A caffeic acid phenethyl ester derivative, its preparation method and uses

Abstract

The present invention discloses a derivative of phenethyl caffeate or a pharmaceutically acceptable salt or stereoisomer thereof, a pharmaceutical composition containing the same. Such compounds have good anti-tumor effects and can effectively inhibit the proliferation of tumor cells. The present invention also discloses a preparation method of such compounds and their use in the preparation of drugs for preventing and / or treating tumors.

Description

Technical Field

[0001] The present invention belongs to the technical field of medicine, and particularly relates to a phenethyl caffeate derivative, a preparation method thereof and uses thereof. Background Art

[0002] Diffuse Large B Cell Lymphoma (DLBCL) is a rapidly growing blood malignancy and one of the most common types of non-Hodgkin lymphoma. The annual incidence of DLBCL in adults is approximately 7-8 cases per 100,000 people. This disease mainly occurs in the elderly, with an average diagnosis age of around 70 years old, but it can also occur at any age.

[0003] Epstein-Barr virus (EBV) is a virus widely present in the population, mainly targeting B lymphocytes. Most people have been infected during puberty and the infection persists throughout life, usually without causing obvious symptoms. However, in some cases, EBV infection can cause some hematological malignancies, such as non-Hodgkin lymphoma, and EBV-positive DLBCL (EBV+DLBCL) is one of them. EBV infection of B cells leads to latent infection. Most EBV-infected B cells are eliminated by cytotoxic T cells and NK cells, but some EBV-infected B cells escape by downregulating antigen expression. Then, they pass through the germinal center and subsequently exist as EBV-infected memory cells. EBV-infected B cells will express viral proteins, such as LMP1, EBNA1, EBNA2, etc. Among them, LMP1 is a transmembrane protein related to cell cycle and apoptosis regulation, promoting the growth of invasive B cells of the virus. EBNA1 is a nuclear antigen protein that plays a key role in the replication and stability maintenance of the viral genome. EBNA2 is involved in the activation of early and late transcription of EBV and plays a role in the expression of other genes. The functions of these proteins are all closely related to the processes of EBV infection, replication and cell transformation, etc.

[0004] The frequency of EBV-positive DLBCL in DLCBL is about 2.5-14.0%, and the incidence is higher in the East Asian population. Most cases occur in patients over 50 years old and mainly male. Compared with EBV-DLBCL, the clinical characteristics of EBV+DLBCL include older age, more advanced clinical stage, higher extranodal involvement rate and poorer physical status, etc.

[0005] EBV+DLBCL is usually treated with R-CHOP, which consists of rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone. The response rates of EBV-positive and EBV-negative patients to first-line treatment (complete remission or partial remission) were 72% and 92.3% respectively (P = 0.006), that is, the response rate of EBV-positive DLBCL patients to first-line treatment was significantly reduced. The 5-year OS and PFS rates of EBV+DLBCL patients were 58.9% and 48.6% respectively. Compared with EBV-negative patients, the OS and PFS of EBV+DLBCL patients were poorer. Therefore, there is an urgent clinical need for new drugs or pharmaceutical compositions for the effective treatment of EBV+DLBCL and DLBCL.

[0006] Caffeic acid phenethyl ester (CAPE) is a natural product commonly found in plant propolis, and its structural formula is as follows:

[0007]

[0008] Its chemical structure is caffeic acid phenethyl ester type. CAPE has a variety of biological activities and is widely used in the fields of medicine, food, cosmetics, etc. Research shows that CAPE has various biological activities such as antioxidant, anti-inflammatory, antibacterial, antiviral, and antitumor, and also has effects such as regulating the immune system, reducing cholesterol, inhibiting platelet aggregation, and promoting skin repair. In recent years, the antitumor effect of CAPE has received extensive attention and research. Experiments have proved that CAPE can induce apoptosis of tumor cells, inhibit the proliferation of tumor cells, and can prevent the invasion and metastasis of tumor cells. In addition, CAPE can also improve the activity of immune cells, enhance the body's immunity, and can reduce the side effects of radiotherapy and chemotherapy.

[0009] Therefore, developing a class of caffeic acid phenethyl ester derivatives is of great significance for the research of new drugs or pharmaceutical compositions for the treatment of EBV+DLBCL and DLBCL. Summary of the Invention

[0010] Object of the Invention: The object of the present invention is to provide an (E)-3-(5,6-dihydroxy-2-naphthyl) acrylate compound of general formula I or a pharmaceutically acceptable salt or stereoisomer thereof:

[0011]

[0012] Wherein,

[0013] R is selected from -H, -F, -Cl, -Br, -I, -CF3, -CCl3, -NO2, -COCH3, -OCOCH3, -CH3, -OCH3, -OC2H5, -NH2, -NHSO2CH3, -SO2NH2 or NHCOCH3;

[0014] n = 1, 2, 3, 4 or 5.

[0015] In some preferred embodiments, the pharmaceutically acceptable salts include acid addition salts formed by the compound of general formula I and the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, citric acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, maleic acid or succinic acid, fumaric acid, salicylic acid, phenylacetic acid, mandelic acid; and also include acid salts formed by the compound of general formula I and inorganic bases.

[0016] In some more preferred embodiments, the pharmaceutically acceptable salts include alkali metal cation salts, alkaline earth metal cation salts and ammonium cation salts.

[0017] The compounds of general formula I of the present invention are preferably the following compounds: phenethyl (E)-3-(5,6-dihydroxy-2-naphthyl)acrylate, phenylpropyl (E)-3-(5,6-dihydroxy-2-naphthyl)acrylate, phenylbutyl (E)-3-(5,6-dihydroxy-2-naphthyl)acrylate, phenylpentyl (E)-3-(5,6-dihydroxy-2-naphthyl)acrylate, phenylhexyl (E)-3-(5,6-dihydroxy-2-naphthyl)acrylate, 2-fluorophenethyl (E)-3-(5,6-dihydroxy-2-naphthyl)acrylate, 2-fluorophenylpropyl (E)-3-(5,6-dihydroxy-2-naphthyl)acrylate, 2-fluorophenylbutyl (E)-3-(5,6-dihydroxy-2-naphthyl)acrylate, 2-fluorophenylpentyl (E)-3-(5,6-dihydroxy-2-naphthyl)acrylate, 2-fluorophenylhexyl (E)-3-(5,6-dihydroxy-2-naphthyl)acrylate.

[0018] The compounds of general formula I of the present invention are further preferably the following compounds:

[0019]

[0020] The above-mentioned compounds of general formula I of the present invention may also exist in the form of their salts, which are converted into the compounds of general formula I in vivo. For example, within the scope of the present invention, according to the processes known in the art, the compounds of the present invention are converted into the form of pharmaceutically acceptable salts and used in the form of salts.

[0021] All tautomeric forms of the compounds of general formula I of the present invention are included within the scope of the present invention. The compounds of the present invention may exist in specific geometric or stereoisomeric forms. There may be additional asymmetric carbon atoms in substituents such as alkyl, and all these isomers and their mixtures are included within the scope of the present invention.

[0022] Another object of the present invention is to provide a method for preparing a compound having general formula I, comprising the following steps:

[0023]

[0024] (1) 6-Bromo-2-naphthol, 1,3-bis(diphenylphosphino)propane, and palladium acetate were uniformly dispersed in DMF. Triethylamine and ethyl acrylate were added successively. Under argon protection, the mixture was heated at 115 °C overnight for reaction. After post-treatment, a crude product was obtained, and the crude product was subjected to silica gel column chromatography to obtain a white solid.

[0025] (2) The above solid was dissolved in ethanol, potassium hydroxide was added, and the mixture was stirred thoroughly. After the reaction was complete, a white solid was obtained.

[0026] (3) The above white solid was dissolved in tetrahydrofuran for standby. The alcohol fragment and triphenylphosphine were dissolved in tetrahydrofuran. Under ice bath conditions, diisopropyl azodicarboxylate was added to the tetrahydrofuran solution of phenethyl alcohol and triphenylphosphine, and the mixture was stirred thoroughly. Then, the initially prepared tetrahydrofuran solution was added to the reaction system in the ice bath, and the system was transferred to room temperature and stirred thoroughly. After post-treatment, a crude product was obtained, and the crude product was subjected to silica gel column chromatography to obtain a white solid.

[0027] (4) The white solid from the previous step was dissolved in DMSO, and 2-iodoxybenzoic acid was added to the reaction system. The reaction system changed from colorless to yellow and finally to orange-red. After post-treatment, an orange-red solid was obtained.

[0028] (5) The above orange-red solid was dissolved in acetonitrile, and an aqueous solution of sodium dithionite was added to the above acetonitrile solution. Under argon protection, the mixture was stirred thoroughly. The reaction system changed from orange-red to light yellow. After post-treatment, a crude product was obtained, and the crude product was separated by column chromatography to obtain a white solid as the target compound.

[0029] The compounds of general formula I of the present invention can all be prepared by the above or similar preparation methods. Appropriate starting materials can be selected according to different substituents. Those skilled in the art should recognize that the above route helps to understand the present invention, but does not limit the content of the present invention. Unless otherwise specified, the variables are defined as mentioned in general formula I.

[0030] Another object of the present invention is to provide a pharmaceutical composition, which comprises a compound of general formula I or a pharmaceutically acceptable salt, stereoisomer thereof, and a pharmaceutically acceptable carrier or excipient.

[0031] The pharmaceutical composition of the present invention can be administered in various known ways, such as orally, parenterally, by inhalation spray, or via an implanted reservoir. The pharmaceutical composition of the present invention can be administered alone or in combination with other anti-tumor drugs. Oral compositions can be any orally acceptable dosage form, including but not limited to tablets, capsules, emulsions, and suspensions, dispersions, and solutions. Commonly used pharmaceutically acceptable carriers or excipients include stabilizers, diluents, surfactants, lubricants, antioxidants, binders, colorants, fillers, emulsifiers, etc.

[0032] Sterile injectable compositions can be formulated using suitable dispersing or wetting agents and suspending agents according to techniques known in the art. Pharmaceutically acceptable carriers and solvents that can be used include water, mannitol, sodium chloride solution, etc.

[0033] The actual dosage level of the active ingredient in the pharmaceutical composition of the present invention can be varied so as to obtain an amount of the active ingredient that is effective in achieving the desired therapeutic response for a particular patient, composition, and mode of administration and that is non-toxic to the patient. The selected dosage level depends on a variety of factors, including the activity of the specific compound or its salt of the present invention used, the route of administration, the time of administration, the excretion rate of the specific composition used, the duration of treatment, other drugs, compounds, and / or materials used in combination with the specific composition used, the age, sex, weight, general health status, and medical history of the patient being treated, and similar factors well known in the medical arts.

[0034] Another object of the present invention is to provide the use of a compound of general formula I or a pharmaceutically acceptable salt or stereoisomer thereof in the preparation of a drug for preventing and / or treating tumors.

[0035] The tumors include Epstein-Barr virus-positive diffuse large B-cell lymphoma, diffuse large B-cell lymphoma, hepatocellular carcinoma, or glioma.

[0036] Beneficial effects:

[0037] The present invention synthesized a class of derivatives of caffeic acid phenethyl ester, called (E)-3-(5,6-dihydroxy-2-naphthyl) acrylate compounds. Pharmacological experiments have proven that the caffeic acid phenethyl ester derivatives of the present invention have good anti-tumor effects and can effectively inhibit the proliferation of tumor cells, showing good prospects in the development of anti-tumor drugs. Specific embodiments

[0038] The preparation method of the compound of general formula I of the present invention will be described below in conjunction with specific examples, but these specific methods do not constitute any limitation to the present invention. The compounds of the present invention can also be conveniently prepared by optionally combining various synthetic methods described in this specification or known in the art, and such combinations can be easily carried out by those skilled in the art to which the present invention pertains.

[0039] The starting materials, reaction reagents, etc. used in the specific embodiments of the present invention are all commercially available. The present invention can be prepared in the form of a salt by using the common salt-forming methods in the art. For example, at room temperature, the compound is dissolved in hydrochloric acid ethanol for reaction to form a hydrochloride; or benzenesulfonic acid is added thereto for reaction to form a benzenesulfonate. Example 32 lists the synthesis method of the hydrochloride of compound I-27. The synthesis of salts of other compounds can refer to this method, or other salts can be formed by using the common methods in the art.

[0040] Synthesis of (E)-phenethyl 3-(5,6-dihydroxy-2-naphthyl)acrylate in Example 1

[0041]

[0042] 6-Bromo-2-naphthol (300 mg, 1.34 mmol), 1,3-bis(diphenylphosphino)propane (DPPP, 54 mg, 0.13 mmol), and palladium acetate (Pd(OAc)2, 30 mg, 0.13 mmol) were placed in a schlenk tube and evenly dispersed in 3 ml of DMF (N,N-dimethylformamide). Triethylamine (541 mg, 5.36 mmol) and ethyl acrylate (1.3 g, 13.40 mmol) were added in sequence. Under argon protection, the reaction was heated at 115 °C overnight. The next day, the reaction was monitored to be complete, and the system was cooled to room temperature. The mixture was extracted with ethyl acetate and water, and the organic phases were combined. The combined organic phase was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and then concentrated under reduced pressure. The crude product was purified by silica gel column chromatography and eluted with a mixed solvent of n-hexane:ethyl acetate (volume ratio 8:1) to obtain a white solid.

[0043] The above solid (300 mg, 1.24 mmol) was dissolved in 4 ml of ethanol, potassium hydroxide (208 mg, 3.72 mmol) was added, and the mixture was stirred vigorously for 3 h. After monitoring the reaction to be complete, 20 ml of water was added for dilution. The aqueous layer was washed 3 times with dichloromethane. The pH of the aqueous layer was adjusted to acidic with 1N hydrochloric acid, and the aqueous layer was extracted with ethyl acetate. The organic phases were combined, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and then concentrated under reduced pressure to obtain a white solid.

[0044] Dissolve the above white solid (500 mg, 2.33 mmol) in 2 ml of tetrahydrofuran (THF) for later use. Then dissolve phenethyl alcohol (2.33 mmol) and triphenylphosphine (TPP, 612 mg, 2.33 mmol) in 4 ml of tetrahydrofuran (THF). Under ice-bath conditions, dropwise add diisopropyl azodicarboxylate (DIAD, 463 mg, 2.33 mmol) to the 4 ml of the tetrahydrofuran solution of phenethyl alcohol and triphenylphosphine. Stir well for 15 min, then dropwise add the initially prepared tetrahydrofuran solution to the reaction system in the ice bath, and transfer the system to room temperature and stir well. After monitoring by TLC that the raw materials have completely reacted, add water to quench the reaction. Extract with ethyl acetate, combine the organic phases, wash the organic phases with saturated sodium chloride solution, dry the obtained organic phases with anhydrous sodium sulfate, and then concentrate under reduced pressure. The crude product is separated by silica gel column chromatography, and eluted with a mixed solvent of n-hexane:ethyl acetate (volume ratio 8:1) to obtain a white solid.

[0045] Dissolve the white solid from the previous step (0.87 mmol) in 2 ml of DMSO, and then add 2-iodoxybenzoic acid (IBX, 269 mg, 0.96 mmol) to the reaction system. Stir well. The reaction system changes from colorless to yellow and finally to orange-red. After monitoring by TLC that the raw materials have completely reacted, add water to quench the reaction. Extract with ethyl acetate, combine the organic phases, wash the organic phases with saturated sodium chloride solution, dry the obtained organic phases with anhydrous sodium sulfate, and then concentrate under reduced pressure to obtain an orange-red solid.

[0046] Dissolve the above orange-red solid in 2 ml of acetonitrile (MeCN). Dissolve sodium dithionite (Na2S2O4, 166 mg, 0.96 mmol) in 2 ml of water. Slowly add the aqueous sodium dithionite solution to the acetonitrile solution of the above orange-red solid. Stir well under argon protection. The reaction system gradually changes from orange-red to light yellow. After monitoring by TLC that the reaction is complete, rotary evaporate under reduced pressure to remove acetonitrile, then extract 3 times with ethyl acetate, combine the organic phases, dry the organic phases with anhydrous sodium sulfate, rotary evaporate to remove ethyl acetate, and then triturate. Separate the obtained triturated sample by column chromatography, and elute with a mixed solvent of n-hexane:ethyl acetate (volume ratio 8:1) to obtain a white solid as the compound.

[0047] 1H NMR (300 MHz, Methanol-d4) δ (ppm) 7.86 - 7.55 (m, 5H), 7.36 - 7.06 (m, 7H), 6.48 (d, J = 15.9 Hz, 1H), 4.39 (t, J = 7.0 Hz, 2H), 3.00 (t, J = 7.0 Hz, 2H); 13C NMR (75 MHz, Methanol-d4) δ 167.55, 145.46, 138.00, 136.25, 130.10, 130.01, 128.91, 128.64, 128.17, 128.07, 126.71, 126.19, 123.47, 118.70, 115.78, 108.81, 64.89, 34.79.; MS (ESI) m / z, 335.12 [M+H] + 。

[0048] Example 2 (E)-3-(5,6-Dihydroxy-2-naphthyl) acrylic acid phenylpropyl ester

[0049]

[0050] Refer to the synthesis method of Example 1.

[0051] 1H NMR (300 MHz, Methanol-d4) δ (ppm) 7.86 - 7.55 (m, 5H), 7.36 - 7.06 (m, 7H), 6.48 (d, J = 15.9 Hz, 1H), 4.39 (t, J = 7.0 Hz, 2H), 3.00 (t, J = 7.0 Hz, 2H), 2.08 (m, 2H); 13C NMR (75 MHz, Methanol-d4) δ 167.55, 145.46, 138.00, 136.25, 130.10, 130.01, 128.91, 128.64, 128.17, 128.07, 126.71, 126.19, 123.47, 118.70, 115.78, 108.81, 64.89, 34.79, 21.06; MS (ESI) m / z, 349.12 [M+H] + 。

[0052] Example 3 (E)-3-(5,6-Dihydroxy-2-naphthyl) acrylic acid phenylbutyl ester

[0053]

[0054] Refer to the synthesis method of Example 1.

[0055] 1H NMR (300 MHz, Methanol-d4) δ (ppm) 7.86 - 7.55 (m, 5H), 7.36 - 7.06 (m, 7H), 6.48 (d, J=15.9 Hz, 1H), 4.39 (t, J=7.0 Hz, 2H), 3.00 (t, J=7.0 Hz, 2H), 1.78 (m, 4H); 13C NMR (75 MHz, Methanol-d4) δ 167.55, 145.46, 138.00, 136.25, 130.10, 130.01, 128.91, 128.64, 128.17, 128.07, 126.71, 126.19, 123.47, 118.70, 115.78, 108.81, 64.89, 34.79, 21.06, 20.56; MS (ESI) m / z, 363.12 [M + H] + 。

[0056] Example 4: Biological Activity

[0057] Test method: A specific small molecule compound was formulated into a 100 mM stock solution.

[0058] For suspension cells Farage and MC116, they were seeded into 96-well plates at a density of 6000 cells / well and 60 μL / well. The corresponding small molecule stock solution was formulated into 600 μM, and the drug-containing medium was serially diluted 3-fold. The serially diluted working solution was added to the wells seeded with cells at a volume of 30 μL / well. After culturing for 48 hours, the absorbance was measured by the CCK8 method, and its IC 50 value was calculated using Graphpad Prism.

[0059] For adherent cells Hep3B and U87, they were seeded into 96-well plates at a density of 3000 cells / well and 100 μL / well. The corresponding small molecule stock solution was formulated into 200 μM, and the drug-containing medium was serially diluted 3-fold. After the cells adhered, the original medium was discarded, and the serially diluted working solution was added to the wells seeded with cells at a volume of 100 μL / well. After culturing for 48 hours, the absorbance was measured by the CCK8 method, and its IC 50 value was calculated using Graphpad Prism. The experimental results are shown in Table 1. CAPE was used as a positive control drug.

[0060] Table 1 IC 50 values (μM) of Example 4 against the anti-proliferative activities of 4 human cancer cell lines

[0061] Compound Farage MC116 Hep3B U87 Example 1 0.873 2.546 48.338 61.584 Example 2 0.998 2.235 49.524 58.452 Example 3 0.924 1.687 50.127 53.696 CAPE 1.525 2.762 58.667 69.935

[0062] The Farage cells were originally derived from a patient with EBV-positive B-cell lymphoma. Therefore, the Farage cells are EBV-positive; the MC116 cells are a type of B lymphocyte and are used in experimental studies of B-lymphocyte-related diseases, including the study of DLBCL; the Hep3B cells are a hepatocellular carcinoma cell line and are widely used in the study of liver cancer; the U87 cells are a human glioblastoma cell line and are widely used in the study of gliomas and cancer.

[0063] As can be seen from Table 1, the compounds of Example 1, Example 2, and Example 3 have IC 50 values lower than CAPE and can effectively inhibit the in vitro growth of EBV+DLBCL, EBV-DLBCL, hepatocellular carcinoma, and glioblastoma cell lines.

[0064] The above experimental results show that the compounds provided by the present invention can effectively inhibit the in vitro growth of EBV+DLBCL, EBV-DLBCL, HCC, and glioblastoma cell lines.

[0065] The above compounds of the present invention and their pharmaceutically acceptable salts can effectively inhibit the in vitro growth of EBV+DLBCL, EBV-DLBCL, HCC, and glioblastoma cell lines and can be used as active ingredients in pharmaceuticals. Therefore, a drug containing the above compound as an active ingredient can be used to prepare a drug for preventing and / or treating tumors.

[0066] As described above, although the present invention has been shown and described with reference to specific preferred embodiments, it should not be construed as a limitation of the present invention itself. Various changes can be made in its form and details without departing from the spirit and scope of the present invention defined by the appended claims.

Claims

1. A method for preparing an (E)-3-(5,6-dihydroxy-2-naphthyl) acrylate compound, characterized in that, It includes the following steps: (1) 6-Bromo-2-naphthol, 1,3-bis(diphenylphosphino)propane and palladium acetate are uniformly dispersed in DMF. Triethylamine and ethyl acrylate are added successively. Under argon protection, the mixture is heated at 115 °C overnight. After post-treatment, a crude product is obtained, and the crude product is subjected to silica gel column chromatography to obtain a white solid; (2) The above solid is dissolved in ethanol, potassium hydroxide is added and stirred well. After the reaction is complete, a white solid is obtained; (3) The above white solid is dissolved in tetrahydrofuran for standby. The alcohol fragment and triphenylphosphine are dissolved in tetrahydrofuran. Under ice bath conditions, diisopropyl azodicarboxylate is added to the tetrahydrofuran solution of phenethyl alcohol and triphenylphosphine, and stirred well. Then the initially prepared tetrahydrofuran solution is added to the reaction system in the ice bath, and the system is transferred to room temperature and stirred well. After post-treatment, a crude product is obtained, and the crude product is subjected to silica gel column chromatography to obtain a white solid; (4) The white solid from the previous step is dissolved in DMSO, and 2-iodoxybenzoic acid is added to the reaction system and stirred well. The reaction system changes from colorless to yellow and finally to orange-red. After post-treatment, an orange-red solid is obtained; (5) The above orange-red solid is dissolved in acetonitrile, and an aqueous solution of sodium dithionite is added to the above acetonitrile solution. Under argon protection, it is stirred well. The reaction system changes from orange-red to light yellow. After post-treatment, a crude product is obtained, and the crude product is separated by column chromatography to obtain a white solid as the target compound; Wherein, R is selected from -H, -F, -Cl, -Br, -I, -CF3, -CCl3, -NO2, -COCH3, -OCOCH3, -CH3, -OCH3, -OC2H5, -NH2, -NHSO2CH3, -SO2NH2 or NHCOCH3; n = 1, 2, 3, 4 or 5.

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