Preparation method and application of steroidal dihydropyrazole thiazole derivatives

Abstract

The present invention discloses a steroidal dihydropyrazole thiazoline derivative, 3β-hydroxy-pregnane-5-en-17β-yl-5'-(substituted phenyl)-1'-(4''-phenyl-[1'',3'']-thiazol-2''-yl)-4',5'-dihydropyrazole-3'-yl. The present invention also discloses the use of the steroidal dihydropyrazole thiazoline derivative. The steroidal dihydropyrazole thiazoline derivative of the present invention has a strong inhibitory effect on nitric oxide (NO) production in RAW 264.7 mouse peritoneal macrophages induced by lipopolysaccharide (LPS), especially compound 4 in the prepared steroidal dihydropyrazole thiazoline derivative has an IC of 0.05. 50 The value is better than that of the positive control methylprednisolone, and the toxicity to cells is low. The steroidal derivative containing a dihydropyrazole thiazoline heterocycle provided by the present invention is a new structural compound, which shows higher activity than existing first-line anti-inflammatory drugs.

Description

Technical Field

[0001] The present invention relates to the fields of medicinal chemistry and pharmacology, and in particular to a novel steroidal dihydropyrazole thiazoline derivative and a preparation method and application thereof. Background Art

[0002] In recent years, nitrogen-containing heterocyclic compounds have shown an increasingly important role in drug research and biology. Among them, compounds containing pyrazolethiazoline heterocycles have strong physiological activity and have attracted particular attention from researchers.

[0003] Steroidal compounds are widely used in medicine, especially steroid hormones. Steroid hormones, such as adrenocortical hormones, can treat or alleviate collagen diseases, as well as difficult-to-treat or critical illnesses like anaphylactic shock. They are also indispensable for treating endocrine disorders such as Addison's disease. Numerous studies in recent decades have demonstrated the expanding medical applications of steroidal drugs. As research into steroid chemistry deepens, scientists have discovered that the introduction of nitrogen atoms or nitrogen-containing functional groups into the steroid nucleus or side chains can result in unique and even superior physiological activities of the resulting steroid compounds, including antibacterial, antiviral, anti-inflammatory, and anti-tumor properties.

[0004] Nitrogen-containing heterocyclic steroids are a class of substances with low toxicity and a wide range of biological activities, including anti-tumor, anti-inflammatory, antibacterial, antihypertensive, and antiparasitic activities. As active drug carriers and the basic backbone structure of drugs, nitrogen-containing heterocyclic steroids have diverse pharmacological effects. For example, the nitrogen-containing heterocyclic steroid abiraterone is currently used in the treatment of advanced prostate cancer, and galeterone has also successfully completed clinical trials for prostate cancer treatment. VN / 85-1 can reduce circulating androgen levels by inhibiting CYP17. Nitrogen-containing heterocyclic steroids, with their low toxicity and broad biological activity, are attracting increasing attention.

[0005]

[0006] Based on this, the present invention introduced nitrogen heterocyclic groups such as thiazoline and dihydropyrazole into steroid derivatives, designed and synthesized a series of novel steroidal dihydropyrazole-thiazoline derivatives, and evaluated their in vitro anti-inflammatory effects on LPS-induced RAW264.7 cell inflammation, hoping to have higher selectivity and lower toxicity. Summary of the Invention

[0007] The present invention has discovered a new class of steroidal derivatives containing aromatic heterocyclic thiazolyl dihydropyrazole units, and these compounds have excellent anti-inflammatory activity. The preparation method of these compounds is simple, the raw materials are readily available, and they are suitable for industrial scale-up reactions. As the resistance of existing anti-inflammatory drugs increases, these compounds can be used as alternative products to prepare the active ingredients of new anti-inflammatory drugs.

[0008] The purpose of the present invention is to solve the deficiencies of the existing technology and overcome the defects of existing clinical steroidal anti-inflammatory drugs such as low activity and high toxicity. By designing novel structural compound molecules, a low-toxic and highly effective aromatic heterocyclic thiazolyl dihydropyrazole steroid derivative is provided as a new type of anti-inflammatory drug, which can be widely used in the preparation of anti-inflammatory drugs.

[0009] Another object of the present invention is to provide a method for preparing the above-mentioned steroidal dihydropyrazole thiazoline derivatives.

[0010] Another object of the present invention is to provide the use of the above-mentioned steroidal dihydropyrazole thiazoline derivatives in the preparation of anti-inflammatory drugs.

[0011] In order to achieve the above-mentioned object of the invention, the present invention adopts the following technical solutions:

[0012] A novel steroidal dihydropyrazole thiazoline derivative, the structural formula of the derivative is shown in formula (I):

[0013] ;

[0014] The steroidal chalcone ester derivative is 3β-hydroxy-pregnane-5-ene-17β-yl-5'-(substituted phenyl)-1'-(4''-phenyl-[1'',3'']-thiazol-2''-yl)-4',5'-dihydropyrazol-3'-yl,

[0015] Wherein, R1 is hydrogen, halogen or C 1～3 Alkyl; R2 is hydrogen, halogen, C 1～3 Alkyl or C 1～3 Alkoxy; R3 is hydrogen, halogen, benzyloxy, mercaptomethyl, amide, C 1～3 Alkyl or C 1～3 Alkoxy.

[0016] Preferably, R1 is hydrogen, fluoro, chloro or methyl.

[0017] Preferably, R2 is hydrogen, fluoro, chloro, bromo, methyl or methoxy.

[0018] Preferably, R3 is hydrogen, fluoro, chloro, bromo, methyl, methoxy, benzyloxy, mercaptomethyl or amide.

[0019] The preparation method of the above-mentioned steroidal dihydropyrazole thiazoline derivatives comprises the following steps:

[0020] Step 1: Mixing a substituted benzaldehyde represented by formula (II), a pregnenolone represented by formula (III), an alkaline solution, and an organic solvent; stirring and reacting at a temperature of 25-36°C for 24-72 hours; filtering, washing, and drying to obtain an intermediate 1; wherein the molar ratio of the substituted benzaldehyde to the pregnenolone is 1:1-1.5;

[0021]

[0022] Step 2: Mix the intermediate 1, thiosemicarbazide and an organic solvent, stir, heat under reflux, and then filter and purify to obtain the intermediate 2;

[0023] Step 3: Mixing the intermediate 2, bromoacetophenone, an alkaline solution and an organic solvent, stirring, heating under reflux, and then filtering and purifying to obtain the steroidal dihydropyrazole thiazoline derivative;

[0024] The alkaline solution is an inorganic strong alkali solution, and the organic solvent is an alcohol solution.

[0025] The preparation method provided by the present invention can successfully prepare the above-mentioned steroidal dihydropyrazole thiazoline derivatives, and the preparation method is simple and has high yield.

[0026] Preferably, the inorganic strong base solution is a NaOH solution or a KOH solution; and the alcohol solution is an anhydrous ethanol solution.

[0027] Preferably, the heating reflux time is determined by tracking the reaction by TLC in step 2 and step 3. The developing solvent ratio of the product in step 3 is ethyl acetate: petroleum ether = 1:1, v / v .

[0028] Common purification methods can be applied to the present invention. Preferably, column chromatography or recrystallization can be used for purification in step 2 and step 3. The eluent for separation by column chromatography is ethyl acetate: petroleum ether = 1:9 to 1:3. v / v .

[0029] Furthermore, in step 3, the purification operation adopts column chromatography, wherein the eluent for separation in the column chromatography is ethyl acetate: petroleum ether = 1:4, v / v .

[0030] The use of the above-mentioned steroidal dihydropyrazole thiazoline derivatives in the preparation of anti-inflammatory drugs is also within the scope of protection of the present invention.

[0031] Preferably, the steroidal dihydropyrazole thiazoline derivatives are used in the preparation of anti-inflammatory drugs.

[0032] Preferably, the anti-inflammatory drug has good anti-inflammatory activity against RAW264.7 cells induced by LPS inflammation.

[0033] Preferably, the steroidal dihydropyrazole thiazoline derivative compound is used in the preparation of anti-inflammatory drugs to eliminate and alleviate inflammatory diseases to combat inflammation caused by NO and inflammatory factors TNF-α and COX-2.

[0034] Furthermore, the compound 4 of the steroidal dihydropyrazole thiazoline derivative: 3β-hydroxy-pregnane-5-ene-17β-yl-5'-(2-chlorophenyl)-1'-(4''-phenyl-[1'',3'']-thiazol-2''-yl)-4',5'-dihydropyrazolyl-3'-yl, is used in the preparation of anti-inflammatory drugs to eliminate and relieve inflammatory diseases, and is used to combat the inflammatory effects of NO and inflammatory factors TNF-α and COX-2, and has an anti-inflammatory effect compared to methylprednisolone.

[0035] Preferably, the anti-inflammatory drug is effective in treating autoinflammatory diseases such as rheumatoid arthritis and systemic lupus erythematosus, and further has therapeutic effects on autoinflammatory diseases such as rheumatoid arthritis and systemic lupus erythematosus.

[0036] Preferably, the anti-inflammatory drug dosage form includes tablets, pills, capsules, injections, suspensions or emulsions.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] The novel steroidal dihydropyrazole thiazoline derivatives provided by the present invention have good anti-inflammatory activity and low cytotoxicity. The steroidal dihydropyrazole thiazoline derivatives have obvious inhibitory effects on LPS-induced NO production in RAW 264.7 cells and inflammatory factors TNF-α and COX-2 released by RAW264.7 cells. In particular, the compound 4 of the steroidal dihydropyrazole thiazoline derivatives proposed by the present invention: 3β-hydroxy-pregnant-5-ene-17β-yl-5'-(2-chlorophenyl)-1'-(4''-phenyl-[1'',3'']-thiazol-2''-yl)-4',5'-dihydropyrazolyl-3'-yl, has an IC 50The values ​​were superior to those of methylprednisolone, and the compounds also exhibited good inhibitory effects on inflammatory factors TNF-α and COX-2 released by RAW264.7 cells, with very low cytotoxicity. The compounds of the present invention possessed excellent anti-inflammatory activity and low cytotoxicity, and are of great significance in the preparation of drugs for the prevention and treatment of inflammatory diseases. In particular, the prepared steroidal dihydropyrazole thiazoline derivative, compound 4, showed higher activity than the existing first-line anti-inflammatory drug methylprednisolone, and has broad application prospects in the preparation of drugs for combating inflammatory diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 is the structural formula of the steroidal dihydropyrazole thiazoline derivatives of the present invention.

[0040] Figure 2 is the structural formula of the substituted benzaldehyde of the present invention.

[0041] Figure 3 is the structural formula of pregnenolone of the present invention.

[0042] Figure 4 is the structural formula of the intermediate 1 of the present invention.

[0043] Figure 5 is the structural formula of intermediate 2 of the present invention.

[0044] Figure 6 This is a bar graph showing the inhibitory effect of compounds 1-16 prepared in Examples 1-16 of the present invention on LPS-induced NO production in RAW264.7 cells.

[0045] Figure 7 This is a bar graph showing the effects of compounds 1-16 prepared in Examples 1-16 of the present invention on LPS-induced RAW264.7 cell viability.

[0046] Figure 8 This is a bar graph showing the inhibitory effect of compound 4 prepared in Example 4 of the present invention on LPS-induced NO production in RAW264.7 cells.

[0047] Figure 9 This is a bar graph showing the effect of compound 4 prepared in Example 4 of the present invention on LPS-induced RAW264.7 cell viability.

[0048] Figure 10 This is a bar graph showing the inhibitory effect of the steroidal dihydropyrazole thiazoline derivatives (corresponding to compound 4 in Example 4) prepared and proposed in the present invention on the inflammatory factor TNF-α released by RAW 264.7 cells induced by LPS.

[0049] Figure 11This is a bar graph showing the inhibitory effect of the steroidal dihydropyrazole thiazoline derivatives (corresponding to compound 4 in Example 4) prepared and proposed in the present invention on the inflammatory factor COX-2 released by RAW 264.7 cells induced by LPS. DETAILED DESCRIPTION

[0050] The technical solution of the present invention is further illustrated below through specific examples.

[0051] These examples are intended to illustrate the present invention only and are not intended to limit its scope. Experimental procedures in the following examples, where specific conditions are not specified, generally followed conventional conditions in the art or the conditions recommended by the manufacturer. Raw materials and reagents used, unless otherwise specified, are commercially available from conventional markets. Any non-substantial variations or substitutions made by those skilled in the art based on the present invention are intended to be within the scope of the present invention.

[0052] Example 1: Synthesis of Compound 1

[0053] Compound 1 provided in this embodiment is: 3β-hydroxy-pregn-5-en-17β-yl-5'-(2-fluorophenyl)-1'-(4''-phenyl-[1'',3'']-thiazol-2''-yl)-4',5'-dihydropyrazol-3'-yl, and its structural formula is as follows:

[0054]

[0055] The preparation method of compound 1 is as follows:

[0056] (1) Preparation of intermediate 1-1:

[0057] Will replace benzaldehyde: (1.24g, 0.01mol), equimolar amounts of pregnenolone Mix 1-1-1 (3.16 g, 0.01 mol), 10% NaOH solution (1 g NaOH dissolved in 10 mL water) and anhydrous ethanol (30 mL), stir and react at 25°C for 48 h, pour into ice water, adjust the pH to neutral, let stand overnight, filter, wash, dry, and recrystallize from ethanol to obtain intermediate 1-1:

[0058]

[0059] (2) Preparation of intermediate 1-2: Thiosemicarbazide (1.83 g, 0.02 mol), intermediate 1 (4.22 g, 0.01 mol), 10% NaOH solution (1 g NaOH dissolved in 10 mL water) and anhydrous ethanol (30 mL) were mixed and heated under reflux with stirring for 8 h. The reaction was monitored by TLC (developer V 石油醚 :V 乙酸乙酯 =2:1), after the reaction, pour into ice water, adjust the pH value to neutral, let stand overnight, filter, wash, dry, and recrystallize from ethanol to obtain intermediate 1-2:

[0060]

[0061] (3) Preparation of Compound 1

[0062] Under stirring, the synthesized intermediate 1-2 (2.48 g, 0.005 mol), bromoacetophenone (2.0 g, 0.01 mol), and anhydrous ethanol (30 mL) were added to a 50 mL round-bottom flask in sequence, and the mixture was heated under reflux and stirred for 8 h. The reaction was monitored by TLC (developer V 石油醚 :V 乙酸乙酯 =1:1), after the reaction is completed, the reaction solution is allowed to stand, a precipitate is precipitated, and the precipitate is allowed to stand, filtered, and the solid is washed with anhydrous ethanol and finally dried in vacuo. The obtained solid is purified by column chromatography to obtain the pure target compound. The eluent for column chromatography separation is ethyl acetate: petroleum ether = 1:9~1:3, v / v .

[0063] White solid powder, yield 55.26%, Mp: 211.5-213.9℃. IR (KBr, cm -1 ): 2929.91(CH), 1631.40, 1601.42 (C=N), 1540.73 (C=C), 1519.02, 1444.45, 1328.45 (CH), 1226.67 (CO), 1046.15 (CO). 1 H-NMR (600 M Hz, δ ppm, CDCl3): 7.66 (d, 2H, J =7.80 Hz, Ar-H), 7.24-7.38 (m, 5H, Ar-H), 7.19-7.22 (m, 1H, Ar-H), 7.07-7.13(m, 2H, Ar-H), 6.80 (s, 1H, thiazole C 5’’ -H), 5.64-5.71 (m, 1H, C 5’-H),5.37(s, 1H, C6-H), 3.53-3.60 (m, 1H, C3α-H), 3.41-3.52 (m, 1H, pyrazoline C 4’ -H2),2.82 -2.98(m, 1H, pyrazoline C 4’ -H2), 2.34-2.42 (m, 1H, C 17 α-H), 2.17-2.32 (m, 3H), 2.01-2.06 (m, 1H), 1.88-1.99(m, 2H), 1.81-1.87(m, 2H), 1.73-1.79(m, 1H), 1.58-1.61(m, 3H), 1.47-1.54(m, 3H), 1.26-1.39(m, 3H), 1.09-1.18(m, 2H), 1.02(s, 3H, C 10 -CH3), 0.94-0.99(m, 1H),0.72 (s, 3H, C 13 -CH3). 13 C-NMR (ppm, CDCl3):166.05, 161.28, 157.30, 151.40, 140.84, 135.07, 128.41(2C), 128.22, 128.17(2C), 127.40(2C), 125.83, 121.42, 115.56, 115.42, 103.19, 71.69, 63.69,56.39, 51.68, 50.11, 47.34, 43.97, 42.26, 38.44, 37.30, 36.58, 32.08, 31.79,31.61, 24.65, 24.49, 21.01, 19.45, 13.47. MS (ESI) (m / z) : 596.30 [M + H] + .HRMS (ESI): m / z [M + H] + calcd for C 37 H 43 FN3OS: 596.3033; found: 596.3122.

[0064] Example 2: Compound 2

[0065] Compound 2 provided in this embodiment is: 3β-hydroxy-pregn-5-en-17β-yl-5'-(3-fluorophenyl)-1'-(4''-phenyl-[1'',3'']-thiazol-2''-yl)-4',5'-dihydropyrazol-3'-yl, and its structural formula is as follows:

[0066]

[0067] The preparation method of compound 2 is the same as that of Example 1 except that R2 of substituted benzaldehyde is fluorine.

[0068] White solid powder, yield 47.81%, Mp: 184.1-187.3℃. IR (KBr, cm -1 ): 2932.97(CH), 1614.99, 1598.00 (C=N), 1551.86 (C=C), 1523.15, 1445.14, 1328.76 (CH), 1281.62 (CO), 1047.69 (CO). 1 H-NMR (600 M Hz, δ ppm, DMSO-d6): 7.68 (d, 2H, J = 7.20 Hz, Ar-H), 7.39-7.43 (m, 1H, Ar-H), 7.30-7.34 (m, 2H, Ar-H), 7.27 (s,1H, thiazole C 5’’ -H),7.22-7.25 (m, 1H, Ar-H), 7.15-7.20 (m, 2H, Ar-H), 7.07-7.12 (m, 1H, Ar-H),5.44-5.50 (m, 1H, C 5’ -H), 5.28 (s, 1H, C6-H), 4.60 (d, 1H, J = 4.20 Hz, C3-OH), 3.61-3.74 (m, 1H, pyrazoline C 4’ -H2), 3.24-3.30 (m, 1H, C3α-H), 2.81 -2.89(m, 1H, pyrazoline C 4’ -H2), 2.42-2.48 (m, 1H, C 17α-H), 2.08-2.19(m, 3H), 1.92-1,99 (m, 2H), 1.85-1.91(m, 1H), 1.77-1.82(m, 1H), 1.65-1.73(m,2H), 1.54-1.61(m, 2H), 1.21-1.47(m, 7H),1.14-1.19(m, 1H), 0.96 (s, 3H, C 10 -CH3), 0.91-0.94(m, 1H),0.66 (s, 3H, C 13 -CH3). 13 C-NMR (ppm, CDCl3):165.85,163.84, 157.31, 151.39, 144.77, 140.82, 135.03, 130.16, 128.40(2C), 127.39,125.83(2C), 122.08, 121.43, 114.40, 113.45, 103.23, 71.72, 63.65, 56.36,51.67, 50.12, 47.00, 43.94, 42.26, 38.44, 37.29, 36.58, 32.07, 31.78, 31.62,26.93, 24.69, 24.48, 20.99, 19.45, 13.44. MS (ESI) (m / z) : 596.30 [M + H] + .HRMS (ESI): m / z [M + H] + calcd for C 37 H 43 FN3OS: 596.3033; found:596.3160.

[0069] Example 3: Compound 3

[0070] Compound 3 provided in this embodiment is: 3β-hydroxy-pregn-5-en-17β-yl-5'-(4-fluorophenyl)-1'-(4''-phenyl-[1'',3'']-thiazol-2''-yl)-4',5'-dihydropyrazol-3'-yl, and its structural formula is as follows:

[0071]

[0072] The preparation method of compound 3 is the same as that of Example 1 except that R3 of substituted benzaldehyde is fluorine.

[0073] Light yellow solid powder, yield 63.32%, Mp:186.4-189.0℃. IR (KBr, cm -1 ): 2930.34(CH), 1631.79, 1603.61 (C=N), 1549.96 (C=C), 1510.52, 1444.28, 1329.56 (CH), 1228.02 (CO), 1047.15 (CO). 1 H-NMR (600 M Hz, δ ppm, DMSO-d6): 7.68 (d, 2H, J = 7.20 Hz, Ar-H), 7.38-7.40 (m, 2H, Ar-H), 7.32-7.34 (m, 2H, Ar-H), 7.27 (s,1H, thiazole C 5’’ -H),7.19-7.25 (m, 3H, Ar-H), 5.44-5.48 (m, 1H, C 5’ -H), 5.28(s, 1H, C6-H), 4.60 (d, 1H, J = 4.20 Hz, C3-OH), 3.55-3.72 (m, 1H, pyrazolineC 4’ -H2), 3.24-3.29 (m, 1H, C3α-H), 2.80 -2.89(m, 1H, pyrazoline C 4’ -H2), 2.39-2.46 (m, 1H, C 17 α-H), 2.08-2.21 (m, 3H), 1.94-1,99 (m, 1H), 1.80-1.91(m, 2H), 1.73-1.79(m, 1H), 1.65-1.72(m, 2H), 1.53-1.61(m, 2H), 1.21-1.50(m, 7H),1.13-1.20(m, 1H), 0.96 (s, 3H, C 10 -CH3), 0.90-0.94(m, 1H),0.67 (s, 3H, C 13 -CH3). 13C-NMR (ppm, CDCl3): 165.96, 157.68, 157.56, 151.39, 140.84, 135.07, 129.01(2C),128.37, 127.34(2C), 125.84(2C), 124.21(2C), 121.45, 115.50, 103.17, 71.72,59.05, 58.47, 56.41, 51.70, 50.12, 46.03, 43.95, 42.27, 38.39, 37.29, 36.58,32.08, 31.71, 26.94, 24.66, 24.48, 21.01, 19.45, 13.32.MS (ESI) (m / z) : 596.35[M + H] + . HRMS (ESI): m / z [M + H] + calcd for C 37 H 43 FN3OS: 596.3033; found:596.3180.

[0074] Example 4: Compound 4

[0075] Compound 4 provided in this embodiment is: 3β-hydroxy-pregn-5-en-17β-yl-5'-(2-chlorophenyl)-1'-(4''-phenyl-[1'',3'']-thiazol-2''-yl)-4',5'-dihydropyrazol-3'-yl, and its structural formula is as follows:

[0076]

[0077] The preparation method of compound 4 is the same as that of Example 1 except that R1 of the substituted benzaldehyde is a chlorine group.

[0078] Yellow solid powder, yield 65.21%, Mp: 119.6-124.2℃. IR (KBr, cm -1 ): 2928.91(CH), 1631.33, 1601.65 (C=N), 1545.36 (C=C), 1519.56, 1443.54, 1328.40 (CH), 1277.76 (CO), 1051.28 (CO). 1 H-NMR (600 M Hz, δ ppm, CDCl3): 7.65 (d, 2H, J=7.20 Hz, Ar-H), 7.41-7.44 (m, 1H, Ar-H), 7.27-7.32 (m, 3H, Ar-H), 7.17-7.24(m, 3H, Ar-H), 6.80 (s, 1H, thiazole C 5’’ -H) , 5.78-5.86 (m, 1H, C 5’ -H), 5.37(s, 1H, C6-H), 3.54-3.68 (m, 1H, C3α-H), 3.47-3.53 (m, 1H, pyrazoline C 4’ -H2),2.71 -2.84(m, 1H, pyrazoline C 4’ -H2), 2.33-2.39 (m, 1H, C 17 α-H), 2.16-2.31 (m,3H), 2.04-2.10 (m, 1H), 1.96-2.02(m, 1H), 1.89-1.95(m, 1H), 1.79-1.88(m, 3H),1.45-1.55(m, 5H), 1.25-1.35(m, 3H), 1.07-1.17(m, 2H), 1.01 (s, 3H, C 10 -CH3),0.95-0.99(m, 1H),0.68 (s, 3H, C 13 -CH3). 13 C-NMR (ppm, CDCl3): 165.77, 157.60,151.46, 140.83, 139.39, 134.99, 132.08, 129.64(2C), 125.81, 128.36, 127.35,127.22, 127.16(2C), 125.86, 121.44, 103.15, 71.73, 61.45, 56.39, 51.67,50.11, 46.22, 43.96, 42.27, 38.40, 37.28, 36.58, 32.06, 31.78, 31.63, 26.93,24.63, 24.48, 20.98, 19.43, 13.37. MS (ESI) (m / z) : 612.30 [M + H] + . HRMS(ESI): m / z [M + H] + calcd for C 37H 43 ClN3OS: 612.2737; found: 612.2862.

[0079] Example 5: Compound 5

[0080] Compound 5 provided in this embodiment is: 3β-hydroxy-pregn-5-en-17β-yl-5'-(3-chlorophenyl)-1'-(4''-phenyl-[1'',3'']-thiazol-2''-yl)-4',5'-dihydropyrazol-3'-yl, and its structural formula is as follows:

[0081]

[0082] The preparation method of compound 5 is the same as that of Example 1 except that R2 of substituted acetophenone is chlorine.

[0083] Light yellow solid powder, yield 57.41%, Mp: 210.8-215.5℃. IR (KBr, cm -1 ): 2933.63(CH), 1631.60, 1598.31 (C=N), 1532.10 (C=C), 1517.31, 1441.95, 1379.29 (CH), 1275.82 (CO), 1045.59 (CO). 1 H-NMR (600 M Hz, δ ppm, CDCl3): 7.67 (d, 2H, J =7.80 Hz, Ar-H), 7.41-7.43 (m, 1H, Ar-H), 7.30-7.36 (m, 4H, Ar-H), 7.22-7.27(m, 2H, Ar-H), 6.81 (s, 1H, thiazole C 5’’ -H), 5.40-5.47 (m, 1H, C 5’ -H),5.39(s, 1H, C6-H), 3.54-3.58 (m, 1H, C3α-H), 3.40-3.53 (m, 1H, pyrazoline C 4’ -H2),2.84 -2.98(m, 1H, pyrazoline C 4’ -H2), 2.38-2.46 (m, 1H, C 17α-H), 2.22-2.35 (m, 3H), 2.05-2.08 (m, 1H), 1.93-2.02(m, 2H), 1.86-1.92(m, 2H), 1.77-1.82(m, 1H),1.60-1.64(m, 3H), 1.52-1.57(m, 3H), 1.30-1.40(m, 4H), 1.12-1.21(m, 2H), 1.04(s, 3H, C 10 -CH3), 0.99-1.03(m, 1H),0.76 (s, 3H, C 13 -CH3). 13 C-NMR (ppm, CDCl3):165.98, 157.32, 151.38, 144.47, 140.83, 135.01, 134.41, 129.95, 128.43(2C),127.69, 127.42(2C), 127.01, 125.86, 124.68, 121.43, 103.31, 71.71, 64.10,56.40, 51.66, 50.11, 47.33, 43.97, 42.27, 38.44, 37.29, 36.58, 32.07, 31.79,31.62, 24.61, 24.48, 21.01, 19.45, 13.46. MS (ESI) (m / z) : 612.30 [M + H] + .HRMS (ESI): m / z [M + H] + calcd for C 37 H 43 ClN3OS: 612.2737; found: 612.2819.

[0084] Example 6: Compound 6

[0085] Compound 6 provided in this embodiment is: 3β-hydroxy-pregn-5-en-17β-yl-5'-(4-chlorophenyl)-1'-(4''-phenyl-[1'',3'']-thiazol-2''-yl)-4',5'-dihydropyrazol-3'-yl, and its structural formula is as follows:

[0086]

[0087] The preparation method of compound 6 is the same as that of Example 1 except that R3 of the substituted benzaldehyde is chlorine.

[0088] Light yellow solid powder, yield 58.73%, Mp: 237.6-243.0℃. IR (KBr, cm -1 ): 2962.65(CH), 1631.85, 1602.44 (C=N), 1553.36 (C=C), 1523.09, 1445.34, 1329.72 (CH), 1296.01 (CO), 1053.41 (CO). 1 H-NMR (600 M Hz, δ ppm, CDCl3): 7.65 (d, 2H, J =7.80 Hz, Ar-H), 7.29-7.34 (m, 6H, Ar-H), 7.22 (t, 1H, Ar-H), 6.79 (s, 1H,thiazole C 5’’ -H), 5.39-5.43 (m, 1H, C 5’ -H),5.37 (s, 1H, C6-H), 3.53-3.58 (m,1H, C3α-H), 3.48-3.52 (m, 1H, pyrazoline C 4’ -H2), 2.78 -2.83(m, 1H, pyrazolineC 4’ -H2), 2.38-2.41 (m, 1H, C 17 α-H), 2.18-2.32 (m, 3H), 2.02-2.06 (m, 1H), 1.92-1.98(m, 2H), 1.84-1.87(m, 2H), 1.73-1.79(m, 1H), 1.57-1.64(m, 3H), 1.46-1.54(m, 3H), 1.27-1.34(m, 3H), 1.08-1.18(m, 2H), 1.02 (s, 3H, C 10 -CH3), 0.95-0.99(m, 1H),0.71 (s, 3H, C 13 -CH3). 13C-NMR (ppm, CDCl3): 165.86, 157.31, 151.40,140.83, 140.67, 135.02, 133.15, 128.78(2C), 128.42, 127.93(2C), 127.42(2C),125.83(2C), 121.42, 103.20, 71.71, 63.51, 56.35, 51.68, 50.12, 47.02, 43.94,42.26, 38.44, 37.29, 36.58, 32.07, 31.70, 31.46, 24.69, 24.48, 20.99, 19.45,13.45. MS (ESI) (m / z) : 612.30 [M + H] + . HRMS (ESI): m / z [M + H] + calcd for C 37 H 43 ClN3OS: 612.2737; found: 612.2826.

[0089] Example 7: Compound 7

[0090] Compound 7 provided in this example is: 3β-hydroxy-pregn-5-en-17β-yl-5'-(3-bromophenyl)-1'-(4''-phenyl-[1'',3'']-thiazol-2''-yl)-4',5'-dihydropyrazol-3'-yl, and its structural formula is as follows:

[0091]

[0092] The preparation method of compound 7 is the same as that of Example 1 except that R2 of the substituted benzaldehyde is a bromine group. The remaining raw materials, amounts and operating steps are the same as those of Example 1. Finally, the following is obtained:

[0093] Yellow solid powder, yield 60.37%, Mp:121.6-124.0℃. IR (KBr, cm -1 ): 2932.13(CH), 1664.74, 1597.33 (C=N), 1544.79 (C=C), 1519.21, 1444.41, 1329.38 (CH), 1277.73 (CO), 1046.01 (CO). 1 H-NMR (600 M Hz, δ ppm, CDCl3): 7.67 (d, 2H, J=7.20 Hz, Ar-H), 7.55 (s, 1H, Ar-H),7.36-7.40 (m, 1H, Ar-H), 7.30-7.33 (m, 3H,Ar-H), 7.20-7.26 (m, 2H, Ar-H), 6.78 (s, 1H, thiazole C 5’’ -H) , 5.53-5.61 (m,1H, C 5’ -H),5.37 (s, 1H, C6-H), 3.53-3.58 (m, 1H, C3α-H), 3.45-3.52 (m, 1H,pyrazoline C 4’ -H2), 2.80 -2.89(m, 1H, pyrazoline C 4’ -H2), 2.38-2.43 (m, 1H, C 17 α-H), 2.18-2.32 (m, 3H), 2.02-2.06 (m, 1H), 1.95-2.00 (m, 2H), 1.84-1.89(m,3H), 1.75-1.80(m, 1H), 1.56-1.66(m, 3H), 1.46-1.53(m, 3H), 1.25-1.37(m, 3H),1.10-1.17(m, 2H), 1.02 (s, 3H, C 10 -CH3), 0.97-1.01(m, 1H),0.71 (s, 3H, C 13 -CH3). 13 C-NMR (ppm, DMSO-d6): 165.65, 159.19, 150.73, 145.02, 141.76, 134.97,131.16, 130.68, 130.39(3C), 128.89, 127.89(2C), 125.94, 122.03, 120.72,104.51, 70.46, 63.61, 56.11, 51.18, 50.10, 46.73, 43.80, 42.68, 38.01, 37.43,36.60, 32.08, 31.88, 31.73, 24.59, 24.49, 20.99, 19.65, 13.42. MS (ESI) (m / z) :658.25 [M + H] + . HRMS (ESI): m / z [M + H] +calcd for C 37 H 43 BrN3OS: 656.2232,658.2231; found: 656.2332,658.2321.

[0094] Example 8: Compound 8

[0095] Compound 8 provided in this embodiment is: 3β-hydroxy-pregn-5-en-17β-yl-5'-(4-bromophenyl)-1'-(4''-phenyl-[1'',3'']-thiazol-2''-yl)-4',5'-dihydropyrazol-3'-yl, and its structural formula is as follows:

[0096]

[0097] The preparation method of compound 8 is the same as that of Example 1 except that R3 of the substituted benzaldehyde is a bromine group. The remaining raw materials, amounts and operating steps are the same as those of Example 1.

[0098] White solid powder, yield 71.05%, Mp: 201.2-205.2℃. IR (KBr, cm -1 ): 2926.65(CH), 1631.67, 1601.95 (C=N), 1550.82 (C=C), 1520.31, 1444.61, 1328.66 (CH), 1278.81 (CO), 1054.38 (CO). 1 H-NMR (600 M Hz, δ ppm, CDCl3): 7.61-7.68 (m,2H, Ar-H), 7.45-7.51 (m, 2H, Ar-H), 7.29-7.33 (m, 2H, Ar-H), 7.25-7.27 (m,2H, Ar-H), 7.20-7.24 (m, 1H, Ar-H),6.79 (s, 1H, thiazole C 5’’ -H), 5.38-5.44(m, 1H, C 5’ -H),5.37 (s, 1H, C6-H), 3.50-3.55 (m, 1H, C3α-H), 3.37-3.44 (m, 1H,pyrazoline C 4’ -H2), 2.78 -2.92(m, 1H, pyrazoline C 4’ -H2), 2.35-2.41 (m, 1H, C 17α-H), 2.18-2.33 (m, 3H), 2.01-2.06 (m, 1H), 1.87-1.99(m, 2H), 1.84-1.87(m,2H), 1.74-1.79(m, 1H), 1.55-1.62(m, 3H), 1.45-1.53(m, 3H), 1.26-1.35(m, 3H),1.08-1.18(m, 2H), 1.02 (s, 3H, C 10 -CH3), 0.95-0.98(m, 1H),0.72 (s, 3H, C 13 -CH3). 13 C-NMR (ppm, CDCl3): 166.10, 157.26, 151.40, 141.57, 140.82, 135.01,131.76(2C), 128.42(2C), 128.26, 127.41(2C), 125.83(2C), 121.43, 103.26,71.72, 63.91, 63.55, 56.39, 51.67, 50.10, 47.44, 46.98, 43.98, 42.27, 38.44,37.29, 36.57, 32.07, 31.78, 31.62, 24.64, 24.48, 20.99, 19.45, 13.48.MS (ESI)(m / z): 658.25 [M + H] + . HRMS (ESI): m / z [M + H] + calcd for C 37 H 43 BrN3OS: 656.2232; found: 656.2318, 658.2305.

[0099] Example 9: Compound 9

[0100] Compound 9 provided in this embodiment is: 3β-hydroxy-pregn-5-en-17β-yl-5'-(2-methylphenyl)-1'-(4''-phenyl-[1'',3'']-thiazol-2''-yl)-4',5'-dihydropyrazol-3'-yl, and its structural formula is as follows:

[0101]

[0102] The preparation method of compound 9 is the same as that of Example 1 except that R1 of the substituted benzaldehyde is methyl.

[0103] Light yellow solid powder, yield 53.29%, Mp: 217.0-222.5℃. IR (KBr, cm -1 ): 2930.07(CH), 1631.45, 1601.55 (C=N), 1549.66 (C=C), 1519.79, 1443.56, 1327.29 (CH), 1278.43 (CO), 1048.20 (CO). 1 H-NMR (600 M Hz, δ ppm, CDCl3): 7.63 (d, 2H, J =7.20 Hz, Ar-H), 7.26-7.30 (m, 2H, Ar-H), 7.13-7.21 (m, 5H, Ar-H), 7.07 (t,1H, Ar-H), 6.78 (s, 1H, thiazole C 5’’ -H), 5.58-5.66 (m, 1H, C 5’ -H),5.37 (s,1H, C6-H), 3.52-3.58 (m, 1H, C3α-H), 3.38-3.51 (m, 1H, pyrazoline C 4’ -H2), 2.70-2.86(m, 1H, pyrazoline C 4’ -H2), 2.52 (s, 3H, Ar-CH3),2.34-2.43 (m, 1H, C 17 α-H), 2.18-2.33 (m, 3H), 2.01-2.06 (m, 1H), 1.89-2.01(m, 2H), 1.81-1.87(m, 2H), 1.73-1.79(m, 1H), 1.54-1.59(m, 3H), 1.47-1.52(m, 3H), 1.26-1.36(m, 3H), 1.07-1.17(m, 2H), 1.02 (s, 3H, C 10 -CH3), 0.95-1.01(m, 1H),0.72 (s, 3H, C 13 -CH3). 13C-NMR (ppm, CDCl3): 165.92, 157.14, 151.41, 140.82, 140.29, 135.16, 134.64,130.39, 128.32(2C), 127.27, 127.18(2C), 126.56, 125.86, 125.57, 121.45,102.99, 71.73, 60.82, 56.41, 51.74, 50.13, 46.31, 43.95, 42.27, 38.43, 37.29,36.58, 32.07, 31.79, 31.62, 24.73, 24.49, 20.99, 19.67, 19.44, 13.41.MS(ESI) (m / z) : 592.35 [M + H] + . HRMS (ESI): m / z [M + H] + calcd for C 38 H 46 N3OS:592.3283; found: 592.3371.

[0104] Example 10: Compound 10

[0105] Compound 10 provided in this embodiment is: 3β-hydroxy-pregn-5-en-17β-yl-5'-(3-methylphenyl)-1'-(4''-phenyl-[1'',3'']-thiazol-2''-yl)-4',5'-dihydropyrazol-3'-yl, and its structural formula is as follows:

[0106]

[0107] The preparation method of compound 10 is the same as that of Example 1 except that R2 of the substituted benzaldehyde is methyl.

[0108] Yellow solid powder, yield 54.72%, Mp: 105.5-108.4℃. IR (KBr, cm -1 ): 2930.28(CH), 1631.19, 1601.87 (C=N), 1541.44 (C=C), 1518.66, 1443.51, 1327.04 (CH), 1277.11 (CO), 1045.54 (CO). 1 H-NMR (600 M Hz, δ ppm, CDCl3): 7.66 (d, 2H,J =7.20 Hz, Ar-H), 7.27-7.32 (m, 2H, Ar-H), 7.15-7.24 (m, 4H, Ar-H), 7.07 (t,1H, Ar-H), 6.77 (s, 1H, thiazole C 5’’ -H) , 5.39-5.45 (m, 1H, C 5’ -H),5.37 (s,1H, C6-H), 3.53-3.57 (m, 1H, C3α-H), 3.37-3.51 (m, 1H, pyrazoline C 4’ -H2), 2.83-2.99(m, 1H, pyrazoline C 4’ -H2), 2.38-2.44 (m, 1H, C 17 α-H), 2.35 (s, 3H, Ar-CH3),2.20-2.33 (m, 3H), 2.02-2.06 (m, 1H), 1.92-1.99(m, 2H), 1.84-1.89(m,2H), 1.75-1.80(m, 1H), 1.57-1.63(m, 3H), 1.48-1.54(m, 3H), 1.27-1.36(m, 3H),1.09-1.18(m, 2H), 1.02 (s, 3H, C 10 -CH3), 0.95-1.01(m, 1H),0.74 (s, 3H, C 13 -CH3). 13 C-NMR (ppm, CDCl3): 166.09, 157.39, 151.38, 142.03, 140.81, 138.17,135.17, 128.49, 128.34(2C), 128.24, 127.46(2C), 127.33, 125.89, 123.55,121.46, 102.98, 71.74, 64.09, 56.38, 51.73, 50.13, 47.11, 43.95, 42.26,38.44, 37.29, 36.58, 32.07, 31.79, 31.62, 24.64, 24.49, 21.52, 21.01, 19.45,13.41.MS (ESI) (m / z) : 592.30 [M + H] + . HRMS (ESI): m / z[M + H] + calcd for C 38 H 46 N3OS: 592.3283; found: 592.3369.

[0109] Example 11: Compound 11

[0110] The compound 11 provided in this embodiment is: 3β-hydroxy-pregn-5-en-17β-yl-5'-(4-methylphenyl)-1'-(4''-phenyl-[1'',3'']-thiazol-2''-yl)-4',5'-dihydropyrazol-3'-yl, and its structural formula is as follows:

[0111]

[0112] The preparation method of compound 11 is the same as that of Example 1 except that R3 of the substituted benzaldehyde is methyl.

[0113] White solid powder, yield 70.22%, Mp: 206.8-208.4℃. IR (KBr, cm -1 ): 2931.70(CH), 1600.61 (C=N), 1544.93 (C=C), 1518.62, 1444.33, 1328.07 (CH), 1278.29(CO), 1046.07 (CO). 1 H-NMR (600 M Hz, δ ppm, CDCl3): 7.67 (d, 2H, J = 7.80 Hz, Ar-H), 7.27-7.33 (m, 4H, Ar-H), 7.20 (t, 1H, Ar-H), 7.16 (d, 2H, J = 7.80 Hz, Ar-H), 6.76 (s, 1H, thiazole C 5’’ -H), 5.38-5.48 (m, 1H, C 5’ -H),5.37 (s, 1H,C6-H), 3.50-3.55 (m, 1H, C3α-H), 3.37-3.42 (m, 1H, pyrazoline C 4’ -H2), 2.91 -2.98(m, 1H, pyrazoline C 4’ -H2), 2.32-2.37 (m, 1H, C 17α-H), 2.32 (s, 3H, Ar-CH3), 2.19-2.30 (m, 3H), 2.02-2.06 (m, 1H), 1.87-1.97(m, 2H), 1.83-1.86(m,2H), 1.74-1.79(m, 1H), 1.54-1.59(m, 2H), 1.41-1.54(m, 4H), 1.27-1.35(m, 2H),1.07-1.17(m, 2H), 1.01 (s, 3H, C 10 -CH3), 0.97-0.98(m, 1H),0.75 (s, 3H, C 13 -CH3). 13 C-NMR (ppm, CDCl3): 166.18, 157.41, 151.26, 140.81, 139.47, 137.07,135.08, 129.33(2C), 128.35, 127.31(2C), 126.48(2C), 125.89(2C), 121.46,102.96, 71.73, 64.16, 56.45, 51.72, 50.11, 47.64, 43.99, 42.25, 38.42, 37.28,36.57, 32.08, 31.79, 31.61, 24.60, 24.48, 21.16, 20.99, 19.43, 13.46.MS(ESI) (m / z) : 592.30[M + H] + . HRMS (ESI): m / z [M + H] + calcd for C 38 H 46 N3OS:592.3283; found: 592.3384.

[0114] Example 12: Compound 12

[0115] Compound 12 provided in this embodiment is: 3β-hydroxy-pregn-5-en-17β-yl-5'-(3-methoxyphenyl)-1'-(4''-phenyl-[1'',3'']-thiazol-2''-yl)-4',5'-dihydropyrazol-3'-yl, and its structural formula is as follows:

[0116]

[0117] The preparation method of compound 12 is the same as that of Example 1 except that R2 of the substituted benzaldehyde is methoxy. The remaining raw materials, amounts and operation steps are the same as those of Example 1. Finally, the following is obtained:

[0118] White solid powder, yield 66.73%, Mp: 173.6-175.3℃. IR (KBr, cm -1 ): 2939.72(CH), 1631.63, 1600.43 (C=N), 1531.76 (C=C), 1518.89, 1467.13, 1326.40 (CH), 1258.85 (CO), 1039.06 (CO). 1 H-NMR (600 M Hz, δ ppm, CDCl3): 7.67 (d, 2H, J =7.80 Hz, Ar-H), 7.25-7.31 (m, 4H, Ar-H), 7.19-7.22 (m, 1H, Ar-H), 6.94-6.98(m, 1H, Ar-H), 6.90-6.93 (m, 1H, Ar-H), 6.77 (s, 1H, thiazole C 5’’ -H), 5.41-5.45 (m, 1H, C 5’ -H),5.37 (s, 1H, C6-H), 3.79 (s, 3H, Ar-OCH3), 3.53-3.57 (m,1H, C3α-H), 3.50-3.52 (m, 1H, pyrazoline C 4’ -H2), 2.82 -2.87(m, 1H, pyrazolineC 4’ -H2), 2.37-2.41 (m, 1H, C 17 α-H), 2.19-2.33 (m, 3H), 2.01-2.06 (m, 1H), 1.93-1.98 (m, 2H), 1.84-1.88(m, 2H), 1.75-1.79(m, 1H), 1.58-1.63(m, 3H), 1.46-1.53(m, 3H), 1.26-1.36(m, 3H), 1.09-1.18(m, 2H), 1.02 (s, 3H, C 10 -CH3), 0.96-1.01(m, 1H),0.71 (s, 3H, C 13 -CH3). 13C-NMR (ppm, CDCl3): 165.94, 159.80, 157.39,151.39, 143.81, 140.83, 135.15, 129.65, 128.35(2C), 127.31, 125.88(2C),121.44, 118.76, 112.91, 112.13, 103.01, 71.72, 64.04, 56.36, 55.25, 51.71,50.13, 47.07, 43.93, 42.27, 38.43, 37.29, 36.58, 32.07, 31.79, 31.63, 24.69,24.49, 20.99, 19.45, 13.43. MS (ESI) (m / z) : 608.35 [M + H] + . HRMS (ESI): m / z [M+H] + calcd for C 38 H 46 N3O2S: 608.3232; found: 608.3318.

[0119] Example 13: Compound 13

[0120] Compound 13 provided in this embodiment is: 3β-hydroxy-pregn-5-en-17β-yl-5'-(4-methoxyphenyl)-1'-(4''-phenyl-[1'',3'']-thiazol-2''-yl)-4',5'-dihydropyrazol-3'-yl, and its structural formula is as follows:

[0121]

[0122] The preparation method of compound 13 is the same as that of Example 1 except that R3 of the substituted benzaldehyde is methoxy. The remaining raw materials, amounts and operation steps are the same as those of Example 1.

[0123] White solid powder, yield 55.78%, Mp: 207.9-210.9℃. IR (KBr, cm -1 ): 2929.72(CH), 1613.07 (C=N), 1545.22 (C=C), 1514.22, 1443.60, 1328.41 (CH), 1247.66(CO), 1045.74 (CO). 1 H-NMR (600 M Hz, δ ppm, CDCl3): 7.66 (d, 2H, J= 7.80 Hz,Ar-H), 7.28-7.35 (m, 4H, Ar-H), 7.19-7.22 (m, 1H, Ar-H), 6.86-6.91 (m, 2H,Ar-H),6.77 (s, 1H, thiazole C 5’’ -H) , 5.38-5.43 (m, 1H, C 5’ -H),5.37 (s, 1H, C6-H), 3.80 (s, 3H, Ar-OCH3),3.50-3.56 (m, 1H, C3α-H), 3.35-3.47 (m, 1H,pyrazoline C 4’ -H2), 2.84 -2.98(m, 1H, pyrazoline C 4’ -H2), 2.32-2.40 (m, 1H, C 17 α-H), 2.18-2.32 (m, 3H), 2.04-2.11 (m, 1H), 1.88-1.98(m, 2H), 1.79-1.85(m,2H), 1.57-1.63(m, 2H), 1.46-1.54(m, 4H), 1.27-1.37(m, 3H), 1.06-1.22(m, 3H),1.02 (s, 3H, C 10 -CH3), 0.92-0.96(m, 1H),0.75 (s, 3H, C 13 -CH3). 13 C-NMR (ppm,CDCl3): 166.22, 158.88, 157.26, 151.39, 140.75, 135.19, 134.59, 128.36(2C),127.80, 127.29(2C), 125.88(2C), 121.52, 113.97(2C), 103.01, 71.72, 63.92,56.44, 55.28, 51.73, 50.12, 47.56, 43.99, 42.27, 38.44, 37.29, 36.58, 32.09,31.80, 31.63, 26.94, 24.59, 24.49, 21.01, 19.44, 13.49. MS (ESI) (m / z) : 608.35[M + H] + . HRMS (ESI): m / z [M + H] +calcd for C 38 H 46 N3O2S: 608.3232; found:608.3361.

[0124] Example 14: Compound 14

[0125] Compound 14 provided in this example is: 3β-hydroxy-pregn-5-en-17β-yl-5'-(4-benzyloxyphenyl)-1'-(4''-phenyl-[1'',3'']-thiazol-2''-yl)-4',5'-dihydropyrazol-3'-yl, and its structural formula is as follows:

[0126]

[0127] The preparation method of compound 14 is the same as that of Example 1 except that R3 of the substituted benzaldehyde is benzyloxy. The remaining raw materials, amounts and operation steps are the same as those of Example 1. Finally, the following is obtained:

[0128] White solid powder, yield 67.52%, Mp: 250.4-251.8℃. IR (KBr, cm -1 ): 2924.06(CH), 1631.69, 1611.47 (C=N), 1541.84 (C=C), 1516.29, 1444.07, 1328.42 (CH), 1240.79 (CO), 1024.13 (CO). 1 H-NMR (600 M Hz, δ ppm, CDCl3): 7.67 (d, 2H, J =8.40 Hz, Ar-H), 7.43 (d, 2H, J = 7.80 Hz, Ar-H), 7.36-7.38 (m, 2H, Ar-H), 7.28-7.35 (m, 6H, Ar-H), 7.20-7.22 (m, 1H, Ar-H), 6.96-6.97 (m, 2H, Ar-H) , 6.76(s, 1H, Thiazole C 5’’ -H), 5.39-5.41 (m, 1H, C 5’ -H),5.38 (s, 1H, C6-H), 5.05(s, 2H, OCH2), 3.51-3.55 (m, 1H, C3α-H), 3.36-3.41 (m, 1H, pyrazoline C 4’-H2),2.94 -2.98(m, 1H, pyrazoline C 4’ -H2), 2.34-2.37 (m, 1H, C 17 α-H), 2.21-2.32 (m, 3H), 2.01-2.06 (m, 1H), 1.88-1.95(m, 2H), 1.82-1.86(m, 2H), 1.75-1.79(m, 1H), 1.59-1.63(m, 2H), 1.43-1.53(m, 4H), 1.27-1.34(m, 2H), 1.08-1.18(m, 2H), 1.02(s, 3H, C 10 -CH3), 0.95-0.98(m, 1H),0.75 (s, 3H, C 13 -CH3). 13 C-NMR (ppm, CDCl3):166.23, 158.16, 157.25, 151.39, 140.81, 137.02, 135.19, 134.87, 128.59(2C),128.36(2C), 127.97, 127.85, 127.54(2C), 127.28(2C), 125.88(2C), 121.47,114.91(2C), 103.02, 71.74, 70.08, 63.96, 56.45, 51.73, 50.13, 47.55, 44.01,42.28, 38.44, 37.29, 36.58, 32.09, 31.80, 31.63, 24.59, 24.49, 21.01, 19.44,13.49.MS (ESI) (m / z) : 684.35 [M + H] + . HRMS (ESI): m / z [M + H] + calcd for C 44 H 50 N3O2S: 684.3545; found: 684.3628.

[0129] Example 15: Compound 15

[0130] Compound 15 provided in this example is: 3β-hydroxy-pregn-5-en-17β-yl-5'-(4-mercaptomethylphenyl)-1'-(4''-phenyl-[1'',3'']-thiazol-2''-yl)-4',5'-dihydropyrazol-3'-yl, and its structural formula is as follows:

[0131]

[0132] The preparation method of compound 15 is the same as that of Example 1 except that R3 of the substituted benzaldehyde is a mercaptomethyl group.

[0133] Light yellow solid powder, yield 60.22%, Mp: 125.1-130.0℃. IR (KBr, cm -1 ): 2930.21(CH), 1600.57 (C=N), 1545.09 (C=C), 1519.15, 1443.74, 1328.59 (CH), 1295.89(CO), 1045.77 (CO). 1 H-NMR (600 M Hz, δ ppm, CDCl3): 7.66 (d, 2H, J = 7.20 Hz, Ar-H), 7.29-7.33 (m, 4H, Ar-H), 7.21-7.25 (m, 3H, Ar-H), 6.75 (s, 1H, thiazole C 5’’ -H), 5.50-5.70 (m, 1H, C 5’ -H),5.36 (s, 1H, C6-H), 3.53-3.58 (m,1H, C3α-H), 3.37-3.50 (m, 1H, pyrazoline C 4’ -H2), 2.81 -2.96(m, 1H, pyrazolineC 4’ -H2), 2.47 (s, 3H, Ar-SCH3), 2.35-2.43 (m, 1H, C 17α-H), 2.18-2.34 (m, 3H),2.01-2.06 (m, 1H), 1.92-1.98(m, 2H), 1.83-1.88(m, 2H), 1.75-1.81(m, 1H),1.57-1.63(m, 2H), 1.45-1.54(m, 3H), 1.27-1.36(m, 2H), 1.08-1.19(m, 2H), 1.02(s, 3H, C 10 -CH3), 0.97-1.00(m, 1H),0.73 (s, 3H, C 13 -CH3). 13 C-NMR (ppm, DMSO-d6):165.54, 158.98, 150.74, 141.77, 139.13, 137.51, 135.02, 128.90(2C), 127.87,127.55(2C), 126.44(2C), 125.94(2C), 120.72, 104.23, 70.47, 63.49, 56.07,51.23, 50.07, 46.98, 43.80, 42.70, 37.96, 37.43, 36.59, 32.07, 31.90, 31.72,24.60, 24.49, 21.01, 19.64, 15.13, 13.4.MS (ESI) (m / z): 624.30[M + H] + . HRMS(ESI): m / z [M + H] + calcd for C 38 H 46 N3OS2: 624.3004; found:624.3116.

[0134] Example 16: Compound 16

[0135] Compound 16 provided in this example is: 3β-hydroxy-pregn-5-en-17β-yl-5'-(4-amidophenyl)-1'-(4''-phenyl-[1'',3'']-thiazol-2''-yl)-4',5'-dihydropyrazol-3'-yl, and its structural formula is as follows:

[0136]

[0137] The preparation method of compound 16 is the same as that of Example 1 except that R3 of the substituted benzaldehyde is an amide group.

[0138] White solid powder, yield 54.75%, Mp: 275.4-280.0℃. IR (KBr, cm -1 ): 3476.36(NH), 3361.47 (NH), 2932.09 (CH), 1653.38 (C=O), 1614.92 (C=N), 1533.40 (C=C), 1519.52, 1443.95, 1326.88 (CH), 1278.38 (CO), 1046.11 (CO). 1 H-NMR (600M Hz, δ ppm, DMSO-d6): 7.92 (s, 1H, NH2), 7.87 (d, 2H, J = 8.40 Hz, Ar-H), 7.66(d, 2H, J = 7.20 Hz, Ar-H), 7.40 (d, 2H, J = 8.40 Hz, Ar-H),7.30-7.34 (m, 3H, Ar-H and NH2), 7.27 (s, 1H, thiazole C 5’’ -H) ,7.22 (t, 1H, Ar-H), 5.48-5.52 (m,1H, C 5’ -H), 5.28 (s, 1H, C6-H), 4.60 (d, 1H, J = 4.80 Hz, C3-OH), 3.57-3.63 (m,1H, pyrazoline C 4’ -H2), 3.23-3.29 (m, 1H, C3α-H), 2.82 -2.89(m, 1H, pyrazolineC 4’ -H2), 2.40-2.45 (m, 1H, C 17 α-H), 2.05-2.21 (m, 3H), 1.93-1,98 (m, 1H), 1.84-1.91(m, 1H), 1.78-1.82(m, 1H), 1.73-1.77(m, 1H), 1.64-1.72(m, 2H), 1.51-1.58(m, 2H),1.41-1.47(m, 1H), 1.32-1.39(m, 2H), 1.23-1.30(m, 2H),1.13-1.18(m,1H), 0.95-1.00(m, 1H),0.94 (s, 3H, C10 -CH3), 0.67 (s, 3H, C 13 -CH3). 13 C-NMR (ppm, DMSO-d6): 168.08, 165.85, 158.88, 150.74, 146.02, 141.76, 134.96, 133.82,128.91(2C), 128.32, 127.89(2C), 126.57(2C), 125.89(2C), 120.74, 104.52,70.47, 63.96, 56.10, 51.15, 50.04, 47.47, 43.86, 42.69, 38.11, 37.39, 36.59,32.08, 31.88, 31.70, 24.59, 24.48, 20.98, 19.60, 13.61. MS (ESI) (m / z) : 621.30[M + H] + . HRMS (ESI): m / z [M + H] + calcd for C 38 H 45 N4O2S: 621.3185; found:621.3302.

[0139] Example 17: Culture of RAW264.7 cells and study of the effects of steroidal dihydropyrazole thiazoline derivatives (Compounds 1-16) on the NO release from RAW264.7 cells induced by lipopolysaccharide (LPS) and their in vitro cytotoxicity.

[0140] Mouse mononuclear macrophage RAW264.7 cells were cultured. RAW264.7 mouse macrophages were inoculated in DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin mixture and cultured in a 37°C incubator with a constant humidity and 5% carbon dioxide. Cells were passaged when the cell growth rate in the culture dish reached approximately 70%-80%.

[0141] 1. Experimental methods: The cytotoxicity of steroidal dihydropyrazole thiazoline derivatives was determined by MTT [3-4 (4, 5)-dimethyl-2-thiazol-(2, 5)-phenyl tetrazolium blue bromide] assay; the half-maximal inhibitory concentration (IC50) of steroidal dihydropyrazole thiazoline derivatives on LPS-induced NO release in mouse peritoneal macrophages RAW 264.7 cells was determined by Griess reagent assay. 50 value).

[0142] (1) Preparation of experimental solutions: Weigh 0.1 mmol of each synthesized compound using a 1 / 10,000 analytical balance. Dissolve the compound in DMSO (ultrasonicate for 20 min to completely dissolve it) to a concentration of 10 mmol / L. Store the solution in a sealed container at room temperature in the dark. Prepare appropriate amounts of the solution using cell culture medium to prepare different concentrations, with final concentrations of 1.25, 2.5, 5, 10, 20, 40, and 80 μmol / L. The DMSO content (volume ratio) is less than 0.1%. Store the stock solution in a -20°C refrigerator until ready for use.

[0143] (2) Cell plating: RAW264.7 mouse macrophages were plated at 2×10 4 The cells were seeded in a 96-well plate at a density of 200 μl per well and incubated at 37°C in 5% CO2 for 24 h, after which the old culture medium was removed.

[0144] (3) Drug addition: ① Injury group (no drug): Add 200 μl of LPS (diluted with culture medium) to a final concentration of 1 μg / ml, and incubate at 37°C, 5% CO2 for 24 hours. ② Experimental group (drug + LPS co-action group): First add 100 μl of drugs of different concentrations, with final concentrations of 1.25, 2.5, 5, 10, 20, 40, and 80 μmol / L respectively); 60 minutes later, add 100 μl of LPS (2 μg / ml) to a final concentration of 1 μg / ml. The final volume of the solution in each well is 200 μl, and incubate at 37°C, 5% CO2 for 24 hours. ③ Negative control group (i.e., blank group): Add culture medium containing RAW264.7 cells to a total volume of 200 μl, and incubate at 37°C, 5% CO2 for 24 hours. ④ Positive control group (methylprednisolone + LPS co-action group): first add 100 μl of methylprednisolone of different concentrations, and the final concentrations should be 1.25, 2.5, 5, 10, 20, 40, and 80 μmol / L respectively; after 60 minutes, add 100 μl of LPS (2 ug / ml) to a final concentration of 1 μg / ml. The final volume of the solution in each well is 200 μl, and incubate at 37°C, 5% CO2 for 24 hours.

[0145] (4) MTT cytotoxicity assay (10 μmol / L): After 24 hours, add 10 μl of MTT (5 mg / ml) to each well, shake well, and continue to culture in a CO2 incubator for 4 hours. After that, aspirate the culture medium and add 150 μl of DMSO solution to each well. Place the 96-well plate in the dark and shake on a shaker for 10-15 minutes to fully dissolve the formazan. Then, measure the absorbance of each well at a wavelength of 570 nm using a microplate reader. Cell proliferation rate = [(OD 药物 -OD 空白 ) / (OD阴性对照 -OD 空白 )]×100%.

[0146] (5) Determination of NO content: After 24 hours, 100 μL of the cell supernatant was transferred to another 96-well cell culture plate. 50 μL of the standard reagent with a series of concentration gradients diluted with the standard sample and the supernatant of the cell culture to be tested were added. 50 μL of Griess Regent 1 reagent was added to each well at room temperature and allowed to stand for 10 min. 50 μL of Griess Regent 2 was then added to each well and allowed to react in the dark for 20 min. The absorbance was measured at a wavelength of 540 nm using a microplate reader to obtain a standard curve and determine the NO concentration in the sample to be tested.

[0147] Table 1 below shows the steroidal dihydropyrazole thiazoline derivatives prepared in the present invention and calculates the cell viability and IC50 values ​​of the test compounds (compounds in Examples 1-16) for inhibiting NO concentration, and compares the effects with those of methylprednisolone, an existing first-line clinical anti-inflammatory drug.

[0148] 2. Test results: According to Table 1, Figure 6 It can be seen that most compounds have very significant inhibitory activity on LPS-induced NO production in RAW264.7 cells at the tested concentrations, showing good anti-inflammatory activity, which is significantly better than the positive control methylprednisolone, showing good anti-inflammatory activity.

[0149] To further investigate the activity and toxicity of the compounds, we performed NO inhibition and MTT assays at different concentrations and obtained their corresponding IC 50 Value, through Table 1, Figure 6 The results also show that most of the compounds in the steroidal derivatives containing dihydropyrazole thiazole units of the present invention have a significant inhibitory effect on the NO production induced by LPS in RAW 264.7 cells. The analysis is as follows: As shown in Table 1, the IC values ​​of compounds 2 and 4 in inhibiting NO are 50 The values ​​were 4.11 and 2.59 μmol / L, respectively, which were better than the positive control methylprednisolone (IC 50 =4.79μmol / L); cell viability values ​​corresponding to compounds 2 and 4 (see Figure 7 ) were 110.64% and 104.05%, respectively, showing good anti-inflammatory activity and low cytotoxicity, among which compound 4 obtained in Example 4 had the best anti-inflammatory activity and low cytotoxicity (see Figure 8-Figure 9 ), which has significant research value and application prospects.

[0150]

[0151] Table 1: Inhibitory effect of compounds on LPS-induced NO production and cell viability in RAW 264.7 cells (10 μmol / L)

[0152] Example 18: Effect of compound 4 obtained in Example 4 on inflammatory factors TNF-α and COX-2 secreted by RAW 264.7 cells (ELISA method).

[0153] In order to further clarify the anti-inflammatory activity of this class of compounds, explore the mechanism of inflammatory response of the target compounds, and provide corresponding basic research data for the development of this class of compounds, the present invention selected compound 4: 3β-hydroxy-pregn-5-ene-17β-yl-5'-(2-chlorophenyl)-1'-(4''-phenyl-[1'',3'']-thiazol-2''-yl)-4',5'-dihydropyrazol-3'-yl in the subsequent experimental process, and conducted an experiment to test the compound's inhibitory effect on inflammatory factors TNF-α and COX-2 secreted by RAW 264.7 cells. The experimental results are as follows:

[0154] 1. Experimental method: RAW264.7 mouse macrophages were cultured at 2×10 4 Cells were seeded at a density of 1 μg / ml per well in a 96-well plate. Three groups were used for the experiment: ① Injury group (no drug): 200 μl of LPS (diluted in culture medium) was added to a final concentration of 1 μg / ml, and the cells were incubated at 37°C, 5% CO2 for 24 hours. ② Experimental group (drug + LPS co-treatment): 100 μl of drug at various concentrations was added, with final concentrations of 2.5, 5, 10, and 20 μmol / L. After 60 minutes, 100 μl of LPS was added to a final concentration of 1 μg / ml. The final volume per well was 200 μl, and the cells were incubated at 37°C, 5% CO2 for 24 hours. ③ Negative control group (blank group): 200 μl of culture medium containing RAW264.7 cells was added to the cells, and the cells were incubated at 37°C, 5% CO2 for 24 hours. The cell culture supernatant was then collected. The cell culture supernatant was centrifuged at 5000 g for 10 min at 4°C to remove insoluble matter. The supernatant was collected and stored at -20°C until determination. The cytokine content was determined according to the instructions of the mouse cytokine (COX-2 and TNF-α) ELISA detection kit.

[0155] 2. Test results: Figure 10-11As shown in the results, compared with the control group, the levels of TNF-α and COX-2 released by RAW 264.7 cells after LPS stimulation were significantly increased (P<0.01); high, medium and low (2.5, 5, 10, 20 μmol / L) concentrations of compound 4 co-incubated with LPS-stimulated RAW 264.7 cells had a significant inhibitory effect on the inflammatory factors TNF-α and COX-2 released by RAW 264.7 cells (P<0.01), and the effect was dose-dependent ( Figure 10-11 Compound 4 (steroidal dihydropyrazole thiazoline derivative of the present invention) has good anti-inflammatory activity.

[0156] Compared with the LPS group, * P < 0.05; ** P<0.01. Compared with the negative control group, ## P<0.01.

[0157] The experimental results show that most of the compounds in the steroidal dihydropyrazole thiazoline derivatives provided by the present invention have good anti-inflammatory activity, especially the compound 4 in Example 4 of the steroidal dihydropyrazole thiazoline derivatives of the present invention: 3β-hydroxy-pregn-5-ene-17β-yl-5'-(2-chlorophenyl) -1'-(4''-phenyl-[1'',3'']-thiazol-2''-yl) -4',5'-dihydropyrazolyl-3'-yl IC 50 The value is better than methylprednisolone, and it has a significant inhibitory effect on the inflammatory factors TNF-α and COX-2 released by RAW264.7 cells, and shows low toxicity to cells; analysis shows that compound 4, a steroidal dihydropyrazole thiazoline derivative, has better anti-inflammatory activity than the existing first-line anti-inflammatory drug methylprednisolone while showing the advantage of low toxicity, and has profound development and application value.

[0158] The above description is only a preferred embodiment of the present invention and is not intended to limit the scope of the present invention. Various changes can be made to the above embodiments of the present invention. That is, all simple, equivalent changes and modifications made according to the claims and description of the present invention application fall within the scope of protection of the claims of the present invention. Anything not described in detail in the present invention is conventional technical content. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A use of a steroidal dihydropyrazole thiazole derivative in the preparation of an anti-inflammatory drug, characterized in that: The structural formula of the derivative is shown in formula (I), The anti-inflammatory drug is an anti-inflammatory drug that has an inhibitory effect on LPS-induced NO and inflammatory factors TNF-α and COX-2 produced by RAW264.7 cells.

2. The use of the steroidal dihydropyrazole thiazole derivative according to claim 1 in the preparation of anti-inflammatory drugs, characterized in that: The dosage form of the anti-inflammatory drug is tablets, pills, capsules, injections, suspensions or emulsions.

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