CN106883217B

CN106883217B - Nucleoside base hydroxamic acid derivative compound and preparation method and application thereof

Info

Publication number: CN106883217B
Application number: CN201710212243.5A
Authority: CN
Inventors: 蒋宇扬; 袁梓高; 高春梅; 孙钦升
Original assignee: Shenzhen Graduate School Tsinghua University
Current assignee: Shenzhen Graduate School Tsinghua University
Priority date: 2017-04-01
Filing date: 2017-04-01
Publication date: 2020-05-22
Anticipated expiration: 2037-04-01
Also published as: CN106883217A

Abstract

The invention discloses a nucleoside base hydroxamic acid compound with DNA methyltransferase and/or histone deacetylase inhibitory activity, and a preparation method and application thereof. The structural formula of the nucleoside base hydroxamic acid compound is shown as a formula I, wherein R is a nucleoside base, a nucleoside base with a substituent group or a nucleoside base analogue in DNA and/or RNA; linker is a linking chain connecting R to the hydroxamate functionality, including but not limited to alkyl chains, alkyl chains with heteroatoms, alkyl chains with aromatic rings, alkyl chains with heterocycles. In-vitro cell proliferation experiments show that the compound shown in the formula I can well inhibit the proliferation of leukemia cells K562 and histiocyte lymphoma cells U937. DNA methyltransferase and histone deacetylase inhibition experiments show that the compound shown in the formula I is a compound with the activity of inhibiting DNA methyltransferase and/or histone deacetylase.

Description

Nucleoside base hydroxamic acid derivative compound and preparation method and application thereof

Technical Field

The invention belongs to the field of medicines, and particularly relates to a nucleoside base hydroxamic acid derivative with DNMT and HDAC inhibitory activity, and a preparation method and application thereof.

Background

The occurrence and development of tumors are closely related to both genetic abnormalities and epigenetic changes. Genetic abnormalities include amplification, translocation, deletion, point mutation, and the like of oncogenes or tumor suppressor genes. Epigenetic modifications mainly include modifications of DNA and histone modifications, such as DNA methylation, histone acetylation, deacetylation, ubiquitination, and the like. Epigenetic changes do not involve changes in DNA sequences, and maintain the genetic information of the parent during cell division, but can be reversed under conditions that convert a malignant cell population into normal cells. Therefore, the enzyme which plays a regulating role in the epigenetic inheritance is considered as a new generation target for developing the anti-tumor drugs.

Research shows that highly expressed DNA methyltransferase (DNMT) and Histone Deacetylase (HDAC) in tumor cells respectively catalyze methylation of DNA CpG islands and deacetylation of histone, so that chromatin is contracted to form nucleosomes, cancer suppressor genes cannot be normally expressed, and occurrence and development of tumors are promoted. Currently, many small molecule inhibitors targeting DNMT or HDAC are being developed.

DNA methyltransferases (DNMTs) are predominantly of 3 subtypes in mammals. Wherein DNMT1 plays a role primarily in maintaining the methylation state of the parent during replication; DNMT3A and DNMT3B function to achieve de novo methylation, i.e., to obtain new methylation patterns in the progeny DNA. Small molecule inhibitors developed by taking DNMTs as targets can be divided into nucleoside DNMT inhibitors and non-nucleoside DNMT inhibitors according to different structures and action mechanisms of the small molecule inhibitors. Nucleoside DNMT inhibitors are mainly cytosine nucleoside analogues and s-adenosylmethionine (SAM) analogues, such as azacitidine and decitabine, which are already on the market. Nucleoside DNMT inhibitors need to be incorporated into DNA and then form irreversible covalent bonds with cysteine residues of DNMT to play a role, and have large toxic and side effects. For this reason, more and more non-nucleoside DNMT inhibitors have been developed. The way for obtaining the inhibitor comprises virtual screening (such as RG108, NSC319745 and SGI-1027), natural products (such as EGCG), old drugs and new drugs (such as procaine and procainamide) and optimization and modification of known inhibitors. They act by competitively binding to the active structural site of DNMT or blocking the binding of DNMT to DNA to inhibit DNMT. Although non-nucleoside DNMT inhibitors may reduce toxic and side effects caused by DNA incorporation, the inhibitory activity of the inhibitors is still to be improved, and the DNMT inhibitors are not clinically researched. We speculate that non-nucleoside inhibitors that can generate irreversible covalent bonds with the active site of DNMT may not only have good DNMT inhibition activity, but also avoid the toxic and side effects caused by the incorporation of nucleoside DNMT inhibitors into DNA.

Lysine residues on histones have various apparent modifications, such as methylation, acetylation, deacetylation, ubiquitination, crotonylation, and the like. Among them, Histone Deacetylase (HDAC) catalyzes deacetylation of acetylated lysine to tightly wind a positively charged lysine residue with a negatively charged DNA phosphate backbone, thereby inhibiting expression of cancer suppressor genes. A total of 18 HDACs have been found in mammals, and are classified into four subtypes based on their homology to yeast, wherein type I (HDAC1, HDAC2, HDAC3, HDAC8), type II a (HDAC4, HDAC5, HDAC7, HDAC9), type II B (HDAC6, HDAC10) and type IV (HDAC11) belong to Zn²⁺An ion-dependent HDAC; HDAC type III contains the deacetylase SIRT1-SIRT7, genus NAD⁺Dependent enzymes. As a target for the development of an antitumor drug, many HDAC inhibitors have entered clinical studies, and five HDACs inhibitors (Vorinostat, Romidepsin, Belinostat, Panobinostat, Chidamide) have been marketed. HDAC inhibitors can be generally classified into four classes according to their structure: short chain fatty acids including butyric acid, valproic acid, etc.; hydroxamic acids, including vorinostat (saha) and trichostatin a (tsa), Belinostat, and the like; cyclic tetrapeptides including Apicidin and romidepte;benzamides, including Chidamide and MS-275, and the like. Currently, the development of HDAC subtype selective inhibitors based on HDAC protein subtype differences and the development of multi-target inhibitors based on the synergistic effect of HDACs with other target (e.g., EGFR, Her2, topoisomerase, etc.) inhibitors are two major directions in the development of HDAC inhibitors.

Studies have shown that inhibitors of epigenetic enzymes can make tumor cells more sensitive to certain chemotherapeutic drugs. In some reports, the combination of DNMT inhibitor and HDAC inhibitor with some chemotherapeutic drugs respectively shows obvious synergistic effect.

DNMT and HDAC are closely linked in physiological function, together affecting the expression of cancer suppressor genes. In one aspect, studies have shown that the combination of a low dose of a DNMT inhibitor and an HDAC inhibitor has a synergistic effect. On the other hand, it is found that when the inhibitor of DNMTs or HDACs is used alone, the high expression of the other enzyme can still cause the silencing of tumor cancer suppressor genes, and the compensatory mechanism causes the drug resistance of tumor cells, thus reducing the treatment effect. Although the combined use has the advantage of synergistic effect, the combined use also has some defects, such as more complex pharmacokinetics, drug interaction, more toxic and side effects and the like. However, a single small molecule with multi-target inhibitory activity can effectively avoid these problems, and the development of multi-target inhibitors has become a common drug development strategy.

In conclusion, the inhibitor targeting both DNMT and HDAC may more effectively re-express the silenced anti-cancer gene and make the tumor cells more difficult to generate drug resistance, thereby exerting better anti-tumor effect. To this end we have focused on the development of multi-target inhibitors with both DNMT and HDAC inhibitory activity. Based on the structures of nucleoside DNMT inhibitors and hydroxamic acid HDAC inhibitors, a nucleoside base hydroxamic acid multi-target inhibitor with DNMT and HDAC inhibition activities is developed by adopting a method of combining drug molecular fragments.

Disclosure of Invention

One of the purposes of the invention is to provide a class of nucleoside base hydroxamic acid multi-target inhibitors with DNMT and HDAC inhibitory activity and a preparation method thereof.

The present invention provides hydroxamic acid compounds of formula i:

in the formula I, R is a nucleoside base, a nucleoside base with a substituent group or a nucleoside base analogue in DNA and/or RNA.

Wherein the nucleoside base is selected from any one of the following: adenine, guanine, cytosine, uracil, thymine;

in the nucleoside base with the substituent group, the substituent group is selected from any one of the following groups: halogen (fluorine, chlorine, bromine, iodine, etc.), alkyl (methyl, ethyl, etc.), nitro, acetyl, acryloyl, crotonyl;

the nucleobase analogue is selected from any one of: 5-azacytosine, 2-hydroxypyrimidine, 6-methyladenine;

preferably, R is selected from

In formula I above, Linker is the linking chain linking R to the hydroxamate functionality. The linking chain includes but is not limited to C1-C20 straight alkyl chain, C1-C20 branched alkyl chain, alkyl chain with hetero atom, alkyl chain with aromatic ring, alkyl chain with hetero ring.

In the formula I, when R is cytosine, uracil or derivatives or analogues thereof, a Linker is connected to the nitrogen atom at the 1-position of R; when R is adenine, guanine or their derivatives or analogs, Linker is attached to the nitrogen atom at position 9 of R.

The invention also provides pharmaceutically acceptable salts or tautomers of the compounds shown in the formula I.

Wherein the salt is an inorganic acid salt or an organic acid salt.

The inorganic acid salt is selected from salts formed by any one of the following inorganic acids: hydrochloric acid, sulfuric acid or phosphoric acid.

The organic acid salt is selected from salts formed by any one of the following organic acids: acetic acid, trifluoroacetic acid, malonic acid, citric acid, and p-toluenesulfonic acid.

The present invention also provides hydroxamic acid compounds of formula II:

wherein R is as described above;

t is 0,1, 2 or 3;

m is 2,3, 4, 5, 6, 7 or 8;

n is 0,1, 2 or 3;

in some embodiments, t is 1;

in some embodiments, m is 5 or 6;

in some embodiments, n is 0;

in some embodiments, the sum of m, n, and t is a value from 2 to 10;

the invention also provides pharmaceutically acceptable salts or tautomers of the compounds shown in the formula II.

The present invention also provides hydroxamic acid compounds of the formula III:

wherein R and t are as described above;

m is 2,3, 4, 5 or 6;

in some embodiments, t is 1;

in some embodiments, m is 3,4, 5, or 6;

in some embodiments, the sum of m and t is a value of 3 to 8;

The present invention also provides hydroxamic acid compounds of formula IV:

wherein R is as described above;

y is 0,1, 2 or 3;

z is 1,2 or 3;

g is 2,3, 4, 5, 6, 7 or 8;

x is nitrogen or carbon;

in some embodiments, y is 1;

in some embodiments, z is 2;

in some embodiments, X is nitrogen;

in some embodiments, g is 4, 5, or 6;

in some embodiments, the sum of y, z, and g is a value of 3 to 8;

the invention also provides pharmaceutically acceptable salts or tautomers of the compounds shown in the formula IV.

The present invention also provides hydroxamic acid compounds of the formula V:

wherein R, y, z and X are as described above;

s is 0,1, 2 or 3;

y is a linking chain linking the phenyl ring to the hydroxamate functionality, including but not limited to a bond, alkyl, methylene, ethylene, vinyl, alkoxy, haloalkyl;

in some embodiments, y is 1;

in some embodiments, z is 2;

in some embodiments, X is nitrogen;

in some embodiments, s is 1;

in some embodiments, Y is methylene, ethylene, or vinyl;

the invention also provides pharmaceutically acceptable salts or tautomers of the compounds shown in the formula V.

The invention also provides pharmaceutically acceptable salts or tautomers of the compounds of formula VI.

The compounds may exist in different polymorphic forms.

The hydroxamic acid compound represented by the formula I is preferably any one of the following compounds:

the invention also provides a preparation method of the compound shown in the formula I.

Hydroxamic acid compounds of the above formula I can be prepared by reacting a compound of the formula IA with hydroxylamine or hydroxylamine hydrochloride;

wherein R and Linker are as described above. The reaction is generally carried out in the presence of a condensing agent such as benzotriazole-N, N, N ', N' -tetramethyluronium Hexafluorophosphate (HBTU) and a base such as triethylamine in a solvent such as N, N-Dimethylformamide (DMF) at a temperature of about 0 ℃ to 80 ℃.

Or the compound shown in the formula I can be obtained by firstly reacting the compound shown in the formula IA with hydroxylamine with a protecting group such as O- (tetrahydro-2H-pyran-2-yl) hydroxylamine under the action of a condensing agent to generate a compound shown in the formula IB and then removing the protecting group from the compound shown in the formula IB;

wherein R and Linker are as described above; p is a protecting group of hydroxyl in hydroxylamine. The reaction for preparing formula IB is typically carried out in the presence of a condensing agent such as benzotriazole-N, N, N ', N' -tetramethyluronium Hexafluorophosphate (HBTU) in a solvent such as N, N-Dimethylformamide (DMF) at a temperature of about 0 deg.C to 80 deg.C. Removal of the protecting group of formula IB is typically carried out under acidic conditions such as addition of hydrochloric acid, trifluoroacetic acid or under basic conditions such as addition of sodium hydroxide in a solvent such as tetrahydrofuran at a temperature of about 0 ℃ to 80 ℃.

Alternatively, the compounds of formula I may be prepared by reacting a compound of formula IC with hydroxylamine or hydroxylamine hydrochloride in an alkaline solution;

wherein R and Linker are as described above; a is an alkyl group such as methyl, ethyl or isopropyl. The reaction is carried out in an aqueous solution or an organic solvent selected from at least one of: dimethylformamide, dimethylacetamide, chloroform, dichloromethane, acetone, tetrahydrofuran, acetonitrile, and the like. The base is selected from at least one of: ammonia, triethylamine, diisopropylethylamine, sodium hydroxide, potassium hydroxide, sodium methoxide, sodium ethoxide, sodium tert-butoxide, potassium tert-butoxide, etc. The reaction temperature of the reaction can be 20-80 ℃, and the reaction time can be 0.5-10 hours.

These compounds are commercially available or can be prepared from commercially available compounds using standard methods or using the extended methods of the examples herein, without describing the synthesis of intermediates and starting materials.

The compounds prepared by the invention are proved to be correct and correct by tests of high-resolution mass spectrum, nuclear magnetic resonance, melting point and the like, and are the compounds shown in the general formula I.

It is a further object of the present invention to provide the use of compounds of formula I and pharmaceutically acceptable salts thereof.

The compound shown in the formula I or the pharmaceutically acceptable salt thereof is applied to the following aspects:

1) the application in preparing DNA methyltransferase and/or histone deacetylase inhibitors;

2) the application in preparing eukaryotic tumor cell proliferation inhibitor;

3) the application in preparing the medicine for preventing and/or treating tumor.

The DNA methyltransferases (DNMTs) comprise subtypes known in mammalian cells (DNMT1, DNMT3A, DNMT 3B); the Histone Deacetylases (HDACs) comprise known isoforms in mammalian cells, including but not limited to, mainly HDAC1, HDAC2, HDAC3, HDAC8, HDAC4, HDAC5, HDAC7, HDAC9, HDAC6, HDAC10, HDAC 11.

The eukaryote is a mammal; the tumor cell is a cancer cell; the cancer cell is leukemia cancer cell, breast cancer cell, liver cancer cell, pancreatic cancer cell, lung cancer cell, brain cancer cell, ovarian cancer cell, uterine cancer cell, testicular cancer cell, skin cancer cell, stomach cancer cell, nasopharyngeal carcinoma cell, colon cancer cell, bladder cancer cell or rectal cancer cell; preferably human chronic myelogenous leukemia cells and human histiocytic lymphoma cells.

The leukemia cancer cell is a human Chronic Myelocytic Leukemia (CML) cell line K562, the lymphoma cell is a human histiocyte lymphoma cell U937, the lung cancer cell is a human lung cancer cell NCI-H520, the human brain glioma cell is U251, the melanoma cell is A375, the glioblastoma cell is a human glioblastoma cell A172 and a human brain astrocytoma cell U-118MG, the cervical cancer cell is a human cervical cancer cell line Hela, the nasopharyngeal cancer cell is a nasopharyngeal cancer cell line CNE-2, the liver cancer cell is a human liver cancer cell line HepG2, and the breast cancer cell is a human breast cancer cell MCF-7 and MDA-MB-231.

The tumor is a carcinoma; the cancer is leukemia, lymphoma, lung cancer, melanoma, glioblastoma, cervical cancer, nasopharyngeal cancer, liver cancer, breast cancer, brain cancer, pancreatic cancer, ovarian cancer, uterine cancer, testicular cancer, skin cancer, stomach cancer, colon cancer, bladder cancer or rectal cancer.

The compound shown in the formula I or the pharmaceutically acceptable salt thereof can also be used for preparing medicaments for preventing and/or treating tumors.

The invention also discloses a medicament for preventing and/or treating tumors, which is prepared by using the compound shown in the formula I or the pharmaceutically acceptable salt thereof as an active ingredient and belongs to the protection scope of the invention.

The DNA methyltransferase and/or histone deacetylase inhibitor, the eukaryotic tumor cell proliferation inhibitor and the medicament for preventing and/or treating tumors, which are prepared by using the compound represented by the formula I or the pharmaceutically acceptable salt thereof, can be introduced into the body such as muscle, intradermal, subcutaneous, intravenous and mucosal tissues by injection, spray, nasal drip, eye drop, penetration, absorption, physical or chemical mediated method; or mixed or coated with other materials and introduced into body.

If necessary, one or more pharmaceutically acceptable carriers can be added into the medicine. The carrier includes diluent, excipient, filler, binder, wetting agent, disintegrating agent, absorption enhancer, surfactant, adsorption carrier, lubricant, etc. which are conventional in the pharmaceutical field.

The medicine for preventing and/or treating tumor prepared from the compound shown in the formula I or the pharmaceutically acceptable salt thereof can be prepared into various forms such as injection, tablets, powder, granules, capsules, oral liquid, ointment, cream and the like. The medicaments in various dosage forms can be prepared according to the conventional method in the pharmaceutical field.

The compound provided by the invention is proved to be a potential anti-tumor medicine with inhibitory activity on DNA methyltransferase and histone deacetylase by various tumor cell line tests (including leukemia cells, lymphoma cells and the like) and DNA methyltransferase and histone deacetylase inhibitory activity tests. The compound provided by the invention has the advantages of easily available raw materials and simple preparation method, and experiments prove that the compound has a good anticancer effect and has a good application prospect in the field of design and research of antitumor drugs.

Detailed Description

The present invention will be described below with reference to specific examples, but the scope of the present invention is not limited thereto.

The experimental methods described in the following examples are all conventional methods for organic synthesis unless otherwise specified; the reagents and biomaterials are commercially available, unless otherwise specified.

Example 1

6- (4- ((4-amino-2-oxopyrimidin-1 (2H) -yl) methyl) -1H-1,2, 3-triazol-1-yl) -N-hydroxyhexanamide

Example 1A

6-Azoic acid Ethyl ester

Ethyl 6-bromohexanoate (1eq) was dissolved in DMF and sodium azide (3eq) was added, slowly warmed from room temperature to 80 ℃ and reacted at this temperature overnight. Then ethyl acetate was added, the excess sodium azide was removed by filtration, water was added and extraction was performed with ethyl acetate. The organic phase was washed with water and saturated brine and dried over anhydrous sodium sulfate. Filtration and removal of the solvent by rotary evaporator gave the title compound.¹H NMR(400MHz,DMSO-d6)δ4.05(q,J＝7.1Hz,2H),3.31(t,J＝6.8Hz,2H),2.29(t,J＝7.3Hz,2H),1.60–1.47(m,4H),1.38–1.27(m,2H),1.18(t,J＝7.1Hz,3H).¹³CNMR(101MHz,DMSO)δ173.26,60.18,51.05,33.91,28.46,26.16,24.52,14.64.

Example 1B

4-amino-1- (prop-2-yn-1-ane) pyrimidin-2 (1H) -one

Cytosine (1eq), 3-bromopropyne (3eq) were dissolved in anhydrous DMF, and anhydrous cesium carbonate (2eq) was added, heated to 80 ℃ and reacted overnight. After the reaction was completed, water was added and extracted with ethyl acetate. The organic phase was washed with water and saturated brine and dried over anhydrous magnesium sulfate. The solvent of the organic phase was removed and the title compound was isolated by column chromatography.¹H NMR(400MHz,DMSO-d6)δ7.52(d,J＝7.7Hz,1H),6.64(d,J＝7.8Hz,1H),4.77(d,J＝2.5Hz,2H),3.49(t,J＝2.5Hz,1H).¹³C NMR(101MHz,DMSO)δ166.55,155.61,145.26,94.61,79.96,75.79,37.84.

Example 1C

6- (4- ((4-amino-2-oxopyrimidin-1 (2H) -yl) methyl) -1H-1,2, 3-triazol-1-yl) hexanoic acid ethyl ester

The solution of example 1B (1eq) and the solution of example 1A (1.2eq) were dissolved in a mixed solution of water and tert-butanol (volume ratio 1:1), and sodium ascorbate (0.1eq) and copper sulfate pentahydrate (0.02eq) were added to react at 60 ℃ for 4 hours under nitrogen protection. After completion of the 1B reaction was confirmed by Thin Layer Chromatography (TLC), an appropriate amount of water was added and filtered to give the title compound. Further purification of the compound by water washing or separation and purification of the compound by column chromatography.¹H NMR(400MHz,DMSO-d₆)δ7.99(s,1H),7.67(d,J＝7.1Hz,1H),7.08(br,2H),5.68(s,1H),4.87(s,2H),4.30(t,J＝7.1Hz,2H),4.03(q,J＝7.1Hz,2H),2.26(t,J＝7.3Hz,2H),1.84–1.70(m,2H),1.59–1.46(m,2H),1.32–1.20(m,2H),1.15(t,J＝7.1Hz,3H).¹³C NMR(101MHz,DMSO)δ173.25,146.27,123.98,60.19,49.66,33.83,29.86,25.84,24.34,14.65.

Example 1D

6- (4- ((4-amino-2-oxopyrimidine-1 (2H) -alkyl) methyl) -1H-1,2, 3-triazol-1-alkyl) -N-hydroxyhexanamide

Example 1C (1eq) was dissolved in methanol or DMF and hydroxylamine hydrochloride (5eq) and sodium tert-butoxide (10eq) were added and reacted at room temperature for 8 h. After confirming the completion of the 1C reaction by TLC, the solvent was removed by spin. The title compound was obtained by column chromatography or crystallization (pH 3) in aqueous hydrochloric acid.¹H NMR(400MHz,DMSO-d₆)δ10.38(s,1H),10.12(s,1H),9.59(s,1H),8.56(s,1H),8.14(s,1H),8.08(d,J＝7.5Hz,1H),6.12(d,J＝7.5Hz,1H),5.03(s,2H),4.33(t,J＝7.1Hz,2H),2.19(t,J＝7.3Hz,2H),1.89–1.69(m,2H),1.63–1.43(m,2H),1.39–1.10(m,2H).¹³C NMR (101MHz, DMSO). delta. 174.80,149.75,141.92,124.28,94.22,49.84,44.22,34.00,29.92,25.94,24.38 calculated by High Resolution Mass Spectrometry (HRMS) (ESI) M/z [ M + H]⁺322.1628, found 322.1622.

Example 2

6- (4- ((4-amino-5-fluoro-2-oxopyrimidin-1 (2H) -yl) methyl) -1H-1,2, 3-triazol-1-yl) -N-hydroxyhexanamide

Example 2A

4-amino-5-fluoro-1- (prop-2-yn-1-yl) pyrimidin-2 (1H) -one

The synthesis was the same as in example 1B.¹H NMR(400MHz,DMSO-d₆)δ7.99(d,J＝6.6Hz,1H),7.64(br,2H),4.44(d,J＝2.5Hz,2H),3.36(d,J＝2.5Hz,1H).¹³C NMR(101MHz,DMSO)δ158.31,158.18,153.91,137.54,135.14,129.87,129.57,79.62,76.02,38.14.

Example 2B

6- (4- ((4-amino-5-fluoro-2-oxopyrimidin-1 (2H) -yl) methyl) -1H-1,2, 3-triazol-1-yl) hexanoic acid ethyl ester

The synthesis was the same as in example 1C.¹H NMR(400MHz,DMSO-d₆)δ8.13–7.98(m,2H),7.67(s,1H),7.45(s,1H),4.85(s,2H),4.30(t,J＝7.1Hz,2H),4.02(q,J＝7.1Hz,2H),2.25(t,J＝7.3Hz,2H),1.79–1.75(m,2H),1.52(p,J＝7.4Hz,2H),1.26–1.18(m,2H),1.15(t,J＝7.2Hz,3H).

Example 2C

The synthesis method is the same as example 1.¹H NMR(400MHz,DMSO-d₆)δ10.47(s,1H),10.33(s,1H),10.27(s,1H),9.56(s,1H),8.67(d,J＝1.7Hz,1H),7.88(s,1H),4.95(s,2H),4.26(t,J＝7.2Hz,2H),1.92(t,J＝7.3Hz,2H),1.85–1.68(m,2H),1.57–1.43(m,2H),1.26–1.13(m,2H).¹³CNMR (101MHz, DMSO) delta 171.92,169.45,149.24,146.82,144.11,144.11,139.33,122.98,49.67,37.96,32.55,29.94,26.02,25.01,0.63 calculated High Resolution Mass Spectrometry (HRMS) (ESI) M/z [ M + H]⁺340.1533, found 322.1529.

Example 3

6- (4- ((4-amino-5-methyl-2-oxopyrimidin-1 (2H) -yl) methyl) -1H-1,2, 3-triazol-1-yl) -N-hydroxyhexanamide

Example 3A

4-amino-5-methyl-1- (prop-2-yn-1-yl) pyrimidin-2 (1H) -one

The synthesis was the same as in example 1B.¹H NMR(400MHz,DMSO-d₆)δ7.47(s,1H),7.02(br,2H),4.45(d,J＝2.5Hz,2H),3.31(t,J＝2.5Hz,1H),1.83(s,3H).¹³C NMR(101MHz,DMSO)δ166.22,155.61,142.51,101.92,80.16,75.59,37.53,13.50.

Example 3B

6- (4- ((4-amino-5-methyl-2-oxopyrimidin-1 (2H) -yl) methyl) -1H-1,2, 3-triazol-1-yl) hexanoic acid ethyl ester

The synthesis was the same as in example 1C.¹H NMR(400MHz,DMSO-d₆)δ7.98(s,1H),7.53(s,1H),4.87(s,2H),4.29(t,J＝7.1Hz,2H),4.02(q,J＝7.1Hz,2H),2.25(t,J＝7.4Hz,2H),1.81(s,3H),1.77–1.69(m,2H),1.52(p,J＝7.5Hz,2H),1.33–1.18(m,2H),1.15(t,J＝7.1Hz,3H).

Example 3C

The synthesis method is the same as example 1.¹H NMR(400MHz,DMSO-d₆) δ 8.05(s,1H),7.93(br,2H),7.81(s,1H),4.94(s,2H),4.31(t, J ═ 7.0Hz,2H),1.92(t, J ═ 7.3Hz,2H),1.88(s,3H), 1.82-1.72 (M,2H), 1.56-1.44 (M,2H), 1.27-1.16 (M,2H), High Resolution Mass Spectrometry (HRMS) (ESI) M/z calculated value [ M + H) (ESI) M/z]⁺336.1784, found 336.1782.

Example 4

6- (4- ((6-amino-9H-purin-9-yl) methyl) -1H-1,2, 3-triazol-1-yl) -N-hydroxyhexanamide

Example 4A

9- (prop-2-yn-1-yl) -9H-purin-6-amine

The synthesis was the same as in example 1B.¹H NMR(400MHz,DMSO-d₆)δ8.20(s,1H),8.17(s,1H),7.32(s,2H),5.03(d,J＝2.5Hz,2H),3.49(t,J＝2.5Hz,1H).

Example 4B

6- (4- ((6-amino-9H-purin-9-yl) methyl) -1H-1,2, 3-triazol-1-yl) hexanoic acid ethyl ester

The synthesis was the same as in example 1C.¹H NMR(400MHz,DMSO-d₆)δ8.22(br,2H),8.11(s,1H),7.24(br,2H),5.42(s,2H),4.30(t,J＝7.1Hz,2H),4.01(q,J＝7.1Hz,2H),2.24(t,J＝7.3Hz,2H),1.88–1.68(m,2H),1.62–1.41(m,2H),1.33–1.02(m,5H).

Example 4C

The synthesis method is the same as example 1.¹H NMR(400MHz,DMSO-d₆)δ8.19(s,1H),8.14(s,1H),8.09(s,1H),7.24(s,2H),5.42(s,2H),4.30(t,J＝7.1Hz,2H),2.17(t,J＝7.3Hz,2H),1.85–1.70(m,2H),1.58–1.42(m,2H),1.26–1.13(m,2H).¹³C NMR (101MHz, DMSO). delta. 174.85,156.50,153.08,149.85,142.96,141.16,123.95,119.11,49.79,38.58,33.98,29.91,25.92,24.37 calculation of [ M + H ] M/z by High Resolution Mass Spectrometry (HRMS) (ESI)]⁺346.1740, found 346.1750.

Example 5

6- (4- ((2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) methyl) -1H-1,2, 3-triazol-1-yl) -N-hydroxyhexanamide

Example 5A

1- (prop-2-yn-1-yl) pyrimidine-2, 4(1H,3H) -diones

The synthesis was the same as in example 1B.¹H NMR(400MHz,DMSO-d₆)δ11.41(s,1H),7.70(d,J＝7.9Hz,1H),5.63(d,J＝7.8Hz,1H),4.51(d,J＝2.5Hz,2H),3.45(t,J＝2.5Hz,1H).

Example 5B

6- (4- ((2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) methyl) -1H-1,2, 3-triazol-1-yl) hexanoic acid ethyl ester

The synthesis was the same as in example 1C.¹H NMR(400MHz,DMSO-d₆)δ11.31(s,1H),8.07(s,1H),7.73(d,J＝7.8Hz,1H),5.58(dd,J＝7.9,2.2Hz,1H),4.92(s,2H),4.32(t,J＝7.1Hz,2H),4.03(q,J＝7.1Hz,2H),2.26(t,J＝7.4Hz,2H),1.88–1.72(m,2H),1.59–1.45(m,2H),1.30–1.18(m,2H),1.16(t,J＝7.1Hz,3H).¹³C NMR(101MHz,DMSO)δ173.24,164.18,151.26,145.95,142.78,123.93,101.78,60.19,49.75,42.90,33.82,29.82,25.82,24.33,14.65.

Example 5C

The synthesis method is the same as example 1.¹H NMR(400MHz,DMSO-d₆)δ11.32(s,1H),8.08(s,1H),7.73(d,J＝7.9Hz,1H),5.59(dd,J＝7.8,2.1Hz,1H),4.92(s,2H),4.32(t,J＝7.1Hz,2H),2.19(t,J＝7.4Hz,2H),1.90–1.70(m,2H),1.61–1.41(m,2H),1.33–1.18(m,2H).¹³C NMR (101MHz, DMSO). delta. 174.83,164.20,151.26,145.97,142.77,123.93,101.78,49.79,42.91,33.98,29.92,25.95,24.38 calculation of [ M + H ] M/z by High Resolution Mass Spectrometry (HRMS) (ESI)]⁺323.1468, found 323.1461.

Example 6

4- (4- (4- ((4-amino-2-oxopyrimidin-1 (2H) -yl) methyl) phenyl) -1H-1,2, 3-triazol-1-yl) -N-hydroxybutyramide

Example 6A

4-ethynylbenzyl methylsulphonic acid

Step 1: methyl p-iodobenzoate (1eq) was dissolved in an appropriate amount of THF, a catalytic amount of palladium ditriphenylphosphine dichloride and cuprous iodide, as well as triethylamine (2eq) and trimethylsilylacetylene (2eq) were added, oxygen in the solvent was removed by nitrogen substitution, and then the mixture was reacted overnight at 70 ℃ under nitrogen protection. The precipitate formed by the reaction was removed by filtration through Celite, and 1M THF solution of tetra-t-butylammonium fluoride (2eq) was added to the filtrate and reacted at room temperature for about 1 hour. THF is removed by rotation, water and ethyl acetate are used for extraction, an organic phase is remained, and 4-acetylenyl methyl benzoate is obtained after ethyl acetate is removed by rotation.¹H NMR(400MHz,Chloroform-d)δ7.99(d,J＝8.7Hz,2H),7.55(d,J＝8.4Hz,2H),3.92(s,3H),3.23(s,1H).¹³C NMR(101MHz,CDCl₃)δ166.45,132.12,130.22,129.50,126.81,82.85,80.05,52.30.

Step 2: methyl 4-ethynylbenzoate (1eq) was dissolved in THF, 2.5M lithium aluminum hydride in THF (2eq) was added slowly under ice bath, and the reaction was carried out at room temperature for 2h under nitrogen protection. After the reaction was completed, ice water was slowly added to the reaction system to remove excess lithium aluminum hydride, and then the reaction system was made weakly acidic with 3M aqueous hydrochloric acid. Extraction with diethyl ether gave 4-ethynylbenzyl alcohol.¹H NMR(400MHz,Chloroform-d)δ7.49(d,J＝8.1Hz,2H),7.32(d,J＝8.1Hz,2H),4.70(s,2H),3.07(s,1H).¹³C NMR(101MHz,CDCl₃)δ141.65,132.36,126.77,121.38,83.52,64.91,50.90.

And step 3: 4-ethynylbenzyl alcohol was dissolved in DCM, triethylamine (2eq) and methanesulfonyl chloride (1.2eq) were added slowly at-5 ℃ and reacted at this temperature for 30 minutes, after which it was slowly warmed to room temperature for 2 h. The reaction was quenched with saturated aqueous sodium bicarbonate and the product was extracted with DCM. The organic phase was dried over anhydrous sodium sulfate and the organic solvent was removed to give the title compound. It was used directly in the subsequent reaction without further purification.

Example 6B

4-amino-1- (4-ethynylbenzyl) pyrimidin-2 (1H) -one

Cytosine (1eq), example 6A (1.2eq), and anhydrous cesium carbonate (1.5eq) were dissolved in anhydrous DMF and reacted at room temperature overnight. TLC added water after confirming completion of the cytosine reaction and extracted with DCM. Removal of the solvent from the organic phase afforded the title compound. Further purification can be achieved by recrystallization from ether or by column chromatography.¹H NMR(400MHz,DMSO-d₆)δ7.69(d,J＝7.2Hz,1H),7.44(d,J＝8.2Hz,2H),7.24(d,J＝8.1Hz,2H),7.09(d,J＝31.1Hz,2H),5.67(d,J＝7.2Hz,1H),4.85(s,2H),4.18(s,1H).¹³C NMR(101MHz,DMSO)δ166.50,156.23,146.51,139.49,132.31,128.20,121.15,94.24,83.78,81.28,51.62.

Example 6C

4-azidobutyric acid ethyl ester

The synthesis was the same as in example 1A.¹H NMR(400MHz,Chloroform-d)δ4.15(q,J＝7.1Hz,2H),3.36(t,J＝6.7Hz,2H),2.41(t,J＝7.2Hz,2H),2.00–1.82(m,2H),1.27(t,J＝7.1Hz,3H).¹³C NMR(101MHz,CDCl₃)δ172.73,60.61,50.69,31.23,24.31,14.25.

Example 6D

4- (4- (4- ((4-amino-2-oxopyrimidin-1 (2H) -yl) methyl) phenyl) -1H-1,2, 3-triazol-1-yl) butanoic acid ethyl ester

Synthesized from example 6B and example 6C. The reaction conditions are those adopted in the Click reaction commonly used in organic synthesis processes. Specifically, the synthesis method can refer to example 1C.¹H NMR(400MHz,DMSO-d₆)δ8.57(s,1H),7.80(d,J＝7.8Hz,2H),7.71(d,J＝7.1Hz,1H),7.33(d,J＝7.9Hz,2H),7.10(br,2H),5.71(br,1H),4.88(s,2H),4.42(t,J＝7.0Hz,2H),4.04(q,J＝7.1Hz,2H),2.34(t,J＝7.3Hz,2H),2.21–2.01(m,2H),1.16(t,J＝7.1Hz,3H).¹³C NMR(101MHz,DMSO)δ171.89,145.98,145.82,137.45,129.84,127.99,125.13,121.25,59.86,48.65,48.49,30.32,24.96,13.95.

Example 6E

The synthesis method was the same as in example 1, using example 6D as a reaction raw material.¹H NMR(400MHz,DMSO-d₆)δ10.43(s,1H),8.75(s,1H),8.59(s,1H),8.11(s,1H),7.89(d,J＝7.3Hz,1H),7.82(d,J＝8.0Hz,2H),7.60(s,1H),7.37(d,J＝8.0Hz,2H),5.87(s,1H),4.93(s,2H),4.40(t,J＝6.8Hz,2H),2.16–2.04(m,2H),1.99(t,J＝7.3Hz,2H).¹³C NMR (101MHz, DMSO). delta. 173.54,167.95,164.14,146.91,145.90,136.70,130.08,128.07,125.18,121.26,51.14,48.97,28.91,25.66 calculated by High Resolution Mass Spectrometry (HRMS) (ESI) M/z [ M + H]⁺370.1628, found 368.1471.

Example 7

5- (4- (4- ((4-amino-2-oxopyrimidin-1 (2H) -yl) methyl) phenyl) -1H-1,2, 3-triazol-1-yl) -N-hydroxypentanamide

Example 7A

5-Azidopentanoic acid ethyl ester

The synthesis was the same as in example 1A.¹H NMR(400MHz,Chloroform-d)δ4.06(q,J＝7.1Hz,2H),3.22(t,J＝6.6Hz,2H),2.26(t,J＝7.2Hz,2H),1.68–1.48(m,4H),1.18(t,J＝7.1Hz,3H).

Example 7B

5- (4- (4- ((4-amino-2-oxopyrimidin-1 (2H) -yl) methyl) phenyl) -1H-1,2, 3-triazol-1-yl) pentanoic acid ethyl ester

The synthesis was the same as in example 6D.¹H NMR(400MHz,DMSO-d₆)δ8.55(s,1H),7.79(d,J＝7.3Hz,2H),7.74(s,1H),7.34(d,J＝7.7Hz,2H),7.10(s,1H),4.94(s,2H),4.39(t,J＝6.9Hz,2H),4.03(q,J＝7.1Hz,2H),2.33(t,J＝7.4Hz,2H),1.95–1.73(m,2H),1.54–1.42(m,2H),1.15(t,J＝7.1Hz,3H).¹³C NMR(101MHz,DMSO)δ172.44,145.89,137.39,129.89,128.03,125.10,121.16,59.64,49.07,32.66,28.85,21.33,13.99.

Example 7C

The synthesis method was the same as in example 1, using example 7B as a reaction raw material.¹H NMR(400MHz,DMSO-d₆)δ10.39(s,1H),8.70(s,1H),8.55(s,1H),7.80(d,J＝8.1Hz,2H),7.71(d,J＝7.2Hz,1H),7.33(d,J＝8.0Hz,2H),7.11(s,1H),7.03(s,1H),5.68(d,J＝7.1Hz,1H),4.87(s,2H),4.39(t,J＝7.0Hz,2H),1.99(t,J＝7.3Hz,2H),1.93–1.76(m,2H),1.58–1.40(m,2H).¹³C NMR (101MHz, DMSO) delta 168.56,165.86,155.72,145.87,145.83,137.43,129.87,127.99,125.10,121.15,93.55,50.99,49.12,31.46,29.10,22.02 calculated [ M + H ] M/z for High Resolution Mass Spectrometry (HRMS) (ESI)]⁺384.1784, found 384.1779.

Example 8

6- (4- (4- ((4-amino-2-oxopyrimidin-1 (2H) -yl) methyl) phenyl) -1H-1,2, 3-triazol-1-yl) -N-hydroxyhexanamide

Example 8A

6- (4- (4- ((4-amino-2-oxopyrimidin-1 (2H) -yl) methyl) phenyl) -1H-1,2, 3-triazol-1-yl) hexanoic acid ethyl ester

The synthesis method was the same as in example 6D, using example 1A and example 6B as starting materials.¹H NMR(400MHz,DMSO-d₆)δ8.55(s,1H),7.79(d,J＝7.9Hz,2H),7.71(d,J＝7.1Hz,1H),7.33(d,J＝7.9Hz,2H),7.09(br,2H),5.78(br,1H),4.88(s,2H),4.37(t,J＝7.0Hz,2H),4.02(q,J＝7.1Hz,2H),2.28(t,J＝7.4Hz,2H),1.97–1.78(m,2H),1.69–1.50(m,2H),1.31–1.21(m,2H),1.14(t,J＝7.1Hz,3H).¹³C NMR(101MHz,DMSO)δ172.61,145.88,145.81,137.40,129.91,127.99,125.09,121.11,59.55,49.24,33.18,29.13,25.20,23.73,13.99.

Example 8B

The synthesis method was the same as in example 1, using example 8A as a reaction starting material.¹H NMR(400MHz,DMSO-d₆)δ10.35(s,1H),8.70(s,1H),8.56(s,1H),7.80(d,J＝7.8Hz,2H),7.71(d,J＝7.2Hz,1H),7.33(d,J＝7.8Hz,2H),7.11(s,1H),7.03(s,1H),5.68(d,J＝7.2Hz,1H),4.87(s,2H),4.37(t,J＝7.0Hz,2H),1.93(t,J＝7.3Hz,2H),1.89–1.72(m,2H),1.60–1.45(m,2H),1.32–1.16(m,2H).¹³C NMR (101MHz, DMSO). delta. 168.77,165.87,155.72,145.88,145.83,137.42,129.90,127.99,125.09,121.12,93.55,51.00,49.28,31.93,29.21,25.36,24.37 calculated values of [ M + H ] M/z for High Resolution Mass Spectrometry (HRMS) (ESI)]⁺398.1941, found 398.1940.

Example 9

7- (4- (4- ((4-amino-2-oxopyrimidin-1 (2H) -yl) methyl) phenyl) -1H-1,2, 3-triazol-1-yl) -N-hydroxyheptanamide

Example 9A

7-Azidoheptanoic acid ethyl ester

The synthesis was the same as in example 1A.¹H NMR(400MHz,Chloroform-d)δ4.11(q,J＝7.1Hz,2H),3.25(t,J＝6.9Hz,2H),2.29(t,J＝7.5Hz,2H),1.72–1.50(m,4H),1.47–1.29(m,4H),1.24(t,J＝7.2Hz,3H).¹³C NMR(101MHz,CDCl₃)δ173.70,60.29,51.41,34.24,28.72,28.68,26.44,24.82,14.30.

Example 9B

7- (4- (4- ((4-amino-2-oxopyrimidin-1 (2H) -yl) methyl) phenyl) -1H-1,2, 3-triazol-1-yl) heptanoic acid ethyl ester

The synthesis was the same as in example 6D.¹H NMR(400MHz,DMSO-d₆)δ8.56(s,1H),7.79(d,J＝7.4Hz,2H),7.75(s,1H),7.34(d,J＝7.7Hz,2H),7.12(s,1H),4.93(s,2H),4.37(t,J＝7.1Hz,2H),4.02(q,J＝7.1Hz,2H),2.25(t,J＝7.3Hz,2H),1.84(t,J＝7.1Hz,2H),1.50(t,J＝7.2Hz,2H),1.37–1.20(m,4H),1.15(t,J＝7.1Hz,3H).¹³C NMR(101MHz,DMSO)δ172.68,145.87,137.35,129.94,128.02,125.10,121.10,59.52,49.33,33.28,29.29,27.66,25.39,24.11,14.01.

Example 9C

The synthesis method was the same as in example 1, using example 9B as a reaction raw material.¹H NMR(400MHz,DMSO-d₆)δ10.34(s,1H),8.69(s,1H),8.57(s,1H),7.80(d,J＝8.1Hz,2H),7.71(d,J＝7.2Hz,1H),7.33(d,J＝8.0Hz,2H),7.11(s,1H),7.03(s,1H),5.68(d,J＝7.2Hz,1H),4.86(s,2H),4.37(t,J＝7.0Hz,2H),1.92(t,J＝7.3Hz,2H),1.88–1.76(m,2H),1.54–1.38(m,2H),1.37–1.13(m,4H).¹³C NMR(101MHz,DMSO) delta 168.89,165.86,155.72,145.88,145.83,137.41,129.90,127.99,125.10,121.09,93.55,50.99,49.36,32.05,29.37,27.85,25.47,24.80. calculated by High Resolution Mass Spectrometry (HRMS) (ESI) M/z [ M + H ]]⁺412.2097, found 412.2090.

Example 10

7- (4- (4- ((4-amino-5-fluoro-2-oxopyrimidin-1 (2H) -yl) methyl) phenyl) -1H-1,2, 3-triazol-1-yl) -N-hydroxyheptanamide

Example 10A

4-amino-1- (4-ethynylbenzyl) -5-fluoropyrimidin-2 (1H) -one

The synthesis was the same as in example 6B.¹H NMR(400MHz,DMSO-d₆)δ8.10(d,J＝6.7Hz,1H),7.45(d,J＝8.2Hz,2H),7.29(d,J＝8.2Hz,2H),4.81(s,2H),4.18(s,1H).

Example 10B

7- (4- (4- ((4-amino-5-fluoro-2-oxopyrimidin-1 (2H) -yl) methyl) phenyl) -1H-1,2, 3-triazol-1-yl) heptanoic acid ethyl ester

The synthesis was the same as in example 6D.¹H NMR(400MHz,DMSO-d₆)δ8.57(s,1H),8.11(d,J＝6.6Hz,1H),7.80(d,J＝8.0Hz,2H),7.67(s,1H),7.44(s,1H),7.37(d,J＝8.1Hz,2H),4.83(s,2H),4.38(t,J＝7.0Hz,2H),4.03(q,J＝7.1Hz,2H),2.26(t,J＝7.3Hz,2H),1.92–1.74(m,2H),1.61–1.42(m,2H),1.36–1.20(m,4H),1.16(t,J＝7.1Hz,3H).¹³C NMR(101MHz,DMSO)δ173.30,158.12,157.99,154.60,146.46,137.56,131.06,130.75,130.62,128.70,125.71,121.74,60.13,51.82,49.94,33.88,29.89,28.27,25.99,24.72,14.62.¹⁹F NMR(376MHz,DMSO)δ-169.55.

Example 10C

The synthesis method was the same as in example 1, using example 10B as a reaction raw material.¹H NMR(400MHz,DMSO-d₆)δ10.33(s,1H),8.66(s,1H),8.57(s,1H),8.11(d,J＝6.7Hz,1H),7.80(d,J＝8.2Hz,2H),7.67(s,1H),7.44(s,1H),7.36(d,J＝8.2Hz,2H),4.83(s,2H),4.37(t,J＝7.1Hz,2H),1.92(t,J＝7.4Hz,2H),1.88–1.76(m,2H),1.57–1.39(m,2H),1.33–1.19(m,4H).¹³C NMR (101MHz, DMSO) delta 169.56,158.14,158.02,154.62,146.50,137.58,137.44,135.04,131.08,130.78,130.65,128.72,125.74,121.73,51.85,49.99,32.68,30.00,28.48,26.10,25.43 calculated by High Resolution Mass Spectrometry (HRMS) (ESI) M/z [ M + H]⁺430.2003, found 430.2010.

Example 11

7- (4- (4- ((4-amino-5-methyl-2-oxopyrimidin-1 (2H) -yl) methyl) phenyl) -1H-1,2, 3-triazol-1-yl) -N-hydroxyheptanamide

Example 11A

4-amino-1- (4-ethynylbenzyl) -5-methylpyrimidin-2 (1H) -one

The synthesis was the same as in example 6B.¹H NMR(400MHz,DMSO-d₆)δ7.55(s,1H),7.44(d,J＝8.1Hz,2H),7.25(d,J＝8.1Hz,2H),4.83(s,2H),4.17(s,1H),1.81(s,3H).

Example 11B

7- (4- (4- ((4-amino-5-methyl-2-oxopyrimidin-1 (2H) -yl) methyl) phenyl) -1H-1,2, 3-triazol-1-yl) heptanoic acid ethyl ester

The synthesis was the same as in example 6D.¹H NMR(400MHz,DMSO-d₆)δ8.56(s,1H),7.79(d,J＝7.9Hz,2H),7.58(s,1H),7.34(d,J＝8.0Hz,2H),4.86(s,2H),4.37(t,J＝7.0Hz,2H),4.03(q,J＝7.1Hz,2H),2.26(t,J＝7.3Hz,2H),1.95–1.69(m,5H),1.56–1.45(m,2H),1.32–1.21(m,4H),1.16(t,J＝7.1Hz,3H).¹³C NMR(101MHz,DMSO)δ172.68,165.38,155.56,145.89,143.16,137.48,129.90,128.02,125.09,121.08,59.52,50.81,49.33,33.28,29.29,27.66,25.39,24.11,14.00,12.89.

Example 11C

The synthesis method was the same as in example 1, using example 11B as a reaction raw material.¹H NMR(400MHz,DMSO-d₆)δ10.33(s,1H),8.66(s,1H),8.56(s,1H),7.79(d,J＝7.8Hz,2H),7.56(s,1H),7.33(d,J＝7.9Hz,2H),7.21(s,1H),6.72(s,1H),4.85(s,2H),4.37(t,J＝7.1Hz,2H),1.92(t,J＝7.3Hz,2H),1.90–1.74(m,5H),1.54–1.41(m,2H),1.35–1.19(m,4H).¹³C NMR (101MHz, DMSO) delta 169.56,166.17,156.28,146.53,143.72,138.17,130.51,128.65,125.72,121.69,51.45,49.99,32.68,30.00,28.48,26.10,25.43,13.56 calculated values of [ M + H ] M/z by High Resolution Mass Spectrometry (HRMS) (ESI)]⁺426.2254, found 426.2254.

Example 12

7- (4- (4- ((6-amino-9H-purin-9-yl) methyl) phenyl) -1H-1,2, 3-triazol-1-yl) -N-hydroxyheptanamide

Example 12A

9- (4-ethynylbenzyl) -9H-purin-6-amine

The synthesis was the same as in example 6B.¹H NMR(400MHz,DMSO-d₆)δ8.26(s,1H),8.13(s,1H),7.45(d,J＝8.0Hz,2H),7.29(d,J＝8.0Hz,2H),7.26(br,2H),5.39(s,2H),4.19(s,1H).

Example 12B

7- (4- (4- ((6-amino-9H-purin-9-yl) methyl) phenyl) -1H-1,2, 3-triazol-1-yl) heptanoic acid ethyl ester

The synthesis was the same as in example 6D.¹H NMR(400MHz,DMSO-d₆)δ8.55(s,1H),8.29(s,1H),8.16(s,1H),7.80(d,J＝8.0Hz,2H),7.39(d,J＝8.0Hz,2H),7.29(s,2H),5.39(s,2H),4.37(t,J＝7.0Hz,2H),4.02(q,J＝7.1Hz,2H),2.25(t,J＝7.3Hz,2H),1.94–1.74(m,2H),1.59–1.43(m,2H),1.31–1.21(m,4H),1.15(t,J＝7.1Hz,3H).¹³C NMR(101MHz,DMSO)δ172.68,155.79,152.39,149.40,145.73,140.75,136.40,130.29,128.04,125.28,121.20,59.52,49.34,45.88,33.27,29.27,27.65,25.36,24.10,14.00.

Example 12C

The synthesis method was the same as in example 1, using example 12B as a reaction raw material.¹H NMR(400MHz,DMSO-d₆)δ10.32(s,1H),8.65(s,1H),8.55(s,1H),8.28(s,1H),8.15(s,1H),7.80(d,J＝7.9Hz,2H),7.38(d,J＝8.0Hz,2H),7.25(s,2H),5.39(s,2H),4.36(t,J＝7.1Hz,2H),1.91(t,J＝7.2Hz,2H),1.88–1.72(m,2H),1.58–1.39(m,2H),1.38–1.09(m,4H).¹³C NMR (101MHz, DMSO) delta 168.90,155.91,152.54,149.41,145.74,140.67,136.43,130.29,128.04,125.28,121.18,118.61,49.36,45.87,32.04,29.36,27.84,25.46,24.79 high resolution Mass Spectrometry (HRM)S) (ESI) M/z calculation [ M + H]⁺436.2209 found 436.2203

Example 13

7- (4- (4- ((2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) methyl) phenyl) -1H-1,2, 3-triazol-1-yl) -N-hydroxyheptanamide

Example 13A

1- (4-ethynylbenzyl) pyrimidine-2, 4(1H,3H) -dione

The synthesis was the same as in example 6B.¹H NMR(400MHz,DMSO-d₆)δ11.36(s,1H),7.77(d,J＝7.8Hz,1H),7.47(d,J＝7.8Hz,2H),7.29(d,J＝8.0Hz,2H),5.61(d,J＝7.9Hz,1H),4.88(s,2H),4.20(s,1H).¹³C NMR(101MHz,DMSO)δ163.51,150.89,145.45,137.66,131.87,127.55,120.91,101.35,83.03,80.87,49.96.

Example 13B

7- (4- (4- ((2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) methyl) phenyl) -1H-1,2, 3-triazol-1-yl) heptanoic acid ethyl ester

The synthesis was the same as in example 6D.¹H NMR(400MHz,DMSO-d₆)δ11.35(s,1H),8.57(s,1H),7.82(d,J＝7.9Hz,2H),7.79(d,J＝7.8Hz,1H),7.37(d,J＝7.9Hz,2H),5.61(d,J＝7.9Hz,1H),4.89(s,2H),4.37(t,J＝7.0Hz,2H),4.02(q,J＝7.1Hz,2H),2.25(t,J＝7.2Hz,2H),1.94–1.76(m,2H),1.56–1.45(m,2H),1.34–1.22(m,4H),1.15(t,J＝7.1Hz,3H).¹³C NMR(101MHz,DMSO)δ172.67,163.52,150.93,145.77,145.44,136.19,130.27,127.97,125.27,121.20,101.28,59.52,49.99,49.35,33.27,29.29,27.66,25.38,24.11,14.01.

Example 13C

The synthesis method was the same as in example 1, using example 13B as a reaction raw material.¹H NMR(400MHz,DMSO-d₆)δ11.35(s,1H),10.33(s,1H),8.66(s,1H),8.58(s,1H),7.83(d,J＝8.1Hz,2H),7.79(d,J＝7.8Hz,1H),7.37(d,J＝8.0Hz,2H),5.61(d,J＝7.8Hz,1H),4.89(s,2H),4.38(t,J＝7.0Hz,2H),1.92(t,J＝7.3Hz,2H),1.89–1.75(m,2H),1.56–1.40(m,2H),1.38–1.16(m,4H).¹³C NMR (101MHz, DMSO). delta. 168.92,163.53,150.93,145.78,145.46,136.20,130.26,127.97,125.28,121.19,101.29,49.99,49.38,32.05,29.37,27.85,25.47,24.80 calculated by High Resolution Mass Spectrometry (HRMS) (ESI) M/z [ M + H]⁺413.1937, found 413.1945.

Example 14

(E) -3- (4- ((4- ((4-amino-2-oxopyrimidin-1 (2H) -yl) methyl) piperidin-1-yl) methyl) phenyl) -N-hydroxyacrylamide

Example 14A

4- ((p-toluenesulfonate) methyl) piperidine-1-carboxylic acid tert-butyl ester

Tert-butyl 4- (hydroxymethyl) piperidine-1-carboxylate (1eq) was dissolved in anhydrous pyridine and p-toluenesulfonyl chloride (1.1eq) was added slowly at 0 ℃. After which it was warmed to room temperature and the reaction stirred at this temperature for 16 h. After the reaction, water was added to the reaction system, and extraction was performed with ethyl acetate. The organic phase was washed with 5% aqueous hydrochloric acid, water and saturated brine in this order. The organic phase was then dried over anhydrous sodium sulfate. After removal of the drying agent by filtration, the solvent of the organic phase was removed to give the title compound. The product is used in the subsequent reaction without purification.

Example 14B

4- ((4-amino-2-oxopyrimidin-1 (2H) -yl) methyl) piperidine 1-carboxylic acid tert-butyl ester

Cytosine (1eq), example 14A (1.1eq), and anhydrous cesium carbonate (1.1eq) were dissolved in anhydrous DMF and reacted at 80 ° overnight. After the reaction was completed, cesium carbonate was removed by filtration, and DMF was removed by centrifugation. Water and ethyl acetate were added for extraction. The organic phase is freed of the solvent to give the title compound. The target compound is further purified by recrystallization from ether or column chromatography.¹H NMR(400MHz,DMSO-d₆)δ7.51(d,J＝4.4Hz,1H),6.97(d,J＝32.6Hz,2H),5.60(d,J＝7.1Hz,1H),3.97–3.82(m,2H),3.53–3.47(m,2H),2.64(br,2H),1.93–1.81(m,1H),1.53–1.20(m,11H),1.12–0.94(m,2H).

Example 14C

4-amino-1- (piperidin-4-ylidene) pyrimidin-2 (1H) -one hydrochloride

Example 14B (1eq) was dissolved in THF, 3M aqueous hydrochloric acid (10eq) was added and the reaction was allowed to proceed at room temperature for 3 h. And (4) after the reaction is finished, removing the solvent in the reaction system by spinning. Methanol was added again to dissolve the product, after which methanol was removed by spinning. Repeating 2-3 times to obtain the title compound.¹H NMR(400MHz,DMSO-d₆)δ9.72(s,1H),9.02(br,1H),8.82(br,1H),8.63(s,1H),8.01(d,J＝7.6Hz,1H),6.10(d,J＝7.6Hz,1H),3.68(d,J＝7.2Hz,2H),3.24(d,J＝12.5Hz,2H),2.79(q,J＝11.9Hz,2H),2.07–1.87(m,1H),1.74(d,J＝13.4Hz,2H),1.38(q,J＝10.9Hz,2H).

Example 14D

(E) -methyl 3- (4- ((4- ((4-amino-2-oxopyrimidin-1 (2H) -yl) methyl) piperidin-1-yl) methyl) phenyl) acrylate

Example 14C (1eq) was dissolved in DMF or acetonitrile and triethylamine (3eq) and methyl p-bromomethyl cinnamate (1.1eq) were added. The reaction was carried out at room temperature overnight. The solvent was removed by rotation and the column chromatography separated to give the title compound.¹H NMR(400MHz,DMSO-d₆)δ7.89–7.55(m,3H),7.51(d,J＝7.1Hz,1H),7.33(d,J＝7.8Hz,2H),6.95(br,2H),6.61(d,J＝16.0Hz,1H),5.59(d,J＝7.1Hz,1H),3.72(s,3H),3.50(d,J＝7.1Hz,2H),3.46(s,2H),2.77(d,J＝10.9Hz,2H),1.86(t,J＝11.3Hz,2H),1.77–1.57(m,1H),1.46(d,J＝12.6Hz,2H),1.31–1.09(m,2H).¹³C NMR(101MHz,DMSO)δ167.22,166.36,156.40,146.99,144.91,129.59,128.75,117.78,93.19,62.39,54.52,53.28,51.94,35.29,29.76.

Example 14E

The synthesis was performed in the same manner as in example 1, using example 14D as a starting material.¹H NMR(400MHz,DMSO-d₆) δ 10.75(s,1H),9.04(s,1H), 7.80-7.46 (M,3H),7.43(d, J ═ 15.7Hz,1H), 7.39-7.12 (M,2H),7.00(s,1H),6.91(s,1H),6.44(d, J ═ 15.7Hz,1H),5.59(d, J ═ 7.1Hz,1H),3.50(d, J ═ 7.0Hz,2H),3.44(br,2H),2.78(br,2H),1.86(br,2H),1.69(br,1H),1.46(br,2H),1.22(br,2H), High Resolution Mass Spectrometry (HRMS) (ESI) M/z calculated value [ M + H ]/[ M ]/[ calculated value]⁺384.2036, found 384.2028.

Example 15

(E) -3- (4- ((4- ((4-amino-5-fluoro-2-oxopyrimidin-1 (2H) -yl) methyl) piperidin-1-yl) methyl) phenyl) -N-hydroxyacrylamide

Example 15A

4- ((4-amino-5-fluoro-2-oxopyrimidin-1 (2H) -yl) methyl) piperidine 1-carboxylic acid tert-butyl ester

The synthesis was performed as in example 14B.¹H NMR(400MHz,Chloroform-d)δ7.40(d,J＝5.2Hz,1H),4.11(s,2H),3.74–3.57(m,2H),2.76–2.54(m,2H),2.11–1.90(m,1H),1.68–1.55(m,2H),1.43(s,9H),1.25–1.06(m,2H).

Example 15B

4-amino-5-fluoro-1- (piperidin-4-methylidene) pyrimidin-2 (1H) -one hydrochloride

The synthesis was the same as in example 14C.¹H NMR(400MHz,DMSO-d₆)δ9.18(s,1H),9.04(br,1H),8.94–8.62(m,2H),8.41(d,J＝6.6Hz,1H),3.60(d,J＝7.2Hz,2H),3.28–3.15(m,2H),2.91–2.70(m,2H),2.09–1.90(m,1H),1.83–1.65(m,2H),1.49–1.30(m,2H).

Example 15C

(E) -methyl 3- (4- ((4- ((4-amino-5-fluoro-2-oxopyrimidin-1 (2H) -yl) methyl) piperidin-1-yl) methyl) phenyl) acrylate

The synthesis was the same as in example 14D.¹H NMR(400MHz,DMSO-d₆)δ7.92(d,J＝6.7Hz,1H),7.81–7.47(m,4H),7.34(s,2H),6.62(d,J＝16.1Hz,1H),3.72(s,3H),3.62–3.35(m,4H),2.96–2.63(m,2H),1.96–1.78(m,2H),1.77–1.61(m,1H),1.57–1.39(m,2H),1.36–1.06(m,2H).

Example 15D

In the following examples15C as the starting material, the synthesis method was the same as in example 1.¹H NMR(400MHz,DMSO-d₆) δ 10.74(s,1H),9.04(s,1H),7.91(d, J ═ 6.8Hz,1H), 7.74-7.47 (M,3H),7.43(d, J ═ 15.8Hz,1H), 7.40-7.11 (M,3H),6.43(d, J ═ 15.8Hz,1H), 3.63-3.37 (M,4H), 2.88-2.68 (M,2H), 1.97-1.73 (M,2H), 1.73-1.58 (M,1H), 1.58-1.33 (M,2H), 1.33-1.06 (M,2H), High Resolution Mass Spectrometry (HRMS) (ESI) M/z calculated value [ M + H [, ] M + H (1H) ]]⁺402.1941, found 402.1942.

Example 16

(E) -3- (4- ((4- ((4-amino-5-methyl-2-oxopyrimidin-1 (2H) -yl) methyl) piperidin-1-yl) methyl) phenyl) -N-hydroxyacrylamide

Example 16A

4- ((4-amino-5-methyl-2-oxopyrimidin-1 (2H) -yl) methyl) piperidine 1-carboxylic acid tert-butyl ester

The synthesis was performed as in example 14B.¹H NMR(400MHz,DMSO-d₆)δ7.39(s,1H),7.11(br,1H),6.65(br,1H),3.91(d,J＝13.1Hz,2H),3.48(d,J＝7.2Hz,2H),2.70–2.56(m,2H),1.90–1.83(m,1H),1.80(s,3H),1.49–1.42(m,2H),1.39(s,9H),1.11–0.92(m,2H).

Example 16B

4-amino-5-methyl-1- (piperidin-4-methylidene) pyrimidin-2 (1H) -one hydrochloride

The synthesis was the same as in example 14C.¹H NMR(400MHz,DMSO-d₆)δ9.01(s,1H),8.81(s,1H),8.65(s,2H),7.87(s,1H),3.62(d,J＝7.2Hz,2H),3.24(d,J＝12.6Hz,2H),2.79(q,J＝11.7Hz,2H),2.05–1.96(m,1H),1.90(s,3H),1.72(dd,J＝13.7,3.4Hz,2H),1.50–1.31(m,2H).

Example 16C

(E) -methyl 3- (4- ((4- ((4-amino-5-methyl-2-oxopyrimidin-1 (2H) -yl) methyl) piperidin-1-yl) methyl) phenyl) acrylate

The synthesis was the same as in example 14D.¹H NMR(400MHz,DMSO-d₆)δ7.84–7.58(m,3H),7.46–7.26(m,3H),7.08(s,1H),6.62(d,J＝16.0Hz,2H),3.72(s,3H),3.57–3.37(m,4H),2.88–2.69(m,2H),2.04–1.82(m,2H),1.79(s,3H),1.75–1.59(m,1H),1.56–1.38(m,2H),1.31–1.12(m,2H).

Example 16D

The synthesis method was the same as in example 1, using example 16C as a starting material.¹H NMR(400MHz,DMSO-d₆) δ 10.76(s,1H),9.04(s,1H), 7.83-7.17 (M,6H),7.09(s,1H),6.65(s,1H),6.44(d, J ═ 15.7Hz,1H), 3.73-3.36 (M,4H), 2.91-2.68 (M,2H), 2.06-1.72 (M,5H), 1.72-1.61 (M,1H), 1.56-1.35 (M,2H), 1.31-1.05 (M,2H), High Resolution Mass Spectrometry (HRMS) (ESI) calculated value [ M + H) M/z [/M/z ]]⁺398.2192, found 398.2193.

Example 17

(E) -3- (4- ((4- ((6-amino-9H-purin-9-yl) methyl) piperidin-1-yl) methyl) phenyl) -N-hydroxyacrylamide

Example 17A

4- ((6-amino-9H-purin-9-yl) methyl) piperidine-1-carboxylic acid tert-butyl ester

The synthesis was performed as in example 14B.¹H NMR(400MHz,DMSO-d₆)δ8.13(s,1H),8.10(s,1H),7.21(s,2H),4.04(d,J＝7.1Hz,2H),3.91(d,J＝13.0Hz,2H),2.78–2.55(m,2H),2.15–1.96(m,1H),1.45(d,J＝12.8Hz,2H),1.38(s,9H),1.17–0.99(m,2H).

Example 17B

9- (piperidin-4-ylidene) -9H-purin-6-amine hydrochloride

The synthesis was the same as in example 14C.¹H NMR(400MHz,DMSO-d₆)δ9.69(s,1H),9.20(s,1H),9.15–8.86(m,2H),8.58(s,1H),8.57(s,1H),4.20(d,J＝7.1Hz,2H),3.38–3.09(m,2H),2.86–2.66(m,2H),2.23–2.03(m,1H),1.76–1.58(m,2H),1.54–1.35(m,2H).

Example 17C

(E) -methyl 3- (4- ((4- ((6-amino-9H-purin-9-yl) methyl) piperidin-1-yl) methyl) phenyl) acrylate

The synthesis was the same as in example 14D.¹H NMR(400MHz,Methanol-d₄)δ8.19(s,1H),8.11(s,1H),7.68(d,J＝16.1Hz,1H),7.58(d,J＝8.0Hz,2H),7.38(d,J＝8.0Hz,2H),6.53(d,J＝16.0Hz,1H),4.14(d,J＝7.1Hz,2H),3.78(s,3H),3.61(br,2H),3.08–2.86(m,2H),2.24–2.03(m,2H),2.03–1.89(m,1H),1.70–1.53(m,2H),1.50–1.35(m,2H).

Example 17D

The synthesis method was the same as in example 1, using example 17C as a starting material.¹H NMR(400MHz,Methanol-d₄)δ819(s,1H),8.10(s,1H),7.55(d, J ═ 15.9Hz,1H),7.51(d, J ═ 7.8Hz,2H),7.35(d, J ═ 7.9Hz,2H),6.45(d, J ═ 15.7Hz,1H),4.13(d, J ═ 7.1Hz,2H),3.56(s,2H),2.93(d, J ═ 11.5Hz,2H),2.05(t, J ═ 11.7Hz,2H), 2.01-1.89 (M,1H), 1.64-1.50 (M,2H), 1.48-1.31 (M,2H), High Resolution Mass Spectrometry (HRMS) (ESI) M/z calculated value [ M + H ]/[ ESI ], (ESI)]⁺408.2148, found 408.2146.

Example 18

(E) -3- (4- ((4- ((2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) methyl) piperidin-1-yl) methyl) phenyl) -N-hydroxyacrylamide

Example 18A

4- ((2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) methyl) piperidine-1-carboxylic acid tert-butyl ester

The synthesis was performed as in example 14B.¹H NMR(400MHz,DMSO-d₆)δ11.25(s,1H),7.61(d,J＝7.8Hz,1H),5.53(d,J＝7.8Hz,1H),3.93(d,J＝13.0Hz,2H),3.54(d,J＝7.3Hz,2H),2.66(br,2H),1.96–1.77(m,1H),1.52(d,J＝12.9Hz,2H),1.39(s,9H),1.17–0.89(m,2H).

Example 18B

1- (piperidin-4-ylidene) pyrimidine-2, 4(1H,3H) -dione hydrochloride

The synthesis was the same as in example 14C.¹H NMR(400MHz,DMSO-d₆)δ11.29(s,1H),8.84(s,1H),8.56(s,1H),7.65(d,J＝7.8Hz,1H),5.57(dd,J＝7.8,2.3Hz,1H),3.59(d,J＝7.1Hz,2H),3.24(d,J＝12.8Hz,2H),2.93–2.68(m,2H),2.10–1.83(m,1H),1.70(d,J＝13.1Hz,2H),1.47–1.20(m,2H).

Example 18C

(E) -methyl 3- (4- ((4- ((2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) methyl) piperidin-1-yl) methyl) phenyl) acrylate

The synthesis was the same as in example 14D.¹H NMR(400MHz,DMSO-d₆)δ11.17(s,1H),7.73–7.56(m,4H),7.33(d,J＝7.9Hz,2H),6.61(d,J＝16.1Hz,1H),5.52(d,J＝7.8Hz,1H),3.72(s,3H),3.54(d,J＝7.1Hz,2H),3.46(s,2H),2.87–2.69(m,2H),1.97–1.81(m,2H),1.75–1.58(m,1H),1.58–1.43(m,2H),1.29–1.12(m,2H).

Example 18D

The synthesis method was the same as in example 1, using example 18C as a starting material.¹H NMR(400MHz,DMSO-d₆) δ 11.24(s,1H),10.76(s,1H),9.04(s,1H),7.61(d, J ═ 7.8Hz,1H),7.52(d, J ═ 7.6Hz,2H),7.44(d, J ═ 15.8Hz,1H),7.35(s,2H),6.44(d, J ═ 15.9Hz,1H),5.53(dd, J ═ 7.8,2.2Hz,1H), 3.84-3.36 (M,4H), 3.03-2.69 (M,2H), 2.13-1.78 (M,2H), 1.78-1.63 (M,1H), 1.62-1.37 (M,2H), 1.37-1.02 (M,2H), high-resolution ESI (ms) (M/z + hrm + H)]⁺385.1876 found 385.1871.

Example 19

6- (4- ((4-amino-2-oxopyrimidin-1 (2H) -yl) methyl) piperidin-1-yl) -N-hydroxyhexanamide

Example 19A

6- (4- ((4-amino-2-oxopyrimidin-1 (2H) -yl) methyl) piperidin-1-yl) hexanoic acid ethyl ester

Example 14C (1eq), ethyl 6-bromohexanoate (2eq), and potassium iodide (0.5eq) were dissolved in absolute ethanol and reacted at reflux overnight. TLC confirmed the 14C reaction was complete and the organic solvent was removed by spin and column chromatographed to give the title compound.¹H NMR(400MHz,DMSO-d₆)δ7.56(d,J＝7.1Hz,1H),7.05(br,2H),5.64(d,J＝7.1Hz,1H),4.06(q,J＝7.1Hz,2H),3.54(d,J＝6.9Hz,2H),3.51–3.40(m,2H),3.07–2.91(m,2H),2.90–2.71(m,2H),2.34(t,J＝7.1Hz,2H),2.01–1.86(m,1H),1.80–1.59(m,4H),1.60–1.36(m,4H),1.18(t,J＝7.1Hz,3H).

Example 19B

The synthesis method was the same as in example 1, using example 19A as a starting material.¹H NMR(400MHz,DMSO-d₆) δ 7.52(d, J ═ 7.1Hz,1H),7.08(s,1H),6.90(s,1H),5.62(d, J ═ 7.0Hz,1H),3.49(d, J ═ 7.2Hz,2H),2.80(d, J ═ 10.9Hz,2H),2.18(t, J ═ 7.3Hz,2H),1.88(t, J ═ 7.4Hz,2H),1.73(t, J ═ 10.9Hz,2H), 1.69-1.59 (M,1H), 1.56-1.28 (M,6H), 1.28-0.99 (M,4H), High Resolution Mass Spectrometry (HRMS) (ESI) M/z calculated value [ M + H ]/, [ M,6H ], ]]⁺338.2192, found 338.2190.

Example 20 MTT method cell proliferation inhibitory Activity test

The MTT method is adopted in the in vitro cell proliferation inhibition experiment, and the following two cell lines are adopted: human Chronic Myelocytic Leukemia (CML) cell line K562 and human histiocytic lymphoma cell U937.

K562 and U937 are suspension cells, cultured in RPIM-1640 culture medium containing 10% fetal calf serum at 37 deg.C with 5% CO₂Culturing under conventional conditions.

The specific operation is as follows:

first, the compounds (i.e., samples) obtained by the preparation of examples 1 to 19 were prepared as DMSO (dimethyl sulfoxide) solutions having a compound concentration of 5mM/L, respectively, and then the obtained solutions were subjected to gradient dilution to obtain a series of sample solutions having concentration gradients.

Subsequently, K562 cells or U937 cells were collected at 1.5X 10 in the logarithmic growth phase⁵The cell density of one/mL was seeded in a 96-well plate at 99. mu.L/well, and then 1. mu.L of the sample solution was added to each well to give final concentrations of 0.01, 0.05, 0.1, 0.5, 1, 5, 10,25, 50. mu.M, respectively. Three multiple wells are set for each sample and each concentration, and a positive control group, a negative control group and a blank control group are set, wherein positive control drugs including histone deacetylase inhibitor SAHA and DNA methyltransferase inhibitor SGI-027 are respectively added into the positive control group, DMSO with the concentration being the same as that of the sample solution is added into the negative control group, and cells are not added into the blank control group. After 72 hours of action, MTT solution was added at 10. mu.L/well, after further incubation for 4 hours, centrifugation was carried out at 2000rpm at 4 ℃ for 5 minutes, the supernatant was aspirated, dimethyl sulfoxide (DMSO) was added at 100. mu.L/well, the incubation was carried out at 37 ℃ for about 10 minutes, followed by shaking with a micro-shaker for about 5 minutes to dissolve the crystals completely, and the OD was measured at 490nm with a microplate reader.

The cell proliferation Inhibition Rate (Inhibition Rate, IR%) was calculated by the following formula:

IR% ((negative control OD-sample OD)/(negative control OD-blank OD) × 100%),

the in vitro cell proliferation inhibitory activity of the compound prepared by the invention is obtained by calculation, and the result is shown in table 1. Wherein, IC₅₀Greater than 50. mu.M indicated as "-", IC₅₀Greater than 10. mu.M and less than 50. mu.M are indicated by "+", IC₅₀Greater than 5 μ M and less than 10 μ M denoted "+", IC₅₀Greater than 1 μ M and less than 5 are indicated by "+++", and less than 1 is indicated by "+++". The results show that most compounds have better inhibition effect on the proliferation of U937 than K562. Wherein the IC of compounds 10, 11,13 and 15 for U937₅₀IC of less than 5. mu.M, Compounds 12 and 17 against U937₅₀Less than 1. mu.M, exhibits a good antitumor proliferative effect.

TABLE 1 inhibitory Activity of the Compounds prepared in examples 1 to 19 on in vitro tumor cell proliferation

Note: IC (integrated circuit)₅₀The median inhibitory concentration is indicated.

Example 21 test of DNA methyltransferase inhibitory Activity

Using isotopically labelled S-adenosylmethionine: (³H-SAM) test compounds were tested for their inhibitory activity against DNA methyltransferase (DNMT1, DNMT3A, DNMT3B) at 50 μ M and against S-adenosylhomocysteine (SAH). Mixing the compound with a certain amount of corresponding DNA methyltransferase subtype, incubating at room temperature for 15min, adding synthetic biotin-labeled oligonucleotide substrate and³H-SAM and react for 4H at room temperature. The reaction was then transferred to streptavidin-coated high-throughput well plates and allowed to react for 1h at room temperature. Radioactivity was obtained by liquid scintillation counting. The inhibitory activity of the compound on the enzyme was obtained by comparison with a negative control group (inhibitor-free group). The inhibition rate of the compound on DNMT under 50. mu.M condition is shown in Table 2. Wherein the inhibition ratio is less than 20% as "-", the inhibition ratio is more than 20% and less than 50% as "+", the inhibition ratio is more than 50% and less than 80% as "+ +", and the inhibition ratio is more than 80% as "+ + + + +". Test results show that most compounds have an inhibiting effect on DNA methyltransferase, and have different inhibition rates on different subtypes of the DNA methyltransferase. Wherein the compounds 6, 7, 8,9 and 11 have a greater inhibitory effect on DNMT1 than on DNMT3A/3B, while the compounds 16 and 17 have a greater inhibitory activity on DNMT3A/B than on DNMT 1.

TABLE 2 DNA methyltransferase inhibitory Activity of the Compounds prepared in examples 1-19

Example 22 Histone deacetylase inhibitory Activity assay

Two isoforms of HDAC1 and HDAC6 of the histone deacetylase family were studied to test the inhibitory activity of compounds against histone deacetylase, with ten concentrations of each compound being setGradient, triplicate wells, and control with the marketed HDAC inhibitor SAHA. Firstly, dissolving a compound into a reaction buffer solution, then adding a buffer solution containing histone deacetylase in a certain volume, incubating for 15 minutes at room temperature, then adding trypsin and acetylated peptide buffer solution as reaction substrates to start deacetylation reaction, simultaneously enabling the concentration of the compound and the enzyme content to reach set values, gently mixing for 60 seconds, then incubating at room temperature, and recording the dynamic parameters of the reaction under certain excitation light and emission light wavelengths within 1 hour. The inhibitory activity of the compound on the enzyme is obtained by comparing with a negative control group (non-inhibitor group), and the half Inhibitory Concentration (IC) of the compound on histone deacetylase is calculated₅₀). The results are shown in Table 3, in which IC₅₀Greater than 1000nM as "-", IC₅₀Greater than 100nM and less than 1000nM are indicated by "+", IC₅₀Greater than 50 and less than 100nM as "+ +", IC₅₀Greater than 10 and less than 50nM as indicated by "+++", IC₅₀Less than 10nM is indicated by "+ ++". From the test results, the IC of most compounds inhibiting HDAC1/6 is known₅₀Values of less than 100nM, where compounds 9,10, 11, 12 and 13 had single digit nanomolar-scale activity comparable to that of the marketed drug SAHA.

TABLE 3 Histone deacetylase inhibitory Activity of the Compounds prepared in examples 1-19

Claims

1. A nucleoside base hydroxamic acid compound shown in formula I or a pharmaceutically acceptable salt or tautomer thereof,

wherein:

r is a nucleobase, a nucleobase with a substituent group or a nucleobase analogue;

linker is a linking chain connecting R with the hydroxamate functionality,

linker is selected from:

wherein

t is 1,2 or 3;

m is 1,2,3, 4, 5, 6, 7 or 8;

y is 0,1, 2 or 3;

z is 1,2 or 3;

g is 0,1, 2,3, 4, 5, 6, 7 or 8;

x is selected from nitrogen or carbon;

y is a linking chain linking the phenyl ring to the hydroxamate functionality selected from the group consisting of alkyl, methylene, alkenyl, ethylene, vinyl, alkoxy, haloalkyl;

the compound shown in the formula I is selected from any one of the following compounds:

2. the compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein: the pharmaceutically acceptable salt is an inorganic acid salt or an organic acid salt;

the inorganic acid salt is selected from salts formed by any one of the following inorganic acids: hydrochloric acid, sulfuric acid and phosphoric acid;

3. Use of a compound of formula i as defined in any one of claims 1 to 2, or a pharmaceutically acceptable salt or tautomer thereof, for the preparation of a product as defined below:

1) DNA methyltransferase and/or histone deacetylase inhibitors;

2) an inhibitor of proliferation of eukaryotic tumor cells;

3) a medicine for preventing and/or treating tumor.

4. Use according to claim 3, characterized in that:

the DNA methyltransferases include subtypes known in mammalian cells, DNMT1, DNMT3A and DNMT 3B;

the histone deacetylase comprises the known isoforms HDAC1, HDAC2, HDAC3, HDAC8, HDAC4, HDAC5, HDAC7, HDAC9, HDAC6, HDAC10 and HDAC11 in mammalian cells;

the eukaryote is a mammal;

the tumor cell is a cancer cell;

the cancer cell is leukemia cancer cell, lymphoma cell, lung cancer cell, human brain glioma cell, melanoma cell, glioblastoma cell, cervical cancer cell, nasopharyngeal cancer cell, liver cancer cell, breast cancer cell, brain cancer cell, pancreatic cancer cell, ovarian cancer cell, uterine cancer cell, testicular cancer cell, skin cancer cell, stomach cancer cell, colon cancer cell, bladder cancer cell or rectal cancer cell;

the leukemia cancer cells are human Chronic Myelogenous Leukemia (CML) cell line K562;

the lymphoma cell is human histiocyte lymphoma cell U937;

the lung cancer cells are human lung cancer cells NCI-H520 and A549;

the human brain glioma cell is U251;

the melanoma cancer cell is A375;

the glioblastoma cell is a human glioblastoma cell A172 or a human brain astrocytoma cell U-118 MG;

the cervical cancer cell is a human cervical cancer cell line Hela;

the nasopharyngeal carcinoma cell is a human nasopharyngeal carcinoma cell strain CNE-2;

the liver cancer cell is a human liver cancer cell strain HepG 2;

the breast cancer cells are human breast cancer cells MCF-7 and MDA-MB-231;

the tumor is a carcinoma;

the cancer is leukemia, lymphoma, lung cancer, melanoma, glioblastoma, cervical cancer, nasopharyngeal cancer, liver cancer, breast cancer, brain cancer, pancreatic cancer, ovarian cancer, uterine cancer, testicular cancer, skin cancer, stomach cancer, colon cancer, bladder cancer or rectal cancer.

5. A product, the active ingredient of which is a compound of formula I according to any one of claims 1-2, or a pharmaceutically acceptable salt or tautomer thereof;

wherein the product is: