HK1209735B - Hydantoin derivative - Google Patents

Description

Hydantoin derivatives

Technical Field

The present invention relates to a pharmaceutical preparation containing as an active ingredient a hydantoin derivative having high metabolic stability and exhibiting a potent PTH-like action.

Background

Parathyroid hormone (PTH) binds to PTH1 receptor (PTH 1R) which is a G protein-coupled receptor (GPCR), activates G protein, and then activates at least 1 signaling cascade in cyclic amp (camp)/protein kinase a cascade and the like. PTH acts on bone and kidney target cells, and is known as a hormone that regulates calcium (Ca) and phosphorus (Pi) homeostasis (non-patent document 1). PTH-based maintenance of serum Ca levels is primarily through direct or indirect effects on the gut, bone and kidney. PTH promotes reabsorption of Ca by renal tubules and inhibits excretion of Ca from the body to the outside. In addition, the increase in the synthesis of enzymes that convert vitamin D into active vitamin D in the kidney contributes to the promotion of Ca absorption from the digestive tract by active vitamin D. PTH indirectly promotes differentiation of osteoclasts by osteoblasts and the like, thereby promoting release of Ca from bone. The action of PTH is thought to be exerted mainly by activation of receptors by adenylate cyclase and/or phospholipase C (due to cAMP production by binding to PTH 1R).

In humans, PTH preparations [ PTH (1-34) and PTH (1-84) ] have a potent bone forming effect, inducing a significant increase in bone density and bone strength. At present, most of osteoporosis therapeutic agents available for human use are bone resorption inhibitors, and only PTH preparations are bone formation agents that positively increase bone density. Although PTH preparations are considered to be one of the most effective means for treating osteoporosis (non-patent document 2), they are peptides and therefore need to be administered invasively. Therefore, it is desired to create a pharmaceutical agent having a PTH-like action and capable of non-invasive administration.

Hypoparathyroidism is a metabolic disease that exhibits hypocalcemia and hyperpi-emia resulting from insufficient PTH secretion from the parathyroid gland, and many symptoms that follow. In the treatment of hypoparathyroidism, active vitamin D preparations and Ca agents are used, but since the regulation mechanism depending on PTH does not work, a sufficient therapeutic effect cannot be obtained. In addition, active vitamin D preparations cause an increase in urinary Ca excretion, and thus are indicated to increase the risk of renal disorders in long-term treatment. In order to solve these problems, the efficacy of a supplementation therapy relying on a PTH preparation for the disease is improved, but in order to obtain a sufficient drug effect, it is tried to perform an invasive administration for a plurality of times over 1 day and a continuous administration using a pump (non-patent document 3). Therefore, in the treatment of hypoparathyroidism, it is also desired to create an agent which has a PTH-like action and can be administered non-invasively.

Further, in the treatment of diseases such as bone fracture, nondynamic bone disease, cartilage growth insufficiency, subchondral chondrogenesis, osteomalacia, osteoarthritis, arthritis, thrombocytopenia, hyperphosphatemia, and tumorous calcinosis, a non-invasive administrable agent having a PTH-like action is also expected.

Under the above circumstances, the present applicant has found that a compound represented by the following general formula (a) or a pharmacologically acceptable salt thereof is useful as a compound having a PTH-like action (preferably, PTH1R agonist), and is useful as a preventive and/or therapeutic agent or stem cell mobilization for osteoporosis, bone fracture, osteomalacia, arthritis, thrombocytopenia, hypoparathyroidism, hyperphosphatemia, tumorous calcinosis, or the like, and has previously applied for a patent (patent document 1).

[ solution 1]

[ in the formula, W, X, Y, m, n, R₁、R₂、R₃₃、R₃₄Refer to patent document 1 ].

However, in order to create a clinically valuable drug capable of being administered invasively, consideration is given to the direct action on the target, and also to the in vivo dynamics such as absorption, distribution, metabolism, and excretion of the drug. In particular, for oral administration, a drug having a strong cAMP production ability through human PTH1R and a PTH-like action with high metabolic stability to human liver microparticles is desired.

In order to provide a drug that can be orally administered to a human, a method of orally administering the drug by an in vivo test using a model animal and confirming the effect is generally used. As a model animal, for example, thyroid gland parathyroid gland extirpation (TPTX) rats are known for hypoparathyroidism. In order to discover a therapeutic agent having a strong PTH-like action and high metabolic stability and acting orally against hypoparathyroidism, a method for verifying the action under oral administration using a TPTX rat model was effective in discovering a compound that acts on rat PTH1R and is stable against rat metabolic enzymes.

In the current treatment of hypoparathyroidism, the treatment target range of serum Ca concentration is set to a range (7.6 to 8.8 mg/dL) slightly lower than the lower limit of the normal range (non-patent document 4). Since the normal range of serum Ca concentration in rats is about 10 mg/dL and is at the same level as that in humans, achieving a serum Ca concentration in a range from the therapeutic target range (7.6 to 8.8 mg/dL) in humans to the lower limit (11.2 mg/dL) of hypercalcemia in humans in rat pathologic models is important for verifying the therapeutic effect.

Documents of the prior art

Patent document

Patent document 1 International publication No. 2010/126030

Non-patent document

Non-patent document 1 Kronenberg, H.M., In Handbook of Experimental Pharmacology, Mundy, G.R., and Martin, T.J., (eds.), pp.185-201, Springer-Verlag, Heidelberg (1993)

Non-patent document 2, Tashjian and Gagel, J. Bone Miner. Res. 21:354-

Non-patent document 3, Rejnmark et al, Osteporosis int. Published 0nline: 27November 2012

Non-patent document 4: Winer KK et al, J. Clin. Endocrinol. Metab. 88(9): 4214-.

Disclosure of Invention

Problems to be solved by the invention

The present invention has found that a compound having a strong PTH-like action and high metabolic stability is useful for the treatment of diseases such as hypoparathyroidism which can be treated by PTH-like action.

Means for solving the problems

Under the circumstances described above, the present inventors have conducted extensive studies and found that the hydantoin derivatives of the present invention newly found exhibit strong cAMP-producing ability in cells in which human PTH1R is forcibly expressed and have high stability against the metabolism of human liver microparticles. The present inventors have also found for the first time that the compound of the present invention exhibits a strong cAMP-producing ability in cells forcibly expressing rat PTH1R, has high stability against metabolism in rat hepatocytes, and restores the serum Ca concentration to a therapeutic target range of 7.6 to 8.8mg/dL at an administration amount of 30 mg/kg in a TPTX rat model by oral administration. The results of this model animal show that the compound represented by the general formula (1) showing a strong effect against human PTH1R and high stability in liver microparticles is useful as a therapeutic agent for hypoparathyroidism.

Namely, the present invention relates to the following aspects.

[ 1] A compound represented by the following general formula (1) or a pharmacologically acceptable salt thereof;

[ solution 2]

In the formula (I), the compound is represented by,

r1 and R2 are each independently, provided that not both R1 and R2 are hydrogen atoms:

1) a hydrogen atom,

2) A halogen atom,

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

4) Alkoxy groups having 1 to 2 carbon atoms which may be substituted with 1 to 5 fluorine atoms; alternatively, the first and second electrodes may be,

r1 and R2 are groups represented by the following formula:

[ solution 3]

(wherein each represents a bonding site to a phenyl moiety.)

And R3 and R4 are each independently a methyl group which may be substituted with 1 to 3 fluorine atoms; alternatively, the first and second electrodes may be,

r3 and R4 form a ring having 3 to 6 carbon atoms (here, one of the carbon atoms forming the ring may be replaced by an oxygen atom; a sulfur atom; or a nitrogen atom which may be replaced by a methyl group). Angle (c)

In the present invention, from among the compounds represented by the above general formula (1), compounds in which the combination of R1 and R2 is a trifluoromethyl group and a hydrogen atom, and R3 and R4 together with the carbon atom to which they are bonded are a cyclopentyl ring are excluded.

[ 2] the compound according to [ 1] or a pharmacologically acceptable salt thereof, wherein R1 and R2 are selected from the group consisting of:

1) r1 is a hydrogen atom or a halogen atom, and R2 is a hydrogen atom, a trifluoromethyl group or a trifluoromethoxy group (wherein, the case where R1 and R2 are both hydrogen atoms is excluded);

2) r1 is trifluoromethyl or trifluoromethoxy, and R2 is hydrogen or halogen;

3) r1 and R2 are groups represented by the following formula, which are bonded to each other:

[ solution 4]

(wherein each represents a site bonded to a phenyl moiety.)

And R3 and R4 are methyl; alternatively, the first and second electrodes may be,

r3 and R4 form, together with the carbon atom to which they are bonded, a ring selected from:

[ solution 5]

(wherein < lambda > represents a site bonded to an imidazolidine-2, 4-dione moiety).

[ 3] the compound according to [ 1] or a pharmacologically acceptable salt thereof, wherein R1 and R2 are selected from the group consisting of:

1) r1 is trifluoromethoxy and R2 is fluorine atom;

2) r1 is a bromine atom, and R2 is a hydrogen atom;

3) r1 is trifluoromethyl, and R2 is a fluorine atom;

4) r1 is a fluorine atom, and R2 is trifluoromethoxy;

5) r1 is trifluoromethyl, and R2 is a hydrogen atom;

6) r1 is a hydrogen atom, and R2 is trifluoromethoxy;

7) r1 and R2 are groups represented by the following formula:

[ solution 6]

(wherein each represents a site bonded to a phenyl moiety.)

And R3 and R4 are methyl; alternatively, the first and second electrodes may be,

r3 and R4 form, together with the carbon atom to which they are bonded, a ring selected from:

[ solution 7]

(wherein < lambda > represents a site bonded to an imidazolidine-2, 4-dione moiety).

[ 4] the compound according to [ 1] or a pharmacologically acceptable salt thereof, wherein R3 and R4 are methyl groups.

[ 5] the compound according to [ 1] or a pharmacologically acceptable salt thereof, wherein R3 and R4 form, together with the carbon atom to which they are bonded, a ring selected from the following rings:

[ solution 8]

(wherein < lambda > represents a site bonded to an imidazolidine-2, 4-dione moiety).

[ 6] the compound according to [ 1] or a pharmacologically acceptable salt thereof, wherein the compound is selected from the group consisting of:

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

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

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

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

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

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

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

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

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

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

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

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

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

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

A pharmaceutical composition comprising as an active ingredient a compound according to any one of [ 1] to [ 9] or a pharmacologically acceptable salt thereof.

[ 11] the pharmaceutical composition according to [ 10] for oral administration.

A pharmaceutical composition for activating an intracellular cAMP response, which comprises the compound according to any one of [ 1] to [ 9] or a pharmacologically acceptable salt thereof as an active ingredient.

[ 13] A prophylactic or therapeutic agent for osteoporosis, bone fracture, nonmotile bone disease, cartilage growth insufficiency, oligochondrogenesis, osteomalacia, osteoarthritis, arthritis, thrombocytopenia, hypoparathyroidism, hyperphosphatemia, or tumorous calcinosis, or a stem cell mobilizer, which comprises the compound according to any one of [ 1] to [ 9] or a pharmacologically acceptable salt thereof as an active ingredient.

A method for the prevention or treatment of a disease or for the mobilization of stem cells, wherein the disease is osteoporosis, bone fracture, unpowered bone disease, cartilage insufficiency, hypochondriasis, osteomalacia, osteoarthritis, arthritis, thrombocytopenia, hypoparathyroidism, hyperphosphatemia, or tumorous calcinosis, comprising: a pharmaceutically effective amount of a composition containing the compound according to any one of [ 1] to [ 9] or a pharmacologically acceptable salt thereof is administered to a patient in need of prevention or treatment of osteoporosis, bone fracture, nondynamic bone disease, cartilage growth insufficiency, oligochondrogenesis, osteomalacia, osteoarthritis, arthritis, thrombocytopenia, hypoparathyroidism, hyperphosphatemia, or tumorous calcinosis, or stem cell mobilization.

Use of the compound according to any one of [ 15] [ 1] to [ 9] or a pharmacologically acceptable salt thereof for the production of a prophylactic or therapeutic agent for treating osteoporosis, bone fracture, nondynamic bone disease, cartilage growth insufficiency, oligochondrogenesis, osteomalacia, osteoarthritis, arthritis, thrombocytopenia, hypoparathyroidism, hyperphosphatemia, or tumorous calcinosis; or in the manufacture of a stem cell mobilizer.

The compound according to any one of [ 16] to [ 1] to [ 9] or a pharmacologically acceptable salt thereof, for use in the treatment or prevention of osteoporosis, bone fracture, nondynamic bone disease, cartilage growth insufficiency, oligochondrogenesis, osteomalacia, osteoarthritis, arthritis, thrombocytopenia, hypoparathyroidism, hyperphosphatemia, or tumorous calcinosis, or in the mobilization of stem cells.

Further, the present invention provides a method for treating a condition treatable by a PTH-like action, represented by hypoparathyroidism, by administering the general formula (1) or a salt thereof.

ADVANTAGEOUS EFFECTS OF INVENTION

The present invention provides a hydantoin derivative having a strong PTH-like action and high metabolic stability. The use of the hydantoin derivative can treat disorders caused by PTH-like action, such as hypoparathyroidism.

Drawings

FIG. 1 is a graph showing the mean change in serum Ca concentration up to 24 hours after administration of each compound when the compound was orally administered to a TPTX rat model at a dose of 30 mg/kg.

Detailed Description

The present invention relates to hydantoin derivatives and use thereof. The present inventors synthesized the compound represented by the above formula (1) or a pharmacologically acceptable salt thereof for the first time, and found that the compound or a salt thereof is a compound having a strong PTH-like action and high metabolic stability.

The "alkyl group" in the present specification is a 1-valent group derived by removing 1 arbitrary hydrogen atom from an aliphatic hydrocarbon, and includes a hydrocarbon group or a partial group of a hydrocarbon group structure containing hydrogen and carbon atoms, in which a hetero atom or an unsaturated carbon-carbon bond is not contained in the skeleton. The alkyl group includes linear and branched structures. The alkyl group is preferably an alkyl group having 1 or 2 carbon atoms. Specific examples of the alkyl group include a methyl group and an ethyl group, and a methyl group is preferable.

The "alkoxy group" in the present specification means an oxy group to which the "alkyl group" defined above is bonded, and is preferably an alkoxy group having 1 or 2 carbon atoms. Specifically, examples thereof include methoxy and ethoxy groups, and methoxy is preferable.

In the present specification, "B which may be substituted with a" means that any hydrogen atom in B may be substituted with any number of a.

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

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

In the present specification, the term "onium" in the chemical formula means a bonding site.

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

The term "PTH-like action" as used herein refers to an activity of generating intracellular cAMP (cAMP: cyclic adenosine monophosphate) by acting on a PTH receptor or acting on a signal transmission system via the PTH receptor.

In the present invention, the presence or absence of "a strong PTH-like action" or "a strong PTH-like action" can be confirmed, for example, by assaying cAMP signaling and measuring cAMP signaling activity according to the method described in J.bone.Miner. Res.14: 11-20, 1999. Specifically, for example, according to the method described in test example 1, the concentration of each compound showing 20% cAMP signaling activity (EC 20) or 50% cAMP signaling activity (EC 50) was measured using a commercially available cAMP EIA kit (e.g., Biotrack cAMP EIA system, GE health care) with respect to the amount of cAMP production in cells that strongly express human PTH1R, with cAMP signaling activity at the time of administration of 100nM of human PTH (1-34) being taken as 100%. The "strong PTH-like effect" or "PTH-like effect" in the present invention means, for example, that the value (μ M) of EC20 measured by the above-mentioned method is preferably 5.0 or less, more preferably 3.0 or less, and further preferably 2.0 or less. In the case of EC50, for example, the value (μ M) measured by the above-described method is preferably 25.0 or less, more preferably 15.0 or less, and further preferably 10.0 or less.

In addition, the presence or absence of "high metabolic stability" or "high metabolic stability" can be confirmed using conventional assay methods. For example, confirmation can be performed using hepatocytes, small intestine cells, liver microparticles, small intestine microsomes, liver S9, and the like. Specifically, for example, it can be confirmed by measuring the stability of the compound in liver microparticles according to the description of T Kronbach et al (Oxidation of midzolam and triazolam by human liverochrome P450IIIA4. mol. Pharmacol, 1989, 36(1), 89-96). More specifically, the confirmation can be performed by the method described in test example 3. The "high metabolic stability" or "high metabolic stability" in the present invention means, for example, that the value of the clearance rate (μ L/min/mg) in the metabolic stability test using human liver microparticles described in the above test examples is preferably 60 or less, more preferably 40 or less, and still more preferably 35 or less. In particular, in the above formula (1), high metabolic stability can be obtained except for the case where the combination of R1 and R2 is a trifluoromethyl group and a hydrogen atom, and R3 and R4 together with the carbon atom to which they are bonded are a cyclopentyl ring.

The compound of the present invention may be an episome, but a pharmacologically acceptable salt is also included in the present invention. Examples of such "salt" include inorganic acid salts, organic acid salts, inorganic base salts, organic base salts, and acidic or basic amino acid salts.

Preferred examples of the inorganic acid salt include hydrochloride, hydrobromide, sulfate, nitrate, phosphate and the like, and preferred examples of the organic acid salt include acetate, succinate, fumarate, maleate, tartrate, citrate, lactate, stearate, benzoate, methanesulfonate, benzenesulfonate, p-toluenesulfonate and the like.

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

Preferable examples of the acidic amino acid salt include, for example, an aspartic acid salt and a glutamic acid salt, and preferable examples of the basic amino acid salt include, for example, an arginine salt, a lysine salt, and an ornithine salt.

The compounds of the present invention may be allowed to stand in the atmosphere, and may sometimes absorb moisture, carry adsorbed water, or form hydrates, and such hydrates also belong to the salts of the present invention.

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

In the present specification, the structural formula of a compound is sometimes represented as a certain isomer for convenience, but the present invention includes all geometric isomers, optical isomers, stereoisomers, tautomers and other isomers and isomer mixtures formed depending on the structure of the compound, and is not limited to the structural formula described for convenience, and may be any isomer or mixture. Therefore, although the optically active form and the racemic form having an asymmetric carbon atom in the molecule may be present in the compound of the present invention, the present invention is not limited thereto, and includes all of them.

The present invention includes all isotopic compounds (isotopes) of the compound represented by formula (1). Isotopic compounds of the present invention are those in which at least 1 atom is replaced by an atom having the same atomic number (proton number) but different mass number (sum of the numbers of protons and neutrons). Examples of the isotope contained in the compound of the present invention include a hydrogen atom, a carbon atom, a nitrogen atom, an oxygen atom, a phosphorus atom,Sulfur atom, fluorine atom, chlorine atom, etc., each of which comprises²H、³H、¹³C、¹⁴C、¹⁵N、¹⁷O、¹⁸O、³¹P、³²P、³⁵S、¹⁸F、³⁶Cl, and the like. In particular, it is possible to use, for example,³H、¹⁴radioactive isotopes such as C, which exhibit radioactive decay, are useful in performing in vivo tissue distribution tests and the like of drugs or compounds. The stable isotope does not decay, and the amount of the stable isotope present hardly changes, and is free from radioactivity, and therefore, it can be safely used. Isotopic compounds of the present invention can be converted by conventional methods by substituting reagents used in synthesis with reagents containing the corresponding isotopes.

In the compound of the present invention, there may be a crystal polymorph, but there is no particular limitation, and any single crystal polymorph or a crystal polymorph mixture may be used.

The present invention relates to compounds including prodrugs thereof. Prodrugs are derivatives of the compounds of the present invention which have chemically or metabolically decomposable groups and, after administration to the body, revert to the original compound and exert their original pharmacological effects, including non-covalent bond-based complexes and salts.

The compound represented by the above formula (1) of the present invention is preferably as follows.

Wherein R1 and R2 are selected from the following combinations:

1) r1 is a hydrogen atom or a halogen atom, and R2 is a hydrogen atom, a trifluoromethyl group or a trifluoromethoxy group (wherein, the case where R1 and R2 are both hydrogen atoms is excluded);

2) r1 is trifluoromethyl or trifluoromethoxy, and R2 is hydrogen or halogen;

3) r1 and R2 are groups represented by the following formula, which are bonded to each other:

[ solution 9]

(wherein, each represents a site bonded to a phenyl moiety.)

And R3 and R4 are methyl;

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

[ solution 10]

(wherein < lambda > represents a site bonded to an imidazolidine-2, 4-dione moiety).

The compound represented by the above formula (1) of the present invention is more preferably as follows.

Wherein R1 and R2 are selected from the following combinations:

1) r1 is trifluoromethoxy and R2 is fluorine atom;

2) r1 is a bromine atom, and R2 is a hydrogen atom;

3) r1 is trifluoromethyl, and R2 is a fluorine atom;

4) r1 is a fluorine atom, and R2 is trifluoromethoxy;

5) r1 is trifluoromethyl, and R2 is a hydrogen atom;

6) r1 is a hydrogen atom, and R2 is trifluoromethoxy;

7) r1 and R2 are groups represented by the following formula:

[ solution 11]

(wherein, each represents a site bonded to a phenyl moiety.)

And R3 and R4 are methyl;

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

[ solution 12]

(wherein < lambda > represents a site bonded to an imidazolidine-2, 4-dione moiety).

The compound represented by the above formula (1) of the present invention is more preferably a compound selected from the group consisting of the following compounds 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-en-8-yl) sulfonyl) ethyl) -3, 5-dimethylphenyl) -5, 5-dimethylimidazolidine-2, 4-dione,

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

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

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

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

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

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

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

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

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

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

Among the above compounds 1 to 11, the compounds 6, 7 and 8 are more preferable.

The compound represented by the above formula (1) or a pharmacologically acceptable salt thereof of the present invention is useful as a compound having a PTH-like action (preferably, PTH1R agonist), and is useful for the prevention and/or treatment of osteoporosis, bone fracture, nondynamic bone disease, cartilage insufficiency, oligochondrogenesis, osteomalacia, osteoarthritis, arthritis, thrombocytopenia, hypoparathyroidism, hyperphosphatemia, or tumorous calcinosis, or for the mobilization of stem cells.

The compound or a salt thereof according to the present invention can be prepared into preparations such as tablets, powders, fine granules, coated tablets, capsules, syrups, buccal preparations, inhalants, suppositories, injections, ointments, eye drops, nose drops, ear drops, cataplasms, and lotions by a method generally used. The pharmaceutical preparation can be prepared by a conventional method using excipients, binders, lubricants, coloring agents, flavoring agents, stabilizers, emulsifiers, absorption enhancers, surfactants, pH adjusters, preservatives, antioxidants, and the like, which are generally used in the preparation, and optionally, ingredients generally used as raw materials for pharmaceutical preparations.

For example, in order to produce an oral preparation, the compound of the present invention or a pharmacologically acceptable salt thereof, an excipient, and optionally a binder, a disintegrant, a lubricant, a coloring agent, a flavoring agent, and the like are added, and then the mixture is prepared into powder, fine granules, tablets, coated tablets, capsules, and the like by a conventional method.

Examples of the components include animal and vegetable oils such as soybean oil, beef tallow, and synthetic glyceride; hydrocarbons such as liquid paraffin, squalane, and solid paraffin; ester oils such as octadecyl myristate and isopropyl myristate; higher alcohols such as cetostearyl alcohol, behenyl alcohol, etc.; a silicone resin; a silicone oil; surfactants such as polyoxyethylene fatty acid esters, sorbitan fatty acid esters, glycerin fatty acid esters, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene hydrogenated castor oils, and polyoxyethylene polyoxypropylene block copolymers; water-soluble polymers such as hydroxyethyl cellulose, polyacrylic acid, carboxyvinyl polymer, polyethylene glycol, polyvinylpyrrolidone, and methyl cellulose; 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 silicic anhydride, magnesium aluminum silicate and aluminum silicate, and purified water.

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

Examples of the binder include polyvinyl alcohol, polyvinyl ether, methyl cellulose, ethyl cellulose, gum arabic, tragacanth, gelatin, shellac, hydroxypropylmethyl cellulose, hydroxypropyl cellulose, polyvinylpyrrolidone, polypropylene glycol, a seeding and seeding polyoxyethylene, a meglumine, and the like.

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

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

As the coloring agent, a coloring agent that is allowed to be added to a pharmaceutical product can be used, and as the flavoring agent, cocoa powder, menthol, aromatic powder, peppermint oil, borneol, cinnamon powder, and the like can be used.

The above-mentioned tablets or granules may be coated with sugar, or may be coated with other coatings as required. In addition, in the production of a liquid preparation such as syrup or an injection preparation, a pH adjuster, a dissolving agent, an isotonizing agent, and the like, and if necessary, a dissolution assistant, a stabilizer, and the like are added to the compound of the present invention or a pharmacologically acceptable salt thereof, and the preparation is prepared by a conventional method.

The method for producing the external preparation is not limited, and the preparation can be produced by a conventional method. That is, as base materials used for preparing a pharmaceutical preparation, various materials generally used in medicines, pharmaceutical analogs (quasi drugs), cosmetics, and the like can be used. Specific examples of the base material used include animal and vegetable oils, mineral oils, ester oils, waxes, higher alcohols, fatty acids, silicone oils, surfactants, phospholipids, alcohols, polyols, water-soluble polymers, clay minerals, purified water, and the like, and further, if necessary, a pH adjuster, an antioxidant, a chelating agent, a preservative and antifungal agent, a coloring agent, a perfume, and the like may be added.

Further, components having differentiation inducing activity, blood flow promoters, antiseptics, anti-inflammatory agents, cell activators, vitamins, amino acids, moisturizers, keratolytic agents, and the like may be blended as necessary. The amount of the base material added is an amount that is a concentration set in the usual production of an external preparation.

When the compound of the present invention, a salt thereof, or a hydrate thereof is administered, the form thereof is not particularly limited, and oral administration or non-oral administration can be performed by a commonly used method. For example, the composition can be administered in the form of preparations such as tablets, powders, granules, capsules, syrups, buccal preparations, inhalants, suppositories, injections, ointments, eye drops, nasal drops, ear drops, cataplasms, and lotions. In particular, the compound of the present invention is suitable for oral administration because of its excellent cAMP signaling activity and metabolic stability.

The dose of the drug according to the present invention can be appropriately selected depending on the degree of symptoms, age, sex, body weight, administration form, species of seeds and salts, specific species of diseases, and the like.

The administration amount varies significantly depending on the kind of disease, the degree of symptoms of the disease, the age, sex, sensitivity to drugs, etc. of the patient, and is usually about 0.03 to 1000 mg, preferably 0.1 to 500 mg, more preferably 0.1 to 100 mg, per 1 day, and 1 to several times per 1 day as an adult.

In the production of the compound of the present invention represented by the above formula (1), the raw material compound may be a salt, hydrate or solvate, which may vary depending on the starting material, the solvent used, etc., and is not particularly limited as long as the reaction is not inhibited.

The solvent used is not particularly limited, as long as it is a solvent that can dissolve the starting material to some extent without inhibiting the reaction, and that can be varied depending on the starting material, the reagent, and the like.

Various isomers (e.g., geometric isomers, asymmetric carbon-based optical isomers, rotamers, stereoisomers, tautomers, etc.) can be purified and separated by using a common separation means (e.g., recrystallization, diastereomer salt method, enzyme partition method, various chromatographies (e.g., thin layer chromatography, column chromatography, high performance liquid chromatography, gas chromatography, etc.)).

When the compound of the present invention is obtained in the form of an isolated form, it can be converted into a state of a salt which the compound can form or a hydrate thereof according to a conventional 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 an isolated form of the compound according to a conventional method.

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

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

General Synthesis methods

The compounds of the present invention can be synthesized using a variety of methods. Some of the synthetic methods are illustrated using the following schemes. The routes are exemplary, and the invention is not limited to the chemical reactions and conditions explicitly described. In the following schemes, some substituents are excluded for clarity of illustration, but they are not intended to be limited to the disclosed schemes. The representative compounds of the present invention can be synthesized using appropriate intermediates, known compounds and reagents. R1, R2, R3 and R4 in the formula in the following general synthetic method are the same as R1, R2, R3 and R4 in the compound represented by the above general formula (1) (the compound represented by the formula 1 in the following general synthetic method).

The compound (formula 1) of the present invention can be synthesized by the following production methods (method a and method B).

Route 1 (method A)

[ solution 13]

。

Route 1 is the following procedure: the carboxylic acid derivative (1) and the amino-amide derivative (2) are condensed to obtain an amide-amide derivative (3), and then a spiroimidazolone ring is constructed by intramolecular cyclization to obtain a hydantoin derivative (formula 1).

Step 1 is a method of reacting carboxylic acid derivative (1) with amino-amide derivative (2). Examples of the coupling reagent include N, N' -Dicyclohexylcarbodiimide (DCC), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), O- (7-azabenzotriazol-1-yl) -1, 1,3, 3-tetramethyluronium Hexafluorophosphate (HATU), and 4- (4, 6-dimethoxy-1, 3, 5-triazin-2-yl) -4-methylmorpholinium chloride N-hydrate (DMT-MM). As the base, triethylamine or N, N-diisopropylethylamine may be mentioned. If desired, 4- (dimethylamino) pyridine (DMAP) can be used as the catalyst. Examples of suitable solvents include dichloromethane and N, N-dimethylformamide. Suitable reaction solvents when DMT-MM is used include methanol, ethanol and acetonitrile. The reaction is carried out at a temperature of, for example, 0 ℃ to room temperature for 0.5 to 24 hours. The obtained amide-amide derivative (3) can be isolated by a conventional technique and, if necessary, purified by crystallization or chromatography.

Step 2 is the following method: the amide-amide derivative (3) is cyclized in an appropriate solvent such as ethanol, t-butanol or dimethyl sulfoxide in the presence of an appropriate base such as an aqueous sodium hydroxide solution or potassium t-butoxide. The reaction is carried out at room temperature to reflux for 1 to 24 hours. The hydantoin derivative (formula 1) thus obtained can be isolated by a conventional technique and, if necessary, purified by crystallization or chromatography.

The amino-amide derivative (2) shown in scheme 1 can be synthesized from the piperidone derivative (4). Scheme 2 shows a synthesis method of amino-amide derivative (2).

Route 2

[ solution 14]

。

Step 3 is Strecker synthesis of derivatizing piperidone derivative (4) to amino-nitrile derivative (5). That is, the piperidone derivative (4) is reacted with sodium cyanide or potassium cyanide, and ammonium chloride or ammonium acetate in an appropriate solvent such as methanol, ethanol, or tetrahydrofuran in the presence or absence of water. The reaction is carried out at room temperature to 80 ℃ for 2 to 72 hours. The obtained amino-nitrile derivative (5) can be isolated by a conventional technique and, if necessary, purified by crystallization or chromatography.

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

Step 5 is at H₂A method of hydrogenating the olefin of the compound (6) in an inert solvent such as methanol, ethanol, trifluoroethanol, dimethylformamide or dimethylacetamide in the presence of a catalyst such as palladium on carbon or palladium on carbon hydroxide under an atmosphere. The reaction temperature is from room temperature to 80 ℃ and may be under pressure. The obtained amino-amide derivative (2) can be isolated by a conventional technique and, if necessary, purified by crystallization or chromatography.

The piperidone derivative (4) shown in scheme 2 can be synthesized from a known ketal-vinylsulfonyl derivative (7) and hydantoin-arylbromide derivative (8). Scheme 3 shows a synthesis method of piperidone derivative (4).

Route 3

[ solution 15]

。

Step 6 is the following method: in N₂Under the atmosphere, a phosphine ligand such as tri-tert-butylphosphine tetrafluoroborate and an appropriate base such as methyldicyclohexylamine are added to N-methyl-2-piperidone (NMP) in the presence of a palladium catalyst such as tris (dibenzylideneacetone) dipalladium (0) or bis (dibenzylideneacetone) palladium) The ketal-vinylsulfonyl derivative (7) and the hydantoin-arylbromide derivative (8) are coupled in an appropriate solvent, whereby the ketal-arylvinylsulfonyl derivative (9) is synthesized. The reaction temperature is between 90 ℃ and reflux temperature. The ketal-arylvinylsulfonyl derivative (9) thus obtained can be isolated by a conventional technique and, if necessary, purified by crystallization or chromatography.

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

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

Route 4

[ solution 16]

。

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

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

Step 10 is the following method: the aryl bromide-amino acid derivative (12) is reacted with sodium cyanate under acidic conditions to synthesize a hydantoin-aryl bromide derivative (8). The solvent is, for example, a mixed solvent of acetic acid and dichloromethane, and the reaction temperature is room temperature to 60 ℃ and the reaction time is 1 to 24 hours. The hydantoin-aryl bromide derivative (8) thus obtained is isolated by a conventional technique and, if necessary, purified by crystallization or chromatography.

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

Route 5

[ solution 17]

。

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

Step 12 is the following method: the iminohydantoin derivative (17) is synthesized by reacting an arylamino-nitrile derivative (16) with 2, 2, 2-trichloroacetyl isocyanate in an appropriate solvent such as methylene chloride, adding a reagent such as methanol, water, or triethylamine, and reacting under heating. The obtained iminohydantoin derivative (17) can be isolated by a conventional technique and, if necessary, purified by crystallization or chromatography.

Step 13 is the following method: the iminohydantoin derivatives (17) are converted under acidic conditions into hydantoin-arylbromide derivatives (8). For example, the synthesis can be carried out in an acetic acid-water solvent under a heating condition of about 65 ℃ for a reaction time of about 1 to 6 hours. The hydantoin-aryl bromide derivative (8) thus obtained is isolated by a conventional technique and, if necessary, purified by crystallization or chromatography.

Route 6 is the following procedure: the vinyl sulfonamide derivative (18) is subjected to Heck reaction with the hydantoin-aryl bromide derivative (8) in the presence of a metal catalyst, and then the olefin of the compound (19) is subjected to catalytic hydrogenation to obtain a hydantoin derivative (formula 1).

Route 6 (method B)

[ solution 18]

。

The reaction of step 14 may be according to the method of step 6, and the reaction of step 15 may be according to the method of step 5, to synthesize the hydantoin derivative (formula 1). The hydantoin derivative (formula 1) thus obtained can be isolated by a conventional technique and, if necessary, purified by crystallization or chromatography.

The vinylsulfonamide derivative (18) used in step 14 can be synthesized by referring to scheme 2, scheme 3, and scheme 12 of WO2010/126030 (a 1).

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

Examples

The present invention will be described in further detail with reference to the following examples and test examples, but the present invention is not limited to these. All starting materials and reagents were obtained from commercial suppliers or synthesized using well known methods. For the¹For H-NMR spectra, Me was used₄Si was used as an internal standard substance or 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, and m ═ multiplet) without using an internal standard substance. Mass spectrometry was carried out using a mass spectrometer, ZQ2000 (manufactured by Waters), SQD (manufactured by Waters), or 2020 (manufactured by Shimazu).

Example 1

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

(reaction 1-1)

[ solution 19]

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

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

(reactions 1-2)

[ solution 20]

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

(reactions 1 to 3)

[ solution 21]

A mixture of 8- (vinylsulfonyl) -1, 4-dioxa-8-azaspiro [4.5] decane (431 mg, 1.85 mmol), 1- (4-bromo-3, 5-dimethylphenyl) -5, 5-dimethylimidazolidine-2, 4-dione (575 mg, 1.85 mmol), tris (dibenzylideneacetone) dipalladium (0) (508 mg, 0.55 mmol), tri-tert-butylphosphine tetrafluoroborate (165 mg, 0.55 mmol) and methyldicyclohexylamine (2.1 ml, 9.25 mmol) in N-methyl-2-pyrrolidone (18.5 ml) was stirred at 110 ℃ under a nitrogen stream for 2 hours. After the reaction mixture was cooled, 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 resulting residue was purified by amino silica gel column chromatography (dichloromethane-methanol) to give (E) -1- (4- (2- (1, 4-dioxa-8-azaspiro [4.5] decan-8-ylsulfonyl) vinyl) -3, 5-dimethylphenyl) -5, 5-dimethylimidazolidine-2, 4-dione (584 mg, 68%).

(reactions 1 to 4)

[ solution 22]

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

(reactions 1 to 5)

[ solution 23]

To a solution of (E) -1- (3, 5-dimethyl-4- (2- ((4-oxopiperidin-1-yl) sulfonyl) vinyl) phenyl) -5, 5-dimethylimidazolidine-2, 4-dione (994 mg, 2.37 mmol) in methanol (24 ml) at room temperature were added potassium cyanide (188 mg, 2.84 mmol) and ammonium acetate (237 mg, 3.08 mmol) in that order. Heating and stirring the mixture at 60-70 ℃ for 3 hours. After the reaction mixture was cooled, 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 resulting residue was purified by silica gel column chromatography (dichloromethane-ethyl acetate) to give (E) -4-amino-1- ((4- (5, 5-dimethyl-2, 4-dioxoimidazolidin-1-yl) -2, 6-dimethylstyryl) sulfonyl) piperidine-4-carbonitrile (681 mg, 68%).

(reactions 1 to 6)

[ solution 24]

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

(reactions 1 to 7)

[ solution 25]

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

(reactions 1 to 8)

[ solution 26]

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

(reactions 1 to 9)

[ solution 27]

To a mixture of 1- ((4- (5, 5-dimethyl-2, 4-dioxoimidazolidin-1-yl) -2, 6-dimethylbenzyl) sulfonyl) -4- (4-fluoro-3- (trifluoromethoxy) benzamide) piperidine-4-carboxamide (150 mg, 0.223 mmol) in tert-butanol (2.5 ml) and ethanol (2.5 ml) at 0 ℃, potassium tert-butoxide (75 mg, 0.670 mmol) was added. The mixture was heated and stirred at 50 ℃ for 1.5 hours under a nitrogen stream. 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 resulting residue was purified by silica gel column chromatography (dichloromethane-methanol) to give 1- (4- (2- ((2- (4-fluoro-3- (trifluoromethoxy) phenyl) -4-oxo-1, 3, 8-triazaspiro [4.5] dec-1-en-8-yl) sulfonyl) ethyl) -3, 5-dimethylphenyl) -5, 5-dimethylimidazolidine-2, 4-dione (118 mg, 81%).

The following compounds of examples were synthesized by the same procedures as in reactions 1 to 8 and reactions 1 to 9 of example 1 using appropriate carboxylic acid starting materials, reagents, and solvents.

(Compound 2-5)

。

Example 2

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

(reaction 2-1)

[ solution 28]

A mixture of 2- (3- (trifluoromethyl) phenyl) -8- (vinylsulfonyl) -1, 3, 8-triazaspiro [4.5] dec-1-en-4-one (150 mg, 0.387 mmol), 1- (4-bromo-3, 5-dimethylphenyl) -5, 5-dimethylimidazolidine-2, 4-dione (169 mg, 0.542 mmol), bis (dibenzylideneacetone) palladium (45 mg, 0.077 mmol), tri-tert-butylphosphine tetrafluoroborate (22 mg, 0.077 mmol) and methyldicyclohexylamine (0.123 ml, 0.581 mmol) in N-methyl-2-pyrrolidone (0.97 ml) synthesized according to the method described in scheme 2, scheme 3 and scheme 12 of WO2010/126030 (A1) was heated and stirred at 100 ℃ for 1 hour under a nitrogen stream. After the reaction mixture was cooled, 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 give (E) -1- (3, 5-dimethyl-4- (2- ((4-oxo-2- (3- (trifluoromethyl) phenyl) -1, 3, 8-triazaspiro [4.5] dec-1-en-8-yl) sulfonyl) vinyl) phenyl) -5, 5-dimethylimidazolidine-2, 4-dione (197 mg, 82%).

(reaction 2-2)

[ solution 29]

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

Example 3

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

(reaction 3)

[ solution 30]

1- (3, 5-dimethyl-4- (2- ((4-oxo-2- (4- (trifluoromethoxy) phenyl) -1, 3, 8-triazaspiro [4.5] dec-1-en-8-yl) sulfonyl) ethyl) phenyl) -5, 5-dimethylimidazolidine-2, 4-dione (compound 7) was synthesized by the same procedure as in example 2 using appropriate starting materials and solvents.

Example 4

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

(reaction 4-1)

[ solution 31]

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

(reaction 4-2)

[ solution 32]

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

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

(reaction 4-3)

[ solution 33]

A mixture of 1- (4-bromo-3, 5-dimethylphenyl) -4-imino-1, 3-diazaspiro [4.4] nonan-2-one (crude product obtained in the preceding reaction) in acetic acid (1.0 ml) and water (0.25 ml) was stirred with heating at 65 ℃ for 1.5 hours under a nitrogen stream. Further, acetic acid (1.0 ml) and water (0.25 ml) were added to the mixture, and the mixture was heated and stirred at 65 ℃ for 17 hours under a nitrogen stream. After cooling the reaction mixture, it was diluted with water, adjusted to pH 8 with saturated aqueous sodium bicarbonate 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 resulting residue was purified by silica gel column chromatography (ethyl acetate-hexane) to give 1- (4-bromo-3, 5-dimethylphenyl) -1, 3-diazaspiro [4.4] nonane-2, 4-dione (121 mg).

(reaction 4-4)

[ chemical 34]

By using appropriate starting materials and solvents, 1- (3, 5-dimethyl-4- (2- ((4-oxo-2- (4- (trifluoromethoxy) phenyl) -1, 3, 8-triazaspiro [4.5] dec-1-en-8-yl) sulfonyl) ethyl) phenyl) -1, 3-diazaspiro [4.4] nonane-2, 4-dione (compound 8) was obtained in the same manner as in example 2.

Example 5

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

(reaction 5-1)

[ solution 35]

Tert-butyl 1- (3, 5-dimethyl-4- (2- ((4-oxo-2- (4- (trifluoromethoxy) phenyl) -1, 3, 8-triazaspiro [4.5] dec-1-en-8-yl) sulfonyl) ethyl) phenyl) -2, 4-dioxo-1, 3, 8-triazaspiro [4.5] decane-8-carboxylate was obtained in the same manner as in example 4 using 4-oxopiperidine-1-carboxylate as a starting material and an appropriate solvent.

(reaction 5-2)

[ solution 36]

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

(reaction 5-3)

[ solution 37]

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

Example 6

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

(reaction 6)

[ solution 38]

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

Example 7

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

(reaction 7-1)

[ solution 39]

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

(reaction 7-2)

[ solution 40]

To a mixture of 1- ((4-bromo-3, 5-dimethylphenyl) amino) cyclopropanecarboxylic acid (198 mg, 0.697 mmol) in acetic acid (3 ml) and dichloromethane (1.5 ml) was added potassium cyanate (424 mg, 5.23 mmol) at room temperature. After the mixture was stirred at room temperature for 1 hour, it was stirred at 60 ℃ for 2 hours under heating. After the reaction mixture was adjusted to pH 8 with a saturated aqueous sodium bicarbonate 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 resulting residue was purified by silica gel column chromatography (ethyl acetate-hexane) to give 4- (4-bromo-3, 5-dimethylphenyl) -4, 6-diazaspiro [2.4] heptane-5, 7-dione (49 mg, 23%).

(reaction 7-3)

[ solution 41]

The same procedures as in example 2 were repeated except for using appropriate starting materials and solvents to give 4- (3, 5-dimethyl-4- (2- ((4-oxo-2- (4- (trifluoromethoxy) phenyl) -1, 3, 8-triazaspiro [4.5] dec-1-en-8-yl) sulfonyl) ethyl) phenyl) -4, 6-diazaspiro [2.4] heptane-5, 7-dione (compound 11).

Example 8

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

(reaction 8)

[ solution 42]

By using appropriate starting materials and solvents, 1- (3, 5-dimethyl-4- (2- ((4-oxo-2- (3- (trifluoromethyl) phenyl) -1, 3, 8-triazaspiro [4.5] dec-1-en-8-yl) sulfonyl) ethyl) phenyl) -1, 3-diazaspiro [4.4] nonane-2, 4-dione was obtained in the same manner as in example 2.

Test examples

The results of the cAMP production ability test using human PTH1R, the cAMP production ability test using rat receptor, the metabolic stability test using human liver microparticles, the metabolic stability test using rat hepatocytes, the serum Ca concentration increase effect test using a TPTX rat model, and the like, with respect to the compound of the present invention are shown in test examples 1 to 5. As a comparative compound, a compound described in WO2010/126030a1 shown in table 2 was used.

Test example 1: determination of in vitro cAMP Signaling Activity of Compounds in human PTH1R

(peptide)

Human PTH (1-34) and calcitonin were purchased from the peptide research institute (Osaka, Japan) and dissolved in 10mM acetic acid to 1mM and stored in a refrigerator at-80 ℃.

(cell culture)

Cells were cultured at 37 ℃ in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (Hyclone), 100 units/ml penicillin G, and 100. mu.g/ml streptomycin sulfate (Invitrogen Corp) under a humidified atmosphere containing 5% CO 2.

LLC-PK 1 cells which do not express PTH1R and LLC-PK 1 cells which each express 9.5 × 10 are subjected to overexpression⁵HKRK-B7 of personal PTH1R was used in cAMP signaling assay (Takasu et al, J. bone. Miner. Res. 14:11-20, 1999).

(cAMP stimulation)

At 1X10⁵Cells/well HKRK-B7 or LLC-PK 1 cells were seeded in 96-well plates and incubated overnight. The next day, 50. mu.l of cAMP assay buffer (DMEM, 2mM IBMX, 0.2mg/ml bovine serum albumin, 35mM Hepes-NaOH, pH 7.4) containing human PTH (1-34) or compound was then added, placed in an incubator at 37 ℃ and incubated for 20 minutes. After removal of the medium, the cells were washed 1 time with 100. mu.l of cAMP assay buffer. To freeze the cells, the plates were placed on dry ice powder and then removed from the dry ice. Cells were melted using 40. mu.l of 50mM HCl and frozen again on dry ice. Using commercially available cAMP EIA kit (Biotrack cAMP EIA system, GE health care) was used to measure the amount of cAMP produced in cells.

(calculation of 20% effective concentration (EC 20) and 50% effective concentration (EC 50) in the determination of cAMP-inducing ability in vitro.)

The analysis was performed using a variable-gradient-based sigmoidal dose response equation, in which the cAMP signaling activity of human PTH (1-34) at 100nM was taken as 100%, and the concentration at which each compound exhibited 20% or 50% of the cAMP signaling activity was calculated as EC20 or EC 50.

The results in HKRK-B7 cells are shown in Table 3.

Note that the extent of cAMP response in LLC-PK 1 cells was lower than in HKRK-B7 cells.

Test example 2: determination of in vitro cAMP Signaling Activity of Compounds in rat PTH1R

Instead of HKRK-B7 cells, LLC-PK 46-RATO-PTH 1R cells overexpressing rat PTH1R, which were established in the field of pharmacy, were used for the measurement in the same manner as in test example 1.

The results obtained using LLC-PK 46-RATO-PTH 1R cells are shown in Table 4.

There is a good correlation between EC20 values for in vitro cAMP signaling activity in rat PTH1 receptor and EC20 values for in vitro cAMP signaling activity in human PTH 1R. With regard to EC50 values, there is a good correlation between rats and humans.

Test example 3: metabolic stability test Using human liver microparticles

Human liver microsomes and compounds or comparative examples were incubated in 0.1M phosphate buffer (pH 7.4) in the presence of NADPH at 37 ℃ for the prescribed time. The concentration of the parent compound was measured at each reaction time by LC/MS/MS, and the intrinsic clearance (. mu.L/min/mg protein) was calculated from the slope of the residual rate with respect to the reaction time.

< analysis Condition >

Compound concentration: 1 μ M

Microsome: 0.5mg/mL

NADPH：1mM

Reaction time: 0.5, 15 and 30 minutes.

The results are shown in Table 5. The compounds 1 to 11 showed high metabolic stability to human liver microparticles compared to comparative examples 1 to 6.

Test example 4: metabolic stability test Using rat hepatocytes

Hepatocytes were prepared from rat (SD, male) liver by collagenase reflux method. The example compound or the comparative example was added, and the mixture was incubated at 37 ℃ for a predetermined period of time, followed by addition of a reaction-terminated solution. The concentration of the parent compound at each reaction time was measured by LC/MS/MS, and the intrinsic clearance (. mu.L/10) was calculated from the slope of the residual rate with respect to the reaction time⁶cells/min）。

< analysis Condition >

Cell concentration 1 × 10⁶cells/mL

Compound concentration: 1 μ M

Culture medium: williams' medium E

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

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

The results are shown in Table 6. The compounds of compounds 2,4, 5, 7, 8, 9, 10, and 11 showed improved metabolic stability of rat hepatocytes as compared with those of comparative examples 1, 2, 3, 5, and 6.

Test example 5: serum Ca concentration elevation in TPTX rat model

Female Crl at 4 weeks of age was obtained from japan チャールス, seeded リバー florists (thick wood breeding center): CD (SD) rats are acclimatized for 1 week at 20-26 ℃ under standard laboratory conditions with a humidity of 35-75%. Rats were allowed to freely ingest tap water and standard rodent feed (CE-2) (Japanese クレア Co., Ltd.) containing 1.1% calcium, 1.0% phosphoric acid and 250IU/100g of vitamin D3.

TPTX was administered to 5-week-old rats. A Sham operation (Sham) was performed on a portion of the individuals. For TPTX rats used for this, individuals with serum Ca concentrations of less than 8mg/dL 4 days after surgery were selected. After 5 days of surgery, the groups were divided into 8 TPTX groups and 1 Sham group, 5 per group, based on serum Ca concentration and body weight measured 4 days after surgery. Only solvent was administered to the Sham group and TPTX-Vehicle group at a dose of 10 mL/kg. To TPTX-each analyte group, each analyte was dissolved in a solvent in an amount of 30 mg/10mL/kg and administered orally. For the composition of the solvent, the following solvents were used: a solvent having a pH of 10 was prepared from 10% dimethyl sulfoxide (Wako pure chemical industries, Ltd.), 10% Cremophor EL (シグマアルドリッチジャパン, Kyoho), 20% hydroxypropyl-. beta. -cyclodextrin (Nippon food chemical Co., Ltd.), and glycine (Wako pure chemical industries, Ltd.). For each group, blood was collected beforehand (Pre blood collection) immediately before administration, and blood was collected 2, 6, 10, and 24 hours after administration to measure the serum Ca concentration. Each group of blood collections was taken from the jugular vein under isoflurane inhalation anesthesia.

Determination of Ca in serum. Serum obtained from collected blood was measured by centrifugation using an automatic analyzer TBA-120 FR (manufactured by Easter Chinensis メディカルシステムズ Co., Ltd.).

Statistical analysis was performed on animal trials. Data are expressed as mean ± Standard Error (SE). Statistical significance was determined using the SAS preclinical package (ver.5.00.010720, SAS Institute Japan, Tokyo, Japan). A p-value of less than 0.05 was considered statistically significant. T-test by 2 groups showed: (# P < 0.05 for the TPTX-Vehicle group, # P < 0.05 for the group of comparative example 1, and ^ P < 0.05 for the group of comparative example 2.

For Pre values of serum Ca concentrations, 9.9mg/dL for the Sham group and 5.3-6.2 mg/dL for the TPTX groups. The average value of the change amount with respect to Pre is shown in fig. 1 with respect to the serum Ca concentration up to 24 hours after administration of each compound. In addition, the peak of serum Ca concentration after administration of each compound was 6 hours or 10 hours after administration in all compounds.

Compound 6, compound 7 and compound 8, which have high metabolic stability of rat hepatocytes, exhibited large positive changes from the pre value, and a strong serum Ca concentration-increasing effect was observed when they were orally administered. On the other hand, the positive change amounts to the Pre value of compound 1, comparative example 1 and comparative example 2, which have low metabolic stability of rat hepatocytes, are smaller than those of compound 6, compound 7 and compound 8. In particular, compound 7 and compound 8 were statistically significant compared to comparative examples 1 and 2.

In addition, compound 6, compound 7 and compound 8, which have high metabolic stability of rat hepatocytes, showed values of 7.8 to 8.5mg/dL as maximum values of the respective compounds at 6 hours or 10 hours after administration, and reached a therapeutic target range of 7.6 to 8.8mg/dL for serum Ca concentration in patients with hypoparathyroidism. On the other hand, in all the measurement times, the compound 1 with low metabolic stability of rat hepatocytes, comparative example 1 and comparative example 2 did not reach the range of the therapeutic target.

From the above test results, it was confirmed that when compound 6, compound 7 and compound 8 (which exhibit strong cAMP signaling activity in cells forcibly expressing rat PTH1R and high stability with respect to metabolism in rat hepatocytes) were orally administered to rats, a strong serum Ca concentration-increasing effect was confirmed. These compounds have cAMP signaling activity in cells that forcibly express human PTH1R, have higher metabolic stability of human liver microparticles than the comparative compounds, and are expected to have a good therapeutic effect on patients with hypoparathyroidism when administered orally. Furthermore, the compound represented by the general formula (1), which exhibits cAMP signaling activity in cells that forcibly express human PTH1R and exhibits metabolic stability of human liver microparticles to the same extent as in compound 6, compound 7 and compound 8, is expected to have a good therapeutic effect on patients with hypoparathyroidism.

INDUSTRIAL APPLICABILITY

By the present invention, a compound having a strong PTH-like action and high metabolic stability can be provided. The present invention also provides a drug for the prevention and/or treatment of osteoporosis, bone fracture, nondynamic bone disease, cartilage growth insufficiency, oligochondrogenesis, osteomalacia, osteoarthritis, arthritis, thrombocytopenia, hypoparathyroidism, hyperphosphatemia, tumorous calcinosis, and the like, or for the mobilization of stem cells.

Claims (9)

1. A compound or a pharmacologically acceptable salt thereof, wherein the compound is selected from the group consisting of:

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

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.

2. A compound which is 1- (3, 5-dimethyl-4- (2- ((4-oxo-2- (4- (trifluoromethoxy) phenyl) -1, 3, 8-triazaspiro [4.5] dec-1-en-8-yl) sulfonyl) ethyl) phenyl) -5, 5-dimethylimidazolidine-2, 4-dione, or a pharmacologically acceptable salt thereof.

3. A pharmaceutical composition comprising the compound according to any one of claims 1 to 2 or a pharmacologically acceptable salt thereof as an active ingredient.

4. The pharmaceutical composition of claim 3, wherein the pharmaceutical composition is for oral administration.

5. The pharmaceutical composition according to claim 3, wherein the pharmaceutical composition is a tablet, powder, fine granule, capsule, syrup, buccal, inhalant, suppository, injection, ointment, eye drop, nasal drop, ear drop, cataplasm or lotion.

6. The pharmaceutical composition of claim 3, wherein the pharmaceutical composition is a coated tablet or an ocular ointment.

7. A pharmaceutical composition for activating intracellular cAMP response, comprising the compound according to any one of claims 1 to 2 or a pharmacologically acceptable salt thereof as an active ingredient.

8. An agent for preventing or treating osteoporosis, bone fracture, nondynamic bone disease, cartilage growth insufficiency, oligochondrogenesis, osteomalacia, osteoarthritis, arthritis, thrombocytopenia, hypoparathyroidism, hyperphosphatemia, or tumorous calcinosis, or an agent for mobilizing stem cells, which comprises the compound according to any one of claims 1 to 2 or a pharmacologically acceptable salt thereof as an active ingredient.

9. Use of a compound according to any one of claims 1 to 2 or a pharmacologically acceptable salt thereof for the production of a prophylactic or therapeutic agent for treating osteoporosis, bone fracture, nondynamic bone disease, cartilage insufficiency, oligochondrogenesis, osteomalacia, osteoarthritis, arthritis, thrombocytopenia, hypoparathyroidism, hyperphosphatemia, or tumorous calcinosis; or in the manufacture of a stem cell mobilizer.