CN108147951B - Phenyl alkene compound and preparation method and application thereof - Google Patents

Phenyl alkene compound and preparation method and application thereof Download PDF

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CN108147951B
CN108147951B CN201810044975.2A CN201810044975A CN108147951B CN 108147951 B CN108147951 B CN 108147951B CN 201810044975 A CN201810044975 A CN 201810044975A CN 108147951 B CN108147951 B CN 108147951B
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包骏
陈海铭
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Abstract

The invention discloses a phenyl alkenyl compound, which at least comprises 5- (4- (4- (3, 5-dihydroxy styrene) with the following structural formulaYl) phenoxy) styryl) -1, 3-benzenediol compound. The invention also discloses a preparation method and application of the 5- (4- (4- (3, 5-dihydroxyl styryl) phenoxy) styryl) -1, 3-benzenediol compound with the structural formula. The 5- (4- (4- (3, 5-dihydroxystyryl) phenoxy) styryl) -1, 3-benzenediol compounds disclosed herein inhibit abnormal growth of B-cells by reducing AKT/mTOR signaling.

Description

Phenyl alkene compound and preparation method and application thereof
Technical Field
The invention relates to the technical field of preparation of medicines for treating autoimmune diseases and B-cell malignant tumors, in particular to a phenyl alkenyl compound and a preparation method and application thereof.
Background
The development of B cells in bone marrow can go through several stages, progenitor B cells (pro-B cells), pre-B cells, immature B cells, activated B cells and mature B cells. The major changes in the differentiation stages of B cells in the bone marrow are the rearrangement of immunoglobulin genes and the expression of membrane surface markers. B cells also undergo selection during developmental differentiation to remove non-functional gene rearrangement B cells and autoreactive B cells, forming a pool of surrounding mature B cells. At this stage, mature B cells, after antigen stimulation, can continue to differentiate into antibody-synthesizing and secreting plasma cells, an antigen-dependent differentiation stage. B cell mutations have been found to cause cancer and autoimmune diseases. Research has shown that activation of the PI3K/mTOR signaling pathway increases B cell survival and function, prolongs T cell-independent antigen exposure, and promotes production of antibodies, including autoantibodies and tumor-associated M protein, by mature B cells.
Numerous studies have demonstrated that autoimmune pathological damage is the result of an autoimmune response, leading to the production of autoantibodies by B-cells. Autoimmune diseases, i.e., the autoantibodies of the immune system attack and destroy the host's own organs and tissues, and there is no cure to date. B cells play an important role in autoimmune diseases, and researches show that some transcription factors in the B cells cause abnormal development of the B cells, so that the diseases are driven.
Although B-cell malignancies patients are initially available for many new treatment regimens, almost all patients develop resistance to currently available treatment regimens.
Disclosure of Invention
In order to find a new compound for inhibiting the growth of B cell malignant tumor and improve the curative effect of the anti-tumor medicament. The invention screens, separates and purifies a large amount of compounds of Chinese herbal medicines, and discovers a compound for inhibiting abnormal growth of B-cells, namely a phenylalkenyl compound, through in vivo and in vitro cell and animal experiments. The disclosed phenylalkenyl compounds can inhibit abnormal growth of B-cells by reducing AKT/mTOR signaling.
The first object of the present invention is to provide a styryl compound comprising at least a 5- (4- (4- (3, 5-dihydroxystyryl) phenoxy) styryl) -1, 3-benzenediol compound of the following structural formula (1):
Figure BDA0001550627490000021
the second object of the present invention is to provide a method for preparing the above-mentioned phenylalkenyl compound, comprising the steps of:
the specific reaction formula is as follows:
Figure BDA0001550627490000022
the second object of the present invention is to provide a method for preparing the above-mentioned phenylalkenyl compound, which is extracted from plants or Chinese herbs. The Chinese herbal medicine is preferably rhubarb.
The third purpose of the invention is to provide the application of the above-mentioned phenyl alkenyl compound in the preparation of drugs for treating autoimmune diseases. The autoimmune disease is one of rheumatoid arthritis, systemic lupus erythematosus, sjogren's syndrome, lung and kidney syndrome, autoimmune hepatitis, primary biliary cirrhosis, Crohn's disease, and ulcerative colitis.
The fourth purpose of the invention is to provide the application of the phenylalkenyl compound in preparing medicines for treating B-cell malignant tumors.
The fourth invention of the invention is to provide the application of the above-mentioned phenyl alkene compounds in preparing medicines for inhibiting the growth of B-cell malignant tumors.
The B-cell malignant tumor is one of lymphoma, multiple myeloma and chronic lymphocytic leukemia.
The fourth purpose of the invention is to provide the application of the phenyl alkene compound in preparing a medicament for inhibiting the signal transduction of AKT/mTOR1 in B-cells.
The 5- (4- (4- (3, 5-dihydroxystyryl) phenoxy) styryl) -1, 3-benzenediol compounds disclosed herein inhibit abnormal growth of B-cells by reducing AKT/mTOR signaling.
Drawings
FIG. 1 shows 2- (4- (3, 5-dimethoxystyryl) phenyl) malonate (C)35H32O8) Schematic diagram of the structural formula.
FIG. 2 is C28H22O5Experimental graphs of inhibition of proliferation of B-cell malignant (Raji) cells: cell proliferation experiments in the figure show C28H22O5Inhibit B-cell malignancy (Raji) cells to proliferate and exhibit a dose-dependent relationship.
FIG. 3 is C28H22O5Experimental picture of significant inhibition of proliferation of B-cell malignant tumor (RPMI8226) cells: cell proliferation experiments in the figure show C28H22O5Obviously inhibit B-cell malignant tumor (RPMI8226) cells to proliferate and show a dose-dependent relationship.
FIG. 4 is C28H22O5、C35H32O8A graphical representation of the 24 hour inhibition of A20B-cell proliferation by compound.
FIG. 5 is C28H22O5、C35H32O8A graphical representation of the inhibition of A20B-cell proliferation by compound for 48 hours.
FIG. 6 is C28H22O5、C35H32O8A graphical representation of the inhibition of A20B-cell proliferation by compound for 72 hours.
FIG. 7 is C28H22O5Schematic of compound cells inducing apoptosis of Raji B-cells and RPMI1640 multiple myeloma cells for 72 hours.
FIG. 8 is a schematic diagram of the weight test of experimental model mice. Blank pair in the figureGroup (I), group (II) for pseudo-operation, group (III) for model, group (IV) for cyclophosphamide and group C28H22O5Group (V), C) for administration of 100mg/kg28H22O550mg/kg administration group (VI), C28H22O5Group (VII) administered at 25 mg/kg. The method adopts an intraperitoneal injection mode, the administration is carried out for 1 time every day, the administration volume is 0.1mL/10g, the intraperitoneal injection of a sham operation group is carried out with the same volume of normal saline, and the administration of cyclophosphamide is carried out for 1 time every week and 2 times in total. Mice in cyclophosphamide administration group (IV) had significant weight loss (P)<0.001), whereas the body weight of the mice of the (C28H22O5) compound-administered group was not substantially changed.
FIG. 9 is a schematic diagram of tumor volume determination in experimental model mice. In the figure (C)28H22O5) Compared with the control group, the tumor volume of the mice of the compound administration group is obviously reduced (P)<0.01)。
FIG. 10 is a schematic diagram of tumor weight measurement in experimental model mice. In the figure (C)28H22O5) Compared with the control group, the tumor volume of the mice of the compound administration group is obviously reduced (P)<0.01)。
Fig. 11A is a schematic of H & E staining experimental model murine pathology examination (control model group) (1X 400).
FIG. 11B is C28H22O5(100mg/kg) administration group schematic (1X400), in which experimental model mouse tumor cells are significantly reduced.
FIG. 11C is C28H22O5(50mg/kg) administration group schematic (1X400) in which experimental model mice show a reduction in tumor cells.
FIG. 11D is C28H22O5(25mg/kg) dosing group diagram (1X 400). In the figure, experimental model mice show a mild reduction in tumor cells.
FIG. 12 is a schematic diagram of the measurement of thymus index of experimental model mouse, in which the high dose (C) is measured28H22O5) Compared with the control group, the thymus index of the mice in the compound administration group is obviously increased (P)<0.01), and the chemotherapeutic drug cyclophosphamide administration group (IV)) Compared with the control group, the thymus index of the cyclophosphamide administration group is obviously reduced (P)<0.01)。
FIG. 13 is a schematic diagram of spleen index determination of an experimental model mouse. High dose in the figure (C)28H22O5) Compared with the control group, the spleen index of the mice in the compound administration group is obviously increased (P)<0.01) and the spleen index of the cyclophosphamide-administered group (IV) as a chemotherapeutic agent was significantly decreased (P) as compared with that of the control group<0.01)。
FIG. 14 is a schematic diagram of the measurement of white blood cells of an experimental model mouse. High dose in the figure (C)28H22O5) The leukocyte index of mice in the compound administration group is obviously increased (P) compared with that of mice in the control group in the administration group experimental model<0.01)。
FIG. 15 is a schematic illustration of the red blood cell assay in experimental model mice at high dose (C)28H22O5) The red blood cell index of mice in the compound administration group is reduced (P) compared with that of the control group in the experimental model of the administration group<0.05), medium and low dose (C)28H22O5) The cell index of the administration group was not changed. While the cell index of the cyclophosphamide-administered chemotherapy drug group (IV) was decreased (P) as compared with that of the control group<0.05)。
FIG. 16 is a schematic diagram of gene expression experiments. In the figure, reverse transcription polymerase chain reaction (RT-PCR) was used to detect the expression of AKT1, AKT2, AKT3, Cyclin D1, mTOR, and P27kip genes. The experimental results show that28H22O5) The compound obviously inhibits the expression of AKT1 gene, but does not affect the expression of AKT2 and AKT3 gene. At the same time (C)28H22O5) The compounds also affected the expression of the mTOR, Cyclin D1, P27kip gene associated with AKT 1. (C)28H22O5) The compound obviously inhibits the expression of mTOR and Cyclin D1 genes.
Detailed Description
The invention relates to a styrenyl compound, which at least comprises a 5- (4- (4- (3, 5-dihydroxystyryl) phenoxy) styryl) -1, 3-benzenediol compound with the following structural formula:
Figure BDA0001550627490000051
the 5- (4- (4- (3, 5-dihydroxystyryl) phenoxy) styryl) -1, 3-benzenediol compound of the above formula is prepared according to the following reaction formula:
Figure BDA0001550627490000052
the 5- (4- (4- (3, 5-dihydroxystyryl) phenoxy) styryl) -1, 3-benzenediol compound of the structural formula is prepared by the following method:
1. preparation of C9-1
The reaction formula is as follows:
Figure BDA0001550627490000053
feeding:
Figure BDA0001550627490000054
Figure BDA0001550627490000061
operation and phenomena:
putting SM125.0 g into a 500ml single-mouth bottle, adding 250ml DCM, stirring and dispersing uniformly, then dripping 46.0g of thionyl chloride at room temperature, adding 5 drops of catalytic amount of DMF after dripping, and heating to reflux after dripping;
after stirring and reacting for 5 hours, monitoring the reaction by TLC (ethyl acetate), and concentrating the system at the external temperature of 50 ℃ under reduced pressure until the system is dry to obtain a yellow solid crude product;
to the crude yellow solid was added 100ml of DCM and rewound to dryness;
adding 100ml DCM into the crude solid product again, and carrying out rotary banding again until the mixture is dry;
adding 25ml of DCM and 25ml of THF into the solid crude product, dissolving and sealing for later use;
adding 28g of N, O-dimethylhydroxylamine hydrochloride into a 1000ml single-neck bottle, adding 300ml of DCM, stirring and dispersing, adding 72.0g of DIEA, and transferring the system into an ice water bath to cool after the addition is finished;
when the internal temperature is 0-5 ℃, dropwise adding a prepared dichloromethane solution of SM1 after chlorination, wherein the internal temperature is controlled to be less than 10 ℃ in the dropwise adding process;
after dripping, removing the ice water bath, and naturally heating to room temperature for reaction;
the reaction was stirred for 2 hours and confirmed by TLC (EA/PE-1/3) to be complete;
after the reaction is finished, transferring the system into a separating funnel for separating liquid, and separating out an organic phase;
the organic phase was washed successively with 0.5N HCl (100 ml. times.2), 0.5N NaOH (100 ml. times.2) and saturated brine (100 ml. times.1), and the organic phase was separated;
50g of anhydrous sodium sulfate is added into the organic phase to be dried and dehydrated for 0.5 hour;
filtering, concentrating the filtrate at 50 deg.C under reduced pressure to dryness to obtain pale yellow oil 30g, with yield of 90%. (theoretical yield: 85-95%)
Controlling the reaction process:
1) acyl chloride reaction process control
The checking method comprises the following steps: TLC method;
thin-layer plate: GF 254;
developing agent: ethyl acetate;
the preparation method of the test solution comprises the following steps: adding a small amount of anhydrous methanol into a reaction solution, shaking for 10 seconds, spotting on a plate, taking methanol solution of SM1 as a reference, and spotting on the same silica gel plate;
the color development method comprises the following steps: after the development, observing under an ultraviolet lamp (254 nm);
monitoring the reaction end point: raw material SM 1: rf ═ 0, methyl ester: rf is 0.8, and it is required that the spots of the raw materials in the reaction solution are substantially eliminated.
2) C9-1 reaction process control
The checking method comprises the following steps: TLC method;
thin-layer plate: GF 254;
developing agent: ethyl acetate: petroleum ether is 1: 3;
the preparation method of the test solution comprises the following steps: adding a small amount of anhydrous methanol into a reaction solution, shaking for 10 seconds, spotting on a plate, comparing the methyl ester solution in the acyl chloride monitoring process, and spotting on the same silica gel plate;
the color development method comprises the following steps: after the development, observing under an ultraviolet lamp (254 nm);
monitoring the reaction end point: methyl ester: rf 0.5, C9-1: rf is 0.4, and it is required that the spot of the methyl ester in the reaction solution is substantially eliminated.
2. Preparation of C9-2
The reaction formula is as follows:
Figure BDA0001550627490000071
feeding:
Figure BDA0001550627490000072
operation and phenomena:
transferring the 34.0g C9-1 oily substance into a 1000ml three-neck flask by using 300ml THF, introducing argon, and transferring the system into a cold bath to control the internal temperature to be less than-20 ℃;
slowly dripping Red-Al (70% in tolumen) into the system and controlling the internal temperature to be less than-10 ℃ all the time;
after completion of the dropwise addition, the reaction was carried out at-5 ℃ to 0 ℃ for 2 hours, and completion of the reaction was confirmed by TLC (EA/PE) ═ 1/3);
adding 10(v/m) EA into the system for dilution, and then cooling the system to about-10 ℃;
slowly dripping 0.5N HCl 400ml for quenching reaction, and stirring at about 0 ℃ for 0.5h after dripping;
filtering to remove insoluble substances, transferring the filtrate to a separating funnel, and separating an organic layer;
the organic layer was washed with 0.5N HCl (100 ml. times.2), pure water (100 ml. times.2) and saturated brine (100 ml. times.1) in this order;
the organic phase was dried over 20.0g of anhydrous sodium sulfate and dehydrated for 1 hour;
filtering to remove the drying agent, and concentrating the filtrate at an external temperature of 50 ℃ under reduced pressure until the filtrate is dry to obtain yellow oily matter;
adding 80ml of petroleum ether into the yellow oily matter, pulping and stirring for 2 hours;
filtering, and rinsing a filter cake by using 25ml of petroleum ether;
the filter cake was air-dried at 45 ℃ for 15 hours to give 16.7g of a yellow solid in 74.9% yield. (expected yield: 70-80%).
C9-2 reaction process control:
the checking method comprises the following steps: TLC method;
thin-layer plate: GF 254;
developing agent: ethyl acetate: petroleum ether is 1: 3;
the preparation method of the test solution comprises the following steps: adding a small amount of reaction solution into a proper amount of water for quenching, adding ethyl acetate for extraction, taking an ethyl acetate layer for spotting, taking an ethyl acetate solution of C9-1 for comparison, and spotting on the same silica gel plate;
the color development method comprises the following steps: after the development, observing under an ultraviolet lamp (254 nm);
monitoring the reaction end point: c9-1: rf 0.4, C9-2: rf is 0.6, and it is required that the spot of C9-1 in the reaction solution substantially disappears.
3. Preparation of C9-4
The reaction formula is as follows:
Figure BDA0001550627490000081
feeding:
Figure BDA0001550627490000091
operation and phenomena:
putting 40.0g of 3, 5-dimethoxy benzyl bromide into a 250ml single-mouth bottle, adding 87.0g of triethyl phosphite at room temperature, heating to 100 ℃ after the addition, and stirring for reaction;
after 3 to 4 hours of reaction, completion of the reaction was confirmed by TLC (EA/PE ═ 1/3);
the reaction mixture was concentrated to dryness at an external temperature of 70 ℃ under reduced pressure using an oil pump to give 49.0g of a reddish brown oil in 98% yield. (expected yield: 90-100%)
Controlling the reaction process of the intermediate C9-4:
the checking method comprises the following steps: TLC method;
thin-layer plate: GF 254;
developing agent: ethyl acetate: petroleum ether is 1: 3;
the preparation method of the test solution comprises the following steps: diluting a small amount of reaction solution with appropriate amount of ethyl acetate, spotting on a plate, comparing with ethyl acetate solution of 3, 5-dimethoxy benzyl bromide, and spotting on the same silica gel plate;
the color development method comprises the following steps: after the development, observing under an ultraviolet lamp (254 nm);
monitoring the reaction end point: 3, 5-dimethoxybromobenzyl: rf 0.8, C9-4: rf is 0.1, and the spot of 3, 5-dimethoxybromobenzyl in the reaction solution is required to be basically disappeared.
4. Preparation of C9-3
The reaction formula is as follows
Figure BDA0001550627490000092
Feeding:
Figure BDA0001550627490000101
operation and phenomena:
transferring C9-431.0 g into a 250ml three-neck flask by using 100ml of DMF, introducing argon, and controlling the internal temperature of the system to be 0-5 ℃ by using ice water bath;
adding 6.4g of NaH (60%) in batches, and controlling the internal temperature to be less than 10 ℃;
after the addition, naturally heating to room temperature, and stirring for reaction for 0.5 h;
cooling the reaction system to 5-10 ℃ again, and dropwise adding C9-28.05 g dissolved in 30ml of DMF, wherein the internal temperature is controlled to be less than 15 ℃ in the dropwise adding process;
after dripping, removing the ice water bath, and naturally heating to room temperature;
after stirring at room temperature for 2 to 3 hours, the end of the reaction was confirmed by TLC (EA/PE) ═ 1/5;
after the reaction is finished, cooling the system to 0-5 ℃;
adding 250ml of EA (ethylene-vinyl acetate) diluted reaction solution into the system, and then adding 100ml of pure water to quench the reaction;
transferring the system to a 1000ml separating funnel, adding 400ml EA for extraction, and separating an organic phase;
the organic phase was washed successively with pure water (200 ml. times.2) and saturated brine (200 ml. times.1);
the organic phase was dried over 25.0g of anhydrous sodium sulfate for 0.5 hour;
filtering to remove the drying agent, and concentrating the filtrate at an external temperature of 50 ℃ under reduced pressure until the filtrate is dried to obtain 27.0g of yellow oily matter;
dissolving the oily substance with 200ml EA, adding about 80.0g of 100-200-mesh silica gel, uniformly stirring, concentrating at an external temperature of 50 ℃ under reduced pressure until the oily substance is dried, then loading the mixture on a dry column, and purifying by adopting silica gel column chromatography, wherein an eluent is DCM/PE (DCM/PE) ═ 1/1;
collecting the target product eluent, concentrating at 50 deg.C under reduced pressure to dry to obtain light yellow transparent oily substance, and solidifying to obtain light yellow solid 15.0g with yield of 85%. (expected yield: 80-90%)
Controlling the reaction process of the intermediate C9-3:
the checking method comprises the following steps: TLC method;
thin-layer plate: GF 254;
developing agent: ethyl acetate: petroleum ether is 1: 5;
the preparation method of the test solution comprises the following steps: quenching a small amount of reaction solution with appropriate amount of water, extracting with ethyl acetate, spotting the ethyl acetate layer on a plate, collecting ethyl acetate solution of C9-2, and spotting on the same silica gel plate;
the color development method comprises the following steps: after the development, observing under an ultraviolet lamp (254 nm);
monitoring the reaction end point: c9-2: rf 0.4, C9-3: rf is 0.5, and it is required that the C9-2 spot in the reaction solution substantially disappears.
5. Preparation of C9
The reaction formula is as follows:
Figure BDA0001550627490000111
feeding:
Figure BDA0001550627490000112
operation II:
putting C9-313.0 g into a 2000ml single-mouth bottle, adding 650ml DCM, stirring and dispersing, controlling the temperature of the system by using an ice water bath, beginning to drop BBr3 when the temperature is lower than 10 ℃, and keeping the internal temperature lower than 10 ℃ all the time;
after dropping, the ice-water bath was removed, the reaction was stirred at room temperature for 1 hour, and then the end of the reaction was confirmed by TLC (EA/PE: 1/5 and EA);
after the reaction is finished, cooling the reaction system to 0-5 ℃, adding 500ml EA to dilute the reaction solution, and then slowly dropwise adding 150ml saturated sodium bisulfite and 50ml pure water to quench the reaction;
transferring the system to a 3000ml separating funnel, adding 1000ml EA into the system for extraction, and separating an organic phase;
the organic phase was washed with saturated sodium hydrogen sulfite (400 ml. times.1), pure water (400 ml. times.1) and saturated brine (400 ml. times.1) in this order;
adding 100g of anhydrous sodium sulfate into the organic phase, drying and dehydrating for 0.5 hour;
filtering to remove the drying agent, concentrating the filtrate at an external temperature of 50 ℃ until 200ml of the remaining 100-: EA/PE 1/2;
collecting target product eluate, concentrating at 40 deg.C under reduced pressure to obtain light yellow solid, and pulping the solid with mixed solvent of EA/PE (ether-ether) 1/1 for 1-2 hr;
filtering, rinsing the filter cake with 50ml PE, vacuum drying the filter cake at 40 deg.C for 5 hours to obtain 1.04g of white solid with 8.7% yield. (expected yield: 7-15%)
③ controlling the reaction process of the intermediate C9:
the checking method comprises the following steps: TLC method;
thin-layer plate: GF 254;
developing agent: ethyl acetate: petroleum ether 1:5 and ethyl acetate;
the preparation method of the test solution comprises the following steps: quenching a small amount of reaction solution with appropriate amount of methanol, spotting on a plate, and spotting with ethyl acetate solution of C9-3 on the same silica gel plate;
the color development method comprises the following steps: after the development, observing under an ultraviolet lamp (254 nm);
monitoring the reaction end point:
ethyl acetate: petroleum ether-1: 5 system: c9-3: rf 0.5, C9: rf is 0, and the C9-3 spot in the reaction solution is required to be substantially disappeared;
ethyl acetate system: c9-3: rf 1, C9: when Rf is 0.2 and the content of C9 specks in the reaction mixture is required to be about 10 to 20%, the reaction can be stopped.
The 5- (4- (4- (3, 5-dihydroxystyryl) phenoxy) styryl) -1, 3-benzenediol compound with the structural formula has the functions of inhibiting the growth of B-cell malignant tumors and the signal conduction of AKT/mTOR1 in B-cells, can be applied to the preparation of medicines for treating autoimmune diseases and B-cell malignant tumors, and is specifically as follows:
b-cell malignancies are common hematological malignancies, and although most patients respond to current first-line therapy, they have a high recurrence rate and poor prognosis and remain one of the refractory malignancies. There is an increasing trend in the incidence of B cell malignancies such as Multiple Myeloma (MM) and B cell non-hodgkin's lymphoma (B-NHL) patients.
The present inventors first examined C28H22O5(5- (4- (4- (3, 5-dihydroxystyryl) phenoxy) styryl) -1, 3-benzenediol compound of structural formula) on cell proliferation of B cell tumors.
The results of MTS or MTT measurement in cell proliferation experiments show that: c28H22O5Significant inhibition of lymphoma cell line (Raji) (FIG. 2) and Multiple Myeloma (MM) at 48 hours) Tumor cell proliferation of cell line (RPMI1640) (fig. 3). (RajiIC50 at 8. mu.M and RPMI8226IC50 at 4. mu.M) (FIGS. 2 and 3).
C28H22O5Also significantly inhibited B cell proliferation of lymphoma cell line (A20). Different concentrations of C28H22O5Control group C35H32O8(structural formula shown in figure 1) (0.25-64. mu.g/mL) after treating mouse lymphoma cells (A20)24, 48, 72, 96h, the effect of the drug on the growth was observed, and the results are shown in figure 4-6, C28H22O5The compound shows obvious cytotoxic effect under the concentration of 0.5-64 mu g/mL, and compared with a control group, the growth inhibition rate is more than 90%, but the dose dependence is not obvious.
C35H32O8Inhibiting effect is compared with C28H22O5Weak and also exhibits no significant dose-time dependence.
C28H22O5、C35H32O8Half inhibition of A20 cells is shown in FIGS. 4-6, C28H22O5Has obvious effect of inhibiting A20 cell proliferation in vitro.
In vivo pharmacodynamic evaluation selection C28H22O5Is the object of study. In apoptosis experiments, C28H22O5Raji and RPMI1640 tumor cells were significantly induced to undergo apoptosis, and the induced apoptosis showed a drug concentration-dependent curve (FIG. 7).
C28H22O5Pharmacodynamic evaluation of in vivo anti-lymphoma transplantation model:
experimental animals: male BALB/c mice, weighing 18-20g, were purchased from Beijing Wittingle laboratory animal technology, Inc. (animal certification number: SCXK (Jing) 2009-0017). Experiments are carried out in the experimental animal barrier environment of Chinese medicine basic theory research institute of Chinese academy of traditional Chinese medicine [ SYXK (Jing) 2010-0032 ], and humane care is given according to the 3R principle used by experimental animals.
Molding: a method for preparing a B-cell lymphoma transplantation model by subcutaneous modeling comprises the following steps: after 24h of moldingThe group is divided into 7 groups, each group comprises 10-12 mice, and the groups respectively comprise: blank control group (I), false operation group (II), model group (III), cyclophosphamide group (IV) and C28H22O5Group (V), C) for administration of 100mg/kg28H22O550mg/kg administration group (VI), C28H22O5Group (VII) administered at 25 mg/kg. The method adopts an intraperitoneal injection mode, the administration is carried out for 1 time every day, the administration volume is 0.1mL/10g, the intraperitoneal injection of a sham operation group is carried out with the same volume of normal saline, and the administration of cyclophosphamide is carried out for 1 time every week and 2 times in total.
Detection indexes are as follows: body weight, tumor volume, thymus index, spleen index, blood routine, pathology examination.
As a result: the increase in body weight of the model group was significant with the growth of the tumor after modeling, and after 4 weeks, the body weight of the model group was higher than that of each group (fig. 8); the body weight gain of the CP group and the high dose group was lower than that of the model group and the sham operation group, and the difference was statistically significant compared with that of the sham operation group and the model group (FIG. 8). After 4 weeks of administration, the small animal ultrasonic imaging system detects the tumor volume, and the result shows that the cyclophosphamide group (IV) does not form obvious tumor, C28H22O5The tumor volume of the compound (V) in the administration group of 100mg/kg is obviously reduced compared with that in the model group, and the compound has statistical significance (P) compared with the model group<0.01) (fig. 9). The mice were sacrificed and the tumor tissue was stripped and weighed, and the results showed that the tumor weight in the model group was significantly heavier than that in each dose group, but not statistically significant (fig. 10). H&Pathological examination of E staining revealed that tumor tissue cells in the model group had uneven size, deep-stained nuclei, abundant blood vessels (FIG. 11A), and C28H22O5The 100mg/kg group (V) showed a decrease in tumor tissue blood vessels, poor blood supply, a reduction in nuclei compared to the other groups, a partially vacuolated change (FIG. 11B), a decrease in tumor tissue blood vessels (FIG. 11C) in the 50mg/kg group (VI), and a deep staining of tumor cell nuclei in the 25mg/kg group (VII), no decrease in tumor tissue blood vessels, and a change in tumor tissue was not significant compared to the model group (FIG. 11D).
And (4) conclusion: c28H22O5Has the function of resisting mouse lymphoma in vivo and in vitro. The in vivo antitumor effect may be combined with the improvement of bodyThe immune function is related.
Autoimmune diseases including Systemic Lupus Erythematosus (SLE) Rheumatoid Arthritis (RA), type I diabetes and Multiple Sclerosis (MS) have been studied for decades. Autoimmunity is caused by abnormal B cell and T cell recognition of self tissues as antigens, resulting in the production of autoantibodies by B cells. B cell function is very relevant to autoimmune disease activity, including SLE, RA, scleroderma, type I diabetes and MS. Tumor necrosis factor alpha (TNF α) has long been recognized in autoimmune diseases. TNF α, IL10 and IL6 play different roles in the development of autoimmune diseases. B cells circulating in the body by the potentially harmful immune system are not permanently inactivated and demonstrate that these harmful B cells can be reactivated and attack the body's own tissues.
C28H22O5Evaluation of the pharmacological efficacy against autoimmune diseases in vivo:
the production of autoantibodies by B cells plays an important role in autoimmune diseases, and some transcription factors in B cells cause abnormal development of B cells, thereby driving the development of autoimmune diseases. Applicant's examination C28H22O5Effect on proliferation of B cells. The results of MTS or MTT measurement in cell proliferation experiments show that: c28H22O5Tumor cell proliferation was significantly inhibited at 48 hours for lymphoma cells (Raji) (FIG. 2) and Multiple Myeloma (MM) B cell line (RPMI1640) (FIG. 3). (RajiIC50 at 8. mu.M, RPMI8226IC50 at 4. mu.M) (FIGS. 2, 3).
C28H22O5Also significantly inhibited B cell proliferation of lymphoma cell line (A20). Different concentrations of C28H22O5、C35H32O8The effect of the drug on the growth of the mouse lymphoma cells (A20) was observed 24, 48, 72, 96h after treatment (0.25-64. mu.g/mL) and the results are shown (FIGS. 4-6). Further experimental animals also confirmed C28H22O5Effects on the mouse immune system. The test method selects the in vivo anti-lymphoma transplantation model. The method comprises the following steps: blank control group (I), false operation group (II), model group (III),cyclophosphamide group (IV), C28H22O5Group (V), C) for administration of 100mg/kg28H22O550mg/kg administration group (VI), C28H22O5Group (VII) administered at 25 mg/kg. The method adopts an intraperitoneal injection mode, the administration is carried out for 1 time every day, the administration volume is 0.1mL/10g, the intraperitoneal injection of a sham operation group is carried out with the same volume of normal saline, and the administration of cyclophosphamide is carried out for 1 time every week and 2 times in total.
Detection indexes are as follows: thymus index, spleen index, blood convention.
The experimental result shows that C28H22O5The (100mg/kg) administration group (V) obviously improves the thymus index, and the thymus index of each treatment group is reduced compared with that of the blank group and the sham operation group, C28H22O5The thymus index was higher in the (100mg/kg) administered group (V) than in each group, and was statistically significant compared with the model group (FIG. 12). Spleen index of each administration group was increased as compared with that of the model group, C28H22O5(100mg/kg) administration group (V), C28H22O5The 50mg/kg administration group (VI) was statistically significant compared with the model group (FIG. 13).
C28H22O5Group (V), C) for administration of 100mg/kg28H22O5The WBC number was significantly higher in the (50mg/kg) administered group (VI) than in each group, and was statistically significant compared with the model group (FIG. 14). The model group and each administration group had a reduced RBC count compared to the sham operation group, and had statistical significance (fig. 15).
Investigation of mechanism of action
Determine C28H22O5Inhibit B-cell proliferation and induction, and we further examined C28H22O5The mechanism of action of the compound. Molecules involved in the AKT and NF-. kappa.B signaling systems are important factors in B cell development, proliferation, and metabolism. We further examined the gene expression of molecules involved in the AKT-mTOR signaling pathway including AKT1, AKT2, AKT3, mTOR, Cyclin D1, and P27kip gene expression, and showed that C28H22O5The gene expression of AKT1 was significantly reduced, but AKT2 and AKT3 were unchanged. Reduced expression of AKT Cyclin D1, P27kipThere was no change (fig. 16). Experimental suggestion C28H22O5The compounds affect B-cell metabolism through the AKT-mTOR signaling system.

Claims (5)

1. A styryl compound, characterized by a 5- (4- (4- (3, 5-dihydroxystyryl) phenoxy) styryl) -1, 3-benzenediol compound of the following structural formula:
Figure FDA0002758963150000011
2. the process for producing a phenylalkenyl compound according to claim 1, wherein the reaction formula is as follows:
Figure FDA0002758963150000012
3. use of the phenylalkenyl compound of claim 1 for the preparation of a medicament for the treatment of an autoimmune disease; the autoimmune disease is one of rheumatoid arthritis, systemic lupus erythematosus, sjogren's syndrome, lung and kidney syndrome, autoimmune hepatitis, primary biliary cirrhosis, Crohn's disease, and ulcerative colitis.
4. The use of the phenylalkenyl compound of claim 1 in the preparation of a medicament for the treatment of a B-cell malignancy; the application is the application in preparing the medicine for inhibiting the growth of B-cell malignant tumor; the B-cell malignant tumor is one of lymphoma, multiple myeloma and chronic lymphocytic leukemia.
5. Use of the phenylalkenyl compound of claim 1 for the preparation of a medicament for inhibiting the signaling of AKT/mTOR1 in B-cells.
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