Synthesis, evaluation and structural investigations of potent purple acid phosphatase inhibitors as drug leads for osteoporosis

European Journal of Medicinal Chemistry 182 (2019) 111611

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European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech

Research paper

Synthesis, evaluation and structural investigations of potent purple acid phosphatase inhibitors as drug leads for osteoporosis Daniel Feder a, 1, Meng-Wei Kan a, 1, Waleed M. Hussein a, b, Luke W. Guddat a, Gerhard Schenk a, c, **, Ross P. McGeary a, * a b c

The University of Queensland, School of Chemistry and Molecular Biosciences, Brisbane, QLD, 4072, Australia Helwan University, Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Ein Helwan, Helwan, Egypt The University of Queensland, Sustainable Minerals Institute, 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: Received 9 April 2019 Received in revised form 31 July 2019 Accepted 9 August 2019 Available online 13 August 2019

Purple acid phosphatases (PAPs) are binuclear hydrolases that catalyze the hydrolysis of phosphorylated substrates under acidic to neutral conditions. Elevated serum concentrations of PAP are observed in patients suffering from osteoporosis, identifying this enzyme as a potential target for the development of novel therapeutic agents to treat this disease. a-Alkoxy-substituted naphthylmethylphosphonic acid derivatives have been identified previously as molecules that bind with high affinity to PAPs, and docking studies suggest that longer alkyl chains may increase the binding affinities of such compounds. Here, we synthesized several derivatives and tested their inhibitory effect against pig and red kidney bean PAPs. The most potent inhibitor within this series is the octadecyl derivative, which has a Ki value of ~200 nM. Crystal structures of the dodecyl and octadecyl derivatives bound to red kidney bean PAP show that the length of the alkyl chain influences the ability of the phosphonate group to interact directly with the bimetallic center. These structures represent the first examples of potent inhibitors bound to a PAP that have drug-like properties. This study provides a starting point for the development of much needed new treatments for osteoporosis. © 2019 Elsevier Masson SAS. All rights reserved.

Keywords: Purple acid phosphatase X-ray crystallography Enzyme inhibitors Metallohydrolases Chemotherapeutics Osteoporosis Drug development

1. Introduction Purple acid phosphatases (PAPs) are binuclear metalloenzymes (i.e. they require two metal ions for catalytic activity) found in animals, plants and fungi [1e3]. The PAP active site contains two metal ions that are used to hydrolyze phosphate esters and anhydrides under acidic to neutral conditions [2]. The phosphatase reaction catalyzed by the PAPs is given in Equation (1). 2 RO-PO23 þ H2O / ROH þ HPO4

(1)

PAPs can dephosphorylate small substrates such as para-nitrophenyl phosphate (pNPP), adenosine 50 -diphosphate (ADP),

* Corresponding author. ** Corresponding author. The University of Queensland, School of Chemistry and Molecular Biosciences, Brisbane, QLD, 4072, Australia. E-mail addresses: [email protected] (G. Schenk), [email protected] (R.P. McGeary). 1 These authors contributed equally. https://doi.org/10.1016/j.ejmech.2019.111611 0223-5234/© 2019 Elsevier Masson SAS. All rights reserved.

adenosine 50 -triphosphate (ATP) and phosphotyrosine, as well as larger substrates such as the phosphoproteins osteopontin and sialoprotein [4e8]. Several biological roles for the PAPs have been suggested. In sows, PAP is expressed at very high levels during gestation leading to the conclusion that the enzyme plays an important role in iron transfer (hence it is also referred to as uteroferrin) [9]. In humans, two isoforms of PAP are observed arising from a post-translational modification. Isoform 5a represents the intact polypeptide chain. The removal of a loop (termed the repression loop) in 5a by lysosomal proteases leads to isoform 5b. Isoform 5a has been implicated in the inflammatory response of antigen-presenting cells [10] and isoform 5b has been shown to participate in osteoclastic bone resorption in bone tissue [4,11,12]. In plants it is speculated that PAP may play a role in mobilizing organic phosphates in the soil during phosphate starvation [13e17], but they may also be involved in the immune response or in survival under oxygen stress through generation of reactive oxygen species (ROS) [16e21]. PAPs are the only known metallohydrolases that require a heterovalent bimetallic center of the form FeIII-MII (where M ¼ Fe, Zn

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or Mn) for optimal activity [22,23]. They can be distinguished from other acid phosphatases by their characteristic purple/pink color in concentrated solutions, due to the formation of a charge transfer complex between a conserved tyrosine ligand and FeIII [2]. In PAPs from animal sources the second metal ion is generally a FeII which can be reversibly oxidized, leading to an inactive form of the enzyme [24,25]. In plants, the majority of enzymes appear to use ZnII as the second metal ion, however, MnII and FeII have also been observed to be present [26,27]. Mammalian PAPs are highly conserved across different species, with over 85% amino acid sequence identity [1,2,4,28e32]. They generally share a basic isoelectric point (pI 7.6e9.5) and optimal enzymatic activity at acidic pH [2]. Despite having similar catalytic activities, plant PAPs are less than 20% identical in their overall amino acid sequence when compared to mammalian PAPs. Furthermore, their quaternary structures also vary; while mammalian PAPs are monomers consisting of a single domain of ~35 kDa, available structures of plant PAPs illustrate the homodimeric nature of these enzymes. Each subunit of these plant PAPs consists of a ~55 kDa polypeptide chain. While the function of the N-terminal domain remains obscure, the C-terminal domain of plant PAPs is structurally very similar to mammalian PAPs and contains the catalytically essential binuclear metal center [7,28e36]. The amino acid residues coordinating the two metal ions are invariant across all known PAPs, including putative bacterial PAPs and a human enzyme that resembles plant rather than other mammalian PAPs (but the function of which has not yet been established) [37,38]. Osteoporosis is the most common bone disease in humans [39] and constitutes a worldwide health problem [40]. The increasing rates of mortality and morbidity as well as the cost of medical care associated with this disease [41e45] illustrate that improved treatments for osteoporosis are in great and urgent demand. Interestingly, despite compelling evidence that suggests a strong correlation between elevated expression of PAP and osteoporosis [4,46e51], there is no current treatment of osteoporosis that targets PAP [52]. The potential of PAP as a target for anti-osteoporotic drugs emerged from an observation in humans that showed elevated PAP levels in the bloodstream directly correlate with the onset and/or progression of osteoporosis [53]. Furthermore, in mice, the overexpression of PAP leads to a decrease in bone density, consistent with the onset of osteoporosis [46], while in mice lacking the PAPencoding gene an increase in bone mineralization occurs, consistent with osteopetrosis, the opposite phenotype of osteoporosis [54]. In the past, inhibitor design for PAPs was hampered by the absence of crystal structures with a suitable inhibitor bound to the active site. Here, we have succeeded, for the first time, in elucidating the structure of a complex between a PAP and a potent inhibitor that has drug-like properties. We chose PAP from red kidney bean (rkbPAP) for structural studies because (i) it crystallizes easily, (ii) the metal-binding residues in its active site are identical to mammalian PAPs, and (iii) mammalian PAPs can only be crystallized in their inactive form and/or in the presence of a phosphate anion, which coordinates directly with the metal center.

this series have large hydrophobic groups substituted at the aposition (Fig. 1a). A subsequent study tested the inhibitory activity of a group of alkyl-phosphonic acids with metal-binding groups such as thiol, carboxylate or phosphonate against rkbPAP (Fig. 1b) [56]. These compounds were less potent, with IC50 values ranging from 80 to 3000 mM, but they did nonetheless demonstrate that the conjugation of metal binding moieties can improve the binding affinities of the compounds by up to 100 times. This led to our group designing and testing a suite of a-alkoxy-substituted naphthylmethylphosphonic acids and a-amino-substituted naphthylmethylphosphonic acids that inhibit pig PAP and rkbPAP with Ki values as low as 4e17 mM (Fig. 1 c-d) [50,57]. In another effort to synthesize new PAP inhibitors, our group designed a suite of phosphotyrosine-containing tripeptides, which exhibit inhibitory activity against several mammalian and plant PAPs in the mid-micromolar range [58]. Despite the significant inhibitory potency of these compounds their high molecular weights, low solubilities and susceptibility to cleavage by cellular proteases have limited their therapeutic potential. More recently, we employed a fragment-based screening approach and three low molecular weight inhibitors of PAPs with good ligand efficiencies and Ki values in the low micromolar range were discovered [51]. Structure-based design approaches and in silico docking were then employed using both pig PAP and rkbPAP to elaborate these inhibitors and synthesize derivatives with improved binding affinities [36,59]. Although these studies contributed to the diversification of the library of promising PAP inhibitors, they did not result in compounds with a dramatic improvement in binding affinity. Consequently, there is currently still no medicinally relevant PAP inhibitor with a Ki value in the nanomolar range. 2.1. In silico rational design The structures of pig PAP and human PAP reveal two interesting features in the vicinity of the active site, a small hydrophobic cleft and a long groove positioned under the repression loop (Fig. 2). While in rkbPAP the region corresponding to this hydrophobic cleft in the mammalian PAPs is formed by His 295, His 296 and R258,

2. Results and discussion In an early effort to design potent PAP inhibitors, a substrate analog discovery approach was employed [55]. Specifically, a series of phosphonate compounds were tested for their effect on human PAP isolated from the spleen of a patient suffering from hairy cell leukemia. Molecules built on the scaffold of 1naphthylmethylphosphonic acid inhibited the enzyme with IC50 values in the low micromolar range. The most potent inhibitors in

Fig. 1. Phosphonates that inhibit PAPs. (a) The initial a-alkoxy-substituted naphthylmethylphosphonic acid lead demonstrated that large hydrophobic groups substituted in the a position enhance the inhibition of human PAP [55]. (b) A promising alkyl phosphonic acid compound featuring a carboxylate substituent exerts considerable inhibition of rkbPAP [56]. (ced) a-Alkoxy and a-amino-substituted naphthylmethylphosphonic acid derivatives synthesized previously in our group were shown to be potent inhibitors of pig PAP and rkbPAP [50,57].

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Fig. 2. Surface representations of the active sites of (a) pig PAP, (b) human PAP and (c) rkbPAP. Metals are shown as green spheres. The hydrophobic cleft (yellow line) and the groove (green line), identified in the mammalian PAPs, are indicated. Although the hydrophobic cleft is not present in rkbPAP we demonstrated previously that the naphthalene group can bind in an equivalent pocket (i.e. the naphthalene binding site - NBS) [36]. Furthermore, an elongated patch corresponding to a similar feature in mammalian PAPs is also present in the plant enzyme. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

thus providing a hydrophilic environment. A recently published crystal structure of rkbPAP has demonstrated that a naphthalene moiety can bind in this cleft via p-cation interactions [36]. Furthermore, although plant PAPs do not have a repression loop a hydrophobic patch resembling the groove found in their mammalian counterparts is present (Fig. 2). We thus hypothesized that in addition to the metal ion center both the cleft and the long groove may be exploited to improve the binding of inhibitors to PAPs in general. Since previous studies (see above) demonstrated that aalkoxy-substituted naphthylmethylphosphonic acid derivatives are promising leads for the development of PAP inhibitors, several representatives in their R and S configurations (Fig. 3) were docked into the active sites of human PAP, pig PAP and rkbPAP. The six variants differ in the length of their alkyl (R) chains in an attempt to exploit the long groove for enhanced binding interactions. Employing the program Molegro Virtual Docker (MVD [60]) the relative binding energies of the docked compounds could be qualitatively compared using calculated binding energies (i.e. MolDock Score and ligand efficiency; Table 1). The docking scores of human PAP and pig PAP are similar (within ~10% deviation) for all compounds, irrespective of stereochemistry. In contrast, lower scores were obtained for rkbPAP (up to

Fig. 3. General structure of the a-alkoxy-substituted naphthylmethylphosphonic acid series that were docked into the active sites of human PAP, pig PAP and rkbPAP.

~25% deviation from pig PAP) for all but compound 2. For pig PAP the predicted binding affinity appears to increase with increasing chain length, with the highest scores obtained for 5 and 6. While some variations between R and S isomers are observed there appears to be no clear trend. For human PAP the docking scores also improve with increasing chain length but they appear to level off (or reach a shallow maximum) by compound 3, which has 16 carbon atoms). Again, no clear trend with respect to the stereochemistry is observed. In contrast, there is no correlation between chain length and docking scores for rkbPAP. Although the docking scores do not identify a particular compound or stereoisomer as the preferred candidate ((R)-4, (S)-5 and (S)-2 are the compounds with the optimal scores for human PAP, pig PAP and rkbPAP, respectively) the majority of the obtained binding poses support our prediction that the alkyl chains interact with the elongated groove near the metal center (Fig. 4). 2.2. Synthesis Based on the encouraging docking results the six racemic compounds 1e6 were synthesized following the procedure previously established in our group [50]. The first two steps in the synthetic pathway include the conversion of a-naphthaldehyde (7) into the corresponding diethyl phosphite derivative (8) using diethyl phosphite and diisopropyl ethylamine (DIPEA). Compound 8 was then treated with mesyl chloride (MsCl) and DIPEA to obtain the mesylate 9, in quantitative yield (Scheme 1). The substitution of the mesylate group with alkoxy groups required, in the case of starting material 4, acetylation of the excess alcohol with acetic anhydride before product 13 could be purified. The acetylated alcohols had much larger retention factor (Rf) values compared to the products and were thus easily removed by flash chromatography, resulting in a yield of 63% for product 13. For compounds 10e12, 14

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Table 1 Docking scores of compounds 1e6 for human, pig and rkbPAP. Both R and S isomers were compared. MolDock Scorea and Ligand Efficiencyb (both in kcal/mol)

Compound Number

isomer

Pig PAP

Human PAP

rkbPAP

1

R S R S R S R S R S R S

187.22/-6.69 172.44/6.16 159.42/-5.31 190.73/-6.36 180.71/-5.65 197.64/-6.18 192.21/-5.65 196.40/-5.78 193.93/-5.39 214.84/-5.97 200.73/-5.28 200.53/-5.28

173.99/-6.21 172.81/6.17 173.82/-5.79 182.09/-6.07 191.72/-5.99 192.06/-6.00 194.24/-5.71 190.64/-5.61 175.74/-4.88 187.78/-5.10 180.90/-4.75 184.51/-4.86

161.80/-5.78 159.01/-5.68 165.21/-5.51 203.30/-6.78 149.70/-4.68 144.66/-4.52 153.44/-4.51 154.36/-4.54 161.37/-4.48 150.69/-4.19 148.41/-3.91 167.00/-4.39

2 3 4 5 6

a Each docking performed in MVD is associated with a MolDock score. This score is calculated by energy minimization. Binding energies of the compounds are related to the magnitude of these scores, i.e. larger negative values indicate better binding energies [60]. b The ligand efficiency is calculated by dividing the MolDock Score with the number of heavy atoms [60].

Fig. 4. Predicted binding orientations of the R (left) and S (right) isomers of compounds 1e6 (color by ID) to pig PAP (a), human PAP (b) and rkbPAP (c). Overall, the most promising compounds appear to have longer alkyl chains (>14 carbons), but all are predicted to exploit the binuclear metal center in the active site, in most cases the hydrophobic cleft (to bind the naphthalene moiety) and the long groove for the alkyl chain. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

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Scheme 1. Reagents and Conditions: (a) diethyl phosphite, DIPEA, r.t., 18 h, 100%; (b) MsCl, DIPEA, CH2Cl2, r.t., 3 d. 93%; (c) ROH, DIPEA, MeCN, D, 18 h. 10 (36%), 11 (36%), 12 (39%), 13 (63%), 14 (22%), 15 (34%); (d) distilled TMSCl, dry NaI, dry MeCN, 40  C, 18 h. 1 (91%), 2 (94%), 3 (94%), 4 (100%), 5 (87%), 6 (100%).

and 15 the acetylation step was not required. For the final ester cleavage reaction to convert the phosphonate diesters 10e15 to the corresponding target phosphonic diacids 1e6, the diesters were treated with freshly distilled TMSCl and NaI in dry acetonitrile (MeCN) at 40  C under anhydrous conditions. 3. Inhibition studies The dissociation constants for competitive and uncompetitive inhibitor binding (i.e. Kic and Kiuc, respectively) were determined for both pig PAP and rkbPAP in presence of compounds 1e6 at pH 4.9 (Table 2). In order to enhance the solubility of the compounds 25% DMSO was included in the assay conditions (both pig PAP and rkbPAP tolerate DMSO concentrations up to 30%; unpublished data). For both pig PAP and rkbPAP only the competitive mode of inhibition is relevant for each compound tested. Notably the decylderivative from the first series (Fig. 1c) inhibited pig PAP in both competitive and uncompetitive modes (Kic 17 mM and Kiuc 20 mM, respectively [50]). In comparison, compounds 1e6 inhibit both pig PAP and rkbPAP solely competitively. The majority of the compounds tested here are stronger inhibitors than their derivatives with shorter alkyl chains, where the inhibition constants or IC50 values for rkbPAP and pig PAP range from the high micromolar to the low micromolar range as the chain length is increased from C6 to C10 [50] (Fig. 1). For pig PAP, 4 (with a C18 alkyl chain) is the most potent inhibitor with a Ki ~200 nM. Extending the chain length beyond C18 leads to a dramatic weakening of the inhibitory potency. While this result appears to contradict the docking results (which indicated that 6 may be the most potent for pig PAP; Table 1) the decreasing inhibitory effect observed experimentally may be related to the solubility of 5 and in particular 6. Nonetheless, the observation that an elongation of the

Table 2 Competitive inhibition constants Kic (mM) of pig PAP and rkbPAP for compounds 1 to 6.a Enzyme

Compound 1

2

3

4

5

6

Pig PAP rkbPAP

0.7 ± 0.2 4.5 ± 1.9

1.1 ± 0.4 0.2 ± 0.1

0.6 ± 0.2 0.4 ± 0.2

0.2 ± 0.1 0.5 ± 0.2

4.4 ± 1.5 0.8 ± 0.2

62 ± 39 14 ± 5

a Kinetic data were analyzed by non-linear regression using the general inhibition equation which accommodates both the competitive and uncompetitive mode of binding for compounds 1 to 6. Only the competitive mode was relevant in our study.

alkyl chain length up to C18 improves the inhibitory potency of the compounds supports the hypothesis that the fatty acid chain may interact with the hydrophobic patch close to the bimetallic metal center. A similar trend is observed for rkbPAP, although here the most potent inhibitor is compound 2 (with a C14 alkyl chain) and a further elongation leads to a more gradual decline in the inhibitory effect. For both pig PAP and rkbPAP compounds 4 and 2, respectively, are the most effective inhibitors yet developed for any PAP, indicating that the multipronged attack on the metal center, the naphthalene binding site and the hydrophobic patch is a successful strategy to develop promising drug leads. The similarity of the inhibition constants suggest that rkbPAP is a suitable model system to study the interactions between mammalian PAPs and potential drug leads. In an attempt to visualize the binding interactions of compounds 1 to 6, rkbPAP crystals were soaked with a solution containing 5 mM of each of the racemates. 4. Crystallographic investigations of inhibitor binding The soaking of rkbPAP crystals with the racemic mixtures of compounds 1 and 4 led to crystals that were suitable for X-ray diffraction studies. Data were collected to 2.30 Å and 2.20 Å, respectively (Table 3). The crystals have the same space group as described for previously determined structures of rkbPAP [51]. Crystallographic parameters are summarized in Table 3. There are two dimers (subunits A-D) in each asymmetric unit of the crystals. The overall fold of the four polypeptides strongly resembles those of the previously determined structures of free rkbPAP and the phosphate- and sulfate-bound complexes of this enzyme (rmsd values for all Ca atoms <0.37 Å) [35,51]. The focus of this study is on subunit A, the subunit where the highest quality electron density is observed in both structures, though the structural features of the polypeptides in the other three subunits are similar as far as observable. The electron density in the active site indicated that both compounds are bound close to the metal center (Figs. 5 and 6). Interestingly, the R-stereoisomer of compound 1 ((R)-1) and the Sstereoisomer of compound 4 ((S)-4) fit best into the Polder omit maps demonstrating that there may be some selective binding in the active site despite the lack of an apparent trend. Polder maps were chosen for this study since they are optimal for visualization of inhibitors obscured by bulk solvent scaling and since they provide improved interpretive powers compared to simulated annealing omit maps [61]. For both inhibitors the naphthalene ring

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Table 3 Data collection and refinement statistics. Diffraction data

rkbPAP-4 (6OFD)

rkbPAP-1 (6OF5)

Resolution Range (Å) Observations (I>s(I)) Unique reflections (I>s(I)) Completeness (%) Mean Rpimb Multiplicity CC(1/2) Crystal parameters Space group Unit cell lengths (Å)

20.05e2.20 726,576 139,709 99.8 (100.0)a 13.6 (2.7) 0.048 (0.293) 5.2 (4.5) 0.997 (0.763)

19.99e2.30 676,902 121,046 97.9 (89.9) 11.6 (4.5) 0.055 (0.185) 5.6 (5.6) 0.994 (0.874)

P 31 2 1 a ¼ b ¼ 126.17 c ¼ 298.04 a ¼ b ¼ 90 g ¼ 120

P 31 2 1 a ¼ b ¼ 126.81 c ¼ 298.21 a ¼ b ¼ 90 g ¼ 120

0.155 0.199 0.008 0.951

0.180 0.231 0.009 1.050

95.27 0.30

94.96 0.50

Unit cell angles ( ) Refinement Rworkc Rfreec rmsd bond lengths (Å) rmsd bond angles (o) Ramachandran statistics Favored regions (%) Outlier regions (%) a

Values in parentheses are for the outer resolution shells. Rpim is a measure of the quality of the data after averaging the multiple measurements and Rpim ¼ Shkl [n/(n-1)]1/2 Si jIi(hkl)ej/Shkl Si Ii(hkl), where n is the multiplicity. c Rwork ¼ SjjFobsj-jFcalcjj/SjFobsj, Rwork is calculated based on the reflections used in the refinement (95% of the total data) and Rfree is calculated using the remaining 5% of the data. b

is bound in the pocket formed by Arg258, His295 and His296 (NBS in Fig. 2), as predicted by the docking studies (Fig. 4). However, for the phosphonate moiety and alkyl chain variations in the binding interactions are observed for the two inhibitors, as described in detail below. The structure of the rkbPAP-(S)-4 complex is shown in Fig. 5. The phosphonate moiety of (S)-4 is bound to the active site of rkbPAP in two different conformations. In one conformation (conformer A; 0.53 occupancy; average B factor of 55.73) a phosphonate oxygen atom of the inhibitor forms a hydrogen bond with the metalbridging hydroxide group of rkbPAP (2.6 Å). The same oxygen atom also forms a hydrogen bond with the side chain amino group of Asn201 (2.7 Å) and a weak electrostatic interaction with ZnII (3.2 Å). Another phosphonate oxygen atom forms a strong hydrogen bond with the side chain imidazole nitrogen atom of His296 (2.3 Å), and the third phosphonate oxygen forms a hydrogen bond with the side chain imidazole nitrogen atom of His202 (2.9 Å) and the metal-bridging hydroxide group (2.7 Å). This observed mode of binding of the phosphonate moiety is similar to that observed for sulfate to rkbPAP, a mode that was suggested to model the incoming substrate in the pre-catalytic enzyme-substrate complex [35]. In addition, the positively charged guanidino group of Arg258 from the adjoining subunit in the rkbPAP dimer is oriented away from the inhibitor, allowing it to form hydrophobic and van der Waals interactions (3.6e4.9 Å) with the naphthalene group of the inhibitor. The naphthalene group is further stabilized by forming p-cation and van der Waals interactions with the positively charged side-chain imidazole group of His295 (3.0e4.9 Å) and hydrophobic interactions with the side chain imidazole group of His296 (3.7e4.9 Å). The alkyl chain forms hydrophobic and van

Fig. 5. (a) Ribbon and stick representation and Polder omit map (>3.63 s) of conformers A and B of (S)-4 (white and grey carbons, respectively) in the active site of rkbPAP (light blue carbons). Metal ions are shown as spheres with FeIII in yellow and ZnII in cyan. (b) Surface and stick representation of conformers A and B of (S)-4 (white and brown carbons, respectively) in the active site of rkbPAP (grey surface), metal ions are shown as green spheres and marked with an asterisk. The alkyl chain of (S)-4 stabilizes Lys7 from the Nterminal domain of rkbPAP by forming hydrophobic interactions with its sidechain, resulting in a more defined hydrophobic patch similar to the groove under the repression loop in the mammalian enzyme. For clarity of presentation, this figure is inverted relative to (a). (c & d) Ribbon and stick representation of (S)-4 conformers A and B (white and brown carbons, respectively) in the active site of rkbPAP (light blue carbons). Metal ions are shown as spheres with Fe(III) in yellow and Zn(II) in cyan. Hydrogen bonds are in dashed cyan and bonds to metal ions are in dashed yellow (created with PyMOL [62]). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

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Fig. 6. (a) Ribbon and stick representation and Polder omit map (>3.61 s) of (R)-1 (grey carbons) in the active site of rkbPAP (light blue carbons). Metal ions are shown as spheres with FeIII in yellow and ZnII in cyan. (b) Surface and stick representation of (R)-1 (brown carbons) in the active site of rkbPAP (grey surface). Metal ions are shown as green spheres and marked with an asterisk. The alkyl chain of (R)-1 folds in on the inhibitor and forms hydrophobic interactions with the naphthalene ring (c) Ribbon and stick representation of (R)-1 (brown carbons) in the active site of rkbPAP (light blue carbons). Metal ions are shown as spheres with FeIII in yellow and ZnII in cyan. Hydrogen bonds are in dashed cyan and bonds to metal ions are in dashed yellow (created with PyMOL [62]). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

der Waals interactions with the side-chains of His202 (3.9e4.9 Å), Tyr365 (3.2e5.0 Å) and Arg170 (3.0e4.7 Å), all within 10 Å of the catalytic center, and with the side-chains of Val367 (3.6e4.6 Å), Lys7 (3.6e4.9 Å) and Arg9 (3.7e4.7 Å), which are >10 Å away from the catalytic center. In the second conformation of (S)-4 (conformer B; 0.47 occupancy; average B factor of 56.17), the phosphonate group faces away from the bimetallic center, with one phosphonate oxygen atom forming a weak hydrogen bond with a nitrogen of the side chain imidazole group of His202 (3.3 Å). Another phosphonate oxygen forms hydrogen bonds with nitrogen atoms of the side chain imidazole groups of His295 (2.7 Å) and His296 (3.1 Å). A third phosphonate oxygen forms a strong hydrogen bond with the oxygen in the side chain phenol group of Tyr365 (2.3 Å) and a weaker hydrogen bond (3.1 Å) with a nearby water molecule. The remaining interactions between conformer B and rkbPAP are similar to those of conformer A. Interestingly; both conformations stabilize Lys7 from the N-terminal domain of rkbPAP, thus forming a more compact hydrophobic patch for the alkyl chain of the inhibitor. For both conformers the extensive network of interactions is in agreement with the potent inhibitory effect exerted by compound 4 (Ki ~500 nM). The structure of the rkbPAP in complex with (R)-1 (0.72 occupancy; average B factor of 52.90) is shown in Fig. 6. In contrast to (S)-4, the phosphonate group of (R)-1 coordinates directly to the metal center, displacing the metal-bridging hydroxide group. One phosphonate oxygen atom coordinates to ZnII (2.4 Å) and forms a hydrogen bond with the side chain amino group of Asn201 (2.8 Å).

Another phosphonate oxygen atom coordinates to FeIII (1.8 Å), thus forming a m-1,3 complex as observed for the structures of rkbPAP and pig PAP with tetraoxo anions such as phosphate or vanadate [31,32,35]. The third phosphonate oxygen is hydrogen-bonded to the oxygen in the backbone carbonyl group of His323 (2.4 Å). Since it is positioned between the metal ions, 2.7 Å from ZnII and 2.9 Å from FeIII, the phosphonate adopts a tripodal conformation in the catalytic center that resembles that of the proposed transition state of the phosphoroylytic reaction [2,31e33,35]. The oxygen in the ester linkage of the inhibitor forms a hydrogen bond with a nitrogen in the side chain imidazole group of His202 (3.0 Å). The naphthalene ring is nestled in the NBS and forms p-cation interactions with the side chain imidazole nitrogens of His295 and His296 (2.9e3.8 Å), similar to the binding interactions observed for (S)-4. The first four carbon atoms of the alkyl chain form hydrophobic interactions with the side chain phenol group of Tyr365 (2.8e4.0 Å) and the guanidino group of Arg170 in a manner similar to that observed in the rkbPAP-(S)-4 complex (Fig. 5). However, subsequently the alkyl chain of (R)-1 does not trace along hydrophobic patch as observed for (S)-4, but instead stacks on top of the naphthalene group in a conformation stabilized via a series of hydrophobic interactions (3.3e3.7 Å) within the inhibitor. The side chain guanidino group of Arg258 adopts a similar orientation as in the rkbPAP-(S)-4 complex but instead of interacting with the naphthalene group it is involved in hydrophobic interactions with the eleventh and twelfth carbon atoms of the alkyl chain (2.9e4.0 Å). The observation that the shorter alkyl chain of (R)-1

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does not form as many interactions with rkbPAP as (S)-4 is in agreement with its ~10-fold reduced inhibitory potency (Table 2).

6.2. Diethyl hydroxy(naphthalen-1-yl)methylphosphonate (8) [50].

5. Conclusions PAPs belong to the group of bimetallic hydrolases and various members of this group have emerged as potential drug targets to treat a range of human disorders. The NiII-dependent urease, for instance, may be targeted to treat disorders of the digestive tract [63e65], while the FeII- and/or MnII-dependent cyclic diesterase Rv0805 is a promising new target to combat tuberculosis [66]. To date few clinically relevant inhibitors for this group of enzymes have emerged, largely because the active sites are frequently close to the surface of the protein and/or the bimetallic catalytic centers have similar geometries that render the design of selective inhibitors more difficult. The difficulty in designing potent and selective inhibitors of clinical relevance for bimetallic hydrolases is powerfully illustrated by reviewing the attempts to develop suitable inhibitors for metallo-b-lactamases (MBLs). MBLs have emerged as a major concern for global health since they are capable of inactivating most of the currently used b-lactam-based antibiotics [3,67]. A large number of inhibitors have been developed (see reference [68] for a recent review and references [69e73] for some recent examples), but none has yet reached clinical application. The role of PAP in bone resorption has been well established since studies with transgenic mice demonstrated that the level of PAP expression is directly correlated with bone density; mice overexpressing PAP show an osteoporotic phenotype [46], while knockout mice display features characteristic of osteopetrosis [54]. Similarly to MBLs no inhibitors of clinical suitability for PAP has yet been reported [52]. However, the discovery of a series of a-alkoxysubstituted naphthylmethylphosphonic acids as strong inhibitors of pig PAP and rkbPAP was encouraging as these molecules provided an easy handle to improve both potency and specificity through modification of the alkoxy chain [50,57]. Indeed, our kinetic measurements indicated that side chains with 14 (compound 2) to 18 (compound 4) carbon atoms in the alkyl chain are ideal to optimize the inhibitory effect for both pig PAP and rkbPAP (Table 2). The visualization of the structures of rkbPAP with two of these compounds bound (Figs. 5 and 6) provides, for the first time, insight into the interactions between a clinically promising lead and a PAP. The structures demonstrate that several structural features can be exploited to enhance the potency of the inhibitor into the nanomolar range. The challenge ahead is to either modify these compounds further to address their relatively modest solubility in an aqueous environment, and/or develop suitable delivery strategies. 6. Experimental 6.1. Synthesis All NMR experiments were recorded on Bruker AVANCE 500, 400 or 300 MHz spectrometers. Chemical shifts are reported in parts per million (ppm) on a d-scale, and referenced to the residual solvent peak (1H 7.24 ppm, 13C 77.0 ppm for CDCl3). Coupling constants (J) are reported in Hertz. Multiplicities of the peaks are abbreviated as follows: s for singlet, d for doublet, t for triplet, q for quartet, td for triplet of doublet, dd for doublet of doublet, m for multiplet and br for broad signals. Melting points were determined on a Stuart SMP11 Melting Point apparatus and are uncorrected. Low- and high-resolution EI-MS were measured on a Finnigan MAT 900 XL-Trap mass spectrometer in positive ionisation mode. LR-ESI were recorded on a Bruker HCT 3D Ion Trap and HR-ESI were performed on a Bruker MicrOTof-Q with the DIONEX Ultimate 3000 LC in positive electrospray ionisation mode.

1-Naphthaldehyde (7, 5.000 g, 32.0 mmol), diethyl phosphite (4.863 g, 35.2 mmol) and DIPEA (12.41 g, 96.0 mmol) were combined to give a brown solution which was stirred at room temperature overnight. Petroleum ether (100 mL) was added to the reaction mixture. After collecting the solids by suction filtration, they were washed with petroleum ether (100 mL), before drying in vacuo to give the product as an off-white solid (9.421 g, 100%); Rf ¼ 0.57 (EtOAc); m.p. 114e115  C, lit. m.p. 114e115  C [74]; 1H NMR (400 MHz, CDCl3) d 8.07 (1H, d, J8.4, Ar-H), 7.87e7.79 (3H, m, Ar-H), 7.52e7.44 (3H, m, Ar-H), 5.85 (1H, d, JP-H11.4, CHP), 4.08e3.78 (3H, m), 3.78e3.73 (1H, m) (2  CH2), 1.20 (3H, t, J7.0, CH3), 1.03 (3H, t, J7.0 CH3); 13C NMR (100 MHz, CDCl3) d133.6 (d, JP-C1.94, quaternary Ar), 132.6 (d, JP-C2.0, quaternary Ar), 130.8 (d, JP-C6.1, quaternary Ar), 128.8 (d, JP-C3.4, Ar), 128.7, 126.1, 125.6 (3  Ar), 125.4 (d, JP-C6.0, Ar), 125.3 (d, JP-C3.5, Ar), 123.6 (Ar), 66.3 (d, JP-C160.2, CHP), 63.4 (d, JPC7.1, CH2), 63.1 (d, JP-C7.3, CH2), 16.3 (d, JP-C5.8, CH3), 16.2 (d, JP-C5.6, CH3). NMR data are in agreement with that previously reported [50]. 6.3. (Diethoxyphosphoryl) (naphthalen-1-yl)methyl methanesulfonate (9) [50].

MsCl (5.255 g, 45.87 mmol) was added dropwise to a solution of diethyl hydroxyl (naphthalen-1-yl)methylphosphonate (8) (9.000 g, 30.58 mmol) in DCM (150 mL) at 0  C. DIPEA (7.906 g, 61.17 mmol) was added dropwise while stirring the reaction vigorously. The reaction was left to warm to room temperature under N2 with stirring for 3 days. The reaction mixture was partitioned between water (250 mL) and Et2O (350 mL). The aqueous layer was extracted with Et2O (2  200 mL). The combined organic layers were washed with saturated NH4Cl (200 mL), brine (200 mL), then dried with Na2SO4. Evaporation of solvent in vacuo gave the solid as an orange solid. The product decomposed on attempted chromatography on silica, but could be recrystallized from toluene to give an off-white solid (10.59 g, 93%); Rf ¼ 0.43 (70% EtOAc in petroleum ether); m.p. 93e94  C, lit. m.p. 94e95  C [50]; 1H NMR (400 MHz, CDCl3) d 8.18 (1H, d, J8.5, Ar-H), 7.89e7.82 (3H, m, Ar-H), 7.60e7.47 (3H, m, Ar-H), 6.54 (1H, d, JP-H16.3, CHP), 4.20e4.15 (2H, m), 4.00e3.94 (1H, m), 3.78e3.72 (1H, m) (2  CH2), 2.72 (3H, s, CH3SO3), 1.29 (3H, td, JH-H7.0, JP-H0.5, CH2CH3), 1.00 (3H, td, JH-H7.1, JP-H0.5, CH2CH3); 13C NMR (100 MHz, CDCl3) d133.7 (d, JP-C1.3, Ar), 130.5 (d, JP-C5.6, Ar), 130.4 (d, JP-C2.9, Ar), 128.9 (Ar), 127.8 (d, JP-C1.2, Ar), 127.7 (d, Ar, JP-C5.5), 127.0, 126.2 (2  Ar), 125.2 (d, Ar, JP-C2.8), 123.3 (Ar), 74.5 (d, JP-C170.4, CHP), 64.3 (d, JP-C7.0, CH2), 63.8 (d, JPC6.7, CH2), 39.5 (CH3SO3), 16.4 (d, JP-C5.8, CH3), 16.0 (d, JP-C5.8, CH3). NMR data are in agreement with that previously reported [50]

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6.4. Diethyl dodecyloxy(naphthalen-1-yl)methylphosphonate (10)

9

29.52, 29.45, 29.43, 29.2, 25.9, 22.6 (12  CH3(CH2)12CH2O), 16.3 (d, JP-C5.8, OCH2CH3), 16.1 (d, JP-C5.8, OCH2CH3), 14.0 (CH3(CH2)13O); ESI-MS m/z 513 (M þ Na)þ HRMS calculated for C29H47O4PNaþ 513.3104, found 513.3119. 6.6. Diethyl hexadecyloxy(naphthalen-1-yl)methylphosphonate (12)

To a solution of (diethoxyphosphoryl) (naphthalen-1-yl)methyl methanesulfonate (9) (1.505 g, 4.040 mmol) in MeCN (2 mL) was added 1-dodecanol (2.259 g, 12.12 mmol), and DIPEA (0.7833 g, 6.051 mmol). The reaction was stirred under reflux and N2 overnight. The solvent was removed in vacuo. The residue was dissolved in EtOAc (30 mL), washed with 5% HCl (2  30 mL), NaHCO3 (2  30 mL), water (30 mL) and dried over Na2SO4. The crude product was purified by flash chromatography (40% EtOAc in petroleum ether) to yield the pure product as a yellow oil (0.6737 g, 36%); Rf ¼ 0.38 (40% EtOAc in petroleum ether); 1H NMR (400 MHz, CDCl3) d 8.18 (1H, d, J8.4, Ar-H), 7.80e7.75 (3H, m, Ar-H), 7.47e7.41 (3H, m, Ar-H), 5.38 (1H, d, JP-H16.8, CHP), 4.07e3.91 (3H, m) and 3.77e3.73 (1H, m) (2  OCH2CH3), 3.43e3.40 (2H, m, CH3(CH2)10CH2O), 1.56e1.55 (2H, m, CH3(CH2)9CH2CH2O), 1.21e1.14 (21H, br m, CH3(CH2)9, OCH2CH3), 1.01 (3H, td, JH-H7.0, JP-H0.4, OCH2CH3), 0.83 (3H, t, J7.1, CH3(CH2)11O); 13C NMR (100 MHz, CDCl3) d133.5 (d, JP-C2.0, quaternary Ar), 131.4 (d, JP-C5.0, quaternary Ar), 131.0 (d, JP-C1.9, quaternary Ar), 128.6 (d, JP-C3.5, Ar), 128.4 (Ar), 126.4 (d, JP-C6.5, Ar), 125.7, 125.4 (2  Ar), 125.1 (d, JP-C3.4, Ar), 123.8 (Ar), 75.7 (d, JP-C170.2, CHP), 71.0 (d, JP-C13.8, CH3(CH2)10CH2O), 62.9 (d, JP-C7.0, OCH2CH3), 62.7 (d, JP-C6.7, OCH2CH3), 31.7, 29.5, 29.44, 29.42, 29.37, 29.36, 29.14, 29.12, 26.0, 22.5 (10  CH3(CH2)10CH2O), 16.2 (d, JP-C5.8, OCH2CH3), 16.0 (d, JP-C5.9, OCH2CH3), 13.9 (CH3(CH2)11O); ESI-MS m/z 485 (M þ Na)þ HRMS calculated for C27H43O4PNaþ 485.2791, found 485.2751. 6.5. Diethyl tetradecyloxy(naphthalen-1-yl)methylphosphonate (11)

The general procedure in synthesising diethyl dodecyloxy(naphthalen-1-yl) methylphosphonate (10) was followed with (diethoxyphosphoryl) (naphthalen-1-yl)methyl methanesulfonate (9) (1.504 g, 4.038 mmol), 1-hexadecanol (3.053 g, 12.59 mmol), and DIPEA (0.7829 g, 6.057 mmol) in MeCN (2 mL). The crude product was purified by flash chromatography (40% EtOAc in petroleum ether) to yield the pure product as a yellow oil (0.8193 g, 39%); Rf ¼ 0.49 (40% EtOAc in petroleum ether); 1H NMR (400 MHz, CDCl3) d 8.19 (1H, d, J8.4, Ar-H), 7.83e7.78 (3H, m, Ar-H), 7.50e7.44 (3H, m, Ar-H), 5.40 (1H, d, JP-H16.7, CHP), 4.09e3.93 (3H, m) and 3.79e3.75 (1H, m) (2  OCH2CH3), 3.46e3.42 (2H, m, CH3(CH2)14CH2O), 1.58e1.56 (2H, m, CH3(CH2)13CH2CH2O), 1.29e1.16 (29H, br m, CH3(CH2)13, OCH2CH3), 1.04 (3H, td, JH-H6.7, JP13 H0.3, OCH2CH3), 0.85 (3H, t, J6.9, CH3(CH2)15O); C NMR (100 MHz, CDCl3) d133.6 (d, JP-C2.0, quaternary Ar), 131.5 (d, JP-C5.1, quaternary Ar), 131.0 (d, JP-C1.6, quaternary Ar), 128.7 (d, JP-C3.6, Ar), 128.5 (Ar), 126.5 (d, JP-C6.5, Ar), 125.9, 125.5 (2  Ar), 125.2 (d, JP-C3.3, Ar), 124.0 (Ar), 75.8 (d, JP-C169.6, CHP), 71.1 (d, JP-C13.8, CH3(CH2)14CH2O), 63.0 (d, JP-C7.0, OCH2CH3), 62.8 (d, JP-C6.7, OCH2CH3), 31.8, 29.59, 29.56, 29.49, 29.47, 29.3, 25.9, 22.6 (14  CH3(CH2)14CH2O), 16.3 (d, JP-C5.8, OCH2CH3), 16.1 (d, JP-C5.8, OCH2CH3), 14.0 (CH3(CH2)15O); ESI-MS m/ z 541 (M þ Na)þ HRMS calculated for C31H51O4PNaþ 541.3417, found 541.3435. 6.7. Diethyl octadecyloxy(naphthalen-1-yl)methylphosphonate (13)

The general procedure in synthesising diethyl dodecyloxy(naphthalen-1-yl) methylphosphonate (10) was followed with (diethoxyphosphoryl) (naphthalen-1-yl)methyl methanesulfonate (9) (1.502 g, 4.034 mmol), 1-tetradecanol (3.458 g, 16.13 mmol), and DIPEA (0.7821 g, 6.051 mmol) in MeCN (2 mL). The crude product was purified by flash chromatography (40% EtOAc in petroleum ether) to yield the pure product as a yellow oil (0.7150 g, 36%); Rf ¼ 0.46 (40% EtOAc in petroleum ether); 1H NMR (400 MHz, CDCl3) d 8.19 (1H, d, J8.4, Ar-H), 7.83e7.77 (3H, m, Ar-H), 7.49e7.43 (3H, m, Ar-H), 5.40 (1H, d, JP-H16.8, CHP), 4.09e3.93 (3H, m) and 3.78e3.75 (1H, m) (2  OCH2CH3), 3.44e3.41 (2H, m, CH3(CH2)12CH2O), 1.58e1.56 (2H, m, CH3(CH2)11CH2CH2O), 1.25e1.16 (25H, br m, CH3(CH2)11, OCH2CH3), 1.03 (3H, td, JH-H6.6, JP13 H0.5, OCH2CH3), 0.85 (3H, t, J6.9, CH3(CH2)13O); C NMR (100 MHz, CDCl3) d133.6 (d, JP-C2.0, quaternary Ar), 131.5 (d, JP-C5.3, quaternary Ar), 131.0 (d, JP-C1.5, quaternary Ar), 128.7 (d, JP-C3.6, Ar), 128.5 (Ar), 126.4 (d, JP-C6.6, Ar), 125.8, 125.5 (2  Ar), 125.2 (d, JP-C3.3, Ar), 123.9 (Ar), 75.6 (d, JP-C170.5, CHP), 71.1 (d, JP-C13.7, CH3(CH2)12CH2O), 63.0 (d, JP-C7.0, OCH2CH3), 62.8 (d, JP-C6.7, OCH2CH3), 31.8, 29.6, 29.53,

The general procedure in synthesising diethyl dodecyloxy(naphthalen-1-yl) methylphosphonate (10) was followed with (diethoxyphosphoryl) (naphthalen-1-yl)methyl methanesulfonate (9) (1.502 g, 4.033 mmol), 1-octadecanol (3.273 g, 12.09 mmol), and DIPEA (0.7819 g, 6.049 mmol) in MeCN (2 mL). After the solvent was removed in vacuo, in order to acetylate the excess 1octadecanol, the residue was dissolved in DCM (50 mL). Pyridine (5.7 mL) and acetic anyhydride (5.7 mL) was added and stirred under N2 at RT for 1 h. MeOH was added to quench the reaction and stirred for a further 30 min. After the solvent was removed in vacuo, the reside was dissolved in EtOAc (30 mL), washed with 5% HCl (2  30 mL), NaHCO3 (2  30 mL), water (30 mL) and dried over Na2SO4. The crude product was purified by flash chromatography (40% EtOAc in petroleum ether) to yield the pure product as a yellow oil (1.400 g, 63%); Rf ¼ 0.56 (40% EtOAc in petroleum ether);

10

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1 H NMR (400 MHz, CDCl3) d 8.19 (1H, d, J8.3, Ar H), 7.85e7.73 (3H, m, Ar-H), 7.51e7.46 (3H, m, Ar-H), 5.40 (1H, d, JP-H16.8, CHP), 4.07e3.93 (3H, m) and 3.79e3.74 (1H, m) (2  OCH2CH3), 3.47e3.42 (2H, m, CH3(CH2)16CH2O), 1.60e1.56 (2H, m, CH3(CH2)15CH2CH2O), 1.31e1.17 (33H, br m, CH3(CH2)15, OCH2CH3), 1.04 (3H, t, J7.1, OCH2CH3), 0.86 (3H, t, J6.9, CH3(CH2)17O); 13C NMR (100 MHz, CDCl3) d 133.7 (d, JP-C2.0, quaternary Ar), 131.6 (d, JP-C5.2, quaternary Ar), 131.1 (d, JP-C1.9, quaternary Ar), 128.8 (d, JP-C3.6, Ar), 128.6 (Ar), 126.5 (d, JP-C6.6, Ar), 125.9, 125.6 (2  Ar), 125.3 (d, JPC3.4, Ar), 124.0 (Ar), 75.6 (d, JP-C170.2, CHP), 71.2 (d, JP-C13.7, CH3(CH2)16CH2O), 63.1 (d, JP-C7.0, OCH2CH3), 62.9 (d, JP-C6.8, OCH2CH3), 31.9, 29.67, 29.63, 29.57, 29.54, 29.3, 26.0, 22.7 (16  CH3(CH2)16CH2O), 16.4 (d, JP-C5.9, OCH2CH3), 16.2 (d, JP-C5.9, OCH2CH3), 14.1 (CH3(CH2)17O); ESI-MS m/z 569 (M þ Na)þ HRMS calculated for C33H55O4PNaþ 569.3730, found 569.3750.

6.8. Diethyl icosyloxy(naphthalen-1-yl)methylphosphonate (14)

ether) to yield the pure product as a yellow oil (0.608 g, 34%); Rf ¼ 0.48 (40% EtOAc in petroleum ether); 1H NMR (400 MHz, CDCl3) d 8.19 (1H, d, J8.4, Ar-H), 7.84e7.79 (3H, m, Ar-H), 7.50e7.45 (3H, m, Ar-H), 5.40 (1H, d, JP-H16.8, CHP), 4.07e3.94 (3H, m) and 3.80e3.75 (1H, m) (2  OCH2CH3), 3.47e3.42 (2H, m, CH3(CH2)20CH2O), 1.60e1.56 (2H, m, CH3(CH2)19CH2CH2O), 1.32e1.16 (41H, br m, CH3(CH2)19, OCH2CH3), 1.04 (3H, t, J7.1, OCH2CH3), 0.86 (3H, t, J6.9, CH3(CH2)19O); 13C NMR (100 MHz, CDCl3) d 133.6 (d, JP-C2.0, quaternary Ar), 131.6 (d, JP-C5.2, quaternary Ar), 131.1 (d, JP-C1.7, quaternary Ar), 128.8 (d, JP-C3.5, Ar), 128.5 (Ar), 126.5 (d, JP-C6.5, Ar), 125.9, 125.5 (2  Ar), 125.2 (d, JP-C3.3, Ar), 124.0 (Ar), 75.1 (d, JP-C170.4, CHP), 71.2 (d, JP-C13.8, CH3(CH2)20CH2O), 63.1 (d, JP-C7.0, OCH2CH3), 62.9 (d, JP-C6.9, OCH2CH3), 31.9, 29.65, 29.60, 29.54, 29.51, 29.3, 25.9, 22.6 (20  CH3(CH2)20CH2O), 16.3 (d, JP-C5.8, OCH2CH3), 16.1 (d, JP-C5.8, OCH2CH3), 14.1 (CH3(CH2)21O); ESI-MS m/ z 625 (M þ Na)þ HRMS calculated for C37H63O4PNaþ 625.4356, found 625.4342. 6.10. Dodecyloxy(naphthalen-1-yl)methylphosphonic acid (1)

The general procedure in synthesising diethyl dodecyloxy(naphthalen-1-yl) methylphosphonate (10) was followed with (diethoxyphosphoryl) (naphthalen-1-yl)methyl methanesulfonate (9) (1.051 g, 2.823 mmol), 1-icosanol (2.538 g, 8.501 mmol), and DIPEA (0.727 g, 5.626 mmol) in MeCN (1 mL). The crude product was purified by flash chromatography (40% EtOAc in petroleum ether) to yield the pure product as a yellow oil (0.350 g, 22%); Rf ¼ 0.51 (40% EtOAc in petroleum ether); 1H NMR (400 MHz, CDCl3) d 8.19 (1H, d, J8.4, Ar-H), 7.84e7.79 (3H, m, Ar-H), 7.50e7.45 (3H, m, Ar-H), 5.40 (1H, d, JP-H16.8, CHP), 4.07e3.94 (3H, m) and 3.79e3.75 (1H, m) (2  OCH2CH3), 3.47e3.42 (2H, m, CH3(CH2)18CH2O), 1.58e1.56 (2H, m, CH3(CH2)17CH2CH2O), 1.29e1.17 (37H, br m, CH3(CH2)17, OCH2CH3), 1.04 (3H, td, JH-H6.6, JP13 H0.5, OCH2CH3), 0.86 (3H, t, J6.9, CH3(CH2)19O); C NMR (100 MHz, CDCl3) d 133.6 (d, JP-C2.0, quaternary Ar), 131.6 (d, JP-C5.2, quaternary Ar), 131.1 (d, JP-C1.6, quaternary Ar), 128.8 (d, JP-C3.6, Ar), 128.6 (Ar), 126.5 (d, JP-C6.6, Ar), 125.9, 125.5 (2  Ar), 125.3 (d, JP-C3.4, Ar), 124.0 (Ar), 75.3 (d, JP-C170.0, CHP), 71.2 (d, JP-C13.8, CH3(CH2)18CH2O), 63.1 (d, JP-C7.0, OCH2CH3), 62.9 (d, JP-C6.9, OCH2CH3), 31.9, 29.7, 29.61, 29.54, 29.52, 29.3, 26.0, 22.6 (18  CH3(CH2)18CH2O), 16.3 (d, JP-C5.8, OCH2CH3), 16.2 (d, JP-C5.8, OCH2CH3), 14.1 (CH3(CH2)19O); ESI-MS m/ z 597 (M þ Na)þ HRMS calculated for C35H59O4PNaþ 597.4043, found 597.4028.

Redistilled TMSCl (0.591 g, 5.44 mmol) was added to a solution of diethyl dodecyloxy(naphthalen-1-yl)methylphosphonate (10) (0.503 g, 1.087 mmol), anhydrous NaI (0.820 g, 5.47 mmol) in MeCN (5 mL), then stirred at 40  C under argon for 18 h. After removing MeCN in vacuo, the residue was extracted in EtOAc (3  10 mL), washed with Na2S2O3 (2  10 mL) and brine before being dried over saturated Na2SO4, then evaporated to dryness in vacuo to give the title compound as a waxy solid (0.4036 g, 91%); 1H NMR (400 MHz, CDCl3): d 8.04 (1H, d, J8.4, Ar-H), 7.81 (1H, d, J8.1, Ar-H), 7.72 (1H, d, J8.2, Ar-H), 7.62 (1H, dd, J6.7, J2.6, Ar-H), 7.44e7.35 (3H, m, Ar-H), 5.20 (1H, d, JP-H16.8, CHP), 3.37e3.28 (2H, m, CH2O), 1.56e1.45 (2H, br m, CH2CH2O), 1.30e1.18 (18H, br m, OCH2CH2(CH2)9), 0.86 (3H, t, J6.8, CH3(CH2)11O); 13C NMR (100 MHz, CDCl3): d 133.6 (quaternary Ar), 131.6 (d, JP-C4.9, quaternary Ar), 130.9 (quaternary Ar), 128.6, 128.5 (2  Ar), 126.7 (d, JP-C6.2, Ar), 125.9, 125.4, 125.2, 124.0 (4  Ar), 74.7 (d, JP-C176.1, CHP), 71.6 (d, JP-C12.93, OCH2), 31.9, 29.67, 29.65, 29.64, 29.61, 29.57, 29.46 29.36, 25.9, 25.8, 22.7 (10  CH3(CH2)10CH2O), 14.1 (CH3); ESI-MS m/z 405 (M - H)- HRMS calculated for C23H34O4P405.2200, found 405.2189. 6.11. Tetradecyloxy(naphthalen-1-yl)methylphosphonic acid (2)

6.9. Diethyl docosyloxy(naphthalen-1-yl)methylphosphonate (15)

The general procedure in synthesising diethyl dodecyloxy(naphthalen-1-yl) methylphosphonate (10) was followed with (diethoxyphosphoryl) (naphthalen-1-yl)methyl methanesulfonate (9) (1.097 g, 2.945 mmol), 1-docosanol (3.197 g, 9.787 mmol), and DIPEA (0.816 g, 6.315 mmol) in MeCN (1 mL). The crude product was purified by flash chromatography (40% EtOAc in petroleum

The general procedure in synthesising dodecyloxy(naphthalen1-yl)methylphosphonic acid (1) was followed with redistilled TMSCl (0.598 g, 5.50 mmol), diethyl tetradecyloxy(naphthalen-1yl)methylphosphonate (11) (0.540 g, 1.101 mmol), anhydrous NaI (0.825 g, 5.51 mmol) in MeCN (5 mL). The product was tetradecyloxy(naphthalen-1-yl)methylphosphonic acid as an off-white solid (0.4503 g, 94%); m.p. 56e59  C; 1H NMR (400 MHz, CDCl3): d 8.05

D. Feder et al. / European Journal of Medicinal Chemistry 182 (2019) 111611

(1H, d, J8.5, Ar-H), 7.80 (1H, d, J7.6, Ar-H), 7.74 (1H, d, J8.2, Ar-H), 7.64 (1H, dd, J6.9, J3.0, Ar-H), 7.46e7.36 (3H, m, Ar H), 5.22 (1H, d, JP-H16.9, CHP), 3.38e3.29 (2H, m, CH2O), 1.56e1.49 (2H, br m, CH2CH2O), 1.33e1.20 (22H, br m, OCH2CH2(CH2)11), 0.89 (3H, t, J7.0, CH3(CH2)13O); 13C NMR (100 MHz, CDCl3): d133.5 (d, JP-C1.9, quaternary Ar), 131.6 (d, JP-C5.1, quaternary Ar), 131.3 (d, JP-C2.2, quaternary Ar), 128.6, 128.5 (2  Ar), 126.7 (d, JP-C6.3, Ar), 125.9, 125.4, 125.2, 124.0 (4  Ar), 74.8 (d, JP-C172.3, CHP), 71.5 (d, JP-C12.7, OCH2), 31.9, 29.71, 29.68, 29.66, 29.65, 29.61, 29.59, 29.57, 29.46, 29.37, 25.8, 22.7 (12  CH3(CH2)12CH2O), 14.1 (CH3); ESI-MS m/z 433 (M H)- HRMS calculated for C25H38O4P 433.2513, found 433.2505.

11

31.9, 29.73, 29.67, 29.60, 29.43, 29.37, 25.9, 25.8, 22.7 (16  CH3(CH2)16CH2O), 14.5 (CH3); ESI-MS m/z 489 (M - H)- HRMS calculated for C29H46O4P 489.3139, found 489.3125. 6.14. Icosyloxy(naphthalen-1-yl)methylphosphonic acid (5)

6.12. Hexadecyloxy(naphthalen-1-yl)methylphosphonic acid (3)

The general procedure in synthesising dodecyloxy(naphthalen1-yl)methylphosphonic acid (1) was followed with redistilled TMSCl (0.493 g, 4.54 mmol), diethyl hexadecyloxy(naphthalen-1yl)methylphosphonate (12) (0.471 g, 0.908 mmol), anhydrous NaI (0.681 g, 4.54 mmol) in MeCN (5 mL). The product was hexadecyloxy(naphthalen-1-yl)methylphosphonic acid as an off white solid (0.389 g, 94%); m.p. 66e68  C; 1H NMR (400 MHz, CDCl3): d 8.07 (1H, d, J8.6, Ar-H), 7.81 (1H, d, J8.4, Ar-H), 7.75 (1H, d, J8.2, ArH), 7.65 (1H, dd, J6.8, J2.5, Ar-H), 7.47e7.37 (3H, m, Ar H), 5.24 (1H, d, JP-H16.9, CHP), 3.38e3.27 (2H, m, CH2O), 1.57e1.50 (2H, br m, CH2CH2O), 1.35e1.22 (26H, br m, OCH2CH2(CH2)13), 0.91 (3H, t, J7.0, CH3(CH2)15O); 13C NMR (100 MHz, CDCl3): d 133.6 (d, JP-C2.0, quaternary Ar), 131.6 (d, JP-C5.1, quaternary Ar), 131.3 (d, JP-C2.2, quaternary Ar), 128.6, 128.5 (2  Ar), 126.7 (d, JP-C5.8, Ar), 126.0, 125.4, 125.2, 124.0 (4  Ar), 75.1 (d, JP-C174.3, CHP), 71.5 (d, JP-C13.0, OCH2), 31.9, 29.72, 29.67, 29.60, 29.45, 29.37, 25.9, 25.8, 22.7 (14  CH3(CH2)14CH2O), 14.1 (CH3); ESI-MS m/z 461 (M - H)- HRMS calculated for C27H42O4P 461.2826, found 461.2809.

The general procedure in synthesising dodecyloxy(naphthalen1-yl)methylphosphonic acid (1) was followed with redistilled TMSCl (0.198 g, 1.819 mmol), diethyl icosyloxy (naphthalen-1-yl) methylphosphonate (14) ((0.203 g, 0.353 mmol), anhydrous NaI (0.265 g, 1.765 mmol) in MeCN (1.8 mL). The product was icosyloxy(naphthalen-1-yl) methylphosphonic acid as an off white solid (0.160 g, 87%); m.p. 55e57  C; 1H NMR (500 MHz, CDCl3): d 8.08 (1H, d, J7.7, Ar- H), 7.82 (1H, d, J7.7, Ar-H), 7.78 (1H, d, J7.9, Ar-H), 7.57 (1H, dd, J6.3, J2.1, Ar-H), 7.47e7.40 (3H, m, Ar-H), 5.20 (1H, d, JPH16.0, CHP), 3.39e3.25 (2H, m, CH2O), 1.56e1.42 (2H, br m, CH2CH2O), 1.33e1.21 (34H, br m, OCH2CH2(CH2)17), 0.90 (3H, t, J7.1, CH3(CH2)19O); 13C NMR (125 MHz, CDCl3): d 133.5 (quaternary Ar), 131.6 (quaternary Ar), 131.5 (quaternary Ar), 128.5, 128.4 (2  Ar), 126.4 (d, JP-C5.6, Ar), 1265.9, 125.4, 125.3, 124.3 (4  Ar), 74.9 (d, JPC172.2, CHP), 71.4 (d, JP-C12.8, OCH2), 31.9, 29.76, 29.72, 29.68, 29.60, 29.44, 29.39, 29.37, 25.94, 25.91, 25.81, 25.6, 22.7 (18  CH3(CH2)18CH2O), 14.1 (CH3); ESI-MS m/z 517 (M - H)- HRMS calculated for C31H50O4P 517.3452, found 517.3446. 6.15. Docosyloxy(naphthalen-1-yl)methylphosphonic acid (6)

6.13. Octadecyloxy(naphthalen-1-yl)methylphosphonic acid (4)

The general procedure in synthesising dodecyloxy(naphthalen1-yl)methylphosphonic acid (1) was followed with redistilled TMSCl (0.249 g, 2.29 mmol), diethyl octadecyloxy (naphthalen-1yl)methylphosphonate (13) (0.250 g, 0.457 mmol), anhydrous NaI (0.343 g, 2.29 mmol) in MeCN (5 mL). The product was octadecyloxy(naphthalen-1-yl) methylphosphonic acid as a yellow solid (0.224 g, 100%); m.p. 60e63  C; 1H NMR (400 MHz, CDCl3): d 8.09 (1H, d, J8.3, Ar-H), 7.82 (1H, d, J8.2, Ar-H), 7.77 (1H, d, J8.23, Ar-H), 7.61 (1H, dd, J6.8, J2.2, Ar-H), 7.47e7.40 (3H, m, Ar H), 5.19 (1H, d, JP-H17.0, CHP), 3.34e3.27 (2H, m, CH2O), 1.60e1.52 (2H, br m, CH2CH2O), 1.31e1.20 (30H, br m, OCH2CH2(CH2)15), 0.89 (3H, t, J7.0, CH3(CH2)17O); 13C NMR (100 MHz, CDCl3): d 133.3 (d, JP-C1.9, quaternary Ar), 131.5 (d, JP-C5.1, quaternary Ar), 131.3 (d, JP-C2.1, quaternary Ar), 128.6, 128.5 (2  Ar), 126.4 (d, JP-C5.5, Ar), 126.0, 125.4, 125.2, 124.0 (4  Ar), 75.4 (d, JP-C176.2, CHP), 71.2 (d, JP-C13.2, OCH2),

The general procedure in synthesising dodecyloxy(naphthalen1-yl)methylphosphonic acid (1) was followed with redistilled TMSCl (0.464 g, 4.270 mmol), diethyl docosyloxy (naphthalen-1-yl) methylphosphonate (15) (0.507 g, 0.840 mmol), NaI (0.846 g, 5.644 mmol) in MeCN (4.2 mL). The product was docosyloxy(naphthalen-1-yl) methylphosphonic acid as an off-white solid (0.459 g, 100%); m.p. 55e58  C; 1H NMR (400 MHz, CDCl3): d 8.00 (1H, d, J8.2, Ar-H), 7.78 (1H, d, J8.3, Ar-H), 7.72 (1H, d, J8.2, Ar H), 7.58 (1H, br m, Ar-H), 7.43e7.33 (3H, m, Ar-H), 5.18 (1H, d, JP-H16.9, CHP), 3.36e3.28 (2H, m, CH2O), 1.45e1.39 (2H, br m, CH2CH2O), 1.30e1.16(38H, br m, OCH2CH2(CH2)19), 0.88 (3H, t, J7.0, CH3(CH2)21O); 13C NMR (100 MHz, CDCl3): d 133.5 (quaternary Ar), 131.5 (d, JP-C5.2, quaternary Ar), 130.7 (quaternary Ar), 128.7, 128.5 (2  Ar), 126.7 (d, JP-C6.0, Ar), 126.0, 125.4, 125.2, 123.9 (4  Ar), 74.5 (d, JP-C172.2, CHP), 71.6 (d, JP-C12.8, OCH2), 31.9, 29.73, 29.71, 29.66, 29.59, 29.42, 29.36, 25.8, 22.7, 21.0 (20  CH3(CH2)20CH2O), 14.2 (CH3); ESI-MS m/z 545 (M - H)- HRMS calculated for C33H54O4P 545.3765, found 545.3749. 7. Enzyme purification and kinetic assays Pig PAP was extracted from the uterine fluid of a pregnant sow and purified by ion-exchange chromatography and gel filtration on

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D. Feder et al. / European Journal of Medicinal Chemistry 182 (2019) 111611

a Sephadex G-75 following a previously established procedure [75]. SDS-PAGE analysis confirmed the purity of pig PAP. Purified pig PAP was stored at 20  C in 0.1 M acetate bffer at pH 4.9. Protein concentration was determined by measuring the absorbance at 280 nm, where an A280 of 1.41 corresponds to a concentration of 1 mg/mL (28.6 mM). Pig PAP was fully reduced to the catalytically active hetetrovalent FeIIIFeII form to ascertain maximum activity prior to its use in kinetic assays. Reduction of pig PAP was performed by incubating it with 0.77 mM b-mercaptoethanol for 10 min at 37  C. The procedure to extract rkbPAP was adapted from a well-established protocol [76]. Purified rkbPAP was stored at 4  C in 0.5 M NaCl. Protein concentration was determined by measuring the absorbance at 280 nm, where an A280 of 2.1 corresponds to a concentration of 1 mg/mL (9.1 mM). The general procedure for kinetic inhibition assays was adapted from a method described in the literature [77]. p-NPP was used as the substrate since its hydrolysis product, para-nitrophenol, has a characteristic intense yellow color, the formation of which can be conveniently measured at 405 nm with a Varian Cary50 UVeVis spectrophotometer. The rates of product formation by pig PAP and rkbPAP were determined at 25  C at their respective optimal pH, i.e. pH 4.9 for pig PAP (0.1 M acetate buffer) and pH 6.2 for rkbPAP (0.1 M MES buffer). The relevant extinction coefficients at these pH values are Dε405 ¼ 342.9 mol1 L cm1 and Dε405 ¼ 1834.4 mol1 L cm1, respectively. Substrate concentrations were in the range from 1 to 15 mM. The enzyme concentrations were 8.4 nM for both enzymes. All the assays contained 25% DMSO to ascertain solubility for the inhibitors. Data analysis was performed using WinCurveFit (Kevin Raner software). For the determination of inhibition constants, the concentrations of the inhibitors were varied from 0.5 mM to 50 mM, and the data were analyzed by non-linear regression using Equation (2) for general inhibition:

v¼

½Sð1 þ

Vmax ½S ½I Kiuc Þ þ Km ð1

þ

½1 Kic Þ

(2)

X-ray diffraction data were collected at the Australian Synchrotron MX1 beam-line and were scaled and merged using XDS [78]. The structures were solved by difference Fourier using the coordinates from a previously solved rkbPAP structure (4DHL [51]) as starting model. Refinement was undertaken using PHENIX [79]. WinCoot [80] was used for electron density fitting and model building. Omit electron density figures were created with CCP4MG [81] and other figures were created with MVD [60] unless otherwise specified in the figure legend. Author contributions DF collected the X-ray data, and together with LWG solved and refined crystal structures. DF also interpreted structural data and performed in silico simulations. M-WK synthesized and characterized the compounds, conducted inhibition studies, purified and crystallized the enzyme. The crystal soaking experiment was designed and performed by DF and M-WK. All authors contributed to the writing of the manuscript, and LWG, GS and RPM were responsible for the experimental design and overall supervision of the project progress and manuscript writing. Acknowledgments The X-ray data were obtained at the Australian Synchrotron. The assistance of Tom Caradoc-Davies and Alan Riboldi-Tunnicliffe during data collection is much appreciated.We thank Dr. Tri Le for assistance with NMR studies and Mr.Graham MacFarlane for MS measurements. This work was funded by Australian Research Council (DP0986292) and (DP150104358). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.ejmech.2019.111611. References

Here, Vmax is the maximum rate and Km is the Michaelis constant, [S] and [I] are the concentrations of substrate and inhibitor respectively, and Kic and Kiuc represent the equilibrium dissociation constants for competitive and uncompetitive mode of binding respectively. 8. Docking studies Docking studies were undertaken with MVD (47) using the MolDock SE algorithm. The ligand search space was confined to a sphere (16 Å radius) originating from the metal center of PAP for compounds 1e2 and a larger sphere (22 Å radius) for compounds 3e6. The receptor coordinates used were those of the crystal structure of the corresponding PAP in complex with phosphate (1WAR [29] for human PAP, 1UQ6 [31] for pig PAP and 4KBP [7] for rkbPAP) with the phosphate anion omitted. Water molecules were removed from all coordinate files prior to docking. 9. Crystallization and structure determination The crystallization solution contained 2.3 M ammonium sulfate, 0.1 M sodium acetate, pH 4.0, and the final enzyme concentration was 11.9 mg/mL; rkbPAP crystals were grown using the hanging drop diffusion method. Subsequently, crystals were soaked in a solution containing 0.1 M sodium citrate, pH 5.0, 0.1 M lithium chloride, 25% polyethylene glycol 3350, 20% isopropyl alcohol, 10% glycerol and 5 mM of the racemate of each inhibitor.

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