CN106928303B - A kind of spiro-compound and its preparation method and application - Google Patents

Abstract

The invention relates to a spiro compound, a preparation method and application thereof, and belongs to the technical field of steroid compounds. The spiro compound I and its hydrate are used as an active ingredient, and a prodrug, a pharmaceutically acceptable salt or a pharmaceutically acceptable carrier is used to form a pharmaceutical composition. Applying the invention to anti-tumor and other fields, it has a strong ability to inhibit cancer cells and provides a lead compound for the development of new anti-tumor drugs.

Description

A kind of spiro compound and its preparation method and application

technical field

The invention relates to a spiro compound, a preparation method and application thereof, and belongs to the technical field of steroid compounds.

Background technique

Steroid drugs are widely used in the field of pharmaceutical industry and have various pharmacological activities. Androstenedione derivatives belong to steroidal drugs in aromatase inhibitors, which are similar in structure to androstenedione and testosterone, the endogenous substrates of aromatase, and are irreversibly bound to them through covalent bonds. Cause permanent inactivation of the enzyme, thereby inhibiting the synthesis of estrogen, exemestane, anastrozole, etc. are currently the main clinical drugs. The chemical name of Exemestane (VI, Exemestane) is 6-methylene-androst-1,4-diene-3,17-dione, which is an irreversible aromatase inhibitor developed by the Italian company Pharmacia&Upjoin The drug was first launched in the UK in 1999. Its molecular formula is C ₂₀ H ₂₄ O ₂ , and its structural formula is shown in formula VI:

Relying on the similar structure with the natural substrate of steroidal aromatase - androstenedione, it irreversibly binds to its active site and permanently inactivates it, so that the hormone level in the body can be rapidly and continuously reduced to achieve the therapeutic purpose. US4808616 reports the preparation method of 6-alkylene-androst-1,4-diene-3,17-dione derivative and its application in medicinal chemistry. CN101312743A discloses a method for (S)-6-methyloxaalkyl exemestane compound for inhibiting the growth of cancer cells or tumor cells.

It can be predicted that compounds with a structure similar to androstenedione are likely to have pharmacological activity, and further research and development of new and effective therapeutic drugs is of great significance.

Based on this, this application is made.

Contents of the invention

Aiming at the above defects in the existing anti-tumor technology, the present application firstly provides a new spiro compound, which is a derivative of androstenediones and has anti-tumor activity.

In order to achieve the above object, the technical scheme adopted by the application is as follows:

A spiro compound, the spiro compound is androstenedione derivatives and their hydrates, prodrugs and pharmaceutically acceptable salts, the structural formula of which is:

For the convenience of description below, it will be denoted as I.

The anti-tumor test in vitro shows that the spiro compound has obvious tumor-inhibiting effect. The cell lines used in the experiment are international tumor cell lines—human liver cancer cell HepG2 and human ovarian cancer cell SK-OV-3.

Simultaneously, the present application also provides a kind of preparation method of aforementioned spiro compound, 6-(N-methyl-N-phenyl)-aminomethyl-4-ene-3,17-androstadione (IV ) dissolved, added dropwise to concentrated hydrochloric acid, reacted at a temperature lower than 15°C for 2 hours, and then filtered to obtain the crude product, which can be purified by column chromatography to obtain the refined spiro compound (I).

The above reaction process can be expressed as:

Further, as a preference:

The solvent is ethanol, and the amount of ethanol used in the reaction is 1500-2000 ml/mol 6-(N-methyl-N-phenyl)-aminomethyl-4-ene-3,17-androstadione . More preferably, the amount of ethanol is 1600-1800 ml/mol 6-(N-methyl-N-phenyl)-aminomethyl-4-ene-3,17-androstadione.

The concentration of the concentrated hydrochloric acid is greater than or equal to 20%, preferably, the concentration of the concentrated hydrochloric acid is 36%, an industrial product.

The amount of concentrated hydrochloric acid is 500-1000mL/mol 6-(N-methyl-N-phenyl)-aminomethyl-4-ene-3,17-androstadione, preferably, the concentrated The dosage of hydrochloric acid is 600-700 ml/mol 6-(N-methyl-N-phenyl)-aminomethyl-4-ene-3,17-androstadione.

The dropwise temperature is 0-20°C, and the reaction temperature is 0-20°C. More preferably, the dropwise temperature is 5-15°C, and the reaction temperature is 5-15°C; 1-3 hours, more preferably, the reaction time after the dropwise addition is 2-3 hours.

The spiro compound provided by the present application has obvious tumor-inhibiting effect, and the experimental method is carried out by the internationally accepted MTT method.

The present application also provides a pharmaceutical composition, including the spiro compound and/or its hydrate, prodrug, pharmaceutically acceptable salt or pharmaceutically acceptable carrier as an active ingredient. The above-mentioned spiro compound is formulated with pharmaceutical excipients and pharmaceutically or physiologically acceptable carriers, and is prepared into any pharmaceutical dosage form by conventional preparation techniques.

The compound of the invention has a strong ability to inhibit cancer cells, provides a lead compound for developing new antitumor drugs, and is of great significance.

Description of drawings

Fig. 1 is the liquid chromatography-mass spectrometry diagram of the product under the preparation conditions of Example 1 of the present application;

Fig. 2 is the NMR figure (hydrogen spectrum) of product under the preparation condition of embodiment 1 of the present application;

Fig. 3 is the NMR figure (carbon spectrum) of the product under the preparation conditions of Example 1 of the present application;

Fig. 4 is the NMR figure (carbon spectrum, partially enlarged) of the product under the preparation conditions of Example 1 of the present application;

Figure 5 is the NMR image (DEPT135) of the product under the preparation conditions of Example 1 of the present application;

Figure 6 is the NMR image of the product under the preparation conditions of Example 1 of the present application (DEPT135, partially enlarged).

Detailed ways

Analytical instruments and equipment used in the examples: liquid mass spectrometry, Thermo LCQ-Fleet, column: HypersilGOLD 150*2.1, electrospray ionization source (ES I); nuclear magnetic resonance instrument, AVANCE DMX Ⅱ Ⅰ 400M (TMS internal standard, Bruker company) ; High performance liquid chromatography: Agilent Technologies 1200Series; Infrared spectrometer, NICOLET360FT-IR (Nicolet Instruments, Inc., USA).

In this example, the preparation method of the raw material 6-(N-methyl-N-phenyl)-aminomethyl-4-ene-3,17-androstanedione (IV) can refer to the patent of Cao Ruiwei et al. ( CN105017370), its reaction synthesis process can be described as follows:

Embodiment 1: the preparation of spiro compound I

Dissolve 40.7 grams of compound IV (0.1 mole) in 170 milliliters of ethanol, add dropwise to 80 milliliters of 36% concentrated hydrochloric acid at 0-5°C, react at 5-10°C for 2 hours after the dropwise, filter to obtain a khaki solid , 23.5 grams after drying, yield 78.9%. The crude product can be purified by column chromatography (mobile phase: ethyl acetate: petroleum ether = 1:3) to obtain the refined product, which is a yellow solid after purification, with a melting point of 180-183°C.

Product structure characterization:

MS: 596

¹³ CNMR (δ, ppm, 100MHz, CDCl ₃ ):

220.66, 220.26 (C17, C17'); 199.86, 196.15 (C1', C1); 182.12, 166.60 (C3', C3); 137.41, 122.68 (C2, C2');

55.25 (CH, C10'); 50.98 (CH, C10); 50.41 (CH, C14'); 48.47 (CH, C14); 47.50, 47.33 (CH ₀ , C13,13').

42.74 (CH ₀ , C7', spiro carbon):

40.22 (CH ₀ , C4); 38.59 (CH ₀ , C4′); 38.31 (CH ₂ ); 37.48 (CH ₂ ); 36.22 (CH ₂ ); 35.78 (two CH ₂ , C5, 5′); 35.34 ( CH); 34.75 (CH ₂ ); 34.32, 34.27 (two CH ₂ , C16, 16'); 33.65 (one CH and one CH ₂ stacked, C6'); 31.23 (CH ₂ , C12'); 31.10 (CH ₂ , C12); 30.27 (CH); 24.82 (CH ₂ ); 22.25 (CH ₃ , C18'); 21.73, 21.66 (two CH ₂ , C15, 15'); 20.40, 20.33 (two CH ₂ , C11 , 11'); 18.22 (CH ₃ , C18); 13.71 (two CH ₃ , C19, 19').

Embodiment 2: the spiro compound shown in the reaction preparation formula (I) under different conditions

Dissolve 40.7 grams of compound IV (0.1 mole) in a certain amount of ethanol, add it dropwise to a certain amount of 36% concentrated hydrochloric acid at a certain temperature, react for a certain period of time within a certain temperature range after the drop, filter to obtain the product, and dry it Weigh and calculate the yield, the results are shown in Table 1.

Product yield under different preparation conditions of table 1

The product nuclear magnetic data obtained under the corresponding conditions of each serial number in the above-mentioned embodiment 2 is the same as that of embodiment 1.

The yield, material ratio, and synthetic conditions of the above-mentioned Table 1 are comprehensively considered, and the conclusions are as follows:

(1) The impact of the amount of ethanol added: In this application, ethanol, as a solvent, will directly affect the reaction efficiency and reaction rate of the product. Under the same conditions, the ethanol addition was tested, and the results showed that when the ethanol addition was lower than 1500mL/mol raw material (compound IV, that is: 6-(N-methyl-N-phenyl)-aminomethyl-4- ene-3,17-androstadione, the same below), because the amount of organic solvent is too low, the dissolution of organic matter in the reaction system is not enough, can not meet the needs of the reaction, so the yield is lower than 30% (see Table 1 for details middle serial number 1-2); along with the increase of ethanol addition, when its addition exceeds 1500mL/mol raw material, reaction tends towards fully gradually, especially when ethanol addition is between 1500-2000mL/mol raw material, especially in When the raw material is 1600-1800mL/mol, because the raw material can be fully dissolved, the reaction can be fully carried out, the stability of the product is gradually increased, the product quality is good, and the yield can reach more than 56% (for details, refer to the serial number 3-14 in Table 1 ); continue to increase the addition of ethanol, although the complete dissolution of the raw material has been ensured, but when the addition of ethanol is greater than 2000mL/mol raw material, because the system concentration is low, the yield is reduced instead (see the sequence number 15-16 in Table 1 for details ). Therefore, the amount of ethanol added is suitably controlled at 1500-2000mL/mol raw material, especially when the added amount of ethanol is controlled at 1600-1800mL/mol raw material, the reaction efficiency is the best.

(2) The impact of concentrated hydrochloric acid addition: in the reaction process, concentrated hydrochloric acid is one of reaction raw material, under the same condition, the concentrated hydrochloric acid addition is tested, and the result shows, when the concentrated hydrochloric acid addition is lower than 500mL/mol raw material (compound IV, namely: 6-(N-methyl-N-phenyl)-aminomethyl-4-ene-3,17-androstadione, the same below), due to insufficient amount of acid, resulting in insufficient reaction, thus Yield is lower than 40% (specifically referring to serial number 17 in table 1); Along with the increase of concentrated hydrochloric acid addition, when its addition exceeds 500mL/mol raw material, reaction tends towards fully gradually, especially when concentrated hydrochloric acid addition is between When the raw material is 600-800mL/mol, the yield is better % (for details, refer to the sequence number 18-22 in Table 1); continue to increase the addition of concentrated hydrochloric acid, although the amount of acid is satisfied, but due to the increase in the total volume of the system, the concentration of the raw material Reduced, resulting in a decline in yield (see No. 23-24 in Table 1 for details). Therefore, the added amount of concentrated hydrochloric acid is suitably controlled at 500-1000mL/mol raw material, especially when the concentrated hydrochloric acid added amount is controlled at 600-800mL/mol raw material, the reaction efficiency is the best.

(3) The influence of titration temperature and reaction temperature: During the reaction, the titration temperature affects the selectivity, and the reaction temperature affects the reactivity. Under the same conditions, the titration temperature and the reaction temperature were tested, and the results showed that when the titration temperature was lower than 0° C., the reaction was slow because the temperature was too low, so the yield was lower than 40% (for details, refer to the serial number 25- 28); as the titration temperature increases, when it exceeds 0°C, the reaction gradually tends to be stable, especially when the titration temperature is between 5-15°C, the yield is more stable (see the serial number 29-35 in Table 1 for details) ); continue to increase the titration temperature, and when it is higher than 20° C., the impurities will increase and the yield will decrease (for details, refer to the serial number 36-39 in Table 1).

At the same time, it can also be seen from the parallel experiments in Table 1 that for the convenience of the control of the experimental conditions, the reaction temperature should be the same as the titration temperature, the titration temperature should be controlled at 0-20°C, and the reaction temperature should be controlled at 0-20°C, especially When the titration temperature is appropriately controlled at 5-15°C and the reaction temperature is appropriately controlled at 5-15°C, the reaction effect is the best.

(4) Influence of reaction time: Reaction time is a key factor of whether the reaction is sufficient or not. Under the same conditions, the reaction time is tested, and the results show that when the reaction time is lower than 1h, the reaction is incomplete and the yield is low (see sequence number 40 in Table 1 for details); as the reaction time increases, when it exceeds 1h , the reaction gradually tends to be sufficient, especially when the reaction time is between 1.5-3h, the yield is more stable (see the serial number 41-44 in Table 1 for details); continue to prolong the reaction time, the yield does not increase, and the result is not Ideal (refer to No. 45-46 in Table 1 for details)

Embodiment 3: the influence of hydrochloric acid concentration on finished product

Dissolve 40.7 g of compound IV (0.1 mol) in 170 mL of ethanol, add dropwise to 60 mL of concentrated hydrochloric acid with different mass concentrations at 10°C, react at 10°C for 2 hours after dropping, filter to obtain the product, and weigh it after drying , the finished product is tested, and the results are shown in Table 2.

The impact of table 2 different concentrations of concentrated hydrochloric acid on the product

As can be seen from Table 2, under the same preparation conditions, when the concentration ≥ 20%, the finished spiro compound is prepared, however, for the concentration lower than 35%, especially when the concentration is between 20-35%, due to insufficient acidity , the reason for insufficient acid content leads to low yield; when the concentration is higher than 37°C, although the yield is good, it is difficult to achieve considering the difficulty of the source; while the concentration of industrial hydrochloric acid is about 36%, the source is convenient and the price is low. Inexpensive, and when the concentration of hydrochloric acid is between 35-37%, especially when the concentration of hydrochloric acid is 36%, the various indicators of the product reach the best.

Embodiment 4: the application of spiro compound

In vitro anti-tumor activity assay experiment:

The experiment was carried out by the internationally accepted MTT method, and the in vitro tumor cell inhibitory activity test of the spiro compound I was carried out: first, ² ×104 logarithmic growth phase human liver cancer cells HepG2 or human ovarian cancer cells were inoculated on a 96-well cell plate SK-OV-3, 5 duplicate wells, after the cells adhere to the wall, add different concentrations of samples to be tested, and set up 7 drug concentration gradients (unit ug/mL), the concentrations are: 1.0, 5.0, 10, 20, Compound I sample groups at 30, 40 and 50; after 72 hours, add 20uL of 5mg/mL MTT solution to the corresponding wells of the 96-well plate, continue to incubate for 3 hours, discard the supernatant in the well plate, and add 150uL of DMSO to dissolve , using a microplate reader to detect the absorbance at a wavelength of 490nm and calculate the half inhibitory concentration IC ₅₀ of the test sample on cell growth. The experimental results show that the compound has obvious inhibitory effect on the two tumor cell lines, and it provides a lead compound for the development of new anti-tumor drugs, which is of great significance.

The IC ₅₀ of human liver cancer cell HepG2 was 11.45ug/mL; the IC ₅₀ of human ovarian cancer cell SK-OV-3 was 18.65ug/mL.

The above results show that the spiro compound I has inhibitory effect on these two human tumor cell lines.

The above content is a further detailed description of the technical solutions provided in conjunction with the preferred embodiments of the present invention. It cannot be determined that the specific implementation of the present invention is only limited to the above descriptions. For those of ordinary skill in the technical field of the present invention, On the premise of not departing from the inventive concept of the present invention, some simple deductions or substitutions can also be made, which should be regarded as belonging to the protection scope of the present invention.

Claims (10)

1. a spiro compound, is characterized in that, its structural formula is: . 2. The preparation method of a kind of spiro compound as claimed in claim 1, is characterized in that: 6-(N-methyl-N-phenyl)-aminomethyl-4-ene-3,17-androster After the diketone is dissolved, it is added dropwise into concentrated hydrochloric acid, and after the reaction is completed below 20°C, the crude product is obtained by filtration, and the crude product is purified to obtain a refined spiro compound. 3. the preparation method of a kind of spiro compound as claimed in claim 2 is characterized in that: described solvent is ethanol, and the ethanol amount used in the reaction is 1500-2000 milliliter/mol 6-(N-methyl- N-phenyl)-aminomethyl-4-ene-3,17-androstandione. 4. The preparation method of a spiro compound as claimed in claim 2 or 3, characterized in that: the amount of ethanol is 1600-1800 ml/mol 6-(N-methyl-N-phenyl)- Aminomethyl-4-ene-3,17-androstadione. 5. the preparation method of a kind of spiro compound as claimed in claim 2, is characterized in that: described concentrated hydrochloric acid concentration ≥ 20%. 6. the preparation method of a kind of spiro compound as claimed in claim 2 or 5 is characterized in that: described concentrated hydrochloric acid is the industrial product of 36%. 7. the preparation method of a kind of spiro compound as claimed in claim 2 is characterized in that: the consumption of described concentrated hydrochloric acid is 500-1000mL/ mol 6-(N-methyl-N-phenyl)-amine methyl yl-4-ene-3,17-androstanedione. 8. The preparation method of a spiro compound as claimed in claim 2, characterized in that: the temperature during the dropping is 0-20°C. 9. The preparation method of a spiro compound as claimed in claim 2, characterized in that: after the dropping, the reaction temperature is 0-20° C., and the reaction time is 1-3 hours. 10. The application of the spiro compound as claimed in claim 1, characterized in that, the spiro compound is used as an active ingredient, and then prepared with a prodrug and a pharmaceutically acceptable salt or a pharmaceutically acceptable carrier to form a pharmaceutical composition .