Synthesis of oxovanadium complexes and their apoptosis-inducing activity in leukemia cells

Article abstract of DOI:10.1248/cpb.60.508

Vanadium complexes with different ligands were synthesized and evaluated for antiproliferative activity on U937 cells. The alkyl chain length of the ligands affected the antiproliferative activity, and two complexes - 3b and 4 - exhibited strong activities with IC₅₀ values of 6.02 and 3.90 μM respectively. Annexin V staining and DNA ladder formation indicated that these complexes induced apoptosis in U937 cells.

Full text of DOI:10.1248/cpb.60.508

508
Regular Article
Chem. Pharm. Bull. 60(4) 508–512 (2012)
Vol. 60, No. 4
Synthesis of Oxovanadium Complexes and Their Apoptosis-Inducing
Activity in Leukemia Cells
Tomoko Yamaguchi,a Shinya Watanabe,b Yuriko Matsumura,a Yoshikazu Tokuoka,b and
,a
Akihiro Yokoyama*
a Department of Materials and Life Science, Faculty of Science and Technology, Seikei University; 3–3–1 Kichijoji-
b
kitamachi, Musashino, Tokyo 180–8633, Japan: and Department of Medical Technology, Faculty of Biomedical
Engineering, Toin University of Yokohama; 1614 Kurogane-cho, Aoba-ku, Yokohama 225–8503, Japan.
Received December 1, 2011; accepted January 14, 2012; published online January 17, 2012
Vanadium complexes with different ligands were synthesized and evaluated for antiproliferative activity
on U937 cells. The alkyl chain length of the ligands affected the antiproliferative activity, and two complex-
—
—
es 3b and 4 exhibited strong activities with IC50 values of 6.02 and 3.90μM respectively. Annexin V stain-
ing and DNA ladder formation indicated that these complexes induced apoptosis in U937 cells.
Key words apoptosis; vanadyl complex; leukemia cell
Necrosis and apoptosis are two major mechanisms of cell tion reduces the polarity of metals, vanadyl complexes can
death. Necrosis is a pathological death process, which involves permeate the lipid bilayer of cell membranes more easily than
cellular swelling, organelle dysfunction, mitochondrial col- the inorganic salts of VOSO4.6)
lapse, and ultimately cellular disintegration.1) In contrast, apo-
The apoptosis-inducing activity of vanadium complexes has
ptosis is a programmed cell death and is characterized by cell also been demonstrated in some papers. For example, oxova-
shrinkage, plasma and nuclear membrane blebbing, organelle nadium complexes such as bis(maltolato)oxovanadium with an
relocalization and compaction, chromatin condensation, and O4 coordination mode, and VO(Me-phen)2 with an N4 coordi-
production of membrane-enclosed particles containing intra- nation mode induced apoptosis in B cell lines and human can-
2)
cer cell lines respectively.9–13) However, the apoptosis-inducing
“
”
cellular material known as apoptotic bodies.
Cancer is presently the most lethal of human diseases. activities of vanadium complexes with other coordination
Some anticancer drugs function by inducing the apoptotic modes have seldom been reported.
process in cancer cells.3) Since resistance to apoptosis is a
In this paper, we report the synthesis, antiproliferative ac-
hallmark of cancer cells, one focus of anticancer drug dis- tivity, and apoptosis-inducing activity of vanadium complexes
covery is the identification of compounds that activate the with the O4 coordination mode (1a,b, 2a–c), N2O2 coordina-
apoptotic cascade.
tion mode (3a,b), and S2O2 coordination mode (4). In addition,
There is a small number of reports describing the pharma- the effect of the hydrophobicity of the ligands on apoptosis-in-
cological properties of vanadium complexes; such properties ducing activity is also discussed. We used the 3-hydroxy-1,2-
include insulin-mimetic and anticancer activities.4,5) Vanadyl dimethyl-4(1H)-pyridinone unit as a ligand of 1a, because it
complexes with heterocyclic bidentate ligands have been has previously been shown to have high tumor-specificity.14)
synthesized and evaluated for their insulin-mimetic activi- The platinum(II) complex with the 3-hydroxypyridine unit of
ties.6) The pharmacological properties of vanadium complexes 2 has been found to show significant anticancer activity.15) The
depend on the oxidation states of vanadium, which can vary 8-hydroxyquinoline unit of 3 was chosen due to its antipro-
from −1 to +5.7) For example, vanadyl compounds, which liferative activity on mouse melanoma cells.16) The 2-phenyl-
contain vanadium with an oxidation state of +4, are less toxic 3-hydroxy-4(1H)-quinolinethione unit of 4 was reported to
than compounds containing vanadium at an oxidation state of show cytotoxic activity in human malignant cell lines.17)
+5. When healthy rats were treated with vanadate complexes,
“
”
most of the vanadium existed in vanadyl state.8) Since chela-
*To whom correspondence should be addressed. e-mail: ayokoyama@st.seikei.ac.jp
© 2012 The Pharmaceutical Society of Japan

April 2012
509
Results and Discussion
Synthesis of Oxovanadium Complexes The oxovana-
dium complexes 1a,b were synthesized as reported previ-
ously (Chart 1).18) As shown in Chart 2, 1-alkyl-3-hydroxy-
2-(1H)-pyridinones 6a–c were prepared by the reaction of
3-methoxy-2(1H)-pyridone with a corresponding alkyl iodide,
followed by treatment with boron tribromide.19) Reaction of
6a–c with VOSO4 yielded 2a–c. 8-Hydroxyquinoline deriva-
tives 7 were prepared from 8-hydroxy-2-methylquinoline by
chlorosulfonation, followed by amidation (Chart 3).20) The
oxovanadium complex 3 was obtained by the reaction of 7
with VO(acac)2. The synthesis of 4 is described in Chart
4. N-Methylanthranilic acid was treated with 2-bromo-4′-
methoxyacetophenone in the presence of K2CO3 to give
p-methoxyphenacyl N-methylanthranilate 8. Treatment of 8
with polyphosphoric acid (PPA) yielded the cyclized product
9, whose carbonyl group was converted into a thiocarbonyl
group using P2S5. Finally, treatment of 10 with VO(acac)2
yielded the target complex 4.
Chart 1
Inhibition of Cancer Cell Proliferation Antiproliferative
activities of the oxovanadium complexes 1a,b, 2a–c, 3a,b,
and 4 were evaluated by testing the viability of U937 cells
in the presence of these compounds. The results are sum-
marized in Fig. 1. The activity order was as follows: 2a<2
≪
b<2c<3a<1a 1b<3b≈4, and the complexes 1b, 3b, and 4
Chart 2
showed significant activity. Moreover, when the activities of
complexes with similar ligands were compared, the activity
order was 1a<1b, 2a<2b<2c, and 3a<3b, indicating that the
antiproliferative activity of the complex increased with length
of the alkyl chain of the ligand. As Uckun and co-workers
had discussed in the study to explore the effect of ligands of
oxovanadium(IV) complexes on the cytotoxic activity against
cancer cells,9) the ligands may affect the ability of the com-
plexes to generate reactive oxygen species, to permeate the
cell membrane, and/or to interact with DNA, which would
result in the cell death. Especially, the higher antiproliferative
activities of the oxovanadium complexes with longer alkyl
chains would be due to the improved cell permeable nature of
the complexes induced by the increased lipophilicity. Similar
effects of alkyl chain have been reported in the study of anti-
proliferative activities of dinuclear Ru-arene complexes,21) bis-
galloyl derivatives,22) and alkyl and alkenyl trisulfides.23)
The 50% inhibitory activity (IC50) values of the complexes
1b, 3b, and 4, which possessed higher antiproliferative activ-
Chart 3
Chart 4

510
Vol. 60, No. 4
ity, are shown in Table 1. The values were between 3–26μM, cleosomal DNA fragmentation is a characteristic feature of the
and the cytotoxic activity increased in the order 1b 3b<4. In apoptotic process.25) As shown in Fig. 3, typical DNA frag-
≪
contrast, the IC50 value reported in the study of insulin-mi- mentation ladders were detected in U937 cells treated with 3b
metic activity of 1b was 2.3mM,18) which was about 100-fold and 4. Together, these data confirm that cell death induced by
higher concentration than the value for the antiproliferative complexes 3b and 4 in U937 cells was due to apoptosis.
activity reported in Table 1. Therefore, if the concentration
of the compounds is sufficiently low, these compounds are
thought to exhibit the antiproliferative activity without show-
ing the insulin-mimetic activity.
Conclusion
Hydroxyazines and their vanadium(IV) complexes were
synthesized. Complexes 3b and 4 exhibited high cytotoxic-
Induction of Apoptosis in U937 Cells On the basis of the ity against U937 cells with IC50 values of 6.02 1.30μM and
data shown above, further biological evaluation focused on the 3.90 1.23μM, respectively. Annexin V staining and agarose
complexes 1b, 3b, and 4. To determine whether the cell death gel electrophoresis experiments indicated that complexes 3b
induced by these complexes was apoptotic or necrotic, U937 and 4 induced apoptosis in U937 cells.
cells were treated with these complexes and then stained with
annexin V and propidium iodide (PI).24) In this approach, apo-
Experimental
ptotic cells exhibit green fluorescence due to annexin V as-
General Procedures IR spectra were recorded with a
sociated with externalized phosphatidylserine, while necrotic JASCO FT/IR-470. 1H-NMR spectra were obtained on a
cells exhibit red fluorescence due to nuclear staining with PI. JEOL JNM-LA400D NMR spectrometer. Tetramethylsilane
As shown in Fig. 2, U937 cells treated with complexes 1b, 3b, was used as an internal reference. Thin layer chromatogra-
and 4 exhibited green fluorescence, indicating that the cell phy (TLC) and column chromatography were conducted on
death induced by these complexes was apoptotic rather than Merck Silica Gel 60 F254 and Merck Silica Gel 60 (70–230
necrotic.
mesh), respectively. The elemental analysis was carried out
To confirm that the cell death associated with these com- using a Perkin-Elmer 2400 analyzer series II or a Euro Vector
plexes was due to apoptosis, agarose gel electrophoresis analy- EUROEA3000-Dual. All chemicals were of reagent grade and
sis of the DNA of treated U937 cells was carried out; internu- used without further purification. Stained cells were observed
with a Nikon ECLIPS ETE300 fluorescence microscope.
Electrophoresis was performed with an ADVANCE Mupid-
Scope. An Annexin V-Fluorescein Staining Kit was obtained
from Wako Pure Chemical Industries Ltd. The compounds
1a,1b,18) 6a–c,19) 2a,26) and 5-(chlorosulfonyl)-8-hydroxy-
2-methylquinoline20) were prepared according to the previ-
ously reported methods.
Fig. 1. Antiproliferative Activities of the Oxovanadium Complexes
(50μM) Evaluated by the Viability of U937 Cells (%)
Control cells were treated with dimethylsulfoxide (DMSO). *p<0.05 vs. DMSO
’
control, and Student s t-test (two-tailed) for unpaired data was used to determine
statistical significance. The values are expressed as means S.D., n=3.
Table 1. IC50 Values in U937 Cells
Compound
IC50 (μM)
1b
3b
4
26.24 2.22
6.02 1.30*
3.90 1.23*
Fig. 3. Agarose Gel Electrophoresis of DNA Fragmentation Induced
in U937 Cells by Vanadyl Complexes Visualized under UV Light with
Ethidium Bromide Staining
The values are expressed as means S.D.; n=3; *p<0.05 vs. 1b.
#1, DNA marker; #2, DMSO; #3, 1b; #4, 3b; #5, 4.
Fig. 2. Annexin V and PI Staining of U937 Cells Treated with (A) DMSO Control, (B) 1b, (C) 3b, and (D) 4

April 2012
511
Bis(1-ethyl-1,2-dihydro-2-oxo-3-pyridinolato)oxovana-
Synthesis of Bis[5-(N,N-dipentylsulfamoyl)-2-methyl-
dium(IV) (2b) Complexes 2b and 2c were synthesized from 8-quinolinato]oxovanadium(IV) (3b) Complex 3b was
6b and 6c, respectively, according to the procedure used for synthesized from 7b according to the procedure for the syn-
the synthesis of 2a.26) 2b: green solid, 45% yield, mp: 146.7 C thesis of 3a and produced an 83% yield. A brown powder was
°
−1
(decomp.). IR (KBr) cm−1: 2989 (νC–H), 1624, 1560, 1536, 1462 produced, mp 171.8 C (decomp.). IR (KBr) cm : 1566, 1503,
°
(νC=C), 1365 (δC–H), 971 (νV=O), 737, 720 (γC–H). Anal. Calcd 1458 (νC–C), 1378 (δO–H), 1305, 1151 (νS=O), 992 (νV=O), 792
for C15H18N2O5V·0.1H2O: C, 46.55; H, 5.02; N, 7.75. Found: C, (γC–H). Anal. Calcd for C40H58N4O7S2V: C, 58.45; H, 7.11; N,
46.57; H, 5.09; N, 7.69.
6.82. Found: C, 58.64; H, 7.39; N, 6.80.
Bis(1,2-dihydro-2-oxo-1-propyl-3-pyridinolato)oxo-
Synthesis of p-Methoxyphenacyl N-Methylanthranilate
°
vanadium(IV) (2c) Gray solid, 34% yield, mp 210.4 C (de- (8) To
a solution of N-methylanthranilic acid (2.03g,
comp.). IR (KBr) cm−1: 2960 (νC–H), 1620, 1520, 1466 (νC=C), 13.5mmol) in N,N-dimethylformamide (DMF) (30mL), K2CO3
971 (νV=O), 758, 652 (γC–H). Anal. Calcd for C17H22N2O5V: C, (2.48g, 17.9mmol) was added, and the reaction mixture was
°
heated to 90 C for 1h and stirred. The mixture was cooled
51.76; H, 5.43; N, 7.74. Found: C, 51.76; H, 5.32; N, 7.37.
Synthesis of 5-(N,N-Dimethylsulfamoyl)-8-hydroxy-2- to room temperature, and α-bromo-p-methoxyacetophenone
methylquinoline (7a) Dimethylamine was bubbled into dry (1.48g, 6.48mmol) was added. After stirring for 30min at
°
tetrahydrofuran (THF; 60mL) for 15min at room tempera- room temperature, the reaction mixture was heated to 50 C
ture, and then 5-(chlorosulfonyl)-8-hydroxy-2-methylquinoline for 30min, cooled to room temperature, and then poured
(508mg, 1.9mmol) was added in small amounts over 3h at over ice in H2O (500g). The precipitated solid was collected
room temperature. The reaction mixture was stirred for an- by filtration, washed with water, and then dried. The residue
other 1h at room temperature with continuous bubbling of was recrystallized from ethanol to yield the colorless solid 8
1
°
dimethylamine. The solvent was removed by use of a rotary (1.68g, 87%), mp 104–106 C. H-NMR (CDCl3) δ: 2.89 (3H,
evaporator, and the remaining dimethylamine was removed by d, J=5.1Hz), 3.88 (3H, s), 5.46 (2H, s), 6.62 (1H, t, J=8.3Hz),
dissolving the residue in CH2Cl2 (50mL) followed by rotary 6.67 (1H, d, J=8.3Hz), 6.97 (2H, d, J=8.3Hz), 7.40 (1H, t,
evaporation (repeated three times). The residue was purified J=8.3Hz), 7.54 (1H, s), 7.96 (2H, d, J=8.3Hz), 8.05 (1H, d,
by column chromatography on silica gel with CHCl3–MeOH J=8.3Hz). IR (KBr) cm−1: 3374 (νN–H), 2996 (νC–H), 1680
(20/1) to yield 7a as a colorless solid (472mg, 48%), mp (νC=O), 1604 (νN–H), 1574 (νC=O), 1318 (νC–N), 1173, 1020 (νC–O),
112.5–114.8 C. 1H-NMR (CDCl3) δ: 2.75 (6H, s), 2.76 (3H, 741, 690 (γC–H).
°
s), 7.18 (1H, d, J=8.3Hz), 7.47 (1H, d, J=8.8Hz), 8.10 (1H, d,
Synthesis of 3-Hydroxy-2-(p-methoxyphenyl)-1-methyl-
J=8.3Hz), 8.97 (1H, d, J=8.8Hz). IR (KBr) cm−1: 3336 (νO–H), 4(1H)-quinolinone (9) Compound 8 (1.54g, 5.16mmol) was
1471 (νC–H), 1372 (νC–O), 1330, 1155 (νS=O), 1205 (δO–H), 835 added to polyphosphoric acid (10g) that had been pre-heated
°
°
(γC–H). Anal. Calcd for C12H14N2O3S: C, 54.12; H, 5.30; N, to 120 C. The reaction mixture was stirred at 120 C for 5h,
10.52. Found: C, 54.07; H, 5.44; N, 10.30. cooled to room temperature, and then diluted with cold water
5-(N,N-Dipentylsulfamoyl)-8-hydroxy-2-methylquinoline (100mL). The pH of the aqueous mixture was adjusted to 7
(7b) Dipentylamine (7mL, 0.03mol) was added to a solution by addition of aqueous 2M KOH. The resulting precipitates
of 5-(chlorosulfonyl)-8-hydroxy-2-methylquinoline (492mg, were collected by filtration and washed with water. The crude
1.9mmol) in dry THF (40mL) at room temperature, and the product was purified by column chromatography on silica gel
mixture was stirred at room temperature overnight. After the (CHCl3–acetone–EtOH=100:5:1) to yield a pale yellow solid
1
°
solvent had evaporated, CHCl3 (50mL) was added to the resi- 9 (316mg, 22%), mp 210 C (decomp.). H-NMR (CDCl3) δ:
due, and the mixture was washed with 1M HCl (50mL), and 3.63 (3H, s), 3.89 (3H, s), 7.08 (2H, d, J=9.0Hz), 7.37 (2H, d,
then dried over anhydrous Na2SO4. The crude product was J=9.0Hz), 7.39 (1H, t, J=8.8Hz), 7.57 (1H, d, J=8.8Hz), 7.68
purified by column chromatography on silica gel with CHCl3 (1H, t, J=8.8Hz), 8.56 (1H, d, J=8.8Hz). IR (KBr) cm−1: 3222
°
to yield 7b as a colorless solid (571mg, 80%), mp 58.8–59.1 C. (νO–H), 1619 (νC=O), 1433, 1363 (δC–H), 1308 (νC–N), 1246 (δO–H),
1H-NMR (CDCl3) δ: 0.78 (6H, t, J=7.3Hz), 1.05–1.25 (8H, m), 1174, 1037 (νC–O), 768 (γC–H).
1.45 (4H, quint, J=7.3Hz), 2.75 (3H, s), 3.18 (4H, t, J=7.3Hz),
Synthesis of 3-Hydroxy-2-(p-methoxyphenyl)-1-methyl-
7.14 (1H, d, J=8.3Hz), 7.46 (1H, d, J=8.8Hz), 8.10 (1H, 4(1H)-quinolinethione (10) Et3N (1.4mL, 10mmol) was
d, J=8.3Hz), 8.84 (1H, d, J=8.8Hz). IR (KBr) cm−1: 3367 added to a mixture of 9 (208mg, 0.74mmol) and P2S5 (380mg,
(νO–H), 1602, 1567, 1504 (νC–C), 1468 (δC–H), 1378 (νC–O), 1302 1.71mmol) in dry CH3CN (30mL) in an ice-bath. The reaction
(νS=O), 1205 (δO–H), 1145 (νS=O), 795 (γC–H). Anal. Calcd for mixture was stirred for 24h at room temperature and then
C20H30N2O3S: C, 63.46; H, 7.99; N, 7.40. Found: C, 63.73; H, concentrated in vacuo. The residue was purified by column
8.19; N, 7.39.
chromatography on silica gel (CHCl3/acetone/EtOH=100/5/1)
°
Synthesis of Bis[5-(N,N-dimethylsulfamoyl)-2-methyl- to yield the yellow solid 10 (145mg, 66%), mp: 196 C (de-
8-quinolinato]oxovanadium(IV) (3a) To a solution of 7a comp.). 1H-NMR (CDCl3) δ: 3.83 (3H, s), 3.91 (3H, s), 7.11 (2H,
(204mg, 0.77mmol) in CH3CN–H2O (4/1; 50mL), a solution d, J=6.8Hz), 7.40 (2H, d, J=6.8Hz), 7.56 (1H, ddd, J=2.7, 5.0,
of VO(acac)2 (103mg, 0.39mmol) in CH3CN (20mL) was 8.5Hz), 7.71–7.72 (2H, m), 8.75 (1H, s), 9.02 (1H, d, J=8.3Hz).
added, and the reaction mixture was refluxed for 2h and then IR (KBr) cm−1: 3061 (νC–H), 1330 (νC–N), 1205 (δO–H), 1111
cooled to room temperature. The resulting precipitates were (νC=S), 847, 755 (γC–H). Anal. Calcd for C17H15NO2S·0.5H2O:
collected by suction filtration to give 3a as a brown powder C, 66.64; H, 5.26; N, 4.57. Found: C, 66.84; H, 5.09; N, 4.51.
−1
°
(177mg, 77%), mp 262.1 C (decomp.). IR (KBr) cm : 1564,
Synthesis of Bis[1,4-dihydro-1-(p-methoxyphenyl)-2-
1501, 1459 (νC–C), 1378 (δO–H), 1305, 1152 (νS=O), 994 (νV=O), methyl-4-thioxo-3-quinolinolato]oxovanadium(IV) (4) To
794 (γC–H). Anal. Calcd for C24H26N4O7S2V: C, 48.24; H, 4.39;
a
mixture of compound 10 (174.5mg, 0.58mmol) in
N, 9.38. Found: C, 48.35; H, 4.39; N, 9.36.
CH3CN–H2O (4:1) (25mL), a solution of VO(acac)2 (78.0mg,

512
Vol. 60, No. 4
0.29mmol) in CH3CN–H2O (4:1; 5mL) was added. The reac- 15min at room temperature, and the cells were observed by
tion mixture was refluxed overnight, and then cooled to room fluorescence microscopy.
temperature. The precipitates were collected by filtration and
washed several times with H2O and then Et2O to give 4 as
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a brown solid (132mg, 69%), mp 325 C (decomp.). IR (KBr)
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C, 61.90; H, 4.28; N, 4.25. Found: C, 62.11; H, 4.23; N, 4.23.
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DNA Fragmentation Analysis by Electrophoresis U937
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°
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°
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incubated at −20 C for 24h, followed by centrifugation at
15000rpm for 20min. The precipitated DNA was dissolved
in Tris–EDTA (5μL) buffer and subjected to electrophoresis
using a 2% agarose gel and Tris–acetate–EDTA buffer at 50V.
The DNA fragmentation pattern was visualized with a UV
transilluminator.
Annexin V and PI Staining U937 cells (0.2×106 cells)
were suspended in 4.95mL of RPMI 1640 medium contain-
ing 5% penicillin-streptomycin and 10% fetal bovine serum,
and treated with 50μL of 2.5mM vanadyl complex in DMSO
to give a final concentration of 12.5μM. Cells were incubated 24) Engeland M. V., Nieland L. J. W., Rameakers F. C. S., Schutte B.,
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Sakurai H., Chem. Lett., 29, 866–867 (2000).
°
at 37 C under an atmosphere of 5% CO2–95% air for 24h,
washed with cold PBS, collected by centrifugation, and lysed
in annexin V-fluorescein (2μL), binding buffer (100μL), and
propidium iodide (2μL). The suspension was incubated for