CN106866773A - One group of preparation method and application with active anticancer pyrazolyl steroid derivative - Google Patents

Abstract

The invention discloses a preparation method and application of a group of pyrazolyl steroid derivatives with anticancer activity, which belongs to the technical field of drug synthesis. Phenylhydrazone 2 and 8 are generated under the catalysis of phosphorus oxychloride, and then ring-closing reaction occurs under the catalysis of phosphorus oxychloride to obtain compounds 3 and 9 with pyrazole rings, and then hydrolyzed to obtain deformyl compounds 4 and 10, in sodium borohydride Restore down to 6. Intermediates 4 and 10 were aminated and reduced with amines under the catalysis of sodium triacetoxyborohydride to obtain 5a‑e and 11a‑e. The route of the present invention is novel, high in yield and easy to separate, which is the optimal route for preparing such compounds.

Description

Preparation method and application of a group of pyrazolyl steroid derivatives with anticancer activity

technical field

The invention belongs to the technical field of drug synthesis, and relates to a preparation method and application of a group of pyrazolyl steroid derivatives with anticancer activity.

Background technique

Cancer has always been the greatest threat to human health. Although the current prevention and treatment methods have been developed, the number of patients dying of cancer is still increasing every year. The treatment methods of existing drugs can no longer meet the needs of disease treatment, and there is an urgent need to screen out new anticancer drugs to deal with variable diseases. Based on the advantages of low toxicity of steroid drugs, high utilization rate, and resistance to drug resistance, more and more steroid drugs have been developed.

Contents of the invention

The object of the present invention is to provide a group of preparation methods and applications of pyrazolyl steroid derivatives with anticancer activity.

Its specific technical plan is:

A group of pyrazolyl steroid derivatives with anticancer activity, the chemical structural formula is:

A method for preparing pyrazolyl steroid derivatives with anticancer activity according to the present invention, comprising the following steps:

(1) Dissolve 6.32g of pregnenolone in a 250mL eggplant-shaped flask with 150mL of glacial acetic acid, stir until it is completely dissolved, then add phenylhydrazine in batches, stir and ultrasonically until dissolved, measure 3.46mL of triethylamine at room temperature and slowly Slowly drop into the reaction system, after half an hour, the drop was completed, stirred at room temperature for 6 hours until solids were precipitated, filtered with suction, washed with glacial acetic acid, and dried to obtain the target compound 2 and 87.8g, which can be directly used in the next step without separation ;

(2) First prepare visemier reagent: take a fully dry round bottom flask, add 20mL of dried DMF solution inwardly, slowly add phosphorus oxychloride solution 4.65mL, 50mmol inwardly with stirring, and dropwise inward after half an hour, The temperature should be kept at 0°C throughout the period. The mixture was stirred and reacted for 20 min for later use. Dissolve 10 mmol of the substrate steroid phenylhydrazone in 30 mL of anhydrous DMF, and slowly drop it into the prepared visemier reagent at 0°C. This process takes 30 minutes. After the drop is completed, continue to stir for 24 hours to check that the reaction is complete. At this point, the reaction system was poured into a saturated sodium bicarbonate solution, stirred until solids were precipitated, the crude product of the target product was obtained by suction filtration, and then dissolved in dichloromethane for extraction three times, the obtained organic phases were combined and dried, filtered, column fractionated, petroleum Ether: ethyl acetate = 4: 1, to obtain 3 g of pure products of target compounds 3 and 9;

(3) Take 5 mmol of pyrazolylpregnenol, dissolve it in a mixed solution of 20 mL of tetrahydrofuran and 20 mL of methanol, then add 5.5 mmol of potassium carbonate, 759 mg, stir ultrasonically until it is completely dissolved, heat and reflux for 2 hours, and detect the reaction until it is complete, and then use The solvent was removed by a rotary evaporator, extracted three times with ethyl acetate, the organic phases were combined, and the column fractionated to obtain the target compound 4 and 104.8mmol;

(4) Take 0.5 mmol of deformylated pyrazolylpregnenol and dissolve it in a mixed solution of 10 mL of tetrahydrofuran and 10 mL of methanol, then add 38 mg of sodium borohydride, 1 mmol, in batches, and react at room temperature for 1 hour. Quenched with acetic acid, then extracted three times with ethyl acetate, combined the organic matter, and separated the phases, petroleum ether: ethyl acetate = 1: 1, and obtained 60.48 mmol of the target product;

(5) Take a 25 mL dry round bottom flask, dissolve 1 mmol of deformylated pyrazolylpregnenol in 15 mL of dehydrated 1,2-dichloroethane, add 1.1 mmol of aniline derivatives under stirring, and then Add sodium triacetylborohydride in batches, stir at room temperature for 8 hours, and check the reaction. After the reaction is complete, add saturated sodium bicarbonate solution to quench, then extract three times with dichloromethane, combine the organic phases, column fraction, dichloromethane: Methanol=10:1, 0.75mmol of aminated reduction products 5a-e and 11a-e were obtained.

The application of the pyrazolyl steroid derivatives with anticancer activity in the process of preparing medicines for treating human lung adenocarcinoma, cervical cancer and human breast cancer.

Compared with prior art, the beneficial effect of the present invention is:

1 The three pyrazolyl steroid derivatives of the present invention are brand new compounds, and their preparation methods are also successfully applied for the first time in the synthesis of such compounds.

2 The three derivatives of the present invention have good inhibition on the proliferation of three tumor cells (A549 (human lung adenocarcinoma cell), Hela (cervical cancer cell line), MCF7 (human breast cancer cell)) and 293T cells Activity, its IG ₅₀ is between 0.53uM-7.51uM.

3 The route of the present invention is novel, high in yield and easy to separate, which is the optimal route for preparing such compounds.

Description of drawings

Figure 1 is the preparation route a of pyrazolyl steroid derivatives, wherein, (a) Phenylhydrazine, AcOH, rt; (b) POCl ₃ , DMF, 0°C; (c) K ₂ CO ₃ , THF/H ₂ O , rt; (d) R-NH ₂ , DCE, NaBH(AcO) ₃ , rt; (e) MeOH/THF, NaBH ₄ , rt;

Figure 2 is the preparation route b of pyrazolyl steroid derivatives, in which, (f) Phenylhydrazine, AcOH, rt; (g) POCl ₃ , DMF, 0°C-40°C; (h) K ₂ CO ₃ , THF/ _H2O , rt; (i) R- _NH2 , DCE, NaBH(AcO) ₃ , rt.

detailed description

The technical solution of the present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments.

First, under the catalysis of pregnenolone 1, 5, 16-diengegesterone and phenylhydrazine acetic acid, phenylhydrazone 2 and 8 are generated, and then a ring-closing reaction occurs under the catalysis of phosphorus oxychloride to obtain a pyrazole ring Compounds 3 and 9 were then hydrolyzed to yield the deformylated compounds 4 and 10, which were reduced to 6 under sodium borohydride reduction. The intermediate products 4 and 10 were aminated and reduced with amines under the catalysis of sodium triacetoxyborohydride to obtain 5a-e and 11a-e. See Table 1 for details of 5a-e and 11a-e.

Table 1

Example 1

Preparation of Pyrazolyl Steroid Derivatives of Formula (I) by Chemical Synthesis

(1) Dissolve 6.32g (20mmol) of pregnenolone in a 250mL eggplant-shaped flask with 150mL of glacial acetic acid, stir until it is completely dissolved, then add phenylhydrazine (22mmol) in batches, stir and ultrasonically until dissolved, and measure 3.46mL (25mmol) triethylamine was slowly dripped into the reaction system at normal temperature, and the dripping was completed after half an hour, and stirred at normal temperature for 6h until solids were precipitated, filtered by suction, washed with glacial acetic acid, and dried to obtain about 7.8g of target compounds 2 and 8, This product was directly used in the next reaction without isolation.

(2) First prepare the visemier reagent: take a well-dried round-bottomed flask, add 20 mL of dried DMF solution, slowly add phosphorus oxychloride solution (4.65 mL, 50 mmol) dropwise under stirring, drop after half an hour After that, the temperature should be kept at 0°C during the whole period. The mixture was stirred and reacted for 20 min for later use. Dissolve the substrate steroidal phenylhydrazone (10mmol) in 30mL of anhydrous DMF, slowly drop it into the prepared visemier reagent at 0°C, this process takes 30min, continue stirring for 24h after the dripping, and detect the reaction to completely. Now, the reaction system is poured into a saturated sodium bicarbonate solution, stirred until solids are precipitated, the crude product obtained by suction filtration is then dissolved in dichloromethane and extracted three times, the resulting organic phases are combined and dried, filtered, column fractionated (petroleum Ether: ethyl acetate = 4: 1) to obtain about 3 g of pure target compounds 3 and 9.

(3) Dissolve 5 mmol of pyrazolylpregnenol in a mixed solution of 20 mL of tetrahydrofuran and 20 mL of methanol, then add potassium carbonate (5.5 mmol, 759 mg), and stir ultrasonically until completely dissolved. Heat to reflux for 2 h, check the reaction until it is complete, then remove the solvent with a rotary evaporator, extract three times with ethyl acetate, combine the organic phases, and obtain about 4.8 mmol of the target compounds 4 and 10 by column separation.

(4) Dissolve 0.5 mmol of deformylated pyrazolylpregnenol in a mixed solution of 10 mL of tetrahydrofuran and 10 mL of methanol, then add sodium borohydride (38 mg, 1 mmol) in batches, react at room temperature for 1 hour, and detect that the reaction is complete Quenched with glacial acetic acid, then extracted three times with ethyl acetate, combined the organic matter, and separated the phases (petroleum ether: ethyl acetate = 1:1) to obtain about 0.48 mmol of the target product 6.

(5) Take a 25 mL dry round bottom flask, dissolve 1 mmol of deformylated pyrazolylpregnenol in 15 mL of dehydrated 1,2-dichloroethane, add 1.1 mmol of aniline derivatives under stirring, and then Add sodium triacetylborohydride in batches, stir at room temperature for 8h, detect the reaction, add saturated sodium bicarbonate solution to quench after the reaction is complete, then extract three times with dichloromethane, combine the organic phases, column fraction (dichloromethane: Methanol=10:1) to obtain about 0.75 mmol of aminated reduction products 5a-e and 11a-e.

Example 2

The structural identification of the compounds, the structures of the three synthesized compounds were confirmed by ¹ H-NMR, ¹³ C-NMR and high-resolution mass spectrometry (HRMS).

¹ H-NMR, ¹³ C-NMR and HRMS data are as follows:

17β-(1-phenyl-4-((methylamino)methyl)-3-pyrazolyl)androst-3β-ol(5a)

5a, white solid, Yield: 88%, mp 204-206°C; ¹ H-NMR (CDCl ₃ & CD ₃ OD, 500MHz): δ (ppm) 8.09 (s, 1H), 7.67 (d, 2H, J=8.5 Hz), 7.43(t, 2H, J=7.0Hz), 7.26(t, 1H, J=7.5Hz), 3.90-3.80(m, 2H), 3.58-3.53(m, 1H), 2.74(t, 1H , J=9.5Hz), 2.53(s, 3H, CH ₃ ), 2.47-2.40(m, 1H), 2.01-1.93(m, 1H), 1.78-1.70(m, 4H), 1.56-1.54(m, 3H), 1.40-1.10(m, 12H), 1.00-0.93(m, 2H), 0.81(s, 3H, CH ₃ ), 0.73-0.67(m, 1H), 0.64(s, 3H, CH ₃ ); ¹³ C-NMR (CDCl ₃ &CD ₃ OD, 125MHz): δ(ppm) 152.48, 140.02, 129.29, 129.29, 127.45, 126.18, 118.91, 118.91, 115.61, 70.81, 56.33, 54.53, 48.39, 44.91, 44.37 38.56, 37.74, 37.02, 35.99, 35.53, 33.17, 32.07, 31.04, 28.65, 26.66, 24.43, 21.07(CH ₃ ), 13.33(CH ₃ ), 12.22(CH ₃ ); ₃₀ H ₄₄ N ₃ O ⁺ (M+H) ⁺ 462.34789, found 462.34738.

17β-(1-phenyl-4-((ethylamino)methyl)-3-pyrazolyl)androst-3β-ol(5b)

5b, white solid, Yield: 80%, mp 246-248°C; ¹ H-NMR (DMSO-d, 500MHz): δ (ppm) 8.66 (s, 1H), 7.72-7.70 (m, 2H), 7.50 ( t, 2H, J=7.0, 8.5Hz), 7.30(t, 1H, J=7.5Hz), 4.43(s, br, 1H), 4.01-3.92(m, 2H), 2.98(dq, 2H, J= 2.0, 7.5Hz), 2.92(t, 1H, J=9.5Hz), 2.40-2.33(m, 1H), 1.96-1.91(m, 1H), 1.70-1.60(m, 4H), 1.53-1.41(m , 3H), 1.37-1.06(m, 12H), 1.24(t, 3H, J=7.5Hz), 0.96-0.88(m, 2H), 0.74(s, 3H, CH ₃ ), 0.71-0.65(m, 1H), 0.55 (s, 3H, CH ₃ ); ¹³ C-NMR (DMSO-d, 125MHz): δ (ppm) 152.71, 139.98, 130.13, 130.13, 129.58, 126.65, 118.52, 118.52, 114.09, 69.79, 56.18 , 54.58, 47.58, 44.98, 44.67, 42.06, 40.32, 38.67, 37.95, 37.21, 36.08, 35.69, 32.32, 31.86, 28.88, 26.62, 24.57, 21.16, 13.85( _CH3CH ), 12.65(CH37 _{) 3} ); HRMS(ESI) m/z calcd for C ₃₁ H ₄₆ N ₃ O ⁺ (M+H) ⁺ 476.36354, found 476.36313.

17β-(1-phenyl-4-((propylamino)methyl)-3-pyrazolyl)androst-3β-ol(5c)

5c, light brown solid, Yield: 76%, mp 160-162°C; ¹ H-NMR (DMSO-d, 500MHz): δ (ppm) 8.28 (s, 1H), 7.74-7.73 (m, 2H), 7.45 (t, 2H, J=7.5, 8.0Hz), 7.30(t, 1H, J=7.5Hz), 3.90(s, br, 1H), 3.63-3.61(m, 2H), 3.38-3.32(m, 1H ), 2.81(t, 1H, J=10.0Hz), 2.56(t, 2H, J=7.5Hz), 2.37-2.30(m, 1H), 1.68-1.58(m, 4H), 1.55-1.32(m, 5H), 1.28-1.04(m, 10H), 0.94-0.90(m, 2H), 0.87(t, 3H, J=7.5Hz), 0.74(s, 3H, CH ₃ ), 0.70-0.65(m, 1H ), 0.58 (s, 3H, CH ₃ ); ¹³ C-NMR (DMSO-d, 125MHz): δ (ppm) 151.54, 139.74, 129.32, 129.32, 126.64, 125.25, 120.35, 117.52, 117.52, 69.23, 55.67, _{( _} CH ₃ ); HRMS (ESI) m/z calcd for C ₃₂ H ₄₈ N ₃ O ⁺ (M+H) ⁺ 490.37919, found 490.37924.

17β-(1-phenyl-4-((isopropylamino)methyl)-3-pyrazolyl)androst-3β-ol(5d)

5d, white solid, Yield: 83%, mp 216-218°C; ¹ H-NMR (CDCl ₃ & CD ₃ OD, 500MHz): δ (ppm) 7.94 (s, 1H), 7.67-7.65 (m, 2H), 7.41(t, 2H, J=7.0, 8.5Hz), 7.23(t, 1H, J=7.5Hz), 3.70(m, 2H), 3.57-3.53(m, 1H), 2.92-2.97(m, 1H) , 2.72(t, 1H, J=10.0Hz), 2.47-2.39(m, 1H), 2.00-1.94(m, 1H), 1.80-1.69(m, 4H), 1.60-1.54(m, 3H), 1.45 -1.32(m, 3H), 1.35-1.28(m, 6H), 1.15(d, 6H, J=6.5Hz, 2CH ₃ ), 1.12-0.86(m, 4H), 0.81(s, 3H, CH ₃ ) , 0.74-0.69 (m, 1H), 0.66 (s, 3H, CH ₃ ); ¹³ C-NMR (CDCl ₃ & CD ₃ OD, 125MHz): δ (ppm) 152.13, 140.34, 129.29, 129.29, 126.40, 125.83, 119.70, 118.84, _118.84 , 70.99, 56.42, 54.59, 48.66, 44.99, 44.80, 40.62, 38.74, 37.92, 37.10, 36.06, 35.61, 32.15, 31.21, 29.69, 28.74, 215.43, 24.9 (1CH) CH ₃ ), 21.16, 13.49 (CH ₃ ), 12.33 (CH ₃ ); HRMS (ESI) m/z calcd for C ₃₂ H ₄₈ N ₃ O ⁺ (M+H) ⁺ 490.37919, found 490.37918.

17β-(1-phenyl-4-((butylamino)methyl)-3-pyrazolyl)androst-3β-ol(5e)

5e, white solid, Yield: 83%, mp 138-140°C; ¹ H-NMR (DMSO-d&CDCl ₃ , 500MHz): δ (ppm) 8.35 (s, 1H), 7.73 (d, 2H, J=8.0Hz ), 7.46(t, 2H, J=7.5, 8.5Hz), 7.24(t, 1H, J=7.5Hz), 3.73(m, 2H), 3.37-3.33(m, 1H), 2.84(t, 1H, J=9.5Hz), 2.70(t, 2H, J=7.5Hz), 2.38-2.32(m, 1H), 1.69-1.62(m, 4H), 1.51-1.47(m, 4H), 1.34-1.11(m , 12H), 1.08-0.88(m, 6H), 0.88(t, 3H, J=7.5Hz), 0.75(s, 3H, CH ₃ ), 0.70-0.66(m, 1H), 0.58(s, 3H, CH ₃ ); ¹³ C-NMR (DMSO-d&CDCl ₃ , 125MHz): δ(ppm) 151.78, 139.71, 129.40, 129.40, 127.30, 125.52, 118.38, 117.72, 117.72, 69.30, 55.74, 54.04, 44481, 4.7 , 44.14, 41.98, 38.13, 37.77, 36.69, 35.56, 35.16, 31.77, 31.32, 29.94, 28.38, 26.24, 24.08, 21.17, 20.65, 13.73 (CH ₃ ), 13.36 (CH ₃ ), 12.12 (CH ₃ ) (ESI)m/z calcd for C ₃₃ H ₅₀ N ₃ O ⁺ (M+H) ⁺ 504.39484, found 504.39404.

17β-(1-phenyl-4-((methylamino)methyl)-3-pyrazolyl)androst-5, 16-dienes-3β-ol(11a)

11a, white solid, Yield: 80%, mp 108-110°C; ¹ H-NMR (CDCl ₃ & CD ₃ OD, 500MHz): δ (ppm) 8.23 (s, 1H), 7.74-7.72 (m, 2H), 7.45(t, 2H, J=7.5Hz, 8.5Hz), 7.28(t, 1H, J=7.5Hz), 5.93-5.92(m, 1H), 5.40-5.39(m, 1H), 4.00(m, 2H ), 3.52-3.46(m, 1H), 3.36(s, 1H), 2.58(s, 3H), 2.41-2.25(m, 4H), 2.17-2.06(m, 1H), 1.88-1.75(m, 3H ), 1.72-1.64(m, 2H), 1.58-1.26(m, 5H), 1.15-0.87(m, 9H), 1.15(s, 3H, CH ₃ ), 1.08(s, 3H, CH ₃ ); ¹³ C-NMR (CDCl ₃ & CD ₃ OD, 125MHz): δ(ppm) 148.34, 147.13, 141.57, 139.99, 129.87, 129.59, 129.59, 127.72, 126.56, 121.39, 118.72, 118.72, 114.74, 7028, 80.57 , 43.84, 42.12, 37.41, 36.96, 35.47, 33.04, 32.59, 31.82, 31.41, 30.67, 21.15 (CH ₃ ), 19.43 (CH ₃ ), 16.35 (CH ₃ ); HRMS (ESI) m/z calcd for C ₃₀ H ₄₀ N ₃ O ⁺ (M+H) ⁺ 458.31659, found 458.31656.

17β-(1-phenyl-4-((ethylamino)methyl)-3-pyrazolyl)androst-5, 16-dienes-3β-ol(11b)

11b, white solid, Yield: 79%, mp 106-108°C; ¹ H-NMR (CDCl ₃ & CD ₃ OD, 500MHz): δ (ppm) 7.86 (s, 1H), 7.62-7.60 (m, 2H), 7.36-7.32(m, 2H), 7.16(t, 1H, J=7.5Hz), 5.84(s, 1H), 5.30(s, 1H), 3.73-3.67(m, 3H), 3.42-3.38(m, 1H), 2.67-2.62(m, 2H), 2.43-2.40(m, 2H), 2.27-2.14(m, 3H), 2.05-1.97(m, 2H), 1.79-1.67(m, 3H), 1.63- 1.58(m, 3H), 1.47-1.34(m, 3H), 1.12-0.80(m, 11H), 1.07(t, 3H, CH ₃ ,, J=7.5Hz), 1.04(s, 3H, CH ₃ ) , 1.00(s, 3H, CH ₃ ); ¹³ C-NMR (CDCl ₃ &CD ₃ OD, 125MHz): δ(ppm) 147.78, 147.67, 141.51, 140.22, 129.43, 129.43, 128.95, 126.11, 125.97, 121.38, 120.18 , 118.46, 118.46, 71.43, 56.95, 50.84, 48.40, 43.86, 43.51, 42.09, 37.36, 36.90, 35.59, 32.45, 31.79, 31.37, 30.63, 21.14, 19.38(CH ₃ ), 16.29(CH _{3 3} ); HRMS(ESI) m/z calcd for C ₃₁ H ₄₂ N ₃ O ⁺ (M+H) ⁺ 472.33224, found 472.33211.

177-(1-phenyl-4-((propylamino)methyl)-3-pyrazolyl)androst-5, 16-diennes-3β-ol(11c)

11c, white solid, Yield: 62%, mp 130-132°C; ¹ H-NMR (CDCl ₃ & CD ₃ OD, 500MHz): δ (ppm) 8.06 (s, 1H), 7.72-7.70 (m, 2H), 7.43(t, 2H, J=7.5Hz, 8.5Hz), 7.25(t, 1H, J=7.5Hz), 5.93-5.92(m, 1H), 5.39(d, 1H, J=5.0Hz), 3.90- 3.83(m, 2H), 3.52-3.47(m, 2H), 2.69(t, 2H, J=7.5Hz), 2.48-2.46(m, 1H), 2.34-2.22(m, 3H), 2.15-2.06( m, 2H), 1.87-1.75(m, 3H), 1.68-1.40(m, 9H), 1.14-1.04(m, 8H), 1.14(s, 3H, _CH3 ), 1.08(s, 3H, _CH3 ), 0.95 (t, 3H, J=7.5Hz); ¹³ C-NMR (CDCl ₃ & CD ₃ OD, 125MHz): δ (ppm) 147.96, 147.51, 141.51, 140.13, 129.46, 129.46, 129.23, 127.70, 126.12, _121.39 , 118.53, 118.53, 118.33, 71.49, 56.98, 50.84, 50.47, 48.43, 43.35, 42.14, 37.35, 36.90, 35.53, 32.49, 31.79, 31.43, 30.62, 21.92, 19.12, ₁₉ ), 11.59 (CH ₃ ); HRMS (ESI) m/z calcd for C ₃₂ H ₄₄ N ₃ O ⁺ (M+H) ⁺ 486.34789, found 486.34793.

17β-(1-phenyl-4-((isopropylamino)methyl)-3-pyrazolyl)androst-5, 16-dienes-3β-ol(11d)

77d, white solid, Yield: 73%, mp 202-204°C; ¹ H-NMR (CDCl ₃ & CD ₃ OD, 500MHz): δ (ppm) 8.57 (s, 1H), 7.76-7.74 (m, 2H), 7.43(t, 2H, J=7.5Hz, 8.5Hz), 7.27(t, 1H, J=7.5Hz), 5.89-5.88(m, 1H), 5.39(d, 1H, J=5.0Hz), 4.13- 4.06(m, 2H), 3.52-3.46(m, 1H), 3.36(s, 1H), 3.27-3.22(m, 1H), 2.36-2.25(m, 4H), 2.17-2.05(m, 2H), 1.86-1.75(m, 3H), 1.71-1.62(m, 3H), 1.56-1.49(m, 3H), 1.37(d, 3H, J=6.5Hz), 1.34(d, 3H, J=6.5Hz) , 1.15-1.03(m, 8H), 1.15(s, 3H, CH ₃ ), 1.07(s, 3H, CH ₃ ); ¹³ C-NMR (CDCl ₃ & CD ₃ OD, 125MHz): δ(ppm) 147.96, 146.53，141.06，139.38，129.72，129.07，129.07，128.19，126.14，120.87，118.30，118.30，112.51，71.04，56.64，50.36，48.60，48.05，41.67，38.41，36.90，36.45，34.82，32.10，31.31，30.96， 30.15, 20.60, 19.25, 18.96(CH ₃ ), 18.83(CH ₃ ), 15.91(CH ₃ ); HRMS(ESI) m/z calcd for C ₃₂ H ₄₄ N ₃ O ⁺ (M+H) ⁺ 486.34789, found 486.34750.

177-(1-phenyl-4-((butylamino)methyl)-3-pyrazolyl)androst-5, 16-dienes-3β-ol(11e)

11e, glasslike solid, Yield: 89%, mp 100-102°C; ¹ H-NMR (CDCl ₃ & CD ₃ OD, 500MHz): δ (ppm) 7.95 (s, 1H), 7.71-7.69 (m, 2H), 7.43(t, 2H, J=7.5Hz, 8.5Hz), 7.24(t, 1H, J=7.5Hz), 5.93-5.94(m, 1H), 5.40(d, 1H, J=5.0Hz), 3.82- 3.74(m, 2H), 3.50-3.48(m, 1H), 3.37(s, 1H), 2.67(t, 2H, 7.5Hz), 2.51-2.49(m, 1H), 2.35-2.26(m, 3H) , 2.14-2.07(m, 2H), 1.89-1.80(m, 3H), 1.72-1.67(m, 3H), 1.52-1.26(m, 8H), 1.15-1.05(m, 8H), 1.14(s, 3H, CH ₃ ), 1.05 (s, 3H, CH ₃ ), 0.94 (t, 3H, J=7.5Hz); ¹³ C-NMR (CDCl ₃ & CD ₃ OD, 125MHz): δ (ppm) 147.82, 147.70, 141.50，140.23，129.44，129.44，128.96，126.19，125.97，121.41，120.11，118.48，118.48，71.49，56.97，50.85，49.07，48.40，44.08，42.11，37.35，36.90，35.59，32.46，31.80，31.60，31.40， 30.63, 21.14, 20.52, 19.40(CH ₃ ), 16.32(CH ₃ ), 13.95(CH ₃ ); HRMS(ESI) m/z calcd for C ₃₃ H ₄₆ N ₃ O ⁺ (M+H) ⁺ 500.36354, found 500.36353.

Example 3

Anti-tumor cell proliferation activity of pyrazolyl steroid derivatives of formula (I) of the present invention

Adopt RSB method to measure the pyrazolyl steroid derivative of formula (I) of the present invention to four kinds of cells of A549 (human lung adenocarcinoma cell), Hela (cervix cancer cell line), MCF7 (human liver cancer cell line), 293T cell line proliferation inhibitory activity.

Specific method: Accurately weigh 1-3mg of the compound to be tested, use DMSO as solvent, prepare a solution with a concentration of 10mMol/L, let it stand at room temperature for half an hour until the sample is completely dissolved, and store it for later use. Take A549, Hela, 293T, MCF7 cells in the logarithmic growth phase, wash and digest with trypsin to make a cell suspension, count the cells and dilute to an appropriate concentration, and add the cell suspension evenly to a 96-well plate, each well 100ul, three replicates were set for each group, and negative control and positive control wells were set at the same time. Take a certain amount of the pre-configured compound to be tested and add it to a 96-well plate. The compound starts from 50uM, and the compound is diluted 3 times, with a total of 9 gradients, and then the treated cells are placed in a 5% carbon dioxide concentration, 37 ℃ incubator Cultured in medium for 72h. After the incubation period, terminate the incubation, remove the supernatant, add 100ul of cold 10% TCA to each well, fix at 4°C for 2h, rinse repeatedly with double distilled water 5 times, and dry in an oven at 50°C for about half an hour. After it dries, add 100ul of SRB solution prepared by glacial acetic acid, stain at room temperature for half an hour, rinse with 1% glacial acetic acid 5 times to remove non-specifically bound dyes, this step needs to be performed quickly to prevent dyes that have specifically bound to proteins break down. Dry in an oven at 50°C for half an hour, add 150ul Tris solution to dissolve and pat to mix, measure the absorbance at A540nm with a microplate reader, calculate the inhibition rate, and calculate the IC ₅₀ with EXCEl, the results are shown in Table 2.

Anti-cell proliferation activity IC ₅₀ (μmol/L) of some compounds in Table 2

The results of table 2 illustrate that pyrazolyl steroid derivatives have good cellular effects on A549 (human lung adenocarcinoma cells), Hela (cervix cancer cell line), MCF7 (human breast cancer cells), and 293T cells. toxicity.

The above is only a preferred specific embodiment of the present invention, and the scope of protection of the present invention is not limited thereto. Any person familiar with the technical field within the technical scope disclosed in the present invention can obviously obtain the simplicity of the technical solution. Changes or equivalent replacements all fall within the protection scope of the present invention.

Claims (3)

1. A group of pyrazolyl steroid derivatives with anticancer activity, characterized in that the chemical structural formula is: 2. a preparation method with anticancer activity pyrazolyl steroid derivatives according to claim 1, characterized in that, comprising the following steps: (1) Dissolve 6.32g of pregnenolone in a 250mL eggplant-shaped flask with 150mL of glacial acetic acid, stir until it is completely dissolved, then add phenylhydrazine in batches, stir and ultrasonically until dissolved, measure 3.46mL of triethylamine at room temperature and slowly Slowly drop into the reaction system, after half an hour, the drop was completed, stirred at room temperature for 6 hours until solids were precipitated, filtered with suction, washed with glacial acetic acid, and dried to obtain 7.8g of target compounds 2 and 8, which were directly used in the next step without separation reaction; (2) First prepare visemier reagent: take a fully dry round bottom flask, add 20mL of dried DMF solution inwardly, slowly add phosphorus oxychloride solution 4.65mL, 50mmol inwardly with stirring, and dropwise inward after half an hour, During the whole period, the temperature should be kept at 0°C; the mixture should continue to stir and react for 20 minutes before use; dissolve 10 mmol of the substrate steroid phenylhydrazone in 30 mL of anhydrous DMF, and slowly drop it into the prepared visemier reagent at 0°C , this process takes 30min, continue to stir for 24h after dripping, and detect the reaction until complete; at this time, pour the reaction system into saturated sodium bicarbonate solution, stir until solid precipitates, obtain the crude product of the target product by suction filtration, and then dissolve it in distilled water Methyl chloride was extracted three times, and the obtained organic phases were combined and dried, filtered, column separated, petroleum ether: ethyl acetate = 4:1, and 3 g of pure target compounds 3 and 9 were obtained; (3) Take 5 mmol of pyrazolylpregnenol, dissolve it in a mixed solution of 20 mL of tetrahydrofuran and 20 mL of methanol, then add 5.5 mmol of potassium carbonate, 759 mg, stir ultrasonically until it is completely dissolved, heat and reflux for 2 hours, and detect the reaction until it is complete, and then use The solvent was removed by a rotary evaporator, extracted three times with ethyl acetate, the organic phases were combined, and the column was separated to obtain 4.8 mmol of target compounds 4 and 10; (4) Take 0.5 mmol of deformylated pyrazolylpregnenol and dissolve it in a mixed solution of 10 mL of tetrahydrofuran and 10 mL of methanol, then add 38 mg of sodium borohydride, 1 mmol, in batches, and react at room temperature for 1 hour. Quenched with acetic acid, then extracted three times with ethyl acetate, combined the organic matter, separated the phases, petroleum ether: ethyl acetate = 1:1, and obtained 0.48 mmol of the target product 6; (5) Take a 25mL dry round bottom flask, dissolve 1mmol of deformylated pyrazolylpregnenol in 15mL of dehydrated 1,2-dichloroethane, add 1.1mmol of aniline derivative under stirring, and then Add sodium triacetylborohydride in batches, stir at room temperature for 8 hours, and detect the reaction. After the reaction is complete, add saturated sodium bicarbonate solution to quench, then extract with dichloromethane three times, combine the organic phases, column fraction, dichloromethane: Methanol=10:1, 0.75mmol of aminated reduction products 5a-e and 11a-e were obtained. 3. The application of the pyrazolyl steroid derivatives with anticancer activity according to claim 1 in the preparation of drugs for the treatment of human lung adenocarcinoma, cervical cancer and human breast cancer.