CN116217585A - Podophyllotoxin/epipodophyllotoxin derivative, preparation method and application thereof - Google Patents

Podophyllotoxin/epipodophyllotoxin derivative, preparation method and application thereof Download PDF

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CN116217585A
CN116217585A CN202111458566.5A CN202111458566A CN116217585A CN 116217585 A CN116217585 A CN 116217585A CN 202111458566 A CN202111458566 A CN 202111458566A CN 116217585 A CN116217585 A CN 116217585A
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podophyllotoxin
compound
trimethoxyphenyl
naphtho
dioxol
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黄国正
杨婷婷
张欣
金鑫
林凯歌
王妮娜
曹建国
高婷
刘姿
马亮
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Anhui University of Technology AHUT
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Abstract

The invention relates to the technical field of chemical synthesis, in particular to a podophyllotoxin/epipodophyllotoxin derivative, a preparation method and application thereof. The compounds have better inhibition effect on cancer cells. The invention is an effective way for modifying the structure of podophyllotoxin/epipodophyllotoxin to synthesize new compounds, and the synthesized and prepared piperazine group-containing podophyllotoxin/epipodophyllotoxin derivative is expected to be applied to the field of antitumor drugs.

Description

Podophyllotoxin/epipodophyllotoxin derivative, preparation method and application thereof
Technical Field
The invention relates to the technical field of chemical synthesis, in particular to a podophyllotoxin/epipodophyllotoxin derivative, a preparation method and application thereof.
Background
Cancer is a generic term for a group of diseases that affect normal function in any part of the body, and one of the features is the rapid production of abnormal cells that grow beyond their normal boundaries and can invade nearby parts of the body and spread to other organs. The latter is called metastasis, which is one of the leading causes of death from cancer. Cancer has become the second leading cause of death worldwide following cardiovascular disease as a non-infectious disease. From a global perspective, 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 fruit and vegetable intake, lack of exercise, use of tobacco, and drinking. Overweight and obesity are associated with various types of cancer, such as esophageal, colorectal, breast, endometrial and renal cancers, and excessive consumption of red meat and marinated meat may increase the risk of colorectal cancer. Concurrent alcohol use is a risk factor for a variety of cancers, including oral, pharyngeal, laryngeal, esophageal, liver, colorectal and breast cancers. The risk of developing cancer increases with increasing alcohol intake. If people drink a large amount of wine and smoke a large amount of cigarettes at the same time, the risk of suffering from various cancers is greatly increased. Since cancer has no obvious symptoms at early stage and is difficult to be found, is difficult to eradicate by treatment, is easy to metastasize and relapse, and the treatment process of the cancer has great harm to human body, so far, no way for completely curing the cancer has been found. For cancer treatment, a treatment mode of combining a conservative treatment, an operation treatment and the like is adopted in most cases.
Anticancer drugs play an indispensable role in the course of cancer treatment, and currently common anticancer drugs include hormones, antibiotics, alkylating agents, natural plant sources, and the like. Plants are important components in nature. It is estimated that there are approximately 350000 plant species present, of which 287655 have been identified until 2004. The plant is a natural source of the anti-tumor drug, the research of the anti-tumor drug from the plant is increasingly paid attention to at present, the structural modification of the natural product from the traditional Chinese medicine is a main direction of the development of the anti-tumor drug, and the plant not only provides a new drug with a unique chemical structure, but also provides a lead compound for creating a more ideal new drug. Therefore, searching for high-efficiency anticancer drugs from plants is one of the current approaches for solving cancers. Terpenes, flavones and the like extracted and separated from plants have other biological activities at the same time, and have profound significance in the aspects of treating diabetes, cardiovascular diseases and the like. Paclitaxel, camptothecine and the like widely used for resisting cancer in recent years belong to high-efficiency anticancer drugs extracted from plants.
Although natural products exhibit biological activity, they have not evolved to cure human diseases, and therefore most natural products do not have activity, selectivity and pharmacokinetic properties as clinical drugs, often requiring modification, removal and introduction of functional groups or chiral centers, and even modification of the basic backbone for structural optimization. The diversity of biosynthesis is limited by the organism's own biosynthetic pathways, whereas chemical synthesis can make a wider variety of modifications and alterations to the compound than biosynthesis.
Podophyllotoxin lignin is an important product with cytotoxicity, insecticidal, antifungal, antiviral, antiinflammatory, neurotoxic, immunosuppressive, antirheumatic, antispasmodic and hypolipidemic effects. Podophyllotoxin has been widely studied and reported as a major active ingredient in podophyllum plants. Podophyllotoxin was originally isolated from the roots and rhizomes of Podophyllum plants and is a naturally occurring aryltetralin lactone compound containing 4 consecutive chiral centers and 4 nearly planar rings. The podophyllotoxin can effectively inhibit herpesvirus and mitosis in metaphase, and can be used for treating toxic diseases. The tincture with more than 98 percent of podophyllotoxin being prepared into 0.5 percent has obvious curative effect on condyloma acuminatum. The epipodophyllotoxin and its beta-configuration derivatives show remarkable anticancer effects by inhibiting tubulin polymerization and topoisomerase II, respectively, resulting in cell cycle arrest and inhibition of mitotic spindle microtubule formation. Podophyllotoxin binds to tubulin and inhibits microtubule polymerization, thereby disrupting spindle filament formation. Because podophyllotoxins have strong side effects, modification of the podophyllotoxin structure through chemical structure derivatization is a viable solution for improving the activity and reducing the toxicity of the podophyllotoxin. (Pan Jianlin, wang Yanan, chen Yaozu. Synthesis of epipodophyllotoxin carboxylate and in vitro antitumor Activity [ J ]. Pharmaceutical Proc 1997,32 (12): 898-901.)
Since the 50 s of the 20 th century, scholars have made a number of structural alterations to podophyllotoxins, and some podophyllotoxin/epipodophyllotoxin derivatives such as etoposide and teniposide have been used in cancer treatment, showing their efficacy as recognized anticancer drugs. Regarding structural modification of podophyllotoxin, more researches on the 4-position of the C ring are carried out, the obtained progress is also outstanding, and currently, various C-4 derivatives of podophyllotoxin enter clinical researches such as NPF, GL331 and TOP-53. (Zhang Fumin, tian) Synthesis of novel 4' -nor epipodophyllotophyllum derivatives and their anticancer Activity [ J ]. Chemical journal, 2002,60 (4): 720-724.) C-4 carbon-, oxygen-and nitrogen-containing derivatives are widely synthesized. Chinese patent No. CN202110690191.9 reports the synthesis of C-site nitrogen-containing derivatives; chinese patent No. 202110543290.4 reports the synthesis of C-4 oxygen-containing derivatives benzene sulfonamide benzene butyric acid podophyllotoxin ester derivatives and the application thereof in anticancer drugs, which all obtain derivatives with better antitumor activity.
Cinnamic acid is one of the main chemical components of Chinese medicinal cinnamon, and has the activities of resisting bacteria, increasing leucocyte, li Dan, resisting cancer and the like. Cinnamic acid and its derivatives can be used for producing local anesthetic, bactericide and hemostatic, and can be widely used in medicines, fragrances, pesticides, plastics, photosensitive resins, etc. Natural products often exhibit excellent anti-tumor cell activity after cinnamic acid derivatization. Therefore, the C-4 position of the podophyllotoxin is subjected to structural modification, and cinnamic acid compounds are introduced to help to synthesize compounds with ideal activity, so that new molecules with anti-tumor activity are discovered. (Yang Gujiang, lei Jing, li Gang, mo Xiaojiang. Synthesis of cinnamoyl oxyphosphonate derivatives and antitumor Activity [ J ]. Pharmacology, 2016,51 (3): 420-424.).
In view of the above drawbacks, the present inventors have finally achieved the present invention through long-time studies and practices.
Disclosure of Invention
The invention aims to solve the problem of how to chemically synthesize podophyllotoxin/epipodophyllotoxin derivatives and screen out compounds with better inhibiting activity on various cancer cells, and provides the podophyllotoxin/epipodophyllotoxin derivatives, a preparation method and application thereof.
In order to achieve the above purpose, the invention discloses a podophyllotoxin/epipodophyllotoxin derivative, which has the following structural general formula:
Figure BDA0003388851080000031
wherein R is phenyl or phenyl containing substituent, and the configuration of the 4-position is R or S.
Is any one of the following compounds:
Figure BDA0003388851080000032
compound 1a is (5 r,5ar,8as,9 r) -9- (4- ((E) -3- (4-nitrophenyl) acryloyl) piperazin-1-yl) -5- (3, 4, 5-trimethoxyphenyl) -5, 8a, 9-tetrahydrofuran [3',4':6,7] naphtho [2,3-d ] [1,3] dioxol-6 (5 aH) -one;
Figure BDA0003388851080000041
compound 1b was (5 r,5ar,8as,9 s) -9- (4- ((E) -3- (4-nitrophenyl) acryloyl) piperazin-1-yl) -5- (3, 4, 5-trimethoxyphenyl) -5, 8a, 9-tetrahydrofuran [3',4':6,7] naphtho [2,3-d ] [1,3] dioxol-6 (5 aH) -one;
Figure BDA0003388851080000042
compound 2a is (5 r,5ar,8as,9 r) -9- (4- ((E) -3- (3, 4-dichlorophenyl) acryloyl) piperazin-1-yl) -5- (3, 4, 5-trimethoxyphenyl) -5, 8a, 9-tetrahydrofuran [3',4':6,7] naphtho [2,3-d ] [1,3] dioxol-6 (5 aH) -one;
Figure BDA0003388851080000043
compound 2b was (5 r,5ar,8as,9 s) -9- (4- ((E) -3- (3, 4-dichlorophenyl) acryloyl) piperazin-1-yl) -5- (3, 4, 5-trimethoxyphenyl) -5, 8a, 9-tetrahydrofuran [3',4':6,7] naphtho [2,3-d ] [1,3] dioxol-6 (5 aH) -one;
Figure BDA0003388851080000051
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compound 3a is (5 r,5ar,8as,9 r) -9- (4- ((E) -3- ((4-trifluoromethyl) phenyl) acryloyl) piperazin-1-yl) -5- (3, 4, 5-trimethoxyphenyl) -5, 8a, 9-tetrahydrofuran [3',4':6,7] naphtho [2,3-d ] [1,3] dioxol-6 (5 aH) -one;
Figure BDA0003388851080000052
compound 3b was (5 r,5ar,8as,9 s) -9- (4- ((E) -3- ((4-trifluoromethyl) phenyl) acryloyl) piperazin-1-yl) -5- (3, 4, 5-trimethoxyphenyl) -5, 8a, 9-tetrahydrofuran [3',4':6,7] naphtho [2,3-d ] [1,3] dioxol-6 (5 aH) -one;
Figure BDA0003388851080000053
compound 4a is (5 r,5ar,8as,9 r) -9- (4- ((E) -3- (4-methylphenyl) acryloyl) piperazin-1-yl) -5- (3, 4, 5-trimethoxyphenyl) -5, 8a, 9-tetrahydrofuran [3',4':6,7] naphtho [2,3-d ] [1,3] dioxol-6 (5 aH) -one;
Figure BDA0003388851080000061
compound 4b was (5 r,5ar,8as,9 s) -9- (4- ((E) -3- (4-methylphenyl) acryloyl) piperazin-1-yl) -5- (3, 4, 5-trimethoxyphenyl) -5, 8a, 9-tetrahydrofuran [3',4':6,7] naphtho [2,3-d ] [1,3] dioxol-6 (5 aH) -one;
Figure BDA0003388851080000062
compound 5a is (5 r,5ar,8as,9 r) -9- (4-cinnamoyl piperazin-1-yl) -5- (3, 4, 5-trimethoxyphenyl) -5, 8a, 9-tetrahydrofuran [3',4':6,7] naphtho [2,3-d ] [1,3] dioxol-6 (5 aH) -one;
Figure BDA0003388851080000063
compound 5b is (5 r,5ar,8as,9 s) -9- (4-cinnamoyl piperazin-1-yl) -5- (3, 4, 5-trimethoxyphenyl) -5, 8a, 9-tetrahydrofuran [3',4':6,7] naphtho [2,3-d ] [1,3] dioxol-6 (5 aH) -one;
Figure BDA0003388851080000071
compound 6a is (5 r,5ar,8as,9 r) -9- (4- ((E) -3- (4-bromophenyl) acryloyl) piperazin-1-yl) -5- (3, 4, 5-trimethoxyphenyl) -5, 8a, 9-tetrahydrofuran [3',4':6,7] naphtho [2,3-d ] [1,3] dioxol-6 (5 aH) -one;
Figure BDA0003388851080000072
compound 6b is (5R, 5aR,8aS, 9S) -9- (4- ((E) -3- (4-bromophenyl) acryloyl) piperazin-1-yl) -5- (3, 4, 5-trimethoxyphenyl) -5, 8a, 9-tetrahydrofuran [3',4':6,7] naphtho [2,3-d ] [1,3] dioxol-6 (5 aH) -one.
The invention also discloses a preparation method of the podophyllotoxin/epipodophyllotoxin derivative, which comprises the following steps:
s1: dissolving podophyllotoxin powder in acetonitrile, adding sodium iodide powder, cooling the reaction system to 0 ℃, stirring, and dropwise adding methanesulfonic acid;
s2: after the reaction system in the step S1 is completely reacted, sequentially adding barium carbonate powder and triethylamine, adding piperazine powder when the pH value of the system reaches 7-8, adding water into the reaction system for quenching after overnight reaction at room temperature, and filtering;
s3: extracting, washing, drying an organic phase, spin-drying and evaporating a crude product obtained after the reaction system obtained in the step S2 to obtain a podophyllotoxin/epipodophyllotoxin piperazine intermediate product;
s4: adding a carboxylic acid compound into dichloromethane for dissolution, then sequentially adding the podophyllotoxin/epipodophyllotoxin piperazine intermediate product obtained in the step S3, 2- (7-aza-benzotriazol) -N, N, N ', N' -tetramethyl urea hexafluorophosphate and diisopropylethylamine, stirring at room temperature, after the reaction is completed, quenching the reaction with water, extracting, washing, drying an organic phase, spin-drying, and separating and purifying a crude product obtained after evaporating to dryness to obtain the podophyllotoxin/epipodophyllotoxin derivative.
The carboxylic acid compound in the step S2 is cinnamic acid or cinnamic acid analogue.
The extraction solvent in the steps S3 and S4 is ethyl acetate.
The organic phase washed in steps S3 and S4 is saturated saline.
The dry organic phase in steps S3 and S4 is anhydrous sodium sulfate.
The column chromatography eluent in the step S3 is dichloromethane and methanol, and the volume ratio is dichloromethane: methanol=1 to 4:1.
the column chromatography eluent in the step S4 is petroleum ether and ethyl acetate, and the volume ratio is petroleum ether: ethyl acetate = 1:1 to 2.
The invention also discloses application of the podophyllotoxin/epipodophyllotoxin derivative in preparing an anti-tumor medicament.
Compared with the prior art, the invention has the beneficial effects that:
1. the synthesis method is simple, the conversion efficiency is high, and the separation and purification are easy;
2. the MTT method is adopted to test the activity of the in vitro anti-tumor cells of the synthesized derivative, and the result shows that most of the compounds show obvious anti-tumor activity. The compounds 1a, 1b, 2b, 3b, 4a, 4b, 5b and 6b have better effect, and have IC (integrated circuit) on 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 50 The values were all lower than the positive control drug etoposide. Wherein, the compounds 1b, 3b, 4b and 5b have optimal anti-tumor activity, and are IC for 4 different human cancer cell lines 50 Values were all below 1 μm;
3. the podophyllotoxin/epipodophyllotoxin derivative can be applied to the preparation of antitumor drugs.
Detailed Description
The above and further technical features and advantages of the present invention are described in more detail below.
Example 1
Figure BDA0003388851080000091
Synthesis of (5R, 5aR,8 aS) -9- (piperazin-1-yl) -5- (3, 4, 5-trimethoxyphenyl) -5, 8a, 9-tetrahydrofuran [3',4':6,7] naphtho [2,3-d ] [1,3] dioxol-6 (5 aH) -one (Compound B):
828mg of podophyllotoxin powder (A) was dissolved in 100mL of acetonitrile, 900mg of sodium iodide powder was added, the reaction system was cooled to 0℃and stirred, and 576mg of methanesulfonic acid was added dropwise. After the reaction was completed, 1.18g of barium carbonate powder and 606mg of triethylamine were sequentially added, the pH of the system reached 7-8, and then 190mg of piperazine powder was added. After overnight reaction at room temperature, water quenching and filtering are added into the reaction system. The reaction system was extracted with ethyl acetate, and 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 is separated and purified by a silica gel column chromatography method to obtain the intermediate product (B) of podophyllotoxin/epipodophyllotoxin piperazine with the yield of 56 percent.
The nuclear magnetic data are as follows:
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 2 H),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)。
example 2
Synthesis of (5R, 5aR,8aS, 9R) -9- (4- ((E) -3- (4-nitrophenyl) propenoyl) piperazin-1-yl) -5- (3, 4, 5-trimethoxyphenyl) -5, 8a, 9-tetrahydrofuran [3',4':6,7] naphtho [2,3-d ] [1,3] dioxol-6 (5 aH) -one (Compound 1 a) and (5R, 5aR,8aS, 9S) -9- (4- ((E) -3- (4-nitrophenyl) propenoyl) piperazin-1-yl) -5- (3, 4, 5-trimethoxyphenyl) -5, 8a, 9-tetrahydrofuran [3',4':6,7] naphtho [2,3-d ] [1,3] dioxol-6 (5 aH) -one (Compound 1 b):
23.2mg of 4-nitrocinnamic acid is added into 5mL of dichloromethane to be dissolved, 43.2mg of 2- (7-azabenzotriazol) -N, N, N ', N' -tetramethyl urea hexafluorophosphate, 19.5mg of diisopropylethylamine and 49.3mg of podophyllotoxin/epipodophyllotoxin piperazine intermediate (B) are sequentially added, the mixture is uniformly mixed, stirred at room temperature, after the reaction is completed, the mixture is quenched with water to react, extracted with ethyl acetate, washed with saturated saline solution, dried with anhydrous sodium sulfate and finally the ethyl acetate is spun dry. The crude product obtained after evaporation is separated and purified by a silica gel column chromatography method to obtain the target compounds 1a and 1b.
Figure BDA0003388851080000101
The nuclear magnetic data are as follows:
25.9mg,yield 39.4％,yellow solid,melting point 162.7℃-163.5℃.
1 H NMR(400MHz,CDCl 3 )δ8.23(t,J＝4.9Hz,2H,h-H,j-H),7.77–7.54(m,3H,d-H,g-H,k-H),6.95(d,J＝15.5Hz,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 2 H),4.57(d,J＝4.4Hz,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 3 H),3.73(s,6H,3’-OC 3 H,5’-OC 3 H),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)
13 C NMR(101MHz,CDCl 3 )δ175.08(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’-OCH 3 ),56.40(3’-OCH 3 ,5’-OCH 3 ),43.90(C-2),41.94(C-1),39.48(C-3).
HRMS(ESI)calcd for C 35 H 36 N3O 10 [M+H] + :658.2395,found 658.2388。
Figure BDA0003388851080000111
the nuclear magnetic data are as follows:
27.3mg,yield 41.5％,yellow solid,melting point 169.8℃-170.9℃.
1 H NMR(400MHz,Chloroform-d)δ8.25–8.17(m,2H,h-H,j-H),7.69(d,J＝15.6Hz,1H,d-H),7.68–7.54(m,2H,g-H,k-H),7.41(s,1H,8-H),6.98(d,J＝15.5Hz,1H,c-H),6.53(s,1H,5-H),6.33(s,2H,2’-H,6’-H),5.98(dd,J＝11.3,1.3Hz,2H,16-C 2 H),4.58(d,J＝3.3Hz,1H,1-H),4.54(dd,J＝8.4,5.9Hz,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 3 H,5’-OC 3 H),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).
13 C NMR(101MHz,CDCl 3 )δ174.16(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’-OCH 3 ),56.27(3’-OCH 3 ,5’-OCH 3 ),46.41(C-2),43.98(C-1),33.33(C-3).
HRMS(ESI)calcd for C 35 H 36 N3O 10 [M+H] + :658.2395,found 658.2383。
example 3
Synthesis of (5R, 5aR,8aS, 9R) -9- (4- ((E) -3- (3, 4-dichlorophenyl) acryloyl) piperazin-1-yl) -5- (3, 4, 5-trimethoxyphenyl) -5, 8a, 9-tetrahydrofuran [3',4':6,7] naphtho [2,3-d ] [1,3] dioxol-6 (5 aH) -one (Compound 2 a) and (5R, 5aR,8aS, 9S) -9- (4- ((E) -3- (3, 4-dichlorophenyl) acryloyl) piperazin-1-yl) -5- (3, 4, 5-trimethoxyphenyl) -5, 8a, 9-tetrahydrofuran [3',4':6,7] naphtho [2,3-d ] [1,3] dioxol-6 (5 aH) -one (Compound 2 b):
26.0mg of 3, 4-dichloro cinnamic acid is added into 5mL of dichloromethane to be dissolved, 43.2mg of 2- (7-aza-benzotriazol) -N, N, N ', N' -tetramethyl urea hexafluorophosphate, 19.5mg of diisopropylethylamine and 49.3mg of podophyllotoxin/epipodophyllotoxin piperazine intermediate (B) are sequentially added, the mixture is uniformly mixed and stirred at room temperature, after the reaction is completed, the mixture is quenched by water, extracted by ethyl acetate, washed by saturated saline solution, dried by anhydrous sodium sulfate, and finally the ethyl acetate is dried by spin. The crude product obtained after evaporation is separated and purified by a silica gel column chromatography method to obtain the target compounds 2a and 2b.
Figure BDA0003388851080000121
The nuclear magnetic data are as follows:
24.5mg,yield 39.9％,white solid,melting point 169.1℃-170.0℃.
1 H NMR(400MHz,CDCl 3 )δ7.91(d,J＝15.5Hz,1H,d-H),7.49(d,J＝8.4Hz,1H,j-H),7.43(t,J＝1.7Hz,1H,k-H),7.22(d,J＝2.0Hz,1H,g-H),6.79(d,J＝15.5Hz,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＝7.3,1.4Hz,2H,16-C 2 H),4.56(d,J＝5.4Hz,1H,1-H),4.45–4.22(m,2H,4-H,11a-H),3.92(d,J＝3.9Hz,1H,11b-H),3.79(s,3H,4’-OC 3 H),3.73(d,J＝1.4Hz,6H,3’-OC 3 H,5’-OC 3 H),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).
13 C NMR(101MHz,CDCl 3 )δ175.12(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’-OCH 3 ),56.39(3’-OCH 3 ,5’-OCH 3 ),43.89(C-2),41.93(C-1),39.49(C-3).
HRMS(ESI)calcd for C 35 H 35 Cl 2 N 2 O 8 [M+H] + :681.1765,found 681.1756。
Figure BDA0003388851080000131
the nuclear magnetic data are as follows:
30.7mg,yield 45.1％,light yellow solid,melting point 125.2℃-125.4℃.
1 H NMR(400MHz,CDCl 3 )δ7.91(d,J＝15.5Hz,1H,d-H),7.50(d,J＝8.4Hz,1H,j-H),7.43(d,J＝2.1Hz,1H,k-H),7.41(s,1H,8-H),7.24(m,1H,g-H),6.81(d,J＝15.5Hz,1H,c-H),6.52(s,1H,5-H),6.33(s,2H,2’-H,6’-H),5.98(dd,J＝10.5,1.3Hz,2H,16-C 2 H),4.58(d,J＝3.7Hz,1H,1-H),4.54(dd,J＝8.3,5.8Hz,1H,4-H),4.03–3.91(m,2H,11a-H,11b-H),3.80(s,3H,4’-OC 3 H),3.72(s,6H,3’-OC 3 H,5’-OC 3 H),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).
13 C NMR(101MHz,CDCl 3 )δ174.19(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’-OCH 3 ),56.26(3’-OCH 3 ,5’-OCH 3 ),46.43(C-2),44.01(C-1),33.31(C-3).
HRMS(ESI)calcd for C 35 H 35 Cl 2 N 2 O 8 [M+H] + :681.1765,found 681.1758。
example 4
Synthesis of (5R, 5aR,8aS, 9R) -9- (4- ((E) -3- ((4-trifluoromethyl) phenyl) propen-1-yl) -5- (3, 4, 5-trimethoxyphenyl) -5, 8a, 9-tetrahydrofurano [3',4':6,7] naphtho [2,3-d ] [1,3] dioxol-6 (5 aH) -one (Compound 3 a) and (5R, 5aR,8aS, 9S) -9- (4- ((E) -3- ((4-trifluoromethyl) phenyl) propen-1-yl) -5- (3, 4, 5-trimethoxyphenyl) -5, 8a, 9-tetrahydrofurano [3',4':6,7] naphtho [2,3-d ] [1,3] dioxol-6 (5 aH) -one (Compound 3 b):
25.9mg of 4-trifluoromethyl cinnamic acid is added into 5mL of dichloromethane to be dissolved, 43.2mg of 2- (7-aza-benzotriazol) -N, N, N ', N' -tetramethyl urea hexafluorophosphate, 19.5mg of diisopropylethylamine and 49.3mg of podophyllotoxin/epipodophyllotoxin piperazine intermediate (B) are sequentially added, the mixture is uniformly mixed, and then stirred at room temperature, after the reaction is completed, the mixture is quenched with water to react, extracted with ethyl acetate, washed with saturated saline solution, dried with anhydrous sodium sulfate, and finally the ethyl acetate is spun dry. The crude product obtained after evaporation is separated and purified by a silica gel column chromatography method to obtain target compounds 3a and 3b.
Figure BDA0003388851080000141
The nuclear magnetic data are as follows:
27.3mg,yield 40.1％,light yellow solid,melting point 129.1℃-130.4℃.
1 H NMR(400MHz,CDCl 3 )δ7.67(d,J＝15.6Hz,1H,d-H),7.64–7.55(m,4H,g-H,h-H,j-H,k-H),6.90(d,J＝15.5Hz,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 2 H),4.57(d,J＝5.5Hz,1H,1-H),4.44–4.23(m,2H,4-H,11a-H),3.92(d,J＝3.9Hz,1H,11b-H),3.80(s,3H,4’-OCH 3 ),3.74(s,6H,3’-OC 3 H,5’-OC 3 H),3.69–3.49(m,4H,4×a-H),3.14(dd,J＝14.0,5.4Hz,1H,2-H),2.93–2.73(m,1H,3-H),2.74–2.35(m,4H,4×a-H).
13 C NMR(101MHz,CDCl 3 )δ175.09(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＝31.2Hz,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＝226.6Hz,Ph-CF 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’-OCH 3 ),56.38(3’-OCH 3 ,5’-OCH 3 ),43.89(C-2),41.92(C-1),39.48(C-3).
HRMS(ESI)calcd for C 36 H 36 F 3 N 2 O 8 [M+H] + :681.2418,found 681.2413。
Figure BDA0003388851080000151
the nuclear magnetic data are as follows:
29.6mg,yield 43.5％,light yellow solid,melting point 122.5℃-122.7℃.
1 H NMR(400MHz,CDCl 3 )δ7.67(d,J＝15.2Hz,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＝15.5Hz,1H,c-H),6.52(s,1H,5-H),6.33(s,2H,2’-H,6’-H),5.98(dd,J＝11.1,1.5Hz,2H,16-C 2 H),4.58(d,J＝3.4Hz,1H,1-H),4.54(dd,J＝8.4,5.9Hz,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 3 H,5’-OC 3 H),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).
13 C NMR(101MHz,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＝32.8Hz,C-i),130.61(C-10),128.03(C-g,C-k),125.96(C-h,C-j),120.00(q,J＝228.3Hz,Ph-CF 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’-OCH 3 ),56.30(3’-OCH 3 ,5’-OCH 3 ),46.45(C-2),44.04(C-1),33.36(C-3).
HRMS(ESI)calcd for C 36 H 36 F 3 N 2 O 8 [M+H] + :681.2418,found 681.2410.
example 5
Synthesis of (5R, 5aR,8aS, 9R) -9- (4- ((E) -3- (4-methylphenyl) propenoyl) piperazin-1-yl) -5- (3, 4, 5-trimethoxyphenyl) -5, 8a, 9-tetrahydrofuran [3',4':6,7] naphtho [2,3-d ] [1,3] dioxol-6 (5 aH) -one (compound 4 a) and (5R, 5aR,8aS, 9S) -9- (4- ((E) -3- (4-methylphenyl) propenoyl) piperazin-1-yl) -5- (3, 4, 5-trimethoxyphenyl) -5, 8a, 9-tetrahydrofuran [3',4':6,7] naphtho [2,3-d ] [1,3] dioxol-6 (5 aH) -one (compound 4 b):
19.4mg of 4-methyl cinnamic acid is added into 5mL of dichloromethane to be dissolved, 43.2mg of 2- (7-aza-benzotriazol) -N, N, N ', N' -tetramethyl urea hexafluorophosphate, 19.5mg of diisopropylethylamine and 49.3mg of podophyllotoxin/epipodophyllotoxin piperazine intermediate (B) are sequentially added, the mixture is uniformly mixed, and then stirred at room temperature, after the reaction is completed, the mixture is quenched with water to react, extracted with ethyl acetate, washed with saturated saline solution, dried with anhydrous sodium sulfate, and finally the ethyl acetate is spun dry. The crude product obtained after evaporation is separated and purified by a silica gel column chromatography method to obtain target compounds 4a and 4b.
Figure BDA0003388851080000161
The nuclear magnetic data are as follows:
23.8mg,yield 37.9％,white solid,melting point 145.8℃-146.0℃.
1 H NMR(400MHz,CDCl 3 )δ7.63(d,J＝15.4Hz,1H,d-H),7.39(d,J＝7.8Hz,2H,h-H,j-H),7.21–7.12(m,2H,g-H,k-H),6.77(d,J＝15.4Hz,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＝5.6Hz,2H,16-C 2 H),4.56(d,J＝5.5Hz,1H,1-H),4.43–4.24(m,2H,4-H,11a-H),3.91(d,J＝3.9Hz,1H,11b-H),3.80(s,3H,4’-OCH 3 ),3.74(s,6H,3’-OC 3 H,5’-OC 3 H),3.72–3.63(m,4H,4×a-H),3.14(dd,J＝14.0,5.6Hz,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 3 H).
13 C NMR(101MHz,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’-OCH 3 ),56.38(3’-OCH 3 ,5’-OCH 3 ),43.90(C-2),41.93(C-1),38.74(C-3),21.54(Ph-CH 3 ).
HRMS(ESI)calcd for C 36 H 39 N 2 O 8 [M+H] + :627.2701,found 627.2697。
Figure BDA0003388851080000171
the nuclear magnetic data are as follows:
25.4mg,yield 40.5％,white solid,melting point 178.9℃-179.3℃.
1 H NMR(400MHz,CDCl 3 )δ7.64(d,J＝15.4Hz,1H,d-H),7.42(s,1H,8-H),7.41(d,J＝7.9Hz,2H,h-H,j-H),7.18(d,J＝7.8Hz,2H,g-H,k-H),6.79(d,J＝15.4Hz,1H,c-H),6.52(s,1H,5-H),6.33(s,2H,2’-H,6’-H),5.98(dd,J＝11.0,1.3Hz,2H,16-C 2 H),4.57(d,J＝3.8Hz,1H,1-H),4.54(dd,J＝8.9,6.5Hz,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 3 H,5’-OC 3 H),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 3 H).
13 C NMR(101MHz,CDCl 3 )δ174.23(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’-OCH 3 ),56.23(3’-OCH 3 ,5’-OCH 3 ),46.42(C-2),44.02(C-1),33.29(C-3),21.55(Ph-CH 3 ).
HRMS(ESI)calcd for C 36 H 39 N 2 O 8 [M+H] + :627.2701,found 627.2697.
example 6
Synthesis of (5R, 5aR,8aS, 9R) -9- (4-cinnamoylpiperazin-1-yl) -5- (3, 4, 5-trimethoxyphenyl) -5, 8a, 9-tetrahydrofuran [3',4':6,7] naphtho [2,3-d ] [1,3] dioxol-6 (5 aH) -one (Compound 5 a) and (5R, 5aR,8aS, 9S) -9- (4-cinnamoylpiperazin-1-yl) -5- (3, 4, 5-trimethoxyphenyl) -5, 8a, 9-tetrahydrofuran [3',4':6,7] naphtho [2,3-d ] [1,3] dioxol-6 (5 aH) -one (Compound 5 b):
17.8mg of cinnamic acid is taken and added into 5mL of dichloromethane for dissolution, then 43.2mg of 2- (7-aza-benzotriazol) -N, N, N ', N' -tetramethyl urea hexafluorophosphate, 19.5mg of diisopropylethylamine and 49.3mg of podophyllotoxin/epipodophyllotoxin piperazine intermediate (B) are sequentially added, the mixture is uniformly mixed, and then the mixture is stirred at room temperature, after the reaction is completed, the mixture is quenched by water, extracted by ethyl acetate, washed by saturated saline solution, dried by anhydrous sodium sulfate and finally the ethyl acetate is dried by spin. The crude product obtained after evaporation is separated and purified by a silica gel column chromatography method to obtain target compounds 5a and 5b.
Figure BDA0003388851080000181
The nuclear magnetic data are as follows:
25.4mg,yield 41.5％,light yellow solid,melting point 121.3℃-122.1℃.
1 H NMR(400MHz,CDCl 3 )δ7.65(d,J＝15.4Hz,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＝15.4Hz,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＝4.4Hz,2H,16-C 2 H),4.55(d,J＝4.3Hz,1H,1-H),4.42–4.10(m,2H,4-H,11a-H),3.91(d,J＝3.9Hz,1H,11b-H),3.79(s,3H,4’-OCH 3 ),3.73(s,6H,3’-OC 3 H,5’-OC 3 H),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).
13 C NMR(101MHz,CDCl 3 )δ175.17(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’-OCH 3 ),56.37(3’-OCH 3 ,5’-OCH 3 ),43.89(C-2),41.92(C-1),39.50(C-3).
HRMS(ESI)calcd for C 35 H 37 N 2 O 8 [M+H] + :613.2544,found 613.2539.
Figure BDA0003388851080000191
the nuclear magnetic data are as follows:
27.2mg,yield 44.4％,white solid,melting point 212.9℃-213.4℃.
1 H NMR(400MHz,CDCl 3 )δ7.67(d,J＝15.4Hz,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＝15.4Hz,1H,c-H),6.53(s,1H,5-H),6.35(s,2H,2’-H,6’-H),5.99(d,J＝11.0Hz,2H,16-C 2 H),4.58(d,J＝3.7Hz,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 3 H,5’-OC 3 H),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).
13 C NMR(101MHz,CDCl 3 )δ174.24(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’-OCH 3 ),56.25(3’-OCH 3 ,5’-OCH 3 ),46.45(C-2),44.04(C-1),33.31(C-3).
HRMS(ESI)calcd for C 35 H 37 N 2 O 8 [M+H] + :613.2544,found 613.2539.
example 7
Synthesis of (5R, 5aR,8aS, 9R) -9- (4- ((E) -3- (4-bromophenyl) acryloyl) piperazin-1-yl) -5- (3, 4, 5-trimethoxyphenyl) -5, 8a, 9-tetrahydrofuran [3',4':6,7] naphtho [2,3-d ] [1,3] dioxol-6 (5 aH) -one (Compound 6 a) and (5R, 5aR,8aS, 9S) -9- (4- ((E) -3- (4-bromophenyl) acryloyl) piperazin-1-yl) -5- (3, 4, 5-trimethoxyphenyl) -5, 8a, 9-tetrahydrofuran [3',4':6,7] naphtho [2,3-d ] [1,3] dioxol-6 (Compound 6 b):
27.2mg of 4-bromocinnamic acid is added into 5mL of dichloromethane to be dissolved, then 43.2mg of 2- (7-azabenzotriazol) -N, N, N ', N' -tetramethylurea hexafluorophosphate, 19.5mg of diisopropylethylamine and 49.3mg of podophyllotoxin/epipodophyllotoxin piperazine intermediate (B) are sequentially added, the mixture is uniformly mixed, and then stirred at room temperature, after the reaction is completed, the mixture is quenched with water to react, extracted with ethyl acetate, washed with saturated saline solution, dried with anhydrous sodium sulfate, and finally the ethyl acetate is spun dry. The crude product obtained after evaporation is separated and purified by a silica gel column chromatography method to obtain target compounds 6a and 6b.
Figure BDA0003388851080000201
The nuclear magnetic data are as follows:
27.6mg,yield 39.9％,light yellow solid,melting point 243.7℃-244.2℃.
1 H NMR(400MHz,CDCl 3 )δ7.66(d,J＝15.5Hz,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＝15.4Hz,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＝7.1,1.3Hz,2H,16-C 2 H),4.56(d,J＝5.4Hz,1H,1-H),4.43–4.27(m,2H,4-H,11a-H),3.92(d,J＝3.9Hz,1H,11b-H),3.80(s,3H,4’-OCH 3 ),3.74(s,6H,3’-OC 3 H,5’-OC 3 H),3.68–3.41(m,4H,4×a-H),3.14(dd,J＝14.0,5.4Hz,1H,2-H),2.97–2.67(m,1H,3-H),2.68–2.32(m,4H,4×a-H).
13 C NMR(101MHz,CDCl 3 )δ175.13(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’-OCH 3 ),56.40(3’-OCH 3 ,5’-OCH 3 ),43.92(C-2),41.95(C-1),39.53(C-3).
HRMS(ESI)calcd for C 35 H 36 BrN 2 O 8 [M+H] + :691.1650,found 691.1643.
Figure BDA0003388851080000211
the nuclear magnetic data are as follows:
30.3mg,yield 43.8％,light yellow solid,melting point 199.2℃-199.6.
1 H NMR(400MHz,CDCl 3 )δ7.57(d,J＝15.4Hz,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＝15.4Hz,1H,c-H),6.50(s,1H,5-H),6.31(s,2H,2’-H,6’-H),5.96(dd,J＝10.0,1.4Hz,2H,16-C 2 H),4.55(d,J＝3.7Hz,1H,1-H),4.51(dd,J＝8.6,6.4Hz,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 3 H,5’-OC 3 H),3.68–3.52(m,2H,2×a-H),2.80(dd,J＝9.4,3.3Hz,2H,2-H,3-H),2.70–2.50(m,4H,4×a-H).
13 C NMR(101MHz,CDCl 3 )δ174.21(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’-OCH 3 ),56.28(3’-OCH 3 ,5’-OCH 3 ),46.47(C-2),44.06(C-1),33.34(C-3).
HRMS(ESI)calcd for C 35 H 36 BrN 2 O 8 [M+H] + :691.1650,found 691.1640.
screening 12 podophyllotoxin/epipodophyllotoxin derivatives for anticancer activity.
Antitumor activity experimental procedure:
(1) Inoculation of cells
a. Cells are subjected to pancreatin digestion and then are centrifugally collected, 1mL of DMEM complete medium is used for resuspension, and single cell suspension is prepared by full blowing;
b. 50. Mu.L of the cell suspension was aspirated, diluted 8-fold with 350. Mu.L of PBS buffer, and cell counted with a cell counting plate to prepare 3X 10 according to the result of the counting 4 individual/mL single cell suspension;
c. inoculating the cells into a 96-well plate, wherein each well contains 100 mu L of single cell suspension, so that the number of inoculated cells is 3000;
d. placing a 96-well plate into CO 2 In an incubator at 37℃with 5% CO 2 Cells were cultured for 24 hours under saturated humidity.
(2) Treatment of cells with Compounds
a. After the cells are cultured for 24 hours, preparing compounds with different concentration gradients according to the difference of the inhibition effects of different compounds on the cells;
b. from CO 2 Taking out the 96-well plate from the incubator, carefully sucking the culture medium in the wells, adding 100 mu L of compound solution, and repeating 3 wells for each concentration;
c. put the 96-well plate back to CO 2 The incubator was continued to cultivate for 72 hours.
(3) MTT assay
a. After cell culture for 70h, 10. Mu.L of MTT solution at a concentration of 5mg/mL was added to each well, and the 96-well plate was returned to CO 2 Culturing in an incubator for 2 hours;
b. taking out the 96-well plate after two hours, carefully sucking out the culture medium, adding 150 mu L of DMSO solution into each well, fully dissolving blue-violet formazan crystals generated by the reaction, and placing the mixture in a small oscillator for oscillation for 5 minutes;
c. the absorbance of each well was measured at OD 492nm of the enzyme-linked immunosorbent assay.
IC of each compound against non-small cell lung cancer A549, breast cancer MCF-7, prostate cancer PC-3 and cervical cancer HeLa, and normal human liver cell HL-7702 50 As shown in table 1.
TABLE 1 results of anti-tumor Activity of Compounds (1 a-6a,1b-2 b)
Figure BDA0003388851080000221
Figure BDA0003388851080000231
According to the invention, podophyllotoxin/epipodophyllotoxin is taken as a substrate, and a piperazine group is introduced into the 4-position of a C ring through nucleophilic substitution reaction, so that a podophyllotoxin/epipodophyllotoxin-piperazine intermediate is obtained. The intermediate is a diastereoisomer mixture comprising the product in the 4-R, S configuration of the C ring, and is a pair of racemates. Then, the obtained podophyllotoxin/epipodophyllotoxin-piperazine intermediate is taken as a substrate, reacts with a cinnamic acid compound to obtain cinnamic acid derivatives of the podophyllotoxin/epipodophyllotoxin, and carries out structural identification on the compounds through physicochemical properties and various spectrum methods. In the screening of anticancer activity, etoposide is used as a positive control, and the inhibition effect of the etoposide 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 is measured by adopting an MTT method. The compounds 1a, 1b, 2b, 3b, 4a, 4b, 5b and 6b have better effect, and have IC (integrated circuit) on 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 50 The values were all lower than the positive control drug etoposide. Wherein, the compounds 1b, 3b, 4b and 5b have optimal anti-tumor activity, and are IC for 4 different human cancer cell lines 50 The values were all below 1. Mu.M.
The foregoing description of the preferred embodiment of the invention is merely illustrative of the invention and is not intended to be limiting. It will be appreciated by persons skilled in the art that many variations, modifications, and even equivalents may be made thereto without departing from the spirit and scope of the invention as defined in the appended claims.

Claims (10)

1. A podophyllotoxin/epipodophyllotoxin derivative characterized by the general structural formula:
Figure FDA0003388851070000011
wherein R is phenyl or phenyl containing substituent, and the configuration of the 4-position is R or S.
2. A podophyllotoxin/epipodophyllotoxin derivative according to claim 1, which is any one of the following compounds:
Figure FDA0003388851070000012
/>
Figure FDA0003388851070000021
wherein,,
compound 1a is (5 r,5ar,8as,9 r) -9- (4- ((E) -3- (4-nitrophenyl) acryloyl) piperazin-1-yl) -5- (3, 4, 5-trimethoxyphenyl) -5, 8a, 9-tetrahydrofuran [3',4':6,7] naphtho [2,3-d ] [1,3] dioxol-6 (5 aH) -one;
compound 1b was (5 r,5ar,8as,9 s) -9- (4- ((E) -3- (4-nitrophenyl) acryloyl) piperazin-1-yl) -5- (3, 4, 5-trimethoxyphenyl) -5, 8a, 9-tetrahydrofuran [3',4':6,7] naphtho [2,3-d ] [1,3] dioxol-6 (5 aH) -one;
compound 2a is (5 r,5ar,8as,9 r) -9- (4- ((E) -3- (3, 4-dichlorophenyl) acryloyl) piperazin-1-yl) -5- (3, 4, 5-trimethoxyphenyl) -5, 8a, 9-tetrahydrofuran [3',4':6,7] naphtho [2,3-d ] [1,3] dioxol-6 (5 aH) -one;
compound 2b was (5 r,5ar,8as,9 s) -9- (4- ((E) -3- (3, 4-dichlorophenyl) acryloyl) piperazin-1-yl) -5- (3, 4, 5-trimethoxyphenyl) -5, 8a, 9-tetrahydrofuran [3',4':6,7] naphtho [2,3-d ] [1,3] dioxol-6 (5 aH) -one;
compound 3a is (5 r,5ar,8as,9 r) -9- (4- ((E) -3- ((4-trifluoromethyl) phenyl) acryloyl) piperazin-1-yl) -5- (3, 4, 5-trimethoxyphenyl) -5, 8a, 9-tetrahydrofuran [3',4':6,7] naphtho [2,3-d ] [1,3] dioxol-6 (5 aH) -one;
compound 3b was (5 r,5ar,8as,9 s) -9- (4- ((E) -3- ((4-trifluoromethyl) phenyl) acryloyl) piperazin-1-yl) -5- (3, 4, 5-trimethoxyphenyl) -5, 8a, 9-tetrahydrofuran [3',4':6,7] naphtho [2,3-d ] [1,3] dioxol-6 (5 aH) -one;
compound 4a is (5 r,5ar,8as,9 r) -9- (4- ((E) -3- (4-methylphenyl) acryloyl) piperazin-1-yl) -5- (3, 4, 5-trimethoxyphenyl) -5, 8a, 9-tetrahydrofuran [3',4':6,7] naphtho [2,3-d ] [1,3] dioxol-6 (5 aH) -one;
compound 4b was (5 r,5ar,8as,9 s) -9- (4- ((E) -3- (4-methylphenyl) acryloyl) piperazin-1-yl) -5- (3, 4, 5-trimethoxyphenyl) -5, 8a, 9-tetrahydrofuran [3',4':6,7] naphtho [2,3-d ] [1,3] dioxol-6 (5 aH) -one;
compound 5a is (5 r,5ar,8as,9 r) -9- (4-cinnamoyl piperazin-1-yl) -5- (3, 4, 5-trimethoxyphenyl) -5, 8a, 9-tetrahydrofuran [3',4':6,7] naphtho [2,3-d ] [1,3] dioxol-6 (5 aH) -one;
compound 5b is (5 r,5ar,8as,9 s) -9- (4-cinnamoyl piperazin-1-yl) -5- (3, 4, 5-trimethoxyphenyl) -5, 8a, 9-tetrahydrofuran [3',4':6,7] naphtho [2,3-d ] [1,3] dioxol-6 (5 aH) -one;
compound 6a is (5 r,5ar,8as,9 r) -9- (4- ((E) -3- (4-bromophenyl) acryloyl) piperazin-1-yl) -5- (3, 4, 5-trimethoxyphenyl) -5, 8a, 9-tetrahydrofuran [3',4':6,7] naphtho [2,3-d ] [1,3] dioxol-6 (5 aH) -one;
compound 6b is (5R, 5aR,8aS, 9S) -9- (4- ((E) -3- (4-bromophenyl) acryloyl) piperazin-1-yl) -5- (3, 4, 5-trimethoxyphenyl) -5, 8a, 9-tetrahydrofuran [3',4':6,7] naphtho [2,3-d ] [1,3] dioxol-6 (5 aH) -one.
3. A process for the preparation of a podophyllotoxin/epipodophyllotoxin derivative according to claim 1 or 2, comprising the steps of:
s1: dissolving podophyllotoxin powder in acetonitrile, adding sodium iodide powder, cooling the reaction system to 0 ℃, stirring, and dropwise adding methanesulfonic acid;
s2: after the reaction system in the step S1 is completely reacted, sequentially adding barium carbonate powder and triethylamine, adding piperazine powder when the pH value of the system reaches 7-8, adding water into the reaction system for quenching after overnight reaction at room temperature, and filtering;
s3: extracting, washing, drying an organic phase, spin-drying and evaporating a crude product obtained after the reaction system obtained in the step S2 to obtain a podophyllotoxin/epipodophyllotoxin piperazine intermediate product;
s4: adding a carboxylic acid compound into dichloromethane for dissolution, then sequentially adding the podophyllotoxin/epipodophyllotoxin piperazine intermediate product obtained in the step S3, 2- (7-aza-benzotriazol) -N, N, N ', N' -tetramethyl urea hexafluorophosphate and diisopropylethylamine, stirring at room temperature, after the reaction is completed, quenching the reaction with water, extracting, washing, drying an organic phase, spin-drying, and separating and purifying a crude product obtained after evaporating to dryness to obtain the podophyllotoxin/epipodophyllotoxin derivative.
4. A process for producing a podophyllotoxin/epipodophyllotoxin derivative according to claim 3, wherein the carboxylic acid compound in step S2 is cinnamic acid or an analogue of cinnamic acid.
5. A process for producing a podophyllotoxin/epipodophyllotoxin derivative according to claim 3, wherein the extraction solvent used in the steps S3 and S4 is ethyl acetate.
6. A process for producing a podophyllotoxin/epipodophyllotoxin derivative according to claim 3, wherein the washing organic phase in the steps S3 and S4 is saturated saline.
7. A process for producing a podophyllotoxin/epipodophyllotoxin derivative according to claim 3, wherein the dried organic phase in the steps S3 and S4 is anhydrous sodium sulfate.
8. A process for preparing a podophyllotoxin/epipodophyllotoxin derivative according to claim 3, wherein the column chromatography eluent in step S3 is dichloromethane and methanol in a volume ratio of dichloromethane: methanol=1 to 4:1.
9. a process for preparing a podophyllotoxin/epipodophyllotoxin derivative according to claim 3, wherein the column chromatography eluent in step S4 is petroleum ether and ethyl acetate in a volume ratio of petroleum ether: ethyl acetate = 1:1 to 2.
10. Use of the podophyllotoxin/epipodophyllotoxin derivative according to claim 1 or 2 for the preparation of an antitumor drug.
CN202111458566.5A 2021-12-02 2021-12-02 Podophyllotoxin/epipodophyllotoxin derivative, preparation method and application thereof Pending CN116217585A (en)

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