# Antitumor Activity of 6-(cyclohexylamino)-1,3-dimethyl-5(2-pyridyl)furo[2,3-d]pyrimidine-2,4(1H,3H)-dione and Its Ti(IV), Zn(II), Fe(III), and Pd(II) Complexes on K562 and Jurkat Cell Lines

Authors: Fahmideh Shabani; Shahriar Ghammamy; Khayroallah Mehrani; Mohammad Bagher Teimouri; Masoud Soleimani; Saeid Kaviani
Journal: Bioinorganic Chemistry and Applications
 (2008)

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## Abstract

(6-(cyclohexylamino)-1,3-dimethyl-5(2-pyridyl)furo[2,3-d]pyrimidine-2,4(1H,3H)-dione) abbreviated as CDP was synthesized and characterized. Ti(IV), Zn(II), Fe(III), and Pd(II) metal complexes of this ligand are prepared by the reaction of salts of Ti(IV), Zn(II), Fe(III), and Pd(II) with CDP in acetonitrile. Characterization of the ligand and its complexes was made by microanalyses, FT-IR,1H NMR, 13C NMR, and UV-Visible spectroscopy. All complexes were characterized by several techniques using elemental analysis (C, H, N), FT-IR, electronic spectra, and molar conductance measurements. The elemental analysis data suggest the stoichiometry to be 1:1 [M:L] ratio formation. The molar conductance measurements reveal the presence of 1:1 electrolytic nature complexes. These new complexes showed excellent antitumor activity against two kinds of cancer cells that are K562 (human chronic myeloid leukemia) cells and Jurkat (human T lymphocyte carcinoma) cells.


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## Body

## 1. Introduction

Nitrogen-containing ligands such as Schiff
bases and their metal complexes played an important role in the development of  coordination chemistry, resulting in an enormous number of publications,
ranging from pure synthetic work to physicochemical [1] and biochemically
relevant studies of metal complexes [2–6] and found wide
range of applications. Other kinds of nitrogen-containing ligands are
well-known pyrimidine systems such as purine analogues that exhibit a wide
range of biological activities. Fused pyrimidine compounds are valued not
only for their rich and varied chemistry, but also for many important
biological properties. Among them, the furopyrimidine ring system, because of a
formal isoelectronic relationship with purine, is of special biological
interest. It has numerous pharmacological and agrochemical applications, namely,
antimalarials, antifolates, and antivirus, as well as
potential radiation protection agents. Recently, some furopyrimidines were
shown to be potent vascular endothelial growth factor receptor2 (VEGFR2) and
epidermal growth factor receptor (EGFR) inhibitors. Because of the importance
of furo[2,3-d]pyrimidine derivatives, several methodologies for synthesizing
them have already been developed. However, many of the synthetic protocols
reported so far prolonged reaction times, harsh reaction suffer from
disadvantages, such as relying on multistep reactions, needing anhydrous conditions, low yields, use of metal-containing
reagents, and special instruments or starting materials. Therefore, the
development of new and
efficient methods for the preparation of furo[2,3-d]pyrimidine derivatives is
still strongly desirable [7].Pyrimidines represent
a very interesting class of compounds because of their wide applications in
pharmaceutical, phytosanitary, analytical, and industrial aspects, for example,
as antibacterial, fungicide [8], anti-inflammatory, antihelmintics,
antitubercular, anti-HIV, antidegenerative and hypothermic activities [8], and herbicides [9], and
have biological activities [10–14].It has long been known that metal ions involve in
biological processes of life and have been subject of interest. The modes of
action of these metal ions are often complex but are believed to involve
bonding to the heteroatoms of the heterocyclic residues of biological
molecules, that is, proteins, enzymes, nucleic acids, and so forth [15].From these points of view, it is interesting to study
different types of transition metal complexes of these biologically active
ligands. In this paper, the synthesis, characterization, and antitumor
properties of a number of the first row transition metal complexes with one of
the above ligands have been studied.


## 2. Materials and Methods

### 2.1. Chemicals and Reagents

N,N′-dimethylbarbituric
acid, 2-pyridinecarbaldehyde, titanium(VI) tetra fluoride, zinc(II) acetate
dihydrate, iron(III) chloride hexahydrate, and palladium chlorides were Merck chemicals (Darmstadt, Germany) and were used without further purification. Organic
solvents were reagent grade. Electronic spectra were recorded by Camspec UV-Visible
spectrophotometer model Wpa bio Wave S2 100. The IR spectra were recorded using
FT-IR Bruker Tensor 27 spectrometer. H1-NMR and C13-NMR
were recorded on a Bruker AVANCE DRX 500 spectrometer. All the chemical shifts
are quoted in ppm using the high-frequency positive convention; H1
and C13-NMR spectra were referenced to external SiMe4.
The percent composition of elements was obtained from the Microanalytical
Laboratories, Department of Chemistry, OIRC, Tehran.


### 2.2. Cell Culture

The human chronic myeloid leukemia—K562 cell line—and the human T
lymphocyte carcinoma-Jurkat cell line, used for  treatment with the
drugs, were provided. K562 and Jurkat cells were grown at37°C in an
atmosphere containing 5%CO2, with RPMI-1640 Medium HEPES
Modification with L-glutamine and 25 mM HEPES (Sigma-Aldrich Chemie GmbH, Germany) supplemented
with 10% heat-inactivated fetal bovine serum (FBS) (Gibco, Carlsbad, Calif, USA), 2.7% sodium bicarbonate, and
500 mg/L ampicillin.


## 2.1. Chemicals and Reagents

N,N′-dimethylbarbituric
acid, 2-pyridinecarbaldehyde, titanium(VI) tetra fluoride, zinc(II) acetate
dihydrate, iron(III) chloride hexahydrate, and palladium chlorides were Merck chemicals (Darmstadt, Germany) and were used without further purification. Organic
solvents were reagent grade. Electronic spectra were recorded by Camspec UV-Visible
spectrophotometer model Wpa bio Wave S2 100. The IR spectra were recorded using
FT-IR Bruker Tensor 27 spectrometer. H1-NMR and C13-NMR
were recorded on a Bruker AVANCE DRX 500 spectrometer. All the chemical shifts
are quoted in ppm using the high-frequency positive convention; H1
and C13-NMR spectra were referenced to external SiMe4.
The percent composition of elements was obtained from the Microanalytical
Laboratories, Department of Chemistry, OIRC, Tehran.


## 2.2. Cell Culture

The human chronic myeloid leukemia—K562 cell line—and the human T
lymphocyte carcinoma-Jurkat cell line, used for  treatment with the
drugs, were provided. K562 and Jurkat cells were grown at37°C in an
atmosphere containing 5%CO2, with RPMI-1640 Medium HEPES
Modification with L-glutamine and 25 mM HEPES (Sigma-Aldrich Chemie GmbH, Germany) supplemented
with 10% heat-inactivated fetal bovine serum (FBS) (Gibco, Carlsbad, Calif, USA), 2.7% sodium bicarbonate, and
500 mg/L ampicillin.


## 3. Experimental

### 3.1. Synthesis of the CDP Ligand

To a solution ofN,N′-dimethylbarbituric
acid (0.78 g,
5.0 mmol) and 2-pyridinecarbaldehyde (0.54 g, 5.0 mmol) in DMF (3 mL) in a
screw-capped vial was added cyclohexyl
isocyanide (0.55 g,
5.0 mmol) via a syringe and was shaken for 1 minute. The reaction mixture was then kept for about 30 minutes
at room temperature (25°C) and the completion of reaction was confirmed
by TLC (EtOAc-hexane 1:2). Then, the resulting crystals were filtered and washed with diethyl ether
(20 mL) to yield as light pink crystals
(1.42 g,
80%). The dried product thus
obtained showed a single spot on TLC and was pure enough for all analytical
purposes [7] (see Figure 1).Figure 1
Synthesis
route of CDP ligand.


#### 3.1.1. Analysis of CDP Ligand

Yield, 80%. Mp135.2–137.5°C; Anal.
Calcd. C19H21N4O3: C, 64.58;
H, 5.94; N, 15.86. Found: C, 64.92; H, 6.29; N, 16.08. H1 NMR (CDCl3):
4.40(1H, d, NH), 8.40 and 7.60(1H, d, pyridine), 7.55 and 7.26(1H, d,
pyridine), 3.61(3H, s, NCH3), 3.45(3H, s, NCH3), 1.38(2H,
m, cyclohexan), 1.63(2H, m, cyclohexan), 2.01(1H, m, cyclohexan); C13 NMR (CDCl3):
158.63(C4), 154.51(NCON), 153.42(C2), 136.27(C6),
122.3(C3), 149.9, 149.4, 147.2, and 118.4(pyridine), 52.34, 33.92,
and 24.49(cyclohexan). IR (KBr, cm−1):
3276 w, 1664 w, 1593 w, 1449 m, 1266 w, 1120 w, 610, and 742 s. UV-vis (MeCN): λmax260 nm (ε 120),
336 nm (ε 110).


### 3.2. Synthesis of the Metal Complexes: General Method

A solution of metal salt dissolved in
acetonitrile was added
gradually to a stirred acetonitrile solution of the ligand (CDP), in the molar
ratio 1:1 (metal:ligand). The reaction mixture was further stirred for 2–4 hours to ensure the completion and
precipitation of the formed complexes. The precipitated solid complexes were
filtered and washed several times with 50% (v/v)  ethanol/water to remove any traces of the
unreacted starting materials. Finally, the complexes were washed with diethyl
ether and dried in vacuum desiccators over anhydrousCaCl2.


#### 3.2.1. Analysis of Ti(C19H20N4O3)F4

Yield, 85%. Anal. Calcd. Ti(C19H20N4O3)F4: C, 47.89;
H, 4.20; N, 11.76. Found: C, 48.2; H, 4.37; N, 12.1. H1 NMR (DMSO):
9(1H, d, pyridine), 8.30(1H, d, pyridine), 7.1(1H, d, pyridine), 3.53(3H, s, NCH3),
3.01(3H, s, NCH3), 1.08–2.5(2H and 1H, m, cyclohexan); IR (KBr, cm−1):
1689 s, 1625 m, 1453 m, 1246 w, 1153 w, 774 s, 667 w, and 603 s. UV-vis (MeCN): λmax377 nm (ε 54), 497 nm (ε 28).


#### 3.2.2. Analysis of Zn(C19H20N4O3)(OAC)2

Yield,
60%. Anal. Calcd. Zn(C19H20N4O3)(OAC)2: C, 42.57;
H, 3.73; N, 10.45. Found: C, 43.5; H, 3.86; N, 10.82. H1 NMR (DMSO):
8.30(1H, d, pyridine), 8.06(1H, d, pyridine), 7.5(1H, d, pyridine), 3.3(3H, s,
NCH3), 3(3H, s, NCH3), 1.2–2.35(2H and 1H, m,
cyclohexan); IR (KBr, cm−1): 1691 s, 1625 m, 1439 w, 1246 w, 1158 w, and 425 s. UV-vis (MeCN): λmax262 nm (ε 110),
302 nm (ε 100), 344 nm (ε 78), and 415 nm (ε 60).


#### 3.2.3. Analysis of Fe(C19H20N4O3)Cl3

Yield, 75%. Anal. Calcd. Fe(C19H20N4O3)Cl3: C, 44.32;
H, 3.88; N, 10.88. Found: C, 44.86; H, 4.18; N, 11.38. H1 NMR
(DMSO): 9.67(1H, d, pyridine), 9.02(1H, d, pyridine), 7.95(1H, d, pyridine),
3.7(3H, s, NCH3), 3.1(3H, s, NCH3), 1.5–2.8(2H and 1H, m,
cyclohexan); IR (KBr, cm−1): 1600 w, 1546 w, 1444 w, 1154 s, 514 m, and 599 s. UV-vis (MeCN): λmax257 nm (ε 280),
314 nm (ε 156), 363 nm (ε 126), 440 nm (ε 60), and 494 nm (ε 28).


#### 3.2.4. Analysis of Pd(C19H20N4O3)Cl2

Yield, 88%.
Anal. Calcd. Fe(C19H20N4O3)Cl3: C, 43.07;
H, 3.77; N, 10.57. Found: C, 43.45; H, 3.95; N, 10.89. H1 NMR
(DMSO): 8.31(1H, d, pyridine), 7.55(1H, d, pyridine), 7.37(1H, d, pyridine), 2.55(3H,
s, NCH3), 2.5(3H, s, NCH3), 1.18–2.07(2H and 1H, m,
cyclohexan); IR (KBr, cm−1): 1649 s, 1546 m, 1467 m, 1266 m, 1142 m, and 495 m. UV-vis (MeCN): λmax261 nm (ε 220), 307
nm (ε 130), 442 nm (ε 118), and 660 nm (ε 75).


### 3.3. Cytotoxicity Studies

CDP ligand and Ti(C19H20N4O3)F4, Zn(C19H20N4O3)
(OAC)2, Fe(C19H20N4O3)Cl3,
and Pd(C19H20N4O3)Cl2 complexes arefive  compounds which were
assayed for cytotoxicity in vitro against K562 (human chronic myeloid leukemia)
cells and Jurkat (human T lymphocyte carcinoma) cells.The two cell lines were provided by the Pasteur
Institute in Iran.
The procedure for cytotoxicity studies was similar to that reported earlier [16].
Briefly, in order to calculate the concentration of each drug that produces a
50% inhibition of cell growth (IC50), 190 mL of cell suspension 4×105 cell/mL) was exposed to various concentrations of ligand and complexes
dissolved in sterile DMSO. The final concentration of DMSO in the growth medium
was 2% (v/v) or lower, concentrations without effect on cell replication [17, 18].After the incubation periods 72 hours for all cell
lines, the cell concentrations were determined both in control and in
drug-treated cultures. All experiments were done  for six times (see Figure2).(a) Tumor cell
after 72 h without Pd(C19H20N4O3)Cl2 compound. (b) tumor cell after 72 h with Pd(C19H20N4O3)Cl2 compound.
(a)(b)


## 3.1. Synthesis of the CDP Ligand

To a solution ofN,N′-dimethylbarbituric
acid (0.78 g,
5.0 mmol) and 2-pyridinecarbaldehyde (0.54 g, 5.0 mmol) in DMF (3 mL) in a
screw-capped vial was added cyclohexyl
isocyanide (0.55 g,
5.0 mmol) via a syringe and was shaken for 1 minute. The reaction mixture was then kept for about 30 minutes
at room temperature (25°C) and the completion of reaction was confirmed
by TLC (EtOAc-hexane 1:2). Then, the resulting crystals were filtered and washed with diethyl ether
(20 mL) to yield as light pink crystals
(1.42 g,
80%). The dried product thus
obtained showed a single spot on TLC and was pure enough for all analytical
purposes [7] (see Figure 1).Figure 1
Synthesis
route of CDP ligand.


### 3.1.1. Analysis of CDP Ligand

Yield, 80%. Mp135.2–137.5°C; Anal.
Calcd. C19H21N4O3: C, 64.58;
H, 5.94; N, 15.86. Found: C, 64.92; H, 6.29; N, 16.08. H1 NMR (CDCl3):
4.40(1H, d, NH), 8.40 and 7.60(1H, d, pyridine), 7.55 and 7.26(1H, d,
pyridine), 3.61(3H, s, NCH3), 3.45(3H, s, NCH3), 1.38(2H,
m, cyclohexan), 1.63(2H, m, cyclohexan), 2.01(1H, m, cyclohexan); C13 NMR (CDCl3):
158.63(C4), 154.51(NCON), 153.42(C2), 136.27(C6),
122.3(C3), 149.9, 149.4, 147.2, and 118.4(pyridine), 52.34, 33.92,
and 24.49(cyclohexan). IR (KBr, cm−1):
3276 w, 1664 w, 1593 w, 1449 m, 1266 w, 1120 w, 610, and 742 s. UV-vis (MeCN): λmax260 nm (ε 120),
336 nm (ε 110).


## 3.1.1. Analysis of CDP Ligand

Yield, 80%. Mp135.2–137.5°C; Anal.
Calcd. C19H21N4O3: C, 64.58;
H, 5.94; N, 15.86. Found: C, 64.92; H, 6.29; N, 16.08. H1 NMR (CDCl3):
4.40(1H, d, NH), 8.40 and 7.60(1H, d, pyridine), 7.55 and 7.26(1H, d,
pyridine), 3.61(3H, s, NCH3), 3.45(3H, s, NCH3), 1.38(2H,
m, cyclohexan), 1.63(2H, m, cyclohexan), 2.01(1H, m, cyclohexan); C13 NMR (CDCl3):
158.63(C4), 154.51(NCON), 153.42(C2), 136.27(C6),
122.3(C3), 149.9, 149.4, 147.2, and 118.4(pyridine), 52.34, 33.92,
and 24.49(cyclohexan). IR (KBr, cm−1):
3276 w, 1664 w, 1593 w, 1449 m, 1266 w, 1120 w, 610, and 742 s. UV-vis (MeCN): λmax260 nm (ε 120),
336 nm (ε 110).


## 3.2. Synthesis of the Metal Complexes: General Method

A solution of metal salt dissolved in
acetonitrile was added
gradually to a stirred acetonitrile solution of the ligand (CDP), in the molar
ratio 1:1 (metal:ligand). The reaction mixture was further stirred for 2–4 hours to ensure the completion and
precipitation of the formed complexes. The precipitated solid complexes were
filtered and washed several times with 50% (v/v)  ethanol/water to remove any traces of the
unreacted starting materials. Finally, the complexes were washed with diethyl
ether and dried in vacuum desiccators over anhydrousCaCl2.


### 3.2.1. Analysis of Ti(C19H20N4O3)F4

Yield, 85%. Anal. Calcd. Ti(C19H20N4O3)F4: C, 47.89;
H, 4.20; N, 11.76. Found: C, 48.2; H, 4.37; N, 12.1. H1 NMR (DMSO):
9(1H, d, pyridine), 8.30(1H, d, pyridine), 7.1(1H, d, pyridine), 3.53(3H, s, NCH3),
3.01(3H, s, NCH3), 1.08–2.5(2H and 1H, m, cyclohexan); IR (KBr, cm−1):
1689 s, 1625 m, 1453 m, 1246 w, 1153 w, 774 s, 667 w, and 603 s. UV-vis (MeCN): λmax377 nm (ε 54), 497 nm (ε 28).


### 3.2.2. Analysis of Zn(C19H20N4O3)(OAC)2

Yield,
60%. Anal. Calcd. Zn(C19H20N4O3)(OAC)2: C, 42.57;
H, 3.73; N, 10.45. Found: C, 43.5; H, 3.86; N, 10.82. H1 NMR (DMSO):
8.30(1H, d, pyridine), 8.06(1H, d, pyridine), 7.5(1H, d, pyridine), 3.3(3H, s,
NCH3), 3(3H, s, NCH3), 1.2–2.35(2H and 1H, m,
cyclohexan); IR (KBr, cm−1): 1691 s, 1625 m, 1439 w, 1246 w, 1158 w, and 425 s. UV-vis (MeCN): λmax262 nm (ε 110),
302 nm (ε 100), 344 nm (ε 78), and 415 nm (ε 60).


### 3.2.3. Analysis of Fe(C19H20N4O3)Cl3

Yield, 75%. Anal. Calcd. Fe(C19H20N4O3)Cl3: C, 44.32;
H, 3.88; N, 10.88. Found: C, 44.86; H, 4.18; N, 11.38. H1 NMR
(DMSO): 9.67(1H, d, pyridine), 9.02(1H, d, pyridine), 7.95(1H, d, pyridine),
3.7(3H, s, NCH3), 3.1(3H, s, NCH3), 1.5–2.8(2H and 1H, m,
cyclohexan); IR (KBr, cm−1): 1600 w, 1546 w, 1444 w, 1154 s, 514 m, and 599 s. UV-vis (MeCN): λmax257 nm (ε 280),
314 nm (ε 156), 363 nm (ε 126), 440 nm (ε 60), and 494 nm (ε 28).


### 3.2.4. Analysis of Pd(C19H20N4O3)Cl2

Yield, 88%.
Anal. Calcd. Fe(C19H20N4O3)Cl3: C, 43.07;
H, 3.77; N, 10.57. Found: C, 43.45; H, 3.95; N, 10.89. H1 NMR
(DMSO): 8.31(1H, d, pyridine), 7.55(1H, d, pyridine), 7.37(1H, d, pyridine), 2.55(3H,
s, NCH3), 2.5(3H, s, NCH3), 1.18–2.07(2H and 1H, m,
cyclohexan); IR (KBr, cm−1): 1649 s, 1546 m, 1467 m, 1266 m, 1142 m, and 495 m. UV-vis (MeCN): λmax261 nm (ε 220), 307
nm (ε 130), 442 nm (ε 118), and 660 nm (ε 75).


## 3.2.1. Analysis of Ti(C19H20N4O3)F4

Yield, 85%. Anal. Calcd. Ti(C19H20N4O3)F4: C, 47.89;
H, 4.20; N, 11.76. Found: C, 48.2; H, 4.37; N, 12.1. H1 NMR (DMSO):
9(1H, d, pyridine), 8.30(1H, d, pyridine), 7.1(1H, d, pyridine), 3.53(3H, s, NCH3),
3.01(3H, s, NCH3), 1.08–2.5(2H and 1H, m, cyclohexan); IR (KBr, cm−1):
1689 s, 1625 m, 1453 m, 1246 w, 1153 w, 774 s, 667 w, and 603 s. UV-vis (MeCN): λmax377 nm (ε 54), 497 nm (ε 28).


## 3.2.2. Analysis of Zn(C19H20N4O3)(OAC)2

Yield,
60%. Anal. Calcd. Zn(C19H20N4O3)(OAC)2: C, 42.57;
H, 3.73; N, 10.45. Found: C, 43.5; H, 3.86; N, 10.82. H1 NMR (DMSO):
8.30(1H, d, pyridine), 8.06(1H, d, pyridine), 7.5(1H, d, pyridine), 3.3(3H, s,
NCH3), 3(3H, s, NCH3), 1.2–2.35(2H and 1H, m,
cyclohexan); IR (KBr, cm−1): 1691 s, 1625 m, 1439 w, 1246 w, 1158 w, and 425 s. UV-vis (MeCN): λmax262 nm (ε 110),
302 nm (ε 100), 344 nm (ε 78), and 415 nm (ε 60).


## 3.2.3. Analysis of Fe(C19H20N4O3)Cl3

Yield, 75%. Anal. Calcd. Fe(C19H20N4O3)Cl3: C, 44.32;
H, 3.88; N, 10.88. Found: C, 44.86; H, 4.18; N, 11.38. H1 NMR
(DMSO): 9.67(1H, d, pyridine), 9.02(1H, d, pyridine), 7.95(1H, d, pyridine),
3.7(3H, s, NCH3), 3.1(3H, s, NCH3), 1.5–2.8(2H and 1H, m,
cyclohexan); IR (KBr, cm−1): 1600 w, 1546 w, 1444 w, 1154 s, 514 m, and 599 s. UV-vis (MeCN): λmax257 nm (ε 280),
314 nm (ε 156), 363 nm (ε 126), 440 nm (ε 60), and 494 nm (ε 28).


## 3.2.4. Analysis of Pd(C19H20N4O3)Cl2

Yield, 88%.
Anal. Calcd. Fe(C19H20N4O3)Cl3: C, 43.07;
H, 3.77; N, 10.57. Found: C, 43.45; H, 3.95; N, 10.89. H1 NMR
(DMSO): 8.31(1H, d, pyridine), 7.55(1H, d, pyridine), 7.37(1H, d, pyridine), 2.55(3H,
s, NCH3), 2.5(3H, s, NCH3), 1.18–2.07(2H and 1H, m,
cyclohexan); IR (KBr, cm−1): 1649 s, 1546 m, 1467 m, 1266 m, 1142 m, and 495 m. UV-vis (MeCN): λmax261 nm (ε 220), 307
nm (ε 130), 442 nm (ε 118), and 660 nm (ε 75).


## 3.3. Cytotoxicity Studies

CDP ligand and Ti(C19H20N4O3)F4, Zn(C19H20N4O3)
(OAC)2, Fe(C19H20N4O3)Cl3,
and Pd(C19H20N4O3)Cl2 complexes arefive  compounds which were
assayed for cytotoxicity in vitro against K562 (human chronic myeloid leukemia)
cells and Jurkat (human T lymphocyte carcinoma) cells.The two cell lines were provided by the Pasteur
Institute in Iran.
The procedure for cytotoxicity studies was similar to that reported earlier [16].
Briefly, in order to calculate the concentration of each drug that produces a
50% inhibition of cell growth (IC50), 190 mL of cell suspension 4×105 cell/mL) was exposed to various concentrations of ligand and complexes
dissolved in sterile DMSO. The final concentration of DMSO in the growth medium
was 2% (v/v) or lower, concentrations without effect on cell replication [17, 18].After the incubation periods 72 hours for all cell
lines, the cell concentrations were determined both in control and in
drug-treated cultures. All experiments were done  for six times (see Figure2).(a) Tumor cell
after 72 h without Pd(C19H20N4O3)Cl2 compound. (b) tumor cell after 72 h with Pd(C19H20N4O3)Cl2 compound.
(a)(b)


## 4. Results and Discussion

### 4.1. Preparation for Ligand, CDP, and Ti(IV), Zn(II), Fe(III), and Pd(II) Complexes

The
reaction of Ti(IV), Zn(II), Fe(III), and Pd(II) salts with the ligand, CDP,
results in the formation of [ML] for M = Ti(IV), Zn(II), Fe(III), and Pd(II).
All complexes are quite stable and could be stored without any appreciable
change. All complexes were characterized by several techniques using elemental
analyze (C, H, N), FT-IR, electronic spectra, and molar conductance
measurements. The elemental analysis data suggest the stoichiometry to be 1:1
[M:L] ratio formation. The molar conductance measurements reveal the presence
of 1:1 electrolytic nature complexes. The complexes Ti(C19H20N4O3)F4, Zn(C19H20N4O3)(OAC)2,
and Pd(C19H20N4O3)Cl2 do not have
sharp melting points but decompose above 237°C, 290°C, and
331°C, respectively, but Fe(C19H20N4O3)Cl3 complex has 153°C–155°C
melting point. They are insoluble in common organic solvents, such as ethanol,
methanol, chloroform, or acetone; however, they are soluble in DMSO and DMF. Their
structures were characterized by elemental analysis, H1NMR and IR. Their
elemental analyses are in accord with their proposed formula. The spectral data
of the complexes have good relationship with the literature data.


### 4.2. Cytotoxicity Assays In Vitro

CDP
ligand and Ti(C19H20N4O3)F4, Zn(C19H20N4O3)
(OAC)2,
Fe(C19H20N4O3)Cl3, and Pd(C19H20N4O3)Cl2 complexes
have been tested against two human cancer cell lines: K562 and Jurkat. The IC50 cytotoxicity values of the complexes were compared to those found for the
starting organic bases as well as  for some of the anticancer agents used
nowadays, that are  cisplatin and oxaplatin compounds [19].The general method used for testing on antitumor
properties of these compounds is the standard testing method that has been
previously described in greater detail.After preincubation
lasting for 12 hours at37°C in a 5%CO2 atmosphere and 100% humidity, the tested compounds in the concentration ranges of
0.1–250 μM for CDP, of 0.1–150 μM for Ti(C19H20N4O3)F4, of 0.1–100 μM for Zn(C19H20N4O3)(OAC)2, of  0.1–200 μM for Fe(C19H20N4O3)Cl3,
and of 0.1–97 μM for Pd(C19H20N4O3)Cl2 were added.
The incubation lasted  for
72 hours and at the end of this period IC90 and IC50 of the dead cells and
live cells were measured by trypan blue. The mechanism by which these complexes act as
antitumor agents is apoptosis. IC90 and IC50 values that
are the compounds concentrations lethal for 90% and 50% of the tumor cells were
determined both in control and in compounds concentrations lethal for both in
compounds-treated cultures. The compounds were first dissolved in DMSO and then
filtrated. The corresponding 50% and 90% inhibitory doses (IC50 and IC90)
values are shown in Table 1.Table 1
72-hourIC50 and IC90 values (μM) obtained for CDP and three CDP complexes.
ComplexesIC50 for cell lineIC90 for cell lineK562JurkatK562JurkatCDP>110>110——Ti(C19H20N4O3)F4>70>50>100>100Zn(C19H20N4O3)(OAC)2>100>100>150>150Fe(C19H20N4O3)Cl3>45>40>120>120Pd(C19H20N4O3)Cl2>36>33>85>80


## 4.1. Preparation for Ligand, CDP, and Ti(IV), Zn(II), Fe(III), and Pd(II) Complexes

The
reaction of Ti(IV), Zn(II), Fe(III), and Pd(II) salts with the ligand, CDP,
results in the formation of [ML] for M = Ti(IV), Zn(II), Fe(III), and Pd(II).
All complexes are quite stable and could be stored without any appreciable
change. All complexes were characterized by several techniques using elemental
analyze (C, H, N), FT-IR, electronic spectra, and molar conductance
measurements. The elemental analysis data suggest the stoichiometry to be 1:1
[M:L] ratio formation. The molar conductance measurements reveal the presence
of 1:1 electrolytic nature complexes. The complexes Ti(C19H20N4O3)F4, Zn(C19H20N4O3)(OAC)2,
and Pd(C19H20N4O3)Cl2 do not have
sharp melting points but decompose above 237°C, 290°C, and
331°C, respectively, but Fe(C19H20N4O3)Cl3 complex has 153°C–155°C
melting point. They are insoluble in common organic solvents, such as ethanol,
methanol, chloroform, or acetone; however, they are soluble in DMSO and DMF. Their
structures were characterized by elemental analysis, H1NMR and IR. Their
elemental analyses are in accord with their proposed formula. The spectral data
of the complexes have good relationship with the literature data.


## 4.2. Cytotoxicity Assays In Vitro

CDP
ligand and Ti(C19H20N4O3)F4, Zn(C19H20N4O3)
(OAC)2,
Fe(C19H20N4O3)Cl3, and Pd(C19H20N4O3)Cl2 complexes
have been tested against two human cancer cell lines: K562 and Jurkat. The IC50 cytotoxicity values of the complexes were compared to those found for the
starting organic bases as well as  for some of the anticancer agents used
nowadays, that are  cisplatin and oxaplatin compounds [19].The general method used for testing on antitumor
properties of these compounds is the standard testing method that has been
previously described in greater detail.After preincubation
lasting for 12 hours at37°C in a 5%CO2 atmosphere and 100% humidity, the tested compounds in the concentration ranges of
0.1–250 μM for CDP, of 0.1–150 μM for Ti(C19H20N4O3)F4, of 0.1–100 μM for Zn(C19H20N4O3)(OAC)2, of  0.1–200 μM for Fe(C19H20N4O3)Cl3,
and of 0.1–97 μM for Pd(C19H20N4O3)Cl2 were added.
The incubation lasted  for
72 hours and at the end of this period IC90 and IC50 of the dead cells and
live cells were measured by trypan blue. The mechanism by which these complexes act as
antitumor agents is apoptosis. IC90 and IC50 values that
are the compounds concentrations lethal for 90% and 50% of the tumor cells were
determined both in control and in compounds concentrations lethal for both in
compounds-treated cultures. The compounds were first dissolved in DMSO and then
filtrated. The corresponding 50% and 90% inhibitory doses (IC50 and IC90)
values are shown in Table 1.Table 1
72-hourIC50 and IC90 values (μM) obtained for CDP and three CDP complexes.
ComplexesIC50 for cell lineIC90 for cell lineK562JurkatK562JurkatCDP>110>110——Ti(C19H20N4O3)F4>70>50>100>100Zn(C19H20N4O3)(OAC)2>100>100>150>150Fe(C19H20N4O3)Cl3>45>40>120>120Pd(C19H20N4O3)Cl2>36>33>85>80


## 5. Conclusion

It is clear
from the above discussion that Ti(IV), Zn(II), Fe(III) and Pd(II) complexes
and CDP ligand offer a new outlook for chemotherapy. The results
of antitumor activity show that the metal complexes exhibit antitumor
properties and it is important to note that they show enhanced inhibitory
activity compared to the parent ligand. The
mechanism by which these complexes act as antitumor agents is apoptosis. It has
also been proposed that concentration plays a vital role in increasing the
degree of inhabitation.



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*Source: 501021-2009-01-26.xml*