阅读说明：本技术 具有磷酸二酯酶4b抑制活性的吲唑杂环类化合物 (Indazole heterocycles having phosphodiesterase 4B inhibitory activity ) 是由 王进欣 左琳飞 徐正文 王有志 顾勤兰 于 2021-07-19 设计创作，主要内容包括：本发明公开了一类含有吲唑杂环骨架的化合物及其应用,属于药物化学领域。所述含有吲唑杂环骨架的化合物对PDE4B具有较好的抑制活性,可作为PDE4B抑制剂,用于制备预防或治疗与PDE4B相关疾病的药物,包括慢性阻塞性肺病、哮喘、皮炎、银屑病等炎症相关疾病的药物以及阿兹海默症、抗焦虑、改善认知等中枢神经疾病的药物。(The invention discloses a compound containing an indazole heterocyclic skeleton and application thereof, belonging to the field of pharmaceutical chemistry. The compound containing indazole heterocyclic skeleton has good inhibitory activity on PDE4B, can be used as a PDE4B inhibitor, and is used for preparing medicaments for preventing or treating diseases related to PDE4B, including medicaments for treating inflammation-related diseases such as chronic obstructive pulmonary disease, asthma, dermatitis and psoriasis, and medicaments for treating central nervous diseases such as Alzheimer's disease, anxiety and cognition.)

1. A substituted indazole heterocyclic compound characterized by: the structural formula is shown as a formula I,

wherein:

R₁selected from C1-C6 alkyl, C2-C6 alkenyl, C2-C6 carboxyl, C1-C3 difluoromethyl, C1-C33 trifluoromethyl or substituted benzyl, wherein the substituent of the benzyl in the substituted benzyl is selected from F, Cl, Br, I and CF₃CN, hydroxyl, methoxyl, methyl, amino, nitryl, sulfydryl, carboxyl, ethenyl, C4-C8 straight-chain alkyl or branched-chain alkyl, aryl, heterocyclic radical;

R₂is aryl or heterocyclic aryl, the aryl is selected from unsubstituted, mono-substituted or poly-substituted phenyl, naphthyl or biphenyl, the mono-substituted or poly-substituted substituent is selected from halogen, cyano, nitro, hydroxyl, amino, carboxyl, carbomethoxy or carbethoxy, the heterocyclic aryl is selected from unsubstituted, mono-substituted or poly-substituted pyrimidine, imidazole, pyrazole, pyridine, triazole, thiazole, thiophene, indole, indazole or benzimidazole, and the mono-substituted or poly-substituted substituent is selected from halogen, cyano, nitro, hydroxyl, amino, carboxyl, carbomethoxy or carbethoxy;

w, X, Y is selected from C, N, O or S, Z is C or N, and the position of substituent X is selected from ortho, para or meta of W;

R₃is selected from C1-C6 alkyl, C2-C6 carboxyl, phenyl, heterocyclic aryl or benzyl, the phenyl, the heterocyclic aryl and the benzyl are unsubstituted, mono-substituted or di-substituted, and the mono-substituted or di-substituted substituent is selected from F, Cl, Br, I, CF₃CN, hydroxyl, methoxyl, methyl, amino, nitryl, sulfydryl, carboxyl, ethenyl, C4-C8 straight-chain alkyl or branched-chain alkyl, aryl, heterocyclic radical.

2. The compound of claim 1, wherein: selected from the following compounds:

3. use of a compound of claim 1 or an optical isomer, enantiomer, diastereomer, racemate, racemic mixture or a pharmaceutically acceptable salt or prodrug thereof for the manufacture of a medicament for the prevention and/or treatment of diseases associated with PDE 4B.

4. Use according to claim 3, characterized in that: the PDE 4B-related diseases include inflammatory diseases and central nervous diseases.

5. Use according to claim 4, characterized in that: the inflammatory diseases include chronic obstructive pulmonary disease, asthma, dermatitis, psoriasis; the central nervous system disorder includes Alzheimer's disease, anxiety resistance, and cognition improvement.

Technical Field

The invention belongs to the technical field of pharmaceutical chemistry, and particularly relates to an indazole heterocyclic compound with phosphodiesterase 4B inhibitory activity.

Background

Chronic Obstructive Pulmonary Disease (COPD) is a common, preventable and treatable disease characterized by persistent respiratory symptoms and airflow limitation, usually due to airway and/or alveolar abnormalities caused by significant exposure to toxic particles or gases. Currently, COPD is the fourth most fatal disease in the world, with over 300 million people dying from COPD each year, accounting for about 6% of the worldwide deaths (Global initial for respiratory obstructive disease, updated 2019). According to the latest statistics, chronic obstructive pulmonary disease has become the third chronic disease in China, which is second to hypertension and diabetes, and about 1 million patients with chronic obstructive pulmonary disease (Lancet.2018; 391(10131):1706-1717) exist. With the increasing air pollution and the aging population, the prevalence rate of COPD is continuously increased in recent years, which not only causes long-term pain and affliction to patients, but also causes heavy economic burden to families and society.

The conventional chronic obstructive pulmonary disease treatment drugs which are commonly used clinically can be divided into bronchodilators and anti-inflammatory drugs according to the pharmacological activity of the drugs. The bronchodilators are the dominant, and include long-acting and short-acting muscarinic receptor antagonists such as ipratropium bromide and tiotropium bromide, long-acting and short-acting beta 2-adrenoceptor agonists, and combinations of bronchodilators and anti-inflammatory glucocorticoids. However, these treatment regimens only improve COPD symptoms, reduce acute exacerbation rates and reduce mortality, provide relief from COPD symptoms, and are not radical, resulting in an inevitable, sustained decline in lung function. Therefore, there is an urgent need for specific therapeutic drugs for COPD in the clinic, and the research and discovery of novel therapeutic drugs for COPD are still hot spots at present.

Since COPD is a complex and comprehensive disease, a variety of mechanisms are involved in the pathogenesis, including inflammatory response, oxidative stress, mitochondrial dysfunction, aging, protease-antiprotease imbalance, and the like, which are interconnected and interact to form the complexity, persistence, and refractory of COPD. Intensive research and development has been conducted on the inflammatory mechanism of COPD. Phosphodiesterase 4 (PDE 4) is widely expressed in a variety of inflammatory and immune cells, and specifically hydrolyzes Cyclic adenosine monophosphate (cAMP), which plays an important role in inflammatory responses. The PDE4 inhibitor can increase cAMP level in vivo, inhibit release of inflammatory factor, promote production of anti-inflammatory mediator, and exert anti-inflammatory effect by inhibiting hydrolysis of PDE 4. The Roflumilast is clinically used for treating COPD, has obvious anti-inflammatory effect, and can inhibit monocytes, macrophages, T cells and the like from releasing inflammatory mediators such as TNF-alpha, interleukin, chemotactic factors and the like. However, the serious side effects of nausea, vomiting and the like exist in the inhibitor, so that the clinical application of the PDE4 inhibitor is limited. A large number of researches show that in human body, subtype B of phosphodiesterase 4 (PDE4B) is related to inflammatory reaction and is involved in the release of various inflammatory mediators in the body, and subtype D is closely related to the generation of side effects such as nausea and vomiting, so that a new idea is provided for finding a PDE4 inhibitor with low side effect, namely designing the PDE4B inhibitor can reduce the influence of the side effect and promote further clinical application.

The protein sequences of the four isoform catalytic domains of PDE4 are highly homologous, and inhibitors acting on the catalytic domains do not produce isoform selectivity, whereas most classical PDE4 inhibitors have been reported to act on the catalytic domain. The novel PDE4 inhibitor mode of action reported in recent years, the inhibitor acts with the catalytic domain and the regulatory sequence simultaneously, so that the regulatory sequence can stabilize the closed conformation of the protein, prevent cAMP from entering and play an inhibiting role.

Accordingly, the invention develops design and synthesis research on PDE4B inhibitors aiming at a regulatory sequence CR3, and designs and synthesizes a compound with PDE4B inhibition activity.

Disclosure of Invention

The invention aims to provide a compound containing indazole heterocyclic skeleton and having phosphodiesterase 4B inhibition activity and application thereof.

In order to achieve the purpose of the invention, the invention adopts the following technical scheme:

a substituted indazole heterocyclic compound is a compound shown as a formula I or an optical isomer, an enantiomer, a diastereomer, a racemate, a racemic mixture or a pharmaceutically acceptable salt or prodrug thereof,

wherein:

R₁is selected from one of C1-C6 alkyl, C2-C6 alkenyl, C2-C6 carboxyl, C1-C3 difluoromethyl, C1-C3 trifluoromethyl or substituted benzyl, wherein the substituent of the benzyl in the substituted benzyl is selected from F, Cl, Br, I, CF₃CN, hydroxyl, methoxyl, methyl, amino, nitryl, sulfydryl, carboxyl, ethenyl, C4-C8 straight-chain alkyl or branched-chain alkyl, aryl, heterocyclic radical;

R₂is aryl or heterocyclic aryl, the aryl is selected from unsubstituted, mono-substituted or poly-substituted phenyl, naphthyl or biphenyl, the mono-substituted or poly-substituted substituent is selected from halogen, cyano, nitro, hydroxyl, amino, carboxyl, carbomethoxy or carbethoxy, the heterocyclic aryl is selected from unsubstituted, mono-substituted or poly-substituted pyrimidine, imidazole, pyrazole, pyridine, triazole, thiazole, thiophene, indole, indazole or benzimidazole, the mono-substituted or poly-substituted substituent is selected from halogen, cyano, nitro or benzimidazoleA hydroxyl group, an amino group, a carboxyl group, a carbomethoxy group or an carbethoxy group;

w, X, Y is selected from C, N, O or S, Z is C or N, and the position of X substituent is selected from ortho, para or meta of W;

R₃is selected from C1-C6 alkyl, C2-C6 carboxyl, phenyl, heterocyclic aryl or benzyl, the phenyl, the heterocyclic aryl and the benzyl are unsubstituted, mono-substituted or di-substituted, and the mono-substituted or di-substituted substituent is selected from F, Cl, Br, I, CF₃CN, hydroxyl, methoxyl, methyl, amino, nitryl, sulfydryl, carboxyl, ethenyl, C4-C8 straight-chain alkyl or branched-chain alkyl, aryl, heterocyclic radical.

Further, the compound is selected from the following compounds:

the application of the compound serving as a PDE4B inhibitor in preparing medicines for preventing and/or treating diseases related to PDE 4B.

The PDE 4B-related diseases include inflammatory diseases and central nervous diseases.

The inflammatory diseases include chronic obstructive pulmonary disease, asthma, dermatitis, psoriasis; the central nervous system disorder includes Alzheimer's disease, anxiety resistance, and cognition improvement.

The substituted indazole heterocyclic compound is proved to have good inhibitory activity on PDE4B through modern pharmacological scientific research, and is applicable to diseases related to PDE 4B.

Drawings

FIG. 1 is a graph showing the TNF-a inhibitory activity of the compounds of the examples.

Detailed Description

To further illustrate the present invention, a series of examples are given below, which are purely illustrative and are intended to be a detailed description of the invention only and should not be understood as limiting the invention.

Example 1

Preparation of 3- ((3- (5-chlorothien-2-yl) -1-isopropyl-1H-indazol-5-yl) amino) propionic acid (I-1)

Step 1. Synthesis of 3-bromo-1-isopropyl-5-nitro-1H-indazole

1.00g (4.14mmol) of 3-bromo-5-nitro-1H-indazole and 331.2mg (8.28mmol) of sodium hydride (content: 60%) are added to a 250mL eggplant-shaped bottle, 120mL of anhydrous N, N-dimethylformamide is added under ice-bath conditions to dissolve the mixture, the mixture is stirred for 0.5H and then moved to room temperature, 446. mu.L (4.54mmol) of 2-iodopropane is added, and the reaction is stirred at room temperature for 24H. The reaction solution was quenched by adding 3 times the volume of water, extracted with ethyl acetate several times, the organic phases were combined and washed with water for 2 times, and the organic phase was dried over anhydrous sodium sulfate. The organic phase was concentrated, and then subjected to sand preparation and column chromatography purification (petroleum ether: dichloromethane: 4:1) to obtain 435mg of a white solid with a yield of 41.4%.

¹H-NMR(500MHz,DMSO-d6):δ8.44(d,1H),8.28(dd,J＝9.3,2.0Hz,1H),8.02(d,J＝9.3Hz,1H),5.14(m,1H),1.51(d,J＝6.6Hz,6H).

Step 2. Synthesis of 3- (5-chlorothiophene-2-yl) -1-isopropyl-5-nitro-1H-indazole

510.0mg (2.00mmol) of the starting material was placed in a 50mL two-necked round-bottomed flask, and 486.0mg (3.00mmol) of 5-chlorothiophene-2-boronic acid was addedAnd 37.0mg (0.05mmol) [1,1' -bis (diphenylphosphino) ferrocene ]]Palladium dichloride (Pd (dppf) Cl₂) Dissolving the mixture in 16mL of toluene and 8mL of methanol, and adding Na₂CO₃424mg (4.00mmol), vacuum pumping 10min with a water pump, nitrogen protection and reflux at 110 ℃. After about 14h the starting material was reacted by TLC. The reaction mixture was filtered with suction, and the filtrate was purified by column chromatography (petroleum ether: dichloromethane: 8:1) to obtain 295mg of a yellow solid in a yield of 46.0%.

¹H-NMR(400MHz,CDCl₃):δ8.91(dd,J＝2.1,0.6Hz,1H),8.29(dd,J＝9.3,2.1Hz,1H),7.50(d,J＝9.3Hz,1H),7.44(d,J＝3.9Hz,1H),7.02(d,J＝3.9Hz,1H),4.88(m,1H),1.64(d,J＝6.7Hz,6H).

Step 3, synthesizing 3- (5-chlorothiophene-2-yl) -1-isopropyl-1H-indazole-5-amine

200mg (0.80mmol) of the starting material was placed in a 100mL eggplant type bottle, 148mg (2.64mmol) of reduced iron powder and 353mg (6.60mmol) of ammonium chloride were added thereto, and the mixture was dissolved in 8mL of ethanol and 4mL of water and reacted at 80 ℃ for about 1 hour. TLC detection raw material has completely reduced, filter insoluble impurities, filtrate with dichloromethane extraction 3 times, combined organic phase, anhydrous sodium sulfate after drying concentrated, drying, weighing, crude product about 165mg, yield about 90%.

Step 4. Synthesis of 3- ((3- (5-chlorothien-2-yl) -1-isopropyl-1H-indazol-5-yl) amino) propionic acid (I-1)

165mg of the crude product from the previous step was taken out and placed in a 10mL round-bottom flask, and 45. mu.L (0.66mmol) of acrylic acid and 4mL of toluene were added and dissolved, followed by stirring and refluxing at 110 ℃. After 24 hours of reaction, the reaction was stopped by detecting the remaining portion of the starting material by TLC. Transferring the reaction solution to a 100mL eggplant-shaped bottle, adding 6mL toluene for dilution, adding a large amount of dilute sodium hydroxide solution, adjusting the pH of the reaction solution to be alkaline, stirring uniformly, standing for layering, and taking the lower-layer water phase. The aqueous phase was washed 2 times with toluene and 2 times with diethyl ether. Adding 15% diluted hydrochloric acid into the water phase, adjusting pH to 5-6, allowing the water phase to become turbid, extracting with ethyl acetate for 3 times, mixing the organic phases, drying with anhydrous sodium sulfate, concentrating, and recrystallizing to obtain solid of about 30mg with yield of about 12.5%.

M.P.165-168℃,¹H-NMR(300MHz,DMSO-d6)：δ7.50(d,J＝9.0Hz,1H),7.43(d,J＝3.9Hz,1H),7.17(d,J＝3.9Hz,1H),6.91(d,J＝9.1Hz,1H),6.84(s,1H),4.87(m,1H),3.33(t,J＝7.0Hz,2H),2.56(t,J＝6.6Hz,2H),1.46(d,J＝6.6Hz,6H).

Example 2

Preparation of 3- ((3- (5-chlorothien-2-yl) -1-isopropyl-1H-indazol-5-yl) amino) butanoic acid (I-2)

Referring to the synthesis of I-1, about 16mg of solid was obtained with a yield of about 12.8%.

M.P.170-172℃,¹H-NMR(300MHz,DMSO-d6):δ8.07–7.62(m,2H),7.38(d,J＝3.7Hz,2H),7.13(d,J＝3.8Hz,1H),4.89(dt,J＝12.8,6.1Hz,1H),3.85(s,1H),2.68(d,J＝15.8Hz,1H),2.55–2.40(m,1H),1.37(d,J＝6.4Hz,6H),1.20(d,J＝5.9Hz,3H)；ESI-MS(m/z):378.1[M+H]⁺,376.1[M-H]^-.

Example 3

Preparation of 3- ((3- (5-chlorothien-2-yl) -1-isopropyl-1H-indazol-5-yl) amino) -2-methylpropanoic acid (I-3)

Referring to the synthesis of I-1, about 9mg of solid was obtained with a yield of about 7.2%.

M.P.170-174℃,¹H-NMR(300MHz,DMSO-d6):δ7.50(d,J＝9.0Hz,1H),7.42(d,J＝3.8Hz,1H),7.16(d,J＝3.8Hz,1H),6.94(d,J＝9.1Hz,1H),6.87(s,1H),4.87(m,1H),3.51(s,1H),3.11(dd,J＝12.7,6.3Hz,1H),2.71(dd,J＝12.9,6.5Hz,1H),1.46(d,J＝6.5Hz,6H),1.24(d,J＝9.2Hz,3H)；ESI-MS(m/z):378.2[M+H]⁺,376.2[M-H]^-.

Example 4

Preparation of (3- (5-chlorothien-2-yl) -1-isopropyl-1H-indazol-5-yl) glycine (I-4)

Step a. Synthesis of (3- (5-chlorothien-2-yl) -1-isopropyl-1H-indazol-5-yl) glycine ethyl ester

About 190mg of the crude product was taken and placed in a 15mL round-bottom flask, and 65mg (0,79mmol) of sodium acetate and 83. mu.L (0.79mmol) of ethyl chloroacetate were added thereto, dissolved in 6mL of ethanol, and the reaction was heated with stirring at 120 ℃. After 24h of reaction, TLC detection shows that the raw materials still remain, and the reaction is stopped. The reaction mixture was spun to dryness, and then subjected to sand purification by column chromatography (petroleum ether: ethyl acetate: 16:1 and 8:1) to obtain 120mg of a pale yellow solid with a yield of 48.1%.

Step b. Synthesis of (3- (5-chlorothien-2-yl) -1-isopropyl-1H-indazol-5-yl) glycine (I-4)

A15 mL round-bottom flask was charged with 120mg (0.32mmol) of the starting material and 60mg (1.28mmol) of sodium hydroxide, dissolved in 3mL of methanol and 2mL of water, and stirred at 50 ℃. After 2h of reaction TLC check that the starting material has been hydrolysed. Adding 15% diluted hydrochloric acid to adjust pH to neutral, separating out solid, filtering, dissolving again, and recrystallizing to obtain pure product about 20mg with yield of 17.9%.

M.P.130-133℃,¹H-NMR(300MHz,DMSO-d6):δ7.52(d,J＝9.1Hz,1H),7.41(d,J＝3.9Hz,1H),7.18(d,J＝3.9Hz,1H),6.99(dd,J＝9.0,2.0Hz,1H),6.80(d,J＝1.7Hz,1H),4.88(m,1H),3.92(s,2H),1.46(d,J＝6.6Hz,6H)；ESI-MS(m/z):348.1[M-H]^-.

Example 5

Preparation of 1- ((3- (5-chlorothien-2-yl) -1-isopropyl-1H-indazol-5-yl) carbamoyl) cyclopropane-1-carboxylic acid (I-5)

Step a. Synthesis of 1- ((3- (5-chlorothien-2-yl) -1-isopropyl-1H-indazol-5-yl) carbamoyl) cyclopropane-1-carboxylic acid (I-5)

A50 mL round-bottom flask was charged with 22mg (0.165mmol) of 1, 1-dicyclopropyldarboxylic acid and 23. mu.L (0.165mmol) of triethylamine, dissolved in anhydrous tetrahydrofuran, and the reaction was stirred for 0.5h under ice-bath. Add slowly 12. mu.L (0.165mmol) of thionyl chloride and continue the reaction for 0.5 h. 48mg (0.165mmol) of the starting material dissolved in anhydrous tetrahydrofuran was slowly added dropwise thereto, followed by reaction for 2 hours. TLC detects that the raw materials react completely, spin-dries the reaction solution, adds toluene to dissolve, then adds a large amount of dilute sodium hydroxide solution, adjusts the pH of the reaction solution to alkalinity, stirs evenly and then stands for layering, and takes the lower aqueous phase. The aqueous phase was washed 2 times with toluene and 2 times with diethyl ether. Adding 15% diluted hydrochloric acid into the water phase, adjusting pH to 5-6, allowing the water phase to become turbid, extracting with ethyl acetate for 3 times, mixing the organic phases, drying with anhydrous sodium sulfate, concentrating, and recrystallizing to obtain solid 25mg with yield of 37.5%.

M.P.140-144℃,¹H-NMR(300MHz,DMSO-d6):δ10.74(s,1H),8.46(s,1H),7.74(d,J＝9.1Hz,1H),7.56(dd,J＝9.1,1.5Hz,1H),7.40(d,J＝3.9Hz,1H),7.23(d,J＝3.9Hz,1H),4.99(m,1H),1.48(m,10H)；ESI-MS(m/z):402.1[M-H]^-.

Example 6

Preparation of 3- ((3- (5-chlorothien-2-yl) -1- (2, 2-difluoroethyl) -1H-indazol-5-yl) amino) propionic acid (I-6)

Step a. Synthesis of 3-bromo-1- (2, 2-difluoroethyl) -5-nitro-1H-indazole

1.00g (4.14mmol) of 3-bromo-5-nitro-1H-indazole, 273. mu.L (6.20mmol) of 1, 1-difluoro-2-iodoethane and 1.14g (8.26mmol) of potassium carbonate were put in a 250mL eggplant-shaped bottle, dissolved in 100mL of anhydrous acetonitrile, and reacted for 24 hours with stirring under reflux. The TLC detected the reaction was almost complete, the reaction was filtered and then directly made into sand, purified by column chromatography (petroleum ether: dichloromethane: 4:1) to give about 850mg of a pale yellow solid with 67.1% yield.

¹H-NMR(300MHz,Chloroform-d):δ8.63(dd,J＝2.1,0.6Hz,1H),8.38(dd,J＝9.3,2.1Hz,1H),7.55(d,J＝9.3Hz,1H),6.19(tt,J＝55.1,4.2Hz,1H),4.75(td,J＝13.4,4.2Hz,2H).

Step b. Synthesis of 3- (5-chlorothien-2-yl) -1- (2, 2-difluoroethyl) -5-nitro-1H-indazole

486.0mg of raw material was placed in a 50mL two-necked round-bottomed flask, 386.0mg (2.38mmol) of 5-chlorothiophene-2-boronic acid, 337mg (3.18mmol) of sodium carbonate and 35.0mg (0.05mmol) of [1,1' -bis (diphenylphosphino) ferrocene ] dichloropalladium (Pd (dppf) Cl2) were added, the mixture was dissolved in 16mL of toluene and 8mL of methanol, Na2CO 3337 mg (3.18mmol) was added, the mixture was evacuated by a water pump for 10min, and the mixture was refluxed at 110 ℃ under nitrogen protection. After about 20h the starting material was reacted by TLC. The reaction mixture was filtered, the filtrate was washed with sand, and purified by column chromatography (petroleum ether: dichloromethane: 8:1) to give a yellow solid (200 mg) in a yield of 36.7%.

¹H-NMR(300MHz,Chloroform-d):δ8.63(dd,J＝2.1,0.6Hz,1H),8.38(dd,J＝9.3,2.1Hz,1H),7.55(d,J＝9.3Hz,1H),6.19(tt,J＝55.1,4.2Hz,1H),4.75(td,J＝13.4,4.2Hz,2H).

Step c, synthesizing 3- (5-chlorothien-2-yl) -1- (2, 2-difluoroethyl) -1H-indazol-5-amine

100mg (0.30mmol) of the above-mentioned raw material was put in a 100mL eggplant type bottle, 67mg (1.20mmol) of reduced iron powder and 214mg (4.0mmol) of ammonium chloride were added thereto, and the mixture was dissolved in 8mL of ethanol and 4mL of water and reacted at 80 ℃ for about 1 hour. TLC detection raw material has completely reduced, filter insoluble impurities, filtrate with dichloromethane extraction 3 times, combined organic phase, anhydrous sodium sulfate drying after concentration, drying, weighing, crude product about 85mg, yield about 90%.

Step d. Synthesis of 3- ((3- (5-chlorothien-2-yl) -1- (2, 2-difluoroethyl) -1H-indazol-5-yl) amino) propanoic acid (I-6)

85mg of the crude product from the previous step was taken out and placed in a 10mL round-bottom flask, 18. mu.L (0.27mmol) of acrylic acid and 4mL of toluene were added and dissolved, and the reaction was stirred at 110 ℃ and refluxed. After 24 hours of reaction, the reaction was stopped by detecting the remaining portion of the starting material by TLC. Transferring the reaction solution to a 100mL eggplant-shaped bottle, adding 6mL toluene for dilution, adding a large amount of dilute sodium hydroxide solution, adjusting the pH of the reaction solution to be alkaline, stirring uniformly, standing for layering, and taking the lower-layer water phase. The aqueous phase was washed 2 times with toluene and 2 times with diethyl ether. Adding 15% diluted hydrochloric acid into the water phase, adjusting pH to 5-6, allowing the water phase to become turbid, extracting with ethyl acetate for 3 times, mixing the organic phases, drying with anhydrous sodium sulfate, concentrating, and recrystallizing to obtain solid of about 10mg with yield of about 9.0%.

M.P.180-182℃¹H-NMR(300MHz,DMSO-d6):δ7.57–7.48(m,2H),7.20(d,J＝3.9Hz,1H),6.96(d,J＝9.1Hz,1H),6.87(s,1H),6.40(tt,J＝54.9,3.6Hz,1H),4.86(td,J＝15.0,2.9Hz,2H),3.32(s,2H),2.56(t,J＝6.6Hz,2H)；ESI-MS(m/z):386.1[M+H]⁺,384.0[M-H]^-.

Example 7

Preparation of 3- ((3- (5-chlorothien-2-yl) -1- (2,2, 2-trifluoroethyl) -1H-indazol-5-yl) amino) propionic acid (I-7)

Referring to the synthesis of I-6, about 11mg of solid was obtained with a yield of about 9.7%.

M.P.168-170℃¹H-NMR(300MHz,DMSO-d6)δ7.64–7.54(m,2H),7.22(d,J＝3.9Hz,1H),7.02(dd,J＝9.1,1.7Hz,1H),6.94(s,1H),5.40(q,J＝9.1Hz,2H),3.36(t,J＝6.7Hz,2H),2.57(t,J＝6.7Hz,2H)；ESI-MS(m/z):404.2[M+H]⁺,402.1[M-H]^-.

Example 8

Preparation of 3- ((3- (5-chlorothien-2-yl) -1-isopropyl-1H-indazol-5-yl) amino) propionic acid (I-8)

Referring to the synthesis of I-1, about 25mg of solid was obtained with a yield of about 13.8%.

M.P.164-166℃,¹H-NMR(300MHz,DMSO-d6):δ7.66(d,J＝9.0Hz,1H),7.51(d,J＝3.9Hz,1H),7.41(s,1H),7.22(m,2H),4.42(q,J＝7.1Hz,2H),3.45(t,J＝6.9Hz,2H),2.65(t,J＝6.9Hz,2H),1.39(t,J＝7.2Hz,3H)；ESI-MS(m/z):350.2[M+H]⁺,348.1[M-H]^-.

Example 9

Preparation of 3- ((3- (3-chlorophenyl) -1-isopropyl-1H-indazol-5-yl) amino) propionic acid (I-9)

Referring to the synthesis of I-1, about 28mg of solid was obtained with a yield of about 15.1%.

M.P.170-171℃¹H-NMR(300MHz,DMSO-d6):δ7.97–7.77(m,4H),7.58(t,J＝8.0Hz,1H),7.52–7.45(m,1H),7.41(d,J＝8.3Hz,1H),5.07(p,J＝6.5Hz,1H),3.52(t,J＝7.0Hz,2H),2.71(t,J＝7.0Hz,2H),1.54(d,J＝6.6Hz,6H)；ESI-MS(m/z):358.2[M+H]⁺,356.2[M-H]^-.

Example 10

Preparation of (3- (5-chlorothien-2-yl) -1- (2, 2-difluoroethyl) -1H-indazol-5-yl) glycine (I-10)

Referring to the synthesis of I-4, a solid was obtained in a yield of about 24mg of 25.9%.

M.P.173-175℃,¹H-NMR(300MHz,DMSO-d6):δ7.64(d,J＝9.2Hz,1H),7.54(d,J＝3.5Hz,1H),7.22(d,J＝8.3Hz,3H),6.42(t,J＝54.7Hz,1H),4.91(t,J＝15.0Hz,2H),4.07(s,2H).

Example 11

Preparation of 3- (5- ((carboxymethyl) amino) -3- (5-chlorothien-2-yl) -1H-indazol-1-yl) propionic acid (I-11)

Referring to the synthesis of I-4, about 30mg of solid was obtained with a yield of 28.2%.

M.P.111-115℃,¹H-NMR(300MHz,DMSO-d6):δ7.67(dd,J＝9.0,4.1Hz,1H),7.51(d,J＝4.0Hz,1H),7.41(d,J＝10.0Hz,1H),7.29(d,J＝9.0Hz,1H),7.23(d,J＝3.9Hz,1H),4.61(t,J＝6.5Hz,2H),4.12(s,2H),2.93(t,J＝6.5Hz,2H).

Example 12

Preparation of (3- (5-chlorothien-2-yl) -1- (3- (methoxycarbonyl) benzyl) -1H-indazol-5-yl) glycine (I-12)

Referring to the synthesis of I-4, about 30mg of solid was obtained in 24.8% yield.

¹H-NMR(300MHz,DMSO-d6):δ7.84(d,J＝8.3Hz,2H),7.70(d,J＝9.1Hz,1H),7.54(d,J＝4.0Hz,1H),7.46(dd,J＝4.6,1.9Hz,2H),7.39(s,1H),7.26(dd,J＝9.8,2.6Hz,2H),5.73(s,2H),4.11(s,2H)；HRMS(ESI-TOF):called for C₂₁H₁₆ClN₃O₄S[M+H]⁺442.0550,found442.0627.

Example 13

Preparation of 1- ((3- (5-chlorothien-2-yl) -1- (2, 2-difluoroethyl) -1H-indazol-5-yl) carbamoyl) cyclopropane-1-carboxylic acid (I-13)

Referring to the synthesis of I-5, about 30mg of solid was obtained with a yield of 25.8%.

M.P.152-155℃,¹H-NMR(300MHz,DMSO-d6):δ10.76(s,1H),8.54–8.46(m,1H),7.76(d,J＝9.1Hz,1H),7.62(dd,J＝9.1,1.8Hz,1H),7.47(d,J＝4.0Hz,1H),7.27(d,J＝3.9Hz,1H),6.46(tt,J＝54.7,3.4Hz,1H),4.97(td,J＝15.2,3.2Hz,2H),1.47(d,J＝3.2Hz,4H)；HRMS(ESI-TOF):called for C₁₈H₁₄ClF₂N₃O₃S[M+H]⁺426.0412,found 426.0487.

Example 14

Preparation of 1- (3- (3-chlorophenyl) -1- (2, 2-difluoroethyl) -1H-indazol-5-yl) -5-oxopyrrolidine-3-carboxylic acid (I-14)

Referring to the synthesis of I-6, about 28mg of a solid was obtained with a yield of 21.9%.

M.P.174-178℃，¹H NMR(400MHz,DMSO-d₆):δ12.62(s,1H),8.17(s,1H),7.94–7.93(m,2H),7.83(d,J＝1.6Hz,2H),7.59(t,J＝8.1Hz,1H),7.53–7.50(m,1H),6.51(tt,J＝54.7,3.5Hz,1H),5.03(td,J＝15.2,3.4Hz,2H),4.21–4.09(m,2H),3.40(tt,J＝8.9,4.9Hz,1H),2.84–2.71(m,2H).HRMS(ESI-TOF):called for C₂₀H₁₆ClF₂N₃O₃[M+H]⁺420.0848,found 420.0980.

Example 15

Preparation of 2- (4- (((3- (3-chlorophenyl) -1- (2, 2-difluoroethyl) -1H-indazol-5-yl) amino) methyl) phenyl) acetic acid (I-15)

Step a. Synthesis of 2- (4- (((3- (5-chlorothien-2-yl) -1- (2, 2-difluoroethyl) -1H-indazol-5-yl) amino) methyl) phenyl) acetic acid (I-15)

100mg of the crude product was taken in a 10ml round-bottom flask, and 42mg (0.26mmol) of 2- (4-formylphenyl) acetic acid, 97mg (0.38mmol) of dihydropyridine and 21ul (0.28mmol) of trifluoroacetic acid, together with an appropriate amount of molecular sieves, dissolved in 5ml of dichloromethane and stirred at 45 ℃ under reflux. After 12h of reaction, TLC detects that the raw materials are reacted completely, and the reaction is stopped. The reaction mixture was filtered under suction, and the filtrate was purified by column chromatography (dichloromethane: methanol: 100:1) to obtain 30mg of a pale yellow solid with a yield of 20.4%.

M.P.134-136℃，¹H NMR(400MHz,DMSO-d₆):δ12.27(s,1H),7.79–7.78(m,1H),7.76(d,J＝7.9Hz,1H),7.54–7.50(m,2H),7.43(ddd,J＝8.0,2.0,0.9Hz,1H),7.38(d,J＝8.0Hz,2H),7.23(d,J＝8.0Hz,2H),7.02(dd,J＝9.1,1.9Hz,1H),6.87(d,J＝1.6Hz,1H),6.43(tt,J＝54.8,3.6Hz,2H),4.87(td,J＝15.0,3.5Hz,2H),4.32(s,2H),3.53(s,2H).HRMS(ESI-TOF):called for C₂₄H₂₀ClF₂N₃O₂[M+H]⁺456.1212,found 456.1295.

Example 16

Preparation of 4- ((3- (5-chlorothien-2-yl) -1- (2, 2-difluoroethyl) -1H-indazol-5-yl) amino) phenylacetic acid (I-16)

Step a. Synthesis of ethyl 2- (4- ((3- (5-chlorothien-2-yl) -1- (2, 2-difluoroethyl) -1H-indazol-5-yl) amino) phenyl) acetate

150mg of the crude product was taken out and put into a 25ml two-necked flask, 97mg (0.40mmol) of ethyl p-bromophenylacetate and 5ml of toluene were added and dissolved, and 261mg (0.80mmol) of cesium carbonate, 9mg (0.04mmol) of palladium acetate and 50mg (0.08mmol) of BINAP were added, and the system was evacuated, protected with nitrogen and refluxed at 120 ℃. After 24h of reaction, TLC detects that the raw materials are reacted completely, and the reaction is stopped. The reaction mixture was spin-dried, directly granulated, and purified by column chromatography (petroleum ether: ethyl acetate: 8:1) to give about 55mg of a brown oil, yield 29.0%.

Synthesis of 4- ((3- (5-chlorothien-2-yl) -1- (2, 2-difluoroethyl) -1H-indazol-5-yl) amino) phenylacetic acid (I-16)

A15 ml round bottom flask was charged with 100mg (0.21mmol) of the starting material and 47mg (0.84mmol) of potassium hydroxide, dissolved in 3ml of methanol and 2ml of water, and stirred with heating at 100 ℃. After 2h of reaction TLC check that the starting material has been hydrolysed. Adjusting pH to neutral with 15% diluted hydrochloric acid, precipitating solid, extracting with ethyl acetate for 3 times, mixing organic phases, drying with anhydrous sodium sulfate, concentrating, and recrystallizing to obtain solid 27mg with yield of 28.7%.

M.P.119-122℃，¹H NMR(400MHz,DMSO-d₆):δ12.23(s,1H),8.20(s,1H),7.70(d,J＝9.0Hz,1H),7.61(s,1H),7.44(d,J＝3.9Hz,1H),7.32–7.29(m,1H),7.22(d,J＝3.8Hz,1H),7.13(d,J＝8.3Hz,2H),7.03(d,J＝8.3Hz,2H),6.45(tt,J＝54.6,3.0Hz,1H),4.94(td,J＝15.1,2.9Hz,2H),3.46(s,2H).HRMS(ESI-TOF):called for C₂₁H₁₆ClF₂N₃O₂S[M+H]⁺448.0620,found448.0733.

Example 17

Preparation of 2- (5- ((3- (5-chlorothien-2-yl) -1- (2, 2-difluoroethyl) -1H-indazol-5-yl) amino) pyridin-2-yl) acetic acid (I-17)

Referring to the synthesis of I-16, about 21mg of a solid was obtained in a yield of 23.1%.

M.P.115-118℃，¹H NMR(400MHz,DMSO-d₆):δ12.32(s,1H),8.41(s,1H),8.27(d,J＝2.8Hz,1H),7.74(d,J＝9.0Hz,1H),7.65(d,J＝2.0Hz,1H),7.47(dd,J＝11.1,3.4Hz,2H),7.34–7.31(m,1H),7.23–7.20(m,2H),6.46(tt,J＝54.7,3.6Hz,1H),4.95(td,J＝15.3,3.6Hz,2H),3.65(s,2H).HRMS(ESI-TOF):called for C₂₀H₁₅ClF₂N₄O₂S[M+H]⁺449.0572,found 449.0832.

Example 18

Preparation of methyl 2- (4- ((3- (5-chlorothien-2-yl) -1- (2, 2-difluoroethyl) -1H-indazol-5-yl) amino) -2-fluorophenyl) acetate (I-18)

Step a. Synthesis of methyl 2- (4- ((3- (5-chlorothien-2-yl) -1- (2, 2-difluoroethyl) -1H-indazol-5-yl) amino) -2-fluorophenyl) acetate (I-18)

150mg of the crude product was taken out and placed in a 25ml two-necked flask, 107mg (0.43mmol) of methyl 2- (4-bromo-2-fluorophenyl) acetate and 5ml of toluene were added and dissolved, and 312mg (0.96mmol) of cesium carbonate, 22mg (0.1mmol) of palladium acetate and 125mg (0.2mmol) of BINAP were added, and the system was evacuated, protected with nitrogen and stirred at 120 ℃ under reflux. After 24h of reaction, TLC detects that the raw materials are reacted completely, and the reaction is stopped. The reaction mixture was spin-dried, directly granulated, and purified by column chromatography (petroleum ether: ethyl acetate: 8:1) to give about 35mg of a brown oil in 22.4% yield.

¹H NMR(400MHz,DMSO-d₆):δ8.43(s,1H),7.75–7.72(m,1H),7.68(d,J＝1.7Hz,1H),7.49(d,J＝4.0Hz,1H),7.35(dd,J＝9.0,2.0Hz,1H),7.22(d,J＝3.9Hz,1H),7.18–7.13(m,1H),6.82(dd,J＝8.3,2.1Hz,1H),6.76(dd,J＝12.5,2.1Hz,1H),6.46(tt,J＝54.7,3.5Hz,1H),4.95(td,J＝15.2,3.2Hz,2H),3.63(s,3H),3.60(s,2H).HRMS(ESI-TOF):called for C₂₁H₁₆ClF₂N₃O₂S[M+H]⁺480.0682,found 480.0807.

Example 19

In vitro PDE4B enzyme inhibition Activity assay

PDE4B enzyme inhibitory activity was assessed by reacting fluorescently labeled cAMP as a substrate with PDE4B1, and measuring the intensity of fluorescence polarization signal of the remaining substrate to indirectly determine the effect of the compound on the enzyme activity. Specific results are shown in table 1.

PDE4B inhibitory Activity of the Compounds of the examples in Table 1

Name of Compound	1 μ M inhibition rate	Inhibition rate of 10. mu.M
			Control group: rolipram	82％	99％
I-1	14％	52％
			I-2	20％	61％
I-3	28％	67％
			I-4	58％	89％
I-5	32％	74％
			I-6	47％	81％
I-7	18％	49％
			I-8	38％	78％
I-9	10％	46％
			I-10	75％	94％
I-11	62％	84％
			I-12	11％	52％
I-13	13％	49％
			I-14	46％	91％
I-15	37％	81％
			I-16	25％	76％
I-17	22％	65％
			I-18	18％	56％

As shown in the above table, the compounds of the present invention have good PDE4B inhibitory activity.

Example 20

In vitro cell activity TNF-alpha inhibition assay

The RAW264.7 macrophages obtained by isolation were inoculated in a 96-well plate in RPMI 1640 medium containing 10% fetal bovine serum and 1% penicillin/streptomycin. Control and experimental groups were formed by adding DMSO-dissolved rolipram and test compounds (1uM and 10uM) to each well. The inoculated cells were placed at 37 ℃ in 5% CO₂Incubation in a saturated humidity incubator, and addition of LPS solution (0.1. mu.g. mL) to each well after 2 hours^－1)10 μ L of the solution was placed in CO₂Incubate in incubator for 24 h. And taking out the 96-well plate after 24h, sucking out the culture solution of each well to a 1.5mL centrifuge tube, centrifuging for 20min, and taking the supernatant for measuring the content of the TNF-alpha. As shown in FIG. 1, the compound showed a good effect on TNF-a inhibitor (p)<0.001)。

The above description is only of the preferred embodiments of the present invention, and it should be noted that: it will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the principles of the invention and these are intended to be within the scope of the invention.