KR101318656B1 - Pharmaceutical Compositions Comprising Anti-asthmatic 3-Aminopyrrolidine Derivatives - Google Patents

Abstract

상기 화학식 1에서 R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ 및 R₉는 본 명세서에서 정의된 바와 같다.The present invention relates to a pharmaceutical composition or a food composition comprising as an active ingredient a compound of formula 1 having a anti-asthmatic activity or a pharmaceutically acceptable salt thereof.
[Formula 1]

In Formula 1, R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , R ₇ , R ₈ And R ₉ is as defined herein.

Description

Pharmaceutical Compositions Comprising Anti-asthmatic 3-Aminopyrrolidine Derivatives

The present invention relates to pharmaceutical compositions and food compositions for the prevention, improvement of symptoms and treatment of asthma diseases comprising 3-aminopyrrolidine derivatives, which are synthetic low molecular weight substances, as active ingredients.

Asthma is an inflammatory disease of the airways that involves various inflammatory cells and mediators, such as increased eosinophils in the airways, excessive mucin secretion, wheezing, airway hyperresponsiveness and reversible airway obstruction. It is a respiratory disease characterized by. In addition, depending on the duration and severity of asthma, it may progress to irreversible airway obstruction, which is caused by airway remodeling due to chronic inflammation. In recent decades, the pathogenesis of asthma has been established as a chronic bronchial inflammatory disease, thus characteristic features of the immune system, namely Th2 cells and their associated cytokines, interleukin-4 (IL-4). , Interleukin-5 (IL-5) and interleukin-13 (IL-13) are known to play an important role in differentiation and proliferation of inflammatory cells (ST Holgate, R. Polosa, Nature Review Immunology , vol 8, 2008, p.218). Therefore, the suppression of this mechanism is proceeding as a major research direction for the development of asthma treatment.

In general, the treatment of asthma uses drug therapy to alleviate the symptoms. The drugs used can be classified into two categories, bronchodilators and anti-inflammatory drugs. Bronchodilators are the most widely used fast-acting or long-acting beta 2 agonists, and other anticholinergic and xanthine preparations. As an anti-inflammatory agent, corticosteroid preparations are most widely used, and there are other leukotriene receptor antagonists and mast cell stabilizers. In addition, a combination therapy of bronchodilator and anti-inflammatory agent is performed for proper therapeutic effect, and many combination products have been developed and used clinically.

Agents, collectively referred to as therapeutic agents for asthma, are generally formulated by oral, inhalable, and various other routes of administration. In particular, inhalation-type preparations are designed to be administered in the form of a therapeutic dose of inhalable form through the airway, which has the advantage of directly acting on the target organ to increase the therapeutic effect and locally act to reduce systemic side effects. Devices manufactured for this purpose can be classified into metered dose inhalers (MDIs) and dry powder inhalers (DPIs), including the most classic nebulizers.

Clinical treatment guidelines for asthma typically consist of the selection of appropriate agents and the adjustment of the dose to alleviate the symptoms. In other words, one or more drugs are selectively administered according to the severity of symptoms, and one of the most widely used drugs is a steroidal drug for the main purpose of anti-inflammatory action. However, steroidal drugs, despite their excellent anti-inflammatory action, have serious side effects due to imbalance of the immune system upon prolonged administration, and are ineffective or ineffective for some patients. In addition, patients with chronic persistent asthma rarely develop airway hyperresponsiveness despite years of steroid treatment. There are even reports that steroid drugs can increase the synthesis of collagen and induce pulmonary fibrosis (GS Warshamana, S. Martinez, et al., American Journal of Physiology , 274, 1998, p. 499).

In conclusion, asthma disease can be controlled through various medications, including symptoms, but cure is almost impossible. Therefore, there is an urgent need to develop drugs that have less physiological side effects and at the same time have more effective therapeutic effects.

The present invention has been made to solve the above problems, an object of the present invention is to provide a pharmaceutical composition comprising a novel compound of the 3-aminopyrrolidine structure having anti-asthmatic activity.

The present invention also aims to provide a food composition comprising the compound.

A pharmaceutical composition for use in the prevention, symptom amelioration or treatment of asthma diseases of the present invention for achieving the above object is characterized in that it comprises a compound of the formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient do:

In Formula 1,

R ₁ is independently a hydrogen atom, C ₁ -C ₃ alkyl, phenyl, benzyl, benzoyl, benzenesulfonyl, C ₁ -C ₃ alkylcarbonyl, C ₃ -C ₇ cycloalkyl,

It may be selected from the group consisting of;

R ₁₀ , R ₁₁ and R ₁₂ can be independently selected from the group consisting of hydrogen atom, C ₁ -C ₃ alkyl, phenyl, and benzyl group;

All benzene groups included as part of R ₁ may independently have a plurality of substituents selected from the group consisting of C ₁ -C ₃ alkyl, C ₁ -C ₂ haloalkyl, a halogen atom, and a cyano group;

R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , R ₇ , R ₈ and R ₉ can be independently selected from a hydrogen atom and C ₁ -C ₃ alkyl;

R ₆ and R ₇ , R ₈ and R ₉ can be independently selected from a carbonyl group;

The halogen is selected from the group consisting of fluorine, chlorine and bromine atoms.

In addition, the compound of Formula 1 of the present invention

N-{[1- (2- (1- (phenylaminocarbonyl) -piperazin-4-yl) ethyl) -pyrrolidin- (3R) -yl-carbamoyl] -methyl} -3-trifluoro Chloromethylbenzamide,

N-{[1- (2- (1- (p-tolylaminocarbonyl) -piperazin-4-yl) -ethyl) -pyrrolidin- (3R) -yl-carbamoyl] -methyl} -3 Trifluoromethylbenzamide,

N-{[1- (2- (1- (4-Chlorophenylaminocarbonyl) -piperazin-4-yl) -ethyl) -pyrrolidin- (3R) -yl-carbamoyl] -methyl}- 3-trifluoromethylbenzamide,

N-{[1- (2- (1- (methoxycarbonyl) -piperazin-4-yl) ethyl) -pyrrolidine- (3R) -yl-carbamoyl] -methyl} -3-trifluoro Chloromethylbenzamide,

N-{[1- (2- (1- (ethoxycarbonyl) -piperazin-4-yl) ethyl) -pyrrolidin- (3R) -yl-carbamoyl] -methyl} -3-trifluoro Chloromethylbenzamide,

N-{[1- (2- (4-benzoylpiperazin-1-yl) ethyl) -pyrrolidin- (3R) -yl-carbamoyl] -methyl} -3-trifluoromethylbenzamide,

N-{[1- (2- (4- (2-methylbenzoyl) piperazin-1-yl) ethyl) -pyrrolidine- (3R) -yl-carbamoyl] -methyl} -3-trifluoro Methylbenzamide,

N-{[1- (2- (4- (3-methylbenzoyl) piperazin-1-yl) ethyl) -pyrrolidine- (3R) -yl-carbamoyl] -methyl} -3-trifluoro Methylbenzamide,

N-{[1- (2- (4- (4-Methylbenzoyl) piperazin-1-yl) ethyl) -pyrrolidin- (3R) -yl-carbamoyl] -methyl} -3-trifluoro Methylbenzamide,

N-{[1- (2- (4- (4-fluorobenzoyl) piperazin-1-yl) ethyl) -pyrrolidine- (3R) -yl-carbamoyl] -methyl} -3-trifluoro Chloromethylbenzamide,

N-{[1- (2- (4- (4-cyanobenzoyl) piperazin-1-yl) ethyl) -pyrrolidine- (3R) -yl-carbamoyl] -methyl} -3-trifluoro Chloromethylbenzamide,

N-{[1- (2- (4- (4-ethylbenzoyl) piperazin-1-yl) ethyl) -pyrrolidin- (3R) -yl-carbamoyl] -methyl} -3-trifluoro Methylbenzamide,

N-{[1- (2- (4- (phenylsulfonyl) piperazin-1-yl) ethyl) -pyrrolidin- (3R) -yl-carbamoyl] -methyl} -3-trifluoromethyl Benzamide,

N-{[1- (2- (4-propionylpiperazin-1-yl) ethyl) -pyrrolidin- (3R) -yl-carbamoyl] -methyl} -3-trifluoromethylbenzamide,

N-{[1- (2- (4-benzylpiperazin-1-yl) ethyl) -pyrrolidin- (3R) -yl-carbamoyl] -methyl} -3-trifluoromethylbenzamide,

N-{[1- (1- (4-benzoylpiperazin-1-yl) propan-2-yl) -pyrrolidin- (3R) -yl-carbamoyl] -methyl} -3-trifluoromethyl Benzamide,

N-{[1- (3- (4-benzoylpiperazin-1-yl) butan-2-yl) -pyrrolidin- (3R) -yl-carbamoyl] -methyl} -3-trifluoromethyl Benzamide,

N-{[1- (2- (4-benzoylpiperazin-1-yl) propan-1-yl) -pyrrolidin- (3R) -yl-carbamoyl] -methyl} -3-trifluoromethyl Benzamide,

N-{[1- (1- (4- (Benzoylpiperazin-1-yl) butan-2-yl) -pyrrolidin- (3R) -yl-carbamoyl] -methyl} -3-trifluoromethyl Benzamide,

N-{[1- (2- (4-benzoylpiperazin-1-yl) butyl) -pyrrolidin- (3R) -yl-carbamoyl] -methyl} -3-trifluoromethylbenzamide,

N-{[1- (2- (4-benzoylpiperazin-1-yl) -2-methylpropyl) -pyrrolidine- (3R) -yl-carbamoyl] -methyl} -3-trifluoromethyl Benzamide,

N-{[1- (1- (4-Benzoylpiperazin-1-yl) -2-methylpropan-2-yl) -pyrrolidin- (3R) -yl-carbamoyl] -methyl} -3- Trifluoromethylbenzamide,

N-{[1- (3- (4- (3-chlorophenyl) piperazin-1-yl) butan-2-yl) -pyrrolidin- (3R) -yl-carbamoyl] -methyl} -3 Trifluoromethylbenzamide,

N-{[1- (1- (1- (4-phenylpiperazin-1-yl) propan-2-yl) -pyrrolidin- (3R) -yl-carbamoyl] -methyl} -3-trifluoromethyl Benzamide,

N-{[1- (1- (4- (cyclohexylpiperazin-1-yl) propan-2-yl) -pyrrolidin (3R) -yl-carbamoyl] -methyl} -3-trifluoro Methylbenzamide,

N-{[1- (2- (4- (2-hydroxybutyl) piperazin-1-yl) ethyl) -pyrrolidine- (3R) -yl-carbamoyl] -methyl} -3-trifluoro Chloromethylbenzamide,

N-{[1- (2- (4- (2-hydroxy-2-methylpropyl) piperazin-1-yl) ethyl) -pyrrolidin- (3R) -yl-carbamoyl] -methyl}- 3-trifluoromethylbenzamide,

N-{[1- (2- (4- (2-hydroxy-2-phenylethyl) piperazin-1-yl) ethyl) -pyrrolidin- (3R) -yl-carbamoyl] -methyl}- 3-trifluoromethylbenzamide,

N-{[1- (2- (4- (2- (4-chlorophenyl) -2-hydroxyethyl) piperazin-1-yl) ethyl) -pyrrolidin- (3R) -yl-carbamoyl ] -Methyl} -3-trifluoromethylbenzamide,

N-{[1- (2- (4-benzoylpiperazin-1-yl) -2-oxoethyl) -pyrrolidine- (3R) -yl-carbamoyl] -methyl} -3-trifluoromethyl Benzamide,

N-{[1- (2- (4- (4-Methylbenzoyl) piperazin-1-yl) -2-oxoethyl) -pyrrolidin- (3R) -yl-carbamoyl] -methyl} -3 Trifluoromethylbenzamide,

N-{[1- (2- (4- (4-ethylbenzoyl) piperazin-1-yl) -2-oxoethyl) -pyrrolidin- (3R) -yl-carbamoyl] -methyl} -3 Trifluoromethylbenzamide,

N-{[1- (2- (4- (3-Methylbenzoyl) piperazin-1-yl) -2-oxoethyl) -pyrrolidin- (3R) -yl-carbamoyl] -methyl} -3 Trifluoromethylbenzamide,

N-{[1- (2- (4- (2-Methylbenzoyl) piperazin-1-yl) -2-oxoethyl) -pyrrolidin- (3R) -yl-carbamoyl] -methyl} -3 Trifluoromethylbenzamide,

N-{[1- (2- (4-phenylpiperazin-1-yl) -2-oxoethyl) -pyrrolidine- (3R) -yl-carbamoyl] -methyl} -3-trifluoromethyl Benzamide,

N-{[1- (2- (4- (Benzooxycarbonyl) piperazin-1-yl) -2-oxoethyl) -pyrrolidine- (3R) -yl-carbamoyl] -methyl} -3 Trifluoromethylbenzamide,

N-{[1- (2- (4-benzoyl-2,5-dimethylpiperazin-1-yl) ethyl) -pyrrolidine- (3R) -yl-carbamoyl] -methyl} -3-trifluoro Chloromethylbenzamide,

N-{[1- (2- (4- (2-chlorobenzoyl) piperazin-1-yl) ethyl) -pyrrolidin- (3R) -yl-carbamoyl] -methyl} -3-trifluoro Methylbenzamide,

N-{[1- (2- (4- (2-fluorobenzoyl) piperazin-1-yl) ethyl) -pyrrolidine- (3R) -yl-carbamoyl] -methyl} -3-trifluoro Chloromethylbenzamide, and

N-{[1- (2- (4- (2,5-Difluorobenzoyl) piperazin-1-yl) ethyl) -pyrrolidin- (3R) -yl-carbamoyl] -methyl} -3 Trifluoromethylbenzamide

It may be a compound selected from the group consisting of.

In addition, the pharmaceutical composition of the present invention may be inhaled in the method of administration.

In addition, the pharmaceutical compositions of the present invention may improve airway inflammation.

In addition, the pharmaceutical compositions of the present invention can inhibit airway remodeling.

In addition, the pharmaceutical compositions of the present invention may improve airway hypersensitivity.

On the other hand, the food composition for improving the asthma disease of the present invention is characterized in that it comprises a compound of the formula (1) or a salt acceptable as a food thereof as an active ingredient.

The pharmaceutical composition of the present invention comprising the compound of formula 1 as an active ingredient significantly reduced inflammation, mucus secretion, eosinophil increase around the bronchus and blood vessels of the asthma-induced group. In addition, interleukin-4 (IL-4), interleukin-5 (IL-5) and interleukin-13 (IL-13) were all significantly reduced in relation to Th2 cytokine production, which is closely related to the mechanism of asthma. Airway obstruction has also been shown to be reduced and may be useful for treating, preventing or ameliorating asthma disease. In addition, the inhibitory effect on CYP450 was relatively low, and it was confirmed that the compound is a safe compound having little cardiac toxicity and cytotoxicity. Furthermore, the compound of Formula 1 may also be included in food compositions such as health functional foods and used to improve and prevent symptoms of asthma.

1 is a histopathological analysis picture by H & E staining of lung tissue.
Figure 2 is a histopathological analysis picture by PAS staining of lung tissue.
Figure 3 is a photograph of the inflammatory cell count of bronchoalveolar lavage fluid (BALF).
4 is a result of analyzing the expression level of cytokines extracted from lung tissue.
5 is a graph measuring the change in airway hypersensitivity by methacholine.

Hereinafter, preferred embodiments of the present invention will be described in detail. In the following description, numerous specific details, such as specific elements, are set forth in order to provide a thorough understanding of the present invention, and it is to be understood that the present invention may be practiced without these specific details, It will be obvious to those who have knowledge of. In the following description of the present invention, a detailed description of known functions and configurations incorporated herein will be omitted when it may make the subject matter of the present invention rather unclear.

The present invention provides pharmaceutical compositions and food compositions for use in the prevention, amelioration of symptoms or treatment of asthma diseases, including compounds of formula (1), or isomers thereof and pharmaceutically acceptable salts thereof:

[Formula 1]

In Formula 1,

R ₁ is independently a hydrogen atom, C ₁ -C ₃ alkyl, phenyl, benzyl, benzoyl, benzenesulfonyl, C ₁ -C ₃ alkylcarbonyl, C ₃ -C ₇ cycloalkyl,

(2)

,

,

(3)

, 및

, And

[Formula 4]

It may be selected from the group consisting of;

R ₁₀ , R ₁₁ and R ₁₂ can be independently selected from the group consisting of hydrogen atom, C ₁ -C ₃ alkyl, phenyl, and benzyl group;

All benzene groups included as part of R ₁ may independently have a plurality of substituents selected from the group consisting of C ₁ -C ₃ alkyl, C ₁ -C ₂ haloalkyl, a halogen atom, and a cyano group;

R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , R ₇ , R ₈ and R ₉ can be independently selected from a hydrogen atom and C ₁ -C ₃ alkyl;

R ₆ and R ₇ , R ₈ and R ₉ can be independently selected from a carbonyl group;

The halogen is selected from the group consisting of fluorine, chlorine and bromine atoms.

The present invention relates to a pharmaceutical composition and a food composition comprising a compound of Formula 1, which exhibits a therapeutic effect against asthma diseases through anti-inflammatory and immune function regulation, wherein the pharmaceutically acceptable salts, geometric or optical isomers of the compound, Hydrates, solvates or polymorphs.

Pharmaceutically acceptable salts refer to acid addition salts or base addition salts which can be prepared by conventional organic synthesis methods depending on the specific functional groups of the compounds presented herein. At this time, the added acid or base is harmless to the human body or relatively less toxic and can be used in the manufacture of a drug. If the compounds of the present invention are basic, an appropriate amount of acid must be added for the salt to form. Preferred acid addition salts include inorganic acids such as hydrochloric acid, bromic acid, nitric acid, sulfuric acid and iodic acid, acetic acid, citric acid, fumaric acid, galactamic acid, malonic acid, maleic acid, succinic acid, tartaric acid, methanesulfonic acid, lactic acid, oxalic acid and propionic acid. Organic acids such as salicylic acid, mandelic acid, and phthalic acid. In addition, when the compound of the present invention is acidic, an appropriate amount of base must be added to form a salt. Preferred base addition salts here include ammonium, sodium, potassium, calcium, magnesium, organoammonium and the like.

Meanwhile, the compounds presented in the present invention may be unsolvated or hydrates containing water or solvates including organic solvents used in conventional organic synthesis processes, and pharmaceutical compositions and food compositions including such anhydrides, hydrates, or solvates. Are all considered to be within the scope of the present invention.

Meanwhile, the compounds presented in the present invention may exhibit amorphous or multiple polymorphisms in a solid state, and pharmaceutical compositions and food compositions including such amorphous or individual polymorphisms are all within the scope of the present invention. Seen to be.

The compounds presented in the present invention can be prepared in a number of ways by conventional organic synthesis techniques. In addition, the 3-aminopyrrolidine structure commonly included in the compounds may be introduced in a number of ways depending on commercially available raw materials. Specifically, compounds according to the present invention may be synthesized according to the methods shown in Schemes 1 to 6 below.

According to Scheme 1 below, a common process for the preparation of intermediate compounds for the preparation of the compounds presented in the present invention is presented. The raw material used to introduce the 3-aminopyrrolidine structure is (3R)-(-)-1-benzyl-3-aminopyrrolidine such as compound (3). Wherein carbon 3 of the pyrrolidine ring exhibits chirality, and all compounds including the 3-aminopyrrolidine structure presented in the present invention are limited to the (R) -form based on the nomenclature of compound (3) .

[Reaction Scheme 1]

According to Scheme 1, Compound (2) can be synthesized by reacting Compound (1), which is a commercially available starting material, with basic glycine, an amino acid, under basic and aqueous conditions. The method of reacting compound (3), which is a derivative of the chiral 3-aminopyrrolidine described above with compound (2), to form an amide bond can be made according to a number of known methods. In this specification, isobutyl chloroformate is exemplified as a method of activating the carboxylic acid of compound (2), wherein the base is N-methylmorpholine (NMM). ) Can be easily produced. The N-benzyl group of compound (4) means a protecting group, which must deprotect the benzyl group in order to prepare N-alkylation and other N-substituents in the pyrrolidine structure. As a conventional method for this, compound (5) can be easily prepared using hydrogen gas under a palladium catalyst (Pd (OH) ₂ ). Compound (5) thus prepared can be utilized as a major intermediate for preparing compounds of various structures exhibiting the anti-asthmatic effect presented in the present invention.

On the other hand, all the intermediate compounds prepared according to the method described in Scheme 1 can be obtained in the solid state, specifically solidifying the desired compound in a non-polar solvent, the present invention provides a manufacturing method that is easy for industrial mass production to provide.

Synthesis of compound (9) as a therapeutic agent for asthma presented in the present invention can be made according to Scheme 2 below.

[Reaction Scheme 2]

Using compound (5) prepared according to the method of Scheme 1 as a starting material, protected with t-butoxycarbonyl group (Boc) and R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , R ₇ , R ₈ and R ₉ are reacted with substituted piperazinylethane mesylate (6) under basic conditions to give an intermediate compound (7). Wherein R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , R ₇ , R ₈ and R ₉ are as defined herein. The intermediate (7) is subjected to deprotection reaction in ethanol saturated with hydrochloric acid to obtain the piperazine intermediate (8) in hydrochloride form. This compound can be reacted with various reagents under basic conditions to prepare compound (9) as a therapeutic agent for asthma in the present invention.

Scheme 3

The method of Scheme 3 may also be used to prepare compound (9) as an agent for treating asthma presented in the present invention. Using compound (5) prepared according to the method of Scheme 1 as a starting material, an intermediate compound was reacted under basic conditions using an epoxy compound or chloroethanol derivative substituted with R ₆ , R ₇ , R ₈ and R _9. 10) to manufacture. Wherein R ₆ , R ₇ , R ₈ and R ₉ are as defined herein.

Intermediate compound (11) can be obtained from the ethanol derivative (10) by reaction with mesyl chloride (MsCl) in basic conditions, whereby the compound (11) is purified, separated or not purified as necessary, and the following reaction is carried out. You can proceed. In order to prepare the desired compound (9) in the present invention, the intermediate (11) is reacted with a piperazine derivative (12) in which R ₁ , R ₂ , R ₃ , R ₄ and R ₅ are substituted under basic conditions. The piperazine derivative (12) used at this time may take the form of a hydrochloride salt as needed, and R ₁ , R ₂ , R ₃ , R ₄ and R ₅ are as defined herein.

Another method for preparing compound (9) as the asthma therapeutic agent presented in the present invention is as described in Scheme 4 below.

[Reaction Scheme 4]

Using the piperazine derivative (12) as a starting material, an intermediate compound (13) is prepared by reacting under basic conditions using an epoxy compound or a chloroethanol derivative substituted with R ₆ , R ₇ , R ₈ and R ₉ . Wherein R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , R ₇ , R ₈ and R ₉ are as defined herein. Intermediate compound (14) can be obtained by reacting with mesyl chloride under basic conditions from ethanol derivative (13), which compound (14) can be subjected to the next reaction without purification, separation or purification as necessary. Intermediate (14) is reacted with pyrrolidine intermediate (5) under basic conditions to produce compound (9) desired in the present invention.

Another method for preparing compound (9) as a therapeutic agent for asthma presented in the present invention is as described in Scheme 5 below.

[Reaction Scheme 5]

Using the piperazine derivative (12) as a starting material, an intermediate compound (15) is prepared under basic conditions using a 2-chloroketone derivative substituted with R ₆ , R ₇ and R ₈ . Wherein R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , R ₇ and R ₈ are as defined herein. The intermediate compound (15) can be subjected to reductive amination reaction with the pyrrolidine intermediate (5) to produce the target compound (9). At this time, the reducing agent used may be variously selected according to a number of known methods, but in this specification, N-alkyl using sodium triacetoxyborohydride (NaBH (OAc) ₃ ), which is a relatively reducing reducing agent. The easy preparation of a pyrrolidine compound is illustrated.

Meanwhile R ₆ and R ₇ , R ₈ and R ₉ of Formula 1 as defined herein independently include a carbonyl group, and such a compound (20) as a therapeutic agent for asthma may be prepared according to the method illustrated in Scheme 6 below. .

[Reaction Scheme 6]

According to this method, R ₈ and R ₉ are formed under basic conditions, starting with piperazine (16) protected with a t-butoxycarbonyl group (Boc) and substituted with R ₂ , R ₃ , R ₄ and R ₅ . Compound (17) is prepared by reaction with a substituted 2-chloroacetyl chloride derivative. Wherein R ₂ , R ₃ , R ₄ , R ₅ , R ₈ and R ₉ are as defined herein. Compound (18) can be prepared from compound (17) via an intermediate reaction with intermediate (5) under basic conditions, and the protecting group is removed under acidic conditions under a suitable solvent to give compound (19). Various halogen reagents can be prepared from intermediate compound (19) to prepare compound (20) as an asthma therapeutic agent provided herein.

Compounds as therapeutic agents for asthma according to the present invention according to the methods shown in Schemes 1 to 6 are as follows.

Example    Chemical name

1 N-{[1- (2- (1- (phenylaminocarbonyl) -piperazin-4-yl) ethyl) -pyrrolidin- (3R) -yl-

Carbamoyl] -methyl} -3-trifluoromethylbenzamide

2 N-{[1- (2- (1- (p-tolylaminocarbonyl) -piperazin-4-yl) ethyl) -pyrrolidine- (3R)-

Yl-carbamoyl] -methyl} -3-trifluoromethylbenzamide

3 N-{[1- (2- (1- (4-chlorophenylaminocarbonyl) -piperazin-4-yl) ethyl) -pyrrolidine

-(3R) -yl-carbamoyl] -methyl} -3-trifluoromethylbenzamide

4 N-{[1- (2- (1- (methoxycarbonyl) -piperazin-4-yl) ethyl) -pyrrolidin- (3R) -yl-carba

Moyl] -methyl} -3-trifluoromethylbenzamide

5 N-{[1- (2- (1- (ethoxycarbonyl) -piperazin-4-yl) ethyl) -pyrrolidin- (3R) -yl-carba

Moyl] -methyl} -3-trifluoromethylbenzamide

6 N-{[1- (2- (4-benzoylpiperazin-1-yl) ethyl) -pyrrolidin- (3R) -yl-carbamoyl] -methyl}

-3-trifluoromethylbenzamide

7 N-{[1- (2- (4- (2-methylbenzoyl) piperazin-1-yl) ethyl) -pyrrolidin- (3R) -yl-carbamo

Yl] -methyl} -3-trifluoromethylbenzamide

8 N-{[1- (2- (4- (3-methylbenzoyl) piperazin-1-yl) ethyl) -pyrrolidin- (3R) -yl-carbamo

Yl] -methyl} -3-trifluoromethylbenzamide

9 N-{[1- (2- (4- (4-methylbenzoyl) piperazin-1-yl) ethyl) -pyrrolidin- (3R) -yl-carbamo

Yl] -methyl} -3-trifluoromethylbenzamide

10 N-{[1- (2- (4- (4-fluorobenzoyl) piperazin-1-yl) ethyl) -pyrrolidin- (3R) -yl-carba

Moyl] -methyl} -3-trifluoromethylbenzamide

11 N-{[1- (2- (4- (4-cyanobenzoyl) piperazin-1-yl) ethyl) -pyrrolidin- (3R) -yl-carbamo

Yl] -methyl} -3-trifluoromethylbenzamide

12 N-{[1- (2- (4- (4-ethylbenzoyl) piperazin-1-yl) ethyl) -pyrrolidin- (3R) -yl-carbamo

Yl] -methyl} -3-trifluoromethylbenzamide

13 N-{[1- (2- (4- (phenylsulfonyl) piperazin-1-yl) ethyl) -pyrrolidin- (3R) -yl-carbamoyl]-

Methyl} -3-trifluoromethylbenzamide

14 N-{[1- (2- (4-propionylpiperazin-1-yl) ethyl) -pyrrolidin- (3R) -yl-carbamoyl] -meth

Tyl} -3-trifluoromethylbenzamide

15 N-{[1- (2- (4-benzylpiperazin-1-yl) ethyl) -pyrrolidin- (3R) -yl-carbamoyl] -methyl} -3-

Trifluoromethylbenzamide

16 N-{[1- (1- (4-benzoylpiperazin-1-yl) propan-2-yl) -pyrrolidin- (3R) -yl-carbamo

Yl] -methyl} -3-trifluoromethylbenzamide

17 N-{[1- (3- (4-benzoylpiperazin-1-yl) butan-2-yl) -pyrrolidin- (3R) -yl-carbamoyl]-

Methyl} -3-trifluoromethylbenzamide

18 N-{[1- (2- (4-benzoylpiperazin-1-yl) propan-1-yl) -pyrrolidin- (3R) -yl-carbamo

Yl] -methyl} -3-trifluoromethylbenzamide

19 N-{[1- (1- (4-benzoylpiperazin-1-yl) butan-2-yl) -pyrrolidin- (3R) -yl-carbamoyl]-

Methyl} -3-trifluoromethylbenzamide

20 N-{[1- (2- (4-benzoylpiperazin-1-yl) butyl) -pyrrolidin- (3R) -yl-carbamoyl] -meth

Tyl} -3-trifluoromethylbenzamide

21 N-{[1- (2- (4-benzoylpiperazin-1-yl) -2-methylpropyl) -pyrrolidin- (3R) -yl-carbamo

Yl] -methyl} -3-trifluoromethylbenzamide

22 N-{[1- (1- (4-benzoylpiperazin-1-yl) -2-methylpropan-2-yl) -pyrrolidin- (3R) -yl-ka

Barmoyl] -methyl} -3-trifluoromethylbenzamide

23 N-{[1- (3- (4- (3-chlorophenyl) piperazin-1-yl) butan-2-yl) -pyrrolidin- (3R) -yl-ka

Barmoyl] -methyl} -3-trifluoromethylbenzamide

24 N-{[1- (1- (4- (phenylphenylazin-1-yl) propan-2-yl) -pyrrolidin- (3R) -yl-carbamoyl]-

Methyl} -3-trifluoromethylbenzamide

25 N-{[1- (1- (4-cyclohexylpiperazin-1-yl) propan-2-yl) -pyrrolidin (3R) -yl-carbamo

Yl] -methyl} -3-trifluoromethylbenzamide

26 N-{[1- (2- (4- (2-hydroxybutyl) piperazin-1-yl) ethyl) -pyrrolidin- (3R) -yl-carbamo

Yl] -methyl} -3-trifluoromethylbenzamide

27 N-{[1- (2- (4- (2-hydroxy-2-methylpropyl) piperazin-1-yl) ethyl) -pyrrolidine- (3R)-

Yl-carbamoyl] -methyl} -3-trifluoromethylbenzamide

28 N-{[1- (2- (4- (2-hydroxy-2-phenylethyl) piperazin-1-yl) ethyl) -pyrrolidin- (3R) -yl

-Carbamoyl] -methyl} -3-trifluoromethylbenzamide

29 N-{[1- (2- (4- (2- (4-chlorophenyl) -2-hydroxyethyl) piperazin-1-yl) ethyl) -pyrroli

Din- (3R) -yl-carbamoyl] -methyl} -3-trifluoromethylbenzamide

30 N-{[1- (2- (4-benzoylpiperazin-1-yl) -2-oxoethyl) -pyrrolidin- (3R) -yl-carbamo

Yl] -methyl} -3-trifluoromethylbenzamide

31 N-{[1- (2- (4- (4-methylbenzoyl) piperazin-1-yl) -2-oxoethyl) -pyrrolidin- (3R) -yl-

Carbamoyl] -methyl} -3-trifluoromethylbenzamide

32 N-{[1- (2- (4- (4-ethylbenzoyl) piperazin-1-yl) -2-oxoethyl) -pyrrolidin- (3R) -yl-

Carbamoyl] -methyl} -3-trifluoromethylbenzamide

33 N-{[1- (2- (4- (3-methylbenzoyl) piperazin-1-yl) -2-oxoethyl) -pyrrolidin- (3R) -yl-

Carbamoyl] -methyl} -3-trifluoromethylbenzamide

34 N-{[1- (2- (4- (2-methylbenzoyl) piperazin-1-yl) -2-oxoethyl) -pyrrolidin- (3R) -yl-

Carbamoyl] -methyl} -3-trifluoromethylbenzamide

35 N-{[1- (2- (4-phenylpiperazin-1-yl) -2-oxoethyl) -pyrrolidin- (3R) -yl-carbamoyl]-

Methyl} -3-trifluoromethylbenzamide

36 N-{[1- (2- (4- (benzoxycarbonyl) piperazin-1-yl) -2-oxoethyl) -pyrrolidin- (3R) -yl

-Carbamoyl] -methyl} -3-trifluoromethylbenzamide

37 N-{[1- (2- (4-benzoyl-2,5-dimethylpiperazin-1-yl) ethyl) -pyrrolidin- (3R) -yl-carba

Moyl] -methyl} -3-trifluoromethylbenzamide

38 N-{[1- (2- (4- (2-chlorobenzoyl) piperazin-1-yl) ethyl) -pyrrolidin- (3R) -yl-carbamo

Il] -methyl} -3-trifluoromethylbenzamide,

39 N-{[1- (2- (4- (2-fluorobenzoyl) piperazin-1-yl) ethyl) -pyrrolidin- (3R) -yl-carba

Moyl] -methyl} -3-trifluoromethylbenzamide

40 N-{[1- (2- (4- (2,5-difluorobenzoyl) piperazin-1-yl) ethyl) -pyrrolidin- (3R) -yl-

Carbamoyl] -methyl} -3-trifluoromethylbenzamide

In addition, the pharmaceutical compositions of the present invention may be made by any suitable formulation means capable of exhibiting the effects of the present invention. That is, the pharmaceutical composition of the present invention includes formulations such as oral, intravenous or intramuscular injections, skin patches, suppositories, nasal or oral inhalants, intraperitoneal injections, etc., and are configured as pharmaceutical forms suitable for other active ingredients and administration purposes. Among them, it is particularly preferable that the suction type. Oral preparations include tablets, effervescent tablets, capsules, granules, powders, sustained-release tablets, sustained-release capsules and the like. It may also be administered as a pharmaceutical form, including formulations such as intravenous or intramuscular injections, suspensions, suppositories, skin patches, intraperitoneal injections, intranasal delivery devices, and the like, suitable for other active ingredients and administration purposes.

In order to prepare a preferred pharmaceutical composition using the compound of the present invention as an active ingredient, it may be formulated to include physiologically resistant excipients, diluents, adjuvants, coating materials, antioxidants, aromatic substances. Specific examples of excipients and auxiliaries are gelatin, sucrose, lactose, lecithin, pectin, starch, cyclodextrin, cyclodextrin derivatives, dextran, polyvinylpyrrolidone, polyvinyl acetate, gum arabic, alginic acid, tylose, talc, ricoh Fordium, silicic acid, calcium hydrogen phosphate, cellulose, methoxypropylcellulose, methylcellulose, hydroxypropylmethylcellulose, hydroxypropylmethylcellulose phthalate, saturated or unsaturated fatty acids, vegetable glycerol esters, polyglycerol esters, alcohols, polyethylene glycols, Esters of aliphatic alcohols, glycols, glycerol, diethylene glycol, propylene glycol, sorbitol, mannitol, saturated or unsaturated fatty acids. Specific examples of supplements or disintegrants that may additionally be used are cross-linked polyvinylpyrrolidone, sodium carboxymethyl starch, sodium carboxymethylcellulose, microcrystalline cellulose. It is also possible to use tablet coatings, specific examples of which are ester polymers and copolymers of acrylic acid, methacrylic acid, methacrylic acid, zein, ethylcellulose, ethylcellulose succinate, shellac, citric acid esters, tartar esters, glycerol , Glycerol esters, polyethylene glycols. On the other hand, an appropriate amount of organic solvent may be used within water or a physiologically acceptable range for the preparation of the desired formulation or suspension. Particularly in liquid formulations, preservatives, antioxidants and fragrance enhancers can be used, specific examples of which include potassium sorbate, methyl 4-hydroxybenzoate, propyl 4-hydroxybenzoate, ascorbic acid, peppermint oil, and the like. . Solvents and emulsifiers, such as polyvinylpyrrolidone, polysorbate 80, may be used as needed for preferred formulation.

When the pharmaceutical composition comprising the compound having the structure of Formula 1 as presented in the present invention as an active ingredient is used as a medicine for treating asthma in humans, the daily dosage of the active ingredient is 0.01 ng / kg to 1,000 mg / kg. Range, preferably 0.01 μg / kg to 1 mg / kg. It consists of forms suitable for the mode of administration chosen depending on the severity of the asthma disease and the subject of administration. That is, it can be administered according to the dosage regimen once to 5 times a day, and can be administered alone or in combination with other drugs as necessary.

In addition, the pharmaceutical composition of the present invention can improve airway inflammation and airway hypersensitivity, and can suppress airway remodeling, which is effective for the treatment of asthma disease, improvement and prevention of symptoms.

Specifically, a budesonide, a steroidal drug, budesonide is used as a control and a composition of the compound of Formula 1 presented in the present invention using an asthma mouse animal model induced by ovalbumin (OVA). In order to examine the effect on inflammation, the lung tissue of the mouse was observed histopathologically. H and E staining (hematoxilin & eosin staining) and PAS (periodic acid-Schiff staining) of the injured lung tissue, as a result of the treatment of the composition of the compound of the present invention in the peribronchial and perivascular inflammation induced asthma Significantly decreased compared to the group. In addition, the composition treatment group of the compound presented in the present invention reduced mucin secretion to a similar level compared to the control group.

In addition, the eosinophils and other inflammatory cell numbers of bronchoalveolar lavage fluids (BALF) were measured to determine the effect of the composition of the compounds presented on the inflammatory cells. As a result, the composition treatment group of the compounds presented in the present invention more effectively reduced the increase in eosinophils compared to the control group.

In addition, in order to determine the effect of the composition of the compounds presented in the present invention on Th2 cytokine production closely related to the mechanism of asthma development, proteins extracted from biological tissues were analyzed molecularly. As a result, interleukin-4 (IL-4), interleukin-5 (IL-5), and interleukin-13 (IL-13) were all significantly reduced in the composition treatment group of the compound of the present invention compared to the asthma-induced group. .

In addition, to determine the effect of the composition of the compound of the present invention on airway hypersensitivity, the change in airway obstruction according to the concentration of methacholine (methacholin) was measured using whole body plethysmography. As a result, the Pehn percentage change value increased by methacholine in the composition treatment group of the compound of the present invention was significantly reduced compared to the asthma-induced group.

On the other hand, the food composition for improving the asthma disease of the present invention is characterized in that the compound of formula (1) or a food acceptable salt thereof as an active ingredient, the food composition comprises a health functional food. As such, the compound of Chemical Formula 1 included in a food composition such as health functional food may be used to improve and prevent symptoms of asthma disease.

The invention is illustrated more specifically through the following examples. However, the scope of the present invention is not limited to these examples.

Example

Intermediates according to the methods described in the following preparations can be used for the synthesis of compounds as therapeutic agents for asthma of the present invention.

Manufacturing example  One :

(3- Trifluoromethylbenzoylamino ) -Acetic acid

0.763 g (10.16 mmol) of glycine was suspended in 20 ml of acetonitrile and 12.7 ml (25.40 mmol, 2.5 eq.) Of 2M NaOH aqueous solution was added. After cooling to 0-3 ° C., 2.12 g (10.16 mmol, 1.0 eq.) Of 3- (trifluoromethyl) -benzoyl chloride was slowly added dropwise with dilution in 4 ml of acetonitrile. The mixture was stirred at the same temperature for 1 hour and then adjusted to pH = 2-3 with 3N aqueous hydrochloric acid solution. After standing at room temperature, the upper organic solution was separated, and the lower aqueous solution was extracted three times with 20 ml of ethyl acetate. The organic solutions thus obtained were combined, dried over anhydrous magnesium sulfate, and the solvent was removed and concentrated under reduced pressure. The residue was solidified with toluene, filtered and washed with normal hexane to afford 2.28 g (91%) of the title compound as a white solid.

¹ H NMR (400MHz, DMSO-d ₆ ) 3.94 (2H, d), 7.74 (1H, t), 7.93 (1H, d), 8.16 (1H, d), 8.20 (1H, s), 9.12 (1H, t)

Manufacturing example  2 :

N-[(1- Benzylpyrrolidine -(3R) -day- Carbamoyl ) - methyl ] -3- Trifluoromethylbenzamide

10.74 g (43.4 mmol) of (3-trifluoromethylbenzoylamino) -acetic acid and 6.58 g (65.10 mmol, 1.5 eq.) Of N-methylmorpholine described in Preparation Example 1 were dissolved in 80 ml of tetrahydrofuran under argon gas. I was. After cooling to −10 ° C., 7.11 g (52.08 mmol, 1.2 eq.) Of isobutyl chloroformate was diluted in 10 ml of tetrahydrofuran and slowly added dropwise to the reaction solution. The mixture was stirred at the same temperature for 15 minutes, and 8.03 g (45.57 mmol, 1.1 eq.) Of (3R)-(-)-1-benzyl-3-aminopyrrolidine was slowly added dropwise to 10 ml of tetrahydrofuran. After stirring at −10 ° C. for 1 hour, 100 ml of purified water was added thereto. The organic layer was recovered by three successive extractions with 100 ml of ethyl acetate. It was dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The residue was solidified with t-butyl methyl ether to give 12.18 g (69%) of the desired compound as a white solid.

¹ H NMR (400 MHz, DMSO-d ₆ ) 1.62-1.66 (1H, m), 2.26-2.36 (2H, m), 2.54-2.59 (1H, m), 2.63-2.66 (1H, m), 2.89-2.93 (1H, m), 3.62 (2H, d), 4.10 (2H, d), 4.46-4.90 (1H, m), 6.45 (1H, br s), 7.15 (1H, br s), 7.29-7.34 (5H , m), 7.59 (1H, t), 7.78 (1H, d), 8.00 (1H, d), 8.11 (1H, s)

Manufacturing example  3:

N- ( Pyrrolidine -(3R) -day- Carbamoylmethyl ) -3- Trifluoromethylbenzamide

9.00 g (22.20 mmol) of N-[(1-benzylpyrrolidin- (3R) -yl-carbamoyl) -methyl] -3-trifluoromethylbenzamide described in Preparation Example 2 were dissolved in 45 ml of methanol 0.05 g (catalyst amount) of palladium hydroxide (Pd (OH) ₂ ) was added thereto. The reaction solution was stirred overnight at room temperature under an atmosphere of hydrogen gas at 1 atmosphere. The reaction solution was filtered using diatomaceous earth, and the filtrate was recovered and concentrated under reduced pressure. The obtained residue was purified by chromatography on silica gel (mobile phase: methanol) to give 5.74 g (82%) of the title compound as a white solid.

¹ H NMR (400 MHz, DMSO-d ₆ ) 1.47-1.52 (1H, m), 1.86-1.91 (1H, m), 2.68-2.74 (1H, m), 2.79-2.84 (1H, m), 2.86-2.91 (1H, m), 3.16 (2H, s), 3.86 (2H, d), 4.05-4.13 (1H, m), 7.73 (1H, t), 7.93 (1H, d), 8.00 (1H, d), 8.17 (1H, d), 8.22 (1H, s), 8.98 (1H, t)

Manufacturing example  4 :

N-{[1- (2- (4- tert - Butoxycarbonylpiperazine -1-yl) ethyl)- Pyrrolidine -(3R) -day- Carbamoyl ] -Methyl} -3- Trifluoromethylbenzamide

1.00 g (3.17 mmol) of N- (pyrrolidin- (3R) -yl-carbamoylmethyl) -3-trifluoromethylbenzamide described in Preparation Example 3 and 1.32 g (9.52 mmol, 3.0 eq.) Of potassium carbonate. ) Into 25 ml of acetonitrile and 0.98 g (3.17 mmol, 1.0 eq.) Of tert-butyl 4- (2- (methylsulfonyloxy) ethyl) piperazine-1-carboxylate diluted in 5 ml of acetonitrile. Was slowly added dropwise. After warming to room temperature to 80 ℃ and stirred for 24 hours, 25 ml of purified water was added and extracted three times with 25 ml of ethyl acetate. The combined organic layers were dried over anhydrous magnesium sulfate, and then concentrated under reduced pressure. The residue obtained was purified by chromatography on silica gel (mobile phase: dichloromethane / methanol = 10: 1) to obtain 1.14 g (68%) of the title compound as a white solid. It was.

¹ H NMR (400 MHz, CDCl ₃ ) 1.45 (9H, s), 1.64-1.72 (1H, m), 2.28-2.80 (12H, m), 2.95-3.04 (1H, m), 3.36-3.52 (4H, m ), 4.07-4.13 (2H, m), 4.47-4.52 (1H, m), 6.73-6.84 (1H, m), 7.28-7.32 (1H, m), 7.58 (1H, t), 7.77 (1H, d ), 8.02 (1H, d), 8.12 (1H, s)

Manufacturing example  5:

N-{[1- (2- (piperazin-1-yl) ethyl)- Pyrrolidine -(3R) -day- Carbamoyl ] - methyl } -3- Trifluoromethylbenzamide  Hydrochloride

N-{[1- (2- (4-tert-butoxycarbonylpiperazin-1-yl) ethyl) -pyrrolidin- (3R) -yl-carbamoyl] -methyl} as described in Preparation Example 4. 0.65 g (1.23 mmol) of 3-trifluoromethylbenzamide was dissolved in 3 ml of ethanol saturated with hydrochloric acid. After stirring at room temperature for 1.5 hours, the mixture was concentrated under reduced pressure, dried under high vacuum overnight, and 0.56 g (98%) of the title compound was obtained as a yellow syrup.

MS (M + 1) ⁺ : 428.4

Manufacturing example  6:

N-{[1- (2-hydroxypropyl)- Pyrrolidine -(3R) -day- Carbamoyl ] - methyl } -3- Trifluoromethylbenzamide

200 mg (0.63 mmol) of N- (pyrrolidin- (3R) -yl-carbamoylmethyl) -3-trifluoromethylbenzamide and 263 mg (1.90 mmol, 3.0 eq.) Of potassium carbonate described in Preparation Example 3 were prepared. It was suspended in 10 ml of acetonitrile at room temperature. 122 mg (2.09 mmol, 3.0 eq.) Of propylene oxide was added, and the mixture was heated to reflux and stirred overnight. After cooling to room temperature, 15 ml of purified water was added, followed by extraction twice with 15 ml of ethyl acetate. The organic layers were combined, dried over anhydrous magnesium sulfate, and then concentrated under reduced pressure. The resulting residue was purified by chromatography on silica gel (mobile phase: dichloromethane / methanol = 5: 1) to give 154 mg (65%) of the title compound as a yellow solid.

¹ H NMR (400 MHz, DMSO-d ₆ ) 1.02 (3H, d), 1.48-1.59 (1H, m), 2.00-2.10 (1H, m), 2.22-2.40 (4H, m), 2.50-2.58 (2H , m), 3.15 (2H, d), 3.60-3.71 (1H, m), 3.85 (2H, d), 4.08-4.20 (2H, m), 4.30 (1H, t), 7.73 (1H, t), 7.91 (1H, d), 8.05-8.12 (1H, m), 8.16 (1H, d), 8.21 (1H, s), 8.95-9.01 (1H, m)

Manufacturing example  7:

N-{[1- (2- Hydroxybutyl ) - Pyrrolidine -(3R) -day- Carbamoyl ] - methyl } -3- Trifluoromethylbenzamide

200 mg (0.63 mmol) of N- (pyrrolidin- (3R) -yl-carbamoylmethyl) -3-trifluoromethylbenzamide described in Preparation Example 3, 263 mg (1.90 mmol, 3.0 eq.) Of potassium carbonate and 110 mg (45%) of the title compound as a yellow liquid were obtained in the same manner as in Preparation Example 6, using 229 mg of 1,2-epoxybutane (3.17 mmol, 5.0 eq.).

¹ H NMR (400 MHz, DMSO-d ₆ ) 0.85 (3H, t), 1.40-1.60 (2H, m), 2.00-2.10 (1H, m), 2.30-2.42 (4H, m), 2.59-2.69 (2H , m), 3.85 (2H, d), 4.08 (2H, q), 4.11-4.20 (1H, m), 4.22 (1H, t), 7.73 (1H, t), 7.91 (1H, d), 8.07 ( 1H, d), 8.16 (1H, d), 8.21 (1H, s), 8.96 (1H, t)

Manufacturing example  8 :

N-{[1- (2-hydroxy-2- Methyl propyl ) - Pyrrolidine -(3R) -day- Carbamoyl ] - methyl } -3- Trifluoromethylbenzamide

1.00 g (3.17 mmol) of N- (pyrrolidin- (3R) -yl-carbamoylmethyl) -3-trifluoromethylbenzamide described in Preparation Example 3, 1.32 g (9.55 mmol, 3.0 eq.) Of potassium carbonate. ) And isobutylene oxide 690 mg (9.57 mmol, 3.0 eq.) To obtain 700 mg (57%) of the target compound as a yellow solid in the same manner as in Preparation Example 6.

¹ H NMR (400 MHz, DMSO-d ₆ ) 1.08 (6H, s), 1.50-1.60 (1H, m), 1.99-2.09 (1H, m), 2.28-2.37 (2H, m), 2.45-2.53 (2H , m), 2.69-2.80 (2H, m), 3.88 (2H, d), 4.05 (1H, s), 4.11-4.20 (1H, m), 7.74 (1H, t), 7.92 (1H, d), 8.04 (1H, d), 8.18 (1H, d), 8.23 (1H, s), 8.99 (1H, t)

Manufacturing example  9:

(4- (2-hydroxy-2- Methyl propyl ) Piperazin-1-yl) Phenylmethanone

500 mg (2.63 mmol) of 1-benzoylpiperazine and 1.10 g (7.96 mmol, 3.0 eq.) Of potassium carbonate were suspended in 20 ml of acetonitrile at room temperature. 570 mg (7.90 mmol, 3.0 eq.) Of isobutylene oxide was added thereto, followed by heating to reflux for stirring overnight. After cooling to room temperature, 30 ml of purified water was added and extracted twice with 30 ml of ethyl acetate. The organic layers were combined, dried over anhydrous magnesium sulfate, and then concentrated under reduced pressure. The obtained residue was purified by chromatography on silica gel (mobile phase: dichloromethane / methanol = 20: 1) to give 276 mg (40%) of the title compound as a pale yellow solid.

¹ H NMR (400 MHz, DMSO-d ₆ ) 1.10 (6H, s), 2.23 (2H, s), 2.43-2.52 (2H, m), 2.53-2.62 (2H, m), 3.35-3.45 (2H, m ), 3.55-3.65 (2H, m), 4.13 (1H, s), 7.35-7.38 (2H, m), 7.43-7.46 (3H, m)

Manufacturing example  10:

1- (4- Benzoylpiperazine -1-yl) propan-2-one

0.10 g (0.53 mmol) of 1-benzoylpiperazine and 0.22 g (1.58 mmol, 3.0 eq.) Of potassium carbonate were added to 5 ml of acetonitrile, and 0.05 g (0.53 mmol, 1.0 eq.) Of chloroacetone was slowly added dropwise at room temperature. . After stirring for 1 hour, 5 ml of purified water was added thereto, and the mixture was extracted three times with 10 ml of ethyl acetate. The organic layer was collected, dried over anhydrous magnesium sulfate, and then concentrated under reduced pressure. The residue was purified by chromatography on silica gel (mobile phase: dichloromethane / methanol = 10: 1) to obtain 0.11 g (83%) of the title compound as a yellow syrup. It was.

¹ H NMR (400 MHz, CDCl ₃ ) 2.18 (3H, s), 2.41-2.69 (4H, m), 3.27 (2H, s), 3.42-3.54 (2H, m), 3.81-3.93 (2H, m), 7.40-7.46 (5H, m)

Manufacturing example  11:

3- (4- Benzoylpiperazine -1-yl) butan-2-one

Preparation Example 10 using 0.05 g (0.26 mmol) of 1-benzoylpiperazine, 0.11 g (0.79 mmol, 3.0 eq.) Of potassium carbonate, and 0.03 g (0.26 mmol, 1.0 eq.) Of 3-chloro-2-butanone In the same manner, 0.05 g (73%) of the target compound as a yellow syrup was obtained.

¹ H NMR (400MHz, CDCl ₃ ) 1.17 (3H, d), 2.25 (3H, s), 2.38-2.72 (4H, m), 3.20 (1H, q), 3.38-3.53 (2H, m), 3.74- 3.89 (2H, m), 7.40-7.48 (5H, m)

Manufacturing example  12:

tert -Butyl 4- (2- Chloroacetyl ) Piperazin-l- Carboxylate

2.00 g (10.74 mmol) of tert-butyl 1-piperazinecarboxylate and 1.30 g (12.89 mmol, 1.2 eq.) of triethylamine were dissolved in 25 ml of dichloromethane under argon gas. After cooling to 3 ° C., 1.33 g (11.81 mmol, 1.1 eq.) Of chloroacetyl chloride was diluted in 5 ml of dichloromethane and slowly added dropwise to the reaction solution. After stirring at 3 ° C. for 1 hour, 20 ml of purified water was added, the organic layer was separated, and the aqueous layer was extracted once with 40 ml of dichloromethane. The organic layers were combined, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. The residue was purified by chromatography on silica gel (mobile phase: ethyl acetate / hexane = 1: 2) to give 2.00 g (71%) of the title compound as a yellow syrup.

¹ H NMR (400 MHz, CDCl ₃ ) 1.48 (9H, s), 3.45 (2H, t), 3.51 (4H, s), 3.61 (2H, t), 4.09 (2H, s)

Manufacturing example  13:

N-{[1- (2- (4- tert - Butoxycarbonylpiperazine -1-yl) -2-oxo-ethyl)- Pyrrolidine -(3R) -day- Carbamoyl ] - methyl } -3- Trifluoromethylbenzamide

1.00 g (3.17 mmol) of N- (pyrrolidin- (3R) -yl-carbamoylmethyl) -3-trifluoromethylbenzamide described in Preparation Example 3 and 1.32 g (9.52 mmol, 3.0 eq.) Of potassium carbonate. ) Was added to 25 ml of acetonitrile, and 0.83 g (3.17 mmol, 1.0 eq.) Of tert-butyl 4- (2-chloroacetyl) piperazine-1-carboxylate described in Preparation 12 was diluted with 5 ml of acetonitrile. Slowly added dropwise at room temperature. After warming to 80 ° C. at room temperature and stirring for 2 hours, 25 ml of purified water was added thereto and extracted three times with 25 ml of ethyl acetate. The combined organic layers were dried over anhydrous magnesium sulfate, and then concentrated under reduced pressure. The residue obtained was purified by chromatography on silica gel (mobile phase: dichloromethane / methanol = 10: 1) to obtain 1.12 g (65%) of the title compound as a white solid. It was.

¹ H NMR (400 MHz, CDCl ₃ ) 1.48 (9H, s), 1.72-1.81 (1H, m), 2.17-2.29 (1H, m), 2.52-2.62 (1H, m), 2.73-2.82 (1H, m ), 2.85-2.95 (1H, m), 3.00 (1H, q), 3.39 (2H, d), 3.40-3.46 (4H, m), 3.47-3.55 (4H, m), 4.11-4.23 (2H, m) ), 4.46-4.52 (1H, m), 7.27-7.36 (1H, m), 7.44 (1H, d), 7.59 (1H, t), 7.77 (1H, d), 8.03 (1H, d), 8.13 ( 1H, s)

Manufacturing example  14:

N-{[1- (2- (piperazin-1-yl) -2-oxo-ethyl)- Pyrrolidine -(3R) -day- Carbamoyl ] - methyl } -3- Trifluoromethylbenzamide  Hydrochloride

N-{[1- (2- (4-tert-butoxycarbonylpiperazin-1-yl) -2-oxo-ethyl) -pyrrolidin- (3R) -yl-carba as described in Preparation Example 13 Moyl] -methyl} -3-trifluoromethylbenzamide 1.12 g (2.07 mmol) was dissolved in 5 ml of ethanol saturated with hydrochloric acid. After stirring at room temperature for 1.5 hours, the mixture was concentrated under reduced pressure, dried under high vacuum overnight, and 0.93 g (94%) of the title compound was obtained as a yellow syrup.

MS (M + 1) ⁺ 478.7

By using the intermediate prepared in the preparation example, the active ingredient of the present invention for treating asthma can be prepared as follows. However, the scope of the present invention is not limited to the following examples, of course.

Example  One :

N-{[1- (2- (1- ( Phenylaminocarbonyl ) -Piperazin-4-yl) ethyl)- Pyrrolidine -(3R) -day- Carbamoyl ] -Methyl} -3- Trifluoromethylbenzamide

N-{[1- (2- (piperazin-1-yl) ethyl) -pyrrolidin- (3R) -yl-carbamoyl] -methyl} -3-trifluoromethylbenz described in Preparation Example 5 50 mg (0.11 mmol) of amide hydrochloride and 40 mg (0.32 mmol, 3.0 eq.) Of N, N-diisopropylethylamine and 10 mg (0.11 mmol, 1.0 eq.) Of phenyl isocyanate were dissolved in 5 ml of dichloromethane under argon gas. After stirring for 2 hours at room temperature, 10 ml of purified water was added, the organic layer was separated, and the aqueous layer was extracted once with 10 ml of dichloromethane. The organic layers were combined, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. The residue was purified by chromatography on silica gel (mobile phase: dichloromethane / methanol = 10: 1) to give 50 mg (67%) of the title compound as a white solid.

¹ H NMR (400 MHz, CDCl ₃ ) 1.67-1.78 (1H, m), 2.28-2.40 (2H, m), 2.48-2.59 (7H, m), 2.60-2.78 (2H, m), 2.84 (1H, d ), 3.03-3.12 (1H, m), 3.45-3.57 (4H, m), 4.05-4.20 (2H, m), 4.47-4.52 (1H, m), 6.60 (1H, s), 7.05 (1H, d ), 7.23 (2H, d), 7.32 (1H, d), 7.32-7.36 (1H, m), 7.58 (1H, t), 7.77 (1H, d), 8.01 (1H, d), 8.11 (1H, s)

Example  2 :

N-{[1- (2- (1- (p- Tolylaminocarbonyl ) -Piperazin-4-yl) -ethyl)- Pyrrolidine -(3R) -day- Carbamoyl ] - methyl } -3- Trifluoromethylbenzamide

N-{[1- (2- (piperazin-1-yl) ethyl) -pyrrolidin- (3R) -yl-carbamoyl] -methyl} -3-trifluoromethylbenz described in Preparation Example 5 Example 1 using 50 mg (0.11 mmol) of amide hydrochloride, 40 mg (0.32 mmol, 3.0 eq.) Of N, N-diisopropylethylamine, and 14 mg (0.11 mmol, 1.0 eq.) Of isocyanic acid m-tolyl ester In the same manner, 45 mg (75%) of the title compound as a white solid were obtained.

¹ H NMR (400 MHz, CDCl ₃ ) 1.63-1.75 (1H, m), 2.23-2.30 (2H, m), 2.32 (3H, s), 2.51-2.60 (7H, m), 2.62-2.73 (2H, m ), 2.79 (1H, d), 3.02 (1H, t), 3.51 (4H, t), 4.08-4.15 (2H, m), 4.47-4.55 (1H, m), 6.37 (1H, s), 6.74 ( 1H, d), 6.86 (1H, d), 7.11 (1H, d), 7.14-7.21 (2H, m), 7.23 (1H, s), 7.59 (1H, t), 7.78 (1H, d), 8.01 (1H, d), 8.11 (1H, s)

Example  3:

N-{[1- (2- (1- (4- Chlorophenylaminocarbonyl ) -Piperazin-4-yl) -ethyl)- Pyrrolidine -(3R)- Carbamoyl ] - methyl } -3- Trifluoromethylbenzamide

N-{[1- (2- (piperazin-1-yl) ethyl) -pyrrolidin- (3R) -yl-carbamoyl] -methyl} -3-trifluoromethylbenz described in Preparation Example 5 Example 1 using 50 mg (0.11 mmol) of amide hydrochloride, 40 mg (0.32 mmol, 3.0 eq.) Of N, N-diisopropylethylamine, and 17 mg (0.11 mmol, 1.0 eq.) Of isocyanic acid 4-chlorophenyl ester In the same manner as the 50 mg (83%) of the target compound as a white solid.

¹ H NMR (400 MHz, CDCl ₃ ) 1.67-1.78 (1H, m), 2.28-2.40 (2H, m), 2.48-2.59 (7H, m), 2.60-2.78 (2H, m), 2.84 (1H, d ), 3.03-3.12 (1H, m), 3.45-3.57 (4H, m), 4.05-4.20 (2H, m), 4.47-4.52 (1H, m), 6.60 (1H, s), 7.05 (1H, d ), 7.23 (2H, d), 7.32 (1H, d), 7.33-7.36 (1H, m), 7.58 (1H, t), 7.77 (1H, d), 8.01 (1H, d), 8.11 (1H, s)

Example  4 :

N-{[1- (2- (1- ( Methoxycarbonyl ) -Piperazin-4-yl) ethyl)- Pyrrolidine -(3R) -day- Carbamoyl ] - methyl } -3- Trifluoromethylbenzamide

N-{[1- (2- (piperazin-1-yl) ethyl) -pyrrolidin- (3R) -yl-carbamoyl] -methyl} -3-trifluoromethylbenz described in Preparation Example 5 50 mg (0.11 mmol) of amide hydrochloride and 60 mg (0.43 mmol, 4.0 eq.) Of potassium carbonate were suspended in 3 ml of acetonitrile at room temperature, and then 10 mg (0.11 mmol, 1.0 eq.) Of methyl chloroformate was diluted in 1 ml of acetonitrile. Slowly added. The mixture was heated to reflux, stirred for 1 hour, and then concentrated under reduced pressure. 10 ml of purified water was added to the obtained residue, followed by extraction twice with 10 ml of ethyl acetate. The organic layers were combined, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. The obtained residue was purified by chromatography on silica gel (mobile phase: dichloromethane / methanol = 5: 1) to obtain 40 mg (80%) of the target compound as a white white solid.

¹ H NMR (400 MHz, CDCl ₃ ) 1.68-1.79 (1H, m), 2.29-2.40 (2H, m), 2.41-2.49 (4H, m), 2.53-2.57 (2H, m), 2.58-2.64 (1H , m), 2.68-2.77 (2H, m), 2.80-2.91 (1H, m), 3.05-3.17 (1H, m), 3.44-3.56 (4H, m), 3.70 (3H, s), 4.06-4.18 (2H, m), 4.47-4.58 (1H, m), 6.62-6.84 (1H, m), 7.12-7.21 (1H, m), 7.60 (1H, t), 7.78 (1H, d), 8.04 (1H d), 8.13 (1H, s)

Example  5:

N-{[1- (2- (1- ( Ethoxycarbonyl ) -Piperazin-4-yl) ethyl)- Pyrrolidine -(3R) -day- Carbamoyl ] - methyl } -3- Trifluoromethylbenzamide

N-{[1- (2- (piperazin-1-yl) ethyl) -pyrrolidin- (3R) -yl-carbamoyl] -methyl} -3-trifluoromethylbenz described in Preparation Example 5 40 mg of the target compound as a white solid in the same manner as in Example 4 using 50 mg (0.11 mmol) of amide hydrochloride, 60 mg (0.43 mmol, 4.0 eq.) Of potassium carbonate, and 10 mg (0.11 mmol, 1.0 eq.) Of ethyl chloroformate ( 74%) was obtained.

¹ H NMR (400 MHz, CDCl ₃ ) 1.26 (3H, t), 1.63-1.78 (1H, m), 2.23-2.38 (2H, m), 2.41-2.49 (4H, m), 2.50-2.57 (2H, m ), 2.58-2.73 (3H, m), 2.80 (1H, d), 2.97-3.08 (1H, m), 3.42-3.53 (4H, m), 4.11-4.20 (4H, m), 4.48-4.53 (1H) , m), 7.17 (1H, d), 7.41-7.48 (1H, m), 7.57 (1H, t), 7.76 (1H, d), 8.02 (1H, d), 8.12 (1H, s)

Example  6:

N-{[1- (2- (4- Benzoylpiperazine -1-yl) ethyl)- Pyrrolidine -(3R) -day- Carbamoyl ] - methyl } -3- Trifluoromethylbenzamide

N-{[1- (2- (piperazin-1-yl) ethyl) -pyrrolidin- (3R) -yl-carbamoyl] -methyl} -3-trifluoromethylbenz described in Preparation Example 5 80 mg (70%) of the target compound as a white solid in the same manner as Example 4 using 100 mg (0.22 mmol) of amide hydrochloride, 120 mg (0.86 mmol, 4.0 eq.) Of potassium carbonate, and 30 mg (0.22 mmol, 1.0 eq.) Of benzoyl chloride. ) Was obtained.

¹ H NMR (400 MHz, CDCl ₃ ) 1.57-1.72 (1H, m), 2.08-2.21 (1H, m), 2.22-2.36 (2H, m), 2.37-2.46 (1H, m), 2.48-2.67 (8H , m), 2.89 (1H, t), 3.36-3.52 (2H, m), 3.71-3.88 (2H, m), 4.11 (2H, d), 4.37-4.51 (1H, m), 6.64 (1H, d ), 7.32-7.47 (6H, m), 7.58 (1H, t), 7.77 (1H, d), 8.00 (1H, d), 8.11 (1H, s)

MS (M + 1) ⁺ 532.2

Example  7:

N-{[1- (2- (4- (2- Methylbenzoyl ) Piperazin-1-yl) ethyl)- Pyrrolidine -(3R) -day- Carbamoyl ] - methyl } Refluoromethylbenzami De

N-{[1- (2- (piperazin-1-yl) ethyl) -pyrrolidin- (3R) -yl-carbamoyl] -methyl} -3-trifluoromethylbenz described in Preparation Example 5 50 mg (0.11 mmol) of amide hydrochloride and 60 mg (0.43 mmol, 4.0 eq.) Of potassium carbonate are suspended in 3 ml of acetonitrile at room temperature, and then 18 mg (0.12 mmol, 1.1 eq.) Of o-toluyl chloride is diluted in 1 ml of acetonitrile. Was added slowly. The mixture was heated to reflux, stirred for 1 hour, and then concentrated under reduced pressure. 10 ml of purified water was added to the obtained residue, followed by extraction twice with 10 ml of ethyl acetate. The organic layers were combined, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. The obtained residue was purified by chromatography on silica gel (mobile phase: dichloromethane / methanol = 5: 1) to give 42 mg (71%) of the target compound as an off-white solid.

¹ H NMR (400 MHz, DMSO-d ₆ ) 1.50-1.60 (1H, m), 2.00-2.10 (1H, m), 2.19 (3H, s), 2.22-2.55 (10H, m), 2.60-2.70 (2H , m), 3.07-3.11 (2H, m), 3.58-3.66 (2H, m), 3.84 (2H, d), 4.10-4.19 (1H, m), 7.11 (1H, d), 7.19-7.31 (3H , m), 7.72 (1H, t), 7.91 (1H, d), 8.11-8.18 (2H, m), 8.21 (1H, s), 8.99 (1H, t)

Example  8 :

N-{[1- (2- (4- (3- Methylbenzoyl ) Piperazin-1-yl) ethyl)- Pyrrolidine -(3R) -day- Carbamoyl ] - methyl } Refluoromethylbenzami De

N-{[1- (2- (piperazin-1-yl) ethyl) -pyrrolidin- (3R) -yl-carbamoyl] -methyl} -3-trifluoromethylbenz described in Preparation Example 5 45 mg of the target compound as a yellow solid in the same manner as in Example 7, using 50 mg (0.11 mmol) of amide hydrochloride, 60 mg (0.43 mmol, 4.0 eq.) Of potassium carbonate and 18 mg (0.12 mmol, 1.1 eq.) Of m-toluyl chloride. (77%) was obtained.

¹ H NMR (400 MHz, DMSO-d ₆ ) 1.08-1.18 (1H, m), 1.20-1.30 (1H, m), 1.50-1.60 (1H, m), 2.00-2.10 (1H, m), 2.32 (3H , s), 2.35-2.46 (8H, m), 2.58-2.68 (2H, m), 3.23-3.33 (2H, m), 3.52-3.62 (2H, m), 3.84 (2H, d), 4.10-4.20 (1H, m), 7.12-7.17 (2H, m), 7.22-7.32 (2H, m), 7.73 (1H, t), 7.91 (1H, d), 8.10-8.18 (2H, m), 8.21 (1H , s), 8.99 (1 H, t)

Example  9:

N-{[1- (2- (4- (4- Methylbenzoyl ) Piperazin-1-yl) ethyl)- Pyrrolidine -(3R) -day- Carbamoyl ] - methyl } Refluoromethylbenzami De

N-{[1- (2- (piperazin-1-yl) ethyl) -pyrrolidin- (3R) -yl-carbamoyl] -methyl} -3-trifluoromethylbenz described in Preparation Example 5 38 mg of the target compound as a white white solid in the same manner as in Example 7, using 50 mg (0.11 mmol) of amide hydrochloride, 60 mg (0.43 mmol, 4.0 eq.) Of potassium carbonate and 18 mg (0.12 mmol, 1.1 eq.) Of p-toluyl chloride. (65%) was obtained.

¹ H NMR (400 MHz, DMSO-d ₆ ) 1.08-1.30 (2H, m), 1.50-1.60 (1H, m), 2.00-2.10 (1H, m), 2.32 (3H, s), 2.33-2.42 (8H , m), 2.58-2.68 (2H, m), 3.25-3.35 (2H, m), 3.50-3.60 (2H, m), 3.84 (2H, d), 4.10-4.20 (1H, m), 7.20-7.30 (4H, m), 7.73 (1H, t), 7.91 (1H, d), 8.11-8.18 (2H, m), 8.21 (1H, s), 8.99 (1H, t)

Example  10:

N-{[1- (2- (4- (4- Fluorobenzoyl ) Piperazin-1-yl) ethyl)- Pyrrolidine -(3R) -day- Carbamoyl ] - methyl } -3- Trifluoromethylbenzamide

N-{[1- (2- (piperazin-1-yl) ethyl) -pyrrolidin- (3R) -yl-carbamoyl] -methyl} -3-trifluoromethylbenz described in Preparation Example 5 Target compound as an off-white solid in the same manner as Example 7 using 50 mg (0.11 mmol) of amide hydrochloride, 60 mg (0.43 mmol, 4.0 eq.) Of potassium carbonate and 19 mg (0.12 mmol, 1.1 eq.) Of 4-fluorobenzoyl chloride. 33 mg (55%) were obtained.

¹ H NMR (400 MHz, DMSO-d ₆ ) 1.50-1.60 (1H, m), 2.00-2.10 (1H, m), 2.30-2.50 (10H, m), 2.60-2.68 (2H, m), 3.25-3.35 (2H, m), 3.50-3.60 (2H, m), 3.84 (2H, d), 4.10-4.20 (1H, m), 7.22-7.30 (2H, m), 7.41-7.48 (2H, m), 7.73 (1H, t), 7.91 (1H, d), 8.12 (1H, d), 8.16 (1H, d), 8.21 (1H, s), 8.98 (1H, t)

Example  11:

N-{[1- (2- (4- (4- Cyanobenzoyl ) Piperazin-1-yl) ethyl)- Pyrrolidine -(3R) -day- Carbamoyl ] - methyl } -3- Trifluoromethylbenzamide

N-{[1- (2- (piperazin-1-yl) ethyl) -pyrrolidin- (3R) -yl-carbamoyl] -methyl} -3-trifluoromethylbenz described in Preparation Example 5 Target compound as an off-white solid in the same manner as in Example 7, using 50 mg (0.11 mmol) of amide hydrochloride, 60 mg (0.43 mmol, 4.0 eq.) Of potassium carbonate and 20 mg (0.12 mmol, 1.1 eq.) Of 4-cyanobenzoyl chloride. 42 mg (70%) were obtained.

¹ H NMR (400 MHz, DMSO-d ₆ ) 1.52-1.62 (1H, m), 2.00-2.10 (1H, m), 2.30-2.55 (10H, m), 2.60-2.70 (2H, m), 3.19-3.25 (2H, m), 3.58-3.63 (2H, m), 3.84 (2H, d), 4.10-4.20 (1H, m), 7.55 (2H, d), 7.73 (1H, t), 7.88-7.94 (3H , m), 8.12-8.18 (2H, m), 8.21 (1H, s), 8.99 (1H, t)

Example  12:

N-{[1- (2- (4- (4- Ethylbenzoyl ) Piperazin-1-yl) ethyl)- Pyrrolidine -(3R) -day- Carbamoyl ] - methyl } Refluoromethylbenzami De

N-{[1- (2- (piperazin-1-yl) ethyl) -pyrrolidin- (3R) -yl-carbamoyl] -methyl} -3-trifluoromethylbenz described in Preparation Example 5 48 mg of the target compound as a white white solid in the same manner as in Example 7, using 50 mg (0.11 mmol) of amide hydrochloride, 60 mg (0.43 mmol, 4.0 eq.) Of potassium carbonate and 20 mg (0.12 mmol, 1.1 eq.) Of 4-ethylbenzoyl chloride. (79%) was obtained.

¹ H NMR (400 MHz, DMSO-d ₆ ) 1.08-1.28 (5H, m), 1.50-1.60 (1H, m), 2.00-2.10 (1H, m), 2.32-2.47 (10H, m), 2.57-2.68 (4H, m), 3.50-3.60 (2H, m), 3.84 (2H, d), 4.10-4.19 (1H, m), 7.23-7.32 (4H, m), 7.73 (1H, t), 7.84 (1H , d), 7.91 (1H, d), 8.16 (1H, d), 8.21 (1H, s), 8.99 (1H, t)

Example  13:

N-{[1- (2- (4- ( Phenylsulfonyl ) Piperazin-1-yl) ethyl)- Pyrrolidine -(3R) -day- Carbamoyl ] - methyl } -3-trifluoromethylbenzamide

N-{[1- (2- (piperazin-1-yl) ethyl) -pyrrolidin- (3R) -yl-carbamoyl] -methyl} -3-trifluoromethylbenz described in Preparation Example 5 34 mg of the target compound as a yellow solid in the same manner as in Example 7 using 50 mg (0.11 mmol) of amide hydrochloride, 60 mg (0.43 mmol, 4.0 eq.) Of potassium carbonate and 21 mg (0.12 mmol, 1.1 eq.) Of benzenesulfonyl chloride 55%) was obtained.

¹ H NMR (400 MHz, DMSO-d ₆ ) 1.08-1.30 (2H, m), 1.48-1.56 (1H, m), 1.96-2.06 (1H, m), 2.25-2.60 (10H, m), 2.80-2.90 (4H, m), 3.82 (2H, d), 4.08-4.15 (1H, m), 7.62-7.68 (2H, m), 7.70-7.76 (4H, m), 7.91 (1H, d), 8.05-8.13 (1H, m), 8.15 (1H, d), 8.20 (1H, s), 8.94-9.00 (1H, m)

Example  14:

N-{[1- (2- (4- Propionylpiperazine -1-yl) ethyl)- Pyrrolidine -(3R) -day- Carbamoyl ] - methyl } -3- Trifluoromethylbenzamide

N-{[1- (2- (piperazin-1-yl) ethyl) -pyrrolidin- (3R) -yl-carbamoyl] -methyl} -3-trifluoromethylbenz described in Preparation Example 5 42 mg (81) of the target compound as a yellow solid in the same manner as in Example 7 using 50 mg (0.11 mmol) of amide hydrochloride, 60 mg (0.43 mmol, 4.0 eq.) Of potassium carbonate and 11 mg (0.12 mmol, 1.1 eq.) Of propionyl chloride. %) Was obtained.

¹ H NMR (400 MHz, DMSO-d ₆ ) 0.95 (3H, t), 1.50-1.60 (1H, m), 2.00-2.10 (1H, m), 2.25-2.40 (10H, m), 2.48-2.58 (2H , m), 2.58-2.68 (2H, m), 3.32-3.42 (4H, m), 3.84 (2H, d), 4.10-4.20 (1H, m), 7.73 (1H, t), 7.91 (1H, d) ), 8.12 (1H, d), 8.16 (1H, d), 8.21 (1H, s), 8.99 (1H, t)

Example  15:

N-{[1- (2- (4- Benzylpiperazine -1-yl) ethyl)- Pyrrolidine -(3R) -day- Carbamoyl ] - methyl } -3- Trifluoromethylbenzamide

N-{[1- (2- (piperazin-1-yl) ethyl) -pyrrolidin- (3R) -yl-carbamoyl] -methyl} -3-trifluoromethylbenz described in Preparation Example 5 0.042 g (75) of the target compound as a yellow solid in the same manner as in Example 7 using 50 mg (0.11 mmol) of amide hydrochloride, 60 mg (0.43 mmol, 4.0 eq.) Of potassium carbonate and 14 mg (0.11 mmol, 1.0 eq.) Of benzyl chloride %) Was obtained.

¹ H NMR (400 MHz, CDCl ₃ ) 1.63-1.79 (1H, m), 2.26-2.39 (2H, m), 2.40-2.57 (10H, m), 2.61-2.77 (3H, m), 2.91 (1H, d ), 3.08-3.14 (1H, m), 3.51 (2H, s), 4.08-4.17 (2H, m), 4.50-4.58 (1H, m), 7.23-7.35 (6H, m), 7.39 (1H, d ), 7.58 (1H, t), 7.77 (1H, d), 8.03 (1H, d), 8.13 (1H, s)

Example  16:

N-{[1- (1- (4- Benzoylpiperazine -1-yl) propan-2-yl)- Pyrrolidine -(3R) -day- Carbamoyl ] - methyl } Refluoromethylbenzami De

100 mg (0.32 mmol) of N- (pyrrolidin- (3R) -yl-carbamoylmethyl) -3-trifluoromethylbenzamide described in Preparation Example 3 and 1- (4-benzoyl as described in Preparation Example 10 80 mg (0.32 mmol, 1.0 eq.) Of piperazin-1-yl) propan-2-one was dissolved in 10 ml of dichloroethane. 200 mg (0.95 mmol, 3.0 eq.) Of sodium triacetoxyborohydride was added thereto, stirred at room temperature for 5 hours, and then 50 ml of saturated aqueous sodium bicarbonate solution was added and extracted with 30 ml of ethyl acetate. The organic layer was separated, dried over anhydrous magnesium sulfate, and purified by chromatography on silica gel (mobile phase: dichloromethane / methanol = 25: 1) to obtain 120 mg (70%) of the title compound as a pale yellow solid.

¹ H NMR (400 MHz, DMSO-d ₆ ) 1.00-1.09 (3H, m), 1.48-1.60 (1H, m), 1.96-2.10 (1H, m), 2.12-2.23 (1H, m), 2.24-2.55 (10H, m), 2.65-2.81 (2H, m), 3.53-3.66 (2H, m), 3.87 (2H, d), 4.07-4.17 (1H, m), 7.34-7.38 (2H, m), 7.42 -7.48 (3H, m), 7.74 (1H, t), 7.92 (1H, d), 8.03-8.12 (1H, m), 8.17 (1H, d), 8.22 (1H, s), 8.93-9.03 (1H , m)

MS (M + 1) ⁺ 546.4

Example  17:

N-{[1- (3- (4- Benzoylpiperazine -1-yl) butan-2-yl)- Pyrrolidine -(3R) -day- Carbamoyl ] - methyl } -3-trifluoromethylbenzamide

50 mg (0.19 mmol) of 3- (4-benzoylpiperazin-1-yl) butan-2-one as described in Preparation Example 11 and N- (pyrrolidine- (3R) -yl-carba as described in Preparation Example 3 120 mg (0.38 mmol, 2.0 eq.) Of moylmethyl) -3-trifluoromethylbenzamide were dissolved in 5 ml of dichloroethane. 200 mg (0.96 mmol, 5.0 eq.) Of sodium triacetoxyborohydride was added thereto, stirred at room temperature for 24 hours, and then 25 ml of saturated aqueous sodium bicarbonate solution was added and extracted with 15 ml of ethyl acetate. The organic layer was separated, dried over anhydrous magnesium sulfate, and purified by chromatography on silica gel (mobile phase: dichloromethane / methanol = 25: 1) to obtain 40 mg (37%) of the title compound as a pale yellow solid.

¹ H NMR (400 MHz, DMSO-d ₆ ) 0.92 (3H, d), 0.96 (3H, d), 1.48-1.62 (1H, m), 1.95-2.08 (1H, m), 2.31-2.48 (9H, m ), 2.53-2.81 (3H, m), 3.48-3.67 (2H, m), 3.87 (2H, d), 4.08-4.17 (1H, m), 7.34-7.39 (2H, m), 7.43-7.48 (3H , m), 7.75 (1H, t), 7.93 (1H, d), 7.98 (1H, dd), 8.18 (1H, d), 8.22 (1H, s), 9.00 (1H, t)

MS (M + 1) ⁺ 560.6

Example  18:

N-{[1- (2- (4- Benzoylpiperazine -1-yl) propan-1-yl)- Pyrrolidine -(3R) -day- Carbamoyl ] - methyl } Refluoromethylbenzami De

400 mg (1.07 mmol) of N-{[1- (2-hydroxypropyl) -pyrrolidin- (3R) -yl-carbamoyl] -methyl} -3-trifluoromethylbenzamide described in Preparation Example 6 And 0.18 ml (1.29 mmol, 1.2 eq.) Of triethylamine were dissolved in 20 ml of dichloromethane and cooled to 3 ° C. under argon gas. 135 mg (1.18 mmol, 1.1 eq.) Of methanesulfonyl chloride were slowly added to the reaction solution, followed by stirring at the same temperature for 30 minutes. 20 ml of purified water was added, the organic layer was separated, and concentrated under reduced pressure. 10 ml of acetonitrile was added to the residue to dissolve it, and then 444 mg (3.21 mmol) of potassium carbonate and 202 mg (1.06 mmol) of 1-benzoylpiperazine were added thereto. After stirring for 2 hours at room temperature, 15 ml of saturated aqueous sodium chloride solution was added to the reaction solution, and extracted twice with 15 ml of ethyl acetate. The organic layers were combined, dried over anhydrous magnesium sulfate, and then concentrated under reduced pressure. The resulting residue was purified by chromatography on silica gel (mobile phase: dichloromethane / methanol = 5: 1 and 1: 1) to give 30 mg (51%) of the title compound as a pale yellow solid.

¹ H NMR (400 MHz, DMSO-d ₆ ) 1.05 (3H, s), 1.55-1.65 (1H, m), 2.00-2.10 (1H, m), 2.15-2.25 (1H, m), 2.28-2.55 (9H , m), 2.70-2.85 (2H, m), 3.17 (1H, d), 3.54-3.65 (2H, m), 3.87 (2H, d), 4.10-4.20 (1H, m), 7.35-7.40 (2H , m), 7.40-7.47 (3H, m), 7.74 (1H, t), 7.92 (1H, d), 8.14 (1H, s), 8.18 (1H, d), 8.23 (1H, s), 9.01 ( 1H, t)

MS (M) ⁺ 545.6

Example  19:

N-{[1- (1- (4- Benzoylpiperazine -1-yl) butan-2-yl)- Pyrrolidine -(3R) -day- Carbamoyl ] - methyl } -3-trifluoromethylbenzamide

100 mg (0.38 mmol) of 1- (4-benzoylpiperazin-1-yl) butan-2-one and N- (pyrrolidin- (3R) -yl-carbamoylmethyl) -3-described in Preparation Example 3 360 mg (1.15 mmol, 3.0 eq.) Of trifluoromethylbenzamide was dissolved in 15 ml of dichloroethane. 490 mg (2.31 mmol, 6.0 eq.) Of sodium triacetoxyborohydride was added thereto, stirred at room temperature for 3 hours, and then 50 ml of saturated aqueous sodium bicarbonate solution was added and extracted with 50 ml of ethyl acetate. The organic layer was separated, dried over anhydrous magnesium sulfate, and purified by chromatography on silica gel (mobile phase: dichloromethane / methanol = 25: 1) to obtain 150 mg (70%) of the title compound as a pale yellow solid.

¹ H NMR (400 MHz, DMSO-d ₆ ) 0.84 (3H, t), 1.42-1.61 (3H, m), 1.98-2.08 (1H, m), 2.22-2.29 (1H, m), 2.30-2.52 (10H , m), 2.63-2.83 (2H, m), 3.53-3.67 (2H, m), 3.87 (2H, d), 4.08-4.19 (1H, m), 7.34-7.40 (2H, m), 7.42-7.48 (3H, m), 7.74 (1H, t), 7.92 (1H, d), 8.02 (1H, d), 8.18 (1H, d), 8.22 (1H, s), 8.99 (1H, t)

MS (M + 1) ⁺ 560.7

Example  20:

N-{[1- (2- (4- Benzoylpiperazine -1-yl) butyl)- Pyrrolidine -(3R) -day- Carbamoyl ] - methyl } -3- Trifluoromethylbenzamide

415 mg (1.07 mmol) of N-{[1- (2-hydroxybutyl) -pyrrolidin- (3R) -yl-carbamoyl] -methyl} -3-trifluoromethylbenzamide as described in Preparation Example 7 To give 21 mg (35%) of the title compound as a yellow solid in the same manner as in Example 18.

¹ H NMR (400 MHz, DMSO-d ₆ ) 0.84 (3H, t), 1.40-1.52 (2H, m), 1.98-2.08 (1H, m), 2.22-2.30 (1H, m), 2.30-2.58 (10H , m), 2.65-2.86 (3H, m), 3.50-3.66 (2H, m), 3.87 (2H, d), 4.10-4.18 (1H, m), 7.34-7.48 (5H, m), 7.74 (1H) , t), 7.92 (1H, d), 8.05 (1H, d), 8.18 (1H, d), 8.23 (1H, s), 9.90 (1H, t)

MS (M + 1) ⁺ 560.8

Example  21:

N-{[1- (2- (4- Benzoylpiperazine Yl) -2- Methyl propyl ) - Pyrrolidine -(3R) -day- Carbamoyl ] - methyl } Refluoromethylbenzami De

415 mg of N-{[1- (2-hydroxy-2-methylpropyl) -pyrrolidin- (3R) -yl-carbamoyl] -methyl} -3-trifluoromethylbenzamide described in Preparation Example 8 (1.07 mmol) of 27 mg (45%) of the title compound as a pale yellow solid were obtained in the same manner as in Example 18.

¹ H NMR (400 MHz, DMSO-d ₆ ) 0.80-0.90 (1H, m), 0.92-1.00 (6H, m), 1.15-1.25 (1H, m), 1.51-1.60 (1H, m), 1.92-2.08 (1H, m), 2.35-2.62 (6H, m), 2.65-2.88 (2H, m), 3.20-3.32 (2H, m), 3.50-3.62 (2H, m), 3.85-3.90 (2H, m) , 4.00-4.18 (1H, m), 7.35-7.40 (2H, m), 7.40-7.48 (3H, m), 7.74 (1H, t), 7.93 (1H, d), 8.02 (1H, d), 8.18 (1H, d), 8.23 (1H, s), 8.98-9.03 (1H, m)

Example  22:

N-{[1- (1- (4- Benzoylpiperazine Yl) -2- Methyl propane -2 days)- Pyrrolidine -(3R) -day- Carbamoyl ] -Methyl} -3- Trifluoromethylbenzamide

60 mg (0.23 mmol) and 28 mg (0.25 mmol, 1.1 eq.) Of (4- (2-hydroxy-2-methylpropyl) piperazin-1-yl) phenylmethanone described in Preparation Example 9 were diluted with It was dissolved in 5 ml of methane and cooled to 3 ° C. under argon gas. 29 mg (0.25 mmol, 1.1 eq.) Of methanesulfonyl chloride was slowly added to the reaction solution, followed by stirring at the same temperature for 1.5 hours. 5 ml of purified water was added, the organic layer was separated, and concentrated under reduced pressure. 5 ml of acetonitrile was added to the obtained residue to dissolve it, and then 76 mg (0.55 mmol) of potassium carbonate and N- (pyrrolidin- (3R) -yl-carbamoylmethyl) -3-trifluoro described in Preparation Example 3 58 mg (0.18 mmol) of chloromethylbenzamide was added thereto. After stirring for 3 hours at room temperature, 10 ml of saturated aqueous sodium chloride solution was added to the reaction solution, and extracted twice with 10 ml of ethyl acetate. The organic layers were combined, dried over anhydrous magnesium sulfate, and then concentrated under reduced pressure. The residue obtained was purified by chromatography on silica gel (mobile phase: dichloromethane / methanol = 5: 1 and 1: 1) to give 57 mg (45%) of the title compound as a pale yellow solid.

¹ H NMR (400 MHz, DMSO-d ₆ ) 0.92-1.00 (6H, m), 1.50-1.60 (1H, m), 1.92-2.08 (1H, m), 2.30-2.60 (8H, m), 2.64-2.78 (1H, m), 2.80-2.88 (1H, m), 3.30-3.62 (4H, m), 3.83-3.90 (2H, m), 4.05-4.18 (1H, m), 7.35-7.40 (2H, m) , 7.40-7.47 (3H, m), 7.74 (1H, t), 7.92 (1H, d), 8.04 (1H, d), 8.18 (1H, d), 8.23 (1H, s), 9.06-9.13 (1H , m)

Example  23:

N-{[1- (3- (4- (3- Chlorophenyl ) Piperazin-1-yl) butan-2-yl)- Pyrrolidine -(3R) -day- Carbamoyl ] -Methyl} -3- Trifluoromethylbenzamide

100 mg (0.37 mmol) of 3- (4- (3-chlorophenyl) piperazin-1-yl) butan-2-one and N- (pyrrolidin- (3R) -yl-carbamoyl described in Preparation Example 3 240 mg (0.75 mmol, 2.0 eq.) Of methyl) -3-trifluoromethylbenzamide were dissolved in 10 ml of dichloroethane. 480 mg (2.25 mmol, 6.0 eq.) Of sodium triacetoxyborohydride was added thereto, stirred at room temperature for 7 hours, and then 30 ml of saturated aqueous sodium bicarbonate solution was added and extracted with 20 ml of ethyl acetate. The organic layer was separated, dried over anhydrous magnesium sulfate, and purified by chromatography on silica gel (mobile phase: dichloromethane / methanol = 25: 1) to obtain 140 mg (66%) of the title compound as a white solid.

¹ H NMR (400 MHz, DMSO-d ₆ ) 0.91-1.05 (6H, m), 1.48-1.64 (1H, m), 1.97-2.19 (1H, m), 2.32-2.41 (1H, m), 2.53-2.85 (9H, m), 3.05-3.20 (4H, m), 3.87 (2H, d), 4.07-4.20 (1H, m), 6.76 (1H, d), 6.87 (1H, d), 6.91 (1H, s ), 7.19 (1H, t), 7.74 (1H, t), 7.93 (1H, d), 7.97-8.03 (1H, m), 8.18 (1H, d), 8.23 (1H, s), 9.02 (1H, s)

Example  24:

N-{[1- (1- (4- Phenylpiperazine -1-yl) propan-2-yl)- Pyrrolidine -(3R) -day- Carbamoyl ] - methyl } -3-trifluoromethylbenzamide

100 mg (0.46 mmol) of 1- (4-phenylpiperazin-1-yl) propan-2-one, N- (pyrrolidin- (3R) -yl-carbamoylmethyl) -3- as described in Preparation Example 3 130 mg (55) of the target compound as a white solid in the same manner as in Example 23 using 290 mg (0.92 mmol, 2.0 eq.) Of trifluoromethylbenzamide and 580 mg (2.75 mmol, 6.0 eq.) Of sodium triacetoxyborohydride. %) Was obtained.

¹ H NMR (400 MHz, DMSO-d ₆ ) 1.06 (3H, t), 1.53-1.62 (1H, m), 2.00-2.09 (1H, m), 2.17-2.25 (1H, m), 2.41-2.62 (8H , m), 2.70-2.83 (2H, m), 3.07-3.15 (4H, m), 3.87 (2H, d), 4.10-4.20 (1H, m), 6.77 (1H, t), 6.91 (2H, d ), 7.20 (2H, t), 7.74 (1H, t), 7.93 (1H, d), 8.09 (1H, d), 8.18 (1H, d), 8.23 (1H, s), 8.98 (1H, t)

Example  25:

N-{[1- (1- (4- Cyclohexylpiperazine -1-yl) propan-2-yl)- Pyrrolidine (3R) -day- Carbamoyl ] - methyl } -3- Trifluoromethylbenzamide

100 mg (0.45 mmol) of 1- (4-cyclohexylpiperazin-1-yl) propan-2-one, N- (pyrrolidin- (3R) -yl-carbamoylmethyl)-described in Preparation Example 3 100 mg of the desired compound as a white solid in the same manner as in Example 23 using 280 mg (0.89 mmol, 2.0 eq.) Of 3-trifluoromethylbenzamide, 570 mg (2.67 mmol, 6.0 eq.) Of sodium triacetoxyborohydride (43%) was obtained.

¹ H NMR (400 MHz, DMSO-d ₆ ) 1.00 (3H, t), 1.03-1.21 (6H, m), 1.50-1.61 (2H, m), 1.68-1.79 (4H, m), 2.01-2.05 (1H , m), 2.06-2.12 (1H, m), 2.13-2.19 (1H, m), 2.30-2.41 (4H, m), 2.42-2.51 (7H, m), 2.65-2.82 (2H, m), 3.87 (2H, d), 4.08-4.17 (1H, m), 7.74 (1H, t), 7.93 (1H, d), 8.06 (1H, d), 8.18 (1H, d), 8.23 (1H, s), 8.97 (1H, t)

Example  26:

N-{[1- (2- (4- (2- Hydroxybutyl ) Piperazin-1-yl) ethyl)- Pyrrolidine -(3R) -day- Carbamoyl ] - methyl } -3- Trifluoromethylbenzamide

N-{[1- (2- (piperazin-1-yl) ethyl) -pyrrolidin- (3R) -yl-carbamoyl] -methyl} -3-trifluoromethylbenz described in Preparation Example 5 100 mg (0.19 mmol) of amide hydrochloride and 129 mg (0.93 mmol, 5.0 eq.) Of potassium carbonate were suspended in 3 ml of acetonitrile. 13 mg (0.19 mmol) of 2-ethyloxirane was added thereto, and the mixture was stirred and refluxed at 80 ° C. overnight, followed by cooling to room temperature. 15 ml of purified water was added, followed by extraction three times with 15 ml of ethyl acetate. The organic layers were combined, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. The obtained residue was purified by chromatography on silica gel (mobile phase: dichloromethane / methanol = 1: 1) to give 73 mg (78%) of the title compound as a white solid.

MS (M + 1) ⁺ 500.6

Example  27:

N-{[1- (2- (4- (2-hydroxy-2- Methyl propyl ) Piperazin-1-yl) ethyl)- Pyrrolidine -(3R) -day- Carbamoyl ] - methyl } -3- Trifluoromethylbenzamide

N-{[1- (2- (piperazin-1-yl) ethyl) -pyrrolidin- (3R) -yl-carbamoyl] -methyl} -3-trifluoromethylbenz described in Preparation Example 5 62 mg (67) of the target compound as a white solid in the same manner as in Example 26 using 100 mg (0.19 mmol) of amide hydrochloride, 129 mg (0.93 mmol, 5.0 eq.) Of potassium carbonate and 13 mg (0.19 mmol) of 2,2-dimethyloxirane. %) Was obtained.

MS (M + 1) ⁺ 500.6

Example  28:

N-{[1- (2- (4- (2-hydroxy-2- Phenylethyl ) Piperazin-1-yl) ethyl)- Pyrrolidine -(3R) -day- Carbamoyl ] - methyl } -3- Trifluoromethylbenzamide

N-{[1- (2- (piperazin-1-yl) ethyl) -pyrrolidin- (3R) -yl-carbamoyl] -methyl} -3-trifluoromethylbenz described in Preparation Example 5 63 mg (62%) of the target compound as a yellow solid in the same manner as in Example 26, using 100 mg (0.19 mmol) of amide hydrochloride, 129 mg (0.93 mmol, 5.0 eq.) Of potassium carbonate and 22 mg (0.19 mmol) of 2-phenyloxirane. Obtained.

MS (M + 1) ⁺ 548.3

Example  29:

N-{[1- (2- (4- (2- (4- Chlorophenyl )-2- Hydroxyethyl ) Piperazin-1-yl) ethyl)- Pyrrolidine -(3R) -day- Carbamoyl ] - methyl } -3- Trifluoromethylbenzamide

N-{[1- (2- (piperazin-1-yl) ethyl) -pyrrolidin- (3R) -yl-carbamoyl] -methyl} -3-trifluoromethylbenz described in Preparation Example 5 Target compound as a white solid in the same manner as in Example 26, using 100 mg (0.19 mmol) of amide hydrochloride, 129 mg (0.93 mmol, 5.0 eq.) Of potassium carbonate and 23 mg (0.19 mmol) of 2- (4-chlorophenyl) oxirane 77 mg (71%) were obtained.

MS (M) ⁺ 582.3

Example  30:

N-{[1- (2- (4- Benzoylpiperazine Yl) -2- Oxoethyl ) - Pyrrolidine -(3R) -day- Carbamoyl ] - methyl } Refluoromethylbenzami De

N-{[1- (2- (piperazin-1-yl) -2-oxo-ethyl) -pyrrolidin- (3R) -yl-carbamoyl] -methyl} -3- as described in Preparation Example 14 100 mg (0.21 mmol) of trifluoromethylbenzamide hydrochloride and 120 mg (0.83 mmol, 4.0 eq.) Of potassium carbonate were suspended in 10 ml of acetonitrile at room temperature, followed by 30 mg (0.21 mmol, 1.0 eq.) Of benzoyl chloride in 1 ml of acetonitrile. Diluted in and added slowly. The mixture was heated to reflux, stirred for 1 hour, and then concentrated under reduced pressure. 10 ml of purified water was added to the obtained residue, followed by extraction twice with 10 ml of ethyl acetate. The organic layers were combined, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. The obtained residue was purified by chromatography on silica gel (mobile phase: dichloromethane / methanol = 25: 1) to give 70 mg (61%) of the target compound as a white solid.

¹ H NMR (400 MHz, DMSO-d ₆ ) 1.53-1.68 (1H, m), 2.01-2.11 (1H, m), 2.37-2.48 (2H, m), 2.66 (1H, d), 2.73 (1H, t ), 3.42-3.68 (10H, m), 3.84 (2H, d), 4.10-4.21 (1H, m), 7.38-7.41 (2H, m), 7.42-7.48 (3H, m), 7.72 (1H, t) ), 7.91 (1H, t), 8.12 (1H, d), 8.16 (1H, d), 8.21 (1H, s), 8.99 (1H, t)

MS (M + 1) ⁺ 546.2

Example  31:

N-{[1- (2- (4- (4- Methylbenzoyl Piperazin-1-yl) -2- Oxoethyl ) - Pyrrolidine -(3R) -day- Carbamoyl ] -Methyl} -3- Trifluoromethylbenzamide

N-{[1- (2- (piperazin-1-yl) -2-oxo-ethyl) -pyrrolidin- (3R) -yl-carbamoyl] -methyl} -3- as described in Preparation Example 14 White in the same manner as in Example 30, using 50 mg (0.10 mmol) of trifluoromethylbenzamide hydrochloride, 60 mg (0.42 mmol, 4.0 eq.) Of potassium carbonate, 20 mg (0.10 mmol, 1.0 eq.) Of 4-methylbenzoyl chloride. 20 mg (34%) of the title compound as a solid were obtained.

¹ H NMR (400 MHz, DMSO-d ₆ ) 1.18 (3H, t), 1.53-1.66 (1H, m), 2.01-2.13 (1H, m), 2.38-2.47 (2H, m), 2.58-2.78 (4H , m), 3.38-3.47 (4H, m), 3.48-3.62 (4H, m), 3.84 (2H, d), 4.09-4.21 (1H, m), 7.26 (2H, d), 7.32 (2H, d) ), 7.72 (1H, t), 7.90 (1H, d), 8.11 (1H, d), 8.13 (1H, d), 8.21 (1H, s), 8.99 (1H, t)

Example  32:

N-{[1- (2- (4- (4- Ethylbenzoyl Piperazin-1-yl) -2- Oxoethyl ) - Pyrrolidine -(3R) -day- Carbamoyl ] -Methyl} -3- Trifluoromethylbenzamide

N-{[1- (2- (piperazin-1-yl) -2-oxo-ethyl) -pyrrolidin- (3R) -yl-carbamoyl] -methyl} -3- as described in Preparation Example 14 White in the same manner as in Example 30, using 50 mg (0.10 mmol) of trifluoromethylbenzamide hydrochloride, 60 mg (0.42 mmol, 4.0 eq.) Of potassium carbonate, 20 mg (0.10 mmol, 1.0 eq.) Of 4-ethylbenzoyl chloride. 20 mg (33%) of the title compound as a solid were obtained.

¹ H NMR (400 MHz, DMSO-d ₆ ) 1.18 (3H, t), 1.53-1.65 (1H, m), 2.01-2.12 (1H, m), 2.37-2.48 (2H, m), 2.57-2.78 (4H , m), 3.25-3.35 (2H, m), 3.38-3.47 (4H, m), 3.48-3.65 (4H, m), 3.84 (2H, d), 4.11-4.22 (1H, m), 7.26 (2H) , d), 7.32 (2H, d), 7.72 (1H, t), 7.90 (1H, d), 8.11 (1H, d), 8.16 (1H, d), 8.21 (1H, s), 8.99 (1H, t)

Example  33:

N-{[1- (2- (4- (3- Methylbenzoyl Piperazin-1-yl) -2- Oxoethyl ) - Pyrrolidine -(3R) -day- Carbamoyl ] -Methyl} -3- Trifluoromethylbenzamide

N-{[1- (2- (piperazin-1-yl) -2-oxo-ethyl) -pyrrolidin- (3R) -yl-carbamoyl] -methyl} -3- as described in Preparation Example 14 White in the same manner as in Example 30, using 50 mg (0.10 mmol) of trifluoromethylbenzamide hydrochloride, 60 mg (0.42 mmol, 4.0 eq.) Of potassium carbonate, 20 mg (0.10 mmol, 1.0 eq.) Of 3-methylbenzoyl chloride 25 mg (43%) of the title compound as a solid were obtained.

¹ H NMR (400 MHz, DMSO-d ₆ ) 1.62-1.75 (1H, m), 2.05-2.18 (1H, m), 2.34 (3H, s), 2.54-2.64 (2H, m), 2.68-2.97 (4H , m), 3.42-3.80 (8H, m), 3.87 (2H, d), 4.16-4.28 (1H, m), 7.20 (1H, d), 7.23 (1H, s), 7.27 (1H, d), 7.34 (1H, t), 7.74 (1H, t), 7.92 (1H, d), 8.18 (2H, d), 8.23 (1H, s), 9.04 (1H, t)

Example  34:

N-{[1- (2- (4- (2- Methylbenzoyl Piperazin-1-yl) -2- Oxoethyl ) - Pyrrolidine -(3R) -day- Carbamoyl ] -Methyl} -3- Trifluoromethylbenzamide

N-{[1- (2- (piperazin-1-yl) -2-oxo-ethyl) -pyrrolidin- (3R) -yl-carbamoyl] -methyl} -3- as described in Preparation Example 14 White in the same manner as in Example 30, using 50 mg (0.10 mmol) of trifluoromethylbenzamide hydrochloride, 60 mg (0.42 mmol, 4.0 eq.) Of potassium carbonate, 20 mg (0.10 mmol, 1.0 eq.) Of 2-methylbenzoyl chloride. 20 mg (34%) of the title compound as a solid were obtained.

¹ H NMR (400 MHz, DMSO-d ₆ ) 1.62-1.82 (1H, m), 2.05-2.20 (1H, m), 2.22 (3H, s), 2.70-3.00 (6H, m), 3.17-3.22 (3H , m), 3.52-3.78 (5H, m), 3.87-3.93 (2H, m), 4.16-4.32 (1H, m), 7.20-7.38 (4H, m), 7.74 (1H, t), 7.93 (1H) , t), 8.18 (1H, d), 8.23 (1H, s), 8.24-8.32 (1H, m), 9.02-9.09 (1H, m)

Example  35:

N-{[1- (2- (4- Phenylpiperazine Yl) -2- Oxoethyl ) - Pyrrolidine -(3R) -day- Carbamoyl ] - methyl } -3-trifluoromethylbenzamide

50 mg (0.16 mmol) of N- (pyrrolidin- (3R) -yl-carbamoylmethyl) -3-trifluoromethylbenzamide and 90 mg (0.63 mmol, 4.0 eq.) Of potassium carbonate described in Preparation Example 3, 40 mg (49%) of the target compound as a white solid were obtained by the same method as Example 30, using 40 mg (0.16 mmol, 1.0 eq.) Of 2-chloro-1- (4-phenylpiperazin-1-yl) ethanone. It was.

¹ H NMR (400 MHz, DMSO-d ₆ ) 1.57-1.68 (1H, m), 2.01-2.11 (1H, m), 2.37-2.48 (2H, m), 2.62-2.71 (1H, m), 2.78 (1H , t), 3.01-3.16 (4H, m), 3.27-3.32 (2H, m), 3.46-3.56 (2H, m), 3.58-3.67 (2H, m), 3.85 (2H, d), 4.11-4.22 (1H, m), 6.79 (1H, t), 6.93 (2H, d), 7.21 (2H, t), 7.72 (1H, t), 7.91 (1H, d), 8.16-8.18 (2H, m), 8.21 (1H, s), 8.98-9.03 (1H, m)

Example  36:

N-{[1- (2- (4- ( Benzoxicarbonyl Piperazin-1-yl) -2- Oxoethyl ) - Pyrrolidine -(3R) -day- Carbamoyl ] - methyl } -3- Trifluoromethylbenzamide

50 mg (0.16 mmol) of N- (pyrrolidin- (3R) -yl-carbamoylmethyl) -3-trifluoromethylbenzamide and 90 mg (0.63 mmol, 4.0 eq.) Of potassium carbonate described in Preparation Example 3, 40 mg (44%) of the target compound as a white solid were obtained by the same method as Example 30 using 30 mg (0.16 mmol, 1.0 eq.) Of benzyl 4- (2-chloroacetyl) piperazine-1-carboxylate.

¹ H NMR (400 MHz, DMSO-d ₆ ) 1.54-1.65 (1H, m), 2.01-2.10 (1H, m), 2.38-2.43 (1H, m), 2.44-2.47 (1H, m), 2.62-2.70 (1H, m), 2.73 (1H, t), 3.26-3.34 (4H, m), 3.38-3.45 (4H, m), 3.46-3.53 (2H, m), 3.85 (2H, d), 4.12-4.18 (1H, m), 5.08 (2H, s), 7.28-7.37 (5H, m), 7.72 (1H, t), 7.90 (1H, d), 8.13 (1H, d), 8.16 (1H, d), 8.22 (1H, s), 9.01 (1H, t)

Example  37:

N-{[1- (2- (4- Benzoyl -2,5-dimethylpiperazin-1-yl) ethyl)- Pyrrolidine -(3R) -day- Carbamoyl ] - methyl } -3- Trifluoromethylbenzamide

200 mg (0.56 mmol) of N-{[1- (2-hydroxyethyl) -pyrrolidine- (3R) -yl-carbamoyl] -methyl} -3-trifluoromethylbenzamide and 68 mg of triethylamine 0.69 mmol, 1.2 eq.) Was dissolved in 10 ml of dichloromethane and cooled to 3 ° C. under argon gas. 70 mg (0.61 mmol, 1.1 eq.) Of methanesulfonyl chloride was slowly added to the reaction solution, followed by stirring at the same temperature for 1 hour. 10 ml of purified water was added, the organic layer was separated, and concentrated under reduced pressure. 10 ml of acetonitrile was added to the residue to dissolve it, and then 247 mg (1.78 mmol) of potassium carbonate and 114 mg (0.45 mmol) of 1-benzoyl-2,5-dimethylpiperazine hydrochloride were added thereto. After stirring for 4 hours at room temperature, 20 ml of saturated aqueous sodium chloride solution was added to the reaction solution, and extracted twice with 20 ml of ethyl acetate. The organic layers were combined, dried over anhydrous magnesium sulfate, and then concentrated under reduced pressure. The residue obtained was purified by chromatography on silica gel (mobile phase: dichloromethane / methanol = 5: 1 and 1: 1) to give 159 mg (51%) of the title compound as a pale yellow solid.

MS (M + 1) ⁺ 560.4

Example  38:

N-{[1- (2- (4- (2- Chlorobenzoyl ) Piperazin-1-yl) ethyl)- Pyrrolidine -(3R) -day- Carbamoyl ] - methyl } -3- Trifluoromethylbenzamide

N-{[1- (2- (piperazin-1-yl) ethyl) -pyrrolidin- (3R) -yl-carbamoyl] -methyl} -3-trifluoromethylbenz described in Preparation Example 5 92 mg of the target compound as a white white solid in the same manner as in Example 7, using 200 mg (0.40 mmol) of amide hydrochloride, 221 mg (1.60 mmol, 4.0 eq.) Of potassium carbonate, 77 mg (0.44 mmol, 1.1 eq.) Of 2-chlorobenzoyl chloride (41%) was obtained.

¹ H NMR (400 MHz, DMSO-d ₆ ) 1.50-1.60 (1H, m), 2.00-2.11 (1H, m), 2.28-2.39 (4H, m), 2.39-2.46 (6H, m), 2.59-2.68 (2H, m), 3.08-3.13 (2H, m), 3.57-3.68 (2H, m), 3.85 (2H, d), 4.10-4.20 (1H, m), 7.35 (1H, d), 7.39-7.46 (2H, m), 7.53 (1H, d), 7.74 (1H, t), 7.92 (1H, d), 8.12 (1H, d), 8.18 (1H, d), 8.22 (1H, s), 8.99 ( 1H, t)

Example  39:

N-{[1- (2- (4- (2- Fluorobenzoyl ) Piperazin-1-yl) ethyl)- Pyrrolidine -(3R) -day- Carbamoyl ] - methyl } -3- Trifluoromethylbenzamide

N-{[1- (2- (piperazin-1-yl) ethyl) -pyrrolidin- (3R) -yl-carbamoyl] -methyl} -3-trifluoromethylbenz described in Preparation Example 5 Target compound as an off-white solid in the same manner as Example 4 using 200 mg (0.40 mmol) of amide hydrochloride, 277 mg (2.00 mmol, 5.0 eq.) Of potassium carbonate, 76 mg (0.48 mmol, 1.2 eq.) Of 2-fluorobenzoyl chloride 143 mg (65%) were obtained.

¹ H NMR (400 MHz, DMSO-d ₆ ) 1.50-1.65 (1H, m), 2.00-2.14 (1H, m), 2.30-2.50 (6H, m), 2.60-2.72 (2H, m), 3.10-3.20 (4H, m), 3.60 (2H, t), 3.88 (2H, t), 4.05-4.20 (3H, m), 7.25-7.40 (3H, m), 7.45-7.52 (1H, m), 7.72 (1H , t), 7.99 (1H, d), 8.10 (1H, d), 8.18 (1H, d), 8.22 (1H, s), 8.99 (1H, t)

Example  40:

N-{[1- (2- (4- (2,5- Difluorobenzoyl ) Piperazin-1-yl) ethyl)- Pyrrolidine -(3R) -day- Carbamoyl ] - methyl } -3- Trifluoromethylbenzamide

N-{[1- (2- (piperazin-1-yl) ethyl) -pyrrolidin- (3R) -yl-carbamoyl] -methyl} -3-trifluoromethylbenz described in Preparation Example 5 White white solid in the same manner as in Example 4 using 200 mg (0.40 mmol) of amide hydrochloride, 277 mg (2.00 mmol, 5.0 eq.) Of potassium carbonate, 85 mg (0.48 mmol, 1.2 eq.) Of 2,5-difluorobenzoyl chloride 120 mg (53%) of the target compound were obtained.

¹ H NMR (400 MHz, DMSO-d ₆ ) 1.50-1.62 (1H, m), 2.00-2.15 (1H, m), 2.30-2.48 (8H, m), 2.55-2.70 (2H, m), 3.12-3.20 (4H, m), 3.60 (2H, s), 3.88 (2H, d), 4.10-4.20 (1H, m), 7.30-7.40 (3H, m), 7.75 (1H, t), 7.95 (1H, d ), 8.10 (1H, d), 8.18 (1H, d), 8.20 (1H, s), 8.98 (1H, t)

Example  41: Inducing Asthma and Drug Administration in Laboratory Animals

In the present invention, in order to verify the therapeutic effect of the compound having the structure of Formula 1 for asthma, an asthma mouse animal model induced by egg white albumin (ovalbumin, OVA) was used. In addition, in order to compare the drug composition and the composition of the compound of Formula 1 shown in the present invention was used as a control compound budesonide (budesonide). Drug administration of each of the compositions and controls presented in the present invention was applied to the local airway topical inhalation regimen by means of an ultrasound nebulizer.

Specifically, BALB / c mice (female, 5 weeks old, average 20 g, multibody science) were purified for 7 days in sterile animals and then used for testing. 100 μl of phosphate buffer solution (PBS, pH 7.4) suspended in egg white albumin (0.8 mg, Sigma, USA), aluminum hydroxide (160 mg) and magnesium hydroxide (160 mg) in 6-week-old BALB / c mice 2 Airway sensitization was performed by intraperitoneal infusion twice at weekly intervals. The substance used as the test drug was N-{[1- (2- (4-benzoylpiperazin-1-yl) ethyl) -pyrrolidin- (3R) -yl-carbamoyl] among the compounds presented herein. -Methyl} -3-trifluoromethylbenzamide (Example 6) and budesonide as a control drug.

Each test group was configured and performed as shown in Table 1 below.

Test History by Animal Group Test group 2-week prayer sensitization
(1,14 days
Dosing, 2 times) Additional antigens
(21,22,23 after sensitization
First dose, 3 times) drug injection
(20,21,22,23,
Dosing on day 24, 5) Negative Control (Nor) - - - EXAMPLES Single Administration Group (Examples) - - Example Budesonide alone group (BUD) - - Budesonide Asthma-induced (OVA) Egg white albumin Egg white albumin - Treatment group (OVA + Example) Egg white albumin Egg white albumin Example Positive control (OVA + BUD) Egg white albumin Egg white albumin Budesonide

In the method of administration of the drug, the drugs used in the treatment group and the positive control group, respectively, are prepared in a phosphate buffer solution and placed in an ultrasonic nebulizer (ultrasonic nebulizer, Nescosonic UN51, Japan), the nebulizer rate 1.5 ml / min, the nebulization air flow) After saturation for 0.5 min at 17 L / min, the mice were placed in a closed container (3 L volume, hemispherical, made by Olmedikus) and inhaled. The actual dose of drug through inhalation was calculated as mean dose level (D) as follows.

[Equation 1]

D (μg / kg) = C (μg / L) x t (min) x MV (ml / min) / m (g)

C: average spray concentration,

t: daily inhalation time,

MV: average breath volume per minute (m ^0.75 x 2.1),

m: mean mouse weight (20 g)).

At this time, the average spray concentration (C) was calculated as follows.

&Quot; (2) "

C (μg / L) = concentration of solution administered (μg / ml) x spray rate (ml / min) x 0.8 / spray stream (L / min)

The composition of each drug was configured as shown in Table 2 below.

Composition of Drugs for Animal Testing drug Manufacture solution Concentration of solution
(μg / ml) Average dosage
(D, μg / kg, 5 doses based) Example Phosphate buffer solution
(PBS, pH 7.4) 0.53 5.51 Budesonide Phosphate buffer solution
(PBS, pH 7.4) 1.25 13.4

Phosphoric acid buffer solution (1% OVA, 50 ml) suspended in egg white albumin three times 21, 22, and 23 days after sensitization was sprayed for 30 minutes using an ultrasonic nebulizer for asthma-induced, treated and positive controls. Inhaled. In addition, the compound of the example and budesonide were sprayed for 30 minutes using an ultrasonic nebulizer for the single administration group, the treatment group, and the positive control group for 5 minutes on 20, 21, 22, 23, and 24 days after sensitization. In the treatment group and the positive control group, the antigen was first inhaled for 30 minutes, and then 30 minutes later, the drug solution was again inhaled for 30 minutes. On the other hand, all mice were used five mice each.

Example  42: Lung tissue  Sampling and Histopathological Analysis

In order to determine the effect of the composition of the compound of the present invention on bronchial inflammation, the lung tissue of the mouse was observed histopathologically. Specifically, after 72 hours after the last antigen administration, mice treated as in Example 41 were lethal using an excess pentobarbital, and then bronchial incision was performed. Lung tissue was removed, fixed in 10% formaldehyde solution, paraffin-treated into flakes, and tissues were observed by H & E staining (hematoxilin & eosin staining) and PAS staining (periodic acid-Schiff staining). As shown in FIG. 1 (H & E staining) and FIG. 2 (PAS staining), the negative control group (Nor), the Example alone treatment group (Example) and the budesonide alone treatment group (BUD) showed relatively normal findings. In contrast, egg white albumin asthma-induced group (OVA) significantly increased inflammatory cell infiltration around the bronchus and around blood vessels. In addition, in the Example treatment group (OVA + Example) and the budesonide positive control group (OVA + BUD), inflammation around the bronchi and perivascular vessels induced by egg white albumin was significantly reduced, and the example treatment group was the budesonide positive control group. Mucin secretion was reduced to a level similar to.

Example  43: Alveolar lavage, inflammatory cell analysis

In order to determine the effect of the composition of the compounds presented in the present invention on inflammatory cells, eosinophils and other inflammatory cell numbers of bronchoalveolar lavage fluids (BALF) were measured. Specifically, after 72 hours after the last antigen administration, mice treated as in Example 41 were lethal using an excess pentobarbital, and then bronchial incision was performed. Bronchoalveolar lavage fluid was obtained by inhalation three times each of 0.7 ml, 0.7 ml, and 0.6 ml by inserting a catheter into the trachea (total 2 ml). The bronchial alveolar lavage fluid was added to the cell compact obtained by centrifugation (1000 rpm, 8 minutes), and the saline solution was added again to loosen and fixed to the slide. The number of cells except dead cells was counted (hemocytometer) by staining with trypan blue, and the resulting inflammatory cells were separated and analyzed by type (diff-quik staining). As shown in Table 3 and FIG. 3, the egg white albumin asthma-induced group (OVA) was compared to the total control group and eosinophils compared to the negative control group (Nor), the Example alone treatment group (Example) and the budesonide alone treatment group (BUD). (eosinophil) increased significantly. In addition, the eosinophil cell number increased by egg white albumin airway sensitization tended to decrease significantly in the Example treatment group (OVA + Example) and the budesonide positive control group (OVA + BUD). In particular, the Example treatment group reduced the increase in eosinophils more effectively than the budesonide positive control group (#P <0.05 vs. SAL + SAL, * P <0.05 vs. SAL + OVA).

Inflammatory cell analysis results by test group (Unit: 10 ⁴ cells / ml ± SEM) Test group Total cell Macrophage Eosan Neutral sphere Monocyte Nor 9.72 ± 0.96 9.67 ± 0.94 0.05 ± 0.05 0.00 ± 0.00 0.00 ± 0.00 Example 8.61 ± 0.59 8.58 ± 0.59 0.02 ± 0.02 0.00 ± 0.00 0.01 ± 0.00 BUD 6.24 ± 1.00 6.10 ± 0.95 0.54 ± 0.54 0.08 ± 0.08 0.00 ± 0.00 OVA 22.86 ± 1.62 8.05 ± 1.32 10.20 ± 2.06 0.32 ± 0.07 0.13 ± 0.07 OVA + Example 12.6 ± 0.81 5.70 ± 0.92 5.82 ± 0.98 0.10 ± 0.03 0.01 ± 0.01 OVA + BUD 15.5 ± 0.57 6.24 ± 0.56 8.51 ± 0.14 1.37 ± 1.23 0.03 ± 0.02

Example  44: molecular biological analysis

In order to investigate the effect of the composition of the compounds presented in the present invention on Th2 cytokine production which is closely related to the mechanism of asthma development, proteins extracted from biological tissues were analyzed molecularly. Specifically, the protein extracted from the lung tissue is electrophoretically separated on a polyacrylamide SDS gel, and then reacted with an antibody, visualized through a color reagent, and analyzed by interleukin-4 (IL- 4), expression of interleukin-5 (IL-5) and interleukin-13 (IL-13) was quantified. As shown in FIG. 4, no expression of interleukin was observed in the negative control group (Nor), the Example treatment group (Example) and the budesonide treatment group (BUD), and the egg white albumin asthma-induced group (OVA). ) Significantly increased the expression of cytokines. In addition, the Example treatment group (OVA + Example) showed a significant decrease in cytokine expression compared to the asthma-induced group (OVA).

Example  45: airway hypersensitivity alveolar hyperresponsivity , AHR )

In order to determine the effect of the composition of the compound presented in the present invention on airway hypersensitivity, the change in airway obstruction according to the concentration of methacholine (methacholin) was measured using whole body plethysmography. Specifically, the numerical values (enhanced pause, Pehn) reflecting airway obstruction as a mathematical parameter in the whole body plethysmograph box were measured while maintaining the free and conscious movement of the experimental animals without anesthesia. Initial bronchial hypersensitivity was checked with saline, and airway resistance was measured by increasing the concentration of methacholine to saline, 2.5, 5.0, 10.0, and 25.0 mg / ml (saline solution). As shown in Table 4 and FIG. 5, the Pehn value increased by methacholine was significantly reduced in the Example treatment group (OVA + Example) compared to the asthma-induced group (OVA), and also negative control group (Nor) and budeso It was confirmed to be reduced to the same level as the positive positive control (OVA + BUD) (#P <0.05 vs. SAL + SAL, * P <0.05 vs. SAL + OVA).

Airway hypersensitivity measurement results by test group (Unit: Pehn percentage) Test group Saline
(saline) Methacholine Concentration (mg / ml) 2.5 5 10 25 Nor 0.83 ± 0.08 1.46 ± 0.27 1.46 ± 0.13 2.89 ± 0.32 2.90 ± 0.29 Example 0.82 ± 0.05 1.03 ± 0.18 1.78 ± 0.31 2.84 ± 0.32 3.52 ± 0.30 BUD 0.66 ± 0.04 1.39 ± 0.24 2.60 ± 0.49 2.84 ± 0.36 3.33 ± 0.35 OVA 0.71 ± 0.05 1.49 ± 0.32 4.44 ± 1.65 11.11 ± 2.07 16.73 ± 2.80 OVA + Example 0.94 ± 0.03 1.22 ± 0.20 1.77 ± 0.20 2.71 ± 0.32 3.47 ± 0.39 OVA + BUD 0.60 ± 0.05 1.09 ± 0.14 2.51 ± 0.51 1.74 ± 0.21 2.60 ± 0.31

Example  46: Preparation of Inhalation Pharmaceutical Composition

Compositions were prepared for the inhalation administration using a quantitative spray inhaler (MDI) in the configuration shown in Table 5 below (based on 60 doses of clinically effective dose for humans).

Composition of Composition for Inhalation Administration ingredient Volume Formulation Example 2.7 mg Dry powder with a particle size of 5 μm or less HFA-134a (1,1,1,2-tetrafluoroethane) 1.35 g Powder propellant

On the other hand, for the compounds presented in the present invention, the effectiveness as a pharmaceutical active ingredient can be verified through appropriate pharmacological tests. Specifically, the CYP450 inhibition is evaluated to predict the interaction between drugs, and the binding force to the hERG K ⁺ ion channel is estimated to predict cardiac toxicity. Cytotoxicity tests on cell lines can be performed to predict the toxicity as a drug in advance.

More specific methods for each pharmacological test are as described below.

Example  47: CYP450  Inhibition evaluation

Inhibition screening (P450-GloTM, Promega) was analyzed for 5 out of 11 CYP450 enzymes (CYP3A4, 2C9, 2C19, 2D6, 1A2). Luciferin assay reagent was dissolved in buffer to prepare membranes for each CYP enzyme. 6.25 µl of the compound was prepared at 4 times the concentration, and 6.25 µl of a reaction mixture of an appropriate substrate and enzyme was added for each type of enzyme, and left for 10 minutes. 12.5 μl of CYP enzyme NADPH regenerating reagent was added for 30 minutes, and then 25 μl of luciferin measuring reagent was added and reacted for 20 minutes to measure fluorescence in Fusion α.

Compounds as therapeutic agents for asthma presented in the present invention showed binding ability in the range of 10 μM to 50% or less of the concentration of the compound with respect to the five CYP450 enzymes described above in performing the CYP450 inhibitory evaluation. It can be seen that it is relatively low.

Example  48: hERG  K ⁺ Cardiac toxicity  evaluation

The hERG K ⁺ ion channel (channel) binding capacity of the drug was performed by the radioligand [ ³ H] Astemizole substitution assay (hERG binding screening). The drug was added to a buffer containing hERG cell membrane (2.5 μg / well) and [ ³ H] -Astemizole (4 nM) to make a 0.2 ml reaction mixture, which was allowed to react at room temperature for 60 minutes. This was quickly filtered and washed with a Filtermat-A (Wallac) filter pre-soaked with 0.3% polyethyleneimine using an Inotech harvester, and then the filter was covered with MeltiLex, sealed in a sample bag, dried and RI values were measured with Beta (Plus). The inhibition obtained from the experiment was calculated by nonlinear regression analysis (GraphPad Prism Program) to calculate the binding inhibition (IC ₅₀ ), and 0.1 μM of astemizole was used for nonspecific binding measurement.

As a result of performing the hERG K ⁺ cardiotoxicity evaluation on the compounds as the asthma therapeutic agent presented in the present invention, it can be expected that the compound exhibits a binding force in the range of 50% or less at a concentration of 10 μM of the compound and thus is a safe compound having little cardiac toxicity.

Example  49: Cytotoxicity Assessment ( cell viability  ; cytotoxicity )

To determine the degree of cytotoxicity of the test compound, MTS (3- (4,5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) -2H on THP-1 cells -tetrazolium, inner salt, Promega, USA) method. The MTS method is a sensitive method that can measure the viability, proliferation and activity of the cell and utilizes the ability of mitochondrial dehydrogenase to convert yellow MTS into insoluble formazan during metabolism of living cells. THP-1 cells at 37 ° C., 5% CO ₂ Proliferation was done in RPMI-1640 supplemented with 10% fetal bovine serum in an incubator. Cell density was maintained between 0.5 x 10 ⁶ cells / ml. THP-1 cells were dispensed into 96-well plates at 1 × 10 ⁵ cells / ml and the test compounds were treated with 1, 10 μM and 20 μl of 317 μg / ml of MTS was treated 24 hours after 24 hours. . After 1 hour, the absorbance of the ELISA reader was measured at 490 nm, and the activity of the cells was calculated by using the absorbance of the formazan crystal of the experimental group for the control group as shown in Equation 2 below.

&Quot; (3) &quot;

% Cell viability = (Absorbance of experimental wells / Absorbance of control wells) × 100

In addition to the THP-1 cell lines exemplified above, the present invention performed cytotoxicity tests on various cell lines, for example, HepG2, NIH 3T3, CHO-K1, HEK 293, and the test method thereof was the same as or similar to that described above. Can be done. As a result of measuring the cytotoxicity of the test compound, the cell viability was more than 50% for all cell lines in the 10 μM treated group of the test compound compared to the untreated control group. Can be.

Test results for the compounds of the present invention by the above pharmacological test protocol are described as Table 6 below.

Pharmacological examination Example 6 Compound Example 7 Compound CYP450 Inhibition,% @ 10μM:
3A4
2C9
2C19
2D6
1A2
6.5
23.6
14.2
8.5
23.5
28.2
19.0
9.5
-3.5
6.6 Cell viability,% @ 10μM:
HepG2
NIH 3T3
CHO-K1
HEK 293
73.5
70.6
84.6
59.3
105.8
73.0
80.1
66.9

In order to administer the compound of the present invention to the human body, a representative pharmaceutical method will be further described by way of example as follows. Compounds A, B, and C shown below refer to substances presented as active ingredients for the treatment of asthma in the present invention.

Composition 1 (unit: mg per tablet)

Compound A: 100.00

Lactose: 183.00

Sodium Lauryl Sulfate (SLS): 18.00

Polyvinyl Pyrrolidone (PVP): 15.00

Sodium croscarmellose: 18.00

Microcrystalline Cellulose: 60.00

Magnesium Stearate: 6.00

Total Quantity: 400.00

Composition 2 (unit: mg per tablet)

Compound B: 200.00

Lactose: 203.75

Sodium Lauryl Sulfate (SLS): 15.00

Polyvinyl Pyrrolidone (PVP): 12.50

Sodium croscarmellose: 15.00

Microcrystalline Cellulose: 50.00

Magnesium Stearate: 3.75

Total Quantity: 500.00

Composition 3 (unit: mg per tablet)

Compound C: 100.00

Lactose: 213.00

Sodium Lauryl Sulfate (SLS): 12.00

Polyvinyl Pyrrolidone (PVP): 10.00

Sodium croscarmellose: 12.00

Microcrystalline Cellulose: 50.00

Magnesium Stearate: 3.00

Total Quantity: 400.00

While the present invention has been described in connection with what is presently considered to be practical exemplary embodiments, it is to be understood that the invention is not limited to the disclosed embodiments, but, on the contrary, Of course it is possible. Accordingly, the scope of the present invention should not be construed as being limited to the above-described embodiments, but should be determined by equivalents to the appended claims, as well as the following claims.

Claims (7)

A pharmaceutical composition for use in the prevention, amelioration of symptoms or treatment of asthma diseases comprising the compound of formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient:
[Formula 1]

In Chemical Formula 1,
R ₁ may be independently selected from the group consisting of hydrogen atom, C ₁ -C ₃ alkyl, phenyl, benzyl, benzoyl, benzenesulfonyl, C ₁ -C ₃ alkylcarbonyl, C ₃ -C ₇ cycloalkyl;
All benzene groups included as part of R ₁ may independently have substituents selected from the group consisting of C ₁ -C ₃ alkyl, C ₁ -C ₂ haloalkyl, a halogen atom, and a cyano group;
R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , R ₇ , R ₈ and R ₉ are hydrogen atoms;
The halogen is selected from the group consisting of fluorine, chlorine and bromine atoms. The method of claim 1, wherein the compound of Formula 1
N-{[1- (2- (4-benzoylpiperazin-1-yl) ethyl) -pyrrolidin- (3R) -yl-carbamoyl] -methyl} -3-trifluoromethylbenzamide,
N-{[1- (2- (4- (2-methylbenzoyl) piperazin-1-yl) ethyl) -pyrrolidine- (3R) -yl-carbamoyl] -methyl} -3-trifluoro Methylbenzamide,
N-{[1- (2- (4- (3-methylbenzoyl) piperazin-1-yl) ethyl) -pyrrolidine- (3R) -yl-carbamoyl] -methyl} -3-trifluoro Methylbenzamide,
N-{[1- (2- (4- (4-Methylbenzoyl) piperazin-1-yl) ethyl) -pyrrolidin- (3R) -yl-carbamoyl] -methyl} -3-trifluoro Methylbenzamide,
N-{[1- (2- (4- (4-fluorobenzoyl) piperazin-1-yl) ethyl) -pyrrolidine- (3R) -yl-carbamoyl] -methyl} -3-trifluoro Chloromethylbenzamide,
N-{[1- (2- (4- (4-cyanobenzoyl) piperazin-1-yl) ethyl) -pyrrolidine- (3R) -yl-carbamoyl] -methyl} -3-trifluoro Chloromethylbenzamide,
N-{[1- (2- (4- (4-ethylbenzoyl) piperazin-1-yl) ethyl) -pyrrolidin- (3R) -yl-carbamoyl] -methyl} -3-trifluoro Methylbenzamide,
N-{[1- (2- (4- (phenylsulfonyl) piperazin-1-yl) ethyl) -pyrrolidin- (3R) -yl-carbamoyl] -methyl} -3-trifluoromethyl Benzamide,
N-{[1- (2- (4-propionylpiperazin-1-yl) ethyl) -pyrrolidin- (3R) -yl-carbamoyl] -methyl} -3-trifluoromethylbenzamide,
N-{[1- (2- (4-benzylpiperazin-1-yl) ethyl) -pyrrolidin- (3R) -yl-carbamoyl] -methyl} -3-trifluoromethylbenzamide,
N-{[1- (2- (4- (2-hydroxybutyl) piperazin-1-yl) ethyl) -pyrrolidine- (3R) -yl-carbamoyl] -methyl} -3-trifluoro Chloromethylbenzamide,
N-{[1- (2- (4- (2-hydroxy-2-methylpropyl) piperazin-1-yl) ethyl) -pyrrolidin- (3R) -yl-carbamoyl] -methyl}- 3-trifluoromethylbenzamide,
N-{[1- (2- (4- (2-chlorobenzoyl) piperazin-1-yl) ethyl) -pyrrolidin- (3R) -yl-carbamoyl] -methyl} -3-trifluoro Methylbenzamide,
N-{[1- (2- (4- (2-fluorobenzoyl) piperazin-1-yl) ethyl) -pyrrolidine- (3R) -yl-carbamoyl] -methyl} -3-trifluoro Chloromethylbenzamide, and
N-{[1- (2- (4- (2,5-Difluorobenzoyl) piperazin-1-yl) ethyl) -pyrrolidin- (3R) -yl-carbamoyl] -methyl} -3 Trifluoromethylbenzamide
Pharmaceutical composition for use in the prevention, improvement of symptoms or treatment of asthma disease, characterized in that the compound selected from the group consisting of. The method according to claim 1,
Pharmaceutical composition for use in the prevention, amelioration of symptoms or treatment of asthma disease, characterized in that the administration method of the pharmaceutical composition is inhalation. delete delete delete A food composition for improving asthma disease, comprising as an active ingredient a compound of formula (1) or a food salt thereof as an active ingredient:
[Formula 1]

In Chemical Formula 1,
R ₁ may be independently selected from the group consisting of hydrogen atom, C ₁ -C ₃ alkyl, phenyl, benzyl, benzoyl, benzenesulfonyl, C ₁ -C ₃ alkylcarbonyl, C ₃ -C ₇ cycloalkyl;
All benzene groups included as part of R ₁ may independently have substituents selected from the group consisting of C ₁ -C ₃ alkyl, C ₁ -C ₂ haloalkyl, a halogen atom, and a cyano group;
R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , R ₇ , R ₈ and R ₉ are independently hydrogen atoms;
The halogen is selected from the group consisting of fluorine, chlorine and bromine atoms.

Images