CN107778243B - Preparation and application of 8-bit long-chain fatty acyloxy substituted diterpenoid alkaloid - Google Patents

Abstract

The invention relates to a Chinese medicinal composition prepared from Sinkiang aconiteAconitum sinchiangenseW.t. Wang) to produce 8-bit long-chain fatty acyloxy substituted diterpenoid alkaloids; also relates to a method for synthesizing the compound by a chemical method; experiments prove that the compounds can be used as antibacterial or anti-tumor preparations.

Description

Preparation and application of 8-bit long-chain fatty acyloxy substituted diterpenoid alkaloid

Technical Field

The invention relates to a Chinese medicinal composition prepared from Sinkiang aconiteAconitum sinchiangenseW.t. Wang) (accession number: no. 20150316-1) a method for producing 8-bit long-chain fatty acyloxy substituted diterpene alkaloids; the invention also relates to a method for synthesizing the compound by a chemical method; the invention also relates to application of the compounds in preparing antibacterial or antitumor preparations, and belongs to the field of pharmacy.

Background

The 20 th century is the prosperous period of human science and technology development, and modern medicine also has a lot of breakthrough development in the 100 years. Unfortunately, cancer has quietly replaced the status of infectious diseases and nutritional diseases in human beings, and is a second killer after cardiovascular and cerebrovascular diseases in terms of harming human health. According to the statistics of the World Health Organization (WHO), about 1000 million people suffer from cancer and about 700 million people die worldwide every year, so that the abuse of cancer is not visible. The chemically synthesized anticancer drugs of the last century have been widely used in clinical practice, but with the deeper and deeper understanding of their side effects, the search for anticancer components from natural drugs has been the focus of more and more pharmaceutical workers.

Diterpenoid alkaloids are important natural compounds, more than 800 kinds of currently known natural diterpenoid alkaloids are distributed in seven genera of five families, and are mainly present in aconitum (A)Aconitium) Delphinium genus (A)Delphinium) And meadowsweet (Spirea). In recent decades, some aconitum medicinal plants have been studied with certain success, and some active ingredients such as lappaconitine (lappaconitine A), 3-acetyl aconitine (3-acetyl aconitine) and kusnezoff monkshood root (crassicauline A) have been applied clinically as novel non-addictive analgesic and anti-inflammatory drugs. Therefore, the deep research on chemical components of the plants is very necessary, and not only can a new high-efficiency low-toxicity medicine be expected to be searched from the plants, a basis is provided for developing and utilizing the superior medicinal plant resource in China, but also an important theoretical basis is provided for phytochemistry taxonomy.

Sinkiang monkshood root (monkshood root)Aconitum sinchiangenseW.T. Wang), belonging to the genus Aconitum of the family Ranunculaceae, produced in Xinjiang and preferring to grow at an elevation of 2000-2500 mSpruceUnder forest and mountain landMeadowIn the zone, the roots contained various alkaloid components, but no antibacterial and antitumor components were found. The invention separates 8-bit long-chain fatty acyloxy substituted diterpenoid alkaloid compounds, synthesizes the compounds by a chemical method, and discovers in the activity research of derivatives of the compounds that the compounds have stronger bacteriostatic action and stronger broad-spectrum anti-tumor action.

Disclosure of Invention

The invention aims to provide a compound with unique structure and good antibacterial and antitumor activity. The structure is shown as formula I (see figure 1)

The method is characterized in that: r₁，R₃，R₄，R₈Hydrogen, hydroxy, C1-6 alkoxy such as methoxy or ethoxy, C1-6 alkanoyloxy such as acetyl; r₂，R₆，R₉，R₁₀Is hydrogen, hydroxy or C1-6 alkanoyloxy such as acetyl, R₅Is a saturated or unsaturated long-chain fatty acyloxy group having 14 to 24 carbon atoms such as linoleyl acyloxy, oleoyloxy, palmitoyloxy, eicosapentaenoyloxy, R₇Is hydrogen or hydroxy or benzoyloxy or p-methoxybenzoyloxy.

Preferred compounds of formula I according to the invention are those in which R is₁，R₃，R₄，R₈Preferably methoxy, R₂，R₆，R₉，R₁₀Preferably hydrogen or hydroxy, R₅Preferably an unsaturated long-chain fatty acyloxy group having 18 carbon atoms such as linoleyloxy group, oleoyloxy group, palmitoyloxy group, eicosapentaenoic acyloxy group, R₇Preferably a benzoyloxy group.

Preferred compounds of formula I according to the invention are selected from the following compounds (see fig. 2):

the compound of the formula I can be obtained by extracting rhizomes of aconitum sinkiangense to obtain an extract of diterpenoid alkaloids with long-chain fatty acyloxy substituted at 8 positions, and then separating and purifying the extract by methods such as silica gel column chromatography, preparative thin layer chromatography and the like.

The compound of the formula I can be prepared by acylating 8-site OH in a structure with long-chain fatty acid or acyl chloride thereof by using corresponding diterpene alkaloid as a raw material by a chemical method.

The following examples of the present invention are exemplified by using Sinkiang aconite (A), (B), (C)Aconitum sinchiangenseW.t. Wang) (accession number: no. 20150316-1), examples of the preparation of inventive compounds I of the formula.

In the following examples of the present invention, examples of derivatives in which 8-position is substituted with a long-chain fatty acyloxy group using diterpene alkaloids as a raw material are given.

The specific implementation scheme is as follows:

drawings

FIG. 1 is a structural formula of a compound of formula I.

FIG. 2 is a structural formula of a preferred compound of formula I according to the present invention.

FIG. 3 is the structural formula of compound I.

FIG. 4 is a flow chart of the extraction of total alkaloids from Aconitum Sinkiangense hand.

FIG. 5 is the structural formula of Compound II.

FIG. 6 shows NMR data (C) for Compound I, II¹H:400MHz; ¹³C:100MHz，in CDCl₃)

FIG. 7 is the structural formula of Compound III.

Figure 8 is a preparation scheme for compound III.

FIG. 9 is the structural formula of Compound IV.

Figure 10 is a preparation scheme for compound IV.

FIG. 11 is the structural formula of Compound V.

Figure 12 is a preparation scheme for compound V.

FIG. 13 is the structural formula of Compound VI.

Figure 14 is a scheme for the preparation of compound VI.

FIG. 15 is the structural formula of Compound VII.

Figure 16 is a preparation scheme for compound VII.

FIG. 17 is the structural formula of Compound VIII.

FIG. 18 is a preparation scheme of Compound VIII.

FIG. 19 is the structural formula of Compound IX.

Figure 20 is a scheme for the preparation of compound IX.

FIG. 21 shows the results of IC50 (. mu.M) inhibition of five tumor cells by compounds I-IX.

Example 1: the structural formula of compound I indicated in the examples below (see fig. 3):

1 preparation of extract

Extracting Sinkiang aconite by acid-soluble alkali-precipitation methodAconitum sinchiangenseW.T. Wang) root part, the specific flow is shown in figure 4.

Dried and sliced rhizome of aconitum sinense kuntze is 20 Kg, soaked overnight in 200L of 3% hydrochloric acid, and percolated until the percolate has no alkaloid reaction. Collecting percolate, and passing through cation exchange resin column for full exchange. Air drying the resin, alkalifying with 10% ammonia water to pH of > 9, and ultrasonic extracting with 75% ethanol for 3 times (mass/volume ratio of 1: 20) for 1 hr each time. Filtering to remove resin, mixing filtrates, concentrating under reduced pressure to obtain extract without alcohol smell to obtain total alkaloids (brown solid, 210.65 g).

2 separation and purification of Compound I

Collecting 210.65 g of above total alkaloids, performing silica gel H column chromatography, and purifying with CHCl₃Gradient elution with MeOH (200:1-6:1) afforded nine fractions A-I. The A fraction was further chromatographed on silica gel column H using CHCl₃MeOH (100: 1-6:1) gradient elution afforded Compound I (yellow oily liquid, 150 mg).

Compound I is a yellow oily liquid of formula C₅₀H₇₅NO₁₀. ESI-MS m/z: 850[M+H]⁺, ¹H-NMR(CDCl₃ , 400 MHz)δ: 1.09 (3H , t , J =7.2 Hz, N-CH₂CH₃ ), 3.17, 3.29 , 3.30, 3.77 (each 3H , s, 4 ×OCH₃), 4.87 (1H ,d, J =4.0 Hz, H-14β), 4.49 (1H , d, J =2.8 Hz, H-15β), 3.99 (1H , d , J = 8 Hz, H-6β), 3.65 (1H, d , J = 8.0 Hz, H-18α), 3.35 (1H , d, J = 8.0 Hz, H-18β); 5.38 (4H, m, H-9′′′,6′′′, 11′′′, 12′′′), 8.05 (2H, d, J =8.0 Hz, H-2′,6′), 7.58 (1H , t , J =8.0 Hz,H-4′), 7.46 (2H , t , J =8.0 Hz, H-3′,5′). ¹³C NMR (100 Hz，CDCl₃): see fig. 6.

Example 2: the structural formula of compound II referred to in the examples below (see fig. 5):

1 preparation of extract

The same as in example 1.

2 separation and purification of Compound II

Collecting 210.65 g of above total alkaloids, performing silica gel H column chromatography, and purifying with CHCl₃Gradient elution with MeOH (200:1-6:1) afforded nine fractions A-I. The A fraction was further chromatographed on silica gel column H using CHCl₃MeOH (100: 1-6:1) gradient elution afforded Compound II (yellow oily liquid, 150 mg).

Compound II was a yellow oily liquid with optical rotation +28.53 (c,1.00, CHCl3), IR (KBr) cm^-1: 3453, 2924, 1724, 1631, 1278, 1096; HR-ESI-MS m/z:882.5458 [M+H]⁺; calcd C₅₀H₇₆NO₁₂, 882.5368 ; ¹H NMR and ¹³C NMR is shown in FIG. 6.

Example 3: the structural formula of compound III referred to in the examples below (see fig. 7):

a process for the preparation of compound III:

monoacetylaconitine (100 mg, 0.175 mmol) was placed in a 50 ml round bottom flask and dissolved in 10 ml dry CHCl₃Then, 0.5 mL of acetic anhydride and 5 drops of pyridine were added and the mixture was stirred at 25 ℃ for 3 hours. Adding concentrated ammonia water to adjust pH in ice bath>Continuing to use CHCl₃(10 ml. times.2) the aqueous layer was extracted and combined with CHCl₃Extract, anhydrous Na₂SO₄Drying, and recovering solvent under reduced pressure to obtain residue. The residue was dissolved in 10 mL of dry CHCl₃DMAP (50 mg, 0.41 mmol) was added thereto, and the mixture was dissolved with stirring, and 70 mg (0.236 mmol) of linoleoyl chloride was added dropwise thereto, followed by stirring at 25 ℃ for 12 hours. Column chromatography on silica gel (3 g) eluting with chloroform-methanol (150:1) afforded compound III (yellow amorphous powder, 90 mg, 59% yield) (see fig. 8). Compound III yellow oily liquid C₅₂H₇₇NO₁₀, ESI-MS m/z: 876 [M+H]⁺. ¹H NMR (400 MHz, CDCl₃) δ: 0.89 (3H, t, J = 7.2 Hz, CH₃), 1.08 (3H, t, J = 6 Hz, N-CH₂CH₃), 1.28-1.36 (22H, CH₂×11), 1.45, 1.79 (each 1H, m, CH₂), 2.21 (3H, s, OAc), 3.30, 3.30, 3.38, 3.69 3.45 (ech 3H, s, OCH₃×4), 4.95 (1 H, d, J = 8 Hz), 5.39 (4H, m, H-9′′′,10′′′, 12′′′, 13′′′), 7.42 (2H, d, J = 8.0 Hz, 2’, 6’), 7.54 (1H, t, J = 8.0 Hz, 4’), 8.15 (2H, t, J = 8.0 Hz, 3’, 5’).

Example 4: the structural formula of compound IV referred to in the examples below (see fig. 9):

a process for the preparation of compound IV:

monoacetylaconitine (100 mg, 0.175 mmol) was placed in a 50 ml round bottom flask and dissolved in 10 ml dry CHCl₃Then, 0.5 mL of acetic anhydride and 5 drops of pyridine were added and the mixture was stirred at 25 ℃ for 3 hours. Adding concentrated ammonia water to adjust pH in ice bath>Continuing to use CHCl₃(10 ml. times.2) the aqueous layer was extracted and combined with CHCl₃Extract, anhydrous Na₂SO₄Drying, and recovering solvent under reduced pressure to obtain residue. The residue was dissolved in 10 mL of dry CHCl₃DMAP (50 mg, 0.41 mmol) was added thereto, and the mixture was dissolved with stirring, and 80 mg (0.271 mmol) of linolenyl chloride was added dropwise thereto, followed by stirring at 25 ℃ for 12 hours. Column chromatography on silica gel (3 g) eluting with chloroform-methanol (150:1) afforded compound IV (yellow amorphous powder, 110 mg, 72% yield) (see fig. 10). Compound IV yellow oily liquid C₅₂H₇₅NO₁₀, ESI-MS m/z: 874 [M+H]⁺. ¹H NMR (400 MHz, CDCl₃) δ: 0.89 (3H, t, J = 7.2 Hz, CH₃), 1.08 (3H, t, J = 6 Hz, N-CH₂CH₃), 1.27-1.34 (20H, CH₂×10), 1.45, 1.79 (each 1H, m, CH₂), 2.20 (3H, s, OAc), 3.30, 3.30, 3.38, 3.69 3.45 (ech 3H, s, OCH₃×4), 4.95 (1 H, d, J = 8 Hz), 5.57 (6H, m, H-9′′′,10′′′, 12′′′, 13′′′, 15′′′, 16′′′), 7.42 (2H, d, J = 8.0 Hz, 2’, 6’), 7.54 (1H, t, J = 8.0 Hz, 4’), 8.15 (2H, t, J = 8.0 Hz, 3’, 5’).

Example 5: the structural formula of compound V indicated in the examples below (see FIG. 11)

A process for the preparation of compound V:

monoacetylaconitine (100 mg, 0.175 mmol) was placed in a 50 ml round bottom flask and dissolved in 10 ml dry CHCl₃Then, 0.5 mL of acetic anhydride and 5 drops of pyridine were added and the mixture was stirred at 25 ℃ for 3 hours. Adding concentrated ammonia water to adjust pH in ice bath> 9Continue to use CHCl₃(10 ml. times.2) the aqueous layer was extracted and combined with CHCl₃Extract, anhydrous Na₂SO₄Drying, and recovering solvent under reduced pressure to obtain residue. The residue was dissolved in 10 mL of dry CHCl₃DMAP (50 mg, 0.41 mmol) was added thereto, and the mixture was dissolved with stirring, and 80 mg (0.267 mmol) of oleoyl chloride was added dropwise thereto, followed by stirring at 25 ℃ for 12 hours. Column chromatography on silica gel (3 g) eluting with chloroform-methanol (150:1) afforded compound V (yellow amorphous powder, 95 mg, 62% yield) (see fig. 12). Compound V yellow oily liquid C₅₂H₇₉NO₁₀, ESI-MS m/z: 878 [M+H]⁺. ¹H NMR (400 MHz, CDCl₃) δ: 0.89 (3H, t, J = 7.2 Hz, CH₃), 1.08 (3H, t, J = 6 Hz, N-CH₂CH₃), 1.25-1.38 (24H, CH₂×12), 1.45, 1.79 (each 1H, m, CH₂), 2.21 (3H, s, OAc), 3.30, 3.30, 3.38, 3.69 3.45 (ech 3H, s, OCH₃×4), 4.95 (1 H, d, J = 8 Hz), 5.36 (2H, m, H-9′′′,10′′′), 7.42 (2H, d, J = 8.0 Hz, 2’, 6’), 7.54 (1H, t, J = 8.0 Hz, 4’), 8.15 (2H, t, J = 8.0 Hz, 3’, 5’).

Example 6: the structural formula of compound VI indicated in the examples below (see FIG. 13)

A process for the preparation of compound VI:

monoacetylaconitine (100 mg, 0.175 mmol) was placed in a 50 ml round bottom flask and dissolved in 10 ml dry CHCl₃Then, 0.5 mL of acetic anhydride and 5 drops of pyridine were added and the mixture was stirred at 25 ℃ for 3 hours. Adding concentrated ammonia water to adjust pH in ice bath>Continuing to use CHCl₃(10 ml. times.2) the aqueous layer was extracted and combined with CHCl₃Extract, anhydrous Na₂SO₄Drying, and recovering solvent under reduced pressure to obtain residue. The residue was dissolved in 10 mL of dry CHCl₃DMAP (50 mg, 0.41 mmol) was added thereto, and the mixture was dissolved with stirring, and 70 mg (0.255 mmol) of palmitoyl chloride was added dropwise thereto, followed by stirring at 25 ℃ for 12 hours. Column chromatography on silica gel (3 g) eluting with chloroform-methanol (150:1) afforded compound VI (yellow amorphous powder, 100 mg, 70% yield) (see fig. 14). Compound VI yellow oily liquid C₅₀H₇₇NO₁₀, ESI-MS m/z: 852 [M+H]⁺. ¹H NMR (400 MHz, CDCl₃) δ: 0.89 (3H, t, J = 7.2 Hz, CH₃), 1.08 (3H, t, J = 6 Hz, N-CH₂CH₃), 1.28-1.36 (26H, CH₂×13), 1.45, 1.79 (each 1H, m, CH₂), 2.21 (3H, s, OAc), 3.30, 3.30, 3.38, 3.69 3.45 (ech 3H, s, OCH₃×4), 4.95 (1 H, d, J = 8 Hz), 7.42 (2H, d, J = 8.0 Hz, 2’, 6’), 7.54 (1H, t, J = 8.0 Hz, 4’), 8.15 (2H, t, J = 8.0 Hz, 3’, 5’).

Example 7: the structural formula of compound VII indicated in the examples below (see FIG. 15)

Preparation of compound VII:

monoacetylaconitine (100 mg, 0.175 mmol) was placed in a 50 ml round bottom flask and dissolved in 10 ml dry CHCl₃Then, 0.5 mL of acetic anhydride and 5 drops of pyridine were added and the mixture was stirred at 25 ℃ for 3 hours. Adding concentrated ammonia water to adjust pH in ice bath>Continuing to use CHCl₃(10 ml. times.2) the aqueous layer was extracted and combined with CHCl₃Extract, anhydrous Na₂SO₄Drying, and recovering solvent under reduced pressure to obtain residue. The residue was dissolved in 10 mL of dry CHCl₃DMAP (50 mg, 0.41 mmol) was added thereto, and the mixture was dissolved with stirring, and 85 mg (0.262 mmol) of eicosapentaenoic acid chloride was added dropwise thereto, followed by stirring at 25 ℃ for 12 hours. Column chromatography on silica gel (3 g) eluting with chloroform-methanol (150:1) afforded compound VII (yellow amorphous powder, 90 mg, 57% yield) (see fig. 16). Compound VII yellow oily liquid C₅₄H₇₅NO₁₀, ESI-MS m/z: 898 [M+H]⁺. ¹H NMR (400 MHz, CDCl₃) δ: 0.89 (3H, t, J = 7.2 Hz, CH₃), 1.08 (3H, t, J = 6 Hz, N-CH₂CH₃), 1.25-1.38 (16H, CH₂×8), 1.45, 1.79 (each 1H, m, CH₂), 2.21 (3H, s, OAc), 3.30, 3.30, 3.38, 3.69 3.45 (ech 3H, s, OCH₃×4), 4.95 (1 H, d, J = 8 Hz), 5.57 (6H, m, H-5′′′,6′′′, 8′′′, 9′′′, 11′′′, 12′′′, 14′′′, 15′′′, 17′′′, 18′′′), 7.42 (2H, d, J = 8.0 Hz, 2’, 6’), 7.54 (1H, t, J = 8.0 Hz, 4’), 8.15 (2H, t, J = 8.0 Hz, 3’, 5’).

Example 8: the structural formula of the compound VIII indicated in the following examples (see FIG. 17)

Preparation method of compound VIII:

8-O-deacetylaconitine (100 mg, 0.166 mmol) was placed in a 50 mL round bottom flask and dissolved in 10 mL dry CHCl₃Then, 0.5 mL of acetic anhydride and 5 drops of pyridine were added and the mixture was stirred at 25 ℃ for 3 hours. Adding concentrated ammonia water to adjust pH in ice bath>Continuing to use CHCl₃(10 ml. times.2) the aqueous layer was extracted and combined with CHCl₃Extract, anhydrous Na₂SO₄Drying, and recovering solvent under reduced pressure to obtain residue. The residue was dissolved in 10 mL of dry CHCl₃DMAP (50 mg, 0.41 mmol) was added thereto, and the mixture was dissolved with stirring, and 70 mg (0.236 mmol) of linoleoyl chloride was added dropwise thereto, followed by stirring at 25 ℃ for 12 hours. Column chromatography on silica gel (3 g) eluting with chloroform-methanol (150:1) afforded compound VIII (yellow amorphous powder, 90 mg, 57% yield) (see FIG. 18). Compound VIII yellow oily liquid C₅₄H₇₉NO₁₃, ESI-MS m/z: 950 [M+H]⁺. ¹H NMR (400 MHz, CDCl₃) δ: 0.87 (3H, t, J = 7.2 Hz, CH₃), 1.02 (3H, t, J = 6 Hz, N-CH₂CH₃), 1.28-1.37 (22H, CH₂×11), 1.45, 1.79 (each 1H, m, CH₂), 2.23, 2.21 (each 3H, s, OAc×2), 3.30, 3.37,3.42, 3.65 (ech 3H, s, OCH₃×4), 4.93 (1 H, d, J = 8 Hz), 5.37 (4H, m, H-9′′′,10′′′, 12′′′, 13′′′), 7.42 (2H, d, J = 8.0 Hz, 2’, 6’), 7.54 (1H, t, J = 8.0 Hz, 4’), 8.15 (2H, t, J = 8.0 Hz, 3’, 5’).

Example 9: the structural formula of compound IX indicated in the examples below (see FIG. 19)

Preparation of compound IX:

8-deacetyl-yunaconitine (100 mg, 0.162 mmol) was placed in a 50 ml round bottom flask and dissolved in 10 ml dry CHCl₃Then, 0.5 mL of acetic anhydride and 5 drops of pyridine were added and the mixture was stirred at 25 ℃ for 3 hours. Adding concentrated ammonia water to adjust pH in ice bath>Continuing to use CHCl₃(10 ml. times.2) the aqueous layer was extracted and combined with CHCl₃Extract, anhydrous Na₂SO₄Drying, and recovering solvent under reduced pressure to obtain residue. The residue was dissolved in 10 mL of dry CHCl₃DMAP (50 mg, 0.41 mmol) was added thereto, and the mixture was dissolved with stirring, and 70 mg (0.236 mmol) of linoleoyl chloride was added dropwise thereto, followed by stirring at 25 ℃ for 12 hours. Column chromatography on silica gel (3 g) eluting with chloroform-methanol (150:1) afforded compound IX (yellow amorphous powder, 100 mg, 67% yield) (see fig. 20). Compound IX yellow oily liquid C₅₃H₇₉NO₁₂, ESI-MS m/z: 922 [M+H]⁺. ¹H NMR (400 MHz, CDCl₃) δ: 0.88 (3H, t, J = 7.2 Hz, CH₃), 1.03 (3H, t, J = 6 Hz, N-CH₂CH₃), 1.28-1.37 (22H, CH₂×11), 1.45, 1.79 (each 1H, m, CH₂), 2.23 (3H, s, OAc), 3.30, 3.30, 3.37, 3.42, 3.83 (ech 3H, s, OCH₃×5), 4.94 (1 H, d, J = 8 Hz), 5.39 (4H, m, H-9′′′,10′′′, 12′′′, 13′′′), 7.42 (2H, d, J = 8.0 Hz, 2’, 6’), 7.54 (1H, t, J = 8.0 Hz, 4’), 8.15 (2H, t, J = 8.0 Hz, 3’, 5’).

Example 10:

testing of in vitro antitumor activity:

1. experimental sample and experimental method

Activity experiments were performed using each of the compounds prepared in examples 1 to 9.

The IC of the compounds I-IX on five tumor cell lines was determined by MTS method using cisplatin as positive control₅₀The value is obtained.

The MTS method is used for detecting the cell activity principle: MTS is a novel MTT analogue, is called 3- (4, 5-dimethylthiozol-2-yl) -5 (3-methoxy-methoxyphenyl) -2- (4-sulfopheny) -2H-tetrazolium, and is a yellow dye. Succinate dehydrogenase in the mitochondria of living cells can metabolize and reduce MTS to generate soluble Formazan (Formazan) compounds, and the content of the Formazan can be measured at 490nm by using an enzyme labeling instrument. Since the formazan production amount is generally proportional to the number of living cells, the number of living cells can be estimated from the optical density OD value.

The experimental method comprises the following steps:

1) sample preparation: 1 mg of each compound prepared in examples 1 to 9 was dissolved in 1 mL of a weakly acidic buffer solution (pH 5.5 to 7) having a predetermined pH, and the resulting solution was stored under refrigeration, and a blank buffer solution having a corresponding pH was used as a diluent and a blank control.

2) Seeding of cells: preparing single cell suspension by using culture solution (DMEM or RMPI 1640) containing 10% fetal calf serum, inoculating 3000-15000 cells in each hole to a 96-well plate, wherein each hole has 100ul volume, and the adherent cells are inoculated and cultured 12-24 hours in advance.

3) Adding a solution of the test compound: leukemia HL-60, Lung cancer A-549 and liver cancer SMMC-7721 cells were rescreened at concentrations of 113.4uM, 22.68uM, 4.54uM, 0.91uM, 0.18uM, and breast cancer MCF-7 and colon cancer SW480 cells were rescreened at concentrations of 113.4uM, 28.35uM, 7.09uM, 1.77uM, 0.44uM, with a final volume of 200ul per well, with 3 replicates per treatment.

4) Color development: culturing at 37 ℃ for 48 hours, removing culture solution in the adherent cells, and adding 20ul of MTS solution and 100ul of culture solution in each hole; discarding 100ul of culture supernatant from the suspension cells, and adding 20ul of MTS solution into each well; setting 3 blank multiple wells (mixed solution of 20ul MTS solution and 100ul culture solution), and continuously incubating for 2-4 hours to ensure that the light absorption value is measured after the reaction is fully performed.

5) Colorimetric: selecting a 492nm wavelength, reading the light absorption value of each hole by a multifunctional microplate reader (MULTISKAN FC), recording the result, drawing a cell growth curve by taking the concentration as an abscissa and the cell survival rate as an ordinate after data processing, and calculating the IC50 value of the compound by using a two-point method (Reed and Muench method). 6) Positive control compound: cisplatin (DDP) is taken as a positive compound in each experiment, a cell growth curve is drawn by taking the concentration as the abscissa and the cell survival rate as the ordinate, and the IC of the compound is calculated by using a two-point method (Reed and Muench method)₅₀The value is obtained.

2. Experimental results (see FIG. 21)

Conclusion

The compounds I-IX have obvious tumor cell strain inhibiting effect, and can be used as antitumor agents for antitumor research.

Example 11:

testing of in vitro antitumor activity:

1. experimental sample and experimental method

Activity experiments were performed using each of the compounds prepared in examples 1 to 9.

In vitro antimicrobial Activity test

Determination of Minimum Inhibitory Concentration (MIC)

1 Experimental sample and experimental method

An activity test was conducted using each of the compounds prepared in examples 1-2.

MIC values for compounds I and II were determined by the broth microdilution method (BMD).

1) Sample preparation: 1 mg of each compound prepared in examples 1 to 9 was dissolved in 1 mL of a weakly acidic buffer solution (pH 5.5 to 7) having a predetermined pH, and the resulting solution was stored under refrigeration as a stock solution, and a blank buffer solution having a corresponding pH was used as a diluent and a blank control.

2 course of experiment

Preparation of bacterial liquid

100ul of glycerol standard bacteria are inoculated into 3 mL of LB broth, the broth is placed in an incubator at 37 ℃ for 24 hours, then staphylococcus aureus is streaked on a yolk high-salt agar plate, and escherichia coli is streaked on a Macconka agar plate. Culturing in a 37 ℃ incubator for 12-16 h, respectively selecting single colonies, inoculating in 3 mL LB broth, and culturing for later use.

Preparation of drug micro-dilution drug sensitive box

Adding MH broth 100uL into each well of a 96-well enzyme label plate, then adding 100uL of drug solution with the concentration of 1 mg/mL into the first well, carrying out serial dilution according to a 2-fold dilution method, adding drugs into the first to eighth wells, and using no drug into the twelfth well as a growth control so as to divide the final drug concentration of the first to eighth wells into 1 mg/mL, 0.5 mg/mL, 0.25 mg/mL, 0.125 mg/mL, 0.0625 mg/mL, 0.0313 mg/mL and 0.0156 mg/mL, thus preparing the microdilution drug sensitive cassette.

Inoculation of bacteria

The concentration of the extract prepared by direct bacterial liquid method is equal to 0.50 wheatAdding 50 uL of bacterial suspension of turbidimetric bacteria into each well of the micro-dilution drug sensitive box, so that the final bacterial liquid concentration without the wells is 5 multiplied by 10⁵ CFU/mL, diluting the bacterial liquid, after inoculation within 15 min, culturing for 16-18 h in a constant temperature box at 37 ℃, repeating for 4 bacteria each time, and simultaneously setting 3 positive drug control groups, a bacteria positive control group (only bacteria have no liquid medicine), and a negative control group (buffer solution and bacterial liquid with the same pH). The culture medium was clarified, and the lowest concentration of compound contained in the bacteria-free growth well was visually observed and recorded as the MIC value.

3. Results of the experiment

Minimum Inhibitory Concentration (MIC)

The MICs of compounds I-IX for E.coli were: 0.147, 0.142, 0.285, 0.286, 0.285, 0.293, 0.279, 0.132, 0.136 umol/mL. It can be seen that compounds I-IX are sensitive to E.coli.

4. Conclusion

The compounds I-IX have obvious escherichia coli bacteriostasis effect, and can be used as bacteriostat for bacteriostasis research.

Claims (1)

1. The use of the following compounds or salts thereof for the preparation of an anti-tumor and/or anti-E.coli formulation:

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