Download - Biotin-guided anticancer drug delivery with acidity ... · Biotin-guided anticancer drug delivery with acidity-triggered drug release ... DMSO-d6 in a Varian 300 and 400 MHz NMR spectrometer.

S1

Supporting Information for

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

Soyeon Park,a Eunjin Kim,b Won Young Kim,a Chulhun Kang*,b and Jong Seung Kim*,a

aDepartment of Chemistry, Korea University, Seoul, 136-701, KoreabThe School of East-West Medical Science, Kyung Hee University, Yongin, 446-701, Korea

Corresponding E-mails: [email protected] (C. Kang); [email protected] (J. S. Kim)

Electronic Supplementary Material (ESI) for ChemComm.This journal is © The Royal Society of Chemistry 2015

mailto:[email protected]

mailto:[email protected]

S2

Experimental Section

Synthetic Materials, methods and instrumentations.

2-chloroethanol (Fluka), sodium azide (Aldrich), biotin (Aldrich), EDC.HCl (GLS), 4-

dimethylaminopyridine (Aldrich), 4-nitrosalicyclic acid (Aldrich), 1-butanol (Aldrich), N,N'-

dicyclohexylcarbodiimide (Fluka), propargyl bromide (TCI), potassium carbonate (Aldrich),

trifluoroacetic acid (Acros), tert-butyl carbazate (Alfa aesor), HATU (Iris Biotech), N,N-

diisopropylethylamine (Aldrich), L-ascorbic acid sodium salt (TCI), copper sulphate

pentahydrate (Duksan), doxorubicin·HCl (Aldrich) were received and were used without

further purification. Most of the reactions were carried out under nitrogen atmosphere. Only

compound 7 was synthesized under argon atmosphere. Compounds 1, 3 and prodrug 9

containing the hydrazone bond were synthesized according to previously reported

procedures. Silica 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 ESI mass spectra were obtained using a

LC/MS-2020 Series (Shimadzu). 1H and 13C NMR spectra were collected in CDCl3 or

DMSO-d6 in a Varian 300 and 400 MHz NMR spectrometer. All chemical shifts are reported

in ppm value using the peak of TMS as an internal reference.

Spectroscopic measurements.

Stock solutions of prodrug 9 and free doxorubicin were prepared in DMSO. 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, HepG2 (human hepatocellular carcinoma) and, WI 38 (human lung fibroblast)

were used. HepG2 and WI-38 cell lines were cultured in RPMI 1640 containing 10% FBS

and 1% penicillin-streptomycin. Cells were grown at 37°C under a humidified atmosphere

containing 5% CO2.

S3

Synthesis

HOCl NaN3, H2O

60 Co HON3

S

NHHN

O

O

OS

NHHN

O

OHO

EDCI, DMAP, DMF, rtN3

OH

O

OHO2N+ OH

DMAP, DCCTHF, reflux

O

O

OHO2N

Br

K2CO3, ACN, rt

O

O

OO2N

TFA, DCM OH

O

OO2N

NH2NHBocHATU, DIPEA, DMF

NH

O

OO2N

HN

O

O

2, Ascorbic acid sodium saltCuSO4

.5H2O, DMF, Ar, rtNH

O

OO2N

HN

O

O

N NN

O

O

S

NH

NH

O

TFA, DCM NH

O

OO2N

NH2

N NN

O

O

S

NH

NH

O

Doxorubicin

NH

O

OO2N

N

N NN

O

O

S

NH

NH

O

OH

OHOH

OO

O

OHOO

HONH2

1 2

3

4 5 6

7

8

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 NaN3 (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%). CAUTION: Handling this low molar mass azide is hazardous as

[nC+nO]/nN = 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.

S4


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·HCl (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 MgSO4 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%) 1H NMR (CDCl3, 300 MHz): δ 6.29 (s, 1H), 5.98 (s, 1H), 4.47–

4.43 (m, 1H), 4.27–4.23 (m, 1H), 4.19 (t, J = 5.01 Hz, 2H), 3.44 (t, J = 5.22 Hz, 2H), 3.13–

3.07 (m, 1H), 2.88–2.82 (m, 1H), 2.71–2.67 (m, 2H), 2.34 (t, J = 7.44 Hz, 2H), 1.59–1.69 (m,

4H), 1.37–1.45 (m, 2H). 13C (CDCl3, 100 MHz): 173.6, 163.8, 63.1, 62.1, 55.6, 50.0, 40.8,

33.9, 28.4, 24.9 ppm. ESI-MS: m/z calcd for C12H19N5O3S [M+Na+], 336.11; found 336.


DCC (1.10 equiv, 1.86 g, 9.00 mmol) dissolved in dry THF (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). 1H NMR (300 MHz, CDCl3): δ 11.27 (s, 1H), 7.94 (d, J = 8.61

Hz, 1H), 7.78 (s, 1H), 7.67 (d, J = 8.64 Hz, 1H), 1.64 (s, 9H).


To a solution of Compound 3 (1.00 equiv, 1.50 g, 6.27 mmol) in CH3CN (15.0 mL), K2CO3

(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) 1H NMR (300 MHz, CDCl3): δ 7.94 (d, J = 2.0 Hz, 1H),

7.88 (dd, J = 8.57, 1.97 Hz, 1H), 7.81 (d, J = 8.40 Hz, 1H), 4.87 (d, J = 2.44 Hz, 2H), 2.60 (t,

S5

J = 2.39 Hz, 1H), 1.60 (s, 9H). 13C NMR (100 MHz, CDCl3): 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.


Compound 4 (850 mg, 3.07mmol) was dissolved in 8.00 mL of CH2Cl2 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). 1H NMR (300 MHz, CDCl3): δ 7.97 (d, J = 1.74 Hz, 1H), 7.93 (s, 1H),

7.91 (d, J = 1.74 Hz, 1H), 4.90 (d, J = 2.40 Hz, 2H), 2.63 (t, J = 2.45 Hz, 1H). 13C NMR (100

MHz, CDCl3): 169.8, 154.5, 147.4, 139.2, 129.6, 116.8, 109.1, 79.7, 79.4, 57.1 ppm. ESI-MS:

m/z calcd for C10H7NO5 [M-H+], 220.03; found 219.9.


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), HATU (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 MgSO4 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%). 1H

NMR (300 MHz, CDCl3): δ 9.36 (s, 1H), 8.39 (d, J = 8.52 Hz, 1H), 8.00 (dd, J = 4.93, 2.02

Hz, 1H), 7.97 (d, J = 2.02 Hz, 1H), 6.98 (s, 1H), 5.01 (d, J = 2.40 Hz, 2H), 2.70 (t, J = 2.40

Hz, 1H), 1.51 (s, 9H). 13C NMR (100 MHz, CDCl3): 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. ESI-MS: m/z calcd for C15H17N3O6 [M-

H+], 334.11; found 333.95.


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

CuSO4.·5H2O (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

S6

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 MgSO4 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%). 1H NMR (300 MHz, CDCl3): δ 8.22 (d, J = 8.63 Hz, 1H),

8.06 (s, 1H), 7.92 (dd, J = 8.63, 1.92 Hz, 1H), 6.38 (s, 1H), 5.57 (s, 2H), 4.70-4.59 (m, 2H),

4.55-4.44 (m, 3H), 4.37-4.28 (m, 1H), 3.14 (dd, J = 7.20, 4.56 Hz, 1H), 2. 92 (dd, J = 13.3,

4.79 Hz, 1H), 2.71 (d, J = 12.7 Hz, 1H), 2.27 (t, J = 7.08 Hz, 2H), 1.62-1.56 (m, 4H), 1.5 (s,

9H), 0.92-0.80 (m, 2H). 13C NMR (100 MHz, CDCl3): 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. ESI-MS: m/z calcd for C27H36N8O9S [M+Na+], 671.23;

found 671.30.


Compound 7 (550 mg, 0.850 mmol) was dissolved in a mixture of 8.00 mL CH2Cl2. 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). 1H NMR (300 MHz, CDCl3): δ 8.22 (d, J = 8.68 Hz, 1H), 8.04 (d, J =

1.77 Hz, 1H), 7.98 (s, 1H), 7.91 (dd, J = 8.68, 1.77 Hz, 1H), 5.49 (s, 2H), 4.70 (t, J = 4.61 Hz,

2H), 4.56-4.49 (m, 3H), 4.35-4.29(m, 1H), 3.21-3.11 (m, 1H), 2.92 (dd, J = 12.7, 4.96 Hz,

1H), 2.72 (d, J = 12.7 Hz, 1H), 2.30 (t, J = 6.91 Hz, 2H), 1.69-1.50 (m, 4H), 1.45-1.32 (m,

2H). 13C NMR (100 MHz, CDCl3): 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. ESI-

MS: m/z calcd for C22H28N8O7S [M+Na+], 571.18; found 571.25.


Doxorubicin·HCl (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). 1H NMR (300 MHz, DMSO-d6):

δ 8.28-8.20 (m, 1H), 7.94-7.89 (m, 2H), 7.88-7.85 (m, 1H), 7.83-7.77 (m, 2H), 7.61-7.53 (m,

S7

1H), 5.55 (s, 1H), 5.43 (d, J = 9.16 Hz, 2H), 5.25 (t, J = 13.9 Hz, 2H), 4.58 (br, 2H), 4.49-

4.43 (br, 1H), 4.39-4.31 (m, 1H), 4.28-4.22 (m, 2H), 4.07 (br, 1H), 3.93 (s, 3H), 3.08-2.93 (m,

4H), 2.76 (br, 2H), 2.23-2.10 (m, 4H), 1.91-1.80 (m, 2H), 1.75-1.65 (m, 2H), 1.57-1.49 (m,

2H), 1.43-1.31 (m, 4H), 1.25-1.13 (m, 4H), 1.04 (s, 3H). 13C NMR (100 MHz, DMSO-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. ESI-MS: m/z calcd for C49H55N9O17S [M+H+] 1074.34, [M+Na+] 1096.

34; found 1074. 40, 1096.40.

S8

H-NMR, 13C-NMR and MS Analyses:

SpinWorks 2.5: STANDARD 1H OBSERVE

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

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

file: D:\nmr\Bio-Azi.fid\fid block# 1 expt: "s2pul"transmitter freq.: 300.176336 MHztime domain size: 17984 pointswidth: 4500.45 Hz = 14.992688 ppm = 0.250247 Hz/ptnumber of scans: 16

freq. of 0 ppm: 300.174854 MHzprocessed size: 32768 complex pointsLB: 0.000 GB: 0.0000

HN

NH

S

O

O

ON3

Fig. S1 1H NMR spectrum of compound 2 in CDCl3.

SpinWorks 2.5: Std proton

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

173

.561

5

163

.824

1

63.

1274

62.

1466

60.

3156

55.

6302

49.

9976

40.

7910

33.

9176

28.

5274

28.

4403

24.

8738

file: C:\Users\sws\Desktop\NMR\13C biotin-azide.fid\fid block# 1 expt: "s2pul"transmitter freq.: 100.572632 MHztime domain size: 63750 pointswidth: 24509.80 Hz = 243.702520 ppm = 0.384468 Hz/ptnumber of scans: 1260


HN

NH

S

O

O

ON3

Fig. S2 13C NMR spectrum of compound 2 in CDCl3.

S9

Fig. S3 Mass spectrum of compound 2.


PPM 10.0 9.0 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0

1.02

5

0.69

1

0.99

7

0.89

9

9.22

6

11.2

741

7.95

62 7

.9272

7.78

58 7

.6837

7.67

94 7

.6764

7.65

46 7

.6473

7.26

40

1.64

42

-0.0

000

file: C:\Users\a\Desktop\소소 \NMR data\140826-step1-new.fid\fid block# 1 expt: "s2pul"transmitter freq.: 300.176336 MHztime domain size: 17984 pointswidth: 4500.45 Hz = 14.992688 ppm = 0.250247 Hz/ptnumber of scans: 60



S

NHHN

O

O

ON3

O

O

OHO2N

S10


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

2.29

2

0.99

9

10.2

5

1.08

9

1.32

6

1.05

3

7.95

20 7

.9455

7.90

13 7

.8948

7.87

33 7

.8667

7.83

02 7

.8021

7.26

41

4.87

22 4

.8642

2.60

89 2

.6009

2.59

29

1.60

34

0.00

00





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

164.1

025

156.8

678

150.1

702

131.8

467

129.3

167

116.2

350

109.2

141

83.0

641

77.5

666

77.3

470

77.2

487

77.1

365

76.9

306

57.4

167

28.3

521

file: C:\Users\a\Desktop\소소 \NMR data\140902-sy-step2-13C.fid\fid block# 1 expt: "s2pul"transmitter freq.: 100.572632 MHztime domain size: 63750 pointswidth: 24509.80 Hz = 243.702520 ppm = 0.384468 Hz/ptnumber of scans: 2368



O

O

OO2N

O

O

OO2N

S11


PPM 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

3.13

0

2.07

9

1.24

4

7.97

94 7

.9735

7.93

07 7

.9140

7.90

82

7.28

29

4.91

09 4

.9029

3.46

38

2.63

99 2

.6317

2.62

42

0.00

00 -

0.001

1




PPM 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 3

.130

2.07

9

1.24

4

7.97

94 7

.9735

7.93

07 7

.9140

7.90

82

7.28

29

4.91

09 4

.9029

3.46

38

2.63

99 2

.6317

2.62

42

0.00

00 -

0.001

1




PPM 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

3.13

0

2.07

9

1.24

4

7.97

94 7

.9735

7.93

07 7

.9140

7.90

82

7.28

29

4.91

09 4

.9029

3.46

38

2.63

99 2

.6317

2.62

42

0.00

00 -

0.001

1




PPM 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

3.13

0

2.07

9

1.24

4

7.97

94 7

.9735

7.93

07 7

.9140

7.90

82

7.28

29

4.91

09 4

.9029

3.46

38

2.63

99 2

.6317

2.62

42

0.00

00 -

0.001

1


freq. of 0 ppm: 300.174847 MHzprocessed size: 32768 complex pointsLB: 0.000 GB: 0.0000Fig. S7 1H NMR spectrum of compound 5 in CDCl3.


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

169.8

044

154.4

700

147.4

403

139.2

189

129.5

908

116.8

444

109.0

999

79.7

560

79.4

172

57.0

519

file: C:\Users\a\Desktop\소소 \NMR data\140917-step3-13C.fid\fid block# 1 expt: "s2pul"transmitter freq.: 100.573110 MHztime domain size: 63750 pointswidth: 24509.80 Hz = 243.701362 ppm = 0.384468 Hz/ptnumber of scans: 896



OH

O

OO2N

OH

O

OO2N

S12



PPM 9.0 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0

0.99

9

2.16

6

2.17

3

1.11

8

0.94

2

9.22

0

1.13

8

9.36

79 9

.3661

8.40

28 8

.3745

8.01

22 8

.0065

7.99

67 7

.9899

7.96

84 7

.9616

7.26

89

6.98

18

5.01

91 5

.0111

2.70

96 2

.7017

2.69

36

1.51

09

-0.0

000

file: C:\Users\a\Desktop\소소 \NMR data\140918-step4.fid\fid block# 1 expt: "s2pul"transmitter freq.: 300.176336 MHztime domain size: 17984 pointswidth: 4500.45 Hz = 14.992688 ppm = 0.250247 Hz/ptnumber of scans: 80


OH

O

OO2N

NH

O

OO2N

HN

O

O

S13


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

162.1

084

155.6

922

155.0

808

150.7

500

134.0

769

125.6

464

117.0

542

108.5

550

82.4

104

78.8

109

77.5

683

77.2

503

76.9

331

76.0

481

57.8

407

28.3

790


freq. of 0 ppm: 100.562073 MHzprocessed size: 65536 complex pointsLB: 0.000 GB: 0.0000Fig. S11 13C NMR spectrum of compound 6 in CDCl3.


NH

O

OO2N

HN

O

O

NH

O

OO2N

HN

O

O

S14


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

2.39

0

2.35

6

1.10

0

0.90

0

1.99

4

1.04

1

1.48

4

1.20

6

3.45

3

1.26

5

1.25

8

1.43

7

1.38

3

9.08

4

4.54

3

2.11

0

8.23

91 8

.2104

8.05

82 7

.9423

7.93

61 7

.9136

7.90

75

6.37

89

5.57

04 5

.5613

5.19

51 4

.6597

4.65

80 4

.6563

4.64

21 4

.5083

4.50

33 4

.5027

4.50

08 4

.4982

4.49

01

4.33

96 4

.3284

4.32

64 4

.3245

4.32

27

3.15

40 3

.1519

3.13

86

2.94

67 2

.9304

2.90

33 2

.8876

2.73

32 2

.6906

2.28

89 2

.2650

2.23

96

1.61

48 1

.6127

1.61

06 1

.6082

1.60

62 1

.6018

1.59

11 1

.5678

1.56

61 1

.5627

1.54

94 1

.5038

0.88

45 0

.8824

0.88

12 0

.8798

0.87

74 0

.8569

0.85

46 0

.8526

0.00

11




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

173.3

027

164.4

112

163.5

097

156.4

913

155.8

075

150.7

180

142.4

435

133.3

279

126.5

932

124.8

475

116.7

208

108.9

569

81.7

998

77.6

150

77.2

969

76.9

785

64.2

654

62.2

657

62.1

164

60.3

583

55.8

649

49.6

679

40.7

796

33.5

705

29.8

990

28.4

416

28.3

440

28.2

650

24.7

692



NH

O

OO2N

HN

O

O

N NN

O

O

S

NH

NH

O

NH

O

OO2N

HN

O

O

N NN

O

O

S

NH

NH

O

S15



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

0.93

8

0.99

4

0.77

5

0.88

5

1.53

9

2.00

2

3.15

8

1.10

6

2.37

9

1.78

3

1.46

8

2.40

2

1.78

7

4.15

3

4.23

2

8.22

32 8

.1948

8.03

10 8

.0248

7.98

45 7

.9291

7.92

30 7

.9007

7.89

44

7.32

98

6.22

05

6.04

49

5.48

81

5.32

26

4.70

66 4

.6919

4.67

47 4

.5337

4.51

91 4

.5039

4.32

54 4

.3106

4.29

96 4

.2845

3.16

33 3

.1458

3.13

64 3

.1304

3.12

14 3

.1199

2.92

73 2

.9111

2.88

41 2

.8681

2.72

72 2

.6850

2.30

63 2

.2826

2.25

87

2.08

15 2

.0512

2.03

40

1.60

01 1

.5548

1.54

97 1

.5394

1.53

03 1

.5260

1.52

20 1

.5197

1.51

78

1.37

97 1

.3777

1.36

43 1

.3626

1.36

06 1

.3567

1.35

49 1

.3536

1.34

81 1

.3451

1.32

87

0.00

07



NH

O

OO2N

HN

O

O

N NN

O

O

S

NH

NH

O

NH

O

OO2N

NH2

N NN

O

O

S

NH

NH

O

S16


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

173.3

670

164.7

831

164.4

853

156.2

922

150.4

520

142.0

927

133.0

858

126.5

093

124.5

031

116.5

704

108.4

388

63.1

181

62.2

011

60.3

155

55.8

153

49.7

176

40.5

070

33.5

381

29.8

317

28.3

944

28.2

100

24.6

397




NH

O

OO2N

NH2

N NN

O

O

S

NH

NH

O

NH

O

OO2N

NH2

N NN

O

O

S

NH

NH

O

S17


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

3.23

1

2.24

0

2.40

0

2.42

8

3.07

0

1.43

1

1.56

4

2.56

3

2.52

8

1.29

4

1.76

8

0.97

0

2.00

6

0.54

4

0.56

6

1.92

8

1.99

2

1.44

9

1.33

9

1.15

9

4.62

7

4.75

9

2.65

2

2.35

8

2.50

2

4.51

8

8.26

25 8

.2605

8.21

28 8

.2112

7.90

99 7

.9083

7.90

68 7

.9038

7.87

45 7

.8729

7.87

08 7

.8568

7.85

54 7

.8539

7.85

25 7

.8508

7.81

35 7

.8119

7.81

03 7

.8090

7.59

26 7

.5909

7.58

30 7

.5817

7.57

99 7

.5785

7.57

55 7

.5736

7.57

18 7

.5694

7.56

79 7

.5665

7.56

49 7

.5630

6.15

09

5.92

41

5.61

80 5

.6168

5.61

51 5

.5546

5.55

31 5

.4667

5.46

57 5

.4641

5.46

30 5

.4606

5.45

94 5

.4571

5.45

55 5

.4531

5.45

16 5

.4507

5.44

85 5

.4458

5.44

44 5

.4422

5.40

50 5

.4027

5.28

87 5

.2875

5.28

62 5

.2848

5.28

38 5

.2826

5.28

13 5

.2789

5.27

63 5

.2581

5.25

69 5

.2546

5.25

28 5

.2518

5.24

97 5

.2482

5.24

69 5

.2425

5.24

14 5

.2396

5.21

86 5

.2166

4.59

15 4

.5896

4.55

20 4

.5506

4.54

87 4

.4626

4.45

75 4

.4560

4.45

34 4

.4522

4.45

10 4

.3518

4.35

05 4

.3493

4.34

79 4

.2538

4.07

68 4

.0746

4.07

30

3.93

23

3.00

72 3

.0054

2.77

22 2

.7708

2.76

94 2

.7559

2.75

33 2

.7519

2.74

99 2

.7453

2.74

34 2

.7420

2.74

06

2.20

34 2

.2014

2.20

02 2

.1977

2.19

61 2

.1910

2.18

63 2

.1851

2.18

36 2

.1828

2.18

11 2

.1744

2.17

29 2

.1702

2.16

88 2

.1669

2.16

54 2

.1643

2.13

50 2

.1307

2.12

97 2

.1281

1.87

10 1

.8696

1.86

71 1

.8662

1.86

46 1

.8634

1.86

06 1

.8598

1.85

82

1.70

35 1

.7018

1.70

01 1

.6981

1.69

68 1

.6954

1.69

22 1

.6895

1.53

54 1

.5343

1.51

94 1

.5165

1.51

41 1

.5132

1.51

06 1

.5091

1.50

78 1

.5068

1.50

54 1

.5038

1.50

20 1

.5006

1.49

87 1

.4970

1.37

15 1

.3703

1.36

91 1

.3678

1.36

67 1

.1717

1.03

84

file: C:\Users\a\Desktop\소소 \NMR data\141103-final-proton.fid\fid block# 1 expt: "s2pul"transmitter freq.: 399.932783 MHztime domain size: 26264 pointswidth: 6410.26 Hz = 16.028334 ppm = 0.244070 Hz/ptnumber of scans: 56

freq. of 0 ppm: 399.930384 MHzprocessed size: 65536 complex pointsLB: 0.000 GB: 0.0000Fig. S19 1H NMR spectrum of prodrug 9 in DMSO-d6.


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

187.0

488

186.9

778

173.1

630

163.4

399

161.4

209

159.9

734

158.5

634

156.4

049

155.1

478

150.4

793

142.7

298

136.8

722

136.4

742

135.9

941

135.8

374

135.3

078

133.4

316

128.1

108

126.3

700

120.5

307

119.6

421

116.5

956

111.2

340

111.1

244

100.0

501

99.9

762

72.4

681

71.9

148

66.8

966

66.7

705

65.5

922

62.7

007

61.6

829

59.8

673

57.2

411

56.0

094

49.3

594

47.2

114

33.6

874

28.5

928

24.9

570

15.8

310

file: C:\Users\a\Desktop\소소 \NMR data\141104-final-13c.fid\fid block# 1 expt: "s2pul"transmitter freq.: 100.573110 MHztime domain size: 63750 pointswidth: 24509.80 Hz = 243.701362 ppm = 0.384468 Hz/ptnumber of scans: 20672

freq. of 0 ppm: 100.562551 MHzprocessed size: 65536 complex pointsLB: 0.000 GB: 0.0000Fig. S20 13C NMR spectrum of prodrug 9 in DMSO-d6.

NH

O

OO2N

N

N NN

O

O

S

NH

NH

O

OH

OHOH

OO

O

OHOO

HONH2

NH

O

OO2N

N

N NN

O

O

S

NH

NH

O

OH

OHOH

OO

O

OHOO

HONH2

S18

Fig. S21 Mass spectrum of prodrug 9.

NH

O

OO2N

N

N NN

O

O

S

NH

NH

O

OH

OHOH

OO

O

OHOO

HONH2

S19

Fig. S22 Mass spectra of prodrug 9 (5 at pH 5.0 after 3 days at 37℃. Mass peak of 𝜇𝑀)compound 8 shown at [M+K+] and mass peak of Doxorubicin shown at [M+Na+] (graph = cationic measurement).

O NO2HN

O

H2N

NNN

HN

HN

S

OO

O

Chemical Formula: C22H28N8O7SMolecular Weight: 548.57

OOH

OOH

OH

O

O

O O

OHNH2

OH

Chemical Formula: C27H29NO11Molecular Weight: 543.52

Dox + Na+

Compound 8 + K+