In Vitro and In Vivo Anticalcification Effects of Novel Bishydroxyiminophosphonates zyxwv GERSHON GOLOMB*', ADA SCHLOSSMAN*, YAEL EITAN*, HANAN SAADEH*, JOEL M. VAN GELDER*, AND ELI BREUER' Received June zyxwvutsrqp 27, 1991, from the zyxwvuts 'Department of Pharmacy and the *Department of Pharmaceutical Chemistry, School of Pharmacy, The Hebrew University of Jerusalem, Jerusalem 97 720, Israel. Accepted for publication January 13, 1992. Abstract zyxwvutsrqp 0 Some geminal bisphosphonates are used clinically for a number of important bone- and/or calcium-related diseases; however, side effects and lack of selectivity impede their wide use. This work reports the synthesis and evaluation of bishydroxyiminophosphonates (e.g., adipoyl- and suberoylbisphosphonate dioximes). These com- pounds significantlyinhibited hydroxyapatiteformation and dissolution in vitro and the calcification of bioprosthetic tissue implanted subdermally in rats. The compounds reported in this paper are less active than the structurally related bisacylphosphonates. The results of this work indi- cate that the introduction of oxime groups adjacent to the phosphonic function in long-chain bisphosphonates confers calcium interaction capabilities and that complete ionizability of a bisphosphonate may enhance its biological activity. Several pathological conditions involve irregularities in calcium metabolism (e.g., bone-related diseases such as Pag- et's disease, osteoporosis, and osteolysis in bone cancer), all of which are characterized by excessive destruction of the bone by resorption.1 On the other hand, ectopic calcification is characterized by the deposition of calcium phosphate in a number of clinically important diseases (e.g., atherosclerosis, kidney and renal calculus, arthritis, and bioprosthetic heart valve calcification2-3). Bisphosphonates are a relatively new class of drugs436 that have been used in the treatment of these diseases. Their development followed the recognition that endogenous inorganic pyrophosphate is a physiological reg- ulator of calcification and bone resorption. Because inorganic pyrophosphate (characterized by a P-0-P-type structure) hydrolyzes rapidly in the body, stable analogues were sought. Such analogues were found -20 years ago in the long-known class of geminal bisphosphonates (compounds of the general structure P-C-PQJ). These compounds can be considered pyrophosphate analogues in which the oxygen atom linking the two phosphorus atoms has been replaced by a carbon atom. Like pyrophosphate, geminal bisphosphonates inhibit both mineralization and bone resorption; therefore, there has been a great deal of interest in their clinical application. A number of such geminal bisphosphonates have been approved for clinical use (see structures), namely, pamidronate (ami- nohydroxypropylidene bisphosphonate; ABP) etidronate (hy- droxyethylidene bisphosphonate; HEBP), and clodronate (dichloromethylene bisphosphonate; ClMBP). The biological effects of the geminal bisphosphonates are attributed mainly to their strong affinity to calcium phos- phate crystals, although direct interaction with osteoclasts and their influence on a variety of biochemical pathways have also been demonstrated.4 However, side effects and the lack of selectivity impede their wide use.4.6 Most of the research to date in this field has been confined solely to side-chain-modified derivatives of P-C-P-type bisphospho- nates.6 In contrast to geminal bisphosphonates, monophos- phonates, vicinal bisphosphonates (compounds character- zyxwvu Pamidronate (ABP) Etidronate (HEBP) Clodronate (CMBP) ,OH HO, ti ti n = 4, AdBPDOx n = 6, zyxwvu SuBPDOx ProPOx ized by a P-C-C-P-type of structure), and compounds in which the distance between the phosphoryl groups is greater [P-(0,-P; n > 21 are inactive with respect to calcium-related disorders.4-5 On the other hand, addition of potential donor groups adjacent to the phosphonate func- tion in phosphonic acids and esters endows these com- pounds with the ability to form bidentate chelates with transition metals7.S and calcium.9 Consequently, it has been hypothesized10 that long-chain bisphosphonates might be made active by introducing additional donor groups into them at positions adjacent to the phosphonic functions. Recently, we reported on the synthesis and anticalcification properties of bisacylphosphonates.10 These compounds interacted with hydroxyapatite (HAP) forma- tion and dissolution in vitro and significantly inhibited the pathological calcification of bioprosthetic heart valve tissue cusps in vivo. We extended our synthetic effort to other types of structures that can reasonably be expected to interact with HAP and calcium and yield new biologically active compounds. In vitro and in vivo testing of new chemical classes of bisphosphonates would provide increased understanding of the relationship between chemical structure and biological activity and might stimulate the development of new generations of such drugs. We report here on the synthesis and anticalcification prop- erties of bishydroxyiminophosphonates (BHIP) that were studied in vitro and in vivo. Experimental Section General-Elemental analyses were performed by the Analytical Laboratories of the Hebrew University, Givat-Ram, Jerusalem. IR spectra were determined on an Analect ETIR spectrometer. Nuclear magnetic resonance (NMR) spectra were obtained on a Varian VXR-300s or a Bruker-WH-300 instrument; 'H NMR and 31P NMR spectra were recorded in deuteriochloroform or in deuterium oxide solutions. Chemical shifts are reported in parts per million from tetramethylsilane or 3-(trimethylsilyl)-l-propane-sulfonic acid (TSP) as internal standard in 'H NMR and from 10% H3P0, as external standard in 31P NMR (positive chemical shifts are at low field with respect to the standards). Atomic absorption spectroscopic and W- visible spectrophotometric analyses were performed on Perkin Elmer 403 and Spectronic 1001 (Milton Roy) instruments, respectively. 1004 zyxwvutsrqponm I Journal of Pharmaceutical Sciences Vol. 87, No. 70, October 7992 0022-3549/92/7000- 7 ~02.50/0 z 0 7992, American PharmaceuticalAssociation