CN113444084A

CN113444084A - Anti-tumor compound and preparation and application thereof

Info

Publication number: CN113444084A
Application number: CN202010224405.9A
Authority: CN
Inventors: 苟少华; 王园江
Original assignee: Southeast University
Current assignee: Southeast University
Priority date: 2020-03-26
Filing date: 2020-03-26
Publication date: 2021-09-28
Also published as: WO2021190142A1

Abstract

The invention relates to an anti-tumor compound and preparation and application thereof, which are compounds for inhibiting CK2 protein kinase and cancer cell dryness, and provide a novel compound shown as a formula II by introducing an amino-group-containing, hydroxyl-group-containing, sulfhydryl-group-containing or halogen-atom-containing aliphatic chain or heterocyclic structure fragment in a known CK2 inhibitor (CX-4945) parent structure in the modes of an amido bond, an ester bond and a thioester bond; the compound has obvious anticancer activity, and the cytotoxic activity of partial compounds on cancer cells, the inhibition capacity on CK2 protein kinase and the selectivity on CK2 protein are superior to those of parent compounds; in addition, some compounds have strong inhibition capability on cancer cell dryness, and are beneficial to overcoming drug resistance; compounds of the structures shown in formulas II-A, II-B, II-C, II-D and II-E:

the compound has good inhibition effect on the proliferation of cancer cells, can inhibit the dryness of the cancer cells, and can be used for preparing antitumor drugs.

Description

Anti-tumor compound and preparation and application thereof

Technical Field

The invention belongs to the technical field of medicines, and relates to a novel compound with CK2 protein kinase and cancer cell dryness inhibition capability; also relates to a preparation method of the compounds and application of the compounds in the preparation of antitumor drugs.

Background

Protein kinase 2, also known as Casein kinase 2(CK2), is a pleiotropic and highly conserved second messenger-independent serine/threonine protein kinase that is widely present in the cytoplasm and nucleus of eukaryotic cells. CK2 is composed of two catalytic isoforms (CK2 α/CK2 α ') and two regulatory isoforms (β), forming three tetramers (α α α β β, α α' β, α 'α' β β). There is a great deal of evidence that CK2 plays a very important role in a variety of cellular activities, its phosphorylated substrates of more than 300 play an important role in the processes of cell growth, proliferation, apoptosis and cancerization, and it regulates multiple anti-apoptotic pathways and leading signaling cascades, including PI3K/AKT signaling pathway, Wnt signaling cascade, NF- κ B transcription and DNA damage response, etc.

CK2 has different critical roles in different stages of the cell cycle. In an in vitro animal cell cycle experiment, the addition of the CK2 inhibitor can inhibit the cell cycle process, and the animal cell cycle activity needs the participation and the regulation of CK 2. Meanwhile, experiments prove that the over-expressed CK2 can inhibit programmed death caused by the anti-cancer drug. Since the phenomenon of CK2 overexpression exists in various drug-resistant cancer cells, the inhibition of CK2 can not only promote the apoptosis of the cancer cells, but also increase the sensitivity of the cancer cells to anticancer drugs. CK2 is overexpressed in cancer cells in a variety of cancers, such as head and neck, breast, colon, kidney, lung, leukemia, and prostate cancers. In addition, CK2 has a direct link to poor prognosis and progression of cancer. Thus, CK2 has become a very promising target for cancer therapy.

The homology across the human protein kinase family is relatively high, especially in the core of its catalytic domain. Most small molecule protein kinase inhibitors achieve inhibitory effects primarily through interaction with the conserved ATP binding site of the kinase target. Due to the presence of larger amino acid residues, the ATP binding site of CK2 is small relative to the binding sites of other kinases, which necessitates the design of ATP-competitive inhibitors of synthetic CK2 that meet two requirements, i.e., high specificity and small molecular weight.

CK2 protein was first published in 1954, and the important role of CK2 protein in cell growth regulation was elucidated (J.biol.chem.1954, 211, 969-one 980). At the foundationIn the above, small molecule inhibitors targeting the protein, such as benzimidazole derivatives (J.biol.chem.1986, 261, 3414-3419; bioorg.Med.chem.2003, 11, 3997-4002), flavonoid derivatives (Curr.Top.Med.chem.2011, 11, 1340-1351), coumarin derivatives (Curr.Pharmaceut.Des.2004, 10, 3797-3811), anthraquinone derivatives (Eur.J.Med.chem.2010, 45, 292-297), pyrazoline triazine derivatives (bioorg.chem.Lett.2007, 17, 4191-4195), carboxylic acid derivatives (ChemMed Biochem2007, 8, 129-139), non-competitive inhibitors (J.Med.chem.chem.2019, 62, 2017-2016-3047-2, 3049, 2, etc.) did not appear successively until 1986. However, only the small molecule CK2 inhibitor CX-4945 (formula I) developed by Cylene pharmaceuticals using benzonaphthyridine as the parent is currently under phase I/II clinical study. Although CX-4945 exhibits a broad spectrum of antitumor activity and excellent CK2 kinase inhibitory activity, there are some significant drawbacks such as the inhibition rate (IC) of CX-4945 against CK2₅₀) At 1nM, but also nanomolar (IC) to the other 12 kinases₅₀) Furthermore, its inhibition of CLK2 is even stronger than that of CK2, leading to severe off-target toxicity. Therefore, it is of great interest to develop a novel CK2 inhibitor with excellent activity and high selectivity based on CX-4945.

In recent years, with the development of tumor biology, clinical treatment of tumors has advanced significantly, but most malignant tumors remain untreatable, with metastasis and recurrence being the most major challenges of current tumor therapy. Cancer Stem Cells (CSCs), also known as cancer stem cells or tumor stem cells, are cells in tumors that have the ability to self-renew and produce heterogeneous tumor cells, and are also considered to be the "motive force" in tumor tissue that actually drives tumor development and progression, as well as the root cause of tumor recurrence and metastasis. Because cancer stem cell populations exist in tumor tissues, the cancer stem cell populations have strong self-renewal and differentiation capacity, and can maintain malignant proliferation, invasion, drug resistance, metastasis, relapse and the like of tumors by regulating multiple signal pathways such as Wnt/beta-catenin, Notch, Hedgehog, BMI-1 and the like. Recently, the tumor stem cell theory has received increased attention, and has succeeded in isolating cancer stem cells in hematological tumors and a variety of solid tumors. There is increasing evidence that conventional chemotherapeutic drugs, including targeted drugs, are effective in killing differentiated cancer cells but are ineffective in cancer stem cells at effective doses, some of the cancer stem cells even promote the growth of cancer stem cells, and the very small amount of cancer stem cells can cause drug resistance and tumor recurrence and metastasis, reducing the efficacy of many anticancer drugs. Therefore, it would be beneficial to develop a class of drugs that can inhibit both normal cancer cells and cancer stem cells.

Disclosure of Invention

The technical problem is as follows: the invention aims to provide an anti-tumor compound, and preparation and application thereof, namely a compound capable of inhibiting CK2 protein kinase and cancer cell dryness, and preparation and application thereof.

The invention provides a novel compound by introducing aliphatic chain or heterocyclic structure fragments containing amino, hydroxyl, sulfhydryl or halogen atoms in the parent structure of a known CK2 inhibitor (CX-4945) by means of amide bonds, ester bonds, thioester bonds and the like. The compound has obvious anticancer activity, and the in vitro anticancer activity and the enzyme activity of partial compounds are superior to those of parent compounds, and the compound is found to be capable of effectively inhibiting the dryness of cancer cells.

The technical scheme is as follows: the invention provides compounds of the structures shown in formulas II-A, II-B, II-C, II-D and II-E:

wherein:

r in the formula II-A₁is-NH₂、-OH、-SO₂CH₃、-CH₂CHF₂、-CH₂C≡CH、-(CH₂)₂OH、-(CH₂)₂NH₂、-(CH₂)₂Br、-(CH₂)₂CO₂CH₃、-(CH₂)₂CO₂H、-(CH₂)₂N(CH₃)₂、-(CH₂)₃OH、-(CH₂)₃NH₂、-(CH₂)₃CO₂CH₃、

R in the formula II-B₂is-CH₂CHF₂、-(CH₂)₂OH、-(CH₂)₂F、-(CH₂)₂I、-(CH₂)₂CHCl₂、-(CH₂O)₂(CH₂)₂OH、

R in the formula II-C₃Is- (CH)₂)₂SH；

R in the formula II-D₄is-C₂H₅、-(CH₂)₂SCH₃、-OCH₂CH(CH₃)₂、

R in the formula II-E₅Is composed of

The compound prepared by the invention is preferably any one or more of the following compounds 1-31:

compound 1: 5- ((3-chlorophenyl) amino) benzo [ c ] [2,6] naphthyridine-8-carbohydrazide

Compound 2: n-hydroxy-5- ((3-chlorophenyl) amino) benzo [ c ] [2,6] naphthyridine-8-carboxamide

Chemical 3: n-methanesulfonyl-5- ((3-chlorophenyl) amino) benzo [ c ] [2,6] naphthyridine-8-carboxamide

Compound 4: n- (2-hydroxyethyl) -5- ((3-chlorophenyl) amino) benzo [ c ] [2,6] naphthyridine-8-carboxamide

Compound 5: n- (2-aminoethyl) -5- ((3-chlorophenyl) amino) benzo [ c ] [2,6] naphthyridine-8-carboxamide

Compound 6: n- (2-bromoethyl) -5- ((3-chlorophenyl) amino) benzo [ c ] [2,6] naphthyridine-8-carboxamide

Compound 7: 3- (5- ((3-chlorophenyl) amino) benzo [ c ] [2,6] naphthyridine-8-amido) propionic acid methyl ester

Compound 8: 3- (5- ((3-chlorophenyl) amino) benzo [ c ] [2,6] naphthyridine-8-amido) propanoic acid

Compound 9: n- (2- (dimethylamino) ethyl) -5- ((3-chlorophenyl) amino) benzo [ c ] [2,6] naphthyridine-8-carboxamide

Compound 10: n- (2, 2-difluoroethyl) -5- ((3-chlorophenyl) amino) benzo [ c ] [2,6] naphthyridine-8-carboxamide

Compound 11: n- (2-propyl-1-yne) -5- ((3-chlorophenyl) amino) benzo [ c ] [2,6] naphthyridine-8-carboxamide

Compound 12: n- (3-hydroxypropyl) -5- ((3-chlorophenyl) amino) benzo [ c ] [2,6] naphthyridine-8-carboxamide

Compound 13: n- (3-aminopropyl) -5- ((3-chlorophenyl) amino) benzo [ c ] [2,6] naphthyridine-8-carboxamide

Compound 14: 4- (5- ((3-chlorophenyl) amino) benzo [ c ] [2,6] naphthyridine-8-amido) butanoic acid methyl ester

Compound 15: 4- (5- ((3-chlorophenyl) amino) benzo [ c ] [2,6] naphthyridine-8-amido) butanoic acid

Compound 16: n- (2-Morpholinoethyl) -5- ((3-chlorophenyl) amino) benzo [ c ] [2,6] naphthyridine-8-carboxamide

Compound 17: n- (2- (1-pyrrolidinyl) ethyl) -5- ((3-chlorophenyl) amino) benzo [ c ] [2,6] naphthyridine-8-carboxamide

Compound 18: n- (1-piperidinyl) ethyl) -5- ((3-chlorophenyl) amino) benzo [ c ] [2,6] naphthyridine-8-carboxamide

Compound 19: 2-hydroxyethyl-5- ((3-chlorophenyl) amino) benzo [ c ] [2,6] naphthyridine-8-carboxylate

Compound 20: 2-fluoroethyl-5- ((3-chlorophenyl) amino) benzo [ c ] [2,6] naphthyridine-8-carboxylate

Compound 21: 2-iodoethyl-5- ((3-chlorophenyl) amino) benzo [ c ] [2,6] naphthyridine-8-carboxylate

Compound 22: 2, 2-Difluoroethyl-5- ((3-chlorophenyl) amino) benzo [ c ] [2,6] naphthyridine-8-carboxylate

Compound 23: 3, 3-Dichloropropyl-5- ((3-chlorophenyl) amino) benzo [ c ] [2,6] naphthyridine-8-carboxylate

Compound 24: 2- (2- (2-Hydroxyethoxy) ethoxy) ethyl-5- ((3-chlorophenyl) amino) benzo [ c ] [2,6] naphthyridine-8-carboxylate

Compound 25: 2- (2-piperidinyl) ethyl-5- ((3-chlorophenyl) amino) benzo [ c ] [2,6] naphthyridine-8-carboxylate

Compound 26: s- (2-mercaptoethanol-based) -5- ((3-chlorophenyl) amino) benzo [ c ] [2,6] naphthyridine-8-carboxylic acid thioester

The reaction of a compound with 27: n' -propionyl-5- ((3-chlorophenyl) amino) benzo [ c ] [2,6] naphthyridine-8-carbohydrazide

Compound 28: n' - (3- (methylthio) chloropropionyl) -5- ((3-chlorophenyl) amino) benzo [ c ] [2,6] naphthyridine-8-carbohydrazide

Compound 29: isobutyl 2- (5- ((3-chlorophenyl) amino) benzo [ c ] [2,6] naphthyridine-8-carbonyl) hydrazine-1-carboxylate

Compound 30: n' - (5- ((3-chlorophenyl) amino) benzo [ c ] [2,6] naphthyridine-8-carbonyl) -3-morpholinopropane-1-sulfonylhydrazide

Compound 31: n' - (5- (3-1, 2-dithiolanyl) pentanol) -5- ((3-chlorophenyl) amino) benzo [2,6] naphthyridine-8-carbohydrazide

The synthesis method of the compound is mainly synthesized by using CX-4945 or a precursor thereof as a raw material.

The synthesis method of CX-4945 reference literature comprises the steps of firstly using 3-bromoisonicotinic acid as an initial raw material, carrying out esterification reaction, carrying out Suzuki coupling reaction with 2-amino-4-carbomethoxyphenyl borate to obtain an intermediate 5, 6-dihydrobenzo [ c ] [2,6] naphthyridine-8-carboxylic acid methyl ester, carrying out chlorination reaction with phosphorus oxychloride, carrying out substitution reaction with m-chloroaniline, and finally hydrolyzing to obtain a product. The specific synthetic route is as follows:

a.EtOH,H₂SO₄,reflux,24～48h；b.Cs₂CO₃,Pd(dppf)Cl₂,N₂,100℃,30～48h；

c.POCl₃,110℃,4h；d.NMP,110℃,8h；e.1.5N NaOH,70℃。

the compound of formula II-A can be obtained by exchange reaction or condensation reaction, and the specific synthetic route is as follows:

the compound of formula II-B can be obtained by esterification, and the specific synthetic route is as follows:

the compound of formula II-C can be obtained by esterification of a thiol, and the specific synthetic route is as follows:

the compound of formula II-D can be obtained by amide condensation reaction of hydrazide and carboxylic acid, and the specific synthetic route is as follows:

the compound of formula II-E can be obtained by amide condensation reaction of hydrazide and sulfonic acid, and the specific synthetic route is as follows:

wherein R is₁、R₂、R₃、R₄、R₅As mentioned above, HATU stands for condensing agent 2- (7-benzotriazole oxide) -N, N, N ', N' -tetramethyluronium hexafluorophosphate, TBTU stands for condensing agent O-benzotriazole-N, N, N ', N' -tetramethyluronium tetrafluoroborate, DIPEA stands for N, N-diisopropylethylamine, and DMF stands for solvent N, N-dimethylformamideAmine, Reflux for Reflux, r.t. for room temperature.

Has the advantages that:

(I) in vitro antitumor Activity and enzymatic Activity of Compounds

The cytotoxic activity of the compounds of the present invention against various cancer cells and normal cells was determined, and their inhibitory activity against CK2 and CLK2 proteins was examined. As can be seen from table 1 and fig. 1:

(1) compared with CX-4945, the cytotoxic activity of the compound 1 on cancer cells, the inhibition capability on CK2 protein kinase and the selectivity on CK2 protein, which are obtained by introducing hydrazino groups into CX-4945 parent skeletons through amido bonds, are improved; when hydroxylamine is introduced, the obtained compound 2 has higher cytotoxic activity but poorer enzymatic activity; when the methanesulfonamide group was introduced, the obtained compound 3 had a reduced cytotoxic activity and enzyme activity as compared with CX-4945. Thus, introduction of a hydrogen bond donor of a smaller group through an amide bond can increase the cytotoxic activity, enzymatic activity and selectivity of the parent compound for the CK2 protein.

(2) The cytotoxic activity of compounds (e.g. compounds 4, 5, 7, 9) incorporating a C2 fatty chain via an amide bond in the CX-4945 parent backbone is generally higher than that of compounds incorporating a C3 fatty chain (e.g. compounds 11, 12, 13, 14, 15), and the activity of the terminal group of the C2 fatty chain being a hydroxyl group is the best. For example, the compound 4 has higher cytotoxic activity, enzyme activity and selectivity to CK2 protein compared with CX-4945, and simultaneously reduces the toxicity to normal liver cells LO 2; when the amino group is used, although the activity of the related compound on CK2 is improved, the cytotoxic activity and the selectivity on CK2 protein are poor. In addition, compounds obtained by introducing an N-containing heterocyclic ring through a C2 aliphatic chain (compounds 16, 17 and 18), such as a morpholine ring, a pyrrole ring and a piperidine ring, can improve the cytotoxic activity of the compounds, wherein the compounds obtained by introducing the morpholine ring have higher cytotoxic activity, enzyme activity and selectivity on CK2 protein. Therefore, when a C2 fatty chain is introduced into the CX-4945 carboxyl terminal through an amido bond and the fatty chain terminal contains a group capable of providing a hydrogen bond acceptor, the cytotoxic activity, the enzymatic activity and the selectivity to CK2 protein of the obtained compound are high.

(3) Compounds in which the C2 fatty chain is introduced via an ester bond at the end of CX-4945 carboxylic acid generally enhance the cytotoxic activity of the parent compound. When the terminal group of the introduced C2 aliphatic chain is smaller and can be used as a hydrogen bond acceptor, the cytotoxic activity, the enzymatic activity and the selectivity to CK2 protein of the obtained compound are better. For example, in compound 20, the C2 fatty chain ends with a fluorine atom having a small atomic radius and a strong electronegativity, which can act as a hydrogen bond acceptor, and the activity is significantly superior to that of the parent compound and other compounds. The compound 19, in which the end of the C2 fatty chain is hydroxyl, retains the original cytotoxic activity of the parent compound and increases the activity and selectivity on CK2 protein. Similarly, the inhibiting activity and selectivity of the compound on CK2 protein can be increased by introducing a pyridine ring into a C2 aliphatic chain. The sulfur atom radius is larger than that of oxygen atom, and the thioester bond is less stable than the oxygen ester bond, so that the compound (compound 26) obtained by introducing thiol through the thioester bond only retains the cytotoxic activity of the original parent compound, but has poor enzymatic activity.

(4) The cytotoxicity and the enzyme activity of the compounds (compounds 26, 27, 28, 29, 30 and 31) obtained by structurally modifying a hydrazide group as an active group on the basis of the compound 1 are general.

(II) ability of Compound to inhibit cancer cell dryness

(1) The inhibition ability of part of compounds of the invention on the siccatness of HCT-116 primary cancer cells is detected by a flow cytometer, and the data in Table 2 show that the detected compounds can effectively inhibit the activity of ALDH1 in HCT-116 primary cancer cells, and are obviously superior to CX-4945, and the inhibition rates of

compounds

4 and 20 on ALDH1 are higher than that of BBI608 which is a cancer cell siccatness inhibitor.

(2) The expression of the stem cell markers CD44 and CD133 in HCT-116 primary cancer cells was determined for

compounds

4 and 20. As can be seen from the flow analysis result of FIG. 2, after HCT-116 primary cancer cells are incubated with the sample for 24h, the inhibition rates of

compounds

4 and 20 on the stem cell marker CD44/CD133 reach 86.97% and 76.05%, respectively, which are obviously higher than 56.70% of compound CX-4945, especially the inhibition rate of compound 4 is higher than BBI608 (82.26%) which is a cancer cell dryness inhibitor, and the compound shows strong cancer cell dryness inhibition activity.

(3) Related genes in stem cell inhibition pathways Wnt/beta-catenin and Hedgehog of

compounds

4 and 20 were analyzed by qRT-PCR. As shown in FIG. 3, the compound 4 can better promote the expression of DKK1 gene in Wnt signaling pathway in HCT-116 primary cancer cells, simultaneously inhibit downstream beta-catenin gene, further inhibit the Wnt signaling pathway, and has the inhibition effect obviously superior to that of the compound 20 and far superior to that of CX-4945. In the Hedgehog signaling pathway, compound 4 can inhibit the upstream PTCH1 gene and the downstream Gli1 gene simultaneously, so that the Hedgehog signaling pathway is inhibited, and the inhibition effect is better than that of compound 20 and CX-4945. Compound 20 also inhibited the Wnt signaling pathway and the Hedgehog signaling pathway better than CX-4945. The compound 4 has higher inhibition on stem cell related genes OCT4, SOX2 and Nanog than CX-4945 and BBI608, while the compound 20 has the same inhibition on sternness gene as BBI608 but better inhibition on CX-4945. These results indicate that

compounds

4 and 20 can inhibit the cancer cell sternness through the Wnt signaling pathway and the Hedgehog signaling pathway, and the inhibition abilities of both are obviously better than those of the parent compound CX-4945, wherein the inhibition of compound 4 on the cancer cell sternness is even better than that of the cancer cell sternness inhibitor BBI 608.

In conclusion, a series of compounds with higher CK2 inhibition activity than CX-4945, such as

compounds

1, 4, 5, 7, 12, 16, 19, 20, 21 and 25, are obtained by introducing different active groups at the tail end of a CX-4945 parent structure. Among them, compounds 1, 4, 16, 19, 20, 21 and 25 have less inhibitory activity against CLK2 than CX-4945, and have better selectivity, especially for compound 20, with a selectivity factor as high as 5.28. Most of the tested compounds have good inhibition effect on the proliferation of 5 kinds of cancer cells, which is better than or equal to CX-4945, and the toxicity to normal liver cells LO2 is mostly lower than that of the parent compound. Through the determination of the inhibition of cancer cell dryness by 7 compounds with CK2 selectivity higher than CLK2, the

compounds

4 and 20 are found to be effective in inhibiting the dryness of primary cancer cells, and the activity of the compounds is even higher than that of a known cancer cell dryness inhibitor BBI 608.

Research shows that the compound of the present invention has obvious anticancer activity, partial compound has cytotoxic activity to cancer cell, inhibition to CK2 protein kinase and selectivity to CK2 protein superior to that of the parent compound, and may be used in preparing antitumor medicine.

Drawings

FIG. 1 shows the inhibitory activity of some compounds on CK2 and CLK2 protein kinases.

FIG. 2. inhibition of the stem cell marker CD44/CD133 in HCT-116 primary cancer cells by representative compounds, (a) flow cytogram of samples, (b) inhibition rate of samples.

FIG. 3. Effect of representative compounds on Wnt and Hedgehog signaling pathways and expression levels of stem cell-associated genes.

Detailed Description

The present invention will be described in further detail with reference to the following examples, which are not intended to limit the scope of the invention.

The invention provides a novel compound shown as a formula II by introducing an amino-group, hydroxyl, sulfydryl or halogen atom-containing aliphatic chain or heterocyclic structure fragment in a known CK2 inhibitor (CX-4945) parent structure in the modes of an amido bond, an ester bond and a thioester bond;

compounds of the structures shown in formulas II-A, II-B, II-C, II-D and II-E:

wherein:

R in the formula II-C₃Is- (CH)₂)₂SH；

R in the formula II-E₅Is composed of

All reagents in the following examples were analytically pure. Nuclear magnetic data used for structural characterization of the compounds are measured by a Bruker ARX-600 nuclear magnetic resonance apparatus, and the internal standard is TMS; high resolution mass spectra were determined using an Agilent6224 TOF LC/MS instrument.

In the invention, the known compound CX-4945 and the precursor compound thereof are prepared by the method reported in the references (J.Med.chem.2011, 54, 635-654), and are verified by nuclear magnetic hydrogen spectroscopy.

CX-4945：¹H NMR(600MHz,DMSO-d₆)δ12.04(s,1H),10.30(s,1H),10.16(s,1H),9.08(d,J＝5.8Hz,1H),9.00-8.97(m,1H),8.94(d,J＝8.5Hz,1H),8.31-8.28(m,2H),8.12(d,J＝8.2Hz,1H),8.03(dd,J＝8.4,1.5Hz,1H),7.45(t,J＝8.1Hz,1H),7.17(d,J＝7.9Hz,1H)ppm.

CX-4945 precursor:¹H NMR(600MHz,DMSO-d₆)δ10.09(s,1H),9.91(s,1H),8.94(d,J＝5.6Hz,1H),8.79(d,J＝8.5Hz,1H),8.77(d,J＝5.6Hz,1H),8.34(t,J＝1.9Hz,1H),8.17(dd,J＝6.3,1.4Hz,1H),7.90(dd,J＝8.4,1.7Hz,1H),7.42(t,J＝8.1Hz,1H),7.12(dd,J＝7.9,1.4Hz,1H),3.92(s,1H)ppm.

(I) preparation of Compounds

Example 1: preparation of Compound 1

A50 mL single vial was charged with 1.09g (3mmol) of CX-4945 precursor and 20mL of methanol, and 0.15g (3mmol) of hydrazine hydrate was slowly added dropwise at room temperature, followed by heating and refluxing for 72h to complete the reaction. Cooling, precipitating a large amount of solid, filtering, washing a filter cake by using 3X 10mL of ice methanol to obtain 1.05g of bright yellow solid product with the yield of 97.1%.¹H NMR(600MHz,DMSO-d₆)δ10.14(s,1H),10.08(s,1H),9.63(s,1H),8.95(d,J＝6.0Hz,1H),8.80(d,J＝12.0Hz,1H),8.55(d,J＝6.0Hz,1H),8.33(t,J＝1.8Hz,1H),8.21(d,J＝1.6Hz,1H),8.07(d,J＝6.0Hz,1H),7.91(dd,J＝12.0,1.6Hz,1H),7.42(t,J＝6.0Hz,1H),7.12(dd,J＝8.0,1.4Hz,1H),4.63(s,2H)ppm.¹³C NMR(150MHz,DMSO-d₆)δ165.77,150.47,148.13,147.69,143.76,142.40,134.73,133.35,130.52,127.52,126.26,124.17,123.00,122.83,122.60,121.47,120.57,119.56,116.79ppm.HR-MS(m/z)(ESI):calcd for C₁₉H₁₄ClN₅O[M+H]⁺:364.0965；found:364.0967.

Example 2: preparation of Compound 2

Adding NH into a 200mL single-mouth bottle₂OH HCl 29.17g (420mmol) and 150mL methanol. Dissolving KOH35.2g (627mmol) in 75mL of methanol, adding dropwise the above solution at 0 deg.C, stirring at 0 deg.C for 30min, filtering, and removing insoluble solid to obtain methanol solution containing hydroxylamine. In a 30mL single vial, 0.36g (1mmol) of CX-4945 precursor and 10mL of the above-prepared methanol solution containing hydroxylamine were added, stirred at room temperature for 1h, TLC detected to complete reaction, the solvent was spun off, 10mL of water was added to dissolve the solid, and a yellow solid was precipitated by adjusting the pH of the solution to 7 with 2M HCl. And (3) carrying out suction filtration, washing a filter cake for three times by using 3X 10mL of deionized water to obtain a crude product, and recrystallizing to obtain 0.332g of a yellow solid product with the yield of 91.2%.¹H NMR(600MHz,DMSO-d₆)δ11.52(s,1H),10.16(s,1H),9.65(s,1H),9.19(s,1H),8.97(d,J＝5.4Hz,1H),8.83(d,J＝8.4Hz,1H),8.57(d,J＝5.2Hz,1H),8.33(s,1H),8.15(d,J＝1.2Hz,1H),8.07(d,J＝8.0Hz,1H),7.85(d,J＝1.2Hz,1H),7.43(t,J＝8.0Hz,1H),7.13(dd,J＝7.8,1.2Hz,1H)ppm.¹³C NMR(150MHz,DMSO-d₆)δ164.19,150.52,148.13,147.73,143.74,142.35,134.16,133.27,130.53,127.49,126.08,124.19,122.95,122.90,122.64,121.54,120.60,119.60,116.81ppm.HR-MS(m/z)(ESI):calcd for C₁₉H₁₃ClN₄O₂[M+H]⁺:365.0805；found:365.0800.

Example 3: preparation of Compound 3

CX-49450.174 g (0.5mmol), methanesulfonamide 0.048g (0.5mmol), HATU0.228g (0.6mmol), DIPEA 0.129g (1mmol) and anhydrous DMF 5mL were added to a 10mL single-neck flask, respectively, and stirred at 45 ℃ for 24h, monitored by TLC until the reaction was complete, the solvent was dried in vacuo, purified by silica gel column chromatography, and eluted with dichloromethane and methanol (100:1-50:1) to give 0.093g of a yellow solid product in 43.5% yield.¹H NMR(600MHz,DMSO-d₆)δ13.26(s,1H),10.19(s,1H),9.69(s,1H),9.01(d,J＝5.5Hz,1H),8.87(d,J＝8.4Hz,1H),8.60(d,J＝5.6Hz,1H),8.33(s,1H),8.28(s,1H),8.10(d,J＝8.2Hz,1H),7.99(d,J＝8.1Hz,1H),7.45(t,J＝8.1Hz,1H),7.16-7.13(d,J＝7.8Hz,1H),2.51(s,3H)ppm.¹³C NMR(150MHz,DMSO-d₆)δ150.71,150.60,148.30,148.09,143.76,142.33,133.24,130.62,128.72,127.42,124.70,124.47,123.13,123.08,122.80,122.67,120.57,119.61,116.86,63.26ppm.HR-MS(m/z)(ESI):calcd for C₂₀H₁₅ClN₄O₃S[M+H]⁺:427.0631；found:427.0643.

Example 4: preparation of Compound 4

Prepared using CX-4945 and 2-aminoethanol as described in reference example 3, to give the product as a yellow solid in 72.1% yield.¹H NMR(600MHz,DMSO-d₆)δ10.18(s,1H),9.89(s,1H),8.97(d,J＝5.4Hz,1H),8.84(t,J＝6.0Hz,2H),8.77(d,J＝5.4Hz,1H),8.35(s,1H),8.27(s,1H),8.21(d,J＝8.2Hz,1H),8.00(d,J＝8.2Hz,1H),7.45(t,J＝8.0Hz,1H),7.14(d,J＝7.6Hz,1H),4.91(t,J＝5.6Hz,1H),3.61(q,J＝5.8Hz,2H),3.43(dd,J＝11.2,5.4Hz,2H)ppm.¹³C NMR(150MHz,DMSO-d₆)δ166.40,150.61,148.05,147.64,143.77,142.51,135.92,133.19,130.46,127.52,126.40,124.25,123.38,122.71,122.56,121.46,120.70,119.73,117.16,60.20,42.88ppm.HR-MS(m/z)(ESI):calcd for C₂₁H₁₇ClN₄O₂[M+H]⁺:393.1118；found:393.1120.

Example 5: preparation of Compound 5

Prepared using CX-4945 and ethylenediamine, according to the method described in example 3, to give the product as a yellow solid with a yield of 81.6%.¹H NMR(600MHz,DMSO-d₆)δ10.12(s,1H),9.60(s,1H),8.95(d,J＝5.4Hz,1H),8.81(dd,J＝11.2,7.0Hz,2H),8.53(d,J＝5.6Hz,1H),8.24(t,J＝1.8Hz,1H),8.20(d,J＝1.4Hz,1H),8.09(d,J＝8.2Hz,1H),7.93(dd,J＝8.2,1.4Hz,1H),7.42(t,J＝8.2Hz,1H),7.11(d,J＝7.8Hz,1H),3.45(dd,J＝12.6,6.4Hz,2H),3.35(m,2H),1.94-1.88(m,2H)ppm.¹³C NMR(150MHz,DMSO-d₆)δ166.29,150.46,148.07,147.66,143.75,142.37,135.96,133.26,130.52,127.48,126.25,124.13,123.30,122.74,122.62,121.45,120.65,119.65,116.78,37.98,29.60ppm.HR-MS(m/z)(ESI):calcd for C₂₁H₁₈ClN₅O[M+H]⁺:393.1278；found:393.1280.

Example 6: preparation of Compound 6

A10 mL single-neck flask was charged with CX-49450.174 g (0.5mmol), 2-bromoethylamine hydrobromide 0.102g (0.5mmol), HATU0.228g (0.6mmol), DIPEA 0.194g (1.5mmol) and anhydrous DMF 5mL, stirred at 50 deg.C for 24h, TLC monitored for completion of the reaction, the solvent was concentrated, purified by silica gel column chromatography using dichloromethane and methanol (100:1-50:1) as eluents to give 0.139g of yellow solid product in 61.4% yield.¹H NMR(600MHz,DMSO-d₆)δ10.20(s,1H),9.67(s,1H),9.12(t,J＝5.0Hz,1H),9.00(d,J＝5.4Hz,1H),8.82(d,J＝3.8Hz,1H),8.59(d,J＝5.4Hz,1H),8.29(s,1H),8.28(s,1H),8.13(d,J＝8.0Hz,1H),7.97(d,J＝8.2Hz,1H),7.45(t,J＝8.0Hz,1H),7.15(d,J＝7.6Hz,1H),4.85(t,J＝5.2Hz,2H),3.88-3.84(m,2H)ppm.¹³C NMR(150MHz,DMSO-d₆)δ166.69,152.29,150.61,148.16,147.77,143.79,142.37,135.05,133.28,130.55,127.50,126.41,124.25,123.36,122.87,122.71,120.76,119.76,116.84,79.74,38.61ppm.HR-MS(m/z)(ESI):calcd for C₂₁H₁₆BrClN₄O[M+H]⁺:455.0274；found:455.0272.

Example 7: preparation of Compound 7

Prepared using CX-4945 and beta-alanine methyl ester hydrochloride as described in reference example 6 to give the product as a pale yellow solid with a yield of 65.4%.¹H NMR(600MHz,DMSO-d₆)δ10.18(s,1H),9.66(s,1H),8.99(d,J＝5.6Hz,1H),8.88(t,J＝5.4Hz,1H),8.85(d,J＝8.4Hz,1H),8.58(d,J＝5.6Hz,1H),8.28(t,J＝1.8Hz,1H),8.22(d,J＝1.4Hz,1H),8.10(dd,J＝8.2,1.2Hz,1H),7.93(dd,J＝8.4,1.6Hz,1H),7.45(t,J＝8.2Hz,1H),7.15(dd,J＝7.8,1.2Hz,1H),3.65(s,3H),3.58(dd,J＝12.6,6.8Hz,2H),2.68(t,J＝7.0Hz,2H)ppm.¹³C NMR(150MHz,DMSO-d₆)δ172.28,166.39,150.55,148.15,147.73,143.76,142.37,135.72,133.27,130.55,127.51,126.31,124.21,123.33,122.81,122.68,121.58,120.71,119.71,116.82,51.89,36.13,34.02ppm.HR-MS(m/z)(ESI):calcd for C₁₉H₁₄ClN₅O[M+H]⁺:435.1224；found:435.1220.

Example 8: preparation of Compound 8

In a 30mL single-neck flask, 70.434 g (1mmol) of compound, LiOH. H₂O0.105 g (2.5mmol), methanol 10mL and pure water 5mL, and stirred at 60 ℃ overnight. The solvent was spun dry to give a yellow solid, 30mL of water was added to dissolve the solid, the pH of the solution was adjusted to 5 with 1M HCl to precipitate a yellow solid, which was filtered, washed with water (3X 10mL), and dried to give 0.39g of a yellow solid product in 91.0% yield.¹H NMR(600MHz,DMSO-d₆)δ12.37(s,1H),10.15(s,1H),9.63(s,1H),8.96(s,1H),8.84(s,1H),8.81(d,J＝7.4Hz,1H),8.55(s,1H),8.24(t,J＝1.8Hz,1H),8.21(d,J＝1.2Hz,1H),8.09(d,J＝6.8Hz,1H),7.92(dd,J＝7.2,1.8Hz,1H),7.42(d,J＝6.8Hz,1H),7.13(d,J＝6.4Hz,1H),3.54(dd,J＝10.6,5.8Hz,2H),2.58(t,J＝6.4Hz,2H)ppm.¹³C NMR(150MHz,DMSO)δ173.63,166.29,150.52,148.12,147.69,143.75,142.37,135.77,133.27,130.54,127.50,126.30,124.18,123.32,122.76,122.65,121.53,120.68,119.68,116.80,36.31,34.40ppm.HR-MS(m/z)(ESI):calcd for C₂₂H₁₇ClN₄O₃[M+H]⁺:421.1067；found:421.1060.

Example 9: preparation of Compound 9

Prepared using CX-4945 and N, N-dimethylethylenediamine according to the method described in example 3 to give the product as a yellow solid in 69.8% yield.¹H NMR(600MHz,DMSO-d₆)δ10.18(s,1H),9.67(s,1H),8.98(d,J＝5.6Hz,1H),8.84(d,J＝8.4Hz,1H),8.72(t,J＝5.4Hz,1H),8.59(d,J＝5.6Hz,1H),8.28(t,J＝1.8Hz,1H),8.23(d,J＝1.4Hz,1H),8.11(dd,J＝8.4,1.0Hz,1H),7.94(dd,J＝8.4,1.6Hz,1H),7.45(t,J＝8.2Hz,1H),7.15(dd,J＝7.8,1.4Hz,1H),3.45(dd,J＝12.8,6.6Hz,2H),2.53-2.51(m,2H),2.25(s,6H)ppm.¹³C NMR(150MHz,DMSO-d₆)δ166.19,150.54,148.13,147.70,143.77,142.39,135.94,133.27,130.54,127.53,126.29,124.20,123.35,122.76,122.66,121.50,120.71,119.70,116.83,58.59,45.64,37.92ppm.HR-MS(m/z)(ESI):calcd for C₂₃H₂₂ClN₅O[M+H]⁺:420.1591；found:420.1595.

Example 10: preparation of Compound 10

Prepared as described in example 3 using CX-4945 and 2, 2-difluoroethylamine to give the product as a yellow solid in 61.8% yield.¹H NMR(600MHz,DMSO-d₆)δ10.18(s,1H),9.99(s,1H),9.28(s,1H),8.96(d,J＝4.6Hz,1H),8.86(d,J＝8.2Hz,1H),8.83(d,J＝4.6Hz,1H),8.37(s,1H),8.29(s,1H),8.22(d,J＝7.4Hz,1H),8.01(d,J＝7.8Hz,1H),7.44(t,J＝7.8Hz,1H),7.14(d,J＝7.2Hz,1H),6.23(t,J＝56.2Hz,1H),3.77(t,J＝14.8Hz,2H)ppm.¹³C NMR(150MHz,DMSO-d₆)δ166.98,150.76,148.05,147.77,143.80,142.50,134.89,133.17,130.42,127.44,126.57,124.37,123.31,122.90,122.59,121.87,120.80,119.82,117.29,115.09(t,J＝240.2Hz),42.25(t,J＝26.6Hz)ppm.HR-MS(m/z)(ESI):calcd for C₂₁H₁₅ClF₂N₄O[M+H]⁺:413.0980；found:413.0967.

Example 11: preparation of Compound 11

Prepared as described in CX-4945 and propargylamine with reference to example 3, to give the product as a yellow solid in 76.4% yield.¹H NMR(600MHz,DMSO-d₆)δ10.17(s,1H),9.96(s,1H),9.36(t,J＝5.2Hz,1H),8.96(d,J＝5.4Hz,1H),8.85(d,J＝8.4Hz,1H),8.82(d,J＝5.5Hz,1H),8.40(s,1H),8.28(s,1H),8.23(d,J＝8.2Hz,1H),8.00(d,J＝8.3Hz,1H),7.45(t,J＝8.1Hz,1H),7.15(d,J＝7.8Hz,1H),4.18(d,J＝3.2Hz,2H),3.21(s,1H)ppm.¹³C NMR(150MHz,DMSO-d₆)δ166.06,150.67,148.01,147.69,143.78,142.50,135.11,133.17,130.40,128.23,127.42,126.52,124.31,123.26,122.83,122.54,121.73,120.71,119.74,117.23,81.83,73.37,29.12ppm.HR-MS(m/z)(ESI):calcd for C₂₂H₁₅ClN₄O[M+H]⁺:387.1012；found:387.1011.

Example 12: preparation of Compound 12

Prepared as described in reference to example 3 using CX-4945 and 3-amino-1-propanol to give the product as a yellow solid in 79.8% yield.¹H NMR(600MHz,DMSO-d₆)δ10.19(s,1H),9.67(s,1H),8.99(d,J＝5.6Hz,1H),8.85(d,J＝8.4Hz,1H),8.77(t,J＝5.6Hz,1H),8.59(d,J＝5.6Hz,1H),8.28(s,1H),8.23(s,1H),8.12(dd,J＝8.2,1.0Hz,1H),7.95(dd,J＝8.4,1.6Hz,1H),7.45(t,J＝8.2Hz,1H),7.15(dd,J＝7.8,1.2Hz,1H),4.54(t,J＝5.2Hz,1H),3.52(q,J＝6.2Hz,2H),3.40(dd,J＝12.8,7.0Hz,2H),1.75(p,J＝6.6Hz,2H)ppm.¹³C NMR(150MHz,DMSO-d₆)δ166.27,150.53,148.15,147.70,143.78,142.39,136.06,133.27,130.56,127.54,126.27,124.19,123.35,122.77,122.66,121.46,120.68,119.69,116.84,59.13,37.22,32.92ppm.HR-MS(m/z)(ESI):calcd for C₂₂H₁₉ClN₄O₂[M+H]⁺:407.1269；found:407.1264.

Example 13: preparation of Compound 13

Prepared as described in example 3 using CX-4945 and 1, 3-diaminopropane to give the product as a yellow solid in 57.3% yield.¹H NMR(600MHz,DMSO-d₆)δ10.19(s,1H),10.01(s,1H),8.96(d,J＝5.6Hz,1H),8.91(t,J＝5.4Hz,1H),8.87(d,J＝3.8Hz,1H),8.38(s,1H),8.25-8.21(m,2H),8.04(s,1H),7.99(d,J＝8.4Hz,1H),7.44(t,J＝8.2Hz,1H),7.14(d,J＝7.8Hz,1H),3.36(dd,J＝12.6,6.4Hz,2H),3.18(dd,J＝12.8,6.2Hz,2H),1.76-1.70(m,2H),1.22(s,2H)ppm.¹³C NMR(150MHz,DMSO-d₆)δ166.25,161.58,150.71,148.07,147.68,143.82,142.56,135.91,133.16,130.41,127.55,126.34,124.30,123.21,122.79,122.56,120.77,119.82,117.38,37.53,35.51,29.61ppm.HR-MS(m/z)(ESI):calcd for C₂₂H₂₀ClN₅O[M+H]⁺:406.1434；found:406.1431.

Example 14: preparation of Compound 14

Prepared by CX-4945 and methyl 4-aminobutyrate hydrochloride according to the method described in example 3, and gives 0.147g of an orange solid product with a yield of 65.8%.¹H NMR(600MHz,DMSO-d₆)δ10.17(s,1H),9.94(s,1H),8.95(d,J＝6.0Hz,1H),8.87-8.83(m,2H),8.81(d,J＝5.0Hz,1H),8.34(s,1H),8.22(s,1H),8.19(d,J＝8.0Hz,1H),7.95(d,J＝7.8Hz,1H),7.43(t,J＝7.8Hz,1H),7.13(d,J＝7.4Hz,1H),3.59(s,3H),3.35(dd,J＝12.6,6.8Hz,2H),2.41(t,J＝6.8Hz,2H),1.87-1.81(m,2H)ppm.¹³C NMR(150MHz,DMSO-d₆)δ173.68,166.36,150.69,148.03,147.66,143.79,142.52,135.91,133.17,130.46,127.54,126.30,124.29,123.35,122.76,122.59,121.49,120.78,119.81,117.30,51.75,39.15,31.36,24.94ppm.HR-MS(m/z)(ESI):calcd for C₂₄H₂₁ClN₄O₃[M+H]⁺:449.1380；found:449.1386.

Example 15: preparation of Compound 15

Prepared according to the method described in example 8 to give the product as a yellow solid in 89.6% yield.¹H NMR(600MHz,DMSO-d₆)δ12.43(s,1H),10.18(s,1H),9.66(d,J＝5.3Hz,1H),8.99(d,J＝5.4Hz,1H),8.84(d,J＝8.4Hz,1H),8.81(t,J＝6.4Hz,1H),8.58(d,J＝5.4Hz,1H),8.27(s,1H),8.24(s,1H),8.13(d,J＝8.5Hz,1H),7.96(d,J＝8.3Hz,1H),7.46(t,J＝8.1Hz,1H),7.15(d,J＝7.5Hz,1H),3.38(dd,J＝12.6,6.5Hz,2H),2.35(t,J＝7.3Hz,2H),1.87-1.81(m,2H)ppm.¹³C NMR(150MHz,DMSO-d₆)δ166.06,150.67,148.01,143.78,142.50,135.11,133.17,130.40,128.23,127.42,126.52,124.31,123.26,122.83,122.54,121.73,120.71,119.74,117.23,81.83,73.37,29.12ppm.HR-MS(m/z)(ESI):calcd for C₂₃H₁₉ClN₄O₃[M+H]⁺:435.1224；found:435.1221.

Example 16: preparation of Compound 16

Prepared as described in example 3 using CX-4945 and 4- (2-aminoethyl) morpholine to give the product as a yellow solid in 68.7% yield.¹H NMR(600MHz,DMSO-d₆)δ10.16(s,1H),9.98(s,1H),8.94(d,J＝5.6Hz,1H),8.84(d,J＝5.2Hz,2H),8.82(s,1H),8.38(s,1H),8.22(s,1H),8.19(d,J＝8.3Hz,1H),7.96(d,J＝8.2Hz,1H),7.42(t,J＝8.1Hz,1H),7.12(dd,J＝7.8,0.9Hz,1H),3.62(m,4H),3.49(m,2H),3.38(m,4H),2.61(m,2H)ppm.¹³C NMR(150MHz,DMSO-d₆)δ166.30,150.71,148.04,147.67,143.80,142.56,135.89,133.16,130.42,127.53,126.36,124.31,123.32,122.79,122.55,121.53,120.79,119.81,117.37,66.36,63.29,57.60,53.54ppm.HR-MS(m/z)(ESI):calcd for C₂₅H₂₄ClN₅O₂[M+H]⁺:462.1697；found:462.1695.

Example 17: preparation of Compound 17

Prepared using CX-4945 and N- (2-aminoethyl) pyrrolidine, according to the method described in example 3, to give the product as a yellow solid in 72.8% yield.¹H NMR(600MHz,DMSO-d₆)δ10.19(s,1H),9.69(s,1H),9.00(d,J＝5.6Hz,1H),8.97(d,J＝5.2Hz,1H),8.88(d,J＝8.4Hz,1H),8.58(d,J＝5.6Hz,1H),8.26(d,J＝4.2Hz,2H),8.05(d,J＝8.0Hz,1H),7.96(d,J＝8.2Hz,1H),7.44(t,J＝8.2Hz,1H),7.16(d,J＝7.8Hz,1H),3.66(dd,J＝10.8,4.8Hz,2H),3.37(m,6H),1.96(m,4H)ppm.¹³C NMR(150MHz,DMSO-d₆)δ166.99,150.73,148.20,147.90,143.79,142.32,135.29,133.29,130.59,127.49,126.43,124.32,123.43,122.92,122.83,121.85,120.90,119.89,116.88,54.06,53.91,36.50,23.00ppm.HR-MS(m/z)(ESI):calcd for C₂₅H₂₄ClN₅O[M+H]⁺:446.1747；found:446.1750.

Example 18: preparation of Compound 18

Prepared as described in example 3 using CX-4945 and N- (2-aminoethyl) piperidine to give the product as a yellow solid in 77.1% yield.¹H NMR(600MHz,DMSO-d₆)δ10.19(s,1H),9.69(s,1H),9.00(d,J＝5.4Hz,2H),8.88(d,J＝8.4Hz,1H),8.59(d,J＝5.6Hz,1H),8.27(s,1H),8.25(d,J＝0.8Hz,1H),8.05(d,J＝8.2Hz,1H),7.96(dd,J＝8.4,1.0Hz,1H),7.45(t,J＝8.2Hz,1H),7.16(dd,J＝7.8,1.2Hz,1H),3.70(dd,J＝11.8,5.8Hz,2H),3.59(t,J＝5.8Hz,2H),3.30(t,J＝6.2Hz,2H),2.97(t,J＝6.4Hz,2H),1.91-1.61(m,6H)ppm.¹³C NMR(150MHz,DMSO-d₆)δ167.00,151.59,148.20,147.90,143.79,142.31,135.24,133.28,130.59,129.32,126.43,124.32,123.39,122.96,122.83,121.19,120.89,119.88,116.88,55.62,52.81,34.77,22.99,21.68ppm.HR-MS(m/z)(ESI):calcd for C₂₆H₂₆ClN₅O[M+H]⁺:460.1904；found:460.1900.

Example 19: preparation of Compound 19

CX-49450.174 g (0.5mmol), TBTU 0.193g (0.6mmol) and anhydrous DMF 5mL are respectively added into a 10mL single-neck flask, stirred for 15min at 50 ℃, added with TEA 0.101g (1.0mmol), then stirred for 15min, added with glycol 0.064g (1mmol) and reacted for 24h, TLC monitors the reaction to be complete, the solvent is concentrated and purified by silica gel column chromatography, and the eluent is dichloromethane and methanol (100:1-50:1), thus obtaining yellow solid product 0.170g, and the yield is 86.5%.¹H NMR(600MHz,DMSO-d₆)δ10.17(s,1H),9.95(s,1H),8.99(d,J＝5.6Hz,1H),8.88(d,J＝8.5Hz,1H),8.79(d,J＝5.6Hz,1H),8.32(t,J＝2.0Hz,1H),8.27(d,J＝1.6Hz,1H),8.18(dd,J＝8.2,1.3Hz,1H),8.01(dd,J＝8.4,1.7Hz,1H),7.45(t,J＝8.1Hz,1H),7.15(dd,J＝7.9,1.4Hz,1H),5.12(t,J＝5.4Hz,1H),4.39-4.35(m,2H),3.78(dd,J＝9.4,4.8Hz,2H)ppm.¹³C NMR(150MHz,DMSO-d₆)¹³C NMR(151MHz,DMSO)δ166.18,150.85,148.21,148.16,143.76,142.34,133.15,131.11,130.52,128.59,127.27,124.62,124.41,123.23,123.15,122.71,120.79,119.85,117.26,67.36,59.52ppm.HR-MS(m/z)(ESI):calcd for C₂₁H₁₆ClN₃O₃[M+H]⁺:394.0958；found:394.0961.

Example 20: preparation of Compound 20

Prepared using CX-4945 and 2-fluoroethanol according to the procedure described in example 19 to give the product as a yellow solid in 82.8% yield.¹H NMR(600MHz,DMSO-d₆)δ10.11(s,1H),9.64(s,1H),8.97(d,J＝5.4Hz,1H),8.82(d,J＝8.2Hz,1H),8.54(d,J＝5.2Hz,1H),8.22(s,1H),8.20(s,1H),8.07(d,J＝8.0Hz,1H),7.94(d,J＝8.2Hz,1H),7.44(t,J＝8.0Hz,1H),7.14(dd,J＝7.8,1.2Hz,1H),4.88-4.86(m,1H),4.80-4.77(m,1H),4.65-4.62(m,1H),4.59-4.57(m,1H)ppm.¹³C NMR(150MHz,DMSO-d₆)δ165.79,150.59,148.15,148.11,143.62,142.15,133.19,130.51,130.46,128.53,127.08,124.44,124.18,123.20,123.18,122.70,120.64,119.64,116.72,82.22(d,J＝166.2Hz),64.72(d,J＝18.2Hz)ppm.HR-MS(m/z)(ESI):calcd for C₂₁H₁₅ClFN₃O₂[M+H]⁺:396.0915；found:396.0911.

Example 21: preparation of Compound 21

Prepared using CX-4945 and 2-iodoethanol as described with reference to example 19 to give the product as a yellow solid in 77.3% yield.¹H NMR(600MHz,DMSO-d₆)δ10.16(s,1H),10.04(s,1H),8.98(d,J＝5.6Hz,1H),8.82(d,J＝8.5Hz,1H),8.52(dd,J＝8.3,1.0Hz,1H),8.34(s,1H),8.26(d,J＝8.2Hz,1H),8.02(d,J＝1.4Hz,1H),7.73(dd,J＝8.4,1.5Hz,1H),7.48(t,J＝8.1Hz,1H),7.16(dd,J＝7.9,1.2Hz,1H),5.11-5.08(t,J＝8.2Hz,2H),4.78(t,J＝7.8Hz,2H).¹³C NMR(150MHz,DMSO-d₆)δ165.62,152.34,150.89,148.19,143.68,142.39,133.15,130.50,130.26,128.49,127.20,124.66,124.04,123.26,123.17,122.71,120.89,119.95,117.39,79.06,63.61.HR-MS(m/z)(ESI):calcd for C₂₁H₁₅ClF₂N₄O[M+H]⁺:503.9975；found:503.9980.

Example 22: preparation of Compound 22

Prepared using CX-4945 and 2, 2-difluoroethanol according to the procedure described in example 19, to give the product as a yellowish solid in a yield of 65.7%.¹H NMR(600MHz,DMSO-d₆)δ10.10(s,1H),9.90(s,1H),8.96(d,J＝5.6Hz,1H),8.83(d,J＝8.5Hz,1H),8.76(d,J＝5.4Hz,1H),8.31(d,J＝1.7Hz,1H),8.19(d,J＝1.7Hz,1H),8.16-8.14(m,1H),7.93(dd,J＝8.4,1.8Hz,1H),7.44(t,J＝8.2Hz,1H),7.14(dd,J＝7.8,1.2Hz,1H),6.51(tt,J＝54.2,3.2Hz,1H),4.68(td,J＝15.2,3.2Hz,2H)ppm.¹³C NMR(150MHz,DMSO-d₆)δ165.21,150.86,148.21,148.12,143.70,142.25,133.12,130.41,129.78,128.69,127.07,124.63,124.17,123.51,123.37,122.69,120.82,119.82,117.19,114.12(t,J＝238.9Hz),63.04(t,J＝26.4Hz).HR-MS(m/z)(ESI):calcd for C₂₁H₁₄ClF₂N₃O₂[M+H]⁺:414.0821；found:414.0820.

Example 23: preparation of Compound 23

Prepared as described in example 19 using CX-4945 and 3, 3-dichloropropanol to give the product as a yellow solid in 61.5% yield.¹H NMR(600MHz,DMSO)δ10.14(s,1H),10.02(s,1H),8.97(d,J＝5.6Hz,1H),8.87(d,J＝3.6Hz,1H),8.86(s,1H),8.39(t,J＝1.8Hz,1H),8.24(d,J＝1.4Hz,1H),8.17(dd,J＝8.2,1.2Hz,1H),7.98(dd,J＝8.4,1.6Hz,1H),7.43(t,J＝8.2Hz,1H),7.14(dd,J＝7.8,1.2Hz,1H),4.84-4.80(m,1H),4.70(dd,J＝11.8,4.6Hz,1H),4.65(dd,J＝11.8,6.2Hz,1H),4.14(t,J＝5.2Hz,2H)ppm.¹³C NMR(150MHz,DMSO-d₆)δ165.47,150.91,148.21,148.17,143.75,142.34,133.12,130.40,130.23,128.72,127.17,124.67,124.27,123.44,123.36,122.67,120.86,119.87,117.38,65.77,58.78,46.48ppm.HR-MS(m/z)(ESI):calcd for C₂₂H₁₆Cl₃N₃O₂[M+H]⁺:460.0386；found:460.0380.

Example 24: preparation of Compound 24

Prepared using CX-4945 and triethylene glycol according to the method described in example 19 to give a yellow solid product with a yield of 72.6%.¹H-NMR(600MHz,DMSO-d₆)δ10.12(s,1H),9.63(s,1H),8.98(d,J＝6.0Hz,1H),8.82(d,J＝6.0Hz,1H),8.54(d,J＝6.0Hz,1H),8.27(t,J＝2.0Hz,1H),8.20(d,J＝1.6Hz,1H),8.07(dd,J＝8.2,1.4Hz,1H),7.94(dd,J＝8.4,1.8Hz,1H),7.45(t,J＝8.1Hz,1H),7.15(dd,J＝6.0,1.4Hz,1H),4.57(t,J＝6.0Hz,1H),4.47(t,J＝6.0Hz,2H),3.83(t,J＝6.0Hz,2H),3.66(dd,J＝5.8,3.8Hz,2H),3.58(dd,J＝5.8,3.8Hz,2H),3.48(dd,J＝10.4,4.8Hz,2H),3.44(dd,J＝7.8,3.0Hz,2H)ppm.¹³C NMR(150MHz,DMSO-d₆)δ165.97,150.65,148.22,148.16,143.67,142.19,133.22,130.88,130.54,128.53,127.19,124.49,124.25,123.21,123.15,122.73,120.67,119.67,116.78,72.86,70.41,70.20,68.84,64.79,60.69ppm.HR-MS(m/z)(ESI):calcd for C₂₅H₂₄ClN₃O₅[M+H]⁺:482.1482；found:482.1483.

Example 25: preparation of Compound 25

Prepared as described in example 19 using CX-4945 and 2- (2-hydroxyethyl) pyridine to give the product as a yellow solid in 77.2% yield.¹H NMR(600MHz,DMSO-d₆)δ10.15(s,1H),10.02(s,1H),8.97(s,1H),8.87(d,J＝7.0Hz,1H),8.83(s,1H),8.61(s,1H),8.36(s,1H),8.27(s,1H),8.18(d,J＝6.1Hz,1H),8.00(d,J＝6.7Hz,1H),7.88(s,1H),7.57(d,J＝5.3Hz,1H),7.43(s,1H),7.39(s,1H),7.13(d,J＝5.9Hz,1H),5.50(s,2H),3.40(s,1H),3.18(s,1H)ppm.¹³C NMR(150MHz,DMSO-d₆)δ165.78,156.03,150.96,149.74,148.25,143.82,142.36,137.58,133.11,130.64,130.47,128.69,127.23,124.71,124.32,123.63,123.44,122.68,122.50,122.10,120.83,119.88,118.27,117.41,67.41,49.03ppm.HR-MS(m/z)(ESI):calcd for C₂₆H₁₉ClN₄O₂[M+H]⁺:455.1275；found:455.1269.

Example 26: preparation of Compound 26

Prepared using CX-4945 and 1, 2-ethanedithiol as described in reference example 19, and provided the product as a yellow solid in 42.7% yield.¹H NMR(600MHz,DMSO-d₆)δ10.15(s,1H),9.94(s,1H),8.93(d,J＝5.6Hz,1H),8.81(s,1H),8.80(d,J＝3.2Hz,1H),8.38(s,1H),8.19(dd,J＝8.2,1.2Hz,1H),7.70(d,J＝2.8Hz,1H),7.48(dd,J＝8.2,1.2Hz,1H),7.40(t,J＝8.1Hz,1H),7.11(dd,J＝7.8,0.8Hz,1H),3.16(d,J＝3.2Hz,1H),3.05(t,J＝5.2Hz,2H),2.99-2.96(m,2H)ppm.¹³C NMR(150MHz,DMSO-d₆)δ170.11,150.64,147.87,147.45,143.71,142.50,138.26,133.13,130.43,127.57,125.65,124.10,123.16,122.88,122.48,120.60,120.01,119.66,117.24,49.03,35.23ppm.HR-MS(m/z)(ESI):calcd for C₂₁H₁₆ClN₃OS₂[M+H]⁺:426.0501；found:426.0507.

Example 27: preparation of Compound 27

In a 10mL single-neck flask, 10.182 g (0.5mmol), 0.037g (0.5mmol) propionic acid, HATU0.228g (0.6mmol), 0.129g (1.0mmol) DIPEA and 5mL anhydrous DMF were added, and the mixture was stirred at 50 ℃ CAfter 24h-48h, TLC was monitored to complete the reaction, the solvent was concentrated and purified by column chromatography on silica gel eluting with dichloromethane and methanol (80:1-30:1) to give 0.088g of yellow solid product in 43.1% yield.¹H NMR(600MHz,DMSO-d₆)δ10.58(s,1H),10.20(s,1H),10.03(s,2H),8.98(d,J＝5.4Hz,1H),8.89(t,J＝8.1Hz,2H),8.42(s,1H),8.29(s,1H),8.22(d,J＝8.0Hz,1H),7.96(d,J＝8.0Hz,1H),7.44(t,J＝8.0Hz,1H),7.14(d,J＝7.6Hz,1H),2.24(dd,J＝15.0,7.4Hz,2H),1.09(t,J＝7.5Hz,3H)ppm.¹³C NMR(150MHz,DMSO-d₆)δ172.85,165.60,150.80,148.13,143.78,142.52,133.93,133.18,130.43,127.47,126.71,124.44,123.30,123.04,122.60,121.99,120.75,119.79,117.39,27.04,10.20ppm.HR-MS(m/z)(ESI):calcd for C₂₂H₁₈ClN₅O₂[M+H]⁺:420.1227；found:420.1219.

Example 28: preparation of Compound 28

Prepared as described in reference to example 27 using compound 1 and 3-methylthiopropionic acid, to give the product as a yellow solid in 46.3% yield.¹H NMR(600MHz,DMSO-d₆)δ10.67(s,1H),10.18(s,2H),10.03(s,1H),8.96(d,J＝5.5Hz,1H),8.87(dd,J＝6.9,4.2Hz,2H),8.41(s,1H),8.28(d,J＝1.5Hz,1H),8.21(d,J＝7.3Hz,1H),7.95(dd,J＝8.4,1.5Hz,1H),7.42(t,J＝8.1Hz,1H),7.12(dd,J＝7.9,1.3Hz,1H),2.73(t,J＝7.3Hz,2H),2.55(t,J＝7.3Hz,2H),2.10(s,3H)ppm.¹³C NMR(150MHz,DMSO-d₆)δ170.49,165.51,150.79,148.10,147.85,143.76,142.52,133.80,133.16,130.41,127.45,126.76,124.43,123.31,123.02,122.58,122.01,120.74,119.78,117.41,34.06,29.36,15.19ppm.HR-MS(m/z)(ESI):calcd for C₂₃H₂₀ClN₅O₂S[M+H]⁺:466.1104；found:466.1109.

Example 29: preparation of Compound 29

Prepared as described in example 27 using compound 1 and isobutyl chloroformate to give the product as a yellow solid in 57.8% yield.¹H NMR(600MHz,DMSO-d₆)δ10.62(s,1H),10.20(s,1H),10.02(s,1H),9.33(s,1H),8.98(d,J＝4.5Hz,1H),8.90(d,J＝8.3Hz,1H),8.87(s,1H),8.43(s,1H),8.28(s,1H),8.21(d,J＝6.3Hz,1H),7.96(d,J＝6.7Hz,1H),7.44(t,J＝7.4Hz,1H),7.14(d,J＝7.2Hz,1H),3.86(d,J＝4.2Hz,2H),1.22(m,1H),0.94(d,J＝4.3Hz,4H),0.84(s,2H)ppm.¹³C NMR(150MHz,DMSO-d₆)δ166.22,157.06,150.81,148.13,147.89,143.78,142.49,133.78,133.18,130.43,127.45,126.68,124.44,123.23,123.10,122.61,122.06,120.76,119.79,117.36,70.91,28.14,19.32ppm.HR-MS(m/z)(ESI):calcd for C₂₄H₂₂ClN₅O₃[M+H]⁺:464.1489；found:464.1476.

Example 30: preparation of Compound 30

Prepared using compound 1 and 3- (N-morpholine) propanesulfonic acid in the manner described for reference example 27 to give the product as a yellow solid in 35.1% yield.¹H NMR(600MHz,DMSO-d₆)δ10.06(s,1H),9.94(s,1H),8.89(d,J＝5.5Hz,1H),8.78(s,1H),8.77(d,J＝2.0Hz,1H),8.30(s,1H),8.19(d,J＝8.1Hz,1H),7.98(d,J＝1.3Hz,1H),7.83(dd,J＝8.4,1.3Hz,1H),7.41(t,J＝8.1Hz,1H),7.11(dd,J＝7.8,1.2Hz,1H),3.39(m,6H),3.09(m,6H),1.23(t,J＝6.3Hz,2H).¹³C NMR(150MHz,DMSO-d₆)δ165.09,158.34,150.87,147.79,147.52,144.16,142.41,133.11,130.42,127.41,125.42,124.09,123.71,123.38,122.58,120.93,120.80,119.82,117.29,49.03,46.39,38.17,29.48,19.02ppm.HR-MS(m/z)(ESI):calcd for C₂₆H₂₇ClN₆O₄S[M+H]⁺:555.1581；found:555.1584.

Example 31: preparation of Compound 31

Prepared using compound 1 and lipoic acid as described with reference to example 27, gave a yellow solid product in 43.7% yield.¹H NMR(600MHz,DMSO-d₆)δ10.55(s,1H),10.20(s,1H),10.07(s,2H),8.97(d,J＝5.6Hz,1H),8.92(d,J＝5.7Hz,1H),8.89(d,J＝8.6Hz,1H),8.43(t,J＝1.9Hz,1H),8.29(d,J＝1.6Hz,1H),8.24(dd,J＝8.2,1.2Hz,1H),7.96(dd,J＝8.4,1.6Hz,1H),7.44(t,J＝8.1Hz,1H),7.14(dd,J＝7.9,1.4Hz,1H),3.65(ddd,J＝12.3,8.3,6.2Hz,1H),3.24-3.19(m,1H),3.18-3.12(m,2H),2.45(td,J＝12.5,6.3Hz,1H),2.25(t,J＝7.2Hz,2H),1.91(dq,J＝13.5,6.8Hz,1H),1.77-1.70(m,1H),1.63-1.58(m,2H),1.49-1.42(m,2H)ppm.HR-MS(m/z)(ESI):calcd for C₂₇H₂₆ClN₅O₂S₂[M+H]⁺:552.1294；found:552.1300.

(II) cytotoxic Activity test of Compounds

The compounds of the invention were tested for cytotoxic activity using the MTT method. Cells in the logarithmic growth phase were counted and plated in 96-well plates at about 8000-10000 cells per well. Culturing overnight, and administering after cell adherence, wherein an administration group and a control group are respectively arranged. The compounds to be tested were formulated as stock solutions in DMSO and diluted to a range of concentrations in cell culture medium just prior to use, with the final DMSO concentration not exceeding 4% o (the same experiment below). Each concentration was provided with 3 multiple wells. After adding the drug, the mixture is cultured for 72h, 20 mu L of MTT with the concentration of 5mg/mL is added, the mixture is incubated for 4h at 37 ℃, the supernatant is removed, and 150 mu L of DMSO is added for dissolution. Measuring the OD value of each hole by using a microplate reader at 490 wavelength, calculating the inhibition rate, and calculating the IC by making a concentration-inhibition rate curve₅₀The value is obtained.

The compounds of the present invention were tested for their cytotoxic activity against human prostate cancer cell PC-3, colon cancer cell HCT-116, breast cancer cell MCF-7, colon cancer cell HT-29, bladder cancer cell T24 and normal liver cell LO2, with CX-4945 as a positive control. Observing the inhibition of the compound on the growth of tumor cells under different concentrations, and calculating the inhibition rate and IC thereof₅₀The cytotoxic activity of the compounds was evaluated and the results are shown in table 1.

(III) in vitro enzyme Activity test of Compounds

The inhibitory activity of the compound of the present invention and CX-4945 against CK2 and CLK2 proteins was examined using commercial human casein kinase 2(CK2) and human cell division cycle-like kinase 2(CLK2) kits. The CK2 kit, the CLK2 kit, the CK2 protein and the CLK2 protein are all purchased from Shanghai Sheng industries, Ltd. Compounds were tested for CK2 and CLK2 enzyme activity according to the experimental methods provided in the CK2 and CLK2 kit instructions, and the results are shown in table 1 and fig. 1.

TABLE 1 cytotoxic and enzymatic Activity of Compounds

^aNd denotes no detection;

^bSF denotes the selection factor, IC₅₀(CLK2)/IC₅₀(CK2)。

(IV) test for inhibition of cancer cell dryness by Compounds

The activity of the partial compound of the invention on the sternness inhibition of HCT-116 primary cancer cells was tested by flow cytometry using a commercial acetaldehyde dehydrogenase 1(ALDH1) kit and CD44 and CD133 antibodies. ALDH1 kit, CD44, CD133 antibody were purchased from Shanghai Biyuntian Biotechnology Co., Ltd and HCT-116 primary tumor was provided by local hospital.

ALDH1 enzyme Activity assay

According to the experimental method provided by the ALDH1 detection kit, the inhibitory activity of the

compounds

1, 4, 16, 19, 20, 21 and 25 on ALDH1 is detected, CX-4945 and a known cancer cell sternness inhibitor BBI608 are selected as positive controls, and the results are shown in Table 2.

TABLE 2 inhibition of the sternness of HCT-116 primary cancer cells by some compounds

2. Stem cell marker assay

Counting cells in logarithmic growth phase, inoculating the cells in a 6-well culture plate, culturing until the cells are attached to the wall, adding samples, and taking a blank group as a control. After 24h, the cells were collected by centrifugation, 300. mu.L of PBS, 1. mu.L of CD44-FITC antibody and 1. mu.L of CD133-PE antibody were added to each tube, incubated for 30min in the dark, transferred to a 2mL centrifuge tube and measured by flow cytometry. The expression conditions of stem cell markers CD44 and CD133 in cells after

compounds

4 and 20 are respectively incubated with HCT-116 primary cancer cells for 24 hours are determined by a flow cytometer, CX-4945 and BBI608 are selected as positive controls,

3. real-time fluorescence quantitative analysis

Related genes in stem cell inhibition pathways Wnt/beta-catenin and Hedgehog and the influence on expression of the related genes of the stem cells by the

compounds

4 and 20 are analyzed through qRT-PCR, and CX-4945 and BBI608 are selected as positive controls.

The experimental method comprises the following steps:

(1) primer sequences were designed using Primer5, β -actin as an internal reference, and the Primer information is shown in table 3 below:

TABLE 3 primer sequences

(2) And (3) detecting the expression conditions of related genes in Wnt/beta-catenin and Hedgehog signal channels and stem cell related genes OCT4, SOX2 and Nanog in the cells after the HCT-116 primary cancer cells and the sample are incubated for 24h by RT-PCR.

The experimental method comprises the following steps:

(i) HCT-116 Primary cancer cell Total RNA extraction

Total RNA in cells was extracted according to the experimental method provided in the Karrol RNA Reagent catalog No. K3102, and the RNA purity and concentration were determined by UV spectrophotometer: taking 1 mu L of RNA solution, diluting by 100 times, and measuring the light absorption values at 260nm and 280nm by an ultraviolet spectrophotometer to calculate the OD260/280 ratio and the solution concentration. The required ratio is between 1.8 and 2.0, and the RNA concentration is more than 500 mu g/mL (in operation, reagents, vessels and consumables are treated without RNase).

(ii) Reverse transcription to synthesize cDNA

Carrying out conventional reverse transcription reaction by using OligdT as a downstream primer; the first strand cDNA was synthesized by reverse transcription according to the following reaction system:

adopting RNase-free deionized water to fix the volume to 20 mu L; the system reacts for 1h at 42 ℃, then AMV is inactivated for 5min at 95 ℃, and the product is placed on ice for subsequent tests.

(iii) Real-time fluorescent quantitative PCR (qRT-PCR)

Using cDNA product synthesized by reverse transcription as template, carrying out quantitative analysis on Wnt and Hedgehog signal path and stem cell related gene expression quantity in HCT-116 primary cancer cell according to the following reaction system, using beta-actin as reference gene, and using ddH₂Setting a negative control (taking beta-catenin as an example) by taking O as a template:

the above system is reacted on a PCR instrument according to the following procedures respectively: beta-catenin and beta-actin, pre-denaturating for 5min at 94 ℃, entering three-step circulation (30 cycles of 94-40 s, 52-40 s and 72-40 s), and finally extending for 10min at 72 ℃.

The real-time fluorescence quantitative analysis is performed by using 2^-△△CTThe method comprises the steps of carrying out four multi-hole parallel experiments on each gene of each sample, selecting experimental data, namely, eliminating numerical values with larger errors, and taking the average value of the residual numerical values as final experiment retention data, wherein the experimental data are shown in figure 3.

Claims

1. An anti-tumor compound is characterized in that fatty chains or heterocyclic structural fragments containing amino, hydroxyl, sulfydryl or halogen atoms are introduced into a parent structure of a known CK2 inhibitor (CX-4945) in the modes of amido bonds, ester bonds and thioester bonds, so that a novel compound shown as a formula II is provided;

compounds of the structures shown in formulas II-A, II-B, II-C, II-D and II-E:

wherein:

R in the formula II-C₃Is- (CH)₂)₂SH；

R in the formula II-E₅Is composed of

2. The class of anti-neoplastic compounds of claim 1, wherein the compound is preferably any one or more of the following compounds 1-31:

Compound 3: n-methanesulfonyl-5- ((3-chlorophenyl) amino) benzo [ c ] [2,6] naphthyridine-8-carboxamide

3. A process for preparing the antineoplastic compound according to claim 1 or 2, wherein said compound is synthesized from CX-4945 or its precursor,

CX-4945 is synthesized by using 3-bromoisonicotinic acid as initial material and through esterification and Suzuki coupling reaction with 2-amino-4-carbomethoxyphenyl borate to obtain intermediate 5, 6-dihydrobenzo [ c ]][2,6]Naphthyridine-8-carboxylic acid methyl ester is subjected to chlorination reaction with phosphorus oxychloride, substitution reaction with m-chloroaniline and hydrolysis to obtain the product. The specific synthetic route is as follows:

c.POCl₃,110℃,4h；d.NMP,110℃,8h；e.1.5N NaOH,70℃。

4. the process for preparing a class of anti-tumor compounds according to claim 1, wherein (i) the compound of formula II-a is obtained by exchange reaction or condensation reaction, the specific synthetic route is as follows:

(ii) the compound of formula II-B can be obtained by esterification, and the specific synthetic route is as follows:

(iii) the compound of formula II-C can be obtained by esterification of a thiol, and the specific synthetic route is as follows:

(iv) the compound of formula II-D can be obtained by amide condensation reaction of hydrazide and carboxylic acid, and the specific synthetic route is as follows:

(v) the compound of formula II-E can be obtained by amide condensation reaction of hydrazide and sulfonic acid, and the specific synthetic route is as follows:

wherein R is₁、R₂、R₃、R₄、R₅As mentioned before, HATU stands for the condensing agent 2- (7-benzotriazole oxide) -N, N '-tetramethyluronium hexafluorophosphate, TBTU stands for the condensing agent O-benzotriazole-N, N' -tetramethyluronium tetrafluoroborate, DIPEA stands for N, N-diisopropylethylamine, DMF stands for the solvent N, N-dimethylformamide, Reflux stands for Reflux, r.t. stands for room temperature.

5. The use of the antitumor compound of claim 1, wherein the compound has good effects on cancer cell proliferation, inhibition of CK2 protein kinase and selectivity of CK2 protein, and can inhibit cancer cell dryness, and can be used for preparing antitumor drugs.