CN116284188A

CN116284188A - Double-substrate inhibitor of DNA methyltransferase DNMT1 and application thereof

Info

Publication number: CN116284188A
Application number: CN202310074289.0A
Authority: CN
Inventors: 杨娜; 苏波; 孙霁雪; 袁龙啸; 于海燕; 杨泽坤
Original assignee: Nankai University
Current assignee: Nankai University
Priority date: 2023-02-07
Filing date: 2023-02-07
Publication date: 2023-06-23

Abstract

The invention provides a DNA methyltransferase DNMT1 double-substrate inhibitor and application thereof, wherein the DNA methyltransferase DNMT1 double-substrate inhibitor is a compound with an adenosine structure as a parent nucleus, as shown in a formula I or pharmaceutically acceptable salt thereof. The in vitro and cell level DNA methylation experiments prove that the DNA methyltransferase DNMT1 double-substrate inhibitor has good inhibition effect on the DNMT1 methylation level on the molecular and cell level, can be used as an effective inhibitor of DNMT1 of various cancer treatment targets, and can be used as a candidate or lead compound for development to prepare related medicaments.

Description

Double-substrate inhibitor of DNA methyltransferase DNMT1 and application thereof

Technical Field

The invention belongs to the field of pharmaceutical chemistry, and particularly relates to a DNA methyltransferase DNMT1 double-substrate inhibitor and application thereof.

Background

DNA 5-methylcytosine methylation (modification of a methyl group on carbon number 5 of cytosine) is an important epigenetic modification in mammals that is involved in regulating a variety of biological processes. Such as genomic imprinting, X-chromosome inactivation, transcriptional repression, embryo development, and the like. The major class of proteins that mediate the DNA methylation process is the family of DNA methyltransferases (DNA methyltransferases, DNMTs). The catalytic mechanism is: by transferring the methyl group of the catalytic cofactor S-adenosylmethionine (SAM) to the carbon atom at position 5 of cytosine, while the demethylated SAM is reduced to S-adenosyl homocysteine (SAH), SAH is a natural DNMTs inhibitor in organisms. DNMTs family members include DNMT1, DNMT3A, DNMT B, and the catalytically inactive structural subunit DNMT3L. Wherein DNMT1 is the first discovered subtype in DNMTs family and is the most abundant subtype, plays a role in maintaining methylation, and plays a vital role in resisting passive demethylation caused by cell mitosis and maintaining methylation patterns established in early embryo development; DNMT3A/3B mediates de novo methylation of DNA, which is critical for establishing methylation patterns in germ cells and early embryonic cells; DNMT3L itself is catalytically inactive and assists its catalytic function by binding to DNMT 3A/3B.

About 60-70% of CpG sites in humans are methylated and cell type specific DNA methylation patterns are established. The correct methylation pattern is critical to maintaining normal cell development. Alterations in methylation patterns are often associated with the occurrence and progression of disease. In recent years, more and more studies have shown that DNA hypermethylation is associated with a variety of cancers including breast cancer, rectal cancer, bladder cancer, and the like. In vitro experimental results show that cancer cells can be promoted to apoptosis by applying DNMT1 inhibitor or knocking out DNMT1 gene. It is widely believed that this is due to the demethylation of the DNA promoter region, resulting in reactivation of the oncogene. Currently, drugs targeting DNMT1, such as the nucleoside analogs decitabine, gemcitabine, etc., are on the market. Although the drugs can inhibit the hypermethylation of DNA in cancer cells to a large extent, the drugs act in a way of irreversibly and covalently binding with DNMT1, and if used for a long time, various toxic and side effects on human bodies are easy to generate, and even life-threatening possibility exists. Therefore, it is important to develop a novel DNMT1 inhibitor which has high selectivity and activity.

Existing DNMT1 inhibitors fall into two main categories. The first class is nucleoside analogues, which have relatively similar chemical skeletons and modes of action, i.e., they "capture" DNMT1 by mimicking the participation of cytosine in the DNA synthesis process, forming covalent complexes (as shown in FIG. 1), and finally achieving the effect of inhibiting DNMT1 methylation. Among these compounds, various drugs represented by decitabine and gemcitabine have been put into clinical use for the treatment of myelodysplastic syndrome, acute myelogenous leukemia, and the like. It is easy to find out from the action mechanism, the medicine can be embedded into human genome along with DNA replication, irreversibly combined with DNMT1, and extremely easy to generate serious toxic and side effects, and if the medicine is used in large dose, the medicine can cause symptoms such as neurotoxicity, somnolence, aphasia, hemiplegia and the like. The compounds lack selectivity to DNMTs family due to nucleoside analogues. In addition, the compound has the problems of unstable pharmacokinetic property, short half-life period and the like, and brings a plurality of inconveniences to medicine storage and transportation. At present, the traditional Chinese medicine composition is only used for coping with non-solid tumors in clinic, and has a narrow application range. The second is non-nucleoside drugs which exert competitive binding effects by mimicking the binding pattern of the cofactor SAM in the active pocket of DNMT1, e.g. the SAM demethylated product SAH is a class of inhibitors. Over the last two decades, several research teams have explored this, and have obtained a range of potential potent inhibitors against DNMT1.

In 2006, sielecki, p.et al obtained the first active prominent non-nucleoside DNMT1 inhibitor-RG 108 by virtual screening. Further research results show that it can inhibit bacterial DNA methyltransferase M.Sss I and in vitro activity IC ₅₀ Nanomolar grades can be achieved. Although RG108 exhibits good killing ability against several classes of cancer cells, there is no significant reduction in intracellular methylation levels suggesting that it may exert antitumor activity through other pathways. In 2011, medina-Franco, J.L. et al obtained a natural product-SGI-1027 with DNMT1 inhibition by means of virtual screening, which can greatly reduce methylation level at the cellular level and 90% at a concentration of 25. Mu.M. In 2014, shijie Chen et al obtained a novel carbazole DNMT1 selective inhibitor DC-05 based on computer virtual screening (which occupies SAM binding pocket). It has high activity (IC) ₅₀ =10.3 μm) and good selectivity for DNMT1 (poor inhibitory activity against various methyltransferases such as DNMT3A, DNMT B, histone methylase G9a, SUV39H1, MLL1, SET7/9 and PRMT 1). They then performed a molecular structural similarity search on DC-05 and identified that compound DC_501 (IC ₅₀ ＝2.5μM)、DC_517(IC ₅₀ =1.7 μm), they have higher inhibitory activity than dc_05. The three effective compounds have remarkable cancer cell proliferation inhibition effect. In 2017, san Jose-Ene riz, E et al designed based on experience and structure to synthesize a double-target inhibitor CM-272[ G9a (IC) ₅₀ ＝8nM)and DNMT1(IC ₅₀ ＝382nM)]Exhibit good efficacy and low toxicity in mice.

Although the above studies have achieved exciting results, no related drug molecules have been marketed through clinical studies to date. Although non-nucleoside drugs avoid potential toxic side effects caused by irreversible binding of cytidine analogs, it is considered that such drugs are designed to exert their inhibitory effect by occupying SAM binding sites, and SAM binding sites are also present in humans for various protein methyltransferases, which may result in poor selectivity of such drugs and affect the results of clinical trials.

Disclosure of Invention

In view of the above, the present invention utilizes molecular virtual screening and molecular backbone transition to screen and design a dual substrate inhibitor for DNMT1, which can occupy both SAM/SAH binding sites and substrate binding pockets. And a series of derivatives are modified and synthesized by taking the derivative as a lead compound. The derivatives are subjected to in vitro biochemical level and intracellular activity research to obtain modification products with in vitro and intracellular activities, and the structure-activity relationship research is carried out with the assistance of molecular docking. The invention provides a DNA methyltransferase DNMT1 double-substrate inhibitor and application thereof.

Specifically, the invention provides a DNA methyltransferase DNMT1 double-substrate inhibitor, which has the technical scheme that:

a DNA methylase DNMT1 dual substrate inhibitor, characterized by: a compound represented by structure I having an adenosine parent nucleus or a pharmaceutically acceptable salt thereof:

wherein R1 is selected from any one of the following structures:

r2 is selected from any one of the following structures:

wherein R1 and R2 are required to be substituted simultaneously; are each independently selected from the structures described above.

Further, the DNA methyltransferase DNMT1 dual-substrate inhibitor is a compound with a structure shown in any one of formulas II to X or pharmaceutically acceptable salt thereof:

fourteen DNA methyltransferase DNMT1 dual-substrate inhibitors taking nucleosides as a mother nucleus are designed and improved based on screening of a DNMT1 cofactor SAM/SAH binding pocket and a substrate cytosine binding pocket. The fourteen DNMT1 double-substrate inhibitors taking nucleoside as a mother nucleus can inhibit methylation reaction of DNMT1 and DNA in vitro, and are effective inhibitors of DNMT1 protein.

Further, the DNA methyltransferase DNMT1 dual-substrate inhibitor is used for inhibiting the activity of DNMT1 protein.

Further, the DNA methyltransferase DNMT1 dual substrate inhibitor inhibits the activity of DNMT1 protein in a non-covalent manner.

Further, the DNA methyltransferase DNMT1 is a human DNMT1.

Furthermore, the DNA methyltransferase DNMT1 double-substrate inhibitor can effectively inhibit DNMT1 protein from playing a methylation function to different degrees in 20 mu M concentration of a compound body with a structure shown in formulas II to X, wherein the inhibition rate of III, IV, V, VI is equivalent to that of a positive control RG108, and the inhibition rate of all the compounds is superior to SAH.

Further, the compound having the structure shown in formula X has an activity of inhibiting DNMT1 methylation reaction on HEK-293T cells and has better cell activity compared with SAH.

The invention also provides a pharmaceutical composition, which comprises the DNA methyltransferase DNMT1 double-substrate inhibitor and pharmaceutically acceptable auxiliary materials, diluents, carriers or the combination thereof.

The invention also provides an application of the DNA methyltransferase DNMT1 dual-substrate inhibitor or the pharmaceutical composition in preparing antitumor drugs.

The medicine can inhibit DNMT1 to catalyze DNA methylation, and further up regulate cancer gene expression, so as to treat cancer-related diseases.

Compared with the prior art, the DNA methyltransferase DNMT1 double-substrate inhibitor and the application thereof have the following advantages:

(1) The invention utilizes a virtual screening means to obtain fragment molecules which can well occupy DNMT1 substrate binding pockets, then combines the fragment molecules with SAH molecules based on the idea of combinatorial chemistry, and utilizes a virtual skeleton transition means to virtually optimize the combined molecular structure, thereby finally obtaining two DNMT1 double-substrate inhibitors with good external activity, namely formulas II and III. DNMT1 dual substrate inhibitors are of great importance for increasing affinity and selectivity for DNMT1. Mainly shows higher activity and smaller toxic and side effects.

(2) A series of derivatives are modified and synthesized by taking II and III as lead compounds. Through in vitro activity detection and cell activity experiments on HEK-293T cells, compounds with higher activity at the cell level than SAH, namely the formula X as described above, are finally obtained.

(3) The DNA methyltransferase DNMT1 double-substrate inhibitor provided by the invention is used as an effective double-substrate inhibitor for resisting DNMT1 proteins of various cancer targets, and can be used as a candidate or lead compound for further development to prepare related medicaments.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention. In the drawings:

FIG. 1 is a schematic diagram showing the principle of action of nucleoside DNMT1 inhibitors by covalently binding DNMT 1;

FIG. 2 is a schematic diagram of the structure of a DNMT1 non-nucleoside inhibitor with higher reported activity;

FIG. 3 is a surface view of a DNMT1 SAM/SAH binding pocket and a substrate binding pocket;

FIG. 4 shows the molecular structure of fragments occupying the binding pocket of DNMT1 substrate and the binding pattern with DNMT1 obtained by screening;

FIG. 5 is a schematic diagram of the concept of a lead compound obtained by combining the concept of combinatorial chemistry with the design of framework transitions;

FIG. 6 shows two-dimensional structures of two lead compound molecules-II and III obtained by combining the combination chemistry ideas with the framework transition design;

FIG. 7 is a hydrogen nuclear magnetic resonance spectrum of compound II, III;

FIG. 8 is a diagram showing the binding patterns of formulas II, III and DNMT 1;

FIG. 9 is a schematic diagram of the in vitro activity detection principle of DNMT 1;

FIG. 10 shows the fluorescence intensity of DNMT1 in vitro activity assay as a function of time;

FIG. 11 is a graph showing peak positions of DNMT1 isolated using molecular sieves;

FIG. 12 is a SDS-PAGE electrophoresis obtained after purification of DNMT1 protein expression;

FIG. 13 is a schematic diagram of the principle of detection of DNMT1 dual substrate inhibitor cell activity;

FIG. 14 shows the results of a DNMT1 dual substrate inhibitor cell activity assay;

FIG. 15 shows the quantitative determination of molecular X-cell activity;

FIG. 16 is a diagram showing a binding mode between a molecule (formula X) having a good cell activity and DNMT1.

Detailed Description

Unless otherwise defined, all terms used herein have meanings conventionally understood by those skilled in the art, and for the convenience of understanding of the present invention, some of the terms used herein shall be defined to have the following meanings.

As used in the specification and in the claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "cell" includes a plurality of cells, including mixtures thereof.

All numerical designations such as pH, temperature, time, concentration, including ranges, are approximations. It is to be understood that the term "about" is not always preceded by the explicit recitation of all numerical designations. It is also to be understood that the agents described herein are merely examples and that equivalents thereof are known in the art, although not always explicitly recited.

Except where specifically indicated, the various reagents referred to herein are all commercially available.

The present invention will be described in detail with reference to the following examples and drawings.

1. Method for obtaining lead Compound

1. Based on the three-dimensional structure of the DNMT1 substrate binding pocket, fragment molecules that bind well at this location were obtained using means of virtual screening. As shown in fig. 2 and 3.

2. FIG. 3 shows a DNMT1 substrate binding pocket and a SAM/SAH binding pocket. The screened fragment molecules (as shown in fig. 4 and 5) are spliced with the SAH molecules by using the idea of combinatorial chemistry so that the binding sites of the SAH and the substrate can be occupied simultaneously.

3. As shown in FIG. 6, the lead compound molecules with better virtual scoring and better in vitro activity, namely formulas II and III, are obtained by optimizing the mode of virtual skeleton transition.

2. Lead compound optimization

1. In order to improve the capability of the lead compound to cross cell membranes, polar functional groups are deleted on the basis of the lead compound, and a series of structural derivatives are designed and synthesized;

2. molecular docking means are used to predict the ability of the engineered product to bind DNMT1. Fig. 8 shows a 3D, 2D binding pattern diagram of the initially optimised binding DNMT1 of formulae II, III.

TABLE one results of scoring the end-to-end of the engineering product and DNMT1 molecule

Compounds	Docking score(kcal/mol)
		SAH	-11.019
II	-11.545
		III	-13.627
IV	-11.866
		V	-10.598
VI	-11.591
		VII	-12.134
VIII	-11.121
		IX	-10.733
X	-12.644

3. Compound synthesis method and characterization data

Example 1: (S) -2-amino-4- ((R) -3-amino-3-carboxypropyl) ((2R, 3S,4R, 5R) -5- (6-amino-9H-purin-9-yl) -3, 4-dihydroxytetrahydrofuran-2-yl) methyl) amino) butanoic acid (Compound II)

(1) 2- ((Boc) amino) -4-oxobutanoic acid tert-butyl ester

4-t-Butoxycarbonylamino-4-oxobutanoic acid (300 mg,1.04 mmol) and methyl iodide (148 mg,1.04 mmol) were dissolved in DMF (6 ml) and reacted overnight at room temperature to give the product 1- (t-butyl) 4-methyl (t-butoxycarbonyl) aspartic acid. This was then added together with NaBH4 (39 mg,1.04 mmol) to an appropriate volume of methanol solution, reacted at room temperature for 6 hours, DMP (4 ml) was added to the obtained reaction product, and the reaction was carried out at room temperature overnight to give tert-butyl 2- ((tert-butoxycarbonyl) amino) -4-oxobutyrate as a product.

(2) 9- (3 aR,4R,6 aR) -6-aminomethyl-2, 2-dimethyltetrahydrofuran [3,4-d ] [1,3] dioxol-4-yl) -9H-purin-6-amine

(3 aR,4R,6 aR) -6- (6-amino-9H-purin-9-yl) -2, 2-dimethyltetrahydrofuran [3,4-d ] [1,3] dioxol-4-methanol (300 mg,0.98 mmol), 4-methylbenzenesulfonyl chloride (186 mg,0.98 mmol) was dissolved in pyridine (6 ml), then 1, 3-dioxoisoindolin-2-ester (143 mg,0.98 mmol), hydrazine (35 mg,1.1 mmol) were added and the reaction was carried out overnight at room temperature to give 9- (3 aR,4R,6 aR) -6-aminomethyl-2, 2-dimethyltetrahydrofuran [3,4-d ] [1,3] dioxol-4-yl) -9H-purin-6-amine.

(3) (2R, 3S,4R, 5R) -5- (6-amino-9H-purin-9-yl) -3, 4-dihydroxytetrahydrofuran-2-yl) methyl) azadiyl bis (2-aminobutyrate

200mg of each of the products obtained in

steps

1 and 2 of example 1 was dissolved in 20ml of DCE, and 4ml of TFA was added thereto, followed by reflux under heating, and after four hours of reaction, 4ml of water was added thereto, and stirring was continued. The progress of the reaction was monitored by thin layer chromatography. The solvent was removed by rotary evaporation of the reaction mixture, 4ml of methanol was added for dissolution, an appropriate amount of diethyl ether was added, suction filtration was performed, and the precipitate was collected. Separating and purifying by silica gel column chromatography, wherein the eluent is prepared from dichloromethane in the following proportion: methanol: ammonia = 1:1:0.1. the obtained target product is white powder, and the yield is between 30 and 32 percent. HRMS (ESI-TOF) m/z 468.210[ M+H ]] ⁺ 。 ¹ H NMR(400MHz,D ₂ O):δ8.08(d,J＝3.1Hz,1H),7.95(d,J＝3.0Hz,1H),5.88(t,J＝3.9Hz,1H),4.62(d,J＝4.4Hz,9H),4.18(dq,J＝31.9,5.5,4.3Hz,2H),3.63(q,J＝4.9,3.4Hz,2H),3.16–2.45(m,6H),1.93(ddt,J＝48.7,14.8,7.1Hz,4H).

Example 2: (R) -2-amino-5- ((R) -3-amino-3-carboxypropyl) ((2R, 3S,4R, 5R) -5- (6-amino-9H-purin-9-yl) -3, 4-dihydroxytetrahydrofuran-2-yl) methyl) amino) pentanoic acid (Compound III)

(1) 2- [ bis (t-Butoxycarbonyl) amino ] -5-t-butyloxopentanoate

2- (Boc) amino-5-methoxy-5-oxopentanoic acid (300 mg,1.15 mmol), tert-butyl 2, 2-trichloroacetimidate (251 mg,1.15 mmol) was dissolved in dichloromethane (6 ml), followed by the addition of di-tert-butyl dicarbonate (251 mg,1.15 mmol), sodium borohydride (48 mg,1.27 mmol) and DMP (4 ml) followed by reaction overnight at room temperature to give 2- [ bis (Boc) amino ] -5-tert-butyloxopentanoate.

(2) (2R, 3S,4R, 5R) -5- (6-amino-9H-purin-9-yl) -3, 4-dihydroxytetrahydrofuran-2-yl) methyl) azadiyl bis (2-aminobutyrate

250mg of each of the products obtained in step 2 of example 1 and step 1 of example 2 was dissolved in 20ml of DCE, and 4ml of TFA was added thereto, and the mixture was refluxed with heating, and after reacting for four hours, 4ml of water was added thereto, and stirring was continued. The progress of the reaction was monitored by thin layer chromatography. The solvent was removed by rotary evaporation of the reaction mixture, 4ml of methanol was added for dissolution, an appropriate amount of diethyl ether was added, suction filtration was performed, and the precipitate was collected. Separating and purifying by silica gel column chromatography, wherein the eluent is prepared from dichloromethane in the following proportion: methanol: ammonia = 1:1:0.1. the obtained target product is white powder, and the yield is between 30 and 32 percent. HRMS (ESI-TOF) m/z 482.220[ M+H ]] ⁺ 。 ¹ H NMR(400MHz,D ₂ O):

δ8.10 (s, 1H), 7.99 (s, 1H), 5.90 (d, j=4.6 hz, 1H), 4.65 (t, j=5.0 hz, 9H), 4.20 (dt, j=24.9, 5.6hz, 2H), 3.88-3.37 (m, 2H), 3.23-2.47 (m, 6H), 2.22-1.06 (m, 6H). Example 3: (S) -2-amino-5- (((R) -4-amino-4-carboxybutyl) ((2R, 3S,4R, 5R) -5- (6-amino-9H-purin-9-yl) -3, 4-dihydroxytetrahydrofuran-2-yl) methyl) amino) pentanoic acid (Compound IV)

(1) Tert-butyl (R) -5- ((((3 aR,4R,6 aR) -6- (6-amino-9H-purin-9-yl) -2, 2-dimethyltetrahydrofuran [3,4-d ] [1,3] dioxo-4-yl) methyl) amino) -2- (bis (tert-butoxycarbonyl) amino) pentanoate

9- ((3 aR,4R,6 aR) -6- (aminomethyl) -2, 2-dimethyltetrahydrofuran [3,4-d ] [1,3] dioxo-4-yl) -9H-purin-6-amine (150 mg,0.49 mmol), (S) -2- (bis (t-butoxycarbonyl) amino) -5-oxopentanoic acid tert-butyl ester (20 mg,0.49 mmol) was dissolved in DCM (3 mL) and the reaction stirred at room temperature for 1 hour. The temperature of the reaction system was lowered to 0℃and sodium triacetoxyborohydride (310 mg,1.47 mmol) was added to the reaction system in portions. The reaction was stirred at room temperature overnight. The reaction was quenched by addition of an appropriate amount of saturated aqueous sodium bicarbonate solution, the mixture was extracted with DCM, the organic phase was washed successively with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by column chromatography over silica gel to give tert-butyl (R) -5- (((((3 aR,4R,6 aR) -6- (6-amino-9H-purin-9-yl) -2, 2-dimethyltetrahydrofuran [3,4-d ] [1,3] dioxo-4-yl) methyl) amino) -2- (bis (tert-butoxycarbonyl) amino) pentanoate (170 mg, yield: 52%) as a white solid.

(2) Tert-butyl (S) -5- ((((3 aR,4R,6 aR) -6- (6-amino-9H-purin-9-yl) -2, 2-dimethyltetrahydrofuran [3,4-d ] [1,3] dioxo-4-yl) methyl) ((R) -4- (bis (tert-butoxycarbonyl) amino) -5- (tert-butoxy) -5-oxopentyl) amino) -2- (bis (tert-butoxyacyl) amino) pentanoate

Referring to the procedure of example 3, step (1), using tert-butyl (R) -5- ((((3 aR,4R,6 aR) -6- (6-amino-9H-purin-9-yl) -2, 2-dimethyltetrahydrofuran [3,4-d ] [1,3] dioxo-4-yl) methyl) amino) -2- (bis (tert-butoxycarbonyl) amino) pentanoate (170 mg,0.25 mmol), tert-butyl (S) -2- (bis (tert-butoxycarbonyl) amino) -5-oxopentanoate (10 mg,0.25 mmol), sodium triacetoxyborohydride (155 mg,0.75 mmol) as starting material gave (2) tert-butyl (S) -5- ((((3 aR,4R,6 aR) -6- (6-amino-9H-purin-9-yl) -2, 2-dimethyltetrahydrofuran [3,4-d ] [1,3] dioxo-4-yl) methyl) ((R) -4- (tert-butoxycarbonyl) amino) -5-oxopentanoate as a white solid (147 mg, 5 mg).

(3) (S) -2-amino-5- (((R) -4-amino-4-carboxybutyl) ((2R, 3S,4R, 5R) -5- (6-amino-9H-purin-9-yl) -3, 4-dihydroxytetrahydrofuran-2-yl) methyl) amino) pentanoic acid

Tert-butyl (S) -5- ((((3 aR,4R,6 aR) -6- (6-amino-9H-purin-9-yl) -2, 2-dimethyltetrahydrofuran [3, 4-d)][1,3]Dioxol-4-yl) methyl) ((R) -4- (bis (t-butoxycarbonyl) amino) -5- (t-butoxy) -5-oxopentyl) amino) -2- (bis (t-butoxyacyl) amino) pentanoate (147 mg,0.14 mmol) was dissolved in 1, 4-dioxane (3 mL), 4M/L aqueous hydrochloric acid (0.1 mL) was added, and stirred overnight at 50 ℃. The reaction solution was neutralized to pH neutral with 4M/L aqueous sodium hydroxide, concentrated under reduced pressure, dried and purified by column chromatography on silica gel to give (S) -2-amino-5- (((R) -4-amino-4-carboxybutyl) ((2R, 3S,4R, 5R) -5- (6-amino-9H-purin-9-yl) -3, 4-dihydroxytetrahydrofuran-2-yl) methyl) amino) pentanoic acid (84 mg, yield: 60%) white solid. HRMS (ESI-TOF) m/z 497.2462[ M+H ]] ⁺ 。 ¹ H NMR(400MHz,D ₂ O):8.24(s,1H),8.19(s,1H),6.04(d,J＝4.7Hz,1H),4.80(t,J＝5.1Hz,1H),4.47–4.33(m,2H),3.67(h,J＝2.6Hz,2H),3.52(s,2H),3.16(d,J＝8.3Hz,4H),1.80(s,8H)。

Example 4: (R) -2-amino-5- ((((2R, 3S,4R, 5R) -5- (6-amino-9H-purin-9-yl) -3, 4-dihydroxytetrahydrofuran-2-yl) methyl) (4-carboxybutyl) amino) pentanoic acid (Compound V)

(1) Tert-butyl (S) -5- ((((3 aR,4R,6 aR) -6- (6-amino-9H-purin-9-yl) -2, 2-dimethyltetrahydrofuran [3,4-d ] [1,3] dioxo-4-yl) methyl) (5- (tert-butoxy) -5-oxopentyl) amino) -2- (bis (tert-butoxycarbonyl) amino) pentanoate

Referring to the procedure of example 3, step (1), tert-butyl (R) -5- ((((3 aR,4R,6 aR) -6- (6-amino-9H-purin-9-yl) -2, 2-dimethyltetrahydrofuran [3,4-d ] [1,3] dioxo-4-yl) methyl) amino) -2- (bis (tert-butoxycarbonyl) amino) pentanoate (170 mg,0.25 mmol), tert-butyl 5-oxopentanoate (43 mg,0.25 mmol) and sodium triacetoxyborohydride (155 mg,0.75 mmol) were used as starting materials to give tert-butyl (S) -5- ((((3 aR,4R,6 aR) -6- (6-amino-9H-purin-9-yl) -2, 2-dimethyltetrahydrofuran [3,4-d ] [1,3] dioxo-4-yl) methyl) (5- (tert-butoxy) -5-oxopentyl) amino) -2- (bis (tert-butoxycarbonyl) pentanoate (150 mg) as a white solid in a yield of 72% white solid.

(2) (R) -2-amino-5- ((((2R, 3S,4R, 5R) -5- (6-amino-9H-purin-9-yl) -3, 4-dihydroxytetrahydrofuran-2-yl) methyl) (4-carboxybutyl) amino) pentanoic acid

Referring to example 3, step (3), tert-butyl (S) -5- ((((3 aR,4R,6 aR) -6- (6-amino-9H-purin-9-yl) -2, 2-dimethyltetrahydrofuran [3, 4-d) was used][1,3]Dioxoethanol-4-yl) methyl) (5- (tert-butoxy) -5-oxopentyl) amino) -2- (bis (tert-butoxycarbonyl) amino) pentanoate (150 mg,0.18 mmol), 4M/L aqueous hydrochloric acid as starting material gave (R) -2-amino-5- ((((2R, 3S,4R, 5R) -5- (6-amino-9H-purin-9-yl) -3, 4-dihydroxytetrahydrofuran-2-yl) methyl) (4-carboxybutyl) amino) pentanoic acid (43 mg, yield: 50%) of a white solid. HRMS (ESI-TOF) M/z482.2352[ M+H ]] ⁺ 。 ¹ H NMR(400MHz,D ₂ O):8.26(s,1H),8.20(s,1H),6.07(d,J＝4.6Hz,1H),4.86(t,J＝5.0Hz,1H),4.50–4.36(m,2H),3.69(t,J＝5.8Hz,1H),3.43(h,J＝5.3Hz,2H),3.13–2.99(m,4H),2.11(t,J＝7.1Hz,2H),1.91–1.71(m,4H),1.64–1.40(m,4H)。

Example 5: (R) -2-amino-5- ((((2R, 3S,4R, 5R) -5- (6-amino-9H-purin-9-yl) -3, 4-dihydroxytetrahydrofuran-2-yl) methyl) (5-hydroxypentyl) amino) pentanoic acid (Compound VI)

(1) Tert-butyl (S) -5- ((((3 aR,4R,6 aR) -6- (6-amino-9H-purin-9-yl) -2, 2-dimethyltetrahydrofuran [3,4-d ] [1,3] dioxo-4-yl) methyl) (5- ((tert-butyldimethylsilyl) oxy) pentyl) amino) -2- (bis (tert-butoxycarbonyl) amino) pentanoate

Referring to the procedure of example 3, step (1), using tert-butyl (R) -5- ((((3 aR,4R,6 aR) -6- (6-amino-9H-purin-9-yl) -2, 2-dimethyltetrahydrofuran [3,4-d ] [1,3] dioxan-4-yl) methyl) amino) -2- (bis (tert-butoxycarbonyl) amino) pentanoate (170 mg,0.25 mmol), 5- ((tert-butyldimethylsilyl) oxy) valeraldehyde (54 mg,0.25 mmol), sodium triacetoxyborohydride (155 mg,0.75 mmol) as starting material gave tert-butyl (S) -5- ((((3 aR,4R,6 aR) -6- (6-amino-9H-purin-9-yl) -2, 2-dimethyltetrahydrofuran [3,4-d ] [1,3] dioxan-4-yl) methyl) (5- ((tert-butyldimethylsilyl) oxy) amino) -2- (bis (tert-butoxycarbonyl) amino) pentanoate as a white solid (149 mg).

(2) (R) -2-amino-5- ((((2R, 3S,4R, 5R) -5- (6-amino-9H-purin-9-yl) -3, 4-dihydroxytetrahydrofuran-2-yl) methyl) (5-hydroxypentyl) amino) pentanoic acid

Referring to example 3, step (3), tert-butyl (S) -5- ((((3 aR,4R,6 aR) -6- (6-amino-9H-purin-9-yl) -2, 2-dimethyltetrahydrofuran [3, 4-d) was used][1,3]Dioxy-4-yl) methyl) (5- ((tert-butyldimethylsilyl) oxy) pentyl) amino) -2- (bis (tert-butoxycarbonyl) amino) pentanoate (149 mg,0.17 mmol), 4M/L aqueous hydrochloric acid as starting material gave (R) -2-amino-5- ((((2R, 3S,4R, 5R) -5- (6-amino-9H-purin-9-yl) -3, 4-dihydroxytetrahydrofuran-2-yl) methyl) (5-hydroxypentyl) amino) pentanoic acid (54 mg, yield: 68%) of a white solid. HRMS (ESI-TOF) m/z 468.2562[ M+H ]] ⁺ 。 ¹ H NMR(400MHz,D ₂ O):8.28(s,1H),8.20(s,1H),6.04(d,J＝4.3Hz,1H),4.32(q,J＝6.9,5.7Hz,2H),3.62(t,J＝5.9Hz,1H),3.46(tt,J＝6.5,3.4Hz,2H),3.03–2.88(m,2H),2.65(dt,J＝30.6,10.4Hz,4H),1.80(q,J＝7.1Hz,2H),1.62(p,J＝7.5Hz,2H),1.35(dd,J＝14.2,7.4Hz,4H),1.16(tt,J＝15.0,7.1Hz,2H)。

Example 6: (R) -2-amino-5- ((((2R, 3S,4R, 5R) -5- (6-amino-9H-purin-9-yl) -3, 4-dihydroxytetrahydrofuran-2-yl) methyl) (5-aminopentyl) amino) pentanoic acid (Compound VII)

(1) Tert-butyl (S) -5- ((((3 aR,4R,6 aR) -6- (6-amino-9H-purin-9-yl) -2, 2-dimethyltetrahydrofuran [3,4-d ] [1,3] dioxo-4-yl) methyl) (5- (1, 3-dioxoisoindol-2-yl) pentyl) amino) -2- (bis (tert-butoxycarbonyl) amino) pentanoate

Referring to the procedure of example 3, step (1), using tert-butyl (R) -5- ((((3 aR,4R,6 aR) -6- (6-amino-9H-purin-9-yl) -2, 2-dimethyltetrahydrofuran [3,4-d ] [1,3] dioxo-4-yl) methyl) amino) -2- (bis (tert-butoxycarbonyl) amino) pentanoate (170 mg,0.25 mmol), 5- (1, 3-dioxoisoindol-2-yl) valeraldehyde (54 mg,0.25 mmol), sodium triacetoxyborohydride (155 mg,0.75 mmol) as starting material gave tert-butyl (S) -5- ((((3 aR,4R,6 aR) -6- (6-amino-9H-purin-9-yl) -2, 2-dimethyltetrahydrofuran [3,4-d ] [1,3] dioxo-4-yl) methyl) (5- (1, 3-dioxo-isoindol-2-yl) pentanoate) as a white solid (167 mg).

(2) (R) -2-amino-5- ((((2R, 3S,4R, 5R) -5- (6-amino-9H-purin-9-yl) -3, 4-dihydroxytetrahydrofuran-2-yl) methyl) (5-aminopentyl) amino) pentanoic acid

Tert-butyl (S) -5- ((((3 aR,4R,6 aR) -6- (6-amino-9H-purin-9-yl) -2, 2-dimethyltetrahydrofuran [3, 4-d)][1,3]Dioxy-4-yl) methyl) (5- (1, 3-dioxoisoindol-2-yl) pentyl) amino) -2- (bis (t-butoxycarbonyl) amino) pentanoate (167 mg,0.18 mmol) was dissolved in ethanol (3 mL), hydrazine hydrate (90 mg,2.88 mmol) was added, refluxing at 78℃for 3 hours, the resulting by-product was removed by filtration, the filtrate was dried and the operation was repeated until no white floc was produced. The crude product was dissolved in 1, 4-dioxane (3 mL) and 4M/L hydrochloric acid was addedThe aqueous solution (0.1 mL) was stirred overnight at 50 ℃. The reaction solution was neutralized to pH neutrality with 4M/L aqueous sodium hydroxide solution, concentrated under reduced pressure, dried and loaded onto a silica gel column to obtain (R) -2-amino-5- ((((2R, 3S,4R, 5R) -5- (6-amino-9H-purin-9-yl) -3, 4-dihydroxytetrahydrofuran-2-yl) methyl) (5-aminopentyl) amino) pentanoic acid (42 mg, yield: 50%) as a white solid. HRMS (ESI-TOF) m/z 467.2719. ¹ H NMR(400MHz,D ₂ O):8.29(s,1H),8.21(s,1H),6.03(d,J＝4.5Hz,1H),4.28(td,J＝9.5,8.5,3.7Hz,2H),3.27(s,1H),2.96–2.70(m,4H),2.49(ddd,J＝17.5,11.9,7.1Hz,4H),1.57–1.07(m,10H)ppm。

Example 7: (2R, 3R,4S, 5R) -2- (6-amino-9H-purin-9-yl) -5- ((bis (5-aminopentyl) amino) methyl) tetrahydrofuran-3, 4-diol (Compound VIII)

(1) 2- (5- (((3 ar,4r,6 ar) -6- (6-amino-9H-purin-9-yl) -2, 2-dimethyltetrahydrofuran [3,4-d ] [1,3] dioxo-4-yl) methyl) amino) pentyl) isoindole-1, 3-dione

Referring to the procedure of example 3, step (1), starting from 9- ((3 ar,4r,6 ar) -6- (aminomethyl) -2, 2-dimethyltetrahydrofuran [3,4-d ] [1,3] dioxo-4-yl) -9H-purin-6-amine (150 mg,0.49 mmol), 5- (1, 3-dioxoisoindol-2-yl) valeraldehyde (107 mg,0.49 mmol), sodium triacetoxyborohydride (310 mg,1.47 mmol) gives 2- (5- (((3 ar,4r,6 ar) -6- (6-amino-9H-purin-9-yl) -2, 2-dimethyltetrahydrofuran [3,4-d ] [1,3] dioxo-4-yl) methyl) amino) pentyl) isoindole-1, 3-dione (112 mg, yield: 44%) of a white solid.

(2) 2,2' - (3 ar,4r,6 ar) -6- (6-amino-9H-purin-9-yl) -2, 2-dimethyltetrahydrofuran [3,4-d ] [1,3] dioxo-4-yl) methyl) azadiyl) bis (pentane-5, 1-diyl)) bis (isoindole-1, 3-dione

According to the method of example 3, step (1), starting from 2- (5- (((3 aR,4R,6 aR) -6- (6-amino-9H-purin-9-yl) -2, 2-dimethyltetrahydrofuran [3,4-d ] [1,3] dioxo-4-yl) methyl) amino) pentyl) isoindole-1, 3-dione (112 mg,0.21 mmol), 5- (1, 3-dioxoisoindol-2-yl) valeraldehyde (48 mg,0.21 mmol), sodium triacetoxyborohydride (130 mg,0.63 mmol) gives 2,2' - (3 aR,4R,6 aR) -6- (6-amino-9H-purin-9-yl) -2, 2-dimethyltetrahydrofuran [3,4-d ] [1,3] dioxo-4-yl) methyl) azadiyl) bis (pentane-5, 1-diyl) bis (isoindol-1, 3-dione) (108 mg, yield: 70%) white solid.

(3) (2R, 3R,4S, 5R) -2- (6-amino-9H-purin-9-yl) -5- ((bis (5-aminopentyl) amino) methyl) tetrahydrofuran-3, 4-diol

Reference to example 7 procedure of step (3) as 2,2' - (3 aR,4R,6 aR) -6- (6-amino-9H-purin-9-yl) -2, 2-dimethyltetrahydrofuran [3, 4-d)][1,3]Dioxy-4-yl) methyl azadiyl) bis (pentane-5, 1-diyl) bis (isoindole-1, 3-dione) (108 mg,0.15 mmol), hydrazine hydrate (75 mg,2.4 mmol), 4M/L aqueous hydrochloric acid (0.1 mL) as starting material gave (2R, 3R,4S, 5R) -2- (6-amino-9H-purin-9-yl) -5- ((bis (5-aminopentyl) amino) methyl) tetrahydrofuran-3, 4-diol (32 mg, yield: 50%) of a white solid. HRMS (ESI-TOF) m/z 437.2984. ¹ H NMR(400MHz,D ₂ O):δ＝8.27(s,1H),8.19(s,1H),6.00(d,J＝4.6Hz,1H),4.86(t,J＝4.8Hz,2H),4.29–4.19(m,2H),2.89–2.71(m,2H),2.52(t,J＝7.2Hz,4H),2.43(dd,J＝10.0,5.9Hz,3H),1.38–1.21(m,8H),1.10(tq,J＝13.2,6.9Hz,4H)。

Example 8:5,5' - ((((2R, 3S,4R, 5R) -5- (6-amino-9H-purin-9-yl) -3, 4-dihydroxytetrahydrofuran-2-yl) methyl) azetidinyl) dipentamic acid (Compound IX)

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(1) 5- ((((3 aR,4R,6 aR) -6- (6-amino-9H-purin-9-yl) -2, 2-dimethyltetrahydrofuran [3,4-d ] [1,3] dioxo-4-yl) methyl) amino) pentanoic acid tert-butyl ester

Referring to the procedure of example 3, step (1), starting from 9- ((3 aR,4R,6 aR) -6- (aminomethyl) -2, 2-dimethyltetrahydrofuran [3,4-d ] [1,3] dioxo-4-yl) -9H-purin-6-amine (150 mg,0.49 mmol), tert-butyl 5-oxopentanoate (85 mg,0.49 mmol) sodium triacetoxyborohydride (310 mg,1.47 mmol) gives tert-butyl 5- ((((3 aR,4R,6 aR) -6- (6-amino-9H-purin-9-yl) -2, 2-dimethyltetrahydrofuran [3,4-d ] [1,3] dioxo-4-yl) methyl) amino) pentanoate (68 mg, yield: 30%) as a white solid.

(2) Di-tert-butyl 5,5' - ((((3 aR,4R,6 aR) -6- (6-amino-9H-purin-9-yl) -2, 2-dimethyltetrahydrofuran [3,4-d ] [1,3] dioxo-4-yl) methyl) azepinyl) dipentainate

According to the method of example 3, step (1), using tert-butyl 5- ((((3 aR,4R,6 aR) -6- (6-amino-9H-purin-9-yl) -2, 2-dimethyltetrahydrofuran [3,4-d ] [1,3] dioxo-4-yl) methyl) amino) pentanoate (68 mg,0.15 mmol), tert-butyl 5-oxopentanoate (25 mg,0.15 mmol), sodium triacetoxyborohydride (93 mg,0.45 mmol) as a starting material, 5' - (((3 aR,4R,6 aR) -6- (6-amino-9H-purin-9-yl) -2, 2-dimethyltetrahydrofuran [3,4-d ] [1,3] dioxo-4-yl) methyl) azadiyl) dipenta-ate (33 mg, yield: 36%) as a white solid.

(3) 5,5' - ((((2R, 3S,4R, 5R) -5- (6-amino-9H-purin-9-yl) -3, 4-dihydroxytetrahydrofuran-2-yl) methyl) azo-diyl) dipentyl acid

Referring to the procedure of example 3, step (3), 5' - ((((3 aR,4R,6 aR) -6- (6-amino-9H-purin-9-yl) -2, 2-dimethyltetrahydrofuran [3, 4-d) was used][1,3]Di-tert-butyl-di-oxo-4-yl-methyl) azepinyl-dipentamate, 4M/L aqueous hydrochloric acid as starting material gave 5,5' - (((2R, 3S,4R, 5R) -5- (6-amino-9H-purin-9-yl) -3, 4-dihydroxytetrahydrofuran-2-yl) methyl) azepinyl) dipentamate (6 mg, yield: 23%) white solid. HRMS (ESI-TOF) m/z 467.2246[ M+H ]] ⁺ 。 ¹ H NMR(400MHz,D ₂ O) 8.24 (s, 1H), 8.19 (s, 1H), 6.06 (d, j=4.6 hz, 1H), 4.89 (t, j=4.9 hz, 2H), 4.46 (dq, j=15.7, 4.9hz, 2H), 3.70-3.54 (m, 2H), 3.21 (t, j=8.2 hz, 4H), 2.14 (t, j=7.2 hz, 2H), 1.67-1.57 (m, 4H), 1.48 (p, j=7.9 hz, 4H). Example 9: (2R, 3R,4S, 5R) -2- (6-amino-9H-purin-9-yl) -5- ((bis (5-hydroxypentyl) amino) methyl) tetrahydrofuran-3, 4-diol (Compound X)

(1) 9- ((3 aR,4R,6 aR) -6- (((5- ((tert-butyldimethylsilyl) oxy) pentyl) amino) methyl) -2, 2-dimethyltetrahydrofuran [3,4-d ] [1,3] dioxo-4-yl) -9H-purin-6-amine

Referring to the procedure of example 3, step (1), starting from 9- ((3 aR,4R,6 aR) -6- (aminomethyl) -2, 2-dimethyltetrahydrofuran [3,4-d ] [1,3] dioxan-4-yl) -9H-purin-6-amine (150 mg,0.49 mmol), 5- ((tert-butyldimethylsilyl) oxy) valeraldehyde (106 mg,0.49 mmol), sodium triacetoxyborohydride (310 mg,1.47 mmol) gave 9- ((3 aR,4R,6 aR) -6- (((5- ((tert-butyldimethylsilyl) oxy) pentyl) amino) methyl) -2, 2-dimethyltetrahydrofuran [3,4-d ] [1,3] dioxan-4-yl) -9H-purin-6-amine (102 mg, yield: 41%) as a white solid.

(2) 9- ((3 aR,4R,6 aR) -6- ((bis (5- ((tert-butyldimethylsilyl) oxy) pentyl) amino) methyl) -2, 2-dimethyltetrahydrofuran [3,4-d ] [1,3] dioxo-4-yl) -9H-purin-6-amine

Referring to the procedure of example 3, step (1), starting from 9- ((3 aR,4R,6 aR) -6- (((5- ((tert-butyldimethylsilyl) oxy) pentyl) amino) methyl) -2, 2-dimethyltetrahydrofuran [3,4-d ] [1,3] dioxan-4-yl) -9H-purin-6-amine (102 mg,0.20 mmol), 5- ((tert-butyldimethylsilyl) oxy) valeraldehyde (43 mg,0.20 mmol), sodium triacetoxyborohydride (124 mg,0.60 mmol) gave 9- ((3 aR,4R,6 aR) -6- ((bis (5- ((tert-butyldimethylsilyl) oxy) pentyl) amino) methyl) -2, 2-dimethyltetrahydrofuran [3,4-d ] [1,3] dioxan-4-yl) -9H-purin-6-amine (99 mg, 70%) as a white solid.

(3) (2R, 3R,4S, 5R) -2- (6-amino-9H-purin-9-yl) -5- ((bis (5-hydroxypentyl) amino) methyl) tetrahydrofuran-3, 4-diol

Referring to the method of example 3, step (3), 9- ((3 aR,4R,6 aR) -6- ((bis (5- ((tert-butyldimethylsilyl) oxy) pentyl) amino) methyl) -2, 2-dimethyltetrahydrofuran [3,4-d][1,3]Dioxy-4-yl) -9H-purin-6-amine (99 mg,0.14 mmol), 4M/L aqueous hydrochloric acid to give (2R, 3R,4S, 5R) -2- (6-amino-9H-purin-9-yl) -5- ((bis (5-hydroxypentyl) amino) methyl) tetrahydrofuran-3, 4-diol (28 mg, yield: 45%) white solid. HRMS (ESI-TOF) m/z 439.2661[ M+H ]] ⁺ 。 ¹ H NMR(400MHz,D ₂ O):8.25(s,1H),8.20(s,1H),6.05(d,J＝4.6Hz,1H),4.92(t,J＝4.6Hz,1H),4.50–4.40(m,2H),3.67–3.48(m,3H),3.42(s,2H),3.17(t,J＝8.2Hz,4H),1.57(s,5H),1.27(d,J＝85.0Hz,8H)。

4. In vitro activity detection and verification of the modified compound

1. Expression purification of DNMT1 protein

The target gene fragment of the coding human DNMT1-728-1600 (the sequence of the DNMT1 coding 728-1600 residues) is inserted into the multicloning site of the pFastbac-GST vector, so that the prokaryotic amplified recombinant plasmid inserted into DNMT1 (728-1600) is constructed.

The recombinant plasmid was transferred into E.coli DH 5. Alpha. And spread on LB solid medium containing 100. Mu.g/ml ampicillin, and cultured at 37℃for 8-12h. Single colonies were picked and cultured overnight in LB medium to extract plasmids.

After sequencing, the recombinant plasmid was transferred to E.coli DH10bac, spread on LB solid medium containing 10. Mu.g/ml tetracycline, 7. Mu.g/ml gentamicin, 50. Mu.g/ml kanamycin, 40. Mu.g/ml IPTG, 100. Mu.g/ml X-gal, and cultured at 37℃for 48 hours.

White single colonies were picked and recombinant bacmid was extracted using isopropanol precipitation.

The extracted bacmid is transfected into insect cells SF21 with the assistance of liposome, amplified and passaged, and third-generation virus suspension is collected. A number of insect cells SF21 were infested with a third generation virus suspension.

After waiting 3 days, insect cells were collected, cells were sonicated by suspending them in ice-cold buffer (20mM Tris pH8.0,1M NaCl,1mM DTT,1mM PMSF), cell debris was removed by high-speed centrifugation, and the supernatant was collected. The target protein was then purified using a GST column. After the supernatant was sufficiently bound to the GST magnetic beads, washing was performed using a washing buffer (20 mM Tris-HCl pH8.0,1M NaCl,1mM DTT) to remove the hetero-proteins not bound to the magnetic beads. Finally, the target protein was eluted using elution buffer (20 mM Tris-HCl pH8.0,1M NaCl,1mM DTT). And adding an appropriate amount of PreScission Protease protease into the eluted protein solution for enzyme digestion to remove GST labels on the fusion protein. After concentrating the protein after label removal, further separation and purification were performed with molecular sieve Superdex 200 (GE Healthcare). The peak corresponding to DNMT1 protein was collected (FIG. 11), validated by SDS-PAGE, concentrated, reduced in salt, and finally sub-packaged and frozen at-80 ℃. FIG. 12 is a SDS-PAGE electrophoresis obtained after purification of DNMT1 protein expression.

2. Compound screening and activity testing

Detection principle:

referring to FIG. 9, the enzyme activity detection method adopted by the invention is based on an enzyme coupling mode, DNMT1 can specifically identify a semi-methylated CpG recognition site in a substrate hairpin DNA through SAM-dependent methylation, and catalyze cytosine to methylcytosine. At this point, the fully methylated CpG sites are specifically recognized and cleaved by the Gla I endonuclease. Resulting in separation of the originally proximal 5 'and 3' ends of the hairpin DNA. A fluorescent group is present at the 5 'end and a fluorescence quenching group is present at the 3' end. With the separation of the 5 'end and the 3' end, the fluorescent group is far away from the fluorescence quenching group, and the fluorescence intensity of the reaction system is increased. The intensity of fluorescence increase is positively correlated with the degree of DNA methylation.

Experimental operation:

experimental group: SAM, test compound, bufferA, DNA were added sequentially to a 96-well plate black plate to a final volume of 90. Mu.L. Wherein the buffer A comprises the following components: 100mM NaCl,10mM Tris-HCl 7.5,5mM MgCl ₂ 1mM DTT,5% glycerol, 0.1mg/ml BSA. Pre-equilibrated at 37 ℃ for 20-25 minutes. 10 microliters of reaction solution is added into each hole, the reaction solution consists of buffer A, gla I and DNMT1, and the concentration or content of each component finally reaches the following standard after the addition: DNMT1:80nM, gla I:1U, SAM: 20. Mu. M, DNA:50nM, test compound: 20. Mu.M.

Control group: test compounds were replaced with equal volumes of DMSO, the remaining conditions being the same as the experimental group.

Experimental results:

table II shows the inhibition of DNMT1 by the modified product at a final concentration of 20. Mu.M. From the results, it can be seen that the nine DNMT1 dual substrate inhibitors of formulas II-X exhibited moderate and superior inhibition of DNMT1, most of the activity exceeded that of the product SAH, with III, IV, V, VI inhibition comparable to the positive control RG108, and all compounds had better inhibition than SAH.

In vitro Activity detection results of Table II Compounds (formulas II-X)

Compounds	inhibition rate in 20μM(％)
		RG108	77.9
SAH	52.3
		II	62.7
III	74.4
		IV	81.5
V	73.5
		VI	73.4
VII	68.1
		VIII	65.1
IX	60.6
		X	64.0

Qualitative activity detection assay for DNA methylation on HEK-293T cells

Detection principle:

as shown in FIG. 13, the DNMT1 dual substrate inhibitor cell activity detection method used in the present invention is a method indicated by a luciferase reporter gene. First, the promoter region regulating the downstream luciferase gene was methylated in vitro and then transfected into HEK-293T cells. The ability of the methylated promoter to initiate transcription is inhibited, so that luciferase is expressed in low amounts after entry into HEK-293T cells; the DNMT1 inhibitor applied at this time inhibits DNMT1 methyltransferase activity in HEK-293T cells, and after several cell divisions, the level of promoter methylation upstream of the reporter gene is reduced. Accordingly, the ability of the downstream fluorescent reporter gene to initiate transcription is enhanced, and the fluorescence intensity is increased.

Experimental operation:

first, the TA promoter of the PGL-GFP fluorescence reporter vector is subjected to in vitro mutation, and the original capability of enhancing the transcription of the downstream reporter gene is destroyed. And a CMV sequence which is subjected to in vitro methylation treatment of M.Sss I is inserted into the upstream multi-cloning site of the GFP fluorescent gene.

HEK-293T cells were grown in the presence of 10%Culturing in DMEM medium containing fetal bovine serum and 1% penicillin-streptomycin at 37deg.C and 5% CO ₂ Is maintained in the environment of (a).

When cells were covered 80% -90% in 60mm dishes, they were transfected with the following transfection mixture: 6 μg luciferase reporter, 20 μg PEI Max.

48h after transfection, digestion with trypsin was performed. Inoculating into 96-well plate to ensure that the number of cells per well is controlled between 10000-30000. Placed at 37℃with 5% CO ₂ And (3) in the incubator for 6 hours, the inoculated HEK-293T cells are fully and adhibited for growth.

Test drugs diluted to different concentrations were applied to 96-well plates inoculated with HEK-293T cells to achieve final drug concentrations of 0, 3.2, 6.3, 12.5, 25, 50, 100 μm in the medium. The blank group added an equal volume of DMSO to the culture broth.

After 24 hours of administration, the culture solution in the 96-well plate was aspirated and rinsed twice with phosphate buffer. To this, 100. Mu.l of cell lysate was added and incubated on ice for 30min.

And adding luciferase reaction liquid into each hole, and detecting the fluorescence intensity within the range of 360-700nm in an enzyme-labeling instrument. Three replicates were performed at room temperature. Wherein the components of the luciferase reaction solution are as follows: 20. Mu.M ATP, 20mM MgCl ₂ 20. Mu.M fluorescein. The reaction solution buffer comprises the following components: 500mM NaCl ₂ 、20mM HEPES 8.0、1mM DTT。

Detection result:

as can be seen from FIG. 10, the compound of formula X has better cellular activity on HEK-293T cells than SAH, which is a non-nucleoside inhibitor of DNMT1. Fig. 13 furthermore shows the 2D binding pattern with DNMT1.

Quantitative Activity detection assay for DNA methylation on B2-11 cells

Detection principle:

the DNMT1 double-substrate inhibitor cell activity detection method adopted by the invention is a method for indicating a stably transfected luciferase reporter gene. First, a promoter region regulating a downstream EGFP (enhanced luciferase) gene is methylated in vitro, transfected into B2-11 cells, and a stably transfected cell line is obtained through resistance screening. Upon administration of the DNMT1 inhibitor, the methylation level of the reporter promoter decreases, thereby attenuating inhibition of downstream EGFP, with a concomitant increase in fluorescence intensity.

Experimental operation:

first, EGFP-resistance genes methylated at the promoter region were transfected into B2-11 cells. Stably transfected cell lines were obtained through several rounds of resistance selection in the presence of neomycin.

The stably transfected B2-11 cells obtained by the selection were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37℃and 5% CO ₂ Is maintained in the environment of (a).

Cells were plated at 1 x 10 cells per well ⁶ Is inoculated into six-hole plates, placed at 37 ℃ and 5% CO ₂ And (5) placing in an incubator for 6 hours to enable the incubator to be fully adhered.

Test drugs diluted to different concentrations were applied to six well plates inoculated with B2-11 cells to achieve final drug concentrations of 8, 16, 32, 64, 128, 256. Mu.M in the medium. The blank group added an equal volume of DMSO to the culture broth. After 4d of administration, the supernatant was aspirated, trypsinized for 90s, centrifuged to discard the supernatant, and the underlying cells were resuspended in 1ml PBS. The resuspended cells were subjected to single cell fluorescence intensity recording with a detection cell flow cytometer, 10000 cells were aspirated each time, and PMT voltage was set at 200V.

Detection result:

as can be seen from FIG. 15, the compound represented by formula X has better cell activity, EC, on B2-11 cells than SAH, which is a non-nucleoside inhibitor of DNMT1 ₅₀ 129.3. Mu.M.

Claims

1. A DNA methyltransferase DNMT1 dual substrate inhibitor, characterized by: a compound represented by structure I having an adenosine parent nucleus or a pharmaceutically acceptable salt thereof:

wherein R1 is selected from any one of the following structures:

r2 is selected from any one of the following structures:

2. the DNA methyltransferase DNMT1 dual substrate inhibitor according to claim 1, wherein: wherein R1 and R2 are required to be substituted simultaneously; are each independently selected from the structures described above.

3. The DNA methyltransferase DNMT1 dual substrate inhibitor according to claim 1 or 2, characterized in that: which is a compound having a structure represented by any one of formulas II to X or a pharmaceutically acceptable salt thereof:

4. a DNA methyltransferase DNMT1 dual substrate inhibitor according to any one of claims 1-3, wherein: which is used to inhibit the activity of DNMT1 proteins; preferably, it inhibits the activity of the DNMT1 protein in such a way that it occupies both the SAH and the substrate cytosine binding cavity.

5. The DNA methyltransferase DNMT1 dual substrate inhibitor according to claim 4, wherein: the DNMT1 protein is human DNMT1.

6. The DNA methyltransferase DNMT1 dual-substrate inhibitor according to claim 5, wherein the compounds having the structures of formulae ii to X can effectively inhibit the methylation of DNMT1 protein to a different extent at a concentration of 20 μm, wherein the inhibition rate of III, IV, V, VI is comparable to that of positive control RG108, and the inhibition rate of all the compounds is superior to SAH.

7. The compound of claim 3 having the structure shown in X has activity in inhibiting DNMT1 methylation in HEK-293T cells and has better cellular activity than SAH.

8. A pharmaceutical composition comprising the DNA methyltransferase DNMT1 dual substrate inhibitor of any one of claims 1-3, and a pharmaceutically acceptable adjuvant, diluent, carrier, or combination thereof.

9. Use of a DNA methyltransferase DNMT1 dual substrate inhibitor according to any one of claims 1-3 or a pharmaceutical composition according to claim 8 for the preparation of an anti-tumour agent.

10. The use according to claim 9, characterized in that: the diseases related to cancer include, but are not limited to leukemia, breast cancer, colon cancer, lung cancer, bladder cancer.