CN118146176A

CN118146176A - GPR133/ADGRD1 agonist, preparation method and application thereof

Info

Publication number: CN118146176A
Application number: CN202410263919.3A
Authority: CN
Inventors: 孙金鹏; 于晓; 杨帆; 肖鹏; 杨照; 朱孔凯; 平玉奇; 王明威; 杨娜; 李越
Original assignee: Shandong University
Current assignee: Shandong University
Priority date: 2024-03-08
Filing date: 2024-03-08
Publication date: 2024-06-07

Abstract

The invention relates to the field of pharmaceutical chemistry, in particular to a novel small molecule compound with a general formula (I), and discloses a synthetic route, a synthetic method and application of the compound. The compound has excellent agonistic activity of a G protein coupled receptor GPR133, can activate Gs/cAMP downstream of GPR133/ADGRD1 to rapidly improve skeletal muscle strength, can improve balance capacity, and is further developed as a potential medicament for treating or preventing muscle-related diseases and vestibular dysfunction diseases.

Description

GPR133/ADGRD1 agonist, preparation method and application thereof

Technical Field

The invention belongs to the field of pharmaceutical chemistry, and particularly relates to a G protein coupled receptor GPR133/ADGRD1 agonist, a synthesis method and application thereof in improving skeletal muscle strength, improving balance capacity and treating or preventing muscle related diseases and vestibular dysfunction diseases.

Background

The disclosure of this background section is only intended to increase the understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art already known to those of ordinary skill in the art.

The vestibular organ is located in the inner ear and comprises an elliptical sac, a balloon and three semicircular canals. Sensory information of peripheral vestibular organs, visual systems and proprioceptive systems is integrated through the vestibular centers of the brainstem, cerebellum and cerebral cortex, maintaining the balance and orientation of the body by vestibulo-ocular reflex (VOR), vestibulo-spinal reflex (VSR). Damage or lesions of the peripheral or central vestibular structure can lead to vestibular dysfunction (vestibular dysfunction, VD), i.e. problems of the inner ear and brain, clinically peripheral vestibular lesions are mainly manifested by dizziness/dizziness (verigo/dizziness), central vestibular lesions are mainly manifested by balance dysfunction, not only limit the daily activity capacity of patients, but also increase the risk of falling attacks, seriously affect the quality of life and social functions of patients, and increase psychological burden and mental stress of patients.

Vestibular dysfunction disease is one of three major problems in outpatient clinics with extremely high clinical morbidity. The investigation result shows that the prevalence rate of vestibular dysfunction disease in China is 5.0-9.0%, and nearly 35% of people in the United states over 40 years experience vestibular dysfunction. Of the population over 65 years old, elderly with a history of dizziness have vestibular dysfunction up to 30%, even up to 85% of elderly over 80 years old. At present, the main way of improving the autonomic nerve function of a patient is to eliminate the lost water, so as to improve the microcirculation of the inner ear. Although many studies have been conducted on vestibular dysfunction diseases, there are no specific drugs to cure the diseases, and most of the drugs (steroids, diuretics, etc.) on the market are anti-motion sickness, anti-nausea drugs or middle ear injection drugs to reduce the onset of dizziness and hearing loss, but the recurrence rate is high, statistically more than 30%. Thus, further research in this area would be helpful in developing methods and medicaments for the prevention and treatment of vestibular dysfunction.

G-protein coupled receptors (GPCRs) are the most successful class of drug receptors in the history of drug development, and 30-40% of drugs used in modern clinics all target G-protein coupled receptors. There are several GPCR ligands in the 100 most popular drugs worldwide, and such targets have great potential in drug development. GPCRs act as a large class of membrane receptors capable of sensing mechanical forces, and their sensing of force is largely carried out by the family of adhesion-type receptors, of which G-protein coupled receptor 133 (GPR 133) is an important member. GPR133 can be used as an important acting target point for designing a drug lead targeting vertigo diseases and developing drugs, and no effective compound aiming at the target point is reported at present.

Androgens are among the typical steroid hormones, such as 5α -dihydrotestosterone (5α -DHT), which regulate many important functions such as expression of male secondary sex characteristics or development of bones and muscles. 5α -DHT acts as a driving factor for bone and muscle formation, increasing bone density (BMD), enhancing bone strength, and promoting skeletal muscle growth to increase muscle strength. 5α -DHT not only regulates transcriptional function by direct interaction with nuclear Androgen Receptor (AR), but also initiates acute physiological responses within seconds to minutes, such as regulating muscle strength, relaxing penile erectile tissue, initiating rapid Ca ²⁺ signaling in macrophages, and stimulating glucose in islets to stimulate insulin secretion. In addition, 5α -DHT plays an important regulatory role in energy metabolism, inflammatory response, sexual function, cognitive effect, canceration, and the like. But 5α -DHT can induce a variety of adverse effects and is associated with increased risk of many diseases such as androgenic alopecia, polycystic ovary syndrome, metabolic syndrome, cardiovascular disease and prostate disease.

Screening data and cell-based reporter experiments showed that GPR133 can be activated by androgens, including endogenous DHT and its derivatives, the steroid mefenodone, and is highly potent. High quality life is a dream of human beings, but the problems of weak muscles, weak bones and the like are becoming reality. Thus, identification of the androgen membrane receptor GPR133 for great therapeutic potential in increasing muscle strength by sensing androgens, and the discovery that highly potent GPR133 agonists increase muscle strength without causing selective androgen adverse effects is important for a range of human diseases (e.g., wasting syndrome, osteoporosis, or sarcopenia).

Disclosure of Invention

An object of the present invention is to provide a compound having the general structural formula (I) as follows:

Wherein R ₁ is selected from one of-OCH ₃、-OCH₂CH₃、-NHCH₃ or-OCH ₂CH₂ OH;

R ₂ is selected from one of-CH ₃、-CH₂CH₃、-COCH₃, cyclopropyl, -CH ₂ Ph or-COPh;

R ₃ is selected from Wherein R ₄、R₅、R₆、R₇、R₈ is independently selected from one of-H, -F, -Cl, -Br, -CH ₃、-OCH₃、-COOH、-NO₂, tert-butyl and trifluoromethyl.

Further, derivatives of the general formula (I) include pharmaceutically acceptable salts, solvates, hydrates or crystals thereof.

Further, the numbers and structural formulas of the specific compounds represented by the general formula (I) are shown in Table 1.

TABLE 1 Structure of Compounds of formula (I)

Another object of the present invention is to provide a process for the preparation of the compounds of general formula (I) according to the invention, the synthetic route being as follows:

Further, the synthesis steps are as follows:

the compound A is subjected to amino protection to obtain an intermediate B, then the intermediate B is reacted with the compound C under the action of a catalyst to obtain an intermediate D, the intermediate D is reacted with trifluoroacetic acid to obtain an intermediate E, the E is reacted with different carboxylic acid compounds F to obtain a compound shown in a general formula (I), the E is reacted with bromoacetyl bromide to obtain an intermediate G, and the intermediate G is reacted with different compounds H to obtain the compound shown in the general formula (I).

Further, the synthesis steps are as follows:

Step 1: heating and refluxing the compound of the formula A and di-tert-butyl dicarbonate in a solvent to react, and protecting amino on a benzene ring to obtain an intermediate compound of the formula B;

Step 2: dissolving the intermediate compound B with a solvent, sequentially adding piperazine compound C, tris (dibenzylideneacetone) dipalladium, 2-dicyclohexylphosphine-2 ',4',6' -triisopropyl biphenyl and cesium carbonate, and heating and refluxing under the protection of nitrogen to obtain an intermediate compound D;

step 3: dissolving the intermediate compound of the formula D, adding trifluoroacetic acid under ice bath, and stirring at room temperature to obtain an intermediate compound of the formula E;

Step 4: dissolving different carboxylic acid compounds F, and carrying out amide condensation reaction on the dissolved carboxylic acid compounds F and an intermediate compound of the formula E under the conditions of alkali DIPEA and a condensing agent HATU to obtain a compound of the general formula (I);

Step 5: dissolving an intermediate compound of the formula E and bromoacetyl bromide in a solvent, and reacting under the catalysis of base triethylamine to obtain an intermediate compound of the formula G;

Step 6: dissolving an intermediate compound of the formula G and a phenol compound H, and reacting at room temperature under the catalysis of potassium carbonate to obtain a compound of the general formula (I).

In response to the deficiencies mentioned in the background art, the inventors found that the compound AP-970/43482503 (AP-970 or AP-503 for short) can activate Gs-cAMP signaling pathway downstream of GPR133/ADGRD1 in HEK293 cells based on the study that the GPR133/ADGRD1 receptor is expressed on the vestibular system of mice and that the vestibular dysfunction occurs in GPR133/ADGRD1 knockout mice in early studies.

Another object of the present invention is to use the structure of 5 alpha-DHT-GPR 133-Gs complex as a template, and the inventor finds that the compound AP-970/43482503 (AP-503 for short) is a potent, selective and AR-inactive GPR133 agonist, wherein the agonist AP-503 selectively activates Gs channels of GPR133, and EC ₅₀ is 1.21+/-0.06 nM by reasonably designing forward and reverse screening strategies. To the inventors' knowledge, this is the most effective synthetic agonist developed so far for aGPCR. Moreover, the derivatives of AP-503 obtained by structural modification of AP-503 according to the structure of AP-503 also exhibit strong agonistic activity of GPR 133. Furthermore, the inventors found that AP-503 retains muscle strengthening effects by activating GPR133 and ameliorates certain androgen adverse effects compared to 5α -DHT.

Another object of the invention is the use of the compounds of the invention as GPR133/ADGRD1 agonists, and further the use of the compounds of the invention for the preparation of a GPR133/ADGRD1 agonist drug. The inventor experiment proves that the compound provided by the invention can activate Gs-cAMP signal channel downstream of GPR133/ADGRD1 to be used as an agonist of G protein coupled receptor 133 (GPR 133)/ADGRD 1.

Further, the use of the compounds of the invention for improving the balance. The compound of the invention is applied to the preparation of drugs for improving balance ability and related diseases.

Further, the use of the compounds of the present invention for the prevention and/or treatment of vestibular dysfunction disorders. The application of the compound in preparing a medicament for preventing and/or treating vestibular dysfunction diseases.

Further, the application of the compound in preparation of the medicament for improving muscle strength is provided. Furthermore, the muscle strength improving medicine can be a muscle increasing medicine, and also can be a medicine for preventing or treating muscular atrophy, muscle weakness and bone weakness.

The beneficial effects are that:

1. The invention provides a small molecular compound targeting a G protein coupled receptor GPR133, which is disclosed for the first time, can obviously strengthen muscles and is used for treating or preventing related diseases such as muscle weakness, bone weakness and the like.

2. The compound has very excellent GPR133 agonistic activity, especially the application of the compound AP-970/43482503 in vestibular function, and can be used for preparing medicines for preventing or/and treating vestibular dysfunction diseases and improving or/and balancing capacity.

3. The GPR133 agonist AP-970/43482503 disclosed by the invention maintains the muscle strengthening effect by activating GPR133, improves certain androgen adverse reactions, and has great application potential in treating or preventing related diseases such as muscle weakness, bone weakness and the like.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention.

FIG. 1 shows the activity of compound AP-970/43482503 in the examples for the activation of GPR133/ADGRD 1;

FIG. 2 shows that compound AP-970/43482503 prevents or/and treats vestibular dysfunction disease and improves or/and enhances balance ability through GPR133/ADGRD1 by rotating stick and VOR experiments in examples;

FIG. 3 shows the change in endogenous cAMP levels in EDL after 8 week old WT and Gpr133 ^-/- mice were treated with control or AP-503 (2 mg/kg, intramuscular injection) for 30 minutes without AR antagonist ORM-15341 (10 mg/kg) (14 in each group, 7 in males, 7 in females);

FIG. 4 is a quantitative analysis of drop latency in 8 week old WT or Gpr133 ^-/- mice without or with NF449 (10 mg/kg) in suspension experiments with control drug or AP-503 min (n = 14 mice per group, including 7 male mice and 7 female mice);

FIG. 5 shows grip performance quantification of 8 week old WT and Gpr133 ^-/- mice (14 mice per group, including 7 male mice and 7 female mice) when grip performance testing was performed 30 minutes after administration of control drug or AP-503 without or with NF449 (10 mg/kg).

Detailed Description

The following describes in detail the examples of the present invention, which are implemented on the premise of the technical solution of the present invention, and detailed embodiments and specific operation procedures are given, but the scope of protection of the present invention is not limited to the following examples.

Example 1: preparation of methyl 3- (2, 4-dimethylphenoxy) acetamido) -4- (4-ethylpiperazin-1-yl) benzoate (AP-503)

Preparation of the compound of formula 2:

Methyl 3-amino-4-bromobenzoate 1 (1.0 equiv.) is dissolved in 30mL of methanol, di-tert-butyl dicarbonate (5.0 equiv.) is added, reflux reaction is performed at 70℃for 12h, after completion of the reaction, methanol is removed under reduced pressure, and purification by silica gel (200-300 mesh) column chromatography is performed to give the compound of formula 2.

Preparation of the compound of formula 4:

A25 mL round bottom flask was charged with compound 2 (1.0 equiv.) and dissolved in 8mL toluene, N-ethylpiperazine 3 (2.0 equiv.), tris (dibenzylideneacetone) dipalladium (0.03 equiv.), 2-dicyclohexylphosphine-2 ',4',6' -triisopropylbiphenyl (2.0 equiv.), cesium carbonate (2.5 equiv.), and nitrogen gas was added to the flask, the reaction system was sealed with nitrogen gas, refluxed at 110℃for 12 hours, and after completion of the reaction, insoluble salts were filtered, the filtrate was dried by spin, and purified by silica gel (200-300 mesh) column chromatography to give the compound of formula 4.

Preparation of the compound of formula 5:

Compound 4 (1.38 equiv.) is dissolved in 3mL of dichloromethane, trifluoroacetic acid (1 mL) is added at 0 ℃ and stirred at room temperature for 2h, after the reaction is completed, the pH is adjusted to be alkaline with saturated sodium bicarbonate solution, extraction is performed with dichloromethane, the organic phase is washed with saturated brine, dried over anhydrous sodium sulfate, filtered and spun-dried, and purification by silica gel column chromatography is performed to obtain compound of formula 5.

Preparation of Compound AP-503:

The compound 2, 4-dimethylbenzeneoxyacetic acid 6 (1.5 equiv.) is dissolved in 2ml of N, N-dimethylformamide, N, N-diisopropylethylamine (DIPEA, 1.1 equiv.) is added and stirred for 5min, then urea N, N, N ', N' -tetramethyl-O- (7-azabenzotriazol-1-yl) hexafluorophosphate (HATU, 1.1 equiv.) is added and stirred for 10min, finally compound 5 (1.0 equiv.) is added and reacted at 60℃for 8h, after completion, ethyl acetate, water extraction, washing the organic phase with saturated brine, drying over anhydrous sodium sulfate, filtration spin-drying, column chromatography purification gives compound AP-503 as a brown oil with a yield 68.6％.¹H NMR(600MHz,CDCl₃)δ9.20(brs,1H),9.05(d,J＝1.9Hz,1H),7.81(dd,J＝8.3,2.0Hz,1H),7.17(d,J＝8.3Hz,1H),7.02(brs,1H),6.95(d,J＝9.0Hz,1H),6.73(d,J＝8.3Hz,1H),4.67(s,2H),3.91(s,3H),2.91(s,4H),2.51(dd,J＝14.5,7.2Hz,2H),2.36(s,3H),2.27(s,3H),2.24(d,J＝22.6Hz,4H),1.12(t,J＝7.3Hz,3H).

Example 2: preparation of 3- (2- ((2- (4-ethylpiperazin-1-yl) -5- (methoxycarbonyl) phenyl) amino) -2-oxoethoxy) -5-methylbenzoic acid (AP-A-7)

Preparation of the compound of formula 7:

In one vial, bromoacetyl bromide (1 equiv.) is dissolved in 10mL dichloromethane. In another vial, compound 5 (1 equiv.) and Et ₃ N (1 equiv.) are dissolved in 10mL dichloromethane. The two reaction mixtures were stirred separately in an ice bath for 5min. The second mixed solution was added dropwise to the first bromoacetyl bromide solution at 0 ℃ and stirred for 12h from 0 ℃ to room temperature, and the solution was poured into an ice bath. The aqueous layer was extracted with dichloromethane, the organic layer was washed with brine, dried over anhydrous sodium sulfate, filtered and spin-dried, and purified by silica gel column chromatography to give compound 7.

Preparation of Compound AP-A-7:

Compound 8 (3-hydroxy-5-methylbenzoic acid, 1.05 equiv.), compound 7 (1.0 equiv.) and potassium carbonate (3.0 equiv.) were dissolved in N, N-dimethylformamide (1.5 mL/mmol), and the mixture was stirred at room temperature for 4 hours. After completion of the reaction by TLC, the mixture was extracted with dichloromethane and water. The organic layer was dried over anhydrous magnesium sulfate, filtered and concentrated in vacuo. Then recrystallizing the crude product from ethanol to obtain compound AP-A-7 as pale yellow solid with purity 98.44％.¹H NMR(400MHz,DMSO-d₆)δ9.38(s,1H),8.85(d,J＝1.9Hz,1H),7.72(dd,J＝8.3,2.0Hz,1H),7.45(s,2H),7.34(d,J＝8.4Hz,1H),7.20(s,1H),4.81(s,2H),3.84(s,3H),3.32(s,4H),2.86(t,J＝4.5Hz,4H),2.41(dd,J＝14.5,7.3Hz,2H),2.36(s,3H),1.01(t,J＝7.2Hz,3H).

Example 3: preparation of 2-hydroxyethyl 3- (2- (3, 5-dimethylphenoxy) acetamido) -4- (4-ethylpiperazin-1-yl) benzoate (AP-A-10)

Preparation of the compound of formula 10:

A compound of formula 9 was prepared in the same manner as in example 1, substituting compound 6 with equimolar 3, 5-dimethylphenoxyacetic acid. The raw material 9 was dissolved in a mixed solvent of THF: H ₂ o=1:1, lithium hydroxide (5 equiv.) was added while stirring at room temperature, after TLC detection, the pH was adjusted to be acidic (about 3) with 1M HCl, ethyl acetate was added and water was extracted three times, the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated, and the residue was purified by column chromatography to give the compound 10.

Preparation of Compound AP-A-10:

Compound 10 (1.0 equiv.) was dissolved in dichloromethane, thionyl chloride solution (1.5 equiv.) was added dropwise, the addition was completed, refluxed for 2h at 70 ℃, after tlc monitoring reaction was completed, and returned to room temperature, the solvent was dried by spin, and residue 11 was directly used for the next reaction.

Ethylene glycol (1.0 equiv.) was dissolved in anhydrous pyridine, triethylamine (5.0 equiv.) was added at room temperature under nitrogen protection, compound 11 (4.5 equiv.) was dissolved in anhydrous dichloromethane, and slowly added dropwise to the reaction system, and the reaction was continued at 60℃for 4 hours. TLC monitoring the completion of the reaction, quenching with water, adding 4M hydrochloric acid to adjust pH to 1 after the reaction has returned to room temperature, extracting with ethyl acetate, combining the organic phases, washing with saturated saline solution, drying with anhydrous sodium sulfate, filtering, spin-drying the solvent, and separating and purifying by column chromatography to give compound AP-A-10 as a yellow solid with purity of 99.76％.¹H NMR(400MHz,CDCl₃)δ9.46(s,1H),9.09(d,J＝2.0Hz,1H),7.84(dd,J＝8.3,2.0Hz,1H),7.21(d,J＝8.3Hz,1H),6.70(s,1H),6.66(s,2H),4.63(s,2H),4.46(dd,J＝5.4,3.8Hz,2H),3.96(s,2H),2.93(s,4H),2.58(s,2H),2.55–2.46(m,4H),2.31(s,6H),1.14(t,J＝7.2Hz,3H).

Example 4: preparation of 3- (2- (3, 5-dimethylphenoxy) acetamido) -4- (4-ethylpiperazin-1-yl) -N-methylbenzamide (AP-A-11)

Compound 10 (1.5 equiv.) is dissolved in 2ml of N, N-dimethylformamide, N-diisopropylethylamine (DIPEA, 1.1 equiv.) is added and stirred for 5min, then urea N, N' -tetramethyl-O- (7-azabenzotriazol-1-yl) hexafluorophosphate (HATU, 1.1 equiv.) is added and stirred for 10min, finally methylamine (1.0 equiv.) is added and reacted at 60 ℃ for 8h, after completion, extracted with ethyl acetate, water, the organic phase is washed with saturated brine, dried over anhydrous sodium sulfate, filtered and spun dry, and purified by column chromatography to give compound AP-a-11 as a yellowish solid of purity 99.86％.¹H NMR(400MHz,CDCl₃)δ9.60(s,1H),8.77(d,J＝2.1Hz,1H),7.70(dd,J＝8.3,2.1Hz,1H),7.24(d,J＝8.3Hz,1H),6.70(s,1H),6.66(s,2H),6.35(s,1H),4.62(s,2H),3.00(d,J＝4.8Hz,3H),2.90(t,J＝4.6Hz,4H),2.57(s,2H),2.50(dd,J＝14.5,7.2Hz,4H),2.31(s,6H),1.13(t,J＝7.2Hz,3H).

Example 5: preparation of Compounds of formula 13

3,4, 5-Trimethylphenol (1.05 equiv.), ethyl bromoacetate (1.0 equiv.) and potassium carbonate (3.0 equiv.) are dissolved in N, N-dimethylformamide, and the mixture is stirred at 60℃for 12 hours. After completion of the reaction by TLC, the mixture was extracted with dichloromethane and water. The organic layer was dried over anhydrous magnesium sulfate, filtered and concentrated in vacuo. Purification by silica gel column chromatography gave compound 12.

Compound 12 (1.0 equiv.) was dissolved in a mixed solvent of THF: H ₂ o=1:1, lithium hydroxide (5 equiv.) was added with stirring at room temperature, after TLC detection was completed, pH was adjusted to be acidic (about 3) with 1M HCl, ethyl acetate and water were added and extracted three times, the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was purified by column chromatography to give compound 13.

3- (2- (2-Chlorophenoxy) acetamido) -4- (4-ethylpiperazin-1-yl) benzoic acid methyl ester (AP-1)

Following the same procedure as in example 1, substituting compound 6 with equimolar o-chlorophenoxyacetic acid gave compound AP-1 as a tan oil in yield 42.8％.¹H NMR(600MHz,CDCl₃)δ9.33(s,1H),9.01(d,J＝2.0Hz,1H),7.83(dd,J＝8.3,2.0Hz,1H),7.44(dd,J＝7.9,1.6Hz,1H),7.28(dd,J＝7.9,1.3Hz,1H),7.20(d,J＝8.3Hz,1H),7.03(td,J＝7.7,1.3Hz,1H),6.99(dd,J＝8.3,1.2Hz,1H),4.74(s,2H),3.91(s,3H),2.94(s,4H),2.59(s,4H),2.51(s,2H),1.11(t,J＝6.9Hz,3H).

3- (2- (4-Chlorophenoxy) acetamido) -4- (4-ethylpiperazin-1-yl) benzoic acid methyl ester (AP-2)

According to the same manner as in example 1, compound 6 was replaced with equimolar p-chlorophenoxyacetic acid to give compound AP-2 as a yellowish solid in a yield 38.9％.¹H NMR(600MHz,CDCl₃)δ9.41(s,1H),9.05(d,J＝1.8Hz,1H),7.83–7.81(m,1H),7.33–7.30(m,2H),7.21(dd,J＝8.2,3.6Hz,1H),6.99–6.95(m,2H),4.64(s,2H),3.91(s,2H),2.94(s,5H),2.50(s,6H),1.25(s,3H).

3- (2, 4-Dichlorophenoxy) acetamido) -4- (4-ethylpiperazin-1-yl) benzoic acid methyl ester (AP-3)

According to the same manner as in example 1, compound 6 was replaced with equimolar 2, 4-dichlorophenoxyacetic acid to give compound AP-3 as a brown oil in a yield 45.3％.¹H NMR(600MHz,DMSO-d₆)δ9.15(s,1H),8.75(s,1H),7.73(dd,J＝8.4,2.0Hz,1H),7.68(d,J＝2.6Hz,1H),7.42(dd,J＝8.9,2.6Hz,1H),7.32(d,J＝8.4Hz,1H),7.26(d,J＝8.9Hz,1H),4.91(s,2H),3.84(s,3H),2.89(s,4H),2.51(s,4H),2.42(s,2H),1.02(t,J＝7.1Hz,3H).

3- (2- (4-Bromophenoxy) acetamido) -4- (4-ethylpiperazin-1-yl) benzoic acid methyl ester (AP-4)

Following the same procedure as in example 1 substituting compound 6 with equimolar p-bromophenoxyacetic acid, compound AP-4 was obtained as a yellowish solid in yield 38％.¹H NMR(600MHz,DMSO-d₆)δ9.29(s,1H),8.78(s,1H),7.72(dd,J＝8.3,2.0Hz,1H),7.55–7.52(m,2H),7.32(d,J＝8.4Hz,1H),7.07(d,J＝9.0Hz,2H),4.78(s,2H),3.84(s,3H),2.87(s,4H),2.51(s,4H),2.42(s,2H),1.03(t,J＝7.1Hz,3H).

Methyl 4- (4-ethylpiperazin-1-yl) -3- (2- (4-fluorophenoxy) acetamido) benzoate (AP-5)

Following the same procedure as in example 1 substituting compound 6 with equimolar amounts of p-fluorophenoxyacetic acid, compound AP-5 was obtained as a tan solid in yield 22.9％.¹H NMR(600MHz,DMSO-d₆)δ9.33(s,1H),8.80(s,1H),7.72(dd,J＝8.3,2.0Hz,1H),7.33(d,J＝8.4Hz,1H),7.22–7.18(m,2H),7.14–7.09(m,2H),4.75(s,2H),3.84(s,3H),2.88(s,4H),2.51(s,4H),2.45–2.39(m,2H),1.03(t,J＝6.6Hz,3H).

Methyl 4- (4-ethylpiperazin-1-yl) -3- (2- (3-methoxyphenoxy) acetamido) benzoate (AP-6)

According to the same manner as in example 1, compound 6 was replaced with equimolar 3-methoxyphenoxyacetic acid to give compound AP-6 as a yellowish solid in yield 48％.¹H NMR(600MHz,DMSO-d₆)δ9.33(s,1H),8.84(d,J＝1.8Hz,1H),7.72(dd,J＝8.3,2.0Hz,1H),7.33(d,J＝8.4Hz,1H),7.25(t,J＝8.2Hz,1H),6.67(dt,J＝7.3,2.2Hz,2H),6.62(dd,J＝8.1,2.1Hz,1H),4.75(s,2H),3.84(s,3H),3.76(s,3H),2.86(t,J＝4.6Hz,4H),2.51(d,J＝0.7Hz,4H),2.37(q,J＝7.2Hz,2H),1.01(t,J＝7.2Hz,3H).

Methyl 3- (2- (4- (tert-butyl) phenoxy) acetamido) -4- (4-ethylpiperazin-1-yl) benzoate (AP-7)

According to the same manner as in example 1, compound 6 was replaced with equimolar 4-tert-butylphenoxyacetic acid to give Compound AP-7 in the form of yellowish crystals with a yield of 73.7％.¹H NMR(600MHz,DMSO-d₆)δ9.34(s,1H),8.86(s,1H),7.72(dd,J＝8.3,1.8Hz,1H),7.36(d,J＝8.7Hz,2H),7.33(d,J＝8.3Hz,1H),7.01(d,J＝8.7Hz,2H),4.72(s,1H),3.84(s,3H),2.83(t,J＝4.6Hz,4H),2.45(s,4H),2.37(q,J＝7.1Hz,2H),1.26(s,9H),1.01(t,J＝7.2Hz,3H).

Methyl 4- (4-ethylpiperazin-1-yl) -3- (2- (4- (trifluoromethyl) phenoxy) acetamido) benzoate (AP-8)

According to the same manner as in example 1, compound 6 was replaced with equimolar 2- (4-trifluoromethyl) phenoxyacetic acid to give compound AP-8 as a yellowish solid in yield 29.2％.¹H NMR(600MHz,DMSO-d₆)δ9.32(s,1H),8.77(s,1H),7.74(d,J＝9.0Hz,2H),7.73–7.72(m,1H),7.32(d,J＝8.4Hz,1H),7.28(d,J＝8.6Hz,2H),4.88(s,2H),3.84(s,3H),2.86(t,J＝4.6Hz,4H),2.46(s,4H),2.37(q,J＝7.1Hz,2H),1.00(t,J＝7.2Hz,3H).

Methyl 4- (4-ethylpiperazin-1-yl) -3- (3- (p-toluenesulfonyloxy) propylamino) benzoate (AP-9)

According to the same manner as in example 1, compound 6 was replaced with equimolar 3- (4-tolyloxy) propionic acid to give compound AP-9 as a yellowish solid in yield 40.8％.¹H NMR(600MHz,CDCl₃)δ8.95(d,J＝1.3Hz,1H),8.57(s,1H),7.78(dd,J＝8.3,1.9Hz,1H),7.16(d,J＝8.3Hz,1H),7.08(d,J＝8.3Hz,2H),6.83(d,J＝8.5Hz,2H),4.34(t,J＝5.9Hz,2H),3.88(s,3H),2.94(t,J＝4.6Hz,4H),2.88(t,J＝5.9Hz,2H),2.55(s,2H),2.39(q,J＝7.2Hz,2H),2.28(s,3H),1.09(t,J＝7.2Hz,3H).

Methyl 4- (4-Acetylpiperazin-1-yl) -3- (2- (4-fluorophenoxy) acetamido) benzoate (AP-10)

According to the same manner as that of example 1, compound 3 was replaced with equimolar 1-acetylpiperazine and compound 6 was replaced with equimolar p-fluorophenoxyacetic acid to obtain compound AP-10 as a yellow solid in a yield 75.6％.¹H NMR(600MHz,CDCl₃)δ9.41(s,1H),9.07(d,J＝1.7Hz,1H),7.83(dd,J＝8.3,1.6Hz,1H),7.16(d,J＝8.3Hz,1H),7.05(t,J＝8.5Hz,2H),6.94–6.91(m,2H),4.64(s,2H),3.91(s,3H),3.73(s,2H),3.52–3.49(m,2H),2.86(t,J＝4.9Hz,4H),2.13(s,3H).

Methyl 4- (4-Acetylpiperazin-1-yl) -3- (2- (3, 4-dimethylphenoxy) acetamido) benzoate (AP-11)

According to the same manner as in example 1, compound 3 was replaced with equimolar 1-acetylpiperazine and compound 6 was replaced with equimolar 3, 4-dimethylphenoxy acetic acid to obtain compound AP-11 as a yellowish solid in a yield of 91.9％.¹H NMR(600MHz,CDCl₃)δ9.41(s,1H),9.07(s,1H),7.83–7.80(m,1H),7.14(dd,J＝8.2,2.1Hz,1H),7.08(d,J＝7.8Hz,1H),6.77(s,1H),6.70(d,J＝8.0Hz,1H),4.64(s,2H),3.91(s,3H),3.69(s,2H),3.46(s,2H),2.92(dd,J＝14.5,2.0Hz,2H),2.83(s,2H),2.25(s,3H),2.21(s,3H),2.11(s,3H).

Methyl 4- (4-Acetylpiperazin-1-yl) -3- (2- (3, 5-dimethylphenoxy) acetamido) benzoate (AP-12)

According to the same manner as in example 1, compound 3 was replaced with equimolar 1-acetylpiperazine and compound 6 was replaced with equimolar 3, 5-dimethylphenoxy acetic acid to obtain compound AP-12 as a yellow solid in yield 75.6％.¹H NMR(600MHz,CDCl₃)δ9.39(s,1H),9.08(d,J＝1.9Hz,1H),7.82(dd,J＝8.3,1.9Hz,1H),7.14(d,J＝8.3Hz,1H),6.71(s,1H),6.59(s,2H),4.65(s,2H),3.91(s,3H),3.69(s,2H),3.47–3.44(m,2H),2.85–2.80(m,4H),2.30(s,6H),2.11(s,3H).

Methyl 4- (4-acetylpiperazin-1-yl) -3- (2, 4-dichlorophenoxy) acetamido) benzoate (AP-13)

According to the same manner as that of example 1, compound 3 was replaced with equimolar 1-acetylpiperazine and compound 6 was replaced with equimolar 2, 4-dichlorophenoxyacetic acid to obtain compound AP-13 as a yellow solid in yield 75.6％.¹H NMR(600MHz,CDCl₃)δ9.34(s,1H),8.99(d,J＝1.5Hz,1H),7.84(dd,J＝8.3,1.7Hz,1H),7.45(d,J＝2.3Hz,1H),7.27(d,J＝2.4Hz,1H),7.16(d,J＝8.4Hz,1H),6.94(d,J＝8.8Hz,1H),4.70(s,2H),3.91(s,3H),3.75(s,2H),3.58(d,J＝4.4Hz,2H),2.88(dd,J＝10.3,5.1Hz,4H),2.12(s,3H).

Methyl 4- (4-acetylpiperazin-1-yl) -3- (2- (3-methoxyphenoxy) acetamido) benzoate (AP-14)

According to the same manner as in example 1, compound 3 was replaced with equimolar 1-acetylpiperazine and compound 6 was replaced with equimolar 3-methoxyphenoxyacetic acid to obtain compound AP-14 as a yellow solid in a yield of 75.6％.¹H NMR(600MHz,CDCl₃)δ9.40(s,1H),9.07(d,J＝1.7Hz,1H),7.81(dd,J＝8.3,1.8Hz,1H),7.24(t,J＝8.3Hz,1H),7.15(d,J＝8.3Hz,1H),6.61(dd,J＝8.3,2.0Hz,1H),6.57(dd,J＝8.2,2.2Hz,1H),6.53(t,J＝2.2Hz,1H),4.66(s,2H),3.91(s,3H),3.79(s,3H),3.71(s,2H),3.50–3.47(m,2H),2.84(dd,J＝10.9,6.4Hz,4H),2.11(s,3H).

Methyl 4- (4-acetylpiperazin-1-yl) -3- (2- (4- (tert-butyl) phenoxy) acetamido) benzoate (AP-15)

According to the same manner as that of example 1, compound 3 was replaced with equimolar 1-acetylpiperazine and compound 6 was replaced with equimolar 4-tert-butylphenoxyacetic acid to give compound AP-15 as a yellow solid in yield 75.6％.¹H NMR(600MHz,CDCl₃)δ9.45(s,1H),9.09(d,J＝1.9Hz,1H),7.82(dd,J＝8.3,2.0Hz,1H),7.37–7.35(m,2H),7.15(d,J＝8.3Hz,1H),6.92–6.90(m,2H),4.66(s,2H),3.91(s,3H),3.69(s,2H),3.48–3.45(m,2H),2.83(dd,J＝10.8,5.9Hz,5H),2.80(s,2H),2.11(s,3H),1.30(s,9H).

Methyl 4- (4-acetylpiperazin-1-yl) -3- (2- (4- (trifluoromethyl) phenoxy) acetamido) benzoate (AP-16)

According to the same manner as that of example 1, compound 3 was replaced with equimolar 1-acetylpiperazine and compound 6 was replaced with equimolar 2- (4-trifluoromethyl) phenoxyacetic acid to obtain compound AP-16 as a yellow solid in yield 75.6％.¹H NMR(600MHz,CDCl₃)δ9.38(s,1H),9.07(s,1H),7.84(d,J＝8.2Hz,1H),7.64(d,J＝8.3Hz,2H),7.18(t,J＝7.4Hz,1H),7.07(d,J＝8.3Hz,2H),4.73(s,2H),3.91(s,3H),3.75(s,2H),3.47(s,2H),2.88–2.83(m,4H),2.12(s,4H).

Methyl 4- (4-acetylpiperazin-1-yl) -3- (2- (p-toluenesulfonyloxy) acetamido) benzoate (AP-17)

According to the same manner as in example 1, compound 3 was replaced with equimolar 1-acetylpiperazine and compound 6 was replaced with equimolar p-methylphenoxy acetic acid to obtain compound AP-17 as a yellowish solid in a yield of 86.9％.¹H NMR(600MHz,CDCl₃)δ9.42(s,1H),9.08(d,J＝1.7Hz,1H),7.82(dd,J＝8.3,1.8Hz,1H),7.14(d,J＝8.0Hz,3H),6.87(d,J＝8.4Hz,2H),4.64(s,2H),3.91(s,3H),3.69(s,2H),3.46(s,2H),2.85–2.82(m,4H),2.31(s,3H),2.11(s,3H).

3- (2, 4-Dimethylphenoxy) acetamido) -4- (4-methylpiperazin-1-yl) benzoic acid methyl ester (AP-18)

Following the same procedure as in example 1 substituting compound 3 with equimolar N-methylpiperazine, compound AP-18 was obtained in the form of a brown oil in a yield 72.8％.¹H NMR(600MHz,CDCl₃)δ9.25(s,1H),9.04(d,J＝1.9Hz,1H),7.81(dd,J＝8.3,2.0Hz,1H),7.18(d,J＝8.3Hz,1H),7.02(s,1H),6.95(d,J＝8.3Hz,1H),6.73(d,J＝8.3Hz,1H),4.66(s,2H),3.91(s,3H),2.85(t,J＝4.7Hz,4H),2.38(s,3H),2.28(s,3H),2.26(s,3H).

3- (2, 4-Dibromophenoxy) propylamino) -4- (4-ethylpiperazin-1-yl) benzoic acid methyl ester (AP-19)

Following the same procedure as in example 1 substituting compound 6 with equimolar 2- (2, 4-dibromophenoxy) propionic acid, compound AP-19 was obtained in the form of a yellowish solid in yield 58.6％.¹H NMR(600MHz,CDCl₃)δ9.17(s,1H),8.97(d,J＝1.7Hz,1H),7.81(dd,J＝8.3,1.8Hz,1H),7.73(d,J＝2.3Hz,1H),7.37(dd,J＝8.8,2.3Hz,1H),7.17(d,J＝8.3Hz,1H),6.81(d,J＝8.8Hz,1H),4.77(q,J＝6.8Hz,1H),3.90(s,3H),2.88(s,2H),2.83(s,2H),2.53(s,2H),2.45(td,J＝12.5,5.2Hz,2H),1.72(d,J＝6.8Hz,3H),1.09(t,J＝7.2Hz,3H).

4- (4-Ethylpiperazin-1-yl) -3- (2-phenoxypropylamino) benzoic acid methyl ester (AP-20)

Following the same procedure as in example 1 substituting compound 6 with equimolar 2-phenoxypropionic acid, compound AP-20 was obtained in the form of a brown oil in a yield 71.6％.¹H NMR(600MHz,CDCl₃)δ9.32(s,1H),9.05(d,J＝1.8Hz,1H),7.79(dd,J＝8.3,1.8Hz,1H),7.31(t,J＝8.0Hz,2H),7.16(d,J＝8.3Hz,1H),7.03(d,J＝7.4Hz,1H),7.00(d,J＝8.1Hz,2H),4.80(q,J＝6.8Hz,1H),3.90(s,3H),2.82(d,J＝25.7Hz,4H),2.48–2.37(m,4H),1.68(d,J＝6.8Hz,3H),1.08(t,J＝7.2Hz,3H).

Methyl 4- (4-ethylpiperazin-1-yl) -3- (2- (4-methoxyphenoxy) propylamino) benzoate (AP-21)

Following the same procedure as in example 1 substituting compound 6 with equimolar 2- (4-methoxyphenoxy) propionic acid, compound AP-21 was obtained in the form of a brown oil with a yield of 53.4％.¹H NMR(600MHz,CDCl₃)δ9.27(s,1H),9.03(s,1H),7.79(d,J＝8.3Hz,1H),7.17(d,J＝8.3Hz,1H),6.93(dd,J＝9.0,2.5Hz,2H),6.87–6.83(m,2H),4.69(dd,J＝13.3,6.5Hz,1H),3.90(s,3H),3.77(s,3H),2.93(s,4H),2.80(s,3H),2.68–2.57(m,4H),1.66–1.64(m,2H),1.16(t,J＝7.1Hz,3H).

3- (2- (4-Chlorophenoxy) propylamino) -4- (4-ethylpiperazin-1-yl) benzoic acid methyl ester (AP-22)

Following the same procedure as in example 1 substituting compound 6 with equimolar 2- (4-chlorophenoxy) propionic acid, compound AP-22 was obtained in the form of a brown oil with a yield of 64.2％.¹H NMR(600MHz,CDCl₃)δ9.26(s,1H),9.03(d,J＝1.8Hz,1H),7.79(dd,J＝8.3,1.9Hz,1H),7.27(d,J＝9.0Hz,2H),7.17(d,J＝8.3Hz,1H),6.94(d,J＝9.0Hz,2H),4.75(q,J＝6.8Hz,1H),3.90(s,3H),2.84(d,J＝19.5Hz,4H),2.50–2.39(m,4H),1.67(d,J＝6.8Hz,3H),1.10(t,J＝7.2Hz,3H).

3- (2- (2-Chlorophenoxy) propylamino) -4- (4-ethylpiperazin-1-yl) benzoic acid methyl ester (AP-23)

Following the same procedure as in example 1, compound 6 was replaced with equimolar 2- (2-chlorophenoxy) propionic acid to give compound AP-23 as a yellowish oil in yield 52.3％.¹H NMR(600MHz,CDCl₃)δ9.29(s,1H),9.00(d,J＝1.6Hz,1H),7.80(dd,J＝8.3,1.6Hz,1H),7.42(d,J＝7.9Hz,1H),7.21(t,J＝7.8Hz,1H),7.16(d,J＝8.3Hz,1H),6.99(t,J＝7.7Hz,1H),6.96(d,J＝8.2Hz,1H),4.82(q,J＝6.8Hz,1H),3.90(s,3H),2.85(d,J＝14.2Hz,4H),2.50(s,2H),2.45–2.40(m,2H),1.72(d,J＝6.8Hz,3H),1.07(t,J＝7.2Hz,3H).

Methyl 4- (4-ethylpiperazin-1-yl) -3- (2- (o-tolyloxy) propanamino) benzoate (AP-24)

Following the same procedure as in example 1 substituting compound 6 with equimolar 2- (2-methylphenoxy) propionic acid, compound AP-24 was obtained as a yellowish solid in yield 59.1％.¹H NMR(600MHz,CDCl₃)δ9.19(s,1H),9.04(d,J＝1.9Hz,1H),7.79(dd,J＝8.3,2.0Hz,1H),7.19(d,J＝7.4Hz,1H),7.14(d,J＝8.3Hz,1H),7.11(t,J＝7.8Hz,1H),6.93(t,J＝7.4Hz,1H),6.80(d,J＝8.2Hz,1H),4.74(q,J＝6.8Hz,1H),3.90(s,3H),2.79(s,4H),2.38(s,3H),2.30(dt,J＝19.3,7.1Hz,4H),1.70(d,J＝6.8Hz,3H),1.04(t,J＝7.2Hz,3H).

3- (2- (3-Chlorophenoxy) propylamino) -4- (4-ethylpiperazin-1-yl) benzoic acid methyl ester (AP-25)

Following the same procedure as in example 1 substituting compound 6 with equimolar 2- (3-chlorophenoxy) propionic acid, compound AP-25 was obtained in the form of a tan oil in yield 51.5％.¹H NMR(600MHz,CDCl₃)δ9.27(s,1H),9.04(s,1H),7.79(d,J＝8.3Hz,1H),7.23(t,J＝8.2Hz,1H),7.17(d,J＝8.3Hz,1H),7.06(s,1H),7.02(d,J＝8.0Hz,1H),6.86(dd,J＝8.3,2.1Hz,1H),4.79(q,J＝6.7Hz,1H),3.90(s,3H),2.85(s,4H),2.51–2.39(m,4H),1.69(d,J＝6.7Hz,3H),1.11(t,J＝7.2Hz,3H).

Methyl 4- (4-Benzoylpiperazin-1-yl) -3- (2- (3, 5-dimethylphenoxy) acetamido) benzoate (AP-A-1)

According to the same manner as that of example 1, compound 3 was replaced with equimolar 1-benzoylpiperazine and compound 6 was replaced with equimolar 3, 5-dimethylphenoxy acetic acid to obtain compound AP-A-1 as a yellowish solid having a purity of 98.36％.¹H NMR(400MHz,DMSO-d₆)δ9.33(s,1H),8.78(d,J＝1.8Hz,1H),7.73(dd,J＝8.3,2.0Hz,1H),7.49–7.46(m,3H),7.41–7.39(m,2H),7.35(d,J＝8.4Hz,1H),6.69(s,2H),6.65(s,1H),4.73(s,2H),3.84(s,3H),3.66(s,2H),3.28(s,2H),2.87(s,4H),2.23(s,6H).

Methyl 4- (4-benzoylpiperazin-1-yl) -3- (2- (o-tolyloxy) acetamido) benzoate (AP-A-2)

According to the same manner as that of example 1, compound 3 was replaced with equimolar 1-benzoylpiperazine and compound 6 was replaced with equimolar o-methylphenoxy acetic acid to obtain compound AP-A-2 as a yellow solid having a purity of 99.58％.¹H NMR(400MHz,DMSO-d₆)δ9.25(s,1H),8.75(d,J＝1.8Hz,1H),7.73(dd,J＝8.4,2.0Hz,1H),7.46(dd,J＝5.0,1.7Hz,3H),7.39(dd,J＝6.6,3.1Hz,2H),7.34(d,J＝8.4Hz,1H),7.23(d,J＝7.2Hz,1H),7.15(t,J＝7.8Hz,1H),6.96(d,J＝8.1Hz,1H),6.91(t,J＝7.4Hz,1H),4.80(s,2H),3.84(s,3H),3.65(s,2H),3.30–3.24(m,2H),2.86(s,4H),2.33(s,3H).

3- (4, 6-Dimethylbenzofuran-2-carboxamide) -4- (4-ethylpiperazin-1-yl) benzoic acid methyl ester (AP-A-3)

According to the same manner as in example 1, compound 3 was replaced with equimolar 1-benzoylpiperazine, and compound 6 was replaced with equimolar 4, 6-dimethylcoumaric acid to give compound AP-A-3 as a tan solid of purity 99.08％.¹H NMR(400MHz,CDCl₃)δ9.56(s,1H),9.12(d,J＝2.0Hz,1H),7.83(dd,J＝8.3,2.0Hz,1H),7.60(d,J＝0.9Hz,1H),7.24(d,J＝8.3Hz,1H),7.14(s,1H),6.97(s,1H),3.92(s,3H),3.06(t,J＝4.4Hz,4H),2.79(s,4H),2.62(dd,J＝14.0,7.0Hz,2H),2.54(s,3H),2.49(s,4H),1.21(t,J＝7.2Hz,3H).

3- (2- (3, 5-Dimethylphenoxy) -2-methylpropylamino) -4- (4-ethylpiperazin-1-yl) benzoic acid methyl ester (AP-A-4)

According to the same manner as in example 1, compound 3 was replaced with equimolar 1-benzoylpiperazine and compound 6 was replaced with equimolar 2- (3, 5-dimethylphenoxy) -2-methyl-propionic acid to give compound AP-a-4 as an orange-yellow solid with a purity of 99.68％.¹H NMR(400MHz,CDCl₃)δ9.57(s,1H),9.10(d,J＝2.0Hz,1H),7.79(dd,J＝8.3,2.0Hz,1H),7.17(d,J＝8.3Hz,1H),6.70(s,1H),6.59(s,2H),3.90(s,3H),2.85(t,J＝4.3Hz,4H),2.37(dd,J＝14.4,7.2Hz,4H),2.25(s,8H),1.62(s,6H),1.04(t,J＝7.2Hz,3H).

Methyl 4- (4-Benzylpiperazin-1-yl) -3- (2- (3, 5-dimethylphenoxy) acetamido) benzoate (AP-A-5)

According to the same manner as that of example 1, compound 3 was replaced with equimolar 1-benzylpiperazine and compound 6 was replaced with equimolar 3, 5-dimethylphenoxy acetic acid to give compound AP-A-5 as a white solid of purity 98.64％.¹H NMR(400MHz,DMSO-d₆)δ9.35(s,1H),8.86(d,J＝1.9Hz,1H),7.71(dd,J＝8.3,2.0Hz,1H),7.36–7.27(m,6H),6.76(s,2H),6.70(s,1H),4.71(s,2H),3.84(s,3H),3.51(s,2H),2.86(t,J＝4.5Hz,4H),2.49(s,4H),2.29(s,6H).

Methyl 4- (4-cyclopropylpiperazin-1-yl) -3- (2- (3, 5-dimethylphenoxy) acetamido) benzoate (AP-A-6)

According to the same manner as in example 1, compound 3 was replaced with equimolar 1-cyclopropylpiperazine and compound 6 was replaced with equimolar 3, 5-dimethylphenoxy acetic acid to give compound AP-A-6 as a yellowish solid having a purity of 99.20％.¹H NMR(400MHz,DMSO-d₆)δ9.38(s,1H),8.87(d,J＝1.9Hz,1H),7.70(dd,J＝8.3,2.1Hz,1H),7.31(d,J＝8.4Hz,1H),6.76(s,2H),6.68(s,1H),4.72(s,2H),3.84(s,3H),2.82–2.78(m,4H),2.67(d,J＝1.8Hz,4H),2.25(s,6H),1.65(dt,J＝9.8,3.3Hz,1H),0.48–0.43(m,2H),0.35–0.30(m,2H).

3-Chloro-5- (2- ((2- (4-ethylpiperazin-1-yl) -5- (methoxycarbonyl) phenyl) amino) -2-oxoethoxy) benzoic acid (AP-A-8)

According to the same manner as in example 2, compound 8 was replaced with equimolar 3-chloro-5-hydroxybenzoic acid to give compound AP-A-8 as a brown yellow solid having a purity 98.87％.¹H NMR(400MHz,DMSO-d₆)δ9.38(s,1H),8.78(s,1H),7.73(dd,J＝8.3,1.9Hz,1H),7.58(dd,J＝6.0,1.4Hz,2H),7.46(s,1H),7.33(d,J＝8.3Hz,1H),4.89(s,2H),3.84(s,3H),2.90(s,4H),2.58(s,4H),2.46(d,J＝7.1Hz,2H),1.03(t,J＝7.1Hz,3H).

3- (2- (3, 5-Dimethylphenoxy) propylamino) -4- (4-ethylpiperazin-1-yl) benzoic acid methyl ester (AP-A-9)

According to the same manner as in example 1, compound 6 was replaced with equimolar 2- (3, 5-dimethylphenoxy) propionic acid to give Compound AP-A-9 as a pale yellow solid of purity 96.50％.¹H NMR(400MHz,DMSO-d₆)δ9.31(s,1H),8.80(d,J＝2.0Hz,1H),7.70(dd,J＝8.3,2.1Hz,1H),7.30(d,J＝8.4Hz,1H),6.69(s,2H),6.66(s,1H),4.98(q,J＝6.6Hz,1H),3.84(s,3H),2.79(dt,J＝12.1,6.1Hz,4H),2.43–2.29(m,6H),2.23(s,6H),1.53(d,J＝6.7Hz,3H),1.00(t,J＝7.2Hz,3H).

Ethyl 4- (4-benzoylpiperazin-1-yl) -3- (2- (3, 5-dimethylphenoxy) acetamido) benzoate (AP-a-12)

According to the same manner as that of example 1, compound 1 was replaced with equimolar ethyl 3-amino-4-bromobenzoate, compound 3 was replaced with equimolar 1-benzoylpiperazine, and compound 6 was replaced with equimolar 3, 5-dimethylphenoxyacetic acid to give compound AP-A-12 as a white solid of purity 99.63％.¹H NMR(400MHz,DMSO-d₆)δ9.33(s,1H),8.77(d,J＝1.8Hz,1H),7.72(dd,J＝8.3,2.0Hz,1H),7.47(dd,J＝5.0,1.7Hz,3H),7.42–7.39(m,2H),7.34(d,J＝8.4Hz,1H),6.69(s,2H),6.64(s,1H),4.73(s,2H),4.30(q,J＝7.1Hz,2H),3.68(s,4H),2.87(s,4H),2.23(s,6H),1.31(t,J＝7.1Hz,3H).

Methyl 4- (4-ethylpiperazin-1-yl) -3- (2- (3-methyl-5-nitrophenoxy) acetamido) benzoate (AP-A-13)

According to the same manner as in example 2, compound 8 was replaced with equimolar 3-methyl-5-nitrophenol to give compound AP-A-13 as a white solid having a purity of 99.62％.¹H NMR(400MHz,CDCl₃)δ9.50(s,1H),9.07(d,J＝1.9Hz,1H),7.83(dd,J＝8.3,2.0Hz,1H),7.78–7.73(m,2H),7.24(d,J＝8.3Hz,1H),7.17(s,2H),4.72(s,2H),3.91(s,3H),2.96(t,J＝4.5Hz,4H),2.64(s,2H),2.53(dd,J＝14.3,7.1Hz,4H),2.48(s,3H),1.14(t,J＝7.2Hz,3H).

Methyl 3- (4- (3, 5-dimethylphenoxy) butyrylamino) -4- (4-ethylpiperazin-1-yl) benzoate (AP-A-14)

According to the same manner as in example 1, compound 6 was replaced with equimolar 3, 5-dimethylbenzeneoxybutyric acid to obtain Compound AP-A-14 as a yellowish solid having a purity 99.57％.¹H NMR(400MHz,CDCl₃)δ8.97(s,1H),8.27(s,1H),7.77(dd,J＝8.3,2.0Hz,1H),7.17(d,J＝8.3Hz,1H),6.58(s,1H),6.51(s,2H),4.03(t,J＝5.7Hz,2H),3.89(s,3H),2.92(s,4H),2.63(t,J＝7.0Hz,6H),2.50(dd,J＝14.3,7.1Hz,2H),2.26(s,6H),2.25–2.20(m,2H),1.14(t,J＝7.2Hz,3H).

Methyl 4- (4-ethylpiperazin-1-yl) -3- (2- (3, 4, 5-trimethylphenoxy) acetamido) benzoate (AP-A-15)

The compound of formula 5 and the compound of formula 13 were prepared by the same procedure as in example 1, and compound AP-A-15 was obtained by condensation reaction as a pale yellow solid with purity 99.49％.¹H NMR(400MHz,CDCl₃)δ9.46(s,1H),9.07(d,J＝2.0Hz,1H),7.81(dd,J＝8.3,2.0Hz,1H),7.19(d,J＝8.3Hz,1H),6.70(s,2H),4.62(s,2H),3.90(s,3H),2.92(t,J＝4.7Hz,4H),2.56(s,2H),2.49(q,J＝7.2Hz,4H),2.28(s,6H),2.12(s,3H),1.12(t,J＝7.2Hz,3H).

Methyl 4- (4-ethylpiperazin-1-yl) -3- (2- (3-methoxy-5-methylphenoxy) acetamido) benzoate (AP-A-16)

According to the same preparation method of the compound AP-A-15, 3,4, 5-trimethylphenol is replaced by equimolar 3-methoxy-5-methylphenol. White solid of purity of 99.29％.¹H NMR(400MHz,CDCl₃)δ9.43(s,1H),9.07(d,J＝2.0Hz,1H),7.81(dd,J＝8.3,2.0Hz,1H),7.20(d,J＝8.3Hz,1H),6.48–6.38(m,3H),4.63(s,2H),3.91(s,3H),3.79(s,3H),2.92(t,J＝4.7Hz,4H),2.56(s,2H),2.48(q,J＝7.2Hz,4H),2.32(s,3H),1.12(t,J＝7.2Hz,3H).

Example 6: compound AP-970/43482503 has GPR133/ADGRD1 activating ability

The stock solution of the compound to be tested (DMSO is dissolved) is prepared into a HEK293 cell with working concentration of 10^-11M、10^-10M、10^-9M、10^- ⁸M、10^-7M、10^-6M、10^-5M、10^-4M, in which GPR133/ADGRD1 and G protein are overexpressed by using HBSS, the HEK293 cell with the empty vector pcDNA3.1 is expressed as a negative control by stimulating the compound with different concentrations (10 ^-11-10^-4 M), and the fluorescence value is recorded in real time by using a multifunctional enzyme-labeled instrument, so that the results are plotted by statistical analysis, and the result is shown in figure 1. Experimental results indicate that compound AP-970/43482503 (abbreviated as AP-503 or AP-970) can activate Gs-cAMP signaling pathway downstream of GPR133/ADGRD1, and EC ₅₀ is 1.21+ -0.06 nM. The agonistic activity of other AP-503 derivatives on GPR133 is shown in Table 1 below.

Agonistic activity of compounds of table 1 on GPR133

Compounds	EC₅₀(nM)	Compounds	EC₅₀(nM)	Compounds	EC₅₀(nM)
						AP-503	1.21±0.06	AP-14	5.42±0.12	AP-A-3	2.46±0.55
AP-1	5.32±0.12	AP-15	1.01±0.04	AP-A-4	5.36±0.38
						AP-2	11.25±0.24	AP-16	15.52±1.34	AP-A-5	4.21±0.24
AP-3	12.41±0.26	AP-17	7.36±0.14	AP-A-6	8.06±0.36
						AP-4	21.23±0.67	AP-18	41.35±3.86	AP-A-7	9.26±0.34
AP-5	31.21±0.96	AP-19	40.06±3.81	AP-A-8	7.02±0.16
						AP-6	22.11±0.84	AP-20	3.01±0.04	AP-A-9	9.82±0.40
AP-7	6.21±0.12	AP-21	51.71±4.06	AP-A-10	8.21±0.38
						AP-8	11.32±0.22	AP-22	12.26±0.36	AP-A-11	22.67±0.96
AP-9	21.03±1.56	AP-23	11.21±0.34	AP-A-12	30.64±1.36
						AP-10	4.01±0.80	AP-24	6.38±0.29	AP-A-13	26.40±1.26
AP-11	6.39±0.14	AP-25	3.03±0.18	AP-A-14	20.52±1.45
						AP-12	21.21±2.44	AP-A-1	2.54±0.09	AP-A-15	3.06±0.23
AP-13	3.31±0.07	AP-A-2	6.80±0.36	AP-A-16	5.21±0.30

Example 7: influence of Compound AP-970/43482503 on Balancing function and vestibular dysfunction disease

8 Week male WT and Adgrd1 ^-/- mice of C57BL/6J were randomly grouped, 6. Mu.g/(kg.d) of vasopressin acetate was continuously injected intraperitoneally for 10d, and 0.1 mg/(kg.d) of aldosterone was injected intraperitoneally for 5d, respectively, to construct a model of vestibular dysfunction, while 1mg/kg of compound AP-970 was administered intraperitoneally. The results were plotted for statistical analysis of the mice held on the rotating rod for 3min at 8 rpm, as shown in fig. 2. At the same time, the results of the mice Vestibular Ocular Reflex (VOR) test, with the results of the statistical analysis of the Gain values (1.0 Hz) plotted in FIG. 2, were examined. Experimental results show that after modeling of the vasopressin acetate (VP) and the Aldosterone (ALD), the vestibular balance function of the mice is obviously impaired; compound AP-970 may improve vestibular balance dysfunction in mice by activating GPR133/ADGRD1 receptor, and may even improve or/and normal vestibular balance function in mice. The effect of other AP-503 derivatives on vestibular balance function is shown in Table 2 below.

Effects of compounds of Table 2 on vestibular balance function

Example 8: effect of compound AP-970/43482503 on cAMP levels in mouse skeletal muscle

The effect of AP-503 on cAMP production and muscle strength in mouse extensor digitorum longus (extensor digitorum longus, EDL) was examined using ELISA kit. Treatment of AP-503 in EDL of female and male mice for 30min significantly increased cAMP levels, rapidly increased skeletal muscle strength, compared to DHT treatment, while pre-incubation of AR antagonist ORM-15341 was unaffected, as shown in FIG. 3.

Example 9: effect of Compound AP-970/43482503 on suspension time in mice

WT or Gpr133 ^-/- mice were placed on a wire platform and then inverted and hung 750 mm above a landing box filled with cotton and wood chips to protect the mice from any potential injury. The time the mice were dropped from the inverted platform (up to 60 seconds each time) was recorded. Mice were subjected to three consecutive trials with a 5 minute rest period between each trial. The analysis was performed using an average residence time of three times, the results of which are shown in figure 4.

Example 10: effect of Compound AP-970/43482503 on mouse grip

An automatic grip dynamometer (Bioseb) was used to measure the muscle grip of the mice. WT or Gpr133 ^-/- mice held the forepaws with the tails and the hindpaws placed on the grid. The tail of the mouse was gently pulled at a speed of 2-3cm/s and the maximum grip value was recorded. Each mouse was subjected to 5 consecutive trials with 5 minutes of rest between each trial. The analysis was performed using the average grip strength of the three highest readings, the results are shown in figure 5.

The DHT analogue AP-503 (intramuscular injection stimulation for 30 min) was further shown to rapidly increase muscle strength by activating GPR133 by both suspension experiments recording the drop time of the mice and grip force experiments recording the grip force of the mice. Can be used as a muscle-increasing medicament, and can also be used for further developing medicaments for preventing or treating muscular atrophy, muscular weakness and skeletal weakness.

It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.

Claims

1. A compound having the general structural formula (I) as follows:

2. The compound of claim 1, wherein the derivative of the compound comprises a pharmaceutically acceptable salt, a solvent compound, a hydrate, or a crystal thereof.

3. The compound according to claim 1, wherein the specific compound represented by the general formula (I) has the following number and structural formula:

4. the method for preparing a compound according to claim 1, wherein the synthetic route is as follows:

5. the method of claim 4, wherein the synthesizing step comprises:

6. The preparation method according to claim 4, wherein the detailed synthesis steps are as follows:

7. The use of a compound according to claim 1 as an agonist of GPR133/ADGRD 1.

8. The use of claim 7, wherein the compound activates the Gs-cAMP signaling pathway downstream of GPR133/ADGRD1 to exert an agonistic effect.

9. The use of a compound according to claim 1 for the preparation of a medicament for improving balance and related diseases.

10. The use of a compound according to claim 1 for the preparation of a medicament for the prophylaxis and/or treatment of vestibular dysfunction.

11. Use of a compound according to claim 1 for the preparation of a medicament for increasing muscle strength.

12. The use according to claim 11, wherein the muscle-strength-enhancing agent is a muscle-increasing agent, an agent for preventing or treating muscular atrophy, muscular weakness, skeletal weakness.