WO2020192348A1

WO2020192348A1 - Phenyl allylidene cyclohexenone derivatives and preparation method and use

Info

Publication number: WO2020192348A1
Application number: PCT/CN2020/076878
Authority: WO
Inventors: 凌勇; 杨圣菊; 张延安; 刘季; 凌长春; 李洋阳; 刘思群; 贾启新; 明古旭; 吴红梅
Original assignee: 南通大学
Priority date: 2019-03-22
Filing date: 2020-02-27
Publication date: 2020-10-01
Also published as: CN110002987A; AU2020213346A1; AU2020213346B2; CN110002987B

Abstract

Disclosed are phenyl allylidene cyclohexenone derivatives with the structure shown in general formula I. In combination with structural characteristics and structure-function relationship of piplartine, amino bonds are replaced with double bonds, and active groups are reserved, aromatic substituents are changed, novel phenyl allylidene cyclohexenone derivatives targeting thioredoxin reductase (TrxR) for tumor tissue overexpression are designed, the defects of multiple piplartine synthesis steps and requirement for expensive metal catalysts are overcome, and the anti-tumor activity of the compounds is improved. Research results indicate that the compounds have strong inhabitation effects on proliferation of tumor cells and can significantly increase the ROS level in the tumor cells and enhance the anti-tumor effect thereof.

Description

Phenyl allyl cyclohexenone derivative, preparation method and application

Technical field

The present invention relates to the field of biomedicine, in particular to a class of phenyl allyl cyclohexenone derivatives and preparation methods, pharmaceutical compositions containing these derivatives and pharmaceutical applications with TrxR inhibitory activity, especially in preparation Application of anti-tumor drugs.

Background technique

The World Health Organization (WHO) report shows that malignant tumors have long become one of the world's major diseases and are rapidly becoming the world's number one "killer disease", seriously threatening human health and lives. According to WHO statistics, in the past 30 years, the world cancer incidence rate has increased at an average annual rate of 3-5% and it is expected that by 2020, the world cancer incidence rate will increase by 50% compared to 2008, that is, 15 million new cancer patients will be added every year . Not only that, the global death toll from cancer is also rising rapidly, and it has become the world's number one deadly disease. And it is estimated that by 2030, the global cancer deaths will reach 13.2 million. Cancer has become a global challenge and problem, and the fight against cancer has a long way to go.

Reactive oxygen species (ROS) is a general term for chemically active oxygen metabolites and their derivatives generated after molecular oxygen is reduced by a single electron. ROS can be divided and non-radical type radical, wherein the radical type mainly containing superoxide anion (O ₂ ^-), hydroxyl radical (HO ·) and the like, non-radical type ROS are mainly hydrogen peroxide (H ₂ O ₂ ), ozone (O ₃ ), pernitrate, etc. Under normal physiological conditions, a variety of ROS scavenging systems exist in cells, such as superoxide dismutase (SOD1, SOD2, SOD3), glutathione peroxidase, catalase (CAT) and glutathione Peptides (GSH), glutaredoxin, antioxidant proteins (peroxiredoxins) and thioredoxins, etc., can make the production and elimination of ROS in the body reach a dynamic balance, so that the normal functions of cells are not affected (Trachootham D, Alexandre J, Huang P. Nature Reviews Drug Discovery, 2009, 8, 579-591). It has been reported that ROS levels increase in a variety of cancer cells. For example, leukemia cells isolated from blood samples of patients with chronic lymphocytic leukemia or hairy cell leukemia have increased ROS production compared with normal lymphocytes (Zhou Y, Hileman EO, Plunkett W, et al. Blood, 2003, 101, 4098-4104). The level of oxidative damage products such as oxidized DNA bases and lipid peroxides in solid tumors increases.

Studies have shown that when the intracellular ROS level reaches a threshold, it will trigger a series of reactions and lead to cell death. Compared with normal cells, tumor cells have higher ROS levels (Fruehauf J P, Meyskens F L. Clinical Cancer Research, 2007, 13, 789-794). Inducing ROS production or inhibiting the antioxidant system to increase the level of ROS in tumor cells is considered an effective anti-tumor strategy. In 2011, Raj et al. found that piperamide can upregulate ROS in tumor cells without affecting the ROS in normal cells, thereby achieving the purpose of selectively killing tumor cells (Raj L, Ide T, Gurkar AU, et al. Nature 2011) ,475,231-234). Studies have shown that piperamide is targeted to regulate thioredoxin protease (TrxR) to regulate the redox effect and the dynamic balance of reactive oxygen species, resulting in a decrease in the level of GSH in tumor cells, an increase in GSSG levels, and ultimately an increase in the concentration of ROS in tumor cells. High, causing apoptosis or necrosis.

It is true that people have found that piperamide has good anti-tumor activity, but it also has some shortcomings that limit its clinical application. First of all, the activity of piper amide is not high enough, and the specific mechanism of action has not been fully elucidated; secondly, the raw materials of piper amide extracted from plants are limited, and the medicinal resources needed for production are quite consumed; in addition, the artificially synthesized piper amide, its The preparation process is complex, requires expensive metal catalysts, and the reaction yield is low. Therefore, it is necessary to carry out structural derivatization and structural optimization of piperamide, and then screen out anti-tumor compounds with strong targeting, high efficiency, low toxicity, and easy synthesis.

Summary of the invention

The present invention designs and synthesizes a novel phenyl allyl cyclohexenone derivative with TrxR inhibitory activity based on the structural characteristics of piper amide, taking into account the biological activity, drug-making properties, and ease of synthesis, and retains PL active sites The C2-C3 double bond and C7-C8 double bond not only have significant inhibitory activity on a variety of human tumor cells and drug-resistant tumor cells, but also have less damage to normal cells and can selectively kill tumor cells. The preliminary research mechanism shows that the compound of the present invention can inhibit TrxR enzyme activity, increase the level of ROS in tumor cells, cause tumor cell membrane damage, induce tumor cell apoptosis, and promote the anti-tumor activity of the compound of the invention.

The specific technical scheme of the present invention is as follows:

A class of phenyl allyl cyclohexenone derivatives with the structure shown in general formula I:

Wherein, R represents one or more substituents on the benzene ring, selected from H, hydroxyl, halogen group, amino, nitro, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkylamine One or more of the group, C1-C6 acyloxy group, C1-C6 methoxy ether; X represents H, halogen group, CN or C1-C6 alkyl group. Preferably, the R represents H, Br, NO ₂ , OCH ₃ , F, CH ₃ , Cl, N(CH ₃ ) ₂ , OH, O(CH ₂ ) ₂ OCH ₃ , O(CH ₂ ) ₂ O( One or more of CH ₂ ) ₂ OCH ₃ or OAc.

Preferably, the substitution position of the R on the benzene ring is one or more of the 2, 3, and 4 positions.

Preferably, the R represents H, 4-F, 4-Cl, 4-Br, 2-NO ₂ , 4-NO ₂ , 3-OH, 2-OCH ₃ , 4-OCH ₃ , 4-CH ₃ , 3-CH ₃ , 4-N(CH ₃ ) ₂ , 4-OH-3-OCH ₃ , 4-OAc-3-OCH ₃ , 3-O(CH ₂ ) ₂ OCH ₃ , 3-OCH ₃ -4- O(CH ₂ ) ₂ OCH ₃ , 3-OCH ₃ -4-O(CH ₂ ) ₂ O(CH ₂ ) ₂ OCH ₃ , X represents H, Cl, Br, CN, CH ₃ .

The preferred structure of the compound of the above general formula is shown in Table 1:

Table 1 Partial compound codes of general formula I and their corresponding structures

I ₁ : (E)-6-((E)-3-phenylallyl)cyclohex-2-enone;

I ₂ : (E)-6-((E)-3-(4-bromophenyl)-allyl)cyclohex-2-enone;

I ₃ : (E)-6-((E)-3-(2-nitrophenyl)allyl)cyclohex-2-enone;

₁₄ : (E)-6-((E)-3-(4-nitrophenyl)allyl)cyclohex-2-enone;

I ₅ : (E)-6-((E)-3-(3-hydroxyphenyl)allyl)cyclohex-2-enone;

I ₆ : (E)-6-((E)-3-(2-methoxyphenyl)allyl)cyclohex-2-enone;

I ₇ : (E)-6-((E)-3-(4-methoxyphenyl)allyl)cyclohex-2-enone;

I ₈ : (E)-6-((E)-3-(4-fluorophenyl)allyl)cyclohex-2-enone;

I ₉ : (E)-6-((E)-3-(4-methylphenyl)allyl)cyclohex-2-enone;

I ₁₀ : (E)-6-((E)-3-(4-chlorophenyl)allyl)cyclohex-2-enone;

I ₁₁ : (E)-6-((E)-3-(4-dimethylaminophenyl)allyl)cyclohex-2-enone;

I ₁₂ : (E)-6-((E)-3-(4-hydroxy-3-methoxyphenyl)allyl)cyclohex-2-enone;

I ₁₃ : (E)-6-((E)-3-(3-(2-methoxyethoxy)phenyl)allyl)cyclohex-2-enone;

I ₁₄ : (E)-6-((E)-3-(3-methoxy-4-(2-methoxyethoxy)phenyl)allyl)cyclohex-2-enone ；

I ₁₅ : (E)-6-((E)-3-(3-methoxy-4-(2-(2-methoxyethoxy)ethoxy)phenyl)allyl) Cyclohex-2-enone;

I ₁₆ : (E)-6-((E)-3-(3-methoxy-4-acetylphenyl)allyl)cyclohex-2-enone;

I ₁₇ : (E)-6-((Z)-2-chloro-3-phenyl)allyl)cyclohex-2-enone;

I ₁₈ : (E)-6-((Z)-2-bromo-3-phenyl)allyl)cyclohex-2-enone;

I ₁₉ : (E)-6-((Z)-2-cyano-3-phenyl)allyl)cyclohex-2-enone.

I ₂₀ : (E)-6-((Z)-2-methyl-3-phenyl)allyl)cyclohex-2-enone.

Another object of the present invention is to provide a preparation method of the compound of general formula I of the present invention, as follows:

The substituted or unsubstituted cinnamaldehyde and cyclohexen-2-one are prepared by the Adol condensation reaction under the catalysis of a catalyst, and the structure of the substituted or unsubstituted cinnamaldehyde is:

R represents one or more substituents on the benzene ring, selected from H, hydroxyl, halogen group, amino, nitro, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkylamino, One or more of C1-C6 acyloxy and C1-C6 methoxy ethers; X represents H, halogen group, CN or C1-C6 alkyl. Preferably, the R represents H, Br, NO ₂ , OCH ₃ , F, CH ₃ , Cl, N(CH ₃ ) ₂ , OH, O(CH ₂ ) ₂ OCH ₃ , O(CH ₂ ) ₂ O( One or more of CH ₂ ) ₂ OCH ₃ or OAc.

Preferably, the catalyst is selected from one or more of triphenylphosphorus, TiCl ₄ , trimethylsilimidazole (TMSI), and magnesium hydrogen sulfate.

A specific preparation method specifically involves dissolving cyclohexen-2-one and triphenylphosphorus in anhydrous dichloromethane, adding a TiCl ₄ dichloromethane solution at -40 to -78°C, slowly dropping Add the cinnamaldehyde solution dissolved in dichloromethane. After the addition is completed, the reaction returns to 0-30°C, and the reaction is continued for 10-12h. Add an appropriate amount of 10% K ₂ CO ₃ solution to make the pH of the reaction solution = 8-10. Phenyl allyl cyclohexenone derivative.

The synthetic route is as follows:

Another object of the present invention is to provide the application of the cyclohexenone derivatives of the present invention in the preparation of drugs with TrxR inhibitory activity. The drug with TrxR inhibitory activity becomes a drug for treating and/or preventing cancer. Preferably, the cancer is selected from liver cancer, colon cancer, gastric cancer, breast cancer or cervical cancer.

The compound of the present invention can be formulated alone or in combination with one or more pharmaceutically acceptable carriers to provide medicine. For example, solvents, diluents, etc., can also be used for oral administration dosage forms, such as capsules, dispersible powders, tablets, granules, etc. Various dosage forms of the pharmaceutical composition of the present invention can be prepared according to methods well known in the pharmaceutical field. These pharmaceutical preparations may contain, for example, 0.05% to 90% by weight of the active ingredient in combination with the carrier, and more commonly about 15% to 60% by weight of the active ingredient. The dosage of the compound of the present invention can be 0.005-5000 mg/kg/day, and the dosage can also exceed this dosage range according to the severity of the disease or the different dosage forms.

The compound of the present invention can self-assemble into nanoparticles to improve activity alone, or be combined with other anti-tumor drugs such as alkylating agents (such as cyclophosphamide or chlorambucil), antimetabolites (such as 5-fluorouracil or hydroxyurea), topological Isomerase inhibitors (such as camptothecin), mitotic inhibitors (such as paclitaxel or vinblastine), DNA inserters (such as doxorubicin) combined with self-assembled nanoparticles to improve activity, and can also be combined with radiotherapy. These other anti-tumor drugs or radiotherapy can be administered at the same time or at different times with the compounds of the present invention. These combination therapies can produce synergistic effects to help improve the therapeutic effect.

The invention combines the structural characteristics, structure-activity relationship and pharmacophore model of the anti-tumor drug piperamide, based on the piperamide, uses the theory of bioelectronic isosteres, and uses cinnamaldehyde with different substituents as raw materials to design and synthesize New phenyl allyl cyclohexenone derivatives with TrxR inhibitory activity have been developed to simplify their synthetic route and facilitate mass production. They have studied their inhibitory effects on TrxR targets and malignant tumor cells. A variety of tumor cells (including liver cancer, breast cancer, gastric cancer, colon cancer, cervical cancer, etc.) have a strong and selective inhibitory effect on the proliferation, and can significantly inhibit TrxR enzyme activity. In addition, the compound of the present invention has a certain concentration of Normal cells are less damaged, and can induce the expression of ROS in tumor cells, and coordinately promote tumor cell apoptosis or necrosis.

detailed description

In order to further clarify the present invention, a series of examples are given below. These examples are completely illustrative. They are only used to describe the present invention in detail and should not be construed as limiting the present invention.

Example 1 Preparation of (E)-6-((E)-3-phenylallyl)cyclohex-2-enone (I ₁ )

Dissolve cyclohexen-2-one (0.48g, 5.0mmol) and triphenylphosphonium (1.31g, 5.0mmol) in 50ml of anhydrous dichloromethane, and add 5ml of 1mol/L TiCl at -50℃ Dichloromethane solution of _4. After 15 minutes, use a constant pressure funnel to slowly add 30ml of dichloromethane-dissolved cinnamaldehyde (1.32g, 10.0mmol) solution. After half an hour, the reaction returns to room temperature and the reaction is continued for about 12 hours. , TLC monitoring, after the reaction is complete, add an appropriate amount of 10% K ₂ CO ₃ solution and continue to stir for about 5 minutes, make the pH of the reaction solution = 9, then extract with dichloromethane (50ml×2), and wash with saturated brine (50mL) Extract the obtained dichloromethane layer, collect the dichloromethane layer, dry with anhydrous sodium sulfate, concentrate, and purify by column chromatography (EA:PE=1:3 as the eluent) to obtain 0.90 g of yellow solid product with a yield 86%. I ₁ spectrum data: ESI-MS (m/z): 211[M+H] ⁺ ; ¹ H NMR (DMSO-d ₆ , 400MHz): δ7.46 (m, 2H, Ar-H), 7.32 (m, 3H, Ar-H), 7.12 (m, 1H, CH), 6.96 (m, 3H, CH), 6.19 (d, 1H, J = 16.8 Hz, CH), 2.88 (m, 2H, CH ₂ ), 2.44 (m, 2H, CH ₂ ). ¹³ C NMR (CDCl ₃ , 100 MHz): δ188.18,141.07,140.25,135.63,134.28,133.77,131.06,131.02,128.73,128.57,128.31,127.16,125.02,25.45,21.00.

(E)-6-((E)-3-(4-bromophenyl)-allyl)cyclohex-2-enone (I ₂ ) preparation

Referring I ₁ synthesis method, 4-bromo - cinnamaldehyde (2.22g, 10.0mmol) Alternatively cinnamaldehyde (1.32g, 10.0mmol), cyclohexene-2-one (0.48g, 5.0mmol) to give The yellow solid product was 1.20 g, yield: 83%. I ₂ spectrum data: ESI-MS (m/z): 289[M+H]+; ¹ H NMR (DMSO-d ₆ , 400MHz): δ (CDCl ₃ , 400MHz) 7.46 (m, 2H, Ar -H), 7.34 (m, 2H, Ar-H), 7.28 (d, 1H, J = 18.4 Hz, CH), 7.07 (m, 1H, CH), 7.01 (m, 1H, CH), 6.87 (m , 1H, CH), 6.22 (m, 1H, CH), 2.91 (m, 2H, CH ₂ ), 2.48 (m, 2H, CH ₂ ). ¹³ C NMR (CDCl ₃ , 100 MHz): δ188.15, 149.06, 138.62, 135.62, 133.81, 131.22, 131.02, 128.51, 128.39, 123.69, 123.56, 122.65, 25.59, 25.20.

(E)-6-((E)-3-(2-nitrophenyl)allyl)cyclohex-2-enone (I ₃ ) preparation

Referring I ₁ synthesis method, the 2-nitro - cinnamaldehyde (1.81g, 10.0mmol) Alternatively cinnamaldehyde (1.32g, 10.0mmol), cyclohexene-2-one (0.48g, 5.0mmol) the reaction, 1.10 g of yellow solid product was obtained, yield: 86%. I ₃ spectrum data: ESI-MS (m/z): 256[M+H] ⁺ ; ¹ H NMR (CDCl ₃ , 400MHz): δ7.98 (m, 1H, CH), 7.72 (m, 1H ,Ar-H),7.61(m,1H,Ar-H),7.45(m,1H,Ar-H),7.42(m,1H,CH),7.31(d,1H,J=16.8Hz,CH) , 7.06 (m, 1H, CH), 7.05 (m, 1H, CH), 6.25 (m, 1H, CH), 2.93 (m, 2H, CH ₂ ), 2.50 (m, 2H, CH ₂ ). ¹³ C NMR (CDCl ₃ , 100 MHz): δ181.98, 149.46, 161.76, 147.99, 136.11, 134.35, 133.22, 133.05, 132.35, 131.02, 128.84, 128.37, 127.69, 124.85, 25.54, 25.43.

Preparation of (E)-6-((E)-3-(4-nitrophenyl)allyl)cyclohex-2-enone (I ₄ )

Referring I ₁ synthesis method, the 4-nitro - cinnamaldehyde (1.81g, 10.0mmol) Alternatively cinnamaldehyde (1.32g, 10.0mmol), cyclohexene-2-one (0.48g, 5.0mmol) the reaction, 0.84 g of yellow solid product was obtained, with a yield of 66%. I ₄ spectrum data: ESI-MS (m/z): 256[M+H] ⁺ ; ¹ H NMR (CDCl ₃ , 400MHz): δ8.18 (m, 2H, Ar-H), 7.58 (m ,2H,Ar-H),7.20(m,2H,CH),7.03(m,1H,CH),6.94(d,1H,J=15.7Hz,CH),6.21(m,1H,CH),2.91 (m, 2H, CH ₂ ), 2.49 (m, 2H, CH ₂ ). ¹³ C NMR (CDCl ₃ , 100 MHz): δ187.86, 149.49, 142.91, 136.99, 136.57, 132.80, 130.98, 127.37, 127.13, 124.11, 25.56, 25.40.

(E)-6-((E)-3-(3-hydroxyphenyl)allyl)cyclohex-2-enone (I ₅ ) preparation

Referring I ₁ synthesis method, the 3-hydroxy - cinnamic aldehyde (1.48g, 10.0mmol) Alternatively cinnamaldehyde (1.32g, 10.0mmol), cyclohexene-2-one (0.48g, 5.0mmol) to give 0.91 g of yellow solid product, yield: 80%. The I ₅ spectrum data is: ESI-MS(m/z): 227[M+H] ⁺ .

(E)-6-((E)-3-(2-methoxyphenyl)allyl)cyclohex-2-enone (I ₆ ) preparation

With reference to the synthesis method of I ₁ , the 2-methoxy-cinnamaldehyde (1.69g, 10.0mmol) was substituted for cinnamaldehyde (1.32g, 10.0mmol) and reacted with cyclohexen-2-one (0.48g, 5.0mmol) , Obtain 0.97 g of yellow solid product, yield: 81%. I ₆ spectrum data: ESI-MS (m/z): 241[M+H] ⁺ ; ¹ H NMR (DMSO-d ₆ , 400MHz): δ7.53 (m, 1H, CH), 7.35 (m ,1H,Ar-H),7.32(m,1H,CH),7.27(m,1H,Ar-H),7.12(m,1H,CH),7.00(m,1H,CH),6.98(m, 1H, Ar-H), 6.91 (m, 1H, Ar-H), 6.21 (d, 1H, J = 18.4 Hz, CH), 3.86 (s, 3H, CH ₃ ), 2.91 (m, 2H, CH ₂ ), 2.47 (m, 2H, CH ₂ ). ¹³ C NMR (CDCl ₃ , 100 MHz): δ 188.42, 157.37, 149.14, 148.97, 135.65, 133.07, 131.16, 129.90, 127.32, 125.69, 123.80, 120.82, 111.01, 55.43, 25.43, 25.22.

Preparation of (E)-6-((E)-3-(4-methoxyphenyl)allyl)cyclohex-2-enone (I ₇ )

Referring to the synthesis method of I ₁ , the 4-methoxy-cinnamaldehyde (1.67g, 10.0mmol) was substituted for cinnamaldehyde (1.32g, 10.0mmol) and reacted with cyclohexen-2-one (0.48g, 5.0mmol) , Obtain 0.98 g of yellow solid product, yield: 82%. I ₇ spectrum data: ESI-MS (m/z): 241[M+H] ⁺ ; ¹ H NMR (CDCl ₃ , 400MHz): δ7.44 (m, 2H, Ar-H), 7.30 (m ,1H,CH),7.0(m,1H,CH),6.94(m,4H,Ar-H,CH), 6.21(m,1H,CH),7.80(m,3H,CH ₃ ), 2.90(m , 2H, CH ₂ ), 2.47 (m, 2H, CH ₂ ). ¹³ C NMR (CDCl ₃ , 100 MHz): δ 188.41, 160.20, 149.19, 140.22, 135.03, 132.59, 131.21, 129.50, 128.58, 121.07, 114.33, 55.51, 25.39, 18.55.

(E)-6-((E)-3-(4-fluorophenyl)allyl)cyclohex-2-enone (I ₈ ) preparation

Referring I ₁ synthesis method, the 4-fluoro - cinnamic aldehyde (1.58g, 10.0mmol) Alternatively cinnamaldehyde (1.32g, 10.0mmol), cyclohexene-2-one (0.48g, 5.0mmol) to give 0.95 g of yellow solid product, yield: 83%. The I ₈ spectrum data is: ESI-MS (m/z): 229[M+H] ⁺ ; ¹ H NMR (DMSO-d ₆ , 400MHz): δ7.46 (m, 2H, Ar-H), 7.28 (m, 1H, CH), 7.04 (m, 2H, Ar-H), 6.96 (m, 1H, CH), 6.94 (m, 1H, CH), 6.91 (m, 1H, CH), 6.21 (m, 1H, CH), 2.91 (m, 2H, CH ₂ ), 2.48 (m, 2H, CH ₂ ). ¹³ C NMR (CDCl ₃ , 100MHz): δ188.18, 164.22, 161.76, 148.95, 138.85, 134.20, 132.96, 131.02, 131.25, 128.68, 122.69, 116.08, 115.60, 25.50, 25.32.

(E)-6-((E)-3-(4-methylphenyl)allyl)cyclohex-2-enone (I ₉ ) preparation

I refer to the synthesis method _1, 4-methyl - cinnamaldehyde (1.46g, 10.0mmol) Alternatively cinnamaldehyde (1.32g, 10.0mmol), cyclohexene-2-one (0.48g, 5.0mmol) the reaction, 0.90 g of yellow solid product was obtained, with a yield of 80%. I ₉ spectrum data: ESI-MS (m/z): 225[M+H] ⁺ ; ¹ H NMR (DMSO-d ₆ , 400MHz): δ7.54 (m, 2H, Ar-H), 7.22 (m, 3H, Ar-H, CH), 7.14 (m, 2H, CH), 7.03 (d, 1H, J = 15.3 Hz, CH), 6.10 (m, 1H, CH), 2.92 (m, 2H, CH ₂ ), 2.44 (m, 2H, CH ₂ ), 2.32 (s, 3H, CH ₃ ). ¹³ C NMR (DMSO-d ₆ , 101 MHz): δ 187.63, 151.05, 140.41, 138.89, 134.34, 134.27, 134.12, 130.63, 129.77, 127.67, 123.10, 25.47, 25.24, 21.40.

Preparation of (E)-6-((E)-3-(4-chlorophenyl)allyl)cyclohex-2-enone (I ₁₀ )

Referring I ₁ synthesis method, the 4-chloro - cinnamaldehyde (1.66g, 10.0mmol) Alternatively cinnamaldehyde (1.32g, 10.0mmol), cyclohexene-2-one (0.48g, 5.0mmol) to give 1.06 g of yellow solid product, yield: 87%. I ₁₀ spectral data: ESI-MS (m/z): 256[M+H] ⁺ ; ¹ H NMR (DMSO-d ₆ , 400MHz): δ7.68 (m, 2H, Ar-H), 7.44 (m, 2H, Ar-H), 7.35 (m, 1H, CH), 7.15 (m, 3H, CH), 6.11 (m, 1H, CH), 2.94 (m, 2H, CH ₂ ), 2.45 (m ,2H,CH ₂ ). ¹³ C NMR (CDCl ₃ , 100 MHz): δ187.67, 151.32, 138.78, 135.96, 135.49, 133.55, 133.48, 130.57, 129.33, 129.23, 124.91, 25.50, 25.32.

Preparation of (E)-6-((E)-3-(4-dimethylaminophenyl)allyl)cyclohex-2-enone (I ₁₁ )

Referring to the synthesis method of I ₁ , the 4-dimethylamino-cinnamaldehyde (1.75g, 10.0mmol) was substituted for cinnamaldehyde (1.32g, 10.0mmol) and reacted with cyclohexen-2-one (0.48g, 5.0mmol) , Obtain 0.97 g of red solid product, yield: 77%. I ₁₁ spectrum data: ESI-MS (m/z): 254[M+H] ⁺ ; ¹ H NMR (CDCl ₃ , 400MHz): δ 7.38 (m, 1H, CH), 7.33 (m, 2H ,Ar-H), 6.97 (m, 1H, CH), 6.88 (m, 1H, CH), 6.68 (m, 2H, Ar-H), 6.19 (m, 1H, CH), 3.00 (s, 6H, CH ₃ ), 2.89 (m, 2H, CH ₂ ), 2.45 (m, 2H, CH ₂ ). ¹³ C NMR (CDCl ₃ , 101 MHz): δ188.24, 150.85, 148.49, 141.34, 135.87, 131.30, 128.55, 124.90, 118.79, 112.11, 40.26, 25.33, 25.10.

(E)-6-((E)-3-(4-hydroxy-3-methoxyphenyl)allyl)cyclohex-2-enone (I ₁₂ ) preparation

Referring to the synthetic method of I ₁ , the 4-hydroxy-3-methoxy-cinnamaldehyde (1.78g, 10.0mmol) was substituted for cinnamaldehyde (1.32g, 10.0mmol), and cyclohexen-2-one (0.48g, 5.0 mmol) reaction to obtain 1.13 g of yellow solid product, yield: 88%. I ₁₂ spectral data: ESI-MS (m/z): 257[M+H] ⁺ ; ¹ H NMR (DMSO-d ₆ , 400MHz): δ7.30 (m, 1H, CH), 7.01 (m ,3H,Ar-H),6.90(m,3H,CH),6.21(m,1H,CH),5.93(s,1H,OH),3.94(s,3H,CH ₃ ),2.90(m,2H , CH ₂ ), 2.48 (m, 2H, CH ₂ ). ¹³ C NMR (CDCl ₃ , 101 MHz): δ188.24, 150.85, 148.49, 141.34, 135.87, 131.30, 128.55, 124.90, 118.79, 112.11, 40.26, 25.33, 25.10.

(E)-6-((E)-3-(3-(2-methoxyethoxy)phenyl)allyl)cyclohex-2-enone (I ₁₃ ) preparation

Referring I ₁ synthesis method, 3- (2-methoxyethoxy) - cinnamic aldehyde (2.06g, 10.0mmol) Alternatively cinnamaldehyde (1.32g, 10.0mmol), cyclohexene-2-one ( 0.48g, 5.0mmol) to obtain 1.08g yellow solid product, yield: 76%. The I ₁₃ spectrum data is: ESI-MS (m/z): 285 [M+H] ⁺ .

(E)-6-((E)-3-(3-methoxy-4-(2-methoxyethoxy)phenyl)allyl)cyclohex-2-enone (I ₁₄ ) Preparation

Referring I ₁ synthesis method, the 3-methoxy-4- (2-methoxyethoxy) - cinnamic aldehyde (2.36g, 10.0mmol) Alternatively cinnamaldehyde (1.32g, 10.0mmol), and cyclohexyl En-2-one (0.48 g, 5.0 mmol) was reacted to obtain 1.10 g of yellow solid product, yield: 70%. The I ₁₄ spectrum data is: ESI-MS (m/z): 315[M+H] ⁺ .

(E)-6-((E)-3-(3-methoxy-4-(2-(2-methoxyethoxy)ethoxy)phenyl)allyl)cyclohexyl- Preparation of 2-enone (I ₁₅ )

Referring I ₁ synthesis method, the 3-methoxy-4- (2- (2-methoxyethoxy) ethoxy - cinnamaldehyde (2.80g, 10.0mmol) Alternatively cinnamaldehyde (1.32g, 10.0 mmol), react with cyclohexen-2-one (0.48g, 5.0mmol) to obtain 1.27g of yellow solid product, yield: 71%. I ₁₅ spectrum data is: ESI-MS(m/z): 359 [M+H] ⁺ .

(E)-6-((E)-3-(3-methoxy-4-acetylphenyl)allyl)cyclohex-2-enone (I ₁₆ ) preparation

Referring I ₁ synthesis method, the 3-methoxy-acetyl - cinnamaldehyde (2.20g, 10.0mmol) Alternatively cinnamaldehyde (1.32g, 10.0mmol), cyclohexene-2-one (0.48g , 5.0mmol) to obtain 1.16g yellow solid product, yield: 78%. I ₁₆ spectral data: ESI-MS (m/z): 299[M+H] ⁺ ; ¹ H NMR (CDCl ₃ , 400MHz): δ7.23 (m, 1H, CH), 7.03 (m, 1H ,CH),6.96(m,4H,Ar-H,CH),6.84(d,J=15.3Hz,1H,CH),6.16(m,1H,CH),3.82(s,3H,CH ₃ ), 2.86 (m, 1H, CH ₂ ), 2.42 (m, 1H, CH ₂ ), 2.26 (s, 3H, CH ₃ ). ¹³ C NMR (CDCl ₃ , 101 MHz): δ 188.06, 168.83, 151.23, 149.14, 140.14, 139.48, 135.67, 134.01, 133.97, 131.01, 123.25, 123.03, 119.69, 110.66, 55.88, 25.34, 25.30.

(E)-6-((Z)-2-chloro-3-phenyl)allyl)cyclohex-2-enone (I ₁₇ ) preparation

Referring I ₁ synthesis method, the α- chloro cinnamic aldehyde (1.66g, 10.0mmol) Alternatively cinnamaldehyde (1.32g, 10.0mmol), cyclohexene-2-one (0.48g, 5.0mmol) to give 0.98 g of yellow solid product, yield: 80%. The I ₁₇ spectrum data is: ESI-MS (m/z): 245[M+H] ⁺ .

(E)-6-((Z)-2-bromo-3-phenyl)allyl)cyclohex-2-enone (I ₁₈ ) preparation

Referring I ₁ synthesis method, the α- bromo cinnamic aldehyde (2.11g, 10.0mmol) Alternatively cinnamaldehyde (1.32g, 10.0mmol), cyclohexene-2-one (0.48g, 5.0mmol) to give The yellow solid product was 1.18 g, yield: 82%. I ₁₈ spectral data: ESI-MS (m/z): 289[M+H] ⁺ ; ¹ H NMR (CDCl ₃ , 400MHz): δ7.66 (m, 2H, Ar-H), 7.36 (m ,3H,Ar-H),7.20(m,1H,CH),7.03(m,1H,CH),6.91(s,1H,CH),6.20(m,1H,CH),3.03(m,2H, CH ₂ ), 2.45 (m, 2H, CH ₂ ).

(E)-6-((Z)-2-cyano-3-phenyl)allyl)cyclohex-2-enone (I ₁₉ ) preparation

Referring I ₁ synthesis method, the α- cyano-cinnamic aldehyde (1.57g, 10.0mmol) Alternatively cinnamaldehyde (1.32g, 10.0mmol), cyclohexene-2-one (0.48g, 5.0mmol) to give The yellow solid product is 0.75 g, yield: 64%. I ₁₉ spectral data: ESI-MS (m/z): 236[M+H] ⁺ ; ¹ H NMR (CDCl ₃ , 400MHz): δ7.38 (m, 7H, Ar-H), 7.03 (m , 1H, CH), 6.64 (s, 1H, CH), 3.02 (m, 2H, CH ₂ ), 2.42 (m, 2H, CH ₂ ).

(E)-6-((Z)-2-methyl-3-phenyl)allyl)cyclohex-2-enone (I ₂₀ ) preparation

Referring I ₁ synthesis method, the α- methyl-cinnamaldehyde (1.46g, 10.0mmol) Alternatively cinnamaldehyde (1.32g, 10.0mmol), cyclohexene-2-one (0.48g, 5.0mmol) to give 0.87 g of yellow solid product, yield: 78%. I ₂₀ spectral data: ESI-MS (m/z): 224[M+H] ⁺ ; ¹ H NMR (CDCl ₃ , 400MHz): δ7.32 (m, 4H, Ar-H), 7.23 (m ,1H,Ar-H),7.16(s,1H,CH),6.99(m,1H,CH),6.61(s,1H,CH),6.18(m,1H,CH),2.99(m,2H, CH ₂ ), 2.40 (m, 2H, CH ₂ ), 2.09 (s, 3H, CH ₃ ). ¹³ C NMR (CDCl ₃ , 101 MHz): δ189.00, 149.09, 139.72, 136.96, 134.59, 133.93, 133.51, 130.84, 129.23, 128.21, 127.17, 26.71,25.71, 18.57.

Example 2 Using MTT method to determine the inhibition rate of tumor cell and normal cell proliferation of the compound of the present invention

The anti-proliferative activity of the compound of the present invention on four human cancer cell lines was evaluated by using the tetramethylazole blue colorimetry (MTT) in vitro anti-tumor test. Piperamine (PL) was used as a positive control drug. Human cancer cell lines: liver cancer cell SMMC7721, colon cancer cell HCT116, gastric cancer cell HGC-27, human cervical cancer cell Hela, human normal cell: human gastric mucosal epithelial cell GES-1.

The experimental method is as follows: Take a bottle of cells that are in good condition in the exponential growth phase, add 0.25% trypsin to digest, make the adherent cells fall off, and prepare a suspension containing 2×10 ⁴ to 4×10 ⁴ cells per ml. The cell suspension was inoculated on a 96-well plate, 180 μL per well, and placed in a constant temperature CO ₂ incubator for 24 hours. Change the liquid, add test compounds I ₁ -I ₂₀ (compounds are dissolved in DMSO and then diluted with PBS, the concentration of test compound is 12.5 μM), 20 μL per well, and incubated for 72 hours. Add MTT to a 96-well plate, 20μL per well, and react in an incubator for 4 hours. Aspirate the supernatant, add DMSO, 150 μL per well, and shake on a plate shaker for 5 minutes. Measure the absorbance of each well with an enzyme-linked immunoassay at a wavelength of 570nm to calculate the cell inhibition rate. The experimental results are shown in Table 2.

Cell inhibition rate=(OD value of negative control group-OD value of test substance group)/OD value of negative control group×100%.

The compound of the present invention has undergone a series of tumor cell anti-proliferation activity tests. The results of pharmacological experiments show (see Table 2) that the compound I ₁ -I _{20 of the} present invention has a strong inhibitory effect on the proliferation of most tumor cells at a concentration of 12.5 μM. In particular, the inhibitory activity of some compounds was significantly better than that of the positive control drug pyramine (PL). However, the compounds I ₁ to I _{20 of the} present invention are significantly less cytotoxic to human normal gastric mucosal cells GES-1 at the same concentration than tumor cells, indicating that the compounds of the present invention not only have significant anti-tumor activity on tumor cells, but also Low cytotoxicity and certain tumor cell selectivity.

Table 2 Inhibition rate% of some compounds of the present invention on human tumor cells and normal cells (12.5μM)

ND: not detected

Example 3 Determination of Intracellular ROS Level

The ROS-Glo hydrogen peroxide test method (Promega, Southampton, UK) measures ROS changes by directly detecting H ₂ O ₂ levels in cells. The cells were seeded into a 96-well cell culture plate and cultured with the test drug (0.01-12.5μM) for 24 hours. Add hydrogen peroxide substrate solution to each well and incubate at 37°C for 6 hours in a constant temperature CO ₂ incubator. After the incubation, add ROS-Glo detection solution to each well and incubate at room temperature for 20 minutes. Fluorescence is detected by BioTek Synergy HT multi-mode microplate reader.

I ₄ to I ₈ , I ₁₁ to I ₁₅ , I ₁₈ , and I _{19 among the} compounds of general formula I of the present invention are selected as representatives, and the ROS levels in tumor cells are tested. DCFH-DA was used as a fluorescent probe to determine the changes of ROS in human cervical cancer Hela cells after drug treatment. The changes in fluorescence intensity can quantitatively reflect the intracellular ROS levels. The results show that the compounds I ₄ ～I ₈ , I ₁₁ ～I ₁₅ , I ₁₈ , and I _{19 of the} present invention can significantly increase the ROS content in Hela cells at 12.5 μM, which is 3.7 to 8.9 times that of the control group, which is better than the positive control drug PL (3.2 times the control group).

Example 4 Study on the inhibitory activity of the compound of the present invention on TrxR

The effect of the tested drug (12.5μM) on TrxR activity was evaluated by the TrxR activity test kit (BioVision, Milpitas, CA, USA). Simply put, the cell line is dissolved in a centrifuge tube with 1x buffer solution and then ice-bathed for 20 minutes, and then centrifuged at 10,000×g 4°C for 15 minutes. The supernatant is transferred to a new centrifuge tube, and the protein concentration is calculated by the Bio-Rad protein test method. The sample is diluted to 2X working concentration with buffer solution. Prepare two wells (with and without inhibitor) for each sample in triplicate. The reaction buffer and the reaction buffer with inhibitors were prepared according to the instructions. Before reading, use the BioTek Synergy HT multi-mode microplate reader to measure the absorbance at 412nm wavelength every 20 seconds within 5 minutes after shaking.

The experimental results show that compounds I ₁ to I ₂₀ have significant inhibitory activity on TrxR at a concentration of 12.5 μM. The inhibitory activity data are shown in Table 3. Most of the compounds show stronger or comparable inhibitory activity than the positive control drug stubmenine The activity indicates that the phenylallyl cyclohexenone derivative of the present invention has good TrxR inhibitory activity, which is consistent with its anti-tumor activity.

Table 3 Inhibition rate of TrxR in vitro of some compounds of the present invention% (12.5μM)

Claims

A class of phenyl allyl cyclohexenone derivatives with the structure shown in general formula I:

Among them, R represents H, hydroxyl, halogen group, amino, nitro, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkylamino, C1-C6 acyloxy, C1- One or more of C6 methoxy ethers; X represents H, halogen group, CN or C1-C6 alkyl.
The phenyl allyl cyclohexenone derivative according to claim 1, wherein the R represents H, F, Cl, Br, NO 2 , OCH 3 , CH 3 , N(CH 3 ) 2 , OH, O(CH 2 ) 2 OCH 3 , O(CH 2 ) 2 O(CH 2 ) 2 OCH 3 , OAc.
The phenyl allyl cyclohexenone derivative according to claim 1, characterized in that the substitution position of the R on the benzene ring is one or more of the 2, 3, and 4 positions.
The phenyl allyl cyclohexenone derivative according to claim 1, wherein the R represents H, 4-F, 4-Cl, 4-Br, 2-NO 2 , 4-NO 2 , 3-OH, 2-OCH 3 , 4-OCH 3 , 4-CH 3 , 3-CH 3 , 4-N(CH 3 ) 2 , 4-OH-3-OCH 3 , 4-OAc-3-OCH 3 , 3-O(CH 2 ) 2 OCH 3 , 3-OCH 3 -4-O(CH 2 ) 2 OCH 3 , 3-OCH 3 -4-O(CH 2 ) 2 O(CH 2 ) 2 OCH 3 , X represents H, Cl, Br, CN, CH 3 .
The phenyl allyl cyclohexenone derivative according to claim 1, wherein the phenyl allyl cyclohexenone derivative is selected from the following:

R stands for H, 4-F, 4-Cl, 4-Br, 2-NO 2 , 4-NO 2 , 3-OH, 2-OCH 3 , 4-OCH 3 , 4-CH 3 , 3-CH 3 , 4-N(CH 3 ) 2 , 4-OH-3-OCH 3 , 4-OAc-3-OCH 3 , 3-O(CH 2 ) 2 OCH 3 , 3-OCH 3 -4-O(CH 2 ) 2 OCH 3 , 3-OCH 3 -4-O(CH 2 ) 2 O(CH 2 ) 2 OCH 3 , X represents H;

Alternatively, R representatives H, X representative of Cl, Br, CN or CH 3.
The preparation method of the phenylallyl cyclohexenone derivative according to any one of claims 1 to 5, characterized in that the substituted or unsubstituted cinnamaldehyde and cyclohexen-2-one are catalyzed by a catalyst It is prepared by Adol condensation reaction, and the substituted or unsubstituted cinnamaldehyde structural formula is:
R represents H, hydroxyl, halogen group, amino, nitro, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkylamino, C1-C6 acyloxy, C1-C6 One or more of methoxy ethers; X represents H, halogen group, CN or C1-C6 alkyl.
The preparation method according to claim 6, characterized in that the catalyst is selected from one or more of triphenylphosphonium, TiCl 4 , trimethylsilimidazole, and magnesium hydrogen sulfate.
The preparation method according to claim 6, characterized in that the preparation method specifically comprises dissolving cyclohexen-2-one and triphenylphosphorus in anhydrous dichloromethane, at -40 to -78°C Add the dichloromethane solution of TiCl 4 and slowly add the cinnamaldehyde solution dissolved in dichloromethane dropwise. After the addition is complete, the reaction returns to 0-30℃, and the reaction is continued for 10-12h. Add an appropriate amount of 10% K 2 CO 3 solution to make The pH of the reaction solution is 8-10, and the phenyl allyl cyclohexenone derivative is obtained.
The use of the phenyl allyl cyclohexenone derivative according to any one of claims 1 to 5 in the preparation of TrxR inhibitory drugs.
The application according to claim 9, characterized in that the drug with TrxR inhibitory activity is a drug for treating and/or preventing cancer.
The use according to claim 10, wherein the cancer is selected from liver cancer, colon cancer, gastric cancer, breast cancer or cervical cancer.