CN109939114B

CN109939114B - Meropenem intermediate related compound and application thereof in antitumor drugs

Info

Publication number: CN109939114B
Application number: CN201910335646.8A
Authority: CN
Inventors: 潘庆华; 钱广; 朱杰; 朱红薇
Original assignee: Jiangsu Hankuo Biological Co ltd; Jiaxing University
Current assignee: Jiaxing University
Priority date: 2019-04-24
Filing date: 2019-04-24
Publication date: 2021-09-03
Anticipated expiration: 2039-04-24
Also published as: CN109939114A

Abstract

The invention provides a meropenem intermediate related compound and application thereof in preparing an anti-tumor medicament. The related compounds have the following chemical formula (I):

Description

Meropenem intermediate related compound and application thereof in antitumor drugs

Technical Field

The invention belongs to the technical field of medicines, and relates to application of meropenem intermediate related compounds in development of antitumor drugs.

Background

Cancer has been one of the most difficult diseases to deal with in terms of life health of modern people. According to the statistics of the world health organization (WH0), about 1000 million people with cancer and about 700 million people with cancer death worldwide have become second killers of people with cardiovascular diseases. Therefore, the search for effective anti-cancer drugs is the focus of most cancer therapists. In the research of anticancer drugs, the research and clinical application of chemotherapeutic drugs have been greatly advanced. Chemically synthesized anticancer drugs such as metal complex platinum complex, gallium salt, organic germanium compound, tin complex and the like have the characteristics of strong anticancer property, wide antitumor spectrum and the like, but have the defects of large toxic and side effects, high manufacturing cost, poor water solubility and the like.

Meropenem is called top-level antibiotic, is a synthetic broad-spectrum carbapenem antibiotic, and is suitable for infection of adults and children caused by single or multiple bacteria sensitive to meropenem; and is also the first choice medicine for serious infection in hospital, mixed infection mainly comprising G-bacteria, multiple drug-resistant bacteria infection and enzyme-producing bacteria infection. Also can be used for treating pneumonia including nosocomial pneumonia, urinary tract infection, gynecological infection (such as endometritis and pelvic inflammatory disease), skin soft tissue infection, meningitis, septicemia, etc. The antibacterial mechanism is to generate antibacterial action by inhibiting the synthesis of bacterial cell walls, and meropenem easily penetrates the cell walls of most gram-positive and gram-negative bacteria to achieve the action target point of Penicillin Binding Protein (PBPS). In addition to metallo beta-lactamases, they are more stable against hydrolysis by most beta-lactamases, including penicillinase and cephalosporinase produced by gram-positive and gram-negative bacteria. Meropenem is not suitable for the treatment of methicillin-resistant staphylococcal infections and sometimes exhibits cross-resistance to other carbapenem-resistant strains. In vitro experiments show that, for some isolated strains of pseudomonas aeruginosa, the combination of meropenem and aminoglycoside antibiotics can generate synergistic effect. The above is the main discovery of the activity of meropenem and intermediates thereof at home and abroad so far. Currently, the research on meropenem in the field focuses on synthesis, and few reports on the biological activity of related compounds exist.

Disclosure of Invention

The invention provides a new application of a meropenem intermediate related compound, and the meropenem intermediate related compound has antitumor activity and application potential in preparation of antitumor drugs.

The invention provides the following technical scheme:

the invention provides an application of meropenem intermediate related compound in preparing antitumor drugs, wherein the related compound has the following chemical formula (I):

such tumors include, but are not limited to, breast cancer, liver cancer, gastric cancer, or leukemia. The meropenem intermediate related compound can be applied to preparation of antitumor drugs for treating or preventing breast cancer, liver cancer, gastric cancer or leukemia. According to the requirement, the anti-tumor medicine can contain pharmaceutically-allowable auxiliary materials, such as carriers, diluents, excipients and the like.

The invention also provides a meropenem intermediate related compound, which has the following chemical formula (I):

the name is 3- (1-oxypyryl) -spiro [2H-1,3-benzoxazine-2,1' -cyclohexan]-4(3H)-one。

The inventor of the application separates the byproduct (namely the compound of the chemical formula (1)) in the process of synthesizing the meropenem, and experiments prove that the byproduct has the in vitro anti-tumor cytotoxic activity.

The invention also provides a preparation method of the meropenem intermediate related compound, wherein the compound of the chemical formula (I) is obtained by separation from the technical process of preparing meropenem or a meropenem intermediate by using the compound M1; the compound M1 has the following formula (II):

the process for preparing meropenem or meropenem intermediate by using the compound M1 can be carried out by adopting the existing process in the field, for example, the corresponding process described in documents such as CN201510535498.6, CN201610812495.7 and CN 201611257633.6. The preparation method of the invention is mainly different in that the byproduct compound of the chemical formula (I) is separated in the process.

In the preparation method of the present invention, the process includes a step of preparing a compound M3 using a compound M1, the compound of formula (I) being produced in the step, and the compound M3 having the following formula (IV). The meropenem intermediate M3, having the name 3- (2-chloride, -1-oxypyr) -spiro [2H-1,3-benzoxazine-2,1' -cyclohexan ] -4(3H) -one, has the following chemical formula:

the present inventors have found that in the preparation of meropenem or a meropenem intermediate, a side reaction of producing a compound of formula (I) of the present invention (hereinafter referred to as compound (I)) is accompanied in the preparation of meropenem intermediate M3 using compound M1 of formula (II), and the by-product is separated therefrom. The step of preparing compound M3 using compound M1 preferably comprises: the compound M1 and chloropropionyl chloride are contacted and reacted to generate a product A containing the compound M3 and the compound of the chemical formula (I). In the preparation method of the present invention, the process for preparing meropenem intermediate M3 (i.e., compound M3) from compound M1 can be performed by using the existing processes in the art, for example, by referring to the corresponding processes described in documents CN201510535498.6, CN201610812495.7, CN201611257633.6, etc., which are not described in detail herein. For example, in some preferred embodiments, the compound M1 and the chloropropionyl chloride are contacted in the presence of an acid-binding agent and an aprotic solvent to produce the compound M3, and the compound of formula (I) is concomitantly produced. The acid-binding agent is selected from one or a combination of more than two of organic base and inorganic base, the organic base preferably comprises one or a combination of more than two of triethylamine, pyridine, N-diisopropylethylamine, 4-dimethylaminopyridine, triethanolamine and quaternary ammonium salt, the inorganic base preferably comprises one or a combination of more than two of potassium carbonate, ammonium carbonate and sodium carbonate, and the aprotic solvent preferably comprises one or a combination of more than two of toluene, xylene, benzene, chlorobenzene, nitrobenzene, acetone, dimethylformamide, tetrahydrofuran, dichloromethane, chloroform and carbon tetrachloride. The reaction temperature of the compound M1 and chloropropionyl chloride can be referred to the temperature conditions of the prior art, for example, the contact reaction is carried out at a temperature of 30-80 ℃. The molar ratio of the compound M1, chloropropionyl chloride and the acid binding agent can be referred to the proportion of the prior art, and is, for example, 1.0: 1.0-2.5: 0.5 to 3.0; more preferably 1.0: 1.4-1.6: 0.8 to 1.4, some embodiments are 1.0: 1.4: 1.2 or 1.0: 1.6: 1.4. the mass ratio of compound M1 to aprotic solvent can also be compared with the ratios of the prior art, for example 1.0: 1.0 to 5.0, more preferably 1.0: 3.0 to 5.0, in some embodiments 1.0: 3.1 or 1.0: 4.4.

in some preferred embodiments of the invention, the compound of formula (I) is isolated by: and (3) carrying out contact reaction on the product A and 4-AA, condensing the compounds M3 and 4-AA to generate meropenem mother nucleus 4BMA to obtain a product B containing the compound of the formula (I) and the 4BMA, and separating the product B to obtain the compound of the formula (I). The reaction process for preparing the meropenem mother nucleus 4BMA by adding the 4-AA and condensing with the meropenem intermediate M3 can also be carried out according to the prior art, for example, according to the corresponding processes described in documents such as CN201510535498.6, CN201610812495.7, CN201611257633.6 and the like; the compound of the chemical formula (I) which is the target of the invention can be separated out later by adding 4-AA to condense the meropenem intermediate M3 with the compound. The separation preferably adopts column chromatography separation, more preferably silica gel column chromatography, and the compound of the chemical formula (I) is obtained after the separation; the eluent is preferably the combination of petroleum ether and other solvents, and the other solvents are selected from one or the combination of more than two of dichloromethane, trichloromethane and acetone; preferably, the eluent is petroleum ether and dichloromethane in a volume ratio of 1:2-6 (e.g. 1:4), or petroleum ether and chloroform in a volume ratio of 1:2-5(1:3), or petroleum ether and acetone in a volume ratio of 3-7:1 (e.g. 5: 1).

The compound M1 can be prepared by reacting salicylamide with cyclohexanone, and the preparation of the compound M1 by reacting salicylamide with cyclohexanone is a mature technology in the field, and is not particularly described; for example, refer to the corresponding process in the literature "synthesis of 1 β -methylcarbapenem bicyclic parent nucleus, zui, etc., journal of chinese medical industry, 2014,41 (12)"; as an example, salicylamide and 1.5 times the amount of cyclohexanone are subjected to a synthesis reaction in a p-toluenesulfonic acid solution, and the resulting compound of formula (III) is subjected to intramolecular dehydration to obtain compound M1.

For ease of understanding, an exemplary synthetic route for formula (I) is provided below:

the obtained compound of the chemical formula (I) is white needle crystal; molecular formula C₁₆H₁₉O₃N; the molecular weight is 273; nuclear magnetism (CDCl)₃)：¹H-NMR(400MHz)：δ7.93(1H,d,J＝8Hz),7.50(1H,dd),7.08(1H,dd),6.96(1H,d,J＝8Hz),2.83(2H,q),2.26(2H,m),2.04(2H,m),1.67(5H,m),1.25,(4H,m+t)；¹³C-NMR(100MHz):δ181.8,162.8,155.1,135.6,128.2,122.2,117.4,117.1,94.9,35.0,33.1,24.3,22.3,9.6。

In vitro experiments prove that the meropenem intermediate related compound (I) has obvious in vitro cytotoxic activity on breast cancer cells (HL-60), liver cancer cells (hepG-2), gastric cancer cells (AGS) and leukemia cells (P388). Experimental study of ED₅₀The effective standard is not more than 20 mug/ml, and the result shows that the compound (I) has stronger inhibitory activity to each tumor cell strain and has the potential of preparing antitumor drugs; ED of said Compound (I) against Breast cancer cells (HL-60)₅₀ED of 8.75. mu.g/ml against hepatoma cells (hepG-2)₅₀6.52 μ g/ml, ED against gastric cancer cells (AGS)₅₀ED at 4.20. mu.g/ml for leukemia cells (P388)₅₀It was 4.33. mu.g/ml.

Detailed Description

In order to better understand the technical solution of the present invention, the following examples are further provided to illustrate the present invention, but the present invention is not limited to the following examples. All documents cited herein are incorporated by reference.

The reagents used in the experiment are all analytically pure and can be obtained commercially.

Example 1

Preparation of a compound of formula (I):

step 1), carrying out a synthesis reaction of salicylamide and 1.5 times of cyclohexanone in volume ratio in a p-toluenesulfonic acid solution (5%) (reaction temperature room temperature, reaction time 3h), and carrying out intramolecular dehydration on the generated compound of the chemical formula (III) (the intramolecular dehydration operation is as follows: heating to 60 ℃ in 20% dilute sulfuric acid, reacting for 1h) to generate a compound M1 which is easy to precipitate, naturally precipitating a compound M1, and drying at 60 ℃ to obtain a compound M1. (Compound M1 can also be prepared by directly referring to the corresponding process in the literature of "1 beta-methylcarbapenem bicyclic parent nucleus synthesis, Zhang Ling, etc., Chinese medicine industry journal, 2014,41 (12)", or by commercially available)

Step 2), carried out in example 1 with reference to patent document CN201510535498.6, 12g of compound M1 and 10g of 2-chloropropionyl chloride were reacted in 10g of pyridine and 75ml of toluene solution at 40 ℃ for 5 hours to give 3- (1-oxypyr) -spiro [2H-1, 3-benzoxane-2, 1 '-cyclohexoxan ] -4(3H) -one (i.e., the compound of formula (I) and 3- (2-chlororide, -1-oxypyr) -spiro [2H-1, 3-benzoxane-2, 1' -cyclohexoxan ] -4(3H) -one (i.e., the compound M3 described above); then 4-AA (4-acetoxyazetidinone) is added for reaction, the compound M3 and 4-AA are condensed by reaction, and the reaction is followed by HPLC until the peak of 4-AA disappears. (this step can also be carried out with reference to the embodiment of CN 201610812495.7.)

Step 3), separating the reaction product obtained in the step 2) by silica gel column chromatography, adopting 300-400 mesh silica gel GF254, and using petroleum ether with the volume ratio of 1:4 as eluent: and collecting the flow part of the compound (I) by dichloromethane, and concentrating by a rotary evaporator to obtain the compound (I) (or the compound (I) for short) of the chemical formula.

The obtained compound (I) is white needle crystal; nuclear magnetism (CDCl)₃)：¹H-NMR(400MHz)：δ7.93(1H,d,J＝8Hz),7.50(1H,dd),7.08(1H,dd),6.96(1H,d,J＝8Hz),2.83(2H,q),2.26(2H,m),2.04(2H,m),1.67(5H,m),1.25,(4H,m+t)。¹³C-NMR(100MHz):δ181.8,162.8,155.1,135.6,128.2,122.2,117.4,117.1,94.9,35.0,33.1,24.3,22.3,9.6。

Example 2 in vitro cytotoxicity assay

Cell lines: 4 tumor cell lines HL-60 (Wuhan Puceno Life technologies, Inc.), hepG-2 (Wuhan Puceno Life technologies, Inc.), AGS (Wuhan Puceno Life technologies, Inc.) and P388 (Shanghai vivid ran Biotechnologies, Inc.);

culture solution: 4ml of 10% by mass calf serum RPMI1640 medium containing 25mM HEPES, 0.2% (w/v) sodium bicarbonate and 100. mu.g/ml kanamycin.

The cell line was inoculated into the culture medium at 37 ℃ with 5% CO₂Cultured under the conditions of (1) for 24 hours. The cell suspension after trypsinization was added to a 96-well plate at a cell concentration of 2X 10⁴A hole. The tumor cells were added with the test component (i.e., Compound (I)) or the positive control (5-fluorouracil (Shanghai Hainan Biotechnology Co., Ltd.) at different concentrations by preparing a culture medium of 400. mu.g/ml, 200. mu.g/ml, 100. mu.g/ml, 50. mu.g/ml, 25. mu.g/ml, 12.5. mu.g/ml, 6.25. mu.g/ml, and 3.125. mu.g/ml, incubating at 37 ℃ for 72 hours, fixing with ice-cold 50% (mass concentration) trichloroacetic acid, staining with 0.4% (mass concentration) SRB, and measuring the absorbance at 562nm after the dye had dissolved.

ED₅₀Conversion of the half-cell inhibitory drug concentration according to dose-Effect Data (ED)₅₀＝log^-1[Xm-i(∑p-0.5)]Where Xm is the maximum concentration logarithm and Σ p is the sum of the apoptosis rates of the respective groups). Each experiment was repeated three times with an absorption difference of less than 5%, ED₅₀The difference is less than 30%. By ED₅₀Less than or equal to 20 mu g/ml is used as an effective standard.

TABLE 1 Compound (I) in vitro cytotoxicity test ED₅₀Data of

The experimental results show that: the compound (I) has stronger inhibitory activity on each tumor cell strain and has the potential of being used for preparing antitumor drugs; ED of said Compound (I) against Breast cancer cells (HL-60)₅₀ED of 8.75. mu.g/ml against hepatoma cells (hepG-2)₅₀6.52. mu.g/ml, the compound (I) showed less inhibition of the first two cancer cells than the positive control (5-fluorouracil for HL-60 cellsED of cells₅₀6.43 mu g/ml, but still has stronger inhibitory activity; ED on hepG-2 cells₅₀5.97. mu.g/ml); the compound (I) is useful for treating gastric cancer cell (AGS) ED₅₀4.20 mug/ml, which is better than a positive control drug (5-fluorouracil ED)₅₀25.86. mu.g/ml); ED on leukemia cells (P388)₅₀4.33 mug/ml, which is better than a positive control drug (5-fluorouracil ED)₅₀75.40. mu.g/ml).

It will be appreciated by those skilled in the art that modifications or adaptations to the invention may be made in light of the teachings of the present specification. Such modifications or adaptations are intended to be within the scope of the present invention as defined in the claims.

Claims

1. The application of meropenem intermediate related compound in preparing antitumor drugs, wherein the related compound has the following chemical formula (I):

wherein the tumor is selected from liver cancer, gastric cancer or leukemia.