ZA200204194B - Phosphonate compounds. - Google Patents

Description

PHOSPHONATE COMPOUNDS ]

FIELD OF THE INVENTION

The present invention relates to novel phosphonate compounds, compositions containing them, processes for producing them, and their use for treating a variety of medical disorders, e.g., osteoporosis and other disorders of bone metabolism, cancer, viral infections, and the like.

BACKGROUND OF THE INVENTION

Phosphonate compounds have long been known to provide a variety of therapeutic benefits. A particular class of therapeutically beneficial phosphonate compounds are the bisphosphonates, i.c., pyrophosphate analogs wherein the central oxygen atom of the pyrophosphate bond is replaced by carbon. Various substituent groups may be attached to this central carbon atom to produce derivative bisphosphonate compounds having various degrees of pharmacological potency.

These derivatives have the general structure:

IT

HO—P— C—P—OH

LLL wherein R, and Ry, may independently be selected from hydroxyl, amino, sulfhydryl, halogen, or a variety of alkyl or aryl groups, or a combination of such groups, which may be further substituted. Examples include Eridronate, wherein R, is CH; and Ry is

OH; Clodronate, dichloromethylene bisphosphonic acid (C1;MDP), wherein R, and

Ry, are C1, Pamidronate, 3-amino-1-hydroxypropylidene bisphosphonic acid, wherein

R, is ethylamino and Ry, is hydroxyl; Alendronate, 4-amino-1-hydroxybutylidene bisphosphonic acid, wherein R, is propylamino and Ry, is hydroxyl; Olpadronate, 3- dimethylamino-1-hydroxypropylidene bisphosphonic acid, wherein R, is dimethylaminoethyl and Ry}, is hydroxyl; and amino-olpadronate (IG-9402), 3-(N,N- dimethylamino)-1-aminopropylidene bisphosphonate, wherein R, is N,N- dimethylaminoethyl and Ry is NH,.

Bisphosphonates and their substituted derivatives have the intrinsic property : of inhibiting bone resorption in vivo. Bisphosphonates also act by inhibiting apoptosis (programmed cell death) in bone-forming cells. Indications for their use therefore include the treatment and prevention of osteoporosis, treatment of Paget’s disease, metastatic bone cancers, hyperparathyroidism, rheumatoid arthritis, algodistrophy, sterno-costo-clavicular hyperostosis, Gaucher’s disease, Engleman’s disease, and certain non-skeletal disorders. (Papapoulos, S. E., in Osteoporosis, R.

Marcus, D. Feldman and J. Kelsey, eds., Academic Press, San Diego, 1996. p. 1210,

Table 1).

Although bisphosphonates have therapeutically beneficial properties, they suffer from pharmacological disadvantages as orally administered agents. One drawback is low oral availability: as little as 0.7% to 5% of an orally administered dose is absorbed from the gastrointestinal tract. Oral absorption is further reduced when taken with food. Further, it is known that some currently available bisphosphonates, e.g., FOSAMAX™ (Merck; alendronate sodium), SKELID™ (Sanofi, tiludronate) and ACTONE™ (Procter and Gamble, risedronate) have local : toxicity, causing esophageal irritation and ulceration. Other bisphosphonates, like amino-olpadronate, lack anti-resorptive effects (Van Beek, E. et al., J. Bone Miner

Res 11(10):1492-1497 (1996) but inhibit osteocyte apoptosis and are able to stimulate net bone formation (Plotkin, L. et al., J Clin Invest 104(10):1363-1374 (1999) and

U.S. Patent No. 5,885,973). It would therefore, be useful to develop chemically modified bisphosphonate derivatives that maintain or enhance the pharmacological activity of the parent compounds while eliminating or reducing their undesirable side effects.

In addition to bisphosphonates, monophosphonates are also known to provide therapeutic benefits. One class of therapeutically beneficial monophosphonates are the antiviral nucleotide phosphonates, such as, for example, cidofovir, cyclic cidofovir, adefovir, tenofovir, and the like, as well as the 5'-phosphonates and methylene phosphonates of azidothymidine, ganciclovir, acyclovir, and the like. In compounds of this type, the 5'-hydroxyl of the sugar moiety, or its equivalent in acyclic nucleosides (ganciclovir, penciclovir, acyclovir) which do not contain a complete sugar moiety, is replaced with a phosphorus-carbon bond. In the case of the methylene phosphonates, a methylene group replaces the 5' -hydroxyl or its : equivalent, and its carbon atom is, in turn, covalently linked to the phosphonate.

Various AZT structures are presented below, including compounds contemplated for use in the practice of the present invention. AZT itself is shown on the left.

Compound A is AZT-monophosphate which has the usual phosphodiester link between the sugar and the phosphate. In contrast, in compounds B (AZT 5’- phosphonate) and C (AZT 5’-methylene phosphonate), the 5°-hydroxyl of 3’-azido, 2’,3’-dideoxyribose is absent and has been replaced by either a phosphorus-carbon bond (AZT phosphonate) or by a methylene linked by a phosphorus- carbon bond (AZT methylene phosphonate). Compounds B and C are examples of compounds useful in the practice of the present invention. fo 0 lo 0 va sO SE. 8 val 07 N ° 0” N 0 0” N o 0“ °N

Ho HO-P-0—) o hice) (HORPCH; o

OH OH

Na Na Na N3

AZT AZT 5'phosphate AZT 5'phosphonate AZT 5-methylene- phosphonate

A B C

Compounds of this type may be active as antiproliferative or antiviral nucleotides. Upon cellular metabolism, two additional phosphorylations occur to form the nucleotide phosphonate diphosphate which represents the equivalent of nucleoside triphosphates. Antiviral nucleotide phosphonate diphosphates are selective inhibitors of viral RNA or DNA polymerases or reverse transcriptases. That is to say, their inhibitory action on viral polymerases is much greater than their degree of inhibition of mammalian cell DNA polymerases «, and y or mammalian RNA polymerases. Conversely, the antiproliferative nucleotide phosphonate diphosphates inhibit cancer cell DNA and RNA polymerases and may show much lower selectivity versus normal cellular DNA and RNA polymerases. Since nucleotide phosphonates are poorly absorbed from the GI tract they frequently require parenteral administration (e.g. cidofovir). Furthermore, the negatively charged phosphonate

2 wo 01/39724 4 PCT/US00/33079 moiety may interfere with cellular penetration, resulting in reduced activity as : y antivirals or antiproliferatives. Invention compounds may surprisingly overcome the disadvantages of this class of agents.

Pharmacologically active agents of antiviral phosphonates are known; the following U.S. Patents describe other approaches for nucleotide phosphonate analogs: 5,672,697 (Nuleoside-5’-methylene phosphonates), 5,922,695 (Antiviral phosphonomethoxy nucleotide analogs), 5,977,089 (Antiviral phosphonomethoxy nucleotide analogs), 6,043,230 (Antiviral phosphonomethoxy nucleotide analogs), 6,069,249. The preparation and use of alkylglycerol phosphates covalently linked to non-phosphonate containing drugs having amino, carboxyl, hydroxyl or sulfhydryl functional groups have previously been disclosed. These prodrugs optionally comprise a linker group or one or two additional phosphates esters between the drug and the alkyl glycerol phosphate (U.S. Patent No. 5,411,947 and U.S. Patent

Application Serial No. 08/487,081). Partial esters of chloromethanediphosphonic acid are known (U.S. Pat. No. 5,376,649) and dianhydrides of clodronate have been reported (Ahimark, et al., J Med Chem 42: 1473-1476 (1999). However, the partial 3 esters were found to not release the active bisphosphonate by chemical or biochemical conversion (Niemi, R. et al., J Chrom B 701:97-102 (1997)). Prodrugs comprising alkylglycerol phosphate residues attached to antiviral nucleosides (U.S. Patent No. 5,223,263) or phosphono-carboxylates (U.S. Patent No. 5,463,092) have also been described.

There is, therefore, a continuing need for less toxic, more effective pharmaceutical agents to treat a variety of disorders, such as those caused by viral infection and inappropriate cell proliferation, e.g., cancer. Thus, itis an object of the present invention to develop chemically modified phosphonate derivatives of : pharmacologically active agents, ¢.g., antiviral and anti-neoplastic pharmaceutical agents. These modified derivatives increase the potency of the parent compound while minimizing deleterious side effects when administered to a subject in need thereof.

BRIEF DESCRIPTION OF THE INVENTION

The invention provides analogs of phosphonate compounds. Phosphonate compounds contemplated for use in accordance with the invention include those that decrease bone resorption or inhibit osteoblast or osteocyte apoptosis, as well as those that improve the bioactivity, selectivity, or bioavailability of nucleotide phosphonate analogs which are useful for the treatment of cancer, various viral infections, and the like. Invention compounds comprise phosphonates covalently linked (directly or indirectly through a linker molecule) to a substituted or unsubstituted alkylglycerol, alkylpropanediol, alkylethanediol, or related moiety. In another aspect of the present invention, there are provided pharmaceutical formulations containing the analogs of phosphonate compounds described herein.

In accordance with another aspect of the present invention, there are provided avariety of therapeutic methods, e.g., methods for treating or preventing bone resorption in a mammal, methods for increasing bone formation by preventing osteoblast and osteocyte apoptosis, methods for increasing bone mass and strength, methods for treating viral infections, methods for treating disorders caused by inappropriate cell proliferation, e.g., cancer, and the like.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 summarizes the effect of a compound according to the invention, 1-

O-hexadecyloxypropane alendronate, on dexamethasone-induced apoptosis of MLO- Y4 osteocytic cells. Bars represent the mean + SD of 3 independent measurements.

Open bars represent the absence of dexamethasone and darkened bars represent the presence of 10 M dexamethasone.

Figure 2 summarizes the effect of a compound according to the invention, 1-

O-hexadecyloxypropane alendronate, on dexamethasone-induced apoptosis of calvarial cells. Bars represent the mean + SD of 3 independent measurements. Gray bars represent the absence of dexamethasone and black bars represent the presence of 10M dexamethasone.

Ce WO 01/39724 6 PCT/US00/33079

DETAILED DESCRIPTION OF THE INVENTION

The phosphonate compounds of the invention have the structure:

I Raz

H— i Xa = 0—@L)}i—Rs

Ry Ry’ wherein:

R; and R;’ are independently -H, optionally substituted -O(C;i- Cas)alkyl, -O(C;-Caa)alkenyl, -O(C;- Cas)acyl, -S(C;-Caa)alkyl, -S(C;-Ca4)alkenyl, or -S(C1-Ca4)acyl, wherein at least one of R; and R,’ are not —H, and wherein said alkenyl or acyl moieties optionally have 1 to 6 double bonds, 3 R; and R;’ are independently -H, optionally substituted -O(C;- Cy)alkyl, -O(C;-Cr)alkenyl, -S(C,-Cr)alkyl, -S(C,-C;)alkenyl, -O(C:1- Cr)acyl, -S(Ci-Cr)acyl, -N(C1-Cr)acyl, -NH(C,-Cr)alkyl, -N((C,-Cy)alkyl),, oxo, halogen, -NH,, -OH, or -SH;

Rj; is a pharmaceutically active phosphonate, bisphosphonate or a phosphonate derivative of a pharmacologically active compound, linked to a functional group on optional linker L or to an available oxygen atom on C°;

X, when present, is:

Ra +3

Ry

L is a valence bond or a bifunctional linking molecule of the formula -J-(CR)-G-, wherein t is an integer from 1 to 24, J and G are independently -0-, -S-, -C(0)O-, or -NH-, and R is -H, substituted or unsubstituted alkyl, or alkenyl;

m is an integer from 0 to 6; and nisOorl.

In preferred embodiments, m = 0, 1 or 2. In these preferred embodiments, R; and R,’ are preferably H, and the prodrugs are then ethanediol, propanediol or butanediol derivatives of a therapeutic phosphonate. A preferred ethanediol phosphonate species has the structure

I

H— | - CH,— o— L)i—Rs a R; 1 wherein Ry, Ry’, R3, L, and n are as defined above.

A preferred propanediol species has the structure:

I

H— i —CH;—CH,— O0—(L);—R;

R/ whereinm=1 and R;, R/, Rs, L and n are as defined above in the general formula.

A preferred glycerol species has the structure:

Uo

H— i Sa 7 — C*H,— O0—(1L)i—R;3

Ry OH wherein m = 1, R, = H, Ry’ = OH, and R; and R,” on C® are both -H. Glycerol is an optically active molecule. Using the stereospecific numbering convention for glycerol, the sn-3 position is the position which is phosphorylated by glycerol kinase.

In compounds of the invention having a glycerol residue, the -(L),-R3 moiety may be joined at either the sn-3 or sn-1 position of glycerol.

SS 8 PCT/US00/33079 . In all species of the pharmacologically active agents of the invention, R; is preferably an alkoxy group having the formula -O-(CH,),-CH3, wherein t is 0-24.

More preferably t is 11-19. Most preferably tis 15 or 17.

Preferred R; groups include bisphosphonates that are known to be clinically useful, for example, the compounds:

Etidronate: 1-hydroxyethylidene bisphosphonic acid (EDHP);

Clodronate: dichloromethylene bisphosphonic acid (C1, MDP);

Tiludronate: chloro-4-phenylthiomethylene bisphosphonic acid;

Pamidronate: 3-amino-1-hydroxypropylidene bisphosphonic acid (ADP);

Alendronate: 4-amino-1-hydroxybutylidene bisphosphonic acid;

Olpadronate: 3-dimethylamino-1-hydroxypropylidene bisphosphonic acid (dimethyl-APD);

Ibandronate: 3-methylpentylamino-1-hydroxypropylidene bisphosphonic acid (BM 21.0955);

EB-1053: 3-(1-pyrrolidinyl)-1-hydroxypropylidene bisphosphonic acid;

Risedronate: 2-(3-pyridinyl)-1-hydroxy-ethylidene bisphosphonic acid; - Amino-Olpadronate: 3-(N,N-diimethylarino-1-aminopropylidene) bisphosphonate (IG9402), and the like.

Rj; may also be selected from a variety of phosphonate-containing nucleotides (or nucleosides which can be derivatized to their corresponding phosphonates), which are also contemplated for use herein. Preferred nucleosides include those useful for treating disorders caused by inappropriate cell proliferation such as 2-chloro- deoxyadenosine, 1-B-D-arabinofuranosyl-cytidine (cytarabine, ara-C), fluorouridine, fluorodeoxyuridine (floxuridine), gemcitabine, cladribine, fludarabine, pentostatin (2’-deoxycoformycin), 6-mercaptopurine, 6-thioguanine, and substituted or unsubstituted 1-B-D-arabinofuranosyl-guanine (ara-G), 1-B-D-arabinofuranosyl- adenosine (ara-A), 1-B-D-arabinofuranosyl-uridine (ara-U), and the like.

Nucleosides useful for treating viral infections may also be converted to their corresponding 5'-phosphonates for use as an R3 group. Such phosphonate analogs typically contain either a phosphonate (-PO3;H,) or a methylene phosphonate (-CH,-

POH) group substituted for the 5’-hydroxyl of an antiviral nucleoside. Some examples of antiviral phosphonates derived by substituting -PO;H, for the 5'- hydroxyl are: 0 Hakimelahi, G. H.; 3'-azido-3',5'- ry Moosavi-Movahedi, dideoxythymidine-5'- Q 0” °N A. A; Sadeghi, M. M; phosphonic acid (HOk No | Tsay, S-C.; Hwy, J. R. (AZT phosphonate) J. Med. Chem. 1995,

Ns 38:4648-4659.

Oo 3',5'-dideoxythymidine-2'- vy ene-5'-phosphonic acid Q 07 nN Ibid. (d4T phosphonate) (HOR 0

NH, Kofoed, T., Ismail, A. 2',3',5'-trideoxycytidine-5'- 1) E. A. A.; Pedersen, E. phosphonic acid HOR 0“ ON B.; Nielsen, C. Bull. (ddC phosphonate) 0 Soc. Chim. Fr. 1997,

EN 134: 59-65.

Kim, C. U.; Luh, B.

Y.; Misco, P. F.; 9-[3-(phosphono- TH Bronson, J. J.; methoxy)propyl]adenine o ox) Hitchcock, M. J. M.; (Adefovir) (HORR._O Ghazzouli, I.; Martin,

J. C. J. Med. Chem. 1990, 33: 1207-1213.

Some examples of antiviral phosphonates derived by substituting ~CH,-POsH; for the 5'-hydroxyl are:

Ce WO 01/39724 10 PCT/US00/33079 : Huffman, J. H.; Sidwell, R.

Ganciclovir 0 ry W.; Morrison, A G,; phosphonate (HO)R o] N” "NH, Coombs, J., Reist, E. J.

Le Nucleoside Nucleotides,

OH 1994, 13: 607-613. 0

Acyclovir 9 Sp Ibid. phosphonate WOR] NZ NH,

Smee, D. F.; Rest, E. J.

Ganciclovir cyclic Io ®s Antimicrob. Agents phosphonate Hot q Nz Chemother. 1996, 40: 1964- so . 1966 : Ha 3 thia-2'3- fe Kraus, J. L.; Nucleosides dideoxycytidine-5'- ¢ 0 Nucleotides, 1993, 12: 157- phosphonic acid ae 162

S

Other preferred antiviral nucleotide phosphonates contemplated for use in the practice of the invention are derived similarly from antiviral nucleosides including ddA, ddl, ddG,L-FMAU, DXG, DAPD, L-dA, L-dI, L-(d)T, L-dC, L-dG, FTC, penciclovir, and the like.

Additionally, antiviral phosphonates such as cidofovir, cyclic cidofovir, adefovir, tenofovir, and the like, may be used as an R; group in accordance with the present invention.

Certain compounds of the invention possess one or more chiral centers, e.g. in the sugar moieties, and may thus exist in optically active forms. Likewise, when the compounds contain an alkenyl group or an unsaturated alkyl or acyl moiety there exists the possibility of cis- and trans- isomeric forms of the compounds. Additional asymmetric carbon atoms can be present in a substituent group such as an alkyl group.

The R- and S- isomers and mixtures thereof, including racemic mixtures as well as . mixtures of cis- and trans-isomers are contemplated by this invention. All such isomers as well as mixtures thereof are intended to be included in the invention. Ifa particular stereoisomer is desired, it can be prepared by methods well known in the art by using stereospecific reactions with starting materials that contain the asymmetric centers and are already resolved or, alternatively, by methods that lead to mixtures of the stereoisomers and resolution by known methods.

Many phosphonate compounds exist that can be derivatized according to the invention to improve their pharmacologic activity, or to increase their oral absorption, such as, for example, the compounds disclosed in the following patents, each of which are hereby incorporated by reference in their entirety: U.S. Patent Nos. 3,468,935 (Etidronate), 4,327,039 (Pamidronate), 4,705,651 (Alendronate), 4,870,063 (Bisphosphonic acid derivatives), 4,927,814 (Diphosphonates), 5,043,437 (Phosphonates of azidodideoxynucleosides), 5,047,533 (Acyclic purine phosphonate nucleotide analogs), 5,142,051 (N-Phosphonylmethoxyalkyl derivatives of pyrimidine and purine bases), 5,183,815 (Bone acting agents), 5,196,409 (Bisphosphonates), 5,247,085 (Antiviral purine compounds), 5,300,671 (Gem-diphosphonic acids), 5,300,687 (Trifluoromethylbenzylphosphonates), 5,312,954 (Bis- and tetrakis- phosphonates), 5,395,826 (Guanidinealkyl-1,1-bisphosphonic acid derivatives), 5,428,181 (Bisphosponate derivatives), 5,442,101 (Methylenebisphosphonic acid derivatives), 5,532,226 (Trifluoromethybenzylphosphonates), 5,656,745 (Nucleotide analogs), 5,672,697 (Nuleoside-5’-methylene phosphonates), 5,717,095 (Nucleotide analogs), 5,760,013 (Thymidylate analogs), 5,798,340 (Nucleotide analogs), 5,840,716 (Phosphonate nucleotide compounds), 5,856,314 (Thio-substituted, nitrogen-containing, heterocyclic phosphonate compounds), 5,885,973 (olpadronate), 5,886,179 (Nucleotide analogs), 5,877,166 (Enantiomerically pure 2-aminopurine phosphonate nucleotide analogs), 5,922,695 (Antiviral phosphonomethoxy nucleotide analogs), 5,922,696 (Ethylenic and allenic phosphonate derivatives of purines), 5,977,089 (Antiviral phosphonomethoxy nucleotide analogs), 6,043,230 (Antiviral phosphonomethoxy nucleotide analogs), 6,069,249 (Antiviral phosphonomethoxy nucleotide analogs); Belgium Patent No. 672205 (Clodronate); European Patent No.

753523 (Amino-substituted bisphosphonic acids); European Patent Application : 186405 (geminal diphosphonates); and the like.

Certain bisphosphonate compounds have the ability to inhibit squalene synthase and to reduce serum cholesterol levels in mammals, including man.

Examples of these bisphosphonates are disclosed, for example, in U.S. Patent Nos. 5,441,946 and 5,563,128 to Pauls et al. Phosphonate derivatives of lipophilic amines, both of which are hereby incorporated by reference in their entirety. Analogs of these squalene synthase inhibiting compounds according to the invention, and their use in the treatment of lipid disorders in humans are within the scope of the present invention. Bisphosphonates of the invention may be used orally or topically to prevent or treat periodontal disease as disclosed in U.S. Pat. No. 5,270,365, hereby incorporated by reference in its entirety.

As used herein, the term “alkyl” refers to a monovalent straight or branched "chain or cyclic radical of from one to twenty-four carbon atoms, including methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-hexyl, and the like.

As used herein, "substituted alkyl" comprises alkyl groups further bearing one or more substituents selected from hydroxy, alkoxy (of a lower alkyl group), mercapto (of a lower alkyl group), cycloalkyl, substituted cycloalkyl, heterocyclic, substituted heterocyclic, aryl, substituted aryl, heteroaryl, substituted heteroaryl, aryloxy, substituted aryloxy, halogen, trifluoromethyl, cyano, nitro, nitrone, amino, amido, -C(O)H, acyl, oxyacyl, carboxyl, carbamate, sulfonyl, sulfonamide, sulfuryl, and the like.

As used herein, "alkenyl" refers to straight or branched chain hydrocarbyl groups having one or more carbon-carbon double bonds, and having in the range of about 2 up to 24 carbon atoms, and "substituted alkenyl" refers to alkenyl groups further bearing one or more substituents as set forth above.

As used herein, "aryl" refers to aromatic groups having in the range of 6 up to 14 carbon atoms and "substituted aryl” refers to aryl groups further bearing one or more substituents as set forth above.

As used herein, "heteroaryl" refers to aromatic groups containing one or more : heteroatoms (e.g., N, O, S, or the like) as part of the ring structure, and having in the range of 3 up to 14 carbon atoms and "substituted heteroaryl" refers to heteroaryl groups 5S further bearing one or more substituents as set forth above.

As used herein, the term “bond” or “valence bond” refers to a linkage between atoms consisting of an electron pair.

As used herein, the term “pharmaceutically acceptable salts” refers to both acid and base addition salts.

As used herein, the term “prodrug” refers to derivatives of pharmaceutically active compounds that have chemically or metabolically cleavable groups and become the pharmaceutically active compound by solvolysis or under in vivo physiological conditions.

Phosphonate analogs, comprising therapeutically effective phosphonates (or phosphonate derivatives of therapeutically effective compounds) covalently linked by ahydroxyl group to a 1-O-alkyglycerol, 3-O-alkylglycerol, 1-S-alkylthioglycerol, or alkoxy-alkanol, may be absorbed more efficiently in the gastrointestinal tract than are the parent compounds. An orally administered dose of the analog is taken up intact from the gastrointestinal tract of a mammal and the active drug is released in vivo by the action of endogenous enzymes. Phosphonate analogs of the invention may also have a higher degree of bioactivity than the corresponding underivatized compounds.

The compounds of the present invention are an improvement over alkylglycerol phosphate prodrugs described in the prior art because the phosphonate- containing moiety is linked directly to the alkyl-glycerol or the alkoxy-alkanol moiety and because the presence of the phosphonate bond prevents enzymatic conversion to the free drug. Other linkers between these groups can be present in the improved analogs. For example, bifunctional linkers having the formula -0-(CH;),-C(0)0O-,

wherein n is 1 to 24, can connect the phosphonate to the hydroxyl group of the : alkoxy-alkanol or alkylglycerol moiety.

The foregoing allows the phosphonate of the invention to achieve a higher degree of oral absorption. Furthermore, cellular enzymes, but not plasma or digestive tract enzymes, will convert the conjugate to a free phosphonate. A further advantage : of the alkoxy-alkanol phosphonates is that the tendency of co-administered food to reduce or abolish phosphonate absorption is greatly reduced or eliminated, resulting in higher plasma levels and better compliance by patients.

Compounds of the invention can be administered orally in the form of tablets, capsules, solutions, emulsions or suspensions, inhaled liquid or solid particles, microencapsulated particles, as a spray, through the skin by an appliance such as a transdermal patch, or rectally, for example, in the form of suppositories. The lipophilic prodrug derivatives of the invention are particularly well suited for transdermal absorption administration and delivery systems and may also be used in toothpaste. Administration can also take place parenterally in the form of injectable solutions.

The compositions may be prepared in conventional forms, for example, capsules, tablets, aerosols, solutions, suspensions, or together with carriers for topical applications. Pharmaceutical formulations containing compounds of this invention can be prepared by conventional techniques, e.g., as described in Remington’s

Pharmaceutical Sciences, 1985.

The pharmaceutical carrier or diluent employed may be a conventional solid or liquid carrier. Examples of solid carriers are lactose, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid, or lower alkyl ethers of cellulose.

Examples of liquid carriers are syrup, peanut oil, olive oil, phospholipids, fatty acids, fatty acid amines, polyoxyethylene or water. The carrier or diluent may include any sustained release material known in the art, such as glyceryl monostearate or distearate, alone or mixed with a wax.

If a solid carrier is used for oral administration, the preparation may be tabletted or placed in a hard gelatin capsule in powder or pellet form. The amount of solid carrier will vary widely, but will usually be from about 25 mg to about 1 gm. If a liquid carrier is used, the preparation may be in the form of a syrup, emulsion, soft gelatin capsule, or sterile injectable liquid such as an aqueous or non-aqueous liquid : suspension or solution.

Tablets are prepared by mixing the active ingredient (that is, one or more compounds of the invention), with pharmaceutically inert, inorganic or organic carrier, diluents, and/or excipients. Examples of such excipients which can be used for tablets are lactose, maize starch or derivatives thereof, talc, stearic acid or salts thereof. Examples of suitable excipients for gelatin capsules are vegetable oils, waxes, fats, semisolid, and liquid polyols. The bisphosphonate prodrugs can also be : made in microencapsulated form. . 15 - For nasal administration, the preparation may contain a compound of the . invention dissolved or suspended in a liquid carrier, in particular, an aqueous carrier, for aerosol application. The carrier may contain solubilizing agents such as propylene glycol, surfactants, absorption enhancers such as lecithin or cyclodextrin, or preservatives.

Pharmaceutical compositions of this invention for parenteral injection comprise pharmaceutically acceptable sterile aqueous or non-aqueous liquids, dispersions, suspensions or emulsions as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use.

Suitable excipients for the preparation of solutions and syrups are water, polyols, sucrose, invert sugar, glucose, and the like. Suitable excipients for the preparation of injectable solutions are water, alcohols, polyols, glycerol, vegetable oils, and the like.

The pharmaceutical products can additionally contain any of a variety of added components, such as, for example, preservatives, solubilizers, stabilizers,

wetting agents, emulsifiers, sweeteners, colorants, flavorings, buffers, coating agents, : antioxidants, diluents, and the like.

Optionally, the pharmaceutical compositions of the invention may comprise a compound according to the general formula combined with one or more compounds exhibiting a different activity, for example, an antibiotic or other pharmacologically active material. Such combinations are within the scope of the invention.

This invention provides methods of treating mammalian disorders related to bone metabolism, viral infections, inappropriate cell proliferation, and the like. The methods particularly comprise administering to a human or other mammal in need thereof a therapeutically effective amount of the prodrugs of this invention.

Indications appropriate to such treatment include senile, post-menopausal or steroid- induced osteoporosis, Paget's disease, metastatic bone cancers, hyperparathyroidism, rheumatoid arthritis, algodystrophy, sterno-costoclavicular hyperostosis, Gaucher’s : - disease, Engleman’s disease, certain non-skeletal disorders and periodontal disease, human immunodeficiency virus (HIV), influenza, herpes simplex virus (HSV), human herpes virus 6, cytomegalovirus (CMV), hepatitis B virus, Epstein-Barr virus (EBV), : varicella zoster virus, lymphomas, hematological disorders such as leukemia, and the like.

In accordance with one aspect of the invention, there are provided methods of preventing or treating bone loss in mammals, especially humans, which method comprises administering to the human or mammal a therapeutically effective amount of the compounds of this invention. The bone resorption inhibiting bisphosphonate prodrugs of the invention are useful therapeutically to oppose osteoclast-mediated bone resorption or bone loss in conditions wherein the bisphosphonate from which the prodrug is prepared has been found efficacious. Indications appropriate to such treatment include osteoporosis, particularly in postmenopausal women, the osteoporosis that accompanies long-term glucocortcoid therapy, and Paget’s disease of bone. The bisphosphonate compound clodronate (Ostac, Boehringer-Mannheim,

Mannheim, Germany) has also been found to reduce osseous as well as visceral metastases in breast cancer patients at high risk for distant metastases (Diel, IJ. et al.

(1998) New Engl. J. Med. 339(60 357-363). Efficacy of the bisphosphonate prodrugs of the invention can be evaluated according to the same methods as for the parent compound. These comprise comparative measurement of bone mineral density of the lumber spine, femoral neck, trochanter, forearm and total body, together with measurements of vertebral fractures, spinal deformities and height in osteoporosis, bone scans or radiographic identification of bone lesions in metastatic disease, and the like. . : In accordance with another aspect of the invention, there are provided methods for increasing bone mass and strength in mammals, especially humans, by administering bone anabolism-promoting compounds of the invention which inhibit osteoblast and osteocyte apoptosis, leading to greater net rates of bone formation, while not substantially altering osteoclast functions (Plotkin et al., J Clin Invest 104:1363-1374 (1999) and Van Beek et al., J Bone Min Res 11:1492 (1996)). . In accordance with yet another aspect of the invention, there are provided ; methods for treating disorders caused by viral infections. Indications appropriate to such treatment include susceptible viruses such as human immunodeficiency virus (HIV), influenza, herpes simplex virus (HSV), human herpes virus 6, cytomegalovirus (CMV), hepatitis B and C virus, Epstein-Barr virus (EBV), varicella zoster virus, and diseases caused by orthopox viruses (e.g., variola major and minor, vaccinia, smallpox, cowpox, camelpox, monkeypox, and the like), ebola virus, papilloma virus, and the like.

In accordance with still another aspect of the invention, there are provided methods for treating disorders caused by inappropriate cell proliferation, e.g. cancers, such as melanoma, lung cancers, pancreatic cancer, stomach, colon and rectal cancers, prostate and breast cancer, the leukemias and lymphomas, and the like. Anti-cancer compounds which can be converted to their nucleotide phosphonates for use as compounds of this invention include, but are not limited to, cytarabine (ara-C), fluorouridine, fluorodeoxyuridine (floxuridine), gemcitibine, cladribine, fludarabine, pentostatin (2'-deoxycoformycin), 6-mercaptopurine and 6-thioguanine and substituted or unsubstituted ara-adenosine (ara-A), ara-guanosine (ara-G), and ara-

uridine (ara-U). Anticancer compounds of the invention may be used alone or in : combination with other antimetabolites or with other classes of anticancer drugs such as alkaloids, topoisomerase inhibitors, alkylating agents, antitumor antibiotics, and the like.

The prodrugs of the invention can be administered orally, parenterally, topically, rectally, and through other routes, with appropriate dosage units, as desired.

As used herein, the term “parenteral” refers to subcutaneous, intravenous, intra-arterial, intramuscular or intravitreal injection, or infusion techniques.

The term “topically” encompasses administration rectally and by inhalation spray, as well as the more common routes of the skin and mucous membranes of the mouth and nose and in toothpaste.

The term “effective amount” as applied to the phosphonate prodrugs of the : invention is an amount that will prevent or reverse the disorders noted above.

Particularly with respect to disorders associated with bone metabolism, an effective amount is an amount that will prevent, attenuate, or reverse abnormal or excessive bone resorption or the bone resorption that occurs in the aged, particularly post- menopausal females or prevent or oppose bone metastasis and visceral metastasis in breast cancer.

With respect to disorders associated with viral infections or inappropriate cell proliferation, e.g., cancer, the “effective amount” is determined with reference to the recommended dosages of the antiviral or anticancer parent compound. The selected dosage will vary depending on the activity of the selected compound, the route of administration, the severity of the condition being treated, and the condition and prior medical history of the patient being treated. However, it is within the skill of the art to start doses of the compound(s) at levels lower than required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, the effective daily dose may be divided into multiple doses for purposes of administration, for example, two to four doses per day. It will be understood, however, that the specific dose level for any particular patient will depend on a variety of factors, including the body weight, general health, diet, time, and route of administration and combination with other drugs, and the severity of the disease being treated. : Generally, the compounds of the present invention are dispensed in unit . dosage form comprising 1% to 100% of active ingredient. The range of therapeutic } dosage is from about 0.01 to about 1,000 mg/kg/day with from about 0.10 mg/kg/day . to 100 mg/kg/day being preferred, when administered to patients, e.g., humans, as a drug. Actual dosage levels of active ingredients in the pharmaceutical compositions of this invention may be varied so as to administer an amount of the active compound(s) that is effective to achieve the desired therapeutic response for a particular patient. } 15 A number of animal experiments have shown the efficacy of bisphosphonates in preventing bone loss under experimental conditions designed to mimic relevant = clinical disorders. Based on these studies, several small animal model systems are available for evaluating the effects of bisphosphonates. These tests are also useful for measuring the comparative efficacy of the bisphosphonate prodrugs of the invention.

The evaluation of bisphosphonate therapy typically requires the determination of femoral ash weight and bone mass, measured, for example as trabecular bone volume, between groups of treated and untreated animals. Thompson, D. et al. (1990) J. Bone and Mineral Res. 5(3):279-286, discloses use of such methods for evaluating the inhibition of bone loss in immobilized rats that were treated with aminohydroxybutane bisphosphonate. Yamamoto, M. et al. (1993) Calcif Tissue Int 53:278-282 induced hyperthyroidism in rats to produce bone changes similar to those in hyperthyroid humans, and compared bisphosphonate-treated and untreated groups biochemically, based on osteocalcin measurement, and by histomorphometric analysis, including differences in cancellous bone volume, and histological comparison of osteoid, osteoclast and osteoblast surfaces in bone sections. Seedor,

J.G. et al. (1991) J. Bone and Mineral Res. 6(4):339-346 describes studies of the effect of alendronate in opposing bone loss in overactomized rats by femoral ash weight and histomorphometric analysis of tibial trabecular volume. The Schenk assay, comprising histological examination of the epiphyses of growing rats, can also : be used as a screening assay. An exemplary screening test for evaluating the bone resorption opposing effects of the compounds of the invention in laboratory rats made osteopenic by various strategies is provided in Example 14.

Compounds of the invention can be prepared in a variety of ways, as generally depicted in Schemes I-VI. The general phosphonate esterification methods described below are provided for illustrative purposes only and are not to be construed as limiting this invention in any manner. Indeed, several methods have been developed for direct condensation of phosphonic acids with alcohols (see, for example, RC.

Larock, Comprehensive Organic Transformations, VCH, New York, 1989, p. 966 and references cited therein). Isolation and purification of the compounds and intermediates described in the examples can be effected, if desired, by any suitable separation or purification procedure such as, for example, filtration, extraction, d 15 crystallization, flash column chromatography, thin-layer chromatography, distillation = ~ or a combination of these procedures. Specific illustrations of suitable separation and isolation procedures are in the examples below. Other equivalent separation and isolation procedures can of course, also be used.

Scheme I outlines a synthesis of bisphosphonate prodrugs that contain a primary amino group, such as pamidronate or alendronate. Example 1 provides conditions for a synthesis of 1-O-hexadecyloxypropyl-alendronate (HDP-alendronate) or 1-O-hexadecyloxypropyl-pamidronate (HDP-pamidronate). In this process, a mixture of dimethyl 4-phthalimidobutanoyl phosphonate (1b, prepared as described in

U.S. patent 5,039,819) and hexadecyloxypropyl methyl phosphite (2) in pyridine solution is treated with triethylamine to yield bisphosphonate tetraester 3b which is purified by silica gel chromatography. Intermediate 2 is obtained by transesterification of diphenyl phosphite as described in Kers, A., Kers, I., Stawinski,

J., Sobkowski, M., Kraszewski, A. Synthesis, April 1995, 427-430. Thus, diphenyl phosphite in pyridine solution is first treated with hexadecyloxypropan-1-ol, then with methanol to provide compound 2.

Scheme 0 : 29 (Cro t-soon =2 0 2 n =3 0

OHO dine, trieth SAT + Pyne. memveTine CL por Boon

Ol (o] ot n, 6h 0O=P-0CHj3

CH3(CHz)s! ( Ha)s “OCH, lo} O(CH2)30(CH,)15CH3 2 Jab bromo- frimethylisilane,

CH4CN, 2h o]

OHO 1. hydrazine, OH 0

FN (Crake Jo 2 (NH) MeOH/1 4-dioxane (1:4) N—(CHp)—C—P(OH), : 2. MeOH/EtOH/NH O=P-OH

O(CH2)30(CHg)15CH3 3 lo] O(CH2)O(CHy)15CHs 5a n = 2: 1-O-hexadecylpropanediol-3-pamidronate 4a-b 5b n = 3: 1-O-hexadecylpropanediol-3-alendronate

An important aspect of the process is that other long chain alcohols may be used in place of hexadecyloxypropan-1-ol to generate the various compounds of this invention. Treatment of intermediate 3b with bromotrimethylsilane in acetonitrile cleaves the methyl esters selectively to yield monoester 4b. Treatment of 4b with hydrazine in a mixed solvent system (20% methanol/80% 1,4-dioxane) results in removal of the phthalimido protecting group as shown. The desired alendronate prodrug is collected by filtration and converted to the triammonium salt by treatment with methanolic ammonia.

Scheme II illustrates a synthesis of analogs of bisphosphonates lacking a primary amino group. In this case the process steps are similar to those of Scheme 1 except that protection with a phthalimido group and subsequent deprotection by hydrazinolysis are unnecessary.

Scheme II pyridine, HO 0

Bs trimethy] phosphite Q9 yamine CH;(CHy);,0 RC——P(OCHj3)

R-C-Cl ——> R-C—P(OCH;); > ono] _ + —p=0

O(CH,)1+CH; OCH, cHiom] ? bromotrimethylsil orn | ping ylsilane,

OCH,

H

CH;3(CH2): coo,

CH;0 I

OH

Bisphosphonates having 1-amino groups, such as amino-olpadronate, may be converted to analogs according to the invention prodrugs using a slightly modified process shown in Scheme III. - E Scheme Ill 3 (CHy)N—(CH,),—C=N NH, O 1 1. HCl (dry) (CHN—(CHp),~GPIOCH:) + 0 2. dimethyl phosphite O=P-OCH,

CH(CHy)ysO(CH, OPH O(CH_)30(CHg)15CH,

OCH, 3 2 bromotrimethyisilane,

CHsCN

NH: © (CHaIN-—(CHy),~C——PIOH),

O=P-OH

O(CHz)sO(CH,)15CHs

HDP-amino-olpadronate

Treatment of a mixture of compound 2 and 3-(dimethylamino)propionitrile with dry HC1 followed by addition of dimethyl phosphite affords tetraester 3 which, after demethylation with bromotrimethylsilane, yields hexadecyloxypropyl-amino- olpadronate.

Scheme IV illustrates synthesis of a bisphosphonate analog where the lipid group is attached to a primary amino group of the parent compound rather than as a . phosphonate ester.

Scheme IV 0 oro

OH 0 Hel (CHG P(OCH)

Crema {boom re, O=P(OCHs), fo) O=P(OCHs), MeOH + 0 0

CH3(CH,)150(CH,)30C(CH,),C—OH occ, pyridine © 9 w OHO

CFCHahsO(CHIOC(CHg)2 CN (CHa) G—P(OCHa),

O=P(OCHz),

TMS-Br,

CH4CN @ 94 OHO

CHs(CHhsO(CHOC(CH CN (Cro) OP(O):

O=P(OH),

Scheme V illustrates a general synthesis of alkylglycerol or alkylpropanediol analogs of cidofovir, cyclic cidofovir, and other phosphonates. Treatment of 2,3- isopropylidene glycerol, 1, with NaH in dimethylformamide followed by reaction with an alkyl methanesulfonate yields the alkyl ether, 2. Removal of the isopropylidene group by treatment with acetic acid followed by reaction with trityl chloride in pyridine yields the intermediate 3. Alkylation of intermediate 3 with an alkyl halide results in compound 4. Removal of the trityl group with 80% aqueous acetic acid affords the O,0-dialkyl glycerol, 5. Bromination of compound 5 followed by reaction with the sodium salt of cyclic cidofovir or other phosphonate-containing nucleotide yields the desired phosphonate adduct, 7. Ring-opening of the cyclic adduct is accomplished by reaction with aqueous sodium hydroxide. The preferred propanediol species may be synthesized by substituting 1-O-alkylpropane-3-ol for : compound 5 in Scheme V. The tenofovir and adefovir analogs may be synthesized by substituting these nucleotide phosphonates for cCDV in reaction (f) of Scheme V.

Similarly, other nucleotide phosphonates of the invention may be formed in this manner.

Scheme V

OH OR, OR, OR; OR, OR, a = oc 2a foro fou 2 for 2 fon << I< OTrityt OTrityl OH Br h | 2 3 4 FH 4 []

NH, NH, ro ® . 5 mo] 0 No g no] bo) No o-PoHa., a o-PeH, *Na'0 AN

H z

Reagents: a) NaH, R,0S0.,Me, DMF; b) 80% aq acetic acid; c) Trityl chloride, pyridine; d) NaH, R;- Br,

DMF; e) CBr, triphenyiphosphine, THF; f) cyclic cidofovir (DCMC salt), DMF; g) 0.5 N NaOH

Scheme VI illustrates a general method for the sythesis of nucleotide phosphonates of the invention using 1-O-hexadecyloxypropyl-adefovir as the example. The nucleotide phosphonate (5 mmol) is suspended in dry pyridine and an alkoxyalkanol or alkylglycerol derivative (6 mmol) and 1,3-dicyclohexylcarbodiimde (DCC, 10 mmol) are added. The mixture is heated to reflux and stirred vigorously until the condensation reaction is complete as monitored by thin-layer chromatography. The mixture is then cooled and filtered. The filtrate is concentrated under reduced pressure and the residue s adsorbed on silica gel and purified by flash column chromatography (elution with approx. 9:1dichloromethane/methanol) to yield the corresponding phosphonate monoester.

NH, Scheme VI

L_o._P(©OH), DCC, pyridine I + reflux NT TN Q oo F~0(CH,);0(CH,)15CH3

CHa{CH,);50(CH2)s0H OH

HDP-adefovir

The invention will now be described in greater detail by reference to the following non-limiting examples.

EXAMPLE 1

Synthesis of 1-O-hexadecylpropanediol-3-alendronate

A. Hexadecyloxypropyl methyl phosphite (b)

Hexadecyloxypropy! methyl phosphite was prepared using the method described in: Kers, A., Kers, 1., Stawinski, J., Sobkowski, M., Kraszewski, A.

Synthesis April 1995, 427-430. To a solution of diphenylphosphite (14 g, 60 mmol) in pyridine (50 mL) maintained at 0°C was slowly added to a solution of hexadecyloxypropan-1-ol (6.0 g, 20 mmol) in pyridine (25 mL). The mixture was stirred one hour before anhydrous methanol (10 mL) was added. After stirring an additional hour, the solvent was evaporated the residue was adsorbed on silica gel and chromatographed, using gradient elution (hexanes to 20% ethyl acetate/80% hexanes), to afford pure compound 2 as a waxy, low-melting solid (4.5 g, 60% yield). 'H NMR (CDCl) 6 6.79 (d, 1H, J = 696 Hz), 4.19 (q, 2H), 3.78 (d, 3H), 3.51 (t, 3H), 3.40 (t, 2H), 1.95 (pent, 2H), 1.25 (broad s, 28H), 0.88 (t, 3H).

B. Hexadecyloxypropyl trimethyl 4-phthalimidobutanoyl phosphonate (3b)

To a mixture of dimethyl 4-phthalimidobutanoyl phosphonate (1b, 3.0 g, 7.9 mmol, prepared as described in U.S. Patent 5,039,819) and hexadecyloxypropyl methyl phosphite (2, 2.9 g, 9 mmol) in pyridine (50 mL) was added triethylamine (0.2 g, 2 mmol). The mixture was stirred 5 hours at room temperature, then the solvent was removed in vacuo. The residue was adsorbed on silica gel and chromatographed

‘We p— 26 PCT/US00/33079 (ethyl acetate) to give compound 3b (3.5 g, 63%) as a viscous oil. 'H NMR (CDCl) 57.84 (d, 2H), 7.72 (d, 2H), 4.45 (m, 1H), 4.27 (m, 4H), 4.15 (q, 2H), 3.68 (s, 3H), 3.84 (s, 3H), 3.71 (t, 2H), 3.51 (m, 2H), 3.38 (t, 2H), 2.04 (m, 2H), 1.94 (pent., 2H), 1.54 (m, 2H), 1.25 (broad s, 28H), 0.88 (t, 3H). >'P NMR (22.54 (doublet), 21.22 (quartet)).

C. Hexadecyloxypropyl 4-phthalimidobutanoyl phosphonate (4b)

Compound 3b from above (3.0 g, 4.3 mmol) was dissolved in dry acetonitrile (50 mL) and cooled to 0°C. A solution of bromotrimethylsilane (3.9 g, 25.5 mmol) in acetonitrile (25 mL) was added slowly then the solution was stirred an additional 2 hours. The mixture was then poured slowly into crushed ice. The precipitate that formed was collected by vacuum filtration and dried in vacuo to give 1.2 g of 4b (42% yield). 'H NMR (DMSO-d¢) 7.86 (m, 4H), 3.99 (q, 2H), 3.66 — 3.55 (m, 1H), : 3.54 (m, 2H), 3.35 (t, 2H), 3.27 (t, 2H), 1.89 — 1.80 (m, ), 1.72 (pent., 2H), 1.53 — 1.40 . 15 (m, 2H), 1.22 (broad s, 28H), 0.85 (t, 3H). *'P NMR (21.51(doublet), 19.50 z (doublet)). ; D. 1-O-Hexadececylpropanediol-3-alendronate (Sb) : ) Compound 4b (300 mg, 0.45 mmol) was dissolved in a mixture of 1,4-dioxane - 20 (20 mL) and methanol (5 mL). Anhydrous hydrazine was then added and the mixture was stirred at room temperature for 4 hours. The precipitate that separated was collected by vacuum filtration and rinsed with 1,4-dioxane. The solid was then suspended in ethanol and methanolic ammonia (3 mL) was added. After stirring for 10 minutes the resulting solid was collected by filtration, rinsed with ethanol and dried under vacuum to yield 220 mg HDP-alendronate (5b) as the triammonium salt.

Analysis by FT-IR indicated removal of the phthalimido protecting group.

Electrospray MS m/e 532 (MH+), 530 (MH).

EXAMPLE 2

Synthesis of 1-O-hexadecylpropanediol-3-pamidronate (5a) 1-O-hexadecylpropanediol-3-pamidronate is prepared in an analogous manner (according to Scheme 1) except that 3-phthalimidopropanoic acid is used to prepare dimethyl 3-phthalimidopropanoyl phosphonate (1a). Compound 1a is condensed with

2 to yield the trimethyl bisphosphonate 3a. Deprotection as in Steps C and D above yields HDP-pamidronate as shown.

EXAMPLE 3

Synthesis of 1-O-Octadecyl-2-O-methyl sn-glycero-3-alendronate

Prodrugs with lipophilic groups other than hexadeclyoxypropyl are prepared by substituting various long-chain alcohols for hexadecyloxypropan-1-ol in Step A of

Example 1. For example, reaction of 1-O-octadecyl-2-O-methyl-sn-glycerol with diphenylphosphite in pyridine followed by treatment with methanol gives 1-O- octadecyl-2-O-methyl-sn-glyceryl methyl phosphite. Condensation of this dialkylphosphite with phosphonate 1b, followed by deprotection steps C and D gives 1-O-Octadecyl-2-O-methyl-sr-glycero-3-alendronate. Scheme 2 illustrates a synthesis of other bisphosphonate conjugates which do not have a primary amino group in the side chain. In this case protection with a phthalimido group and deprotection by hydrazinolysis are unnecessary.

EXAMPLE 4

Synthesis of HDP-amino-olpadronate

Scheme 3 illustrates the synthesis of 1-amino bisphosphonate conjugates.

Using compound 2 from Example 1, 3-(dimethylamino)propionitrile, and procedures described in: Orlovskii, V. V.; Vovsi, B.A. J. Gen Chem. USSR (Engl. Transl.) 1976, 46: 294-296, the bisphosphonate trimethyl ester 3 is prepared. Demethylation with bromotrimethylsilane as described in step C of Example 1 provides HDP-amino- olpadronate.

EXAMPLE 5

Synthesis of 1-O-Hexadecylpropanediol-3-succinyl-alendronate

Scheme 4 illustrates the synthesis of a bisphosphonate conjugate wherein the lipid group is attached to a primary amino group of the parent compound.

Tetramethyl-(4-phthalimido-1-hydrobutylidene)bisphosphonate (2.0 g, 4.4 mmol) was dissolved in 0.2M methanolic hydrazine (100 mL), and the solution stirred at room temperature for 3 days. The mixture was concentrated to half its volume when a solid started separating. The solid was filtered off and the filtrate concentrated to dryness.

Proton NMR showed this compound to be tetramethyl-(4-amino-1- . hydroxybutylidene)bisphosphonate. This was dried over phosphorus pentoxide at 50°C overnight. To a suspension of 1.2 g of the compound in a mixture of pyridine (25 mL) and N,N-dimethylformamide (25 mL) was added 3-succinyl-1- hexdeclyoxypropane (1.76 g, 4.4 mmol). Dicyclohexyl carbodiimide (2.52 g, 12.21 mmol) was added and the mixture stirred at room temperature for two days. The mixture was filtered; the filtrate was absorbed on silica gel and flash chromatographed with an increasing gradient of methanol in dichloromethane (0%-20%) to yield succinylated compound. This was deblocked with trimethylsilyl bromide in acetronitrile to yield the title compound which was purified by crystallization from methanol.

EXAMPLE 6

Synthesis of Adefovir Hexadecyloxypropyl and 1-O-Octadecyl-sn-glyceryl Esters

To a mixture of adefovir 1.36 g, 5 mmol) and 3-hexadecyloxy-1-propanol (1.8 g, 6 mmol) in dry pyridine was added DCC (2.06 g, 10 mmol). The mixture was heated to reflux and stirred 18h then cooled and filtered. The filtrate was concentrated under reduced pressure and the residue was applied to a short column of silica gel. Elution of the column with 9:1 dichloromethane/methanol yielded hexadecyloxypropyl-adefovir (HDP-ADV) as a white powder.

To a mixture of adefovir ( 1.36 g, 5 mmol) and 1-O-octadecyl-sn-glycerol (2.08 g, 6 mmol) in dry pyridine (30 mL) was added DCC (2.06 g, 10 mmol). The mixture was heated to reflux and stirred overnight then cooled and filtered. The filtrate was concentrated under reduced pressure and the residue was applied to a column of silica gel. Elution of the column with a 9:1 dichloromethane/methanol yielded 1-O-octadecyl-sn-glyceryl-3-adefovir.

EXAMPLE 7

Synthesis of AZT-phosphonate Hexadecyloxypropyl Ester

The phosphonate analog of AZT (3’-Azido-3'-5'-dideoxythymidine-5'- phosphonic acid) was synthesized using the published procedure: Hakimelahi, G. H.;

Moosavi-Movahedi, A. A.; Sadeghi, M. M.; Tsay, S-C.; Hwu, J. R. Journal of

Medicinal Chemistry, 1995 38, 4648-4659.

The AZT phosphonate ( 1.65 g, 5 mmol) was suspended in dry pyridine (30 mL), then 3-hexadecyloxy-1-propanol (1.8 g, 6 mmol) and DCC (2.06 g, 10 mmol) were added and the mixture was heated to reflux and stirred for 6h, then cooled and filtered. The filtrate was concentrated under reduced pressure and the residue was applied to a column of silica gel. Elution of the column with a 9:1 dichloromethane/methanol yielded 3’-azido-3'-5'-dideoxythymidine-5’'- phosphonic acid, hexadecyloxypropyl ester.

EXAMPLE 8

Synthesis of the Hexadecyloxypropyl, Octadecyloxypropyl, Octadecyloxyethyl and Hexadecyl Esters of Cyclic Cidofovir

To a stirred suspension of cidofovir (1.0 g, 3.17 mmol) in N,N-DMF (25 mL) was added N, N-dicyclohexyl-4-morpholine carboxamidine (DCMC, 1.0 g, 3.5 mmol).

The mixture was stirred overnight to dissolve the cidofovir. This clear solution was then charged to an addition funnel and slowly added (30 min.) to a stirred, hot pyridine solution (25 mL, 60 °C) of 1,3-dicyclohexyl carbodiimide (1.64 g, 7.9 mmol). This reaction mixture was stirred at 100 °C for 16 h then cooled to room temperature, and the solvent was removed under reduced pressure. The residue was adsorbed on silica gel and purified by flash column chromatography using gradient elution (CH,Cl; + MeOH). The UV active product was finally eluted with 5:5:1

CH,Cl,/MeOH/H,0 Evaporation of the solvent gave 860 mg of a white solid. The

Hand *'P NMR spectrum showed this to be the DCMC salt of cyclic cidofovir (yield = 44 %).

To a solution of cyclic cidofovir (DCMC salt) (0.5 g, 0.8 mmol) in dry DMF (35 mL) was added 1-bromo-3-hexadecyloxypropane (1.45 g, 4 mmol) and the mixture was stirred and heated at 80 °C for 6h. The solution was then concentrated in vacuo and the residue adsorbed on silica gel and purified by flash column chromatography using gradient elution (CH,Cl, + EtOH). The alkylated product was

Claims (22)

‘yo. WO 01739724 42 PCT/US00/33079 WHAT IS CLAIMED IS:

1. A phosphonate compound having the structure: I’ Ry H— i — X)g— 7 O—@Lyi—R; Ry Ry wherein: R; and R,’ are independently -H, optionally substituted -O(C-Caa)alkyl, -O(C;-Ca4)alkenyl, -O(C1-Caa)acyl, -S(C1-Caa)alkyl, -S(C1-Ca4)alkenyl, or -S(C;-Ca4)acyl, wherein at least one of R; and R;’ are not —H, and wherein said alkenyl or acyl optionally have 1 to about 6 double bonds, R; and R;’ are independently -H, optionally substituted -0(C-Cr)alkyl, -O(C,-Cr)alkenyl, -S(C,-Cs)alkyl, -S(C,-Cy)alkenyl, -O(C-Cr)acyl, -S(C;-Cr)acyl, -N(C;-C7)acyl, -NH(C;-C7)alkyl, -N((C1-Cr)alkyl), oxo, halogen, -NH,, -OH, or -SH; Rs is a phosphonate derivative of a pharmacologically active compound linked to a functional group on optional linker L or to an available oxygen atom on C% X, when present, is: Ra t+ R,' L is a valence bond or a bifunctional linking molecule of the formula

-J-(CR2)-G-, wherein t is an integer from 1 to 24, J and G are independently -O-, -S-, -C(0O)O-, or -NH-, and R is -H, substituted or unsubstituted alkyl, or alkenyl; m is an integer from 0 to 6; and nisOorl.

2. The phosphonate compound according to claim 1, wherein R; is a ~bisphosphonate.

3. The phosphonate compound according to claim 2, wherein the bisphosphonate is alendronate, etidronate, tiludronate, ibandronate, EB-1053, pamidronate, olpadronate, amino-olpadronate, clodronate, or risedronate.

4. The phosphonate compound according to claim 1, wherein Rj is a phosphonate derivative of an antiviral nucleoside.

5. The phosphonate compound according to claim 4, wherein said phosphonate derivative is adefovir, cidofovir, cyclic cidofovir, or tenofovir.

6. The phosphonate compound according to claim 4, wherein said phosphonate derivative is a derivative of azidothymidine (AZT).

7. The phosphonate compound according to claim 1, wherein R3 is a phosphonate derivative of an anti-neoplastic nucleoside.

8. The phosphonate compound according to claim 7, wherein said phosphonate is a derivative of cytosine arabinoside, gemcitabine, 5-fluorodeoxyuridine riboside, S-fluorodeoxyuridine deoxyriboside, 2-chlorodeoxyadenosine, fludarabine, or 1-B-D-arabinofuranosyl-guanine.

9. A pharmaceutical composition comprising a phosphonate compound according to claim 1 and a pharmaceutically acceptable carrier therefor. :

oo tw

. :

10. A phosphonate compound according to claim 1, for use in a method for treating osteoporosis in a mammal.

11. A phosphonate compound according to claim 1, for use in a method for augmenting bone mineral density in a mammal.

12. A phosphonate compound according to claim 1, for use in a method for preventing osteoblast and osteocyte apoptosis in a mammal.

13. A phosphonate compound according to claim 1, for use in a method for treating a viral infection in a mammal.

14. A phosphonate compound according to claim 1, for use in a method for treating a growing neoplasm in a mammal.

. 15. A phosphonate compound according to claim 1, for use in a method for modulating cell proliferation in a subject.

16. The use of a phosphonate compound according to claim 1 in the manufacture of a medicament for use in a method for treating osteoporosis in a mammal.

17. The use of a phosphonate compound according to claim 1 in the manufacture of a medicament for use in a method for augmenting bone mineral density in a mammal.

18. The use of a phosphonate compound according to claim 1 in the manufacture of a medicament for use in a method for preventing osteoblast and osteocyte apoptosis in a mammal.

19. The use of a phosphonate compound according to claim 1 in the manufacture of a medicament for use in a method for treating a viral infection in a mammal.

20. The use of a phosphonate compound according to claim 1 in the manufacture of a medicament for use in a method for treating a growing neoplasm in a mammal. AMENDED SHEET 30.09.2003

- EN

21. The use of a phosphonate compound according to claim 1 in the manufacture of a medicament for use in a method for modulating cell proliferation in a subject.

22. A phosphonate compound according to claim 1 specifically as herein described with reference to any one of the illustrative examples. ’ AMENDED SHEET 30.09.2003