AU2020213346B2

AU2020213346B2 - Phenylallyl cyclohexenone derivatives and their preparation method and application

Info

Publication number: AU2020213346B2
Application number: AU2020213346A
Authority: AU
Inventors: Qixin JIA; Yangyang Li; Changchun LING; Yong Ling; Ji LIU; Siqun LIU; Guxu MING; Hongmei WU; Shengju Yang; Yan'an ZHANG
Original assignee: Nantong University
Current assignee: Nantong University
Priority date: 2019-03-22
Filing date: 2020-02-27
Publication date: 2020-12-17
Anticipated expiration: 2040-02-27
Also published as: CN110002987A; AU2020213346A1; CN110002987B; WO2020192348A1

Abstract

The present invention discloses a class of phenylallyl cyclohexenone derivatives with the general structure shown in general formula I: 0 5 R I The present invention combines the structural characteristics and structure-activity relationships of piperlongumine, replacing the amide bond with a double bond, while retaining the active group, changing the aromatic ring substituents, so as to design a 10 novel type of phenylallyl cyclohexenone derivatives targeting the highly expressed thioredoxin reductase (TrxR) in tumor tissues. It overcomes the shortcomings of multiple synthesis steps and expensive metal catalysts for the preparation of piperlongumine, and improves the anti-tumor activity of piperlongumine. These research results show that the compounds of the present invention have strong 15 inhibitory effects on the proliferation in many tumor cells, and can significantly increase the level of ROS in tumor cells to enhance their antitumor effects.

Description

Phenylallyl cyclohexenone derivatives and their preparation

method and application

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

Background technique The World Health Organization (WHO) report shows that malignant tumors have long been one of the major global diseases and are rapidly becoming the number one "killer disease" in the world, seriously threatening human health and life. According to WHO statistics, in the past three decades, the world's cancer incidence rate has been increasing at an average annual rate of 3-5%. It is expected that by 2020, the world cancer incidence rate will increase by 50% compared with 2008, that is, 15 million new cancer patients will be added each year. Not only that, the number of cancer-related deaths in the world has also risen rapidly, and it has become the world's leading cause of death. 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 the general term for chemically active oxygen metabolites and their derivatives produced by the reduction of molecular oxygen by a single electron. ROS can be divided into free radicals and non-free radicals, of which free radicals mainly include superoxide anion (02-), hydroxyl radical (HO.), etc. Non-free radical ROS mainly include hydrogen peroxide (H 2 0 2 ), Ozone (03), 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 Peptide (GSH), glutaredoxin, peroxiredoxins, and thioredoxin, etc., can make the generation and elimination of ROS in the body reach a dynamic balance, so that the normal functions of the 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 are increased in various 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 to normal lymphocytes (Zhou Y, Hileman EO, Plunkett W, et al. Blood, 2003, 101, 4098-4104). The levels of oxidative damage products such as oxidized DNA bases, lipid peroxides in solid tumors increase. Studies have shown that when the level of ROS in a cell reaches a threshold, a series of reactions will be triggered and cause cell death. Compared with normal cells, tumor cells have higher ROS levels (Fruehauf J P, Meyskens FL. Clinical Cancer Research,

2007, 13, 789-794). Inducing ROS production or inhibiting the antioxidant system to increase ROS levels in tumor cells is considered an effective anti-tumor strategy. In 2011, Raj et al. found that piperlongumine can upregulate the ROS in tumor cells without affecting the ROS of normal cells, thereby achieving the purpose of selectively killing tumor cells. Studies have shown that piperlongumine is targeted to modulate thioredoxin (TrxR) to regulate the dynamic balance of redox and reactive oxygen species, resulting in the decrease of GSH levels and increase of GSSG levels in tumor cells, which eventually leads to the increase of ROS concentration in tumor cells and causing apoptosis or necrosis. It is true that at present people have found that piperlongumine (PL) has good antitumor activity, but it also has some shortcomings, which limit its clinical application. Firstly, the activity of piperlongumine is not high enough, and the specific mechanism of action has not been fully elucidated; secondly, the raw materials of piperlongumine extracted from plants are limited, and the consumption of medicinal materials required for production is quite large; in addition, the preparation process of the synthetic piperlongumine is complicated, requiring expensive metal catalysts, and the reaction yield is low. Therefore, it is necessary to derivatize and optimize the structure of piperlongumine, and then screen out anti-tumor compounds with strong targeting, high efficiency and low toxicity, and easy to synthesize. It is an object of the present invention to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative. Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field.

Summary of the invention

According to a first aspect the invention provides a phenylallyl cyclohexenone derivative with the general structure shown in general formula I: 0

R

I where R represents one or more substituents on the benzene ring, selected from one or more of H, hydroxyl, halogen group, amino, nitro, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkylamine group, C1-C6 acyloxy group and C1-C6 methoxy ether; X represents H, halogen group, CN or Cl-C6 alkyl.

According to the structural characteristics of piperlongumine and taking into account three aspects of biological activity, medicinal properties, and ease of synthesis, the present invention designed and synthesized new phenylallyl cyclohexenone derivatives with TrxR inhibitory activity, retaining the PL active site C2-C3 double bond and C7-C8 double bond. Not only does it have significant inhibitory activity against a variety of human-derived tumor cells and drug-resistant tumor cells, but it also has 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 tumor cell ROS level, cause tumor cell membrane damage, induce tumor cell apoptosis, and promote the anti-tumor activity of the compound. The specific technical solution of the present invention is as follows: A class of phenylallyl cyclohexenone derivatives with the structure shown in general formula I : 0

R

I Where R represents one or more substituents on the benzene ring, selected from one or more of H, hydroxyl, halogen group, amino, nitro, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkylamine group, C1-C6 acyloxy group and C1-C6 methoxy ether; X represents H, halogen group, CN or Cl-C6 alkyl. Preferably, R represents one or more of H, Br, NO 2 , OCH3 , F, CH3 , Cl, N(CH 3 ) 2 , OH, O(CH 2 ) 2 0CH 3 , O(CH 2) 2 O(CH 2) 2 0CH 3 or OAc. Preferably, the substitution position of R in the benzene ring is one or more of the 2, 3, and 4 positions. Preferably, 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-OCH3, 3-O(CH 2) 2 0CH3 , 3-OCH 3 -4-O(CH 2)2 0CH 3 , 3-OCH 3-4-0 (CH 2 ) 2 (CH 2 ) 2 0CH 3 , X represents H, Cl, Br, CN, CH3 .

The preferred structure of the compound of the above general structure is shown in Table 1: Table 1 Partial compound codenames of general formula I and their corresponding structures 0

R -x

Compd. R X

Ii H H

12 4-Br H

13 2-NO 2 H

14 4-NO 2 H

15 3-OH H

16 2-OCH 3 H

17 4-OCH 3 H

18 4-F H

19 4-CH 3 H

Iio 4-Cl H

Iii 4-N(CH 3) 2 H

112 4-OH-3-OCH 3 H

113 3-O(CH 2) 20CH 3 H

114 3-OCH 3-4-O(CH 2) 20CH 3 H

Iis 3-OCH 3-4-O(CH 2) 20(CH 2) 2 0CH 3 H

116 4-OAc-3-OCH 3 H

117 H Cl

118 H Br

119 H CN

120 H CH3

Ii: (E)-6-((E)-3-phenylallyl)cyclohex-2-enone; 12: (E)-6-((E)-3-(4-bromophenyl)-allyl)cyclohex-2-enone; 13: (E)-6-((E)-3-(2-nitrophenyl)allyl)cyclohex-2-enone; 14: (E)-6-((E)-3-(4-nitrophenyl)allyl)cyclohex-2-enone; Is: (E)-6-((E)-3-(3-hydroxyphenyl)allyl)cyclohex-2-enone; 16: (E)-6-((E)-3-(2-methoxyphenyl)allyl)cyclohex-2-enone; 17: (E)-6-((E)-3-(4-methoxyphenyl)allyl)cyclohex-2-enone; 18: (E)-6-((E)-3-(4-fluorophenyl)allyl)cyclohex-2-enone; 19: (E)-6-((E)-3-(4-methylphenyl)allyl)cyclohex-2-enone; Iio: (E)-6-((E)-3-(4-chlorophenyl)allyl)cyclohex-2-enone; Iii: (E)-6-((E)-3-(4-dimethylaminophenyl)allyl)cyclohex-2-enone; 112: (E)-6-((E)-3-(4-hydroxy-3-methoxyphenyl)allyl)cyclohex-2-enone; 113: (E)-6-((E)-3-(3-(2-methoxyethoxy)phenyl)allyl)cyclohex-2-enone; 114: (E)-6-((E)-3-(3-methoxy-4-(2-methoxyethoxy)phenyl)allyl)cyclohex-2-enone;

115: (E)-6-((E)-3-(3-methoxy-4-(2-(2-methoxyethoxy)ethoxy)phenyl)allyl) ring Hex-2-enone; 116: (E)-6-((E)-3-(3-methoxy-4-acetylphenyl)allyl)cyclohex-2-enone;

117: (E)-6-((Z)-2-chloro-3-phenyl)allyl)cyclohex-2-enone; 118: (E)-6-((Z)-2-bromo-3-phenyl)allyl)cyclohex-2-enone; I19: (E)-6-((Z)-2-cyano-3-phenyl)allyl)cyclohex-2-enone; 120: (E)-6-((Z)-2-methyl-3-phenyl)allyl)cyclohex-2-enone. Another object of the present invention is to provide a method for preparing the compound of formula I of the present invention, as follows: Prepared by Adol condensation reaction of substituted or unsubstituted cinnamaldehyde and cyclohexene-2-one under catalyst catalysis, the substituted or

R- CHO unsubstituted cinnamaldehyde structural formula is: . Where R represents one or more substituents on the benzene ring, selected from one or more of H, hydroxyl, halogen group, amino, nitro, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkylamine group, C1-C6 acyloxy group and C1-C6 methoxy ether; X represents H, halogen group, CN or Cl-C6 alkyl. Preferably, R represents one or more of H, Br, NO 2 , OCH3 , F, CH3 , Cl, N(CH 3 ) 2 , OH, O(CH 2 ) 2 0CH 3 , O(CH 2) 2 O(CH 2) 2 0CH 3 or OAc. Preferably, the substitution position of R in the benzene ring is one or more of the 2, 3, and 4 positions. Preferably, 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-OCH3, 3-O(CH 2) 2 0CH3

, 3-OCH 3 -4-O(CH 2)2 0CH 3 , 3-OCH 3-4-0 (CH 2 ) 2 (CH 2 ) 2 0CH 3 , X represents H, Cl, Br, CN, CH3 .

Preferably, the catalyst is selected from one or more of triphenylphosphine, TiC 4

, trimethylsilane imidazole (TMSI), and magnesium bisulfate. The preparation method, specifically, cyclohexen-2-one and triphenylphosphine were dissolved in anhydrous dichloromethane, then TiCl4 in dichloromethane was added at -40 to -78°C, and the cinnamaldehyde dissolved in dichloromethane was slowly added. After the dropwise addition was completed, the reaction was restored to 0-30°C, and the reaction was continued for 10-12h. An appropriate amount of 10% K2 C03 solution was added to make the pH of the reaction solution to 8-10. The reaction yielded a phenylallyl cyclohexenone derivative. The synthetic route is as follows: 0 R- CH O TiCI 4 ,PPh3 CHO 0CM -~ X

Another object of the present invention is to provide the use of the cyclohexenone derivatives with TrxR inhibitory activity in the present invention. Compounds of the invention with TrxR inhibitory activity become drugs for treating and/or preventing cancer. Preferably, the cancer is selected from liver cancer, colon cancer, stomach 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 medicines, such as solvents, diluents, etc. It can also be used for oral administration, 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 combined with the carrier, and more commonly between about 15% to 60% by weight of the active ingredient. The dosage of the compound of the present invention may be 0.005 to 5000 mg/kg/day, and the dosage may exceed the dosage range according to the severity of the disease or the dosage form. The compound of the present invention can be self-assembled into nanoparticles to improve activity, or combined with other anti-tumor drugs such as alkylating agents (such as cyclophosphamide or chlorambucil), antimetabolites (such as 5-fluorouracil or hydroxyurea), topologies Isomerase inhibitors (such as camptothecin), mitotic inhibitors (such as paclitaxel or vinblastine), DNA intercalators (such as doxorubicin) to self-assemble nanoparticles to improve activity. It can also be used in combination with radiation therapy. These other anti-tumor drugs or radiotherapy can be administered simultaneously with the compounds of the present invention or at different times. These combined therapies can produce a synergistic effect and help to improve the therapeutic effect. The present invention combines the structural characteristics, structure-activity relationship and pharmacophore model of the anti-tumor drug piperlongumine, based on piperlongumine, utilizing the theory of bioisosterism, using cinnamaldehyde with different substituents as raw materials, so as to design and synthesize a new type of phenylallyl cyclohexenone derivatives with TrxR inhibitory activity, which simplifies the synthetic route and facilitates mass production. Studying its inhibitory effect on TrxR target and malignant tumor cells, it was found that the compounds not only have strong and selective inhibition on the proliferation of various tumor cells (including liver cancer, breast cancer, gastric cancer, colon cancer, cervical cancer, etc.), but also can effectively inhibit the activity of TrxR enzyme. In addition, the compounds of the present invention have less damage to normal cells at a certain concentration, and can induce the expression of ROS in tumor cells, and synergistically promote apoptosis or necrosis of tumor cells.

Detailed description In order to further clarify the present invention, a series of examples are given below. These examples are completely illustrative, and 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 (Ii) 0 R- OX TiCI 4 ,PPh 3 CHO K> DOM

Cyclohexene-2-one (0.48 g, 5.0 mmol) and triphenylphosphine (1.31 g, 5.0 mmol) were dissolved in 50 ml of anhydrous dichloromethane, then 5ml I1 M TiCl 4 in dichloromethane was added at -50°C. After 15 min, 30 ml of a solution of cinnamaldehyde (1.32 g, 10.0 mmol) dissolved in dichloromethane was slowly added using a constant pressure funnel. After half an hour of dropping, the reaction returned to room temperature, and was continued for about 12 h. TLC monitoring was performed. After the reaction was completed, an appropriate amount of 10% K 2 C0 3 solution was added and stirring was continued for about 5 min to make the pH of the solution to 9. Then, it was extracted with dichloromethane (50 ml x 2), and the extracted dichloromethane layer was washed with saturated brine (50 mL). The dichloromethane layer was collected, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography (EA: PE = 1:3 as an eluent) to obtain 0.90 g of a yellow solid product with a yield of 86%. The spectral data of Ii is: ESI-MS (m/z): 211 [M+H]*; 'H NMR (DMSO-d, 400 MHz): 6 7.46 (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, CH2), 2.44 (m, 2H, CH 2 ); 1 3 C NMR (CDCl 3 , 100 MHz): 6 188.18, 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. Preparation of (E)-6-((E)-3-(4-bromophenyl)-allyl)cyclohex-2-enone (12) According to the synthesis method ofIi, 4-bromo-cinnamaldehyde (2.22 g, 10.0 mmol) was substituted for cinnamaldehyde (1.32 g, 10.0 mmol) to obtain a yellow solid product 1.20 g, yield: 83%. The spectral data of12 is: ESI-MS (m/z): 289 [M+H]+; IH NMR (DMSO-d, 400 MHz): 6 (CDCl 3 ,400 MHz) 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, CH2), 2.48 (m, 2H, CH2 ); 1 3 C NMR (CDCl 3 , 100 MHz): 6 188.15, 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. Preparation of (E)-6-((E)-3-(2-nitrophenyl)allyl)cyclohex-2-enone (13) According to the synthesis method ofIi, 2-nitro-cinnamaldehyde (1.81 g, 10.0 mmol) was substituted for cinnamaldehyde (1.32 g, 10.0 mmol) to obtain a yellow solid product 1.10 g, yield: 86%. The spectral data of 13 is: ESI-MS (m/z): 256 [M+H]*; 1 H NMR(CDC 3 ,400 MHz): 6 7.98 (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= 16.8 Hz, CH), 7.06 (m, 1H, CH), 7.05 (m, 1H, CH), 6.25 (m, 1H, CH), 2.93 (m, 2H, CH2),2.50 (m, 2H, CH2 ); 3I C NMR (CDCl 3 , 100 MHz): 6 181.98, 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 (14) According to the synthesis method ofIi, 4-nitro-cinnamaldehyde (1.81 g, 10.0 mmol) was substituted for cinnamaldehyde (1.32 g, 10.0 mmol) to obtain a yellow solid product 0.84 g, yield: 66%. The spectral data of 14 is: ESI-MS (m/z): 256 [M+H]*; 1 H NMR(CDC 3 , 400 MHz): 68.18 (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= 15.7 Hz, CH), 6.21 (m, 1H, CH), 2.91 (m, 2H, CH 2), 2.49 (m, 2H, CH2 ); 1 3 C NMR (CDCl 3 , 100 MHz): 6 187.86, 149.49, 142.91, 136.99, 136.57, 132.80, 130.98, 127.37, 127.13, 124.11, 25.56, 25.40.

Preparation of(E)-6-((E)-3-(3-hydroxyphenyl)allyl)cyclohex-2-enone (15) According to the synthesis method ofIi, 3-hydroxy-cinnamaldehyde (1.48 g, 10.0 mmol) was substituted for cinnamaldehyde (1.32 g, 10.0 mmol) to obtain a yellow solid product 0.91 g, yield: 80%. The spectral data of 1 is: ESI-MS (m/z): 227

[M+H]*. Preparationof(E)-6-((E)-3-(2-methoxyphenyl)allyl)cyclohex-2-enone(16) According to the synthesis method ofIi, 2-methoxy-cinnamaldehyde (1.69 g, 10.0 mmol) was substituted for cinnamaldehyde (1.32 g, 10.0 mmol) to obtain a yellow solid product 0.97 g, yield: 81%. The spectral data of 16 is: ESI-MS (m/z): 241

[M+H]*; 'H NMR (DMSO-d, 400 MHz): 6 7.53(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, CH3), 2.91(m, 2H, CH2 ),2.47(m, 2H, CH 2 ); 1 3C NMR (CDCl 3 , 100 MHz): 6 188.42, 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 (17) According to the synthesis method ofIi, 4-methoxy-cinnamaldehyde (1.67 g, 10.0 mmol) was substituted for cinnamaldehyde (1.32 g, 10.0 mmol) to obtain a yellow solid product 0.98 g, yield: 82%. The spectral data of 17 is: ESI-MS (m/z): 241

[M+H]*; 'HNMR(CDC 3,400 MHz): 6 7.44 (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, CH3),2.90 (m, 2H, CH2 ),2.47 (m, 2H, CH 2 ); 13 C NMR (CDCl 3 , 100 MHz): 6 188.41, 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. Preparation of (E)-6-((E)-3-(4-fluorophenyl)allyl)cyclohex-2-enone (18) According to the synthesis method ofIi, 4-fluoro-cinnamaldehyde (1.58 g, 10.0 mmol) was substituted for cinnamaldehyde (1.32 g, 10.0 mmol) to obtain a yellow solid product 0.95 g, yield: 83%. The spectral data of 18 is: ESI-MS (m/z): 229 [M+H]*; 1 H NMR (DMSO-d,400 MHz): 6 7.46(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 ); 1 3 C NMR (CDCl 3 , 100 MHz): 6 188.18, 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. Preparation of (E)-6-((E)-3-(4-methylphenyl)allyl)cyclohex-2-enone (19) According to the synthesis method ofIi, 4-methyl-cinnamaldehyde (1.46g, 10.Ommol) was substituted for cinnamaldehyde (1.32 g, 10.0 mmol) to obtain a yellow solid product 0.90 g, yield: 80%. The spectral data of 19 is: ESI-MS (m/z): 225 [M+H]*; H NMR (DMSO-d,400 MHz): 67.54 (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, CH2), 2.32 (s, 3H, CH3 ); 1 3 C NMR (DMSO-d 6 , 101 MHz): 6 187.63, 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 (Iio) According to the synthesis method ofIi, 4-chloro-cinnamaldehyde (1.66g, 10.0mmol) was substituted for cinnamaldehyde (1.32 g, 10.0 mmol) to obtain a yellow solid product 1.06 g, yield: 87%. The spectral data of Iio is: ESI-MS (m/z): 256 [M+H]*; H NMR (DMSO-d,400 MHz): 67.68 (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, CH2),2.45 (m, 2H, CH2 ); "C NMR (CDCl3 , 100 MHz): 6187.67, 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 (Iii) According to the synthesis method ofIi, 4-dimethylamino-cinnamaldehyde (1.75g, 10.0mmol) was substituted for cinnamaldehyde (1.32 g, 10.0 mmol) to obtain a yellow solid product 0.97 g, yield: 77%. The spectral data of Iii is: ESI-MS (m/z): 254

[M+H]*; 'H NMR (CDCl 3,400 MHz): 67.38 (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, CH2), 2.45 (m, 2H, CH 2 ); 13 C NMR (CDCl 3 , 101 MHz): 6 188.24, 150.85, 148.49, 141.34, 135.87, 131.30, 128.55, 124.90, 118.79, 112.11, 40.26, 25.33, 25.10. Preparation of(E)-6-((E)-3-(4-hydroxy-3-methoxyphenyl)allyl)cyclohex-2-enone (112) According to the synthesis method of Ii, 4-hydroxy-3-methoxy-cinnamaldehyde (1.78g, 10.0mmol) was substituted for cinnamaldehyde (1.32 g, 10.0 mmol) to obtain a yellow solid product 1.13 g, yield: 88%. The spectral dataof112 is: ESI-MS (m/z): 257 [M+H]*;H NMR (DMSO-d,400 MHz): 6 7.30 (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, CH3),2.90 (m, 2H, CH2 ),2.48 (m, 2H, CH 2 ); 1 3 C NMR (CDCl 3 , 101 MHz): 6 188.24, 150.85, 148.49, 141.34,135.87,131.30,128.55, 124.90,118.79,112.11, 40.26,25.33, 25.10. Preparation of (E)-6-((E)-3-(3-(2-methoxyethoxy)phenyl)allyl)cyclohex-2-enone (113) According to the synthesis method ofIi, 3-(2-methoxyethoxy)-cinnamaldehyde (2.06 g, 10.0 mmol) was substituted for cinnamaldehyde (1.32 g, 10.0 mmol) to obtain a yellow solid product 1.08 g, yield: 76%. The spectral data of113 is: ESI-MS (m/z): 285 [M+H]*. Preparation of (E)-6-((E)-3-(3-methoxy-4-(2-methoxyethoxy)phenyl)allyl)cyclohex-2-enone(114) According to the synthesis method of Ii, 3-methoxy-4-(2-methoxyethoxy)-cinnamaldehyde (2.36 g, 10.0 mmol) was substituted for cinnamaldehyde (1.32 g, 10.0 mmol) to obtain a yellow solid product 1.10 g, yield: 70%. The spectral data ofI14 is: ESI-MS (m/z): 315 [M+H]*. Preparation of (E)-6-((E)-3-(3-methoxy-4-(2-(2-methoxyethoxy)ethoxy)phenyl)allyl)cyclohex-2-eno ne (Iis) According to the synthesis method of Ii, 3-methoxy-4-(2-(2-methoxyethoxy)ethoxy-cinnamaldehyde (2.80g, 10.0mmol) was substituted for cinnamaldehyde (1.32 g, 10.0 mmol) to obtain a yellow solid product 1.27 g, yield: 71%. The spectral data ofI15 is: ESI-MS (m/z): 359 [M+H]*. Preparation of (E)-6-((E)-3-(3-methoxy-4-acetylphenyl)allyl)cyclohex-2-enone (116) According to the synthesis method ofIi, 3-methoxy-4-acetyl-cinnamaldehyde (2.20g, 10.0mmol) was substituted for cinnamaldehyde (1.32 g, 10.0 mmol) to obtain a yellow solid product 1.16 g, yield: 78%. The spectral data of116 is: ESI-MS (m/z):

299 [M+H]*; 'H NMR (CDCl 3 ,400 MHz): 67.23 (m, 1H, CH), 7.03 (m, 1H, CH), 6.96 (m, 4H, Ar-H, CH), 6.84 (d, J= 15.3 Hz, 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, CH3 ); 1 3 C NMR (CDCl 3

, 101 MHz): 6 188.06,168.83,151.23,149.14,140.14,139.48,135.67,134.01,133.97, 131.01, 123.25, 123.03, 119.69, 110.66, 55.88, 25.34, 25.30. Preparation of (E)-6-((Z)-2-chloro-3-phenyl)allyl)cyclohex-2-enone (I17) According to the synthesis method ofIi, a-chlorocinnamaldehyde (1.66 g, 10.0 mmol) was substituted for cinnamaldehyde (1.32 g, 10.0 mmol) to obtain a yellow solid product 0.98 g, yield: 80%. The spectral data of117 is: ESI-MS (m/z): 245 [M+H]*. Preparation of (E)-6-((Z)-2-bromo-3-phenyl)allyl)cyclohex-2-enone (Ii8) According to the synthesis method ofIi, a-Bromocinnamaldehyde (2.11g, 10.0mmol) was substituted for cinnamaldehyde (1.32 g, 10.0 mmol) to obtain a yellow solid product 1.18 g, yield: 82%. The spectral data of Iis is: ESI-MS (m/z): 289 [M+H]*; 1 H NMR (CDCl3 ,400 MHz): 6 7.66 (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, CH2 ). Preparation of (E)-6-((Z)-2-cyano-3-phenyl)allyl)cyclohex-2-enone (I19) According to the synthesis method ofIi, a-cyanocinnamaldehyde (1.57g, 10.0 mmol) was substituted for cinnamaldehyde (1.32 g, 10.0 mmol) to obtain a yellow solid product 0.75 g, yield: 64%. The spectral data of Iig is: ESI-MS (m/z): 236 [M+H]*; 1 H NMR (CDC 3,400 MHz): 67.38 (m, 7H, Ar-H), 7.03 (m, 1H, CH), 6.64 (s, 1H, CH), 3.02 (m, 2H, CH 2),2.42 (m, 2H, CH2 ). Preparation of (E)-6-((Z)-2-methyl-3-phenyl)allyl)cyclohex-2-enone (120) According to the synthesis method ofIi, a-Methylcinnamaldehyde (1.46g, 10.mmol) was substituted for cinnamaldehyde (1.32 g, 10.0 mmol) to obtain a yellow solid product 0.87 g, yield: 78%. The spectral data of 120 is: ESI-MS (m/z): 224 [M+H]*; 1 H NMR (CDCl3 ,400 MHz): 6 7.32 (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, CH2 ), 2.09 (s, 3H, CH 3 ); 1 3 C NMR (CDCl 3 , 101 MHz): 6 189.00, 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 The MTT method was used to determine the tumor cell and normal cell proliferation inhibition rate of the compounds of the present invention The anti-proliferative activity of the compounds of the present invention against four human cancer cell lines was evaluated by the in vitro anti-tumor test using the tetramethylazazole blue colorimetry method (MTT). Piperlongumine (PL) was used as a positive control drug. Human cancer cell line: 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 in an exponential growth phase, add 0.25% trypsin to digest, make the adherent cells fall off, and make a suspension containing 2x104~4x104 cells per ml. The cell suspension was inoculated on a 96-well plate, 180 L per well, and incubated in a constant temperature C02 incubator for 24 hours. Change the medium, add test compounds 11-120 (the compounds were dissolved in DMSO and diluted with PBS, the test compounds concentration is 12.5 M), 20 L per well, and culture for 72 hours. Add MTT to a 96-well plate, 20 L per well, and react for 4 hours in an incubator. Aspirate the supernatant, add DMSO, 150 L per well, and shake for 5 minutes on a plate shaker. The absorbance of each well was measured with an enzyme-linked immunoassay at a wavelength of 570 nm, and the cell inhibition rate was calculated. The experimental results are shown in Table 2. Cell inhibition rate = (OD value of negative control group-OD value of test group)/ OD value of negative control group x 100%. The compounds of the present invention have undergone a series of tests on the anti-proliferative activity of tumor cells. The results of pharmacological experiments (see Table 2) show that the compounds 11-120 of the present invention have a strong inhibitory effect on the proliferation of most tumor cells at a concentration of 12.5 [M. In particular, the inhibitory activity of some compounds was significantly better than that of the positive control drug piperlongumine (PL). However, the cytotoxicity of the compounds 11~20 of the present invention on human normal gastric mucosal cells GES-1 at the same concentration is significantly lower than that of tumor cells, indicating that the compounds of the present invention not only have significant antitumor activity against tumor cells, but also have low toxicity to normal cells, with certain tumor cell selectivity. Table 2 Inhibition rate of some compounds of the present invention on human tumor cells and normal cells (12.5 [M) Compounds SMMC7721 HCT116 HGC27 Hela GES-1 PL 70.2 69.6 72.3 66.4 47.5 Ii 76.3 69.8 78.9 78.1 30.3 12 72.1 76.4 80.2 79.8 32.6 13 75.9 73.3 78.9 76.4 27.5 14 78.8 74.6 80.7 74.9 28.2 15 79.6 89.7 86.1 87.6 ND 16 73.8 75.5 79.4 74.9 ND 17 77.3 79.5 81.8 78.4 ND 18 76.8 83.8 80.1 73.9 ND 19 69.8 65.8 74.6 72.6 ND Iio 81.1 87.5 84.6 81.9 ND Iii 89.6 82.0 90.9 85.1 26.8 112 80.8 83.7 84.1 83.5 25.9 113 85.2 91.4 84.7 80.8 26.3 114 84.3 88.7 90.8 86.1 24.7 Iis 86.7 91.2 93.5 87.8 25.1 116 71.6 75.4 79.0 73.7 27.2 117 74.2 83.1 78.9 72.3 30.6 118 72.4 74.5 79.4 ND 33.0 119 ND 86.3 89.6 ND 28.5 120 ND 77.3 78.9 ND 23.8

ND: Not detected

Example 3 Determination of intracellular ROS level ROS-Glo hydrogen peroxide test (Promega, Southampton, UK). ROS changes were measured by directly detecting H 2 0 2 levels in cells. Cells were seeded into 96-well cell culture plates and incubated with test drugs (0.01~12.5 M) for 24 hours. Add hydrogen peroxide substrate solution to each well and incubate in a constant temperature C02 incubator at 37°C for 6 hours. After incubation, add ROS-Glo probe solution to each well and incubate for 20 minutes at room temperature. Fluorescence was detected by BioTek Synergy HT multi-mode microplate reader. 14~I8, 11115, 118, I19in the compounds of general formula I of the present invention were selected as representatives, and their ROS levels in tumor cells were tested. DCFH-DA was used as a fluorescent probe to measure the change of ROS in human cervical cancer Hela cells after drug treatment. The changes in fluorescence intensity can quantitatively reflect the intracellular ROS level. The results showed that the compounds of the present invention 14~I8, Iii~is,118, I19 at 12.5 pM can significantly increase the ROS content in Hela cells, which is 3.7-8.9 times that of the control group, better than the positive control drug PL (3.2 times that of the control group).

Example 4 Study on TrxR inhibitory activity of the compounds in the present invention The effect of the tested drugs (12.5 M) on TrxR activity was evaluated by the TrxR activity test kit (BioVision, Milpitas, CA, USA). Briefly, the cell line was dissolved in a centrifuge tube with 1x buffer solution, then ice-cooled for 20 minutes, and then centrifuged at 10000xg at 4°C for 15 minutes. The supernatant was transferred to a new centrifuge tube, and the protein concentration was calculated by the Bio-Rad protein test method. Samples were diluted to 2 x working concentration with buffer solution. Prepare two wells (with and without inhibitors) in triplicate for each sample. The reaction buffer and the reaction buffer added with inhibitor were prepared according to the instructions. The absorbance was measured at a wavelength of 412 nm every 20 seconds within 5 minutes after shaking with BioTek Synergy HT multi-mode microplate reader before reading. The experimental results show that compounds 11~20 have significant inhibitory activity against TrxR at a concentration of 12.5 [M. The inhibitory activity data are shown in Table 3. Most compounds showed stronger or equivalent inhibitory activity than the positive control drug PL. It is suggested that the phenylallyl cyclohexenone derivatives of the present invention have good TrxR inhibitory activity, which is consistent with their anti-tumor activity. Table 3 In vitro TrxR inhibition rate of some compounds of the present invention (12.5 [M) Compd. Inhibition(%) Compd. Inhibition(%)

PL 86.7 I 11 90.7

Ii 83.9 1 12 95.1

12 89.7 113 94.8 1I3 88.9 114 97.3 1I4 90.6 115 96.6 1I5 95.1 116 89.2 16 92.5 117 87.6 1I7 89.8 118 90.6 18 93.4 119 93.8 1I9 86.5 1I20 85.3

110o 91.2

Claims

1. A phenylallyl cyclohexenone derivative with the general structure shown in general formula I: 0

N R Rx

2. The phenylallyl cyclohexenone derivative according to claim 1, wherein R represents H, F, Cl, Br, NO 2, OCH 3, CH3 , N(CH 3) 2, OH, O(CH 2) 2 0CH3

, O(CH 2) 2 O(CH 2) 20CH 3 , 0Ac.

3. The phenylallyl cyclohexenone derivatives according to claim 1 or claim 2, wherein the substitution position of R in the benzene ring is one or more of the 2, 3, and 4 positions.

4. The phenylallyl cyclohexenone derivative according to claim 1, wherein 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-OCH3, 3-O(CH 2) 2 0CH3

, 3-OCH 3 -4-O(CH 2)2 0CH 3 , 3-OCH 3-4-0 (CH 2) 2 (CH 2)2 0CH 3 and X represents H, Cl, Br, CN, CH 3 .

5. The phenylallyl cyclohexenone derivative according to claim 1, selected from the following: 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-OCH3, 3-O(CH 2) 2 0CH 3 ,

3-OCH 3 -4-O(CH 2)2 0CH 3 , 3-OCH 3 -4-0 (CH 2) 2 (CH2) 20CH 3 and X represents H; or R represents H and X represents Cl, Br, CN or CH3 .

6. A method for preparing a phenylallyl cyclohexenone derivative according to any one of the preceding claims, wherein the phenylallyl cyclohexenone derivative is prepared by Adol condensation reaction under catalysis of a substituted or unsubstituted cinnamaldehyde and cyclohexene-2-one, wherein the substituted or

R- CHO unsubstituted cinnamaldehyde structural formula is: , where R represents one or more of H, hydroxyl, halogen group, amino, nitro, C1-C6 alkyl,

C1-C6 alkoxy, C1-C6 alkylamine group, C1-C6 acyloxy group and C1-C6 methoxy ether; X represents H, halogen group, CN or Cl-C6 alkyl.

7. The preparation method according to claim 6, wherein the catalyst is selected from triphenylphosphine and/or TiC 4

. 8. The preparation method according to claim 6, wherein cyclohexen-2-one and triphenylphosphine are dissolved in anhydrous dichloromethane, then TiCl4 in dichloromethane is added at -40 to -78°C, and the cinnamaldehyde dissolved in dichloromethane is slowly added and after addition is completed, the reaction temperature is restored to 0-30°C, and the reaction continued for 10-12h and an appropriate amount of 10% K2 C3 solution is added to make the pH of the reaction solution to 8-10.

9. The use of a compound of any one of claims 1 to 5 in the manufacture of a medicament for the treatment of a disorder which is modulated by TrxR inhibition.

10. The use according to claim 9 wherein the disorder which is modulated by TrxR inhibition is cancer.

11. The use according to claim 10,wherein the cancer is selected from liver cancer, colon cancer, stomach cancer, breast cancer or cervical cancer.

12. A method of treating a disorder which is modulated by TrxR inhibition comprising the step of administering to a subject in need thereof an effective amount of a compound of any one of claims I to 5.

13. The method according to claim 12 wherein the disorder which is modulated by TrxR inhibition is cancer.

14. The method according to claim 13,wherein the cancer is selected from liver cancer, colon cancer, stomach cancer, breast cancer or cervical cancer.