1
Supporting Information
Synthesis and Εvaluation of Αnticancer Αctivity in Cells of
Novel Water Soluble Stoichiometric Pegylated FullereneDoxorubicin Conjugates
George E. Magoulasa, Marina Bantzib, Danai Messarib, Chrisostomi Gialelic,
Despoina Barbouric, Athanassios Giannisd, Nikolaos Karamanosc, Dionissios
Papaioannoua, and Konstantinos Avgoustakis*b
Konstantinos Avgoustakis: avgoust@upatras.gr
a
Laboratory of Synthetic Organic Chemistry, Department of Chemistry, University of
Patras, 26504 Patras, Greece
b
c
Department of Pharmacy, University of Patras, 26504 Patras, Greece
Laboratory of Biochemistry, Department of Chemistry, University of Patras, 26504
Patras, Greece
d
Institut für Organische Chemie, Fakultät für Chemie und Mineralogie, Universität
Leipzig, Johannisallee 29, 04103 Leipzig, Germany
Contents
Extended structures of conjugates 7a, 7b and 8 ........................................................................ 2
Experimental procedures ........................................................................................................... 2
UV-Vis measurements .............................................................................................................. 5
Thermogravimetric analysis (TGA) .......................................................................................... 6
1
H-NMR spectra of conjugates 7a, 7b and 8 ............................................................................. 8
Effect of C60-PEG construct 26 on MCF-7 cells ....................................................................... 9
Solubilities in water of selected intermediates and final products ............................................ 9
An example of a C60-DOX conjugate (A) which is not soluble in water in the absence of a
PEG moiety ............................................................................................................................. 10
References ............................................................................................................................... 10
2
Extended structures of conjugates 7a, 7b and 8
O
O
OH
OH
O
O
O
OH
O
OH
O
OH
OH
O
HO
HO
O
O
O
O
HO NH
HO NH
O
O
O
O
O
O
OMe
O
OMe
O
O
N
O
O
N
H
O
OMe
N
O
N
H
n
7a
O
OMe
n
7b
HO
O
OH
HO
O
O
OH O
O
N
N
O
O
N
NH
OH
O
OMe
O
N
O
O
O
HO
OH
O
O
NH
OH
O
OH
O
HO
O
N
O
N
H
O
OMe
O
n
8
O
Experimental procedures
3-(2-(-2-Hydroxyethoxy)ethoxy)-4-methoxybenzaldehyde (11)  To an ice-cold
solution of isovanillin 10 (0.76 g, 5.0 mmol), diethylene glycol (0.95 ml, 10 mmol)
and Ph3P (2.62 g, 10 mmol) in THF (10 ml), DIAD (1.94 ml, 10 mmol) was added
dropwise over 10 min. The reaction mixture was stirred at 0 oC for 15 min and for 1h
at ambient temperature. Then, it was evaporated to dryness and subjected directly to
FCC using gradient elution 30-70 % EtOAc in PhMe.
3
Yield: 0.67 g (56%); colorless oil; Rf (PhMe/EtOAc 3:7 v/v): 0.10; IR (thin film,
CHCl3, cm-1): 3418, 2936, 2876, 1681, 1586, 1514, 1436, 1270, 1138, 1072, 1020,
724; MS (ESI, 30eV): m/z 279.36 [M+K], 263.37 [M+Na], 241.41 [M+H]; 1H NMR
(d6-DMSO): δ 9.84 (s, 1H), 7.54 (dd, J = 2.0 and 8.4 Hz, 1H), 7.40 (d, J = 2.0 Hz,
1H), 7.19 (d, J = 8.0 Hz, 1H), 4.62 (distorted t, J = 5.6 Hz, 1H), 4.22-4.18 (m, 2H),
3.79 (s, 3H), 3.78-3.76 (m, 2H), 3.54-3.47 (m, 2H);
13
C NMR (d6-DMSO): δ 191.8,
153.9, 149.7, 130.2, 126.5, 112.6, 110.1, 72.9, 69.1, 68.6, 60.7, 56.0.
4-Methoxy-3-(prop-2-yn-1-yloxy)benzaldehyde (12)[S1]  To a suspension of
isovanillin 10 (0.76 g, 5.0 mmol) and K2CO3 (2.76 g, 20 mmol) in acetone (25 ml),
propargyl chloride (1.08 ml, 15 mmol) was added. The resulting mixture was refluxed
for 5h, then evaporated to dryness and the resulting residue was partitioned between
EtOAc and water. The aqueous phase was re-extracted thrice with EtOAc and the
combined organic phases were dried over Na2SO4 and evaporated to dryness. Pure
aldehyde 12 was obtained after FCC purification.
Yield: 0.70 g (74%); white solid, m.p: 66-67 oC; Rf (PhMe/EtOAc 95:5 v/v): 0.28; IR
(KBr, cm-1): 3232, 2972, 2838, 2125, 1674, 1600, 1586, 1510, 1438, 1264, 1138,
1014, 812, 758; MS (ESI, 30eV): m/z 213.52 [M+Na]; 1H NMR (CDCl3): δ 9.86 (s,
1H), 7.55 (d, J = 2.0 Hz, 1H), 7.52 (dd, J = 2.0 and 8.0 Hz, 1H), 7.00 (d, J = 8.0 Hz,
1H), 4.83 (d, J = 2.4 Hz, 1H), 3.97 (s, 3H), 2.54 (t, J = 2.4 Hz, 1H);
13
C NMR
(CDCl3): δ 190.7, 154.9, 147.3, 129.9, 127.4, 111.9, 110.9, 77.7, 76.5, 56.6, 56.2.
Benzyl 2-(2-tert-butoxy-2-oxoethylamino)acetate (14) [S2] To an ice-cold solution
of benzyl ester 13 (1.00 g, 5.0mmol) in DMF (7 ml), iPr2NEt (2.18 ml, 12.5 mmol)
was added and the resulting solution was stirred for 15 min. Then, tert-butyl
bromoacetate (0.81 ml, 5.5 mmol) was added and the reaction mixture was stirred at
ambient temperature for 12 h and diluted with EtOAc. The organic phase was washed
twice with a 5% aq. solution of NaHCO3, several times with water, dried over Na2SO4
and evaporated to dryness. Pure diester 14 was obtained after FCC purification.
Yield: 0.85 g (61%); colorless oil; Rf (PhMe/EtOAc 1:1 v/v): 0.25; IR (thin film,
CHCl3, cm-1): 1742, 1728, 1674, 1426, 1147, 741; MS (ESI, 30eV): m/z 559.39
[2M+H], 302.26 [M+Na], 280.24 [M+H]; 1H NMR (CDCl3): δ 7.30-7.19 (m, 5H),
5.08 (s, 2H), 3.41 (s, 2H), 3.26 (s, 2H), 1.99 (br. s, 1H), 1.37 (s, 9H);
13
CNMR
4
(CDCl3): δ171.6,170.9, 135.5, 128.5 (two C), 128.3, 128.2 (two C), 81.3, 66.5, 50.8,
50.1, 28.0 (three C).
2-(2-tert-Butoxy-2-oxoethylamino)acetic acid (15) [S2]  To a solution of diester 14
(0.8 g, 2.86 mmol) in MeOH (15 ml), 10% Pd/C (80 mg) was added and the mixture
was hydrogenated for 1 h. Then, the mixture was filtered through a celite pad, the
filtrate was evaporated to dryness and the resulting residue was triturated with Et2O.
The precipitate was filtered and dried.
Yield: 0.50 g (93%); white solid, m.p: 158-161 oC; Rf (EtOAc/n-BuOH/gl.AcOH/H2O
5:3:1:1 v/v/v/v): 0.29; IR (KBr, cm-1): 3446, 2976, 3100-2700, 1748, 1634, 1584,
1398, 1368, 1310, 1260, 1222, 1152, 1094; MS (ESI, 30eV): m/z 417.36 [2M+K],
401.36 [2M+Na], 228.41 [M+K], 212.45 [M+Na], 190.47 [M+H], 134.47 [(M+H)CH2=CMe2]; 1H NMR (d6-DMSO): δ 3.37 (s, 2H), 3.23 (s, 2H), 1.42 (s, 9H);
13
C
NMR (d6-DMSO): δ 171.9, 170.3, 81.2, 49.9, 49.8, 28.2 (three C).
4-(Azidomethyl)benzoic acid (23)[S3]  To a solution of bromide 22 (1.07 g, 5
mmol) in DMF (5 ml), NaN3 (0.38 g, 5.5 mmol) was added and the reaction mixture
was heated at 60 oC for 3 h. The reaction mixture was allowed to attain ambient
temperature and water (25 ml) was added. The resulting white precipitate was filtered
and dried under vacuo to afford acid 23.
Yield: 0.78 g (88%); white solid, m.p: 128-130oC; Rf (PhMe/EtOAc1:1 v/v): 0.28; IR
(KBr, cm-1): 3300-2500, 2110, 1682, 1610, 1426, 1294, 1180, 754; MS (ESI, 30eV):
m/z 393.22 [2M+K], 377.16 [2M+Na], 355.20 [2M+H], 216.19 [M+K], 178.18
[M+H]; 1H NMR (d6-DMSO): δ 13.01 (br.s, 1H), 7.96 (d, J = 8.0 Hz, 2H), 7.48 (d, J
= 8.0 Hz, 2H), 4.56 (s, 2H); 13C NMR (d6-DMSO): δ 167.5, 141.1, 131.0, 130.2 (two
C), 128.8 (two C), 53.6.
4-(Azidomethyl)-N,N-bis(2-hydroxyethyl)benzamide (24)  To an ice-cold
solution of acid 23 (0.5 g, 2.8mmol) and diethanolamine (0.35 g, 3.36 mmol) in DMF
(3 ml), Et3N (1.17 ml, 8.4 mmol) and HBTU (1.48 g, 3.9 mmol) were added
sequentially. The reaction mixture was stirred at ambient temperature for 1 h and then
diluted with EtOAc. The organic phase was washed once with a 5% aq. solution of
NaHCO3 and twice with water, dried over Na2SO4 and evaporated to dryness. Pure
amide 24 was obtained after FCC purification.
5
Yield: 0.57 g (77%); colorless oil; Rf (CHCl3/MeOH 9:1 v/v): 0.22; IR (thin film
CHCl3, cm-1): 3316, 2934, 2870, 2100, 1610, 1458, 1240, 1068, 843; MS (ESI,
30eV): m/z 551.40 [2M+Na], 287.63 [M+Na], 265.58 [M+H]; 1H NMR (CDCl3): δ
7.48 (d, J = 8.4 Hz, 2H), 7.32 (d, J = 8.4 Hz, 2H), 4.34 (s, 2H), 3.87 (unresolved t,
2H), 3.67-3.60 (m, 4H), 3.38 (unresolved t, 2H);
C NMR (CDCl3): δ 173.3, 137.1,
13
136.1, 128.2 (two C), 127.8 (two C), 60.4, 60.0, 54.3, 53.3, 49.4.
((4-(Azidomethyl)benzoyl)azanediyl)bis(ethane-2,1-diyl)
bis(4-nitrophenyl)
dicarbonate (25) To an ice-cold solution of amide 24 (0.5 g, 1.89 mmol) and Et3N
(2.0 ml, 14.25 mmol) in THF (60 ml), a solution of 4-nitrophenyl chloroformate (1.15
g, 5.7 mmol) in THF (30 ml) was added dropwise over 1 h. The reaction mixture was
further stirred at ambient temperature overnight. The resulting precipitate was filtered
off and the filtrate was evaporated and subjected to FCC to give pure compound 25.
Yield: 0.69 g (61%); light yellow oil; Rf (PhMe/EtOAc 8:2 v/v): 0.17; IR (thin film
CHCl3, cm-1): 2102, 1766, 1638, 1594, 1524, 1212, 858, 758; MS (ESI, 30eV): m/z
633.14 [M+K], 617.16 [M+Na], 595.28 [M+H]; 1H NMR (CDCl3): δ 8.27 (d, J = 8.8
Hz, 4H), 7.48 (d, J = 8.0 Hz, 2H), 7.40 (d, J = 8.8 Hz, 4H), 7.39-7.35 (m, 2H), 4.684.57 (m, 2H), 4.42-4.35 (m, 2H), 4.38 (s, 2H), 4.04-3.94 (m, 2H), 3.85-3.74 (m, 2H);
C NMR (CDCl3): δ 172.4, 155.2 (two C), 152.3 (four C), 145.5 (two C), 137.4,
13
135.4, 128.3 (two C), 127.2 (two C), 125.3 (four C), 121.7 (two C), 66.8, 66.0, 54.1,
48.5, 44.8.
UV-Vis measurements
Four solutions of identical concentration (c = 2.48 x 10-5 M) of DOX·HCl, C60-adduct
26, conjugate 7b and conjugate 8 in water were prepared and their absorbance was
measured (Figure S1A). Conjugates 7b and 8 (green and purple lines) showed an
absorbance peak at around 480 nm which is characteristic for the doxorubicin moiety
(blue line). A comparison of the curves for conjugates 7b and 8 (Figure S1B), shows
that conjugate 8 presented an absorption value almost twice as high compared to the
absorption value of conjugate 7b at the same concentration. Obviously, the absorption
6
of intermediate 26 (red line) at that area is responsible for the declined pattern
observed in both conjugates. Thus, these results offer a solid indication that conjugate
8 bears two DOX units attached per C60 molecule.
Figure S1. UV-Vis spectra of DOX (blue curve), intermediate 26 (red curve) and
conjugates 7b (green curve) and 8 (purple curve).
Thermogravimetric analysis (TGA)
As it can be seen from the TGA analysis (Figure S2A), pure DOX·HCl showed a
significant weight loss at the temperature range of 190-250 oC which is consistent
with the DOX.HCl melting point at 216 oC (decomp.). The same behavior was
observed at the thermograms of both conjugates 7b and 8 incorporating one and two,
respectively, covalently bound DOX molecules. Most importantly, a greater weight
loss was observed, at the afore mentioned temperature range, for conjugate 8 which
could be attributed to the fact that this compound bears two DOX units (Figure S2B).
Also, the TGA analysis clearly exhibits two degradation steps for the thermal
decomposition of conjugates 7b and 8 corresponding to the degradation of DOX and
PEG segments respectively.
7
Figure S2. TGA of C60 (black curve), MeO-PEG-NH2 (blue curve), intermediate 26
(green curve), conjugate 7b (pink curve), conjugate 8 (purple curve) and DOX·HCl
(red curve).
8
1H-NMR
spectra of conjugates 7a, 7b and 8
Figure S3. 1H-NMR spectra for conjugates 7a (A), 7b (B) and 8. Peaks at δ ca. 3.37
and 1.24 ppm correspond to the methoxy group of PEG-chain (single star) and the
methyl group of the sugar moiety of DOX (two stars), respectively.
9
Effect of C60-PEG construct 26 on MCF-7 cells
The antiproliferative effect of C60-PEG on MCF-7 cells, measured by the WST-1
assay, is shown in Figure S4.
Figure S4. Antiproliferative effect of C60-PEG on MCF-7 cells.
Solubilities in water of selected intermediates and final products
10
An example of a C60-DOX conjugate (A) which is not soluble in water in the
absence of a PEG moiety
References
[S1] Zammit SC, Cox AJ, Gow RM, Zhang Y, Gilbert RE, Krum H, Kelly DJ,
Williams SJ. Evaluation and optimization of antifibrotic activity of cinnamoyl
anthranilates. Bioorg. Med. Chem. Lett. 2009; 19(24): 7003-7006.
[S2] Biondi L, Giannini E, Filira F, Gobbo M, Negri L, Rocchi R. [D-Ala2]deltorphin I peptoid and retropeptoid analogues: synthesis, biological activity and
conformational investigations, J. Pept. Science 2004; 10(9): 578-587.
[S3] Di Antonio M, Biffi G, Mariani A, Raiber E-A, Rodriguez R, Balasubramanian
S. Selective RNA Versus DNA G-Quadruplex Targeting by In Situ Click Chemistry.
Angew. Chem. Int. Ed. 2012; 51(44): 11073-11078.