AU7976300A - Novel trisubstituted pyridine compounds - Google Patents

Description

WO 01/25204 PCT/SEOO/01859 NOVEL TRISUBSTITUTED PYRIDINE COMPOUNDS 5 Technical field In general, the present invention relates to a selective inhibitor of mammalian osteoclast cell activity, processes for its preparation and pharmaceutical compositions comprising the same as well as methods of treatment, where said selective inhibitor is administered to a lo human or animal patient. More specifically, the present invention relates to a selective inhibitor of vacuolar H-ATPase in mammalian osteoclast cells. Background art is Diseases associated with loss of bone mass, i.e. conditions involving bone resorption, are known to be caused by over activity of osteoclast cells. It is known that certain compounds, usually structurally related to bafilomycin, are inhibitors of vacuolar H ATPase in osteoclast cells, thereby being potentially useful for treatment of said diseases, see e.g. WO 91/06296. WO 98/01443, WO 98/01423, WO 98/01436, WO 9S/01445 and 20 WO 96/21644. Furthermore, certain quinolines and benzimidazoles are also claimed to be inhibitors of vacuolar H~-ATPase, see e.g. WO 97/14681 and WO 97/102219. Recent evidence suggests that isoforms of the 116 kDa ("a") subunit of vacuolar H--ATPase exist. At present, it appears that there are three subtypes of "a" in vertebrate species, and they are denoted "a 1", "a2" and "a3", respectively. Of these, "a l" has been cloned from rat and 25 bovine brain and may represent the ubiquitous intracellular isoform of "a" (see Perin, M. S., Fried, V. A., Stone. D. K., Xie, X. S. and Sudhof, T. C., Journal of Biological Chemistry, 266(6). 3877-81 (1991) and Peng, S. B., Crider, B. P., Xie, X. S. and Stone, D. K., Journal of Biological Chemistry, 269(25), 17262-6 (1994)). Solely on the basis of its sequence homology, the subtype "a2" (TJ6 mouse immunosuppressor factor). is thought to 30 be an isoform of"a" (Lee, C., Ghoshal, K. and Beaman, K. D., Molecular Immunology, 27(11), 1137-44 (1990)). The third subtype, i.e. "a3" (EMBL accession number U45285). has been cloned from a human osteoclastoma cDNA library and suggested to be an WO 01/25204 PCT/SEOO/01859 2 osteoclast-specific isoform of "a" (Li, Y. P., Chen, W. and Stashenko, P., Biochemical & Biophysical Research Communications, 218(3). 813-21 (1996)). In summary, none of the compounds disclosed in the prior art above provides sufficient s therapeutic efficiency in the treatment of disorders related to bone resorption. Thus, there is a demand in the art for new therapeutic agents against such disorders. Disclosure of the invention 10 Surprisingly, novel compounds with excellent therapeutical effect against physiological disorders involving inter alia bone resorption have now been found. More specifically, said compounds comprise a selective inhibitor of mammalian osteoclast cell activity, wherein said inhibitor comprises a trisubstituted pyridine compound. Since the selective inhibitor of the present invention has been found to inhibit vacuolar H*-ATPase, such as vacuolar H is ATPase in osteoclast cells, it is thereby therapeutically efficient against physiological disorders involving bone resorption. According to the present invention, the trisubstituted pyridine compounds have the general formula I: R2 20 Ri R3 (I) R, Rs R4 R 25 N R R R 6 R1 7 wherein R 1 is selected from the group consisting of 30 (a) H; (b) O-alkyl having 1-3 carbon atoms; (c) R l+ R- form a five membered ring containing at least one 0, S and/or N: WO 01/25204 PCT/SE0O/01859 3 (d) alkyl having 1-3 carbon atoms and (e) S-methyl or S- ethyl: R-2 is selected from the group consisting of s (a) H; (b) straight chain. branched or cyclic saturated or unsaturated alkyl having 1-6 carbon atoms: (c) R 1 2R 13 , wherein R 1 2 and R 13 are either independently selected from the group consisting of H: a straight chain, branched or cyclic saturated or unsaturated alkyl 1o having 1-6 carbon atoms, optionally substituted with O-alkyl, NH-alkyl, N-dialkyl, N pyrrolidinyl, N-piperidinyl, N-morpholinyl or N-piperazinyl; and aryl selected from the group consisting of phenyl, imidazolyl, pyridinyl or pyrrolyl; or together form a five or six membered saturated or unsaturated ring, optionally containing 0, S, and/or N; is (d) straight chain, branched or cyclic saturated or unsaturated O-alkyl having 1-6 carbon atoms optionally substituted with O-alkyl, NH-alkyl, N-dialkyl, N-pyrrolidinyl, N piperidinyl, N-morpholinyl or N-piperazinyl; (e) straight chain, branched or cyclic saturated or unsaturated S-alkyl having 1-6 carbon atoms optionally substituted with O-alkyl , NH-alkyl, N-dialkyl, N-pyrrolidinyl, N 20 piperidinyl:

R

3 -RI 1 are selected from the group consisting of (a) H (b) alkyl having 1-3 carbon atoms 25 (c ) 0-alkyl having 1-3 carbon atoms (d) S-methyl or S- ethyl wherein at least one of RI-R 3 , R 4

-R

7 and R 8 -R 1 1, respectively, is not H or a pharmaceutical acceptable salt thereof.

WO 01/25204 PCT/SE00/01859 4 In another preferred embodiment, R2 is S-Me, NR2R 1 3 , 0-alkyl or alkyl having 1-6 carbon atoms; R , R-. R 4 and R 8 are H; and R 5 , R 6 , R 7 , R 9 . R 10 and R I I are straight chain, branched or cyclic saturated or unsaturated O-alkvl having 1-3 carbon atoms. 5 In yet another preferred embodiment, R2 is S-Me, NR 1 2R 13 , 0-alkyl or alkyl having 1-6 carbon atoms; R 1 , R3. R 4 . R 5 , R8 and R 9 are H; and R 6 . R7, R 10 and R I I are straight chain, branched or cyclic saturated or unsaturated 0-alkyl having 1-3 carbon atoms. In still another preferred embodiment, R-2 is S-Me, NR 1 2R 13 , 0-alkyl or alkyl having 1-6 1o carbon atoms; RI, R 3 . R , R 6 , R 7 , R 9 ., R 10 , R 1 I are H: and R 4 and R 8 are straight chain, branched or cyclic saturated or unsaturated 0-alkyl having 1-3 carbon atoms. In a particularly preferred embodiment of the present invention, said trisubstituted pyridine compound is selected from the group consisting of: is 4-(4-pyrrolidin- I -vl-phenyl)-2,6-bis-(3,4,5-trimethoxy-phenyl)-pyridine; {4-[2,6-bis-(3,4,5-trimethoxy-phenyl)-pyridin-4-y]-phenyl } -dimethyl-amine; {4-[2,6-bis-(3,4-dimethoxy-phenyl)-pyridin-4-yl]-phenoxy}-propyl)-dimethyl amine; and 4-(4-ethyl-phenyll-2.6-bis-(3 ,4,5-trimethoxy-phenyl)-pyridine. 20 The present invention also relates to a process for the preparation of a trisubstituted pyridine compound according to any one of the embodiments set forth above. In one embodiment of said preparation, a suitably substituted benzaldehyde is heated with 25 ammonium acetate and a suitably substituted acetophenone, optionally in the presence of a solvent, during 2-24 h. In another embodiment, a suitably substituted chalcone is heated with ammonium acetate and a suitably substituted acetophenone, optionally in the presence of a solvent, during 30 2-24 h.

WO 01/25204 PCT/SEOO/01859 5 In yet another embodiment. a suitably substituted aromatic aldehyde or chalcone is reacted with a suitably substituted N-(diphenylphosphinyl)-1-phenylethaneimine in the presence of a base, preferably t-BuOK, in an aprotic solvent. s In the preparation of trisubstituted pyridine compounds according to the present invention, the following references provide useful guidance concerning e.g. suitable reaction conditions and proper selection of reagents: Weiss, J. Am. Chem. Soc., 74, 200 (1952); Tewari, R.S., Dubey, A.K., Indian J. Chem. Sect. B, 19(2), 153-154 (1980); oa Kobayashi, T., Kakiuchi, H., Kato, H., Bull. Chem. Soc. Jpn., 64(2), 392-395 (1991 Kobayashi, T., Kawate, H., Kakiuchi, H., Kato, H., Bull. Chem. Soc. Jpn., 63(7), 1937-1942 (1990); Tewari, R.S., Awasthi. A.K., Synthesis, 4, 314-315 (1981). is Scheme 1 below illustrates general synthetic pathways for the preparation of the trisubstituted pyridine compounds of the present invention. By guidance of inter alia known reference literature (vide supra), the synthesis of suitable starting materials is readily accomplished by a person skilled in the art.

WO 01/25204 PCTSEOO/01859 6 P:- Ph

R

4 0 R 4 Ph

R

6

R

6

NH

4 OAc t--BuOK HOAc <Rbenzene II 20 R9 SNR R5 Rio R6 0 R11RR7 25 400 45 hRS 0R S Rem N. S thsso mPoud h th geerlfomla WO 01/25204 PCT/SEOO/01859 7 General procedures for the synthesis of compounds represented by the formula I are outlined below. 5 Procedure la: A suitably substituted benzaldehyde and a suitably substituted acetophenone (molar ratio 2:1) are heated with ammonium acetate in acetic acid for 2-24 h. The desired compound may be isolated by crystallisation or column chromatogtraphy on silica gel. 10 Procedure ib: A suitably substituted chalcone, prepared by standard methods, and a suitably substituted acetophenone derivative (molar ratio 1:1), such as e.g. phenacyl-pyridinium bromide, phenacyl-dimethylsulfonium bromide or dimethyl-(2-oxo-2-phenyl-ethyl)-sulfonium betaine), are heated with ammonium acetate in acetic acid for 2-24 h. The desired is compound may be isolated by crystallisation or column chromatography on silica gel. Procedure 2: A suitably substituted N-(diphenylphosphinvl)-2-phenyl-1-azaallyl anion, generated from N-(diphenylphosphinyl)- I -phenylethaneimine under basic conditions (t-BuOK), is reacted 20 with an aromatic aldehyde (molar ratio 2:1) or a chalcone (molar ratio 1:1). The desired compound may be isolated by crystallisation or column chromatogtraphy on silica gel. Furthermore, the present invention relates to a trisubstituted pyridine compound with the general formula I for use as a pharmaceutical. 25 Thus, the present invention also relates to a pharmaceutical composition comprising a trisubstituted pyridine compound according to any one of the embodiments set forth above as active ingredient in association with a pharmaceutically acceptable adjuvant, diluent or carrier. 30 In said pharmaceutical composition, the amount of said active ingredient per dosage unit is generally within the range of about I to 1 000 mg, preferably 1-300 mg.

WO 01/25204 PCT/SEOO/01859 8 Moreover, the amount of said active ingredient is typically within the range of about 0.1 to 95% by weight of said pharmaceutical composition. s Additionally, the present invention is also related to the use of a trisubstituted pyridine compound according to any one of the previously outlined embodiments in the manufacture of a medicament for use in therapeutic or prophylactic treatment of a human or animal body. 10 In a preferred embodiment, said use is related to a medicament intended for treatment involving inhibition of vacuolar H+-ATPase, preferably vacuolar H+-ATPase in osteoclast cells. In another preferred embodiment of said use, the medicament is intended for treatment 15 involving inhibition of vacuolar H+-ATPase containing the isoform a3, said vacuolar H+ ATPase preferably being present in osteoclast cells. In yet another preferred embodiment, said medicament is intended for treatment involving inhibition of bone resorption, or is intended for treatment and/or prevention of diseases 20 related to increased bone resorption, preferably osteoporosis. In still another preferred embodiment of said use, the medicament is intended for treatment of Paget's disease of bone, hyperparathyroidism, malignant neoplasms, parodontal diseases, prosthetic and/or implant related bone loss, tumours, AIDS and disorders related 25 thereto, Alzheimer's disease, angiogenesis, atherosclerosis, rheumatoid arthritis, diabetic retinopathy, psoriasis or diabetes. The present invention is also related to a method for inhibiting vacuolar H~-ATPase, preferably vacuolar H -ATPase in osteoclast cells, or to a method for inhibiting vacuolar 30 H-ATPase containing the isoform a3, said vacuolar H+-ATPase preferably being present in osteoclast cells, wherein any one of said methods comprises administering to a human or WO 01/25204 PCT/SEOO/01859 9 animal patient a therapeutically effective amount of a trisubstituted pyridine compound according to any one of the embodiments outlined above. Moreover, the present invention is related to a method for inhibiting bone resorption, or to s a method for treatment and/or prevention of diseases related to increased bone resorption, preferably osteoporosis, wherein any one of said methods comprises administering to a human or animal patient a therapeutically effective amount of a trisubstituted pyridine compound according to any one of the embodiments set forth above. 10 Furthermore, the present invention concerns a method for treatment of Pa2et's disease of bone, hyperparathyroidism, malignant neoplasms, parodontal diseases, prosthetic and/or implant related bone loss, tumours, AIDS and disorders related thereto, Alzheimer's disease, angiogenesis, atherosclerosis, rheumatoid arthritis, diabetic retinopathy, psoriasis or diabetes, wherein said method comprises administering to a human or animal patient a 15 therapeutically effective amount of a trisubstituted pyridine compound according to any one of the previously outlined embodiments. The typical daily dose of the active ingredient varies within a wide range and will depend on various factors such as e.g. the individual requirement of each patient. the route of 2o administration and the disease. However, oral and parenteral doses will usually be in the range of 1 to 1000 mg, preferably 1-300 mg, per day of the active ingredient. In another aspect, the present invention relates to pharmaceutical compositions containing at least one compound according to the invention, or a therapeutically acceptable salt 25 thereof, as active ingredient. In cases where the active ingredient contains a basic nitrogen, the pharmaceutically acceptable salts include acid addition salts. Acids that form therapeutically acceptable salts are e.g. hydrohalogen acids, such as hydrochloric acid, sulphuric acid, phosphoric acid, nitric acid, aliphatic, alicyclic, aromatic or heterocyclic carboxyl or sufphonic acids, such as formic acid, acetic acid, propionic acid. succinic acid. 30 glycolic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, maleic acid, hydroxymaleic acid, pyruvic acid, p-hydroxybensoic acid, embonic acid, WO 01/25204 PCT/SEOO/01859 10 methanesulphonic acid, ethanesulphonic acid, hydroxyethanesulphonic acid. halogenbensensulphonic acid, toluenesulphonic acid and naphtalenesulphonic acid. As outlined above, there is provided a trisubstituted pyridine compound for use as a 5 pharmaceutical. Thus, it may be used in pharmaceutical compositions for oral, intravenous, topical, intraperitoneal or subcutaneous administration, in association with one or more pharmaceutically acceptable carriers, diluents or adjuvants that are well known in the art. The pharmaceutical composition of the invention may be administered topically, in the l0 form of solutions, suspensions or systemically, e.g. by oral administration in the form of tablets, capsules, syrups, powders or granules or by parenteral administration in the form of solutions or suspensions, or by subcutaneous administration or by rectal administration in the form of suppositories or transdermally. 15 Experimental part Preparation of compounds illustrating the invention. Example 1. Synthesis of 4-(4-Pyrrolidin-1-yl-phenyl)-2,6-bis-(3,4,5-trimethoxy-phenyl)-pyridine 60 mg (0.34 mmol) 4-N-pyrrolidinyl-benzaldehyde, 144 mg (0.69 mmol) 3.4,5-trimethoxy 20 acetophenone and 58 mg (0.75 mmol) ammonium acetate were stirred at 80 C for 4 h. The crude product was filtered and recrystallized from warm ethanol/CH 2 Cl 2 . 42 mg of the title compound was obtained. 'H NMR (400 MHz, CDCl 3 ) 5-values in ppm: 2.07 (m, 4H); 3.38 (t, 4H); 3.93 (s, 6H); 3.99 (s, 12H); 6.70 (d, 2H); 7.43 (s, 4H); 7.68 (d, 2H); 7.77 (s, 2H). 25 Example 2. Synthesis of {4-[2,6-Bis-( 3,4,5-trimethoxy-phenyl)-pyridin-4-yl]-phenyl}-dimethyl-amine was performed according to example 1. The crude compound was purified by column chromatography on silica gel. 'H NMR (600 MHz, CDC13) 5-values in ppm: 3.06 (s, 6H); 3.93 (s, 6H); 3.99 (s, 12H): 6.86 (d, 2H); 7.43 (s, 4H); 7.68 (d, 2H); 7.77 (s, 2H). 30 WO 01/25204 PCT/SE00/01859 11l Example 3. Synthesis of {4-[2,6-Bis-(3,4-dimethoxy-phenvl)-pyridin-4-yl]-phenoxy}-propyl) dimethyl-amine was performed according to example 1. The crude compound was purified by column 5 chromatography on silica gel. 'H NMR (500 MHz, CDCI) 5-values in ppm: 2.17 (dt, 2H); 2.52(s, 6H); 2.83 (t, 2H). 3.96 (s, 6H); 4.03 (s, 6H); 4.12 (t. 2H); 7.00 (d. 2H); 7.04 (d, 2H); 7.69 (d, 2H); 7.70 (dd. 2H); 7.75( s, 2H); 7.86 (d, 2H). Example 4. 10 Synthesis of 4-(4-Ethvl-phenyl)-2,6-bis-(3,4,5 -trimethoxy-phenyl)-pyridine was performed according to example 1. The crude compound was purified by column chromatography on silica gel. 'H NMR (400 MHz, CDCl,) a-values in ppm: 1.28 (t, 3H); 2.73 (q, 2H); 3.90 (s, 6H); 3.96 (s, 12H); 7.36 (d, 2H); 7.40 (s, 4H); 7.65 (d, 2H); 7.76 (s, 2H). I5 BIOLOGICAL TESTS In vitro experiments Preparation of membrane vesicles containing vacuolar H+-ATPase Chicken medullary bone 20 Membrane vesicles were prepared from egg-laying hens after 14 days of calcium-deprived diet, as previously described (Mattsson, J.P., Lorentzon, P., Wallmark, B., and Keeling, D.J., Biochim. Biophys. Acta, 1146(1), 106-12 (1993)), with some modifications. Briefly, the medullary bone scraped from the long bones was resuspended in isolation buffer (1 25 ml/g medullary bone) containing 5 mM Hepes/Tris, pH 7.4, 250 mM sucrose and 1 mM EGTA, minced using a pair of scissors, diluted 1:10 (w/v) in isolation buffer and homogenised in a polytron homogeniser. The homogenate was filtered through a 250 pm nylon mesh. Membrane vesicles were then obtained by differential centrifugation (2000 x g for 10 min, 10,000 x g for 20 min and 40,000 x g for I h). The final pellet was 30 resuspended in isolation buffer (0.4 ml/g medullary bone) by 20 passes using a teflon/glass homogenizer, snap frozen in MeOH/dry ice and then stored at -70 0

C.

WO 01/25204 PCT/SEOO/01859 12 Bovine brain Fresh bovine brains were obtained from a local slaughter house. Membrane vesicles were prepared from whole brain exactly as described for the medullary bone membrane vesicles. 5 Human osteoclastoma Human osteoclastoma tumours were obtained courtesy of Dr. Bjarm Gunterberg (Sahlgrenska hospital. Sweden). A portion of each tumour was snap frozen in liquid nitrogen and stored at -70'C. Membrane vesicles were prepared exactly as described for the medullary bone membrane vesicles, except that protease inhibitors (0.2 mM AEBSF, io 15.4 pOM aprotinin, 3.6 iM bestatin, 8.8 ptM leupeptin) were included in the isolation buffer. Measurement of ATP-dependent proton transport Proton transport in membrane vesicles was measured in a 96-well plate reader using the is weak base acridine orange (Mattsson, J.P., Lorentzon, P., Wallmark, B., and Keeling, D.J., Biochim. Biophys. Acta, 1146(1), 106-12 (1993)). Test substances (dissolved in DMSO) or DMSO (control) were added to the wells of a 96-well plate, followed by the addition of 220 jil acridine-orange buffer (final concentrations: 5 mM Hepes/Tris, pH 7.4. 125 mM KCI, 3 mM MgS0 4 , 0.25 mM DTT, I ptM valinomycin and 5 p.tM acridine orange) and 20 membrane vesicles (10-50 pg protein). After 10 min incubation with mixing, reactions were started by the addition of Tris-ATP (pH 7.4) to a final concentration of 3 mM, and the proton transport was monitored by measurement of acridine orange absorbance (A 4 9 0 ) quenching in a Molecular Devices plate reader for 2 minutes. The initial rate of proton transport, taken as the maximum rate decrease in acridine orange absorbance, was 25 calculated using Molecular devices Softmax software. IC 5 0 values were obtained from dose-response curves constructed using the 4-parameter logistic equation. Measurement of bone resorption by 45Ca release from neonatal mouse calvarial (skull) bones 30 Measurement of bone resorption by 4 5Ca release from mouse calvaria was performed as described (Mattsson, J.P., Vudnanen, K., Wallmark, B., and Lorentzon, P., Biochim. Biophys. Acta, 1065(2), 26 1-8 (1991)), albeit with some modifications. Pregnant mice WO 01/25204 PCT/SEOO/01859 13 were injected with a 4 -Ca -solution (0.25 ml 120 pCi/ml, s.c.). day 2 and I before partus. When the new-born mice were 5 to 8 days old, they were killed by decapitation and the calvaria were dissected out and cut into four equally sized pieces. The pieces were placed in petri dishes containing incubation medium (I mM L-glutamine, 100 U/ml penicillin, 100 5 Ig/ml streptomycin, I mg/ml albumin and I FTM indomethacin) with or without 10 nM PTH. and preincubated in a CO: incubator (5% CO 2 in air) for 20-24 h at 37 0 C. The calvaria pieces were then transferred to a 24-well plate containing fresh incubation medium. After 24 h, an aliquot (400 pl) of the incubation medium was analysed for the content of 4 5Ca in a Microbeta (Wallac) scintillation counter (Control CPM). The calvaria 10 pieces were transferred to fresh incubation medium and incubated with or without test substance and after another 24 h incubation, an aliquot (400 pl) of the incubation medium was analysed for the content of 4 "Ca (Compound CPM). The resorption ratio between the control period and the compound period (compound CPM/control CPM) was calculated and dose-response curves constructed using the 4-parameter logistic equation. 15 Protein determination Protein was determined according to Bradford (Bradford, M.M., Anal. Biochem., 72, 248-54 (1976)) using Bio Rad's protein assay kit and y-globulin as a standard.

Claims (23)

1. A trisubstituted pyridine compound comprising a structure with the general formula I: R2 5 R1 R3 () R N 4 R 10 Rio R6 R1 7 wherein Rl is selected from the group consisting of (a) H; 15 (b) O-alkyl having 1-3 carbon atoms; (c) R 1 + R2 form a five membered rung containing at least one 0. S and/or N; (d) alkyl having 1-3 carbon atoms and (e) S-methyl or S- ethyl. 20 R 2 is selected from the group consisting of (a) H; (b) straight chain, branched or cyclic saturated or unsaturated alkyl having 1-6 carbon atoms; (c) R 12 R 1 3 , wherein R 1 2 and R 13 are either independently selected from the group 25 consisting of H; a straight chain, branched or cyclic saturated or unsaturated alkyl having 1-6 carbon atoms, optionally substituted with O-alkyl, NH-alkyl, N-dialkyl, N pyrrolidinyl, N-piperidinyl, N-morpholinyl or N-piperazinyl; and aryl selected from the group consisting of phenyl, imidazolyl, pyridinyl or pyrrolyl; or together form a five or six membered saturated or unsaturated ring, optionally containing 0, S, and/or 30 N: WO 01/25204 PCT/SE00/01859 15 (d) straight chain, branched or cyclic saturated or unsaturated 0-alkyl having 1-6 carbon atoms optionally substituted with 0-alkyl, NH-alkyl, N-dialkyl, N-pyrrolidinyl, N piperidinyl, N-morpholinyl or N-piperazinyl; (e) straight chain, branched or cyclic saturated or unsaturated S-alkyl having 1-6 carbon 5 atoms optionally substituted with 0-alkyl , NH-alkyl, N-dialkyl, N-pyrrolidinyl, N piperidinyl; R 3 -RI I are selected from the group consisting of (a) H 10 (b) alkyl having 1-3 carbon atoms (c ) 0-alkyl having 1-3 carbon atoms (d) S-methyl or S- ethyl wherein at least one of R I-R 3 , R 4 -R 7 and R 8 -R 1 1, respectively, is not H or a pharmaceutically acceptable salt thereof I5

2. A trisubstituted pyridine compound according to claim 1, wherein R2 is S-Ne, NR 1 2R 13 , 0-alkyl or alkyl having 1-6 carbon atoms; R I, R 3 , R4 and R8 are H; and R 5 , R 6 , R 7 , R 9 , R 10 and RI I are straight chain, branched or cyclic saturated or 20 unsaturated 0-alkyl having 1-3 carbon atoms.

3. A trisubstituted pyridine compound according to any one of claims 1-2, wherein R2 is S-Me, NR 1 2R 13 , 0-alkyl or alkyl having 1-6 carbon atoms; R 1 , R 3 , R4, R 5 , R 8 and R 9 are H; and 25 R 6 , R 7 , R 10 and RI I are straight chain, branched or cyclic saturated or unsaturated 0 alkyl having 1-3 carbon atoms.

4. A trisubstituted pyridine compound according to any one of claims 1-3, wherein R 2 is S-Me, NR 12 R 1 3 , 0-alkyl or alkyl having 1-6 carbon atoms; 30 R 1 , R 3 , R 5 , R 6 , R 7 , R 9 , R 10 , RI I are H; and WO 01/25204 PCT/SEOO/01859 16 R 4 and R 8 are straight chain, branched or cyclic saturated or unsaturated O-alkyl having 1-3 carbon atoms.

5. A trisubstituted pyridine compound according to any one of the preceding claims, wherein said compound is selected from the group consisting of: 4-(4-pyrrolidin- 1 -vl-phenyl )-2.6-bis-(3 ,4,5-trimethoxy-phenyl)-pyridine; {4-[2,6-bis-(3,4.5-trimethoxy-phenyl)-pyridin-4-yl]-phenyI -dimethyl-amine; {4 [2,6-bis-(3,4-dimethoxy-phenyl)-pyridin-4-yl]-phenoxy} -propyl)-dimethyl-amine; 4-(4-ethyl-phenyl)-2,6-bis-( 3,4,5-trimethoxy-phenyl)-pyridine. 10

6. A process for the preparation of a trisubstituted pyridine compound according to any one of claims 1-5, wherein a suitably substituted benzaldehvde is heated with ammonium acetate and a suitably substituted acetophenone, optionally in the presence of a solvent, during 2-24 h. 15

7. A process for the preparation of a trisubstituted pyridine compound according to any one of claims 1-5, wherein a suitably substituted chalcone is heated with ammonium acetate and a suitably substituted acetophenone, optionally in the presence of a solvent, during 2-24 h. 20

8. A process for the preparation of a trisubstituted pyridine compound according to any one of claims 1-5, wherein a suitably substituted aromatic aldehyde or chalcone is reacted with a suitably substituted N-(diphenylphosphinyl)-l-phenylethaneimine in the presence of a base, preferably t-BuOK, in an aprotic solvent. 25

9. A trisubstituted pyridine compound according to any one of claims 1-5 for use as a pharmaceutical.

10. A pharmaceutical composition comprising a trisubstituted pyridine compound 30 according to any one of claims 1-5 as active ingredient in association with a pharmaceutically acceptable adjuvant, dilutent or carrier. WO 01/25204 PCT/SEOO/01859 17

11. A pharmaceutical composition according to claim 10. wherein the amount of said active ingredient per dosage unit is within the range of about I to 1 000 mg, preferably 1 300 mg. s

12. A pharmaceutical composition according to any one of claims 10- 11, wherein the amount of said active ingredient is within the range of about 0.1 to 95% by weight of said pharmaceutical composition.

13. Use of a trisubstituted pyridine compound according to any one of claims 1-5 in the I manufacture of a medicament for use in therapeutic or prophylactic treatment of a human or animal body.

14. Use according to claim 13, wherein the medicament is intended for treatment involving inhibition of vacuolar H+-ATPase, preferably vacuolar H+-ATPase in osteoclast 15 cells.

15. Use according to claim 13, wherein the medicament is intended for treatment involving inhibition of vacuolar H+-ATPase containing the isoform a3, said vacuolar H+ ATPase preferably being present in osteoclast cells. 20

16. Use according to claim 13, wherein the medicament is intended for treatment involving inhibition of bone resorption.

17. Use according to claim 13, wherein the medicament is intended for treatment and/or 25 prevention of diseases related to increased bone resorption, preferably osteoporosis.

18. Use according to claim 13, wherein the medicament is intended for treatment of Paget's disease of bone, hyperparathyroidism, malignant neoplasms, parodontal diseases, prosthetic and/or implant related bone loss, tumours, AIDS and disorders related thereto, 30 Alzheimer's disease, angiogenesis, rheumatoid arthritis, diabetic retinopathy, psoriasis or diabetes. WO 01/25204 PCT/SEOO/01859 18

19. A method for inhibiting vacuolar H'-ATPase, preferably vacuolar H~-ATPase in osteoclast cells, wherein said method comprises administering to a human or animal patient a therapeutically effective amount of a trisubstituted pyridine compound according to any one of claims 1-5.

20. A method for inhibiting vacuolar H--ATPase containing the isoform a3, said vacuolar H+-ATPase preferably being present in osteoclast cells, wherein said method comprises administering to a human or animal patient a therapeutically effective amount of a trisubstituted pyridine compound according to any one of claims 1-5. 10

21. A method for inhibiting bone resorption which comprises administering to a human or animal patient a therapeutically effective amount of a trisubstituted pyridine compound according to any one of claims 1-5. is

22. A method for treatment and/or prevention of diseases related to increased bone resorption, preferably osteoporosis, which comprises administering to a human or animal patient a therapeutically effective amount of a trisubstituted pyridine compound according to any one of claims 1-5. 20

23. A method for treatment of Paget's disease of bone, hyperparathyroidism, malignant neoplasms, parodontal diseases, prosthetic and/or implant related bone loss, tumours, AIDS and disorders related thereto, Alzheimer's disease, angiogenesis, rheumatoid arthritis, diabetic retinopathy, psoriasis or diabetes, wherein said method comprises administering to a human or animal patient a therapeutically effective amount of a 25 trisubstituted pyridine compound according to any one of claims 1-5