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WO1998017267A9 - Compositions et methodes pour traiter les etats associes a un deficit osseux - Google Patents

Compositions et methodes pour traiter les etats associes a un deficit osseux

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Publication number
WO1998017267A9
WO1998017267A9 PCT/US1997/018864 US9718864W WO9817267A9 WO 1998017267 A9 WO1998017267 A9 WO 1998017267A9 US 9718864 W US9718864 W US 9718864W WO 9817267 A9 WO9817267 A9 WO 9817267A9
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WO
WIPO (PCT)
Prior art keywords
alkyl
nrcr
independently
compound
bone
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PCT/US1997/018864
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English (en)
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WO1998017267A1 (fr
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Publication date
Application filed filed Critical
Priority to JP51952998A priority Critical patent/JP2001510450A/ja
Priority to EP97912787A priority patent/EP0973513A4/fr
Priority to AU49889/97A priority patent/AU4988997A/en
Priority to US09/297,188 priority patent/US6649631B1/en
Publication of WO1998017267A1 publication Critical patent/WO1998017267A1/fr
Publication of WO1998017267A9 publication Critical patent/WO1998017267A9/fr

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  • the invention relates to compositions and methods for use in limiting undesired bone loss in a vertebrate at risk of such bone loss, in treating conditions that are characterized by undesired bone loss or by the need for bone growth, in treating fractures, and in treating cartilage disorders. More specifically, the invention concerns the use of specific classes of compounds identified or characterized by a high throughput screening assay.
  • Bone is not a static tissue. It is subject to constant breakdown and resynthesis in a complex process mediated by osteoblasts, which produce new bone, and osteoclasts, which destroy bone. The activities of these cells are regulated by a large number of cytokines and growth factors, many of which have now been identified and cloned. Mundy has described the current knowledge related to these factors (Mundy, G.R. Clin Orthop 324:24-28, 1996; Mundy, G.R. J Bone Miner Res 8:S505-10, 1993). Although there is a great deal of information available on the factors which influence the breakdown and resorption of bone, information on growth factors which stimulate the formation of new bone is more limited.
  • bovine bone tissue itself is a storehouse for factors which have the capacity for stimulating bone cells.
  • extracts of bovine bone tissue obtained from slaughterhouses contain not only structural proteins which are responsible for maintaining the structural integrity of bone, but also biologically active bone growth factors which can stimulate bone cells to proliferate.
  • these latter factors are transforming growth factor ⁇ , the heparin-binding growth factors (acidic and basic fibroblast growth factor), the insulin-like growth factors (insulin-like growth factor I and insulin-like growth factor II), and a recently described family of proteins called bone morphogenetic proteins (BMPs) All of these growth factors have effects on other types of cells, as well as on bone cells
  • the BMPs are novel factors in the extended transforming growth factor ⁇ superfamily They were first identified by Wozney J et al. Science (1988) 242 1528- 34, using gene cloning techniques, following earlier descriptions characterizing the biological activity in extracts of demineralized bone (Urist M Science (1965) 150 893- 99) Recombinant BMP2 and BMP4 can induce new bone formation when they are injected locally into the subcutaneous tissues of rats (Wozney J Molec ReprodDev (1992) 32 160-67) These factors are expressed by normal osteoblasts as they differentiate, and have been shown to stimulate osteoblast differentiation and bone nodule formation in vitro as well as bone formation in vivo (Harris S et al J. Bone Miner Res (1994) 9 855-63) This latter property suggests potential usefulness as therapeutic agents in diseases which result in bone loss
  • the cells which are responsible for forming bone are osteoblasts As osteoblasts differentiate from precursors to mature bone-forming cells, they express and secrete a number of enzymes and structural proteins of the bone matrix, including Type-1 collagen, osteocalcin, osteopontin and alkaline phosphatase (Stein G et al. Curr Opm Cell Biol ( 1990) 2 1018-27, Harris S et al. ( 1994), supra) They also synthesize a number of growth regulatory peptides which are stored in the bone matrix, and are presumably responsible for normal bone formation These growth regulatory peptides include the BMPs (Harris S et al.
  • BMPs 1, 2, 3, 4, and 6 are expressed by cultured cells prior to the formation of mineralized bone nodules (Harris S et al. (1994), supra)
  • the BMPs are expressed by cultured osteoblasts as they proliferate and differentiate
  • BMPs are potent stimulators of bone formation in vitro md in vivo
  • Receptors for the bone morphogenetic proteins have been identified in many tissues, and the BMPs themselves are expressed in a large va ⁇ ety of tissues in specific temporal and spaiial patterns This suggests that BMPs may have effects on many tissues other than bone, potentially limiting their usefulness as therapeutic agents when administered systemically.
  • BMPs since they are peptides, they would have to be administered by injection.
  • bone fractures where it would be desirable to stimulate bone growth and to hasten and complete bone repair.
  • Agents that enhance bone formation would also be useful in facial reconstruction procedures.
  • Other bone deficit conditions include bone segmental defects, periodontal disease, metastatic bone disease, osteolytic bone disease and conditions where connective tissue repair would be beneficial, such as healing or regeneration of cartilage defects or injury.
  • Also of great significance is the chronic condition of osteoporosis, including age-related osteoporosis and osteoporosis associated with postmenopausal hormone status.
  • Other conditions characterized by the need for bone growth include primary and secondary hype ⁇ arathyroidism, disuse osteoporosis, diabetes-related osteoporosis, and glucocorticoid-related osteoporosis.
  • the compounds of the present invention may modulate metabolism, proliferation and/or differentiation of normal or aberrant cells or tissues.
  • Bone fractures are still treated exclusively using casts, braces, anchoring devices and other strictly mechanical means. Further bone deterioration associated with postmenopausal osteoporosis has been decreased or prevented with estrogens or bisphosphonates.
  • US Patent 5, 280, 040 discloses a class of compounds which are 3, 4-diaryl chromans. These compounds can be considered derivatives of 2,3,4 triphenyl butanol, where the hydroxy at the 1 -position forms an ether with the ortho position of the phenyl group substituted at the 4-position of the butanol.
  • the parent 3, 4-diaryl chromans do not contain nitrogen atoms in the aromatic moieties or their linkers.
  • a preferred compound, centchroman contains a nitrogen substituent only in one of the substituents on a phenyl moiety.
  • R a is a non-interfering substituent
  • m is an integer of 0-4
  • each dotted line represents an optional ⁇ -bond
  • each Z is independently N, NR, O, S, CR or CR 2 , where each R is independently H or alkyl (1-6C);
  • X is O, S, SO or SO 2 ;
  • L is a flexible linker;
  • Ar 2 is a substituted or unsubstituted 6-membered aromatic ring; or:
  • R a is a non-interfering substituent
  • n is an integer of 0 and 5
  • L is a flexible linker which does not contain nitrogen or is a constrained linker
  • Ar 2 is a substituted or unsubstituted phenyl or a substituted or unsubstituted naphthyl.
  • compositions which can ameliorate the effects of abnormalities in bone formation or resorption.
  • the present invention expands the repertoire of compounds useful for limiting or treating bone deficit conditions, and for other uses that should be apparent to those skilled in the art from the teachings herein.
  • the invention provides compounds that can be administered as ordinary pharmaceuticals and have the metabolic effect of enhancing bone growth or inhibiting reso ⁇ tion.
  • the compounds of the invention can be identified using an assay for their ability to activate control elements associated with bone anabolic factors.
  • the invention is directed to methods and compositions for treating bone disorders, which methods and compositions use, as active ingredients, compounds wherein two aromatic systems are coupled so as to be spaced apart from each other by about 1.5 to about 15 Angstroms.
  • the thus-linked systems (including the linker coupling them) preferably include at least one nitrogen atom.
  • the compounds useful in the invention can be described as having the formula Ar 1 -linker- Ar 2 , wherein each of Ar 1 and Ar 2 is independently an aromatic system and the linker portion of the formula spaces Ar 1 and Ar 2 apart by a distance of approximately 1.5-15 Angstroms.
  • Ar 1 , Ar 2 and the linker may optionally be substituted with non interfering substituents.
  • the compounds of the invention contain at least one additional heteroatom selected from the group consisting of N, S and O, independent of any substituent.
  • the invention is directed to a method to treat a condition in a vertebrate animal characterized by a deficiency in, or need for, bone growth replacement and/or an undesirable level of bone reso ⁇ tion, which method comprises administering to a vertebrate subject in need of such treatment an effective amount of certain compounds of the formula:
  • Ar'-L-Ar 2 wherein each of Ar 1 and Ar 2 is independently substituted or unsubstituted phenyl, substituted or unsubstituted naphthyl, a substituted or unsubstituted aromatic system containing a 6-membered heterocycle, or a substituted or unsubstituted aromatic system containing a 5-membered heterocycle; and L is a linker that provides spacing of 1.5- 15 A.
  • the invention relates to pharmaceutical compositions for use in the method, and to the compounds for use in preparing a medicament for use in the method.
  • Figure 1 gives a schematic representation of the compounds used as active ingredients in the methods and compositions of the invention.
  • Figure 2 shows the dose response curve for a positive control compound, designated 59-0008.
  • Figures 3 and 4 show illustrative compounds of the invention and the results obtained with them in an in vitro test for stimulation of bone growth.
  • Figures 5 A, 5B and 5C show structures and results of a screening assay for a group of compounds which varies the parameters of lead compound 59-0072.
  • Figures 6 A, 6B and 6C show structures and results of a screening assay for a group of compounds which varies the parameters of lead compound 50-0197.
  • Figure 7 shows structures and results of a screening assay for a group of compounds which varies the parameters of lead compound 59-0145.
  • Figures 8 A, 8B and 8C show structures and results of a screening assay for a group of compounds which varies the parameters of lead compound 59-0045.
  • Figure 9 shows the results in an ex vivo calvarial assay for various compunds of the invention.
  • Figure 10 shows the increase in bone volume effected by subcutaneous administration of compound 59-0145 in the OVX in vivo assay.
  • Figure 11 is a graphical representation of percent increase in trabecular bone in ovariectomized rats treated with compound 59-0145.
  • Figure 12 presents graphs showing results of qCT and bone histomorphometri and serum osteocalcin levels in rats treated with compound 59-0145.
  • Figure 13 (41 pages) is a list of compounds used in screening for bone mo ⁇ hogenic activity according to the screening assay set forth herein.
  • a rapid throughput screening test for compounds capable of stimulating expression of a reporter gene linked to a BMP promoter is described in WO96/38590 published 5 December 1996, the contents of which are inco ⁇ orated herein by reference.
  • This assay is also described as a portion of a study of immortalized murine osteoblasts (derived from a mouse expressing a transgene composed of a BMP2 promoter driving expression of T-antigen) in Ghosh-Choudhery, N. et al. Endocrinology (1996) 137:331-39.
  • the immortalized cells were stably transfected with a plasmid containing a luciferase reporter gene driven by a mouse BMP2 promoter (-2736/114 bp), and responded in a dose-dependent manner to recombinant human BMP2.
  • the assay utilizes cells transformed permanently or transiently with constructs in which the promoter of a bone mo ⁇ hogenetic protein, specifically BMP2 or BMP4, is coupled to a reporter gene, typically luciferase. These transformed cells are then evaluated for the production of the reporter gene product; compounds that activate the BMP promoter will drive production of the reporter protein, which can be readily assayed. Over 40,000 compounds have been subjected to this rapid screening technique, and only a very small percentage are able to elicit a level of production of luciferase 5-fold greater than that produced by vehicle. Compounds that activate the BMP promoter share certain structural characteristics not present in inactive compounds.
  • BMP promoter-active compounds are useful in promoting bone or cartilage growth, and thus in the treatment of vertebrates in need of bone or cartilage growth.
  • BMP promoter-active compounds can be examined in a variety of other assays that test specificity and toxicity
  • nonBMP promoters or response elements can be linked to a reporter gene and inserted into an appropriate host cell Cytotoxicity can be determined by visual or microscopic examination of BMP promoter- and/or nonBMP promoter-reporter gene-containing cells, for instance.
  • nucleic acid and/or protein synthesis by the cells can be monitored
  • tissues may be removed and examined visually or microscopically, and optionally examined in conjunction with dyes or stains that facilitate histologic examination
  • dyes or stains that facilitate histologic examination
  • limit or “limiting” and “treat” or “treatment” are interchangeable terms
  • the terms include a postponement of development of bone deficit symptoms and/or a reduction in the severity of such symptoms that will or are expected to develop.
  • the terms further include ameliorating existing bone or cartilage deficit symptoms, preventing additional symptoms, ameliorating or preventing the underlying metabolic causes of symptoms, preventing or reversing bone reso ⁇ tion and/or encouraging bone growth.
  • the terms denote that a beneficial result has been conferred on a vertebrate subject with a cartilage, bone or skeletal deficit, or with the potential to develop such deficit
  • bone deficit is meant an imbalance in the ratio of bone formation to bone reso ⁇ tion, such that, if unmodified, the subject will exhibit less bone than desirable, or the subject's bones will be less intact and coherent than desired. Bone deficit may also result from fracture, from surgical intervention or from dental or periodontal disease
  • cartilage defect is meant damaged cartilage, less cartilage than desired, or cartilage that is less intact and coherent than desired.
  • Representative uses of the compounds of the present invention include: repair of bone defects and deficiencies, such as those occuring in closed, open and nonunion fractures, prophylactic use in closed and open fracture reduction; promotion of bone healing in plastic surgery, stimulation of bone ingrowth into noncemented prosthetic joints and dental implants; elevation of peak bone mass in premenopausal women; treatment of growth deficiencies; treatment of peridontal disease and defects, and other tooth repair processes; increase in bone formation during distraction osteogenesis; and treatment of other skeletal disorders, such as age-related osteoporosis, postmenopausal osteoporosis, glucocorticoid-induced osteoporosis or disuse osteoporosis and arthritis.
  • the compounds of the present invention can also be useful in repair of congenital, trauma-induced or surgical resection of bone (for instance, for cancer treatment), and in cosmetic surgery. Further, the compounds of the present invention can be used for limiting or treating cartilage defects or disorders, and may be useful in wound healing or tissue repair.
  • Bone or cartilage deficit or defect can be treated in vertebrate subjects by administering compounds of the invention which have been identified through suitable screening assays and which exhibit certain structural characteristics.
  • the compositions of the invention may be administered systemically or locally.
  • the compounds herein are formulated for parenteral (e.g., intravenous, subcutaneous, intramuscular, intraperitoneal, intranasal or transdermal) or enteral (e.g., oral or rectal) delivery according to conventional methods.
  • parenteral e.g., intravenous, subcutaneous, intramuscular, intraperitoneal, intranasal or transdermal
  • enteral e.g., oral or rectal
  • Intravenous administration will be by a series of injections or by continuous infusion over an extended period.
  • Administration by injection or other routes of discretely spaced administration will generally be performed at intervals ranging from weekly to once to three times daily.
  • the compounds disclosed herein may be administered in a cyclical manner (administration of disclosed compound; followed by no administration; followed by administration of disclosed compound, and the like). Treatment will continue until the desired outcome is achieved.
  • pharmaceutical formulations will include a compound of the present invention in combination with a pharmaceutically acceptable vehicle, such as saline, buffered saline, 5% dextrose in water, borate-buffered saline containing trace metals or the like.
  • Formulations may further include one or more excipients, preservatives, solubilizers, buffering agents, albumin to prevent protein loss on vial surfaces, lubricants, fillers, stabilizers, etc.
  • compositions for use within the present invention can be in the form of sterile, nonpyrogenic liquid solutions or suspensions, coated capsules, suppositories, lyophilized powders, transdermal patches or other forms known in the art.
  • Local administration may be by injection at the site of injury or defect, or by insertion or attachment of a solid carrier at the site, or by direct, topical application of a viscous liquid.
  • the delivery vehicle preferably provides a matrix for the growing bone or cartilage, and more preferably is a vehicle that can be absorbed by the subject without adverse effects. Delivery of compounds herein to wound sites may be enhanced by the use of controlled-release compositions, such as those described in WIPO publication WO 93/20859, which is inco ⁇ orated herein by reference in its entirety. Films of this type are particularly useful as coatings for prosthetic devices and surgical implants. The films may, for example, be wrapped around the outer surfaces of surgical screws, rods, pins, plates and the like. Implantable devices of this type are routinely used in orthopedic surgery.
  • the films can also be used to coat bone filling materials, such as hydroxyapatite blocks, demineralized bone matrix plugs, collagen matrices and the like.
  • a film or device as described herein is applied to the bone at the fracture site. Application is generally by implantation into the bone or attachment to the surface using standard surgical procedures.
  • the biodegradable films and matrices may include other active or inert components.
  • agents that promote tissue growth or infiltration such as growth factors.
  • Exemplary growth factors for this pu ⁇ ose include epidermal growth factor (EGF), fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), transforming growth factors (TGFs), parathyroid hormone (PTH), leukemia inhibitory factor (LLF), and insulin-like growth factors (IGFs).
  • agents that promote bone growth such as bone morphogenetic proteins (U.S. Patent No. 4,761,471; PCT Publication WO 90/11366), osteogenin (Sampath et al. Proc. Natl.
  • Biodegradable films or matrices include calcium sulfate, tricalcium phosphate, hydroxyapatite, polylactic acid, polyanhydrides, bone or dermal collagen, pure proteins, extracellular matrix components and combinations thereof. Such biodegradable materials may be used in combination with nonbiodegradable materials, to provide desired mechanical, cosmetic or tissue or matrix interface properties.
  • Alternative methods for delivery of compounds of the present invention include use of ALZET osmotic minipumps (Alza Corp., Palo Alto, CA); sustained release matrix materials such as those disclosed in Wang et al. (PCT Publication WO 90/11366), electrically charged dextran beads, as disclosed in Bao et al. (PCT Publication WO 92/03125); collagen-based delivery systems, for example, as disclosed in Ksander et al. Ann. Surg. (1990) 211(3).288-94, methylcellulose gel systems, as disclosed in Beck et al. J. Bone Min. Res. (1991) 6(11): 1257-65; and alginate-based systems, as disclosed in Edelman et al. Biomaterials (1991) 12.619-26.
  • Other methods well known in the art for sustained local delivery in bone include porous coated metal protheses that can be impregnated and solid plastic rods with therapeutic compositions inco ⁇ orated within them.
  • the compounds of the present invention may also be used in conjunction with agents that inhibit bone reso ⁇ tion.
  • Antiresorptive agents such as estrogen, bisphosphonates and calcitonin, are preferred for this pu ⁇ ose
  • the compounds disclosed herein may be administered for a period of time (for instance, months to years) sufficient to obtain correction of a bone deficit condition. Once the bone deficit condition has been corrected, the vertebrate can be administered an antiresorptive compound to maintain the corrected bone condition.
  • the compounds disclosed herein may be adminstered with an anti-reso ⁇ tive compound in a cyclical manner (administration of disclosed compound, followed by anti-reso ⁇ tive, followed by disclosed compound, and the like).
  • Aqueous suspensions may contain the active ingredient in admixture with pharmacologically acceptable excipients, comprising suspending agents, such as methyl cellulose; and wetting agents, such as lecithin, lysolethicin or long-chain fatty alcohols.
  • suspending agents such as methyl cellulose
  • wetting agents such as lecithin, lysolethicin or long-chain fatty alcohols.
  • the said aqueous suspensions may also contain preservatives, coloring agents, flavoring agents and sweetening agents in accordance with industry standards.
  • Preparations for topical and local application comprise aerosol sprays, lotions, gels and ointments in pharmaceutically appropriate vehicles which may comprise lower aliphatic alcohols, polyglycols such as glycerol, polyethylene glycol, esters of fatty acids, oils and fats, and silicones.
  • the preparations may further comprise antioxidants, such as ascorbic acid or tocopherol, and preservatives, such as p-hydroxybenzoic acid esters.
  • Parenteral preparations comprise particularly sterile or sterilized products.
  • Injectable compositions may be provided containing the active compound and any of the well known injectable carriers. These may contain salts for regulating the osmotic pressure.
  • the osteogenic agents can be incorporated into liposomes by any of the reported methods of preparing liposomes for use in treating various pathogenic conditions.
  • the present compositions may utilize the compounds noted above inco ⁇ orated in liposomes in order to direct these compounds to macrophages, monocytes, other cells and tissues and organs which take up the liposomal composition.
  • the liposome-incorporated compounds of the invention can be utilized by parenteral administration, to allow for the efficacious use of lower doses of the compounds.
  • Ligands may also be incorporated to further focus the specificity of the liposomes.
  • Suitable conventional methods of liposome preparation include, but are not limited to, those disclosed by Bangham, A.D. et al. J Mol Biol (1965) 23:238-252, Olson, F. et al. Biochim Biophys Acta (1979) 557:9-23, Szoka, F. et al. Proc Natl
  • the liposomes may be made from the present compounds in combination with any of the conventional synthetic or natural phospholipid liposome materials including phospholipids from natural sources such as egg, plant or animal sources such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, sphingomyelin, phosphatidylserine, or phosphatidylinositol.
  • natural sources such as egg, plant or animal sources such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, sphingomyelin, phosphatidylserine, or phosphatidylinositol.
  • Synthetic phospholipids that may also be used, include, but are not limited to: dimyristoylphosphatidylcholine, dioleoylphosphatidylcholine, dipalmitoylphosphatidylcholine and distearoylphosphatidycholine, and the corresponding synthetic phosphatidylethanolamines and phosphatidylglycerols.
  • Cholesterol or other sterols, cholesterol hemisuccinate, glycolipids, cerebrosides, fatty acids, gangliosides, sphingolipids, l,2-bis(oleoyloxy)-3-(trimethyl ammonio) propane (DOTAP), N-[l- (2,3-dioleoyl) propyl-N,N,N-trimethylammonium chloride (DOTMA), and other cationic lipids may be incorporated into the liposomes, as is known to those skilled in the art.
  • the relative amounts of phospholipid and additives used in the liposomes may be varied if desired.
  • the preferred ranges are from about 60 to 90 mole percent of the phospholipid; cholesterol, cholesterol hemisuccinate, fatty acids or cationic lipids may be used in amounts ranging from 0 to 50 mole percent.
  • the amounts of the present compounds inco ⁇ orated into the lipid layer of liposomes can be varied with the concentration of their lipids ranging from about 0.01 to about 50 mole percent.
  • the liposomes with the above formulations may be made still more specific for their intended targets with the incorporation of monoclonal antibodies or other ligands specific for a target.
  • monoclonal antibodies to the BMP receptor may be inco ⁇ orated into the liposome by linkage to phosphatidylethanolamine (PE) inco ⁇ orated into the liposome by the method of Leserman, L. et al. Nature (1980) 288:602-604.
  • PE phosphatidylethanolamine
  • Veterinary uses of the disclosed compounds are also contemplated. Such uses would include limitation or treatment of bone or cartilage deficits or defects in domestic animals, livestock and thoroughbred horses.
  • the compounds described herein can also modify a target tissue or organ environment, so as to attract bone- forming cells to an environment in need of such cells.
  • the compounds of the present invention may also be used to stimulate growth of bone-forming cells or their precursors, or to induce differentiation of bone-forming cell precursors, either in vitro or ex vivo.
  • precursor cell refers to a cell that is committed to a differentiation pathway, but that generally does not express markers or function as a mature, fully differentiated cell.
  • meenchymal cells or “mesenchymal stem cells” refers to pluripotent progenitor cells that are capable of dividing many times, and whose progeny will give rise to skeletal tissues, including cartilage, bone, tendon, ligament, marrow stroma and connective tissue (see A. Caplan J. Orthop. Res.
  • osteoogenic cells includes osteoblasts and osteoblast precursor cells. More particularly, the disclosed compounds are useful for stimulating a cell population containing marrow mesenchymal cells, thereby increasing the number of osteogenic cells in that cell population. In a preferred method, hematopoietic cells are removed from the cell population, either before or after stimulation with the disclosed compounds. Through practice of such methods, osteogenic cells may be expanded. The expanded osteogenic cells can be infused (or reinfused) into a vertebrate subject in need thereof.
  • a subject's own mesenchymal stem cells can be exposed to compounds of the present invention ex vivo, and the resultant osteogenic cells could be infused or directed to a desired site within the subject, where further proliferation and/or differentiation of the osteogenic cells can occur without immunorejection.
  • the cell population exposed to the disclosed compounds may be immortalized human fetal osteoblastic or osteogenic cells. If such cells are infused or implanted in a vertebrate subject, it may be advantageous to "immunoprotect" these nonself cells, or to immunosuppress (preferably locally) the recipient to enhance transplantation and bone or cartilage repair.
  • an "effective amount" of a composition is that amount which produces a statistically significant effect.
  • an “effective amount” for therapeutic uses is the amount of the composition comprising an active compound herein required to provide a clinically significant increase in healing rates in fracture repair; reversal of bone loss in osteoporosis; reversal of cartilage defects or disorders; prevention or delay of onset of osteoporosis; stimulation and/or augmentation of bone formation in fracture nonunions and distraction osteogenesis; increase and/or acceleration of bone growth into prosthetic devices; and repair of dental defects.
  • Such effective amounts will be determined using routine optimization techniques and are dependent on the particular condition to be treated, the condition of the patient, the route of administration, the formulation, and the judgment of the practitioner and other factors evident to those skilled in the art.
  • the dosage required for the compounds of the invention (for example, in osteoporosis where an increase in bone formation is desired) is manifested as a statistically significant difference in bone mass between treatment and control groups. This difference in bone mass may be seen, for example, as a 5-20% or more increase in bone mass in the treatment group.
  • Other measurements of clinically significant increases in healing may include, for example, tests for breaking strength and tension, breaking strength and torsion, 4-point bending, increased connectivity in bone biopsies and other biomechanical tests well known to those skilled in the art.
  • the dosage of the compounds of the invention will vary according to the extent and severity of the need for treatment, the activity of the administered compound, the general health of the subject, and other considerations well known to the skilled artisan. Generally, they can be administered to a typical human on a daily basis on an oral dose of about 0.1 mg/kg- 1000 mg/kg, and more preferably from about 1 mg/kg to about 200 mg/kg. The parenteral dose will appropriately be 20-100% of the oral dose.
  • the osteogenic activity of the compounds used in the methods of the invention can be verified using in vitro screening techniques, such as the assessment of transcription of a reporter gene coupled to a bone morphogenetic protein-associated promoter, as described above, or in alternative assays such as the following:
  • Neonatal Mouse Calvarial Assay In vitro This assay is similar to that described by Go wen M. & Mundy G. J Immunol
  • the bones are removed from the incubation media and fixed in 10% buffered formalin for 24-48 hours, decalcified in 14% EDTA for 1 week, processed through graded alcohols; and embedded in paraffin wax.
  • Three ⁇ m sections of the calvaria are prepared. Representative sections are selected for histomo ⁇ hometric assessment of bone formation and bone reso ⁇ tion. Bone changes are measured on sections cut 200 ⁇ m apart. Osteoblasts and osteoclasts are identified by their distinctive mo ⁇ hology.
  • auxiliary assays can be used as controls to determine nonBMP promoter- mediated effects of test compounds.
  • mitogenic activity can be measured using screening assays featuring a serum-response element (SRE) as a promoter and a luciferase reporter gene. More specifically, these screening assays can detect signalling through SRE-mediated pathways, such as the protein kinase C pathway. For instance, an osteoblast activator SRE-luciferase screen and an insulin mimetic SRE-luciferase screen are useful for this pu ⁇ ose. Similarly, test compound stimulation of cAMP response element (CRE)-mediated pathways can also be assayed.
  • CRE cAMP response element
  • auxiliary assays In vivo Assay of Effects of Compounds on Murine Calvarial Bone Growth Male ICR Swiss white mice, aged 4-6 weeks and weighing 13-26 gm, are employed, using 4-5 mice per group. The calvarial bone growth assay is performed as described in PCT application WO 95/24211. Briefly, the test compound or appropriate control vehicle is injected into the subcutaneous tissue over the right calvaria of normal mice.
  • the control vehicle is the vehicle in which the compound was solubilized, and is PBS containing 5% DMSO or is PBS containing Tween (2 ⁇ l 10 ml).
  • the animals are sacrificed on day 14 and bone growth measured by histomo ⁇ hometry. Bone samples for quantitation are cleaned from adjacent tissues and fixed in 10% buffered formalin for 24-48 hours, decalcified in 14% EDTA for 1-3 weeks, processed through graded alcohols; and embedded in paraffin wax. Three to five ⁇ m sections of the calvaria are prepared, and representative sections are selected for histomo ⁇ hometric assessment of the effects on bone formation and bone resorption.
  • Sections are measured by using a camera lucida attachment to trace directly the microscopic image onto a digitizing plate. Bone changes are measured on sections cut 200 ⁇ m apart, over 4 adjacent lxl mm fields on both the injected and noninjected sides of the calvaria. New bone is identified by its characteristic woven structure, and osteoclasts and osteoblasts are identified by their distinctive morphology. Histomorphometry software (OsteoMeasure, Osteometrix, Inc., Atlanta) is used to process digitizer input to determine cell counts and measure areas or perimeters.
  • Prototypical dosing may be accomplished by subcutaneous, intraperitoneal or oral administration, and may be performed by injection, sustained release or other delivery techniques. The time period for administration of test compound may vary (for instance, 28 days as well as 35 days may be appropriate).
  • An exemplary, in vivo subcutaneous dosing assay may be conducted as follows: In a typical study, 70 three-month-old female Sprague-Dawley rats are weight- matched and divided into seven groups, with ten animals in each group.
  • test compound positive control compound, PBS, or vehicle alone is administered subcutaneously once per day for 35 days. All animals are injected with calcein nine days and two days before sacrifice (two injections of calcein administered each designated day). Weekly body weights are determined. At the end of the 35-day cycle, the animals are weighed and bled by orbital or cardiac puncture. Serum calcium, phosphate, osteocalcin, and CBCs are determined. Both leg bones (femur and tibia) and lumbar vertebrae are removed, cleaned of adhering soft tissue, and stored in 70% ethanol for evaluation, as performed by peripheral quantitative computed tomography (pqCT; Ferretti, J.
  • pqCT peripheral quantitative computed tomography
  • test compounds also be tested in acute ovariectomized animals (prevention model) using an in vivo dosing assay.
  • assays may also include an estrogen- treated group as a control.
  • An exemplary subcutaneous dosing assay is performed as follows:
  • a typical study 80 three-month-old female Sprague-Dawley rats are weight- matched and divided into eight groups, with ten animals in each group. This includes a baseline control group of animals sacrificed at the initiation of the study; three control groups (sham ovariectomized (sham OVX) + vehicle only; ovariectomized (OVX) + vehicle only; PBS-treated OVX); and a control OVX group that is administered a compound known to promote bone growth. Three dosage levels of the compound to be tested are administered to the remaining three groups of OVX animals. Since ovariectomy (OVX) induces hyperphagia, all OVX animals are pair-fed with sham OVX animals throughout the 35 day study.
  • OVX ovariectomy
  • test compound positive control compound, PBS, or vehicle alone is administered subcutaneously once per day for 35 days.
  • test compound can be formulated in implantable pellets that are implanted for 35 days, or may be administered orally, such as by gastric gavage. All animals, including sham OVX/vehicle and OVX/vehicle groups, are injected intraperitoneally with calcein nine days and two days before sacrifice (two injections of calcein administered each designated day, to ensure proper labeling of newly formed bone). Weekly body weights are determined. At the end of the 35-day cycle, the animals' blood and tissues are processed as described above.
  • Lead compounds may also be tested in chronic OVX animals (treatment model).
  • An exemplary protocol for treatment of established bone loss in ovariectomized animals that can be used to assess efficacy of anabolic agents may be performed as follows. Briefly, 80 to 100 six month old female, Sprague-Dawley rats are subjected to sham surgery (sham OVX) or ovariectomy (OVX) at time 0, and 10 rats are sacrificed to serve as baseline controls. Body weights are recorded weekly during the experiment. After approximately 6 weeks of bone depletion (42 days), 10 sham OVX and 10 OVX rats are randomly selected for sacrifice as depletion period controls. Of the remaining animals, 10 sham OVX and 10 OVX rats are used as placebo-treated controls.
  • the remaining OVX animals are treated with 3 to 5 doses of test drug for a period of 5 weeks (35 days).
  • a group of OVX rats can be treated with an agent such as PTH, a known anabolic agent in this model (Kimmel et al. Endocrinology (1993) 132: 1577-84).
  • PTH a known anabolic agent in this model
  • the proximal left and right tibiae are used for pqCT measurements, cancellous bone mineral density (BMD) (gravimetric determination), and histology, while the midshaft of each tibiae is subjected to cortical BMD or histology.
  • BMD cancellous bone mineral density
  • the femurs are prepared for pqCT scanning of the midshaft prior to biomechanical testing.
  • LV lumbar vertebrae
  • LV2 are processed for BMD (pqCT may also be performed);
  • LV3 are prepared for undecalcified bone histology; and LV4 are processed for mechanical testing.
  • Ar 2 spaced apart by a linker at a distance of 1.5- 15 A, and may preferably contain at least one nitrogen atom.
  • Figure 1 A summary of the structural features of the compounds included within the invention is shown in Figure 1.
  • Ar 1 and Ar 2 may include various preferred embodiments. These are selected from the group consisting of a substituted or unsubstituted aromatic ring system containing a 5-membered heterocycle; a substituted or unsubstituted aromatic ring system containing a six-membered heterocycle; a substituted or unsubstituted naphthalene moiety; and a substituted or unsubstituted benzene moiety.
  • Ar 1 and Ar 2 There are 16 possible combinations of these embodiments, if Ar 1 and Ar 2 are considered distinguishable. As will be clear, however, the designation of one aromatic system as Ar 1 and the other as Ar 2 is arbitrary; thus there are only ten possible combinations.
  • Ar 1 and Ar 2 are designated separately with the realization that the choice is arbitrarily made. All linkers described herein if not palindromic, are considered to link Ar 1 to Ar 2 or vice-versa whether or not the complementary orientation is explicitly shown (as it is in some cases). Thus, if Ar 1 and Ar 2 are different and a linker is specified as -CONR-, it is understood that also included is the linker -NRCO- when the designations Ar 1 and Ar 2 are retained.
  • the noninterfering substituents on the aromatic system represented by Ar 1 and the noninterfering substituents on the aromatic system represented by Ar 2 are represented in the formulas herein by R and R b , respectively. Generally, these substituents can be of wide variety.
  • alkyl 1-6C, preferably lower alkyl 1-4C
  • alkenyl 1-6C, preferably 1-4C
  • alkynyl 1-6C, preferably 1-4C
  • R a and R b may also include halogens, (e.g.
  • R is alkyl (1-6C) or is H.
  • substituents may contain R as a substitute for R wherein R' is aryl (6- IOC) or alkylaryl (6-15C) or aryl alkyl (6-15C). Where R a or R b substituents are in adjacent positions in the aromatic system, they may combine to form a ring.
  • rings may be included in substituents which contain sufficient carbon and heteroatoms to provide this possibility.
  • the choice of noninterfering substituents depends on the overall nature of the system. For example, in compounds of the invention wherein two pyridine rings are linked through a saturated flexible linker, a CF 3 substituent para to the linker in each of the pyridine rings is particularly preferred.
  • a quinoline is coupled through a flexible conjugated or nonconjugated linker to a phenyl substituent or to a naphthyl substituent, an amino group para to the linker in the phenyl or naphthyl moiety is preferred. Particularly preferred amino groups are dimethylamino and diethylamino.
  • substituents on the phenyl moiety include alkoxy or alkylthio in combination with halo, in particular, chloro. Also preferred is the presence of a diethylamino group in the phenyl moiety para to the position that is coupled to the linker. In general, the presence of a substituent in the phenyl moiety para to the position of joinder to the linker is preferred.
  • preferred noninterfering substituents include hydrocarbyl groups of 1-6C, including saturated and unsaturated, linear or branched hydrocarbyl as well as hydrocarbyl groups containing ring systems; halo groups, alkoxy, hydroxy, amino, monoalkyl- and dialkylamino where the alkyl groups are 1-6C, CN, CF 3 , OCF 3 and COOR, and the like.
  • R a and R b may typically be 0-4 (m) or 0-5 (n) depending on the available positions in the aromatic system, preferred embodiments include those wherein the number of R is 0, 1 or 2 and of R b is 0, 1, 2 or 3, particularly 1 or 2
  • the linker group, L may be a covalent bond or any group having a valence of at least two and covering a linear distance of from about 1 5 to about 15 Angstroms, including those that contain cyclic moieties, that meet this spatial requirement
  • Useful linkers are divided, by definition herein, into three general categories (1) flexible nonconjugating linkers, (2) flexible conjugating linkers, and (3) constrained linkers
  • the preferred choice of linker will depend on the choices for Ar 1 and Ar 2
  • flexible nonconjugating linkers are those that link only one position of Ar 1 to one position of Ar 2 , and provide only a single covalent bond or a single chain between Ar 1 and Ar 2
  • the chain may contain branches, but may not contain ⁇ -bonds (except in the branches) or cyclic portions in the chain
  • the linker atoms in the chain itself rotate freely around single covalent bonds, and thus the linker has more than two degrees of freedom
  • Particularly useful flexible nonconjugating linkers, besides a covalent bond are those of the formulas -NR-, -CR 2 -, -S-, or -O-, wherein R is H or alkyl (1-6C), more preferably H or lower alkyl (1-4C) and more preferably H
  • Also contemplated are those of the formulas -NRCO-, -CONR-, -CR 2 S-, -SCR 2 -, -OCR 2 -, -CR 2 O-, -NRNR-, -CR 2
  • Constrained linkers are those that have more than one point of attachment to either or both Ar 1 and Ar 2 and, thus, generally allow for only one degree of freedom. Constrained linkers most frequently form fused 5- or 6-membered cyclic moieties with Ar 1 and/or Ar 2 where either Ar 1 or Ar 2 has at least one substituent appropriately positioned to form a second covalent bond with the linker, e.g., where Ar 2 is a phenyl group with a reactive, ortho-positioned substituent, or is derivatized to the linker directly at the ortho position.
  • Ar 1 is an aromatic system containing a 5-membered heterocycle, of the formula: or
  • Z is S, O, NR or -CR 2 in formula (la) or CR in formula (2a), where each R is independently H or alkyl (1-6C), the dotted line represents an optional ⁇ -bond, each R a is independently a noninterfering substituent as defined above, and m is an integer of 0-4.
  • Ar 2 is phenyl, naphthyl, or an aromatic system containing a 5- or 6- membered heterocyclic ring. All may be unsubstituted or substituted with noninterfering substituents, R b .
  • Ar 2 is an aromatic system containing a six-membered heterocycle
  • the formula of said system is preferably:
  • each Z is independently a heteroatom selected from the group consisting of S, O and N; or is CR or CR 2 , the dotted lines represent optional ⁇ -bonds, each R b is independently a noninterfering substituent, and m is an integer of 0-4, with the proviso that at least one Z must be a heteroatom.
  • Ar 2 in these compounds may also have the formula where R b is a noninterfering substituent as defined above and n is an integer from 0 to 5.
  • Ar 2 when Ar 2 is naphthyl, it may contain 0-5 R b substitutions.
  • Ar 2 when Ar 2 is an aromatic system containing a 5-membered heterocycle, preferred forms are those as described for Ar 1 .
  • Ar 1 is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl
  • each R a is a noninterfering substituent
  • m is an integer of 0-4
  • the dotted line represents an optional ⁇ bond
  • Z is O, S, NR or CR in formula (1) or is CR in formula (2) wherein each R is independently H or alkyl (1-6C).
  • L is a flexible conjugating or nonconjugating linker.
  • Ar 2 is preferably a substituted or unsubstituted aromatic system containing a 5-membered heterocycle or is
  • each R b is independently halo, OR, SR, NR 2 , NO, NO 2 , OCF 3 or CF 3 wherein R is H or alkyl (1-6C), or R b comprises an aromatic system.
  • Preferred compounds in this group are 59-0100, 59-103, 59-104, 59-105 and 59-106 (See Figure 13).
  • Z is S
  • Ar 2 is preferably a substituted or unsubstituted aromatic system containing a 6-membered heterocycle or is of the formula
  • R b is a noninterfering substituent and n is an integer of 0-5; and/or L is
  • each R b is independently halo, OR, SR, NR 2 , NO, NO 2 , OCF 3 or CF 3 wherein R is H or alkyl (1-6C) or R b comprises an aromatic system.
  • Preferred compounds in this group include compounds 59-002, 59-0070, 59-0072, 59-0099, 59-0102, the benzothiazole counterpart of 59-0104, 59-0144,
  • Z can also be CR, CR 2 or O; here it is also preferred that Ar 2 is
  • each R b is independently halo, OR, SR, NR 2 , NO, NO 2 , OCF 3 or CF 3 wherein R is H or alkyl (1-6C) or R b comprises an aromatic system.
  • a preferred compound is 896-5005. (See Figure 4)
  • the compounds wherein Ar 1 is la or 2a as above may also contain a constrained linker.
  • preferred Z is S or NR; and/or those wherein L is selected from the group consisting of
  • R b is a noninterfering substituent and m is 0-4.
  • each R b is independently halo, OR, SR, NR 2 , NO, NO 2 , OCF 3 or CF 3 wherein R is H or alkyl (1-6C) or R b comprises an aromatic system.
  • R is H or alkyl (1-6C) or R b comprises an aromatic system.
  • a preferred compound is 59-0124. (See Figure 13)
  • Ar 1 is of the formula
  • each R a is independently a noninterfering substituent or is H and Z is NR, S or O, wherein R is alkyl (1-6C) or H, especially where Z is S and/or wherein Ar 2 is
  • R b is independently halo, OR, SR, NR 2 , NO, NO 2 , OCF 3 or CF 3 wherein R is H or alkyl (1-6C) or R b comprises an aromatic system.
  • R a is a noninterfering substituent
  • m is an integer of 0-4
  • each dotted line represents an optional ⁇ -bond
  • each Z is independently N, NR, CR or CR 2 , where each R is independently H or alkyl (1-6C) with the proviso that at least one Z is N or NR.
  • each R b is independently halo, OR, SR, NR , NO, NO 2 , OCF 3 or CF 3 wherein R is H or alkyl (1-6C) or R b comprises an aromatic system.
  • Ar 1 has formula (4a) or (5a) and wherein Ar 2 is substituted or unsubstituted quinolyl or naphthyl of the formula
  • each R b is a noninterfering substituent and m is 0-4.
  • each R b is independently halo, OR, SR, NR 2 , NO, NO 2 , OCF 3 or CF 3 wherein R is H or alkyl (1-6C) or R b comprises an aromatic system and m is 0, 1 or 2.
  • R is H or alkyl (1-6C) or R b comprises an aromatic system and m is 0, 1 or 2.
  • the compounds 59-0089, 59-0090, 59-0092 or 59-0094 are particularly preferred.
  • Ar 1 is also preferably
  • R b is a noninterfering substituent and n is an integer of 0-5.
  • R b is independently halo, OR, SR, NR 2 , NO, NO 2 , OCF 3 or CF 3 wherein R is H or alkyl (1-6C) or R b comprises an aromatic system, in particular compounds 59-203, 59-285 or 59-286. (See Figure 13)
  • L can also be a constrained linker.
  • Ar 1 is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl
  • each R a is independently a noninterfering substituent
  • m is an integer of 0-4
  • each Z is independently N or CR, where R is H or alkyl (1-6C), with the proviso that at least one Z must be N and at least one Z must be CR.
  • L is preferably a flexible conjugating or nonconjugating linker, and/or wherein Ar 2 is
  • each R b is independently a noninterfering substituent, and in (vi) each Z is independently N or CR, where R is H or alkyl (1-6C), with the proviso that at least one Z must be a N and at least one Z must be CR.
  • R is H or alkyl (1-6C)
  • each R a is a noninterfering substituent
  • n is an integer of 0 and 5
  • L is a flexible linker that contains at least one nitrogen.
  • Ar 2 is of the formula
  • each R b is independently halo, OR, SR, NR 2 , NO, NO 2 , OCF 3 or CF 3 wherein R is H or alkyl (1-6C) or R b comprises an aromatic system.
  • Ar 1 and Ar 2 are defined by specifying Ar 1 and Ar 2 , and L.
  • Ar 1 is an aromatic system containing a 5-membered heterocyclic ring
  • Ar 2 is 2-methoxy-4- methylthiophenyl was used as a lead compound and variations of the structure studied.
  • Figure 5 shows representative compounds synthesized to analyze the effects of the nature of the linker, various alternatives of Ar 1 wherein Z is O, NR or S, and the effect of substitution on the phenyl moiety, as well as the heterocycle.
  • Figure 5 gives the structures of these compounds, along with their maximum activity as compared to 59-0008 at 10 ⁇ M (the maximum for 59-0008) in the in vitro bone growth stimulation assay as well as the concentration at which 50% of maximum stimulation of the BMP promoter was obtained (EC 5 o). See Example 1 for the details of this assay.
  • Preferred compounds in this group which perform better than 59-0008 in the screening assay, included 59-0450, 59-0459, 59-0480, and 59-0483.
  • Ar 1 is 3-carboxyphenyl was studied using 59-0045 as the lead compound.
  • Figure 8 shows the compounds synthesized in this series. Under the circumstances of this assay, analogs wherein R b was, instead of a nitrogen-containing moiety, F, Cl, or OMe were inactive.
  • Preferred compounds in this series are 59-0096 and 59-0098. 59-0098 is very active in the ex vivo calvarial assay described above. Synthesis of the Compounds Useful in the Invention
  • the 2T3-BMP-2-LUC cells a stably transformed osteoblast cell line described in Ghosh-Choudhury et al. Endocrinology (1996) 137:331-39, referenced above, was employed.
  • the cells were cultured using ⁇ -MEM, 10% FCS with 1% penicillin/streptomycin and 1% glutamine ("plating medium"), and were split 1 :5 once per week.
  • plating medium 10% FCS with 1% penicillin/streptomycin and 1% glutamine
  • the cells were resuspended in a plating medium containing 4% FCS, plated in microtiter plates at a concentration of 5 x 10 3 cells (in 50 ⁇ l)/well, and incubated for 24 hours at 37°C in 5% CO 2 .
  • the treated cells were incubated for 24 hours at 37°C and 5% CO 2 .
  • the medium was then removed, and the cells were rinsed three times with PBS.
  • 25 ⁇ l of IX cell culture lysing reagent (Promega #E153A) was added to each well and incubated for at least ten minutes.
  • the plates/samples could be frozen at this point.
  • To each well was added 50 ⁇ l of luciferase substrate (Promega #E152A; 10 ml Promega luciferase assay buffer per 7 mg Promega luciferase assay substrate).
  • Luminescence was measured on an automated 96-well luminometer, and was expressed as either picograms of luciferase activity per well or as picograms of luciferase activity per microgram of protein.
  • compound 59-0008 (3-phenylazo-lH-4,l,2-benzothiadiazine) exhibited a pattern of reactivity, as shown in Figure 2.
  • the activity for compound 59-0008 was maximal at a concentration of approximately 3-10 ⁇ M and, more particularly, at about 3 ⁇ M, and thus provided a response of approximately 175 light emission units.
  • other tested compounds were evaluated at various concentrations, and these results were compared to the results obtained for 59-0008 at 10 ⁇ M (which value was normalized to 100). For instance, any tested compound in Figure 3 and Figure 4 that showed greater activity than 10 ⁇ M of 59-0008 would result in a value over 100.
  • the invention compound 59-0203 shows consistent increases over the 0.1-50 mg/kg/day range at a somewhat lower level than that obtained with FGF. Similar results are obtained when new bone width in microns is measured. There was no new bone present in the control group. 59-0203 caused new bone formation at all doses, with a significant increase at 25-50 mg/kg/day. New bone as percentage of the total bone area was about 45% for the FGF positive control and from about 15% to 30% over the range of 0.1-50 mg/kg/day for 59-0203. There was no new bone present in the negative control.
  • Example 3 Ex vivo Calvarial Bone Growth Assay A number of compounds, in particular, those studied in connection with lead compounds classified as hydrazone/hydrazides (H) exemplified by 59-0045, benzothiazoles (T) exemplified by 59-0104, bis-pyridines (P) exemplified by 59-0145, and quinolines/quinoxalines (Q) exemplified by 59-0197, were tested in the ex vivo calvarial assay described hereinabove. The results of this assay are shown in Figure 9.
  • histomo ⁇ hotometry and osteoblast numbers are measured and effects are measured on an arbitrary scale from 1-3: i.e., 1, 1+, 2-, 2, 2+, 3-, 3, wherein 1 denotes "inactive.”
  • FGF scores 2-3 are measured and effects are measured on an arbitrary scale from 1-3: i.e., 1, 1+, 2-, 2, 2+, 3-, 3, wherein 1 denotes "inactive.”
  • FGF scores 2-3 for example,
  • the scores are assigned to bone formation on the ectocranial periosteal surface.
  • the area immediately surrounding midline suture is excluded from analysis.
  • a score of " 1 " is the bone forming activity seen in control cultures containing BGJb media + 0.1% bovine serum albumin.
  • the periosteal surface is covered by one layer of osteoblasts (at about 50% of the bone surface, with the remaining 50% being covered by bone lining cells).
  • a score of "1-” is assigned if less than 50% of the periosteal surface is covered by osteoblasts due to inhibitory activity or minor toxicity of the agents being tested.
  • a score of "1+” is given if over 50% of the surface is covered by osteoblasts.
  • a moderate increase in bone forming activity 20-40% of the periosteal surface is covered by up to two layers of osteoblasts. A score of "2-" is given if less than 20% of the surface is covered by two layers and "2+” if more than 40% of the surface is covered by two layers of osteoblasts.
  • a score of "3" is the bone forming activity seen in control cultures containing BGJb media + 0.1% BSA +10% fetal bovine serum.
  • More than 20% of the periosteal surface is covered by three layers of osteoblasts.
  • the cells appear plump (size can exceed 100 ⁇ m2).
  • a score of "3-” is given if less than 20% of the periosteal surface is covered by three layers of osteoblasts and or osteoblast size is less than 100 ⁇ m2.
  • a score of "3+" has never been observed.
  • Example 4 Stimulation of Bone Growth in Ovariectomized Rats The compound 59-0145 was tested at various concentrations in the OVX assay conducted as described above. The increase in bone volume was measured by two different groups; one group found 5 ⁇ g/kg/day of 59-0145 gave 21% increase over control whereas the second group found a 71% increase. At 50 ⁇ g/kg/day, the first group found a 31% increase, and the second a 54% increase. In another experiment, the lumbar vertebrae were measured and the above dosages of 59-0145 were shown to provide a beneficial effect, as shown in Figure 10. In another experiment, 3 month old Sprague Dawley rats were ovariectomized and depleted for six weeks.
  • treatment was started with subcutaneous administration of compound 59-0145. The treatment continued for 10 weeks. At the end of the 10 weeks animals were sacrificed, bones were collected for qCT measurements and histology; serum was also collected for osteocalcin determinations.
  • Figure 11 shows the percentage increase in trabecular bone (proximal tibia) compared to the placebo-treated group in chronic ovariectomized rats after 10 weeks of treatment.
  • Compound 59-0145 causes significant increase in trabecular bone at doses of 50-500 ⁇ g/kg/day.
  • Figure 12 shows results of qCT and bone histomorphometry in proximal tibia in the first two panels, as well as serum osteocalcin levels at the time of sacrifice as a percentage increase compared to control group (OVX placebo-treated group).
  • Example 5 Chondrogenic Activity Compounds 59-008, 59-0102 and 50-0197 were assayed for effects on the differentiation of cartilage cells, as compared to the action of recombinant human BMP-2. Briefly, a mouse clonal chondrogenic cell line, TMC-23, was isolated and cloned from costal cartilage of transgenic mice containing the BMP-2 gene control region driving SV-40 large T-antigen, generated as described in Ghosh-Choudhury et al Endocrinology 137:331-39, 1996.
  • TMC-23 cells were cultured in DMEM/10% FCS, and were shown to express T-antigen, and also to produce aggrecan (toluidine blue staining at pH 1.0) and Type-II collagen (immuno staining) by 7 days after confluence.
  • aggrecan toluidine blue staining at pH 1.0
  • Type-II collagen immuno staining
  • Compounds of the invention represented by exemplary Compound 59-0234, wherein Z is O, L is -CH CH-, and Ar 2 is 2,4-dimethyoxy-phenyl, including Compounds 59-0211 and 59-0233, were prepared according to the following procedure describing synthesis of Compound 59-0234. Briefly, to a N,N-dimethylformamide (DMF) solution of 2-methylbenzoxazole (1 mmol) and 2,4-dimethoxybenzaldehyde (1 mmol) was added lithium t-butoxide (2 mmol). The reaction mixture was heated at 130°C for 3h. After cooling to room temperature, the reaction mix was poured into ether and washed several times with water.
  • DMF N,N-dimethylformamide
  • b Azo derivatives may be obtained by reaction of 2- aminoquinolines with aldehydes, Morimoto, T , et al, Chem Pharm Bull (1977) 25 1607-09, Renault, J , et al, Hebd Seances Acad Sci, Ser C (1975) 280 1041- 43, and Lugovkin, B P , Zh Obshch Khim (1972) 42 966-69 c
  • Imino derivatives may be obtained by reaction of 2- formylquinohnes with anilines, Tran Quoc Son, et al , (1983) 21 22-26, Hagen, V et al.
  • conjugated linkers can be formed by bromination of the olefin of 50-0197 with Br 2 in AcOH followed by elimination with DBU as set forth in Zamboni et al. JMed Chem (1992) 35 3832-44 H Analogs having the constrained linker depicted below
  • R alkyl, OH
  • Exemplary Compound 59-0145 can be synthesized according to the following method Briefly, a mixture of 2-chloro-5-trifluoromethylpyridine (15 mmol), ethylenediamine (6 mmol), and diisopropylethylamine (18 mmol) was heated at reflux for 18 h After cooling to room temperature, the solid mass was triturated with dichloromethane. The product was filtered and then suspended in hot EtOAc:CHCl 3 (50:50, 800 mL) and filtered to remove insoluble material. The volume was reduced to -200 mL by heating on a steam bath. On standing, crystals of pure product were deposited.
  • the product was purified by flash column chromatography over silica gel eluting with 5 % methanol in dichloromethane.
  • the pure product was obtained as a red to purple powder.
  • the compounds of the invention are produced by substituting for at least one phenyl group the appropriate heterocycle.
  • exemplary Compound 59-0045 can be synthesized using standard procedures for the synthesis of phenyl hydrazones of aromatic aldehydes, as described in any organic textbook.
  • the synthesis of exemplary Compound 59-0045 may be performed as follows. Briefly, a suspension of 3- hydrazinobenzoic acid (1 mmol), p-dimethylaminobenzaldehyde (1 mmol), and AcOH (50 ⁇ L) in EtOH H 2 O (4 mL 1 mL) was heated at 105°C in a sealed vial for 3 h After cooling, a bright yellow solid was removed by filtration The solid was washed with cold MeOH and then with ether to give pure product
  • Exemplary Compound 59-0096 and related, exemplary Compounds 59- 0098, 59-0095, 59-0107, 59-0108, 59-0109, 59-0110 and 59-0200 may be synthesized according to the following general procedure Briefly, the appropriate carboxylic acid (1 mmol) and HATU ([O-(7-azabenzotriazol-l-yl)-l, l,3,3-tritetramethyluronium hexafluorophosphate], 1 mmol) were dissolved in 5 mL of anhydrous DMF in a vial and the solution was stirred at room temperature (RT) Diisopropylethyamine (3 mmol) was added dropwise to the above stirred solution and the mixture was stirred for 15 min 3 -Hydrazinobenzoic acid (1 mmol) was then added all at once to the above stirred mixture and the mixture was stirred overnight at RT It was then diluted with 50 mL of cold water with vigorous stirring and

Abstract

Des composés qui contiennent deux systèmes aromatiques liés de manière covalente par un élément de liaison comprenant un ou plusieurs atomes, ou par un 'élément de liaison' défini comme comportant une liaison covalente per se, qui écarte lesdits systèmes aromatiques d'une distance comprise entre 1,5 et 15 Å, sont efficaces pour traiter des états associés à un déficit osseux. Les composés peuvent être administrés à des vertébrés, seuls ou associés à des agents supplémentaires favorisant la croissance osseuse ou inhibant la résorption osseuse. Pour déterminer leur activité avant de les administrer, il est possible de les cribler en étudiant leur capacité à effectuer la transcription d'un gène marqueur couplé à un promoteur associé à une protéine morphogénétique osseuse, et/ou leur capacité à stimuler la croissance de la calotte crânienne dans les systèmes de modèles animaux.
PCT/US1997/018864 1996-10-23 1997-10-23 Compositions et methodes pour traiter les etats associes a un deficit osseux WO1998017267A1 (fr)

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AU49889/97A AU4988997A (en) 1996-10-23 1997-10-23 Compositions and methods for treating bone deficit conditions
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