The development of bisphosphonates for therapeutic uses, and bisphosphonate structure-activity consideration

Recent progress in the synthesis of major constituents of the methyl-1,1-bisphosphonate family is reviewed. These compounds are important precursors of the corresponding bisphosphonic acid with, in many cases, remarkable pharmacologically interesting properties. The literature has been fully covered over the last two decades.


Introduction
2][3][4][5][6][7][8] The BPs are synthetic organic compounds characterized by a P-C-P backbone structure.They are chemically stable analogues of the endogenous metabolites, inorganic pyrophosphates (PPi) (Figure 1).Unlike PPi, BPs are resistant to breakdown by enzymatic hydrolysis.They bind to bone minerals and inhibit the resorption of living bone.The biological effects of BPs on calcium metabolism were originally ascribed to their physicochemical effects to impede the dissolution of hydroxyapatite crystals.
Although such effects may contribute to their overall action, their effects on cells are probably of greater importance, particularly for the more potent compounds.1b,6,8 The marked structureactivity relationship observed among more complex compounds indicate that the pharmacophore required for maximal activity depends not only upon the bisphosphonate moiety but also on key additional features, especially nitrogen-substitution in alkyl or heterocyclic side chains. 6 In clinical medicine, several BPs (e.g., etidronate, clodronate, pamidronate, alendronate, risedronate and ibandronate) are established as effective treatments for bone diseases such as Paget's disease (a condition in which patches of bone become softened and enlarged), myeloma, and bone metastases.In addition, etidronate and alendronate are approved for the prevention and treatment of osteoporosis.Both can increase bone mass and produce a reduction in fracture rates to approximately half of control rates at the spine, hip and other sites in post-menopausal women.
The clinical pharmacology of BPs is characterized by low intestinal absorption but inhibition of bone resorption by being highly selectively taken up and adsorbed to mineral surfaces in bone, where they interfere with the action of the bone-resorping cells known as osteoclasts.It is likely that BPs are internalized by osteoclasts and interfere with specific biochemical processes, and thereby induce programmed cell death or apoptosis.The molecular mechanisms by which these effects are brought about are becoming clearer. 9,10][3][4][5][6][7][8][9][10] Nevertheless, no parallel effort has been placed on reviewing the synthesis of these compounds.In this review, the syntheses and chemical reactions of BPs are thoroughly discussed, whereas biological and technological applications are only indicated briefly.The emphasis is on information added to this topic from 1990 to 2008.The authors have attempted the review to be encyclopedic with respect to the topics and chemistry, but not in citing all examples of every reaction.

Technological Applications of Bisphosphonates
The early uses of BPs were industrial, mainly as corrosion inhibitors, as complexing agents in the textile, fertilizer and oil industries, as well as for many other industrial processes. 9Their use as, "water softeners" was based on their ability to inhibit calcium carbonate precipitation, as do polyphosphates, and has been applied in domestic and industrial water installations.It is only in the past three decades that the BPs have been developed as drugs for use in various diseases of calcium metabolism.

Biological Activities of Bisphosphonates
The BPs have been known to chemists since the middle of the 19 th century, and the first synthesis dates back to 1865 in Germany. 11Etidronate-the first BP to be used in humans for the treatment of Paget's disease- 12 was synthesized just over 100 years ago. 13In the early 1960s, William Neuman and Herbert Fleisch 14 were studying mechanisms of calcification induced by collagen, and showed that body fluids such as plasma and urine contained inhibitors of calcification.Since it had been known since the 1930s that trace amounts of polyphosphates were capable of acting as water softeners by inhibiting the crystallization of calcium salts, such

Structural-activity relationships
Studies of the relationships between bisphosphonate structure and anti-resorptive potency suggest that the ability of BPs to inhibit bone resorption is dependent on two separate properties of the bisphosphonate molecule.The two-phosphonate groups, together with a hydroxyl group at the R 1 position, impart high affinity for bone mineral and act as a "bone hook", allowing rapid and efficient targeting of BPs to bone mineral surfaces.Once localized within bone, the structure and three-dimensional conformation of the R 2 side chain (as well as the phosphonate groups in the molecule) determine the biological activity of the molecular targets.The understanding of what these molecular targets might be has become much clearer as a result of recent work.In the following sketch (Figure 2), we summarize the correlation between moieties and the potency, while Table 1 indicates the most BP-drugs on the market.

Biochemical bases for the mechanisms of action of BPs
Although our bones seem solid and stable, they actually undergo constant renewal.Specialized cells called osteoclasts draw used calcium out of the bones while other cells called osteoblasts replace it.In some instances (for example after the menopause), this process can get out of balance.Calcium starts to leach out of bones faster than it can be replaced, leading to a brittlebone disease called osteoporosis.BP-drugs reduce this problem by reducing the activity of osteoclasts and slowing the loss of calcium from the bones.3][24][25][26] The effect on the osteoclast leads to a decrease in bone turnover, and is secondary to the inhibition of bone resorption.Several studies 22,24,[27][28][29][30] indicate that BPs can be classified into at least two groups, with different modes of action.BPs that most closely resemble PPi (such as Clodronate and Etidronate, Figure 3) can be metabolically incorporated into non-hydrolyzable analogues of ATP-dependent intracellular enzymes.The more potent, nitrogen-containing BPs (such as Zoledronate and YH529, Figure 4) are not metabolized in this way but they act on liver-enzymes function, which explains the loss of osteoclast activity and induction of apoptosis. 31,32These different modes of action might account for subtle differences between compounds in terms of their clinical effects.

Synthesis
The need to develop more powerful anti-resorptive agents has generated a diverse spectrum of BPs with unique pharmacological activities, as well as uses for other purposes.The desire to obtain unique BPs has stimulated numerous synthetic strategies as alternatives to the established methods, namely the Arbusov reaction and the condensation of PCl 3 with an acid.Nevertheless, the preparation of bisphosphonates could be achieved through many available methods via application of phosphorus reagents on saturated and unsaturated systems.

Bisphosphonates from halo-substrates
The classical strategy method to synthesize substituted-bisphosphonates has included two steps.The first is an Arbusov reaction between an halo-substrate and trialkyl phosphite, followed by an addition of dialkyl phosphonate on the produced monophosphonate (Scheme 1).Later, this method is developed, and a one-pot synthesis of symmetric bisphosphonic esters could be obtained, without the use of dialkyl phosphonate, by introducing a protic reagent, which would remove the unused monophosphonate.The other methodology is to apply Wittig-Horner reagents, such as tetraalkyl methane-1,1bisphosphonate, with halo-substrates to synthesize the requested bisphosphonates (Scheme 1). 33

Scheme 3
Methane-1,1-bisphosphonates bearing fluorine substituents are widely used in the design of new drugs to improve their lipophilicity and to modify their pharmacological properties.The first preparation of fluorinated bisphosphonates was achieved by Burton et al. 38 On treating bromodifluoromethyldibutyl phosphonate, 8, with sodium dibutyl phosphonate, 2c, in hexane, the tetrabutyl-difluoromethylene bisphosphonate, 9, was isolated in 47% yield (Scheme 4).The bisphosphonate 10 (Figure 5), which is an analog of 9, could also be obtained via direct reaction of CF 2 Br 2 with excess sodium diethyl phosphonate (2b).However, this route gave lower yields, and the product-isolation is more difficult owing to increased formation of side products. 38,39 urthermore, a novel type of fluorinated bisphosphonates could be prepared.When the trifluoro-acetimidoyl chlorides 11a,b were allowed to react with diethyl phosphonate (2b), the α-(sulfonylamino)trifluoroethylidene-bisphosphonates (14a,b) were formed as outlined in Scheme 5. 40 In the manner shown in Schemes 4 and 5, several BPs and their relevant BP-acids were synthesized in moderate to high yields. 4,41,42P(OEt) The most common Wittig-Horner reagents used for this purpose are tetra-alkylmethane-1,1-bis-phosphonates, 15a,b, which were prepared easily (~40% yield) by treating a trialkyl phosphite with dibromo-(or di-iodo) methane according to Scheme 6. 43 Then, metallated bisphosphonate esters, 16, react with a series of alkyl halides 17 to prepare the substituted esters 18. Dealkylation of the ester moieties was performed by hydrolysis with water or aqueous alkali to give methylene-1,1-bisphosphonic acids 19 as in Scheme 6. 35 In another report, the heterocyclic bisphosphonate 21 could be prepared in 78 % yield by the nucleophilic substitution of 2-(3-bromopropyl)-1-iso-indolinone 20 with the bis-phosphonate reagents 15a,b (Scheme 7).33  The methane-1,1-bisphosphonate esters 15 were treated with base (KOCMe 3 ) and then with FClO 3 to prepare several mono-26 and di-α-fluorinated methane-bisphosphonates 27 (Scheme 9).44 Furthermore, Chaleix and Lecouvey, 45 performed an efficient synthetic route for preparation of a new family of aldehyde-bisphosphonates as shown in Scheme 10.Two simple and efficient one-pot procedures for the synthesis of a series of α-branched Nheterocycle-substituted methane-1,1-bisphosphonates were reported recently by Abdou et al. 46 The first method involved two steps.The first was the preparation of the corresponding diethyl cyanomethyl phosphonate derivatives C in ~70% yield from the reaction of the lithium salt of diethyl cyanomethyl phosphonate, B, with the parent halo-compound A. Further reaction of sodium dialkyl phosphonates D on these adducts, followed by acid hydrolysis, afforded the corresponding bisphosphonic acids (BP-acids) E in ~42% (Scheme 11).In a second approach, the same halo-compounds were treated with 15b to give the requisite BPs (Scheme 11).

Scheme 17
The treatment of N-alkyl lactams 49a,b with excess LDA, subsequent addition of diethyl phosphorochloridite, and oxidation of the reaction mixture with H 2 O 2 allowed synthesis of several lactone bisphosphonates, 50, in good yield (Scheme 18). 49Similarly, when the same reaction conditions were applied to the six-membered lactam ring 51, the bisphosphonate 52 was isolated in a moderate yield as in Scheme 19. 49 Scheme 18

Scheme 19
Preparation of bis-aminomethylene bisphosphonates through a double insertion-reaction of 1 on diamines is also reported. 62

Diazo-compounds.
Vinylidene bisphosphonate 57 is another phosphorus reagent, which could be prepared by heating tetraalkyl methylene-1,1-bisphosphonate in toluene solution of paraformaldehyde and diethylamine to give the methylene bisphosphonate A, which when heated for 24 h in toluene in the presence of p-TSA converted to ethylidene bisphosphonate 57.At 0 ºC, the addition of diazomethane to a solution of 57a, in ether gave, after concentration, the pyrazoline bis-phosphonate, 58, as the sole product in nearly quantitative yield.However, upon standing for one week at room temperature or during attempted distillation, 58 was converted quantitatively into the propenylidene-diphosphonate 59 as in Scheme 21. 63 On the other hand, the reaction of ethyl diazo-acetate, 60, with the vinylidene bisphosphonate 57a in ether afforded the un-rearranged pyrazoline 61 as the crude product, but upon silica gel chromatography a 1,3-proton shift occurred to give 62 as shown in Scheme 22. 64 In another report, the diazo-substrates, 63, combined with 57a in ether at 22 ºC, and stirring overnight, to give the pyrazoline bisphosphonates, 64.The latter products were found to be more stable. 64 White and Fritzen 66 elaborated the new asymmetrical carbanion 69 for preparation a novel series of pyrimidine bisphosphonate esters.Treatment of the phosphinite 68 with BuLi in tetrahydrofuran gave the dimerization product 69 in 56% yield.This reagent underwent Cmethylenation by treatment with paraformaldehyde and triethylamine, followed by p- toluenesulfonic acid, and then reaction with the corresponding lithiated pyrimidinone derivatives 70 to give the products 71 (Scheme 24).

Bisphosphonates from substrates bearing an activated carbon-carbon double bond
Michael addition reaction of methylene-1,1-bisphosphonate 15 to alkylidene (or arylidene) substrates has been used as a convenient tool for the preparation of the heterocyclic substituted-1,1-bisphosphonates in different yields.Hydrolysis of BP-products by conc.hydrochloric acid produced the corresponding 1,1-bisphosphonic acids (Scheme 25). 67

Scheme 26
In a systematic study, 1,1-bisphosphonates bearing S-, and N-heterocycles (75-77, Figure 8) were prepared in reasonable yields (47 to 72%) by a simple one-pot reaction, and were isolated exclusively in the Z-configuration. 67ARKAT USA, Inc.Later, the same authors used the same route to introduce another series of heteroarylmethylene-bisphosphonates (BPs) via Michael addition reaction of substituted arylidene thiazoles with the Wittig-Horner reagent 15b.The article was offered to generalize an easy route for the transformation of easily available starting materials to the title BPs and the related BPacids (78-80, Figure 9) in satisfactory yields.In addition, the protocol demonstrates an efficient site selective method for making addition products in high yields from arylidenes and methyl-1,1-bisphosphonate under microwave conditions. 68n the other hand, 2-substituted 1,1-cyclopropanediyl-bisphosphonates, 82, were prepared by reacting bromomethylene bisphosphonate 81 with electron deficient alkenes, as Michael acceptors, in the presence of thallium-(I) ethoxide.Bisphosphonates, 82, were converted into the corresponding free acids 83 by treatment with chlorotrimethylsilane in the presence of potassium iodide followed by treatment with water (Scheme 27). 69he synthesis of geminal bisphosphonates 85 could also be attained through the photochemical radical addition of tetraethyl phenylselenomethylene bisphosphonate, 84, prepared in situ from deprotonation of tetraethyl methylene bisphosphonate 15b with NaH, followed by addition of phenylselenium chloride (PhSeCl), to a variety of monosubstituted alkenes (Scheme 28). 70

Bisphosphonates from substrates-bearing carbonyl functions
Bisphosphonates derived from substrates bearing carbonyl functions, in most cases, are αhydroxy-bisphosphonates.These BPs have much biological interest since it is well known that when the R1 side chain (attached to the geminal carbon atom of the P-(R1)C(R2)-P group) is a hydroxyl group, the ability of the BPs to bind to the hydroxyapatite crystals, and to prevent both crystal growth, and dissolution is enhanced.Furthermore, the presence of a hydroxyl group at the R1-position increases the affinity for calcium (and thus for bone mineral), due to the ability of BPs to chelate calcium ions by tridentate rather than bidentate binding.71a 5.4.1 Acids, acid chlorides, acid anhydrides, and esters.The synthesis of α-hydroxy bisphosphonic esters (HBP) involved three steps.The first is the formation of the acid chloride, which by an Arbusov reaction with a trialkyl phosphite produced an α-keto-phosphonate 86.Further dialkyl phosphonate (or another mole of trialkyl phosphite) to the keto-phosphonate yielded the hydroxy-bisphosphonates 87.Acidic hydrolysis yields the corresponding bisphosphonic acid (BP-acid) 88.In some cases, phosphorous acid (H 3 PO 3 ) added to the acid chloride to give, directly, the BP-acids (Scheme 29). 712-(4-Aminocyclohexyl)-1-hydroxyethane-1,1-bisphosphonic acid, 90, was readily prepared from the parent acid.Thus, when a mixture of (4-aminocyclohexyl)acetic acid 89 and phosphorus trichloride (PCl 3 ) in toluene was treated with phosphonic acid afforded 90 in 45% yield (Scheme 30). 72Similarly, the bisphosphonic acid 92 was prepared from the substituted acid 91 (Scheme 31). 73 HCl (conc) 1 .

Scheme 31
Particularly pure 1-hydroxy-1,1-bisphosphonic acids or their sodium salts 93 were prepared in a high-yield, one-pot procedure (Scheme 32). 74,75The hydroxyvinyl phosphonate 94 with dimethyl phosphonate 2a gave the hydroxy-bisphosphonates (HBP) 95 (Scheme 33). 76A modified, efficient one-pot method to prepare hydroxy bisphosphonic esters without using a dialkyl phosphonate, by introducing a protic reagent, was reported.Valeryl chloride 96 was added to benzyl phosphite derivatives to give the hydroxy bis-phosphonate benzyl esters 97a-d.A protic reagent such as methanol was then added, and the solution stirred for one hour at ambient temperature to give BP-acids 98 (Scheme 34). 77This allowed the synthesis of 97a-d in excellent yields, except for 97d, R= OCH 3 , where a 35% yield was obtained.

Scheme 34
In another instance, the hydroxymethylene-bisphosphonic acids 100 were prepared by a simple and efficient one-pot procedure. 78,79Thus, treatment of acyl chlorides with two equivalents of tris-(trimethylsilyl) phosphite at RT leads to the tetrakis-(trimethylsilyl) ester of 1trimethylsiloxy-1,1-bisphosphonic acids 99.Hydrolysis of 99 was carried out in methanol at room temperature for 1 h, to produce the free acids, 100, as in Scheme 35.

Miscellaneous methods
The need to develop more powerful BP-drugs has generated a diverse spectrum of compounds with unique pharmacological activities, as well as uses for other industrial purposes.The desire to obtain unique BPs has stimulated numerous synthetic strategies as alternatives to the previous established methods.Recently, it has been focused on the sulfur-containing bisphosphonic acids as a chondroprotective therapy for the treatment of arthritis, particularly rheumatoid arthritis.The first example in this area is mercapto-ethyl-1,1-bisphosphonic acid (HSEDP ® , Figure 11), which has demonstrated remarkable activity. 92ater, a series of novel S-BP-acids-that contain a latent (or free) thiol group with other chemical moieties of potential anti-catabolic pharmacology were designed and synthesized by a one-pot approach.The properties of these products in the rat adjuvant model of arthritis, and the synthetic routes to these and related compounds were described in detail. 93For example, 5phenyl-3H-3-thioxo-1,2,4-dithiazole (115) was treated with three equivalents of 15b in a mixture of CHCl 3 containing LiOH solution (0.5 M) at room temperature, to give BPs-acids 116b and 117b after treatment of the produced BPs 116a and 117a with conc.HCl (Scheme 43). 93ynthesis of the bisphosphonates 118a-121a (Figure 12), and their relevant acids 118b-121b, were also accomplished by applying 15b on cyclic-, and acyclic cis-disulfide, as well as on electron deficient N=C-function in anils, under phase-transfer basic catalysis.

OH
Furthermore, a process for producing a 1-alkylthio-or arylthio-methane-bisphosphonic acids 124 by conducting the coupling reaction of the disulfides 122 with Horner reagent 15c in the presence of a metallic oxide (MgO), followed by acidic hydrolysis of the formed BPs, 123, was reported (Scheme 44). 94On the other hand, the synthesis of 2-(2-mercaptoethylamino)ethylidene-1,1-bis-phosphonic acid (125) is described by Alferiev et al. 95 via nucleophilic addition of cystamine to vinylidine-bisphosphonic acids followed by reduction of disulfide bond with trimethylphosphine (Scheme 45).

Major Current Uses of Bisphosphonates
The pronounced selectivity of BPs for bone rather than other tissues is the basis of their value in clinical practice.Their preferential uptake by and adsorption to mineral surfaces in bone brings them into close contact with osteoclasts that lead to the loss of osteoclast activity and induction of apoptosis and death of osteoclasts. 96,97So, they are used in the following applications: -Bone scanning. 1,4Inhibition of calcification, e.g., heterotopic bone formation, and dental calculus. 2,5RKAT USA, Inc.
-And recently in the prevention of bone loss and erosions in rheumatoid arthritis. 92,99 Ps can reduce the pain associated with a variety of painful diseases. 100, 101

Newer and Potential Clinical Indications for Uses of BPs
][102] -Use in children with osteogenesis imperfect and other osteopenic disorders. 102Use after cardiac or liver transplantation.
-Extended use in cancers to optimize anti-tumor effects and survival.
-Prevention of bone loss and erosions in rheumatoid arthritis. 92Possible applications in other joint diseases, such as osteoarthritis. 92Reduction of bone loss associated with periodontal disease.
-Prevention of loosening of joint prostheses.
-BPs appear to be useful lead compounds of novel anti-amebic and anti-malarial drugs. 103BPs can provide benefits to patients with prostate cancer throughout the course of their disease. 104Bioassay results raise the possibility that BP inhibit cancer growth in organs other than bone. 105

Conclusions
In summary, the discovery and the development of the BPs as a major class of drugs for treatment of bone diseases has been a fascinating saga that is not yet completed.Despite the synthesis of hundreds of compounds, no clear-cut structure-effect relationship has been unravelled up to now, we can shed light on some results of several studies on structure-activity consideration: ][3][4] • It was generally found that high anti-resorptive potency required a hydroxyl group at the carbon atom between phosphonate groups (C1). 17 The Sulfur containing analogs, have demonstrated remarkable activity in the rat adjuvant arthritis model. 92,93 The results suggested that BPs can be divided into two distinct categories in terms of their effects on inflammatory macrophages: (1) BPs that can metabolized and which inhibit the inflammatory response of macrophages, that possessing potential anti-arthritics properties, and Figure 1

Table 1 .
Most applicable BP-drugs and their structures *Indicates BPs already approved for one or more countries.Pamidronate is the most extensively used drug for Paget's disease.