Calix[4]arene methylenebisphosphonic acids as calf intestine alkaline phosphatase inhibitors

Article abstract of DOI:10.1039/b409526j

Calix[4]arenes bearing one or two methylenebisphosphonic acid fragments were prepared via addition of diethylphosphite to the parent calix[4]arene aldehydes. The resulting compounds displayed stronger inhibition of calf intestine alkaline phosphatase than simple methylenebisphosphonic or 4-hydroxyphenyl methylenebisphosphonic acids. The action of these phosphorylated calix[4]arenes is concordant with partial mixed-type inhibition. The inhibition constants K_i and K_i′ for the calix[4]arene bis(methylenebisphosphonic) acid in Tris-HCl buffer at pH 9 are 0.38 μM and 2.8 μM respectively. The replacement of the phosphoric acid moieties on the macrocycle with diethylphosphonates results in a sharp decrease of its inhibitory action. Preorganizing phosphonic acid fragments using a calixarene platform therefore provides a promising approach for the design of efficient alkaline phosphatase inhibitors.

Full text of DOI:10.1039/b409526j

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A R T I C L E
Calix[4]arene methylenebisphosphonic acids as calf intestine
alkaline phosphatase inhibitors
Andriy I. Vovk,*a Vitaly I. Kalchenko,b Sergey A. Cherenok,b Valery P. Kukhar,a
Oxana V. Muzychkaa and Myron O. Lozynskyb
a
Institute of Bioorganic Chemistry and Petrochemistry, NAS of Ukraine, 02094, Kyiv-94,
Ukraine. E-mail: vovk@bpci.kiev.ua; Fax: +38-044-573 2552
Institute of Organic Chemistry, NAS of Ukraine, 02094, Kyiv-94, Ukraine
b
Received 24th June 2004, Accepted 10th September 2004
First published as an Advance Article on the web 5th October 2004
Calix[4]arenes bearing one or two methylenebisphosphonic acid fragments were prepared via addition
of diethylphosphite to the parent calix[4]arene aldehydes. The resulting compounds displayed stronger
inhibition of calf intestine alkaline phosphatase than simple methylenebisphosphonic or 4-hydroxyphenyl
methylenebisphosphonic acids. The action of these phosphorylated calix[4]arenes is concordant with partial mixed-
type inhibition. The inhibition constants Ki and Ki for the calix[4]arene bis(methylenebisphosphonic) acid in Tris-
HCl buffer at pH 9 are 0.38 lM and 2.8 lM respectively. The replacement of the phosphoric acid moieties on the
macrocycle with diethylphosphonates results in a sharp decrease of its inhibitory action. Preorganizing phosphonic
acid fragments using a calixarene platform therefore provides a promising approach for the design of efficient
alkaline phosphatase inhibitors.
We hypothesized that the preorganization of two fragments
Introduction
of methylenebisphosphonic acid at the wide rim of calix[4]arene
(Fig. 1) would result in improved affinity, and hence inhibitory
activity, through their simultaneous coordination with metal
ions at the binding site of alkaline phosphatase. Herein we
report on the synthesis and inhibitory activity of calix[4]arene
bisphosphonic acids 2 and 3 (Fig. 1).
With our increasing understanding of the role of phosphoryla-
tion in regulating biochemical events, there is growing interest
in the bioactivities associated with natural and synthetic
phosphonic acids.1 The mode of action of these compounds
is generally associated with the metabolic pathways involving
monoalkylphosphates.
Non-specific alkaline phosphatases catalyze phosphoryl
transfer in the hydrolysis or the transphosphorylation of phos-
phate monoesters.2,3 These are involved in a variety of biological
phenomena, including recent studies demonstrating their height-
ened expression in osteoblasts,4 with possible consequences for
osteoporotic patients. Single crystal X-ray analyses have revealed
the presence of a magnesium and two zinc ions in the active site
of the alkaline phosphatase of Escherichia coli, which are able
to bind inorganic phosphate,5 vanadate,6 phosphonoacetic
or mercaptomethylphosphonic acids.7 Mammalian alkaline
phosphatases display greater variety in their structural features,
including a diversity of loop regions located near the active site.8
The hydrophobic isoforms of these enzymes bear lipophilic
anchors of importance to their membrane-associated function.9
Despite their structural similarities with natural monoalkyl-
phosphates, alkylphosphonic acids are only weak inhibitors
of alkaline phosphatases. By taking advantage of the greater
affinity of sulfur for the metal ions in the active site, the intro-
duction of a thiol group into the structure of the phosphonic
acid was found to considerably improve the binding of the
inhibitor to bovine alkaline phosphatase.10 Methylenebisphos-
phonic acid 1, however, causes only insignificant inhibition of
alkaline phosphatase.10
The preorganization of several high affinity groups on the
surface of a molecular platform is often used in the design of
highly efficient and selective ligands and receptors. Calixarenes
represent a very commonly used class of molecular platforms
readily available through the cyclocondensation of p-substituted
phenols with formaldehyde.11 They were recently employed for
the preorganization of multiple biologically relevant groups
such as amino acids and small peptides.12 Hence peptidocalix-
arenes are able to complex cytochrome C and other proteins,13
while calixarenes bearing several permethylammonium residues
interact with nucleotides and nucleic acids.14 Amphiphilic
sulfo- and sulfonatocalix[n]arenes were demonstrated to bind to
bovine serum albumin.15
Fig. 1 Molecular design of novel alkaline phosphatase inhibitors.
Results and discussion
Synthesis
Methylenebisphosphonates are conventionally obtained by
the Arbuzov reaction of geminal dihaloalkanes with alkyl-
phosphites.16 4-Hydroxy-3,5-di-tert-butylbenzaldehyde has been
reported to react with two equivalents of dialkylphosphite in
the presence of diethylamine as a base to give 4-hydroxy-3,5-di-
tert-butylphenyl methylenebisphosphonate in moderate yield.17
We employed a slightly modified procedure for the synthesis of
calix[4]arene bisphosphonic acids 2 and 3 as well as the model
4-hydroxyphenyl methylenebisphosphonic acid 4 from the
readily available mono- and diformyl calixarenes 5 and 618 and
4-hydroxybenzaldehyde 7, respectively.
Scheme 1 highlights the sequence of events in the trans-
formation. The reaction of 4-hydroxybenzaldehyde 7 with
3 1 6 2
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 3 1 6 2 – 3 1 6 6
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 4

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one equivalent of sodium diethylphosphite generated in situ
in dioxane gives hydroxyphosphonate 8 in 80% yield. Subse-
quently 8 is converted quantitatively to bisphosphonate 9 by
a large excess of (EtO)2P(O)Na in diethylphosphite/dioxane
solution.19 A possible mechanism for this reaction involves the
base-mediated elimination of hydroxide ion from hydroxyphos-
phonate 8, yielding the highly reactive quinonemethide 10, to
which the addition of diethylphosphite leads to bisphosphonate
9. The treatment of either benzaldehyde or anisaldehyde under
the same conditions fails to give the respective bisphosphonates,
inagreementwiththeproposedintermediates.19 Theuseof alarge
excess of sodium diethylphosphite in dioxane/diethylphosphite
allows the conversion of aldehyde 7 to bisphosphonate 9 in a
single step. The standard dealkylation of bisphosphonate 9 with
Me3SiBr and subsequent methanolysis of the silyl esters affords
bisphosphonic acid 416b in almost quantitative yield.
of the bridges indicative of the syn orientation of all four
aromatic rings. These doublets are separated by 0.75–0.85 ppm,
indicating a C2v-symmetrical pinched cone conformation.22
Although two pinched cone conformations are theoretically
possible for molecules 3 and 12 – one with quasi-parallel and the
other with quasi-coplanar arylbisphosphonate fragments – the
1H NMR spectrum does not allow us to distinguish these two
structures.
Assay results
The inhibitory activities of compounds 1–4 and 12 were
investigated in the calf intestine alkaline phosphatase catalyzed
hydrolysis of p-nitrophenylphosphate. The Km and Vmax values
for the control reaction, and apparent values Km and Vmax for
the reactions at fixed inhibitor concentrations were determined
from the Lineweaver–Burk plots (Fig. 2). The influence of com-
pounds 1–4 and 12 on the enzyme activity is in agreement with
mixed-type or partial mixed-type inhibition. The correlations
between Km and Vmax and the concentration of compounds 1,
4 and 12 are linear, whereas in the case of compounds 2 and 3,
there are negative deviations from linearity (Fig. 3). Thus com-
pounds 1–4 and 12 not only result in a reduction of the apparent
substrate–enzyme affinity, but also affect the ability of the
enzyme–substrate complex to undergo its transformation into
the reaction products via full or partial inhibition (Scheme 3).
The dissociation constants of the enzyme inhibitor (Ki) and
the enzyme–substrate inhibitor (Ki) complexes with 1, 4 and
12 were calculated using the kinetic model for mixed-type
inhibition (k2 = 0). In the case of compounds 2 and 3, Ki and
Ki values were obtained according to the kinetics for partial
mixed-type inhibition (k2/k2 < 1). The rate of the formation of
p-nitrophenol during hydrolysis of p-nitrophenylphosphate in
the presence of an inhibitor (Scheme 3) is given by eqn. (1):23
Scheme 1 Synthesis of 4-hydroxyphenyl methylenebisphosphonic
acid 4. Reagents and conditions: (i) (EtO)2P(O)H, sodium metal; (ii) 1)
bromotrimethylsilane, room temperature, 30 h, 2) MeOH, 50 °C, 2 h.
′
′
(k2 + k2[I]/ Ki )[E][S]
Under identical conditions, calixarene aldehydes 5 and
6 provided bisphosphonate 11 and bis(bisphosphonate) 12
respectively (Scheme 2), with hydroxyphosphonates 13 and
1420 as intermediates.21 Calixarene bisphosphonate 11 and
bis(bisphosphonate) 12 were converted quantitatively to the
corresponding acids 2 and 3 by subsequent treatment with
Me3SiBr and methanol (Scheme 2).
(1)
V =
′
KS (1+ [I ]/ Ki ) + [S](1+ [I ]/ Ki )
The ratios Vmax/Vmax and Km/Km can be expressed (KS  Km)
by eqn. (2) and eqn. (3).24
′
Ki
1
The 1H NMR spectra of calix[4]arenes 2, 3, 11 and 12 contain
pairs of AB doublets (J = 13 Hz) for the methylene protons
(2)
′
1/(Vmax /Vmax −1) =
+
′
′
(k2 / k2 −1)[I] k2 / k2 −1
Scheme 2 Synthesis of calix[4]arene methylenebisphosphonic acids 2 and 3.
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 3 1 6 2 – 3 1 6 6
3 1 6 3

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Table 1 Inhibition constants for the inhibitors 1–4 and 12 of calf
intestine alkaline phosphatasea
Inhibitor
Ki/lM
Ki/lM
1
2
67 ± 5
2.5
0.38
22 ± 4
820 ± 180
750 ± 100
46
2.8
290 ± 110
8500 ± 2100
3
4
12
a 0.1 M Tris-HCl buffer, pH 9; Km value 36 ± 9 lM.
In the active site of the bacterial alkaline phosphatase, the
distance separating the two zinc cations is known to be 3.9 Å,
whereas the magnesium ion is positioned at a distance of 5 Å
and 7 Å respectively from each of these atoms.5 The active site
of calf intestine alkaline phosphatase is believed to share these
structuralfeatures.8 Themechanismof catalysisbyalkalinephos-
phatase includes the formation of the enzyme–substrate com-
plex, phosphoryl transfer from this complex to a nucleophilic
serine group, the hydrolysis of the covalent phosphorylated
enzyme intermediate, the formation of a non-covalent enzyme–
phosphate complex and the release of inorganic phosphate.3,5,8
Presumably, the activity of compounds 1–4 is a consequence of
their coordination to the cations in the binding site. The lipo-
philicity of the calix[4]arene scaffold may tighten the binding
of 2 and 3 through interactions with non-polar residues in the
vicinity of the catalytic domain. The higher inhibitory activity
of bis(bisphosphonic) acid 3 compared to bisphosphonic acid
2 can be attributed to better metal chelation or to synergistic
interactions with two of the metal ions. A plausible alternative
synergy may be the result of additional electrostatic interactions
between the anions of 3 and a positively charged arginine3,5 at
the enzyme’s active site. The main contribution to the stability of
the enzyme–inhibitor complex remains that of the phosphoryl
groups of calix[4]arenes 2 and 3, as the protected analogous
phosphonate 12 displays no inhibitory activity.
Fig. 2 Lineweaver–Burk graphical representation of calf intestine
alkaline phosphatase inhibition by calix[4]arene 3. The reaction
mixtures contain 0.1 M Tris-HCl buffer (pH 9) and various substrate
concentrations. The concentrations of the inhibitor were 0 (), 1.1 lM
(), 2 lM (), 3 lM (), 4 lM () and 5 lM ().
In conclusion, calix[4]arenes prove to be versatile molecular
platforms for the construction of efficient alkaline phosphatase
inhibitors. The efficient inhibitory activity of calixarene 3 is the
consequence of the preorganization of two methylenebisphos-
phonic acid substructures. The efficient coordination of 3 with
the metal cations in the enzyme’s active site and the additional
van der Waals interactions brought about by the scaffold itself
are probably responsible for its high activity.
Fig. 3 Dependencies of Km () and Vmax () values on the concen-
tration of calix[4]arene 3.
Experimental
Materials
1H and 31P NMR spectra were recorded on a VXP 300 spectro-
meter operating at 300 MHz and 121.5 MHz respectively. The
chemical shifts are reported using internal tetramethylsilane
and external 85% H3PO4 as references. The melting points were
determined on a Boetius apparatus and are uncorrected. Bromo-
trimethylsilane was freshly distilled. All reactions were carried
out under dry argon. Formylcalix[4]arenes 5 and 6 were synthe-
sized according to known procedures.25 Calf intestine alkaline
phosphatase was purchased from Fluka and used without
additional purification. The activity of enzyme was 1.1 U mg−1.
p-Nitrophenylphosphate was obtained from Sigma.
Scheme 3
′
Ki
′
1
′
1/(Km / Km −1) =
+
(3)
′
Ki / Ki −1 [I](Ki / Ki −1)
The inhibition constants Ki and Ki are obtained from the
ordinate intercept and the slope of the plot of {(Km/Km)−1}−1
versus 1/[I]. The ordinate intercept of plot of {(Vmax/Vmax)−1}−1
versus 1/[I] gives the relative value k2/k2.
The inhibition constants for calix[4]arene 2 were found to be
considerably lower than those of methylenebisphosphonic acid
1. Remarkably, the Ki value for calix[4]arene bisphosphonic
acid 2 is about 10 times smaller than that of model compound
4 (Table 1). As expected, though, it is calixarene 3, bearing two
methylenebisphosphonic acid motifs, which displays the stron-
gest inhibition with Ki = 0.38 lM, Ki = 2.8 lM and the relative
value k2/k2 = 0.3. The affinity of alkaline phosphatase for
calix[4]arene 3 is approximately two orders of magnitude higher
than for p-nitrophenylphosphate, and three orders of magnitude
higher than for the protected calixarene 12 (Table 1).
General procedure for synthesis of bisphosphonates 9, 11 and 12
To diethyl phosphite (5 ml) was cautiously added sodium
metal (0.3 g, 13 mmol) in small portions at room temperature.
Compound 5 (1 mmol), 7 (1 mmol) or 6 (0.5 mmol) was added
to the resulting solution, as appropriate. The reaction mixture
was stirred at room temperature for 48 h and then quenched
with water (100 ml) and extracted with chloroform (3 × 50 ml).
The chloroform layer was concentrated under vacuum and the
residue was washed with hexane and dried in vacuo. Yields were
almost quantitative.
3 1 6 4
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 3 1 6 2 – 3 1 6 6

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Tetraethyl 4-hydroxyphenylmethylene-1,1-bisphosphonate 9
J 7.5 Hz, OCH2CH2CH3), 2.05 (m, 4 H, OCH2CH2CH3), 3.25 (t,
2 H, J 25 Hz, P2CH), 3.43 (d, 4 H, J 13.2 Hz, ArCH2eq), 3.93 (t,
4 H, J 6.9 Hz, OCH2CH2CH3), 4.21 (d, 4 H, ArCH2ax, J 13.2 Hz),
6.58 (t, 2 H, J 7.5 Hz, p-ArH), 6.92 (d, 4 H, J 7.5 Hz, m-ArH);
7.26 (s, 4 H, m-ArH), 8.46 (s, 2 H, ArOH); dP (121.421 MHz;
DMSO-d6; H3PO4) 18.61.
White powder. Yield 99%, mp 88–89 °C (from hexane) (mp
89 °C16a). Found: C, 47.45; H, 6.75; P, 16.41. C15H26O7P2 requires
C, 47.37; H, 6.89; P, 16.29%; dH (300 MHz; CDCl3; Me4Si) 1.05,
1.28 (two t, 6 H + 6 H, J 6.9 Hz, diastereotopic POCH2CH3),
3.61 (t, 1 H, J 25 Hz, P2CH), 3.89, 4.04, 4.14 (three m, 2 H + 2
H + 4 H, diastereotopic POCH2CH3); 6.80 (d, 2 H, J 7.5 Hz,
ArH), 7.21 (d, 2 H, J 7.5 Hz, ArH), 8.42 (s, 1 H, ArOH); dP
(121.421 MHz; CDCl3; H3PO4) 20.4.
4-Hydroxyphenylmethylene-1,1-bisphosphonic acid 4
Light powder. Yield 98%, mp 148–150 °C (from methanol).
Found: C, 31.45; H, 3.64; P, 23.22. C7H10O7P2 requires C, 31.36;
H, 3.76; P, 23.11%. dH (300 MHz; DMSO-d6; Me4Si) 3.46 (t, 1 H,
J 25 Hz, P2CH), 6.67 (d, 2 H, J 7.5 Hz, ArH), 7.26 (d, 2 H, J
7.5 Hz, ArH); dP (121.421 MHz; DMSO-d6; H3PO4) 18.5; m/z
(FAB MS) 537.9 ([2M + H]+, 10%), 269.0 ([M + H]+, 30).
5-Bis(diethoxyphosphoryl)methyl-25,27-dipropoxycalix[4]-
arene 11
Light oil. Yield 98%. Found: C, 61.42; H, 5.80; P, 9.15.
C43H56O10P2 requires C1, 61.58; H, 5.91; P, 9.07%. dH (300 MHz;
CDCl3; Me4Si) 0.86, 1.22 (two t, 6 H + 6 H, J 6.9 Hz, diastereo-
topic POCH2CH3), 1.24 (t, 6 H, J 7.5 Hz, OCH2CH2CH3), 2.04
(m, 4 H, OCH2CH2CH3), 3.36, 3.38 (two d, 2 H + 2 H, J 13.2 Hz,
ArCH2eq), 3.58 (t, 1 H, J 25 Hz, P2CH), 3.70, 3.91 (two m, 2
H + 2 H, diastereotopic POCH2CH3), 3.98 (t, 4 H, JHH 6.9 Hz,
OCH2CH2CH3), 4.07 (m, 4H, diastereotopic POCH2CH3), 4.30
(d, 4 H, J 3.2 Hz, ArCH2ax); 6.88, 6.91, 7.06 (three d, 2 H + 2
H + 2 H, J 7.5 Hz, m-ArH), 6.67 (m, 3H, p-ArH), 7.20 (s, 2H,
m-ArH), 8.15, 8.21 (two s, 1 H + 1 H, ArOH); dP (121.421 MHz;
CDCl3; H3PO4) 19.7; m/z (FAB MS) 795.5 ([M + H]+, 100%).
General procedure for the enzymatic assays of alkaline
phosphatase activity
The influence of compounds 1–4 and 12 on the rate of p-nitro-
phenylphosphate hydrolysis catalyzed by alkaline phosphatase
was determined in 0.1 M Tris-HCl buffer at pH 9. The kinetics
of inhibition were studied at various concentrations for each
inhibitor: 1: 50 lM, 100 lM, 150 lM, 200 lM; 2 and 4: 10 lM,
20 lM, 30 lM, 40 lM; 3: 1.1 lM, 2 lM, 3 lM, 4 lM; 5 lM;
5: 250 lM, 500 lM, 750 lM, 1000 lM. The mixtures contain-
ing the buffer, the substrate (0.08–1.0 mM) and inhibitor were
incubated for 5 min at 23 °C, and reactions were initiated by the
addition of enzyme (3 lg ml−1). The generation of p-nitrophenol
during the hydrolysis of p-nitrophenylphosphate was measured
by the increase of absorbance at 410 nm, using a molar absorp-
tion coefficient of 18300 M−1 cm−1.
5,17-Bis[bis(diethoxyphosphoryl)methyl]-25,27-
dipropoxycalix[4]arene 12
Light oil. Yield 99%. Found: C, 57.69; H, 7.15; P, 11.53.
C52H76O16P4 requires C, 57.77; H, 7.09; P, 11.46%. dH
(300 MHz; CDCl3; Me4Si) 0.84, 1.24 (two t, 12 H + 12 H, J
6.9 Hz, diastereotopic POCH2CH3), 1.31 (t, 6 H, J 7.5 Hz,
OCH2CH2CH3), 3.60 (t, 2 H, J 25 Hz, P2CH), 2.05 (m, 4H,
OCH2CH2CH3), 3.38 (d, 4 H, J 13.2 Hz, ArCH2eq), 3.71,
3.89 (two m, 4 H + 4 H, diastereotopic POCH2CH3), 3.98 (t,
4 H, J 6.9 Hz, OCH2CH2CH3), 4.08 (m, 8 H, diastereotopic
POCH2CH3), 4.28 (d, 4 H, J 13.2 Hz, ArCH2ax), 6.61 (t, 2 H, J
7.5 Hz, p-ArH), 6.87 (d, 4 H, J 7.5 Hz, m-ArH); 7.22 (s, 4 H,
m-ArH), 8.13 (s, 2 H, ArOH); dP (121.421 MHz; CDCl3; H3PO4)
19.36; m/z (FAB MS) 1081.4 ([M + H]+, 100%).
Acknowledgements
The authors thank Dr Alexander Shivanyuk (IOC,
NASU), Mr Liam Palmer (The Scripps Research Institute,
La Jolla, CA) and Dr Adel Rafai Far for helpful discussions.
VK and SC thank STCU for support through grant RUS-09.
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5-Bis(dihydroxyphosphoryl)methyl-25,27-dipropoxycalix[4]-
arene 2
Light powder. Yield 99%, mp 132–134 °C (from methanol).
Found: C, 64.85; H, 7.15; P, 7.82. C35H40O10P2 requires C, 64.98;
H, 7.10; P, 7.79%. dH (300 MHz; DMSO-d6; Me4Si) 1.31 (t, 6 H, J
7.5 Hz, OCH2CH2CH3), 2.04 (m, 4 H, OCH2CH2CH3), 3.37, 3.44
(two d, 4H, J 13.2 Hz, ArCH2eq), 3.25 (t, 1 H, J 25 Hz, P2CH),
3.98 (wide s, 4H, OCH2CH2CH3), 4.19 (d, 4 H, J 13.2 Hz,
ArCH2ax), 6.57, 6.78 (two t, 2 H + 1 H, J 7.5 Hz, p-ArH), 7.1
(m, 2 H + 2 H + 2 H, m-ArH); 7.23 (s, 2 H, m-ArH), 8.46,
8.52 (two s, 1 H + 1 H, ArOH); dP (121.421 MHz; DMSO-d6;
H3PO4)18.2; m/z (FAB MS) 683.4 ([M + H]+, 75%).
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5,17-Bis[bis(dihydroxyphosphoryl)methyl]-25,27-dipropoxy-
calix[4]arene 3
Light powder. Yield 99%, mp 145–147 °C (from methanol).
Found: C, 50.61; H, 5.12; P, 14.52. C36H44O16P2 requires C, 50.48;
H, 5.18; P, 14.46%. dH (300 MHz; DMSO-d6; Me4Si) 1.31 (t, 6 H,
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 3 1 6 2 – 3 1 6 6
3 1 6 5

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