CA2073448A1

CA2073448A1 - 1-azetidyl and 1-hexamethylenimine alkyl of aryl bisphosphonic acids and their use as pharmacological agents

Info

Publication number: CA2073448A1
Application number: CA002073448A
Authority: CA
Inventors: Raymond D. Youssefyeh; Daniel L. Cheney; Christopher J. Burns
Original assignee: Individual
Current assignee: Rhone Poulenc Rorer International Holdings Inc
Priority date: 1990-01-12
Filing date: 1991-01-03
Publication date: 1991-07-13
Also published as: EP0510066A4; WO1991010646A1; EP0510066A1; JPH05507061A; AU7165791A

Abstract

This invention relates to compounds having: (A) a bisphosphonic acid endgroup; (B) an azetidine- or hexamethylenimine-derived azacyclic endgroup; and (C) an alkyl and/or aryl group linking the bisphosphonic acid and azacyclic groups through the nitrogen atom of the azacyclic group. These compounds are useful as agents for regulating the metabolism of polyvalent ions, particularly calcium. This invention relates also to pharmaceutical compositions including said compounds and to methods of treating conditions for which said metabolism is involved by using said compounds.

Description

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ALKYL OR ARYL BISPHOSPHONIC ACIDS
AND THEIR USE AS PHARMACOLOGICAL AGENTS
.
This application is a continuation-in-part application of application Serial No. 07/464,423 filed on January 12, 1990.
, , .
FIELD OF THE INVENTION
This invention relates to novel bisphosphonic acids, to pharmaceutical 1~ compositions containing bisphosphonic acids, and to the use of such compositions in regulating calcium metabolism in mammals.

The metabolism of polyvalent ions, particularly calcium and phosphate ions, plays an important role in many physiological processes. Consequently, 20 abnormal metabolism of these ions has been implicated in many physiological disorders in humans and other animals.

A common and exemplary manifestation of abnorrnal calcium metabolism involves the mineral balance of bone tissue. Normally, bones are 25 living tissues which constantly undergo calcium resorption (from the bone) and calcium deposition (to the bone), a dual process resulting in what is called "bone turnover". During growth, the rate of calcium deposition exceeds resorption, while in normal adults, the two processes are in equilibrium.
Abnormal calcium metabolism results in a change in the rate of calcium 30 resorption and/or deposition, leading to bone loss, calcium deposits or an excessive rate of bone turnover.

Examples in humans of conditions with which abnormal calcium metabolism has been implicated include conditions associated with bone 3~ turnover, such as osteoporosis, osteodystrophia fibrosa, tumor-induced osteolytic processes, diseases of the sollagen and the skeletal systems, Morbus Paget (Paget's disease), Morbus Bechterew (Bechterew's disease), SUBST~UTE SHET

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WO 91/10646 ~ PCl /US91/0005 periodontitis, bursitis fibrodysplasia, ankylosing spondylitis, ectopic calcifications, hyperparathyroidism, osteopenia and osteomalacia, and also conditions not generally associated with bone turnover, such as cardiovascular disorders (e.g., arteriosclerosis), tendinitis and neuritis.

Normally, calcium metabolism is regulated primarily by Vitamin D3, parathyroid hormone and other hormones such as calcitonin. Consequently, treatment of calcium metabolism disorders has been dominated by the use of ; these naturally-occurring compounds and analogs thereof, most commonly 10 calcitonin. The therapeutic effect of calcitonin treatment, however, is shortlived, as it is rapidly metabolized in the body, having a half-life of only about 10 minutes. Furthermore, calcitonin cannot be administered orally.

Prior developmental work has led to the recognition that certain types of 15 compounds are effective in regulating calcium metabolism in mammals and are thus suitable as active ingredients in pharmaceutical compositions which.
are effective in treating or preventing the above-identified disorders and other: . disorders. Examples of classes of such compounds include bisphosphonic acids, antibiotics such as mithramycin (piicamycin) and the aforementioned 20 calcitonin and Vitamin D3. The present invention relates to the use of bisphosphonic acids in the treatment of disorders of the aforementioned type.

In addition to their use in the treatment of said disorders, it has been surprisingly found that bisphosphonic acids exhibit antiproliferating activity 25 towards macrophages.

Macrophages are immune cells which are ubiquitous throughout tissues, including bone. As immune cells, macrophages play an essential role in inflammatory joint disease, releasing a large number of destructive agents, 30 such as lipid mediators, hydrolytic enzymes, proteases, and peroxides all of which cause injury to neighboring connective and soft tissues. IL-1 secretion by macrophages induces synovial cells and chondrocytes to produce large quantities of prostaglandins and proteases which turther contribute to this degenerative process. Therefore, compounds inhibiting macrophage 3~ proliferation are thought to be of benefit in the treatment of this degenerative process.

W~STITUTE SHET

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Compared with calcitonin, the use of bisphosphonic acids provides several advantayes. For example, bisphosphonic acids are not metabolized at an appreciable rate in the body, leading to a longer duration of activity. In addition, they can be administered orally.
REPORTED DEVELOPMENTS
The following publications disclose the use of various bisphosphonic acids to regulate calcium metabolism. Dialkylaminoalkyl bisphosphonic acids are disclosed in U.S. Patent Nos. 4,064,164; 4,134,969; and 4,624,947.
1 0 Dialkylaminocycloalkyl bisphosphonic acids are disclosed in U.S. Patent No.
4,719,203. Azacycloalkyl-2,2-bisphosphonic acids are disclosed in U.S.
Patent Nos. 3,941,772; 3,988,443; 4,034,086; 4,086,334; 4,108,961; and 4,117,090. Pyrrolidinyl-2-methyl bisphosphonic acid is disclosed in U.S.
Patent No. 4,267,108. Heteroaromatic alkylbisphosphonic acids are disclosed in U.S. Patent Nos. 4,503,049; 4,687,767; and 4,777,163.
.
Soviet Union Patent No. 1002300 discloses 1-hydroxy-3-(1-piperidinyl)-propylidine-1,1-bisphosphonic acid. PCT Application No. DK89/00071 ~ .
discloses 1-hydroxy-3-(1-pyrrolidinyl)propylidine-1,1-bisphosphonic acids. -Australian Patent Application No. 81451/87 discloses aromatically substituted (1-azacycloalkyl) alkyl bisphosphonic acids. -International Publication No. W089/09775 discloses N-heferocyclic propylidene-1,1-bisphosphonic acids, such as 1-hydroxy-3-(1'-pyrrolidinyl)- : -propylidene-1,1-bisphosphonic acid for use of influencing calcium metabolism.

SUMMARY OF THE INVENTION
In accordance with the present invention, there is provided a compound comprising: (A) a bisphosphonic acid endgroup; (B) an azetidine- or hexamethylenimine-derived azacyclic endgroup; and (C) an alkyl and/or aryl group linking the bisphosphonic acid azacyclic groups through the nitrogen atom of the azacyclic group. More specifically, there is included within the scope of the present invention 1-azetidins or 1-hexamethylenimine alkyl or aryl bisphosphonic acids which are pharmaceutically active in regulating calcium metabolism and inhibiting macrophage proliferation in mammals, and to therapeutic compositions comprising said compounds.

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Preferred compounds within the scope of the present invention are azetidinyl alkylidene bisphosphonates and hexamethyleniminyl alkylidene bisphosphonates.
Bisphosphonic acids within the scope of the present invention can be prepared by bisphosphorylation of their corresponding carboxylic acids using phosphorous acid and/or phosphorous trichloride.
4: .
The present invention relates also to a pharmaceutical composition comprising, in admixture with a pharmaceutically acceptable carrier, a pharmaceutically-effective amount of a bisphosphonic acid compound(s) within the scope of the present invention.

Still another aspect of the present invention relates to pharmacological methods comprising the administration of an effective amount of the above-mentioned pharmaceutical composition to human or other animal patients in need of therapy for disorders which are capable of being treated by regulation of calcium metabolism. Such therapy includes providing antiinflammatory activity, inhibiting bone resorption and treating arthritic conditions.

Some advantages which flow from the practice of the present invention include ease of manufacture, resulting in the availability of large quantities of : pure compound, good activity in inhibiting bone resorption and a long duration of therapeutic activity.

DETAILED DESCRIPTION OFTHE INVENTION
Unless otherwise indicated, the following terms shall be understood to have the following meanings.
"Alkyl" means a saturated aliphatic hydrocarbon which may be either straight- or branched-chained containing from about 1 to about 8 carbon atoms.

"Lower alkyl" means an alkyl group as above, having 1 to about 4 carbon atoms.

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"Aryl~ means a ~ to 7 membered unsaturated cyclic organic group which can be homocyclic or heterocyciic.

"Alkoxy" means an alkyl-oxy group in which "alkyl" is as previously 5 described.

As mentioned above, the compounds of the present invention can be considered as having three essential groups, namely, two endgroups joined by a linking group.
The bisphosphonic acid endgroup of the compounds of the present invention can be derived from -CH[PO(OH)2]2 in which each of the hydrogen atoms of the group is subject to substitution as defined herein. For simplicity,all such compounds (unsubstituted and substituted) are referred to herein as 15 "bisphosphonic acids~. Preferably, the bisphosphonic acid endgroup is hydroxylated and is derived from -COH[PO(OH)2]2, again, with any of the hydrogen atoms of the group subject to substitution.

The æacyclic endgroup of the compounds of the present invention can 20 be a 4- or 7-membered ring containing one nitrogen atom, and can be fully or partially hydrogenated. Examples of 4-membered rings are 1-azetine (C3H5N) and æetidine (trimethylenimine, C3H7N). Examples of 7-membered rin3s are azepine (C6H7N), hexamethylenimine (C6H13N) and those related compounds having intermediate states of hydrogenation. Preferably, the æacyclic group is 25 fully hydrogenated. Optionally, one or more of the carbon atoms of the ring can contain one or more substituent groups.

The aforesaid bisphosphonic acid and æacyclic groups are bonded together by means of an alkyl or aryl bridge, provided that when the æacyclic 30 ring contains an aryl substituent, the bridging group must be aryl. The atomsthrough which the two endgroups are connected are the nitrogen atom of the æacyclic ring and the carbon atom of the bisphosphonic acid group.
Preferably, the bridging group is alkyl, particularly straight chain lower alkyl.
The most preferred bridging group is ethyl. Optionally, the bridging group can 35 contain a substituent group. Preferred substituent groups include hydroxy, amino or substituted amino, alkyl, cyclic alkyl, heterocyclic alkyl, aryl, heteroaryl, aralkyl, alkoxy, aryloxy, aralkoxy, halogen, CF3, carboxy carbonyl SU~S~ NET

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and alkali metal. Optionally, vicinal substituent groups can form a cyclic ring.The most preferred substituent is hydroxy.

A preferred class of bisphosphonic acids for use in the practice of the 5 present invention has the structure:
o ~ I (OR )2 (CRR)n N--Y--C--X
~ / Il(OR )2 o or a salt or ester thereof, wherein n is 3 or 6; X is H, OH, amino, substituted 10 amino, alkyl, aryl, aralkyl, halo or O-Y; each Y is independently alkyl, cyclic alkyl, aryl or aralkyl; each R is independently H, halogen, CF3, OH, alkoxy, amino, substituted amino, carboxy, carbonyl, alkyl, cyclic alkyl, heterocyclic alkyl, aryl or heteroaryl; vicinal R groups can optionally form a cyclic group or a double bond; and each R' is independently H, alkali metal, alkyl, aryl or aralkyl;
15 with the proviso that Y is aryl when R is aryl or heteroaryl.

A preferred class of bisphosphonic acids for use in the practice of the present invention are those having either of structures 11 or 111 below, or a salt or ester thereof, and wherein n is from 0 to about 3, each R is independently 20 halogen, OH, alkoxy, amino, substituted amino or alkyl, and each R' is independently H, alkali metal or alkyl.

n ~\ I (OR )2 < N-alkyl-C--OH 11 \/ Il(OR )2 2~

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o Rn 1~\ I (OR )2 N-alkyl-f--OH 111 Il(OR )2 Another preferred class of bisphosphonic acids for use in the practice of the present invention has either of structures ll or lll above, or a salt or ester thereof, wherein n is 0 and each R' is either H or alkali metal, most preferablysodium.

Certain of the compounds of the present invention may exist in enolic or tautomeric forms, and all of these forms are considered to be included within the scope of this invention.

Bisphosphonic acids included in the compositions of this invention may be useful in the form of free bases, and also in the form of salts, esters and as hydrates. All forms are within the scope of the invention.
; 15 Acid addition salts may be formed and are simply a more convenient form for use; in practice, use of the salt form inherently amounts to use of thebase form. The acids which can be used to prepare the addition salts include preferably those which produce, when combined with the free base, pharmaceutically acceptable salts, that is, salts whose ions are non-toxic to the animal organism in pharmaceutical doses of the salts, so that the beneficial pharmacological properties inherent in the free base are not vitiated by side effects ascribable to the ions.

Although pharmaceutically acceptable salts of said compound are preferred, all addition salts are use as sources of the free base form even if the particular salt ~ se is desi - d only as an intermediate product as, for example, when the salt is formed only for purposes of purification and identification, or when it is used as an intermediate in preparing a pharmaceutically acceptable salt by ion exchange procedures.

Pharmaceutically acceptable salts of the compounds useful in the practice of this invention include, for example, those derived from the following SUBS~TUTE SN~r , - , - , ,::. , - , ., . ; : ~ :

WO 91/10646 ~C~ 8 PCI/US91/00û5 acids: mineral acids such as hydrochloric acid, sulfuric acid, phosphoric acid and sulfamic acid; and organic acids such as acetic acid, citric acid, lactic acid, tartaric acid, malonic acid, methanesulfonic acid ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, cyclohexylsulfamic acid, quinic 5 acid, and the like. The corresponding acid addition salts comprise the following: hydrochloride, sulfate, phosphate, sulfamate, acetate, citrate, lactate, tartarate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, cyclohexylsulfamate and quinate, respectively.

The acid addition salts of the bisphosphonic acids of the present invention are prepared either by dissolving the free base in aqueous or aqueous-alcohol solution or other suitable solvents containing the appropriate acid and isolating the salt by evaporating the solution, or by reacting the freebase and acid in an organic solvent, in which case the salt separates directly or 15 can be obtained by concentration of the solution.

Speaking generally, bisphosphonic acids within the scope of the present invention can be prepared by the reaction of the corresponding carboxylic acid with phosphorous acid and phosphorous trichloride in 20 chlorobenzene at about 80 to about 1 00C.

The compounds of the present invention can be administered to a mammalian host in a variety of forms adapted to the chosen route of administration, i.e., orally, or parenterally. Parenteral administration in this25 respect includes administration by the following routes: intravenous, intramuscular, subcutaneous, intraocular, intrasynovial, transepithelially including transdermal, ophthalmic, sublingual and buccal; topically including ophthalmic, dermal, ocular, rectal and nasal inhalation via insufflation and aerosol and rectal systemic.
The active compound may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsules, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet. For oral therapeutic 35 administration, the active compound may be incorporated with excipient and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and SUeSTITUTE SHET ~ -.. . . . . ~ ~ ... . .. .
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preparations may, of course, be varied and, for example, may conveniently be between about 1 to about 10% of the weight of the unit. The amount of active compound in such therapeutically useful compositions is such that a suitable dosage will be obtained. Preferred compositions or preparations according to 5 the present invention are prepared so that an oral dosage unit form for humans contains between about 2 and 100 mg of active compound.

The tablets, troches, pills, capsules and the like may also contain the following: A binder such as gum tragacanth, acacia, corn starch or gelatin;
10 excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; a sweetening agent such as sucrose, lactose or saccharin; or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring.

When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier.

Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both. A syrup or elixir may contain the active compound sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compound may be incorporated into sustained-release preparations and formulations.
.

The active compound may also be administered parenterally or intraperiotoneally. Soiutions of the active compound as a free base or pharmacologically acceptable salt can be prepared in water suitab!y mixed with a surfactant such as hydroxypropylcellulose. Dispersion can also be prepared in glycerol, iiquid polyethylene glycols, and mixtures thereof and i~
oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

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WO 91/10646~ o PCI/US91/0005 The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It may be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable 10 mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, 15 chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be accomplished by the use of an agent effective in delaying absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions are prepared by incorporating the active compound in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various 25 sterilized active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze drying technique which yield a powder of the active ingredient plus any 30 additional desired ingredient from previously sterile-filtered solution thereof.

The therapeutic compounds of this invention may be administered to a mammal alone or in combination with pharmaceutically acceptable carriers, as noted above, the proportion of which is determined by the solubility and 3~ chemical nature cf the compound, chosen route of administration and standard pharmaceutical practice.

SU~STITUTE SHEET

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2 ~ 7 3 ~ L1 8 Pharmaceutical compositions containing the pharmacologically active bisphosphonic acids of the present invention are believed to function by binding with polyvalent ions, for example, calcium and iron. This affinity of the compounds of the resent invention for calcium allows them to be used in the 5 treatment of diseases or disorders where abnormal calcium metabolism has been implicated. Furthermore, due to this affinity for calcium, and hence bone and other calcified tissues, the compounds of the present invention may be useful as vehicles for carrying other agents to such tissues.

Pharmaceutical compositions containing the compounds of the present invention can be used for the general purpose of inhibiting bone resorption, and are believed to be particularly suitable for treating or preventing conditions such as arthritis (via inhibition of macrophage proliferation), osteoporosis, osteopenia, osteomalacia, Paget's disease, hypocalcemia- and hypercalcemia-related conditions.

The dosage of the present therapeutic agents which will be most suitable for prophylaxis or treatment will vary with the form of administration,the particular compound chosen and the physiological characteristics of the particular patient under treatment. Generally, small dosages will be used initially and if necessary, will be increased by small in¢rements until the optimum effect under the circumstances is reached. The therapeutic human dosage, based on physiological studies using rats, will generally be from about 0.01 mg to about 10 mg/kg of body weight per day or from about 0.4 mg to about 19 or and higher although - may be administered in several different dosage units from once to severa, limes a day. Oral administration requires higher dosages.

EXAMPLES
Erbodiments of the present invention are described in the following non-limiting examples which include a description of pharmacological test procedures believed to correlate to therapeutic activity in humans and other animals.

Examples 1 and 2 illustrate the preparation of bisphosphonic acids within the scope of the present invention. Example 1 is prepared from a 4-SUISTITUTE SHET
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membered azacyclic ring, namely, azetidine, and Example 2 is prepared fro a 7-membered azacyclic ring, namely, hexarnethylenimine.

Example 1 1-Hydroxy-3-(1-azetidinyl)-propylidene-1.1-bisphosphoniç acid. monosodium salt . Ethyl 1-azetidine proprionate (5g, 0.032 mol) was dissolved in 10 M
hydrochloric acid (600 ml). The resulting solution was stirred at room temperature for 2 hours and concentrated to yield 5.3g of 1-azetidine propionic 1 0 acid hydrochloride, a whi~e crystalline product. A 100 ml, three-necked flask was equipped with a condenser, mechanical stirrer, and an argon inlet. Dry chlorobenzene (10 ml), phosphorous acid (2.5g, 0.030 mol) and 1-azetidine propionic acid hydrochloride (3.0g, 0.018 mol) were added thereto and mechanically stirred for 15 minutes at 80C. Phosphorous trichloride (3.7g, 1 5 0.027 mol) was then added dropwise, and the mixture was stirred at 80C foran additional 2 hours. After cooling to room temperature, deionized water (20.
ml) was added, and the mixture stirred for 2 hours. A biphase mixture formed which was filtered through a bed of Celite, and the chlorobenzene was ~ removed by extraction with ether. Acetone (500 ml) was added to the aqueous 20 phase, and the crude product came out of solution as a gum. Inorganic .: phosphorous impurities were removed by passing the crude product over - ~--- Amberlite-120 cation exchange resin (115g) and eluting with water. A small ~, amount of starting carboxylic acid was detected by 13C and was remsved by dissoiving the mixture in water and adjusting the pH of the medium to 5.5 with 1M sodium hydroxide solution. The sodium salt of the bisphosphonic acid was selectively precipitated by the addition of methanol and acetone (1 :1, 500 ml) and isolated by filtration, yielding 2.1g (40%) of product. The compound had a melting (decomposition) point of 250C and the following elemental analysis:
C,18.01 %; H, 5.56%; and N, 3.42%. The theoretical numbers (for the composition C6H14N NaO7P2 5.75 H2O) are C, 18.03%; H, 6.40%; and N, 3.50%.
:
Example 2 1-Hydroxy-3-(1 -hexamethyleniminyl)-propylidin~-1.1-bisphosphonic acid. monosodium salt Ethyl 1-hexamethyleniminepropionate (6.8g, 0.034 mol) was treated with concentrated hydrochloric acid (500 ml) for 2 hours at 20C and SUISTITUTE SHEET

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concentrated at 40C on a rotary evaporator to yield 7.2g of 1-hexamethyleniminepropionic acid hydrochloride, a white crystalline product.
The propionic acid (6g, 0.029 moi), phosphoric acid (4.3g, 0.052 mol) and anhydrous chlorobenzene (20 ml) were mechanically stirred for 15 minutes at 5 100C under a nitrogen atmosphere. Phosphorous trichloride (7.129, 0.052 mol) was added dropwise, and heating was continued for 5 hours. Deionized water (30 ml) was added and after refluxing for 4 hours, the resulting orange suspension was filtered through a bed of Celite. Chlorobenzene was extracted out with ether. The crude product was precipitated from the aqueous phase by 1 0 the addition of acetone, and impurities removed by passage over Amberlite-120 cation exchange resin (145g) in water. The sodium salt was precipitated by dissolving the bisphosphonic acid in water (10 ml), adjusting the pH of the solution to 5.5 with 2 N sodium hydroxide and adding acetone (500 ml).
Filtration yielded 1.39 of a white powder. The compound melted 1 5 (decomposed) between 238 and 242C and had the following elemental analysis: C, 2969%; H, 6.09%; and N, 3.75%. The theoretical numbers (for the composition C6H20N NaO7P2-1.4 H2O) are C, 29.68%; H, 5.94%; and N, 4.13%.

20 Activity Tests - Inhibition of Bone Resorption The assay chosen to test the new bisphosphonates for their inhibitory activity of bone resorption consists of measuring the preventive effect on the hypercalcemia induced by a retinoid in rats, according to the following method of Trechsel et al., J. Clin. Invest. 80:1679-1686 (1987).
Male Wistar rats weighing about 1609 were thyroparathyroidectomized.
Five days later the effectiveness of the operation was controlled by measuring calcemia after a night fasting (day 0). From that day on, each of the animals was given the same quantity of food. The animals received daily, for 3 days, 2 30 subcutaneous injections, one containing 25 ~,19 of ethyl p-[(E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthyl)-1-propenyl]benzoate, a synthetic retinoid obtained from Hoffman-La Roche & Co., AG, Basel, the other one the bisphosphonate (bisphosphonic acid sodium salt) to be tested. Additionally, all animals were given 2 1l9 of thyroxine the first and last day of treatment.
35 Twenty-four hours after the last injection of the retinoid and the bisphosphonate, and after one night of fasting, blood was taken by retroorbital , .
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punctu~re under ether anesthesia (day 3). Plasma calcium was then analyzed by means of atomic absorption.

During all these experiments, the animals received water ad libitum.

5 The diet given was Kliba 331, which contains 1.09 Ca/100g, 0.89 Pi/100g and - 800 I.U. of vitamin D3/kg.

- The results are calculated using the following method. Calcemia is measured at day 0 and at day 3, and the latter value is subtracted from the 10 former to yield the change, ~Ca. As a control, the retinoid alone is administered, and this value of ~Ca is used as standard. The value for ~Cas for the test compounds is calculated and expressed as a percentage of the value of ~Ca for the control. This latter value is an index of the inhibitory activity. A value of 0% means no inhibition, 100% a complete inhibition and 15 values over 100% indicate that besides complete inhibition of the retinoid induced hypercalcemia, the bisphosphonate decreased calcemia below the .
level present before the retinoid administration. Generally speaking, inhibitory- values higher than about 50% are considered significant.

2~ The results of this test work are summarized in Table I below.

. TABLE I
Concentration 25~m~!~!g. (mg/kg) % InhibitiDQ
Control 0.01 89.17 0.1 155.7 2 0.01 54.17 2 0.1 131.1 The foregoing results indicate that the bisphosphonic acids of the present invention exhibit significant activity in inhibiting bone resorption when 35 administered in concentrations at least as low as 0.01 mg/kg of animal body weight, and that at dosages at least as low as 0.1 mg/kg, the compounds can completely reverse the retinoid-induced hypercalcemia.

Antiproliferative activity of bisphosphonic acids toward macrophages 40 were tested by the following method which is essentially based on the method SUeSIITUTE SHEEI

'O 91/10646 1 ~i 2 a PCr/US91/0~)055 :
of Cecchini et al., Journal of Bone & Mineral Res., Vol.2, No. 2, pp. 135-142 - (1 987).

Proliferation of Bone Marrow Cells as Monitored by 5 Incorporation of Tritiated Thvmidine (l3H]-TdR)/lnhibitiQn of Macropha~es Bone marrow cells (BMC) obtained by separation on a density . centrifugation medium, Iympholyte M (Ficoll~ 5400 and sodium diatrizoate, density = 1.0875 + 0.0005 at 25C, Cedonlaime Lab. Ltd., Ontario, Canada), were used as target cells. Bisphosphonates were added as concentrated stock 1 Q solutions in phosphate buffered saline (PBS) individually to each well (2.5% of : the final volume) to reach the final desired concentration. The bisphosphonates (Sample 1: 1-hydroxy-3-(1'-pyrrolidinyl)propyiidene-1,1-bisphosphonic acid disclosed in International Publication No.: W089/09775;
Sample 2: 1-hydroxy-3-(1-æetidinyl)propylidine-1,1-bisphosphonic acid of the ;~ 15 present invention) were present throughout the entire test period. BMC (20 x ; 104/ml) were suspended in Dulbecco's Modified Eagle's medium (DMEM) containing 30%(v/v) L-cell conditioned medium. 0.2 mllwell of all suspension was plated in 96-well plates and the cultures incubated for a total of 96 hours with the addition of [3H]-TdR (0.5 IlCi/well) during the last 24 hours. At the end 20 of the culture period, the cells, after being Iysed with 0.0~% (wlv) SDS, were harvested onto glass fiber filters with a cell harvestor (Skranton, Tecnomara AG, Zurich, Switzerland) and [3H]-TdR incorporation was determined by liquid scintillation spectrometry. Controls, not containing the bisphosphonates, were ran simultaneously with the bisphosphonate samples.
The results of two comparative tests are shown in Table ll and Table lll, wherein IC50 values and % Inhibition denote inhibition of macrophage proliferation.

TABLE ll~
IC50 Ratio of % Inhibition IC50 Sample2 over Sample No.(2.5 ~,lM)Micromolar Sample 1 Control A
Sample 1 31 13.0 Sample 2 58 2.5 SII~STIIUTE SH~T
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6 ~ 16 PCr/US91/000~' ~S
, .
TABLE lll~
ICso Ratio of % Inhibition IC50 Sample 2 over Sample No. (2.5 ~,lM! Micromol~r Sample 1 Control A - -Sample 1 37 45 5.0 Sample 2 67 0.9 (~ It is to be noted that while the absolute values of lCso vary from one experiment to another, the ratio of ICso values remain about the same.) 1 ~
The foregoing results indicate that the bisphosphonic acids of the ~ .
present invention, as illustrated by the test rsults on 1-hydroxy-3-(1-azetidinyl)propylidine-1,1-bisphosphonic acid (Sampie 2), exhibit several-fold higher activity in inhibiting macrophage proliferation than the prior art 20 compounds tested simultaneously.

8UBSTITUTE SHE~T

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Claims

We Claim:

1. A compound of the structure 1 or a salt or ester thereof, wherein n is 3 or 6; X is H, OH, amino, substituted amino, alkyl, aryl, aralkyl, halo or O-Y; each Y is independently alkyl, cyclic alkyl, aryl or aralkyl; each R is independently H, halogen, CF3, OH, alkoxy, amino, substituted amino, carboxy, carbonyl, alkyl, cyclic alkyl, heterocyclic alkyl, aryl or heteroaryl; vicinal R groups together can form a cyciic group or a double bond; and each R' is independently H, alkali metal, alkyl, aryl or aralkyl;
with the proviso that Y is aryl when R is aryl or heteroaryl.

2. A compound according to Claim 1, wherein n is 3.

3. A comound according to Claim 1, wherein n is 6.

4. A comound according to Claim 1, wherein X is OH.

5. A compound according to Claim 4 wherein Y is alkyl.

6. A compound according to Claim 5, wherein each R' is independently H
or Na.

7. A compound according to Claim 6, wherein each R is H.

8. A compound acording to Claim 7, which is 1-hydroxy-3-(1-azetidinyl)propylidine-1,1-bisphosphonic acid or a sodium salt thereof.

9. A compound according to Claim 7, which is 1-hydroxy-3-(1-hexamethyleniminyl)propylidine-1,1-bisphosphonic acid or a sodium salt thereof.

10. A pharmaceutical composition comprising in admixture with a pharmaceutically acceptable carrier a therapeutically effective amount of compound(s) of Claim 1, and which is capable of regulating calcium metabolism in mammals.

11. A pharmaceutical composition according to Claim 10 wherein said compound is effective in inhibiting bone resorption.

12. A pharmaceutical composition according to Claim 10 wherein said compound has antiinflammatory properties.

13. A method of treating calcium metabolism disorders comprising the administration to a human or other animal in need of such treatment a calcium metabolism regulating effective amount of a pharmaceutical composition according to Claim 10.

14. A method of inhibiting bone resorption comprising the administration to a human or other animal in need of such treatment a bone resorption inhibiting effective amount of a pharmaceutical composition according to Claim 10.

15. A method of treating inflammation comprising the administration to a human or other animal in need of such treatment an effective antiinflammatory amount of a pharmaceutical composition according to Claim 10.

16. A method of treating arthritic conditions comprising the administration to a human or other animal in need of such treatment an effective arthritic amount of a pharmaceutical composition according to Claim 10.