Synthesis and evaluation of oxovanadium(iv) complexes of Schiff-base condensates from 5-substituted-2-hydroxybenzaldehyde and 2-substituted- benzenamine as selective inhibitors of protein tyrosine phosphatase 1B

Article abstract of DOI:10.1039/c2dt30198a

Five oxovanadium(iv) complexes, which were divided into two groups, [V ^IVO(bhbb, nhbb)(H₂O)₂] (tridentate ligands: H₂bhbb = 2-(5-bromo-2-hydroxylbenzylideneamino)benzoic acid, 1; H₂nhbb = 2-(5-nitro-2

Full text of DOI:10.1039/c2dt30198a

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PAPER
Synthesis and evaluation of oxovanadium(IV) complexes of Schiff-base
condensates from 5-substituted-2-hydroxybenzaldehyde and 2-substituted-
benzenamine as selective inhibitors of protein tyrosine phosphatase 1B†
a
a
a
a
a
b
b
Hong Han, Liping Lu,* Qingming Wang, Miaoli Zhu,* Caixia Yuan, Shu Xing* and Xueqi Fu
Received 27th January 2012, Accepted 9th July 2012
DOI: 10.1039/c2dt30198a
IV
Five oxovanadium(IV) complexes, which were divided into two groups, [V O(bhbb, nhbb)(H O) ]
2
2
(
tridentate ligands: H bhbb = 2-(5-bromo-2-hydroxylbenzylideneamino)benzoic acid, 1; H nhbb = 2-(5-
2 2
IV
nitro-2-hydroxylbenzylideneamino)benzoic acid, 2) and [V O(cpmp, bpmp, npmp) ] (bidentate ligands:
2
Hcpmp = 4-chloro-2-((phenylimino)methyl)phenol, 3; Hbpmp = 4-bromo-2-((phenylimino)methyl)-
phenol, 4; Hnpmp = 4-nitro-2-((phenylimino)methyl) phenol, 5) have been prepared and characterized by
elemental analysis, infrared, UV-visible and electrospray ionization mass spectrometry. The coordination
IV
in [V O(bhbb)(H O) ] (1) was confirmed by X-ray crystal structure analysis. The oxidation state of V(IV)
2
2
1
with d configuration in 1–5 was confirmed by EPR. The speciation of VO/H bhbb in methanol–aqueous
2
solution was investigated by potentiometric pH titrations. The result indicated that the main species were
IV
−
IV
2−
[
V O(bhbb)(OH)] and [V O(bhbb)(OH) ] at the pH range 7.0–7.4. The structure–activity
2
relationship of the vanadium complexes in inhibiting protein tyrosine phosphatases (protein tyrosine
phosphatase 1B, PTP1B; T-cell protein tyrosine phosphatase, TCPTP; megakaryocyte protein-tyrosine
phosphatase, PTP-MEG2; Src homology phosphatase 1, SHP-1 and Src homology phosphatase 2, SHP-2)
was investigated. The oxovanadium(IV) complexes were potent inhibitors of PTP1B, TCPTP, PTP-MEG2,
SHP-1 and SHP-2, but exhibited different inhibitory abilities over different PTPs. Complexes 2 and 4
displayed better selectivity to PTP1B over the other four PTPs. Kinetic data showed that complex 2
inhibited PTP1B, TCPTP and SHP-1 with a noncompetitive inhibition mode, but a classical competitive
inhibition mode for PTP-MEG2 and SHP-2. The results demonstrated that both the structures of
vanadium complexes and the conformations of PTPs influenced PTP inhibition activity. The proper
modification of the organic ligand moieties may result in screening potent and selective vanadium-based
PTP1B inhibitors.
Introduction
As potential therapeutic targets, PTPs have attracted great atten-
tion in both academia and industry in past decades.
6
–8
Protein tyrosine phosphatases (PTPs) are a large family of sig-
naling enzymes that play an important role in signal transduction
and regulation of cellular processes. Together with protein
kinases, they control the level of phosphorylation of cells. Many
human diseases, such as diabetes, obesity, cancer and immune
disorders, are involved in the dysregulation of PTP activities.
PTP1B has been demonstrated to play key roles in the nega-
tive regulation of signaling pathways mediated by insulin recep-
tors and leptin receptors. The PTP1B gene-null mice have
increased insulin sensitivity and obesity resistance, even on a
high-fat diet. Importantly, the PTP1B knockout mice are con-
sidered to be normal and do not show any other phenotypic
1
2
–6
9
characteristics. PTP1B antisense oligonucleotide lowers PTP1B
protein expression, normalizes blood glucose and improves
1
0
a
insulin sensitivity in diabetic mice.
Some data suggest
Institute of Molecular Science, Key Laboratory of Chemical Biology
1
1–14
and Molecular Engineering of the Education Ministry, Shanxi
University, Taiyuan, Shanxi 030006, P.R. China.
that PTP1B regulates insulin sensitivity in humans.
PTP1B
has therefore emerged as a potential pharmaceutical target for
the treatment of type II diabetes and obesity. Great efforts have
been made in screening potent and selective PTP1B inhibitors in
the last two decades. However, the common architecture of a
PTP active site (i.e., pTyr-binding pocket) impedes the develop-
ment of selective PTP1B inhibitors. Therefore, it is a real chal-
lenge to design and exploit potent and specific PTP1B
E-mail: luliping@sxu.edu.cn, miaoli@sxu.edu.cn
b
Edmond H. Fischer Signal Transduction Laboratory, College of Life
Sciences, Jilin University, Changchun 130023, P.R. China.
E-mail: xingshu@jlu.edu.cn
†
Electronic supplementary information (ESI) available. CCDC 862832
(1). Crystallographic data in CIF and BVS calculations for complex 1
and the experimental and simulation isotopic distribution patterns of
ESI-MS spectra of complexes 1–5. For ESI and crystallographic data in
CIF or other electronic format see DOI: 10.1039/c2dt30198a
15,16
inhibitors.
11116 | Dalton Trans., 2012, 41, 11116–11124
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Vanadium is useful in the treatment of both type I and type II
diabetes in various animal models, as well as in limited human
and supported by Bruker Company. The X-ray data were col-
lected on a Bruker SMART APEX 1K CCD diffractometer.
Bioactivity assays (IC50 values) of the complexes were carried
out on SpectraMax M5 Multi-Mode Microplate Readers (Mole-
1
7–22
trials.
Vanadium complexes with appropriate organic
ligands can improve absorption, tissue uptake and potency, and
decrease toxicity of the metal. Organic vanadium complexes
exhibit high insulin-mimetic potential with low side effects com-
pared to inorganic vanadium. A bis(maltolato)oxovanadium(IV)
28–31
cular Devices, USA), as previously described.
Synthesis of the complexes
(
BMOV) derivative was tested on phase II clinical trials as an
22
antidiabetic drug. The inhibition of PTPs that dephosphorylate
the insulin receptor, resulting in prolongation of insulin signal-
ing, is involved in the mechanism of vanadium’s insulin-sensitiz-
ing effects.
vanadium complexes can inhibit PTP1B. Pervanadate inhibits
The synthesis of Schiff bases and oxovanadium(IV) complexes
–5 followed the routes described in Scheme 1. Tridentate and
1
bidentate Schiff base ligands were synthesized from an equi-
molar mixture of 5-X-salicylaldehyde (X = Cl, Br and NO2)
with anthranilic acid or aniline by use of the respective pro-
23,24
In vitro experiments have demonstrated that
35
PTP1B by irreversibly oxidizing the catalytic cysteine to a SO H
3
cedures previously reported. Next, the object complexes were
group, while vanadate and vanadium complexes competitively
synthesized by the Schiff bases reacted with VOSO ·xH O in
4
2
24–32
inhibit PTP1B.
However, few studies have investigated the
methanol solution.
inhibition against other PTPs and the selectivity of vanadium
complexes. It is worth mentioning that the latest research shows
metal-based PTP selective inhibitors exhibit consistent selectiv-
2
-((5-Bromo-2-oxybenzylidene)amino)benzoato-diaqua-oxova-
nadium(IV) (1). 0.14 g (1.0 mmol) anthranilic acid and 0.16 g
2.0 mmol) NaOAc were thoroughly dissolved in 10 mL of
water with a constant stirring. To it 0.20 g (1.0 mmol) 5-bromo-
salicylaldehyde in 10 mL of absolute ethanol was added drop-
3
3,34
(
ity in cells as for in vitro results,
suggesting that in vitro
screened metal-based selective PTP inhibitors may work in vivo.
Thus, it is promising to carry out a wide investigation into
screening PTP1B selective inhibitors among vanadium com-
plexes in order to improve the potency of vanadium-based anti-
diabetic drugs and decrease their side effects. As a matter of fact,
our recent study revealed that several vanadium complexes do
wise. After several minutes, 0.23 g (1.0 mmol) of VOSO ·xH O
4
2
in 3.0 mL of water was added dropwise. The reaction mixture
was heated under refluxing for 3 h. After cooling slowly, the
brown precipitates were separated out. The separated compound
was filtered, washed thoroughly with absolute ethanol and water,
and then dried in a vacuum desiccator with P O . Yield 41%,
element analysis (EA) for 1(C H BrNO V), calcd/found(%): C
2
8,30
display some selectivity against PTP1B,
inspiring us to
screen potent and selective vanadium-based PTP1B inhibitors by
modifying the ligands of vanadium complexes.
2 5
14 12
6
3
9.93/39.75, H 2.87/3.14, N 3.33/3.28; IR (s = strong, m =
medium, w = weak): ν 1577s (1604s in H bhbb), ν
VvO
In this report, a series of oxovanadium complexes of Schiff
bases have been synthesized and well characterized. Their inhibi-
tory effects against five PTPs are evaluated. The results show
these complexes are potent PTPs inhibitors and have certain
selectivity against PTP1B.
CvN
2
1
9
82m (995 cm− in VOSO ); ESI-MS(m/z, a negative mode, see
4
Table 1 and ESI†), observed molecular ion peak (I%):
−
4
(
20.86(38%) for [1 − H] 420.92; UV-Vis(DMSO): λmax/nm
4
ε × 10 /M cm−1), 264(2.37), 290–310(1.45), 412(0.85).
−1
A brown crystal of 1 was obtained by slow evaporation of the
Experimental section
reaction solution at room temperature after two weeks.
-((5-Nitro-2-oxybenzylidene)amino)benzoato-diaqua-oxova-
Materials and methods
2
nadium(IV) (2). Following the same procedures as described in
synthesis of 1, received 2 with 5-nitro-salicylaldehyde instead of
All reagents and solvents were obtained from commercial
sources and used without further purification unless specially
noted. Double distilled water was used to prepare buffer
solutions.
5
-bromo-salicylaldehyde. Yield 30%, EA for 2 (C H N O V),
14 12 2 8
calcd/found(%): C 43.43/43.85, H 3.12/3.01, N 7.23/7.34; IR:
νCvN 1562s (1611s in H nhbb), ν 952m; ESI-MS(m/z, a
2
VvO
negative mode, see Table 1 and ESI†), observed molecular ion
−
Physical measurement
peak (I%): 385.94(38%) for [2 − H] 386.00; UV-Vis(DMSO):
4
−1
−1
λmax/nm (ε × 10 /M cm ), 262(1.26), 307(1.38), 376(1.79).
The C, H and N analyses were performed on a VARI-EL elemen-
tal analyzer. IR spectra on KBr pellets were recorded on a Shi-
madzu FT IR-8300 spectrometer in the range of 4000–400 cm−
Bis(4-chloro-2-((phenylimino)methyl)phenolate)-oxovana-
dium(IV) (3). Hcpmp was precipitated by the condensation of
5-chloro-salicylaldehyde and aniline in absolute ethanol. 3.13 g
(20.0 mmol) 5-chloro-salicylaldehyde was dissolved in 30 mL
of absolute ethanol. To this, 1.8 mL (20.0 mmol) of aniline was
added dropwise with a constant stirring. The reaction mixture
was heated under refluxing for 3 h. After cooling slowly, the
light orange needle crystals were separated out. The separated
compound was filtered, washed thoroughly with absolute ethanol
and dried in a vacuum desiccator with P O . Yield 82%, EA for
1
(KBr disks). The electronic spectra were recorded on a Hewlett-
Packard HP-8453 Chemstation spectrophotometer. UV-Vis
−5
spectra of titrations of ligands (2 ml, 5 × 10 M) with VOSO4
3
(
2.5 × 10− M) were used in the study in DMSO–aqueous solu-
tion (1 : 9) at room temperature. Electrospray ionization mass
spectra (ESI-MS) were recorded with a micrOTOF-Q instrument
(Bruker) in methanol solution. The EPR spectrum was obtained
in solid and DMSO solution at 110 K on a Bruker-ER 200-
D-SRC spectrometer. The EPR spectrum of a frozen sample was
simulated by use of the WinEPR SimFonia program developed
2
5
Hcpmp(C H ClNO), calcd/found(%): C 67.39/67.54, H 4.35/
4.35, N 6.05/5.82; IR: νCvN 1614s. A 0.46 g sample (2.0 mmol)
13 10
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IV
IV
IV
IV
2 2
Scheme 1 Schematic routes of the synthesis of the complexes 1–5, [V O(bhbb)](1), [V O(nhbb)](2), [V O(cpmp) ](3), [V O(bpmp) ](4),
IV
2
[V O(npmp) ] (5).
of the ligand in 15.0 mL of absolute ethanol was heated under
refluxing until thoroughly dissolved and 0.45 g (2.0 mmol) of
N 10.20/9.95; IR: νCvN 1560s, νVvO 947m; ESI-MS(m/z, a
positive mode, see Table 1 and ESI†), observed molecular ion
+
VOSO in 10.0 mL of water was added dropwise with constant
peak (I%): 549.07(100%) for [5] 549.06; UV-Vis(DMSO):
4
4
−1
−1
stirring. The reaction mixture was adjusted to pH = 7 with
NaOH solution and then it was heated under refluxing for 4 h.
After cooling slowly, the green precipitates were separated out.
The separated compound was filtered, washed thoroughly with
absolute ethanol and water, and then dried in a vacuum desicca-
tor with P O . Yield 84%, EA for 3(C H Cl N O V), calcd/
λmax/nm (ε × 10 /M cm ), 261(1.95), 298(1.65), 364(2.97),
429(1.48).
X-ray crystallography
2
5
26 18
2 2 3
A single crystal of the complex 1·1.5H O was mounted on glass
2
found(%): C 59.11/59.20, H 3.43/3.47, N 5.30/5.19; IR: νCvN
559s, νVvO 973m; ESI-MS(m/z, a positive mode, see Table 1
and ESI†), observed molecular ion peak (I%): 528.04(94%) for
fibers for data collection. Cell parameters and an orientation
matrix for data collection were obtained by least-square refine-
ment of diffraction data from 6213 reflections with 2.41–25.26°
1
+
4
−1
−1
[
3 + H] 528.02; UV-Vis(DMSO): λ /nm (ε × 10 /M cm ),
max
of θ for 1·1.5H O using a Bruker SMART APEX 1K CCD auto-
2
2
62(1.78), 344(1.33).
matic diffractometer. Data were collected at 298 K using MoKα
radiation (λ = 0.71073 Å) and the ω-scan technique, and cor-
Bis(4-bromo-2-((phenylimino)methyl)phenolate)-oxovana-
36
rected for Lorentz and polarization effects (SADABS). The
dium(IV) (4). Following the same procedures as described in
synthesis of Hcpmp, received Hbpmp with 5-bromo-salicylalde-
hyde instead of 5-chloro-salicylaldehyde. Hbpmp yield 91%,
EA for Hbpmp(C H BrNO), calcd/found(%): C 56.55/56.58,
37
structures were solved by direct methods (SHELXS-97) and
2
subsequent difference Fourier maps and then refined on F by a
full-matrix least-squares procedure using anisotropic displace-
13
10
37
ment parameters. After several cycles of refinement, hydrogen
H 3.65/3.69, N 5.07/5.11; IR: νCvN 1614s. 0.46 g (2.0 mmol) of
Hbpmp in 10.0 mL of absolute ethanol was thoroughly dissolved
with constant stirring, 0.16 g (2.0 mmol) NaOAc was added to
the solution, and then 0.23 g (1.0 mmol) of VOSO ·xH O in
atoms attached to C atoms were located at their calculated
2
positions with C(sp )–H 0.93 and were refined using a riding
model. H atoms attached to O atoms in 1·1.5H O were located
2
4
2
from difference Fourier maps and their global U value refined.
iso
3
.0 mL of water was added dropwise with constant stirring. The
38
Molecular graphics are from Ortep3.
reaction mixture was refluxed for 3 h. After cooling slowly, the
green precipitates 4 were separated out. The separated compound
was filtered, washed thoroughly with absolute ethanol and water,
and then dried in a vacuum desiccator with P O . Yield 70%,
Potentiometric titration
2
5
EA for 4(C H Br N O V), calcd/found(%): C 50.60/50.60,
pH-Potentiometric titration was performed to study the species
formed in solution. The experiment was carried out in 20%
methanol–water (by volume) due to the poor solubility of
26
18
2 2 3
H 2.94/2.98, N 4.54/4.48; IR: νCvN 1559s, νVvO 982m;
ESI-MS (m/z, a positive mode, see Table 1 and ESI†), observed
+
molecular ion peak (I%): 616.92(48%) for [4] 617.18; UV-Vis
H bhbb and its complexes in pure water. The ionic strength of
2
4
−1
−1
(
DMSO): λmax/nm (ε × 10 /M cm ), 263(1.88), 344(1.43).
the solutions was maintained by 0.2 M NaCl. The protonation
constants of ligands and the stability constants of the complexes
VOSO4 with ligands were determined by pH-potentiometric
titrations of 40 mL samples containing the ligand being acidified
by HCl. High-purity nitrogen gas was used to remove carbon
dioxide and molecular oxygen from samples and to provide an
inert gas atmosphere during all measurements. Measurements of
the pH values were carried out at 298 ± 0.2 K on a PHS-3TC pH
meter with a combined glass electrode. The electrode was
Bis(4-nitro-2-(( phenylimino)methyl)phenolate)-oxovana-
dium(IV) (5). Following the same procedures as described in
synthesis of 3, received Hnpmp and 5 with 5-nitro-salicylalde-
hyde instead of 5-chloro-salicylaldehyde. Hnpmp yield 91%,
EA for Hnpmp(C H N O ), calcd/found(%): C 64.46/64.45,
13 10 2 3
H 4.16/4.10, N 11.56/11.14; IR: νCvN 1620s. 5 yield 30%, EA for
(C H N O V), calcd/found(%): C 56.84/56.60, H 3.30/3.36,
5
2
6 18 4 7
11118 | Dalton Trans., 2012, 41, 11116–11124
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a
Table 1 Characteristics of complexes
IR (cm−
ν
1
)
UV-Vis
DMSO,λmax/nm (ε × 10 /M cm−1
ESI-MS (m/z) isotopic peaks
d
b
c
4
−1
Complex, yield (%)
CvN,νVvO
EPR (g)
)
Species calcd/found
−
1
2
3
4
5
, 41%
, 30%
, 84%
, 70%
, 30%
1577s, 982s
1562s, 952s
1559s, 973s
1559s, 982s
1560s, 947s
1.981
1.976
2.011
2.023
1.975
264(2.37), 290–310(1.45), 412(0.85)
262(1.26), 307(1.379), 376(1.786)
262(1.78), 344(1.33)
263(1.88), 344(1.43)
261(1.95), 298(1.65), 364(2.97), 429(1.48)
[1 − H] 417.92–423.93/417.90–423.86
−
[2 − H] 385.00–389.00/385.56–388.92
+
[3 + H] 526.02–533.01/526.02–533.02
+
[4 + H] 613.62–622.32/613.92–621.92
+
[5 + H] 547.98–552.07/548.06–552.07
a
Element analysis: see experimental section. 995 cm−1 in VOSO
b
c
d
4
, s for strong. In solid at 110 K. In methanol.
calibrated by standard buffer solutions. The titrations were per-
formed with a carbonate-free NaOH solution of known concen-
tration (0.09495 M) using a microsyringe under a nitrogen
atmosphere in a jacketed vessel. The protonation constants of
ligands and the equilibrium constants of the complexes were cal-
The inhibiting kinetic analysis was performed according to
eqns (1) and (2) for competitive and noncompetitive inhibition
modes, respectively, where Vmax is the maximum initial velocity,
Km for the corresponding Michaelis–Menten constant, S for the
substrate, I for the inhibitor and K for the inhibition constant at
i
3
9
culated with the aid of the SUPERQUAD program. The cor-
varied substrate concentrations, derived from the slope of the
rection ionization constants of water in 20% methanol–water
Lineweaver–Burk plots.
4
0
+
mixtures were taken from Wooley et al., namely pK
= [H ]
ꢀ
ꢁ
M/W
−
−
−
−
1
Km
½Iꢀ
1
1
[
A ] = 14.10, where A is OH and CH O . The overall for-
3
¼
1 þ
þ
ð1Þ
ð2Þ
ν
Vmax
Ki
S
Vmax
mation constant of the complex is denoted as the logarithm of
p
q
r
βpqr = [VO H bhbb H ]/[VO] [H bhbb] [H] . The conventional
p
2
q
r
2
ꢀ
ꢁ
ꢀ
ꢁ
1
Km
½Iꢀ
1
1
½Iꢀ
Ki
notation has been used: negative indices for H in the formulas
indicate the dissociation of groups that do not deprotonate in the
absence of either V(IV)O coordination or hydroxo ligands. The
¼
1 þ
þ
Vmax
1 þ
ν
Vmax
Ki
S
Inhibition constants were determined by the measurement of
initial hydrolysis rates at different concentrations of substrate and
inhibitor. The apparent Michaelis–Menten constant (Kapp) values
measured at the various inhibitor concentrations were plotted
2
+
following hydroxo species of VO were taken into account in
+
the calculations: [VO(OH)] (log β10-1
=
−5.94) and
2+
41
−
[
−
(VO) (OH) ] (log β20-2 = −6.95), [VO(OH) ] (log β
18.0) and [(VO) (OH) ] (log β20-5 = −22.0). All solutions
were freshly prepared before use. As in our previous report, the
cumulative stability constants (βpqr) of oxovanadium complex
species in the V(IV)O/H bhbb system were log β = 14.97(4),
=
2
2
3
42
10-3
−
2
5
against concentration of the inhibitor to calculate the K values.
30
i
For the K value of each PTP, the measurements were indepen-
i
dently repeated at least three times.
2
111
log β110 = 9.44(9), log β11-1 = 4.94(4), log β11-2 = −2.91(4) and
log β11-3 = −13.15(5).
Results and discussion
Synthesis and general aspects
Protein tyrosine phosphatase inhibition assays
The oxovanadium complexes were prepared from a typical syn-
thetic procedure, in which oxovandium(IV) sulfate is reacted
in situ with dianionic or monoanionic Schiff bases in methanol–
aqueous solution, as shown in Scheme 1, in which the intermedi-
ate tridentate Schiff bases are not isolated. All of the vanadium
complexes are elucidated on the basis of the EAs, IR, EPR,
UV-Vis and ESI-MS. Selected characteristics of the complexes
are listed in Table 1. The complexes are remarkably soluble in
DMSO, soluble in methanol and insoluble in water. The elemen-
tal analysis data for 1–5 are consistent with the compositions of
the desired products. They are confirmed by an ESI-MS study
(see Table 1 and ESI†). Complex 1 is further supported by X-ray
single crystal diffraction in the forthcoming discussion. In the
infrared spectra, the absorption bands at ca. 1559–1577 cm−
can be assigned to the CvN vibration involving coordination
through the nitrogen of the azomethine group. The VvO stretch-
ing frequencies of the complexes occur in the range of
947–982 cm− , which is consistent with the previously reported
range observed for the majority of oxovanadium(IV)
Human PTPs were expressed and purified as described
43–46
previously.
PTP activities were measured using p-nitro-
phenol phosphate (pNPP) as the substrate. The assays were per-
formed in 20 mM 3-morpholinopropanesulfonic acid (MOPS)
buffer, pH 7.2, containing 50 mM NaCl and 2 mM GSH (L-glu-
tathione(reduced)). Complexes 1–5 were dissolved in DMSO
−2
(
10 M) and diluted to various concentration gradients, and
further diluted 10 times into enzyme-MOPS buffer solutions for
activity evaluations. Inhibition assays were performed in the
same buffer on a 96-well plate in 100 μL volumes. Namely,
1
8
0 μL of complex with various concentrations was mixed to
2 μL enzyme solution for 30 min. Then 2 μL of pNPP (0.1 M)
1
substrate was added to initiate enzyme reactions. After incu-
bation for 30 min at room temperature, the reactions were termi-
nated by the addition of 6 μL of 2 M NaOH. The optical density
at 405 nm was measured on a microplate reader. IC50 values
were obtained by fitting the concentration-dependent inhibition
curves by use of the Origin program. All data points were
carried out in triplicate. Solutions of the oxovanadium com-
plexes were all freshly prepared before each experiment.
1
2
8–31
complexes.
The electronic absorption spectra of the oxo-
vanadium(IV) complexes in DMSO show a progress of ligand-to-
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Table 2 Crystallographic data of complex 1
1
·1.5H
862832
12BrNO
448.12
2
O
CCDC number
Formula
C
14
H
6 2
V·1.5H O
Formula weight
Wavelength (Å)
Temperature (K)
Crystal system
Space group
a (Å)
0.71073(Mo Kα)
298(2)
monoclinic
C2/c
33.835(4)
7.374(1)
13.086(2)
92.497(2)
3261.8(7)
0.20 × 0.15 × 0.10
8
b (Å)
c (Å)
β (°)
V (Å )
3
Crystal size (mm)
Z
D
μ (mm
−3
calc (g cm
)
1.825
3.099
−
1
)
F
000
1792
θ range (°)
Limiting indices, h
K
1.2–25.59
−40 ≤ h ≤ 40
−8 ≤ k ≤ 8
−15 ≤ l ≤ 15
1.029
17390
3051
Fig. 1 EPR spectra of complex 1, (a) powder sample at 110 K, (b)
frozen sample in DMSO at 110 K and (c) the simulated spectrum of
frozen sample.
L
Goodness-of-fit
Reflections collected
Reflections unique
R
int
0.0354
0.0245
0.0320
0.998
metal charge transfer (LMCT) from occupied π(n) orbitals of the
chelated ligand to empty d orbitals of the vanadium near
Final R [I > 2σ(I)]
R (all data)
Completeness
3
44–429 nm (Table 1).
3
Largest diff. peak and hole (e Å ).
0.298, −0.354
In order to ascertain the oxidation state of the vanadium, elec-
tron paramagnetic resonance was employed. As shown in Fig. 1,
the X-band EPR spectra of complex 1 of the solid powder and
frozen sample in DMSO at 110 K, which exhibits an axially
symmetrical signal of tetravalent vanadium in solution, split into
Table 3 Coordinated geometry of complex 1
Bond length, Å
Bond angle, °
1
a number of hyperfine lines which originate from the d electron
interaction with a nuclear spin I = 7/2. The spectrum displays
well-resolved V (I = 7/2) hyperfine lines. Furthermore, bond
V1–O1 1.585(2) O1–V1–O2 101.6(1) O2–V1–N1 91.6(1)
V1–O2 1.957(2) O1–V1–O3 102.7(1) O3–V1–N1 88.1(1)
V1–O3 1.985(2) O2–V1–O3 155.6(1) O5–V1–N1 169.8(1)
51
valence sum (BVS) calculations show a value of 4.14 for the V
V1–O5 2.056(2) O1–V1–O5 93.9(1)
V1–N1 2.078(2) O2–V1–O5 87.3(1)
V1–O6 2.248(2) O3–V1–O5 88.7(1)
N1–C7 1.295(3) O1–V1–N1 96.3(1)
N1–V1–O6 84.3(1)
O1–V1–O6 179.4(1)
O2–V1–O6 78.5(1)
O3–V1–O6 77.2(1)
O5–V1–O6 85.5(1)
C7–N1–C8 117.7(2)
47
center. This value is clearly within the stipulated error limit.
Thus, an oxidation state of +4 is assigned to vanadium (see
ESI†). This agrees with the result of EPR analysis.
Molecular structure of complex 1
single bonds. The lattice water molecules in the complex 1 show
intermolecular H-bonding interactions with coordinated water and
the carboxylate oxygen of the Schiff base (Fig. 2 and Table 4).
Complex 1·1.5H O crystallizes in space groups C2/c. Some para-
2
meters on the crystal data and structure determination are sum-
marized in Table 2. Coordination parameters of vanadium are
shown in Table 3 and the structure is shown in Fig. 2.
The structure of the complex consists of a monomeric oxo-
vanadium(IV) species with a VO moiety bonded by two
chelate rings of one dianionic tridentate Schiff base, namely, V1/
Chemistry in solution
2
+
The interaction of the ligands with the oxovanadium(IV) was ana-
lyzed by use of UV-Vis titration experiments in order to explore
the structure of the complex in aqueous solution. Here, we take
O2/C2/C1/C7/N1 and V1/N1/C8/C13/C14/O3. The complex has
IV
a V O N distorted octahedron geometry with a VvO distance
H nhbb and Hnpmp as representatives of tridentate and bidentate
2
5
of 1.585(2) Å, typical for a double bond. The Schiff base is
bound through the phenolate oxygen (O2), the imine nitrogen
ligands. UV-Vis titration of complexes 2 and 5 were performed
in aqueous solution including 10% DMSO. As shown in Fig. 3,
IV
(N1) and the carboxylate oxygen (O3) of the ligand, which
ligands H nhbb and Hnpmp and V O ion easily form the com-
2
cooperate with O5 atom of water to form the equatorial plane
and O1 bond to V1 and O6 (water) atoms located at the axial
positions. Due to the Jahn–Teller effect in a d electron configur-
plexes [VO(nhbb)] (1 : 1) and [VO(npmp) ] (2 : 1), respectively,
2
in which the ratio of tridentate and bidentate Schiff bases
1
IV
binding to V O is 1 : 1 and 2 : 1. The results agree with those in
ation of vanadium(IV), the V1–O6 bond trans to the VvO group
is significantly longer (2.248(2) Å) than the other two V–O
bonds (Table 3). The V–O distances involving the Schiff bases
and water are in range of 1.957(2)–2.056(2) Å in 1, typical for
the solid structure studies of complexes 2 and 5.
The five complexes were further characterized by ESI-MS in
methanol solution. As shown in Table 1 and ESI,† species corre-
sponding to the molecular ion are observed for all of the
11120 | Dalton Trans., 2012, 41, 11116–11124
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Fig. 2 An ORTEP view of complex 1 showing atom labeling with
3
0% probability thermal ellipsoids, double dash lines for H-bonds.
a
Table 4 Hydrogen bonds in 1 (Å, °)
D–H⋯A
d(D–H)
d(H⋯A)
d(D⋯A)
∠(DHA)
O5–H5⋯O8
0.86(4)
0.83(4)
0.75(3)
0.75(3)
0.83(3)
0.79(4)
0.95(5)
0.93
1.86(4)
1.88(4)
2.00(4)
2.10(4)
1.91(3)
2.17(4)
1.95(5)
2.41
2.714(3)
2.671(2)
2.743(3)
2.839(3)
2.721(3)
2.950(3)
2.897(3)
3.116(3)
173(4)
156(3)
169(3)
172(4)
166(3)
169(3)
177(4)
133
i
O5–H5B⋯O3
ii
O6–H6⋯O7
iii
O6–H6B⋯O4
O7–H7A⋯O8
O8–H8A⋯O4
iv
v
O8–H8B⋯O2
vi
C3–H3⋯O1
a
Symmetry codes: (i) −x + 1, y, −z + 1/2; (ii) x, y − 1,z; (iii) x, −y,
z + 1/2; (iv) x, −y + 1, z + 1/2; (v) −x + 1,−y + 1, −z + 1; (vi) x, 1 − y,
1
/2 + z.
Fig. 3 UV-Vis spectra of titrations of Hnpmp (up) and H
(
2
nhbb
bottom) (2 mL, 5 × 10− M) with VOSO
(5 × 10 M) used in the
4
5
−3
complexes. The compositions of the complexes deduced from
the elemental analysis are confirmed by the ESI-MS study and
the corresponding parent peaks in the ESI-MS exclude immedi-
study in DMSO–aqueous solution (1 : 9) at room temperature.
4
8
ate hydrolysis of the complexes in methanol solution.
In addition, the data of the potentiometric titration of the
IV
system of V O cation and H bhbb shows the species distri-
2
bution of the complexes formed in aqueous solution, which is
relevant to their bioactivities. On the basis of these data, the
species distribution diagram as a function of pH is shown in
Fig. 4. The distribution curves suggest that the oxovanadium
−
2−
species [VO(bhbb)(OH)] and [VO(bhbb)(OH) ] are predomi-
2
nant in the pH range 7.0–7.4.
Recombinant PTPs inhibition assays
The five complexes were tested for their abilities in inhibiting
PTP1B, TCPTP, PTP-MEG2, SHP-1 and SHP-2 by use of pNPP
as the substrate. The IC50 values are listed in Table 5. The results
show that almost all of the five complexes display strong inhi-
bition against PTP1B, though their inhibitory abilities vary over
different PTPs. Complexes 1 and 2 with a ligand:V ratio of
Fig. 4 Species distribution as a function of pH for VO-H bhbb (1 : 1,
2
3
0
0
.75 mM) systems. Coordinated water molecules were omitted.
1
: 1 have slightly stronger inhibitory effects against PTP1B
Na [VO(Glu) (CH OH)] and the most ternary oxovanadium(IV)
2 2 3
(
2
IC , 0.21–0.23 μM) than 3–5 possessing a ligand:V ratio of
: 1 (IC , 0.69–0.93 μM). The potency of PTP1B inhibitions of
50
complexes of amino-acid Schiff bases and polypyridyl deriva-
tives, as well as BMOV (Table 5). In both 1 : 1 complexes, 2
shows better selectivity against PTP1B over the other four PTPs.
The inhibitory ability against PTP1B is about 5, 3, 8 and 20-fold
stronger than that against TCPTP, PTP-MEG2, SHP-1 and
5
0
the five complexes are obviously weaker than that of our pre-
viously reported ternary oxovanadium(IV) complexes of ONO-
donor Schiff bases and polypyridyl derivatives, but equivalent to
This journal is © The Royal Society of Chemistry 2012
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a
Table 5 IC50(S.D. )(μM) of oxovanadium complexes on five PTPs
SHP-2, while complex 1 displays a similar inhibitory effect over
PTP1B and TCPTP, but about 5, 15 and 250-fold stronger than
over PTP-MEG2, SHP-1 and SHP-2. In the three 2 : 1 com-
plexes, complex 4 seems to be more selective in inhibiting
PTP1B. The potency against PTP1B is more efficient by about
b
PTP1B
TCPTP
PTP-MEG2
SHP-1
SHP-2
Ref.
1
2
3
4
0.21(2)
0.23(1)
0.80(9)
0.69(3)
0.93(9)
0.030
0.19(3)
1.3(1)
8.3(7)
1.9(3)
1.4(1)
0.054
0.97(6)
0.60(9)
3.6(4)
2.9(4)
3.2(6)
3.0(3)
1.9(2)
1.0(2)
6.5(3)
2.5(5)
0.26
49(9)
4.2(4)
2.3(6)
35(3)
2.6(2)
R1
R1
R1
R1
R1
30
3
, 4, 10 and 50-fold than against TCPTP, PTP-MEG2, SHP-1
and SHP-2. Complex 3 exhibits comparable inhibitory ability
against PTP1B and SHP-1, and about 10, 3 and 2-fold stronger
inhibition against TCPTP, PTP-MEG2 and SHP-2, while
complex 5 shows lower selectivity with comparable inhibition
against PTP1B and TCPTP, and about only 2 to 3-fold stronger
against the other three. Our previous study shows that ternary
oxovanadium(IV) complexes of ONO-donor Schiff bases and
5
c
d
e
S1
S2
S3
0.86(2)
0.29
49
31
0.34
0.21
a
b
c
All data points were carried out in triplicates. R1 for this work. S1 =
VO(SAA)(bpy)]. S2 = BMOV. S3 = Na [VO(Glu) (CH OH)].
2 2 3
d
e
[
Fig. 5 Lineweaver–Burk plots of 1/v (min mM−1) vs. the reciprocal of the pNPP concentrations (mM−1) at five fixed concentrations of complex 2
for PTP1B (A), TCPTP (B), SHP-1 (C), SHP-2 (D) and PTP-MEG2 (E), error bars represent ±S.D. Inset: determination of K for complex 2 inhibiting
i
PTP1B, TCPTP, SHP-1, SHP-2 and PTP-MEG2, respectively.
11122 | Dalton Trans., 2012, 41, 11116–11124
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polypyridyl derivatives selectively inhibit PTP1B 2-fold and
0-fold stronger than TCPTP and SHP-1, but Na [VO-
21171109), the Specialized Research Fund for the Doctoral
Program of Higher Education (20111401110002), the Scientific
Research Foundation for the Returned Overseas Chinese Scho-
lars, State Education Ministry (20093602), the China Scholar-
ship Council, the Natural Science Foundation of Shanxi
Province (20051013, 2010011011-2, 2011011009-1 and
2011021006-2), Shanxi Scholarship Council of China
(2012-004) and Instrumental Analysis Foundation of Large
Scale Instruments of Shanxi University, China.
1
2
(
Glu) (CH OH)] almost equally inhibit PTP1B, TCPTP, SHP-1
2 3
and HePTP (hematopoietic tyrosine phosphatase). Obviously,
complexes 2 and 4 show better selectivity to PTP1B. All of
these results illustrate that the ligands of vanadium complexes
influence the inhibitory effects of different PTPs. Properly modi-
fying the organic ligand moieties on vanadium may result in
screening potent and selective vanadium-based PTP1B
inhibitors.
Kinetic analysis of recombinant PTPs inhibition
Notes and references
Complex 2 was further chosen to investigate the inhibition mode
of the five PTPs because of its stronger potency and better selec-
tivity against PTP1B. As shown in Fig. 5, for PTP1B, TCPTP
and SHP-1, the Lineweaver–Burk double-reciprocal plot of the
kinetic data of complex 2 shows that the lines converge at an
intersection on the x-axis left of the y-axis, implying a noncom-
petitive inhibition mode versus pNPP, while the lines converge at
an intersection on the y-axis above the x-axis for PTP-MEG2
and SHP-2, indicating a classical competitive inhibition mode
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1
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1
In summary, we have synthesized and characterized five oxo-
vanadium(IV) complexes. Biochemical assays demonstrate that
the oxovanadium(IV) complexes are potent inhibitors of PTP1B,
TCPTP, PTP-MEG2, SHP-1 and SHP-2, but exhibit different
inhibitory abilities over different PTPs. Complexes 2 and 4
display better selectivity to PTP1B over the other four PTPs.
Kinetic data show complex 2 inhibits PTP1B, TCPTP and
SHP-1 with a noncompetitive inhibition mode, but a classical
competitive inhibition mode for PTP-MEG2 and SHP-2. The
results demonstrate that both the structures of vanadium com-
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activity. Properly modifying the organic ligand moieties on
vanadium may result in screening potent and selective
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