mPEG–CURCUMIN CONJUGATES WITH ANTICANCER ACTIVITY
5209
activated PEG succinimidyl ester. The yield was
94.3%; IR (KBr): νmax 2883, 1771, 1736, 1627, 1588,
1511, 1466, 1359, 1341, 1279, 1240, 1145, 1101, 1060,
IR (KBr): νmax 3500, 1626, 1601, 1504, 1427, 1261,
1026 cm−1; 1H NMR (500 MHz, CDCl3): δ 3.95 (6H, s),
5.80 (1H, s) 6.48 (2H, d, J = 15.70 Hz), 6.94 (2H, d, J
= 8.10 Hz), 7.05 (2H, d, J = 1.80 Hz), 7.13 (2H, dd, J =
8.13 and 1.80 Hz), 7.59 (2H, d, J = 15.70 Hz); 13C NMR
(125.76 MHz, CDCl3): *55.96, 101.16, 109.62, 114.82,
121.77, 122.86, 127.69, 140.50, 146.77, 147.83, 183.25;
HRMS calculated for C21H21O6 [M + H+]: 369.1338;
found 369.1335.
959, 841, 528 cm−1 1H NMR (CDCl3, 500 MHz) δ
;
2.06 (2H, m), 2.50 (2H, t, J = 7.18 Hz), 2.64 (2H, t,
J = 7.18 Hz), 3.35 (s, 3H), 3.45–3.70 (s + m, repeat
ethylene units), 3.75 (2H, t, J = 4.90 Hz), 3.84 (s,
3H), 3.92 (s, 3H), 4.23 (2H, t, J = 4.90 Hz), 5.81 (s,
1H), 6.47 (1H, d, J = 15.76 Hz), 6.53 (1H, d, J = 15.80
Hz), 6.90 (1H, d, J = 8.30 Hz,), 7.01 (1H, d, J = 8.30
Hz), 7.03 [1H, s(br)], 7.09 (1H, d, J = 1.84 Hz), 7.11
(1H, dd, J = 8.30 and 1.80 Hz), 7.12 (1H, dd, J = 8.30
and 1.80 Hz), 7.57 (1H, d, J = 15.78 Hz,), 7.58 (1H, d,
J = 15.82 Hz); 13C NMR (CDCl3, 125 MHz): δ 20.14,
29.95, 32.94, 55.86, 55.93, 58.98, 63.56, 69.09, 70.52
(repeat ethylene units), 71.89, 101.46, 109.71, 111.35,
114.92, 120.95, 121.72, 123.00, 123.18, 124.24,
127.47, 134.05, 139.35, 141.10, 146.88, 148.08,
151.31, 170.77, 172.83, 181.76, 184.48; Calculated
MW = 2684.061, MALDI–TOF MS = 2684.146.
Synthesis of mPEG2000–Curcumin Conjugates (2a–2c)
The
general
procedure
for
synthesis
of
mPEG2000–curcumin conjugates (2a–2c) is shown in
Scheme 1. An activated mPEG2000 succinimidyl ester
(mPEG2000–NHS, 3a–3c, 50 mg, 0.022 mmol) was
dissolved in dry CH2Cl2 (5 mL) and the solution was
added to a mixture of curcumin (1, 79 mg, 0.22 mmol)
and 4-(N,N-dimethylamino)pyridine (DMAP; 13 mg,
0.11 mmol) in CH2Cl2 (10 mL) in a round bottomed
flask. The reaction was monitored by TLC. The
mixture was stirred for 24 h at room temperature
until no mPEG2000–NHS was detected by TLC. After
the reaction was complete, glacial acetic acid (5 :L)
was added to neutralize DMAP. Subsequently, the
reaction mixture was dried on a rotary evaporator to
remove the excess of glacial acetic acid and CH2Cl2.
The residue was dissolved in methanol (2 mL),
loaded onto a Sephadex LH-20 column (60 × 2 cm),
and eluted with methanol. The clear methanolic
solution was evaporated to afford a bright yellow,
water-soluble solid product of mPEG2000–curcumin
conjugate.
mPEG2000–methylcarboxyl–curcumin (PMC, 2c):
mPEG2000–methylcarboxyl–NHS (3c) was used as
an activated PEG succinimidyl ester. The yield was
93.8% yield; IR (KBr): νmax 2883, 1779, 1628, 1589,
1511, 1466, 1359, 1341, 1279, 1240, 1145, 1100, 1060,
960, 841, 528 cm−1; 1H NMR (CDCl3, 500 MHz): *3.35
(3H, s), 3.45–3.75 (s + m, repeat ethylene units), 3.84
(3H, s), 3.92 (3H, s), 4.42 (2H, s), 5.81 (1H, s), 6.46 (1H,
d, J = 15.91 Hz), 6.53 (1H, d, J = 15.92 Hz), 6.90 (1H,
d, J = 8.30 Hz), 7.03 [1H, s (br)], 7.05 (1H, d, J = 8.30
Hz), 7.09 (1H, d, J = 1.80 Hz), 7.10 (1H, d, J = 8.30 and
1.80 Hz), 7.13 (1H, d, J = 8.30 and 1.80 Hz), 7.57 (1H,
d, J = 15.90 Hz), 7.58 (1H, d, J = 15.86 Hz); 13C NMR
(CDCl3, 125 MHz): * 55.68, 55.85, 58.94, 68.28, 70.47
(repeat ethylene units), 71.83, 101.46, 109.68, 111.34,
114.93, 120.86, 121.60, 122.98, 123.24, 124.32, 127.35,
134.21, 140.53, 146.90, 148.11, 151.14, 139.14, 141.13,
168.34, 181.57, 184.53; Calculated MW = 2671.023,
MALDI–TOF MS = 2670.875.
mPEG2000–succinyl–curcumin
(PSC,
2a):
mPEG2000–succinyl–NHS (3a) was used as an
activated PEG succinimidyl ester. The yield was
90.8%; IR (KBr): νmax 2883, 1771, 1736, 1627, 1588,
1511, 1466, 1359, 1341, 1279, 1240, 1204, 1144, 1100,
1060, 960, 841, 528 cm−1; 1H NMR (CDCl3, 500 MHz):
δ 2.76 (2H, t, J = 6.80Hz,), 2.90 (2H, t, J = 6.80Hz,),
3.35 (3H, s), 3.46–3.76 (repeat ethylene units, s + m),
3.68 (2H, t, J = 4.92 Hz,), 3.84 (3H, s), 3.92 (3H, s),
4.25 (2H, t, J = 4.92 Hz,), 5.81 (1H, s), 6.47 (1H, d,
J = 15.72 Hz), 6.53 (1H, d, J = 15.80 Hz), 6.91 (1H,
d, J = 8.31 Hz), 7.03 [1H, s(br)], 7.05 (1H, d, J = 8.33
Hz), 7.09 (1H, d, J = 1.80 Hz,), 7.11 (1H, dd, J = 8.31
and 1.80 Hz), 7.12 (1H, dd, J = 8.32 and 1.80 Hz),
7.57 (1H, d, J = 15.76 Hz), 7.58 (1H, d, J = 15.80
Hz,); 13C NMR (CDCl3, 125 MHz): δ 28.91, 29.06,
55.94, 58.99, 63.93, 69.02, 70.51 (repeat ethylene
units), 71.89, 101.47, 109.70, 111.43, 114.91, 120.92,
121.73, 123.01, 123.24, 124.26, 127.49, 134.08,
139.34, 141.11, 146.87, 148.06, 151.33, 170.22,
171.97, 181.75, 184.47; Calculated MW = 2646.574,
MALDI–TOF MS = 2646.858.
Chemical Stability of mPEG2000–Curcumin Conjugates
in a Buffer Solution
Stock solutions of curcumin in methanol and
mPEG2000–curcumin conjugates in water were pre-
pared at 40 :M. The stock solutions were diluted with
0.1 M potassium phosphate buffer (pH 7.4) to give a
final concentration of 4 :M. The solution was left to
stand at 37◦C for 24 h. The amount of curcumin or con-
jugate was determined at appropriate time intervals
using a previously reported HPLC method with some
modifications.46 Chromatography was performed us-
ing a gradient system with an autosampler temper-
ature of 15◦C, a column temperature of 33◦C, a flow
rate of 2.0 mL/min, and a detection wavelength of 400
nm. Gradient elution consisted of eluents A (2%, v/
v aqueous acetic acid) and B (acetonitrile). The elu-
tion program was optimized and conducted as follows:
mPEG2000–glutaryl–curcumin
(PGC,
2b):
mPEG2000–glutaryl–NHS (3b) was used as an
DOI 10.1002/jps
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 12, DECEMBER 2011