Dichloroacetic acid coupled diphenylethane compound as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of medicine synthesis, and particularly relates to a dichloroacetic acid coupled diphenylethane compound as well as a preparation method and application thereof. The structure of the dichloroacetic acid coupled diphenylethane compound is shown as a general formula (I), R1, R2 and R3 are respectively selected from but not limited to any one of-OH,-OMe and H, R4 is selected from but not limited to any one of-OMe and-OEt, R5 and R6 are selected from chlorine atoms or fluorine atoms, m is 0, and n is a positive integer selected from 1-8 or 11-12. The dichloroacetic acid coupled diphenylethane compound disclosed by the invention is stable in structure, low in toxicity, simple and convenient in preparation process and high in yield, does not need to be stored in a dark place, has obvious anti-tumor activity on various tumor cells cultured in vitro, and is obvious in in-vivo anti-tumor effect. General formula (I): # imgabs0 #

Description

[0001] This invention is a divisional application based on the Chinese invention patent application with application date of December 31, 2021, application number CN202111665548.4, and invention name “Dichloroacetic acid coupled diphenylethane compounds, preparation methods and applications thereof”. Technical Field

[0002] The invention belongs to the technical field of drug synthesis, and in particular relates to a dichloroacetic acid-coupled diphenylethane compound and a preparation method and application thereof. Background Art

[0003] According to data provided by the World Health Organization, cancer is still the second leading cause of death in the world. Due to the shortcomings of traditional cancer treatments, targeted tumor drugs have become a hot topic of research. Targeted tumor drugs use a single compound to act on two or more different biological targets to enhance the effectiveness of tumor inhibition while achieving high selectivity and low toxicity. However, the efficacy of targeted tumor drugs varies. Although some of them have strong specificity, their tumor inhibition efficacy needs to be improved. Modifying an existing anti-tumor drug in order to obtain a more active drug is usually a high-risk project that costs hundreds of millions of dollars, takes a ten-year R&D cycle, and faces various eliminations such as efficacy and approval.

[0004] In 1956, German chemist Warburg discovered that even under aerobic conditions, cancer cells still tend to use aerobic glycolysis rather than the oxidative phosphorylation pathway used by normal cells. This special phenomenon is called the "Warburg effect". Studies have shown that pyruvate dehydrogenase kinase (PDK) is a necessary substance for glycolysis, so inhibiting this enzyme will reverse the Warburg effect, and dichloroacetic acid (DCA) is a PDK inhibitor. Sodium dichloroacetate is a small molecule compound that has been used to treat lactic acidosis for many years. In recent years, studies have found that DCA can act on the energy metabolism pathway of tumor cells by inhibiting PDK, promoting tumor cell oxidative phosphorylation, inducing apoptosis, and inhibiting tumor growth. Through continuous research, there are now several PDK inhibitors containing dichloroacetic acid structures, such as: VER-246608, 2-Chloro-N-(2-methylphenyl)propanamide, Mitaplatin, etc. However, there are some problems in the clinical application of DCA. For example, the efficacy of DCA is not high in clinical trials, and some patients who use the drug experience symptoms such as pain and numbness. Currently, there is no legal DCA drug for sale in my country. Therefore, it is necessary to find new DCA-derived candidate drug compounds with high anti-tumor activity and low toxicity. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a new compound with anti-tumor activity and a preparation method and application thereof.

[0006] Diphenylethane compounds, such as Erianin, are tubulin-targeted drugs with good anti-tumor activity. Dichloroacetic acid compounds have the activity of PDK inhibitors, but their efficacy is not high in clinical experiments, and some patients may also experience pain, numbness or toxic side effects. This study modifies the structure of dichloroacetic acid to improve anti-tumor activity and reduce toxic side effects while maintaining its targeting effect. Previous studies have attempted to modify or transform dichloroacetic acid and its derivatives using many other compound groups (including natural products), but the results were not ideal, including unstable compounds, short half-life, poor solubility, too strong toxicity, poor anti-tumor activity, long metabolic time, too high preparation cost, and too harsh process conditions. The present invention finds that diphenylethane compounds such as Erianin, etc., combined with dichloroacetic acid in a certain way, have good anti-tumor activity and low toxicity. Therefore, the present invention combines natural product diphenylethane compounds with energy-targeted drug sodium dichloroacetate in order to obtain anti-tumor active compounds with multiple targets.

[0007] The present invention provides a dichloroacetic acid coupled diphenylethane compound, the structure of which is as shown in the general formula (I):

[0008]

[0009] in,

[0010] R1, R2 and R3 are independently selected from any one of -OH, -OMe, H, R4 is selected from any one of -OMe, -OEt, R5 and R6 are selected from chlorine atoms or fluorine atoms, m is 0, and n can be selected from 1, 2, 3, 4, 6, 7, 8, 11 or 12.

[0011] Optionally, R4 is selected from -OMe or -OEt.

[0012] The present invention also provides a pharmaceutical composition, wherein the active ingredient of the pharmaceutical composition is the above-mentioned dichloroacetic acid coupled diphenylethane compound.

[0013] Optionally, the pharmaceutical composition is composed of an effective dose of the above-mentioned dichloroacetic acid-coupled diphenylethane compound and a pharmaceutically acceptable carrier.

[0014] Optionally, the pharmaceutical preparation is selected from but not limited to the following dosage forms: lyophilized powder, powder, injection, liposome, emulsion, microcapsule, suspension or solution for intravenous administration; granules, tablets, capsules or syrup for oral administration; or suppository.

[0015] Optionally, the pharmaceutical composition is a drug that inhibits tumor cell proliferation, growth or damage, or eliminates tumor cells.

[0016] Optionally, the pharmaceutical composition is suitable for humans or animals, wherein the animals are selected from but not limited to monkeys, rats, mice, rabbits, cats, dogs, cows, horses, sheep or pigs.

[0017] On the other hand, the present invention also provides a method for preparing the above-mentioned dichloroacetic acid coupled diphenylethane derivative. When one or more of R1, R2 and R3 is -OH, the synthetic route is as follows:

[0018]

[0019] The specific steps are as follows:

[0020] (1) When more than one of R1, R2 and R3 is -OH, first The hydroxyl group in is protected with benzyl chloride to obtain the corresponding benzyl protected product;

[0021] (2) The benzyl protected product and triphenylphosphine ylide Wittig reaction was performed to obtain benzyl-protected stilbene compounds;

[0022] (3) reducing and deprotecting the benzyl-protected stilbene compound to obtain the corresponding diphenylethane compound;

[0023] (4) When dichloroacetic acid is directly coupled with diphenylethane, It can be condensed with the corresponding diphenylethane compounds to obtain a series of dichloroacetic acid coupled diphenylethane compounds.

[0024] In the above preparation method, when R1, R2 and R3 are not -OH, the step of protecting with benzyl chloride is omitted and the reaction is directly carried out with A Wittig reaction occurs.

[0025] In the above preparation method, when dichloroacetic acid and diphenylethane are combined with amino acids of different chain lengths, the following steps are included:

[0026] (1) Amino acids of different chain lengths (n = 1, 2, 3, 4, 6, 7, 8, 11, 12) were prepared by dichlorothionyl to obtain the corresponding amino acid methyl ester hydrochloride (n=1, 2, 3, 4, 6, 7, 8, 11, 12);

[0027] (2) Amino acid methyl ester hydrochlorides of different chain lengths were mixed with Condensation to give compound (n=1, 2, 3, 4, 6, 7, 8, 11, 12);

[0028] (3) Compound Hydrolysis and demethylation to obtain the corresponding acid (n=1, 2, 3, 4, 6, 7, 8, 11, 12);

[0029] (4) (n=1, 2, 3, 4, 6, 7, 8, 11, 12) are condensed with corresponding diphenylethane compounds to obtain a series of dichloroacetic acid-coupled diphenylethane compounds.

[0030] On the other hand, the present invention provides the use of the dichloroacetic acid coupled diphenylethane compound in the preparation of anti-tumor drugs. The various tumors mainly include: lung cancer, non-small cell lung cancer, liver cancer, pancreatic cancer, gastric cancer, bone cancer, esophageal cancer, breast cancer, prostate cancer, testicular cancer, colon cancer, ovarian cancer, bladder cancer, cervical cancer, melanoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland cancer, sebaceous gland cancer, papillary carcinoma, papillary adenocarcinoma, cystic adenocarcinoma, cystic carcinoma, medullary carcinoma, bronchial carcinoma, osteocellular carcinoma, epithelial carcinoma, bile duct cancer, choriocarcinoma, embryonal carcinoma, seminoma, Wilms' cancer, glial cell carcinoma, astrocytoma, medulloblastoma, craniopharyngioma, ependymal tumor, pinealoma, hematoblastoma, vocal cord neuroma, meningioma, neuroblastoma, optic neuroblastoma, retinoblastoma, neurofibroma, fibrosarcoma, fibroblastoma, fibroma, fibroadenoma, fibrochondroma, fibrocystic tumor, fibromyxoma, fibroosteoma, fibromyxosarcoma, fibropapillary tumor, myxosarcoma, myxocystoma, myxochondroma, myxochondrosarcoma, myxochondrosarcoma, myxochondral fibrosarcoma, myxadenoma, myxoblastoma, liposarcoma, lipoma, lipoadenoma, lipoblastoma, lipochondroma, lipofibroma, lipovascular tumor, myxolipomas, chondrosarcomas, chondromas, chondromyomas, chordomas, chorioadenoma, chorioepithelioma, chorioblastoma, osteosarcoma, osteoblastoma, osteochondrofibroma, osteochondrosarcoma, osteochondroma, bone cystoma, osteodentinoma, osteofibroma, osteofibrosarcoma, angiosarcoma, angiolipoma, angiochondroma, angioblastoma, angiokeratoma, angioglioma, angioendothelioma, angiofibroma, angiomyoma, angiolipoma, angiolymphangioma, angiolipomyoma, angiomyolipomas, angiomyoneuromas, blood vessels Angiomyxoma, angioreticuloendothelioma, lymphangiosarcoma, lymphogranuloma, lymphangioma, lymphoma, lymphomyxoma, lymphosarcoma, lymphangiofibroma, lymphocytoma, lymphoepithelioma, lymphoblastoma, endothelioma, endothelioma, synovioma, synovialoma, mesothelioma, connective tissue tumor, Ewing's tumor, leiomyoma, leiomyosarcoma, leiomyoma, leiomyofibroma, rhabdoid tumor, rhabdomyoma, rhabdomyosarcoma, rhabdomyxoma, acute lymphatic leukemia, acute myeloid leukemia, chronic disease cell, polycythemia, lymphoma, multiple myeloma.

[0031] The present invention relates to the therapeutic use of the dichloroacetic acid coupled diphenylethane compound. The indications include tumors (cancer) or diseases caused by abnormal cell proliferation.

[0032] Optionally, the anti-tumor drug targets tumors or neoplasms arising from one or more of gastric cancer cells, lung cancer cells, cervical cancer cells, intestinal cancer cells, breast cancer cells or liver cancer cells.

[0033] Optionally, the anti-tumor drug includes a drug or agent that alleviates or inhibits the proliferation of tumor cells.

[0034] Optionally, the dichloroacetic acid-coupled diphenylethane compound is added to the culture environment of tumor cells cultured in vitro to control the number of tumor cells.

[0035] Optionally, the concentration range of the dichloroacetic acid-coupled diphenylethane compound in the cell culture medium is 0.1-10 μM (mol / L).

[0036] The present invention also provides a reagent for inhibiting cell proliferation, which contains the dichloroacetic acid coupled diphenylethane compound. Optionally, the cells are tumor cells cultured in vitro.

[0037] The experimental results show that the dichloroacetic acid-coupled diphenylethane compound of the present invention has an anti-tumor activity comparable to or better than that of Erianin or sorafenib.

[0038] Erianin, English name is 2-Methoxy-5-[2-(3,4,5-trimethoxyphenyl)ethyl]benzolol, referred to as Erianin, CAS number is 95041-90-0, chemical formula C 18 H 22 O5. Erianin is extracted from the whole plant of Dendrobium, an orchid plant. It is a Chinese herbal medicine monomer with anti-tumor effects. Although there is still some controversy about its anti-tumor effect, it has been confirmed that it has a good proliferation inhibition effect on certain tumor cells.

[0039] Sorafenib is a new multi-targeted oral drug for treating tumors. It can be used to treat gastrointestinal stromal tumors and metastatic renal cells that do not respond to or cannot tolerate standard therapies. It can selectively target the receptors of certain proteins and is believed to play a role as a molecular switch in the process of tumor growth. It has obtained the "fast track" approval for the above indications granted by the FDA in the United States and has been used as a commonly used anti-tumor drug in clinical practice in my country.

[0040] The tumor treating drug provided by the invention contains an effective dose of dichloroacetic acid coupled diphenylethane compound or dichloroacetic acid coupled diphenylethane compound and a pharmaceutically acceptable carrier.

[0041] In the present invention, LD is the abbreviation of lethal dose, which refers to the minimum dose of a substance that can kill an organism. Depending on the dosage of the drug, the overall mortality rate of the experimental subjects varies, and can usually be divided into different dosages such as half lethal dose, absolute lethal dose, minimum lethal dose and maximum tolerated dose.

[0042] IC used in the present invention 50 The half-inhibitory concentration (or half inhibition rate) refers to the concentration of the inhibitor when the "reaction" is inhibited by half. In terms of anti-tumor, it can be understood as the concentration of the drug used when the number of tumor cells is halved.

[0043] As used in the present invention, "therapeutically effective amount" or "effective dose" refers to a dose of dichloroacetic acid-coupled diphenylethane compounds that is non-toxic but sufficient to achieve the desired biological, therapeutic and / or preventive results. The result can be a reduction and / or alleviation of the signs, symptoms or inducements of the disease, or any other desired change in the biological system. For example, an "effective dose" for therapeutic use is the amount of dichloroacetic acid-coupled diphenylethane compounds as disclosed herein, themselves, or a composition containing the dichloroacetic acid-coupled diphenylethane compounds herein, required to produce a significant improvement in the clinical disease. The appropriate effective dose in any individual case can be determined by a person of ordinary skill in the art using conventional tests. Therefore, the effective dose of dichloroacetic acid-coupled diphenylethane compounds used in the present invention refers to an amount that can achieve the desired oral use of dichloroacetic acid-coupled diphenylethane compounds.

[0044] "Pharmaceutically acceptable" means that a substance is not biologically or otherwise undesirable, i.e., the substance can be administered to a subject without causing undesirable biological effects or interacting in a deleterious manner with any ingredients of a composition in which it is contained.

[0045] The term "treatment" and its grammatical synonyms include achieving a therapeutic effect and / or a prophylactic effect. A therapeutic effect means the alleviation or eradication of the underlying condition for which treatment is being performed. A therapeutic effect is also achieved by the eradication or alleviation of one or more physiological symptoms associated with the underlying condition, such that an improvement is observed in the patient, despite the fact that the patient may still be afflicted with the underlying condition. For a prophylactic effect, the methods of the present invention may be practiced on patients clinically diagnosed with a tumor, or on patients reporting one or more physiological symptoms of these conditions, although this condition may not have been diagnosed, or the compositions of the present invention may be administered to these patients.

[0046] As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier used for treatment, i.e., administration, including various excipients and diluents. The term refers to pharmaceutical carriers that are not necessary active ingredients themselves and have no excessive side effects after administration. Suitable carriers are those known to those of ordinary skill in the art. Pharmaceutically acceptable carriers in drugs may include liquids such as water, saline, glycerol, and tween. In addition, auxiliary substances such as disintegrants, wetting agents, emulsifiers, pH buffer substances, etc. may also be present in these carriers.

[0047] The drug for treating tumors provided by the present invention can be prepared into various dosage forms according to different administration routes. These dosage forms can be administered in one of the following ways: oral administration, topical administration (skin patch, subcutaneous implant), parenteral administration, such as subcutaneous and intramuscular administration, or administration via an explanted reservoir. Among them, oral administration is preferred for treating tumors.

[0048] In the present invention, when the dichloroacetic acid-coupled diphenylethane compounds of the present invention are used for therapeutic purposes or as medicines, a safe and effective amount of the dichloroacetic acid-coupled diphenylethane compounds is administered to rats, wherein the safe and effective amount for treating tumors is usually at least about 80 mg / kg body weight, and in most cases does not exceed 100 mg / kg body weight, and a preferred dose is about 40-80 mg / kg body weight; of course, the specific dose is based on humans as an example, and factors such as the patient's age, weight, natural health status, nutritional status, compound activity strength, administration, and metabolic rate must also be considered, which are all knowledge and skills within the skill range of those skilled in the art and will not be elaborated herein.

[0049] Before administration, the dichloroacetic acid-coupled diphenylethane compound can be mixed with sodium carboxymethylcellulose, physiological saline or any other suitable solution known to those skilled in the art. The dichloroacetic acid-coupled diphenylethane compound preparation can be administered in the form of solid (freeze-dried) or liquid preparation as required.

[0050] The present invention combines sodium dichloroacetate, a representative drug of energy-targeted PDK (phosphatidylinositol-dependent kinase) inhibitors, with diphenylethane compounds with VEGFR2 targeting. In the ethoxy diphenylethane compounds of the present invention, the two benzene rings are connected by a single bond, which can greatly improve the stability of the drug and reduce the toxicity. The preparation process is simpler and does not require column chromatography separation. The process yield is significantly improved, the loss of raw materials is greatly reduced, and the unit synthesis process cost is also greatly reduced. The stability of the drug is improved, and it does not need to be stored in the dark, which brings great convenience in storage and practical application. The ethoxy diphenylethane compounds of the present invention have obvious anti-tumor activity on a variety of tumor cells cultured in vitro, and can reach or even exceed the anti-tumor effect of Erianin or Sonafenib.

[0051] Specific example method

[0052] The invention discloses a dichloroacetic acid coupled diphenylethane compound having a structure as shown in general formula (I), a preparation method and an application thereof. The dichloroacetic acid coupled diphenylethane compound of the invention is coupled with dichloroacetic acid at the 4' position of the B aromatic ring of diphenylethane, and at the same time, the 3, 4 and 5 positions of its A ring are respectively replaced by methoxy, hydroxyl or fluorine atoms; the 3' position of its B ring is replaced by methoxy, ethoxy, trifluoromethyl, difluoromethyl or difluoroethyl; and the chlorine atom of dichloroacetic acid at the 4' position of its B ring is replaced by a fluorine atom or a bromine atom. In addition, dichloroacetic acid and diphenylethane are combined with amino acids of different chain lengths to obtain a compound with good anti-tumor activity.

[0053] The technical solution of the present invention is further explained below in conjunction with preferred examples.

[0054] Example 1. Preparation of 2-methoxy-5-(3,4,5-trimethoxyphenethyl)phenyl 2,2-dichloroacetate (referred to as 7a)

[0055]

[0056] Under nitrogen protection, add 10g (19.05mmol) of 3-benzyloxy-4-methoxybenzyl chloride triphenylphosphonium salt to a 100mL three-necked flask equipped with a thermometer and dried, add 50mL of anhydrous tetrahydrofuran, put it in an ice-salt bath, and after the temperature cools to 0°C, slowly add 3.22g (28.57mmol) of potassium tert-butoxide, and continue stirring for 30min. Then slowly add a tetrahydrofuran solution (10mL) of 3,4,5-trimethoxybenzaldehyde, and the addition is completed in about 20min. Then the reaction device is naturally slowly heated to room temperature and the reaction is continued for 4 hours. Close the reaction device, remove tetrahydrofuran by rotary evaporation, add 60mL of water and 100mL of ethyl acetate, extract and separate the ethyl acetate layer, and extract the water layer with 60mL of ethyl acetate × 2; combine the ethyl acetate layers and dry them over anhydrous sodium sulfate. Remove the solvent by vacuum distillation to obtain a yellow solid, and separate 5.89g of cis-trans product by rapid column chromatography. Then mix it with palladium carbon, add 24mL of ethanol, replace with hydrogen three times, and stir the reaction for 6h. Use diatomaceous earth to filter palladium carbon, separate by rapid paper chromatography, and obtain 3.21g product 5a. Take 500mg of Erianin, add 8mL of dry dichloromethane and 0.327mL of triethylamine, and stir in an ice-salt bath for 10min. Then slowly add 0.166mL of dichloroacetyl chloride dropwise, and continue to react for 1.5 hours. After the reaction is completed, add 20mL of water to quench, 40mL of dichloromethane, extract and separate the organic phase, and extract the water layer with dichloromethane 20×2. Combine the organic phases, dry over anhydrous sodium sulfate, and distill under reduced pressure to obtain a yellow oil, which is separated by rapid paper chromatography to obtain 586mg of white solid with a yield of 86.91%.

[0057] 1H NMR (400MHz, CDCl3) δ7.01(d,J=8.3Hz,1H),6.93(s,1H),6.89(d,J=8.4Hz,1H),6.33(s,2H),6.19(s,1H),3.81(d,J=5.0Hz,12H),2.84(s,4H); 13 C NMR (101MHz, CDCl3) δ162.68,153.15,149.04,138.93,137.10,136.35,134.54,1 27.77,121.99,112.72,105.53,77.33,64.09,60.92,56.19,56.12,38.26,36.98.

[0058] The above results show that this method successfully prepared 2-methoxy-5-(3,4,5-trimethoxyphenethyl)phenyl 2,2-dichloroacetate.

[0059] Example 2. Preparation of 2-ethoxy-5-(3,4,5-trimethoxyphenethyl)phenyl 2,2-dichloroacetate (7b)

[0060]

[0061] The synthesis method was the same as 7a, except that 3-benzyloxy-4-ethoxybenzyl chloride triphenylphosphonium salt was used instead of 3-methoxy-4-benzyloxybenzyl chloride triphenylphosphonium salt to obtain 526 mg of yellow oil 7b with a yield of 78.88%.

[0062] 1 H NMR (400MHz, CDCl3) δ6.98(dd,J=8.3,1.8Hz,1H),6.92(d,J=1.8Hz,1H),6.88(d,J=8.3Hz,1H),6.33( s,2H),6.19(s,1H),4.03(q,J=7.0Hz,2H),3.82(d,J=5.3Hz,9H),2.84(s,4H),1.37(t,J=7.0Hz,3H); 13 C NMR (101MHz, CDCl3) δ162.69,153.11,148.35,137.23,134.41,127.71,121.92,113.71,105.58,64.77,64.08,60.95,56.14,38.25,37.00.

[0063] Example 3. Preparation of 2-methoxy-4-(3,4,5-trimethoxyphenethyl)phenyl 2,2-dichloroacetate (7c)

[0064]

[0065] The synthesis method was the same as 7a, except that 3-methoxy-4-benzyloxybenzyl chloride triphenylphosphonium salt was used instead of 3-benzyloxy 4-methoxy-benzyl chloride triphenylphosphonium salt to obtain 565 mg of yellow oil 7c with a yield of 83.83%.

[0066] 1H NMR (400MHz, CDCl3) δ7.07 (d, J=7.5Hz, 1H), 6.75 (dd, J=7.5, 1.4Hz, 1H), 6.71 (t, J=1.2Hz,1H),6.47(s,2H),6.27(s,1H),3.89(s,2H),3.83(s,6H),,2.84(s,4H); 13 C NMR (101MHz, CDCl3) δ165.17,153.21,153.19,150.72,150.72,150.67,150.66,141.99,141.97,141.94 ,141.92,136.80,136.75,136.71,136.31,135.64,135.59,122.06,122.04,122.02,122.00,121.98,12 1.26,121.20,121.18,121.15,113.50,113.48,113.46,113.44,113.41,105.94,105.92,105.89,105.87,105.84,63.17,60.79,56.12,56.05,37.64,37.62,37.61,37.59,37.55,37.02,36.99,36.96,36.94.

[0067] Example 4. Preparation of 3-(3,4,5-trimethoxyphenethyl)phenyl 2,2-dichloroacetate (7d)

[0068]

[0069] The synthesis method was the same as 7a, except that 3-benzyloxybenzyl chloride triphenylphosphonium salt was used instead of 3-benzyloxy 4-methoxy-benzyl chloride triphenylphosphonium salt to obtain 506 mg of yellow oil 7d with a yield of 73.12%.

[0070] 1H NMR (400MHz, CDCl3) δ7.15(dt,J=7.4,1.0Hz,1H),7.09–7.03(m,1H),6.48(d,J=1.1Hz,1H),3.82(d,J=2.7Hz,4H),2.89(m,2H); 13C NMR (101MHz, CDCl3) δ163.64,153.10,150.70,150.68,150.64,150.62,136.71,136.38,136.36,134.59,134.56,134.53,130.44,130 .41,130.38,121.74,121.73,121.72,121.71,106.55,106.52,63.07,60.80,56.12,38.09,38.06,38.04,37.66,37.63,37.62,37.60.

[0071] Example 5. Preparation of 4-(3,4,5-trimethoxyphenethyl)phenyl 2,2-dichloroacetate (7e)

[0072]

[0073] The synthesis method was the same as 7a, except that 4-benzyloxybenzyl chloride triphenylphosphonium salt was used instead of 3-benzyloxy 4-methoxy-benzyl chloride triphenylphosphonium salt to obtain 513 mg of yellow oil 7e with a yield of 74.10%.

[0074] 1H NMR (400MHz, CDCl3) δ7.15(dt,J=7.4,1.0Hz,1H),7.09–7.03(m,1H),6.48(d,J=1.0Hz,1H),3.82(d,J=2.0Hz,4H),2.89(m,2H); 13 C NMR (101MHz, CDCl3) δ163.64,153.11,153.09,150.70,150.68,150.64,150.62,136.7 6,136.71,136.66,136.37,134.56,130.45,130.42,130.40,130.39,130.37,121.77, 121.76,121.75,121.74,121.71,121.70,121.69,121.68,106.59,106.56,106.54,106.51,106.49,63.07,60.80,56.12,38.09,38.07,38.06,38.03,37.65,37.63,37.60.

[0075] Example 6. Preparation of 5-(3,5-dimethoxyphenethyl)-2-methoxyphenyl 2,2-dichloroacetate (7f)

[0076]

[0077] The synthesis method was the same as 7a, except that 3,5-dimethoxybenzaldehyde was used instead of 3,4,5-trimethoxybenzaldehyde to obtain 557 mg of yellow oil 7f with a yield of 80.45%.

[0078] 1 H NMR (400MHz, CDCl3) δ6.92(m,1H),6.38(m,1H),3.88(s,1H),3.77(s,2H),2.91(m,2H); 13 C NMR (101MHz, CDCl3) δ163.83, 160.88 (dd, J=2.7, 1.4Hz), 149.52 (dd, J=7.1, 2.6Hz), 143.73 (dd d,J=5.0,3.7,1.8Hz),141.13(dd,J=7.2,1.0Hz),134.52(dt,J=7.4,1.7Hz),123.20(dddt,J=9 .0,6.3,3.2,1.4Hz),120.37(ddd,J=6.5,3.6,1.4Hz),112.56(tt,J=7.4,2.5Hz),107.29(dt,J =6.0, 2.9Hz), 98.06 (td, J = 5.9, 2.4Hz), 63.17, 56.00, 55.31, 37.41 (ddd, J = 10.3, 4.6, 2.3Hz).

[0079] Example 7. 5-(3,4-dimethoxyphenethyl)-2-methoxyphenyl 2,2-dichloroacetate (7 g)

[0080]

[0081] The synthesis method was the same as 7a, except that 3,4-dimethoxybenzaldehyde was used instead of 3,4,5-trimethoxybenzaldehyde. 571 mg of yellow oil was obtained, with a yield of 82.47%.

[0082] 1 H NMR (400MHz, CDCl3) δ6.92 (m, 4H), 6.81 (m, 4H), 6.27 (s, 1H), 3.88 (s, 3H), 3.83 (d, J = 1.3Hz, 8H), 2.85 (d, J = 1.5Hz, 5H); 13C NMR (101MHz, CDCl3) δ163.83,149.75,149.73,149.69,149.54,149.51,147.8 6,147.84,141.50,141.43,135.16,134.64,134.58,123.24,123.22,123.20,1 23.18,121.50,121.48,121.45,120.41,120.40,120.38,120.36,120.35,120.32,112.62,112.50,112.10,63.17,56.00,55.91,55.88,37.76,37.69,37.30.

[0083] Example 8. Preparation of 2-methoxy-5-(3-methoxyphenethyl)phenyl 2,2-dichloroacetate (7h)

[0084]

[0085] The synthesis method was the same as 7a, except that 3-methoxybenzaldehyde was used instead of 3,4,5-trimethoxybenzaldehyde to obtain 684 mg of yellow oil 7h with a yield of 95.80%.

[0086] 1 H NMR (400MHz, CDCl3) δ7.23(m,1H),6.92(m,1H),6.90(m,2H),6.79(m,3H),6.27(s,1H),3.87(s,2H),3.78(s,2H),2.89(m,4H); 13 C NMR (101MHz, CDCl3) δ163.86,159.73,159.68,159.66,149.67,149.65,143.39,143.37,143.36,143.35,143.34,141.97 4,134.64,134.62,134.58,129.43,129.40,129.36,129.35,129.34,123.66,123.58,120.48,120.47,120.39,115.09,115.06,115.04,115.02,114.98,114.46,114.34,113.97,113.83,112.53,112.49,112.48,112.45,63.17,56.00,36.78,36.32.

[0087] Example 9. Preparation of 2-methoxy-5-(4-methoxyphenethyl)phenyl 2,2-dichloroacetate (7i)

[0088]

[0089] The synthesis method was the same as 7a, except that 3-methoxybenzaldehyde was used instead of 3,4,5-trimethoxybenzaldehyde to obtain 659 mg of yellow oil 7i with a yield of 92.20%.

[0090] 1 H NMR (400MHz, CDCl3) δ7.23(m,1H),6.91(m,1H),6.89(m,2H)6.80(m,3H),6.28(s,1H),3.87(s,2H),3.79(s,2H),2.83(s,4H); 13 C NMR (101MHz, CDCl3) δ163.86,158.96,158.88,149.65,149.59,141.97,141.96,134.64,134.45,130.84,130.78,,123.69,123.60,1 23.58,120.07,120.04,119.98,113.87,112.53,112.48,112.46,112.42,63.17,56.00,55.32,37.46,37.44,37.35,37.28,,37.21.

[0091] Example 10. Preparation of 2-methoxy-5-(3,4,5-trimethoxyphenethyl)phenyl 3-(2,2-dichloroacetamide)acetate (8a)

[0092]

[0093] Add 80 mL of dry dichloromethane and 8.3 mL of triethylamine (59.74 mmol) to 3 g (23.89 mmol) of glycine methyl ester hydrochloride, and stir in an ice-salt bath for 15 min. Slowly add dichloroacetyl chloride dropwise, and continue to react and stir for 1.5 hours. After the reaction is completed, add 20 mL of water to quench, 40 mL of dichloromethane, extract and separate the organic phase, and extract the water layer with dichloromethane 20×2. Combine the organic phases, dry over anhydrous sodium sulfate, and distill under reduced pressure to obtain a yellow oil, which is separated by rapid paper chromatography to obtain 3.98 g of compound 11a. Add 30 mL of methanol: water = 4:1 to 3 g (15 mmol) of compound 11a, and then add 718 mg (30 mmol) of lithium hydroxide and react for 6 hours. After the reaction is completed, remove the solvent by distillation under reduced pressure, add 40 mL of water, adjust the pH to 3-4 with 2M hydrochloric acid, and then add 90 mL of ethyl acetate, extract and separate the organic phase, and extract the water layer with ethyl acetate 60×2. The organic phases were combined, dried over anhydrous sodium sulfate, and distilled under reduced pressure to obtain a yellow oil, which was separated by rapid paper chromatography to obtain 1.6 g of compound 6a. 150 mg (0.806 mmol) of compound 6a was added to 3 ml of dry dichloromethane, 368 mg (0.967 mmol) of HATU was added, and 0.199 mL (1.21 mmol) of DIPEA was added after stirring in an ice-salt bath for 10 min. After stirring for 15 min, 257 mg of Erianin was added. After reacting for 3 hours, the white solid was filtered off and separated by rapid column chromatography to obtain 254 mg of compound 8a with a yield of 64.79%.

[0094] 1 H NMR (400MHz, CDCl3) δ7.00(d,J=8.4Hz,1H),6.89(d,J=8.6Hz,2H),6.34(s,2 H),6.02(s,1H),4.39(d,J=5.2Hz,2H),3.81(d,J=4.5Hz,12H),2.84(s,4H); 13 C NMR (101MHz, CDCl3) δ167.30,164.41,153.09,149.00,138.89,137.23,136.16,134.4 7,127.33,122.55,112.40,105.43,66.00,60.93,56.10,56.06,41.84,38.29,36.99.

[0095] Example 11. Preparation of 2-methoxy-5-(3,4,5-trimethoxyphenethyl)phenyl 3-(2,2-dichloroacetamide)propanoate (8b)

[0096]

[0097] The synthesis method was the same as compound 8a, except that alanine methyl ester hydrochloride was substituted for glycine methyl ester hydrochloride to obtain 294 mg of compound 8b with a yield of 78.34%.

[0098] 1 H NMR(400MHz, CDCl3) δ6.99(dd,J=8.4,2.2Hz,1H),6.88(d,J=8.4Hz,1H),6.84(d,J=2.1H z,1H),6.34(s,2H),6.01(s,1H),3.84–3.77(m,12H),3.69(q,J=6.1Hz,2H),2.83(s,6H); 13 C NMR (101MHz, CDCl3) δ170.16,164.42,153.06,149.05,139.18,137.30,136.13,134.4 0,126.99,122.75,112.35,105.55,66.49,60.86,56.07,38.20,36.93,36.03,33.45.

[0099] Example 12. Preparation of 2-methoxy-5-(3,4,5-trimethoxyphenethyl)phenyl 4-(2,2-dichloroacetamide)butyrate (8c)

[0100]

[0101] The synthesis method was the same as compound 8a, except that γ-aminobutyric acid methyl ester hydrochloride was substituted for glycine methyl ester hydrochloride to obtain 182 mg of compound 8c with a yield of 50.48%.

[0102] 1 H NMR (400MHz, CDCl3) δ7.46(t,J=7.1Hz,1H),6.94–6.88(m,1H),6.92–6.83(m,2H),6.51(t,J=0.9Hz,2H),6.28(s,1H),3. 87(s,2H),3.82(d,J=5.7Hz,9H),3.26(q,J=7.2Hz,2H),2.87–2.79(m,4H),2.43(t,J=7.1Hz,2H),1.78(p,J=7.1Hz,2H); 13C NMR (400MHz, CDCl3) δ171.52,,168.36,153.29,149.60,140.73,136.79,134.51,134.48,123. 48,123.41,120.76,112.50,105.89,60.75,56.13,40.17,36.63,32.44,32.41,24.90,24.81.

[0103] Example 13. Preparation of 2-methoxy-5-(3,4,5-trimethoxyphenethyl)phenyl 6-(2,2-dichloroacetamide)hexanoate (8d)

[0104]

[0105] The synthesis method was the same as compound 8a, except that aminocaproic acid methyl ester hydrochloride was substituted for glycine methyl ester hydrochloride to obtain 227 mg of compound 8d with a yield of 67.54%.

[0106] 1 H NMR (400MHz, CDCl3) δ6.97(dd,J=8.3,2.1Hz,1H),6.90–6.81(m,3H),6.35(s,2H),5.95(s,1H),3.83–3.78(m,12H),3.34(q, J=6.7Hz,2H),2.83(s,4H),2.58(t,J=7.3Hz,2H),1.78(p,J=7.4Hz,2H),1.63(p,J=7.2Hz,2H),1.47(tt,J=9.6,5.9Hz,2H); 13 CNMR(101MHz, CDCl3)δ171.76,164.31,153.05,149.32,139.51,137.46,134.32,126.74,122.86 ,112.35,105.43,66.61,60.93,56.08,56.05,40.19,38.31,37.02,33.79,28.74,26.10,24.51.

[0107] Example 14. In vitro inhibitory activity on tumor cell proliferation

[0108] The cells in the logarithmic growth phase were used for the experiment. The cells were digested, counted, and made into 1×10 5Cell suspension of 100 μL / well was inoculated in a 96-well plate (100 μL / well), and cultured in a 37°C, 5% CO2 incubator for 24 hours; the test substance containing the corresponding concentration was added to each well, and a negative control group and a blank group were set up at the same time, with 3 replicates in each group; the plate was placed in the incubator for 72 hours, and the cell morphology of each group was observed under a microscope, 10 μL of CCK8 solution was added to each well, and the cells were incubated in the cell incubator for another 4 hours, the absorbance was measured at 450 nm, and the cell proliferation inhibition rate was calculated, and P<0.05 was considered statistically significant. The results are shown in Table 1.

[0109] 2. Experimental results

[0110] The experiments showed that compounds 7a and 8d had good inhibitory activity against HepG2 cells, among which compound 7b had a better HepG2 inhibition rate than Erianin.

[0111] Table 1: In vitro activity evaluation of some examples of compounds on HepG2 cell lines

[0112]

[0113] Among them, *P<0.05, **p<0.01.

[0114] Example 15. CCK-8 method to test the anti-tumor activity of compounds against various tumor cells

[0115] 1. Test methods

[0116] The cells in the logarithmic growth phase were used for the experiment. The cells were digested, counted, and made into 1×10 5 A cell suspension of 100 μL / well was inoculated into a 96-well plate (100 μL / well) and cultured in a 37°C, 5% CO2 incubator for 24 hours; the test substance containing the corresponding concentration was added to each well, and a negative control group and a blank group were set up at the same time, with 3 replicates in each group; after the plate was cultured in the incubator for 72 hours, the cell morphology of each group was observed under a microscope, 10 μL of CCK8 solution was added to each well, and the cells were continued to be incubated in the cell culture incubator for 4 hours, and the absorbance was measured at 450 nm. Eight concentration gradients were taken in the experiment to test the inhibition rate of the corresponding concentrations. The data were analyzed using GraphPad Prism5.0 statistical software. The t-test was used for comparison between two groups, and the One-way Anova (Dunnett) was used for comparison between multiple groups. P<0.05 was considered statistically significant, and the IC 50 The results are shown in Table 2.

[0117] 2. Experimental results

[0118] Table 2: In vitro activity evaluation of some examples of compounds against various tumor cell lines

[0119]

[0120] The results of the above in vitro antitumor activity evaluation of tumor cells showed that dichloroacetic acid-coupled diphenylethane compounds had a wide range of inhibitory activity against a variety of tumor cell lines, including human gastric cancer SGC7901 cell line, human lung adenocarcinoma A549 cell line, human cervical cancer Hela cell line, human breast cancer MCF-7 cell line, human liver cancer HepG2 cell line, and human colon cancer SW480 cell line. Its IC 50 The value is 0.5160 μM / mL, compared with the IC 50 The value is 8.3 μM / mL, which is a large difference in activity, indicating that the introduction of dichloroacetic acid into compound 7a on the basis of Erianin greatly improves the inhibitory activity of the compound on Hela cells.

[0121] From the perspective of in vitro anti-tumor comparison, compound 7a is more selective for HepG2 liver cancer cell line. Its IC 50 The value is 0.0489 μM / mL, compared with the IC 50 The value was 0.0728μM / mL, and the activity was nearly doubled.

[0122] Compound 8d has better inhibitory activity against human gastric cancer SGC7901 cell line and human breast cancer MCF-7 cell line than Erianin. 50 The value is 0.2883 μM / mL, while the IC 50 The value was 0.0728 μM / mL. The inhibitory effect of compound 8d on Hela cells was also significantly better than that of Erianin.

[0123] The above results show that the dichloroacetic acid-coupled diphenylethane compounds of the present invention have strong anti-tumor activity, which is significantly better than the positive control Erianin, and has good application development prospects.

[0124] Example 16. Acute toxicity experiment of single oral administration in mice

[0125] 1. Experimental Methods

[0126] ICR mice (SPF grade) were used in the experiment. Before oral gavage, the experimental animals were fasted overnight. The experiment was conducted with a dose of 5000mg / kg·bw, and 20 animals (weight 19-22g, half male and half female) were poisoned and randomly divided into groups according to body weight. The highest dose of the tested drug was 5000mg / kg, and a single oral gavage was administered. The animals were observed once at 0.5h, 1h, 2h, 4h, and 24h, and the mortality rate was recorded. After that, the animals were observed once a day and the mortality rate was recorded. This lasted for 154 days. On the 15th day, the surviving mice were killed for pathological anatomy.

[0127] 2. Experimental results

[0128] No animals died after single oral administration or high, medium and low doses of the drug (LD>5000mg / kg). No obvious residual drug solution was found after autopsy. No abnormalities in the heart, lungs, spleen, kidneys and other organs were found after autopsy of mice. The test drug has good safety.

[0129] Example 17. Experiment on the inhibition rate of tumors in nude mice by intragastric administration

[0130] 1. Experimental Methods

[0131] The human liver cancer cell HepG2 xenograft tumor model was established by inoculating the human liver cancer cell line HepG2 into the subcutaneous part of the armpit of nude mice. HepG2 cells in the logarithmic growth phase were taken and inoculated into the subcutaneous part of the right armpit of 55 nude mice under sterile conditions. The diameter of the transplanted tumor was measured with a vernier caliper. When the tumor grew to 100 mm 3 48 tumor-bearing nude mice with good growth status and uniform tumor size were selected and randomly divided into 8 groups, 6 in each group, namely model group, 7a low-dose group (50 mg / kg), 7a medium-dose group (100 mg / kg), 7a high-dose group (150 mg / kg), and sorafenib group (50 mg / kg). The model group animals were gavaged with equal volume of solvent once a day; the 7a low-dose group, 7a medium-dose group, and 7a high-dose group were gavaged once a day at 50 mg / kg, 100 mg / kg, and 150 mg / kg, respectively, for a total of 14 times; the sorafenib group was gavaged once a day at 50 mg / kg, for a total of 14 times. The anti-tumor effect of the test substance was dynamically observed by measuring the tumor diameter. The tumor diameter was measured every other day, and the nude mouse body weight was weighed while measuring the tumor diameter. The mice were killed on the 15th day, the tumor mass was surgically removed and weighed, the tumor tissue was preserved in a liquid nitrogen tank, and the tumor inhibition rate (%) was calculated. Tumor inhibition rate (%) = (tumor weight of model group - tumor weight of drug-treated group) / tumor weight of model group × 100%.

[0132] 2. Experimental results

[0133] According to the dosing regimen, the above compounds can significantly inhibit the growth of tumor transplants in tumor-bearing nude mice. Around the 11th day after administration, a trend of tumor shrinkage can be observed in the low-dose group; on the 9th day, a trend of tumor shrinkage can be observed in the medium-dose group; and on the 7th day, a significant trend of tumor shrinkage can be observed in the high-dose group.

[0134] Table 3. Inhibitory effect of compound 7a on the growth of human hepatoma cell HepG2 transplanted tumors in nude mice

[0135]

[0136] The above results show that the compounds of the present invention can significantly inhibit the growth of tumors in vivo and the tumors tend to shrink. As the dosage of the compounds increases, the rate of tumor shrinkage increases. Sorafenib is a multi-targeted oral anti-tumor drug currently widely used in clinical practice. The tumor inhibition rate of the compounds of the present invention is better than that of sorafenib and has better safety (LD50>5000mg / kg).

[0137] The above-mentioned related explanations and descriptions of the embodiments are for the convenience of those skilled in the art to understand and apply the present invention. It is obvious that those familiar with the art can easily make various modifications to these contents and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above-mentioned related explanations and descriptions of the embodiments. Improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the protection scope of the present invention.

Claims

1. A dichloroacetic acid coupled diphenylethane compound, characterized in that: Its structure is shown in general formula (I): in, R1, R2 and R3 are each selected from any one of -OH, -OMe and H, R4 is selected from any one of -OMe and -OEt, R5 and R6 are selected from chlorine atoms or fluorine atoms, m is 0, n is a positive integer selected from 1-8 or 11-12.

2. The dichloroacetic acid coupled diphenylethane compound according to claim 1, characterized in that: The dichloroacetic acid coupled diphenylethane compound is selected from:

3. A pharmaceutical composition, characterized in that The active ingredient of the pharmaceutical composition is the dichloroacetic acid-coupled diphenylethane compound described in claim 1.

4. The pharmaceutical composition according to claim 3, characterized in that The pharmaceutical composition is composed of an effective dose of the dichloroacetic acid-coupled diphenylethane compound according to claim 1 and a pharmaceutically acceptable carrier.

5. The pharmaceutical composition according to claim 3, characterized in that The pharmaceutical preparation is selected from but not limited to the following dosage forms: lyophilized powder, powder, injection, liposome, emulsion, microcapsule, suspension or solution for intravenous administration; granules, tablets, capsules or syrup for oral administration; or suppository.

6. The pharmaceutical composition according to claim 3, characterized in that The pharmaceutical composition is a drug for inhibiting the proliferation, growth or damage of tumor cells and eliminating tumor cells.

7. The method for preparing dichloroacetic acid coupled diphenylethane compounds according to claim 1, characterized in that: When more than one of R1, R2 and R3 is -OH, the preparation method comprises the following steps: (1) The hydroxyl group in is protected with benzyl chloride to obtain the corresponding benzyl protected product; (2) The benzyl protected product and triphenylphosphine ylide Wittig reaction was performed to obtain benzyl-protected stilbene compounds; (3) reducing and deprotecting the benzyl-protected stilbene compound to obtain the corresponding diphenylethane compound; (4) When m = 0, The product is condensed with the corresponding diphenylethane compound to obtain dichloroacetic acid coupled diphenylethane compound or dichloroacetic acid coupled stilbene compound.

8. The preparation method according to claim 7, characterized in that: When R1, R2 and R3 are not -OH, the step of protecting with benzyl chloride is omitted and the reaction is directly carried out with A Wittig reaction occurs.

9. The use of dichloroacetic acid coupled with diphenylethane compounds according to claim 1, characterized in that: The dichloroacetic acid-coupled diphenylethane compound is used in the preparation of an anti-tumor drug, wherein the anti-tumor drug is directed against tumors or neoplasms originating from one or more of gastric cancer cells, lung cancer cells, cervical cancer cells, intestinal cancer cells, breast cancer cells or liver cancer cells.

10. A reagent for inhibiting cell proliferation, characterized in that The reagent contains the dichloroacetic acid coupled diphenylethane compound according to claim 1.

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