A podophyllotoxin / epipodophyllotoxin derivative, its preparation method and its application

Abstract

The present invention relates to the field of chemical synthesis technology, and specifically relates to a podophyllotoxin / epipodophyllotoxin derivative, a preparation method and an application thereof. Taking podophyllotoxin as a substrate, a piperazine group is first introduced at the 4-position of the C ring through a nucleophilic substitution reaction to obtain a podophyllotoxin / epipodophyllotoxin-piperazine intermediate, which is a mixture of diastereoisomers containing R and S configuration products at the 4-position of the C ring. Then, using the obtained podophyllotoxin / epipodophyllotoxin-piperazine intermediate as a substrate, it reacts with a cinnamic acid compound to obtain a cinnamic acid derivative of podophyllotoxin / epipodophyllotoxin. This type of compound has a good inhibitory effect on cancer cells. The present invention is an effective way to structurally modify podophyllotoxin / epipodophyllotoxin to synthesize new compounds, and the synthesized podophyllotoxin / epipodophyllotoxin derivatives containing piperazine groups are expected to be applied in the field of anti-tumor drugs.

Description

Technical Field

[0001] The present invention relates to the technical field of chemical synthesis, and particularly relates to a podophyllotoxin / epipodophyllotoxin derivative, a preparation method thereof, and an application thereof. Background Art

[0002] Cancer is a general term for a group of various diseases that can affect the normal functions of any part of the body. One of its characteristics is the rapid generation of abnormal cells. At the same time, these cells grow beyond their normal boundaries and can invade adjacent parts of the body and spread to other organs. The latter is called metastasis, and metastasis is a major cause of cancer-related deaths. As a non-communicable disease, cancer has become the second leading cause of death globally after cardiovascular diseases. Globally, nearly one-sixth of human deaths are caused by cancer, and approximately one-third of cancer deaths are caused by five major behavioral and dietary risk factors: high body mass index, low intake of fruits and vegetables, lack of exercise, tobacco use, and alcohol consumption. Overweight and obesity are associated with various types of cancer, such as esophageal cancer, colorectal cancer, breast cancer, endometrial cancer, and kidney cancer. Excessive consumption of red meat and pickled meat may increase the risk of colorectal cancer. At the same time, alcohol use is a risk factor for multiple cancers, including oral cancer, pharyngeal cancer, laryngeal cancer, esophageal cancer, liver cancer, colorectal cancer, and breast cancer. The risk of developing cancer increases with the increase in alcohol intake. If people smoke heavily while drinking a large amount of alcohol, the risk of developing multiple cancers will increase significantly. Since cancer has no obvious symptoms in the early stage and is not easily detected, it is difficult to be eradicated by treatment, is prone to metastasis and recurrence, and the treatment process of cancer causes great harm to the human body. Therefore, so far, no way to completely cure cancer has been found. Most treatments for cancer adopt a combination of conservative treatment, surgical treatment, etc.

[0003] Anticancer drugs play an indispensable role in the process of cancer treatment. Currently, common anticancer drugs include hormonal drugs, antibiotic drugs, alkylating agents, natural plant sources, etc. Plants are an important component of nature. It is estimated that there are approximately 350,000 existing plant species, and until 2004, 287,655 of these species had been identified. Plants are a natural source of antitumor drugs. Currently, the research on antitumor drugs derived from plants has been increasingly emphasized. The structural modification of natural products from traditional Chinese medicine is the main direction for the development of antitumor drugs. Plants not only provide new drugs with unique chemical structures but also provide lead compounds for the creation of more ideal new drugs. Therefore, searching for highly effective anticancer drugs from plants is one of the ways for people to solve cancer at present. Terpenoids, flavonoids, etc. extracted and isolated from plants also have other biological activities, such as having far-reaching significance in the treatment of diabetes, cardiovascular diseases, etc. Paclitaxel, camptothecin, etc., which are widely used in cancer treatment in recent years, all belong to highly effective anticancer drugs extracted from plants.

[0004] Although natural products exhibit biological activities, they have not evolved to cure human diseases. Therefore, most natural products do not possess the activity, selectivity, and pharmacokinetic properties required of clinical drugs. Structural optimization often involves modifying, removing, and introducing functional groups or chiral centers, and even altering the basic skeleton. The diversity of biosynthesis is restricted by the organism's own biosynthetic pathways, while chemical synthesis allows for more extensive modification and transformation of compounds compared to biosynthesis.

[0005] Podophyllotoxin lignans are a class of important products with cytotoxic, insecticidal, antifungal, antiviral, anti-inflammatory, neurotoxic, immunosuppressive, antirheumatic, antispasmodic, and hypolipidemic effects. As the main active ingredient of Podophyllum plants, podophyllotoxin has been extensively studied and reported. Initially isolated from the roots and rhizomes of Podophyllum plants, podophyllotoxin is a naturally occurring aryltetrahydronaphthalide lactone compound containing four contiguous chiral centers and four nearly planar rings. Podophyllotoxin can effectively inhibit herpes viruses, suppress mitosis in the metaphase of cells, and is used for venereal diseases. A tincture containing more than 98% podophyllotoxin at a concentration of 0.5% shows significant efficacy in treating condyloma acuminata. Podophyllotoxin and its β-configurational derivative, epipodophyllotoxin, exhibit significant anticancer effects by inhibiting tubulin polymerization and topoisomerase II, respectively, leading to cell cycle arrest and inhibition of the formation of mitotic spindle microtubules. Podophyllotoxin can bind to tubulin and inhibit microtubule polymerization, thereby disrupting the formation of spindle fibers. Due to the strong side effects of podophyllotoxin, structural modification of podophyllotoxin through chemical structure derivation is a feasible approach to enhance its activity and reduce its toxicity. (Pan Jianlin, Wang Yanguang, Chen Yaozu. Synthesis and in vitro antitumor activity of epipodophyllotoxin carboxylates [J]. Acta Pharmaceutica Sinica, 1997, 32(12): 898-901.)

[0006] Since the 1950s, scholars have carried out a large number of structural modifications on podophyllotoxin. Some derivatives of podophyllotoxin / epipodophyllotoxin, such as etoposide and teniposide, have been used in cancer treatment, showing their efficacy as recognized anti-cancer drugs. Regarding the structural modification of podophyllotoxin, more research has been conducted on the 4-position of the C-ring, and more prominent progress has been made. At present, many derivatives of podophyllotoxin at the C-4 position have entered clinical research, such as NPF, GL331, and TOP-53, etc. (Zhang Fumin, Tian Xuan. Synthesis and anti-cancer activity of new 4'-demethylepipodophyllum derivatives [J]. Acta Chimica Sinica, 2002, 60(4): 720-724.) Carbon-containing, oxygen-containing, and nitrogen-containing derivatives at the C-4 position have been widely synthesized. Chinese invention patent CN202110690191.9 reported the synthesis of nitrogen-containing derivatives at the C-position; Chinese invention patent CN202110543290.4 reported the synthesis of a class of oxygen-containing derivatives at the C-4 position, phenylsulfonamide phenylbutyric acid podophyllotoxin esters derivatives and their application in anti-cancer drugs, and derivatives with good anti-tumor activity have been obtained.

[0007] Cinnamic acid is one of the main chemical components of Chinese medicine cinnamon bark, and has activities such as antibacterial, increasing white blood cells, anti-inflammatory, and anti-cancer. Cinnamic acid and its derivatives can be used to produce local anesthetics, bactericides, hemostatic drugs, and are widely used in pharmaceuticals, spices, pesticides, plastics, and photosensitive resins, etc. Natural products often show excellent anti-tumor cell activity after being derived from cinnamic acid. Therefore, carrying out structural modification on the C-4 position of podophyllotoxin and introducing cinnamic acid compounds helps to synthesize compounds with relatively ideal activities and discover new molecules with anti-tumor activities. (Yang Jiaqiang, Lei Jing, Li Gang, Wan Xiaoqiang. Synthesis and anti-tumor activity of cinnamoyloxyphosphonate derivatives [J]. Acta Pharmaceutica Sinica, 2016, 51(3): 420-424.).

[0008] In view of the above defects, the creators of the present invention finally obtained the present invention through long-term research and practice. Summary of the Invention

[0009] The purpose of the present invention is to solve how to chemically synthesize podophyllotoxin / epipodophyllotoxin derivatives and screen out compounds with good inhibitory activity against a variety of cancer cells, and provides a podophyllotoxin / epipodophyllotoxin derivative, a preparation method and its application.

[0010] To achieve the above purpose, the present invention discloses a podophyllotoxin / epipodophyllotoxin derivative, and the structural general formula is:

[0011]

[0012] Among them, R is a phenyl group or a phenyl group with substituents, and the configuration at the 4-position is R or S.

[0013] Any one of the following compounds:

[0014]

[0015] Compound 1a is (5R,5aR,8aS,9R)-9-(4-((E)-3-(4-nitrophenyl)acryloyl)piperazin-1-yl)-5-(3,4,5-trimethoxyphenyl)-5,8,8a,9-tetrahydrofuro[3',4':6,7]naphtho[2,3-d][1,3]dioxol-6(5aH)-one;

[0016]

[0017] Compound 1b is (5R,5aR,8aS,9S)-9-(4-((E)-3-(4-nitrophenyl)acryloyl)piperazin-1-yl)-5-(3,4,5-trimethoxyphenyl)-5,8,8a,9-tetrahydrofuro[3',4':6,7]naphtho[2,3-d][1,3]dioxol-6(5aH)-one;

[0018]

[0019] Compound 2a is (5R,5aR,8aS,9R)-9-(4-((E)-3-(3,4-dichlorophenyl)acryloyl)piperazin-1-yl)-5-(3,4,5-trimethoxyphenyl)-5,8,8a,9-tetrahydrofuro[3',4':6,7]naphtho[2,3-d][1,3]dioxol-6(5aH)-one;

[0020]

[0021] Compound 2b is (5R,5aR,8aS,9S)-9-(4-((E)-3-(3,4-dichlorophenyl)acryloyl)piperazin-1-yl)-5-(3,4,5-trimethoxyphenyl)-5,8,8a,9-tetrahydrofuro[3',4':6,7]naphtho[2,3-d][1,3]dioxol-6(5aH)-one;

[0022]

[0023] Compound 3a is (5R,5aR,8aS,9R)-9-(4-((E)-3-((4-trifluoromethyl)phenyl)acryloyl)piperazin-1-yl)-5-(3,4,5-trimethoxyphenyl)-5,8,8a,9-tetrahydrofuro[3',4':6,7]naphtho[2,3-d][1,3]dioxol-6(5aH)-one;

[0024]

[0025] Compound 3b is (5R,5aR,8aS,9S)-9-(4-((E)-3-((4-trifluoromethyl)phenyl)acryloyl)piperazin-1-yl)-5-(3,4,5-trimethoxyphenyl)-5,8,8a,9-tetrahydrofuro[3',4':6,7]naphtho[2,3-d][1,3]dioxol-6(5aH)-one;

[0026]

[0027] Compound 4a is (5R,5aR,8aS,9R)-9-(4-((E)-3-(4-methylphenyl)acryloyl)piperazin-1-yl)-5-(3,4,5-trimethoxyphenyl)-5,8,8a,9-tetrahydrofuro[3',4':6,7]naphtho[2,3-d][1,3]dioxol-6(5aH)-one;

[0028]

[0029] Compound 4b is (5R,5aR,8aS,9S)-9-(4-((E)-3-(4-methylphenyl)acryloyl)piperazin-1-yl)-5-(3,4,5-trimethoxyphenyl)-5,8,8a,9-tetrahydrofuro[3',4':6,7]naphtho[2,3-d][1,3]dioxol-6(5aH)-one;

[0030]

[0031] Compound 5a is (5R,5aR,8aS,9R)-9-(4-cinnamoylpiperazin-1-yl)-5-(3,4,5-trimethoxyphenyl)-5,8,8a,9-tetrahydrofuro[3',4':6,7]naphtho[2,3-d][1,3]dioxol-6(5aH)-one;

[0032]

[0033] Compound 5b is (5R,5aR,8aS,9S)-9-(4-cinnamoylpiperazin-1-yl)-5-(3,4,5-trimethoxyphenyl)-5,8,8a,9-tetrahydrofuro[3',4':6,7]naphtho[2,3-d][1,3]dioxol-6(5aH)-one;

[0034]

[0035] Compound 6a is (5R,5aR,8aS,9R)-9-(4-((E)-3-(4-bromophenyl)acryloyl)piperazin-1-yl)-5-(3,4,5-trimethoxyphenyl)-5,8,8a,9-tetrahydrofuro[3',4':6,7]naphtho[2,3-d][1,3]dioxol-6(5aH)-one;

[0036]

[0037] Compound 6b is (5R,5aR,8aS,9S)-9-(4-((E)-3-(4-bromophenyl)acryloyl)piperazin-1-yl)-5-(3,4,5-trimethoxyphenyl)-5,8,8a,9-tetrahydrofuro[3',4':6,7]naphtho[2,3-d][1,3]dioxol-6(5aH)-one.

[0038] The present invention also discloses a preparation method of the above podophyllotoxin / epipodophyllotoxin derivatives, comprising the following steps:

[0039] S1: Take podophyllotoxin powder and dissolve it in acetonitrile, add sodium iodide powder, cool the reaction system to 0 °C and stir, and dropwise add methanesulfonic acid;

[0040] S2: After the reaction system in step S1 reacts completely, successively add barium carbonate powder and triethylamine, add piperazine powder when the pH value of the system reaches 7-8, quench the reaction by adding water to the reaction system after reacting overnight at room temperature, and filter;

[0041] S3: Extract and wash the reaction system obtained in step S2, dry the organic phase, spin-dry it, separate and purify the crude product obtained after evaporation to obtain a podophyllotoxin / epipodophyllotoxin piperazine intermediate;

[0042] S4: Dissolve the carboxylic acid compound in dichloromethane, then successively add the podophyllotoxin / epipodophyllotoxin piperazine intermediate obtained in step S3, 2-(7-azabenzotriazol)-N,N,N',N'-tetramethyluronium hexafluorophosphate, and diisopropylethylamine, stir at room temperature, quench the reaction with water after the reaction is complete, extract, wash, dry the organic phase, spin-dry it, separate and purify the crude product obtained after evaporation to obtain a podophyllotoxin / epipodophyllotoxin derivative.

[0043] The carboxylic acid compound in step S2 is cinnamic acid or an analogue of cinnamic acid.

[0044] The extraction solvent in steps S3 and S4 is ethyl acetate.

[0045] The organic phase washing in steps S3 and S4 is saturated brine.

[0046] In the steps S3 and S4, the drying organic phase is anhydrous sodium sulfate.

[0047] In the step S3, the column chromatography eluent is dichloromethane and methanol, and the volume ratio is dichloromethane:methanol = 1-4:1.

[0048] In the step S4, the column chromatography eluent is petroleum ether and ethyl acetate, and the volume ratio is petroleum ether:ethyl acetate = 1:1-2.

[0049] The present invention also discloses the application of the above podophyllotoxin / epipodophyllotoxin derivatives in the preparation of anti-tumor drugs.

[0050] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0051] 1. The synthesis method is simple, the conversion efficiency is high, and it is easy to separate and purify;

[0052] 2. The MTT method was used to test the in vitro anti-tumor cell activity of the synthesized derivatives. The results showed that most of the compounds exhibited obvious anti-tumor activity. Compounds 1a, 1b, 2b, 3b, 4a, 4b, 5b and 6b had better effects, and the IC 50 values for 4 different human cancer cell lines, including breast cancer MCF-7, prostate cancer PC-3, non-small cell lung cancer A549, and cervical cancer Hela, were all lower than those of the positive control drug etoposide. Among them, compounds 1b, 3b, 4b, and 5b had the best anti-tumor activity, and the IC 50 values for 4 different human cancer cell lines were all lower than 1 μM;

[0053] 3. The podophyllotoxin / epipodophyllotoxin derivatives of the present invention can be applied to the preparation of anti-tumor drugs. Detailed implementation mode

[0054] The above and other technical features and advantages of the present invention will be described in more detail below.

[0055] Example 1

[0056]

[0057] (5R,5aR,8aS)-9-(Piperazin-1-yl)-5-(3,4,5-trimethoxyphenyl)-5,8,8a,9-tetrahydrofuro[3',4':6,7]naphtho[2,3-d][1,3]dioxol-6(5aH)-one (Compound B) was synthesized:

[0058] 828 mg of podophyllotoxin powder (A) was dissolved in 100 mL of acetonitrile, 900 mg of sodium iodide powder was added, the reaction system was cooled to 0 °C and stirred, and 576 mg of methanesulfonic acid was added dropwise. After the reaction was complete, 1.18 g of barium carbonate powder and 606 mg of triethylamine were added successively. The pH value of the system reached 7 - 8, and then 190 mg of piperazine powder was added. After reacting overnight at room temperature, water was added to the reaction system to quench the reaction, and then filtration was carried out. The reaction system was extracted with ethyl acetate, the combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. The crude product obtained after evaporation to dryness was separated and purified by silica gel column chromatography to obtain the podophyllotoxin / epipodophyllotoxin piperazine intermediate (B) with a yield of 56%.

[0059] The NMR data are as follows:

[0060] 1 H NMR(400MHz,CDCl 3 )δ7.42(s,4α - 8 - H),6.69(s,4β - 8 - H),6.49(d,1H,5 - H),6.34(s,4α - 2’ - H,4α - 6’ - H),6.25(s,4β - 2’ - H,4β - 6’ - H),5.99–5.92(m,2H,16 - C H 2 ),4.66–4.31(m,2H,1 - H,4 - H),4.03–3.82(m,2H,11a - H,11b - H),3.80(d,3H,4’ - OCH 3 ),3.72(d,6H,3’ - OCH 3 ,5’ - OCH 3 ),3.14 - 2.76(m,2H,2 - H,3 - H),3.25 - 1.82(m,4H,4×a - H),2.62 - 2.44(m,4H,4×a - H).

[0061] Example 2

[0062] (5R,5aR,8aS,9R)-9-(4-((E)-3-(4-Nitrophenyl)acryloyl)piperazin-1-yl)-5-(3,4,5-trimethoxyphenyl)-5,8,8a,9-tetrahydrofuro[3',4':6,7]naphtho[2,3-d][1,3]dioxol-6(5aH)-one (Compound 1a) and (5R,5aR,8aS,9S)-9-(4-((E)-3-(4-Nitrophenyl)acryloyl)piperazin-1-yl)-5-(3,4,5-trimethoxyphenyl)-5,8,8a,9-tetrahydrofuro[3',4':6,7]naphtho[2,3-d][1,3]dioxol-6(5aH)-one (Compound 1b) Synthesis:

[0063] 23.2 mg of 4-nitrocinnamic acid was added to 5 mL of dichloromethane for dissolution, and then 43.2 mg of 2-(7-azabenzotriazol)-N,N,N',N'-tetramethyluronium hexafluorophosphate, 19.5 mg of diisopropylethylamine, and 49.3 mg of podophyllotoxin / epipodophyllotoxin piperazine intermediate (B) were successively added. After mixing evenly, the mixture was stirred at room temperature. After the reaction was complete, the reaction was quenched with water, extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, and finally the ethyl acetate was evaporated to dryness. The crude product obtained after evaporation was separated and purified by silica gel column chromatography to obtain the target compounds 1a and 1b.

[0064]

[0065] The NMR data are as follows:

[0066] 25.9 mg, yield 39.4%, yellow solid, melting point 162.7 °C - 163.5 °C.

[0067] 1 H NMR (400 MHz, CDCl 3 ) δ 8.23 (t, J = 4.9 Hz, 2H, h-H, j-H), 7.77–7.54 (m, 3H, d-H, g-H, k-H), 6.95 (d, J = 15.5 Hz, 1H, c-H), 6.62 (s, 1H, 8-H), 6.52 (s, 1H, 5-H), 6.25 (s, 2H, 2’-H, 6’-H), 6.09–5.85 (m, 2H, 16-C H 2 ), 4.57 (d, J = 4.4 Hz, 1H, 1-H), 4.44–4.24 (m, 2H, 4-H, 11a-H), 3.93 (s, 1H, 11b-H), 3.79 (s, 3H, 4’-OC H 3 ), 3.73 (s, 6H, 3’-OC H 3 , 5’-OC H 3 ), 3.68–3.43 (m, 4H, 4×a-H), 3.22–3.02 (m, 1H, 2-H), 2.95–2.82 (m, 1H, 3-H), 2.62–2.44 (m, 4H, 4×a-H)

[0068] 13 C NMR (101 MHz, CDCl 3) δ 175.08 (C-13), 164.46 (C-b), 152.68 (C-3’, C-5’), 148.31 (C-6), 148.28 (C-7), 147.00 (C-i), 141.51 (C-f), 140.39 (C-d), 137.38 (C-4’), 135.59 (C-1’), 132.79 (C-9), 128.45 (C-g, C-k), 127.58 (C-10), 124.29 (C-h, C-j), 121.32 (C-c), 110.61 (C-5), 109.91 (C-8), 108.43 (C-2’, C-6’), 101.68 (C-16), 68.50 (C-11), 62.59 (C-4), 60.91 (4’-O C H 3 ), 56.40 (3’-O C H 3 , 5’-O C H 3 ), 43.90 (C-2), 41.94 (C-1), 39.48 (C-3).

[0069] HRMS(ESI) calcd for C 35 H 36 N3O 10 [M + H] + : 658.2395, found 658.2388。

[0070]

[0071] The NMR data are as follows:

[0072] 27.3 mg, yield 41.5%, yellow solid, melting point 169.8℃ - 170.9℃.

[0073] 1 H NMR (400 MHz, Chloroform-d) δ 8.25–8.17 (m, 2H, h-H, j-H), 7.69 (d, J = 15.6 Hz, 1H, d-H), 7.68–7.54 (m, 2H, g-H, k-H), 7.41 (s, 1H, 8-H), 6.98 (d, J = 15.5 Hz, 1H, c-H), 6.53 (s, 1H, 5-H), 6.33 (s, 2H, 2’-H, 6’-H), 5.98 (dd, J = 11.3, 1.3 Hz, 2H, 16-C H 2 ), 4.58 (d, J = 3.3 Hz, 1H, 1-H), 4.54 (dd, J = 8.4, 5.9 Hz, 1H, 4-H), 4.06–3.89 (m, 2H, 11a-H, 11b-H) 3.80 (s, 3H, 4’-OCH 3 ), 3.73 (s, 6H, 3’-OC H 3 , 5’-OC H 3 ), 3.73–3.62 (m, 2H, 2×a-H), 2.87–2.74 (m, 2H, 2-H, 3-H), 2.75–2.54 (m, 4H, 4×a-H).

[0074] 13 C NMR (101 MHz, CDCl 3 ) δ 174.16 (C-13), 164.61 (C-b), 152.71 (C-3’, C-5’), 148.28 (C-i), 147.96 (C-6), 147.70 (C-7), 141.43 (C-f), 140.57 (C-d), 137.23 (C-4’), 135.84 (C-1’), 132.57 (C-9), 130.52 (C-10), 128.47 (C-g, C-k), 124.27 (C-h, C-j), 121.17 (C-c), 110.19 (C-5), 108.21 (C-2’, C-6’), 107.29 (C-8), 101.53 (C-16), 71.22 (C-4), 68.48 (C-11), 60.89 (4’-O C H 3 ), 56.27 (3’-O C H 3 , 5’-O C H 3 ), 46.41 (C-2), 43.98 (C-1), 33.33 (C-3).

[0075] HRMS (ESI) calcd for C 35 H 36 N3O 10 [M + H] + : 658.2395, found 658.2383。

[0076] Example 3

[0077] Synthesis of (5R,5aR,8aS,9R)-9-(4-((E)-3-(3,4-dichlorophenyl)acryloyl)piperazin-1-yl)-5-(3,4,5-trimethoxyphenyl)-5,8,8a,9-tetrahydrofuro[3',4':6,7]naphtho[2,3-d][1,3]dioxol-6(5aH)-one (Compound 2a) and (5R,5aR,8aS,9S)-9-(4-((E)-3-(3,4-dichlorophenyl)acryloyl)piperazin-1-yl)-5-(3,4,5-trimethoxyphenyl)-5,8,8a,9-tetrahydrofuro[3',4':6,7]naphtho[2,3-d][1,3]dioxol-6(5aH)-one (Compound 2b):

[0078] 26.0 mg of 3,4-dichlorocinnamic acid was added to 5 mL of dichloromethane for dissolution, and then 43.2 mg of 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate, 19.5 mg of diisopropylethylamine, and 49.3 mg of podophyllotoxin / epipodophyllotoxin piperazine intermediate (B) were successively added. After mixing evenly, the mixture was stirred at room temperature. After the reaction was complete, the reaction was quenched with water, extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, and finally the ethyl acetate was evaporated to dryness. The crude product obtained after evaporation was separated and purified by silica gel column chromatography to obtain the target compounds 2a and 2b.

[0079]

[0080] The NMR data are as follows:

[0081] 24.5 mg, yield 39.9%, white solid, melting point 169.1 °C - 170.0 °C.

[0082] 1 H NMR (400 MHz, CDCl 3 ) δ 7.91 (d, J = 15.5 Hz, 1H, d-H), 7.49 (d, J = 8.4 Hz, 1H, j-H), 7.43 (t, J = 1.7 Hz, 1H, k-H), 7.22 (d, J = 2.0 Hz, 1H, g-H), 6.79 (d, J = 15.5 Hz, 1H, c-H), 6.63 (s, 1H, 8-H), 6.51 (s, 1H, 5-H), 6.25 (s, 2H, 2'-H, 6'-H), 5.97 (dd, J = 7.3, 1.4 Hz, 2H, 16-C H 2 ), 4.56 (d, J = 5.4 Hz, 1H, 1-H), 4.45–4.22 (m, 2H, 4-H, 11a-H), 3.92 (d, J = 3.9 Hz, 1H, 11b-H), 3.79 (s, 3H, 4’-OC H 3 ), 3.73 (d, J = 1.4 Hz, 6H, 3’-OC H 3 , 5’-OC H 3 ), 3.70–3.58 (m, 4H, 4×a-H), 3.25–3.02 (m, 1H, 2-H), 2.96–2.69 (m, 1H, 3-H), 2.65–2.34 (m, 4H, 4×a-H).

[0083] 13 C NMR (101 MHz, CDCl 3 ) δ 175.12 (C-13), 164.90 (C-b), 152.65 (C-3’, C-5’), 148.27 (C-6), 146.98 (C-7), 137.85 (C-i), 137.32 (C-4’), 135.75 (C-f), 135.64 (C-1’), 135.34 (C-d), 132.73 (C-9), 132.30 (C-h), 130.13 (C-j), 128.46 (C-g), 127.62 (C-10), 127.51 (C-k), 120.56 (C-c), 110.55 (C-5), 109.94 (C-8), 108.40 (C-2’, C-6’), 101.66 (C-16), 68.53 (C-11), 62.57 (C-4), 60.90 (4’-O C H 3 ), 56.39 (3’-O C H 3 , 5’-O C H 3 ), 43.89 (C-2), 41.93 (C-1), 39.49 (C-3).

[0084] HRMS (ESI) calcd for C 35 H 35 Cl 2 N 2 O 8 [M + H] + : 681.1765, found 681.1756。

[0085]

[0086] The NMR data are as follows:

[0087] 30.7 mg, yield 45.1%, light yellow solid, melting point 125.2 °C - 125.4 °C.

[0088] 1 H NMR (400 MHz, CDCl 3 ) δ 7.91 (d, J = 15.5 Hz, 1H, d-H), 7.50 (d, J = 8.4 Hz, 1H, j-H), 7.43 (d, J = 2.1 Hz, 1H, k-H), 7.41 (s, 1H, 8-H), 7.24 (m, 1H, g-H), 6.81 (d, J = 15.5 Hz, 1H, c-H), 6.52 (s, 1H, 5-H), 6.33 (s, 2H, 2’-H, 6’-H), 5.98 (dd, J = 10.5, 1.3 Hz, 2H, 16-C H 2 ), 4.58 (d, J = 3.7 Hz, 1H, 1-H), 4.54 (dd, J = 8.3, 5.8 Hz, 1H, 4-H), 4.03–3.91 (m, 2H, 11a-H, 11b-H), 3.80 (s, 3H, 4’-OC H 3 ), 3.72 (s, 6H, 3’-OC H 3 , 5’-OC H 3 ), 3.71–3.60 (m, 2H, 2×a-H), 2.88–2.75 (m, 2H, 2-H, 3-H), 2.74–2.57 (m, 4H, 4×a-H).

[0089] 13 C NMR (101 MHz, CDCl 3 ) δ 174.19 (C-13), 165.09 (C-b), 152.73 (C-3’, C-5’), 147.95 (C-6), 147.69 (C-7), 138.04 (C-i), 137.23 (C-f), 135.84 (C-4’), 135.82 (C-1’), 135.34 (C-d), 132.58 (C-h), 132.27 (C-9), 130.60 (C-j), 130.15 (C-10), 128.52 (C-g), 127.54 (C-k) 120.49 (C-c), 110.18 (C-5), 108.19 (C-2’, C-6’), 107.35 (C-8), 101.52 (C-16), 71.54 (C-4), 68.51 (C-11), 60.91 (4’-O C H 3 ), 56.26 (3’-O CH 3 , 5’-O C H 3 ), 46.43 (C-2), 44.01 (C-1), 33.31 (C-3).

[0090] HRMS(ESI) calcd for C 35 H 35 Cl 2 N 2 O 8 [M + H] + : 681.1765, found 681.1758。

[0091] Example 4

[0092] (5R, 5aR, 8aS, 9R)-9-(4-((E)-3-((4-Trifluoromethyl)phenyl)acryloyl)piperazin-1-yl)-5-(3,4,5-trimethoxyphenyl)-5,8,8a,9-tetrahydrofuro[3',4':6,7]naphtho[2,3-d][1,3]dioxol-6(5aH)-one (Compound 3a) and (5R, 5aR, 8aS, 9S)-9-(4-((E)-3-((4-Trifluoromethyl)phenyl)acryloyl)piperazin-1-yl)-5-(3,4,5-trimethoxyphenyl)-5,8,8a,9-tetrahydrofuro[3',4':6,7]naphtho[2,3-d][1,3]dioxol-6(5aH)-one (Compound 3b) Synthesis:

[0093] Take 25.9 mg of 4-trifluoromethylcinnamic acid and add it to 5 mL of dichloromethane for dissolution. Then, add 43.2 mg of 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate, 19.5 mg of diisopropylethylamine, and 49.3 mg of podophyllotoxin / epipodophyllotoxin piperazine intermediate (B) in sequence. After mixing evenly, stir at room temperature. After the reaction is complete, quench the reaction with water, extract with ethyl acetate, wash with saturated brine, dry over anhydrous sodium sulfate, and finally rotary evaporate the ethyl acetate. The crude product obtained after evaporation is separated and purified by silica gel column chromatography to obtain the target compounds 3a and 3b.

[0094]

[0095] The NMR data is as follows:

[0096] 27.3 mg, yield 40.1%, light yellow solid, melting point 129.1℃ - 130.4℃.

[0097] 11H NMR (400 MHz, CDCl 3 ) δ 7.67 (d, J = 15.6 Hz, 1H, d-H), 7.64–7.55 (m, 4H, g-H, h-H, j-H, k-H), 6.90 (d, J = 15.5 Hz, 1H, c-H), 6.62 (s, 1H, 8-H), 6.51 (s, 1H, 5-H), 6.25 (s, 2H, 2’-H, 6’-H), 5.97 (dd, 2H, 16-C H 2 ), 4.57 (d, J = 5.5 Hz, 1H, 1-H), 4.44–4.23 (m, 2H, 4-H, 11a-H), 3.92 (d, J = 3.9 Hz, 1H, 11b-H), 3.80 (s, 3H, 4’-OCH 3 ), 3.74 (s, 6H, 3’-OC H 3 , 5’-OC H 3 ), 3.69–3.49 (m, 4H, 4×a-H), 3.14 (dd, J = 14.0, 5.4 Hz, 1H, 2-H), 2.93–2.73 (m, 1H, 3-H), 2.74–2.35 (m, 4H, 4×a-H).

[0098] 13 13C NMR (101 MHz, CDCl 3 ) δ 175.09 (C-13), 164.87 (C-b), 152.65 (C-3’, C-5’), 148.27 (C-6), 146.98 (C-7), 141.38 (C-d), 138.67 (C-f), 137.35 (C-4’), 135.62 (C-1’), 132.75 (C-9), 130.96 (q, J = 31.2 Hz, C-i), 127.99 (C-g, C-k), 127.61 (C-10), 125.89 (C-h, C-j), 119.53 (C-c), 119.18 (q, J = 226.6 Hz, Ph- C F 3 ), 110.56 (C-5), 109.92 (C-8), 108.42 (C-2’, C-6’), 101.66 (C-16), 68.50 (C-11), 62.56 (C-4), 60.89 (4’-O C H 3 ), 56.38 (3’-O C H 3 , 5’-O C H 3 ), 43.89 (C-2), 41.92 (C-1), 39.48 (C-3).

[0099] HRMS(ESI) calcd for C 36 H 36 F 3 N 2 O 8 [M + H] + : 681.2418, found 681.2413。

[0100]

[0101] The NMR data are as follows:

[0102] 29.6 mg, yield 43.5%, light yellow solid, melting point 122.5℃ - 122.7℃.

[0103] 1 H NMR(400 MHz, CDCl 3 ) δ 7.67 (d, J = 15.2 Hz, 1H, d - H), 7.64–7.58 (m, 4H, g - H, h - H, j - H, k - H), 7.41 (s, 1H, 8 - H), 6.91 (d, J = 15.5 Hz, 1H, c - H), 6.52 (s, 1H, 5 - H), 6.33 (s, 2H, 2’ - H, 6’ - H), 5.98 (dd, J = 11.1, 1.5 Hz, 2H, 16 - C H 2 ), 4.58 (d, J = 3.4 Hz, 1H, 1 - H), 4.54 (dd, J = 8.4, 5.9 Hz, 1H, 4 - H), 4.04–3.86 (m, 2H, 11a - H, 11b - H), 3.80 (s, 3H, 4’ - OCH 3 ), 3.73 (s, 6H, 3’ - OC H 3 , 5’ - OC H 3 ), 3.70–3.52 (m, 2H, 2×a - H), 2.90–2.72 (m, 2H, 2 - H, 3 - H), 2.74–2.46 (m, 4H, 4×a - H).

[0104] 13 C NMR(101 MHz, CDCl 3) δ174.15 (C-13), 165.07 (C-b), 152.77 (C-3’, C-5’), 147.98 (C-6), 147.72 (C-7), 141.60 (C-d), 138.69 (C-f), 137.36 (C-4’), 135.84 (C-1’), 133.15 (C-9), 131.46 (q, J=32.8 Hz, C-i), 130.61 (C-10), 128.03 (C-g, C-k), 125.96 (C-h, C-j), 120.00 (q, J=228.3 Hz, Ph- C F 3 ), 118.87 (C-c), 110.20 (C-5), 108.31 (C-2’, C-6’), 107.35 (C-8), 101.52 (C-16), 71.54 (C-4), 68.54 (C-11), 60.89 (4’-O C H 3 ), 56.30 (3’-O C H 3 , 5’-O C H 3 ), 46.45 (C-2), 44.04 (C-1), 33.36 (C-3).

[0105] HRMS(ESI) calcd for C 36 H 36 F 3 N 2 O 8 [M + H] + : 681.2418, found 681.2410.

[0106] Example 5

[0107] (5R, 5aR, 8aS, 9R)-9-(4-((E)-3-(4-methylphenyl)acryloyl)piperazin-1-yl)-5-(3,4,5-trimethoxyphenyl)-5,8,8a,9-tetrahydrofuro[3',4':6,7]naphtho[2,3-d][1,3]dioxol-6(5aH)-one (Compound 4a) and (5R, 5aR, 8aS, 9S)-9-(4-((E)-3-(4-methylphenyl)acryloyl)piperazin-1-yl)-5-(3,4,5-trimethoxyphenyl)-5,8,8a,9-tetrahydrofuro[3',4':6,7]naphtho[2,3-d][1,3]dioxol-6(5aH)-one (Compound 4b) Synthesis:

[0108] 19.4 mg of 4-methylcinnamic acid was added to 5 mL of dichloromethane for dissolution, and then 43.2 mg of 2-(7-azabenzotriazol)-N,N,N',N'-tetramethyluronium hexafluorophosphate, 19.5 mg of diisopropylethylamine, and 49.3 mg of podophyllotoxin / epipodophyllotoxin piperazine intermediate (B) were added in sequence. After mixing evenly, the mixture was stirred at room temperature. After the reaction was complete, the reaction was quenched with water, extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, and finally the ethyl acetate was rotary evaporated. The crude product obtained after evaporation was separated and purified by silica gel column chromatography to obtain the target compounds 4a and 4b.

[0109]

[0110] The NMR data are as follows:

[0111] 23.8 mg, yield 37.9%, white solid, melting point 145.8 °C - 146.0 °C.

[0112] 1 H NMR (400 MHz, CDCl 3 ) δ 7.63 (d, J = 15.4 Hz, 1H, d-H), 7.39 (d, J = 7.8 Hz, 2H, h-H, j-H), 7.21–7.12 (m, 2H, g-H, k-H), 6.77 (d, J = 15.4 Hz, 1H, c-H), 6.63 (s, 1H, 8-H), 6.51 (s, 1H, 5-H), 6.26 (s, 2H, 2’-H, 6’-H), 5.96 (d, J = 5.6 Hz, 2H, 16-C H 2 ), 4.56 (d, J = 5.5 Hz, 1H, 1-H), 4.43–4.24 (m, 2H, 4-H, 11a-H), 3.91 (d, J = 3.9 Hz, 1H, 11b-H), 3.80 (s, 3H, 4’-OCH 3 ), 3.74 (s, 6H, 3’-OC H 3 , 5’-OC H 3 ), 3.72–3.63 (m, 4H, 4×a-H), 3.14 (dd, J = 14.0, 5.6 Hz, 1H, 2-H), 2.90–2.81 (m, 1H, 3-H), 2.57–2.38 (m, 4H, 4×a-H), 2.36 (s, 3H, Ph-C H 3 ).

[0113] 13 C NMR (101 MHz, CDCl 3)δ175.14 (C-13), 165.72 (C-b), 152.65 (C-3’, C-5’), 148.23 (C-6), 146.96 (C-7), 143.28 (C-i), 140.19 (C-d), 137.35 (C-4’), 135.68 (C-1’), 132.71 (C-9’), 132.50 (C-f), 129.67 (C-h, C-j), 127.87 (C-g, C-k), 127.73 (C-10), 115.76 (C-c), 110.50 (C-5), 109.96 (C-8), 108.45 (C-2’, C-6’), 101.62 (C-16), 68.55 (C-11), 62.52 (C-4), 60.87 (4’-O C H 3 ), 56.38 (3’-O C H 3 , 5’-OCH 3 ), 43.90 (C-2), 41.93 (C-1), 38.74 (C-3), 21.54 (Ph- C H 3 ).

[0114] HRMS(ESI) calcd for C 36 H 39 N 2 O 8 [M + H] + : 627.2701, found 627.2697。

[0115]

[0116] The NMR data are as follows:

[0117] 25.4 mg, yield 40.5%, white solid, melting point 178.9℃ - 179.3℃.

[0118] 1 H NMR (400 MHz, CDCl 3 )δ7.64 (d, J = 15.4 Hz, 1H, d-H), 7.42 (s, 1H, 8-H), 7.41 (d, J = 7.9 Hz, 2H, h-H, j-H), 7.18 (d, J = 7.8 Hz, 2H, g-H, k-H), 6.79 (d, J = 15.4 Hz, 1H, c-H), 6.52 (s, 1H, 5-H), 6.33 (s, 2H, 2’-H, 6’-H), 5.98 (dd, J = 11.0, 1.3 Hz, 2H, 16-CH 2 ), 4.57 (d, J = 3.8 Hz, 1H, 1-H), 4.54 (dd, J = 8.9, 6.5 Hz, 1H, 4-H), 4.05–3.88 (m, 2H, 11a-H, 11b-H), 3.80 (s, 3H, 4’-OCH 3 ), 3.73 (s, 6H, 3’-OC H 3 , 5’-OC H 3 ), 3.72–3.60 (m, 2H, 2×a-H), 2.95–2.70 (m, 2H, 2-H, 3-H), 2.72–2.51 (m, 4H, 4×a-H), 2.36 (s, 3H, Ph-C H 3 ).

[0119] 13 C NMR (101 MHz, CDCl 3 ) δ 174.23 (C-13), 165.87 (C-b), 152.70 (C-3’, C-5’), 147.93 (C-6), 147.64 (C-7), 143.42 (C-i), 140.23 (C-d), 137.16 (C-4’), 135.85 (C-1’), 132.55 (C-9), 132.46 (C-f), 130.69 (C-10), 129.68 (C-h, C-j), 127.88 (C-g, C-k), 115.68 (C-c), 110.13 (C-5), 108.14 (C-2’, C-6’), 107.35 (C-8), 101.49 (C-16), 71.57 (C-4), 68.50 (C-11), 60.90 (4’-O C H 3 ), 56.23 (3’-O C H 3 , 5’-O C H 3 ), 46.42 (C-2), 44.02 (C-1), 33.29 (C-3), 21.55 (Ph- C H 3 ).

[0120] HRMS (ESI) calcd for C 36 H 39 N 2 O 8 [M + H] + : 627.2701, found 627.2697.

[0121] Example 6

[0122] (5R,5aR,8aS,9R)-9-(4-Cinnamoylpiperazin-1-yl)-5-(3,4,5-trimethoxyphenyl)-5,8,8a,9-tetrahydrofuro[3',4':6,7]naphtho[2,3-d][1,3]dioxol-6(5aH)-one (Compound 5a) and (5R,5aR,8aS,9S)-9-(4-Cinnamoylpiperazin-1-yl)-5-(3,4,5-trimethoxyphenyl)-5,8,8a,9-tetrahydrofuro[3',4':6,7]naphtho[2,3-d][1,3]dioxol-6(5aH)-one (Compound 5b) Synthesis:

[0123] Take 17.8 mg of cinnamic acid and add it to 5 mL of dichloromethane for dissolution. Then, successively add 43.2 mg of 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate, 19.5 mg of diisopropylethylamine, and 49.3 mg of podophyllotoxin / epipodophyllotoxin piperazine intermediate (B). After mixing evenly, stir at room temperature. After the reaction is complete, quench the reaction with water, extract with ethyl acetate, wash with saturated brine, dry over anhydrous sodium sulfate, and finally rotary evaporate the ethyl acetate. The crude product obtained after evaporation is separated and purified by silica gel column chromatography to obtain the target compounds 5a and 5b.

[0124]

[0125] The NMR data are as follows:

[0126] 25.4 mg, yield 41.5%, light yellow solid, melting point 121.3 °C - 122.1 °C.

[0127] 1 H NMR (400 MHz, CDCl 3 ) δ 7.65 (d, J = 15.4 Hz, 1H, d-H), 7.58–7.45 (m, 2H, h-H, j-H), 7.36–7.33 (m, 3H, g-H, k-H, i-H), 6.81 (d, J = 15.4 Hz, 1H, c-H), 6.62 (s, 1H, 8-H), 6.50 (s, 1H, 5-H), 6.25 (s, 2H, 2'-H, 6'-H), 5.95 (d, J = 4.4 Hz, 2H, 16-C H 2 ), 4.55 (d, J = 4.3 Hz, 1H, 1-H), 4.42–4.10 (m, 2H, 4-H, 11a-H), 3.91 (d, J = 3.9 Hz, 1H, 11b-H), 3.79 (s, 3H, 4'-OCH 3 ), 3.73 (s, 6H, 3'-OCH 3 , 5’-OC H 3 ), 3.68–3.36 (m, 4H, 4×a-H), 3.18–3.02 (m, 1H, 2-H), 2.93–2.77 (m, 1H, 3-H), 2.76–2.33 (m, 4H, 4×a-H).

[0128] 13 C NMR (101MHz, CDCl 3 ) δ 175.17 (C-13), 165.53 (C-b), 152.64 (C-3’, C-5’), 148.25 (C-6), 146.96 (C-7), 143.19 (C-d), 137.30 (C-4’), 135.68 (C-1’), 135.28 (C-9), 132.70 (C-10), 129.84 (C-f), 128.94 (C-h, C-j), 127.87 (C-g, C-k), 127.68 (C-i), 116.94 (C-c), 110.52 (C-5), 108.40 (C-2’, C-6’), 105.36 (C-8), 101.64 (C-16), 68.54 (C-11), 62.55 (C-4), 60.89 (4’-O C H 3 ), 56.37 (3’-O C H 3 , 5’-O C H 3 ), 43.89 (C-2), 41.92 (C-1), 39.50 (C-3).

[0129] HRMS (ESI) calcd for C 35 H 37 N 2 O 8 [M + H] + : 613.2544, found 613.2539.

[0130]

[0131] The NMR data are as follows:

[0132] 27.2 mg, yield 44.4%, white solid, melting point 212.9℃ - 213.4℃.

[0133] 1 H NMR (400MHz, CDCl 3) δ 7.67 (d, J = 15.4 Hz, 1H, d-H), 7.56–7.46 (m, 2H, h-H, j-H), 7.43 (s, 1H, 8-H), 7.41–7.34 (m, 3H, g-H, k-H, i-H), 6.84 (d, J = 15.4 Hz, 1H, c-H), 6.53 (s, 1H, 5-H), 6.35 (s, 2H, 2’-H, 6’-H), 5.99 (d, J = 11.0 Hz, 2H, 16-C H 2 ), 4.58 (d, J = 3.7 Hz, 1H, 1-H), 4.55 (dd, 1H, 4-H), 4.06–3.86 (m, 2H, 11a-H, 11b-H), 3.81 (s, 3H, 4’-OCH 3 ), 3.74 (s, 6H, 3’-OC H 3 , 5’-OC H 3 ), 3.68–3.52 (m, 2H, 2×a-H), 2.94–2.75 (m, 2H, 2-H, 3-H), 2.72–2.52 (m, 4H, 4×a-H).

[0134] 13 C NMR (101 MHz, CDCl 3 ) δ 174.24 (C-13), 165.71 (C-b), 152.73 (C-3’, C-5’), 147.95 (C-6), 147.68 (C-7), 143.43 (C-d), 137.19 (C-4’), 135.86 (C-1’), 135.24 (C-9), 132.57 (C-f), 130.68 (C-10), 129.93 (C-i), 128.98 (C-h, C-j), 127.91 (C-g, C-k), 116.83 (C-c), 110.17 (C-5), 108.15 (C-2’, C-6’), 107.37 (C-8), 101.52 (C-16), 71.59 (C-4), 68.54 (C-11), 60.92 (4’-O C H 3 ), 56.25 (3’-O C H 3 , 5’-O C H 3 ), 46.45 (C-2), 44.04 (C-1), 33.31 (C-3).

[0135] HRMS (ESI) calcd for C 35 H 37 N 2 O 8[M+H] + : 613.2544, found 613.2539.

[0136] Example 7

[0137] Synthesis of (5R,5aR,8aS,9R)-9-(4-((E)-3-(4-bromophenyl)acryloyl)piperazin-1-yl)-5-(3,4,5-trimethoxyphenyl)-5,8,8a,9-tetrahydrofuro[3',4':6,7]naphtho[2,3-d][1,3]dioxol-6(5aH)-one (Compound 6a) and (5R,5aR,8aS,9S)-9-(4-((E)-3-(4-bromophenyl)acryloyl)piperazin-1-yl)-5-(3,4,5-trimethoxyphenyl)-5,8,8a,9-tetrahydrofuro[3',4':6,7]naphtho[2,3-d][1,3]dioxol-6(5aH)-one (Compound 6b):

[0138] Take 27.2 mg of 4-bromocinnamic acid and add it to 5 mL of dichloromethane for dissolution. Then, successively add 43.2 mg of 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate, 19.5 mg of diisopropylethylamine, and 49.3 mg of podophyllotoxin / epipodophyllotoxin piperazine intermediate (B). After mixing evenly, stir at room temperature. After the reaction is complete, quench the reaction with water, extract with ethyl acetate, wash with saturated brine, dry over anhydrous sodium sulfate, and finally rotary evaporate the ethyl acetate. The crude product obtained after evaporation is separated and purified by silica gel column chromatography to obtain the target compounds 6a and 6b.

[0139]

[0140] The NMR data is as follows:

[0141] 27.6 mg, yield 39.9%, light yellow solid, melting point 243.7 °C - 244.2 °C.

[0142] 1 H NMR (400 MHz, CDCl 3 ) δ 7.66 (d, J = 15.5 Hz, 1H, d-H), 7.58–7.43 (m, 2H, h-H, j-H), 7.42–7.31 (m, 2H, g-H, k-H), 6.82 (d, J = 15.4 Hz, 1H, c-H), 6.63 (s, 1H, 8-H), 6.51 (s, 1H, 5-H), 6.25 (s, 2H, 2'-H, 6'-H), 5.97 (dd, J = 7.1, 1.3 Hz, 2H, 16-CH 2 ), 4.56 (d, J = 5.4 Hz, 1H, 1-H), 4.43–4.27 (m, 2H, 4-H, 11a-H), 3.92 (d, J = 3.9 Hz, 1H, 11b-H), 3.80 (s, 3H, 4’-OCH 3 ), 3.74 (s, 6H, 3’-OC H 3 , 5’-OC H 3 ), 3.68–3.41 (m, 4H, 4×a-H), 3.14 (dd, J = 14.0, 5.4 Hz, 1H, 2-H), 2.97–2.67 (m, 1H, 3-H), 2.68–2.32 (m, 4H, 4×a-H).

[0143] 13 C NMR (101 MHz, CDCl 3 ) δ 175.13 (C-13), 165.54 (C-b), 152.67 (C-3’, C-5’), 148.27 (C-6), 146.99 (C-7), 143.19 (C-d), 137.37 (C-4’), 135.68 (C-1’), 135.31 (C-f), 132.74 (C-9), 129.84 (C-i), 128.95 (C-h, C-j), 127.88 (C-g, C-k), 127.71 (C-10), 116.98 (C-c), 110.54 (C-5), 109.96 (C-8), 108.46 (C-2’, C-6’), 101.65 (C-16), 68.55 (C-11), 62.58 (C-4), 60.90 (4’-O C H 3 ), 56.40 (3’-O C H 3 , 5’-O C H 3 ), 43.92 (C-2), 41.95 (C-1), 39.53 (C-3).

[0144] HRMS (ESI) calcd for C 35 H 36 BrN 2 O 8 [M + H] + : 691.1650, found 691.1643.

[0145]

[0146] The NMR data are as follows:

[0147] 30.3 mg, yield 43.8%, light yellow solid, melting point 199.2 °C - 199.6 °C.

[0148] 1 H NMR (400 MHz, CDCl 3 ) δ 7.57 (d, J = 15.4 Hz, 1H, d-H), 7.54–7.44 (m, 2H, h-H, j-H), 7.40 (s, 1H, 8-H), 7.38–7.29 (m, 2H, g-H, k-H), 6.82 (d, J = 15.4 Hz, 1H, c-H), 6.50 (s, 1H, 5-H), 6.31 (s, 2H, 2’-H, 6’-H), 5.96 (dd, J = 10.0, 1.4 Hz, 2H, 16-C H 2 ), 4.55 (d, J = 3.7 Hz, 1H, 1-H), 4.51 (dd, J = 8.6, 6.4 Hz, 1H, 4-H), 4.04–3.86 (m, 2H, 11a-H, 11b-H), 3.78 (s, 3H, 4’-OCH 3 ), 3.71 (s, 6H, 3’-OC H 3 , 5’-OC H 3 ), 3.68–3.52 (m, 2H, 2×a-H), 2.80 (dd, J = 9.4, 3.3 Hz, 2H, 2-H, 3-H), 2.70–2.50 (m, 4H, 4×a-H).

[0149] 13 C NMR (101 MHz, CDCl 3 ) δ 174.21 (C-13), 165.69 (C-b), 152.75 (C-3’, C-5’), 147.96 (C-6), 147.69 (C-7), 143.40 (C-d), 137.26 (C-4’), 135.85 (C-f), 135.28 (C-1’), 132.60 (C-9), 130.70 (C-i), 129.91 (C-10), 128.98 (C-h, C-j), 127.91 (C-g, C-k), 116.89 (C-c), 110.18 (C-5), 108.21 (C-2’, C-6’), 107.38 (C-8), 101.52 (C-16), 71.59 (C-4), 68.56 (C-11), 60.92 (4’-O C H 3 ), 56.28 (3’-O C H 3 , 5’-O CH 3 ), 46.47 (C-2), 44.06 (C-1), 33.34 (C-3).

[0150] HRMS(ESI) calcd for C 35 H 36 BrN 2 O 8 [M + H] + : 691.1650, found 691.1640.

[0151] Twelve podophyllotoxin / epipodophyllotoxin derivatives were screened for anticancer activity.

[0152] Antitumor activity experimental procedure:

[0153] (1) Cell seeding

[0154] a. After digesting the cells with trypsin, centrifuge to collect the cells, resuspend them with 1 mL of complete DMEM medium, and pipette thoroughly to prepare a single-cell suspension;

[0155] b. Pipette 50 μL of the cell suspension, add it to 350 μL of PBS buffer solution to dilute 8-fold, count the cells using a cell counting chamber, and prepare a 3×10 4 cells / mL single-cell suspension according to the counting results;

[0156] c. Seed the cells into a 96-well plate, add 100 μL of the single-cell suspension to each well, so that the number of seeded cells in each well is 3000;

[0157] d. Place the 96-well plate in a CO 2 incubator and culture the cells at 37°C, 5% CO 2 and saturated humidity for 24 hours.

[0158] (2) Treat the cells with the compound

[0159] a. After culturing the cells for 24 h, prepare different concentration gradients of the compound according to the differences in the inhibitory effects of different compounds on the cells;

[0160] b. Take out the 96-well plate from the CO 2 incubator, carefully aspirate the medium in the wells, add 100 μL of the compound solution, and have 3 replicate wells for each concentration;

[0161] c. Put the 96-well plate back into the CO 2 incubator and continue to culture for 72 h.

[0162] (3) MTT assay

[0163] a. After culturing the cells for 70 h, add 10 μL of MTT solution with a concentration of 5 mg / mL to each well, and place the 96-well plate back into the CO 2 incubator for continued culturing for 2 h;

[0164] b. After two hours, take out the 96-well plate, carefully aspirate the culture medium, add 150 μL of DMSO solution to each well, fully dissolve the blue-violet formazan crystals formed by the reaction, and place it on a small oscillator and shake for 5 min;

[0165] c. Measure the absorbance of each well at 492 nm using an enzyme-linked immunosorbent assay detector.

[0166] The IC 50 values of each compound against non-small cell lung cancer A549, breast cancer MCF-7, prostate cancer PC-3, cervical cancer HeLa, and normal human liver cells HL-7702 are as shown in Table 1.

[0167] Table 1 Antitumor activity results of compounds (1a-6a, 1b-2b)

[0168]

[0169]

[0170] In the present invention, taking podophyllotoxin / epipodophyllotoxin as the substrate, first a piperazine group is introduced at the 4-position of the C ring through a nucleophilic substitution reaction to obtain a podophyllotoxin / epipodophyllotoxin-piperazine intermediate. This intermediate is a mixture of diastereoisomers containing R and S configuration products at the 4-position of the C ring and is a pair of racemates. Then, using the obtained podophyllotoxin / epipodophyllotoxin-piperazine intermediate as the substrate, reacting with cinnamic acid compounds to obtain cinnamic acid derivatives of podophyllotoxin / epipodophyllotoxin, and the structures of these compounds are identified by physical and chemical properties and various spectroscopic methods. In the anti-cancer activity screening, taking etoposide as the positive control, the inhibitory effects on breast cancer MCF-7, prostate cancer PC-3, non-small cell lung cancer A549, cervical cancer Hela, and normal human liver cells HL-7702 are determined by the MTT method. Compounds 1a, 1b, 2b, 3b, 4a, 4b, 5b, and 6b have good effects, and the IC 50 values against 4 different human cancer cell lines, including breast cancer MCF-7, prostate cancer PC-3, non-small cell lung cancer A549, and cervical cancer HeLa, are all lower than those of the positive control drug etoposide. Among them, compounds 1b, 3b, 4b, and 5b have the best anti-tumor activity, and the IC 50 values against 4 different human cancer cell lines are all lower than 1 μM.

[0171] The above are only the preferred embodiments of the present invention, which are illustrative rather than restrictive to the present invention. Those skilled in the art understand that many changes, modifications or even equivalents can be made within the spirit and scope defined by the claims of the present invention, but all of them will fall within the protection scope of the present invention.

Claims

1. A podophyllotoxin derivative, characterized in that, it is any one of the following compounds: wherein, Compound 1a is (5R,5aR,8aS,9R)-9-(4-((E)-3-(4-nitrophenyl)acryloyl)piperazin-1-yl)-5-(3,4,5-trimethoxyphenyl)-5,8,8a,9-tetrahydrofuro[3',4':6,7]naphtho[2,3-d][1,3]dioxol-6(5aH)-one; Compound 1b is (5R,5aR,8aS,9S)-9-(4-((E)-3-(4-nitrophenyl)acryloyl)piperazin-1-yl)-5-(3,4,5-trimethoxyphenyl)-5,8,8a,9-tetrahydrofuro[3',4':6,7]naphtho[2,3-d][1,3]dioxol-6(5aH)-one; Compound 2a is (5R,5aR,8aS,9R)-9-(4-((E)-3-(3,4-dichlorophenyl)acryloyl)piperazin-1-yl)-5-(3,4,5-trimethoxyphenyl)-5,8,8a,9-tetrahydrofuro[3',4':6,7]naphtho[2,3-d][1,3]dioxol-6(5aH)-one; Compound 2b is (5R,5aR,8aS,9S)-9-(4-((E)-3-(3,4-dichlorophenyl)acryloyl)piperazin-1-yl)-5-(3,4,5-trimethoxyphenyl)-5,8,8a,9-tetrahydrofuro[3',4':6,7]naphtho[2,3-d][1,3]dioxol-6(5aH)-one; Compound 3a is (5R,5aR,8aS,9R)-9-(4-((E)-3-((4-trifluoromethyl)phenyl)acryloyl)piperazin-1-yl)-5-(3,4,5-trimethoxyphenyl)-5,8,8a,9-tetrahydrofuro[3',4':6,7]naphtho[2,3-d][1,3]dioxol-6(5aH)-one; Compound 3b is (5R,5aR,8aS,9S)-9-(4-((E)-3-((4-trifluoromethyl)phenyl)acryloyl)piperazin-1-yl)-5-(3,4,5-trimethoxyphenyl)-5,8,8a,9-tetrahydrofuro[3',4':6,7]naphtho[2,3-d][1,3]dioxol-6(5aH)-one; Compound 4a is (5R,5aR,8aS,9R)-9-(4-((E)-3-(4-methylphenyl)acryloyl)piperazin-1-yl)-5-(3,4,5-trimethoxyphenyl)-5,8,8a,9-tetrahydrofuro[3',4':6,7]naphtho[2,3-d][1,3]dioxol-6(5aH)-one; Compound 4b is (5R,5aR,8aS,9S)-9-(4-((E)-3-(4-methylphenyl)acryloyl)piperazin-1-yl)-5-(3,4,5-trimethoxyphenyl)-5,8,8a,9-tetrahydrofuro[3',4':6,7]naphtho[2,3-d][1,3]dioxol-6(5aH)-one; Compound 5a is (5R,5aR,8aS,9R)-9-(4-cinnamoylpiperazin-1-yl)-5-(3,4,5-trimethoxyphenyl)-5,8,8a,9-tetrahydrofuro[3',4':6,7]naphtho[2,3-d][1,3]dioxol-6(5aH)-one; Compound 5b is (5R,5aR,8aS,9S)-9-(4-cinnamoylpiperazin-1-yl)-5-(3,4,5-trimethoxyphenyl)-5,8,8a,9-tetrahydrofuro[3',4':6,7]naphtho[2,3-d][1,3]dioxol-6(5aH)-one; Compound 6a is (5R,5aR,8aS,9R)-9-(4-((E)-3-(4-bromophenyl)acryloyl)piperazin-1-yl)-5-(3,4,5-trimethoxyphenyl)-5,8,8a,9-tetrahydrofuro[3',4':6,7]naphtho[2,3-d][1,3]dioxol-6(5aH)-one; Compound 6b is (5R,5aR,8aS,9S)-9-(4-((E)-3-(4-bromophenyl)acryloyl)piperazin-1-yl)-5-(3,4,5-trimethoxyphenyl)-5,8,8a,9-tetrahydrofuro[3',4':6,7]naphtho[2,3-d][1,3]dioxol-6(5aH)-one.

2. A method for preparing a podophyllotoxin derivative as claimed in claim 1, wherein, it comprises the following steps: S1: Dissolve podophyllotoxin powder in acetonitrile, add sodium iodide powder, cool the reaction system to 0 °C and stir, and add methanesulfonic acid dropwise; S2: After the reaction system in step S1 reacts completely, add barium carbonate powder and triethylamine in sequence. When the pH value of the system reaches 7-8, add piperazine powder. After reacting overnight at room temperature, add water to quench the reaction system and filter; S3: Extract and wash the reaction system obtained in step S2, dry the organic phase, spin-dry it, separate and purify the crude product obtained after evaporation to obtain a podophyllotoxin piperazine intermediate; S4: Dissolve the carboxylic acid compound in dichloromethane, and then add the podophyllotoxin piperazine intermediate obtained in step S3, 2-(7-azabenzotriazol)-N,N,N',N'-tetramethyluronium hexafluorophosphate, and diisopropylethylamine in sequence, stir at room temperature. After the reaction is complete, quench the reaction with water, extract, wash, dry the organic phase, spin-dry it, separate and purify the crude product obtained after evaporation to obtain a podophyllotoxin derivative.

3. A method for preparing a podophyllotoxin derivative as claimed in claim 2, wherein, In steps S3 and S4, the extraction solvent is ethyl acetate.

4. A method for preparing a podophyllotoxin derivative as described in claim 2, wherein, in steps S3 and S4, the organic phase washing liquid is saturated sodium chloride solution.

5. A method for preparing a podophyllotoxin derivative as described in claim 2, wherein, in steps S3 and S4, the organic phase drying agent is anhydrous sodium sulfate.

6. A method for preparing a podophyllotoxin derivative as described in claim 2, wherein, in step S3, column chromatography is used for separation, and the column chromatography eluent is dichloromethane and methanol, with a volume ratio of dichloromethane:methanol = 1-4:

1.

7. A method for preparing a podophyllotoxin derivative as described in claim 2, wherein, in step S4, column chromatography is used for separation, and the column chromatography eluent is petroleum ether and ethyl acetate, with a volume ratio of petroleum ether:ethyl acetate = 1:1-2.

8. An application of a podophyllotoxin derivative as described in claim 1 in the preparation of an anti-tumor drug, wherein, the tumor types are breast cancer MCF-7, prostate cancer PC-3, non-small cell lung cancer A549 or cervical cancer HeLa.

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