|||||||III US005583122A United States Patent (19) 11) Patent Number: 5,583,122 Benedict et al. 45) Date of Patent: Dec. 10, 1996

54) PHARMACEUTICAL COMPOSITIONS 100718 2/1984 European Pat. Off.. CONTAINING GEMINAL DIPHOSPHONATES 0170228 2/1986 European Pat. Off.. 2343476 4/1975 Germany. 75) Inventors: James J. Benedict, Norwich, N.Y.; 3. yals SNY shristopher M. Perkins, Cincinnati,- 53-59674 5/1978 Japan.ermany. O 54-135724 10/1979 Japan. (73) Assignee: The Procter & Gamble Company, 5.3 As E. Kingdom. Cincinnati, Ohio OTHER PUBLICATIONS 21 Appl. No.: 806,155 C.A. vol. 93(1980) 93: 232711f, Nissan Chem. Ind. (22 Filed: Dec. 6, 1985 C.A. vol.93(1980) 93: 199,239h, Nissan Chem. Ind. ( Worms Q.A. vol. 77 (1972) 130478N. Related U.S. Application Data C.A. vol. 94(1981) 94:11638f Nissan Chem. Ind. - - Francis & Martodam, "Chemical, Biochemical, & Medicinal (63) Contain-in-part of Ser. No. 684,543, Dec. 21, 1984, Properties of the Diphosphonates', in The Role of Phospho nates in Living Systems (CRC Press; Hilderbrand editor), pp. (51) Int. Cl...... C07F 9/38; C07F 9/58; 55-96 (1983). A61K 31/675 Unterspann, "Experimental Examinations on the Suitability 52 U.S. C...... 514/89; 546/22 of Organoaminomethane-bis-Phosphonic Acids for 58) Field of Search ...... 514/89; 546/23, Bone-Scintigraphy by Means of Tc-99m in Animals”, Eur. 546/22 J. Nucl. Med., vol. 1, pp. 151-154 (1976). Sologub et al., "Synthesis and Intramolecular Electronic (56) References Cited Interactions in Molecules of U.S. PATENT DOCUMENTS Tetrachloropyridyl-4-carbonimidoyl Dichloride and Its Derivatives", Khim. Geterotsikl. Soedin, 1983, (6), pp. 3. E. EM one sea as no saaa out onesi; 798-800; abstract attached Chem. Abs. 99:122,246Z. swVas Talc1S ...... -- 66 3,941.772E. 3/1976A. W.E.R.E."Pl M.Eg, phorusMaier, 'OrganophosphorusSulfur, vol. 11 (3), Compounds.pp. 311-22 (1981):ES5"Phos abstract 3,957,1601 I as 5/1976 Plogeroger et al...... 20546/6 attachedhed Chem.Ch Abst. 96:52398n,96:52398 plusl article.icl 3,960,888 6/1976 Ploger et al...... 548/412 Ploeger et al., "Preparation of 1-Aminoalkylidene Diphos 3,979,385 9/1976 Wollmann et al...... 544/57 phonic Acids”, Z. Anorg. Allg. Chem, vol. 389 (2), pp. 3,988,443 10/1976 Ploger et al...... 514/79 119-28 (1972); (original and English translation attached). 4,034,086 7/1977 Ploger et al...... 514/91 Grapov et al., "Reaction of Ethyl-N-2-pyridylimidoformate 392, SE strial et al. 26.s with Dialkylphosphites” Zh. Obshch. Khim, vol. 57 (7), pp. 43 4965 1/1979 Schmidt-Dunker ...... 424/49 1655-1657 (1980); abstract attached Chem. Abst. 93:220, 4,239,695 12/1980 Chai et al...... 260,502.5 855t, 4,267,108 5/1981 Blum et al. .. 260/326.61 Bandurina et al., "Synthesis of Some Aminophosphonic 4,304,734 12/1981 Jary et al...... 562/13 Acids and Their Antineoplastic Activity", Khim.-Farm. Zh., 4407,761 10/1983 Blum et al. .. 562/13 vol. 12 (11), pp. 35-37 (1978); Chem. Abst. 90:115,081c. 4,447,255 5/1984 Suzuki et al...... 71/86 4,447,256 5/1984 Suzuki et al...... 7/86 Primary Examiner-Alan L. Rotman 4473,560 9/1984 Biere et al...... 514/95 Attorney, Agent, or Firm-K. W. Zerby; David L. Suter 4,503,049 3/1985 Biere et al...... 514/80 4,608,368 8/1986 Blum et al...... 54/107 57 ABSTRACT 4,621,077 11/1986 Rosini et al...... 514/108 4,687,767 8/1987 Bosies et al...... 514/89 Pharmaceutical compositions, useful for treating abnormal 4,687,768 8/1987 Benedict et al. . ... 514/102 calcium and phosphate metabolism, which contain geminal

4,696,920 9/1987 Bentzen et al...... diphosphonic acid compounds; and a method of treating 4,719,203 1/1988 Bosies et al...... 514/108 diseases characterized by abnormal calcium and phosphate 4,777,163 10/1988 Bosies et al...... 54/80 metabolism utilizing these pharmaceutical compositions. FOREIGN PATENT DOCUMENTS 88359 9/1983 European Pat. Off.. 23 Claims, No Drawings 5,583,122 1. 2 PHARMACEUTICAL COMPOSITIONS efforts in this area. Paget's disease and heterotopic ossifi CONTAINING GEMINAL DIPHOSPHONATES cation are currently successfully treated with EHDP. The diphosphonates tend to inhibit the resorption of bone tissue, CROSS REFERENCE TO RELATED which is beneficial to patients suffering from excessive bone APPLICATION loss. However, EHDP, APD and many other prior art diphos This application is a Continuation-in-Part of prior appli phonates have the propensity of inhibiting bone mineraliza cation Ser. No. 684,543, filed Dec. 21, 1984, now aban tion when administered at high dosage levels. doned. It is believed that mineralization inhibition is predomi nantly a mass related physico-chemical effect, whereas 10 resorption inhibition results from a biological interaction TECHNICAL FIELD with the cells. It is therefore desirable to develop more This invention relates to pharmaceutical compositions biologically potent diphosphonate compounds that can be containing compounds which are useful in treating or pre administered at low dosage levels which cause little or no venting diseases characterized by abnormal calcium and mineralization inhibition, thereby resulting in a wider mar phosphate metabolism, in particular those which are char 15 gin of safety. Low dosage levels are also desirable to avoid acterized by abnormal bone metabolism. This invention the gastro-intestinal discomfort (like diarrhea) sometimes further relates to a method of treating or preventing diseases associated with oral administration of large quantities of characterized by abnormal calcium and phosphate metabo diphosphonates. lism using pharmaceutical compositions of the present It is therefore an object of this invention to provide high invention. 20 potency compositions for the treatment and prophylaxis of abnormal calcium and phosphate metabolism. It is a still BACKGROUND OF THE INVENTION further object of this invention to provide an improved method for treating diseases characterized by abnormal A number of pathological conditions which can afflict calcium and phosphate metabolism. warm-blooded animals involve abnormal calcium and phos 25 phate metabolism. Such conditions may be divided into two broad categories. BACKGROUND ART 1. Conditions which are characterized by anomalous U.S. Pat. No. 3,683,080, issued Aug. 8, 1972, to Francis, mobilization of calcium and phosphate leading to general or discloses compositions comprising polyphosphonates, in specific bone loss or excessively high calcium and phos 30 particular diphosphonates, and their use in inhibiting anoma phate levels in the fluids of the body. Such conditions are lous deposition and mobilization of calcium phosphate in Sometimes referred to herein as pathological hard tissue animal tissue. demineralizations. Japanese Patent 80-98,193, issued Jul. 25, 1980, to Nissan 2. Conditions which cause or result from deposition of Kygaku Kagyo K.K. discloses pyridyl ethane diphosphonic calcium and phosphate anomalously in the body. These 35 acid, S-(pyridyl)-thiomethane diphosphonic acid, and the conditions are sometimes referred to herein as pathological derivatives with halogen or alkyl group substitution on the calcifications. pyridyl ring. These compounds are used as post-emergence The first category includes osteoporosis, a condition in herbicides. which bone hard tissue is lost disproportionately to the Japanese Patent 80-98,105, issued Jul. 25, 1980, to Nissan development of new hard tissue. Marrow and bone spaces 40 Chemical Industries, discloses N-(3-pyridyl)-aminomethane become larger, fibrous binding decreases, and compact bone diphosphonic acid, and the derivatives with halogen or alkyl becomes fragile. Osteoporosis can be subclassified as meno group substitution on the pyridyl ring, for use as herbicides. pausal, senile, drug induced (e.g., adrenocorticoid, as can Various N-(pyridyl)-aminomethane diphosphonates are also occur in steroid therapy), disease induced (e.g., arthritic and disclosed in West German Patent 2,831,578, issued Feb. 1, tumor), etc., however, the manifestations are essentially the 45 1979 to Fumio, for use as herbicides. same. Another condition in the first category is Paget's European Patent Application 100,718 (Sanofi SA), pub disease (osteitis deformans). In this disease, dissolution of lished Feb. 15, 1984, discloses various alkyl diphosphonates normal bone occurs which is then haphazardly replaced by which are -substituted by a sulfide attached to a 5- or soft, poorly mineralized tissue such that the bone becomes 50 6-membered nitrogen- or sulfur-containing heterocycle. deformed from pressures of weight bearing, particularly in These compounds are used as anti-inflammatory and anti the tibia and femur. Hyperparathyroidism, hypercalcemia of rheumatic drugs. malignancy, and osteolytic bone metastases are conditions British Patent Application 2,004,888, published Apr. 11, also included in the first category. 1979, discloses N-(3-methyl-2-picolyl)-aminomethane and The second category, involving conditions manifested by 55 related compounds for use in herbicidal compositions. anomalous calcium and phosphate deposition, includes W. Ploger et al., Z. Anorg. Allg. Chem, 389, 119 (1972), myositis ossificans progressiva, calcinosis universalis, and discloses the synthesis of N-(4-pyridyl)-aminomethane such afflictions as arthritis, neuritis, bursitis, tendonitis and diphosphonic acid. No properties or utility of the compound other inflammatory conditions which predispose involved are disclosed. tissue to deposition of calcium phosphates. 60 Polyphosphonic acids and their pharmaceutically-accept SUMMARY OF THE INVENTION able salts have been proposed for use in the treatment and prophylaxis of such conditions. In particular diphospho The present invention relates to pharmaceutical compo nates, like ethane-1-hydroxy-1,1-diphosphonic acid sitions comprising: (EHDP), propane-3-amino-1-hydroxy-1,1-diphosphonic 65 (a) from about 0.001 mg P to about 600 mg P of a geminal acid (APD), and dichloromethane diphosphonic acid diphosphonic acid compound, or its pharmaceutically-ac (CMDP) have been the subject of considerable research ceptable salt or ester, in which the diphosphonic acid 5,583,122 3 4 containing carbon is linked directly, or via a chain of length The linkage from the diphosphonic acid-containing car from 1 to about 5 atoms, to a 6-membered aromatic ring bon to the ring may be direct through a single bond, or by containing one or more nitrogen atoms with the parts of said a chain of length of from 1 to about 5 atoms. The chain may compound being comprised as follows: be all carbon atoms, a nitrogen atom or nitrogen-containing chain, an oxygen atom or oxygen-containing chain, or a said ring may be unsubstituted or substituted with one or 5 selenium atom or selenium-containing chain. The carbon more substituents selected from the group consisting of and nitrogen atoms in the chains may, independently, be substituted and unsubstituted alkyl (saturated or unsatur unsubstituted or substituted with one (or one or two in the ated) having from 1 to about 6 carbon atoms, substituted and case of carbon atoms) substituted or unsubstituted alkyl unsubstituted aryl, substituted and unsubstituted benzyl, (saturated or unsaturated) having from 1 to about 4 carbon hydroxy, halogen, carbonyl, alkoxy, nitro, amido, amino, 10 atoms (methyl and ethyl being preferred). The nitrogen substituted amino, carboxylate, and combinations thereof; atoms in the chains may also be substituted with an acyl said linking chain may be all carbon atoms, a nitrogen group (e.g., -COCH). Unsubstituted carbon and nitrogen atom or nitrogen-containing chain, an oxygen atom or atoms in the chain are preferred. Also preferred are chains oxygen-containing chain, or a selenium atom or selenium one atom in length, i.e., -CH2-, -NH-, and -O-. containing chain, with said chain being unsubstituted or 15 The carbon atom which has the phosphonate groups substituted on the nitrogen and/or carbon atoms, indepen attached to it may be unsubstituted (i.e., a hydrogen atom), dently, with one or more substituted or unsubstituted alkyl or substituted with amino, substituted amino, amido, (saturated or unsaturated) having from 1 to about 4 carbon hydroxy, alkoxy, halogen, carboxylate, substituted or unsub atoms, and said nitrogen atom also may be substituted with stituted alkyl (saturated or unsaturated) having from 1 to an acyl group; 20 about 6 carbon atoms, substituted or unsubstituted aryl, or said diphosphonate-containing carbon may be unsubsti substituted or unsubstituted benzyl. For the compounds in tuted or substituted with substituted or unsubstituted alkyl which the phosphonate-containing carbon is linked to the (saturated or unsaturated) having from 1 to about 6 carbon ring via an oxygen, selenium, or nitrogen-containing chain, atoms, substituted or unsubstituted aryl, substituted or and that oxygen, selenium, or nitrogen atom is bonded unsubstituted benzyl, amino, substituted amino, amido, 25 directly to the phosphonate containing carbon, then the hydroxy, alkoxy, halogen or carboxylate, except where said substituent on the phosphonate-containing carbon may be diphosphonate-containing carbon is directly bonded to a substituted or unsubstituted alkyl (saturated or unsaturated) nitrogen, selenium, or oxygen atom in the linking chain, then having from 1 to about 6 carbon atoms, substituted or the substituents may be substituted or unsubstituted alkyl unsubstituted aryl, or substituted or unsubstituted benzyl. (saturated or unsaturated) having from 1 to about 6 carbon 30 Thus, diphosphonic acid compounds to be included in the atoms, substituted or unsubstituted aryl, or substituted or pharmaceutical compositions of the present invention have unsubstituted benzyl, and the structure: (b) a pharmaceutical carrier. The invention further encompasses a method of treating 35 R poh, diseases characterized by abnormal calcium and phosphate metabolism, comprising administering to a human or animal Ra-Z f Q g-pot: R2 R2 R in need of such treatment a safe and effective amount of a f diphosphonic acid-containing composition of the present invention. 40 wherein Q is oxygen, -NR-, selenium, or a single bond, DETAILED DESCRIPTION OF THE preferred being oxygen, -NR-, or a single bond; m+n is INVENTION an integer from 0 to about 5, with m+n=0 or 1 preferred for Q being oxygen, selenium, or -NR-, and m+n=1 or 2 This invention relates to pharmaceutical compositions, preferred otherwise, Z is a ring selected from the group preferably in unit dosage form, comprising a pharmaceutical 45 consisting of pyridine, pyridazine, pyrimidine, and pyrazine, carrier and a safe and effective amount of geminal diphos with preferred being pyrimidine, and especially pyridine; R. phonic acid compounds, or their pharmaceutically-accept is hydrogen, substituted or unsubstituted amino, amido, able salts and esters, in which the diphosphonic acid hydroxy, alkoxy, halogen, carboxylate, substituted or unsub containing carbon is linked to a 6 membered aromatic ring stituted alkyl (saturated or unsaturated) having from 1 to containing one or more nitrogen atoms. Preferred rings are 50 about 6 carbon atoms, substituted or unsubstituted aryl, or pyridine, pyridazine, pyrimidine, and pyrazine. Most pre substituted or unsubstituted benzyl, except that when n=0 ferred are pyrimidine, and especially pyridine. The rings and Q is oxygen, selenium, or -NR-then R is hydrogen, may be unsubstituted or substituted with one or more substituted or unsubstituted alkyl (saturated or unsaturated) substituents selected from the group consisting of substi having from 1 to about 6 carbon atoms, substituted or tuted and unsubstituted alkyl (saturated or unsaturated) 55 unsubstituted aryl, or substituted or unsubstituted benzyl, having from 1 to about 6 carbon atoms, substituted and with R being hydrogen, chloro, amino, methyl, or hydroxy unsubstituted aryl (e.g., phenyl and naphthyl), substituted preferred; each R is, independently, hydrogen, or substi and unsubstituted benzyl, hydroxy, halogen, carbonyl (e.g., tuted or unsubstituted alkyl (saturated or unsaturated) having -CHO and -COCH), alkoxy (e.g., methoxy and ethoxy), from 1 to about 4 carbon atoms, with R being hydrogen nitro, amido (e.g., -NHCOCH), amino, substituted amino 60 preferred; R is one or more substituents selected from the (e.g., dimethylamino, methylamino, and diethylamino), car group consisting of hydrogen, substituted or unsubstituted boxylate (e.g., -OCOCH), and combinations thereof. The alkyl (saturated or unsaturated) having from 1 to about 6 rings may be fused with other rings, e.g., benzene fused with carbon atoms, substituted and unsubstituted aryl, substituted pyridine (e.g., quinoline), and cyclohexane fused with pyri and unsubstituted benzyl, hydroxy, halogen, carbonyl, dine (e.g., 5,6,7,8-tetrahydroquinoline). Additional substitu- 65 alkoxy, nitro, amido, amino, substituted amino, carboxylate, ents could be substituted or unsubstituted sulfide, sulfoxide, and combinations thereof, with preferred being hydrogen, sulfate, or sulfone. methyl, amino, chloro, methoxy, nitro, hydroxy and combi 5,583,122 5 6 nations thereof; R is hydrogen, substituted or unsubstituted -continued alkyl (saturated or unsaturated) having from 1 to about 4 carbon atoms, or acyl (i.e., the amide of the nitrogen), with & N (i \ OH: preferred being hydrogen, methyl, or ethyl; and pharmaceu R3 N-C--C-PO3H2; tically-acceptable salts and esters of these compounds. N1 R4 R Finally, for any of the R, R2, R, or R substituents which r are themselves substituted, the substitution on these sub O r stituents may be any one or more of the above substituents, preferred being methyl, ethyl, amino, chloro, nitro, methoxy, H POH hydroxy, acetamido, and acetate. 10 R3 O f i-pO3H2; More specifically, the diphosphonic acid compounds, and H R their pharmaceutically-acceptable salts and esters, to be r included in the pharmaceutical compositions of the present N invention are of the structure: O 15

poli, R3 R3-Z g-pot: O No g-pot: R2 R y H R1 f 20 N

Or or wherein for the four preceding structures n=0 or 1; R is R2 R2 PO3H2 hydrogen, chloro, amino, or hydroxy when n=1, and R is hydrogen when n=0; R is one or more substituents selected R-z------Poti. 25 from the group consisting of hydrogen, methyl, amino, chloro, methoxy, nitro, hydroxy, and combinations thereof; and R is hydrogen, methyl, or ethyl. or Specific examples of compounds which may be utilized in compositions of the present invention include; R2 R2 PO3H2 30 N-(2-pyridyl)-aminomethane diphosphonic acid; R-z---0-1l---pot: N-(2-(5-amino)-pyridyl)-aminomethane diphosphonic acid; N-(2-(5-chloro)-pyridyl)-aminomethane diphosphonic acid; l, , , N-(2-(5-nitro)-pyridyl)-aminomethane diphosphonic acid; r n N-(2-(3,5-dichloro)-pyridyl)-aminomethane diphosphonic wherein m-n, Z, R, R2, R, and R are as described above. 35 acid; Generally preferred diphosphonic acid compounds, and N-(4-pyridyl)-N-ethyl-aminomethane diphosphonic acid; their pharmaceutically acceptable salts and esters, to be N-(2-(3-picolyl)-aminomethane diphosphonic acid; included in the pharmaceutical compositions of the present N-(2-(4-picolyl)-aminomethane diphosphonic acid; invention are of the structure: N-(2-(5-picolyl)-aminomethane diphosphonic acid; 40 N-(2-(6-picolyl))-aminomethane diphosphonic acid; N-(2-(3,4-lutidine))-aminomethane diphosphonic acid; H PO3H2 N-(2-(4.6-lutidine))-aminomethane diphosphonic acid; N-(2-pyrimidyl)-aminomethane diphosphonic acid; R3 g {-pot: N-(4-(2,6-dimethyl)-pyrimidyl)-aminomethane diphospho N H r R1 45 nic acid; N-(2-(4,6-dihydroxy)-pyrimidyl)-aminomethane diphos O phonic acid; N-(2-(5-methoxy)-pyridyl)-aminomethane diphosphonic N It poh, acid; R3 C C-PO3H2; 50 N-(2-pyridyl)-2-aminoethane-1,1-diphosphonic acid; N-(2-(3-picolyl)-2-aminoethane-1,1-diphosphonic acid; N1, , , N-(3-pyridyl)-2-amino-1-chloroethane-1,1-diphosphonic acid; wherein for both structures above m+n=1 or 2; R is N-(2-(4-picolyl)-2-amino-1-hydroxy-ethane-1,1-diphos hydrogen, chloro, amino, or hydroxy; R is one or more 55 phonic acid; substituents selected from the group consisting of hydrogen, (2-pyridyl)-methane diphosphonic acid; methyl, amino, chloro, nitro, methoxy, hydroxy, and com (3-pyridyl)-aminomethane diphosphonic acid; binations thereof; or (2-pyridyl)-chloromethane diphosphonic acid; (4-pyridyl)-hydroxymethane diphosphonic acid; 60 2-(2-pyridyl)-ethane-1,1-diphosphonic acid; H foll, 2-(3-pyridyl)-ethane-1,1-diphosphonic acid; R3 N i-roll; 2-(4-pyridyl)-ethane-1,1-diphosphonic acid; R4 H R 2-(2-pyridyl)-1-amino-ethane-1,1-diphosphonic acid; N 2-(2-pyrimidyl)-1-hydroxy-ethane-1,1-diphosphonic acid; 65 2-(2-(3-picolyl))-1-chloro-ethane-1,1-diphosphonic acid; 2-(2-(4-methoxy)-pyridyl)-ethane-1,1-diphosphonic acid; 1-(2-pyridyl)-propane-2,2-diphosphonic acid; 5,583,122 7 8 2-(2-pyridyl)-1-chloro-ethane-1,1-diphosphonic acid; Compounds having the general formula 2-(2-pyridyl)-1-hydroxy-ethane-1,1-diphosphonic acid; 2-(3-pyridyl)-1-hydroxy-ethane-1,1-diphosphonic acid; 2-(4-pyridyl)-1-hydroxy-ethane-1,1-diphosphonic acid; : pott, 3-(3-pyridyl)-1-hydroxy-propane-1,1-diphosphonic acid; R3-Z f g-poh, O-(2-pyridyl)-2-oxa-ethane-1,1-diphosphonic acid; R2 OH O-(2-pyridyl)-oxamethane diphosphonic acid; O-(2-pyrimidyl)-oxamethane diphosphonic acid; (whereinnis an integer of from 1 to about 5, preferably n=1; O-(2-(4-amino)-pyridyl)-oxamethane diphosphonic acid; and Z, R and R are as described hereinbefore, with O-(2-pyrimidyl)-2-oxa-ethane-1,1-diphosphonic acid; 10 preferred Z being pyrimidine and especially pyridine, pre O-(2-(3-picolyl)-2-oxa-ethane-1,1-diphosphonic acid; ferred R being hydrogen, and preferred R being one or O-(2-(3-picolyl)-oxamethane-diphosphonic acid; more substituents selected from the group consisting of O-(2-pyridyl)-1-hydroxy-2-oxa-ethane-1,1-diphosphonic hydrogen, methyl, amino, chloro, nitro, methoxy, hydroxy, and combinations thereof) are best prepared as follows: acid; 15 Synthesis of 2-(2-pyridyl)-1-hydroxy-ethane-1,1-diphos O-(4-pyridyl)-1-amino-2-oxa-ethane-1,1-diphosphonic phonic acid acid; and pharmaceutically-acceptable salts and esters A 3-neck round-bottom flask fitted with a reflux con thereof. denser and a magnetic stir bar is charged with 6.94 grams Preferred compounds are (0.04 mole) 2-pyridine acetic acid, 9.84 grams (0.14 mole) N-(2-(5-amino)-pyridyl)-aminomethane diphosphonic acid; 20 phosphorus acid, and 150 ml of chlorobenzene. This reac N-(2-(5-chloro)-pyridyl)-aminomethane diphosphonic acid; tion mixture is heated on a boiling water bath, and 16.5 N-(2-(3-picolyl)-aminomethane diphosphonic acid; grams (0.12 mole) phosphorus trichloride is added dropwise N-(2-(4-picolyl)-aminomethane diphosphonic acid; with stirring. This reaction mixture is heated for 2/2 hours N-(2-(5-picolyl)-aminomethane diphosphonic acid; during which time a viscous yellow oil forms. The reaction N-(2-(6-picolyl)-aminomethane diphosphonic acid; 25 mixture is then cooled in an ice bath and the chlorobenzene N-(2-(3,4-lutidine))-aminomethane diphosphonic acid; solution is decanted off from the solidified product. The N-(2-pyrimidyl)-aminomethane diphosphonic acid; reaction flask containing this solidified product is charged N-(2-pyridyl)-2-aminoethane-1,1-diphosphonic acid; with 150 ml of water and heated in a boiling water bath for 2-(2-pyridyl)-ethane-1,1-diphosphonic acid; several hours. The hot solution is then filtered through Celite 2-(3-pyridyl)-ethane-1,1-diphosphonic acid; 30 545(E). 300 ml of methanol is added to the warm filtrate 2-(4-pyridyl)-ethane-1,1-diphosphonic acid; solution, and a precipitate develops. After cooling in ice for 2-(2-pyridyl)-1-hydroxy-ethane-1,1-diphosphonic acid; 1 hour, the precipitate is filtered off and then washed with 2-(3-pyridyl)-1-hydroxy-ethane-1,1-diphosphonic acid; methanol/water (1/1 volume/volume), methanol, and ether, 2-(4-pyridyl)-1-hydroxy-ethane-1,1-diphosphonic acid; and air dried. The product may be recrystallized from hot 35 water. Yield is approximately 5.9 grams (52%). The sample O-(2-(3-picolyl)-oxamethane-diphosphonic acid; and phar is characterized by P-31 and C-13 NMR. maceutically-acceptable salts and esters thereof. By "pharmaceutically-acceptable salts and esters' as used The diphosphonate compounds to be included in the herein is meant hydrolyzable esters and salts of the diphos pharmaceutical compositions of the present invention can be phonate compounds which have the same general pharma made using the synthetic methods disclosed in Japanese cological properties as the acid form from which they are Patent 80-98,193 (Jul 25, 1980, to Nissan Kygaku Kagyo derived, and which are acceptable from a toxicity viewpoint. K.K.), Japanese Patent 80-98,105 (Jul. 25, 1980, to Nissan Pharmaceutically-acceptable salts include alkali metal Chemical Industries), West German Patent 2,831,578 (Feb. (sodium and potassium), alkaline earth metal (calcium and 1, 1979, to Fumio), and W. Ploger et al., Z. Anorg. Allg. magnesium), non-toxic heavy metal (stannous and indium), Chem., 389, 119 (1972), the disclosures of which are incor 45 and ammonium and low molecular weight substituted porated herein by reference. The aminoethane diphosphonic ammonium (mono-, di- and triethanolamine) salts. Preferred acid compounds, however, are best prepared as follows: compounds are the sodium, potassium, and ammonium salts. Synthesis of N-(2-(3-picolyl)aminoethane DP By "pharmaceutical carrier' as used herein is meant one The above-named compound is prepared via a typical or more compatible solid or liquid filler diluents or encap Michael reaction between tetraethyl vinyldiphosphonate and 50 sulating substances. By "compatible' as used herein is meant that the components of the composition are capable of 2-amino-3-picoline. (See H. O. House, Modern Synthetic being commingled without interacting in a manner which Reaction 2nd Ed. W. A. Benjamin Inc. p. 595-623, the would substantially decrease the pharmaceutical efficacy of disclosure of which is incorporated herein by reference.) the total composition under ordinary use situations. To a solution of 1.62 g (15 mmol) of 2-amino-3-picoline 55 Some examples of substances which can serve as phar in tetrahydrofuran at 5° C. was added 4.50 g (15 mmol) maceutical carriers are sugars such as lactose, glucose and tetraethyl vinyldiphosphonate. The reaction mixture was sucrose; starches such as corn starch and potato starch; stirred at room temperature for 16 hours. Evaporation of the cellulose and its derivatives, such as sodium carboxymeth solvent and chromatography (acetone/hexane, 4/1) of the ylcellulose, ethylcellulose, cellulose acetate; powdered product on silica gel gave pure tetraethyl N-(2-(3-picolyl)- 60 tragacanth; malt, gelatin, talc, stearic acid; magnesium stear 2-aminoethane diphosphonate. P-31 NMR of the pure tet ate, calcium sulfate, vegetable oils, such as peanut oil, raethyl ester in CDCI shows a resonance at 22.1 ppm. The cottonseed oil, sesame oil, olive oil, corn oil and oil of ester was hydrolyzed in refluxing 6N HCl overnight. The theobroma, polyols such as propylene glycol, glycerin, product showed a P-31 NMR signalin DO at pH=12 of 19.0 Sorbitol, mannitol, and polyethylene glycol; agar; alginic ppm. 65 acid; pyrogen-free water, isotonic saline; and phosphate N-(2-pyridyl)-2-aminoethane DP and N-(2-(5-picolyl)-2- buffer solutions, as well as other non-toxic compatible aminoethane DP were prepared in an identical manner. substances used in pharmaceutical formulations. Wetting 5,583,122 10 agents and lubricants such as sodium lauryl sulfate, as well EXAMPLE II as coloring agents, flavoring agents, lubricants, excipients, tableting agents, stabilizers, anti-oxidants and preservatives, Tablets are prepared by conventional methods, formulated can also be present. Other compatible pharmaceutical addi as follows: tives and actives (e.g., vitamin D or vitamin D metabolites, and mineral supplements) may be included in the pharma Ingredient mg per tablet ceutical compositions of the present invention. N-(2-pyrimidyl) AMDP 25.00 The choice of a pharmaceutical carrier to be used in Lactose 40.00 conjunction with the diphosphonates of the present compo Starch 2.50 sitions is basically determined by the way the diphosphonate 10 Magnesium stearate 1.00 is to be administered. If the compound is to be injected, the preferred pharmaceutical carrier is sterile, physiological The above tablets administered orally twice daily for 6 saline, the pH of which has been adjusted to about 7.4. months substantially reduce bone resorption in a patient However, the preferred mode of administering the diphos weighing approximately 70 kilograms afflicted with 15 osteoporosis. Similar results are obtained when the N-(2- phonates of the present invention is orally, and the preferred pyrimidyl) AMDP, or its pharmaceutically-acceptable salt or unit dosage form is therefore tablets, capsules and the like, ester, in the above-described tablets is replaced with comprising from about 0.1 mg P to about 600 mg P of the N-(2-(5-amino)-pyridyl)-aminomethane diphosphonic acid; diphosphonic acid compounds described herein. Pharma N-(2-(5-chloro)-pyridyl)-aminomethane diphosphonic acid; ceutical carriers suitable for the preparation of unit dosage 20 N-(2-(3-picolyl)-aminomethane diphosphonic acid; forms for oral administration are well known in the art. Their N-(2-(4-picolyl)-aminomethane diphosphonic acid; selection will depend on secondary considerations like taste, N-(2-(5-picolyl)-aminomethane diphosphonic acid; cost, shelf stability, which are not critical for the purposes of N-(2-(6-picolyl))-aminomethane diphosphonic acid; N-(2-(3,4-lutidine))-aminomethane diphosphonic acid; the present invention, and can be made without difficulty by N-(2-pyridyl)-2-aminoethane-1,1-diphosphonic acid; a person skilled in the art. The pharmaceutical carrier 25 2-(2-pyridyl)-ethane-1,1-diphosphonic acid; employed in conjunction with the diphosphonates of the 2-(3-pyridyl)-ethane-1,1-diphosphonic acid; present invention is used at a concentration sufficient to 2-(4-pyridyl)-ethane-1,1-diphosphonic acid; provide a practical size to dosage relationship. Preferably, 2-(2-pyridyl)-1-hydroxy-ethane-1,1-diphosphonic acid; the pharmaceutical carrier comprises from about 0.01% to 2-(3-pyridyl)-1-hydroxy-ethane-1,1-diphosphonic acid; about 99.99% by weight of the total composition. 30 2-(4-pyridyl)-1-hydroxy-ethane-1,1-diphosphonic acid; O-(2-(3-picolyl)-oxamethane-diphosphonic acid; or the pharmaceutically-acceptable salts or esters thereof. EXAMPLE I EXAMPLE III Capsules are prepared by conventional methods, com 35 Injectable solutions are prepared by conventional meth prised as follows: ods using 1.0 ml of either physiological saline or water solution and 3.5 mg of 2-(2-pyridyl)-ethane-1,1-diphospho Ingredient Mg per capsule nic acid, adjusted to pH=7.4. N-(2-(3-picolyl) AMDP 100 (as mg P) 40 One injection, one time daily for 4 days results in appre Starch 55.60 ciable alleviation of hypercalcemia of malignancy in Sodium lauryl sulfate 2.90 patients weighing approximately 70 kilograms. Similar results are obtained when the 2-(2-pyridyl)- ethane-1,1diphosphonic acid in the above-described treat The above capsules administered orally twice daily for 6 45 ment is replaced with months substantially reduce bone resorption in a patient N-(2-(5-amino)-pyridyl)-aminomethane diphosphonic acid; weighing approximately 70 kilograms afflicted with N-(2-(5-chloro)-pyridyl)-aminomethane diphosphonic acid; osteoporosis. Similar results are obtained when the N-(2- N-(2-(3-picolyl)-aminomethane diphosphonic acid; (3-picolyl)-aminomethane diphosphonic acid, or its phar N-(2-(4-picolyl))-aminomethane diphosphonic acid; maceutically-acceptable salt or ester, in the above-described 50 N-(2-(5-picolyl)-aminomethane diphosphonic acid; capsules is replaced with N-(2-(6-picolyl)-aminomethane diphosphonic acid; N-(2-(5-amino)-pyridyl)-aminomethane diphosphonic acid; N-(2-(3,4-lutidine))-aminomethane diphosphonic acid; N-(2-(5-chloro)-pyridyl)-aminomethane diphosphonic acid; N-(2-pyrimidyl)-aminomethane diphosphonic acid; N-(2-(4-picolyl)-aminomethane diphosphonic acid; N-(2-pyridyl)-2-aminoethane-1,1-diphosphonic acid; N-(2-(5-picolyl)-aminomethane diphosphonic acid; 55 2-(3-pyridyl)-ethane-1,1-diphosphonic acid; N-(2-(6-picolyl)-aminomethane diphosphonic acid; 2-(4-pyridyl)-ethane-1,1-diphosphonic acid; N-(2-(3,4-lutidine))-aminomethane diphosphonic acid; 2-(2-pyridyl)-1-hydroxy-ethane-1,1-diphosphonic acid; N-(2-pyrimidyl)-aminomethane diphosphonic acid; 2-(3-pyridyl)-1-hydroxy-ethane-1,1-diphosphonic acid; N-(2-pyridyl)-2-aminoethane-1,1-diphosphonic acid; 2-(4-pyridyl)-1-hydroxy-ethane-1,1-diphosphonic acid; 2-(2-pyridyl)-ethane-1,1-diphosphonic acid; 60 O-(2-(3-picolyl)-oxamethane-diphosphonic acid; or phar 2-(3-pyridyl)-ethane-1,1-diphosphonic acid; maceutically-acceptable salts or esters thereof. 2-(4-pyridyl)-ethane-1,1-diphosphonic acid; The compositions of the present invention are useful in 2-(2-pyridyl)-1-hydroxy-ethane-1,1-diphosphonic acid; the treatment of abnormal calcium and phosphate metabo 2-(3-pyridyl)-1-hydroxy-ethane-1,1-diphosphonic acid; lism. Other diphosphonic acids and their pharmaceutically 2-(4-pyridyl)-1-hydroxy-ethane-1,1-diphosphonic acid; 65 acceptable salts have been proposed for use in the treatment O-(2-(3-picolyl))-oxamethane-diphosphonic acid; or the and prophylaxis of such conditions. In particular, ethane-1- pharmaceutically-acceptable salts or esters thereof. hydroxy-1,1-diphosphonic acid (EHDP), propane-3-amino 5,583,122 11 12 1-hydroxy-1,1-diphosphonic acid (APD), and dichlo predispose involved tissue to diposition of calcium phos romethane diphosphonic acid (ClMDP) have been the phate. subject of considerable research efforts in this area. By “human or lower animal afflicted with or at risk to However, the compositions of the present invention are osteoporosis' as used herein is meant a subject diagnosed as generally more biologically potentin inhibiting bone resorp suffering from one or more of the various forms of tion than the art-disclosed diphosphonates. Thus, the com osteoporosis, or a subject belonging to a group known to positions of the present invention may provide one or more have a significantly higher than average chance of develop of the following advantages over the art-disclosed diphos ing osteoporosis, e.g., postmenopausal women, men over the phonates of (1) being more potent in inhibiting bone resorp age of 65, and persons being treated with drugs known to tion; (2) possessing less potential for inhibition of bone 10 cause osteoporosis as a side effect (such as adrenocorticoid). mineralization, since mineralization inhibition is believed to By "safe and effective amount” as used herein is meant an be predominantly a mass related physico-chemical effect; amount of a compound or composition high enough to (3) having generally a wider margin of safety (i.e., wider significantly positively modify the condition to be treated, dosing interval between the lowest effective antiresorptive but low enough to avoid serious side effects (at areasonable dose and the lowest dose producing mineralization inhibi 15 benefit/risk ratio), within the scope of sound medical judg tion); (4) allowing lower oral dosages to be administered, ment. The safe and effective amount of diphosphonates will thereby avoiding the gastro-intestinal discomfort (like diar vary with the particular condition being treated, the age and rhea) sometimes associated with higher dosages of diphos physical condition of the patient being treated, the severity phonates; and (5) having potential for flexibility of dosing of the condition, the duration of treatment, the nature of methods. 20 concurrent therapy, and the specific diphosphonate Another aspect of this invention is a method for treating employed. However, single dosages can range from about or preventing diseases characterized by abnormal calcium 0.001 mg P to about 3500 mg P, or from about 0.1 micro and phosphate metabolism, in particular those which are grams P/kg of body weight to about 500 mg P/kg of body characterized by abnormal bone metabolism, in persons at weight. Preferred single dosages are from about 0.1 mg Pto risk to such disease, comprising the step of administering to 25 about 600 mg P, or from about 0.01 to about 50 mg P/kg of persons in need of such treatment a safe and effective body weight. Up to about four single dosages per day may amount of a diphosphonic acid-containing composition of be administered. Daily dosages greater than about 2000 mg the present invention. P/kg are not required to produce the desired effect and may The preferred mode of administration is oral, but other produce undesirable side effects. The higher dosages within modes of administration include, without limitation, trans 30 this range are, of course, required in the case of oral dermal, mucosal, sublingual, intramuscular, intravenous, administration because of limited absorption. intraperitoneal, and subcutaneous administration, as well as topical application. Schenk Model By "abnormal calcium and phosphate metabolism' as The compounds were evaluated for in vivo bone resorp used herein is meant (1) conditions which are characterized 35 tion inhibition and mineralization inhibition in an animal by anomalous mobilization of calcium and phosphate lead model system known in the field of bone metabolism as the ing to general or specific bone loss, or excessively high Schenk Model. The general principles of this model system calcium and phosphate levels in the fluids of the body; and are disclosed in Shinoda et al., Calcif. Tissue Int., 35, 87-99 (2) conditions which cause or result from deposition of (1983); and in Schenket al., Calcif. Tissue Res. 11, 196-214 calcium and phosphate anomalously in the body. The first 40 (1973), the disclosures of which are incorporated herein by category includes, but is not limited to, osteoporosis, Pagets reference. disease, hyperparathyroidism, hypercalcemia of malig Materials and Methods nancy, and osteolytic bone metastases. The second category Animals includes, but is not limited to, myositis ossificans progres Preweaning 17-day-old (30 gms) male Sprague Dawley siva, calcinosis universalis, and such afflictions as arthritis, 45 neuritis, bursitis, tendonitis and other inflammatory condi rats (Charles River Breeding Laboratories) were shipped tions which predispose involved tissue to deposition of with their mothers and placed in plastic cages with their calcium phosphates. mothers upon arrival. At 21 days of age, pups receiving Rat By "person at risk', or "person in need of such treatment', Chow and water ad libitum were randomly allocated into as used herein is meant any human or lower animal which 50 treatment groups comprising five animals per group, except suffers a significant risk of abnormal calcium and phosphate for control animals receiving saline vehicle which had 10 metabolism if left untreated, and any human or lower animal rats per group. On day 0 and again on day 1 all animals were diagnosed as being afflicted with abnormal calcium and given a subcutaneous injection of Calcein (Sigma) as a 1% phosphate metabolism. For example, postmenopausal solution in 0.9% NaCl solution to label the skeleton. women; persons undergoing certain steroid therapy; persons 55 Dose Solutions and Dosing Procedure on certain anti-convulsant drugs; persons diagnosed as hav All solutions were prepared for subcutaneous injection in ing Pagets disease, hyperparathyroidism, hypercalcemia of 0.9% normal saline and adjusted to pH 7.4 using NaOH malignancy, or osteolytic bone metastases, persons diag and/or HCl. Dose solution calculation was made by consid nosed as suffering from one or more of the various forms of ering the mass of powder (based on molecular weight, osteoporosis; persons belonging to a population group 60 hydration) of the active material in mg/kg (body weight) that known to have a significantly higher than average chance of corresponds to mgP/kg. Concentrations were based on dos developing osteoporosis, e.g., postmenopausal women, men ing 0.2 ml/100g body weight. Initially, all compounds were over age 65, and persons being treated with drugs known to administered at 0.1, 1.0 and 10.0 mg P/kg/day for 7 days. cause osteoporosis as a side effect, persons diagnosed as Compounds showing activity at 0.1 mg P/kg/day were then suffering from myositis ossificans progressiva or calcinosis 65 tested at logarithmic decrements down to 0.001 mg P/kg/ universalis; and persons afflicted with arthritis, neuritis, day. Adjustments in dosage based on changes in body weight bursitis, tendonitis and other inflammatory conditions which were made on a daily basis. 5,583,122 13 14 Necropsy, Tissue Processing and Histomorphometry On day 8 after the start of dosing, all animals were TABLE II sacrificed by CO asphyxiation. Tibias were dissected free Mineralization Inhibition (Schenk Model)) and placed in 70% ethyl alcohol. One tibia was dehydrated Lowest tested dosage in graded ethanol solutions and embedded in methyl meth producing a statistically acrylate using a rapid procedure described in Boyce et al., significant widening of Lab. Investig., 48, 683-689 (1983), the disclosures of which Diphosphonate epiphyseal growth plate are incorporated herein by reference. The tibia was sectioned Compound (mg P/Kg) longitudinally through the metaphyseal area (Leitz(8) saw 10 EHDP 10 microtome at 150). Specimens were stained on one surface APD 10 CMDP - with silver nitrate and mounted on microscope slides for N-(2-pyridyl) AMDP* 0.1 evaluation with a Quantimet Image Analyzer (Cambridge N-(4-pyridyl)-N-ethyl AMDP* -1) Instruments, Inc.) using both incandescent and ultraviolet N-(2-(3-picolyl)) AMDP* -) illumination. Metaphyseal trabecular bone content was mea N-(2-(4-picolyl)) AMDP* 0.1 5 N-(2-(5-picolyl)) AMDP* 0.1 sured in the region between the fluorescent label and the N-(2-(6-picolyl)) AMDP* -) growth plate: expressed as percent of total area (bone-- N-(2-pyrimidyl) AMDP* 1.0 marrow). Epiphyseal growth plate width was obtained as the - mean value of 10 equally-spaced measurements across the 1) section. 20 - = No plate widening observed at highest dose tested (highest dose tested Statistical evaluation of data was made using parametric is 10 mg P/kg/day unless otherwise indicated) and non-parametric analysis of variance and Wilcoxons rank = Highest dose evaluated is 1 mg P/kg/day (compound lethally toxic at 10 mg Pikg/day) sum test to determine a statistically significant effect com EHDP = ethane-1-hydroxy-1,1-DP pared to control animals. 25 APD = 3-aminopropane-1-hydroxy-1,1-DP The Schenk model provided data for in vivo bone resorp CMDP = Dichloromethane DP AMDP = aminomethane diphosphonic acid, where the ring is attached to the tion inhibition by the compounds. The lowest effective all (antiresorptive) dose ("LED") for representative compounds EDP = ethane-1,1-diphosphonic acid, where the ring is attached at the 2 position of the ethane tested, as determined by the Schenk model, are provided in * = Compounds included in pharmaceutical compositions of the present Table I. 30 invention TABLE I Thyroparathyroidectomized (TPTX) Rat Model The compounds were evaluated for in vivo bone resorp Lowest Effective (Antiresorptive) Dose tion inhibition potency by an animal model system known as Schenk 35 the thyroparathyroidectomized (TPTX) rat model. The gen Diphosphonate Compound LED (mg P/kg) eral principles of this model system are disclosed in Russell EHDP 1.0 et al., Calcif. Tissue Research, 6, 183-196 (1970), and in CIMDP 1.0 Muhlbauer and Fleisch, Mineral Electrolyte Metab., 5, APD 0.1 296–303 (1981), the disclosures of which are incorporated N-(2-pyridyl) AMDP* 0.01 N-(2-(5-chloro)-pyridyl) AME)P* 0.01 40 herein by reference. The basic biochemical concept of the N-(2-(3-picolyl)) AMDP* 0.001 TPTX system is inhibition of the parathyroid hormone N-(2-(4-picolyl)) AMDP* 0.001 (PTH)-induced rise in serum and ionized calcium levels by N-(2-(5-picolyl)) AMDP* 0.001 the respective bone active polyphosphonates. N-(2-(6-picolyl)) AMDP* 0.001 N-(2-pyrimidyl) AMDP* 0.001 Materials and Methods N-(4-pyridyl)-N-ethyl AMDP* 0.1 45 Materials 2-(2-pyridyl) EDP* 0.01 2-(3-pyridyl) EDP* 0.01 Low calcium and low phosphorous diets used were pre 1-(2-pyridyl) propyl DP* 10 pared by Teklad() Test Diets (Harlan Industries, Madison, Wis. 53711; Order #TD82195) in a pellet form of approxi EHDP = ethane-1-1 hydroxy-1,1-DP mately 0.18% calcium and 0.22% phosphorous. The diets CMDP = dichloromethane DP 50 APD = 3-aminopropane-1-hydroxy-1,1-DP contained all the essential vitamins and minerals required for AMDP = aminomethane diphosphonic acid, where the ring is attached to the the rat, with the exception of calcium and phosphorous. The alle, calcium and phosphorous levels of the pellets were verified * = Compounds included in pharmaceutical compositions of the present invention. analytically (Procter & Gamble Co., Miami Valley Labora EDP = ethane-1,1-diphosphonic acid, where the ring is attached at the 2 tories, Cincinnati, Ohio). position of the ethane. 55 Propyl DP = propane-2,2-diphosphonic acid PTH was acquired as a powdered bovine extract (Sigma Chemical Co., P. O. Box 14508, St. Louis, Mo., order Diphosphonate compounds which have a bone mineral #P-0892, Lot #72F-9650) at an activity of 138 USP units per ization inhibiting effect cause widening of the epiphyseal mg. PTH was prepared in 0.9% saline such that the final growth plate, since matrix production continues but miner concentration was 100 U.S.P.?ml. All solutions were filtered alization is impeded. The widening of the epiphyseal growth 60 through a #4 Whatman Filter Paper and refiltered through a plate as observed in the Schenk model is, therefore, a 0.45 um Metricel(B) filter. measure of the mineralization inhibiting effect of the diphos Dose solutions and Dosing Procedure phonate compound tested. All solutions of compounds to be tested for bone resorp The lowest tested dosages producing a statistically sig 65 tion inhibition potency were prepared for subcutaneous nificant widening of epiphyseal growth plate for compounds injection in 0.9% normal saline and adjusted to pH 7.4 using tested are given in Table II. NaOH and/or HCl. Dose solution calculation was made by 5,583,122 15 16 considering the mass of powder (based on molecular weight, phonates tend to inhibit resorption of bone materials, the hydration) of the active material in mg/kg (body weight) that animals pretreated with polyphosphonate showed a rise in corresponds to mg P/kg. Concentrations were based on serum calcium level after PTH challenge which was less dosing 0.2 ml/100 grams of body weight. Initially, all than that found in control animals which had been treated compounds were administered at 0.01, 0.1, and 1.0 mg with saline vehicle instead. The lowest dose at which the P/kg/day for 4 days. Where necessary the test was repeated, polyphosphonate is capable of inhibiting bone resorption, as whereby the animals were administered with 0.5 LED in evidenced by a decreased rise in serum calcium upon PTH order to refine the determination of LED. Adjustments in challenge, is a measure of the bone resorption inhibition dosage based on changes in body weight were made on a potency of the polyphosphonate. The LED values of the daily basis. 10 bone resorption inhibition potency of representative com Animals pounds as determined by the TPTX rat model are presented In this study 50 male Wistar rats weighing approximately in Table III. 150-160 grams were thyroparathyroidectomized surgically by the breeder (Charles River Breeding Laboratories). All TABLE III rats were double housed on arrival in suspended cages with 5 Purina Laboratory Rodent Chow(E) and tap water ad libitum. Lowest Effective (Antiresorptive) Dose After acclimation to the laboratory environment for 3-5 TPTX days, the rats were placed on a low calcium, low phospho Diphosphonate Compound LED (mg P/kg) rous (0.18%/0.22%) diet (Teklad(E)) and given 2% (WIV) EHDP 1.0 calcium gluconate supplemented deionized water via water 20 ClMDP 1.0 bottles. APD 0.1 N-(2-pyridyl) AMDP* O.O. Method N-(2-(5-amino)-pyridyl) AMDP 0.0 On day four of low-calcium diet all rats were anesthetized N-(2-(5-chloro)-pyridyl) AMDP* O.O. with Ketaset(B (Ketamine Hydrochloride, 100 mg/ml, Bris N-(2-(5-nitro)-pyridyl) AMDP* 0.1 25 N-(2-(5-carboxy)-pyridyl) AMDP N tol Myers), 0.10 ml/100 grams of body weight, weighed and N-(2-(3,5-dichloro)-pyridyl) AMDP* 1.0 then bled from the retro-orbital venous plexus for serum N-(4-pyridyl)-N-ethyl AMDP* 0.1 total calcium analysis using Flame Atomic Absorption N-(2-(3-picolyl)) AMDP* 0.002 N-(2-(4-picolyl)) AMDP* 0.001 (FAA). All rats weighing less than 180grams were elimi N-(2-(5-picolyl)) AMDP* 0.001 nated from the study. Animals were then randomized statis 30 N-(2-(6-picolyl)) AMDP* 0.01 tically such that the mean total serum calcium for each group N-(2-(3.4-lutidine)) AMDP* 0.01 was the same. Only rats deemed hypocalcemic (total serum N-(2-(4,6-lutidine) AMDP* 0.01) calciums8.0 mg/dl) were placed in study groups compris N-(2-pyrimidyl) AMDP* 0.01 N-(4-(2,6-dimethyl)-pyrimidyl) AMDP* 1.0 ing six animals per group. N-(2-(4,6-dihydroxy)-pyrimidyl) AMDP* 0.011) Treatments with the various experimental compounds N-(2-pyridyl) AEDP* 0.01 35 N-(2-(3-picolyl) AEDP* 10 commenced on day 6 and lasted through day 9 of the study 2-(2-pyridyl) EDP* 0.01 (at 1:00 PM. each day). Dose solutions were prepared to be 2-(3-pyridyl) EDP* 0.01 given at a constant rate of 0.2 ml/100 grams of body weight 2-(4-pyridyl) EDP* 0.1 subcutaneously in the ventral skin flap where the hind leg 1-(2-pyridyl) propyl DP 10 meets the torso. All rats were weighed and dosed daily. A 25 2-(2-pyridyl)-1-chloroethane DP' 0. gauge %" needle was used to administer drug, alternating 40 O-(2-pyridyl)-oxamethane DP 1.0 dose sites daily. On day 8, animals were changed to deion O-(2-(3-picolyl))-oxamethane DP* 0.1 ized, distilled water via water bottles. On day 9 all rats were N = no activity at any of the dosage levels tested fasted in the afternoon at approximately 4:00 P.M. On day 10 EHDP = ethane-1-hydroxy-1,1-DP CMDP = dichloromethane DP of study no treatment was given. In the morning a 600 ul 45 APD = 3-aminopropane-1-hydroxy-1,1-DP sample of whole blood was collected from each rat in AMDP = aminomethane diphosphonic acid, where the ring is attached to the Microtainer (B-D#5060) serum separater tubes for serum ae AEDP = 2-aminoethane-1,1-diphosphonic acid, where the ring is attached to total calcium (FAA). Two 125 ul samples of heparinized the amine whole blood were also collected to be used for ionized EDP = ethane-1,1-diphosphonic acid, where the ring is attached at the 2 calcium analysis. Immediately following blood collection all position of the ethane 50 propyl DP = propane-2,2-diphosphonic acid rats were weighed and injected with bovine parathyroid * = Compounds included in pharmaceutical compositions of the present hormone subcutaneously at a rate of 75 USP (filtered) per invention 100 grams of body weight. Blood sampling for total and = activity level questionable due to lack of dose response ionized calcium was repeated three and one-half hours post-PTH injection. 55 EXAMPLE IV All pre- and post-PTH total and ionized calciums were statistically analyzed for significance compared to PTH Patients weighing approximately 70 kilograms who are alone (control) using Students t-test, analysis of variance, clinically diagnosed as suffering from hypercalcemia of and their non-parametric equivalents. The post minus pre malignancy are administered 0.7 mg P of 2-(2-pyridyl)- change and % change were also determined on calcium 60 ethane-1,1-diphosphonic acid, or its pharmaceutically-ac levels and pre-drug vs post-drug body weights. ceptable salt or ester, by a 2/2 hour intravenous infusion one The physiological effect of the PTH challenge is a rise in time daily for 4 days. This treatment results in an appreciable serum calcium level, with peak activity observed at three alleviation of the hypercalcemia of malignancy. and one-half hours. Since the hormonal and dietary controls Similar results are obtained when the 2-(2-pyridyl)- of calcium metabolism are minimized in the TPTX model, 65 ethane-1,1-diphosphonic acid in the above-described treat an observed increase in serum calcium level is presumably ment is replaced with the result of resorption of bone material. Since polyphos N-(2-(5-amino)-pyridyl)-aminomethane diphosphonic acid; 5,583,122 17 18 N-(2-(5-chloro)-pyridyl)-aminomethane diphosphonic acid; 3. A diphosphonic acid compound, or a pharmaceutically N-(2-(3-picolyl)-aminomethane diphosphonic acid; acceptable salt or ester thereof, having the structure: N-(2-(4-picolyl)-aminomethane diphosphonic acid; N-(2-(5-picolyl)-aminomethane diphosphonic acid; N-(2-(6-picolyl)-aminomethane diphosphonic acid; R foll, N-(2-(3,4-lutidine))-aminomethane diphosphonic acid; R3-Z g-pot: N-(2-pyrimidyl)-aminomethane diphosphonic acid; R2 OH N-(2-pyridyl)-2-aminoethane-1,1-diphosphonic acid; 2-(3-pyridyl)-ethane-1,1-diphosphonic acid; 10 wherein Z is a pyridine ring n is 1; R is hydrogen, or a 2-(4-pyridyl)-ethane-1,1-diphosphonic acid; substituted or unsubstituted, saturated or unsaturated hydro 2-(2-pyridyl)-1-hydroxy-ethane-1,1-diphosphonic acid; carbon chain having from 1 to 4 carbon atoms; and R is 2-(3-pyridyl)-1-hydroxy-ethane-1,1-diphosphonic acid; hydrogen, a substituted or unsubstituted, saturated or unsat 2-(4-pyridyl)-1-hydroxy-ethane-1,1-diphosphonic acid; urated hydrocarbon chain having from 1 to 6 carbon atoms, O-(2-(3-picolyl)-oxamethane-diphosphonic acid; or phar 15 substituted or unsubstituted benzyl, hydroxy, halogen, maceutically-acceptable salts or esters thereof. C-C alkoxy, amino, substituted amino, substituted or What is claimed is: unsubstituted phenyl, substituted or unsubstituted naphthyl, 1. A diphosphonic acid compound, or a pharmaceutically carbonyl, nitro, amido, or carboxylate; and wherein said substituted R2 and R groups are independently substituted acceptable salt or ester thereof, having the structure: 20 with methyl, ethyl, amino, chloro, nitro, methoxy, hydroxy, acetamido, or acetate. R. poli, 4. A diphosphonic and acid compound, or pharmaceuti R-z----Poh, cally-acceptable salt or ester thereof, which is 2-(3-pyridyl)- R4 R2 R 1-hydroxyethane diphosphonic acid. 5. A pharmaceutical composition comprising: wherein Z is a pyridine ring; R is hydrogen substituted or (a) a geminal diphosphonic acid compound, or a pharma unsubstituted amino, amido, hydroxy, C-C alkoxy, halo ceutically-acceptable salt or ester thereof, at a level gen, carboxylate, a substituted or unsubstituted, a saturated providing from 0.001 to 600 mg of phosphorus in said or unsaturated hydrocarbon chain having from 1 to 6 carbon 30 composition, wherein said diphosphonic acid com atoms, substituted or unsubstituted phenyl, or substituted or pound is of the formula: unsubstituted benzyl; R is hydrogen, or a substituted or unsubstituted, saturated or unsaturated hydrocarbon chain having from 1 to 4 carbon atoms; R is hydrogen, a R. R. Foti, 35 R3-Z f N f g-pot: substituted or unsubstituted, saturated or unsaturated hydro R2 R4 R1 R1 carbon chain having from 1 to 6 carbon atoms, substituted r r or unsubstituted benzyl, hydroxy, halogen, C-C alkoxy, amino, substituted amino, substituted or unsubstituted phe wherein Z is a pyridine ring; m+n is an integer from 0 to 5; nyl, substituted or unsubstituted naphthyl, carbonyl, nitro, R is hydrogen, substituted or unsubstituted amino, amido, amido, or carboxylate; and R is hydrogen, a substituted or hydroxy, C-C alkoxy, halogen, carboxylate, a substituted unsubstituted, saturated or unsaturated hydrocarbon chain or unsubstituted, saturated or unsaturated hydrocarbon chain having from 1 to 4 carbon atoms, or acetyl; and wherein said having from 1 to 6 carbon atoms, substituted or unsubsti substituted R, R2, R and Ra groups are independently tuted phenyl, or substituted or unsubstituted benzyl, except substituted with methyl, ethyl, amino, chloro, nitro, meth 45 that when n=0, then R is hydrogen, a substituted or unsub oxy, hydroxy, acetamido, or acetate. stituted, saturated or unsaturated hydrocarbon chain having 2. A diphosphonic acid compound, or a pharmaceutically from 1 to 6 carbon atoms, substituted or unsubstituted acceptable salt or ester thereof, having the structure: phenyl, or substituted or unsubstituted benzyl; R is hydro gen, or a substituted or unsubstituted, saturated or unsatur poli, 50 ated hydrocarbon chain having from 1 to 4 carbon atoms; R. is hydrogen, a substituted or unsubstituted, saturated or R3-Z f g-poh, unsaturated hydrocarbon chain having from 1 to 6 carbon R2 OH 2. atoms, substituted or unsubstituted benzyl, hydroxy, halo gen, C-C alkoxy, amino, substituted amino, substituted or wherein Z is a pyridine ring; n is 0 or 1; R is hydrogen, or 55 unsubstituted phenyl, substituted or unsubstituted naphthyl, a substituted or unsubstituted, saturated or unsaturated carbonyl, nitro, amido, or carboxylate; and R is hydrogen, hydrocarbon chain having from 1 to 4 carbon atoms; and R. a substituted or unsubstituted, saturated or unsaturated is hydrogen, a substituted or unsubstituted, saturated or hydrocarbon chain having from 1 to 4 carbon atoms, or unsaturated hydrocarbon chain having from 1 to 6 carbon acetyl, atoms, substituted or unsubstituted benzyl, hydroxy, halo (b) a pharmaceutically-acceptable carrier. gen, C-C alkoxy, amino, substituted amino, substituted or 6. A pharmaceutical composition comprising: unsubstituted phenyl, substituted or unsubstituted naphthyl, (a) a geminal diphosphonic acid compound, or a pharma carbonyl, nitro, amido, or carboxylate; and wherein said ceutically-acceptable salt or ester thereof, at a level substituted R2 and R groups are independently substituted 65 providing from 0.001 to 600 mg of phosphorus in said with methyl, ethyl, amino, chloro, nitro, methoxy, hydroxy, composition, wherein said diphosphonic acid com acetamido, or acetate. pound is of the formula: 5,583,122 19 20 R. POH2 R. poli, R3 -Z O f–Pott, R3-Z g-pot: R2 R R2 R f wherein Z is a pyridine ring; n is 0 or 1; R is hydrogen, wherein Z is a pyridine ring; m+n is an integer from 0 substituted or unsubstituted amino, amido, hydroxy, C1-C to 5; R is hydrogen, substituted or unsubstituted alkoxy, halogen, carboxylate, a substituted or unsubstituted, amino, amido, hydroxy, C-C alkoxy, halogen, car O saturated or unsaturated hydrocarbon chain having from 1 to boxylate, a substituted or unsubstituted, saturated or 6 carbon atoms, substituted or unsubstituted phenyl, or unsaturated hydrocarbon chain having from 1 to 6 substituted or unsubstituted benzyl; R2 is hydrogen, or a carbon atoms, substituted or unsubstituted phenyl, or substituted or unsubstituted, saturated or unsaturated hydro substituted or unsubstituted benzyl, except that when 15 carbon chain having from 1 to 4 carbon atoms; and R is hydrogen, a substituted or unsubstituted, saturated or unsat n=0, then R is hydrogen, a substituted or unsubsti urated hydrocarbon chain having from 1 to 6 carbon atoms, tuted, saturated or unsaturated hydrocarbon chain hav substituted or unsubstituted benzyl, hydroxy, halogen, ing from 1 to 6 carbon atoms, substituted or unsubsti C-C alkoxy, amino, substituted amino, substituted or tuted phenyl, or substituted or unsubstituted benzyl; R2 20 unsubstituted phenyl, substituted or unsubstituted naphthyl, is hydrogen, or a substituted or unsubstituted, saturated carbonyl, nitro, amido, or carboxylate; and wherein said or unsaturated hydrocarbon chain having from 1 to 4 substituted R, R2 and R groups are independently substi carbon atoms: and R is hydrogen, a substituted or tuted with methyl, ethyl, amino, chloro, nitro, methoxy, unsubstituted, saturated or unsaturated hydrocarbon 25 hydroxy, acetamido, or acetate; and chain having from 1 to 6 carbon atoms, substituted or (b) a pharmaceutically-acceptable carrier. unsubstituted benzyl, hydroxy, halogen, C-C alkoxy, 12. A pharmaceutical composition according to claim 11, amino, substituted amino, substituted or unsubstituted wherein n=1. phenyl, substituted or unsubstituted naphthyl, carbonyl, 13. A pharmaceutical composition according to claim 11, nitro, amido, or carboxylate; and 30 wherein said diphosphonic acid compound is of the formula: (b) a pharmaceutically-acceptable carrier. 7. A pharmaceutical composition according to claim 5, H POH wherein m+n=0. R 8. A pharmaceutical composition according to claim 6, 35 3 N g C-POH wherein m+n=0. H P R 9. A pharmaceutical composition according to claim 7, wherein said diphosphonic acid compound is of the formula: wherein n=0 or 1; R is hydrogen, chloro, amino, or hydroxy; and R is hydrogen, methyl, amino, chloro, meth 40 oxy, hydroxy, or nitro. 14. A pharmaceutical composition according to claim 12, R3 O). N-C-POHoil, wherein said diphosphonic acid compound is selected from N the group consisting of 2-(2-pyridyl)-ethane-1,1-diphospho R4 Ril 45 nic acid; 2-(3-pyridyl)-ethane-1,1-diphosphonic acid; 2-(4- pyridyl)-ethane-1,1-diphosphonic acid; 2-(2-pyridyl)-hy wherein R is hydrogen; R is hydrogen, methyl, amino, droxyethane-1,1-diphosphonic acid; 2-(3-pyridyl)- chloro, methoxy, nitro, or hydroxy, and R is hydrogen, hydroxyethane-1,1-diphosphonic acid; and 2-(4-pyridyl)- methyl, or ethyl. hydroxyethane-1,1-diphosphonic acid. 10. A pharmaceutical composition according to claim 8, 50 15. A pharmaceutical composition according to claim 14, wherein said diphosphonic acid compound is of the formula: wherein said diphosphonic acid compound is 2-(2-pyridyl)- ethane-1,1-diphosphonic acid. poh, 16. A pharmaceutical composition according to claim 14, R3 -G- o--Poh, 55 wherein said diphosphonic acid compound is 2-(3-pyridyl)- N R1 hydroxyethane diphosphonic acid. 17. A method of treating diseases associated with abnor mal calcium and phosphate metabolism, comprising admin wherein R is hydrogen, and R is hydrogen, methyl, amino, istering to a person in need of such treatment a safe and chloro, methoxy, nitro, or hydroxy. 60 11. A pharmaceutical composition comprising: effective amount of a composition of claim 5. 18. A method of treating diseases associated with abnor (a) a geminal diphosphonic acid compound or a pharma mal calcium and phosphate metabolism, comprising admin ceutically-acceptable salt or ester thereof, at a level istering to a person in need of such treatment a safe and providing from 0.001 to 600 milligrams phosphorus in 65 effective amount of a composition of claim 6. said composition, wherein said compound is of the 19. A method of treating diseases associated with abnor formula: mal calcium and phosphate metabolism, comprising admin 5,583,122 21 22 istering to a person in need of such treatment a safe and 22. A method of treating diseases associated with abnor effective amount of a composition of claim 7. mal calcium and phosphate metabolism, comprising admin 20. A method of treating diseases associated with abnor istering to a person in need of such treatment a safe and mal calcium and phosphate metabolism, comprising admin effective amount of a composition of claim 15. istering to a person in need of such treatment a safe and 23. A method of treating diseases associated with abnor effective amount of a composition of claim 12. mal calcium and phosphate metabolism, comprising admin 21. A method of treating diseases associated with abnor istering to a person in need of such treatment a safe and mal calcium and phosphate metabolism, comprising admin effective amount of a composition of claim 16. istering to a person in need of such treatment a safe and effective amount of a composition of claim 14. ck k k >k : UNITED STATES PATENT AND TRADEMARK OFFICE CERTIFICATE OF CORRECTION

PATENT NO. : 5,583,122 Page 1 of 1 APPLICATIONNO. : 06/806155 DATED : December 10, 1996 INVENTOR(S) : Benedict, James J, and Perkins, Christopher M It is certified that error appears in the above-identified patent and that said Letters Patent is hereby corrected as shown below:

Col. 18, line 22, please delete “and”

Signed and Sealed this First Day of August, 2006 WDJ

JON. W. DUDAS Director of the United States Patent and Trademark Office