Selenium cyanopregnenolone amide compound and its application

Abstract

The present invention relates to the technical field of pharmaceutical compounds and specifically discloses a selenocyanine pregnenol ketamide compound having the following general structural formula: #imgabs0#. The selenocyanine pregnenol ketamide compound of the present invention exhibits strong inhibitory activity against human breast cancer cells, cervical cancer cells, ovarian cancer cells, and human liver cancer cells. Furthermore, the selenocyanine pregnenol ketamide compound of the present invention also exhibits strong inhibitory effects against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci (VRE). The selenocyanine pregnenol ketamide compound of the present invention has low toxicity to humans and exhibits strong inhibitory effects against cancer cells and various drug-resistant bacteria at low concentrations. It can be used to prepare cancer treatment drugs and as an antibacterial agent against drug-resistant bacteria.

Description

Technical Field

[0001] The present invention relates to the technical field of medicinal compounds, and in particular to a selenocyanopregnenolone amide compound and application thereof. Background Art

[0002] Cancer, characterized by uncontrolled cell proliferation, is a major threat to human health and one of the leading causes of death worldwide. Conquering malignant tumors is a major challenge facing modern medicine and one of the greatest challenges facing the pharmaceutical industry today. Currently, a wide range of anti-cancer drugs are emerging and gaining clinical application. However, most clinically used anti-cancer drugs have significant side effects. For example, conventional platinum-based anticancer drugs, for example, are known to cause significant toxic side effects and drug resistance in human tissues, limiting their widespread use. Therefore, the development of highly effective, low-toxic anti-cancer drugs remains a research hotspot and of vital importance. On the other hand, cancer patients have weak resistance and immunity, allowing pathogens to invade with minimal care, leading to bacterial infections. Bacterial infections are a major cause of disease progression or death in cancer patients. Bacterial infection is also one of the most common infectious diseases for the normal population. It has certain hazards and can easily cause damage to the functions of various organs. For bacterial infections, antibacterial drugs including penicillins, cephalosporins, and cephamycins are usually used for treatment. Due to continuous excessive use, drug-resistant bacteria are currently increasing, and some pathogenic bacteria have become resistant to multiple antibacterial drugs. For example, methicillin-resistant Staphylococcus aureus (MRSA) is a bacterium that is resistant to multiple antibacterial drugs, and vancomycin-resistant enterococci (VRE) is resistant to vancomycin. Both of these are serious hospital infection pathogens. The development of new and highly effective antibacterial compounds as alternatives to antibacterial drugs plays an important role in the treatment of drug-resistant pathogenic bacterial infections.

[0003] Steroidal compounds are essential and physiologically active substances in the human body. Many possess physiological functions such as hormones, anti-inflammatory properties, and anti-tumor effects, playing a crucial role in maintaining normal human physiology and preventing and treating diseases. Currently, certain steroidal drugs are clinically used in cancer treatment, including estramustine sodium phosphate for the treatment of advanced prostate cancer, exemestane for the treatment of postmenopausal breast cancer, abiraterone acetate for the treatment of recurrent prostate cancer (CRPC), and 2-methoxyestradiol, an anticancer drug used as monotherapy for the treatment of stable or recurrent myeloma and prostate cancer. All of these are derived through structural modification of steroidal molecules. The modification of steroidal compounds and the study of their physiological activities are hot topics in medicinal chemistry.

[0004] Selenium cyanide is a type of pharmacophore with good physiological activity. When introduced into organic compounds, it often makes the organic compounds have various biological interactions, including conferring special selective antitumor activity and antioxidant properties on the substrate. Selenium cyanide pharmacophores have been proven to be effective in preventing and treating various cancers both in vitro and in vivo. These effects include DNA binding, changes in gene expression (including growth factors and pro- / anti-apoptotic genes), and changes in cyclooxygenase-2 (COX-2) activity. Existing literature (Pang Liping, Synthesis and Biological Activity of Steroidal Selenium Compounds) reports that in the presence of hydrochloric acid, organic solvents and nitrite, selenocyanate and pregnenolone introduce a selenocyanide functional group at the α position of the carbonyl group to obtain 21-selenocyanide pregnenolone and 17-selenocyanide pregnenolone and their derivatives. The synthesis process is simple, but 17-selenocyanide pregnenolone has a good inhibitory effect on tumor cells, while most other compounds have weaker inhibitory effects on tumor cells in vitro (IC 50 >100), and its inhibitory effect is significantly lower than that of the existing drug abiraterone. It has some antibacterial effect on some bacteria, but does not reach the antibacterial effect of existing antibacterial drugs. Further integration of steroids and selenocyanide, structural modification, and synthesis of novel structural compounds will facilitate the discovery of new antibacterial and anti-tumor drugs, and are expected to further enhance the antibacterial effect against bacteria and the inhibition of cancer cell proliferation. Summary of the Invention

[0005] In view of the above shortcomings, the present invention provides a new class of selenocyanine pregnenolone amide compounds with both anti-tumor activity and antibacterial activity. The specific technical solution is as follows:

[0006] A selenocyanopregnenolone amide compound having the following general structural formula:

[0007]

[0008] Wherein, R1 is H, CH3 or CH2CH3, and R is any one of the following structural formulas 1-19:

[0009]

[0010] Preferably, in the above-mentioned selenocyanine pregnenolone amide compound, R1 is H, CH3 or CH2CH3, and R is any one of the structural formulas 12-19.

[0011] On the other hand, the present invention also provides the use of the above-mentioned selenocyanine pregnenolone amide compound in the preparation of anticancer drugs.

[0012] Preferably, in the above application, the selenocyanine pregnenolone amide compound is used as an active ingredient to prepare drugs for treating cervical cancer, ovarian cancer, liver cancer or breast cancer.

[0013] On the other hand, the present invention also provides the use of the above-mentioned selenocyanine pregnenolone amide compound in the preparation of antibacterial drugs.

[0014] Preferably, in the above application, the selenocyanine pregnenolone amide compound is used to prepare a drug against methicillin-resistant Staphylococcus aureus or vancomycin-resistant Enterococcus.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] 1. The selenocyanine pregnenolone amide compounds of the present invention exhibit strong inhibitory activity against human cervical cancer cell lines, human breast cancer cell lines, human ovarian cancer cell lines and human liver cancer cell lines. The IC values ​​of most compounds against these tumor cell lines are 50 The values ​​are all below 10μmol / L, which can effectively inhibit cancer cells at low concentrations. Compared with the control drug abiraterone, its inhibition rate on cancer cells is increased by 5-10 times.

[0017] 2. The selenocyanine pregnenolone amide compound of the present invention can effectively kill methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE) at low concentrations, wherein the compound pregnenolone-20-(3'-selenocyaninemethyl)benzamide (I 13 ) The minimum inhibitory concentration (MIC) against MRSA was 2 μg / mL, and the minimum inhibitory concentration (MIC) against VRE was 1 μg / mL, which was significantly better than the control drugs vancomycin and ampicillin, and had a strong antibacterial effect.

[0018] 3. The selenocyanine pregnenol ketone amide compound of the present invention can be used as a drug for treating cancer and bacterial infections, which contains the above-mentioned selenocyanine pregnenol ketone amide compound and a pharmaceutically acceptable adjuvant. The drug can be prepared into the form of injection, tablet, pill, capsule, suspension or emulsion, and its administration route can be oral, or by subcutaneous, intravenous or intramuscular injection. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] To more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are some embodiments of the present invention, and those skilled in the art can derive other drawings based on these drawings without inventive effort.

[0020] Figure 1 Compound I in Application Example 2 of the present invention 13Figure 3. Inhibitory effect on two drug-resistant bacteria (unit: μg / mL): (a) Inhibitory effect on MRSA, the minimum inhibitory concentration (MIC) is 2 μg / mL; (b) Inhibitory effect on VRE, the minimum inhibitory concentration (MIC) is 1 μg / mL.

[0021] Figure 2 Compound I in Application Example 3 of the present invention 12 Effects on the growth of zebrafish breast cancer transplanted tumor cells: (a) Effects on the growth of tumor area in zebrafish; (b) Effects on the fluorescence intensity of tumor in zebrafish. DETAILED DESCRIPTION

[0022] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited by the specific embodiments. Unless otherwise defined, all technical terms used hereinafter have the same meaning as those generally understood by those skilled in the art. The technical terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of protection of the present invention. Unless otherwise specified, the various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or can be prepared by existing methods.

[0023] A selenocyanopregnenolone amide compound having a structure of general formula (I):

[0024]

[0025] Wherein: R1 is H, CH3 or CH2CH3, and R is any one of the following structural formulas 1-19:

[0026]

[0027] The synthetic route of selenocyanopregnenolone amide compound is:

[0028]

[0029] Wherein, X is any one of the following structures:

[0030]

[0031] In the above synthesis, when R1 is CH3 or CH2CH3, 3-methoxypregnenolone or 3-ethoxypregnenolone is used to replace pregnenolone in step 1.

[0032] The preparation method of the selenocyanine pregnenolone amide compound specifically comprises the following steps:

[0033] Step 1: Weigh pregnenolone, 3-methoxypregnenolone or 3-ethoxypregnenolone into an eggplant-shaped flask, add ethanol to dissolve it, add sodium acetate trihydrate during the heating process, stir for 10-40 minutes, then add hydroxylamine hydrochloride, stir at 60-90°C for 2-5 hours, and stop the reaction after tracking by TLC until there is no raw material. Most of the solvent is decompressed and spun out, extracted with ethyl acetate, washed with water, saturated NaCl, saturated NaHCO3, and dried over anhydrous Na2SO4. Finally, the organic phase is spun out, and the residual liquid is separated by column chromatography to obtain compound II;

[0034] Step 2: Weigh compound II into an eggplant-shaped flask, add ethanol and stir to dissolve it, then add sodium cyanoborohydride, platinum pentachloride and sodium bisulfate respectively, and stir at room temperature for 2-6 hours. Stop the reaction after TLC tracking until there is no starting material; filter out most of the salt, then evaporate the solvent under reduced pressure, and separate the residue by column chromatography to obtain compound III;

[0035] Step 3: Weigh compound III into an eggplant-shaped flask, add dichloromethane and stir to dissolve, then add triethylamine. Stir at 0-5°C for 10-30 minutes, then dropwise add the corresponding acid chloride. Stir for 20-50 minutes, then transfer to room temperature and continue stirring for 2-5 hours. Stop the reaction when no starting material is detected by TLC. Evaporate the solvent, dissolve in ethyl acetate, wash with water and saturated NaCl, and dry over anhydrous Na2SO4. Finally, remove the solvent under reduced pressure, and separate the crude product by column chromatography to obtain compound IV.

[0036] Step 4: Weigh compound IV into an eggplant-shaped flask, add DMF, and stir to dissolve. Then, add 1-3.5 equivalents (based on the molar amount of compound IV) of a potassium selenocyanate solution dissolved in distilled water. Stir and react at 50-90°C under argon and in the dark for 5-20 hours. Stop the reaction when no starting material is detected by TLC. Evaporate the solvent, extract with ethyl acetate, and combine the organic layers, wash with water and saturated NaCl, and dry over anhydrous Na2SO4. Evaporate the organic phase, and separate the crude product by column chromatography to obtain the target product I, which is the selenocyanine pregnenolone amide compound.

[0037] Example 1

[0038] A selenocyano pregnenolone amide compound, specifically pregnenolone-20-(4′-selenocyano) butanamide I3, has the structure:

[0039]

[0040] A method for preparing a selenocyanopregnenolone amide compound (I3) comprises the following steps:

[0041] (1) 4.0 mmol of pregnenolone was weighed into an eggplant-shaped flask, 30 mL of ethanol was added and stirred to dissolve, and then heated. During the heating process, 8 mmol of sodium acetate trihydrate was added, and after stirring for 10 min, 8 mmol of hydroxylamine hydrochloride was added. The mixture was stirred at 80°C for 4 h. The reaction was stopped by thin layer chromatography (TLC) until no starting material was found. The solvent was evaporated under reduced pressure, and the organic layers were combined after extraction with ethyl acetate, washed with water, saturated NaCl, and saturated NaHCO3, respectively, and dried over anhydrous Na2SO4. Finally, the organic phase was spun off, and the residue was separated by column chromatography to obtain 1.0 g of a colorless oil, namely, compound II, with a yield of 77%.

[0042] (2) 3.0 mmol of compound II was weighed into an eggplant-shaped flask, 30 mL of ethanol was added, and the mixture was stirred to dissolve. 12.0 mmol of NaBH3CN, 53.0 mmol of MoCl, and 9.0 mmol of NaHSO4·H2O were then added, respectively. The mixture was stirred at room temperature for 3 h. The reaction was stopped when the starting material disappeared, as monitored by TLC. Most of the salt was removed by column chromatography, and the solvent was removed under reduced pressure. Product III was isolated by solid column chromatography, with a melting point of 189-191°C and a yield of 68.6%.

[0043] (3) 0.60 mmol of compound III was weighed into an eggplant-shaped flask, 15 mL of dichloromethane was added and stirred to dissolve it, followed by the addition of 2.0 mmol of triethylamine. The mixture was stirred at 0°C for 15 min, and 0.75 mmol of 4-chlorobutyryl chloride was added dropwise. After stirring for 30 min, the mixture was transferred to room temperature and stirred for 3.5 h. The reaction was stopped by TLC until the starting material disappeared. The solvent was evaporated under reduced pressure, the mixture was dissolved in ethyl acetate, washed with water and saturated NaCl, and dried over anhydrous Na2SO4. Finally, the solvent was removed under reduced pressure, and the crude product was separated by column chromatography to obtain the target product IV3 in a yield of 66.6%.

[0044] (4) 0.60 mmol of compound IV3 was weighed and placed in an eggplant-shaped flask. 15 mL of DMF was added and stirred to dissolve. 0.90 mmol of potassium selenocyanate solution dissolved in 1 mL of distilled water was then added. The reaction was stirred at 80°C under argon and in the dark for 15 h. The reaction was stopped when no starting material was observed by TLC. Most of the solvent was distilled off under reduced pressure, and the mixture was extracted with ethyl acetate. The organic layers were combined, washed with water and saturated NaCl, and dried over anhydrous Na2SO4. Finally, the organic phase was distilled off under reduced pressure, and the crude product was separated by column chromatography to obtain product I3 in a yield of 72.9%.

[0045] In the above steps, the solvent is removed by vacuum distillation using a rotary evaporator, so that the liquid is distilled out at a lower temperature, avoiding the decomposition of some unstable compounds under high temperature conditions, and at the same time, the solvent can be distilled out better.

[0046] The method of stopping the reaction by tracking the point of no raw material by TLC is as follows: the reaction of the stirring mixture is tracked by thin layer chromatography, the reactants are added dropwise onto the thin layer plate using a capillary, and after drying, the reactants are developed using a developing agent, and then the dried thin layer plate is placed under ultraviolet light for observation. When no raw material points are observed, the reaction is completed.

[0047] The column chromatography separation in the above steps is silica gel column chromatography separation, and the developing solvent for column chromatography separation in step (1) is V EA :V PE =1:2, eluent is V EA :V PE =1:3; the developing solvent for column chromatography separation in step (2) is V MeOH :V DCM =1:10, eluent is V MeOH :V DCM =1:15; the developing solvent for column chromatography separation in step (3) is V EA :V PE =1:1, eluent is V EA :V PE =1:2; the developing solvent for column chromatography separation in step (4) is V EA :V PE =1:1, eluent is V EA :V PE =1:3-1:2.

[0048] Selenium cyanopregnenolone amide compounds I1-I2, I4-I 19 By using 2-chloroacetyl chloride, 3-chloropropionyl chloride, 5-chlorovaleryl chloride, 6-chlorohexanoyl chloride, 7-chloroheptanoyl chloride, 8-chlorooctanoyl chloride, 9-chlorononanoyl chloride, 10-chlorodecanoyl chloride, 11-chloroundecanoyl chloride, 12-chlorododecanoyl chloride, 2-chloromethylbenzoyl chloride, 3-chloromethylbenzoyl chloride, 4-chloromethylbenzoyl chloride, 5-chloromethylpyridine-2-carbonyl chloride, 4-chloromethylpyridine-2-carbonyl chloride, 6-chloromethylpyridine-2-carbonyl chloride, 6-chloromethylpyridine-6-carbonyl chloride and 3-chloromethylquinoline-2-carbonyl chloride to replace 4-chlorobutyryl chloride in step 3, compounds IV1-IV2, IV4-IV5 and IV6 were prepared. 19 , in step (4), IV1-IV2, IV4-IV 19 Substituting compound IV3, other steps and parameters were the same as in Example 1 to prepare the obtained product.

[0049] Compound I1-I 19 The chemical yields are: I1: 70.9%; I2: 74.2%; I3: 72.9%; I4: 40.4%; I5: 65.3%; I6: 69.2%; I7: 63.1%; I8: 66.5%; I9: 50.1%; I10 :78.8%;I 11 :72.4%;I 12 :52.0%;I 13 :64.5%;I 14 :61.3%;I 15 :76.1%;I 16 :69.9%;I 17 :65.4%;I 18 :58.3%;I 19 :75.8%.

[0050] In step 1, 3-methoxypregnenolone or 3-ethoxypregnenolone is used instead of pregnenolone as the starting material, and the operations of steps 1, 2, 3 and 4 are repeated to obtain a selenocyanopregnenolone amide compound in which R1 is CH3 or CH2CH3.

[0051] The selenocyanopregnenolone amide compound I of the present invention is composed of two diastereomers in different ratios because the amino group is connected to a chiral carbon. The NMR data of some representative compounds are shown below: 20-(2-selenocyanoacetamide)pregnenolone (I1)

[0052] Compound I1 has a melting point of 192-193°C and a diastereoisomer ratio of 2:1 (epim-1:epim-2).

[0053] Isomer-1: 1 H NMR(600MHz,Chloroform-d)δ:5.83(d,J=6.0Hz,1H,-NH),5.35(s,1H,C6-H),4.01-3.93(m,1H,C20-H),3.91-3.82(m,2H,1'-CH2-SeCN),3.55-3.50(m,1H, C3-H),2.30(dd,1H,J=9.2,3.6Hz,C7-H),2.23(t,J=8.8,7.6Hz,1H,C4-H),1. 12(d,J=4.4Hz,1.94H,21-CH3),1.00(s,3H,19-CH3),0.71(s,1.94H,18-CH3); 13C NMR(150MHz, CDCl3)δ:164.46(C=O),140.75(5-C),121.46(6-C),101.98(SeCN),71.73,56.16,56.05,50.01,48 .45,42.26,42.24,39.60,37.23,36.48,32.07,31.75,31.74,31.61,26.70,23.97,21.22,20.98,19.38,12.39.

[0054] Isomer-2: 1 H NMR(600MHz,Chloroform-d)δ:5.91(d,J=6.0Hz,1H,-NH),5.35(s,1H,C6-H),4.01-3.93(m,1H,C20-H),3.91-3.82(m,2H,C24-H),3.55-3.50(m,1H,C3- H),2.30(dd,J=9.2,3.6Hz,1H,C7-H),2.23(t,J=8.8,7.6Hz,1H,C4-H),1.20 (d,J=4.4Hz,0.96H,21-CH3),1.00(s,3H,19-CH3),0.74(s,0.96H,18-CH3); 13 C NMR(150MHz, CDCl3)δ:164.82(C=O),140.78(5-C),121.40(6-C),102.01(SeCN),71.73,56.68,56.32,49.98,49.39,42.24,4 1.96,39.04,37.23,36.48,31.77,31.69,31.61,29.69,26.65,24.05,21.45,20.86,19.38,12.13; HREIMS:m / z465.2019[M+H] + (calcd for C 24 H 37 N2O2Se, 465.2015).

[0055] 20-(4-selenocyanobutyramide)pregnenolone (I3)

[0056] Compound I3 has a melting point of 108-109°C and a diastereoisomer ratio of 2:1 (epim-1:epim-2).

[0057] Epimer-1: 1H NMR(600MHz,Chloroform-d)δ:5.44(d,J=9.1Hz,0.71H,-NH),5.35-5.34

[0058] (m,1H,C6-H),4.00-3.94(m,1H,C20-H),3.54-3.49(m,1H,C3-H),3.24-3.10(m,2H,4-CH2),2.33(t,J=7.1Hz,2H,3'-CH2),1.08(d,J=6.4Hz,3H,21-CH3),1.00(s,3H,19-CH3),0.70(s,2.04H,18-CH3); 13 C NMR(150MHz,CDCl3)δ:169.55(C=O),140.82(5-C),121.41(6-C),102.02(SeCN),71.69,56.76,56.21,50.05,47.21,42.25,41.85,39.62,37.25,36.49,35.01,31.79,31.75,31.60,29.20,26.79,26.37,24.02,21.54,21.00,19.40,12.39.

[0059] Epimer-2: 1 H NMR(600MHz,Chloroform-d)δ:5.53(d,J=9.1Hz,0.39H,-NH),5.35-5.34

[0060] (m,1H,C6-H),4.00-3.94(m,1H,C20-H),3.54-3.49(m,1H,C3-H),3.24-3.10(m,2H,4-CH2),2.33(t,J=7.1Hz,2H,3'-CH2),1.08(d,J=6.4Hz,3H,21-CH3),1.00(s,3H,19-CH3),0.73(s,0.96H,18-CH3); 13C NMR(150MHz, CDCl3)δ:169.85(C=O),140.82(5-C),121.44(6-C),102.10(SeCN),71.69,56.76,56.50,50.00,47.99,41.85,3 9.08,37.25,36.49,35.01,31.75,31.70,31.60,29.14,26.69,26.37,24.02,21.74,21.54,20.88,19.39,12.16; HREIMS:m / z 493.2334[M+H] + (calcd for C 26 H 41 N2O2Se,493.2328).

[0061] 20-(5-selenocyanovaleramide)pregnenolone (I4)

[0062] Compound I4 has a melting point of 83-84°C and a diastereoisomer ratio of 2.5:1 (epim-1:epim-2).

[0063] Epimer-1: 1 H NMR(600MHz,Chloroform-d)δ:5.34(d,J=5.6Hz,1H,-NH),5.30-5.28(m,

[0064] 1H,C6-H),4.00-3.94(m,1H,C20-H),3.58-3.46(m,1H,C3-H),3.07(t,J=7.3Hz,2H,4'-CH2),2.31-2.23(m,2H,4-CH 2),2.18(t,J=7.3Hz,2H,1'-CH2),1.07(d,J=6.2Hz,2.59H,21-CH3),1.00(s,3H,19-CH3),0.71(s,2.14H,18-CH3); 13 C NMR(150MHz, CDCl3)δ:170.40(C=O),140.81(5-C),121.44(6-C),101.63(SeCN),71.73,56.28,56.25,50.08,47.15,42.26 ,39.60,37.25,36.50,35.93,31.81,31.76,31.62,30.54,29.15,26.81,24.77,24.03,21.56,21.00,19.39,19.38,12.34.

[0065] Epimer-2:1 H NMR(600MHz,Chloroform-d)δ:5.34(d,J=5.6Hz,1H,-NH),5.30-5.28(m,

[0066] 1H,C6-H),4.00-3.94(m,1H,C20-H),3.58-3.46(m,1H,C3-H),3.07(t,J=7.3Hz,2H,4'-CH2),2.31-2.23(m,2H,4-CH 2),2.18(t,J=7.3Hz,2H,1'-CH2),1.10(d,J=6.2Hz,0.41H,21-CH3),1.00(s,3H,19-CH3),0.74(s,0.86H,18-CH3); 13 C NMR(150MHz, CDCl3)δ:170.72(C=O),140.81(5-C),121.44(6-C),101.69(SeCN),71.73,56.79,56.28,53.44,50.08,47.87,41.8 6,39.10,37.25,36.50,35.93,31.81,31.71,31.62,30.41,29.15,26.75,24.96,24.03,21.78,20.88,19.18,12.16; HREIMS:m / z 507.2490[M+H] + (calcd forC 27 H 43 N2O2Se,507.2484).

[0067] 20-(11-selenocyanide undecylamide) pregnenolone (I 10 )

[0068] Compound I 10 Melting point: 97-98℃, this compound is a single compound.

[0069] 1H NMR(600MHz,Chloroform-d)δ:5.34(d,J=5.3Hz,1H,-NH),5.22(d,J=9.1Hz,1H,C6-H),3.98-3.93(m,1H,C20-H),3.54-3.49(m,1H,C3-H) ,3.05(t,J=7.4Hz,2H,10'-CH2),2.12(t,J=6.0Hz,2H,1'-CH2),1.06(d,J=6.5Hz,3H,21-CH3),1.00(s,3H,19-CH3),0.71(s,3H,18-CH3); 13 C NMR(150MHz, CDCl3)δ:171.46(C=O),140.78(5-C),121.49(6-C),101.63(S eCN),71.72,56.36,56.30,50.11,46.97,42.28,42.27,39.52,37.29,37.1 9,36.50,31.83,31.77,31.64,30.83,29.65,29.35,29.31,29.29,29.27,2 9.10,28.83,26.86,25.63,24.06,21.61,21.00,19.40,12.29; HREIMS:m / z 591.3428[M+H] + (calcd forC 33 H 55 N2O2Se,591.3423).

[0070] 20-(4-selenocyanomethylbenzamide)pregnenolone (I 12 )

[0071] Compound I 12 Melting point: 142-143°C, the ratio of epimers is 1.5:1 (epim-1:epim-2).

[0072] Epimer-1: 1 H NMR(600MHz,Chloroform-d)δ:7.76(d,J=8.1Hz,1.20H,2'and 6'-Ph-H),

[0073] 7.43(d,J=8.1Hz,1.20H,3'and 5'-Ph-H),6.03(d,J=9.3Hz,1H,-NH),5.34(d,J=3.1Hz,1H,C6-H),4.29(s,1.28H,7'-CH2),4.22-4.18(m,1H,C20-H),3.53-3.49(m,1H,C3-H),1.17(d,J=6.5Hz,3H,21-CH3),0.97(s,1.95H,19-CH3),0.76(s,1.85H,18-CH3); 13 CNMR(100MHz,CDCl3)δ:164.95(C=O),140.81(5-C),138.98(4'-Ph-C),135.08(1'-Ph-C),129.20(3'and 5'-Ph-C),127.62(2'and 6'-Ph-C),121.41(6-C),101.51(SeCN),71.69,56.57,56.26,53.45,50.08,47.59,42.24,39.48,37.25,36.50,36.46,31.94,31.79,31.76,31.60,29.69,26.82,24.06,21.45,21.06,19.37,12.42.

[0074] Epimer-2: 1 H NMR(600MHz,Chloroform-d)δ:7.73(d,J=8.1Hz,0.80H,2'and 6'-Ph-H),7.41(d,J=8.1Hz,0.80H,3'and 5'-Ph-H),6.03(d,J=9.3Hz,1H,-NH),5.34(d,J=3.1Hz,1H,C6-H),4.29(s,0.72H,7'-CH2),4.22-4.18(m,1H,C20-H),3.53-3.49(m,1H,C3-H),1.17(d,J=6.5Hz,3H,21-CH3),1.01(s,1.05H,19-CH3),0.78(s,1.15H,18-CH3); 13C NMR (100MHz, CDCl3) δ: 165.60 (C=O), 140.81 (5-C), 138.89 (4'-Ph-C), 135.47 (1'-Ph-C), 129.13 (3'and 5'-Ph-C), 127.65 (2'and 6'-Ph-C),121.44(6-C),101.49(SeCN),71.71,56.77,56.26,53.45,50.04,48.47,42.31,41.97, 39.10,37.25,36.50,31.82,31.63,29.69,26.73,24.04,21.70,20.89,19.37,12.24; HREIMS:m / z 541.2332[M+H]+(calcd forC 30 H 41 N2O2Se,541.2328).

[0075] 20-(4-selenocyanomethylbenzamide)pregnenolone (I 13 )

[0076] Compound I 13 Melting point: 154-156°C, the ratio of epimers is 2.8:1 (epim-1:epim-2).

[0077] Epimer-1:1H NMR(600MHz,Chloroform-d)δ:7.72(s,0.78H,2'-Ph-H),7.60(d,J=7.7Hz,1H,6'-Ph-H),7 .43(d,J=7.8Hz,1H,4'-Ph-H),7.37(t,J=7.8Hz,1H,5'-Ph-H),6.01(d,J=9.2Hz,1H,-NH), 5.36-5.24(m,1H,C6-H),4.24(s,1.49H,7'-CH2),4.17-4.09(m,1H,C20-H),3.46-3.41(m, 1H,C3-H),1.11(d,J=6.4Hz,3H,21-CH3),0.90(s,2.28H,19-CH3),0.69(s,2.28H,18-CH3); 13C NMR(150MHz,CDCl3)δ:164.00(C=O),139.79(5-C),135.33(3'-Ph-C),134.79(1'-Ph-C),130.62(4'-Ph-C),128.41(2'-Ph-C),126.75(5'-Ph-C),125.66(6'-Ph-C),120.40(6-C),100.78(SeCN),70.67,55.48,55.21,49.03,46.58,41.22,38.39,36.23,35.45,31.15,30.78,30.75,30.57,28.67,25.78,23.04,20.43,20.03,18.35,11.42.

[0078] Epimer-2: 1 H NMR(600MHz,Chloroform-d)δ:7.66(s,0.22H,2'-Ph-H),7.60(d,J=7.7Hz,1H,6'-Ph-H),7.43(d,J=7.8Hz,1H,4'-Ph-H),7.37(t,J=7.8Hz,1H,5'-Ph-H),6.01(d,J=9.2Hz,1H,-NH),5.36-5.24(m,1H,C6-H),4.22(s,0.51H,7'-CH2),4.17-4.09(m,1H,C20-H),3.46-3.41(m,1H,C3-H),1.11(d,J=6.4Hz,3H,21-CH3),0.94(s,0.72H,19-CH3),0.71(s,0.72H,18-CH3); 13 C NMR(150MHz,CDCl3)δ:164.69(C=O),139.79(5-C),135.22(3'-Ph-C),135.20(1'-Ph-C),130.51(4'-Ph-C),128.38(2'-Ph-C),126.57(5'-Ph-C),125.94(6'-Ph-C),120.40(6-C),100.90(SeCN),70.67,55.75,55.66,49.01,47.56,40.95,38.08,36.23,35.48,31.11,30.78,30.69,30.57,28.67,25.78,23.04,20.65,19.88,18.35,11.23;HREIMS:m / z 541.2333[M+H] + (calcd for C 30H 41 N2O2Se,541.2328).

[0079] Application Example 1: In vitro inhibition of tumor cell proliferation by selenocyanine pregnenolone amide compounds

[0080] The MTT method was used to test the anti-proliferation activity of the selenocyanine pregnenolone amide compound of the present invention on tumor cells, and its cytotoxicity on human cervical cancer cell line (HeLa), human ovarian cancer cell line (SK-OV-3), human liver cancer cell line (HepG-2) and human breast cancer cell line (MCF-7, T47D). Different concentrations of selenocyanine pregnenolone amide compound were added to the cells in the logarithmic growth phase cultured in 96-well plates, and three parallel tests were performed at the same time and compared with the control group. After 72 hours of culture, MTT was added and its absorbance was measured. The concentration of the compound that inhibited the growth and proliferation of tumor cells by 50% was calculated, and the IC value was used as the value. 50 The results are shown in Table 1.

[0081] As shown in Table 1, the selenocyanine pregnenolone amide compound of the present invention has an IC of 0.0447 for human cervical cancer cells, human ovarian cancer cells, human liver cancer cells, and human breast cancer cells. 50 The values ​​are basically less than 10μM, which is significantly better than the similar steroidal anti-tumor drug abiraterone hydrochloride. In addition, in addition to the breast cancer cell MCF-7, the inhibitory activity of the selenocyanine pregnenolone amide compound of the present invention on other tumor cells is also significantly better than the similar steroidal anti-tumor drug 2-methoxyestradiol. For example, the inhibitory IC of compound numbered I3 on ​​human cervical cancer cell line (HeLa) is 50 The IC value of inhibition against human ovarian cancer cell line (SK-OV-3) is 3.67μM. 50 The IC value of inhibition on liver cancer cell line (HEPG2) was 5.39μM. 50 The value is 12.7μM, and the cytotoxicity against human breast cancer cell lines (MCF-7, T47D) is 5.97 and 5.71μM respectively. Compared with the control steroid antitumor drug 2-methoxyestradiol, the inhibitory IC of 2-methoxyestradiol on human cervical cancer cell line (HeLa) is 50 The inhibitory IC value is 8.6μM against human ovarian cancer cell line (SK-OV-3) 50 The IC value of inhibition on liver cancer cell line (HEPG2) is 16.5μM. 50 The value is 13.8 μM, and the cytotoxicity to human breast cancer cell lines (MCF-7, T47D) is >70 and 2.3 μM respectively. The above results show that the selenocyanine pregnenolone amide compound of the present invention has a very significant growth and proliferation inhibitory effect on tumor cell lines.

[0082] Table 1 In vitro inhibition of tumor cell growth and proliferation activity of selenocyanine pregnenolone amide compounds (IC 50 ,μmol / L)

[0083]

[0084]

[0085] Application Example 2: In vitro inhibition of methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococci by selenocyanine pregnenolone amide compounds

[0086] The selenocyanine pregnenol ketone amide compound of the present invention was used to perform an inhibitory activity test on methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE), and the test results were expressed as minimum inhibitory concentration (MIC, μg / mL). Specifically: the MIC of the compound was determined by the broth dilution method, and this was used as an indicator to evaluate the antibacterial activity of the compound. The selenocyanine pregnenol ketone amide compound was dissolved in DMSO solution to prepare a 10 mg / mL compound stock solution for standby use. 1.28 μL of the compound stock solution was added to a 96-well plate (repeated 6 groups), followed by 98.72 μL of TSB culture medium, and multiple dilutions were performed using a two-fold dilution method to obtain 50 μL of compound solutions of different concentrations. Then 50 μL of 2×10 5 CFU / mL bacterial solution, so that the final concentration of bacteria is 1×10 5 CFU / mL, the final concentration of the compound is 1-64μg / mL. After adding a small amount of sterile water to all the outermost wells of the 96-well plate, incubate at 37°C for 18 hours. The lowest concentration corresponding to the well with a clear solution is the MIC. The minimum inhibitory concentration of selenocyanide pregnenolone amide compounds against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE) is shown in Table 2. Among them, compound I 13 MIC of two drug-resistant bacteria Figure 1 shown.

[0087] From the data in Table 2, it can be seen that the selenocyanine pregnenolone amide compounds of the present invention have a significant growth inhibitory effect on methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE), especially compound I 12 , I 13 and I 14 Their minimum inhibitory concentrations against these two drug-resistant bacteria were significantly better than those of the positive controls vancomycin and ampicillin. Moreover, these compounds also had a significant growth inhibitory effect on both drug-resistant bacteria. Figure 1 It can be seen that compound I 13It has a very significant growth inhibitory effect on both drug-resistant bacteria, which is better than the positive controls vancomycin and ampicillin. In summary, it can be seen that the selenocyanine pregnenolone amide compound of the present invention has a strong growth inhibitory effect on drug-resistant bacteria and can be used as an alternative antibacterial drug.

[0088] Table 2 Minimum inhibitory concentration (MIC, μg / mL) of selenocyanine pregnenolone amide compounds against MRSA and VRE

[0089]

[0090]

[0091] Application Example 3: Inhibition of the Growth of Breast Cancer Cell Transplants in Zebrafish by Selenium Cyanopregnenolone Amide Compounds

[0092] The growth inhibition effect of some selenocyanine pregnenolone amide compounds described in the present invention on breast cancer (MCF-7) cell transplanted tumors in zebrafish was tested. 12 For example.

[0093] Cell staining: Breast cancer (MCF-7) cells were cultured in T75 cell flasks. After confluence, the cells were washed three times with PBS and stained with the red fluorescent dye CM-DiI (Hanks dilution). The cells were incubated at 37°C for 3 min and then incubated in a refrigerator at 4°C for 15 min.

[0094] Breast Cancer Cell Transplantation: Place the collected breast cancer cell suspension in serum-free cell culture medium and keep on ice until ready. Breast cancer cells were microinjected into the mid-yolk sac of 2-dpf wild-type AB zebrafish, with 500-800 cells injected per tail, to establish a breast cancer cell xenograft zebrafish model. After successful modeling, zebrafish were placed in a 28°C larval incubator to recover for 1 hour before being placed in a tri-gas incubator for continued culture.

[0095] Experimental grouping: 20 hours after transplantation of MCF-7 cells, zebrafish with consistent cell fluorescence were selected and divided into two groups: solvent control group and compound I 12 The zebrafish larvae in the solvent control group were injected with 10 nL of 1% DMSO solution per tail, and compound I was administered. 12 Each fish in the drug group was injected with 10 nL of compound I at a concentration of 5.8 μM. 12 Solution, Compound I 12 The solution was prepared by first mixing compound I with DMSO. 12 Dissolved and then diluted to 5.8 μM with deionized water, wherein the DMSO content is 1%.

[0096] Compound I 12 Effect on zebrafish transplant tumor growth: Breast cancer cells were transplanted into wild-type AB zebrafish larvae. 24 hours and 72 hours later, the zebrafish yolk sac was observed and photographed using a stereofluorescence microscope. The fluorescence intensity and tumor cell area of ​​red fluorescent-labeled tumor cells in the zebrafish yolk sac were calculated. Image J software was used to analyze and calculate the fluorescence intensity and tumor cell area of ​​the tumor cells in the yolk sac of the zebrafish larvae. Comparison of the solvent control group and compound I 12 There was no significant difference between the drug groups to evaluate the effect of compound I 12 Effects on tumor cell growth in zebrafish breast cancer xenografts.

[0097] Compound I 12 Effects on the growth of zebrafish breast cancer xenograft tumor cells Figure 2 As shown, Figure 2 The tumor area growth fold and tumor cell fluorescence intensity growth fold refer to the growth fold of the tumor area and fluorescence intensity corresponding to 5 dpf in the figure compared to the tumor area and fluorescence intensity corresponding to 3 dpf. 12 The tumor area growth fold and tumor cell fluorescence intensity growth fold of the group were significantly lower than those of the solvent control group Vehicle (p < 0.001), indicating that compound I 12 It has an inhibitory effect on the growth of human breast cancer cell (MCF-7) zebrafish transplanted tumors.

[0098] The foregoing descriptions of specific exemplary embodiments of the present invention are for purposes of illustration and description. These descriptions are not intended to limit the invention to the precise forms disclosed, and it is apparent that many variations and modifications are possible in light of the foregoing teachings. The exemplary embodiments have been selected and described for the purpose of explaining the specific principles of the invention and their practical application, thereby enabling those skilled in the art to realize and utilize a variety of exemplary embodiments of the invention and various options and modifications. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. A selenocyanopregnenolone amide compound, characterized in that: It has the following general structural formula: Wherein, R1 is H, and R is any one of the following structural formulas 1-19: 。 2. The selenocyanine pregnenolone amide compound according to claim 1, characterized in that The R1 is H, and R is any one of the structural formulas 12-19.

3. Use of the selenocyanine pregnenolone amide compound according to claim 1 or 2 in the preparation of anticancer drugs, characterized in that: The selenocyanine pregnenolone amide compound is used as an active ingredient to prepare drugs for treating cervical cancer, ovarian cancer, liver cancer or breast cancer.

4. Use of the selenocyanopregnenolone amide compound according to claim 1 or 2 in the preparation of antibacterial drugs, characterized in that: The selenocyanine pregnenolone amide compound is used for preparing drugs for resisting methicillin-resistant Staphylococcus aureus or vancomycin-resistant enterococci.

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