Diethylalkylsulfonamido(4-methoxyphenyl)methyl)phosphonate/phosphonic acid derivatives act as acid phosphatase inhibitors: synthesis accompanied by experimental and molecular modeling assessments

Article abstract of DOI:10.1080/14756366.2016.1230109

Purple acid phosphatases (PAPs) are binuclear metallo-hydrolases that have been isolated from various mammals, plants, fungi and bacteria. In mammals, PAP activity is associated with bone resorption and can lead to bone metabolic disorders such as osteopo

Full text of DOI:10.1080/14756366.2016.1230109

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Diethylalkylsulfonamido(4-
methoxyphenyl)methyl)phosphonate/phosphonic
acid derivatives act as acid phosphatase inhibitors:
synthesis accompanied by experimental and
molecular modeling assessments
Nahid Alimoradi, Mohammad Reza Ashrafi-Kooshk, Mohsen Shahlaei,
Shabnam Maghsoudi, Hadi Adibi, Ross P. McGeary & Reza Khodarahmi
To cite this article: Nahid Alimoradi, Mohammad Reza Ashrafi-Kooshk, Mohsen
Shahlaei, Shabnam Maghsoudi, Hadi Adibi, Ross P. McGeary & Reza Khodarahmi (2016):
Diethylalkylsulfonamido(4-methoxyphenyl)methyl)phosphonate/phosphonic acid derivatives
act as acid phosphatase inhibitors: synthesis accompanied by experimental and molecular
modeling assessments, Journal of Enzyme Inhibition and Medicinal Chemistry, DOI:
10.1080/14756366.2016.1230109
To link to this article: http://dx.doi.org/10.1080/14756366.2016.1230109
Published online: 21 Oct 2016.
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Date: 24 October 2016, At: 00:49

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY, 2016
http://dx.doi.org/10.1080/14756366.2016.1230109
RESEARCH ARTICLE
Diethylalkylsulfonamido(4-methoxyphenyl)methyl)phosphonate/phosphonic acid
derivatives act as acid phosphatase inhibitors: synthesis accompanied by
experimental and molecular modeling assessments
a
b
c
b
d
ꢀ
ꢀ
Nahid Alimoradi , Mohammad Reza Ashrafi-Kooshk , Mohsen Shahlaei , Shabnam Maghsoudi , Hadi Adibi ,
Ross P. McGearye and Reza Khodarahmib
b
aStudent Research Committee, Kermanshah University of Medical Sciences, Kermanshah, Iran; Medical Biology Research Center, Kermanshah
c
University of Medical Sciences, Kermanshah, Iran; Nano Drug Delivery Research Center, Kermanshah University of Medical Sciences,
d
Kermanshah, Iran; Pharmaceutical Sciences Research Center, Faculty of Pharmacy, Kermanshah University of Medical Sciences, Kermanshah,
e
Iran; The University of Queensland, School of Chemistry and Molecular Biosciences, St. Lucia, QLD, Australia
ABSTRACT
ARTICLE HISTORY
Received 25 March 2016
Revised 16 July 2016
Accepted 18 August 2016
Published online 21 Septem-
ber 2016
Purple acid phosphatases (PAPs) are binuclear metallo-hydrolases that have been isolated from various
mammals, plants, fungi and bacteria. In mammals, PAP activity is associated with bone resorption and can
lead to bone metabolic disorders such as osteoporosis; thus human PAP is an attractive target to develop
anti-osteoporotic drugs. The aim of the present study was to investigate inhibitory effect of synthesized
diethylalkylsulfonamido(4-methoxyphenyl)methyl)phosphonate/phosphonic acid derivatives as potential
red kidney bean PAP (rkbPAP) inhibitors accompanied by experimental and molecular modeling assess-
ments. Enzyme kinetic data showed that they are good rkbPAP inhibitors whose potencies improve with
increasing alkyl chain length. Hexadecyl derivatives, as most potent compounds (Ki ¼ 1.1 mM), inhibit
rkbPAP in the mixed manner, while dodecyl derivatives act as efficient noncompetitive inhibitor. Also, ana-
lysis by molecular modeling of the structure of the rkbPAP–inhibitor complexes reveals factors, which may
be important for the determination of inhibition specificity.
KEYWORDS
Acid phosphatase; enzyme
inhibition; phosphonic acid;
synthesis
Introduction
organic phosphate esters in soil during germination11, and mam-
malian PAPs have been linked with iron transport during preg-
nancy12. Both may be involved in bacterial killing since they are
able, at least in vitro, to generate reactive oxygen species via
Fenton chemistry as a response against pathogens9,13,14. The asso-
ciation of mammalian PAP activity with osteoporosis2,15–17 has
prompted investigators, around the world, to examine its potential
as a therapeutic target for the treatment of this disease, and to
develop potent inhibitors of this enzyme17,18. It is known that PAP
is secreted into the bone-resorptive space in mammals2, and PAP
activity has long been associated with bone resorption19. In add-
ition, transgenic mice in which the PAP enzyme was over-
expressed developed osteoporosis due to increased bone resorp-
tion20, whereas the corresponding knockout mice exhibited the
opposite condition (osteopetrosis), further implicating the role of
PAP in this process4, and establishing PAP as a drug target in the
treatment of osteoporosis21. Although clinical treatments are avail-
able for osteoporosis, notably bisphophonates, which inhibit farne-
sylpyrophosphate synthase in osteoclast22, but these drugs have
significant side effects and compliance issues. Also, a number of
PAPs inhibitors including fluoride23, and several tetrahedral inor-
ganic oxyanions, including phosphate, arsenate, vanadate, tung-
state and molybdate7,24 have been discovered, but most of these
do not possess drug-like properties and so are not suitable for fur-
ther modifications that may convert them into chemotherapeutic
agents. Myers et al.25 have described a number of simple phos-
Purple acid phosphatase (PAP; E.C.3.1.3.2) is a binuclear hydrolase
that has been isolated from various mammals and plants1.
Mammalian PAP is also known as tartrate-resistant acid phosphat-
ase2 or osteoclastic acid phosphatase3. These enzymes are distin-
guished from other acid phosphatases by their characteristic purple
color in concentrated solution4. PAPs catalyze the hydrolysis of
phosphorylated substrates under neutral to acidic conditions in
vivo1. Several crystal structures of PAPs have been described,
including those from plants (red kidney bean and a sweet potato
PAP) as well as from mammals (pig, human and rat)5 and references
90–93 therein. The enzyme structures from the two plant organisms
are homodimers consisting of 55 kDa subunits, while the mamma-
lian PAPs are 35 kDa monomers6–9. Despite the apparent difference
between the structures of plant and animal PAPs, and the low
degree of overall amino acid sequence homology between
enzymes from different kingdoms, their catalytic sites display a
remarkable similarity5 and references 2, 89, 107 therein (Figure 1)
and include two metal centers, which are coordinated by seven
invariant ligands (one aspartate, one tyrosine and one histidine for
Fe3þ and two histidines and one asparagine for Zn2þ), with an
aspartate residue bridging the two ions10. Due to the structural
similarity in their catalytically relevant active sites, all PAPs are
believed to employ highly conserved mechanistic strategies1.
The biological roles of PAPs are diverse and depend on the
host organism. Plant PAPs are believed to assist in mobilizing phonates with pendant metal-binding groups, such as thiol,
CONTACT Hadi Adibi
Kermanshah, Iran; Reza Khodarahmi
hadibi@kums.ac.ir
Pharmaceutical Sciences Research Center, Faculty of Pharmacy, Kermanshah University of Medical Sciences,
rkhodarahmi@kums.ac.ir, rkhodarahmi@mbrc.ac.ir Medical Biology Research Center, Kermanshah University of Medical
Sciences, Kermanshah, Iran
ꢀ
These authors had equally contributed.
ß 2016 Informa UK Limited, trading as Taylor & Francis Group

2
N. ALIMORADI ET AL.
O
P
O
P
O
P
EtO
EtO
EtO
EtO
NH
NH2
HO
HO
NH
CHO
OMe
SO 2R
SO 2R
(CH3)3SiCl, NaI
(i-Pr)2NEt, N2
(EtO)2POH, NH3
RSO2Cl, N2
(i-Pr)2NEt
CHCl3
3a-3d
4a-4d
OMe
OMe
OMe
2
1
3a,4a: R= n-Hexyl, 3b,4b: n-Octyl, 3c,4c: n-Dodecyl, 3d,4d: n-Hexadecyl
Scheme 1. Synthesis of diethylalkylsulfonamido(4-methoxyphenyl)methyl)phosphonate/phosphonic acid derivatives.
purification of the intermediate and final compounds. Melting
points were determined on a Kofler hot stage apparatus (Vienna,
Austria) and are uncorrected. H NMR spectra were recorded using
phosphonate and carboxylate, and these have shown inhibitory
activity against red kidney PAP (rkbPAP), in the range 80–3000 mM.
Moreover, several modified phosphotyrosine containing tripeptides
have also shown inhibitory activity toward several mammalian and
plant PAPs, with IC50 values in the mid-micromolar range21. More
recently, a-alkoxynaphthylmethylphosphonic acids and acyl deriva-
tives of a-aminonaphthylmethylphosphonic acid have also been
shown to be PAP inhibitors with Ki and IC50 values in the low
micromolar range18,26. These molecules were designed as the
derivatives of 1-naphthylmethylphosphonic acid, a PAP inhibitor
previously reported by Schwender et al.3 A number of phospho-
nate- and carboxylate-containing derivatives of these substrates
have been synthesized, and these displayed mid- to low-micromo-
lar inhibitory potency against several mammalian and plant
PAPs21.
1
a Bruker 500 spectrometer (Bruker, Rheinstatten, Germany), and
chemical shifts are expressed as d (ppm) with tetramethylsilane
(TMS) as an internal standard. Coupling constants (J) are reported
in hertz and peak multiplicities described as singlet (s), doublet
(d), triplet (t), multiplet (m) or broad (br). The IR spectra were
obtained on a Shimadzu 470 (Shimadzu, Tokyo, Japan) spectro-
photometer (potassium bromide disks). The mass spectra were run
on a Finnigan TSQ-70 spectrometer (Finnigan) at 70 eV.
General procedure for the syntheses and spectral data of the
synthesized compounds
General procedure for the synthesis of diethyl(amino(4-methoxy-
The current study is particularly novel and significant because to
the best of our knowledge, no previous similar studies have been phenyl)methyl)phosphonate (2)
reported on possible inhibitory potential of diethylalkylsulfona- This compound was prepared according to the general procedure
described in the literature3,18,26,27. NH4OAc (7.70 g, 0.10 mol) was
mido(4-methoxyphenyl)methyl)phosphonate/phosphonic
acid
derivatives on the PAP enzyme. Based on previous lead com- added to EtOH (20 mL) in the round-bottomed flask containing
pound(s) and rational drug design, different sulfonyl derivatives of the activated molecular sieves, followed by the addition of p-
alkylsulfonamido(4-methoxyphenyl)methyl)phosphonic acid with methoxybenzaldehyde (1) (13.60 mL, 0.10 mol) and diethyl phos-
varying lengths of alkyl chains were synthesized and evaluated as phite (12.90 mL, 0.10 mol). The reaction mixture was stirred at
PAP inhibitors. The sulfonyl and alkyl groups were introduced for 60 ꢁC under nitrogen atmosphere for 48 h. Once it has cooled to
two reasons: (i) the sulfonyl group was expected to improve the room temperature, conc. HCl was added to the resulting mixture
water solubility of these compounds; (ii) McGeary et al. have previ- to acidify it to pH 1, and then was washed with Et2O (50 mL).
ously reported that PAP inhibitors bearing an alkoxy group with Addition of 2 M NaOH to the aqueous layer changed the pH to
long alkyl chain were superior to those that did not, suggesting 11, and the product was extracted with dichloromethane (100 mL)
effective hydrophobic interactions between the alkyl group of the to give the free base of diethyl (amino(naphthalen-1-yl)methyl)-
phosphonate27 as yellow oil. The product was later treated with
HCl gas in EtOH (10 mL)–Et2O (10 mL). Et2O (20 mL) was further
inhibitors and nonpolar surface residue near the enzyme’s active
site18; (iii) a nonclassical bioisosteric replacement was made in
added to the resulting solution, and the solvent was evaporated
to obtain the hydrochloride salt of diethyl compound 2 as white
solid.
which the acyl moiety was changed to sulfonamide one. Therefore,
inhibitors of general structures in Scheme 1 with C6, C8, C12 and C16
alkyl chains were examined. Moreover, to evaluation of coordin-
ation of phosphonate moiety to the bimetal center, the ethyl
groups were attached to the oxygen atoms of phosphonate. The
aim of the current study was also to expand this working frame-
work through suggesting a molecular modeling component to
advance the mechanistic understanding of PAP–drug interactions.
General procedure for the preparation of diethyl (alkylamido(4-
methoxyphenyl)methyl)phosphonates (3a–3d)
This compound was prepared according to the general procedure
described in the literature27.
A solution of DIPEA (2.60 mL,
15.2 mmol) and the hydrochloride salt 2 (0.50 g, 1.52 mmol) in
CH2Cl2 (5 mL) were then added dropwise to the sulfonyl chloride
and the reaction mixture was stirred under argon N2 for 24 h. The
crude product was purified using silica flash column chromatog-
raphy (10–60% EtOAc in light petroleum) to give 3a–3h.
Materials and methods
Chemistry
All starting materials, reagents, and solvents were purchased
from Merck Finnigan (Germany). The purity of the synthesized
compounds was confirmed by thin layer chromatography (TLC)
using various solvents of different polarities. Merck silica gel 60
General procedure for the preparation of (alkylamido(4-methoxy-
phenyl)methyl)phosphonic acids (4a–4d)
F254 plates were applied for analytical TLC. Column chromatog- This compound was prepared according to the general procedure
raphy was performed on Merck silica gel (70–230 mesh) for described in the literature27. NaI (5 equiv) and distilled MeCN

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
3
(5 mL) were added to the corresponding diethyl ester 3a–3d (stretch C¼C, aromatic), 1512 (bending NH), 1458 (bending CH2),
(1 equiv). Distilled TMSCl (5 equiv) was then added to the mixture,
1396 (bending CH3), 1311, 1176 (stretch O¼S¼O), 1211(stretch
which was then stirred at 40 ꢁC under N2 for 18 h. MeCN was
C–O), 1041 (stretch C–O, OCH3), 948 (stretch CH, oop), 729 (rocking
removed in vacuo. The precipitate was then extracted with EtOAc
(3 ꢂ 10 mL), washed with NaS2O3 (10 mL), water and NaCl
(2 ꢂ 10 mL), followed by drying with NaSO4. The product was then
co-evaporated with toluene (3 ꢂ 5 mL), and purified under high
vacuum to give the corresponding phosphonic acids 4a–4d.
CH2), 547 (bending NH).
1H NMR (CDCl3, 300 MHz): d (ppm): 7.55–7.53 (dd, J ¼ 2.1,
2.1 Hz, 1H, NH), 7.38–7.36 (dd, J ¼ 1.2 Hz, 2H, C(2,6)H aromatic),
6.81–6.79 (d, J ¼ 5.1 Hz, 2H, C(3,5)H aromatic), 5.51–5.45 (dd, J ¼ 5.7,
ꢀ
5.7 Hz, H, CH–P), 4.13–4.07 (m, 2H, C(1,1 )H–OP), 3.85–3.82 (m, 1H,
ꢀ
C(1)H–OP), 3.73 (s, 3H, CH3–O), 3.66–3.61 (m, 1H, C(1 )H–OP), 2.17
(t, J ¼ 4.2 Hz, 2H, CH2–S), 1.54 (t, J ¼ 6.9 Hz, 2H, CH2–CH2S), 1.28 (t,
Diethyl(amino(4-methoxyphenyl)methyl)phosphonate (2)
Molecular weight: 204 (g/mol), FT-IR (KBr, cmꢃ1): 3429 (stretch NH),
2935 (stretch CH, aromatic), 2904 (stretch CH, aliphatic), 1608
(stretch C¼C, aromatic), 1519 (bending NH), 1462 (bending CH2),
1388 (bending CH3), 1242 (stretch C–O), 1026 (stretch C–O, OCH3),
948 (stretch CH, oop), 547 (bending NH).
J ¼ 4.2 Hz, 3H, C(2)H3–CH2O), 1.16 (bro, J ¼ 7.5 Hz, 10H, CH ali-
ꢀ
phatic), 1.04 (t, J ¼ 4.2 Hz, 3H, C(2 )H3–CH2O), 0.83 (t, J ¼ 6.9 Hz, 3H,
CH3–RS).
13C NMR (CDCl3, 75 MHz):
d
(ppm): 13.9 (CH3–RS), 16.1
ꢀ
(C(2 )H3 ethoxy), 16.3 (C(2)H3 ethoxy), 22.5 & 22.6 & 29.1 & 29.2
ꢀ
& 29.4 & 31.7 & 36.2 (CH2 alkyl), 48.4(C(1 )H2 ethoxy), 49.6 (C(1)H2
ethoxy), 55.1 (P–CH–N), 62.8 (S–CH2), 63.1 (CH3 methoxy), 113.8
(C3,5 phenyl), 127.3 (C2,6 phenyl), 129.4 (C1 phenyl), 159.2 (C4
phenyl).
Diethyl(hexylsulfonamido(4-methoxyphenyl)methyl)phosphonate
(3a)
Molecular weight: 421 (g/mol), FT-IR (KBr, cmꢃ1): 3437 (stretch NH),
2931 (stretch CH, aromatic), 2873 (stretch CH, aliphatic), 1612
(stretch C¼C, aromatic), 1516 (bending NH), 1465 (bending CH2),
1392 (bending CH3), 1311, 1188 (stretch O¼S¼O), 1249 (stretch
C–O), 1041 (stretch C–O, OCH3), 952 (stretch CH, oop), 551 (bend-
ing NH).
Diethyl(hexadecylsulfonamido(4-methoxyphenyl)methyl)phospho-
nate (3d)
Molecular weight: 561 (g/mol), FT-IR (KBr, cmꢃ1): 3433 (stretch NH),
2920 (stretch CH, aromatic), 2850 (stretch CH, aliphatic), 1608
(stretch C¼C, aromatic), 1512 (bending NH), 1465 (bending CH2),
1400 (bending CH3), 1307, 1184 (stretch O¼S¼O), 1249 (stretch
C–O), 1053 (stretch C–O, OCH3), 968 (stretch CH, oop), 721 (rocking
CH2), 536 (bending NH).
1H NMR (CDCl3, 300 MHz): d (ppm): 7.68–7.66 (d, J ¼ 6 Hz, 1H,
NH), 7.39–7.36 (dd, J ¼ 1.5, 1.5 Hz, 2H, C(2,6)H aromatic), 6.80–6.78
(d, J ¼ 6.6 Hz, 2H, C(3,5)H aromatic), 5.52–5.44 (dd, J ¼ 7.2, 7.2 Hz,
ꢀ
H, CH–P), 4.15–4.04 (m, 2H, C(1,1 )H–OP), 3.88–3.79 (m, 1H,
1H NMR (CDCl3, 300 MHz): d (ppm): 7.36–7.33 (dd, J ¼ 1.5,
1.8 Hz, 2H, C(2,6)H aromatic), 6.85–6.83 (d, J ¼ 6.3 Hz, 2H, C(3,5)H aro-
matic), 6.60–6.56 (dd, J ¼ 3.3, 3.3 Hz, 1H, NH), 5.47–5.40 (dd, J ¼ 7.2,
ꢀ
C(1)H–OP), 3.72 (s, 3H, CH3–O), (m, 1H, C(1 )H–OP), 2.16 (t,
J ¼ 5.7 Hz, 2H, CH2–S), 1.53 (t, J ¼ 5.4 Hz, 2H, CH2–CH2S), 1.28 (t,
J ¼ 5.4 Hz, 3H, C(2)H3–CH2O), 1.15 (bro, J ¼ 7.5 Hz, 10H, CH ali-
ꢀ
7.2 Hz, H, CH–P),4.13–4.07 (m, 2H, C(1,1 )H–OP), 3.92–3.86 (m, 1H,
ꢀ
phatic), 1.03 (t, J ¼ 5.7 Hz, 3H, C(2 )H3–CH2O), 0.80 (t, J ¼ 5.4 Hz,
ꢀ
C(1)H–OP), 3.70 (s, 3H, CH3–O), 3.68–3.64 (m, 1H, C(1 )H–OP), 2.19
3H, CH3–RS).
(t, J ¼ 6 Hz, 2H, CH2–S), 1.58 (t, J ¼ 5.7 Hz, 2H, CH2–CH2S), 1.29 (t,
J ¼ 5.1 Hz, 3H, C(2)H3–CH2O), 1.23 (bro, J ¼ 7.5 Hz, 26H, CH ali-
Diethyl((4-methoxyphenyl)(octylsulfonamido)methyl)phosphonate
(3b)
ꢀ
phatic), 1.07 (m, J ¼ 5.1 Hz, 3H, C(2 )H3–CH2O), 0.85 (t, J ¼ 5.4 Hz,
3H, CH3–RS).
Molecular weight: 449 (g/mol), FT-IR (KBr, cmꢃ1): 3437 (stretch NH),
2927 (stretch CH, aromatic), 2858 (stretch CH, aliphatic), 1612
(stretch C¼C, aromatic), 1512 (bending NH), 1465 (bending CH2),
1396 (bending CH3), 1303, 1215 (stretch O¼S¼O), 1246 (stretch
C–O), 1037(stretch C–O, OCH3), 948(stretch CH, oop), 721 (rocking
CH2), 551 (bending NH).
(Hexylsulfonamido(4-methoxyphenyl)methyl)phosphonic acid (4a)
Molecular weight: 365 (g/mol), FT-IR (KBr, cmꢃ1): 3433 (stretch NH),
2931 (stretch CH, aromatic), 2866 (stretch CH, aliphatic), 1612
(stretch C¼C, aromatic), 1516 (bending NH), 1462 (bending CH2),
1392 (bending CH3), 1303, 1188 (stretch O¼S¼O), 1045 (stretch
C–O, OCH3), 952 (stretch CH, oop), 775 (rocking CH2), 559 (bending
NH).
1H NMR (CDCl3, 300 MHz): d (ppm): 7.54–7.49 (dd, J ¼ 3.6,
3.6 Hz, 1H, NH), 7.39–7.35 (dd, J ¼ 2.1, 2.1 Hz, 2H, C(2,6)H aromatic),
6.81–6.78 (dd, J ¼ 9 Hz, 2H, C(3,5)
H
aromatic), 5.53–5.46 (dd,
ꢀ
1H NMR (CH3OH-d4, 300 MHz): d (ppm): 7.38–7.35 (d, J ¼ 8.4 Hz,
2H, C(2,6)H aromatic), 6.90–6.86 (d, J ¼ 4.2 Hz, 2H, C(3,5)H aromatic),
5.46–5.29 (dd, J ¼ 21, 21 Hz, H, CH-P), 3.77 (s, 3H, CH3–O), 2.28 (t,
J ¼ 7.2 Hz, 2H, CH2–S), 1.59 (t, J ¼ 5.7 Hz, 2H, CH2–CH2S), 1.25 (bro,
6H, CH aliphatic), 0.85 (t, J ¼ 5.4 Hz, 3H, CH3–RS).
J ¼ 20.7 Hz, H, CH–P), 4.14–4.04 (m, 2H, C(1,1 )H–OP), 3.88–3.80 (m,
ꢀ
1H, C(1)H–OP), 3.73 (s, 3H, CH3–O), 3.68–3.59 (m, 1H, C(1 )H–OP),
2.17 (t, J ¼ 7.8 Hz, 2H, CH2–S), 1.54 (t, J ¼ 6.9 Hz, 2H, CH2–CH2S),
1.28 (t, J ¼ 7.2 Hz, 3H, C(2)H3–CH2O), 1.16 (bro, J ¼ 7.5 Hz, 10H, CH
ꢀ
aliphatic), 1.04 (t, J ¼ 7.2 Hz, 3H, C(2 )H3-CH2O), 0.81 (t, J ¼ 6.9 Hz,
3H, CH3–RS).
MS (m/z, %): 365 (M), 364 (M-1, 4), 336 (M-29, 1), 310(M-57, 2),
284 (M-81, 9), 216 (M-148, 2), 163 (M-201, 18), 148 (M-216, 11),
136 (M-229, 26), 106 (M-259, 9), 91 (M-274, 13), 83 (M-282, 49), 72
(M-293, 78), 56 (M-311, 76), 43 (M-292, 97).
13C NMR (CDCl3, 75 MHz): d (ppm): 13.9 (CH3–RS), 16.1 (C(2 )H3
ꢀ
ethoxy), 16.3 (C(2)H3 ethoxy), 22.5 & 22.6 & 29.1 & 29.2 & 31.7 & 36.1
ꢀ
(CH2 alkyl), 48.1(C(1 )H2 ethoxy), 49.7 (C(1)H2 ethoxy), 55.0 (P–CH–N),
62.8 (S–CH2), 63.1 (CH3 methoxy), 113.7 (C3,5 phenyl), 127.3 (C2,6
phenyl), 129.4 (C1 phenyl), 159.2 (C4 phenyl).
((4-Methoxyphenyl)(octylsulfonamido)methyl)phosphonic acid (4b)
Molecular weight: 393 (g/mol), FT-IR (KBr, cmꢃ1): 3606 (stretch NH),
2927 (stretch CH, aromatic), 2858 (stretch CH, aliphatic), 1612
(stretch C¼C, aromatic), 1516 (bending NH), 1462 (bending CH2),
Diethyl(dodecylsulfonamido(4-methoxyphenyl)methyl)phosphonate
(3c)
Molecular weight: 505 (g/mol), FT-IR (KBr, cmꢃ1): 3444 (stretch NH),
2924 (stretch CH, aromatic), 2854 (stretch CH, aliphatic), 1651 1396 (bending CH3), 1307, 1184 (stretch O¼S¼O), 1041 (stretch

4
N. ALIMORADI ET AL.
C–O, OCH3), 960 (stretch CH, oop), 729 (rocking CH2), 547 (bending the protein samples on a 12% slab gel. Protein concentration was
determined by Lowry method28.
NH).
1H NMR (CH3OH-d4, 300 MHz):
d (ppm): 7.38–7.36 (dd,
J ¼ 1.2 Hz, 2H, C(2,6)H aromatic), 6.9–6.87 (dd, J ¼ 6.6 Hz, 2H, C(3,5)
H
Enzyme assay
aromatic), 5.45–5.30 (dd, J ¼ 15.9, 5.9 Hz, H, CH–P), 3.76 (s, 3H,
CH3–O), 2.29–2.26 (m, 2H, CH2–S), 1.59 (t, J ¼ 5.1 Hz, 2H,
CH2–CH2S), 1.25 (bro, 10H, CH aliphatic), 0.88 (t, J ¼ 2.4 Hz, 3H,
CH3–RS).
Inhibition assays for rkbPAP was performed in 96-well multi-titer
plates using a microplate reader. Kinetic measurements were taken
at pH 4.9 (0.1 M sodium citrate buffer with 5% DMSO) at 25 ꢁC
using p-nitrophenyl phosphate (p-NPP) as the substrate at concen-
trations of 1, 2, 4, 6, 8, 10, 12, 14, 18 and 24 mM29. The hydrolysis
of p-NPP by PAP produces p-nitrophenol (p-NP), which has a char-
acteristic intense yellow color at 405 nm with e ¼ 343
Mꢃ1 cmꢃ1 18,26. The concentration of enzyme used was ꢄ60 nM,
while at least eight concentrations of each compound ranged
from 50 to 2000 mM. The IC50 values obtained by “Dose–response
inhibition/variable slopes” equation that was performed using
GraphPad Prism version 5.00 for Windows (GraphPad Software,
San Diego CA, www.graphpad.com). The experimental data are
representative example of three independent experiments. In most
cases, the standard deviations were almost within 5% of the
experimental values, except for some inhibition data points where
standard errors of means were within 10% of the measured values.
p < .05 was considered statistically significant.
MS (m/z, %): 393 (M), 321 (M-71, 12), 313 (M-80, 39), 299 (M-94,
17), 204 (M-189, 5), 176 (M-217, 19), 164 (M-229, 18), 148 (M-245,
25), 135 (M-258, 100).
(Dodecylsulfonamido(4-methoxyphenyl)methyl)phosphonic
(4c)
acid
Molecular weight: 449 (g/mol), FT-IR (KBr, cmꢃ1): 3410 (stretch NH),
2920 (stretch CH, aromatic), 2850 (stretch CH, aliphatic), 1612
(stretch C¼C, aromatic), 1512 (bending NH), 1462 (bending CH2),
1392 (bending CH3), 1307, 1184 (stretch O¼S¼O), 1053 (stretch
C–O, OCH3), 952 (stretch CH, oop), 725 (rocking CH2), 559 (bending
NH).
1H NMR (CH3OH-d4, 300 MHz):
d (ppm): 7.37–7.36 (dd,
J ¼ 4.8 Hz, 2H, C(2,6)H aromatic), 6.88 (t, J ¼ 5.1 Hz, 2H, C(3,5)H aro-
matic), 5.45–5.31 (dd, J ¼ 12.51, 12.57 Hz, H, CH–P), 3.76 (s, 3H,
CH3–O), 2.27 (t, J ¼ 1.8 Hz, 2H, CH2–S), 1.58 (t, J ¼ 5.7 Hz, 2H,
CH2–CH2S), 1.25 (bro, 18H, CH aliphatic), 0.88 (t, J ¼ 5.4 Hz, 3H,
CH3–RS).
MS (m/z, %): 450 (M þ 1), 368(M ꢃ 81, 25), 281 (M ꢃ 169, 12),
233 (M ꢃ 217, 17), 218 (M ꢃ 232, 13), 163 (M ꢃ 286, 33), 148 (M-
301, 40), 135 (M ꢃ 314, 100).
Determination of the inhibition mode and constants
To determination of inhibition mode, the Michaelis–Menten con-
stant (Km) and maximum velocity (Vmax) and inhibition constants
(Ki and KI) values were obtained using Equation (1)29,30 in the pres-
ence of at least four concentrations of each compound which
were selected from concentration–activity curves. The data were
analyzed by nonlinear regression using the program GraphPad
Prism
(Hexadecylsulfonamido(4-methoxyphenyl)methyl)phosphonic acid
(4d)
Molecular weight: 505 (g/mol), FT-IR (KBr, cmꢃ1): 3603 (stretch OH),
3356 (stretch NH), 2916 (stretch CH, aromatic), 2850 (stretch CH,
aliphatic), 1612 (stretch C¼C, aromatic), 1512 (bending NH), 1465
(bending CH2), 1392 (bending CH3), 1311, 1180 (stretch O¼S¼O),
1037 (stretch C–O, OCH3), 952 (stretch CH, oop), 721 (rocking CH2),
555 (bending NH).
Vmax½Sꢅ
v ¼
;
(1)
½Sꢅð1 þ ½Iꢅ=KIÞ þ Kmð1 þ ½Iꢅ=KiÞ
where Ki and KI are the equilibrium dissociation constants for com-
petitive and uncompetitive inhibitor binding, respectively, while, v,
[S] and [I] represent the rate of product formation, the substrate
and the inhibitor concentration, respectively29. It has been found
that there are two inhibitor constants: Ki, which defined as the dis-
sociation constant of the enzyme–inhibitor complex, and KI, which
defined as the dissociation constant of the enzyme–substrate–inhi-
bitor complex. If Ki <KI, the type of enzyme inhibition was defined
as competitive-noncompetitive and if Ki >KI, the type of enzyme
inhibition was defined as uncompetitive–noncompetitive. The type
of enzyme inhibition was defined as pure noncompetitive inhib-
ition when Ki ¼KI30–32. For better visual presentation of data of
inhibition mode, the Lineweaver–Burk plots were also provided.
1H NMR (CH3OH-d4, 300 MHz): d (ppm): 7.36–7.34 (d, J ¼ 4.8 Hz,
2H, C(2,6)H aromatic), 6.86–6.84 (d, J ¼ 4.8 Hz, 2H, C(3,5)H aromatic),
3.74 (s, 3H, CH3–O), 2.27 (s, 2H, CH2–S), 1.57 (s, 2H, CH2–CH2S),
1.25 (s, Hz, 26H, CH aliphatic), 0.86 (t, J ¼ 5.4 Hz, 3H, CH3–RS).
MS (m/z, %): 505 (M), 425 (M ꢃ 80, 27), 338 (M ꢃ 167, 2), 316
(M ꢃ 189, 12), 302 (M ꢃ 203, 17), 289 (M ꢃ 217, 9), 282 (M ꢃ 223, 6),
224 (M ꢃ 281, 77), 212 (M ꢃ 293, 8), 204 (M ꢃ 301, 25), 190
(M ꢃ 315, 100).
Enzyme preparation and purification
Docking studies
PAP from red kidney bean (rkbPAP) was purified following a previ-
ously published protocol8. Briefly, red kidney beans (Phaseolus vul-
garis), provided from local market, were ground and suspended in
0.5 M sodium chloride solution. The suspension was filtered
through a muslin cloth, followed by ethanol fractionation and
ammonium sulfate precipitation. The purple enzyme solution was
dialyzed against several changes of 0.25 M NaCl over a period of
several days. The purple precipitate, which formed in the dialysis
bag was collected by centrifugation. The resulting preparation
stored at 4 ꢁC in 0.5 M NaCl. The protein identity and purity was
tested by SDS–polyacrylamide gel electrophoresis (SDS–PAGE) and
The structures of molecules were drawn and optimized using
HyperChem (HyperCube Inc., Gainesville, FL). Semi-empirical AM1
method with Polak–Ribiere algorithm until the root mean square
gradient of 0.01 kcal molꢃ1 was used as the optimization method.
The crystal structure of rkbPAP retrieved from the Protein Data
Bank (PDB code: 2QFR) was used. Then, after determining Kollman
united atom charges33 and merging nonpolar hydrogens, rotatable
bonds were assigned. Using autoGrid tool, the grid maps (one for
each atom type in the ligand, and one for electrostatic interac-
tions) were constructed adequately large to include the active site
by comparing with molecular weight protein markers, by loading of enzyme as well as significant regions of the surrounding

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
5
surface. In all the cases, a grid map of 60 points in each Cartesian enzymes, the similarities in substrate binding pockets, and the
direction besides a grid-point spacing of 0.375 Å (a quarter of the
carbon–carbon single bond) were generated. By the ligand loca-
tion in the complex of ligand–enzyme, the maps were centered
on the binding site (Zn atom of the enzyme), searching effective
interactions with the functional groups. Based on Lamarckian
Genetic Algorithm34, using the pseudo-Solis and Wets local search
method35, AutoDock Tools was employed to produce both grid
and docking parameter files, that is .gpf and .dpf files. Applying
0.5 Å clustering tolerance to construct clusters of the closest com-
pounds, the initial coordinates of the ligand were used as the ref-
erence structure. Finally, docking results (enzyme–inhibitor
complex) were visualized using Viewer Lite 4.2.
similarities of inhibition constants for a range of structurally differ-
ent inhibitors reported for several animal (including human) and
plant PAPs18,26. The rkbPAP was purified from red kidney bean
according to the method described by Beck et al.8 The SDS–PAGE
analysis is shown in Figure 1, which clearly indicates the reason-
able purity of the isolated PAP. The densitometry of bands indi-
cates that the mass fraction of the purified enzyme is around 0.90
(ꢄ90% purity). It is noteworthy that the plant enzymes are mostly
dimeric glycoproteins, and have subunit molecular masses of
50–60 kD. As shown in the figure, differential glycosylation of the
subunits, leading to a heterogeneity in the molecular mass of the
subunits, has been reported for kidney bean PAP, which is a
dimeric glycoprotein (molecular mass 110 kD) with an inter-sub-
unit disulfide linkage11. Activity staining on PAGE gel also showed
that only the ꢄ50 kDa bands had esterase activity, confirming that
there is no contaminant phosphatase enzymes other than desired
PAP (data not shown).
The isolated rkbPAP was assayed using p-NPP as a substrate.
The obtained Km and Vmax by fitting data to the Michaelis–Menten
equation were 12.6 1.4 and 48 6, respectively. We also
designed/synthesized and evaluated the catalytic effects of a num-
ber of new inhibitor candidates of rkbPAP, based on the alkylsulfo-
namido(4-methoxyphenyl)methyl)phosphonic acid scaffold. The
inhibitory effects of derivatives 3a–d and 4a–d as well as tartrate
as the positive control were tested against rkbPAP at pH 4.938. In
order to identifying the PAP inhibition mode of test compounds
and determining apparent kinetic parameters, the kinetic data
were analyzed using Equation (1). The IC50 and inhibition constant
(Ki and KI) values of test compounds have been summarized in
Table 1. As it was expected, PAP activity was weakly inhibited
by tartrate, which is in good agreement with previous
reports11,38–40. Most of the synthesized compounds demonstrated
their inhibitory potentials on PAP in a dose-dependent manner.
Results and discussion
Chemistry
The conversion of 4-methoxybenzaldehyde (1) into diethyl
(amino(4-metoxybenzyl)-methyl)phosphonate was achieved by
heating 1 with a mixture of ammonium acetate and diethyl
phosphite over activated molecular sieves in ethanol, according
to a literature procedure reported for the synthesis of diethyl
a-aminobenzylphosphonate from benzaldehyde (Scheme 1)36.
Introducing hydrogen chloride gas to the free amine product
gave its corresponding hydrochloride salt
2 in 23% overall
yield36. Alkylation of 2 to give the sulfonamides 3a–d was
achieved with adding appropriate sulfonyl chlorides and DIPEA
to 2 in chloroform. The reaction mixture was stirred under nitro-
gen atmosphere for 24 h. The final synthetic step to produce
the newly designed inhibitors 4a–d required the cleavage of the
phosphonate ester bonds of 3a–d to give their corresponding
free phosphonic acids. This was achieved by stirring 3a–d with
a mixture of potassium iodide and trimethylsilyl chloride in
chloroform at 25 ꢁC for 24 h that giving the desired products in
moderate yields.
Table 1. Kinetic data for inhibitors against rkbPAP at pH 4.9.
Inhibitor name
IC50 (lM)
Ki (lM)
KI (lM)
Enzyme purification and biological activity measurements
3a and 3b
Very weak inhibitor
–
–
3c
3d
759
92
202 11
211
36
–
103 5
8.2 1.4
8
3
Although human PAP (hPAP) would be the best choice for our
purposes, hPAP can only be obtained in minute quantities using a
13
1
4a and 4b
Very weak inhibitor
–
baculoviral recombinant expression system37. However, as stated 4c
421
13
3315
95
9
4d
1.1 0.2
783 41
earlier, rkbPAP is a valid model of hPAP (see Figure 1), as indicated
by the highly conserved active sites across these different
Tartrate
–
Figure 1. (Left) Active site of all known PAPs has an Fe3þ in the active site, which coordinates with an invariant tyrosine ligand, producing a ligand to metal CT transi-
tion giving rise to the characteristic purple color of the enzyme. PAPs active site also contains a divalent metal ion which may be either Zn2þ, Mn2þ or Fe2þ. The seven
metal ion-coordinating residues are invariant among PAPs from different sources. Residue labels refer to the sequences of red kidney bean PAP and pig PAP (in brack-
ets or parentheses). Taken from Schenk et al.5 (Right) SDS–PAGE illustrates PAP purity (lane 1). M, molecular size markers.

6
N. ALIMORADI ET AL.
Among
the
evaluated
compounds,
3d
(IC50 ¼92 mM, other on the 1/S (x) axis, which is characteristic of noncompeti-
Ki ¼ 13 1 mM), 4c (IC50 ¼421 mM, Ki ¼ 95 9 mM) and 4d tive inhibition, so Ki ꢇKI. Such noncompetitive mode inhibition
has also been previously reported for hexyl-derivative of a-alkox-
(IC50 ¼13 mM, Ki ¼ 1.1 0.2 mM), were the most potent inhibitors; of
ynaphthylmethylphosphonic acid18. For longer chain-length
inhibitors (3d and 4d), the lines of the Lineweaver–Burke plot
which 3d and 4d were recognized as potent inhibitors of PAP
even stronger than the standard inhibitor11,38–40. As can be seen,
intersect at a single point between x- and y-axis, close to the
1/S axis, indicate that PAP activity was inhibited with mixed
manner by these compounds. On the other hand, since Ki <KI,
the exact mechanism of inhibition is competitive–noncompeti-
tive18,23,26,31. In agreement with this mode change, McGeary
et al. reported that longer alkyl chains of a-alkoxynaphthylme-
thylphosphonic acid derivatives inhibit rkbPAP and pPAP with
mixed (competitive–noncompetitive) manner18. This behavior
may reflect a stronger anchoring effect of the longer alkyl
chains into the groove adjacent to the active site of the
enzyme, which would favor partially competitive inhibition.
Furthermore, the replacement of the diethyl phosphonate group
of series 3 by phosphonate in series 4 has a minor decrement
effect on the inhibitory effect of compound and does not alter
the mode of inhibition, since probably this moiety is not bind/
coordinate to bimetal/binuclear center.
the Ki values are in reasonable agreement with the IC50 values,
comparable at least with the best of the naphthalenemethylphos-
phonic acid derivatives3,18. Also, the 3a, 3b, 4a and 4b exhibited
negligible inhibitions of PAP, in millimolar range. Thus, these com-
pounds were excluded from further studies (determination of
inhibition mode).
Although an in depth SAR analysis is currently impossible, it
appears that a major improve in rkbPAP inhibitory activity corre-
lates with increasing the asymmetric hydrophobicity of the com-
pounds. It appears that inhibitor potency increases with increase
the length of alkyl chain in alkylsulfonamide moieties (Table 1
and Scheme 1), so that the largest effect on inhibition was
achieved with the hexadecyl moiety. Substitution at the dodecyl
(in 3c/4c) with hexadecyl (in 3d/4d) greatly decrease the inhib-
ition constant of the inhibitor, suggesting an extremely large
hydrophobic or sterically tolerant region near/not into the phos-
phate binding site of the enzyme as well as a possible prefer-
ence for the long-chain acyl (ꢆ C12) derivatives. McGeary et al.
was reported a similar trend for the rkbPAP and pig PAP
enzymes, that is, the longer the alkyl chain the more potent
inhibitor of PAPs18.
Molecular docking studies
Molecular docking studies on binding modes are essential to eluci-
date key structural characteristics and interactions and they pro-
vide helpful data for designing effective PAP inhibitors41. Hence,
to make the rational design of novel and more selective PAP
inhibitors possible, molecular docking was carried out on PAP
binding pocket using a set of PAP inhibitors shown in Scheme 1.
As well as RMSD cluster analysis, AutoDock also uses binding free
energy assessment to assign the best binding conformation.
The inhibition modes of the compounds can be elicited by
comparing values of Ki and KI and represented by
Lineweaver–Burke plot. Typical Lineweaver–Burk plots for inhibi-
tory activity of 3c and 4d as representative have been demon-
strated in Figure 2. If the Lineweaver–Burke plot shows
crisscross lines at a single point on the 1/V (y) axis, it is indica-
tive of competitive inhibition. Also, the trend lines of Energies estimated by AutoDock are described by intermolecular
Lineweaver–Burke plot for 3c and 4c compounds intersect each energy (including van der Waals, hydrogen bonding, desolvation,
(A)
4
3
2
1
0
80
60
40
20
0
-0.06
-0.03
0
0.03
-0.2
0
0.2
0.4
0.6
0.8
1
1.2
-1
1/[p-NPP](mM
)
(B)
0.8
0.6
0.4
0.2
0
16
12
8
4
-0.09
-0.06
-0.03
0
0.03
0
-0.2
0
0.2
0.4
0.6
0.8
1
1.2
-1
1/[p-NPP](mM
)
Figure 2. Typical Lineweaver–Burk plots for inhibitory activity of synthetic compounds against rkbPAP. The data represent the average of 3–5 experiments.
(A) Lineweaver–Burk plot of rkbPAP activity in the absence (ꢈ) and the presence of 300 (ꢁ), 600 (~) and 1200 lM of 3c (~). (B) Lineweaver–Burk plot of rkbPAP activ-
ity in the absence (·) and the presence of 10 (ꢁ), 20 (~) and 40 lM of 4d (~).

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
7
Figure 3. The three-dimensional representation of docking result of 3d in the close vicinity of rkbPAP binding pocket (left). Surface and stick representation of the pre-
dicted binding mode of 3d to the enzyme based on docking simulations (right).
Figure 4. The two-dimensional representation of docking result of 3d compound with binding pocket of rkbPAP.
Table 2. Interaction, van der Waals and electrostatic energies for selected resi-
dues of PAP and 3d.
and electrostatic energies), internal energy, and torsional free
energy42. Among these calculated energies by AutoDock, the first
Interaction
energy (kcal/mol)
VDW interaction
energy (kcal/mol)
Electrostatic interaction
energy (kcal/mol)
two provide the docking energy, while the sum of the first and
the third items account for the binding energy. Among all interac-
tions occurring in the active site, the electrostatic interaction
between the ligand and the enzyme is the most significant,
Residue
PHE206
GLU299
GLY200
ꢃ12.1
ꢃ11.3
ꢃ7.45
ꢃ7.32
ꢃ3.67
ꢃ2.60
ꢃ1.22
ꢃ0.27
ꢃ3.78
ꢃ3.86
ꢃ0.18
0.02
ꢃ8.30
ꢃ7.47
ꢃ7.27
ꢃ7.35
ꢃ2.01
ꢃ17.8
ꢃ0.60
0.0
because in most cases it can assign the strength of binding and ASP169
the exact position of the inhibitor in the binding site energy42
.
TYR256
SER287
MET285
LEU289
ꢃ1.66
15.2
The docking results show that all of the studied compounds
occupy an almost similar space in the binding site. Also, the calcu-
lated binding affinities using computational modeling correlate
well with measured inhibition constants (results not shown).
Unexpectedly, modeling suggests that the phosphonate moiety of
the 3d inhibitor does not bind to the dimetal center in the active
site of rkbPAP. Furthermore, the alkyl chain of the inhibitor binds
to the groove on the surface of the enzyme. Other part of mole-
cules including polar atoms and aromatic portions are placed on
the hydrophilic patch around the active site, which is in good
agreement with the obtained inhibition modes. The best possible
binding mode of 3d as the most biologically active compound in
the rkbPAP binding site is illustrated in a three-dimensional
(Figure 3) and two-dimensional space (Figure 4).
ꢃ0.62
ꢃ0.27
energetic influences of critical residues on the binding. The resi-
dues were separately studied, and the Van der Waals, electrostatic
and interaction energies of these residues and 3d were deter-
mined. These include Phe-206, Glu-299, Gly-200, Asp-169, Tyr-256,
Ser-287, Met-285 and Leu-289 (Table 2). Energy of interaction
between individual residues and ligand was calculated with the
“Calculate Interaction Energy” protocol encoded in Discovery
Studio. The complexes were typed with CHARMM force field. All
parameters used were kept at their default settings. The residue-
based decomposition of interaction energies identified several
critical residues of PAP. Table 2 lists the calculated energies contri-
butions of these key residues of interest. As can be seen, major
On the other hand, to obtain a detailed description of the
effects of individual residues on binding affinity, a per-residue favorable energy contributions originate predominantly from Phe-
decomposition of the total energy was carried out to evaluate the 206 and Glu-299. The methoxy phenyl core of 3d is anchored by

8
N. ALIMORADI ET AL.
an aromatic stacking interaction with Phe-206, whereas Glu-299 is
one of major contributor of van der Waals interactions.
2. Oddie G, Schenk G, Angel N, et al. Structure, function, and
regulation of tartrate-resistant acid phosphatase. Bone
2000;27:575–84.
As it can be concluded from Figures 3, 4 and Table 2, the
major part of the binding cavity of rkbPAP is hydrophilic, which
allows for the tight binding of polar substrates and inhibitors. A
long apolar groove adjacent to the active site was identified as an
additional potential point of interaction of hydrophobic aliphatic
chain and is required for recognition and directionality of the sub-
strate aliphatic chain functionality18. Also, hydrogens of NH2 group
of Asn-201 side chain and sulfonamide group of ligand, also
hydrogens of NH2 group of Phe-206 backbone and oxygen of
phosphonate group of ligand form hydrogen bonds (3.12 and
2.90 Å, respectively). It is was interesting to note that the aliphatic
chain orients inside the binding cavity and interacts by both
hydrophobic as well as hydrophilic interactions (Table 2) with
amino acids such as Leu-289 and Met-303. With respect to the
docking results (Figures 3 and 4) Ser-252, Tyr-256, Ser-287, Met-
303 and Leu-289 assemble one hydrophobic cluster, which accom-
modates the aliphatic hydrophobic chain moiety of the ligand.
Taking literature26 into account, similar binding orientations of
inhibitors in the active sites of pig, red kidney bean and hPAP,
may support the contention that the inhibitory potencies obtained
using rkbPAP can be extrapolated to hPAP.
3. Schwender CF, Beers SA, Malloy E, et al. 1-naphthylmethyl-
phosphonic acid derivatives as osteoclastic acid phosphatase
inhibitors. Bioorg Med Chem Lett 1995;5:1801–6.
4. Hayman AR, Jones SJ, Boyde A, et al. Mice lacking tartrate-
resistant acid phosphatase (Acp 5) have disrupted endo-
chondral ossification and mild osteopetrosis. Development
1996;122:3151–62.
ꢀ
5. Schenk G, Mitic N, Hanson GR, Comba P. Purple acid phos-
phatase: a journey into the function and mechanism of a
colorful enzyme. Coord Chem Rev 2013;257:473–82.
6. Bernhardt PV, Schenk G, Wilson GJ. Direct electrochemistry
of porcine purple acid phosphatase (uteroferrin).
Biochemistry 2004;43:10387–92.
7. Wang DL, Holz RC, David SS, et al. Electrochemical proper-
ties of the diiron core of uteroferrin and its anion com-
plexes. Biochemistry 1991;30:8187–94.
8. Beck JL, McConachie LA, Summors AC, et al. Properties of a
purple phosphatase from red kidney bean: a zinc-iron metal-
loenzyme. BBA-Protein Struct M 1986;869:61–8.
9. Leung EW, Teixeira M, Guddat LW, et al. Structure, function
and diversity of plant purple acid phosphatases. Curr Topic
Plant Biol 2007;8:21–31.
10. Schenk G, Guddat L, Ge Y, et al. Identification of mamma-
lian-like purple acid phosphatases in a wide range of plants.
Gene 2000;250:117–25.
11. Cashikar AG, Kumaresan R, Rao NM. Biochemical character-
ization and subcellular localization of the red kidney bean
purple acid phosphatase. Plant Physiol 1997;114:907–15.
12. Nuttleman PR, Roberts RM. Transfer of iron from uteroferrin
(purple acid phosphatase) to transferrin related to acid phos-
phatase activity. J Biol Chem 1990;265:12192–9.
Conclusions
To the best of our knowledge, there are no published reports in
the literature on the application of alkylsulfonamidophenyl)me-
thyl)phosphonic acid derivatives as potential PAP inhibitors. In the
current work, we identified these new derivatives of alkylsulfona-
mido(4-methoxyphenyl)methyl)phosphonic acid, as potent inhibi-
tors of rkbPAP. The hexadecyl derivative (4d, with Ki value of
1.1 mM) was found to be the most active compared to compounds
contain shorter alkyl chains. In addition, it was illustrated that the
inhibitor bound in the vicinity of the enzyme active site showed
mixed mode inhibition due to partial competition whit substrate.
Considering the crucial role(s) of mammalian PAPs in bone metab-
olism, such inhibitors are anticipated to be promising lead com-
pounds in the development of new anti-osteoporotic
chemotherapeutics.
13. Sibille J-C, Doi K, Aisen P. Hydroxyl radical formation and
iron-binding proteins. Stimulation by the purple acid phos-
phatases. J Biol Chem 1987;262:59–62.
€ €
14. Raisanen SR, Alatalo SL, Ylipahkala H, et al. Macrophages
overexpressing tartrate-resistant acid phosphatase show
altered profile of free radical production and enhanced cap-
acity of bacterial killing. Biochem Biophys Res Commun
2005;331:120–6.
15. Boonen S. Bisphosphonate efficacy and clinical trials for
postmenopausal osteoporosis: similarities and differences.
Bone 2007;40:S26–31.
16. Halleen JM, Tiitinen SL, Ylipahkala H, et al. Tartrate-resistant
acid phosphatase 5b (TRACP 5b) as a marker of bone resorp-
tion. Clin Lab 2006;52:499–510.
Acknowledgement
This work was performed in partial fulfillment of the requirements
for Pharm. D. of N. Alimoradi, in Faculty of Pharmacy, Kermanshah
University of Medical Sciences, Kermanshah, Iran.
17. Vella P, McGeary RP, Gahan LR, Schenk G. Tartrate-resistant
acid phosphatase: a target for anti-osteoporotic chemothera-
peutics. Curr Enzyme Inhib 2010;6:118–29.
Disclosure statement
The authors declare that they have no conflict of interests.
18. McGeary RP, Vella P, Mak JY, et al. Inhibition of purple acid
phosphatase with alpha-alkoxynaphthylmethylphosphonic
acids. Bioorg Med Chem Lett 2009;19:163–6.
Funding
19. Susi F, Goldhaber P, Jennings JM. Histochemical and bio-
chemical study of acid phosphatase in resorbing bone in
culture. Am J Physiol 1966;211:959–62.
20. Angel NZ, Walsh N, Forwood MR, et al. Transgenic mice
overexpressing tartrate-resistant acid phosphatase exhibit an
The financial support [Grant No. 92047] from the Research Council
of Kermanshah University of Medical Sciences is gratefully
acknowledged.
increased rate of bone turnover.
J Bone Miner Res
2000;15:103–10.
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