Synthesis and evaluation of copper complexes of Schiff-base condensates from 5-substituted-2-hydroxybenzaldehyde and 2-substituted-benzenamine as selective inhibitors of protein tyrosine phosphatases

Article abstract of DOI:10.1016/j.ica.2013.05.018

Five copper complexes, [Cu(bhbb,chbb,nhbb)(H₂O)_n] (tridentate-ligands: H₂bhbb = 2-(5-bromo-2-hydroxylbenzylideneamino) benzoic acid, 1; H₂chbb = 2-(5-chloro-2-hydroxylbenzylideneamino) benzoic acid, 2; H₂

Full text of DOI:10.1016/j.ica.2013.05.018

Accepted Manuscript
Synthesis and evaluation of copper complexes of Schiff-base condensates from
5-substituted-2-hydroxybenzaldehyde and 2-substituted-benzenamine as selec‐
tive inhibitors of protein tyrosine phosphatases
Ruiting Zhu, Liping Lu, Miaoli Zhu, Hong Han, Caixia Yuan, Shu Xing, Xueqi
Fu
PII:
S0020-1693(13)00291-0
http://dx.doi.org/10.1016/j.ica.2013.05.018
ICA 15473
DOI:
Reference:
To appear in:
Inorganica Chimica Acta
Received Date:
Revised Date:
Accepted Date:
22 February 2013
9 May 2013
16 May 2013
Please cite this article as: R. Zhu, L. Lu, M. Zhu, H. Han, C. Yuan, S. Xing, X. Fu, Synthesis and evaluation of
copper complexes of Schiff-base condensates from 5-substituted-2-hydroxybenzaldehyde and 2-substituted-
benzenamine as selective inhibitors of protein tyrosine phosphatases, Inorganica Chimica Acta (2013), doi: http://
dx.doi.org/10.1016/j.ica.2013.05.018
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1 Cover Page
2
3
4
5
Synthesis and evaluation of copper complexes of Schiff-base
condensates from 5-substituted-2-hydroxybenzaldehyde and
2-substituted-benzenamine as selective inhibitors of protein tyrosine
phosphatases
6
7
Ruiting Zhua, Liping Lu*a, Miaoli Zhu*,a,c, Hong Hana, Caixia Yuana, Shu Xing*b,
Xueqi Fub
8
9
aInstitute of Molecular Science, the Key Laboratory of Chemical Biology and
Molecular Engineering of Education Ministry, Shanxi University, Taiyuan, 030006,
10 China (miaoli@sxu.edu.cn)
11 bKey Laboratory for Molecular Enzymology and Enginnering of Ministry of
12 Education, College of Life Sciences, Jilin University, Changchun 130012, China
13 (xingshu@jlu.edu.cn)
14 cState Key Laboratory of Coordination Chemistry, Nanjing University, Nanjing
15 210093, P.R. China
16
17
18 Corresponding Author's Name and Address:
19 Drs Liping Lu and Miaoli Zhu, Professors
20 Institute of Molecular Science, Shanxi University, 92 Wucheng Road, Taiyuan,
21 Shanxi 030006, People's Republic of China
22 *Corresponding author: E-mail: miaoli@sxu.edu.cn ; Tel: +86-351-7017974
1

1 Index Abstract
2
3
4
5
Synthesis and evaluation of copper complexes of Schiff-base
condensates from 5-substituted-2-hydroxybenzaldehyde and
2-substituted-benzenamine as selective inhibitors of protein tyrosine
phosphatases
6
7
Ruiting Zhu, Liping Lu*, Miaoli Zhu*, Hong Han, Caixia Yuan, Shu Xing*, Xueqi Fu
8
9
Five copper complexes of Schiff-base condensates from 5-substituted-2-hydroxy-
10 benzaldehyde and 2-substituted- benzenamine were synthesized and characterized.
11 The copper complexes noncompetitively inhibit PTP1B, TCPTP, PTP-MEG2 and
12 SHP-1, but do not inhibit SHP-2. Complex 5 exhibits very strong inhibition and better
13 selectivity against PTP1B (IC50=0.059 µM).
14
15
16
17
2

1
2
3
4
Synthesis and evaluation of copper complexes of Schiff-base
condensates from 5-substituted-2-hydroxybenzaldehyde and
2-substituted-benzenamine as selective inhibitors of protein tyrosine
phosphatases
5
6
Ruiting Zhua, Liping Lu*a, Miaoli Zhu*,a,c, Hong Hana, Caixia Yuana, Shu Xing*b,
Xueqi Fub
7
8
9
aInstitute of Molecular Science, the Key Laboratory of Chemical Biology and
Molecular Engineering of Education Ministry, Shanxi University, Taiyuan, 030006,
China (miaoli@sxu.edu.cn)
10 bKey Laboratory for Molecular Enzymology and Enginnering of Ministry of
11 Education, College of Life Sciences, Jilin University, Changchun 130012, China
12 (xingshu@jlu.edu.cn)
13 cState Key Laboratory of Coordination Chemistry, Nanjing University, Nanjing
14 210093, P.R. China
15
16 Keywords: copper complexes, protein tyrosine phosphatases, selective inhibitor,
17 Schiff base.
18
19
20
3

1
Abstract
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3
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5
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9
Five
copper
complexes,
[Cu(bhbb,chbb,nhbb)(H2O)n]
(tridentate-ligands:
H2bhbb=2-(5-bromo-2-hydroxylbenzylideneamino)benzoic acid, 1; H2chbb=2-(5-
chloro-2-hydroxylbenzylideneamino)benzoic acid, 2; H2nhbb=2-(5-nitro-2-hydroxyl-
benzylideneamino)benzoic acid, 3) and [Cu(cpmp,npmp)2] (bidentate-ligands:
Hcpmp=4-chloro-2-((phenylimino)methyl)phenol, 4; Hnpmp=4-bromo-2-((phenyl-
imino)methyl)phenol, 5) have been prepared and characterized by EA, IR, EPR
UV-vis, and ESI-MS. Structure-activity relationship of copper complexes in inhibiting
protein tyrosine phosphatases (protein tyrosine phosphatase 1B, PTP1B; T-cell
10 protein tyrosine phosphatase, TCPTP; megakaryocyte protein-tyrosine phosphatase,
11 PTP-MEG2; Src homology phosphatase 1, SHP-1 and Src homology phosphatase 2,
12 SHP-2) is investigated. Inhibitory activities of complexes against the five PTPs
13 indicate that they potently inhibit PTP1B, TCPTP, PTP-MEG2 and SHP-1, but do not
14 inhibit SHP-2. In the complexes, 5 exhibits very strong inhibition (IC50, 0.059 μM)
15 and better selectivity against PTP1B while 1 and 2 show very strong inhibition
16 (IC50=0.089 and 0.067 μM) and a little selectivity against TCPTP. Compared with the
17 oxovanadium(IV) complexes of same ligands, the copper complexes increase the
18 inhibitory ability against TCPTP, PTP-MEG2 and SHP-1 but decrease the inhibition
19 against SHP-2. For complex 5, the inhibition over PTP1B, TCPTP, PTP-MEG2 and
20 SHP-1 are all improved about 5~15-fold compared with the oxovanadium(IV)
21 complex. The results demonstrate that both the ligand structures and the center metals
22 influence the inhibition and selectivity against different PTPs.
4

1
1. Introduction
2
3
4
5
6
7
8
9
Protein tyrosine phosphatases (PTPs) are a large family of signaling enzymes
playing a critical role in signal transduction and regulation of cellular processes. Many
human diseases such as diabetes, obesity, cancer, and immune disorders are involved
in the dysregulation of PTP activities [1-5]. PTPs have therefore emerged as potential
therapeutic targets and attracted great interests in both academia and industrial in past
decades [6-8]. PTP inhibitors are thus expected as promising targeted therapeutic
drugs and dozens of new PTP inhibitors are reported each year [9, 10]. In recent years,
one important progress is the investigation of metal-based PTPs inhibitors [11].
10 Except vanadium complexes that their insulin-sensitizing effects have been
11 demonstrated to be at least partly related to the inhibition of PTPs [12-20], other metal
12 complexes such as Au, Sb, Fe, Ru complexes are reported to potently inhibit PTPs
13 and result in the increase of cellular phosphorylation [21-25].
14
Copper complexes are widely explored as anticancer agents and expected relatively
15 lower side effects than traditional platinum-based drugs. One of the main mechanisms
16 proposed to explain the anticancer activity of copper complexes is the DNA damage
17 by generating reactive oxygen species[26, 27]. Recent researches indicate copper
18 complexes inhibit many enzymes such as topoisomerase II [28], chymotrypsin-like
19 activity of proteasome[29], urease[30] and so on. Our study demonstrates that copper
20 complexes are potent PTPs inhibitors [31]. The structures of the ligands coordinated
21 to copper influence the inhibitory effects and the selectivity against different PTPs
22 [32-36]. The in vitro screened copper complex exhibits consistent selectivity as in
5

1
2
cells [32], suggesting that in vitro screened metal-based selective PTP inhibitors may
work in vivo.
3
4
5
6
7
8
9
Our previous study shows that copper complexes of Schiff base ligands containing
3,5-substituted-4-salicylideneamino-3,5-dimethyl-1,2,4-triazole,
[Cu(RSal-dmta)2],
potently inhibit PTPs and exhibit some selectivity against PTP1B. In order to further
check the influence of the ligands on PTPs inhibition, a series of copper complexes of
Schiff bases condensated from 5-substituted-2-hydroxybenzaldehyde and
2-substituted-benzenamine have been synthesized and well characterized. Their
inhibitory effects against five PTPs are evaluated. The results show these complexes
10 are also potent PTPs inhibitors and one exhibits some selectivity against PTP1B.
11 2. Experimental
12 2.1. Materials and physical measurements
13
All reagents and solvents were obtained from commercial sources and used without
14 further purification unless specially noted. Double distilled water was used to prepare
15 buffer solutions.
16
The C, H and N analyses were performed on a VARI-EL elemental analyzer. IR
17 spectra on KBr pellets were recorded on a Shimadzu FT IR-8300 spectrometer in the
18 range of 4000-400 cm-1(KBr disks). The electronic spectra were recorded on a
19 Hewlett-Packard HP-8453 Chemstation spectrophotometer. Electrospray ionization
20 mass spectra (ESI-MS) were recorded with a Quattro Micro API instrument (Waters,
21 USA) in methanol solution. EPR spectrum was obtained in solid and DMSO solution
6

1
2
3
at 110 K on a Bruker-ER 200-D-SRC spectrometer. Bioactivity assays (IC50 values)
of the complexes were carried out on a SpectraMax M5 Multi-Mode Microplate
Readers (Molecular Devices, USA) as previously described [15-17, 20].
4
2.2. Synthesis of the complexes
5
6
7
8
9
The synthesis of Schiff bases and copper complexes 1 - 5 was described in Scheme
1. Tridentate and bidentate Schiff base ligands were synthesized from an equimolar
mixture of 5-X-salicylaldehyde (X = Cl, Br and NO2) and anthranilic acid or aniline
using previously reported procedures [19, 37], respectively. Next, the object
compouds were synthesized by the Schiff bases reacting with Cu(OAc)2 H2O in
10 water/ethanol solution.
11
12
Scheme 1. Schematic routes of the synthesis of the ligands and complexes 1-5
13 2.2.1. 2-(5-bromo-2-hydroxylbenzylidene-amino)benzoic acid (H2bhbb) and
14 [Cu(bhbb)(H2O)] (1)
7

1
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3
4
5
6
7
8
9
H2bhbb: a 20.0 mmol of 5-bromosalicylaldehyde was thoroughly dissolved in 20
mL of absolute ethanol with a constant stirring. To it 20.0 mmol of anthranilic acid in
20 mL of absolute ethanol was added dropwise. The reaction mixture was heated
under refluxing for 3 h. After cooling slowly, the yellow precipitates were separated
out. The separated compound was filtered, washed thoroughly with absolute ethanol,
and then dried in a vacuum desiccator with P2O5. Yield 74%, element analysis for
H2bhbb (C14H10BrNO3): Calcd. (%) C 52.52, H 3.15, N 4.38; found (%) C 52.59, H
3.11, N 4.04; IR (υ/cm-1, s = strong, m = medium, w = weak): νC=O 1620s, νC=N 1604s,
νC-O 1226w.
10
[Cu(bhbb)(H2O)]: a 2.0 mmol of H2bhbb was thoroughly dissolved in 15 mL of
11 absolute ethanol with a constant stirring. To it 0.399 g (2.0 mmol) of Cu(OAc)2 H2O
12 in 10 mL of water was added dropwise and the pH of the reaction solution was
13 adjusted to neutral by use of 0.1 M NaOH solution. The reaction mixture was heated
14 under refluxing for 4 h. After cooling slowly, the green precipitates were separated
15 out. The separated compound was filtered, washed thoroughly with absolute ethanol
16 and water, and then dried in a vacuum desiccator with P2O5. Yield 83%, element
17 analysis for 1(C14H10BrCuNO4): Calcd. (%) C 42.07, H 2.52, N 3.50; found (%) C
18 42.11, H 2.24, N 3.39; IR: νC=O 1597s,
1564s, νC-O 1329m; EPR: g = 2.126;
C=N
19 ESI-MS(m/z, a positive mode): observed molecular ion peak in methanol 414.42 for
20 [1-H2O+CH3OH+H]+ (Calcd. 414.72); UV-Vis(DMSO): max/nm (ε 104/M-1cm-1), 264
21 (1.926), 420 (0.788).
8

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3
4
5
6
7
2.2.2. 2-(5-chloro-2-hydroxylbenzyl-idene-amino)benzoic acid (H2chbb) and
[Cu(chbb)(H2O)] (2)
H2chbb: following the same procedures as described in synthesis of H2bhbb,
received orange H2chbb with 5-chlorosalicylaldehyde instead of 5-bromo-
salicylaldehyde. Yield 80%, element analysis for H2chbb (C14H10ClNO3): Calcd. (%)
C 60.99, H 3.66, N 5.08; found (%) C 61.039, H 3.60, N 4.69; IR: νC=O 1620s, νC=N
1604s, νC-O 1228w.
8
9
[Cu(chbb)(H2O)]: following the same procedures as described in synthesis of 1,
received 2 with H2chbb instead of H2bhbb. Yield 82%, element analysis for
10 2(C14H10ClCuNO4), Calcd. (%) C 47.34, H 2.84, N 3.94; found (%) C 47.28, H 3.12,
11 N 3.90; IR(cm-1): νC=O 1520s,
1597s, νC-O 1314w; EPR: g = 2.126; ESI-MS(m/z,
C=N
12 a positive mode): observed molecular ion peak in methanol 436.75 for
13 [2+H2O+2CH3OH+H]+ (Calcd. 437.03); UV-Vis(DMSO): max/nm (ε 104/M-1cm-1),
14 263 (1.857), 421 (0.778).
15 2.2.3. 2-(5-nitro-2-hydroxylbenzylidene-amino)benzoic acid (H2nhbb) and
16 [Cu(nhbb)(H2O)2] (3)
17
H2nhbb: following the same procedures as described in synthesis of H2bhbb,
18 received orange-yellow H2chbb with 5-nitrosalicylaldehyde instead of
19 5-bromosalicylaldehyde. Yield 89%, element analysis for H2nhbb (C14H10N2O5):
20 Calcd. (%) C 58.74, H 3.52, N 9.79; found (%) C 58.17, H 3.49, N 9.32; IR: νC=O
21 1641s, νC=N 1611s, νC-O 1244m.
9

1
2
3
4
5
6
[Cu(nhbb)(H2O)2]: following the same procedures as described in synthesis of 1,
received 3 with H2nhbb instead of H2bhbb. Yield 80%, Element analysis for
3(C14H12CuN2O7): Calcd. (%) C 43.81, H 3.15, N 7.30; found (%) C 44.02, H 2.92, N
7.39; IR (υ/cm-1): νC=O 1597s, νC=N 1562s, νC-O 1336s; EPR: g = 2.125; ESI-MS(m/z, a
positive mode): observed molecular ion peak in methanol 384.67 for [3+H]+ (Calcd.
384.81); UV-Vis(DMSO): max/nm (ε 104/M-1cm-1), 270 (1.798), 387 (2.094).
7
2.2.4. 4-chloro-2-((phenylimino)methyl)phenol (Hcpmp) and [Cu(cpmp)2] (4)
8
9
Hcpmp: a 20.0 mmol of 5-chlorosalicylaldehyde was thoroughly dissolved in 30
mL of absolute ethanol with a constant stirring. To it 20.0 mmol of aniline in 20 mL
10 of absolute ethanol was added dropwise. The reaction mixture was heated under
11 refluxing for 3 h. After cooling slowly, the orange-yellow precipitates were separated
12 out. The separated compound was filtered, washed thoroughly with absolute ethanol,
13 and then dried in a vacuum desiccator with P2O5. Yield 82%, element analysis for
14 Hcpmp (C13H10ClNO): Calcd. (%) C 67.39, H 4.35, N 6.05; found (%) C 67.54, H
15 4.35, N 5.82; IR: νC=N 1614s, νC-O 1276s.
16
[Cu(cpmp)2]: a 2.0 mmol of Hcpmp in 15 mL of absolute ethanol was heated under
17 refluxing until thoroughly dissolved and 0.399 g (2.0 mmol) of Cu(OAc)2 H2O in 10
18 mL of water was added dropwise with a constant stirring. The reaction mixture was
19 adjusted to neutral pH with NaOH solution, and then it heated under refluxing for 4 h.
20 After cooling slowly, the brown-yellow precipitates were separated out. The separated
21 compound was filtered, washed thoroughly with absolute ethanol and water, and then
10

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4
5
dried in a vacuum desiccator with P2O5. Yield 38%, Element analysis for
4(C26H18Cl2CuN2O2), Calcd. (%) C 58.60, H 3.44, N 5.26; found (%): C 59.49, H
3.46, N 5.34; IR: νC=N 1557s, νC-O 1333s; EPR: g = 2.085; ESI-MS (m/z, a positive
mode): observed molecular ion peak in methanol 546.17 for [4+Na]+ (Calcd. 545.99);
UV-Vis (DMSO): max/nm (ε 104/M-1cm-1), 265 (2.556), 390 (1.181).
6
2.2.5. 4-nitro-2-((phenylimino)methyl)phenol (Hnpmp) and [Cu(npmp)2] (5)
7
8
9
Hnpmp: following the same procedures as described in synthesis of Hcpmp,
received orange-yellow Hnpmp with 5-nitrosalicylaldehyde instead of
5-chlorosalicylaldehyde. Yield 91%, element analysis for Hnpmp (C13H10N2O3):
10 Calcd. (%) C 64.46, H 4.16, N 11.56; found (%) C 64.45, H 4.10, N 11.14; IR: νC=N
11 1620s, νC-O 1288w.
12
[Cu(npmp)2]: Following the same procedures as described in synthesis of 4,
13 received 5 with Hnpmp instead of Hcpmp, yield 54%, element analysis for
14 5(C26H18CuN4O6 4.5H2O), Calcd.（%） C 49.80, H 4.34, N 8.93; found (%): C 49.86,
15 H 3.76, N 8.9; IR (υ/cm-1): νC=N 1555s, νC-O 1336s; EPR: g = 2.091; ESI-MS (m/z, a
16 positive mode): observed molecular ion peak in methanol 578.83 for [5+CH3OH+H]+
17 (Calcd. 579.04); UV-Vis(DMSO): max/nm (ε 104/M-1cm-1), 264 (1.758), 375 (2.986).
18 2.3. Protein tyrosine phosphatase inhibition assays
19
Human PTPs were expressed and purified as described previously [15, 33, 38-40].
20 PTP activities were measured by use of pNPP as the substrate. The assays were
11

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5
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performed in 20 mM MOPS buffer (pH 7.2, 50 mM NaCl). The complexes 1 - 5 were
dissolved in DMSO (1.0 10-2 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, 10 µl of complex with various concentrations were mixed to 83 µl
enzyme solution for 30 min. Then 2 µl of pNPP (0.1 M) substrate was added to
initiate enzyme reactions. After incubation for 30 min at room temperature, the
reactions were terminated by the addition of 5 µl of 2 M NaOH. The optical density at
405 nm was measured on a microplate reader. IC50 values were obtained by fitting the
10 concentration-dependent inhibition curves using the Origin program. All data points
11 were carried out in triplicates. Solutions of the copper complexes were all freshly
12 prepared before each experiment.
13
The inhibiting kinetic analysis was performed according to Eq. (1) for
14 noncompetitive inhibition mode, where Vmax is the maximum initial velocity, Km for
15 the corresponding Michaelis-Menten constant, S for the substrate, I for the inhibitor,
16 Ki for the inhibition constant at varied substrate concentrations, derived from the slope
17 of the Lineweaver-Burk plots.
1
Km
[I] 1
1
[I]
18
=
(1+
)
+
(1+
)
(1)
ν
Vmax
Ki
S
Vmax
Ki
19
Inhibition constants were determined by measuring initial hydrolysis rates at
20 different concentrations of substrate and inhibitor. The apparent Kapp values measured
12

1
2
at the various inhibitor concentrations were plotted against concentration of the
inhibitor to calculate the Ki values.
3
4
5
6
7
8
9
Phosphatase inhibition of cell extracts was carried out as described in reference
[36]. C6 rat glioma cells were harvested after growing in DMEM medium
supplemented with 10% fetal bovine serum, 100 units/ml of penicillin and 100 lg/ml
of streptomycin for 2 days. After washed with 0.85% NaCl (pH 7.4), cells were lysed
by sonication in 20 mM MOPS, 50 mM NaCl, pH 7.2, 0.002% PMSF. Cell lysates
were cleared by centrifugation at 15,000g for 30 min. The protein concentration of the
cell lysates was estimated using the Bradford Method. Next, 82 μl of cell lysates with
10 protein concentration of 1.55 mg/ml were treated with 30 μM complexes 1-5 for 1 h at
11 310 K. Then, 2μl of pNPP (0.1 M) substrate was added. After incubation for 30 min at
12 310 K, the reactions were terminated by the addition of 6 μl of 2 M NaOH. The A405
13 was measured on a microplate reader.
14 3. Results and Discussion
15 3.1. Synthesis and general aspects
16
The copper complexes are prepared from a typical synthetic procedure, in which
17 Cu(OAc)2 H2O is reacted with dianionic or monoanionic Schiff bases in ethanol
18 aqueous as shown in Scheme 1. The complexes are soluble in DMSO and DMF,
19 slightly soluble in methanol, and almost insoluble in water as well. The compositions
20 of the complexes deduced from the elemental analysis were confirmed by ESI-MS
21 study (see section experimental). In the infrared spectra, the strong bands at ca. 1555
22 ~ 1597 cm-1, assignable to the C=N stretching frequency, shifted 7 ~ 57 cm-1 and
13

1
2
3
clearly indicates that C=N was coordinated to copper for these complexes. Crystal
structure of complex 4 had been determined by the single crystal X-ray diffraction
method with monoclinic, space group P
and cell parameters: a = 13.142(7), b =
21 / n
4
9.7950(9), c = 9.7028(15) Å; = 110.923(2)°, which agreed with a reported result
5
6
7
8
9
[41], CCDC refcode YIDFEW in CSD [42]. It revealed the structure of 4 was
mononuclear and had perfectly planar trans-tetra-coordinated [CuO2N2].
Unfortunately, we failed to get better data of X-ray single crystal diffracting for the
other four complexes. From CSD [42], similar crystal structures were disclosed, such
as [Cu(hmbb)(H2O)] (CCDC refcode NUVBOV, H2hmbb = 2-(2-hydroxy-3-methoxy-
10 benzylideneamino)benzoic acid) [43], [Cu(hbb)(H2O)2] (CCDC refcode WEHWEM,
11 H2hbb = 2-(2- hydroxylbenzylideneamino)benzoic acid) [44], which revealed that the
12 molecular structures in 1 & 2 or 3 with tridentate ligands had distorting planar
13 tetra-coordinated [CuO3N] or square-pyramidal coordinated geometries [CuO4N].
14 Complexes 1 - 5 have also been characterized by electron paramagnetic resonance
15 (EPR). The gev values of five complexes were in range of 2.085 ~ 2.126 (see section
16 experimental), showing that all copper cations had d9 electron configuration.
17
In order to explore the stoichiometries of the ligand bonding to metal cation in
18 aqueous solution, the analysis of UV-Vis titration is employed. Here we take H2bhbb
19 and Hnpmp as representatives of tridentate and bidentate ligands. UV-Vis titration of
20 complexes 1 and 5 are performed in 10% DMSO aqueous solution. As shown in
21 Figure 1, ligands H2bhbb or Hnpmp and Cu2+ ion easily form the stable complexes
22 [Cu(bhbb)] and [Cu(npmp)2], respectively, in which the ratio of tridentate and
14

1
2
bidentate Schiff bases binding to Cu2+ is 1 1 and 2:1. The results agree with those
deduced from the elemental analysis and ESI-MS studies of complexes 1 and 5.
3
4
5
Figure 1 UV-Vis spectra of titrations of H2bhbb (left) and Hnpmp (right) (2ml, 5×10-5M) with
Cu2+ ion (5×10-3M) used in the study in 10% DMSO aqueous solution at room temperature.
6
7
8
9
The stabilities of the five complexes in PTP activity assay buffer are further
investigated. The results are shown in Figure 2 with complexes 1 and 5 as
representatives. The UV-Vis spectra are almost not changed in 2 h when the
complexes are added to the buffer, suggesting the complexes are stable in the solution.
10
11
12
Figure 2 UV-Vis spectra of complexes 1 (left) (5×10-5M) and 5 (right) (1×10-5M) in PTP activity
assay buffer in 2 h at room temperature. UV-Vis spectra are recorded every five minutes.
13 3.2. Inhibition of PTPs
14
The five ligands and complexes as well as CuCl2 were tested for their abilities in
15 inhibiting PTP1B, TCPTP, PTP-MEG2, SHP-1 and SHP-2 by use of pNPP as the
15

1
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3
4
5
6
7
8
9
substrate. The IC50 values for the complexes and CuCl2 inhibiting the five PTPs and
the comparison with other complexes are listed in Table 1. The results tell us that
almost all the five complexes and CuCl2 display strong inhibition against PTP1B,
TCPTP, PTP-MEG2 and SHP-1, but do not inhibit SHP-2, similar to Schiff bases
copper complexes of [Cu(RSal-dmta)2][33]. However, the ligands do not inhibit any
PTP (the data not shown here). Obviously, CuCl2 inhibits PTP1B, TCPTP,
PTP-MEG2 and SHP-1 with similar potency. However, the five copper complexes
exhibit some changes in the inhibition. In the three complexes possessing ligand/Cu2+
ratio of 1:1, 1 and 2 exhibit strongest inhibition against TCPTP with IC50 of 0.089 and
10 0.067 M, about 2 ~ 10-fold stronger than against SHP-1, PTP1B and PTP-MEG2
11 while 3 have not obvious selectivity over the four PTPs, suggesting substitute nitro
12 group for chloride or bromine slightly affect inhibition against different PTPs. The
13 selectivity of 1 and 2 against TCPTP is obviously different from [Cu(RSal-dmta)2]
14 complexes which either inhibit PTPs in ligand/Cu2+ ratio of 1:1 but have selectivity
15 against PTP1B over other four PTPs, demonstrating substitute anthranilic acid for
16 3,5-dimethyl-1,2,4-triazole changes the selectivity. For the complexes with
17 ligand/Cu2+ ratio of 2 1, 5 exhibits about 4-fold stronger inhibition against PTP1B
18 (IC50, 0.059 M) than against TCPTP, PTP-MEG2 and SHP-1(IC50, 0.21 ~ 0.24 M),
19 displaying very strong inhibition and better selectivity against PTP1B, similar to
20 [Cu(RSal-dmta)2] complexes. This similarity seems show that substitute anthranilic
21 acid for 3,5-dimethyl-1,2,4-triazole do not obviously influence the inhibitory potency
22 and selectivity in these bidentate Schiff base ligands. However, complex 4 with only a
16

1
2
3
4
5
6
7
8
9
substitute chloride for nitro group displays weaker inhibition against 1B than other
three PTPs. All the results illustrate that the structures of ligands influence the
inhibitory effects and selectivity of copper complexes over different PTPs but the
influence is complicated. To screen potent and selective copper-based PTPs inhibitors
against specific PTP by properly modification of the organic ligand moieties is a
challenge.
Our previous researches indicate that oxovanadium(IV) complexes which have
same ligands with these copper complexes (Table 1) show better selectivity to PTP1B
[19]. Comparing the copper complexes with the oxovanadium(IV) complexes with
10 same ligands, it is not difficult to find that the copper complexes increase the
11 inhibitory ability against TCPTP, PTP-MEG2 and SHP-1 but decrease the inhibition
12 against SHP-2. For complex 5, the inhibition over PTP1B, TCPTP, PTP-MEG2 and
13 SHP-1 are all improved about 5 ~ 15-fold compared with the oxovanadium(IV)
14 complexes with same ligands. The results suggest the metal ions of coordination
15 sphere influence the inhibition of PTPs. Thus, both the structures of ligands and the
16 metal ions of metal complexes influence the inhibition against different PTPs and the
17 selectivity.
18
Table 1 IC50(S.D.)(μM) of metal complexes on five PTPs.
SHP-2
Ref
Compoud
PTP1B
TCPTP
PTP-MEG2
SHP-1
[Cu(bhbb)(H2O)], 1
[Cu(chbb)(H2O)], 2
[Cu(nhbb)(H2O)2], 3
[Cu(cpmp)2], 4
[Cu(npmp)2], 5
CuCl2
0.26(1)
0.19(1)
0.41(2)
0.46(3)
0.059(5)
0.14
0.089(19)
0.067(5)
0.12(3)
0.17(1)
0.24(2)
0.15
0.80(1)
0.13(1)
0.16(3)
0.11(2)
0.21(1)
0.20
0.15(6)
0.38(3)
0.25(4)
0.21(5)
0.20(3)
0.18
>100
>100
>100
>100
>100
>1000
49(9)
4.2(4)
2.3(6)
This work
This work
This work
This work
This work
This work
[19]
[VO(bhbb)(H2O)2]
[VO(nhbb)(H2O)2]
[VO(cpmp)2]
0.21(2)
0.23(1)
0.80(9)
0.19(3)
1.3(1)
0.97(6)
0.60(9)
3.6(4)
3.0(3)
1.9(2)
1.0(2)
[19]
[19]
8.3(7)
17

[19]
[19]
[33]
[VO(bpmp)2]
[VO(npmp)2]
0.69(3)
0.93(9)
1.9(3)
1.4(1)
2.9(4)
3.2(6)
6.5(3)
2.5(5)
35(3)
2.6(2)
0.038(5) -
0.091(6)
0.069(9) -
0.23(5)
0.12(8) -
0.26(4)
0.27(8) -
0.50(5)
[Cu(RSal-dmta)2]
>100
1
2
3
4
5
6
7
8
9
Complex 1 as a representative of five complexes was chosen to further investigate
the inhibition mode of the four PTPs. As shown in Figure 3, for PTP1B, TCPTP,
PTP-MEG2 and SHP-1, the Lineweaver–Burk double-reciprocal plot of the kinetics
data of complex 1 all show the lines converged at an intersection on the x-axis left the
y-axis, implying a noncompetitive inhibition mode versus pNPP. The apparent
inhibition constants (Ki) for PTP1B, TCPTP, PTP-MEG2 and SHP-1 are calculated to
be 0.13, 0.28, 0.22 and 0.46 M, respectively (Figure 2, inset). The noncompetitive
inhibition modes of complex 1 agree with the copper complexes of
[Cu2(phen)2(ida)(NO3)]NO3 which exhibits obvious selectivity against TCPTP[32],
10 but differ from copper complexes of [Cu(RSal-dmta)2] which exhibit competitive
11 inhibition modes and selectivity against PTP1B[33], suggesting the binding sites of
12 copper complexes bonding to PTPs will be changed with the structural changes of
13 copper complexes, and may be related to their selectivity.
14
18

1
2
3
4
5
Figure 3. Lineweaver–Burk plots of 1/v (min mM-1) vs. the reciprocal of the pNPP concentrations
(mM-1) at five fixed concentrations of complex 1 for PTP1B (A), TCPTP (B), PTP-MEG2 (C),
and SHP-1 (D). Inset: determination of Ki for complex 1 inhibiting PTP1B, TCPTP, PTP-MEG2,
and SHP-1, respectively.
6
7
8
9
Additionally, the phosphatase inhibitory activity on cell extracts from C6 rat glioma
cells was evaluated. As shown in Figure 4, complexes 1 - 5 inhibit about 35% - 55%
phosphatase activity at 30 μM. Complexes 3 and 5 show stronger inhibition. The
inhibitory effects are slightly weaker than the copper complexes with
10 multi-benzimidazole derivatives [36], but comparable to quinquedentate binuclear
11 copper complexes [35]. This seems not consistent with the inhibition over the
12 recombinant PTPs since these Schiff base copper complexes exhibit evidently
13 stronger inhibition against SHP-1 or SHP-1 and PTP-MEG2 compared with the two
14 type copper complexes respectively, and they should then have stronger inhibitory
15 effects in inhibit cellular total phosphatases from cell extracts. The fact is that there
16 are various phosphatases such as PTPs which are far more than the five, protein
17 serine/threonine phosphatases, acidic and alkaline phosphatases existing in cell
18 extracts. Different copper complexes may produce different phosphatase inhibitory
19 effects by inhibiting different phosphatases activity. Although the Schiff base copper
20 complexes exhibit stronger inhibition against PTP1B, TCPTP, SHP-1 and PTP-MEG2,
19

1
2
3
4
5
6
7
8
9
they may weakly inhibit other phosphatases, and then display weaker inhibition
against cell extracts compared with the copper complexes with multi-benzimidazole
derivatives. On the other hand, the protein components in cell extracts are
complicated. Many metal complexes including some copper complexes are illustrated
to bind to various proteins such as topoisomerase II, proteasome, carbonic
anhydrases[28, 29, 45]. The interaction of copper complexes with these protein will
decrease the inhibitory ability over PTPs. Other non-protein components in cell
extracts may also complex with copper complexes and block the inhibition on PTPs.
Thus, the PTP inhibition against cell extracts is intricate.
10
11
12
Figure 4. The inhibitory effects of complexes 1-5 on phosphatases from cell extracts, C for
control.
13 4. Conclusion
14
In summary, we have synthesized and characterized five copper complexes of
15 Schiff-base
condensates
from
5-substituted-2-hydroxybenzaldehyde
and
16 2-substituted-benzenamine. Complexes 1-3 have the 1 1 molar ratio of tridentate
17 ligands Cu2+ while complexes 4-5 possess 2:1 molar ratio of bidentate ligands
20

1
2
3
4
5
6
7
8
9
Cu2+. The inhibitory activities of the complexes against PTP1B, TCPTP, PTP-MEG2,
SHP-1 and SHP-2 indicate that the complexes potently inhibit PTP1B, TCPTP,
PTP-MEG2 and SHP-1, but do not inhibit SHP-2. In the complexes, 5 with a ratio of
2 1 ligand/copper exhibits very strong inhibition and better selectivity against PTP1B
while 1 and 2 show very strong inhibition and a little selectivity against TCPTP.
Compared with the oxovanadium(IV) complexes with same ligands, the copper
complexes increase the inhibitory ability against TCPTP, PTP-MEG2 and SHP-1 but
decrease the inhibition against SHP-2. For complex 5, the inhibition over PTP1B,
TCPTP, PTP-MEG2 and SHP-1 are all improved about 5~15-fold. Usually, copper
10 and vanadium complexes inhibit PTPs with competitive, non competitive or mixed
11 inhibition models, implying they unactivate the enzymes by reversibly binding to
12 them not oxidating SH to SO3 at catalytic domain[13, 15, 17, 19, 31, 33-36, 46]. Thus,
13 the differences in the PTP inhibition between the two complexes more possibly result
14 from the dissimilarity of coordination sphere for the two metals.
15
The results demonstrate both the structures of ligands and the metal ions of
16 coordination sphere influence the inhibition and the selectivity against different PTPs.
17 Acknowledgements
18
This work was supported financially by the National Natural Science Foundation of
19 China (21001070, 21171109 and 21271121), SRFDP (20111401110002 and
20 20121401110005), the Natural Science Foundation of Shanxi Province of China
21 (2010011011-2, 2011011009-1 and 2011021006-2) and the Shanxi Scholarship
22 Council of China (2012-004).
21

1
2
3
4
5
6
7
8
9
Appendix A. Abbreviations
CSD
Cambridge Structural Database
ESI-MS
Hcpmp
Hnpmp
H2bhbb
H2chbb
H2nhbb
Kapp
electrospray ionization mass spectra
4-chloro-2-((phenylimino)methyl)phenol
4-bromo-2-((phenylimino)methyl)phenol
2-(5-bromo-2-hydroxylbenzylideneamino)benzoic acid
2-(5-chloro-2-hydroxylbenzylideneamino)benzoic acid
2-(5-nitro-2-hydroxylbenzylideneamino)benzoic acid
apparent Michaelis-Menten constant
inhibition constant
10 Ki
11 Km
Michaelis-Menten constant,
12 MOPS
13 pNPP
14 PTP1B
15 PTP-MEG2
16 PTPs
17 SHP-1
18 SHP-2
19 TCPTP
3-morpholinopropanesulfonic acid
p-nitrophenol phosphate
protein tyrosine phosphatase 1B
megakaryocyte protein-tyrosine phosphatase
protein tyrosine phosphatases
Src homology phosphatase 1
Src homology phosphatase 2
T-cell protein tyrosine phosphatase
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26

1 Index Abstract
2
3
4
5
Synthesis and evaluation of copper complexes of Schiff-base
condensates from 5-substituted-2-hydroxybenzaldehyde and
2-substituted-benzenamine as selective inhibitors of protein tyrosine
phosphatases
6
7
Ruiting Zhu, Liping Lu*, Miaoli Zhu*, Hong Han, Caixia Yuan, Shu Xing*, Xueqi Fu
8
9
27

1
2
3
Synthesis and evaluation of copper complexes of Schiff-base condensates from
5-substituted-2-hydroxybenzaldehyde and 2-substituted- benzenamine as
selective inhibitors of protein tyrosine phosphatases
4
Liping Lu, Ruiting Zhu, Miaoli Zhu*, Hong Han, Caixia Yuan, Shu Xing, Xueqi Fu*
5
6
7
Highlights
•Schiff-base copper complexes were synthesized and characterized.
8
9
•The complexes noncompetitively inhibit PTP1B, TCPTP, PTP-MEG2 & SHP-1 but
SHP-2.
10 •Complex 5 exhibits very strong inhibition and better selectivity against PTP1B.
11
12
13
28