CN110540566A - Astragalin derivative and application thereof - Google Patents

Abstract

Description

Astragalin derivative and application thereof

Technical Field

The invention relates to the field of medicines, and particularly relates to an astragalin derivative, a preparation method thereof, and application of the astragalin derivative as a CDK2 kinase inhibitor.

Background

CDKs are key proteins for regulating cell cycle, and the abnormal expression of CDKs is closely related to the occurrence and development of tumors. Of the 13 proteins of the CDKs family, CDK2 is a kinase essential for cells to undergo mitosis. Mutation of the CDK2 gene can cause over-activation of the CDK2 gene, and abnormal expression of CDK2 is frequently found in tumor tissues such as breast cancer, ovarian cancer and bladder cancer. Therefore, finding a CDK2 kinase inhibitor with higher selectivity and less side effects, thereby preventing the proliferation of tumor cells, has become a new direction for treating tumors.

Various small molecule CDK2 inhibitors with different structure types have been reported AT home and abroad, and currently, CDK2 inhibitors entering clinical research are Adenosine Triphosphate (ATP) combination inhibitors of CDK2, such as currently only marketed palbociclib, and clinical stage II/III R-roscovitine, dinaciclib, flavopiridol, AT-7519 and the like, which have better therapeutic effects on breast cancer, leukemia, lymphoma and the like. However, some inhibitors have a variety of side effects in clinical trials and are forced to terminate the trial. In conclusion, the discovery of a new class of CDK2 inhibitors is of great interest for the subsequent development of such drugs, whereas derivatives of drug molecules of natural origin are a safer and more reliable source of drug discovery.

The inventors previously confirmed through reverse targeting, molecular docking and in vitro experiments that the CDK2 kinase is a new target protein for astragalin. The half inhibition of astragalin to CDK2 kinase has an IC50 value of 7.42 mu M, and can remarkably inhibit the expression of CDK2 kinase in A431 human epidermal and squamous carcinoma cells. However, the inventor finds that astragalin still has a hydrophobic cavity in a CDK2 binding pocket and cannot be sufficiently bound, so that a bulky hydrophobic side chain group is linked on an active site of astragalin by a chemical synthesis method, the interaction force of astragalin and CDK2 kinase can be increased, and the astragalin can become a novel CDK2 kinase to be applied to development and preparation of anti-cancer drugs. At present, few reports are made on the synthesis of astragalin derivatives, and the astragalin derivatives with biological activity are not reported. Therefore, the synthesis of astragalin derivatives with CDK2 kinase inhibitory activity is necessary, and a new choice can be provided for developing new stable and reliable anticancer drugs with CDK2 kinase inhibitors.

Disclosure of Invention

aiming at the problems, the most active reaction part in astragalin is on the phenolic hydroxyl of the benzene ring side chain, bromocarboxylic acid bornyl alcohol (or bornylamine) with different carbon chain lengths (2-6 carbons) is linked to the oxygen atom of the phenol side chain of astragalin according to the volume size and the radius of a cavity, so that the interaction force with CDK2 kinase is enhanced.

The technical scheme of the invention is as follows: an astragalin derivative is 4' -O-2- { N- [ (1S,3R,4S) -4,7, 7-trimethyl-3-bicyclo [2.2.1] heptanyl ] acetamido } astragalin, and is shown as a structural formula I:

The preparation method of astragalin derivative 4' -O-2- { N- [ (1S,3R,4S) -4,7, 7-trimethyl-3-bicyclo [2.2.1] heptylalkyl ] acetamido } astragalin comprises the following steps:

s1, adding (S) -norbomylamine and triethylamine into a dry dichloromethane solution in an argon environment, slowly dropwise adding bromoacetyl bromide at room temperature, and continuously stirring for 24 hours to obtain a reaction solution A;

S2, dissolving potassium carbonate, dicyclohexylcarbodiimide and astragalin compounds in methanol and stirring for 1 hour;

And S3, dropwise adding the reaction liquid A obtained in the step S1 into the reaction system of the step S2, continuously reacting for 3 hours at room temperature, filtering the reaction liquid after the reaction is finished, concentrating the solution, and separating and purifying the concentrated solution by silica gel column chromatography to obtain a yellow solid compound, namely the astragalin derivative shown in the formula I.

Preferably, in step S1, the molar ratio of (S) -norbomylamine, triethylamine and bromoacetyl bromide is 1: 1: 0.5, the volume of the dichloromethane solution is 30 ml.

Preferably, in step S2, the molar ratio of potassium carbonate, dicyclohexylcarbodiimide and astragalin is 1: 1: 0.5, the volume of the methanol is 30 ml.

Preferably, the elution reagent for silica gel column chromatography in S3 is chloroform/methanol ═ 1: (3-6).

preferably, the astragalin derivative 4' -O-2- { N- [ (1S,3R,4S) -4,7, 7-trimethyl-3-bicyclo [2.2.1] heptanyl ] acetamido } astragalin is used as a CDK2 kinase inhibitor.

Preferably, the spectral information of the astragalin derivative 4' -O-2- { N- [ (1S,3R,4S) -4,7, 7-trimethyl-3-bicyclo [2.2.1] heptanyl ] acetamido } astragalin is as follows: 1H NMR (500MHz, CD3OD, ppm) 8.11(2H, d, J ═ 8.5),6.96(2H, d, J ═ 8.5),6.27(1H, s),6.44(1H, s),5.28(1H, d, J ═ 7.5),4.83(2H, d, J ═ 1.5),3.74(1H, m),3.60(1H, m),3.34(1H, m), 3.26-3.50 (4H, m),1.87(1H, dd, J ═ 13.3,9.1),1.52-1.80(4H, m),1.23-1.35(1H, m),1.10-1.22(1H, m),0.94(3H, s),0.83-0.86(6H, m); 13C NMR (125MHz, CD3OD, ppm) 179.7,166.1,164.97,163.2,161.7,159.2,158.6,135.6,132.4,122.9,116.2,105.9,104.2,100.0,94.8,78.6,78.2,75.9,71.5,62.8,57.12,48.69,47.21,45.01,43.00,39.01,35.95,27.10,20.33,20.19, 11.82; [ α ]27D ═ 42.7 ° (methanol, c ═ 0.2); HRMS, calculated C33H39NO12 m/z 641.2502; found 641.2045.

The invention has the beneficial effects that: the astragalin derivative with biological activity is prepared and obtained for the first time, and has a strong inhibition effect on CDK2 kinase, so that the astragalin derivative has the potential to become an anti-cancer drug of a novel CDK2 kinase inhibitor. The preparation method of astragalin derivatives disclosed by the invention is high in yield, and can be used for research and development of new medicines and expanded production.

Drawings

In the figure 1, a, b and c are astragalin derivatives Z2, Z4, Z6 and CDK2 kinase molecule docking simulation 2D diagrams respectively;

FIG. 2(a) is a simulated 3D map of astragalin binding to CDK2 kinase (red circles are marked as hydrophobic cavities), (b) is a simulated 3D map of designed astragalin derivative Z2 binding to CDK2 kinase;

FIG. 3 is a schematic diagram of the synthesis of astragalin derivative Z2;

FIG. 4 is a graph comparing the inhibitory effect of astragalin derivative Z2 on CDK2 kinase at different concentrations.

Detailed Description

The present invention is further illustrated by the following examples.

example 1 computer-aided design of astragalin derivatives Z1-Z6

In order to improve the binding force and anticancer activity of astragalin to CDK2 kinase, and based on the knowledge of the binding mechanism of astragalin to CDK2 kinase, it is pointed out that a hydrophobic cavity in a CDK2 binding pocket of astragalin is not sufficiently bound, as shown in fig. 2(a), a hydrophobic side chain group with a large volume may be linked to the active site of astragalin by a chemical synthesis method, so as to increase the interaction force between astragalin and CDK2 kinase. The most active reaction site in astragalin is on the phenolic hydroxyl of the benzene ring side chain, bromocarboxylic acid bornyl alcohol (or bornyl amine) with different carbon chain lengths (2-6 carbons) is designed to be linked to the side chain oxygen atom of astragalin phenol according to the volume size and radius of the cavity. To verify the CDK2 kinase binding capacity and inhibitory activity of such derivatives. Virtually 6 astragalin derivatives Z1-Z6 expressed by the general formula (II) are designed, as shown in Table 1, wherein X is NH or O atom, and n is 1-5 alkylene.

TABLE 1 Structure of derivatives Z1-Z6

Name of derivative	X	n
			Z1	O	2
Z2	N	2
			Z3	N	3
Z4	N	4
			Z5	N	5
Z6	N	6

Example 2 evaluation of binding Capselagin derivative Z1-Z6

The method comprises the following steps: the binding ability of Z1-Z6 was evaluated by molecular docking with DS software (see FIG. 1).

as a result: z1, Z3 and Z5 were unable to dock effectively with CDK2 kinase, Z2, Z4 and Z6 were able to dock effectively with CDK2 kinase, and Z2 derivatives were able to form more interaction with CDK2 kinase (see fig. 2). in DS-simulated molecular docking, Z2 was also found to be able to indeed fill the hydrophobic pocket of CDK2 kinase, forming a stronger binding compared to astragalin. The binding energy of Z2 to CDK2 kinase was also calculated using the SwissDock molecular docking platform and found to be significantly higher than astragalin (Z2 is-9.18 kcal/mol, astragalin is-8.42 kcal/mol).

Example 3 astragalin derivative Z2 synthesis

the method comprises the following steps: as shown in figure 3 of the drawings,

1) (S) -norbomylamine (0.012mol) and triethylamine (0.012mol) were added to a solution of 30ml of dry dichloromethane in an argon atmosphere, and bromoacetyl bromide (0.024mol) was slowly added dropwise thereto at room temperature, followed by further stirring for 24 hours to obtain a reaction solution A.

2) Potassium carbonate (0.016mol) and a compound of dicyclohexylcarbodiimide (0.016mol) and astragalin (0.008mol) were dissolved in 30ml of methanol and stirred for 1 hour.

3) Dropwise adding the reaction liquid A in the step 1 into the reaction system in the step 2, continuously reacting for 3 hours at room temperature, filtering the reaction liquid after the reaction is finished, concentrating the solution, and separating and purifying by silica gel column chromatography (gradient elution is carried out by chloroform/methanol which is 1: 4) to obtain a yellow solid, namely a compound Z2, wherein Z2 is named as 4' -O-2- { N- [ (1S,3R,4S) -4,7, 7-trimethyl-3-bicyclo [2.2.1] heptylalkyl ] acetamido } astragalin, and the yield is 37%.

Example 4 astragalin derivative Z2 in vitro CDK2 kinase Activity inhibition assay

the method comprises the following steps: CDK2 is a Ser/Thr protein kinase that requires the consumption of ATP in substrate phosphorylation while producing an equivalent amount of ADP. Therefore, an ADP-GloTMMax detection kit is selected, and the kit quantitatively detects ADP generated in the reaction based on a luminescence method, so that the ATP kinase activity is detected. In the experiment, the blank control without adding astragalin derivative Z2 and enzyme is used, and the positive control without adding astragalin derivative Z2 and enzyme is used.

1) P-CDK2 was established to purify the protein and activate in vitro with > 95% purity.

2) ADP generated by astragalin derivatives with different concentrations in the reaction 6 is detected by an ADP-GloTMMax detection kit: the reaction is carried out in a 96-well plate, the total reaction system is 5 mu L, and the reaction totality comprises astragalin derivative Z21 mu L (DMSO content is 5%), activated P-CDK2 purified protein (2 mu L, 78nmol/L), ATP (0.75 mu L, 75 mu mol/L), and pseudosubstrate polypeptide HHASPRK (1.25 mu L, 250 mu mol/L). And (3) uniformly mixing the reactants, reacting for 10min at room temperature, adding 5 mu of LADP-GloTM reagent to terminate the reaction, incubating for 55min at room temperature to consume the residual ATP in the reaction system, adding 10 mu of ADP-GloTM Max detection reagent, incubating for 1h at room temperature, converting ADP into ATP and coupling a fluorescence signal. And detecting the fluorescence signal value by using a microplate reader.

3) The inhibition was calculated from the ratio of the difference in fluorescence signal to the blank signal value, and the IC50 curve was fitted with Graphpad prism6 and the IC50 value was calculated.

As a result:

After 6 concentrations of astragalin derivative Z2 were tested for CDK2 kinase inhibition, its IC50 was calculated to be 4.24 μ M and showed significant dose dependence as the compound concentration increased, as shown in fig. 4.

The IC50 value of astragalin derivative Z2 is compared with 14.90 mu M of astragalin, which shows that the inhibition effect is obviously improved.

In conclusion, the method for designing and synthesizing the corresponding CDK2 kinase inhibitor by computer assistance is reliable and effective, and a lead compound 4' -O-2- { N- [ (1S,3R,4S) -4,7, 7-trimethyl-3-bicyclo [2.2.1] heptyl ] acetamido } astragalin with remarkable CDK2 kinase inhibitory activity is found by the method, so that a research basis is provided for the research of related anti-cancer drugs.

Claims (3)

1. An astragalin derivative is characterized in that the astragalin derivative is 4' -O-2- { N- [ (1S,3R,4S) -4,7, 7-trimethyl-3-bicyclo [2.2.1] heptylalkyl ] acetamido } astragalin, and is shown in a structural formula I:

2. The use of an astragalin derivative according to claim 1, as a CDK2 kinase inhibitor.

3. The use of astragalin derivative according to claim 2, wherein the astragalin derivative has an IC50 value of 4.24 μ M as CDK2 kinase inhibitor.