Synthesis, characterization and cytotoxicity studies of platinum(II) complexes with reduced amino acid ester Schiff-bases as ligands

Article abstract of DOI:10.1016/j.ica.2014.04.036

A series of platinum(II) complexes with reduced amino acid esters Schiff-bases as ligands were prepared as potential anticancer agents and characterized by NMR, IR spectroscopy, elemental analysis and molar conductivity. These compounds were tested for their stability and DNA interaction with salmon sperm DNA by ultraviolet spectrum. The cytotoxicities of these complexes were validated against HL-60, BGC-823, Hela, and HepG2 cell lines by the MTT test. The complexes 5e and 5f exhibited better cytotoxic activity than cisplatin against HepG2 and BGC-823 cell lines, respectively.

Full text of DOI:10.1016/j.ica.2014.04.036

Accepted Manuscript
Synthesis, characterization and cytotoxicity studies of platinum(II) complexes
with reduced amino acid ester Schiff-bases as ligands
Li-Jun Li, Bin Fu, Yu Qiao, Cheng Wang, Ying-Ying Huang, Chuan-Chuan Liu,
Chao Tian, Jian-Long Du
PII:
S0020-1693(14)00241-2
http://dx.doi.org/10.1016/j.ica.2014.04.036
ICA 15975
DOI:
Reference:
Inorganica Chimica Acta
To appear in:
Received Date:
Revised Date:
Accepted Date:
22 October 2013
18 April 2014
30 April 2014
Please cite this article as: L-J. Li, B. Fu, Y. Qiao, C. Wang, Y-Y. Huang, C-C. Liu, C. Tian, J-L. Du, Synthesis,
characterization and cytotoxicity studies of platinum(II) complexes with reduced amino acid ester Schiff-bases as
ligands, Inorganica Chimica Acta (2014), doi: http://dx.doi.org/10.1016/j.ica.2014.04.036
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Synthesis, characterization and cytotoxicity studies of platinum(II)
complexes with reduced amino acid ester Schiff-bases as ligands
Li-Jun Li*, Bin Fu ,Yu Qiao, Cheng Wang, Ying-Ying Huang, Chuan-Chuan Liu, Chao Tian,
Jian-Long Du*
Chemical Biology Key Laboratory of Hebei Province, Hebei University, Baoding 071002, P. R. China
Abstract: A series of platinum(II) complexes with reduced amino acid esters Schiff-bases as ligands
were prepared as potential anticancer agents and characterized by NMR, IR spectroscopy, elemental
analysis and molar conductivity. These compounds were tested for their stability and DNA interaction
with salmon sperm DNA by ultraviolet spectrum. The cytotoxicities of these complexes were validated
against HL-60, BGC-823, Hela, and HepG2 cell lines by the MTT test. The complexes 5e and 5f
exhibited better cytotoxic activity than cisplatin against HepG2 and BGC-823 cell lines, respectively.
Key words: platinum(II) complexes, synthesis, characterize, reduced amino acid esters
Schiff-bases, DNA interaction, antitumor activity
1. Introduction
Much interest in developing metal based drugs appeared in the mid-1960s [1]. As frequently used
anticancer agents, platinum containing compounds have a unique place in metal coordination chemistry
[2,3]. Almost five decades after Rosenberg's accidental discovery of cisplatin [4,5], the antitumor
activity of platinum based drugs still captures the attention of scientists worldwide, and new potential
drugs are being synthesized and investigated [6-10]. Despite its wide application as a chemotherapeutic
agent, cisplatin exhibits several main disadvantages: narrow range of activity, acquired resistance and
toxicity. These side effects limit the use of cisplatin in some cancers. Recently, great efforts have been
devoted to overcoming these main limits. Up to now, apart from cisplatin, metal-based anticancer
agents that are in worldwide clinical use include carboplatin and oxaliplatin [11].
Over the last few decades, transition metal complexes have been well studied for their application as
artificial nucleases, because of their diverse structural features and the possibility to tune their redox
potential through the choice of ligands [12-14]. Transition metal complexes containing Schiff base
ligands and their reduced products are often used as artificial chemical nucleases, these complexes have
proved to be efficient DNA cleavage reagents [15,16]. Amino acids, as the basic unit of proteins and
related enzymes, perform critical biological roles including neurotransmitters and transport in the body,
are one of the essential and indispensable nutrients in vivo. When they are introduced to the drug
molecules after esterified, the drug lipophilicity, toxicity and bioavailability will be improved. Most of
the model studies of the metal complexes of Schiff-base ligands bearing salicylaldehyde and amino
acid esters have focused on the binding mode of these ligands [17,18]. Structural studies on the metal
complexes of reduced Schiff-base ligands derived from various amino acid esters and salicylaldehyde,
are well documented [19,20]. Compared to the Schiff bases, the corresponding reduced Schiff bases are
expected to be more stable and adaptable to form conformationally flexible 5- or 6-membered rings
upon complexation as they are not constrained to be planar. Numerous papers on transition metal
complexes of reduced Schiff bases derived from amino acids or amino acid esters have been reported
[21,22], however, information on the corresponding derivatives of platinum(II) is still rare. Herein, a
series of platinum(II) complexes with reduced amino acid esters Schiff-bases as ligands were
synthesized and characterized, their interaction with salmon sperm DNA was investigated by
1

ultraviolet spectrum, and their antiproliferative activity against a panel of human tumor cell lines from
solid tumors including HL-60, BGC-823, Hela, and HepG2 cell lines has also been evaluated. It
appears possible that the mechanism of activity is very similar to that of cisplatin since the ester ligand
can be easily substituted by other ligands and thus there are two leaving groups.
2. Experimental
2.1. Materials
All reagents and chemicals were purchased from commercial sources and used as received.
Salicylaldehyde and K2PtCl4 were of Chemical Grade, L-amino acids were of analytical grade,
MTT and salmon sperm DNA were from Sigma. Four different human carcinoma cell lines:
HL-60 (immature granulocyte leukemia), Hela (human epithelial cancer), BGC-823 (human gastro
carcinoma) and HepG2 (human hepatocellular carcinoma) were obtained from American Type
Culture Collection.
2.2. Instrumentation and measurement
Elemental analysis were determined on a Exeter Analytical CE-440 elemental analyzer. The IR
1
spectra were recorded using KBr pellets and a Thermo Nicolet 380 spectrophotometer. The H
NMR spectra were recorded in DMSO-d6 on a Bruker AVIII 600 NMR spectrometer. The mass
spectra was measured by Bruker apex-ultra 7.0T. The interaction between DNA and complexes
was measured on UV-3400 Toshniwal spectrophotometer. Molar conductances were measured at
room temperature in 1×10-3 M methanol using a DDS-12DW type conductivity meter.
2.3. Synthesis of ligands
5-Chlorosalicylaldehyde
To a three-neck flask, equipped with a reflux condenser, thermometer, a nitrogen source and
magnetic stirrer was added parachlorophenol (16 mmol), sodium hydroxide (100 mmol), distilled water
(40 mL), chloroform (7.0 mL). The mixture was heated to reflux for 8 h. After cooling, the reaction
mixture was acidified to pH 2~3 with 2M hydrochloric acid, and extracted with ethyl acetate. The ethyl
acetate extract was treated with vacuum distillation, dried and obtained crude 5-chlorosalicylaldehyde.
The crude product was purified by column chromatography gave white solid product (2.158 g, 29.6 %):
1
mp: 90-91 ºC. IR (KBr, pellet) υ/cm-1: 3225, 1685, 1566, 1470, 1275, 1155, 885, 831, 650, 535. H
NMR (600M Hz, DMSO-d6), δ: 9.873 (s, 1 H, -CHO), 7.354-7.365 (m, 2H, Ar-H), 6.911-6.927 (d, 1H,
Ar-H).
5-Bromosalicylaldehyde
5-Bromosalicylaldehyde was synthesized according to a published procedure [23]. To a
three-neck flask, equipped with a thermometer and magnetic stirrer was added glacial acetic acid
(8 mL), salicylaldehyde (18.40 mmol), hydrobromic acid (6 mL). The mixture was heated to 35 ºC
for 90 minutes and NaClO3 was slowly added in dropwise, milky precipitate was then appeared.
The reaction mixture was then recrystallized from ethanol and the white crystalline solid was
collected by filtration and washed with a bit of ethanol. The product was dried to obtain
5-bromosalicylaldehyde (1.53 g, 42.9%): mp: 104-105 ºC. IR (KBr, pellet) υ/cm-1: 3445, 1669,
1
1563, 1465, 1273, 1159, 888, 828, 696, 533. H NMR (600M Hz, DMSO-d6), δ: 10.211 (s, 1 H,
2

-CHO), 6.986-7.732 (m, 3 H, Ar-H).
General procedure of ligands synthesis
Amino acid ester were synthesized according to a published procedure [24]. The synthesis of
reduced amino acid ester Schiff bases were according to one-pot. Amino acid ester (4.68 mmol) was
added to the round bottom flask containing dichloromethane (20 mL), then triethylamine (4.75 mmol)
and a certain amount of anhydrous magnesium sulfate were also added to the round bottom flask at
room temperature, stirring for 1 h. And then salicylaldehydethe (4.80 mmol) was added to the
dichloromethane solution with dropwise, stirring for another 36 h at room temperature. Methanol
solution (10 mL) containing sodium borohydride (7.02 mmol) was then added to the above solution,
stirred for 3 h. Rotary evaporation, wash three times. The reduced amino acid ester Schiff bases were
purified by column chromatography gave the white solid or colorless oil product.
Methyl 2-(2-hydroxybenzylamino)-4-methylpentanoate (4a)
1
Pale yellow oil. Yield 73.0%. H NMR (600 MHz, DMSO-d6): δ 7.057-7.102 (m, 2H, Ar-H),
6.716-6.744 (m, 2H, Ar-H), 3.791 (d, 1H, N-CH2), 3.636 (s, 3H, CH3), 3.611 (d, 1H, N-CH2), 3.259 (t,
1H, N-CH), 1.649-1.728 (m, 1H, CH), 1.391-1.497 (m, 2H, CH2), 0.870 (d, 3H, CH3), 0.826 (d, 3H,
CH3); 13C NMR (DMSO-d6) δ: 16.5 (C-3’, C-4’), 27.4 (C-2’), 42.1 (C-7), 50.3 (-OCH3), 65.4 (C-8),
115.6 (C-3), 120.7 (C-5), 123.9 (C-1), 128.1 (C-4), 129.0 (C-6), 156.8 (C-2), 173.1 (C-9). IR (KBr):
3316 (N-H), 2956, 1735 (C=O), 1586, 1254 (ph-O), 1150, 992, 840, 754 cm-1. Anal. Calc. for
C14H21NO3: C, 66.91; H, 8.42; N, 5.57. Found: C, 66.78; H,8.56; N, 5.46.
Methyl 2-(2-hydroxybenzylamino)propanoate (4b)
1
Pale yellow oil. Yield 33.6%. H NMR (600 MHz, DMSO-d6): δ 7.198 (t, 1H, Ar-H), 6.983 (d, 1H,
Ar-H), 6.871 (d, 1H, Ar-H), 6.800 (t, 1H, Ar-H), 4.101 (d, 1H, N-CH2), 3.813 (d, 1H, N-CH2), 3.792 (s,
3H, CH3) 3.467 (q, 1H, N-CH), 1.389 (d, 3H, CH3); 13C NMR (DMSO-d6) δ: 16.3 (C-5’), 47.5 (C-7),
50.2 (-OCH3), 57.8 (C-8), 116.3 (C-3), 121.2 (C-5), 124.6 (C-1), 129.2 (C-4), 130.6 (C-6), 157.4 (C-2),
172.8 (C-9). IR (KBr): 3430 (N-H), 2951, 1723 (C=O), 1569, 1256 (ph-O), 1160, 977, 864, 751 cm-1.
Anal. Calc. for C11H15NO3: C, 63.14; H, 7.23; N, 6.69. Found: C, 63.44; H, 7.51; N, 6.32.
Methyl 3-hydroxy-2-(2-hydroxybenzylamino)propanoate (4c)
Pale yellow oil. Yield 36.3%. 1H NMR (600 MHz, DMSO-d6): δ 7.104 (d, 1H, Ar-H), 7.071 (t, 1H,
Ar-H), 6.720-6.745 (m, 2H, Ar-H), 3.826 (d, 1H, N-CH2), 3.668 (d, 1H, N-CH2), 3.635 (s, 3H, CH3),
3.606 (d, 2H, CH2), 3.324 (t, 1H, N-CH); 13C NMR (DMSO-d6) δ: 43.2 (C-7), 51.4 (-OCH3), 64.5 (C-8),
65.8 (C-6’), 117.6 (C-3), 121.4 (C-5), 126.2 (C-1), 129.3 (C-4), 130.5 (C-6), 158.9 (C-2), 173.4 (C-9).
IR (KBr): 3426 (N-H), 2955, 1735 (C=O), 1569, 1257 (ph-O), 1174, 1042, 871, 757 cm-1. Anal. Calc.
for C11H15NO4: C, 58.66; H, 6.71; N, 6.22. Found: C,58.89; H, 6.45; N, 6.52.
Methyl 2-(2-hydroxybenzylamino)-3-(1H-indol-3-yl)propanoate (4d)
1
White solid. Yield 53.5%. mp 105-106 °C H NMR (600 MHz, DMSO-d6): δ 10.852 (s, 1H, NH),
7.452 (d, 1H, Ar-H), 7.336 (d, 1H, Ar-H), 7.121 (s, 1H, Ar-H), 7.046-7.078 (m, 3H, Ar-H), 6.971 (t, 1H,
Ar-H), 6.692-6.721 (m, 2H, Ar-H), 3.796 (d, 1H, N-CH2), 3.654 (d, 1H, N-CH2), 3.556 (s, 1H, N-CH),
3.545 (s, 3H, CH3), 3.028-3.095 (m, 2H, CH2), 2.836 (s, 1H, NH); 13C NMR (DMSO-d6) δ: 31.5
(C-15’), 42.8 (C-7), 50.6 (-OCH3), 64.7 (C-8), 111.6 (C-13’), 112.5 (C-8’), 115.7 (C-3), 119.8 (C-12’),
120.9 (C-5), 121.1 (C-10’), 121.9 (C-11’), 122.8 (C-7’),124.6 (C-1), 128.5 (C-4), 129.7 (C-6), 131.6
(C-9’), 136.4 (C-14’), 157.3 (C-2), 172.8 (C-9). IR (KBr): 3438 (N-H), 2955, 1739 (C=O), 1568, 1241
(ph-O), 1176, 1006, 868, 735 cm-1. Anal. Calc. for C19H20N2O3: C, 70.35; H, 6.21; N, 8.64. Found: C,
70.64; H, 5.95; N, 8.91.
Methyl 2-(5-bromo-2-hydroxybenzylamino)-3-phenylpropanoate (4e)
3

1
Pale yellow oil. Yield 57.0%. H NMR (600 MHz, DMSO-d6): δ 7.281 (t, 2H, Ar-H), 7.224 (dd, 2H,
Ar-H), 7.186(d, 3H, Ar-H), 6.676 (d, 1H, Ar-H), 3.709 (d, 1H, N-CH2), 3.584 (d, 1H, N-CH2), 3.556 (s,
3H, CH3), 3.463 (t, 1H, N-CH), 2.892 (dd, 2H, CH2); 13C NMR (DMSO-d6) δ: 39.1 (C-21’), 47.7
(C-7), 51.7 (-OCH3), 62.4 (C-8), 110.8 (C-5), 117.9 (C-3), 126.8 (C-18’), 127.8 (C-1), 128.7
(C-20’, C-16’), 129.8 (C-19’, C-17’), 130.3 (C-4), 131.6 (C-6), 137.9 (C-15’), 156.5 (C-2), 174.4
(C-9). IR (KBr): 3431 (N-H), 2953, 1725 (C=O), 1578, 1259 (ph-O), 1179, 1073, 867, 748 cm-1. Anal.
Calc. for C17H18BrNO3: C, 56.06; H, 4.98; N, 3.85. Found: C, 56.35; H, 4.82; N, 3.97.
Methyl 2-(5-chloro-2-hydroxybenzylamino)-4-methylpentanoate (4f)
1
Pale yellow oil. Yield 87.6%. H NMR (600 MHz, DMSO-d6): δ 7.203 (d, 1H, Ar-H), 7.087 (dd, 1H,
Ar-H), 6.746 (d, 1H, Ar-H), 3.732 (d, 1H, N-CH2), 3.625 (s, 3H, CH3), 3.580 (d, 1H, N-CH2), 3.237 (t,
1H, N-CH), 1.673-1.741 (m, 1H, CH), 1.383-1.490 (m, 2H, CH2), 0.876 (d, 3H, CH3), 0.835 (d, 3H,
CH3); 13C NMR (DMSO-d6) δ: 22.3 (C-3’, C-4’), 23.6 (C-2’), 42.5 (C-1’), 42.0 (C-7), 50.2 (-OCH3),
59.1 (C-8), 117.1 (C-3), 125.8 (C-1), 126.3 (C-5), 128.6 (C-4), 129.7 (C-6), 155.4 (C-2), 173.2 (C-9).
IR (KBr): 3441 (N-H), 2957, 1735 (C=O), 1569, 1259 (ph-O), 1174, 999, 871, 719 cm-1. Anal. Calc.
for C14H20ClNO3: C, 58.84; H, 7.05; N, 4.90. Found: C, 58.62; H, 6.75; N, 5.21.
2.4. Complexes synthesis
Platinum complexes 5a-5f were synthesized with the following method: K2PtCl4 (0.0482 mmol) was
added to a CH3OH/H2O (4 mL, v/v = 1/1) solution of reduced Schiff bases 4a-4f (0.0461 mmol) in the
room temperature, the mixture was adjusted to pH = 8~9 in the beginning, then stirred for 24 h and the
pH was down to about 7. The solution was heated in vacuo and recrystallized from CH3OH/CH2Cl2
under the protection of N2.
Pt[methyl 2-(2-hydroxybenzylamino)-4-methylpentanoate]Cl (5a)
1
Yellow solid. Yield 70.2%. H NMR (600 MHz, DMSO-d6): δ 7.061-7.106 (m, 2H, Ar-H), 6.715 (t,
2H, Ar-H), 3.801 (d, 1H, N-CH2), 3.647 (d, 1H, N-CH2), 3.604 (s, 3H, CH3), 3.262 (t, 1H, N-CH),
1.642-1.720 (m, 1H, CH), 1.386-1.492 (m, 2H, CH2), 0.873 (d, 3H, CH3), 0.830 (d, 3H, CH3); 13
C
NMR (DMSO-d6) δ: 16.7 (C-3’, C-4’), 27.8 (C-2’), 38.9 (C-7), 50.5 (-OCH3), 63.2 (C-8), 115.2 (C-3),
120.9 (C-5), 123.6 (C-1), 128.5 (C-4), 128.7 (C-6), 155.4 (C-2), 174.8 (C-9). IR (KBr): 3441 (N-H),
2957, 1649 (C=O), 1438, 1284 (ph-O), 1173, 786, 649, 620 (Pt-OAr), 520 (Pt-N), 412 (Pt-O=C) cm-1.
ESI-MS: 504.0671 [M + Na]+.Anal. Calc. for C14H20ClNO3Pt: C, 34.97; H, 4.19; N, 2.91. Found: C,
35.15; H, 4.04; N, 2.84. Λm = 48 S·cm2·mol-1.
Pt[methyl 2-(2-hydroxybenzylamino)propanoate]Cl (5b)
1
Yellow solid. Yield 78.9%. H NMR (600 MHz, DMSO-d6): δ 7.201 (t, 1H, Ar-H), 6.989 (d, 1H,
Ar-H), 6.8761 (d, 1H, Ar-H), 6.806 (t, 1H, Ar-H), 4.113 (d, 1H, N-CH2), 3.820 (d, 1H, N-CH2), 3.798
(s, 3H, CH3), 3.472 (q, 1H, N-CH), 1.392 (d, 3H, CH3); 13C NMR (DMSO-d6) δ: 16.4 (C-5’), 39.6
(C-7), 50.4 (-OCH3), 55.2 (C-8), 116.1 (C-3), 122.3 (C-5), 124.3 (C-1), 129.4 (C-4), 130.5 (C-6), 156.1
(C-2), 175.3 (C-9). IR (KBr): 3440 (N-H), 2957, 1653 (C=O), 1439, 1267 (ph-O), 1117, 756, 649, 617
(Pt-OAr), 524 (Pt-N), 418 (Pt-O=C) cm-1. ESI-MS: 462.0195 [M + Na]+. Anal. Calc. for
C11H14ClNO3Pt: C, 30.11; H, 3.22; N, 3.19. Found: C, 30.39; H, 2.97; N, 3.02. Λm = 56S·cm2·mol-1.
Pt[methyl 3-hydroxy-2-(2-hydroxybenzylamino)propanoate]Cl (5c)
1
Yellow solid. Yield 68.9%. H NMR (600 MHz, DMSO-d6): δ 7.107 (d, 1H, Ar-H), 7.067 (t, 1H,
Ar-H), 6.724-6.750 (m, 2H, Ar-H), 3.833 (d, 1H, N-CH2), 3.676 (d, 1H, N-CH2), 3.637 (s, 3H, CH3),
3.611 (d, 2H, CH2), 3.332 (t, 1H, N-CH); 13C NMR (DMSO-d6) δ: 40.5 (C-7), 51.9 (-OCH3), 61.7 (C-8),
65.0 (C-6’), 117.2 (C-3), 120.8 (C-5), 125.6 (C-1), 129.5 (C-4), 129.9 (C-6), 156.7 (C-2), 176.6 (C-9)
4

IR (KBr): 3440 (N-H), 2957, 1638 (C=O), 1421, 1265 (ph-O), 1173, 787, 648, 620 (Pt-OAr), 520
(Pt-N), 420 (Pt-O=C) cm-1. ESI-MS: 478.0145 [M + Na]+. Anal. Calc. for C11H14ClNO4Pt: C, 29.05; H,
3.10; N, 3.08. Found: C, 29.24; H, 2.95; N, 3.27. Λm = 58 S·cm2·mol-1.
Pt[methyl 2-(2-hydroxybenzylamino)-3-(1H-indol-3-yl)propanoate]Cl (5d)
1
Yellow solid. Yield 78.5%. H NMR (600 MHz, DMSO-d6): δ 10.856 (s, 1H, NH), 7.457 (d, 1H,
Ar-H), 7.340 (d, 1H, Ar-H), 7.126 (s, 1H, Ar-H), 7.054-7.086 (m, 3H, Ar-H), 6.977 (t, 1H, Ar-H),
6.699-6.730 (m, 2H, Ar-H), 3.810 (d, 1H, N-CH2), 3.669 (d, 1H, N-CH2), 3.566 (s, 1H, N-CH), 3.550
(s, 3H, CH3), 3.034-3.102 (m, 2H, CH2), 2.849 (s, 1H, NH) ; 13C NMR (DMSO-d6) δ: 31.2 (C-15’),
39.3 (C-7), 50.4 (-OCH3), 60.2 (C-8), 110.9 (C-13’), 112.3 (C-8’), 115.4 (C-3), 119.5 (C-12’), 120.6
(C-5), 120.8 (C-10’), 121.7 (C-11’), 122.6 (C-7’),124.4 (C-1), 128.1 (C-4), 129.5 (C-6), 131.5 (C-9’),
136.4 (C-14’), 155.1 (C-2), 174.3 (C-9). IR (KBr): 3441 (N-H), 2957, 1645 (C=O), 1420, 1286 (ph-O),
1173, 786, 648, 620 (Pt-OAr), 520 (Pt-N), 418 (Pt-O=C) cm-1. ESI-MS: 577.0630 [M + Na]+. Anal.
Calc. for C19H19ClN2O3Pt: C, 41.20; H, 3.46; N, 5.06. Found: C, 41.38; H, 3.20; N, 5.24. Λm = 55
-1
·cm2
S
·mol .
Pt[methyl 2-(5-bromo-2-hydroxybenzylamino)-3-phenylpropanoate]Cl (5e)
1
Yellow solid. Yield 68.8%. H NMR (600 MHz, DMSO-d6): δ 7.287 (t, 2H, Ar-H), 7.229 (dd, 2H,
Ar-H), 7.192 (d, 3H, Ar-H), 6.680 (d, 1H, Ar-H), 3.720 (d, 1H, N-CH2), 3.596 (d, 1H, N-CH2), 3.560 (s,
3H, CH3), 3.473 (t, 1H, N-CH), 2.895 (dd, 2H, CH2); 13C NMR (DMSO-d6) δ: 39.2 (C-21’), 45.1
(C-7), 51.8 (-OCH3), 60.1 (C-8), 111.2 (C-5), 117.6 (C-3), 126.8 (C-18’), 127.7 (C-1), 128.8
(C-20’, C-16’), 129.8 (C-17’, C-19’), 130.4 (C-4), 131.4 (C-6), 137.9 (C-15’), 155.4 (C-2), 177.5
(C-9). IR (KBr): 3442 (N-H), 2957, 1656 (C=O), 1420, 1267 (ph-O), 1173, 786, 649, 620 (Pt-OAr),
524 (Pt-N), 422 (Pt-O=C) cm-1. ESI-MS: 615.9609 [M + Na]+. Anal. Calc. for C17H17BrClNO3Pt: C,
34.39; H, 2.89; N, 2.36. Found: C, 34.51; H, 2.68; N, 2.52. Λm = 53 S·cm2·mol-1.
Pt[methyl 2-(5-chloro-2-hydroxybenzylamino)-4-methylpentanoate]Cl (5f)
1
Yellow solid. Yield 87.6%. H NMR (600 MHz, DMSO-d6): δ 7.207 (d, 1H, Ar-H), 7.092 (dd, 1H,
Ar-H), 6.750 (d, 1H, Ar-H), 3.744 (d, 1H, N-CH2), 3.629 (s, 3H, CH3), 3.589 (d, 1H, N-CH2), 3.245 (t,
1H, N-CH), 1.677-1.746 (m, 1H, -CH-), 1.386-1.492 (m, 2H, CH2), 0.882 (d, 3H, CH3), 0.839 (d, 3H,
-CH3) ; 13C NMR (DMSO-d6) δ: 22.3 (C-3’, C-4’), 23.1 (C-2’), 41.3 (C-1’), 40.5 (C-7), 50.4 (-OCH3),
57.2 (C-8), 116.7 (C-3), 125.8 (C-1), 126.2 (C-5), 128.5 (C-4), 129.7 (C-6), 153.2 (C-2), 174.8 (C-9).
IR (KBr): 3442 (N-H), 2957, 1644 (C=O), 1438, 1270 (ph-O), 1173, 788, 649, 619 (Pt-OAr), 518
(Pt-N), 422 (Pt-O=C) cm-1. ESI-MS: 538.0279 [M + Na]+. Anal. Calc. for C14H19Cl2NO3Pt: C, 32.63; H,
3.72; N, 2.72. Found: C, 32.84; H, 3.50; N, 2.80. Λm = 50S·cm2·mol-1.
2.5. Determination of UV absorption spectra
An UV-3400 Toshniwal spectrophotometer emitting UV light was used mainly between 200~400 nm.
The salmon sperm DNA (M = 208.8 g/mol) was dissolved in Tris-HCl (pH = 7.5) buffer solution, and
rested in 24 h at 4 °C. Then complexes (5c and 5f) were added in buffer solution in different
concentrations (ccomplex : cDNA = 0.1, 0.3 and 0.5), and rested in 24 h at 4 °C. The UV absorption spectra
was determined at room temperature (△t = 1 s, n = 3).
2.6. Cell culture
Four different human carcinoma cell lines: HL-60, Hela, BGC-823 and HepG-2 were cultured in
RPMI-1640 medium supplemented with 10% fetal bovine serum, 100 units/mL of penicillin and 100
µg/mL of streptomycin. Cells were maintained at 37 °C in a humidified atmosphere of 5% CO2 in air.
5

2.7. Solutions
The complexes were dissolved in DMSO at a concentration of 5 mM as stock solution, and diluted in
culture medium at concentrations of 0.1, 1.0, and 10 µM as working solution. To avoid DMSO toxicity,
the concentration of DMSO was less than 0.1% (v/v) in all experiments.
2.8. Cytotoxicity analysis
The cells harvested from exponential phase were seeded equivalently into a 96-well plate, and then
the complexes were added to the wells to achieve final concentrations. Control wells were prepared by
addition of culture medium. Wells containing culture medium without cells were used as blanks. All
experiments were performed in quintuplicate. The MTT assay was performed as described by
Mosmann for HL-60 [25]. Upon completion of the incubation for 44 h, stock MTT dye solution (20 mL,
5 mg/mL) was added to each well. After 4 h incubation, 2-propanol (100 mL) was added to solubilize
the MTT formazan. The OD of each well was measured on a microplate spectrophotometer at a
wavelength of 570 nm. The IC50 value was determined from plot of % viability against dose of
compounds added.
3. Results and discussion
3.1. Synthesis and characterization
Ligands 4a-4f were synthesized from salicylaldehyde, 5-bromosalicylaldehyde and
5-chlorosalicyladehyde with amino esters 1a-1f. The platinum(II) complexes 5a-5f have been prepared
by the reaction of K2PtCl4 with reduced amino acid esters Schiff-bases 4a-4f in a mixture of methanol
and water at room temperature (Scheme 1).
R1
O
N
OCH3
CHO
OH
O
CH2Cl2
Et3N
R1
R2
OH
R1
R2
.
OCH3 HCl
NH2
1
2
R1
8
3
OR2
O
O
9
NH
Pt
7
NH OCH3
OH
2
6
1
NaBH4
K2PtCl4
CH3OH/H2O
R2
O
R2
Cl
5
4
3
4
5
a: R1 = i-Bu R2 = H; b: R1 = Me R2 = H; c: R1 = CH2OH R2 = H
d: R1 = (1H-indol-3-yl)methanide R2 = H; e: R1 = Bn R2 = Br; f: R1 = i-Bu R2 = Cl
11'
17'
16'
18'
10'
9'
1'
2'
12'
13'
6'
5'
CH3
15'
3'
22'
R1 =
19'
OH
8'
7'
4'
14'
HN
21'
20'
Scheme 1. Synthetic pathway for the preparation of compounds.
The bands at about 3316~3441 cm-1 in the IR spectra of 4a-4f can be assigned to the (N-H)
stretching frequencies, which shifted to the higher wave number (3440~3442 cm-1) upon complexation
in 5a-5f. A carbonyl (C=O) vibration band which appeared in the IR spectra of ligands (1723~1739
cm-1) similarly underwent a shift into lower frequency (1638~1656 cm-1) in their Pt(II) complexes. The
bands for the phenolic group (C-O) stretching frequency observed in the region 1241~1260 cm-1 of free
6

ligands shifted to higher frequency (1265~1286 cm-1) in their complexes. On the other hand, new bands
appeared at about 617~620, 518~524 and 412~422 cm-1 could be assigned to υPt-O=C, υPt-N, and υPt-OAr
,
respectively. The IR data revealed that nitrogen atoms of the imino groups and oxygen atoms of the
carbonyl groups and phenolic groups were coordinated to the metal ion (Table 1).
Although the overall pattern of the 1H NMR spectra of 5a-5f resemble very closely to that of the free
ligands, almost all the signals have been shifted to lower fields upon coordination. In the 13C NMR
analysis, most of the characteristic signals for the complexes changed slightly, however signals of
5a-5e observed at 38.9–45.1 ppm, 55.2–63.2 ppm and 153.2–156.7 ppm are assignable to the carbon
atoms (C7, 8, 2) respectively, which have been shifted to higher fields upon coordination. What’s more,
the C9 signals for all the complexes have been shifted to lower fields which proved the -C=O
coordination to the Pt(II) ion. All the platinum complexes were characterized by ESI-MS, the
results showed the presence of [M+Na]+ peaks were in good agreement with their formula weights and
relative fragment peaks. Elemental analysis confirmed the structures of the target compounds further.
The conductivity data of all complexes were also measured. The molar conductance values of
-1
·cm2
platinum(II) complexes in deionized methanol are in the range of 40~60 S
complexes are electroneutral [30].
·mol . Therefore, all
Table 1.
Main IR absorptions of ligands and complexes (cm-1)
υ (N-H)
3316
3430
3426
3438
3431
3441
3441
3440
3440
3441
3442
3442
υ (C=O-)
1735
1723
1735
1739
1725
1735
1649
1653
1638
1645
1656
1644
υ (ph-O)
1254
1256
1257
1241
1260
1259
1284
1267
1265
1286
1267
1270
υ (Pt-OAr)
υ (Pt-N)
υ (Pt-O=C)
4a
4b
4c
4d
4e
4f
412
418
420
418
422
422
520
524
520
520
524
518
620
617
620
620
620
619
5a
5b
5c
5d
5e
5f
3.2. DNA Binding Study
Interaction of the complexes with DNA has been studied with UV spectroscopy in order to
investigate the possible binding modes [26]. Generally speaking, there are three main noncovalent
binding modes between antitumor drugs with DNA: Electrostatic binding, intercalations between base
pairs and binding into the groove of DNA. In the spectral range 200~400 nm, DNA with different
concentration of complexes (5c and 5f) was examined. As the concentration of complex increased,
the absorbance value increased and maximum absorption wavelength occurred blue-shifted. It might be
due to the complex was inserted into the DNA double helix structure and stacking with DNA base pairs
where π-electron accumulation occurred. The results indicated that hyperchromicity occurred after
coordination, thus the binding mode of complexes with DNA may be intercalation [27-29].
7

3.3. Stability of Complexes
Because the stability of platinum-based compounds is one of the key criteria for being an anticancer
candidate, complex 5f was evaluated by UV spectral analysis at different times in 1% DMSO/99% H2O
(Figure 1). According to the results in Figure 1, no detectable significant changes of the absorption
bands of complex 5f have been observed within 18 h, which demonstrates the excellent stability of the
determined complex under the test conditions [31].
Figure 1
3.4. In Vitro Cytotoxicity Assay
In order to find possible structure-activity relationships, the in vitro cytotoxicity of complexes 5a-5f
was evaluated against four human cancer cell lines including HL-60, BGC-823, Hela, and HepG2. For
comparison purposes, the cytotoxicity of cisplatin, a standard antitumor drug, was also evaluated under
the same conditions. The IC50 values (the concentration that inhibited in 50% the cellular proliferation)
of the studied complexes are presented in Table 2. It was noted that all complexes showed
dose-dependent antiproliferative effect with a lower IC50 value (<50 µM) toward investigated cancer
cell lines. Moreover, of all the studied complexes, complex 5f displayed the best cytotoxicity against
selected carcinoma cell lines, it showed better cytotoxicity against BGC-823 than cisplatin, but slightly
less activity than cisplatin on HL-60, Hela and HepG2. When tested against HepG2 cell line, 5e
showed better activity than cisplatin, other complexes exhibited comparable cytotoxicity to 5e, but a
slightly less than cisplatin. Compared 5a with 5f , the antitumor activity increased with the introduce of
electron-withdrawing groups at the C4 position of the benzene ring. The complexes (5a, 5b, 5c and 5d)
demonstrated less active cytotoxicity than cisplatin against the tested cell lines, all the complexes had
less active cytotoxicity against HL-60 and Hela cell lines in compared with cisplatin.
Table 2.
The cytotoxicity of complexes in vitro (IC50)
IC50 (µM)
Complex
HL-60
40.56
17.32
24.44
21.68
22.09
4.08
BGC-823
20.59
15.62
15.99
26.96
9.78
Hela
47.41
38.91
41.75
43.12
10.28
9.92
HepG2
30.57
23.80
24.13
28.00
18.45
21.46
20.60
5a
5b
5c
5d
5e
6.36
5f
Cisplain
2.29
6.48
4.41
4. Conclusion
In conclusion, some platinum(II) complexes with reduced Schiff bases derived from amino acid ester
as ligands were synthesized and characterized. All complexes were tested for their DNA interaction
ability with the salmon sperm DNA through ultraviolet spectrum, the results revealed that the binding
mode of complexes with DNA may be intercalation after coordination. The in vitro antitumor activities
of the platinum complexes have been validated against the HL-60, BGC-823, Hela, and HepG2 cell
lines by the MTT assay, the results showed that the complexes 5a-5f have selectivity against tested
8

carcinoma cell lines. The complex 5f was the most effective agent among these complexes,
furthermore its antitumor activity is better than that of cisplatin against BGC-823 cell line. The
structure-activity relationship has been analyzed, and this study would be helpful in designing new
platinum anticancer drugs.
Acknowledgements
The work was financially supported by the Natural Science Foundation of Hebei Province (No.
B2012201109, No. B2010000223), and Science & Technology Pillar Program of Hebei Province
(No. 12211212).
References
[1] B. Lippert, Coord. Chem. Rev. 182 (1999) 263.
[2] L.R. Kelland, N.P. Farrell, Platinum-Based Drugs in Cancer Therapy, Humana Press, New Jersey,
2000.
[3] H.M. Pinedo, J.H. Schornagel, Platinum and Other Metal Coordination Compounds in Cancer
Chemotherapy 2, Plenum Press, New York, 1996.
[4] B. Rosenberg, L. VanCamp, J.E. Trosko, V.H. Mansour, Nature 222 (1969) 385.
[5] B. Lippert, in: B. Lippert (Ed.), Cisplatin Chemistry and Biochemistry of a Leading Anticancer
Drug, Wiley-VCH, Weinheim, 1999, pp. 29-70.
[6] S.D. Schaefer, J.D. Post, L.G. Close, C.G. Wright, Cancer 56 (1985) 1934.
[7] M.P. Goren, R.K. Wright, M.E. Horowitz, Cancer Chemother. Pharmacol. 18 (1986) 69.
[8] D.S. Alberts, J.K. Noel, Anticancer Drugs 6 (1995) 369.
[9] M. Kartalou, J.M. Essigmann, Mutat. Res. 478 (2001) 23.
[10] M.A. Fuertes, M. Alonso, J.M. Pérez, Chem. Rev. 103 (2003) 645.
[11] M.A. Jakupec, M. Galanski, V.B. Arion, Ch.G. Hartinger, B.K. Keppler, Dalton Trans (2008) 183.
[12] P.U. Maheswari, S. Roy, S. Barends, B. Kozlevcar, P. Gamez, J. Reedijk, J. Am. Chem. Soc. 128
(2006) 710.
[13] J. Qian, W. Gu, H. Liu, F.X. Gao, L. Feng, S.P. Yan, D.Z. Liao, P. Cheng, Dalton Trans 11 (2007)
1060.
[14] J.L. Tian, L. Feng, W. Gu, G.J. Xu, S.P. Yan, D.Z. Liao, Z.H. Jiang, P. Cheng, Inorg. Biochem. 101
(2007) 96.
[15] J.H. Li, J.T. Wang, Z.W. Mao, L.N. Ji, J. Coord. Chem. 62 (2009) 446.
[16] T.D. Tullius, J.A. Greenbaum, Curr. Opin. Chem. Biol. 9 (2005) 127.
[17] K. Tatsumoto, A.E. Martell, R.J. Motekaitis, J. Am. Chem. Soc. 103 (1981) 6197.
[18] K. Korhonen, R. Hamalainen, U. Turpeinen, Acta. Crystallogr. Sect. C. 40 (1984) 1175.
[19] R. Ganguly, B. Sreenivasulu, Vittal J J. Coord. Chem. Rev. 252 (2008) 1027.
[20] B. Sreenivasulu, M. Vetrichelvan, F. Zhao, S. Gao, J.J. Vittal, Eur. J. Inorg. Chem. (2005) 4635.
[21] X.F. Ma, D.D. Li, J.L. Tian, Y.Y. Kou, S.H. Yan, Transit. Metal. Chem. 34 (2009) 475.
[22] C. Gao, X. Ma, J. Tian, D. Li, S. Yan, J. Coord. Chem. 63 (2010) 115.
[23] C. Elzbieta, E. Zygmunt, K. Romuald, Dissertation. Pharm. 15 (1964) 369.
[24] Jiabo Li and Yaowu Sha. Molecules 13 (2008) 1111.
[25] T. Mosmann, J. Immunol. Methods 65 (1983) 55.
9

[26] A. Skladanowski, P. Bozko, M. Sabisz, Chem. Rev. 109 (2009) 2951.
[27] M. Wang, L.R. Hou, Z.N. Gao, T.L. Yang, Chinese J Appl Chem. 25 (2008) 1186.
[28] L.N. Ji, Q.L. Zhang, J.G. Liu, Sci. China (Ser. B), 31 (2001) 193.
[29] P.R. Cao, M.M. McHugh, T. Melendy, T. Beerman,Mol. Cancer Ther. 2 (2003) 651.
[30] W.J. Geary, Coord. Chem Rev. 7 (1971) 81.
[31] Jian Zhao, Shaohua Gou, Yanyan Sun, Lei Fang, Zhimei Wang, Inorg. Chem. 51 (2012) 10317.
10

2.5
2.0
1.5
1.0
0.5
0.0
0.5h
1h
6h
18h
200
300
400
wavelength (nm)
Figure 1. Ultraviolet spectrum of stability of complex 5f
11

Synthesis, characterization and cytotoxicity studies of platinum(II)
complexes with reduced amino acid ester Schiff-bases as ligands
Li-Jun Li*, Bin Fu ,Yu Qiao, Cheng Wang, Ying-Ying Huang, Chuan-Chuan Liu, Chao Tian,
Jian-Long Du*
Chemical Biology Key Laboratory of Hebei Province, Hebei University, Baoding 071002, P. R. China
R1
O
N
OCH3
CHO
OH
O
CH2Cl2
Et3N
R1
R2
OH
R2
.
HCl
OCH3
NH2
1
2
3
R1
R1
OCH3
O
O
NH
NH OCH3
OH
Pt
NaBH4
K2PtCl4
CH3OH/H2O
R2
O
R2
Cl
4
5
12

Synthesis, characterization and cytotoxicity studies of platinum(II)
complexes with reduced amino acid ester Schiff-bases as ligands
Li-Jun Li*, Bin Fu, Yu Qiao, Cheng Wang, Ying-Ying Huang, Chuan-Chuan Liu, Chao Tian,
Jian-Long Du*
Chemical Biology Key Laboratory of Hebei Province, Hebei University, Baoding 071002, P. R. China
ꢀ
ꢀ
ꢀ
ꢀ
We synthesized six platinum(II) complexes which were rarely reported.
All complexes were prepared with reduced amino acid esters Schiff-bases as ligands.
Some complexes showed better activity than cisplatin.
The complex we synthesized showed good stability.
13