CN110317137B

CN110317137B - 9, 10-anthraquinone compound or pharmaceutically acceptable salt thereof and pharmaceutical application thereof

Info

Publication number: CN110317137B
Application number: CN201810273938.9A
Authority: CN
Inventors: 周璐; 黄科; 叶德泳; 沈瑛; 江露露; 王鹏辉; 李慧逖; 张晓丹; 楚勇; 梁倩
Original assignee: Fudan University
Current assignee: Fudan University
Priority date: 2018-03-29
Filing date: 2018-03-29
Publication date: 2022-04-19
Anticipated expiration: 2038-03-29
Also published as: CN110317137A

Abstract

The invention belongs to the field of pharmaceutical chemistry, and relates to a 9, 10-anthraquinone compound and a pharmaceutical application thereof, in particular to a 9, 10-anthraquinone compound, an application thereof in preparing phosphoglyceromutase inhibitors and an application thereof in preparing drugs for treating cancers. In particular discloses an application of a 9, 10-anthraquinone compound shown as a formula I structure, a pharmaceutically acceptable salt thereof or a pharmaceutical composition taking the compound as an effective active ingredient in preparing medicaments for preventing and treating tumors, the compound can inhibit phosphoglyceromutase activity and reduce cell metabolism level, can be used for treating diseases such as solid tumors, blood tumors and the like, and the related tumors are pancreatic cancer, lung cancer, liver cancer, stomach cancer, esophageal cancer,Intestinal cancer, breast cancer, cervical cancer, leukemia, and melanoma.

Description

9, 10-anthraquinone compound or pharmaceutically acceptable salt thereof and pharmaceutical application thereof

Technical Field

The invention belongs to the field of pharmaceutical chemistry, relates to a 9, 10-anthraquinone compound and a pharmaceutical application thereof, and particularly relates to a 9, 10-anthraquinone compound or a pharmaceutically acceptable salt thereof, an application thereof in preparing a phosphoglyceromutase inhibitor and an application thereof in preparing a medicament for treating cancer.

Background

The data show that cancer is the first killer of human life health. According to the data of '2012 Chinese health statistics treatise', malignant tumors are ranked first in 2011 in ten-year-old Chinese disease mortality. The world health organization report shows that 1270 million new cancer patients are added all over the world in 2008, 760 million new cancer patients die of cancer, particularly in developing countries, the number of new cancer cases reaches 56%, and the global cancer incidence is supposed to increase by 50% by 2020, namely 1500 million new cancer patients are added each year. Moreover, cancer deaths have increased dramatically worldwide, and the number may increase to 1320 thousands in 2030.

It was reported that in the last 30 th century, the german scientist Warburg found that there was a metabolic difference between tumor and normal human tissues, and that tumor cells prefer inefficient energy production through glycolytic pathway (1 molecule of glucose produces about 4 molecules of ATP) and produce a large amount of lactic acid, rather than energy production in mitochondria through normal oxidative phosphorylation pathway (1 molecule of glucose produces 36 molecules of ATP), in the presence or absence of sufficient oxygen, and this metabolic property makes the rate of sugar consumption of tumor cells much higher than that of normal cells. The phenomenon of tumor cells with an enhanced capacity dependence on glycolytic pathways is called "Warburg effect", Warburg thus awarding the 1931 Nobel prize. However, the mechanism of action has not been fully understood in the art to date.

Research shows that glycolysis pathway is the first step of sugar metabolism in all biological cells and is the necessary pathway for organisms to perform energy metabolism, and the pathway comprises ten steps of reaction, wherein a plurality of enzymes participate in precisely regulating the circulation and balance of the pathway. More and more researches show that the metabolic abnormality of the pathway is closely related to cancer (Science, 2010; 330(6009):1340-1344.), and the increase or decrease of some key enzyme activities in glycolytic pathway in cancer cells compared with normal cells (hexokinase, pyruvate kinase M2 and the like) can cause the metabolic abnormality of the whole pathway, so that the concentration of metabolic intermediates is changed, and the activity of other metabolic networks influenced by the metabolic intermediates is abnormal. In recent years, the metabolic pathway has received more and more scientific research attention, and the protein of pyruvate kinase M2(PKM2) subtype, as a star protein, has become a great hotspot of cancer research. Researchers believe that these key enzymes in glycolytic pathway can be used as important candidate targets for drug therapy of cancer, and small molecular compounds acting on target enzymes in the pathway can regulate and control the switch of glycolytic pathway, and may change the metabolic network of tumor so as to inhibit the growth and development of tumor. Small molecules that activate pyruvate kinase M2 have been reported to inhibit the growth and development of cancer cells, providing a new research approach that regulates specific target enzyme activities in the glycolytic metabolic pathway to treat cancer (nat. chem. biol. 2012; 8(10): 839-47).

Studies have shown that phosphoglycerate mutase (PGM) is an important enzyme in the glycolytic pathway, and can reversibly catalyze the production of 2-phosphoglycerate (2PG) from 3-phosphoglycerate (3PG), and that 2, 3-diphosphoglycerate (2,3-BPG) is an activator of this enzyme, and can phosphorylate histidine at position 11 of the active residue to accelerate the reaction. The enzyme exists in two subtypes, with subtype m being present in muscle tissue and subtype b being present in other tissues. The research shows that the phosphoglycerate mutase of the b-subtype (PGAM1) presents a high expression state in tumor cells, the enzyme can regulate the concentration of a substrate 3-phosphoglycerate and a product 2-phosphoglycerate, and the equilibrium is shifted to the direction of 2-phosphoglycerate under the high expression and high activity state; further studies have found that 3-phosphoglycerate, the substrate, is an inhibitor of 6-phosphogluconate dehydrogenase (6PGD) and 2-phosphoglycerate, the product, is an activator of phosphoglycerate dehydrogenase (PHGDH), and that modulating the activity of PGAM1 can affect the activity of the pentose phosphate pathway (the target enzyme is 6-phosphogluconate dehydrogenase) and the serine synthesis pathway (the target enzyme is phosphoglycerate dehydrogenase) (Cancer cell. 2012; 22(5): 585-600). The pentose phosphate pathway is an important pathway for synthesizing nucleotide and generating NADPH by cells, provides a necessary nucleotide skeleton and an electron donor NADPH for cell division and proliferation, and the flux increase of the serine synthesis pathway can improve the amino acid synthesis rate and promote cell division (Nature.2011; 476(7360):346-350), and the two pathways have extremely important significance in cell biosynthesis and division and proliferation.

The research results comprehensively show that PGAM1 is in a very important position in the metabolic network, the enzyme itself as an enzyme participating in glycolysis can influence the rate of glycolysis pathway, influence the digestion, absorption and energy generation of tumor to sugar, and can also regulate the concentration of substrate and product so as to influence the activity of other metabolic pathways, so that an inhibitor (including siRNA and small molecular compounds) only acting on PGAM1 can inhibit two biosynthesis pathways (ribose and amino acid synthesis pathways) of Cancer cells by influencing the concentration of product and substrate, thereby being capable of inhibiting the growth of Cancer cells extremely effectively, and being considered as a strategy of treating Cancer ' Shierdi ' bird ' (Cancer cell.2012; 22(5): 565-. The research result not only provides new information for understanding a complex tumor metabolic network, but also provides an important anti-tumor strategy: the inhibitor acting on PGAM1 alone can inhibit tumor energy metabolism and biosynthesis pathway simultaneously, and inhibit tumor growth.

The association of PGAM1 with cancer has only been mentioned occasionally in recent studies, and this enzyme has been recognized in recent two years as a novel drug target in the metabolic network of cancer (nat. Rev. drug. Discov.2013; (12): 829-46). In early studies, some groups conducted the mechanism of glycolysis pathway studies using PGAM1 substrate analogues as inhibitors, and these compounds were far from practical use from the activity and pharmaceutical point of view. Recently, a patent application reports that PGAM1 small-molecule inhibitor PGMI-004A is derived from a lead compound of sodium Alizarin Red S (hereinafter referred to as Red S), can show excellent capacity of inhibiting tumor growth on cells and animal models, and has no obvious toxic and side effects (Cancer cell.2012; 22(5): 585-600). However, the in vitro activity of the compound is only on the micromolar level, the activity on the cellular level is only on the dozens of micromolar level, and the drug forming standard is not reached yet.

Based on the basis of the prior art, the inventor intends to provide a 9, 10-anthraquinone compound and a pharmaceutical application thereof, and particularly relates to a 9, 10-anthraquinone compound or a pharmaceutically acceptable salt thereof, an application thereof in preparing phosphoglyceromutase inhibitors and an application thereof in preparing drugs for treating cancers.

Disclosure of Invention

The invention aims to overcome the defects and shortcomings of the prior art, provides a 9, 10-anthraquinone compound and a pharmaceutical application thereof, and particularly relates to the 9, 10-anthraquinone compound or a pharmaceutically acceptable salt thereof, an application thereof in preparing phosphoglyceromutase inhibitors and an application thereof in preparing medicines for treating cancers.

The first purpose of the invention is to provide 9, 10-anthraquinone compounds or pharmaceutically acceptable salts thereof; the 9, 10-anthraquinone compound is a compound with a structure shown in a formula (I) or a salt thereof,

in the formula

R₁Is hydroxy, 2-oxyacetic acid or 2-oxyacetic ester;

R_{2 is}Hydroxy, alkoxy (less than 12 carbons), ester groups, 2-oxyacetic acid esters, 2-oxyacetamides, and amide derivatives;

R₃selected from hydrogen and substituted sulfonamido; r₃Can be in the 3-position or 4-position as shown in structural formula (I);

R₄selected from the group consisting of alkyl (less than 12 carbons), cycloalkyl, amino, N-azacycloalkyl, phenyl, pyridyl, naphthyl, thiazole, biphenyl, quinolyl and substituted derivatives thereof wherein the substituent of the substituted derivative is selected from the group consisting of F, Cl, Br, I, alkyl, alkoxy, NO₂，CN，-CF₃，-OCF₃Any one to five substituents of hydroxyl, ester, amino, amido and substituted amino;

R₅selected from the group consisting of hydrogen, methyl, alkyl, cycloalkyl and substituted derivatives of the foregoing;

R₂and R₅Can be subjected to linking cyclization, the number of atoms connected is 2-4, and the connecting atoms are oxygen atoms or carbon atoms.

In the present invention, R₁、R₂When it is hydroxy, R₃Is a 3-substituted sulfonamide derivative.

In the present invention, R₃In the case of 3-substituted sulfonamide derivatives, R₄Selected from the group consisting of alkyl (less than 12 carbons), cycloalkyl, amino, N-azacycloalkyl, phenyl, pyridyl, naphthyl, thiazole, biphenyl, quinolyl, isoquinolyl and substituted derivatives thereof, wherein the substituents of the substituted derivatives are selected from the group consisting of F, Cl, Br, I, alkyl, alkoxy, NO₂，CN，-CF₃，-OCF₃Any one to five substituents of hydroxyl, ester, amino, N-azacycloalkyl, amido and substituted amino.

In the present invention, R₃Is a 3-substituted sulfonamide derivative, R₄When substituted by phenyl, the substituents on the phenyl ring are selected from F, Cl, Br, I, alkyl, alkoxyBasic group, NO₂，CN，-CF₃，-OCF₃Any one to five substituents of hydroxyl, ester, amino, N-azacycloalkyl, amido and substituted amino.

In the present invention, compounds of formula I-1 to formula I-62 can be further described:

in the invention, part of compounds contain acidic groups such as carboxyl, hydroxyl and the like which can be salified with alkali, and part of compounds contain basic groups such as amino and the like which can be salified with acid, and salts of derivatives can be formed by adopting common means. Including organic acid salts such as acetate, citrate, fumarate, maleate, oxalate, malate, citrate, succinate, tartrate, lactate, camphorsulfonate, benzenesulfonate, p-toluenesulfonate, methanesulfonate, trifluoroacetate, trifluoromethanesulfonate, etc.; inorganic acid salts such as hydrohalic acid (hydrofluoric, hydrochloric, hydrobromic, hydroiodic), sulfate, phosphate, nitrate, and the like. Or with amino acids such as glutamic acid or aspartic acid to form glutamate or aspartate.

The solvate of the 9, 10-anthraquinone compound is also in the protection scope of the invention, and the solvent is preferably water, ethanol or methanol.

The second purpose of the invention is to provide the application of the 9,10 anthraquinone compound shown in the formula (I) in the preparation of a phosphoglycerate mutase small-molecule inhibitor. The invention adopts an enzyme-linked quantitative detection method reported in the literature to measure the inhibition activity of 9, 10-anthraquinone compounds shown in formula (I) on phosphoglycerate mutase (Cancer cell.2012; 22(5):585-600), and calculates the change of the inhibition agent on the activity of catalyzing 3PG to 2PG by phosphoglycerate mutase through the change of the NADH content.

An activity test experiment based on an enzyme-linked method shows that the 9, 10-anthraquinone compound shown in the formula (I) has submicron-molar phosphoglycerate mutase inhibition activity and is an effective component for inhibiting phosphoglycerate mutase; the inhibitory activity of the compounds on phosphoglycerate mutase measured by the enzyme-linked assay is shown in table 1.

The invention further aims to provide the application of the 9, 10-anthraquinone compound shown in the formula (I) and the salt or solvate thereof in treating cancers, and the in vitro antitumor activity test of various tumor cells shows that the compound has the antitumor activity of submicromolar level, and the activity result is shown in the table 1.

Experiments prove that compared with a phosphoglycerate mutase small-molecule inhibitor PGMI-004A in the prior art, the compound disclosed by the invention has obviously higher inhibitory activity on phosphoglycerate mutase than PGMI-004A. The compound has the same structure as a PGMI-004A mother nucleus, but the activity of the synthesized novel compound is obviously improved due to the optimization of the substituent group at the 3-position of the anthraquinone ring, which is beyond the range which can be expected, so that the compound reported by the invention is considered to be novel.

The above medicine may further comprise one or more pharmaceutically acceptable carriers, which include conventional diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption enhancers, surfactants, adsorption carriers, lubricants, etc. in the pharmaceutical field, and if necessary, flavoring agents, sweeteners, etc.

The 9, 10-anthraquinone compounds provided by the invention are phosphoglyceromutase inhibitors with novel structures, have submicron-molar-level molecular level inhibition activity, have good potential and application prospects, and can be further prepared into medicines for treating cancers, wherein the cancers are pancreatic cancer, lung cancer, liver cancer, gastric cancer, esophageal cancer, intestinal cancer, breast cancer, cervical cancer, leukemia or melanoma.

Detailed Description

Example 1

Preparation of diethyl 2,2' - ((9, 10-dioxo-9, 10-dihydroanthracene-1, 2-yl) dioxo) diacetate (1)

A100 mL round-bottom flask was charged with 1, 2-dihydroxyanthracene-9, 10-dione (1.2g, 5mmol), potassium carbonate (1.66g, 12mmol), ethyl bromoacetate (2g, 12mmol) and N, N-dimethylformamide (50mL), reacted at 80 ℃ and stopped by TLC detection of disappearance of the starting material. After the reaction mixture was cooled to room temperature, the reaction mixture was added dropwise to about 350mL of 10% hydrochloric acid, extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, and separated by silica gel column chromatography (petroleum ether: ethyl acetate 2:1) to obtain 0.89g of a yellow solid in 43% yield. MS (ESI) (M/z):413.1(M-H)^-.¹HNMR(400MHz,DMSO-d₆)δ8.13–8.07(m,2H),7.98(d,J＝8.7Hz,1H),7.90–7.81(m,2H),7.50(d,J＝8.8Hz,1H),5.04(s,2H),4.71(s,2H),4.17(qd,J＝4.2,7.2Hz,4H),1.20(td,J＝2.6,7.2Hz,6H).。

Example 2

Preparation of 2,2' - ((9, 10-dioxo-9, 10-dihydroanthracene-1, 2-yl) dioxo) diacetic acid (2)

A50 mL round-bottomed flask was charged with 1(206mg,0.5mmol), 0.5M aqueous lithium hydroxide (8mL), and methanol (8mL), reacted at 30 ℃ and the reaction was stopped by TLC detection of disappearance of the starting material spotShould be used. Methanol was removed under reduced pressure, and the reaction solution was added dropwise to about 10% hydrochloric acid to adjust pH 1-2 to precipitate 169mg of a yellow solid in 95% yield. MS (ESI) (M/z) 355.0(M-H)^-.¹HNMR(400MHz,DMSO-d₆)δ12.71(brs,2H),8.09(dt,J＝1.4,7.3Hz,2H),7.96(d,J＝8.7Hz,1H),7.84(m,2H),7.46(d,J＝8.8Hz,1H),4.93(s,2H),4.62(s,2H).。

Example 3

Preparation of 2- ((1-hydroxy-9, 10-dioxo-9, 10-dihydroanthracen-2-yl) oxy) -N, N-dimethylacetamide (3)

First, preparation of Ethyl 2- ((1-hydroxy-9, 10-dioxo-9, 10-dihydroanthracene-1, 2-yl) oxy) acetate (HKb-2)

HKb-2 was prepared in the same manner as in 1 except that the amount of potassium carbonate was changed to 0.83g and the amount of ethyl bromoacetate was changed to 1g, to obtain an orange solid with a yield of 48%.

Preparation of 2' - ((1-hydroxy-9, 10-dioxo-9, 10-dihydroanthracene-1, 2-yl) oxy) acetic acid (HKb-3)

HKb-3 was prepared as described for 2, giving an orange solid with a yield of 95%.

A50 mL round-bottomed flask was charged with HKb-3(149mg,0.5mmol), dimethylaminochloride (31mg,0.68mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide (120mg,0.6mmol), 1-hydroxybenzotriazole (80mg,0.6mmol), N-diisopropylethylamine (175. mu.L, 1mmol), and dried N, N-dimethylformamide (10mL), reacted at room temperature, and stopped by TLC detection of disappearance of the starting material spot. Handle barThe reaction solution was added dropwise to about 140mL of water, and the resulting mixture was filtered with suction to obtain 114mg of an orange solid in a yield of 70%. MS (ESI) (M/z) 324.0(M-H)^-.¹H NMR(400MHz,DMSO-d₆)δ12.68(s,1H),8.22–8.05(m,2H),7.93–7.81(m,2H),7.62(d,J＝8.5Hz,1H),7.24(d,J＝8.5Hz,1H),5.03(s,2H),2.96(s,3H),2.80(s,3H).。

Example 4

Preparation of N- (3, 4-dihydroxy-9, 10-dioxo-9, 10-dihydroanthracen-2-yl) -4-methylbenzenesulfonamide (4)

Preparation of 3-nitro-1, 2-dihydroxyanthracene-9, 10-dione (HKb-10)

1, 2-dihydroxyanthracene-9, 10-dione (5g,20.8mmol) and glacial acetic acid (350mL) are added into a 1L round-bottom flask, the mixture is stirred for about 10 minutes at 50 ℃,1.5 mL of concentrated nitric acid is dropwise added, the reaction is stopped when a TLC detection raw material point disappears, after the reaction solution is returned to room temperature, a yellow solid is obtained by suction filtration, and the crude product can be directly used for the next reaction without purification, and the crude yield is 70%.

Preparation of 3-amino-1, 2-dihydroxyanthracene-9, 10-dione (HKb-11)

Adding HKb-10(1.75g,6.14mmol) and absolute ethyl alcohol (350mL) into a 1L round-bottom flask, stirring at room temperature, sequentially adding tin powder (10.5g,341mmol) and stannous chloride dihydrate (12.5g,55.4mmol), dropwise adding 50.4mL concentrated hydrochloric acid, stopping reaction when TLC detects that a raw material point disappears, removing the tin powder by suction filtration, concentrating under reduced pressure to remove part of ethanol, dropwise adding the rest reaction liquid into 1L water, suction filtration and vacuum drying to obtain a black solid, wherein the crude product can be directly used for the next reaction without purification, and the crude yield is 90%.

Preparation of N- (3, 4-dihydroxy-9, 10-dioxo-9, 10-dihydroanthracene-2-yl) -4-methylbenzenesulfonamide (4)

To a 25mL round bottom flask was added HKb-11(255mg,1mmol) and dry pyridine (5mL), stirred at room temperature, p-toluenesulfonyl chloride (286mg,1.5mmol) was added slowly and reacted for 4 h. The reaction solution was added dropwise to 10% hydrochloric acid, pH was adjusted to 1-2, extraction was performed with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, and subjected to silica gel column chromatography (dichloromethane) to obtain an orange-red solid 205mg, with a yield of 50%. MS (ESI) (M/z) 408.0(M-H)^-.¹HNMR(400MHz,DMSO-d₆)δ12.59(brs,1H),10.90(brs,1H),10.25(brs,1H),8.21–8.09(m,2H),7.93–7.85(m,2H),7.80(d,J＝8.2Hz,2H),7.76(s,1H),7.40(d,J＝8.0Hz,2H),2.35(s,3H).。

Example 5

Preparation of N- (3, 4-dihydroxy-9, 10-dioxo-9, 10-dihydroanthracen-2-yl) -4-nitrobenzenesulfonamide (5)

5 the procedure of reaction No. 5 was followed in 4, p-toluenesulfonyl chloride in reaction No. 5 was replaced by p-nitrobenzenesulfonyl chloride as an orange solid in 50% yield. MS (ESI) (M/z) 439.0(M-H)^-.¹HNMR(400MHz,DMSO-d₆)δ12.58(brs,1H),10.95(brs,1H),10.76(brs,1H),8.38(d,J＝8.2Hz,2H),8.22–8.04(m,4H),7.95–7.83(m,2H),7.70(s,1H).。

Example 6

Preparation of N- (3, 4-dihydroxy-9, 10-dioxo-9, 10-dihydroanthracen-2-yl) methanesulfonamide (6)

6 preparation method 4, 4 reaction three in the p-toluene sulfonyl chloride was changed to methane sulfonyl chloride, dark yellow solid, yield 45%. MS (ESI) (M/z) 332.0(M-H)^-.¹HNMR(400MHz,DMSO-d₆)δ12.59(brs,1H),10.91(brs,1H),9.50(brs,1H),8.10(dt,J＝5.0,17.2Hz,2H),7.90–7.76(m,2H),7.72(s,1H),3.08(s,3H).。

Example 7

Preparation of N- (3, 4-dihydroxy-9, 10-dioxo-9, 10-dihydroanthracen-2-yl) benzenesulfonamide (7)

7 preparation method 4, 4 reaction three in the p-toluene sulfonyl chloride was changed to benzene sulfonyl chloride, dark red solid, yield 55%. MS (ESI) (M/z) 394.0(M-H)^-.¹HNMR(400MHz,DMSO-d₆)δ12.52(brs,1H),10.83(brs,1H),10.29(brs,1H),8.14–8.03(m,2H),7.83(q,J＝4.9Hz,4H),7.68(s,1H),7.55(dt,J＝7.6,15.5Hz,3H).。

Example 8

Preparation of N- (3, 4-dihydroxy-9, 10-dioxo-9, 10-dihydroanthracen-2-yl) thiophene-2-sulfonamide (8)

8 the p-toluenesulfonyl chloride in reaction three, reaction 4, was replaced with thiophene-2-sulfonyl chloride as a yellow solid in 50% yield as in 4. MS (ESI) (M/z) 400.0(M-H)^-.¹H NMR(400MHz,DMSO-d₆)δ12.51(brs,1H),10.64(brs,2H),8.16–8.01(m,2H),7.93–7.72(m,4H),7.59(d,J＝3.7Hz,1H),7.10(t,J＝4.5Hz,1H).。

Example 9

Preparation of N- (3, 4-dihydroxy-9, 10-dioxo-9, 10-dihydroanthracen-2-yl) -4-trifluoromethylbenzenesulfonamide (9)

9 preparation method 4, 4 reaction three in the p-toluene sulfonyl chloride was changed to three methyl benzene sulfonyl chloride, yellow solid, yield 25%. MS (ESI) (M/z) 462.0(M-H)^-.¹HNMR(400MHz,DMSO-d₆)δ12.57(brs,1H),10.87(brs,1H),10.64(brs,1H),8.19–8.09(m,2H),8.06(d,J＝8.3Hz,2H),7.98(d,J＝8.4Hz,2H),7.92–7.83(m,2H),7.70(s,1H).。

Example 10

Preparation of N- (3, 4-dihydroxy-9, 10-dioxo-9, 10-dihydroanthracen-2-yl) -4-cyanobenzenesulfonamide (10)

10 preparation method 4, 4 reaction three in the p-toluene sulfonyl chloride was changed to p-cyanobenzene sulfonyl chloride, dark red solid, yield 60%. MS (ESI) (M/z) 419.0(M-H)^-.¹HNMR(400MHz,DMSO-d₆)δ12.57(brs,1H),10.74(brs,2H),8.19–8.10(m,2H),8.07(d,J＝8.1Hz,2H),7.99(d,J＝8.2Hz,2H),7.93–7.84(m,2H),7.68(s,1H).。

Example 11

Preparation of N- (3, 4-dihydroxy-9, 10-dioxo-9, 10-dihydroanthracen-2-yl) naphthalene-2-sulfonamide (11)

The preparation method of 11 is the same as 4, the p-toluenesulfonyl chloride in the reaction III of 4 is replaced by naphthalene-2-sulfonyl chloride, and dark red solid is producedThe rate was 65%. MS (ESI) (M/z) 444.0(M-H)^-.¹H NMR(400MHz,DMSO-d₆)δ12.45(brs,1H),10.58(brs,2H),8.48(s,1H),8.15–7.66(m,9H),7.60(p,J＝7.0Hz,2H).。

Example 12

Preparation of N- (3, 4-dihydroxy-9, 10-dioxo-9, 10-dihydroanthracen-2-yl) naphthalene-1-sulfonamide (12)

12 preparation method 4, 4 reaction three in the p-toluene sulfonyl chloride was replaced by naphthalene-1-sulfonyl chloride, yellow solid, yield 65%. MS (ESI) (M/z) 444.0(M-H)^-.¹H NMR(400MHz,DMSO-d₆)δ12.50(brs,1H),10.82(brs,1H),10.71(brs,1H),8.78(d,J＝8.6Hz,1H),8.27–8.16(m,2H),8.06(dd,J＝7.4,13.0Hz,3H),7.80(qd,J＝3.6,7.8Hz,2H),7.72–7.58(m,4H).。

Example 13

Preparation of N- (3, 4-dihydroxy-9, 10-dioxo-9, 10-dihydroanthracen-2-yl) -4-fluorobenzenesulfonamide (13)

13 preparation method 4, 4 reaction three in p-toluene sulfonyl chloride was changed to p-fluorobenzene sulfonyl chloride, yellow solid, yield 62%. MS (ESI) (M/z) 412.0(M-H)^-.¹HNMR(400MHz,DMSO-d₆)δ12.52(brs,1H),10.78(brs,1H),10.37(brs,1H),8.13–8.05(m,2H),7.94–7.78(m,4H),7.67(s,1H),7.39(t,J＝8.7Hz,2H).。

Example 14

Preparation of 4-chloro-N- (3, 4-dihydroxy-9, 10-dioxo-9, 10-dihydroanthracen-2-yl) benzenesulfonamide (14)

14 preparation method 4, 4 reaction three in the p-toluene sulfonyl chloride was changed to p-chlorobenzene sulfonyl chloride, yellow solid, yield 56%. MS (ESI) (M/z) 427.8(M-H)^-.¹H NMR(400MHz,DMSO-d₆)δ12.57(brs,1H),10.91(brs,1H),10.43(brs,1H),8.18–8.07(m,2H),7.93–7.80(m,4H),7.74–7.60(m,3H).。

Example 15

Preparation of N- (3, 4-dihydroxy-9, 10-dioxo-9, 10-dihydroanthracen-2-yl) quinoline-8-sulfonamide (15)

15 preparation method 4, 4 reaction three in the p-toluene sulfonyl chloride was changed to quinoline-8-sulfonyl chloride, dark red solid, yield 40%. MS (ESI) (m)/z):445.0(M-H)^-.¹HNMR(400MHz,DMSO-d₆)δ12.42(brs,1H),11.10(brs,1H),9.42(brs,1H),9.05(d,J＝4.1Hz,1H),8.52(d,J＝8.3Hz,1H),8.40(d,J＝7.3Hz,1H),8.28(d,J＝8.2Hz,1H),8.06(t,J＝6.4Hz,2H),7.89–7.76(m,3H),7.75–7.66(m,2H).。

Example 16

Preparation of N- (3, 4-dihydroxy-9, 10-dioxo-9, 10-dihydroanthracen-2-yl) cyclohexylsulfonamide (16)

16 preparation method 4, 4 reaction three in p-toluene sulfonyl chloride was changed to cyclohexyl sulfonyl chloride, orange solid, yield 60%. MS (ESI) (M/z) 400.0(M-H)^-.¹HNMR(400MHz,DMSO-d₆)δ12.65(brs,1H),10.99(brs,1H),9.46(brs,1H),8.19–8.07(m,2H),7.91–7.79(m,3H),3.05(tt,J＝3.5,12.0Hz,1H),2.05(d,J＝12.4Hz,2H),1.72(d,J＝12.4Hz,2H),1.54(d,J＝12.1Hz,1H),1.38(qd,J＝3.3,12.6Hz,2H),1.25–1.00(m,3H).。

Example 17

Preparation of N- (3, 4-dihydroxy-9, 10-dioxo-9, 10-dihydroanthracen-2-yl) -3, 4-dimethoxybenzenesulfonamide (17)

17 was prepared as in 4 by replacing p-toluenesulfonyl chloride in reaction three, 4, with 3, 4-dimethoxybenzenesulfonyl chloride as a dark red solid in 45% yield. MS (ESI) (M/z) 454.0(M-H)^-.¹H NMR(400MHz,DMSO-d₆)δ12.57(s,1H),10.91(s,1H),10.09(s,1H),8.14–8.04(m,2H),7.89–7.78(m,2H),7.74(s,1H),7.51–7.42(m,2H),7.09(d,J＝8.6Hz,1H),3.78(s,3H),3.76(s,3H).。

Example 18

Preparation of N- (3, 4-dihydroxy-9, 10-dioxo-9, 10-dihydroanthracen-2-yl) butane-1-sulfonamide (18)

18 preparation of 4 As in 4, p-toluenesulfonyl chloride from reaction three 4 was replaced by butane-1-sulfonyl chloride as an orange solid in 39% yield. MS (ESI) (M/z) 374.0(M-H)^-.¹HNMR(400MHz,DMSO-d₆)δ12.61(brs,1H),10.95(brs,1H),9.55(brs,1H),8.18–8.07(m,2H),7.91–7.80(m,2H),7.76(s,1H),3.21–3.13(m,2H),1.66(p,J＝7.7Hz,2H),1.32(h,J＝7.4Hz,2H),0.80(t,J＝7.3Hz,3H).。

Example 19

Preparation of N- (3, 4-dihydroxy-9, 10-dioxo-9, 10-dihydroanthracen-2-yl) - [1, 1' -biphenyl ] -4-sulfonamide (19)

The preparation method of 19 is the same as 4, p-toluenesulfonyl chloride in the reaction III of 4 is replaced by [1, 1' -biphenyl ]]-4-sulfonyl chloride, orange red solid, yield 60%. MS (ESI) (M/z) 470.0(M-H)^-.¹H NMR(400MHz,DMSO-d₆)δ12.53(brs,1H),10.70(brs,1H),10.55(brs,1H),8.15–8.03(m,2H),7.93(d,J＝8.3Hz,2H),7.88–7.77(m,4H),7.76(s,1H),7.66(d,J＝7.6Hz,2H),7.39(dt,J＝7.4,26.0Hz,3H).。

Example 20

Preparation of N- (3, 4-dihydroxy-9, 10-dioxo-9, 10-dihydroanthracen-2-yl) morpholine-4-sulfonamide (20)

20 was prepared as in 4 by replacing p-toluenesulfonyl chloride in reaction three, 4, with morpholine-4-sulfonyl chloride as a dark red solid in 49% yield. MS (ESI) (M/z) 403.0(M-H)^-.¹HNMR(400MHz,DMSO-d₆)δ12.62(brs,1H),10.93(brs,1H),9.81(brs,1H),8.13(dt,J＝4.9,18.3Hz,2H),7.91–7.81(m,2H),7.80(s,1H),3.52(t,J＝4.5Hz,4H),3.06(t,J＝4.6Hz,4H).。

Example 21

Preparation of N- (3, 4-dihydroxy-9, 10-dioxo-9, 10-dihydroanthracen-2-yl) -2, 4-dimethoxybenzenesulfonamide (21)

21 was prepared as in 4 by replacing p-toluenesulfonyl chloride in reaction three, 4, with 2, 4-dimethoxybenzenesulfonyl chloride as a yellow solid in 66% yield. MS (ESI) (M/z) 454.0(M-H)^-.¹H NMR(400MHz,DMSO-d₆)δ12.59(brs,1H),11.00(brs,1H),9.08(brs,1H),8.17–8.05(m,2H),7.90–7.80(m,2H),7.76(s,1H),7.71(d,J＝8.8Hz,1H),6.66(d,J＝2.3Hz,1H),6.60(dd,J＝2.3,8.8Hz,1H),3.82(s,3H),3.76(s,3H).。

Example 22

Preparation of N- (3, 4-dihydroxy-9, 10-dioxo-9, 10-dihydroanthracen-2-yl) -N ', N' -dimethylsulfodiamine (22)

22 preparation of same as 4, 4 reaction three in the p-toluene sulfonyl chloride was replaced by two amino sulfonyl chloride, orange solid, 45% yield. MS (ESI) (M/z) 361.0(M-H)^-.¹HNMR(400MHz,DMSO-d₆)δ12.68(brs,1H),10.95(brs,1H),9.74(brs,1H),8.18(d,J＝18.5Hz,2H),7.92(s,2H),7.84(s,1H),2.77(s,6H).。

Example 23

Preparation of 2, 4-dichloro-N- (3, 4-dihydroxy-9, 10-dioxo-9, 10-dihydroanthracen-2-yl) benzenesulfonamide (23)

23 preparation method 4, 4 reaction three in the p-toluene sulfonyl chloride was changed to 2, 4-two benzene sulfonyl chloride, orange solid, yield 60%. MS (ESI) (M/z) 461.8(M-H)^-.¹H NMR(400MHz,DMSO-d₆)δ12.56(brs,1H),10.94(brs,1H),10.50(brs,1H),8.14–8.02(m,2H),7.95(d,J＝8.6Hz,1H),7.89–7.78(m,3H),7.62–7.57(m,2H).。

Example 24

Preparation of 4-chloro-N- (3, 4-dihydroxy-9, 10-dioxo-9, 10-dihydroanthracen-2-yl) -3-trifluoromethylbenzenesulfonamide (24)

24 preparation method 4, 4 reaction three in the p-toluene sulfonyl chloride was changed to 4-chloro-3-three methyl benzene sulfonyl chloride, yellow solid, yield 71%. MS (ESI) (M/z) 495.8(M-H)^-.¹HNMR(400MHz,DMSO-d₆)δ12.55(brs,1H),10.96(brs,1H),10.61(brs,1H),8.26(d,J＝2.2Hz,1H),8.16–8.07(m,2H),8.03(dd,J＝2.2,8.4Hz,1H),7.92(d,J＝8.5Hz,1H),7.91–7.80(m,2H),7.64(s,1H).。

Example 25

Preparation of N- (3, 4-dihydroxy-9, 10-dioxo-9, 10-dihydroanthracen-2-yl) -4-fluoro-3-trifluoromethylbenzenesulfonamide (25)

25 preparation method 4, 4 reaction three in the p-toluene sulfonyl chloride was changed to 4-fluoro-3-three methyl benzene sulfonyl chloride, yellow solid, yield 50%. MS (ESI) (M/z) 480.0(M-H)^-.¹HNMR(400MHz,DMSO-d₆)δ12.55(brs,1H),10.94(brs,1H),10.55(brs,1H),8.24(dd,J＝2.2,6.7Hz,1H),8.17–8.07(m,3H),7.92–7.81(m,2H),7.72(t,J＝9.6Hz,1H),7.65(s,1H).。

Example 26

Preparation of 5-chloro-N- (3, 4-dihydroxy-9, 10-dioxo-9, 10-dihydroanthracen-2-yl) thiophene-2-sulfonamide (26)

26 is prepared by the same method as 44 the p-toluenesulfonyl chloride in the third reaction is replaced by 5-chlorothiophene-2-sulfonyl chloride, and the yellow solid is obtained with the yield of 52 percent. MS (ESI) (M/z) 433.8(M-H)^-.¹HNMR(400MHz,DMSO-d₆)δ12.61(brs,1H),10.98(brs,1H),10.69(brs,1H),8.22–8.12(m,2H),7.94–7.86(m,2H),7.75(s,1H),7.48(d,J＝4.1Hz,1H),7.22(d,J＝4.1Hz,1H).。

Example 27

Preparation of 2,4, 6-trichloro-N- (3, 4-dihydroxy-9, 10-dioxo-9, 10-dihydroanthracen-2-yl) benzenesulfonamide (27)

27 preparation method 4, 4 three reaction in the same way for toluene sulfonyl chloride was changed to 2,4, 6-three chlorobenzene sulfonyl chloride, yellow solid, yield 10%. MS (ESI) (M/z) 495.8(M-H)^-.¹HNMR(400MHz,DMSO-d₆)δ12.57(brs,1H),10.91(brs,1H),10.69(brs,1H),8.17–8.05(m,2H),7.88-7.83(m,4H),7.61(s,1H).。

Example 28

Preparation of 2, 6-dichloro-N- (3, 4-dihydroxy-9, 10-dioxo-9, 10-dihydroanthracen-2-yl) benzenesulfonamide (28)

28 preparation method 4, 4 reaction three in the p-toluene sulfonyl chloride was changed to 2, 6-two benzene sulfonyl chloride, red solid, yield 12%. MS (ESI) (M/z) 461.8(M-H)^-.¹HNMR(400MHz,DMSO-d₆)δ12.54(brs,1H),10.93(brs,1H),10.48(brs,1H),8.06(dt,J＝5.7,14.9Hz,2H),7.86–7.75(m,2H),7.59(d,J＝8.6Hz,3H),7.50(t,J＝8.1Hz,1H).。

Example 29

Preparation of N- (3, 4-dihydroxy-9, 10-dioxo-9, 10-dihydroanthracen-2-yl) -4-trifluoromethoxybenzenesulfonamide (29)

29 preparation method 4, 4 reaction three in the p-toluene sulfonyl chloride was changed to 4-three methoxy benzene sulfonyl chloride, yellow solid, yield 41%. MS (ESI) (M/z) 478.0(M-H)^-.¹H NMR(400MHz,DMSO-d₆)δ12.58(brs,1H),10.91(brs,1H),10.48(brs,1H),8.19–8.09(m,2H),8.02–7.96(m,2H),7.92–7.82(m,2H),7.71(s,1H),7.58(dd,J＝1.1,9.1Hz,2H).。

Example 30

Preparation of N- (3, 4-dihydroxy-9, 10-dioxo-9, 10-dihydroanthracen-2-yl) -2-trifluoromethoxybenzenesulfonamide (30)

30 preparation method 4, 4 reaction three in the p-toluene sulfonyl chloride was changed to 2-three methoxy benzene sulfonyl chloride, yellow solid, yield 40%. MS (ESI) (M/z) 477.8(M-H)^-.¹H NMR(400MHz,DMSO-d₆)δ12.59(brs,1H),10.97(brs,1H),10.34(brs,1H),8.21–8.06(m,2H),7.97(dd,J＝1.8,8.0Hz,1H),7.93–7.83(m,2H),7.80–7.72(m,1H),7.68(s,1H),7.60–7.48(m,2H).。

Example 31

Preparation of 4-cyclohexyl-N- (3, 4-dihydroxy-9, 10-dioxo-9, 10-dihydroanthracen-2-yl) benzenesulfonamide (31)

31 preparation method 4, 4 reaction three in the p-toluene sulfonyl chloride was changed to 4-cyclohexyl benzene sulfonyl chloride, orange solid, yield 52%. MS (ESI) (M/z) 476.0(M-H)^-.¹H NMR(400MHz,DMSO-d₆)δ12.55(brs,1H),10.92(brs,1H),10.34(brs,1H),8.17–8.06(m,2H),7.91–7.72(m,5H),7.42(d,J＝8.2Hz,2H),2.53(t,J＝3.1Hz,1H),1.68(dd,J＝10.8,26.2Hz,5H),1.42–1.10(m,5H).。

Example 32

Preparation of N- (3, 4-dihydroxy-9, 10-dioxo-9, 10-dihydroanthracen-2-yl) -2,3,4,5, 6-pentafluorobenzenesulfonamide (32)

32 was prepared as in 4 by replacing p-toluenesulfonyl chloride from reaction three, 4,5, 6-pentafluorobenzenesulfonyl chloride, in the form of an orange solid in 15% yield. MS (ESI) (M/z):483.8(M-H)^-.¹H NMR(400MHz,DMSO-d₆)δ12.57(brs,1H),10.91(brs,2H),8.19–8.09(m,2H),7.92–7.83(m,2H),7.61(s,1H).。

Example 33

Preparation of 4-tert-butyl-N- (3, 4-dihydroxy-9, 10-dioxo-9, 10-dihydroanthracen-2-yl) benzenesulfonamide (33)

33 preparation method as 4, 4 reaction three in the p-toluene sulfonyl chloride was changed to 4-tert butyl benzene sulfonyl chloride, orange solid, yield 65%. MS (ESI) (M/z) 450.0(M-H)^-.¹H NMR(400MHz,DMSO-d₆)δ12.54(brs,1H),10.87(brs,1H),10.24(brs,1H),8.12–8.03(m,2H),7.86–7.73(m,5H),7.57(d,J＝8.3Hz,2H),1.20(s,9H).。

Example 34

Preparation of N- (3, 4-dihydroxy-9, 10-dioxo-9, 10-dihydroanthracen-2-yl) -4 '-fluoro- [1, 1' -biphenyl ] -4-sulfonamide (34)

34 the preparation method is the same as 4, p-toluenesulfonyl chloride in the 4-reaction step is changed into 4 '-fluoro- [1, 1' -biphenyl]-4-sulfonyl chloride, orange solid, yield 53%. MS (ESI) (M/z) 488.0(M-H)^-.¹HNMR(400MHz,DMSO-d₆)δ12.55(brs,1H),10.91(brs,1H),10.34(brs,1H),8.14–8.03(m,2H),7.93(d,J＝8.2Hz,2H),7.84(dd,J＝2.7,7.9Hz,4H),7.78–7.68(m,3H),7.26(t,J＝8.7Hz,2H).。

Example 35

Preparation of 4 '-chloro-N- (3, 4-dihydroxy-9, 10-dioxo-9, 10-dihydroanthracen-2-yl) - [1, 1' -biphenyl ] -4-sulfonamide (35)

35 the preparation method is the same as 4, p-toluenesulfonyl chloride in the 4-reaction step is changed into 4 '-chloro- [1, 1' -biphenyl]-4-sulfonyl chloride, yellow solid, yield 55%. MS (ESI) (M/z) 504.0(M-H)^-.¹HNMR(400MHz,DMSO-d₆)δ12.56(brs,1H),10.91(brs,1H),10.37(brs,1H),8.15–8.06(m,2H),7.96–7.81(m,6H),7.76(s,1H),7.71(d,J＝8.6Hz,2H),7.50(d,J＝8.3Hz,2H).。

Example 36

Preparation of N- (3, 4-dihydroxy-9, 10-dioxo-9, 10-dihydroanthracen-2-yl) -2-fluorobenzenesulfonamide (36)

36 preparation method 4, 4 reaction three in the p-toluene sulfonyl chloride was changed to 2-fluorobenzene sulfonyl chloride, yellow solid, yield 42%. MS (ESI) (M/z) 412.0(M-H)^-.¹H NMR(400MHz,DMSO-d₆)δ12.56(brs,1H),10.90(brs,1H),10.44(brs,1H),8.16–8.03(m,2H),7.90–7.75(m,3H),7.71–7.62(m,2H),7.40(t,J＝9.4Hz,1H),7.32(t,J＝7.7Hz,1H).。

Example 37

Preparation of N- (3, 4-dihydroxy-9, 10-dioxo-9, 10-dihydroanthracen-2-yl) -2, 3-difluorobenzenesulfonamide (37)

37 was prepared as in 4 by replacing p-toluenesulfonyl chloride in reaction three, 4, with 2, 3-difluorobenzenesulfonyl chloride as a dark red solid in 52% yield. MS (ESI) (M/z) 412.0(M-H)^-.¹H NMR(400MHz,DMSO-d₆)δ12.57(brs,1H),10.80(brs,2H),8.20–8.08(m,2H),7.93–7.83(m,2H),7.76(q,J＝8.6Hz,1H),7.66(s,1H),7.59(t,J＝7.0Hz,1H),7.41–7.31(m,1H).。

Example 38

Preparation of 3-bromo-N- (3, 4-dihydroxy-9, 10-dioxo-9, 10-dihydroanthracen-2-yl) benzenesulfonamide (38)

38 was prepared as in 4 by replacing p-toluenesulfonyl chloride in reaction three, 4, with 3-bromobenzenesulfonyl chloride as a yellow solid in 54% yield. MS (ESI) (M/z):473.8(M-H)^-.¹H NMR(400MHz,DMSO-d₆)δ12.54(brs,1H),10.93(brs,1H),10.48(brs,1H),8.15–8.05(m,2H),8.04(s,1H),7.88–7.77(m,4H),7.65(s,1H),7.51(t,J＝8.0Hz,1H).。

Example 39

Preparation of 2-bromo-N- (3, 4-dihydroxy-9, 10-dioxo-9, 10-dihydroanthracen-2-yl) benzenesulfonamide (39)

39 para-toluenesulfonyl chloride from reaction three, 4, was replaced with 2-bromobenzenesulfonyl chloride as was 4, a dark yellow solid, 46% yield. MS (ESI) (M/z) 471.8(M-H)^-.¹HNMR(400MHz,DMSO-d₆)δ12.57(brs,1H),11.05(brs,1H),10.19(brs,1H),8.14–8.00(m,3H),7.88–7.79(m,3H),7.61–7.48(m,3H).。

Example 40

Preparation of 2-iodo-N- (3, 4-dihydroxy-9, 10-dioxo-9, 10-dihydroanthracen-2-yl) benzenesulfonamide (40)

40 preparation method 4, 4 reaction three in the p-toluene sulfonyl chloride was changed to 2-iodine benzene sulfonyl chloride, dark yellow solid, yield 48%. MS (ESI) (M/z) 519.8(M-H)^-.¹HNMR(400MHz,DMSO-d₆)δ12.59(brs,1H),11.13(brs,1H),9.98(brs,1H),8.15–8.01(m,4H),7.88–7.80(m,2H),7.60(s,1H),7.56(td,J＝1.2,7.7Hz,1H),7.28(td,J＝1.6,7.6Hz,1H).。

EXAMPLE 41

Preparation of N- (3, 4-dihydroxy-9, 10-dioxo-9, 10-dihydroanthracen-2-yl) -3-methoxybenzenesulfonamide (41)

41 para-toluenesulfonyl chloride from reaction three, reaction 4, was replaced with 3-methoxybenzenesulfonyl chloride as orange solid in 51% yield as in 4. MS (ESI))(m/z):424.0(M-H)^-.¹H NMR(400MHz,DMSO-d₆)δ12.55(brs,1H),10.91(brs,1H),10.31(brs,1H),8.14–8.03(m,2H),7.88–7.78(m,2H),7.70(s,1H),7.50–7.38(m,3H),7.16(dt,J＝2,8Hz,1H),3.76(s,3H).。

Example 42

Preparation of Ethyl 2- ((4- ((4-chlorophenyl) sulfonylamino) -1-hydroxy-9, 10-dioxo-9, 10-dihydroanthracen-2-yl) oxy) acetate (42)

HKb-2 is prepared by the first reaction of HKb-6.

II, preparation of Ethyl 2- ((1-hydroxy-4-nitro-9, 10-dioxo-9, 10-dihydroanthracene-1, 2-yl) oxy) acetate (HKb-12)

HKb-12 preparation method is the same as the first reaction method of preparation 4, in which 1, 2-dihydroxyanthracene-9, 10-dione is changed to ethyl 2- ((1-hydroxy-9, 10-dioxo-9, 10-dihydroanthracene-1, 2-yl) oxy) acetate (HKb-2), to obtain yellow solid with 73% yield.

Preparation of Ethyl 2- ((4-amino-1-hydroxy-9, 10-dioxo-9, 10-dihydroanthracene-1, 2-yl) oxy) acetate (HKb-18)

HKb-18 was prepared by the same procedure as that of preparation 4, wherein 3-nitro-1, 2-dihydroxyanthracene-9, 10-dione (HKb-10) was replaced by ethyl 2- ((1-hydroxy-4-nitro-9, 10-dioxo-9, 10-dihydroanthracene-1, 2-yl) oxy) acetate (HKb-12) to give a purple solid with a crude yield of 93%.

Fourthly, preparing ethyl 2- ((4- ((4-chlorphenyl) sulfamide) -1-hydroxy-9, 10-dioxo-9, 10-dihydroanthracene-2-

Yl) oxy) acetate (42)

42 can be prepared by the same procedure as 4, except that p-toluenesulfonyl chloride in reaction III of preparation 4 is replaced by p-chlorobenzenesulfonyl chloride, and 3-amino-1, 2-dihydroxyanthracene-9, 10-dione (HKb-11) is replaced by ethyl 2- ((4-amino-1-hydroxy-9, 10-dioxo-9, 10-dihydroanthracene-1, 2-yl) oxy) acetate (HKb-18), as a red solid, in 6% yield. MS (ESI) (M/z):514.2(M-H)^-.¹H NMR(400MHz,DMSO-d₆)δ13.31(s,1H),12.39(s,1H),8.14(d,J＝7.2Hz,2H),7.89(p,J＝7.3Hz,2H),7.81(d,J＝8.3Hz,2H),7.59(d,J＝8.3Hz,2H),7.26(s,1H),5.04(s,2H),4.19(q,J＝7.1Hz,2H),1.21(t,J＝7.1Hz,3H).。

Example 43

Preparation of 2- ((4- ((4-chlorophenyl) sulfonylamino) -1-hydroxy-9, 10-dioxo-9, 10-dihydroanthracen-2-yl) oxy) acetic acid (43)

43 preparation method As in reaction two of preparation 3, ethyl 2- ((1-hydroxy-9, 10-dioxo-9, 10-dihydroanthracen-1, 2-yl) oxy) acetate (HKb-2) was changed to ethyl 2- ((4- ((4-chlorophenyl) sulfonamido) -1-hydroxy-9, 10-dioxo-9, 10-dihydroanthracen-2-yl) oxy) acetate (42) as a red solid in 95% yield. MS (ESI) (M/z) 485.8(M-H)^-.¹H NMR(400MHz,DMSO-d₆)δ13.30(s,1H),12.46(s,1H),8.12(d,J＝6.5Hz,2H),7.85(dt,J＝7.4,23.9Hz,4H),7.57(d,J＝8.2Hz,2H),7.29(s,1H),4.93(s,2H).。

Example 44

Preparation of Ethyl 2- ((3- ((4-chlorophenyl) sulfonylamino) -1-hydroxy-9, 10-dioxo-9, 10-dihydroanthracen-2-yl) oxy) acetate (44)

44 preparation method and 3 preparation method reaction one, 1, 2-dihydroxy anthracene-9, 10-diketone is changed into 4-chlorine-N- (3, 4-dihydroxy)Yl-9, 10-dioxo-9, 10-dihydroanthracen-2-yl) benzenesulfonamide (14) as a yellow solid in 24% yield. MS (ESI) (M/z) 514.0(M-H)^-.¹H NMR(400MHz,DMSO-d₆)δ12.65(s,1H),10.51(s,1H),8.14–8.01(m,2H),7.92–7.78(m,4H),7.69(s,1H),7.63(d,J＝8.3Hz,2H),4.77(s,2H),4.10(q,J＝7.1Hz,2H),1.14(t,J＝7.2Hz,3H).。

Example 45

Preparation of 2- ((3- ((4-chlorophenyl) sulfonylamino) -1-hydroxy-9, 10-dioxo-9, 10-dihydroanthracen-2-yl) oxy) acetic acid (45)

45 as in 2, diethyl 2,2' - ((9, 10-dioxo-9, 10-dihydroanthracen-1, 2-yl) dioxo) diacetate (1) was replaced by ethyl 2- ((3- ((4- ((chlorophenyl) sulfonamido) -1-hydroxy-9, 10-dioxo-9, 10-dihydroanthracen-2-yl) oxy) acetate (44) as a yellow solid in 95% yield. MS (ESI) (M/z) 486.0(M-H)^-.¹H NMR(400MHz,DMSO-d₆)δ12.67(s,1H),8.14–8.05(m,2H),7.92–7.82(m,4H),7.70(s,1H),7.66(d,J＝8.6Hz,2H),4.75(s,2H).。

Example 46

Preparation of 4-chloro-N- (4-hydroxy-3-methoxy-9, 10-dioxo-9, 10-dihydroanthracen-2-yl) benzenesulfonyl chloride (46)

A25 mL round-bottomed flask was charged with 4-chloro-N- (3, 4-dihydroxy-9, 10-dioxo-9, 10-dihydroanthracen-2-yl) benzenesulfonamide (14) (215mg,0.5mmol), potassium carbonate (70mg,0.5mmol), potassium iodide (70mg,12mmol) and N, N-dimethylformamide (2.5mL), reacted at room temperature, and the reaction was stopped by TLC detection of disappearance of the starting material spot. After the reaction mixture was cooled to room temperature, the reaction mixture was added dropwise to about 20mL of 10% hydrochloric acid, extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, and separated by silica gel column chromatography (petroleum ether: dichloromethane ═ 1:9) to obtain 115mg of a yellow solid with a yield of 52%. MS (ESI) (m/z):442.0(M-H)^-.¹HNMR(400MHz,DMSO-d₆)δ12.66(brs,1H),11.00(brs,1H),8.26–8.12(m,2H),7.97–7.87(m,2H),7.68(q,J＝8.5Hz,4H),7.42(s,1H),3.18(s,3H).。

Example 47

Preparation of 4-chloro-N- (4-hydroxy-3-methoxy-9, 10-dioxo-9, 10-dihydroanthracen-2-yl) -N-methylbenzenesulfonyl chloride (47)

47 was prepared by the same method as 46, with a room temperature of 40 ℃ to give an orange solid with a yield of 50%. MS (ESI) (M/z) 456.0(M-H)^-.¹HNMR(400MHz,DMSO-d₆)δ12.73(s,1H),8.22–8.07(m,2H),7.95–7.86(m,2H),7.70(q,J＝8.5Hz,4H),7.35(s,1H),3.74(s,3H),3.17(s,3H).。

Example 48

Preparation of 4- ((4-chlorophenyl) sulfonyl) -12-hydroxy-3, 4-dihydro-2H-anthrac [2,3-b ] [1,4] oxazine-6, 11-dione (48)

48, the ethyl bromoacetate was replaced with 1, 2-dibromoethane to give an orange solid in 32% yield. MS (ESI) (M/z) 456.0(M + H)⁺.¹HNMR(400MHz,DMSO-d₆)δ12.59(s,1H),8.13(t,J＝8.2Hz,2H),8.07(s,1H),7.93–7.83(m,2H),7.79(d,J＝8.4Hz,2H),

7.67(d,J＝8.3Hz,2H),4.01(dd,J＝4.5,16.7Hz,4H).。

Example 49

Preparation of 4-chloro-N- (3- (2- (1, 3-dioxoisoindol-2-yl) ethoxy) -4-hydroxy-9, 10-dioxo-9, 10-dihydroanthracen-2-yl) benzenesulfonamide (49)

Preparation of 2- (2-bromoethyl) isoindole-1, 3-dione

The preparation method of 2- (2-bromoethyl) isoindole-1, 3-dione is the same as that of 1, the amount of ethyl bromoacetate is changed to 1, 2-dibromoethane, and potassium carbonate is changed to potassium hydroxide, so that white solid is obtained, and the yield is 73%.

Secondly, preparing 4-chloro-N- (3- (2- (1, 3-dioxo isoindol-2-yl) ethoxy) -4-hydroxy-9, 10-dioxo-9, 10-dihydro

Anthracene-2-yl) benzenesulfonamide (49)

49 is prepared by the same method as 1, and ethyl bromoacetate is replaced by 2- (2-bromoethyl) isoindole-1, 3-dione to obtain an orange solid with a yield of 65%. MS (ESI) (M/z) 601.0(M-H)^-.¹HNMR(400MHz,DMSO-d₆)δ12.54(s,1H),10.64(s,1H),8.05(d,J＝6.6Hz,2H),7.90–7.68(m,9H),7.63(d,J＝8.3Hz,2H),4.22(t,J＝5.6Hz,2H),3.95(t,J＝5.6Hz,2H).。

Example 50

Preparation of 4-chloro-N- (3- (2-hydrazide-2-ethoxy) -4-hydroxy-9, 10-dioxo-9, 10-dihydroanthracen-2-yl) benzenesulfonyl chloride (50)

44(20mg,0.04mmol), hydrazine hydrate (85%, 5. mu.L, 0.16mmol) and ethanol (1.5mL) were added to a 5mL round bottom flask, and the mixture was refluxed at 76 ℃ and stopped by TLC detection of disappearance of the starting material spot, and the reaction solution was precipitated as a yellow solid, which was filtered with suction to obtain 14mg, yield 72%. MS (ESI) (M/z) 500.0(M-H)^-.¹H NMR(400MHz,DMSO-d₆)δ12.93(brs,1H),8.41–7.34(m,9H),4.51(s,2H).。

Example 51

Preparation of 3- ((4-chlorophenyl) sulfonylamino) -1-hydroxy-9, 10-dioxo-9, 10-dihydroanthracen-2-yl methanesulfonate (51)

14(107mg,0.25mmol), N-diisopropylethylamine (50. mu.L, 0.3mmol) and pyridine (1.5mmol) were added to a 5mL round-bottomed flask, followed by addition of methanesulfonyl chloride (23. mu.L, 0.3mmol) and reaction at room temperature for 4 h. The reaction solution was dropwise added to 10% hydrochloric acid, pH was adjusted to 1-2, extraction was performed with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, and subjected to silica gel column chromatography (dichloromethane) to obtain 83mg of a yellow solid, with a yield of 65%. MS (ESI) (M/z) 505.8(M-H)^-.¹H NMR(400MHz,DMSO-d₆)δ12.74(s,1H),8.11–8.02(m,2H),7.97(d,J＝8.6Hz,2H),7.87–7.80(m,2H),7.71(d,J＝8.3Hz,2H),7.59(s,1H),3.60(s,3H).。

Example 52

Preparation of 3- ([1, 1' -biphenyl ] -4-sulfonylamino) -1-hydroxy-9, 10-dioxo-9, 10-dihydroanthracen-2-yl acetate (52)

52 was prepared as in 4 by replacing p-toluenesulfonyl chloride in reaction three, 4, with acetyl chloride and HKb-11 with 19 as a yellow solid in 42% yield. MS (ESI) (M/z) 470.1(M-Ac)^-.¹H NMR(400MHz,DMSO-d₆)δ12.50(s,1H),11.21(brs,1H),8.13–8.03(m,2H),8.02–7.77(m,7H),7.66(d,J＝7.6Hz,2H),7.39(dt,J＝7.4,24.8Hz,3H),2.31(s,3H).。

Example 53

Preparation of N- (3, 4-dihydroxy-9, 10-dioxo-9, 10-dihydroanthracen-2-yl) -4- (piperidin-1-yl) benzenesulfonamide (53)

Firstly, preparing N- (3, 4-dihydroxy-9, 10-dioxo-9, 10-dihydroanthracene-2-yl) -4-iodobenzene sulfonamide

The preparation method of N- (3, 4-dihydroxy-9, 10-dioxo-9, 10-dihydroanthracene-2-yl) -4-iodobenzene sulfonamide is the same as that of 4, p-toluenesulfonyl chloride in the reaction step three of 4 is replaced by p-iodobenzene sulfonyl chloride, so that yellow solid is obtained, and the yield is 54%.

II, preparation of N- (3, 4-dihydroxy-9, 10-dioxo-9, 10-dihydroanthracen-2-yl) -4- (piperidin-1-yl) benzenesulfonamide (53)

N- (3, 4-dihydroxy-9, 10-dioxo-9, 10-dihydroanthracen-2-yl) -4-iodobenzenesulfonamide (52.1mg,0.1mmol), tris (dibenzylideneacetone) dipalladium (9.2mg,0.01mmol), (+ -) -2,2 '-bis- (diphenylphosphino) -1,1' -binaphthyl (9.3mg,0.015mmol), sodium tert-butoxide (48.1mg,0.5mmol) and piperidine (1mL) were added to a 5mL round-bottomed flask, and vacuum and nitrogen were sequentially applied three times, the reaction was carried out at 80 ℃ and the disappearance of the starting material point was detected by TLC to stop the reaction. After the reaction solution was cooled to room temperature, the reaction solution was added dropwise to about 20mL of 10% hydrochloric acid, extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, and subjected to silica gel column chromatography (dichloromethane) to obtain 33mg of an orange solid in a yield of 68%. MS (ESI) (M/z) 477.0(M-H)^-.¹HNMR(400MHz,DMSO-d₆)δ8.15–8.03(m,2H),7.88–7.79(m,2H),7.76(s,1H),7.61(d,J＝8.7Hz,2H),6.92(d,J＝8.8Hz,2H),3.43–3.14(m,8H),1.04(t,J＝7.0Hz,2H).。

Example 54

Preparation of N- (3, 4-dihydroxy-9, 10-dioxo-9, 10-dihydroanthracen-2-yl) -4- (4-methylpiperazin-1-yl) benzenesulfonamide (54)

54 was prepared as in 53, with the piperidine in reaction two being replaced with methylpiperazine as a dark yellow solid in 42% yield. MS (ESI) (M/z) 492.0(M-H)^-.¹HNMR(400MHz,DMSO-d₆)δ10.75(brs,2H),8.15(dd,J＝6.5,15.5Hz,2H),7.95–7.84(m,2H),7.80(s,1H),7.75(d,J＝8.6Hz,2H),7.12(d,J＝8.7Hz,2H),3.38(s,4H),3.23(s,4H),2.76(s,3H).。

Example 55

Preparation of N- (3, 4-dihydroxy-9, 10-dioxo-9, 10-dihydroanthracen-2-yl) -4- (3, 5-dimethylpiperidin-1-yl) benzenesulfonamide (55)

55 was prepared as in 53, substituting piperidine in reaction two for 3, 5-dimethylpiperidine in the form of an orange solid in 63% yield. MS (ESI) (M/z) 505.0(M-H)^-.¹HNMR(400MHz,DMSO-d₆)δ12.56(brs,1H),10.81(brs,1H),9.83(brs,1H),8.17–8.05(m,2H),7.90–7.80(m,2H),7.76(s,1H),7.60(d,J＝8.9Hz,2H),6.94(d,J＝8.9Hz,2H),3.80(d,J＝12.2Hz,2H),2.24(t,J＝12.2Hz,2H),1.67(d,J＝12.9Hz,1H),1.58–1.41(m,2H),1.04(t,J＝7.0Hz,1H),0.80(d,J＝6.6Hz,6H).。

Example 56

Preparation of N- (3, 4-dihydroxy-9, 10-dioxo-9, 10-dihydroanthracen-2-yl) -4- (pyrrolidin-1-yl) benzenesulfonamide (56)

56 was prepared as in 53, substituting piperidine in reaction two for pyrrolidine, as a yellow solid, 65% yield. MS (ESI) (M/z) 463.0(M-H)^-.¹H NMR(400MHz,DMSO-d₆)δ12.60(brs,1H),10.83(brs,1H),9.80(brs,1H),8.20–8.08(m,2H),7.93–7.84(m,2H),7.82(s,1H),7.66(d,J＝8.8Hz,2H),6.56(d,J＝8.7Hz,2H),3.24(t,J＝6.4Hz,4H),1.96–1.84(m,4H).。

Example 57

Preparation of N- (3, 4-dihydroxy-9, 10-dioxo-9, 10-dihydroanthracen-2-yl) -3- (pyrrolidin-1-yl) benzenesulfonamide (57)

57 was prepared as in 53, replacing piperidine in reaction two with pyrrolidine and N- (3, 4-dihydroxy-9, 10-dioxo-9, 10-dihydroanthracen-2-yl) -4-iodobenzenesulfonamide with 38 as a dark red solid in 51% yield. MS (ESI) (M/z) 463.0(M-H)^-.¹H NMR(400MHz,DMSO-d₆)δ12.55(brs,1H),10.87(brs,1H),10.13(brs,1H),8.14–8.04(m,2H),7.88–7.78(m,2H),7.75(s,1H),7.27(t,J＝7.9Hz,1H),7.06–6.98(m,2H),6.67(d,J＝8.2Hz,1H),3.16(t,J＝6.4Hz,4H),1.96–1.83(m,4H).。

Example 58

Preparation of 4- (azetidin-1-yl) -N- (3, 4-dihydroxy-9, 10-dioxo-9, 10-dihydroanthracen-2-yl) benzenesulfonamide (58)

58 was prepared as in 53, replacing piperidine in reaction two with azetidine as an orange solid in 63% yield. MS (ESI) (M/z) 449.0(M-H)^-.¹HNMR(400MHz,DMSO-d₆)δ12.58(brs,1H),10.82(brs,1H),9.81(brs,1H),8.17–8.08(m,2H),7.92–7.82(m,2H),7.77(s,1H),7.63(d,J＝8.8Hz,2H),6.38(d,J＝8.8Hz,2H),3.85(t,J＝7.3Hz,4H),2.27(p,J＝7.3Hz,2H).。

Example 59

Preparation of N- (3, 4-dihydroxy-9, 10-dioxo-9, 10-dihydroanthracen-2-yl) -2- (pyrrolidin-1-yl) benzenesulfonamide (59)

36(143mg,0.34mmol), sodium tert-butoxide (166mg,1.7mmol) and pyrrolidine (2mL) were added to a 10mL round bottom flask, reacted at 90 ℃ and stopped by TLC detection of disappearance of the starting material spot. After the reaction solution was cooled to room temperature, the reaction solution was added dropwise to about 20mL of 10% hydrochloric acid, extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, and subjected to silica gel column chromatography (dichloromethane) to obtain 103mg of an orange solid with a yield of 65%. MS (ESI) (M/z) 463.0(M-H)^-.¹HNMR(400MHz,DMSO-d₆)δ12.58(brs,1H),11.20(brs,1H),9.56(brs,1H),8.16–8.02(m,2H),7.88–7.78(m,3H),7.69(s,1H),7.47(t,J＝7.8Hz,1H),7.34(d,J＝8.1Hz,1H),7.07(t,J＝7.6Hz,1H),3.16(t,J＝6.0Hz,4H),1.85(t,J＝6.0Hz,4H).。

Example 60

Preparation of N- (3, 4-dihydroxy-9, 10-dioxo-9, 10-dihydroanthracen-2-yl) -4-dimethylaminobenzenesulfonamide (60)

Preparation of 4-dimethylaminobenzenesulfonyl chloride

4-dimethylaminobenzenesulfonic acid (402mg,2mmol) is added to 5.5mL of dry dichloromethane, stirred to dissolve, and then phosphorus pentachloride (458mg,2.2mmol) is slowly added and stirred for 1.5h, and the reaction can be directly carried out without purification.

II, preparing N- (3, 4-dihydroxy-9, 10-dioxo-9, 10-dihydroanthracene-2-yl) -4-dimethylaminobenzenesulfonamide

60 the preparation method is the same as 4, the p-toluenesulfonyl chloride in the third reaction step 4 is replaced by the 4-dimethylaminobenzenesulfonyl chloride reaction liquid in the previous step, red solid is obtained, and the yield is 20%.¹HNMR(400MHz,DMSO-d₆)δ12.59(brs,1H),10.79(brs,1H),9.79(brs,1H),8.23–8.11(m,2H),7.95–7.79(m,3H),7.67(d,J＝8.6Hz,2H),6.74(d,J＝8.6Hz,3H),2.95(s,6H).。

Example 61

Preparation of 2- (N- (3, 4-dihydroxy-9, 10-dioxo-9, 10-dihydroanthracene-2-yl) sulfonamide) phenyl acetate (61) one, preparation of sodium 2-acetoxybenzenesulfonate

Sodium 2-hydroxybenzenesulfonate (2g,10mmol) and acetyl chloride (10mL) were added to a dry 50mL round-bottomed flask, reacted at 50 ℃ for 5h, and concentrated under reduced pressure to remove acetyl chloride, which was allowed to proceed to the next reaction without purification.

Preparation of 2- (sulfonyl chloride) phenyl acetate

Adding thionyl chloride (10mL) and one drop of N, N-dimethylformamide into the sodium 2-acetoxy benzene sulfonate obtained in the previous step, reacting for 5 hours, concentrating under reduced pressure to remove thionyl chloride, washing the residual liquid with 5mL of toluene, and concentrating under reduced pressure to remove toluene three times to obtain a gray oily liquid, wherein the crude product can be directly subjected to the next step without purification. Thirdly, preparing 2- (N- (3, 4-dihydroxy-9, 10-dioxo-9, 10-dihydroanthracene-2-yl) sulfamide) phenyl acetate

61 was prepared as in 4 by replacing p-toluenesulfonyl chloride in reaction three, 4, with the 2- (sulfonylchloro) phenylacetate obtained in the previous step as a dark yellow solid in 40% yield. MS (ESI) (M/z) 463.0(M-H)^-.¹HNMR(400MHz,DMSO-d₆)δ8.07–7.96(m,2H),7.83–7.70(m,4H),7.63(s,1H),7.22(d,J＝8.5Hz,2H),2.14(s,3H).。

Example 62

Preparation of N- (6-bromo-3, 4-dihydroxy-9, 10-dioxo-9, 10-dihydroanthracen-2-yl) -4-methylbenzenesulfonamide (62)

Preparation of 6/7-bromo-1, 2-dihydroxyanthracene-9, 10-dione

Adding aluminum chloride (50g) and sodium chloride (11g) into a 250mL round-bottom flask, stirring at 125 ℃ to a molten state, adding 4-bromophthalic anhydride (7.38g, 32.5mmol) and catechol (2.75g,25mmol), gradually heating to 185 ℃, stirring for 4h, cooling a reaction system, adding into ice hydrochloric acid, refluxing at 100 ℃ for 1h, extracting with ethyl acetate, washing with saturated saline, drying with anhydrous sodium sulfate, and separating by silica gel column chromatography (petroleum ether: ethyl acetate: 17: 3) to obtain 1.12g of an orange solid with the yield of 15%.

Preparation of 6/7-bromo-1, 2-dihydroxy-3-nitroanthracene-9, 10-dione

6/7-bromo-1, 2-dihydroxy-3-nitroanthracene-9, 10-dione is prepared by the same method as 3-nitro-1, 2-dihydroxyanthracene-9, 10-dione (HKb-10), and 6/7-bromo-1, 2-dihydroxyanthracene-9, 10-dione obtained in the previous step is replaced with 1, 2-dihydroxyanthracene-9, 10-dione in HKb-10 reaction to obtain yellow solid with crude yield of 60%.

Preparation of 3-amino-6/7-bromo-1, 2-dihydroxyanthracene-9, 10-dione

3-amino-6/7-bromo-1, 2-dihydroxyanthracene-9, 10-dione is prepared by the same method as 3-amino-1, 2-dihydroxyanthracene-9, 10-dione (HKb-11), and HKb-10 in HKb-11 reaction is replaced by 6/7-bromo-1, 2-dihydroxy-3-nitroanthracene-9, 10-dione obtained in the previous step, to obtain a black solid with a crude yield of 80%.

Fourthly, preparing N- (6-bromo-3, 4-dihydroxy-9, 10-dioxo-9, 10-dihydroanthracene-2-yl) -4-methylbenzenesulfonamide

The preparation method of N- (6-bromo-3, 4-dihydroxy-9, 10-dioxo-9, 10-dihydroanthracene-2-yl) -4-methylbenzenesulfonamide is the same as that of N- (3, 4-dihydroxy-9, 10-dioxo-9, 10-dihydroanthracene-2-yl) -4-methylbenzenesulfonamide (4), HKb-11 in the 4 reaction step is replaced by the 3-amino-6/7-bromo-1, 2-dihydroxyanthracene-9, 10-dione obtained in the previous step, and finally, yellow solid is obtained by silica gel column chromatography separation (dichloromethane) and preparative high performance liquid phase separation (methanol: water: 13:7), and the yield is 15%. MS (ESI) (M/z) 488.0(M-H)^-.¹H NMR(400MHz,DMSO-d₆)δ12.50(brs,1H),10.20(brs,1H),9.74(brs,1H),8.00(s,1H),7.90–7.83(m,2H),7.78(d,J＝8.2Hz,2H),7.70(s,1H),7.34(d,J＝8.0Hz,2H),2.30(s,3H).。

Example 63

The 9, 10-anthraquinone compound of the invention tests the in vitro enzyme inhibition activity of PGAM1

The enzyme-linked assay is used for determining the inhibitory activity of the small molecule inhibitor on PGAM1, and the specific reaction steps are as follows:

3-phosphoglycerate (3PG) is taken as a substrate, the substrate is converted into lactic acid through the combined action of four enzymes of phosphoglycerate mutase1(PGAM1), enolase, Pyroltate Kinase (PK) and Lactate Dehydrogenase (LDH), and a molecule reduced for amino-adenine dinucleotide (NADH) is oxidized to generate Nicotinamide Adenine Dinucleotide (NAD) in the last step of reaction⁺) We measured the inhibitory ability of small molecules by measuring the change in NADH signal (λ 340 nm). Since the enzyme-linked reaction involves 4 enzymes, in order to verify the specificity and specificity of the action of the small molecular compound, 2-phosphoglycerate (2PG) is used as a substrate, and enolase, PK and LDH are used as catalytic enzymes at the same time, so as to determine the reaction rate as a negative control, thereby excluding false positive small molecular compounds which can inhibit the following 3 target enzymes.

The main reagents of the experiment are as follows:

recombinant PGAM1 (purified by E.coli expression), Enolase (Sigma-Aldrich), PKM2(Sigma-Aldrich), LDH (Sigma-Aldrich), Tris-HCl (national drug group chemical Co., Ltd.), KCl (national drug group chemical Co., Ltd.), MgCl₂(national pharmaceutical group chemical Co., Ltd.), ADP (Sigma-Aldrich), NADH (Sigma-Aldrich).

The experimental steps are as follows:

the formulation contained 100mM Tris-HCl,100mM KCl,5mM MgCl₂1mM ADP,0.2mM ADH,5mg/ml PGAM1,0.5units/ml enolase,0.5units/ml pyruvate kinase M2, and0.1units/ml LDH, and adding the final concentrationThe reaction was started with 2mM 3PG and the reduction of ultraviolet absorption (OD:340nm) by oxidation of NADH was taken as the activity of PGAM 1. Control experiments were also performed, with the formulation containing 100mM Tris-HCl,100mM KCl,5mM MgCl₂1mM ADP,0.2mM ADH,0.5units/ml enolase,0.5units/ml pyroltate kinase M2, and0.1units/ml LDH, and 2PG was added to a final concentration of 2mM to initiate the reaction, and the activity as enolase, PKM2 and LDH was decreased by ultraviolet absorption (OD:340nm) due to oxidation of NADH, thereby excluding false positives.

Example 64

Experiment for inhibiting tumor cell viability in vitro by using 9, 10-anthraquinone compounds

Cell culture:

h1299 was cultured in RPMI 1640 medium containing 10% total bone serum (FBS) and 100units/mL penicillin, 100mg/mL streptomycin. The culture conditions are as follows: 37 ℃ and 5% CO₂。

Cell viability assay:

1X 10 per well in 96-well plates³Cells were incubated 24h later with drug-containing medium for 72h, medium removed, medium with 0.5mg/mLMTT added, incubated 4h, medium removed, 200. mu.L DMSO added, and UV absorbance (OD:570nm) read. Inputting the corresponding drug concentration and OD value into GraphPadprism 7 software, and calculating the half Effective Concentration (EC)₅₀). The activity of other tumor cells (PANC-1, K562) was determined similarly to H1299.

TABLE 1 inhibitory Activity of Compounds of formula I on phosphoglycerate mutase and different tumor cell lines

Median inhibitory concentration IC₅₀：*:>100μM,**:10-100μM,***:1-10μM,****:<1μM。

Claims

1. A 9, 10-anthraquinone compound shown in the structure of formula (I) or pharmaceutically acceptable salt thereof;

in the formula (I), the compound is shown in the specification,

R₁selected from hydroxyl;

R₂selected from hydroxyl;

R₃substituted at the 3-position as shown in formula (I);

wherein R is₄Selected from the group consisting of alkyl of less than 12 carbon atoms, phenyl, pyridyl, naphthyl, biphenyl, and substituted versions of the foregoing;

wherein the substituents of said substituted radicals are selected from the group consisting of F, Cl, Br, I, NO₂，CN，-CF₃，-OCF₃Any one to five substituents of hydroxyl and amino;

R₅selected from hydrogen;

R₆selected from hydrogen or halogen.

2. A compound selected from the following structures:

3. a pharmaceutical composition comprising the 9, 10-anthraquinone compound according to any one of claims 1 to 2, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

4. Use of the 9, 10-anthraquinone compounds, their pharmaceutically acceptable salts and their pharmaceutical compositions with pharmaceutically acceptable carriers according to any one of claims 1-2 in the preparation of phosphoglycerate mutase small molecule inhibitors.