Novel Compounds and Pharmaceutical Compositions

Abstract

To provide novel compounds as degradation inducers targeting hematopoietic prostaglandin D synthase.SOLUTION: The present invention is characterized by linking with a ligand of E3 ubiquitin ligase and a ligand of hematopoietic prostaglandin D synthase. The ligand for E3 ubiquitin ligase is, for example, pomalidomide, and the ligand for hematopoietic prostaglandin D synthase is, for example, TFC-007. Cardiac hypertrophy and myocardial damage in muscular dystrophy myocardial inflammation model mice are suppressed.SELECTED DRAWING: Figure 7

Description

[Technical field]

[0001] The present invention relates to a novel compound and a pharmaceutical composition containing the novel compound as an active ingredient. [Background technology]

[0002] Prostaglandin H produced from arachidonic acid by the action of cyclooxygenase (COX) 2 (P.G.H. 2 ) is converted to PGD by the action of various PG synthesis enzymes. 2 or PGE 2 Among them, PGD 2 Not only does it have various physiological effects such as platelet aggregation and sleep induction, but it also undergoes further metabolism to produce the cyclopentenone structure J 2 It is known that these compounds are converted into PGs of the PG type (Non-Patent Document 1).

[0003] Prostaglandin D 2 (PGD 2 Excessive production of prostaglandin D is associated with various diseases such as allergies, sleep disorders, and Duchenne muscular dystrophy. Hematopoietic prostaglandin (PG) D synthase (H-PGDS) is an enzyme that synthesizes PGD. 2 H-PGDS is one of the enzymes that produces PGD (Non-Patent Documents 2 to 5). Mast cells, which play a key role in food allergies, strongly express H-PGDS along with physiologically active substances such as histamine and serotonin that cause inflammation, and secrete large amounts of PGD. 2 In addition, in skeletal muscles of Duchenne muscular dystrophy, H-PGDS produces PGD 2 is actively produced, and secondary inflammation mediated by DP1 or DP2 receptors expands muscle necrosis, resulting in collective muscle necrosis.

[0004] H-PGDS has attracted attention as a drug discovery target for the above-mentioned diseases, and it has been reported that inhibitors targeting H-PGDS are useful for the treatment of allergic reactions and inflammatory reactions (Non-Patent Document 6). However, since problems such as the inability to obtain the desired therapeutic effect depending on the patient's condition have been reported in clinical trials (Non-Patent Document 7), the development of drugs with a new mechanism of action is required.

[0005] In recent years, innovative molecular target drugs that can induce the degradation of target proteins by utilizing the intracellular ubiquitin-proteasome system (UPS) have been developed at home and abroad. These drugs are called PROTAC (Proteolysis targeting chimeras) (Non-Patent Document 8) and SNIPER (specific and non-genetic inhibitor of apoptosis protein-dependent protein erasers) (Non-Patent Document 9), and are chimeric molecules (X-Y) in which a target protein ligand (X) and a ubiquitin ligase ligand (Y) are linked by a linker (-). PROTAC and SNIPER physically bring the target protein and the ubiquitin ligase closer together, causing the target protein to be polyubiquitinated and induced to be degraded by the proteasome. It has also been reported that PROTAC and SNIPER can continuously suppress the cellular responses caused by the target protein compared to conventional inhibitors (Non-Patent Document 10). PROTAC and SNIPER are expected as one of the drug discovery modalities, and in particular, the development targeting proteins related to cancer is active. However, no degradation inducer targeting H-PGDS has been reported yet, and the effectiveness of RROTAC and SNIPER targeting H-PGDS has not been demonstrated either.

Prior Art Documents

Non-Patent Documents

[0006]

Non-Patent Document 1

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

Non-Patent Document 8

Non-Patent Document 9

Non-Patent Document 10

Summary of the Invention

Problems to be Solved by the Invention

[0007] The present invention has been made in view of such problems, and an object thereof is to provide a novel compound as an inducer of degradation targeting H-PGDS and a pharmaceutical composition containing the novel compound as an active ingredient.

Means for Solving the Problems

[0008] The novel compound according to the present invention is characterized in that a ligand of an E3 ubiquitin ligase and a ligand of hematopoietic prostaglandin D synthase are connected.

[0009] The pharmaceutical composition according to the present invention uses the novel compound according to the present invention as an active ingredient and is a pharmaceutical composition for preventing and / or treating allergic diseases.

[0010] The pharmaceutical composition according to the present invention uses the novel compound according to the present invention as an active ingredient and is a pharmaceutical composition for preventing and / or treating inflammatory diseases.

Effects of the Invention

[0011] According to the present invention, a novel compound as a degradation inducer targeting H-PGDS can be obtained. Further, a pharmaceutical composition containing the novel compound as an active ingredient can be obtained.

Brief Description of the Drawings

[0012]

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Embodiments for Carrying Out the Invention

[0013] Hereinafter, embodiments of the present invention will be specifically described with reference to the accompanying drawings. However, the embodiments are for facilitating the understanding of the principle of the present invention, and the scope of the present invention is not limited to the following embodiments. Other embodiments in which those skilled in the art appropriately substitute the configurations of the following embodiments are also included in the scope of the present invention.

[0014] As shown in Fig. 1(a), the novel compound according to the present invention is one in which a ligand of an E3 ubiquitin ligase and a ligand of hematopoietic prostaglandin D synthase are connected by a linker.

[0015] Drugs that can induce the degradation of target proteins by utilizing the intracellular UPS are called PROTACs and SNIPERs, and are chimeric molecules (X-Y) in which a target protein ligand (X) and a ubiquitin ligase ligand (Y) are connected by a linker (-). PROTAC physically brings the target protein and the ubiquitin ligase closer, so that the target protein is polyubiquitinated and undergoes degradation induction by the proteasome. The inventor of the present case synthesized a novel compound (X-Y) in which a ligand (X) of hematopoietic prostaglandin D synthase and a ligand (Y) of an E3 ubiquitin ligase are connected by a linker (-), and found as a new finding that such a novel compound efficiently degrades the H-PGDS protein, and completed the present invention based on such a fact.

[0016] In addition, as shown in Fig. 1(b), the novel compound according to the present invention is one in which a ligand of an E3 ubiquitin ligase and a ligand of hematopoietic prostaglandin D synthase are connected without an intervening linker.

[0017] It is a chimeric molecule (XY) in which a target protein ligand (X) and a ubiquitin ligase ligand (Y) are connected without an intervening linker. The inventor of the present case synthesized a novel compound (XY) in which a ligand (X) of hematopoietic prostaglandin D synthase and a ligand (Y) of E3 ubiquitin ligase are connected without an intervening linker, and found as a new finding that such a novel compound efficiently degrades the H-PGDS protein, and completed the present invention based on such a fact.

[0018] H-PGDS is highly expressed in the placenta, lung, fetal liver, and lymph nodes in humans, and is also expressed in the brain, thymus, heart, spleen, and bone marrow. PGD synthesized by H-PGDS 2 has been suggested to be closely involved in the progression of allergic reactions. In addition to allergic reactions, induction of H-PGDS has been observed in necrotic muscles of patients with Duchenne muscular dystrophy and polymyositis, and in microglial cells activated by traumatic brain injury.

[0019] As the ligand of hematopoietic prostaglandin D synthase, it is preferable to use a substance having a specific binding action to hematopoietic prostaglandin D synthase. Here, specific means having a binding action to hematopoietic prostaglandin D synthase and substantially no binding action to lipocalin-type prostaglandin D synthase. As the ligand of hematopoietic prostaglandin D synthase, a compound candidate having an inhibitory action on H-PGDS can be obtained by a screening method.

[0020] The ligand of hematopoietic prostaglandin D synthase is not particularly limited, and examples include those shown below, and preferably TFC-007. ·TFC-007···N-(4-(4-(Morpholine-4-carbonyl)piperidin-1-yl)phenyl)-2-phenoxypyrimidine-5-carboxamide ·TAS-204···(N-Methoxy-N-methyl)-4-(5-benzoylbenzimidazol-2-yl)-3,5-dimethylpyrrole-2-carboxamide hydrochloride ·TAS-205···4-(1-Methyl-1H-pyrrole-2-carbonyl)-N-(4-(4-(morpholine-4-carbonyl)piperidin-1-yl)phenyl)piperazine-1-carboxamide ·4-((1-Methylpyrrol-2-yl)-carbonyl)-N-(1-(4-(3-(1,2,3-triazol-1-yl)-propyl)-phenyl)-piperidin-4-yl)-1-piperazinecarboxamide ·4-((1-Methylpyrrol-2-yl)-carbonyl)-N-(1-(4-(3-morpholino-3-oxopropen-1-yl)-phenyl)-piperidin-4-yl)-1-piperazinecarboxamide ·4-((1-Methylpyrrol-2-yl)-carbonyl)-N-(1-(4-(3-morpholino-3-oxopro pyl)-phenyl)-piperidin-4-yl)-1-piperazinecarboxamide ·6-(4-(4-((1-Methylpyrrol-2-yl)-carbonyl)-1-piperazinecarbamoyl)-piperidin-1-yl)-nicotinic acid ·4-((1-Methylpyrrol-2-yl)-carbonyl)-N-(1-(5-(4-morpholinylcarbonyl)pyridin-2-yl)-piperidin-4-yl)-1-piperazinecarboxamide ·4-((1-Ethylpyrrol-2-yl)-carbonyl)-N-(1-(4-(2-morpholinoethylcarbamoyl)phenyl)-piperidin-4-yl)-1-piperazinecarboxamide ·4-((1-Ethylpyrrol-2-yl)-carbonyl)-N-(1-(4-(2-(1,2,3-triazol-1-yl)-ethyl)-phenyl)-piperidin-4-yl)-1-piperazinecarboxamide · 4-(4-(4-((1-Methylpyrrol-2-yl)-carbonyl)-1-piperazinecarboxamoyl)piperidin-1-yl)-benzoic acid · 4-((1-Methylpyrrol-2-yl)-carbonyl)-N-(1-(4-(pyridin-3-ylmethylcarbamoyl)phenyl)-piperidin-4-yl)-1-piperazinecarboxamide · 4-((1-Methylpyrrol-2-yl)-carbonyl)-N-(1-(4-(2-morpholinoethylcarbamoyl)-phenyl)-piperidin-4-yl)-1-piperazinecarboxamide · 4-((1-Methylpyrrol-2-yl)-carbonyl)-N-(1-(4-(4-morpholinylcarbonyl)phenyl)-piperidin-4-yl)-1-piperazinecarboxamide · 4-((1-Methylpyrrol-2-yl)-carbonyl)-N-(1-(4-(1-piperidinylcarbonyl)phenyl)-piperidin-4-yl)-1-piperazinecarboxamide · 4-((1-Methylpyrrol-2-yl)-carbonyl)-N-(1-(4-(1-pyrrolidinylcarbonyl)phenyl)-piperidin-4-yl)-1-piperazinecarboxamide · 4-((1-Methylpyrrol-2-yl)-carbonyl)-N-(1-(4-(2-(1,2,3-triazol-1-yl)-ethyl)-phenyl)-piperidin-4-yl)-1-piperazinecarboxamide · 4-((1-Methylpyrrol-2-yl)-carbonyl)-N-(1-(4-(3-(1,2,4-triazol-1-yl)-propyl)-phenyl)-piperidin-4-yl)-1-piperazinecarboxamide · 4-((1-Methylpyrrol-2-yl)-carbonyl)-N-(1-(4-(3-(3,5-dimethyl-1,2,4-triazol-1-yl)-propyl)-phenyl)-piperidin-4-yl)-1-piperazinecarboxamide Ubiquitination is a reaction in which the C-terminal glycine of ubiquitin is isopeptide-bonded to the lysine residue of a substrate by the action of three enzymes: ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin ligase (E3). Among these enzymes, it is the E3 ubiquitin ligase that determines which substrate to add ubiquitin to.

[0021] The E3 ubiquitin ligase is not particularly limited. For example, it includes cereblon (CRBN), MDM2, APC, UBR5, SOCS, LNX1, BIRC2, BIRC3, BIRC4, CBX4, CBLL1, HACE1, HECTD1, HECTD2, HECTD3, HECTD4, HECW1, HECW2, HERC1, HERC2, HERC3, HERC4, HERC5, HERC6, HUWE1, ITCH, NEDD4, NEDD4L, PPIL2, PRPF19, PIAS1, PIAS2, PIAS3, PIAS4, RANBP2, RNF4, RBX1, SMURF1, SMURF2, STUB, TOPORS, TRIP12, UBE3A, UBE3B, UBE3C, UBE3D, UBE4A, UBE4B, UBOX5, UBR5, WWP1, WWP2, or Parkin, etc. Preferably, it is cereblon (CRBN).

[0022] Ligands of the E3 ubiquitin ligase include pomalidomide, lenalidomide, thalidomide, or indisulam, etc. Preferably, it is pomalidomide.

[0023] The linker is not particularly limited as long as it can connect the ligand of hematopoietic prostaglandin D synthase and the ligand of the E3 ubiquitin ligase. For example, it includes a polyethylene glycol linker or an alkyl linker, etc. Preferably, it is a polyethylene glycol linker. The number of constituent units of the linker is not particularly limited. For example, it is 1 to 10.

[0024] In the case where the ligand (X) of hematopoietic prostaglandin D synthase is TFC-007, the ligand (Y) of E3 ubiquitin ligase is pomalidomide, and the constituent unit of the linker (-) is a polyethylene glycol linker, the novel compound according to the present invention is shown below. Here, the number of constituent units of the polyethylene glycol linker is not particularly limited. For example, n is 1 to 10, preferably 1 to 5, more preferably 1 or 2, and most preferably 1.

[0025]

Chemical formula

[0026] When n = 1, it may be described as PROTAC(H-PGDS)-6 and is shown below.

[0027]

Chemical formula

[0028] When n = 2, it may be described as PROTAC(H-PGDS)-5.

[0029] When n = 3, it may be described as PROTAC(H-PGDS)-4.

[0030] When n = 4, it may be described as PROTAC(H-PGDS)-3.

[0031] When n = 5 and N-methyl pomalidomide, it may be described as PROTAC(H-PGDS)-2.

[0032] When n = 5, it may be described as PROTAC(H-PGDS)-1 and is shown below.

[0033]

Chemical formula

[0034] In the novel compound according to the present invention, when the ligand (X) of hematopoietic prostaglandin D synthase is TFC-007, the ligand (Y) of E3 ubiquitin ligase is pomalidomide, and they are connected without a linker, it is shown below (in this specification, the following compound may be described as PROTAC(H-PGDS)-7).

[0035]

Chemical formula

[0036] In the novel compound according to the present invention, when the ligand (X) of hematopoietic prostaglandin D synthase is TAS-205, the ligand (Y) of E3 ubiquitin ligase is pomalidomide, and they are connected without a linker, it is shown below (in this specification, the following compound may be described as PROTAC(H-PGDS)-10).

[0037]

Chemical formula

[0038] In the present invention, by physically bringing the target protein H-PGDS and ubiquitin ligase close to each other, H-PGDS is polyubiquitinated and undergoes degradation induction by the proteasome. Specifically, when PROTAC(H-PGDS)-1, PROTAC(H-PGDS)-7, or PROTAC(H-PGDS)-10 is added to cells expressing H-PGDS, the TFC-007 site in the PROTAC(H-PGDS)-1 or PROTAC(H-PGDS)-7 structure binds to the H-PGDS protein in the cell, or the TAS-205 site in the PROTAC(H-PGDS)-10 structure binds to the H-PGDS protein. Then, the pomalidomide site binds to ubiquitin ligase (cereblon), which is an enzyme present in the cell. As a result, H-PGDS and ubiquitin ligase (cereblon) are brought close to each other, H-PGDS is polyubiquitinated, and is degraded by the proteasome. That is, the novel compound according to the present invention functions as a degradation inducer targeting H-PGDS.

[0039] The pharmaceutical composition according to the present invention is a pharmaceutical composition for preventing and / or treating allergic diseases, having the novel compound according to the present invention as an active ingredient. The allergic diseases are not particularly limited, and examples include atopic dermatitis, bronchial asthma, pollinosis, allergic rhinitis, sinusitis, otitis media, or allergic conjunctivitis, etc.

[0040] The pharmaceutical composition according to the present invention is a pharmaceutical composition for preventing and / or treating inflammatory diseases, having the novel compound according to the present invention as an active ingredient. The inflammatory diseases are not particularly limited, and examples include muscular dystrophy, myositis, chronic obstructive arterial disease, rheumatoid arthritis, osteoarthritis, multiple sclerosis, amyotrophic lateral sclerosis, inflammatory bowel disease, chronic obstructive pulmonary disease, interstitial pneumonia, hypersensitivity pneumonia, or eosinophilic pneumonia, etc. Muscular dystrophy includes, for example, facioscapulohumeral muscular dystrophy, Duchenne muscular dystrophy, or Becker muscular dystrophy, preferably Duchenne muscular dystrophy.

[0041] As used herein, "prevention" includes suppressing and delaying the onset of a disease, and includes not only prevention before the onset of the disease but also prevention against recurrence of the disease after treatment. On the other hand, "treatment" includes curing symptoms, improving symptoms, and suppressing the progression of symptoms.

[0042] The pharmaceutical composition according to the present invention can be formulated and administered as a drug according to a known method. For example, it can be administered orally or parenterally to humans or mammals as a liquid as it is or as a drug in an appropriate dosage form. The dosage for humans is not particularly limited, but for example, it can be 0.01 mg / kg to 50 mg / kg.

[0043] The drug may contain a preservative that inhibits the growth of microorganisms or a buffer that helps maintain the pH within an acceptable range. Preservatives include sodium azide, octadecyl dimethyl benzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol. Buffers are phosphoric acid, citric acid, and other organic acids.

[0044] In addition, the drug may contain, for example, an excipient, a stabilizer, a chelating agent such as EDTA, a salt, or an antibacterial agent. Additionally, it can contain antioxidants such as ascorbic acid and methionine, proteins such as polypeptides, serum albumin, gelatin, or non-specific immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine, monosaccharides, disaccharides, and other carbohydrates such as glucose, mannose, or dextrin, and saccharides such as sucrose, mannitol, trehalose, or sorbitol.

Example

[0045] 1. Synthesis of PROTAC(H-PGDS)-1 and PROTAC(H-PGDS)-2 According to the following synthetic procedure, PROTAC(H-PGDS)-1 was synthesized by binding TFC-007, a ligand of H-PGDS, and a ubiquitin ligase ligand (pomalidomide) via a polyethylene glycol linker.

[0046]

Chemical formula

[0047] In the above synthetic procedure, Compounds 1 and 3 were synthesized by previously reported methods.

[0048] For the synthesis of Compound 2, to a solution of Compound 1 (1.51 g, 5.99 mmol) in N,N-dimethylformamide were added N-Boc piperazine (1.69 g, 8.99 mmol), EDCI (1.40 g, 7.19 mmol), and HOBt·H 2 O (1.02 g, 6.59 mmol), and the mixture was stirred at room temperature for 6 hours. Then, the reaction solution was poured into water, and the resulting solid was collected by filtration and dried to obtain a yellow solid. The obtained solid was dissolved in ethanol (60 ml) without purification, palladium carbon (91.2 mg) was added, and the mixture was stirred overnight under a hydrogen atmosphere. The reaction solution was filtered through celite to obtain reddish-brown solid 2 (2.14 g, 92%).

[0049] For the synthesis of Compound 4, Compound 2 (2.10 g, 5.40 mmol), Compound 3 (1.17 g, 6.49 mmol), EDCI (1.25 g, 6.49 mmol), and HOBt·H 2 O (1.02 g, 6.59 mmol) were dissolved in N,N-dimethylformamide (20 mL), and the mixture was stirred at room temperature for 6 hours. Then, the reaction solution was poured into water, and the resulting solid was collected by filtration and dried under vacuum to obtain gray solid 4 (1.14 g, 36%).

[0050] For the synthesis of Compound 5, Compound 4 (20.7 mg, 0.0352 mmol) in CH 2 Cl2 To the solution (1 mL), trifluoroacetic acid (TFA, 40.2 mg, 0.352 mmol) was added, and the mixture was stirred at room temperature for 2 hours. The reaction solution was evaporated under reduced pressure, and the obtained solid was purified by NH silica gel column chromatography (CH 2 Cl 2 / MeOH = 10 / 1) to obtain a colorless transparent oily compound 5 (19.3 mg, quant.).

[0051] In the synthesis of PROTAC(HPGDS)-1, to the N,N-dimethylformamide solution (2.5 mL) of compound 5 (21.2 mg, 0.0436 mmol), commercially available compound 6 (21.9 mg, 0.0371 mmol), EDCI (12.0 mg, 0.0653 mmol), DIPEA (11.0 mg, 0.0653 mmol), and HOBt (11.0 mg, 0.0872 mmol) were added, and the mixture was stirred at room temperature for 2 days. The reaction solution was purified by HPLC (0.1% TFA MeCN / H 2 O = 15 : 85 to 50 : 50, 40 min) to obtain a white solid PROTAC(HPGDS)-1 (2.88 mg, 2.78 μMmol, 7.5%).

[0052] Also, according to the following synthetic procedure, PROTAC(H-PGDS)-2 into which N-Methylated pomalidomide that cannot bind to ubiquitin ligase was introduced was synthesized as a negative control.

[0053]

Chemical Structure

[0054] In the above synthesis procedure, in the synthesis of compound 8, to a solution of compound 5 (56.7 mg, 0.119 mmol) in N,N-dimethylformamide (2.0 ml), commercially available compound 7 (36.2 mg, 0.108 mmol), EDCI (64.2 mg, 0.324 mmol), and DIPEA (49.2 mg, 0.324 mmol) were added, and the mixture was stirred at room temperature for 1.5 hours. Then, after distilling off the reaction solution under reduced pressure, saturated aqueous sodium bicarbonate was added and the mixture was extracted with ethyl acetate. The organic layer was washed with saturated brine, dried over sodium sulfate, filtered, and then distilled off under reduced pressure. The resulting solid was purified by silica gel column chromatography (CH 2 Cl 2 / MeOH = 10 / 1) to obtain white solid 8 (23.7 mg, 25%).

[0055] In the synthesis of compound 9, compound 8 (23.7 mg, 0.0295 mmol) was dissolved in methanol (4 ml), then palladium carbon (6.9 mg) was added, and the mixture was stirred under a hydrogen atmosphere for 24 hours. The reaction solution was filtered through celite, and the filtrate was distilled off under reduced pressure to obtain white solid 9 (20.3 mg, 66%).

[0056] In the synthesis of PROTAC(HPGDS)-2, to a solution of compound 9 (28.9 mg, 0.0276 mmol) in dimethyl sulfoxide (1 mL), compound 10 (9.7 mg, 0.0308 mmol) and DIPEA (15.6 mg, 0.0772 mmol) were added, and the mixture was stirred at 90 °C for 30 minutes. The reaction solution was cooled and stirred at room temperature for 6 hours. The resulting solid was purified by HPLC (0.1% TFA MeCN / H 2 O = 10 : 90 to 90 : 10, 40 min) to obtain white solid PROTAC(HPGDS)-2 (1.2 mg, 4.2%).

[0057] 2. Evaluation of H-PGDS degradation activity and mechanism analysis of the above compounds PROTAC (H-PGDS)-1 (1 - 1000 nM) was added to human KU812 cells, which are H-PGDS-expressing cells, and after incubation at 37°C for 3 - 24 hours, the amount of H-PGDS protein was evaluated by Western blot. As a result, it was revealed that PROTAC (H-PGDS)-1 decreased H-PGDS protein in a concentration-dependent and time-dependent manner.

[0058] The results are shown in Figure 2. Figure 2 shows the results of examining the degradation induction using PROTAC (H-PGDS)-1. KU812 cells were treated with PROTAC (H-PGDS)-1 (0, 1, 10, 100, 1000 nM) and cultured for 3 / 6 / 24 hours. The amount of H-PGDS protein was corrected with the amount of β-Actin protein as an internal standard, and evaluated as a relative value with the DMSO control set to 100. The experiment was performed 3 times, and the bar graph represents the mean ± standard deviation.

[0059] Next, the mechanism of the decrease in H-PGDS treated with PROTAC (H-PGDS)-1 was investigated. First, the turnover of H-PGDS after treatment with PROTAC (H-PGDS)-1 was examined. When KU812 cells were treated with CHX, a protein synthesis inhibitor, the amount of H-PGDS protein was maintained up to 6 hours in control cells, while the amount of H-PGDS protein decreased dramatically within 6 hours in cells treated with PROTAC (H-PGDS)-1. The results are shown in Figure 3. Figure 3 shows the results of examining the turnover of H-PGDS protein using CHX. KU812 cells were treated with CHX (10 μg / ml) in the presence of PROTAC (H-PGDS)-1 (0, 100 nM) and cultured for 0 / 1 / 2 / 4 / 6 hours. The amounts of H-PGDS and cyclin-B1 proteins were corrected with the amount of β-Actin protein as an internal standard, and evaluated as relative values with the value at the treatment time of 0 set to 100. The experiment was performed 3 times, and the bar graph represents the mean ± standard deviation.

[0060] Furthermore, when examining the mRNA level of H-PGDS in KU812 cells, no effect was observed upon the addition of PROTAC(H-PGDS)-1, indicating that PROTAC(H-PGDS)-1 is a degrader of H-PGDS protein. The results are shown in Figure 4. Figure 4 shows the expression level of H-PGDS mRNA in KU812 cells upon the addition of PROTAC(HPGDS)-1. KU812 cells were treated with PROTAC(H-PGDS)-1 (0, 1, 10, 100, 1000 nM) and cultured for 6 hours. The protein amount of H-PGDS was corrected with the protein amount of β-Actin as an internal standard, and evaluated as a relative value with the DMSO control set to 100. The experiment was conducted 3 times, and the bar graph represents the mean ± standard deviation. Student's t-test (two-tailed test) was used to evaluate the significant difference, and * represents P < 0.01 compared to the control.

[0061] Subsequently, to examine the effects of TFC-007 and pomalidomide on H-PGDS protein, when a mixture of TFC-007 and pomalidomide (1 μM) was added, no decrease in H-PGDS protein was observed. The results are shown in Figure 5(a). Figure 5(a) shows KU812 cells treated with PROTAC(H-PGDS)-1 (1 μM) or a TFC-007 / pomalidomide mixture (described as Ligand mix, 1 μM each) and cultured for 6 hours. This revealed that the novel compound conjugating the two ligands (TFC-007 and pomalidomide) is important for the degradation of H-PGDS protein.

[0062] In addition, to examine the necessity of recruiting cereblon involved in the degradation of H-PGDS protein, a competitive inhibition assay was performed by co-adding an excessive amount of pomalidomide (10 μM) to PROTAC(H-PGDS)-1 (100 nM). The results are shown in Fig. 5(b). Fig. 5(b) shows the effect of PROTAC(H-PGDS)-1 on the decrease in H-PGDS protein when an excessive amount of pomalidomide (10 μM) was co-added, and the KU812 cells were cultured for 6 hours after drug treatment. In the presence of an excessive amount of pomalidomide, a decrease in H-PGDS protein by PROTAC(H-PGDS)-1 was not observed, suggesting that the binding of the pomalidomide moiety of PROTAC(H-PGDS)-1 to cereblon is important for the proteolysis of H-PGDS.

[0063] Furthermore, to examine the involvement of the UPS in the degradation of H-PGDS protein by PROTAC(H-PGDS)-1, the amount of H-PGDS protein was measured when a proteasome inhibitor (MG132) or a ubiquitin-activating enzyme inhibitor (MLN7243) was co-added. The results are shown in Fig. 5(c). Fig. 5(c) shows the effect of PROTAC(H-PGDS)-1 on the decrease in H-PGDS protein in the co-addition of a proteasome inhibitor (MG132) or a ubiquitin-activating enzyme inhibitor (MLN7243) in KU812 cells, and the KU812 cells were cultured for 6 hours after drug treatment. The amount of H-PGDS protein was corrected with the amount of β-Actin protein as an internal standard, and evaluated as a relative value with the control set to 100. The experiment was performed 3 times, and the bar graph represents the mean ± standard deviation. The significance was evaluated by Student's t-test (two-tailed test), and * represents P < 0.01 compared to the control. The addition of various inhibitors suppressed the degradation of H-PGDS protein induced by PROTAC(H-PGDS)-1, suggesting that the UPS is involved in the degradation of H-PGDS protein.

[0064] 3. Evaluation of the binding ability of PROTAC(H-PGDS)-1 and PROTAC(H-PGDS)-2 to H-PGDS As described above, it was revealed that PROTAC(H-PGDS)-1 is a potent inducer of H-PGDS proteolysis via the UPS. However, since PROTAC(H-PGDS)-1 contains the TFC-007 structure that inhibits the enzymatic activity of H-PGDS, it may also inhibit the enzymatic activity of H-PGDS. In the future, in order to evaluate the contribution of the proteolysis-inducing activity in examining the inhibitory effect of PGD production by PROTAC(H-PGDS)-1, the binding ability was evaluated using PROTAC(H-PGDS)-2 composed of TFC-007 and N-Methylated pomalidomide (a pomalidomide derivative without binding activity to cereblon). 2 First, the binding activity of PROTAC(H-PGDS)-1 and PROTAC(H-PGDS)-2 to H-PGDS and the proteolytic activity of the H-PGDS protein were evaluated. The results are shown in Fig. 6(a). Fig. 6(a) shows the binding evaluation to H-PGDS by a fluorescence polarization assay using Prostaglandin D Synthase FP-Based Inhibitor Screening Assay Kit-Green (Cayman). As a result of evaluating the binding affinity of each compound to H-PGDS by a fluorescence polarization assay, TFC-007, PROTAC(H-PGDS)-1, and PROTAC(H-PGDS)-2 showed equivalent affinities (0.32 μM, 0.32 μM, 0.30 μM) for H-PGDS (R

[0065] = 0.9942, R 2 = 0.9841, R 2 = 0.9683, and for HQL-79, a specific inhibitor for H-PGDS, R 2 = 0.9569). 2 = 0.9569).

[0066] In addition, the degradation of H-PGDS protein was investigated. The results are shown in Fig. 6(b). Fig. 6(b) shows the evaluation of H-PGDS degradation activity by PROTAC(H-PGDS)-2. KU812 cells were treated with PROTAC(H-PGDS)-1 (100 nM) or PROTAC(H-PGDS)-2 (100 nM) and cultured for 6 hours. The protein amount of H-PGDS was corrected with the protein amount of β-Actin as an internal standard, and evaluated as a relative value with the DMSO control set to 100. The experiment was performed 3 times, and the bar graph represents the mean ± standard deviation. Student's t-test (two-tailed test) was used to evaluate the significant difference, and * represents P < 0.01 compared to the control. As shown in Fig. 6(b), PROTAC(H-PGDS)-2 did not decrease the amount of H-PGDS protein.

[0067] From the above, it is suggested that PROTAC(H-PGDS)-2 is a compound that has the enzyme inhibitory activity of H-PGDS but no degradation-inducing activity, and it was considered to be a negative control compound when examining the inhibitory effect on PGD 2 production by PROTAC(H-PGDS)-1.

[0068] 4. Synthesis of PROTAC(H-PGDS)-3 to PROTAC(H-PGDS)-10 Using PROTAC(H-PGDS)-1 as a lead, PROTAC(H-PGDS)-3 to PROTAC(H-PGDS)-7 with different numbers of PEGs were designed and synthesized. In addition, compound PROTAC(H-PGDS)-7 without a linker and the corresponding negative control PROTAC(H-PGDS)-8 were synthesized by the same route. Also, compound PROTAC(H-PGDS)-10 without a linker was synthesized. Furthermore, PROTAC(H-PGDS)-9, which linked Pomalidomide and a TFC-007 analog, was synthesized.

[0069]

Chemical Structure

[0070] The synthesis procedure is shown below.

[0071]

Chem.

[0072] NMR was performed using an ECZ600 (JEOL). Chemical shift values (δ, ppm) were corrected using the residual solvent signal (DMSO-d 6 : 2.49 for 1 1H NMR, 39.5 for 13 13C NMR).

[0073] Analytical HPLC was performed using Inertsil® WP300 C18, 5 μm, 4.6 mm × 250 mm, solvent A: 0.1% TFA / water, solvent B: 0.1% TFA / MeCN, gradient: 10 - 90% gradient of solvent B over 30 min, flow rate: 2 mL / min, 40°C General Procedure A The amine compound was dissolved in dimethylformamide, and compound 5 (1.5 eq) and diisopropylethylamine (1.5 eq) were added. The reaction was carried out under microwave irradiation at 80°C for 40 min, stirred at 50°C for 42 h, then the reaction mixture was cooled and filtered. Purification was performed by HPLC (gradient 30 - 60% MeCN - H 2 2O containing 0.1% TFA in 40 min).

[0074] General Procedure B The amine compound was dissolved in dimethylformamide, and compound 5 or 7 (3 eq) and diisopropylethylamine (3 eq) were added. The reaction was carried out under microwave irradiation at 80°C for 2 h. The reaction mixture was cooled and filtered. Purification was performed by HPLC (gradient 30 - 60% MeCN - H 2 2O containing 0.1% TFA in 40 min).

[0075] Synthesis of PROTAC(H-PGDS)-6 Compound 4 (32.2 mg, 53.5 μmol) was dissolved in dimethylformamide (0.5 mL), and PROTAC(H-PGDS)-6 was obtained as a yellow solid (14.5 mg, 32%) according to the method of General Procedure B. 1 H NMR (600 MHz, DMSO-d 6) : δ 11.1 (s, 1H), 10.5 (br, 1H), 9.10 (s, 2H), 7.78 - 7.71 (br, 3H), 7.58 (t, J = 7.2 Hz, 1H), 7.47 (t, J = 8.4 Hz, 2H), 7.38 (br, 1H), 7.29 (t, J = 7.2 Hz, 1H), 7.25 (d, J = 8.4 Hz, 2H), 7.13 (d, J = 9.0 Hz, 1H), 7.40 (d, J = 7.8 Hz, 1H), 6.57 (br, 1H), 5.05 (dd, J = 7.2, 5.4 Hz, 1H), 3.69 - 3.60 (m, 6H), 3.53 - 3.39 (m, 10H), 3.26 (br, 1H), 2.99 - 2.84 (m, 2H), 2.61 (br, 3H), 2.18 - 2.12 (m, 1H), 2.11 - 2.06 (m, 1H), 2.06 - 1.99 (m, 1H), 1.86 (br, 4H). 13 C NMR (151 MHz, DMSO-d 6 ) : δ 172.8, 172.0, 170.1, 169.0 (2C), 167.3, 165.8, 161.7, 159.8 (2C), 152.5, 146.4 (2C), 136.3 (2C), 132.1, 132.1, 129.8 (2C), 125.6, 123.9 (2C), 121.6 (2C), 121.4 (2C), 117.4 (2C), 114.6, 68.6 (2C), 66.8, 66.5, 55.0, 48.6 (6C), 41.6, 31.0 (2C), 26.9, 22.1. HRMS (ESI) m / z calculated for C 45 H 48 N 9 O 9 [M + H] + 858.3570 found 858.3563. HPLC purity: >98% (t R = 13.0 min).

[0076] Synthesis of PROTAC(H-PGDS)-5 Compound 3 (9.2 mg, 14.2 μmol) was dissolved in dimethylformamide (0.5 mL), and PROTAC(H-PGDS)-5 was obtained as a yellow solid (1.2 mg, 9%) according to the method of General Procedure A. HRMS (ESI) m / z calculated for C 47 H 52 N 9 O 10 [M + H] + 902.3832 found 902.3838. HPLC purity: >93% (t R = 13.1 min).

[0077] Synthesis of PROTAC(H-PGDS)-4 Compound 2 (18.2 mg, 26.4 μmol) was dissolved in dimethylformamide (0.5 mL), and PROTAC(H-PGDS)-4 was obtained as a yellow solid (3.0 mg, 12%) according to the method of General Procedure B. 1 H NMR (600 MHz, DMSO-d 6) : δ 11.1 (s, 1H), 10.5 (br, 1H), 9.10 (s, 2H), 7.74 (br, 3H), 7.57 (t, J = 18.0 Hz, 1H), 7.47 (t, J= 7.8 Hz, 2H), 7.32 (br, 1H), 7.29 (t, J = 7.2 Hz, 1H), 7.25 (d, J= 7.2 Hz, 2H), 7.14 (d, J = 8.4 Hz, 1H), 7.03 (d, J = 7.2 Hz, 1H), 6.59 (br, 1H), 5.05 (dd, J = 7.8, 5.4 Hz, 1H), 3.68-3.63 (m, 2H), 3.62-3.34 (m, 24H), 2.90-2.84 (m, 1H), 2.61-2.55 (m, 5H), 2.04-1.99 (m, 1H), 1.84 (br, 4H). 13 C NMR (151 MHz, DMSO-d 6 ) : δ 172.8, 172.1, 170.1, 168.9 (2C), 167.3, 165.7, 161.6, 159.8 (2C), 152.5, 146.4 (2C), 136.2 (2C), 132.1, 129.8 (2C), 129.3, 125.6, 123.9 (2C), 121.6 (2C), 121.4 (2C), 117.5 (2C), 115.2, 69.8-69.7 (5C), 68.9, 67.0, 66.7, 54.9, 48.5 (6C), 41.7, 31.0 (2C), 27.1, 22.1. HRMS (ESI) m / z calculated for C 49 H 56 N 9 O 11 946.4094 [M + H] + found 946.4098. HPLC purity: >85% (t R = 13.3 min).

[0078] Synthesis of PROTAC(H-PGDS)-3 Compound 1 trifluoroacetate (30.5 mg, 36.0 μmol) was dissolved in dimethylformamide (0.5 mL), and PROTAC(H-PGDS)-3 was obtained as a yellow solid (1.0 mg, 3%) according to the method of General Procedure A. HRMS (ESI) m / z calculated for C 51 H 60 N 9 O 12 [M + H] + 990.4356 found 990.4332. HPLC purity: >97% (t R = 13.4 min).

[0079] Synthesis of PROTAC(H-PGDS)-7 Using compound 6 (20 mg, 41.1 μmol) and compound 5, PROTAC(H-PGDS)-7 was obtained as a yellow solid (10.7 mg, 35%) according to the method of General Procedure B. 1 H NMR (600 MHz, DMSO-d 6 ) : δ 11.1 (s, 1H), 10.5 (br, 1H), 9.09 (s, 2H), 7.72 (m, 4H), 7.47 (t, J = 8.4 Hz, 2H), 7.40 (d, J = 6.6 Hz, 1H), 7.35 (d, J = 8.4 Hz, 1H), 7.29 (t, J= 7.8 Hz, 1H), 7.25 - 2.22 (m, 3H), 5.10 (dd, J = 7.2, 5.4 Hz, 1H), 3.75 (br, 2H), 3.67 (br, 4H), 3.34 (br, 2H), 3.26 (br, 2H), 3.15 (br, 1H), 2.98 (br, 1H), 3.15 (br, 1H), 2.90 - 2.84 (m, 1H), 2.60 - 2.51 (m, 2H), 2.02 (br, 1H), 1.86 (br, 4H). 13 C NMR (151 MHz, DMSO-d6 ) : δ 172.7, 169.9, 166.9, 166.3, 165.7 (2C), 161.5, 159.7 (2C), 152.5, 149.3 (2C), 135.9 (2C), 133.5, 129.7 (2C), 125.5, 123.9, 123.8 (2C), 121.5 (2C), 121.3 (2C), 116.9 (2C), 115.2, 51.1 (2C), 48.9 (2C), 48.7 (2C), 44.8, 41.0, 30.9 (2C), 27.1, 21.7. HRMS (ESI) m / z calculated for C 40 H 39 N 8 O 7 [M + H] + 743.2936 found 743.2939 HPLC purity: >94% (t R = 12.6 min).

[0080] Synthesis of PROTAC(H-PGDS)-8 Compound 6 (30 mg, 61.7 μmol) and Compound 7 were dissolved in dimethylformamide (0.50 mL), and PROTAC(H-PGDS)-8 was obtained as a yellow solid (10.7 mg, 22%) according to the method of General Procedure B. 1 H NMR (600 MHz, DMSO-d 6) : δ 10.4 (broad, 1H), 9.09 (singlet, 2H), 7.73 (triplet, J = 7.2 Hz, 1H), 7.68 (broad, 2H), 7.47 (triplet, J = 7.8 Hz, 2H), 7.40 (doublet, J = 7.8 Hz, 1H), 7.36 (doublet, J = 9.0 Hz, 1H), 7.29 (triplet, J = 6.6 Hz, 1H), 7.25 (doublet, J = 8.4 Hz, 2H), 7.16 (broad, 1H), 5.18 (doublet of doublets, J = 8.4, 4.8 Hz, 1H), 3.75 - 3.67 (multiplet, 8H), 3.34 (singlet, 2H), 3.27 (singlet, 2H), 3.01 (singlet, 3H), 2.97 - 2.91 (multiplet, 2H), 2.76 (doublet, J = 16.2 Hz, 1H), 2.60 - 2.53 (multiplet, 2H), 2.04 (broad, 1H), 1.82 (broad, 4H). 13 C NMR (151 MHz, DMSO-d 6 ) : δ 172.3, 170.2, 167.5, 166.9, 166.3 (2C), 161.8, 160.3 (2C), 153.1, 149.9 (2C), 136.5 (2C), 134.4, 130.3 (2C), 126.2, 124.6, 124.5 (2C), 122.2 (2C), 122.0 (2C), 117.5 (2C), 115.8, 51.7 (2C), 49.9 (2C), 45.39 (2C), 43.68, 41.6, 31.7 (2C), 27.9, 27.2, 21.7. HRMS (ESI) m / z calculated for C 41 H 41 N 3 O 5 757.3087 [M + H] + found 757.3090. HPLC purity: >98% (t R = 13.9 min).

[0081] Synthesis of Compound 10 To a solution of compound 8 (238 mg, 0.858 mmol) in anhydrous DMF (4 mL) were added compound 9 (204 mg, 0.944 mmol), EDCI hydrochloride (181 mg, 0.944 mmol), and HOBt (145 mg, 0.944 mmol). The mixture was stirred at room temperature for 6 hours, and then water was added. The reaction mixture was filtered, and the solid was washed with water. The obtained solid was dried under vacuum to give compound 10 (288 mg, 71%) as a gray solid. 1 H NMR (600 MHz, DMSO-d 6 ) : δ 10.3 (s, 1H), 9.08 (s, 2H), 7.57 (d, J = 9.0 Hz, 2H), 7.46 (t, J = 7.8 Hz, 2H), 7.29 (t, J = 7.8 Hz, 1H), 7.24 (d, J = 7.8 Hz, 2H), 6.96 (d, J = 9.0 Hz, 2H), 3.45 (br, 4H), 3.06 (br, 4H), 1.41 (s, 9H). 13 C NMR (151 MHz, DMSO-d 6 ) : δ 165.6, 161.1, 159.6 (2C), 153.8, 152.6, 147.6, 130.8, 129.8 (2C), 125.6, 124.1, 121.6 (2C), 121.4 (2C), 116.1 (2C), 79.0, 48.6 (4C), 28.0 (3C). HRMS (ESI): m / z calcd. for C 26 H 30 N 5 O 4 [M+H] + 476.2292 found 476.2294.

[0082] Synthesis of compound 11 To a solution of compound 10 (50 mg, 0.105 mmol) in dichloromethane (1.5 mL) was added trifluoroacetic acid (0.5 mL), and the mixture was stirred at room temperature. After 4 hours, the solvent was evaporated under reduced pressure. The resulting residue was purified by amino silica gel chromatography (dichloromethane:methanol = 1:0 to 9:1) to obtain compound 11 (38.4 mg, 97%) as a pale brown solid. 1 H NMR (600 MHz, DMSO-d 6 ) : δ 10.2 (s, 1H), 9.08 (s, 2H), 7.56 (d, J = 9.0 Hz, 2H), 7.46 (t, J = 7.8 Hz, 2H), 7.29 (t, J = 7.8 Hz, 1H), 7.24 (d, J = 7.8 Hz, 2H), 6.91 (d, J = 9.0 Hz, 2H), 3.00 (br, 4H), 2.82 (br, 4H). 13 C NMR (151 MHz, DMSO-d 6 ) : δ 165.6, 161.0, 159.6 (2C), 152.6, 148.5, 130.2, 129.8 (2C), 125.6, 124.2, 121.6 (2C), 121.4 (2C), 115.4 (2C), 49.6 (2C), 45.6 (2C). HRMS (ESI): m / z calcd. for C 21 H 22 N 5 O 2 [M+H] + 376.1768 found 376.1767.

[0083] Synthesis of PROTAC(H-PGDS)-9 To a solution of compound 11 (38.4 mg, 0.103 mmol) in anhydrous DMF (1 mL) were added compound 5 (85.1 mg, 0.307 mmol) and diisopropylethylamine (53.5 μL, 0.307 mmol), and the mixture was reacted at 80 °C for 2 hours under microwave irradiation. The solvent was distilled off under reduced pressure. The obtained residue was purified by HPLC (30 - 60% in 40 min) to give PROTAC(H - PGDS)-9 (12.1 mg, 17%) as a yellow solid. 1 H NMR (600 MHz, DMSO - d 6 ) : δ 11.1(s, 1H), 10.3 (s, 1H), 9.09 (s, 2H), 7.73 (t, J = 7.2 Hz, 1H), 7.60 (d, J = 9.0 Hz, 2H), 7.47 (t, J = 7.2 Hz, 2H), 7.40 (t, J = 7.2 Hz, 2H), 7.29 (t, J = 7.26 Hz, 1H), 7.25 (d, J = 8.4 Hz, 2H), 7.03 (d, J = 9.0 Hz, 2H), 5.11 (dd, J = 7.8 Hz, 5.4Hz, 1H), 3.39 (br, 12H). 13 C NMR (151 MHz, DMSO - d 6 ) : δ 172.8,170.0,167.0,166.4,165.6, 161.1, 159.6 (2C), 152.6, 149.5, 147.6, 136.0, 133.7, 130.7, 129.8 (2C), 125.6, 124.2, 123.8, 121.6 (2C), 121.4, 116.8, 115.8, 115.1, 54.9, 50.4, 48.8 (2C), 48.6 (2C), 30.9, 29.0, 22.1. HRMS (ESI): m / z calcd. for C 34 H 30 N 7 O 6 [M + H] + 632.2252 found 632.2245.

[0084] Synthesis of PROTAC(H-PGDS)-10 According to the following synthetic procedure, PROTAC(H-PGDS)-10, which binds TAS-205, a ligand of H-PGDS, and a ubiquitin ligase ligand (pomalidomide), was synthesized by the method of General Procedure B.

[0085]

Chemical Structure

[0086] 1 H NMR (600 MHz, CD 3 OD): δ 7.68 (t, J = 7.2 Hz, 2H), 7.60 (d, J = 9.0 Hz, 2H), 7.47 (t, J = 7.2 Hz, 2H), 7.42 (d, J = 7.2 Hz, 1H), 7.33 (d, J = 8.4 Hz, 1H), 7.21 (d, J = 8.4 Hz, 1H), 6.83 (t, J = 1.8 Hz, 1H), 6.41 (dd J = 1.8, 3.6 Hz, 1H), 6.08 (dd, J = 3.0, 3.6 Hz, 1H), 5.08 - 5.10 (m, 1H), 3.17 - 3.80 m, 23H), 2.81 - 2.86 (m, 1H), 2.68 - 2.76 (m, 2H), 2.07 - 2.19 (m, 6H).

[0087] Analytical HPLC: Inertsil® WP300 C18, 5 μm, 4.6 mm × 250 mm, solvent A: 0.1% TFA / water, solvent B: 0.1% TFA / MeCN, gradient: 10 - 90% gradient of solvent B over 30 min, flow rate: 2 mL / min, 40°C HRMS (ESI) m / z calculated for C49H56N9O11 763.3442 [M + H]+ found 763.3462. HPLC purity: >98% (tR = 10.8 min).

[0088] 5. Evaluation of H-PGDS degradation activity and mechanism analysis of the above compound Compounds PROTAC(H-PGDS)-1, PROTAC(H-PGDS)-3 to PROTAC(H-PGDS)-10 synthesized were added to H-PGDS-expressing cells (human KU812 cells) and incubated at 37 °C for 6 hours. Then, the amount of H-PGDS protein was evaluated by Western blot. The involvement of UPS in the degradation of H-PGDS by the compounds was evaluated by co-adding a proteasome inhibitor (MG132) and a ubiquitin-activating enzyme inhibitor (MLN7243). Furthermore, the binding ability of PROTAC(H-PGDS)-7 to PROTAC(H-PGDS)-9 to H-PGDS was evaluated by a fluorescence polarization assay using Prostaglandin D Synthase FP-Based Inhibitor Screening Assay Kit-Green (Cayman).

[0089] The effects on the production of prostaglandins (PGs) (D2, E2, F2α) were evaluated by adding PROTAC(H-PGDS)-7 and PROTAC(H-PGDS)-8 to human KU812 cells and incubating at 37 °C for 6 hours. Then, to remove PROTAC(H-PGDS)-7 and PROTAC(H-PGDS)-8 from the culture medium, a washing operation (after centrifugation, removing the supernatant and adding a culture medium without PROTAC(H-PGDS)-7 and PROTAC(H-PGDS)-8) was repeated 3 times. Then, a calcium ionophore (A23187) was added to induce the production of PGs, and the PGs released into the culture medium in 10 minutes were quantitatively analyzed by LC-MS / MS method for evaluation.

[0090] The protocol for in vivo evaluation was as follows. To examine the inhibitory effect on dystrophic dilated cardiomyopathy, Duchenne muscular dystrophy (DMD) model mice (mdx mice, C57BL / 6 background, 9 - 10 weeks old, male) were used. According to the method of Anderson et al. [Anderson JE, Liu L, Kardami E, The effects of hyperthyroidism on muscular dystrophy in the mdx mouse : Greater dystrophy in cardiac and soleus muscle. Muscle Nerve 17(1) : 64 - 73. 1994], 3,4,3'-triiodo-L-thyronine (T3, Nacalai Tesque, Inc.) was subcutaneously administered to mdx mice at a dose of 2 mg / kg / day for 2 weeks to induce dilated cardiomyopathy accompanied by cardiac hypertrophy and apical fibrosis.

[0091] PROTAC(HPGDS)-7 or the HPGDS inhibitor (TFC-007) was subcutaneously administered once a day for 2 weeks simultaneously with the start of T3 administration. The effects of T3 administration on dilated cardiomyopathy in mdx mice were evaluated by measuring the organ weight of the heart (weight ratio to body weight), the concentration of cardiac troponin I (cTnI) in plasma, which is a biochemical marker of myocardial inflammation, the mRNA expression levels of inflammatory cytokines (TNF-α, TGF-β) in cardiac tissue by quantitative PCR, and the CD11β, a macrophage infiltration marker, by Western blot.

[0092] PROTAC(H-PGDS)-1, PROTAC(H-PGDS)-3 to PROTAC(H-PGDS)-7 (10 pM to 10 nM) were added to human KU812 cells and incubated at 37°C for 6 hours. After that, the amount of H-PGDS protein was evaluated by Western blot. As a result, it was revealed that PROTAC(H-PGDS)-1, PROTAC(H-PGDS)-3 to PROTAC(H-PGDS)-7 decreased the H-PGDS protein in a concentration-dependent manner (Figure 7). Figure 7 shows the relationship between the linker length of the compound and the H-PGDS degradation activity. The amount of H-PGDS protein was corrected with the amount of β-Actin protein as an internal standard and evaluated as a relative value with the DMSO control set to 100. The experiment was conducted 3 times, and the bar graph represents the mean ± standard deviation. As shown in Figure 7, PROTAC(H-PGDS)-7, which has no linker structure in particular, had high H-PGDS degradation activity even at 100 pM.

[0093] Figure 8 shows the evaluation of H-PGDS degradation activity by PROTAC(H-PGDS)-10. KU812 cells were treated with PROTAC(H-PGDS)-10 (10 pM - 10000 pM) and cultured for 6 hours, and then the effect on the amount of H-PGDS protein was evaluated. The amount of H-PGDS protein was corrected with the amount of β-Actin protein as an internal standard and evaluated as a relative value with the DMSO control set to 100. The experiment was conducted 3 times, and the bar graph represents the mean ± standard deviation. It was revealed that PROTAC(H-PGDS)-10 decreased the H-PGDS protein in a concentration-dependent manner (Figure 8). Therefore, it was found that not only TFC-007 but also the compound obtained by connecting TAS-205 to pomalidomide showed H-PGDS degradation-inducing activity as a ligand for H-PGDS.

[0094] Subsequently, the effects of TFC-007 and Pomalidomide on H-PGDS protein were examined. KU812 cells were treated with PROTAC(H-PGDS)-7 (100 pM) or a TFC-007 / Pomalidomide mixture (100 pM each), cultured for 6 hours, and then the effect on the amount of H-PGDS protein was evaluated. Figure 9 shows the necessity of ligand conjugation for the degradation of H-PGDS by PROTAC(H-PGDS)-7. The amount of H-PGDS protein was corrected with the amount of β-Actin protein as an internal standard and evaluated as a relative value with the DMSO control set to 100. The experiment was conducted 3 times, and the bar graph represents the mean ± standard deviation. As shown in Figure 9, no decrease in H-PGDS protein was observed upon treatment with the TFC-007 / Pomalidomide mixture, indicating that the chimeric compound conjugated with the two ligands (TFC-007 and Pomalidomide) is important for the degradation of H-PGDS protein.

[0095] Also, the necessity of recruiting cereblon involved in the degradation of H-PGDS protein was examined. KU812 cells were treated by co-adding an excessive amount of Pomalidomide (1 μM) to PROTAC(H-PGDS)-7 (100 pM), cultured for 6 hours, and then the effect on the amount of H-PGDS protein was evaluated. Figure 9 is a diagram for evaluating the effect of PROTAC(H-PGDS)-7 on the decrease in H-PGDS protein when an excessive amount of pomalidomide (1 μM) was co-added. The amount of H-PGDS protein was corrected with the amount of β-Actin protein as an internal standard and evaluated as a relative value with the DMSO control set to 100. The experiment was conducted 3 times, and the bar graph represents the mean ± standard deviation. As a result of the competitive inhibition assay, no decrease in H-PGDS protein by PROTAC(H-PGDS)-7 was observed in the presence of an excessive amount of Pomalidomide. It was suggested that the binding of the Polmalidomide site of PROTAC(H-PGDS)-7 to cereblon is important for the proteolysis of H-PGDS (Figure 10).

[0096] Furthermore, to investigate the involvement of the UPS in the degradation of H-PGDS by PROTAC (H-PGDS)-7, the amount of H-PGDS protein was measured when a proteasome inhibitor (MG132) or a ubiquitin-activating enzyme inhibitor (MLN7243) was co-added. Figure 11 is a diagram for evaluating the effect of co-addition of a proteasome inhibitor (MG132, 10 μM) or a ubiquitin-activating enzyme inhibitor (MLN7243, 10 μM) on the decrease in H-PGDS protein by PROTAC (H-PGDS)-7 in KU812 cells. The protein amount of H-PGDS was corrected with the protein amount of β-Actin as an internal standard and evaluated as a relative value with the DMSO control set to 100. The experiment was performed three times, and the bar graph represents the mean ± standard deviation. The addition of various inhibitors suppressed the degradation of H-PGDS protein induced by PROTAC (H-PGDS)-7, suggesting that the UPS is involved in the degradation of H-PGDS protein (Figure 11). The degradation of H-PGDS protein by PROTAC (H-PGDS)-7 is proteasome-, ubiquitin-, and ubiquitin ligase (cereblon)-dependent, similar to PROTAC (H-PGDS)-1 found so far.

[0097] 6. Evaluation of the binding ability of PROTAC (H-PGDS)-7 to H-PGDS PROTAC(H-PGDS)-7 was revealed to be a potent inducer of H-PGDS proteolysis via the UPS. However, since PROTAC(H-PGDS)-7 contains the TFC-007 structure that inhibits the enzymatic activity of H-PGDS, it may also inhibit the enzymatic activity of H-PGDS. In order to examine the effect of PROTAC(H-PGDS)-7 on PGD2 production in the future, the binding activity of PROTAC(H-PGDS)-8, which is composed of TFC-007 and N-Methylated pomalidomide (a Pomalidomide derivative without binding activity to cereblon), useful for evaluating the contribution of proteolysis-inducing activity, to H-PGDS and the proteolytic activity of H-PGDS protein were evaluated. Figure 12 is a diagram showing the binding evaluation to H-PGDS by fluorescence polarization assay. As a result of evaluating the binding affinity of each compound to H-PGDS by fluorescence polarization assay, TFC-007, PROTAC(H-PGDS)-7, and PROTAC(H-PGDS)-8 showed equivalent affinities (0.15 μM, 0.14 μM, 0.14 μM) to H-PGDS (Figure 12).

[0098] In addition, the degradation of H-PGDS protein was examined. KU812 cells were treated with PROTAC(H-PGDS)-7 (10 pM - 10000 pM) or PROTAC(H-PGDS)-8 (10 pM - 10000 pM), cultured for 6 hours, and then the effect on the amount of H-PGDS protein was evaluated. The amount of H-PGDS protein was corrected with the amount of β-Actin protein as an internal standard and evaluated as a relative value with the DMSO control set to 100. The experiment was performed 3 times, and the bar graph represents the mean ± standard deviation. Figure 13 is a diagram showing the evaluation of H-PGDS degradation activity by PROTAC(H-PGDS)-8. PROTAC(H-PGDS)-8 did not decrease the amount of H-PGDS protein (Figure 13). It is considered that PROTAC(H-PGDS)-7 has proteolysis-inducing activity and enzymatic inhibitory activity for H-PGDS, while PROTAC(H-PGDS)-8 has only H-PGDS enzymatic inhibitory activity.

[0099] From the above, it is suggested that PROTAC(H-PGDS)-8 is a compound that has the enzyme inhibitory activity of H-PGDS but no degradation-inducing activity, and it is considered to be a negative control compound when examining the inhibitory effect of PGD2 production by PROTAC(H-PGDS)-7. Figure 14 is a diagram showing the evaluation of the binding to H-PGDS by fluorescence polarization assay. Figure 15 is a diagram showing the evaluation of the H-PGDS degradation activity by PROTAC(H-PGDS)-9. KU812 cells were treated with PROTAC(H-PGDS)-9 (10 pM - 10000 pM), cultured for 6 hours, and then the effect on the amount of H-PGDS protein was evaluated. The amount of H-PGDS protein was corrected with the amount of β-Actin protein as an internal standard, and evaluated as a relative value with the DMSO control set to 100. The experiment was performed 3 times, and the bar graph represents the mean ± standard deviation. PROTAC(H-PGDS)-9 and its ligand site compound 11 showed an affinity equivalent to that of TFC-007 for H-PGDS (Figure 14), but PROTAC(H-PGDS)-9 did not decrease the amount of H-PGDS protein (Figure 15). Since PROTAC(H-PGDS)-9 contains the structure excluding the piperazine site of PROTAC(H-PGDS)-7, it became clear that the piperazine site of PROTAC(H-PGDS)-7 is necessary for the degradation activity.

[0100] KU812 cells were treated with PROTAC(H-PGDS)-7, PROTAC(H-PGDS)-8 or the HPGDS inhibitor TFC-007 for 6 hours. After removing the compounds in the culture medium, KU812 cells were stimulated with calcium ionophore (A23187) to induce prostaglandin production. When KU812 cells were stimulated with A23187, the production of PGD 2 , PGE 2 , PGF 2α was enhanced compared to non-stimulated cells. After pre-treating with TFC-007 (10 - 100 nM) for 6 hours and then stimulating the cells with A23187 after removing TFC-007 from the culture medium, PGD 2 , PGE 2 , PGF 2αProduction was not different from that from cells pretreated with a solvent. On the other hand, when cells from which PROTAC(H-PGDS)-7 (10 - 100 nM) had been removed from the culture medium after 6-hour pretreatment were stimulated with A23187, PGD 2 production was significantly suppressed at any dose. On the other hand, PGE 2 , PGF 2α production was significantly enhanced (Figure 16). The experiment was performed 3 times, and the bar graph represents the mean ± standard deviation.

[0101] When cells from which PROTAC(H-PGDS)-8 (10 - 100 nM) had been removed from the culture medium after 6-hour pretreatment were stimulated with A23187, PGD 2 production was significantly suppressed when pretreated at 30 and 100 nM, and PGE 2 , PGF 2α production was enhanced (Figure 16).

[0102] When 3,4,3'-triiodo-L-thyronine (T3), a thyroid hormone, was subcutaneously administered to Duchenne muscular dystrophy model mice (mdx mice) at 2 mg / kg / day for 2 weeks, obvious cardiac hypertrophy developed compared to mdx mice not administered T3. Cardiac hypertrophy was compared by the value obtained by dividing the wet weight of the heart of each mouse by the body weight. The experiment was performed 3 - 5 times, and the bar graph represents the mean ± standard deviation. When PROTAC(H-PGDS)-7 (78 mg / kg / day) or TFC-007 (30 mg / kg / day) was subcutaneously administered for 2 weeks starting at the time of T3 administration, cardiac hypertrophy was suppressed compared to mice administered the solvent, and the inhibitory effect of PROTAC(H-PGDS)-7 was equal to or greater than that of TFC-007 (Figure 17).

[0103] mdx mice were administered T3 (2 mg / kg / day) for 2 weeks to induce myocardial inflammation. When measuring plasma cardiac troponin I (cTnI), one of the myocardial damage markers, mice administered T3 at 2 mg / kg / day showed a clear increase in plasma cTnI levels compared to mdx mice not administered T3. Subcutaneous administration of PROTAC(H-PGDS)-7 (78 mg / kg / day) or TFC-007 (30 mg / kg / day) for 2 weeks starting from the onset of T3 administration decreased the plasma cTnI level compared to mice administered the vehicle (Figure 18). The experiment was performed 3 - 5 times, and the bar graph represents the mean ± standard deviation.

[0104] The mRNA levels of TNF-α, an inflammatory cytokine, TGF-β, which promotes fibrosis, or CD11b, an infiltration marker for macrophages and monocytes, in the hearts of mdx mice that developed cardiac hypertrophy after administration of T3 at 2 mg / kg / day were examined by quantitative PCR.

[0105] Administration of T3 at 2 mg / kg / day to mdx mice for 2 weeks clearly increased the mRNA expression of TNF-α, TGF-β, and CD11b compared to mdx mice not administered T3, strongly suggesting that inflammation and fibrosis accompanied by infiltration of monocytes and macrophages were progressing in the myocardium. Subcutaneous administration of PROTAC(H-PGDS)-7 (78 mg / kg / day) or TFC-007 (30 mg / kg / day) for 2 weeks starting from the onset of T3 administration suppressed the expression levels of these mRNAs, and the effect of PROTAC(H-PGDS)-7 was stronger (Figure 19). The experiment was performed 3 - 5 times, and the bar graph represents the mean ± standard deviation.

[0106] From the above results, it was revealed that PROTAC(H-PGDS)-7 suppresses the progression of the disease in dystrophic cardiomyopathy developed by continuous administration of T3 to mdx mice. As described above, not only PROTAC(H-PGDS)-7 but also PROTAC(H-PGDS)-10 has been found to exhibit H-PGDS degradation-inducing activity, and PROTAC(H-PGDS)-10 is also effective against muscular dystrophy, particularly Duchenne muscular dystrophy.

Industrial Applicability

[0107] It can be used as a therapeutic agent for allergic diseases and inflammatory diseases.

Claims

**Claim 1** A novel compound characterized by being represented by the following chemical formula. 【Chemical 1】 **Claim 2** A novel compound characterized by being represented by the following chemical formula. 【Chemical 2】 **Claim 3** A novel compound characterized by being represented by the following chemical formula (n is an integer from 1 to 10). [Chemical Formula 3] **Claim 4** A novel compound characterized by being represented by the following chemical formula. 【Chemical Formula 4】 **Claim 5** A degrading inducer targeting hematopoietic prostaglandin D synthase, characterized by being represented by the following chemical formula. [Chemical Formula 5] **Claim 6** A degrading inducer targeting hematopoietic prostaglandin D synthase, characterized by being represented by the following chemical formula. 【Chemical Formula 6】 **Claim 7** A degrading inducer targeting hematopoietic prostaglandin D synthase, characterized by being represented by the following chemical formula. [Chemical Formula 7]

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