Progesterone spiro compound and nano preparation thereof, and preparation method and application of progesterone spiro compound and nano preparation

Abstract

The invention relates to a progesterone spiro compound, a nano preparation thereof, a preparation method and application, the progesterone spiro compound is a spiro dimer of progesterone, and the structural formula of the progesterone spiro compound is as shown in formula (I). The progesterone spiro compound (I) and the hydrate thereof are used as active ingredients and matched with a prodrug, a pharmaceutically acceptable salt or a pharmaceutically acceptable carrier to form a nano preparation, the progesterone spiro compound (I) and the hydrate thereof are applied to the fields of tumor resistance and the like and have high cancer cell inhibition capacity, and a lead compound is provided for developing new anti-tumor drugs. # imgabs0 #

Description

Technical Field

[0001] The invention relates to a progesterone 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. Progesterone (Ⅱ, progesterone), also known as progesterone and progesterone, has a chemical name of pregnane-4-ene-3,20-dione. It is a first-line drug for the treatment and prevention of pregnancy loss and habitual abortion. It is also used for dysmenorrhea, excessive menstrual bleeding or metrorrhagia, amenorrhea, premenstrual syndrome, menstrual disorders caused by ovulation arrest, breast disease, etc. Clinical studies have found that the indications of progesterone are not limited to gynecological diseases, but can also be used to treat male-related diseases, renal colic, ureteral stones and refractory liver cirrhosis ascites, abdominal colic, brain trauma, asthma, diuresis and other diseases. Recent studies have shown that progesterone also has the effects of protecting the central nervous system, inhibiting epileptic seizures, and improving symptoms of chronic respiratory failure, diabetes and sleep apnea syndrome. The molecular formula of progesterone is C 21 H 30 O 2 , whose structural formula is shown in formula (II):

[0003]

[0004] Chinese patent application CN108159058A reports that progesterone and its pharmaceutically acceptable derivatives have the effect of inhibiting the expression of hypoxia-inducible factor HIF-1α, which is of great significance in inhibiting airway remodeling. They can also be used to prepare drugs for preventing or treating cancer, abdominal aortic aneurysm (AAA) disease, bone atrophy during aging, improving the long-term survival of cancer patients, rheumatoid arthritis, chronic lung disease, pregnancy-related diseases and inflammatory bowel disease.

[0005] It can be foreseen that compounds with progesterone-like structures are likely to have pharmacological activity, and the further development of new and effective therapeutic drugs is of great significance. Summary of the invention

[0006] In view of this, in view of the above-mentioned defects existing in the existing anti-tumor technology, the present invention provides a new progesterone spiro compound and a nano preparation thereof, which is a derivative of progesterone and has anti-tumor activity.

[0007] In order to achieve the above object, the present invention provides a progesterone spiro compound, which is a progesterone derivative and a hydrate, a prodrug and a pharmaceutically acceptable salt thereof. The progesterone spiro compound is a spiro dimer of progesterone, and its structural formula is shown in formula (I):

[0008]

[0009] In the preferred technical solution of the progesterone spiro compound of the present invention, preferably, the progesterone spiro compound is of chiral R type at the central carbon of the spiro ring.

[0010] In vitro anti-tumor tests show that the progesterone spirocyclic compound has a significant inhibitory effect on the proliferation of breast cancer cells. The cell lines used in the in vitro anti-tumor experiments are internationally common tumor cell lines - human breast cancer cells MCF7, lung cancer resistant to osimertinib HCC827R and lung cancer sensitive to osimertinib HCC827S.

[0011] According to a second aspect of the present invention, the present invention provides a nanoformulation (ie, a pharmaceutical composition), wherein the nanoformulation comprises the progesterone spirocyclic compound.

[0012] In the preferred technical solution of the nanoformulation of the present invention, preferably, the progesterone spiro compound is a nanomicelle of the progesterone spiro compound and a solid lipid nanoparticle of the progesterone spiro compound.

[0013] In the preferred technical solution of the nanoformulation of the present invention, preferably, the particle size of the nanomicelles of the progesterone spiro compound is 57.3±6.4 nm, and the particle size of the solid lipid nanoparticles of the progesterone spiro compound is 185.7±9.4 nm.

[0014] In the preferred technical solution of the nanoformulation of the present invention, preferably, the effective dose of the nanomicelles of the progesterone spiro compound is 50 μM, and the effective dose of the solid lipid nanoparticles of the progesterone spiro compound is 5 μM.

[0015] According to the third aspect of the present invention, the present invention also provides a method for preparing a progesterone spiro compound as shown in formula (I), the preparation method comprising: dissolving 6-(N-methyl-N-phenyl)-aminomethyl-pregnane-4-ene-3,20-dione (IV) with dichloromethane, dropping trifluoroacetic acid, and then keeping the reaction warm until the reaction is completed, after most of the trifluoroacetic acid is evaporated under reduced pressure, extracting and washing, and concentrating to dryness to obtain a crude progesterone spiro compound. The crude progesterone spiro compound can be purified by column chromatography and other methods to obtain a fine progesterone spiro compound. The above reaction process can be expressed as:

[0016]

[0017] According to the fourth aspect of the present invention, the present invention also provides a use of a progesterone spirocyclic compound in the preparation of a drug for preventing tumors.

[0018] Experiments show that the progesterone spirocyclic compound provided by the present invention has a significant tumor inhibition effect, and the experimental method adopts the internationally used CCK8 method for detection.

[0019] The progesterone spiro compound of the present invention has low water solubility and poor oral bioavailability. To address this defect, we adopt two measures: 1) preparing a derivative oxime with greater polarity to increase its water solubility and bioavailability; 2) preparing a nanoformulation of the compound, including the progesterone spiro compound and / or its hydrate, prodrug, and pharmaceutically acceptable salt or pharmaceutically acceptable carrier as an active ingredient. The above-mentioned progesterone spiro compound is prepared with pharmaceutical excipients and pharmaceutically or physiologically acceptable carriers, and prepared into any pharmaceutical dosage form by conventional preparation technology.

[0020] The progesterone spirocyclic compound and the nano-preparation thereof of the present invention have strong ability to inhibit cancer cells, provide a leading compound for the development of new anti-tumor drugs, and are of great significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a LC-MS graph of the product prepared under the conditions of Example 1 of the present invention;

[0022] Figure 2 This is the NMR (hydrogen spectrum) of the product prepared under the conditions of Example 1 of the present invention;

[0023] Figure 3 The NMR (carbon spectrum) of the product prepared under the conditions of Example 1 of the present invention;

[0024] Figure 4 This is the NMR image (carbon spectrum, partially enlarged) of the product prepared under the conditions of Example 1 of the present invention;

[0025] Figure 5 This is the NMR image (DEPT135) of the product prepared under the conditions of Example 1 of the present invention;

[0026] Figure 6 This is the NMR image of the product prepared under the conditions of Example 1 of the present invention (DEPT135, partial enlargement);

[0027] Figure 7 This is a single crystal derivative diagram of the product prepared under the conditions of Example 1 of the present invention;

[0028] Figure 8 is the inhibition rate of tumor cells at a single concentration of the product under the preparation conditions of Example 1 of the present invention;

[0029] Fig. 9 The inhibition rates of different dosage forms of the product under the preparation conditions of Example 1 of the present invention on related tumor cells. DETAILED DESCRIPTION

[0030] 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.

[0031] 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).

[0032] Example 1: Preparation of 6-(N-methyl-N-phenyl)-aminomethyl-pregnane-4-ene-3,20-dione (IV)

[0033] In this embodiment, the preparation method of the raw material 6-(N-methyl-N-phenyl)-aminomethyl-pregnane-4-ene-3,20-dione (IV) is described by the following reaction formula:

[0034]

[0035] 55 g of raw material progesterone (II), 325 ml of anhydrous tetrahydrofuran, 50 ml of anhydrous ethanol, 60 ml of triethyl orthoformate and 1.3 g of p-toluenesulfonic acid were stirred in a four-necked flask equipped with a thermometer and mechanical stirring at 40°C for 6 hours to carry out enol etherification reaction to obtain reaction solution 1 [reaction solution containing compound (III)].

[0036] Then, 31 g of N-methylaniline and 40 ml of paraformaldehyde were added to the reaction solution 1, and the mixture was stirred and subjected to Mannich reaction for 7 hours to obtain reaction solution 2. Triethylamine was added to reaction solution 2 to neutralize the reaction solution. The solvent tetrahydrofuran was evaporated under reduced pressure below 50°C, and 130 ml of ethyl acetate was added. The mixture was stirred and dissolved at 50°C, cooled to -10°C for crystallization, and stirred for 5 hours. The mixture was filtered and dried to obtain 57.6 g of a light yellow solid compound (IV) with a yield of 76%.

[0037] Example 2: Preparation of progesterone spirocyclic compound (I)

[0038] Dissolve 21.7 g of compound (IV) (0.05 mol) in 100 ml of dichloromethane, then add 30 ml of trifluoroacetic acid dropwise at 5°C, raise the temperature to 15°C and stir for 40 hours. Distill off most of the trifluoroacetic acid under reduced pressure. Add 190 ml of dichloromethane and 250 ml of water, stir and separate. Wash the organic layer three times with deionized water. Concentrate the organic layer to dryness to obtain 14.4 g of crude product [crude compound (I)], with a yield of 86.0%.

[0039] The crude product can be purified by column chromatography (mobile phase: ethyl acetate: petroleum ether = 1:2) and other methods to obtain a fine product. After column purification, 9.88 g of a white solid compound (I) was obtained, with a yield of 59%.

[0040]

[0041] Compound (I) structural characterization: MS: 652.46

[0042]

[0043] 13 CNMR (101MHz, CDCl 3)δ209.44(C19,C),209.42(C40,C),200.03(C2,C),196.29(C23,C),182.73(C25,C),166.96( C4,C),137.39(C3,C),122.57(C24,CH),63.59(C17,CH),63.38(C38,CH),56.04(C11,CH),55. 57(C32,CH),55.17(C10,CH),48.17(C31,CH),43.95(C28,C,spirocyclic carbon),43.86(C33,C),42.86(C12,C),40.15(C26,C),39.54(C29,CH2),38.62(C8,CH2),38.52(C5,C),38.49(C34,CH2),38.46(C 13,CH2),36.28(C27,CH2),35.79(C9,CH),34.83(C42,CH2),34.39(C1,CH2),34.30(C6,CH2), 33.82(C7,CH),33.74(C22,CH2),31.56(C43,CH3),31.53(C46,CH3),30.67(C30,CH),24.82(C 21,CH2),24.41(C15,CH2),24.23(C36,CH2),22.82(C37,CH2),22.74(C16,CH2),22.24(C48,C H3),21.11(C14,CH2),21.08(C35,CH2),18.12(C47,CH3),13.36(C41,CH3),13.35(C20,CH3).

[0044] Example 3: Preparation of oxime of progesterone spirocyclic compound (I) derivative

[0045] 13.04 g (0.02 mol) of progesterone spirocyclic compound (I) and 200 mL of 95% ethanol were added to a 500 mL three-necked flask, and heated to dissolve under stirring. Then 5.46 g (0.04 mol) of sodium acetate trihydrate and 3.96 g (0.058 mol) of hydroxylamine hydrochloride were added, and the mixture was stirred at 60°C for 90 min. After the reaction was completed, the reaction solution was poured into ice water to precipitate solids, which were filtered to obtain a yellowish brown solid, which was dried in a vacuum oven at 50°C to obtain 12.80 g of a crude product with a yield of 83.5%.

[0046] The crude product can be purified by column chromatography (mobile phase: ethyl acetate: petroleum ether = 1:1) and other methods to obtain a fine product. After column purification, 9.38 g of a white solid was obtained, with a yield of 67.5%.

[0047] Example 4: Application of progesterone spirocyclic compound (I)

[0048] Single concentration screening of progesterone spirocyclic compound (I) on tumor cell proliferation inhibition, this test uses the internationally used CCK8 method for detection.

[0049] Experimental method: First, the compound was dissolved in a certain amount of NMP to prepare a 20mM stock solution, and then diluted to different concentrations according to the experimental needs. Use 0.25% trypsin containing EDTA to digest the 2-3 generations of tumor cells and count them using 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 inhibition rate (%) of the drug on cancer cell proliferation can be calculated by the following formula: [1-(OD T -OD B ) / (OD C -OD B )]×100%.OD T OD C and OD B The experimental group, control group and blank group are the absorbance values. The experimental results are shown in Table 2 and Figure 8 Said:

[0050] Table 2 Inhibition rate of products on three tumor cells at single concentration (single experiment repeated 4 times)

[0051]

[0052]

[0053] From Table 2 and Figure 8 It can be seen that the cell lines used in the in vitro anti-tumor experiment are internationally common tumor cell lines - human breast cancer cell MCF7, lung cancer resistant to osimertinib HCC827R and lung cancer sensitive to osimertinib HCC827S. The experimental results show that the progesterone spirocyclic compound has a significant inhibitory effect on the proliferation of breast cancer cells.

[0054] Example 5: Preparation of nanomicelles of progesterone spirocyclic compound (I)

[0055] The preparation process is as follows: weigh the nano-micelle prescription materials according to the prescription in Table 3 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 the anhydrous ethanol. -1 The sodium phosphate solution (5mM, pH7.4) 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 minutes to obtain the initial micelle product. The initial product was subjected to ultrasonic dispersion treatment (power and time: 200W×2min+400W×6min, working 1s and resting 1s), and then passed through 0.80 and 0.22μm microporous filter membranes in turn to obtain the progesterone spirocyclic compound (I) nanomicelles.

[0056] The experimental results show that the particle size of the progesterone spirocyclic compound (I) nanomicelles is 57.3±6.4 nm, the potential is -16.4±2.3 mV, and the drug encapsulation efficiency is 98.3±1.1%.

[0057] Table 3: Progesterone spirocyclic compound (I) nanomicelle formulation

[0058]

[0059] Example 6: Preparation of solid lipid nanoparticles of progesterone spirocyclic compound (I)

[0060] The preparation process is as follows: according to the prescription in Table 4, weigh the solid lipid nanoparticles and place them in a vial, add 2 mL of anhydrous ethanol, stir and dissolve in a 65°C water bath, and wait until the prescription materials and drugs are completely dissolved. Then, add 5 mL·S -1 Inject 10 mL of sodium phosphate solution (5 mM, pH 7.4) preheated to 65°C into the membrane material at a speed of 100 °C. Stir in a 65°C water bath for 20 min to obtain a solid lipid nanoparticle preliminary product, and evaporate to remove ethanol. The preliminary product is 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 turn to obtain progesterone spirocyclic compound (I) solid lipid nanoparticles.

[0061] The experimental results showed that the particle size of the progesterone spirocyclic compound (I) solid lipid nanoparticles was 185.7±9.4 nm, the potential was -13.5±2.6 mV, and the drug encapsulation efficiency was 95.3±1.4%.

[0062] Table 4: Progesterone spirocyclic compound (I) solid lipid nanoparticle formulation

[0063]

[0064] Example 7: In vitro antitumor effects of nanomicelles and solid lipid nanoparticles of progesterone spirocyclic compound (I)

[0065] Single concentration screening of the inhibition of tumor cell proliferation by nanomicelles and nanoemulsions of progesterone spirocyclic compound (I) was performed using the internationally accepted CCK8 assay.

[0066] Experimental method: First, the progesterone spirocyclic compound (I) nanomicelles and nanoemulsion 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 tumor cells of the 2nd and 3rd generations of culture, and the cells were counted by an automatic cell counter. The tumor cells were inoculated in a 96-well transparent cell culture plate at a number of 5000 cells per well and 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. 9 shown.

[0067] Depend on Fig. 9The results show that the nano-micelles and solid lipid nanoparticles of the progesterone spiro compound (I) have a certain proliferation inhibitory effect on three types of tumor cells (human breast cancer cells MCF7, lung cancer resistant to osimertinib HCC827R and lung cancer sensitive to osimertinib HCC827S) at drug concentrations of 50 μM and 5 μM, and each group is significantly better than the inhibitory effect of the free progesterone spiro compound (I) in Example 4. This is because the progesterone spiro compound (I) is poorly soluble in water. After being prepared into nano-micelles and solid lipid nanoparticles, the water dispersibility of the progesterone spiro compound (I) can be significantly improved. At the same drug concentration, the inhibitory effect of the nano-micelle group of the progesterone spiro compound (I) on the three types of tumor cells is significantly better than that of the solid lipid nanoparticle group. The reason for this phenomenon is that the size of the progesterone spiro compound (I) nano micelles is much smaller than that of the solid lipid nanoparticle group. The nano micelles can quickly diffuse and penetrate the cell membrane, efficiently deliver the progesterone spiro compound (I) into tumor cells, and inhibit tumor cell proliferation. Therefore, the nano drug preparation of the progesterone spiro compound (I) has good development value and application prospects.

[0068] 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 progesterone spirocyclic compound, characterized in that: The progesterone spiro compound is a spiro dimer of progesterone, and its structural formula is shown in formula (I):

2. The progesterone spiro compound according to claim 1, characterized in that The progesterone spirocyclic compound is a chiral R-type spirocyclic central carbon.

3. A nano preparation, characterized in that: The nanoformulation comprises the progesterone spiro compound according to claim 1 or 2.

4. The nanoformulation according to claim 3, characterized in that The progesterone spiro compound is a nano micelle of the progesterone spiro compound and a solid lipid nanoparticle of the progesterone spiro compound.

5. The nanoformulation according to claim 4, characterized in that The particle sizes of the nano micelles of the progesterone spiro compound are 57.3±6.4 nm, and the particle sizes of the solid lipid nanoparticles of the progesterone spiro compound are 185.7±9.4 nm.

6. The nanoformulation according to any one of claims 3-4, characterized in that: The effective dosage of the nano micelles of the progesterone spiro compound is 50 μM, and the effective dosage of the solid lipid nanoparticles of the progesterone spiro compound is 5 μM.

7. A method for preparing the progesterone spirocyclic compound according to claim 1 or 2, characterized in that: After dissolving 6-(N-methyl-N-phenyl)-aminomethyl-pregnane-4-ene-3,20-dione (IV) 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 progesterone spirocyclic compound (I) is obtained.

8. Use of the progesterone spirocyclic compound according to any one of claims 1 to 2 and the nanoformulation according to any one of claims 3 to 6 in the preparation of a drug for preventing tumors.

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