CN106946974B - Ursolic amide derivative containing pyrazole heterocycle and synthesis and application thereof - Google Patents

Abstract

The present invention provides the general structural formula of a class of ursamide derivatives containing a pyrazole heterocycle. The present invention further provides a synthesis route and synthesis steps of a class of pyrazole heterocycle-containing ursamide derivatives. The present invention also provides the use of a class of pyrazole heterocycle-containing ursamide derivatives in preparing a medicament for treating tumors. The pyrazole heterocycle-containing ursamide derivatives provided by the invention and their synthesis and application have obvious killing effect on tumor cell growth through in vitro anti-tumor activity test, and have the development potential of new anti-tumor drugs.

Description

A class of ursamide derivatives containing pyrazole heterocycle and their synthesis and application

technical field

The invention belongs to the technical field of chemical pharmacy, and relates to a class of ursamide derivatives containing pyrazole heterocycles, synthesis and application thereof, and in particular to a class of ursamide derivatives containing pyrazole heterocycles and a synthesis method thereof, which are used in the treatment of The application of tumor drugs.

Background technique

Modern medicine has proved that tumors are caused by various physiological and biochemical factors such as environmental pollution, chemical pollution, ionizing radiation, free radical toxins, microorganisms (bacteria, fungi, viruses, etc.) and their metabolic toxins, genetic characteristics, endocrine imbalance, and immune dysfunction. Under the action, some cells in the local tissue lose their normal regulation of their growth at the gene level, and a new organism is formed by clonal abnormal proliferation. Clinically, tumor treatment mainly adopts various measures such as surgery, radiation, chemotherapy, immunization and traditional Chinese medicine. Chemotherapy is chemical drug treatment, which belongs to the systemic treatment of tumors. At present, more than 80 kinds of chemotherapy drugs have been used in clinical practice, and the cure rate of chemotherapy for various malignant tumors is more than 50%. Anti-tumor drugs currently in clinical use can be divided into five categories: alkylating agents, platinum compounds, antibiotics, natural medicines and hormones. Among them, about 1/3 of the drugs come from plants. Antitumor active ingredients isolated from natural plants, representative drugs include paclitaxel, vincristine, teniposide, etc. Natural anti-tumor drugs have broad anti-cancer spectrum, high selectivity and small toxic and side effects, and have a very broad development prospect in clinical application. At present, the extraction and isolation of anti-tumor active ingredients from natural plants, and the development of natural anti-tumor drugs with less toxic and side effects, high specificity and strong lethality, have become a hot spot in the research of anti-tumor drugs at home and abroad in recent years.

Ursolic acid (UA) is a kind of ursane-type pentacyclic triterpenoids existing in natural plants. It has various biological activities such as sedation, anti-inflammatory, liver protection and blood lipid lowering. The main active ingredient of Chinese herbal medicine. In recent years, it has been reported that ursolic acid has a significant inhibitory effect on the proliferation of various tumor cells, and has a definite effect of inducing tumor cell apoptosis. However, the development of ursolic acids in drug development is limited due to their poor solubility, unclear targets and low bioavailability. In recent years, with the re-emergence of research on the druggability of natural products and their analogs, the structural modification of ursolic acid by chemical means to reduce side effects, improve biological activity and improve its bioavailability has once again become a terpenoid natural product hotspots in drug development.

According to the structural statistics of the FDA-approved drugs by Professor Njardarson of Cornell University in 2014, it was found that 59% of the drug molecules contained a nitrogen atom heterocyclic structure. Therefore, the introduction of nitrogen-atom heterocyclic structures based on the structure of complex natural products is an important strategy for their structural modification. Among them, the pyrazole heterocycle is an important structural skeleton of active drug molecules, and is widely used in the structural modification of natural products and the design and synthesis of active drug molecules. Therefore, it is necessary to conduct further research and discussion on ursolic acid.

SUMMARY OF THE INVENTION

In view of the above-mentioned shortcomings of the prior art, the object of the present invention is to provide a class of ursamide derivatives containing pyrazole heterocycles and their synthesis and application, which are used to inhibit the growth of tumor cells and have novel antitumor drugs. development potential.

In order to achieve the above-mentioned purpose and other related purposes, a first aspect of the present invention provides a class of ursamide derivatives containing pyrazole heterocycles, and the derivatives have the general structural formula shown in the following formula 8:

In the formula,

R ¹ is selected from hydrogen (H-), C ₁ -C ₄ alkyl, C ₃ -C ₆ cycloalkyl, aryl, arylalkyl;

R ² is selected from hydrogen (H-), C ₁ -C ₄ alkyl, haloalkyl, C(O)OR'; said R' is C ₁ -C ₄ alkyl;

R ³ is selected from hydrogen (H-), C ₁ -C ₆ alkyl, C ₃ -C ₆ cycloalkyl, haloalkyl, cycloalkylalkyl, alkoxyalkyl, optionally substituted arylalkyl , heterocyclyl alkyl;

R ⁴ is C ₁ -C ₆ alkyl, C ₃ -C ₆ cycloalkyl, haloalkyl, cycloalkylalkyl, alkoxyalkyl, optionally substituted arylalkyl, heterocyclylalkyl;

Alternatively, the ^R3 and R4 ^together with the bridged nitrogen atom form a substituted or unsubstituted heterocycloalkyl.

Preferably, the C ₁ -C ₄ alkyl group in R ¹ is methyl (CH ₃ -) or isopropyl (iPr-).

Preferably, the C ₃ -C ₆ cycloalkyl in the R ¹ is cyclopentyl.

Preferably, the arylalkyl group in R ¹ is benzyl (Bn-).

Preferably, the aryl group in R ¹ is phenyl (Ph-), 4-bromophenyl (4-BrC ₆ H ₅ -), 3-fluorophenyl (3-FC ₆ H ₅ -), 4- Cyanophenyl ( ₄ - _CNC6H5- ) or ₄ -carboxyphenyl ( ₄ - _CO2HC6H5- ).

Preferably, the C ₁ -C ₄ alkyl group in the R ² is methyl (CH ₃ -).

Preferably, the haloalkyl group in R ² is trifluoromethyl (CF ₃ -).

Preferably, C(O)OR' in the R ² is CO ₂ Et-.

Preferably, the C ₁ -C ₆ alkyl group in the R ³ is isopropyl (isopropyl-), n-hexyl (n-hexyl-) or n-butyl (n-butyl-).

Preferably, the C ₃ -C ₆ cycloalkyl in the R ³ is cyclohexyl (cyclopentyl-) or cyclobutyl (cyclobutyl-).

Preferably, the cycloalkylalkyl group in R ³ is cyclopropylmethyl-.

)。Preferably, the haloalkyl group in the R ³ is 2,2,2-trifluoroethyl (2,2,2-trifluoroethyl-,

).

)。Preferably, the alkoxyalkyl group in the R ³ is 3-isopropoxypropyl- (3-isopropoxypropyl-,

).

)或4-甲氧基苯乙基((4-methoxyphenyl)ethyl-，

)。Preferably, the arylalkyl substituted in the R ³ is 4-fluorobenzyl ((4-fluorophenyl)methyl-,

) or 4-methoxyphenylethyl ((4-methoxyphenyl)ethyl-,

).

)、4-吡啶甲基(4-pyridylmethyl-，

)、2-呋喃甲基(2-furanylmethyl-，

)或3-吗啉基丙基(3-morpholinopropyl-，

)。Preferably, the heterocyclyl alkyl in the R ³ is 2-pyrrolidin-1-yl)ethyl-,

), 4-pyridylmethyl (4-pyridylmethyl-,

), 2-furanylmethyl-,

) or 3-morpholinopropyl (3-morpholinopropyl-,

).

Preferably, the C ₁ -C ₆ alkyl group in R ⁴ is isopropyl (isopropyl-), n-hexyl (n-hexyl-) or n-butyl (n-butyl-).

Preferably, the C ₃ -C ₆ cycloalkyl group in R ⁴ is cyclopentyl (cyclopentyl-), cyclohexyl (cyclopentyl-) or cyclobutyl (cyclobutyl-).

Preferably, the cycloalkylalkyl group in R ⁴ is cyclopropylmethyl-.

Preferably, the haloalkyl group in R ⁴ is 2,2,2-trifluoroethyl (2,2,2-trifluoroethyl-).

Preferably, the alkoxyalkyl group in R ⁴ is 3-isopropoxypropyl-.

Preferably, the arylalkyl group substituted in R ⁴ is 4-fluorobenzyl ((4-fluorophenyl)methyl-) or 4-methoxyphenethyl ((4-methoxyphenyl)ethyl-).

Preferably, the heterocyclyl alkyl group in R ⁴ is 2-pyrrolidin-1-yl)ethyl-, 4-pyridylmethyl-, 2-furylmethyl (2-furanylmethyl-) or 3-morpholinopropyl-.

中的一种。Preferably, the heterocycloalkyl group formed by said ^R3 and R4 ^together with the bridged nitrogen atom is selected from

one of the.

Preferably, the class of ursamide derivatives containing a pyrazole heterocycle is compound LB-1, compound LB-2, compound LB-3, compound LB-4, compound LB-5, compound LB-6, compound LB-7, compound LB-8, compound LB-9, compound LB-10, compound LB-11, compound LB-12, compound LB-13, compound LB-14 or compound LB-15, wherein, the compound of The R ¹ , R ² , R ³ , R ⁴ groups are shown in Table 1 below.

Table 1. List of R ¹ , R ² , R ³ , R ⁴ groups for compounds

The second aspect of the present invention provides a kind of synthetic method of ursamide derivatives containing pyrazole heterocycle, and its synthetic route is as follows:

Specifically include the following steps:

a) Using ursolic acid (1) as a raw material, the C-28 carboxyl group of ursolic acid (1) is protected by a benzyl group to obtain an intermediate (2);

Preferably, in step a), the ursolic acid (1) is commercial ursolic acid.

Preferably, in step a), the benzyl protection is to mix ursolic acid (1) with potassium carbonate (K ₂ CO ₃ ), N,N-dimethylamide (DMF), benzyl bromide (BnBr) After heating reaction, the obtained mixture was cooled to room temperature, water was added to precipitate a solid product, and the solid product was filtered, washed and dried to obtain intermediate (2).

More preferably, the molar (mol) ratio of the ursolic acid (1) and potassium carbonate added is 1:1-3. Further preferably, the molar (mol) ratio of the ursolic acid (1) and potassium carbonate added is 1:1.5.

More preferably, the molar (mol) ratio of the ursolic acid (1) and N,N-dimethylamide added is 1:9-11. Further preferably, the molar (mol) ratio of the ursolic acid (1) and N,N-dimethylamide added is 1:10.

More preferably, the molar (mol) ratio of the ursolic acid (1) and benzyl bromide added is 1:1-2. Further preferably, the molar (mol) ratio of the ursolic acid (1) and benzyl bromide added is 1:1.5.

More preferably, the conditions of the heating reaction are as follows: the reaction temperature is 50-70° C.; and the reaction time is 3-5 h. Further preferably, the conditions of the heating reaction are: the reaction temperature is 60° C.; the reaction time is 4 h.

More preferably, the room temperature is 20-25°C.

More preferably, the ratio of the added mass (mg) of the ursolic acid (1) to the added volume (mL) of the water is 450-470:40-60. Further preferably, the ratio of the added mass (mg) of the ursolic acid (1) to the added volume (mL) of the water is 460:50.

Preferably, in step a), the yield of the intermediate (2) is 90-94%.

More preferably, in step a), the washing is performed multiple times with water.

b) adding PCC to the intermediate (2) to carry out oxidation reaction, oxidizing the C-3 hydroxyl group of the intermediate (2) to form a carbonyl group to obtain the intermediate (3);

Preferably, in step b), the oxidation reaction is to dissolve the intermediate (2) in dichloromethane and then cool it to below 0° C., add PCC and stir at room temperature to carry out the oxidation reaction, and filter and concentrate the obtained reaction product. , after separation and purification, intermediate (3) was obtained.

More preferably, the ratio of the added mass (mg) of the intermediate (2) to the added volume (mL) of dichloromethane is 440-460:40-60. Further preferably, the ratio of the added mass (mg) of the intermediate (2) to the added volume (mL) of dichloromethane is 450:50.

More preferably, the molar ratio of the intermediate (2) and PCC added is 1:1.2-3. Further preferably, the molar ratio of the intermediate (2) and PCC added is 1:1.5. The PCC is a complex salt of pyridine and CrO ₃ in hydrochloric acid solution.

More preferably, the stirring time is 11-13h. Further preferably, the stirring time is 12h.

Preferably, in step b), the yield of the intermediate (3) is 83-85%.

c) Add the ester compound to the intermediate (3) under basic conditions to react, so that the -CO-R ² substituent is formed on the α-position of the carbonyl group at the C-3 position of the intermediate (3) to obtain the intermediate ( 4);

Preferably, in step c), the reaction is to dissolve the intermediate (3) in tetrahydrofuran (THF) and then cool it to below 0° C., add basic compounds and ester compounds, and stir and mix the reaction at room temperature to obtain The reaction product was quenched by adding water, and after extraction, washing, drying, filtration, concentration, separation and purification, intermediate ( ⁴ ) was obtained. definition.

More preferably, the ratio of the added mass (mg) of the intermediate (3) to the added volume (mL) of tetrahydrofuran is 250-350:15-25. Further preferably, the ratio of the added mass (mg) of the intermediate (3) to the added volume (mL) of tetrahydrofuran is 300:20.

More preferably, the basic compound is sodium methoxide.

More preferably, the molar ratio of the intermediate (3) to the basic compound is 1:1-2. Further preferably, the molar ratio of the intermediate (3) and the basic compound added is 1:1.2.

More preferably, the ester compound is selected from one of ethyl formate, ethyl acetate and ethyl trifluoroacetate.

More preferably, the molar ratio of the intermediate (3) to the ester compound is 1:1-2. Further preferably, the molar ratio of the intermediate (3) and the ester compound added is 1:1.0-1.5.

More preferably, the stirring time is 3-5h. Further preferably, the stirring time is 4h.

More preferably, the quenching reaction is to remove water-soluble substances in the reaction product that do not need to participate in subsequent reactions by adding water.

More preferably, the reaction conditions of the extraction are as follows: the extraction reagent is ethyl acetate, the extraction times are 3-4 times, and the amount of the extraction reagent is 25-35 ml. Further preferably, the reaction conditions of the extraction are as follows: the extraction reagent is ethyl acetate, the extraction times are 3 times, and the amount of the extraction reagent is 30 ml.

More preferably, the washing is performed for multiple times with water and salt in sequence.

More preferably, the drying is carried out using anhydrous sodium sulfate.

Preferably, in step c), the yield of the intermediate (4) is 65-75%.

d) Condensing intermediate (4) with a hydrazine compound, dehydrating the carbonyl group at position C-3 of intermediate (4) to form a pyrazole heterocycle of the R ² substituent to obtain intermediate (5);

Preferably, in step d), in the condensation reaction, the intermediate (4) and the hydrazine compound are dissolved in an organic solvent, heated and stirred for reaction and then cooled to room temperature, and the obtained reaction product is concentrated, washed, separated and purified, Intermediate (5) in which R ¹ and R ² have the same definitions as in the compound of formula 8 is obtained.

More preferably, the hydrazine compound is selected from one of alkylhydrazine, arylhydrazine, and heteroarylhydrazine. Further preferably, the hydrazine compound is selected from one of phenylhydrazine, p-toluenehydrazine, p-chlorophenylhydrazine, p-bromophenylhydrazine, p-carboxyphenylhydrazine, and p-cyanophenylhydrazine.

More preferably, the molar ratio of the intermediate (4) to the hydrazine compound is 1:1-2. Further preferably, the molar ratio of the intermediate (4) and the hydrazine compound added is 1:1.

More preferably, the organic solvent is ethanol.

More preferably, the ratio of the added mass (mg) of the intermediate (4) to the added volume (mL) of the organic solvent is 310-330:15-25. Further preferably, the ratio of the added mass (mg) of the intermediate (4) to the added volume (mL) of the organic solvent is 320:20.

More preferably, the conditions for the heating and stirring reaction are: the heating temperature is 80-90° C., and the stirring time is 11-13 h.

Further preferably, the conditions of the heating and stirring reaction are as follows: the heating temperature is 85° C. and the stirring time is 12 h.

More preferably, the washing is performed multiple times with water.

Preferably, in step d), the yield of the intermediate (5) is 80-90%.

e) hydrogenation of intermediate (5) is carried out to remove the benzyl protecting group on the carboxyl group at position C-28 of intermediate (5) to obtain intermediate (6);

Preferably, in step e), in the hydrogenation reaction, the intermediate (5) and the catalyst are dissolved in an organic solvent, and then hydrogen is introduced for the reaction, and the obtained reaction product is filtered, washed, concentrated, stirred and slurried, and filtered again. , and drying to obtain the desired intermediate (6). In the intermediate (6), R ¹ and R ² have the same definitions as in the compound of formula 8.

More preferably, the catalyst is a 10 wt% palladium-carbon mixture (the weight ratio of Pd to C is 10:90).

More preferably, the mass ratio of the intermediate (5) to the catalyst added is 30-40:45-55. Further preferably, the mass ratio of the intermediate (5) to the catalyst added is 35:50.

More preferably, the organic solvent is methanol.

More preferably, the ratio of the added mass (mg) of the intermediate (5) to the added volume (mL) of the organic solvent is 30-40:15-25. Further preferably, the ratio of the added mass (mg) of the intermediate (5) to the added volume (mL) of the organic solvent is 35:20.

More preferably, the hydrogen gas is fed under normal pressure.

More preferably, the catalytic hydrogenation reaction is carried out under normal pressure.

More preferably, the washing is performed multiple times with methanol.

More preferably, the re-filtration is a vacuum filtration method.

Preferably, in step e), the yield of the intermediate (6) is 85-90%.

f) acyl-chloride the C-28 carboxyl group in the intermediate (6) to obtain the intermediate (7);

Preferably, in step f), the acid chlorination is to dissolve the intermediate (6) in anhydrous dichloromethane and cool it to below 0°C, and then sequentially add oxalyl chloride, N,N-dimethylamide (DMF) ), stir and mix the reaction at room temperature, concentrate and dry to obtain intermediate (7), in which R ¹ and R ² have the same definitions as in the compound of formula 8.

More preferably, the ratio of the added mass (g) of the intermediate (6) to the added volume (ml) of anhydrous dichloromethane is 1:2-10.

More preferably, the cooling is performed in an ice-water bath to below 0°C.

More preferably, the molar ratio of the intermediate (6) to oxalyl chloride is 1:1-2.

More preferably, the molar ratio of the intermediate (6) and N,N-dimethylamide added is 1:0.01-0.1.

More preferably, the stirring time is 5-7h. Further preferably, the stirring time is 6h.

g) The C-28 carboxylic acid chloride of the intermediate (7) forms an amide bond with an amine compound to obtain the desired compound (8).

Preferably, in step g), forming an amide bond is to dissolve the intermediate (7) in N,N-dimethylamide (DMF), add basic compounds and amine compounds in sequence, and stir and mix at room temperature After reaction, concentration, separation and purification, stirring and beating, and drying, the desired compound (8) is obtained.

More preferably, the ratio of the added mass (g) of the intermediate (7) to the added volume (ml) of N,N-dimethylamide (DMF) is 1:2-10.

More preferably, the basic compound is triethylamine (Et ₃ N) or N,N-diisopropylethylamine (iPr ₂ NEt).

More preferably, the molar ratio of the intermediate (7) to the basic compound is 1:1-2.

More preferably, the general structural formula of the amine compound is: R ³ R ⁴ NH, where R ³ and R ⁴ have the same definitions as in the compound of formula 8.

More preferably, the molar ratio of the amount of the intermediate (7) and the amine compound added is 1:1-1.5.

More preferably, in step e) or g), the stirring and beating is stirring and beating the residue through diethyl ether.

Preferably, in step a), b), c) or e), the filtration is to filter the reaction product through diatomaceous earth to remove insoluble matter.

Preferably, in step b), c), d), e), f) or g), the concentration is to concentrate the reaction product by pressure distillation.

Preferably, in step b), c), d) or g), the separation and purification is to separate and purify the reaction product through silica gel column chromatography.

Preferably, in step a), e), f) or g), the drying is vacuum drying, and the conditions of the vacuum drying are respectively 55-65° C. and vacuum degree 0.005-0.015Pa. The vacuum drying is a conventional drying method in which an oil pump is used for vacuuming.

The third aspect of the present invention provides the use of a class of ursamide derivatives containing a pyrazole heterocycle in the preparation of a medicament for treating tumors.

Preferably, the tumor is cervical cancer or lung cancer.

More preferably, the cells of the tumor are one or more of cervical cancer cell Hela or lung cancer cell SPC-A-1.

The mechanism of preparing the ursamide derivatives containing a pyrazole heterocycle for the treatment of tumors is to kill tumor cells, thereby inhibiting their activity, thereby inhibiting the growth of tumor cells.

A fourth aspect of the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of a class of pyrazole heterocycle-containing ursamide derivatives.

As mentioned above, a class of ursolic acid derivatives containing pyrazole heterocycles provided by the present invention and their synthesis and application, through the structural modification of the ursolic acid A ring, a series of ursolic acid A ring with Ursamide derivatives of pyrazole heterocycles. The antitumor activity study in vitro shows that this ursolic acid triterpenoid derivative has obvious inhibitory effect on the growth of tumor cells, and has the potential for the development of new antitumor drugs.

Description of drawings

Figure 1 is a schematic diagram showing the inhibitory activity of a class of pyrazole heterocycle-containing ursamide derivatives on tumor cells HeLa in different time periods.

Fig. 2 is a schematic diagram showing the inhibitory activity of a class of pyrazole heterocycle-containing ursamide derivatives on tumor cells SPC-A-1 in different time periods.

Detailed ways

The present invention will be further described below with reference to specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the protection scope of the present invention.

The embodiments of the present invention are described below through specific specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

The reagents used in the following examples are all commonly used reagents in the art, and the used instruments are all commonly used instruments in the art.

Example 1

Dissolve 460 mg of ursolic acid (1) and 280 mg of anhydrous potassium carbonate in 10 ml of N,N-dimethylamide, add 300 mg of BnBr, and heat the reaction mixture at about 60° C. for 4 hours until the reaction is complete. The mixture was cooled to room temperature, 50 mL of _H2O was added, and a white solid was precipitated. The obtained solid was filtered, washed with water, and dried in vacuum to obtain 0.50 g of intermediate (2), and the yield of intermediate (2) was 92%.

The spectral identification results of intermediate (2) are as follows:

¹ H NMR (500MHz, CDCl ₃ ): δ=7.36-7.29 (m, 5H), 5.24-5.23 (m, 1H), 5.10 (d, J=12.5Hz, 1H), 4.98 (d, J=12.5Hz) ,1H),3.20(dd,J＝11.0,4.5Hz,1H),2.04-1.98(m,1H),1.89-1.76(m,3H),1.73-1.67(m,2H),1.64-1.55(m ,4H),1.53-1.44(m,6H),1.37-1.26(m,6H),1.07(s,3H),1.05-1.02(m,2H),0.98(s,3H),0.93(d,J =6.0Hz,3H),0.89(s,3H),0.85(d,J=6.5Hz,3H),0.78(s,3H),0.64(s,3H).

¹³ C NMR (125 MHz, CDCl ₃ ): δ=177.3, 138.1, 136.3, 128.4 (2C), 128.1 (2C), 127.9, 125.7, 79.0, 66.0, 55.2, 52.9, 48.1, 47.5, 42.0, 39.5, 39.1, 38.8,38.7,38.6,36.9,36.6,33.0,30.6,28.1,27.9,27.2,24.2,23.5,23.2,21.2,18.3,17.0(2C),15.6,15.4.

The synthesis process is as follows:

Example 2

Dissolve 450 mg of intermediate (2) in 50 mL of anhydrous dichloromethane, cool to below 0°C, add 355 mg of PCC to react, and the mixture is slowly raised to room temperature and stirred for about 12 hours until the reaction is complete. The reaction mixture was filtered through celite to remove insolubles, the organic phase was concentrated under pressure, and the residue was separated and purified by silica gel column chromatography to obtain 375 mg of intermediate (3), which was a white solid, and the intermediate (3) was a white solid. Yield 84%.

The spectral identification results of intermediate (3) are as follows:

¹ H NMR (500MHz, CDCl ₃ ): δ=7.37-7.29 (m, 5H), 5.25 (t, J=3.5Hz, 1H), 5.11 (d, J=12.5Hz, 1H), 4.99 (d, J ＝12.5Hz, 1H), 2.57-2.50(m, 1H), 2.40-2.34(m, 1H), 2.27(d, J＝11.5Hz, 1H), 2.04-1.98(m, 1H), 1.92-1.87( m,3H),1.82-1.68(m,3H),1.64-1.54(m,3H),1.50-1.41(m,5H),1.35-1.28(m,4H),1.10-1.07(m,1H), 1.08(s,6H),1.03(d,J＝9.0Hz,6H),0.93(d,J＝6.0Hz,3H),0.85(d,J＝6.5Hz,3H),0.68(s,3H).

¹³ C NMR (125MHz, CDCl ₃ ): δ=177.2, 138.2, 136.3, 128.4(2C), 128.1(2C), 127.9, 125.4, 66.0, 55.2, 52.9, 48.1, 47.4, 46.7, 42.1, 39.4, 39.3, 39.1, 38.8, 36.6, 36.5, 34.2, 32.5, 30.6, 27.9, 26.5, 24.2, 23.5, 23.3, 21.4, 21.1, 19.5, 17.0, 16.9, 15.2.

The synthesis process is as follows:

Example 3

300 mg of intermediate (3) was dissolved in 20 mL of anhydrous tetrahydrofuran, the reaction solution was cooled to 0° C., 45 mg of sodium methoxide and then 45 mg of ethyl formate were added thereto. The reaction mixture was stirred at room temperature for about 4 hours until the reaction was complete. A small amount of water was added to quench the reaction and the mixture was extracted three times with ethyl acetate (3 x 30 ml). The combined organic phases were washed successively with water and brine, dried over anhydrous sodium sulfate, and filtered. The organic phase was concentrated, and the residue was rapidly separated by silica gel column chromatography to obtain intermediate (4a).

The spectral identification results of intermediate (4a) are as follows:

¹ H NMR (500MHz, CDCl ₃ ): δ=14.92 (br s, 1H), 8.57 (s, 1H), 7.37-7.30 (m, 5H), 5.28 (t, J=3.3Hz, 1H), 5.12 ( d, J=12.5Hz, 1H), 4.99 (d, J=12.5, 1H), 2.32-2.28 (m, 2H), 2.05-1.92 (m, 4H), 1.82-1.69 (m, 3H), 1.65- 1.56(m,2H),1.50-1.43(m,3H),1.39-1.26(m,5H),1.18(s,3H),1.14-1.10(m,2H),1.11(s,3H),1.08( s,3H),0.94(d,J=6.5Hz,3H),0.89(s,3H),0.86(d,J=6.5Hz,3H),0.68(s,3H).

¹³ C NMR (125MHz, CDCl ₃ ): δ=177.2, 138.1, 136.3, 128.4(2C), 128.2(2C), 127.9, 125.4, 105.8, 66.0, 53.0, 52.0, 48.1, 45.5, 42.2, 40.1, 39.4, 39.3, 39.1, 38.8, 36.6, 36.2, 32.3, 30.6, 28.4, 27.9, 24.2, 23.4, 23.3, 21.1, 20.9, 19.4, 17.0, 16.9, 14.6.

HRMS(ESI): m/z[M+H] ⁺ calcd for C ₃₈ H ₅₂ O ₄ : 572.3866; found: 572.3871.

The synthesis process is as follows:

Example 4

300 mg of intermediate (3) was dissolved in 20 mL of anhydrous tetrahydrofuran, the reaction solution was cooled to 0°C, 45 mg of sodium methoxide was added thereto, and then 55 mg of ethyl acetate was added. The reaction was stirred at room temperature for about 4 hours until the reaction was complete, and a small amount of water was added to quench the reaction. The mixture was extracted three times with ethyl acetate (3 x 30 mL). The combined organic phases were washed with water, washed with brine, dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated by pressure distillation, and the residue was rapidly separated by silica gel column chromatography to obtain intermediate (4b).

The synthesis process is as follows:

Example 5

300 mg of the intermediate (3) was dissolved in 20 mL of anhydrous tetrahydrofuran, the reaction solution was cooled to 0°C, 45 mg of sodium methoxide and then 55 mg of ethyl trifluoroacetate were added thereto. The reaction was stirred at room temperature for about 4 hours until the reaction was complete, and a small amount of water was added to quench the reaction. The mixture was extracted three times with ethyl acetate (3 x 30 mL). The combined organic phases were washed with water, washed with brine, dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated by pressure distillation, and the residue was rapidly separated by silica gel column chromatography to obtain intermediate (4c).

The synthesis process is as follows:

Example 6

Dissolve 320 mg of intermediate (4a) and 95 mg of 4-cyanophenylhydrazine hydrochloride in 20 mL of ethanol, heat to 80° C. and stir the reaction for about 12 hours until the reaction is completed. The reaction solution was cooled to room temperature, the solvent was distilled off under reduced pressure, the residue was washed with water several times, and the initial product was further purified by silica gel column chromatography to obtain 315 mg of intermediate (5), intermediate (5) was a white solid, intermediate (5). ) in 84% yield.

The spectral identification results of intermediate (5) are as follows:

¹ H NMR (500 MHz, CDCl ₃ ): δ=7.78 (d, J=8.5 Hz, 2H), 7.55 (d, J=8.5 Hz, 2H), 7.34 (s, 1H), 7.37-7.31 (m, 5H) ),5.32(t,J＝3.0Hz,1H),5.12(d,J＝12.5,1H),4.98(dJ＝12.5Hz,1H),4.14-4.09(m,1H),2.77(d,J＝ 15.5Hz, 1H), 2.31(d, J=11.0Hz, 1H), 2.15(d, J=15.5Hz, 1H), 2.05-1.99(m, 3H), 1.90-1.84(m, 1H), 1.82- 1.60(m,4H),1.50-1.47(m,3H),1.39-1.24(m,6H),1.12-1.08(m,1H),1.09(s,3H),1.03(s,3H),1.02( s,3H),0.95(s,3H),0.93(s,3H),0.88(d,J＝3.9Hz,3H),0.69(s,3H).

¹³ C NMR (125MHz, CDCl ₃ ): δ=177.3, 146.7, 146.5, 139.5, 138.1, 136.4, 132.6, 130.0, 128.5(2C), 128.2(2C), 128.0, 125.7, 118.1, 115.1, 112.9, 66.1, 58.5,54.6,53.1,48.3,46.4,42.2,39.5,39.2,38.9,38.0,37.1,36.7,34.7,32.6,30.7,29.7,28.0,24.3,23.4,23.3,22.8,21.2,19.2,18.5,17.0, 16.9, 15.5.

HRMS(ESI): m/z[M+H] ⁺ calcd for C ₄₅ H ₅₅ N ₃ O ₂ : 669.4294; found: 669.4283.

The synthesis process is as follows:

Example 7

Dissolve 35 mg of intermediate (5) and 50 mg of 10% Pd/C in 20 mL of methanol, and carry out catalytic hydrogenation at atmospheric pressure. After the reaction was completed, the palladium carbon was removed by filtration and washed with methanol for several times. The filtrate was concentrated under pressure, the obtained residue was stirred and slurried with ether, filtered, and the solid was dried in vacuo to obtain 25 mg of compound (6), which was a white solid, and the yield of compound (6) was 86%.

The spectral identification results of compound (6) are as follows:

¹ H NMR (500 MHz, CDCl ₃ ): δ=10.95 (br s, 1H), 7.75 (d, J=8.5 Hz, 2H), 7.52 (d, J=8.5 Hz, 2H), 7.37 (s, 1H) ,5.32(t,J＝3.5Hz,1H),2.64(d,J＝15.0Hz,1H),2.23(d,J＝11.5Hz,1H),2.14(d,J＝15.5Hz,1H),2.05 -1.99(m,3H),1.90-1.84(m,1H),1.72-1.66(m,4H),1.53-1.49(m,3H),1.42-1.26(m,6H),1.15-1.12(m, 1H), 1.10(s, 3H), 1.03(s, 3H), 0.99(s, 3H), 0.95(d, J=6.5Hz, 3H), 0.93(s, 3H), 0.89(d, J=6.0 Hz,3H),0.84(s,3H).

¹³ C NMR (125MHz, CDCl ₃ ): δ=183.6, 146.6, 146.2, 139.3, 137.8, 132.5(2C), 129.9(2C), 125.6, 117.9, 115.0, 112.9, 54.4, 52.6, 47.9, 46.3, 42.1, 39.4, 39.1, 38.7, 37.9, 36.9, 36.6, 34.6, 32.3, 30.6, 29.5, 27.9, 24.0, 23.4, 23.2, 22.7, 21.1, 19.1, 16.9, 16.7, 15.4.

HRMS(ESI): m/z[M+H] ⁺ calcd for C ₄₄ H ₆₃ N ₄ O ₂ : 579.3825; found: 579.3816.

The synthesis process is as follows:

Example 8

100 mg of intermediate (6) was dissolved in 20 mL of anhydrous CH ₂ Cl ₂ , cooled to below 0° C., and 25 μL of (COCl) ₂ and 30 mg of catalytic DMF were added in sequence. The reaction mixture was stirred at room temperature for 6 hours, the solvent was evaporated under reduced pressure, and the residue was vacuum-dried to obtain intermediate (7), which was directly subjected to the next reaction without purification.

The synthesis process is as follows:

The preparation of embodiment 9 compound LB-1

100 mg of intermediate (7) was taken and dissolved in 10 mL of anhydrous DMF, and 35 μL of Et ₃ N and 0.22 mmol of R ¹ R ² NH ₂ were sequentially added. The mixture was stirred at room temperature until the reaction was complete. The solvent was evaporated under high vacuum and reduced pressure. The residue was rapidly purified by silica gel column chromatography. The obtained product was slurried with methyl tert-butyl ether. The collected solid was dried to obtain 102 mg of compound LB-1. Compound LB-1 was a white solid. The yield of compound LB-1 was 89%.

The spectral identification results of compound LB-1 are as follows:

¹ H NMR (500MHz, Chloroform-d)δ=7.85-7.70(m, 2H), 7.59-7.49(m, 2H), 7.38(s, 1H), 6.79(t, J=5.2Hz, 1H), 5.40 (t, J=3.6Hz, 1H), 3.50 (dt, J=16.5, 5.6Hz, 1H), 3.40–3.23 (m, 1H), 2.73 (m, 7H), 2.17 (d, J=15.0Hz, 1H), 2.09 (m, 2H), 2.05–1.95 (m, 2H), 1.95–1.82 (m, 5H), 1.82–1.59 (m, 3H), 1.56–1.37 (m, 5H), 1.36–1.23 ( m, 4H), 1.13 (s, 4H), 1.04 (d, J=15.8Hz, 6H), 0.99–0.93 (m, 5H), 0.92 (d, J=6.5Hz, 3H), 0.90–0.83 (m ,5H)ppm.

HRMS(ESI): m/z[M+H] ⁺ calcd for C ₄₄ H ₆₂ N ₅ O: 676.4954; found: 676.4957.

The synthesis process is as follows:

Example 10 Preparation of compound LB-2

The preparation method is the same as the preparation method of compound LB-1. The yield of compound LB-2 was 72%.

The spectral identification results of compound LB-2 are as follows:

¹ H NMR (500MHz, Chloroform-d)δ=7.93-7.63(m, 2H), 7.63-7.45(m, 2H), 7.39(s, 1H), 5.83(d, J=6.5Hz, 1H), 5.39 (t, J=3.6Hz, 1H), 4.12 (m, 1H), 2.67 (d, J=14.9Hz, 1H), 2.18 (d, J=14.9Hz, 1H), 2.13–2.04 (m, 2H) , 2.03–1.82 (m, 5H), 1.81–1.57 (m, 8H), 1.57–1.40 (m, 7H), 1.38–1.28 (m, 2H), 1.14 (s, 4H), 1.04 (d, J= 12.9Hz, 6H), 0.97(s, 7H), 0.94–0.82(m, 7H) ppm.

HRMS(ESI): m/z[M+H] ⁺ calcd for C ₄₃ H ₅₉ N ₄ O: 647.4689; found: 647.4694.

The synthesis process is as follows:

Example 11 Preparation of compound LB-3

The preparation method is the same as the preparation method of compound LB-1. The yield of compound LB-3 was 72%.

The spectral identification results of compound LB-3 are as follows:

¹ H NMR (500MHz, Chloroform-d)δ=8.57(d,J=5.0Hz,2H),7.78(d,J=8.2Hz,2H),7.54(d,J=8.3Hz,2H),7.39( s, 1H), 7.20 (d, J=5.0Hz, 2H), 6.34 (dt, J=11.5, 5.8Hz, 1H), 5.40 (t, J=3.5Hz, 1H), 4.62 (dt, J=15.5 ,7.7Hz,1H),4.19(dd,J＝15.6,5.2Hz,1H),2.64(dd,J＝15.0,5.6Hz,1H),2.21–2.12(m,1H),2.12–1.90(m, 6H), 1.82(m, 1H), 1.71(m, 2H), 1.62-1.47(m, 5H), 1.41-1.28(m, 4H), 1.15(s, 4H), 1.04(d, J=13.7Hz ,6H),0.99(d,J＝5.2Hz,3H),0.96–0.89(m,6H),0.76(s,3H)ppm.

HRMS(ESI): m/z[M+H] ⁺ calcd for C ₄₄ H ₅₆ N ₅ O: 670.4485; found: 670.4494.

The synthesis process is as follows:

Example 12 Preparation of compound LB-4

The preparation method is the same as the preparation method of compound LB-1. The yield of compound LB-4 was 75%.

The spectral identification results of compound LB-4 are as follows:

¹ H NMR (500MHz, Chloroform-d)δ=7.81-7.73(m,2H), 7.57-7.51(m,2H), 7.41-7.35(m,2H), 6.33(m,1H), 6.27(q, J=5.3Hz, 1H), 6.20 (d, J=3.3Hz, 1H), 5.38 (t, J=3.5Hz, 1H), 4.44–4.32 (m, 2H), 2.64 (dd, J=14.9, 6.1 Hz, 1H), 2.15 (d, J=14.9, 5.1 Hz, 1H), 2.09–1.97 (m, 3H), 1.97–1.87 (m, 2H), 1.78 (m, 1H), 1.75–1.64 (m, 3H), 1.57–1.46 (m, 5H), 1.44–1.34 (m, 2H), 1.33–1.21 (m, 2H), 1.12 (m, 4H), 1.03 (d, J=12.7Hz, 6H), 0.97 (d,J＝2.6Hz,3H),0.93(s,3H),0.90(d,J＝6.5Hz,3H),0.74(s,3H)ppm.

HRMS(ESI): m/z[M+H] ⁺ calcd for C ₄₃ H ₅₅ N ₄ O ₂ : 659.4325; found: 659.4327.

The synthesis process is as follows:

Example 13 Preparation of compound LB-5

The preparation method is the same as the preparation method of compound LB-1. The yield of compound LB-5 was 83%.

The spectral identification results of compound LB-5 are as follows:

¹ H NMR (500MHz, Chloroform-d)δ=7.79-7.73(m, 2H), 7.57-7.51(m, 2H), 7.38(s, 1H), 5.33(d, J=3.6Hz, 1H), 3.63 (d, J=17.4Hz, 4H), 2.65 (d, J=15.0Hz, 1H), 2.53–2.49 (m, 1H), 2.43–2.36 (m, 5H), 2.31 (s, 4H), 2.15 ( d, J=15.2Hz, 2H), 2.11–2.05 (m, 2H), 1.82–1.75 (m, 2H), 1.72 (m, 2H), 1.54–1.49 (m, 3H), 1.42 (qd, J= 9.7,8.2,4.8Hz,3H),1.32-1.25(m,1H),1.12(s,4H),1.09-1.00(m,7H),1.00-0.94(m,6H),0.92(d,J= 6.4Hz,3H),0.85(d,J=9.3Hz,3H)ppm.

HRMS(ESI): m/z[M+H] ⁺ calcd for C ₄₃ H ₆₀ N ₅ O: 662.4798; found: 662.4810.

The synthesis process is as follows:

Example 14 Preparation of compound LB-6

The preparation method is the same as the preparation method of compound LB-1. The yield of compound LB-6 was 72%.

The spectral identification results of compound LB-6 are as follows:

¹ H NMR (500MHz, Chloroform-d) δ=7.77(d, J=8.2Hz, 2H), 7.54(d, J=8.2Hz, 2H), 7.38(s, 1H), 6.43(t, J=5.3 Hz, 1H), 5.39(d, J=3.5Hz, 1H), 3.76(t, J=4.7Hz, 4H), 3.45(m, 1H), 3.11(m, 1H), 2.70–2.62(m, 1H) ), 2.49–2.39 (m, 6H), 2.17 (d, J=15.2Hz, 1H), 2.13–1.96 (m, 4H), 1.83 (m, 1H), 1.78–1.66 (m, 5H), 1.51 ( m, 4H), 1.42 (m, 3H), 1.31–1.25 (m, 3H), 1.14 (s, 4H), 1.04 (d, J=15.4Hz, 6H), 0.97 (d, J=12.1Hz, 6H) ),0.93(d,J＝6.4Hz,3H),0.86(s,3H)ppm.

HRMS(ESI): m/z[M+H] ⁺ calcd for C ₄₅ H ₆₄ N ₅ O ₂ : 706.5060; found: 706.5086.

The synthesis process is as follows:

Example 15 Preparation of compound LB-7

The preparation method is the same as the preparation method of compound LB-1. The yield of compound LB-7 was 78%.

The spectral identification results of compound LB-7 are as follows:

¹ H NMR (500 MHz, Chloroform-d) δ=7.78 (d, J=8.0 Hz, 2H), 7.55 (d, J=8.1 Hz, 2H), 7.39 (s, 1H), 7.25 (dd, J=8.5 ,5.4Hz,2H),7.03(t,J＝8.5Hz,2H),6.17(t,J＝5.4Hz,1H),5.33(t,J＝3.5Hz,1H),4.50(dd,J＝14.6 ,5.9Hz,1H),4.21(dd,J＝14.5,4.9Hz,1H),2.69-2.60(m,1H),2.16(d,J＝15.0Hz,1H),2.10-1.89(m,5H) ,1.80(m,1H),1.71(m,3H),1.58–1.45(m,5H),1.40(m,2H),1.30(m,2H),1.14(s,4H),1.05(d,J =11.9Hz,6H),0.98(s,3H),0.94(s,3H),0.91(d,J=6.3Hz,3H),0.76(s,3H)ppm.

HRMS(ESI): m/z[M+H] ⁺ calcd for C ₄₅ H ₅₆ FN ₄ O: 687.4438; found: 687.4445.

The synthesis process is as follows:

Example 16 Preparation of compound LB-8

The preparation method is the same as the preparation method of compound LB-1. The yield of compound LB-8 was 85%.

The spectral identification results of compound LB-8 are as follows:

¹ H NMR (500MHz, Chloroform-d)δ=7.82-7.71(m,2H),7.59-7.50(m,2H),7.40(s,1H),7.19-7.08(m,2H),6.93-6.87( m, 2H), 5.89(dd, J=7.0, 3.7Hz, 1H), 5.04(t, J=3.4Hz, 1H), 3.83(s, 3H), 3.72(m, 1H), 3.26–3.11(m ,1H),2.82(m,1H),2.71–2.65(m,1H),2.61(d,J=15.0Hz,1H),2.14(d,J=15.0Hz,1H),1.94(m,3H) ,1.77-1.59(m,6H),1.54-1.34(m,7H),1.33-1.24(m,4H),1.09(s,4H),1.04(d,J＝10.4Hz,6H),0.97(s ,4H),0.93(s,3H),0.71(s,3H)ppm.

HRMS(ESI): m/z[M+H] ⁺ calcd for C ₄₆ H ₅₉ N ₄ O ₂ : 713.4795; found: 713.4796.

The synthesis process is as follows:

Example 17 Preparation of compound LB-9

The preparation method is the same as the preparation method of compound LB-1. The yield of compound LB-9 was 81%.

The spectral identification results of compound LB-9 are as follows:

¹ H NMR (500 MHz, Chloroform-d) δ=7.77 (d, J=8.2 Hz, 2H), 7.54 (d, J=8.2 Hz, 2H), 7.39 (s, 1H), 5.97 (dd, J=6.9 ,4.3Hz,1H),5.42(d,J＝3.5Hz,1H),3.28(dt,J＝13.4,6.8Hz,1H),2.79(m,4.2Hz,1H),2.66(d,J＝15.0 Hz, 1H), 2.18 (d, J=15.0Hz, 1H), 2.10 (m, 2H), 2.04–1.85 (m, 3H), 1.82–1.62 (m, 5H), 1.59–1.37 (m, 7H) ,1.37–1.22(m,2H),1.14(s,4H),1.04(d,J=13.8Hz,6H),0.97(d,J=8.1Hz,6H),0.95–0.90(m,9H), 0.86(s,3H)ppm.

HRMS(ESI): m/z[M+H] ⁺ calcd for C ₄₂ H ₅₉ N ₄ O: 635.4689; found: 635.4694.

The synthesis process is as follows:

Example 18 Preparation of compound LB-10

The preparation method is the same as the preparation method of compound LB-1. The yield of compound LB-10 was 76%.

The spectral identification results of compound LB-10 are as follows:

¹ H NMR (500MHz, Chloroform-d)δ=7.83-7.71(m, 2H), 7.60-7.48(m, 2H), 7.39(s, 1H), 6.18(dd, J=7.4, 4.9Hz, 1H) ,5.46(t,J＝3.6Hz,1H),4.22-4.11(m,1H),3.59(m,1H),2.67(d,J＝14.9Hz,1H),2.18(d,J＝15.0Hz, 1H), 2.11 (m, 2H), 2.08–2.03 (m, 1H), 1.99 (m, 1H), 1.86 (m, 1H), 1.80–1.72 (m, 2H), 1.72–1.63 (m, 2H) ,1.60-1.46(m,5H),1.46-1.36(m,2H),1.33-1.26(m,2H),1.15(m,4H),1.04(d,J＝14.6Hz,6H),0.98(d , J=11.3Hz, 6H), 0.93 (d, J=6.4Hz, 3H), 0.84 (s, 3H) ppm.

HRMS(ESI): m/z[M+H] ⁺ calcd for C ₄₀ H ₅₂ F ₃ N ₄ O: 661.4093; found: 661.4098.

The synthesis process is as follows:

Example 19 Preparation of compound LB-11

The preparation method is the same as the preparation method of compound LB-1. The yield of compound LB-11 was 82%.

The spectral identification results of compound LB-11 are as follows:

HRMS(ESI): m/z[M+H] ⁺ calcd for C ₄₂ H ₅₇ N ₄ O ₂ : 649.4482; found: 649.4470.

The synthesis process is as follows:

Example 20 Preparation of compound LB-12

The preparation method is the same as the preparation method of compound LB-1. The yield of compound LB-12 was 76%.

The spectral identification results of compound LB-12 are as follows:

¹ H NMR (500MHz, Chloroform-d) δ=7.77(d, J=8.0Hz, 2H), 7.54(d, J=8.2Hz, 2H), 7.39(s, 1H), 6.03(t, J=5.1 Hz,1H),5.44(d,J＝3.7Hz,1H),3.22(dt,J＝13.0,6.3Hz,1H),2.90(ddd,J＝13.8,7.6,4.2Hz,1H),2.67(d , J=14.9Hz, 1H), 2.18 (d, J=15.0Hz, 1H), 2.11 (dq, J=7.6, 3.8, 3.0Hz, 2H), 2.03–1.86 (m, 3H), 1.82–1.65 ( m, 4H), 1.61–1.36 (m, 7H), 1.34–1.25 (m, 2H), 1.15 (s, 4H), 1.04 (d, J=14.2Hz, 6H), 0.98 (d, J=8.6Hz, 6H),0.93(d,J=6.4Hz,3H),0.88(s,4H),0.51(dt,J=8.1,1.9Hz,2H),0.26-0.12(m,2H)ppm.

HRMS(ESI): m/z[M+H] ⁺ calcd for C ₄₂ H ₅₇ N ₄ O: 633.4532; found: 633.4539.

The synthesis process is as follows:

Example 21 Preparation of compound LB-13

The preparation method is the same as the preparation method of compound LB-1. The yield of compound LB-13 was 76%.

The spectral identification results of compound LB-13 are as follows:

¹ H NMR (500MHz, Chloroform-d)δ=7.86-7.70(m, 2H), 7.62-7.45(m, 2H), 7.39(s, 1H), 5.97-5.84(m, 1H), 5.41(t, J=3.6Hz, 1H), 3.31 (dq, J=13.4, 6.8Hz, 1H), 3.07 (dtd, J=11.6, 7.1, 4.4Hz, 1H), 2.66 (d, J=14.9Hz, 1H), 2.18 (d, J=15.0Hz, 1H), 2.14–2.07 (m, 2H), 1.99 (td, J=13.6, 4.0Hz, 1H), 1.95–1.85 (m, 2H), 1.82–1.65 (m, 4H), 1.59–1.38 (m, 10H), 1.36–1.28 (m, 8H), 1.14 (s, 4H), 1.04 (d, J=14.2Hz, 6H), 0.97 (d, J=6.0Hz, 6H) ),0.94–0.88(m,6H),0.87(s,3H)ppm.

HRMS(ESI): m/z[M+H] ⁺ calcd for C ₄₄ H ₆₃ N ₄ O: 663.5002; found: 663.5005.

The synthesis process is as follows:

Example 22 Preparation of compound LB-14

The preparation method is the same as the preparation method of compound LB-1. The yield of compound LB-14 was 76%.

The spectral identification results of compound LB-14 are as follows:

¹ H NMR (500MHz, Chloroform-d)δ=7.84-7.68(m, 2H), 7.61-7.46(m, 2H), 7.38(s, 1H), 6.39(t, J=5.1Hz, 1H), 5.39 (t, J=3.7Hz, 1H), 3.60–3.52 (m, 2H), 3.50–3.44 (m, 1H), 3.41–3.34 (m, 1H), 3.24 (dq, J=13.3, 5.7Hz, 1H) ), 2.66(d, J＝15.0Hz, 1H), 2.17(d, J＝15.0Hz, 1H), 2.09(m, 2H), 2.03–1.94(m, 2H), 1.81(m, 2H), 1.74 (m, 4H), 1.52 (m, 4H), 1.45–1.37 (m, 3H), 1.20 (dd, J=6.1, 1.5Hz, 8H), 1.13 (s, 4H), 1.04 (d, J=14.9 Hz, 6H), 1.00–0.94(m, 7H), 0.91(d, J=6.5Hz, 3H), 0.87(s, 3H) ppm.

HRMS(ESI): m/z[M+H] ⁺ calcd for C ₄₄ H ₆₃ N ₄ O ₂ : 679.4951; found: 679.4964.

The synthesis process is as follows:

Example 23 Preparation of compound LB-15

The preparation method is the same as the preparation method of compound LB-1. The yield of compound LB-15 was 76%.

The spectral identification results of compound LB-14 are as follows:

¹ H NMR (500MHz, Chloroform-d)δ=7.83-7.71(m, 2H), 7.60-7.51(m, 2H), 7.39(s, 1H), 5.34(d, J=3.8Hz, 1H), 3.47 (s, 3H), 2.65 (d, J=15.0Hz, 1H), 2.56 (d, J=11.2Hz, 1H), 2.21–1.99 (m, 4H), 1.93–1.69 (m, 7H), 1.56– 1.48(m,3H),1.47-1.37(m,3H),1.34-1.24(m,4H)1.13(s,4H),1.04(d,J=15.7Hz,6H),0.97(d,J=6.3 Hz,3H),0.96–0.91(m,6H),0.91–0.85(m,2H),0.85(s,3H)ppm.

HRMS(ESI): m/z[M+H] ⁺ calcd for C ₄₂ H ₅₇ N ₄ O: 633.4532; found: 633.4539.

The synthesis process is as follows:

Example 24 In vitro antitumor activity of ursamide derivatives

The anti-tumor activity of arbutamide derivatives was studied, tumor cells HeLa and SPC-A-1 were selected as detection cells, and CCK-8 colorimetry was used as detection method. Cell inhibition rate.

Preparation of experimental liquid: Dissolve the test sample with a small amount of DMSO to prepare a stock solution, that is, prepare a stock solution according to 1000 times the highest concentration of the test. The stock solution was stored in a -20°C refrigerator for later use.

Culture of human cervical cancer cells (HeLa) and human lung cancer cells (SPC-A-1): they are adherent growth cells, routinely cultured in DMEM medium (containing 10% fetal bovine serum, penicillin and streptomycin), placed in Cultured in a 37°C, 5% CO ₂ incubator, and passaged every 3-5 d. Take the tumor cells in the logarithmic growth phase and adjust the cell suspension concentration to 1-1.5×10 ³ cells/mL. Add 100 μL of cell suspension to each well of a 96-well plate, and place it in a 37°C, 5% CO ₂ incubator for 24 h.

After culturing for 24h, the liquid medicine was added according to the design. The test liquids were added to each well, and 6 parallel wells were set for each concentration. The experiment was divided into drug test group (adding different test drugs respectively), control group (adding only culture medium and cells, no test drug) and blank group (adding only culture medium, no cells and test drug). The 96-well plate after dosing was cultured in a 37°C, 5% _CO2 incubator.

After drug treatment for 24h/48h/72h, add 10 μL of CCK-8 solution to each well, and place the culture plate in an incubator for 1-4 hours. The OD value of each well at 450 nm was measured with a microplate reader, and the cell inhibition rate was calculated.

Cell inhibition rate (%)=(OD value of control group-OD value of experimental group)/(OD value of control group-OD value of blank well)×100%

The measured cell inhibition rates are shown in Figures 1-2 and Tables 2-3, indicating the inhibitory effects of these compounds on HeLa and SPC-A-1 cells.

It can be seen from the above experiments that the ursamide derivatives of the present invention have obvious killing effect on human cervical cancer cells (HeLa) and human lung cancer cells (SPC-A-1), and have significant tumor cell inhibiting activity.

Table 2. Killing effect of ursamide derivatives on HeLa cells

Table 3. Killing effect of ursamide derivatives on SPC-A-1 cells

Therefore, the present invention effectively overcomes various shortcomings in the prior art and has high industrial utilization value.

The above-mentioned embodiments merely illustrate the principles and effects of the present invention, but are not intended to limit the present invention. Anyone skilled in the art can modify or change the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or changes made by those with ordinary knowledge in the technical field without departing from the spirit and technical idea disclosed in the present invention should still be covered by the claims of the present invention.

Claims (12)

1. a compound is characterized in that, described compound is compound LB-2, compound LB-3, compound LB-4, compound LB-6, compound LB-7, compound LB-8, compound LB-9, compound LB-10, Compound LB-11, Compound LB-12, Compound LB-13, Compound LB-14 or Compound LB-15, in, The LB-2 has the following structural formula:

The LB-3 has the following structural formula:

The LB-4 has the following structural formula:

The LB-6 has the following structural formula:

The LB-7 has the following structural formula:

The LB-8 has the following structural formula:

The LB-9 has the following structural formula:

The LB-10 has the following structural formula:

The LB-11 has the following structural formula:

The LB-12 has the following structural formula:

The LB-13 has the following structural formula:

The LB-14 has the following structural formula:

The LB-15 has the following structural formula:

The compound is used for preparing a medicine for treating tumors; the tumor is cervical cancer or lung cancer; the cells of the tumor are one or more of cervical cancer cell Hela or lung cancer cell SPC-A-1. 2. the synthetic method of a kind of compound according to claim 1, its synthetic route is as follows:

Specifically include the following steps: a) ursolic acid (1) is protected by benzyl group to obtain intermediate (2); b) adding PCC to intermediate (2) to carry out oxidation reaction to obtain intermediate (3); c) adding the intermediate (3) to the ester compound under basic conditions for reaction to obtain the intermediate (4); d) subjecting intermediate (4) to a condensation reaction with a hydrazine compound to obtain intermediate (5); e) hydrogenation of intermediate (5) to obtain intermediate (6); f) acid chloride of intermediate (6) to obtain intermediate (7); g) forming an amide bond between the intermediate (7) and the amine compound to obtain the desired compound (8); R ¹ is 4-cyanophenyl; R ² is hydrogen; R ³ is hydrogen; R ⁴ is cyclopentyl, 4-pyridylmethyl, 2-furanmethyl, 3-morpholinopropyl, 4-fluoro Benzyl, 4-methoxyphenethyl, isopropyl, 2,2,2-trifluoroethyl, cyclopropylmethyl, n-hexyl, 3-isopropoxypropyl; the R ³ and R4 ^together with the bridged nitrogen atom to form a heterocycloalkyl group is

3. the synthetic method of compound according to claim 2, is characterized in that, comprises one or more in following technical characteristic: i) In step a), the benzyl protection is to mix ursolic acid (1) with potassium carbonate, N,N-dimethylamide and benzyl bromide, and then carry out a heating reaction, and after the obtained mixture is cooled to room temperature, Add water to separate out the solid product, and after the solid product is filtered, washed and dried, intermediate (2) is obtained; ii) In step b), the oxidation reaction is to dissolve the intermediate (2) in dichloromethane and then cool it to below 0°C, add PCC and stir at room temperature to carry out the oxidation reaction, filter the obtained reaction product, concentrate, After separation and purification, intermediate (3) is obtained; iii) In step c), the reaction is to dissolve the intermediate (3) in tetrahydrofuran and then cool it to below 0°C, add basic compounds and ester compounds, stir and mix the reaction at room temperature, and add water to the obtained reaction product. The quenching reaction is carried out, and after extraction, washing, drying, filtration, concentration, separation and purification, intermediate (4) is obtained, in the intermediate (4), R ² has the same definition as in the compound of formula 8; iv) In step d), in the condensation reaction, the intermediate (4) and the hydrazine compound are dissolved in an organic solvent, heated and stirred to react and then cooled to room temperature, and the obtained reaction product is concentrated, washed, separated and purified to obtain Intermediate (5), in the intermediate (5), R ¹ and R ² have the same definitions as in the compound of formula 8; ⅴ) In step e), the hydrogenation reaction is to dissolve the intermediate (5) and the catalyst in an organic solvent, and then introduce hydrogen for the reaction, filter, wash, and concentrate the obtained reaction product, stir and make a slurry, filter again, Drying to obtain the desired intermediate (6), in the intermediate (6), R ¹ and R ² have the same definitions as in the compound of formula 8; ⅵ) In step f), the acid chlorination is to dissolve the intermediate (6) in anhydrous dichloromethane and cool it to below 0°C, then add oxalyl chloride and N,N-dimethylamide in sequence, and at room temperature Under stirring and mixing reaction, concentrated and dried to obtain intermediate (7), in the intermediate (7), R ¹ and R ² have the same definitions as in the compound of formula 8; ⅶ) In step g), the forming of the amide bond is to dissolve the intermediate (7) in N,N-dimethylamide, add the basic compound and the amine compound in turn, stir and mix the reaction at room temperature, concentrate, After separation and purification, stirring and beating, and drying, the desired compound (8) is obtained. 4. the synthetic method of compound according to claim 3, is characterized in that, in step a), also comprises one or more in following technical characteristic: A1) the mol ratio that described ursolic acid (1) and potassium carbonate add is 1:1-3; A2) the mol ratio that described ursolic acid (1) and N,N-dimethylamide add is 1:9-11; A3) the mol ratio that described ursolic acid (1) and benzyl bromide add is 1:1-2; A4) The conditions of the heating reaction are: the reaction temperature is 50-70°C; the reaction time is 3-5h; A5) The ratio of the mass of the ursolic acid (1) added to the volume of the water added in mg/mL is 450-470:40-60. 5. the synthetic method of compound according to claim 3, is characterized in that, in step b), also comprises one or more in following technical characteristic: B1) The ratio mg/mL of the mass that the intermediate (2) adds and the volume that dichloromethane adds is 440-460:40-60; B2) The mol ratio of the intermediate (2) and PCC added is 1:1.2-3; B3) The stirring time is 11-13h. 6. the synthetic method of compound according to claim 3, is characterized in that, in step c), also comprises one or more in following technical characteristic: C1) the mass that described intermediate (3) adds and the ratio of the volume mg/mL that tetrahydrofuran adds are 250-350:15-25; C2) the basic compound is sodium methoxide; C3) the mol ratio that described intermediate (3) adds to basic compound is 1:1-2; C4) described ester compound is selected from a kind of in ethyl formate, ethyl acetate, ethyl trifluoroacetate; C5) the mol ratio that described intermediate (3) and ester compound add is 1:1-2; C6) described stirring time is 3-5h; C7) The reaction conditions of the extraction are as follows: the extraction reagent is ethyl acetate, the extraction times are 3-4 times, and the amount of the extraction reagent is 25-35 ml. 7. the synthetic method of compound according to claim 3, is characterized in that, in step d), also comprises one or more in following technical characteristic: D1) the hydrazine compound is selected from the one in alkyl hydrazine, aryl hydrazine, heteroaryl hydrazine; D2) The molar ratio of the intermediate (4) and the hydrazine compound added is 1:1-2; D3) the organic solvent is ethanol; D4) the ratio of the mass that described intermediate (4) adds and the volume that organic solvent adds are 310-330:15-25; D5) The conditions for the heating and stirring reaction are: the heating temperature is 80-90° C., and the stirring time is 11-13 h. 8. the synthetic method of compound according to claim 3, is characterized in that, in step e), also comprises one or more in following technical characteristic: E1) The catalyst is a 10wt% palladium-carbon mixture, and the weight ratio of Pd and C is 10:90; E2) the ratio of the mass added by the intermediate (5) and the catalyst is 30-40:45-55; E3) described organic solvent is methanol; E4) The ratio of the added mass of the intermediate (5) to the added volume mg/mL of the organic solvent is 30-40:15-25. 9. the synthetic method of compound according to claim 3, is characterized in that, in step f), also comprises one or more in following technical characteristic: F1) The ratio of the mass that the intermediate (6) adds and the volume g/mL that anhydrous dichloromethane adds are 1:2-10; F2) the mol ratio that described intermediate (6) and oxalyl chloride add is 1:1-2; F3) The molar ratio of the intermediate (6) and N,N-dimethylamide added is 1:0.01-0.1; F4) The stirring time is 5-7h. 10. the synthetic method of compound according to claim 3, is characterized in that, in step g), also comprises one or more in following technical characteristic: G1) The ratio of the mass added by the intermediate (7) and the volume g/mL added by N,N-dimethylamide is 1:2-10; G2) the basic compound is triethylamine or N,N-diisopropylethylamine; G3) The molar ratio of the intermediate (7) and the basic compound added is 1:1-2; G4) The general structural formula of the amine compound is: R ³ R ⁴ NH, where R ³ and R ⁴ have the same definitions as in the compound of formula 8; G5) The molar ratio of the amount of the intermediate (7) and the amine compound added is 1:1-1.5. 11. Use of the compound according to claim 1 in the preparation of a medicament for the treatment of tumors; the tumor is cervical cancer or lung cancer; the cells of the tumor are cervical cancer cells Hela or lung cancer cells SPC-A-1. one or more. 12. A pharmaceutical composition comprising a therapeutically effective amount of a compound of claim 1.

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