Penicillin inhibitors of purple acid phosphatase

Article abstract of DOI:10.1016/j.bmcl.2012.01.123

Purple acid phosphatases (PAPs) are binuclear metallohydrolases that have a multitude of biological functions and are found in fungi, bacteria, plants and animals. In mammals, PAP activity is linked with bone resorption and over-expression can lead to bone disorders such as osteoporosis. PAP is therefore an attractive target for the development of drugs to treat this disease. A series of penicillin conjugates, in which 6-aminopenicillanic acid was acylated with aromatic acid chlorides, has been prepared and assayed against pig PAP. The binding mode of most of these conjugates is purely competitive, and some members of this class have potencies comparable to the best PAP inhibitors yet reported. The structurally related penicillin G was shown to be neither an inhibitor nor a substrate for pig PAP. Molecular modelling has been used to examine the binding modes of these compounds in the active site of the enzyme and to rationalise their activities.

Full text of DOI:10.1016/j.bmcl.2012.01.123

Bioorganic & Medicinal Chemistry Letters 22 (2012) 2555–2559
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Penicillin inhibitors of purple acid phosphatase
Faridoon a,b, Waleed M. Hussein a,c, Nazar Ul Islam b, Luke W. Guddat a, Gerhard Schenk a,d
,
Ross P. McGeary a,e,
⇑
a The University of Queensland, School of Chemistry and Molecular Biosciences, Brisbane, Qld 4072, Australia
b Institute of Chemical Sciences, University of Peshawar, Peshawar-25120, Pakistan
c Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Helwan University, Ein Helwan, Egypt
d National University of Ireland-Maynooth, Department of Chemistry, Maynooth, Co. Kildare, Ireland
e 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 metallohydrolases that have a multitude of biological
functions and are found in fungi, bacteria, plants and animals. In mammals, PAP activity is linked with
bone resorption and over-expression can lead to bone disorders such as osteoporosis. PAP is therefore
an attractive target for the development of drugs to treat this disease. A series of penicillin conjugates,
in which 6-aminopenicillanic acid was acylated with aromatic acid chlorides, has been prepared and
assayed against pig PAP. The binding mode of most of these conjugates is purely competitive, and some
members of this class have potencies comparable to the best PAP inhibitors yet reported. The structurally
related penicillin G was shown to be neither an inhibitor nor a substrate for pig PAP. Molecular modelling
has been used to examine the binding modes of these compounds in the active site of the enzyme and to
rationalise their activities.
Received 4 January 2012
Revised 26 January 2012
Accepted 30 January 2012
Available online 4 February 2012
Keywords:
Purple acid phosphatase (PAP)
Osteoporosis
Penicillin
Inhibition assays
Crown Copyright Ó 2012 Published by Elsevier Ltd. All rights reserved.
Purple acid phosphatases (PAPs)1 are binuclear metallohydro-
lases which are associated with disorders such as osteoporosis,2–4
hairy cell leukaemia,5 Gaucher disease,6 and AIDS.7 These enzymes
have a characteristic purple colour due to a charge transfer transi-
tion between a tyrosine phenolate and Fe(III) in the active site.8
PAPs catalyse the hydrolysis of various phosphorylated substrates
at neutral to acidic pH values according to the equation:1
Many small anions including fluoride25–28 and tetrahedral oxoa-
nions such as phosphate, arsenate, vanadates,29 tungstate and
molybdate30,31 have been reported as weak PAP inhibitors. Simple
phosphonate-containing molecules with pendant metal binding
groups such as carboxylate and thiol,32 and several modified
phosphotyrosine-containing tripeptides33 are also PAP inhibitors.
Recently, our group reported a series of
phosphonic acids which inhibit pig PAP (pPAP) with Ki values as
low as 17 M, and inhibit red kidney bean PAP (rkbPAP) with Ki
values as low as 4
M.20 We have also reported the activities of
structurally related compounds, acylated -aminonaphthylmethyl-
phos phonic acids, which have Ki values as low as 8 M against pPAP
and 5
M against rkbPAP.22
a-alkoxynaphthylmethyl
RO-PO23ꢀ þ H2O ! ROH þ HPO2ꢀ
4
l
l
Mammalian PAPs are monomeric with a molecular mass of
35 kDa and contain a redox active Fe(III)–Fe(III/II) centre in their
active site.9,10 Plant PAPs are homodimeric with a molecular mass
of 110 kDa and a redox inactive Fe(III)–Zn(II) or Fe(III)–Mn(II)
centre.1,11–13 The mammalian enzyme is secreted by osteoclasts14
into the bone-resorptive space, where it is required for normal bone
resorption.15 Osteoporosis, a decrease in bone mineral density,
occurs when the rate of bone resorption exceeds that of bone forma-
tion, creating an imbalance in the dynamic bone remodelling pro-
cess.16 The association of elevated PAP levels in the serum with
the onset of osteoporosis14,17–19 has prompted us to develop potent
inhibitors of this enzyme.20–22 Whilst clinical treatments are
available for osteoporosis, particularly bisphosphonates which inhi-
bit farnesyl pyrophosphate synthase in osteoclasts,23,24 these drugs
have significant side effects and compliance issues.
a
l
l
We report here our efforts at identifying potent inhibitors of
pPAP by exploring the inhibitory effects of penicillin conjugates.
H
N
R
H
N
H
H2N
O
S
S
Me
Me
Me
Me
a
O
N
O
CO2H
CO2H
1
2a-h
a, R = 4-(PhCO); b, R = 4-(2-ClC6H4CO); c, R = 4-(4-ClC6H4CO);
d, R = 4-(3-NO2C6H4CO); e, R = 4-(4-NO2C6H4CO); f, R = 3-(4-MeC6H4CO);
g, R = 3-(piperidinylcarbonyl); h, R = 4-OAc
⇑
Corresponding author. Tel.: +61 7 3365 3955; fax: +61 7 3346 3249.
E-mail address: r.mcgeary@uq.edu.au (R.P. McGeary).
Scheme 1. Reagents and conditions: (a) ArCOCl, 2% aq NaHCO3, 0 °C, acetone, 2–4 h
71% (2a); 65% (2b); 51% (2c); 60% (2d); 66% (2e); 53% (2f); 51% (2g); 53% (2h).
0960-894X/$ - see front matter Crown Copyright Ó 2012 Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2012.01.123

2556
Faridoon et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2555–2559
O
O
CO2H
a
b
Cl
CO2H
Cl
Cl
Me
Scheme 2. Synthesis of the aryl carboxylic acid used for the preparation of penicillin conjugate 2c. Reagents and conditions: (a) PhMe, P2O5–SiO2,
AcOH, , 4 h (35%).
D (49%); (b) Jones reagent,
D
O
O
HO2C
CO2Me
CO2Me
CO2H
N
N
a
b
Scheme 3. Synthesis of the aryl carboxylic acid used for the preparation of penicillin conjugate 2g. Reagents and conditions: (a) Me3COCl, Et3N, piperidine, CH2Cl2, rt, 18 h
(78%); (b) LiOHꢁH2O, MeOH–H2O, rt, 18h (80%).
Merck scientists pioneered the investigation of b-lactams as
potential therapeutics other than for their common use as antibiot-
ics. b-Lactams have been identified as irreversible inactivators of
many classes of serine and cysteine proteases, including porcine
pancreatic elastase, human neutrophil elastase, Escherichia coli
signal peptidase, prostate-specific antigen, cathepsin G, thrombin,
chymotrypsin, trypsin, plasmin, human cytomegalovirus protease,
poliovirus and human rhinovirus 3C proteases, and papain.34 The
application of b-lactams as reversible enzyme inhibitors has been lit-
tle explored, although reversible b-lactam inhibitors of human leu-
cocyte elastase and porcine pancreatic elastase,35 carboxypeptidase
A,36 and galactosidase37 have been reported. The structural and
functional similarities between PAPs and the b-lactam-degrading
metallo-b-lactamases (both enzymes are binuclear metallohydro-
lases38) prompted us to examine whether suitably functionalised
b-lactam antibiotics might inhibit the activity of PAP. Here, we dis-
close that penicillin conjugates (2a–h) are good inhibitors of pPAP,
comparable in potency to the most effective PAP inhibitors reported
to date.
The conjugates 2a–h were prepared by acylation of the sensi-
tive 6-aminopenicillanic acid (6-APA) 1 with different aromatic
acid chlorides in a mixture of aqueous sodium bicarbonate solution
and acetone (Scheme 1). Compound 2i is commercially available
penicillin G.
Compound 2a was prepared using commercially available 4-
benzoylbenzoic acid. The syntheses of the aromatic carboxylic
acids required to prepare compounds 2b and 2d–f have been re-
ported by us elsewhere.39 The preparation of the aromatic carbox-
ylic acid required for the synthesis of compound 2c is shown in
Scheme 2, and the synthesis of the carboxylic acid used in the
preparation of compound 2g is shown in Scheme 3. 4-Acetoxyben-
zoic acid, required for the synthesis of compound 2h, was prepared
by acetylation of 4-hydroxybenzoic acid40 (see Supplementary
data).
compound 2i, penicillin G, has no inhibitory effect on pPAP. This
is surprising, given the close structural similarity between com-
pound 2i and the inhibitory compounds listed in Table 1.
Table 1
Kinetic data for inhibitors against pPAP at pH 4.9a
b
c
Inhibitors
Kic
(
l
M) Kiuc (lM)
O
H
N
H
N
d
S
2a 26 12
—
—
—
—
Me
Me
O
O
O
CO2H
Cl
O
H
N
H
N
S
2b 32
2c 12
2d 23
2e 17
8
4
8
7
Me
Me
O
CO2H
O
H
H
S
N
Cl
Me
Me
O
N
O
CO2H
O
H
N
H
S
Me
Me
NO2
O
N
O
CO2H
The inhibitory effects of the penicillin conjugates 2a–i were
tested against pPAP at pH 4.9 using a standard spectrophotometric
assay, with para-nitrophenyl phosphate as substrate, as described
elsewhere.20,22,25–29 The determined competitive (Kic) and uncom-
petitive (Kiuc) inhibition constants are listed in Table 1.
For inhibitors 2a–f,h, only competitive inhibition was observed
against pPAP. We found that penicillin conjugates with para-
substituted 4-benzoylbenzoic acid side chains (2c and 2e) are the
O
H
H
N
S
—
—
O2N
Me
Me
O
N
O
CO2H
Me
most potent, with Kic values of 12 lM and 17 lM, respectively.
H
H
N
To assess the effect of a nitrogen heterocyclic ring on the inhibitory
activity, we replaced the terminal aromatic ring of 2f with piperi-
dine in compound 2g. However, the kinetic data showed that 2g
has reduced inhibitory potency and, unlike the other inhibitors
listed in Table 1, both competitive and uncompetitive inhibition
are observed. Another intriguing result shown in Table 1 is that
S
Me
Me
2f 60 59
O
O
N
O
CO2H

Faridoon et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2555–2559
2557
To investigate the possible binding modes of the most potent
inhibitor, 2c, to pPAP, in silico docking was employed using Molegro
Virtual Docker.41 The pPAP crystal structure (PDB Code: 1UTE)42
was used in this study. To prepare the crystal structure for docking,
the Molegro Virtual Docker was used to define the active site, set
charges of +3 for Fe1 and +2 for Fe2, and to assign Phe 56, Gln 151
and Phe 244 as flexible residues. PyMol was used to visualise the
final docking results.
Table 1 (continued)
b
c
Inhibitors
Kic (lM) Kiuc (lM)
H
N
H
N
N
O
S
Me
Me
2g 241 207 491 558
O
O
CO2H
The lowest energy binding orientation of 2c in the active site of
pPAP is shown in Figure 1. Modelling suggests that 2c binds close
to the two metal ions via its carboxylate group, with one oxygen
bridging the metal ions (with O–M distances of 2.0 Å (Fe1) and
3.3 Å (Fe2)) and the other oxygen atom of the carboxylate group in
2c binding weakly to the two metal ions (with O–M distances of
4.0 Å (Fe1) and 3.3 Å (Fe2)). In addition, the oxygen of the carbonyl
group in the b-lactam ring of 2c forms a hydrogen bond to the NH
group of the imidazole side chain of His 223 (N–O distance 2.6 Å)
and there is another hydrogen bond between the nitrogen atom of
the b-lactam ring and the NH group of the imidazole side chain of
His 195 (N–N distance 3.2 Å). There is also a hydrophobic interaction
between the benzyl group of Phe 244 and the chlorobenzene moiety
of 2c. Importantly, modelling predicts a favourable interaction be-
tween the negatively charged carboxylate side-chain of Asp 246
AcO
H
N
H
N
S
Me
Me
2h 53 17
—
—
O
O
CO2H
H
N
H
N
S
Me
Me
2i
—
O
O
CO2H
a
b
c
Values standard errors of at least two replicates—see Supplementary data.
Kic, competitive inhibition constant.
Kiuc, uncompetitive inhibition constant.
No significant effect.
d
Figure 1. Surface view of the enzyme active site for the highest Molegro Virtual Docker score conformation of pPAP with 2c docked into the active site. Atom colours are as
follows: blue—nitrogen, red—oxygen, white—carbon (on pPAP), sulfur—yellow, green—carbon, cyan—chlorine (on inhibitor), orange—iron active site metals.
Figure 2. Surface view of the enzyme active site for the highest Molegro Virtual Docker score conformation of pPAP with penicillin G (2i) docked into the active site. Atom
colours are as follows: blue—nitrogen, red—oxygen, white—carbon (on pPAP), sulfur—yellow, green—carbon, orange—iron active site metals.

2558
Faridoon et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2555–2559
and the partial positive charge on the biaryl carbonyl carbon; the
distance between this carbon atom and the oxygen atom of the
Asp 246 carboxylate group is 4.8 Å.
Acknowledgments
This work was supported by an Australian Research Council
Grant (DP0986292). Mr. Faridoon is the recipient of a scholarship
from the Pakistan Higher Education Commission (International Re-
search Support Initiative Program).
The proposed binding mode of 2c within the active site of pPAP
through the carboxylate group differs from the mode of binding of
b-lactam antibiotics in the active site of the structurally related
metallo-b-lactamases. In these structures the oxygen atom of the
carbonyl group of the b-lactam is believed to bind to one of the
two zinc ions, activating it for nucleophilic attack by a proximal
metal-bridging hydroxide.43 The modelling shown in Figure 1 is
therefore consistent with our findings that inhibitor 2c is compet-
itive, and is not a substrate for pPAP.
Modelling also led to some suggestions as to why penicillin G
(2i) is not an inhibitor of pPAP. Figure 2 shows the lowest energy
binding orientation of 2i with pPAP. It can be seen that 2i does
not bind in the active site of pPAP, preferring a stabilising electro-
static interaction between the side chain of Arg 27 and the carbox-
ylate group of the 2i (N–O distance 3.5 Å); the b-lactam carbonyl
oxygen also interacts with the Arg 27 side chain. This leaves the ac-
tive site of pPAP exposed. As noted above, we suggest that a con-
tributing factor for the potency of inhibitor 2c is the favourable
interaction between the biaryl carbonyl group and the side chain
of Asp 246 (Fig. 1). Penicillin G (2i) lacks this carbonyl group, and
so cannot interact in this way with the side chain of Asp 246. It
is notable that the most potent of our inhibitors listed in Table 1
bear the same 1,4-dicarbonylbenzene moiety.
Supplementary data
Supplementary data (experimental procedures for the prepara-
tion of compounds 2a–h and details of the kinetic assayed per-
formed) associated with this article can be found, in the online
version, at doi:10.1016/j.bmcl.2012.01.123.
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Again this modelling is consistent with our observations that 2i
is not an inhibitor, nor was it a substrate for pPAP under our assay
conditions. The stability of b-lactams towards pPAP was further
confirmed using the metallo-b-lactamase substrate, cephem CEN-
TA 3. This compound generates the intensely yellow 3-carboxy-
4-nitrobenzenethiolate anion on hydrolysis of its b-lactam ring.
No such colour developed when CENTA was incubated with pPAP
under our assay conditions, confirming that this compound is not
a substrate for pPAP.
H
H
N
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O
N
S
O
CO2H
NO2
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potency could be achieved by incorporating into these molecules
hydrophobic naphthalenes to maximise interactions with surface
hydrophobic patches on the enzyme.22 Efforts are underway to
co-crystallise inhibitor 2c with pPAP to validate our modelling
results.
´
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In summary, 6-aminopenicillanic acid has been coupled with a
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