CN112194668B - Compound containing coumarin spirooxazoline and preparation method and application thereof - Google Patents

Abstract

The invention provides a coumarin spirooxazoline compound containing coumarin and a preparation method and application thereof, and the coumarin spirooxazoline compound is a coumarin spiroderivative formed by introducing oxazoline heterocycle at the C4 site of a coumarin parent nucleus, and has the structural formula:

wherein R is¹Any one selected from hydrogen, alkyl, alkoxy, halogen, nitro and hydroxyl; r is²Selected from any one of alkyl, substituted alkyl, aryl, substituted aryl, aliphatic heterocycle and aromatic heterocycle. The preparation method can be realized by a one-pot method, the product yield can reach 55-85%, and the method is economical and efficient; the compound of the invention shows higher inhibitory activity on human gastric cancer cell MGC 803.

Description

Compound containing coumarin spirooxazoline and preparation method and application thereof

Technical Field

The invention relates to the technical field of medicines, in particular to a coumarin spirooxazoline-containing compound and a preparation method and application thereof.

Background

The coumarin core skeleton is present in a large number of compounds with significant biological activity and is an important fragment of many natural and synthetic antitumor drugs. The coumarin 3, 4-position polycyclic compound glauppalol extracted and separated from plant Glaucidium palmatum can promote tubulin polymerization and enhance the activity of taxol on KB cell. The coumarin derivative Irusostat (STX-64) is currently in phase I clinical study for advanced prostate cancer, and in phase II clinical study for endometrial and advanced breast cancer. The oxazoline heterocycle and the derivative structure thereof have obvious pharmacological and biological activities, can be used as an intermediate for drug synthesis, are concerned and play important roles as a pharmacophore in new drug research.

However, to date, the structural types and related biological activities of spiro compounds containing coumarin core skeleton have been studied only in limited ways, and only three types of dihydrocoumarin spiro compounds have been reported.

Disclosure of Invention

One of the objects of the present invention is to propose a novel compound containing a coumarin spirooxazoline structure.

The second purpose of the invention is to provide a preparation method of the compound.

The invention also aims to provide an application method of the compound.

In order to achieve one of the above purposes, the invention firstly provides the following technical scheme:

a coumarin spirooxazoline-containing compound having a structural formula as shown in formula (i):

wherein R is¹Any one selected from hydrogen, alkyl, alkoxy, halogen, nitro and hydroxyl;

R²selected from any one of alkyl, substituted alkyl, aryl, substituted aryl, aliphatic heterocycle and aromatic heterocycle.

As shown in formula (I), the compound of the invention has coumarin spiro oxazoline, and oxazoline heterocycle is introduced at C4 position of coumarin parent nucleus to obtain a novel coumarin spiro derivative.

In the above embodiments, the hydrocarbon group means a saturated or unsaturated, chain-like and/or cyclic organic group containing only carbon and hydrogen.

The substituted hydrocarbyl refers to a hydrocarbyl group having a substituent on the main chain or branch chain.

The aryl refers to a group having a benzene ring structure in the main chain and/or branch chain.

The substituted aryl group refers to an aryl group having a substituent on the main chain and/or the branch chain.

The aromatic heterocycle refers to an aryl group which has a cyclic structure consisting of hetero elements (other elements except carbon and hydrogen) and carbon elements on a main chain or a branched chain;

the aliphatic heterocyclic ring refers to heterocyclic compounds other than aromatic heterocyclic ring.

Preferably, in the formula (I), R¹Selected from H, 7-Me, 7-OMe 7-OCH₂CH₃、7-OH、6-NO₂Or 6-Br.

In the structural formula (I), R²Selected from Ph, 4-FPh, 3-ClPh, 2-MePh, 4-OMePh, 4-OCOMePh, 4-C (Me)₃Ph、

or-C₆H₁₃Any one of the above.

Wherein Ph represents phenyl, Me represents methyl, the number preceding the group represents the position of a substituent on the phenyl, e.g. 4-OMePh represents the fourth position on the phenyl ring substituted by-OCH₃Substituted phenyl, 4-F represents phenyl substituted with-F at the fourth position on the phenyl ring, 4-OCOMePh represents phenyl substituted with-OCOMe at the fourth position on the phenyl ring, and so on.

Preferably, the structural formula (i) is selected from any one of the following (1) to (20):

(1)R¹＝H,R²＝Ph；

(2)R¹＝H,R²＝4-FPh；

(3)R¹＝H,R²＝4-ClPh；

(4)R¹＝H,R²＝3-ClPh；

(5)R¹＝H,R₂＝2-MePh；

(6)R¹＝H,R²＝4-OCOMePh；

(7)R¹＝H,R²＝4-OMePh；

(8)R¹＝H,R²＝4-C(Me)₃Ph；

(9)R¹＝H,

(10)R¹＝H,

(11)R¹＝H,

(12)R¹＝H,

(13)R¹＝H,R²＝C₆H₁₃；

(14)R¹＝7-CH₂CH₃,R²＝Ph；

(15)R¹＝7-Me,R²＝Ph；

(16)R¹＝7-OMe,R²＝Ph；

(17)R¹＝7-OCH₂CH₃,R²＝Ph；

(18)R¹＝7-OH,R²＝Ph；

(19)R¹＝6-NO₂,R²＝Ph；

(20)R¹＝6-Br,R²＝Ph。

in order to achieve the second purpose, the invention provides the following technical scheme:

the preparation method of the compound comprises the following steps:

carrying out free radical cycloaddition reaction on a compound shown as a formula (II) and a compound shown as a formula (III) under the action of a catalyst to prepare a compound shown as a formula (I);

wherein R is¹、R²Is selected from any one of R¹And/or R²；

The ratio of the amount of the compound represented by the formula (II) to the amount of the compound represented by the formula (III) is (1-2): 1. Preferably, the mass ratio of the compound shown in the formula (II) to the compound shown in the formula (III) (1-1.5) is 1.

The preparation process is carried out in a solvent.

Preferably, the solvent is selected from one or more of toluene, benzene and chlorobenzene.

The reaction temperature in the preparation process is 60-100 ℃. Preferably, the reaction temperature is 80-90 ℃.

The reaction time of the preparation process is 2.5-10 h. Preferably, the reaction time is 6 to 8 hours.

The preparation process is carried out in an air environment.

The catalyst is selected from monovalent copper salts. Preferably, the catalyst is selected from cuprous chloride, CuCl.

Further, the amount of the catalyst is 10 to 100 mol% of the compound represented by the formula (III). Preferably, the catalyst is used in an amount of 10 to 20 mol% based on the compound represented by the formula (III).

Wherein the compound shown in the formula (II) is prepared by reacting coumarin with azido trimethyl silane.

Such as when R¹When the compound is H, the compound (II), namely the 4-azido coumarin, can be prepared by the following preparation process:

the specific process can be as follows: dissolving 2.5mol of trimethylsilyl azide in a dry solvent dichloromethane in a reaction vessel, adding 0.5mol of acetic acid, stirring for 20min, adding 0.5mol of coumarin and 0.1mol of triethylamine, stirring at room temperature for 18-28h in an air environment, removing the solvent dichloromethane under reduced pressure after the reaction is finished, and separating by using column chromatography to obtain 4-azido coumarin.

In order to achieve the third purpose, the invention provides the following technical scheme:

the coumarin spirooxazoline-containing compound or the coumarin spirooxazoline-containing compound prepared by the preparation method is applied to the preparation of cancer treatment drugs.

Further, the drug is used for inhibiting gastric cancer cells.

In particular, the medicament is used for inhibiting gastric cancer cells MGC 803.

The invention has the following beneficial effects:

(1) the invention obtains a novel coumarin-containing spirooxazoline compound;

(2) the preparation method can be realized by a one-pot method, the product yield can reach 55-85%, and the method is economical and efficient;

(3) the compound of the invention shows higher inhibitory activity to human gastric cancer cell MGC803, and provides an important opportunity for finding coumarin spirooxazoline lead compounds for resisting gastric cancer.

Detailed Description

The present invention is described in detail with reference to the following examples, but it should be understood that the examples are only for illustrative purposes and are not intended to limit the scope of the present invention. All reasonable variations and combinations that fall within the spirit of the invention are intended to be within the scope of the invention.

The following examples prepare the target product (I) by the following preparation procedure:

taking 4-azido coumarin (II) and terminal olefin (III) as substrates, 10-20 mol% cuprous salt such as CuCl as a catalyst, taking aprotic solvent such as toluene as a solvent, reacting for 8-10 hours at 70-80 ℃, and obtaining a product (I) through 1, 3-dipolar cycloaddition, cyclocracking denitrification and copper-catalyzed air oxidation dehydrocyclization processes,

example 1

Preparation of R by the following procedure¹＝H,R²Ph coumarin (C)₄) Spirooxazoline compounds:

(1) adding 2.5mol of trimethylsilyl azide and 25-30mL of dry dichloromethane into a 100mL reaction bottle, adding 0.5mol of acetic acid after dissolving, stirring for 20min, then adding 0.5mol of coumarin and 0.1mol of triethylamine, stirring for 18h at room temperature in an air environment, removing the solvent dichloromethane under reduced pressure after the reaction is finished, and separating by using a column chromatography to obtain the product 4-azido coumarin.

(2) 0.5mmol of styrene, 0.6mmol of 4-azido coumarin, 0.05mmol of CuCl (0.1equiv) and 5.0mL of toluene serving as a solvent are sequentially added into a reaction tube, the mixture is put into an oil bath kettle at the temperature of 80 ℃ in the air for reaction for 8 to 10 hours, after the reaction is finished, the mixture is cooled, the solvent toluene is removed under reduced pressure, and a product 5' -phenyl-5 ' H-spiro [ coumarin-4, 2' -oxazoline ] -2-ketone (A) is obtained by separation through column chromatography, wherein the yield is 79%;

the product was obtained as a pale yellow solid with a melting point of 87.1-89.9 ℃ as determined by SGW X-4 digital melting point apparatus.

Subjecting the product A to nuclear magnetism (¹H NMR and¹³c NMR), the results are as follows:

¹H NMR(500MHz,CDCl₃):δ8.01(d,J＝6.2Hz,1H),7.41(dd,J＝7.1,1.8Hz,1H),7.38–7.13(m,8H),5.72(d,J＝6.0Hz,1H),3.44(d,J＝12.5Hz,1H),2.90(d,J＝12.3Hz,1H)ppm；

¹³C NMR(125MHz,CDCl₃):δ167.0,158.9,145.3,137.8,134.1,128.7,128.2,126.8,126.2,124.2,121.8,120.0,40.88ppm。

and performing high-resolution mass spectrum characterization on the product A, wherein the result is as follows:

HRMS m/z(ESI)calcd for C₁₇H₁₄NO₃[M+H]⁺280.0968,found:280.0961。

according to¹H NMR、¹³C NMR and high resolution mass spectrum data comparison can determine that the product has a structural formula shown as a formula A.

Examples 2 to 20

Referring to the specific process of example 1, the target products (I) of examples 2-20 can be prepared by the foregoing preparation process as shown in table 1 below, respectively:

TABLE 1 target products (I) of examples 2-20

The nuclear magnetism and high resolution mass spectrum characterization can verify that the product has the structure shown in the formula (I), and the yield of the obtained product is 55-86%.

Taking the product of example 3 as an example, the product is a light yellow solid, and the melting point of the product is 81.2-83.5 ℃ as measured by an SGW X-4 digital melting point instrument.

The nuclear magnetic characterization result is as follows:

¹H NMR(500MHz,CDCl₃):8.01(d,J＝12.5Hz,1H),7.47–7.19(m,8H),5.72(d,J＝12.3Hz,1H),3.43(d,J＝24.9Hz,1H),2.91(d,J＝24.7Hz,1H)ppm；

¹³C NMR(125MHz,CDCl₃):δ167.0,158.9,145.3,135.3,134.5,134.1,128.5,128.3,126.2,124.2,121.8,120.0,40.9ppm。

the high resolution mass spectrum characterization result is as follows:

HRMS m/z(ESI)calcd for C₁₇H₁₃ClNO₃[M+H]⁺314.0578,found:314.0573。

according to¹H NMR、¹³C NMR and high resolution mass spectrum data comparison, and the structural formula is determined as follows:

example 21

The products were tested for tumor inhibition:

taking MGC-803 (human gastric cancer cell), T24 (human bladder cancer cell), HepG-2 (liver cancer cell) and A549 (human non-small cell lung cancer cell) in DMEM medium containing 10% fetal bovine serum and 1% double antibody at 37 deg.C and 5% CO₂And 95% air CO₂Culturing in an incubator. After 3 washes with PBS, cells were digested with 0.25% trypsin and digested with 10% serum medium. The cells were suspended in a medium containing 10% serum and gently pipetted to prepare a single cell suspension. By cell counting, an appropriate amount of cell suspension was diluted with 10% medium and inoculated into a 96-well plate180. mu.L per well, 2X 10 cells per well⁴-4×10⁴. The samples to be tested were added to 96-well plates seeded with different tumor cells at 20. mu.L per well to give a final drug concentration of 20. mu.M, and primary screening was performed. Selecting samples with good selection effect after primary screening, selecting different concentration gradients for screening, and calculating IC₅₀Each group is provided with 9 multiple holes. In the control group, 20. mu.L of a culture medium containing 10% DMSO was added to each well and cultured for 48 hours. After 48h of MTT drug addition, 10. mu.L of 5mg/mL MTT solution in CO was added to each well₂The incubator continues to culture for 4 h. The medium in the wells was carefully removed by pipetting, 100. mu.L of DMSO was added to each well, and the wells were shaken on a shaker for 10min to completely dissolve the formazan crystals. The absorbance of each well was then measured using a microplate reader (490 nm). The inhibition rate of each sample on tumor cells was calculated according to the following formula: inhibition ratio (%) ═ 1-A_{Sample (I)}/A_ControlX 100%. Plotting different concentrations of the same sample on the growth inhibition rate of tumor cells to obtain a dose response curve, and calculating the half inhibition concentration IC of the sample according to a linear regression equation₅₀The results shown in table 2 below were obtained (average + positive and negative standard deviations for each data):

TABLE 2 IC of different samples on different cancer cells₅₀Value of

As can be seen from the biological activity test data in the above table, the compounds obtained in examples 2, 3, 16 and 20 of the present invention all showed higher activity inhibition effect on human gastric cancer cell MGC803, and the same tests performed on the compounds of other examples showed similar anti-tumor effect. Therefore, the compounds obtained in examples 1 to 20 can be used for preparing a medicament for treating cancer.

The above examples are merely preferred embodiments of the present invention, and the scope of the present invention is not limited to the above examples. All technical schemes belonging to the idea of the invention belong to the protection scope of the invention. It should be noted that modifications and embellishments within the scope of the invention may be made by those skilled in the art without departing from the principle of the invention, and such modifications and embellishments should also be considered as within the scope of the invention.

Claims (7)

1. A coumarin spirooxazoline-containing compound is characterized in that a coumarin spirocyclic derivative formed by introducing oxazoline heterocycle at the C4 position of a coumarin parent nucleus; it has a structural formula shown as formula (I):

wherein R is¹Selected from H, 7-Me, 7-OMe 7-OCH₂CH₃、7-OH、6-NO₂Or 6-Br;

R²selected from Ph, 4-FPh, 3-ClPh, 2-MePh, 4-OMePh, 4-OCOMePh, 4-C (Me)₃Ph、

Any one of (a);

wherein Ph represents a phenyl group, Me represents a methyl group, and the number preceding the group represents the position of a substituent on the phenyl group.

2. The coumarin spirooxazoline-containing compound according to claim 1, wherein structural formula (I) is selected from any one of the following (1) to (17):

(1)R¹＝H,R²＝Ph；

(2)R¹＝H,R²＝4-FPh；

(3)R¹＝H,R²＝4-ClPh；

(4)R¹＝H,R²＝3-ClPh；

(5)R¹＝H,R₂＝2-MePh；

(6)R¹＝H,R²＝4-OCOMePh；

(7)R¹＝H,R²＝4-OMePh；

(8)R¹＝H,R²＝4-C(Me)₃Ph；

(9)R¹＝H,

(10)R¹＝H,

(11)R¹＝7-CH₂CH₃,R²＝Ph；

(12)R¹＝7-Me,R²＝Ph；

(13)R¹＝7-OMe,R²＝Ph；

(14)R¹＝7-OCH₂CH₃,R²＝Ph；

(15)R¹＝7-OH,R²＝Ph；

(16)R¹＝6-NO₂,R²＝Ph；

(17)R¹＝6-Br,R²＝Ph。

3. the method for preparing a coumarin spirooxazoline-containing compound according to claim 1 or 2, characterized by comprising the following steps:

carrying out free radical cycloaddition reaction on a compound shown in a formula (II) and a compound shown in a formula (III) under the action of a catalyst to prepare a compound shown in a formula (I);

wherein R is¹Selected from H, 7-Me, 7-OMe 7-OCH₂CH₃、7-OH、6-NO₂Or 6-Br;

R²selected from Ph, 4-FPh, 3-ClPh, 2-MePh, 4-OMePh, 4-OCOMePhh、4-C(Me)₃Ph、

Any one of (a);

the catalyst is selected from cuprous salts, and the dosage of the catalyst is 10-100 mol% of the compound shown in the formula (III).

4. The method for preparing a coumarin spirooxazoline-containing compound, according to claim 3, wherein the ratio of the amount of the compound represented by the formula (II) to the amount of the compound represented by the formula (III) is 1-2: 1.

5. The method of claim 3, wherein the addition reaction conditions are as follows:

in a solvent selected from one or more of toluene, benzene and chlorobenzene;

the reaction temperature is 60-100 ℃;

the reaction time is 2.5-10 h.

6. The method of claim 3, wherein the compound of formula (II) is prepared by reacting coumarin with azido trimethylsilane.

7. Use of a coumarin spirooxazoline-containing compound in the preparation of a medicament for the treatment of cancer, according to claim 1 or 2.