Antipruritic drugs using PAC1 receptor antagonists

Abstract

To provide a novel antipruritic agent that has an action mechanism different from conventional antipruritic agents.SOLUTION: This invention provides an antipruritic agent containing a compound represented by the formula (I) in the figure, where R1 is a hydrogen atom, a C1-2-alkoxy group, or the like, and R2 is a hydrogen atom, a C2-6-alkenyloxy group, or the like.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to an antipruritic agent using a PAC1 receptor antagonist.

Background Art

[0002] Itching is one of the skin sensations experienced daily. However, due to the recent increase in the number of patients with allergic diseases and the significant aging of the population in developed countries, itching associated with dry skin, itching associated with systemic diseases such as kidney diseases and liver diseases, etc., an increase in various pruritic diseases is expected.

[0003] The first choice for itching treatment is antihistamines, but they are ineffective for itching associated with skin diseases such as atopic dermatitis and psoriasis, and itching associated with systemic diseases such as kidney diseases and liver diseases. For pruritus associated with atopic dermatitis, topical steroid drugs and topical tacrolimus drugs are used. However, topical steroid drugs have skin striae / atrophy and an increase in skin infections associated with reduced local immunity, and topical tacrolimus drugs cause local burning sensation, and about 30% of patients do not obtain sufficient effects. For such patient groups, short-term oral administration of steroids and cyclosporine is inevitably carried out, but serious adverse effects are a concern and they cannot be used for a long period of time.

[0004] This year, a new drug (dupilumab) has appeared for patients with high severity who do not sufficiently respond to treatment with topical steroid / tacrolimus drugs. This drug is a humanized anti-human IL-4 / IL-13 receptor monoclonal antibody and is said to be effective in about 40% of patients with high severity, but it is a biopharmaceutical and is expensive (the patient's out-of-pocket expense is tens of thousands of yen / month). Regarding psoriasis, biopharmaceuticals (humanized anti-human IL-17 receptor A monoclonal antibody) have also been introduced in recent years, but they are also expensive therapeutic drugs.

[0005] For pruritus in patients with renal failure undergoing dialysis and patients with liver failure, the κ-opioid receptor agonist nalflurafine has been used since 2009 and shows a high efficacy rate (effective in 70% of patients), but about 30% of patients do not obtain sufficient effects.

[0006] Therefore, the development of new antipruritic drugs with a mechanism of action different from these drugs and available at low cost is still desired.

[0007] PACAP (Pituitary Adenylate Cyclase Activating Polypeptide) is a neuropeptide isolated from sheep hypothalamus and structurally determined in 1989 using rat pituitary adenylate cyclase activity as an index. It causes mechanical allodynia (a phenomenon of feeling pain even when touched) via spinal cord PAC1 receptors (Non-Patent Document 1), but it is not clear what kind of pain it is involved in clinically (in humans).

[0008] At the animal experiment (mouse / rat) level, it has been suggested that PACAP is involved in peripheral nerve (spinal nerve) neuropathic pain (SNL model) (Non-Patent Document 2), but it is not clear which PACAP receptor (there are at least three types of PACAP receptors: PAC1, VPAC1, and VPAC2) is involved.

[0009] Patent Document 1 describes that PACAP has a sweat secretion promoting effect and is useful as a prophylactic or therapeutic agent for dry skin.

[0010] Non-Patent Document 3 shows that compounds PA-8 and PA-9 extracted from an existing compound database with over 4 million registered items have PAC1 receptor antagonistic and analgesic effects along with their structures, but their antipruritic effects have not been examined.

[0011] Compound PA-8 is a compound represented by the following formula (A):

Chemical formula

Chemical formula

[0012] Non-Patent Documents 4 and 5 describe the following formula (C):

Chemical Formula

Prior Art Documents

Patent Documents

[0013]

Patent Document 1

Non-Patent Documents

[0014]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Summary of the Invention

Problems to be Solved by the Invention

[0015] An object of the present invention is to provide a new antipruritic agent having an action mechanism different from that of conventional antipruritic agents.

Means for Solving the Problems

[0016] In order to solve the above problems, the present inventors have found that a specific compound has an excellent antipruritic effect among PAC1 receptor antagonist candidate compounds, and have thus completed the present invention.

[0017] That is, the gist of the present invention is as follows. (1) The following formula (I):

Chemical formula

Chemical formula

[0018] The antipruritic agent of the present invention contains a PAC1 receptor antagonist as an active ingredient, has a mechanism of action different from that of current antipruritic agents such as antihistamines, steroids, and immunosuppressive agents, and can avoid the adverse effects seen in steroids and immunosuppressive agents.

[0019] In addition, since the antipruritic agent of the present invention has action points both in the periphery (intradermal) and the central nervous system (posterior horn of the spinal cord), it has high efficacy and may reduce the need for combination therapy, thus avoiding the occurrence of unexpected adverse effects associated with combination therapy.

[0020] Since more than 80% of the elderly have dry skin and half of them complain of itching, the need for antipruritic agents for itching associated with dry skin in Japan, a super-aged society, is high. The antipruritic agent of the present invention can be expected to improve the quality of life (QOL) of the elderly with itching associated with dry skin. [Brief Description of the Drawings]

[0021]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13

Figure 14

Mode for Carrying Out the Invention

[0022] Hereinafter, the present invention will be described in detail.

[0023] In the formula (I), R 1 or R 2 Examples of the C 1-6 -alkyl group represented by are, for example, methyl group, ethyl group, propyl group, isopropyl group, butyl group, isobutyl group, sec-butyl group (1-methylpropyl group), tert-butyl group, pentyl group, isopentyl group, 1-ethylpropyl group, hexyl group, cyclopropyl group, cyclobutyl group, cyclopentyl group, cyclohexyl group.

[0024] In the formula (I), R 1 or R 2 Examples of the C 1-6 -alkoxy group represented by are, for example, methoxy group, ethoxy group, propoxy group, isopropoxy group, butoxy group, isobutoxy group, sec-butoxy group, tert-butoxy group, pentyloxy group, isopentyloxy group, hexyloxy group, cyclopropyloxy group, cyclobutyloxy group, cyclopentyloxy group, cyclohexyloxy group.

[0025] In the formula (I), R 1 or R 2 Examples of the C 2-6 -alkenyloxy group represented by are, for example, vinyloxy group, 1-propenyloxy group, allyloxy group, 1-butenyloxy group, 2-butenyloxy (crotyloxy) group, pentyloxy group, 3-methyl-2-butenyloxy (prenoxy) group, hexyloxy group.

[0026] In the formula (I), R 1 or R 2Examples of the halogen atom represented by [ [ ID = 0 ] ] include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom.

[0027] In the formula (I), R 1 or R 2 Examples of the C 1-6 -haloalkyl group represented by [ [ ID = 10 ] ] include a trifluoromethyl group.

[0028] In the formula (I), R 1 or R 2 Examples of the C 1-6 -haloalkoxy group represented by [ [ ID = 20 ] ] include a trifluoromethoxy group.

[0029] In the formula (I), R 1 or R 2 The phenyl group represented by [ [ ID = 28 ] ] may be substituted with one or more substituents selected from a C 1-6 -alkyl group, a C 1-6 -alkoxy group, a methylenedioxy group, a C 2-6 -alkenyloxy group, an aralkyloxy group (for example, a benzyloxy group, a 4-methylbenzyloxy group, a 3-methylbenzyloxy group, a 2-methylbenzyloxy group, a 4-fluorobenzyloxy group, a 3-fluorobenzyloxy group, a 4-chlorobenzyloxy group, a 3-chlorobenzyloxy group), a halogen atom, a C 1-6 -haloalkyl group, a C 1-6 -haloalkoxy group, a substituted or unsubstituted phenyl group, an acyl group (for example, a C 1-6 -aliphatic acyl group such as a formyl group, an acetyl group, a propanoyl group, a butanoyl group, a pentanoyl group, a hexanoyl group; an aroyl group such as a benzoyl group, a toluoyl group), an acyloxy group (for example, a C 1-6 -aliphatic acyloxy group such as a formyloxy group, an acetoxy group, a propanoyloxy group, a butanoyloxy group, a pentanoyloxy group, a hexanoyloxy group; an aroyl oxy group such as a benzoyloxy group, a toluoyloxy group), a hydroxyl group, a carboxyl group, an acetamido group, a carbamoyl group, a cyano group, a nitro group, etc.

[0030] As the compound represented by the formula (I), R 1 is a C 1-6 -alkoxy group or a C 1-6 -haloalkoxy group (e.g., trifluoromethoxy group), and the compound is preferred.

[0031] The indazolyl group substituted with a halogen atom represented by R in the formula (II) is not particularly limited as long as it is an indazolyl group substituted with at least one halogen atom (preferably a chlorine atom) selected from a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom, but preferably a 3-indazolyl group substituted with at least one chlorine atom.

[0032] Examples of the aralkyl group represented by R in the formula (II) include a benzyl group and a phenethyl group.

[0033] The phenyl group and the aralkyl group represented by R in the formula (II) are C 1-6 -alkyl groups (e.g., methyl group, ethyl group, propyl group, isopropyl group, butyl group, isobutyl group, sec-butyl group (1-methylpropyl group), tert-butyl group, pentyl group, isopentyl group, 1-ethylpropyl group, hexyl group, cyclopropyl group, cyclobutyl group, cyclopentyl group, cyclohexyl group), C 1-6 -alkoxy groups (e.g., methoxy group, ethoxy group, propoxy group, isopropoxy group, butoxy group, isobutoxy group, sec-butoxy group, tert-butoxy group, pentyloxy group, isopentyloxy group, hexyloxy group, cyclopropyloxy group, cyclobutyloxy group, cyclopentyloxy group, cyclohexyloxy group), methylenedioxy group, C 2-6 -alkenyloxy groups, aralkyloxy groups (e.g., benzyloxy group, 4-methylbenzyloxy group, 3-methylbenzyloxy group, 2-methylbenzyloxy group, 4-fluorobenzyloxy group, 3-fluorobenzyloxy group, 4-chlorobenzyloxy group, 3-chlorobenzyloxy group), halogen atoms (fluorine atom, chlorine atom, bromine atom, iodine atom), C 1-6 -haloalkyl groups, C1-6 - a haloalkoxy group, a substituted or unsubstituted phenyl group, an acyl group (e.g., a formyl group, an acetyl group, a propanoyl group, a butanoyl group, a pentanoyl group, a hexanoyl group, etc., C 1-6 - aliphatic acyl group; aroyl group such as benzoyl group, toluoyl group), an acyloxy group (e.g., a formyloxy group, an acetoxy group, a propanoyloxy group, a butanoyloxy group, a pentanoyloxy group, a hexanoyloxy group, etc., C 1-6 - aliphatic acyloxy group; aroyloxy group such as benzoyloxy group, toluoyloxy group), a hydroxyl group, a carboxyl group, an acetamido group, a carbamoyl group, a cyano group, a nitro group, etc. may be substituted with one or more substituents selected from the group consisting of.

[0034] As the salt of the compound represented by the formula (I) or (II), a pharmaceutically acceptable salt is preferred. For example, inorganic acids such as hydrochloric acid, sulfuric acid, phosphoric acid, hydrobromic acid, hydroiodic acid, nitric acid, pyrosulfuric acid, metaphosphoric acid, or organic acids such as citric acid, benzoic acid, acetic acid, propionic acid, fumaric acid, maleic acid, sulfonic acid (e.g., methanesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid), etc. And salts thereof.

[0035] Examples of the solvate of the compound represented by the formula (I) or (II) or a salt thereof include hydrates.

[0036] The compound represented by the formula (I) can be produced, for example, according to the methods described in Shi, D. Q. et al. J. Heterocyclic Chem. 2009, 46, 1331-1334, or Tu, S. et al. Bioorg. Med. Chem. Lett. 2006, 16, 3578-3581, as shown below.

[0037]

Chemical formula

[0038] That is, the target compound (I) can be produced by reacting the corresponding aromatic aldehyde compound, 2,4-diamino-6-hydroxypyrimidine (alias, 2,6-diaminopyrimidin-4(3H)-one), and Meldrum's acid (i) in water in the presence of triethylbenzylammonium chloride under heating, (ii) under heating under microwave irradiation, or (iii) in an organic solvent under heating.

[0039] The compound represented by the formula (II) can be produced, for example, according to the method described in JP-T-2006-510596 as follows.

[0040]

Chemical formula

[0041] That is, the target compound (II) can be produced by reacting the corresponding amine compound with itaconic acid to convert it into γ-lactam carboxylic acid and then reacting it with histamine in the presence of a condensing agent (for example, carbodiimide).

[0042] To purify the product obtained as described above, commonly used methods such as column chromatography using silica gel or the like as a carrier or a recrystallization method using methanol, ethanol, chloroform, dimethyl sulfoxide, n-hexane-ethyl acetate, water, etc. may be used. Examples of the elution solvent for column chromatography include methanol, ethanol, chloroform, acetone, hexane, dichloromethane, ethyl acetate, and mixed solvents thereof.

[0043] The above-mentioned compound can be formulated as an antipruritic agent in combination with a conventional pharmaceutical carrier. The dosage form is not particularly limited and can be appropriately selected and used as needed. Oral preparations such as tablets, capsules, granules, fine granules, powders, sustained-release preparations, solutions, suspensions, emulsions, syrups, elixirs, etc., and parenteral preparations such as injections and suppositories can be mentioned.

[0044] Oral preparations are produced by conventional methods using, for example, starch, lactose, sucrose, mannitol, carboxymethyl cellulose, inorganic salts, etc. In addition to these, binders, disintegrants, surfactants, lubricants, fluidity promoters, flavoring agents, coloring agents, fragrances, etc. can be appropriately added.

[0045] Examples of binders include starch, dextrin, gum arabic, gelatin, hydroxypropyl starch, methyl cellulose, sodium carboxymethyl cellulose, hydroxypropyl cellulose, crystalline cellulose, ethyl cellulose, polyvinylpyrrolidone, macrogol, etc.

[0046] Examples of disintegrants include starch, hydroxypropyl starch, sodium carboxymethyl cellulose, calcium carboxymethyl cellulose, carboxymethyl cellulose, low-substituted hydroxypropyl cellulose, etc.

[0047] Examples of surfactants include sodium lauryl sulfate, soybean lecithin, sucrose fatty acid ester, polysorbate 80, etc.

[0048] Examples of lubricants include talc, waxes, hydrogenated vegetable oils, sucrose fatty acid esters, magnesium stearate, calcium stearate, aluminum stearate, polyethylene glycol, etc.

[0049] Examples of fluidity promoters include light anhydrous silicic acid, dried aluminum hydroxide gel, synthetic aluminum silicate, magnesium silicate, etc.

[0050] The injection is manufactured according to conventional methods, and generally, distilled water for injection, physiological saline, glucose aqueous solution, olive oil, sesame oil, peanut oil, soybean oil, corn oil, propylene glycol, polyethylene glycol, etc. can be used as diluents. Furthermore, if necessary, bactericides, preservatives, stabilizers, isotonic agents, soothing agents, etc. may be added. Also, from the perspective of stability, after filling into vials or the like, it can be frozen, the moisture can be removed by ordinary lyophilization technology, and the liquid preparation can be re-prepared from the lyophilized product immediately before use. The proportion of the compound of formula (I) or (II) in the injection can vary between 5 and 50% by weight, but is not limited thereto.

[0051] Examples of other parenteral preparations include suppositories for rectal administration, external preparations for topical administration (e.g., eye drops, ointments, pastes), etc., which are manufactured according to conventional methods.

[0052] The formulated antipruritic drug varies depending on the dosage form, administration route, etc. For example, it can be administered 1 to 4 times a day for a period of 1 week to 3 months.

[0053] In order to exhibit the expected effect as an oral preparation, although it varies depending on the patient's age, weight, and degree of the disease, in the case of an ordinary adult, as the weight of the compound of formula (I) or (II), for example, 0.1 to 1000 mg, preferably 1 to 500 mg, is appropriately taken in divided doses several times a day.

[0054] In order to exhibit the expected effect as a parenteral preparation, although it varies depending on the patient's age, weight, and degree of the disease, in the case of an ordinary adult, as the weight of the compound of formula (I) or (II), for example, 0.1 to 1000 mg, preferably 1 to 500 mg, is appropriately administered by intravenous injection, intravenous drip, subcutaneous injection, intramuscular injection, intraperitoneal administration, intrathecal (subarachnoid space) administration.

[0055] The antipruritic agent of the present invention can be used for treating and / or preventing dry skin diseases such as dry skin and xeroderma, allergic dermatitis such as contact dermatitis and atopic dermatitis, psoriasis, itching caused by insect bites, plant dermatitis, and athlete's foot.

[0056] This specification includes the contents described in the specification and drawings of Japanese Patent Application No. 2019-034313, which is the basis of the priority of this application.

Example

[0057] Hereinafter, the present invention will be described in more detail with reference to examples, but the scope of the present invention is not limited thereto.

[0058] [Example 1] Synthesis of pyrido[2,3-d]pyrimidine derivatives

Chemical formula

[0059] Literature 1) According to the method described in the literature, under an Ar atmosphere, commercially available aldehydes (1a, b, d, e, f, g, j, k, m, n, o, p, q, r) or literature-known aldehydes (1c, h, i, l, s, t) (1.00 mmol) were added sequentially to a solution of 2,4-diamino-6-hydroxypyrimidine (0.67 mmol) and Meldrum's acid (1.00 mmol) in ethylene glycol (0.5 mL) at room temperature, and the mixture was stirred at 100 °C for 20 hours. After cooling, the reaction solution was filtered and further washed with methanol (0.5 mL × 3) to obtain pale yellow crystals 2a~u.

[0060] 2-Amino-5-(4-ethoxy-3-methylphenyl)-5,8-dihydro-3H,6H-pyrido[2,3-d]pyrimidine-4,7-dione (2a) Yield: 46%; mp: >300 ℃; IR (KBr): 3455, 3166, 2856, 2699, 1578, 1477 cm -1 ; 11H NMR (400 MHz, DMSO-d6): δ 10.55 (1H, br s), 10.01 (1H, s), 6.90 (1H, d, J = 2.4 Hz), 6.84 (1H, dd, J = 8.8, 2.4 Hz), 6.77 (1H, d, J = 8.8 Hz), 6.51 (2H, br s), 4.00 (1H, d, J = 7.6 Hz), 3.95 (2H, q, J = 7.0 Hz), 2.88 (1H, dd, J = 16.0, 7.6 Hz), 2.42 (1H, d, J = 16.0 Hz), 2.07 (3H, s), 1.29 (3H, t, J = 7.0 Hz); 13 13C NMR (100 MHz, DMSO-d6): δ 171.10, 161.42, 156.45, 155.20, 154.95, 135.08, 128.74, 125.49, 124.61, 111.22, 92.07, 63.14, 32.12, 16.21, 14.83; MS (EI) m / z 314(M+).

[0061] 2-Amino-5-(4-ethoxy-3-methoxyphenyl)-5,8-dihydro-3H,6H-pyrido[2,3-d]pyrimidine-4,7-dione (2b) Yield: 37%; mp: >300 ℃; IR (KBr): 3462, 3309, 2850, 2743, 1582, 1521 cm -1 ; 11H NMR (400 MHz, DMSO-d6): δ 10.57 (1H, br s), 10.02 (1H, s), 6.84 (1H, d, J = 2.3 Hz), 6.78 (1H, d, J = 8.7 Hz), 6.53 (1H, dd, J = 8.7, 2.3 Hz), 6.50 (2H, br s), 4.05 (1H, d, J = 7.7 Hz), 3.92 (2H, q, J = 6.9 Hz), 3.69 (3H, s), 2.88 (1H, dd, J = 16.4, 7.7 Hz), 1.27 (3H, t, J = 6.9 Hz); 13 13C NMR (100 MHz, DMSO-d6): δ 171.34, 161.47, 156.43, 155.01, 148.89, 146.63, 136.09, 117.65, 112.90, 111.24, 92.03, 63.73, 55.38, 38.71, 32.44, 14.81; MS (EI) m / z 330 (M+).

[0062] 2-Amino-5-(3-methoxy-4-propoxyphenyl)-5,8-dihydro-3H,6H-pyrido[2,3-d]pyrimidine-4,7-dione (2c) Yield: 38%; mp: >300 ℃; IR (KBr): 3450, 3167, 2959, 2876, 1652, 1591 cm -1 ; 11H NMR (400 MHz, DMSO-d6): δ 10.57 (1H, br s), 10.03 (1H, br s), 6.83 (1H, d, J = 2.3 Hz), 6.79 (1H, d, J = 8.4 Hz), 6.52 (1H, dd, J = 8.4, 2.3 Hz), 6.51 (2H, br s), 4.05 (1H, d, J = 7.9 Hz), 3.82 (2H, t, J = 7.1 Hz), 3.69 (3H, s), 2.88 (1H, dd, J = 16.4, 7.9 Hz), 1.67 (2H, sext, J = 7.1 Hz), 0.92 (3H, t, J = 7.1 Hz); 13 13C NMR (100 MHz, DMSO-d6): δ 171.22, 161.69, 158.44, 157.50, 146.831, 145.48, 137.33, 117.79, 113.05, 111.44, 99.65, 69.86, 55.57, 38.73, 32.52, 22.20, 10.53; MS (EI) m / z 344 (M+).

[0063] 2-Amino-5-(3,4-diethoxyphenyl)-5,8-dihydro-3H,6H-pyrido[2,3-d]pyrimidine-4,7-dione (2d) Yield: 41%; mp: >300 ℃; IR (KBr): 3188, 2977, 2868, 1653, 1635 cm -1 ; 11H NMR (400 MHz, DMSO-d6): δ 10.57 (1H, s), 10.01 (1H, s), 6.81 (1H, d, J = 1.8 Hz), 6.78 (1H, d, J = 8.8 Hz), 6.53 (1H, dd, J = 8.8, 1.8 Hz), 6.50 (2H, br s), 4.03 (1H, d, J = 7.8 Hz), 3.93 (2H, q, J = 6.9 Hz), 3.92 (2H, q, J = 6.9 Hz), 2.87 (1H, dd, J = 16.0, 7.8 Hz), 1.28 (3H, t, J = 6.9 Hz), 1.26 (3H, t, J = 6.9 Hz); 13 13C NMR (100 MHz, DMSO-d6): δ 171.27, 161.47, 156.42, 154.97, 148.11, 146.87, 136.19, 117.94, 113.45, 112.69, 92.07, 63.83, 63.78, 38.66, 32.37, 14.82; MS (EI) m / z 344 (M+).

[0064] 2-Amino-5-(3,4-difluorophenyl)-5,8-dihydro-3H,6H-pyrido[2,3-d]pyrimidine-4,7-dione (2e) Yield: 34%; mp: >300 ℃; IR (KBr): 3471, 3161, 1646, 1592 cm -1 ; 1 1H-NMR (400 MHz, DMSO-d6): δ 10.62 (1H, br s), 10.13 (1H, br s), 7.32 (1H, dt, J = 10.8, 8.4 Hz), 7.18 (1H, ddd, J = 10.8, 8.4, 2.4 Hz), 6.95 (1H, m), 6.57 (2H, br s), 4.12 (1H, d, J = 7.2 Hz), 2.94 (1H, dd, J = 16.3, 7.2 Hz); 1313C NMR (125 MHz, DMSO-d6): δ 170.86, 161.41, 156.68, 155.24, 149.02, 147.17, 141.45, 122.88, 117.41 (d, J = 17.0 Hz), 115.62 (d, J = 17.0 Hz), 91.05, 38.22, 32.24; MS (EI) m / z 292 (M+).

[0065] 2-Amino-5-(3-bromo-4-ethoxyphenyl)-5,8-dihydro-3H,6H-pyrido[2,3-d]pyrimidine-4,7-dione (2f) Yield: 40%; mp: >300 ℃; IR (KBr): 3458, 3080, 2863, 2751, 1540, 1475 cm -1 ; 1 1H NMR (400 MHz,DMSO-d6): δ 10.57 (1H, br s), 10.09 (1H, s), 7.30 (1H, d, J = 2.4 Hz), 7.06 (1H, dd, J = 8.0, 2.4 Hz), 6.99 (1H, d, J = 8.0 Hz), 6.56 (2H, br s), 4.09 (1H, m), 4.04 (2H, q, J = 7.1 Hz), 2.90 (1H, dd, J = 16.2, 6.7 Hz), 1.31 (3H, t, J = 7.1 Hz); 13 13C NMR (100 MHz, DMSO-d6): δ170.93, 161.39, 156.55, 155.10, 153.29, 137.33, 130.87, 126.80, 113.77, 110.89, 91.53, 64.39, 38.47, 31.82, 14.58; MS (EI) m / z 379 (M+).

[0066] 2-Amino-5-(3-ethoxy-4-methoxyphenyl)-5,8-dihydro-3H,6H-pyrido[2,3-d]pyrimidine-4,7-dione (2g) Yield: 45%; mp: >300 ℃; IR (KBr): 3461, 3160, 2841, 1591, 1516 cm -1 ; 1 1H NMR (400 MHz, DMSO-d6): δ 10.58 (1H, br s), 10.03 (1H, s), 6.82 (1H, d, J = 2.4 Hz), 6.80 (1H, d, J = 8.4 Hz), 6.55 (1H, dd, J = 8.4, 2.4 Hz), 6.50 (1H, br s), 4.04 (1H, d, J = 7.8 Hz), 3.96 - 3.88 (2H, m), 3.68 (3H, s), 2.87 (1H, dd, J = 16.0, 7.8 Hz), 1.28 (3H, t, J = 7.2 Hz); 13 13C NMR (100 MHz, DMSO-d6): δ 171.30, 161.55, 156.51, 155.00, 147.88, 147.66, 136.10, 117.81, 112.20, 111.90, 92.11, 63.68, 55.54, 38.72, 32.42, 14.83; MS (EI) m / z 330 (M+).

[0067] 5-(3 - Allyloxy - 4 - methoxyphenyl)-2 - amino - 5,8 - dihydro - 3H,6H - pyrido[2,3 - d]pyrimidine - 4,7 - dione (2h) Yield: 35%; mp: >300 ℃; IR (KBr): 3462, 3169, 1636, 1617, 1591 cm -1 ; 11H NMR (400 MHz, DMSO-d6): δ 10.58 (1H, br s), 10.02 (1H, s), 6.84 (1H, d, J = 2.0 Hz), 6.81 (1H, d, J = 8.4 Hz), 6.57 (1H, dd, J = 8.4, 2.0 Hz), 5.99 (1H, ddd, J = 17.6, 10.4, 5.2 Hz), 5.36 (1H, dd, J = 17.6, 2.0 Hz), 5.22 (1H, dd, J = 10.4, 2.0 Hz), 4.46 (2H, d, J = 5.2 Hz), 4.03 (1H, d, J = 7.2 Hz), 3.68 (3H, s), 2.87 (1H, dd, J = 15.8, 7.2 Hz); 13 13C NMR (100 MHz, DMSO-d6): δ 171.16, 158.72, 156.11, 154.99, 147.79, 147.55, 136.10, 133.95, 118.27, 117.88, 112.81, 112.09, 92.02, 69.10, 55.65, 32.41; MS (EI) m / z 342 (M+).

[0068] 2-Amino-5-(4-methoxy-3-propoxyphenyl)-5,8-dihydro-3H,6H-pyrido[2,3-d]pyrimidine-4,7-dione (2i) Yield: 41%; mp: >300 ℃; IR (KBr): 3463, 3160, 2846, 1695, 1591, 1539, 1516 cm -1 ; 11H NMR (400 MHz, DMSO-d6): δ 10.58 (1H, br s), 10.03 (1H, s), 6.82 (1H, d, J = 2.4 Hz), 6.80 (1H, dd, J = 8.4, 2.4 Hz), 6.55 (1H, d, J = 8.4 Hz), 6.51 (2H, br s), 4.04 (1H, d, J = 7.2 Hz), 3.84 - 3.81 (2H, m), 3.67 (3H, s), 2.87 (1H, dd, J = 16.4, 7.2 Hz), 1.68 (2H, sext, J = 6.8 Hz), 0.94 (3H, t, J = 6.8 Hz); 13 13C NMR (100 MHz, DMSO-d6): δ 171.24, 161.48, 156.43, 154.96, 148.04, 147.68, 136.14, 117.78, 112.27, 112.04, 92.06, 69.63, 55.61, 38.66, 32.38, 22.12, 10.46; MS (EI) m / z 344 (M+).

[0069] 2-Amino-5-(3-ethoxyphenyl)-5,8-dihydro-3H,6H-pyrido[2,3-d]pyrimidine-4,7-dione (2j): Yield: 43%; mp: >300 ℃; IR (KBr): 3447, 3170, 2854, 1592, 1539 cm -1 ; 11H NMR (400 MHz, DMSO-d6): δ 10.58 (1H, br s), 10.06 (1H, s), 7.15 (1H, t, J = 7.8 Hz), 6.72 (1H, dd, J = 7.8, 2.4 Hz), 6.70 (1H, dd, J = 7.8, 2.4 Hz), 6.66 (1H, d, J = 2.4 Hz), 6.53 (2H, br s), 4.07 (1H, d, J = 7.4 Hz), 3.94 (2H, q, J = 6.0 Hz), 2.91 (1H, dd, J = 16.3, 7.4 Hz), 1.28 (3H, t, J = 6.0 Hz); 13 13C NMR (100 MHz, DMSO-d6): δ 171.04, 161.40, 158.57, 156.59, 155.03, 145.32, 129.28, 118.56, 113.19, 111.58, 91.65, 62.78, 38.50, 32.86, 14.65; MS (EI) m / z 300 (M+).

[0070] 5-(3-Allyloxyphenyl)-2-amino-5,8-dihydro-3H,6H-pyrido[2,3-d]pyrimidine-4,7-dione (2k) Yield: 45%; mp: >300 ℃; IR (KBr): 3085, 2855, 2727, 1520 cm -1 ; 11H NMR (400 MHz, DMSO-d6): δ 10.58 (1H, br s), 10.06 (1H, s), 7.15 (1H, t, J = 7.6 Hz), 6.76 (1H, dd, J = 7.6, 2.4 Hz), 6.71 (2H, m), 6.53 (2H, br s), 6.00 (1H, ddd, J = 17.4, 10.6, 5.5 Hz), 5.36 (1H, dd, J = 17.4, 1.6 Hz), 5.23 (1H, dd, J = 10.6, 1.6 Hz), 4.48 (2H, d, J = 5.5 Hz), 4.07 (1H, d, J = 7.5 Hz), 3.89 (1H, dd, J = 16.2, 7.5 Hz); 13 13C NMR (100 MHz, DMSO-d6): δ 171.09, 161.41, 158.26, 156.58, 155.06, 145.35, 133.75, 129.48, 118.78, 117.56, 113.43, 112.03, 91.60, 68.11, 38.50, 32.86; MS (EI) m / z 312 (M+).

[0071] 2-Amino-5-(3-chloro-4-ethoxyphenyl)-5,8-dihydro-3H,6H-pyrido[2,3-d]pyrimidine-4,7-dione (2l) Yield: 30%; mp: >300 ℃; 1 1H NMR (400 MHz, DMSO-d6): δ 10.60 (1H, br s), 10.09 (1H, s), 7.15(1H, s), 7.02 (2H, s), 6.55 (2H, br s), 4.06 (1H, d, J = 8.0 Hz), 4.05 (2H, q, J = 7.0 Hz), 2.91 (1H, dd, J = 16.0, 8.0 Hz), 1.31 (3H, t, J = 7.0 Hz); 1313C NMR (100 MHz, DMSO-d6): δ 171.0, 161.38, 156.52, 155.11, 153.38, 136.81, 127.90, 126.09, 121.07, 113.90, 91.53, 64.28, 38.42, 31.87, 14.57.

[0072] 2-Amino-5-(3-chlorophenyl)-5,8-dihydro-3H,6H-pyrido[2,3-d]pyrimidine-4,7-dione (2m) Yield: 35%; mp: >300 ℃; 1 1H NMR (400 MHz, DMSO-d6): δ 10.61 (1H, br s), 10.11 (1H, s), 7.30(1H, t, J = 8.0 Hz), 7.24 (1H, d, J = 8.0 Hz), 7.16 (1H, s), 7.11 (1H, d, J = 8.0 Hz), 6.57 (2H, br s), 4.13 (1H, d, J = 8.0 Hz), 2.95 (1H, dd, J = 16.0, 8.0 Hz); 13 13C NMR (100 MHz, DMSO-d6): δ 170.85, 161.42, 156.76, 155.18, 146.35, 133.07, 130.46, 126.49, 126.41, 125.20, 91.61, 38.24, 32.72.

[0073] 2-Amino-5-(3-bromophenyl)-5,8-dihydro-3H,6H-pyrido[2,3-d]pyrimidine-4,7-dione (2n) Yield: 25%; mp: >300 ℃; 1H NMR (400 MHz, DMSO-d6): δ 10.61 (1H, br s), 10.12 (1H, s), 7.38(1H, d, J = 8.0, 1.8 Hz), 7.31 (1H, t, J = 1.8 Hz), 7.24 (1H, t, J = 8.0 Hz), 7.14 (1H, d, J= 8.0 Hz), 6.57 (2H, br s), 4.12 (1H, d, J = 8.0 Hz), 2.95 (1H, dd, J = 16.2, 8.0 Hz); 13 C NMR (100 MHz, DMSO-d6): δ 170.81, 161.40, 156.74, 155.18, 146.63, 130.77, 129.38, 129.30, 125.57, 121.78, 91.03, 38.25, 32.71.

[0074] 2-Amino-5-(3-trifluoromethoxyphenyl)-5,8-dihydro-3H,6H-pyrido[2,3-d]pyrimidine-4,7-dione (2o): Yield: 23%; mp: >300 ℃; 1 H NMR (400 MHz, DMSO-d6): δ 10.64 (1H, br s), 10.15 (1H, s), 7.40(1H, t, J = 8.0 Hz), 7.17 (2H, t, J = 8.0 Hz), 7.12 (1H, s), 6.58 (2H, br s), 4.18 (1H, d, J= 7.2 Hz), 2.97 (1H, dd, J = 16.2, 7.2 Hz); 13 C NMR (100 MHz, DMSO-d6): δ 171.26, 161.71, 156.92, 155.40, 148.72, 146.70, 130.66, 120.22 (q, J = 254.60 Hz), 119.21, 119.08, 116.41, 91.23, 38.31, 32.81.

[0075] 2-Amino-5-(3-methoxyphenyl)-5,8-dihydro-3H,6H-pyrido[2,3-d]pyrimidine-4,7-dione (2p) Yield: 35%; mp: >300 ℃; 1 H NMR (400 MHz, DMSO-d6): δ 10.60 (1H, br s), 10.07 (1H, s), 7.17(1H, t, J = 7.6 Hz), 6.75 (1H, dd, J = 7.6, 2.4 Hz), 6.71 (1H, d, J = 2.4 Hz), 6.70 (1H, s),6.54 (2H, br s), 4.08 (1H, d, J = 7.2 Hz), 3.69 (3H, s), 2.92 (1H, dd, J = 16.0, 7.2 Hz); 13 C NMR (100 MHz, DMSO-d6): δ 171.24, 161.45, 159.34, 156.54, 155.11, 145.32, 129.53, 118.56,112.85, 111.29, 91.65, 54.91, 38.51, 32.89.

[0076] 2-Amino-5-(3-propoxyphenyl)-5,8-dihydro-3H,6H-pyrido[2,3-d]pyrimidine-4,7-dione (2q) Yield: 32%; mp: >300 ℃; 1 H NMR (400 MHz, DMSO-d6): δ 10.58 (1H, br s), 10.05 (1H, s), 7.15(1H, t, J = 8.0 Hz), 6.74-6.67 (3H, m), 6.52 (2H, br s), 4.07 (1H, d, J = 8.0 Hz), 3.84 (2H,t, J = 6.4 Hz), 2.91 (1H, dd, J = 16.4, 8.0 Hz), 1.69 (2H, sext, J = 6.4 Hz), 0.94 (3H, t, J= 6.4 Hz); 1313C NMR (100 MHz, DMSO-d6): δ 171.09, 161.47, 158.76, 156.62, 155.05, 145.34, 129.50, 118.54, 112.19, 111.74, 91.68, 68.76, 38.52, 32.89, 22.05, 10.47.

[0077] 2-Amino-5-biphenyl-3-yl-5,8-dihydro-3H,6H-pyrido[2,3-d]pyrimidine-4,7-dione (2r) Yield: 37%; mp: >300 °C; 1 1H NMR (400 MHz, DMSO-d6): δ 10.58 (1H, br s), 10.09 (1H, s), 7.53(2H, dd, J = 8.0, 1.2 Hz), 7.42 (5H, m), 7.32 (2H, t, J = 8.0 Hz), 7.01 (1H, d, J = 8.0 Hz),6.51 (2H, brs), 4.17 (1H, d, J = 7.6 Hz), 2.95 (1H, dd, J = 16.0, 7.6 Hz); 13 13C NMR (100 MHz,DMSO-d6): δ 171.38, 161.86, 155.22, 144.66, 140.62, 140.46, 129.37, 129.21, 127.71, 126.94,126.85, 125.65, 125.20, 91.95, 62.93, 38.71, 33.17.

[0078] 2-Amino-5-(3-ethylphenyl)-5,8-dihydro-3H,6H-pyrido[2,3-d]pyrimidine-4,7-dione (2s) Yield: 37% ; mp: >300 °C; 1H NMR (400 MHz, DMSO-d6): δ 10.60 (1H, br s), 10.07 (1H, s), 7.15 (1H, t, J = 8.0 Hz), 7.01 (1H, d, J = 8.0 Hz), 7.00 (1H, s), 6.91 (1H, d, J = 8.0 Hz), 6.53 (2H, br s), 4.08 (1H, d, J = 7.2 Hz), 2.93 (1H, dd, J = 16.6, 7.2 Hz), 2.52 (2H, q, J = 7.6 Hz), 1.13 (3H, t, J = 7.6 Hz); 13 C NMR (100 MHz, DMSO-d6): δ 171.27, 161.52, 156.60, 155.09, 143.76, 128.43, 126.14, 125.87, 123.66, 91.75, 62.82, 32.95, 28.24, 15.61.

[0079] 2-Amino-5-(3-propylphenyl)-5,8-dihydro-3H,6H-pyrido[2,3-d]pyrimidine-4,7-dione (2t) Yield: 18%; mp: >300 ℃; 1 H NMR (400 MHz, DMSO-d6): δ 10.57 (1H, br s), 10.05 (1H, s), 7.14(1H, t, J = 7.6 Hz), 6.98 (1H, d, J = 7.6 Hz), 6.97 (1H, s), 6.91 (1H, d, J = 7.6 Hz), 6.52 (2H, br s), 4.07 (1H, d, J = 8.0 Hz), 2.92 (1H, dd, J = 16.2, 8.0 Hz), 1.52 (2H, sext, J = 7.2 Hz), 0.86 (3H, t, J = 7.2 Hz); 13¹³C NMR (100 MHz, DMSO-d6): δ 171.20, 161.48, 156.59, 155.04, 143.66, 142.24, 128.32, 126.63, 126.43, 123.73, 91.75, 38.87, 37.38, 32.90, 24.18, 13.74.

[0080] 2-Amino-5-(3-butylphenyl)-5,8-dihydro-3H,6H-pyrido[2,3-d]pyrimidine-4,7-dione (2u) Yield: 30%; mp: >300 ℃; 1 ¹H NMR (400 MHz, DMSO-d6): δ 10.57 (1H, br s), 10.01 (1H, s), 7.13(1H, t, J = 7.6 Hz), 6.98 (1H, d, J = 7.6 Hz), 6.97 (1H, s), 6.90 (1H, d, J = 7.6 Hz), 6.52 (2H, br s), 4.07 (1H, d, J = 8.0 Hz), 2.92 (1H, dd, J = 16.4, 8.0 Hz), 1.48 (2H, quin, J = 7.4 Hz), 1.27 (2H, sext, J = 7.4 Hz), 0.87 (3H, t, J = 7.4 Hz); 13 ¹³C NMR (100 MHz, DMSO-d6): δ 171.16, 161.46, 156.59, 155.04, 143.66, 142.42, 128.33, 126.59, 126.39, 123.67, 91.76, 38.70, 34.93, 33.20, 32.90, 21.82, 13.82.

[0081] 1) Tu, S. et al. Bioorg. Med. Chem. Lett. 2006, 16, 3578-3581. 2) Muskinja, J. et al. Med. Chem. Res. 2016, 25, 1744-1753. 3) McDonald, B. et al. Org. Lett. 2015, 17, 98-101. 4) U.S. Patent Application Publication No. 2015 / 0210682 5) Special Publication No. 2010-526138 6) Wang, B. et al. Eur. J. Org. Chem. 2009, 22, 3688-3692. 7) WO2008 / 136756 8) Shi, DQ et al. J. Heterocyclic Chem. 2009, 46, 1331-1334.

[0082] [Example 2] Synthesis of PA-9 derivatives [ka]

[0083] literature 1) According to the method described in JP-A-2006-510596, amines 1a-d (1.0 eq) and itaconic acid (1.0 eq) were mixed at room temperature under an Ar atmosphere and gradually heated from 60 to 150°C. After heating at 150°C for 30 minutes, the mixture was cooled to room temperature to obtain carboxylic acids 2a-d as pale yellow solids. Dicyclohexylcarbodiimide (DCC) (1.2 eq), 1-hydroxybenzotriazole (HOBt) (1.2 eq), and histamine (1.2 eq) were sequentially added to a DMF solution of carboxylic acids 2a-d (1.0 eq) at room temperature and stirred for 15 hours. The crude product obtained by distilling off the solvent was purified by silica gel chromatography (CHCl:MeOH = 5:1) and further washed with EtOAc:MeOH = 5:1 (0.5 mL × 3), to obtain 3a-d as white solids.

[0084] 1-(4-chloro-1H-indazol-3-yl)-5-oxo-3-pyrrolidinecarboxylic acid (2a) 11H NMR (400 MHz, Pyridine-d5): δ 10.74 (1H, br s), 7.73 (1H, d, J = 2.1 Hz), 7.45 (1H, d, J = 8.6 Hz), 7.15 (1H, dd, J = 2.1, 8.6 Hz), 4.65 (1H, dd, J = 13.0, 7.6 Hz), 4.44 (1H, t, J = 7.6 Hz), 3.40 (1H, quint, J = 7.6 Hz), 2.77 (1H, dd, J = 17.6, 7.6 Hz), 2.60 (1H, dd, J = 17.6, 7.6 Hz).

[0085] 1-(5-chloro-1H-indazol-3-yl)-5-oxo-3-pyrrolidinecarboxylic acid (2b) 1 1H NMR (400 MHz, Pyridine-d5): δ 11.53 (1H, br s), 7.38 (1H, d, J = 8.4 Hz), 7.14 (1H, dd, J = 8.4, 7.2 Hz), 6.93 (1H, d, J = 7.2 Hz), 4.67 (1H, dd, J = 13.0, 7.5 Hz), 4.52 (1H, t, J = 7.5 Hz), 3.39 (1H, quint, J = 7.5 Hz), 2.76 (1H, dd, J = 17.5, 7.5 Hz), 2.57 (1H, dd, J = 17.5, 7.5 Hz).

[0086] 1-(6-chloro-1H-indazol-3-yl)-5-oxo-3-pyrrolidinecarboxylic acid (2c) 1H NMR (400MHz, Pyridine-d5): δ 11.63 (1H, br s), 7.71 (1H, d, J = 9.0 Hz), 7.48 (1H, d, J = 1.7 Hz), 6.88 (1H, dd, J = 9.0, 1.7 Hz), 4.65 (1H, dd, J = 13.0, 7.7 Hz), 4.44 (1H, t, J =7.7 Hz), 3.40 (1H, quint, J = 7.7 Hz), 2.68 (1H, dd, J = 17.1, 7.7 Hz), 2.60 (1H, dd, J = 17.1, 7.7 Hz).

[0087] 1-(7-chloro-1H-indazol-3-yl)-5-oxo-3-pyrrolidinecarboxylic acid (2d) 1 H NMR (400MHz, Pyridine-d5): δ 11.66 (1H, br s), 7.67 (1H, d, J = 8.7 Hz), 7.30 (1H, d, J = 7.3 Hz), 6.86 (1H, dd, J = 8.7, 7.3 Hz), 4.71 (1H, dd, J = 13.4, 7.4 Hz), 4.48 (1H, t, J =7.4 Hz), 3.42 (1H, quint, J = 7.4 Hz), 2.78 (1H, dd, J = 17.6, 7.4 Hz), 2.61 (1H, dd, J = 17.6, 7.4 Hz).

[0088] N-[2-(1H-imidazol-4-yl)ethyl]-1-(4-chloro-1H-indazol-3-yl)-5-oxo-3-pyrrolidinecarboxamide (3a) mp: 190-191 ℃; IR (KBr): 3566, 3437, 3306, 1695, 1636, 1558, 1508 cm -1 ; 11H NMR (400 MHz, Pyridine-d5): δ 9.00 (1H, br s), 7.96 (1H, s), 7.61 (1H, d, J = 7.4 Hz), 7.14 (1H, t, J = 7.4 Hz), 7.15(1H, s), 6.98 (1H, d, J = 7.4 Hz), 4.72 (1H, t, J = 9.1 Hz), 3.96 - 3.85 (3H, m), 3.26 (1H, dd, J = 15.6, 9.1 Hz), 3.11 (2H, t, J = 6.8 Hz), 2.78 (1H, dd, J = 15.6, 9.1 Hz); 13 13C NMR (100 MHz, DMSO-d6): δ 169.56, 168.62, 147.59, 134.59, 131.61, 126.70, 123.80, 119.28, 116.97, 115.67, 107.11, 48.23, 36.47, 32.61, 26.57.

[0089] N-[2-(1H-imidazol-4-yl)ethyl]-1-(5-chloro-1H-indazol-3-yl)-5-oxo-3-pyrrolidinecarboxamide (3b) Yield: 24% over two steps; mp: 208 - 209 ℃; IR (KBr): 3735, 3649, 3097, 1684, 1653, 1558, 1508 cm -1 ; 11H NMR (400 MHz, Pyridine-d5): δ 9.04 (1H, br s), 8.05 (1H, s), 7.69 (1H, d, J = 9.6 Hz), 7.68 (1H, s), 7.27 (1H, d, J = 9.6 Hz), 7.71(1H, s), 5.09 (1H, dd, J = 13.6, 8.4 Hz), 4.73 (1H, t, J = 8.4 Hz), 3.94 - 3.86 (3H, m), 3.28 (1H, dd, J = 14.8, 8.4 Hz), 3.11 (2H, t, J = 6.6 Hz), 2.84 (1H, dd, J = 14.8, 8.4 Hz); 13 13C NMR (100 MHz, DMSO-d6): δ 169.63, 168.21, 145.55, 134.50, 133.79, 131.92, 127.15, 123.19, 118.50, 118.39, 116.85, 108.13, 48.33, 38.747, 36.33, 32.92, 26.53.

[0090] N-[2-(1H-imidazol-4-yl)ethyl]-1-(6-chloro-1H-indazol-3-yl)-5-oxo-3-pyrrolidinecarboxamide (3c) Yield: 17% over two steps; mp: 196 - 198 ℃; IR(KBr): 3290, 3213, 3101, 1683, 1636, 1558, 1508cm -1 ; 1H NMR (400MHz, Pyridine-d5): δ 9.12 (1H, br s), 8.30 (1H, s), 7.83 (1H, s), 7.66 (1H, d, J = 9.2 Hz), 7.25 (1H, s), 6.96 (1H, d, J = 9.2 Hz), 5.03 (1H, dd, J = 13.0, 8.3 Hz), 4.71 (1H, t, J = 8.3 Hz), 3.91-3.84 (3H, m), 3.27 (1H, dd, J = 15.2, 8.3 Hz), 3.12 (2H, t, J = 6.4 Hz), 2.87 (1H, dd, J = 15.2, 8.3 Hz); 13 C NMR (100 MHz, DMSO-d6): δ 169.79, 168.28, 147.25, 134.03, 132.74, 132.42, 131.35, 121.77, 119.93, 116.53, 115.03, 106.65, 48.23, 38.21,36.25, 32.92, 25.47.

[0091] N-[2-(1H-imidazol-4-yl)ethyl]-1-(7-chloro-1H-indazol-3-yl)-5-oxo-3-pyrrolidinecarboxamide (3d) Yield: 36% over two steps; mp: 238-239 ℃; IR(KBr): 3319, 3231, 3213, 1663, 1636, 1558, 1508cm -1 ; 1H NMR (400MHz, Pyridine-d5): δ 9.02 (1H, br s), 8.49 (1H, s), 7.76 (1H, s), 7.52 (1H, d, J = 7.5 Hz), 7.40 (1H, d, J = 7.5 Hz), 6.98 (1H, s), 6.88 (1H, t, J = 7.5 Hz), 5.13 (1H, dd, J = 14.6, 8.5 Hz), 4.81 (1H, t, J = 8.5 Hz), 3.95-3.86 (3H, m), 3.26 (1H, dd, J = 16.2, 8.5 Hz), 3.11 (2H, t, J = 6.8 Hz), 2.83 (1H, dd, J = 16.2, 8.5 Hz); 13 C NMR (100 MHz, DMSO-d6): δ 169.56, 168.32, 144.32, 134.51, 133.83, 133.30, 125.80, 120.48, 119.42, 119.04, 116.84, 109.42, 48.36, 38.77, 36.32, 32.95, 26.57.

[0092] literature 2) According to JP-A No. 2012-529476, itaconic acid (1.2 eq) was added to an aqueous solution (3 mL) of amines 1e–j (1.0 eq) at room temperature under an Ar atmosphere, and the mixture was heated to reflux with stirring for 20 hours. After cooling to room temperature, the precipitated solid was collected by filtration to obtain carboxylic acids 2e–j. To a mixed solution of carboxylic acids 2e–j (1.0 eq) in CHCl and DMF, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) (1.2 eq), 4-dimethylaminopyridine (DMAP) (0.1 eq), and histamine (1.2 eq) were added sequentially at room temperature and stirred for 15 hours. The solvent was evaporated, and the resulting crude product was purified by silica gel chromatography (CHCl:MeOH = 10:1) to obtain 3e–j as a white solid.

[0093] N-[2-(1H-imidazol-4-yl)ethyl]-1-phenyl-5-oxo-3-pyrrolidinecarboxamide (3e) Yield: 63%; mp: 149 - 151 °C; IR (KBr): 3675, 3306, 1678, 1643, 1558 cm -1 ; 1 1H NMR (400 MHz, Pyridine - d5): δ 9.11 (1H, t, J = 5.8 Hz), 7.90 (1H, s), 7.72 (2H, d, J = 8.4 Hz), 7.28 (2H, t, J = 8.4 Hz), 7.09 (1H, s), 7.06 (1H, t, J = 8.4 Hz), 4.13 (1H, dd, J = 9.6, 8.3 Hz), 3.93 (1H, t, J = 8.3 Hz), 3.83 (2H, q, J = 7.5 Hz), 3.47 (1H, quint, J = 8.3 Hz), 3.14 (1H, dd, J = 16.7, 8.3 Hz), 3.08 (2H, t, J = 7.5 Hz), 2.84 (1H, dd, J = 16.7, 8.3 Hz); 13 13C NMR (100 MHz, DMSO - d6): δ 172.16, 171.96, 139.22, 134.58, 133.95, 128.70, 124.00, 119.36, 116.83, 50.73, 38.66, 35.66, 35.43, 26.38.

[0094] N - [2 - (1H - imidazol - 4 - yl)ethyl] - 1 - (4 - methylphenyl) - 5 - oxo - 3 - pyrrolidinecarboxamide (3f) Yield: 93%; mp: 176 - 178 °C; IR (KBr): 3306, 3088, 1675, 1639, 1556 cm -1 ; 11H NMR (400 MHz, Pyridine-d5): δ 9.10 (1H, t, J = 5.8 Hz), 7.91 (1H, s), 7.64 (2H, d, J = 7.2 Hz), 7.08 (2H, t, J = 7.2 Hz), 7.07 (1H, s), 4.14 (1H, dd, J = 9.6, 8.0 Hz), 3.92 (1H, t, J = 8.0 Hz), 3.83 (2H, q, J = 7.2 Hz), 3.46 (1H, quint, J = 8.0 Hz), 3.14 (1H, dd, J = 17.6, 8.0 Hz), 3.08 (2H, t, J = 7.2 Hz), 2.84 (1H, dd, J = 17.6, 8.0 Hz), 2.13 (3H, s); 13 13C NMR (100 MHz, DMSO-d6):δ 172.17, 172.10, 137.00, 134.86, 134.44, 133.26, 129.30, 119.58, 117.04, 51.00, 39.17, 36.02, 35.84, 27.01, 20.63.

[0095] 1-(4-chlorophenyl)-N-[2-(1H-imidazol-4yl)ethyl]- 5-oxo-3-pyrrolidinecarboxamide (3g) Yield: 93%; mp: 196-198 ℃; IR (KBr): 3119, 3017, 1695, 1647, 1558 cm -1 ; 11H NMR (400 MHz, Pyridine-d5): δ 9.16 (1H, t, J = 5.2 Hz), 7.95 (1H, s), 7.71 (2H, d, J = 8.4 Hz), 7.30 (2H, d, J = 8.4 Hz), 7.11 (1H, s), 4.10 (1H, dd, J = 9.4, 8.3 Hz), 3.92 (1H, t, J = 8.3 Hz), 3.83 (2H, q, J = 6.7 Hz), 3.50 (1H, quint, J = 8.3 Hz), 3.11 (1H, dd, J = 17.5, 8.3 Hz), 3.09 (2H, t, J = 5.7 Hz), 2.85 (1H, dd, J = 17.5, 8.3 Hz); 13 13C NMR (100 MHz, DMSO-d6): δ 172.20, 171.68, 137.90, 134.43, 133.95, 128.36, 127.49, 120.60, 116.62, 50.47, 38.74, 35.62, 35.30, 26.52.

[0096] N-[2-(1H-imidazol-4-yl)ethyl]-1-(4-fluorophenyl)-5-oxo-3-pyrrolidinecarboxamide (3h) Yield: 94%; mp: 232 - 234 ℃; IR (KBr): 3140, 3126, 1688, 1645, 1570 cm -1; 1H NMR (400MHz, Pyridine-d5): δ 9.13 (1H, t, J = 4.8 Hz), 7.72-7.68 (2H, m), 7.92 (1H, s), 7.11 (1H, s), 7.09-7.04 (2H, m), 4.12 (1H, dd, J = 12.2, 8.7 Hz), 3.92 (1H, t, J = 8.7 Hz), 3.84 (2H, t, J = 7.0 Hz), 3.49 (1H, quint, J = 8.7 Hz), 3.14 (1H, dd, J = 17.2, 8.7 Hz), 3.09 (2H, t, J = 7.0 Hz), 2.85 (1H, dd, J = 17.2, 8.7 Hz); 13 C NMR (100 MHz, DMSO-d6): δ 172.05, 171.90, 158.44 (d, J = 240.3 Hz), 135.64, 134.62, 134.14, 121.35 (d, J = 7.6 Hz), 116.81, 115.25 (d, J = 22.0 Hz), 50.92, 38.92, 35.68, 35.58, 26.72.

[0097] N-[2-(1H-imidazol-4-yl)ethyl]-1-(4-methoxyphenyl)-5-oxo-3pyrrolidinecarboxamide (3i) Yield: 95%; mp: 151-153 ℃; IR (KBr): 3239, 3075, 1684, 1635, 1568 cm -1 ; 1H NMR (400 MHz, Pyridine-d5): δ 9.08 (1H, t, J = 5.2 Hz), 7.91 (1H, s), 7.68 (2H, d, J = 8.6 Hz), 7.10 (1H, s), 6.92 (2H, d, J = 8.6 Hz),4.16 (1H, dd, J = 9.4, 8.4 Hz), 3.93 (1H, t, J = 8.4 Hz), 3.84 (2H, q, J = 7.6 Hz), 3.62 (3H, s), 3.46 (1H, quint, J = 8.4 Hz), 3.15 (1H, dd, J = 17.1, 8.4 Hz), 3.09 (2H, t, J = 7.6 Hz), 2.84 (1H, dd, J = 17.1, 8.4 Hz); 13 C NMR (100 MHz, DMSO-d6): δ 171.97, 171.57, 155.80, 134.65, 134.26, 132.41, 121.20, 116.80, 113.82, 55.20, 51.03, 38.95, 35.68, 35.62, 26.81.

[0098] N-[2-(1H-imidazol-4-yl)ethyl]-1-(4-cyanophenyl)-5-oxo-3-pyrrolidinecarboxamide (3j) Yield: 69%; mp: 211-212 ℃; IR (KBr): 3151, 3019, 2231, 1703, 1646, 1558 cm -1 ; 11H NMR (400 MHz, Pyridine-d5): δ 9.17 (1H, t, J = 5.2 Hz), 7.92 (1H, s), 7.83 (2H, d, J = 8.8 Hz), 7.60 (2H, d, J = 8.8 Hz), 7.11 (1H, s), 4.12 (1H, dd, J = 9.6, 8.4 Hz), 3.96 (1H, t, J = 8.4 Hz), 3.84 (2H, t, J = 7.0 Hz), 3.51 (1H, quint, J = 8.4 Hz), 3.16 (1H, dd, J = 17.2, 8.4 Hz), 3.10 (2H, t, J = 7.0 Hz), 2.88 (1H, dd, J = 17.2, 8.4 Hz); 13 13C NMR (100 MHz, DMSO-d6): δ 173.28, 171.75, 143.03, 134.67, 133.06, 119.03, 118.93, 105.53, 51.51, 38.98, 36.03, 35.39, 26.85.

[0099] Literature 1) According to JP-T 2006-510596, amine 1k~m (1.0 eq) and itaconic acid (1.0 eq) were mixed at room temperature under an Ar atmosphere and gradually heated from 60 °C to 150 °C. After heating at 150 °C for 30 minutes, it was cooled to room temperature to obtain carboxylic acid 2k~m. EDC (1.2 eq), DMAP (0.1 eq) and histamine (1.2 eq) were sequentially added to a mixed solution of carboxylic acid 2k~m (1 eq) in CH2Cl2 and DMF at room temperature, and stirred for 15 hours. The crude product obtained by distilling off the solvent was purified by silica gel chromatography (CH2Cl2:MeOH = 10:1) to obtain 3k~m as white, yellow and red foamy solids, respectively.

[0100] N-[2-(1H-imidazol-4-yl)ethyl]-1-(2-hydroxyphenyl)-5-oxo-3pyrrolidinecarboxamide (3k) Yield: 40%; IR (KBr): 3651, 3265, 3213, 1684, 1670, 1558 cm -1 ; 1 H NMR (400 MHz, Pyridine-d5): δ 9.08 (1H, br s), 7.91 (1H, s), 7.42 (1H, d, J = 8.8 Hz), 7.21-7.15 (2H, m), 7.10 (1H, s), 6.92-6.88 (1H, m), 4.30 (1H, dd, J = 9.2, 7.5 Hz), 4.10 (1H, t, J = 7.5 Hz), 3.85 (2H, q,J = 6.4 Hz), 3.47 (1H, quint, J = 7.5 Hz), 3.13 (1H, dd, J = 16.4, 7.5 Hz), 3.06 (2H, t, J =6.4 Hz), 2.83 (1H, dd, J = 16.4, 7.5 Hz); 13 C NMR (100 MHz, DMSO-d6): δ 172.52, 172.22, 152.77, 134.68, 128.30, 128.24, 125.48, 119.12, 118.74, 116.87, 116.74, 51.68, 38.98, 37.09, 34.39, 26.85.

[0101] N-[2-(1H-imidazol-4-yl)ethyl]-1-(3-hydroxyphenyl)-5-oxo-3pyrrolidinecarboxamide (3l) Yield: 58%; IR (KBr): 3790, 3439, 3337, 1684, 1653, 1558 cm -1 ; 11H NMR (400 MHz, Pyridine-d5): δ 8.97 (1H, br s), 8.09 (1H, s), 7.93 (1H, s), 7.28 (1H, t, J = 8.2 Hz), 7.20 (1H, d, J =8.2 Hz), 7.13 (1H, s), 6.98 (1H, d, J = 8.2 Hz), 4.29 (1H, dd, J = 9.0, 8.3 Hz), 3.98 (1H, t, J = 8.3 Hz), 3.89 (2H, q, J = 6.9 Hz), 3.36 (1H, quint, J = 8.3 Hz), 3.21 (1H, dd, J = 17.1, 8.3 Hz), 3.09 (2H, t, J = 6.9 Hz), 2.82 (1H, dd, J = 17.1, 8.3 Hz); 13 13C NMR (100 MHz, DMSO-d6): δ 172.07, 171.93, 157.55, 140.28, 134.68, 129.38, 111.17, 109.78, 106.63, 50.84, 39.00, 35.99, 35.54, 26.89.

[0102] N-[2-(1H-imidazol-4-yl)ethyl]-1-(4-hydroxyphenyl)-5-oxo-3pyrrolidinecarboxamide (3m) Yield: 54%; IR (KBr): 3585, 3251, 3190, 1684, 1653, 1558 cm -1 ; 1H NMR (400 MHz, Pyridine-d5): δ 11.49 (1H, br s), 8.97 (1H, br s), 7.93 (1H, s), 7.76 (2H, dd, J = 8.6, 2.4 Hz), 7.15 (2H, dd, J = 8.6, 2.4 Hz), 7.14 (1H, s), 4.29 (1H, td, J = 8.4, 2.1 Hz), 3.95 (1H, td, J = 8.4, 2.1 Hz), 3.90 (2H, q, J = 6.3 Hz), 3.39 (1H, quint, J = 8.4 Hz), 3.22 (1H, ddd, J = 16.7,8.4, 2.1 Hz), 3.10 (2H, t, J = 6.3 Hz), 2.84 (1H, ddd, J = 16.7, 8.4, 2.1 Hz); 13 C NMR (100 MHz, DMSO-d6): δ 172.52, 171.86, 154.61, 135.20, 131.48, 122.05, 115.61, 51.67, 39.49, 36.23, 36.09, 27.40.

[0103] literature 1) According to the method described in JP 2006-510596 A, amine 1n (1.0 eq) and itaconic acid (1.0 eq) were mixed at room temperature under an Ar atmosphere and gradually heated from 60 to 150°C. After heating at 150°C for 30 minutes, the mixture was cooled to room temperature to obtain carboxylic acid 2n. To a solution of carboxylic acid 2n (1.0 eq) in CHCl and DMF, DCC (1.2 eq), HOBt (1.2 eq), and histamine (1.2 eq) were added sequentially at room temperature and stirred for 15 hours. The solvent was evaporated to obtain a crude product, which was purified by silica gel chromatography (CHCl:MeOH = 10:1) to obtain 3n as a white solid.

[0104] N-[2-(1H-imidazol-4-yl)ethyl]-1-(1H-pyrazol-3-yl)-5-oxo-3-pyrrolidinecarboxamide (3n) Yield: 30%; mp: 213 - 211 °C; IR (KBr): 3676, 3320, 3203, 1689, 1652, 1635, 1557 cm -1 ; 1 1H NMR (400 MHz, Pyridine - d5): δ 12.24 (1H, br s), 9.00 (1H, br s), 8.03 (1H, s), 7.88 (1H, s), 7.56 (1H, s), 7.10 (1H, s), 4.78 (1H, dd, J = 12.6, 8.1 Hz), 4.26 (1H, t, J = 8.1 Hz), 3.82 (2H, q, J = 6.7 Hz), 3.69 (1H, quint, J = 8.1 Hz), 3.22 (1H, dd, J = 15.3, 8.1 Hz), 3.06 (2H, t, J = 6.7 Hz), 2.72 (1H, dd, J = 15.3, 8.1 Hz); 13 13C NMR (100 MHz, DMSO - d6): δ 169.67, 168.26, 162.33, 138.77, 138.73, 134.64, 134.53, 89.01, 47.04, 38.87, 36.72, 33.00, 26.97.

[0105] Literature 1) According to JP - T 2006 - 510596, under an Ar atmosphere, amine 1o - r (1.0 eq) and itaconic acid (1.0 eq) were mixed at room temperature and gradually heated from 60 °C to 150 °C. After heating at 150 °C for 30 minutes, it was cooled to room temperature and purified by silica gel chromatography (CH2Cl2:MeOH = 30:1) to obtain carboxylic acid 2o - r. To a mixed solution of carboxylic acid 2o - r (1.0 eq) in CH2Cl2 and DMF, DCC (1.2 eq), HOBt (1.2 eq) and histamine (1.2 eq) were sequentially added at room temperature and stirred for 15 hours. The crude product obtained by evaporating the solvent was purified by silica gel chromatography (CH2Cl2:MeOH = 10:1) to obtain 3o - r as a yellow solid or white foamy solid.

[0106] N-[2-(1H-imidazol-4yl)ethyl]-5-oxo-1-(phenylmethyl)- 3-pyrrolidinecarboxamide (3o) Yield: 29%; IR (KBr): 3271, 3155, 1670, 1652, 1558 cm -1 ; 1 1H NMR (400MHz, Pyridine-d5): δ 8.85 (1H, br), 7.92 (1H, s), 7.33 - 7.25 (5H, m), 7.08 (1H, s), 4.56 (1H, d, J = 14.6 Hz), 4.48 (1H, d, J = 14.6 Hz), 3.85 (2H, q, J = 6.5 Hz), 3.67 (1H, t, J = 8.0 Hz), 3.39 (1H, t, J = 8.0 Hz), 3.25 (1H, quint, J = 8.0 Hz), 3.10 (1H, dd, J =17.0, 8.0 Hz), 3.05 (2H, t, J = 6.5 Hz), 2.72 (1H, dd, J = 17.0, 8.0 Hz).

[0107] N-[2-(1H-imidazol-4-yl)ethyl]-1-[(2-hydroxyphenyl)methyl]-5-oxo-3-pyrrolidinecarboxamide (3p) Yield: 30%; IR (KBr): 3748, 3738, 3651, 1684, 1653, 1558 cm -1 ; 1H NMR (400MHz, Pyridine-d5):δ 11.36 (1H, br s) 8.81 (1H, t, J = 5.8 Hz), 7.84 (1H, d, J = 1.2 Hz), 7.34 (1H, dd, J = 7.5, 1.3 Hz), 7.13 (1H, td, J = 7.5, 1.3 Hz), 7.08 (1H, dd, J = 7.5, 1.3 Hz), 7.03 (1H, s), 6.81 (1H, td, J = 1.3, 7.5 Hz), 4.74 (1H, d, J = 15.2 Hz), 4.65 (1H, d, J = 15.2 Hz), 3.82 (1H, dd, J = 9.6, 8.2 Hz), 3.77 (2H, q, J = 6.6 Hz), 3.57 (1H, t, J = 8.2 Hz), 3.22 (1H, quint,J = 8.2 Hz), 3.03 (1H, dd, J = 16.5, 8.2 Hz), 2.99 (2H, t, J = 6.6 Hz), 2.63 (1H, dd, J = 16.5, 8.2 Hz); 13 C NMR (100 MHz, DMSO-d6): δ 172.57, 172.18, 155.26, 134,67, 128.78, 128.38, 122.58, 119.03, 115.21, 49.63, 40.57, 38.94, 35.93, 33.91, 26.87.

[0108] N-[2-(1H-imidazol-4-yl)ethyl]-1-[(3-hydroxyphenyl)methyl]-5-oxo-3-pyrrolidinecarboxamide (3q) Yield: 40%; IR (KBr): 3734, 3647, 3623, 1684, 1653, 1558 cm -1 ; 11H NMR (400 MHz, Pyridine-d5): δ 8.83 (1H, br s), 7.93 (1H, s), 7.24 (2H, d, J = 7.9 Hz), 7.22 (1H, s), 7.09 (1H, s), 7.08(1H, d, J = 7.9 Hz), 6.88 (1H, d, J = 7.9 Hz), 4.57 (1H, d, J = 14.6 Hz), 4.48 (1H, d, J = 14.6 Hz), 3.84 (2H, q, J = 6.1 Hz), 3.72 (1H, t, J = 8.1 Hz), 3.45 (1H, t, J = 8.1 Hz), 3.23 (1H, quint, J = 8.1 Hz), 3.09 (1H, dd, J = 16.0, 8.1 Hz), 3.05 (2H, t, J = 6.1 Hz), 2.67 (1H, dd, J = 16.0, 8.1 Hz); 13 13C NMR (100 MHz, DMSO-d6): δ 172.31, 172.05, 157.63, 138.14, 134.66, 134.18, 129.55, 118.17, 116.90, 114.41, 114.30, 49.14, 45.31, 38.88, 35.89, 26.78.

[0109] N-[2-(1H-imidazol-4-yl)ethyl]-1-[(4-hydroxyphenyl)methyl]-5-oxo-3-pyrrolidinecarboxamide (3r) Yield: 29%; IR (KBr): 3651, 3271, 3213, 1663, 1653, 1558 cm -1 ; 11H NMR (400 MHz, Pyridine-d5): δ 8.85 (1H, br s), 7.91 (1H, s), 7.28 (2H, d, J = 7.6 Hz), 7.10 (2H, d, J = 7.6 Hz), 4.55 (1H, d, J = 14.6 Hz), 4.44 (1H, d, J = 14.6 Hz), 3.91 - 3.71 (2H, m), 3.70 (1H, dd, J = 8.8, 7.9 Hz), 3.45 (1H, t, J = 7.9 Hz), 3.26 (1H, quint, J = 7.9 Hz), 3.11 (1H, dd, J = 15.0, 7.9 Hz), 3.06 (2H, t, J = 7.4 Hz), 2.71 (1H, dd, J = 15.0, 7.9 Hz); 13 13C NMR (100 MHz, DMSO-d6) : δ 172.15, 156.66, 134.68, 129.07, 126.80, 115.30, 48.93, 44.83, 38.94, 35.83, 34.03, 26.89.

[0110] 1) Japanese Patent Publication No. 2006 - 510596 2) Japanese Patent Publication No. 2012 - 529476 3) Saczewski, F. et al. Bioorg. Med. Chem. 2011, 19, 321 - 329 4) Mestichelli, P. et al. Org. Lett. 2013, 15, 5448 - 5451 5) Commercially available, Aurora Building Blocks, A17.818.885 6) Commercially available, Aurora Building Blocks, A21.884.126

[0111] [Example 3] Evaluation of drug efficacy using a serotonin (5-HT) - induced pruritus model mouse (Figure 1) 5-HT (50 nmol, 10 μL) was injected intradermally into the lumbar region (id). Biting, licking, or scratching at the injection site was measured as pruritic behavior, and the duration of this behavior was measured every 5 minutes. Intrathecal pre-administration of PA-8 (0.1 or 1 nmol) (30 minutes prior to administration) almost completely suppressed 5-HT-induced pruritic behavior. In contrast, pre-administration of the peptide PACAP receptor antagonist PACAP6-38 (P6-38: 100 pmol) had no significant effect. SLN indicates the effect of administering saline instead of 5-HT. ACSF stands for artificial cerebrospinal fluid, and is the solvent for P6-38 and PA-8.

[0112] [Example 4] Evaluation of drug efficacy using a mouse model of dry skin pruritus This test was performed with reference to the method of Miyamoto et al. (Miyamoto T., Nojima H., Shinkado T., et al.: Itch-associated response induced by experimental dry skin in mice, Jpn. J. Pharmacol., 88, 285-292, 2002), which is a published method for evaluating itch in animals.

[0113] Five-week-old ICR mice (40 mice) were individually housed in cages and allowed to acclimate for one week. Three days before the start of the experiment, the rostral dorsal hair was shaved (treatment method A). The 40 shaved mice were divided into five groups of eight mice each: a PA-8 3 mg / kg oral (po) administration group, a 10 mg / kg oral (po) administration group, a 30 mg / kg oral (po) administration group, a comparison group, and a control group.

[0114] <PA-8投与群> In the PA-8 administration group, mice subjected to hair removal treatment (treatment method A) were anesthetized with ether, and a 2×2 cm cotton soaked with a mixture of acetone (A): diethyl ether (E) (1:1) was applied to the rostral back for 15 seconds, and then a cotton soaked with ion-exchanged water (W) was applied for 30 seconds [hereinafter, this series of treatments is abbreviated as AEW treatment (treatment method B)]. The AEW treatment was performed 10 times over 5 days at 8-hour intervals twice a day. On the day after the AEW treatment, PA-8 was orally administered at 3 mg / kg, 10 mg / kg, or 30 mg / kg, and behavioral observation was carried out for 1 hour starting from 30 minutes later (treatment method C).

[0115] <Comparison group> In the comparison group, the AEW treatment (treatment method B) was performed on the mice subjected to hair removal treatment (treatment method A), and a 10% aqueous DMSO solution (solvent) was orally administered.

[0116] <Control group> In the control group (Naive), only the hair removal treatment (treatment method A) was performed. The outline of the procedure of the behavioral experiment using dry skin itching model mice is shown in Figure 2, and the results of evaluating the scratching time of each group are shown in Figure 3.

[0117] From Figure 3, it can be seen that by orally administering PA-8, the scratching behavior induced by AEW treatment was significantly suppressed.

[0118] [Example 5] Pharmacodynamic evaluation 1 using PACAP receptor-expressing cultured cells Using mouse PAC1 receptor-expressing CHO cells (PAC1 / CHO cells) and mouse VPAC1 receptor-expressing CHO cells (VPAC1 / CHO cells), the effects of each compound on the phosphorylation of CREB (cAMP-responsive element-binding protein) caused by PACAP stimulation were examined by Western blot analysis using an anti-phosphorylated CREB (pCREB) antibody.

[0119] Specifically, cells were treated with 10 pM to 10 nM of compounds (PA-8, compound 2j, and compound 2o) and their solvent DMSO (VEH: phosphate buffer containing 0.1% DMSO) for 30 minutes, after which 1 nM of PACAP was added, and protein was recovered 30 minutes after PACAP stimulation.

[0120] Figure 4A shows data from PAC1 / CHO cells. PA-8 inhibited CREB phosphorylation induced by PACAP (1 nM) at concentrations ranging from 10 pM to 10 nM (n = 5; Figure 4A). In contrast, in studies using VPAC1 / CHO cells, PACAP (1 nM) did not inhibit CREB phosphorylation induced by CREB at concentrations ranging from 10 pM to 10 nM (n = 3; Figure 4B). Thus, PA-8 is a selective antagonist of the PAC1 receptor.

[0121] Figure 4C shows the effect of compound 2j (10 pM-10 nM) in PAC1 / CHO cells, which inhibited CREB phosphorylation induced by PACAP (1 nM) in a concentration-dependent manner (n = 4). Figure 4D shows the effect of compound 2o (10 pM-10 nM). Compound 2o also inhibited CREB phosphorylation induced by PACAP (1 nM) at the concentrations used (10 pM-10 nM) (n = 4). In particular, compound 2o exhibited a slightly stronger inhibitory effect on CREB phosphorylation than PA-8.

[0122] The PA-8 used in the above experiments was a commercially available compound purchased from Namiki Shoji Co., Ltd.

[0123] [Example 6] Drug efficacy evaluation 2 using cultured cells expressing PACAP receptor The effects of each compound on the PACAP-induced phosphorylation of CREB (cAMP-responsive element-binding protein) were examined by Western blot analysis using anti-phospho-CREB (pCREB) antibody in CHO cells expressing the mouse PAC1 receptor (PAC1 / CHO cells). Figures 5A and 5B show the effects of PA-9 and compound 3d (10 pM-10 nM), respectively. Both compounds inhibited PACAP (1 nM)-induced CREB phosphorylation in a concentration-dependent manner (n = 4-5).

[0124] [Example 7] Effect of Intradermal Administration of PACAP to the Lumbar Region When PACAP (1 - 1,000 pmol) was administered intradermally to the lumbar region (10 μL), scratching behaviors such as biting, licking, and scratching the administration site were observed in a dose - dependent manner. However, such an effect was not observed with vasoactive intestinal peptide (VIP) (100 pmol). Therefore, this effect is considered to be mediated through the PAC1 receptor (Figure 6).

[0125] [Example 8] The scratching behavior induced by intradermal administration of PACAP is suppressed by co - intradermal administration of PA - 8 or subcutaneous pretreatment with naloxone (NTX).

[0126] The biting, licking, and scratching behaviors induced by intradermal administration of PACAP (100 pmol) to the lumbar region (10 μL) were dose - dependently suppressed by co - intradermal administration of PA - 8 (0.01 - 1 nmol) (Figure 7A). SLN indicates intradermal administration of physiological saline. DMSO (10% solution, dissolved in physiological saline) is the solvent for PA - 8. Also, when the opioid receptor antagonist naloxone (NTX: 1 mg / kg) was administered subcutaneously 15 minutes before intradermal administration of PACAP, the induction of scratching behavior by intradermal administration of PACAP (100 pmol) was significantly suppressed (Figure 7B). SLN indicates administration of physiological saline instead of NTX.

[0127] [Example 9] The scratching behavior induced by intradermal administration of PACAP is suppressed by co - intradermal administration of PA - 8 or compound 2o.

[0128] The biting, licking, and scratching behaviors induced by intradermal administration of PACAP (100 pmol) to the lumbar region (10 μL) were dose - dependently suppressed by co - intradermal administration of PA - 8 (0.01 - 1 nmol) or compound 2o (0.01 - 1 nmol) (Figure 8). SLN indicates intradermal administration of physiological saline. VEH (10% DMSO solution, dissolved in physiological saline) is the solvent for PA - 8 and compound 2o.

[0129] [Example 10] The scratching behavior induced by intradermal administration of PACAP is suppressed by co-intradermal administration of PA-9 or compound 3d.

[0130] The biting, licking, and scratching behaviors induced by intradermal administration of PACAP (100 pmol) into the lumbar region (10 μL) are dose-dependently suppressed by co-intradermal administration of PA-9 (0.01 - 1 nmol) or compound 3d (0.01 - 1 nmol) (Figure 9). SLN indicates intradermal administration of physiological saline. VEH (10% DMSO solution, dissolved in physiological saline) is the solvent for PA-9 and compound 3d.

[0131] [Example 11] The scratching behavior induced by intradermal administration of chloroquine is suppressed by pre-intrathecal administration of PA-8 or PA-9, but not by co-intradermal administration.

[0132] Chloroquine (100 μg / 10 μL) was injected intradermally (i.d.) into the lumbar region. The reactions of biting, licking, and scratching the injection site were regarded as scratching behavior, and the behavior time was measured every 5 minutes. Pre-intrathecal administration (10 minutes before, i.t.) of PA-8 (1 nmol) or PA-9 (1 nmol) almost completely suppressed the chloroquine-induced scratching behavior, but co-intradermal administration (Co-i.d.) did not suppress it (Figure 10). DMSO (10% solution, dissolved in physiological saline) is the solvent for PA-8 or PA-9.

[0133] [Example 12] The scratching behavior induced by intradermal administration of Compound 48 / 80 is suppressed by pre-intrathecal administration of PA-8 or PA-9, but not by co-intradermal administration.

[0134] Compound 48 / 80 (100 μg / 10 μL) was injected intradermally into the lumbar region (id). Biting, licking, or scratching at the injection site was considered pruritic behavior, and the duration of this behavior was measured every 5 min. Intrathecal pre-administration (10 min before i) of PA-8 (1 nmol) or PA-9 (1 nmol) almost completely inhibited Compound 48 / 80-induced pruritic behavior, but simultaneous intradermal administration (Co-id) did not (Figure 11). DMSO (10% solution, dissolved in saline) was the solvent for PA-8 or PA-9.

[0135] [Example 13] Evaluation of drug efficacy using atopic dermatitis model mice This study was conducted with reference to the method of Kitamura et al. (Kitamura A., Takata R., Aizawa S., et al.: A murine model of atopic dermatitis can be generated by painting the dorsal skin with hapten twice 14 days apart, Scientific Rep., 8:5988, 2018), a published method for evaluating pruritus in animals.

[0136] Thirty 5-week-old ICR mice were individually housed and acclimated for one week. Three days before the start of the experiment, their lower backs were shaved. On the first day of the experiment, tape stripping was performed to disrupt the skin barrier, and 100 μL of 0.15% 2,4-dinitrofluorobenzene (DNFB) solution [acetone / olive oil (3:1)] was applied to the shaved lower backs of the mice using a pipette. Seven days later, the 0.15% DNFB solution was applied again, and two hours later, video observation was performed for one hour. The 30 shaved mice were divided into five groups of six mice each: PA-8 30 mg / kg oral (po), compound 2o 30 mg / kg oral (po), PA-9 30 mg / kg oral (po), compound 3d 30 mg / kg oral (po), and vehicle (VEH: oral administration of 10% DMSO aqueous solution). Oral administration was performed 30 minutes before video observation.

[0137] The procedure for the behavioral experiment using atopic dermatitis model mice is outlined in Figure 12, and the results of evaluating the scratching time for each group are shown in Figure 13. Figure 13 shows that oral administration of PA-8, PA-9, or their derivatives (compounds 2o and 3d) significantly suppressed the scratching behavior induced by DNFB treatment.

[0138] [Example 14] Evaluation of drug efficacy using psoriasis model mice This test was performed with reference to the method of van der Fits et al. (van der Fits L., Mourits S., Voerman JSA., et al.: Imiquimod-Induced Psoriasis-Like Skin Inflammation in Mice Is Mediated via the IL-23 / IL-17 Axis, J. Immunol., 182, 5836-5845, 2009), which is a published test system for evaluating pruritus in animals.

[0139] Twenty 5-week-old ICR mice were individually housed and allowed to acclimate for one week. The lumbar region was shaved three days prior to the start of the study. Commercially available imiquimod cream (5% Veselna Cream; Mochida Pharmaceutical) was applied to the shaved lumbar skin of the mice at 62.5 mg once daily for five days to create an imiquimod-induced psoriasis model. Twenty mice were shaved and divided into five groups (four mice each): PA-8 30 mg / kg oral (po), Compound 2o 30 mg / kg oral (po), PA-9 30 mg / kg oral (po), Compound 3d 30 mg / kg oral (po), and vehicle (VEH: oral administration of 10% DMSO aqueous solution). Oral administration was performed 30 minutes prior to video observation.

[0140] The results of evaluating the scratching time for each group are shown in Figure 14. Figure 14 shows that oral administration of PA-8, PA-9, or their derivatives (compounds 2o and 3d) significantly suppressed the scratching behavior induced by topical application of imiquimod.

[0141] All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

Claims

1. An antipruritic agent containing a compound represented by the following formula (I): 【Chemical 1】 (wherein R １ is a C １－２ - alkoxy group, a C ２－６ - alkenyloxy group or a C １－６ - haloalkoxy group; R ２ is a hydrogen atom or a C ２－６ - alkenyloxy group.) or a salt thereof or a solvate thereof.

2. The antipruritic agent according to Claim 1, for treating and / or preventing itching associated with contact dermatitis, atopic dermatitis, dry skin and / or psoriasis.

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