CN109134470B

CN109134470B - Selenium-containing compound and application thereof

Info

Publication number: CN109134470B
Application number: CN201710450505.1A
Authority: CN
Inventors: 贺贤然; 李少磊
Original assignee: Shenzhen Fushan Biotechnology Co ltd
Current assignee: Shenzhen Fushan Biotechnology Co ltd
Priority date: 2017-06-15
Filing date: 2017-06-15
Publication date: 2021-04-16
Anticipated expiration: 2037-06-15
Also published as: CN109134470A

Abstract

The invention relates to application of a riboflavin selenium derivative shown as a formula I in preparing a cancer chemopreventive agent.

Description

Selenium-containing compound and application thereof

Technical Field

The invention belongs to the field of biological medicine, and particularly relates to a selenium-containing compound and a preparation method and application thereof.

Background

Cancer is the leading cause of illness and death worldwide, and is expected to increase as modern technology extends life expectancy. During the life of a cell, small changes in DNA called "mutations" occasionally occur. Among these mutations, some mutations (referred to as "silent mutations") do not result in any substantial change in cell function, while others may alter the mode of action of the cell. Various mechanisms can prevent cells that have mutated from continuing the cell cycle and if genetic errors are not corrected, these cells will "suicide" through a process called "apoptosis". However, if mutations occur in proteins involved in cell cycle regulation, this can lead to uncontrolled cell proliferation (known as tumor formation), which can further progress to cancer.

Cancer cells often have adverse effects on the body. Cancer can spread by invasion of adjacent tissues by malignant tumor cells, and can also spread by a process known as "metastasis" in which malignant cells detach from the tumor mass and spread to distant sites. Cancer appears in many different types of tissues in multiple forms and can be characterized by its degree of invasion and invasiveness.

Cancer occurs as a mass of abnormal tissue in a living host organism, which receives nutrients from the host without relying on host hyperproliferation and destroys the host organism. The human organ is composed of a large number of cells. Cancer occurs when normal cells of the human body become abnormal cells and the abnormal cells divide and proliferate without examination. Although genetic factors are closely related to the onset of cancer, environmental factors also have a significant impact on whether an individual develops cancer. Cancer is particularly prevalent in developed countries. It has been reported that the causes of cancer are increased use of pesticides, insecticides, etc. (and thus the amount of such substances remaining in foods) and consumption of processed foods containing additives such as food preservatives and colorants, increased pollution of water, soil and air, stress of modern life, reduction of activities, obesity caused by greasy dietary habits, and the like. In recent years, it has also been pointed out that cancer is caused when the cell signaling system of normal cells fails, when cancer genes are activated, or when cancer suppressor genes fail.

Various cancer treatment methods exist, such as surgical treatment, chemotherapy and radiotherapy. The surgical treatment method effectively removes cancer at an early stage, but has disadvantages in that organs have to be removed from time to time, which causes side effects, and there is uncertainty in spreading cancer to other organs. Radiation therapy is advantageous for effectively treating cancer occurring in a particular organ, but has the following disadvantages: exposure of the patient to other cancer risks due to radiation, failure to prevent the spread of cancer cells to other organs, and significant pain to the patient during treatment. Chemotherapy is generally performed using anticancer drugs, but it is known that toxicity of anticancer drugs acts not only on cancer cells but also on normal cells of patients, causing side effects. Therefore, development of new anticancer drugs having higher cancer cell selectivity and as little toxicity as possible is desired.

Selenium is a trace element essential for life activities of the body. In recent years, studies have been made on selenium compounds, particularly organic selenium compounds, in an attempt to find compounds having anticancer or antitumor activities therefrom. For example, EI-Baulomy et al [ K El-Baulomy, Drugs Future,1997, 22(5): 539-545 ] found that benzyl selenium cyanide exhibited anti-tumor effects in a mouse model of DMBA-induced breast cancer. Benzyl selencyanide has a higher anticancer activity than sodium selenite, but has a strong off-taste itself and has side effects that cause significant weight loss in patients.

The research shows that the action mechanism of ebselen is mainly to inhibit the activity of target enzyme thioredoxin reductase and regulate the downstream signal conduction path and the anti-tumor apoptosis path thereof to realize the anti-tumor action of the drug, and the bioactivity and low toxicity of ebselen may be related to the cyclic selenamide structure or the benzisoselenone-containing heterocyclic ring (H J Reich, et al J.Am.chem.Soc., 1987, 109(18):5549-, has synergistic effect and activity superior to that of ebselen.

Despite the discovery of the above organic selenium compounds, the existing organic selenium compounds still have the problems of further improved anticancer efficacy, limited anticancer spectrum, and limited structural types of compounds, and are far from meeting the increasing demands of human beings for cancer prevention and treatment. Therefore, the development of more effective anticancer drugs, especially organic selenium compounds, has been urgently needed.

Therefore, there is still an urgent need in the art for a novel cancer prophylactic or therapeutic agent having a good effect.

Disclosure of Invention

Through a great deal of experimental research, the inventor unexpectedly finds that the selenium-containing organic compound has unexpected biological activity for preventing and treating cancers. The compounds are useful in the treatment and/or prevention of various cancers.

The present invention provides a compound of formula I:

in another aspect, the present invention also provides a process for the preparation of a compound of formula I as described above, said process comprising the steps of:

(i) converting the compound of formula II into compound III,

(ii) hydrolyzing the compound of formula III to provide compound IV; and

(iii) amination of the compound of formula IV affords the compound of formula I.

In the preparation process of the present invention, the compound of formula IV is preferably reacted with 2-selenoethylamine hydrochloride in step (iii) to give compound I.

In the preparation process of the present invention, the compound of the formula II is preferably reacted with tert-butyl 2-bromoacetate in step (i) to give the compound III.

In a particularly preferred embodiment, the compounds of formula I are prepared by the following method: firstly, reacting a compound shown in a formula II with tert-butyl 2-bromoacetate to obtain a compound III, then reacting the compound III with trifluoroacetic acid in a dichloromethane solvent to obtain a compound IV, and then reacting the compound IV with 2-selenoethylamine hydrochloride in the dichloromethane solvent to obtain a compound I.

In the above reaction, the compound II can be obtained commercially or by reacting riboflavin with an acetic anhydride/pyridine mixture.

The reaction conditions for the above reaction are preferably as follows: the molar ratio of the riboflavin to the acetic anhydride is 1: 1-4, the reaction temperature is 0-50 ℃, and the reaction time is 24 hours; the molar ratio of the compound shown in the formula II to the tert-butyl 2-bromoacetate is 1: 1-2, the reaction temperature is 0-40 ℃, and the reaction time is 10-20 hours; the molar ratio of the compound III to trifluoroacetic acid is 1: 2-5, the reaction temperature is 0-50 ℃, and the reaction time is 5-10 hours; the molar ratio of the compound IV to the 2-selenocyanoethylamine hydrochloride is 1: 1.2-2.0, the reaction temperature is 0-40 ℃, and the reaction time is 2-12 hours.

The reaction formula is as follows:

the preparation method of the invention is simple, has high yield, and can easily prepare the compound of the formula I.

In another aspect of the invention, there is provided a pharmaceutical composition comprising a compound of formula I of the invention or a pharmaceutically acceptable salt thereof and optionally pharmaceutically acceptable excipients and/or carriers. In the pharmaceutical composition of the present invention, other pharmaceutically active ingredients may be further included in addition to the compound of formula I of the present invention or a pharmaceutically acceptable salt thereof. The pharmaceutical compositions of the invention may be prepared by conventional techniques, for example as described in Remington: the method described in The Science and Practice of Pharmacy, 19 th edition, 1995, which is incorporated herein by reference. The compositions may be presented in conventional forms, such as capsules, tablets, aerosols, solutions, suspensions or topical application forms.

Typical compositions comprise a compound of formula I of the present invention or a salt thereof and a pharmaceutically acceptable excipient or carrier. For example, the active compound is typically mixed with a carrier, or diluted by a carrier, or enclosed within a carrier which may be in the form of an ampoule, capsule, sachet (sachet), paper or other container. When the active compound is mixed with a carrier, or when the carrier serves as a diluent, the carrier can be a solid, semi-solid, or liquid material that serves as a carrier, excipient, or medium for the active compound. The active compound may be adsorbed on a particulate solid carrier (e.g. contained in a sachet). Some examples of suitable carriers are water, salt solutions, alcohols, polyethylene glycols, polyhydroxyethoxylated castor oil, peanut oil, olive oil, gelatin, lactose, terra alba, sucrose, dextrin, magnesium carbonate, sugars, cyclodextrins, amylose, magnesium stearate, talc, gelatin, agar, pectin, acacia, stearic acid or lower alkyl ethers of cellulose, silicic acid, fatty acids, fatty acid amines, fatty acid mono-and diglycerides, pentaerythritol fatty acid esters, polyoxyethylene, hydroxymethylcellulose and polyvinylpyrrolidone. Similarly, the carrier or diluent may include any sustained release material known in the art, such as glyceryl monostearate or glyceryl distearate alone or with a wax.

The formulations may be mixed with adjuvants which do not deleteriously react with the active compound. These additives may include wetting agents, emulsifying and suspending agents, salts for influencing osmotic pressure, buffering and/or coloring substances, preservatives, sweeteners or flavorings. The composition may also be sterilized, if desired.

The route of administration may be any route which is effective for the transport of a compound of formula I of the present invention to the appropriate or desired site of action, for example the oral, nasal, pulmonary, buccal, subcutaneous, intradermal, transdermal or parenteral routes, for example the rectal, depot (depot), subcutaneous, intravenous, intraurethral, intramuscular, intranasal, ophthalmic solutions or ointments route, the oral route being preferred.

If a solid carrier is used for oral administration, the formulation may be tableted, placed in a hard gelatin capsule as a powder or pellet, or it may be in the form of a troche (troche) or lozenge. If a liquid carrier is used, the formulation may be in the form of a syrup, emulsion, soft gelatin capsule, or sterile injectable liquid, such as an aqueous or non-aqueous liquid suspension or solution.

Injectable dosage forms typically comprise an aqueous or oily suspension, which may be formulated using suitable dispersing or wetting agents and suspending agents. Injectable forms may be in the form of a solution phase or a suspension prepared with a solvent or diluent. Acceptable solvents or carriers include sterile water, ringer's solution, or isotonic saline solution. Alternatively, sterile oils may be employed as a solvent or suspending agent. Preferably, the oil or fatty acid is non-volatile and comprises a natural or synthetic oil, a fatty acid, a monoglyceride, diglyceride, or triglyceride.

For injection, the formulation may also be a powder suitable for reconstitution with a suitable solution as described above. Examples of these include, but are not limited to, freeze-dried, spin-dried or spray-dried powders, amorphous powders, granules, precipitates or microparticles. For injections, the formulation may optionally include stabilizers, pH modifiers, surfactants, bioavailability modifiers, and combinations of these agents. The compounds may be formulated for parenteral administration by injection, for example by bolus injection or continuous infusion. Unit dosage forms for injection may be in ampoules or in multi-dose containers.

The formulations of the present invention may be designed to provide rapid, sustained or delayed release of the active ingredient after administration to a patient by methods well known in the art. Thus, the formulation may also be formulated for controlled release or slow release.

The compounds of formula I of the present invention are effective over a wide dosage range. For example, in the treatment of adults, a dose of about 0.05 to about 5000mg, preferably about 1 to about 2000mg, more preferably about 2 to about 2000mg per day may be used. Typical dosages are from about 10mg to about 1000mg per day. When selecting a patient treatment regimen, it may often be necessary to start with a higher dose and reduce the dose when the condition is controlled. The precise dosage will depend upon the mode of administration, the desired treatment, the form of administration, the subject to be treated and the weight of the subject to be treated, as well as the preferences and experience of the attending physician or veterinarian.

Typically, the compounds of formula I of the present invention are dispensed in unit dosage forms containing from about 0.05mg to about 1000mg of the active ingredient per unit dose and a pharmaceutically acceptable carrier.

In general, a dosage form suitable for oral, nasal, pulmonary or transdermal administration comprises from about 125 μ g to about 1250mg, preferably from about 250 μ g to about 500mg, more preferably from about 2.5mg to about 250mg of said compound of formula I in admixture with a pharmaceutically acceptable carrier or diluent.

The dosage form may be administered once daily, or more than once daily, e.g., twice daily or three times daily. Alternatively, the dosage form may be administered less frequently than once daily, for example every other day or weekly, if deemed appropriate by the prescribing physician.

The pharmaceutical compositions of the present invention may be in the form of tablets, capsules, powders, granules, lozenges, liquids or gels. Tablets and capsules for oral administration may be in a form suitable for unit dose administration and may contain conventional excipients, such as: binders such as syrup, gum arabic, gelatin, sorbitol, tragacanth, polyvinylpyrrolidone (PVP); fillers such as lactose, sugars, corn flour, calcium phosphate, sorbitol or glycine; tablet lubricants such as magnesium stearate, silicon dioxide, talc, polyethylene glycol or silicon dioxide; disintegrants such as potato starch; acceptable lubricants such as sodium lauryl sulfate. The tablets may be coated according to known methods of conventional pharmaceutical practice. Oral liquid preparations may be in the form of aqueous or oily suspensions, solutions, emulsions, syrups or tinctures, or may be presented as a dry substance for reconstitution with water or other suitable vehicle before use. These liquid preparations may contain conventional additives such as suspending agents (e.g., sorbitol, syrup, methyl cellulose, glucose syrup, gelatin, hydrogenated edible fats and oils). Emulsifying agents (e.g. lecithin, sorbitol monooleate or acacia), non-aqueous vehicles (including edible oils such as almond oil, fractionated coconut oil, fats and oils such as glycerol, propylene glycol or ethanol), preservatives (e.g. methyl or propyl p-hydroxybenzoic acid or sorbic acid), and if desired conventional flavouring or colouring agents.

The dosage may vary with the method and dosage form of administration, as well as the age, weight, condition and sensitivity of the patient. In the case of oral administration, an effective daily dosage range, for example, may be from 20mg to 1 g. Single dose units containing a compound of formula I or a pharmaceutically acceptable salt thereof in an amount of from 20mg to 200mg may conveniently be employed to meet the daily dosage requirements. The dosages and dosage units employed may be outside the ranges set forth above.

The percentage of active substance in the pharmaceutical composition of the present invention is variable because the pharmaceutical formulation must be formulated in a suitable proportion of the dosage to achieve the desired therapeutic effect. In general, the pharmaceutical preparations according to the invention should be administered orally or by injection in an amount of 1 to 15 mg of the compound of the formula I per 70kg of body weight per day. The following examples are for the purpose of illustrating certain aspects of the invention and should not be construed as limiting the scope of the invention in any way.

Detailed Description

Examples

Examples of synthetic preparations

Example 1: synthesis of Compounds of formula II

Riboflavin (1g, 2.66mmol) was dissolved in pyridine (10ml) in a three-necked flask, acetic anhydride (1.356g, 13.3mmol) was added, and the mixture was stirred for 16 hours to complete the reaction by TLC. The reaction solution was dissolved in 30 mL of water, the aqueous phase was extracted with methylene chloride (10 mL. times.3), and the combined organic phases were washed with saturated brine (20 mL. times.1) and dried over anhydrous sodium sulfate. The solvent was removed by distillation under the reduced pressure, and the resulting crude product was purified by column chromatography (ethyl acetate: petroleum ether (v/v) ═ 20:1) to give 721mg of a yellow powdery solid in 50% yield.

Nuclear magnetic resonance¹H NMR(400MHz,CDCl₃)δ:8.60(s,1H),8.03(s,1H),7.57(s,1H),5.68～5.66(d,1H),5.46～5.39(m,2H),4.91(s,1H),4.45～4.42(m,1H),4.27～4.22(m,1H),2.57(s,3H),2.45(s,3H),2.30(s,3H),2.22(s,3H),2.08(s,3H),1.76(s,4H).

MS[ESI]Calculated value (C)₂₅H₂₈N₄O₁₀)⁺545.18, found: 545.40.

example 2: synthesis of Compounds of formula III

Compound II (670mg) was dissolved in anhydrous DMF (5ml) in a three-hole flask, potassium carbonate (204mg) and potassium iodide (20mg) were added, and after stirring for 30 minutes, t-butyl bromoacetate (1.68g) was added, and after the addition, the reaction mixture was heated to 40 ℃ to continue the reaction for 16 hours. TLC showed the reaction was complete, after the reaction was cooled to room temperature, water (10mL) was added, the aqueous phase was extracted with ethyl acetate (15 mL × 3), and the combined organic phases were washed with saturated brine (20mL × 1) and dried over anhydrous sodium sulfate. The solvent was removed by distillation under the reduced pressure, and the resulting crude product was purified by column chromatography (ethyl acetate: petroleum ether ═ 5:1) to give a yellow powdery solid (770 mg) in 95% yield.

Nuclear magnetic resonance¹H NMR(400MHz,CDCl₃)δ:8.02(s,1H),7.55(s,1H),5.66～5.64(d,1H),5.38～5.41(m,2H),4.94～4.92(m,1H),4.71(s,2H),4.43～4.40(m,1H),4.27～4.22(m,1H),2.55(s,3H),2.43(s,3H),2.28(s,3H),2.20(s,3H),2.06(s,3H),1.75(s,4H),1.46(s,9H).

MS[ESI]Calculated value (C)₃₁H₃₈N₄O₁₂)⁺659.25, found: 659.31.

example 3: synthesis of Compounds of formula IV

A100 ml three-neck flask is added with compound III (200mg,0.3mmol) and 1ml of anhydrous dichloromethane, and then cooled to 0 ℃ by introducing argon gas, 0.3ml of trifluoroacetic acid is added, and the mixture is heated to 50 ℃ and stirred for 5 h. After the reaction was completed, the reaction solution was poured into ice water, pH was adjusted to 5 with a saturated sodium bicarbonate solution, dichloromethane was added for extraction, the collected organic phase was washed with saturated brine and then with water, the collected organic phase was dried over anhydrous magnesium sulfate, suction filtration, vacuum drying, and column chromatography was evaporated to dryness (ethyl acetate: ethanol ═ 1:1(V: V)) to obtain 120mg of a pale yellow solid (compound IV), yield: 66 percent.

Nuclear magnetic resonance¹H NMR(400MHz,D₂O)δ:7.82(s,1H),7.74(s,1H),5.67-5.65(m,1H),5.53(br t,J＝6,1H),5.49-4.45(m,1H),5.10(br s,2H),4.55(s,2H),4.51(dd,J₁＝12；J₂＝3Hz,1H),4.38(dd,J₁＝12；J₂＝6Hz,1H),2.59(s,3H),2.46(s,3H),2.26(s,3H),2.24(s,3H),2.03(s,3H),1.73(s,3H).

MS[ESI]Calculated value (C)₂₇H₃₀N₄O₁₂)⁺603.19; measured value 603.21

Example 4: synthesis of Compounds of formula I

A25 ml two-neck flask was charged with compound IV (100mg, 0.16mmol) and 20ml of anhydrous dichloromethane, stirred at room temperature, successively charged with carbodiimide (37.1mg, 0.18mmol), 1-hydroxybenzotriazole (24.3mg, 0.18mmol) and 2-selenocyanulethylamine hydrochloride, purged with nitrogen, stirred at room temperature for 6.0 hours, TLC monitored for completion of the reaction, distilled under reduced pressure to remove the solvent, chromatographed on silica gel with ethyl acetate: petroleum ether 1:10(V: V) was used as a mobile phase to give 90mg of yellow solid (compound I) in 76% yield.

Nuclear magnetic resonance¹H NMR(400MHz,CDCl₃)δ:8.04(s,1H),7.57(s,1H),5.67～5.65(d,1H),5.56(s,2H),5.43(s,2H),4.43～4.40(d,1H),4.28～4.22(m,3H),3.90～3.86(t,2H),3.18～3.14(t,2H),3.04～3.00(t,2H),2.56(s,3H),2.43(s,3H),2.36(s,3H),2.29(s,3H),2.20(s,3H),2.14(s,2H),2.06(s,3H).

MS[ESI]Calculated value (C)₃₀H₃₆N₆O₁₁)⁺735.60; measured value 735.62

In vitro pharmacology

Experimental example 1 cytotoxic Activity experiment

Tumor cells were taken at log phase 3X 10 per well⁴Inoculating on a 96-well plate, adding 200 μ L of DMEM medium to each well, removing the supernatant after 12h, then adding drugs to multiple wells according to blank group and drug adding group (with concentration of 0,2.5,5,10,20,30 and 50 μ M respectively), wherein the compound of formula I prepared in example 4 is added to the drug adding group, and removing the supernatant after culturing for 24 h. mu.L of MTT-containing solution prepared by dissolving MTT (thiazole blue, Biyuntian reagent) in phosphate buffer (PBS, pH 7.3) at a concentration of 0.5mg/mL was added thereto and cultured for 4 hours, and then 100. mu.L of dimethyl sulfoxide (DMSO) was added to each well, followed by measuring the OD (optical density) value at 570nm on a microplate reader with shaking for 1 hour. Fitting and calculating the gradient dosage of each tumor cell line and the corresponding proliferation inhibition rate to obtain a nonlinear regression equation, IC₅₀The value is the amount of the drug added when the tumor cell line proliferation inhibition rate is 50%. Wherein, the proliferation inhibition ratio = (blank OD value-administered OD value)/blank OD value. ) The results show that the activity of the tumor cells is obviously reduced after the medicine is added. In this experiment, the cytotoxic activity of human esophageal cancer cells ECA109, TE-13 and SKGT-4 were examined, and the above cell lines were purchased from ATCC, and the specific results are shown in Table 1.

TABLE 1

The experimental result shows that the compound I prepared in the example 4 has cytotoxic activity to different tumor cells, especially has good cytotoxic activity to ECA109 cell strains, and can play a significant role in tumor cell inhibition.

Experimental example 2 quinone reductase Induction experiment

Logarithmic phase mouse hepatoma cells (Hepa1c1c7, purchased from ATCC) were collected at 3X 10 cells per well⁴Inoculating on 96-well plate, adding 200 μ L DMEM medium to each well, removing supernatant after 12h, adding compound I prepared in example 4 to multiple wells, and repeatingAfter the control group and the drug-added group were cultured for 24 hours, the supernatant was discarded. Adding 4' -bromoflavone (0.106mg) as positive control drug-adding group; dimethyl sulfoxide (0.106mg) was added as a negative control group; blank was 200 μ L DMEM medium without drug. After 24h incubation, the supernatant was discarded. Digitonin was added to each of the drug-addition group of compound I, the positive control drug-addition group, the negative control group, and the blank group to lyse the cells, 200 μ L of a solution containing MTT was added and cultured for 5min, the MTT solution was formed by dissolving MTT (thiazole blue, picnic reagent) in phosphate buffer solution (PBS, pH 7.3) at a concentration of 0.5mg/mL, and the IR value was measured at 550nm on a microplate reader.

Fold induction: IR ═ [ (additivated OD value-blank OD value)/(negative control OD value-blank OD value) ]/(1-inhibition of proliferation%); wherein, the proliferation inhibition ratio = (blank group OD value-additive group OD value)/blank group OD value. The results showed that the IR values at the administration concentrations of compound I prepared in example 4 were 2.52 (the IR value of the positive control 4' -bromoflavone was 2.21), respectively, and the results showed that the compound of formula I was able to effectively induce the expression of quinone reductase, and was considered to have a cancer preventive effect at the initial stage of tumorigenesis. Specific results are shown in table 2.

TABLE 2

^a4' -Bromoflavone as positive control

The experimental result shows that the IR value of the compound I prepared in example 4 is greater than that of the positive control drug 4' -bromoflavone, which indicates that the compound of the formula I has good quinone reductase induction activity and better cancer prevention effect at the initial stage of tumor.

Claims

1. A compound of formula I:

or a pharmaceutically acceptable salt thereof.

2. A process for preparing the compound of claim 1, comprising the steps of:

(i) converting the compound of formula II into compound III,

(ii) hydrolyzing the compound of formula III to provide compound IV; and

3. The process according to claim 2, wherein in step (i) the compound of formula II is reacted with tert-butyl 2-bromoacetate to give compound III.

4. The process of claim 2, wherein the compound of formula IV is reacted with 2-selenoethylamine hydrochloride in step (iii) to provide the compound of formula I.

5. A pharmaceutical composition comprising a compound of formula I as claimed in claim 1 or a pharmaceutically acceptable salt thereof, and optionally a pharmaceutically acceptable excipient and/or carrier.

6. Use of a compound of formula I according to claim 1 or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment or prevention of cancer.

7. The use of claim 6, wherein the cancer is selected from the group consisting of esophageal cancer, gastric cancer, breast cancer, prostate cancer, cervical cancer, liver cancer, lung cancer, and colon cancer.