Steroid derivatives with structural diversity and their chemical-microbial catalytic preparation method and application

Abstract

The application discloses a kind of structural diversity steroidal ketone derivatives and its chemical-microorganism catalytic combined preparation method and application, belong to biological medicine preparation technical field.The structural diversity steroidal ketone derivatives described in the application include four big types of steroidal derivatives, such as sterol conversion into steroidal ketone derivative, pyrimidine derivative, thiazoline derivative or bis-hydrazone derivative.Firstly, four structural types of sterol derivatives are prepared by chemical method with dehydroepiandrosterone as raw material;Secondly, microbial cell catalysis is used, i.e., new mycobacterium aurum strain is used to catalyze oxidation of sterol and isomerization to obtain novel steroidal ketone derivatives.The chemical-microorganism cell catalytic combined method of the application can realize the preparation of compounds that cannot be obtained by partial chemical method, and the microbial cell catalysis step has mild conditions, simple separation and extraction process, and is environment-friendly, which provides a new strategy for constructing novel steroidal derivative molecular library.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological drug preparation, and particularly relates to a method for preparing novel steroidal derivatives with structural diversity by using a chemical-microorganism cell catalytic cascade strategy, the novel steroidal derivatives with structural diversity prepared by the method, and application of the novel steroidal derivatives with structural diversity. BACKGROUND

[0002] Steroids account for a large proportion in the field of modern drug molecules, and are widely used for anti-inflammatory, anticancer, antiviral, and treatment of cardiovascular diseases, human organ transplant rejection, and various senile diseases. Meanwhile, sterols are widely present in nature, and are found in plants, animals, yeasts, and fungi, and are a class of metabolites widely present in animals and plants and playing an important role in the life process. With continuous in-depth research, more and more steroidal derivatives are designed and prepared, and show broad pharmacological activities.

[0003] Applicants disclosed a series of novel sterol derivatives in the literatures “Synthesis of novel steroid derivatives derived from dehydroepiandrosterone as potential anticancer agents”, “Discovery of novel isatin-dehydroepiandrosterone conjugates as potential anticancer agents”, “Synthesis and evaluation of steroidal thiazole conjugates as potential antiviral agents”, “Steroidal [17,16-d]pyrimidines derived from dehydroepiandrosterone: A convenient synthesis, antiproliferation activity, structure-activity relationships, and role of heterocyclic moiety”, and “Synthesis and bioevaluation of novel steroidal isatin conjugates derived from epiandrosterone / androsterone”, and found that the compounds have significant anticancer or antiviral functions, for example:

[0004]

[0005] In order to further develop the biological functions based on the natural steroidal skeleton molecules, the application is proposed. SUMMARY

[0006] The application adopts the chemical-microbial catalysis combination method to perform molecular modification on specific parts of part of sterol derivatives, and realizes efficient and stereoselective preparation of a series of novel steroidal derivatives. The chemical-microbial cell catalysis combination method can realize the preparation of compounds that cannot be obtained by part of chemical methods, and the microbial cell catalysis step has mild conditions, simple separation and extraction process, and environmental friendliness, thereby providing a new strategy for constructing a molecular library of novel steroidal derivatives.

[0007] Based on the above reasons, in view of the problems or defects in the prior art, the purpose of the application is to provide a steroidal derivative with structural diversity and a chemical-microbial catalysis combination preparation method and application thereof, so as to solve or at least partially solve the above technical defects in the prior art.

[0008] In order to achieve the above first purpose of the application, the technical scheme adopted by the application is as follows:

[0009] The steroidal derivative with structural diversity provided by the application includes four types of steroidal derivatives, i.e., sterol converted steroidal derivatives, pyrimidine derivatives, thiazoline derivatives, and dihydrazone derivatives.

[0010] Further, in the above technical scheme, the molecular structure formula of the steroidal derivative is shown in the following formula one, wherein: R 1 is one of Ph, 2-Cl-Ph, 2-F-Ph, 4-CF3-Ph, 2-CF3-Ph, 4-MeO-Ph, 2-Py, 3-Py, 4-Py, 4-Cl-Ph, 4-F-Ph, 4-MeS-Ph, 3,4,5-(MeO)3-Ph, etc.

[0011]

[0012] Further, in the above technical scheme, in the preferred embodiment of the application, the steroidal derivative includes but is not limited to each representative compound shown in the following formula two, and the application will not be listed again due to the consideration of the length and simplicity.

[0013]

[0014] Further, in the above technical scheme, the molecular structure formula of the pyrimidine derivative is shown in the following formula three, wherein: R 2It is one of Ph, 2-Cl-Ph, 2-F-Ph, 4-CF3-Ph, 2-CF3-Ph, 4-MeO-Ph, 2-Py, 3-Py, 4-Py, 4-MeS-Ph, 3,4,5-(MeO)3-Ph, etc.;

[0015]

[0016] Furthermore, in the preferred embodiment of the present invention, the pyrimidine derivatives described above include, but are not limited to, the representative compounds shown in Formula 4. Due to space limitations and for the sake of brevity, the present invention will not exhaustively list them.

[0017]

[0018] Furthermore, in the above technical solution, the molecular structural formula of the thiazoline derivative is shown in Formula 5 below, wherein: R 3 It is one of H, 2-F, 2-Cl, 4-F, 4-Cl, 4-Br, 4-MeO, 4-Me, 4-CN, etc.;

[0019]

[0020] Furthermore, in the preferred embodiment of the present invention, the thiazoline derivatives described above include, but are not limited to, the representative compounds shown in Formula 6. Due to space limitations and for the sake of brevity, the present invention will not exhaustively list them.

[0021]

[0022] Furthermore, in the above technical solution, the molecular structural formula of the dihydrazone derivative is shown in Formula 7 below, wherein: R 4 It is one of Ph, 3-CF3-Ph, 2,6-F2-Ph, 4-Br-Ph, 4-Cl-Ph, 4-F-Ph, 2-Cl-Ph, 2-F-Ph, 2-OH-Ph, 3-Indole, 3-ClPy, etc.

[0023]

[0024] Furthermore, in the preferred embodiment of the present invention, the above-mentioned technical solution includes, but is not limited to, the representative compounds shown in Formula 8. Due to space limitations and for the sake of brevity, the present invention will not exhaustively list them.

[0025]

[0026] The second object of the present application is to provide a method for preparing the above-mentioned sterone derivatives by using a chemical-microbial catalysis combined method. First, four types of sterol derivatives are prepared by using a chemical method with dehydroepiandrosterone as a raw material; second, a microbial cell catalysis, i.e., a new Mycobacterium aurum strain is used to catalyze the oxidation of sterols and isomerization to convert into novel sterone derivatives.

[0027] The specific reaction formula of the above-mentioned microbial cell catalysis is shown in the following formula nine:

[0028]

[0029] Specifically, the synthesis route of the above-mentioned sterone derivatives of the present application is shown in the following formula ten:

[0030]

[0031] The specific preparation method of the above-mentioned sterone derivatives includes the following steps:

[0032] 1) Activating the bacterial strain: the Mycobacterium aurum is streaked on LB solid plate culture medium, and incubated at 37°C for 3-5 days until single colonies are grown on the LB solid plate;

[0033] 2) Preparing the seed liquid: the single colony strain activated on the above-mentioned LB plate is picked into a small volume of LB liquid culture medium test tube, and incubated at 150-250 rpm and 30-37°C for 2-4 days until the OD 600nm is 1.5-2.0, and then transferred to a large volume of LB liquid culture medium, and incubated at 150-250 rpm and 30-37°C for 2-4 days, and then transferred to MnC01 seed culture medium at a 10-20% inoculation amount (V / V) for expansion, and incubated at 150-250 rpm and 30-37°C for 2-4 days to obtain the seed liquid;

[0034] 3) Preparing the sterol derivative 1: the sterol derivative 1 is prepared by using a chemical synthesis method with dehydroepiandrosterone (DHEA) as a raw material;

[0035] 4) Microbial cell catalysis reaction: the seed liquid prepared in step 2) is taken in a flask of MnC02G fermentation medium, the liquid loading coefficient is 10-30% (V / V), the inoculation amount is 5-20% (V / V), 0.2-1 mmol of the sterol compound 1, 2-hydroxy-β-cyclodextrin 0.5-2 wt%, and incubated at 150-250 rpm and 25-40°C for 2-4 days, then 2-4 volumes of ethyl acetate are added and stirred for 3-6 hours, and then extracted, separated, concentrated and purified by silica gel column chromatography to obtain the sterone derivative.

[0036] Furthermore, in the above technical solution, the formulation and preparation method of the liquid LB (Luria-Bertani) culture medium are as follows: 10g tryptone, 5g yeast extract, and 10g NaCl. Weigh the above reagents and dissolve them in double-distilled water, then bring the volume to 1L. Autoclave at 121℃ for 30 minutes, cool, and store at 4℃ for later use.

[0037] Furthermore, in the above technical solution, the formulation and preparation method of the solid LB (Luria-Bertani) medium are as follows: 1.3% (13g) agar is added to 1L of liquid LB medium, and the medium is autoclaved at 121℃ for 30min. After cooling, it is stored at 4℃ for later use.

[0038] Further, in the above technical solution, the formulation and preparation method of the MnCO1 seed culture medium are as follows: 20g glycerol, 2g citric acid, 0.05g ferric ammonium citrate, 0.5g K2HPO4, 0.5g MgSO4·7H2O, 2g (NH4)2SO4, and 2g NaNO3. Weigh the above reagents and dissolve them in double-distilled water, then bring the volume to 1L (pH 7.5). Autoclave at 121℃ for 30 minutes, and store at room temperature for later use.

[0039] Furthermore, in the above technical solution, the formulation and preparation method of the MnCO2G fermentation medium are as follows: 10g glucose, 2g citric acid, 0.05g ferric ammonium citrate, 0.5g K2HPO4, 0.5g MgSO4·7H2O, 2g (NH4)2SO4, and 2g NaNO3. Weigh the above reagents and dissolve them in double-distilled water, then bring the volume to 1L (pH 7.5). Autoclave at 115℃ for 30 minutes, and store at room temperature for later use.

[0040] Furthermore, in the above technical solution, the sterol compound 1 is prepared by a chemical method using dehydroepiandrosterone (DHEA) as a raw material. The specific steps are as follows: Dehydroepiandrosterone (DHEA) is dissolved in an organic alcohol solvent according to the specified ratio, and then R is added. 1 CHO and NaOH were reacted at room temperature, and the reaction was monitored by TLC until the reaction was completed. After the reaction was completed, ice water was added to the reaction solution and the mixture was stirred continuously. The precipitate was filtered and dried to obtain a powdered product, which was then purified by silica gel column chromatography to obtain the sterol compound.

[0041] Preferably, in the above technical solution, the molar ratio of dehydroepiandrosterone to benzaldehyde is 1:(1-2).

[0042] Preferably, in the above technical solution, the organic alcohol solvent is anhydrous methanol.

[0043] Preferably, the above technical solution, the molar ratio of dehydroepiandrosterone to NaOH is 1:10.

[0044] Preferably, the above technical solution, R 1 is one of Ph, 2-Cl-Ph, 2-F-Ph, 4-CF3-Ph, 2-CF3-Ph, 4-MeO-Ph, 2-Py, 3-Py, 4-Py, 4-Cl-Ph, 4-F-Ph, 4-MeS-Ph, 3,4,5-(MeO)3-Ph) and the like.

[0045] Specifically, the synthesis route of the above-mentioned pyrimidine derivative of the present application is shown in formula eleven:

[0046]

[0047] The specific preparation method of the above-mentioned pyrimidine derivative includes the following steps:

[0048] Steps 1)-3) are the same as the preparation method of steroidal ketone derivatives;

[0049] Step 4) preparation of sterol derivative 2: dissolving the sterol derivative 1 prepared in step 3) in tert-butyl alcohol, then adding guanidine nitrate and potassium tert-butoxide according to the ratio, heating and refluxing overnight, then adding ice water to the reaction solution and stirring constantly, then adjusting the pH to 7-8, filtering and drying to obtain the precipitate, and purifying by silica gel column chromatography to obtain the sterol derivative 2;

[0050] Step 5) taking 0.2-1 mmol of the sterol derivative 2 prepared in step 4) in MnCO2G fermentation medium, adding the seed liquid prepared in step 2), the liquid loading coefficient is 10-30% (V / V), the inoculation amount is 5-20% (V / V), 2-hydroxy-β-cyclodextrin is 0.5-2 wt%, 150-250 rpm, 25-40℃ for 2-4d, then adding 2-4 times the volume of ethyl acetate and stirring for 3-6h, then extracting, separating, concentrating and purifying by silica gel column chromatography to obtain the pyrimidine derivative.

[0051] Further, the above technical solution, the molar ratio of the sterol derivative 1 to guanidine nitrate in step 4) is 1:4.

[0052] Further, the above technical solution, the molar ratio of the sterol derivative 1 to potassium tert-butoxide in step 4) is 1:3.

[0053] Specifically, the synthesis route of the above-mentioned thiazoline derivative of the present application is shown in formula twelve:

[0054]

[0055] The specific preparation method of the thiazoline derivative described above comprises the following steps:

[0056] Steps 1)-2) are the same as the preparation method of the steroidal ketone derivative;

[0057] Step 3) preparation of sterol derivative 3: dehydroepiandrosterone is dissolved in anhydrous ethanol, then N-substituted thiosemicarbazide is added in proportion, heated to reflux at 60-70 DEG C overnight, and the reaction is tracked by TLC until the reaction is completed; after the reaction is completed, ice water is added to the reaction solution and stirred constantly, the precipitate is filtered and dried to obtain a powdery product, and the sterol derivative 3 is purified by silica gel column chromatography;

[0058] Step 4) preparation of sterol derivative 4: the sterol derivative 3 prepared in step 3) is dissolved in anhydrous ethanol, then substituted α-bromoacetophenone is added in proportion, heated to reflux at 60-70 DEG C overnight, and the reaction is tracked by TLC until the reaction is completed; after the reaction is completed, ice water is added to the reaction solution and stirred constantly, the precipitate is filtered and dried, and the sterol derivative 4 is purified by silica gel column chromatography;

[0059] Step 5) 0.2-1 mmol of the sterol derivative 4 prepared in step 4) is added to a MnCO2G fermentation medium, seed liquid prepared in step 2) is added, the liquid loading coefficient is 10-30% (V / V), the inoculation amount is 5-20% (V / V), 2-hydroxy-β-cyclodextrin is 0.5-2 wt%, the culture is carried out at 150-250 rpm and 25-40 DEG C for 2-4 days, after the fermentation is completed, 2-4 times the volume of ethyl acetate is added and stirred for 3-6 hours, then extraction separation, concentration and purification by silica gel column chromatography are carried out to obtain the thiazoline derivative.

[0060] Further, in the technical scheme, the molar ratio of dehydroepiandrosterone to N-substituted thiosemicarbazide in step 3) is 1: (1-2).

[0061] Further, in the technical scheme, the molar ratio of the sterol derivative 3 to 2-bromoacetophenone in step 4) is 1: (1-2).

[0062] Further, in the technical scheme, the substituent in the substituted α-bromoacetophenone in step 4) is one of H, 2-F, 2-Cl, 4-F, 4-Cl, 4-Br, 4-MeO, 4-Me, 4-CN and the like.

[0063] Specifically, the synthesis route of the bishydrazone derivative described above is shown in formula XIII:

[0064]

[0065] The specific preparation method of the bishydrazone derivative described above comprises the following steps:

[0066] Step 1) - 2) are the same as the preparation method of the steroidal ketone derivative;

[0067] Step 3) Preparation of sterol derivative 5: dehydroepiandrosterone is dissolved in anhydrous ethanol, then hydrazine hydrate is added in proportion, and hydrazine sulfate dissolved in deionized water is added in proportion, and the reaction is stirred at room temperature. The reaction is tracked by TLC until the reaction is completed; after the reaction is completed, ice water is added to the reaction solution and stirred constantly, the precipitate is filtered and dried to obtain a powdery product, and the product is purified by silica gel column chromatography to obtain the sterol derivative 5;

[0068] Step 4) Preparation of sterol derivative 6: the sterol derivative 5 prepared in step 3) is dissolved in anhydrous ethanol, then R 4 CHO is added in proportion, and the reaction is tracked by TLC until the reaction is completed; after the reaction is completed, the solvent is concentrated, and the crude product is separated at 0-4°C, and then purified by silica gel column chromatography to obtain the sterol derivative 6;

[0069] Step 5) 0.2-1 mmol of the sterol derivative 6 prepared in step 4) is taken in the MnC02G fermentation medium, and the seed solution prepared in step 2) is added, the liquid loading coefficient is 10-30% (V / V), the inoculation amount is 5-20% (V / V), 2-hydroxy-β-cyclodextrin is 0.5-2 wt%, 150-250 rpm, 25-40°C for 2-4 days, then 2-4 volumes of ethyl acetate is added and stirred for 3-6 hours, then extracted, separated, concentrated and purified by silica gel column chromatography to obtain the bishydrazone derivative.

[0070] Further, in the above technical solution, the molar ratio of dehydroepiandrosterone to hydrazine sulfate in step 3) is 10:0.1.

[0071] Further, in the above technical solution, the molar ratio of dehydroepiandrosterone to hydrazine sulfate in step 3) is 1:5.

[0072] Further, in the above technical solution, the molar ratio of the sterol derivative 5 in step 4) to R 4 CHO is 1:(10-15).

[0073] Further, in the above technical solution, R 4 CHO is R 4 Ph, 3-CF3-Ph, 2,6-F2-Ph, 4-Br-Ph, 4-Cl-Ph, 4-F-Ph, 2-Cl-Ph, 2-F-Ph, 2-OH-Ph, 3-Indole, 3-ClPy, etc.

[0074] The third object of the present application is to provide the use of the above-mentioned steroidal derivatives in the preparation of a cancer cell inhibiting drug.

[0075] Further, in the above technical solution, the steroidal derivative is preferably compound h, 2b or 4a.

[0076] Further, in the above technical solution, the cancer cell is preferably human melanoma cell line A875 and / or hepatocarcinoma cell line Huh7.

[0077] Compared with the prior art, the present application has the following beneficial effects:

[0078] (1) The present application provides a method for preparing a plurality of novel structural type steroidal derivatives, which specifically uses dehydroepiandrosterone as a raw material to prepare corresponding sterol derivative intermediates by chemical synthesis design, and then catalyzes the sterol derivative intermediates by microorganism cells to efficiently prepare novel steroidal derivatives containing steroidal units.

[0079] (2) The present application uses the oxidation-reduction and isomerization of cholesterol oxidase, isomerase and dehydrogenase in Mycobacterium neoaurum microorganism cells, which has strong selectivity, mild catalytic conditions, simple extraction process, environmental friendliness, good substrate adaptability, and can realize the conversion of four types of sterol derivatives, i.e., steroidal-aldehyde condensation product, steroidal-pyrimidine derivative, steroidal-thiazoline derivative, and steroidal-bishydrazine, and provides a new strategy for constructing a novel steroidal derivative molecular library. DETAILED DESCRIPTION

[0080] The present application provides a method for preparing a plurality of novel structural type steroidal derivatives by using a chemical-microorganism catalytic cascade reaction strategy. First, four structural type sterol derivatives are prepared by using dehydroepiandrosterone as a raw material by a chemical method; second, Mycobacterium neoaurum Tsukamura 3503 (ATCC25795, i.e., CCTCC AA2018059) is used to catalyze the oxidation of sterol and isomerization to novel steroidal derivatives.

[0081] The present application will be further described in detail by the following examples.

[0082] For a better understanding of the present application without restricting the scope thereof, all numbers expressing quantities of ingredients, percentages, and other numerical values used in the specification and claims herein are to be understood as being modified in all instances by the term "about". Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that can vary depending upon the desired properties sought to be obtained by the present application. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0083] The equipment and raw materials used in the present application can be purchased from the market or are commonly used in the art. The methods in the following examples are conventional methods in the art, unless otherwise specified.

[0084] The microorganism source involved in the following examples of the present application: China Center for Type Culture Collection (Wuhan University), Mycobacterium neoaurum Tsukamura 3503, accession number: CCTCC AA2018059.

[0085] Example 1

[0086] The specific synthesis route of a sterone derivative a in this example is shown in the following formula XIV:

[0087]

[0088] The sterone derivative a described above in this example is prepared by the following method, comprising the following steps:

[0089] 1) The culture medium used in the experiment:

[0090] Liquid LB (Luria-Bertani) medium: 10 g of tryptone, 5 g of yeast powder, 10 g of NaCl. Weigh the above reagents, dissolve them in double distilled water, and make up to 1 L. Autoclave at 121°C for 30 min, and store at 4°C after cooling.

[0091] Solid LB (Luria-Bertani) medium: Add 1.3% (13 g) agar to 1 L of liquid LB medium, autoclave at 121°C for 30 min, and store at 4°C after cooling.

[0092] MnC01 seed medium: 20 g of glycerol, 2 g of citric acid, 0.05 g of ferric ammonium citrate, 0.5 g of K2HPO4, 0.5 g of MgSO4·7H2O, 2 g of (NH4)2SO4, and 2 g of NaNO3. Weigh the above reagents, dissolve them in double distilled water, and make up to 1 L (pH 7.5). Autoclave at 121°C for 30 min, and store at room temperature.

[0093] MnC02G fermentation medium: glucose 10 g, citric acid 2 g, ferric ammonium citrate 0.05 g, K2HPO40.5 g, MgS04·7H20 0.5 g, (NH4)2S04 2 g, NaNO32 g. The above reagents were weighed and dissolved in double distilled water and made up to 1 L (pH 7.5), autoclaved at 115 °C for 30 min and stored at room temperature for use.

[0094] 2) Activation of the bacterial strain: Mycobacterium neoaurum Tsukamura 3503 was streaked on LB solid plate medium and incubated at 37 °C for 4 days until single colonies appeared on the LB solid plate.

[0095] 3) Preparation of seed broth: single colonies of the activated strain were picked from the LB plate and inoculated in 5 mL of LB liquid medium in a test tube and incubated at 200 rpm and 30 °C for 3 days until the OD 600nm was 1.5. The culture was transferred to 50 mL of LB liquid medium and incubated at 200 rpm and 30 °C for 3 days. The culture was then transferred to MnC02G seed medium at a 10% inoculum (V / V) and incubated at 200 rpm and 30 °C for 3 days.

[0096] 4) 4 mmol of dehydroepiandrosterone (DHEA) was dissolved in 25 mL of methanol, followed by the addition of 4.4 mmol of benzaldehyde and 40 mmol of NaOH. The reaction was carried out at room temperature and the progress of the reaction was monitored by TLC until completion. After completion of the reaction, 40 mL of ice water was added to the reaction mixture and stirred continuously. The precipitate was filtered and dried to obtain a powder. The intermediate a-1 was purified by silica gel column chromatography and obtained as a white powder with a yield of 93%. MS (ESI) m / z 377.5 (M+H) + , calcd. for C 26 H 32 O2 m / z = 376.2.

[0097] 5) 0.5 mmol of the intermediate a-1 was added to a 2 L flask containing 160 mL of MnC02G fermentation medium, followed by the addition of 40 mL of seed broth and 1.5 g of 2-hydroxy-β-cyclodextrin. The reaction was carried out at 200 rpm and 30 °C for 3 days. After completion of the fermentation, 400 mL of ethyl acetate was added and stirred for 4 h. The product was extracted, separated, concentrated and purified by silica gel column chromatography to obtain the target product a as a pale yellow powder with a conversion rate of 94.7%. The spectral data of the compound a obtained are as follows:

[0098] 1H NMR (600 MHz, CDC13): δ 7.52 (d, J = 7.4 Hz, 2H), 7.44 (s, 1H, =CH-20), 7.40 (t, J = 7.4 Hz, 2H), 7.35 (t, J = 7.3 Hz, 1H), 5.74 (s, 1H, =CH-4), 2.90 (dd, J = 15.7, 6.5, 1.6 Hz, 1H), 2.50 - 2.39 (m, 3H), 2.38 - 2.31 (m, 2H), 2.06-2.02 (m, 2H), 2.01 - 1.96 (m, 1H), 1.88 - 1.82 (m, 1H), 1.75-1.68 (m, 2H), 1.57-1.49 (m, 1H), 1.42 - 1.32 (m, 2H), 1.23 (s, 3H, CH3), 1.17-1.10 (m, 1H), 1.06 - 1.01 (m, 1H), 1.00 (s, 3H, CH3);

[0099] 13 C NMR (150 MHz, CDC13): δ 209.08, 199.28, 170.18, 135.64, 135.59, 133.50, 130.42, 129.44, 128.81, 124.32, 53.96, 49.11, 47.38, 38.81, 35.74, 34.94, 34.00, 32.65, 31.50, 31.07, 29.32, 20.46, 17.49, 14.50;

[0100] MS (ESI) m / z 375.7 (M+H) + , calcd. for C 26 H 30 O2 m / z = 374.

[0101] Example 2

[0102] A specific synthetic route of the pyrimidine derivative 2a of this example is shown in Formula XV as follows:

[0103]

[0104] The pyrimidine derivative 2a of this example is prepared by the following method, comprising the following steps:

[0105] Step 1) -4) are the same as in Example 1.

[0106] 5) Take 1 mmol of intermediate a-1 dissolved in 15 mL of tert-butyl alcohol, then add 4 mmol of guanidine nitrate and 3 mmol of potassium tert-butoxide, heat to reflux and react overnight, after the reaction is completed, add 50 mL of ice water to the reaction liquid and continuously stir, then adjust the pH to 7-8, suction filter and dry to obtain a precipitate, and purify by silica gel column chromatography to obtain intermediate a-2 as a white powder with a yield of 96%, MS (ESI) m / z 416.6 (M+H) + , calcd. for C 27 H 33 N3O m / z = 415.3.

[0107] 6) Take 0.5 mmol of intermediate 2a-1 and add it to a 2L flask containing 160 mL of MnCO2G fermentation medium, add 40 mL of seed liquid and 2 g of 2-hydroxy-β-cyclodextrin, cultivate at 250 rpm and 37°C for 2d, after the fermentation is completed, add 500 mL of ethyl acetate and stir for 6h, then extract, separate, concentrate and purify by silica gel column chromatography to obtain the target product 2a as a gray-white powder with a conversion rate of 61.0%. The spectral data of the obtained compound 2a are as follows:

[0108] 1 H NMR (600 MHz, CDCl3): δ 7.83 (dd, J = 7.3, 2.2 Hz, 2H), 7.51 (dd, J = 7.9, 2.5 Hz, 3H), 5.75 (s, 1H, =CH-4), 5.34 (s, 2H, NH2), 2.80 (dd, J = 14.4, 6.5 Hz, 1H), 2.73-2.67 (m, 1H), 2.47-2.32 (m, 4H), 2.21 (d, J = 7.8 Hz, 1H), 2.08-2.04 (m, 1H), 2.02-1.92 (m, 3H), 1.80-1.72 (m, 2H), 1.66-1.62 (m, 2H), 1.59-1.54 (m, 2H), 1.25 (s, 3H, CH3), 1.14 (s, 3H, CH3);

[0109] 13 C NMR (150 MHz, CDCl3): δ 199.40, 170.18, 130.80, 128.93, 128.78, 128.69, 125.43, 124.42, 124.41, 120.13, 54.90, 54.08, 46.28, 38.87, 35.74, 34.55, 34.04, 32.70, 32.67, 31.53, 29.84, 20.63, 17.47, 17.10;

[0110] MS (ESI) m / z 414.6 (M+H) + , calcd. for C 27 H 31 N3O m / z = 413.3.

[0111] Example 3

[0112] The specific synthetic route of one thiazoline derivative 3a of this example is shown in the following formula sixteen:

[0113]

[0114] The thiazoline derivative 3a described above of this example is prepared by the following method, comprising the following steps:

[0115] Steps 1)-3) are the same as Example 2.

[0116] 4) Take 10 mmol dehydroepiandrosterone dissolved in 25 mL of ethanol, then add 11 mmol of the corresponding N-substituted thiosemicarbazide, heat to reflux at 60-70°C overnight, track the reaction by TLC until the reaction is complete. After the reaction is completed, 60 mL of ice water is added to the reaction liquid and stirred constantly, the precipitate is filtered and dried to obtain a powdery product, and purified by silica gel column chromatography to obtain the intermediate 3a-1 as a white powder with a yield of 92%, MS (ESI) m / z 376.5 (M+H) + , calcd. for C 21 H 33 N3OS m / z = 375.2.

[0117] 5) Take 1 mmol of the above intermediate 3a-1 dissolved in 10 mL of anhydrous ethanol, then add 1.1 mmol of substituted 2-bromoacetophenone, heat to reflux at 60-70°C overnight, track the reaction by TLC until the reaction is complete. After the reaction is completed, 20 mL of ice water is added to the reaction liquid and stirred constantly for 30 min, the precipitate is filtered and dried, and purified by silica gel column chromatography to obtain the intermediate 3a-2 as a grayish white powder with a yield of 93%, MS (ESI) m / z 506.8 (M+H) + , calcd. for C 30 H 39 N3O2S m / z = 505.3.

[0118] 6) Take 3a-2 intermediate 0.5 mmol into a 2 L flask containing 160 mL of MnC02G fermentation medium, add seed liquid 40 mL and 2-hydroxy-β-cyclodextrin 2 g, cultivate at 200 rpm and 37 °C for 3 d, after fermentation, add 400 mL of ethyl acetate and stir for 5 h, then extract, separate, concentrate and purify by silica gel column chromatography to obtain the target product 3a as a light yellow powder, with a conversion rate of 50.7%. The spectral data of the obtained compound 3a are as follows:

[0119] 1 H NMR (600 MHz, CDC13): δ 7.27 (d, J = 2.8 Hz, 1H), 7.26-7.25 (m, 1H), 6.96-6.93 (m, 2H), 5.82 (s, 1H, N-C=CH), 5.74 (s, 1H, =CH-4), 3.84 (s, 3H, Ph-OCH3), 3.29 (s, 3H, NCH3), 2.72-2.55 (m, 2H), 2.45-2.29 (m, 4H), 2.11-2.04 (m, 2H), 1.96-1.92 (m, 1H), 1.87-1.82 (m, 1H), 1.75-1.67 (m, 3H), 1.55-1.50 (m, 1H), 1.47-1.40 (m, 2H), 1.22 (s, 3H, CH3), 1.21-1.17 (m, 1H), 1.11 (dd, J = 13.2, 3.3 Hz, 1H), 1.06-1.01 (m, 1H), 0.98 (s, 3H, CH3);

[0120] 13 C NMR (150 MHz, CDC13): δ 199.89, 174.42, 171.47, 168.21, 160.18, 140.71, 130.15, 124.02, 123.58, 114.16, 98.37, 55.49, 54.18, 53.14, 44.18, 38.82, 35.72, 35.23, 34.20, 34.06, 33.60, 32.88, 31.49, 27.78, 23.47, 20.74, 17.47, 17.15;

[0121] MS (ESI) m / z 504.7 (M+H) + , calcd. for C 30 H 37 N3O2S m / z = 503.3.

[0122] Example 4

[0123] The specific synthesis route of the dihydrazone derivative 4a of the present embodiment is shown in the following formula seventeen:

[0124]

[0125] The dihydrazone derivative 4a described above is prepared by the following method, comprising the following steps:

[0126] Steps 1)-3) are the same as in Embodiment 2.

[0127] 4) Take 10 mmol dehydroepiandrosterone and dissolve it in 25 mL of ethanol, then add 50 mmol of hydrazine hydrate, then add hydrazine sulfate dissolved in 5 mL of water (0.1 mmol), stir the reaction at room temperature, and track the reaction by TLC until the reaction is complete. After the reaction is completed, add 40 mL of ice water to the reaction liquid and continuously stir, filter the precipitate and dry to obtain a powder, and purify by silica gel column chromatography to obtain the intermediate 4a-1 as a white powder with a yield of 84%, MS (ESI) m / z 303.6 (M+H) + , calcd. for C 19 H 30 N2O m / z = 302.2.

[0128] 5) Take 1 mmol of intermediate 4a-1 and dissolve it in 10 mL of ethanol, then add 11 mmol of the corresponding 4-trifluoromethylbenzaldehyde, heat to reflux at 40-50°C overnight, and track the reaction by TLC until the reaction is complete. After the reaction is completed, concentrate the solvent and separate at 0-4°C to obtain the crude product, and purify by silica gel column chromatography to obtain the intermediate 4a-2 as a yellow powder with a yield of 91%, MS (ESI) m / z 459.5 (M+H) + , calcd. for C 27 H 33 F3N2O m / z = 458.3.

[0129] 6) Take 0.5 mmol of 4a-2 intermediate and add it to a 2L Erlenmeyer flask containing 160 mL of MnCO2G fermentation medium, add 40 mL of seed liquid and 2-hydroxy-β-cyclodextrin 2g, cultivate at 200 rpm and 30°C for 3d, after the fermentation is completed, add 600 mL of ethyl acetate and stir for 4h, then extract, separate, concentrate and purify by silica gel column chromatography to obtain the target product 4a as a brown powder with a conversion rate of 90.6%. The spectral data of the obtained compound 4a are as follows:

[0130] 1H NMR (600 MHz, CDC13): δ 8.28 (s, 1H, N=CH), 7.84 (d, J = 8.1 Hz, 2H), 7.64 (d, J = 8.2 Hz, 2H), 5.73 (s, 1H, =CH-4), 2.66-2.60 (m, 1H), 2.47-2.38 (m, 3H), 2.36-2.30 (m, 2H), 2.11-2.00 (m, 3H), 1.98-1.92 (m, 2H), 1.88-1.84 (m, 1H), 1.73-1.67 (m, 3H), 1.57-1.41 (m, 4H), 1.20 (s, 3H, CH3), 1.01 (s, 3H, CH3);

[0131] 13 C NMR (150 MHz, CDC13): δ 199.39, 183.14, 170.72, 170.41, 155.83, 137.97, 125.71, 124.24, 53.92, 52.78, 50.94, 44.51, 38.78, 35.79, 35.25, 34.00, 31.38, 30.85, 28.03, 23.29, 21.84, 20.41, 17.47, 16.78;

[0132] MS (ESI) m / z 457.6 (M+H) + , calcd. for C 27 H 31 F3N2O m / z = 456.2.

[0133] Example 5

[0134] The preparation of other steroidal ketone derivatives in the present application can refer to the basic synthesis method described in the above Examples 1-4, and different chemical raw materials can be selected according to the structural characteristics of the substituents of the compounds described in the foregoing text, so that other compounds can be conveniently prepared.

[0135] The spectral data of the compounds listed in the foregoing technical solutions are as follows:

[0136] Compound b is a light yellow powder, and the conversion rate is 86.1%. The spectral data of the obtained compound b are as follows:

[0137] 1H NMR (600 MHz, DMSO-d6): δ 7.74 (dd, J = 6.9, 2.5 Hz, 1H, =CH-20), 7.60 - 7.54 (m, 2H), 7.46 - 7.41 (m, 2H), 5.64 (s, 1H, =CH-4), 2.71-2.67 (m, 1H), 2.62 - 2.56 (m, 1H), 2.44 - 2.38 (m, 2H), 2.28 (dd, J = 11.4, 3.0 Hz, 1H), 2.18 - 2.14 (m, 1H), 1.99 - 1.91 (m, 2H), 1.85-1.80 (m, 2H), 1.65 - 1.58 (m, 2H), 1.50 - 1.45 (m, 1H), 1.35-1.30 (m, 2H), 1.19 (s, 3H, CH3), 1.08 - 0.99 (m, 2H), 0.95 (s, 3H, CH3);

[0138] 13 C NMR (150 MHz, DMSO-d6): δ 207.84, 198.01, 170.50, 138.80, 134.42, 132.72, 130.90, 130.36, 129.86, 127.49, 127.20, 123.34, 53.03, 47.95, 47.07, 38.31, 35.05, 34.06, 33.62, 31.77, 30.98, 30.43, 28.49, 19.90, 16.91, 13.99;

[0139] MS (ESI) m / z 409.6 (M+H) + , calcd. for C 26 H 29 ClO2 m / z = 408.2.

[0140] Compound c as a pale yellow powder with a conversion of 90.6%. The spectral data of the obtained compound c are as follows:

[0141] 1H NMR (600 MHz, CDC13): δ 7.65 (s, 1 H, =CH-20), 7.53 (t, J = 7.5 Hz, 1 H), 7.37-7.33 (m, 1 H), 7.18 (t, J = 7.5 Hz, 1 H), 7.13-7.09 (m, 1 H), 5.75 (s, 1 H, =CH-4), 2.81 (dd, J = 15.9, 5.3 Hz, 1 H), 2.49-2.41 (m, 3 H), 2.38-2.33 (m, 2 H), 2.07-1.99 (m, 3 H), 1.86 (dd, J = 11.2, 3.0 Hz, 1 H), 1.76-1.71 (m, 2 H), 1.58-1.51 (m, 1 H), 1.44-1.31 (m, 3 H), 1.24 (s, 3 H, CH3), 1.15-1.10 (m, 1 H), 1.02 (s, 3 H, CH3);

[0142] 13 C NMR (150 MHz, CDC13): δ 208.75, 199.63, 170.43, 137.85, 131.371, 131.31, 125.72, 124.59, 124.36, 116.39, 116.25, 54.16, 49.13, 47.76, 39.05, 35.98, 35.16, 34.25, 32.89, 31.72, 31.28, 29.55, 20.69, 17.74, 14.70;

[0143] MS (ESI) m / z 393.4 (M+H) + , calcd. for C 26 H 29 FO2 m / z = 392.2.

[0144] Compound d was obtained as an off-white powder with 100% conversion. The spectral data of compound d obtained are as follows:

[0145] 1H NMR (600 MHz, CDC13): δ 7.65 (d, J = 7.6 Hz, 2H), 7.61 (d, J = 8.0 Hz, 2H), 7.43 (s, 1H, =CH-20), 5.75 (s, 1H, =CH-4), 2.88 (dd, J = 15.9, 6.4 Hz, 1H), 2.52 - 2.41 (m, 3H), 2.35 (d, J = 16.1 Hz, 2H), 2.06 - 1.99 (m, 3H), 1.89 - 1.83 (m, 1H), 1.74-1.69 (m, 2H), 1.57-1.50 (m, 1H), 1.43 - 1.35 (m, 2H), 1.24 (s, 3H, CH3), 1.16 - 1.11 (m, 1H), 1.05 (d, J = 10.6 Hz, 1H), 1.01 (s, 3H, CH3);

[0146] 13 C NMR (150 MHz, CDC13): δ 208.65, 199.33, 170.03, 139.03, 138.01, 131.67, 130.38, 125.73, 125.70, 124.38, 53.92, 48.96, 47.50, 38.81, 35.75, 34.92, 34.00, 32.60, 31.45, 31.03, 29.30, 20.43, 17.49, 14.45;

[0147] MS (ESI) m / z 443.5 (M+H) + , calcd. for C 27 H 29 F302 m / z = 442.5.

[0148] Compound e was obtained as an off-white powder with a conversion of 89.4%. The spectral data of the obtained compound e are as follows:

[0149] 1H NMR (600 MHz, CDC3): δ 7.73 (s, 1H, =CH-20), 7.72 (d, J = 8.0 Hz, 1H), 7.59 - 7.54 (m, 2H), 7.45 (t, J = 7.3 Hz, 1H), 5.73 (s, 1H, =CH-4), 2.69 (dd, J = 15.8, 6.3, 1.5 Hz, 1H), 2.44 - 2.29 (m, 5H), 2.07-1.94 (m, 3H), 1.86-1.80 (m, 1H), 1.76-1.69 (m, 3H), 1.57-1.50 (m, 1H), 1.44 - 1.35 (m, 2H), 1.23 (s, 3H, CH3), 1.13 - 1.08 (m, 1H), 1.02 (s, 3H, CH3);

[0150] 13 C NMR (150 MHz, CDC13): δ 207.98, 199.44, 170.19, 138.98, 131.69, 130.00, 129.31, 128.72, 126.38, 126.34, 124.34, 53.84, 48.86, 47.74, 38.79, 35.72, 34.90, 34.01, 32.61, 31.39, 30.98, 29.82, 28.96, 20.41, 17.49, 14.38;

[0151] MS (ESI) m / z 443.4 (M+H) + , calcd. for C 27 H 29 F3O2 m / z = 442.5.

[0152] Compound f was obtained as a pale yellow powder with a conversion of 88.6%. The spectral data of the obtained compound f are as follows:

[0153] 1H NMR (600 MHz, CDC13): δ 7.45 (d, J = 8.4 Hz, 2H), 7.39 (s, 1H, =CH-20), 7.25 (d, J = 8.7 Hz, 2H), 5.75 (s, 1H, =CH-4), 2.90-2.85 (m, 1H), 2.51 (s, 3H, SCH3), 2.48-2.40 (m, 3H), 2.39-2.33 (m, 2H), 2.07-2.04 (m, 2H), 2.01-1.97 (m, 1H), 1.89-1.83 (m, 1H), 1.74-1.70 (m, 2H), 1.57-1.51 (m, 1H), 1.46-1.37 (m, 2H), 1.24 (s, 3H, CH3), 1.15 (dd, J = 13.1, 3.7 Hz, 1H), 1.06-1.02 (m, 1H), 1.00 (s, 3H, CH3);

[0154] 13 C NMR (150 MHz, CDC13): δ 209.25, 199.48, 170.33, 141.10, 134.73, 133.08, 132.01, 130.82, 125.92, 124.33, 53.94, 49.11, 47.37, 38.82, 35.73, 34.90, 34.02, 32.67, 31.48, 31.06, 29.82, 29.40, 17.50, 15.25, 14.55;

[0155] MS (ESI) m / z 421.3 (M+H) + , calcd. for C 27 H 32 O2S m / z = 420.6.

[0156] Compound g, as a light yellow powder, conversion rate was 82.9%. The spectral data of the obtained compound g are as follows:

[0157] 1H NMR (600 MHz, CDC13): δ 8.70 (d, J = 3.9 Hz, 1H), 7.71 (t, J = 7.2 Hz, 1H), 7.44 (d, J = 7.8 Hz, 1H), 7.39 (s, 1H, =CH-20), 7.22 (dd, J = 7.2, 4.8 Hz, 1H), 5.75 (s, 1H, =CH-4), 3.36 (dd, J = 17.3, 5.4 Hz, 1H), 2.59 - 2.51 (m, 1H), 2.48 - 2.40 (m, 2H), 2.38 - 2.33 (m, 2H), 2.07-1.98 (m, 3H), 1.88-1.83 (m, 1H), 1.75-1.71 (m, 2H), 1.57 - 1.51 (m, 1H), 1.43 - 1.32 (m, 2H), 1.24 (s, 3H, CH3), 1.17 - 1.12 (m, 1H), 1.06-1.03 (m, 1H), 1.00 (s, 3H, CH3);

[0158] 13 C NMR (150 MHz, CDC13): δ 209.75, 199.45, 170.50, 154.91, 149.95, 140.14, 136.61, 130.97, 126.95, 124.30, 123.06, 54.03, 48.81, 47.56, 38.86, 35.79, 35.03, 34.05, 32.76, 31.49, 31.15, 29.78, 20.51, 17.54, 14.51;

[0159] MS (ESI) m / z 376.5 (M+H) + , calcd. for C 25 H 29 NO2 m / z = 375.2.

[0160] Compound h was a light yellow powder with a conversion of 68.1%. The spectral data of the obtained compound h are as follows:

[0161] 1H NMR (600 MHz, CDC13): δ 8.82 (s, 1H, N=CH), 8.67-8.52 (m, 1H), 7.83 (d, J = 7.7 Hz, 1H), 7.38 (s, 1H, =CH-20), 7.42 (d, J = 14.3 Hz, 1H), 7.40-7.33 (m, 1H), 5.76 (s, 1H), 2.90 (dd, J = 15.8, 5.8 Hz, 1H), 2.52-2.42 (m, 3H), 2.38-2.34 (m, 2H), 2.08-1.99 (m, 3H), 1.90-1.85 (m, 1H), 1.77-1.70 (m, 2H), 1.58-1.51 (m, 1H), 1.43-1.37 (m, 2H), 1.25 (s, 3H, CH3), 1.18-1.13 (m, 1H), 1.06 (dd, J = 11.5, 3.4 Hz, 1H), 1.02 (s, 3H, CH3);

[0162] 13 C NMR (150 MHz, CDC13): δ 208.27, 199.22, 169.90, 150.72, 149.39, 137.94, 137.19, 129.50, 124.30, 53.82, 48.89, 47.38, 38.70, 35.65, 34.81, 33.90, 32.49, 31.33, 30.93, 29.26, 20.33, 17.39, 14.36;

[0163] MS (ESI) m / z 376.4 (M+H) + , calcd. for C 25 H 29 NO2 m / z = 375.2.

[0164] Compound i was a light yellow powder with a conversion of 75.4%. The spectral data of the obtained compound i are as follows:

[0165] 1H NMR (600 MHz, CDC13): δ 8.69 (s, 2H), 7.46 (s, 2H), 7.35 (s, 1H, =CH-20), 5.76 (s, 1H, =CH-4), 2.92 (d, J = 10.0 Hz, 1H), 2.54-2.41 (m, 3H), 2.39-2.34 (m, 2H), 2.08-2.00 (m, 3H), 1.88 (d, J = 10.4 Hz, 1H), 1.77-1.71 (m, 2H), 1.57-1.51 (m, 1H), 1.46-1.38 (m, 2H), 1.25 (s, 3H, CH3), 1.17 (d, J = 13.4 Hz, 1H), 1.07 (dd, J = 11.3, 2.9 Hz, 1H), 1.02 (s, 3H, CH3);

[0166] 13 C NMR (150 MHz, CDC13): δ 208.06, 199.30, 169.79, 148.97, 144.47, 141.36, 129.80, 124.50, 100.12, 53.91, 48.77, 47.61, 38.82, 35.79, 34.96, 34.03, 32.59, 31.43, 31.09, 29.84, 20.43, 17.53, 14.44;

[0167] MS (ESI) m / z 376.4 (M+H) + , calcd. for C 25 H 29 NO2 m / z = 375.2.

[0168] Compound j was obtained as a pale yellow powder with a conversion of 93.5%. The spectral data of the obtained compound j are as follows:

[0169] 1H NMR (600 MHz, CDC13): δ 7.45 (d, J = 8.5 Hz, 2H), 7.38 (s, 2H), 7.37 (s, 1H, =CH-20), 5.75 (s, 1H, =CH-4), 2.86 (dd, J = 15.7, 6.4, 1H), 2.50 - 2.40 (m, 3H), 2.38-2.32 (m, 2H), 2.09-2.03 (m, 2H), 2.01 - 1.96 (m, 1H), 1.89-1.83 (m 1H), 1.74-1.70 (m, 2H), 1.57-1.50 (m, 1H), 1.43 - 1.34 (m, 2H), 1.24 (s, 3H, CH3), 1.18-1.11 (m, 1H), 1.06-1.02 (m, 1H), 1.00 (s, 3H, CH3); 13 C NMR (150 MHz, CDC13): δ 208.98, 199.44, 170.20, 136.10, 135.39, 134.03, 132.18, 131.57, 129.12, 124.37, 53.92, 49.01, 47.42, 38.81, 35.73, 34.90, 34.02, 32.64, 31.44, 31.04, 29.28, 20.43, 17.50, 14.50;

[0170] MS (ESI) m / z 409.4 (M+H) + , calcd. for C 26 H 29 Cl02 m / z = 408.2.

[0171] Compound k was a light yellow powder with a conversion rate of 73.7%. The spectral data of the obtained compound k are as follows:

[0172] 1 H NMR (600 MHz, CDC13): δ 7.56 - 7.48 (m, 2H), 7.38 (s, 1H, =CH-20), 7.15-7.05 (m, 2H), 5.73 (s, 1H, =CH-4), 2.84 (dd, J = 15.6, 6.4 Hz, 1H), 2.48 - 2.32 (m, 5H), 2.10 - 1.93 (m, 4H), 1.88-1.81 (m, 1H), 1.74 - 1.66 (m, 2H), 1.55 - 1.49 (m, 1H), 1.40 - 1.33 (m, 2H), 1.22 (s, 3H, CH3), 1.16 - 1.11 (m, 1H), 0.98 (s, 3H, CH3); 13C NMR (150 MHz, CDC13): δ 209.01, 199.36, 170.20, 135.12, 132.29, 132.24, 131.73, 124.26, 116.01, 115.87, 53.85, 48.99, 47.32, 38.74, 35.66, 34.82, 33.95, 32.58, 31.38, 30.98, 29.14, 20.37, 17.42, 14.44;

[0173] MS (ESI) m / z 393.4 (M+H) + , calcd. for C 26 H 29 FO2 m / z = 392.5.

[0174] Compound 2b was a light yellow powder with a conversion rate of 48.0%. The spectral data of the obtained compound 2b were as follows:

[0175] 1 H NMR (600 MHz, CDC13): δ 7.46 (dd, J = 6.0, 3.1 Hz, 1H), 7.41 - 7.35 (m, 3H), 5.73 (s, 1H, =CH-4), 5.42 (s, 2H, NH2), 2.49 - 2.36 (m, 5H), 2.35 - 2.25 (m, 2H), 2.20 (d, J = 11.8 Hz, 1H), 2.07 - 2.03 (m, 1H), 1.90 - 1.84 (m, 2H), 1.80 - 1.71 (m, 2H), 1.65 - 1.55 (m, 3H), 1.24 (s, 3H, CH3), 1.09 (s, 3H, CH3), 1.08 - 1.05 (m, 1H);

[0176] 13 C NMR (150 MHz, CDC13): δ 199.37, 183.39, 170.41, 161.78, 159.68, 136.60, 132.15, 130.34, 130.32, 129.88, 127.04, 124.15, 122.43, 54.36, 54.07, 46.07, 38.75, 35.62, 34.38, 33.92, 32.58, 31.39, 29.71, 28.19, 20.55, 17.32, 17.01;

[0177] MS (ESI) m / z 448.5 (M+H) + , calcd. for C 27 H 30 ClN3O m / z = 447.2.

[0178] Compound 2c was obtained as a light yellow powder with a conversion rate of 40.4%. The spectral data of the obtained compound 2c are as follows:

[0179] 1 H NMR (600 MHz, CDC13): δ 7.58 (t, J = 7.3 Hz, 1H), 7.43 (dd, J = 13.4, 6.6 Hz, 1H), 7.26 (t, J = 7.5 Hz, 1H), 7.17 - 7.12 (m, 1H), 5.73 (s, 1H, =CH-4), 5.38 (s, 2H, NH2), 2.53 - 2.28 (m, 7H), 2.19 (d, J = 12.0 Hz, 1H), 2.07 - 2.03 (m, 1H), 1.94 - 1.87 (m, 2H), 1.77 - 1.70 (m, 2H), 1.65 - 1.52 (m, 3H), 1.25 (s, 3H, CH3), 1.11 (s, 3H, CH3), 1.08 - 1.06 (m, 1H);

[0180] 13 C NMR (150 MHz, CDC13): δ 198.36, 182.39, 169.46, 161.07, 159.63, 157.97, 130.34, 129.95, 123.47, 123.11, 123.11, 121.55, 115.08, 53.35, 53.04, 45.00, 37.73, 34.58, 33.37, 32.90, 31.65, 30.38, 28.68, 27.49, 19.52, 16.30, 16.00;

[0181] MS (ESI) m / z 432.5 (M+H) + , calcd for C 27 H 30 FN3O m / z = 431.2.

[0182] Compound 2d was obtained as a light yellow powder with a conversion rate of 56.7%. The spectral data of the obtained compound 2d are as follows:

[0183] 1H NMR (600 MHz, CDC13): δ 7.93 (d, J = 8.1 Hz, 2H), 7.74 (d, J = 8.2 Hz, 2H), 5.75 (s, 1H, =CH-4), 5.54 (s, 2H, NH2), 2.79-2.67 (m, 2H), 2.47-2.31 (m, 4H), 2.23-2.20 (m, 1H), 2.07-2.04 (m, 1H), 1.99-1.92 (m, 2H), 1.80-1.73 (m, 2H), 1.71-1.62 (m, 2H), 1.59-1.54 (m, 2H), 1.25 (s, 3H), 1.13 (s, 3H, CH3), 1.10-1.07 (m, 1H, CH3);

[0184] 13 C NMR (150 MHz, CDC13): δ 199.41, 170.20, 161.74, 161.72, 129.88, 128.97, 126.73, 126.70, 126.68, 124.40, 120.48, 54.96, 54.13, 46.11, 38.87, 35.74, 34.54, 34.04, 32.76, 32.66, 31.54, 29.84, 29.75, 20.64, 17.46, 17.16;

[0185] MS (ESI) m / z 482.5 (M+H) + , calcd. for C 28 H 30 F3N3O m / z = 481.2.

[0186] Compound 2e, as a light yellow powder, conversion rate was 46.8%. The spectral data of the resulting compound 2e are as follows:

[0187] 1 H NMR (600 MHz, CDC13): δ 7.76 (d, J = 7.8 Hz, 1H), 7.63 (t, J = 7.4 Hz, 1H), 7.56 (t, J = 7.7 Hz, 1H), 7.34 (d, J = 7.5 Hz, 1H), 5.73 (s, 1H, =CH-4), 5.41 (s, 2H, NH2), 2.45-2.34 (m, 3H), 2.32-2.20 (m, 4H), 2.07-2.04 (m, 1H), 1.85-1.72 (m, 4H), 1.71-1.64 (m, 1H), 1.63-1.55 (m, 3H), 1.24 (s, 3H, CH3), 1.11-1.08 (m, 1H), 1.07 (s, 3H, CH3);

[0188] 13 C NMR (150 MHz, CDC13): δ 199.35, 170.29, 161.04, 131.91, 129.71, 129.08, 128.19, 126.69, 126.65, 124.66, 124.17, 121.91, 54.44, 54.02, 46.18, 38.73, 35.61, 34.34, 33.92, 32.52, 31.34, 29.71, 27.66, 20.53, 17.31, 16.98;

[0189] MS (ESI) m / z 482.5 (M+H) + , calcd. for C 28 H 30 F3N3O m / z = 481.2.

[0190] Compound 2f was obtained as an off-white powder with a conversion rate of 43.6%. The spectral data of the obtained compound 2f are as follows:

[0191] 1 H NMR (600 MHz, CDC13): δ 7.76 (d, J = 8.5 Hz, 2H), 7.39 - 7.30 (m, 2H), 5.75 (s, 1H, =CH-4), 5.40 (s, 2H, NH2), 2.77 (d, J = 5.1 Hz, 1H), 2.68 (dd, J = 14.3, 11.9 Hz, 1H), 2.53 (s, 3H, SCH3), 2.47 - 2.32 (m, 4H), 2.20-2.18 (m, 1H), 2.08-2.04 (m, 1H), 1.99 (dd, J = 7.9, 5.0 Hz, 2H), 1.94 - 1.90 (m, 1H), 1.77-1.72 (m, 2H), 1.67-1.61 (m, 2H), 1.57 - 1.52 (m, 2H), 1.25 (s, 3H, CH3), 1.11 (s, 3H, CH3);

[0192] 13 C NMR (150 MHz, CDC13): δ 199.48, 170.43, 129.47, 128.98, 128.93, 125.94, 124.35, 123.77, 121.48, 119.74, 97.27, 54.99, 54.17, 45.93, 38.89, 35.73, 34.54, 34.05, 32.82, 32.72, 31.56, 29.84, 20.67, 17.47, 17.12, 15.41;

[0193] MS (ESI) m / z 460.5 (M+H) + , calcd. for C 28 H 33 N3OS m / z = 459.2.

[0194] Compound 2g as a light yellow powder with a conversion rate of 90.8%. The spectral data of the obtained compound 2g are as follows:

[0195] 1 H NMR (600 MHz, CDC13): δ 8.70 (dd, J = 4.7, 0.8 Hz, 1H), 8.15 (d, J = 7.9 Hz, 1H), 7.79 (td, J = 7.7, 1.8 Hz, 1H), 7.31 (ddd, J = 7.5, 4.8, 1.1 Hz, 1H), 5.74 (s, 1H, =CH-4), 5.24 (s, 2H, NH2), 3.19 (dd, J = 15.4, 6.4 Hz, 1H), 2.73 (dd, J = 15.4, 12.2 Hz, 1H), 2.49 - 2.39 (m, 2H), 2.37-2.30 (m, 2H), 2.20 - 2.16 (m, 1H), 2.06 - 2.01 (m, 2H), 1.92-1.87 (m, 1H), 1.77 - 1.69 (m, 2H), 1.68 - 1.56 (m, 2H), 1.55 - 1.48 (m, 2H), 1.24 (s, 3H, CH3), 1.16 - 1.10 (m, 1H), 1.06 (s, 3H, CH3);

[0196] 13 C NMR (150 MHz, CDC13): δ 199.59, 184.78, 170.90, 162.26, 158.19, 156.38, 149.19, 136.69, 124.17, 124.06, 123.16, 121.55, 54.64, 54.24, 45.58, 38.87, 35.69, 34.54, 34.04, 32.87, 31.61, 30.17, 29.80, 20.68, 17.42, 17.14;

[0197] MS (ESI) m / z 415.5 (M+H) + , calcd. for C 26 H 30 N4O m / z = 414.2.

[0198] Compound 2h as a light yellow powder with a conversion rate of 80.9%. The spectral data of the obtained compound 2h are as follows:

[0199] 1 H NMR (600 MHz, CDC13): δ 9.01 (s, 1H, N=CH), 8.67 (d, J = 3.8 Hz, 1H), 8.17 (dt, J = 7.9, 1.9 Hz, 1H), 7.42 (dd, J = 7.8, 4.8 Hz, 1H), 5.74 (s, 1H, =CH-4), 5.36 (s, 2H, NH2), 2.79 (dd, J = 14.2, 6.5 Hz, 1H), 2.70 (dd, J = 14.2, 11.9 Hz, 1H), 2.49 - 2.41 (m, 2H), 2.38 - 2.31 (m, 2H), 2.22 - 2.17 (m, 1H), 2.07 - 2.03 (m, 1H), 1.98 - 1.91 (m, 2H), 1.78 - 1.70 (m, 2H), 1.64 (dd, J = 13.0, 3.9 Hz, 1H), 1.57-1.53 (m, 2H), 1.43-1.39 (m, 1H), 1.24 (s, 3H, CH3), 1.11 (s, 3H, CH3), 1.11-1.07 (m, 1H);

[0200] 13 C NMR (150 MHz, CDC13): δ 199.52, 184.25, 170.47, 162.47, 157.72, 150.51, 149.49, 136.01, 133.64, 124.30, 123.68, 120.25, 55.00, 54.13, 45.87, 38.84, 35.68, 34.45, 34.02, 32.67, 31.50, 30.28, 29.81, 20.62, 17.42, 17.14;

[0201] MS (ESI) m / z 415.5 (M+H) + , calcd. for C 26 H 30 N4O m / z = 414.2.

[0202] Compound 2i was obtained as a light yellow powder with a conversion rate of 73.9%. The spectral data of the obtained compound 2i are as follows:

[0203] 1H NMR (600 MHz, CDC13): δ 8.76 (s, 2H), 7.73 (s, 2H), 5.75 (s, 1H, =CH-4), 5.30 (s, 2H, NH2), 2.80 (dd, J = 14.3, 6.5 Hz, 1H), 2.73 - 2.67 (m, 1H), 2.48 - 2.41 (m, 2H), 2.36 (dd, J = 22.5, 9.3 Hz, 2H), 2.20 (dd, J = 9.8, 3.3 Hz, 1H), 2.09 - 2.05 (m, 1H), 2.01 - 1.92 (m, 2H), 1.79 - 1.61 (m, 4H), 1.59 - 1.53 (m, 2H), 1.44-1.40 (m, 1H), 1.24 (s, 3H, CH3), 1.11 (s, 3H, CH3);

[0204] 13 C NMR (150 MHz, CDC13): δ 199.37, 184.75, 170.19, 162.25, 157.35, 149.93, 145.21, 124.48, 124.26, 120.68, 54.80, 54.02, 45.79, 38.75, 35.60, 34.39, 33.93, 32.65, 32.55, 31.43, 30.19, 29.72, 17.33, 17.08;

[0205] MS (ESI) m / z 415.5 (M+H) + , calcd. for C 26 H 30 N4O m / z = 414.2.

[0206] Compound 2j was obtained as an off-white powder with a conversion rate of 40.6%. The spectral data of the obtained compound 2j are as follows:

[0207] 1H NMR (600 MHz, CDC13): δ 7.84 (d, J = 8.8 Hz, 2H), 7.01 (d, J = 8.8 Hz, 2H), 5.75 (s, 1H, =CH-4), 5.34 (s, 2H, NH2), 3.87 (s, 3H, OCH3), 2.80 (dd, J = 14.3, 6.5 Hz, 1H), 2.69 (dd, J = 14.1, 11.9 Hz, 1H), 2.49 - 2.32 (m, 4H), 2.22 - 2.18 (m, 1H), 2.08 - 2.04 (m, 1H), 2.01 - 1.98 (m, 1H), 1.95-1.90 (m, 1H), 1.80 - 1.72 (m, 2H), 1.67 - 1.52 (m, 4H), 1.25 (s, 3H, CH3), 1.12 (s, 3H, CH3), 1.10 - 1.07 (m, 1H);

[0208] 13 C NMR (150 MHz, CDC13): δ 199.42, 194.34, 170.27, 161.72, 130.40, 124.38, 119.30, 114.31, 107.23, 100.12, 55.60, 54.92, 54.11, 46.13, 38.88, 35.73, 34.54, 34.05, 32.69, 31.54, 30.08, 29.84, 20.64, 17.47, 17.08;

[0209] MS (ESI) m / z 444.6 (M+H) + , calcd. for C 28 H 33 N3O2 m / z = 443.3.

[0210] Compound 2k was a light yellow powder with a conversion rate of 94.5%. The spectral data of the obtained compound 2k were as follows:

[0211] 1H NMR (600 MHz, CDC13): δ 7.03 (s, 2H, H-Ph), 5.74 (s, 1H, =CH-4), 5.35 (s, 2H, NH2), 3.92 (s, 6H), 3.89 (s, 3H), 2.81 (dd, J = 14.1, 6.4 Hz, 1H), 2.67 (dd, J = 14.1, 11.9 Hz, 1H), 2.48 - 2.31 (m, 4H), 2.21 - 2.15 (m, 1H), 2.07-2.04 (m, 1H), 1.97-1.89 (m, 2H), 1.79 - 1.48 (m, 6H), 1.24 (s, 3H, CH3), 1.12 (s, 3H, CH3), 0.88-0.85 (m, 1H), 1.09-1.06 (m, 1H);

[0212] 13 C NMR (150 MHz, CDC13): δ 199.40, 184.08, 170.33, 162.07, 153.19, 139.56, 124.48, 124.22, 124.00, 119.52, 105.79, 60.98, 56.30, 54.90, 54.01, 45.78, 38.76, 35.58, 34.35, 33.93, 32.72, 32.58, 31.45, 29.90, 29.72, 20.52, 17.32, 17.02;

[0213] MS (ESI) m / z 504.6 (M+H) + , calcd. for C 30 H 37 N3O4 m / z = 503.3.

[0214] Compound 3b was obtained as a light yellow powder with a conversion rate of 57.8%. The spectral data of the obtained compound 3b are as follows:

[0215] 1H NMR (600 MHz, CDC13): δ 7.44 (dd, J = 13.3, 6.0 Hz, 1H), 7.31 (td, J = 7.4, 1.6 Hz, 1H), 7.21 (t, J = 7.5 Hz, 1H), 7.15 (t, J = 9.0 Hz, 1H), 5.98 (s, 1H, N-C=CH), 5.74 (s, 1H, =CH-4), 3.26 (s, 3H, NCH3), 2.75 - 2.57 (m, 2H), 2.47 - 2.40 (m, 2H), 2.37 - 2.29 (m, 2H), 2.11 - 2.04 (m, 2H), 1.96-1.92 (m, 1H), 1.88-1.83 (m, 1H), 1.76 - 1.66 (m, 4H), 1.52 (dd, J = 12.7, 3.5 Hz, 1H), 1.47 - 1.41 (m, 2H), 1.23 (s, 3H, CH3), 1.11 (dd, J = 13.1, 3.0 Hz, 1H), 1.05 - 1.02 (m, 1H), 0.98 (s, 3H, CH3);

[0216] 13 C NMR (150 MHz, CDC13): δ 199.71, 171.30, 161.04, 159.38, 131.81, 131.79, 131.60, 124.74, 124.72, 124.08, 116.22, 116.08, 54.29, 53.26, 44.31, 38.89, 35.82, 35.36, 34.30, 34.10, 32.93, 31.59, 29.84, 23.55, 20.84, 17.55, 17.22;

[0217] MS (ESI) m / z 492.6 (M+H) + , calcd. for C 29 H 34 FN3OS m / z = 491.2.

[0218] Compound 4b, as a light yellow powder, conversion rate was 95.5%. The spectral data of the obtained compound 4b are as follows:

[0219] 1H NMR (600 MHz, CDC13): δ 8.45 (s, 1H, N=CH), 7.00 (t, J = 8.5 Hz, 1H), 6.95 (t, J = 8.5 Hz, 2H), 5.75 (s, 1H, =CH-4), 2.66-2.58 (m, 2H), 2.46-2.41 (m, 2H), 2.38-2.30 (m, 3H), 2.11-2.02 (m, 3H), 1.96-1.93 (m, 1H), 1.89-1.85 (m, 1H), 1.76-1.71 (m, 3H), 1.55-1.43 (m, 4H), 1.23 (s, 3H, CH3), 1.03 (s, 3H, CH3);

[0220] 13 C NMR (150 MHz, CDC13): δ 199.70, 170.96, 162.57, 160.91, 153.77, 131.68, 124.18, 112.16, 112.02, 54.03, 52.79, 44.52, 38.82, 35.81, 35.27, 34.07, 33.74, 32.83, 31.48, 28.12, 23.32, 20.79, 17.52, 16.78;

[0221] MS (ESI) m / z 425.4 (M+H) + , calcd. for C 26 H 30 F2N2O m / z = 424.2.

[0222] Compound 4c was a light yellow powder with a conversion rate of 84.1%. The spectral data of the obtained compound 4c were as follows:

[0223] 1H NMR (600 MHz, CDC13): δ 9.96 (s, 1H, NH), 7.85 (d, J = 7.5 Hz, 1H), 7.30 (t, J = 7.6 Hz, 1H), 6.99 (t, J = 7.6 Hz, 1H), 6.93 (d, J = 7.5 Hz, 1H), 5.74 (s, 1H, =CH-4), 2.56 (dd, J = 19.5, 8.8 Hz, 1H), 2.49 - 2.27 (m, 6H), 2.19 (d, J = 10.9 Hz, 1H), 2.05 (dd, J = 9.4, 3.6 Hz, 1H), 1.93 - 1.83 (m, 2H), 1.77 - 1.69 (m, 3H), 1.60 - 1.53 (m, 2H), 1.47 - 1.41 (m, 1H), 1.26 (d, J = 7.2 Hz, 1H), 1.21 (s, 3H, CH3), 1.12 - 1.08 (m, 1H), 1.06 (s, 3H, CH3);

[0224] 13 C NMR (150 MHz, CDC13): δ 199.81, 178.49, 171.02, 166.46, 146.95, 143.65, 133.07, 128.61, 124.07, 122.92, 117.13, 111.28, 53.78, 52.65, 45.04, 38.71, 35.65, 35.08, 33.96, 33.72, 32.68, 31.26, 28.10, 23.27, 20.65, 17.40, 16.65;

[0225] MS (ESI) m / z 430.4 (M+H) + , calcd. for C 27 H 31 N3O2 m / z = 429.2.

[0226] Compound 4d was converted with 88.7% yield. The spectral data of the obtained compound 4d are as follows:

[0227] 1H NMR (600 MHz, CDC13): δ 8.23 (s, 1H, N=CH), 7.61 (d, J = 8.4 Hz, 2H), 7.53 (d, J = 8.4 Hz, 2H), 5.75 (s, 1H, =CH-4), 2.69-2.59 (m, 2H), 2.47-2.40 (m, 3H), 2.08-2.04 (m, 2H), 1.99-1.93 (m, 2H), 1.88-1.85 (m, 2H), 1.74-1.70 (m, 3H), 1.57-1.54 (m, 2H), 1.51-1.44 (m, 3H), 1.23 (s, 3H, CH3), 1.01 (s, 3H, CH3);

[0228] 13 C NMR (150 MHz, CDC13): δ 199.63, 182.97, 161.35, 156.50, 132.26, 132.06, 130.08, 129.58, 124.19, 54.09, 52.84, 44.50, 38.83, 35.84, 35.30, 34.07, 33.83, 32.83, 31.52, 29.84, 28.08, 23.34, 17.53, 16.81;

[0229] MS (ESI) m / z 467.5 (M+H) + , calcd. for C 26 H 31 BrN2O m / z = 466.2.

[0230] Example 6

[0231] This example determined the in vitro cytotoxic activity of all steroidal derivatives a-4d. The MTT colorimetric method was used to determine the inhibitory effect of the proliferation of human melanoma cell line A875, hepatoma cell line Huh7, and the antitumor drug 5-FU as a positive control, and the experimental results are shown in Table 1 below:

[0232] Table 1 In vitro cytotoxic activity of novel steroidal derivatives on human tumor cells

[0233]

[0234]

[0235] a IC 50 – Concentration at which the inhibition rate is 50%;

[0236] b Abbreviations: A875 - human melanoma cell line; Huh-7 - human hepatoma cell line;

[0237] c FU-5-fluorouracil, as a positive control.

[0238] As shown in Table 1, most of the compounds have obvious inhibitory activity on the tested cancer cells, especially the compounds h, 2b and 4a, which have significant anticancer activity on both A875 and Huh-7 cancer cells, with IC 50 values of 2.15±0.41, 1.34±0.08 and 1.80±0.71 μg / mL, and 4.90±1.82, 3.19±1.77 and 2.42±0.44 μg / mL, respectively, which are significantly better than the positive control drug 5-FU, showing a more obvious application prospect.

[0239] The content of the present application is not limited to the above-mentioned embodiments, and any improvement or replacement that can be conceived by those skilled in the art without departing from the essential content of the present application falls within the scope of the present application.

Claims

1. The application of a sterone derivative in the preparation of drugs that inhibit cancer cells; The sterone derivative is compound h; The cancer cells are human melanoma cell line A875 and / or liver cancer cell line Huh7; The molecular structure of compound h is shown below: 。 2. The application according to claim 1, characterized in that: The method for preparing the sterone derivative includes the following steps: 1) Activation of bacterial strain: Mycobacterium neoaurum Tsukamura 3503 was streaked onto solid LB medium and incubated at 37°C for 3-5 days until single colonies grew on the solid LB medium; the Mycobacterium neoaurum Tsukamura 3503 was deposited at the China Center for Type Culture Collection, accession number: CCTCC AA 2018059; 2) Preparation of seed culture: Pick the single-clone strain activated on the solid LB medium above and place it in a small volume of liquid LB medium test tube. Incubate at 150-250 rpm and 30-37℃ for 2-4 days until OD. 600nm The inoculum was 1.5-2.0, transferred to large volume liquid LB medium, and cultured at 150-250 rpm and 30-37℃ for 2-4 days. Then, it was transferred to MnCO1 seed medium at an inoculum rate of 10-20% V / V for expansion culture and cultured at 150-250 rpm and 30-37℃ for 2-4 days to obtain the seed culture. 3) Preparation of Sterol Derivative 1: Sterol Derivative 1 was prepared by chemical synthesis using dehydroepiandrosterone (DHEA) as a raw material; the chemical structural formula of Sterol Derivative 1 is as follows: , where R is ; 4) Microbial cell catalytic reaction: Take the seed liquid prepared in step 2) into an Erlenmeyer flask of MnCO2G fermentation medium, with a filling coefficient of 10-30% V / V, an inoculum size of 5-20% V / V, 0.2-1 mmol of sterol derivative 1,2-hydroxy-β-cyclodextrin 0.5-2 wt%, and culture at 150-250 rpm and 25-40℃ for 2-4 days. After fermentation, add 2-4 times the volume of ethyl acetate and stir for 3-6 hours. Then extract, separate, concentrate and purify by silica gel column chromatography to obtain the sterone derivative. The liquid LB medium consisted of: 10g tryptone, 5g yeast extract, and 10g NaCl. Solid LB medium is prepared by adding 13g of agar to 1L of liquid LB medium, autoclaving at 121℃ for 30min, cooling and storing at 4℃ for later use. The MnC01 seed culture medium is as follows: 20g glycerol, 2g citric acid, 0.05g ferric ammonium citrate, 0.5g K2HPO4, 0.5g MgSO4·7H2O, 2g (NH4)2SO4, and 2g NaNO3. Weigh the above reagents and dissolve them in double-distilled water and bring the volume to 1L. The pH is 7.

5. Autoclave at 121℃ for 30 minutes and store at room temperature for later use. The fermentation medium for MnCO2G is as follows: 10g glucose, 2g citric acid, 0.05g ferric ammonium citrate, 0.5g K2HPO4, 0.5g MgSO4·7H2O, 2g (NH4)2SO4, and 2g NaNO3. Weigh out the above reagents and dissolve them in double-distilled water and bring the volume to 1L. The pH is 7.

5. Sterilize by autoclaving at 115℃ for 30 minutes and store at room temperature for later use.

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