Purple acid phosphatase inhibitors as leads for osteoporosis chemotherapeutics

Article abstract of DOI:10.1016/j.ejmech.2018.08.004

Purple acid phosphatases (PAPs) are metalloenzymes that catalyse the hydrolysis of phosphate esters under acidic conditions. Their active site contains a Fe(III)Fe(II) metal centre in mammals and a Fe(III)Zn(II) or Fe(III)Mn(II) metal centre in plants. In humans, elevated PAP levels in serum strongly correlate with the progression of osteoporosis and metabolic bone malignancies, which make PAP a target suitable for the development of chemotherapeutics to combat bone ailments. Due to difficulties in obtaining the human enzyme, the corresponding enzymes from red kidney bean and pig have been used previously to develop specific PAP inhibitors. Here, existing lead compounds were further elaborated to create a series of inhibitors with K_i values as low as ～30 μM. The inhibition constants of these compounds were of comparable magnitude for pig and red kidney bean PAPs, indicating that relevant binding interactions are conserved. The crystal structure of red kidney bean PAP in complex with the most potent inhibitor in this series, compound 4f, was solved to 2.40 ? resolution. This inhibitor coordinates directly to the binuclear metal centre in the active site as expected based on its competitive mode of inhibition. Docking simulations predict that this compound binds to human PAP in a similar mode. This study presents the first example of a PAP structure in complex with an inhibitor that is of relevance to the development of anti-osteoporotic chemotherapeutics.

Full text of DOI:10.1016/j.ejmech.2018.08.004

Accepted Manuscript
Purple acid phosphatase inhibitors as leads for osteoporosis chemotherapeutics
Waleed M. Hussein, Daniel Feder, Gerhard Schenk, Luke W. Guddat, Ross P.
McGeary
PII:
S0223-5234(18)30659-7
10.1016/j.ejmech.2018.08.004
EJMECH 10615
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 23 April 2018
Revised Date: 14 June 2018
Accepted Date: 1 August 2018
Please cite this article as: W.M. Hussein, D. Feder, G. Schenk, L.W. Guddat, R.P. McGeary, Purple acid
phosphatase inhibitors as leads for osteoporosis chemotherapeutics, European Journal of Medicinal
Chemistry (2018), doi: 10.1016/j.ejmech.2018.08.004.
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ACCEPTED MANUSCRIPT
Graphical Abstract Revised Manuscript EJMECH-D-18-01046

ACCEPTED MANUSCRIPT
Purple acid phosphatase inhibitors as leads for osteoporosis
chemotherapeutics
a,b
a
a
a
Waleed M. Hussein , Daniel Feder , Gerhard Schenk , Luke W. Guddat and Ross P.
McGeary
a,*
a
The University of Queensland, School of Chemistry and Molecular Biosciences, Brisbane, QLD 4072, Australia
b
Helwan University, Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Ein Helwan,
Helwan, Egypt
*Corresponding author: E-mail address r.mcgeary@uq.edu.au
Key Words: Purple
acid
phosphatase,
Tartrate-resistant
acid
phosphatase,
Metallohydrolases,
Chemotherapeutics, Osteoporosis.
Abstract:
Purple acid phosphatases (PAPs) are metalloenzymes that catalyse the hydrolysis of
phosphate esters under acidic conditions. Their active site contains a Fe(III)Fe(II) metal centre
in mammals and a Fe(III)Zn(II) or Fe(III)Mn(II) metal centre in plants. In humans, elevated
PAP levels in serum strongly correlate with the progression of osteoporosis and metabolic
bone malignancies, which make PAP a target suitable for the development of
chemotherapeutics to combat bone ailments. Due to difficulties in obtaining the human
enzyme, the corresponding enzymes from red kidney bean and pig have been used previously
to develop specific PAP inhibitors. Here, existing lead compounds were further elaborated to
create a series of inhibitors with K values as low as ~30 µM. The inhibition constants of these
i
compounds were of comparable magnitude for pig and red kidney bean PAPs, indicating that
relevant binding interactions are conserved. The crystal structure of red kidney bean PAP in
complex with the most potent inhibitor in this series, compound 4f, was solved to 2.40 Å
resolution. This inhibitor coordinates directly to the binuclear metal centre in the active site
as expected based on its competitive mode of inhibition. Docking simulations predict that this
compound binds to human PAP in a similar mode. This study presents the first example of a
PAP structure in complex with an inhibitor that is of relevance to the development of anti-
osteoporotic chemotherapeutics.
1
. Introduction
Purple acid phosphatases (PAPs, also referred to as tartrate-resistant acid phosphatases) are
metalloenzymes found in animals, plants and fungi [1-5]. They utilise a binuclear metal centre
to catalyse the hydrolysis of phosphate esters and anhydrides under acidic conditions, with an
optimal pH ~5 [6-12]. The overall reaction is given in Equation 1.
1

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PAPs are non-specific enzymes that can dephosphorylate diverse substrates, including ATP,
ADP and para-nitrophenyl phosphate (pNPP) [1-4, 13], as well as phosphoproteins such as
osteopontin and bone sialoprotein [14-15]. Several roles for mammalian PAPs have been
suggested. Pig PAP found in the allantoic fluid of pregnant sows is believed to transport iron
from the mother to the foetus during gestation [16], and therefore the enzyme is also referred
to as uteroferrin [6, 17-19]. In humans, PAP function appears to be tissue-specific; the
enzyme is involved in the inflammatory response of antigen-presenting cells [20], as well as
in bone resorptive processes in osteoclasts [15, 21]. The diverse roles of mammalian PAP are
associated with the enzyme’s bifunctional character; apart from its hydrolytic activity it can
also act as a Fenton catalyst due to the presence of its redox active Fe(III)Fe(II/III) centre
[
22]. It has been hypothesised that the reactive oxygen species (ROS)-generating activity of
PAP could play a role in collagen degradation [23]. Although the substrate(s) of PAP in bone
tissue is unknown, studies with transgenic mice clearly established the enzyme’s function in
bone turnover. Mice overexpressing the PAP gene are found to be osteoporotic [14] and show
an increase in bone turnover [24]. In contrast, mice deficient in PAP display a phenotype
characteristic of osteopetrosis [24], with increased bone mineral density, abnormal
ossification [14] and defects in the resorption and mineralisation of growing bone [25]. Thus,
PAP has emerged as a target for inhibitors that may lead to the development of novel
treatments for osteoporosis, bone malignancies and metabolic bone diseases.
In the absence of effective recombinant expression systems for human PAP the enzymes
extracted from pig uterine fluid (i.e. uteroferrin) and red kidney bean PAP (rkbPAP) have
been used as models to test the efficacy of inhibitors. Despite the modest sequence similarity
between rkbPAP and mammalian PAPs [26-27] its inclusion into an inhibitor design program
is warranted on the basis of similar (i) substrate specificities, (ii) interactions with known
2

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inhibitors and (iii) mechanism [19, 28-29]. A number of thiol and phosphonate compounds
with IC values 80–3000 µM have been developed [30]; similar IC values were reported for
5
0
50
a series of phosphotyrosine-containing tripeptides [28]. Our group reported a series of α-
alkoxynaphthylmethylphosphonic acids [31], acyl derivatives of α-aminonaphthylmethyl
phosphonic acid [32] and acyl derivatives of 6-aminopenicillanic acid [33] as inhibitors of
PAP with K and IC values in the low micromolar range. We also employed a fragment-
i
50
based screening approach to identify three potential inhibitor leads; crystal structures
provided insight about the mode of their binding in the active site of rkbPAP [27]. Here, we
used these fragments as starting points for the design of a new series of inhibitors with
improved binding interactions.
2
. Results and Discussion
TM
Inhibitor synthesis: In a previous study, four fragments from a Maybridge library were
identified as promising inhibitor leads for PAPs [27]. The molecular structures of these
compounds (i.e. 1, 2, 3 and 4a) are shown in Figure 1. The crystal structures of rkbPAP in
complex with compounds 2 and 4a (competitive inhibitors with K values of 43 µM and 340
i
µM for rkbPAP and 59 µM and 42 µM pig PAP, respectively [27]) demonstrate that the active
site of the enzyme offers numerous structural features to allow further elaboration of inhibitor
molecules with high affinity and specificity (Figure 1).
3

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(a)
OH
O
O CH3
OH
CH3
N
1
2
O
O
CH3
OH
O
N
N
S
H C
3
H C
3
S
3
4a
TM
Figure 1: (a) Four fragments identified from a Maybridge compound library as PAP inhibitors. (b) Crystal
structure of rkbPAP (yellow surface) in complex with compound 4a (as sticks with cyan carbons), and (c) in
complex with 2.Water molecules are shown as red spheres [27].
Using compounds 2 and 4a as starting points, we generated two series of derivatives (i.e. 7a-b
and 4b-h, respectively). To synthesise the two monocyclic alkoxy benzyl ether derivatives
(7a-b) of compound 2 (i.e. the benzofuran) the 2-alkoxy benzaldehydes (6a-b) were first
prepared by alkylation of salicylaldehyde (5) using alkyl halides and potassium carbonate in
DMF solution, followed by sodium borohydride reduction to the corresponding benzyl
alcohols [34] (Scheme 1).
4

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Scheme 1. Reagents and Conditions: (a) RX, K CO , DMF, r.t., 36 h. 6a (86%), 6b (84%); (b) NaBH ,
2
3
4
2
M, NaOH, MeOH, r.t., 4 h. 7a (74%); 7b (85%).
The preparation of compounds 4a-f first required the synthesis of the intermediates 11a-f, as
outlined in Scheme 2. Following the method described by Gududuru et al. [35], the
hydrochloride salt of the methyl ester of
L-cysteine (9), which was first prepared by refluxing
L
-cysteine (8) with SOCl in methanol, was reacted with a series of aromatic aldehydes 10a-f
2
to give the corresponding thiazolidine methyl ester intermediates 11a-f.
O
O
a
HS
OH
HS
OCH3
NH2
NH HCl
2.
8
9
O
O
N
OCH3
OCH3
S
NH
S
CHO
Ar
0a-f
b
c
Ar
11a-f
Ar
12a-f
1
O
N
OH a, Ar = p-tolyl
b, Ar = Ph
d
S
c, Ar = 4-methoxyphenyl
d, Ar = 4-chlorophenyl
e, Ar = 2-chlorophenyl
f, Ar = naphth-1-yl
Ar
a-f
4
Scheme 2. Reagents and Conditions: (a) SOCl , MeOH, ∆, 3 h, 100%; (b) 9, NaHCO , EtOH, H O,
2
3
2
r.t., 14 h. 11a (65%); 11b (74%); 11c (81%); 11d (57%); 11e (79%); 11f (37%); (c) N-
5

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bromosuccinimide, benzoyl peroxide, CCl , ∆, 14 h. 12a (23%), 12b (26%), 12c (17%), 12d (19%),
4
1
2e (32%), 12f (16%); (d) 2 M NaOH, MeOH, 0 ºC, 2 h. 4a (98%), 4b (80%), 4c (84%), 4d (100%),
e (100%), 4f (100%).
4
The thiazole carboxylic acid derivatives 4a-f were prepared in two steps from their
corresponding thiazolidine intermediates 11a-f, by oxidation of 11a-f using N-
bromosuccinimide (NBS) and benzoyl peroxide, followed by basic hydrolysis of the methyl
esters 12a-f, using the method described by Gududuru et al. [35] (Scheme 2).
The syntheses of the thiazole dicarboxylic acids 4g-h are outlined in Scheme 3. Following the
method described by Ashram [36], the two aromatic aldehydes 13 and 15 were prepared by
refluxing the 2-hydroxy aromatic aldehydes 5 and 14, respectively, with ethyl 2-bromoacetate
in the presence of potassium carbonate in acetone. The two aromatic aldehydes, 13 and 15,
then reacted with compound 9 to give the corresponding thiazolidine derivatives 11g-h in
1
2% and 77% yields, respectively. Oxidation of 11g-h to the thiazoles 12g-h, followed by
saponification, gave the carboxylic acids 4g-h [35] (Scheme 3).
6

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Scheme 3. Reagents and Conditions: (a) BrCH CO Et, K CO , Me CO, ∆, 4 h. 13 (86%), 15 (48%);
2
2
2
3
2
(
b) 9, NaHCO , EtOH, H O, r.t., 14 h. 11g (12%), 11h (77%); (c) N-bromosuccinimide, benzoyl
3 2
peroxide, CCl , ∆, 14 h. 12g (17%), 12h (8%); (d) 2 M NaOH, MeOH, r.t., 4 h. 4g (73%), 4h (80%).
4
Additionally, a series of thiazolidine-4-carboxylic acid derivatives 16a-c and 16f-j were
synthesised for testing against both rkb and pig PAP enzymes to investigate the importance of
the unsaturation of the thiazole ring for the activity of the compounds. Treating L-cysteine (8)
with a number of aromatic aldehydes 10a-c and 10f-j in ethanol solution gave the
corresponding thiazolidine-4-carboxylic acid derivatives 16a-c and 16f-j, as mixtures of cis
and trans isomers, in good yields [35] (Scheme 4).
7

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O
a, Ar = p-tolyl
b, Ar = Ph
OH
c, Ar = 4-methoxyphenyl
f, Ar = naphth-1-yl
g, Ar = 2-hydroxynaphth-1-yl
h, Ar = 4-hydroxyphenyl
i, Ar = 2-hydroxyphenyl
j, Ar = 2-furyl
S
NH
CHO
Ar
a
Ar
16a-c and 16f-j
1
0a-c and 10f-j
Scheme 4. Reagents and Conditions: (a)
L-Cysteine (8), EtOH, r.t., 5 h. 16a (63%), 16b (66%), 16c
(
52%), 16f (68%), 16g (70%), 16h (64%), 16i (93%), 16j (66%).
Inhibition assays: The inhibitory effects of compounds 7a-b and 4a-h were tested using a
standard kinetic assay with the chromophoric substrate para-nitrophenyl phosphate (pNPP);
both rkbPAP and fully reduced pig PAP were used. In the first round of assessment,
percentage inhibition was measured at a fixed concentration (i.e. 100 µM) of inhibitor. This
initial screen indicated that compound 4f was the most promising lead; both pig PAP and
rkbPAP activities were reduced to approximately half the value measured in its absence.
Since accurate measurement of the binding affinity of a possible inhibitor also depends on its
mode of binding and its competition with a substrate of the reaction, several of the above
compounds were tested in inhibition assays where the concentrations of both the substrate and
inhibitors were varied. Relevant parameters are summarised in Table 1. K and K represent
ic
iuc
the inhibitor dissociation constants for the enzyme-inhibitor and enzyme-substrate-inhibitor
complex, respectively (i.e. competitive vs uncompetitive inhibition constants). Compounds
7
a-b and 4g-h exhibited little or no inhibitory activity against either pig or rkbPAP
8

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Table 1: Kinetic data for inhibitors 4a-f against fully reduced pig PAP and rkbPAP at pH 4.9.
Compound
Enzyme
Pig PAP
rkbPAP
Pig PAP
rkbPAP
Pig PAP
rkbPAP
Pig PAP
rkbPAP
Pig PAP
rkbPAP
Pig PAP
rkbPAP
K (µM)
K (µM)
ic
iuc
4
4
a
a
42 ± 14
340 ± 130
190 ± 62
-
-
-
4
4
b
b
-
-
4
4
c
c
120 ± 35
400 ± 210
49 ± 13
-
770 ± 700
4
4
d
d
-
530 ± 480
630 ± 570
920 ± 630
33 ± 15
-
4
4
e
e
350 ± 180
-
4
4
f
f
110 ± 69
-
185 ± 75
For each of the compounds 4a – 4f their inhibitory effects on pig PAP and rkbPAP are within
the same order of magnitude, but in each case the binding is stronger for the pig enzyme, and
in most cases the mode of inhibition is competitive. The exceptions are compounds 4e and 4f
that also have a contribution from an uncompetitive binding mode (characterised by K ) in
iuc
pig PAP. However, overall only compound 4f has a binding affinity (based on the magnitude
of respective K values) that is modestly better than that of the parental compound 4a (Table
i
1
).
The thiazolidine-4-carboxylic acid derivatives 16a-c and 16f-j were tested against a fully
reduced pig PAP enzyme at 100 µM concentrations using 5 mM pNPP as a substrate. This
9

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series of compounds did not show any inhibition effect, except for compound 16k, that
displays modest (~40%) inhibition. Consequently, this series of compounds was not further
investigated.
Crystallographic investigations of inhibitor binding
Crystallographic investigations into the binding mode of compound 4f in complex with rkbPAP
were undertaken wherein a rkbPAP crystal was grown and then soaked with a solution containing
4
f. The crystal diffracted to 2.4 Å resolution (Table 2) and has the same space group as described
previously [27], and with the asymmetric unit consisting of two dimers of rkbPAP; the monomers
are denoted A through D After fitting of the polypeptide, amino acid side chains, metal ions and
.
solvent molecules, 4f was modelled into the difference electron densities of Polder omit maps.
Polder maps were chosen for this study since they are optimal for visualisation of weak binding
inhibitors obscured by bulk solvent scaling [37]. Compound 4f was modelled in subunit A (>
4
.75 σ) and subunit B (> 5.24 σ) (Figure 2A,B). In subunits C and D, the difference electron
densities indicate the presence of a sulfate anion bound to the metal centre.
1
0

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Figure 2: (A) Polder (Fo-Fc) omit electron density map (> 5.24 σ) and cartoon/stick representation showing the fit
of the inhibitor 4f (magenta carbons) to the difference electron density in subunit B of rkbPAP (light blue carbons),
the metals are shown as green spheres (B) Surface and stick representation of the active site of rkbPAP (turquoise
surface) in complex with 4f (CPK colouring sticks and blue surface), metals are shown as black spheres. For clarity
of presentation, this view is rotated by 180º relative to 2(A). (C) Surface and stick representation showing the result
of MVD predicting the binding mode of 4f (green sticks) to rkbPAP superimposed onto the crystal structure of
rkbPAP (turquoise surface) in complex with 4f (yellow sticks); the metals are shown as black spheres (D) Surface
and stick representation showing the result of MVD predicting the binding mode of 4f (CPK colouring sticks, blue
surface) to human PAP (purple surface); the metals are shown as black spheres.
The inhibitor binds through its carboxylate group in a µ-1,3 bidentate mode to the metal ions in
the active site, more closely to Fe(III) (1.7 Å) than to Zn(II) (2.2 Å) (Figure 2A). The sulfur atom
in the thiazole ring of the inhibitor forms a hydrogen bond with the hydroxyl group of the
adjacent Y365 side chain (3.2 Å). The nitrogen atom in the thiazole ring forms a hydrogen bond
(3.0 Å) with a nitrogen atom in the imidazole group of H296, and the thiazole ring forms π-cation
1
1

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interactions with H295 and H296 (3.0-4.0 Å). The naphthalene ring, while not as well resolved as
the thiazole ring (likely to be due to some structural flexibility), forms π-cation interactions with
the guanidino group of R258 from the adjacent subunit in the rkbPAP dimer (3.3-4.0 Å) and with
the imidazole group of H295 (2.90-3.70 Å). In comparison to the binding interactions of
compound 4a (Figure 3) both the carboxylate and thiazole moieties are positioned similarly in the
two molecules, suggesting that the naphthalene ring in 4f enhances inhibitor binding despite not
being locked in place by non-covalent associations. This indicates further improvements are
possible to improve potency.
Figure 3: Stereo superimposition of the crystal structure of rkbPAP with 4a bound to the active site of subunit B
(
PDB ID: 4DHL, yellow carbons) and rkbPAP with 4f bound to the active site of subunit B (cyan carbons) to the
active site. Substitution of the p-tolyl group with a p-naphthyl group picks up an additional interaction with H295.
Table 2: Data collection and refinement statistics for the rkbPAP-4f complex
Data collection
Temperature
100
a
Resolution range (Å)
Total number of reflections
Total number unique
Completeness (%)
43.26 - 2.40 (2.49 - 2.40)
467895 (30571)
107272 (10656)
99.4 (97.5)
0.083 (0.280)
10.9 (3.4)
^
Rmerge
Mean I/σI
CC(1/2)
0.98 (0.93)
1
2

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Mosaicity (°)
0.27
Unit cell lengths (Å)
Space group
a = b = 126.21 c = 297.12
P 3 2 1
1
Refinement
Total number of atoms
Number of water molecules
Wilson B-factor (Å )
15,764
1,225
33.66
0.172
0.223
0.009
0.957
2
*
Rfree
Rwork
#
RMS bonds (Å)
RMS angles (°)
Ramachandran statistics (%)
Favoured
Allowed
Outlier
95.02
4.04
0.94
*
Rwork = Σ|F |-|F |/Σ|F | and is calculated using 95% of the total reflections, and
o c o
#
Rfree uses the remaining 5% of the reflections. ^Rmerge = Σ Σ |I (hkl)-
I(hkl)|/Σ Σ I (hkl). Values in parentheses are for the highest resolution shell.
hkl
i i
a
hkl
i i
The occupancy and B-factors of the inhibitor and the neighbouring residues have been refined.
The occupancy is close to 0.9 (0.84 on average) for the inhibitor and the B-factors for the
inhibitor are, on average, approximately double that of the neighbouring residues (data not
shown). This suggests that the inhibitor is stabilised in the active site but not necessarily bound
with high affinity. The K value of 185 ± 75 for this compound with rkb PAP supports this.
i
Docking studies
Active site comparison of human, pig and red kidney bean PAPs
To further validate the use of red kidney bean PAP and pig PAP as model systems for human
PAP, we carried out a three dimensional structure-based alignment. It shows that the position of
the metal ions and the three-dimensional arrangement of the metal-coordinating residues are
completely conserved across the three species and that the amino acids that form the surface of
1
3

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the active site share 100% sequence identity between the human and pig enzymes (Figure 4).
This makes pig PAP an ideal model for human PAP inhibitor design.
A
B
Figure 4: (A) Superimposition of the structure of pig PAP with two conformations of phosphate bound (PDB ID:
UQ6, cyan) and recombinant human PAP with bound phosphate (PDB ID: 1WAR, yellow). All active site residues
are fully conserved. (B) Structure-based alignment of the active sites of pig PAP (PDB ID: 5UQ6, cyan) and rkbPAP
PDB ID: 4KBP, magenta) with phosphate omitted, highlighting that most residues surrounding the active site are
5
(
conserved or semi-conserved between the two enzymes. M is either Fe(II/III) in pig and human PAPs or Zn(II) in
rkbPAP. P represents inorganic phosphate.
i
Docking studies were undertaken to predict the binding mode of 4f to human PAP (Figure 2C-D).
Firstly, the accuracy of the docking algorithm was gauged by verifying its ability to predict the
correct binding mode of 4f to rkbPAP. As seen previously [27], the MolDock Simplex Evolution
(SE) algorithm can predict the correct binding mode of an inhibitor to rkbPAP with reasonable
accuracy (Figure 2C). The same algorithm was used to dock 4f to human PAP (Figure 2D). In the
optimal conformation the carboxylate group of the inhibitor coordinates bidentately to the two
metals in the active site as observed in the crystal structure of the rkbPAP-4f complex (Figure
2
A). The thiazole ring of the inhibitor forms hydrophobic interactions with the side-chain of
conserved N89 (N201 in rkbPAP). The thiazole sulfur is oriented towards a groove in the
interface between the conserved metal ligating residues N89 and H221 (N201 and H325 in
rkbPAP) and the mammalian repression loop (N142, S143 and D144, spatially equivalent Y365
1
4

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in rkbPAP), whereby it is stabilised by van der Waals interactions with the side-chains of these
residues and by a hydrogen bond with the side chain amino group of Q149. The remainder of the
thiazole ring is stabilised by π-cation interactions with H90 and H193 (H202 and H296 in
rkbPAP, respectively). The naphthalene ring fits into a hydrophobic wedge formed by the
sidechains of F54 and F242. Pose binding energies obtained from Molegro Virtual Docker
(MVD) algorithm are -89.51 kcal/mol and -56.61 kcal/mol for the interactions of 4f with human
PAP and rkbPAP, respectively, which suggests that this inhibitor binds tighter to the human
enzyme, in good agreement with the enhanced affinity of 4f for pig PAP when compared to
rkbPAP (Table 1). The major contribution to the enhanced affinity of 4f to the human enzyme is
due to a stronger interaction of the naphthalene ring with the hydrophobic side chains of F54 and
F242 when compared to R258 in rkbPAP; however, the thiazole portion of the inhibitor also
contributes to the difference due to its interaction with the repression loop that is characteristic
for mammalian PAPs.
1
5

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3
. Conclusion
The role of PAPs in bone resorption has been well established since studies of transgenic
mice demonstrated that abnormal expression levels of this enzyme are associated with bone
disorders; overexpression of PAP leads to an osteoporotic phenotype, while a knockout
displays features characteristic of osteopetrosis [15]. While these observations make PAP a
target for the development of chemotherapeutics to combat osteoporosis, little effort has been
directed towards the design of specific and potent inhibitors of this enzyme. Though a recent
paper used a small molecule screening library to identify a family of carboxamides as low
micromolar inhibitors of human PAP [5], the majority of inhibitors discovered to date are
simple anions (e.g. phosphate, vanadate or sulfate [1-4, 8-9, 19, 29] that target the bimetallic
metal centre. Since the bimetallic metal centre and its seven amino acid ligands are well
conserved amongst a range of metallohydrolases (as an example, enzymes such as the
diesterases GpdQ and Rv0805 have an active site that is nearly identical to that of PAPs [38-
3
9] it is not surprising that inhibitors targeting mainly the metal centre are non-specific. More
complex molecules that may exploit structural features in the outer sphere of the PAP active
site were developed but their precise binding modes had not been explored, largely due to the
lack of crystallographic data. In this respect, the crystal structures of several small fragments
in complex with rkbPAP provided, for the first time, detailed insight into how potential
inhibitor leads may interact with a PAP [27]. Here, two of these fragments (2 and 4a in
Figure 1a) were used as starting points for further elaboration. Of significance is that our
structure activity relationship (SAR) results indicate that most of the thiazolidine derivatives
of 16 are ineffective inhibitors of PAPs. The exception is compound 16k, bearing a
naphthalene ring. Upon oxidising its thiazolidine ring to the corresponding thiazole while
retaining the naphthalene ring leads to compound 4f. This compound displays improved
1
6

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binding affinity, and its structure in complex with rkbPAP guides further modifications to
enhance the binding specificity and potency (Figure 2).
To date it has not been possible to obtain crystal structures of catalytically active forms of a
mammalian PAP. Available structures contain an inactive di-Fe(III) centre, mostly in
complex with phosphate [40-41]. In contrast, plant PAPs crystallise readily in their
catalytically active form, largely due to the fact that they contain redox-inactive Fe(III)Zn(II)
or Fe(III)Mn(II) centres [8-11, 13, 42-43]. Consequently, in particular rkbPAP has been
employed as a model to investigate the effect of inhibitors on PAPs. The observation of
reasonably conserved (i) catalytic properties, including substrate specificities, mechanism and
inhibition by non-specific inhibitors, and (ii) amino acid side chains in the vicinity of the
active site validate the selection of rkbPAP as a model for mammalian PAPs [1-4]. Indeed,
all compounds tested here have comparable inhibitory effects for pig PAP and rkbPAP, albeit
their binding to the mammalian enzyme is always more potent (Table 1). In order to gain
insight into how 4f binds to the human enzyme in silico docking was employed. The
methodology was initially tested using rkbPAP to demonstrate that the predicted (in silico)
mode of binding of 4f is virtually identical to that observed experimentally (in the crystal
structure; Figure 2). Indeed, the inhibitor is predicted to bind better to the mammalian
enzyme, in part due to additional interactions between this compound and the repression loop
that is characteristic for mammalian PAPs [40]. This predicted improvement in binding is
reflected in the lower K of pig PAP for 4f when compared to rkbPAP (Table 1).
i
In summary, this study provides the first crystallographic insight into a rationally designed
inhibitor for PAP. The structure provides guidance for the further elaboration of PAP
inhibitors that may find applications in the treatment of conditions associated with elevated
levels of this enzyme, including not only osteoporosis but potentially also AIDS
1
7

ACCEPTED MANUSCRIPT
encephalopathy [44], Gaucher’s disease [45], hairy cell leukemia [46], Alzheimer’s disease
[
47] and bone metastases [48-49].
4
. Experimental
4
.1. Enzyme preparation and Purification
Purple acid phosphatase from red kidney bean (rkbPAP) was purified following a previously
published protocol [50]. Briefly, red kidney beans (Phaseolus vulgaris) were ground in a
Waring blender and suspended in 0.5 M sodium chloride. The suspension was filtered through
a muslin cloth, followed by ethanol fractionation and ammonium sulfate precipitation and
further purified by ion-exchange chromatography using a CM-cellulose column followed by
gel filtration on a Sephadex S-300 column. The resulting preparation was concentrated to 23.8
mg⁄mL using a Millipore Amicon centrifugal concentrator and stored at 4 ºC in 0.5 M sodium
chloride. Pig PAP was extracted from the uterine fluid of a pregnant sow and purified by ion-
exchange chromatography using CM-cellulose followed by gel filtration on a Sephadex G-75
o
[
51]. Purified pig PAP was concentrated to 8.1 mg⁄mL and stored at 20 C in 100 mM acetate
buffer at pH 4.9. Protein concentrations were determined by measuring the absorbance at 280
nm using extinction coefficients of 1.41 for a 1 mg⁄mL solution (28.6 µM) of pig PAP and 2.1
for a 1 mg⁄mL solution (9.1 µM) of rkbPAP. SDS-PAGE analysis showed that the enzymes
were >95% pure.
4
.2. Crystallisation, soaking and cryoprotection
Crystallisation of rkbPAP was achieved using previously determined conditions [42]. Once
crystals had reached a size of ~0.1 mm in all three dimensions, an equivalent volume of
cryoprotectant containing the inhibitor was added to the hanging drop. This solution consisted
of 0.1 M sodium citrate pH 5.0, 0.1 M lithium chloride, 25% polyethylene glycol 3350, 20%
isopropyl alcohol, 10% glycerol and 4 mM of 4f. This was introduced 4 days before the data
1
8

ACCEPTED MANUSCRIPT
collection. For cryoprotection, a crystal from the above preparation was soaked in
cryoprotectant (containing the inhibitor) for 10 s before placing it in the cryostream (100 K).
4
.3. Data collection & analysis
Data were collected using a FR-E X-ray generator (voltage: 45 kV, current: 45 mA) and
recorded using a Raxis IV++ imaging plate at UQ. CuKα X-rays (wavelength: 1.54 Å) were
used for these diffraction studies. The program Rigaku Crystal clear 2.0 [52] was used to
integrate and scale reflections and Scala in CCP4 was used to scale and merge the data [53].
Initial protein model for refinements was that of the crystal structure of rkbPAP in complex
with 4a (PDB access code: 4DHL) with the inhibitor removed from the active site.
Refinements and building of Polder maps were performed using PHENIX [54] and model
building was undertaken using Coot [55]. Figure 2A was produced using CCP4MG [56] and
Figures 2B-D were produced using MVD [57]. Coordinates and structure factors for the
complex have been deposited in the Protein Data Bank with access code 6G46.
4
.4. Computational docking studies
Docking studies were undertaken with MVD [57] using the MolDock SE algorithm with
flexible residues using Tabu clustering and softened potentials. The ligand search space was
confined to a 9 Å sphere originating from the metal centre of PAP. For rkbPAP, the receptor
coordinates used were those of the crystal structure of the enzyme in complex with the
inhibitor obtained here. For human PAP, the coordinates used were those of recombinant
human PAP in complex with phosphate (PDB code 1WAR) [40] with the phosphate anion
omitted from the active site. Water molecules were removed from all coordinate files prior to
docking.
4
.5. Enzyme kinetics
1
9

ACCEPTED MANUSCRIPT
Inhibition assays with both pig PAP and rkbPAP were performed in 96 well 400 µL multi-
titre plates using a UV/Vis multiplate spectrophotometer [29]. Prior to its use in kinetic assays
pPAP was fully reduced to the heterovalent Fe(III)Fe(II) state to ascertain maximum activity
o
by incubating it with 0.77 mM β-mercaptoethanol for ten minutes at 37 C. Kinetic
o
measurements were carried out at pH 4.9 (0.1 M acetate buffer in 25% DMSO) at 25 C using
para-nitrophenyl phosphate (pNPP) as substrate at different concentrations (1, 3, 5, 7.5, 10
and 12.5 mM). The rate of product (p-nitrophenol) formation was measured at λ = 405 nm (ε
-1
-1
=
343 M cm ) [13]. Enzyme concentration used was 12 nM, while the concentrations for
tested compounds ranged from 50 µM to 300 µM. The data were analysed by non-linear
regression using the general inhibition equation (equation 2) and the program WinCurveFit
(Kevin Raner software).
Equation 2
In this equation, K and K represent the equilibrium dissociation constants for competitive
ic
iuc
and uncompetitive inhibitor binding, respectively, while Vmax, K , [S] and [I] represent the
M
maximum rate of product formation, the Michaelis constant, substrate concentration and
inhibitor concentration, respectively.
4
.6. Experimental chemistry
Light petroleum (LP, b.p. 40-60 °C) was distilled before use. Flash chromatography was
carried out with Merck Kieselgel 60 as described by Still [58]. NMR experiments were
recorded on 300, 400 and 500 MHz spectrometers (Bruker, Rheinstetten, Germany).
Chemical shifts are reported in parts per million (ppm) on a δ scale, relative to the solvent
peak (CDCl δ 7.24, δ 77.0; (CD ) SO δ 2.49, δ 39.5; MeOD δ 3.30, δ 49.0). Coupling
3
H
C
3 2
H
C
H
C
2
0

ACCEPTED MANUSCRIPT
constants (J) are reported in hertz and peak multiplicities described as singlet (s), doublet (d),
triplet (t), quartet (q), septet (sept), multiplet (m), or broad (br). High resolution electrospray
ionisation accurate mass measurements were recorded in positive and negative mode on a
quadrupole – time of flight instrument (Bruker) with an ESI source. Accurate mass
measurements were carried out with external calibration using sodium formate as reference
calibrant and/or Agilent tune mix (mw > 500). Low and high resolution electron impact
ionisation mass measurements were recorded using perfluorokerosene-H as reference
calibrant. For TLC staining, Ce(SO ) and/or KMnO were used. Ce(SO ) dye ingredients:
4
2
4
4 2
2
.5 g phosphomolybdic acid; 1 g ceric sulfate; 100 mL water and 8 mL concentrated sulfuric
acid. KMnO dye ingredients: 3 g potassium permanganate; 20 g potassium carbonate; 5 mL
4
5
% sodium hydroxide and 300 mL water.
.6.1. General method for preparation of 2-alkoxy benzaldehydes (6a-b) [59]
4
Salicylaldehyde (5) (3.66 g, 3.2 mL, 30 mmol) was added to a suspension of potassium
carbonate (8.29 g, 60 mmol) in DMF (50 mL). Alkyl halide was then added dropwise and the
solution was stirred for 36 h under an atmosphere of argon. Water (150 mL) was added and
the mixture was extracted with diethyl ether (3 x 50 mL). The combined organic layers were
washed with 2M NaOH (3 x 50 mL), dried over Na SO , filtered and evaporated in vacuo to
2
4
afford the 2-alkoxy benzaldehyde 6a-b.
.6.1.1. 2-Ethoxybenzaldehyde (6a) [59]
4
2
1

ACCEPTED MANUSCRIPT
The alkyl halide used was ethyl iodide (1 eq, 4.68 g, 2.4 mL, 30 mmol), which afforded 6a as
1
a yellow oil (3.85 g, 86%). R : 0.51 (10% EtOAc in LP, UV and KMnO dip). H NMR (300
f
4
MHz, CDCl ) δ 1.38 (3H, t, J 7.0 Hz), 4.06 (2H, q, J 7.0 Hz), 6.87 – 6.93 (2H, m), 7.43 – 7.47
3
1
3
(
1H, m), 7.71 (1H, dd, J 1.8 Hz, J 7.5 Hz), 10.41 (1H, s); C NMR (75 MHz, CDCl ) δ 14.4,
3
6
4.0, 112.4, 120.3, 124.6, 128.0, 135.8, 161.2, 189.8. NMR spectra are in agreement with
those reported by Leardini [59].
4
.6.1.2. 2-Isopropoxybenzaldehyde (6b) [59]
The alkyl halide used was isopropyl bromide (1.1 eq, 4.06 g, 3.1 mL, 33 mmol), which
afforded 6b as a yellow oil (4.1 g, 84%). R : 0.59 (10% EtOAc in LP, UV and KMnO dip).
f
4
1
H NMR (400 MHz, CDCl ) δ 1.34 (6H, d, J 6.0 Hz), 4.62 (1H, sept, J 6.0 Hz), 6.90 – 6.95
3
13
(
2H, m), 7.43 – 7.48 (1H, m), 7.77 (1H, dd, J 1.9 Hz, J 7.6 Hz), 10.45 (1H, s); C NMR (100
MHz, CDCl ) δ 21.8, 70.9, 113.9, 120.2, 125.6, 128.1, 135.6, 160.5, 190.0. NMR spectra are
3
in agreement with those reported by Leardini [59].
4
.6.2. General method for preparation of 2-alkoxy benzyl alcohols (7a-b)
2
2

ACCEPTED MANUSCRIPT
These compounds 7a-b were prepared using the general method described by Zartler and
Sharpiro [34]. A solution of sodium borohydride (0.37 eq, 28 mg, 0.74 mmol) in 2M NaOH
(40 µL) diluted with water (360 µL) was added dropwise to a solution of the 2-alkoxy
o
benzaldehyde (6a-b) (2 mmol) in methanol (2 mL) at 18 – 25 C. The reaction mixture was
stirred for 4 h at room temperature and then evaporated in vacuo. Methanol (2 x 10 mL) was
added and evaporated in vacuo. HCl (5%, 10 mL) was added to the residue which was
extracted with diethyl ether (2 x 20 mL). The combined organic layers were dried over
Na SO , filtered and evaporated in vacuo to afford 7a-b.
2
4
4
.6.2.1. 2-Ethoxybenzyl alcohol (7a)
The crude product was purified by silica flash column chromatography (10% EtOAc in LP) to
afford 7a as a colourless oil (224 mg, 74%) R : 0.28 (10% EtOAc in LP, UV and KMnO4
f
1
dip). H NMR (300 MHz, CDCl ) δ 1.41 (3H, t, J 7.0 Hz), 3.18 (1H, s, OH), 4.02 (2H, q, J
3
7
.0 Hz), 4.68 (2H, s), 6.83 (1H, d, J 8.2 Hz ), 6.92 (1H, dt, J 0.8 Hz, J 7.4 Hz), 7.21 – 7.32
1
3
(
2H, m); C NMR (75 MHz, CDCl ) δ 14.5, 61.0, 63.2, 110.7, 120.1, 128.0, 128.2, 129.1,
3
1
56.2. NMR spectra are in agreement with those reported by Wang [60].
4
.6.2.2. 2-Isopropoxybenzyl alcohol (7b)
2
3

ACCEPTED MANUSCRIPT
1
Colorless oil (282 mg, 85%) R : 0.43 (10% EtOAc in LP, UV and KMnO dip). H NMR
f
4
(
500 MHz, CDCl ) δ 1.36 (6H, d, J 6.1 Hz), 2.92 (1H, br s, OH), 4.61 (1H, sept, J 6.2 Hz),
3
4
.67 (2H, s), 6.89 (1H, d, J 8.3 Hz), 6.92 (1H, dt, J 0.9 Hz, J 7.4 Hz), 7.23 – 7.29 (2H, m) in
1
3
agreement with that reported by Fukatsu et al. [61]; C NMR (125 MHz, CDCl ) δ 22.0,
3
6
2.0, 70.0, 112.4, 120.3, 128.5, 128.6, 130.0, 155.6.
4
.6.3. General method for preparation of 2-aryl-1,3-thiazolidine methyl esters (11a-f)
These compounds 11a-f were prepared using the general method described by Gududuru et
al. [35].
Thionyl chloride (8.3 mL, 110 mmol) was added dropwise under an atmosphere of argon to a
solution of
refluxed for 3 h then evaporated in vacuo, then co-evaporated with toluene (2 x 5 mL) to
afford the hydrochloride salt of the methyl ester of -cysteine (9) as white solid. One-sixth of
L-cysteine (8) (9.00 g, 74 mmol) in 150 mL MeOH. The reaction mixture was
L
this material (9) (12.4 mmol) was dissolved in water/ ethanol (1:1) (15 mL). Sodium
hydrogen carbonate (1.14 g, 13.6 mmol) was added and, after 10 min, the aromatic aldehyde
(10a-f) (12.38 mmol) was added and the reaction mixture was stirred for 14 h. The ethanol
was evaporated in vacuo and the aqueous residue was extracted with DCM (50 mL). The
2
4

ACCEPTED MANUSCRIPT
organic layer was washed with water (25 mL), dried over Na SO , filtered and evaporated in
2
4
vacuo to afford the crude products 11a-f.
.6.3.1. (2R/2S, 4R)-Methyl 2-(p-tolyl)thiazolidine-4-carboxylate (11a)
4
The crude product was purified by silica flash column chromatography (10% EtOAc in LP) to
afford a mixture of cis and trans isomers of 11a as a yellow oil (1.90 g, 65%) R : 0.31 (20%
f
1
EtOAc in LP, UV and Ce(SO ) dip). H NMR (300 MHz, CDCl ) δ 2.32 (s) and 2.34 (s)
4
2
3
integrate for 3H, 2.72 (1H, br s, NH), 3.06 – 3.21 (1H, m), 3.32 – 3.52 (1H, m), 3.77 (s) and
.76 (s) integrate for 3H, 3.96 (0.6H, dd, J 7.1 Hz, J 8.9 Hz), 4.21 (0.4H, dd, J 5.7 Hz, J 7.1
3
1
3
Hz), 5.52 (0.6H, s), 5.77 (0.4H, s), 7.11 – 7.17 (2H, m), 7.35 – 7.42 (2H, m); C NMR (75
MHz, CDCl ) δ 20.8, 20.9, 37.8, 38.9, 52.16, 52.22, 64.1, 65.2, 70.5, 72.2, 126.6, 127.0,
3
1
28.8, 129.0, 134.9, 137.3, 137.8, 138.2, 171.3, 171.9 in agreement with that described by
Paul and Korytnyk [62].
.6.3.2. (2R/2S, 4R)-Methyl 2-phenylthiazolidine-4-carboxylate (11b) [35]
4
2
5

ACCEPTED MANUSCRIPT
The crude product was purified by silica flash column chromatography (10% EtOAc in LP) to
afford cis and trans mixture of 11b as a yellow oil (2.055 g, 74%) R : 0.31 (20% EtOAc in
f
1
LP, UV and Ce(SO ) dip). H NMR (300 MHz, CDCl ) δ 2.65 (1H, br s, NH), 3.11 (1H, dd,
4
2
3
J 9.0 Hz, J 10.2 Hz), 3.20 (0.5H, dd, J 6.0 Hz, J 10.8 Hz), 3.38 (0.5H, t, J 10.5 Hz), 3.41 –
3
.49 (1H, m), 3.79 (1.5H, s), 3.80 (3H, s), 3.99 (1H, dd, J 7.2 Hz, J 9.0 Hz), 4.21 (0.5H, dd, J
.0 Hz, J 6.9 Hz), 5.56 (1H, s), 5.82 (0.5H, s), 7.23 – 7.40 (5H, m), 7.47 – 7.52 (3H, m) in
6
1
3
agreement with that described by Gududuru et al. [35]; C NMR (75 MHz, CDCl ) δ 38.0,
3
3
9.1, 52.4, 52.5, 64.2, 65.4, 70.7, 72.5, 126.8, 127.3, 127.8, 128.3, 128.57, 128.60, 138.1,
41.0, 171.5, 172.1.
1
4
.6.3.3. (2R/2S, 4R)-Methyl 2-(4-methoxyphenyl)thiazolidine-4-carboxylate (11c)
The crude product was purified by silica flash column chromatography (10-60% EtOAc in
LP) to afford cis and trans mixture of 11c as a yellow oil (2.525 g, 81%) R : 0.19 (20%
f
+
EtOAc in LP, UV and Ce(SO ) dip). ESI – MS, m/z: 276 [M + Na] . HRMS calculated for
4
2
+
1
C H NNaO S 276.0665, found 296.0654. H NMR (500 MHz, CDCl ) δ 3.08 (dd, J 8.9
1
2
15
3
3
Hz, J 10.3 Hz) and 3.20 (dd, J 5.5 Hz, J 10.7 Hz) integrate for 1H, 3.36 (dd, J 7.1 Hz, J 10.7
Hz) and 3.43 (dd, J 7.1 Hz, J 10.3 Hz) integrate for 1H, 3.766 (s), 3.770 (s), 3.78 (s) and 3.79
(
s) integrate for 6H (2 x CH ), 3.95 (0.7H, dd, J 7.2 Hz, J 9.0), 4.21 (0.3H, dd, J 5.5 Hz, J 7.2
3
1
3
Hz), 5.50 (s) and 5.74 (s) integrate for 1H, 6.83 – 6.88 (2H, m), 7.38 – 7.44 (2H, m); C
NMR (125 MHz, CDCl ) δ 38.0, 39.2, 52.5, 52.6, 55.27, 55.29, 64.2, 65.4, 70.6, 72.3, 113.7,
3
2
6

ACCEPTED MANUSCRIPT
1
14.0, 128.2, 128.7, 130.1, 132.9, 159.3, 159.8, 171.6, 172.3. NMR spectra are in agreement
with those reported by Anwar and Moloney [63].
.6.3.4. (2R/2S, 4R)-Methyl 2-(4-chlorophenyl)thiazolidine-4-carboxylate (11d)
4
The crude product was purified by silica flash column chromatography (10-50% EtOAc in
LP) to afford cis and trans mixture of 11d as a yellow oil (1.80 g, 57%) R : 0.31 (20% EtOAc
f
+
in LP, UV and Ce(SO ) dip). ESI – MS, m/z: 280 [M + Na] . HRMS calculated for
4
2
+
1
C H ClNNaO S 280.0169, found 280.0171. H NMR (300 MHz, CDCl ) δ 2.68 (1H, br s,
1
1
12
2
3
NH), 3.06 – 3.18 (1H, m), 3.35 (dd, J 7.1 Hz, J 10.6 Hz) and 3.44 (dd, J 7.1 Hz, J 10.3 Hz)
integrate for 1H, 3.78 (s) and 3.79 (s) integrate for 3H, 3.96 (0.6H, dd, J 7.1 Hz, J 8.9), 4.13
(0.4H, t, J 6.5 Hz), 5.54 (s) and 5.77 (s) integrate for 1H, 7.26 – 7.34 (2H, m), 7.40 – 7.47
1
3
(
2H, m); C NMR (75 MHz, CDCl ) δ 38.0, 39.1, 52.48, 52.52, 64.0, 65.4, 69.8, 71.7, 128.2,
3
1
28.4, 128.7, 128.8, 133.4, 134.3, 136.7, 139.9, 171.4, 172.0.
4
.6.3.5. (2R/2S, 4R)-Methyl 2-(2-chlorophenyl)thiazolidine-4-carboxylate (11e)
2
7

ACCEPTED MANUSCRIPT
The crude product was purified by silica flash column chromatography (10-50% EtOAc in
LP) to afford cis and trans mixture of 11e as a yellow oil (2.50 g, 79%) R : 0.37 (20% EtOAc
f
+
in LP, UV and Ce(SO ) dip ESI – MS, m/z: 280 [M + Na] . HRMS calculated for
4
2
+
1
C H ClNNaO S 280.0169, found 280.0174. H NMR (300 MHz, CDCl ) δ 2.88 (1H, t, J
1
1
12
2
3
1
1
4
7
0.4 Hz, NH), 3.03 – 3.14 (1H, m), 3.33 (dd, J 6.5 Hz, J 10.6 Hz) and 3.46 (dd, J 6.9 Hz, J
0.2 Hz) integrate for 1H, 3.80 (s) and 3.81 (s) integrate for 3H, 3.99 (dd, J 4.0 Hz, J 6.9) and
.25 (t, J 6.7 Hz) integrate for 1H, 5.94 (s) and 6.08 (s) integrate for 1H, 7.15 – 7.40 (4H, m),
1
3
.57 (dd, J 1.7 Hz, J 7.6 Hz) and 7.71 (dd, J 2.1 Hz, J 7.5 Hz) integrate for 1H; C NMR (75
MHz, CDCl ) δ 37.4, 38.9, 52.5, 52.6, 65.4, 68.3, 126.5, 126.8, 127.3, 128.1, 128.5, 129.7,
3
1
29.8, 132.9, 133.7, 135.8, 140.0, 171.5, 172.0.
4
.6.3.6. (2R/2S, 4R)-Methyl 2-(naphth-1-yl)thiazolidine-4-carboxylate (11f)
The crude product was purified by recrystallisation from diethyl ether to afford cis and trans
mixture of 11f as white needles (1.232 g, 37%) R : 0.33 (20% EtOAc in LP, UV and
f
+
+
Ce(SO ) dip). ESI – MS, m/z: 296 [M + Na] . HRMS calculated for C H NNaO S
4
2
15 15
2
1
2
96.0716, found 296.0724. H NMR (300 MHz, CDCl ) δ 2.85 (1H, br s, NH), 3.12 (dd, J 9.3
3
Hz, J 10.2 Hz) and 3.20 (dd, J 6.3 Hz, J 10.5 Hz) integrate for 1H, 3.39 (dd, J 6.6 Hz, J 10.5
Hz) and 3.51 (dd, J 7.2 Hz, J 10.5 Hz) integrate for 1H, 3.80 (s) and 3.82 (s) integrate for 3H,
4
.13 (dd, J 7.2 Hz, J 9.3 Hz) and 4.36 (t, J 6.5 Hz) integrate for 1H, 6.30 (s) and 6.49 (s)
integrate for 1H, 7.40 – 7.58 (3H, m), 7.75 – 7.97 (3H, m), 8.13 (d, J 8.4) and 8.20 (d, J 8.4)
1
3
integrate for 1H; C NMR (75 MHz, CDCl ) δ 37.9, 38.7, 52.6, 64.7, 65.6, 67.9, 69.3, 122.5,
3
2
8