Arene-ruthenium(II) complexes containing 5-fluorouracil-1-methyl isonicotinate: Synthesis and characterization of their anticancer activity

Article abstract of DOI:10.1016/j.ica.2012.02.046

To integrate the respective advantages exhibited by half-sandwich arene-Ru(II) fragments and 5-FU derivatives, a multifunctional ligand that links isonicotinic acid and 5-Fu (5-fluorouracil) through an ester bond for achieving the enhanced effect and an organoruthenium(II) complex containing this ligand were prepared and characterized by standard analytical techniques. The cytotoxicity of these organoruthenium(II) compounds containing 5-fluorouracil-1-methyl isonicotinate against BEL-7402 human cancer cells was moderately improved, which implies synergistic action of 5-Fu and the half-sandwich-structured arene-Ru(II). In addition, the organoruthenium(II) compounds containing isonicotinic acid and methyl isonicotinate are also discussed, and the crystal structure of [(η⁶-p-cymene)RuCl ₂(methyl isonicotinate)] (4) was determined; its piano-stool structure is analyzed in this paper.

Full text of DOI:10.1016/j.ica.2012.02.046

Inorganica Chimica Acta 388 (2012) 78–83
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Arene-ruthenium(II) complexes containing 5-fluorouracil-1-methyl
isonicotinate: Synthesis and characterization of their anticancer activity
⇑
⇑
Kuan-Guan Liu, Xiao-Qing Cai , Xian-Chuan Li, Da-An Qin, Mao-Lin Hu
College of Chemistry and Materials Engineering, Wenzhou University, Wenzhou 325035, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
To integrate the respective advantages exhibited by half-sandwich arene-Ru(II) fragments and 5-FU
derivatives, a multifunctional ligand that links isonicotinic acid and 5-Fu (5-fluorouracil) through an ester
bond for achieving the enhanced effect and an organoruthenium(II) complex containing this ligand were
prepared and characterized by standard analytical techniques. The cytotoxicity of these organorutheni-
um(II) compounds containing 5-fluorouracil-1-methyl isonicotinate against BEL-7402 human cancer cells
was moderately improved, which implies synergistic action of 5-Fu and the half-sandwich-structured
Received 30 December 2011
Received in revised form 22 February 2012
Accepted 28 February 2012
Available online 13 March 2012
Keywords:
Bioorganometallic
arene-Ru(II). In addition, the organoruthenium(II) compounds containing isonicotinic acid and methyl
6
isonicotinate are also discussed, and the crystal structure of [(
g
-p-cymene)RuCl
2
(methyl isonicotinate)]
5
-Fluorouracil (5-Fu)
(
4) was determined; its piano-stool structure is analyzed in this paper.
Organoruthenium
Crystal structure
Anticancer
Ó 2012 Elsevier B.V. All rights reserved.
1
. Introduction
-Fluorouracil (5-Fu) has been used in the treatment of various
the areas of diagnosis and therapy [16]. To integrate the advanta-
ges exhibited by half-sandwich arene-Ru(II) fragments and by 5-
Fu derivatives [17], the arene-Ru(II) complex coupled with 5-fluo-
rouracil-1-methyl isonicotinate was synthesized for the first time,
and its anticancer activity was tested in vitro against HL-60 and
BEL-7402 human cancer cells. The arene-Ru(II) complex coupled
with methyl isonicotinate was also prepared, and its piano-stool
structure was analyzed in this paper.
5
cancers, but its strong toxicities to the gastric system, intestinal
mucosa and bone marrow have limited its clinical application [1].
To solve these problems and further develop more efficient, low-
toxicity drugs, scientists have committed to the modification of
5
-FU through the introduction of nucleoside analogues [2], short
peptides [3,4], glucose [5], polymers [6], porphyrins [7], sulfona-
mides [8], or stable nitroxide radicals [9] into the molecules. For
example, a variety of sulfonyl 5-Fu derivatives [4] and 5-Fu-
dipeptide derivatives [10] have recently been synthesized, and their
biological activities were evaluated in vitro. Building on those suc-
cessful cases, we here report the design of a multifunctional ligand
that links isonicotinic acid and 5-Fu through an ester bond to
achieve enhanced effects.
Because of their preferential accumulation in cancer cells and
their low toxicity, arene-Ru(II) complexes have recently attracted
increasing interest as another potential anticancer bioorganome-
tallic agent [11–15]. Beyond the traditional view that ligands are
merely metal binders, various ligands are increasingly being devel-
oped to enhance the applicability of metal-based agents. Tailored,
multifunctional ligands for metal-based medicinal agents offer
exciting potential and may play an integral role in muting the
toxicity of metallodrugs; they would therefore positively impact
2
. Experimental
2
2
.1. Syntheses of complexes
.1.1. Synthesis of methyl isonicotinate (1)
A mixture of isonicotinic acid (3.1 g, 25 mmol), absolute metha-
nol (16 mL, excess) and conc. sulfuric acid (2 mL, excess) was heated
to reflux for 12 h. After the solution cooled, it was slowly added to
2
reached 7–8. The above process was performed under continuous
stirring. The product, an ester, was extracted twice with ether. The
extract liquor was dried with an excess of anhydrous sodium sulfate
0% aqueous sodium carbonate solution until the pH of the mixture
(
approximately 3 g) for 12 h. The distillation of the dried ester ex-
1
tract yielded yellow liquid 1 (2.52 g, 18.4 mmol, 73.5%). H NMR
CDCl , 500 MHz, 25 °C) d 8.78 (2H, dd, J = 4.5, 1.5 Hz, C2HC6H-
Py); 7.84 (2H, dd, J = 4.5, 1.5 Hz, C3HC5H-Py), 3.96 (3H, s, OCH ).
C@O; 1561.74
(
3
3
IR (KBr): 3035.66
m
CH–Py; 2955.03
m
CH—CH3 ; 1733.03
m
d
1
(N–CH in the plane); 1438.27 C@C; 1286.06
120.43 dCH; 1066.14 dCH; 965.79 dCH Py; 847.68 dC–C out of the plane
m
m
Py ring; 1202.88
m
C–O
;
;
⇑
Corresponding authors. Tel.: +86 577 8866 1902; fax: +86 577 8668 9300.
E-mail addresses: cxq@wzu.edu.cn (X.-Q. Cai), hu403cn@yahoo.com.cn
(
M.-L. Hu).
758.10 dC–C in the plane; 709.09 dPy.
0
020-1693/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.ica.2012.02.046

K.-G. Liu et al. / Inorganica Chimica Acta 388 (2012) 78–83
79
2.1.2. Synthesis of 5-fluorouracil-1-methyl isonicotinate (2)
Me, CHMe
2
). 13C NMR (DMSO-d
6
, 126 MHz, 25 °C) d 165.08
The synthesis of compound 2 was performed in three steps: the
(COOMe); 150.70 (C2C6, Py); 136.68 (C4, Py); 122.42 (C3C5, Py);
106.32, 100.01, 86.29, 85.44, 83.01, 81.39 (C , cymene); 52.70
(Me, COOMe); 29.93 (CHMe ); 21.46 (Me, CHMe ); 17.83 (Me). IR
(KBr): 3058.85 CH–Py; 2961.02 CO2 ; 1732.33
1553.86 C@N in pyridine ring; 1423.32 C@C in pyridine ring; 1292.26
ring; 1210.36
C–C out of the plane; 769.17 dC–C in the plane; 696.71 dPy
first step was the preparation of 1,3-bis(hydroxymethyl)-5-fluoro-
uracil (5-Fu(CH OH) ), in which 5-Fu (1.94 g, 15 mmol) and 37%
formaldehyde solution (10 mL, excess) were heated for 6 h at
6 4
H
2
2
2
2
m
m
CH—CH2 ; 2362.59
m
m
C@O
;
6
5
0 °C. Removal of the solvent and excess formaldehyde resulted in
-Fu(CH OH) in quantitative yield. In the second step, isonicotinic
OH) in the
m
m
m
Py
2
2
mC–O; 1122.21 dCH;1057.35 dCH; 985.67 dCH Py; 868.64
acid (1.24 g, 10 mmol) was coupled with 5-Fu(CH
2
2
d
.
presence of dicyclohexylcarbodiimide (DCC) (3.11 g, 15 mmol)
and N-hydroxysuccinimide (HO-Su) (1.38 g, 12 mmol) in DMF
6
2.1.5. Synthesis of [(
g
2
-p-cymene)RuCl (5-fluorouracil-1-methyl
(
35 mL) at room temperature for 24 h. The progress of the reaction
isonicotinate)] (5)
was monitored by TLC. In the last step, 20 mL of 5% Na CO solution
2
3
Analogous to the procedure described in Section 2.1.3, com-
pound 5 was obtained from [Ru(g -p-cymene)Cl ] (M) (0.12 g,
2 2
0.2 mmol) and 5-fluorouracil-1-methyl isonicotinate (0.107 g,
0.4 mmol). The yield of the product, a yellow–brown powder,
was 0.174 g (0.30 mmol), which corresponds to a yield of 75%.
6
was added to hydrolyze the 3-hydroxymethyl group in the 5-Fu, and
the solution was subsequently treated with concentrated HCl until a
pH of 5–6 was reached. After the product was filtered and dried with
an infrared lamp, white powder 2 (1.1 g, 4.15 mmol, 42%) was
obtained. The melting point of the white power was 209.6–
The melting temperature of compound 5 was greater than
1
1
2
8
10.1 °C; H NMR (DMSO-d
.81 (2H, dd, J = 4.4, 1.6 Hz, C2HC6H-Py); 8.25 (1H, d, J = 6.6 Hz,
6
, 500 MHz, 25 °C) d 12.01 (1H, S, NH);
200 °C; H NMR (DMSO-d
6
, 500 MHz, 25 °C) d 12.03 (1H, S, NH);
8.82 (2H, dd, J = 4.4, 1.6 Hz, C2HC6H-Py); 8.25 (1H, d, J = 6.6 Hz,
C6H-5-Fu); 7.84 (2H, dd, J = 4.4, 1.6 Hz, C3HC5H-Py); 5.87 (2H, s,
C6H-5-Fu); 7.85 (2H, dd, J = 4.4, 1.6 Hz, C3HC5H-Py); 5.86 (2H, s,
1
3
OCH
57.53 (JC–F = 26.1 Hz, C4, 5-Fu); 150.87 (C2, 5-Fu); 149.36 (C2C6,
Py); 139.64 (JC–F = 230.7 Hz, C5, 5-Fu); 136.05 (C4, Py); 129.45
2
N). C NMR (DMSO-d
6
, 126 MHz, 25 °C) d = 164.17 (COO);
OCH
2
N); 5.81 (4H, dd, J = 20.4, 6.4 Hz, C
6
H
4
); 2.84 (sept, 1H,
); 2.09 (s, 3H, Me); 1.20 (6H, dd, J = 2.1, 4.8 Hz, Me, CHMe ).
, 126 MHz, 25 °C) d = 164.09 (COO); 157.42
1
CHMe
C NMR (DMSO-d
2
2
1
3
6
(
(
1
J
C–F = 34.5 Hz, C6, 5-Fu); 122.72 (C3C5, Py); 71.83 (OCH
KBr): 3327.73 N–H; 3040.11 CH–Py; 2927.72, 2849.44
729.33 C@O; 1626.31
plane); 1451.20 C@C; 1408.47
C–O; 1110.22 dCH; 962.16 dCH Py; 856.28 dC–C out of the plane
2
N). IR
CH—CH2
;
(JC–F = 26.1 Hz, C4, 5-Fu); 150.80 (C2, 5-Fu); 149.27 (C2C6, Py);
139.57 (JC–F = 230.6 Hz, C5, 5-Fu); 135.98 (C4, Py); 129.35
(JC–F = 34.5 Hz, C6, 5-Fu); 122.62 (C3C5, Py); 106.36, 100.05,
m
m
m
m
mC@O in pyrimidine ring; 1574.34 d(N–CH in the
m
m
C@C in pyrimidine ring; 1243.47
m
C–F
;
86.31, 85.46, 83.01, 81.45 (C
(CHMe ); 21.45 (Me, CHMe ); 17.81 (Me). IR (KBr): 3326.26
3134.66 CH–Py; 2929.20 CO2 ; 1668.86
idine ring; 1577.91 C@N in pyridine ring; 1408.53
C–F; 876.20 dC–C out of the plane; 812.17 dRu–N; 761.86 dC–C
6 4 2
H , cymene); 71.76 (OCH N); 29.93
1
7
162.07
m
;
2
2
m
N–H
C@O in pyr-
C@C in pyridine ring
;
57.65 dC–C in the plane; 650.27, 557.97 dPy
.
m
m
CH—CH2 ; 2359.43
m
m
m
m
;
6
2
.1.3. Synthesis of [(
g
-p-cymene)RuCl
(M) (0.12 g, 0.2 mmol) and isonicotinic
acid (0.049 g, 0.4 mmol) in anhydrous CH Cl (10 mL) were re-
2
(isonicotinic acid)] (3)
1245.29 m
6
[
Ru(g
-p-cymene)Cl
]
2 2
in the plane; 644.28, 548.84 dPy; 487.39 dRu–Cl.
2
2
fluxed under argon protection for 6 h. The solution was kept over-
night and then filtered to remove a small amount of insoluble
substance under reduced pressure. The brown–red solution was
slowly condensed to ca. 1 mL on a rotary evaporator. After the
solution was cooled À5 °C, a brown–red product formed. This
product was collected, washed with petroleum ether several times,
and dried under reduced pressure. The brown–red powder is noted
2.2. Instruments
The melting points were measured with an X-4 melting point
apparatus. Infrared spectra (IR) were recorded on a Perkin–Elmer
2000 system in the range of 4000–400 cm using the KBr disk
technique. Nuclear magnetic resonance (NMR) spectra were ob-
tained on a Bruker AVANCE-500 spectrometer with DMSO-d
À1
6
or
1
as substance 3. Yield: 0.16 g (0.37 mmol, 93.2%); m.p.: >200 °C; H
CDCl as the solvent. The crystallographic data were collected on
3
NMR (DMSO-d
C2HC6H-Py); 7.84 (2H, dd, J = 4.5, 1.5 Hz, C3HC5H-Py); 5.79 (2H,
dd, J = 20.4, 6.2 Hz, C ); 5.50 (2H, dd, J = 24.7, 5.9 Hz, C );
); 2.01 (s, 3H, Me); 1.19 (6H, dd, J = 2.1,
6
, 500 MHz, 25 °C) d 8.98 (2H, dd, J = 4.5, 1.5 Hz,
a Bruker Smart-Apex CCD diffractometer. TGA/DSC analyses were
performed on a NETZSCH STA 449C unit. The thermal properties
of complexes 2 and 5 were studied by means of simultaneous
TGA/DSC analyses at a heating rate of 5 °C/min from room temper-
ature to 800 °C under a nitrogen atmosphere. All reagents were
purchased commercially and were used without further purifica-
tion. Antimicrobial activities were evaluated at The National Center
for Drug Screening in Shanghai, China.
6
H
4
6 4
H
2
4
1
.82 (sept, 1H, CHMe
2
13
.8 Hz, Me, CHMe
66.13 (COOH); 150.54 (C2C6, Py);138.09 (C4, Py); 123.08 (C3C5,
, cymene);
); 17.83 (Me). IR (KBr):
CH–Py; 2964.90
C@N in pyridine ring; 1423.84
Py ring; 1196.21
CH Py; 815.21 dC–C out of the plane; 745.84 dC–C in the plane; 683.03
Py; 522.48 dRu–Cl
2
).
6
C NMR (DMSO-d , 126 MHz, 25 °C) d
6 4
Py); 106.36, 100.05, 86.32, 85.46, 82.92, 81.46 (C H
2
3
m
1
d
d
9.93 (CHMe
450.66
C@O; 1605.01
284.59
2 2
); 21.46 (Me, CHMe
O–H, –COOH; 3062.07
m
m
mCH—CH2 ; 1727.79
m
m
C@C in pyridine ring
;
2.3. Crystal-structure determination and refinement
m
mC–O; 1129.82 dCH; 1013.05 dCH; 978.98
An X-ray-quality crystal of the complex 4 was grown from
dichloromethane diffusion by petroleum ether at room tempera-
ture, and X-ray crystallographic data were recorded by mounting
.
6
3
2.1.4. Synthesis of [(
g
-p-cymene)RuCl
2
(methyl isonicotinate)] (4)
a red–brown single-crystal of complex 4 (0.44 Â 0.27 Â 0.20 mm )
Analogous to the procedure described in Section 2.1.3, com-
onto a glass fiber. Diffraction data were collected at 25(±2) °C using
6
pound 4 was obtained from [Ru(
g
-p-cymene)Cl
2
]
2
(M) (0.245 g,
graphite-monochromated MoK
a (k = 0.071073 nm) radiation with
0
.4 mmol) and methyl isonicotinate (0.11 g, 0.8 mmol). The final
an -scan technique. Determinations of the crystal class, orienta-
x
product, a yellow powder, weighed 0.28 g (0.64 mmol), which cor-
tion matrix, and cell dimensions were performed according to the
established procedures. Lorentz polarization and absorption correc-
tions were applied [18]. Empirical absorption corrections were
performed with the SADABS program. Most of the non-hydrogen
atoms were located using direct methods, and the remainder were
derived by subsequent Fourier syntheses. All non-hydrogen atoms
were refined anisotropically, and all hydrogen atoms were held
responds to a yield of 80%. The melting point of the yellow product
1
was determined to be greater than 200 °C. H NMR (DMSO-d
6
,
5
(
C
(
00 MHz, 25 °C) d 8.98 (2H, dd, J = 4.5, 1.5 Hz, C2HC6H-Py); 7.84
2H, dd, J = 4.5, 1.5 Hz, C3HC5H-Py); 5.79 (2H, dd, J = 20.4, 6.2 Hz,
); 5.50 (2H, dd, J = 24.7, 5.9 Hz, C ); 3.91 (3H, s, OCH ); 2.82
sept, 1H, CHMe ); 2.01 (s, 3H, Me); 1.21 (6H, dd, J = 2.1, 4.8 Hz,
H
6 4
H
6 4
3
2

8
0
K.-G. Liu et al. / Inorganica Chimica Acta 388 (2012) 78–83
Table 1
Crystallographic data and structure refinement summary for complex 4.
5.87 ppm was assigned to a 1-methyl group, and the two dd peaks
at 8.81 and 7.84 ppm (J = 4.4, 1.6 Hz), respectively, are again in
agreement with two different types of protons of the pyridine ring.
Comparable with complex 1, protons of the methyl group in each
compound are discrepant for the electronic pulling effect of N,
but protons of the pyridine ring are consistent with each other.
The structure is further confirmed by C NMR. The values of the
C4,C5, and C6 of 5-Fu coupling with the F atom are 26.1, 230.7,
and 34.5 Hz, respectively, which also agree with the results of
other 5-Fu derivates [4,10].
Empirical formula
Formula weight
T (K)
17 2 2
C H21Cl NO Ru
443.32
298(2)
Crystal system
Space group
Unit-cell dimensions
a (Å)
b (Å)
c (Å)
monoclinic
P21/c
1
3
16.641(3)
15.469(3)
7.3064(12)
90
102.544(3)
90
1836.0(5)
4
1.604
a
(°)
b (°)
(°)
Like other arene-Ru(II) complexes with pyridine derivatives
c
[25–27], the synthesis of complexes 3–5 by reaction of the starting
3
6
V (Å )
material, dichloro(
g
-p-cymene)ruthenium dimer (M), with the
Z
D
pyridine ligand in a ratio of 1:2 at room temperature is straightfor-
ward (as shown in Scheme 2).
calc (mg/m3)
À1
Absorption coefficient (mm
)
1.152
Complexes 3–5 are stable in air at room temperature and are
soluble in most organic solvents, including methanol, chloroform,
and dichloromethane, but complex 5 is only soluble in dimethyl-
formamide (DMF) and dimethylsulfoxide (DMSO). These results
indicate that the solubility of arene-Ru(II) complexes is related to
the solubility of their ligands. All of the above complexes have been
F(000)
896
1.25–25
Theta range for data collection (°)
Index ranges
À10 6 h 6 19, À18 6 k 6 18,
À8 6 l 6 8
Reflections collected/unique (Rint
Completeness to 2h = 25.00 (%)
Maximum and minimum
transmission
)
9497/3231 (0.0287)
99.9
0.8023 and 0.6311
1
13
confirmed by IR, H NMR, and C NMR. Complexes 3–5 exhibited
Refinement method
full-matrix least-squares on F2
3231/0/212
1.244
1
13
similar analytical IR, H NMR and C NMR results, which is consis-
Data/restraints/parameters
Goodness-of-fit (GOF) on F2
tent with the fact that they have a common structure of a half-
6
sandwich of
g
-p-cymene-Ru(II)Cl
2
and isonicotinate. The infrared
Final R indices [I > 2
R indices (all data)
Largest difference peak and hole
r(I)]
R
R
1
= 0.0614, wR
= 0.0707, wR
2
= 0.1469
= 0.1572
1
2
spectra (IR) of complexes 3–5 exhibit broad bands at approxi-
À1
À1
À1
0.873 and À0.594
mately 3058–3134 cm
1
,
2929–2964 cm
,
1727–1732 cm ,
À3
(
e Å
)
À1
À1
553–1605 cm , and 1408–1423 cm , which correspond to the
stretching frequencies of the C–H bonds of the aromatic ring, the
C–H bonds of methyl groups, the C@O bond of the carbonyl group,
the C@N bond of the pyridine ring, and the C@C bond of the pyri-
dine ring, respectively. Typically, the O–H stretching of complex 3
stationary and included in the final stage of full-matrix least-
squares refinement based on F using the SHELXS-97 [19], SHELXL-97
2
[
20] and SHELXTL [21] program packages. Pertinent crystallographic
À1
appears at approximately 3450 cm , which is in line with the –
data are presented in Table 1.
COOH group. The NH stretching of complex 5 occurs at approxi-
À1
mately 3326 cm , which is in line with complex 2. The proton
2.4. In vitro cytotoxicity assay experiment
NMR spectra of all three arene-Ru(II) complexes show a slight
downfield shift compared to the spectrum of the free ligand, espe-
cially the proton near N in the pyridine ring, which indicates that
the nitrogen atom coordinated with ruthenium due to the deshiel-
The antitumor activities of compounds 2, 5 and [Ru(g
6-p-
cymene)Cl ] (M) were evaluated in vitro by the SRB method for
2 2
BEL-7402 cells and by the MTT method for HL-60 cells at The
National Center for Drug Screening in Shanghai, China. All the com-
plexes were dissolved in DMSO for testing. The IC50 value was
calculated using SPSS according to the literature [22].
13
ding effect of the central metal [28]. C NMR spectra of complexes
3
–5 show nearly the same peaks as the spectrum of the free ligand,
13
which demonstrates that the deshielding effect in C NMR can be
ignored in this coordinated condition.
3
. Results and discussion
3.1. Thermal analysis (TGA/DSC)
Consistent with the literature [23], compound 1 was obtained in
The TGA of compound 2 in Fig. 1 shows that there are two main
mass-loss steps during the decomposition of the compound. The
first step occurs between 174 and 272 °C, and the second step
occurs between 446 and 591 °C. The DSC results for compound 2
show endothermic peaks at 202 and 490 °C. The first peak indicates
that compound 2 produces one stable catabolite, and the second
peak indicates the possible phase transition during which the com-
pound is completely decomposed.
a yield of 73.5%. The yellow-oil complex is air stable and soluble as
well as stable in most organic solvents, including diethyl ether,
dichloromethane, and chloroform. The 1H NMR and IR spectros-
copy results of compound 1 are consistent with its formulation.
As shown in Scheme 1, the synthesis of compound 2 is a three-
step process [24], which has a final yield of 42%. The white-powder
complex 2 is air stable, but it is insoluble in most organic solvents
except dimethylformamide (DMF) and dimethylsulfoxide (DMSO).
For compound 5, the TGA in Fig. 2 shows one main step of
weight loss from 193 to 437 °C during which the compound
1
13
The H NMR, C NMR and IR spectroscopy results are consistent
with its formulation. The infrared spectra (IR) of complex 2 exhibit
2
decomposes. The final decomposition product is RuO . The total
À1
broad bands at approximately 3327, 1729, 1626, and 1451 cm
,
weight loss of 75.5% is close to the calculated value of 76.7%. The
DSC trace of compound 5 shows one endothermic peak at 202 °C
and one exothermic peak at 490 °C, which indicates a possible
phase transition during the decomposition process.
which correspond to the stretching frequencies of the N–H bond
of N3 of the 5-Fu ring, the C@O bond of the ester group, the C@O
bond of the 5-Fu ring, and the C@C and C@N bonds of the pyridine
ring, respectively. Complex 2 shows a singlet at 12.01 ppm and a
doublet at 8.25 ppm, which correspond to the proton of 5-Fu(N3)
and the proton of 5-Fu(C6), respectively. The value of the proton
3.2. Single-crystal X-ray analysis of complex 4
3
of 5-Fu(C6) coupling with the F atom ( JFC@CH) is 6.6 Hz, which
Complex 4 crystallizes in the monoclinic space group P21/c, and
agrees with results of other 5-Fu derivates [4,10]. A singlet at
the crystal structure is built of two independent molecules in the

K.-G. Liu et al. / Inorganica Chimica Acta 388 (2012) 78–83
81
Scheme 1. The synthesis route for compound 2.
2
Scheme 2. The synthesis route for [(g (isonicotinic acid derivatives)].
6-p-cymene)RuCl
Fig. 1. Simultaneous TGA/DSC curves of compound 2 performed under an argon
Fig. 2. Simultaneous TGA/DSC curves of compound 5 performed under an argon
atmosphere. The flow rate of nitrogen was 100.0 mL/min.
atmosphere. The flow rate of nitrogen was 100.0 mL/min.
asymmetric unit. The relative orientation of the molecules is de-
picted in Fig. 3, which shows the displacement ellipsoids of the
non-hydrogen atoms. Selected important bond lengths and bond
angles for complex 4 are listed in Table 2. The ruthenium atom is
in a formally six-coordinate environment, and the structure of
complex 4 adopts a distorted piano-stool type of geometry with an-
gles around the ruthenium of 83.89(14)° (N(1)–Ru(1)–Cl(2)),
g
6-arene-Ru(II) complexes [26,29]. The Ru–Cl(1) and Ru–Cl(2) bond
lengths of 2.4108(18) Å and 2.4111(17) Å, respectively, are on the
order of those in related arene-ruthenium(II) compounds [26,29,
30]. The pyridine Ru–N(1) distance is 2.128(5) Å in the range of
Ru–N distances [26,30]. The dihedral angle formed by the pyridine
plane and the
those in analogous compounds [26]. In the structure of complex 4,
two short intramolecular interactions exist, which can be consid-
ered as weak C(15)–H(15)Á Á ÁCl(1) and C(10)–H(10A)Á Á ÁCl(2) hydro-
gen bonds [26] (DÁ Á ÁA distances 2.7402(24) Å and 2.8317(14) Å, D–
HÁ Á ÁA angles 111.730(442)° and 116.556(549)°).
6
g
-p-cymene is 41.546(203)°, which is in line with
8
The
7.03(13)° (N(1)–Ru(1)–Cl(1)), and 87.40(7)° (Cl(1)–Ru(1)–Cl(2)).
6
g -p-cymene ring is planar and the Ru–C distances are almost
equal, with an average bond length of 2.179(6) Å (range 2.157(6)–
6
2
.206(7) Å). The Ru(1) to
g -p-cymene centroid distance is
1
.663 Å, which is consistent with the values reported for other

8
2
K.-G. Liu et al. / Inorganica Chimica Acta 388 (2012) 78–83
Table 3
Percentage of inhibition of HL-60 cell growth (%).
À1
Compound
Concentration (mol L )
À4
À5
À6
À7
À8
10
10
10
10
10
2
M
5
15.7
48.7
6.8
88.6
61.8
16.6
0
0
93.9
45.7
13.1
0
1.3
93.5
20.2
9.8
12.6
9.4
74.6
20.0
18.5
1.6
0
12.5
25.7
a
Adriamycin
5
b
-Fu
a
Adriamycin is the compound of the control experiment.
The percentage of 5-Fu was from previously acquired data.
b
Table 4
Percentage of inhibition of BEL-7402 cell growth (%).
Fig. 3. ORTEP drawing of compound 4 showing the atom numbering scheme.
À1
Compound
Concentration (mol L
)
À4
À5
À6
À7
À8
10
10
10
10
10
Table 2
2
M
5
86.2
83.4
87.1
90.4
81.7
37.0
10.9
41.8
90.6
71.9
16.8
4.2
29.7
85.6
66.4
10.4
4.5
3.5
34.1
21.7
0
4.1
0
23.2
0
Selected bond lengths (Å) and angles (°) for compound 4.
Bond lengths
a
Adriamycin
5
Ru(1)–N(1)
Ru(1)–Cl(1)
Ru(1)–Cl(2)
Ru(1)–C(5)
Ru(1)–C(9)
2.128(5)
Ru(1)–C(8)
Ru(1)–C(6)
Ru(1)–C(4)
Ru(1)–C(7)
2.177(6)
2.183(7)
2.185(6)
2.206(7)
b
-Fu
2.4108(18)
2.4111(17)
2.157(6)
a
Adriamycin is the compound of the control experiment.
The percentage of 5-Fu was from previously acquired data.
b
2.167(6)
Bond angles
N(1)–Ru(1)–C(5)
N(1)–Ru(1)–C(9)
N(1)–Ru(1)–C(8)
N(1)–Ru(1)–C(6)
N(1)–Ru(1)–C(4)
N(1)–Ru(1)–C(7)
Cl(1)–Ru(1)–Cl(2)
N(1)–Ru(1)–Cl(1)
N(1)–Ru(1)–Cl(2)
C(5)–Ru(1)–C(9)
C(5)–Ru(1)–C(8)
C(9)–Ru(1)–C(8)
C(5)–Ru(1)–C(6)
C(9)–Ru(1)–C(6)
C(8)–Ru(1)–C(6)
C(5)–Ru(1)–C(4)
C(9)–Ru(1)–C(4)
C(8)–Ru(1)–C(4)
152.7(3)
91.4(2)
94.7(2)
159.3(2)
114.8(3)
121.8(2)
87.40(7)
87.03(13)
83.89(14)
67.6(3)
79.6(3)
38.0(3)
37.1(3)
79.5(3)
66.7(3)
38.3(3)
37.5(3)
68.3(3)
C(6)–Ru(1)–C(4)
C(5)–Ru(1)–C(7)
C(9)–Ru(1)–C(7)
C(8)–Ru(1)–C(7)
C(6)–Ru(1)–C(7)
C(4)–Ru(1)–C(7)
C(5)–Ru(1)–Cl(1)
C(9)–Ru(1)–Cl(1)
C(8)–Ru(1)–Cl(1)
C(6)–Ru(1)–Cl(1)
C(4)–Ru(1)–Cl(1)
C(7)–Ru(1)–Cl(1)
C(5)–Ru(1)–Cl(2)
C(9)–Ru(1)–Cl(2)
C(8)–Ru(1)–Cl(2)
C(6)–Ru(1)–Cl(2)
C(4)–Ru(1)–Cl(2)
C(7)–Ru(1)–Cl(2)
68.2(3)
67.9(3)
68.3(3)
37.2(3)
37.5(3)
81.6(3)
89.7(2)
122.2(2)
160.1(2)
113.6(2)
92.88(19)
150.4(2)
123.1(2)
149.9(2)
112.5(2)
94.6(2)
Table 5
The IC50
(
l
M)c values of complexes 2 and 5 and [Ru(
g
6-p-cymene)Cl
2
]
2
(M) against
HL-60 and BEL-7402 human cancer cells.
Compound
HL-60
BEL-7402
22.4
2
M
5
>200
>200
>200
0.05
74.3
d
40.6
8.1
0.15
0.84
a
Adriamycin
b
5-Fu
a
b
c
Adriamycin is the compound of the control experiment.
The percentage of 5-Fu was from previously acquired data.
IC50 : the 50% concentration of inhibition.
Not optimal solution.
161.3(2)
88.98(19)
d
4
. Conclusions
The first organoruthenium(II) complex to contain the
3.3. In vitro cytotoxicity assessment
g
6-p-cym-
ene ligand and 5-fluorouracil-1-methyl isonicotinate was prepared
and characterized by standard analytical techniques. The cytotoxic-
ity of this complex against BEL-7402 human cancer cells was found
to be moderate. In addition, organoruthenium(II) compounds that
The percentage of inhibition of the growth of HL-60 and BEL-
7
402 human cancer cells is presented in Tables 3 and 4. The IC50
6
(l
M) values of complexes 2, 5 and [Ru(
g
-p-cymene)Cl
2
]
2
(M)
against HL-60 and BEL-7402 human cancer cells are presented in
Table 5. All the compounds tested in this research are less cytotoxic
than both adriamycin (used as a control) and 5-Fu (determined in
our earlier experiments) in both cell lines. The inhibition of all the
compounds against the HL-60 cells is different from that against
the BEL-7402 cell lines, which suggests that these compounds pos-
sibly have different inhibition mechanisms against various tumor
cells [31]. Although there were less inhibiting effect on the growth
contain isonicotinic acid and methyl isonicotinate were also
6
discussed, and the crystal structure of [(
g
2
-p-cymene)RuCl (methyl
isonicotinate)] (4) was reported in this work. The results of this
work suggest that the utilization of the synergistic action of
coupling 5-Fu with the half-sandwich arene-Ru(II) structure by a
coordinating method provides a promising direction. Work along
this direction is being pursed in our lab.
À4
À8
À1
of HL-60 cells over the concentrations range of 10 –10 mol L
,
the newly prepared complexes 2 and 5 showed a moderate inhib-
iting effect on the growth of BEL-7402 cells, where their IC50 values
Acknowledgments
are 22.4 and 8.1
of other arene-Ru(II) complexes with other pyridine derivatives
27], the IC50 value of compound 5, which is the first arene-Ru(II)
complex to be coupled with 5-fluorouracil-1-methyl isonicotinate,
provides a promising direction that we can develop further.
lM, respectively. More importantly, a comparison
We acknowledge financial support by the National Natural
Science Foundation of China (No. 21071111) and the Natural
Science Foundation of Zhejiang Province for Distinguished Young
Students (No. 2010R424048).
[

K.-G. Liu et al. / Inorganica Chimica Acta 388 (2012) 78–83
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