Exploration of α-aminophosphonate N-derivatives as novel, potent and selective inhibitors of protein tyrosine phosphatases

Article abstract of DOI:10.1016/j.ejmech.2012.01.038

Seventeen α-aminophosphonates are synthesized. Their compositions and structures are established by EA, UV, FT-IR, ¹H NMR, ¹³C NMR, ³¹P NMR and ESI-MS. Compounds 1-4 are confirmed by X-ray crystallography. PTP inhibition shows compounds 1-5, 12, 15 are moderate competitive inhibitors with some selectivity. The most potent inhibitor is compound 5 with the lowest IC₅₀ value about 6.64 μM against PTP1B, about 2-fold and 25-fold stronger than against TCPTP and PTP-MEG2 while it doesn't inhibit SHP-1 and SHP-2. The binding constant of 5 to PTP1B is 2.23 × 10⁵ M^-1 and binding ratio approximates 1:1. Cell viability and apoptosis assays indicate 5 is cell permeable with lower cytotoxicity. The results indicate α-aminophosphonates are possibly developed to effective and selective inhibitors of PTPs.

Full text of DOI:10.1016/j.ejmech.2012.01.038

European Journal of Medicinal Chemistry 49 (2012) 354e364
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Exploration of
a
-aminophosphonate N-derivatives as novel, potent and selective
inhibitors of protein tyrosine phosphatases
,
c
,
a
a,
a
b
b,
***
Qingming Wang a, Miaoli Zhu a , Ruiting Zhu , Liping Lu
, Caixia Yuan , Shu Xing , Xueqi Fu
,
*
**
Yuhua Mei c, Qingwei Hang c
a Institute of Molecular Science, Key Laboratory of Chemical Biology and Molecular Engineering of the Education Ministry, Shanxi University, Taiyuan, Shanxi 030006,
People’s Republic of China
b Edmond H. Fischer Signal Transduction Laboratory, College of Life Sciences, Jilin University, Changchun 130023, People’s Republic of China
c State Key Laboratory of Coordination Chemistry, Nanjing University, Nanjing, 210093, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
Seventeen a-aminophosphonates are synthesized. Their compositions and structures are established by
EA, UV, FT-IR, 1H NMR, 13C NMR, 31P NMR and ESI-MS. Compounds 1e4 are confirmed by X-ray crys-
tallography. PTP inhibition shows compounds 1e5, 12, 15 are moderate competitive inhibitors with some
Received 17 December 2011
Received in revised form
14 January 2012
Accepted 17 January 2012
Available online 24 January 2012
selectivity. The most potent inhibitor is compound 5 with the lowest IC50 value about 6.64 mM against
PTP1B, about 2-fold and 25-fold stronger than against TCPTP and PTP-MEG2 while it doesn’t inhibit SHP-
1 and SHP-2. The binding constant of 5 to PTP1B is 2.23 ꢀ 105 Mꢁ1 and binding ratio approximates 1:1.
Cell viability and apoptosis assays indicate 5 is cell permeable with lower cytotoxicity. The results
Keywords:
indicate
a
-aminophosphonates are possibly developed to effective and selective inhibitors of PTPs.
a
-Aminophosphonate
Ó 2012 Elsevier Masson SAS. All rights reserved.
Protein tyrosine phosphatases
Competitive inhibitor
Selective inhibitor
Cell viability
Apoptosis
1. Introduction
phosporylation, which makes for the pathogenesis of several
human diseases, such as cancers, autoimmune disorders, diabetes
Protein tyrosine phosphatases play an important role in cata-
lyzing protein tyrosine dephosphorylation and modulating several
cellular signal transduction pathways [1]. It is known that the
dysregulation of PTPs activities could cause aberrant tyrosine
and obesity [2]. It has been demonstrated that PTP1B acts as
a negative regulator of insulin and leptin-mediated processes [3].
TCPTP is crucial for inflammation control. TCPTP null mice develop
progressive systemic inflammatory disease as shown by chronic
myocarditis, gastritis, nephritis, and sialadenitis as well as elevated
serum interferon-
g and tumor necrosis factor a in vivo [4].
Abnormal expression of many PTPs (SHP-2, PTP1B, TCPTP, PRL-3,
CDC14, LMW-PTP, Cdc25, etc.) is found in various cancers [5].
Because of the role of PTPs in the disease onset and progression,
PTP inhibitors especially that can selectively inhibit the particular
PTP are therefore highly desirable as promising targeted thera-
peutic drugs [5b,6].
Abbreviations: DMEM, Dulbecco’s modified Eagle’s medium; EA, elemental
analysis; ESI-MS, electrospray ionization mass spectrometry; FBS, fetal bovine
serum; GSH, glutathione; IC50, the half maximal inhibitory concentration; MOPS, 3-
(N-morpholino)propanesulfonic acid; MTT, 3-[4,5-Dimethylthiazo-2-yl]-2,5-
diphenpyltetra-zolium bromide; p-NPP, p-nitrophenol phosphate; PI, propidium
iodide; PMSF, phenylmethane-sulfonyl fluoride; PTP, protein tyrosine phosphatase;
PTP1B, protein tyrosine phosphatase 1B; PTP-MEG2, megakaryocyte protein tyro-
sine phosphatase; SHP-1, src homology phosphatase 1; SHP-2, src homology
phosphatase 2; TCPTP, T-cell protein tyrosine phosphatase.
* Corresponding author. Institute of Molecular Science, Shanxi University, 92
Wucheng Road, Taiyuan 030006, People’s Republic of China. Tel.: þ86 351 8902618,
7017974, 7018755.
** Corresponding author.
*** Corresponding author.
As mimics of natural amino acids, a-aminophosphonic acids and
relative derivatives are currently attracting a great deal of interest
in medicinal chemistry due to their important biological effects [7].
They are reported to have antibacterial activities [8], to act as
inhibitors of enzymes such as rennin, HIV proteases, serine prote-
ases and so on [9]. Some studies demonstrated that phosphorous
compounds were effective PTP1B inhibitors [10]. Moreover, our
E-mail addresses: miaoli@sxu.edu.cn (M. Zhu), luliping@sxu.edu.cn (L. Lu), fxq@
jlu.edu.cn (X. Fu).
recent research indicated that dinuclear copper complexes of
a-
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2012.01.038

Q. Wang et al. / European Journal of Medicinal Chemistry 49 (2012) 354e364
355
shown in Scheme 1, we obtained these compounds 1e4 by
two steps: a) the Schiff bases were firstly prepared in
a condensation of different substituted aromatic amines and
an aromatic aldehyde in ethanol solvent by refluxing equi-
molar amounts of reagents; b) reaction of Schiff bases with
a diethyl phosphonate in ethanol solvent by refluxing. The
rigid linkers were achieved using substituted aromatic
amines in the first step.
aminophosphonate containing bisphenol can potently inhibit PTPs
with some selectivity [11]. Therefore,
also be developed as PTPs inhibitors.
a-aminophosphonate may
From crystal structures of PTP1B [12], the active-site cysteine
(Cys215) situated at the amino-terminus of -4 is at the base of
a
a cleft on the surface of the protein formed by residues of the PTP1B
signature motif (residues from His214 to Arg222). The bell-
mouthed cleft has a diameter in range of 10e18 Å. Thus, a sterical
tetrahedral phosphonate, ePO(OR)2, is selected in order to block up
ii) Compound 5 was gotten in one-pot with a three-component
system using a starting material of phosphonic acid, salicy-
laldehyde and 4-aminophenol by the Mannich-type reaction
shown in Scheme 2.
the active-site. Based on this point,
a-aminophosphonate N-
derivatives, o-cresoleCH(eNHR)ePO(OR1)(OR2), are good candi-
dates for our design: i) R ¼ rigid group, R1 ¼ R2 ¼ ethyl; ii) R ¼ rigid
group, R1 ¼ R2 ¼ H; iii) R ¼ flexible group, R1 ¼ H, R2 ¼ ethyl; iv)
R ¼ flexible side chains from proteinogenic amino acids, R1 ¼ H,
R2 ¼ ethyl. For situations i) and ii), it can engender significant rigid
effect and thus influence the structural assembly with an appro-
priate plug to decrease their contact between substrate and Cys215.
In the light of this, we synthesized and characterized seventeen a-
aminophosphonate compounds and investigated their inhibitory
effect against PTPs.
iii) Next group of o-cresol
a
-aminophosphonate N-derivatives
-amino-
via flexible e(CH2)ne (n ¼ 2,3,6) connecting two
a
alkylphosphonate fragments was synthesized by the proce-
dure of KabachnikeFields reaction in one-pot with a three-
component system consisting of salicylaldehyde, diamine of
fatty alkyl groups (for 6e8) and diethyl phosphonate in
ethanol solvent by refluxing from Scheme 3. This is a conve-
nient synthetic procedure without precursors being sepa-
rated. The intermediate Schiff bases were not isolated, but
were reacted in situ with the other reagent. Here, the flexible
linkers consist of fatty alkyl groups of two or three or six
carbon atoms.
2. Results and discussion
2.1. Synthesis of o-cresol a-aminophosphonate N-derivatives
iv) A last group of o-cresol a-aminophosphonate N-derivatives
with flexible proteinogenic amino acid residue was synthe-
sized by the procedure of KabachnikeFields reaction in one-
pot with a three-component system consisting of salicy-
In the synthesis of aminophosphonic acids, Pudovik reaction as
well as other pathways, like Mannich-type reaction and
KabachnikeFields reaction are powerful and direct methods for
construction of PeCeN bonds [13e17]. The Pudovik reaction
involves the addition of the PeH bond of phosphonic acid esters to
laldehyde, sodium salt of
phosphonate in ethanol solvent by refluxing. As shown in
Scheme 4, o-cresol -aminophosphonate N-derivatives 9e17
are acquired under the condition of sodium hydroxide to
L-amino acid (for 9e17) and diethyl
a
Schiff bases and forms an asymmetric center in a-position to the
phosphorus atom if there are two different substituents in C-
terminal. Expediently, the KabachnikeFields condensation reaction
occurs in one-pot with a three-component system consisting of
a carbonyl compound, an amine and a hydrophosphoryl compound
in organic solvents or in ionic liquids under MW irradiation by
increase the solubility of
time.
L-amino acids and short the reacting
catalyst.
type reaction using phosphorous acid, aldehyde compound, and
amines as reactants. In our blueprint of synthesis, four kinds of
a
-Aminophosphonic acids are also achieved by a Mannich-
2.2. Characterization of o-cresol a-aminophosphonate N-
derivatives 1e17
a
-
aminophosphonate N-derivatives were prepared, namely these
compounds exhibited characteristic either the rigid structure with
All the compounds were elucidated on the basis of the EAs, IR,
UV, NMR (1H, 13C, & 31P) and ESI-MS. The elemental analysis data for
1e17 were consistent with the compositions of desired products.
Some of them were further supported by X-ray single crystal
diffraction in coming expatiation. All the compounds showed
absorption bands of infrared in the region 3182e3440, 2882e2982,
1221e1231 and 970e985 cmꢁ1 for stretch vibrations of NeH, CaeH,
one
connecting two
aminoalkylphosphonate with proteinogenic amino acid side
chains. To form the -aminoalkylphosphonate fragment,
a
-aminoalkylphosphonate fragment or the flexible structure
a-aminoalkylphosphonate fragments or one a-
a
PeCHR1eNHR (R1 ¼ o-cresol), the synthetic routes shown in
Schemes 1e4.
P]O and PeCa, respectively [15,16,18,19]. The UV spectral data of a-
aminophosphonate compounds indicated that there are several
absorptions in ranges of 216e369 nm, respectively.
i) A method for the synthesis of o-cresol a-aminophosphonate
N-derivatives with the rigid structures was through Pudovik
reaction or Mannich-type reaction. According to the strategy
The 31P NMR spectroscopy is the most precise method and a very
convenient tool for determining the structure of the
Scheme 1. The Synthesis of the rigid a-aminophosphonate N-derivatives 1e4 in two-step by Pudovik reaction.

356
Q. Wang et al. / European Journal of Medicinal Chemistry 49 (2012) 354e364
Scheme 2. The Synthesis of the rigid a-aminophosphonic acid N-derivatives 5 using Mannich-type reaction in one-pot fashion.
Scheme 3. The Synthesis of the flexible a-aminophosphonate N-derivatives 6e8 in one-pot fashion by KabachnikeFields reaction.
organophosphorus compounds. Chemical shifts of 31P NMR spectra
in the diesters of H-phosphonic acid and -aminophosphonic acids
101 MHz, CDCl3 21.13 ppm) [22]. The 31P NMR spectra of
monoester -aminophosphonic acids 6e17 have signals with the
a
a
are in range of 0e15 and 15e25 ppm referenced to 85% H3PO4 [14].
The 31P NMR spectra of all the compounds show one single peak
and indicate that there are three kinds of the phosphorus-
containing compounds (see Experimental section). The 31P NMR
chemical shifts of 10.79, 10.37, 11.25, 14.77, 10.99 &10.76, 17.97&
17.31, 10.86, 19.185, 20.46, 17.65 & 16.88, 17.86 ppm, respectively.
The 31P NMR spectrum of
a-aminophosphonic acids 5 without
ester group has signal with the chemical shift of 2.29 ppm, which
is smaller than 7.54 ppm in N-(phosphonomethyl)-N-methyl-
glycine, MeN-(CH2COOH)(CH2PO(OH)2) [17b]. Further evidence
for the formation of desired compounds was also confirmed
from 13C NMR spectra, which proved to be a diagnostic tool for
the positional elucidation of the carbon. The chemical shifts of
spectra of diester a-aminophosphonates 1e4 have signals with the
chemical shifts of 22.37e24.68 ppm. The values agree with other
diester aminophosphonates, such as diethyl (1R)-1-{[(carbobenzy-
loxy]amino}ethylphosphonate (31P NMR, DMSO-d6 25.89 ppm)
[20], (R,S)-a-phenylethylaminodiethoxy-phosphos-phonylmethyl-
4-hydroxylbenzene (31P NMR at 121 MHz, CDCl3 25.01 ppm) [21],
carbons (PeC, AreC, AreCeN,
& AreCeO) showed peaks
p-[N-Methyl(dietoxy- phosphonyl)(2-furyl)]toluidine (31P NMR at
around the 44.64e56.28, 113.26e137.51, 143.63e147.28 and
Scheme 4. The Synthesis of the flexible a-aminophosphonate N-derivatives 9e17 with natural L-amino acid side chains in one-pot fashion by KabachnikeFields reaction.

Q. Wang et al. / European Journal of Medicinal Chemistry 49 (2012) 354e364
357
154.46e157.13 ppm regions, respectively. The mass spectrum of
compounds showed relative molecular ion peaks as well.
SHP-2 but not PTP-MEG2. The most potent inhibitor is compound 5
with the lowest IC50 value about 6.64 M against PTP1B, nearly the
m
same capacity compared with other phosphate compounds [10b].
The inhibitory effect of compound 5 on PTP1B is about 2-fold
stronger than against TCPTP and about 25-fold stronger than
against PTP-MEG2, displaying selectivity against PTP1B, as well as
TCPTP. Compounds 1e4 show less potency against PTP1B, TCPTP
and PTP-MEG2. Meanwhile, the substitute groups and their posi-
tions also slightly influence the inhibitory effects. Compared 1e3,
we can see that compound 2 with p-Br moiety improves potency
about 4 and 2-fold to 1 and 3 respectively, consistent with that
incorporation eBr moiety can increase the potency [24]. And
compound 4 with substituent Cl atom at 2-position of aniline
increases the inhibitory effect of 3-fold than compound 1 having
substituent Cl atom at 4-position. The results indicate that by
2.3. Crystal structures of 1e4
In order to comprehensively understand the structural features
of 1e17, the a-aminophosphonates were attempted to grow their
crystals in several solvents or mix-solvents. Suitable crystals for
x-ray single crystal diffraction were only obtained from DMF (1)
and ethanol (2e4). Crystallographic data are listed in Table 1. Also
the X-ray structures of the o-cresol
derivatives 1e4 are shown in Figs. 1e4.
a-aminophosphonates N-
The crystal structures of 1e4 are triclinic or monoclinic system
with the space group Pꢁ1, C2/c, Pꢁ1 and P21/c, respectively. Seen
from Figs. 1e4, all P atoms have tetrahedral geometries involving
two O-ethyl groups, one Ca atom, and a double bond O atom. In the
structures, Ca atoms are chiral centers, which are responsible for
the existence of optically activity. The location of NeH group is in
opposite directions with OeH of 2-hydroxyphenyl in the four
compounds. The CeP and P]O bond lengths are almost compa-
rable with the similar structures [23]. The intramolecule CaeH/O1
hydrogen bond makes the configurations of the molecules more
rigid. The dihedral angles both planes (aniline and 2-
hydroxyphenyl groups) for 1e4 are located in the range of 80.8(2)
to 89(1) (Supplementary material), especially the dihedral angles in
4 closed to a right angle. The max outer-diameters of molecular Van
der Waals interaction in 1e4 are about 15 Å, which is coincident
with the cleft on the surface of the protein formed by residues of
the PTP1B signature motif (residues from His214 to Arg222).
rational design,
a-aminophosphonate compounds are possibly
developed to effective and selective inhibitors of some PTPs.
2.4.2. Kinetic analysis of PTP1B and PTP-MEG2 inhibition
In order to elucidate the inhibition modes of PTP1B and PTP-
MEG2 by the a-aminophosphonate, compounds 2, 5 (for PTP1B)
and 12 (for PTP-MEG2) were chosen for kinetic studies as their
representative compounds because of their lower IC50 values. Six
different concentrations of the substrate p-NPP (0.2, 0.3, 0.5,1.0, 2.0,
8.0 mM for 2 and 5; 0.2, 0.3, 0.5, 1.0, 2.0, 4.0 mM for 12) and five
different concentrations of the inhibitors (0, 60, 120, 160, 240
for 2 & 12; 0, 0.1, 1, 4, 8 M for 5) were used in the steady state
kinetics assays. Fig. shows the LineweavereBurk double-
mM
m
5
reciprocal plot of the kinetics data in the presence of various
concentrations of compound 2, 5 and 12. It appears that the lines
converged at an intersection on the y-axis above the x-axis,
implying a competitive inhibition mode versus p-NPP. The Ki values
of compound 2, 5 and 12 are determined to be 96.3, 4.4 and
2.4. Biological evaluation of
2.4.1. PTPs inhibition assays
a-aminophosphonate
102.2 mM, respectively.
The inhibitory activities of all the a-aminophosphonates on five
PTPs were measured using p-NPP as the substrate. As illustrated in
Table 2, in the seventeen compounds, only seven exhibit inhibition
activity. Their inhibitions against different PTPs are varied.
Compounds 1e5 inhibit PTP1B, TCPTP and PTP-MEG2, but do not
inhibit SHP-1 and SHP-2 even at 10 mM. Compound 12 inhibits the
five PTPs, while compound 15 inhibits PTP1B, TCPTP, SHP-1 and
2.5. Fluorescence spectra and binding constants of PTP1B with 5
The interaction of PTP1B and compound 5 was further investi-
gated by fluorescence. The run for PTP1B/5 system had shown
major decrease in fluorescence intensity of PTP1B at 335 nm after
Table 1
Crystallographic data for 1e4.
1$C3H7NO
2
3$C2H5OH
4$H2O
CCDC
Formula
Mr
Temperature (K)
Crystal system
Space group
a(Ǻ)
790024
C17H21ClNO4P$C3H7NO
442.86
298(2)
Triclinic
Pꢁ1
790025
C17H21BrNO4P
414.23
298(2)
Monoclinic
C2/c
31.715(6)
7.459(1)
30.349(5)
90
148.475(8)
90
790026
C17H21N2O6P$C2H5OH
426.4
298(2)
Triclinic
Pꢁ1
790027
C17H21ClNO4P$H2O
387.78
298(2)
Monoclinic
P21/c
15.1078 (13)
33.094 (3)
8.0216 (7)
90
99.880 (2)
90
3951.1 (6)
8
1.304
8.180(2)
11.188(2)
14.126(3)
109.29(3)
96.41(3)
104.07(3)
1157.1(4)
2
9.6267 (19)
10.073 (2)
12.885 (3)
99.72 (4)
111.19 (3)
98.95 (3)
1116.0 (4)
2
B(Ǻ)
C(Ǻ)
a
(ꢂ)
(ꢂ)
b
g
(ꢂ)
V(Ǻ3)
3753.7(12)
8
1.466
Z
Dcalc(g cmꢁ3
F(000)
)
1.271
468
1.269
452
1696
1632
Goodness of fit
Reflections collect.
Reflections unique
Rint
1.07
5343
3854
0.022
1.024
9465
3297
0.042
1.055
5138
3730
0.021
1.061
20496
6900
0.125
R1, wR2 [I > 2
R1, wR2 (all data)
s
(I)]
0.094,0.230
0.132,0.254
94.5
0.440, ꢁ0.214
0.045,0.112
0.083,0.129
99.9
0.537,ꢁ0.564
0.071,0.204
0.087,0.219
94.7
0.769,ꢁ0.274
0.068,0.182
0.092,0.202
99.0
0.751,ꢁ0.614
Completeness (%)
Largest diff. peak (e Ǻ3) and hole

358
Q. Wang et al. / European Journal of Medicinal Chemistry 49 (2012) 354e364
Fig. 1. X-ray structure of the 1$DMF with a 30% probability of thermal ellipsoids.
Fig. 3. X-ray structure of the 3$C2H5OH with a 30% probability of thermal ellipsoids
(dot line for H-bond).
addition the inhibitor 5 (Fig. 6), agreeing to our previous study that
the intrinsic fluorescence of PTP1B was quenched by addition of
metal complexes [11a,25]. The binding constant and stoichiometry
of PTP1B and 5 were estimated through the static quenching
equation [26].
dependent manner at higher concentrations (Fig. 7A), suggesting
a weaker anti-proliferation ability. For the two cells, 5 do not
exhibit obviously different in anti-proliferation. Apoptosis assay
was carried out in HepG2 cells. The result shows that 5 hardly
F0 ꢁ F
induces apoptosis even at 50
increases to 100 M, only a very small amount of cells are apoptotic
and necrotic (about 7.2 and 1.9% respectively), but when the
concentration increases to 300 M, the total apoptotic and necrotic
cells increase to about 69.7 and 22.7% respectively (Fig. 7B). These
results demonstrate 5 is cell permeable, but with lower cytotox-
icity, maybe due to its potent inhibition only for PTP1B and TCPTP.
mM, and when the concentration
lg
¼ lg KA þ n lg½Qꢃ
m
F
where F and F0 are current and initial fluorescence intensities,
respectively. Q is the quencher (compound 5), KA is the binding
constant and n stands for the stoichiometry of PTP1B and 5. The
binding constant is calculated to be 2.23 ꢀ 105 Mꢁ1, comparable to
m
that (2.27 ꢀ 105
M
ꢁ1) deduced from Ki, and binding ratio approx-
imates 1:1.
3. Conclusions
2.6. The influence of 5 on cell viability and apoptosis
A
series of novel a-aminophosphonate N-derivatives are
synthesized and characterized. The structures of compounds 1e4
are determined by X-ray crystal analysis. PTPs inhibition tests
reveal that some of the a-aminophosphonate N-derivatives inhibit
PTPs moderately while compound 5 is a potent and selective
inhibitor of PTP1B and TCPTP. The substitute groups and their
positions slightly influence the inhibitory effects. Kinetic analysis
indicates that these compounds are competitive inhibitors of PTPs.
Cell viability and apoptosis assays show 5 is cell permeable, but
with lower cytotoxicity, maybe due to its potent inhibition only for
The effect of 5 on cell viability and apoptosis of HepG2 and MCF7
cells was further investigated. MTT assay reveals that the treatment
of 5 with 48 h decreases the viability of the two cells in dose-
PTP1B and TCPTP. These results suggest by rational design, a-ami-
nophosphonates are possibly developed to potent, selective and
cell permeable inhibitors of some PTPs.
4. Experimental
4.1. General methods
All reagents and solvents were purchased commercially and
used without further purification. Deionized water was used for all
chemical experiments and double-distilled water was used to
prepare buffer solutions as well as biological evaluation. pH values
of buffer solutions were measured using a PHS-3TC pH meter. All
reactions were magnetically stirred. Elemental analyses (EAs) were
carried out with
a VARI-EL elemental analyzer. IR spectra
(4000e400 cmꢁ1) were recorded using a Shimadzu Fourier trans-
form IR-8300 spectrometer in KBr disks. The electronic absorption
spectra and Ki were taken on a HewlettePackard HP-8453 Chem-
station spectrophotometer. 1H NMR spectra were recorded at RT
with a DRX 300 instrument at 300 MHz 31P NMR spectra were
Fig. 2. X-ray structure of the 2 with a 30% probability of thermal ellipsoids.

Q. Wang et al. / European Journal of Medicinal Chemistry 49 (2012) 354e364
359
Fig. 4. X-ray structure of the 4$H2O with a 30% probability of thermal ellipsoids (dot line for H-bond).
recorded with the same spectrometer at 121.49 MHz with H3PO4
(85%) as external standard. 13C NMR spectra were recorded at RT
with a DRX 500 instrument at 125.77 MHz (defined position of
methanol and DMSO as 49.00 and 39.50 ppm, respectively). Elec-
trospray ionization mass spectra (ESI-MS, negative mode) were
recorded with a Quattro Micro API instrument (Waters, USA) in
methanol/water solution. Fluorometric experiments were carried
out on a Cary Eclipse Spectrophotometer. The X-ray data were
collected using a Bruker SMART APEX 1K CCD diffractometer.
Bioactivity assays of the compounds were carried out with a Bio-
Following Scheme 2, compound 5 was obtained. A salicyalde-
hyde (15 mmol) in 10 mL of ethanol solution was added dropwise
into the stirring 30 mL of ethanol solution containing 4-
hydroxyphenylamine (10 mmol). The reacting solution was
refluxed for 6 h with constant stirring, and then a 20 mL of ethanol
solution of phosphonic acid (10 mmol) was added dropwise into
the resulting solution. Refluxed for about 42 h and removed out
part solvent on a rotary evaporator. The orange solid was obtained,
recrystallized from ethanol/water (1:1).
According to Scheme 3,
a-aminophosphonates 6e8 were
Rad model 550 microplate reader for A405
.
prepared using one-pot synthesis: salicyaldehyde (40 mmol) in
a 20 mL of ethanol solution was added dropwise into the stirred
solution of 20 mmol of hexane-1,6-diamine (6) or propane-1, 3-
diamine (7) or ethane-1,2-diamine (8) in ethanol (20 mL). The
resulting solution was refluxed for 2e4 h with constant stirring.
Then a diethyl phosphonate (40 mmol) in a 20 mL of ethanol
solution was added dropwise into the solution. Refluxed for about
24e32 h and the light yellow solids were obtained, recrystallized
from ethanol/water (1:1).
4.2. Synthesis of a-aminophosphonates
According to Scheme 1,
a-aminophosphonates 1e4 were
prepared by two-step procedure: (1) equimolar amounts
a
(0.02 mol) of amine and salicylaldehyde were mixed in a 30e40 mL
of ethanol solution and refluxed for 2e4 h. After the reacting
mixture were cooled to room temperature, the generated Schiff
bases were crystallized from solution (Yield: 1a 86.6%, 2a 96%, 3a
90%, 4a 96%); (2) a 10e20 mL of ethanol solution containing
15e50 mmol of diethyl phosphonate was added dropwise into the
stirred solution of an equimolar amount of Schiff base compounds
in a 30 mL of ethanol. And then, the mixture was refluxed for
12e24 h with constant stirring. The solvent was removed by
a rotary evaporator. The oily residue was dissolved in 10e20 mL of
N, N-dimethylformamide (1) or ethanol (2e4), and block crystal
(1e4) were collected after a week, recrystallized from ethanol/
water (1:1).
Alao following to Scheme 4, a-aminophosphonates 9e17 were
prepared by one-pot synthesis: 20 mmol of glycine (9) (alanine
(10), threonine (11), serine (12), tryptophan (13), cystine (14)
histidine(15), phenylalanine (16), tyrosine (17)) and sodium
hydroxide (20 mmol) in 50 mL of ethanol solution, heated to reflux
in order to dissolve completely. Then 20 mL of ethanol solution
containing salicyaldehyde (20 mmol) was added dropwise into the
upper solution. The reacting solution was refluxed for 3e4 h with
constant stirring, and then a 20 mL of ethanol solution of diethyl
phosphonate (20 mmol) was added dropwise into the resulting
solution. After refluxed for about 10e20 h, the light yellow solids
were obtained, recrystallized from ethanol/water (1:1).
4.2.1. Diethyl(4-chlorophenylamino)(2-hydroxyphenyl)
methylphosphonate (1)
Table 2
IC50(S.D.)/m
Ma of some compounds on five PTPs.
Yield: 6.84 g, 32%. Colorless crystals were obtained from DMF.
EAs: calcd/found (%) for C17H21ClNO4P$0.8C3H7NO (1$0.8DMF): C
PTP1Bb
TCPTPb
PTP-MEG2b
SHP-1b
SHP-2b
1
2
3
4
5
12
15
373(43)
91.7(16)
217(18)
137(21)
6.64(53)
135(19)
334(35)
627(61)
51.1(45)
239(12)
178(25)
14.4(22)
123(14)
178(20)
347(21)
173(10)
291(21)
146(9)
160(9)
68.6(58)
>10 mM
>10 mM
>10 mM
>10 mM
>10 mM
>10 mM
129(11)
167(16)
>10 mM
>10 mM
>10 mM
>10 mM
>10 mM
191(29)
210(24)
54.41/54.80, H 6.26/5.94, N 5.89/5.90. IR (cmꢁ1): 3290
n
(OeH,
(PeOeC), 973
102/Mꢁ1cmꢁ1): 220(162),
252(146), 278(43), 305(18), 31P NMR (D2O/ethanol, ppm):
23.72.
13C NMR (CD3OD, ppm):
16.64, 16.69, 16.87, 16.92 (eCH3), 49.90
NeH), 2982
n(CaeH), 1221 n(P]O), 1053 and 1025 n
(CeP). UV (ethanol, lmax/nm,
ε
ꢀ
d
d
(eCHeP), 64.62, 64.68, 64.71, 64.77 (OeCH2e), 116.10, 116.17,
120.91,120.93,123.53,123.56,129.82,129.86,130.22,130.24 (AreC),
147.17, 147.28 (AreCeN), 156.77 (AreCeO), 164.93 (DMF). 1H NMR
a
Triplicate.
b
IC50 > 10 mM for compounds 6e11, 13e14 and 16e17.

360
Q. Wang et al. / European Journal of Medicinal Chemistry 49 (2012) 354e364
Fig. 6. The fluorescence emission spectra of PTP1B/5 system. Insert presents the
fluorescence intensity (>) at 335 nm plotted against the ratio of 5/PTP1B, and the plot
(-) for the binding constant (K) and stoichiometry (n) determination. Conditions:
pH ¼ 7.2, lex ¼ 282 nm, 310 K.
Fig. 5. LineweavereBurk plot of 1/rate (min
m
Mꢁ1) vs the reciprocal of the p-NPP (mM)
M), 5 (B, [PTP1B] ¼ 6 M)
M), insets for relative values of Ki determination.
Fig. 7. (A) Inhibitory effect of 5 on the viability of HepG2 and MCF7 cells for 48 h
evaluated by MTT. Each value represents the mean ꢄ SD, n ¼ 6, ***p < 0.001, (5 vs
control). (B) Results of flow cytometric cellular apoptosis of HepG2 treated with 5. Each
value represents the mean ꢄ SD, n ¼ 3. *p < 0.05, **p < 0.01, ***p < 0.001, (5 vs
control).
at five fixed concentrations of compounds 2 (A, [PTP1B] ¼ 6
m
m
and 12 (C, [PTP-MEG2] ¼ 46
m

Q. Wang et al. / European Journal of Medicinal Chemistry 49 (2012) 354e364
361
(CD3OD, ppm):
d
0.95e1.12 (t, 6H, 2CH3), 6.51e7.20 (m, 8H, aromatic
4.2.6. N1,N6-Di(ethylhydrogen1-(2-hydroxyphenyl)
H), 5.20 (m, 2H, eOCH2e), 4.81 (s, 1H, NHe), 4.03 (d, 1H, eCHe).
methylenephosphonate)hexane-1,6-diamine (6)
Exact mass for 1: 369.09, ESI-MS: [1-H]ꢁ (m/z, 368.08).
Following
Ref.
[11b].
EAs:
calcd/found(%)
for
C24H38N2O8P2$2H2O (6$2H2O): C 49.65/50.08, H 7.29/7.20, N 4.83/
4.2.2. Diethyl(4-bromophenylamino)(2-hydroxyphenyl)
methylphosphonate (2)
4.95; IR (cmꢁ1): 3433
1075 & 1049 (PeOeC), 950
n(OeH, NeH), 2964 n(CaeH), 1221 n(P]O),
n
n
(CeP). UV (water, lmax/nm, ε ꢀ 102/
Yield: 2.065 g, 30%. Block colorless crystals were obtained from
ethanol. EAs: calcd/found (%) for C17H21BrNO4P (2): C 49.29/49.41,
M
ꢁ1cmꢁ1): 220(0.74), 280(0.42). 31P NMR (D2O/water, ppm):
d
10.63. 13C NMR (D2O, ppm):
d 15.92, 15.97 (eCH3), 24.66, 24.69,
H 5.11/5.07, N 3.38/3.36. IR (cmꢁ1): 3417 and 3182
n
(OeH, NeH),
24.87, 24.91, 25.06, 26.35, 39.26 (eCH2e), 46.60, 46.79 (eNHCH2e),
54.72, 55.83 (PeC), 62.27, 62.33 (OeCH2e), 116.14, 117.01, 120.43,
120.57, 129.97, 130.00, 130.52, 133.60, 137.51 (AreC), 154.70, 154.74
2982
985
n(CaeH), 1221 n(P]O), 1045 and 1011 n(PeOeC), 980 and
n
(CeP). UV (ethanol, lmax/nm, ε ꢀ 102/Mꢁ1cmꢁ1): 223(77),
253(143), 305(21), 383(0.4), 31P NMR (D2O/ethanol, ppm):
d
24.68.
(AreCeO). 1H NMR (D2O, ppm):
d 6.70e7.50 (m, 8H, aromatic H),
13C NMR (CD3OD, ppm):
d
16.64, 16.69, 16.87, 16.91 (eCH3), 49.82
3.61 (q, 4H, 2eOCH2e), 4.60 (m, 2H, 2eNHe), 3.45 (s, 2H, 2-CHe),
2.74 (m, 4H, 2-CH2e(N)e), 0.90e1.41(t, 14H, e(CH2)4 & 2-CH3).
Exact mass for 6: 544.21, ESI-MS: [6-H]ꢁ (m/z, 543.25).
(PeC), 64.64, 64.70, 64.73, 64.78 (OeCH2e), 110.64, 116.16, 116.18,
116.58, 120.92, 120.95, 123.46, 129.81, 129.85, 130.23, 130.25, 132.78
(AreC), 147.58, 147.70 (AreCeN), 156.71, 156.77 (AreCeO). 1H NMR
(CD3OD, ppm):
d
1.06e1.23 (t, 6H, 2-CH3), 6.56e7.02 (m, 8H,
4.2.7. N1,N3-Di(ethylhydrogen1-(2-hydroxyphenyl)
aromatic H), 5.31 (m, 2H, eOCH2-), 4.90 (s, 1H, NH-), 4.12 (d, 1H,
methylphosphonate)propane-1,3-diamine (7)
eCHe). Exact mass for 2: 414.23, ESI-MS: [2-H]ꢁ (m/z, 413.92).
Following
Ref.
[11a].
EAs:
calcd/found
(%)
for
C21H32N2O8P2$0.5H2O (7$0.5H2O): C 49.32/49.10, H 6.50/6.62, N
4.2.3. Diethyl(4-nitrophenylamino)(2-hydroxyphenyl)
methylphosphonate (3)
5.48/5.50; IR (cmꢁ1): 3421
n(OeH, NeH), 2979 n(CaeH), 1231 n(P]
O), 1074 & 1048 (PeOeC), 947
n
n
(CeP). UV (water, lmax/nm, ε ꢀ 102/
Yield: 1.895 g, 64%. Block yellow crystals were received from
ethanol. EAs: calcd/found (%) for C17H20N2O6P (3): C 53.83/53.97, H
M
ꢁ1cmꢁ1): 219(0.94), 276(0.51). 31P NMR (D2O/water, ppm):
d
10.22. 13C NMR (D2O, ppm):
d 15.89, 15.94 (eCH3), 21.46, 21.61
5.31/5.61, N 7.39/7.43. IR (cmꢁ1): 3273
n
(OeH, NeH), 2982
n
(CaeH),
(eCH2e), 43.40, 43.46, 43.55, 43.60 (eNHCH2e), 54.27, 55.39, 55.53
(PeC), 62.33, 62.38 (OeCH2e), 116.13, 116.35, 116.38, 116.42, 120.69,
129.95, 130.78 (AreC), 154.46, 154.50 (AreCeO). 1H NMR (CD3OD,
1230 n(P]O), 1056 and 1011 n(PeOeC), 970 n(CeP). UV (ethanol,
lmax/nm, ε ꢀ 102/Mꢁ1cmꢁ1): 220(189), 281(42), 369(215), 31P NMR
(D2O/ethanol, ppm):
d
22.37. 13C NMR (CD3OD, ppm):
d
16.65, 16.70,
ppm): d 6.70e7.20 (m, 8H, aromatic H), 4.43 (q, 4H, 2eOCH2e), 2.74
16.88, 16.92 (eCH3), 49.69 (PeC), 64.74, 64.80, 64.96, 65.02
(OeCH2e), 113.26, 116.27, 121.04, 122.87, 127.03, 130.04, 130.07,
130.65 (AreC), 139.60 (AreCeNO2), 154.59, 154.69, 156.61, 156.67
(m, 2H, 2eNHe), 3.57 (s, 2H, 2-CHe), 1.79 (m, 4H, 2-CH2e(N)e),
1.20 (q, 2H, eCH2e), 0.94e1.05 (t, 6H, 2-CH3). Exact mass for 7:
502.16, ESI-MS: [7-H]ꢁ (m/z, 501.17).
(AreCeN, AreCeO). 1H NMR (CD3OD, ppm):
d 1.08e1.27 (t, 6H, 2-
CH3), 6.74e7.49 (m, 8H, aromatic H), 5.31 (m, 2H, eOCH2e), 4.94
(s, 1H, NHe), 4.22 (d, 1H, eCHe). Exact mass for 3: 380.11, ESI-MS:
[3-H]ꢁ (m/z, 379.08).
4.2.8. N1,N2-Di(ethylhydrogen1-(2-hydroxyphenyl)
methylphosphonate)ethane-1,2-diamine (8)
Following Ref. [11b]. EAs: calcd/found (%) for C20H30N2O8P2 (8),
C 49.18/49.39, H 6.19/6.30, N 5.74/5.79; IR (cmꢁ1): 3420
n
(OeH,
4.2.4. Diethyl(2-chlorophenylamino)(2-hydroxyphenyl)
methylphosphonate (4)
NeH), 2980 n(CeH), 1228 n(P]O), 1082, 1048 n(PeOeC), 953
n
(PeC). UV (ethanol, lmax/nm, ε ꢀ 102/Mꢁ1cmꢁ1): 220(0.67),
Yield: 2.325 g, 42%. Block colorless crystals were obtained from
ethanol. EAs: calcd/found(%) for C17H21ClNO4P (4): C 55.22/55.12, H
278(0.41). 31P NMR (D2O/DMSO, ppm): 11.52. 13C NMR ((CD3)2SO,
d
ppm):
61.87 (OeCH2e), 116.98, 117.52, 121.52, 131.41, 132.59 (AreC), 157.13
(AreCeO). 1H NMR (D2O, ppm):
6.48e6.82 (m, 8H, aromatic H),
d 17.85, 17.90 (eCH3), 44.64 (PeC, eNHCH2CH2NHe), 64.49,
5.72/5.97, N 3.79/3.77. IR (cmꢁ1): 3430
n
(OeH, NeH), 2982
n
(CaeH),
1226
n
(P]O), 1035 and 1015
n
(PeOeC), 980 and 970
n
(CeP). UV
d
(ethanol, lmax/nm, ε ꢀ 102/Mꢁ1cmꢁ1): 243(42), 285(16), 212(53),
4.06 (q, 4H, 2eOCH2-), 2.77 (m, 2H, 2eNHe), 3.25 (s, 2H, 2-CHe),
1.43 (m, 4H, 2-CH2e(N)e), 0.803 (t, 6H, 2-CH3). Exact mass for 8:
488.15, ESI-MS: [8-H]ꢁ (m/z, 487.25).
31P NMR (D2O/ethanol, ppm):
d
23.25. 13C NMR (CD3OD, ppm):
d
16.64, 16.68, 16.86, 16.90 (eCH3), 50.11 (PeC), 64.81, 64.87, 64.99,
65.04 (OeCH2e), 113.92, 116.30, 119.82, 120.98, 120.99, 122.95,
122.96, 129.01, 129.29, 129.32, 130.23, 130.41, 130.44 (AreC), 143.63,
143.75 (AreCeN), 156.87, 156.93 (AreCeO). 1H NMR (CD3OD):
4.2.9. Sodium 2-((2-hydroxyphenyl)(phosphono))methylamino)
acetateN3-di(ethylhydrogen (9)
d
1.06e1.24 (t, 6H, 2-CH3), 6.56e7.02 (m, 8H, aromatic H), 5.36 (m,
Yield: 3.84 g, 66%. EAs: calcd/found (%) for C11H15NNaO6P$1.2H2O
2H, eOCH2e), 4.89 (s, 1H, NH-), 4.12 ppm (d, 1H, eCHe). Exact mass
for 4: 369.09, ESI-MS: [4-H]ꢁ (m/z, 368.03).
(9$1.2H2O): C 39.70/39.87, H 5.27/5.46, N 4.21/4.37. IR (cmꢁ1): 3442
n
(OeH, NeH), 2980 n(CaeH), 1236 n(P]O), 1071 and 1045
n
(PeOeC), 955
n
(CeP). UV (water, lmax/nm, ε ꢀ 102/Mꢁ1cmꢁ1):
4.2.5. (2-Hydroxyphenyl)(4-hydroxyphenylamino)
217(72), 276(23), 31P NMR (D2O/water, ppm):
d
14.77. 13C NMR (D2O,
methylphosphonic acid (5)
ppm): d 15.97, 16.01, 16.79 (eCH3), 49.18, 49.27 (PeC), 56.91, 57.36,
Yield 1.7134 g, 58%. EAs: calcd/found(%) for C13H14NO5P$0.5CH3
58.03 (NHeC), 62.28, 62.33 (OeCH2e), 116.22, 117.37, 118.22, 118.26,
120.13, 120.29, 129.81, 130.36, 130.40, 133.41, 134.63, 137.55 (AreC),
155.44, 155.48 (AreCeO), 174.49 (COONa). 1H NMR (D2O, ppm):
-
CH2OH (5$0.5CH3CH2OH): C 52.97/52.89, H 4.93/4.78, N 4.54/4.40. IR
(cmꢁ1): 3417 and 3442
n(OeH, NeH), 2882 n(CaeH), 1224 n(P]O),
985
n
(CeP). UV (ethanol, lmax/nm, ε ꢀ 102/Mꢁ1cmꢁ1): 232(206),
d 6.78e7.18 (m, 4H, aromatic H), 4.39 (q, 2H, eOCH2e), 3.77 (s, 1H,
270(126), 350(216), 31P NMR (D2O/ethanol, ppm):
d
2.29. 13C NMR
eCHe), 3.55 (d, 2H, eCH2e (N)e), 3.21 (m, H, eNHe), 1.05e1.11 (t,
(CD3OD, ppm):
d 49.67 (PeC), 116.76, 117.20, 117.68, 117.97, 120.30,
3H, eCH3). Exact mass for 9: 311.05, ESI-MS: [9-Na]ꢁ (m/z, 288.08).
120.34, 121.87, 123.62, 125.19, 128.81, 130.92, 133.55, 134.03,
141.13 (Ar-C), 158.55, 161.49, 162.29 (AreCeN, AreCeO). 1H NMR
4.2.10. Sodium 2-((2-hydroxyphenyl)(phosphono))methylamino)
propanoate (10)
(CDCl3, ppm):
d 6.70e7.72 (m, 8H, aromatic H), 4.85 (s, 1H, NH-),
3.15 (d, 1H, eCHe). Exact mass for 5: 295.23, ESI-MS: [5-H]ꢁ (m/z,
Yield: 4.15 g, 68%. EAs: calcd/found (%) for C12H17NNaO6P$0.7H2O
294.17).
(10$0.7H2O): C 42.66/42.68, H 5.58/5.44, N 4.15/3.98. IR (cmꢁ1):

362
Q. Wang et al. / European Journal of Medicinal Chemistry 49 (2012) 354e364
3422
n
n
(OeH, NeH), 2979
n
(CaeH), 1241
n
(P]O), 1079 and 1025
eCH2-NH-), 0.99 (q, 3H, eCH3). Exact mass for 13: 440.11, ESI-MS:
[13-Na]ꢁ (m/z, 417.17).
(PeOeC), 955 (CeP). UV (water, lmax/nm, ε ꢀ 102/Mꢁ1cmꢁ1):
217(46), 279(18), 31P NMR (D2O/water, ppm): 11.26. 13C NMR (D2O,
d
ppm):
d
15.80, 15.92, 15.96, 16.81 (eCH3), 54.36, 55.42 (PeC), 56.49,
4.2.14. Sodium 2-((ethoxyphosphono)(2-hydroxyphenyl)
57.04, 57.09, 57.35 (NHeC), 62.49, 62.54 (OeCH2e), 116.20, 116.29,
116.32, 130.42, 130.73, 130.83 (AreC), 154.85, 154.89 (AreCeO),
methylamino)-3-mercapto-propanoate (14)
Yield:
C10H13NNaO6PS$0.5H2O (14$0.5H2O): C 35.35/35.37, H 4.95/4.80, N
3.82/3.75. IR (cmꢁ1): 3412
(OeH, NeH), 2973 (CaeH), 1235 (P]
3.45
g,
51%.
EAs:
calcd/found
(%)
for
174.30 (COONa). 1H NMR ((CD3)2SO, ppm):
d 6.99e7.41 (m, 4H,
aromatic H), 3.90 (q, 2H, eOCH2e), 3.51 (m, H, eNHe), 3.85 (s,
1H, eCHe), 3.62 (m, 1H, eCH(CO)),1.43 (d, 3H, eCH3(CH2)e),1.16 (t,
3H, eCH3). Exact mass for 10: 325.07, ESI-MS: [10-Na]ꢁ (m/z,
302.08).
n
n
n
O), 1081 and 1046 n(PeOeC), 953 n(CeP). UV (water, lmax/nm,
ε ꢀ 102/Mꢁ1cmꢁ1): 278(17), 319(1.9), 31P NMR (D2O/H2O, ppm):
d
19.18. 13C NMR (D2O/CD3OD, ppm):
d 16.64, 16.69, 17.73 (eCH3),
27.08 (eCH2SH), 49.53, 58.38, 59.96 (PeC, NHeC), 63.97 (O-
CH2e), 106.78, 112.94, 115.24, 118.58, 120.64, 121.56, 123.24,
126.27, 131.20, 132.38, 137.56 (Ar-C), 155.39 (AreCeO), 171.09
4.2.11. Sodium 2-((2-hydroxyphenyl)(phosphono))methylamino)-
3-hydroxy-butanoate (11)
Yield: 3.92 g, 55%. EAs: calcd/found (%) for C13H19NNaO7P (11): C
(COONa). 1H NMR ((CD3)2SO, ppm):
d 6.56e7.19 (m, 4H, aromatic
43.95/43.99, H 5.39/5.47, N 3.94/4.12. IR (cmꢁ1): 3412
n
(OeH, NeH),
H), 4.1 (q, 2H, eOCH2e), 2.81 (m, 1H, eNHe), 3.77 (m, 1H, O]
CeCHe(N)-), 3.15 (s, 1H, eCHe), 2.53 (m, 1H, SH-CH2),1.38 (t, 1H,
eSH), 1.01 (t, 3H, eCH3). Exact mass for 14: 357.04, ESI-MS: [14-
Na]ꢁ (m/z, 334.67).
2986
754
n(CaeH), 1235 n(P]O), 1079 and 1035 n(PeOeC), 958 n(CeC),
n
(CeP). UV (H2O,lmax/nm, ε ꢀ 102/Mꢁ1cmꢁ1): 223(77), 216(63),
278(18), 31P NMR(D2O/H2O, ppm):
d
10.99, 10.76. 13C NMR (D2O,
ppm):
d 15.90, 15.95, 17.03, 19.42, 19.49, 19.89 (eCH3), 47.71
(eCH2e), 55.53 (PeC), 56.27, 56.66, 57.36, 57.78, 60.26 (NHeC),
62.36, 62.41 62.45, 62.68, 62.73 (OeCH2e), 65.21, 65.77, 65.80,
66.11, 66.17, 67.86, 67.92, 68.40, 68.43 (CHeOH), 115.53, 115.57,
115.61, 116.21, 117.01, 117.05, 120.62, 120.78, 120.85, 130.54, 130.72,
130.87, 130.97, 131.08 (AreC), 154.52, 154.62, 154.66, 154.92, 154.97
(AreCeO), 170.03, 170.80, 170.90, 171.11, 172.70 (COONa). 1H NMR
4.2.15. Sodium 2-((2-hydroxyphenyl)(phosphono))methylamino)-
3-(1H-imidazol-5-yl)- propanoate trihydrate (15)
Yield: 1.89 g, 26%. EAs: calcd/found (%) for C15H19N3NaO6P$3H2O
(15$3H2O): C 50.57/50.90, H 5.35/5.36, N 12.90/12.84. IR (cmꢁ1):
3422
n(OeH, NeH), 2986 n(CaeH), 1226 n(P]O), 1071 and 1029
n(PeOeC), 955
n
(CeP). UV (DMSO, lmax/nm, ε ꢀ 102/Mꢁ1cmꢁ1):
(D2O, ppm):
d
6.86e7.28 (m, 4H, aromatic H), 4.57 (d, 1H,
278(28), 31P NMR (D2O/H2O, ppm): 20.46. 13C NMR (D2O/CD3OD/
d
eCH(OH)eCHe), 4.13 (q, 2H, eOCH2e), 3.49 (m,1H, eNHe), 3.95 (s,
1H, eCHe), 3.79(d, 1H, eCH(CO)), 1.21 (d, 3H, eCH(OH)eCH3), 1.11
(t, 3H, eCH3). Exact mass for 11: 355.08, ESI-MS: [11-Na]ꢁ (m/z,
332.09).
(CD3)2SO, ppm): d 18.18, 22.34, 22.59 (eCH3), 49.73, 50.89, 53.17,
56.55 (PeC, NHeC, OeCH2e), 117.51, 117.74, 118.40, 122.07, 122.21,
123.47, 125.87, 125.98, 131.82, 132.22, 134.24, 134.37, 136.71 (AreC),
156.38 (AreCeO), 169.87, 170.29 (COONa). 1H NMR (D2O, ppm):
d
6.79e7.70 (m, 6H, aromatic H), 5.71 (q, 2H, eOCH2-), 4.55 (m, 1H,
4.2.12. Sodium 2-((2-hydroxyphenyl)(phosphono))methylamino)-
3-hydroxy-propanoate (12)
O]CeCHe(N)e), 3.88 (m, 1H, eNHe), 3.52 (s, 1H, eCHe), 2.94 (m,
2H, eCHeCH2e), 1.06 (t, 3H, eCH3). Exact mass for 15: 391.09, ESI-
MS: [15-Na]ꢁ (m/z, 368.07).
Yield: 3.92 g, 62%. EAs: calcd/found (%) for C12H17NNaO7P$H2O
(12$H2O): C 40.12/39.99, H 5.33/5.54, N 3.90/4.02. IR (cmꢁ1): 3442
n
n
(OeH, NeH), 2980
n
(CaeH), 1236
n
(P]O), 1071 and 1045
4.2.16. Sodium 2-((2-hydroxyphenyl)(phosphono))methylamino)-
3-phenyl-propanoate (16)
(PeOeC), 955
n
(CeP). UV (water, lmax/nm, ε ꢀ 102/Mꢁ1cmꢁ1):
223(77), 253(143), 305(21), 383(0.4), 31P NMR (D2O/H2O, ppm):
Yield: 2.37 g, 32%. EAs: calcd/found (%) for C18H21NNaO6P
d
17.97, 17.37. 13C NMR (D2O, ppm):
d 15.91, 15.95, 15.99, 16.77
(16): C 53.87/53.87, H 5.27/5.35, N 3.49/3.41. IR (cmꢁ1): 3422
(eCH3), 56.28 (PeC), 57.10, 57.36, 58.24, 58.86, 60.08, 61.12, 61.88,
61.93 (NHeC), 62.40, 62.43, 62.45, 62.48, 63.24, 63.32 (O-CH2e,
eCHe), 116.15, 116.25, 117.54, 120.42, 120.53, 130.07, 130.31,
130.45, 130.69, 130.73 (AreC), 155.35, 155.39 (AreCeO), 172.30,
n
(OeH, NeH), 2980 n(CaeH), 1228 n(P]O), 1085 and 1055
n
(PeOeC), 948
n
(CeP). UV (DMSO, lmax/nm, ε ꢀ 102/Mꢁ1cmꢁ1):
279(25), 31P NMR(D2O/H2O, ppm):
d
17.65, 16.88. 13C NMR (D2O/
CD3OD, ppm):
d 16.72, 16.77 (eCH3), 49.51, 61.35 (PeC, NHeC),
173.89 (COONa). 1H NMR (D2O, ppm):
d
6.71e7.16 (m, 4H, aromatic
64.53 (OeCH2e), 116.95, 121.90, 129.15, 129.20, 130.04, 130.14,
130.28, 130.34, 131.76, 132.83, 134.40 (AreC), 155.86 (AreCeO),
H), 4.31 (q, 2H, eOCH2e), 4.11 (d, 2H, eCH2eOH), 3.21 (m, H,
eNHe), 3.55 (s, 1H, eCHe), 2.92 (d, 2H, eCH (CO)), 1.05 (t, 3H,
eCH3). Exact mass for 12: 341.06, ESI-MS: [12-Na]ꢁ (m/z, 318.08).
170.65 (COONa). 1H NMR (D2O, ppm):
d 6.28e6.91 (m, 8H,
aromatic H), 4.06 (q, 2H, eOCH2e), 4.01 (m, 1H, O]CeCHe(N)e),
3.29 (m, 1H, eCH (CO)), 3.33 (s, 1H, eCHe), 3.14 (m, 1H, eNHe),
2.43e2.78 (m, 2H, eCH2eNHe), 1.07 (q, 3H, eCH3). Exact mass
for 16: 401.10, ESI-MS: [16-Na]ꢁ (m/z, 378.08).
4.2.13. Sodium 2-((2-hydroxyphenyl)(phosphono))methylamino)-
3-(1H-indol-3-yl)- propanoate (13)
Yield: 3.57 g, 41%. EAs: calcd/found (%) for C20H22N2NaO6P (13):
C 54.55/54.69, H 5.04/5.18, N 6.36/6.29. IR (cmꢁ1): 3413
n
(OeH,
4.2.17. Sodium 2-((2-hydroxyphenyl)(phosphono))methylamino-3-
(4-hydroxyphenyl)-propanoate (17)
NeH), 2979 n(CaeH), 1236 n(P]O), 1079 and 1043 n(PeOeC), 952
n
(CeP). UV (water, lmax/nm, ε ꢀ 102/Mꢁ1cmꢁ1): 223(77), 216(146),
Yield: 1.50 g, 20%. EAs: calcd/found (%) for C18H21NNaO7P
280(46), 31P NMR (D2O/H2O, ppm):
d
10.86. 13C NMR (D2O, ppm):
(17): C 51.80/51.89, H 5.07/5.07, N 3.36/3.43. IR (cmꢁ1): 3413
n(OeH,
d
15.94, 15.97, 16.02, 16.07, 16.80 (eCH3), 55.29, 56.31, 57.18, 57.40,
NeH), 2973 n(CaeH), 1236 n(P]O), 1072 and 1031 n(PeOeC),
58.29, 58.90, 60.12, 60.98, 61.16, 61.92, 61.97, 62.15, 62.43, 62.45,
62.48, 62.51, 62.63, 63.28, 63.36 (PeC, NHeC, O-CH2e), 116.20,
116.21, 116.29, 116.68, 116.87, 117.62, 120.44, 120.57, 120.63, 124.10,
128.88, 129.16, 129.69, 130.00, 130.09, 130.11, 130.32, 130.34, 130.45,
130.48, 130.73, 130.77 (AreC), 154.85, 154.89, 155.38, 155.42
(AreCeO), 172.32, 173.93 (COONa). 1H NMR (D2O, ppm):
951
n
(CeP). UV (DMSO, lmax/nm, ε ꢀ 102/Mꢁ1cmꢁ1): 279(31), 31P
NMR (D2O/H2O, ppm):
d
17.86. 13C NMR (D2O/CD3OD/(CD3)2SO,
ppm): d 16.75, 16.80 (eCH3), 49.71, 61.41, 64.52, 64.66, (PeC, NHeC,
OeCH2e), 114.93, 116.97, 117.22, 121.92, 129.18, 129.21, 130.06,
130.16, 130.30, 130.36, 131.79, 132.84, 134.44 (AreC), 155.86
(AreCeO), 170.65 (COONa). 1H NMR (D2O, ppm):
d 6.91e7.19 (m,
d
5.90e6.98 (m, 9H, aromatic H), 3.29 (q, 2H, eOCH2e), 2.99 (m, H,
6H, aromatic H), 4.71 (q, 2H, eOCH2e), 3.97 (s, 1H, eCHe), 3.53 (d,
1H, O]CeCHe(N)e), 3.18 (m, 1H, eNHe), 2.77 (m, 2H,
eNHe), 3.98 (s, 1H, eCHe), 3.28 (m, 1H, eCH(CO)e), 2.68 (m, 2H,

Q. Wang et al. / European Journal of Medicinal Chemistry 49 (2012) 354e364
363
eCHeCH2e), 1.18 (t, 3H, eCH3). Exact mass for 17: 417.10, ESI-MS:
After exposure, the cells in each well were added with 20
[4,5-Dimethylthiazo-2-yl]-2,5-diphenpyltetra-zolium
m
L the 3-
[17-Na]ꢁ (m/z, 394.08).
bromide
(MTT, 5 mg/mL) and incubated at 37 ꢂC for 4 h, then the medium and
4.3. X-ray crystallography
MTT were removed and 150 mL DMSO was added to each well. The
optical density (OD) was obtained at 490 nm with microplate
reader. Each data point was collected by averaging that of six wells.
The cell viability rate was calculated by the following equation:
Cell viability (%) ¼ T/C ꢀ 100.
All the data of the compounds 1e4 were collected on a Bruker
Smart Apex II diffractometer equipped with 1 K CCD instrument by
using
a graphite monochromator utilizing Mo-Ka radiation
(l
¼ 0.71073 Å) at room temperature. Cell parameters were deter-
mined using SMART software [27]. Data reduction and corrections
were performed using SAINTPlus. Absorption corrections were
made via SADABS [28]. The structuresweresolved by direct methods
with the program SHELXS-97 and refined by full-matrix least-
squares methods on all F2 data with SHELXL-97 [29]. The non-H
atoms were refined anisotropically. Hydrogen atoms attached C
were added theoretically and treated as riding on the concerned
atoms. The final cycle of full-matrix least-squares refinement was
based on observed reflections and variable parameters.
4.7. Cell apoptosis assessment
HepG2 cells were treated with 50, 100 and 300
Then they were harvested and washed twice with ice-cold 0.85%
NaCl and resuspended in 500
L of 1 ꢀ binding buffer containing
L of Annexin V-FITC and 5 L of PI (KeyGEN Biotech.). After
mM 5 for 48 h.
m
5
m
m
15 min incubation in the dark at room temperature, cells were
analyzed for annexin V binding within 1 h with a flow cytometer.
The number of viable, early apoptotic, late apoptotic and necrotic
cells were quantitated by annexin V bindng and PI uptake.
4.4. PTPs inhibition assay
Acknowledgments
Five human PTPs (PTP1B, TCPTP, PTP-MEG2, SHP-1 and SHP-2)
were expressed and purified as described previously [30].
This work was supported financially by the National Natural
Science Foundation of China (21001070 and 21171109), the Scien-
tific Research Foundation for the Returned Overseas Chinese
Scholars, State Education Ministry (20093602), the Natural Science
Foundation of Shanxi Province (2010011011-2, 2011011009-1 and
2011021006-2), Graduate Innovation Foundation of Shanxi Prov-
ince of China and Instrumental Analysis Foundation of Large Scale
Instruments of Shanxi University of China.
The inhibitory effects and Kinetics analysis of the
a-amino-
phosphonates against PTPs were measured similarly as described
previously, using p-nitrophenol phosphate (p-NPP) as the substrate
[11,25,30]. Briefly, the assays were performed on a 96-well plate in
20 mM MOPS buffer, pH 7.2, containing 50 mM NaCl. 10
compound at various concentrations was mixed with PTPs solution
(82 L of p-NPP (0.1 M) substrate was
L) for 35 min at 37 ꢂC. Then 2
added. After incubation for about 30 min, the assays were termi-
nated by the addition of 6 L of 2 M NaOH. The A405 was measured
mL of the
m
m
m
Appendix. Supplementary data
on a microplate reader. IC50 values were obtained by fitting the
concentration-dependent inhibition curves using the Origin
program (OriginLab, Northampton, MA).
Supplementary data related to this article can be found online at
doi:10.1016/j.ejmech.2012.01.038.
The inhibiting kinetic analysis was carried out according to the
LineweavereBurk plot analysis. Phosphatase activities were
measured at a fixed enzyme (PTP1B and PTP-MEG2) concentration
while concentrations of the substrate (p-NPP) and the inhibitor
(compound 2, 5 & 12) were varied. The data were fitted using Origin
program to generate the LineweavereBurk plot.
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