RU2772958C2 - Nitrogen-containing 6-element cyclic compounds - Google Patents

Abstract

FIELD: pharmaceutics.

SUBSTANCE: invention relates to a compound represented by the general formula (1), where R¹ is -H or halogen; Ar¹ is any substituent selected from a group G¹, which can be substituted with the same or different 1-3 substituents selected from a group consisting of -F and methyl (provided that structures represented by the formula 2 are excluded), in which the group G¹ is a group consisting of structures of the formula 3 (a and b are the direction of attachment); Ar² is any substituent selected from a group G², which can be substituted with the same or different 1-3 substituents selected from a group consisting of cyano, -Cl, methyl, methoxy and phenyl (provided that structures of the formula 4 are excluded), in which the group G² is a group consisting of phenyl, thienyl, furyl and thiazolyl; and * is asymmetric carbon, or its pharmaceutically acceptable salt. The invention also relates to a pharmaceutical composition having excellent agonistic activity relatively to EP₄ receptor, containing the effective amount of the compound according to the invention or its pharmaceutically acceptable salt, as an active ingredient, and a pharmaceutically acceptable carrier. The invention also relates to a pharmaceutical composition for the therapeutic treatment and/or stimulation of fracture healing, for stimulation of bone fusion during spinal surgery, containing the effective amount of the compound according to the invention or its pharmaceutically acceptable salt, as an active ingredient, and a pharmaceutically acceptable carrier.

EFFECT: compounds having excellent agonistic activity relatively to EP₄ receptor.

21 cl, 10 tbl, 39 ex

Description

Technical field

[0001]

The present invention relates to new nitrogen-containing 6-membered cyclic compounds and drugs, using them as an active ingredient.

State of the art

[0002]

A bone fracture is a condition in which a bone is partially or completely broken or deformed by an external force resulting from an accident or a fall. Bone fractures are divided into complete fracture (fracture) and incomplete fracture (fracture), simple fracture (there is one line of fracture) and comminuted fracture (complex fracture of the bone), closed fracture (the broken part does not leave the body) and open fracture (the broken part comes out from the body) or the like. Bone fractures pose serious problems for the daily life of patients, and their healing takes a significant long period of time, although this depends on the broken part and the presence or absence of displacement (dislocation) of the bone. The condition of bone fusion without correction of the dislocation is referred to as an "improperly fused fracture". As conditions for bone fracture, although it depends on the site and type of bone fracture, conditions such as "delayed union" may occur, in which union is not achieved even 3-9 months after injury due to various factors such as aging, diabetes and smoking, and "non-union", in which healing is not achieved even 9 months after the injury, and arrest of the fracture healing process is suspected. In cases such as malunion, delayed healing, and nonunion, pain or discomfort occurs, normal functional healing of the fractured parts is not achieved, and consequently, the quality of life of patients with a fracture is markedly reduced.

[0003]

Especially serious problems in the treatment of fractures are fractures accompanying osteoporosis. Fractures accompanying osteoporosis often occur in the appendicular and vertebral metaphyses, specifically, hip fracture, vertebral compression fracture, distal radius fracture, and proximal humerus fracture are considered as the four major fractures seen in osteoporosis. Fractures accompanying osteoporosis have the problem that their repair is difficult due to bone fragility, and even if osteosynthesis proceeds, sufficient stability can hardly be obtained, and improper fixation causes malunion, delayed healing, and also nonunion. In addition, since daily activity is limited during the period of fracture treatment, a negative spiral occurs, which prevents the reduction of bone mass and muscle atrophy progresses, and they again cause a fall and a fracture. In particular, the delay in the normal healing of a vertebral body fracture or femoral neck fracture forces patients to be bedridden. The incidence of various and critical complications such as muscle weakness, joint contracture, pressure ulcer, dementia, urinary tract infection, and cardiopulmonary hypofunction accompanying systemic inactivity in these patients is extremely high, and a significant reduction in survival after injury has been reported (non-patent document 1).

[0004]

As described above, fractures, especially fractures accompanying osteoporosis, cause a deterioration in the quality of life, severe complications, as well as a significant impact on the prognosis of vital functions, and therefore they create extremely serious social problems, such as an increase in the cost of medical care and care costs. Fracture treatment is currently performed by restoring the bone to its anatomically normal position and performing fixation to achieve healing to a functional level before injury, to the extent possible, using the normal bone repair mechanism to prevent complications such as malunion, delayed healing, and nonunion.

[0005]

As treatments positively promoting fracture healing, ultrasound devices for fracture treatment are used, and as therapeutic agents, bone morphogenetic protein (BMP) preparations, parathyroid hormone preparations, fibroblast growth factor (FGF) preparations, and the like are used or attempted to be clinically used. However, despite the use or attempts to use this variety of drugs as mentioned above, the number of patients with bone diseases such as fracture is increasing every year, for example, the number of patients with fracture of the femoral neck was estimated to be 1,700,000 worldwide. in 1990 and is projected to increase to 6,300,000 in 2050. In this regard, it is desirable to develop new innovative drugs that have a preventive and/or therapeutic effect in bone diseases such as fractures.

[0006]

Prostaglandin E2 (hereinafter abbreviated as PGE ₂ ) is known to perform various physiological functions such as analgesic and oxytocytic effects, and is also well known to play an important role in bone metabolism. When PGE ₂ is added to the bone marrow cell culture system, the activity of alkaline phosphatase, which is a marker of calcified bone node formation and osteoblast differentiation, is increased. It has also been found that when PGE ₂ is actually administered to laboratory animals such as rats or humans, osteogenesis is increased and bone mass is increased. In addition, when PGE ₂ is topically administered to bone as a sustained release preparation, osteogenesis is enhanced at the injection site, and therefore, a systemic or local effect of positive osteogenesis promotion can be expected for PGE ₂ .

[0007]

However, since PGE ₂ exhibits side effects such as pain-inducing action and oxytocytic action as described above, which should be avoided in long-term administration, there is a need for a selective PGE ₂ derivative that is safe and effective for bone tissue. For example, as PGE ₂ receptors, for mouse, rat, dog, human, and the like, four species, EP ₁ , EP ₂ , EP ₃ and EP ₄ receptors, have been described so far, and since their expression sites and intracellular signal transduction systems for activation are different, compounds selective for each subtype have been created.

[0008]

Among the four types of receptors affected by PGE ₂ , the EP ₂ and EP ₄ receptors are thought to play an important role in bone metabolism in cells and animals, and both are coupled to the Gs protein to increase cAMP in osteoblasts. To date, EP ₂ selective agonists, EP ₄ selective agonists and EP ₂ /EP ₄ agonists have been developed, and a significant effect on osteogenesis or fracture healing has been demonstrated in animal models by systemic or local administration. As compounds that act on PGE receptors, for example, the compounds described in Patent Documents 1-8 are known.

Related Links

Patent Documents

[0009]

Patent Document 1: International Patent Publication WO02/24647

Patent Document 2: International Patent Publication WO02/42268

Patent Document 3: International Patent Publication WO03/007941

Patent Document 4: International Patent Publication WO03/035064

Patent Document 5: International Patent Publication WO2004/063158

Patent Document 6: International Patent Publication WO2004/085430

Patent Document 7: US Patent No. 6747037

Patent Document 8: International Patent Publication WO2006/080323

Non-Patent Documents

[0010]

Non-Patent Document 1: C. Cooper et al., Am. J. Epidemiol., 137, 1001-1005, 1993

The essence of the present invention

Purpose Achieved by the Present Invention

[0011]

The object achieved by the present invention is to provide a novel compound having excellent EP ₄ receptor agonist activity. Preferably, it is an object of the present invention to provide a novel compound having excellent selective EP ₄ receptor agonist activity. Another object of the present invention is to provide a novel compound useful as an active ingredient in a medicament for the prophylactic and/or therapeutic treatment of a disease associated with EP ₄ receptor agonism, for example a novel compound useful as an active ingredient in a medicament for the therapeutic treatment of a fracture or stimulation of fracture healing. Yet another object of the present invention is to provide a medicament containing this compound.

Ways to achieve the goal

[0012]

In order to achieve the above objects, the inventors of the present invention have made various studies. As a result, they found that the compounds of the present invention represented by the following formula (1) have excellent EP ₄ agonist activity, in particular, the compounds according to one embodiment of the present invention have excellent EP ₄ receptor selective agonist activity, and these compounds are suitable for the prophylactic and/or therapeutic treatment of a disease associated with EP ₄ receptor agonism, for example, therapeutic treatment and/or promotion of fracture healing, and the present invention has been completed. It is considered preferable to provide a compound having selective EP ₄ receptor agonist activity for the following reasons. Namely, while the EP ₄ receptor is observed in osteoblasts and osteoclasts in human cultured osteoblasts and bone tissue, no expression of the EP ₂ receptor has been found (P. Sarrazin, G et al. Prostaglandins Leukot. Essent. Fatty Acids, 64, 203-210, 2001; I. Fortier et al., Prostaglandins Leukot. Essent. Fatty Acids, 70, 431-439, 2004) and, therefore, consider that the most important target of PGE ₂ for action in bone tissues is the EP ₄ receptor, and a selective agonist The EP ₄ receptor may be a safe and effective drug for bone regeneration. Of course, compounds having EP ₂ agonistic activity are not excluded from the compounds of the present invention.

[0013]

Thus, the present invention relates to the following.

[1] The compound represented by the following general formula (1):

[Formula 1]

[where in formula (1),

R ¹ represents -H or halogen;

Ar ¹ is any substituent selected from the group G ¹ which may be substituted by 1-3 identical or different substituents selected from the group consisting of -F and methyl (provided that

[formula 2]

excluded),

in which the group G ¹ is a group consisting of

[formula 3]

(a and b represent the direction of attachment);

Ar ² is any substituent selected from the group G ² which may be substituted by 1-3 identical or different substituents selected from the group consisting of cyano, -Cl, methyl, methoxy and phenyl (provided that

[formula 4]

excluded),

in which the G ² group is a group consisting of phenyl, thienyl, furyl and thiazolyl; and

* represents an asymmetric carbon],

or its salt.

[0014]

[2] The compound or salt thereof according to [1] above, wherein R ¹ is -H, -Cl, or -Br.

[3] The compound or salt thereof according to [2] above, wherein Ar ¹ is any substituent selected from the group consisting of

[formula 5]

.

.

[0015]

[3-2] The compound or salt thereof according to [1] or [2] above, wherein Ar ¹ is any substituent selected from the group consisting of

[formula 6]

.

.

[0016]

[3-3] The compound or salt thereof according to [1] or [2] above, wherein Ar ¹ is any substituent selected from the group consisting of

[formula 7]

.

.

[0017]

[4] The compound or salt thereof according to [2] above, wherein Ar ¹ is

[formula 8]

.

.

[0018]

[4-2] The compound or salt thereof according to [1] or [2] above, wherein Ar ¹ is

[formula 9]

.

.

[0019]

[4-3] The compound or salt thereof according to [1] or [2] above, wherein Ar ¹ is

[formula 10]

.

.

[4-4] The compound or salt thereof according to [1] or [2] above, wherein Ar ¹ is

[formula 11]

.

.

[0020]

[5] The compound or salt thereof according to [3] or [4] above, wherein Ar ² is any substituent selected from the group consisting of

[formula 12]

.

.

[0021]

[5-2] The compound or salt thereof according to any one of [1]-[4-4] above, wherein Ar ² is any substituent selected from the group consisting of

[formula 13]

.

.

[0022]

When the numbers of cited clauses are indicated by a range such as [1]-[4-4] above, and this range includes the clause indicated by a number having an additional number, such as [3-2], this means that the clause is also cited , indicated by a number having a sub-number such as [3-2]. The same applies to the following definitions.

[0023]

[6] The compound or salt thereof according to [3] or [4] above, wherein Ar ² is any substituent selected from the group consisting of

[formula 14]

.

.

[0024]

[6-2] The compound or salt thereof according to any one of [1]-[4-4] above, wherein Ar ² is any substituent selected from the group consisting of

[formula 15]

.

.

[0025]

[7] The compound or salt thereof according to [3] or [4] above, wherein Ar ² is any substituent selected from the group consisting of

[formula 16]

.

.

[0026]

[7-2] The compound or salt thereof according to any one of [1]-[4-4] above, wherein Ar ² is any substituent selected from the group consisting of

[formula 17]

.

.

[0027]

[7-3] The compound or salt thereof according to any one of [1]-[4-4] above, wherein Ar ² is any substituent selected from the group consisting of

[formula 18]

.

.

[0028]

[7-4] The compound or salt thereof according to any one of [1]-[4-4] above, wherein Ar ² is

[formula 19]

.

.

[7-5] The compound or salt thereof according to any one of [1]-[4-4] above, wherein Ar ² is

[formula 20]

.

.

[0029]

[7-6] The compound or salt thereof according to any one of [1]-[4-4] above, wherein Ar ² is

[formula 21]

.

.

[7-7] The compound or salt thereof according to any one of [1]-[4-4] above, wherein Ar ² is [Formula 22]

.

.

[0030]

[8] The compound or salt thereof according to [7] above, wherein R ¹ is -H.

[8-2] The compound or salt thereof according to any one of [1]-[7-7] above, wherein R ¹ is -H.

[0031]

[9] The compound or salt thereof according to [7] above, wherein R ¹ is -Cl.

[9-2] The compound or salt thereof according to any one of [1]-[7-7] above, wherein R ¹ is -Cl.

[0032]

[10] The compound or salt thereof according to [7] above, wherein R ¹ is -Br.

[10-2] The compound or salt thereof according to any one of [1]-[7-7] above, wherein R ¹ is -Br.

[0033]

[11] The compound or salt thereof according to [1] above, wherein R ¹ is -H, -Cl, or -Br;

Ar ¹ is any substituent selected from the group consisting of

[formula 23]

; и

; and

Ar ² is any substituent selected from the group consisting of

[formula 24]

.

.

[0034]

[11-2] The compound or salt thereof according to [1] above, wherein R ¹ is -H, -Cl or -Br;

Ar ¹ is any substituent selected from the group consisting of

[formula 25]

; и

; and

Ar ² is any substituent selected from the group consisting of

[formula 26]

.

.

[0035]

[11-3] The compound or salt thereof according to [1] above, wherein R ¹ is -H, -Cl or -Br;

Ar ¹ is any substituent selected from the group consisting of

[formula 27]

; и

; and

Ar ² is any substituent selected from the group consisting of

[formula 28]

.

.

[0036]

[11-4] The compound or salt thereof according to [1] above, wherein R ¹ is -H, -Cl or -Br;

Ar ¹ is any substituent selected from the group consisting of

[formula 29]

; и

; and

Ar ² is any substituent selected from the group consisting of

[formula 30]

.

.

[0037]

[12] Any compound selected from the following group, or a salt thereof;

[formula 31]

.

.

[0038]

[13] The compound below, or a salt thereof;

[formula 32]

.

.

[0039]

[14] The compound below, or a salt thereof;

[formula 33]

.

.

[0040]

[15] The compound below, or a salt thereof;

[formula 34]

.

.

[0041]

[16] The compound below, or a salt thereof;

[formula 35]

.

.

[0042]

[17] The compound below, or a salt thereof;

[formula 36]

.

.

[0043]

[18] The compound below, or a salt thereof;

[formula 37]

.

.

[0044]

[19] The compound below, or a salt thereof;

[formula 38]

.

.

[0045]

[20] A drug containing a compound according to any of [1] to [19] above, or a pharmaceutically acceptable salt thereof, as an active ingredient.

[21] The drug according to [20] above, which is intended for the prophylactic and/or therapeutic treatment of a disease associated with EP ₄ receptor agonism.

[0046]

[22] The drug according to [20] above, which is intended to stimulate osteogenesis.

[23] The drug according to [20] above, which is intended for therapeutic treatment and/or promotion of fracture healing.

[24] The drug according to [20] above, which is intended for therapeutic treatment and/or promotion of healing of a bone defect.

[0047]

[25] The drug according to [20] above, which is intended to stimulate bone fusion.

[25-2] The drug according to [25] above, which is designed to stimulate bone fusion during spinal fixation.

[26] An EP ₄ agonist containing a compound according to any of [1]-[19] above, or a pharmaceutically acceptable salt thereof, as an active ingredient.

[27] A pharmaceutical composition for the therapeutic treatment of a fracture, which contains a compound according to any of [1]-[19] above, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[28] Preparation of microspheres containing a compound according to any of [1]-[19] above, or a pharmaceutically acceptable salt thereof, and a lactic/glycolic acid copolymer.

[0048]

[29] The compound according to any one of [1]-[19] above, or a pharmaceutically acceptable salt thereof, which is used for the therapeutic treatment of a fracture.

[30] A method for therapeutically treating a fracture in a mammal, which comprises the step of administering an effective amount of a compound of any of [1] to [19] above, or a pharmaceutically acceptable salt thereof, to the mammal.

Effect of the present invention

[0049]

"Compounds represented by formula (1) and salts thereof" (hereinafter also simply referred to as "compounds of the present invention") have excellent EP ₄ receptor agonist activity. The compounds of the present invention can be used as an active ingredient in a medicament for the prophylactic and/or therapeutic treatment of a disease associated with EP ₄ receptor agonism, for example, therapeutic treatment and/or promotion of fracture healing. As another embodiment, the compounds of the present invention can be used as a reagent having EP ₄ receptor agonist activity.

Embodiments of the present invention

[0050]

In the present invention, hereinafter, the present invention will be explained specifically.

In the present invention, a carbon atom can be simply represented as “C”, a hydrogen atom as H”, an oxygen atom as “O”, a sulfur atom as “S”, and a nitrogen atom as N”. In addition, the carbonyl group can be simply represented as “-C(O)-”, the carboxyl group as “-COO-”, the sulfinyl group as “-S(O)-”, the sulfonyl group as “-S(O) ₂ - ”, an ether bond as “-O-”, and a thioether bond as “-S-” (each “-” in these groups denotes a bond).

[0051]

In the present invention, alkyl having 1-4 carbon atoms means methyl, ethyl, propyl, butyl or its isomer [normal (n), iso, secondary (sec), tertiary (tert) and the like].

[0052]

In the present invention, acyl containing 2-6 carbon atoms means acetyl, propanoyl, butanoyl, pentanoyl, hexanoyl, or an isomer thereof.

[0053]

In the present invention, alkoxy containing 1-4 carbon atoms means methoxy, ethoxy, propoxy, butoxy, or an isomer thereof.

In the present invention, halo refers to fluorine (-F), chlorine (-Cl), bromine (-Br), or iodine (-I).

[0054]

In the present invention, all isomers are included unless otherwise indicated. For example, alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylene, alkenylene, and alkynylene include straight and branched groups. In addition, any of the isomers based on the double bond, ring or condensed ring (E- or Z-isomers, or cis- or trans-isomers), isomers based on the presence of asymmetric carbon and the like (R- or S-isomer, isomer, based on the α- or β-configuration, enantiomers, diastereomers and the like), optically active substances exhibiting optical rotation (D- or L-isomers or d- or l-isomers), isomers, based on polarity in chromatographic separation ( isomers of high polarity or isomers of low polarity), balanced compounds, rotational isomers, mixtures of these isomers in arbitrary proportions and racemates are included in the scope of the present invention.

[0055]

In the present invention, as is clear to a person skilled in the art, the symbol is:

[formula 39]

indicates that the bond is behind the plane (i.e., α-configuration), symbol:

[formula 40]

indicates that the bond is in front of the plane (i.e., β-configuration), symbol:

[formula 41]

denotes the α-configuration or β-configuration, or a mixture thereof, and the symbol:

[formula 42]

denotes a mixture of α-configuration and β-configuration, unless otherwise indicated.

[0056]

In the present invention, hereinafter, the compounds represented by formula (1) or salts thereof will be described in detail.

R ¹ is, for example, -H or halogen. According to another embodiment, R ¹ is, for example, -H, -Cl or -Br. According to yet another embodiment, R ¹ is, for example, -H.

[0057]

Ar ¹ is, for example, any substituent selected from the group G ¹ which may be substituted by 1-3 identical or different substituents selected from the group consisting of -F and methyl (provided that

[formula 43]

excluded), in which the group G ¹ is a group consisting of

[formula 44]

(a and b represent the direction of attachment).

[0058]

According to another embodiment of this group consisting of -F and methyl above, the group consists of, for example, -F, and according to yet another embodiment, the group consists of, for example, methyl.

[0059]

According to another embodiment of the group G ¹ , the group consists of, for example,

[formula 45]

.

.

[0060]

In another embodiment, Ar ¹ , Ar ¹ is, for example, any substituent selected from the group consisting of

[formula 46]

.

.

[0061]

According to yet another embodiment of Ar ¹ , Ar ¹ is, for example, any substituent selected from the group consisting of

[formula 47]

.

.

[0062]

According to yet another embodiment of Ar ¹ , Ar ¹ is, for example,

[formula 48]

.

.

[0063]

According to yet another embodiment of Ar ¹ , Ar ¹ is, for example,

[formula 49]

.

.

[0064]

According to yet another embodiment of Ar ¹ , Ar ¹ is, for example,

[formula 50]

.

.

[0065]

When Ar ¹ is substituted with 1-3 identical or different substituents selected from the group consisting of -F and methyl, in another embodiment, Ar ¹ is substituted with, for example, 1 or 2 identical or different substituents selected from the group consisting of -F and methyl, and according to yet another embodiment, Ar ¹ is substituted, for example, with one of -F or methyl. Unsubstituted Ar ¹ is also one of the preferred embodiments.

[0066]

Ar ² is, for example, any substituent selected from the group G ² which may be substituted by 1-3 identical or different substituents selected from the group consisting of cyano, -Cl, methyl, methoxy and phenyl (provided that

[formula 51]

excluded).

[0067]

The G ² group above is a group consisting of phenyl, thienyl, furyl and thiazolyl.

According to another embodiment of this group consisting of cyano, -Cl, methyl, methoxy and phenyl, the group consists of, for example, cyano.

According to another embodiment of the G ² group, G ² is, for example, a group consisting of thienyl and furyl.

[0068]

According to yet another embodiment of the G ² group, the group consists of, for example, thienyl.

In another embodiment, Ar ² , Ar ² is, for example, any substituent selected from the group consisting of

[formula 52]

.

.

[0069]

According to yet another embodiment of Ar ² , Ar ² is, for example, any substituent selected from the group consisting of

[formula 53]

.

.

[0070]

According to yet another embodiment of Ar ² , Ar ² is, for example, any substituent selected from the group consisting of

[formula 54]

.

.

[0071]

According to yet another embodiment of Ar ² , Ar ² is, for example, any substituent selected from the group consisting of

[formula 55]

.

.

[0072]

According to yet another embodiment of Ar ² , Ar ² is, for example,

[formula 56]

.

.

[0073]

According to yet another embodiment of Ar ² , Ar ² is, for example,

[formula 57]

.

.

[0074]

According to yet another embodiment of Ar ² , Ar ² is, for example,

[formula 58]

.

.

[0075]

According to yet another embodiment of Ar ² , Ar ² is, for example,

[formula 59]

.

.

[0076]

When Ar ² is substituted with 1-3 identical or different substituents selected from the group consisting of cyano, -Cl, methyl, methoxy and phenyl, according to another embodiment, Ar ² is substituted, for example, with 1 or 2 identical or different substituents selected from a group consisting of cyano, -Cl, methyl, methoxy and phenyl, and according to yet another embodiment, Ar ² is substituted, for example, with 1 cyano, -Cl, methyl, methoxy or phenyl. Unsubstituted Ar ² is also one of the preferred embodiments.

[0077]

As specific compounds falling within the scope of the present invention, the following compounds can be exemplified.

[formula 60]

[0078]

As yet other examples of compounds falling within the scope of the present invention, the following compounds can be exemplified.

[formula 61]

[0079]

In this specification, "compounds represented by formula (1)" generally means compounds represented by formula (1) in free form. Examples of their salts include the following salts.

[0080]

The type of the salt of the compounds represented by the formula (1) is not particularly limited, and it may be an acid addition salt or a base addition salt, and may be an intramolecular counterion. In particular, when the salt is used as an active ingredient of a drug, the salt is preferably a pharmaceutically acceptable salt. When the description is given for use as a medicine in this specification, the salt of the compounds represented by formula (1) is usually understood as a pharmaceutically acceptable salt. Acid addition salts include, for example, inorganic acid addition salts such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid and phosphoric acid and organic acid addition salts such as formic acid, acetic acid, propionic acid, oxalic acid , malonic acid, succinic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, citric acid, malic acid, tartaric acid, dibenzoyl tartaric acid, mandelic acid, maleic acid, fumaric acid, aspartic acid and glutamic acid. As base addition salts, for example, addition salts of an inorganic base such as sodium, potassium, magnesium, calcium and aluminum, addition salts of organic bases such as methylamine, 2-aminoethanol, arginine, lysine and ornithine and the like can be mentioned. However, the type of salt is not limited to these, and, of course, it can be appropriately selected by those skilled in the art.

[0081]

The compounds of the present invention may be in the form of a hydrate. Compounds of the present invention may be in the anhydride form

The compounds of the present invention may be in the form of a solvate. The compounds of the present invention may also be in a form other than the solvate.

[0082]

The compounds of the present invention may be in the form of a crystal. The compounds of the present invention may also be in amorphous form.

More specifically, the compounds of the present invention include anhydrides and forms other than solvates, "compounds represented by formula (1)", their hydrates and/or solvates, and their crystals.

[0083]

The compounds of the present invention also include anhydrides and forms other than solvates, "salts of the compounds represented by formula (1)", hydrates and/or solvates of salts, and crystals thereof.

[0084]

The compounds of the present invention may also be a pharmaceutically acceptable prodrug of the "compounds represented by formula (1)". A pharmaceutically acceptable prodrug is a compound containing a group that can be exchanged for an amino group, a hydroxyl group, a carboxyl group, or the like by solvolysis or under physiological conditions. For example, as a group that forms a prodrug for a hydroxy group or an amino group, for example, an acyl group and an alkoxycarbonyl group can be exemplified. As a group that forms a prodrug for a carboxyl group, for example, a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a sec-butyl group, a t-butyl group can be exemplified. , an amino group, a methylamino group, an ethylamino group, a dimethylamino group, and a diethylamino group.

[0085]

This prodrug can be obtained, for example, by suitably introducing a group that forms a prodrug in any of the compounds of the present invention at one or more arbitrary groups selected from a hydroxyl group and an amino group, using a prodrug-forming reagent such as the corresponding halide, in a conventional manner, then, if necessary, by suitable isolation and purification of the compound in a conventional manner. A group that forms a prodrug can also be suitably introduced into the compound of the present invention at the carboxyl group using a prodrug-forming reagent such as an appropriate alcohol or amine in a conventional manner.

[0086]

The compounds of the present invention may contain an asymmetric carbon. The spatial configuration of this asymmetric carbon is not particularly limited, and it may be an S-configuration or an R-configuration, or a mixture of both. Any optically active substances, starting from a given asymmetric carbon, in pure form, in the form of stereoisomers such as diastereoisomers, arbitrary mixtures of stereoisomers, racemates and the like, fall within the scope of the compounds of the present invention.

[0087]

In particular, the spatial configuration of the asymmetric carbon indicated by "*" in the formula (1) is not specifically limited. However, the configuration shown below is one of the preferred embodiments.

[formula 62]

When Ar ¹ is a benzene ring among G ¹ groups, the steric configuration in the above formula is the S configuration, and when Ar ¹ is a thiophene ring among the G ¹ groups, the steric configuration in the above formula is the R configuration.

[0088]

<Methods for Producing Compounds of the Present Invention>

The compounds of the present invention are novel compounds not described in the literature. Although the compounds of the present invention can be obtained, for example, by the following methods, the method for producing the compounds of the present invention is not limited to the following methods.

[0089]

Although the reaction time in each of the reactions is not specifically limited, the course of the reactions can be easily controlled by the analysis methods described below, and therefore, the reactions can be stopped when the maximum yield of the target substance is reached. Each of the reactions can be carried out under an inert gas atmosphere, such as under a stream of nitrogen or under a stream of argon, if desired. When protection with a protecting group and subsequent deprotection is necessary in each of the reactions, the reactions can be appropriately carried out using the methods described below.

[0090]

Examples of the protecting group used in the present invention include the following groups: protecting groups for carboxyl group (-COOH), protecting groups for hydroxyl group (-OH), protecting groups for alkynyl group, protecting groups for amino group (-NH ₂ ) and similar.

[0091]

Examples of a protecting group for a carboxyl group include, for example, alkyl containing 1-4 carbon atoms, alkenyl containing 2-4 carbon atoms, alkyl containing 1-4 carbon atoms and substituted alkoxy containing 1-4 carbon atoms, alkyl containing 1-4 carbon atoms and substituted with 1-3 halogens, and the like. Specific examples include methyl, ethyl, t-butyl, allyl, methoxyethyl, trichloroethyl, and the like.

[0092]

Examples of protecting groups for a hydroxyl group include, for example, alkyl containing 1-4 carbon atoms, alkenyl containing 2-4 carbon atoms, alkyl containing 1-4 carbon atoms and substituted alkoxy containing 1-4 carbon atoms, alkyl containing 1-4 carbon atoms and substituted with 1-3 halogens, silyl substituted with three identical or different alkyls containing 1-4 carbon atoms or phenyls, tetrahydropyranyl, tetrahydrofuryl, propargyl, trimethylsilylethyl group and the like. Specific examples include methyl, ethyl, t-butyl, allyl, methoxymethyl (MOM), methoxyethyl (MEM), trichloroethyl, phenyl, methylphenyl, chlorophenyl, benzyl, methylbenzyl, chlorobenzyl, dichlorobenzyl, fluorobenzyl, trifluoromethylbenzyl, nitrobenzyl, methoxyphenyl, N-methylaminobenzyl , N, N-dimethylaminobenzyl, phenacyl, trityl, 1-ethoxyethyl (EE), tetrahydropyranyl (THP), tetrahydrofuryl, propargyl, trimethylsilyl (TMS), triethylsilyl (TES), tert-butyldimethylsilyl (TBDMS), tert-butyldiphenylsilyl (TBDPS) , acetyl (Ac), pivaloyl, benzoyl, allyloxycarbonyl (Alloc), 2,2,2-trichloroethoxycarbonyl (Troc), and the like.

[0093]

Examples of alkynyl protecting groups include trimethylsilyl, 2-hydroxy-2-propyl, and the like.

Examples of amino protecting groups include, for example, benzyl, methylbenzyl, chlorobenzyl, dichlorobenzyl, fluorobenzyl, trifluoromethylbenzyl, nitrobenzyl, methoxyphenyl, N-methylaminobenzyl, N,N-dimethylaminobenzyl, phenacyl, acetyl, trifluoroacetyl, pivaloyl, benzoyl, allyloxycarbonyl, 2 ,2,2-trichloroethoxycarbonyl, benzyloxycarbonyl, tert-butoxycarbonyl (Boc), 1-methyl-1-(4-biphenyl)ethoxycarbonyl (Bpoc), 9-fluorenylmethoxycarbonyl, benzyloxymethyl (BOM), 2-(trimethylsilyl)ethoxymethyl (SEM) and the like.

[0094]

By removing these protecting groups simultaneously with the preparation or step by step during the preparation or at the final stage, the protected compounds can be converted to the target compounds. The protection and deblocking reactions can be carried out according to known methods, such as those described, for example, in Protective groups in Organic Synthesis published by John Wiley and Sons (2007) and the like, and can be carried out, for example, by methods (1)-(6 ) below and the like.

[0095]

(1) The alkaline hydrolysis deblocking reaction is carried out, for example, by reacting the protected compound with a base in a polar solvent. Examples of the base used in this reaction include, for example, alkali metal bases such as sodium hydroxide, potassium hydroxide, lithium hydroxide, barium hydroxide, calcium hydroxide, sodium carbonate, potassium carbonate, sodium methoxide, and potassium t-butoxide, and organic bases. such as triethylamine. For example, they can generally be used in an amount of 1-20 mole fold, preferably 1-10 mole fold, relative to the reactant when an alkali metal base is used, or from 1 mole fold to a greater excess when an organic base is used. With regard to the reaction solvent, it is generally preferred to carry out the reaction in an inactive medium that does not inhibit the reaction, preferably a polar solvent. Examples of the polar solvent include water, methanol, ethanol, tetrahydrofuran, dioxane and the like, and they can be used as a mixture if necessary. With regard to the reaction temperature, a suitable temperature is selected, for example -10° C. to the boiling point of the solvent. The reaction time is, for example, typically 0.5-72 hours, preferably 1-48 hours when an alkali metal base is used, or typically 5 hours-14 days when an organic base is used. However, the progress of the reaction can be followed by thin layer chromatography (TLC), high performance liquid chromatography (HPLC) or the like, and accordingly, the reaction can usually be terminated when the maximum yield of the target compound is obtained.

[0096]

(2) The deblocking reaction under acidic conditions is carried out, for example, in an organic solvent (dichloromethane, chloroform, dioxane, ethyl acetate, anisole and the like) in the presence of an organic acid (acetic acid, trifluoroacetic acid, methanesulfonic acid, p-toluenesulfonic acid and the like), Lewis acid (boron tribromide, boron trifluoride, aluminum bromide, aluminum chloride and the like) or inorganic acid (hydrochloric acid, sulfuric acid and the like) or mixtures thereof (hydrobromic acid/acetic acid and the like) at a temperature of -10-100°C. There is also a method of adding ethanethiol, 1,2-ethanethiol or the like as an additive.

[0097]

(3) The hydrogenolysis deblocking reaction is carried out, for example, in a solvent [ether type solvents (tetrahydrofuran, dioxane, dimethoxyethane, diethyl ether and the like), alcohol type solvents (methanol, ethanol and the like), benzene type solvents (benzene, toluene and the like) , ketone type solvents (acetone, methyl ethyl ketone and the like), nitrile type solvents (acetonitrile and the like), amide type solvents (dimethylformamide and the like), ester type solvents (ethyl acetate and the like), water, acetic acid, a mixture of two or more types of data solvents and the like] in the presence of a catalyst (palladium/charcoal powder, platinum oxide (PtO ₂ ), activated nickel and the like) and a source of hydrogen such as standard pressure or pressurized hydrogen gas, ammonium formate, or hydrazine hydrate at a temperature of -10- 60°C.

[0098]

(4) The silyl group deblocking reaction is carried out, for example, using tetra-n-butylammonium fluoride or the like in a water-miscible organic solvent (tetrahydrofuran, acetonitrile and the like) at -10 to 60°C.

[0099]

(5) The deblocking reaction using a metal is carried out, for example, in an acidic solvent (acetic acid, buffer pH 4.2-7.2, mixtures of this solution and an organic solvent such as tetrahydrofuran) in the presence of zinc powder with or without sonication at -10-60°C.

[0100]

(6) The deblocking reaction using a metal complex is carried out, for example, in an organic solvent (dichloromethane, dimethylformamide, tetrahydrofuran, ethyl acetate, acetonitrile, dioxane, ethanol, and the like), water, or a mixture thereof, in the presence of a trapping agent (tributyltin hydride, triethylsilane, dimedone, morpholine, diethylamine, pyrrolidine and the like), an organic acid (acetic acid, formic acid, 2-ethylhexanoic acid and the like) and/or an organic acid salt (sodium 2-ethylhexanoate, potassium 2-ethylhexanoate and the like) in the presence of or the absence of a phosphine-type reagent (triphenylphosphine and the like), using a metal complex [tetrakistriphenylphosphine palladium (0), bis(triphenylphosphine)palladium(II) dichloride, palladium(II) acetate, tris(triphenylphosphine)rhodium(I) chloride and the like] at temperature -10-60°C.

[0101]

The compounds of the present invention represented by formula (1) can be obtained, for example, according to the following reaction methods. In the following diagrams, "stage" refers to each stage, for example, "stage 1-1" refers to stage 1-1.

Stage 1-1

[formula 63]

The compounds represented by formula (1) can be obtained by deprotecting the compound represented by formula (2) [in formula (2), "Pro ¹ " is a carboxyl protecting group in formula (1)] to the protecting group Pro ¹ . The deblocking reaction can be carried out according to known methods, such as those described in Protective groups in Organic Synthesis published by John Wiley and Sons (2007) and the like.

[0102]

Pro ¹ is not particularly limited as long as it is a carboxyl protecting group above, and examples thereof include, for example, alkyl having 1 to 4 carbon atoms.

[formula 64]

[0103]

Stage 1-2

The compounds represented by formula (2) can be obtained by deblocking the compound represented by formula (3) [in formula (3), "Pro ² " is the protecting group of the hydroxyl group in formula (1), and "Pro ¹ " has the same meaning as as defined above] for the protecting group of the compound represented by formula (3). The deblocking reaction can be carried out according to known methods, such as those described in Protective groups in Organic Synthesis published by John Wiley and Sons (2007) and the like.

[0104]

Although Pro ² is not particularly limited as long as it is the above hydroxyl protecting group, Pro ² is preferably a group other than TMS in order to selectively deblock Pro ² with respect to TMS in formula (5). Examples of Pro ² include, for example, a tert-butyl group, a MOM group, a MEM group, a THP group, an acetyl group, and a TBDMS group.

[0105]

Stage 1-3

The compounds represented by formula (3) can be obtained by condensing the compound represented by formula (4) [in formula (4), "Pro ¹ " and "Pro ² " have the same meanings as defined above] and the compound represented by formula ( 11) [in formula (11), "hal ¹ " is bromine or iodine] in the presence of a base and a palladium catalyst. As for the amount of the compound represented by formula (11) used in the reaction of the compound represented by formula (4) and the compound represented by formula (11), 1/5 to 20 equivalents, preferably 1/2 to 10 equivalents, more preferably 1- 5 equivalents of the compound represented by formula (11) can be used relative to the compound represented by formula (4). However, the amount of the compound represented by formula (11) to be used can be appropriately determined in consideration of the purity, yield, purification efficiency, and the like of the compound represented by formula (4).

[0106]

As the base, for example, cesium carbonate, sodium carbonate, potassium carbonate and the like can be used, and cesium carbonate is preferred. As for the amount of base to be used, it can be used in an amount of an equivalent to an excess amount, for example, 1 to 10 equivalents, preferably 1 to 5 equivalents, relative to the compound represented by formula (4) which serves as the starting compound.

[0107]

As the palladium catalyst, for example, commercially available catalysts such as tetrakis(triphenylphosphine)palladium, tetrakis(methyldiphenylphosphine)palladium, dichlorobis(triphenylphosphine)palladium, dichlorobis(tri-o-tolylphosphine)palladium, dichlorobis(tricyclohexylphosphine)palladium, dichlorobis( triethylphosphine)palladium, palladium acetate, palladium chloride, bis(acetonitrile)palladium chloride, bis(dibenzylideneacetone)palladium, tris(dibenzylideneacetone)dipalladium and bis(diphenylphosphinoferrocene)palladium chloride can be added to the reaction system as is or as a catalyst separately prepared and isolated from palladium acetate, tris(dibenzylideneacetone) dipalladium or the like, and an arbitrary ligand. The catalyst that is considered to actually participate in the reaction can be obtained in the reaction system by mixing palladium acetate, tris(dibenzylideneacetone)dipalladium or the like and an arbitrary ligand. The valency of palladium can be 0 or +2. In particular, bis(acetonitrile)palladium chloride may be mentioned as a preferred example.

[0108]

As a ligand used to produce a palladium catalyst from an arbitrary ligand, phosphine ligands such as triphenylphosphine, tri(o-tolyl)phosphine, tri(cyclohexyl)phosphine, tri(tert-butyl)phosphine, dicyclohexylphenylphosphine, 1 ,1'-bis(di-tert-butylphosphino)ferrocene, 2-dicyclohexylphosphino-2'-dimethylamino-1,1'-biphenyl, 2-(di-tert-butylphosphino)biphenyl, 2-(dicyclohexylphosphino)biphenyl, 2, 2'-bis(diphenylphosphino)-1,1'-binaphthyl, xantphos, and tri(tert-butyl)phosphine. Also exemplified are 2-dicyclohexylphosphino-2',6'-dimethoxybiphenyl, 2-dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl, 1,2,3,4,5-pentamethyl-1'-(di -tert-butylphosphino)ferrocene) and the like, and 2-dicyclohexyl-2',4',6'-triisopropylbiphenyl may be preferably mentioned.

[0109]

Although the equivalent amount of the palladium catalyst used may be an equivalent amount or a catalytic amount, it is 0.01 mol% or more, more preferably, in particular 0.10 to 50.0 mol% based on the amount of the starting compound. Examples of the solvent used for the reaction include, for example, N,N-dimethylformamide, N,N-dimethylacetamide, acetonitrile, xylene, toluene, 1,4-dioxane and tetrahydrofuran, and preferred examples include acetonitrile. Two or more types of these solvents can also be used as a mixture. With regard to the reaction temperature, the reaction can be carried out usually at -40-100°C, preferably at -20°C-60°C. Although the reaction time is not particularly limited, it is, for example, usually 0.5-48 hours, preferably 1-24 hours.

[0110]

Stage 1-4

The compounds represented by formula (4) can be obtained by selectively deblocking the TMD of the compound represented by formula (5) [in formula (5), "Pro ¹ " and "Pro ² " have the same meanings as defined above]. The deblocking reaction can be carried out according to known methods, such as those described in Protective groups in Organic Synthesis published by John Wiley and Sons (2007) and the like.

[0111]

Specifically, the compounds can be obtained, for example, by exposing the compound represented by formula (5) to an inorganic base in an organic solvent. As the inorganic base, for example, sodium hydroxide, potassium hydroxide, cesium carbonate, sodium carbonate, potassium carbonate or the like can be used, and potassium carbonate is preferred. As for the amount of base to be used, the base can be used in an amount of an equivalent amount to an excess amount with respect to the compound represented by formula (5) which serves as the starting compound, and the amount is, for example, 1-10 equivalents, preferably 1- 5 equivalents. Examples of the solvent used for the reaction include methanol and ethanol, and preferred examples include methanol. With regard to the reaction temperature, the reaction can be carried out usually at -20-60°C, preferably at 0-40°C. Although the reaction time is not particularly limited, it is, for example, usually 0.5-48 hours, preferably 1-24 hours.

[0112]

Stage 1-5

The compounds represented by formula (5) can be obtained by condensing the compound represented by formula (6) [in formula (6), "Pro ¹ " and "Pro ² " have the same meanings as defined above, and "hal ² " is bromine or iodine] with the compound represented by formula (13) in an organic solvent in the presence of an inorganic base. Connections can be obtained in the same way as the connection of stage 1-3. In this preparation, the compound represented by formula (13) can be used in the amount of 1/5 to 20 equivalents, preferably 1/2 to 10 equivalents, more preferably 1 to 5 equivalents, relative to the compound represented by formula (6).

[0113]

Stage 1-6

The compounds represented by formula (6) can be obtained by protecting the hydroxyl group of the compound represented by formula (7) [in formula (7), "Pro ¹ " and "hal ² " have the same meanings as defined above]. The protection reaction for the hydroxyl group can be carried out according to known methods, for example, the methods described in Protective groups in Organic Synthesis published by John Wiley and Sons (2007) and the like. Although the hydroxyl protecting group is not particularly limited, as long as the above hydroxyl protecting group is selected, for example, tert-butyl group, MOM group, MEM group, THP group, acetyl group, TBDMS group and the like can be used.

[0114]

Stage 1-7

The compounds represented by formula (7) can be obtained by exposing the compound represented by formula (9) to a reducing agent [in formula (9), "Pro ¹ " and "hal ² " have the same meanings as defined above] in an organic solvent. As the reducing agent, for example, sodium borohydride, lithium borohydride, triacetoxyborohydride, cyanoborohydride and the like can be used, and sodium borohydride is preferred. As for the amount of the reducing agent to be used, it can be used in an amount of 1/4 equivalent to an excess amount with respect to the compound represented by the formula (9) which serves as the starting compound, and the amount is, for example, 1/4-10 equivalents, preferably 1-5 equivalents. Examples of the organic solvent used for the reaction include methanol, ethanol, isopropanol, and a mixed solvent thereof with tetrahydrofuran, and preferred examples include methanol. With regard to the reaction temperature, the reaction can be carried out usually at -20-60°C, preferably at 0-40°C. Although the reaction time is not particularly limited, it is, for example, usually 0.5-48 hours, preferably 1-24 hours.

[0115]

Stage 1-8

The compounds represented by formula (9) can be obtained by exposing the compound represented by formula (14) [in formula (14), "hal ² " has the same meaning as defined above] to the compound represented by formula (10) [in formula (10 ), "Pro ¹ " has the same meaning as defined above]. As for the amount of the compound represented by formula (14) to be used, it can be used in an amount of an equivalent amount to an excess amount with respect to the compound represented by formula (10) that serves as a starting compound, and the amount is, for example, an equivalent amount -10 equivalents, preferably 1-5 equivalents. Examples of the solvent used for the reaction include methanol, ethanol, isopropanol, and their mixed solvent with water, and preferred examples include ethanol. With regard to the reaction temperature, the reaction can be carried out usually at 0-120°C, preferably at 40-100°C. Although the reaction time is not particularly limited, it is, for example, usually 0.5-48 hours, preferably 1-24 hours.

[0116]

Stage 1-9

[formula 65]

The compounds represented by formula (3) can also be obtained from the compound represented by formula (6) [in formula (6), "Pro ¹ ", "Pro ² " and "hal ² " have the same meanings as defined above], and the compound represented by the general formula (12) in the same manner as the compound of the method of Step 1-3. As for the amount of the compound represented by formula (12) used here, it can be used in the amount of 1/5 to 20 equivalents, preferably 1/2 to 10 equivalents, more preferably 1 to 5 equivalents, relative to the compound represented by formula (6 ).

[0117]

Stage 1-10

[formula 66]

The compounds represented by formula (2) can also be obtained from the compound represented by formula (8) [in formula (8), "Pro ¹ " has the same meaning as defined above] in the same manner as the method of steps 1-7.

[0118]

Stage 1-11

[formula 67]

The compounds represented by formula (8) are obtained by condensation of the compound represented by formula (9) in which "hal ² " is an iodine atom [in formula (9), "Pro ¹ " has the same meaning as defined above] and the compounds represented by formula (12) in the presence of a base, a copper catalyst, and a palladium catalyst. As for the amount of the compound represented by formula (12) used in the reaction of the compound represented by formula (9) and the compound represented by formula (12), it can be used in the amount of 1/5 to 20 equivalents, preferably 1/2 to 10 equivalents , more preferably 1 to 5 equivalents, relative to the compound represented by formula (9), but it can be appropriately determined in view of the purity, yield, purification efficiency and the like of the compound represented by formula (8).

[0119]

As for the base, for example, triethylamine, diethylamine, diisopropylamine, diisopropylethylamine, morpholine, piperidine, pyridine and the like can be used, and diester amines are preferred. As for the amount of base to be used, it can be used in an amount of an equivalent amount to an excess amount, such as 1 to 10 equivalents, preferably 1 to 5 equivalents, with respect to the compound represented by formula (9) which serves as the starting compound.

[0120]

Examples of the copper catalyst include, for example, copper(I) iodide, copper(I) bromide, copper(I) chloride and the like, and copper(I) iodide is preferred.

Although the equivalent amount of the copper catalyst to be used may be an equivalent amount or a catalytic amount, it is preferably 0.01 mol% or more, particularly preferably 0.10-50.0 mol%, relative to the starting compound.

[0121]

As the palladium catalyst, for example, commercially available catalysts such as tetrakis(triphenylphosphine)palladium, tetrakis(methyldiphenylphosphine)palladium, dichlorobis(triphenylphosphine)palladium, dichlorobis(tri-o-tolylphosphine)palladium, dichlorobis(tricyclohexylphosphine)palladium, dichlorobis( triethylphosphine)palladium, palladium acetate, palladium chloride, bis(acetonitrile)palladium chloride, bis(dibenzylideneacetone)palladium, tris(dibenzylideneacetone)dipalladium and bis(diphenylphosphinoferrocene)palladium chloride can be added to the reaction system as is or as a catalyst separately prepared and isolated from palladium acetate, tris(dibenzylideneacetone)dipalladium or the like, and an arbitrary ligand. The catalyst that is considered to actually participate in the reaction can be obtained in the reaction system by mixing palladium acetate, tris(dibenzylideneacetone)dipalladium or the like, and an arbitrary ligand. The valency of palladium can be 0 or +2. In particular, tetrakis(triphenylphosphine)palladium may be mentioned as a preferred example. When the palladium catalyst is prepared from an arbitrary ligand, the same ligands as the ligands exemplified for Step 1-3 can be used.

[0122]

Although the equivalent amount of the palladium catalyst to be used may be an equivalent amount or a catalytic amount, it is preferably 0.01 mol% or more, more preferably, especially 0.10-50.0 mol% relative to the starting compound.

[0123]

Examples of the solvent used for the reaction include, for example, N,N-dimethylformamide, N,N-dimethylacetamide, acetonitrile, xylene, toluene, 1,4-dioxane, tetrahydrofuran and the like, or the reaction can be carried out without a solvent. The reaction carried out without solvent is a preferred example. As for the reaction temperature, the reaction can be carried out usually at -40-100°C, preferably at -20-60°C. Although the reaction time is not particularly limited, it is, for example, usually 0.5-48 hours, preferably 1-24 hours.

[0124]

Stage 1-12

[formula 68]

The compounds represented by formula (8) can also be obtained from the compound represented by formula (10) [in formula (10), "Pro ¹ " has the same meaning as defined above] and the compound represented by formula (15) by the same in a manner such as the method of steps 1-8.

[formula 69]

[0125]

Stage 2-1

Compounds represented by formula (10) can be obtained by treating a compound represented by formula (A1) with a base [in formula (A1), "Pro ¹ " has the same meaning as defined above] in an organic solvent. As the base, for example, sodium hydroxide, potassium hydroxide, cesium carbonate, sodium carbonate, sodium hydrogen carbonate, potassium carbonate and the like can be used, and sodium hydrogen carbonate is preferred. The base can be used in an amount of an equivalent amount to an excess amount, for example, 1 to 20 equivalents, preferably 1 to 10 equivalents, relative to the compound represented by formula (10) which serves as the starting compound. Sodium iodide can be used as an additive, and can be used in an amount of an equivalent amount to an excess amount, for example, 1-10 equivalents, preferably 1-5 equivalents, relative to the compound represented by formula (10) which serves as the starting compound. Examples of the organic solvent used for the reaction include N,N-dimethylformamide, N,N-dimethylacetamide, acetonitrile, toluene, tetrahydrofuran, 1,4-dioxane, diethyl ether and their mixed solvent, and preferred examples include acetonitrile. With regard to the reaction temperature, the reaction can be carried out usually at 0-100°C, preferably at 20-60°C. Although the reaction time is not particularly limited, it is, for example, usually 0.5-48 hours, preferably 1-24 hours.

[0126]

Stage 2-2

The compounds represented by formula (A1) can be obtained by deblocking the compound represented by formula (A2) [in formula (A2), "Pro ¹ " has the same meaning as defined above] of the protecting group. The deblocking reaction can be carried out according to known methods, such as those described in Protective groups in Organic Synthesis published by John Wiley and Sons (2007) and the like.

[0127]

Stage 2-3

Compounds represented by formula (A2) can be obtained by reacting a chlorothioformic acid ester such as 2-chloroethyl chlorothioformate with a compound represented by formula (A3) [in formula (A3), "Pro ¹ " has the same meaning as defined above] in the presence of grounds. The chlorothioformic acid ester can be used in an amount of an equivalent amount to an excess amount, for example, 1-5 equivalents, preferably 1-2 equivalents, relative to the compound represented by the formula (A2) which serves as the starting compound. As the base to be used, for example, sodium carbonate, potassium carbonate, sodium hydrogen carbonate, cesium carbonate, sodium hydroxide, diisopropylethylamine, triethylamine and the like can be used, and sodium hydrogen carbonate is preferred. Examples of the solvent used for the reaction include dichloromethane, toluene, tetrahydrofuran, 1,4-dioxane, acetonitrile and the like, and dichloromethane is preferred. As for the reaction temperature, the reaction can be carried out at 0-100°C, preferably at 10-30°C. Although the reaction time is not particularly limited, it is, for example, usually 1-24 hours, preferably 2-4 hours.

[0128]

Stage 2-4

Compounds represented by formula (A3) can be obtained by treating a compound represented by formula (A4) with tert-butoxycarbonylhydrazine [in formula (A4), "Pro ¹ " has the same meaning as defined above] in the presence of a base. As the base to be used, for example, sodium carbonate, potassium carbonate, sodium hydrogen carbonate, cesium carbonate, sodium hydroxide, diisopropylethylamine, triethylamine and the like can be used, and sodium hydrogen carbonate is preferred. The amount of base to be used is, for example, 1 to 20 equivalents, preferably 3 to 5 equivalents, relative to the compound represented by formula (A4) which serves as the starting compound. Sodium iodide or the like can be used as an additive. Examples of the solvent used for the reaction include acetonitrile, propionitrile, N,N-dimethylformamide, dimethylsulfoxide, N-methylpyrrolidone and the like, and preferred examples include acetonitrile. With regard to the reaction temperature, the reaction can be carried out usually at room temperature-150°C, preferably at 70°C-100°C. Although the reaction time is not particularly limited, it is, for example, 3-36 hours, preferably 3-18 hours.

[0129]

Stage 2-5

Compounds represented by formula (A4) can be obtained by substituting a bromine atom for the hydroxyl group of a compound represented by formula (A5) [in formula (A5), "Pro ¹ " has the same meaning as defined above]. The bromine atom substitution reaction can be carried out by exposing the compound to carbon tetrabromide, N-bromosuccinimide or the like in the presence of triphenylphosphine or the like. The amount of triphenylphosphine to be used is, for example, 1 to 5 equivalents, preferably 1 to 2 equivalents, relative to the compound represented by formula (A5), which is the parent compound. The amount of carbon tetrabromide or the like to be used is, for example, 1 to 5 equivalents, preferably 1 to 2 equivalents, relative to the compound represented by formula (A5), which is the starting compound. Examples of the solvent used for the reaction include dichloromethane, toluene, tetrahydrofuran, 1,4-dioxane, acetonitrile and the like, and dichloromethane is preferred. With regard to the reaction temperature, the reaction can be carried out usually at -20-40°C, preferably at -10-10°C. Although the reaction time is not particularly limited, it is, for example, usually 3-36 hours, preferably 12-20 hours.

[0130]

Stage 2-6

The compounds represented by formula (A5) can be obtained by deprotecting the hydroxyl group of the compound represented by formula (6A). Deblocking can be performed according to known methods, such as those described in Protective groups in Organic Synthesis published by John Wiley and Sons (2007) and the like.

[0131]

Stage 2-6

The compounds represented by the formula (A5) can be obtained by converting the compound represented by the formula (A6) which is a carboxylic acid into an ester and deblocking by removing the protecting group of the hydroxyl group. The reaction can be carried out in an alcoholic solvent in the presence of an acid. Examples of the acid used for the reaction include sulfuric acid, hydrochloric acid, methanesulfonic acid, p-toluenesulfonic acid, trifluoroacetic acid and the like, and preferred examples include sulfuric acid. As the solvent, methanol, ethanol or the like can be used, and preferred examples include methanol. As for the reaction temperature, the reaction can be carried out usually at room temperature -140°C, preferably at 50-80°C. Although the reaction time is not particularly limited, it is, for example, usually 2-24 hours, preferably 8-16 hours.

[0132]

Stage 2-7

The compounds represented by the formula (A6) can be obtained by exposing the compound represented by the formula (A7) to a strong base and then carbon dioxide. As a strong base, lithium amide such as lithium diisopropylamide or lithium hexamethyldisilazide can be used. When R ¹ is hydrogen, lower alkyl lithium such as n-butyl lithium, sec-butyl lithium and n-propyl lithium can also be used, and lithium diisopropylamide is preferably used. The amount of strong base used is, for example, 1-3 equivalents, preferably 1-2 equivalents, relative to the compound represented by formula (A7), which is the starting compound. Examples of the solvent used for the reaction include tetrahydrofuran, diethyl ether, 1,4-dioxane and the like, and tetrahydrofuran is preferred. With regard to the reaction temperature with a strong base, the reaction can be carried out generally at -100--20°C, preferably at -80--60°C. The subsequent reaction with carbon dioxide or the like can generally be carried out at -40-40°C and preferably -20-10°C. Although the reaction time with a strong base is not particularly limited, it is, for example, 0.2 to 3 hours, preferably 0.5 to 1 hour. Although the reaction time with carbon dioxide or the like is not particularly limited, it is, for example, usually 0.5 to 24 hours, preferably 0.75 to 2 hours.

[0133]

Stage 2-8

The compounds represented by formula (A7) can be obtained by protecting the hydroxyl group of the compound represented by formula (A8) by TBDMS. Protection of the hydroxyl group can be carried out using a method similar to the method of steps 1-6.

[0134]

The compound of formula (A8) in which R ¹ is H is a commercially available compound (2-(thiophen-2-yl)ethanol, Tokyo Chemicals). Therefore, when R ¹ in formula (A8) is H, the following steps are not required.

[0135]

Stage 2-9

The compounds represented by formula (A8) can be obtained by reducing the ester group of the compound represented by formula (A9) [in formula (A9), "Pro ³ " is the carboxyl protecting group of the compound of formula (A8)]. That is, as Pro ³ can be used, for example, alkyl containing 1-4 carbon atoms.

[0136]

As the reducing agent, for example, lithium aluminum hydride, diisobutylaluminum hydride, lithium triethylborane hydride and the like can be used, and lithium aluminum hydride is preferred. The amount of the reducing agent to be used is, for example, 0.5 to 5 mol equivalents, preferably 1 to 2 mol equivalents, relative to the compound represented by formula (A9) which serves as the starting compound.

[0137]

Examples of the solvent used for the reaction include tetrahydrofuran, diethyl ether, toluene, and a mixed solvent thereof, and preferred examples include tetrahydrofuran and diethyl ether. With regard to the reaction temperature, the reaction can be carried out usually at -10-20°C, preferably at -5-5°C. Although the reaction time is not particularly limited, it is, for example, usually 0.08-0.5 hours, preferably 0.15-0.3 hours.

[0138]

Stage 2-10

The compounds represented by formula (A9) can be obtained, for example, by carrying out solvolysis of the compound represented by formula (A10) to an alcohol in the presence of an acid. As the acid, sulfuric acid, methanesulfonic acid, hydrochloric acid and the like can be used, and sulfuric acid is preferred. The amount of sulfuric acid to be used is, for example, 0.0001 to 0.005 mol equivalent, preferably 0.0002 to 0.001 mol equivalent, relative to the compound represented by the formula (A10) which serves as the starting compound. As the alcohol used as the solvent, for example, ethanol, methanol, n-propanol, n-butyl alcohol, isobutyl alcohol and the like can be used. Although the reaction time is not particularly limited, it is, for example, usually 6-48 hours, preferably 16-24 hours.

[0139]

Stage 2-11

Compounds represented by the formula (A10) can be obtained by exposing the compound represented by the formula (A11) to cyanide. As the cyanide, for example, sodium cyanide, potassium cyanide and the like can be used. The amount of cyanine to be used is, for example, 1 to 5 equivalents, preferably 1 to 2 equivalents, relative to the compound represented by formula (A10), which is the parent compound. As the reaction solvent, tetrahydrofuran, acetonitrile, dimethylsulfoxide, N,N-dimethylacetamide, N,N-dimethylformamide and the like can be used, and preferred examples include a mixed solvent of acetonitrile and dimethylsulfoxide. As for the reaction temperature, the reaction can be carried out usually at 0-60°C, preferably 10-40°C. Although the reaction time is not particularly limited, it is, for example, usually 0.5-20 hours, preferably 2-6 hours.

[0140]

Stage 2-12

The compounds represented by the formula (A11) can be obtained by converting the hydroxyl group of the compound represented by the formula (A12) into a bromine atom. The conversion to a bromine atom can be carried out in the same manner as the method of steps 2-5.

[0141]

Stage 2-13

Compounds represented by formula (A12) can be obtained by reducing the carboxyl group of a commercially available compound represented by formula (A13) to a hydroxyl group. As the reducing agent, for example, borane-dimethyl sulfide, borane-tetrahydrofuran and the like can be used, and borane-tetrahydrofuran is preferred. The amount of the reducing agent to be used is, for example, 1 to 5 mol equivalents, preferably 1 to 2 mol equivalents, relative to the compound represented by the formula (A13) which serves as the starting compound.

[0142]

As the solvent used for the reaction, tetrahydrofuran, diethyl ether and the like can be used, and preferred examples include tetrahydrofuran. With regard to the reaction temperature, the reaction can be carried out usually at 0-60°C, preferably at 10-40°C. Although the reaction time is not particularly limited, it is, for example, usually 4-24 hours, preferably 10-18 hours.

[0143]

Examples of a commercially available compound represented by formula (A13) include, for example, 3-chlorothiophene-2-carboxylic acid, 3-bromothiophene-2-carboxylic acid, and the like, and can be purchased from, for example, Sigma-Aldrich.

[0144]

Stage 3-1

[formula 70]

The compounds represented by the formula (15) can be obtained by exposing the compound represented by the formula (W1) to a vinylating agent in an organic solvent. As the vinylating agent, for example, vinyl magnesium bromide, vinyl magnesium chloride, vinyl lithium and the like can be used, and vinyl magnesium bromide and vinyl magnesium chloride are preferred. Vinylmagnesium bromide and vinylmagnesium chloride can be used as a solution in tetrahydrofuran, diethyl ether or toluene, and a tetrahydrofuran solution is preferred. As for the amount of the vinylating agent to be used, the agent can be used in an amount of an equivalent amount to an excess amount, such as 1 to 10 equivalents, preferably 1 to 5 equivalents, relative to the compound represented by the formula (W1) which serves as the starting compound. . Examples of the solvent used for the reaction include toluene, tetrahydrofuran, 1,4-dioxane, diethyl ether, 1,2-dimethoxyethane, and a mixed solvent thereof, and preferred examples include tetrahydrofuran and 1,2-dimethoxyethane. With regard to the reaction temperature, the reaction can be carried out usually at -78-0°C, preferably at -50-0°C. Although the reaction time is not particularly limited, it is, for example, usually 0.5-24 hours, preferably 1-12 hours.

[0145]

Stage 3-2

[formula 71]

The compounds represented by the formula (W1) can be obtained from the compound represented by the formula (W2) [in the formula (W2), "hal ² " has the same meaning as defined above] and the compound represented by the formula (12) in the same way, as the method of steps 1-3. In this reaction, the compound represented by formula (12) can be used in the amount of 1/5 to 20 equivalents, preferably 1/2 to 10 equivalents, more preferably 1 to 5 equivalents, relative to the compound represented by formula (W2).

[0146]

Stage 3-3

[formula 72]

Compounds represented by formula (W2) can be prepared by reacting a compound represented by formula (W3) [in formula (W3), "hal ² " has the same meaning as defined above] with N,O-dimethylhydroxylamine hydrochloride in an organic solvent in the presence of a base and a condensing agent that splits off water. The amount of N,O-dimethylhydroxylamine hydrochloride used in the reaction of the compound represented by formula (W3) and N,O-dimethylhydroxylamine hydrochloride may be an equivalent amount to an excess amount, such as 1 to 10 equivalents, preferably 1 to 5 equivalents, relative to the compound represented by the formula (W3), but the amount can be appropriately determined in view of the purity, yield, purification efficiency, and the like of the compound represented by the formula (W3).

[0147]

As the base, for example, triethylamine, diisopropylethylamine, 1,4-diazabicyclo[2.2.2]octane, N,N-dimethyl-4-aminopyridine and the like can be used, and diisopropylethylamine is preferred. The amount of base to be used may be from an equivalent amount to an excess amount, for example, 1 to 10 equivalents, preferably 1 to 5 equivalents, relative to the sum of the equivalents of the compound represented by the formula (W3) which serves as the starting compound and the equivalents N,O-dimethylhydroxylamine hydrochloride.

[0148]

N, N'-dicyclohexylcarbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, N,N'-diisopropylcarbodiimide, N-cyclohexyl-N'-(2-morpholinoethyl)carbodiimide can be used as a condensing agent that removes water. p-toluenesulfonate, N, N'-carbonyldimidazole, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride, 1H-benzotriazol-1-yloxy-tris(dimethylphosphonium) hexafluorophosphate ), 1H-benzotriazol-1-yloxy-tripyrrolidinophosphonium hexafluorophosphate, O-(benzotriazol-1-yl)-N, N,N',N'-tetramethyluronium hexafluorophosphate, O-(7-azabenzotriazol-1-yl)-N hexafluorophosphate , N,N',N'-tetramethyluronium and the like, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide is preferred. The amount of the water-removing condensing agent to be used may be an equivalent amount to an excess amount, such as 1 to 10 equivalents, preferably 1 to 5 equivalents, relative to the equivalent of the compound represented by the formula (W3) which serves as the starting compound .

[0149]

As an activator, N, N-dimethyl-4-aminopyridine or the like can be added. The amount of activator to be used may be from a catalytic amount to an excess amount, for example, 0.01 to 5 equivalents, preferably 0.1 to 1 equivalents, relative to the equivalent of the compound represented by the formula (W3) which serves as the starting compound .

[0150]

Examples of the solvent used for the reaction include, for example, N,N-dimethylformamide, N,N-dimethylacetamide, acetonitrile, xylene, toluene, 1,4-dioxane, dichloromethane, chloroform, 1,2-dichloroethane, tetrahydrofuran and the like, and preferred examples include dichloromethane. Two or more types of these solvents can also be used as a mixture. With regard to the reaction temperature, the reaction can be carried out usually at 0-100°C, preferably at 20-60°C. Although the reaction time is not particularly limited, it is, for example, usually 0.5-48 hours, preferably 1-24 hours.

[0151]

Stage 3-4

[formula 73]

The compounds represented by formula (14) can be obtained from the compound represented by formula (W2) [in formula (W2), "hal ² " has the same meaning as defined above] in the same manner as the method of step 3-1.

[formula 74]

[0152]

Stage 3-5

The compounds represented by formula (W3) can be obtained by heating the compound represented by formula (W4) [in formula (W4), "hal ² " has the same meaning as defined above] in dilute sulfuric acid. As the dilute sulfuric acid used for the reaction, dilute concentrated sulfuric acid or dilute sulfuric acid can be suitably used, and its concentration is, for example, 0.1-15 mol/l, preferably 1-10 mol/l. The amount of dilute sulfuric acid to be used may be an excess amount for the compound represented by formula (W4) and may be determined in view of yield, purification efficiency, and the like. As for the reaction temperature, the reaction can be carried out usually at 20-100°C, preferably at 60-100°C. Although the reaction time is not particularly limited, it is, for example, usually 0.5-48 hours, preferably 1-24 hours.

[0153]

Among the compounds represented by the formula (W3), 3-bromophenylacetic acid, 3-iodophenylacetic acid and 3-bromo-4-fluorophenylacetic acid are commercially available compounds and can be obtained from Tokyo Chemical Industry. 2-(4-Bromothiophen-2-yl)acetic acid is a commercially available compound and can be obtained from APOLLO.

[0154]

Stage 3-6

Compounds represented by formula (W4) can be obtained by exposing a compound represented by formula (W5) to cyanide [in formula (W5), "hal ² " has the same meaning as defined above]. As the cyanide, sodium cyanide, potassium cyanide, copper cyanide and the like can be used, and sodium cyanide and potassium cyanide are preferred. The amount of cyanide to be used may be an equivalent amount to an excess amount, such as 1-10 equivalents, preferably 1-5 equivalents, relative to the compound represented by the formula (W5) which serves as the starting compound. Examples of the solvent used for the reaction include, for example, methanol, ethanol, isopropanol, water, their mixed solvent and the like, and preferred examples include a 2:1 mixed solvent of ethanol and water. With regard to the reaction temperature, the reaction can be carried out usually at 0-100°C, preferably at 20-100°C. Although the reaction time is not particularly limited, it is, for example, usually 0.5-24 hours, preferably 1-12 hours.

[0155]

Stage 3-7

Compounds represented by formula (W5) can be obtained by bromination of a compound represented by formula (W6) [in formula (W6), "hal ² " has the same meaning as defined above]. Examples of the brominating agent include N-bromosuccinimide and 1,3-dibromo-5,5-dimethylhydantoin, and N-bromosuccinimide is preferred. The amount of the brominating agent to be used may be an equivalent amount to an excess amount, such as 1-10 equivalents, preferably 1-5 equivalents, relative to the compound represented by formula (W6) which serves as the starting compound. Examples of the activator that is added together with the brominating agent include benzoyl peroxide, t-butyl hydroperoxide and azabisisobutyronitrile, and benzoyl peroxide is preferred. The amount of activator to be used may be from a catalytic amount to an excess amount, for example, 0.01-2 equivalents, preferably 0.05-1 equivalents, relative to the compound represented by formula (W6) which serves as the starting compound. Examples of the solvent used for the reaction include, for example, carbon tetrachloride, chloroform, 1,2-dichloroethane, a mixed solvent thereof, and the like, and preferred examples include carbon tetrachloride. With regard to the reaction temperature, the reaction can be carried out usually at 20-90°C, preferably at 60-90°C. Although the reaction time is not particularly limited, it is, for example, usually 0.5-24 hours, preferably 1-12 hours.

[0156]

Examples of the compound represented by formula (W6) include 2-bromo-1,4-dimethylbenzene, 1-bromo-3,5-dimethylbenzene and the like, and can be purchased as commercially available compounds, for example, from Tokyo Chemical Industry.

[0157]

Stage 3-8

[formula 75]

The compounds represented by the formula (12) can be obtained by exposing the compound represented by the formula (W7) with an α-diazophosphate compound together with an inorganic base. Examples of the combination of an α-diazophosphate compound and an inorganic base include, for example, combinations of dimethyl(diazomethyl)phosphate and potassium t-butoxide, dimethyl(diazomethyl)phosphate and sodium t-butoxide, dimethyl(1-diazo-2-oxopropyl)phosphate and potassium carbonate and dimethyl(1-diazo-2-oxopropyl)phosphate and sodium carbonate, and a combination of dimethyl(1-diazo-2-oxopropyl)phosphate and potassium carbonate is preferred. The amount of α-diazophosphate to be used may be an equivalent amount to an excess amount, such as 1-10 equivalents, preferably 1-5 equivalents, relative to the compound represented by the formula (W7) which serves as the starting compound. The amount of inorganic base to be used may be from an equivalent amount to an excess amount, for example 1-5 equivalents, preferably 1-3 equivalents, relative to the α-diazophosphate to be used. Examples of the solvent used for the reaction include, for example, methanol, ethanol, isopropanol, t-butanol, a mixed solvent thereof and the like, and preferred examples include methanol. With regard to the reaction temperature, the reaction can be carried out usually at -20-80°C, preferably at 0-60°C. Although the reaction time is not particularly limited, it is, for example, usually 0.5-24 hours, preferably 1-12 hours.

[0158]

Among the compounds represented by formula (12), 3-ethynylthiophene and 2-ethynylthiophene are commercially available compounds and can be obtained from Tokyo Chemical Industry.

[0159]

Stage 3-9

4-Phenylthiophene-3-carbaldehyde as the compound represented by formula (W7) can be obtained by reacting 4-formylthiophene-3-boronic acid and bromobenzene in a solvent in the presence of a base and a palladium catalyst.

[0160]

As the palladium catalyst, for example, commercially available catalysts such as tetrakis(triphenylphosphine)palladium, tetrakis(methyldiphenylphosphine)palladium, dichlorobis(triphenylphosphine)palladium, dichlorobis(tri-o-tolylphosphine)palladium, dichlorobis(tricyclohexylphosphine)palladium, dichlorobis( triethylphosphine)palladium, palladium acetate, palladium chloride, bis(acetonitrile)palladium chloride, bis(dibenzylideneacetone)palladium, tris(dibenzylideneacetone)dipalladium and bis(diphenylphosphinoferrocene)palladium chloride can be added to the reaction system as is or as a catalyst separately prepared and isolated from palladium acetate, tris(dibenzylideneacetone)dipalladium or the like, and an arbitrary ligand. The catalyst that is considered to actually participate in the reaction can be obtained in the reaction system by mixing palladium acetate, tris(dibenzylideneacetone)dipalladium or the like, and an arbitrary ligand. The valency of palladium can be 0 or +2. In particular, tris(dibenzylideneacetone)dipalladium(0), palladium(II) acetate and the like may be mentioned as preferred examples.

[0161]

As a ligand used in the preparation of a palladium catalyst from an arbitrary ligand, there are examples of phosphine ligands such as triphenylphosphine, tri(o-tolyl)phosphine, tri(cyclohexyl)phosphine, tri(t-butyl)phosphine, dicyclohexylphenylphosphine, 1,1' -bis(di-tert-butylphosphino)ferrocene, 2-dicyclohexylphosphino-2'-dimethylamino-1,1'-biphenyl, 2-(di-tert-butylphosphino)biphenyl, 2-(dicyclohexylphosphino)biphenyl, 2,2'- bis(diphenylphosphino)-1,1'-binaphthyl, xantphos, tri(tert-butyl)phosphine, 2-dicyclohexylphosphino-2',6'-dimethoxybiphenyl, 2-dicyclohexyl-2',4',6'-triisopropylbiphenyl and 1 ,2,3,4,5-pentamethyl-1'-(di-tert-butylphosphino)ferrocene), and preferred examples include 2-dicyclohexylphosphino-2',6'-dimethoxybiphenyl and the like.

[0162]

Although the amount of palladium catalyst equivalents to be used may be an equivalent amount or a catalytic amount, it is preferably 0.01 mol% or more, particularly preferably 0.10-50.0 mol%, relative to the starting compound. Examples of the base include, for example, sodium tert-butoxide, cesium carbonate, potassium phosphate and the like, and potassium phosphate is preferred. The amount of base equivalents to be used may be an equivalent amount or an excess amount, such as 1-5 equivalents, preferably 1-3 equivalents. Examples of the solvent used for the reaction include, for example, ether-type solvents such as tetrahydrofuran, 1,4-dioxane, and 1,2-dimethoxyethane, toluene, N,N-dimethylformamide, N,N-dimethylacetamide, n-butanol, water their mixed solvent and the like, and a mixed solvent of n-butanol and water at a ratio of 5:1 can be mentioned as a preferred example. As for the reaction temperature, the reaction can be carried out usually at -20-120°C, preferably at 0-100°C. Although the reaction time is not particularly limited, it is, for example, usually 0.5-48 hours, preferably 1-24 hours.

[0163]

Methods for obtaining the compounds of the present invention are not limited to the methods described in the present invention. For example, compounds of the present invention may be prepared by modifying or converting substituents of compounds as precursors to compounds of the present invention using one or a combination of two or more of the reactions described in conventional chemistry articles and the like.

[0164]

Examples of the preparation method for the compounds of the present invention which contain an asymmetric carbon include a preparation method based on asymmetric reduction, a method using a commercially available starting compound (or a starting compound that can be obtained by a known method or a method similar to a known method) in which a moiety corresponding to asymmetric carbon, is initially optically active, and the like. There is also a method in which the compound of the present invention or its precursor is isolated as the active isomer in a conventional manner. Examples of this method include, for example, a method using high performance liquid chromatography (HPLC) using a chiral column, classical fractional crystallization for separating optically active substances, including obtaining a salt with an active agent, separation by fractional crystallization or the like, and converting the salt into a compound in free form, and the method includes condensation with an optically active agent, obtaining a diastereomer, followed by separation, purification and decomposition of the resulting diastereomer, and the like. When the precursor is isolated to obtain optically active substances, the optically active compound of the present invention can then be obtained by carrying out the above production methods.

[0165]

When the compound of the present invention contains an acidic functional group such as a carboxyl group, a phenolic hydroxyl group or a tetrazole ring, the compound can be converted into a pharmaceutically acceptable salt (for example, inorganic salts with sodium and the like, or organic salts with triethylamine and the like) by known methods. For example, when an inorganic salt is to be prepared, the compound of the present invention may be dissolved in water containing at least 1 equivalent of a hydroxide, carbonate, bicarbonate, or the like corresponding to the desired inorganic salt. For the reaction, a water-miscible inactive organic solvent such as methanol, ethanol, acetone and dioxane can be mixed. For example, using sodium hydroxide, sodium carbonate, or sodium hydrogen carbonate, a sodium salt solution can be prepared.

[0166]

When the compound of the present invention contains an amino group, another basic functional group, or an aromatic ring which itself has basic properties (for example, a pyridine ring and the like), the compound can also be converted into a pharmaceutically acceptable salt (for example, a salt with an inorganic acid such as hydrochloric acid, or a salt with an organic acid such as acetic acid) by known methods. For example, when it is intended to form a salt with an inorganic acid, a compound of the present invention may be dissolved in water containing at least 1 equivalent of the desired inorganic acid. For the reaction, a water-miscible inactive organic solvent such as methanol, ethanol, acetone and dioxane can be mixed. For example, by using hydrochloric acid, a hydrochloride solution can be obtained.

[0167]

If a solid salt is desired, the solution can be evaporated, or a water-miscible organic solvent having some degree of polarity, such as n-butanol or ethyl methyl ketone, can be added to the solution to form a solid salt.

[0168]

The various compounds described by the present invention can be purified by known methods such as a number of chromatographic methods (column chromatography, flash column chromatography, thin layer chromatography, high performance liquid chromatography).

[0169]

The compounds of the present invention according to one embodiment have EP ₄ agonist activity and can be used as an EP ₄ agonist. That is, the compounds of the present invention according to one embodiment can be used as a drug for the prophylactic and/or therapeutic treatment of a disease associated with EP ₄ receptor agonism. A disease associated with EP ₄ receptor agonism is, more specifically, a disease for which EP ₄ receptor agonism is effective, and more specifically, it is not specifically limited as long as it is a disease that can be prevented and/or treated. an increase in the amount of cAMP synthesized in osteoblasts.

[0170]

EP ₄ agonist activity can be measured, for example, by the methods described below. Thus, a method for confirming the stimulation of cAMP production in a cell expressing the human EP ₄ receptor can be mentioned. As another embodiment, a method for confirming an osteogenesis-promoting effect based on stimulating cAMP production in the presence of a cyclooxygenase 2 (COX-2) inhibitor in rat bone marrow cells can be mentioned. As another embodiment, a method for confirming binding activity to the human EP ₄ receptor can be mentioned. As a method for confirming an osteogenesis-promoting action based on stimulating cAMP production, the method described in Test Example 1 below can be specifically exemplified.

[0171]

EP ₄ agonist activity, which can be confirmed by the method described in Test Example 1, is, for example, 10 nM or less, preferably 1 nM or less, more preferably 0.6 nM or less, even more preferably 0.3 nM or less, particularly preferably 0.1 nM or less, most preferably 0.05 nM or less.

[0172]

The compounds of the present invention according to one embodiment show high specificity (selectivity) for EP ₄ . Selectivity for EP ₄ can be assessed, for example, by performing a measurement of agonist activity and a receptor binding test using cells that express each of the human EP ₁ , EP ₂ and EP ₃ receptors, calculating the ratio of IC ₅₀ values (concentration of the compound of the present invention, at which the binding of [ ³ H]PGE ₂ and the receptor is inhibited by 50%), or Ki values. Specifically, the method described in Test Case 2 can be exemplified.

[0173]

Ratio of IC ₅₀ magnitude (duration)=IC ₅₀ for each receptor/IC ₅₀ for EP ₄

Ki ratio of magnitude (duration)=Ki dissociation constant for each receptor/Ki dissociation constant for EP ₄

[0174]

In order to avoid side reactions, it is preferable that the compounds of the present invention according to one embodiment show high specificity for EP ₄ . For example, the ratio of IC ₅₀ value or Ki value should be 10 times or more, preferably 100 times or more, more preferably 1000 times or more, more preferably 3000 times or more, particularly preferably 10000 times or more.

[0175]

It is also preferred that the compounds of the present invention according to one embodiment selectively act on or bind to the EP ₄ receptor but do not act on or bind to the EP ₁ receptor, EP ₂ receptor and EP ₃ receptor, as well as DP receptor, FP receptor, IP receptor. , TP receptor, PPARα receptor, PPARδ receptor, PPARγ receptor, S1P receptors (e.g. S1P1 receptor, S1P2 receptor, S1P3 receptor and the like), LTB4 receptors (e.g. BLT1, BLT2 and the like), LPA receptor (e.g. LPA1 receptor, LPA2 receptor, LPA3 receptor, and the like), and cannabinoid receptors (eg, CB1 receptor, CB2 receptor, and the like), or act on or bind to them more weakly than act on or bind to the EP ₄ receptor.

The disease associated with EP4 receptor agonism is not particularly limited as long as it is a disease for which _EP4 receptor antagonism is effective, and specific examples include, for example, bone fracture and bone defect.

[0176]

The compounds of the present invention according to one embodiment have an osteogenesis stimulating effect as shown in the test examples mentioned below and are useful as an active ingredient of a drug. The compounds of the present invention according to one embodiment are used, in particular, to therapeutically treat and/or promote healing of a fracture or bone defect, and are preferably used to therapeutically treat and/or promote healing of a fracture. As another embodiment, they are also preferably used to therapeutically treat and/or promote the healing of a bone defect.

[0177]

The suitability of a drug of the present invention according to one embodiment for therapeutic treatment and/or promotion of healing of a fracture or bone defect can be confirmed using a closed fracture model or a partial or longest bone defect model. Specifically, an example is the method described in Test Case 5.

[0178]

It can be expected that the drug of the present invention according to one embodiment will exhibit a systemic bone density increasing action and a bone strength increasing action or a local action simulating bone regeneration/osteoanagenesis. The osteogenesis stimulating effect of the compounds of the present invention according to one embodiment can be assessed, for example, using bone marrow cells isolated from experimental animals such as rats or humans and cultured, and using the amount of calcified bone-like nodules formed, alkaline phosphatase activity, which is a marker of osteoblast differentiation, or the like as a marker. It can also be assessed using pathological animal models such as rat models with reduced bone mass subjected to sciatic nerve resection and oophorectomy or the like, and the density or strength of the appendicular skeletons or the like as a marker. It can also be assessed by using a rat model of a closed fracture of a tubular bone or a model of an incision of a bone obtained by an invasive operation, a model in which a bone defect is created in an arbitrary area or the like, and osteogenesis, bone fusion rate, bone strength of the restored bone, or the like as a marker. .

[0179]

Fracture refers to a condition in which a bone is partially or completely broken or deformed by an external force. The bone that may suffer from a fracture is not specifically limited, as long as it refers to a patient whose bone tissue is damaged, and examples include bones of the face (orbital bone, cheekbone, lower jaw), bones of the trunk (rib, pelvis, cervical vertebra , thoracic vertebra, lumbar vertebra, sacrum, coccygeal bone), bones of the upper limb (scapula, collarbone, humerus, elbow, radius, ulna, scapula, hamatum, metacarpus, phalanx), bones of the lower limb (hip joint, femur, tibia, fibula, ankle joint, calcaneus, scaphoid, metatarsal) and the like, and the target bone can be any part. The type of bone tissue injury is not particularly limited, and stimulating bone fusion in fracture (complete fracture, incomplete fracture, simple fracture, comminuted fracture, and the like) or intentionally cut bone in osteotomy or bone expansion surgery adapted as one of the surgical methods is also included. treatment. Hip fracture, vertebral compression fracture, distal radius fracture, proximal humerus fracture, and the like, for which osteoporosis is the cause, are also included in the scope of the fracture above.

[0180]

Bone defect refers to various bone diseases themselves, such as osteonus, osteomyelitis, traumatic injury, chronic joint disease, prolonged healing after a fracture, and weakness of an artificial joint, or a condition in which a bone defect is formed when a lesion is surgically removed in the treatment of these diseases. Its part is not particularly limited, as long as it refers to a patient who is forced to have a bone defect. Examples include bones of the face (orbital bone, zygoma, lower jaw), bones of the trunk (rib, pelvis, cervical vertebrae, thoracic vertebrae, lumbar vertebrae, sacrum, coccygeal bone), bones of the upper limb (scapula, collarbone, humerus, elbow, radius, ulna, scapula, hamatum, metacarpus, phalanx), lower limb bones (hip, femur, tibia, fibula, ankle, calcaneus, scaphoid, metatarsal) and the like, and the target bone can be any part. The type of bone defect is not particularly limited, and any type of bone defects are included, such as a condition in which the intermediate part of the bone is severely damaged, and a condition in which the bone becomes partially defective due to a comminuted fracture.

[0181]

The drug of the present invention according to one embodiment can be used as a bone fusion stimulating agent at the time of surgical therapeutic interventions. For example, it can be used in spinal fixation (cervical vertebrae, thoracic and lumbar vertebrae), denatured scoliosis surgery, joint replacement, spinal canal expansion, osteotomy, bone expansion surgery, cranial bone defect compensation, cranioplasty, bone supported ilium fixation, heterologous bone grafting, homologous bone grafting, autologous bone grafting and bone graft replacement therapy, and bone repair and/or bone reconstruction after surgical removal of a primary malignant tumor or bone metastasis, which are examples of medical interventions.

[0182]

The drug of the present invention according to one embodiment is preferably used as an osteogenesis stimulating agent. The drug of the present invention, according to one embodiment, is more preferably used for the therapeutic treatment and/or promotion of healing of a fracture or bone defect. In addition, the drug of the present invention according to one embodiment is extremely preferably used for the prophylactic and/or therapeutic treatment of a fracture. In addition, a person skilled in the art will understand that a drug for preventing or suppressing the development of a pathological condition falls within the scope of a drug for prophylactic and/or therapeutic treatment related to the present invention, as the case may be.

[0183]

The drug of the present invention according to one embodiment can be obtained as a drug containing a compound represented by formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient, and for example, a drug containing a compound or a pharmaceutically acceptable salt thereof that is metabolized in a living body, giving a compound represented by formula (1) or a pharmaceutically acceptable salt thereof when administered as a prodrug also falls within the scope of the drug of the present invention.

Although the route of administration of the drug of the present invention according to one embodiment is not particularly limited, the administration regimen can be suitably selected from, for example, oral administration, subcutaneous administration, intradermal administration, intramuscular administration, intravenous administration, pernasal administration, intravaginal administration, intrarectal administration, topical impact on the affected part and the like. Topical administration to the affected part is one of the preferred modes of administration.

[0184]

As a medicine of the present invention, the compound represented by the formula (1) or a pharmaceutically acceptable salt thereof can be used by itself. However, it is preferable to add one or more types of pharmaceutically acceptable carriers to the compound represented by formula (1) or a pharmaceutically acceptable salt thereof to prepare a pharmaceutical composition and administer the composition. In addition, as the active ingredient of the drug of the present invention, a hydrate or solvate of the compound represented by the general formula (I) or a pharmaceutically acceptable salt thereof can be used.

[0185]

Examples of the dosage form used to prepare the above pharmaceutical composition include tablet, powder, granule, syrup, suspension, capsule, inhalant, injection, and the like. To obtain them, various carriers suitable for these preparations are used. For example, examples of a carrier for oral preparations include excipients, binders, lubricants, flow accelerators, and colorants. Examples of the method of using the composition in the form of inhalants include a method of inhaling a powder of a pharmaceutical composition or a liquid dosage form obtained by dissolving or suspending a pharmaceutical composition in a solvent as it is, a method of inhaling an aerosol thereof using a nebulizer called an atomizer or a nubelizer, and the like. When the composition is formulated as an injection, distilled water for injection, physiological saline, aqueous glucose solution, vegetable oil for injection, propylene glycol, polyethylene glycol and the like can usually be used as a diluent. Disinfectants, antiseptics, stabilizing, isotonic, soothing agents and the like can be added as needed. A clathrate compound in which a compound of the present invention is clathrated into a cyclodextrin can be prepared and used as a drug of the present invention.

[0186]

When a drug of the present invention is administered according to one embodiment, a suitable dosage form can be suitably selected and administered by a suitable route. For example, it may be administered orally as a tablet, powder, granule, syrup, suspension, capsule, or the like. The drug may also be administered via the respiratory tract as an inhalation agent. In addition, the drug can be administered subcutaneously, intradermally, intravascularly, intramuscularly or intraperitoneally in the form of an injection, including drip infusion. In addition, the drug can be administered transmucosally as a sublingual tablet, suppository or the like, and can be administered transdermally as a gel, lotion, ointment, cream, spray or the like. In addition, the drug can also be administered as a sustained release drug, such as a sustained release injection or an implant formulation (eg, a film formulation and the like).

[0187]

When the drug of the present invention according to one embodiment is administered locally, the drug can be directly administered to a local site such as a fracture site. In this case, the compound can be directly administered to the local site along with a suitable non-hydrophilic solvent, or the compound can also be formulated in a suitable carrier such as biodegradable polymers and used as a drug, rod-shaped, needle-shaped, spherical, in the form of a film or the like, or in the form of an ointment, cream or gel, or a sustained release preparation by transplantation or drug administration at a local site such as a fracture. Examples of the biodegradable high molecular weight polymer include, for example, polyesters of aliphatic acids (polymers and copolymers of one or more types of α-hydroxycarboxylic acids, hydroxydicarboxylic acids, lactic acid/caprolactone, valerolactone and the like, and mixtures thereof), their derivatives (polylactic acids, polyglycolic acids, block polymers of polyethylene glycol and the like), poly-α-cyanoacrylic acid esters, poly-β-hydroxybutyric acids, polyalkylene salates, polyorthoesters, polyorthocarbonates, polycarbonates, polyamino acids, hyaluronic acid esters, polystyrene groups, polymethacrylic acids, copolymers of acrylic acid and methacrylic acid, polyamino acids , decynstearate, ethylcellulose, cellulose acetate, nitrocellulose, maleic anhydride copolymers, ethylene vinyl acetate copolymers, polyvinyl acetates, polyacrylamides, collagen, gelatin, fibrin, bone meal, bone cement, and the like.

[0188]

A biodegradable high molecular weight polymer may be composed of one type of substance or copolymer, a complex or a simple mixture of two or more types of substances, and its polymerization scheme may be any of random, block and graft polymerizations.

The drug of the present invention according to one embodiment can also be applied or adsorbed on an artificial bone (implant) composed of a highly biocompatible material (metal, calcium, ceramic materials, polymeric materials, and the like), bone prostheses (hydroxyapatite, β-tricalcium phosphate, and the like). ) or the like together with a suitable solvent or carrier, or implanted therein and administered at the local site.

[0189]

The administration period of the drug of the present invention according to one embodiment is not particularly limited. In principle, the drug is administered during the period when it is found that the clinical symptoms of the disease are expressed, and usually continue from a few weeks to one year. However, it is also possible to extend the period of administration depending on pathological conditions, or to continue administration even after recovery from clinical symptoms. The drug can also be prescribed prophylactically at the discretion of the doctor, even if any clinical symptom is not expressed. The dosage of the drug of the present invention according to one embodiment is not particularly limited. When the drug of the present invention is administered directly to a local site such as part of a fracture, 0.01-1000 µg of the active ingredient can be administered to an adult in one administration. As for the frequency of administration in the case above, the drug can be administered at a frequency of every 6 months to every day, and the drug can be preferably administered from once every 3 months to once a month, or once a week.

[0190]

The daily dose and/or single dose, the period of administration and the frequency of administration may be respectively increased or decreased depending on various conditions such as the age, weight, degree of physical health of the patient, the type and severity of the disease to be treated, the route of administration and dosage form ( sustained release property of the carrier for the active ingredient, and the like).

[0191]

When the drug of the present invention according to one embodiment is used for the therapeutic treatment and/or promotion of healing of a fracture or bone defect, or the prophylactic and/or therapeutic treatment of a fracture, the drug of the present invention according to one embodiment can be used together with one or more types of drugs selected from the group consisting of bone activating agents, osteogenesis stimulating agents, bone resorption suppressing agents, bone metabolism enhancing agents, sex hormone preparations and calcium preparations, simultaneously or at different points in time. In addition, the drug of the present invention according to one embodiment can also be prepared as a so-called combination drug together with the drugs exemplified above and then administered. The aforementioned combination drug may be in the form of a complete mixture of active ingredients similar to typical compositions of this type, as well as in the form of a dosage form, kit or package containing an unmixed combination of ingredients, separately administered from two or more containers, each of which contains each active ingredient.

[0192]

Examples of bone activating agents useful in combination with the drug of the present invention according to one embodiment include, for example, vitamin D or vitamin D derivatives such as calcitriol, alfacalcidol, OCT, 2MD and ED-71, examples of osteogenesis stimulating agents, include, for example, menatetrenone, teriparatide, somatropin, insulin-like growth factor-I (IGF-I), bone morphogenetic proteins (BMP), basic fibroblast growth factor (bFGF), transforming growth factor-β (TGF-β), EP ₂ agonist , LRP ₅ agonist, anti-SOST antibody, GSK-3 inhibitor, Dkk1 inhibitor, calcilytics, agents that stimulate growth hormone secretion and the like, examples of agents that inhibit bone resorption include, for example, elkatonin, salmon calcitonin, etidronate, pamidronate, clodronate, alendronate, incadronate, risedronate, minodronate, ibandronate, cathepsin K inhibitors, osteoprotegerin, anti-RANKL antibodies and the like, examples of bone metabolism enhancing agents , include, for example, fluorine, strontium ranelate, ipriflavon, and the like, examples of sex hormone preparations include, for example, estriol, estradiol, conjugated estrogen, progesterone, medroxyprogesterone, testosterone, methyltestosterone, mestanolone, stanozolol, methenolone, nandrolone, selective estrogen receptor modulators ( SERMs: raloxifene, lasofoxifene, bazedoxifene, ospemifene, arzoxifene, CHF4227, PSK-3471 and others), selective androgen receptor modulators (SARMs) and the like, and examples of calcium preparations include, for example, calcium carbonate, calcium lactate, calcium gluconate, calcium acetate , calcium chloride, calcium citrate, calcium hydrogen phosphate, calcium L-aspartate and the like. It can also be used along with various types of drugs for bone diseases that will be created in the future. These combination drugs are not limited, provided that the combinations are clinically relevant.

[0193]

The compounds of the present invention according to one embodiment include compounds showing excellent safety (including various toxicities and pharmacological safety), pharmacokinetic characteristics and the like, and their usefulness as drug active ingredients can be confirmed, for example, by the methods shown below.

[0194]

Examples of tests related to security include, for example, the tests listed below. However, they are not limited to these examples. Examples include cytotoxicity tests (tests using HL60 cells, hepatocytes and the like), genotoxicity tests (Ames test, TK mouse lymphoma test, chromosome aberration test, micronucleus test and the like), skin sensitization tests (Bühler method, GPMT, APT method, LLNA test and the like), skin photosensitivity tests (adjuvant and strip method and the like), eye irritation tests (single instillation, short continuous instillation, repeated instillation and the like), cardiovascular safety pharmacological tests (telemetry method, APD method, hERG inhibition test and the like), pharmacological safety tests for the central nervous system (FOB method, a modified version of the Irwin method and the like), pharmacological safety tests for the respiratory system (measurement method using a device for measuring respiratory function, measurement method using a blood gas analyzer, and the like), general t toxicity tests, reproductive and embryotoxicity tests, and the like.

[0195]

Examples of tests related to pharmacokinetic parameters include, for example, the tests listed below. However, they are not limited to these examples. Examples include cytochrome P450 enzyme inhibition or induction tests, cell permeability tests (tests using CaCO-2 cells, MDCK cells and the like), ATPase drug transporter assay, oral absorption tests, tests to measure changes in blood concentration, tests for metabolism (stability test, metabolic molecular fragment test, reactivity test and the like), dissolution tests (turbidimetric method based dissolution test and the like), and the like.

[0196]

The suitability of the compounds of the present invention according to one embodiment as an active drug ingredient can be confirmed by performing, for example, a cytotoxicity test. Examples of the cytotoxicity test include methods using various cultured cells, for example, HL-60 cells, which are human proleukemia cells, isolated cultured primary hepatocyte cells, neutrophil fraction obtained from human peripheral blood, and the like. Although the test can be performed by the method described below, the method is not limited to the following description. Cells are obtained in the form of suspensions of 105-107 cells/ml, the suspension is added to microtubes or microplates in a volume of 0.01 to 1 ml. A solution dissolving the compound in a volume of 1/100 to 1 times the volume of the cell suspension is added to the suspension, and the cells are cultured in cell culture medium with a final compound concentration of 0.001 to 1000 μM for 30 minutes to several days at 37° C. atmosphere of 5% CO ₂ . After cessation of culture, the percentage of surviving cells is evaluated using the MTT method, WST-1 method (Ishiyama, M., et al., In Vitro Toxicology, 8, page 187, 1995) or the like. By measuring the cytotoxicity of a compound in cells, the suitability of the compound as an active ingredient in a drug can be confirmed.

[0197]

The suitability of the compounds of the present invention according to one embodiment as an active drug ingredient can be confirmed by performing, for example, a genotoxicity test. Examples of the genotoxicity test include the Ames test, the mouse lymphoma TK test, the chromosome aberration test, the micronucleus test, and the like. The Ames test is a method for detecting back mutation by culturing Salmonella or Escherichia bacteria of a specified strain in culture dishes or the like with the addition of a compound (reference to IYAKUSHIN (Notification by the chief of Evaluation and Licensing Division, Pharmaceutical and Medical Safety Bureau, Ministry of Health, Labor and Welfare, Japan), No. 1604, 1999, "Guideline for Genotoxicity Test", II-1. Genotoxicity Test and the like). The mouse lymphoma TK test is a test to detect the possibility of genetic mutations directed to the thymidine kinase L5178Y gene of mouse lymphoma cells (reference to IYAKUSHIN No. 1604, 1999, “Guideline for Genotoxicity Test”, II-3. Mouse Lymphoma TK Test; Clive, D. et al., Mutat Res., 31, pp. 17-29, 1975; Cole, J., et al., Mutat. Res., 111, pp. 371-386, 1983 and the like). Chromosome aberration test is a method for determining chromosome aberration-inducing activity by culturing mammalian cells in the presence of a compound, then after fixing the cells, staining and observing the chromosomes of the cells (Ref. IYAKUSHIN No. 1604, 1999, “Guideline for Genotoxicity Test”, II-2 Chromosomal Aberration Test Utilizing Mammalian Cultured Cells and the like). The micronucleus test is a method for assessing the ability to form micronuclei caused by chromosome aberration, and a rodent method (in vivo test) is available (IYAKUSHIN No. 1604, 1999, “Guideline for Genotoxicity Test”, II-4. Micronucleus Test Using Rodent; Hayashi M. et al., Mutat Res., 312, pp. 293-304, 1994; Hayashi, M. et al., Environ Mol. Mutagen., 35, pp. 234-252, 2000), method of application cultured cells (in vitro test) (Fenech M., et al., Mutat. Res., 147, pp. 29-36, 1985; Miller, B., et al., Mutat. Res., 392, p. 45 -59, 1997) and the like. By ascertaining the genotoxicity of a compound using one or more of these methods, the suitability of the compound as an active ingredient in a drug can be confirmed.

[0198]

The suitability of the compounds of the present invention according to one embodiment as an active drug ingredient can be confirmed by performing, for example, a skin sensitization test. Skin sensitization tests include, as skin sensitization tests using guinea pigs, Buehler method (Buehler, E.V., Arch. Dermatol., 91, pp. 171-177, 1965), GPMT method (maximization method, Magnusson B., et al., J. Invest. Dermatol., 52, pp. 268-276, 1969), APT method (adjuvant and application method (Sato, Y. et al., Contact Dermatitis, 7, pp. 225-237, 1981 )) and the like. In addition, as a skin sensitization test using mice, the LLNA (Regional Lymph Node Response Assay) method (OECD Guideline for the testing of chemicals 429, Skin sensitization 2002; Takeyoshi, M. et al., Toxicol. Lett., 119 ( 3), pp. 203-8, 2001; Takeyoshi, M. et al., J. Appl. Toxicol., 25(2), pp. 129-34, 2005) and the like are available. By ascertaining the ability to sensitize the skin using one or more of these methods, the suitability of a compound as an active ingredient in a drug can be confirmed.

[0199]

The suitability of the compounds of the present invention according to one embodiment as an active drug ingredient can be confirmed by performing, for example, a skin photosensitivity test. Examples of the skin photosensitization test include skin photosensitization test using guinea pigs (reference to "Drug Nonclinical Test Guideline Commentary 2002", Yakuji Nippo published in 2002, 1-9: Skin Photosensitization Test and the like) and the like, and examples of the method include adjuvant strip method (Ichikawa, H. et al., J. Invest. Dermatol., 76, pp. 498-501, 1981), Harber method (Harber, L.C., Arch. Dermatol., 96, pp. 646-653, 1967), Horio's method (Horio, T., J. Invest. Dermatol., 67, pp. 591-593, 1976), Jordan's method (Jordan, W.P., Contact Dermatitis, 8, pp. 109-116, 1982), Kochever's method (Kochever, T. E. et al., J. Invest. Dermatol., 73, pp. 144-146, 1979), Maurer's method (Maurer, T. et al., Br. J. Dermatol., 63 , pp. 593-605, 1980), the Morikawa method (Morikawa, F. et al., “Sunlight and Man”, Tokyo Univ. Press, Tokyo, pp. 529-557, 1974), the Vinson method (Vinson, L.J., J. Soc. Cosm. Chem., 17, pp. 123-130, 1966) and the like. By ascertaining the ability to photosensitize the skin of a compound using one or more of these methods, the suitability of the compound as an active ingredient in a drug can be confirmed.

[0200]

The suitability of the compounds of the present invention according to one embodiment as an active drug ingredient can be confirmed by performing, for example, an eye irritation test. Examples of the eye irritation test include a single instillation test method using rabbit eyes, monkey eyes and the like (single instillation), a short continuous instillation test method (several instillations in a short period of time at equal intervals), a repeated test method instillation (repeated intermittent instillation for several days to 10 days) and the like, and there is a method for assessing symptoms of eye irritation for a certain period of time after instillation according to the improved Draize scale (Fukui, N. et al., Gendai no Rinsho, 4 (7), pp. 277-289, 1970) and the like. By ascertaining eye irritation by a compound using one or more of these methods, the suitability of the compound as an active ingredient in a drug can be confirmed.

[0201]

The suitability of the compounds of the present invention according to one embodiment as an active drug ingredient can be confirmed by performing, for example, a pharmacological test for cardiovascular safety. Examples of a cardiovascular safety pharmacological test include a telemetric method (a method for measuring the effect of administering a compound without anesthesia on an electrocardiogram, heart rate, blood pressure, blood flow, and the like (Electrocardiogram, Echocardiography, Blood Pressure and Pathological Tests of Animals for Fundamental and Clinical Medicine, edited by Sugano S., Tsubone H., Nakada Y., published in 2003, Maruzen), APD method (a method for measuring the action potential retention time of cardiac muscle cells (Muraki, K. et al., AM. J. Physiol., 269 , H524-532, 1995; Ducic, I. et al., J. Cardiovasc. Pharmacol., 30 (1), pp. 42-54, 1997)), hERG inhibition assessment method (patch clamp method (Chachin, M et al., Nippon Yakurigaku Zasshi, 119, pp. 345-351, 2002), binding assay method (Gilbert, JD et al., J. Pharm. Tox. Methods, 50, pp. 187-199, 2004 ), a Rb ⁺ outflow analysis method (Cheng, CS et al., Drug Develop. Indust. Phar m., 28, pp. 177-191, 2002), membrane potential analysis method (Dorn, A. et al., J. Biomol. Screen., 10, pp. 339-347, 2005) and the like. By ascertaining the cardiovascular effects of a compound using one or more of these methods, the suitability of the compound as an active ingredient in a drug can be confirmed.

[0202]

The suitability of the compounds of the present invention according to one embodiment as an active ingredient in a drug can be confirmed by performing, for example, a pharmacological test for central nervous system safety. Examples of a pharmacological safety test for the central nervous system include the FOB method (Battery of Standard Tests, Mattson, J.L. et al., J. American College of Technology, 15 (3), pp. 239-254, 1996)), a modified version of the Irvine method (a method for assessing the observation of general symptoms and behavior (Irwin, S., Comprehensive Observational Assessment (Berl.) 13, pp. 222-257, 1968)) and the like. By ascertaining the central nervous system activity of a compound using one or more of these methods, the suitability of the compound as an active ingredient in a drug can be confirmed.

[0203]

The suitability of the compounds of the present invention according to one embodiment as an active drug ingredient can be confirmed by performing, for example, a pharmacological respiratory safety test. Examples of a pharmacological safety test for the respiratory system include a measurement method using a respiratory function measuring apparatus (method for measuring respiratory rate, lung ventilation unit volume, minute ventilation, and the like, Drorbaugh, J. E. et al., Pediatrics, 16, pp. 81-87 , 1955; Epstein, M.A. et al., Respir. Physiol., 32, pp. 105-120, 1978), measurement method using a blood gas analyzer (method for measuring blood gases, hemoglobin oxygen saturation, and the like, Matsuo, S. , Medicina, 40, pp. 188-, 2003) and the like. By ascertaining the effect on the respiratory system of a compound using one or more of these methods, the suitability of the compound as an active ingredient in a drug can be confirmed.

[0204]

The suitability of the compounds of the present invention according to one embodiment as an active drug ingredient can be confirmed by performing, for example, a general toxicity test. The general toxicity test is a method of oral or intravenous administration of a compound, dissolved or suspended in a suitable vehicle, either single or repeated (over several days) to rodents such as rats and mice or non-rodent species such as monkeys and dogs, and evaluation based on observation of the general condition, clinical-chemical changes, histopathological changes and the like in the animal to which the administration is carried out. By ascertaining the overall toxicity of a compound using these methods, the suitability of the compound as an active ingredient in a drug can be confirmed.

[0205]

The suitability of the compounds of the present invention according to one embodiment as an active drug ingredient can be confirmed by performing, for example, a test for reproductive toxicity and embryotoxicity. The Reproductive Toxicity and Embryotoxicity Test is a test to investigate the effect of a compound's deleterious effects on reproduction and development using rodents such as rats and mice, or non-rodent species such as monkeys and dogs (link to “Drug Nonclinical Test Guideline”). Commentary 2002”, Yakuji Nippo, published on 2002, 1-6: test for reproductive toxicity and embryotoxicity and the like). Examples of a test for reproductive toxicity and embryotoxicity include tests for fertility and early embryogenesis up to nidation, tests for development and maternal functions before and after birth, tests for embryogenesis and fetal development (reference to IYAKUSHIN No. 1834, 2000, Appendix, “Guideline for Drug Toxicity Test”, [3] test for reproductive and embryotoxicity and the like) and the like. By elucidating the reproductive and embryotoxicity of a compound using these methods, the suitability of the compound as an active ingredient in a drug can be confirmed.

[0206]

The suitability of the compounds of the present invention according to one embodiment as an active drug ingredient can be confirmed by performing, for example, the cytochrome P450 enzyme induction and inhibition test (Gomez-Lechon, M.J. et al., Curr. Drug Metab., 5 (5), page 443-462, 2004). Examples of the cytochrome P450 enzyme induction and inhibition test include, for example, an in vitro method for determining whether compounds inhibit cytochrome P450 enzyme activity using cytochrome P450 enzyme of each molecular species purified from cells or obtained using genetic recombination or human P450 microsomal expression system (Miller, V.P. et al., Ann. N.Y. Acad. Sci., 919, pp. 26-32, 2000), a method for measuring changes in cytochrome P450 enzyme expression of each molecular species or enzymatic activity using human liver microsomes or disrupted cell suspension ( Hengstler, J. G. et al., Drug Metab. Rev., 32, pp. 81-118, 2000), a method for extracting PHC from human hepatocytes exposed to a compound and comparing the degree of mRNA expression with the degree to control to study the ability of the compound to induce the enzyme (Kato, M. et al., Drug Metab. Pharmacokinet., 20 (4), pp. 236-243, 2005) and the like. By ascertaining the effect of a compound on inhibition or induction of the cytochrome P450 enzyme using one or more of these methods, the suitability of the compound as an active ingredient in a drug can be confirmed.

[0207]

The suitability of the compounds of the present invention according to one embodiment as an active drug ingredient can be confirmed by performing, for example, a cell permeability test. Examples of a cell permeability test include, for example, a method for measuring cell membrane permeability for a compound in an in vitro cell culture system using CaCO-2 cells (Delie, F. et al., Crit. Rev. Ther. Drug Carrier Syst., 14, pp. 221-286, 1997; Yamashita, S. et al., Eur. J. Pham. Sci., 10, pp. 195-204, 2000; Ingels, F. M. et al., J. Pham. Sci., 92 , pp. 1545-1558, 2003), a method for measuring cell membrane permeability for junction in an in vitro cell culture system using MDCK cells (Irvine, J. D. et al., J. Pham. Sci., 88, pp. 28-33, 1999) and the like. By ascertaining the cellular permeability of a compound using one or more of these methods, the suitability of the compound as an active drug ingredient can be confirmed.

[0208]

The suitability of the compounds of the present invention according to one embodiment as an active drug ingredient can be confirmed by performing, for example, an ATPase drug transporter assay for an ATP-binding cassette (ABC) transporter. Examples of the ATPase drug transporter assay include a method for examining whether a compound is a P-glycoprotein (P-gp) substrate using a P-gp baculovirus expression system (Germann, UA, Methods Enzymol., 292, pp. 427-41, 1998) and similar. Moreover, suitability can also be tested by performing, for example, a transporter test using oocytes from African clawed frogs ( Xenopus laevis ) as the solute transporter (SLC). Transporter assays include a method for examining whether a test compound is an OATP2 substrate using OATP2 expressing oocytes (Tamai I. et al., Pharm. Res., 2001 September; 18(9), 1262-1269) and the like. By ascertaining the effect of a compound on the ABC transporter or SLC transporter, using these methods, the suitability of the compound as an active ingredient in a drug can be confirmed.

[0209]

The suitability of the compounds of the present invention according to one embodiment as an active drug ingredient can be confirmed by performing, for example, an oral absorption test. Examples of the oral absorption test include a method of orally administering a certain amount of a compound dissolved or suspended in a suitable solvent to a rodent, monkey, dog, or the like, and measuring the blood concentration of the compound after oral administration over time, evaluating the blood movement of the compound by oral administration, using LC-MS/MS method (“Newest Mass Spectrometry for Life Science”, Kodansha Scientific, 2002, edited by Harada K. et al and the like) and the like. By ascertaining the oral absorption of a compound using these methods, the suitability of the compound as an active ingredient in a drug can be confirmed.

[0210]

The suitability of the compounds of the present invention according to one embodiment as an active drug ingredient can be confirmed by performing, for example, a test to measure the change in blood concentration. Examples of the test for measuring change in blood concentration include a method of oral or parenteral (for example, intravenous, intramuscular, intraperitoneal, subcutaneous, transdermal, instillation, transnasal, and the like) administering a compound to a rodent, monkey, dog, or the like, and measuring the change in blood concentration of the compound over time after administration using the LC-MS/MS method (“Newest Mass Spectrometry for Life Science”, Kodansha Scientific, 2002, edited by Harada K. et al and the like) and the like. By ascertaining the change in blood concentration of a compound using these methods, the suitability of the compound as an active ingredient in a drug can be confirmed. In the case of, in particular, topical administration among parenteral routes of administration, in order to avoid side reactions, the compounds of the present invention according to one embodiment giving a low blood concentration after administration may be preferred.

[0211]

The suitability of the compounds of the present invention according to one embodiment as an active drug ingredient can be confirmed by performing, for example, a metabolic test. Examples of the metabolic test include a method for testing blood stability (a method for predicting in vivo metabolic clearance based on the rate of metabolism of a compound in liver microsomes of a human or other animal species (ref. to Shou, W.Z. et al., J. Mass Spectrom., 40 (10) 1347-1356, 2005; Li, C. et al., Drug Metab. Dispos., 34(6), 901-905, 2006 and the like), a method for testing for molecular metabolites, a method for testing for reactive metabolites, and the like. By ascertaining the metabolic profile of a compound using one or more of these methods, the suitability of the compound as an active ingredient in a drug can be confirmed.

[0212]

The suitability of the compounds of the present invention according to one embodiment as an active drug ingredient can be confirmed by conducting, for example, a dissolution test. As a method for evaluating water solubility, examples are confirmation of solubility under acidic conditions, neutral conditions or basic conditions, and confirmation of solubility change in the presence or absence of bile acid is also included. Examples of the dissolution test include a dissolution test based on the turbidimetric method (Lipinski, C.A. et al., Adv. Drug Deliv. Rev., 23, pp. 3-26, 1997; Bevan, C.D. et al., Anal. Chem. , 72, pp. 1781-1787, 2000) and the like. By ascertaining the solubility of a compound using these methods, the suitability of the compound as an active ingredient in a drug can be confirmed.

[0213]

The suitability of the compounds of the present invention according to one embodiment as an active ingredient of a drug can be confirmed by inspection of, for example, trauma to the upper gastrointestinal tract, renal dysfunction, and the like. As a pharmacological test for the upper gastrointestinal tract, the effect on the gastric mucosa can be investigated using the rat gastric mucosal injury model. Examples of a pharmacological test for kidney function include a method for measuring renal blood flow and glomerular filtration rate [Physiology, 18th edition, Bunkodo, 1986, chapter 17] and the like. By elucidating the effects of a compound on the upper gastrointestinal tract and kidney function using one or more of these methods, the suitability of the compound as an active drug ingredient can be confirmed.

Examples

[0214]

In the present invention, hereinafter, the present invention will be further specifically explained with reference to examples, reference examples, production examples, and test examples (hereinafter, they may be collectively referred to as "examples and the like"). However, the scope of the present invention is not limited to the following examples and the like.

[0215]

In the examples and the like, pre-coated silica gel 60 F254 (obtained by Merck, item number 5715-1M) was used for thin layer chromatography (TLC). After a run with chloroform:methanol (1:0-1:1), acetonitrile:acetic acid:water (200:1:1-100:4:4) or ethyl acetate:hexane (1:0-0:1), confirmation was performed by UV irradiation (254 nm or 365 nm), or by staining with iodine solution, aqueous potassium permanganate, phosphomolybdic acid (ethanol solution) or the like.

Anhydrous magnesium sulfate or anhydrous sodium sulfate was used to dry the organic solvent.

[0216]

For column chromatography, a Multi Prep YFLC obtained from Yamazen Corporation or a 2-ch parallel purification apparatus “Purif-α2(50F)” obtained from MORITEX Corporation was used. In the case of Multi Prep YFLC, any of Ultra Pack Si-40A, 40B and 40D obtained by Yamazen Corporation was used as a column, and in the case of Purif-α2(50F), PurifPack-Si series obtained by MORITEX Corporation was used as a column .

Silica gel 60N (spherical shape, neutral, 40-100 µm, obtained by Kanto Chemicals) was used for flash column chromatography.

[0217]

Preparative thin layer chromatography (hereinafter also referred to as “PTLC”) was performed using one or more LC Plate Silica Gel 60 F254 (20 x 20 cm, layer thickness 2 mm, including concentration zones (4 cm) obtained by Merck, product number 13793- 1M) depending on the amount of sample.

[0218]

For HPLC purification, a chromatographic preparation and purification apparatus obtained from Waters Japan, Develosil C30-UG-5 (obtained by Nomura Kagaku) or the like was used as a column, and a water-acetonitrile solvent containing 0.1% acetic acid was used in as an eluent.

[0219]

When purification was performed by HPLC, the title compound was obtained by removal of the solvent by lyophilization, unless otherwise indicated. To obtain nuclear magnetic resonance (NMR) spectra, Gemini-300 (FT-NMR, Varian Co., Ltd.) or AL-300 (FT-NMR, obtained by JEOL Co., Ltd.) was used. Deuterated chloroform was used as solvent unless otherwise noted, chemical shifts were measured using tetramethylsilane (TMS) as an internal standard and δ (ppm) was reported and binding constants were J (Hz).

[0220]

For LCMS, the mass spectrum was measured by liquid chromatography-mass spectrometry (LC-MS). Unless otherwise indicated, a single quadrupole mass spectrometer, UPLC/SQD System (obtained by Waters) was used as the mass spectrometer, and measurement was performed by the electrospray ionization (ESI) method. The liquid chromatography apparatus used was an Acquity Ultra Performance LC system obtained by Waters. ACQUITY UPLC BEH C18 (1×50 mm, 1.7 µm, obtained by Waters) was used as a separation column.

[0221]

However, for the LC FLC-1 conditions below, a single quadrupole mass spectrometer, Platform-LC (obtained by Waters) was used as a mass spectrometer, and measurement was performed by an electrospray ionization (ESI) method. As a liquid chromatography apparatus, a 306 PUMP system obtained by GILSON was used. As a separation column, Develosil C30-UG-5 (50×4.6 mm, obtained by Nomura Kagaku) was used.

[0222]

When the LC conditions are mentioned specifically in the examples and reference examples, it means that the measurement was carried out with the following solvent conditions. The m/z symbol denotes mass spectrum data (MH ⁺ , M ⁺ NH ⁴⁺ , or MH- are also indicated).

[0223]

(LC-1) The measurement was carried out under such conditions that the elution was carried out at a flow rate of 0.6 ml/minute using a linear gradient of 5-90% (v/v) solution B [acetonitrile containing 0.1% (v/v ) acetic acid] in solution A [water containing 0.1% (v/v) acetic acid] from 0 minutes to 2.0 minutes, and then a linear gradient of 90-98% solution B in solution A from 2.0 to 2.5 minutes.

[0224]

(LC-6) The measurement was carried out under such conditions that the elution was carried out at a flow rate of 0.6 ml/minute using a linear gradient of 70-90% (v/v) solution B [acetonitrile containing 0.1% (v/v) ) acetic acid] in solution A [water containing 0.1% (v/v) acetic acid] from 0 to 2.0 minutes, and then a linear gradient of 90-98% solution B in solution A from 2.0 to 2 ,5 minutes.

[0225]

(NLC-1) Measurement was carried out under such conditions that elution was performed at a flow rate of 0.6 ml/minute using a linear gradient of 5-90% (v/v) solution B [acetonitrile] in solution A [10 mM aqueous ammonium acetate ] from 0 minutes to 2.0 minutes, and then a linear gradient of 90-98% solution B in solution A from 2.0 to 2.5 minutes.

[0226]

(NLC-6) The measurement was carried out under such conditions that the elution was carried out at a flow rate of 0.6 ml/minute using a linear gradient of 70-90% (v/v) solution B [acetonitrile] in solution A [10 mM aqueous ammonium acetate ] from 0 minutes to 2.0 minutes, and then a linear gradient of 90-98% solution B in solution A from 2.0 to 2.5 minutes.

[0227]

(FLC-1) The measurement was carried out under such conditions that the elution was carried out at a flow rate of o2 ml/minute using a linear gradient of 5-98% (v/v) solution B [acetonitrile containing 0.1% (v/v) acetic acid] in solution A [water containing 0.1% (v/v) acetic acid] 0 to 5 minutes, 98% (v/v) solution B in solution A 5 to 6 minutes, linear gradient 98-5 % (v / v) solution B in solution A from 6 to 6.01 minutes, and 5% (v / v) solution B in solution A from 6.01 to 7.5 minutes.

[0228]

For chiral LC, the retention time was measured by high performance liquid chromatography (HPLC). When the chiral LC conditions are specifically mentioned in the Examples and Reference Examples, it means that the measurement was carried out with the following measurement conditions.

[0229]

(Chiral LC-1) Measurement was performed using CHIRALCEL OD-H (4.6×250 mm, 5, obtained by Daicel Corporation) as a separation column under such conditions that elution was performed at a flow rate of 0.6 ml/minute in as isocratic elution using 5% (v/v) solution B (ethanol) in solution A (n-hexane).

[0230]

(Chiral LC-2) The measurement was performed using CHIRALCEL OJ-H (4.6×250 mm, 5 µm, obtained by Daicel Corporation) as a separation column under such conditions that elution was performed at a flow rate of 1.0 ml/minute using ethanol containing 0.1% (v/v) trifluoroacetic acid.

[0231]

The manufacturers of the reagents used can sometimes be identified with the following abbreviations: Tokyo Chemical Industry Co., Ltd., TCI; Sigma Aldrich Co. LLC., ALDRICH; Kanto Kagaku Co., Inc., KANTO; Wako Pure Chemical Industries Ltd., WAKO; Maybridge Co., Ltd., MAYBRIDGE; APOLLO Co., Ltd., APOLLO; Combi-Blocks Inc., COMBI-BLOCKS; Takasago International Corporation, TAKASAGO; Johnson Matthey Co., Ltd., JOHNSON; Nippon Chemical Industrial Co., Ltd., Nippon Chemical; and Japan EnviroChemicals, Limited, Japan EnviroChemicals.

[0232]

Abbreviations or symbols used in the description have the following meanings: n, normal; i, iso; s, secondary; t, tertiary; c, cyclo; Me, methyl; Et, ethyl; Pr, propyl; Bu, butyl; Pen, pentyl; Hex, hexyl; Hep, heptyl; Ph, phenyl; Bn, benzyl; Py, pyridyl; Ac, acetyl; CHO, formyl; COOH, carboxyl; NO ₂ , nitro; DMA, dimethylamino; NH ₂ , amino; CF ₃ , trifluoromethyl; F, fluorine; Cl, chlorine; Br, bromine; OMe, methoxy; OH, hydroxy; TFA, trifluoroacetyl; SO ₂ sulfonyl; CO, carbonyl; THF, tetrahydrofuran; DMF, N,N-dimethylformamide; DMSO, dimethyl sulfoxide; and DME, dimethoxyethane.

[0233]

Numbers before substituents represent positions of substitution. The numbers given before the hyphenated aromatic ring abbreviations indicate substitution positions on the aromatic rings. The symbol (S) mentioned in compound names and structural formulas indicates that the corresponding asymmetric carbon is in the S-configuration, and (R) indicates that the corresponding asymmetric carbon is in the R-configuration. In addition, when (R) or (S) is not indicated for a compound containing an asymmetric carbon, this means that the compound consists of a mixture of the (R) isomer and the (S) isomer in an arbitrary ratio. Such a compound may be a racemic mixture of the (R)-isomer and the (S)-isomer.

[0234]

When deblocking is required from the process for preparing the compounds of examples, it is carried out according to known methods, such as those described in Protective groups in Organic Synthesis published by John Wiley and Sons (2007).

[0235]

Reference Example A-2: tert-Butyldimethyl(2-(thiophen-2-yl)ethoxy)silane (Intermediate A-2)

[formula 76]

To a solution of 2-(thiophen-2-yl)ethanol (4 g, TCI) in N,N-dimethylformamide (312 ml) were added successively imidazole (4.3 g), tert-butyldimethylchlorosilane (7.05 g) and N, N-dimethyl-4-aminopyridine (763 mg) under ice-cooling, and the mixture was stirred at room temperature for 2.75 hours. Ethyl acetate was added to the reaction mixture, and the organic layer was washed successively with 1 mol/L hydrochloric acid, saturated brine, saturated aqueous sodium hydrogen carbonate, and saturated brine, and then dried. The solvent was evaporated under reduced pressure to give the title compound (7.32 g).

(intermediate A-2: Rf (TLC)=0.70 (hexane:ethyl acetate=4:1))

[0236]

Reference A-3: 5-(2-((tert-butyldimethylsilyl)oxy)ethyl)thiophene-2-carboxylic acid (Intermediate A-3)

[formula 77]

A solution of intermediate A-2 (7.15 g) in tetrahydrofuran (111 ml) was cooled to -78° C. under nitrogen. n-Butyl lithium (2.6 mol/l solution in hexane, 14.3 ml, KANTO) was added dropwise to the reaction mixture, and the mixture was stirred for 0.75 hours as is. The reaction mixture was heated to -5° C., then dry ice (125 g) was added in portions, and after completion of the addition, the reaction mixture was further stirred for 0.75 hours. Saturated aqueous ammonium chloride was added to the reaction mixture, and the mixture was stirred at room temperature. Ethyl acetate was added to the reaction mixture for extraction, and the organic layer was washed successively with saturated aqueous ammonium chloride and saturated brine, and then dried. The solvent was evaporated under reduced pressure to give the title compound (9.03 g).

(intermediate A-3: LCMS m/z 287.0 (MH ⁺ ), retention time 1.35 minutes, LC conditions NLC-1)

[0237]

Reference A-4: Methyl 5-(2-hydroxyethyl)thiophene-2-carboxylate (Intermediate A-4)

[formula 78]

A solution of Intermediate A-3 (9.03 g) in methanol (64 ml) was cooled to 0°C, concentrated sulfuric acid (3.2 ml) was added portionwise to the solution, and the mixture was stirred as is for 5 minutes. The reaction mixture was heated to 70° C., stirred for 24 hours, and then cooled to 0° C., and saturated aqueous sodium hydrogen carbonate was added in portions until the reaction mixture became neutral. Ethyl acetate was added to the extraction reaction mixture, and the organic layer was washed successively with saturated aqueous sodium hydrogen carbonate and saturated brine, and then dried. The solvent was evaporated under reduced pressure, and the residue was purified by column chromatography (hexane/ethyl acetate) to give the title compound (4.61 g).

(intermediate A-4: Rf (TLC)=0.33 (hexane:ethyl acetate=1:1))

[0238]

Reference Example A-5: Methyl 5-(2-bromoethyl)thiophene-2-carboxylate (Intermediate A-5)

[formula 79]

To a solution of intermediate A-4 (4.61 g) in dichloromethane (200 ml) was added triphenylphosphine (9.8 g) and then the mixture was cooled to 0°C. Carbon tetrabromide (12.3 g) was added portionwise to the reaction mixture, and the mixture was warmed to room temperature, and then stirred for 13.5 hours. The reaction mixture was subjected to decompression by evaporating the solvent, and then the residue was purified by column chromatography (hexane/ethyl acetate) to give the title compound (5.4 g).

(intermediate A-5: Rf (TLC)=0.70 (hexane:ethyl acetate=1:1))

[0239]

Reference Example A-6 tert-Butyl 2-(2-(5-(methoxycarbonyl)thiophen-2-yl)ethyl)hydrazinecarboxylate (Intermediate A-6)

[formula 80]

To a solution of Intermediate A-5 (6.2 g) in acetonitrile (125 ml) was added tert-butylcarbazate (16.5 g, TCI), sodium hydrogen carbonate (10.5 g) and sodium iodide (700 mg) in succession, and the mixture was stirred at 90°C for 13 hours. The reaction mixture was cooled to 0°C, ethyl acetate (155 ml) was added to the reaction mixture, and the organic layer was washed successively with 1 mol/l hydrochloric acid, saturated aqueous sodium hydrogen carbonate, and saturated brine, and then dried. The solvent was evaporated under reduced pressure, and the residue was purified by column chromatography (hexane/ethyl acetate) to give the title compound (5.1 g).

(intermediate A-6: LCMS m/z 301.1 (MH ⁺ ), retention time 1.42 minutes, LC conditions NLC-1)

¹ H-NMR (CDCl ₃ ): δ (ppm) 7.64 (1H, d, J=4.0Hz), 6.87 (1H, d, J=4.0Hz), 3.86 (3H, s ), 3.18 (2H, t, J=7.2Hz), 3.02 (2H, t, J=7.2Hz), 2.60-1.90 (2H, br), 1.64 (9H , With)

[0240]

Reference B-3: S-(2-chloroethyl)carbochloridethioate (Intermediate B-3)

[formula 81]

A mixed solution of ethylene sulfide (320 g, TCI) and pyridine (4.3 ml) was cooled in an ice bath under argon atmosphere, triphosgene (474 g, TCI) was added portionwise to the reaction mixture, and the mixture was stirred as is for 4 hours. The reaction mixture was distilled under reduced pressure (0.7-0.8 kPa, 50-52° C.) for purification to give the title compound (281 g).

¹ H-NMR (CDCl ₃ ): δ (ppm) 3.72 (2H, t, J=7.0Hz), 3.30 (2H, t, J=7.0Hz)

[0241]

Reference Example Z-1: tert-Butyl 2-(((2-chloroethyl)thio)carbonyl)-2-(2-(5-(methoxycarbonyl)thiophen-2-yl)ethyl)hydrazinecarboxylate (Intermediate Z-1)

[formula 82]

To a solution of Intermediate A-6 (140.3 g) in dichloromethane (660 ml) was added water (330 ml) and sodium hydrogen carbonate (78.09 g), the mixture was stirred for 10 minutes, and then Intermediate B-3 ( 81.71 g) was added portionwise to the reaction mixture while the temperature inside the reaction mixture flask was maintained at 20-25°C. The mixture was stirred as is for 1 hour, and then the organic layer was washed with saturated brine and dried. The solvent was evaporated under reduced pressure to give the title compound (199.5 g).

(intermediate Z-1: Rf (TLC)=0.43 (hexane:ethyl acetate=2:1))

[0242]

Reference Example Z-2: Methyl 5-(2-(1-(((2-chloroethyl)thio)carbonyl)hydrazinyl)ethyl)thiophene-2-carboxylate (Intermediate Z-2)

[formula 83]

To intermediate Z-1 (199 g) was added a 4 mol/l solution of hydrochloric acid in dioxane (800 ml) and the mixture was stirred at room temperature for 18 hours. The solvent was evaporated under reduced pressure, and then dichloromethane (6 L) and saturated aqueous sodium hydrogen carbonate (2 L) were added to the residue for extraction. The organic layer was washed with saturated brine (2 L) and dried, and then the solvent was evaporated under reduced pressure to give the title compound (172 g).

(intermediate A-4: Rf (TLC)=0.49 (heptane:ethyl acetate=1:1))

[0243]

Reference Example Z-3: Methyl 5-(2-(2-oxo-1,3,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylate (Intermediate Z-3)

[formula 84]

To a solution of intermediate Z-2 (172 g) in acetonitrile (3.4 L) were added sodium hydrogen carbonate (223 g) and sodium iodide (397 g) in succession, and the mixture was stirred at 75° C. for 15 hours. The mixture was further stirred at 83°C for 15 hours and then cooled to room temperature. The reaction mixture was filtered using filter paper, and the residue remaining on the filter paper was washed with acetonitrile (1 L), mixed with the filtrate, and concentrated under reduced pressure. Dichloromethane (3 L) was added to the residue obtained after concentration, and then the mixture was filtered using filter paper, the residue remaining on the filter paper was washed with dichloromethane (1 L), mixed with the filtrate, and concentrated under reduced pressure. The resulting residue was purified by column chromatography (hexane/ethyl acetate), and then concentrated under reduced pressure. The residue was dissolved in ethyl acetate (1 L) by heating, then heptane was added to the solution and the mixture was cooled on ice. The precipitate that formed was collected by filtration to give the title compound (97 g).

¹ H-NMR (CDCl ₃ ): δ (ppm) 7.64 (1H, d, J=3.8Hz), 6.88 (1H, d, J=3.8Hz), 3.86 (3H, s ), 3.85 (2H, t, 7.0Hz), 3.30 (2H, t, J=7.0Hz), 3.25-3.17 (4H, m), 3.16 (2H, t , J=7.0 Hz)

[0244]

Reference Example C-2: 2-(3-Bromophenyl)-N-methoxy-N-methylacetamide (Intermediate C-2)

[formula 85]

A solution of diisopropylethylamine (800 ml) in dichloromethane (1.8 L) was cooled on ice, 3-bromophenylacetic acid (313 g, TCI), N,O-dimethylhydroxylamine hydrochloride (284 g), 1-ethyl-3-( 3-dimethylaminopropyl)carbodiimide (334 g) and N,N-dimethyl-4-aminopyridine (18 g), and the mixture was stirred at room temperature for 12.5 hours. Water (630 ml) and dichloromethane (630 ml) were added to the reaction mixture, and then the organic layer was successively washed twice with 2 mol/l hydrochloric acid (630 ml), and once each with saturated aqueous sodium hydrogen carbonate (630 ml) and saturated brine (630 ml). The organic layer was dried, and then the solvent was evaporated under reduced pressure to give the title compound (372 g).

(intermediate C-2: LCMS m/z 257.9 (MH ⁺ ), retention time 1.37 minutes, LC conditions NLC-1)

¹ H-NMR (CDCl ₃ ): δ (ppm) 7.46-7.44 (1H, m), 7.39-7.36 (1H, m), 7.25-7.15 (2H, m ), 3.74 (2H, s), 3.64 (3H, s), 3.20 (3H, s)

[0245]

Reference Example C-3: 1-(3-Bromophenyl)but-3-en-2-one (Intermediate C-3)

[formula 86]

A solution of Intermediate C-2 (104.2 g) in tetrahydrofuran (2.1 L) was cooled to -45° C. under nitrogen. Vinyl magnesium bromide (1 mol/l solution in tetrahydrofuran, 605 ml, Aldrich) was added to the reaction mixture over 30 minutes, and the mixture was heated to 0° C., and then stirred for 1.5 hours. The reaction mixture was added to a mixture of ice water (1 L) and 2 mol/L hydrochloric acid (1 L) and the mixture was stirred for 1 minute. Isopropyl ether (2 L) was added for extraction, and the organic layer was washed successively with 1 mol/L hydrochloric acid (1 L), water (1 L) and brine (1 L) and dried. The solvent was evaporated under reduced pressure to give the title compound (92.1 g).

(intermediate C-3: Rf (TLC)=0.74 (heptane:ethyl acetate=2:1))

[0246]

Reference Example C-2-2: 2-(3-Iodophenyl)-N-methoxy-N-methylacetamide (Intermediate C-2-2)

[formula 87]

Intermediate C-2-2 was prepared according to the method described in Reference Example C-2 using 3-iodophenylacetic acid (2.85 g) instead of 3-bromophenylacetic acid, and thus the title compound (3.07 g) was obtained .

(intermediate C-2-2: Rf (TLC)=0.42 (hexane:ethyl acetate=1:2))

When the compound was obtained according to the method described above, the amount of reagents, the amount of solvent, the reaction time and the like can be appropriately changed depending on the equivalent amount of the starting compound to be used in light of the prior art to those skilled in the art. The same applies to the following examples.

[0247]

Reference Example C-3-2: 1-(3-Iodophenyl)but-3-en-2-one (Intermediate C-3-2)

[formula 88]

Intermediate C-3-2 was prepared according to the method described in Reference Example C-3 using Intermediate C-2-2 (100 mg) instead of Intermediate C-2, and thus the title compound (58.7 mg).

(intermediate C-3-2: Rf (TLC)=0.60 (hexane:ethyl acetate=1:2))

[0248]

Reference Example Z-4: Methyl 5-(2-(4-(4-(3-bromophenyl)-3-oxobutyl)-2-oxo-1,3,4-thiadiazinan-3-yl)ethyl)thiophene-2 -carboxylate (intermediate Z-4)

[formula 89]

To a solution of Intermediate Z-3 (44.4 g) in ethanol (444 ml) was added Intermediate C-3 (92.1 g) and the mixture was stirred at 110° C. for 40 hours. The reaction mixture was subjected to decompression, evaporating the solvent. The resulting residue was purified by column chromatography (toluene/ethyl acetate) to give the title compound (75.9 g).

(intermediate Z-4: LCMS m/z 511.2 (MH ⁺ ), retention time 1.75 minutes, LC conditions NLC-1)

[0249]

Reference Example Z-4-2: Methyl 5-(2-(4-(4-(3-iodophenyl)-3-oxobutyl)-2-oxo-1,3,4-thiadiazinan-3-yl)ethyl)thiophene -2-carboxylate (intermediate Z-4-2)

[formula 90]

Intermediate Z-4-2 was prepared according to the method described in Reference Example Z-4 using Intermediate C-3-2 (680.2 mg) instead of Intermediate C-3, and thus the title compound (120 .1 mg).

(intermediate Z-4-2: Rf (TLC)=0.50 (hexane:ethyl acetate=1:2), LCMS m/z 559.0 (MH ⁺ ), retention time 1.84 minutes, LC conditions LC- one)

[0250]

Reference Example Z-5: Methyl 5-(2-(4-(4-(3-bromophenyl)-3-hydroxybutyl)-2-oxo-1,3,4-thiadiazinan-3-yl)ethyl)thiophene-2 -carboxylate (intermediate Z-5)

[formula 91]

A solution of intermediate Z-4 (75.7 g) in methanol (1.14 L) was cooled to 0° C. and sodium borohydride (7.47 g) was added portionwise to the solution. The mixture was stirred at 0° C. for 1 hour, and then dilute hydrochloric acid was added portionwise to the reaction mixture until the reaction mixture became neutral. The organic solvent was evaporated under reduced pressure, then ethyl acetate (2 L) was added to the residue, the mixture was cooled to 0°C, saturated aqueous sodium hydrogen carbonate (1 L) was added to the mixture in portions, and the resulting mixture was stirred for 5 minutes. The organic layer was extracted and dried, and then the solvent was evaporated under reduced pressure to give the title compound (71.0 g).

(intermediate Z-5: LCMS m/z 513.15 (MH ⁺ ), retention time 1.70 minutes, LC conditions LC-1)

[0251]

Reference Example Z-14: Methyl 5-(2-(2-oxo-4-(3-oxo-4-(3-(thiophen-3-ylethynyl)phenyl)butyl)-1,3,4-thiadiazinan-3 -yl)ethyl)thiophene-2-carboxylate (intermediate Z-14)

[formula 92]

Diethylamine (78 µl), 3-ethynylthiophene (21 µl), copper(I) iodide (0.8 mg), and tetrakis(triphenylphosphine)palladium (1, 1 mg), and the mixture was stirred at room temperature for 2 hours. In addition, diethylamine (600 µl), copper(I) iodide (1.5 mg), and tetrakis(triphenylphosphine)palladium (2.0 mg) were added successively, and the mixture was stirred at room temperature for 12 hours. Diethyl ether and 1 mol/l hydrochloric acid (0.5 ml) were added to the reaction mixture, and the organic layer was successively washed 5 times with 1 mol/l hydrochloric acid (1 ml), and once with saturated aqueous sodium hydrogen carbonate (0.5 ml ) and dried. The solvent was evaporated under reduced pressure, and the resulting residue was purified by column chromatography (hexane/ethyl acetate) to give the title compound (31.7 mg).

(intermediate Z-14: Rf (TLC)=0.12 (hexane:ethyl acetate=1:2), LCMS m/z 539.1 (MH ⁺ ), retention time 1.95 minutes, LC conditions LC-1)

[0252]

Reference Example Z-17: Methyl 5-(2-(4-(3-hydroxy-4-(3-(thiophen-3-ylethynyl)phenyl)butyl)-2-oxo-1,3,4-thiadiazinan-3 -yl)ethyl)thiophene-2-carboxylate (intermediate Z-17)

[formula 93]

Intermediate Z-17 was prepared according to the method described in Reference Example Z-5 using Intermediate Z-14 (31.7 mg) instead of Intermediate Z-4, thus obtaining the title compound (31.8 mg) ,.

(intermediate Z-17: LCMS m/z 541.1 (MH ⁺ ), retention time 1.90 minutes, LC conditions LC-1)

[0253]

Example 1: 5-(2-(4-(3-Hydroxy-4-(3-(thiophen-3-ylethynyl)phenyl)butyl)-2-oxo-1,3,4-thiadiazinan-3-yl)ethyl )thiophene-2-carboxylic acid

[formula 94]

To a solution of intermediate Z-17 (31.8 mg) in tetrahydrofuran (884 µl) was added water (221 µl) and 2 mol/l aqueous lithium hydroxide (442 µl) and the mixture was stirred at 50°C for 17.5 hours. The reaction mixture was cooled to 0°C, 2 mol/L hydrochloric acid (660 µl) was added to the mixture, and then the resulting mixture was extracted with ethyl acetate. The solvent was evaporated under reduced pressure, and the resulting residue was purified by thin layer chromatography (hexane/ethyl acetate) to give the title compound (22.7 mg).

(LCMS m/z 527.2 (MH ⁺ ), retention time 1.68 minutes, LC conditions LC-1)

[0254]

Reference Example A-10: (3-Bromothiophen-2-yl)methanol (Intermediate A-10)

[formula 95]

A solution of 3-bromothiophene-2-carboxylic acid (3.0 g, Aldrich) in tetrahydrofuran (46 ml) was cooled to 0°C under nitrogen gas, a 1 mol/l solution of borane-tetrahydrofuran was added dropwise to the solution over 15 minutes. complex in tetrahydrofuran (26.1 ml), and then the mixture was stirred at room temperature for 21.5 hours. The reaction mixture was cooled to 0°C, ice water, 1 mol/l hydrochloric acid and ethyl acetate were added to the mixture, and the resulting mixture was stirred. The organic solvent was evaporated under reduced pressure, then ethyl acetate was added to the residue, and the organic layer was washed successively with 1 mol/L hydrochloric acid, saturated aqueous sodium hydrogen carbonate, and saturated brine, and dried. The solvent was evaporated under reduced pressure to give the title compound (2.87 g).

(Intermediate A-10: Rf (TLC)=0.42 (hexane:ethyl acetate=2:1))

[0255]

Reference Example A-11: 3-Bromo-2-(bromomethyl)thiophene (Intermediate A-11)

[formula 96]

A solution of intermediate A-10 (7.14 g) in dichloromethane (169 ml) was cooled to 0°C, triphenylphosphine (13.3 g) and carbon tetrabromide (13.45 g) were added to the solution, and the mixture was stirred at room temperature within 2.75 hours. Saturated aqueous sodium hydrogencarbonate was added to the reaction mixture, and then the organic solvent was evaporated under reduced pressure. Ethyl acetate was added to the residue, and then the organic layer was washed successively with saturated aqueous sodium hydrogen carbonate and saturated brine, and dried. The organic solvent was evaporated under reduced pressure, then a mixed solvent of hexane and ethyl acetate (8:1) was added to the resulting residue to obtain a slurry, and the slurry was filtered through silica gel-coated filter paper. The solvent of the filtrate was evaporated under reduced pressure to give the title compound (9.70 g).

(Intermediate A-11: Rf (TLC)=0.64 (hexane:ethyl acetate=8:1))

[0256]

Reference A-12: 2-(3-Bromothiophen-2-yl)acetonitrile (Intermediate A-12)

[formula 97]

Dimethyl sulfoxide (28 ml) and acetonitrile (140 ml) were added to Intermediate A-11 (9.70 g), the mixture was cooled to 0° C., then sodium cyanide (2.15 g) was added to the mixture, and the resulting mixture stirred at room temperature for 16 hours. Saturated aqueous sodium hydrogencarbonate was added to the reaction mixture, and the mixture was stirred and then concentrated under reduced pressure. The reaction mixture was filtered through celite-coated filter paper and the residue remaining on the celite was washed with ethyl acetate. The filtrate and washing liquid were mixed, and the organic layer was washed successively with saturated aqueous sodium hydrogen carbonate and saturated brine, and dried. The organic solvent was evaporated under reduced pressure, and then the resulting residue was purified by column chromatography (hexane/ethyl acetate) to give the title compound (3.89 g).

(Intermediate A-12: Rf (TLC)=0.18 (hexane:ethyl acetate=8:1))

[0257]

Reference Example A-13: Ethyl 2-(3-bromothiophen-2-yl)acetate (Intermediate A-13)

[formula 98]

Water (0.4 ml) was added to a solution of Intermediate A-12 (3.89 g) in ethanol (32.3 ml), the mixture was cooled to 0°C, and then concentrated sulfuric acid (5.63 ml). The reaction mixture was stirred at 85° C. for 115 hours and then cooled to 0° C. and saturated aqueous sodium hydrogen carbonate was added until the reaction mixture became neutral. Ethyl acetate was added to the reaction mixture, and the resulting mixture was stirred and then concentrated under reduced pressure. Ethyl acetate was added to the reaction mixture, and the organic layer was washed successively with saturated aqueous sodium hydrogen carbonate and saturated brine, and dried. The solvent was evaporated under reduced pressure to give the title compound (4.40 g).

(Intermediate A-13: Rf (TLC)=0.33 (hexane:ethyl acetate=8:1))

[0258]

Reference Example A-14: 2-(3-Bromothiophen-2-yl)ethanol (Intermediate A-14)

[formula 99]

A solution of Intermediate A-13 (4.40 g) in tetrahydrofuran (88.5 ml) was cooled to 0° C. under nitrogen gas, lithium aluminum hydride (672 mg) was added to the solution, and the mixture was stirred for 0.6 hour. To the reaction mixture, ice water, 1 mol/L hydrochloric acid, and ethyl acetate were added, and the resulting mixture was stirred, and then the organic layer was washed successively with 1 mol/L hydrochloric acid, saturated aqueous sodium hydrogen carbonate, and saturated brine, and dried. The solvent was evaporated under reduced pressure, and the resulting residue was purified by column chromatography (hexane/ethyl acetate) to give the title compound (2.69 g).

(Intermediate A-14: Rf (TLC)=0.23 (hexane:ethyl acetate=4:1))

[0259]

Reference Example A-2-2: (2-(3-Bromothiophen-2-yl)ethoxy)(tert-butyl)dimethylsilane (Intermediate A-2-2)

[formula 100]

Intermediate A-2-2 was prepared according to the method described in Reference Example A-2 using Intermediate A-14 (2.69 g) in place of 2-(thiophen-2-yl)ethanol, and thus the title claimed compound (3.93 g).

(intermediate A-2-2: Rf (TLC)=0.76 (hexane:ethyl acetate=4:1))

[0260]

Reference Example A-3-2: 4-Bromo-5-(2-((tert-butyldimethylsilyl)oxy)ethyl)thiophene-2-carboxylic acid (Intermediate A-3-2)

[formula 101]

Intermediate A-3-2 was prepared according to the method described in Reference Example A-3 using Intermediate A-2-2 (293 mg) in place of Intermediate A-2 and lithium diisopropylamide (1.09 mol/l solution in hexane/tetrahydrofuran, 928 μl, KANTO) instead of n-butyllithium (2.6 mol/l solution in hexane, KANTO), and thus the title compound (339 mg) was obtained.

(intermediate A-3-2: Rf (TLC)=0.12 (hexane:ethyl acetate=1:1))

[0261]

Reference Example A-4-2: Methyl 4-bromo-5-(2-hydroxyethyl)thiophene-2-carboxylate (Intermediate A-4-2)

[formula 102]

Intermediate A-4-2 was prepared according to the method described in Reference Example A-4 using Intermediate A-3-2 (377 mg) instead of Intermediate A-3, and thus the title compound (217 mg) was obtained .

(intermediate A-4-2: Rf (TLC)=0.53 (hexane:ethyl acetate=1:1))

[0262]

Reference Example A-5-2: Methyl 4-bromo-5-(2-bromoethyl)thiophene-2-carboxylate (Intermediate A-5-2)

[formula 103]

Intermediate A-5-2 was prepared according to the method described in Reference Example A-5 using Intermediate A-4-2 (217 mg) instead of Intermediate A-4, and thus the title compound (315 mg) was obtained .

(intermediate A-5-2: Rf (TLC)=0.44 (hexane:ethyl acetate=8:1))

[0263]

Reference Example A-6-2: tert-Butyl 2-(2-(3-bromo-5-(methoxycarbonyl)thiophen-2-yl)ethyl)hydrazinecarboxylate (Intermediate A-6-2)

[formula 104]

Intermediate A-6-2 was prepared according to the method described in Reference Example A-6 using Intermediate A-5-2 (315 mg) instead of Intermediate A-5, thus obtaining the title compound (168 mg) .

(intermediate A-5-2: Rf (TLC)=0.48 (hexane:ethyl acetate=1:1))

[0264]

Reference Example Z-1-2: tert-butyl 2-(2-(3-bromo-5-(methoxycarbonyl)thiophen-2-yl)ethyl)-2-(((2-chloroethyl)thio)carbonyl)hydrazinecarboxylate ( intermediate connection Z-1-2)

[formula 105]

To a solution of intermediate A-6-2 (1.98 g) in dichloromethane (13.1 ml) was added sodium hydrogen carbonate (880 mg), the mixture was cooled to 0°C, and then intermediate B-3 ( 993 mg). The resulting mixture was stirred at room temperature for 0.5 hour, then water and ethyl acetate were added to the reaction mixture, and the organic layer was washed successively with saturated aqueous sodium hydrogen carbonate and saturated brine, and dried. The solvent was evaporated under reduced pressure to give the title compound (796 mg).

(intermediate Z-1-2: Rf (TLC)=0.53 (toluene:ethyl acetate=8:1))

[0265]

Reference Example Z-2-2: Methyl 4-bromo-5-(2-(1-(((2-chloroethyl)thio)carbonyl)hydrazinyl)ethyl)thiophene-2-carboxylate (intermediate Z-2-2)

[formula 106]

To intermediate Z-1-2 (796 mg) was added a 4 mol/l solution of hydrochloric acid in dioxane (7.5 ml) and the mixture was stirred at room temperature for 17.6 hours. Ethyl acetate and 5 mol/L aqueous sodium hydroxide were added to the reaction mixture, and then saturated aqueous sodium hydrogen carbonate was added to the mixture until the mixture became basic. The organic layer was washed successively with saturated aqueous sodium hydrogen carbonate and saturated brine, and dried, and then the solvent was evaporated under reduced pressure to give the title compound (594 mg).

(intermediate Z-2-2: Rf (TLC)=0.42 (toluene:ethyl acetate=8:1))

[0266]

Reference Example Z-3-2: Methyl 4-bromo-5-(2-(2-oxo-1,3,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylate (intermediate Z-3-2 )

[formula 107]

To a solution of intermediate Z-2-2 (594 mg) in acetonitrile (14.9 ml) were added sodium bicarbonate (626 mg) and sodium iodide (1.12 g) in succession, and the mixture was stirred at 85° C. for 120 hours . The reaction mixture was cooled to room temperature, and then ethyl acetate and water were added to the mixture for extraction. The organic layer was washed successively with saturated aqueous sodium hydrogen carbonate and saturated brine, and dried, then the solvent was evaporated under reduced pressure, and the resulting residue was purified by column chromatography (hexane/ethyl acetate) to give the title compound (374 mg).

(intermediate Z-3-2: Rf (TLC)=0.13 (hexane:ethyl acetate=2:1))

[0267]

Reference Example C-4: N-Methoxy-N-methyl-2-(3-(thiophen-3-ylethynyl)phenyl)acetamide (Intermediate C-4)

[formula 108]

To a solution of intermediate C-2-2 (1.0 g) in acetonitrile (26 ml) was added successively bis(acetonitrile)palladium chloride (43 mg), 2-dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl (243 mg), cesium carbonate (2.1 g) and 3-ethynylthiophene (650 μl), and the mixture was stirred at 60° C. for 14 hours under nitrogen gas. The reaction mixture was filtered through celite-coated filter paper and the residue remaining on celite was washed with ethyl acetate. The filtrate and washing liquid were mixed, the organic solvent was evaporated under reduced pressure, and then the resulting residue was purified by column chromatography (hexane/ethyl acetate) to give the title compound (850 mg).

(intermediate C-4: Rf (TLC)=0.40 (hexane:ethyl acetate=1:1), LCMS m/z 286.13 (MH ⁺ ), retention time 1.70 minutes, LC conditions LC-1)

[0268]

Reference Example Z-14-2: Methyl 4-bromo-5-(2-(2-oxo-4-(3-oxo-4-(3-(thiophen-3-ylethynyl)phenyl)butyl)-1,3 ,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylate (intermediate Z-14-2)

[formula 109]

A solution of intermediate C-4 (117 mg) in 1,2-dimethoxyethane (2.3 ml) was cooled to 0° C. under nitrogen. Vinyl magnesium bromide (1 mol/l solution in tetrahydrofuran, 620 μl, Aldrich) was added to the reaction mixture, and the resulting mixture was stirred for 3 hours. 2 mol/L hydrochloric acid was added to the reaction mixture, and the resulting mixture was stirred for 1 minute. Ethyl acetate was added to the extraction reaction mixture, the organic layer was dried, and then the solvent was evaporated under reduced pressure. Ethanol (3 ml), water (3 ml) and Intermediate Z-3-2 (100 mg) were added to the resulting residue, and the mixture was stirred at 110° C. for 18 hours. Saturated brine and chloroform were added to the reaction mixture to extract, the organic layer was dried, and then the solvent was evaporated under reduced pressure. The resulting residue was purified by column chromatography (hexane/ethyl acetate) to give the title compound (156.1 mg).

(intermediate Z-14-2: LCMS m/z 617.2 (MH ⁺ ), retention time 2.08 minutes, LC conditions LC-1)

[0269]

Example 2: 4-Bromo-5-(2-(4-(3-hydroxy-4-(3-(thiophen-3-ylethynyl)phenyl)butyl)-2-oxo-1,3,4-thiadiazinan-3 -yl)ethyl)thiophene-2-carboxylic acid

[formula 110]

A solution of intermediate Z-14-2 (156.1 mg) in methanol (3 ml) was cooled to 0° C. and sodium borohydride (17.3 mg) was added portionwise to the solution. The resulting mixture was stirred at 0° C. for 1 hour, and then dilute hydrochloric acid was added portionwise to the mixture until the reaction mixture became neutral. The organic solvent was evaporated under reduced pressure, then ethyl acetate (2 L) was added to the residue, the mixture was cooled to 0°C, saturated aqueous sodium hydrogen carbonate (1 L) was added portionwise to the mixture, and the resulting mixture was stirred for 5 minutes. The organic layer was extracted and dried, and then the solvent was evaporated under reduced pressure. To the resulting residue were added tetrahydrofuran (4.6 ml), methanol (4.6 ml) and 1 mol/l aqueous sodium hydroxide (4.6 ml), and the mixture was stirred at room temperature for 3 hours. The reaction mixture was cooled to 0°C, 2 mol/L hydrochloric acid was added to the reaction mixture, then the resulting mixture was extracted with ethyl acetate, and the organic layer was dried. The solvent was evaporated under reduced pressure, and the resulting residue was purified by column chromatography (chloroform/methanol) to give the title compound (140 mg).

(LCMS m/z 605.1 (MH ⁺ ), retention time 1.78 minutes, LC conditions LC-1)

[0270]

Reference Example Z-14-3: Methyl 5-(2-(2-oxo-4-(3-oxo-4-(3-(thiophen-2-ylethynyl)phenyl)butyl)-1,3,4-thiadiazinan -3-yl)ethyl)thiophene-2-carboxylate (intermediate Z-14-3)

[formula 111]

Intermediate Z-14-3 was prepared according to the method described in Reference Example Z-14 using 2-ethynylthiophene (33.6 mg, MAYBRIDGE) instead of 3-ethynylthiophene, thus obtaining the title compound (32.9 mg) .

(intermediate Z-14-3: LCMS m/z 539.0 (MH ⁺ ), retention time 2.00 minutes, LC conditions LC-1)

[0271]

Example 3: 5-(2-(4-(3-Hydroxy-4-(3-(thiophen-2-ylethynyl)phenyl)butyl)-2-oxo-1,3,4-thiadiazinan-3-yl)ethyl )thiophene-2-carboxylic acid

[formula 112]

A solution of intermediate Z-14-3 (32.9 mg) in methanol (0.6 ml) was cooled to 0° C. and sodium borohydride (3.6 mg) was added portionwise to the solution. The mixture was stirred at 0° C. for 1.5 hours, and then diluted with ethyl acetate, and dilute hydrochloric acid (1.5 ml) was added to the mixture. Saturated aqueous sodium hydrogen carbonate was added to the mixture until the mixture became neutral. The resulting mixture was extracted with ethyl acetate, the organic layer was dried, and then the solvent was evaporated under reduced pressure. To the resulting residue were added tetrahydrofuran (920 μl), water (230 μl) and 2 mol/l aqueous sodium hydroxide (460 μl), and the resulting mixture was stirred at 50°C for 14 hours. The reaction mixture was cooled to 0° C. and left for 5.5 hours, then 2 mol/l hydrochloric acid was added to the mixture, the mixture was extracted with ethyl acetate, and the organic layer was dried. The solvent was evaporated under reduced pressure, and the resulting residue was purified by liquid chromatography (acetonitrile/water) to give the title compound (3.6 mg).

(LCMS m/z 527.0 (MH ⁺ ), retention time 1.79 minutes, LC conditions LC-1)

[0272]

Reference Example Z-6: Methyl 5-(2-(4-(4-(3-bromophenyl)-3-((t-butyldimethylsilyl)oxy)butyl)-2-oxo-1,3,4-thiadiazinan-3 -yl)ethyl)thiophene-2-carboxylate (intermediate Z-6)

[formula 113]

To a solution of intermediate Z-5 (1.0 g) in N,N-dimethylformamide (19.5 ml) was added imidazole (265 mg) and tert-butyldimethylchlorosilane (596 mg) and the resulting mixture was stirred at 30°C within 15 hours. Saturated aqueous sodium hydrogencarbonate was added to the reaction mixture, and the resulting mixture was extracted with ethyl acetate. The organic layer was washed with saturated brine and then dried, the solvent was evaporated under reduced pressure, and the resulting residue was purified by column chromatography (hexane/ethyl acetate) to give the title compound (1.16 g).

(intermediate Z-6: LCMS m/z 627.0 (MH ⁺ ), retention time 2.53 minutes, LC conditions LC-1)

[0273]

Reference Example Z-21: Methyl 5-(2-(4-(3-((tert-butyldimethylsilyl)oxy)-4-(3-((trimethylsilyl)ethynyl)phenyl)butyl)-2-oxo-1,3 ,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylate (intermediate Z-21)

[formula 114]

To a solution of intermediate Z-6 (300 mg) in acetonitrile (15.3 ml) was added successively bis(acetonitrile)palladium chloride (12.4 mg), 2-dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl (68 .3 mg), cesium carbonate (311 mg) and ethynyltrimethylsilane (331 μl), and the resulting mixture was stirred at 60° C. for 19 hours under nitrogen gas. To the reaction mixture were added ethynyltrimethylsilane (199 µl), bis(acetonitrile)palladium chloride (6.2 mg), 2-dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl (34.1 mg), and cesium carbonate (187 mg) , and the resulting mixture was stirred at 60°C for 3.75 hours. The solvent of the reaction mixture was evaporated under reduced pressure, and then the resulting residue was purified by column chromatography (hexane/ethyl acetate) to give the title compound (245 mg).

(intermediate Z-21: LCMS m/z 645.4 (MH ⁺ ), retention time 2.35 minutes, LC conditions LC-6)

[0274]

Reference Example Z-22: Methyl 5-(2-(4-(3-((tert-butyldimethylsilyl)oxy)-4-(3-ethynylphenyl)butyl)-2-oxo-1,3,4-thiadiazinan-3 -yl)ethyl)thiophene-2-carboxylate (intermediate Z-22)

[formula 115]

To a solution of intermediate Z-21 (225 mg) in methanol (3.6 ml) was added potassium carbonate (50 mg) and the mixture was stirred at room temperature for 1 hour. The reaction mixture was filtered and the filtrate was mixed with a methanol wash used to wash the residue remaining on the filter paper and concentrated. The resulting residue was purified by column chromatography (hexane/ethyl acetate) to give the title compound (198 mg).

(intermediate Z-22: LCMS m/z 573.3 (MH ⁺ ), retention time 1.37 minutes, LC conditions LC-6)

[0275]

Example 4: 5-(2-(4-(3-Hydroxy-4-(3-((4-methylthiophen-3-yl)ethynyl)phenyl)butyl)-2-oxo-1,3,4-thiadiazinan- 3-yl)ethyl)thiophene-2-carboxylic acid

[formula 116]

[Step a]

Methyl 5-(2-(4-(3-((tert-butyldimethylsilyl)oxy)-4-(3-((4-methylthiophen-3-yl)ethynyl)phenyl)butyl)-2-oxo-1,3 ,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylate (intermediate Z-7-4)

[formula 117]

To a solution of intermediate Z-22 (10 mg) in acetonitrile (280 µl) was successively added bis(acetonitrile)palladium chloride (0.5 mg), mg), cesium carbonate (6.8 mg), and 3-bromo-4-methylthiophene (9.3 mg, TCI), and the resulting mixture was stirred at 60° C. for 4 hours under nitrogen gas. The reaction mixture was filtered through celite-coated filter paper and the residue remaining on celite was washed with ethyl acetate. The filtrate and washing liquid were mixed, and the organic layer was washed successively with water and brine, and then dried. The solvent was evaporated under reduced pressure, and then the resulting residue was purified by column chromatography (hexane/ethyl acetate) to give the title compound (7.4 mg).

(intermediate Z-7-4: LCMS m/z 699.4 (MH ⁺ ), retention time 2.18 minutes, LC conditions LC-6)

[0276]

[Step b]

5-(2-(4-(3-Hydroxy-4-(3-((4-methylthiophen-3-yl)ethynyl)phenyl)butyl)-2-oxo-1,3,4-thiadiazinan-3-yl )ethyl)thiophene-2-carboxylic acid

A solution of intermediate Z-7-4 (7.4 mg) in tetrahydrofuran (390 μl) was cooled to 0°C, tetrabutylammonium fluoride (1 mol/l solution in tetrahydrofuran, 33 μl) was added to the solution, and the resulting mixture was stirred at room temperature for 2.5 hours. Methanol (390 μl) and 1 mol/l aqueous sodium hydroxide (390 μl) were added to the reaction mixture, and the resulting mixture was stirred at room temperature for 2 hours. 2 mol/L hydrochloric acid (100 µl) and water (400 µl) were added to the reaction mixture, the mixture was extracted 5 times with ethyl acetate (1 ml), and then the organic layer was dried. The solvent was evaporated under reduced pressure, and the resulting residue was purified by column chromatography (chloroform/methanol) to give the title compound (4.9 mg).

(LCMS m/z 541.2 (MH ⁺ ), retention time 1.74 minutes, LC conditions LC-1)

[0277]

Example 5: 5-(2-(4-(4-(3-((2-Chlorothiophenyl-3-yl)ethynyl)phenyl)-3-hydroxybutyl)-2-oxo-1,3,4-thiadiazinan-3 -yl)ethyl)thiophene-2-carboxylic acid

[formula 118]

According to the method described in Example 4, preparation was carried out using 3-bromo-2-chlorothiophene (20.7 mg, TCI) instead of 3-bromo-4-methylthiophene to give the title compound (12.9 mg).

(LCMS m/z 561.1 (MH ⁺ ), retention time 1.77 minutes, LC conditions LC-1)

[0278]

Example 6: 5-(2-(4-(3-Hydroxy-4-(3-((5-methylthiophen-3-yl)ethynyl)phenyl)butyl)-2-oxo-1,3,4-thiadiazinan- 3-yl)ethyl)thiophene-2-carboxylic acid

[formula 119]

[Step a]

Methyl 5-(2-(4-(3-((tert-butyldimethylsilyl)oxy)-4-(3-((5-methylthiophen-3-yl)ethynyl)phenyl)butyl)-2-oxo-1,3 ,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylate (intermediate Z-7-6)

[formula 120]

To a solution of intermediate Z-22 (20 mg) in acetonitrile (1120 μl) was added successively bis(acetonitrile)palladium chloride (0.9 mg), mg), cesium carbonate (13.7 mg) and 3-bromo-5-methylthiophene (18.5 mg, TCI), and the resulting mixture was stirred at 60° C. for 4 hours under nitrogen gas. The reaction mixture was filtered through Celite-coated filter paper, and the residue remaining on Celite was washed with a mixed solvent of chloroform and methanol (9:1). The filtrate and washing liquid were mixed, the solvent was evaporated under reduced pressure, and then the resulting residue was purified by column chromatography (hexane/ethyl acetate) to give the title compound (16.2 mg).

(intermediate Z-7-6: LCMS m/z 699.4 (MH ⁺ ), retention time 2.20 minutes, LC conditions LC-6)

[0279]

[Step b]

5-(2-(4-(3-Hydroxy-4-(3-((5-methylthiophen-3-yl)ethynyl)phenyl)butyl)-2-oxo-1,3,4-thiadiazinan-3-yl )ethyl)thiophene-2-carboxylic acid

A solution of intermediate Z-7-6 (16.2 mg) in tetrahydrofuran (850 μl) was cooled to 0°C, tetrabutylammonium fluoride (1 mol/l solution in tetrahydrofuran, 73 μl) was added, and the mixture was stirred at room temperature for 2.5 hours. 1 mol/L aqueous sodium hydroxide (66 μl) was added to the reaction mixture, and the resulting mixture was stirred at room temperature for 5 hours. 1 mol/L hydrochloric acid (500 µl) was added to the reaction mixture, the resulting mixture was extracted 5 times with ethyl acetate (1 ml), and then the organic layer was washed with saturated brine (500 µl) and dried. The solvent was evaporated under reduced pressure to give the title compound (22.1 mg).

(LCMS m/z 541.2 (MH ⁺ ), retention time 1.76 minutes, LC conditions LC-1)

[0280]

Example 7: 5-(2-(4-(4-(3-((4-Cyanothiophen-3-yl)ethynyl)phenyl)-3-hydroxybutyl)-2-oxo-1,3,4-thiadiazinan-3 -yl)ethyl)thiophene-2-carboxylic acid

[formula 121]

Preparation was carried out according to the method described in Example 6 using 4-bromothiophene-3-carbonitrile (18.9 mg, COMBI-BLOCKS) instead of 3-bromo-5-methylthiophene to give the title compound (3.8 mg).

(LCMS m/z 552.1 (MH ⁺ ), retention time 1.52 minutes, LC conditions LC-1)

[0281]

Example 8: 5-(2-(4-(4-(3-((2-Cyanothiophen-3-yl)ethynyl)phenyl)-3-hydroxybutyl)-2-oxo-1,3,4-thiadiazinan-3 -yl)ethyl)thiophene-2-carboxylic acid

[formula 122]

The preparation was carried out according to the method described in Example 6 using 3-bromothiophene-2-carbonitrile (18.9 mg, APOLLO) instead of 3-bromo-5-methylthiophene to give the title compound (5.5 mg).

(LCMS m/z 552.2 (MH ⁺ ), retention time 1.56 minutes, LC conditions LC-1)

[0282]

Example 9: 5-(2-(4-(3-Hydroxy-4-(3-(thiazol-4-ylethynyl)phenyl)butyl)-2-oxo-1,3,4-thiadiazinan-3-yl)ethyl )thiophene-2-carboxylic acid

[formula 123]

The preparation was carried out according to the method described in Example 6 using 4-bromothiazole (17.2 mg, ALDRICH) instead of 3-bromo-5-methylthiophene to give the title compound (14.3 mg).

(LCMS m/z 528.2 (MH ⁺ ), retention time 1.38 minutes, LC conditions LC-1)

[0283]

Example 10: 5-(2-(4-(4-(3-(furan-3-ylethynyl)phenyl)-3-hydroxybutyl)-2-oxo-1,3,4-thiadiazinan-3-yl)ethyl) thiophene-2-carboxylic acid

[formula 124]

Preparation was carried out according to the method described in Example 6 using 3-bromofuran (15.4 mg, TCI) instead of 3-bromo-5-methylthiophene to give the title compound (13.2 mg).

(LCMS m/z 511.2 (MH ⁺ ), retention time 1.57 minutes, LC conditions LC-1)

[0284]

Example 11: 5-(2-(4-(4-(3-(furan-2-ylethynyl)phenyl)-3-hydroxybutyl)-2-oxo-1,3,4-thiadiazinan-3-yl)ethyl) thiophene-2-carboxylic acid

[formula 125]

Preparation was carried out according to the method described in Example 6 using 2-bromofuran (14.8 mg, ALDRICH) instead of 3-bromo-5-methylthiophene to give the title compound (4.5 mg).

(LCMS m/z 511.2 (MH ⁺ ), retention time 1.58 minutes, LC conditions LC-1)

[0285]

Example 12 5-(2-(4-(4-(3-((5-cyanothiophen-3-yl)ethynyl)phenyl)-3-hydroxybutyl)-2-oxo-1,3,4-thiadiazinan-3 -yl)ethyl)thiophene-2-carboxylic acid

[formula 126]

[Step a]

Methyl 5-(2-(4-(3-((tert-butyldimethylsilyl)oxy)-4-(3-((5-cyanothiophen-3-yl)ethynyl)phenyl)butyl)-2-oxo-1,3 ,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylate (intermediate Z-7-12)

[formula 127]

To a solution of intermediate Z-22 (14 mg) in acetonitrile (400 µl) was successively added bis(acetonitrile)palladium chloride (0.7 mg), mg), cesium carbonate (12.3 mg) and 4-bromothiophene-2-carbonitrile (18.9 mg, COMBI-BLOCKS), and the resulting mixture was stirred at 60° C. for 6 hours under nitrogen gas. The solvent was evaporated under reduced pressure, and then the resulting residue was purified by column chromatography (hexane/ethyl acetate) to give the title compound (6.4 mg).

(intermediate Z-7-12: LCMS m/z 680.4 (MH ⁺ ), retention time 1.71 minutes, LC conditions LC-6)

[0286]

[Step b]

5-(2-(4-(4-(3-((5-Cyanothiophen-3-yl)ethynyl)phenyl)-3-hydroxybutyl)-2-oxo-1,3,4-thiadiazinan-3-yl) ethyl)thiophene-2-carboxylic acid

A solution of intermediate Z-7-12 (6.4 mg) in tetrahydrofuran (330 μl) was cooled to 0°C, tetrabutylammonium fluoride (1 mol/l solution in tetrahydrofuran, 28 μl) was added to the solution, and the resulting mixture was stirred at room temperature for 4 hours. 1 mol/L aqueous sodium hydroxide (30 μl) was added to the reaction mixture, and the mixture was stirred at room temperature for 2 hours. 1 mol/L hydrochloric acid was added to the reaction mixture, the mixture was extracted with ethyl acetate, and then the organic layer was dried. The solvent was evaporated under reduced pressure, and the resulting residue was purified by column chromatography (chloroform/methanol) to give the title compound (0.7 mg).

(LCMS m/z 552.2 (MH ⁺ ), retention time 1.60 minutes, LC conditions LC-1)

[0287]

Example 13: 5-(2-(4-(3-Hydroxy-4-(3-(thiazol-2-ylethynyl)phenyl)butyl)-2-oxo-1,3,4-thiadiazinan-3-yl)ethyl )thiophene-2-carboxylic acid

[formula 128]

Preparation was carried out according to the method described in Example 6 using 2-bromothiazole (16.5 mg, TCI) instead of 4-bromothiophene-2-carbonitrile to give the title compound (0.4 mg).

(LCMS m/z 528.2 (MH ⁺ ), retention time 1.43 minutes, LC conditions LC-1)

[0288]

Example 14 5-(2-(4-(4-(3-((3-cyanothiophen-2-yl)ethynyl)phenyl)-3-hydroxybutyl)-2-oxo-1,3,4-thiadiazinan-3 -yl)ethyl)thiophene-2-carboxylic acid

[formula 129]

[Step a]

Methyl 5-(2-(4-(3-((tert-butyldimethylsilyl)oxy)-4-(3-((3-cyanothiophen-2-yl)ethynyl)phenyl)butyl)-2-oxo-1,3 ,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylate (intermediate Z-7-14)

[formula 130]

To a solution of intermediate Z-22 (16 mg) in acetonitrile (1 ml) was added bis(acetonitrile)palladium chloride (0.7 mg), 2-dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl (4.0 mg), cesium carbonate (10.9 mg) and 2-bromothiophene-3-carbonitrile (15.7 mg, MAYBRIDGE), and the resulting mixture was stirred at 60° C. for 18 hours under nitrogen gas. The reaction mixture was filtered through Celite-coated filter paper, and the residue remaining on Celite was washed with a mixed solvent of chloroform and methanol (9:1). The filtrate and washing liquid were mixed, the solvent was evaporated under reduced pressure, and then the resulting residue was purified by column chromatography (hexane/ethyl acetate) to give the title compound (7.9 mg).

(intermediate Z-7-14: LCMS m/z 680.5 (MH ⁺ ), retention time 2.52 minutes, LC conditions LC-1)

[0289]

[Step b]

5-(2-(4-(4-(3-((3-Cyanothiophen-2-yl)ethynyl)phenyl)-3-hydroxybutyl)-2-oxo-1,3,4-thiadiazinan-3-yl) ethyl)thiophene-2-carboxylic acid

A solution of intermediate Z-7-14 (7.9 mg) in tetrahydrofuran (1 ml) was cooled to 0°C, tetrabutylammonium fluoride (1 mol/l solution in tetrahydrofuran, 0.5 ml) was added to the solution, and the resulting the mixture was stirred at room temperature for 18 hours. Methanol (0.5 ml) and 1 mol/l aqueous sodium hydroxide (0.5 ml) were added to the reaction mixture, and the resulting mixture was stirred at room temperature for 2.5 hours. 1 mol/L hydrochloric acid was added to the reaction mixture, the mixture was extracted with ethyl acetate, and the organic layer was dried. The solvent was evaporated under reduced pressure, and the resulting residue was purified by column chromatography (chloroform/methanol) to give the title compound (3.0 mg).

(LCMS m/z 552.0 (MH ⁺ ), retention time 1.57 minutes, LC conditions LC-1)

[0290]

Example 15: 5-(2-(4-(3-Hydroxy-4-(3-(phenylethynyl)phenyl)butyl)-2-oxo-1,3,4-thiadiazinan-3-yl)ethyl)thiophene-2 -carboxylic acid

[formula 131]

Preparation was carried out according to the method described in Example 14 using bromobenzene (12.3 mg, TCI) instead of 2-bromothiophene-2-carbonitrile to give the title compound (2.4 mg).

(LCMS m/z 521.0 (MH ⁺ ), retention time 1.71 minutes, LC conditions LC-1)

[0291]

Example 16 5-(2-(4-(3-Hydroxy-4-(3-((2-methoxyphenyl)ethynyl)phenyl)butyl)-2-oxo-1,3,4-thiadiazinan-3-yl) ethyl)thiophene-2-carboxylic acid

[formula 132]

Preparation was carried out according to the method described in Example 14 using 1-bromo-2-methoxybenzene (14.7 mg, WAKO) instead of 2-bromothiophene-2-carbonitrile to give the title compound (1.6 mg).

(LCMS m/z 551.3 (MH ⁺ ), retention time 1.64 minutes, LC conditions LC-1)

[0292]

Reference Example A-10-2: (3-Chlorothiophenyl-2-yl)methanol (Intermediate A-10-2)

[formula 133]

A solution of 3-chlorothiophene-2-carboxylic acid (4.47 g, ALDRICH) in tetrahydrofuran (88.1 ml) was cooled to 0°C under nitrogen gas, added dropwise to a solution of a 1 mol/l solution of borane-tetrahydrofuran complex in tetrahydrofuran (49.7 ml), and the resulting mixture was stirred at room temperature for 22 hours. The reaction mixture was cooled to 0°C, methanol was added to the mixture of water and ethyl acetate, and the resulting mixture was stirred. The organic solvent was evaporated under reduced pressure, then ethyl acetate was added to the residue, and the organic layer was washed successively with saturated aqueous sodium hydrogen carbonate and saturated brine, and dried. The solvent was evaporated under reduced pressure to give the title compound (4.62 g).

(intermediate A-10-2: Rf (TLC)=0.40 (hexane:ethyl acetate=2:1))

[0293]

Reference Example A-11-2: 3-Chloro-2-(bromomethyl)thiophene (Intermediate A-11-2)

[formula 134]

A solution of intermediate A-10-2 (4.62 g) in dichloromethane (110 ml) was cooled to 0°C, triphenylphosphine (10.8 g) and carbon tetrabromide (10.9 g) were added to the solution, and the resulting the mixture was stirred at room temperature for 1 hour. Water, brine and ethyl acetate were added to the reaction mixture, the mixture was stirred, and then the organic solvent was evaporated under reduced pressure. Ethyl acetate was added to the residue, and the organic layer was successively washed with saturated brine and dried. The organic solvent was evaporated under reduced pressure, and then a mixed solvent of hexane and ethyl acetate (9:1) was added to the resulting residue to obtain a suspension, and the suspension was filtered through silica gel-coated filter paper. The filtrate solvent was evaporated under reduced pressure to give the title compound (9.09 g).

(intermediate A-11-2: Rf (TLC)=0.56 (hexane:ethyl acetate=8:1))

[0294]

Reference Example A-12-2: 2-(3-Chlorothiophenyl-2-yl)acetonitrile (Intermediate A-12-2)

[formula 135]

Dimethyl sulfoxide (42 ml) and acetonitrile (126 ml) were added to intermediate A-11-2 (9.09 g), the mixture was cooled to 0°C, then sodium cyanide (2.46 g) was added to the mixture, and the resulting As a result, the mixture was stirred at room temperature for 2 hours. Water, saturated brine and ethyl acetate were added to the reaction mixture, the resulting mixture was stirred, and then the organic layer was washed with saturated brine and dried. The solvent was evaporated under reduced pressure, and then the resulting residue was purified by column chromatography (hexane/ethyl acetate) to give the title compound (3.41 g).

(intermediate A-12-2: Rf (TLC)=0.20 (hexane:ethyl acetate=8:1))

[0295]

Reference Example A-13-2: Ethyl 2-(3-chlorothiophen-2-yl)acetate (Intermediate A-13-2)

[formula 136]

To a solution of intermediate A-12-2 (3.41 g) in ethanol (36 ml) was added water (0.46 ml), the mixture was cooled to 0°C, and then concentrated sulfuric acid (6.3 ml). The reaction mixture was stirred at 85° C. for 88 hours, and then cooled to 0° C., and saturated aqueous sodium hydrogen carbonate was added to the mixture until the mixture became neutral. Ethyl acetate was added to the mixture, and the resulting mixture was stirred and then concentrated under reduced pressure. Ethyl acetate was added to the mixture, and the organic layer was washed successively with saturated aqueous sodium hydrogen carbonate and saturated brine, and dried. The solvent was evaporated under reduced pressure to give the title compound (4.56 g).

(intermediate A-13-2: Rf (TLC)=0.31 (hexane:ethyl acetate=8:1))

[0296]

Reference Example A-14-2: 2-(3-Chlorothiophenyl-2-yl)ethanol (Intermediate A-14-2)

[formula 137]

A solution of intermediate A-13-2 (4.56 g) in tetrahydrofuran (108 ml) was cooled to 0° C. under nitrogen gas, lithium aluminum hydride (1.48 g) was added to the solution, and the mixture was stirred for 0.7 hours. Water, diethyl ether and 1 mol/L hydrochloric acid were added to the reaction mixture, the resulting mixture was stirred, and then the organic layer was successively washed with 1 mol/L hydrochloric acid, saturated aqueous sodium hydrogen carbonate and saturated brine, and dried. The solvent was evaporated under reduced pressure to give the title compound (3.67 g).

(intermediate A-14-2: Rf (TLC)=0.13 (hexane:ethyl acetate=4:1))

[0297]

Reference Example A-2-3: (2-(3-Chlorothiophenyl-2-yl)ethoxy)(tert-butyl)dimethylsilane (Intermediate A-2-3)

[formula 138]

Intermediate A-2-3 was prepared according to the method described in Reference Example A-2 using Intermediate A-14-2 (3.67 g) instead of 2-(thiophen-2-yl)ethanol, and thus the claimed the title compound (4.29 g).

(intermediate A-2-3: Rf (TLC)=0.61 (hexane:ethyl acetate=4:1))

[0298]

Reference Example A-3-3: 4-Chloro-5-(2-((tert-butyldimethylsilyl)oxy)ethyl)thiophene-2-carboxylic acid (Intermediate A-3-3)

[formula 139]

Intermediate A-3-3 was prepared according to the method described in Reference Example A-3 using Intermediate A-2-3 (3.03 g) instead of Intermediate A-2, and thus the title compound (3 .41 g).

(intermediate A-3-3: Rf (TLC)=0.11 (hexane:ethyl acetate=4:1))

[0299]

Reference Example A-4-3: Methyl 4-chloro-5-(2-hydroxyethyl)thiophene-2-carboxylate (Intermediate A-4-3)

[formula 140]

Intermediate A-4-3 was prepared according to the method described in Reference Example A-4 using Intermediate A-3-3 (4.35 g) instead of Intermediate A-3, and thus the title compound (2 .23 g).

(intermediate A-4-3: Rf (TLC)=0.38 (hexane:ethyl acetate=1:1))

[0300]

Reference Example A-5-3: Methyl 4-chloro-5-(2-bromoethyl)thiophene-2-carboxylate (Intermediate A-5-3)

[formula 141]

Intermediate A-5-3 was prepared according to the method described in Reference Example A-5 using Intermediate A-4-3 (2.23 g) instead of Intermediate A-4, and thus the title compound (3 .18 g).

(Intermediate A-5-3: Rf (TLC)=0.52 (hexane:ethyl acetate=2:1))

[0301]

Reference Example A-6-3 tert-Butyl 2-(2-(3-chloro-5-(methoxycarbonyl)thiophen-2-yl)ethyl)hydrazinecarboxylate (Intermediate A-6-3)

[formula 142]

Intermediate A-6-3 was prepared according to the method described in Reference Example A-6 using Intermediate A-5-3 (3.18 g) instead of Intermediate A-5, and thus the title compound (3 .29 g).

(intermediate A-5-3: Rf (TLC)=0.24 (hexane:ethyl acetate=2:1))

[0302]

Reference Example Z-1-3: tert-butyl 2-(2-(3-chloro-5-(methoxycarbonyl)thiophen-2-yl)ethyl)-2-(((2-chloroethyl)thio)carbonyl)hydrazinecarboxylate ( intermediate connection Z-1-3)

[formula 143]

Intermediate Z-1-3 was prepared according to the method described in Reference Example Z-1-2 using Intermediate A-6-3 (1.79 g) instead of Intermediate A-6-2, thus obtaining header compound (2.36 g).

(intermediate Z-1-3: Rf (TLC)=0.24 (hexane:ethyl acetate=4:1))

[0303]

Reference Example Z-2-3: Methyl 4-chloro-5-(2-(1-(((2-chloroethyl)thio)carbonyl)hydrazinyl)ethyl)thiophene-2-carboxylate (intermediate Z-2-3)

[formula 144]

Intermediate Z-2-3 was prepared according to the method described in Reference Example Z-2-2 using Intermediate Z-1-3 (2.36 g) instead of Intermediate Z-1-2, and thus header compound (1.73 g).

(intermediate Z-2-3: Rf (TLC)=0.69 (hexane:ethyl acetate=1:1))

[0304]

Reference Example Z-3-3: Methyl 4-chloro-5-(2-(2-oxo-1,3,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylate (intermediate Z-3-3 )

[formula 145]

Intermediate Z-3-3 was prepared according to the method described in Reference Example Z-3-2 using Intermediate Z-2-3 (1.73 g) instead of Intermediate Z-2-2, and thus header compound (1.04 g).

(intermediate Z-3-3: Rf (TLC)=0.23 (hexane:ethyl acetate=1:1))

[0305]

Reference Example Z-4-3: Methyl 4-chloro-5-(2-(4-(4-(3-iodophenyl)-3-oxobutyl)-2-oxo-1,3,4-thiadiazinan-3-yl )ethyl)thiophene-2-carboxylate (intermediate Z-4-3)

[formula 146]

Intermediate Z-4-3 was prepared according to the method described in Reference Example Z-4 using Intermediate Z-3-3 (0.30 g) in place of Intermediate Z-3 and Intermediate C-3-2 (total the amount of Intermediate C-3-2 synthesized and prepared according to the method described in Reference Example C-3-2, using 2.56 g of the starting compound, Intermediate C-2-2) instead of Intermediate C-3, and thus the title compound (0.64 g) was obtained in this way.

(intermediate Z-4-3: Rf (TLC)=0.31 (hexane:ethyl acetate=1:1))

[0306]

Reference Example Z-14-4: Methyl 4-chloro-5-(2-(2-oxo-4-(3-oxo-4-(3-(thiophen-3-ylethynyl)phenyl)butyl)-1,3 ,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylate (intermediate Z-14-4)

[formula 147]

Diethylamine (3.62 ml), 3-ethynylthiophene (93 μl), copper(I) iodide (13.8 mg), and tetrakis(triphenylphosphine)palladium (41 .8 mg), and the mixture was stirred at room temperature for 3.5 hours under nitrogen gas. 1 mol/L hydrochloric acid was added to the reaction mixture, and the mixture was extracted twice with ethyl acetate. The organic layer was washed successively with saturated brine, saturated aqueous sodium hydrogen carbonate and saturated brine, and dried. The solvent was evaporated under reduced pressure, and the resulting residue was purified by column chromatography (chloroform/methanol) to give the title compound (290.4 mg).

(intermediate Z-14-4: LCMS m/z 573.2 (MH ⁺ ), retention time 2.03 minutes, LC conditions LC-1)

[0307]

Reference Example Z-17-17: Methyl 4-chloro-5-(2-(4-(3-hydroxy-4-(3-(thiophen-3-ylethynyl)phenyl)butyl)-2-oxo-1,3 ,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylate (intermediate Z-17-17)

[formula 148]

To a solution of intermediate Z-14-4 (290 mg) in methanol (5 ml) was added tetrahydrofuran (1 ml), the mixture was cooled to 0° C., and sodium borohydride (28.8 mg) was added portionwise to the mixture. The mixture was stirred at room temperature for 1.6 hours, then water was added to the mixture, and 1 N hydrochloric acid was further added in portions to the reaction mixture until the mixture became slightly acidic. The mixture was extracted twice with ethyl acetate, and then the organic layer was washed successively with saturated aqueous sodium hydrogen carbonate and saturated brine, and dried. The solvent was evaporated under reduced pressure, and the resulting residue was purified by column chromatography (chloroform/methanol) to give the title compound (183 mg).

(intermediate Z-17-17: LCMS m/z 575.2 (MH ⁺ ), retention time 1.98 minutes, LC conditions LC-1)

[0308]

Example 17 4-Chloro-5-(2-(4-(3-hydroxy-4-(3-(thiophen-3-ylethynyl)phenyl)butyl)-2-oxo-1,3,4-thiadiazinan-3 -yl)ethyl)thiophene-2-carboxylic acid

[formula 149]

To a solution of intermediate Z-17-17 (183 mg) in tetrahydrofuran (2 ml) was added methanol (2 ml), the mixture was cooled to 0°C, and water (2.38 ml) and 4 mol/l aqueous lithium hydroxide (2.38 ml). The resulting mixture was stirred at room temperature for 1 hour, then water was added to the reaction mixture, and additional 2 N hydrochloric acid was added portionwise to the reaction mixture until the mixture became slightly acidic. The reaction mixture was extracted twice with ethyl acetate, and then the organic layer was washed successively with saturated aqueous sodium hydrogen carbonate and saturated brine, and dried. The solvent was evaporated under reduced pressure, and the resulting residue was purified by column chromatography (chloroform/methanol) to give the title compound (129 mg).

(LCMS m/z 561.25 (MH ⁺ ), retention time 1.72 minutes, LC conditions LC-1)

[0309]

Reference Example C-1: 2-(3-Bromo-4-methylphenyl)acetonitrile (Intermediate C-1)

[formula 150]

To a solution of 2-bromo-1,4-dimethylbenzene (2 g, TCI) in carbon tetrachloride (21.6 ml) was added N-bromosuccinimide (1.06 g) and benzoyl peroxide (56.7 mg) and the mixture was stirred at 85 °C for 1.5 hours. N-bromosuccinimide (1.06 g) and benzoyl peroxide (56.7 mg) were added to the reaction mixture, and the resulting mixture was further stirred at 85° C. for 4.5 hours. The reaction mixture was cooled to room temperature and then filtered through filter paper, and the residue remaining on the filter paper was washed with dichloromethane. The filtrate and washing liquid were mixed, and the solvent was evaporated under reduced pressure. Ethanol (10.8 ml), water (5.4 ml) and potassium cyanide (2.1 g) were added to the resulting residue, and the mixture was stirred at 100° C. for 5 hours. The reaction mixture was cooled to room temperature and extracted with ethyl acetate, and then the organic layer was dried. The solvent was evaporated under reduced pressure, and the resulting residue was purified by column chromatography (hexane/ethyl acetate) to give the title compound (576 mg).

(intermediate C-1: Rf (TLC)=0.58 (hexane:ethyl acetate=2:1))

¹ H-NMR (CDCl ₃ ): δ (ppm) 7.51 (1H, s), 7.24 (1H, d, J=7.5Hz), 7.18 (1H, d, J=7.5Hz ), 3.70 (2H, s), 2.40 (3H, s)

[0310]

Reference Example C-2-4: 2-(3-Bromo-4-methylphenyl)-N-methoxy-N-methylacetamide (Intermediate C-2-4)

[formula 151]

Water (7.1 ml) was added to Intermediate C-1 (300 mg), the mixture was cooled to 0° C., concentrated sulfuric acid (5.7 ml) was added portionwise to the mixture, and then the resulting mixture was stirred at 105° C within 15 hours. The reaction mixture was cooled to room temperature and then ethyl acetate was added to extract. Hexane was added to the aqueous layer for extraction, and then the aqueous layer was further extracted with diethyl ether. The resulting organic layers were mixed and washed with saturated aqueous sodium hydrogen carbonate and dried. The solvent was evaporated under reduced pressure, and N,N-dimethylformamide (14.3 ml), N,O-dimethylhydroxylamine hydrochloride (557 mg), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride were added successively to the resulting residue. (821 mg), N,N-dimethyl-4-aminopyridine (17 mg) and diisopropylethylamine (1.2 ml), and the resulting mixture was stirred at room temperature for 17 hours. Diethyl ether was added to the reaction mixture, and then the organic layer was washed 3 times with 1 mol/L hydrochloric acid and once with saturated brine, and dried. The solvent was evaporated under reduced pressure, and the resulting residue was purified by column chromatography (hexane/ethyl acetate) to give the title compound (221 mg).

(intermediate C-2-4: LCMS m/z 272.3 (MH ⁺ ), retention time 1.57 minutes, LC conditions LC-1)

[0311]

Reference Example Z-4-4: Methyl 5-(2-(4-(4-(3-bromo-4-methylphenyl)-3-oxobutyl)-2-oxo-1,3,4-thiadiazinan-3-yl )ethyl)thiophene-2-carboxylate (intermediate Z-4-4)

[formula 152]

A solution of intermediate C-2-4 (142.6 mg) in dimethoxyethane (2.85 ml) was cooled to 0° C. under nitrogen. Vinyl magnesium bromide (1 mol/l solution in tetrahydrofuran, 790 μl, ALDRICH) was added to the reaction mixture, and the mixture was stirred for 4 hours. 2 mol/L hydrochloric acid was added to the reaction mixture, and the mixture was stirred for 1 minute. Ethyl acetate was added to the extraction mixture and the organic layer was dried. The solvent was evaporated under reduced pressure, ethanol (3 ml), water (3 ml) and intermediate Z-3 (100 mg) were added to the resulting residue, and the mixture was stirred overnight at 110°C. A saturated saline solution was added to the reaction mixture, and the mixture was extracted with chloroform. The organic layer was dried and the solvent was evaporated under reduced pressure. The resulting residue was purified by column chromatography (hexane/ethyl acetate) to give the title compound (151.9 mg).

(intermediate Z-4-4: LCMS m/z 525.1 (MH ⁺ ), retention time 1.87 minutes, LC conditions LC-1)

[0312]

Reference Example Z-6-4: Methyl 5-(2-(4-(4-(3-bromo-4-methylphenyl)-3-((t-butyldimethylsilyl)oxy)butyl)-2-oxo-1,3 ,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylate (intermediate Z-6-4)

[formula 153]

To a solution of intermediate Z-4-4 (152 mg) in methanol (2.9 ml) was added tetrahydrofuran (5 ml), the mixture was cooled to 0° C., and sodium borohydride (16.4 mg) was added to the mixture. The mixture was stirred at 0° C. for 1 hour, and then dilute hydrochloric acid was added to the reaction mixture in portions until the mixture became slightly acidic. The organic solvent was evaporated under reduced pressure, then ethyl acetate was added to the extraction residue, the organic layer was dried, and then the solvent was evaporated under reduced pressure. To the resulting residue were added N,N-dimethylformamide (1.4 ml), imidazole (98 mg) and t-butyldimethylchlorosilane (131 mg) in succession, and the resulting mixture was stirred overnight at room temperature. 2 mol/L hydrochloric acid was added to the reaction mixture, and the mixture was extracted with ethyl acetate. The organic layer was washed successively with water and brine, and then dried. The solvent was evaporated under reduced pressure, and the resulting residue was purified by column chromatography (hexane/ethyl acetate) to give the title compound (165.7 mg).

(intermediate Z-6-4: LCMS m/z 641.2 (MH ⁺ ), retention time 2.02 minutes, LC conditions LC-6)

[0313]

Reference Example Z-7-18: Methyl 5-(2-(4-(3-((tert-butyldimethylsilyl)oxy)-4-(4-methyl-3-(thiophen-3-ylethynyl)phenyl)butyl)- 2-oxo-1,3,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylate (intermediate Z-7-18)

[formula 154]

Intermediate Z-7-18 was prepared according to the method described in Reference Example C-4 using Intermediate Z-6-4 (20.0 mg) instead of Intermediate C-2-2, and thus the title compound was obtained. (23.4 mg).

(intermediate Z-7-18: LCMS m/z 669.3 (MH ⁺ ), retention time 2.25 minutes, LC conditions LC-6)

[0314]

Example 18 5-(2-(4-(3-Hydroxy-4-(4-methyl-3-(thiophen-3-ylethynyl)phenyl)butyl)-2-oxo-1,3,4-thiadiazinan-3 -yl)ethyl)thiophene-2-carboxylic acid

[formula 155]

A solution of intermediate Z-7-18 (23.4 mg) in tetrahydrofuran (0.93 ml) was cooled to 0°C, tetrabutylammonium fluoride (1 mol/l solution in tetrahydrofuran, 93 μl) was added to the solution, and the mixture was stirred at room temperature for 1.5 hours. Tetrabutylammonium fluoride (1 mol/l solution in tetrahydrofuran, 93 μl) was added to the reaction mixture, and the mixture was stirred at room temperature for the next 1.5 hours. Methanol (0.93 ml) and 1 mol/l aqueous sodium hydroxide (0.93 ml) were added to the reaction mixture, and the mixture was stirred overnight at room temperature. 1 mol/L hydrochloric acid was added to the reaction mixture, the mixture was extracted with ethyl acetate, and the organic layer was dried. The solvent was evaporated under reduced pressure, and the resulting residue was purified by column chromatography (chloroform/methanol) to give the title compound (17.3 mg).

(LCMS m/z 541.2 (MH ⁺ ), retention time 1.74 minutes, LC conditions LC-1)

[0315]

Reference Example C-1-2: 2-(3-Bromo-5-methylphenyl)acetonitrile (Intermediate C-1-2)

[formula 156]

Intermediate C-1-2 was prepared according to the method described in Reference Example C-1 using 1-bromo-3,5-dimethylbenzene (4.00 g, TCI) instead of 2-bromo-1,4-dimethylbenzene, and thus the title compound (2.34 g) was obtained in this way.

(intermediate C-1-2: Rf (TLC)=0.64 (hexane:ethyl acetate=2:1))

¹ H-NMR (CDCl ₃ ): δ (ppm) 7.31 (1H, m), 7.28 (1H, m), 7.09 (1H, m), 3.69 (2H, s), 2 .35 (3H, s)

[0316]

Reference Example C-2-5: 2-(3-Bromo-5-methylphenyl)-N-methoxy-N-methylacetamide (Intermediate C-2-5)

[formula 157]

Intermediate C-2-5 was prepared according to the method described in Reference Example C-2-4, using Intermediate C-1-2 (500 mg) instead of Intermediate C-1, and thus the title compound (553 mg).

(intermediate C-2-5: LCMS m/z 272.3 (MH ⁺ ), retention time 1.57 minutes, LC conditions LC-1)

[0317]

Reference Example Z-4-5: Methyl 5-(2-(4-(4-(3-bromo-5-methylphenyl)-3-oxobutyl)-2-oxo-1,3,4-thiadiazinan-3-yl )ethyl)thiophene-2-carboxylate (intermediate Z-4-5)

[formula 158]

Intermediate Z-4-5 was prepared according to the method described in Reference Example Z-4-4 using Intermediate C-2-5 (142.6 mg) instead of Intermediate C-2-4, thus obtaining header compound (149.9 mg).

(intermediate Z-4-5: LCMS m/z 525.1 (MH ⁺ ), retention time 1.88 minutes, LC conditions LC-1)

[0318]

Reference Example Z-6-5: Methyl 5-(2-(4-(4-(3-bromo-5-methylphenyl)-3-((t-butyldimethylsilyl)oxy)butyl)-2-oxo-1,3 ,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylate (intermediate Z-6-5)

[formula 159]

Intermediate Z-6-5 was prepared according to the method described in Reference Example Z-6-4 using Intermediate Z-4-5 (149.9 mg) instead of Intermediate Z-4-4, thus obtaining header compound (163.0 mg).

(intermediate Z-6-5: LCMS m/z 641.3 (MH ⁺ ), retention time 2.00 minutes, LC conditions LC-6)

[0319]

Reference Example Z-7-19: Methyl 5-(2-(4-(3-((tert-butyldimethylsilyl)oxy)-4-(3-methyl-5-(thiophen-3-ylethynyl)phenyl)butyl)- 2-oxo-1,3,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylate (intermediate Z-7-19)

[formula 160]

Intermediate Z-7-19 was prepared according to the method described in Reference Example C-4 using Intermediate Z-6-5 (20 mg) instead of Intermediate C-2-2, and thus the title compound (19 .2 mg).

(intermediate Z-7-19: LCMS m/z 669.3 (MH ⁺ ), retention time 2.25 minutes, LC conditions LC-6)

[0320]

Example 19 5-(2-(4-(3-Hydroxy-4-(3-methyl-5-(thiophen-3-ylethynyl)phenyl)butyl)-2-oxo-1,3,4-thiadiazinan-3 -yl)ethyl)thiophene-2-carboxylic acid

[formula 161]

Preparation was carried out according to the method described in Example 18 using Intermediate Z-7-19 (19.2 mg) instead of Intermediate Z-7-18 to give the title compound (13.5 mg).

(LCMS m/z 541.2 (MH ⁺ ), retention time 1.74 minutes, LC conditions LC-1)

[0321]

Reference Example C-2-6: 2-(3-Bromo-4-fluorophenyl)-N-methoxy-N-methylacetamide (Intermediate C-2-6)

[formula 162]

To a solution of 2-(3-bromo-4-fluorophenyl)acetic acid (500 mg) in dichloromethane (43 ml) was successively added N,O-dimethylhydroxylamine hydrochloride (419 mg), 1-ethyl-3-(3-dimethylaminopropyl) hydrochloride carbodiimide (494 mg), N,N-dimethyl-4-aminopyridine (26 mg) and diisopropylethylamine (1.2 ml), and the resulting mixture was stirred overnight at room temperature. The solvent of the reaction mixture was evaporated under reduced pressure, ethyl acetate was added to the residue, and then the organic layer was washed successively with 1 mol/L hydrochloric acid and saturated brine, and dried. The solvent was evaporated under reduced pressure, and the resulting residue was purified by column chromatography (hexane/ethyl acetate) to give the title compound (449 mg).

(intermediate C-2-6: LCMS m/z 276.2 (MH ⁺ ), retention time 1.37 minutes, LC conditions LC-1)

[0322]

Reference Example Z-4-6: Methyl 5-(2-(4-(4-(3-bromo-4-fluorophenyl)-3-oxobutyl)-2-oxo-1,3,4-thiadiazinan-3-yl )ethyl)thiophene-2-carboxylate (intermediate Z-4-6)

[formula 163]

Intermediate Z-4-6 was prepared according to the method described in Reference Example Z-4-4 using Intermediate C-2-6 (450.5 mg) instead of Intermediate C-2-4, thus obtaining header compound (562.8 mg).

(intermediate Z-4-6: LCMS m/z 529.1 (MH ⁺ ), retention time 1.78 minutes, LC conditions LC-1)

[0323]

Reference Example Z-6-6: Methyl 5-(2-(4-(4-(3-bromo-4-fluorophenyl)-3-((t-butyldimethylsilyl)oxy)butyl)-2-oxo-1,3 ,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylate (intermediate Z-6-6)

[formula 164]

Intermediate Z-6-6 was prepared according to the method described in Reference Example Z-6-4 using Intermediate Z-4-6 (562.8 mg) instead of Intermediate Z-4-4, thus obtaining the title compound (719.7 mg).

(intermediate Z-6-6: LCMS m/z 645.3 (MH ⁺ ), retention time 1.64 minutes, LC conditions LC-6)

[0324]

Reference Example Z-7-20: Methyl 5-(2-(4-(3-((tert-butyldimethylsilyl)oxy)-4-(4-fluoro-3-(thiophen-3-ylethynyl)phenyl)butyl)- 2-oxo-1,3,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylate (intermediate Z-7-20)

[formula 165]

Intermediate Z-7-20 was prepared according to the method described in Reference Example C-4 using Intermediate Z-6-6 (15.0 mg) instead of Intermediate C-2-2, and thus the title compound was obtained. (15.3 mg).

(intermediate Z-7-20: LCMS m/z 673.4 (MH ⁺ ), retention time 1.87 minutes, LC conditions LC-6)

[0325]

Example 20: 5-(2-(4-(4-(4-Fluoro-3-(thiophen-3-ylethynyl)phenyl)-3-hydroxybutyl)-2-oxo-1,3,4-thiadiazinan-3- yl)ethyl)thiophene-2-carboxylic acid

[formula 166]

A solution of intermediate Z-7-20 (15.3 mg) in tetrahydrofuran (345 µl) was cooled to 0°C, tetrabutylammonium fluoride (1 mol/l solution in tetrahydrofuran, 69 µl) was added to the solution, and the mixture was stirred at room temperature within 4 hours. Tetrabutylammonium fluoride (1 mol/l solution in tetrahydrofuran, 70 μl) and tetrahydrofuran (350 μl) were added to the reaction mixture, and the mixture was stirred at room temperature for the next 2.5 hours. Methanol (345 μl) and 1 mol/l aqueous sodium hydroxide (345 μl) were added to the reaction mixture, and the mixture was stirred overnight at room temperature. To the reaction mixture were added 1 mol/l hydrochloric acid and ethyl acetate for extraction, and then the organic layer was washed with 1 mol/l hydrochloric acid, and dried. The solvent was evaporated under reduced pressure, and the resulting residue was purified with an anion exchange resin to give the title compound (15.3 mg).

(LCMS m/z 545.2 (MH ⁺ ), retention time 1.67 minutes, LC conditions LC-1)

[0326]

Reference X-1: 4-Phenylthiophene-3-carbaldehyde (Intermediate X-1)

[formula 167]

To a solution of (4-formylthiophen-3-yl)boronic acid (0.5 g, COMBI-BLOCKS) in n-butanol (32 ml) was successively added bromothiophene (1.0 ml, TCI), water (6.4 ml) , palladium acetate (36 mg), 2-dicyclohexylphosphino-2',6'-dimethoxybiphenyl (132 mg) and potassium phosphate (1.36 g), and the resulting mixture was stirred overnight at 95° C. under nitrogen gas . To the reaction mixture, diethyl ether was added, and then the organic layer was washed with water and dried. The solvent was evaporated under reduced pressure, and the resulting residue was purified by column chromatography (hexane/ethyl acetate) to give the title compound (445 mg).

(Intermediate X-1: LCMS m/z 189.0 (MH ⁺ ), retention time 1.54 minutes, LC conditions LC-1)

[0327]

Reference X-2: 3-Ethynyl-4-phenylthiophene (Intermediate X-2)

[formula 168]

To a solution of dimethyl (1-diazo-2-oxopropyl)phosphate (727 mg, TCI) in methanol (14.9 ml) was added Intermediate X-1 (445 mg) and the mixture was cooled to 0°C. Potassium carbonate (686 mg) was added portionwise to the reaction mixture, and the mixture was stirred overnight at room temperature. Saturated aqueous ammonium chloride was added to the reaction mixture, and the mixture was extracted with diethyl ether. The organic layer was dried, then the solvent was evaporated under reduced pressure, and the resulting residue was purified by column chromatography (hexane/ethyl acetate) to give the title compound (384 mg).

(intermediate X-2: LCMS m/z 185.1 (MH ⁺ ), retention time 1.81 minutes, LC conditions LC-1)

[0328]

Reference Example Z-7-21: Methyl 5-(2-(4-(3-((t-butyldimethylsilyl)oxy)-4-(3-((4-phenylthiophen-3-yl)ethynyl)phenyl)butyl) -2-oxo-1,3,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylate (intermediate Z-7-21)

[formula 169]

Intermediate Z-7-21 was prepared according to the method described in Reference Example C-4 using Intermediate Z-6 (15.0 mg) instead of Intermediate C-2-2 and Intermediate X-2 (9.4 mg) instead of 3-ethynylthiophene, thus obtaining the title compound (15.2 mg).

(intermediate Z-7-21: LCMS m/z 731.21 (MH ⁺ ), retention time 2.41 minutes, LC conditions LC-6)

[0329]

Example 21: 5-(2-(4-(3-Hydroxy-4-(3-((4-phenylthiophen-3-yl)ethynyl)phenyl)butyl)-2-oxo-1,3,4-thiadiazinan- 3-yl)ethyl)thiophene-2-carboxylic acid

[formula 170]

Preparation was carried out according to the method described in Example 18 using Intermediate Z-7-21 (15.2 mg) instead of Intermediate Z-7-18 to give the title compound (5.7 mg).

(LCMS m/z 603.0 (MH ⁺ ), retention time 1.87 minutes, LC conditions LC-1)

[0330]

Reference Example T-2: 2-(4-Bromothiophen-2-yl)-N-methoxy-N-methylacetamide (Intermediate T-2)

[formula 171]

A solution of 2-(4-bromothiophen-2-yl)acetic acid (1.0 g) in dichloromethane (9 ml) was cooled to 0°C, N,O-dimethylhydroxylamine hydrochloride (882 mg), 1-ethyl hydrochloride was added to the solution -3-(3-dimethylaminopropyl)carbodiimide (1.04 g), N,N-dimethyl-4-aminopyridine (55 mg) and diisopropylethylamine (3.38 ml), then further diisopropylethylamine (0.35 ml) was added, and the mixture was stirred at room temperature for 41 hours. The reaction mixture was concentrated, and then ethyl acetate and water were added, 1 mol/L hydrochloric acid was added to partition, and extraction was further carried out twice with ethyl acetate. The organic layer was washed successively with 1 mol/L hydrochloric acid, water, saturated aqueous sodium hydrogen carbonate, water and brine, dried over magnesium sulfate, and concentrated under reduced pressure to give the title compound (891 mg).

(intermediate T-2: LCMS m/z 264.2, 266 (MH ⁺ ), retention time 1.36 minutes, LC conditions LC-1)

[0331]

Reference T-3: 1-(4-Bromothiophen-2-yl)-4-(methoxy(methyl)amino)butan-2-one (Intermediate T-3)

[formula 172]

A solution of intermediate T-2 (200 mg) in 1,2-dimethoxyethane (7 ml) was cooled to 0° C. under nitrogen. Vinyl magnesium bromide (1 mol/l solution in tetrahydrofuran, 1.1 ml, ALDRICH) was added to the reaction mixture, and the mixture was stirred at the same temperature for 2 hours and 50 minutes. 1 mol/L hydrochloric acid was added to the reaction mixture, making the mixture acidic still at 0°C, and then the mixture was extracted 3 times with ethyl acetate. The organic layer was washed with water and brine, dried over magnesium sulfate, and then concentrated under reduced pressure to give the title compound (172.8 mg).

(intermediate T-3: LCMS m/z 292.1, 294 (MH ⁺ ), retention time 1.55 minutes, LC conditions LC-1)

[0332]

Reference Example T-4: Methyl 5-(2-(4-(4-(4-bromothiophen-2-yl)-3-oxobutyl)-2-oxo-1,3,4-thiadiazinan-3-yl)ethyl )thiophene-2-carboxylate (intermediate T-4)

[formula 173]

Intermediate T-3 (172.8 mg) and Intermediate Z-3 (181 mg) were dissolved in ethanol (5 ml), water (5 ml) was added to the solution, and the mixture was stirred at 105° C. for 16 5 o'clock. The reaction mixture was poured into brine diluted with water, and the mixture was extracted 3 times with chloroform. The organic layer was washed with saturated brine and dried over magnesium sulfate, and then the solvent was evaporated under reduced pressure. The resulting residue was purified by column chromatography (n-hexane/ethyl acetate) to give the title compound (112.0 mg).

(intermediate T-4: LCMS m/z 517.0, 519.1 (MH ⁺ ), retention time 1.76 minutes, LC conditions LC-1)

[0333]

Reference Example T-5: Methyl 5-(2-(4-(4-(4-bromothiophen-2-yl)-3-hydroxybutyl)-2-oxo-1,3,4-thiadiazinan-3-yl)ethyl )thiophene-2-carboxylate (intermediate T-5)

[formula 174]

A solution of intermediate T-4 (112 mg) in methanol (2 ml) was cooled to 0° C., sodium borohydride (12.3 mg) was added portionwise to the solution. The reaction mixture was stirred at room temperature for 3 hours, and then poured into water, and the mixture was extracted 3 times with ethyl acetate. The organic layer was washed with water and then with saturated brine and dried over magnesium sulfate, and then the solvent was evaporated under reduced pressure to give the title compound (99.6 mg).

(intermediate T-5: LCMS m/z 519.08, 521.08 (MH ⁺ ), retention time 1.70 minutes, LC conditions LC-1)

[0334]

Reference Example T-6: Methyl 5-(2-(4-(3-acetoxy-4-(4-bromothiophen-2-yl)butyl)-2-oxo-1,3,4-thiadiazinan-3-yl) ethyl)thiophene-2-carboxylate (intermediate T-6)

[formula 175]

A solution of intermediate T-5 (95.7 mg) in dichloromethane (1.8 ml) was cooled to 0°C, acetic anhydride (348 μl) and pyridine (741 μl) were added to the solution, the mixture was stirred at room temperature for 17 hours, then acetic anhydride (348 μl) was added to the mixture, and the resulting mixture was stirred at room temperature for the next 4 hours. Water was added to the reaction mixture, and then the mixture was extracted 3 times with chloroform. The organic layer was washed with saturated aqueous sodium hydrogen carbonate, and then with saturated brine, and dried over magnesium sulfate, and then the solvent was evaporated under reduced pressure. The resulting residue was purified by column chromatography (n-hexane/ethyl acetate) to give the title compound.

(intermediate T-6: LCMS m/z 561.1, 563.1 (MH ⁺ ), retention time 1.89 minutes, LC conditions LC-1)

[0335]

Example 22 5-(2-(4-(3-Hydroxy-4-(4-(thiophen-3-ylethynyl)thiophen-2-yl)butyl)-2-oxo-1,3,4-thiadiazinan-3 -yl)ethyl)thiophene-2-carboxylic acid

[formula 176]

[Step a]

Methyl 5-(2-(4-(3-acetoxy-4-(4-(thiophen-3-ylethynyl)thiophen-2-yl)butyl)-2-oxo-1,3,4-thiadiazinan-3-yl )ethyl)thiophene-2-carboxylate (intermediate T-7)

[formula 177]

To a solution of intermediate T-6 (17.5 mg) in acetonitrile (1 ml) was added successively bis(acetonitrile)palladium chloride (1.2 mg), 2-dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl (6 .7 mg), cesium carbonate (13.2 mg) and 3-ethynylthiophene (5.6 μl), and the resulting mixture was stirred at 60° C. for 17 hours under argon gas. The reaction mixture was filtered through celite-coated filter paper and the residue remaining on the celite was washed with ethyl acetate. The filtrate and washing were mixed, the solvent was evaporated under reduced pressure, and then the resulting residue was purified by column chromatography (n-hexane/ethyl acetate) to give a mixture (18.6 mg) of intermediate T-6 and the title compound. To a solution of the resulting mixture in acetonitrile (2 ml) were added in succession bis(acetonitrile)palladium chloride (1.2 mg), 2-dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl (6.7 mg), cesium carbonate (13.2 mg) and 3-ethynylthiophene (6.14 μl), and the resulting mixture was stirred at 60° C. for 19 hours under argon gas. The reaction mixture was filtered through celite-coated filter paper and the residue remaining on the celite was washed with ethyl acetate. The filtrate and washing liquid were mixed, the solvent was evaporated under reduced pressure, and then the resulting residue was purified by column chromatography (n-hexane/ethyl acetate) to give the title compound (14.2 mg).

(intermediate T-7: LCMS m/z 589.1 (MH ⁺ ), retention time 2.03 minutes, LC conditions LC-1)

[0336]

[Step b]

5-(2-(4-(3-Hydroxy-4-(4-(thiophen-3-ylethynyl)thiophen-2-yl)butyl)-2-oxo-1,3,4-thiadiazinan-3-yl) ethyl)thiophene-2-carboxylic acid

To a solution of intermediate T-7 (14.2 mg) in tetrahydrofuran (0.36 ml) was added 1 mol/l aqueous lithium hydroxide (0.36 ml) and the mixture was stirred overnight at room temperature. The reaction mixture was cooled to 0° C. and 1 mol/l hydrochloric acid (0.36 ml) was added to the mixture. The mixture was diluted with water and extracted 3 times with chloroform, then the organic layer was washed with saturated brine and dried over magnesium sulfate, and the solvent was evaporated under reduced pressure. The resulting residue was purified by column chromatography (chloroform/methanol) to give the title compound (3.5 mg).

(LCMS m/z 533.0 (MH ⁺ ), retention time 1.64 minutes, LC conditions LC-1)

[0337]

Example 23: (S)-5-(2-(4-(3-Hydroxy-4-(3-(thiophen-3-ylethynyl)phenyl)butyl)-2-oxo-1,3,4-thiadiazinan-3 -yl)ethyl)thiophene-2-carboxylic acid

[formula 178]

[Step a]

(S)-Methyl 5-(2-(4-(3-hydroxy-4-(3-(thiophen-3-ylethynyl)phenyl)butyl)-2-oxo-1,3,4-thiadiazinan-3-yl )ethyl)thiophene-2-carboxylate (intermediate Z-17-S)

[formula 179]

Intermediate Z-17 (592 mg) was subjected to HPLC separation using a chiral column (HPLC unit was a preparative purification unit made by Japan Waters, CHIRALCEL AD-H chiral column (Daicel Corporation), eluent ethanol, flow rate 0.5 ml /minute, retention time 14.91 minutes) to give the title compound (194.2 mg).

[0338]

[Step b]

(S)-5-(2-(4-(3-Hydroxy-4-(3-(thiophen-3-ylethynyl)phenyl)butyl)-2-oxo-1,3,4-thiadiazinan-3-yl) ethyl)thiophene-2-carboxylic acid

Preparation was carried out according to the method described in Example 1 using Intermediate Z-17-S (41.4 mg) instead of Intermediate Z-17 to give the title compound (31.8 mg).

(LCMS m/z 527.2 (MH ⁺ ), retention time 1.68 minutes, LC conditions LC-1)

[0339]

Reference Example U: (S)-Methyl 5-(2-(4-(4-(3-bromophenyl)-3-hydroxybutyl)-2-oxo-1,3,4-thiadiazinan-3-yl)ethyl)thiophene -2-carboxylate (intermediate U)

[formula 180]

Intermediate Z-5 (1 g) was subjected to HPLC separation using a chiral column (HPLC unit was a preparative purification unit made by Japan Waters, CHIRALCEL OJ-H chiral column (Daicel Corporation), eluent methanol, flow rate 0.5 ml /minute, retention time 20.72 minutes) to give the title compound (309 mg).

[0340]

Reference Example V-1: (S)-Methyl 5-(2-(4-(4-(3-bromophenyl)-3-((t-butyldimethylsilyl)oxy)butyl)-2-oxo-1,3,4 -thiadiazinan-3-yl)ethyl)thiophene-2-carboxylate (Intermediate V-1)

[formula 181]

Preparation was carried out according to the method described in Reference Example Z-6 using Intermediate U (299.3 mg) instead of Intermediate Z-5 to give the title compound (333.9 mg).

(Intermediate V-1: LCMS m/z 627.35, 629.35 (MH ⁺ ), retention time 1.79 minutes, LC conditions NLC-1)

[0341]

Reference Example V-2: (S)-Methyl 5-(2-(4-(3-((tert-butyldimethylsilyl)oxy)-4-(3-((trimethylsilyl)ethynyl)phenyl)butyl)-2-oxo -1,3,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylate (Intermediate V-2)

[formula 182]

Preparation was carried out according to the method described in Reference Example Z-21, using Intermediate V-1 (333.9 mg) instead of Intermediate Z-6 to give the title compound (91.3 mg).

(Intermediate V-2: LCMS m/z 645.49 (MH ⁺ ), retention time 2.38 minutes, LC conditions NLC-6)

[0342]

Reference Example V-3: (S)-Methyl 5-(2-(4-(3-((tert-butyldimethylsilyl)oxy)-4-(3-ethynylphenyl)butyl)-2-oxo-1,3,4 -thiadiazinan-3-yl)ethyl)thiophene-2-carboxylate (Intermediate V-3)

[formula 183]

Preparation was carried out according to the method described in Reference Example Z-22 using Intermediate V-2 (91.3 mg) instead of Intermediate Z-21 to give the title compound (91.6 mg).

(Intermediate V-3: LCMS m/z 573.45 (MH ⁺ ), retention time 1.41 minutes, LC conditions NLC-6)

[0343]

Example 24: (S)-5-(2-(4-(4-(3-((2-cyanothiophen-3-yl)ethynyl)phenyl)-3-hydroxybutyl)-2-oxo-1,3,4 -thiadiazinan-3-yl)ethyl)thiophene-2-carboxylic acid

[formula 184]

[Step a]

(S)-Methyl 5-(2-(4-(3-((tert-butyldimethylsilyl)oxy)-4-(3-((2-cyanothiophen-3-yl)ethynyl)phenyl)butyl)-2-oxo -1,3,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylate (intermediate Z-24-1)

[formula 185]

To a solution of intermediate V-3 (50.6 mg) in acetonitrile (1 ml) was added successively bis(acetonitrile)palladium chloride (2.0 mg), 2-dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl (11 .2 mg), cesium carbonate (51.1 mg) and 3-bromothiophene-2-carbonitrile (73.8 mg), and the resulting mixture was stirred at 60° C. for 0.75 hours under nitrogen gas. The reaction mixture was filtered through Celite-coated filter paper, and the residue remaining on Celite was washed with a mixed solvent of chloroform and methanol (9:1). The filtrate and washing liquid were mixed, the solvent was evaporated under reduced pressure, and then the resulting residue was purified by column chromatography (hexane/ethyl acetate) to give the title compound (10.1 mg).

(intermediate Z-24-1: LCMS m/z 680.44 (MH ⁺ ), retention time 1.73 minutes, LC conditions NLC-6)

[0344]

[Step b]

(S)-Methyl 5-(2-(4-(4-(3-((2-cyanothiophen-3-yl)ethynyl)phenyl)-3-hydroxybutyl)-2-oxo-1,3,4-thiadiazinan -3-yl)ethyl)thiophene-2-carboxylate (intermediate Z-24-2)

[formula 186]

To a solution of intermediate Z-24-1 (10.1 mg) in tetrahydrofuran (0.5 ml) was added tetrabutylammonium fluoride (1 mol/l solution in tetrahydrofuran, 44.6 μl) and the mixture was stirred at room temperature for 1 hours. The reaction mixture was purified by column chromatography (hexane/ethyl acetate) to give the title compound (9.3 mg).

(intermediate Z-24-2: LCMS m/z 566.35 (MH ⁺ ), retention time 1.77 minutes, LC conditions NLC-6)

[0345]

[Stage c]

(S)-5-(2-(4-(4-(3-((2-Cyanothiophen-3-yl)ethynyl)phenyl)-3-hydroxybutyl)-2-oxo-1,3,4-thiadiazinan- 3-yl)ethyl)thiophene-2-carboxylic acid

To a solution of intermediate Z-24-2 (9.3 mg) in tetrahydrofuran (400 μl) was added 1 mol/l aqueous sodium hydroxide (197 μl) and the mixture was stirred at room temperature for 60 hours. 1 mol/L hydrochloric acid (400 µl) was added to the reaction mixture, the mixture was extracted 3 times with chloroform, and then the organic layer was washed with saturated brine and dried. The solvent was evaporated under reduced pressure. The resulting residue was purified by column chromatography (methanol/chloroform) to give the title compound (7.5 mg).

(LCMS m/z 552.30.2 (MH ⁺ ), retention time 1.23 minutes, LC conditions NLC-1)

[0346]

Example 25: (S)-5-(2-(4-(3-Hydroxy-4-(3-(thiazol-4-ylethynyl)phenyl)butyl)-2-oxo-1,3,4-thiadiazinan-3 -yl)ethyl)thiophene-2-carboxylic acid

[formula 187]

[Step a]

(S)-Methyl 5-(2-(4-(3-((tert-butyldimethylsilyl)oxy)-4-(3-(thiazol-4-ylethynyl)phenyl)butyl)-2-oxo-1,3, 4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylate (intermediate Z-25-1)

[formula 188]

The preparation was carried out according to the method described in example 24, step a, using 4-bromothiazole (28.6 μl) instead of 3-bromothiophene-2-carbonitrile, obtaining the title compound (21.0 mg).

(intermediate Z-25-1: LCMS m/z 656.43 (MH ⁺ ), retention time 1.37 minutes, LC conditions NLC-1)

[0347]

[Step b]

(S)-5-(2-(4-(3-Hydroxy-4-(3-(thiazol-4-ylethynyl)phenyl)butyl)-2-oxo-1,3,4-thiadiazinan-3-yl) ethyl)thiophene-2-carboxylic acid

To a solution of intermediate Z-25-1 (21.0 mg) in tetrahydrofuran (1 ml) was added tetrabutylammonium fluoride (1 mol/l solution in tetrahydrofuran, 92.6 μl) and the mixture was stirred at room temperature for 3 hours. The reaction mixture was purified by column chromatography (hexane/ethyl acetate). To a solution of the resulting intermediate (12.2 mg) in tetrahydrofuran (540 μl) and methanol (270 μl) was added 1 mol/l aqueous sodium hydroxide (270 μl) and the mixture was stirred at room temperature for 16 hours. 1 mol/L hydrochloric acid was added to the reaction mixture, the mixture was extracted 5 times with ethyl acetate, and then the organic layer was washed with saturated brine and dried. The solvent was evaporated under reduced pressure to give the title compound (12.5 mg).

(LCMS m/z 528.29 (MH ⁺ ), retention time 1.08 minutes, LC conditions NLC-1)

[0348]

Example 26: (S)-5-(2-(4-(4-(3-(Furan-3-ylethynyl)phenyl)-3-hydroxybutyl)-2-oxo-1,3,4-thiadiazinan-3- yl)ethyl)thiophene-2-carboxylic acid

[formula 189]

The preparation was carried out according to the method described in Example 25 using 3-bromofuran (31.2 mg) instead of 4-bromothiazole, and then the resulting residue was purified by column chromatography (methanol/chloroform) to give the title compound (5.1 mg ).

(LCMS m/z 511.35 (MH ⁺ ), retention time 1.23 minutes, LC conditions NLC-1)

[0349]

Reference Example Z-27: (E)-Methyl 5-(2-(4-(3-((tert-butyldimethylsilyl)oxy)-4-(3-(2-(thiophen-3-yl)vinyl)phenyl) butyl)-2-oxo-1,3,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylate (intermediate Z-27)

[formula 190]

(E)-4,4,5,5-Tetramethyl-2-(2-(thiophen-3-yl )vinyl)-1,3,2-dioxaborolane (22.6 mg, ALDRICH), bis(triphenylphosphine)palladium chloride (5.6 mg), sodium carbonate (21.1 mg) and water (199 µl), and the resulting as a result, the mixture was stirred at 85° C. for 11 hours under nitrogen gas. Water was added to the reaction mixture, and the mixture was extracted with ethyl acetate. The organic layer was dried, then the solvent was evaporated under reduced pressure, and the resulting residue was purified by column chromatography (hexane/ethyl acetate) to give the title compound (44.5 mg).

(intermediate Z-27: LCMS m/z 657.3 (MH ⁺ ), retention time 1.93 minutes, LC conditions LC-6)

[0350]

Example 27: (E)-5-(2-(4-(3-Hydroxy-4-(3-(2-(thiophen-3-yl)vinyl)phenyl)butyl)-2-oxo-1,3, 4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylic acid

[formula 191]

Preparation was carried out according to the method described in Example 18 using Intermediate Z-27 (24.8 mg) instead of Intermediate Z-7-18 to give the title compound (17.8 mg).

(LCMS m/z 529.1 (MH ⁺ ), retention time 1.63 minutes, LC conditions LC-1)

[0351]

Reference Example Z-14-5: Methyl 4-chloro-5-(2-(2-oxo-4-(3-oxo-4-(3-(pyridin-2-ylethynyl)phenyl)butyl)-1,3 ,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylate (intermediate Z-14-5)

[formula 192]

Diethylamine (850 μl), 2-ethynylpyridine (22.8 μl), copper(I) iodide (2.9 mg) and tetrakis(triphenylphosphine)palladium (3, 9 mg), and the mixture was stirred at room temperature for 15.5 hours under nitrogen gas. The reaction mixture was diluted with ethyl acetate, then the solvent was evaporated, and the resulting residue was purified by column chromatography (toluene/acetonitrile) to give the title compound (47.6 mg).

(intermediate Z-14-5: LCMS m/z 570.425 (MH ⁺ ), retention time 4.86 minutes, LC conditions FLC-1))

[0352]

Example 28 4-Chloro-5-(2-(4-(3-hydroxy-4-(3-(pyridin-2-ylethynyl)phenyl)butyl)-2-oxo-1,3,4-thiadiazinan-3 -yl)ethyl)thiophene-2-carboxylic acid

[formula 193]

To a solution of intermediate Z-14-5 (47.6 mg) in methanol (840 μl) was added portionwise sodium borohydride (4.8 mg). The mixture was stirred at room temperature for 0.5 hour, and then water and ethyl acetate were added to the mixture, and the organic layer was washed successively with saturated aqueous sodium hydrogen carbonate and saturated brine, and dried. The solvent was evaporated under reduced pressure, and then tetrahydrofuran (640 μl), methanol (640 μl) and 2 mol/l aqueous sodium hydroxide (640 μl) were added to the residue. The reaction mixture was stirred at room temperature for 1.8 hours, and then cooled to 0° C., and 2 mol/l hydrochloric acid (640 μl) was added portionwise to the mixture. Ethyl acetate was added to the reaction mixture, and the organic layer was washed with saturated brine and dried. The solvent was evaporated under reduced pressure, and the resulting residue was purified by thin layer chromatography (toluene/ethanol/acetic acid) to give the title compound (15.0 mg).

(LCMS m/z 556.023 (MH ⁺ ), retention time 4.49 minutes, LC conditions FLC-1)

[0353]

Reference Example Z-29-1: 2-tert-butyl 1-(2-chloroethyl) 1-(2-(5-(methoxycarbonyl)thiophen-2-yl)ethyl)hydrazine-1,2-dicarboxylate (Intermediate Z -29-1)

[formula 194]

To a solution of intermediate A-6 (5.0 g) in acetonitrile (165 ml) was added potassium carbonate (0.46 g) and the mixture was cooled to 0°C. 2-Chloroethyl chloroformate (2.07 ml) was slowly added to the mixture, and the resulting mixture was stirred at the same temperature for 1 hour. Water was added to the reaction mixture, and the resulting mixture was warmed to room temperature and extracted 3 times with ethyl acetate. The organic layer was washed with water and then with saturated brine and dried, and then the solvent was evaporated under reduced pressure to give the title compound (8.34 g).

(intermediate Z-29-1: LCMS m/z 307.14, 309.11 (MH ⁺ -Boc), retention time 1.64 minutes, LC conditions LC-1)

[0354]

Reference Example Z-29-2: tert-butyl 3-(2-(5-(methoxycarbonyl)thiophen-2-yl)ethyl)-2-oxo-1,3,4-oxadiazinane-4-carboxylate (Intermediate Z -29-2)

[formula 195]

A solution of intermediate Z-29-1 (8.34 g) in DMF (140 ml) was cooled to 0° C., sodium hydride (55%, 0.87 g) was added portionwise over 30 minutes and the mixture was stirred for 2 hours. The reaction mixture was warmed to room temperature and stirred for 1 hour, then sodium hydride (55%, 0.1 g) was added to the mixture again at 0°C and the mixture was stirred at room temperature for 15 hours. The reaction mixture was cooled to 0°C, water was added, and the resulting mixture was extracted 4 times with ethyl acetate. The organic layer was washed twice with water and once with saturated brine, and dried, and then the solvent was evaporated under reduced pressure. The resulting residue was purified by column chromatography (hexane/ethyl acetate) to give the title compound (1.75 g).

(intermediate Z-29-2: LCMS m/z 371.3 (MH ⁺ ), retention time 1.51 minutes, LC conditions LC-1)

[0355]

Reference Example Z-29-3: Methyl 5-(2-(2-oxo-1,3,4-oxadiazinan-3-yl)ethyl)thiophene-2-carboxylate (intermediate Z-29-3)

[formula 196]

A solution of intermediate Z-29-2 (1.75 g) in dichloromethane (25 ml) was cooled to 0°C, trifluoroacetic acid (12.5 ml) was added to the solution, and the mixture was stirred at room temperature for 1 hour. The reaction mixture was cooled to 0°C, neutralized with 5 M aqueous sodium hydroxide, and then extracted 3 times with chloroform. The organic layer was washed with water and then dried, and the solvent was evaporated under reduced pressure. The residue was purified by a short silica gel column (chloroform, then ethyl acetate), and then the resulting precipitate was washed with diethyl ether to give the title compound (0.90 g).

(intermediate Z-29-3: LCMS m/z 271.2 (MH ⁺ ), retention time 0.94 minutes, LC conditions LC-1)

[0356]

Reference Example Z-29-4: Methyl 5-(2-(4-(4-(3-iodophenyl)-3-oxobutyl)-2-oxo-1,3,4-oxadiazinan-3-yl)ethyl)thiophene -2-carboxylate (intermediate Z-29-4)

[formula 197]

A solution of Intermediate C-2-2 (250 mg) in DME (5 mL) was cooled to 0° C. under nitrogen flow, vinylmagnesium bromide (1 M solution, 1.2 mL) was added dropwise to the solution and the mixture was stirred at the same temperature for 2 hours. The reaction mixture was acidified by adding 2 M hydrochloric acid, and then water was added to the mixture, and the resulting mixture was extracted 3 times with ethyl acetate. The organic layer was washed twice with water and dried, and then the solvent was evaporated under reduced pressure.

To a flask containing intermediate Z-29-3 (110.7 mg) and water (3.3 ml) was added a solution of the residue obtained above in ethanol (3.3 ml) and the mixture was stirred at an internal temperature of 110° C within 15 hours. The reaction mixture was warmed to room temperature and then poured into water, and the resulting mixture was extracted 3 times with ethyl acetate. The organic layer was washed with saturated brine and dried, and then the solvent was evaporated under reduced pressure. The resulting residue was purified by column chromatography (hexane/ethyl acetate) to give the title compound (126.2 mg).

(intermediate Z-29-4: LCMS m/z 541.2 (MH ⁺ ), retention time 1.66 minutes, LC conditions LC-1)

[0357]

Reference Example Z-29-5: Methyl 5-(2-(4-(3-hydroxy-4-(3-iodophenyl)butyl)-2-oxo-1,3,4-oxadiazinan-3-yl)ethyl) thiophene-2-carboxylate (intermediate Z-29-5)

[formula 198]

To a solution of intermediate Z-29-4 (58.1 mg) in methanol (1 ml) was added sodium borohydride (6.1 mg) and the mixture was stirred at room temperature for 1 hour. Water was added to the mixture, and the resulting mixture was extracted 3 times with ethyl acetate. The organic layer was washed with saturated brine and dried, and then the solvent was evaporated under reduced pressure to give the title compound (61.4 mg).

(intermediate Z-29-5: LCMS m/z 545.2 (MH ⁺ ), retention time 1.60 minutes, LC conditions LC-1)

[0358]

Reference Example Z-29-6: Methyl 5-(2-(4-(3-acetoxy-4-(3-iodophenyl)butyl)-2-oxo-1,3,4-oxadiazinan-3-yl)ethyl) thiophene-2-carboxylate (intermediate Z-29-6)

[formula 199]

Intermediate Z-29-5 (61.4 mg) was dissolved in dichloromethane (2 ml), acetic anhydride (0.27 ml) and pyridine (0.23 ml) were added to the solution, and the mixture was stirred at room temperature 5.5 hours. Water was added to the reaction mixture, and the resulting mixture was extracted 3 times with chloroform. The organic layer was washed with water and dried, and then the solvent was evaporated under reduced pressure. The residue was purified by short column chromatography to give the title compound (71.8 mg).

(intermediate Z-29-6: LCMS m/z 587.26 (MH ⁺ ), retention time 1.82 minutes, LC conditions LC-1)

[0359]

Reference Example Z-29-7: Methyl 5-(2-(4-(3-acetoxy-4-(3-(thiophen-3-ylethynyl)phenyl)butyl)-2-oxo-1,3,4-oxadiazinan -3-yl)ethyl)thiophene-2-carboxylate (intermediate Z-29-7)

[formula 200]

Preparation was carried out according to the method described in Reference Example Z-14 using Intermediate Z-29-6 (71.8 mg) instead of Intermediate Z-4-2 to give the title compound (66.4 mg).

(intermediate Z-29-7: LCMS m/z 567.36 (MH ⁺ ), retention time 1.94 minutes, LC conditions LC-1)

[0360]

Example 29: 5-(2-(4-(3-Hydroxy-4-(3-(thiophen-3-ylethynyl)phenyl)butyl)-2-oxo-1,3,4-oxadiazinan-3-yl)ethyl )thiophene-2-carboxylic acid

[formula 201]

The preparation was carried out according to the method described in Example 22, step b, using Intermediate Z-29-7 (66.4 mg) instead of Intermediate T-7 to give the title compound (45.6 mg).

(LCMS m/z 509.2 (MH ⁺ ), retention time 1.53 minutes, LC conditions LC-1)

[0361]

Reference D-2: Methyl 4-(3-bromophenyl)-3-oxobutanoate (Intermediate D-2)

[formula 202]

Potassium monomethylmalonate (0.885 kg), THF (4.077 kg) and magnesium chloride (0.47 kg) were added to the solution, and the mixture was stirred at 50° C. for 10 minutes. To this mixture, the reaction mixture obtained by adding a solution of carbonyldimidazole (0.801 kg) in DMF (4.025 kg) to a solution of 3-bromophenylacetic acid (1.005 kg) in THF (2.023 kg), and the mixture was stirred at room temperature for 1 hour. Additionally, THF (0.508 kg) was added to the reaction mixture, and the resulting mixture was stirred at 50° C. for 30 minutes. The reaction mixture was cooled to room temperature, ethyl acetate (8.071 kg) was added to the mixture, the organic layer was washed twice with 20% aqueous citric acid (6.032 kg), and then the solvent was evaporated under reduced pressure to give a concentrate (2.358 kg). Ethyl acetate (1.011 kg) was added to this concentrate to give a solution containing Intermediate D-2 (3.369 kg). A solution containing intermediate D-2 was mixed with a solution prepared in a similar manner. Ethyl acetate (6.063 kg) was added to this mixture (6.770 kg), and 5% aqueous sodium hydrogen carbonate (10.055 kg) chloride and sodium (0.506 kg) were further added, washing the organic layer. 5% aqueous sodium hydrogen carbonate (10.054 kg) and sodium chloride (0.503 kg) were added, further washing the organic layer. The organic layer was further washed with 20% aqueous sodium chloride (10.064 kg), ethyl acetate (1.01 kg) was added, and then the solvent was evaporated under reduced pressure to give the title compound (2.52 kg).

(LCMS m/z 268.9, 270.9 (MH ^- ), retention time 1.44 minutes, LC conditions NLC-1)

[0362]

Reference D-3: Methyl (S)-4-(3-bromophenyl)-3-hydroxybutanoate (Intermediate D-3)

[formula 203]

To intermediate D-2 (2.52 kg) was added methanol (14.325 kg), water (0.237 kg), and [NH ₂ Me ₂ ][(RuCl((S)-dm-segphos)) ₂ (u-Cl ) ₃ ] (82.46 g, TAKASAGO), the mixture was stirred at 60° C. for 6 hours under a hydrogen atmosphere, and then the solvent was evaporated under reduced pressure. Toluene (20.70 kg) and QuadraSil AP (0.60 kg, JOHNSON) were added to the residue, the mixture was stirred at 60°C for 1 hour, then the reaction mixture was filtered and the filter cake was washed with toluene (2.066 kg). The filtrate solvent was evaporated under reduced pressure to give a concentrate (5.68 kg), and then n-heptane (1.22 kg) was added dropwise to the concentrate at 10°C over 15 minutes, and n-heptane (1.22 kg) was further added dropwise at 5°C over 50 minutes. The reaction mixture was stirred as is at 5°C for 45 minutes, and then filtered. The filter cake was washed with a mixture of n-heptane (0.48 kg) and toluene (0.24 kg) and dried to give the title compound (1.923 kg).

(LCMS m/z 273.0, 275.1 (MH ⁺ ), retention time 1.36 minutes, LC conditions NLC-1)

(Chiral LC: retention time 21.1 minutes, LC conditions Chiral LC-1)

[0363]

Reference Example D-35: Methyl (S)-3-hydroxy-4-(3-(thiophen-3-ylethynyl)phenyl)butanoate (Intermediate D-35)

[formula 204]

To intermediate D-3 (558 g) were added acetonitrile (1386 g), cesium carbonate (665.7 g), X-Phos (48.7 g, Nippon Kagaku), and bis(acetonitrile)palladium(II) dichloride (13 .28 g, TCI) and a solution of 3-ethynylthiophene (287.3 g) in acetonitrile (277.2 g) was added dropwise over 30 minutes under nitrogen. The reaction mixture was stirred at 40°C for 30 minutes and then at 60°C for 90 minutes. The reaction mixture was cooled to room temperature, toluene (1208.3 g) and water (3488.2 g) was added to the mixture, and the resulting mixture was stirred for 20 minutes and then filtered through celite. The filter cake was washed with toluene (2416.3 g), the filtrate was stirred for 10 minutes and then left until it separated into an organic layer and an aqueous layer, and the aqueous layer was removed. The organic layer was washed with water (2232.3 g) and then the solvent was evaporated under reduced pressure to give the title compound (753.9 g).

(LCMS m/z 301.2 (MH ⁺ ), retention time 1.65 minutes, LC conditions NLC-1)

[0364]

Reference Example D-50: Methyl (S)-3-((2-methoxyethoxy)methoxy)-4-(3-(thiophen-3-ylethynyl)phenyl)butanoate (Intermediate D-50)

[formula 205]

To intermediate D-35 (753.5 g) were added toluene (2588.1 g), diisopropylethylamine (334.3 g), and 2-methoxyethoxymethyl chloride (322.2 g) under nitrogen atmosphere, and the mixture was stirred at 80°C within 150 minutes. The reaction mixture was cooled, water (3107.7 g) was added to the mixture, and the resulting mixture was stirred for 10 minutes and then filtered. The filter cake was washed with toluene (1031.6 g), the filtrate was left until it separated into an organic layer and an aqueous layer, and the aqueous layer was removed. The organic layer was washed with water (2071.9 g) and then the solvent was evaporated under reduced pressure to give an oily substance containing Intermediate D-50 (915.8 g). To this oily substance containing Intermediate D-50 (915.4 g) was added methanol (2310.7 g) and Shirasagi A activated carbon (292.3 g, Japan EnviroChemicals) and the mixture was stirred at room temperature for 1 hour and then filtered through filter paper. The filter cake was washed with methanol (928.8 g), then the filtrate was filtered through a membrane filter having a pore diameter of 0.5 μm, and the filter cake was washed with methanol (464.2 g). The filtrate was concentrated, toluene (2479.1 g) and QuadraSil MTU (349.6 g, JOHNSON) were added to the concentrate, and the mixture was stirred at 40° C. for 1 hour and then filtered through filter paper. The filter cake was washed with toluene (1008.0 g), then the filtrate was filtered through a membrane filter having a pore diameter of 0.5 μm, and the filter cake was washed with toluene (503.8 g). The filtrate was concentrated under reduced pressure to give the title compound (710.9 g).

(LCMS m/z 406.2 (M ⁺ NH4 ⁺ ), retention time 1.88 minutes, LC conditions NLC-1)

[0365]

Reference Example D-60: (S)-3-((2-Methoxyethoxy)methoxy)-4-(3-(thiophen-3-ylethynyl)phenyl)butanal (Intermediate D-60)

[formula 206]

Toluene (2740.1 g) was added to intermediate D-50 (355.3 g) under nitrogen and the mixture was cooled to -83°C. To this mixture was added dropwise a 1 mol/l solution of diisobutylaluminum hydride in toluene (571.6 g) over 90 minutes. 1 mol/L hydrochloric acid (1884.1 g) was added to the reaction mixture, the mixture was stirred at room temperature for 1 hour, toluene (64.3 g) was added to the mixture, the resulting mixture was stirred for 10 minutes, and then was left until it separated into an organic layer and an aqueous layer, and the aqueous layer was removed. The organic layer was washed successively with 1 mol/l hydrochloric acid (1004.9 g), 1% aqueous sodium hydrogen carbonate (1997.8 g) and water (1978.1 g), and then filtered through a membrane filter having a pore diameter of 0.5 µm. The filter cake was washed with toluene (214.3 g) to give a toluene solution (4061.0 g) containing the title compound (248.5 g).

(LCMS m/z 376.2 (M ⁺ NH ₄ ⁺ ), retention time 1.80 minutes, LC conditions NLC-1)

[0366]

Reference Example Z-70: Methyl (S)-5-(2-(4-(3-((2-methoxyethoxy)methoxy)-4-(3-(thiophen-3-ylethynyl)phenyl)butyl)-2- oxo-1,3,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylate (intermediate Z-70)

[formula 207]

To intermediate Z-3 (286.3 g) were added under nitrogen atmosphere toluene (1959.6 g), acetic acid (474.5 g) and sodium triacetoxyborohydride (498.5 g). The solvent solution in toluene (7927.0 g) containing Intermediate D-60 (460.28 g) was evaporated under reduced pressure to give a concentrated solution of Intermediate D-60 (1503.9 g) and this solution was added to the reaction mixture at room temperature for 50 minutes. Toluene (245 g) was added to the reaction mixture, and the resulting mixture was stirred for 2 hours and then washed successively with 5% aqueous sodium hydrogen carbonate (7326.6 g), 5% aqueous sodium hydrogen carbonate (7327.2 g) and water. (7071.4 g). The solvent was evaporated under reduced pressure to give the title compound (903.8 g).

(LCMS m/z 629.29 (MH ⁺ ), retention time 2.05 minutes, LC conditions NLC-1)

[0367]

Reference Example Z-17-S: Methyl (S)-5-(2-(4-(3-hydroxy-4-(3-(thiophen-3-ylethynyl)phenyl)butyl)-2-oxo-1,3 ,4-thiadiazinan-3-yl)ethyl)thiophene-2-carboxylate (intermediate Z-17-S)

[formula 208]

Methanol (4293.5 g) and Shirasagi A activated carbon (90.35 g, Japan EnviroChemicals) were added to Intermediate Z-70 (903.5 g), the reaction mixture was stirred at 40°C for 1 hour, and then filtered , and the filter residue was washed with methanol (715.1 g). 36% hydrochloric acid (588.6 g) was added to the filtrate, the mixture was stirred at 40° C. for 6 hours, then toluene (2406.8 g) was added to the mixture, and the resulting mixture was cooled to room temperature. The reaction mixture was washed successively with 5% aqueous sodium hydrogen carbonate (9765.4 g) and water (1685.1 g), and then the solvent was evaporated under reduced pressure. The resulting residue was purified by column chromatography (n-heptane/ethyl acetate) to give the title compound (471.6 g).

(LCMS m/z 541.19 (MH ⁺ ), retention time 1.87 minutes, LC conditions NLC-1)

[0368]

Example 30: (S)-5-(2-(4-(3-Hydroxy-4-(3-(thiophen-3-ylethynyl)phenyl)butyl)-2-oxo-1,3,4-thiadiazinan-3 -yl)ethyl)thiophene-2-carboxylic acid

[formula 209]

THF (2393.3 g) and Shirasagi A activated carbon (94.0 g, Japan EnviroChemicals) were added to intermediate Z-17-S (469.5 g), the reaction mixture was stirred at room temperature for 1 hour, and then filtered and the filter residue washed with THF (1597.1 g). THF (378.9 g), methanol (1421.8 g) and 1 mol/l aqueous sodium hydroxide (1728.2 g) were added to the filtrate, and the mixture was stirred for 17 hours. Toluene (2331.5 g) and water (1346.5 g) were added to the reaction mixture, the mixture was stirred and left until it separated into an organic layer and an aqueous layer, and the organic layer was removed. A mixture of toluene (2332 g), THF (1596 g) and methanol (710 g) was added to the aqueous layer, the resulting mixture was stirred and left until it separated into an organic layer and an aqueous layer, and the organic layer was removed. Then, washing with a mixture of toluene, THF and methanol was carried out 3 times in a similar manner. Toluene (4663.3 g) was added to the aqueous layer, the mixture was stirred and left until it separated into an organic layer and an aqueous layer, and the organic layer was removed. 1 mol/l hydrochloric acid was added to the aqueous layer until the pH of the mixture became 7.0, then ethyl acetate (2425.9 g) was added, additional 1 mol/l hydrochloric acid was added until the pH of the mixture became 2.2, the mixture was stirred for 10 minutes and then left until it separated into an organic layer and an aqueous layer, and the aqueous layer was removed. Ethyl acetate (1347.1 g) and water (897.5 g) were added to the organic layer, the mixture was stirred for 10 minutes, and then left until it separated into an organic layer and an aqueous layer, and the aqueous layer was removed. The organic layer solvent was evaporated under reduced pressure to give a residue containing the title compound (358.6 g). To this residue containing the title compound (358.3 g) was added methanol (1860.0 g) and Shirasagi A activated carbon (47.6 g, Japan EnviroChemicals), the mixture was stirred at room temperature for 1 hour, and then filtered through a membrane filter having a pore diameter of 0.5 μm, and the filter residue was washed with methanol (2231.2 g). Shirasagi A activated carbon (188.6 g, Japan EnviroChemicals) was added to the filtrate, the mixture was stirred at room temperature for 1 hour, and then filtered through filter paper, and the filter residue was washed with methanol (1490.5 g). The filtrate was filtered through a membrane filter having a pore diameter of 0.2 μm, and the filter residue was washed with methanol (743.7 g). Shirasagi A activated carbon (94.0 g, Japan EnviroChemicals) was added to the filtrate, the mixture was stirred at room temperature for 1 hour, and then filtered through a membrane filter having a pore diameter of 0.2 μm, and the filter residue was washed with methanol (2231 .2 g). Shirasagi A activated carbon (94.1 g, Japan EnviroChemicals) was added to the filtrate, the mixture was stirred at room temperature for 1 hour, and then filtered through a membrane filter having a pore diameter of 0.2 μm, and the filter residue was washed with methanol (742 .9 g). The filtrate solvent was evaporated under reduced pressure to give the title compound (160.1 g).

(LCMS m/z 527.2 (MH ⁺ ), retention time 1.26 minutes, LC conditions NLC-1)

(chiral LC: retention time: 21.3 minutes, LC conditions chiral LC-2)

Example 30 is the same as Example 23.

[0369]

Comparative Example 1: 4-(2-(4-(4-(3-Bromophenyl)-3-hydroxybutyl)-2-oxo-1,3,4-thiadiazinan-3-yl)ethyl)benzoic acid

[formula 210]

The title compound can be prepared by the production method described in International Patent Publication WO2006/080323 (Patent Document 8), Example IAH-H072.

[0370]

Receiving Example 1

Dichloromethane (20 ml) was added to poly(lactic-co-glycolic acid)) (RESOMER RG504, obtained by Evonik Industries, 2.0 g) which was dissolved using an ultrasonic cleaning machine and the compound of Example 23 (1.6 mg) , additionally added to the solution, and dissolved. This solution was added in portions to a 0.1% aqueous solution of polyvinyl alcohol (300 ml) stirred at 3000 rpm using a homogenizer (MARK II, obtained by PRIMIX Corporation), and the mixture was stirred at room temperature for 10 minutes, obtaining an oil emulsion in water. This oil-in-water emulsion was stirred at room temperature for 16 hours, dichloromethane was evaporated to solidify the oil phase, and the resulting residue was centrifuged (3000 rpm, 20° C., 15 minutes) using a centrifuge. After removing the supernatant, the pellet was dispersed in a 0.1% (w/v) solution of Tween 80, the dispersion was passed through 53 µm and 20 µm sieves, and the 20 µm sample remaining on the sieves was centrifuged (3000 rpm, 20°C , 15 minutes). The supernatant was removed, purified water was added to the residue, the mixture was centrifuged again (3000 rpm, 20° C., 15 minutes) and the supernatant was removed. The pellet was frozen at -80° C. and dried under reduced pressure (48 hours) to give drug-containing microspheres (1.2 g) having an entrained drug fraction of 0.06%.

[0371]

Receiving Example 2

Dichloromethane (20 ml) was added to poly(lactic-co-glycolic acid)) (RESOMER RG504, obtained by Evonik Industries, 2.0 g) which was dissolved using an ultrasonic cleaning machine, and the compound of example 23 (20 mg) was further added to the solution, and dissolved. This solution was added in portions to a 0.1% aqueous solution of polyvinyl alcohol (300 ml) stirred at 3000 rpm using a homogenizer (MARK II, obtained by PRIMIX Corporation), and the mixture was stirred at room temperature for 10 minutes, obtaining an oil emulsion in water. This oil-in-water emulsion was stirred at room temperature for 16 hours, dichloromethane was evaporated to solidify the oil phase, and the resulting residue was centrifuged (3000 rpm, 20° C., 15 minutes) using a centrifuge. After removing the supernatant, the pellet was dispersed in a 0.1% (w/v) solution of Tween 80, the dispersion was passed through 53 µm and 20 µm sieves, and the 20 µm sample remaining on the sieves was centrifuged (3000 rpm, 20°C , 15 minutes). The supernatant was removed, then purified water was added to the residue, the mixture was centrifuged again (3000 rpm, 20°C, 15 minutes) and the supernatant was removed. The pellet was frozen at -80° C. and dried under reduced pressure (48 hours) to obtain drug-containing microspheres (1.3 g) having an entrained drug fraction of 0.8%.

[0372]

Receiving example 3

Dichloromethane (20 ml) was added to poly(lactic-co-glycolic acid)) (RESOMER RG504, obtained by Evonik Industries, 2.0 g), which was dissolved using an ultrasonic cleaning machine, and the compound of example 23 (124 mg), additionally added to the solution, and dissolved. This solution was added in portions to a 0.1% aqueous solution of polyvinyl alcohol (300 ml) stirred at 3000 rpm using a homogenizer (MARK II, obtained by PRIMIX Corporation), and the mixture was stirred at room temperature for 10 minutes, obtaining an oil emulsion in water. This oil-in-water emulsion was stirred at room temperature for 16 hours, dichloromethane was evaporated to solidify the oil phase, and the resulting residue was centrifuged (3000 rpm, 20° C., 15 minutes) using a centrifuge. After removing the supernatant, the pellet was dispersed in a 0.1% (w/v) solution of Tween 80, the dispersion was passed through 53 µm and 20 µm sieves, and the 20 µm sample remaining on the sieves was centrifuged (3000 rpm, 20°C , 15 minutes). The supernatant was removed, then purified water was added to the residue, the mixture was centrifuged again (3000 rpm, 20°C, 15 minutes) and the supernatant was removed. The precipitate was frozen at -80° C. and dried under reduced pressure (48 hours) to obtain drug-containing microspheres (1.1 g) having an entrained drug fraction of 3.7%.

[0373]

<Test Example 1: Measurement of EP ₄ Agonist Activity>

In order to investigate the EP ₄ receptor agonist activity of the compounds of the present invention, cAMP synthesis was measured in HEK293 cells that were made to stably express the human EP ₄ receptor.

(1) Measurement method

Using the Refseq database, a search was made for the prostaglandin E receptor. As a result, information about the human EP ₄ (NM_000958) receptor gene was obtained. Based on this sequence information, the human EP ₄ receptor gene was cloned by PCR in a conventional manner using human cDNA as a template, and HEK293 cells were obtained, which made the human EP ₄ receptor stably expressed. When cryopreserved cells of a given strain were thawed and used, cells suspended three times or more over a period of time (approximately 1-2 weeks) were used using Dulbecco's Modified Eagle's Medium (Dulbecco's Modified Eagle's Medium can be shortened further to DMEM) containing 10% FBS, 50 units each of penicillin and streptomycin. The subcultured cells were inoculated into wells of a 96-well poly-D-lysine coated plate at a density of 2×104-2.5×104 cells/well and cultured for one day. The medium in the wells was removed by suction, then DMEM (80 μl) was added to each well, and incubation was carried out at 37°C for 15 minutes. Then, 20 µl of assay medium (DMEM containing 100 mM HEPES and 1 mM IBMX) containing PGE ₂ or test compound (at a concentration 5 times the final concentration) was added to each well, starting the reaction. The reaction was carried out at 37°C for 30 minutes, then the medium was removed by suction. Assay/lysis buffer (100 μl) contained in the cAMP screening kit (obtained by Applied Biosystems) was added to terminate the reaction. Then, the reaction mixture was incubated at 37° C. for 30 minutes to obtain a cAMP quantification sample, and the amount of cFMP in the sample was determined according to the method indicated in the cAMP screening kit. By non-linear regression of compound concentration and cAMP amount, the compound concentration required to increase cAMP to 50% of the maximum increase (EC ₅₀ value) was calculated using the Kaleida Graph.

[0374]

(2) Measurement results

As shown in Table 1, the compounds of the present invention showed excellent EP ₄ agonist activity. In particular, they showed excellent EP ₄ agonist activity even compared to the agonist activity of the compound of Comparative Example 1, which is a known compound analogous to the compounds of the present invention.

For compounds for which EP ₄ agonist activity was measured two or more times, average values are shown, if necessary. Example No. shown in the table indicates the number of the example.

[0375]

[Table 1]

Example No. EC ₅₀ (nM) one 0.045 2 0.035 3 0.13 four 0.34 5 0.3 6 0.56 7 0.09 eight 0.08 9 0.09 ten 0.065 eleven 0.09 12 0.6 13 0.34 fourteen 0.3 fifteen 0.63 16 0.4 17 0.026 eighteen 0.1 19 0.1 twenty 0.07 21 0.5 22 0.055 23 0.033 24 0.3 25 0.095 26 0.17 27 0.09 28 0.087 29 0.16 Comparative Example 1 2.5

[0376]

<Test Example 2: Receptor Binding Test Using Cells Expressing Human EP Receptor>

In order to evaluate selectivity for each EP receptor subtype, [ ³ H]PGE ₂ binding inhibition activities of test compounds were measured for cell membranes stably expressing human EP ₂ , human EP ₃ and human EP ₄ receptors.

(1) Measurement method

As membrane fractions of the prostaglandin E receptors EP ₂ , EP ₃ and EP ₄ , 10.0 μg protein/tube each of HTS185M, HTS092M and HTS142M Merck Millipore, respectively, was used. Each membrane fraction was incubated with the reaction mixture (250 μl/tube) containing the test compound and [ ³ H]PGE ₂ at 25°C for 60 minutes. The final concentrations of [ ³ H]PGE ₂ were 2.56 nmol/l in the EP ₂ measurement system, 1.54 nmol/l in the EP ₃ measurement system, and 1.24 nmol/l in the EP ₄ measurement system. After the reaction, the membrane fraction was collected on filter paper using a cell collector, the filter paper was transferred to a measurement tube, and measurement was performed on a liquid scintillation counter.

[0377]

Non-specific binding was defined as binding observed in the presence of an excess (10 μm) of unlabeled PGE ₂ . [ ³ H]PGE ₂ binding inhibition activity of a test compound was measured by adding the test compound at various concentrations. The following buffers were used for the reaction.

EP ₂ buffer: 50 mmol/l HEPES-NaOH (pH 7.4) containing 5 mmol/l MgCl ₂ , 1 mmol/l CaCl ₂ and 0.2% BSA

Buffer for EP ₃ : 50 mmol/l Tris-HCl buffer containing 10 mmol/l MgCl ₂ and 1 mmol/l EDTA

EP ₄ buffer: 50 mmol/l HEPES-NaOH (pH7.4) containing 5 mmol/l MgCl ₂ , 1 mmol/l CaCl ₂ and 0.2% BSA

A dose-response curve for [ ³ H]PGE ₂ binding inhibition activity was plotted for each test compound, and the concentration of test compound that inhibited 50% of PGE ₂ and receptor binding (IC ₅₀ value) was calculated.

[0378]

(2) Measurement results

As shown in Table 2, the compounds of the present invention showed excellent EP ₄ selectivity. Example No. shown in the table indicates the number of the example.

[0379]

[Table 2]

Example No. IC ₅₀ (nM) _{ep2 ep3} EP ₄ 23 >1000 >1000 4.35 24 >1000 >1000 43.0 25 >1000 >1000 10.9 26 >1000 >1000 16.2

[0380]

<Test Example 3: Neo-Osteogenesis Effect on Rat Femur>

In order to investigate the osteogenesis-promoting effect of the compounds of the present invention, the compounds acted on the femurs of rats and the new bone formed was evaluated.

(1) Measurement method

Eight-week-old female SD rats (Charles River Japan) were fixed in the lateral position under triple anesthesia (medetomidine hydrochloride, midazolam, and butorphanol tartrate). After the hair of the left thigh region was shaved with a hair clipper and disinfected with 70% ethanol, in situ cured gel solutions of the test compound, specifically the test compound dissolved in a poly(lactic-co-glycolic acid) block copolymer ( RESOMER RG502H obtained by Evonik Industries)/poly(lactic-co-glycolic acid)-polyethylene glycol (5050 DLG mPEG 5000 obtained by Lakeshore Biomaterials)/N-methyl-2-pyrrolidone (obtained by Wako) (wt. ratio 47%/3%/ 50%) was filled into a 1 ml syringe, a 21G injection needle was attached to the syringe, and the needle was injected transdermally from the quadriceps muscle to the periosteum near the center of the femoral shaft. Then, 100 μg as the amount of the test compound or 50 μl as the injection volume of the solution was injected between the quadriceps muscle and the periosteum, and the injection needle was withdrawn. The rats of the control group were administered only the above-mentioned in situ cured gel solution. One week after administration of the drug solution, the animals were given triple anesthesia, and they were fixed in the supine position and euthanized by bloodletting. The left thigh was removed, peripheral tissues such as muscles were removed, the bone mineral content of the entire femur was measured, the bone mineral analyzer DCS-600EX (manufactured by ALOKA) was used, then it was divided into three parts along the long axis, and the bone mineral content was evaluated. central part (diaphysis). The test was carried out with groups, each of which consisted of 6 animals.

[0381]

(2) Measurement results

In the groups administered with exemplary compounds of the present invention, the bone mineral content of the left femoral shaft increased compared to the control group (Tables 3-5). On the other hand, the content of mineral substances in the bones of the diaphysis of the right femur, into which the drug solution was not injected, did not change. Based on these results, it was confirmed that the compounds of the present invention are useful as a bone-forming agent for topical administration. In all compound administration groups, no death of animals was observed, the side effects observed with PGE ₂ administration were also not observed, and thus it was demonstrated that the compounds of the present invention can be administered safely.

Test results are given for each test.

[0382]

[table 3]

Example No. Mineral content of 1/3 of the central region of the femur (mg) Left femur (with compound injection) Right femur (no connection) Control group 69.4±3.3 62.9±2.2 3 80.2±2.6 66.7±2.7 6 81.3±3.6 64.7±2.4 eight 98.7±4.1 68.0±1.2 13 82.0±3.4 65.5±2.7

[0383]

[table 4]

Example No. Mineral content of 1/3 of the central region of the femur (mg) Left femur (with compound injection) Right femur (no connection) Control group 65.4±1.9 61.6±1.5 ten 78.0±1.5 61.1±0.5 19 85.8±2.3 59.6±1.4 twenty 79.4±5.2 58.7±3.6 22 79.5±1.9 60.3±2.7

[0384]

[table 5]

Example No. Mineral content of 1/3 of the central region of the femur (mg) Left femur (with compound injection) Right femur (no connection) Control group 68.0±2.4 63.4±2.1 23 82.7±6.7 58.9±2.9 24 80.7±4.0 61.5±2.3 25 79.1±4.0 61.0±2.1 26 87.0±1.6 65.9±1.2

[0385]

<Test Example 4: Neo-Osteogenesis Effect on Dog Femur>

In order to investigate the osteogenesis-promoting effect of the compounds of the present invention, when the microspheres containing the test compound were administered near the hip of a dog, the osteogenesis-promoting effect was evaluated by measuring the new bone formed after the administration.

(1) Measurement method

Nine to eleven-month-old female beagle dogs (KITAYAMA LABES) were anesthetized with a 1:1 mixture of ketamine hydrochloride (Ketalar 500 mg, Daiichi Sankyo Propharma Co., Ltd.) and xylazine (2% selactar injection, Bayer Yakuhin, Ltd.) .) at a dose of approximately 0.5 ml/kg, and isoflurane listed in the Japanese Pharmacopoeia (Elucaine, Mylan Pharmaceutical) was used to maintain anesthesia, which was administered with an IMPAC6 inhaler (VetEquip Inc.). After the right hind leg femoral hair was shaved and disinfected, 350 µl of a microsphere suspension prepared by suspending microspheres (prepared according to Preparation Method 1 or 2) containing the test compound (Example 23 compound) in CMC solution was injected transdermally around the periosteum femoral shaft using a 1 ml injection syringe and a 21G injection needle. The test compound doses were 0.01, 0.1, 1.0, 10 and 100 μg/site, and the above microspheres were used in an amount corresponding to each dose. As a control group, a drug liquid obtained by suspending test compound-free microspheres in only 350 µl of CMC solution was administered. Four weeks after administration of the medicinal fluid, the animals were euthanized by bloodletting under anesthesia with sodium pentobarbital (somnopentyl). The femurs of both sides were separated, immersed in 10% neutral buffered formalin solution and stored. The mineral content of the femur was measured using a Discovery bone density x-ray analyzer (manufactured by Toyo Medic). The test was carried out with groups, each of which consisted of 4 dogs.

[0386]

(2) Measurement results

Four weeks after administration of the aforementioned microsphere suspension into the femoral shafts of dogs, the mineral content of the femur increased with doses of 1.0, 10 and 100 μg as administered amount of test compound in a dose-dependent manner compared to the control group (Table 6). Based on these results, it was confirmed that the compounds of the present invention are useful as an osteogenesis stimulating agent for topical administration. For all compound administration groups, no death of animals was observed, side reactions commonly observed with PGE ₂ administration were also not observed, and thus it was demonstrated that the aforementioned microsphere preparation containing the compound of the present invention can be administered safely.

[0387]

[table 6]

Mineral content of 1/3 of the central region of the femur (mg) Left femur (no compound injection) Right femur (with connection insertion) Control group 2.6±0.2 2.9±0.3 0.01 µg 2.9±0.4 2.9±0.4 0.1 µg 2.6±0.1 2.7±0.2 1.0 µg 3.0±0.1 4.1±0.4 10 mcg 2.8±0.3 6.4±0.7 100 mcg 2.7±0.4 8.2±1.1

[0388]

<Test Example 5: Fracture Healing Action in Rat Femoral Closed Fracture Model>

In order to investigate the fracture healing effect of the compounds of the present invention, the effect of injection of microspheres containing the test compound into a fracture site in a rat closed fracture model was confirmed.

(1) Measurement method

Thirteen week old female SD rats (Japan SLC) were fixed in the lateral position under triple anesthesia (medetomidine hydrochloride, midazolam and butorphanol tartrate). After the hair of the area from the left knee to the thigh area was shaved with a hair clipper, and the disinfection treatment was carried out with povidone-iodine listed in the Japanese Pharmacopoeia (isodine solution for animals, 20 mg of povidone-iodine, listed in the Japanese Pharmacopoeia in 1 ml, Meiji Seika Pharma), the skin of the patella and the medial large muscle of the lateral part of the patella was removed, and the patella was displaced from the femoral head. A drill attached to a trephine was placed on the intercondylar fossa of the open femoral head and rotated by hand to make a hole. A Kirschner wire (Mizuho Co., Ltd.) having a diameter of 1.2 mm, previously cut to a length of 31 mm, was inserted into the medullary cavity of the femur through the hole. Then, the left femur was fixed on a three-point bending test fixture of a small benchtop universal testing machine (EZ Test, Shimadzu Corp.), and a closed femoral shaft fracture was induced by dynamic loading. Whether a fracture was successfully created or not was confirmed by confirming the generation of a complete transverse fracture of the femur on an x-ray image obtained with a soft x-ray generator (M-100W, Softex) and a digital x-ray sensor (NX-04, RF Co., Ltd.). The microsphere suspension obtained by suspending the microspheres (prepared according to Production Method 3) containing the test compound (Example 23 compound) in CMC solution was injected into the fracture part in a volume of 100 μl as the injection volume (containing 100 or 300 μg of the test compound). As a control group, a drug liquid obtained by suspending microspheres not containing the aforementioned test compound was administered in CMC solution (100 µl) only. One, two, and three weeks after fracture generation, soft X-rays were taken under isoflurane anesthesia, and callus areas on the X-rays were quantified using Image J. Four weeks after fracture generation, soft X-rays were taken under triple anesthesia, animals were fixed in position lying on his back, euthanized by bloodletting and removed the left femurs. By confirming the presence or absence of continuity of the callus in a blind way on soft x-rays obtained 4 weeks after the generation of the fracture, bone fusion was determined. The femur specimens were cryopreserved prior to the bone strength test, and the torsional strength was measured on the day of the test using a bone strength tester (MZ-500S, Maruto Instrument Co., Ltd.).

This test can also be carried out using dogs instead of rats.

[0389]

(2) Measurement results

In the test compound-administered group, an increase in callus area was observed two weeks after fracture generation and thereafter (Table 7), and the degree of bone fusion determined from X-ray images obtained 25 days after fracture generation was clearly increased (Table 8 ) compared to the control group. For hips isolated from animals in the test compound administration group four weeks after fracture generation, an increase in bone strength as determined by the torsion test (maximum torque) was observed compared to the control group (Table 8). Based on these results, the compounds of the present invention were confirmed to be useful as a bone fusion promoter in a fracture healing process. For all groups of administration of the compound, no death of animals was observed, side reactions usually observed with the administration of PGE ₂ were also not observed, and thus it was demonstrated that the aforementioned microsphere preparation containing the compound of the present invention can be administered safely.

[0390]

[table 7]

Callus area (mm ² ) One week after fracture Two weeks after fracture Three weeks after fracture Control group 1.98±1.52 22.3±4.1 27.4±4.4 100 mcg 5.24±2.87 28.6±4.7 35.9±6.3 300 mcg 6.01±2.75 28.9±3.8 33.8±6.8

[0391]

[table 8]

The degree of bone union after 25 days (%) maximum torque (N∙cm) Control group 61.1 18.8±6.1 100 mcg 90.0 20.4±7.1 300 mcg 85.7 26.3±11.7

[0392]

<Test Example 6: Bone Fusion Stimulating Action in a Canine Postolateral Lumbar Fixation Model>

In order to investigate the bone fusion stimulating action of the compounds of the present invention, the effect of adding microspheres containing the test compound during bone autografting was confirmed using a post-lateral fixation model of the lumbar vertebrae.

(1) Measurement method

Twelve to thirteen month old male beagle dogs (KITAYAMA LABES) were anesthetized with a 1:1 mixture of ketamine hydrochloride (Ketalar 500 mg, Daiichi Sankyo Propharma Co., Ltd.) and xylazine (2% selactar injection, Bayer Yakuhin, Ltd.). ) at a dose of approximately 0.5 ml/kg, and isoflurane listed in the Japanese Pharmacopoeia (elucain obtained by Mylan Pharmaceutical) was inhaled with an IMPAC6 inhaler (VetEquip Inc.) to maintain anesthesia. The animals were fixed in the prone position, the hair on a large area above the right and left posterior iliac neck, the part around the iliac crest and lower back was shaved and disinfected with povidone-iodine specified in the Japanese Pharmacopoeia (Isodine solution for animals 20 mg povidone- iodine specified in the Japanese Pharmacopoeia in 1 ml (Meiji Seika Pharma) and ethanol for disinfection (Wako Pure Chemical Industries) The skin and soft tissues were cut and opened from the posterior superior iliac bone, along the iliac crest using a scalpel, and then the muscles, covering the iliac crest were dissected under the periosteum to expose the iliac crest Approximately 2 g each of the right and left iliac bones were dissected using bone scissors and compression was applied to stop bleeding. broken with bone scissors and thus turned into cuttings of 1 mm in size, the floor teaching 2 g of each of the bone grafts for the right and left parts. Next, the skin was dissected along the spinous process of the lower back with a scalpel, the left and right lumbosacral fascia were dissected, and then the transverse processes of the fourth and fifth lumbar vertebrae were exposed, while the multifidus muscle and the longus muscle were separated and dissected between their muscular sheath. Soft tissues adhering to the transverse processes were removed, and then decortication of the cortical bone on the surface of the transverse processes was performed with an electric drill (OS-40MV2, Osada Electric Co., Ltd.) to prepare a bone graft bed. The bone graft (2 g) obtained above was sufficiently mixed with a microsphere suspension obtained by suspending microspheres containing the test compound (compound of Example 23) in an amount corresponding to 10, 30, or 100 μg of the test compound (prepared according to the method of Preparation 1) in 800 µl of CMC solution, introduced and grafted onto the transverse processes of the fourth and fifth lumbar vertebrae, and the bed of the bone graft between the transverse processes. After autologous bone grafting, the lumbosacral fascia, subcutaneous tissues, and skin were sutured, and the surgical site was disinfected. Twelve weeks after surgery, the animals were sacrificed with an overdose (30 mg/kg) of sodium pentobarbiturate (somnopentyl, Kyoritsuseiyaku Corporation), and then the lumbar vertebrae were isolated. Bone fusion was judged by manually confirming the mobility of the fourth and fifth lumbar vertebrae (manual palpation) in a blind manner, and soft x-ray images were obtained in one direction using Softex M-60 (Softex). Each soft x-ray image was evaluated by one person in a blind way in accordance with the evaluation criteria shown in table 9. The test was performed with groups, each of which consisted of 5 animals.

[0393]

[table 9]

index Evaluation criterion 3 Osteogenesis developed at the graft site, and the graft mass sutured the transverse processes and fused with the transverse processes, vertebral arch, or center with uniform continuity. 2 Osteogenesis developed at the graft site, and the graft mass sutured the transverse processes and fused with the transverse processes, the vertebral arch, or the center, but they did not constitute a single mass, and the continuity between the transverse processes was only partial, or breaks or radiolucent lines were observed in part of the graft mass. one Progressive osteogenesis was observed mainly around the transverse processes, but the graft mass between the transverse processes was not continuous, and certain tears or radiolucent lines interrupting the graft mass were observed. 0 Osteogenesis was not observed and the graft material did not change or was absorbed and disappeared.

[0394]

(2) Measurement results

When samples of the lumbar vertebra were separated and osteogenesis was assessed based on the degree of calcification using soft x-ray images, in the control group where microspheres not containing the test compound were added to the ilium, mobility was observed between the 4th and 5th lumbar vertebrae, but calcification between the transverse processes was not observed on soft x-rays in all five examples. On the other hand, in the group in which microspheres containing the test compound were administered at each of the doses of 10, 30, or 100 μg, it was found that mobility was observed between the 4th and 5th lumbar vertebrae in one example out of five examples, but no mobility was observed in four other examples. On soft x-rays, progression of osteogens and bone continuity between the transverse processes of the 4th and 5th lumbar vertebrae was observed, namely, the continuity score was 2 or higher at all doses. In addition, the index increased depending on the dose (table 10, 2.4±0.5, 2.6±0.5 and 2.8±0.4, respectively).

Based on these results, the compounds of the present invention were confirmed to be useful as an agent for promoting bone fusion in spinal fixation based on autologous bone grafting. In all compound administration groups, no animal death was observed, adverse reactions commonly observed with PGE ₂ administration were also not observed, and thus it was demonstrated that the aforementioned microspheres containing the compound of the present invention can be safely administered during spinal fixation.

[0395]

[table 10]

Control group 10 mcg/site 30 mcg/site 100 mcg/site Manual assessment of bone fusion 0/5 4/5 4/5 4/5 Continuity score based on soft x-ray images
(mean±SD) 0±0 2.4±0.5 2.6±0.5 2.8±0.4

Claims (70)

1. The compound represented by the following general formula (1): [Formula 1]

[where in formula (1) R ¹ represents -H or halogen; Ar ¹ is any substituent selected from the group G ¹ which may be substituted by 1-3 identical or different substituents selected from the group consisting of -F and methyl (provided that [formula 2]

excluded), where the group G ¹ is the group consisting of [formula 3]

(a and b represent the direction of attachment); Ar ² is any substituent selected from the group G ² which may be substituted by 1-3 identical or different substituents selected from the group consisting of cyano, -Cl, methyl, methoxy and phenyl (provided that [formula 4]

excluded), where the G ² group is a group consisting of phenyl, thienyl, furyl and thiazolyl; and * represents an asymmetric carbon], or a pharmaceutically acceptable salt thereof. 2. A compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein R ¹ is -H, -Cl, or -Br. 3. The compound or pharmaceutically acceptable salt thereof according to claim 2, wherein Ar ¹ is any substituent selected from the group consisting of [formula 5]

4. The compound or pharmaceutically acceptable salt thereof according to claim 2, wherein Ar ¹ is [formula 6]

5. A compound or pharmaceutically acceptable salt thereof according to claim 3 or 4, wherein Ar ² is any substituent selected from the group consisting of [formula 7]

6. A compound or pharmaceutically acceptable salt thereof according to claim 3 or 4, wherein Ar ² is any substituent selected from the group consisting of [formula 8]

7. A compound or pharmaceutically acceptable salt thereof according to claim 3 or 4, wherein Ar ² is any substituent selected from the group consisting of [formula 9]

8. A compound or pharmaceutically acceptable salt thereof according to claim 7, wherein R ¹ is -H. 9. A compound or pharmaceutically acceptable salt thereof according to claim 7, wherein R ¹ is -Cl. 10. A compound or a pharmaceutically acceptable salt thereof according to claim 7, wherein R ¹ is -Br. 11. The compound or its pharmaceutically acceptable salt according to claim 1, in which R ¹ represents -H, -Cl or -Br; Ar ¹ is any substituent selected from the group consisting of [formula 10]

Ar ² is any substituent selected from the group consisting of [formula 11]

12. Any compound selected from the following group, or a pharmaceutically acceptable salt thereof: [formula 12]

13. The compound below, or a pharmaceutically acceptable salt thereof: [formula 13]

14. The compound below, or a pharmaceutically acceptable salt thereof: [formula 14]

15. The compound below, or a pharmaceutically acceptable salt thereof: [formula 15]

16. The compound below, or a pharmaceutically acceptable salt thereof: [formula 16]

17. The compound below, or a pharmaceutically acceptable salt thereof: [formula 17]

18. The compound below, or its salt: [formula 18]

19. Pharmaceutical composition with excellent agonist activity against the EP ₄ receptor, containing an effective amount of a compound according to any one of paragraphs. 13-18 or a pharmaceutically acceptable salt thereof, as the active ingredient, and a pharmaceutically acceptable carrier. 20. Pharmaceutical composition for therapeutic treatment and/or stimulation of fracture healing, containing an effective amount of a compound according to any one of paragraphs. 13-18 or a pharmaceutically acceptable salt thereof, as the active ingredient, and a pharmaceutically acceptable carrier. 21. Pharmaceutical composition for stimulation of bone fusion during operations on the spine, containing an effective amount of the compound according to any one of paragraphs. 13-18 or a pharmaceutically acceptable salt thereof, as the active ingredient, and a pharmaceutically acceptable carrier.