TWI686392B - Pharmaceutical composition containing hydantoin derivative - Google Patents

Abstract

The present invention is a drug for inducing assimilation of hard bone and/or cartilage comprising a compound represented by the following general formula (1) or a pharmacologically acceptable salt thereof as an active ingredient:

(In the formula, R1, R2, R3, R4 are synonymous with those who apply for patent scope).

Description

Pharmaceutical composition containing hydantoin derivative

The present invention relates to a medicinal product using hydantoin derivatives having high metabolic stability and exerting a powerful PTH-like effect as an active ingredient, and provides an assimilation effect for hard bone and/or cartilage for prevention, treatment, recovery and Accelerated healing of osteoporosis, bone loss in periodontal disease, alveolar bone defect after tooth extraction, arthrosis deformation, articular cartilage defect, adynamic bone disease, cartilage hypoplasia ( achondrogenesis, hypochondroplasia, osteomalacia, fractures and other diseases.

Parathyroid hormone (PTH) is known to act on target cells of bone or kidney as a hormone that regulates the constant of calcium (Ca) and phosphorus (Pi) (Non-Patent Document 1). The use of PTH to maintain serum calcium levels is mainly performed by direct or indirect effects on the digestive tract, bones, and kidneys. PTH promotes the reabsorption of calcium by the renal tubules and inhibits the excretion of calcium in vivo. In addition, increasing the synthesis of enzymes that convert vitamin D into active vitamin D in the kidney helps promote the absorption of calcium in the digestive tract due to active vitamin D. In addition, PTH indirectly intensifies osteoclast differentiation by osteoblasts and the like, thereby promoting calcium release from bone. It is believed that these PTH effects are mainly exerted through the activation of adenylate cyclase and/or phospholipase C receptors to generate cAMP through the binding of PTH1R.

PTH preparations [PTH(1-34) and PTH(1-84)] have a strong bone assimilation effect in the human body and induce a significant increase in bone density and bone strength. At present, many of the osteoporosis treatment drugs available to humans are bone resorption inhibitors, and the only drug with a bone assimilation function that actively increases bone density is the PTH preparation. In view of this situation, the PTH preparation is regarded as the most effective therapeutic agent for the treatment of osteoporosis (Non-Patent Document 2), but because it is a peptide, it requires invasive administration. Therefore, it is expected to create drugs that have a PTH-like effect and can be administered non-invasively.

Deformable arthritis refers to a degenerative disease characterized by cartilage degeneration, destruction, synovitis, subchondral bone sclerosis, bone spurs, and joint dysfunction caused by chronic pain in systemic joints such as knees, hips, spine, fingers, etc. In the population over 65 years old, more than 40% of the patients suffer from it, which causes a great burden on the medical economy (Non-Patent Document 3, Non-Patent Document 4). The causes of deformed arthritis include physical overload of articular cartilage, inflammation in the synovium and bone marrow, genetic defects in cartilage matrix components, and increased bone metabolism in subchondral bone, etc., but due to the suppression of degeneration and destruction of articular cartilage Of therapeutic drugs are not yet available, and medical demand remains high.

So far, the proteoglycan enzymes (ADAMTS-4, ADAMTS-5, etc.) or matrix metalloproteinases (MMP-3, MMP-9, MMP-13, etc.) that are related to the destruction of the cartilage matrix as the target of therapeutic drugs Patent Document 5) or proinflammatory cytokines (IL-1, IL-6, etc., Non-Patent Document 6), but it has not yet been actually put into use. On the other hand, drugs (Risedronate, calcitonin, non-patent document 7, non-patent document 8) that target metabolic conversion of subchondral bone have also been tested, but they cannot be suppressed Degeneration and destruction of articular cartilage Bad. In addition to this mechanism, in clinical trials of strontium ranelate, which has the promotion effect of hard bone formation and cartilage formation, it shows the effect of inhibiting the destruction of articular cartilage (Non-Patent Document 9), but it has not yet Actually put into use.

However, according to recent studies, it has been pointed out that the pathogenesis of deformed arthritis is the conversion of articular cartilage from permanent cartilage to calcified cartilage, and suppressing the occurrence of this phenomenon has become the target of therapeutic drugs (Non-Patent Document 10). According to this mechanism of action, several agents used to inhibit the final differentiation mechanism of articular cartilage have been shown to inhibit the degradation and destruction of articular cartilage in animal models of arthritic arthritis, and the use of therapeutic drugs based on this mechanism is recommended (Non-Patent Document 11, Non-Patent Document 12).

Under this situation, the applicant of the present case found that the compound represented by the following general formula (A) or its pharmacologically acceptable salt is useful as a compound having a PTH-like effect, preferably a PTH1R agonist, for osteoporosis, fractures, The prevention and/or treatment of osteomalacia, arthritis, thrombocytopenia, hypothyroidism, hyperphosphatemia, or neoplastic calcification, or stem cell drive are useful, and patent applications have been filed (Patent Literature 1 );

(In the formula, W, X, Y, m, n, R ₁ , R ₂ , R ₃₃ , R ₃₄ refer to Patent Document 1)

The creation of high-value clinically non-invasively administered pharmaceuticals must consider the direct effects on the target and the in vivo dynamics of drug absorption, distribution, metabolism, excretion, etc. For this reason, it is desired to have a PTH-like agent having a strong ability to produce cAMP via human PTH1R and having a high metabolic stability to human liver microsomes.

Prior technical literature

Patent Literature

Patent Literature 1: International Publication No. 2010/126030

Non-patent literature

Non-Patent Literature 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 literature 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 Literature 5: Osteoarth Cart 2010; 18:1109-1116

Non-Patent Literature 6: Osteoarth Cart 2013; 21:16-21

Non-Patent Document 7: Arthritis Rheum. 2006; 54(11): 3494-507

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

Non-Patent Document 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 Document 12: Sci Trans Med 2011; 3:101ra93

The purpose of the present invention is to provide hydantoin derivatives which have high metabolic stability and can exert a strong PTH-like effect through non-invasive systemic exposure or local exposure to induce the assimilation of hard bone and cartilage. Prevention, treatment, recovery and accelerated healing of osteoporosis, bone loss in periodontal disease, alveolar bone defect after tooth extraction, deformed arthropathy, articular cartilage defect, non-dynamic bone lesion, cartilage dysplasia (achondroplasia), Methods of hypochondroplasia (hypochondroplasia), osteomalacia, fractures and other diseases.

Under these circumstances, the inventors of this case have worked hard to find out that the newly found hydantoin derivative of the present invention exhibits strong cAMP production ability in cells that forcibly express human PTH1R and has high stability in the metabolism of human liver microsomes . In addition, the inventors of the present invention, by administering the compound of the present invention, found that it can induce the assimilation of hard bone and cartilage and thus can effectively prevent, treat, recover and accelerate the healing of bone loss in osteoporosis, periodontal disease, and tooth extraction Medical composition for groove bone defect, deformed arthropathy, articular cartilage defect, non-dynamic bone disease, cartilage hypoplasia, cartilage hypoplasia, osteomalacia, fracture and other diseases.

That is, the present invention relates to the following.

[式中，R1與R2，在R1與R2不同時為氫原子的條件下，各自獨立為1)氫原子，2)鹵素原子，3)可經1~5個氟原子取代之碳數1~2之烷基，或4)可經1~5個氟原子取代之碳數1~2之烷氧基；或R1與R2彼此鍵結形成下式表示之基：

(式中之各*表示與苯基部分的鍵結位置)；且R3及R4各自獨立為可經1~3個氟原子取代之甲基；或R3與R4和其所鍵結之碳原子共同形成碳數3~6之環(在此，形成環的碳原子的其中之一可經氧原子、硫原子、或可經甲基取代之氮原子所取代)]。 [1] A drug containing a compound represented by the following general formula (1) or a pharmacologically acceptable salt thereof for inducing assimilation of hard bone and/or cartilage:

[In the formula, R1 and R2, under the condition that R1 and R2 are not hydrogen atoms, are each independently 1) hydrogen atom, 2) halogen atom, 3) carbon number that can be substituted by 1 to 5 fluorine atoms. 2 alkyl group, or 4) alkoxy group having 1 to 2 carbon atoms which can 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 the bonding position with the phenyl moiety); and R3 and R4 are each independently a methyl group which may be substituted with 1 to 3 fluorine atoms; or R3 and R4 and the carbon atom to which they are bonded are common Forming a ring having 3 to 6 carbon atoms (here, one of the carbon atoms forming the ring may be substituted with an oxygen atom, a sulfur atom, or a nitrogen atom which may be substituted with a methyl group)].

As the active ingredient compound included in the pharmaceutical composition of the present invention, the compound represented by the aforementioned general formula (1) can be excluded, and the combination of R1 and R2 is trifluoromethyl and a hydrogen atom, and R3 and R4 are bonded thereto The carbon atoms together form a compound of cyclopentane.

(式中，各*表示與苯基部分之鍵結位置)；且R3及R4為甲基；或R3及R4與其所鍵結的碳原子共同形成選自下列的環：

(式中，*表示與咪唑啶-2,4-二酮部分的鍵結位置)。 [2] The pharmaceutical composition according to [1], wherein R1 and R2 of the compound represented by the aforementioned general formula (1) or a pharmacologically acceptable salt thereof are selected from the following combinations: 1) R1 is a hydrogen atom or a halogen atom, And R2 is a hydrogen atom, trifluoromethyl, or trifluoromethoxy (but not including the case where R1 and R2 are both hydrogen atoms); 2) R1 is trifluoromethyl or trifluoromethoxy, and R2 is Hydrogen atom or halogen atom; 3) R1 and R2 are bonded to each other to form a group represented by the following formula:

(In the formula, each * represents a bonding position with a phenyl moiety); and R3 and R4 are methyl groups; or R3 and R4 and the carbon atom to which they are bonded together form a ring selected from the following:

(In the formula, * represents the bonding position with the imidazolidine-2,4-dione moiety).

(式中，各*表示與苯基部分之鍵結位置)；且R3及R4為甲基；或R3及R4與其所鍵結的碳原子共同形成選自下列的環：

(式中，*表示與咪唑啶-2,4-二酮部分的鍵結位置)。 [3] The pharmaceutical composition according to [1], wherein R1 and R2 of the compound represented by the general formula (1) or a pharmacologically acceptable salt thereof are selected from the following combinations: 1) R1 is trifluoromethoxy, 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 trifluoromethyl 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 each other to form a group represented by the following formula:

(In the formula, each * represents a bonding position with a phenyl moiety); and R3 and R4 are methyl groups; or R3 and R4 and the carbon atom to which they are bonded together form a ring selected from the following:

(In the formula, * represents the bonding position with the imidazolidine-2,4-dione moiety).

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

(式中，*表示與咪唑啶-2,4-二酮部分的鍵結位置)。 [5] The pharmaceutical composition according to [1], wherein the compound represented by the general formula (1) or its pharmacologically acceptable salt R3 and R4 together with the carbon atom to which they are bonded form a ring selected from the following:

(In the formula, * represents the bonding position with the imidazolidine-2,4-dione moiety).

[6] The pharmaceutical composition according to [1], wherein the compound or the pharmacologically acceptable salt contained as an active ingredient is selected from the group consisting of: 1-(4-(2-(( 2-(4-fluoro-3-(trifluoromethoxy)phenyl)-4-oxo-1,3,8-triazaspiro[4.5]dec-1-en-8-yl)sulfonate (Acetyl)ethyl)-3,5-dimethylphenyl)-5,5-dimethylimidazolidine-2,4-dione; 1-(4-(2-((2-(3- Bromophenyl)-4-oxo-1,3,8-triazaspiro[4.5]decyl -1-ene-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-ene-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)sulfonamide Group) ethyl)-3,5-dimethylphenyl)-5,5-dimethylimidazolidine-2,4-dione; 1-(4-(2-((2-(2,2 -Difluorobenzo[d][1,3]1,3-dioxo-5-yl)-4-pentoxy-1,3,8-triazaspiro[4.5]dec-1-ene -8-yl)sulfonyl)ethyl)-3,5-dimethylphenyl)-5,5-dimethylimidazolidine-2,4-dione; 1-(3,5-dimethyl Yl-4-(2-((4-oxo-2-(3-(trifluoromethyl)phenyl)-1,3,8-triazaspiro[4.5]dec-1-ene-8 -Yl)sulfonyl)ethyl)phenyl)-5,5-dimethylimidazolidine-2,4-dione; 1-(3,5-dimethyl-4-(2-((4 -Penoxy-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-(trifluoromethyl Oxy)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-(tri Fluoromethoxy)benzene Group)-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- pendant-2-(4-(trifluoromethoxy) Phenyl)-1,3,8-triazaspiro[4.5]dec-1-en-8-yl)sulfonyl)ethyl)phenyl)-4,6-diazaspiro[2.4]heptane Alkane-5,7-dione.

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

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

[9] The pharmaceutical composition of [1], wherein the compound is 1-(3,5-dimethyl-4-(2-((4-oxo-2-(4-(trifluoromethyl Oxy)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 its pharmacologically acceptable salt.

[10] The pharmaceutical composition as described in [1], which can be used for prevention or treatment of osteoporosis, improvement of bone loss in periodontal disease, accelerated healing of alveolar bone defect after tooth extraction, deformable joint Prevention or treatment of morbidity, accelerated healing of articular cartilage defects, prevention or treatment of non-dynamic bone lesions, prevention or treatment of achondroplasia, prevention or treatment of achondroplasia, prevention or treatment of osteomalacia, or fractures The accelerated recovery.

[11] A method for inducing assimilation of hard bone and/or cartilage, including Including the prevention or treatment of osteoporosis, the improvement of bone loss in periodontal disease, the accelerated recovery of alveolar bone defects after tooth extraction, the prevention or treatment of deformed arthropathy, the accelerated recovery of articular cartilage defects, and lack of motivation For the prevention or treatment of osteopathic bone disease, the prevention or treatment of achondroplasia, the prevention or treatment of achondroplasia, the prevention or treatment of osteomalacia, or the accelerated recovery of fractures, it is administered [1] to [9] A pharmacologically effective dose of a compound of any one or a pharmacologically acceptable salt thereof.

[12] The method of [11], wherein the method of inducing assimilation of hard bone and/or cartilage is a method of prevention or treatment of osteoporosis, a method of improving bone loss in periodontal disease, and alveolar bone after tooth extraction Accelerated healing of defects, methods of prevention or treatment of deformed arthrosis, accelerated healing of articular cartilage defects, methods of prevention or treatment of non-dynamic bone lesions, methods of prevention or treatment of achondroplasia, prevention of hypochondrosis Or treatment methods, osteomalacia prevention or treatment methods, or accelerated healing of fractures.

[13] The use of a compound as described in any one of [1] to [9] or a pharmacologically acceptable salt thereof for the preparation or prevention of osteoporosis, improvement of bone loss in periodontal disease, and tooth extraction Accelerated healing of groove defects, prevention or treatment of deformed arthropathy, accelerated healing of articular cartilage defects, prevention or treatment of non-dynamic bone lesions, prevention or treatment of achondroplasia, prevention or treatment of achondroplasia, prevention or treatment Medical composition for osteomalacia or accelerated healing of fractures.

[14] The use of the compound according to any one of [1] to [9] or a pharmacologically acceptable salt thereof to prepare a pharmaceutical composition for inducing assimilation of hard bone and/or cartilage.

[15] A compound as described in any one of [1] to [9] or a pharmacologically acceptable salt thereof, for the prevention or treatment of osteoporosis, the improvement of bone loss in periodontal disease, and the tooth groove after tooth extraction Accelerated healing of bone defects, prevention or treatment of deformed arthrosis, accelerated healing of articular cartilage defects, prevention or treatment of non-dynamic bone lesions, prevention or treatment of achondroplasia, prevention or treatment of achondroplasia, bone Prevention or treatment of softening disease, or accelerated healing of fractures.

In addition, the present invention provides a treatment method for preventing, treating, and/or recovering diseases through assimilation of hard bone and/or cartilage by administering general formula (1) or a pharmacologically acceptable salt thereof.

According to the present invention, the use of hydantoin derivatives having a strong PTH-like effect and high metabolic stability can induce the assimilation of hard bone and/or cartilage to prevent, treat, recover and accelerate the healing of osteoporosis, periodontal Bone loss in the disease, alveolar bone defect after tooth extraction, deformed arthropathy, articular cartilage defect, non-dynamic bone disease, cartilage hypoplasia, cartilage hypoplasia, osteomalacia, fracture, etc.

Figure 1 is a schematic diagram showing the bone mineral density of the lumbar vertebrae and thigh bones of rats after 6 weeks of repeated ovarian removal. That is, this graph shows repeated administration of vehicle, compound 7 or hPTH (1-34) once a day to the ovarian-removed rats within 6 weeks, and measured by dual energy X-ray bone mineral content measuring device The bone mineral density results of the lumbar spine and thigh bone were obtained.

Figure 2 shows the lumbar spine and shin of the normal rat after repeated administration for 4 weeks Schematic diagram of bone density. That is, this figure shows the lumbar spine and lower leg measured by the dual energy X-ray bone mineral content measuring device by repeatedly administering the carrier, compound 7 or hPTH (1-34) once a day to the normal rats within 4 weeks Tibia bone density results.

Figure 3 is a schematic diagram showing the bone density of the mandible of normal rats after repeated administration for 4 weeks. That is, this graph shows the repeated administration of the carrier, compound 7 or hPTH (1-34) once a day to normal rats within 4 weeks, and the measurement of the mandible bone by the dual energy X-ray bone mineral content measuring device Bone density results.

Figure 4 is a schematic diagram showing the inhibitory effect of Compound 7 on the final differentiation of rabbit calf tibial articular chondrocytes. That is, by alkaline phosphatase staining (A) and alizarin red staining (B), the results of the inhibitory effect of compound 7 and hPTH (1-34) on the final differentiation of rabbit calf tibial articular chondrocytes were evaluated.

Figure 5 is a schematic diagram of the amount of proteoglycan synthesis in human chondrocytes. That is, the results of the compound 7 and hPTH (1-34) on the synthesis promoting effect of proteoglycan in human chondrocytes were evaluated.

Figure 6 is a diagram showing the proportion of the affected area of the articular cartilage of the tibia of the rabbit's calf with a partial meniscus model. That is, in the case where the carrier or compound 7 is continuously administered into the joints of the meniscus local excision model rabbit, the ratio of the area of the affected area of the articular cartilage of the calf tibia is measured.

Figure 7 is a graph showing the changes of the articular cartilage of the lower leg tibial articular cartilage of the rabbit with a meniscus partial resection model within two weeks after the operation. That is, in the case where the carrier or compound 7 was continuously administered into the joints of the meniscus local excision model rabbit, the results of changes in the articular cartilage of the calf tibia within two weeks after the operation were visually observed.

Figure 8 shows a representative example of a normal rat after repeated oral administration for 4 weeks An image of the cartilage tissue at the end of the leg bone. That is, this graph shows the histopathological results of the end articular cartilage of the representative thigh bone observed through an optical microscope after repeated oral administration of the carrier or compound 7 to a normal rat once a day for 4 weeks.

Figure 9 shows the average change in serum calcium concentration when the TPTX rat model was orally administered at a dose of 30 mg/kg until 24 hours after administration of each compound.

Figure 10 shows the time of repeated administration of the carrier, compound 7 or hPTH (1-34) to a mature ovary-removed rat once a day for 3 months using a dual-energy X-ray bone mineral content measuring device Image of the results of the bone density of the lumbar spine and thigh bones.

Figure 11 is an image of the results of histological evaluation of the changes in the cartilage of the knee joint after 4 weeks of intra-articular administration of the carrier or compound 7 to the meniscus local excision model rabbit.

The present invention relates to a hydantoin derivative having high metabolic stability and exerting a strong PTH-like action and its use. The inventors of the present invention synthesized the compound represented by the aforementioned formula (1) or its pharmacologically acceptable salt, and found that the compound or its salt is a compound that can induce assimilation of hard bone and/or cartilage.

In this specification, "alkyl" refers to a monovalent group derived from an aliphatic hydrocarbon by removing an arbitrary hydrogen atom, the structure does not contain heteroatoms or unsaturated carbon-carbon bonds, and is a divalent hydrocarbon group containing hydrogen and carbon atoms (hydrocarbyl) or a collection of parts with a hydrocarbyl structure. The alkyl group includes straight-chain and branched-chain structures. The alkyl group is preferably an alkyl group having 1 or 2 carbon atoms. Specific examples of alkyl include methyl and ethyl, Methyl is preferred.

The "alkoxy" in this specification refers to an oxy group bonded to the "alkyl" defined above, preferably an alkoxy group having 1 or 2 carbon atoms. Specifically, for example, methoxy and ethoxy are preferred, and methoxy 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.

In addition, in the present invention, the number of substituents is not limited unless otherwise specified, for example, the number of substituents is 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 etc. .

The "halogen atom" in this specification means a fluorine atom, a chlorine atom, a bromine atom or an iodine atom.

In this specification, "*" in the chemical formula represents the bonding position.

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

In the present specification, "PTH-like action" refers to an activity that acts on a PTH receptor or acts on a signal transmission system via the PTH receptor to produce intracellular cAMP (cAMP: cyclic adenosine-phosphate).

In the present invention, whether it is "strong PTH-like action", "strong PTH-like action", or "having strong PTH-like action", for example, can be described according to J.Bone.Miner.Res.14:11-20,1999 For the method, cAMP signal transmission analysis was performed, and cAMP signal activity was measured to confirm. Specifically, for example, for the amount of cAMP production in cells that are forced to express human PTH1R, refer to the method described in Test Example 1 using a commercially available cAMP EIA kit (eg, Biotrack cAMP EIA system, GE health care) at 100 nM. Humanity The cAMP signal activity at PTH (1-34) was 100%, and the concentration of each compound showing 20% cAMP signal activity (EC20) or 50cAMP signal activity (EC50) was determined. In the present invention, "strong PTH-like action" or "strong PTH-like action" is preferably, for example, the EC20 value (μM) measured according to 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 according to the above method is preferably 25.0 or less, more preferably 15.0 or less, and even more preferably 10.0 or less.

In addition, whether it is "high metabolic stability" or "high metabolic stability" can be confirmed using a general measurement method. For example, hepatocytes, small intestine cells, liver microsomes, small intestine microsomes, liver S9, etc. can be used for confirmation. Specifically, for example, the stability of compounds in liver microsomes can be described in the literature of TKronbach et al. (Oxidation of midazolam and triazolam by human liver cytochrome P450IIIA4. Mol. Pharmacol, 1989, 36(1), 89-96) Measurement to confirm. More specifically, it can be confirmed by the method described in Reference Test Example 3. The "high metabolic stability" or "high metabolic stability" of the present invention, for example, the value of clearance (μl/min/mg) in the metabolic stability test using human liver microsomes described in the above reference test example It is preferably 60 or less, 40 or less, and 35 or less. In particular, in the aforementioned formula (1), the combination of R1 and R2 is a trifluoromethyl group and a hydrogen atom, and the carbon atoms to which R3 and R4 are bonded together form a cyclopentyl ring, high metabolic stability can be obtained .

In addition, whether it is "induced hard bone and/or cartilage assimilation" can be confirmed using a conventional method.

Regarding the induction of hard bone assimilation, for example, after the test compound is continuously administered for a certain period, the bone density can be measured using a general measurement method Degree or bone mass, and compared with the control group to confirm it. Specifically, for example, according to the method described in Takeda et al. (Bone 2013; 53(1): 167-173, a dual-energy X-ray bone mineral content measuring device (eg, DCS-600EX (Aloka Corporation) Measurement). At this time, when the bone density is higher than the solvent control group, it can be considered to have the ability to induce the assimilation of the bone. The compound of the present invention, for example, will be the same as osteoporosis The clinically equivalent dose of hPTH (1-34) used for therapeutic drugs, when administered to the subject to be tested, is preferably one with the same or higher increase in bone density, specifically, for example, the preferred is Bone density increased by 8~12% compared with the solvent control group, and more preferably increased by more than 12%.

Regarding the induction of cartilage assimilation, for example, chondrocytes can be cultured in the presence of the compound of the present invention and confirmed by measuring the production of chondrocyte matrix (eg proteoglycan). In addition, it can be confirmed by measurement whether it is possible to inhibit the final differentiation and calcification of chondrocytes. Specifically, for example, regarding the yield of cartilage matrix, according to the literature of Loeser et al. (Atrh Rheum 2003; 48(8): 2188-2196), the literature of Ab-Rahim et al. (Mol Cell Biochem 20131; 376: 11-20. ). In addition, the inhibition of final differentiation can be evaluated according to the method of Okazaki et al. (Osteoarth Cart 2003; 11(2): 122-32.). At this time, when the cartilage matrix yield or the degree of final differentiation and calcification are higher than those of the control group and inhibited, it may be considered to be induced by cartilage assimilation. The compound of the present invention, for example, is preferred to have an effect equal to or higher than PTH in the production of cartilage matrix and the inhibition of the final differentiation of chondrocytes. Since the compound of the present invention has higher metabolic stability than PTH, it has sufficient effects on the aforementioned conditions, and there are several kinds of I can choose the path. Furthermore, when the yield of cartilage matrix is higher than that of PTH, the above-mentioned conditions can obtain better effects than PTH.

In addition, for example, if the compound to be tested is continuously administered to the target cartilage bone for a certain period of time, the hypertrophy of the articular cartilage and growth plate is confirmed according to histopathological observation, and the effect of inducing cartilage assimilation can be confirmed. Specifically, the cartilage thickness of articular cartilage and growth plate can be calculated histologically. At this time, when the degree of cartilage hypertrophy is increased compared with the control group, the test compound may be considered to induce cartilage assimilation. In particular, when the degree of cartilage hypertrophy is more significant than PTH, it is considered that it is better to have a sufficient effect on the aforementioned condition, and it is more preferable that the test compound is administered orally to exert its effect.

In addition, for example, according to the method of Kikuchi et al. (Osteoarth Cart 1996: 4(2): 99-100) or the method of Sampson et al. (Sci Transl Med 2011; 3: 101ra93), the meniscus is partially excised to make the knee joint unstable. Animals (rodents and non-rodents) that induce deformable arthrosis can continue to administer the test compound for a certain period of time to the articular cartilage of the knee joint through the naked eye or histopathology The degradation state is evaluated and confirmed. At this time, as with PTH, if the degradation of the articular cartilage of the knee joint is suppressed, it can be judged by the effect of the test compound's cartilage assimilation and final differentiation inhibitory effect. Even better, these effects are obtained by oral administration of the test compound.

In addition, for example, according to the method of Wakitani et al. (Bone Joint Surg Br. 1989; 71(1): 74-80.), the test compound is administered to a subject with defective articular cartilage and subchondral bone within a certain period, and It can be evaluated by analyzing the regeneration status of cartilage in the defect. At this time, if better cartilage is observed than the control group The regenerative effect can be considered that the test compound induces the assimilation of cartilage. In particular, when the cartilage regeneration effect is more significant than PTH, it can be considered that it has a sufficient effect on the aforementioned condition and is better, and more preferably, the test compound is administered orally to exert its effect.

These effects can also be demonstrated by measuring PTH-like effects. PTHrP, which activates the PTH receptor PTH1R through paracrine, is an important factor related to the regulation of chondrocyte proliferation and differentiation. It is known to have the effect of inhibiting the final differentiation of chondrocytes and maintaining cartilage tissue (Science 1996; 273:663-666). The evaluation of cartilage assimilation by activation of PTH1R can also be based on methods such as Xie (Human Mol Genet 2012; 21(18): 3941-3955), and the test compound is administered to normal within a certain period of time Or an object with hereditary growth disease, analyze the growth rate of cartilage bone and the hypertrophy of the growth plate in histology. At this time, if the growth rate or growth plate hypertrophy can be proved compared to the control group, the test compound can be judged to have cartilage assimilation. In particular, the effect of the test compound is better than PTH, the better, and the test compound is administered orally to exert the effect.

The compound of the present invention may be a free form or a pharmacologically acceptable salt, which are included in the present invention. Such "salts" include, for example, inorganic acid salts, organic acid salts, inorganic basic salts, organic basic salts, acidic or basic amino acid salts, and the like.

As ideal examples of inorganic acid salts, such as hydrochloride, hydrogen bromide, sulfate, nitrate, phosphate, etc., as ideal examples of organic acid salts, such as acetate, succinate, fumarate, Maleate, tartrate, citrate, lactate, stearate, benzoate, methanesulfonate, benzenesulfonate, p-toluenesulfonate, etc.

Preferable examples of inorganic alkali 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, and ammonium salts. Preferable examples of organic base salts include diethylamine salts , Diethanolamine salt, meglumine salt, N,N-dibenzyl ethylenediamine salt, etc.

As ideal examples of acidic amino acid salts, for example, aspartate, glutamate, etc., as ideal examples of basic amino acid salts, for example, spermine, lysine, ornithine, etc. .

The compound of the present invention sometimes absorbs water when it is placed in the atmosphere, and adheres to or adsorbs water, or becomes a hydrate. Such a hydrate is also included in the salt of the present invention.

Furthermore, the compounds of the present invention sometimes absorb some other solvents and become solvates. Such salts are also salts of compounds of formula (1) and are included in the present invention.

In this specification, the structural formula of the compound, for simplicity, sometimes represents a certain isomer, but the present invention includes all geometric isomers generated in the structure of the compound, optical isomers based on asymmetric carbon, stereo Isomers such as isomers and tautomers and mixtures of isomers are not limited to the description of the formula for simplicity, and either one of the isomers may be a mixture. Therefore, although the compounds of the present invention may have asymmetric carbon atoms in the molecule and may have optically active bodies and racemates, the present invention is not limited and is included in all.

The present invention includes all isomers of the compound represented by formula (1). The isoform of the compound of the present invention is substituted by atoms having at least one atom with the same atomic number (proton number) but different mass numbers (sum of protons and neutrons). As the isotope contained in the compound of the present invention, for example, hydrogen atom, carbon atom, nitrogen atom, oxygen atom, phosphorus atom, sulfur atom, fluorine atom, chlorine atom, etc., including ² H, ³ H, ¹³ C, ¹⁴ C, ¹⁵ N, ¹⁷ O, ¹⁸ O, ³¹ P, ³² P, ³⁵ S, ¹⁸ F, ³⁶ Cl, etc. In particular, radioactive isotopes such as ³ H and ¹⁴ C that emit radiation and collapse are useful for in vivo tissue distribution tests of pharmaceuticals or compounds. The stable isotopes do not collapse, the existing amount is almost unchanged, and there is no radiation ability, so it can be used safely. The isoform of the compound of the present invention can be transformed according to the usual method by replacing the synthetically used test drug with a test drug containing the corresponding isoform.

The compound of the present invention sometimes has polymorphic crystal forms, but is not particularly limited, and any one of the crystal forms may be single or a mixture of crystal forms.

The compound of the present invention includes its prodrug. Prodrug refers to a derivative of a compound of the present invention that has a chemically or metabolically decomposable group and returns to the original compound after administration to the living body, and shows the original drug effect, including complexes and salts without covalent bonds.

(式中，*各表示與苯基部分之鍵結位置)；且R3及R4為甲基；或R3及R4與其所鍵結之碳原子共同 形成選自以下之環：

(式中之*表示與咪唑啶-2,4-二酮部分之鍵結位置)。 The compound represented by the aforementioned formula (1) in the present invention is preferably as follows. In the formula, R1 and R2 are selected from the following combination: 1) R1 is a hydrogen atom or a halogen atom, and R2 is a hydrogen atom, trifluoromethyl, or trifluoromethoxy (except when R1 and R2 simultaneously become hydrogen atoms) ); 2) R1 is trifluoromethyl or trifluoromethoxy, 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, * each represents a bonding position with a phenyl moiety); and R3 and R4 are methyl groups; or R3 and R4 and the carbon atom to which they are bonded together form a ring selected from the following:

(* in the formula represents the bonding position with the imidazolidine-2,4-dione moiety).

As the compound represented by the aforementioned formula (1) of the present invention, more desirable ones are as follows.

(式中，*各代表與苯基之鍵結位置)；且R3及R4為甲基；或R3及R4與其所鍵結之碳原子共同形成選自以下的環：

(式中之*表示與咪唑啶-2,4-二酮部分之鍵結位置)。 In the formula, R1 and R2 are selected from the following combinations: 1) R1 is trifluoromethoxy and R2 is a fluorine atom; 2) R1 is a bromine atom and R2 is a hydrogen atom; 3) R1 is trifluoromethyl and R2 is Fluorine atom; 4) R1 is fluorine atom and R2 is trifluoromethoxy; 5) R1 is trifluoromethyl and R2 is hydrogen atom; 6) R1 is hydrogen atom and R2 is trifluoromethoxy; 7) R1 , R2 is bonded to each other to form a base represented by the following formula;

(In the formula, * each represents a bonding position with a phenyl group); and R3 and R4 are methyl groups; or R3 and R4 and the carbon atom to which they are bonded together form a ring selected from the following:

(* in the formula represents the bonding position with the imidazolidine-2,4-dione moiety).

As the compound represented by the aforementioned formula (1) of the present invention, more preferably A compound selected from the group consisting of the following, or a pharmacologically acceptable salt thereof.

Compound 1: 1-(4-(2-((2-(4-fluoro-3-(trifluoromethoxy)phenyl)-4-oxo-1,3,8-triazaspiro[ 4.5] Dec-1-ene-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)ethane Radical)-3,5-dimethylphenyl)-5,5-dimethylimidazolidine-2,4-dione; compound 5: 1-(4-(2-((2,2-difluoro Benzo[d][1,3]dioxan-5-yl)-4-oxo-1,3,8-triazaspiro[4.5]dec-1-en-8-yl)sulfonamide Yl)ethyl)phenyl)-5,5-dimethylimidazolidine-2,4-dione; compound 6: 1-(3,5-dimethyl-4-(2-((4-side Oxy-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- pendantoxy-2-(4-(trifluoromethoxy Group) 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- pendant-2-(4-(trifluoromethoxy) Phenyl)-1,3,8-triazaspiro[4.5]dec-1-en-8-yl)sulfonyl)ethyl)phenyl)-8-methyl-1,3,8-tri Azaspiro[4.5]decane-2,4-dione; compound 10: 5-(3,5-dimethyl-4-(2-((4-oxo-2-(4-(tri Fluoromethoxy)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 these compounds 1 to 11, compounds 6, 7, and 8 are more desirable.

Such a compound represented by the aforementioned formula (1) of the present invention or its pharmacologically acceptable salt induces the assimilation of hard bone and cartilage, and through this effect, prevents or treats osteoporosis, improves bone loss in periodontal disease, Accelerated healing of alveolar bone defects after tooth extraction, prevention or treatment of deformed arthropathy, accelerated healing of articular cartilage defects, prevention or treatment of non-dynamic bone lesions, prevention or treatment of achondroplasia, hypochondrogenesis Prevention or treatment, prevention or treatment of osteomalacia, or accelerated healing of fractures are useful.

The compound of the present invention or its salt can be formulated into tablets, powders, granules, granules, coated tablets, capsules, syrups, tablets, inhalants, suppositories, injections, ointments, eye ointments according to conventional methods Agents, eye drops, nose drops, ear drops, cataplasm, lotions, etc. Formulations can use commonly used carriers, such as excipients, binders, lubricants, colorants, or Odor correctors, and stabilizers, emulsifiers, absorption enhancers, surfactants, pH adjusters, preservatives, antioxidants, etc. as required, can be blended with ingredients generally used as raw materials for pharmaceutical preparations Formulation.

For example, when manufacturing oral preparations, the compound of the present invention or its pharmacologically acceptable salts and excipients, and, if necessary, binders, disintegrating agents, lubricants, colorants, flavoring and flavoring agents, etc. are added and prepared according to the usual method Granules, fine granules, granules, lozenges, coated lozenges, capsules, etc.

As such ingredients, for example, animal and vegetable oils such as soybean oil, tallow, and synthetic glycerides; hydrocarbons such as liquid paraffin, squalane, and solid paraffin; ester oils such as octyl dodecyl myristate and isopropyl myristate; spermaceti Higher alcohols such as stearyl alcohol and sorbitol; silicone resin; silicone oil; polyoxyethylene fatty acid ester, sorbitan fatty acid ester, glycerin fatty acid ester, polyoxyethylene sorbitan fatty acid ester, polyoxygen Surfactants such as ethylene hardened grate oil, polyoxyethylene polyoxypropylene block copolymer; hydroxyethyl cellulose, polyacrylic acid, carboxyvinyl polymer, polyethylene glycol, polyvinylpyrrolidone, methyl cellulose, etc. Water-soluble polymers; lower alcohols such as ethanol and isopropanol; polyhydric alcohols such as glycerin, propylene glycol, dipropylene glycol, and sorbitol; sugars such as glucose and sucrose; inorganic powders such as anhydrous silicic acid, aluminum magnesium silicate, and aluminum silicate; Refined water, etc.

As an excipient, for example, lactose, corn starch, white sugar, glucose, mannitol, sorbitol, crystalline cellulose, silica, and the like.

As a binder, for example, polyvinyl alcohol, polyvinyl ether, methyl cellulose, ethyl cellulose, gum arabic, tragacanth, gelatin, shellac, hydroxypropyl methyl cellulose, hydroxypropyl cellulose, poly Vinylpyrrolidone, polypropylene glycol, polyoxyethylene, block polymer, meglumine, etc.

As disintegrants, for example starch, agar, gelatin powder, crystals Cellulose, calcium carbonate, sodium bicarbonate, calcium citrate, dextrin, pectin, carboxymethyl cellulose‧calcium, etc.

As the lubricant, for example, magnesium stearate, talc, polyethylene glycol, silicon dioxide, hardened vegetable oil and the like.

As the coloring agent, those which can be added to pharmaceutical products with permission can be used. As the flavoring and flavoring agent, cocoa powder, menthol, fragrant powder, peppermint oil, borneol, and cinnamon powder can be used.

Of course, these tablets, granules can be coated with sugar coating, and other appropriate coating agents as needed. In addition, when manufacturing liquids such as syrups or injection preparations, the compound of the present invention or its pharmacologically acceptable salt may be added with a pH adjuster, a dissolving agent, an isotonicity agent, etc., and a dissolution aid or stabilizer if necessary After the chemical agent etc., it is formulated according to the usual method.

The method for manufacturing the external preparation is not limited, and can be manufactured according to the usual method. That is to say, the base material used in the formulation can be various raw materials commonly used in pharmaceuticals, quasi-pharmaceuticals, cosmetics and the like. As the base material used, specifically, for example, animal and vegetable oils, mineral oils, ester oils, waxes, higher alcohols, fatty acids, silicone oils, surfactants, phospholipids, alcohols, polyols, water-soluble Raw materials such as polymers, clay minerals, purified water, etc. Furthermore, pH adjusting agents, antioxidants, chelating agents, anticorrosive and antifungal agents, coloring materials, fragrances, etc. can be added as needed, but the bases of agents other than the present invention Raw materials are not limited to these.

Furthermore, components having a differentiation-inducing effect, blood flow promoting agents, bactericides, anti-inflammatory agents, cell activating agents, vitamins, amino acids, humectants, keratolytic agents, etc. may be blended as necessary. In addition, the amount of the base material added is an amount that is usually set at the time of manufacturing the external preparation.

When the compound of the present invention, its salt, or these hydrates are administered, the form is not particularly limited, and it may be administered orally or non-orally according to a commonly used method. For example, it can be formulated into tablets, powders, granules, capsules, syrups, tablets, inhalants, suppositories, injections, ointments, eye ointments, eye drops, nasal drops, ear drops, decoction (cataplasm), lotion and other agents and administered.

The administration amount and administration method of the medicine of the present invention can be appropriately selected according to the degree of symptoms, age, sex, body weight, administration form, type of salt, and specific type of disease.

The dosage depends on the patient's disease type, symptom level, patient's age, gender difference, poor sensitivity to the drug, etc. It is usually about 0.03-1000mg per day for adults, preferably 0.1-500mg, more It is better to administer 0.1-100 mg once a day to several times.

The route of administration depends on the patient's disease type, degree of symptoms, patient's age, gender difference, and poor sensitivity to the drug, etc., and is appropriately selected. The method of administration is through non-invasive whole-body exposure or local exposure, as long as the induction effect of assimilation of hard bone and/or cartilage can be obtained, and there is no particular limitation. Such administration methods include, for example, oral administration, intravenous administration, nasal administration, percutaneous administration, pulmonary administration, intra-articular administration, and the like.

When the compound of the present invention represented by the aforementioned formula (1) is produced, the raw material compound and various reagents may form salts, hydrates, or solvates, which vary depending on the starting materials, the solvent used, etc., as long as they do not hinder the reaction. limited.

The solvent used also varies according to the starting materials, reagents, etc., as long as it does not hinder the reaction and can dissolve the starting material to some extent, it is not particularly limited and will not be repeated here.

Various isomers (such as geometric isomers, optical isomers based on asymmetric carbon, rotamers, stereoisomers, tautomers, etc.) can be separated according to common separation methods such as recrystallization, non-mirror image The isomer salt method, enzyme split method, and various chromatography (such as thin layer chromatography, column chromatography, high-speed liquid chromatography, gas chromatography, etc.) are purified and separated.

When the compound of the present invention is obtained in the form of a free form, it can be converted into the state of a salt or these hydrates which the compound can also form according to the usual method. In addition, when the compound of the present invention is obtained in the form of a salt or hydrate of the compound, it can be converted into the free form of the compound according to an ordinary method.

The isolation and purification of the compound of the present invention can be carried out by applying ordinary chemical operations such as extraction, concentration, distillation, crystallization, filtration and recrystallization, and various chromatography.

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

General synthesis

The compounds of the present invention can be synthesized according to various methods, some of which are described in the following scheme. This flow is an illustration, and the present invention is not limited to the explicit chemical reactions and conditions. In the following flow, some substituents are not described for conciseness, but it is not intended to limit the disclosure of the flow. Representative compounds of the present invention can be synthesized using appropriate intermediates, known compounds, and reagents. In the following general synthesis method, R1, R2, R3, R4 in the formula and the compound represented by the aforementioned general formula (1) (the compound represented by the formula 1 in the following general synthesis method) R1, R2, R3, R4 are Same meaning.

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

In Scheme 1, a carboxylic acid derivative (1) is condensed with an amine-amide derivative (2) 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-acylamine derivative (2). As a coupling reagent, for example: N,N'-dicyclohexylcarbodiimide (DCC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC ), O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethylurea hexafluorophosphate (HATU), and 4-(4,6-dimethoxy Yl-1,3,5-triazin-2-yl)-4-methylmorpholine chloride n-hydrate (DMT-MM). Examples of the base include triethylamine or N,N-diisopropylethylamine. If necessary, 4-(dimethylamino)pyridine (DMAP) can be used as a catalyst. Examples of suitable solvents include methylene chloride 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 a general technique, and if necessary, it can also be purified by crystallization or chromatography.

Step 2 is carried out in an appropriate solvent such as ethanol, tertiary butanol, or dimethyl sulfoxide in the presence of a suitable base such as aqueous sodium hydroxide or potassium tertiary butoxide. The method of cyclization of amide-amide derivatives (3). The reaction temperature is, for example, room temperature to reflux conditions, and the reaction time is 1 to 24 hours. The obtained hydantoin derivative (Formula 1) is isolated according to the general technique, and if necessary, it can also be purified by crystallization or chromatography.

The amino-acylamine derivative (2) shown in Scheme 1 can also be synthesized from the piperidinone derivative (4). Scheme 2 shows the synthesis method of the amino-acylamine derivative (2).

Step 3 is to derivatize the piperidone derivative (4) into the amino-nitrile derivative (5) and synthesize it for Strecker. That is, make the piperidone derivative (4) in the presence/absence of water in a suitable solvent such as methanol, ethanol, or tetrahydrofuran with sodium cyanide, or potassium cyanide, and ammonium chloride, or ammonium acetate The method of reaction. 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 according to the general technique, and if necessary, it can be purified by crystallization or chromatography.

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

Step 5 is in a hydrogen atmosphere, in the presence of a catalyst such as palladium carbon or palladium hydroxide carbon, in a passive solvent such as methanol, ethanol, trifluoroethanol, dimethylformamide, or dimethylacetamide. In the method, the olefin of the compound (6) is hydrogenated. The reaction temperature is from room temperature to 80°C, and sometimes the reaction can be carried out under pressure. The obtained amino-acylamine derivative (2) is isolated according to the general technique, and can be purified by crystallization or chromatography if necessary.

The piperidone derivative (4) shown in Scheme 2 can be synthesized from the well-known ketal-vinylsulfonyl derivative (7) and hydantoin-bromoaromatic derivative (g). Scheme 3 shows the synthesis method of piperidone derivative (4).

Step 6 is to add tri-tertiary butylphosphine tetrafluoroborate in the presence of palladium catalysts such as ginseng (dibenzylideneacetone) dipalladium (0) and bis (dibenzylideneacetone) palladium under a nitrogen atmosphere. Appropriate bases such as phosphine ligands, methyldicyclohexylamine, etc., in an appropriate solvent such as N-methyl-2-piperidone (NMP), the ketal-vinylsulfonyl derivative (7) is combined with A method of coupling hydantoin-bromoaromatic derivative (8) to synthesize ketal-arylvinylsulfonyl derivative (9). The reaction temperature is from 90°C to reflux temperature. The obtained ketal-aryl vinyl sulfonyl derivative (9) according to the general technical list If necessary, it can also be purified by crystallization or chromatography.

Step 7 is a method of converting the ketal-arylvinylsulfonyl derivative (9) from an ketal to a ketone in an appropriate solvent such as aqueous tetrahydrofuran in the presence of hydrochloric acid or the like. The reaction temperature is, for example, the boiling point of the solvent, and the reaction time is 1 to 24 hours. The obtained piperidone derivative (4) is isolated according to the general technique, and can be purified by crystallization or chromatography if necessary.

The hydantoin-bromoaromatic hydrocarbon derivative (8) shown in Scheme 3 can be selected from 4-bromo-3,5-dimethylaniline (10) and bromoacetic acid derivative (11) or 2-bromo-5- Iodine-1,3-dimethylbenzene (13) is synthesized with amino acid derivatives (14). Scheme 4 shows the synthesis method of hydantoin-bromoaromatic derivative (8).

Step 8. In the presence of a suitable base such as diisopropylethylamine, in a suitable solvent such as N-methyl-2-piperidone (NMP), 4-bromo-3,5-dimethylaniline (10) A method for alkylation with bromoacetic acid derivative (11). The reaction temperature is, for example, room temperature to 100°C, and the reaction time is 1 to 24 hours. The obtained brominated aromatic hydrocarbon-amino acid derivative (12) is isolated according to the general technique, and can be purified by crystallization or chromatography if necessary.

Step 9 is to use 2-bromo-5- in the presence of a metal catalyst such as copper iodide (1) Iodine-1,3-dimethylbenzene (13) reacts with amino acid derivative (14) to synthesize brominated aromatic hydrocarbon-amino acid derivative (12). The reaction can be carried out in the presence of a suitable base such as diazabicycloundecene (DBU) and a suitable solvent such as N,N-dimethylacetamide (DMA) at a reaction temperature of about 80 to 120°C. The obtained brominated aromatic hydrocarbon-amino acid derivative (12) is isolated according to the general technique, and can be purified by crystallization or chromatography if necessary.

Step 10 is a method of reacting brominated aromatic hydrocarbon-amino acid derivative (12) with sodium cyanate under acidic conditions to synthesize hydantoin-brominated aromatic hydrocarbon derivative (8). The solvent is performed in a mixed solvent such as acetic acid-dichloromethane, the reaction temperature is room temperature to 60°C, and the reaction time is 1 to 24 hours. The obtained hydantoin-bromoaromatic derivative (8) is isolated according to the general technique, and can be purified by crystallization or chromatography if necessary.

The hydantoin-bromoaromatic hydrocarbon derivative (8) shown in Scheme 3 can also be synthesized from 4-bromo-3,5-dimethylaniline (10) and ketone derivative (15). Scheme 5 shows the synthesis method of hydantoin-bromoaromatic derivative (8).

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

Step 12 is to react the arylamino-nitrile derivative (16) in a suitable solvent such as dichloromethane with 2,2,2-trichloroethylenyl isocyanate, and then add methanol, water, triethylamine and other reagents A method of reacting under heating conditions to synthesize iminohydantoin derivative (17). The obtained iminohydantoin derivative (17) is isolated according to the general technique, and can be purified by crystallization or chromatography if necessary.

Step 13 is a method of converting the iminohydantoin derivative (17) into a hydantoin-bromoaromatic derivative (8) under acidic conditions. For example, it can be synthesized in a solvent of acetic acid-water under a heating condition of about 65°C with a reaction time of about 1 to 6 hours. The obtained hydantoin-bromoaromatic derivative (8) is isolated according to the general technique, and can be purified by crystallization or chromatography if necessary.

Process 6 is that after the Heck reaction of the vinylsulfonamide derivative (18) and the hydantoin-bromoaromatic derivative (8) in the presence of a metal catalyst, the olefin of the compound (19) obtained is subjected to contact hydrogenation And the method of obtaining the hydantoin derivative (formula 1).

Process 6 (Method B)

The reaction of step 14 can be synthesized according to the method of step 6 and the reaction of step 15 according to the method of step 5 (Formula 1). The obtained hydantoin derivative (Formula 1) is isolated according to the general technique, and if necessary, it can also be purified by crystallization or chromatography.

The vinyl sulfonamide derivative (18) used in step 14 can be synthesized by referring to scheme 2, scheme 3, and scheme 12 of WO2010/126030(A1).

In addition, all the prior art documents cited in this specification are incorporated in this specification as a reference.

The content of the present invention is described in more detail in the following examples, but the present invention is not limited to this content.

Examples

Example 1: Effect of repeated administration of ovarian-removed rats on bone density after 6 weeks

Female Crl:CD (SD) rats of Japan Charles River Co., Ltd. were acclimated for more than 1 week under standard laboratory conditions of 20-26°C and humidity of 35-75%, and then used in the experiment. Rats were allowed to ingest tap water and standard rodent feed (CE-2) (manufactured by Japan CLEA Co., Ltd.) containing 1.1% calcium, 1.0% phosphoric acid, and 250 IU/100g vitamin D3.

Ovarian removal surgery (OVX) and sham surgery (Sham) were performed on 12-week-old rats. Body weight was measured in the fourth week after the operation, with 6 rats as a group, and the rats were divided into groups so that the average body weight of each group was equal. Starting every other day after grouping, each rat was administered with repeated administration once a day within 6 weeks. The rats in the Sham-Control group were administered orally and subcutaneously, respectively, with the solvent (vehicle) and the subcutaneously administered solvent (PC buffer). The rats in the OVX-Control group were administered orally and subcutaneously with vehicle and PC buffer. To the OVX-Compound 7 rats, the above Compound 7 dissolved in the carrier was orally administered at a dose of 30 mg/kg, and the PC buffer was administered subcutaneously. The rats in the OVX-hPHT (1-34) group were orally administered the carrier, and hPTH (1-34) dissolved in PC buffer was administered subcutaneously at a dose of 0.9 nmol/kg.

In addition, Forteo (registered trademark) used clinically as an osteoporosis treatment drug was administered to 20 μg of AUC (area under the blood concentration-time curve) to the same extent as in humans, as shown when administered to rats The dosage is set to 0.9 nmol/kg. Either group received 5 mL/kg orally and 1 mL/kg subcutaneously. The carrier is 10% dimethyl sulfoxide (Wako Pure Chemical Industries, pH10) prepared by using glycine (made by Wako Pure Chemical Industries, Ltd.) and sodium hydroxide (made by Wako Pure Chemical Industries, Ltd.). (Incorporated company), 10% Kolliphor EL (manufactured by Sigma-Aldrich Japan Co., Ltd.) and 10% hydroxypropyl-β-cyclodextrin (manufactured by Japan Food Chemical Co., Ltd.). For the PC buffer, a pH 5.0 composition prepared from 25 mmol/L phosphate citrate buffer, 100 mmol/L NaCl and 0.05% Tween 80 was used. On the day after the final administration, after taking blood from the abdominal aorta under anaesthesia and euthanizing the rat, dissect and take the waist Vertebral and thigh bones. Store the lumbar vertebrae and thigh bones in 70% ethanol. The bone mineral density of the lumbar spine and thigh bone was measured by a dual-energy X-ray bone mineral content measuring device (DCS-600EX, manufactured by Aloka Corporation). The bone density of the lumbar spine is measured from the 2nd to the 4th lumbar vertebrae, and the bone density of the thigh bone is divided into 10 equal parts along the longitudinal direction to measure the position 3 points away from the knee. The results are shown in Figure 1.

The data is represented by the average value + standard error (SE). The following statistical analysis was performed using SAS pre-clinical package ver5.00 (manufactured by SAS Institute Japan). The significant level is 5% on both sides. Regarding the bone density of the lumbar spine and thigh bones, the t test of 2 groups was used to compare the Sham-Control group with the OVX-Control group (#P<0.05), OVX-Control group and OVX-Compound 7 group Comparison of groups (*P<0.05), and comparison between OVX-Control group and OVX-hPTH(1-34) group (ʃP<0.05).

As shown in Figure 1, regarding the bone density of the thigh bone, the bone density of the OVX-Control group was significantly reduced relative to the Sham-Control group, and the OVX-hPTH(1-34) group of the positive control group was relative to OVX -Control group increased significantly. The increase rate of the OVX-hPTH(1-34) group relative to the OVX-Control group is 8%. At this time, the OVX-Compound 7 group significantly increased relative to the OVX-Control group, with an increase rate of 12%. In addition, regarding the bone density of the lumbar spine, the bone density of the OVX-Control group decreased significantly relative to the Sham-Control group, and the OVX-Compound 7 group did not tend to increase significantly relative to the OVX-Control group. The OVX-hPTH(1-34) group increased significantly compared to the OVX-Control group, with an increase rate of 12%.

As described above, because the bone density of the osteoporosis pathological model OVX rat with repeated oral administration of compound 7 increases, compound 7 is considered to be Prevention, treatment, improvement, and accelerated healing of osteoporosis, bone loss in periodontal disease, alveolar defect after tooth extraction, etc., diseases that require induction of bone assimilation, increase in bone mass, or bone reconstruction are effective. Furthermore, it was confirmed in Reference Test Examples 1 to 5 that the compound represented by general formula (1) has strong PTH-like action and high metabolic stability, and is also considered to be able to pass the bone assimilation due to PTH-like action Produce an increase in bone density. Therefore, the compound represented by the general formula (1) is also considered to be required for the induction of bone assimilation, bone prevention, etc. for the prevention, treatment, improvement, accelerated healing of osteoporosis, bone loss in periodontal disease, and alveolar defects after tooth extraction. Increased amounts or diseases of bone reconstruction are effective.

Example 2: Effect on bone density after repeated administration to normal rats for 4 weeks

Female RccHan: WIST rats of Japan Medical Science Animal Materials Research Institute Co., Ltd. were acclimated for more than one week under standard laboratory conditions of 20 to 26°C and humidity of 30 to 70%, and then used in the experiment. Rats were allowed to ingest tap water and standard rodent feed (CR-LPF) (made by Oriental Yeast Industry Co., Ltd.) freely.

Catheters were fixed intravenously in 8-week-old rats. The catheter is inserted from the thigh vein of the groin, and its front end is extended and fixed to the posterior great vein. The body weight was measured 1 week after the operation, with 10 rats as a group, and the rats were grouped to make the average body weight of each group equal. Starting from the next day after grouping, each rat was administered with repeated administration once a day within 4 weeks. The administration is performed via an infusion pump connected to a fixed catheter (MEDFUSION SYRINGEINFUSION PUMP Model 2001).

Vehicle (Control) was administered intravenously to the Vehicle-Control group. Compound 7 dissolved in the carrier was administered intravenously at 20 mg/kg, 30 mg/kg, and 50 mg/kg, respectively, to form the compound 7-20 mg/kg, 30 mg/kg, and 50 mg/kg groups. For each group, the administration volume was 5 mL/kg and the administration rate was 5 mL/kg/min. The carrier system uses 5% dimethyl sulfoxide (made by Wako Pure Chemical Industries, Ltd.), 25% propylene glycol (made by Kanto Chemical Co., Ltd.)/20% ethanol (made by Pure Chemical Co., Ltd.)/15% hydroxyl Propyl-β-cyclodextrin (manufactured by Japan Food Chemical Co., Ltd.)/300mM glycine (manufactured by Wako Pure Chemical Industries, Ltd.)/192mM sodium hydroxide (manufactured by Wako Pure Chemical Industries, Ltd.) / Composition of physiological saline (manufactured by Otsuka Pharmaceutical Factory Co., Ltd.). On the day after the final administration, blood was taken from the abdominal aorta under anaesthesia and the rat was euthanized, then dissected and the lumbar spine, tibia, and mandible were taken. Store the lumbar spine, shinbone, and mandible in 70% ethanol. The bone mineral density of the lumbar vertebrae (2nd to 4th lumbar vertebrae), lower leg tibia and mandible bone was measured with a dual-energy X-ray bone mineral content measuring device (DCS-600EX, manufactured by Aloka Corporation). The results are shown in Figure 2 and Figure 3.

The data is represented by the average value + standard error (SE). The following statistical analysis was performed using SAS pre-clinical package ver5.00 (manufactured by SAS Institute Japan). The significant level is 5% on both sides. Regarding the bone mineral density of the lumbar spine, tibia, and mandible, the Article-Control group was used as a control group, and the Dunnett-type multiple comparison of the parameters of the 7 groups of 3 doses was performed (*P<0.05).

As shown in FIG. 2, regarding the bone density of the lumbar spine and thigh bone, the bone density of the compound 7 administered group showed a significant dose-dependent bone density increase effect relative to the Vehicle-Control group. In addition, the compound 7-20mg/kg, 30mg/kg, 50mg/kg three groups, relative to the Vehicle-Control group, the increase rate of lumbar spine bone density was 16%, 21%, 25%, the lower leg tibia The increase rate of bone mineral density was 7%, 16% and 19%. In addition, as shown in Figure 3, regarding the bone density of the mandible, the bone density of the compound 7-30 mg/kg group is relative to The Vehicle-Control group shows a significant effect of increasing bone density. The increase rate relative to the Vehicle-Control group is 7%.

As described above, the bone density of the thigh of OVX rats in the osteoporosis pathological model with repeated oral administration of compound 7 increases, and the bone density of the lumbar spine, tibia and mandible of normal rats with repeated intravenous administration of compound 7 increases Therefore, systemic exposure of compound 7 is believed to require the induction of bone assimilation, increase in bone mass, or bone for prevention, treatment, improvement, and accelerated healing of osteoporosis, bone loss in periodontal disease, and alveolar defects after tooth extraction. The reconstructed disease system is effective. Furthermore, it was confirmed in Reference Test Examples 1 to 5 that the compound represented by general formula (1) has strong PTH-like action and high metabolic stability, and is also considered to be able to pass the bone assimilation due to PTH-like action Produce an increase in bone density. Therefore, the compound represented by the general formula (1) is also considered to be required for the induction of bone assimilation, bone prevention, etc. for the prevention, treatment, improvement, accelerated healing of osteoporosis, bone loss in periodontal disease, and alveolar defects after tooth extraction. Increased amounts or diseases of bone reconstruction are effective.

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

After euthanizing the NZW rabbit (4 weeks old, manufactured by Oriential Yeast Industry Co., Ltd.), the articular cartilage of the shin of the lower leg was taken into a 50 mL test tube (manufactured by Becton, Dickinson Co., Ltd., Japan). At this time, PBS (manufactured by Wako Pure Chemical Industries, Ltd.) containing 1% trypsin (manufactured by Wako Pure Chemical Industries, Ltd.) was added, and the soft tissue was digested at 37°C for 1 hour. Next, the supernatant was removed by centrifugation at 1,200 rpm for 5 minutes, and the cartilage tissue was suspended by adding PBS (one), the supernatant was removed by centrifugation at 1,200 rpm for five minutes, and then washed with PBS (one) suspension, and repeated three times. After centrifugation at 1,200 rpm for 5 minutes, the cell pellets were digested in DMEM (manufactured by Life Technologies Japan Co., Ltd.) containing 0.2% Type II collagenase (CLS-2, manufactured by Worthington Biochemical) at 37°C. 3 After 10 hours, 10% v/v fetal bovine serum (manufactured by Life Technologies Japan Co., Ltd.) was added to stop the reaction, and vigorously pipetted with a 10 mL dropper (manufactured by Becton, Dickinson Co., Ltd., Japan) to separate the chondrocytes. Then, centrifuge at 1,200 rpm for 5 minutes, remove the supernatant, add DMEM containing 10% FCS and wash it in suspension, repeat three times, and place the chondrocytes in a 96-well culture dish coated with type I collagen (manufactured by AGC TECHNO GLASS) ), sow at 1 x 10 ⁴ per well. The medium was changed 3 times a week. After the cells were confluent, they contained 100 μg/mL magnesium ascorbyl phosphate n-hydrate (manufactured by Wako Pure Chemical Industries, Ltd.) and 10 mmol/L β-glycerol phosphate 5-hydrate (and Light Pure Pharmaceutical Industry Co., Ltd.), 10% FCS in DMEM. At this time, culture was performed according to the following conditions, and alkaline phosphatase staining (cultured for 14 days) and alizarin red staining (cultured for 21 days) were performed to evaluate the final differentiation of chondrocytes.

(1)Control

(2) BMP-2 100ng/mL

(3) Compound 7 10 ^-7 mol/L

(4) Compound 7 10 ^-6 mol/L

(5) Compound 7 3x10 ^-6 mol/L

(6) Compound 7 10 ^-5 mol/L

(7)PTH(1-34) 10 ^-10 mol/L

(8)PTH(1-34) 10 ^-9 mol/L

(9)PTH(1-34) 10 ^-8 mol/L

(10)PTH(1-34) 10 ^-7 mol/L

Alkaline phosphatase staining is to wash the chondrocytes once with 200mmo1/L Tris-HCl pH8.2 buffer after discarding the medium, according to the Vector Red Alkaline Phosphatase Substrate Kit I, Vector Laboratories Co., Ltd. was stained according to the protocol, and a photo (objective lens 4x) was taken with an inverted microscope (manufactured by Nikon Corporation).

The results showed that the alkaline phosphatase activity of BMP-2 increased. Either compound 7 or PTH(1-34) can inhibit alkaline phosphatase activity in a concentration-dependent manner (Figure 4A).

Alizarin red staining is to wash the chondrocytes twice with PBS after discarding the culture medium, fix it with 100% ethanol (made by Wako Pure Chemical Industries, Ltd.) for 15 minutes, and use 1% alizarin after discarding the ethanol Red S (made by Wako Pure Chemical Industries, Ltd.) was stained for 15 minutes, then washed with distilled water, and photographed with an inverted microscope. The results showed that BMP-2's alizarin red staining significantly increased, promoting calcification. Either compound 7 or PTH(1-34) can inhibit alizarin red staining and calcification in a concentration-dependent manner (Figure 4B).

Example 4: Effect of compound 7 on proteoglycan synthesis of human articular chondrocytes

After purchasing cryopreserved human articular chondrocytes (Lot 2867, manufactured by Cell Applications), thaw in 37°C water, and add 15 mL of Basal Medium containing 10% Growth Supplement to T75 culture flask (CORNING, manufactured by Corning Japan KK) (Growth Medium, manufactured by Cell Applications). The next day, replace the Growth Medium 15mL and incubate for 3 days. Growth Medium was removed, and the cartilage cell layer was washed and removed with HBSS (manufactured by Cell Applications). 1 mL of trypsin/EDTA solution (manufactured by Cell Applications) was added and allowed to stand at room temperature for about 5 minutes to separate the chondrocytes from the flask. Add 10 mL of Neutalizing solution (manufactured by Cell Applications), measure the number of cells using a Bulker-Turk blood cell counting plate, and transfer to a 15 mL test tube for centrifugation (1,200 rpm, 5 minutes, TOMY Seiko Co., Ltd.) to produce chondrocytes. Particles. The supernatant was discarded and modulated into 2x10 ⁶ cells / mL of 1.2% sodium alginate (25mmol / L HEPES / 150mmol / L NaCl solution, pH7.0), with a 22G needle connected to a 1mL syringe (TERUMO ( Co., Ltd.) suck and add ₂ mL of a 102 mmol/L CaCl ₂ aqueous solution, add 5 drops to the wells of a 24-well plate (made by Corning Japan KK), and let stand for 5 minutes to produce beads. After that, it was washed three times with 150 mmol/L sodium chloride solution, cultured in Growth Medium for one day, and replaced with Basal Medium (manufactured by Cell Applications) containing 1% Growth supplement. At this time, the following factors were added to the medium, the medium was changed 3 times a week, and the culture was continued for 13 days.

(1)Control

(2) TGF-β1 10ng/m

(3)PTH(1-34) 10 ^-8 mol/L

(4) Compound 7 10 ^-6 mol/L

(5) Compound 7 10 ^-5 mol/L

After 12 days of cultivation, ³⁵ S logo sulfuric acid (manufactured by PerkinElmer Japan Co., Ltd.) was added to make 370 kBg/well, and after 24 hours, the medium was recovered in a test tube and stored at 4°C. A 55 mmol/L sodium citrate solution (manufactured by Nacalai tesque Co., Ltd., 1 mL/well) was added to the alginic acid gel, incubated at 37° C. for 10 minutes to make it sol, and recovered into microtubes (Eppendorf (share)) (Made by the company) Internal centrifugation (1,200 rpm, 5 minutes) to produce chondrocyte particles. Use 0.2mol/L Tris-HCl (manufactured by Sigma-Aldrich Corporation)/5mol/L CaCl ₂ (Nacalai tesque (share)) containing 1mg/mL actinase E (manufactured by Scientific Research Pharmaceutical Co., Ltd.) at pH 7.8 and 0.5mL (Made by a company), suspended in a microtube, then transferred to a 12-well plate (made by Corning Japan KK) and sealed, and incubated overnight in an incubator (made by an ESPEC company) set at 50°C. Move 0.4mL of this digestive fluid into a test tube, add 250μl of 0.1mg/mL chondroitin sulfate (Wako Pure Chemical Industries Co., Ltd.) aqueous solution, and 2.5mL each of 2mmol/L MgSO ₄ (Wako Pure Chemical Industries System), 0.2mol/L Tris-HCl (manufactured by Sigma-Aldrich)/5mmol/L CaCl ₂ (manufactured by Nacalai tesque Co., Ltd., pH7.8), 1% Cetylpyridinium chrolide (CPC, Wako Pure Chemical Industries Co., Ltd.) Co., Ltd.)/20mmol/L NaCl (made by Nacalai tesque Co., Ltd.), and cultured at 37°C for three hours. Through the suction of the vacuum pump, the solution was filtered with a glass filter (GC-50, manufactured by ADVANTEC), and washed with 1% CPC/20mmol/L NaCl to remove free ³⁵ S logo sulfuric acid. Move the glass filter into the vial for the liquid scintillation counter, and measure with a liquid scintillation counter (TRI-CARB, manufactured by PerkinElmer Japan Co., Ltd.) 5mL of scintillator (Hionic-Fluor, manufactured by PerkinElmer Japan Co., Ltd.) The radioactivity of the liquid.

The remaining 0.1 mL of digestion fluid was used for DNA quantification. The DNA quantification kit (made by CosmoBio Co., Ltd.) buffer 1mL was mixed with 100 μL of coupler and 450 μL of digestion solution, and the fluorescence intensity was measured at an excitation wavelength of 356 nm and a measurement wavelength of 458 nm (Infinite M200, manufactured by Tecan Gropu) .

The standard curve of DNA concentration is the standard solution (100μg/mL) attached to the kit Made by continuously diluting twice. Based on the measurement results of this standard curve, a linear regression formula (Excel, Microsoft) was prepared, and the DNA concentration of the sample was calculated.

The radioactivity of each hole is normalized by DNA content (cpm/μg DNA).

As a result, TGF-β1 of the positive control group showed a 10-fold radioactivity of the solvent control group, indicating that the synthesis amount of proteoglycan increased. PTH (1-34) showed 6 times the radioactivity of the solvent control group. ^10-6 mmol/L of compound 7 showed 3 times the radioactivity of the solvent control group, and ^10-5 mmol/L of compound 7 showed 10 times the radioactivity of the solvent control group (Figure 5). From this result, it is known that Compound 7 has a promoting effect on the synthesis of cartilage matrix in human articular chondrocytes.

Example 5: Effect of compound 7 in a rabbit model of partial meniscus resection

After rearing and acclimating a 12-week-old NZW male rabbit (manufactured by Oriential Yeast Industry Co., Ltd.) for five days, under the anesthesia of isoflurane, the outer skin of the left knee joint was cut, and the lateral collateral ligament and seed bone were cut. The ligament is removed, exposing the lateral meniscus. The central portion of the lateral meniscus was excised with a width of 3-4 mm to make a model of deformed arthropathy (Kikuchi Tetal, Osteoarth Cart 1999; 4(2): 99-110). At this time, connect three polyethylene pipes (PE60, manufactured by Japan Becton and Dickinson Co., Ltd.) connected to three osmotic pumps (2ML1, Durect, Road Cupertino, CA, US) buried under the skin of the left thigh. The front end is fixed in the joint, and the drug solution is continuously administered into the joint. The osmotic pump system is filled with (1) solvent control group (50% dimethyl sulfoxide/50% saline solution v/v), (2) compound 7 at 73.0 μg/mL, and (3) compound 7 at 730 μg/mL Any of them. Seven days after the operation, anesthetize again with isoflurane, and replace the infiltration aid filled with the same medicine as the original transplant Pu. 14 days after the meniscus partial excision operation, the rabbit was euthanized, the thigh bone and the shin tibia were taken, and immersed in 20% neutral buffered formalin to fix it. After that, the roughened surface of the articular cartilage is stained with India ink (Figure 7). A digital microscope (VHX-2000, manufactured by KEYENCE Co., Ltd.) was used to photograph the surface structure of the lower leg tibia, and the area of the ink positive reaction and the total area of the lateral malleolus were measured, and the proportion of the affected area to the entire lateral malleolus was calculated (Figure 6). As a result, it was found that Compound 7 reduced the affected area in a dose-dependent manner. The photograph of the articular cartilage surface of the calf tibia at this time is shown in Figure 4.

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

Female RccHan: WIST rats of Japan Medical Science Animal Materials Research Institute Co., Ltd. were acclimated for more than one week under standard laboratory conditions of 20 to 26°C and humidity of 30 to 70%, and then used in the experiment. Rats were allowed to ingest free rodent feed (CE-2, manufactured by Japan CLEA Co., Ltd.) containing tap water, 1.1% calcium, 1.0% phosphoric acid, and 250 IU/100g vitamin D3.

The body weight of 6-week-old rats was measured, with 10 rats as a group, and the rats were grouped to make the average body weight of each group equal. Starting from the next day after grouping, each rat was administered with repeated administration once a day within 4 weeks. To the Vehicle-Control group, the solvent (carrier) was orally administered. The carrier suspension compound 7 was administered orally at 6 mg/kg, 60 mg/kg, and 600 mg/kg, respectively, to form the compound 7-6 mg/kg, compound 7-60 mg/kg, and compound 7-600 mg/kg groups. The administration volume of any group is 2mL/kg. Propylene glycol (special grade, manufactured by Kanto Chemical Co., Ltd.) was used as a carrier. On the day after the final administration, blood was taken from the abdominal aorta under anaesthesia and the rat was euthanized, then dissected and the thigh bone was taken. The thigh bones were fixed in 10% neutral buffered formalin, and after decalcification, thin paraffin-embedded tissue specimens were prepared (hematoxylin-eosin staining). Observe the histopathology of the end of the femur through the optical microscope. The results are shown in Table 1, and the histological images of representative examples are shown in Figure 8.

As shown in Table 1, the compound 7 group, relative to the Vehicle-Control group, dose-dependently enlarged the growth plate cartilage of the femur. According to the ratio of the tissue image in Fig. 8, the thickness of the growth plate cartilage (arrow) of the representative example is obtained. The individual of Vehicle-Control is approximately 390 μm, and the individual of Compound 7-600 mg/kg is approximately 2940 μm.

As mentioned above, repeated oral administration of Compound 7 hypertrophy of the growth plate cartilage in the femur of rats. These effects are the induction of compound 7 on cartilage assimilation, the inhibition of cartilage final differentiation, or the enhancement of cartilage proliferation ability. Oral administration of compound 7 is considered to be effective in the treatment of deformed arthropathy. Furthermore, it was confirmed in Reference Test Examples 1 to 5 that the compound represented by the general formula (1) has strong PTH-like action and high metabolic stability, and is also considered to be effective against cartilage assimilation due to PTH-like action. The treatment of deformed arthropathy is effective.

Example 7: Repeated administration of 3 months for mature ovarian removal rats Effect on bone density

Female Crl:CD (SD) rats obtained from Charles River (Japan) Co., Ltd. were acclimated for more than 1 week under standard laboratory conditions of 20-26°C and 35-75% humidity for experimental use. Rats were allowed to ingest tap water and standard rodent feed (CE-2) (Japan Clea Co., Ltd.) including 1.1% calcium, 1.0% phosphoric acid, and 250 IU/100 g of vitamin D3.

8-month-old rats were given bilateral ovarian removal (OVX) and sham surgery (Sham). The body weight and lumbar spine bone density were measured at 3 months after the operation, and Sham was divided into 10 groups and OVX rats were divided into 5 groups. The rats were divided into groups and the average body weight and lumbar spine BMD became equal. After grouping, each rat was administered repeatedly once a day for a period of 3 months in the following manner. For rats in the Sham-Control group and OVX-Control group, the solvent (PO carrier) administered orally (PO), the solvent (IV carrier) administered intravenously (IV), and the SC administered subcutaneously (SC) The solvent (PC buffer) was administered for PO, IV, and SC, respectively. For the OVX-compound 7-PO group of rats, the above compound 7 dissolved in the PO carrier was administered at a dose of 30 mg/kg, and the IV carrier and the PC buffer were administered to the IV and SC, respectively. For the OVX-compound 7-IV (low dosage) group of rats, the above compound 7 dissolved in IV carrier was administered IV at a dosage of 3 mg/kg, and PO carrier and PC buffer were subjected to PO and SC Cast. For the OVX-Compound 7-IV (high dose) group of rats, the above compound 7 dissolved in IV carrier was administered IV at a dose of 10 mg/kg, and the PO carrier and PC buffer were subjected to PO and SC gave. For the OVX-hPTH(1-34) group of rats, PO vector and IV vector were separately subjected to PO And IV administration, and SC administration of hPTH (1-34) dissolved in PC buffer at 0.9 nmol/kg.

In addition, Forteo (registered trademark) used clinically as an osteoporosis treatment drug was administered to 20 μg of AUC (area under the blood concentration-time curve) to the same extent as in humans, as shown when administered to rats The dosage is set to 0.9 nmol/kg. Either group was administered with 5 mL/kg PO, 2 mL/kg IV, and 1 mL/kg subcutaneously. PO carrier or IV carrier is 10% dimethyl sulfoxide (pH 10) prepared by using glycine (made by Wako Pure Chemical Industries, Ltd.) and sodium hydroxide (made by Wako Pure Chemical Industries, Ltd.) Wako Pure Chemical Industries, Ltd.), 10% Kolliphor EL (manufactured by Sigma-Aldrich Japan Co., Ltd.), and 10% hydroxypropyl-β-cyclodextrin (manufactured by Japan Food Chemical Co., Ltd.). For the PC buffer, a pH 5.0 composition prepared from 25 mmol/L phosphate citrate buffer, 100 mmol/L NaCl and 0.05% Tween 80 was used. On the day after the final administration, blood was taken from the abdominal aorta under anesthesia and the rat was euthanized, then dissected and the lumbar spine and thigh bones were taken.

Rats that were considered to have abnormalities in the general state during the administration period or at the time of dissection were excluded from analysis. The final number of each group is: 9 in Sham-Control group, 12 in OVX-Control group, 11 in OVX-Compound 7-PO group, and 11 in OVX-Compound 7-IV (low dosage) group 10 OVX-compound 7-IV (high dose) groups and 12 OVX-hPTH (1-34) groups.

Store the lumbar vertebrae and thigh bones in 70% ethanol. The bone mineral density of the lumbar spine and thigh bone was measured by a dual-energy X-ray bone mineral content measuring device (DCS-600EX, manufactured by Aloka Corporation). Lumbar spine bone mineral density was measured in the second to fourth lumbar vertebrae, large The bone density of the thigh bone is to divide the thigh bone into 10 equal parts in the longitudinal direction and measure the position 3 minutes away from the head side of the thigh bone. The results are shown in Figure 10.

The data is represented by the average value + standard error (SE). The following statistical analysis was performed using JMP 9.02 (SAS Institute Inc). The significant level is 5% on both sides. Regarding the bone density of the lumbar spine and thigh bones, the t test of 2 groups was used to compare the Sham-Control group with the OVX-Control group (#P<0.05). In addition, using the OVX-Control group as a control group, a parameter Dunnett type multiple comparison (*P<0.05) was performed on a total of 4 groups administered with hPTH(1-34) in 3 groups administered with compound 7 (*P<0.05) .

As shown in Figure 10, regarding the bone density of the thigh bone, the OVX-Control group showed a significant decrease in bone density relative to the Sham-Control group, and the OVX-hPTH(1-34) group, which is a positive control, relative to OVX- Control group performance has increased significantly. The increase rate of the OVX-hPTH(1-34) group relative to the OVX-Control group was 14.3%. At this time, the OVX-Compound 7-PO group and the OVX-Compound 7-IV (high dosage) group showed a significant increase relative to the OVX-Control group, with the increase rates of 8.8% and 18.7%, respectively.

Regarding lumbar spine bone density, the OVX-Control group showed a significant decrease in bone density compared to the Sham-Control group, and the OVX-hPTH(1-34) group, which is a positive control, showed a significant decrease compared to the OVX-Control group. increase. The increase rate of the OVX-hPTH(1-34) group relative to the OVX-Control group is 22.1%. At this time, the OVX-Compound 7-IV (high dosage) group showed a significant increase relative to the OVX-Control group, each increasing rate was 13.3%.

As described above, because the bone density of the osteoporosis pathological model OVX rat with repeated PO or IV administration of compound 7 increased, compound 7 was recognized For the prevention, treatment, improvement, accelerated healing of osteoporosis, bone loss in periodontal disease, alveolar defects after tooth extraction, etc., diseases that require induction of bone assimilation, increase in bone mass, or bone reconstruction are effective. Furthermore, it was confirmed in Reference Examples 2 and 5 that the compound represented by the general formula (1) also has a strong PTH-like effect, and it is also considered to have an effect of increasing bone density through bone assimilation due to the PTH-like effect. Therefore, the compound represented by the general formula (1) is also considered to be required for the induction of bone assimilation, bone prevention, etc. for the prevention, treatment, improvement, accelerated healing of osteoporosis, bone loss in periodontal disease, and alveolar defects after tooth extraction. Increased amounts or diseases of bone reconstruction are effective.

Example 8: Effect of compound 7 in a rabbit model of partial meniscus resection

Introduce the 13-week-old Kbl: JW male rabbit (Oriential Yeast Industry Co., Ltd.) and domesticate it. After 10 or 11 days, under the isoflurane anesthesia, cut the outer skin of the left knee joint and replace the lateral side The ligament and seed bone ligament are excised to expose the lateral meniscus. The central portion of the lateral meniscus was excised with a width of 3-4 mm to make a model of deformed arthropathy (Kikuchi Tetal, Osteoarth Cart 1999; 4(2): 99-110). Three days after the meniscus partial resection, the carrier or compound 7 was administered into the knee joint at a low dose (0.4 mg), a medium dose (2 mg), and a high dose (10 mg). For the carrier, physiological saline was used, and 400 μL was administered to each individual. 28 days after the administration, the rabbit was euthanized, the thigh bone and tibia were taken, and immersed in 20% neutral buffered formalin containing 0.5% CPC for fixation. Then, after descaling with 10% EDTA solution (pH 7.4), a certain part of the ankles on both sides of the thigh bone and tibia (the cross section of the thigh ankle 5 mm away from the beginning of the knee fossa muscle and the center of the tibia ankle Section cross section) Paraffin sections were made and stained with safranine O (saffron O, fast red and iron hematoxylin multiple staining).

The examination of histopathology was performed using an optical microscope (BHS, Olympus Optical Industries Co., Ltd.). In order to quantitatively evaluate cartilage degeneration using specimens of lateral thigh bone ankle and lateral tibia ankle, according to Colombo et al. (Colombo C et. Al, Arthritis Rheum. 1983; 26: 875-86) Kikuchi et al.’s evaluation criteria (Kikuchi T et. Al, Osteoarthritis and Cartilage. 1995; 4:99-110), for the disappearance of the surface layer, cartilage erosion, roughening/cracking, proteoglycan staining (Saffron O Staining) Decreased, chondrocytes are not arranged properly, chondrocytes disappear, subchondral bone is exposed, and the formation of chamber-like clusters (of chondrocytes) is evaluated according to four stages of +1~+4. The sum of the scores of the 8 items is defined as the comprehensive histology score (referred to as the comprehensive score) (Figure 11). The data is expressed in terms of mean + standard error (SE). For the item-level scoring and comprehensive scoring of the evaluation items in the examination of histopathology, the statistical software SPSS 14.0J (Japan IBM Co., Ltd.) was used to implement the Mann-Whitney U test. The significance level is set at 5 and 1% risk rate.

As a result, in the histopathological examination, with regard to the lateral tibial malleolus cartilage lesions, the composite index of the cartilage lesions in the low-dose group showed a significantly lower value (inhibition of cartilage degeneration) than the solvent control group. The middle-dose and high-dose groups also tended to have low comprehensive scores.

From these, it can be considered that Compound 7 has an inhibitory effect on cartilage degeneration in lateral ankle cartilage lesions.

Reference example

The content of the present invention is described in more detail with the following reference examples and reference test examples, but the present invention is not limited to this content. All starting materials and reagents were obtained from commercial suppliers or synthesized using known methods. ¹ H-NMR spectrum, with Me ₄ Si or not for internal standards, measured using Mercury300 (manufactured by Varian), ECP-400 (manufactured by JEOL), or 400-MR (manufactured by Varian) (s=singlet, d= doublet, t=triplet, brs=broad singlet, m=multiplet). Mass analysis was performed using a mass analyzer, ZQ2000 (manufactured by Waters), SQD (manufactured by Waters), and 2020 (manufactured by Shimazu).

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

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

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

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

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

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

(Reaction 1-3)

8-(vinylsulfonyl)-1,4-dioxa-8-azaspiro[4.5]decane (431mg, 1.85mmol), 1-(4-bromo-3,5-dimethyl Phenyl)-5,5-dimethylimidazolidine-2,4-dione (575mg, 1.85mmol), ginseng (dibenzylideneacetone) dipalladium (0) (508mg, 0.55mmol), gins- A mixture of tributylphosphine tetrafluoroboric acid (165 mg, 0.55 mmol) and methyldicyclohexylamine (2.1 ml, 9.25 mmol) in N-methyl-2-pyrrolidone (18.5 ml) was carried out at 110° C. under nitrogen flow 2 Stir for hours. After cooling the reaction mixture, it was quenched with water and extracted 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 obtained residue was purified by aminosilica column chromatography (dichloromethane-methanol) to obtain (E)-1-(4-(2-(1,4-dioxa-8-azaspiro[4.5 ]Decane-8-ylsulfonyl)vinyl)-3,5-dimethylphenyl)-5,5-dimethylimidazolidine-2,4-dione (584 mg, 68%).

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

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

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

To (E)-1-(3,5-dimethyl-4-(2-((4-oxopiperidin-1-yl)sulfonyl)vinyl)phenyl)-5,5- To a solution of dimethylimidazolidine-2,4-dione (994mg, 2.37mmol) in methanol (24ml), potassium cyanide (188mg, 2.84mmol) and ammonium acetate (237mg, 3.08mmol) were added sequentially at room temperature. . The mixture was heated and stirred at 60-70°C for 3 hours. After cooling the reaction mixture, it was 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 obtained residue was purified by silica gel column chromatography (dichloromethane-ethyl acetate) to obtain (E)-4-amino-1-((4-(5,5-dimethyl-2,4- Dioxo-imidazolidin-1-yl)-2,6-dimethylstyryl)sulfonyl)piperidine-4-carbonitrile (681 mg, 68%).

¹ H-NMR (300 MHz, 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=1.59Hz),7.1(2H,s),7.4(1H,d,J=15.9Hz), 11.2(1H,brs)

To (E)-4-amino-1-((4-(5,5-dimethyl-2,4-bi- pendant imidazolidin-1-yl)-2,6-dimethylstyrene Group) sulfonyl) piperidine-4-carbonitrile (675mg, 1.50mmol) in methanol (7.5ml) and dimethyl sulfoxide (0.195ml) solution, 2N sodium hydroxide was slowly added dropwise sequentially at room temperature Aqueous solution (1.6ml, 1.6mmol) and 30% hydrogen peroxide water (0.2ml, 1.95mmol). The mixture was stirred at room temperature for 1 hour. Ethyl acetate, hexane and saturated aqueous ammonium chloride solution 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-bi- pendant imidazolidin-1-yl) -2,6-dimethylstyryl)sulfonyl)piperidine-4-carboxamide (498 mg, 72%).

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

(E)-4-amino-1-((4-(5,5-dimethyl-2,4-bi- pendant imidazolidin-1-yl)-2,6-dimethylstyrene Carboxyl) sulfonyl) piperidine-4-carboxamide (1.3g, 2.8mmol) and palladium hydroxide/carbon (Pd 20%) (about 50% water wet product) (1.3g) in methanol (21ml)- A mixture of dimethylformamide (7 ml) was stirred under hydrogen atmosphere at room temperature for 4 hours. After filtering and washing the reaction mixture, the filtrate was concentrated under reduced pressure to obtain 4-amino-1-((4-(5,5-dimethyl-2,4-bi- pendant imidazolidine-1- Base)-2,6- Dimethylphenethyl) sulfonyl) piperidine-4-carboxamide (998 mg, 77%).

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

To 4-amino-1-((4-(5,5-dimethyl-2,4-bi- pendant imidazolidin-1-yl)-2,6-dimethylphenethyl)sulfonamide )Piperidin-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) of dimethylformamide (2.5ml) solution, add O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethylurea hexafluorophosphate (HATU) (118 mg, 0.309 mmol). The mixture was stirred at room temperature for 1.5 hours. After quenching the reaction mixture with water, it was extracted with dichloromethane. After the organic layer was washed with saturated brine and washed with anhydrous sodium sulfate, it was concentrated under reduced pressure to obtain 1-((4-(5,5-dimethyl-2,4-bi- pendant imidazolidine- 1-yl)-2,6-dimethylphenethyl)sulfonyl)-4-(4-fluoro-3-(trifluoromethoxy)benzamide) piperidine-4-carboxamide (150mg, 67%).

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

At 1-((4-(5,5-dimethyl-2,4-bi- pendant imidazolidin-1-yl)-2,6-dimethylphenethyl)sulfonyl)-4- (4-fluoro-3-(trifluoromethoxy) benzoyl Amine) piperidine-4-carboxamide (150mg, 0.223mmol) in a mixture of tert-butanol (2.5ml) and ethanol (2.5ml), potassium tert-butoxide (75mg, 0.670mmol) was added at 0°C ). The mixture was heated and stirred at 50°C for 1.5 hours under a nitrogen flow. After cooling the reaction mixture, it was diluted with water, quenched with saturated aqueous ammonium chloride solution, and extracted with dichloromethane. The organic layer was washed with water and saturated brine in this order, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography (dichloromethane-methanol) to obtain 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 (118mg, 81%).

MS (ESI) m/z = 654 (M+H)+. ¹ H-NMR (400 MHz, CD ₃ OD) δ: 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)

Use appropriate carboxylic acid starting materials and reagents. The solvent was subjected to the same operations as Reactions 1-8 and 1-9 of Reference Example 1 to synthesize the compounds of Reference Examples shown below.

(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-ene-8-yl)sulfonyl)ethyl)phenyl)-5,5-dimethylimidazolidine-2,4-dione (Compound 6)

2-(3-(trifluoromethyl)phenyl)-8-(vinylsulfonyl)-1, synthesized according to the methods described in Scheme 2, Scheme 3, and Scheme 12 of WO2010/126030(A1) 3,8-Triazaspiro[4.5]dec-1-en-4-one (150mg, 0.387mmol), 1-(4-bromo-3,5-dimethylphenyl)-5,5-di Methylimidazolidine-2,4-dione (169mg, 0.542mmol), bis(dibenzylideneacetone)palladium (45mg, 0.077mmol), tri-tert-butylphosphine tetrafluoroborate (22mg, 0.077mmol) and A mixture of 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. After cooling the reaction mixture, it was quenched with water and extracted with ethyl acetate. The organic layer was washed with water and saturated brine in this order, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography (ethyl acetate-hexane) to obtain (E)-1-(3,5-dimethyl-4-(2-((4-sideoxy-2 -(3-(trifluoromethyl)phenyl)-1,3,8-triazaspiro[4.5]dec-1-en-8-yl)sulfonyl)vinyl)phenyl)-5, 5-Dimethylimidazolidine-2,4-dione (197mg, 82%).

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

(Reaction 2-2)

(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 (195mg, 0.316mmol) A mixture of 2,2,2-trifluoroethanol (6ml) with palladium hydroxide/carbon (Pd20%) (about 50% water-wet product) (195mg, 0.139mmol) was stirred at room temperature in a hydrogen atmosphere for 14 hours. After filtering the mixture, the filtrate was concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography (ethyl acetate-hexane) to obtain 1-(3,5-dimethyl-4-(2-((4-pentoxy-2-(3- (Trifluoromethyl)phenyl)-1,3,8-triazaspiro[4.5]dec-1-en-8-yl)sulfonyl)ethyl)phenyl)-5,5-dimethyl Imidazolidine-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),1.72-1.81(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- pendant-2-(4-(trifluoromethoxy)phenyl)-1,3,8-triazaspiro [4.5]dec-1-ene-8-yl)sulfonyl)ethyl)phenyl)-5,5-dimethylimidazolidine-2,4-dione (Compound 7)

(Reaction 3)

Using the appropriate starting materials and solvents and following the same procedure as in Example 2, 1-(3,5-dimethyl-4-(2-((4-pentoxy-2-(4-( Trifluoromethoxy)phenyl)-1,3,8-triazaspiro[4.5]dec-1-en-8-yl)sulfonyl)ethyl)phenyl)-5,5-dimethyl Imidazolidine-2,4-dione (compound 7).

MS (ESI) m/z = 636 (M+H)+. ¹ H-NMR (400 MHz, CDCl ₃ ) δ: 1.47 (6H, s), 1.70-1.78 (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- pendant-2-(4-(trifluoromethoxy)phenyl)-1,3,8-triazaspiro [4.5]dec-1-ene-8-yl)sulfonyl)ethyl)phenyl)-1,3-diazaspiro[4.4]nonane-2,4-dione (Compound 8)

In cyclopentanone (42mg, 0.500mmol) and 4-bromo-3,5-dimethylbenzene To a mixture of amine (100 mg, 0.500 mmol) in acetic acid (0.5 ml) was added trimethylsilane cyanide (0.063 ml, 0.500 mmol) at room temperature. The mixture was stirred at room temperature for 1.5 hours under nitrogen flow. The reaction mixture was quenched into 28% ammonia water (1 ml), diluted with water, and extracted with dichloromethane. The organic layer was washed sequentially with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 1-((4-bromo-3,5-dimethylphenyl)amino)cyclopentan Alkanonitrile is the crude product (152 mg).

¹ H-NMR (400 MHz, 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)

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

After adding triethylamine (0.103 ml, 0.742 mmol), water (0.045 ml) and methanol (0.10 ml) to the reaction mixture in this order, the mixture was heated to reflux under a nitrogen flow for 1.5 hours. After cooling the reaction mixture, it was diluted with water, adjusted to pH 5 with 1N aqueous hydrochloric acid solution, and extracted with dichloromethane. The organic layer was washed sequentially with water and saturated brine, 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 crude product.

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

1-(4-Bromo-3,5-dimethylphenyl)-4-imino-1,3-diazaspiro[4.4]nonane-2-one (crude product obtained in the previous reaction ) Of a mixture of acetic acid (1.0 ml) and water (0.25 ml) under a nitrogen stream, heated and stirred at 65° C. for 1.5 hours. After further adding acetic acid (1.0 ml) and water (0.25 ml), the mixture was heated and stirred under a nitrogen stream at 65°C for 17 hours. After cooling the reaction mixture, it was diluted with water, adjusted to pH 8 with saturated aqueous sodium bicarbonate solution, and extracted with ethyl acetate. The organic layer was washed with water and saturated brine in this order, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The obtained 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 (121mg).

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

Using appropriate starting materials and solvents, the same procedure as in Example 2 was used to obtain 1-(3,5-dimethyl-4-(2-((4-pentoxy-2-(4-( Trifluoromethoxy Yl)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 (400 MHz, 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- pendant-2-(4-(trifluoromethoxy)phenyl)-1,3,8-triazaspiro [4.5]dec-1-ene-8-yl)sulfonyl)ethyl)phenyl)-8-methyl-1,3,8-triazaspiro[4.5]decane-2,4-di Ketone (Compound 9)

Using 3-butyl 4-piperoxypiperidine-1-carboxylate as the starting material Material, and using an appropriate solvent, in the same manner as in Reference Example 4 to obtain 1-(3,5-dimethyl-4-(2-((4-pentoxy-2-(4-(tri Fluoromethoxy)phenyl)-1,3,8-triazaspiro[4.5]dec-1-en-8-yl)sulfonyl)ethyl)phenyl)-2,4-bioxy Yl-1,3,8-triazaspiro[4.5]decane-8-carboxylic acid third butyl ester.

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

On 1-(3,5-dimethyl-4-(2-((4- pendant-2-(4-(trifluoromethoxy)phenyl)-1,3,8-triaza Spiro[4.5]dec-1-en-8-yl)sulfonyl)ethyl)phenyl)-2,4-bi- pendantoxy-1,3,8-triazaspiro[4.5]decane- To a mixed solution of 8-carboxylic acid third butyl ester (11.7 mg, 0.015 mmol) in methylene chloride (0.13 ml), trifluoroacetic acid (0.05 ml, 0.673 mmol) was added at room temperature. The mixture was stirred under nitrogen flow at room temperature for 1 hour. The reaction mixture was concentrated under reduced pressure to obtain 1-(3,5-dimethyl-4-(2-((4-sideoxy-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)+.

On 1-(3,5-dimethyl-4-(2-((4- pendant-2-(4-(trifluoromethoxy)phenyl)-1,3,8-triaza Spiro[4.5]dec-1-en-8-yl)sulfonyl)ethyl)phenyl)-1,3,8-triazaspiro[4.5]decane-2,4-dione 2 trifluoro To a mixture of acetate (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 flow. After the reaction mixture was concentrated, the residue was diluted with ethyl acetate. After washing the organic layer with dilute sodium hydroxide aqueous solution, it was dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. The obtained residue was purified by column chromatography (dichloromethane-methanol) to obtain 1-(3,5-dimethyl-4-(2-((4-oxo-2-(4-(tris (Fluoromethoxy)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 (4.5mg, 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- pendant-2-(4-(trifluoromethoxy)phenyl)-1,3,8-triazaspiro [4.5]dec-1-ene-8-yl)sulfonyl)ethyl)phenyl)-2-oxa-5,7-diazaspiro[3.4]octane-6,8-dione ( Compound 10).

(Reaction 6)

Using oxetane-3-one as a starting material and using an appropriate solvent, in the same manner as in Reference Example 4, 5-(3,5-dimethyl-4-(2-(( 4-oxo-2-(4-(trifluoromethoxy)phenyl)-1,3,8-triazaspiro[4.5]dec-1-en-8-yl)sulfonyl)ethane Yl)phenyl)-2-oxa-5,7-diazaspiro[3.4]octane-6,8-dione.

MS (ESI) m/z = 650 (M+H)+. ¹ H-NMR (400 MHz, 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- pendant-2-(4-(trifluoromethoxy)phenyl)-1,3,8-triazaspiro [4.5]dec-1-ene-8-yl)sulfonyl)ethyl)phenyl)-4,6-diazaspiro[2.4]heptane-5,7-dione (Compound 11)

(Reaction 7-1)

Combine 2-bromo-5-iodo-1,3-dimethylbenzene (300 mg, 0.965 mmol), 1-aminocyclopropanecarboxylic acid (195 mg, 1.93 mmol), and copper (I) iodide (37 mg, 0.194 mmol) ) And a mixture of dimethylacetamide (2.6ml) in diazabicycloundecene (0.50ml, 3.35mmol), heated and stirred under a nitrogen flow 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 obtain 1-((4-bromo-3,5-dimethylphenyl)amino)cyclopropanecarboxylic acid ( 219mg, 80%).

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

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

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

Using appropriate starting materials and solvents, the same procedure as in Reference Example 2 was performed to obtain 4-(3,5-dimethyl-4-(2-((4-pentoxy-2-(4- (Trifluoromethoxy)phenyl)-1,3,8-triazaspiro[4.5]dec-1-en-8-yl)sulfonyl)ethyl)phenyl)-4,6-di Azaspiro[2,4]heptane-5,7-dione (Compound 11).

MS(E SI) 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=8.4 Hz), 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-ene-8-yl)sulfonyl)ethyl)phenyl)-1,3-diazaspiro[4.4]nonane-2,4-dione (Compound 12)

(Reaction 8)

Using an appropriate starting material and solvent, and following the same operation as in Reference Example 2, 1-(3,5-dimethyl-4-(2-((4-pentoxy-2-(3- (Trifluoromethyl)phenyl)-1,3,8-triazaspiro[4.5]dec-1-en-8-yl)sulfonyl)ethyl)phenyl)-1,3-diazepine Heterospiro[4.4]nonane-2,4-dione.

MS (ESI) m/z = 646 (M+H)+. ¹ H-NMR (400 MHz, 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 example

For the compounds of the present invention, the ability to produce cAMP via human PTH1R, the ability to produce cAMP via rat receptors, the metabolic stability using human liver microsomes, and the metabolic stability using rat liver cells are greater than TPTX. The test results of the rat model of increasing serum calcium concentration are shown in Test Examples 1 to 5. In addition, as a comparative compound, the compound described in WO2010/126030A1 shown in Table 3 was used.

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

(Peptide)

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

(Cell culture)

Cells were added to a humidified gas containing 5CO ₂ 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). Incubate at 37°C in the environment.

The use of LLC-PK1 cells that do not express PTH1R and one cell of LLC-PK1 that overexpress 9.5×10 ⁵ human PTH1R HKRK-B7 was 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 a 96-well plate at 1×10 ⁵ cells/well and cultured overnight. The next day, 50 μl of cAMP assay buffer (DMEM, 2 mM IBMX, 0.2 mg/ml bovine serum albumin, 35 m MHepes-NaOH, pH 7.4) containing human PTH (1-34) or compound was added and placed at 37°C Incubator for 20 minutes. After removing the medium, the cells were washed once with 100 μl of cAMP analysis buffer. To freeze the cells, the plate was placed on dry ice powder and then removed from dry ice. The cells were melted using 40 μl of 50 mM HCl, and refrozen on dry ice. The amount of cAMP produced in the cells was measured using a commercially available cAMP ElA kit (Biotrack cAMP EIA system, GE healthcare).

(According to the determination of in vitro cAMP inducibility, 20% effective concentration (EC20) and 50% effective concentration (EC50) are calculated)

S-shaped dosage reaction formula made of variable gradient was used for analysis. The cAMP signal activity of 100 nM human PTH (1-34) was taken as 100%, and the concentration of each compound showing 20% or 50% cAMP signal activity was calculated. Defined as EC20 or EC50.

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

In addition, the degree of cAMP response in LLC-PK1 cells was lower than that in HKRK-B7 cells.

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

Replacing HKRK-B7 cells with the excessive performance established by using Chinese and foreign pharmaceuticals The LLC-PK46_RATO_PTH1R cells of mouse PTH1R were measured in the same manner as in Reference Test Example 1.

The results obtained using LLC-PK46_RATO_PTH1R cells are shown in Table 5.

The EC20 value of in vitro cAMP signaling activity in rat PTH1 receptors has a good correlation with the EC20 value of in vitro cAMP signaling activity in human PTH1R. Regarding EC50 values, there is also a good correlation between rats and humans.

Reference Test Example 3: Metabolic stability test using human liver microsomes In 0.1 M phosphate buffer (PH7.4), human liver microsomes were incubated with a compound or a comparative example at 37°C for a predetermined time in the presence of NADPH. The concentration of the parent compound at each reaction time was measured using LC/MS/MS, and the intrinsic clearance rate (μl/min/mg protein) was calculated from the slope of the residual rate relative to the reaction time.

<Analysis conditions>

Compound concentration: 1μM

Microsomes: 0.5mg/mL

NADPH: 1mM

Response time: 0, 5, 15 and 30 minutes

结果如表6。 The results are shown in Table 6. Compared with Comparative Examples 1 to 6, Compounds 1 to 11 showed higher metabolic stability for human liver microsomes.

Reference Test Example 4: Metabolic stability test using rat liver cells

Hepatocytes were prepared from the livers of rats (SD, females) using collagenase reflux method. The compound of the reference example or the comparative example was added, and the reaction stop solution was added after incubating at 37°C for a predetermined time. The concentration of the parent compound at each reaction time was measured using LC/MS/MS, and the intrinsic clearance rate (μL/10 ⁶ cells/min) was calculated from the slope of the residual rate to the reaction time.

<Analysis conditions>

Cell concentration: 1×10 ⁶ cells/mL

Compound concentration: 1μM

Medium: Williams’medium E

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

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

结果如表7。 The results are shown in Table 7. The compounds 2, 4, 5, 6, 7, 8, 9, 10, and 11 have improved metabolic stability of rat liver cells compared to Comparative Examples 1, 2, 3, 5, and 6.

Reference Test Example 5: Serum calcium concentration increase in TPTX rat model

A 4-week-old female Crl:CD (SD) rat was obtained from Japan Charles River Co., Ltd. (Atsugi Feeding Center) and acclimated for 1 week under standard laboratory conditions of 20-26°C and humidity of 35-75%. Rats were allowed to ingest tap water and standard rodent feed (CE-2) (Japan Clea Co., Ltd.) containing 1.1% calcium, 1.0% phosphoric acid, and 250 IU/100 g of vitamin D3.

TPTX was administered to 5-week-old rats. Sham surgery is performed on some individuals. For the TPTX rats used, individuals with a serum calcium concentration of less than 8 mg/dL after 4 days of surgery were selected. Five days after the operation, according to the serum calcium concentration and body weight measured 4 days after the operation, 5 animals were divided into 8 groups and divided into 8 TPTX groups and 1 Sham group. For the Sham group and TPTX-Vehicle group, the solvent was administered orally at a dose of 10 mL/kg. For the TPTX-each sample group, each sample was dissolved in a solvent at a dose of 30 mg/10 mL/kg and administered orally. Solvent composition, using 10% dimethyl sulfoxide (Wako Pure Chemical Industries Co., Ltd.), 10% Cremophor EL (Sigma Aldrich Japan contract company), 20% hydroxypropyl-β-cyclodextrin (Japanese food Chemical Industry Co., Ltd.), Glycine (Wako Pure Chemical Industries Co., Ltd.), adjusted to pH10. Blood was drawn in advance (pre) immediately before the start of each administration, and blood was also drawn 2, 6, 10, and 24 hours after the administration to measure the serum calcium concentration. Each blood draw was performed from the jugular vein under isoflurane inhalation anesthesia.

Determination of serum calcium. The serum obtained by centrifugation of the drawn blood was measured using an automatic analyzer TBA-120FR (Toshiba Medical System Co., Ltd.).

Statistical analysis for animal experiments. Data is average + standard error Poor (SE) said. Statistical significance is determined using SAS pre-clinical software package (Ver.5.00.010720, SAS In statute Japan, Tokyo, Japan). A p value <0.05 is considered statistically significant. According to the t test of 2 groups, it shows #P<0.05 for the TPTX-Vehicle group, *P<0.05 for the group of Comparative Example 1, and ʃP<0.05 for the group of Comparative Example 2.

The Pre value of serum calcium concentration was 9.9 mg/dL in Sham group and 5.3-6.2 mg/dL in TPTX group. For the serum calcium concentration up to 24 hours after administration of each compound, the value obtained by averaging the amount of change from the Pre value is shown in FIG. 9. In addition, the peak of serum calcium concentration after administration of each compound was 6 hours or 10 hours after administration of any compound.

The compound 6, compound 7, and compound 8 with high metabolic stability of rat liver cells have a large amount of change from the positive value of pre, and it is considered to have a strong effect of increasing serum calcium concentration by oral administration. In addition, Compound 1, Comparative Example 1, and Comparative Example 2 with a low metabolic stability of rat liver cells had a smaller positive change from the Pre value than Compound 6, Compound 7, and Compound 8. In particular, Compound 7 and Compound 8 are statistically superior to Comparative Example 1 and Comparative Example 2.

Furthermore, compound 6, compound 7, and compound 8 with high metabolic stability in rat liver cells showed a value of 7.8 to 8.5 mg/dL for the maximum value of each compound 6 hours or 10 hours after the administration. The therapeutic target range of serum calcium concentration in patients with parathyroidism is 7.6~8.8mg/dL. In addition, the compound 1, comparative example 1, and comparative example 2 with low metabolic stability of rat hepatocytes did not reach this therapeutic target range at all measurement times.

From the above test results, it can be seen that the cells that forcibly express rat PTH1R show strong cAMP signaling activity, and the Xie showed high stability of compound 6, compound 7, and compound 8, and it was confirmed in rats that it showed a strong effect of increasing serum calcium concentration under oral administration. These compounds have cAMP signaling activity in cells that forcefully express human PTH1R, and the metabolic stability of human liver microsomes is higher than that of comparative compounds, and oral administration is expected to have a high therapeutic effect on patients with parathyroidism. In addition, the compound represented by general formula (1) that exhibits cAMP signaling activity and human liver microsomal metabolic stability in cells forcibly expressing human PTH1R to the same extent as compound 6, compound 7, and compound 8 is also expected It has a high therapeutic effect for patients with hypothyroidism.

Industrial availability

According to the present invention, a hydantoin derivative having high metabolic stability and exerting a strong PTH-like action is provided to induce assimilation of hard bone and cartilage through non-invasive systemic exposure or local exposure to prevent, treat, recover and Accelerate the healing of osteoporosis, bone loss in periodontal disease, alveolar bone defect after tooth extraction, deformed arthropathy, articular cartilage defect, non-dynamic bone lesion, achondroplasia, achondroplasia, osteomalacia, Medicine for fractures and other diseases.

Claims (6)

Use of a compound selected from the group consisting of the following or a pharmacologically acceptable salt thereof in the preparation of a pharmaceutical composition for preventing or treating hypothyroidism, or inducing assimilation of hard bone and/or cartilage, 1-(4- (2-((2-(4-fluoro-3-(trifluoromethoxy)phenyl)-4-oxo-1,3,8-triazaspiro[4.5]dec-1-ene- 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 -Penoxy-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]-dioxan-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-(trifluoro (Methyl)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-sideoxy-2-(4-(trifluoromethoxy)phenyl)-1, 3,8-triazaspiro[4.5]dec-1-en-8-yl)sulfonyl)ethyl)benzene Radical)-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-diazepine Heterospiro[4.4]nonane-2,4-dione; 1-(3,5-dimethyl-4-(2-((4-sideoxy-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-(trifluoromethyl Oxy)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-(trifluoro Methoxy)phenyl)-1,3,8-triazaspiro[4.5]dec-1-en-8-yl)sulfonyl)ethyl)phenyl)-4,6-diazaspiro [2.4] Heptane-5,7-dione. The use as described in item 1 of the patent application scope, wherein the compound is 1-(3,5-dimethyl-4-(2-((4-oxo-2-(3-(trifluoromethyl Yl)phenyl)-1,3,8-triazaspiro[4.5]dec-1-en-8-yl)sulfonyl)ethyl)phenyl)-5,5-dimethylimidazolidine- 2,4-Dione or its pharmacologically acceptable salt. The use as described in item 1 of the patent application scope, wherein the compound is 1-(3,5-dimethyl-4-(2-((4-oxo-2-(4-(trifluoromethyl Oxy)phenyl)-1,3,8-triazaspiro[4.5]dec-1-en-8-yl)sulfonyl)ethyl)phenyl)-5,5-dimethylimidazolidine -2,4-dione or its pharmacologically acceptable salt. The use as described in item 1 of the patent application scope, wherein the compound is 1-(3,5-dimethyl-4-(2-((4-oxo-2-(4-(trifluoromethyl Oxy)phenyl)-1,3,8-triazaspiro[4.5]dec-1-en-8-yl)sulfonyl)ethyl)benzene Radical)-1,3-diazaspiro[4.4]nonane-2,4-dione or a pharmacologically acceptable salt thereof. The use according to any one of items 1 to 4 of the patent application range, wherein the pharmaceutical composition is used for the prevention or treatment of osteoporosis, the improvement of bone loss in periodontal disease, and the acceleration of alveolar bone defect after tooth extraction Healing, prevention or treatment of deformed arthropathy, accelerated healing of articular cartilage defects, prevention or treatment of non-dynamic bone lesions, prevention or treatment of achondroplasia, prevention or treatment of achondroplasia, prevention of osteomalacia Or treatment, or accelerated healing of fractures. The use according to any one of items 1 to 4 of the patent application range, wherein the pharmaceutical composition is used for the prevention or treatment of hypothyroidism.

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