DK174685B1 - New cyclo-alkane-di:phosphonic acid derivs. - useful for treating and preventing abnormal conditions of calcium and phosphorus metabolism esp. bone conditions - Google Patents

Abstract

Cycloalkanediphosphonic acid derivs. of formulae (I)-(IV) and their salts and esters are new, either X=Y=CH; or one of X and Y= N and the other=CH; R1=H or one or more opt. substd. opt. unsatd. 1-6C alkyl, opt. substd. aryl, opt. substd. benzyl, OH, halogen, opt. substd. NH2, amido, COOH, carbonyl, carboxylate or alkoxy; R2=R1 or NO2. Also new are 3-chlorocyclopentane-; cyclohexane-; cyclohex-2-ene-; cyclohex-3-ene-; and cycloheptane-1,1-diphosphonic acids and their salts and esters. Also new are cyclopropane-; cyclobutane-; cyclopentane-; cyclopentene-; cyclohexane-; cyclohexene-; and cycloheptane-1,1-diphosphonic acids; and indan- and hexahydroindan-2,2-diphosphonic acids; and dihydro-1-pyrindine- and dihydro-2-pyrindine-6,6-diphosphonic acids; and their salts and esters, each ring being substd. by R2.

Description

in DK 174685 B1

The present invention relates to novel monocyclic geminal diphosphonic acids and pharmaceutically acceptable salts thereof and to pharmaceutical compositions containing the novel compounds 5 of the invention.

These compounds are useful in treating or preventing diseases characteristic of abnormal calcium and phosphate metabolism, especially those which are characteristic of abnormal bone metabolism.

There are a number of pathological conditions that may haunt warm-blooded animals and involve abnormal calcium and phosphate metabolism. These modes can be divided into two broad categories.

1. Conditions characterized by abnormal mobilization of calcium and phosphate, which usually lead to a specific bone loss or high 20 calcium and phosphate levels in body fluids. These conditions are sometimes referred to herein as pathological hard tissue demineralizations.

2. Conditions that cause or are the result of abnormal calcium and phosphate deposition in the body. These conditions are sometimes referred to herein as pathological calcifications.

The first category includes osteoporosis, which is a condition in which disproportionately much hard bone tissue is lost relative to the development of new hard tissue. Marrow and bone cavities become larger, the fibrous bond decreases and the compact bone becomes brittle. Osteoporosis can be subdivided into menopause, senile and drug-induced 35 (e.g., adrenocorticoid-induced that can occur with steroid therapy) and disease-induced condition {e.g.

DK 174685 B1 2 arthritis and tumor induced), etc., but the manifestations are essentially the same. Another condition of the first category is Paget's disease (osteitis deformans). In this disease, resolution of normal bone occurs, which is then randomly replaced by soft, poorly mineralized tissue, so that the bone is deformed by weight loading pressure, especially in the tibia and femur. Hyperparathyroidism, hypercalcemia in malignancy and osteolytic bone metastases are conditions that also fall into the first 10 categories.

The second category, which includes conditions that manifest in abnormal calcium and phosphate deposits, includes myositis ossificans 15 progressives, calcinosis universalis, and such disorders as arthritis, neuritis, bursitis, tendonitis and other inflammatory conditions that predispose involved tissues to calcium phosphate deposition. .

Polyphosphonic acid and their pharmaceutically acceptable salts have been proposed for use in the treatment and prophylaxis of these conditions. In particular, diphosphonates, such as ethane-1-hydroxy-1,1-diphosphonic acid (EHDP), propane-3-amino-1-hydroxy-1,1-diphosphonic acid (APD) 25, and dichloromethane diphosphonic acid (Cl2MDP), have been the subject of considerable research efforts within this area. Covenant disease and heterotopic ossification are sometimes successfully treated with EHDP. The diphosphonates tend to prevent the resorption of bone tissue, which is favorable for patients suffering from excessive bone loss. However, ehdp, APD and many other known diphosphonates have the tendency to inhibit bone mineralization when administered in high dose ranges.

It is therefore an object of the present invention to provide novel diphosphonate compounds which inhibit the resorption of bone tissue and have a reduced tendency to inhibit bone mineralization. It is a further object of the present invention to provide compositions for the treatment and prophylaxis of abnormal calcium and phosphate metabolism.

Preparation of tetraethyl ester of xanthan-9,9-diphosphonic acid is described by Mustafa et al. in Ann. , 698, 109 (1966). Synthesis of diphosphone methylene ether of 1,2-dihydroxybenzene is described by Gross et al.

Liebigs Ann.Chem., 707, 35 (1967). none of these references discloses any specific utility of the compounds described therein.

In the specification of US Patent No. 3,683,080 issued to

Francis discloses compositions comprising polyphosphonates, particularly diphosphonates, and their use in inhibiting abnormal deposition and mobilization of calcium phosphate in animal tissues.

20 discloses compositions comprising certain phosphonate compounds in combination with vitamin D-like compounds for use in inhibiting the mobilization of calcium phosphate in animal tissues. Among the phosphonate compounds described therein are cycloalkyl-substituted hydroxymethyl diphosphonates and vicinal diphosphonates of fluorinated cycloalkanes.

In the specification of US Patent No. 3,988,433 issued to

Ploger et al. azacycloalkane-2,2-diphosphonic acids are described. The compounds are stated to be useful as sequestering agents, as per-compound stabilizers, for delaying gypsum hardening, for preventing the formation of tartar and plaque, and for treating diseases associated with abnormal deposition or dissolution of difficultly soluble calcium salts. animal body.

In connection with the present invention, there are provided novel monocyclic geminal diphosphonic acids which are characterized by being selected from cyclobutane-1,1-diphosphonic acids with one or more substituents selected from methyl, methylarino, amino , chloro, hydroxy and methoxy; 3-Chlorocyclopentane-1,1-diphosphonic acid, cyclohexane-1,1-diphosphonic acid, cyclohex-2-ene-1,1-diphosphonic acid, cyclohex-3-ene-1, 1-diphosphonic acid and cycloheptane-1,1-diphosphonic acid and pharmaceutically acceptable salts thereof.

The invention further relates to pharmaceutical compositions comprising (a) from 15 mg P to 600 mg P of a diphosphonic acid compound of the invention, and (b) a pharmaceutical carrier.

The compounds of the present invention fall within the class of geminal, cycloalkyl diphosphonic acids and the pharmaceutically acceptable salts thereof. The cycloalkan moiety of the compounds is cyclobutane, cyclopentane, cyclohexane, cyclohex-2-ene, cyclohex-3-ene or cycloheptane. The ring is optionally substituted with one or more substituents, as indicated above.

By "pharmaceutically acceptable salts" as used herein is meant the hydrolysable salts of the diphosphonic acids which have the same general pharmacological properties as the acid form from which they are derived and which are acceptable from a toxicity point of view. Pharmaceutically acceptable salts include alkali metal (sodium or potassium), alkaline earth metal (calcium and magnesium), non-toxic heavy metal (tin and indium) and ammonium and low molecular weight substituted ammonium (mono-, di- and DK 174685 B1 triethanolamine salts. Preferred compounds are sodium, potassium and ammonium salts.

The compounds of the present invention are useful in treating conditions in humans and animals characterized by abnormal calcium and phosphate metabolism. Other diphosphonates have been proposed for their use, in particular ethane-1-hydroxy-1,1-diphosphonate (EHDP), propane-3-amino-1-hydroxy-1, 1- diphosphonate (APD) and dichloromethane diphosphonic acid (C12MDP).

Although metabolic bone disorders have been successfully treated with the prior art known diphosphonates, 15 ehdp and APD tend to inhibit bone mineralization as well as bone resorption. Administration of these compounds must therefore be carefully monitored to achieve maximum bone resorption inhibition while avoiding inhibition of bone mineralization.

20

It has been found that in vitro, cyclic diphosphonates generally have a greatly reduced tendency for bone mineralization inhibition over EHDP and APD.

It has also been found that in vivo certain cyclic diphosphonates inhibit the resorption of bone tissue. Thus, at equally effective doses to inhibit bone resorption, the compounds of the present invention are expected to inhibit bone mineralization to a lesser extent than that of many known diphosphonates. The compounds of the present invention therefore allow flexibility in the treatment of patients suffering from anomalous calcium and phosphate metabolism. The compounds of the invention are also useful as bone scanning agents after labeling with 99m Technetium.

35 DK 174685 B1 6

Furthermore, the compounds of the invention are useful as sequestering agents for polyvalent metal ions, especially di- and tri-valent metal ions, and can therefore be used for many technical purposes, such as builders 5 in detergents and scavengers, as well as for water treatment. They can also be used as stabilizers for per-compound. Other uses of the diphosphonic acids of the present invention will be appreciated by those skilled in the art.

10

Specific examples of the compounds of the present invention include: 2-methylcyclobutane-1,1-diphosphonic acid 2-hydroxycyclobutane-1,1-diphosphonic acid, 3-chlorocyclopentane-1,1-diphosphonic acid, cyclohexane-1,1-diphosphonic acid, cyclohexane 2-ene-1,1-diphosphonic acid, cyclohex-3-ene-1,1-diphosphonic acid, cycloheptane-1,1-diphosphonic acid and pharmaceutically acceptable salts thereof.

Preferred compounds are 2-methylcyclobutane-1,1-diphosphonic acid and pharmaceutically acceptable salts.

25

Krystalvækstinhiberinqsforsøg

The relative affinity of cyclic diphosphonates to calcified tissue is demonstrated by the crystal growth inhibition test. This test was developed for polyphosphonates to assess their ability to decrease calcium phosphate deposition and has been shown to be indicative of the affinity of these compounds for calcified tissue such as bone. The sample is described in 35 detail by Nancollas et al. in Oral. Biol., 15, 731 (1970).

DK 174685 B1 7

In this test, hydroxyapatite seed crystals are added to a calcium phosphate solution which is supersaturated for induced precipitation of calcium phosphate but metastable for spontaneous precipitation. The graft crystals induce precipitation and crystal growth. Test chemicals are added to the metastable Ca / P solution before inoculation. The effect of the chemicals on the formation of hydroxyapatite induced by the 10 seed crystals has been shown to correlate with the in vivo effects of the chemicals on calcium metabolism.

Formation of calcium phosphate crystals results in the release of hydrogen ions (i.e., pH change).

The rate of crystal growth is monitored by observing the addition of base required to maintain a constant pH. Low concentrations (1 x 6'6 M) of polyphosphonates are capable of inhibiting the formation of calcium phosphate for 20 minutes or longer. The crystal growth inhibition 20 depends on the ability of the polyphosphonates to adsorb onto calcium phosphate crystal cores.

In the test, the time T between the addition of seed crystal and the onset of crystal growth is determined.

The effect of the presence of a diphosphonate compound is calculated as:

Tag = TDP “T Check

30 where TDp is the time course of the experiment with 1 x 10 6 m

of the diphosphonate compound present in the sample solution, T control is the time course of the experiment without diphosphonate, and Tlag is the time delay that results from the presence of the diphosphonate in the solution. For the present purpose, the time delay has been normalized {Tn, where Tn (EHDP) = DK 174685 B1 8 1.0) by dividing the time delay for each connection by that determined for EHDP (τη =

Tiag (test compound) / Tiag (EHDP)). The Tn values for the various compounds are listed in Table I.

5

It has been found that diphosphonates, which in this assay have low Tlag values relative to EHDP, have a relatively low tendency for in vivo bone mineralization inhibition.

10

Table i

Mineral Inhibition (Crystal Growth Inhibition Test) Diphosphonate Compound _Tn_ f

EHDP

APD 2) °> 9

C1MDP3)> 1 Cyclobutane —1,1-DP 0/2 2-Methylcyclobutane —1,1-DP 0/1

Cyclopentane - ~ 1,1-DP 0.3

Cyclopent-3-ene-1,1-DP 0.2 2- Methylcyclopentane -1,1-DP 0.2

2,5-Dimethylcyclopentane — 1,1-DP O, T

3- Hydroxycyclopentane — 1,1-DP 0.3 3-Cbiorocyclopentane — 1,1-DP 0.2

Quinoxalino-2,3-cyclopentane-1,1-DP 0.3

Cyclohexane — 1,1-DP 0.1 20 2-Methylcyclohexane - 1.1 -DP 0.1 tJ-Methylcyclohexane - 1,1-DP 0.2 * = Compounds Useful in Pharmaceutical Compositions of the Invention.

DK 174685 B1 9

Ethane-1-hydroxy-1,1-DP

3-Aminopropane-1-hydroxy-1,1-DP Dichloromethane-DP

5 Pour model

The compounds were assessed for dj vivo bone resorption inhibition and mineralization inhibition in an animal model system known in the field of bone metabolism as the schenk model. The general principles of this model system are described by Shinoda et al. in Calcif. Tissue Int., 35, 87-99 (1983) and af

Schenk et al. in Calcif. Tissue Res., 11, 196-214 (1973).

15 Materials and Methods

Animals

Non-weaned 17-day-old (30 g) male Sprague Dawley rats were purchased with their mothers (from 20 Charles River Breeding Laboratories) and placed on plastic cages with their mothers on arrival. At the age of 21 days, the cubs were randomly assigned to treatment groups that included 5 animals per group and given "Rat Chow" and water ad libitum, except 25 control animals that had 10 rats per group and given saline. On day 0 and again on day 1, all animals were given a subcutaneous injection with Calcein (sigma) as a 1% solution in 0.9% NaCl solution to label the skeleton.

30

Dose solutions and dosing procedures

All solutions were prepared for subcutaneous injection in 0.9% normal saline and adjusted to pH 7.4 using NaOH and / or HCl. Calculation of the solution dose was made from the powder mass of the active substance (based on molecular weight and hydration) in mg / kg (body weight) corresponding to mg P / kg. The concentrations were based on dosage of 0.2 ml / 100 g body weight. Initially, all compounds 5 were administered with 0.1, 1.0 and 10.0 mg P / kg / day for 7 days.

Compounds exhibiting activity at 0.1 mg P / kg / day were then tested in logarithmic reductions down to 0.001 mg p / kg / day. Dose adjustments based on body weight changes were made daily.

10

Necroscopy, Tissue Treatment and Histomorphometry On day 8 after dosing, all animals were killed by CO 2 poisoning. Tibiae were dissected free and placed in 70% ethyl alcohol. One tibia was dehydrated in graduated ethanol solutions and embedded in methyl methacrylate using a rapid method described by Boyce et al. in Lab. Investiq., 48, 683-689 (1983). Tibia was cut in 20 longitudinal sections through the metaphysical area (Leitzø saw microtome at 15 0mm). Subjects were stained on one surface with silver nitrate and placed on microscope glass for evaluation by a quantitative image analyzer (Cambridge Instruments, Inc.) using both clear and ultraviolet illumination. The metaphysical trabecular bone content was determined in the region between the fluorescence label and the growth plate: expressed as a percentage of total area (bone + marrow). The epiphyseal growth plate width was obtained as the mean of 10 determinations performed 30 at equal distances across the section.

Statistical data evaluation was performed using parametric and non-parametric analysis of variance and Wilcoxons' range sum test to determine statistically significant effect on control animals.

DK 174685 B1 11

Diphosphonate compounds which have a bone mineralization inhibitory effect cause a width increase of the epiphyseal growth plate as matrix production continues, but mineralization is inhibited. Therefore, as observed by the Schenk model, the increase in width 5 of the epiphyseal growth plate is a measure of the mineralization inhibition effect of the diphosphonate compounds tested (see Table II).

Table II

Mineralization inhibition (Schenk model)

Lowest tested dose which produces a statistically significant increase in width of the epiphyseal growth plate

Diphosphonate Compound (mg P / kq) ______ 20 EHDP 2) 10 APD 3 * 10 C12MDP 4

Azacyclopentane-2,2-DP 5 ^ 10

Cyclopentane-1,1-DP 3-Chlorocyclopentane-1,1-DP *

2-methylcyclopentane-I, L-DP

* = Compounds usable in pharmaceutical compositions of the present invention.

30 - = no plate width increase was observed at the highest dose tested (the highest dose tested is 10 mg P / kg / day unless otherwise stated).

1) The highest evaluated dose is 1 mg P / kg / day 35 (the compound fatally toxic in 10 mg P / kg / day).

2) Ethane-1-hydroxy-1,1-DP.

3) 3-Aminopropane-1-hydroxy-1,1-DP.

4) dichloromethane dp.

5) A compound disclosed in the disclosure of U.S. Patent No. 3,988,433 to Ploger et al.

5

Of the compounds tested, the known compounds EHDP, APD and azacyclopentane-2,2-DP showed significant plate width increase. None of the compounds of the present invention caused substantial plate width increase. These results are in line with the crystal growth inhibition data (see Table I) indicating that the compounds of the present invention have affinity for calcified tissue.

The Schenk mode also provides data for the in vivo bone resorption inhibition of the compounds. The lowest effective (antiresorptive) dose ("L.E.D.") as determined by the Schenk model is for representative compounds listed in Table IV along with L.E.D. values determined by the thyroparathyroidectomerized (TPTX) rat model.

Thyroparathyroidectomerized (TPTX) rat model The compounds were evaluated for potential in vivo bone resorption inhibition with an animal model system known as the thyroparathyroidectomerized (TPTX) rat model. The general principles of this model system are described by Russell et al. in Calcif. Tissue Research, 30 6, 183-196 (1970) and by Muhlbauer and Fleisch i

Mineral Electrolyte Metab., 5, 296-303 (1981), the contents of which are incorporated herein by reference. The biochemical idea underlying the TPTX system is inhibition of the increase in serum and calcium ion levels of the parathyroid hormone (PTH) by the respective bone active polyphosphonates.

DK 174685 B1 13

Materials and Methods Materials 5 The low calcium and phosphorus diets used were prepared from Teklad0 Test Diets (Harlan Industries, Madison, Wisconsin 53711, order # TD82195) in pill form with approx. 0.18% calcium and 0.22% phosphorus. The diets contained all the essential vitamins and minerals for rats with the exception of calcium and phosphorus. The calcium and phosphorus content of the pills was confirmed by analysis (Procter & Gamblee Co., Miami Valley Laboratories, Cincinnati, Ohio).

15 PTH was obtained as a powdered bovine extract (Sigma Chemical Co., P.0. Box 14508, St. Louis, Missouri, Order No. P0892, Lot No. 72F-9650) with an activity of 138 USP units per mg. PTH was prepared in a 0.9% saline solution such that the final concentration was 20 100 U.S.P./ml. All solutions were filtered through one

Whatman filter paper # 4 and refiltered through a 0.45 mm Metricel00 filter.

Dose solutions and dosing procedure 25

All solutions of compounds to be tested for bone resorption inhibition were prepared for subcutaneous injection in 0.9% normal saline and adjusted to pH 7.4 using NaOH and / or 30 HCl. Calculation of solution doses was made from the powder mass (based on molecular weight and hydration) of the active substance in mg / kg (body weight) corresponding to mg p / kg. The concentrations were based on a dosage of 0.2 ml / 100 g body weight. Initially, all 35 compounds were administered in 0.01, 0.1 and 1.0 mg P / kg / day for 4 days. Where necessary, test 14 DK 174685 B1 was repeated, whereby the animals were administered 0.5 joints for a finer determination of LED. Dose adjustments based on body weight changes were made daily.

5

Animals

For this study, 50 Wistar male rats weighing approx. 150-160 g, thyroid parathyroidectomized surgical 10 by the breeder (Charles River Breeding Laboratories). On arrival, all rats were housed in suspended cages with Purina Laboratory Rodent Chow0 ("rodent food") and tap water ad libitum. After acclimatization to the laboratory environment for 3-5 days, the rats were put on 15 diets of low calcium, low phosphorus content (0.18% / 0.22%), (Teklad00) and via water bottles given deionized water supplemented with 2% (wt. volume) calcium gluconate.

Method On day 4 on a low calcium diet, all rats were anesthetized with Ketaset0 (Ketamine hydrochloride, 100 mg / ml, Bristol Meyers), 0.10 ml / 100 g body weight, weighed 25 and then removed from the retroorbital plexus venosa with for analysis of total serum calcium using flame atomic absorption (FAA). All rats weighing less than 180 g were excluded from the study. The animals were randomized statistically, such that the mean total serum calcium for each group was the same. Only rats assessed as hypocalcemic (total serum calcium H 8.0 mg / dl) were placed in study groups comprising 6 animals per group.

35 DK 174685 B1 15

Treatments with the various experimental compounds began on day 6 and lasted up to day 9 of the study (1pm daily). The dose solutions were prepared to be administered at a constant rate of 0.2 ml / 100 g body weight by subcutaneous administration in the ventral skin patch where the hind leg meets the body All rats were weighed and dosed daily using a 25 gauge 16 mm cannula to administer the drug at various sites from day 10 to day. a change for the animals to deionized distilled water via water bottles.

On day 9, all rats were fasted in the afternoon from approx. pm. At noon, no treatment was given on day 10 of the study. In the morning, a 600 ml whole blood sample was taken from each 15 rat in a Microtainer (B-D No. 5060) serum separation tube for total serum calcium (FAA). Two 125 ml samples of heparinized whole blood were also taken for analysis for ionized calcium. Immediately after blood sampling, all 20 rats were weighed and subcutaneously injected with bovine parathyroid hormone to the extent of USP 75 (filtered) per 100 g of body weight. Blood sampling determination of total and ionized calcium was repeated at 3A hour after PTH injection.

25

All total calcium and calcium ion values before and after PTH were analyzed statistically for significance relative to PTH alone (control) using Student's t test, analysis of variance and their non-parametric equivalents.

The change: "after minus before" and the percentage change was also determined for calcium values and for pre-drug body weight versus post-drug body weight.

35 The physiological effect of the PTH provocation is an increase in the serum calcium level with a peak activity, observed after 3½ hours. As the hormonal and dietary control of calcium metabolism is minimized in the TPTX model, an observed increase in serum calcium level is presumably the result of bone resorption. Since polyphosphonates tend to inhibit resorption of bone materials, the animals pre-treated with polyphosphonate showed an increase in serum calcium levels after PTH provocation that was less than that found in 10 control animals, which had instead been treated with saline vehicle. The lowest dose at which the polyphosphonate is able to inhibit bone resorption as indicated by a slight increase in serum calcium after PTH provocation is a measure of the bone resorption inhibitory ability of the polyphosphonate. LED values for the bone resorption inhibitory ability of representative compounds as determined by the TPTX rat model and the Schenk model are listed in Table IV. The data in Table IV shows that while the diphosphonic acid compounds of the present invention inhibit bone material resorption, there are closely related cyclic diphosphonic acid compounds which do not actually exhibit this property.

DK 174685 B1 17

Table III

Lowest effective (antiresorptive) dose of 5 TPTX Schenk

Diphosphonate Compound (mg P / kq) (mg P / kg) EHDP f M 1.0 AP ° 3. 0.1 0.1 C, 2MDP + 1.0 1/0 Azacyclopentane * -2,2-DP5 'N

Cyclobutane.-1, T-DP N

2- Methylcyclobutane-1 # 1-DP 1τ0

Cydopentane.-1, ϊ-DP N

2-Methylcyclopentane-1, T-DP 10 N

2,5-Dimethylcyclopentane-1,1-DP N

3- Hydroxycyclopentane No. 1,1-DP N

* 3-Chlorocyclopentane r-i, i-DP N j 0

Quinoxalino-2 # 3-cyctopentane — 1,1-DP N

Cyclohexane — 1,1-DP110

2-Methylcyclohexane — 1,1-OP N

Ί-Methylcyclohexane! * ~ L, 1-OP N

Cyclopent-3-ene-1, t-DP N

25 * = Compounds useful in pharmaceutical compositions of the present invention.

- = Not tested N = No activity 30 i) Highest dose evaluated is 1 mg P / kg / day (the compound fatally toxic in 10 mg P / kg / day).

2} Ethane-1-hydroxy-1,1-DP.

3) 3-Aminopropane-1-hydroxy-1,1-DP.

4) Dichloromethane DP.

5) A compound described in the disclosure to US

U.S. Patent No. 3,988,433 to Ploger et al.

DK 174685 B1 18

Synthesis of cyclic diphosphonate compounds

The synthesis reaction is carried out as follows: In a first step, a methane diphosphonate ester in solution is converted to the corresponding carbanion using standard organic-chemical methods. In a second step, a solution of hydrocarbon compound which is suitably activated for a double-nucleophilic substitution is added to this reaction mixture.

Typically, a solution of methane diphosphonate ester will be added to a cold suspension of potassium hydride in an inert organic solvent and the solution will be stirred at room temperature for some time. The suitably activated hydrocarbon will then be added as a solution to the reaction mixture and the whole mixture heated to ca. 80 ° C until completion. After the mixture has been cooled, filtered and concentrated, the concentrate is comatographed on silica gel to give the desired ester. The ester is hydrolyzed by reflux in HCl and the resulting substance is concentrated under vacuum. The residue is dissolved in H 2 O and treated with activated charcoal.

After filtration, the solution is concentrated and the product is finally dried under vacuum. The synthesis of salts and esters of these compounds is achieved by using standard organic chemistry methods well known to those of skill in the art.

30

The following are examples of synthesis of specific cyclic diphosphonates according to the invention.

The compounds were identified by 1 H NMR using Me 4 Si or sodium (2,2-dimethyl-2-35 silapentane-5-sulfonate) as internal standards and by 31 P NMR using H 3 PO 4 as an external standard DK 174685 B1 19 (positive values indicate a chemical shift field downward relative to the reference) by chemical ionization mass spectrometry, by melting point determination and by elemental analysis.

5

Example I

Synthesis of cyclohexane-1,1-diphosphonic acid.

To a stirred, ice-cold suspension of potassium hydride (1.66 g of a 35% mineral oil dispersion, 14.5 mmol) in 20 ml of dry toluene was added dropwise a solution of tetraisopropylmethane diphosphonate (5.00 g, 14.5 mmol) in 50 ml of toluene. After completion of the addition, the ice bath was removed and the clear yellow solution was stirred at room temperature for 1 hour. 1,5-Dibrompentane (1.67 g, 7.3 mmol) was dissolved in 10 ml of toluene and added to the reaction mixture. The mixture was heated to 80 ° C for 18 hours, then cooled in an ice bath, filtered and concentrated. The concentrate was chromatographed (1: 1 hexane: THF eluant) on silica gel to give the desired ester as a white crystalline substance (61% yield based on the dibromide), m.p. 36-38.5 ° C, 1 H NMR (CDCl 3) chemical shift 4.95-4.59 (m, 4H, CH), 2.19-25 1.94 (m, 4H, CH 2), 1.85 -1.51 (m, 6H, CH 2), 1.34 (d, 24H, J = 6 Hz, CH 3); 31 P NMR (CDCl 3) 25.6 ppm, El mass spectrum m / e 412 (M +). Calculated composition for C18H38 ° 6P2: C '52.42; H, 9.29; P, 15.02. Found: C, 52.55; H, 9.43; P, 15.09.

30

A 10% solution of the DP ester in 12N HCl was heated at reflux for 2 hours. The solution was then concentrated in vacuo with the addition of additional H2 O to remove the last traces of HCl. The residue 35 was dissolved in H 2 O and treated with activated charcoal. The charcoal was removed by filtration and the filtrate was concentrated using ether to remove the last traces of H2 O. The product was then dried under vacuum at 50 ° C for 24 hours. Yield: 95%, m.p. 239 ° C; 1 H NMR (D 2 O) chemical displacement 2.04-1.56 (overlap m, 5 CH 2); 31 P NMR (D 2 O) 26.8 ppm, Calculated C 6 H 14 ° 6 P 2: C 29-52, H 5.78; P, 25.38. Found: C, 29.54; H, 6.02, P, 25.19.

Example 11 10

Synthesis of 2-methylcyclobutane-1,1-diphosphonic acid.

Using the procedure of Example I, tetraisopropylmethane diphosphonate was converted to tetraisopropyl (2-methylcyclobutane-1,1-diphosphonate) by reaction with 1,3-dibromobutane at 80 ° C for 4 hours. 31 P NMR (CDCl 3) chemical shift 24.1 ppm.

The ester was hydrolyzed to the corresponding acid using the method described in Example 1. Yield 86.2%.

31 P NMR (D 2 O) chemical shear 24.5 ppm. 13 C NMR (D 2 O) 45.0, 43.1, 41.1 (t), 34.8, 26.4, 22.7 and 18.0 ppm.

Example III

Synthesis of 3-chlorocyclopentane-1,1-diphosphonic acid, a) Preparation of tetraisopropyl (3-hydroxycyclopentane-1,1-3 diphosphonate) tetrahydropyran ether

Using the example described in the procedure described, tetraisopropyl methane diphosphonate was converted to the desired ester in 67% yield by reaction with the tetrahydropyran ether of 1,4-dibromobutan-2-ol at 80 ° C for 18 hours, clear viscous oil 1 H NMR (CDCl 3) chemical DK 174685 B1 21 displacement 5.17-4.58 (m, 5H, OCH_Me2 and OCHO), 4.26-3.95 (m, 1H), 3.86-3.79 (m, 1H), 3.68-3.35 (m, 1H), 2.68-1.79 and 1.99 (m plus s, 6H total), 1.65-1.52 (τη, 6H ), 1.35 (d, 24H, J = 6Hz, CH 3), 31 P NMR (CDCl 3) 26.2, 25.8, 25.6, δ 25.2 ppm; ammonia Cl mass spectrum m / e 499 (MH +).

Calculated composition for C22H4408P2: C / 53 »00, - H, 8.90; P, 12.43. Found: C, 52.70? H, 9.10; P, 12.67.

b) Preparation of tetraisopropyl (3- hydroxycyclopentane-1,1-diphosphonate)

The above-described protected tetraester (9.40 g, 18.9 mmol) was dissolved in 180 ml of Me OH. A catalytic amount (100 mg) of p-toluenesulfonic acid was added and the mixture was stirred for 2 hours at room temperature.

The solution was then concentrated. The residue was dissolved in ether (100 ml), washed with saturated NaHCO3 solution, and then washed with brine. The ether solution was dried, concentrated and then chromatographed (1: 1 THFihexane) on silical gel to give 7.18 g (92%) of

the desired product in the form of a clear oil: 1 H NMR

(CDCl 3) chemical shift 4.88-4.65 (m, 4H, OCH_Me2), 4.72 (s, 1H), 4.56-4.25 (m, 1H), 2.82-2.02 ( m, 4H), 2.02-1.77 (m, 2H), 1.37 and 1.34 (overlapping d, 24H, J = 6 25 and 6 Hz, CH 3), 31 P NMR (CDCl 3) 30, 1, 30.0, 25.4, 25.3 ppm? ammonia Cl mass spectrum m / e 415 (MH +).

c) Preparation of 3-chlorocyclopentane-1,1-diphosphonate from tetraisopropyl- (3- hydroxycyclopentane-1,1-diphosphonate)

Tetraisopropyl - (3hydroxycyclopentane-1,1-diphosphonate) (3.00 g, 7.24 mmol) was dissolved in 50 ml of dry CC14.

Triphenylphosphine (3.80 g, 14.5 mmol) was added and the mixture was heated under reflux for 72 hours. The mixture was filtered, concentrated and the residue was chromatographed (3: 2 hexane: THF) on silica gel to give 2.15 g (69%) of the desired product as a clear viscous oil: 1 H NMR ( CDC13) chemical shift 4.97-4.68 (m, 4H, OCHMe2), 4.24 (m, 1H, J = 7 Hz, CH_OH), 2.65-1.87 (m, 6H, ring CH2 ), 1.36 (d, 24H, J = 6 Hz, CH 3), 31 P NMR (CDCl 3) 25.3, 24.9, 24.4, 24.0; ammonia Cl mass spectrum m / e 433, 435 (MH +).

The ester was then hydrolyzed in a yield of 93% to 10 the corresponding acid using the procedure described in Example I.

Mp. 194 - 195 ° C, 1 H NMR (D 2 O). 4.35 (p, 1H, J = 6Hz,

ClCH), 2.47-1.90 (overlapping m, 6H, CH 2), 31 P NMR (D 2 O) δ 25.8 ppm, · Calculated composition for C 5 H 11 ClO 2 P 2: C, 22.70; H, 4.19; Cl, 13.40, P, 23.42. Found: C, 22.44; H, 4.28; Cl, 10.07, P, 23.44. The indicated analytical data are average values for two different material samples.

The low percentage of cl found was indicative of some hydrolysis of the Cl group during the reaction.

Example IV

Synthesis of cycloheptane-1,1-diphosphonic acid.

25

Using the same procedure as in Example I, tetraisopropylmethane diphosphonic acid was converted to tetraisopropyl (cycloheptane-1,1-diphosphonate) by reaction with 1,6-dibromohexane at 80 ° C for 18 hours. 31 P 30 NMR (toluene) indicated that the reaction solution contained a mixture of the desired tetraisopropyl (cycloheptane-1,1-diphosphonate) (25.3 ppm) and octaisopropyl (octane-1,1,8,8-tetraphosphonate) (22, 1 ppm). The compounds were separated by chromatography (1: 1 hexane: THF eluent) on silica gel and the desired ester was hydrolyzed with 6N hydrochloric acid by reflux as in Example I.

Example V 5

Synthesis of 3-cyclohexene-1,1-diphosphonic acid.

Using the same procedure as in Example I, tetraisopropylmethane diphosphonate was converted to 10 tetraisopropyl (3-cyclohexene / 1,1-diphosphonate) by reaction with 1,5-dichloro-cis-2-pentene. The resulting ester was hydrolyzed as in Example 1 to give the 3-cyclohexene-1,1-diphosphonic acid.

Example VI

Synthesis of 2-cyclohexene-1,1-diphosphonic acid.

Using the same procedure as in Example III 20, tetraethyl methane diphosphonate was converted to tetraethyl (3-hydroxycyclohexane-1,1-diphosphonate) by reaction with the tetrahydropyran ether of 1,5-dibrompentan-2-ol followed by hydrolysis of the ether with p-toluenesulfonic acid. MeOH. The resulting ester was dehydrated to tetraethyl (2-cyclohexene-1,1-diphosphonate) using the procedure described in Vogel: A Textbook of Practical Organic Chemistry 3rd ed. p.243, the contents of which are incorporated herein by reference. The tetraethyl ester was hydrolyzed to the desired 2-cyclohexene-1,1-diphosphonic acid by dropwise addition of trimethylsilyl bromide to a solution of the tetraethyl ester in CC14 at room temperature.

The compounds of the present invention can be used to treat or prevent diseases characterized by abnormal calcium and phosphate metabolism, in particular those characterized by abnormal bone metabolism, in persons at risk of such diseases. The preferred mode of administration is oral, but other modes of administration include transdermal, mucosal, sublingual, intramuscular, intravenous, intraperitoneal and subcutaneous administration as well as topical application.

By "abnormal calcium and phosphate metabolism" as used herein is meant (l) conditions characterized by abnormal calcium and phosphate metabolism leading to general or specific bone loss or excessively high calcium and phosphate levels in body fluids, and (2) ) conditions that cause or result in abnormal calcium and phosphate deposition in the body. The first category includes Paget's disease, hyperparathyroidism, hypercalcemia of malignancy and osteolytic bone metastases. The second category includes myositis ossificans progressiva, calcinosis universalis and such disorders as arthritis, neuritis, bursitis, tendonitis, and other inflammatory conditions that predispose involved tissue to the deposition of calcium phosphoates.

By "person at risk" or "person in need of such treatment" as used herein is meant any human or lower animal exposed to a significant risk of abnormal calcium and phosphate metabolism without treatment, and any any human or lower animal diagnosed to suffer from abnormal calcium and phosphate metabolism. Examples include women after menopause, people undergoing certain steroid therapy, people taking certain anticonvulsants, people diagnosed as having Paget's disease, hyperparathyroidism, hypercalcemia in malignancy or osteolytic bone metastases, people diagnosed with suffering DK 174685 B1 25 of one or more of the various forms of osteoporosis, persons belonging to a population group known to have a significantly higher probability of developing osteoporosis than the average, e.g. women after menopause, men over the age of 65 and people treated with medications known to cause osteoporosis as a side effect, people diagnosed with myositis ossificans progressiva or calcinosis universalis, and people 10 suffering from arthritis , neuritis, bursitis, tendonitis, and other inflammatory conditions that predispose involved tissues to the deposition of calcium phosphate.

By "human or lower animals" suffering from or at risk of osteoporosis "as used herein is meant a subject diagnosed as suffering from one or more of the various forms of osteoporosis, or a subject belonging to a group known to be significantly more likely to develop 20 osteoporosis than the average, such as postmenopausal women, men over 65, and people being treated with medications known to cause osteoporosis as a side effect (such as adrenocorticoids).

By "harmless but effective amount" as used herein is meant within the framework of sound medical judgment a quantity of a compound or composition that is large enough to significantly and positively change the condition to be treated but small enough to that serious 30 side effects are avoided (by a reasonable benefit-risk ratio). The harmless but effective amount of cyclic diphosphonic acids will vary with the particular condition being treated, the age and physical condition of the treated patient, the severity of the condition, the duration of treatment, the nature of concomitant treatment and the specific diphosphonate used. However, single doses may vary from DK 174685 B1 26 approx. 0.1 mg P to approx. 3500 mg P or from approx. o, 01 to approx.

500 mg P / kg body weight. Preferred single doses are from approx. 5 mg p to approx. 600 mg p or from approx. 0.5 to approx. 50 mg P / kg body weight. Up to approx. 4 5 single doses per day. Daily doses of more than approx. 20000 mg P / kg is not required to produce the desired effect and may produce undesirable side effects. Of course, the higher doses in this field are required in the case of oral administration for limited absorption.

Example VIi

Patients weighing approx. 70 kg, and clinically diagnosed to suffer from hypercalcemia by malignancy, 350 mg of P in the form of diphosphonic acid or its pharmaceutically acceptable salt or ester is administered orally twice daily for 3 months. This treatment results in a significant improvement in the malignant hypercalcemia.

Similar results are obtained using other diphosphonic acids of the present invention or their pharmaceutically acceptable salts or esters, e.g. 2-25 methylcyclobutane-1,1-diphosphonic acid, 3-chlorocyclopentane-1,1-diphosphonic acid or cyclohexane-1,1-diphosphonic acid.

By "pharmaceutical carrier" as used in the characterization of the pharmaceutical compositions 30 of the invention is meant one or more compatible, solid or liquid fillers, diluents or encapsulants. By "compatible" as used herein is meant that the components of the composition can be blended without interfering with each other in a manner that will substantially reduce the pharmaceutical utility of the overall composition under usual conditions of use.

DK 174685 B1 27

Some examples of substances which may serve as pharmaceutical carriers are sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch, cellulose and its derivatives such as sodium carboxymethyl cellulose, methyl cellulose, ethyl cellulose, cellulose acetate; pulvertragacanth; malt; gelatin, talc; stearic acid; magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and theobroma oil; polyols such as propylene glycol, glycerine, sorbitol, mannitol and polyethylene glycol; agar, alginic acid, pyrogen-free water; isotonic saline; and phosphate buffer solutions as well as other non-toxic compatible substances used in pharmaceutical formulations. Wetting agents and lubricants such as sodium lauryl sulfate, as well as colorants, flavors and preservatives may also be present. Other compatible additives and active substances may be included in the pharmaceutical compositions of the present invention.

The choice of pharmaceutical carrier for use with the diphosphonic acids of the present invention is first and foremost determined by the way in which the diphosphonic acids are to be administered. If the compound is to be injected, the preferred pharmaceutical carrier is sterile, physiological saline whose pH has been adjusted to ca. 7.4. However, the preferred way of administering the diphosphonates of the present invention is oral administration, and the preferred unit dosage form is therefore tablets, capsules and the like which comprise from ca. 15 mg P to approx. 600 mg P of a diphosphonic acid compound according to the invention.

Pharmaceutical carriers suitable for preparing unit dosage forms intended for oral administration are well known in the art. Their selection will depend on secondary considerations, such as taste, price, and shelf life, which are not critical to the objects of the present invention and which can easily be made by one of ordinary skill in the art. The pharmaceutical carrier used in conjunction with the diphosphonates of the present invention is used at a concentration sufficient to provide a practical size in relation to dose.

Preferably, the pharmaceutical carrier comprises from about 0.1 to approx. 99.9% by weight of the total composition.

Example wine 15 Capsules are prepared by usual procedures following the following prescription: ingredient mg per capsule

Indan-2,2-DP * 350.00 (some P) 20 Starch 55.60

Sodium lauryl sulfate 2.90 * Not according to the invention 25 Orally administered twice daily for 6 months, the above capsules reduce bone resorption in a patient who weighed approx. 70 kg and as a result of osteoporosis, substantially. Similar results are obtained when indan-2,2-DP in the capsules described above is replaced by 30 compounds of the invention, namely 2-methylcyclobutane-1,1-DP, 3-chlorocyclopentane-1, 1-diphosphonic acid and cyclohexane-1.1 -DP or a pharmaceutically acceptable salt of these diphosphonic acid compounds.

Claims (3)

1. Monocyclic geminal diphosphonic acids, characterized in that they are selected from cyclobutane-1, 5-diphosphonic acids with one or more substituents selected from methyl, methylamino, amino, chloro, hydroxy and methoxy; 3-chlorocyclopentane-1,1-diphosphonic acid, cyclohexane-1,1-diphosphonic acid, cyclohex-2-ene-1,1-diphosphonic acid, cyclohex-3-ene-1, 1-diphosphonic acid and cycloheptane-1,1-diphosphonic acid and pharmaceutically acceptable salts thereof. 2. 2-Methylcyclobutane-1,1-diphosphonic acid and pharmaceutically acceptable salts thereof. 15 A pharmaceutical composition comprising: (a) from 15 mg P to 600 mg p of a diphosphonic acid compound according to claim 1 or 2, and 20 (b) a pharmaceutical carrier. 25