AU2020387494B2

AU2020387494B2 - 6-benzylidene-2-aryl ethynyl cyclohexenone derivatives, and preparation method therefor and medical use thereof

Info

Publication number: AU2020387494B2
Application number: AU2020387494A
Authority: AU
Inventors: Yangyang Li; Changchun LING; Yong Ling; Xin Liu; Chi MENG; Jiefei MIAO; Jianqiang QIAN; Hongmei WU; Xudong Xie; Yirong Zhang
Original assignee: Nantong University
Current assignee: Nantong University
Priority date: 2019-11-19
Filing date: 2020-08-26
Publication date: 2022-07-14
Anticipated expiration: 2040-08-26
Also published as: CN111018686A; WO2021098319A1; AU2020387494A1; CN111018686B

Abstract

Disclosed in the present invention are a class of 6-benzylidene-2-aryl ethynyl cyclohexenone derivatives and pharmaceutically acceptable salts thereof, preparation methods therefor, pharmaceutical compositions containing these derivatives, and the medical use thereof, in particular the use thereof in the preparation of drugs for treating malignant tumors. The compounds of the present invention have a certain tumor selectivity, can significantly inhibit tumor proliferation and have a weak inhibition effect on normal cells. The compounds of the present invention can significantly inhibit the activity of a TrxR target and induce an increase in the ROS level in tumor cells, thereby promoting the anti-tumor activity of the compounds of the present invention.

Description

i W O 202 1/0983 19 A 1 ||1||||||||||||||||||||||||||||||||||||||||||||||i|||||||||l|i

- MP ;- tK T , (3)

Description

The preparation methods and medical applications of 6

benzylidene-2-(phenylethynyl) cyclohexenone derivatives Technical field

The invention relating to the field of medicine, discloses the preparation methods

and medical application of a class of 6-benzylidene-2-(phenylethynyl) cyclohexenone

derivatives and their pharmaceutically acceptable salts, and pharmaceutical

compositions containing these derivatives, especially in the preparation of drugs for the

treatment of malignant tumors.

Background technology

Tumor is the second cause of death after cardiovascular disease. Although there

are many kinds of antitumor drugs available in clinic, the existing drugs still cannot

meet the needs of treatment due to the complexity of tumor etiology, drug resistance

and toxic side effects of antitumor drugs. Therefore, it is of great significance to find

new antitumor drugs with high efficacy, strong targeting, weak toxic side effects.

Piperlongumine (PL) belonging to the natural product has a,p-unsaturated ketones,

which can effectively kill various metastatic cancer cells other than normal cells. a,p

Unsaturated ketones are important pharmacodynamic groups in natural products and

synthetic small molecules. They can interact with nucleophilic groups of biological

macromolecules highly expressed in tumors (such as sulfhydryl groups of cysteine

residues), regulate multiple signaling pathways of proliferation and metastasis in tumor

cells to exert anti-tumor effects. Mechanism studies show that PL can selectively

increase the level of ROS and other oxidative stress in cancer cells and inhibit

thioredoxin oxidoreductase (TrxR) result in DNA damage and the promotion of

apoptosis in cancer cells. Optimizing PL structure through skeleton transition and

combining the structure-activity relationship of PL derivatives, designing and

synthesizing The novel 6-benzylidene-2-(phenylethynyl) cyclohexenone derivatives

is designed and synthesized through skeleton transition and combining the structure

activity relationship of PL derivatives to optimize PL structure to improve the inhibitory

activity of these compounds on malignant tumor cells, which is the research and development idea of developing new drugs to inhibit thioredoxin oxidoreductase activity. Contents of the invention In order to further improve the antitumor activity and enzyme inhibitory activity of benzylidene cyclohexenone derivatives, according to the structure-activity relationship of PL and its derivatives, on the basis of a,p-unsaturated ketones, arylacetylene groups were introduced at appropriate positions to form a new dominant skeleton of cyclohexanone. A series of new 6-benzylidene-2-(phenylethynyl) cyclohexanone derivatives with inducing ROS level and targeting TrxR in tumor cells were designed and synthesized. The designed compound not only improves the antitumor activity and target inhibition ability, but also can selectively inhibit the proliferation of tumor cells and cause weak damage to normal cells. Meanwhile, it also enriches the diversity of benzylidene cyclohexenone skeleton. The invention aims to provide the preparation method and medical application of novel 6-benzylidene-2-(phenylethynyl) cyclohexenone derivatives. The specific technical scheme of the invention is as follows: The general structure of 6-benzylidene-2-(phenylethynyl) cyclohexenone derivatives as shown in formula I:

O0 R'

RC

Wherein, R and R'are the same or different and each represents one or more substituents on the corresponding substituted benzene ring, which is selected from H, halogen, OH, NH 2, NO 2, alkyl, haloalkyl, alkoxy, haloalkoxy and alkamine. When R or R'represents multiple substituents, each substituent is the same or different. Preferably, R and R 'are the same or different, each represents one or more substituents on the corresponding substituted benzene ring, which is selected from one or more of H, halogen, OH, NH 2, NO 2 , C1-C6 alkyl group, C1-C6 haloalkyl group, C1-C6 alkoxy group, C1-C6 haloalkoxy group and CI-C6 alkamine group. When R or R' represents multiple substituents, each substituent is the same or different.

Preferably, R and R' are the same or different, each representing one or more

substituents on the corresponding substituted benzene ring, which is selected from H,

F, Cl, Br, I, OH, NH 2, N02, CH 3 , C 2 H, (CH2) 2 CH3 , CH(CH 3) 2, CF 3 , C 2 F 5 , CC13 , CBr 3

, OCH 3 , OC2 H, OCF3 , OCC1 3 , OCBr3 , NHCH 3 , NHC 2H, NC 3H 8. When R or R' represents multiple substituents, each substituent is the same or different.

Preferably, R'represents one or more substituents on the benzene ring, selected from H,

F, Cl, Br, and when R' represents multiple substituents, each substituent is the same or

different.

Preferably, R represents one or more substituents on the benzene ring, selected from

one or more of H, halogen, N02, CH 3 , CF 3 , OCH 3 , OCF3, NHCH 3 . R 'represents

halogen group.

The preferred compound code of the above general formula I and its corresponding

structure are shown in Table 1:

Table 1 Codes of some compounds in formula I and their corresponding structures

Compd. R R

11 H 2'-F

12 4-F 2'-F

13 2-CH 3-4-OCH 3 4'-F 35

14 3-N0 2-4-OCH 3 2'-Cl

15 3-OCH 3 2'-Br

16 3-CF 3 2',4'-di-F

17 4-N(CH 3) 2 2'-F

18 4-OCF 3 2'-F

19 4-OCF 3 2'-Cl

1o 3,4-di-OCH 3 4'-F

Inl 3,4,5-tri-OCH 3 2'-F

112 3,4,5-tri-OCH 3 4'-F

113 3,4,5-tri-OCH 3 2'-Br

114 3,4,5-tri-OCH 3 2',4'-di-F

Another object of the invention is to provide a preparation method of 6-benzylidene-2

(phenylethynyl) cyclohexanone derivatives according to the general formula I of the

invention, comprising the following steps:

(1) Compound 3 is obtained by Aldol condensation reaction of R-substituted

benzaldehyde and cyclohexene-2-one (preferably under the catalysis of TiCl 4 and PPh 3).

R represents one or more substituents on the benzene ring and is selected from one or

more of H, halogen, hydroxyl, amino, nitro, alkyl, haloalkyl, alkoxy, haloalkoxy and

alkamine. When R represents multiple substituents, each substituent is the same or

different;

0 0 CHO + R + R 105I

1 2 3

(2) Compound 3 is reacted with iodine (preferably in a mixed solvent of CCl4: Py= 1:1)

to obtain compound 4;

0 0

R R

34

(3) Compound 4 is coupled with R 'substituted phenylacetylene by Sonogashira

reaction (preferably in the presence of Pd(PPh3) 4 and DIPEA) to obtain a 6

benzylidene-2-(phenylethynyl) cyclohexanone derivatives. R' represents one or more

substituents on the benzene ring, selected from H, halogen, hydroxyl, amino, nitro,

alkyl, haloalkyl, alkoxy, haloalkoxy One or more of the alkyl amino groups. When R

'represents multiple substituents, each substituent is the same or different;

R - +RR 00

R R 4 5 0

The specific synthesis route is as follows: 00 0

R C R- I R 6 ~L DCM,-0O CCI 4, rt 1 2 3

O 0 _0 R' R I + /' _ Pd(PPh3 )4 , DIPEA R-F R DMA, rt, 10min R 4 5

one or more of H, halogen, N02, CH 3 , CF 3 , OCH 3 , OCF3, NHCH 3 . R' represents

halogen group.

Another object of the invention is to provide a pharmaceutical composition comprising

the 6-benzylidene-2-(phenylethynyl) cyclohexenone derivatives or a pharmaceutically

acceptable salt of the invention. Further, it can be composed of a therapeutically

effective dose of a compound of general formula I or a pharmaceutically acceptable salt

thereof and a pharmaceutically acceptable carrier or excipient.

Another object of the invention is to provide the application of the 6-benzylidene-2

(phenylethynyl) cyclohexenone derivatives or pharmaceutically acceptable salt in the

preparation of drugs for thioredoxin oxidoreductase inhibitory activity. The drug with

thioredoxin oxidoreductase inhibitory activity is a drug for preventing and/or treating

cancer which include liver cancer, prostate cancer, colon cancer, gastric cancer or lung cancer.

The compound of the invention can be prepared into a preparation alone or in

combination with one or more pharmaceutically acceptable carriers for drug supply.

For example, solvents, diluents and so on, they can be administered in oral dosage forms,

such as tablets, capsules, dispersible powders, granules, etc. Various dosage forms of

the pharmaceutical composition of the invention can be prepared according to methods

well known in the pharmaceutical field. These pharmaceutical preparations may contain,

for example, 0.05%~90% by weight of active ingredients combined with the carrier,

more commonly about 15%~60% by weight of active ingredients. The dose of the

compound of the invention can be 0.005-5000 mg/kg/day, or it can be used according

to the severity of the disease or different dosage forms and the dose exceeds this dose

range.

The compound of the invention can be used in combination with other antitumor

drugs, such as alkylating agents (such as cyclophosphamide or cisplatin), antimetabolic

drugs (such as 5-fluorouracil or hydroxyurea), topoisomerase inhibitors (such as

camptothecin), mitotic inhibitors (such as paclitaxel or vinblastine), DNA insertion

agents (such as doxorubicin), and radiotherapy. These other antitumor drugs or

radiotherapy may be administered simultaneously with the compounds of the present

invention or at different times. These combined treatments can exert synergistic effects,

which helps to improve the therapeutic efficacy.

The advantages of the invention:

According to the structure-activity relationship of natural product PL and its

derivatives, on the basis of the active fragment a,p-unsaturated ketones, arylacetylene

groups were introduced at appropriate positions to form a new dominant skeleton of

cyclohexanone by the skeleton transition and splicing principle of active structure. A

series of new 6-benzylidene-2-(phenylethynyl) cyclohexanone derivatives with

inducing ROS level and targeting TrxR in tumor cells were designed and synthesized.

The compound of the invention greatly enriches the structural diversity of PL analogues

and expresses the activity of significantly inhibiting many types of tumor cells with

weak inhibitory effect on normal cells, which indicating that the compound of the invention has certain tumor selectivity. Moreover, the compounds of the invention can significantly inhibit the activity of TrxR target and induce the increase of ROS level in tumor cells, so as to promote the antitumor activity of the compound of the invention.

Specific embodiments

In order to further clarify the invention, a series of embodiments are given below.

These embodiments are completely illustrative. They are only used to specifically

describe the invention and should not be known as limitations on the invention.

Embodiment 1: Preparation of (E)-6-benzylidene-2-((2-fluorophenyl) ethynyl)

cyclohex-2-enone (Ii)

(E)-6-benzylidene-cyclohexane-2-enone (3a)

Dissolve the raw material cyclohex-2-en-1-one (0.95 g, 10.0 mmol) in 15 mL of

anhydrous dichloromethane, then add TiC 4 (200 mg) and PPh3 (2.62 g, 10.0 mmol) at

°C. After 15 min, add benzaldehyde (2.12 g, 20.0 mmol) and the mixture was reacted

overnight at room temperature. Upon completion of the reaction, 10% K 2 C03 solution

was added for treatment for 10 min and the organic layer was collected through

concentration in vacuum and further purified by column chromatography to produce

solid 3a (1.69 g), yield: 79%. MS (ESI) m/z: 185 [M + H]*.

(E)-2-iodo-6-benzylidene-cyclohexane-2-enone (4a)

Compound 3a (0.37 g, 2.0 mmol) was dissolved in a mixed solution (16 mL) of

CCl4 and pyridine 1:1. Iodine (1.01 g, 4.0 mmol) was added at room temperature and

stirred away from light for 3 hours. After the reaction was complete, 80 mL of saturated

NH 4 C1 solution was added and the mixture was mixed with ethyl acetate (3x40 mL).

After washing the organic layer with saturated sodium thiosulfate and drying with

anhydrous MgSO4, the residue was concentrated and purified by column

chromatography to obtain solid product 4a (0.76 g), yield: 95%. MS (ESI) m/z: 311 [M

+ H]*.

(E)-6-benzylidene-2-((2-fluorophenyl) ethynyl) cyclohex-2-enone (Ii)

Dissolve compound 4a (0.62 g, 2 mmol) and 3-fluorophenylacetylene (0.72 g, 6 mmol)

in anhydrous 1,4-dioxane (15 mL), add PD(PPh 3) 4 (0.23 g, 0.2 mmol), Cul (76.18 mg,

0.4 mmol) and DIPEA (1.05 ml, 6 mmol) under nitrogen atmosphere, and stir at room temperature for 10 min. After the reaction was complete, saturated NH 4 Cl solution (80 mL) was added and the mixture was mixed with ethyl acetate (3x40 mL). After washing the organic layer with saturated sodium thiosulfate and drying with anhydrous MgSO4, the residue was concentrated and purified by column chromatography to obtain the target compound Ii (0.52 g), yield: 60%. MS (ESI) m/z: 303 [M + H]*.'H NMR (400

MHz, DMSO-d) 6 7.48-7.53 (m, 2H, Ar-H, ArCH), 7.35-7.43 (m, 6H, Ar-H), 7.15

7.19 (m, 2H, Ar-H), 6.43 (m, 1H, C=CH), 2.97 (m, 2H, CH 2), 2.43 (m, 2H, CH2 )o

Embodiment 2: Preparation of (E)-6-(4-fluorobenzylidene)-2-((2-fluorophenyl)

ethynyl) cyclohexan-2-enone (12)

(E)-6-(4-fluorobenzylidene)-cyclohex-2-enone (3b)

Referring to the preparation method of compound 3a, benzaldehyde was replaced

with 3-fluorobenzaldehyde to obtain solid compound 3b, yield: 75%. MS (ESI) m/z:

203 [M + H]*.

(E) - 2-iodo-6 - (4-fluorobenzylidene) - cyclohexane-2-enone (4b)

Referring to the preparation method of compound 4a, 3a was replaced with 3b to

obtain solid compound 4b, yield: 95%. MS (ESI) m/z: 329 [M + H]*.

(E)-6-(4-fluorobenzylidene)-2-((2-fluorophenyl) ethynyl) cyclohex-2-enone (12)

Referring to the preparation method of compound Ii, 4b instead of 4a to obtain the

target compound 12, yield: 65%. MS (ESI) m/z: 321 [M + H]*.'H NMR (400 MHz,

DMSO-d) 67.69 (d, J= 8.4 Hz, 2H, Ar-H), 7.51-7.55 (m, 2H, ArCH), 7.38-7.41 (m,

3H, Ar-H), 7.14-7.16 (m, 2H, Ar-H), 6.41 (m, 1H, C=CH), 2.95 (m, 2H, CH 2), 2.42 (m,

2H, CH2 )o

Embodiment 3: Preparation of (E)-6-(2-methyl-4-methoxy-benzylidene)-2-((4

fluorophenyl) ethynyl) cyclohexan-2-enone (13)

(E)-6-(2-methyl-4-methoxy-benzylidene) cyclohexane-2-enone (3c)

Referring to the preparation method of compound 3a, benzaldehyde was replaced

with 3-methyl-5-methoxybenzaldehyde to obtain solid compound 3c, yield: 68%. MS

(ESI) m/z: 229 [M + H]*.

(E)-2-iodo-6-(2-methyl-4-methoxy-benzylidene) cyclohex-2-enone (4c)

Referring to the preparation method of compound 4a, 3a was replaced with 3c to

obtain solid compound 4c, yield: 95%. MS (ESI) m/z: 355 [M + H]*.

(E)-6-(2-methyl-4-methoxy-benzylidene)-2-((4-fluorophenyl) ethynyl) cyclohexan-2

enone (13)

Referring to the preparation method of compound Ii, 4c instead of 4a and 5

fluorophenylacetylene instead of 3-fluorophenylacetylene to obtain the target

compound 13, yield: 62%. MS (ESI) m/z: 347 [M + H]*. 'H NMR (400 MHz, DMSO

d) 6 7.55 (s, 1H, ArCH), 7.46 (d, J= 8.4 Hz, 2H, Ar-H), 7.10-7.16 (m, 3H, Ar-H), 6.73

(m, 1H, Ar-H), 6.69 (m, 1H, Ar-H), 6.30 (m, 1H, CH=), 3.78 (s, 3H, OCH3 ), 2.84 (m,

2H, CH2 ), 2.35 (m, 2H, CH2), 2.28 (s, 3H, Ar-CH3).

Embodiment 4: Preparation of (E)-6-(3-nitro-4-methoxy-benzylidene)-2-((2

chlorophenyl) ethynyl) cyclohexan-2-enone (14)

(E)-6-(3-nitro-4-methoxy-benzylidene) cyclohex-2-enone (3d)

Referring to the preparation method of compound 3a, benzaldehyde was replaced

with 3-methoxy-4-nitrobenzaldehyde to obtain a solid compound 3d, yield: 76%. MS

(ESI) m/z: 260 [M + H]*.

(E)-2-iodo-6-(3-nitro-4-methoxy-benzylidene) cyclohex-2-enone (4d)

Referring to the preparation method of compound 4a, 3a was replaced with 3d to

obtain solid compound 4d, yield: 95%. MS (ESI) m/z: 386 [M + H]*.

(E)-6-(3-nitro-4-methoxy-benzylidene)-2-((2-chlorophenyl) ethynyl) cyclohexan-2

enone (14)

Referring to the preparation method of compound I, 4d instead of 4a and 3

chlorophenylacetylene instead of 3-fluorophenylacetylene to obtain the target

compound 14, yield: 58%. MS (ESI) m/z: 394 [M + H]*.H NMR (400 MHz, DMSO

d) 6 7.78 (d, J= 2.1 Hz,1H, Ar-H), 7.41-7.49 (m, 3H, Ar-H, ArCH), 7.31-7.33 (m, 2H,

Ar-H), 7.25 (m, 1H, Ar-H), 7.11 (d, J= 8.7 Hz, 1H, Ar-H), 6.36 (m, 1H, CH=), 3.89 (s,

3H, OCH 3),2.92 (m, 2H, CH 2 ),2.37 (m, 2H, CH 2)o

Embodiment 5: preparation of (E)-6-(3-methoxybenzylidene)-2-((2-Bromophenyl)

ethynyl) cyclohexan-2-enone (15)

(E)-6-(3-methoxybenzylidene) cyclohex-2-enone (3e)

Referring to the preparation method of compound 3a, benzaldehyde was replaced

with 4-methoxybenzaldehyde to obtain solid compound 3e, yield: 70%. MS (ESI) m/z:

215 [M + H]*.

(E)-2-iodo-6-(3-methoxybenzylidene) cyclohex-2-enone (4e)

Referring to the preparation method of compound 4a, 3a was replaced with 3e to

obtain solid compound 4e, yield: 95%. MS (ESI) m/z: 341 [M + H]*.

(E)-6-(3-methoxybenzylidene)-2-((2-Bromophenyl) ethynyl) cyclohexan-2-enone (15)

Referring to the preparation method of compound Ii, 4e instead of 4a and 3

bromophenylacetylene instead of 3-fluorophenylacetylene to obtain the target

compound Is, yield: 55%. MS (ESI) m/z: 393 [M + H]*.H NMR (400 MHz, DMSO

d) 6 7.51 (s, 1H, ArCH), 7.40-7.45 (m, 2H, Ar-H), 7.33 (m, 1H, Ar-H), 7.26 (m, 1H, Ar-H), 7.13 (m, 1H, Ar-H), 7.02 (m, 1H, Ar-H), 6.81 (m, 1H, Ar-H), 6.39 (m, 1H,

CH=C), 3.77 (s, 3H, OCH3),2.96 (m, 2H, CH 2),2.37 (m, 2H, CH 2 );

Embodiment 6: Preparation of (E)-6-(3-trifluoromethyl benzylidene)-2-((2,4

difluorophenyl) ethynyl) cyclohexan-2-enone (16)

(E)-6-(3-trifluoromethyl benzylidene) cyclohex-2-enone (3f)

Referring to the preparation method of compound 3a, benzaldehyde was replaced

with 4-trifluoromethylbenzaldehyde to obtain solid compound 3f, yield: 72%. MS (ESI)

m/z: 253 [M + H]*.

(E)-2-iodo-6-(3-trifluoromethylbenzylidene) cyclohex-2-enone (4f)

Referring to the preparation method of compound 4a, 3a was replaced with 3f to

obtain solid compound 4f, yield: 95%. MS (ESI) m/z: 379 [M + H]*.

(E)-6-(3-trifluoromethyl benzylidene)-2-((2,4-difluorophenyl) ethynyl) cyclohexan-2

enone (16) Referring to the preparation method of compound Ii, 4f instead of 4a and 3,5

difluorophenylacetylene instead of 3-fluorophenylacetylene to obtain the target

compound 16, yield: 58%. MS (ESI) m/z: 389 [M + H]*. 'H NMR (400 MHz, DMSO

d) 67.57 (s, 1H, CH=), 7.43-7.46 (m, 3H, Ar-H), 7.19 (d, J= 8.0 Hz, 2H, Ar-H), 7.01

(m, 1H, Ar-H), 6.71 (s, 1H, Ar-H), 6.49 (m, 1H, C=CH), 3.00 (m, 2H, CH2 ), 2.45 (m,

2H, CH2 )o

Embodiment 7: Preparation of (E)-6-(4-(dimethylamino) benzylidene)-2-((2

fluorophenyl) ethynyl) cyclohexan-2-enone (17)

(E)-6-(4-(dimethylamino) benzylidene) cyclohex-2-enone (3g)

Referring to the preparation method of compound 3a, benzaldehyde was replaced

with 5-dimethylaminobenzaldehyde to obtain solid compound 3g, yield: 70%. MS (ESI)

m/z: 228 [M + H]*.

(E)-2-iodo-6-(4-(dimethylamino) benzylidene) cyclohex-2-enone (4g)

Referring to the preparation method of compound 4a, 3a was replaced with 3g to

obtain solid compound 4g, yield: 95%. MS (ESI) m/z: 354 [M + H]*.

(E)-6-(4-(dimethylamino) benzylidene)-2-((2-fluorophenyl) ethynyl) cyclohexan-2

enone (17)

Referring to the preparation method of compound Ii, 4g instead of 4a to obtain the

target compound 17, yield: 60%. MS (ESI) m/z: 346 [M + H]*.

Embodiment 8: Preparation of (E)-6-(4-trifluoromethoxy benzylidene)-2-((2

fluorophenyl) ethynyl) cyclohex-2-enone (18)

(E)-6-(4-trifluoromethoxy benzylidene) cyclohex-2-enone (3h)

Referring to the preparation method of compound 3a, benzaldehyde was replaced

with 5-trifluoromethoxybenzaldehyde to obtain solid compound 3h, yield: 65%. MS

(ESI) m/z: 269 [M + H]*.

(E)-2-iodo-6-(4-trifluoromethoxy benzylidene) cyclohexane-2-enone (4h)

Referring to the preparation method of compound 4a, 3a was replaced with 3h to

obtain solid compound 4h, yield: 95%. MS (ESI) m/z: 395 [M + H]*.

(E)-6-(4-trifluoromethoxy benzylidene)-2-((2-fluorophenyl) ethynyl) cyclohex-2

enone (18)

Referring to the preparation method of compound Ii, 4h instead of 4a to obtain the

target compound 18, yield: 67%. MS (ESI) m/z: 387 [M + H]*. 'H NMR (400 MHz,

DMSO-d) 6 7.52-7.55 (m, 2H, Ar-H, CH=), 7.46 (m, 1H, Ar-H), 7.37 (m, 1H, Ar-H),

7.31 (d,J= 8.0Hz,2H,Ar-H), 7.15 (d,J= 8.0 Hz, 2H,Ar-H), 7.03 (m,1H,Ar-H), 6.43

(m, 1H, C=CH), 2.93-2.99 (m, 2H, CH2),2.38-2.43 (m, 2H, CH 2 )o

Embodiment 9: Preparation of (E)-6-(4-trifluoromethoxy benzylidene)-2-((2

chlorophenyl) ethynyl) cyclohex-2-enone (19)

Referring to the preparation method of compound Ii, 4h instead of 4a and 3

chlorophenylacetylene instead of 3-fluorophenylacetylene to obtain the target

compound 19, yield: 67%. MS (ESI) m/z: 403 [M + H]*.'H NMR (400 MHz, DMSO

d) 67.57 (s, 1H, CH=), 7.47 (m, 1H, Ar-H), 7.31-7.35 (m, 4H, Ar-H), 7.16 (d, J= 8.0

Hz, 2H, Ar-H), 7.05 (m, 1H, Ar-H), 6.48 (m, 1H, C=CH), 2.98 (m, 2H, CH 2), 2.41 (m,

2H, CH2 )o

Embodiment 10: Preparation of (E)-6-(3,4-dimethoxy benzylidene)-2-((4

fluorophenyl) ethynyl) cyclohexan-2-enone (Iio)

(E)-6-(3,4-dimethoxy benzylidene) cyclohex-2-enone (3i)

Referring to the preparation method of compound 3a, benzaldehyde was replaced

with 4,5-dimethoxybenzaldehyde to obtain solid compound 3i, yield: 80%. MS (ESI)

m/z: 245 [M + H]*.

(E)-2-iodo-6-(3,4-dimethoxy benzylidene) cyclohex-2-enone (4i)

Referring to the preparation method of compound 4a, 3a was replaced with 3i to

obtain solid compound 4i, yield: 95%. MS (ESI) m/z: 371 [M + H]*.

(E)-6-(3,4-dimethoxy benzylidene)-2-((4-fluorophenyl) ethynyl) cyclohexan-2-enone

(Iio)

Referring to the preparation method of compound Ii, 4i instead of 4a and 5

fluorophenylacetylene instead of 3-fluorophenylacetylene to obtain the target 1 HNMR (400 MHz, DMSO compound Iio, yield: 62%. MS (ESI) m/z: 363 [M + H]*.

d) 6 7.47-7.51 (m, 3H, Ar-H), 7.27 (m, 1H, CH=), 7.16 (d, J= 8.0 Hz, 2H, Ar-H), 6.97

(m, 2H, Ar-H), 6.43 (m, 1H, C=CH), 3.95 (m, 3H, OCH 3), 3.81 (m, 3H, OCH 3), 3.01

(m, 2H, CH 2),2.39 (m, 2H, CH2 ).

Embodiment 11: Preparation of (E)-6-(3,4,5-trimethoxy benzylidene)-2-((2

fluorophenyl) ethynyl) cyclohexan-2-enone (Iii)

(E)-6-(3,4,5-trimethoxy benzylidene) cyclohex-2-enone (3j)

Referring to the preparation method of compound 3a, benzaldehyde was replaced

with 4,5,6-trimethoxybenzaldehyde to obtain solid compound 3j, yield: 75%. MS (ESI)

m/z: 275 [M + H]*.

(E)-2-iodo-6-(3,4,5-trimethoxy benzylidene) cyclohex-2-enone (4j)

Referring to the preparation method of compound 4a, 3a was replaced with 3j to

obtain solid compound 4j, yield: 95%. MS (ESI) m/z: 401 [M + H]*.

(E)-6-(3,4,5-trimethoxy benzylidene)-2-((2-fluorophenyl) ethynyl) cyclohexan-2

enone (Iii)

Referring to the preparation method of compound Ii, 4j instead of 4a to obtain the

target compound Iii, yield: 60%. MS (ESI) m/z: 393 [M + H]*. 'H NMR (400 MHz,

CDC 3) 6 7.57 (m, 1H, Ar-H), 7.47 (d, J= 1.6 Hz, 1H, Ar-H), 7.38 (m, 1H, CH=), 7.19

(m, 1H, Ar-H), 7.07 (m, 1H, Ar-H), 7.01 (m, 1H, CH=C), 6.54 (m, 2H, Ar-H), 3.82 (s,

3H, OCH 3),3.81 (s, 6H, 2xOCH3 ),3.02 (m, 2H, CH 2),2.49 (m, 2H, CH 2 )o

Embodiment 12: Preparation of (E)-6-(3,4,5-trimethoxy benzylidene)-2-((4

fluorophenyl) ethynyl) cyclohexan-2-enone (112)

Referring to the preparation method of compound Ii, 4j instead of 4a and 5

fluorophenylacetylene instead of 3-fluorophenylacetylene to obtain the target

compound 112, yield: 62%. MS (ESI) m/z: 393 [M + H]*.1 H NMR (400 MHz, CDC 3 )

6 7.48 (m, 2H, Ar-H), 7.36 (s, 1H, CH=C), 7.00-7.08 (m, 3H, Ar-H, CH=), 6.58 (s, 2H,

Ar-H), 3.86 (s, 3H, OCH3), 3.85 (s, 6H, OCH 3), 3.05 (m, 2H, CH 2), 2.52 (m, 2H, CH 2 )o.

Embodiment 13: Preparation of (E)-6-(3,4,5-trimethoxy benzylidene)-2-((2

Bromophenyl) ethynyl) cyclohexan-2-enone (113)

Referring to the preparation method of compound I, 4j instead of 4a and 3

bromophenylacetylene instead of 3-fluorophenylacetylene to obtain the target

compound 113, yield: 55%. MS (ESI) m/z: 453 [M + H]*. 'H NMR (400 MHz, CDC 3 )

6 7.59 (s, 1H, Ar-H), 7.48 (m, 1H, Ar-H), 7.40 (m, 1H, CH=), 7.23 (m,1H, Ar-H), 7.03 (m, 2H, Ar-H, CH=C), 6.57 (s, 2H, Ar-H), 3.84 (s, 3H, OCH 3), 3.82 (s, 6H, 2xOCH 3),

3.05 (m, 2H, CH 2),2.51 (m, 2H, CH2 )o

Embodiment 14: The preparation of (E)-6-(3,4,5-trimethoxy benzylidene)-2-((2,4

difluorophenyl) ethynyl) cyclohexan-2-enone (114)

Referring to the preparation method of compound Ii, 4j instead of 4a and 3,5

difluorophenylacetylene instead of 3-fluorophenylacetylene to obtain the target

compound 114, yield: 60%. MS (ESI) m/z: 411 [M + H]*.

Embodiment 15: Study on the proliferation inhibitory of tumor cells and normal cells

with the compound of the invention by MTT method

The anti-proliferative activity with the compound of the invention on four

human cancer cell lines was evaluated by MTT in vitro and piperlongumine (PL) was

used as the positive control drug. Human cancer cell lines: human liver cancer cell

HepG2, human lung cancer cell H1975, human gastric cancer cell HGC-27, human

prostate cancer cell DU145. Human normal cell: human gastric mucosal epithelial cell

GES-1.

The experimental methods are as follows: Choose a bottle of cells in good condition in

exponential growth period and add 0.25% trypsin for digestion to make the adherent

cells fall off, which was splinted into appropriate cells (2x104-4x104 cells/mL). The

cell suspension was seeded in 96-well plates at 180 L/well and incubated in a constant

temperature C02 incubator for 24 hours. Transfer the solution and add test compounds

11-114 20 M/well (the compounds were dissolved in DMSO and diluted with PBS to

make the concentration of test compound be 20 pM). After being cultured for 72 hours,

add MTT into 96-well plate at 20 L/well and further incubate for 4 h. Suck the

supernatant and add DMSO at 150 [L/well. Then shake on a flat shaker for 5 minutes.

The absorbance of each well was measured at the wavelength of 570 nm by enzyme

linked immunosorbent assay and the cell inhibition rate was calculated.

The pharmacological experiment results are shown in Table 2. The results reveal that

compounds 11-114 (20 pM) of the invention have inhibitory effects on the proliferation

of multiple human tumor cells to varying degrees. Most of the compounds of the

invention have greater inhibitory effects on multiple cancer cells and are more

significant than the positive control drug PL. Particularly, the compounds of the invention exhibit better inhibitory effects on prostate cancer cells and their activity is significantly better than the positive control drug PL. Table 2 inhibitory effects of compounds 11-114 (20 pM) of the invention on some human tumor cells and normal cells

Compd. H1975 DU145 HepG2 HGC27 GES-1 PL 77.9 75.1 72.3 79.0 52.8 I1 71.3 79.2 ND 77.6 29.6 12 73.8 78.2 ND 76.3 33.1

13 81.1 87.3 ND 84.6 30.8 14 89.2 92.8 90.6 95.7 ND 15 91.3 93.2 89.2 93.5 ND 16 93.6 95.8 93.2 95.2 ND

17 93.1 96.5 92.9 94.7 32.6 18 95.4 97.2 95.3 97.6 34.2 19 92.8 98.1 94.8 96.1 31.9 Iio 72.2 77.6 ND 70.5 25.7 11 78.4 85.1 ND 82.6 27.1

112 73.6 79.2 ND 75.7 28.4 113 75.6 83.8 ND 77.2 30.5 114 85.2 84.5 ND 81.5 24.7

Nd: not detected In addition, compounds 11-114 of the invention are also tested the proliferation inhibitory activity of normal gastric cells. The experimental results show that the inhibitory activity of most compounds of the invention on normal gastric cells is significantly weaker than that on tumor cells, which shows that the compound of the invention can selectively inhibit the growth of tumor cells at a certain concentration and has weak damage on normal cells. Embodiment 16: Study on TrxR inhibitory activity of the compound of the invention

The effect of test drug (10 pM) on TrxR activity was evaluated by TrxR activity

test kit (BioVision, Milpitas, CA, USA). The tested cell line HGC27 was dissolved in

1x buffer solution in a centrifuge tube, followed by ice bath for 20 minutes, and then

with 10000xg centrifugation at4C for 15 minutes. The supernatant was transferred to

a new centrifuge tube, and the protein concentration was calculated by Bio-Rad protein

test method. Dilute the sample to 2X working concentration with buffer solution. The

samples adding and test according to the instructions of the kit. Before reading, the

absorbance was measured at 412 nm every 20 seconds within 5 minutes after the shock

with BioTek Synergy HT multimodal enzyme marker.

The experimental results are shown in Table 3. The results indicate that compound

11-114 (10 pM) has significant activity against TrxR, and most compounds have stronger

or equivalent inhibitory activity than the positive control drug PL, suggesting that

compounds 11-114 of the invention have better TrxR inhibitory activity, which is

consistent with their anti-tumor activity.

Table 3 TrxR inhibitory effects of compounds 11-114(10 M) of the invention in vitro

Compd. Inhibition(%) Compd. Inhibition(%)

PL 80.5 18 98.2 I1 81.6 19 96.7

12 85.2 Iio ND

13 87.6 11 92.6

14 90.3 112 90.9 15 88.5 113 89.7

16 91.8 114 ND 17 95.6

Nd: not detected

Embodiment 17: Determination of intracellular ROS level

ROS-Glo hydrogen peroxide assay (Promega, Southampton, UK) measures ROS

changes by detecting H2 0 2 levels directly in cells. The cells were cultured in 96-well cell culture plates and treated with compound (20 M) for 24 hours. Hydrogen peroxide substrate solution was added into the respective well in a constant temperature C02 incubator at 37 °C for 6 hours. After incubation, ROS-Glo detection solution was added to each well and incubated at room temperature for 20 minutes. Fluorescence was detected by BioTek Synergy HT multimodal enzyme marker.

14-19 and 111-112 in the compound of general formula I of the invention were

selected as representatives to test the ROS level in tumor cells at the concentration of

pM. The changes of ROS in human gastric cancer HGC27 cells incubated with drugs

were measured with DCFH-DA as a fluorescent probe. The changes of fluorescence

intensity can quantitatively reflect the level of ROS in cells. The results show that the

compounds 14-19 and II-112(20 pM) of the invention can significantly increase the

expression level of ROS in HGC27 cells, which is 4.2-9.5-fold higher than that of the

control group, and better than the positive control drug PL (3.7-fold higher than that of

the control group).

Table 3 Effects of compounds 14-19 and II-112 of the invention on the expression level

of ROS in tumor cells at the concentration of 20 pM (taking the ROS level of control

group as a unit)

Compd. expression level of ROS Compd. expression level of ROS

PL 3.7 18 9.2 14 4.9 19 9.5

15 4.2 11 8.3

16 7.5 112 7.8

17 8.9

Claims

Claims 1. The general structure of 6-benzylidene-2--(phenylethynyl) cyclohexenone

derivatives as shown in formula I:

R'

Wherein, R and R' are the same or different, each represents one or more substituents

on the corresponding substituted benzene ring, which is selected from one or more of

H, halogen, OH, NH 2, NO 2 , C1-C6 alkyl group, C1-C6 haloalkyl group, C1-C6 alkoxy

group, C1-C6 haloalkoxy group and C1-C6 alkamine group. When R or R' represents

multiple substituents, each substituent is the same or different.
2. The 6-benzylidene-2--(phenylethynyl) cyclohexenone derivatives according to claim

1, which is characterized by:

Wherein, R and R' are the same or different, each representing one or more substituents

on the corresponding substituted benzene ring, which is selected from H, F, Cl, Br, I,

OH, NH 2, NO 2, CH 3, C2H 5 , (CH2) 2CH3 , CH(CH3) 2, CF3 , C 2F, CC13, CBr3, OCH 3 , 0

C 2 H5, OCF 3, OCC 3 , OCBr3, NHCH 3 , NHC 2H, NC 3 H. When R or R' represents

multiple substituents, each substituent is the same or different.
3. The 6- benzylidene -2-(phenylethynyl) cyclohexenone derivatives according to claim

2, which is characterized by:

R' represents one or more substituents on the benzene ring, selected from H, F, Cl, Br,

and when R' represents multiple substituents, each substituent is the same or different.
4. The 6- benzylidene -2-(phenylethynyl) cyclohexenone derivatives according to claim

3, which is characterized in that R and R' in the structure of general formula I are

selected from the following combination:

R=H, R'=2'-F;

or R =4-F, R' = 2'-F;

or R= 2-CH3 -4-OCH 3 , R'= 4'-F: or R = 3-NO 2 -4-OCH 3 , R'= 2'-Cl; or R = 3-OCH 3 , R'= 2'-Br; or R = 3-CF 3 , R'= 2',4'-di-F; or R = 4-N(CH 3) 2 , R'= 2'-F: or R = 4-OCF 3 , R'= 2'-F; or R = 4-OCF 3 , R'= 2'-Cl; or R = 3,4-di-OCH 3 , R'= 4'-F; or R = 3,4,5-tri-OCH 3 , R'= 2'-F: or R = 3,4,5-tri-OCH 3 , R'= 4'-F; or R = 3,4,5-tri-OCH 3 , R'= 2'-Br; or R = 3,4,5-tri-OCH 3 , R'= 2',4'-di-Fo
5. The preparation method of 6-benzylidene-2--(phenylethynyl) cyclohexenone

derivatives according to any one of claims 1-4, which is characterized in that it

comprises the following steps:

(1) Compound 3 is obtained by the condensation reaction of R-substituted

benzaldehyde with cyclohexene-2-one through Adol condensation. R represents one or

more substituents on the benzene ring and is selected from one or more of H, halogen,

OH, NH 2, N02, C1-C6 alkyl group, C1-C6 haloalkyl group, C1-C6 alkoxy group, Cl

C6 haloalkoxy group and C1-C6 alkamine group. When R represents multiple

substituents, each substituent is the same or different;

0 0

RCHO W R

1 2 3 (2) Compound 3 is reacted with iodine to obtain compound 4;

0 0

R -R

3 4

(3) Compound 4 is coupled with R'-substituted phenylacetylene through Sonogashira

reaction to obtain the 6-benzylidene-2-(phenylethynyl) cyclohexenone derivatives according to any one of claims 1-4. R' represents one or more substituents on the benzene ring, selected from H, halogen, OH, NH 2 , N02, C1-C6 alkyl, C1-C6 haloalkyl,

C1-C6 alkoxy, C1-C6 haloalkoxy One or more of the alkylamine groups of C1-C6.

When R'represents multiple substituents, each substituent is the same or different;

00 R -+R' R 5 C 4
6. A pharmaceutical composition that is characterized by comprising the 6-benzylidene

2-(phenylethynyl) cyclohexenone derivatives or a pharmaceutically acceptable salt

according to any one of claims 1-5.
7.Application of the 6-benzylidene-2-(phenylethynyl) cyclohexenone derivatives or a

pharmaceutically acceptable salt prepare for a drug with thioredoxin oxidoreductase

inhibitory activity according to any one of claims 1-4.
8.The application that is characterized in that the drug with thioredoxin oxidoreductase

inhibitory activity is a drug for preventing and/or treating cancer according to claim 7.
9. The application that is characterized in that the cancer include liver cancer, prostate

cancer, colon cancer, gastric cancer or lung cancer according to claim 8.