Biotin-guided anticancer drug delivery with acidity-triggered drug release

Electronic upplementary Material (EI) for ChemComm. This journal is The Royal ociety of Chemistry 2015 upporting Information for Biotin-guided anticancer drug delivery with acidity-triggered drug release oyeon Park, a Eunjin Kim, b Won Young Kim, a Chulhun Kang *, b and Jong eung Kim *, a a Department of Chemistry, Korea University, eoul, 136-701, Korea b The chool of East-West Medical cience, Kyung ee University, Yongin, 446-701, Korea Corresponding E-mails: kangch@khu.ac.kr (C. Kang); jongskim@korea.ac.kr (J.. Kim) 1

Experimental ection ynthetic Materials, methods and instrumentations. 2-chloroethanol (Fluka), sodium azide (Aldrich), biotin (Aldrich), EDC.Cl (GL), 4- dimethylaminopyridine (Aldrich), 4-nitrosalicyclic acid (Aldrich), 1-butanol (Aldrich),,'- dicyclohexylcarbodiimide (Fluka), propargyl bromide (TCI), potassium carbonate (Aldrich), trifluoroacetic acid (Acros), tert-butyl carbazate (Alfa aesor), ATU (Iris Biotech),,diisopropylethylamine (Aldrich), L-ascorbic acid sodium salt (TCI), copper sulphate pentahydrate (Duksan), doxorubicin Cl (Aldrich) were received and were used without further purification. Most of the reactions were carried out under nitrogen atmosphere. nly compound 7 was synthesized under argon atmosphere. Compounds 1, 3 and prodrug 9 containing the hydrazone bond were synthesized according to previously reported procedures. ilica gel 60 (Merck, 0.063~0.2 mm) was used for column chromatography as a stationary phase. Analytical thin layer chromatography was performed using Merck 60 F254 silica gel (precoated sheets, 0.25 mm thick). The EI mass spectra were obtained using a LC/M-2020 eries (himadzu). 1 and 13 C MR spectra were collected in CDCl 3 or DM-d6 in a Varian 300 and 400 Mz MR spectrometer. All chemical shifts are reported in ppm value using the peak of TM as an internal reference. pectroscopic measurements. tock solutions of prodrug 9 and free doxorubicin were prepared in DM. Excitation was carried out at 501 nm with all excitation and emission slit widths at 5 nm. The concentrations of each of the samples were fixed 5 μm at a total volume of 3 ml, while 20 μm samples were used in the UV/Vis spectra. Cell line and cell culture. In this study, epg2 (human hepatocellular carcinoma) and, WI 38 (human lung fibroblast) were used. epg2 and WI-38 cell lines were cultured in RPMI 1640 containing 10% FB and 1% penicillin-streptomycin. Cells were grown at 37 C under a humidified atmosphere containing 5% C 2. 2

ynthesis Cl a 3, 2 3 60 C EDCI, DMAP, DMF, rt 1 2 3 2 + DMAP, DCC TF, reflux 2 Br K 2 C 3, AC, rt 3 2 TFA, DCM 2 2 Boc ATU, DIPEA, DMF 2 4 5 6 2, Ascorbic acid sodium salt Cu 4. 5 2, DMF, Ar, rt 2 7 TFA, DCM 2 2 8 Doxorubicin 2 2 9 Prodrug 9 was synthesized according to this scheme. Preparation and Characterization of 1 A solution of 2-chloroethanol (20.0 g, 248 mmol) and a 3 (48.3 g, 744 mmol) in water (200 ml) was stirred at 60 C for 48 h. After the solution was allowed to cool to room temperature, the crude product was extracted with diethyl ether (3 300 ml) to afford, after evaporation of the solvent under reduced pressure and at room temperature, 2-azidoethanol as a colorless liquid (15.5 g, 71.9%). CAUTI: andling this low molar mass azide is hazardous as [nc+n]/n = 1.42. Therefore, it should be manipulated with extreme caution, and the crude product was thus directly used in the next step without further purification, handling, or storage. 3

Preparation and Characterization of 2 Biotin (2.24 g, 9.19 mmol) was dissolved in 20 ml DMF and a solution of compound 1 (1.00 g, 11.5 mmol) in DMF (3 ml) was added followed by EDC Cl (5.35 g, 34.4 mmol) and, DMAP (9.12 g, 74.6 mmol). After being stirred for 16 h at room temperature, the reaction mixture was concentrated in vacuo and diluted with 30 ml of ethyl acetate and the organic layer was washed by water (3 10 ml), dried over Mg 4 and then concentrated in vacuo. The crude product was purified by column chromatography DCM/methanol (9:1) to obtain the product 1.91 g (66.3%) 1 MR (CDCl 3, 300 Mz): δ 6.29 (s, 1), 5.98 (s, 1), 4.47 4.43 (m, 1), 4.27 4.23 (m, 1), 4.19 (t, J = 5.01 z, 2), 3.44 (t, J = 5.22 z, 2), 3.13 3.07 (m, 1), 2.88 2.82 (m, 1), 2.71 2.67 (m, 2), 2.34 (t, J = 7.44 z, 2), 1.59 1.69 (m, 4), 1.37 1.45 (m, 2). 13 C (CDCl 3, 100 Mz): 173.6, 163.8, 63.1, 62.1, 55.6, 50.0, 40.8, 33.9, 28.4, 24.9 ppm. EI-M: m/z calcd for C 12 19 5 3 [M+a+], 336.11; found 336. Preparation and Characterization of 3 DCC (1.10 equiv, 1.86 g, 9.00 mmol) dissolved in dry TF (12.0 ml) was added dropwise over 7 min to a stirred solution of 4-nitrosalicyclic acid (1.00 equiv, 1.50 g, 8.20 mmol) and DMAP (1.00 eqiuv, 990 mg, 8.20 mmol) in dry tert-butyl alcohol (30.0 ml). The mixture was stirred and heated to reflux overnight. The reaction was monitored by TLC. After the reaction was complete, the solvents were removed under reduced pressure. Then 15 ml of ethyl acetate was added, and the solution was filtered to remove the 1,3-dicyclohexylurea (DCU) formed. After removal of the solvent under reduced pressure, the crude product was purified by column chromatography using ether/ethyl acetate (30:1) as the eluent. Pale yellow crystals, 1.62 g (81.7% yield). 1 MR (300 Mz, CDCl 3 ): δ 11.27 (s, 1), 7.94 (d, J = 8.61 z, 1), 7.78 (s, 1), 7.67 (d, J = 8.64 z, 1), 1.64 (s, 9). Preparation and Characterization of 4 To a solution of Compound 3 (1.00 equiv, 1.50 g, 6.27 mmol) in C 3 C (15.0 ml), K 2 C 3 (2.00 equiv, 1.73 g, 12.5 mmol) and propargyl bromide (1.20 equiv, 0.600 ml, 7.54 mmol) were added. The mixture was stirred for 15 hours at room temperature. The color changed to pale yellow. After removal of the solvent under reduced pressure, the crude product was purified by column chromatography using ethyl acetate/hexane (1:9) as the eluent. Light yellow solid, 2.00 g (97.1% yield) 1 MR (300 Mz, CDCl 3 ): δ 7.94 (d, J = 2.0 z, 1), 7.88 (dd, J = 8.57, 1.97 z, 1), 7.81 (d, J = 8.40 z, 1), 4.87 (d, J = 2.44 z, 2), 2.60 (t, 4

J = 2.39 z, 1), 1.60 (s, 9). 13 C MR (100 Mz, CDCl 3 ): 164.1, 156.9, 150.2, 131.8, 129.3, 116.2, 109.2, 83.1, 77.3, 77.1, 57.4, 28.4 ppm. Preparation and Characterization of 5 Compound 4 (850 mg, 3.07mmol) was dissolved in 8.00 ml of C 2 Cl 2 where trifluoroacetic acid (2.00 ml) was added. The reaction mixture was stirred for 4 hours at room temperature and subsequently the solvent was co-evaporated with toluene and the crude product was purified by column chromatography using DCM/methanol (9:1) as an eluent. White solid, 650 mg (95.8% yield). 1 MR (300 Mz, CDCl 3 ): δ 7.97 (d, J = 1.74 z, 1), 7.93 (s, 1), 7.91 (d, J = 1.74 z, 1), 4.90 (d, J = 2.40 z, 2), 2.63 (t, J = 2.45 z, 1). 13 C MR (100 Mz, CDCl 3 ): 169.8, 154.5, 147.4, 139.2, 129.6, 116.8, 109.1, 79.7, 79.4, 57.1 ppm. EI-M: m/z calcd for C 10 7 5 [M-+], 220.03; found 219.9. Preparation and Characterization of 6 Compound 5 (1.00 equiv, 1.15 g, 4.35 mmol) was dissolved in 15.0 ml DMF. tert-butyl carbazate (1.20 equiv, 688 mg, 5.21 mmol), ATU (1.20 equiv, 1.98 g, 5.21 mmol) and DIPEA (0.940 ml) were added. The reaction mixture was stirred for 5 hours at room temperature. After removal of the solvent under reduced pressure, the crude product was diluted by 30.0 ml of ethyl acetate and the organic layer was washed by water (3 10.0 ml), dried over Mg 4 and then concentrated in vacuum. The crude product was purified by column chromatography ethyl acetate/hexane (1:3) to obtain the product 1.34 g (92.0%). 1 MR (300 Mz, CDCl 3 ): δ 9.36 (s, 1), 8.39 (d, J = 8.52 z, 1), 8.00 (dd, J = 4.93, 2.02 z, 1), 7.97 (d, J = 2.02 z, 1), 6.98 (s, 1), 5.01 (d, J = 2.40 z, 2), 2.70 (t, J = 2.40 z, 1), 1.51 (s, 9). 13 C MR (100 Mz, CDCl 3 ): δ 162.1, 155.7, 155.1, 150.8, 134.1, 125.6, 117.1, 108.6, 82.4, 78.8, 76.0, 57.8, 28.4 ppm. EI-M: m/z calcd for C 15 17 3 6 [M- +], 334.11; found 333.95. Preparation and Characterization of 7 To a solution of compound 6 (1.00 equiv, 264 mg, 0.790 mmol) in DMF (5.00 ml), 2 (1.10 eqiuv, 270 mg, 0.860 mmol) and sodium ascorbate (1.00 equiv, 156 mg, 0.790 mmol) were added. The reaction mixture was degassed for 30 min by purging argon gas. Then Cu 4. 5 2 (1.00 equiv, 197 mg, 0.790 mmol) in DMF (3.00 ml) was added to the reaction mixture and the mixture was stirred for 2 hours. After removal of solvent under 5

reduced pressure, the crude product was diluted by 30.0 ml of ethyl acetate and the organic layer was washed by water (3 10.0 ml), dried over Mg 4 and then concentrated in vacuum. The crude product was purified by column chromatography DCM/methanol (9:1) to obtain the product 380 mg (74.4%). 1 MR (300 Mz, CDCl 3 ): δ 8.22 (d, J = 8.63 z, 1), 8.06 (s, 1), 7.92 (dd, J = 8.63, 1.92 z, 1), 6.38 (s, 1), 5.57 (s, 2), 4.70-4.59 (m, 2), 4.55-4.44 (m, 3), 4.37-4.28 (m, 1), 3.14 (dd, J = 7.20, 4.56 z, 1), 2. 92 (dd, J = 13.3, 4.79 z, 1), 2.71 (d, J = 12.7 z, 1), 2.27 (t, J = 7.08 z, 2), 1.62-1.56 (m, 4), 1.5 (s, 9), 0.92-0.80 (m, 2). 13 C MR (100 Mz, CDCl 3 ): 173.3, 164.4, 163.5, 156.5, 155.8, 150.7, 142.4, 133.3, 126.6, 124.8, 116.7, 108.9, 81.8, 64.3, 62.3, 62.1, 60.4, 55.9, 49.7, 40.8, 33.6, 29.9, 28.4, 28.3, 24.7 ppm. EI-M: m/z calcd for C 27 36 8 9 [M+a+], 671.23; found 671.30. Preparation and Characterization of 8 Compound 7 (550 mg, 0.850 mmol) was dissolved in a mixture of 8.00 ml C 2 Cl 2. And trifluoroacetic acid (2.00 ml). The reaction mixture was stirred for 4 hours at room temperature. The solvent was co-evaporated with toluene and the crude product was purified by column chromatography DCM/methanol (9:1) as the eluent. The product was obtained 330 mg (70.9% yield). 1 MR (300 Mz, CDCl 3 ): δ 8.22 (d, J = 8.68 z, 1), 8.04 (d, J = 1.77 z, 1), 7.98 (s, 1), 7.91 (dd, J = 8.68, 1.77 z, 1), 5.49 (s, 2), 4.70 (t, J = 4.61 z, 2), 4.56-4.49 (m, 3), 4.35-4.29(m, 1), 3.21-3.11 (m, 1), 2.92 (dd, J = 12.7, 4.96 z, 1), 2.72 (d, J = 12.7 z, 1), 2.30 (t, J = 6.91 z, 2), 1.69-1.50 (m, 4), 1.45-1.32 (m, 2). 13 C MR (100 Mz, CDCl 3 ): 173.3, 164.7, 164.4, 156.3, 150.4, 142.1, 133.1, 126.5, 124.5, 116.6, 108.4, 77.8, 63.1, 62.2, 60.3, 55.8, 49.7, 40.5, 33.5, 29.8, 28.4, 24.7 ppm. EI- M: m/z calcd for C 22 28 8 7 [M+a+], 571.18; found 571.25. Preparation and Characterization of 9 Doxorubicin Cl (1.00 equiv, 60.0 mg, 0.110 mmol) and 8 (1.30 equiv, 75.0 mg, 0.140 mmol) were dissolved in anhydrous methanol (15.0 ml) and the mixture was treated with TFA (7.80 μl). After stirring overnight at room temperature in the dark all volatiles were removed under reduced pressure, the residue was redissolved in methanol (5.00 ml) and diethyl ether (35.0 ml) was added to precipitate the product. The red solid was collected by centrifugation and dried in vacuum. 91.0 mg (60.5% yield). 1 MR (300 Mz, DM-d6): δ 8.28-8.20 (m, 1), 7.94-7.89 (m, 2), 7.88-7.85 (m, 1), 7.83-7.77 (m, 2), 7.61-7.53 (m, 6

1), 5.55 (s, 1), 5.43 (d, J = 9.16 z, 2), 5.25 (t, J = 13.9 z, 2), 4.58 (br, 2), 4.49-4.43 (br, 1), 4.39-4.31 (m, 1), 4.28-4.22 (m, 2), 4.07 (br, 1), 3.93 (s, 3), 3.08-2.93 (m, 4), 2.76 (br, 2), 2.23-2.10 (m, 4), 1.91-1.80 (m, 2), 1.75-1.65 (m, 2), 1.57-1.49 (m, 2), 1.43-1.31 (m, 4), 1.25-1.13 (m, 4), 1.04 (s, 3). 13 C MR (100 Mz, DM-d6): 187.0, 186.9, 173.2, 163.4, 161.4, 159.9, 158.5, 157.0, 156.4, 155.1, 150.4, 142.1, 136.9, 136.5, 135.9, 135.8, 135.3, 133.4, 133.0, 128.1, 126.3, 120.5, 119.6, 116.6, 111.3, 111.1, 109.8, 100.0, 72.5, 71.9, 66.9, 66.8, 65.6, 62.7, 61.7, 59.8, 57.2, 56.0, 49.3, 47.3, 47.2, 33.7, 28.5, 24.9, 15.8 ppm. EI-M: m/z calcd for C 49 55 9 17 [M++] 1074.34, [M+a+] 1096. 34; found 1074. 40, 1096.40. 7

-MR, 13 C-MR and M Analyses: pinworks 2.5: TADARD 1 BERVE 0.956 0.914 0.988 2.913 1.629 1.047 1.044 1.812 2.132 4.595 3.083 6.2905 5.9822 4.4777 4.4609 4.4519 4.4360 4.2770 4.2617 4.2519 4.2364 4.2173 4.2004 4.1834 3.4599 3.4426 3.4261 3.3937 3.1399 3.1134 3.0978 3.0762 2.8874 2.8709 2.8445 2.8284 2.7169 2.6750 2.3707 2.3458 2.3209 1.7231 1.7017 1.6775 1.6524 1.6278 1.6020 1.4563 1.4309 1.4053 1.3795 3 PPM 7.2 6.8 6.4 6.0 5.6 5.2 4.8 4.4 4.0 3.6 3.2 2.8 2.4 2.0 1.6 1.2 0.8 0.4-0.0 Fig. 1 1 MR spectrum of compound 2 in CDCl 3. pinworks 2.5: td proton 173.5615 163.8241 63.1274 62.1466 60.3156 55.6302 49.9976 40.7910 33.9176 28.5274 28.4403 24.8738 3 PPM 170.0 160.0 150.0 140.0 130.0 120.0 110.0 100.0 90.0 80.0 70.0 60.0 50.0 40.0 30.0 file: C:\Users\sws\Desktop\MR\13C biotin-azide.fid\fid block# 1 expt: "s2pul" transmitter freq.: 100.572632 Mz time domain size: 63750 points width: 24509.80 z = 243.702520 ppm = 0.384468 z/pt number of scans: 1260 freq. of 0 ppm: 100.562073 Mz processed size: 65536 complex points LB: 0.000 GB: 0.0000 Fig. 2 13 C MR spectrum of compound 2 in CDCl 3. 8

3 Fig. 3 Mass spectrum of compound 2. pinworks 2.5: TADARD 1 BERVE 0.691 1.025 0.899 0.997 9.226 11.2741 7.9562 7.9272 7.7858 7.6837 7.6794 7.6764 7.6546 7.6473 7.2640 1.6442-0.0000 2 PPM 10.0 9.0 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0 Fig. 4 1 MR spectrum of compound 3 in CDCl 3. 9

pinworks 2.5: TADARD 1 BERVE 1.089 1.326 1.053 2.292 0.999 10.25 7.9520 7.9455 7.9013 7.8948 7.8733 7.8667 7.8302 7.8021 7.2641 4.8722 4.8642 2.6089 2.6009 2.5929 1.6034 0.0000 2 PPM 8.4 8.0 7.6 7.2 6.8 6.4 6.0 5.6 5.2 4.8 4.4 4.0 3.6 3.2 2.8 2.4 2.0 1.6 1.2 0.8 0.4-0.0 Fig. 5 1 MR spectrum of compound 4 in CDCl 3. pinworks 2.5: td proton 164.1025 156.8678 150.1702 131.8467 129.3167 116.2350 109.2141 83.0641 77.5666 77.3470 77.2487 77.1365 76.9306 57.4167 28.3521 2 PPM 170.0 160.0 150.0 140.0 130.0 120.0 110.0 100.0 90.0 80.0 70.0 60.0 50.0 40.0 30.0 20.0 10.0 file: C:\Users\a\Desktop\ 소소 \MR data\140902-sy-step2-13c.fid\fid block# 1 expt: "s2pul" transmitter freq.: 100.572632 Mz time domain size: 63750 points width: 24509.80 z = 243.702520 ppm = 0.384468 z/pt number of scans: 2368 freq. of 0 ppm: 100.562073 Mz processed size: 65536 complex points LB: 0.000 GB: 0.0000 Fig. 6 13 C MR spectrum of compound 4 in CDCl 3. 10

pinworks 2.5: TADARD 1 BERVE pinworks 2.5: TADARD 1 BERVE pinworks 2.5: TADARD 1 BERVE pinworks 2.5: TADARD 1 BERVE 7.9794 7.9735 7.9307 7.9140 7.9082 7.2829 7.9794 7.9735 7.9307 7.9140 4.9109 7.9082 4.9029 7.2829 3.4638 7.9794 7.9735 7.9307 7.9140 7.9082 2.6399 2.6317 2.6242 7.2829 4.9109 4.9029 3.4638 0.0000 4.9109-0.0011 4.9029 2.6399 2.6317 2.6242 3.4638 7.9794 7.9735 7.9307 7.9140 7.9082 7.2829 4.9109 4.9029 3.4638 2 3.130 3.130 2.079 3.130 3.130 1.244 2.079 2.079 1.244 PPM 8.0 7.6 7.2 6.8 6.4 6.0 5.6 PPM 5.2 8.0 4.8 7.6 4.4 7.2 4.0 6.8 3.6 PPM 3.2 8.0 2.8 7.6 2.4 7.2 2.0 6.8 1.6 6.4 1.2 6.0 0.8 5.6 0.4 5.2-0.0 4.8 4.4 4.0 3.6 3.2 PPM 6.4 8.0 6.0 7.6 5.6 7.2 5.2 6.8 4.8 6.4 4.4 6.0 4.0 5.6 3.6 5.2 3.2 4.8 2.8 4.4 2.4 2.0 4.0 1.6 3.6 1.2 3.2 Fig. 7 1 MR spectrum of compound 5 in CDCl 3. file: C:\Users\a\Desktop\ 소소 \MR data\140828-step3-new.fid\fid block# 1 expt: file: C:\Users\a\Desktop\ "s2pul" 소소 \MR data\140828-step3-new.fid\fid block# 1 expt: "s2pul" transmitter freq.: 300.176336 Mz file: C:\Users\a\Desktop\ transmitter freq.: 300.176336 소소 \MR data\140828-step3-new.fid\fid Mz block# 1 expt: "s2pul" time domain size: 17984 points transmitter time freq.: domain 300.176336 size: 17984 Mz points width: 4500.45 z = 14.992688 ppm = 0.250247 z/pt time domain width: size: 4500.45 17984 z points = 14.992688 ppm = 0.250247 z/pt number of scans: 64 width: 4500.45 number of z scans: = 14.992688 64 ppm = 0.250247 z/pt number of scans: 64 freq. of 0 ppm: 300.174847 Mz processed size: 32768 complex points LB: 0.000 GB: 0.0000 freq. of 0 ppm: 300.174847 Mz processed freq. of 0 size: ppm: 32768 300.174847 complex Mz points LB: processed 0.000 size: GB: 0.0000 32768 complex poin LB: 0.000 GB: 0.0000 pinworks 2.5: td proton 169.8044 154.4700 147.4403 139.2189 129.5908 116.8444 109.0999 79.7560 79.4172 57.0519 2 PPM 170.0 160.0 150.0 140.0 130.0 120.0 110.0 100.0 90.0 80.0 70.0 60.0 50.0 40.0 30.0 20.0 10.0 file: C:\Users\a\Desktop\ 소소 \MR data\140917-step3-13c.fid\fid block# 1 expt: "s2pul" transmitter freq.: 100.573110 Mz time domain size: 63750 points width: 24509.80 z = 243.701362 ppm = 0.384468 z/pt number of scans: 896 freq. of 0 ppm: 100.562551 Mz processed size: 65536 complex points LB: 0.000 GB: 0.0000 Fig. 8 13 C MR spectrum of compound 5 in CDCl 3. 11

2 Fig. 9 Mass spectrum of compound 5. pinworks 2.5: TADARD 1 BERVE 0.942 1.118 2.173 1.138 2.166 0.999 9.220 9.3679 9.3661 8.4028 8.3745 8.0122 8.0065 7.9967 7.9899 7.9684 7.9616 7.2689 6.9818 5.0191 5.0111 2.7096 2.7017 2.6936 1.5109-0.0000 2 PPM 9.0 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0 Fig. 10 1 MR spectrum of compound 6 in CDCl 3. 12

pinworks 2.5: td proton 162.1084 155.6922 155.0808 150.7500 134.0769 125.6464 117.0542 108.5550 82.4104 78.8109 77.5683 77.2503 76.9331 76.0481 57.8407 28.3790 2 PPM 160.0 150.0 140.0 130.0 120.0 110.0 100.0 90.0 80.0 70.0 60.0 50.0 40.0 30.0 20.0 10.0 Fig. 11 13 C MR spectrum of compound 6 in CDCl 3. 2 Fig. 12 Mass spectrum of compound 6. 13

pinworks 2.5: TADARD 1 BERVE 1.041 1.484 1.206 1.100 1.994 0.900 2.390 3.453 1.265 1.258 1.437 1.383 2.356 4.543 9.084 2.110 8.2391 8.2104 8.0582 7.9423 7.9361 7.9136 7.9075 6.3789 5.5704 5.5613 5.1951 4.6597 4.6580 4.6563 4.6421 4.5083 4.5033 4.5027 4.5008 4.4982 4.4901 4.3396 4.3284 4.3264 4.3245 4.3227 3.1540 3.1519 3.1386 2.9467 2.9304 2.9033 2.8876 2.7332 2.6906 2.2889 2.2650 2.2396 1.6148 1.6127 1.6106 1.6082 1.6062 1.6018 1.5911 1.5678 1.5661 1.5627 1.5494 1.5038 0.8845 0.8824 0.8812 0.8798 0.8774 0.8569 0.8546 0.8526 0.0011 2 PPM 8.8 8.4 8.0 7.6 7.2 6.8 6.4 6.0 5.6 5.2 4.8 4.4 4.0 3.6 3.2 2.8 2.4 2.0 1.6 1.2 0.8 0.4 0.0 Fig. 13 1 MR spectrum of compound 7 in CDCl 3. pinworks 2.5: td proton 173.3027 164.4112 163.5097 156.4913 155.8075 150.7180 142.4435 133.3279 126.5932 124.8475 116.7208 108.9569 81.7998 77.6150 77.2969 76.9785 64.2654 62.2657 62.1164 60.3583 55.8649 49.6679 40.7796 33.5705 29.8990 28.4416 28.3440 28.2650 24.7692 2 PPM 170.0 160.0 150.0 140.0 130.0 120.0 110.0 100.0 90.0 80.0 70.0 60.0 50.0 40.0 30.0 20.0 10.0 Fig. 14 13 C MR spectrum of compound 7 in CDCl 3. file: C:\Users\a\Desktop\ 소소 \MR data\140924-step5-13c.fid\fid block# 1 expt: "s2pul" transmitter freq.: 100.572632 Mz time domain size: 63750 points width: 24509.80 z = 243.702520 ppm = 0.384468 z/pt number of scans: 832 freq. of 0 ppm: 100.562073 Mz processed size: 65536 complex points LB: 0.000 GB: 0.0000 14

2 Fig. 15 Mass spectrum of compound 7. pinworks 2.5: TADARD 1 BERVE 0.775 0.938 0.885 0.994 1.539 2.002 3.158 1.106 2.379 1.783 1.468 2.402 1.787 4.153 4.232 8.2232 8.1948 8.0310 8.0248 7.9845 7.9291 7.9230 7.9007 7.8944 7.3298 6.2205 6.0449 5.4881 5.3226 4.7066 4.6919 4.6747 4.5337 4.5191 4.5039 4.3254 4.3106 4.2996 4.2845 3.1633 3.1458 3.1364 3.1304 3.1214 3.1199 2.9273 2.9111 2.8841 2.8681 2.7272 2.6850 2.3063 2.2826 2.2587 2.0815 2.0512 2.0340 1.6001 1.5548 1.5497 1.5394 1.5303 1.5260 1.5220 1.5197 1.5178 1.3797 1.3777 1.3643 1.3626 1.3606 1.3567 1.3549 1.3536 1.3481 1.3451 1.3287 0.0007 2 2 PPM 8.4 8.0 7.6 7.2 6.8 6.4 6.0 5.6 5.2 4.8 4.4 4.0 3.6 3.2 2.8 2.4 2.0 1.6 1.2 0.8 0.4-0.0 Fig. 16 1 MR spectrum of compound 8 in CDCl 3. 15

pinworks 2.5: td proton 173.3670 164.7831 164.4853 156.2922 150.4520 142.0927 133.0858 126.5093 124.5031 116.5704 108.4388 63.1181 62.2011 60.3155 55.8153 49.7176 40.5070 33.5381 29.8317 28.3944 28.2100 24.6397 2 2 PPM 160.0 150.0 140.0 130.0 120.0 110.0 100.0 90.0 80.0 70.0 60.0 50.0 40.0 30.0 20.0 10.0 Fig. 17 13 C MR spectrum of compound 8 in CDCl 3. 2 2 Fig. 18 Mass spectrum of compound 8. 16

pinworks 2.5: td proton 1.339 1.928 1.159 1.992 1.449 0.566 0.544 0.970 1.768 2.006 2.528 1.294 2.563 1.564 1.431 3.070 4.627 2.428 4.759 2.652 2.358 2.502 4.518 2.400 2.240 3.231 8.2625 8.2605 8.2128 8.2112 7.9099 7.9083 7.9068 7.9038 7.8745 7.8729 7.8708 7.8568 7.8554 7.8539 7.8525 7.8508 7.8135 7.8119 7.8103 7.8090 7.5926 7.5909 7.5830 7.5817 7.5799 7.5785 7.5755 7.5736 7.5718 7.5694 7.5679 7.5665 7.5649 7.5630 6.1509 5.9241 5.6180 5.6168 5.6151 5.5546 5.5531 5.4667 5.4657 5.4641 5.4630 5.4606 5.4594 5.4571 5.4555 5.4531 5.4516 5.4507 5.4485 5.4458 5.4444 5.4422 5.4050 5.4027 5.2887 5.2875 5.2862 5.2848 5.2838 5.2826 5.2813 5.2789 5.2763 5.2581 5.2569 5.2546 5.2528 5.2518 5.2497 5.2482 5.2469 5.2425 5.2414 5.2396 5.2186 5.2166 4.5915 4.5896 4.5520 4.5506 4.5487 4.4626 4.4575 4.4560 4.4534 4.4522 4.4510 4.3518 4.3505 4.3493 4.3479 4.2538 4.0768 4.0746 4.0730 3.9323 3.0072 3.0054 2.7722 2.7708 2.7694 2.7559 2.7533 2.7519 2.7499 2.7453 2.7434 2.7420 2.7406 2.2034 2.2014 2.2002 2.1977 2.1961 2.1910 2.1863 2.1851 2.1836 2.1828 2.1811 2.1744 2.1729 2.1702 2.1688 2.1669 2.1654 2.1643 2.1350 2.1307 2.1297 2.1281 1.8710 1.8696 1.8671 1.8662 1.8646 1.8634 1.8606 1.8598 1.8582 1.7035 1.7018 1.7001 1.6981 1.6968 1.6954 1.6922 1.6895 1.5354 1.5343 1.5194 1.5165 1.5141 1.5132 1.5106 1.5091 1.5078 1.5068 1.5054 1.5038 1.5020 1.5006 1.4987 1.4970 1.3715 1.3703 1.3691 1.3678 1.3667 1.1717 1.0384 2 2 PPM 8.4 8.0 7.6 7.2 6.8 6.4 6.0 5.6 5.2 4.8 4.4 4.0 3.6 3.2 2.8 2.4 2.0 1.6 1.2 0.8 0.4-0.0-0.4 Fig. 19 1 MR spectrum of prodrug 9 in DM-d6. pinworks 2.5: td proton 187.0488 186.9778 173.1630 163.4399 161.4209 159.9734 158.5634 156.4049 155.1478 150.4793 142.7298 136.8722 136.4742 135.9941 135.8374 135.3078 133.4316 128.1108 126.3700 120.5307 119.6421 116.5956 111.2340 111.1244 100.0501 99.9762 72.4681 71.9148 66.8966 66.7705 65.5922 62.7007 61.6829 59.8673 57.2411 56.0094 49.3594 47.2114 33.6874 28.5928 24.9570 15.8310 2 2 PPM 185.0 175.0 165.0 155.0 145.0 135.0 125.0 115.0 105.0 95.0 85.0 75.0 65.0 55.0 45.0 35.0 25.0 15.0 Fig. 20 13 C MR spectrum of prodrug 9 in DM-d6. file: C:\Users\a\Desktop\ 소소 \MR data\141104-final-13c.fid\fid block# 1 expt: "s2pul" transmitter freq.: 100.573110 Mz time domain size: 63750 points width: 24509.80 z = 243.701362 ppm = 0.384468 z/pt number of scans: 20672 freq. of 0 ppm: 100.562551 Mz processed size: 65536 complex points LB: 0.000 GB: 0.0000 17

2 2 Fig. 21 Mass spectrum of prodrug 9. 18

Compound 8 + K + 2 Chemical Formula: C 27 29 11 Molecular Weight: 543.52 Dox + a + 2 Chemical Formula: C 22 28 8 7 Molecular Weight: 548.57 2 Fig. 22 Mass spectra of prodrug 9 (5 μm) at p 5.0 after 3 days at 37. Mass peak of compound 8 shown at [M+K + ] and mass peak of Doxorubicin shown at [M+a + ] (graph = cationic measurement). 19