Synthesis, modelling and kinetic assays of potent inhibitors of purple acid phosphatase Siti Hajar Mohd-Pahmi a , Waleed M. Hussein a , Gerhard Schenk a,b , Ross P. McGeary a,c,⇑ a The University of Queensland, School of Chemistry and Molecular Biosciences, Brisbane, Qld 4072, Australia b National University of Ireland, Department of Chemistry, Maynooth, Co. Kildare, Ireland c The University of Queensland, School of Pharmacy, Brisbane, Qld 4072, Australia article info Article history: Received 29 January 2011 Revised 7 March 2011 Accepted 8 March 2011 Available online 13 March 2011 Keywords: Purple acid phosphatase PAP Phosphonate Osteoporosis abstract Purple acid phosphatases (PAPs) are binuclear metallohydrolases that have been isolated from various mammals, plants, fungi and bacteria. In mammals PAP activity is associated with bone resorption and can lead to bone metabolic disorders such as osteoporosis; thus human PAP is an attractive target to develop anti-osteoporotic drugs. Based on a previous lead compound and rational drug design, acyl deriv- atives of a-aminonaphthylmethylphosphonic acid were synthesised and tested as PAP inhibitors. Kinetic analysis showed that they are good PAP inhibitors whose potencies improve with increasing acyl chain length. Maximum potency is reached when the number of carbons in the acyl chain is between 12 and 14. The most potent inhibitor of red kidney bean PAP is the dodecyl-derivative with K ic =5 lM, while the most potent pig PAP inhibitor is the tetradecyl-derivative with K ic =8 lM, the most potent inhibitor of a mammalian PAP yet reported. Crown Copyright Ó 2011 Published by Elsevier Ltd. All rights reserved. Purple acid phosphatases (PAPs) 1 are binuclear metallohydro- lases which belong to the superfamily protein phosphatases (PPs) 2–4 and have a characteristic purple colour due to a metal–li- gand charge transfer from a tyrosine phenolate to a chromophoric Fe(III) in the active site. 5 They utilise heterovalent metal ions (Fe(III)M(II); M = Fe (mammals), Zn or Mn (plants)) 5–8 in their ac- tive sites to bring about phosphoric ester hydrolysis, 1,6,9 especially at neutral to acidic pH 5,10,11 according to the equation: 12 RO-PO 2 3 þ H 2 O ! ROH þ HPO 2 4 ð1Þ Mammalian PAPs (also known as tartrate-resistant acid phos- phatase (TRAP) 13 or osteoclastic acid phosphatase (OAP) 14 ) are 35 kDa monomeric proteins, while plant PAPs are homodimers with molecular weight of approximately 100–120 kDa, with each subunit connected through a disulfide bridge. 1,15,16 While the se- quence homology between animal and plant PAPs is low, the ami- no acids in the active site, and the spatial arrangements of the active site residues, are highly conserved. 17 For these reasons, all PAPs are believed to use similar mechanistic strategies. 1,5,7,11,15 The biological roles of PAPs are diverse and not yet fully explored. 12 The association of mammalian PAP with osteoporo- sis 15,18–20 has prompted us to examine its potential as a therapeutic target for the treatment of this disease, and to develop potent inhib- itors of this enzyme. 12,21 While clinical treatments are available for osteoporosis, notably bisphophonates which inhibit farnesyl pyrophosphate synthase in osteoclasts, 22,23 these drugs have signifi- cant side effects and compliance issues. Mammalian PAP is secreted into osteoclasts, the giant multinucleated bone resorbing cells, 24,25 where it plays a role in bone turnover by increasing bone resorp- tion. 1,13,15,26–30 Increased bone resorption that exceeds bone forma- tion creates an imbalance in the dynamic bone remodelling process, 31 which is the major factor in osteoporosis development. Evidence of PAP’s role in bone resorption includes the development of osteopetrosis (the opposite phenotype of osteoporosis) in PAP knockout mice, 32 and that over-expression of PAP in transgenic mice results in them becoming osteoporotic. 33 Furthermore, osteoporosis patients have elevated PAP serum levels, 15,34 and reducing PAP activity using a PAP-specific antibody has been shown to hinder osteoclastic bone resorption in an in vitro bone resorptive assay. 26,27 These reports therefore identify human PAP (hPAP) as a very important target for the development of anti-osteoporotic drugs. Fluoride 10,35–37 and a number of simple tetrahedral inorganic oxyanions, such as phosphate, arsenate, vanadates, 38 tungstate and molybdate 26,39 are weak and non-specific inhibitors of PAP. Simple phosphonate-containing molecules with pendant metal- binding groups such as carboxylate, thiol and phosphonate have been shown to inhibit red kidney bean PAP (rkbPAP) with IC 50 values of 80–3000 lM, 40 and several modified phosphotyrosine- containing tripeptides have also shown inhibitory activity towards several mammalian and plant PAPs, with IC 50 values in the mid-micromolar range. 34 Recently our group reported the potent inhibitory activities of a series of a-alkoxynaphthylmethylphos- phonic acids, 1, with K i and IC 50 values against pig PAP (pPAP) 0960-894X/$ - see front matter Crown Copyright Ó 2011 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.bmcl.2011.03.024 ⇑ Corresponding author. Tel.: +61 7 3365 3955; fax: +61 7 3346 3249. E-mail address: r.mcgeary@uq.edu.au (R.P. McGeary). Bioorganic & Medicinal Chemistry Letters 21 (2011) 3092–3094 Contents lists available at ScienceDirect Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl