Biotin-guided anticancer drug delivery with acidity ... · Biotin-guided anticancer drug delivery...
Transcript of Biotin-guided anticancer drug delivery with acidity ... · Biotin-guided anticancer drug delivery...
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
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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.
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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.
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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·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.
Preparation and Characterization of 3
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).
Preparation and Characterization of 4
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,
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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.
Preparation and Characterization of 5
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.
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), 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.
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
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
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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.
Preparation and Characterization of 8
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.
Preparation and Characterization of 9
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,
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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
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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
freq. of 0 ppm: 100.562073 MHzprocessed size: 65536 complex pointsLB: 0.000 GB: 0.0000
HN
NH
S
O
O
ON3
Fig. S2 13C NMR spectrum of compound 2 in CDCl3.
S9
Fig. S3 Mass spectrum of compound 2.
SpinWorks 2.5: STANDARD 1H OBSERVE
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
freq. of 0 ppm: 300.174854 MHzprocessed size: 32768 complex pointsLB: 0.000 GB: 0.0000
Fig. S4 1H NMR spectrum of compound 3 in CDCl3.
S
NHHN
O
O
ON3
O
O
OHO2N
S10
SpinWorks 2.5: STANDARD 1H OBSERVE
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
file: C:\Users\a\Desktop\소소 \NMR data\140827-step2-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: 100
freq. of 0 ppm: 300.174853 MHzprocessed size: 32768 complex pointsLB: 0.000 GB: 0.0000
Fig. S5 1H NMR spectrum of compound 4 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 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
freq. of 0 ppm: 100.562073 MHzprocessed size: 65536 complex pointsLB: 0.000 GB: 0.0000
Fig. S6 13C NMR spectrum of compound 4 in CDCl3.
O
O
OO2N
O
O
OO2N
S11
SpinWorks 2.5: STANDARD 1H OBSERVE
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
file: C:\Users\a\Desktop\소소 \NMR data\140828-step3-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: 64
freq. of 0 ppm: 300.174847 MHzprocessed size: 32768 complex pointsLB: 0.000 GB: 0.0000
SpinWorks 2.5: STANDARD 1H OBSERVE
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
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9
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4
7.97
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07 7
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7.90
82
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29
4.91
09 4
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3.46
38
2.63
99 2
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2.62
42
0.00
00 -
0.001
1
file: C:\Users\a\Desktop\소소 \NMR data\140828-step3-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: 64
freq. of 0 ppm: 300.174847 MHzprocessed size: 32768 complex pointsLB: 0.000 GB: 0.0000
SpinWorks 2.5: STANDARD 1H OBSERVE
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
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3.46
38
2.63
99 2
.6317
2.62
42
0.00
00 -
0.001
1
file: C:\Users\a\Desktop\소소 \NMR data\140828-step3-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: 64
freq. of 0 ppm: 300.174847 MHzprocessed size: 32768 complex pointsLB: 0.000 GB: 0.0000
SpinWorks 2.5: STANDARD 1H OBSERVE
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
file: C:\Users\a\Desktop\소소 \NMR data\140828-step3-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: 64
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.
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 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
freq. of 0 ppm: 100.562551 MHzprocessed size: 65536 complex pointsLB: 0.000 GB: 0.0000
Fig. S8 13C NMR spectrum of compound 5 in CDCl3.
OH
O
OO2N
OH
O
OO2N
S12
Fig. S9 Mass spectrum of compound 5.
SpinWorks 2.5: STANDARD 1H OBSERVE
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
freq. of 0 ppm: 300.174852 MHzprocessed size: 32768 complex pointsLB: 0.000 GB: 0.0000Fig. S10 1H NMR spectrum of compound 6 in CDCl3.
OH
O
OO2N
NH
O
OO2N
HN
O
O
S13
SpinWorks 2.5: Std proton
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
file: C:\Users\a\Desktop\소소 \NMR data\140919-step4-13C.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: 2816
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.
Fig. S12 Mass spectrum of compound 6.
NH
O
OO2N
HN
O
O
NH
O
OO2N
HN
O
O
S14
SpinWorks 2.5: STANDARD 1H OBSERVE
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
file: C:\Users\a\Desktop\소소 \NMR data\140922-step5.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: 200
freq. of 0 ppm: 300.174852 MHzprocessed size: 32768 complex pointsLB: 0.000 GB: 0.0000Fig. S13 1H NMR spectrum of compound 7 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 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
file: C:\Users\a\Desktop\소소 \NMR data\140924-step5-13C.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: 832
freq. of 0 ppm: 100.562073 MHzprocessed size: 65536 complex pointsLB: 0.000 GB: 0.0000Fig. S14 13C NMR spectrum of compound 7 in CDCl3.
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
Fig. S15 Mass spectrum of compound 7.
SpinWorks 2.5: STANDARD 1H OBSERVE
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
file: C:\Users\a\Desktop\소소 \NMR data\140925-step6.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: 200
freq. of 0 ppm: 300.174833 MHzprocessed size: 32768 complex pointsLB: 0.000 GB: 0.0000Fig. S16 1H NMR spectrum of compound 8 in CDCl3.
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
SpinWorks 2.5: Std proton
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
file: C:\Users\a\Desktop\소소 \NMR data\140927-step6-13C.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: 17856
freq. of 0 ppm: 100.562073 MHzprocessed size: 65536 complex pointsLB: 0.000 GB: 0.0000Fig. S17 13C NMR spectrum of compound 8 in CDCl3.
Fig. S18 Mass spectrum of compound 8.
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
SpinWorks 2.5: Std proton
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.
SpinWorks 2.5: Std proton
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+