Testosterone spiro compound and nano preparation, preparation method and application thereof

Abstract

The invention relates to a testosterone spiro compound, a nano preparation thereof, a preparation method and application, the testosterone spiro compound is a testosterone spiro dimer, and the structural formula of the testosterone spiro compound is shown as a formula (Ia). According to the invention, the testosterone spiro compound (I) and the hydrate thereof are used as active ingredients, and are matched with a prodrug, a pharmaceutically acceptable salt or a pharmaceutically acceptable carrier to form a pharmaceutical composition. The testosterone spiro compound (I) has a good proliferation inhibition effect on various tumor cells (breast cancer cells: MCF7; non-small cell lung cancer cells: HCC827R and HCC827S; colorectal cancer cells: HCT116, HCT8, HCT8 / 5FU, HT29, CT26, SW620), and especially have the best effect on human colorectal cancer cells. # imgabs0 #

Description

Technical Field

[0001] The invention relates to a testosterone spirocyclic compound and a nano preparation thereof, as well as a preparation method and application thereof, and belongs to the technical field of steroid compounds. Background Art

[0002] Steroid drugs are widely used in the pharmaceutical industry and have a variety of pharmacological activities. Testosterone (Ⅱ), also known as testosterone, testosterone, and testosterone, is a steroid hormone secreted by the male testicles or female ovaries. The adrenal glands also secrete a small amount of testosterone. In addition to its androgenic effects, testosterone also has a significant effect of promoting protein synthesis, also known as anabolic effects. Therefore, it has the effects of maintaining muscle strength and quality, maintaining bone density and strength, refreshing and improving physical fitness, increasing red blood cell production, promoting angiogenesis, and antagonizing excessive estrogen. The molecular formula of testosterone is C 19 H 28 O2, whose structural formula is shown in formula (II):

[0003]

[0004] So far, a variety of testosterone derivatives have been invented and used clinically. It can be predicted that new testosterone derivatives are likely to have pharmacological activity, and further research and development of new and effective therapeutic drugs is of great significance. Summary of the invention

[0005] In view of this, in view of the above-mentioned defects existing in the existing anti-tumor technology, the present invention first provides a new testosterone spiro compound, and screens the single concentration of tumor cell proliferation inhibition. The testosterone spiro compound has a good proliferation inhibition effect on human colorectal cancer cells.

[0006] In order to achieve the above object, the present invention provides a testosterone spirocyclic compound, which is a testosterone derivative and a hydrate, a prodrug and a pharmaceutically acceptable salt thereof, and its structural formula is shown in formula (I):

[0007]

[0008] Wherein: R is hydrogen, succinic acid monoester, maleic acid monoester, phosphate ester.

[0009] When R is hydrogen, the structural formula is as shown in formula (Ia).

[0010]

[0011] In the preferred technical scheme of the testosterone spiro compound of the present invention, preferably, the testosterone spiro compound is a chiral S-type of the central carbon of the spiro ring.

[0012] The experiment showed that: through single concentration screening for inhibition of tumor cell proliferation, the spirocyclic compound (Ia) had a good proliferation inhibition effect on human colorectal cancer cells.

[0013] According to a second aspect of the present invention, the present invention provides a nanoformulation (ie, a pharmaceutical composition), wherein the nanoformulation comprises the testosterone spirocyclic compound.

[0014] In the preferred technical solution of the nanoformulation of the present invention, preferably, the testosterone spiro compound is a nanoliposome of the testosterone spiro compound and a nanoemulsion of the testosterone spiro compound.

[0015] In the preferred technical solution of the nanoformulation of the present invention, preferably, the particle size of the nanoliposome of the testosterone spiro compound is 184.2±10.8 nm, and the particle size of the nanoemulsion of the testosterone spiro compound is 225.4±5.3 nm.

[0016] In the preferred technical solution of the nanoformulation of the present invention, preferably, the effective dose of the nanoliposome of the testosterone spiro compound is 50 μM, and the effective dose of the nanoemulsion of the testosterone spiro compound is 5 μM.

[0017] According to the third aspect of the present invention, the present invention also provides a method for preparing a testosterone spirocyclic compound as shown in formula (Ia), the preparation method comprising: dissolving 6-(N-methyl-N-phenyl)-aminomethyl-17β-hydroxy (ester) androst-4-ene-3-one (IV) with dichloromethane, dripping trifluoroacetic acid, then keeping the reaction warm until the reaction is completed, removing most of the trifluoroacetic acid under reduced pressure, extracting and washing, and concentrating under reduced pressure to dryness to obtain a crude product of a testosterone spirocyclic compound (Ia). The crude product can be purified by column chromatography and the like to obtain a fine testosterone spirocyclic compound (Ia). The above reaction process can be expressed as:

[0018]

[0019] In addition, in the structural formula shown in formula (I), the preparation method of the compound in which R is respectively a succinic acid monoester includes: adding dry dichloromethane to a testosterone spirocyclic compound (Ia), slowly adding succinic anhydride under stirring, then adding triethylamine, heating to boiling, and after thin layer chromatography shows that there is no testosterone spirocyclic compound (Ia), the reaction is complete and cooled to room temperature. Dichloromethane is evaporated to obtain a viscous liquid, water is added, and extracted with ethyl acetate. The organic phases are combined, dried over anhydrous sodium sulfate, filtered, and the filtrate is evaporated to remove the solvent, and recrystallized with petroleum ether-acetone. Vacuum drying at room temperature gives a white solid.

[0020] And, in the structural formula shown in formula (I), the preparation method of the compound of R being respectively maleic acid monoester includes: add dry ethylene dichloride to testosterone spirocyclic compound (Ia), slowly add maleic anhydride under stirring, then add triethylamine, be heated to boiling, after thin layer chromatography shows no testosterone spirocyclic compound (Ia), react completely, cool to room temperature. Boil off ethylene dichloride, obtain viscous liquid, add water, extract with ethyl acetate. Combine organic phase, add anhydrous sodium sulfate drying, filter, filtrate evaporates solvent, recrystallizes with petroleum ether-acetone. Vacuum drying at room temperature obtains white solid.

[0021] Furthermore, in the structural formula shown in formula (I), the preparation method of the compound in which R is respectively a phosphate ester comprises: adding dry acetone to the testosterone spirocyclic compound (Ia), cooling to under stirring, and simultaneously dropping phosphorus oxychloride and pyridine. The temperature is kept ≤15°C during the dropping process, and after the dropping is completed, the temperature is kept at 5-10°C. After the thin layer chromatography shows that there is no testosterone spirocyclic compound (Ia), the reaction is completed. Add purified water to another reaction bottle, start stirring, and cool to below 30°C. Add the reaction solution of the previous step to the hydrolysis kettle, and control the temperature not to exceed 35°C. After the addition is completed, keep warm at 30-40°C until the thin layer chromatography shows that the hydrolysis is complete. Control the internal temperature ≤35°C, slowly drop liquid alkali into the reaction bottle, and adjust the pH of the reaction solution to 3-4. After the adjustment is completed, add activated carbon and stir at room temperature. Filter to remove the carbon layer. The filtrate is concentrated under reduced pressure. Dilute with a small amount of anhydrous ethanol and dry with anhydrous sodium sulfate. Filter, let the filtrate stand at 0-5°C for crystallization, filter and dry to obtain white crystals.

[0022] According to the fourth aspect of the present invention, the present invention also provides a use of a testosterone spirocyclic compound in the preparation of a drug for preventing tumors.

[0023] Experiments show that the testosterone spirocyclic compound provided by the present invention has a good proliferation inhibition effect on various tumor cells. The experiment adopts the internationally used CCK8 method for detection.

[0024] The present invention prepares a nanometer preparation of the compound, comprising a testosterone spirocyclic compound and / or its hydrate, a prodrug and a pharmaceutically acceptable salt as an active ingredient. The testosterone spirocyclic compound is prepared with a pharmaceutical excipient and a pharmaceutically or physiologically acceptable carrier to form any dosage form in pharmacy by a conventional preparation process.

[0025] The testosterone spiro compound of the present invention has low water solubility and poor oral bioavailability. To address this defect, succinic acid monoesters, maleic acid monoesters and phosphate esters of the compound of the present invention are prepared to increase the molecular polarity, thereby increasing its water solubility and bioavailability. Its preparation method is obtained by reacting a testosterone spiro compound (Ia) with a corresponding acid anhydride or an equivalent.

[0026] The testosterone spirocyclic compound (Ia) has a good proliferation inhibition effect on a variety of tumor cells (breast cancer cells: MCF7; non-small cell lung cancer cells: HCC827R and HCC827S; colorectal cancer cells: HCT116, HCT8, HCT8 / 5FU, HT29, CT26, SW620), especially for human colorectal cancer cells. The testosterone spirocyclic compound of the present invention has a strong ability to inhibit cancer cells, provides a lead compound for the development of new anti-tumor drugs, and is of great significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a LC-MS graph of the product prepared under the conditions of Example 2 of the present invention;

[0028] Figure 2 This is the NMR (hydrogen spectrum) of the product prepared under the conditions of Example 2 of the present invention;

[0029] Figure 3 The NMR (carbon spectrum) of the product prepared under the conditions of Example 2 of the present invention;

[0030] Figure 4 This is the NMR image (carbon spectrum, partially enlarged) of the product prepared under the conditions of Example 2 of the present invention;

[0031] Figure 5 This is the NMR image (DEPT135) of the product prepared under the conditions of Example 2 of the present invention;

[0032] Figure 6 This is the NMR image of the product prepared under the conditions of Example 2 of the present invention (DEPT135, partial enlargement);

[0033] Figure 7 The single crystal diffraction pattern of the product prepared under the conditions of Example 2 of the present invention;

[0034] Figure 8 is the inhibition rate of tumor cells at a single concentration of the product under the preparation conditions of Example 2 of the present invention;

[0035] Fig. 9 This is the inhibition curve of the product on HCT116 cells under the preparation conditions of Example 2 of the present invention;

[0036] Fig.10 This is the inhibition curve of the product on HT-29 cells under the preparation conditions of Example 2 of the present invention;

[0037] Fig.11 The inhibition rate of different dosage forms of nanoliposomes and nanoemulsions of testosterone spirocyclic compound (Ia) on related tumor cells. DETAILED DESCRIPTION

[0038] The present invention will be further described below in conjunction with embodiments and the accompanying drawings. The embodiments of the present invention are only used to illustrate the technical solutions of the present invention, but are not intended to limit the present invention.

[0039] Analytical instruments and equipment used in the examples: LC-MS / MS, ThermoLCQ-Fleet, column: Hypersil GOLD150*2.1, electrospray ionization source (ESI); nuclear magnetic resonance, AVANCED MXⅡⅠ400M (TMS internal standard, Bruker); high performance liquid chromatograph: Agilent Technologies 1200 Series; infrared spectrometer, NICOLET 360 FT-IR (Nicolet Instruments, USA).

[0040] Example 1: Preparation of 6-(N-methyl-N-phenyl)-aminomethyl-17β-hydroxyandrost-4-ene-3-one (IV)

[0041] In this embodiment, the preparation method of the raw material 6-(N-methyl-N-phenyl)-aminomethyl-17β-hydroxyandrost-4-ene-3-one (IV) can be described as follows:

[0042]

[0043] 15 g of raw material testosterone (II), 100 ml of anhydrous tetrahydrofuran, 15 ml of anhydrous ethanol, 15 ml of triethyl orthoformate, and 0.15 g of p-toluenesulfonic acid were stirred in a four-necked flask equipped with a thermometer and mechanical stirring at 40°C for 2 hours to obtain reaction solution 1 [reaction solution containing compound (III)].

[0044] Then, add 6 g of N-methylaniline and 8.5 g of paraformaldehyde to the above reaction solution 1, and continue to stir and react for 7 hours. Add triethylamine to neutralize the reaction solution. Evaporate the solvent tetrahydrofuran under reduced pressure below 50°C, add 30 ml of ethyl acetate, stir and dissolve at 50°C, cool to -10°C, and continue to stir for 5 hours. Filter and dry to obtain a pale yellow solid (IV), 18.5 g of crude product, with a yield of 87%.

[0045] Example 2: Preparation of testosterone spirocyclic compound (Ia)

[0046] 16.3 g of compound IV (0.04 mol) was dissolved in 120 ml of dichloromethane, and then 25 ml of trifluoroacetic acid was added dropwise at 5°C, the temperature was raised and stirred at 15°C for 20 hours, and then the temperature was raised to 20°C for reaction for 8 hours. Most of the trifluoroacetic acid was evaporated under reduced pressure. 150 ml of dichloromethane and 200 ml of water were added, and the layers were stirred. The organic layer was washed three times with deionized water. The organic layer was concentrated to dryness under reduced pressure to obtain 10.8 g of crude product [crude compound (Ia)] with a yield of 89.9%.

[0047] The crude product can be purified by column chromatography (mobile phase: ethyl acetate: petroleum ether = 1:1) and the like to obtain a fine product, which is a white solid compound (Ia) after purification.

[0048]

[0049] Structural characterization of compound (Ia): MS: 600.42

[0050]

[0051] 13 CNMR (101MHz,CDCl3)δ199.99(C2,C),196.29(C24,C),182.71(C26,C),166.98(C4,C),137.39(C3,C),122.65(C25,CH),81.68(C39,CH),81.49(C17,CH),55.42(C33,CH),50.52(C10,CH),50.10(C11,CH),48.53(C32,CH),42.88(C12,C34,C),42.66(C29,C,spirocyclic carbon),40.23(C27,C),39.23(C30,CH2),38.62(C5,C),38.27(C8,CH2),36.37( C35,CH2),36.34(C13,CH2),36.25(C28,CH2),35.93(C9,CH),34.87(C44,CH2),34. 43(C1,CH2),34.33(C23,CH2),33.85(C31,CH),33.77(C6,CH2),30.77(C7,CH),30. 40(C38,C16,CH2),24.88(C22,CH2),23.37(C15,CH2),23.23(C37,CH2),22.29(C42 ,CH3),20.77(C14,CH2),20.70(C36,CH2),18.24(C20,CH3),11.07(C43,C21,CH3).

[0052] Example 3: In vitro antitumor effect of testosterone spirocyclic compound (Ia)

[0053] Single-concentration screening of testosterone spirocyclic compound (Ia) on the inhibition of tumor cell proliferation. This experiment uses the internationally used CCK8 method for detection.

[0054] Experimental method: First, testosterone spirocyclic compound Ia was dissolved in a certain amount of NMP to prepare a 20mM stock solution, which was later diluted to different concentrations according to experimental needs. 0.25% trypsin containing EDTA was used to digest the 2-3 generations of tumor cells and counted by an automatic cell counter. Tumor cells were inoculated in a 96-well transparent cell culture plate at a number of 5000 cells per well and continued to be cultured for 24 hours. The drug was added to the experimental group at a final concentration of 50μM; the control group contained an equal amount of NMP; the blank group had the same composition as the control group but did not contain cells. After continuing to culture for 24 hours, the culture plate was removed; according to the instructions of the CCK-8 kit (Beijing Lanjieke Technology Co., Ltd.), 10μL CCK-8 reagent was added to each well, protected from light, and incubated in a cell culture incubator at 37°C for 1 hour, and then the absorbance value was detected at 562nm using an enzyme reader. The inhibitory rate (%) of the drug on the proliferation of colorectal cancer cells can be calculated by the following formula: [1-(OD T -OD B ) / (OD C -OD B )]×100%.OD T OD C and OD B They are the absorbance values ​​of the experimental group, the control group and the blank group respectively. The experimental results are shown in Table 1 below:

[0055] Table 1

[0056] HCT116 HT29 77.841590 94.496280 76.250240 94.344560 73.033930 93.995600 74.221840 94.101810

[0057] From Table 1 and Figure 8 , Fig. 9 and Fig.10 It can be seen that the IC value of testosterone spirocyclic compound Ia for the inhibition of proliferation of human colorectal cancer cell HCT116 is 50 The IC value for the inhibition of proliferation of human colon cancer cell HT29 is 8.23±0.91μM. 50 The experimental results show that the testosterone spirocyclic compound Ⅰa has a significant inhibitory effect on the proliferation of human colorectal cancer cells HCT116 and HT29.

[0058] Example 4: Preparation of Nanoliposomes of Testosterone Spirocyclic Compound (Ia)

[0059] The preparation process is as follows: weigh the nanoliposome prescription materials according to the prescription in Table 2 and place them in a vial, add 500 μL of anhydrous ethanol and stir to dissolve in a 65°C water bath. After the prescription materials and drugs are dissolved, open the system and continue stirring to evaporate 80% of the anhydrous ethanol. -1The sodium phosphate solution (5 mM, pH 7.4) preheated to the same temperature was injected into the membrane material at a speed of 5 mL. Stirred in a 65°C water bath for 20 min to obtain the initial liposome product. The initial product was subjected to ultrasonic dispersion treatment (power and time: 200 W × 2 min + 400 W × 6 min, working 1 s and resting 1 s), and then passed through 0.80 and 0.22 μm microporous filter membranes in sequence to obtain nanoliposomes of testosterone spirocyclic compound (Ia).

[0060] The experimental results showed that the particle size of the nanoliposomes of the testosterone spirocyclic compound (Ia) was 184.2±10.8 nm, the potential was -9.8±1.4 mV, and the drug encapsulation efficiency was 95.1±2.2%.

[0061] Table 2: Testosterone spirocyclic compound (Ia) nanoliposome formulation

[0062]

[0063] Example 5: Preparation of Nanoemulsion of Testosterone Spirocyclic Compound (Ia)

[0064] The preparation process is as follows: according to the prescription in Table 3, weigh the emulsion prescription materials and place them in a vial, stir and dissolve them in a 65°C water bath, and wait until the prescription materials and drugs are completely dissolved. -1 Sodium phosphate solution (5 mM, pH 7.4) preheated to 65°C was injected into the membrane material at a speed of 10 mL. Stirred in a 65°C water bath for 20 min to obtain a preliminary emulsion. The preliminary product was subjected to ultrasonic dispersion treatment (power and time: 200 W×2 min+400 W×6 min, working for 1 s and resting for 1 s), and then passed through 0.80 and 0.22 μm microporous filter membranes in sequence to obtain a nanoemulsion of testosterone spirocyclic compound (Ia).

[0065] The experimental results showed that the particle size of the nanoemulsion of the testosterone spirocyclic compound (Ia) was 225.4±5.3 nm, the potential was -8.6±1.8 mV, and the drug encapsulation efficiency was 98.6±0.7%.

[0066] Table 3: Testosterone spirocyclic compound (Ia) nanoemulsion formulation

[0067]

[0068] Example 6: In vitro antitumor effects of nanoliposomes and nanoemulsions of testosterone spirocyclic compound (Ia)

[0069] Single concentration screening of nanoliposomes and nanoemulsions of testosterone spirocyclic compound (Ia) on the inhibition of tumor cell proliferation. This experiment uses the internationally accepted CCK8 method for detection.

[0070] Experimental method: First, the testosterone spirocyclic compound (Ia) nanoliposomes and nanoemulsions were prepared at a drug concentration of 1mM, and then diluted to different concentrations according to experimental needs. 0.25% EDTA-containing trypsin was used to digest the 2-3 generations of tumor cells and counted by an automatic cell counter. Tumor cells were inoculated in a 96-well transparent cell culture plate at a number of 5000 cells per well and continued to be cultured for 24 hours. The drug was added to the experimental group at a final concentration of 50μM and 5μM; the control group contained an equal amount of phosphate buffer (5mM, pH7.4); the blank group had the same composition as the control group but did not contain cells. After continuing to culture for 24 hours, the culture plate was removed; according to the instructions of the CCK-8 kit (Beijing Lanjieke Technology Co., Ltd.), 10μL CCK-8 reagent was added to each well, protected from light, and incubated at 37°C in a cell culture incubator for 1 hour, and then the absorbance value was detected at 562nm using an enzyme reader. The inhibitory rate (%) of the drug on the proliferation of colorectal cancer cells can be calculated by the following formula: [1-(OD T -OD B ) / (OD C -OD B )]×100%.OD T OD C and OD B are the absorbance values ​​of the experimental group, control group and blank group respectively. Fig.11 shown.

[0071] The experimental results show that the nanoliposomes and nanoemulsions of the testosterone spiro compound (Ia) have a certain proliferation inhibitory effect on three types of tumor cells (breast cancer cells: MCF7; non-small cell lung cancer cells: HCC827R; colorectal cancer cells: HCT8 / 5FU) at drug concentrations of 50 μM and 5 μM, and each group is significantly better than the inhibitory effect of the free testosterone spiro compound (Ia) in Example 3, especially the nanoliposome group of the testosterone spiro compound (Ia) has a significantly better inhibitory effect on the three types of tumor cells than the nanoemulsion group. This is because the testosterone spiro compound (Ia) is poorly soluble in water. After being prepared into nanoliposomes and nanoemulsions, the water dispersibility of the testosterone spiro compound (Ia) can be significantly improved, and the testosterone spiro compound (Ia) nanoliposomes can quickly fuse with tumor cells, efficiently deliver the testosterone spiro compound (Ia) to tumor cells, and inhibit tumor cell proliferation. Therefore, the nanopharmaceutical preparation of testosterone spirocyclic compound (Ia) has good development value and application prospects.

[0072] The above content is a further detailed description of the technical solution provided in combination with the preferred implementation mode of the present invention. It cannot be determined that the specific implementation of the present invention is limited to the above descriptions. For ordinary technicians in the technical field to which the present invention belongs, they can make several simple deductions or substitutions without departing from the concept of the present invention, which should be regarded as belonging to the protection scope of the present invention.

Claims

1. A testosterone spirocyclic compound, characterized in that The testosterone spiro compound is a spiro dimer of testosterone, and its structural formula is shown in formula (Ia):

2. The testosterone spiro compound according to claim 1, characterized in that The testosterone spirocyclic compound is a chiral S-type of the spirocyclic central carbon.

3. A nano preparation, characterized in that: The nanoformulation comprises the testosterone spiro compound according to any one of claims 1-2.

4. The nanoformulation according to claim 3, characterized in that The testosterone spiro compound is a nanoliposome of the testosterone spiro compound and a nanoemulsion of the testosterone spiro compound.

5. The nanoformulation according to claim 4, characterized in that The particle size of the nanoliposome of the testosterone spiro compound is 184.2±10.8 nm, and the particle size of the nanoemulsion of the testosterone spiro compound is 225.4±5.3 nm.

6. The nanoformulation according to claim 5, characterized in that The effective dosage of the nanoliposome of the testosterone spiro compound is 50 μM, and the effective dosage of the nanoemulsion of the testosterone spiro compound is 5 μM.

7. A method for preparing a testosterone spirocyclic compound according to any one of claims 1-2, characterized in that: 6-(N-methyl-N-phenyl)-aminomethyl-17β-hydroxy(ester)androst-4-ene-3-one (IV) is dissolved in dichloromethane, trifluoroacetic acid is added dropwise, and then the reaction is kept warm until the reaction is completed. After most of the trifluoroacetic acid is evaporated under reduced pressure, extraction and washing are performed, and the testosterone spirocyclic compound (Ia) is obtained.

8. Use of the testosterone spiro compound as claimed in any one of claims 1 to 2 and the nanoformulation as claimed in any one of claims 3 to 6 in the preparation of a drug for preventing tumors.

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