CN109694376B

CN109694376B - Beta-beta2Receptor agonist compounds and their use in the treatment of asthma

Info

Publication number: CN109694376B
Application number: CN201910081428.6A
Authority: CN
Inventors: 陈宏�; 陈群; 张伟; 隋博文; 刘征; 王海强; 蒋巍
Original assignee: Heilongjiang University of Chinese Medicine
Current assignee: Heilongjiang University of Chinese Medicine
Priority date: 2019-01-28
Filing date: 2019-01-28
Publication date: 2019-12-27
Anticipated expiration: 2039-01-28
Also published as: CN109694376A

Abstract

The present invention discloses a novel beta₂Receptor agonist compounds and their use in the treatment of asthma, having the structure of formula (1) wherein R1, R2, R3, X, n and m are as defined in the specification. The compound has better beta₂Receptor agonistic activity, can be used for preventing or treating beta₂Receptor-related diseases, in particular asthma diseases, preferably pediatric asthma diseases. The invention also provides a preparation method of the compound shown in the formula (1) and a pharmaceutical composition containing the compound shown in the formula (1).

Description

Beta-beta2Receptor agonist compounds and their use in the treatment of asthma

Technical Field

The present invention relates to novel beta₂Receptor agonist compounds and their use in the treatment of asthma, more particularly pediatric asthma.

Background

The incidence and mortality of asthma are rising year by year, which makes the research of developing high-efficiency and high-selectivity antiasthmatic drugs very important. Asthma is a chronic inflammatory disease of the airways involving various cells (such as eosinophils, mast cells, lymphocytes, neutrophils, airway epithelial cells, etc.) and cellular components. This chronic inflammation is associated with airway hyperreactivity. Causing recurrent wheezing, dyspnea, chest distress or cough, and the like, and generally showing widely variable reversible airflow limitation, most patients can relieve themselves or through drug treatment. Currently, drug therapy is still the most effective method for preventing and treating bronchial asthma.

β₂Adrenergic receptor agonists (abbreviated as beta)₂Receptor agonists) are biological effects produced by initiating a cascade reaction by increasing intracellular cAMP levels, using guanylate binding regulatory protein (G) -Adenylate Cyclase (AC) as the effector, and using this as the second messenger; on the other hand, it can stimulate calcium pump to make calcium ionEntering the sarcoplasmic reticulum from the cell to store or discharge the cell, and relaxing the smooth muscle by reducing the calcium ion concentration in the cell; beta is a₂The AR agonist can activate adenylate cyclase on sensitized cell membrane of mast cell, increase cAMP in cell, and release Ca²⁺Decrease and thus stabilize cell membranes, thereby inhibiting the release of allergic reaction mediators such as histamine, slow reacting substances, 5-hydroxytryptamine, mitigant, and eotaxin (ECF-A), etc. This not only helps relieve bronchospasm, but also relieves or eliminates congestion and edema of the bronchial mucosa. Thereby making beta₂Receptor agonists are widely used as bronchodilators and are the first choice drugs for controlling asthma attacks.

Disclosure of Invention

The invention mainly aims to provide a novel beta₂A receptor agonist compound having the structure of formula (1), which compound has a preferred beta₂Receptor agonistic activity, can be used for preventing or treating beta₂Receptor-related diseases, in particular asthma.

Wherein:

r1 can be H, halogen, OH, NH₂、NO₂CN, alkyl, cycloalkyl, alkoxy, alkylamino, alkoxyalkyl or haloalkyl;

r2 can be H, halogen, OH, NH₂、NO₂CN, alkyl, alkoxy, alkylamino, alkoxyalkyl or haloalkyl, hydroxyalkyl;

r3 can be H, alkyl, hydroxyalkyl;

x may be O or NH

n can be 0 to 5;

m may be 0 to 5.

Further, R1 is preferably H, halogen, OH, or alkyl, and more preferably H or halogen.

Further, R2 is preferably H, halogen, OH, hydroxyalkyl; more preferably H, OH or hydroxyalkyl;

further, R3 is preferably H;

further, n is preferably 1 to 3, preferably n is 1;

further m is preferably 1 to 3, preferably 2.

Further, the compound of formula (1) is preferably the following compound:

compound 1:

compound 2:

compound 3:

compound 4:

compound 5:

the invention also provides a preparation method of the compound shown in the formula (1).

The preparation method comprises the following steps:

step (1):

reacting a compound shown in the formula (2) with bromoacetyl bromide in an organic solvent in the presence of an organic base to generate a compound shown in the formula (3);

the organic solvent is preferably anhydrous dichloromethane, anhydrous toluene, anhydrous chloroform, anhydrous acetonitrile or anhydrous tetrahydrofuran;

the organic base is preferably pyridine, triethylamine or 4-aminopyridine;

the reaction temperature is preferably from 0 ℃ to room temperature.

The molar ratio of formula (2) to bromoacetyl bromide is preferably 1 (1.1-3); the molar ratio of organic base to bromoacetyl bromide is preferably (1-5): 1.

Step (2):

reacting the compound of formula (3) with the compound of formula (4) in an organic solvent in the presence of an organic base to produce the compound of formula (1).

The organic solvent is preferably N, N-dimethylformamide, N, N-diethylformamide or dimethyl sulfoxide;

the organic base is preferably pyridine, triethylamine or 4-aminopyridine;

the reaction mode is preferably that the compound of the formula (4) is dissolved in an organic solvent and slowly dropped into the solution of the formula (3);

the reaction temperature is preferably from-30 ℃ to room temperature.

The invention also provides a composition containing the compound shown in the formula (1) and a pharmaceutically acceptable carrier. The pharmaceutical composition comprises at least one compound of formula (1) according to the invention as active ingredient and one or more inorganic or organic, solid or liquid pharmaceutically acceptable carriers or excipients. The term "pharmaceutically acceptable" refers to additives or compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction (e.g., dizziness, etc.) when administered to an animal such as a mammal (e.g., a human). Pharmaceutical carriers and excipients may include, but are not limited to, diluents, such as lactose, glucose, mannose, and/or glycerol; a lubricant; polyethylene glycol; binders, such as magnesium aluminum silicate, starch, gelatin, methylcellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone; and, if desired, disintegrating agents, such as starch, agar, alginic acid or a salt thereof such as sodium alginate; and/or adsorbents, colorants, preservatives, stabilizers, flavorants and sweeteners.

The composition can be prepared into pharmaceutical preparations. The compounds of formula (1) may be incorporated as the active ingredient in any of the usual oral dosage forms or topically (powders, ointments or drops) or in the form of sprays or inhalants, including tablets, capsules and liquid preparations (e.g., elixirs and suspensions), containing coloring agents, flavoring agents, stabilizers and taste-masking substances. For mixed oral dosage forms, the compound of formula (1) as an active ingredient may be mixed with various conventional tablet materials (e.g., starch, calcium carbonate, lactose, sucrose, and dicalcium phosphate) to facilitate tableting and encapsulation. The compound of formula (1) may be dissolved or suspended in a pharmaceutically acceptable sterile liquid carrier, such as sterile water, a sterile organic solvent, or a mixture of both. The liquid carrier may be a carrier suitable for injection, such as physiological saline, propylene glycol or aqueous polyethylene glycol solution. In other cases, it can also be prepared by dispersing the micronized active ingredient in an aqueous solution of starch or sodium carboxymethyl cellulose or in a suitable oil. Liquid pharmaceutical formulations (referred to as sterile solutions or suspensions) can be used for intravenous, intramuscular, intraperitoneal or subcutaneous injection.

The invention also provides a compound of formula (1) for the prophylaxis or treatment of beta₂The use of a receptor agonist-related disease, preferably asthma, and further preferably asthma in children.

The compound of formula (1) of the present invention can be used in combination with common asthma drugs, such as budesonide, prednisone, cimetidine, quinolones, macrolides, salbutamol, ipratropium bromide, etc.

The effective amount of the active ingredient of the β 2 receptor agonist of the present invention varies depending on the administration route, the symptoms, age, sex, body weight, and kind of disease of a patient, but is usually in the range of 0.01 to 1000mg/Kg per day for an adult, and the dose for children is halved. The administration frequency is generally 1 to 3 times per day, and if the inhalant is used, it can be used at any time according to the condition of the patient. However, since the dose varies depending on various conditions, an amount smaller than the above dose may be sufficient, and an amount exceeding the above range may be necessary.

Detailed Description

The following embodiments are provided to describe the principle and features of the present invention, and the examples are only for explaining the present invention and not for limiting the scope of the present invention.

Example 1

Step (1):

16.2g (0.1mol) of the compound of formula (5), 2g (0.2mol) of triethylamine and 500ml of anhydrous dichloromethane were added to a 1L round-bottom flask, 100ml of anhydrous dichloromethane containing 2g (0.2mol) of bromoacetyl bromide was slowly added dropwise over 2h with stirring, and the reaction was monitored by TLC until completion. The reaction was quenched by adding 100ml of purified water, followed by liquid separation, collection of the organic layer and concentration under reduced pressure, washing with purified water 3 times, drying over anhydrous sodium sulfate, direct evaporation of the organic solvent, and crystallization in a solvent of dichloromethane and methanol (volume ratio 1:1) to obtain 26.7g of the compound of formula (6) as a white solid (yield 94.6%).

1H-NMR(400MHz,CDCl3)δ7.01(d,2H),6.93(m,2H),4.79(d,1H),4.39(m, 1H)，4.20(t,1H),4.26(s,2H),1.97(m,2H)；13C-NMR(100MHz,CDCl3)δ171.0, 141.2,139.2,128.1,129.6,125.5,125.6,79.5,78.5,71.7,37.6,36.5；ESI-MS：283.12。

Step (2):

27g (0.2mol) of 2-amino-1-phenyl-ethanol, 28g (0.35mol) of pyridine and 500ml of anhydrous dichloromethane were added to a 1L round-bottomed flask, stirred at 0 ℃ and 100ml of an anhydrous dichloromethane solution in which 50g (0.18mol) of the compound of formula (6) was dissolved was slowly dropped into the reaction solution, and the reaction was monitored by TLC until completion of the reaction. After removal of the solvent, column chromatography gave compound 1 as a white solid 44.1g (yield 72.3%).

1H-NMR(400MHz,CDCl3)δ7.19(m，2H)，7.23(m，1H)，7.31(d，2H)， 7.01(d,2H),6.93(m,2H),5.12(s,2H)，4.79(d,1H),4.92(d，2H)，4.85(s，1H)， 4.74(t，1H)，4.39(m,1H)，4.20(t,1H),3.12(s，1H)，1.97(m,2H)；13C-NMR (100MHz,CDCl3)δ171.9,141.2,140.9，139.2,129.6,128.7,128.1,127.6,127.3, 126.8，126.5,125.5,125.6,79.5，79.2,76.7,71.7,58.6,53.4，37.6；ESI-MS：339.39。

Example 2

30g (0.2mol) of 3- (2-amino-1-hydroxy-ethyl) phenol, 28g (0.35mol) of pyridine and 500ml of anhydrous dichloromethane were charged into a 1L round-bottomed flask, stirred at-10 ℃ and 100ml of an anhydrous dichloromethane solution in which 50g (0.18mol) of the compound of formula (6) was dissolved was slowly dropped into the reaction solution, and the reaction was monitored by TLC until the reaction was complete. After removal of the solvent, column chromatography gave compound 1 as a white solid 33.5g (yield 52.4%).

1H-NMR(400MHz,CDCl3)δ7.5(s，1H)，7.02(s，1H)，7.01(d,2H),6.93 (m，2H)，6.85(m，1H)，6.75(m,1H),6.67(d，1H)，4.79(d,1H),5.12(s,2H)， 4.92(d，2H)，4.85(s，1H)，4.74(t，1H)，4.39(m,1H)，4.20(t,1H),3.12(s， 1H)，1.97(m,2H)；13C-NMR(100MHz,CDCl3)δ171.9,157.5，142.2,130.2, 129.6,128.7,128.1,127.6,127.3,126.5,119.9,114.6,114.5,79.5，79.2,76.7,71.7, 58.6,53.4，37.6；ESI-MS：355.38。

Example 3

34g (0.2mol) of 4- (2-amino-1-hydroxy-ethyl) benzene-1, 2-diol, 28g (0.35mol) of pyridine and 500ml of anhydrous dichloromethane were charged into a 1L round-bottomed flask, and stirred at-10 ℃ to slowly drop 100ml of an anhydrous dichloromethane solution containing 50g (0.18mol) of the compound of formula (6) into the reaction solution, and the reaction was monitored by TLC until completion of the reaction. After removal of the solvent, column chromatography gave compound 1 as a white solid 24.3g (yield 35.8%).

1H-NMR(400MHz,CDCl3)δ7.5(s，1H)，7.3(s，1H)，7.11(s，1H)， 7.01(d,2H),6.93(m，2H)，6.58(d，1H)，6.49(d，1H)，5.12(s,2H)，4.79(d, 1H),4.92(d，2H)，4.85(s，1H)，4.74(t，1H)，4.39(m,1H)，4.20(t,1H),3.12 (s，1H)，1.97(m,2H)；13C-NMR(100MHz,CDCl3)δ171.8,144.5，143.4，139.2, 138.8，134.9,130.2,129.6,128.7,127.6,127.3,126.5,119.9，79.5，79.2,76.7,71.7, 58.6,53.4，37.6；ESI-MS：371.38。

Example 4

37g (0.2mol) of 4- (2-amino-1-hydroxy-ethyl) -2-hydroxymethylphenol, 28g (0.35mol) of pyridine and 500ml of anhydrous dichloromethane were charged into a 1L round-bottomed flask, stirred at-10 ℃, and 100ml of an anhydrous dichloromethane solution in which 50g (0.18mol) of the compound of formula (6) was dissolved was slowly dropped into the reaction solution, and the reaction was monitored by TLC until the reaction was complete. After removal of the solvent, column chromatography gave compound 1 as a white solid 24.3g (yield 35.8%).

1H-NMR(400MHz,CDCl3)δ7.10(s，1H)，7.01(d,2H),6.95(s，1H)，6.93 (m，2H)，6.58(d，1H)，6.56(d，1H)，5.12(s,2H)，4.92(s，2H)，4.79(d, 1H),4.92(d，2H)，4.85(s，1H)，4.74(t，1H)，4.39(m,1H)，4.20(t,1H),3.7 (s，1H)，3.12(s，1H)，1.97(m,2H)；13C-NMR(100MHz,CDCl3)δ171.7, 155.0，139.2,138.8，133.7,129.6,128.7,128.2，127.6,127.3,126.5,125.4，116.1， 79.5，79.2,76.7,71.7,60.3，58.6,53.4，37.6；ESI-MS：385.15。

Example 5

37g (0.2mol) of 5- (2-amino-1-hydroxy-ethyl) -2-hydroxymethylphenol, 28g (0.35mol) of pyridine and 500ml of anhydrous dichloromethane were charged into a 1L round-bottomed flask, stirred at-10 ℃, and 100ml of an anhydrous dichloromethane solution in which 50g (0.18mol) of the compound of formula (6) was dissolved was slowly dropped into the reaction solution, and the reaction was monitored by TLC until completion of the reaction. After removal of the solvent, column chromatography gave compound 1 as a white solid (26.8 g, 39.5% yield).

1H-NMR(400MHz,CDCl3)δ7.10(s，1H)，7.12(d,2H),7.05(s，1H)，6.92 (m，2H)，6.61(d，1H)，6.55(d，1H)，5.12(s,2H)，4.92(s，2H)，4.79(d, 1H),4.92(d，2H)，4.85(s，1H)，4.74(t，1H)，4.39(m,1H)，4.20(t,1H),3.7 (s，1H)，3.12(s，1H)，1.97(m,2H)；13C-NMR(100MHz,CDCl3)δ171.7, 155.3，140.1,138.9，133.7,129.6,128.7,128.2，127.6,127.3,126.5,125.4，116.1， 79.5，79.2,76.7,71.7,60.3，58.6,53.4，37.6；ESI-MS：385.15。

Example 6 beta 2 adrenergic Activity assay

Preparation of the test Compounds

The highest dose required to dilute 10 mM/100% DMSO (dimethylsulfoxide) stock solutions of compounds 1-5 in 4% DMSO. This highest dose was used to construct a 10-point semilog dilution curve, all in 4% DMSO. Isoproterenol was used as a standard in each experiment and for the control wells on each plate.

Cell culture

Allowing recombinant expression of human beta₂CHO cells for adrenergic receptors were grown in Dulbeccos MEM/NUT MIX F12(Gibco, 21331-020) supplemented with 10% fetal bovine serum (Sigma, F4135, Lot 90K8404Exp 09/04), 2mM glutamine (Sigma, G7513), 500. mu.g/ml geneticin (Sigma, G7034) and 10. mu.g/ml puromycin (puromycin) (Sigma, P8833). Cells were seeded to obtain about 90% confluency for testing.

Test method

Doses of 25 μ l/well of each compound were transferred to cAMP-Flashplate (NEN, SMP004B) with 1% DMSO as the basal control and 100nM isoproterenol as the highest control. It was diluted 1: 2 by the addition of 25. mu.l/well PBS. Cells were trypsinized (0.25% Sigma, T4049), washed with PBS (Gibco, 14040-⁶Individual cells/ml CHOhB 2. The compounds were incubated with 50. mu.l/well of cells for 1 hour. Cells were then lysed by adding 100. mu.l/well of assay buffer (NEN, SMP004B) containing 0.18. mu. Ci/ml125I-cAMP (NEN, NEX-130) and the plates were incubated at room temperature for an additional 2 hours. The amount of 125I-cAMP bound to Flashplate was quantified using Topcount NXT (Packard) with normal counting efficiency of 1 minuteAmount of the compound (A). Dose response data were expressed as% of isoproterenol activity and fitted using a four parameter sigmoidal fit.

Table 1: beta is a₂Results of the adrenergic agonist Activity test

EXAMPLES Compounds	IC50(nM)
		Compound 1	151.2
Compound 2	72.8
		Compound 3	43.3
Compound 4	25.0
		Compound 5	12.9

As can be seen from the pharmacodynamic experiments, the compounds 1-5 have good beta₂Adrenoreceptor activity.

Example 7 efficacy test of asthma

Experimental animals: 60 healthy clean male BN rats weighing 180-220 g are adopted and divided into a model group and a control group.

Preparing a model:

(1) preparation of asthma model

The model group and the control group are both administered 1ml of mixed solution of 100mg of hydrogenated alumina gel containing egg protein on the first day, and on the 15 th day, 5L/min of 5% egg protein physiological saline solution is atomized and inhaled for 20 minutes to induce asthma attack, 1 time per day for 7 days continuously, and after 7 days of atomization stopping, the mixed solution is continuously excited for 7 days;

animal behavioral observation: after the egg protein is atomized, the rat generates sneezing, coughing, rapid respiration, mouth opening respiration and arch back stagnation, hair color is dry after repeated attacks, and the weight is slowly increased; the inflammation of the large airways is obviously changed, the exudation in the bronchial cavity is increased, and inflammatory cells are obviously increased in the bronchial wall and tissues around the bronchi.

Test compounds 1 to 5 were administered at 0.01mg/Kg three times a day for 3 consecutive days, and after three days, IL-4 and IL-5 in serum of male BN rats were detected using IL-4 and IL-5 kits to observe changes in IL-4 and IL-5 after the drug administration in the model group and the control group.

Table 2 shows the serum IL-4(pg/ml) and IL-5(pg/ml) of the BN rat model group and the control group

Test compounds	IL-4(pg/ml)	IL-5(pg/ml)
			Compound 1	12.1±1.2	23.8±3.6
Compound 2	16.3±0.9	29.4±2.8
			Compound 3	8.6±0.8	18.1±1.6
Compound 4	7.3±1.3	13.1±1.1
			Compound 5	4.9±0.5	9.8±1.4
Control group	38.72±2.1	68.2±6.5

As can be seen from the pharmacodynamic experiments, the compounds 1-5 have good asthma treatment effect.

The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and all simple modifications, alterations and equivalents of the above embodiments according to the technical spirit of the present invention are still within the protection scope of the technical solution of the present invention.

Claims

1. A compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof, characterized in that the formula (1) has the following structure:

wherein:

r1 is selected from H, halogen, OH, NH₂、NO₂Or CN;

r2 is selected from H, halogen, OH, NH₂、NO₂Or CN;

r3 is selected from H;

x is selected from O or NH

n is selected from 1-3;

m is selected from 1-3.

2. A compound according to claim 1, characterized in that R1 is H, halogen or OH.

3. A compound according to claim 1, characterized in that R2 is H, halogen or OH.

4. A compound according to claim 1, characterized in that R3 is H.

5. A compound represented by:

compound 1:

compound 2:

compound 3:

compound 4:

compound 5:

6. a process for the preparation of a compound according to claim 1, characterized in that it comprises the following steps:

step (1):

the organic solvent is anhydrous dichloromethane, anhydrous toluene, anhydrous chloroform, anhydrous acetonitrile or anhydrous tetrahydrofuran;

the organic base is pyridine, triethylamine or 4-aminopyridine;

the reaction temperature is 0-room temperature;

the molar ratio of the formula (2) to the bromoacetyl bromide is 1 (1.1-3); the molar ratio of the organic base to the bromoacetyl bromide is (1-5) to 1;

step (2):

reacting a compound shown in a formula (3) with a compound shown in a formula (4) in an organic solvent in the presence of an organic base to generate a compound shown in a formula (1);

the organic solvent is N, N-dimethylformamide, N, N-diethylformamide or dimethyl sulfoxide;

the organic base is pyridine, triethylamine or 4-aminopyridine;

the reaction temperature is-30 ℃ to room temperature.

7. A composition comprising a compound of any one of claims 1-5 and a pharmaceutically acceptable carrier.

8. Use of a compound according to any one of claims 1 to 5 for the preparation of a medicament for the prophylaxis or treatment of beta₂Use in the manufacture of a medicament for a receptor agonist-related disorder.

9. Use according to claim 8, characterized in that said use is asthma.

10. Use according to claim 8, characterized in that the use is pediatric asthma.