Synthesis, characterization, and anticancer activity of a series of ketone-N4-substituted thiosemicarbazones and their ruthenium(II) arene complexes

Article abstract of DOI:10.1021/ic401362s

A series of ketone-N⁴-substituted thiosemicarbazone (TSC) compounds (L1-L9) and their corresponding [(η⁶-p-cymene)Ru ^II(TSC)Cl]^+/0 complexes (1-9) were synthesized and characterized by NMR, IR, elemental analysis, and HR-ESI-mass spectrometry. The molecular structures of L4, L9, 1-6, and 9 were determined by single-crystal X-ray diffraction analysis. The compounds were further evaluated for their in vitro antiproliferative activities against the SGC-7901 human gastric cancer, BEL-7404 human liver cancer, and HEK-293T noncancerous cell lines. Furthermore, the interactions of the compounds with DNA were followed by electrophoretic mobility spectrometry studies.

Full text of DOI:10.1021/ic401362s

Article
pubs.acs.org/IC
Synthesis, Characterization, and Anticancer Activity of a Series of
Ketone‑N4‑Substituted Thiosemicarbazones and Their Ruthenium(II)
Arene Complexes
Wei Su,*,†,‡ Quanquan Qian,† Peiyuan Li,*,§ Xiaolin Lei,† Qi Xiao,† Shan Huang,† Chusheng Huang,†
and Jianguo Cui†
†Key Laboratory of Beibu Gulf Environment Change and Resources Utilization (Guangxi Teachers Education University), Ministry of
Education, Nanning, China
‡College of Chemistry and Life Science, Guangxi Teachers Education University, 175 Mingxiu East Road, Nanning 530000, China
§College of Pharmacy, Guangxi University of Traditional Medicine,179 Mingxiu East Road, Nanning, China
S
* Supporting Information
ABSTRACT: A series of ketone-N4-substituted thiosemicarbazone (TSC)
compounds (L1−L9) and their corresponding [(η6-p-cymene)RuII(TSC)Cl]+/0
complexes (1−9) were synthesized and characterized by NMR, IR, elemental
analysis, and HR-ESI-mass spectrometry. The molecular structures of L4, L9, 1−
6, and 9 were determined by single-crystal X-ray diffraction analysis. The
compounds were further evaluated for their in vitro antiproliferative activities
against the SGC-7901 human gastric cancer, BEL-7404 human liver cancer, and
HEK-293T noncancerous cell lines. Furthermore, the interactions of the
compounds with DNA were followed by electrophoretic mobility spectrometry
studies.
arene complexes with ethylenediamine (en) as the ligand can
bind to DNA and thus lead to cytotoxicity toward cancer cells.9
In addition, related complexes incorporating the 1,3,5-triaza-7-
phosphatricyclo-[3.3.1.1]-decane (PTA) ligand, e.g., [(η6-p-
cymene)RuII(PTA)Cl2] (RAPTA-C), showed activity against
metastases.10
The Ru(II)-arene complexes of TSCs have also emerged as
an approach to develop promising lead Ru-based therapeutic
agents. Beckford et al. reported the first structurally
characterized ruthenium-arene half-sandwich complexes with
thiosemicarbazone ligands combining the {(η6-p-cymene)RuII}
moiety with 9-anthracenyl-TSC derivatives as ligands.4a These
complexes exhibited good cytotoxic profiles against different
human cancer cell lines, and their biological activities were
apparently modulated by the TSC coligand.11 More recently,
Smith et al. demonstrated the cytotoxicity and antiparasitic
activity of a set of TSC derivatives and their corresponding
Ru(II)-arene complexes.12 Moreover, the pharmacological
activity of the binuclear TSC Ru-arene complexes were also
demonstrated by Gambino and his co-workers.13
INTRODUCTION
■
Thiosemicarbazones (TSCs) have attracted considerable
attention by chemists and biologists because of their wide
range of pharmacological effects; these compounds and their
metal complexes have shown marked antibacterial, antiviral,
antifungal, and, particularly, antitumor activity.1 In recent years,
a number of TSC derivatives have been synthesized, and their
antitumor activity against a broad spectrum of chemo-
therapeutic properties were also evaluated.2 Moreover, the
complexes consisted of transition metals, and TSC ligands
usually possess more potent pharmacological effects than the
thiosemicarbazone ligands alone.3 The biological properties of
the TSC ligands can be modified and improved by the linkage
to transition metal ions.4
The ruthenium-based complexes, which exhibit considerable
promise in antiproliferation activity and lower toxicity than
platinum drugs,5 have been developed in the last two decades.
In particular, the Ru(III) complexes, [HIm][trans-
RuCl4(DMSO)(Im)] (NAMI-A) and [ImH][trans-Ru-
Cl4(Im)2] (KP1019), have progressed into clinical trials with
very promising results.6 The organometallic Ru(II) arene
complexes, with the half-sandwich type structure, have
demonstrated their potential increasingly.7 Their coordination
sites can be filled with various ligands, which offer numerous
possibilities to modulate biological and pharmacological
properties by proper ligand selection.8 For instance, Ru(II)
Although the TSC Ru-arene complexes have been proven to
have potential as effective anticancer drugs, their distinct modes
of action and biological targets are still the focus of active
Received: May 30, 2013
© XXXX American Chemical Society
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2.890 (s, 3H, C(CH3)2), 2.708 (s, 3H, C(CH3)2), 2.267 (s, 3H, p-cym
CCH3), 1.269 (d, 3H, p-cym CH(CH3)2, J = 6.9 Hz), 1.222 (d, 3H, p-
cym CH(CH3)2, J = 6.9 Hz) ppm. Anal. Calcd for C20H27Cl2N3RuS·
0.5H2O: C, 45.97; H, 5.40; N, 8.04. Found: C, 46.24; H, 5.24; N, 8.09.
Single crystals suitable for X-ray diffraction were obtained by
recrystallization in ethanol and hexane (30/70) solution.
research. In this Article, a series of TSC derivatives (L1−L9)
and their corresponding Ru(II)-arene complexes (1−9) were
synthesized and characterized. The cytotoxicity toward the
SGC-7901 human gastric and BEL-7404 human liver cancer
cell lines of these compounds was investigated. In addition, the
interactions of the compounds with DNA were followed by
electrophoretic mobility spectrometry studies.
[(η6-p-Cymene)RuII(L4)Cl]Cl (4). Yield 25.5 mg, (51%). HR-ESI-MS
(MeOH) m/z [Found (Calcd)]: 428.0740 (428.0739) (100%) {[(η6-
p-cymene)Ru(L4)Cl] − HCl}+. IR (cm−1): ν (NH2, NH) 3436, 3256;
1
EXPERIMENTAL SECTION
ν (CN) 1613; ν (CS) 1025. UV−vis (methanol) λ: 231 nm. H
■
Materials. The starting material [(η6-p-cymene)RuCl2]2 was
NMR (300 MHz, CDCl3) δ: 7.96 (br, 2H, NH2), 7.50 (m, 3H, C-
phenyl), 7.30 (br, 1H, NH), 7.24 (m, 2H, C-phenyl), 5.41 (d, 1H, p-
cym, J = 5.7 Hz), 4.94 (d, 1H, p-cym, J = 5.7 Hz), 4.74 (d, 1H, p-cym, J
= 5.7 Hz), 4.63 (d, 1H, p-cym, J = 5.7 Hz), 3.70 (m, 3H, CNCH3),
2.82−2.73 (m, 1H, p-cym CH(CH3)2), 2.03 (s, 3H, p-cym CCH3),
1.25 (d, 3H, p-cym CH(CH3)2, J = 6.9 Hz), 1.18 (d, 3H, p-cym
CH(CH3)2, J = 6.9 Hz) ppm. Anal. Calcd for C19H25Cl2N3RuS·
0.5H2O: C, 44.88; H, 5.15; N, 8.26. Found: C, 45.15; H, 5.02; N, 8.24.
Single crystals suitable for X-ray diffraction were obtained by
recrystallization in chloroform and hexane (30/70) solution.
prepared according to literature methods.11
General Procedure for the Synthesis of Thiosemicarba-
zones. All of the thiosemicarbazones were prepared by previously
reported procedures.11 The relevant thiosemicarabazide (1 mmol) and
ketone (1 mmol) were combined in methanol (5 mL) with acetic acid
(1−2 drops). The mixture was refluxed for 3 h, during which a white
or off-white precipitate appeared. After 3 h, the mixture was allowed to
cool to room temperature, and dried in vacuo. Ligands were further
1
purified by recrystalliztion from methanol. HR-ESI-MS and H NMR
[(η6-p-Cymene)RuII(L5)Cl]Cl (5). Yield 30 mg (58%). HR-ESI-MS
(MeOH) m/z [Found (Calcd)]: 442.1096 (442.0896) (100%) {[(η6-
p-cymene)Ru(L5)Cl] − HCl}+. IR (cm−1): ν (NH2, NH) 3444, 3182,
3117; ν (CN) 1593; ν (CS) 1037. UV−vis (methanol) λ: 342,
data were collected for all ligands.
General Procedure for the Synthesis of [(η6-p-Cymene)-
RuII(TSC)Cl]+/0 Complexes. The [(η6-p-cymene)RuII(TSC)Cl]Cl
complexes were obtained by reacting the dimer [(η6-p-cymene)-
RuCl2]2 and the thiosemicarbazone ligands.7 Thiosemicarbazone (0.1
mmol) and [(η6-p-cymene)RuCl2]2 (0.05 mmol) were combined in
acetone (8 mL). The resultant mixture was stirred and refluxed. After 3
h, the mixture was allowed to cool to room temperature, and the dark
solid formed was filtered. All metal complexes were subsequently
purified by recrystallization from ethanol and hexane. HR-ESI-MS, IR,
1H NMR (300 MHz, CDCl3), and elemental analysis were performed
1
266, 230 nm. H NMR (300 MHz, CDCl3) δ: 12.62 (br, 1H, NNH),
10.31 (br, 1H, NHCH3), 7.89 (s, 2H, C-phenyl), 7.60 (d, 3H, C-
phenyl), 5.29 (d, 1H, p-cym, J = 5.7 Hz), 4.91 (d, 1H, p-cym, J = 5.7
Hz), 4.67 (d, 1H, p-cym, J = 5.7 Hz), 3.85 (d, 1H, p-cym, J = 5.7 Hz),
3.18 (d, 3H, NHCH3, J = 3.6 Hz), 2.95 (m, 3H, CNCH3), 2.69−2.62
(m, 1H, p-cym CH(CH3)2), 2.02 (s, 3H, p-cym CCH3), 1.16 (d, 3H,
p-cym CH(CH3)2, J = 6.9 Hz), 1.11 (d, 3H, p-cym CH(CH3)2, J = 6.9
Hz) ppm. Anal. Calcd for C20H27Cl2N3RuS·0.75CH2Cl2: C, 43.18; H,
4.98; N, 7.28. Found: C, 42.76; H, 4.84; N, 7.29. Single crystals
suitable for X-ray diffraction were obtained by recrystallization in
chloroform and hexane (30/70) solution.
for all metal complexes.
Characterization Data. [(η6-p-Cymene)RuII(L1)Cl]Cl (1). Yield: 20
mg (44%). HR-ESI-MS (MeOH) m/z [Found (Calcd)]: 366.0581
(366.0581) (100%) {[(η6-p-cymene)Ru(L1)Cl] − HCl}+. IR (cm−1):
ν (NH2, NH) 3432, 3252, 3190; ν (CN) 1621; ν (CS) 1045.
UV−vis (methanol) λ: 306, 264, 229 nm. 1H NMR (300 MHz,
CDCl3) δ: 13.336 (br, 1H, NH), 9.390 (br, 1H, NH2), 6.488 (br, 1H,
NH2), 5.664 (d, 1H, p-cym phenyl-H, J = 6.1 Hz), 5.539 (q, 2H, p-cym
phenyl-H, J = 6.2 Hz), 5.386 (d, 1H, p-cym phenyl-H, J = 6.1 Hz),
2.905−2.813 (m, 1H, p-cym CH(CH3)2), 2.855 (s, 3H, C(CH3)2),
2.660 (s, 3H, C(CH3)2), 2.274 (s, 3H, p-cym CCH3), 1.301 (d, 3H, p-
cym CH(CH3)2, J = 7.0 Hz), 1.238 (d, 3H, p-cym CH(CH3)2, J = 7.0
Hz) ppm. Anal. Calcd for C14H22Cl2N3RuS·H2O: C, 37.00; H, 5.32;
N, 9.25. Found: C, 37.1; H, 5.55; N, 9.24. Single crystals suitable for X-
ray diffraction were obtained by recrystallization in ethanol and hexane
(30/70) solution.
[(η6-p-Cymene)RuII(L6)Cl]Cl (6). Yield 30 mg (51%). HR-ESI-MS
(MeOH) m/z [Found (Calcd)]: 504.1038 (504.1053) (100%) {[(η6-
p-cymene)Ru(L6)Cl] − HCl}+. IR (cm−1): ν (NH2, NH) 3440, 3256,
3137; ν (CN) 1621; ν (CS) 1025. UV−vis (methanol) λ: 273,
229 nm. 1H NMR (300 MHz, CDCl3) δ: 13.005 (br, 1H, NH), 12.349
(br, 1H, NH), 7.917 (s, 2H, phenyl-H), 7.648−7.623 (m, 5H, phenyl-
H), 7.440−7.389 (m, 2H, phenyl-H), 7.316−7.291 (m, 1H, phenyl-H),
5.249 (d, 1H, p-cym phenyl-H, J = 5.7 Hz), 4.914 (d, 1H, p-cym
phenyl-H, J = 5.7 Hz), 4.646 (d, 1H, p-cym phenyl-H, J = 5.7 Hz),
3.910 (d, 1H, p-cym phenyl-H, J = 5.7 Hz), 3.043 (s, 3H, CCH3),
2.698−2.591 (m, 1H, p-cym CH(CH3)2), 2.022 (s, 3H, p-cym CCH3),
1.162−1.112 (m, 6H, p-cym CH(CH3)2) ppm. Anal. Calcd for
C25H29Cl2N3RuS·1.5CH2Cl2: C, 45.28; H, 4.59; N, 5.98. Found: C,
45.53; H, 4.44; N, 6.12. Single crystals suitable for X-ray diffraction
were obtained by recrystallization in chloroform and hexane (30/70)
solution.
[(η6-p-Cymene)RuII(L2)Cl]Cl (2). Yield 20 mg,(43%). HR-ESI-MS
(MeOH) m/z [Found (Calcd)]: 380.0737 (380.0738) (100%) {[(η6-
p-cymene)Ru(L2)Cl] − HCl}+. IR (cm−1): ν (NH2, NH) 3452; ν
(CN) 1625; ν (CS) 1086. UV−vis (methanol) λ: 263, 230 nm.
1H NMR (300 MHz, CDCl3) δ: 12.789 (br, 1H, NH), 9.842 (br, 1H,
NH), 5.657 (d, 1H, p-cym phenyl-H, J = 6.0 Hz), 5.513 (s, 2H, p-cym
phenyl-H), 5.398 (d, 1H, p-cym phenyl-H, J = 6.0 Hz), 3.154 (d, 3H,
NHCH3, J = 4.9 Hz), 2.883−2.791 (m, 1H, p-cym CH(CH3)2), 2.834
(s, 3H, C(CH3)2), 2.618 (s, 3H, C(CH3)2), 2.258 (s, 3H, p-cym
CCH3), 1.288 (d, 3H, p-cym CH(CH3)2, J = 7.0 Hz), 1.219 (d, 3H, p-
cym CH(CH3)2, J = 7.0 Hz) ppm. Anal. Calcd for C15H25Cl2N3RuS·
0.5H2O: C, 39.13; H, 5.69; N, 9.13. Found: C, 39.23; H, 5.56; N, 9.42.
Single crystals suitable for X-ray diffraction were obtained by
recrystallization in ethanol and hexane (30/70) solution.
[(η6-p-Cymene)RuII(L7)Cl]Cl (7). Yield 36 mg, (63%). HR-ESI-MS
(MeOH) m/z [Found (Calcd)]: 490.0897 (490.0897) (100%) {[(η6-
p-cymene)Ru(L7)Cl] − HCl}+. IR (cm−1): ν (NH2, NH) 3448, 3256,
3051; ν (CN) 1621; ν (CS) 1123. UV−vis (methanol) λ: 303,
1
229 nm. H NMR (300 MHz, CDCl3) δ: 8.858 (br, 1H, NH), 8.767
(br, 1H, NH2), 7.884 (br, 1H, NH2), 7.616−7.293 (m, 10H, phenyl-
H), 5.385 (d, 1H, p-cym phenyl-H, J = 5.8 Hz), 5.024 (d, 1H, p-cym
phenyl-H, J = 5.8 Hz), 4.778 (d, 1H, p-cym phenyl-H, J = 5.8 Hz),
3.857 (d, 1H, p-cym phenyl-H, J = 5.8 Hz), 3.035−2.966 (m, 1H,
CH(CH3)2), 2.238 (s, 3H, p-cym CCH3), 1.322 (d, 6H, p-cym
CH(CH3)2, J = 6.9 Hz) ppm. Anal. Calcd for C24H27Cl2N3RuS·
0.5H2O: C, 50.52; H, 4.95; N, 7.37. Found: C, 50.51; H, 4.91; N, 7.29.
[(η6-p-Cymene)RuII(L8)Cl]Cl (8). Yield 29.5 mg (50%). HR-ESI-MS
(MeOH) m/z [Found (Calcd)]: 504.1065 (504.1053) (100%) {[(η6-
p-cymene)Ru(L8)Cl] − HCl}+. IR (cm−1): ν (NH2, NH) 3450; ν
(CN) 1634; ν (CS) 1070. UV−vis (methanol) λ: 360, 231 nm.
1H NMR (300 MHz, CDCl3) δ: 12.364 (br, 1H, NH), 10.560 (br, 1H,
NH), 7.881 (s, 2H, phenyl-H), 7.758−7.527 (m, 6H, phenyl-H),
7.307−7.295 (m, 2H, phenyl-H), 5.371 (d, 1H, p-cym phenyl-H, J =
[(η6-p-Cymene)RuII(L3)Cl]Cl (3). Yield 30 mg (57%). HR-ESI-MS
(MeOH) m/z [Found (Calcd)]: 442.0900 (442.0896) (100%) {[(η6-
p-cymene)Ru(L3)Cl] − HCl}+. IR (cm−1): ν (NH2, NH) 3432; ν
(CN) 1642; ν (CS) 1057. UV−vis (methanol) λ: 278, 230 nm.
1H NMR (300 MHz, CDCl3) δ: 13.260 (br, 1H, NH), 11.851 (br, 1H,
NH), 7.576 (d, 2H, phenyl-H, J = 8.0 Hz), 7.421 (t, 2H, phenyl-H, J =
7.7 Hz), 7.321 (d, 1H, phenyl-H, J = 7.4 Hz), 5.618 (d, 1H, p-cym
phenyl-H, J = 5.8 Hz), 5.529 (s, 2H, p-cym phenyl-H), 5.374 (d, 1H, p-
cym phenyl-H, J = 5.9 Hz), 2.842−2.773 (m, 1H, p-cym CH(CH3)2),
B
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Table 2. Selected Bond Lengths (Å) and Angles (deg) in L4, L9, 1−6, and 9
L4
L9
1
2
3
4
5·CHCl3
6·CHCl3
9
C11−S1
C11−N1
C11−N2
N2−N3
Ru1−centroid
Ru1−S1
Ru1−Cl1
1.6822(29)
1.3143(43)
1.3489(37)
1.3828(33)
1.6589(21)
1.3416(26)
1.3677(25)
1.3688(24)
1.7001(45)
1.3166(68)
1.3360(48)
1.3961(45)
1.6935(3)
1.7025(38)
1.3301(39)
1.3362(54)
1.4033(32)
1.6958(2)
1.6983(30)
1.3302(42)
1.3432(36)
1.3949(33)
1.6950(3)
1.6869(34)
1.3245(41)
1.3415(48)
1.4060(32)
1.6968(2)
2.3511(9)
2.4116(9)
2.1633(23)
82.427(71)
86.704(31)
85.768(63)
1.6957(36)
1.3101(63)
1.3469(48)
1.3973(45)
1.6864(3)
1.6877(29)
1.3436(41)
1.3479(41)
1.3918(34)
1.6964(2)
2.3405(9)
2.4067(8)
2.1452(25)
81.916(65)
88.308(30)
84.807(82)
1.7347(26)
1.3694(30)
1.3015(32)
1.4092(26)
1.6976(2)
2.3355(6)
2.4351(8)
2.1240(19)
81.266(51)
87.809(26)
83.667(54)
2.3696(15)
2.3984(12)
2.1307(31)
80.548(86)
86.700(42)
87.861(79)
2.3727(10)
2.4147(8)
2.3727(8)
2.3512(12)
2.4056(13)
2.1587(22)
81.484(88)
86.225(41)
89.868(89)
2.3978(11)
2.1356(20)
80.408(62)
87.703(30)
87.937(81)
Ru1−N3
2.1274(28)
80.963(68)
88.398(32)
86.159(83)
N3−Ru1−S1
S1−Ru1−Cl1
Cl1−Ru1−N3
5.7 Hz), 5.026 (d, 1H, p-cym phenyl-H, J = 5.7 Hz), 4.749 (d, 1H, p-
cym phenyl-H, J = 5.7 Hz), 3.867 (d, 1H, p-cym phenyl-H, J = 5.7 Hz),
3.096 (d, 3H, NHCH3, J = 4.2 Hz), 2.800−2.754 (m, 1H, p-cym
CH(CH3)2), 2.085 (s, 3H, p-cym CCH3), 1.263−1.214 (m, 6H, p-cym
CH(CH3)2) ppm. Anal. Calcd for C25H29Cl2N3RuS·H2O: C, 50.59; H,
5.26; N, 7.08. Found: C, 50.66; H, 5.04; N, 7.08.
Cell Titer 96 aqueous cell proliferation assay (MTT) according to the
instructions provided by the manufacturer (Promega, Madison, WI).
Briefly, cells (1−3 × 104 cells per well) were seeded in 96 wells plates.
One day after seeding, the cells were treated with or without a different
concentration of each compound and reincubated for 72 h. After the
cells were washed with sterile phosphate buffer saline (PBS), 190 μL of
RPMI-1640 and 10 μL of the tetrazolium dye (MTT) (5 mg/mL)
solution were added to each well, and the cells were incubated for an
additional 4 h. The medium was discarded; 200 μL of DMSO was
added to dissolve the purple formazan crystals formed. The
absorbance (A) at 492 nm was measured using a Thermo Scientific
Multiskan MK3.
DNA Cleavage Studies Using Agarose Gel Electrophoresis.
In the gel electrophoresis experiments, supercoiled plasmid pBR322
DNA was treated with compound, and the mixture was incubated for
30 min at 37 °C. The samples were then analyzed by 1.5% agarose gel
electrophoresis in Tris−acetic acid−ethylenediamine tetraacetic acid
buffer. The gel was stained with 0.5 μg mL−1 ethidium bromide before
migration. After electrophoresis at 50 V for 3 h, the gel was
illuminated, and the digital images were analyzed by gel
documentation system (Junyi-Dongfang Co., JY02G).
[(η6-p-Cymene)RuII(L9)Cl] (9). Yield 35 mg (54%). HR-ESI-MS
(MeOH) m/z [Found (Calcd)]: 566.1226 (566.1212) (100%) {[(η6-
p-cymene)Ru(L9)Cl] − Cl}+. IR (cm−1): ν (NH2, NH) 3450, 3264; ν
(CN) 1650; ν (CS) 1050. UV−vis (methanol) λ: 335, 251, 228
1
nm. H NMR (300 MHz, CDCl3) δ: 8.082 (br, 1H, NH), 7.549−
7.517 (m, 3H, phenyl-H), 7.417−7.334 (m, 3H, phenyl-H), 7.296−
7.265 (m, 3H, phenyl-H), 6.982−6.884 (m, 4H, phenyl-H), 6.803−
6.706 (m, 2H, phenyl-H), 5.437 (d, 1H, p-cym phenyl-H, J = 6.0 Hz),
4.974 (d, 1H, p-cym phenyl-H, J = 6.0 Hz), 4.727 (d, 1H, p-cym
phenyl-H, J = 6.0 Hz), 3.842 (d, 1H, p-cym phenyl-H, J = 6.0 Hz),
2.813−2.720 (m, 1H, p-cym CH(CH3)2), 2.080 (s, 3H, p-cym CCH3),
1.193 (d, 3H, p-cym CH(CH3)2, J = 6.9 Hz), 1.152 (d, 3H, p-cym
CH(CH3)2, J = 6.9 Hz) ppm. Anal. Calcd for C30H31Cl2N3RuS·
3CH2Cl2: C, 49.25; H, 5.13; N, 6.89. Found: C, 49.42; H, 5.14; N,
6.75. Single crystals suitable for X-ray diffraction were obtained by
recrystallization in chloroform and hexane (30/70) solution.
Methods and Instrumentantion. NMR spectra were recorded
on a Bruker AV-300 spectrometer at working frequency 300 MHz.
Chemical shifts are expressed in parts per million (δ) values and
coupling constants (J) in Hertz. Mass spectra for the complexes were
recorded on a Waters UPLC XEVO G2 TOF mass spectrometer using
electrospray ionization probe. Elemental analyses were carried out
using an Elementar Vario EL Cube.
RESULTS AND DISCUSSION
■
Synthesis and Characterization. For the study at hand, a
series of ketone-N4-substitued thiosemicarbazones (TSCs) and
their corresponding [(η6-p-cymene)RuII(TSC)Cl]+/0 com-
plexes were synthesized. The thiosemicarbazone ligands L1−
L9 were synthesized via the typical condensation route from
their parent thiosemicarbazides and the appropriate ketone.
Subsequently, the [(η6-p-cymene)RuII(TSC)Cl]+/0 complexes
1−9 were prepared via heating of the TSC ligands with [(η6- p-
cymene)RuCl2]21 (Figure 1).11 The ligands L1−L9 were
characterized by HNMR and HR-ESI-MS, and the complexes
1−9 were characterized with IR, HR-ESI-MS, and elemental
analysis.
X-ray Crystallographic Determination. All reflection data were
collected on a Bruker SMART CCD instrument by using graphite
monochromatic Mo Kα radiation (λ = 0.710 73 Å) at room
temperature. A semiempirical absorption correction by using the
SADABS program was applied, and the raw data frame integration was
performed with SAINT.14 The crystal structures were solved by the
direct method using the program SHELXS-9715 and refined by the
full-matrix least-squares method on F2 for all non-hydrogen atoms
using SHELXTL-9716 with anisotropic thermal parameters. All
hydrogen atoms were located in calculated positions and refined
isotropically, except that the hydrogen atoms of water molecules were
fixed in a difference Fourier map and refined isotropically. The details
of the crystal data were summarized in Table 1, and selected bond
lengths and angles for L4, L9, 1−6, and 9 are listed in Table 2.
Crystallographic data for the structural analysis have been deposited
with the Cambridge Crystallographic Data Center. CCDC reference
numbers follow: 941661 (L4), 941662 (L9), 860112 (1), 883966 (2),
860113 (3), 884189 (4), 941658 (5), 941659 (6), 941660 (9).
Cell Culture and Assay for Cell Viability. SGC-7901 (gastric
carcinoma), BEL-7404 (liver carcinoma), and HEK-293T (human
embryonic kidney) cell lines. Cells were obtained by Commerce. Cells
were grown in RPMI-1640 supplemented with 10% cosmic calf serum
(Hyclone) and antibiotics in a humidified atmosphere of 5% CO2 at 37
°C. The viability of these cells was determined using the colorimetric
The 1H NMR spectra of L1−L9 display the broad peaks due
to the imine protons in the range 6.29−9.50 ppm. There is a
distinct shift in the imine proton for the metalloadducts 1−9 in
comparison with the free proligand L1−L9, demonstrating the
coordination of ruthenium to the ligands. The asymmetry (C1)
of the complexes induces significant modifications to the H
NMR signals of the p-cymene moiety in 1−9. The H NMR
spectra of 1−9 in CDCl3 show a doublet for each of the four p-
cymene ring protons and two doublets for the methyl groups of
the isopropyl moiety. The four proton resonances attributable
to the p-cymene ring of 1−9 in CDCl3 are in the range 3.82−
5.67 ppm. In 1−3, just as shown in Figure 2A, the four proton
resonances attributable to the p-cymene ring are in the range
5.67−5.31 ppm. However, in 4−9, just as shown in Figure
2B,C, one of the four proton resonances attributable to the p-
1
1
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range 4.63−5.39 ppm, indicating that it is typical of ruthenium
arene systems.17 The aforementioned high-field shift of one of
the aromatic protons is likely due to the close vicinity of the
phenyl group in the relevant ketone moiety of the coordinated
ligand, as is confirmed by X-ray diffraction studies (see
below).18
The X-ray crystal structures of acetone thiosemicarbazone
(L1), acetone-N4-methyl- thiosemicarbazone (L2), acetone-N4-
phenylthiosemicarbazone (L3), acetophenone-N4- methylthio-
semicarbazone (L5), acetophenone-N4-phenylthiosemicarba-
zone (L6), and benzophenone thiosemicarbazone (L7) are
known.19 Herein, we have determined the structures of the
remaining ligands from our series, namely acetophenone
thiosemicarbazone (L4) and benzophenone-N4-phenylthiose-
micarbazone (L9) (Figure 3), with the crystals data being
Figure 1. General synthetic route to the TSCs and corresponding
[(η6-p-cymene)RuII(TSC)Cl]+/0 complexes.
Figure 3. ORTEP plots of TSC ligands L4 and L9; thermal ellipsoids
are drawn at 50% probability.
presented in Table 1 and the selected bond lengths (Å) and
angles (deg) being presented in Table 2. The structures of L4
and L9 are solved in the monoclinic space group P2/n and P21/
c, respectively. The pairs of donor atoms, N2 and S1, in an anti
orientation are observed. The C11−S1 bonds, with the length
of 1.659−1.682 Å, are typical of double bonds; that is, the
thioamide tautomer is present.20 The intramolecular H-bonds
(N1−H···N3), 2.198 Å for L4 and 2.143 Å for L9, are also
noted.
The X-ray crystal structures of the Ru-arene complexes 1, 2,
3, 4, 5, 6, and 9 were also determined. Their structures and
atom numbering schemes are shown in Figure 4. The
crystallographic data are listed in Table 1, and selected bond
lengths and angles are presented in Table 2. In all the
complexes, RuII adopts the familiar “three-legged piano-stool”
geometry with the metal center being coordinated by the
aromatic arene ligand, a terminal chloride, and a chelating N,S-
ligand. In all complexes, the arene ligands are essentially planar,
and the distances between the ruthenium atom and the
centroid of the aromatic ring of the arene ligand are in the
range 1.686−1.698 Å. The molecular structures of complexes
Figure 2. 1H NMR spectra of 3 (A), 6 (B), and 9 (C) in CDCl3 in the
range 3.5−6.1 ppm.
cymene ring is strongly shifted to higher fields in the range
3.82−3.92 ppm, whereas the other three doublets are in the
E
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Figure 4. ORTEP plots of complex 1−6 and 9; thermal ellipsoids are drawn at 50% probability. The solvent molecules have been omitted for clarity.
1−6 are similar. The bond distances around the Ru atom vary
over a small range (Ru−S = 2.341−2.373 Å; Ru−N = 2.127−
2.163 Å; Ru−Cl = 2.398−2.415 Å), which are comparable to
those of similar compounds.7 The Cl component of the anion
are interlinked by N1−H···Cl2 (2.264−2.445 Å) and N2−H···
Cl2 (2.265−2.438 Å) hydrogen bonding interactions.
In the crystal structure of [(η6-p-cymene)RuII(L1)Cl]Cl (1),
an intramolecular hydrogen bond is formed between the
F
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coordinated chloridion (Cl2) and the hydrogen from C13
(C13−H···Cl2, 2.714 Å). The crystal structures of 2 and 3
show the similar intramolecular hydrogen bond between Cl1
and the proton of C14, C14−H···Cl1, and the lengths are 2.823
and 2.826 Å, respectively.
In the crystal structures of 4−6, the complexes with the
acetophenone-N4-substituent thiosemicarbazone ligands, the
same T-stacking (edge-to-face) π-interactions are identified
(Figure 4) with regard to the nonbonded interactions between
the aromatic rings (distances from 2.478 to 2.840 Å).21 This is
supported by the aforementioned high-field shift of one of the
aromatic protons in 1H NMR spectra of 4−9 which is likely due
to the close vicinity of the phenyl group in the relevant ketone
moiety of the coordinated ligand. In the crystal structure of 4,
the lengths of C11−S1 and C11−N2 bonds are 1.687 and
1.342 Å, respectively, which are similar to those of L4,
consistent with a thioamide (NCS) resonance form of
the coordinated ligand (Figure 1). The [(η6-p-cymene)-
RuII(L5)Cl]Cl·CHCl3 (5·CHCl3) and [(η6-p-cymene)-
RuII(L6)Cl]Cl·CHCl3 (6·CHCl3) crystallized with one mole-
cule of solvent (CHCl3) in the lattice per molecule of complex,
respectively. Moreover, the crystal structure of 5·CHCl3 shows
intramolecular H-bonding involving the Cl1 and C15 (C15
H···Cl1 2.781 Å).
Figure 6. H-bonding involving the coordinated chloridion and the L9
ligand of [(η6-p-cymene)RuII(L9)Cl]Cl (9) giving rise to dimers in the
unit cell.
activity with an IC50 value of 39 μM against SGC-7901
carcinoma cell lines, and L6 displays the highest activity with an
IC50 value around 37 μM against BEL-7404 carcinoma cell
lines. In comparison to their free ligands, the Ru-arene
complexes demonstrated significantly increased antiproliferative
activity (Table 3). All the complexes exhibit moderate
cytotoxicity against the two cancer cell lines, with the IC50
values in the micromolar range (16−48 μM). Notably, under
the experimental conditions the series of complexes shows
comparable activity to cisplatin and oxaliplatin (16−32 μM to
∼20 μM), and are more cytotoxic than carboplatin, against
BEL-7404 cell line, indicating the good cytotoxicity of these
complexes. The compounds with the N4-substitution of the
phenyl ring (3, 6, and 9), to within experimental errors, appear
to have the higher anticancer activity against the two cell lines
compared to the H and methyl substituted. This may be due to
a structural effect, as the N4-phenyl can adjust the hydro-
phobicity of the compound, which may influence the
interaction with biological targets as the similar mechanism of
action as that for the analogues, [(η6-arene)Ru(en)Cl]+.22
However, the type of ketone which condensates to N1, seems
to be of minor importance for the cytotoxic activity. In order to
assess selectivity of the compounds for tumor cells rather than
normal cell lines, the compounds were also screened for their
antiproliferative effects on the human embryonic kidney (HEK-
293T, a model for healthy cells) cell lines. In most cases of the
compounds, the cytotoxicity is comparable for the tumors and
normal cell lines. Compared to the TSC ligands, most of the
Ru-arene complexes show relatively high cytotoxicity toward
the noncancerous HEK-293T cells, indicating that the
complexes are not selective. It is noteworthy that complex 1,
which is markedly more toxic on the tumor cell lines (IC50 of
∼30 μM) than on the noncancerous HEK-293T cells (IC50 of
>100 μM), shows the most selectivity for tumor cells rather
than normal cell lines in the series of complexes.
In the crystal structure of 9 (Figure 4), the N2 of L9 ligand is
deprotonated, and the complex is neutral. As a consequence of
deprotonation, the C11−S1 bonds (formally double bonds in
the free L9 ligand) lengthen from 1.659 to 1.735 Å (Table 2),
while the C11−N2 bonds shorten from 1.368 to 1.302 Å;
consistent with a dominantly ene-thiolate (NCS)
resonance form of the coordinated ligand (Figure 5). Similar to
Figure 5. Different resonance form of L9 with N2-protonated.
the structures of 4−6, one of the benzene rings from the
benzophenone system of L9 in the crystal structure of 9 is
pointed toward the p-cymene ligand (Figure 4), which results
in the intramolecular CH/π interactions between C7−H
protons and the benzene ring [R(1) = C(19)/C(20)/C(21)/
C(22)/C(23)/C(24)] (distance 2.601 Å). In addition, dimers
formed in the unit cell (Figure 6) through a pair of
intermolecular H-bonds between N1−H, C30−H protons of
L9 and the coordinated chloridion of another molecule, with
distances 2.674 and 2.805 Å, respectively, and Ru−Cl (2.435 Å)
in 9 is slightly longer than that of 1−6 (2.398−2.415 Å) owing
to the formation of dimers.
Cancer Cell Growth Inhibition. The cytotoxicity of the
series of TSC ligands (L1−L9) and their corresponding [(η6-p-
cymene)RuII(TSC)Cl]Cl complexes (1−9) was determined
using the MTT assay on human gastric carcinoma (SGC-7901)
and human liver carcinoma (BEL-7404) cell lines, and for
comparison purposes the cytotoxicity of cisplatin, oxaliplatin,
and carboplatin was evaluated under the same experimental
conditions (Table 3). The IC50 values of the most of the TSC
ligands toward these two carcinoma cells are deemed inactive
(>100 μM). In the series of TSC ligands, L3 shows the highest
It is reported that the Ru-arene complexes containing
N,N-,23 N,O-,18,24 and O,O-chelating25 ligands show activity
toward some human cancer cells with an IC50 value in the
submicromolar or micromolar range (0.2−240 μM), and some
Ru-arene compounds of the RAPTA type26 show IC50 values
from 2 to 49 μM against A2780 human ovarian cancer cells. In
addition, the Ru-arene complexes [(arene)2Ru2(SR)3]Cl with
IC50 values in the nanomolar range (0.03−0.66 μM) are
reported,27 in which the most active complex is more than 100
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Table 3. IC50 Values of TSCs (L1−L9), Complexes (1−9), Cisplatin, Oxaliplatin, and Carboplatin towards SGC-7901, BEL-
7404, and HEK-293T Cell Lines
IC50(μM)
IC50(μM)
TSC
SGC-7901
BEL-7404
HEK-293T
complex
SGC-7901
BEL-7404
HEK-293T
L1
L2
L3
L4
L5
L6
L7
L8
L9
>100
>100
>100
96.1 9.7
>100
>100
>100
1
30.8 5.7
39.9 2.6
17.5 1.6
28.6 3.5
43.4 9.3
46.7 0.6
47.7 1.3
34.4 3.1
17.0 2.9
6.7 0.4
11 0.8
32.0 3.1
28.2 0.6
18.2 3.2
29.1 7.1
29.7 0.5
15.9 2.2
24.5 2.2
21.8 4.4
17.1 4.6
23.1 2.6
20 0.6
>100
2
22 3.5
25 0.9
46 2.5
33 0.4
17 4.3
20 1.3
17 2.2
10 2.0
10 0.7
38.6 4.3
62.2 6.1
>100
97 8.3
41 4.1
68 5.9
>100
3
>100
4
>100
5
>100
37.1 0.1
50.1 4.9
>100
6
>100
51 6.3
>100
7
8
>100
>100
45.4 4.1
>100
9
cisplatin
oxaliplatin
carboplatin
2
0.2
39 2.6
70 9.5
44 3.7
times more cytotoxic than cisplatin toward A2780 and
A2780cisR cells. The in vitro anticancer activity of our
compounds is moderate compared with the above-reported
compounds. However, in vitro anticancer potency appears not
to be a prerequisite in particular for ruthenium drug
candidates;28 i.e., RAPTA compounds exhibit low activity
against cancer cells but possess very good antimetastatic activity
in vivo.10
Biological Assays: Gel Electrophoresis of Compound-
pBR322 Complexes. As DNA is generally a potential target
for arene-ruthenium(II) drugs,29 it is among the critically
important targets in cancer chemotherapy. Most cytotoxic
ruthenium drugs act as DNA intercalators upon coordination to
the appropriate ancillary ligands.30 Thus, the interactions of the
representative TSC ligands (L1−L3) and their corresponding
complexes (1−3) with DNA were investigated. For comparison
purposes, plasmid DNA was incubated in presence of the
representative TSC ligands (L1−L3) and their corresponding
complexes (1−3) for 24 h at 37 °C at the molar ratios (r = 0.4
and 0.8). The cleaving efficiency of all compounds was assessed
by their ability to convert supercoiled pBR322 DNA (form I) to
nicked DNA (form II), while no linear DNA (form III) was
observed, by agarose gel electrophoresis (Figure 7). The DNA
phenomenon. A control experiment showed that SC DNA
(form I) was cleaved by TSC ligands (L1−L3), and formed NC
DNA (form II), indicating a different interaction mode between
TSC ligands and DNA.33 Obviously, the DNA binding activities
of the ruthenium complexes are higher than that of TSC
ligands, which correlates quite well with their cytotoxicity.
CONCLUSIONS
■
In conclusion, nine TSC compounds (L1−L9) and their
corresponding Ru-arene complexes (1−9) have been synthe-
sized and characterized by a variety of physical methods. The
molecular structures of L4, L9, 1−6, and 9 have been
characterized by X-ray crystallography. The in vitro activity of
all the compounds has been evaluated against the SGC-7901
human gastric cancer, BEL-7404 human liver cancer and HEK-
293T noncancerous cell lines, and compared to that of cisplatin,
oxaliplatin, and carboplatin, the well-known antitumor agents.
The TSC compounds exhibited lower antiproliferative
activities, but the Ru-arene complexes were found to exhibit
moderate activities. Especially, some of the Ru-arene complexes
showed IC50 cytotoxicity coefficients similar to those of
cisplatin and oxaliplatin. Compared to the TSC ligands, most
of the Ru-arene complexes show relatively high cytotoxicity
toward the noncancerous HEK-293T cells, indicating that the
complexes are not selective. The results of agarose gel
electrophoresis indicate that the interaction mechanisms with
pBR322 plasmid DNA are different for the TSC ligands and
their corresponding complexes.
ASSOCIATED CONTENT
* Supporting Information
Crystallographic data in CIF format. This material is available
free of charge via the Internet at http://pubs.acs.org.
■
S
Figure 7. Gel electrophoresis of pBR322 plasmid DNA incubated in
presence of (from left to right on each gel) the TSC ligands (L1−L3)
and their corresponding complexes (1−3) for 24 h at the molar ratios
(r = 0.4 and 0.8).
AUTHOR INFORMATION
Corresponding Author
*E-mail: suwmail@163.com (W.S.); lipearpear@163.com
(P.L.). Phone: + 86 771 3908065.
■
cleaving abilities of complexes (1−3) are in concentration-
dependent manners. With increasing concentration of com-
plexes (1−3), the amount of SC DNA (form I) diminishes
gradually whereas the NC DNA (form II) exhibits no obvious
change. The DNA binding results of these complexes are
similar to that of Ru(II)-DMSO-Cl-Chalcone31 and some other
Ru-arene complexes,29b,32 indicating a similar oxidative
mechanism. Surprisingly, the result of agarose gel electro-
phoresis of TSC ligands (L1−L3) shows a different
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
This research was supported by the National Natural Science
Foundation of China (21261005, 51263002, 21203035), Key
■
H
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M.; Li, Y.; Khot, H.; De Kock, C.; Smith, P. J.; Land, K.; Chibalea, K.;
Smith, G. S. Dalton Trans. 2013, 42, 4677−4685.
Project of Chinese Ministry of Education (2010168), and
Program for Excellent Talents in Guangxi Higher Education
Institutions.
(13) (a) Demoro, B.; Sarniguet, C.; Sanchez-Delgado, R.; Rossi, M.;
Liebowitz, D.; Caruso, F.; Olea-Azar, C.; Moreno, V.; Medeiros, A.;
Comini, M. A.; Otero, L.; Gambino, D. Dalton Trans. 2012, 41, 1535−
1543. (b) Demoro, B.; de Almeida, R. F. M.; Marques, F.; Matos, C.
P.; Otero, L.; Pessoa, J. C.; Santos, I.; Rodríguez, A.; Moreno, V.;
Lorenzo, J.; Gambino, D.; Tomaz, A. I. Dalton Trans. 2013, 42, 7131−
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