A thienyl, pyridyl-containing steroidal pyrazole-thiazolone derivative, and a preparation method and application thereof

Abstract

The application discloses a steroid pyrazole-thiazolone derivative containing thiophene and pyridine, a preparation method and application thereof. The steroid pyrazole-thiazolone derivative containing thiophene and pyridine provided by the application can significantly inhibit the production of NO (nitric oxide) of BV-2 microglial cells induced by LPS (lipopolysaccharide), wherein the anti-inflammatory activity of compound 2 is the most significant, the IC50 value of the compound 2 is 2.05 micromoles / L, which is superior to the positive control progesterone, and the compound 2 can inhibit the expression of downstream inflammatory proteins iNOS and COX-2 by inhibiting the NF-kappa B signal pathway, thereby exerting the anti-neuroinflammatory activity, and the compound 2 can be used for preparing a medicine for treating and / or preventing neurodegenerative diseases; the compound 2 is stable in structure, convenient to store, and can be developed as a lead derivative for preventing and treating neurodegenerative diseases (such as Alzheimer's disease), neuroinflammatory diseases and other related diseases.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of drug design and medicinal chemistry, in particular to a kind of containing thiophene, pyridine's steroidal pyrazole-thiazolone derivative and its preparation method and application. BACKGROUND

[0002] Neuroinflammation is a common protective mechanism of the central nervous system (CNS) against various external stimuli and pathogens. However, excessive or persistent neuroinflammatory processes can lead to neuronal damage and cause neurodegeneration-related diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), and amyotrophic lateral sclerosis (ALS). Among them, Alzheimer's disease, with the highest incidence, has been listed by the World Health Organization as one of the five major diseases that seriously endanger human health in the 21st century.

[0003] With the intensification of global population aging and the extension of human lifespan, the number of Alzheimer's disease patients is increasing year by year. It is estimated that by 2030, the number of Alzheimer's disease patients worldwide will reach 74.7 million, which will greatly reduce the quality of life of the relevant population and increase the burden on patients' families and the entire society. Although basic and clinical research has begun to explore the pathogenesis and development of neurodegenerative diseases, there are still many problems to be solved, and the development of therapeutic drugs for neurodegenerative diseases is a top priority.

[0004] Currently, drugs for treating neuroinflammation and neurodegeneration-related diseases in clinical practice can only temporarily relieve symptoms and have side effects, and cannot slow down or stop the disease progression. Therefore, it is of great significance to develop safe and effective new drugs for preventing and treating neurodegenerative diseases.

[0005] In the human body, microglia are a type of mononuclear macrophage in the brain and play an important role in the immune defense and tissue repair response of the central nervous system. The process of neuroinflammation is accompanied by the sustained activation of microglia. Activated microglia can release various pro-inflammatory factors, leading to direct and indirect neurotoxic effects. Under pathological conditions in the brain, microglia are activated and extensively release various pro-inflammatory mediators, including nitric oxide (NO), prostaglandin E2 (PGE2), and pro-inflammatory cytokines such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, and reactive oxygen species (ROS), thereby inducing neuronal cell damage and leading to direct and indirect neurotoxic effects.

[0006] Increasing evidence suggests that inhibiting microglia-mediated neuroinflammation is an important and direct strategy for treating neurodegenerative diseases. Therefore, modulating neuroinflammation is a potential strategy for alleviating and treating neuroinflammation-related diseases.

[0007] Steroids are a large class of secondary metabolites that are widely present in plants and animals. According to research reports, many steroids have various biological activities and great application potential. In particular, some of them have been used in clinical and preclinical studies. Biomedical research shows that some steroids, such as pregnenolone, progestins, dehydroepiandrosterone (DHEA), dihydrotestosterone (DHT), spiro-cyclic epoxy neurosteroid compounds, 22R hydroxycholesterol (SP222) and SP233, are involved in various central nervous system functions, including reducing neuroinflammation. It is suggested that the aromatase-mediated aromatization of pregnenolone and DHEA to estradiol also has neuroprotective effects, and may be part of the neuroprotective endogenous mechanism.

[0008] Nitrogen-containing heterocyclic drugs are ubiquitous due to their physicochemical properties and pharmacological activities, such as anti-tumor, anti-inflammatory and anti-bacterial effects. Element nitrogen is indispensable in the design and development of new drugs. As a very important pharmacophore, dihydropyrazole is an important nitrogen-containing heterocyclic compound, which has been widely used in the medical field due to its high efficiency, low toxicity and multi-directional transformation of ring substituents. In recent years, many new pyrazole drugs have been commercialized, and in-depth research on pyrazole compounds has become one of the hotspots in drug design and synthesis. In addition, many thiazolone derivatives exhibit strong biological activity and low toxicity, but few reports have designed and synthesized steroid compounds containing dihydropyrazole and thiazolone. The combination of the ring substructure of dihydropyrazole and thiazolone may show synergistic anti-inflammatory effects, so the development of safe and effective new drugs for the prevention and treatment of neurodegenerative diseases, steroid pyrazole-thiazole heterocyclic compounds, has become one of the research hotspots in the medical field in China and even the world.

[0009] Molecular hybridization is a commonly used strategy in drug design. Hybrid molecules usually exhibit better activity and lower toxicity. Steroidal 4,5-dihydropyrazole-thiazolone derivatives are a very important pharmacophore with significant therapeutic effects on inflammation. Due to the universality of steroids and pyrazoles and thiazoles in many biological and chemical applications, the development of steroid compounds containing pyrazole and thiazole linkage has important value.

[0010] Based on this, the present application designs and synthesizes a series of novel D-ring substituted steroid pyrazole-thiazolone heterocyclic steroid derivatives containing thiophene and pyridine, and evaluates their in vitro inhibitory anti-inflammatory effects on LPS-induced BV-2 microglia-mediated neuroinflammation, expecting to have better biological activity, higher selectivity and lower toxicity.

[0011] In summary, how to provide a nitrogen-containing heterocyclic steroid derivative with anti-neuroinflammatory activity is a problem that those skilled in the art urgently need to solve. SUMMARY

[0012] The present application is directed to the deficiencies of the prior art, overcomes the low selection of anti-neuroinflammatory drugs available in clinical practice, large side effects and other shortcomings, and provides a new multifunctional anti-neuroinflammatory steroidal pyrazole-thiazolone derivative containing thiophene and pyridine.

[0013] The new steroidal pyrazole-thiazolone derivative containing thiophene and pyridine provided by the present application has good anti-inflammatory activity on LPS-induced BV-2 microglial cells; the derivative is easy to obtain, the preparation method is simple to operate, the raw materials are easy to obtain, the preparation and development cost is low, the structure is stable, the storage is convenient, and the derivative can be widely used in the preparation of anti-neuroinflammatory and neurodegenerative disease drugs. The present application first studies the anti-neuroinflammatory activity of the new steroidal pyrazole-thiazolone derivative containing thiophene and pyridine, and elucidates the mechanism of action.

[0014] Another object of the present application is to provide a preparation method of the steroidal pyrazole-thiazolone derivative containing thiophene and pyridine.

[0015] The 10 compounds obtained by the steroidal pyrazole-thiazolone derivative containing thiophene and pyridine and the preparation method can significantly inhibit the production of NO in LPS-induced BV-2 microglial cells, and the activity of compound 2 is the strongest. Further research on compound 2 shows that compound 2 can inhibit the NF-κB signaling pathway to inhibit inflammatory mediators PGE2, IL-1β and TNF-α and inflammatory proteins iNOS and COX-2, combine and occupy the active site of iNOS to inhibit the production of NO and play an anti-neuroinflammatory activity.

[0016] Another object of the present application is to provide the use and application of the steroidal pyrazole-thiazolone derivative containing thiophene and pyridine in the preparation of anti-neuroinflammatory and neurodegenerative disease drugs.

[0017] To achieve the above-mentioned application purposes, the present application adopts the following technical solutions:

[0018] A steroidal pyrazole-thiazolone derivative containing thiophene and pyridine is 3β-hydroxy-pregn-5-ene-17β-yl-5'-(heterocyclic substituent)-1'-(4''-ketone-[1'',3'']-thiazole-2''-yl)-4',5'-dihydropyrazol-3'-yl, and the structure of the derivative is shown as formula (I):

[0019] ;

[0020] wherein the heterocyclic substituent HET is selected from a thiophene heteroaromatic ring or a pyridine heteroaromatic ring with a carbon atom number of 4-8

[0021] ,

[0022] The heteroaromatic ring can also be substituted by one or more substituents selected from alkyl, alkoxy or halogen having 1-3 carbon atoms.

[0023] When the heterocyclic substituent HET is selected from the substituents are selected from alkyl or halogen having 1-3 carbon atoms;

[0024] When the heterocyclic substituent HET is selected from the substituents are selected from alkoxy or halogen having 1-3 carbon atoms;

[0025] Preferably, the heterocyclic substituent HET is selected from one of the following structures:

[0026]

[0027] Further, the preparation method of the thienyl-pyridyl-containing steroidal pyrazole-thiazolone derivative comprises the following steps:

[0028] Step 1: the pregnenolone shown in formula (II), an organic solvent, a base solution and a heteroaromatic aldehyde derivative HET are mixed, a Claisen-Schmidt aldol condensation reaction is carried out at a temperature of 25-36°C, the reaction is tracked and monitored by TLC (thin layer chromatography) after 24-72 h, the reaction product is precipitated in the form of a solid, the reaction liquid is poured into ice water and dilute hydrochloric acid is added, the pH value of the reaction liquid is adjusted to pH=7 with dilute hydrochloric acid, the solid-liquid precipitate is separated at room temperature and left to stand for 6 hours, and then filtration, washing and drying are carried out to obtain the steroidal chalcone derivative, i.e. intermediate 1, the molar ratio of the substituted heteroaromatic aldehyde derivative HET to the pregnenolone is 1.5:1;

[0029]

[0030] Step 2: the dried steroidal chalcone derivative, i.e. intermediate 1, an organic solvent, a thiosemicarbazide and a base solution are stirred and mixed, a nucleophilic addition reaction is carried out under heating reflux at 80°C, the reaction progress is tracked and monitored by TLC, after the reaction is completed, the reaction liquid is poured into ice water, the pH value of the reaction liquid is adjusted to pH=7 with dilute hydrochloric acid, the mixture is left to stand at room temperature for 6 hours, and then suction filtration and washing with anhydrous ethanol and water are carried out to obtain the thioamide derivative, i.e. intermediate 2.

[0031] The molar ratio of the intermediate 1 to the thiosemicarbazide is 1:3, and 3 mmol of the thiosemicarbazide is added for each 1 mmol of the chalcone derivative, i.e. intermediate 1;

[0032]

[0033] Step 3: the thioamide derivative, i.e. intermediate 2, an organic acid solution, sodium acetate, chloroacetic acid and an organic solvent are mixed, and a ring condensation reaction is carried out by stirring and heating at 80°C under reflux, the progress of the reaction is monitored by TLC, then the reaction solution is concentrated under vacuum, and the target derivative is obtained by purification by column chromatography.

[0034] Preferably, in step 3, the mass ratio of the thioamide derivative, i.e. intermediate 2, to chloroacetic acid is 1:3, and the eluent used in column chromatography is ethyl acetate: petroleum ether = 7:3.

[0035]

[0036] Further, the organic solvent is anhydrous ethanol solution, the organic acid solution is glacial acetic acid solution, the alkali solution is concentrated sodium hydroxide or potassium hydroxide solution, the developing agent is added when the progress of the reaction is monitored by TLC, and the eluent is added when the purification is carried out by column chromatography.

[0037] The preparation method provided by the present application can successfully prepare the above-mentioned steroid pyrazole-thiazolone derivative containing thiophene and pyridine, and has the advantages of simple preparation method and high preparation yield.

[0038] Preferably, the heteroaromatic aldehyde derivative HET in step 1 is one of the following structures:

[0039]

[0040] Preferably, the time of heating under reflux in steps 2 and 3 is determined by monitoring the reaction by TLC.

[0041] The application of TLC in monitoring the reaction can conveniently determine whether the substituted benzaldehyde and pregnenolone are completely consumed, so as to determine whether the reaction is completed. In step 2, the developing agent added when the progress of the reaction is monitored by TLC is petroleum ether: ethyl acetate = 2:1, v / v In step 3, the developing agent added when the progress of the reaction is monitored by TLC is ethyl acetate: petroleum ether = 2:1, v / v

[0042] Preferably, in step 3, the mass ratio of the thioamide derivative, i.e. intermediate 2, to chloroacetic acid is 1:3, and the eluent used in column chromatography is ethyl acetate: petroleum ether = 7:3. v / v ， When the eluent is ethyl acetate: petroleum ether = 7:3, v / v the target derivative with the most ideal purity can be separated.

[0043] The application of the above-mentioned steroid pyrazole-thiazolone derivative containing thiophene and pyridine in preparing anti-inflammatory drugs is also within the protection scope of the present application.​​

[0044] Preferably, the steroidal pyrazole-thiazolidinone derivatives containing thiophene and pyridine are used in the preparation of drugs for the prevention and treatment of neuroinflammatory diseases and neurodegenerative diseases.

[0045] This invention investigated the inhibitory activity of all synthesized compounds against LPS-induced neuroinflammation using the NO assay. 50 The IC50 values ​​of most compounds showed excellent NO-inhibiting activity, and the IC50 values ​​of the obtained compounds were [data missing]. 50 The values ​​ranged from 2.05 μmol / L to 19.88 μmol / L, with compounds 2 and 4 showing the most significant effects, IC50... 50 The values ​​were 2.05 μmol / L and 2.42 μmol / L, respectively, which were superior to the positive control progesterone (3.23 μmol / L). The results indicate that when the heterocyclic substituent on the dihydropyrazole structure is thiophene, the drug's anti-neuritis activity is superior to that when the heterocyclic substituent is pyridine. In summary, the position of the substituent on the dihydropyrazole and the differences in the substituents themselves affect its inhibitory activity against NO. The good anti-neuritis activity of these derivatives provides new ideas and references for the modification of steroidal pyrazole-thiazolone derivatives and the development of anti-neuritis drugs.

[0046] Preferably, the steroidal pyrazole-thiazolidinone derivative containing thiophene and pyridine can inhibit the levels of PGE2, IL-1β and TNF-α in neuroinflammatory cells.

[0047] Preferably, further studies have shown that compound 2 can inhibit the NF-κB signaling pathway to suppress the inflammatory proteins iNOS and COX-2, bind to and occupy the active site of iNOS to inhibit NO production, and exhibit significant anti-neuritis activity.

[0048] Preferably, the thiophene-pyridine-containing steroidal pyrazole-thiazolidinone derivatives are used to prepare drugs that inhibit the activity of nitric oxide synthase and / or cyclooxygenase-2.

[0049] Preferably, the neurodegenerative diseases include, but are not limited to, Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), Huntington's disease (HD), or Parkinson's disease (PD).

[0050] Preferably, the drug dosage form includes tablets, pills, capsules, injections, suspensions, or emulsions.

[0051] As can be seen from the above technical solution, compared with the prior art, the beneficial effects achieved by the present invention are as follows:

[0052] 1. The steroid pyrazole-thiazolone derivative containing thiophene and pyridine provided by the application has obvious inhibitory effect on the NO produced by BV-2 microglial cells induced by LPS, and shows obvious structure-activity correlation, indicating that the steroid pyrazole-thiazolone derivative containing thiophene and pyridine provided by the application can be used as an anti-neurogenic small molecule drug, and has development and application potential in the prevention and treatment of neurodegenerative diseases.

[0053] 2. The steroid pyrazole-thiazolone derivative containing thiophene and pyridine provided by the application can significantly inhibit the production of NO of BV-2 microglial cells induced by LPS, and in particular, the IC50 value of compound 2 (3β-hydroxy-pregn-5-ene-17β-yl-5'-(thiophene-3-yl)-1'-(4''-ketone-[1'',3'']-thiazole-2''-yl)-4',5'-dihydropyrazol-3'-yl) is as low as 2.05 μmol / L, and the anti-neurogenic activity is better than that of progesterone, and can effectively inhibit the release of inflammatory factors PGE2, IL-1β and TNF-α produced by BV-2 microglial cells after LPS stimulation. Further research shows that compound 2 can inhibit the NF-κB signaling pathway to inhibit downstream inflammatory proteins iNOS and COX-2, thereby exerting its anti-neurogenic activity.

[0054] 3. The anti-neurogenic activity of the steroid pyrazole-thiazolone derivative containing thiophene and pyridine is studied for the first time, the mechanism is clarified, the material basis research of the steroid pyrazole-thiazolone derivative is enriched, and powerful data support and reference are provided for the development of new, efficient, low-toxicity and small-side-effect nitrogen heterocyclic steroid derivative anti-neurogenic drugs. The derivative provided by the application has excellent anti-inflammatory activity and lower cytotoxicity, and has important significance in the preparation of drugs for preventing and treating inflammation-related diseases. BRIEF DESCRIPTION OF DRAWINGS

[0055] Figure 1 The structural formula of the steroid pyrazole-thiazolone derivative containing thiophene and pyridine of the application.

[0056] Figure 2 The impact column chart of the inhibitory effect of the compounds 1-10 prepared in Examples 1-10 on the production of NO of BV-2 cells induced by LPS.

[0057] Figure 3 The impact column chart of the effect of the compounds 1-10 prepared in Examples 1-10 on the activity of BV-2 cells induced by LPS.

[0058] Figure 4 The impact column chart of the effect of compounds 2, 4 and progesterone prepared in Examples 1-10 on the inflammatory factor PGE2 secreted by BV-2 cells induced by LPS.

[0059] Figure 5 The effect of the compounds 2, 4 and progesterone prepared in the present application on the secretion of inflammatory factor TNF-α of BV-2 cells induced by LPS is shown in the column chart.

[0060] Figure 6 The effect of the compounds 2, 4 and progesterone prepared in the present application on the secretion of inflammatory factor IL-1β of BV-2 cells induced by LPS is shown in the column chart.

[0061] Figure 7 The effect of the compound 2 (corresponding to compound 2 of Example 2) of the present application on the expression of COX-2 and iNOS in BV-2 cells induced by LPS is shown in the schematic diagram.

[0062] Figure 8 The effect of the compound 2 (corresponding to compound 2 of Example 2) of the present application on the expression of iNOS in BV-2 cells induced by LPS is shown in the column chart.

[0063] Figure 9 The effect of the compound 2 (corresponding to compound 2 of Example 2) of the present application on the expression of COX-2 in BV-2 cells induced by LPS is shown in the column chart. DETAILED DESCRIPTION

[0064] The technical solutions of the present application are further illustrated below through specific examples.

[0065] The examples are only used for illustrating the present application and not used for limiting the scope of the present application. The experimental methods not specified in the following example are generally according to the conventional conditions in the art or according to the conditions suggested by the manufacturers; the raw materials, reagents and the like used, if not specially specified, are all the raw materials and reagents that can be obtained through commercial channels, etc. Any non-essential changes and substitutions made by the person skilled in the art on the basis of the present application all belong to the scope of the present application.

[0066] Example 1: Synthesis of compound 1

[0067] The compound 1 provided in the present example is 3β-hydroxy-pregn-5-ene-17β-yl-5’-(thiophen-2-yl)-1’-(4’’-keto-[1’’’,3’’ ]-thiazol-2’’-yl)-4’,5’-dihydropyrazol-3’-yl, and its structural formula is as follows:

[0068]

[0069] The preparation method of the compound is as follows:

[0070] (1) Preparation of intermediate 1:

[0071] The substituted heteroaromatic aldehyde: 2-thiophene carboxaldehyde (1.12 g, 0.01 mol), an equimolar amount of pregnenolone (3.16 g, 0.01 mol), NaOH solution (3 g NaOH dissolved in 10 mL of water), and anhydrous ethanol (30 mL) were mixed, stirred at 25°C, and subjected to a Claisen-Schmidt aldol condensation reaction for 48 h, and the reaction progress was monitored by TLC. The reaction solution was poured into ice water and diluted hydrochloric acid was added. The pH value of the reaction solution was adjusted to pH = 7 with diluted hydrochloric acid to neutralize it. Solid-liquid precipitation was performed, and the mixture was allowed to stand for 6 h. After solid-liquid separation and filtration, recrystallization was performed using ethanol, and then filtration, washing, and drying were performed to obtain intermediate 1:

[0072]

[0073] (2) Preparation of intermediate 2:

[0074] Amino thiourea (1.83 g, 0.02 mol), intermediate 1 (4.10 g, 0.01 mol), NaOH solution (3 g NaOH dissolved in 10 mL of water), and anhydrous ethanol (30 mL) were mixed, heated to 80°C to reflux, and subjected to a nucleophilic addition reaction for 8 h. The reaction progress was monitored by TLC (developing agent: chloroform:methanol = 10:1). After the reaction was completed, the reaction solution was poured into ice water, and the pH value of the reaction solution was adjusted to pH = 7 with diluted hydrochloric acid to neutralize it. The product was automatically precipitated, and the mixture was allowed to stand at room temperature for 6 h. Subsequently, it was filtered and washed with anhydrous ethanol and water to obtain intermediate 2: v 石油醚 : v 乙酸乙酯 = 2:1), and the reaction was completed. The reaction solution was poured into ice water, and the pH value of the reaction solution was adjusted to pH = 7 with diluted hydrochloric acid to neutralize it. The product was automatically precipitated, and the mixture was allowed to stand at room temperature for 6 h. Subsequently, it was filtered and washed with anhydrous ethanol and water to obtain intermediate 2:

[0075]

[0076] (3) Preparation of compound 1

[0077] Synthesized intermediate 2 (2.42 g, 0.005 mol), chloroacetic acid (2.0 g, 0.01 mol), sodium acetate, glacial acetic acid, and ethanol (30 mL) were sequentially added to a 50 mL round-bottom flask with stirring, and subjected to a ring condensation reaction at 80°C for 8 h. The reaction progress was monitored by TLC (developing agent: chloroform:methanol = 10:1). After the reaction was completed, the reaction solution was concentrated under vacuum, and the target compound 1 was purified by column chromatography. The eluent used in the column chromatography was ethyl acetate: petroleum ether = 7:3, v 石油醚 : v 乙酸乙酯 = 1:2). After the reaction was completed, the reaction solution was concentrated under vacuum, and the target compound 1 was purified by column chromatography. The eluent used in the column chromatography was ethyl acetate: petroleum ether = 7:3, v / v .

[0078] Compound 1: Light yellow solid powder (Yield: 71.22%), M.p.: 252.6-253.5 °C. IR (KBr, cm -1 ): 2961.65 (C-H), 2934.29 (C-H), 1686.05 (C=O), 1614.19 (C=N), 1555.74 (C=C), 1431.22, 1298.90(C-N), 1237.53, 1047.17, 809.94, 696.39. 1 H NMR (600MHz, δppm, CDCl3): 7.19-7.23 (d, J = 4.80 Hz, 1H, thiopHene C5-H), 7.11-7.15 (d, J =3.60 Hz, 1H, thiopHene C3-H), 6.91-6.95 (m, 1H, thiopHene C4-H), 5.89-5.95 (m,1H, pyrazoline C 5’ -H), 5.36 (s, 1H, C6-H of steroid ring), 3.81 (s, 2H,thiazole C 5’’ -H2), 3.50-3.56 (m, 1H, C3α-H), 3.43-3.49 (m, 1H, pyrazoline C 4’ -H2), 3.14-3.21 (m, 1H, pyrazoline C 4’ -H2), 2.36-2.42 (m, 1H, C 17 α-H), 2.29–2.33(m, 1H), 2.20–2.28 (m, 2H), 2.00–2.08 (m, 1H), 1.91–1.98 (m, 2H), 1.81–1.87(m, 2H), 1.75–1.80 (m, 1H), 1.58–1.63 (m, 3H), 1.43–1.57 (m, 4H), 1.30–1.37(m, 2H), 1.15–1.21 (m, 1H), 1.05–1.13 (m, 1H), 1,01 (s, 3H, C 10 -CH3), 0.96–1.00 (m, 1H), 0.77 (s, 3H, C 13 -CH3).13 C NMR (150MHz, δ ppm, CDCl3): 187.91, 178.18, 165.96, 141.78, 140.92, 127.04, 126.74, 125.22, 121.18, 71.50, 58.62, 56.61, 51.90, 49.98, 47.02, 44.43, 42.17, 38.90, 38.43, 37.27, 36.54, 32.06, 31.69, 31.51, 24.78, 24.34, 21.03, 19.44, 13.58. HRMS (ESI): m / z [M+H] + calcd for C 29 H 38 N3O2S2: 524.2327; found: 524.2499.

[0079] Example 2: Compound 2

[0080] The compound 2 provided in this example is: 3β-hydroxy-pregna-5-ene- 17β-yl-5'-(thiophen-3-yl)-l'-(4"-keto-[1'',3'']-thiazol-2''-yl)-4',5'- dihydropyrazolyl-3'-yl, whose structural formula is as follows:

[0081]

[0082] The preparation method of compound 2 is identical to that of Example 1 except that the substituted heteroaromatic aldehyde HET is 3-thiophenecarboxaldehyde. The final product is obtained:

[0083] Compound 2: orange red solid powder (yield: 67.53%), M.p.: 252.5-257.4 °C. IR (KBr, cm -1 ): 2930.79 (C-H), 1685.96 (C=O), 1614.25 (C=N), 1556.51 (C=C), 1436.15, 1284.09 (C-N), 1235.29, 1047.44, 771.23, 679.99. 1 H NMR (600MHz, δ ppm, CDCl3): 7.27-7.32 (m, 1H, thiopHene-H), 7.20 (s, 1H, thiopHene C2-H), 6.91-6.95 (d, J= 4.80 Hz, 1H, thiopHene-H), 5.71-5.76 (m, 1H, pyrazoline C 5’ -H), 5.36 (s, 1H,C6-H of steroid ring), 3.81 (s, 2H, thiazole C 5’’ -H2), 3.48-3.55 (m, 1H, C3α-H), 3.37-3.44 (m, 1H, pyrazoline C 4’ -H2), 2.99-3.06 (m, 1H, pyrazoline C 4’ -H2),2.36-2.41 (m, 1H, C 17 α-H), 2.29–2.33 (m, 1H), 2.19–2.27 (m, 2H), 1.99–2.06 (m,1H), 1.91–1.98 (m, 1H), 1.81–1.88 (m, 3H), 1.74–1.80 (m, 1H), 1.56–1.65 (m,3H), 1.43–1.53 (m, 3H), 1.26–1.37 (m, 3H), 1.14–1.21 (m, 1H), 1.05–1.13 (m,1H), 1,00 (s, 3H, C 10 -CH3), 0.96–0.99 (m, 1H), 0.70 (s, 3H, C 13 -CH3). 13 C NMR(150MHz, δ ppm, CDCl3):187.93, 177.98, 166.40, 149.93, 139.96, 127.13,125.02, 122.54, 121.16, 71.49, 58.93, 56.53, 51.85, 49.96, 46.21, 44.31,42.16, 38.89, 38.46, 37.26, 36.53, 32.02, 31.68, 31.50, 24.72, 24.35, 20.98,19.44, 13.62. HRMS (ESI): m / z [M+H] + calcd for C 29 H 38 N3O2S2: 524.2327; found:524.2397.

[0084] Example 3: Compound 3

[0085] The compound 3 provided in this example is: 3β-hydroxy-pregn-5-ene-17β-yl-5'-(3- methylthiophen-2-yl)-1'-(4"-keto-[1'',3'']-thiazol-2''-yl)-4',5'-dihydropyrazolyl-3'-yl, whose structural formula is as follows:

[0086]

[0087] The preparation method of compound 3 is identical to that of Example 1 except that the substituted heteroaromatic aldehyde HET is 3-methyl-2-thiophenecarboxaldehyde. The final product is obtained as follows:

[0088] Compound 3: orange yellow solid powder (yield: 74.57%), M.p.: 135.2-139.3 °C. IR (KBr, cm -1 ): 2930.66 (C-H), 1734.10 and 1697.82 (C=O), 1614.19 (C=N), 1540.95 (C=C),1435.62, 1273.43 (C-N), 1221.88, 1045.55, 808.14, 712.28. 1 H NMR (600MHz, δppm, CDCl3): 7.04-7.13 (m, 1H, thiopHene-H), 6.70-6.76 (m, 1H, thiopHene-H),5.80-5.88 (m, 1H, pyrazoline C 5’ -H), 5.36 (s, 1H, C6-H of steroid ring), 3.80(s, 2H, thiazole C 5’’ -H2), 3.51-3.59 (m, 1H, C3α-H), 3.42-3.51 (m, 1H,pyrazoline C 4’ -H2), 2.92-3.10 (m, 1H, pyrazoline C 4’ -H2), 2.36-2.50 (m, 1H, C 17α-H), 2.34 (s, 3H, CH3), 2.11–2.28 (m, 3H), 1.91–2.00 (m, 2H), 1.76–1.87 (m,3H), 1.43–1.59 (m, 6H), 1.29–1.38 (m, 2H), 1.15–1.22 (m, 1H), 1.06–1.12 (m,1H), 1.01 (s, 3H, C 10 -CH3), 0.90–1.00 (m, 2H), 0.77 (s, 3H, C 13 -CH3). 13 C NMR(150MHz, δ ppm, CDCl3):187.98, 178.15, 165.77, 140.94, 135.78, 135.52,130.26, 123.12, 121.20, 71.53, 57.54, 56.62, 56.42, 51.98, 50.01, 47.27,46.70, 44.42, 42.18, 39.01, 38.37, 37.28, 36.53, 32.04, 31.71, 31.52, 24.75,24.39, 19.45, 14.26, 13.47. HRMS (ESI): m / z [M+H] + calcd for C 30 H 40 N3O2S2:538.2484; found: 538.2675.

[0089] Example 4: Compound 4

[0090] The compound 4 provided in this example is 3β-hydroxy-pregna-5-ene-17β-yl-5'-(benzothiophen-2-yl)-1'-(4"-keto-[1'',3'']-thiazol-2''-yl)-4',5'-dihydropyrrolyl-3'-yl, whose structural formula is as follows:

[0091]

[0092] The preparation method of compound 4 is identical with that of Example 1 except that the substituted heteroaromatic aldehyde HET is 1-benzothiophene-2-carboxaldehyde. Finally, the following compound 4 is obtained:

[0093] Compound 4: yellow solid powder (yield: 82.17%), M.p.: 161.1-165.7 °C. IR (KBr, cm-1 ): 2930.24 (C-H), 1732.75 and 1697.70 (C=O), 1544.97 (C=C), 1269.50 (C-N),1222.58, 1045.32, 810.02, 725.87. 1 H NMR (600MHz, δ ppm, CDCl3): 7.67-7.79 (m,2H, benzothiopHene-H), 7.28-7.37 (m, 3H, benzothiopHene-H), 5.93-6.03 (m, 1H,pyrazoline C 5’ -H), 5.37 (s, 1H, C6-H of steroid ring), 3.82 (s, 2H, thiazoleC 5’’ -H2), 3.53-3.63 (m, 1H, C3α-H), 3.45-3.52 (m, 1H, pyrazoline C 4’ -H2), 3.08-3.28 (m, 1H, pyrazoline C 4’ -H2), 2.36-2.54 (m, 1H, C 17 α-H), 2.16–2.35 (m, 3H),1.90–2.03 (m, 3H), 1.76–1.88 (m, 3H), 1.42–1.58 (m, 5H), 1.30–1.40 (m, 2H),1.15–1.22 (m, 1H), 1.06–1.14 (m, 1H), 1.02 (s, 3H, C 10 -CH3), 0.89–0.99 (m,2H), 0.78 (s, 3H, C 13 -CH3). 13C NMR (150 MHz, δ ppm, CDCl3): 187.80, 178.26, 166.05, 141.71, 140.91, 139.17, 139.03, 124.86, 124.68, 124.03, 123.17, 122.42, 121.22, 71.58, 59.52, 56.43, 51.97, 50.02, 46.41, 44.46, 42.19, 38.99, 38.18, 37.27, 36.55, 32.06, 31.71, 31.55, 24.76, 24.41, 21.01, 19.45, 13.42. HRMS (ESI): m / z [M+H] + calcd for C 33 H 40 N3O2S2: 574.2484; found: 574.2642.

[0094] Example 5: Compound 5

[0095] The compound 5 provided in this example is 3β-hydroxy-pregn-5-ene-17β-yl-5'-(4- bromothiophene-2-yl)-l'-(4"-keto-[1'',3'']-thiazol-2''-yl)-4',5'-dihydropyrazol-3'-yl, whose structural formula is as follows:

[0096]

[0097] The preparation method of compound 5 is identical to that of Example 1 except that the substituted heteroaromatic aldehyde HET is 4-bromo-2-thiophenecarboxaldehyde. The final product is obtained as follows:

[0098] Compound 5: white solid powder (yield: 76.28%), M.p.: 257.6-258.1 °C. IR (KBr, cm -1 ): 3116.92 (thiopHene-H), 2962.12 (C-H), 2937.66 (C-H), 1705.01 (C=O), 1622.35 (C=N), 1548.90 (C=C), 1270.06 (C-N), 1234.75, 1056.42, 809.19, 747.40. 1H NMR (600MHz, δ ppm, CDCl3): 7.11 (s, 1H, thiopHene C5-H), 6.95 (s,1H, thiopHene C3-H), 5.80-5.88 (m, 1H, pyrazoline C 5’ -H), 5.35 (s, 1H, C6-H ofsteroid ring), 3.84 (s, 2H, thiazole C 5’’ -H2), 3.49-3.57 (m, 2H, C3α-H andpyrazoline C 4’ -H2), 2.95-3.05 (m, 1H, pyrazoline C 4’ -H2), 2.44-2.51 (m, 1H, C 17 α-H), 2.29–2.34 (m, 1H), 2.21–2.26 (m, 1H), 2.11–2.17 (m, 1H), 2.00–2.05 (m,1H), 1.93–1.99 (m, 1H), 1.90–1.93 (m, 1H), 1.83–1.89 (m, 2H), 1.74–1.82 (m,1H), 1.57–1.69 (m, 4H), 1.44–1.54 (m, 3H), 1.30–1.36 (m, 2H), 1.15–1.22 (m,1H), 1.07–1.12 (m, 1H),1.02 (s, 3H, C 10 -CH3), 0.98–1.00 (m, 1H), 0.71 (s, 3H,C 13 -CH3). 13 C NMR (150MHz, δ ppm, CDCl3):187.66, 178.25, 165.84, 142.94, 140.88,128.27, 122.51, 121.23, 109.63, 71.61, 58.36, 56.44, 51.99, 50.00, 46.43,44.44, 42.20, 39.05, 38.21, 37.26, 36.55, 32.01, 31.69, 31.57, 24.64, 24.43,20.91, 19.45, 13.39. HRMS (ESI): m / z [M+H] + calcd for C29 H 37 BrN3O2S2: 602.1432,604.1412; found: 604.1571.

[0099] Example 6: Compound 6

[0100] The compound 6 provided in this example is 3β-hydroxy-pregn-5-ene-17β-yl-5'-(5- chlorothiophen-2-yl)-l'-(4"-keto-[l'',3'']-thiazol-2''-yl)-4',5'-dihydropyrazolyl-3'-yl, whose structural formula is as follows:

[0101]

[0102] The preparation method of compound 6 is identical to that of Example 1 except that the substituted heteroaromatic aldehyde HET is 5-chloro-2-thiophenecarboxaldehyde. The final product is obtained as follows:

[0103] Compound 6: orange solid powder (yield: 74.49%), M.p.: 223.1-225.4 °C. IR (KBr, cm -1 ): 2934.91 (C-H), 1685.41 (C=O), 1613.62 (C=N), 1557.58 (C=C), 1270.24 (C-N), 1238.17, 1060.20, 797.51, 736.63. 1 H NMR (600MHz, δ ppm, CDCl3): 6.87-6.92(d, J = 3.60 Hz, 1H, thiopHene C5-H), 6.71-6.77 (d, J = 4.20 Hz, 1H, thiopHeneC4-H), 5.77-5.82 (m, 1H, pyrazoline C 5’ -H), 5.36 (s, 1H, C6-H of steroidring), 3.82 (s, 2H, thiazole C 5’’ -H2), 3.50-3.56 (m, 1H, C3α-H), 3.41-3.47 (m,1H, pyrazoline C 4’ -H2), 3.09-3.16 (m, 1H, pyrazoline C 4’ -H2), 2.34–2.41 (m, 1H,C 17α-H), 2.29–2.32 (m, 1H), 2.20–2.26 (m, 2H), 2.00–2.07 (m, 1H), 1.90–1.97(m, 2H), 1.81–1.88 (m, 2H), 1.75–1.80 (m, 1H), 1.55–1.64 (m, 3H), 1.46–1.53(m, 3H), 1.30–1.37 (m, 2H), 1.15–1.21 (m, 1H), 1.06–1.13 (m, 1H), 1.02 (s,3H, C 10 -CH3), 0.98–1.00 (m, 1H), 0.75 (s, 3H, C 13 -CH3). 13 C NMR (150MHz, δ ppm,CDCl3):187.81, 178.35, 165.71, 140.86, 140.21, 130.03, 126.10, 125.98,121.23, 71.60, 58.74, 56.61, 51.88, 49.99, 46.50, 44.43, 42.19, 38.95, 38.49,37.27, 36.55, 32.05, 31.68, 31.55, 24.77, 24.34, 21.05, 19.45, 13.70. HRMS(ESI): m / z [M+H] + calcd for C 29 H 37 ClN3O2S2: 558.1937; found: 558.2115.

[0104] Example 7: Compound 7

[0105] The compound 7 provided in this example is 3β-hydroxy-pregna-5-ene-17β-yl-5'-(pyridin-2-yl)-1'-(4"-keto-[1'',3'']-thiazol-2''-yl)-4',5'-dihydropyrrolyl-3'-yl, whose structural formula is as follows:

[0106]

[0107] The preparation method of compound 7 is identical to that of Example 1 except that the substituted heteroaromatic aldehyde HET is 2-pyridinecarboxaldehyde. The final product is obtained:

[0108] Compound 7: Yellow solid powder (Yield: 63.71%), M.p.: 234.5-239.5 °C. IR (KBr, cm -1 ): 2964.19 (C-H), 2934.35 (C-H), 1684.14 (C=O), 1556.76 and 1472.47 (C=C),1438.32, 1355.34, 1297.28 (C-N), 1241.97, 1048.05. 1 H NMR (600MHz, δ ppm, CDCh): 8.49-8.59 (d, J = 4.80 Hz, 1H, pyridine C6-H), 7.62–7.69 (m, 1H,pyridine C4-H), 7.39–7.46 (d, J =7.20 Hz, 1H, pyridine C3-H), 7.16–7.24 (m, 1H,pyridine C5-H), 5.63-5.71 (m, 1H, pyrazoline C 5’ -H), 5.36 (s, 1H, C6-H of steroid ring), 3.80 (s, 2H, thiazole C 5’’ -H2),3.50-3.56 (m, 1H, C3α-H), 3.33-3.45 (m, 2H, pyrazoline C 4’ -H2), 2.37-2.43 (m, 1H, C 17 α-H), 2.21–2.33 (m, 3H),1.99–2.05 (m, 1H), 1.91–1.98 (m, 2H), 1.81–1.87 (m, 2H),1.73–1.80 (m, 1H),1.43–1.56 (m, 5H), 1.27–1.37 (m, 3H), 1.15–1.20 (m, 1H), 1.06–1.11 (m, 1H),1.01 (s, 3H, C 10 -CH3), 0.95–0.99 (m, 1H), 0.74 (s, 3H, C 13 -CH3). 13C NMR (150 MHz, δ ppm, CDCl3): 187.92, 178.12, 167.11, 157.05, 149.94, 140.87, 136.84, 123.22, 122.52, 121.26, 71.54, 63.77, 56.60, 51.85, 50.01, 44.78, 44.46, 42.19, 38.95, 38.32, 37.27, 36.53, 32.03, 31.71, 31.53, 24.58, 24.38, 21.02, 19.44, 13.12. HRMS (ESI): m / z [M+H] + calcd for C 30 H 39 N4O2S: 519.2715; found:519.2872.

[0109] Example 8: Compound 8

[0110] The compound 8 provided in this example is: 3β-hydroxy-pregn-5-ene-17β-yl-5'-(pyridin-3-yl)-1'-(4"-keto-[1'',3'']-thiazol-2''-yl)-4',5'-dihydropyrazolyl-3'-yl, whose structural formula is as follows:

[0111]

[0112] The preparation method of the compound is identical with that of Example 1 except that the substituted heteroaromatic aldehyde HET is 3-pyridinecarboxaldehyde. The final obtained is:

[0113] Compound: white solid powder (yield: 53.11%), M.p.: 268.5-274.1 °C. IR (KBr, cm -1 ): 2964.01 (C-H), 2924.95 (C-H), 1694.24 (C=O), 1619.08 (C=N), 1549.69 (C=C), 1430.93, 1395.35, 1287.71 (C-N), 1234.46, 1070.09. 1 H NMR (600MHz, δ ppm, CDCl3): 8.53-8.57 (d, J= 5.40 Hz, 1H, pyridine C6-H), 8.46 (s, 1H, pyridineC2-H), 7.49–7.53 (d, J = 7.80 Hz, 1H,pyridine C4-H), 7.27–7.32 (m, 1H, pyridineC5-H), 5.61-5.66 (m, 1H, pyrazoline C 5’ -H), 5.36 (s, 1H, C6-H of steroidring), 3.82 (s, 2H, thiazole C 5’’ -H2), 3.59-3.66 (m, 1H, pyrazoline C 4’ -H2),3.50-3.56 (m, 1H, C3α-H),2.84-2.91 (m, 1H, pyrazoline C 4’ -H2), 2.42-2.48 (m,1H, C 17 α-H), 2.28–2.34 (m, 1H), 2.22–2.27 (m, 1H), 2.12–2.19 (m, 1H), 2.00–2.06 (m, 1H), 1.95–1.99 (m, 1H), 1.90–1.94 (m, 1H), 1.83–1.88 (m, 2H),1.77–1.81 (m, 1H), 1.44–1.59 (m, 6H),1.29–1.36 (m, 2H), 1.15–1.20 (m, 1H),1.07–1.12 (m, 1H), 1.02 (s, 3H, C 10 -CH3), 0.97–1.01 (m, 1H), 0.70 (s, 3H, C 13 -CH3). 13C NMR (150 MHz, δ ppm, CDCl3): 187.63, 178.12, 165.82, 149.55, 147.22, 140.88, 135.32, 133.76, 123.80, 121.21, 71.57, 61.07, 56.43, 51.95, 49.98, 46.73, 44.46, 42.20, 39.01, 38.26, 37.26, 36.55, 32.00, 31.68, 31.56, 24.66, 24.43, 20.91, 19.44, 13.41. HRMS (ESI): m / z [M+H] + calcd for C 30 H 39 N4O2S: 519.2715; found: 519.2936.

[0114] Example 9: Compound 9

[0115] The compound 9 provided in this example is 3β-hydroxy-pregn-5-ene-17β-yl-5'-(6- methoxypyridin-2-yl)-l'-(4"-keto-[1'',3'']-thiazol-2''-yl)-4',5'-dihydropyrazolyl-3'-yl, whose structural formula is as follows:

[0116]

[0117] The preparation method of compound 9 is identical to that of Example 1 except that the substituted heteroaromatic aldehyde HET is 6-methoxypyridine-2-carboxaldehyde. The final product is obtained as follows:

[0118] Compound 9: yellow solid powder (yield: 72.38%), M.p.: 233.1-238.1 °C. IR (KBr, cm -1 ): 2931.38 (C-H), 1691.44 (C=O), 1602.74 and 1579.01 (C=N), 1556.17 and 1466.83 (C=C), 1438.15, 1382.12, 1356.21, 1282.50 (C-N), 1236.17, 1039.66. 1HNMR (600MHz, δ ppm, CDCl3): 7.48-7.54 (m, 1H, pyridine-H), 6.91–6.99 (m, 1H,pyridine-H), 6.61–6.67 (m, 1H, pyridine-H), 5.52-5.62 (m, 1H, pyrazoline C 5’ -H), 5.35 (s, 1H, C6-H of steroid ring), 3.83 (s, 3H, OCH3), 3.80 (s, 2H,thiazole C 5’’ -H2), 3.49-3.56 (m, 1H, C3α-H), 3.33-3.47 (m, 1H, pyrazoline C 4’ -H2), 3.12-3.32 (m, 1H, pyrazoline C 4’ -H2), 2.36-2.50 (m, 1H, C 17 α-H), 2.14-2.34(m, 3H),1.74–2.04 (m, 6H), 1.41–1.59 (m, 6H), 1.28–1.38 (m, 2H), 1.14–1.23(m, 1H), 1.04–1.12 (m, 1H), 0.97–1.03 (m, 4H, C 10 -CH3), 0.90–0.96 (m, 1H),0.71 (s, 3H, C 13 -CH3). 13 C NMR (150MHz, δ ppm, CDCl3):187.92, 178.09, 166.57,154.29, 140.94, 139.24, 121.16, 115.18, 114.72, 110.63, 71.48, 63.60, 56.47,53.36, 51.87, 50.01, 44.72, 44.26, 42.16, 38.92, 38.21, 37.26, 36.51, 32.01,31.71, 31.51, 24.71, 24.40, 20.90, 19.43, 13.28. HRMS (ESI): m / z [M+H] + calcdfor C 31 H 41N4O3S: 549.2821; found: 549.2974.

[0119] Example 10: Compound 10

[0120] The compound 10 provided in this example is 3β-hydroxy-pregn-5-ene- 17β-yl-5'-(6-bromopyridine-3-yl)-l'-(4"-keto-[1'',3'']-thiazole-2''-yl)-4',5'- dihydropyrazolyl-3'-yl, whose structural formula is as follows:

[0121]

[0122] The preparation method of compound 10 is identical with that of Example 1 except that the substituted heteroaromatic aldehyde HET is 6-bromo-3-pyridine carboxaldehyde. The final product is obtained as follows:

[0123] Compound 10: orange solid powder (yield: 80.09%), M.p.: 159.4-163.2 °C. IR (KBr, cm -1 ): 2933.82 (C-H), 1698.19 (C=O), 1548.41 and 1456.53(C=C), 1385.12,1273.75 (C-N), 1239.48, 1088.38, 1045.38. 1 H NMR (600MHz, δ ppm, CDCl3): 8.20-8.26 (m, 1H, pyridine-H), 7.43-7.47 (m, 1H, pyridine-H),7.36-7.40 (m, 1H,pyridine-H), 5.55-5.63 (m, 1H, pyrazoline C 5’ -H), 5.35 (s, 1H, C6-H of steroidring), 3.82(s, 2H, thiazole C 5’’ -H2), 3.54-3.66 (m, 1H, pyrazoline C 4’ -H2),3.49-3.53 (m, 1H, C3α-H), 2.80-2.96 (m, 1H, pyrazoline C 4’ -H2), 2.35-2.45 (m,1H, C 17α-H), 2.20–2.33 (m, 3H), 2.07–2.18 (m, 1H), 1.90–2.01 (m, 2H), 1.77–1.85 (m, 3H), 1.53–1.66 (m, 3H), 1.43–1.52 (m, 3H), 1.28–1.41 (m, 3H), 1.14–1.21 (m, 1H), 1.05–1.11 (m, 1H), 0.99 (s, 3H, C 10 -CH3), 0.95–0.98 (m, 1H),0.69 (s, 3H, C 13 -CH3). 13 C NMR (150MHz, δ ppm, CDCl3):187.60, 178.27, 165.95,147.72, 141.77, 140.93, 136.43, 134.93, 128.41, 121.10, 71.46, 60.43, 56.44,51.79, 49.91, 46.74, 44.42, 42.14, 39.03, 38.34, 37.24, 36.52, 32.00, 31.65,31.48, 24.69, 24.37, 20.91, 19.41, 13.59. HRMS (ESI): m / z [M+H] + calcd forC 30 H 38 BrN4O2S:597.1821, 599.1800; found: 599.1955.

[0124] Example 11:

[0125] 1. Culture of BV-2 microglial cells.

[0126] The mouse microglial cell line BV-2 cells were selected for culture. The cell culture medium containing 10% fetal bovine serum was prepared on the basis of DMEM medium, and the BV-2 microglial cells were subcultured in a 5% CO2, 37°C culture bottle. When the adherent cells reached about 70-80% of the bottom area of the culture bottle on the third day, the adherent cells were trypsinized and subcultured into another culture bottle. The recovered BV-2 microglial cells were used as the first generation, and the 3rd-8th generation of BV-2 cells were selected for subsequent experiments.

[0127] 2. Cell viability detection and anti-neuroinflammatory activity evaluation of the thienyl and pyridyl-containing steroidal pyrazole-thiazolone compounds prepared in Examples 1-10.

[0128] The effect of the steroid pyrazole-thiazolone steroid derivative containing thiophene and pyridine on the viability of mouse BV-2 cells was determined by MTT [3-4(4,5)-dimethyl-2-thiazole-(2,5)-phenyl bromide tetrazolium blue] method. The basic principle is that succinate dehydrogenase in the mitochondria of living cells can reduce exogenous MTT to insoluble blue-purple crystalline, which is dissolved by dimethyl sulfoxide (DMSO), and then the absorbance value is determined at 570 nm by an enzyme-labeled instrument, which can indirectly reflect the number of living cells, so as to evaluate the cytotoxicity. The inhibitory effect of the steroid pyrazole-thiazolone steroid derivative containing thiophene and pyridine on the release of NO from mouse BV-2 cells induced by lipopolysaccharide was detected by the method of Griess.

[0129] (1) Cell plating: in a 96-well plate, add BV-2 cell suspension 1×10 5 μl per well, 37°C, 5% CO2, incubate for 24 hours, then aspirate the old culture medium.

[0130] (2) Drug addition: ① Damage group (without drug): add 200 μl of LPS (diluted with culture medium), the final concentration is 1 μg / ml, 37°C, 5% CO2, incubate for 24 hours. ② Test group (drug + LPS co-action group): first add 100 μl of different concentrations of drugs (general operation: the initial screening concentration is set to 10 μmol / L, and the re-screening is set to 0.625, 1.25, 2.5, 5, 10, 20, 40 μmol / L seven gradients); 60 min later, add LPS 100 μl, the final concentration is 1 μg / ml, the final volume of solution per well is 200 μl, 37°C, 5% CO2, incubate for 24 hours. ③ Negative control group (blank group): add culture medium containing BV-2 cells, the total volume is 200 μl, 37°C, 5% CO2, incubate for 24 hours. ④ Positive control group (progesterone + LPS co-action group): first add 100 μl of different concentrations of progesterone, the final concentration should be 0.625, 1.25, 2.5, 5, 10, 20, 40 μmol / L; 60 min later, add LPS 100 ul, the final concentration is 1 μg / ml, the final volume of solution per well is 200 μl, 37°C, 5% CO2, incubate for 24 hours.

[0131] ④ The progesterone selected for the positive control group is a neuroactive steroid. Progesterone and its related metabolite 3α,5α-THPROG have been proven to have multiple functional effects on the nervous system, including myelination, neuroprotection, and neuropathic pain, etc.

[0132] (3) MTT cytotoxicity assay: 24 hours later, 10 μl of MTT (5 mg / ml) was added to each well, shaken and placed in a carbon dioxide incubator for 4 h, then the culture solution was aspirated, 150 μl of DMSO solution was added to each well, the 96-well plate was placed in the dark and shaken on a shaker for 10-15 min to fully dissolve the formazan, and then the absorbance value OD of each well was measured at 570 nm wavelength using an enzyme marker. Cell survival rate = [(OD 药物 - OD 空白 ) / (OD 阴性对照 - OD 空白 )] x 100%.

[0133] (4) In vitro NO inhibition activity IC 50 value determination:

[0134] The compounds with an NO inhibition rate greater than 80% in the preliminary screening were further tested for their in vitro NO inhibition activity IC 50 value. The final concentration of the compounds and the positive control compound (progesterone) was set to 0.625, 1.25, 2.5, 5, 10, 20, 40 μmol / L, then the operation was performed according to the NO detection kit instructions. After 24 h of culture, 50 μL of supernatant was transferred from each well to a new 96-well plate, then 50 μL of Griess reagent I and II was added in turn, shaken and the absorbance value (A 540 ) at 540 nm wavelength was measured using an enzyme marker. The inhibition rate fitting curve of each compound was obtained by Graph Pad Prism 7.0 software processing, and the IC 50 value was calculated. All data were set in triplicate, and the data were expressed as means ± standard deviation (means ± SD).

[0135] The compound and progesterone preliminary screening concentration was 10 μmol / L, and the re-screening concentration was set to 0.625, 1.25, 2.5, 5, 10, 20, 40 μmol / L. Progesterone was used as the positive control group, LPS (1 μg / mL) was used as the negative control group, and DMSO was used as the blank control group. Each treatment was set in triplicate.

[0136] In vitro anti-neuroinflammatory activity and cytotoxicity evaluation:

[0137] As can be seen from Table 1 and Figure 2 , compounds 1-10 have good inhibitory effect on the production of NO in LPS-induced BV-2 cell neuroinflammation in vitro. The IC 50 values of the NO inhibition activity of all compounds range from 2.05 μmol / L to 19.88 μmol / L. Among them, compounds 2 and 4 show the most significant, IC 50The values were 2.05 μmol / L and 2.42 μmol / L, respectively, which were superior to the positive control progesterone (3.23 μmol / L). The results showed that when the heterocyclic substituent on the dihydropyrazole structure was a thiophene structure, the anti-neurogenic activity of the drug was superior to the activity of the heterocyclic substituent being a pyridine structure. This was the first time that such compounds were found to have good activity in anti-neurogenic, providing experimental and theoretical basis for further expanding the research of steroidal pyrazole-thiazolone derivatives in anti-neurogenic and developing new drugs for treating degenerative neurological diseases.

[0138] The test compound concentration was 10 μmol / L for the in vitro cytotoxicity test (Table 1 and Figure 3 ), and the results showed that compounds 2, 3 and 4 had no significant cytotoxicity (cell survival rate > 80%), and the corresponding cell viability values of compounds 2 and 4 were 92.19 and 83.72%, respectively, which indicated that the activity of inhibiting NO of the compounds was not produced by killing cells, and the cytotoxicity results provided feasibility for further exploration of the experiment. Compound 2 showed good anti-inflammatory activity and low cytotoxicity, and had deep development value. Therefore, we further tested the anti-inflammatory indicators (ELISA method) of compounds 2 and 4 to screen more excellent anti-inflammatory compounds.

[0139]

[0140] Table 1: Effect of compounds on LPS-induced BV-2 cell production of NO and cell viability (10 μmol / L)

[0141] 3. Effect of compounds 2, 4 and progesterone on the secretion of inflammatory factors PGE2, TNF-α and IL-1β by BV-2 cells (ELISA method).

[0142] The dynamic changes of active target compounds 2, 4 and progesterone on the expression of LPS-induced BV-2 cell inflammatory mediators PGE2, TNF-α and IL-1β were observed, the mechanism of inflammatory response of the target compounds was discussed, and theoretical reference basis was provided for subsequent compound design and research.

[0143] The BV-2 cells were seeded at 2×10 4Cells were seeded at density in 96-well plates and pretreated for 1 h with compounds 2.5, 5, 10, and 20 μmol / L, respectively, and progesterone. Cells were then stimulated with LPS (1 μg / mL) for 24 h, after which the cell culture supernatant was collected. The supernatant was centrifuged at 5000 g for 10 min at 4 °C to remove insoluble matter, and the supernatant was collected and stored at -20 °C until assay. The levels of secreted PGE2, TNF-α, and IL-1β in the cell culture supernatant were measured using a commercially available ELISA kit according to the provided instructions. The absorbance at 450 nm for each sample was analyzed using a microplate reader.

[0144] Test results: such as Figure 4 , Figure 5 , Figure 6 As shown, compared with the control group, LPS stimulation of BV-2 cells significantly increased the levels of released PGE2, TNF-α, and IL-1β (P<0.01); different concentrations (2.5, 5, 10, 20 μmol / L) of compounds 2, 4, and progesterone, when co-incubated with LPS-stimulated BV-2 cells, significantly inhibited the release of inflammatory factors PGE2, TNF-α, and IL-1β from BV-2 cells (P<0.01), and this effect was dose-dependent. Experimental results showed that compound 2 (3β-hydroxy-pregn-5-en-17β-yl-5'-(thiophen-3-yl)-1'-(4''-keto-[1'',3'']-thiazolyl-2''-yl)-4',5'-dihydropyrazolyl-3'-yl) exhibited the best anti-inflammatory activity and good anti-neuritis activity. We further explored the anti-inflammatory mechanism of this class of compounds by detecting the effect of compound 2 on the expression levels of COX-2 and iNOS proteins using Western blotting.

[0145] Compared with the LPS group, * P < 0.05; ** P < 0.01.

[0146] Compared with the negative control group, ## P < 0.01.

[0147] 4. Effect of compound 2 on the expression levels of COX-2 and iNOS.

[0148] Protein expression was detected using Western blotting, and the specific steps are as follows:

[0149] (1) Protein sample preparation: Place the treated cell sample on ice, add 200 μL RIPA solution (containing 2 μL PMSF), shake every ten minutes, and allow the cells to fully contact the solution. Place on ice for 30 min, centrifuge at 11000 rpm, 4°C for 5 min, collect the supernatant, and dilute the protein sample 5 times with PBS buffer.

[0150] (2) BCA method for determining protein concentration: Dilute the BSA standard with a concentration of 2 mg / mL to prepare a standard curve (0, 0.00625, 0.0125, 0.25, 0.5, 1, 2 mg / mL). According to the number of BSA standard and samples to be tested, prepare BCA working solution in advance, i.e. mix reagent A and reagent B at a volume ratio of 50:1, add 100 μL of BCA working solution to each well, incubate at 37°C for about 5 min in the dark, and use an enzyme label instrument to determine the optical density of each sample at a wavelength of 562 nm. According to the standard curve, determine the protein concentration.

[0151] (3) Protein sample treatment: After diluting the protein sample to the same concentration, add the loading buffer and heat at 97°C for 7 min.

[0152] (4) Preparation of SDS-PAGE gel plate: Prepare 10% separation gel and 5% concentration gel, and stand for more than 30 min.

[0153] (5) Loading and electrophoresis: Assemble the electrophoresis tank and add the electrophoresis buffer. Add each experimental sample and protein Maker to the loading well. Under the condition of constant voltage 90V, electrophorese for 90 min.

[0154] (6) Membrane transfer: After electrophoresis, transfer the protein to a polyvinylidene fluoride membrane (PVDF membrane) under the condition of constant current 300 mA for 120 min.

[0155] (7) Blocking: Take out the PVDF membrane, add about 5 mL of 5% skimmed milk powder, and block at room temperature for 2 h.

[0156] (8) Incubation of primary antibody: Discard the skimmed milk powder, and dilute the primary antibody with 5% skimmed milk powder according to the corresponding proportion in advance, and incubate at 4°C overnight.

[0157] (9) Membrane washing: Recover the primary antibody, and wash the membrane with TBST three times, each time for ten minutes.

[0158] (10) Incubation of secondary antibody: Incubate the pre-diluted secondary antibody at room temperature for 1 h or at 4°C for 2-3 h.

[0159] (11) Membrane washing and color development: Wash with TBST for 3 times, 5 min / time, develop color by ECL chemiluminescence method, and develop and observe using a gel imaging instrument.

[0160] Test results: The effect of compound 2 on the expression of two inflammation-related proteins, cyclooxygenase-2 (COX-2) and inducible NO synthase (iNOS) in LPS-induced BV-2 cells was tested by Western-blot at depths of 2.5, 5 and 10 μmol / L. The results showed that compound 2 exhibited a concentration-dependent effect on the expression of the two proteins in LPS-induced BV-2 cells, as shown in Figures 7-9 .

[0161] Compared with the LPS group, * P < 0.05; ** P < 0.01.

[0162] Compared with the negative control group, ## P < 0.01.

[0163] In summary, the steroidal pyrazole-thiazolone steroid derivative containing thiophene and pyridine provided by the present application has good anti-neuroinflammatory activity in the prevention and treatment of neurodegenerative diseases, and in particular, compound 2: 3β-hydroxy-pregn-5-ene-17β-yl-5'-(thiophen-3-yl)-1'-(4"-keto-[1'',3'']-thiazol-2''-yl)-4',5'-dihydropyrazol-3'-yl, can be developed as a potential lead compound.

[0164] The above description is only the preferred embodiment of the present application, and is not intended to limit the scope of the present application. The above-described embodiments of the present application can be variously changed. That is, any simple, equivalent changes and modifications made according to the content of the claims and the specification of the present application are intended to fall within the scope of the present application. The present application is not described in detail, and is conventional technical content. In addition, it should be understood that, after reading the above description of the present application, those skilled in the art can make various changes or modifications to the present application, and these equivalent forms also fall within the scope of the claims of the present application.

Claims

1. A thienyl-containing steroidal pyrazole-thiazolone derivative, characterized by, The structural formula of the derivative is shown as formula (I), ； characterized in that said HET is selected from one of the following structures: 。 2. Use of a thienyl-containing steroidal pyrazole-thiazolone derivative according to claim 1 for the preparation of a medicament for the treatment of neuroinflammation, characterized in that, The thienyl-containing steroidal pyrazole-thiazolone derivative can inhibit LPS-induced BV 2NO, PGE2, IL 1β and TNF α inflammatory factors, or can inhibit inflammatory proteins iNOS (nitric oxide synthase) and COX 2 (cyclooxygenase 2) by inhibiting NF κB signaling pathway.

3. Use of a thienyl-containing steroidal pyrazole-thiazolone derivative according to claim 2 for the preparation of a medicament for the treatment of neuroinflammation, characterized in that, The dosage form of the drug is granules, tablets, pills, capsules, injections or dispersants.

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