Rational design of selective organoruthenium inhibitors of protein tyrosine phosphatase 1B

Article abstract of DOI:10.1021/ic301884j

Protein tyrosine phosphatases (PTPs) belong to a large family of important regulatory enzymes involved in vital mammalian signaling pathways. Selective inhibitors of PTPs are highly valuable from a therapeutic standpoint given their association with various pathological conditions. One such target is PTP-1B which has previously been linked to diabetes and cancer. However, developing a selective inhibitor against PTP-1B has proven to be daunting because the enzyme shares a high degree of structural homology with TC-PTP, an essential PTP involved in modulating immune functions. To address this challenge, a series of organoruthenium complexes was developed to bind at the PTP substrate-binding site while simultaneously target the peripheral structural space. By capitalizing on the potential difference in the structural environment proximal to the active site between different PTPs, selectivity toward PTP-1B over TC-PTP was improved, paving the way for organoruthenium complexes as selective PTP-1B metalloinhibitors.

Full text of DOI:10.1021/ic301884j

Article
pubs.acs.org/IC
Rational Design of Selective Organoruthenium Inhibitors of Protein
Tyrosine Phosphatase 1B
Jun Xiang Ong,† Chun Wei Yap,‡ and Wee Han Ang*,†
†Department of Chemistry, National University of Singapore, 3 Science Drive 3, Singapore 117543
‡Department of Pharmacy, National University of Singapore, 18 Science Drive 4, Singapore 117543
S
* Supporting Information
ABSTRACT: Protein tyrosine phosphatases (PTPs) belong to a large family of
important regulatory enzymes involved in vital mammalian signaling pathways.
Selective inhibitors of PTPs are highly valuable from a therapeutic standpoint given
their association with various pathological conditions. One such target is PTP-1B
which has previously been linked to diabetes and cancer. However, developing a
selective inhibitor against PTP-1B has proven to be daunting because the enzyme
shares a high degree of structural homology with TC-PTP, an essential PTP involved
in modulating immune functions. To address this challenge, a series of organo-
ruthenium complexes was developed to bind at the PTP substrate-binding site while
simultaneously target the peripheral structural space. By capitalizing on the potential
difference in the structural environment proximal to the active site between different
PTPs, selectivity toward PTP-1B over TC-PTP was improved, paving the way for
organoruthenium complexes as selective PTP-1B metalloinhibitors.
TC-PTP deficient mice at 3−5 weeks of age in vivo, using a
nonselective PTP-1B inhibitor that also acts on TC-PTP can
lead to severe side effects with increased mortality.11
Developing inhibitors with high selectivity toward PTP-1B
and not TC-PTP remains a daunting task.
INTRODUCTION
■
Protein tyrosine phosphatases (PTPs) belong to a large family
of 107 enzymes that play a vital role in the regulation of various
signaling transduction pathways in mammalian systems.1 PTP
enzymes catalyze the dephosphorylation of phosphotyrosine
(pTyr) residues, and in conjunction with protein tyrosine
kinases (PTKs), they are responsible for managing the levels of
phosphorylation within the cells.2 Studies have shown that
dysregulation of PTP can lead to several pathological
conditions including diabetes, obesity, cancer, and autoimmune
disorders.3 Among the members of the PTP family, PTP-1B is a
key negative regulator of the insulin and leptin signaling
pathways associated with obesity and diabetes. PTP-1B is
responsible for dephosphorylation of activated insulin receptor
or insulin receptor substrates in insulin signaling,4 as well as
JAK2 which is downstream of the ObR receptor in the leptin
signaling pathway.5 Cell cultures and gene coding studies have
also shown that aberrant expression of PTP-1B can contribute
to obesity and diabetes.6 Experiments on PTP-1B knockout
mice showed, for instance, that PTP-1B deficiency leads to
increased sensitivity toward insulin and resistance to diet-
induced obesity.7 This suggests that inhibition of PTP-1B can
potentially address insulin resistance and obesity.8
So far, several metal complexes have been investigated as
potential PTP inhibitors. A series of Schiff-base vanadium and
copper complexes was found to be very potent PTP inhibitors.
However, low selectivity was observed between PTP-1B and
TC-PTP.12 These vanadium and copper Schiff-base complexes
exhibited only 2-fold and 3−4-fold selectivity toward PTP-1B
over TC-PTP, respectively. Recently, a library of gold−
phosphine complexes was screened, and several were found
to exhibit good PTP-1B inhibitory activity with varying levels of
selectivity.13 Their mechanisms were not expounded, but given
the affinity of these metal centers, especially Au, toward S-
containing Cys residues within the enzyme active site, it is
possible that the metal centers directly reacted with Cys, thus
accounting for the poor selectivity between the two enzymes.
There are also several other examples of inhibitors covalently
bonded to the active site by design.14 Nevertheless, a
metalloinhibitor that is designed to be selective toward PTP-
1B remains elusive. Meggers showed that highly selective
active-site inhibitors of PTKs can be prepared using a known
kinase inhibitor, staurosporine, as a template.15 By replacing the
glycoside motif with an organoruthenium fragment, improved
inhibitory profiles against specific PTKs were achieved.16 In this
manner, the octahedral Ru(II) framework provided the scaffold
The highly conserved enzymatic active site across the PTP
family poses a major challenge in the design of selective PTP
inhibitors.1a The signature sequence motif CX5R can be found
among the PTP active sites and is responsible for catalyzing the
dephosphorylation of pTyr.2b In addition, PTP-1B shares 80%
structural homology with TC-PTP in their catalytic domains.9
TC-PTP is widely distributed throughout the body and is
responsible for modulating immune functions.10 As observed in
Received: August 28, 2012
© XXXX American Chemical Society
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upon which ligands could be structurally organized.17 This
strategy yielded some of the most efficacious PTK inhibitors
reported.
PFP ligand to Ru(II). In this manner, a panel of structurally
diverse metalloinhibitors was easily assembled using different
predefined metal precursors and PFP ligands which could then
be screened for PTP selectivity.
We were interested in combining these principles with
metal−ligand interactions for rapid assembly to the design of
PTP inhibitors. PTPs present a different challenge since their
active sites are small and they are only capable of binding the
pTyr motif. PTKs, in contrast, have a large cleft capable of
accommodating a bulky ATP substrate. Since PTPs exhibit high
substrate specificities even though their active sites are highly
conserved, we reasoned that the space peripheral to the PTP
active site must be important for their molecular recognition.
Consequently, we sought to develop metalloinhibitors that are
selective toward specific PTPs by capitalizing on the potential
difference in the structural environment proximal to the active
site. In this study, we report a class of organoruthenium PTP
inhibitors with low micromolar level inhibitory constants
toward PTP-1B and exhibit a 10-fold selectivity toward PTP-
1B over TC-PTP.
Synthesis and Characterization. The ligands 1−4 were
synthesized from p-tolualdehyde as shown in Scheme 1. p-
Tolualdehyde was first treated with diethylphosphite in the
presence of a catalytic amount of base to give the hydroxyl-
phosphonate ester P1. Subsequent oxidation of P1 with Dess−
Martin periodinane (DMP) afforded the keto-phosphonate
ester P2. Treatment of compound P2 with diethylaminosulfur-
trifluoride (DAST) resulted in the replacement of the ketone
functional group by geminal fluorine atoms to give the
difluoromethylphosphonate ester P3. It was reported that P3
could be synthesized directly via Shibuya coupling using 4-
iodotoulene and diethyl(bromodifluoromethyl)phosphonate
with Zn dust and CuBr as coupling agents.19 However, after
repeated unsuccessful attempts, the described three-step
approach was adopted. Bromination was first carried out on
P3 with N-bromosuccinimide (NBS) in the presence of
azobisisobutyronitrile (AIBN) as the catalyst. The bromination
reaction resulted in the formation of both monobrominated P4
as the major product, as well as a dibrominated side product
which could not be effectively separated. However, only P4
reacted with the 2-(2-pyridyl)imidazole and 2-(2-pyridyl)-
benzimidazole to afford the desired ligands 1 and 3,
respectively, in good yields. The dibrominated side-product
remained unreacted and was removed by flash-column
chromatography. Treatment of 1 and 3 with N,O-bis-
(trimethylsilyl)trifluoroacetamide (BSTFA) and iodotrimethyl-
silane (TMSI) followed by methanol resulted in the hydrolysis
of the phosphonate ester groups to give phosphonic acids 2 and
4, respectively, as the desired ligands.
RESULTS AND DISCUSSION
■
Design Approach. Due to the highly conserved nature of
the PTP enzymatic active sites, targeting them directly to
achieve selectivity would not be feasible. Our strategy was to
target the structural space at the periphery of the active site in
order to exploit differences in this environment between
different PTPs. The approach was to construct metallo-
inhibitors that could bind to the active site while simultaneously
being able to interact with the structural space proximal to the
active site (Figure 1). Hence, we tethered the phenyl-
The reaction of [(η6-arene)RuCl2]2, where arene = cymene
or 1,3,5-triisopropyl-benzene (TIPB), with 2 equiv of
imidazole-diethylphosphonate ester 1 yielded mononuclear
Ru(II) complexes 1a and 1b, whereas treatment with
imidazole-phosphonic acid 2 yielded 2a and 2b. The reaction
was promoted by the formation of the stable 5-membered
chelate between the bidentate pyridyl-imidazole ligands and the
Ru(II) center. Increased steric encumbrance afforded by the
larger TIPB arene did not adversely affect the formation of the
complexes. After anion exchange using NH4PF6, 1a,b were
obtained as monocationic Ru(II) [PF6] complexes. Anion
exchange was necessary to improve the yields and purity of the
products. Complexes 2a,b were isolated directly from the
reaction mixture without anion exchange. Under similar
reaction conditions, benzimidazole-diethylphosphonate ester 3
and benzimidazole-phosphonic acid 4 gave organoruthenium
complexes 3a,b and 4a,b in good yields. All complexes were
obtained as yellow or orange-yellow solids. Complexes with the
diethylphosphonate ester groups, namely 1a,b and 3a,b, were
soluble in moderately polar organic solvents such as dichloro-
methane, chloroform, methanol, DMSO, and acetone. In
contrast, 2a,b and 4a,b were soluble only in polar solvents
such as methanol and DMSO, presumably due to the highly
polar phosphonic acid groups. Notably, all of the synthesized
Ru(II) complexes were soluble in aqueous conditions to
concentrations exceeding 1 mM, an attribute that was
important in subsequent biological investigations.
Figure 1. Approach to designing Ru(II)-based PTP inhibitors.
difluoromethylphosphonic acid (PFP) motif to bulky organo-
ruthenium scaffolds via imidazole linkers. The PFP motif, being
a nonhydrolyzable mimetic of pTyr, had been previously
reported to bind PTP active sites effectively.18 The organo-
ruthenium fragment comprised ligands capable of interacting
with the peripheral hydrophobic amino-acid residues. The
Ru(II) center, in addition, served a structural role to organize
these ligands in the three-dimensional space. Owing to its
octahedral coordination geometry, the rigid Ru(II) scaffold
offered new structural possibilities not easily achievable in
purely organic molecules. Consequently, we coupled the
organoruthenium fragment and the PFP group with a bidentate
pyridiyl-imidazole linker for improved synthetic expediency in
order to exploit the principle of chelation to rapidly bind the
The compounds were analyzed by 1H, 31P{1H}, and 19F{1H}
NMR, ESI-MS, and RP-HPLC. A distinct feature of the
Ru(II)−arene complexes was the presence of resonances at 5−
B
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Scheme 1
6 ppm due to the aryl-CH protons of the facially bound arene
ligand. The resonances were shifted upfield from the aromatic
region, indicating a more shielded environment in the presence
of the metal center. In addition, the arene protons of the
cymene ligand of 1a−4a could be observed as 4 sets of
doublets, compared to two sets of doublets of the precursor,
due to the presence of diastereomers. Likewise, the six methyl
protons on the isopropyl group were observed as 2 sets of
doublets, as opposed to a set of doublet in the precursor. This
indicated that the ligands were bound at more than 1
coordination site around the metal center. Similar observations
were made on 1b−4b with the TIPB arene ligand. A set of
triple and a set of doublet were observed in the 31P{1H} and
the CF2 group which could yield interfering HF on combustion,
thus giving rise to inaccurate results.20 Instead, we determined
the purity of the newly synthesized compounds to be >95%
using RP-HPLC.
Single crystals of 1b suitable for X-ray diffraction studies were
obtained via layer diffusion of diethyl ether into its methanolic
solution, while 4a was grown by slow evaporation of a
methanolic solution. To the best of our knowledge, these
complexes represent the first reported organoruthenium
complexes containing difluoromethylphosphonate functional
groups. Figures 2 and 3, respectively, depict the structures of 1b
and 4a with atomic numbering. Table 1 shows selected
crystallographic data, and Table 2 shows selected bond lengths
and angles of the complexes. The pyridyl-imidazole and pyridyl-
benzimidazole rings in the complexes were observed to be
largely planar. Complex 4a crystallized as a zwitterionic
structure with a deprotonated monobasic phosphonate group.
The negative charge on the deprotonated phosphonate group
was delocalized between O1−P1−O3 with similar O1−P1 and
O3−P1 bond lengths of 1.481(3) and 1.512(3) Å. The
distances were consistent with a bond order of 1−2. In
2
19F{1H} NMR, respectively, for all the complexes due to JPF
coupling. All the complexes were observed as M+ parent
molecular ions in the ESI-MS and confirmed with MS/MS
fragmentation analysis. However, organic CHN elemental
analyses did not yield results consistent with the molecular
formula of the desired compounds despite using crystalline and
highly purified samples. In comparison, Ru content analyses on
the samples by ICP-OES were within error limits. We
hypothesized that the discrepancy was due to the presence of
C
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a
Table 1. Selected X-ray Crystallographic Data for 1b and 4a
complex
formula
1b·(C2H5)2O
4a·(CH3OH)4
C39H56ClF8N3O4P2Ru
981.33
C34H45ClF2N3O7PRu
813.22
fw
T [K]
293(2)
100(2)
wavelength [Å]
cryst size [mm3]
cryst syst
space group
a [Å]
0.71073
0.71073
0.36 × 0.20 × 0.10
triclinic
0.36 × 0.26 × 0.10
triclinic
P1
̅
P1
̅
9.8539(4)
13.3013(6)
18.0943(8)
110.4200(10)
93.6680(10)
96.1320(10)
2196.78(16)
2
10.653(3)
12.504(3)
14.449(4)
101.457(4)
101.362(4)
92.915(4)
1841.3(8)
2
b [Å]
c [Å]
Figure 2. Molecular representation of 1b; atoms are represented as
thermal ellipsoids at 50% equiprobability. Disordered phosphonate
groups and [PF6]− anion were excluded for clarity.
α [deg]
β [deg]
γ [deg]
V [Å3]
Z
Dc [Mg/m3]
1.484
1.467
μ [mm−1
]
0.566
0.601
θ range [deg]
1.66−27.50
28 789
1.67−27.50
23 737
no. unique data
max, min transm
R indices (all data)
0.4305 and 0.3930
R1 = 0.0524
wR2 = 0.1030
1.042
0.9423 and 0.8126
R1 = 0.0860
wR2 = 0.1524
1.040
GOF on F2
peak/hole [e Å−3
]
0.716 and −0.543
1.767 and −1.120
a
R = Σ∥Fo| − |Fc∥/Σ|Fo|, wR2 = {Σ[w(Fo − Fc2)2]/Σ[w(Fo )2]}1/2
.
2
2
Goodness-of-fit (GOF) = {Σ[w(Fo − Fc2)2]/(n − p)}1/2, where n is
2
the number of data and p is the number of parameters refined.
Table 2. Comparison of Bond Distances [Å] and Angles
[deg] of 1b and 4a
Figure 3. Molecular representation of 4a; atoms are represented as
thermal ellipsoids at 50% equiprobability. Disordered isopropyl group
on the arene ligand was excluded for clarity.
complex
1b
4a
Ru1−N1 [Å]
Ru1−N2 [Å]
Ru1−Cl1 [Å]
av Ru−Carene [Å]
N2−Ru1−N1 [deg]
N2−Ru1−Cl1 [deg]
N1−Ru1−Cl1 [deg]
P1−O1 [Å]
2.099(2)
2.080(2)
2.3861(7)
2.185−2.239
76.32(8)
85.44(6)
83.78(6)
1.443(2)
1.506(4)
1.635(4)
2.103(3)
comparison, the observed protonated O2−P1 bond in the
crystal structure was significantly longer at 1.558(3) Å.
2.078(3)
2.4063(12)
2.163−2.289
75.68(13)
86.12(10)
84.66(10)
1.481(3)
We investigated whether it was possible to hydrolyze the
phosphonate ester groups as a facile entry to 2a,b and 4a,b after
they have been coordinated to the Ru(II)−arene scaffold. The
direct hydrolysis of phosphonate ester groups was reported for
several polyaromatic Ru(II) complexes21 but not Ru(II)−arene
compounds which were more reactive and susceptible to ligand
displacement. Treatment of 1a directly with TMSI followed by
methanol yielded quantitatively an unknown species with a
similar 1H NMR profile to 2a. Disappearance of the ethyl peaks
in the 1H NMR spectrum indicated hydrolysis of the
phosphonate ester group. Other resonances were largely
unchanged, suggesting that organoruthenium scaffold remained
intact. Closer inspection by ESI-MS analysis, however,
suggested that the chloride ligand coordinated to Ru(II) was
displaced by iodide, with a single peak observed at m/z 728 and
corresponding to [(η6-cymene)RuI(2)]+. In contrast, 2a was
previously observed by ESI-MS with a parental molecular ion at
m/z 636. The source of iodide was likely decomposed TMSI
reagent that was used in stoichiometric excess. Although direct
hydrolysis of phosphonate groups was technically feasible,
further steps would be required to convert the iodide ligand
back to chloride, and the method was thus abandoned.
P1−O2 [Å]
P1−O3 [Å]
1.558(3)
1.512(3)
vis spectra over the period, thus indicating good aqueous
stability. In the presence of 1 mM glutathione (GSH), an
endogenous intracellular thiol-containing tripeptide, there was a
significant blue shift in the spectra of both compounds,
suggesting that organoruthenium complexes could potentially
react with these nucleophiles. This shift was suppressed with
the addition of 200 mM NaCl. This suggests that the aquation
of the Ru−Cl bond, not degradation of the imidazole linker or
phosphonate group, was important for their reactivity.22 Under
physiological conditions at high chloride concentrations, the
organoruthenium complexes can be expected to maintain their
good stability even in the presence of nucleophiles. Upon cell
entry, where the chloride levels are lower, reaction with
intracellular nucleophiles may also occur.
Aqueous Stability of Organoruthenium PTP Inhib-
itors. The aqueous stabilities of 3a and 4a were investigated
using UV−vis spectroscopy over a 24 h period in water (pH
7.2) (Figure 4). There were no significant shifts in their UV−
Inhibition of PTP-1B and TC-PTP by Organoruthe-
nium Complexes. The organoruthenium inhibitors were
designed to bind the PTP active sites using their PFP motif as a
nonhydrolyzable mimetic of pTyr.18 At the same time, the
D
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namely 2−2b and 4−4b, were efficacious. This may be
attributed to increased steric encumbrance around active site-
targeting moiety which prevented effective binding to the
enzymatic site. In addition, it rendered the phosphonate group
strongly hydrophobic and lowered their affinity toward the
hydrophilic enzyme pocket.
Because the inhibitor contained a second-row transition
metal with a known affinity toward soft nucleophiles, e.g., Cys,
we investigated whether the Ru(II)−arene fragment could itself
inhibit enzymatic activity directly. Therefore, [(η6-cymene)Ru-
(pyridylimidazole)Cl]PF6 (5) and [(η6-cymene)Ru-
(pyridylbenzimidazole)Cl]PF6 (6), which modeled the organo-
ruthenium-imidazole and organoruthenium-benzimidazole frag-
ments, respectively, and did not contain the PFP motif, were
prepared and evaluated. Their inhibitory activities against both
enzymes were completely abrogated, thus indicating that the
organoruthenium imidazole/benzimidazole components them-
selves cannot inhibit PTP through allosteric interactions. In
addition, it was evident from the single concentration screen
(100 μM) that PFP ligands 2 and 4 were equally efficacious
against both PTP-1B and TC-PTP. After incorporation of the
organoruthenium scaffold, selectivity toward PTP-1B was
drastically improved. A more detailed investigation was carried
out by examining the dose−response of the organoruthenium
inhibitors against both PTP enzymes (Figure 5) by determining
the concentration at which the enzymatic activity was reduced
to 50% level (IC50) versus untreated controls (Table 3). On the
basis of the IC50 values, the metalloinhibitors were 7−10-fold
more effective against PTP-1B than TC-PTP, while uncoordi-
nated PFP ligands were relatively unselective. This improved
selectivity could be due to favorable interactions of the Ru(II)
scaffold with amino-acid residues surrounding the active site in
PTP-1B, thus leading to better binding of the complexes in
PTP-1B.
Docking studies were carried out against PTP-1B (PDB ID:
2F71) using 4 and 4a to establish the mode of binding to the
target enzyme. The structure, reported by Klopfenstein, was
determined at 1.55 Å resolution, and it contained a bound
tetrahydroisoquinolinyl-sulfamic acid inhibitor. The inhibitor
was vacated, and a simulation cell was defined around and
extended for 6 Å beyond the substrate binding site. The solid
state structure of 4a, unambiguously determined using single
crystal X-ray diffraction analyses, was used for the docking
studies. Since the docking software was unable to process a
multivalent metal center, such as Ru, the relative positions of
the cymene ligand were affixed to the benzimidazole ligand by
using a carbon atom to simulate the metal center. The
remaining atoms in the benzimidazole-phosphonic acid frag-
ment were unrestrained. This workaround was not expected to
affect the validity of the docking results significantly since the
Ru(II)−arene fragment served a structural role and since the
Ru atom was also not expected to directly interact with the
protein. Our model also assumed that the cymene ligand was
locked to a specific orientation even though in practice it would
be freely rotatable about the metal−arene centroid axis. The
structure of 4 was obtained by deleting the Ru(II)−arene
fragment of 4a and was not constrained. The docking results
revealed that the energetically favored conformation of 4 and
4a involved their PFP motifs occupying the substrate-binding
sites with the benzimidazole fragments directed toward the
solvent region (Figure 6). These conformations were in good
agreement with our design to position the bulky organo-
ruthenium fragment at the periphery of the substrate-binding
Figure 4. (A) UV−vis spectrum of 4a in H2O. (B) UV−vis spectrum
of complex 4a in 1 mM GSH. (C) UV−vis spectrum of 4a in 1 mM
GSH + 200 mM NaCl. The graphs are plotted in 2 h intervals over a
period of 24 h.
organoruthenium scaffold would interact with amino acid
residues at the periphery of the enzyme active site. By
exploiting differences in this peripheral structural environment
among different PTPs, selectivity toward specific PTPs can be
engineered.
The compounds were subjected to an initial screen against
PTP-1B and TC-PTP (Table 3) using the p-nitrophenylphos-
Table 3. Initial Screening of Inhibitors against PTP-1B and
TC-PTP and IC50 of Selected Compounds
a
screening [% control]
IC50 [μM]
compd
PTP-1B
TC-PTP
PTP-1B
TC-PTP
1a
1b
2
99.3 1.7
99.6 0.7
43.4 1.8
7.8 0.3
99.7 0.7
100.2 0.8
34.8 1.0
45.1 1.2
44.0 1.6
99.4 1.6
100.4 0.6
42.1 2.5
57.4 1.2
56.5 0.6
99.4 1.2
100.1 0.9
72.0 1.1
20.9 1.0
18.7 1.0
45.1 1.0
73.4 1.0
69.1 1.1
2a
2b
3a
3b
4
7.3 0.7
99.1 1.9
99.7 0.6
42.8 1.1
6.7 0.7
68.6 1.1
14.2 1.0
16.6 1.0
72.4 1.0
112.1 1.1
108.7 1.1
4a
4b
5
7.0 0.3
99.5 0.9
100.6 1.3
6
a
Screening was performed at inhibitor concentration of 100 μM.
phate (pNPP). Phosphatases catalyze the hydrolysis of the
phosphate group on pNPP to yield p-nitrophenol, which can be
quantitated by UV−vis spectroscopy at A405. The enzymes were
treated with the inhibitors for 30 min before pNPP was added.
After an incubation period of 30 min, levels of p-nitrophenol
were determined. We took further precautions to mitigate
weighing errors and the purity of metalloinhibitors by
accurately determining Ru content in the stock solutions
using ICP-OES and normalizing the data obtained. In addition,
the same stock of organoruthenium solution was applied
against two different enzymes to minimize errors arising from
sample preparation. On the basis of the initial screen,
compounds containing the diethylphosphonate ester ligands,
namely 1−1b and 3−3b, were ineffective regardless of the
nature of their Ru(II)−arene fragment or linker groups. Indeed,
only compounds containing the phosphonic acid moiety,
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Figure 5. Dose−response curves of inhibition of compounds toward PTP-1B (bold) and TC-PTP (dotted).
a hinge to orientate the bulky benzimidazole group at ca. 120°
from the entrance of the binding site. Because this orientation
would position the isopropyl group on cymene ligand of 4a to
within 3.36 Å of Phe182, we speculated that it would give rise
to favorable π-CH interaction with phenyl ring, thereby
enhancing binding of 4a to the enzyme compared to 4.
However, without further structural evidence, we cannot rule
out other enzyme−substrate interactions arising from ligand-
induced conformation changes that could account for the
improved 4a potency.
On the other hand, the observed diminishing potency of the
inhibitors toward TC-PTP compared to their parent ligands
could be due to steric effects arising from the bulky Ru(II)−
arene scaffold impeding effective binding to the active site. A
similar docking study on TC-PTP could not be performed
since the only crystallographic data of TC-PTP (PDB ID:
1L8K) did not contain a bound inhibitor, and hence, the
residues in the active site were not oriented correctly for
substrate binding. Our preliminary docking study (results not
shown) showed that the phosphonic acid group was not able to
Figure 6. Conformation of parent ligand 4 in PTP-1B substrate
binding site (left); conformation of 4a in PTP-1B substrate binding
site (right). The hydrophobic regions around the phosphatase
substrate binding site are highlighted in yellow.
site using the PFP motif as the directing group. The methylene
group between PFP and the benzimidazole linker also acted as
Figure 7. Lineweaver−Burk plot of 1/v (min μM−1) vs the reciprocal of pNPP concentration (mM−1) at five fixed concentrations of 4a for the
inhibition toward PTP-1B (left) and TC-PTP (right). Ki determination for 4a against PTP-1B and TC-PTP (inset). The value of Ki was determined
from the x-intercept of a plot of the slope of the line from the double-reciprocal plot as a function of inhibitor concentration (μM).
F
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follows: 20−80% B over 20 min where solvent A is H2O + 0.1%
CF3COOH and solvent B is CH3CN + 0.1% CF3COOH.
bind correctly at the active site using this structure of TC-PTP,
and as a result, any data derived would not be sufficiently
accurate.
Synthesis of Hydroxyl-phosphonate Ester (P1). p-Tolualdehyde
(2.34 mL, 20 mmol) was dissolved in THF (10 mL), and
diethylphosphite (3.09 mL, 24 mmol) was added dropwise.
Tetramethylguanidine (0.25 mL, 2 mmol) was added dropwise, and
the reaction mixture was stirred at r.t. for 1 h. The reaction mixture
was diluted with ethyl acetate (15 mL) and washed two times with
HCl (1 M, 10 mL). The organic portion was dried over MgSO4, and
the solvent was removed to give the product as white solid. Yield: 4.65
g (90%). 1H NMR (300 MHz, CDCl3): δ 7.35 (2H, d), 7.15 (2H, d),
4.97 (1H, d), 4.05 (4H, m), 2.34 (3H, s), 1.24 (6H, t). 31P NMR (121
MHz, CDCl3): δ 22.3 (s).
From these experimental results, the presence of the Ru(II)
scaffold in the structures increased potency toward PTP-1B,
while the inhibition toward TC-PTP was diminished, thereby
resulting in pronounced selectivities not observed in the parent
ligands. Unexpectedly, the imidazole analogues were more
potent in the inhibition of TC-PTP as compared to the
benzimidazole counterparts. This suggests that steric effects
could be used to directly tune the efficacy of the inhibitor
toward TC-PTP. Detailed kinetic studies were performed to
elucidate the binding modes of the complexes toward the
enzymes in order to determine whether the observed
differences in inhibition activities arose from different binding
mechanisms toward PTP-1B and TC-PTP. Steady-state kinetic
experiments with each of the enzymes were conducted with six
different concentrations of pNPP and five different concen-
trations of 4a. The Lineweaver−Burk plots (Figure 7)
intersected at the same point on the 1/v axes, indicating a
competitive mode of inhibition versus pNPP with inhibition
constants (Ki) of 7.3 and 80.1 μM against PTP-1B and TC-
PTP, respectively. Taken together, these data implied that 4a
was bound to both PTP-1B and TC-PTP at their substrate
binding sites and that the different inhibition potencies were
unlikely to be a result of different inhibitory mechanisms.
Synthesis of Difluoromethylphosphonate Ester (P3). P1 (516.5
mg, 2 mmol) was dissolved in dichloromethane (10 mL) and cooled
to 0 °C. DMP (1.27 g, 3 mmol) was added to the solution, and the
reaction mixture was stirred at r.t. for 2 h. The reaction mixture was
filtered, and to the filtrate was added a solution containing Na2S2O3
(0.5 M, 15 mL) and NaHCO3 (1 M, 15 mL) and stirred for 30 min.
The solution was then extracted three times with ethyl acetate (20
mL), and the organic portion was dried over MgSO4. The solvent was
removed to give crude P2 as yellow oil. P2 was redissolved in dry
dichloromethane (8 mL) and cooled to 0 °C. DAST (0.53 mL, 4
mmol) was then added dropwise under nitrogen atmosphere. The
reaction mixture was allowed to warm to r.t. and stirred for 12 h.
Thereafter, the mixture was diluted with dichloromethane (10 mL)
and ice-cold NaHCO3 (1 M, 20 mL) and stirred for an additional 30
min. The reaction mixture was extracted three times with dichloro-
methane (15 mL), and the organic portion was dried over MgSO4.
The solvent was removed to give the crude compound as dark-brown
oil and purified by flash column chromatography (2:3 v/v ethyl
acetate:hexane, Rf = 0.7) to give the product as yellow oil. Final yield:
306.1 mg (55%). P2 data follow. 1H NMR (300 MHz, CDCl3): δ 8.17
(2H, d), 7.30 (2H, d), 4.26 (4H, m), 2.39 (3H, s), 1.37 (6H, t). 31P
NMR (121 MHz, CDCl3): δ −0.38 (s). P3 data follow. 1H NMR (300
MHz, CDCl3): δ 7.50 (2H, d), 7.25 (2H, d), 4.20 (4H, m), 2.39 (3H,
s), 1.31 (6H, t). 31P NMR (121 MHz, CDCl3): δ 7.16 (t, JP−F = 118.5
Hz). 19F NMR (282 MHz, CDCl3): δ −31.9 (d, JP−F = 118.5 Hz).
Synthesis of Bromobenzyl Difluoromethylphosphonate Ester
(P4). P3 (322.7 mg, 1.16 mmol), NBS (231.4 mg, 1.30 mmol), and
AIBN (9.5 mg, 58 μmol) were dissolved in carbon tetrachloride (10
mL), and the reaction mixture was refluxed for 3 h. After the reaction,
the reaction mixture was diluted with carbon tetrachloride (10 mL)
and washed once with water (15 mL), NaHCO3 (1 M, 15 mL), and
brine (15 mL). The organic portion was dried over MgSO4 to give the
crude product as pale yellow oil. 1H NMR analysis of the crude
material indicated a mixture of starting material, monobrominated P4,
and the dibrominated side-product in a ratio of 16:67:17%. The crude
product was purified by flash column chromatography (1:2 v/v ethyl
acetate:hexane, Rf = 0.8) to isolate monobrominated compound P4
and dibrominated side-product as mixture of colorless oils. The
mixture was used without further purification for the following step.
P4 data follow. Yield: 220.2 mg (53%). 1H NMR (300 MHz, CDCl3):
δ 7.60 (2H, d), 7.48 (2H, d), 4.49 (2H, s), 4.18 (4H, m), 1.23 (6H, t).
31P NMR (121 MHz, CDCl3): δ 6.75 (t, JP−F = 115.5 Hz). 19F NMR
CONCLUSION
■
In summary, several Ru(II)−arene complexes were rationally
designed and synthesized to be selective inhibitors of PTP-1B.
Only inhibitors containing PFP groups were efficacious.
Addition of the organoruthenium−arene fragments improved
inhibitory activities against PTP-1B while activities against TC-
PTP were diminished, leading to pronounced selectivities not
observed in the parent ligands. Alone, the organoruthenium-
imidazole/benzimidazole fragments were not PTP inhibitors,
thus indicating that they are not solely responsible for the
improved efficacies. Steady-state kinetics and molecular docking
studies showed that the complexes competitively bind to the
enzymes at their active sites. The results, therefore,
demonstrated that molecular recognition of PTPs can be
achieved by targeting both the substrate site and its periphery
by applying organometallic principles of assembly and
structural diversity.
EXPERIMENTAL PROCEDURES
■
Materials and Methods. All reagents were purchased from
commercial vendors and used without further purification. Solvents
were dried and distilled by standard procedures, and reactions were
performed under nitrogen using standard Schlenk techniques. PTP-1B
and TC-PTP were purchased from Sino-Biological. [(η6-cymene)-
RuCl2]2,23 [(η6-TIPB)RuCl2]2,24 and 2-(2-pyridyl)imidazole25 were
(282 MHz, CDCl3): δ −32.5 (d, JP−F = 115.5 Hz).
Synthesis of Imidazole-diethylphosphonate Ester (1). The crude
mixture (0.177 g containing 67% of monobrominated compound P4)
and 2-(2-pyridyl)-imidazole (47.7 mg, 0.331 mmol) were dissolved in
DMF (4 mL), and KtOBu (44.6 mg, 0.40 mmol) in butanol (1 mL)
was added dropwise to the reaction mixture with stirring. The reaction
mixture was stirred at r.t. for 12 h. After the reaction, the solvent was
removed, and the crude compound was purified by column
chromatography (4:1 v/v ethyl acetate/hexane, Rf = 0.35) to give
synthesized according to literature methods. H, 31P, and 19F NMR
1
data were recorded on a Bruker Avance 300 or 400 MHz model.
Chemical shifts are reported in parts per million relative to residual
solvent peaks. Electrospray ionization mass spectra (ESI-MS) were
obtained on a Thermo Finnigan MAT ESI-MS system. UV−vis spectra
were recorded on a Shimadzu UV-1800 UV−vis spectrophotometer.
Determination of Ru levels was carried out by CMMAC (National
University of Singapore) on a Optima ICP-OES (Perkin-Elmer).
Purity of Ru(II) compounds was conducted using analytical HPLC on
a Shimadzu Prominence using a Shimpack VP-ODS C18 (5 μM, 120
Å, 250 mm × 4.60 mm i.d) column at r.t. at a flow rate of 1.0 mL/min
with 254 nm UV detection. The gradient eluent conditions were as
1
product as viscous colorless oil. Yield: 66.9 mg (48%). H NMR (300
MHz, CDCl3): 8.48 (1H, d); 8.20 (1H, d); 7.74 (1H, td); 7.53 (2H,
d); 7.17−7.23 (4H, m); 7.00 (1H, d); 5.98 (2H, s); 4.15 (4H, m); 1.27
(6H, t). 31P NMR (121 MHz, CDCl3): δ 6.80 (t, JP−F = 117.0 Hz). 19
F
NMR (282 MHz, CDCl3): δ −32.4 (d, JP−F = 117.0 Hz). ESI (+ve
mode) m/z 422.1 [M + H+], 444.1 [M + Na+].
G
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Synthesis of Imidazole-phosphonic Acid (2). Ligand 1 (18 mg, 4.3
μmol) was dissolved in dichloromethane (3 mL), and BSTFA (57 μL,
0.214 mmol) was added to the solution. After stirring for 15 min,
TMSI (30 μL, 0.214 mmol) was added to the reaction mixture. After
stirring for an additional 2 h, the solvent was removed and the residue
was coevaporated three times with dichloromethane (4 mL). The
resulting residue was redissolved in dichloromethane (2 mL) followed
by addition of methanol (10 mL) and stirred at r.t. overnight. After the
reaction, solvent was removed and the residue was washed three times
with water (4 mL). The residue was then suspended in water (2 mL),
and NH4OH (1 M, 1 mL) was added dropwise till the residue
dissolved followed by stirring for 30 min. The solvent was removed,
and the residue was washed once with dichloromethane (3 mL) and
twice with diethyl ether (3 mL) to give the product as white solid.
100%). Purity (HPLC): 98.2% (based on chromatogram at 254 nm),
Rt = 12.3 min.
Synthesis of [(η6-TIPB)Ru(1)Cl]PF6 (1b). Complex 1b was obtained
from ligand 1 (11.2 mg, 26.6 μmol) and [(η6-TIPB)RuCl2]2 (10.0 mg,
13.3 μmol) using the same procedure as used for 1a. Yield: 24.0 mg
1
(99%). H NMR (300 MHz, CDCl3): δ 9.14 (1H, d); 7.85 (1H, t);
7.68 (1H, d); 7.56−7.58 (3H, m); 7.48 (1H, t); 7.26 (1H, s), 7.16
(2H, d); 5.70 (1H, d); 5.63 (1H, d); 5.55 (3H, s); 4.17 (4H, m); 2.93
(3H, m); 1.31 (9H, d); 1.25 (9H, d). 31P NMR (121 MHz, CDCl3): δ
−
6.24 (t, JP−F = 115.5 Hz); −143.7 (m, PF6 ). 19F NMR (282 MHz,
−
CDCl3): δ −32.7 (d, JP−F = 115.5 Hz); 3.45 (d, PF6 ). ESI (+ve
mode) m/z 762.3 (M+, 100%). Purity (HPLC): 99.2% (based on
chromatogram at 254 nm), Rt = 15.4 min.
Synthesis of [(η6-cymene)Ru(3)Cl]PF6 (3a). Complex 3a was
obtained from ligand 3 (13.0 mg, 27.6 μmol) and [(η6-cymene)-
RuCl2]2 (8.4 mg, 13.7 μmol) using the same procedure as used for 1a.
1
Yield: 11.0 mg (70%). H NMR (400 MHz, D2O): δ 8.58 (1H, d);
7.88 (1H, td); 7.67 (1H, d); 7.46 (1H, s); 7.43 (2H, d); 7.36 (1H, s);
7.18 (1H, s); 7.03 (2H, d); 5.57 (2H, s). 31P NMR (162 MHz, D2O):
δ 4.98 (t, JP−F = 93.0 Hz). 19F NMR (376 MHz, D2O): δ −106.2 (d,
JP−F = 93.0 Hz). ESI (+ve mode) m/z 366.1 (M + H+); (−ve mode)
m/z 364.2 (M − H−).
1
Yield: 24.3 mg (99%). H NMR (300 MHz, CDCl3): δ 9.16 (1H, d);
7.86−7.94 (3H, m); 7.49−7.67 (6H, m); 7.11 (2H, d); 6.03−6.10
(3H, m); 5.87−5.93 (2H, m); 5.80 (1H, d); 4.15 (4H, m); 2.63 (1H,
m); 2.61 (3H, s); 1.27 (6H, t); 1.04 (6H, d). 31P NMR (121 MHz,
−
CDCl3): δ 6.23 (t, JP−F = 114.4 Hz); −143.7 (m, PF6 ). 19F NMR
Synthesis of Benzimidazole-diethylphosphonate Ester (3). The
crude mixture (0.242 g containing 67% of monobrominated
compound P4) and 2-(2-pyridyl)-benzimidazole (88.1 mg, 0.451
mmol) were dissolved in DMF (4 mL), and KtOBu (60.7 mg, 0.542
mmol) in butanol (1 mL) was added dropwise to the reaction mixture
with stirring. The reaction mixture was stirred at r.t. for 12 h. After the
reaction, the solvent was removed, and the crude compound was
purified by column chromatography (1:1 v/v ethyl acetate/hexane, Rf
= 0.5) to give product as viscous yellow oil. Yield: 131.8 mg (62%). 1H
NMR (400 MHz, CDCl3): 8.60 (1H, d); 8.44 (1H, dd); 7.81−7.88
(2H); 7.50 (2H, d); 7.28−7.33 (4H, m); 7.25 (2H, d); 6.23 (2H, s);
4.12 (4H, m); 1.26 (6H, t). 31P NMR (162 MHz, CDCl3): δ 6.21 (t,
−
(282 MHz, CDCl3): δ −32.6 (d, JP−F = 114.4 Hz); 3.84 (d, PF6 ). ESI
(+ve mode) m/z 742.1 (M+, 100%). Purity (HPLC): 99.6% (based on
chromatogram at 254 nm), Rt = 14.9 min.
Synthesis of [(η6-TIPB)Ru(3)Cl]PF6 (3b). Complex 3b was obtained
from ligand 3 (12.5 mg, 26.5 μmol) and [(η6-TIPB)RuCl2]2 (9.9 mg,
13.2 μmol) using the same procedure as used for 1a. Yield: 24.5 mg
(97%). 1H NMR (300 MHz, CDCl3): 9.16 (1H, d); 8.00 (1H, d); 7.89
(2H, d); 7.52−7.65 (6H, m); 7.10 (2H, d); 6.17 (1H, d); 5.87 (1H,
d); 5.65 (3H, s); 4.14 (4H, m); 2.95 (3H, m); 1.26 (9H, d); 1.22 (9H,
d). 31P NMR (121 MHz, CDCl3): δ 6.26 (t, JP−F = 114.4 Hz); −143.9
−
(m, PF6 ). 19F NMR (282 MHz, CDCl3): δ −32.6 (d, JP−F = 114.4
JP−F = 115.8 Hz). 19F NMR (376 MHz, CDCl3): δ −108.3 (d, JP−F
=
−
Hz); 3.42 (d, PF6 ). ESI (+ve mode) m/z 812.2 (M+, 100%). Purity
115.8 Hz). ESI (+ve mode) m/z 472.4 [M + H+], 494.4 [M + Na+].
Synthesis of Benzimidazole-phosphonic Acid (4). Ligand 2 (30
mg, 63.6 μmol) was dissolved in dichloromethane (3 mL), and BSTFA
(85 μL, 0.318 mmol) was added to the solution. After stirring for 15
min, TMSI (46 μL, 0.318 mmol) was added to the reaction mixture.
After stirring for an additional 2 h, the solvent was removed, and the
residue was coevaporated three times with dichloromethane (4 mL).
The resulting residue was redissolved in dichloromethane (2 mL)
followed by addition of methanol (10 mL) and stirred at r.t. overnight.
After the reaction, solvent was removed, and the residue was washed
three times with water (4 mL). The residue was then suspended in
water (2 mL), and 1 M NH4OH (1 mL) was added dropwise till the
residue dissolved followed by stirring for 30 min. The solvent was
removed, and the residue was washed once with dichloromethane (3
mL) and twice with diethyl ether (3 mL) to give the product as
(HPLC): 98.6% (based on chromatogram at 254 nm), Rt = 17.8 min.
Synthesis of [(η6-cymene)Ru(2)Cl]Cl (2a). [(η6-Cymene)RuCl2]2
(8.5 mg, 14 μmol) was dissolved in methanol (10 mL), and ligand 2
(10.2 mg, 28 μmol) dissolved in water (1 mL) was added. The
reaction mixture was stirred at r.t. overnight. After the reaction, the
solvent was reduced to a small volume (2 mL), and diethyl ether (8
mL) was added to give a yellow precipitate. The yellow precipitate was
collected, washed once with dichloromethane (4 mL), once with ethyl
acetate (4 mL), once with diethyl ether (4 mL), and vacuum-dried.
1
Yield: 16.9 mg (90%). H NMR (400 MHz, CD3OD): 9.37 (1H, d),
7.94−7.98 (2H, m); 7.83 (1H, d); 7.65 (1H, s); 7.57 (2H, d); 7.50
(1H, t); 7.06 (2H, d); 6.06 (1H, d); 5.94 (1H, d); 5.77−5.81 (3H);
5.70 (1H, d); 2.59 (1H, m); 2.18 (3H, s); 1.03 (3H, d); 0.98 (3H, d).
31P NMR (162 MHz, CD3OD): δ 2.96 (t, JP−F = 98.1 Hz). 19F NMR
(376 MHz, CD3OD): δ −109.1 (d, JP−F = 98.1 Hz). ESI (+ve mode)
m/z 636.1 (M+, 100%). Purity (HPLC): 95.6% (based on chromato-
gram at 254 nm), Rt = 10.2 min.
1
colorless solid. Yield: 18.2 mg (69%). H NMR (400 MHz, D2O): δ
8.66 (1H, d); 7.89−8.00 (2H, m); 7.78 (1H, d); 7.55 (2H, d); 7.35−
7.40 (4H, m); 6.99 (2H, d); 5.85 (2H, s). 31P NMR (162 MHz, D2O):
δ 5.05 (t, JP−F = 92.6 Hz). 19F NMR (376 MHz, D2O): δ −106.1 (d,
JP−F = 92.6 Hz). ESI (+ve mode) m/z 416.2 (M + H+); (−ve mode)
m/z 414.3 (M − H−).
Synthesis of [(η6-TIPB)Ru(2)Cl]Cl (2b). Complex 2b was obtained
from ligand 2 (10.2 mg, 28 μmol) and [(η6-TIPB)RuCl2]2 (10.5 mg,
14 μmol) using the same procedure as used for 2a. Yield: 18.3 mg
(88%). 1H NMR (400 MHz, CD3OD): 9.15 (1H, d), 7.72−7.76 (2H,
m); 7.61 (1H, d); 7.43 (1H, s); 7.35 (2H, d); 7.28 (1H, t); 6.84 (2H,
d); 6.06 (1H, d); 5.93 (1H, d); 5.81 (3H, s); 2.87 (3H, m); 1.10 (9H,
d); 1.07 (9H, d). 31P NMR (162 MHz, CD3OD): δ 3.05 (t, JP−F = 99.1
Hz). 19F NMR (376 MHz, CD3OD): δ −109.0 (d, JP−F = 99.1 Hz).
ESI (+ve mode) m/z 706.1 (M+, 100%). Purity (HPLC): 95.5%
(based on chromatogram at 254 nm), Rt = 12.7 min.
Synthesis of [(η6-cymene)Ru(1)Cl]PF6 (1a). Ligand 1 (9.3 mg, 22.1
μmol) was dissolved in methanol (10 mL), and [(η6-cymene)RuCl2]2
(6.7 mg, 11 μmol) was added. The reaction mixture was stirred at r.t.
for 4 h. After the reaction, solid NH4PF6 (3.6 mg, 22.1 μmol) was
added to the reaction mixture and stirred for an additional 30 min. The
solvent was removed, and the residue was redissolved in small amount
of dichloromethane (2 mL) and filtered through Celite. Diethyl ether
(8 mL) was added to give an orange-yellow precipitate. The precipitate
was collected, washed twice with diethyl ether (6 mL), and vacuum-
Synthesis of [(η6-cymene)Ru(4)Cl]Cl (4a). Complex 4a was
obtained from ligand 4 (12.6 mg, 30.3 μmol) and [(η6-cymene)-
RuCl2]2 (9.2 mg, 15.1 μmol) using the same procedure as used for 2a.
1
dried. Yield: 17.6 mg (95%). H NMR (300 MHz, CDCl3): δ 9.24
1
Yield: 20.4 mg (93%). H NMR (400 MHz, CD3OD): 9.54 (1H, d);
(1H, d); 7.85 (1H, t); 7.66−7.69 (2H, m), 7.59 (2H, d); 7.42 (1H, t);
7.17−7.20 (3H, m); 5.85 (1H, d); 5.78 (1H, d); 5.67 (3H, s); 5.60
(1H, d); 4.16 (4H, m); 2.79 (1H, m); 2.19 (3H, s); 1.28 (6H, t); 1.19
8.08−8.16 (3H, m); 7.84 (1H, d), 7.62−7.70 (3H, m); 7.53 (2H, d);
7.02 (2H, d); 6.34 (1H, d); 6.08−6.15 (3H); 6.04 (1H, d); 5.95 (1H,
d); 2.43 (1H, m); 2.27 (3H, s); 0.90 (3H, d); 0.88 (3H, d). 31P NMR
(162 MHz, CD3OD): δ 3.08 (t, JP−F = 98.1 Hz). 19F NMR (376 MHz,
CD3OD): δ −109.2 (d, JP−F = 98.1 Hz). ESI (+ve mode) m/z 686.1
(3H, d); 1.17 (3H, d). 31P NMR (121 MHz, CDCl3): δ 6.21 (t, JP−F
=
−
114.4 Hz); −143.7 (m, PF6 ). 19F NMR (282 MHz, CDCl3): δ −32.7
−
(d, JP−F = 114.4 Hz); 3.81 (d, PF6 ). ESI (+ve mode) m/z 692.1 (M+,
H
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Inorganic Chemistry
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(M+, 100%). Purity (HPLC): 97.4% (based on chromatogram at 254
nm); Rt = 12.2 min.
existing ligand in 2F71, extending 6.0 Å from it. The existing ligand
was then removed. The macro, dock_run.mcr, available in YASARA
was used to perform the docking. This macro uses the AutoDock
version in YASARA to perform 25 docking runs for both parent ligand
4 and 4a.32 The top docked structure for parent ligand 4 and complex
4a were used for the analysis.
Synthesis of [(η6-TIPB)Ru(4)Cl]Cl (4b). Complex 4b was obtained
from ligand 4 (12.5 mg, 30 μmol) and [(η6-TIPB)RuCl2]2 (11.3 mg,
15 μmol) using the same procedure as used for 2a. Yield: 21.4 mg
(90%). 1H NMR (400 MHz, CD3OD): 9.32 (1H, d); 8.09−8.20 (3H,
m); 7.63−7.85 (4H, m); 7.56 (2H, d); 7.07 (2H, d); 6.16 (1H, d);
6.03 (1H, d); 5.91 (3H, s); 2.97 (3H, m); 1.20 (9H, d); 1.17 (9H, d).
31P NMR (162 MHz, CD3OD): δ 3.07 (t, JP−F = 99.5 Hz). 19F NMR
(376 MHz, CD3OD): δ −109.2 (d, JP−F = 99.5 Hz). ESI (+ve mode)
m/z 756.2 (M+, 100%). Purity (HPLC): 98.4% (based on chromato-
gram at 254 nm), Rt = 15.2 min.
PTP Inhibition Assays and Enzymatic Kinetic Studies. PTP
inhibition assays and enzymatic kinetic studies were carried out in
accordance with a reported procedure, using pNPP as the substrate.33
The inhibition assays were performed in buffer (20 mM MOPS, 200
mM NaCl, pH 7.2, 100 μL) on a 96-well plate. Stock solutions of the
inhibitors were prepared in ultrapure water and serially diluted to
concentrations of 0.03−1 mM. Ru concentrations were determined
using ICP-OES. Inhibitors (0.03−1 mM, 10 μL) were incubated with
enzymes (150 nM) in buffer (20 mM MOPS, 200 mM NaCl, pH 7.2,
75 μL) at r.t. for 30 min. The reaction was initiated by the addition of
pNPP (20 mM MOPS, 10 μL) and incubated for further 30 min
before being terminated using the stop buffer (1 M NaOH, 5 μL). A405
was recorded using a microplate reader (Tecan), and IC50 values were
obtained by fitting the concentration-dependent inhibition curves
using Prism (GraphPad). The experiments were carried out in
triplicates. Solutions of the compounds were freshly prepared before
each inhibition assays and Ru levels determined using ICP-OES where
applicable.
Synthesis of [(η6-cymene)Ru(pyridylimidazole)Cl]PF6 (5). 2-(2-
Pyridyl)-imidazole (7.26 mg, 50 μmol) was dissolved in methanol (10
mL), and [(η6-cymene)RuCl2]2 (15.3 mg, 25 μmol) was added. The
reaction mixture was stirred at r.t. for 4 h. After the reaction, solid
NH4PF6 (8.2 mg, 50 μmol) was added to the reaction mixture and
stirred for an additional 30 min. After the reaction, the solvent was
reduced to a small amount (2 mL) and filtered through Celite, and
diethyl ether (8 mL) was added to give an orange-yellow precipitate.
The precipitate was collected, washed twice with diethyl ether (6 mL),
and vacuum-dried. Yield: 26.9 mg (96%). 1H NMR (400 MHz,
CD3OD): δ 9.33 (1H, d); 8.08 (1H, td); 7.97 (1H, d), 7.83 (1H, d),
7.53−7.57 (2H, m); 6.03 (1H, d); 5.94 (1H, d); 5.79 (1H, d); 5.70
(1H, d); 2.62 (1H, m); 2.15 (3H, s); 1.04 (3H, d); 0.99 (3H, d). 31P
Kinetic analysis was performed according to the following rate
equation34
−
NMR (162 MHz, CD3OD): δ −144.5 (m, PF6 ). 19F NMR (376
−
MHz, CD3OD): δ −74.5 (d, PF6 ).
V
max[S]
Syntheis of [(η6-cymene)Ru(pyridylbenzimidazole)Cl]PF6 (6).
Complex 6 was obtained from 2-(2-pyridyl)-benzimidazole (9.8 mg,
50 μmol) and [(η6-cymene)RuCl2]2 (15.3 mg, 25 μmol) using the
v =
[I]
Km 1 +
+ [S]
(
)
Ki
1
same procedure as used for 5. Yield: 28.7 mg (94%). H NMR (400
where v = initial velocity, Vmax is the maximum initial velocity, Km is
the Michaelis constant for the substrate, [S] and [I] for concentrations
of substrate and inhibitor, and Ki is the inhibition constant, derived
from the slope of the Lineweaver−Burk plots.
MHz, CD3OD): δ 9.48 (1H, d); 8.18−8.23 (2H, m); 8.00−8.02 (1H,
m); 7.67−7.71 (2H, m); 7.51−7.58 (2H, m); 6.27 (1H, d); 6.13 (1H,
d); 5.98 (1H, d); 5.95 (1H, d); 2.47 (1H, m); 2.20 (3H, s); 0.92 (3H,
−
d); 0.91 (3H, d). 31P NMR (162 MHz, CD3OD): δ −144.5 (m, PF6 ).
The initial hydrolysis rates were measured at different substrate and
inhibitor concentrations. The reciprocal of the reaction rate was
plotted as a function of the reciprocal of the substrate concentration
for each concentration of the inhibitor. The Ki values measured at the
various inhibitor concentrations were plotted against concentration of
the inhibitor to calculate the inhibition constants.
−
19F NMR (376 MHz, CD3OD): δ −74.5 (d, PF6 ).
Synthesis of [(η6-cymene)RuI(2)]PF6. Complex 1a (8.4 mg, 10
μmol) was dissolved in dichloromethane (5 mL), and TMSI (11 μL,
75 μmol) was added and stirred at r.t. overnight. The solvent was then
evaporated, and to the residue was added methanol (10 mL). The
reaction mixture was stirred at r.t. for 24 h. After the reaction, the
solvent was reduced to ca. 2 mL, and diethyl ether (8 mL) was added
to give a yellow precipitate. The yellow precipitate was collected,
washed once with dichloromethane (6 mL), once with ethyl acetate (6
mL), twice with diethyl ether (6 mL), and vacuum-dried. Yield: 7.1 mg
ASSOCIATED CONTENT
* Supporting Information
■
S
Crystallographic data (CIF) for the complexes 1b and 4a, NMR
spectra, and HPLC chromatograms of Ru(II) complexes. This
material is available free of charge via the Internet at http://
pubs.acs.org.
1
(81%). H NMR (300 MHz, CD3OD): δ 9.40 (1H, d); 7.99 (1H, s);
7.96 (1H, s); 7.89 (1H, d); 7.74 (1H, s); 7.64 (2H, d); 7.51 (1H, t);
7.13 (2H, d); 6.03(1H, d); 5.94 (1H, d); 5.88 (2H, s); 5.84 (1H, d);
5.79 (1H, d); 2.87 (1H, m); 2.47 (3H, s); 1.15 (3H, d); 1.11(3H, d).
31P NMR (121 MHz, CD3OD): δ 3.63 (t, JP−F = 99.0 Hz); −144.0 (m,
AUTHOR INFORMATION
Corresponding Author
*E-mail: chmawh@nus.edu.sg.
−
PF6 ). 19F NMR (282 MHz, CD3OD): δ −33.3 (d, JP−F = 99.0 Hz);
■
−
1.09 (d, PF6 ). ESI (+ve mode) m/z 728.0 (M+, 100%).
X-ray Diffraction Studies. X-ray data were collected with a
Bruker AXS SMART APEX diffractometer using Mo Kα radiation at
223(2) K with the SMART suite of programs.26 Data were processed
and corrected for Lorentz and polarization effects using SAINT
software,27 and for absorption effects using the SADABS software.28
Structural solution and refinement were then carried out using the
SHELXTL suite of programs.29 The structure was solved by Direct
methods. Non-hydrogen atoms were located using difference maps
and were given anisotropic displacement parameters in the final
refinement. All H atoms were put at calculated positions using the
riding model.
Molecular Docking Studies. There were 116 X-ray crystallog-
raphy structures of PTP-1B available in the RCSB Protein Data Bank.
Structure 2F71 was selected due to its high data quality with a
resolution of 1.55 Å, R value of 0.153, and Rfree value of 0.172. The
structure was prepared using YASARA30 by using its CleanAll,
OptHydAll, CorrectIso, CorrectCis functions and then minimized
using AMBER03 forcefield.31 A simulation cell was defined using the
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors thank Lip Lin Koh, Geok Kheng Tan, and Yimian
Hong for performing the X-ray crystallographic analysis. The
funding provided by National University of Singapore and
Ministry of Education (R143-000-411-133 and R143-000-512-
112 to W.H.A.) and National University of Singapore
NUSAGE Bridging Grant (C148-000-005-001 to Y.C.W) is
gratefully acknowledged.
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