Inhibition of purple acid phosphatase with α-alkoxynaphthylmethylphosphonic acids

Article abstract of DOI:10.1016/j.bmcl.2008.10.125

Purple acid phosphatases (PAPs) are binuclear hydrolases that catalyse the hydrolysis of a range of phosphorylated substrates. Human PAP is a major histochemical marker for the diagnosis of osteoporosis. In patients suffering from this disorder, PAP activity contributes to increased bone resorption and, therefore, human PAP is a key target for the development of anti-osteoporotic drugs. This manuscript describes the design and synthesis of derivatives of 1-naphthylmethylphosphonic acids as inhibitors of PAP. The K_i values of these compounds are as low as 4 μM, the lowest reported to date for a PAP inhibitor.

Full text of DOI:10.1016/j.bmcl.2008.10.125

Bioorganic & Medicinal Chemistry Letters 19 (2009) 163–166
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Inhibition of purple acid phosphatase with
acids
a-alkoxynaphthylmethylphosphonic
a
a
a
a
Ross P. McGeary a,b, , Peter Vella , Jeffrey Y. W. Mak , Luke W. Guddat , Gerhard Schenk
*
a The University of Queensland, School of Molecular and Microbial Sciences, Brisbane, Qld 4072, Australia
b The University of Queensland, School of Pharmacy, Brisbane, Qld 4072, Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
Purple acid phosphatases (PAPs) are binuclear hydrolases that catalyse the hydrolysis of a range of phos-
phorylated substrates. Human PAP is a major histochemical marker for the diagnosis of osteoporosis. In
patients suffering from this disorder, PAP activity contributes to increased bone resorption and, therefore,
human PAP is a key target for the development of anti-osteoporotic drugs. This manuscript describes the
design and synthesis of derivatives of 1-naphthylmethylphosphonic acids as inhibitors of PAP. The Ki val-
Received 26 September 2008
Revised 27 October 2008
Accepted 28 October 2008
Available online 5 November 2008
ues of these compounds are as low as 4
l
M, the lowest reported to date for a PAP inhibitor.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Purple acid phosphatase
PAP
Phosphonate
Purple acid phosphatase (PAP; E.C. 3.1.3.2) is a binuclear hydro-
lase that has been isolated from various mammals and plants.1
Mammalian PAP is also known as tartrate-resistant acid phospha-
tase (TRAP)2 or osteoclastic acid phosphatase (OAP).3 PAPs catalyse
the hydrolysis of phosphorylated substrates under neutral to acidic
conditions in vivo, according to the equation:1
centre.1 Due to the structural similarity in their catalytically rel-
evant active sites, all PAPs are believed to employ highly con-
served mechanistic strategies.1
The biological roles of PAPs are diverse and depend on the host
organism. Plant PAPs are believed to assist in mobilising organic
phosphate esters in soil during germination,19 and mammalian
PAPs have been linked with iron transport during pregnancy.20
Both may be involved in bacterial killing since they are able, at
least in vitro, to generate reactive oxygen species (ROS) via Fenton
chemistry as a response against pathogens.13,21–24 Our interest in
PAP as a potential therapeutic target lies in its association with
osteoporosis. It is known that PAP is secreted into the bone-resorp-
tive space in mammals,2 and PAP activity has long been associated
with bone resorption.25 In addition, transgenic mice in which the
PAP enzyme was over-expressed developed osteoporosis due to
increased bone resorption,26 whereas the corresponding knock-
out mice exhibited the opposite condition (osteopetrosis), further
implicating the role of PAP in this process,27 and establishing PAP
as a drug target in the treatment of osteoporosis.28
RO ꢀ PO23ꢀ þ H2O ! ROH þ HPO2ꢀ
4
The characteristic purple colour of PAP is due to a charge transfer
transition between a conserved tyrosine residue and an Fe(III) in
the active site (kmax = 510–560 nm).4–6
Mammalian PAPs are ꢁ35 kDa monomeric proteins with a
redox active Fe(III)–Fe(II/III) centre in their active sites.7,8 While
it is the reduced form that is catalytically active, only the struc-
tures of the inactive diferric forms of PAPs from human (hPAP)9
and pig (pPAP)10 have been determined by X-ray crystallography.
In contrast, most plant PAPs are 110 kDa homodimers with
redox-inactive Fe(III)–Zn(II) or Fe(III)–Mn(II) centres.11–13 The
crystal structures of red kidney bean PAP (rkbPAP)14–16 and
sweet potato PAP17 have been solved. While the sequence
homology between characterised animal and plant PAPs is low,
the amino acids in the active site are highly conserved.18 In the
uninhibited enzyme, the coordination spheres of the two metal
ions have an approximate octahedral arrangement with four
amino acid ligands and possibly two water molecules per metal
Small anions, including fluoride and tetrahedral oxoanions such
as phosphate, vanadate and tungstate are known to inhibit
PAP.1,29–31 Myers and coworkers32 have described a number of
simple phosphonates with pendant metal-binding groups, such
as thiol, phosphonate and carboxylate, and these have shown
inhibitory activity against rkbPAP, in the range 80–3000 lM. Phos-
photyrosyl-containing tripeptides are excellent substrates for PAP.
A number of phosphonate- and carboxylate-containing derivatives
of these substrates have been synthesised, and these displayed
mid- to low-micromolar inhibitory potency against several mam-
malian and plant PAPs.28
* Corresponding author. Tel.: +61 7 3365 3955; fax: +61 7 3346 3249.
E-mail address: r.mcgeary@uq.edu.au (R.P. McGeary).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.10.125

164
R. P. McGeary et al. / Bioorg. Med. Chem. Lett. 19 (2009) 163–166
Schwender and coworkers3 reported the synthesis of a number
of derivatives of 1-naphthylmethylphosphonic acid (1) and their
inhibitory effects against hPAP. Compound 1 was reported to have
an IC50 of 30 lM, whereas no inhibition was detected for its a-
hydroxy analogue (2). The potencies of these compounds could
be increased by the substitution of lipophilic aroyl groups at the
a
-position of 1, to give compounds 3 and 4, with reported IC50s
of 6 and 1.4
l
M, respectively.3
PO3H2
HO PO3H2
1
2
R
R1 PO3H2
3, R = 2-naphthoyl, R1 = H
, R = R1 = benzoyl
4
Figure 1. Highest GOLD score docking conformation of the (R)-isomer of compound
7 in the active site of hPAP (PDB code 1WAR9).
Here, we report the design, synthesis and catalytic effects for a
number of new inhibitors of PAP, based on the 1-naphthylmethyl-
phosphonic acid scaffold. rkbPAP and pPAP were used for kinetic
studies due to their ready availability and ease of purification. In
contrast, hPAP can only be obtained in very small quantities, using
a baculoviral recombinant expression system.33 The validity of
using rkbPAP and pPAP as models of the human enzyme is sup-
ported by a number of factors including (i) the high degree of struc-
tural conservation in their substrate-binding pockets,9,10,14–16 (ii)
their conserved reaction mechanism(s),1 and (iii) the similarity of
inhibition constants for a range of inhibitors reported for several
animal (including human) and plant PAPs (e.g., the fluoride inhibi-
The synthetic route to compounds 5–7 is shown in Scheme 1. All
compounds were synthesised as racemates. Reaction of 1-naph-
thaldehyde (8) with diethyl phosphite in the presence of base
yielded the
a-hydroxyphosphonate diester (9). Attempted Wil-
liamson etherification of 9 with strong base and alkyl iodides sur-
prisingly failed. Treatment of 9 with sodium hydride in THF led
instead to the formation of the diethyl phosphate ester (10); the
product presumably arising via a Brook-type 1,2-rearrangement
of the intermediate alkoxide. Indeed, this type of rearrangement
has been previously observed in 1-naphthylmethyl systems.36,37
Attempts to alkylate
9 under milder conditions, using alkyl
tion constants of rkbPAP, pPAP and hPAP are 170, 120 and 170 lM,
iodides in the presence of either silver oxide38 or bis(tributyltin)
oxide39 were also unsuccessful, yielding only 1-naphthaldehyde
(8) in both cases.
respectively).1,11,17,28–31 Furthermore, mammalian and rkbPAPs
have similar catalytic efficiencies and catalyse the hydrolysis of
similar substrates.1,11,30
Efforts were then directed towards transforming the hydroxyl
group of 9 into a leaving group, suitable for displacement with
Computer modelling was used to identify new compounds
that might act as inhibitors of PAPs. In the search for these mol-
ecules, naphthalen-1-ylmethyl-phosphonic acid (1) and several of
its derivatives were chosen as starting points. The binding orien-
tations of these compounds were assessed by docking them into
the hPAP active site9 GOLD (Genetic Optimisation for Ligand
Docking).34,35 The predicted binding orientations of these com-
pounds were examined. As expected, the phosphonate groups
bound to the binuclear centre, and a long groove adjacent to
the active site was identified as an additional potential point of
HO PO3Et2
O
a
8
9
b
c
interaction (see Fig. 1). Hence, the
a-alkoxy phosphonic acids
5–7 (see below) were designed and docked into the hPAP active
site, with the aim that the long alkyl chain would bind in the
groove. The docking studies suggested that these modified inhib-
itors may indeed bind in such a mode to the active site and the
adjacent groove. Compounds 5–7 were therefore synthesised as
novel candidate PAP inhibitors.
PO Et
O
MsO PO3Et2
3
2
10
11
d
O
PO3H2
O
PO3H2
RO PO3H2
RO PO3Et2
e
5
6
5, R = hexyl
6, R = octyl
7, R = decyl
12, R = hexyl
13, R = octyl
14, R = decyl
O
PO3H2
Scheme 1. Reagents and conditions: (a) diethyl phosphite, DIPEA, neat, rt, 17 h,
100%; (b) NaH, THF, rt, 1 h, 69%; (c) MsCl, DIPEA, DCM, rt, 3 d, 100%; (d) ROH, DIPEA,
MeCN, reflux, 18 h, 68% (12), 41% (13), 24% (14); (e) TMSCl, NaI, MeCN, rt, 18 h, 89%
(5), 100% (6), 66% (7).
7

R. P. McGeary et al. / Bioorg. Med. Chem. Lett. 19 (2009) 163–166
165
Table 2
an alcohol or alkoxide. To that end, 9 was converted to its meth-
Kinetic data for inhibitors 1, 2, 5, 6 and 7 against pig PAP at pH 4.9
anesulfonate ester (11) (Scheme 1) which readily underwent sub-
stitution with long-chain alcohols in hot acetonitrile in the
presence of a base, yielding the desired ethers 12–14. Selective
cleavage of the phosphonate ester groups of 12–14, without rup-
ture of the benzylic ether linkage,40 was achieved using TMSI,
formed in situ from TMSCl and NaI in acetonitrile, according to
Morita et al.,41 yielding the final 1-naphthylmethylphosphonic
acids 5–7. The same reaction conditions were employed to prepare
Inhibitor KM
(mM)
IC50
(mM)
IC50(calc)
(mM)
logIC50
( logSTD)
Kic
(mM)
Kiuc
(mM)
5
6
7
2.65(34) 0.43
1.50(20) 0.03
1.13(11) 0.02
ND
0.04
0.02
ꢀ0.37(1.70)
ꢀ1.5(3)
ND
ND
0.023(8) 0.044(9)
0.017(3) 0.020(4)
ꢀ1.70(3.50)
the
a
-hydroxyphosphonic acid (2) from the corresponding diethyl
the benzylic carbon of 1, to give compound 2, had very little effect
on the binding affinity. Since the estimated IC50 was ꢁ5 mM the Ki
values are expected to be large (>5 mM) and were thus not deter-
mined. However, the addition of an alkyl chain to the hydroxylated
phosphonate derivative led to a substantial increase in binding
affinity. The critical length required to observe a significant effect
is six carbon atoms; although the ‘hexyl’ derivative 5 is still only
weakly binding, the ‘octyl’ derivative 6 displayed a 10-fold reduc-
tion in IC50 value for rkbPAP (Table 1). A further elongation of the
chain to ten carbon atoms (‘decyl’ derivative’) led to another dra-
phosphonic ester (9) (Scheme 2).
The synthesis of 1-naphthylmethylphosphonic acid (1) is
shown in Scheme 3. Chloromethylation of naphthalene (15)
yielded halide 16, which was subjected to an Arbuzov-type reac-
tion with diethyl phosphite to give 17. Again, we found that
Morita’s method41 successfully dealkylated the diester (17) to
give 1, albeit in low yield.
The inhibitory effects of the new compounds were tested with
both rkbPAP and pPAP at pH 6.2 and 4.9, the respective pH opti-
ma28,31 of the two enzymes (Tables 1 and 2). Hydroxylation of
matic reduction in the IC50 value to ꢁ8
lM. These observations
are in good agreement with the in silico docking study (see above),
which indicates that the predicted binding mode of the phospho-
nate compounds involves interactions with a groove in the vicinity
of the binuclear active site.
HO OPO3Et2
HO PO3H2
a
For the ‘hexyl’-, ‘octyl’- and ‘decyl’-derivatives (compounds 5,
6 and 7 respectively), Ki values were measured to determine the
mode of interactions between the enzyme and inhibitor (Table
1). As anticipated, the Ki values are in reasonable agreement with
the IC50 values. The compounds inhibit the catalytic reaction in a
noncompetitive or mixed competitive mode, indicating that they
bind to both the free enzyme and the enzyme-substrate (ES)
complex (see Figure S1 in the Supplementary Section). Kic and
Kiuc represent the inhibitor dissociation constants (inhibition
constants) for the free enzyme and ES complex. If Kic ꢁ Kiuc the
inhibition mode is noncompetitive, if Kic – Kiuc it is of mixed
type.42 For rkbPAP the two inhibition constants for the ‘hexyl’-
derivative 5 are similar (noncompetitive inhibition), and as the
chain length increases both constants become smaller (indicating
increased binding affinity of the inhibitor), but Kic becomes dom-
inant. In case of the ‘decyl’-derivative 7, the inhibitor binds
approximately sevenfold tighter to the free enzyme than the ES
9
2
Scheme 2. Reagents and conditions: (a) TMSCl, NaI, MeCN, rt, 3.5 d, 52%.
Cl
a
15
16
17
b
PO3H2
PO3Et2
c
complex (4 lM vs 30 lM, respectively, for rkbPAP, Table 1) The
IC50 of a particular inhibitor can be calculated from the above
Ki values.43 In general, there is good agreement between these
calculated IC50 values and the experimentally determined ones
(Table 1). Compound 7 is to date the most potent PAP inhibitor
of those tested, and its high water solubility makes it a promis-
ing candidate as a clinical lead.
1
Scheme 3. Reagents and conditions: (a) CH2O, HCl, H3PO4, AcOH, reflux, 6 h, 56%;
(b) diethyl phosphite, Bu4NI, K2CO3, DMF, 64 h, 16%; (c) TMSCl, NaI, MeCN, rt, 16 h,
23%.
The phosphonate derivatives (5–7) have a similar inhibitory
effect on pPAP (Table 2). In terms of the IC50 values a similar
trend as the one observed for the rkbPAP enzyme has been
observed, that is, the longer the alkyl chain the more potent
the inhibitor. However, in contrast to the plant PAP the ‘hexyl’-
derivative 5 is already a good inhibitor of the pig enzyme with
an IC50 value of 0.43 mM.
Table 1
Kinetic data for inhibitors 1, 2, 5, 6 and 7 against rkbPAP at pH 6.2
Inhibitor
KM
(mM)
IC50
(mM)
IC50(calc)
(mM)
logIC50
( logSTD)
Kic
(mM)
Kiuc
(mM)
1
2
5
6
7
5.78(24)
5.31(17)
4.28(54)
3.41(42)
3.10(18)
>7
5
1.0
0.1
0.008
ND
ND
1.4
0.09
0.01
>0.845
0.700
0(0.70)
ND
ND
1.47(39)
0.05(1)
0.0040(7)
ND
ND
1.43(30)
0.129(30)
0.03(1)
In conclusion, we have used computer-aided inhibitor design
to identify a family of
a-alkoxynaphthylmethylphosphonic acids
ꢀ1(2.15)
as potent inhibitors of PAPs. These compounds were synthesised
and assayed with rkbPAP and pPAP. Compound 7 was found to
ꢀ2.10(3.50)
KM, Michaelis constant.
be the most active, with an IC50 value of 20
lM in pPAP and
Kic, Competitive inhibition constant.
Kiuc, Uncompetitive inhibition constant.
8
lM in rkbPAP. These compounds are currently being used in
IC50(calc), Ki and IC50 values are correlated via the following equation:43
crystallisation trials to form complexes with mammalian and
plant PAPs. Future work will address the inhibitory activities of
the individual enantiomers of each inhibitor. The results of these
studies will provide the basis for the next round of rational drug
design.
KM þ ½Sꢂ
ꢀ
ꢁ
IC50
¼
:
½Sꢂ
KM
KIuc KIc
ND, not determined.

166
R. P. McGeary et al. / Bioorg. Med. Chem. Lett. 19 (2009) 163–166
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Mr. Graham MacFarlane for MS measurements. This work was
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a grant from the Australian Research Council
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Supplementary data
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