Calix[4]arene methylenebisphosphonic acids as inhibitors of protein tyrosine phosphatase 1B

Article abstract of DOI:10.1016/j.bmcl.2013.08.040

Calix[4]arenes bearing methylenebisphosphonic or hydroxymethylenebisphosphonic acid fragments at the wide rim of the macrocycle were studied as inhibitors of PTP1B. Some of the inhibitors showed IC ₅₀ values in the micromolar range and good sel

Full text of DOI:10.1016/j.bmcl.2013.08.040

Accepted Manuscript
Calix[4]arene methylenebisphosphonic acids as inhibitors of protein tyrosine
phosphatase 1B
Viacheslav V. Trush, Sergiy O. Cherenok, Vsevolod Yu. Tanchuk, Valery P.
Kukhar, Vitaly I. Kalchenko, Andriy I. Vovk
PII:
S0960-894X(13)00969-4
DOI:
http://dx.doi.org/10.1016/j.bmcl.2013.08.040
BMCL 20778
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
4 July 2013
5 August 2013
7 August 2013
Please cite this article as: Trush, V.V., Cherenok, S.O., Tanchuk, V.Y., Kukhar, V.P., Kalchenko, V.I., Vovk, A.I.,
Calix[4]arene methylenebisphosphonic acids as inhibitors of protein tyrosine phosphatase 1B, Bioorganic &
Medicinal Chemistry Letters (2013), doi: http://dx.doi.org/10.1016/j.bmcl.2013.08.040
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Calix[4]arene methylenebisphosphonic acids as inhibitors of protein
tyrosine phosphatase 1B
a
b
a
a
Viacheslav V. Trush , Sergiy O. Cherenok , Vsevolod Yu. Tanchuk , Valery P. Kukhar , Vitaly I.
b,
a,
Kalchenko *, Andriy I. Vovk *
a
Institute of Bioorganic Chemistry and Petrochemistry, National Academy of Sciences of Ukraine,
Murmanska, 1, 02660, Kyiv-94, Ukraine
b
Institute of Organic Chemistry, National Academy of Sciences of Ukraine,
Murmanska, 5, 02660, Kyiv-94, Ukraine
AB STRACT
ꢀ
alix[4]arenes bearing methylenebisphosphonic or hydroxymethylenebisphosphonic acid fragments
at the wide rim of the macrocycle were studied as inhibitors of PTP1B. Some of the inhibitors
showed IC50 values in the micromolar range and good selectivity in comparison with other protein
tyrosine phosphatases such as TC-PTP, PTPꢁ, LAR, and CD45. Kinetic studies indicated that the
calix[4]arene derivatives influence PTP1B activity as slow-binding inhibitors. Based on molecular
docking results, the binding modes of the macrocyclic bisphosphonates in the active centre of
PTP1B are discussed.
Keywords: Protein tyrosine phosphatase 1B, methylenebisphosphonic acid, calixarene, inhibition,
molecular docking.
*
Corresponding author. Tel.: +38 044 573 2553; fax: +38 044 573 2552 (A.I. Vovk).
E-mail addresses: vik@ioch.kiev.ua (V.I. Kalchenko), vovk@bpci.kiev.ua (A.I. Vovk).

ꢀ
Protein tyrosine phosphatases regulate a number of biochemical processes which proceed
through dephosphorylation of phosphotyrosine residues in proteins, including cell growth,
1
-3
differentiation, metabolism, and movement. Because of importance of protein phosphorylation-
dephosphorylation cycle in living cells, the activities of these enzymes are associated with diabetes,
4
-6
obesity, cancer and other human diseases. Protein tyrosine phosphatase 1B belongs to the
superfamily of protein tyrosine phosphatases and involves in insulin and leptin signal transduction
by dephosphorylation of activated insulin receptor or leptin receptor-associated kinase, JAK2. As a
negative regulator of insulin and leptin signalling, PTP1B is one of the most promising therapeutic
7
,8
targets for treatment of type 2 diabetes.
With increasing understanding of the physiologic role of PTP1B, there is growing interest in
2
,6,9
10
11
designing new inhibitors of this enzyme.
Derivatives of carboxylic, sulfonic acids,
have been identified as PTP1B inhibitors. As bioisosteric
analogues of phosphotyrosine, phosphonic acid derivatives can also be potent inhibitors of
12,13
14-16
heterocyclic
and other compounds
1
7,18
PTP1B.
The biological properties of synthetic phosphonic acids as well as therapeutic potential
1
9,20
of bisphosphonates
demonstrate practical relevance in studying new bioactive phosphonate
compounds. Recent studies have identified the bisphosphonates as possible inhibitors of protein
tyrosine phosphatase in osteoblastic cells, and this enzyme is supposed to be involved in the
21
regulation of the osteoblast proliferation.
Calixarenes represent macrocyclic molecules recently shown to serve as molecular scaffolds
2
2,23
for the design of biologically active compounds.
They have been employed for the covalent
2
4,25
attachment of relevant functional groups
to achieve high affinity for a protein of pharmaceutical
26,27
interest.
For example, some of these functionalized macrocycles exhibit antimicrobial
2
8,29
30,31
activities
and are able to inhibit HIV and HCV infection.
ꢀalixarenes have also been studied
32
33
as inhibitors of histone deacetylase and ꢂ-chymotrypsin. We reported that preorganizing the
phosphonic or methylenebisphosphonic acids into calixarene derivatives provides an efficient
3
4
35
approach for designing inhibitors of alkaline phosphatases, glutathione-S-transferase, and
36
Yersinia protein tyrosine phosphatase. Extending this work, we hypothesized that functionalized
anionic calixarenes might be interesting compounds to be investigated for their complexation with
PTP1B. It should be mentioned that PTP1B exhibit a preference for bis- and triphosphorylated
3
7
peptides in comparison with monophosphorylated ones which were used as the enzyme substrates.
The present research was undertaken to evaluate in vitro the inhibitory activity of
methylenebisphosphonate and hydroxymethylenebisphosphonate derivatives of calix[4]arene
toward PTP1B. We also investigated how these bisphosphonate inhibitors may influence activity of
other human protein tyrosine phosphatases.

ꢁ
ꢀ
alix[4]arene methylenebisphosphonic acids 1 and 2 (Fig. 1) were synthesized according to
3
4
previously developed synthetic protocols. The ꢂ-hydroxymethylenebisphosphonic acids 9 and 10
38-40
were prepared by a simple and efficient one-pot procedure
starting from calix[4]arene
carboxylic acids 3 and 4 (Scheme 1).
Figure 1
Scheme 1
In the first stage, the acids 3 and 4 were converted to corresponding calixarene acyl chlorides
4
1
5
and 6 by reaction with thionyl chloride. In the second stage, the Arbusov reaction of 5 and 6
with tris(trimethylsilyl)phosphite led to formation of ꢂ-ketophosphonic acid silyl esters 7 and 8,
which then transformed into ꢂ-hydroxymethylenebisphosphonic acid silyl esters 9 and 10. Finally,
in the third stage, silyl esters 9 and 10 were converted into free calixarene ꢂ-
42
1
hydroxymethylenebisphosphonic acids 11 and 12 by treatment with methanol (Scheme 1). The H
NMR spectra of calix[4]arene derivatives 11 and 12 display AB spin system doublets of axial and
equatorial protons for the methylene bridges (∆ꢃ = 0.75 – 0.85 ppm, JHH = 13 Hz) indicating C2v-
43
symmetrical pinched cone conformation of the macrocyclic skeleton.
Commercially available recombinant forms of truncated or full length PTP1B were used to
evaluate in vitro inhibitory effects of compounds 1, 2, 11, and 12. The employed substrate was p-
nitrophenyl phosphate or phosphorylated insulin receptor fragment 1142-1153 (pTyr1146) with
amino acid sequence Thr-Arg-Asp-Ile-Tyr[PO
3 2
H ]-Glu-Thr-Asp-Tyr-Tyr-Arg-Lys. The enzymatic
activities were determined by following the changes in concentrations of p-nitrophenol or inorganic
4
4
phosphate released during enzymatic hydrolysis of the substrate. Value of IC50 calculated from a
dose-dependent curve was concentration of the tested compound which decreased the enzyme
activity to 50% after 10 min of incubation of reaction mixture. An apparent K value of 1.7 ± 0.3
m
mM was found for p-nitrophenyl phosphate, and it was reported to be of 0.75 mM by using
4
5
pTyr1146.
Under assay conditions, the inhibition resulted in significant reduction of PTP1B activity at
micromolar concentrations of macrocyclic phosphonate inhibitors 1, 2, 11, and 12. Structurally
more simple bisphosphonates such as 50 ꢄM alendronate (4-amino-1-hydroxybutylidene 1,1-
bisphosphonate)
did
not
exhibit
inhibition
effect,
and
25,27-dipropoxy-26,28-
dihydroxycalix[4]arene backbone did not change the PTP1B activity at the concentrations used (up
to 20 ꢄM). The protected analogues of the calix[4]arene methylenebisphosphonic acids (diethyl
esters of 1 and 2) were also not active. Thus, the higher inhibitory activities of calix[4]arene
methylenebisphosphonates compared to the model compounds can be attributed to synergistic

ꢂ
effects of methylenebisphosphonate fragment and macrocyclic scaffold involved in the interaction
with PTP1B.
Calix[4]arene methylenebisphosphonic acids 1 and 11 bearing one methylenebisphosphonate
or ꢂ-hydroxymethylenebisphosphonate fragment at the wide rim of the macrocycle showed the
highest affinities for the enzyme with IC50 of 1.2 and 1.9 ꢄM, respectively. Introduction of the
second bisphosphonate group into the structure of calix[4]arene leads to analogs 2 and 12 being
slightly less effective inhibitors as compared to the mono-substituted analogues. The presence of
hydroxy groups in the molecule of bis-methylenebisphosphonate 12 can be the reason for its
somewhat lower activity compared to 2 (Table 1). All phosphorylated calix[4]arenes showed
approximately the same effects on PTP1B in reactions containing p-nitrophenyl phosphate or
pTyr1146 as the enzyme substrates. In the last case the IC50 values were found to be 3.7, 3.9, 4.1,
and 7.3 ꢄM for compounds 1, 2, 11, and 12, respectively.
Table 1
Although many of mammalian protein tyrosine phosphatases are characterized by highly
conserved active sites, the bulky macrocyclic methylenebisphosphonate inhibitor appears to be
capable of selective binding to the active center of some of these enzymes. As is seen from Table 1,
the inhibitory activities of compounds tested toward PTP1B are considerably higher than toward
LAR. The other phosphatases examined in this study were inhibited by all calix[4]arene derivatives
with IC50 in the range of 1.8 - 59 ꢄM (Table 1). The macrocyclic inhibitors of PTP1B showed good
selectivity over PTPꢁ (with exception of bis-methylenebisphosphonate 12) and 4-9-fold selectivity
over CD45. Mono-substituted calix[4]arenes 1 and 11 exhibited 8- and 14-fold selectivity over TC-
PTP, which has a high degree of similarity to PTP1B. Bis-substituted compound 2 and 12 showed
only modest selectivity over this enzyme. At the same time, compounds 1 and 11 were found to be
also potent inhibitors of SHP2 which is considered as the first oncogene in the superfamily of
16,46
protein tyrosine phosphatases.
Kinetic evaluation of calix[4]arene methylenebisphosphonates as inhibitors of PTP1B
showed a time-dependent decrease in the reaction rate during the first minutes of product
accumulation. This feature was also observed for TC-PTP, but inhibition of SHP2 was
characterized by linear kinetics. Progress curves were analyzed for the inhibition of PTP1B by
compounds 1 and 11. The reaction was initiated by adding the enzyme to a mixture of substrate and
4
7
inhibitor being in large excess over PTP1B concentration. A pre-steady-state phase was followed
by an equilibrium establishment, and release of product became linear with time. Additional
experiments with dilution of the reaction mixture to reduce the inhibitor concentration showed
partial restoration of the enzyme activity.

ꢃ
48
Two basic mechanisms could explain the reversible slow-binding inhibition. Two-step
mechanism assumes rapid formation of initial enzyme-inhibitor complex that slowly transforms into
the more tightly bound complex. However, the inhibition can be formally treated as a single-step
process if the inhibitor binding is slow and the formation of modified complex is fast. In this case
the initial velocity is independent of inhibitor concentration. The shapes of the curves in Fig. 2A
show that the initial velocity of p-nitrophenol production is practically the same at fixed
concentration of the substrate and various concentrations of the inhibitor. These data indicate that
the slow PTP1B inhibition by calix[4]arene derivatives can be described by a one-step mechanism
of formation of enzyme-inhibitor complex.
Using the experimental kinetic data and the equation for the reversible pseudo-first-order
association of the inhibitor with the enzyme, the observed pseudo-first-order rate constants kobs were
calculated from the slope of a linear plot of ln(P'-P) at 20 s interval against time. According to
possible mechanism of slow binding of the inhibitor to the enzyme (Scheme 2), kobs may be gived
48
by the equation
k
obs = k-3 + k
3
[I] / (1+ [S] / K
and k-1 are association and dissociation rate constants for reversible complex formation
between PTP1B and phosphorylated macrocycle, and K = (k-1 + k ) / k . The values of the rate
constants were calculated from linear plot of kobs against concentration of inhibitor 1 (Fig. 2B),
m
),
where k
1
m
2
1
3
-1 -1
-1
giving k
data for inhibitor 11 gave following values of the constants: k
= 0.68 ꢄM.
3
= 3.3·10 M s , k-3 = 0.0024 s and inhibition constant K
i
= 0.74 ꢄM. Fitting experimental
3
-1 -1
-1
3
= 2.9·10 M s , k-3 = 0.0020 s , and
K
i
Figure 2
In the case of macrocyclic phosphonate 1, the steady-state phase of kinetics appears to be
applicable to calculate the inhibition constant by means of Lineweaver-Burk plot analysis. The
slopes of the straight lines from Fig. 3 linearly depend on the inhibitor concentration. Thus, the
influence of inhibitor 1 on the activity of PTP1B is in agreement with a competitive type. Inhibition
constant determined in such a way was 0.98 ꢄM. These results correspond to the mechanism
consisting of slow binding of the inhibitor in the substrate binding site of PTP1B.
Figure 3
Scheme 2
To analyze the binding mode of macrocyclic phosphonate inhibitors in the active site of
49
PTP1B, computer-simulated docking studies were performed using AutoDock 4.2. X-ray
crystallographic analysis of PTP1B with different ligands reveals significant differences in the
enzyme binding site conformations. In general, PTP1B is represented by more than one hundred

ꢄ
PDB files in the RSCB Protein Data Bank and it is not possible to use all existing crystal structures
of PTP1B for the docking procedure. To find the appropriate structure, we utilized the reported
50
early approach of clustering all available conformations based on similarity of binding profile.
Five centroids, namely 1NL9 and 1PH0 representing structures with open WPD loop and 2CNF,
Q6M, and 2CM8 with closed WPD loop were used as the starting points for docking of
1
compounds 1, 2, 11, and 12. The selection of a particular X-ray crystal structure among the
centroids was done by considering the binding free energy values (ꢅGdoc) and experimental
activities of inhibitors. As a result, binding models of the inhibitors with PTP1B were predicted by
51
using the enzyme structure in complex with isothiazolidinone inhibitor (PDB entry 2CNF). The
ligand was preliminary removed from the binding site of the enzyme, and then calix[4]arene
phosphonate inhibitors in monoanionic forms were examined to explore their binding modes.
The predicted free energies ꢅGdoc for all inhibitors varied between -6.54 and -8.90 kcal/mol
which is in agreement with experimentally obtained activity data. The docking calculations reveal
that
compounds
1,
2,
and
11
exhibit
stronger
binding
affinity
than
bis(hydroxymethylenebisphosphonate) 12. The calix[4]arene are characterized by flexibility at the
level of aromatic cores, and therefore different conformations of macrocyclic inhibitors have been
achieved by their interactions with amino acid environment of the enzyme active site. In the
docking models, all of calix[4]arene derivatives adopt flattened cone conformations with angles
o
between two opposite rings of approximately 110 , while other two aromatic rings are almost
parallel to each other. All macrocyclic inhibitors were observed to be capable of occupying the
active region with interfering with amino acid residues around catalytic Cys215 located in the
binding cavity. The phosphonate oxygens of both phosphonate groups of methylenebisphosphonate
fragment were positioned to make P(O)···H-O-hydrogen bond to Asp181 and P(O)···H-N hydrogen
bonds to Arg221, Ser216, Ala217, and Gly220. Closed conformation of WPD loop provides the
interaction of Phe182 with the upper rim of the macrocycle. In addition, hydrophobic interactions of
propoxy groups on the lower rim of calix[4]arene platform were observed with Lys120, Tyr46,
Arg47, and Asp48. In case of ꢂ-hydroxymethylenebisphosphonates 11 and 12 additional
interactions were found between the oxygen of the ꢂ-hydroxy group and the residue of Gly220. The
second bisphosphonate groups of compound 2 and 12 seem not to be essential for the inhibitor
activity being directed outside from the surface of the active site cavity (Fig. 4).
Figure 4
In
summary,
we
established
that
calix[4]arene
macrocycles
bearing
methylenebisphosphonate or ꢂ-hydroxymethylenebisphosphonate groups on the wide rim exhibit
inhibition of PTP1B and show selectivity over some of other phosphatases. The bisphosphonate

ꢅ
groups as well as macrocyclic structure are essential for the activities of these macrocyclic
compounds. This data provides a basis for further development of calixarene inhibitors of PTP1B
which might be considered as potential drugs.
Acknowledgements
This research was supported by the State Program “Nanotechnology and nanomaterials”
(grants of NAS of Ukraine 5.18.2.8 and 5.16.1.2).

ꢆ
O
P
O
P
O
P
O
P
O
P
Me SiO
OSiMe3
HO
HO
OH
OH
OSiMe3
OSiMe3
3
O
OH
O
Cl
O
OSiMe3
Me SiO
3
OH
OH
OH
OPr PrO
OH
OH
OPr PrO
OH
OH
OPr PrO
OH
OH
OPr PrO
OH
c
OH
OPr PrO
OH
d
a
b
9
11
7
3
5
O
P
O
O
O
O
OSiMe3
OSiMe
HO
OH
OH
OSiMe3
OSiMe3
Me SiO
3
O
OH
O
Cl
O
P
P
P
P
3
HO
Me SiO
3
OH
OH
OH
OPr PrO
OH
OH
OPr PrO
OH
OH
OPr PrO
OH
d
OH
OPr PrO
OH
OH
OPr PrO
OH
b
c
a
OH
OH
OH
OH
OSiMe3
HO
HO
Me SiO
OSiMe3
3
P
P
P
P
P
O
OSiMe3
HO
O
Cl
O
OSiMe3
Me SiO
3
O
O
O
O
O
1
2
4
6
8
10
Scheme 1. Synthesis of calix[4]arene ꢂ-hydroxymethylenebisphosphonic acids. Reagents and
conditions: (a) SOCI , toluene; (b, c) (Me SiO) P, THF; (d) MeOH.
2
3
3

ꢇ
k1
k
2
E + S
ES
E + P
k -1
slow k [I] k -3
3
EI
Scheme 2. Kinetic scheme for possible one-step mechanism of time-dependent inhibition of PTP1B
by calix[4]arene methylenebisphosphonates 1 and 11.

ꢈꢉ
O
P
O
P
HO
HO
OH
OH
O
P
O
P
HO
HO
OH
OH
OH
OPr PrO
OH
OH
OPr PrO
OH
HO
HO
OH
OH
P
P
O
O
1
2
Figure 1. Chemical structures of calix[4]arene methylenebisphosphonic acids 1 and 2. At
-
-
physiological pH, the phosphonic acid derivatives exist in P(O)(OH)O and partially in P(O)(O )
2
anionic forms called as phosphonates.

ꢈꢈ
0
0
0
.25
A
0
.2
.15
0
.1
.05
0
0
200
400
600
800
t, s
B
0.007
0.006
0.005
0.004
0.003
0.002
0.001
0
0
1
2
3
[
I], µM
Figure 2. (A) Progress curves for the hydrolysis of p-nitrophenyl phosphate (2mM) catalyzed by
PTP1B in absence of the inhibitor (upper curve) and in presence of 0.5, 1.0, 1.5, and 2.5 ꢄM
calix[4]arene methylenebisphosphonate 1. (B) Dependence of kobs for the slow enzyme-inhibitor
association on the inhibitor concentration.

ꢈꢀ
2
1
1
0
0
.0
.5
.0
.5
.0
-
0.6
-0.1
0.4
0.9
1.4
1.9
2.4
-
1
1
/[S], mM
Figure 3. Lineweaver-Burk graphical representations of PTP1B inhibition by calix[4]arene
methylenebisphosphonate 1 using the steady-state velocities. p-Nitrophenyl phosphate was used as
a substrate. The inhibitor concentrations were 0 (ꢆ), 0.6 (ꢇ), 1.2 (ꢈ), 1.8 (∆) ꢄM.

ꢈꢁ
Figure 4. Interaction of calix[4]arene methylenebisphosphonate 1 with adjacent amino acid
residues (top panel). Possible binding mode of bis-methylenebisphosphonate 2 to the active site of
PTP1B (bottom panel).

ꢈꢂ
Table 1.
Experimental inhibitory activity (IC50, ꢄM) of calix[4]arene methylenebisphosphonic acids 1, 2, 11,
a
and 12 as inhibitors of ꢉꢊꢉ1ꢋ and other protein tyrosine phosphatases.
Inhibitor
PTP1B
1.2 ± 0.2
2.5 ± 0.2
TC-PTP
9.3 ± 1.9
4.9 ± 1.5
PTPꢁ
13 ± 1
32 ± 7
CD45
5.7 ± 0.9
22 ± 6
SHP2
LAR
>100
>100
≥1 00
1
2
1.8 ± 0.5
2.6 ± 0.6
3
.4 ± 0.3
1
1
2
1.9 ± 0.2
8.3 ± 1.1
27 ± 6
22 ± 2
18 ± 1
12 ± 2
8.4 ± 2.1
59 ± 10
1
9.2 ± 0.4
>100
a
IC50
values are the means of three assays ± standart deviations. Concentrations of p-nitrophenyl
phosphate were 2 mM (PTP1B, TC-PTP, LAR), 1 mM (PTPꢁ), 5 mM (CD45), and 7 mM (SHP2).

ꢈꢃ
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9. Patel, M. B.; Modi , N. R., Raval, J. P.; Menon, S. K. Org. Biomol. Chem. 2012, 10, 1785.
0. Tsou, L. K.; Dutschman, G. E.; Gullen, E. A.; Telpoukhovskaia, M.; Cheng, Y.-C.; Hamilton,
A. D. Bioorg. Med. Chem. Lett. 2010, 20, 2137.
3
3
3
3
3
3
1. Mourer, M.; Psychogios, N.; Laumond, G.; Aubertin, A.-M.; Regnouf-de-Vains, J.-B. Bioorg.
Med. Chem. 2010, 18, 36.
2. Chini, M. G.; Terracciano, S.; Riccio, R.; Bifulco, G.; Ciao, R.; Gaeta, C.; Troisi, F.; Neri, P.
Org. Lett. 2010, 12, 5382.
3. D'Acquarica, I.; Cerreto, A.; Delle Monache, G.; Subrizi, F.; Boffi, A.; Tafi, A.; Forli, S.;
Botta, B. J. Org. Chem. 2011, 76, 4396.
4. Vovk, A.; Kalchenko, V.; Cherenok, S.; Kukhar, V.; Muzychka, O.; Lozynsky, M. Org.
Biomol. Chem. 2004, 2, 3162.
5. Cherenok, S. O.; Yushchenko, O. A.; Tanchuk, V.Yu.; Mischenko, I. M.; Samus, N. V.;
Ruban, O. V.; Matvieiev, Y. I.; Karpenko, J. A.; Kukhar, V. P.; Vovk, A. I.; Kalchenko, V. I.
Arkivok 2012, 4, 278.
3
3
3
6. Vovk, A.I.; Kononets, L.A.; Tanchuk, V.Yu.; Cherenok, S.O.; Drapailo, A.B.; Kalchenko, V.I.;
Kukhar, V.P. Bioorg. Med. Chem. Lett. 2010, 20, 483.
7. Salmeen, A.; Andersen, J. N.; Myers, M. P.; Tonks, N. K.; Barford, D. Mol. Cell. 2000, 6,
1
401.
8. Lecouvey, M.; Mallard, I.; Bailly, T.; Burgarda, R.; Leroux, Y. Tetrahedron Lett. 2001, 42,
475
8
3
9. Migianu, D. M.; Mallard, I.; Bouchemal, N.; Lecouvey, M.; Tetrahedron Lett. 2004, 45, 4511.
0. Migianu-Griffoni, E.; Mbemba, C.; Burgada, R.; Lecercle, D.; Taran, F.; Lecouvey, M.
Tetrahedron, 2009, 65, 1517.
4
41. General procedure for calixarene acyl chlorides 5 and 6. Thionyl chloride (0.2 or 0.3 mmol)
was added to a suspension of calixarene carboxylic acid 3 (0.1 mmol) or 4 (0.1 mmol),

ꢈꢅ
respectively, in toluene (20 mL) under argon. One drop of DMF was added to the reaction
o
mixture as a catalyst. The resulting reaction mixture was heated at 100 C for 2 hours. The
solvent was distilled off under reduced pressure and the residue was washed with hexane to
afford a colourless solid. The obtained acyl chlorides 5 and 6 were used in the next steps
without further purification.
4
2. General procedure for the preparation of calixarene hydroxymethylenebisphosphonic acids 11
and 12. Tris(trimethylsilyl)phosphite (4 mmol or 8 mmol, respectively) was added under
argon to a solution of calixarene acyl chloride 5 or 6 (0.1 mmol) in dry THF (20 mL). The
o
reaction mixture was stirred at 35 C for 8 h. After evaporation of volatile fractions under
reduce pressure, the crude product was stirred with methanol at room temperature (2 h). After
evaporation of solvent under vacuum, the obtained compound was washed with diethyl ether.
Yields 80-86 %.
5-[1-Hydroxy-1,1-bis(dihydroxyphosphoryl)methyl]-25,27-dipropoxycalix[4]arene 11. White
o
1
solid; yield 80%; M.p. 165-167 C. H NMR (ꢌꢍꢀꢎ-d
6
) δ: 8.41, 8.47 (two s, 1H + 1H, ArOH
); 7.19 (s, 2 H, m-ArH), 7.09 (m, 2H + 2H + 2ꢏ, m-ArH); 6.51, 6.73 (two d, 2H + 2H, J 7.5 Hz,
2 2 3
ꢀ-ArH), 4.18 (d, 4H, J 13.2 Hz, ArCH2ax), 3.93 (m, 4H, OCH CH CH ), 3.25, 3.31 (two d, 4H,
31
J 13.2 Hz, ArCH2eq), 2.06 (m, 4H, OCH
2
CH
2
CH
3
), 1.35 (t, 6H, J 7.5 Hz, OCH
2
CH
2
3
CH ). P
+
NMR (ꢌꢍꢀꢎ-d ),
6
ꢐ
δ: 19.9; m/z (FAB MASS) 700 ([M+H] , 75 %). Calculated M 698.6.
5,17-Bis[1-hydroxy-1,1-bis(dihydroxyphosphoryl)methyl]-25,27-dipropoxycalix[4]arene 12.
o
1
White solid; yield 86%. m.p. 187-189 C. H NMR (ꢌꢍꢀꢎ-d
wide s, 4H, ArH); 7.23 (wide s, 4H, ArH); 6.79 (wide s, 2H, ArH); 4.23 (d, 4H, J 13.2 Hz,
ArCH2ax); 3.98 (t, 4H, J 7.5 Hz, OCH CH CH ), 3.36 (d, 4H, J 13.2 Hz, ArCH2eq), 2.05 (m,
CH CH CH O), 1.24 (t, 6ꢏ, J 7.5 Hz, CH CH
90 [M+H] . Calculated ꢍ 888.6
6
) δ: 8.4 (wide s, 2H, PhOH); 7.63
(
2
2
3
3
1
3
2
2
2
3 6
), P NMR (ꢌꢍꢀꢎ-d ) δ19.6. MS (FAB) m/z;
+
8
4
4
3. Grootenhuis, P. D. J.; Kollman, P. A.; Groenen, L. C.; Reinhoudt, D. N.; van Hummel, G. J.;
Ugozzoli, F.; Andreetti, G. D. J. Am. Chem. Soc. 1990, 112, 4165.
4. An accumulation of inorganic phosphate was registered by means of protein tyrosine
phosphatase assay kit. Reaction mixture (0.2 mL) contained 50 mꢍ Bis-Tris (ꢑꢏ 7.2), 1.1 mM
DTT, 0.1 M NaCl, 1 mM EDTA, 0.075 mM pTyr1146, and 40 nM recombinant truncated
ꢉꢊꢉ1ꢋ. Reaction was started by addition of the enzyme. Each test was incubated for 20 min at
o
3
7 ꢀ and then stopped by addition of 50 µL of malachite green solution. The mixture kept for
o
1
0 min at 37 ꢀ followed by adding of 0.25 mL of the twice-distilled water to the final volume
of 0.5 mL. Amount of inorganic phosphate was determined from the standard curve by

ꢈꢆ
measuring the absorbance of solution at 620 nm. Values of absorption in absence of enzyme or
substrate and at presence of inhibitors were taken into account.
45. Maccari, R.; Paoli, P.; Ottana, R.; Jacomelli, M.; Ciurleo, R.; Manao, G.; Steindl, T.; Langer,
T.; Vigorita, M. G.; Camici, G. Bioorg. Med. Chem. 2007, 15, 5137.
4
4
6. Jiang, Z.-X.; Zhang, Z.-Y. Cancer metastasis rev. 2008, 27, 263.
7. The assay solution contained 50 mM Bis-Tris (pH 7.2), 1 vol % of dimethyl sulfoxide, 100
mM NaCl, 1mM DTT, 2mM EDTA, 2 mM p-nitrophenyl phosphate and inhibitor. The mixture
ꢆ
was preincubated for 5 min at 37 C and reaction was initiated by addition of recombinant full
length PTP1B (5 nM in reaction mixture). The generation of p-nitrophenol was measured by
increase of absorbance at 410 nm.
4
4
8. Golicnik, M.; Stojan, J. Biochem. Mol. Biol. Educ. 2004, 32, 228.
9. Morris, G. M.; Huey, R.; Lindstrom, W.; Sanner, M. F.; Belew, R. K.; Goodsell, D. S; Olson,
A. J. J. Comput. Chem. 2009, 16, 2785.
5
5
0. Tanchuk, V. Y.; Tanin, V. O.; Vovk, A. I. Chem. Biol. Drug Des. 2012, 80, 121.
1. Ala, P. J.; Gonneville, L.; Hillman, M.; Becker-Pasha, M.; Yue, E.W.; Douty, B.; Wayland, B.;
Polam, P.; Crawley, M. L.; McLaughlin, E.; Sparks, R. B.; Glass, B.; Takvorian, A.; Combs,
A. P.; Burn, T. C.; Hollis, G. F.; Wynn, R. J. Biol. Chem. 2006, 281, 38013.

ꢈꢇ
Graphical abstract