + calcd ). t R is 3.30 min (99.5% purity) [1-(3-Ethyl-9,10-dimethoxy-1,3,4,6,7,11b-hexahydro-2-pyrido[2,1-a]isoquinolin-2- ylmethyl)-6,7-dimethoxy-3,4-dihydro-1-isoquinolin-2-yl]-2-methyl-4-oxo-but-2-enoic acid (22): After precipitation with 20% diethyl ether in hexanes, compound 22 was further purified by flash chromatography on silica gel using gradient elution beginning with 100% ethyl acetate followed by an eluent mixture of 50% methanol in ethyl acetate to remove all less polar impurities and 100% methanol eluted compound 22. S20

21 Yield. 94%, pale yellow solid, mp = o C; 1 MR (400 Mz, CDCl 3 ) δ 0.84 (3, t, J = 7.4 z), (2, m), (4, m), 1.98 (3, s), 2.17 (1, t, J = 12.7 z), (2, m), (4, m), (3, m), (2, m), 3.84 (9, s), 3.85 (3, s), 4.03 (1, dd, J = 5.8, 13.8 z), 5.73 (1, br d, J = 10.8 z), 6.10 (1, br s), 6.50 (1, s), 6.56 (2, s), 6.57 (1, s), 6.79 (1, br s); 13 C MR (100 Mz, CDCl 3 ) δ 8.7, 10.9, 21.3, 28.7, 36.3, 37.0 (2C), 38.9, 40.0, 45.1 (2C), 48.8 (2C), 56.0, 56.1, 56.3 (2C), 56.4, 109.2, 110.2, 111.2, 111.8, (2C), (3C), (2C), 147.8, 148.0, 148.2, 167.9, 170.2; MALDI-MS m/z , ([C ] + calcd ). t R is 3.72 min (91.5% purity). In order to convert compounds to their respective sodium salt, each compound was dissolved in chloroform (25 ml) and the solution was washed gently with 0.1 a solution (2 x 20 ml). The organic phase was then washed gently with water (1 x 20 ml), brine (2 x 15 ml), dried over anhydrous MgS 4 and the solvent evaporated in-vacuo at a temperature not greater than 45 o C. For compound 24, the residue obtained was further dissolved in ethyl acetate (1.0 ml) and the product precipitated by adding hexanes (15-20 ml). The precipitate was filtered under vacuum and washed with hexanes (15 ml). The solid product obtained was dried under vacuum at 70 o C for 4 hours. The proton and C-13 MR of are identical to that of respectively. S21

22 a Sodium 4-[1-(3-Ethyl-9,10-dimethoxy-1,3,4,6,7,11b-hexahydro-2-pyrido[2,1- a]isoquinolin-2-ylmethyl)-6,7-dimethoxy-3,4-dihydro-1-isoquinolin-2-yl]-4-oxo-butyrate (24): Yield. 88%, white solid, mp = o C; 1 MR (400 Mz, CD 3 D) δ 0.81 (3, t, J = 7.5 z), (3, m), (2, m), (1, m), 2.14 (1, t, J = 12.4 z), 2.29 (1, t, J = 11.6 z), (2, m), (2, m), (3, m), (1, m), (1, m), (1, m), (1, m), (2, m), (2, m), 3.66 (3, s), 3.68 (3, s), 3.69 (3, s), 3.77 (3, s), 3.98 (1, dd, J = 5.0, 13.5 z), 5.66 (1, dd, J = 3.8, 11.6 z), 6.58 (1, s), 6.59 (1, s), 6.60 (1, s), 6.84 (1, s). 13 C MR (100 Mz, CD 3 D) δ 11.2, 24.3, 28.7, 29.6, 30.6, 30.9, 33.6, 37.0, 38.3, 40.0, 49.7 (2C), 52.8, 55.8, 56.5, 56.6, 56.8, 57.0, 64.2, 110.6, 111.9, 113.0, 113.5, 126.6, 127.1, 129.5, 131.7, 149.3, 149.4, 149.5, 149.6, 174.3, 180.9; MALDI-MS m/z , ([C ] + calcd ). t R is 3.05 min (97.5% purity). S22

23 a Sodium 4-[1-(3-Ethyl-9,10-dimethoxy-1,3,4,6,7,11b-hexahydro-2-pyrido[2,1- a]isoquinolin-2-ylmethyl)-6,7-dimethoxy-3,4-dihydro-1-isoquinolin-2-yl]-4-oxo-but-2- enoate (25): Yield. 79%, white solid, mp = o C; 1 MR (400 Mz, CD 3 D) δ 0.86 (3, t, J = 7.1 z), (3, m), (4, m), (1, m), 2.23 (1, t, J = 12.8 z), 2.67 (1, br d, J = 12.9 z), (5, m), (1, m), (3, m), 3.82 (4, s), 3.83 (4, s), 3.86 (4, s), 4.07 (1, dd, J = 2.4, 7.1 z), 5.70 (1, br d, J = 8,6 z), 6.10 (1, dd, J = 3.0, 11.9 z), 6.31 (1, dd, J = 3.0, 11.9 z), 6.49 (1, s), 6.51 (1, s), 6.55 (1, s), 6.87 (1, s); 13 C MR (100 Mz, CDCl 3 ) δ 10.3, 22.7, 26.3, 28.4, 34.7 (2C), 35.4, 38.4, 39.4, 45.3, 48.5 (2C), 55.8, 55.9, 56.1, 56.2, 60.4, 109.3, 109.8, 110.8, 111.6, (2C), (2C), 132.5, 135.7, 147.7, 147.9, 148.2, 148.4, 167.2, 169.5; MALDI-MS m/z , ([C ] + calcd ). t R is 3.30 min (99.5% purity). S23

24 a Sodium 4-[1-(3-Ethyl-9,10-dimethoxy-1,3,4,6,7,11b-hexahydro-2-pyrido[2,1- a]isoquinolin-2-ylmethyl)-6,7-dimethoxy-3,4-dihydro-1-isoquinolin-2-yl]-2-methyl-4-oxobut-2-enoate (26): Yield. 80%, pale yellow solid, mp = o C; 1 MR (400 Mz, CDCl 3 ) δ 0.84 (3, t, J = 7.4 z), (2, m), (4, m), 1.98 (3, s), 2.17 (1, t, J = 12.7 z), (2, m), (4, m), (3, m), (2, m), 3.84 (9, s), 3.85 (3, s), 4.03 (1, dd, J = 5.8, 13.8 z), 5.73 (1, br d, J = 10.8 z), 6.10 (1, br s), 6.50 (1, s), 6.56 (2, s), 6.57 (1, s), 6.79 (1, br s); 13 C MR (100 Mz, CDCl 3 ) δ 8.7, 10.9, 21.3, 28.7, 36.3, 37.0 (2C), 38.9, 40.0, 45.1 (2C), 48.8 (2C), 56.0, 56.1, 56.3 (2C), 56.4, 109.2, 110.2, 111.2, 111.8, (2C), (3C), (2C), 147.8, 148.0, 148.2, 167.9, 170.2; MALDI-MS m/z , ([C ] + calcd ). t R is 3.72 min (91.5% purity). a + - a S24

25 Disodium 2-{2-[1-(3-Ethyl-9,10-dimethoxy-1,3,4,6,7,11b-hexahydro-2-pyrido[2, 1 a]isoquinolin-2-ylmethyl)-6,7-dimethoxy-3,4-dihydro-1-isoquinolin-2-yl]-2-oxoethylidene}-succinate (27): Yield. 79%, yellow solid, mp = o C; 1 MR (400 Mz, DMS-d6) δ 0.83 (3, t, J = 7.4 z), (4, m), (2, m), (2, m), (1, m), 2.11 (1, t, J = 12.5 z), 2.33 (1, br s), (3, m), (4, m), 3.19 (2, s), 3.70 (9, s), 3.80 (3, s), (1, m), 5.62 (1, d, J = 7.8 z), 5.79 (1, br s), 6.63 (1, s), 6.67 (1, s), 6.78 (1, s), 6.82 (1, s); 13 C MR (100 Mz, CDCl 3 ) δ 10.3, 22.7, 22.9, 34.7, 36.1, 38.4, 39.1 (2C), 40.0, 45.6 (2C), 48.2, 51.1, 55.9, 56.1, 56.2, 56.3, 56.6, 108.8, 109.8, 111.1, 111.5, 124.2, 127.3, (2C), 138.2, 147.9, 148.1, 148.2, 148.3, 148.4, 169.9, 171.6, 172.0; MALDI-MS m/z , ([C ] + calcd ). t R is 2.60 min (95.2 % purity). PLC assay for evaluating the p-responsiveness of emetine analogs PLC method 2 was used for this study. Some of the synthetic analogs of emetine (compounds 5, 12, 13, 20-27) were selected to be evaluated for p responsiveness. To each of three different 9 ml vials marked A, B, and C was weighed 2 mg of each analog. Aqueous phosphate solutions (2 ml, 0.1 M) buffered at p s 5.5, 6.5 and 7.4 was added to dissolve the sample in each of vials A, B and C, respectively, affording sample solutions of concentration 1.0 mg/ml. Compounds 5, 12 and 24 were not expected to be p responsive under the slightly acidic conditions employed in this study. owever, in order to confirm our hypothesis they were also evaluated. All samples were incubated at 37 o C and aliquots were removed at various time points and analysed by S25

26 PLC. Data from the chromatograms obtained are shown in Supporting Table 1 and Supporting Table 2. Supporting Table 1: p- Responsiveness of emetine pro-drugs 13, 21, 22, 25, 26 and 27 in aqueous solutions of p 5.5 and 6.5; and stability in aqueous solutions of p 7.4 to 8.0. % Emetine = Percent composition of emetine in the mixture due to acid initiated hydrolysis at different time points after incubation at 37 o C. Compounds %Emetine at p 5.5 at different time points %Emetine at p 6.5 at different time points % Emetine at p 7.4 to 8.0 over 72 hrs 0 hr 24 hrs 48 hrs 0 hr 24 hrs 48 hrs 0 hrs 24 hrs 48hrs % 55.9% 94.5% 0.0% 5.2% 13.2% 0.0% 0.0% 0.0% % % 35.5% 52.1% 0.0% 0.0% 0.0% % % 76.8% 82.7% 0.0% 0.0% 0.0% % 19.2% 27.1% 0.0% 8.2% 17.1% 0.0% 0.0% 0.0% % 61.5% 77.3% 0.0% 24.9% 49.2% 0.0% 0.0% 0.0% % 33.9% 42.1% 0.0% 13.4% 21.1% 0.0% 0.0% 0.0% S26

27 Supporting Table 2: Stability of emetine analogs 5, 12 and 24 in aqueous solutions of p 5.5 and 7.4 over a period of 72 hrs at 37 o C. These compounds are not expected to be p-responsive at mildly acidic p of 5.5 and the duration of incubation employed for this study. % Emetine = Percent composition of emetine in the mixture due to acid initiated hydrolysis at different time points after incubation at 37 o C. Compounds % Emetine at p 5.5 at different time points % Emetine at p 7.4 at different time points 24 hrs 48hrs 72 hrs 24 hrs 48hrs 72 hrs 5 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% % 0.0% 0.0% 0.0% 0.0% 0.0% % 0.0% 3.0% 0.0% 0.0% 0.0% Biological Assays Cytotoxicity Assay General information The human androgen independent and dependent prostate cancer cell lines PC3 and LCaP respectively were purchased from the American Type Culture Collection (Manassas, VA). All cells were grown in cell culture flasks in RPMI culture medium with phenol red (GIBC) supplemented with only 10% fetal bovine serum, 1% L-glutamine and 1% Penicillin- Streptomycin. Cells were cultured in a humidified atmosphere of 95% air and 5% carbon dioxide at 37 o C. To sub-culture cells for experiments, cells growing as monolayer cultures were S27

28 released from the tissue culture flasks by treatment with 0.05% trypson/edta. Cell population density was determined with aid of coulter counter (Beckman) and/or hemocytometer. For all the in-vitro experiments, cells were used during the logarithmic growth phase. Screening of emetine analogs for cytotoxicity in LCaP and PC3 cell lines. For the initial determination of the cytotoxicity of emetine and its analogs in LCaP and PC3, the cells were plated at a density of 6 X 10 3 in 100 µl of medium per well and 2.5 X 10 3 in 100 µl medium per well respectively in 96-well plates. These cells were incubated for 24 hours to allow attachment to the tissue culture plates. Serial dilutions (0.02 mm, 0.1 mm, 0.2 mm, 2 mm, 10 mm, 20 mm) of each drug (emetine and its analogs) were made in sterile DMS and these were further diluted to micromolar scale (0.02 µm, 0.1 µm, 0.2 µm, 2 µm, 10 µm, 20 µm) using RPMI medium. 100 µl of each drug concentration was added in eight replicates to cells in the 96 well-plate providing final drug concentrations of 0.01 µm, 0.05 µm, 0.1 µm, 1 µm, 5 µm, and 10 µm respectively of each drug and uniform DMS concentration of 0.1% v/v in all drug-treated medium. A vehicle and blank control of 0.2% v/v and 0.0% v/v DMS in RPMI medium were made and 100 µl of these was also added in eight replicates to the cells plated in 100 µl of medium in the 96 well-plate. Thus, the vehicle control is 0.1% DMS and blank control is 0% DMS. Each experiment was repeated at least twice. Population of viable cells was determined on day zero and each of the 3 rd, 5 th, and 7 th day of incubating the cells with the drugs using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) cell proliferation assay. S28

29 The Cell Titer 96 on-radioactive Cell Proliferation Assay produced by Promega Corporation was employed for this study. This is based on cellular conversion of a tetrazolium salt into a formazan product that can be measured easily using a 96-well plate reader. To carry out each cell proliferation assay, spent medium was removed from the cells on each of day zero, 3 rd, 5 th and 7 th day. 100 µl of fresh culture medium was added to each well followed by 15 µl Promega Dye Solution. This was incubated for 3 hours at 37 o C followed by addition of 100 µl of Solubilization/Stop Solution to each well and incubation at room temperature for 1 hour. Absorbance was recorded at 570 nm using SpectraMax Plus384 absorbance micro-plate reader (Molecular Devices). In both the vehicle and blank control, the growth of LCaP and PC3 were found to be in the logarithmic phase from day zero to the 7 th day. Further, for each cell line, both the vehicle and blank control have equal quantitative proliferation rate in all assays carried out (Supporting Figure 1). A. B. S29

30 Supporting Figure 1. Growth curve of LCaP (A) and PC3 (B) treated with 0.1% v/v DMS in culture medium (Vehicle control) and 0.0% v/v DMS in RPMI culture medium (Blank control). This is monitored over a 7-day period. This indicates that cell death does not result from 0.1% v/v DMS concentration in RPMI culture medium. Proliferation of these cells is in the logarithmic growth phase. IC 50 values were calculated based on the vehicle control for both cell lines. In vivo toxicity study to establish the safety of the prodrug of emetine in mice Mice were purchased from Taconic. Mouse care and treatment was done in line with the guidelines of the Animal Care and Use Committee of the Johns opkins University School of Medicine. Eight weeks old male Balb/c mice were injected intravenously with different doses of emetine, 13 and 26. Each drug was given to the mice at doses 33 mg/kg, and 100 mg/kg, and each experiment or dosage was done in triplicate. Drug solutions were made in 1% DMS in saline, and mice received 0.2 ml bolus of each dose per day for five days. Mice in the control experiment received 0.2 ml of 1% DMS in saline at every dosage. References 1. Wong, R.; Dolman, S. J. Isothiocyanates from tosyl chloride mediated mediated decomposition of in situ generated dithiocarbamic acid salts. J. rg. Chem. 2007, 72, S30

31 2. Sigurdsson, S. T.; Seeger, B.; Kutzke, U.; Eckstein, F. A mild and simple method for the preparation of isocyanates from aliphatic amines using trichloromethyl chloroformate. synthesis of an isocyanate containing an activated disulfide. J. rg. Chem. 1996, 72, S31