CN104817490B - Dithiocarbamates compound and preparation method and application - Google Patents

Abstract

The invention discloses aminodithiocarbamate compounds, a preparation method and application thereof, and belongs to the field of medicinal chemistry. The structure of the compound of the present invention is shown in formula I. Studies have shown that the compound of the present invention targets PKM2 and is an agonist of PKM2, and it is found that this type of compound has an anti-tumor effect by preventing PKM2 from entering the nucleus, and has a good anti-tumor effect. And it has good selectivity for tumor cells. Therefore, the compound of the invention can be used as an antitumor drug and has broad application value.

Description

Carbamate compound and its preparation method and application

technical field

The present invention relates to compounds and their preparation methods and applications, in particular to novel carbamodithiocarbamate compounds and their preparation methods and applications. These compounds are a new class of PKM2 agonists and can be used as antitumor drugs, belonging to Medicinal Chemistry field.

Background technique

Anticancer drug research has always been one of the hot spots in drug research all over the world. At present, there are many effective anti-tumor drugs, but there are few anti-tumor drugs with good efficacy and low side effects. Therefore, the key problem to be solved in the current research on anti-tumor drugs is to find anti-tumor drugs with good curative effect and low side effects. Taking advantage of the difference between tumor cells and normal cells in the metabolic process, selectively interfering with the key links in the metabolic process of tumor cells, it is possible to achieve the purpose of inhibiting the growth of tumor cells without damaging normal cells.

In the 1920s, Otto Warburg proposed that tumors generate energy using glycolysis rather than the more efficient oxidative phosphorylation. The result of this metabolic regulation is higher glucose consumption and lactate secretion, a feature known as the Warburg effect or aerobic glycolysis. Recently, tumor metabolism has become a hot topic in the field of tumor research, which has brought back attention to a long-ago discovery that all cancer cells, regardless of their tissue of origin, pyruvate kinase M2 subtype ( PKM2) were significantly up-regulated. This discovery quickly positioned PKM2 as a potential target for cancer therapy.

Pyruvate kinase (PK) is an enzyme that catalyzes the final step of glycolysis, converting PEP (phosphoenolpyruvate) to pyruvate and phosphorylating ADP to ATP. There are four isoforms of PK in mammals, and their expression profiles and regulation may reflect the special functions and energy requirements of different tissues in which they are located. These four isoforms are encoded by two genes, PKLR and PKM, whose expression is regulated by tissue-specific promoters and alternative splicing, respectively. PKLR encodes two isoforms, PKL and PKR, which are specifically expressed in liver, kidney and reticulocytes, respectively. Alternative splicing of the PKM gene produces two variants: an M1, which is mainly expressed in skeletal muscle, heart, and brain; and a second, M2, which was originally identified in proliferating and developing embryonic cells. Among them, the M1 subtype is a highly active form that is continuously activated, while PKM2 can be converted between low activity and high activity. In tumor cells, however, PKM2 gradually replaces the tissue-specific PK isoform until it becomes the dominant form. This isoform shift suggests that a specific property of PKM2 promotes tumorigenesis. This conjecture was further confirmed by a study that found that after replacing PKM2 with PKM1 in tumor cells, this persistently activated PK isoform delayed xenograft tumor growth.

Substantial progress has also been made in the study of the role of PKM2 in the abnormal metabolism of cancer. Studies have found that its effects are multifaceted, including the effect on metabolism and the regulation of some genes. It has been suggested that PKM2 is inactive in proliferating cells and that its inactivity contributes to the accumulation of glucose metabolic intermediates. It can be used to synthesize biological macromolecules, thereby promoting the growth and proliferation of cells. In addition to the function of PKM2 as a glycolytic enzyme in the cytoplasm, it is also found in the nucleus. The nuclear localization of PKM2 may be due to the C-terminal NLS sequence, which differs from the classical NLS in that there are not many arginines and lysines. It has been suggested that the translocation mechanism of PKM2 involves the interaction of PKM2 and SUMO-E3PIAS3, which promotes the sumoylation and nuclear translocation of PKM2. The functions of the PKM2s mentioned vary. Nuclear PKM2 is necessary for interleukin-3-stimulated proliferation and apoptosis-stimulated cell death, but the exact mechanism remains unclear. Other studies have proposed that nuclear PKM2 can interact with and activate transcription factors such as β-catenin, Oct-4, etc., and play a role in cell survival and proliferation. Activation of epidermal growth factor receptor induces nuclear translocation of PKM2 and c-SRC-mediated phosphorylation of β-catenin. In the nucleus, PKM2 binds to phosphorylated β-catenin and promotes its transcriptional activity; in particular, induces the expression of cyclingD1, which is essential for EGF-induced cell proliferation. Moreover, the catalytically inactive PKM2 mutant binds β-catenin and translocates to the nucleus, however, it fails to activate the transcription of cycling D1. A more recent study provides new evidence for a role for glycolytically inactive PKM2 in cell proliferation. Dimers of PKM2 have been shown to exist in the nucleus as protein kinases that dephosphorylate the transcription factor Stat 3 with PEP. Interestingly, mutants of PKM2 cannot form tetramers and are therefore less active as a PK, so it prefers the nucleus, where it can promote cell proliferation by enhancing Stat3 phosphorylation. In addition, there are reports on the regulation of cell cycle by nuclear PKM2. Recent studies have found that PKM2 phosphorylates Bub2, thereby helping the latter to form a complex and localize to the centromere to help cells divide. Overall, the nuclear function of PKM2 promotes cell proliferation. These functions of PKM2 can be regarded as the complement of key enzymes of glycolysis. Unlike PKM2, targeting PKM1 to the nucleus does not affect cell growth and survival. Therefore, the nuclear activity of PKM2 may be the real reason for its high expression in tumors.

In recent years, the research on new anti-tumor drugs targeting PKM2 has begun to receive attention internationally, but there are very few domestic studies. At present, some small molecule PKM2 agonists with good activity have been found, but very limited. Moreover, the reported PKM2 agonists only show good activity at the enzyme level, and have no significant anti-tumor activity at the cellular level. Unless the tumor cells are cultured in a culture medium depleted of nutrients such as serine, the existing agonists can show the killing effect on tumor cells. Our group took the lead in establishing a pyruvate kinase M2 subtype (PKM2) enzyme activity screening model in China, and conducted a preliminary screening of our own compound library. From it, we discovered a new class of PKM2 agonists with a new structure, and found for the first time that such agonists also have good anti-tumor activity at the cellular level. And compared with normal cells, it has good selectivity for tumor cells. Further, we synthesized a series of derivatives of lead compounds, evaluated their activity at the enzyme and cell levels, revealed their structure-activity relationship, and optimized a new type of PKM2 agonist that can be used as a candidate drug for further research , and a preliminary mechanism study was carried out on it. It is found that this kind of compound produces anti-tumor effect by preventing PKM2 from entering the nucleus. Our work provides a new framework for the research of new drugs targeting PKM2, and provides new ideas for the discovery of less toxic anti-tumor drugs.

Contents of the invention

The object of the present invention is to provide aminodithiocarbamate compounds and their preparation methods and applications.

In order to achieve the above object, the technical means adopted in the present invention are:

The carbamate compound with the structure of formula I or the pharmaceutically acceptable salt thereof provided by the present invention,

_Wherein R is selected from aryl, substituted aryl, aromatic heterocyclic or substituted aromatic heterocyclic.

In the present invention, preferably, R ₁ is selected from one of the following groups: (1) phenyl; (2) C _1-3 alkoxy, halogen, C _1-3 haloalkane (3) furyl, pyridyl, thienyl, pyrrolyl, thiazolyl, indolyl, coumarinyl, azaindolyl or 3-imidazopyridyl.

In the present invention, preferably, R is selected from phenyl, 3,4 _- dichlorophenyl, 4-fluorophenyl, 2-furyl, 4-chlorophenyl, 4-methoxyphenyl, 4 -Benzyloxyphenyl, 4-nitrophenyl, 4-hydroxyphenyl, 4-cyanophenyl, 2-chlorophenyl, 2-methoxyphenyl, 4-trifluoromethylphenyl, 3,4,5-trimethoxyphenyl, 3,4-difluorophenyl, 3-pyridyl, 2-thienyl, 2-pyrrolyl, 2-thiazolyl, 3-indolyl, 3-indol Soybeinyl, 3-azaindolyl or 3-imidazopyridyl.

The method for preparing the compound or a pharmaceutically acceptable salt thereof is characterized in that the compound of the general formula I can be prepared according to the following method:

Wherein, R ₁ is phenyl.

The method for preparing the compound or a pharmaceutically acceptable salt thereof is characterized in that the compound of the general formula I can be prepared according to the following method:

Wherein, R is 3,4 _- dichlorophenyl, 4-fluorophenyl, 2-furyl, 4-chlorophenyl, 4-methoxyphenyl, 4-benzyloxyphenyl, 4-nitro phenyl, 4-hydroxyphenyl, 4-cyanophenyl, 2-chlorophenyl, 2-methoxyphenyl, 4-trifluoromethylphenyl, 3,4,5-trimethoxybenzene 3,4-difluorophenyl, 3-pyridyl, 2-thienyl, 2-pyrrolyl, 2-thiazolyl, 3-indolyl, 3-coumarinyl, 3-azaindole base or 3-imidazopyridyl.

In the present invention, preferably, the compound of the structure shown in formula II can be prepared according to the following method:

(1) When R ₁ is 3,4-dichlorophenyl, 4-fluorophenyl, 2-furyl, 4-chlorophenyl, 4-methoxyphenyl, 4-benzyloxyphenyl, 4 -Nitrophenyl, 4-hydroxyphenyl, 4-cyanophenyl, 2-chlorophenyl, 2-methoxyphenyl, 4-trifluoromethylphenyl, 3,4,5-trimethoxy When phenylphenyl, 3,4-difluorophenyl, 3-pyridyl, 2-thienyl, 2-pyrrolyl, 2-thiazolyl, 3-indolyl or 3-coumarinyl,

The compound of formula III reacts with paraformaldehyde under the action of catalyst CF ₃ COOH·(iPr) ₂ NH to generate the compound of formula II;

(2) When R ₁ is 3-azaindolyl,

7-azaindole and acetyl chloride are reacted under the action of AlCl3 to form a compound of _formula II structure;

(3) When R ₁ is 3-imidazopyridyl,

2-Aminopyridine is first reacted with N,N-dimethylformamide dimethyl acetal, and the intermediate product is reacted with chloroacetone to generate a compound of formula II.

It should be noted that the synthetic route described above is to illustrate the preparation of the compound of the present invention, and the preparation is by no means limited thereto, that is, other synthetic methods are also feasible, and said method refers to the synthetic method within the general knowledge of the skilled person.

Compounds of the present invention may, if desired, be converted into their pharmaceutically acceptable salts by methods known in the art.

Another object of the present invention is to provide the use of the compounds of the present invention.

The inventors of the present invention have confirmed through experiments that the compound of the present invention targets PKM2 and is an agonist of PKM2, and finds that this type of compound produces an anti-tumor effect by preventing PKM2 from entering the nucleus, has a good anti-tumor effect, and has an anti-tumor effect on tumor cells. There are good options.

Therefore, the present invention proposes the use of the compound or a pharmaceutically acceptable salt thereof in the preparation of an agonist of pyruvate kinase M2 subtype. Wherein, the pyruvate kinase M2 subtype agonist has the effect of preventing the pyruvate kinase M2 subtype from entering the nucleus. and

Use of the compound or a pharmaceutically acceptable salt thereof in the preparation of antitumor drugs. Wherein, the anti-tumor drug is a drug for treating colon cancer, cervical cancer or lung cancer. and

Use of the compound or a pharmaceutically acceptable salt thereof in the preparation of an agonist of the glycolysis pathway.

The preparation method of the compound of table 1 is given in detail in the specific examples of the present invention:

Target compound:

Table 1

Description of drawings

Figure 1 is a diagram of the western detection results of PKM2 in the nucleus and cytoplasm after compound 22 was added to the cells; wherein, Mock is the control group without compound, and NZT is the reported agonist;

Figure 2 is a diagram of compound 22 arresting cell cycle; wherein, (A) compound 22 dose-dependently arrests cells in G2/M phase, (B) structure of compound 22, (C) drug treatment group (1 μM, 5 μM) Compared with the DMSO control group, the ratio of G2/M phase to the whole cell cycle.

detailed description

The present invention will be further described below in conjunction with specific embodiments, and the advantages and characteristics of the present invention will become clearer along with the description. However, the examples are merely exemplary and do not limit the scope of the present invention in any way. Those skilled in the art should understand that the details and forms of the technical solutions of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the protection scope of the present invention.

Explanation of abbreviations appearing in the present invention:

P petroleum ether

E ethyl acetate

Example 1 Synthesis of 3-pyridinemethylcarbamodithioate-(2-benzoyl)ethyl ester (compound 1)

Dissolve 3-aminomethylpyridine (541 mg, 5 mmol) in 25 mL of DCM, add TEA (506 mg, 5 mmol), stir at room temperature for 10 min, add CS ₂ (457 mg, 6 mmol), react at room temperature for 20 min, add 3-chlorobenzene Acetone (843 mg, 5 mmol) was reacted at room temperature for 5 h, the solvent was directly spin-dried under reduced pressure, and column chromatography (P:E=1:2) gave a white solid with a yield of 77.2%. Melting point: 82-84°C.

¹ H NMR(400MHz,DMSO)δ10.50(s,1H),8.70-8.32(m,2H),7.97(d,J=7.4Hz,2H),7.66(ddd,J=10.8,8.5,4.4Hz ,2H),7.53(t,J=7.1Hz,2H),7.37(dd,J=7.7,4.8Hz,1H),4.86(d,J=5.3Hz,2H),3.50(dt,J=11.8, 4.7Hz, 4H).

¹³ C NMR (100MHz, DMSO) δ198.72, 197.82, 149.56, 148.90, 136.65, 135.96, 133.90, 133.40, 129.25, 128.37, 123.98, 47.58, 38.65, 29.32.

Example 2 Synthesis of 3-pyridinemethylcarbamodithioate-[2-(3,4-dichloro-benzoyl)]ethyl ester (compound 2)

Dissolve 3,4-dichloroacetophenone (756mg, 4mmol) in 10mL DMF, add paraformaldehyde (240mg, 8mmol), catalyst CF ₃ COOH·(iPr) ₂ NH (860mg, 4mmol), at 80°C React for 12 hours, cool to room temperature, add 50 mL of water, extract with ethyl acetate (10 mL×3), combine the organic phases, dry over anhydrous sodium sulfate, concentrate, and perform column chromatography (P:E=20:1) to obtain an oil Intermediate (2-1), yield 27.5%. Dissolve 3-aminomethylpyridine (324 mg, 3 mmol) in 20 mL of DCM, add TEA (303 mg, 3 mmol), stir at room temperature for 5 min, add CS ₂ (274 mg, 3.6 mmol), react for 10 min, add intermediate 2-1 , reacted at room temperature for 5 h, evaporated the solvent under reduced pressure, and performed column chromatography (P:E=2:1) to obtain a white solid with a yield of 56.3%. Melting point: 139.3-140.2°C.

Intermediate 2-1

¹ H NMR (400MHz, CDCl ₃ )δ8.14-7.78(m,1H),7.81-7.60(m,1H),7.60-7.37(m,1H),7.15-6.93(m,1H),6.39(dd ,J＝17.1,1.4Hz,1H),6.06-5.70(m,1H).

¹³ C NMR (100MHz, CDCl ₃ ) δ187.61, 136.61, 135.80, 132.35, 130.51, 130.37, 129.79, 129.67, 126.69.

Product 2

¹ H NMR (400MHz, DMSO) δ10.52(q, J=5.3Hz, 1H), 8.77-8.28(m, 2H), 8.37-8.05(m, 1H), 7.92(dd, J=4.9, 3.5Hz ,1H),7.79(td,J=8.3,4.1Hz,1H),7.75-7.58(m,1H),7.60-7.26(m,1H),5.09-4.70(m,2H),3.74-3.39(m ,4H).

¹³ C NMR (100MHz, DMSO) δ197.18, 196.51, 149.08, 148.40, 136.21, 135.47, 132.88, 131.85, 131.06, 129.81, 127.92, 123.46, 47.11, 38.41, 28.64.

HRMS calcd. For C ₁₆ H ₁₅ Cl ₂ N ₂ OS ₂ ⁺ [M+H] ⁺ 384.9997, found: 384.9989.

Example 3 Synthesis of 3-pyridinemethylcarbamodithioate-[2-(4-fluoro-benzoyl)]ethyl ester (compound 3)

Using 4-fluoroacetophenone as a raw material, the target compound was synthesized with reference to the synthetic route of Example 2. Intermediate 3-1 is an oily product with a yield of 46.1%; product 3 is a white solid with a yield of 35.4% and a melting point of 127.4-128.1°C.

Intermediate 3-1

¹ H NMR (400MHz, CDCl ₃ ) δ8.11-7.83 (m, 2H), 7.14 (dt, J=10.6, 9.6Hz, 3H), 6.58-6.23 (m, 1H), 6.12-5.77 (m, 1H ).

¹³ C NMR (100MHz, CDCl ₃ ) δ188.31, 165.96, 163.43, 132.63, 132.60, 130.98, 130.34, 130.25, 129.29, 114.85, 114.64.

Product 3

¹ H NMR(400MHz,DMSO)δ10.60(t,J=5.5Hz,1H),8.81-8.35(m,2H),8.33-7.92(m,2H),7.93-7.56(m,1H),7.46 -7.17(m,3H),4.85(d,J=5.6Hz,2H),3.72-3.40(m,4H).

¹³ C NMR (100MHz, DMSO) δ197.73, 197.32, 166.84, 164.34, 149.57, 148.85, 135.98, 133.44, 131.45, 131.36, 123.94, 116.35, 116.13, 47.53, 45.79, 38.65.

HRMS calcd. For C ₁₆ H ₁₆ FN ₂ OS ₂ ⁺ [M+H] ⁺ 335.0683, found: 384.0680.

Example 4 Synthesis of 3-pyridinemethylcarbamodithioate-[2-(2-furoyl)]ethyl ester (compound 4)

Using 2-acetylfuran as a raw material, the target compound was synthesized with reference to the synthetic route of Example 2. Intermediate 4-1 is an oily product with a yield of 36.5%; product 4 is a white solid with a yield of 63.5% and a melting point of 122.9-123.6°C.

Intermediate 4-1

¹ H NMR (400MHz, CDCl ₃ )δ7.77-7.56(m,1H),7.31-7.24(m,1H),7.19-6.96(m,1H),6.72-6.42(m,2H),6.00-5.73 (m,1H).

¹³ C NMR (100MHz, CDCl ₃ ) δ177.97, 152.91, 146.95, 131.32, 129.38, 118.27, 112.45.

Product 4

¹ H NMR (400MHz, DMSO) δ10.51 (t, J = 5.5Hz, 1H), 8.82-8.26 (m, 2H), 7.99 (dd, J = 1.6, 0.6Hz, 1H), 7.69 (dt, J =7.8,1.9Hz,1H),7.60-7.18(m,2H),6.71(dd,J=3.6,1.7Hz,1H),4.85(d,J=5.6Hz,2H),3.49(t,J= 6.8Hz, 2H), 3.26(t, J=6.8Hz, 2H).

¹³ C NMR (100MHz, DMSO) δ197.54, 186.94, 152.05, 149.55, 148.88, 148.35, 135.96, 133.38, 123.96, 119.10, 113.02, 47.58, 38.15, 29.01.

HRMS calcd. For C ₁₄ H ₁₅ N ₂ O ₂ S ₂ ⁺ [M+H] ⁺ 307.0570, found: 307.0563.

Example 5 Synthesis of 3-pyridinemethylcarbamodithioate-[2-(4-chloro-benzoyl)]ethyl ester (compound 5)

Using 4-chloroacetophenone as a raw material, the target compound was synthesized with reference to the synthetic route of Example 2.

Due to the different substituents, it is difficult to separate the raw material methyl ketone and the formed intermediate Michael acceptor. Therefore, the paraformaldehyde is only removed by rough column chromatography, and the next step reaction is directly carried out without accurate separation. Product 5 is a white solid with a two-step yield of 21.7% and a melting point of 113.7-113.9°C.

¹ H NMR (400MHz, DMSO) δ10.52(t, J=5.2Hz, 1H), 8.77-8.34(m, 2H), 8.22-7.93(m, 2H), 7.70(dt, J=7.8, 1.8Hz ,1H),7.68-7.52(m,2H),7.36(dd,J=7.8,4.8Hz,1H),4.85(d,J=5.3Hz,2H),3.62-3.40(m,4H).

¹³ C NMR (100MHz, DMSO) δ197.75, 197.74, 149.57, 148.88, 138.80, 135.97, 135.31, 133.40, 130.30, 129.34, 123.95, 47.58, 38.72, 29.23.

HRMS calcd. For C ₁₆ H ₁₆ ClN ₂ OS ₂ ⁺ [M+H] ⁺ 351.0387, found: 351.0380.

Example 6 Synthesis of 3-pyridinemethylcarbamodithioate-[2-(4-methoxy-benzoyl)]ethyl ester (compound 6)

Using 4-methoxyacetophenone as a raw material, the target compound was synthesized with reference to the synthetic route of Example 2. Product 6 is a white solid with a two-step yield of 54.1% and a melting point of 135.5-135.8°C.

¹ H NMR (400MHz, DMSO) δ 10.49 (t, J = 5.2Hz, 1H), 8.50 (dd, J = 19.5, 2.4Hz, 2H), 7.94 (d, J = 8.8Hz, 2H), 7.70 ( d,J=7.8Hz,1H),7.36(dd,J=7.7,4.8Hz,1H),7.03(d,J=8.8Hz,2H),4.86(d,J=5.5Hz,2H),3.84( s,3H),3.44(ddd,J=17.6,12.1,5.6Hz,4H).

¹³ C NMR (100MHz, DMSO) δ197.91, 196.99, 163.74, 149.56, 148.89, 135.96, 133.42, 130.71, 129.67, 123.96, 114.40, 56.00, 47.57, 38.21, 29.50.

HRMS calcd. For C ₁₇ H ₁₉ N ₂ O ₂ S ₂ ⁺ [M+H] ⁺ 347.0883, found: 347.0876.

Example 7 Synthesis of 3-pyridinemethylcarbamodithioate-[2-(4-benzyloxy-benzoyl)]ethyl ester (compound 7)

Using 4-benzyloxyacetophenone as a raw material, the target compound was synthesized with reference to the synthetic route of Example 2. Product 7 is a white solid, the two-step yield is 22.0%, and the melting point is 148.3-148.9°C.

¹ H NMR (400MHz, DMSO) δ8.72-8.36 (m, 2H), 7.92 (dd, J = 9.4, 2.3Hz, 2H), 7.69 (dt, J = 7.8, 1.9Hz, 1H), 7.38 (dqd ,J=9.6,8.7,4.3Hz,6H),7.11(d,J=8.9Hz,2H),5.19(s,2H),4.85(s,2H),3.76-3.26(m,4H).

¹³ C NMR (100MHz, DMSO) δ197.84, 197.04, 162.81, 149.46, 148.83, 136.86, 136.00, 133.44, 130.71, 129.80, 128.96, 128.49, 128.21, 124.00, 115.21, 69.49.38, 2

HRMS calcd. For C ₂₃ H ₂₃ N ₂ O ₂ S ₂ ⁺ [M+H] ⁺ 423.1196, found: 423.1194.

Example 8 Synthesis of 3-pyridinemethylcarbamodithioate-[2-(4-nitro-benzoyl)]ethyl ester (compound 8)

Using 4-nitroacetophenone as a raw material, the target compound was synthesized with reference to the synthetic route of Example 2. Product 8 is a light yellow solid, the two-step yield is 26.8%, and the melting point is 153.0-154.1°C.

¹ H NMR (400MHz, DMSO) δ8.69-8.43(m, 2H), 8.34(d, J=8.9Hz, 2H), 8.25-8.09(m, 2H), 7.71(dd, J=7.8, 1.7Hz ,1H),7.38(dd,J=7.8,4.8Hz,1H),4.86(s,2H),3.54(d,J=15.1Hz,4H).

¹³ C NMR (100MHz, DMSO) δ197.52, 197.06, 149.96, 148.95, 148.34, 140.58, 135.53, 132.89, 129.32, 123.84, 123.51, 46.92, 38.83, 28.54.

HRMS calcd. For C ₁₆ H ₁₆ N ₃ O ₃ S ₂ ⁺ [M+H] ⁺ 362.0628, found: 362.0625.

Example 9 Synthesis of 3-pyridinemethylcarbamodithioate-[2-(4-hydroxyl-benzoyl)]ethyl ester (compound 9)

Using 4-hydroxyacetophenone as a raw material, the target compound was synthesized with reference to the synthetic route of Example 2. Product 9 is a white solid, the two-step yield is 11.1%, and the melting point is 166.7-167.2°C.

¹ H NMR (400MHz, DMSO) δ8.75-8.35 (m, 2H), 7.85 (d, J = 8.7Hz, 2H), 7.81-7.60 (m, 1H), 7.38 (dd, J = 7.7, 4.8Hz ,1H),6.87(d,J=8.7Hz,2H),4.85(s,2H),3.88-3.19(m,4H).

¹³ C NMR (100MHz, DMSO) δ197.38, 196.29, 161.99, 148.94, 148.33, 135.50, 132.95, 130.46, 127.89, 123.53, 115.18, 46.86, 37.48, 29.03.

HRMS calcd. For C ₁₆ H ₁₇ N ₂ O ₂ S ₂ ⁺ [M+H] ⁺ 333.0726, found: 333.0718.

Example 10 Synthesis of 3-pyridinemethylcarbamodithioate-[2-(4-cyano-benzoyl)]ethyl ester (compound 10)

Using 4-cyanoacetophenone as a raw material, the target compound was synthesized with reference to the synthetic route of Example 2. The product 10 is a white solid, the two-step yield is 16.9%, and the melting point is 146.7-147.5°C.

¹ H NMR (400MHz, DMSO) δ10.51(t, J=5.5Hz, 1H), 8.64-8.41(m, 2H), 8.11(d, J=8.5Hz, 2H), 8.00(d, J=8.5 Hz,2H),7.69(d,J=7.8Hz,1H),7.36(dd,J=7.7,4.8Hz,1H),4.85(d,J=5.6Hz,2H),3.52(s,4H).

¹³ C NMR (100MHz, DMSO) δ198.22, 197.65, 149.56, 148.90, 139.70, 135.98, 133.37, 133.31, 129.03, 123.97, 118.59, 115.82, 47.60, 39.11, 29.09.

HRMS calcd. For C ₁₇ H ₁₆ N ₃ OS ₂ ⁺ [M+H] ⁺ 342.0729, found: 342.0723.

Example 11 Synthesis of 3-pyridinemethylcarbamodithioate-[2-(2-chloro-benzoyl)]ethyl ester (compound 11)

Using 2-chloroacetophenone as a raw material, the target compound was synthesized with reference to the synthetic route of Example 2. The product 11 is a white solid, the two-step yield is 7.45%, and the melting point is 78.0-78.6°C.

¹ H NMR (400MHz, DMSO) δ10.53(t, J=5.4Hz, 1H), 8.84-8.32(m, 2H), 7.67(dd, J=9.9, 7.8Hz, 2H), 7.58-7.33(m ,4H),4.85(d,J=5.6Hz,2H),3.44(dt,J=12.9,6.7Hz,4H).

¹³ C NMR (100MHz, DMSO) δ201.11, 197.53, 149.54, 148.91, 138.53, 135.94, 133.37, 132.94, 130.98, 130.07, 129.67, 127.95, 123.97, 47.57, 42.47, 29.04.

HRMS calcd. For C ₁₆ H ₁₆ ClN ₂ OS ₂ ⁺ [M+H] ⁺ 351.0387, found: 351.0382.

Example 12 Synthesis of 3-pyridinemethylcarbamodithioate-[2-(2-methoxy-benzoyl)]ethyl ester (compound 12)

Using 2-methoxyacetophenone as a raw material, the target compound was synthesized with reference to the synthetic route of Example 2. Product 12 was a white solid, yield 1.4% in two steps, melting point: 96.2-97.0°C.

¹ H NMR (400MHz, DMSO) δ10.47(t, J=5.5Hz, 1H), 8.62-8.41(m, 2H), 7.69(dt, J=7.8, 1.8Hz, 1H), 7.64-7.46(m ,2H),7.36(dd,J=7.6,4.7Hz,1H),7.17(d,J=8.3Hz,1H),7.11-6.97(m,1H),4.85(d,J=5.6Hz,2H) ,3.86(s,3H),3.58-3.32(m,4H).

¹³ C NMR (100MHz, DMSO) δ199.94, 197.88, 158.92, 149.53, 148.88, 135.93, 134.54, 133.42, 130.04, 127.49, 123.96, 120.96, 113.01, 56.28, 47.54, 43.441.29

HRMS calcd. For C ₁₇ H ₁₉ N ₂ O ₂ S ₂ ⁺ [M+H] ⁺ 347.0883, found: 347.0877.

Example 13 Synthesis of 3-pyridinemethylcarbamodithioate-[2-(4-trifluoromethyl-benzoyl)]ethyl ester (compound 13)

Using 4-trifluoromethylacetophenone as a raw material, the target compound was synthesized with reference to the synthetic route of Example 2. Product 13 was a yellow solid, yield 10.4% in two steps, melting point: 135.8-136.6°C.

¹ H NMR (400MHz, DMSO) δ10.51(t, J=5.5Hz, 1H), 8.69-8.43(m, 2H), 8.16(d, J=8.1Hz, 2H), 7.90(d, J=8.3 Hz, 2H), 7.71(d, J=1.8Hz, 1H), 7.37(dd, J=7.8, 4.8Hz, 1H), 4.85(d, J=5.6Hz, 2H), 3.53(s, 4H).

¹³ C NMR (100MHz, DMSO) δ198.27, 197.68, 149.56, 148.90, 139.65, 135.97, 133.38, 132.96, 129.24, 126.23, 123.97, 109.89, 47.59, 39.08, 29.10.

HRMS calcd. For C ₁₇ H ₁₆ F ₃ N ₂ OS ₂ ⁺ [M+H] ⁺ 385.0651, found: 385.0644.

Example 14 Synthesis of 3-pyridinemethylaminodithiocarbamate-[2-(3,4,5-trimethoxy-benzoyl)]ethyl ester (compound 14)

Using 3,4,5-trimethoxyacetophenone as a raw material, the target compound was synthesized referring to the synthesis route of Example 2. The product 14 is a white solid, the two-step yield is 45.7%, melting point: 85.6-86.8°C.

¹ H NMR (400MHz, DMSO) δ 10.52 (s, J = 5.0Hz, 1H), 8.49 (dd, J = 11.1, 10.2Hz, 2H), 7.70 (d, J = 7.8Hz, 1H), 7.37 ( dd,J=7.6,4.9Hz,1H),7.23(d,J=28.6Hz,2H),4.86(d,J=5.4Hz,2H),3.85(s,6H),3.74(s,3H), 3.61-3.40(m,4H).

¹³ C NMR (100MHz, DMSO) δ197.39, 197.18, 152.76, 149.07, 148.41, 141.95, 135.48, 132.91, 131.54, 123.47, 105.44, 60.12, 56.02, 47.09, 38.10, 29.09.

HRMS calcd. For C ₁₉ H ₂₃ N ₂ O ₄ S ₂ ⁺ [M+H] ⁺ 407.1094, found: 407.1087.

Example 15 Synthesis of 3-pyridinemethylaminodithiocarbamate-[2-(3,4,-difluoro-benzoyl)]ethyl ester (compound 15)

Using 3,4-difluoroacetophenone as a raw material, the target compound was synthesized referring to the synthesis route of Example 2. The product 15 is a white solid, the two-step yield is 24.4%, and the melting point is 128.8-129.7°C.

¹ H NMR (400MHz, DMSO) δ10.51(s, J=4.9Hz, 1H), 8.62-8.35(m, 2H), 7.99(t, J=8.2Hz, 1H), 7.86(d, J=2.1 Hz,1H),7.70(d,J=7.8Hz,1H),7.56(d,J=5.1Hz,1H),7.42-7.28(m,1H),4.86(d,J=5.4Hz,2H), 3.49(tt,J=8.4,4.2Hz,4H).

¹³ C NMR (100MHz, DMSO) δ207.24, 196.61, 151.12, 149.66, 149.45, 149.03, 148.57, 136.05, 135.79, 134.14, 133.38, 123.94, 117.83, 47.51, 38.80, 29.18.

HRMS calcd. For C ₁₆ H ₁₅ F ₂ N ₂ OS ₂ ⁺ [M+H] ⁺ 353.0588, found: 353.0581.

Example 16 Synthesis of 3-pyridinemethylcarbamodithioate-[2-(3-pyridineformyl)]ethyl ester (compound 16)

Using 3-acetylpyridine as a raw material, the target compound was synthesized with reference to the synthetic route of Example 2. Product 16 was a yellow solid, yield 10.0% in two steps, melting point: 95.1-95.9°C.

¹ H NMR (400MHz, DMSO) δ10.52(t, J=5.3Hz, 1H), 9.13(t, J=4.3Hz, 1H), 8.80(dd, J=4.7, 1.4Hz, 1H), 8.50( dd,J=20.1,2.4Hz,2H),8.30(dd,J=6.1,1.8Hz,1H),7.70(d,J=7.8Hz,1H),7.56(dd,J=7.8,4.8Hz,1H ), 7.36(dd, J=7.7, 4.8Hz, 1H), 4.86(d, J=5.5Hz, 2H), 3.53(t, J=4.3Hz, 4H).

¹³ C NMR (100MHz, DMSO) δ198.32, 197.71, 154.06, 149.62, 149.56, 148.89, 135.97, 135.92, 133.39, 131.90, 124.38, 123.97, 47.61, 39.00, 29.05.

HRMS calcd. For C ₁₅ H ₁₆ N ₃ OS ₂ ⁺ [M+H] ⁺ 318.0729, found: 318.0725.

Example 17 Synthesis of 3-pyridinemethylcarbamodithioate-[2-(2-thienyl)]ethyl ester (compound 17)

Using 2-acetylthiophene as a raw material, the target compound was synthesized with reference to the synthesis route in Example 2. Product 17 is a white solid, the two-step yield is 52.8%, melting point: 135.3-136.2°C.

¹ H NMR (400MHz, DMSO) δ10.49 (t, J = 5.3Hz, 1H), 8.56-8.45 (m, 2H), 8.02 (dd, J = 4.9, 1.1Hz, 1H), 7.96 (dd, J =3.8,1.1Hz,1H),7.69(dt,J=7.8,1.8Hz,1H),7.37(ddd,J=7.8,4.8,0.7Hz,1H),7.24(dd,J=4.9,3.8Hz, 1H), 4.85(d, J=5.6Hz, 2H), 3.51(t, J=6.5Hz, 2H), 3.39(dd, J=10.3, 3.6Hz, 2H).

¹³ C NMR (100MHz, DMSO) δ197.62, 191.68, 149.56, 148.90, 143.72, 135.95, 135.52, 134.04, 133.38, 129.28, 123.97, 47.59, 38.89, 29.38.

HRMS calcd. For C ₁₄ H ₁₅ N ₂ OS ₃ ⁺ [M+H] ⁺ 323.0341, found: 323.0339.

Example 18 Synthesis of 3-pyridinemethylcarbamodithioate-[2-(2-pyrrolecarbonyl)]ethyl ester (compound 18)

Using 2-acetylpyrrole as a raw material, the target compound was synthesized with reference to the synthetic route of Example 2. Product 18 was a white solid, yield 18.4% in two steps, melting point: 128.7-129.3°C.

¹ H NMR (400MHz, DMSO) δ11.85(s, 1H), 10.47(t, J=5.3Hz, 1H), 8.57-8.39(m, 2H), 7.69(d, J=7.8Hz, 1H), 7.36(dd,J=7.7,4.8Hz,1H),7.09(s,1H),6.97(s,1H),6.22-6.13(m,1H),4.86(d,J=5.5Hz,2H),3.50 (t,J=6.8Hz,2H),3.18(t,J=6.9Hz,2H).

¹³ C NMR (100MHz, DMSO) δ197.81, 187.76, 149.55, 148.88, 135.95, 133.42, 131.83, 126.09, 123.96, 117.18, 110.33, 47.55, 37.55, 29.85.

HRMS calcd. For C ₁₄ H ₁₆ N ₃ OS ₂ ⁺ [M+H] ⁺ 306.0729, found: 306.0724.

Example 19 Synthesis of 3-pyridinemethylcarbamodithioate-[2-(2-thiazolylformyl)]ethyl ester (compound 19)

Using 2-acetylthiazole as a raw material, the target compound was synthesized with reference to the synthetic route of Example 2. The product 19 is a light yellow solid, the two-step yield is 9.2%, and the melting point is 114.0-114.9°C.

¹ H NMR (400MHz, CDCl ₃ ) δ9.12-8.96 (m, 1H), 8.51-8.36 (m, 2H), 8.02-7.93 (m, 1H), 7.70 (dd, J=7.1, 4.2Hz, 2H ),7.28-7.20(m,1H),5.03-4.89(m,2H),3.77-3.62(m,4H).

¹³ C NMR (100MHz, CDCl ₃ ) δ198.21, 191.90, 166.19, 149.20, 148.81, 144.87, 136.32, 132.58, 126.59, 123.76, 48.09, 38.91, 29.00.

HRMS calcd. For C ₁₃ H ₁₄ N ₃ OS ₃ ⁺ [M+H] ⁺ 324.0294, found: 324.0290.

Example 20 Synthesis of 3-pyridinemethylcarbamodithioate-[2-(3-indoleformyl)]ethyl ester (compound 20)

Using 3-acetylindole as a raw material, the target compound was synthesized with reference to the synthetic route of Example 2. Product 20 is a white solid, yield 5.6% in two steps, melting point: 156.6-157.4°C.

¹ H NMR (400MHz, DMSO) δ11.96(s, 1H), 10.45(t, J=5.5Hz, 1H), 8.56-8.45(m, 2H), 8.33(d, J=3.1Hz, 1H), 8.25-8.13(m,1H),7.75-7.63(m,1H),7.47(dd,J＝6.7,1.7Hz,1H),7.37(dd,J＝7.8,4.8Hz,1H),7.26-7.12( m, 2H), 4.86(d, J=5.6Hz, 2H), 3.55(t, J=6.9Hz, 2H), 3.29(t, J=7.0Hz, 2H).

¹³ C NMR (100MHz, DMSO) δ198.09, 193.35, 149.56, 148.87, 137.06, 135.92, 134.52, 133.45, 125.75, 123.95, 123.28, 122.25, 121.72, 116.48, 112.63, 87.70,

HRMS calcd. For C ₁₈ H ₁₈ N ₃ OS ₂ ⁺ [M+H] ⁺ 356.0886, found: 356.0883.

Example 21 Synthesis of 3-pyridinemethylaminodithiocarbamate-[2-(3-coumarinformyl)]ethyl ester (compound 21)

Using 3-acetylcoumarin as a raw material, the target compound was synthesized with reference to the synthetic route of Example 2. The product 21 is a white solid, the two-step yield is 5.2%, and the melting point is 127.8-128.7°C.

¹ H NMR (400MHz, DMSO) δ10.49(t, J=5.5Hz, 1H), 8.75-8.68(m, 1H), 8.54-8.42(m, 2H), 7.98(dd, J=7.8, 1.2Hz ,1H),7.74(ddd,J=22.0,12.9,4.6Hz,2H),7.50-7.34(m,3H),4.85(d,J=5.6Hz,2H),3.58-3.37(m,4H).

¹³ C NMR (100MHz, DMSO) δ197.72, 196.12, 158.81, 155.08, 149.56, 148.89, 147.89, 135.96, 135.11, 133.38, 131.33, 125.44, 124.25, 123.97, 118.64, 148.60.6

HRMS calcd. For C ₁₉ H ₁₇ N ₂ O ₃ S ₂ ⁺ [M+H] ⁺ 385.0675, found: 385.0672.

Example 22 Synthesis of 3-pyridinemethylcarbamodithioate-[2-(3-N heteroindoleformyl)]ethyl ester (compound 22)

The key of the reaction is the preparation of 3-acetylazaindole (22-I). Dissolve 7-azaindole (590mg, 5mmol) in 80mL DCM, add AlCl ₃ (3.39g, 25mmol), slowly add acetyl chloride (3.4mL, 47.8mmol), react at room temperature for 10h, and place the reaction solution in In an ice bath, slowly add 20 mL of methanol to quench the reaction, concentrate the reaction solution, add 30 mL of water to the residue, adjust the pH to about 4 with 1N NaOH, extract with EtOAc (15 mL×5), dry over anhydrous sodium sulfate, and concentrate. Separation by column chromatography (P:E=1:1) gave a white solid (22-I) with a yield of 82.5%. Using 22-I as a raw material, the target compound was synthesized with reference to the synthetic route of Example 2. Product 22 was a white solid, yield 4.9% in two steps, melting point: 147.8-148.2°C.

22-I

¹ H NMR (400MHz, CDCl ₃ ) δ13.11-12.78(m,1H),8.79-8.69(m,1H),8.47-8.37(m,1H),8.15-7.98(m,1H),7.36-7.28 (m,1H),2.64-2.52(m,3H).

¹³ C NMR (100MHz, CDCl ₃ ) δ193.43, 149.21, 143.54, 132.43, 131.83, 118.96, 118.44, 116.79, 27.15.

Product 22

¹ H NMR (400MHz, DMSO) δ12.50(s, 1H), 10.47(t, J=5.5Hz, 1H), 8.56-8.43(m, 4H), 8.33(dd, J=4.7, 1.6Hz, 1H ),7.74-7.65(m,1H),7.37(dd,J=7.7,4.8Hz,1H),7.26(dd,J=7.9,4.7Hz,1H),4.86(d,J=5.6Hz,2H) ,3.55(t,J=6.8Hz,2H),3.31(d,J=7.0Hz,2H).

¹³ C NMR (100MHz, DMSO) δ197.99, 193.64, 149.56, 149.41, 148.88, 144.72, 135.94, 134.90, 133.43, 130.00, 123.97, 118.59, 118.05, 115.13, 47.55, 398.697.2

HRMS calcd. For C ₁₇ H ₁₇ N ₄ OS ₂ ⁺ [M+H] ⁺ 357.0838, found: 357.0832.

Example 23 Synthesis of 3-pyridinemethylaminodithiocarbamate-[2-(3-imidazopyridineformyl)]ethyl ester (compound 23)

The key of the reaction is the synthesis of raw material 3-acetylimidazopyridine (23-I). Dissolve 2-aminopyridine (1.88g, 20mmol) in 3.82g of N,N-dimethylformamide dimethyl acetal, react at 105°C for 24h, concentrate under reduced pressure, add 35mL of ethanol, chloroacetone (2.08g, 22.7 mmol), reacted at room temperature for 24 h, continued to concentrate under reduced pressure, and separated by column chromatography (P:E=2:1) to obtain a yellow solid (23-I), with a yield of 70.0%. Using 23-I as a raw material, the target compound was synthesized with reference to the synthetic route of Example 2. Product 23 was a white solid, yield 14.0% in two steps, melting point: 164.8-165.6°C.

23-I

¹ H NMR (400MHz, CDCl ₃ )δ9.73-9.47(m,1H),8.46-8.27(m,1H),7.88-7.69(m,1H),7.56-7.42(m,1H),7.25-6.94 (m,1H),2.86-2.35(m,3H).

¹³ C NMR (100MHz, CDCl ₃ ) δ187.25, 148.80, 143.43, 128.96, 128.71, 123.96, 117.69, 114.98, 27.16.

Product 23

¹ H NMR (400MHz, DMSO) δ10.51(t, J=5.1Hz, 1H), 9.54(d, J=6.8Hz, 1H), 8.64(s, 1H), 8.50(dd, J=10.4, 8.8 Hz, 3H), 7.85(d, J=8.9Hz, 1H), 7.70(d, J=7.7Hz, 1H), 7.38(d, J=5.0Hz, 1H), 7.29(t, J=6.7Hz, 1H), 4.86(d, J=5.0Hz, 2H), 3.59(t, J=6.8Hz, 2H), 3.40(t, J=6.8Hz, 2H).

¹³ C NMR (100MHz, DMSO) δ197.14, 187.57, 149.06, 148.40, 143.81, 135.45, 132.89, 132.63, 129.61, 128.04, 123.48, 123.03, 117.44, 115.60, 47.09, 398.383.2

HRMS calcd. For C ₁₇ H ₁₇ N ₄ OS ₂ ⁺ [M+H] ⁺ 357.0838, found: 357.0832.

Test example 1 The agonistic activity evaluation of the compound of the present invention to PKM2

Experimental steps:

PKM2 activity is detected by lactate dehydrogenase (LDH) coupling reaction system, and the catalytic activity of PKM2 can be judged by detecting the decrease of the substrate NADH content. It is known that the substrate NADH can emit 340nm fluorescence and 340nm fluorescence after being excited by ultraviolet light. The reduction is directly proportional to the reduction in NADH. Therefore, the activity of PKM2 is detected by the reduction of NADH per unit time. Add 44ul of substrate-containing reaction system (final concentration: 50mM Tris-Cl pH 8.0, 200mM KCl, 15mM MgCl ₂ , 0.1mMPEP, 4.0mM ADP and 0.2mM NADH) into a 96-well plate, then add 1μl compound and 5μl enzyme mixture (Final concentration: 10 nM hPKM2 and 1 μM of LDH). After incubation for 20 minutes, put the 96-well plate into FlexStation 3 (Molecular Devices), and detect the NADH reading value every 30 seconds for a total of 3-6 minutes. Record the results, and use GraphPad prism to make a dose-effect relationship curve to obtain the results of compound AC ₅₀ .

Result evaluation:

In the present invention, 23 aminodithio compounds were synthesized, and their agonistic activity on PKM2 was detected. The experimental results are shown in Table 2. It can be seen that the activities of these compounds are all below 10 μM, and compound No. 22 reaches 1 μM, which has a strong agonistic activity on PKM2. Moreover, most of these compounds have reached more than 50% in terms of the maximum excitation rate. Among them, compound No. 22 reached 182%.

Table 2 The compounds of the present invention are to the agonist activity assay result of PKM2

^a : %FBP represents the ratio of the maximum activating rate to the activating rate of FBP (normalized to 100%), where FBP is the endogenous agonist of PKM2.

Test Example 2 Evaluation of the inhibitory effect of the compounds of the present invention on the proliferation of different tumor cells

Experimental steps:

Detect cell viability by MTT method, prepare MTS solution and MTS working solution; inoculate cells in 96-well plate (5×10 ³ cells/100 μL), incubator at 37°C, 5% CO ₂ and 95% air environment Incubate for 12 hours; each 96-well plate is set with a blank control group and an administration group, and the administration group is respectively added with a compound solution with a final concentration of 0.625 μM, 1.25 μM, 2.5 μM, 5 μM, 10 μM, and 20 μM, and 3 parallel wells for each concentration , placed in an incubator for 48 hours; add MTS working solution, placed in an incubator for 4 hours, and measure the OD value at a wavelength of 490nm. The cell survival rate was calculated according to the following formula: cell survival rate (%)=OD _{administration group} /OD _{blank control group} ×100%. Repeat the experiment 3 times.

Result evaluation:

The reported agonists have no significant anti-proliferative effect at the cellular level unless the cells are cultured in a serine-glutamine-free medium. However, the compounds of the present invention show stronger effects at the cellular level. In the present invention, 10 compounds with better enzyme activity were selected to detect cell activity by MTT method in three tumor cell lines with high PKM2 expression, HCT116, Hela and H1299. Since there is no suitable PKM2 agonist as a positive control at the cellular level, paclitaxel was selected as a control in the present invention to verify the accuracy of the experiment. The activity data of paclitaxel measured in the experiment is equivalent to the reported activity, indicating that the accuracy and sensitivity of the model test of the present invention are still acceptable. The results are shown in Table 3, the activity range of the compound of the present invention is between 0.64-5.6 μM. Among them, Hela cells were the most sensitive to these compounds, and most of the IC ₅₀ values reached nM level. In order to further explore the selectivity of these compounds on tumor cells, the present invention also detects the activity of BEAS-2B on normal pulmonary bronchial epithelial cells. It can be seen from Table 3 that most of the compounds showed selectivity to tumor cells. These results show that this series of compounds has the potential to become new tumor-selective anti-tumor drugs. This is also consistent with the characteristics of the PKM2 target. Normal cells prefer the highly active form of pyruvate kinase, so activating PKM2 will not damage normal cells.

Table 3 Compounds of the present invention inhibit the growth of HCT116, Hela and H1299 cells IC ₅₀ (μM)

Among them, BEAS-2B is normal pulmonary bronchial epithelial cells.

Test Example 3 Compounds of the present invention inhibit PKM2 from entering the nucleus

1) After knocking down PKM2, compound 22 was added to the cells, and the activity of the compound decreased, indicating that the compound was indeed targeting PKM2.

Experimental procedure: Knock down the PKM2 gene in HCT116 cells with a lentivirus system to obtain two cell lines, shPKM2-1 and shPKM2-2, treat the two lines of cells and normal cells with compound No. 22, and use the MTT method to detect the activity of the compound on the cells .

Results evaluation: From Table 4, it can be found that after knocking down PKM2, the activity of the compound decreased from 2.5 μM to about 8 μM, indicating that the activity of the compound is dependent on the PKM2 pathway, and also indicating that the compound is well targeted to PKM2 this target.

Table 4 Comparison of the activity of compound 22 in normal HCT116 cells and cells after knocking down PKM2

2) Adding compound 22 to the cells inhibits the entry of PKM2 into the nucleus.

Experimental procedure: HCT16 cells were normally cultured to a density of about 60%, replaced with serum-free medium and compound 22 was added at the final concentration of 10 μM. Four hours later, the cells were harvested, washed with PBS, and the nuclei and cytoplasm were separated using the Pulilai Nuclear Plasma Separation Kit. The amount of PKM2 in the cytoplasm was detected by western.

Evaluation of results: Through nucleoplasmic separation, PKM2 distributed in the nucleus and cytoplasm can be observed more clearly. It can be seen from Figure 1 that after administration of No. 22 drug, almost no PKM2 can be observed in the nucleus. In Figure 1, NZT is a representative of the reported agonists, and it can be seen that the existing agonists cannot inhibit PKM2 into the nucleus. This result shows that the mechanism of action of this compound is different from that of previous agonists. It inhibits PKM2 into the nucleus, and then inhibits its pro-proliferation-related protein kinase activity in the nucleus, so as to achieve the anti-tumor effect.

3) After compound 22 was added to HCT116 cells, the cells were obviously arrested in G2/M phase.

Experimental procedure: Use a 6cm culture dish to plant about 1 million HCT16 cells and incubate for 36-48h. Digest with trypsin to make a single cell suspension, rinse with pre-cooled PBS 1-2 times, then add 1ml PBS to resuspend. Add pre-cooled (-20°C) absolute ethanol, fix at 4°C overnight or store at -20°C for a long time. Centrifuge to remove the fixative, and finally add 5ml PBS in advance to facilitate centrifugation, discard the supernatant. Resuspend in 5ml PBS, soak for 15min-30min, and centrifuge to discard the supernatant. Resuspend in 475μl PBS, add to 1.5ml EP tube, add 25μl 20×RNAse A, and incubate at 37°C for 30min. Add 5 μl of PI staining solution, incubate at 4 degrees in the dark for 30 minutes, centrifuge to remove the staining solution, add 500 μl of PBS to resuspend, pass through a cell sieve, and test on the machine.

Result evaluation: Different concentrations of compound 22 (1 μM, 5 μM) were added to HCT116 cells for cell detection. It can be seen from Figure 2 that compound No. 22 can significantly arrest cells in G2/M phase. Compared with the DMSO negative control group, the ratio of G2/M phase of HCT116 cells treated with 1 μM and 5 μM drug increased by 22.74% and 59.75%, respectively (Fig. 2, C).

Claims (9)

1. A carbamate compound having the structure of formula I or a pharmaceutically acceptable salt thereof, Wherein R is selected from phenyl, ₂ -furyl, 4-nitrophenyl, 4-hydroxyphenyl, 4-cyanophenyl, 2-chlorophenyl, 2-methoxyphenyl, 3-pyridine base, 2-thiazolyl or 3-azaindolyl. 2. The method for preparing the compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that, the compound of formula I is prepared according to the following method: Wherein, R ₁ is phenyl. 3. The method for preparing the compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that, the compound of formula I is prepared according to the following method: Wherein, R is ₂ -furyl, 4-nitrophenyl, 4-hydroxyphenyl, 4-cyanophenyl, 2-chlorophenyl, 2-methoxyphenyl, 3-pyridyl, 2 -thiazolyl or 3-azaindolyl. 4. the method according to claim 3 is characterized in that, the compound of structure shown in formula II is prepared by the following method: ( ₁ ) When R is 2-furyl, 4-nitrophenyl, 4-hydroxyphenyl, 4-cyanophenyl, 2-chlorophenyl, 2-methoxyphenyl, 3-pyridyl or 2-thiazolyl, The compound of formula III reacts with paraformaldehyde under the action of catalyst CF ₃ COOH·(iPr) ₂ NH to generate the compound of formula II; (2) When R ₁ is 3-azaindolyl, 7-Azaindole reacts with acetyl chloride under the action of AlCl ₃ to produce 3-acetyl azaindole, and 3-acetyl azaindole reacts with paraformaldehyde in the catalyst CF ₃ COOH·(iPr) ₂ NH The reaction occurs under the action to generate the compound of formula II structure. 5. Use of the compound of claim 1 or a pharmaceutically acceptable salt thereof in the preparation of an agonist of pyruvate kinase M2 subtype. 6 . The application according to claim 5 , wherein the pyruvate kinase M2 subtype agonist has the effect of preventing the pyruvate kinase M2 subtype from entering the nucleus. 7. The application of the compound as claimed in claim 1 or a pharmaceutically acceptable salt thereof in the preparation of antitumor drugs. 8. The application according to claim 7, characterized in that the antineoplastic drug is a drug for treating colon cancer, cervical cancer or lung cancer. 9. Use of the compound of claim 1 or a pharmaceutically acceptable salt thereof in the preparation of an agonist of the glycolysis pathway.