HK1230945B - Hydantoin derivative-containing pharmaceutical composition - Google Patents

Description

Pharmaceutical composition containing hydantoin derivatives

Technical Field

The present invention relates to a pharmaceutical product containing a hydantoin derivative as an active ingredient that has high metabolic stability and exerts a potent PTH-like effect. The pharmaceutical product is intended to provide a drug that induces bone and/or cartilage assimilation and is used to promote the prevention, treatment, recovery, and cure of osteoporosis, bone loss in periodontal disease, alveolar bone damage after tooth extraction, osteoarthritis, articular cartilage damage, adynamic bone disease, achondroplasia, hypochondroma, osteomalacia, and bone fractures.

Background Art

Parathyroid hormone (PTH) acts on the target cells of bone and kidney, and is known as the hormone regulating calcium (Ca) and phosphorus (Pi) homeostasis (non-patent literature 1). Mainly carried out by direct or indirect effect for digestive tract, bone and kidney based on the maintenance of serum Ca concentration level of PTH. PTH promotes the reabsorption of Ca by renal tubules, suppresses Ca excretion in body. In addition, by improving the synthesis of the enzyme of vitamin D converted into active vitamin D in kidney, thereby contributes to promoting the Ca absorption based on active vitamin D from digestive tract. In addition, PTH indirectly promotes the differentiation of osteoclasts by osteoblasts etc., thereby promotes to discharge Ca from bone. It is believed that the effect of above-mentioned PTH is mainly played by the activation (based on cAMP combined with PTH1R) of the receptor based on adenylate cyclase and/or phospholipase C.

In humans, PTH preparations (PTH (1-34) and PTH (1-84)) have potent bone-anabolizing effects, inducing a significant increase in bone density and strength. Currently, most osteoporosis treatments available to humans are bone resorption inhibitors, and PTH preparations are the only drugs with bone-anabolizing effects that actively increase bone density. Therefore, while PTH preparations are considered one of the most effective treatments for osteoporosis (Non-Patent Document 2), they require invasive administration because they are peptides. Therefore, there is a desire for the development of drugs that exhibit PTH-like effects and can be administered non-invasively.

Osteoarthritis (OA) is a degenerative disease characterized by joint dysfunction caused by degeneration and destruction of cartilage in joints throughout the body, including the knee, hip, spine, and fingers, as well as synovitis, sclerosis of the subchondral bone, bone spur formation, and chronic pain. It affects over 40% of the population over 65 years old, placing a significant burden on healthcare economics (Non-Patent Documents 3 and 4). Causes of OA include excessive physical damage to articular cartilage, inflammation of the synovium and bone marrow, genetic factors affecting cartilage matrix components, and hypermetabolism in the subchondral bone. However, therapeutic drugs that inhibit the degeneration and destruction of articular cartilage are not yet commercially available, and medical needs remain high.

At present, as the target of therapeutic drug, the proteoglycanase (ADAMTS-4, ADAMTS-5 etc.), matrix metalloproteinase (MMP-3, MMP-9, MMP-13 etc., non-patent literature 5) and inflammatory cytokine (IL-1, IL-6 etc., non-patent literature 6) relevant to the destruction of cartilage matrix receive attention, but have not yet been put into practical use.On the other hand, the clinical trial of the medicament (risedronate, calcitonin, non-patent literature 7, non-patent literature 8) with the metabolic renewal hyperactivity of subchondral bone as target is also being carried out, but fails to suppress the degeneration destruction of articular cartilage.In addition, in the clinical trial of strontium ranelate that also has bone formation promoting effect and cartilage formation promoting effect concurrently except this mechanism, demonstrated the effect (non-patent literature 9) of suppressing the destruction of articular cartilage, but have not yet been put into practical use.

However, recent studies have shown that the pathogenesis of osteoarthritis involves the conversion of articular cartilage from a permanent cartilage form to calcified cartilage, and its inhibition has attracted attention as a target for therapeutic drugs (Non-Patent Document 10). Based on this mechanism of action, drugs that inhibit the terminal differentiation of articular cartilage by multiple mechanisms have been reported to inhibit the degeneration and destruction of articular cartilage in osteoarthritis model animals, suggesting the potential for practical application of therapeutic drugs based on this mechanism (Non-Patent Documents 11 and 12).

Under the above circumstances, the present applicants have discovered that the compound represented by the following general formula (A) or a pharmacologically acceptable salt thereof is useful as a compound having a PTH-like action (preferably a PTH1R agonist), and is useful for the prevention and/or treatment of osteoporosis, fractures, osteomalacia, arthritis, thrombocytopenia, hypoparathyroidism, hyperphosphatemia, tumoral calcification, etc., or for stem cell mobilization, and have previously filed a patent application (Patent Document 1).

[Wherein, W, X, Y, m, n, R ₁ , R ₂ , R ₃₃ , and R ₃₄ are as described in Patent Document 1].

However, the creation of clinically valuable non-invasively administered drugs requires consideration of the direct action on the target, as well as the in vivo dynamics of the drug, including absorption, distribution, metabolism, and excretion. Therefore, for oral administration, drugs with strong cAMP production via the human PTH1R and high metabolic stability relative to human liver microsomes are desired.

Prior art literature

Patent Literature

Patent Document 1: International Publication No. 2010/126030

Non-patent literature

Non-patent document 1: Kronenberg, H.M., et al., In Handbook of Experimental Pharmacology, Mundy, G.R., and Martin, T.J., (eds), pp.185-201, Springer-Verlag, Heidelberg (1993) [Non-patent document 2: Tashjian and Gagel, J. Bone Miner. Res. 21: 354-365 (2006)

Non-patent literature 3: Sem Arth Rheumatism 2013; 43: 303-13

Non-patent document 4: CPMP/EWP/784/97 Rev. 1. 2010, European Medicines Agency

Non-patent document 5: Osteoarth Cart 2010;18:1109-1116

Non-patent literature 6: Osteoarth Cart 2013; 21: 16-21

Non-patent literature 7: Arthritis Rheum. 2006; 54(11): 3494-507

Non-patent literature 8: J Clin Pharmacol. 2011; 51(4): 460-71

Non-patent literature 9: Ann Rheum Dis. 2013 Feb; 72(2): 179-86

Non-patent literature 10: Arth Rheum 2006; 54(8): 2462-2470

Non-patent literature 11: Nat Med 2009; 15(12): 1421-1426

Non-patent literature 12: Sci Trans Med 2011;3:101ra93.

Summary of the Invention

Problems to be solved by the invention

The present invention provides a method for promoting the prevention, treatment, recovery, and cure of osteoporosis, bone loss in periodontal disease, alveolar bone damage after tooth extraction, osteoarthritis, articular cartilage damage, adynamic bone disease, achondroplasia, hypochondroma, osteomalacia, bone fractures, and the like by non-invasive systemic or local exposure of a hydantoin derivative having high metabolic stability and exerting a potent PTH-like action.

Solutions to the Problem

Under the above circumstances, the present inventors have conducted extensive research and have discovered that the newly discovered hydantoin derivatives of the present invention exhibit potent cAMP production in cells forcibly expressing human PTH1R and are highly stable with respect to metabolism in human liver microsomes. Furthermore, the present inventors have discovered that administration of the compounds of the present invention induces bone and cartilage assimilation, making them useful as pharmaceutical compositions for promoting the prevention, treatment, recovery, and treatment of conditions such as osteoporosis, bone loss in periodontal disease, alveolar bone damage after tooth extraction, osteoarthritis, articular cartilage damage, adynamic bone disease, achondroplasia, hypochondroma, osteomalacia, and bone fractures.

That is, the present invention relates to the following aspects.

[1] A pharmaceutical composition for inducing bone and/or cartilage assimilation, comprising a compound represented by the following general formula (1) or a pharmacologically acceptable salt thereof as an active ingredient;

〔where,

R1 and R2, unless both R1 and R2 are hydrogen atoms, are each independently:

1) Hydrogen atoms,

2) Halogen atoms,

3) an alkyl group having 1 to 2 carbon atoms which may be substituted with 1 to 5 fluorine atoms, or

4) an alkoxy group having 1 to 2 carbon atoms which may be substituted with 1 to 5 fluorine atoms; or

R1 and R2 are bonded to each other to form a group represented by the following formula:

(Each * in the formula represents a bonding site with the phenyl moiety.)

Furthermore, R3 and R4 are each independently a methyl group which may be substituted with 1 to 3 fluorine atoms; or

R3 and R4, together with the carbon atoms to which they are bonded, form a ring having 3 to 6 carbon atoms (herein, one of the carbon atoms forming the ring may be substituted by an oxygen atom, a sulfur atom, or a nitrogen atom which may be substituted by a methyl group).

The compound containing the pharmaceutical composition of the present invention as an active ingredient may be selected from the compounds represented by the above general formula (1), except for the compound wherein the combination of R1 and R2 is a trifluoromethyl group and a hydrogen atom, and R3 and R4 together with the carbon atoms to which they are bonded form a cyclopentyl ring.

[2] The pharmaceutical composition described in [1], wherein R1 and R2 of the compound represented by the general formula (1) or a pharmaceutically acceptable salt thereof can be selected from the following combinations:

1) R1 is a hydrogen atom or a halogen atom, and R2 is a hydrogen atom, a trifluoromethyl group or a trifluoromethoxy group (excluding the case where both R1 and R2 are hydrogen atoms);

2) R1 is a trifluoromethyl group or a trifluoromethoxy group, and R2 is a hydrogen atom or a halogen atom;

3) R1 and R2 are bonded to each other to form a group represented by the following formula:

(Each * in the formula represents a site bonded to the phenyl moiety.)

And, R3 and R4 are methyl; or,

R3 and R4 together with the carbon atom to which they are bonded form a ring selected from the following rings:

(* in the formula represents the site bonded to the imidazolidine-2,4-dione moiety.)

[3] The pharmaceutical composition described in [1], wherein R1 and R2 of the compound represented by the general formula (1) or a pharmaceutically acceptable salt thereof can be selected from the following combinations:

1) R1 is a trifluoromethoxy group, and R2 is a fluorine atom;

2) R1 is a bromine atom, and R2 is a hydrogen atom;

3) R1 is a trifluoromethyl group, and R2 is a fluorine atom;

4) R1 is a fluorine atom, and R2 is a trifluoromethoxy group;

5) R1 is a trifluoromethyl group, and R2 is a hydrogen atom;

6) R1 is a hydrogen atom, and R2 is a trifluoromethoxy group;

7) R1 and R2 are bonded to form a group represented by the following formula:

(Each * in the formula represents a site bonded to the phenyl moiety.)

And, R3 and R4 are methyl; or,

R3 and R4 together with the carbon atom to which they are bonded form a ring selected from the following rings:

(* in the formula represents the site bonded to the imidazolidine-2,4-dione moiety.)

[4] The pharmaceutical composition described in [1], wherein R3 and R4 of the compound represented by the aforementioned general formula (1) or a pharmaceutically acceptable salt thereof are methyl groups.

[5] The pharmaceutical composition described in [1], wherein R3 and R4 of the compound represented by the aforementioned general formula (1) or a pharmaceutically acceptable salt thereof, together with the carbon atoms to which they are bonded, form a ring selected from the following rings:

(* in the formula represents the site bonded to the imidazolidine-2,4-dione moiety.)

[6] The pharmaceutical composition described in [1], which contains a compound selected from the following group or a pharmacologically acceptable salt thereof as an active ingredient:

1-(4-(2-((2-(4-fluoro-3-(trifluoromethoxy)phenyl)-4-oxo(oxo)-1,3,8-triazaspiro[4.5]dec-1-en-8-yl)sulfonyl)ethyl)-3,5-dimethylphenyl)-5,5-dimethylimidazolidine-2,4-dione;

1-(4-(2-((2-(3-bromophenyl)-4-oxo-1,3,8-triazaspiro[4.5]dec-1-en-8-yl)sulfonyl)ethyl)-3,5-dimethylphenyl)-5,5-dimethylimidazolidine-2,4-dione:

1-(4-(2-((2-(4-fluoro-3-(trifluoromethyl)phenyl)-4-oxo-1,3,8-triazaspiro[4.5]dec-1-en-8-yl)sulfonyl)ethyl)-3,5-dimethylphenyl)-5,5-dimethylimidazolidine-2,4-dione;

1-(4-(2-((2-(3-fluoro-4-(trifluoromethoxy)phenyl)-4-oxo-1,3,8-triazaspiro[4.5]dec-1-en-8-yl)sulfonyl)ethyl)-3,5-dimethylphenyl)-5,5-dimethylimidazolidine-2,4-dione;

1-(4-(2-((2-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)-4-oxo-1,3,8-triazaspiro[4.5]dec-1-en-8-yl)sulfonyl)ethyl)-3,5-dimethylphenyl)-5,5-dimethylimidazolidine-2,4-dione;

1-(3,5-dimethyl-4-(2-((4-oxo-2-(3-(trifluoromethyl)phenyl)-1,3,8-triazaspiro[4.5]dec-1-en-8-yl)sulfonyl)ethyl)phenyl)-5,5-dimethylimidazolidine-2,4-dione;

1-(3,5-dimethyl-4-(2-((4-oxo-2-(4-(trifluoromethoxy)phenyl)-1,3,8-triazaspiro[4.5]dec-1-en-8-yl)sulfonyl)ethyl)phenyl)-5,5-dimethylimidazolidine-2,4-dione):

1-(3,5-dimethyl-4-(2-((4-oxo-2-(4-(trifluoromethoxy)phenyl)-1,3,8-triazaspiro[4.5]dec-1-en-8-yl)sulfonyl)ethyl)phenyl)-1,3-diazaspiro[4.4]nonane-2,4-dione;

1-(3,5-dimethyl-4-(2-((4-oxo-2-(4-(trifluoromethoxy)phenyl)-1,3,8-triazaspiro[4.5]dec-1-en-8-yl)sulfonyl)ethyl)phenyl)-8-methyl-1,3,8-triazaspiro[4.5]decane-2,4-dione;

5-(3,5-dimethyl-4-(2-((4-oxo-2-(4-(trifluoromethoxy)phenyl)-1,3,8-triazaspiro[4.5]dec-1-en-8-yl)sulfonyl)ethyl)phenyl)-2-oxa-5,7-diazaspiro[3.4]octane-6,8-dione; and

4-(3,5-dimethyl-4-(2-((4-oxo-2-(4-(trifluoromethoxy)phenyl)-1,3,8-triazaspiro[4.5]dec-1-en-8-yl)sulfonyl)ethyl)phenyl)-4,6-diazaspiro[2.4]heptane-5,7-dione.

[7] The pharmaceutical composition described in [1], wherein the compound is 1-(3,5-dimethyl-4-(2-((4-oxo-2-(3-(trifluoromethyl)phenyl)-1,3,8-triazaspiro[4.5]dec-1-en-8-yl)sulfonyl)ethyl)phenyl)-5,5-dimethylimidazolidine-2,4-dione or a pharmacologically acceptable salt thereof.

[8] The pharmaceutical composition described in [1], wherein the compound is 1-(3,5-dimethyl-4-(2-((4-oxo-2-(4-(trifluoromethoxy)phenyl)-1,3,8-triazaspiro[4.5]dec-1-en-8-yl)sulfonyl)ethyl)phenyl)-5,5-dimethylimidazolidine-2,4-dione or a pharmacologically acceptable salt thereof.

[9] The pharmaceutical composition described in [1], wherein the compound is 1-(3,5-dimethyl-4-(2-((4-oxo-2-(4-(trifluoromethoxy)phenyl)-1,3,8-triazaspiro[4.5]dec-1-en-8-yl)sulfonyl)ethyl)phenyl)-1,3-diazaspiro[4.4]nonane-2,4-dione or a pharmacologically acceptable salt thereof.

[10] A pharmaceutical composition according to [1], wherein the pharmaceutical composition is used for the prevention or treatment of osteoporosis, improvement of bone mass loss in periodontal disease, promotion of recovery of alveolar bone damage after tooth extraction, prevention or treatment of osteoarthritis, promotion of recovery of articular cartilage damage, prevention or treatment of adynamic bone disease, prevention or treatment of achondroplasia, prevention or treatment of hypochondroma, prevention or treatment of osteomalacia, or promotion of recovery of fractures.

[11] A method for inducing bone and/or cartilage assimilation, comprising administering a pharmaceutically effective amount of a compound described in any one of [1] to [9] or a pharmacologically acceptable salt thereof to a patient in need of prevention or treatment of osteoporosis, improvement of bone mass loss in periodontal disease, promotion of recovery of alveolar bone damage after tooth extraction, prevention or treatment of osteoarthritis, promotion of recovery of articular cartilage damage, prevention or treatment of adynamic bone disease, prevention or treatment of achondroplasia, prevention or treatment of hypochondroma, prevention or treatment of osteomalacia, or promotion of recovery of fractures.

[12] The method according to [11], wherein the method for inducing bone and/or cartilage assimilation is a method for preventing or treating osteoporosis, a method for improving bone mass loss in periodontal disease, a method for promoting the recovery of alveolar bone damage after tooth extraction, a method for preventing or treating osteoarthritis, a method for promoting the recovery of articular cartilage damage, a method for preventing or treating adynamic bone disease, a method for preventing or treating achondroplasia, a method for preventing or treating hypochondroma, a method for preventing or treating osteomalacia, or a method for promoting the recovery of fractures.

[13] Use of a compound or a pharmacologically acceptable salt thereof according to any one of [1] to [9] in the manufacture of a pharmaceutical composition for the prevention or treatment of osteoporosis, improvement of bone mass loss in periodontal disease, promotion of recovery of alveolar bone damage after tooth extraction, prevention or treatment of osteoarthritis, promotion of recovery of articular cartilage damage, prevention or treatment of adynamic bone disease, prevention or treatment of achondroplasia, prevention or treatment of hypochondroma, prevention or treatment of osteomalacia, or promotion of recovery of fractures.

[14] Use of the compound described in any one of [1] to [9] or a pharmacologically acceptable salt thereof in the manufacture of a pharmaceutical composition for inducing bone and/or cartilage assimilation.

[15] A compound or a pharmacologically acceptable salt thereof according to any one of [1] to [9], which is used for the prevention or treatment of osteoporosis, improvement of bone mass loss in periodontal disease, promotion of recovery of alveolar bone damage after tooth extraction, prevention or treatment of osteoarthritis, promotion of recovery of articular cartilage damage, prevention or treatment of adynamic bone disease, prevention or treatment of achondroplasia, prevention or treatment of hypochondroma, prevention or treatment of osteomalacia, or promotion of recovery of fractures.

In addition, the present invention provides a method for treating a condition that can be prevented, treated and/or restored by bone and/or cartilage assimilation, said method being carried out by administering the compound of formula (1) or a pharmaceutically acceptable salt thereof.

Effects of the Invention

The present invention can provide a method for inducing bone and/or cartilage assimilation by using a hydantoin derivative having a strong PTH-like action and high metabolic stability, thereby promoting the prevention, treatment, recovery and/or cure of osteoporosis, bone loss in periodontal disease, alveolar bone damage after tooth extraction, osteoarthritis, articular cartilage damage, adynamic bone disease, achondroplasia, hypochondroma, osteomalacia, bone fractures, and the like.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the bone density of the lumbar vertebrae and femur in ovariectomized rats after repeated administration of the drug for 6 weeks. Specifically, the graph shows the results of measuring the bone density of the lumbar vertebrae and femur in ovariectomized rats using a dual X-ray bone mineral density analyzer after repeated administration of vehicle, Compound 7, or hPTH (1-34) once daily for 6 weeks.

Figure 2 shows the bone density of the lumbar vertebrae and lower leg bones in normal rats after repeated administration of the drug for 4 weeks. Specifically, the graph shows the results of bone density measurements of the lumbar vertebrae and lower leg bones in normal rats using a dual X-ray bone mineral density analyzer after repeated administration of vehicle, Compound 7, or hPTH (1-34) once daily for 4 weeks.

Figure 3 shows the bone density of the mandible in normal rats after repeated administration for 4 weeks. Specifically, the graph shows the results of mandibular bone density measurements using a dual X-ray bone mineral density analyzer when normal rats were administered vehicle, Compound 7, or hPTH (1-34) once daily for 4 weeks.

Figure 4 shows the inhibitory effect of Compound 7 on the terminal differentiation of rabbit hamstring articular chondrocytes. Specifically, the graph shows the results of evaluating the inhibitory effect of Compound 7 and hPTH (1-34) on the terminal differentiation of rabbit hamstring articular chondrocytes using alkaline phosphatase staining (A) and Alizarin Red S staining (B).

[ Fig. 5 ] A graph showing the amount of proteoglycan synthesis in human chondrocytes. Specifically, it shows the results of evaluating the effects of Compound 7 and hPTH (1-34) on promoting proteoglycan synthesis in human chondrocytes.

Figure 6 shows the area ratio of the lesioned articular cartilage in the lower leg of a rabbit partial meniscectomy model. Specifically, it shows the results of measuring the area ratio of the lesioned articular cartilage in the lower leg of a rabbit partial meniscectomy model after continuous intra-articular administration of vehicle or compound 7.

Figure 7 shows the macroscopic changes in the articular cartilage of the lower femur in a rabbit partial meniscectomy model two weeks after surgery. Specifically, the figure shows the results of macroscopic observation of the changes in the articular cartilage of the lower femur in a rabbit partial meniscectomy model two weeks after surgery, following continuous intra-articular administration of vehicle or compound 7.

Figure 8 shows histological images of distal femoral articular cartilage in a representative example after repeated oral administration of a vehicle or compound 7 for four weeks to normal rats. Specifically, the images show the results of optical microscopic histopathological observation of distal femoral articular cartilage in a representative example after repeated oral administration of a vehicle or compound 7 for four weeks to normal rats.

FIG9 is a graph showing the average change in serum Ca concentration up to 24 hours after administration of each compound when the compound was orally administered at a dose of 30 mg/kg to a TPTX rat model.

DETAILED DESCRIPTION

The present invention relates to hydantoin derivatives having high metabolic stability and exerting strong PTH-like effects and their use. The present inventors synthesized the compound represented by the above formula (1) or a pharmacologically acceptable salt thereof and found that the compound or the salt thereof induces bone and/or cartilage assimilation.

As used herein, an "alkyl group" is a monovalent group derived from an aliphatic hydrocarbon by removing one arbitrary hydrogen atom. It includes hydrocarbon groups or partial collections of hydrocarbon structures that contain hydrogen and carbon atoms, without heteroatoms or unsaturated carbon-carbon bonds in the backbone. Alkyl groups include both straight-chain and branched structures. Alkyl groups preferably have 1 or 2 carbon atoms. Specific examples of alkyl groups include methyl and ethyl, with methyl being preferred.

In the present specification, an "alkoxy group" refers to an oxy group to which the above-defined "alkyl group" is bonded, and is preferably an alkoxy group having 1 or 2 carbon atoms. Specific examples include a methoxy group and an ethoxy group, and a methoxy group is preferred.

In this specification, "B which may be substituted with A" means that any hydrogen atom in B may be substituted with any number of A's.

In the present invention, the number of substituents is not particularly limited, and examples thereof include 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 substituent.

In the present specification, the "halogen atom" refers to a fluorine atom, a chlorine atom, a bromine atom or an iodine atom.

In this specification, "*" in a chemical formula refers to a bonding site.

The compound represented by the above formula (1) of the present invention has a strong PTH-like effect and high metabolic stability.

The term "PTH-like action" as used herein refers to an activity that acts on a PTH receptor or a signal transduction system via a PTH receptor to generate intracellular cAMP (cAMP: cyclic adenosine monophosphate).

In the present invention, the presence or absence of "strong PTH-like action," "strong PTH-like action," or "having a potent PTH-like action" can be confirmed, for example, by measuring cAMP signaling activity using a cAMP signaling assay according to the method described in J. Bone. Miner. Res. 14:11-20, 1999. Specifically, for example, using a commercially available cAMP EIA kit (e.g., Biotrack cAMP EIA system, GE Healthcare) according to the method described in Reference Test Example 1, the concentration at which each compound exhibits 20% cAMP signaling activity (EC20) or 50% cAMP signaling activity (EC50) is determined, with the cAMP signaling activity upon administration of 100 nM human PTH (1-34) being 100%. "Strong PTH-like action" or "strong PTH-like action" in the present invention means, for example, that the EC20 value (μM) measured by the above method is preferably 5.0 or less, more preferably 3.0 or less, and even more preferably 2.0 or less. In the case of EC50, for example, the value (μM) measured by the above-mentioned method is preferably 25.0 or less, more preferably 15.0 or less, and even more preferably 10.0 or less.

In addition, whether "high metabolic stability" or "high metabolic stability" exists can be confirmed using conventional measurement methods. For example, it can be confirmed using hepatocytes, small intestinal cells, liver microsomes, small intestinal microsomes, liver S9, etc. Specifically, for example, it can be confirmed by measuring the stability of the compound in liver microsomes according to the description of T Kronbach et al. (Oxidation of midazolam and triazolam by human liver cytochrome P450IIIA4. Mol. Pharmacol, 1989, 36 (1), 89-96). More specifically, it can be confirmed according to the method described in Reference Test Example 3. The "high metabolic stability" or "high metabolic stability" of the present invention, for example, means that the clearance rate (μL/min/mg) in the metabolic stability test using human liver microsomes described in the above-mentioned reference test example is preferably 60 or less, more preferably 40 or less, and even more preferably 35 or less. In particular, in the above formula (1), except for the case where the combination of R1 and R2 is a trifluoromethyl group and a hydrogen atom, and R3 and R4 together with the carbon atoms to which they are bonded form a cyclopentyl ring, high metabolic stability can be obtained.

In addition, the presence or absence of "bone and/or cartilage assimilation induction effect" can be confirmed using a known method.

Regarding the induction of bone assimilation, for example, after the test compound is continuously administered for a certain period of time, bone density or bone mass can be measured using conventional measurement methods and confirmed by comparison with a control. Specifically, for example, bone density can be measured using a dual X-ray bone mineral content measurement device (for example, DCS-600EX (Aroka Co., Ltd.)) according to the method described in Takeda's document (Bone 2013; 53 (1): 167-173). In this case, when the bone density is higher than that of the solvent control, it can be considered that bone assimilation is induced. The compound of the present invention preferably shows an increase in bone density that is equal to or greater than the increase in bone density when the subject is administered a clinically equivalent dose of hPTH (1-34) used as an osteoporosis therapeutic drug. Specifically, for example, it is preferred that the bone density increases by 8 to 12% relative to the solvent control, and more preferably increases by 12% or more.

Regarding the induction of cartilage assimilation, for example, chondrocytes can be cultured in the presence of the compound of the present invention and the amount of matrix (e.g., proteoglycan) produced by the chondrocytes can be determined. In addition, it can also be determined by determining whether the final differentiation and calcification of the chondrocytes are inhibited. Specifically, for example, it can be determined according to the method described in the literature of Loeser et al. (Athr Rheum 2003; 48(8): 2188-2196) and the literature of Ab-Rahim et al. (Mol Cell Biochem 2013; 376: 11-20.) on the amount of cartilage matrix production. In addition, regarding the inhibition of terminal differentiation, it can be evaluated according to the method of the literature of Okazaki et al. (Osteoarth Cart 2003; 11(2): 122-32.). At this time, when the amount of cartilage matrix production, final differentiation, and calcification are hyperactive and inhibited compared to the control, it can be considered that cartilage assimilation is induced. For example, the compounds of the present invention preferably have an effect equivalent to or greater than that of PTH in inhibiting cartilage matrix production and terminal chondrocyte differentiation. Compared to PTH, the compounds of the present invention have higher metabolic stability, thus providing sufficient effects for the aforementioned pathological conditions and allowing for multiple administration routes. Furthermore, if a higher effect than PTH is achieved in cartilage matrix production, superior effects compared to PTH can be achieved for the aforementioned pathological conditions.

In addition, for example, by taking the cartilaginous bone of the object of taking the test compound continuously for a certain period of time, it is carried out pathological histological observation, confirming the hypertrophy of articular cartilage and growth plate, and the induction of cartilage assimilation can also be confirmed. Specifically, the thickness of the cartilage of articular cartilage and growth plate can be measured histologically. At this time, when the hypertrophy of cartilage increases compared with the control, it can be considered that the cartilage assimilation of the test compound is induced. Especially when the cartilage hypertrophy is significantly compared with PTH, it is believed that there is a sufficient effect on the aforementioned morbid state, so it is preferred, more preferably, that the test compound is administered orally and has an effect.

Alternatively, for example, according to the method of Kikuchi et al. (Osteoarth Cart 1996; 4(2): 99-110) or the method of Sampson et al. (Sci Transl Med 2011; 3: 101ra93), after partial meniscus removal to destabilize the knee joint and induce osteoarthritis in animals (rodents and non-rodents), the degeneration state of the articular cartilage in the knee joint is evaluated visually or histopathologically, thereby confirming the effect. In this case, as with PTH, if the degeneration of the articular cartilage in the knee joint is inhibited, it can be determined that the test compound has an effect of inhibiting cartilage assimilation and terminal differentiation. More preferably, these effects are obtained by oral administration of the test compound.

Alternatively, for example, according to the method of Wakitani et al. (Bone Joint Surg Br. 1989; 71(1): 74-80.), a test compound can be administered to a subject with articular cartilage and subchondral bone damage for a certain period of time, and the regeneration of the cartilage in the damaged area can be evaluated by analyzing the state of the cartilage. In this case, if a superior cartilage regeneration effect is observed compared to the control, it can be considered that the cartilage assimilation effect of the compound is induced. In particular, when the cartilage regeneration effect is more significant than that of PTH, it is considered that there is a sufficient effect for the aforementioned pathological condition, and therefore, it is preferred, and more preferred, that the test compound be administered orally to exert its effect.

These effects can be confirmed by measuring PTH-like effects. PTHrP, which activates the PTH receptor PTH1R through paracrine secretion, is an important factor involved in regulating the proliferation and differentiation of chondrocytes. It is known to inhibit the terminal differentiation of chondrocytes and maintain cartilage tissue (Science 1996; 273: 663-666.). The cartilage assimilation effect based on the activation of PTH1R can be evaluated by administering the test compound to normal or genetic growth-disordered subjects for a certain period of time according to the method of Xie et al. (Human Mol Genet 2012; 21 (18): 3941-3955), and analyzing the growth rate of cartilaginous bone and the histological growth plate hypertrophy. In this case, if the growth rate and growth plate hypertrophy are confirmed compared to the control, it can be determined that the test compound has a cartilage assimilation effect. In particular, it is preferred that the effect of the non-test compound is superior to that of PTH, and it is more preferred that the test compound has an effect when administered orally.

The compounds of the present invention may be free forms, but pharmacologically acceptable salts are also encompassed by the present invention. Examples of such "salts" include inorganic acid salts, organic acid salts, inorganic base salts, organic base salts, and acidic or basic amino acid salts.

Preferred examples of inorganic acid salts include hydrochlorides, hydrobromides, sulfates, nitrates, and phosphates, and preferred examples of organic acid salts include acetates, succinates, fumarates, maleates, tartrates, citrates, lactates, stearates, benzoates, methanesulfonates, benzenesulfonates, and p-toluenesulfonates.

Preferred examples of inorganic base salts include alkali metal salts such as sodium salts and potassium salts, alkaline earth metal salts such as calcium salts and magnesium salts, aluminum salts, ammonium salts, and preferred examples of organic base salts include diethylamine salts, diethanolamine salts, meglumine salts, and N,N-dibenzylethylenediamine salts.

Preferred examples of acidic amino acid salts include aspartate and glutamate, and preferred examples of basic amino acid salts include arginine, lysine, and ornithine.

When the compound of the present invention is exposed to the atmosphere, it may absorb moisture, carry adsorbed water, or form a hydrate. Such a hydrate also belongs to the salt of the present invention.

Furthermore, the compound of the present invention may absorb other types of solvents to form solvates, and such salts are also included in the present invention as salts of the compound of formula (1).

In this specification, for convenience, the structural formula of a compound may sometimes be expressed as a specific isomer. However, the present invention encompasses all geometric isomers, optical isomers based on asymmetric carbon atoms, stereoisomers, tautomers, and other isomers and mixtures of isomers formed according to the structure of the compound. The present invention is not limited to the structural formula described for convenience and may be any one isomer or a mixture. Therefore, although the compounds of the present invention may sometimes contain optically active forms and racemates having asymmetric carbon atoms in the molecule, the present invention is not limited thereto and encompasses all of them.

The present invention includes all isotopes of the compound represented by formula (1). An isotope of the compound of the present invention is a compound in which at least one atom is replaced by an atom having the same atomic number (number of protons) but a different mass number (the sum of the number of protons and ^neutrons ). Examples of isotopes contained in the compound of the present invention include hydrogen atoms, carbon atoms, nitrogen atoms, oxygen atoms, phosphorus atoms, sulfur atoms, fluorine atoms, chlorine atoms, etc., including ^2H , 3H, ^13C , ^14C , ^15N , ^17O , ^18O , ^31P , ^32P , ^35S , ^18F , ^36Cl , etc. In particular, radioactive isotopes such as ^3H and ^14C that can exhibit radioactive decay are useful when conducting in vivo tissue distribution tests for drugs or compounds. Stable isotopes do not decay, their presence amount hardly changes, and they are also non-radioactive and can therefore be used safely. Isotopic compounds of the compounds of the present invention can be converted by replacing the reagents used in the synthesis with reagents containing corresponding isotopes according to conventional methods.

The compound of the present invention may sometimes exist in crystalline polymorphs, but there is no particular limitation and the compound may be any single crystalline form or a mixture of crystalline forms.

The compounds of the present invention include their prodrugs. Prodrugs are derivatives of the compounds of the present invention that have chemically or metabolically decomposable groups. After administration to the body, they return to the original compound and exert their original pharmacological effects. These include complexes and salts that are not based on covalent bonds.

Preferred examples of the compound represented by the above formula (1) of the present invention include the following.

Wherein, R1 and R2 can be selected from the following combinations:

1) R1 is a hydrogen atom or a halogen atom, and R2 is a hydrogen atom, a trifluoromethyl group or a trifluoromethoxy group (excluding the case where both R1 and R2 are hydrogen atoms);

2) R1 is a trifluoromethyl group or a trifluoromethoxy group, and R2 is a hydrogen atom or a halogen atom;

3) R1 and R2 are bonded to each other to form a group represented by the following formula:

(In the formula, * represents the site bonded to the phenyl moiety.)

Furthermore, R3 and R4 are methyl groups;

or R3 and R4 together with the carbon atom to which they are bonded form a ring selected from the group consisting of:

(* in the formula represents the site bonded to the imidazolidine-2,4-dione moiety.)

As the compound represented by the above formula (1) of the present invention, the following are more preferable.

Wherein, R1 and R2 can be selected from the following combinations:

1) R1 is a trifluoromethoxy group, and R2 is a fluorine atom;

2) R1 is a bromine atom, and R2 is a hydrogen atom;

3) R1 is a trifluoromethyl group, and R2 is a fluorine atom;

4) R1 is a fluorine atom, and R2 is a trifluoromethoxy group;

5) R1 is a trifluoromethyl group, and R2 is a hydrogen atom;

6) R1 is a hydrogen atom, and R2 is a trifluoromethoxy group;

7) R1 and R2 are bonded to form a group represented by the following formula:

(In the formula, * represents the site bonded to the phenyl moiety.)

Furthermore, R3 and R4 are methyl groups;

or R3 and R4 together with the carbon atom to which they are bonded form a ring selected from the group consisting of:

(* in the formula represents the site bonded to the imidazolidine-2,4-dione moiety.)

As the compound represented by the above formula (1) of the present invention, a compound selected from the group consisting of the following compounds or a pharmacologically acceptable salt thereof is further preferred.

Compound 1: 1-(4-(2-((2-(4-fluoro-3-(trifluoromethoxy)phenyl)-4-oxo-1,3,8-triazaspiro[4.5]dec-1-en-8-yl)sulfonyl)ethyl)-3,5-dimethylphenyl)-5,5-dimethylimidazolidine-2,4-dione,

Compound 2: 1-(4-(2-((2-(3-bromophenyl)-4-oxo-1,3,8-triazaspiro[4.5]dec-1-en-8-yl)sulfonyl)ethyl)-3,5-dimethylphenyl)-5,5-dimethylimidazolidine-2,4-dione,

Compound 3: 1-(4-(2-((2-(4-fluoro-3-(trifluoromethyl)phenyl)-4-oxo-1,3,8-triazaspiro[4.5]dec-1-en-8-yl)sulfonyl)ethyl)-3,5-dimethylphenyl)-5,5-dimethylimidazolidine-2,4-dione,

Compound 4: 1-(4-(2-((2-(3-fluoro-4-(trifluoromethoxy)phenyl)-4-oxo-1,3,8-triazaspiro[4.5]dec-1-en-8-yl)sulfonyl)ethyl)-3,5-dimethylphenyl)-5,5-dimethylimidazolidine-2,4-dione,

Compound 5: 1-(4-(2-((2-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)-4-oxo-1,3,8-triazaspiro[4.5]dec-1-en-8-yl)sulfonyl)ethyl)-3,5-dimethylphenyl)-5,5-dimethylimidazolidine-2,4-dione,

Compound 6: 1-(3,5-dimethyl-4-(2-((4-oxo-2-(3-(trifluoromethyl)phenyl)-1,3,8-triazaspiro[4.5]dec-1-en-8-yl)sulfonyl)ethyl)phenyl)-5,5-dimethylimidazolidine-2,4-dione,

Compound 7: 1-(3,5-dimethyl-4-(2-((4-oxo-2-(4-(trifluoromethoxy)phenyl)-1,3,8-triazaspiro[4.5]dec-1-en-8-yl)sulfonyl)ethyl)phenyl)-5,5-dimethylimidazolidine-2,4-dione,

Compound 8: 1-(3,5-dimethyl-4-(2-((4-oxo-2-(4-(trifluoromethoxy)phenyl)-1,3,8-triazaspiro[4.5]dec-1-en-8-yl)sulfonyl)ethyl)phenyl)-1,3-diazaspiro[4.4]nonane-2,4-dione,

Compound 9: 1-(3,5-dimethyl-4-(2-((4-oxo-2-(4-(trifluoromethoxy)phenyl)-1,3,8-triazaspiro[4.5]dec-1-en-8-yl)sulfonyl)ethyl)phenyl)-8-methyl-1,3,8-triazaspiro[4.5]decane-2,4-dione,

Compound 10: 5-(3,5-dimethyl-4-(2-((4-oxo-2-(4-(trifluoromethoxy)phenyl)-1,3,8-triazaspiro[4.5]dec-1-en-8-yl)sulfonyl)ethyl)phenyl)-2-oxa-5,7-diazaspiro[3.4]octane-6,8-dione, and Compound 11: 4-(3,5-dimethyl-4-(2-((4-oxo-2-(4-(trifluoromethoxy)phenyl)-1,3,8-triazaspiro[4.5]dec-1-en-8-yl)sulfonyl)ethyl)phenyl)-4,6-diazaspiro[2.4]heptane-5,7-dione.

Among the above-mentioned compounds 1 to 11, the compounds represented by the above-mentioned formula (1) of the present invention such as compounds 6, 7, and 8, or their pharmacologically acceptable salts, are more preferably used in inducing bone-cartilage assimilation, and through this action, they are useful for the prevention or treatment of osteoporosis, improvement of bone mass loss in periodontal disease, promotion of recovery of alveolar bone damage after tooth extraction, prevention or treatment of osteoarthritis, promotion of recovery of articular cartilage damage, prevention or treatment of adynamic bone disease, prevention or treatment of hypochondroma, or prevention or treatment of osteomalacia.

The compounds or salts thereof of the present invention can be prepared into tablets, powders, fine granules, granules, coated tablets, capsules, syrups, buccal preparations, inhalants, suppositories, injections, ointments, eye ointments, eye drops, nasal drops, ear drops, papules, lotions, and the like using commonly used methods. Carriers commonly used in preparations, such as excipients, binders, lubricants, colorants, flavoring agents, and, if necessary, stabilizers, emulsifiers, absorption enhancers, surfactants, pH adjusters, preservatives, antioxidants, and the like can be used, and ingredients commonly used as raw materials for pharmaceutical preparations can be combined to prepare preparations using conventional methods.

For example, to produce an oral preparation, the compound of the present invention or a pharmacologically acceptable salt thereof and an excipient, as well as a binder, a disintegrant, a lubricant, a colorant, a flavoring agent, etc., are added, and then prepared into a powder, fine granules, granules, tablets, coated tablets, capsules, etc. using conventional methods.

Their components include, for example, animal and vegetable oils such as soybean oil, beef tallow, and synthetic glycerides; hydrocarbons such as liquid paraffin, squalane, and paraffin wax; ester oils such as stearyl myristate and isopropyl myristate; higher alcohols such as cetostearyl alcohol mixture and behenyl alcohol; silicone resins; silicone oils; surfactants such as polyoxyethylene fatty acid esters, sorbitan fatty acid esters, glycerol fatty acid esters, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene hydrogenated castor oil, and polyoxyethylene polyoxypropylene block copolymers; water-soluble polymers such as hydroxyethyl cellulose, polyacrylic acid, carboxyvinyl polymer, polyethylene glycol, polyvinyl pyrrolidone, and methylcellulose; lower alcohols such as ethanol and isopropyl alcohol; polyols such as glycerol, propylene glycol, dipropylene glycol, and sorbitol; sugars such as glucose and sucrose; inorganic powders such as silicic anhydride, magnesium aluminum silicate, and aluminum silicate, and purified water.

Examples of the excipient include lactose, corn starch, saccharide, glucose, mannitol, sorbitol, crystalline cellulose, and silicon dioxide.

Examples of the binder include polyvinyl alcohol, polyvinyl ether, methylcellulose, ethylcellulose, gum arabic, tragacanth, gelatin, shellac, hydroxypropyl methylcellulose, hydroxypropyl cellulose, polyvinyl pyrrolidone, polypropylene glycol-polyoxyethylene-block polymer, and meglumine.

Examples of the disintegrant include starch, agar, gelatin powder, crystalline cellulose, calcium carbonate, sodium hydrogen carbonate, calcium citrate, dextrin, pectin, and carboxymethylcellulose calcium.

Examples of the lubricant include magnesium stearate, talc, polyethylene glycol, silicon dioxide, and hydrogenated vegetable oil.

As coloring agents, coloring agents permitted for addition to pharmaceuticals can be used, and as flavoring agents, cocoa powder, menthol, aromatic powder, peppermint oil, borneol, cinnamon powder, etc. can be used.

The tablets and granules may be coated with sugar coatings, and other coatings may be applied as needed. Furthermore, when preparing liquid preparations such as syrups and injections, a pH regulator, a solubilizing agent, an isotonic agent, and the like are added to the compound of the present invention or a pharmacologically acceptable salt thereof, and, if necessary, a dissolution aid, a stabilizer, and the like, and the preparation is prepared using conventional methods.

There is no restriction on the method when manufacturing external-use agent, conventional method can be utilized to manufacture.That is, as the base raw material used when making preparation, the various raw materials normally used in medicine, drug analogs (quasi drugs), cosmetics etc. can be used.As the base raw material used, specifically, the raw materials such as animal and vegetable oils, mineral oil, ester oil, waxes, higher alcohols, fatty acids, silicone oil, surfactant, phospholipids, alcohols, polyols, water-soluble polymers, clay minerals, purified water can be enumerated, in addition, as required, pH adjusting agent, antioxidant, chelating agent, antiseptic and mildew inhibitor, coloring material, spices etc. can be added, and the base raw material of the external-use agent to which the present invention relates is not limited to these.

In addition, as needed, ingredients with differentiation-inducing effects, blood flow promoters, bactericides, anti-inflammatory agents, cell activators, vitamins, amino acids, moisturizers, keratolytic agents, etc. may also be incorporated. It should be noted that the amounts of the base raw materials added are amounts that achieve concentrations typically set during the manufacture of external preparations.

When administering the compound of the present invention, its salt, or its hydrate, there is no particular limitation on its form, and it can be administered orally or parenterally using commonly used methods. For example, it can be prepared into tablets, powders, granules, capsules, syrups, buccal preparations, inhalants, suppositories, injections, ointments, eye ointments, eye drops, nasal drops, ear drops, cataplasms, lotions, and the like for administration.

The dosage and administration method of the drug of the present invention can be appropriately selected according to the severity of symptoms, age, sex, body weight, administration form, type of salt, specific type of disease, and the like.

The dosage varies significantly depending on the type of disease, severity of symptoms, age, sex, and sensitivity to drugs of the patient. Generally, for adults, about 0.03 to 1000 mg, preferably 0.1 to 500 mg, and more preferably 0.1 to 100 mg are administered per day, divided into one to several doses per day.

The route of administration is appropriately selected based on the type of disease, severity of symptoms, age, gender, and sensitivity to the drug. The method of administration is not particularly limited, as long as the compound of the present invention is non-invasively exposed systemically or locally to induce bone and/or cartilage assimilation. Examples of such administration methods include oral administration, intravenous administration, nasal administration, transdermal administration, pulmonary administration, and intra-articular administration.

In the production of the compound of the present invention represented by the above formula (1), the raw material compounds and various reagents may form salts, hydrates or solvates, which vary depending on the starting materials, the solvent used, etc., and are not particularly limited as long as they do not inhibit the reaction.

The solvent used will, of course, vary depending on the starting materials, reagents, etc., and is not particularly limited as long as it does not inhibit the reaction and dissolves the starting materials to a certain extent.

Various isomers (e.g., geometric isomers, optical isomers based on asymmetric carbons, rotational isomers, stereoisomers, tautomers, etc.) can be purified and separated by using conventional separation means (e.g., recrystallization, diastereomeric salt method, enzyme resolution method, various chromatographic methods (e.g., thin layer chromatography, column chromatography, high performance liquid chromatography, gas chromatography, etc.)).

When the compound of the present invention is obtained in the form of a free form, it can be converted into a salt or a hydrate thereof that the compound can form according to a conventional method. In addition, when the compound of the present invention is obtained in the form of a salt or a hydrate of the compound, it can be converted into the free form of the compound according to a conventional method.

The compounds of the present invention can be isolated and purified by applying conventional chemical operations such as extraction, concentration, distillation, crystallization, filtration, recrystallization, and various chromatography methods.

All prior art documents cited in this specification are incorporated herein by reference.

General synthesis method

The compounds of the present invention can be synthesized using a variety of methods. The following routes illustrate some of the synthesis methods. The routes are examples, and the present invention is not limited to the chemical reactions and conditions clearly described. In the following routes, some substituents are excluded for clarity of description, but it is not intended to limit them to the disclosed routes. The representative compounds of the present invention can be synthesized using appropriate intermediates, known compounds and reagents. R1, R2, R3, and R4 in the formula in the following general synthesis method have the same meaning as R1, R2, R3, and R4 in the compound represented by the above-mentioned general formula (1) (the compound represented by formula 1 in the following general synthesis method).

The compound of the present invention (Formula 1) can be synthesized by the production methods (Method A, Method B) shown below.

Route 1 (Method A)

Route 1 is a method in which a carboxylic acid derivative (1) and an amino-amide derivative (2) are condensed to obtain an amide-amide derivative (3), and then a spiroimidazolone ring is constructed by intramolecular cyclization to obtain a hydantoin derivative (Formula 1).

Step 1 is a method of reacting a carboxylic acid derivative (1) with an amino-amide derivative (2). Examples of coupling reagents include N,N'-dicyclohexylcarbodiimide (DCC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU), and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorphophosphonium chloride n-hydrate (DMT-MM). Examples of bases include triethylamine and N,N-diisopropylethylamine. 4-(Dimethylamino)pyridine (DMAP) can be used as a catalyst as needed. Examples of suitable solvents include dichloromethane and N,N-dimethylformamide. Examples of suitable reaction solvents when using DMT-MM include methanol, ethanol, and acetonitrile. The reaction temperature is, for example, 0°C to room temperature, and the reaction time is 0.5 to 24 hours. The obtained amide-amide derivative (3) is isolated by conventional techniques and, if necessary, purified by crystallization or chromatography.

Step 2 is a method in which an amide-amide derivative (3) is cyclized in the presence of an appropriate base such as aqueous sodium hydroxide or potassium tert-butoxide in an appropriate solvent such as ethanol, tert-butanol, or dimethyl sulfoxide. The reaction temperature is, for example, from room temperature to reflux, and the reaction time is 1 to 24 hours. The resulting hydantoin derivative (Formula 1) is isolated using conventional techniques and, if necessary, purified by crystallization or chromatography.

The amino-amide derivative (2) shown in Scheme 1 can be synthesized from the piperidinone derivative (4). Scheme 2 shows a method for synthesizing the amino-amide derivative (2).

Route 2

Step 3 is a Strecker synthesis method for deriving a piperidinone derivative (4) into an amino-nitrile derivative (5). Specifically, the piperidinone derivative (4) is reacted with sodium cyanide or potassium cyanide and ammonium chloride or ammonium acetate in a suitable solvent such as methanol, ethanol, or tetrahydrofuran in the presence or absence of water. The reaction temperature is, for example, room temperature to 80°C, and the reaction time is 2 to 72 hours. The obtained amino-nitrile derivative (5) is isolated using conventional techniques and, if necessary, purified by crystallization or chromatography.

Step 4 is a method for converting the nitrile group into an amide group using alkaline hydrolysis conditions in the presence of hydrogen peroxide. This reaction can be carried out, for example, with reference to Chemistry-A European Journal (2002), 8(2), 439-450.

Step 5 is a method for hydrogenating the olefin of compound (6) in the presence of a catalyst such as palladium carbon or palladium hydroxide on carbon in an _H2 atmosphere in an inert solvent such as methanol, ethanol, trifluoroethanol, dimethylformamide, or dimethylacetamide. The reaction temperature is between room temperature and 80°C, and the reaction may be carried out under pressure. The obtained amino-amide derivative (2) is isolated by conventional techniques and, if necessary, purified by crystallization or chromatography.

The piperidinone derivative (4) shown in Scheme 2 can be synthesized from a known ketal-vinylsulfonyl derivative (7) and a hydantoin-aryl bromide derivative (8). Scheme 3 shows a method for synthesizing the piperidinone derivative (4).

Route 3

Step 6 is a method in which a ketal-vinylsulfonyl derivative (7) and a hydantoin-aryl bromide derivative (8) are coupled in an appropriate solvent such as N-methyl _- 2-piperidone (NMP) in the presence of a palladium catalyst such as tris(dibenzylideneacetone)dipalladium (0) or bis(dibenzylideneacetone)palladium under a N₂ atmosphere, with the addition of a phosphine ligand such as tri-tert-butylphosphine tetrafluoroborate and an appropriate base such as methyldicyclohexylamine. The reaction is carried out at a temperature between 90°C and reflux. The obtained ketal-arylvinylsulfonyl derivative (9) is isolated by conventional techniques and, if necessary, purified by crystallization or chromatography.

Step 7 is a method in which the ketal-aryl vinylsulfonyl derivative (9) is converted into a ketone in a suitable solvent such as aqueous tetrahydrofuran in the presence of an acid such as hydrochloric acid. The reaction temperature is, for example, the boiling point of the solvent, and the reaction time is 1 to 24 hours. The resulting piperidinone derivative (4) is isolated using conventional techniques and, if necessary, purified by crystallization or chromatography.

Hydantoin-aryl bromide derivatives (8) shown in Scheme 3 can be synthesized from 4-bromo-3,5-dimethylaniline (10) and bromoacetic acid derivatives (11) or 2-bromo-5-iodo-1,3-dimethylbenzene (13) and amino acid derivatives (14). Scheme 4 shows the synthesis of hydantoin-aryl bromide derivatives (8).

Route 4

Step 8 is a method in which 4-bromo-3,5-dimethylaniline (10) is alkylated with a bromoacetic acid derivative (11) in a suitable solvent such as N-methyl-2-piperidone (NMP) in the presence of a suitable base such as diisopropylethylamine. The reaction temperature is, for example, room temperature to 100°C, and the reaction time is 1 to 24 hours. The resulting aryl bromide-amino acid derivative (12) is isolated using conventional techniques and, if necessary, purified by crystallization or chromatography.

Step 9 is a method in which 2-bromo-5-iodo-1,3-dimethylbenzene (13) is reacted with an amino acid derivative (14) in the presence of a metal catalyst such as copper (I) iodide to synthesize an aryl bromide-amino acid derivative (12). The reaction can be carried out in the presence of an appropriate base such as diazabicycloundecene (DBU) in an appropriate solvent such as N,N-dimethylacetamide (DMA) at a reaction temperature of about 80-120°C. The obtained aryl bromide-amino acid derivative (12) is isolated using conventional techniques and, if necessary, purified by crystallization or chromatography.

Step 10 is a method in which an aryl bromide-amino acid derivative (12) is reacted with sodium cyanate under acidic conditions to synthesize a hydantoin-aryl bromide derivative (8). A mixed solvent such as acetic acid-dichloromethane is used, for example. The reaction temperature is from room temperature to 60°C, and the reaction time is from 1 to 24 hours. The obtained hydantoin-aryl bromide derivative (8) is isolated by conventional techniques and, if necessary, purified by crystallization or chromatography.

Hydantoin-aryl bromide derivatives (8) shown in Scheme 3 can also be synthesized from 4-bromo-3,5-dimethylaniline (10) and ketone derivatives (15). Scheme 5 shows the synthesis of hydantoin-aryl bromide derivatives (8).

Route 5

Step 11 is a Strecker synthesis method for deriving a ketone derivative (15) into an arylamino-nitrile derivative (16). Specifically, the ketone derivative (15) is reacted with 4-bromo-3,5-dimethylaniline (10) and trimethylsilyl cyanide in an appropriate solvent such as acetic acid. The reaction can be carried out at room temperature or the like, and the reaction time is about 1 to 3 hours. The obtained arylamino-nitrile derivative (16) is isolated using conventional techniques and, if necessary, purified by crystallization or chromatography.

Step 12 is a method in which an arylamino-nitrile derivative (16) is reacted with 2,2,2-trichloroacetyl isocyanate in a suitable solvent such as dichloromethane, and then a reagent such as methanol, water, and triethylamine are added to react under heating conditions to synthesize an iminohydantoin derivative (17). The obtained iminohydantoin derivative (17) is isolated by conventional techniques and, if necessary, purified by crystallization or chromatography.

Step 13 is a method for converting an iminohydantoin derivative (17) into a hydantoin-aryl bromide derivative (8) under acidic conditions. For example, the synthesis can be carried out in an acetic acid-water solvent under heating conditions of approximately 65°C for a reaction time of approximately 1 to 6 hours. The obtained hydantoin-aryl bromide derivative (8) is isolated using conventional techniques and, if necessary, purified by crystallization or chromatography.

Route 6 is a method in which a vinylsulfonamide derivative (18) is subjected to a Heck reaction with a hydantoin-aryl bromide derivative (8) in the presence of a metal catalyst, and then the olefin of the compound (19) is catalytically hydrogenated to obtain a hydantoin derivative (Formula 1).

Route 6 (Method B)

The reaction in step 14 can be carried out according to the method in step 6, and the reaction in step 15 can be carried out according to the method in step 5, to synthesize a hydantoin derivative (Formula 1). The obtained hydantoin derivative (Formula 1) can be isolated using conventional techniques and purified by crystallization or chromatography as needed.

The vinylsulfonamide derivative (18) used in Step 14 can be synthesized with reference to Scheme 2, Scheme 3, and Scheme 12 of WO2010/126030 (A1).

It should be noted that all prior art documents cited in this specification are incorporated into this specification as reference.

The present invention is further illustrated by the following examples, but is not limited thereto.

Example

Example 1: Effect of repeated administration for 6 weeks on bone density in ovariectomized rats

Female Crl:CD (SD) rats purchased from Japan Chiralsu Liber Co., Ltd. were acclimated for at least one week under standard laboratory conditions at 20-26°C and 35-75% humidity before use in the experiments. The rats were provided with free access to tap water and a standard rodent diet (CE-2) containing 1.1% calcium, 1.0% phosphate, and 250 IU/100g of vitamin D3 (Japan Cure Co., Ltd.).

Twelve-week-old rats underwent bilateral ovariectomy (OVX) and sham surgery (Sham). Body weight was measured four weeks after surgery, and rats were divided into groups of six to achieve equal average body weights. Starting from the second day of grouping, each rat was dosed once daily for six weeks. Rats in the Sham control group were administered an oral solvent (vehicle) and a subcutaneous solvent (PC buffer), respectively. Rats in the OVX control group were administered a vehicle and PC buffer, respectively. Rats in the OVX-Compound 7 group were administered Compound 7 dissolved in the vehicle orally at a dose of 30 mg/kg and PC buffer subcutaneously. Rats in the OVX-hPTH (1-34) group were administered a vehicle orally at a dose of 0.9 nmol/kg and hPTH (1-34) dissolved in PC buffer subcutaneously.

Furthermore, the dose used in rats was set at 0.9 nmol/kg to achieve an AUC (area under the blood concentration-time curve) comparable to that achieved when 20 μg of Forteo (registered trademark), a clinically used osteoporosis treatment, is administered to humans. Each group received an oral dose of 5 mL/kg and a subcutaneous dose of 1 mL/kg. The vehicle used was a composition composed of 10% dimethyl sulfoxide (Wako Pure Chemical Industries, Ltd.), 10% Kolliphor EL (Sigma Aldrich Japan Co., Ltd.), and 10% hydroxypropyl-β-cyclodextrin (Japan Food Chemicals Co., Ltd.), adjusted to pH 10 using glycine (Wako Pure Chemical Industries, Ltd.) and sodium hydroxide (Wako Pure Chemical Industries, Ltd.). PC buffer was prepared with 25mmol/L phosphate-citrate buffer, 100mmol/L NaCl, and 0.05% Tween80 to a pH of 5.0. On the second day after the final administration, blood was collected from the abdominal aorta under anesthesia and the rats were euthanized. An autopsy was performed to remove the lumbar vertebrae and femur. The lumbar vertebrae and femur were stored in 70% ethanol. The bone density of the lumbar vertebrae and femur was measured using a dual X-ray bone mineral density measuring device (DCS-600EX, Aroka Co., Ltd.). The lumbar vertebrae bone density was measured at the 2nd to 4th lumbar vertebrae, and the femoral bone density was measured by dividing the femur into 10 equal parts longitudinally and measuring 3 portions at the distal end of the knee. The results are shown in Figure 1.

Data are presented as mean + standard deviation (SE). The following statistical analysis was performed using SAS Preclinical Manual ver. 5.00 (SAS Institute Japan). The significance level was 5% on both sides. Regarding lumbar spine and femoral bone density, a 2-group t-test was performed to compare the sham-control group with the OVX-control group (#P < 0.05), the OVX-control group with the OVX-compound 7 group (*P < 0.05), and the OVX-control group with the OVX-hPTH (1-34) group (∫P < 0.05).

As shown in Figure 1, in terms of femoral bone density, the OVX-control group showed a significant decrease in bone density relative to the Sham-control group, while the positive control OVX-hPTH (1-34) group showed a significant increase relative to the OVX-control group. The rate of increase in the OVX-hPTH (1-34) group relative to the OVX-control group was 8%. Meanwhile, the OVX-Compound 7 group showed a significant increase relative to the OVX-control group, with an increase rate of 12%. Furthermore, in terms of lumbar spine bone density, the OVX-control group showed a significant decrease in bone density relative to the Sham-control group, while the OVX-Compound 7 group showed no significant decrease relative to the OVX-control group, but showed an increasing trend. The OVX-hPTH (1-34) group showed a significant increase relative to the OVX-control group, with an increase rate of 12%.

As described above, repeated oral administration of compound 7 increased the bone density of OVX rats, a pathological model of osteoporosis. Therefore, compound 7 is believed to be effective in preventing, treating, improving, and promoting recovery of diseases that require induction of bone assimilation, increase of bone mass, or bone remodeling, such as osteoporosis, bone loss in periodontal disease, and alveolar bone damage after tooth extraction. Furthermore, for the compound represented by general formula (1), a strong PTH-like effect and high metabolic stability were confirmed in Reference Test Examples 1 to 5, and it is believed that a bone density-increasing effect can be obtained through bone assimilation based on PTH-like effect. It is believed that the compound represented by general formula (1) is effective in preventing, treating, improving, and promoting recovery of diseases that require induction of bone assimilation, increase of bone mass, or bone remodeling, such as osteoporosis, bone loss in periodontal disease, and alveolar bone damage after tooth extraction.

Example 2: Effect of repeated administration for 4 weeks on bone density in normal rats

Female RccHan:WIST rats purchased from Nippon Medical Science Animal Research Institute Co., Ltd. were acclimated for at least one week under standard laboratory conditions at 20-26°C and 30-70% humidity before use in the experiments. The rats had free access to tap water and a standard rodent diet (CR-LPF) (Oryental Yeast Industry Co., Ltd.).

Eight-week-old rats were placed with an indwelling intravenous catheter. The catheter was inserted into the femoral vein at the circumference of the rat, with the tip extending into the posterior vena cava. Body weight was measured one week after surgery, and the rats were divided into groups of 10 to achieve equal average body weights. Two days after grouping, all rats were repeatedly administered intravenously once daily for four weeks. The indwelling catheter was connected to an infusion pump (MEDFUSION SYRINGEINFUSION PUMP Model 2001) for administration.

The vehicle-control group received intravenous administration of the solvent (vehicle). Compound 7 (20 mg/kg, 30 mg/kg, and 50 mg/kg) groups were administered intravenously at doses of 20 mg/kg, 30 mg/kg, and 50 mg/kg, respectively, dissolved in the vehicle. The administration volume for each group was 5 mL/kg, and the administration rate was 5 mL/kg/minute. The vehicle consisted of 5% dimethyl sulfoxide (Wako Pure Chemical Industries, Ltd.), 25% propylene glycol (Kanto Chemical Co., Ltd.), 20% ethanol (Junsei Chemical Co., Ltd.), 15% hydroxypropyl-β-cyclodextrin (Nippon Food Chemical Co., Ltd.), 300 mM glycine (Wako Pure Chemical Industries, Ltd.), 192 mM sodium hydroxide (Wako Pure Chemical Industries, Ltd.), and normal saline (Otsuka Pharmaceutical Co., Ltd.). On the second day after the final administration, rats were euthanized by collecting blood from the abdominal aorta under anesthesia. Lumbar vertebrae, femurs, and mandibles were collected by autopsy. These lumbar vertebrae, femurs, and mandibles were stored in 70% ethanol. Bone density of the lumbar vertebrae (2nd to 4th lumbar vertebrae), femurs, and mandibles was measured using a dual X-ray bone mineral density analyzer (DCS-600EX, Aroka Co., Ltd.). The results are shown in Figures 2 and 3.

Data are presented as mean ± standard deviation (SE). The following statistical analysis was performed using SAS Preclinical Manual ver. 5.00 (SAS Institute Japan). The significance level was 5% on both sides. Regarding lumbar spine, lower leg, and mandibular bone density, parametric Dunnett-type multiple comparisons were performed with the vehicle-control group as the control group against the three dose groups of compound 7 (*P < 0.05).

As shown in Figure 2, the Compound 7-administered group demonstrated a significant dose-dependent increase in lumbar spine and lower leg bone density compared to the vehicle-control group. Furthermore, the Compound 7-20 mg/kg, 30 mg/kg, and 50 mg/kg groups demonstrated increases in lumbar spine bone density of 16%, 21%, and 25%, respectively, compared to the vehicle-control group, and increases in lower leg bone density of 7%, 16%, and 19%, respectively. Furthermore, as shown in Figure 3, the Compound 7-30 mg/kg group demonstrated a significant increase in mandibular bone density compared to the vehicle-control group, achieving a 7% increase relative to the vehicle-control group.

As described above, repeated oral administration of compound 7 increased the femoral bone density of OVX rats, a pathological model of osteoporosis, and repeated intravenous administration of compound 7 increased the lumbar spine, lower leg bone, and mandibular bone density of normal rats. Therefore, systemic exposure of compound 7 is believed to be effective for the prevention, treatment, improvement, and promotion of recovery of diseases requiring induction of bone assimilation, increase of bone mass, or bone remodeling, such as osteoporosis, bone loss in periodontal disease, and alveolar bone damage after tooth extraction. Furthermore, in Reference Test Examples 1 to 5, a strong PTH-like effect and high metabolic stability were confirmed for the compound represented by general formula (1), and it is believed that a bone density-increasing effect is achieved through bone assimilation based on a PTH-like effect. Therefore, the compound represented by general formula (1) is believed to be effective for the prevention, treatment, improvement, and promotion of recovery of diseases requiring induction of bone assimilation, increase of bone mass, or bone remodeling, such as osteoporosis, bone loss in periodontal disease, and alveolar bone damage after tooth extraction.

Example 3: Inhibitory effect of compound 7 on terminal differentiation of rabbit articular chondrocytes

NZW rabbits (4 weeks old, Oriental Yeast Co., Ltd.) were euthanized, and articular cartilage was harvested from the lower leg bones and transferred to 50 mL test tubes (Beconn Dikinson, Japan). PBS (Nakarai Tesque Co., Ltd.) containing 1% trypsin (Wako Pure Chemical Industries, Ltd.) was added to the tubes, and the soft tissue was digested at 37°C for 1 hour. The mixture was then centrifuged at 1,200 rpm for 5 minutes, the supernatant removed, and the cartilage tissue suspended in PBS (-). This process was repeated three times, followed by centrifugation at 1,200 rpm for 5 minutes, the supernatant removed, and the suspension washed with PBS (-). After centrifugation at 1,200 rpm for 5 minutes, the cells were digested in a cell plate with DMEM (Lifetech Norovirus Digest, Japan Co., Ltd.) containing 0.2% Type II collagenase (CLS-2, Worthington Biochemical Corp.) at 37°C for 3 hours. 10% v/v fetal bovine serum (Lifetech Norovirus Digest, Japan Co., Ltd.) was added to stop the reaction, and chondrocytes were separated by vigorous pipetting using a 10 mL pipette (Japan Biotech Norovirus Digest, Ltd.). After centrifugation at 1,200 rpm for 5 minutes, the supernatant was removed and the cells were suspended and washed three times in DMEM containing 10% FCS. Chondrocytes were then seeded at ^1x104 cells/well in a 96-well culture plate coated with type I collagen (AGC TECHNO GLASS CO., LTD.). The medium was changed three times a week. Once the cells reached confluence, they were cultured in DMEM supplemented with 100 μg/mL magnesium ascorbate phosphate n-hydrate (Wako Pure Chemical Industries, Ltd.), 10 mmol/L β-glycerophosphate pentahydrate (Wako Pure Chemical Industries, Ltd.), and 10% FCS. At this point, the cells were cultured under the following conditions and evaluated for terminal chondrocyte differentiation by alkaline phosphatase staining (14 days of culture) and Alizarin Red staining (21 days of culture).

For alkaline phosphatase staining, after discarding the culture medium, the chondrocytes were washed once with 200 mmol/L Tris-HCl pH 8.2 buffer, stained according to the protocol of the alkaline phosphatase staining reagent (Vector Red Alkaline Phosphatase Substrate Kit I, Vector Laboratories, Inc.), and photographed using an inverted microscope (Nikon Co., Ltd.) (4x objective).

As a result, BMP-2 showed an increase in alkaline phosphatase activity. Compound 7 and PTH (1-34) both inhibited alkaline phosphatase activity in a concentration-dependent manner (Figure 4A).

For Alizarin Red staining, after discarding the culture medium, chondrocytes were washed twice with PBS and fixed with 100% ethanol (Wako Pure Chemical Industries, Ltd.) for 15 minutes. After discarding the ethanol, the cells were stained with 1% Alizarin Red S (Wako Pure Chemical Industries, Ltd.) for 15 minutes, washed with distilled water, and photographed using an inverted microscope. The results showed a significant increase in Alizarin Red staining for BMP-2, indicating the promotion of calcification. Compound 7 and PTH (1-34) both inhibited Alizarin Red staining in a concentration-dependent manner, thereby inhibiting calcification (Figure 4B).

Example 4: Effect of Compound 7 on Proteoglycan Synthesis in Human Articular Chondrocytes

After purchasing cryopreserved human articular chondrocytes (Lot 2867, Cell Applications Inc.), thaw them in a 37°C water bath and add 15 mL of Basal Medium (Growth Medium, Cell Applications Inc.) containing 10% Growth Supplement to a T75 culture flask (CORNING, Corning Japan KK) for culture. The next day, exchange the medium with 15 mL of Growth Medium and culture for 3 days. Remove the Growth Medium and wash the chondrocyte layer with HBSS (Cell Applications Inc.) and remove it. Add 1 mL of trypsin/EDTA solution (Cell Applications Inc.) and let it stand at room temperature for about 5 minutes to peel the chondrocytes from the flask. After adding 10 mL of Neutalizing Solution (Cell Applications Inc.), the cell count was measured using a Bulker-Turk hemacytometer, transferred to a 15 mL test tube, and centrifuged (1,200 rpm, 5 minutes, Tomy Seiko Co., Ltd.) to prepare a chondrocyte plate. The supernatant was discarded, and the cells were prepared to a concentration of 2 x 10 ⁶ cells/mL in 1.2% sodium alginate (25 mmol/L HEPES/150 mmol/L sodium chloride solution, pH 7.0). The cells were aspirated into a 1 mL syringe with a 22G needle (Telmo Co., Ltd.) and dripped 5 drops per well into a 24-well plate (Corning Japan KK) containing 2 mL of a 10 ² mmol/L CaCl ₂ aqueous solution. The cells were allowed to stand for 5 minutes to form beads. Then, the cells were washed three times with 150 mmol/L sodium chloride solution, cultured in Growth Medium for one day, and then exchanged to Basal Medium (Cell Applications Inc.) containing 1% Growth supplement. At this time, the following factors were added to the medium, and the medium was exchanged three times a week for 13 days.

After 12 days of culture, 35S-labeled sulfuric acid (Pakin Elmer Japan Co., Ltd.) was added to a concentration of 370 kBq/well. After 24 hours, the culture medium was collected in a test tube and stored at 4°C. A 55 mmol/L sodium citrate solution (Nacalai Test Co., Ltd., 1 mL/well) was added to the alginate gel and incubated at 37°C for 10 minutes to allow it to become a sol. The solution was then collected in a microtube (Eppendorf Co., Ltd.) and centrifuged (1,200 rpm, 5 minutes) to prepare a chondrocyte plate. The microtube was suspended in 0.5 mL of 0.2 mol/L Tris-HCl (Sigma-Aldrich Co., LLC.)/5 mmol/L CaCl ₂ (Nacalai Test Co., Ltd.), pH 7.8, containing 1 mg/mL actinase E (Kaikan Pharmaceutical Co., Ltd.), transferred to a 12-well plate (Corning Japan KK), sealed, and incubated overnight in an incubator (Especk Co., Ltd.) set at 50°C. 0.4 mL of this digestion solution was transferred to a test tube, and 250 μL of a 0.1 mg/mL aqueous solution of chondroitin sulfate (Wako Pure Chemical Industries, Ltd.), 2 mmol/L MgSO₄ (Wako Pure Chemical Industries, Ltd.), 2.5 mL _each of 0.2 mol/L Tris-HCl (Sigma-Aldrich)/5 mmol/L CaCl₂ (Nacalai Tesque Co., Ltd.), pH 7.8, and 1% cetypyridinium chrolide (CPC, Wako Pure Chemical Industries, Ltd.)/20 mmol/L NaCl (Nacalai Tesque Co., Ltd.) were added. The mixture was incubated at 37°C for 3 hours. The solution was filtered through a glass filter (GC-50, ADVANTEC) under vacuum and washed with 1% CPC/20 mmol/L NaCl to remove free 35S-labeled sulfuric acid. The filter was transferred to a vial for a liquid scintillation counter, 5 mL of scintillant (Hionic-Fluor, manufactured by Parkin Elmer Japan Co., Ltd.) was added, and the radioactivity was measured using a liquid scintillation counter (TRI-CARB, manufactured by Parkin Elmer Japan Co., Ltd.).

The remaining 0.1 mL of the digestion solution was used for DNA quantification. 100 μL of the chromatographic solution and 450 μL of the digestion solution were mixed with 1 mL of a DNA quantification reagent (Cosmo Bio Co., Ltd.) buffer, and the fluorescence intensity at 458 nm was measured under excitation at 356 nm (Infinite M200, Tecan Group Ltd.).

A standard curve of DNA concentration was prepared by diluting the standard solution (100 μg/mL) provided with the kit by 2-fold. A linear regression equation (Excel, Microsoft) was constructed based on the results of the standard curve to calculate the DNA concentration of the sample.

The radioactivity in each well was normalized to the DNA content (cpm/μg DNA).

The results showed that the positive control TGF-β1 showed 10 times the radioactivity of the solvent control, and the synthesis of proteoglycans increased. PTH (1-34) showed 6 times the radioactivity of the solvent control. Compound 7 showed 3 times the radioactivity of the solvent control at 10-6 mol/L and 10 times the radioactivity of the solvent control at 10-5 mol/L (Figure 5). It is clear from these results that compound 7 has the effect of promoting the synthesis of cartilage matrix in any joint chondrocyte.

Example 5: Effect of Compound 7 on Rabbit Meniscectomy Model

Twelve-week-old NZW male rabbits (Olyental Yeast Industry Co., Ltd.) were bred and acclimated for 5 days. Under isoflurane anesthesia, the lateral skin of the left knee joint was incised, and the lateral collateral ligament and sesamum ligament were removed to expose the lateral meniscus. The central part of the lateral meniscus was resected to a width of 3-4 mm to create an osteoarthritis model (Kikuchi T et al, Osteoarth Cart 1999; 4(2): 99-110.). At this time, the front end of three polyethylene tubes (PE60, Japan Bekton Dekinson Co., Ltd.) connected to three osmotic pumps (2ML1, Durect, Road Cupertino, CA, US) buried subcutaneously in the left thigh were left indwelling in the joint, and the drug solution was continuously administered into the joint. The osmotic pump was filled with 1) solvent control (50% dimethyl sulfoxide/50% raw food, v/v), 2) compound 7 3.0 μg/mL, 3) any one of compound 7 30 μg/mL. Isoflurane anesthesia was performed again 7 days after the operation, and the osmotic pump filled with the same agent as the initial transplant was exchanged. 14 days after partial meniscectomy, the rabbit was euthanized, and the thigh bone and calf bone were taken, immersed and fixed in 20% neutral buffered formalin. Then, the roughening of the articular cartilage surface was stained with India ink (Figure 7). A digital microscope (VHX-2000, Keyence Co., Ltd.) was used to photograph the surface structure of the calf bone, and the area of the India ink-positive lesion and the area of the entire lateral malleolus were measured, and the proportion of the lesion to the entire lateral malleolus was calculated (Figure 6). As a result, the surface compound 7 had a smaller lesion area in a dose-dependent manner. A photograph of the articular cartilage surface of the calf bone at this time is shown in Figure 4.

Example 6: Effects on growth plate cartilage of normal rats after repeated oral administration for 4 weeks

Female RccHan:WIST rats purchased from Nippon Medical Science Animal Research Institute Co., Ltd. were acclimated for at least one week under standard laboratory conditions at 20-26°C and 30-70% humidity before use in the experiments. The rats had free access to tap water and a standard rodent diet (CE-2, Nippon Cure Co., Ltd.) containing 1.1% calcium, 1.0% phosphate, and 250 IU/100g of vitamin D3.

The body weights of 6-week-old rats were measured and divided into groups of 10 rats per group to achieve equal average body weights. All rats were dosed once daily for four weeks, starting on the second day of grouping. The vehicle-control group received oral administration of the vehicle (vehicle). The compound 7-6 mg/kg, compound 7-60 mg/kg, and compound 7-600 mg/kg groups received oral administration of compound 7 suspended in the vehicle at doses of 6 mg/kg, 60 mg/kg, and 600 mg/kg, respectively. Each group received a dose of 2 mL/kg. Propylene glycol (special grade, Kanto Chemical Co., Ltd.) was used as the vehicle. On the second day after the final dose, blood was collected from the abdominal aorta under anesthesia, and the rats were euthanized and autopsied to obtain the femur. The femur was fixed in 10% neutral buffered formalin, desalted, and paraffin-embedded thin-section tissue specimens were prepared (hematoxylin and eosin staining). The distal end of the femur specimens was examined histopathologically using a light microscope. The results are shown in Table 1, and histological images of representative examples are shown in FIG8 .

[Table 1]

Histological changes in femoral growth plate cartilage in normal rats after repeated oral administration for 4 weeks

As shown in Table 1, the Compound 7 group exhibited dose-dependent hypertrophy of the femoral growth plate cartilage compared to the vehicle-control group. The width of the growth plate cartilage (arrow) in a representative example based on the reduced scale of the histological image in Figure 8 was approximately 390 μm in the vehicle-control group and approximately 2940 μm in the Compound 7-600 mg/kg group.

As described above, repeated oral administration of Compound 7 resulted in hypertrophy of the growth plate cartilage of the femur of rats. These effects are based on the induction of cartilage assimilation by Compound 7, the inhibition of terminal cartilage differentiation, or the hyperplasia of cartilage, and oral administration of Compound 7 is considered to be effective in the treatment of osteoarthritis. Furthermore, for the compound represented by general formula (1), strong PTH-like effects and high metabolic stability were confirmed in Reference Test Examples 1 to 5, and it is expected that the cartilage assimilation effect based on the PTH-like effect is effective in the treatment of osteoarthritis.

Reference Examples

The present invention is further described in detail with the following reference examples and reference test examples, but the present invention is not limited thereto. All starting materials and reagents were obtained from commercial suppliers or synthesized using known methods. For ¹ H-NMR spectra, Me ₄ Si was used as an internal standard substance, or no internal standard substance was used, and Mercury 300 (varian), ECP-400 (JEOL), or 400-MR (varian) was used for measurement (s = singlet, d = doublet, t = triplet, brs = broad singlet, m = multiplet). Mass spectrometry was measured using a mass spectrometer, ZQ2000 (Waters), SQD (Waters), or 2020 (Shimazu).

Reference Example 1

1-(4-(2-((2-(4-fluoro-3-(trifluoromethoxy)phenyl)-4-oxo-1,3,8-triazaspiro[4.5]dec-1-en-8-yl)sulfonyl)ethyl)-3,5-dimethylphenyl)-5,5-dimethylimidazolidine-2,4-dione (Compound 1).

(Reaction 1-1)

To a solution of 4-bromo-3,5-dimethylaniline (3.47 g, 17.4 mmol) and diisopropylethylamine (5.3 ml, 30.4 mmol) in DMI (13 ml) was added 2-bromoisobutyric acid (3.86 g, 23.1 mmol) at room temperature. The mixture was heated and stirred at 100°C for 1 hour. Furthermore, 2-bromoisobutyric acid (496 mg, 2.97 mmol) and diisopropylethylamine (0.8 ml, 4.59 mmol) were added, and the mixture was heated and stirred at 100°C for 1 hour.

MeOH (52 ml) and 5N aqueous sodium hydroxide solution (52 ml, 260 mmol) were added to the reaction mixture at room temperature, and the mixture was then heated and stirred at 75°C for 1.5 hours. After cooling, the reaction mixture was added with water, adjusted to pH 5 with 1N aqueous potassium hydrogen sulfate, and extracted with ethyl acetate. The organic layer was washed with water, dried over anhydrous magnesium sulfate, and concentrated to obtain 2-((4-bromo-3,5-dimethylphenyl)amino)-2-methylpropanoic acid (5.79 g) as a crude product.

MS (ESI) m/z = 286, 288 (M+H)

(Reaction 1-2)

To a mixture of 2-((4-bromo-3,5-dimethylphenyl)amino)-2-methylpropanoic acid (5.79 g, the compound obtained in Reaction 1-1) in dichloromethane (62 ml) and acetic acid (62 ml) was added sodium cyanate (5.03 g, 59.8 mmol) at room temperature. The mixture was stirred at room temperature for 3 hours. The reaction mixture was added to a saturated aqueous sodium bicarbonate solution (400 ml), further adjusted to pH 7-8 with a 5N aqueous sodium hydroxide solution, and extracted with ethyl acetate. The organic layer was dried over anhydrous magnesium sulfate and then concentrated under reduced pressure. The resulting solid was washed with ethyl acetate-hexane and then dichloromethane-hexane to obtain 1-(4-bromo-3,5-dimethylphenyl)-5,5-dimethylimidazolidine-2,4-dione (3.80 g, 66%).

MS (ESI) m/z = 311, 313 (M+H)

(Reaction 1-3)

A mixture of 8-(vinylsulfonyl)-1,4-dioxa-8-azaspiro[4.5]decane (431 mg, 1.85 mmol), 1-(4-bromo-3,5-dimethylphenyl)-5,5-dimethylimidazolidine-2,4-dione (575 mg, 1.85 mmol), tris(dibenzylideneacetone)dipalladium(0) (508 mg, 0.55 mmol), tri-tert-butylphosphine tetrafluoroborate (165 mg, 0.55 mmol), and methyldicyclohexylamine (2.1 ml, 9.25 mmol) in N-methyl-2-pyrrolidone (18.5 ml) was stirred at 110°C for 2 hours under a nitrogen stream. The reaction mixture was cooled, quenched with water, and extracted with ethyl acetate. The organic layer was washed with water and saturated brine, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. The resulting residue was purified by amino silica gel column chromatography (dichloromethane-methanol) to give (E)-1-(4-(2-(1,4-dioxa-8-azaspiro[4.5]decan-8-ylsulfonyl)vinyl)-3,5-dimethylphenyl)-5,5-dimethylimidazolidine-2,4-dione (584 mg, 68%).

MS (ESI) m/z = 464 (M+H) +

(Reactions 1-4)

To a solution of (E)-1-(4-(2-(1,4-dioxa-8-azaspiro[4.5]decane-8-ylsulfonyl)vinyl)-3,5-dimethylphenyl)-5,5-dimethylimidazolidine-2,4-dione (1.2 g, 2.58 mmol) in tetrahydrofuran (26 ml) was added dropwise 2N aqueous hydrochloric acid solution (26 ml, 52 mmol) over 10 minutes. The mixture was heated and stirred at 60°C for 2 hours. After cooling, the reaction mixture was adjusted to pH 7 with 2N aqueous sodium hydroxide solution and extracted with ethyl acetate. The organic layer was washed with saturated brine, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (dichloromethane-ethyl acetate) to give (E)-1-(3,5-dimethyl-4-(2-((4-oxopiperidin-1-yl)sulfonyl)vinyl)phenyl)-5,5-dimethylimidazolidine-2,4-dione (998 mg, 92%).

MS (ESI) m/z = 420 (M+H) +

(Reactions 1-5)

To a solution of (E)-1-(3,5-dimethyl-4-(2-((4-oxopiperidin-1-yl)sulfonyl)vinyl)phenyl)-5,5-dimethylimidazolidine-2,4-dione (994 mg, 2.37 mmol) in methanol (24 ml) were added potassium cyanide (188 mg, 2.84 mmol) and ammonium acetate (237 mg, 3.08 mmol) in this order at room temperature. The mixture was heated and stirred at 60-70°C for 3 hours. The reaction mixture was cooled, concentrated under reduced pressure, and diluted with ethyl acetate. The organic layer was washed with water and saturated brine in this order, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (dichloromethane-ethyl acetate) to give (E)-4-amino-1-((4-(5,5-dimethyl-2,4-dioxoimidazolidin-1-yl)-2,6-dimethylphenyl)sulfonyl)piperidine-4-carbonitrile (681 mg, 68%).

¹ H-NMR (300MHz, DMSO-d ₆ ) δ: 1.3 (6H, s), 1.7 (2H, m), 2.0 (2H, m), 2.3 (6H, s), 2.7 (2H, s), 2.9 (2H, m), 3.4 (2H, m), 6.9 (1H, d, J=1 5.9Hz), 7.1 (2H, s), 7.4 (1H, d, J = 15.9Hz), 11.2 (1H, brs)

(Reactions 1-6)

To a solution of (E)-4-amino-1-((4-(5,5-dimethyl-2,4-dioxoimidazolidin-1-yl)-2,6-dimethylstyryl)sulfonyl)piperidine-4-carbonitrile (675 mg, 1.50 mmol) in methanol (7.5 ml) and dimethyl sulfoxide (0.195 ml) were slowly added dropwise, in that order, 2N aqueous sodium hydroxide solution (1.6 ml, 1.6 mmol) and 30% aqueous hydrogen peroxide solution (0.2 ml, 1.95 mmol). The mixture was stirred at room temperature for 1 hour. Ethyl acetate, hexane, and saturated aqueous ammonium chloride were added to the reaction mixture. The precipitated solid was collected by filtration, washed, and dried to obtain (E)-4-amino-1-((4-(5,5-dimethyl-2,4-dioxoimidazolidin-1-yl)-2,6-dimethylstyryl)sulfonyl)piperidine-4-carboxamide (498 mg, 72%).

MS (ESI) m/z = 464 (M+H) +

(Reactions 1-7)

A mixture of (E)-4-amino-1-((4-(5,5-dimethyl-2,4-dioxoimidazolidin-1-yl)-2,6-dimethylphenylethyl)sulfonyl)piperidine-4-carboxamide (1.3 g, 2.8 mmol) and palladium hydroxide/carbon (Pd 20%) (approximately 50% water-wet) (1.3 g) in methanol (21 ml)-dimethylformamide (7 ml) was stirred under a hydrogen atmosphere at room temperature for 4 hours. The reaction mixture was filtered and washed, and the filtrate was concentrated under reduced pressure to give 4-amino-1-((4-(5,5-dimethyl-2,4-dioxoimidazolidin-1-yl)-2,6-dimethylphenylethyl)sulfonyl)piperidine-4-carboxamide (998 mg, 77%).

MS (ESI) m/z = 466 (M+H) +

(Reactions 1-8)

To a solution of 4-amino-1-((4-(5,5-dimethyl-2,4-dioxoimidazolidin-1-yl)-2,6-dimethylphenethyl)sulfonyl)piperidine-4-carboxamide (120 mg, 0.258 mmol), 4-fluoro-3-(trifluoromethoxy)benzoic acid (69 mg, 0.309 mmol), and diisopropylethylamine (0.09 ml, 0.516 mmol) in dimethylformamide (2.5 ml) was added O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU) (118 mg, 0.309 mmol). The mixture was stirred at room temperature for 1.5 hours. The reaction mixture was quenched with water and then extracted with dichloromethane. The organic layer was washed with saturated brine and anhydrous sodium sulfate, and then concentrated under reduced pressure to give 1-((4-(5,5-dimethyl-2,4-dioxoimidazolidin-1-yl)-2,6-dimethylphenethyl)sulfonyl)-4-(4-fluoro-3-(trifluoromethoxy)benzamide)piperidine-4-carboxamide (150 mg, 67%).

MS (ESI) m/z = 672 (M+H) +

(Reactions 1-9)

To a mixture of 1-((4-(5,5-dimethyl-2,4-dioxoimidazolidin-1-yl)-2,6-dimethylphenethyl)sulfonyl)-4-(4-fluoro-3-(trifluoromethoxy)benzamide)piperidine-4-carboxamide (150 mg, 0.223 mmol) in tert-butanol (2.5 ml) and ethanol (2.5 ml) at 0°C was added potassium tert-butoxide (75 mg, 0.670 mmol). The mixture was heated and stirred at 50°C under a nitrogen stream for 1.5 hours. The reaction mixture was cooled, diluted with water, quenched with saturated aqueous ammonium chloride, and extracted with dichloromethane. The organic layer was washed with water and saturated brine in that order, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (dichloromethane-methanol) to give 1-(4-(2-((2-(4-fluoro-3-(trifluoromethoxy)phenyl)-4-oxo-1,3,8-triazaspiro[4.5]dec-1-en-8-yl)sulfonyl)ethyl)-3,5-dimethylphenyl)-5,5-dimethylimidazolidine-2,4-dione (118 mg, 81%).

MS(ESI)m/z=654(M+H)+. ¹ H-NMR (400MHz, CD ₃ OD)S: 1.40 (6H, s), 1.71-1.80 (2H, m), 2.00-2.08 (2H, m), 2.43 (6H, s), 3.22 (4H, s), 3.47 -3.57(2H,m),3.80-3.88(2H,m),7.01(2H,s),7.50-7.57(1H,m),7.97-8.04(1H,m),8.05-8.12(1H,m)

The following reference example compounds were synthesized by the same procedures as in Reaction 1-8 and Reaction 1-9 of Reference Example 1 using appropriate carboxylic acid starting materials, reagents, and solvents.

(Compound 2-5)

[Table 2]

Reference Example 2

1-(3,5-Dimethyl-4-(2-((4-oxo-2-(3-(trifluoromethyl)phenyl)-1,3,8-triazaspiro[4.5]dec-1-en-8-yl)sulfonyl)ethyl)phenyl)-5,5-dimethylimidazolidine-2,4-dione (Compound 6).

(Reaction 2-1)

A mixture of 2-(3-(trifluoromethyl)phenyl)-8-(vinylsulfonyl)-1,3,8-triazaspiro[4.5]dec-1-en-4-one (150 mg, 0.387 mmol), 1-(4-bromo-3,5-dimethylphenyl)-5,5-dimethylimidazolidine-2,4-dione (169 mg, 0.542 mmol), bis(dibenzylideneacetone)palladium (45 mg, 0.077 mmol), tri-tert-butylphosphine tetrafluoroborate (22 mg, 0.077 mmol) and methyldicyclohexylamine (0.123 ml, 0.581 mmol) in N-methyl-2-pyrrolidone (0.97 ml) was heated and stirred at 100° C. for 1 hour under a nitrogen flow. The reaction mixture was cooled, quenched with water, and extracted with ethyl acetate. The organic layer was washed sequentially with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (ethyl acetate-hexane) to obtain (E)-1-(3,5-dimethyl-4-(2-((4-oxo-2-(3-(trifluoromethyl)phenyl)-1,3,8-triazaspiro[4.5]dec-1-en-8-yl)sulfonyl)vinyl)phenyl)-5,5-dimethylimidazolidine-2,4-dione (197 mg, 82%).

MS (ESI) m/z = 618 (M+H) +.

(Reaction 2-2)

Under a hydrogen atmosphere, a mixture of (E)-1-(3,5-dimethyl-4-(2-((4-oxo-2-(3-(trifluoromethyl)phenyl)-1,3,8-triazaspiro[4.5]dec-1-en-8-yl)sulfonyl)vinyl)phenyl)-5,5-dimethylimidazolidine-2,4-dione (195 mg, 0.316 mmol) and palladium hydroxide on carbon (Pd 20%) (approximately 50% water-wet) (195 mg, 0.139 mmol) in 2,2,2-trifluoroethanol (6 ml) was stirred at room temperature for 14 hours. The mixture was filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (ethyl acetate-hexane) to give 1-(3,5-dimethyl-4-(2-((4-oxo-2-(3-(trifluoromethyl)phenyl)-1,3,8-triazaspiro[4.5]dec-1-en-8-yl)sulfonyl)ethyl)phenyl)-5,5-dimethylimidazolidine-2,4-dione (121 mg, 62%).

MS(ESI)m/z=620(M+H)+. ¹ H-NMR (400MHz, CD ₃ OD) δ: 1.40 (6H, s), 1.7 2-1.81 (2H, m), 2.00-2.10 (2H, m), 2.44 (6H, s), 3.22 (4H, s), 3.50-3 .58 (2H, m), 3.80-3.88 (2H, m), 7.01 (2H, s), 7.72-7.79 (1H, m), 7.88 -7.94 (1H, m), 8.16-8.23 (1H, m), 8.31 (1H, s)

Reference Example 3

1-(3,5-Dimethyl-4-(2-((4-oxo-2-(4-(trifluoromethoxy)phenyl)-1,3,8-triazaspiro[4.5]dec-1-en-8-yl)sulfonyl)ethyl)phenyl)-5,5-dimethylimidazolidine-2,4-dione (Compound 7).

(Reaction 3)

Using appropriate starting materials and solvents, the same procedures as in Reference Example 2 were followed to synthesize 1-(3,5-dimethyl-4-(2-((4-oxo-2-(4-(trifluoromethoxy)phenyl)-1,3,8-triazaspiro[4.5]dec-1-en-8-yl)sulfonyl)ethyl)phenyl)-5,5-dimethylimidazolidine-2,4-dione (Compound 7).

MS(ESI)m/z=636(M+H)+. ¹ H-NMR (400MHz, CDCl ₃ ) δ: 1.47 (6H, s), 1.7 0-1.78 (2H, m), 2.10-2.19 (2H, m), 2.40 (6H, s), 3.00-3.07 (2H, m), 3 .19-3.25(2H,m), 3.45-3.53(2H,m), 3.81-3.88(2H,m), 6.94(2H,s), 7.35(2H,d,J=8.0Hz), 7.73(1H,brs), 7.93(2H,d,J=8.0Hz), 9.37(1H,brs)

Reference Example 4

1-(3,5-Dimethyl-4-(2-((4-oxo-2-(4-(trifluoromethoxy)phenyl)-1,3,8-triazaspiro[4.5]dec-1-en-8-yl)sulfonyl)ethyl)phenyl)-1,3-diazaspiro[4.4]nonane-2,4-dione (Compound 8).

(Reaction 4-1)

[Chemistry 31]

To a mixture of cyclopentanone (42 mg, 0.500 mmol) and 4-bromo-3,5-dimethylaniline (100 mg, 0.500 mmol) in acetic acid (0.5 ml) at room temperature was added trimethylsilyl cyanide (0.063 ml, 0.500 mmol). The mixture was stirred at room temperature for 1.5 hours under a nitrogen stream. The reaction mixture was quenched by adding 28% aqueous ammonia (1 ml), diluted with water, and extracted with dichloromethane. The organic layer was washed with water and then with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 1-((4-bromo-3,5-dimethylphenyl)amino)cyclopentanecarbonitrile (152 mg) as a crude product.

¹ H-NMR (400MHz, CDCl ₃ ) δ: 1.83-1.92 (4H, m), 2.07-2.15 (2H, m), 2.33- 2.42 (2H, m), 2.37 (6H, m), 3.71 (1H, brs), 6.56 (2H, s)

(Reaction 4-2)

To a solution of 1-((4-bromo-3,5-dimethylphenyl)amino)cyclopentanecarbonitrile (145 mg, 0.495 mmol) in dichloromethane (5 ml) was added 2,2,2-trichloroacetyl isocyanate (0.070 ml, 0.593 mmol) at room temperature. The mixture was stirred at room temperature for 1 hour under a nitrogen stream.

To the reaction mixture, triethylamine (0.103 ml, 0.742 mmol), water (0.045 ml) and methanol (0.10 ml) were added in sequence, and the mixture was heated to reflux under a nitrogen stream for 1.5 hours. After the reaction mixture was cooled, it was diluted with water, adjusted to pH 5 with 1N aqueous hydrochloric acid, and extracted with dichloromethane. The organic layer was washed with water and saturated brine in sequence, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 1-(4-bromo-3,5-dimethylphenyl)-4-imino-1,3-diazaspiro[4.4]nonane-2-one as a crude product.

MS(ESI)m/z=336,338(M+H)+.

(Reaction 4-3)

Under a nitrogen stream, a mixture of acetic acid (1.0 ml) and water (0.25 ml) of 1-(4-bromo-3,5-dimethylphenyl)-4-imino-1,3-diazaspiro[4.4]nonane-2-one (the crude product obtained in the previous reaction) was heated and stirred at 65°C for 1.5 hours. Furthermore, after adding acetic acid (1.0 ml) and water (0.25 ml), the mixture was heated and stirred at 65°C for 17 hours under a nitrogen stream. After cooling the reaction mixture, it was diluted with water, adjusted to pH 8 with a saturated aqueous sodium bicarbonate solution, and extracted with ethyl acetate. The organic layer was washed with water and saturated brine in sequence, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (ethyl acetate-hexane) to obtain 1-(4-bromo-3,5-dimethylphenyl)-1,3-diazaspiro[4.4]nonane-2,4-dione (121 mg).

MS(ESI)m/z=337,339(M+H)+.

(Reaction 4-4)

Using appropriate starting materials and solvents, the same procedures as in Reference Example 2 were followed to obtain 1-(3,5-dimethyl-4-(2-((4-oxo-2-(4-(trifluoromethoxy)phenyl-1,3,8-triazaspiro[4.5]dec-1-en-8-yl)sulfonyl)ethyl)phenyl)-1,3-diazaspiro[4.4]nonane-2,4-dione (Compound 8).

MS(ESI)m/z=662(M+H)+. ¹ H-NMR (400MHz, DMSO-d ₆ ) δ: 1.36-1.44 (2H, m), 1.60-1.70 (4H.m), 1.82-1.91 (2H, m), 1.91-2.06 (4H, m), 2.38 (6H , s), 3.01-3.09 (2H, m), 3.22-3.30 (2H, m), 3.30-3.42 (2H, m), 3.70-3.77 (2H, m), 7.03 (2H, s), 7.57 (2H, d, J = 8.4Hz), 8.14 (2H, d, J = 8.4Hz)

Reference Example 5

1-(3,5-Dimethyl-4-(2-((4-oxo-2-(4-(trifluoromethoxy)phenyl)-1,3,8-triazaspiro[4.5]dec-1-en-8-yl)sulfonyl)ethyl)phenyl)-8-methyl-1,3,8-triazaspiro[4.5]decane-2,4-dione (Compound 9).

(Reaction 5-1)

Using tert-butyl 4-oxopiperidine-1-carboxylate as a starting material and an appropriate solvent, the same procedures as in Reference Example 4 were followed to obtain tert-butyl 1-(3,5-dimethyl-4-(2-((4-oxo-2-(4-(trifluoromethoxy)phenyl)-1,3,8-triazaspiro[4.5]dec-1-en-8-yl)sulfonyl)ethyl)phenyl)-2,4-dioxo-1,3,8-triazaspiro[4.5]decane-8-carboxylate.

MS (ESI) m/z = 777 (M+H) +.

(Reaction 5-2)

To a mixture of tert-butyl 1-(3,5-dimethyl-4-(2-((4-oxo-2-(4-(trifluoromethoxy)phenyl)-1,3,8-triazaspiro[4.5]dec-1-en-8-yl)sulfonyl)ethyl)phenyl)-2,4-dioxo-1,3,8-triazaspiro[4.5]decane-8-carboxylate (11.7 mg, 0.015 mmol) in dichloromethane (0.13 ml) at room temperature was added trifluoroacetic acid (0.05 ml, 0.673 mmol). The mixture was stirred at room temperature under a nitrogen stream for 1 hour. The reaction mixture was concentrated under reduced pressure to give 1-(3,5-dimethyl-4-(2-((4-oxo-2-(4-(trifluoromethoxy)phenyl)-1,3,8-triazaspiro[4.5]dec-1-en-8-yl)sulfonyl)ethyl)phenyl)-1,3,8-triazaspiro[4.5]decane-2,4-dione 2-trifluoroacetate (13.6 mg).

MS (ESI) m/z = 677 (M+H) +.

(Reaction 5-3)

To a mixture of 1-(3,5-dimethyl-4-(2-((4-oxo-2-(4-(trifluoromethoxy)phenyl)-1,3,8-triazaspiro[4.5]dec-1-en-8-yl)sulfonyl)ethyl)phenyl)-1,3,8-triazaspiro[4.5]decane-2,4-dione 2-trifluoroacetate (21.1 mg, 0.022 mmol) in formic acid (0.033 ml) was added 37% aqueous formaldehyde solution (0.055 ml). The mixture was heated and stirred at 80°C for 3 hours under a nitrogen stream. The reaction mixture was concentrated, and the residue was diluted with ethyl acetate. The organic layer was washed with a dilute aqueous sodium hydroxide solution, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. The resulting residue was purified by column chromatography (dichloromethane-methanol) to give 1-(3,5-dimethyl-4-(2-((4-oxo-2-(4-(trifluoromethoxy)phenyl)-1,3,8-triazaspiro[4.5]dec-1-en-8-yl)sulfonyl)ethyl)phenyl)-8-methyl-1,3,8-triazaspiro[4.5]decane-2,4-dione (4.5 mg, 30%).

MS(ESI)m/z=691(M+H)+. ¹ H-NMR (400MHz, CD ₃ OD) δ: 1.76-1.84 (2H, m), 1.92-2.02 (2H, m), 2.02-2.12 (4H, m), 2.38 (3H, s), 2.46 (6H, s), 2 .81-2.88(2H,m), 2.92-3.02(2H,m), 3.23(4H,s), 3.51-3.60(2H,m), 3.72-3.80(2H,m), 7.01(2H,s), 7.48(2H,d,J=8.0Hz), 8.10(2H, d, J＝8.0Hz)

Reference Example 6

5-(3,5-Dimethyl-4-(2-((4-oxo-2-(4-(trifluoromethoxy)phenyl)-1,3,8-triazaspiro[4.5]dec-1-en-8-yl)sulfonyl)ethyl)phenyl)-2-oxa-5,7-diazaspiro[3.4]octane-6,8-dione (Compound 10).

(Reaction 6)

Using oxetan-3-one as a starting material and an appropriate solvent, the same procedures as in Reference Example 4 were followed to obtain 5-(3,5-dimethyl-4-(2-((4-oxo-2-(4-(trifluoromethoxy)phenyl)-1,3,8-triazaspiro[4.5]dec-1-en-8-yl)sulfonyl)ethyl)phenyl)-2-oxa-5,7-diazaspiro[3.4]octane-6,8-dione.

MS (ESI) m/z = 650 (M+H) +. ¹ H-NMR (400MHz, CDCl ₃ ) δ: 1.69-1.77 (2H, m), 2.12-2.22 (2H, m), 2.45 (6H, s), 3.03-3.11 (2H, m), 3.22-3.29 (2H, m), 3.46-3.53 (2H, m), 3.84-3.91 (2H, m), 4.86 (2H, d, J = 7.2Hz), 5.03 (2H, d, J = 7.2Hz), 7.07 (2H, s), 7.35 (2H, d, J = 8.4Hz), 7.98 (2H, d, J=8.4Hz), 8.56 (1H, s), 10.34 (1H, s)

Reference Example 7

4-(3,5-Dimethyl-4-(2-((4-oxo-2-(4-(trifluoromethoxy)phenyl)-1,3,8-triazaspiro[4.5]dec-1-en-8-yl)sulfonyl)ethyl)phenyl)-4,6-diazaspiro[2.4]heptane-5,7-dione (Compound 11).

(Reaction 7-1)

Under a nitrogen stream, a mixture of 2-bromo-5-iodo-1,3-dimethylbenzene (300 mg, 0.965 mmol), 1-aminocyclopropanecarboxylic acid (195 mg, 1.93 mmol), copper (I) iodide (37 mg, 0.194 mmol), and diazabicycloundecene (0.50 ml, 3.35 mmol) in dimethylacetamide (2.6 ml) was heated with stirring at 120° C. for 3 hours. The reaction mixture was purified by silica gel column chromatography (Wakosil C18, acetonitrile-water (0.1% formic acid) to give 1-((4-bromo-3,5-dimethylphenyl)amino)cyclopropanecarboxylic acid (219 mg, 80%).

MS(ESI)m/z=284,286(M+H)+.

(Reaction 7-2)

To a mixture of 1-((4-bromo-3,5-dimethylphenyl)amino)cyclopropanecarboxylic acid (198 mg, 0.697 mmol) in acetic acid (3 ml) and dichloromethane (1.5 ml) was added potassium cyanate (424 mg, 5.23 mmol) at room temperature. The mixture was stirred at room temperature for 1 hour and then heated and stirred at 60°C for 2 hours. The reaction mixture was adjusted to pH 8 with a saturated aqueous sodium bicarbonate solution and extracted with ethyl acetate. The organic layer was washed with water and saturated brine in that order, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate-hexane) to give 4-(4-bromo-3,5-dimethylphenyl)-4,6-diazaspiro[2.4]heptane-5,7-dione (49 mg, 23%).

MS(ESI)m/z=309,311(M+H)+.

(Reaction 7-3)

Using appropriate starting materials and solvents, the same procedures as in Reference Example 2 were followed to obtain 4-(3,5-dimethyl-4-(2-((4-oxo-2-(4-(trifluoromethoxy)phenyl)-1,3,8-triazaspiro[4.5]dec-1-en-8-yl)sulfonyl)ethyl)phenyl)-4,6-diazaspiro[2.4]heptane-5,7-dione (Compound 11).

MS(ESI)m/z=634(M+H)+. ¹ H-NMR (400MHz, DMSO-d ₆ ) δ: 0.99-1.03 (2H, m), 1.19-1.27 (4H, m), 1.58-1.64 (2H, m), 1.81-1.90 (2H, m), 2.35 (6H , s), 2.99-3.04 (2H, m), 3.22-3.29 (2H, m), 3.67-3.73 (2H, m), 6.95 ( 2H, s), 7.56 (2H, d, J = 8.4Hz), 8.12 (2H, d, J = 8.4Hz)

Reference Example 8

1-(3,5-Dimethyl-4-(2-((4-oxo-2-(3-(trifluoromethyl)phenyl)-1,3,8-triazaspiro[4.5]dec-1-en-8-yl)sulfonyl)ethyl)phenyl)-1,3-diazaspiro[4.4]nonane-2,4-dione (Compound 12).

(Reaction 8)

Using appropriate starting materials and solvents, the same procedures as in Reference Example 2 were followed to obtain 1-(3,5-dimethyl-4-(2-((4-oxo-2-(3-(trifluoromethyl)phenyl)-1,3,8-triazaspiro[4.5]dec-1-en-8-yl)sulfonyl)ethyl)phenyl)-1,3-diazaspiro[4.4]nonane-2,4-dione.

MS (ESI) m/z = 646 (M+H) +. ¹ H-NMR (400MHz, DMSO-d ₆ ) δ: 1.40-1.48 (2H, m), 1.62-1.71 (4H, m), 1.88-1.97 (2H, m), 1.97-2.08 (4H, m), 2.41 (6H , s), 3.03-3.10 (2H, m), 2.29-3.34 (2H, m), 3.38-3.47 (2H, m), 3.72-3.79 (2H, m), 7.06 (2H, s), 7.84 (1H, dd, J=7.6, 7.6Hz), 8.02 (1H, d, J=7.6Hz), 8.33 (1H, d, J=7.6Hz), 8.38 (1H, s)

Reference test examples

The results of various tests on the compounds of the present invention, including a cAMP-generating ability test using human PTH1R, a cAMP-generating ability test using rat receptors, a metabolic stability test using human liver microsomes, a metabolic stability test using rat hepatocytes, and a serum Ca concentration-increasing effect test using a TPTX rat model, are shown in Reference Test Examples 1 to 5. As comparative compounds, the compounds described in WO2010/126030A1 shown in Table 3 were used.

Table 3

Reference Test Example 1: Determination of in vitro cAMP signaling activity of compounds in human PTH1R (peptide)

Human PTH (1-34) and calcitonin were purchased from Peptide Research Institute (Osaka, Japan), dissolved in 10 mM acetic acid to 1 mM, and stored in a -80°C freezer.

(Cell Culture)

The cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (Hyclone), 100 units/ml penicillin G, and 100 μg/ml streptomycin sulfate (Invitrogen Corp) at 37° C. in a humidified atmosphere containing 5% CO 2 .

LLC-PK1 cells not expressing PTH1R and HKRK-B7 cells overexpressing 9.5×10 ⁵ human PTH1R per LLC-PK1 cell were used for cAMP signaling analysis (Takasu et al., J. Bone. Miner. Res. 14: 11-20, 1999).

(cAMP stimulation)

HKRK-B7 or LLC-PK1 cells were seeded in 96-well plates at 1×10 ⁵ cells/well and incubated overnight. The next day, 50 μl of cAMP assay buffer (DMEM, 2 mM IBMX, 0.2 mg/ml bovine serum albumin, 35 mM Hepes-NaOH, pH 7.4) containing human PTH (1-34) or compound was added and placed in an incubator at 37°C for 20 minutes. After removing the culture medium, the cells were washed once with 100 μl of cAMP assay buffer. In order to freeze the cells, the culture plate was placed on dry ice powder and then removed from the dry ice. The cells were melted using 40 μl of 50 mM HCl and frozen again on dry ice. The amount of intracellular cAMP produced was determined using a commercially available cAMPEIA kit (Biotrack cAMP EIA system, GE health care).

(Calculation of 20% effective concentration (EC20) and 50% effective concentration (EC50) in the in vitro cAMP inducibility assay.)

The analysis was performed using a sigmoidal dosage response formula with a variable gradient. The cAMP signaling activity of human PTH (1-34) at 100 nM was defined as 100%, and the concentration at which each compound showed 20% or 50% of the cAMP signaling activity was calculated as EC20 or EC50.

The results in HKRK-B7 cells are shown in Table 4.

It should be noted that the degree of cAMP response in LLC-PK1 cells was lower than that in HKRK-B7 cells.

Table 4

Reference Test Example 2: Determination of in vitro cAMP signaling activity of compounds in rat PTH1R

Measurement was performed in the same manner as in Reference Test Example 1 using LLC-PK46_RATO_PTH1R cells overexpressing rat PTH1R established by Chugai Pharmaceutical instead of HKRK-B7 cells.

Table 5 shows the results using LLC-PK46_RATO_PTH1R cells.

There is a good correlation between the EC20 value of in vitro cAMP signaling activity at the rat PTH1 receptor and the EC20 value of in vitro cAMP signaling activity at the human PTH1R. There is also a good correlation between the EC50 values in rats and humans.

Table 5

Reference Test Example 3: Metabolic Stability Test Using Human Liver Microparticles

Human liver microsomes were incubated with compounds or comparative examples in 0.1 M phosphate buffer (pH 7.4) in the presence of NADPH at 37°C for the specified time. The parent compound concentration at each reaction time was measured using LC/MS/MS, and the intrinsic clearance (μL/min/mg protein) was calculated from the slope of the residual rate versus reaction time.

＜Analysis conditions＞

Compound concentration: 1 μM

Microsomes: 0.5 mg/mL

NADPH: 1mM

Reaction time: 0, 5, 15 and 30 minutes.

The results are shown in Table 6. Compounds 1 to 11 showed higher metabolic stability against human liver microsomes than those of Comparative Examples 1 to 6.

Table 6

Reference Test Example 4: Metabolic Stability Test Using Rat Hepatocytes

Hepatocytes were prepared from rat (SD, ♀) liver using the collagenase refluxing method. Reference Example compounds or comparative examples were added, incubated at 37°C for the specified time, and then reaction termination solution was added. Parent compound concentrations at various reaction times were measured using LC/MS/MS, and intrinsic clearance (μL/10 ⁶ cells/min) was calculated from the slope of the residual rate versus reaction time.

＜Analysis conditions＞

Cell concentration: 1×10 ⁶ cells/mL

Compound concentration: 1 μM

Culture medium: Williams' medium E

Reaction time: 0, 15, 30, 60, 120 and 240 minutes

Reaction termination solution: acetonitrile/2-propanol (4/6, v/v).

The results are shown in Table 7. Compounds 2, 4, 5, 7, 8, 9, 10, and 11 showed improved metabolic stability in rat hepatocytes compared to the compounds of Comparative Examples 1, 2, 3, 5, and 6.

Table 7

Reference Test Example 5: Effect of increasing serum Ca concentration in TPTX rat model

Four-week-old female Crl:CD (SD) rats were obtained from Japan Charls Reiber Co., Ltd. (Atsugi Breeding Center) and acclimated for one week under standard laboratory conditions at 20-26°C and 35-75% humidity. The rats were given free access to tap water and a standard rodent diet (CE-2) containing 1.1% calcium, 1.0% phosphate, and 250 IU/100g of vitamin D3 (Japan Charia Co., Ltd.).

TPTX was administered to 5-week-old rats. A sham operation (Sham) was performed on some individuals. For the TPTX rats used, individuals with serum Ca concentrations less than 8 mg/dL 4 days after surgery were selected. 5 days after surgery, based on the serum Ca concentrations and body weights measured 4 days after surgery, the rats were divided into 8 TPTX groups and 1 Sham group, with 5 rats in each group. The Sham group and the TPTX-vehicle group were only administered with a solvent at a dosage of 10 mL/kg. Each analyte in the TPTX-analyte group was dissolved in a solvent at a dosage of 30 mg/10 mL/kg and administered orally. The following solvent composition was used: 10% dimethyl sulfoxide (Wako Pure Chemical Industries, Ltd.), 10% Cremophor EL (Sigma Aldrich Japan Co., Ltd.), 20% hydroxypropyl-β-cyclodextrin (Nippon Food Chemicals Co., Ltd.), and glycine (Wako Pure Chemical Industries, Ltd.) adjusted to a pH of 10. For each group, pre-blood collection was performed immediately before administration (pre-blood collection) and 2, 6, 10, and 24 hours after administration to measure serum calcium levels. Blood was collected from the jugular vein under isoflurane inhalation anesthesia for each group.

Measurement of Serum Ca: Serum obtained from the collected blood by centrifugation was measured using an automatic analyzer TBA-120FR (Toshiba Medical Systems Co., Ltd.).

Statistical analysis was performed on the animal experiments. Data are expressed as mean ± standard error (SE). Statistical significance was determined using the SAS Preclinical Package (Ver. 5.00.010720, SAS Institute Japan, Tokyo, Japan). A p-value of less than 0.05 was considered statistically significant. A t-test of two groups showed: #P < 0.05 relative to the TPTX-vehicle group, *P < 0.05 relative to the group of Comparative Example 1, ∫P < 0.05 relative to the group of Comparative Example 2.

The Pre value of serum Ca concentration was 9.9 mg/dL in the sham group and 5.3 to 6.2 mg/dL in each TPTX group. The average value of the change in serum Ca concentration relative to the Pre value up to 24 hours after administration of each compound is shown in Figure 1. Furthermore, the peak of serum Ca concentration after administration of each compound occurred 6 or 10 hours after administration for all compounds.

Compounds 6, 7, and 8, which have high metabolic stability in rat hepatocytes, showed large positive changes relative to the Pre value, demonstrating a strong serum Ca concentration-raising effect upon oral administration. On the other hand, Compound 1, Comparative Example 1, and Comparative Example 2, which have low metabolic stability in rat hepatocytes, showed smaller positive changes relative to the Pre value than Compound 6, 7, and 8. Compounds 7 and 8 were particularly statistically significant relative to Comparative Examples 1 and 2.

Furthermore, Compound 6, Compound 7, and Compound 8, which have high metabolic stability in rat hepatocytes, exhibited maximum values of 7.8 to 8.5 mg/dL at 6 or 10 hours after administration, reaching the therapeutic target range of 7.6 to 8.8 mg/dL for serum calcium concentration in patients with hypoparathyroidism. On the other hand, Compound 1, Comparative Examples 1, and Comparative Examples 2, which have low metabolic stability in rat hepatocytes, did not reach this therapeutic target range at any of the measurement times.

The above test results confirmed that when Compounds 6, 7, and 8 (which exhibit strong cAMP signaling activity in cells forcibly expressing rat PTH1R and high metabolic stability relative to rat hepatocytes) were orally administered to rats, a strong serum Ca level-elevating effect was confirmed. These compounds exhibit cAMP signaling activity in cells forcibly expressing human PTH1R and have higher metabolic stability in human liver microsomes than the comparative compounds. Therefore, oral administration can be expected to have a favorable therapeutic effect on patients with hypoparathyroidism. Furthermore, compounds represented by general formula (1), which exhibit cAMP signaling activity in cells forcibly expressing human PTH1R and metabolic stability in human liver microsomes to the same extent as Compounds 6, 7, and 8, can also be expected to have a favorable therapeutic effect on patients with hypoparathyroidism.

Industrial Applicability

The present invention provides a drug for promoting the prevention, treatment, recovery, and cure of osteoporosis, bone loss in periodontal disease, alveolar bone damage after tooth extraction, osteoarthritis, articular cartilage damage, adynamic bone disease, achondroplasia, hypochondroma, osteomalacia, fractures, etc. by non-invasive systemic or local exposure of hydantoin derivatives that have high metabolic stability and exert strong PTH-like effects.

Claims (1)

1. The use of a compound or a pharmacologically acceptable salt thereof in the manufacture of a pharmaceutical composition for improving bone loss in periodontal disease, promoting recovery of alveolar bone damage after tooth extraction, or promoting recovery of articular cartilage damage, wherein the compound is 1-(3,5-dimethyl-4-(2-((4-oxo-2-(4-(trifluoromethoxy)phenyl)-1,3,8-triazaspiro[4.5]dec-1-en-8-yl)sulfonyl)ethyl)phenyl)-1,3-diazaspiro[4.4]nonane-2,4-dione.