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    • 专利摘要:
    Process for producing a regioisomeric mixture of two derivatives of (11R, 17R) -11-epi-fistularin-3, inhibitory mixture of human leukemia cells. The present invention relates to a specific extractive/synthetic process for obtaining a regioisomeric mixture of two derivatives of (11R, 17R) -11-epi-fistularin-3, from the biomass of the marine sponge Aplysina (= Verongia) Aerophobia, as well as its use in various therapies as inhibitors of human leukemia cells. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2680044A1
    申请号:ES201700216
    申请日:2017-03-02
    公开日:2018-09-03
    发明作者:Francisco Javier TOLEDO MARANTE；Francisco Jesús ESTÉVEZ ROSAS；Alba GONZÁLEZ BENTOVICS
    申请人:Universidad de las Palmas de Gran Canaria；
    IPC主号:
    专利说明:

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    boiling (<70 ° C). Never use methanol or ethanol, as they give undesirable reactions with the bioactive substances that you want to extract.
    d) Remove the solvent in a rotary evaporator at 60 ° C in the water bath and at 3 mm Hg of vacuum to obtain a first blackish residue with a semi-solid appearance.
    e) Re-extract the hot biomass using a Soxhlet device (at least ten siphoned). Again, said extraction can be carried out with any volatile aprotic solvent (acetone, ethyl acetate, dichloromethane), with a low boiling point (<70 ° C). Never use methanol or ethanol, as they give undesirable reactions with the substances that you want to extract. The size of the Soxhlet apparatus and the amount of solvent are adapted to the amount of biomass, depending on the amount of extract to be obtained.
    f) Remove the solvent in a rotary evaporator at 60 ° C in the water bath and at 3 mm Hg of vacuum to obtain a second semi-solid brown residue, which meets the one obtained in point (d) above. The yield on the total waste (that of the two wastes together) usually ranges in the range 2-5% (w / w of dry biomass).
    g) The total residue obtained in section (f) is dissolved in the minimum amount of chloroform in a pear-shaped flask (rotary evaporator flask) and silica gel is added for chromatographic heads (silica gel for column chromatography of 0.06-0.2 mm of grain size) in the proportion 2 grams of gel per gram of extract, and the solvent is evaporated in a rotary evaporator (3 mm Hg, 60 ° C) to obtain a "chromatographic head".
    h) A chromatographic column is mounted with silica gel for chromatographic column with grain size of 0.04-0.06 mm. The dimensions of the column depend on the amount of extract obtained in section (f), which is the same as saying that the size appropriate to the amount of "chromatographic head" obtained in section (g). For reference, use the following proportion: 25 grams of silica gel per gram of total residue obtained in (f).
    i) The above chromatographic column is eluted with hexane and mixtures of hexane and ethyl acetate (100: 0, 95: 5, 90:10, 85:15, 80: 20,… 0: 100), changing the polarity (this is the proportion) each liter of eluent collected. The fractions collected will have a volume proportional to the size of the chromatographic column.
    j) Fractions eluting from the chromatographic column with the hexane-ethyl acetate mixture (30:70) are monitored by analytical thin layer chromatography (silica gel, 0.25 mm, hexane: acetone 6: 4 v / v) to identify the presence of (11R, 17R) -11-epi-fistularina-3 (1) (greenish-brown color, Rf = 0.74). Fractions containing only (11R, 17R) -11-epifistularin-3 (1) are combined and evaporated in a rotary evaporator. The result of the procedure is to obtain the desired substance (1) separated and purified.
    k) The fractions that in section (j) turn out to be mixtures of the compound (1) and other substances in the TLC analysis can be rechromatographed according to section (h) to obtain additional amounts of (11R, 17R) -11- epi-fistularin-3 (1).
    l) The regioisomeric mixture of 2 + 3 derivatives (in a 1: 1 ratio) is then obtained by reacting 3.00 grams of (11R, 17R) -11-epi-fistularin-3 (1) with 3.58 g of chloride of p-bromobenzoyl and 15 mL of pyridine, cooling with an ice bath. After 24 hours of reaction, 10 g of crushed ice and 10 mL of water are added. It is then extracted three times in a separatory funnel with 30 mL of diethyl ether (x3) and the extracts are washed with 2N HCI first and with a 5% NaHCO3 solution afterwards. Subsequent drying over anhydrous sodium sulfate followed by filtration and rotary evaporation yields 3,394 g of the reaction product. This last product is dissolved in 150 mL of
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    25
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    dichloromethane in a pear-shaped flask for rotary evaporator, 10 g of silica gel for chromatographic column (0.06-0.2 mm in grain size) are added, mixed well and dried. The chromatographic head thus obtained is applied to a wet chromatographic column containing 93 g of silica gel for a chromatographic column of 0.04-0.06 mm in grain size. The column is eluted with mixtures of hexane and acetone 95: 5, 90:10, 85:15, etc. The 2 + 3 stereoisomer mixture is identified by analytical thin layer chromatography on silica gel eluting with 6: 4 hexane-acetone and developing with oleum (greenish brown spots of Rf = 0.76). The spectroscopic data of substances 2 and 3 are given in Tables 1 and 2.
    The proportions of the materials used in this procedure are:  Marine sponge biomass [Aplysina (= Verongia) aerophoba] between 10.50% and a
    11.50%  Hexane between 38.00% and 38.80%  Acetone between 21.5% and 22.30%  Ethyl acetate between 34.10% and 35.00%  Dichloromethane between 0.80% and 1.20%  Silica gel for chromatography in column (0.06-0.2 mm) between 0.14% and 0.18%  Silica gel for column chromatography (0.04-0.06 mm) between 1.60% and 2.10
    %  P-Bromobenzoyl chloride between 0.01% and 0.03%  Pyridine between 0.06% and 0.10%  Diethyl ether between 0.30% and 0.40%  2N aqueous hydrochloric acid between 0.25% and 0.30%  5% aqueous sodium bicarbonate (w / w) between 0.20% and 0.35%  Anhydrous sodium sulfate between 0.04% and 0.06%
    The product obtained according to the aforementioned procedure is also described, characterized in that the main component is a mixture (1: 1) of the synthetic regioisomers 2 and 3 (Figures 1, 3 and 4), a mixture that is cytotoxic against several lines Human leukemia cells. Characteristics such as chromatographic constants, spectroscopic data and antileukemic activity data are given in the following sections (m-p).
    m) A compound obtained according to the previous procedure selected from the group consists of:
    The antileukemic (11R, 17R) -11-epi-fistularina-4 (2, Figures 1 and 3) .- It turns out to be a brown powder that presents in TLC (silica gel, 0.25 mm thickness) an Rf = 0.74 when eluted with hexane - acetone (6: 4) and it is revealed on the thin layer (oleum, 120 ° C) in daylight as a brown-greenish spot (Table 3). The spectrum of 1HRMN at 300 MHz (Table 1) and that of 13C-NMR at 75 MHz (Table 1), enriched by two-dimensional experiments confirm that this regioisomer has structure 2 (Figures 1 and 3), a formula that is New in the bibliography.
    Position (Figure 3) 1 H-NMR13C-NMR
    one 3.91 (1H, s)73.53 (CH)
    2 ---112.50 (C)
    3 ---147.70 (C)
    4 ---121.10 (C)
    5 6.56 (1H, s)131.10 (CH)
    6 ---90.70 (C)
    7 Overlapping38.50 (CH2)
    8 ---153.50 (C)
    9 ---160.20 (C)
    10 Overlapping42.36 (CH2)
    eleven 4.06 (1H, m)67.99 (CH)
    12 3.83 (2H, m)75.35 (CH2)
    13 ---151.60 (C)
    14.14 ' ---117.30 (C)
    15.15 ' 7.59 (2H, s)130.50 (CH)
    16 ---141.50 (C)
    17 4.68 (1H, m)69.34 (CH)
    18 3.30 (2H, m)46.25 (CH2)
    fifteen' 7.81 (2H, s)146.61; 146.51 (CH)
    2 ', 4' ---126.95 (C)
    3' ---154.97 (C)
    6 ' ---131.21 (C)
    7 ' 3.17 (2H, s)42.96 (CH2)
    8 ' ---158.44 (C)
    9 ' ---171.64 (C)
    OCH3 3.63 (6H, s)59.59 (CH3)
    1-OH 6.30 (s wide)---
    11-OH 5.27 (wide s)---
    17-OH 5.74 (s wide)---
    NH 8.41 (1H, t)---
    NH 8.46 (1H, t)---
    = N-OH 3.34 (1H, overlap)---
    Table 1.- 1 H-NMR and 13 C-NMR spectra of the (11R, 17R) -11-epi-fistularin-4 (2) in DMSO-D6.
    5 n) Another compound obtained according to the previous procedure selected from the group consists of:
    The antileukemic (11R, 17R) -11-epi-fistularin-5 (3, Figures 1 and 4). It turns out to be a brown powder that presents in TLC (silica gel, 0.25 mm thickness) an Rf = 0.74 when eluted with
    10 hexane - acetone (6: 4) and is revealed on the thin layer (oleum, 120 ° C) in daylight as a brown-greenish spot (Table 3). The 1HRMN spectrum at 300 MHz (Table 2) and the 13C-NMR spectrum at 75 MHz (Table 2), enriched by two-dimensional experiments confirm that this regioisomer has structure 3 (Figures 1 and 4), which is New in the bibliography.
    15 Table 2.- 1 H-NMR and 13 C-NMR spectra of the (11R, 17R) -11-epi-fistularin-5 (3) in DMSO-D6.
    Position (Figure 4) 1 H-NMR13C-NMR
    1.5 7.81 (2H, s)146.61; 146.51 (CH)
    2.4 ---126.91; 126.98 (C)
    3 ---154.92 (C)
    6 ---131.21 (C)
    7 3.17 (2H, s)42.96 (CH2)
    8 ---158.50 (C)
    9 ---171.64 (C)
    10 Overlapping42.36 (CH2)
    eleven 4.06 (1H, m)67.99 (CH)
    12 3.83 (2H, m)75.35 (CH2)
    13 ---151.60 (C)
    14.14 ' ---117.30 (C)
    15.15 ' 7.59 (2H, s)130.50 (CH)
    16 ---141.50 (C)
    17 4.68 (1H, s)69.34 (CH)
    18 3.30 (2H, m)46.25 (CH2)
    one' 3.91 (1H, s)73.48 (CH)
    2' ---112.50 (C)
    3' ---147.7 (C)
    4' ---121.10 (C)
    5' 6.59 (1H, s)131.10 (CH)
    6 ' ---90.70 (C)
    7 ’ Overlapping38.50 (CH2)
    8 ' ---153.50 (C)
    9 ' ---160.20 (C)
    OCH3 3.63 (6H, s)59.59 (CH3)
    1’-OH 6.40 (s wide)---
    11-OH 5.27 (wide s)---
    17-OH 5.74 (s wide)---
    NH 8.41 (1H, t)---
    NH 8.46 (1H, t)---
    = N-OH 3.34 (overlap)---
    o) TLC analysis of both bioactive substances.
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    Table 4. Effects of the 1: 1 mixture of (11R, 17R) -11-epi-fistularin-4 (2) and (11R, 17R) -11-epifistularin-5 (3) on the growth of three cell lines of human leukemia Cells were cultured for 72 hours, and IC50 values were deduced from graphs as shown in Figure 5. The data shown represents the average value of three independent experiments with three determinations in each. The etoposide was used as a positive control. Preferred Embodiment of the Invention
    A preferred embodiment of the invention can be summarized in the following steps:
    a) Collect 2 kg of biomass from the marine sponge Aplysina (= Verongia) aerophoba) that grows naturally on the coast of the Canary Islands between 10 and 30 meters deep (Figure 2).
    b) Dry the biomass in a vacuum oven (3 mm Hg, 60 ° C, 48 hours).
    c) Extract the biomass by cold maceration with 5 L of acetone.
    d) Remove the solvent in a rotary evaporator at 60 ° C in the water bath and at 3 mm Hg of vacuum to obtain a first blackish residue with a semi-solid appearance.
    e) Re-extract the hot biomass using a Soxhlet device (at least ten siphoned). Again said extraction is performed with acetone. The size of the Soxhlet apparatus can be such that it has a cabin for the biomass container cartridge of about 200 mL and the amount of solvent, about 5 L (applied in successive extractions of about 500 mL each).
    f) Remove the solvent containing the different extracts in a rotary evaporator, at 60 ° C in the water bath, and 3 mm Hg of vacuum to obtain a second semi-solid brown residue, which meets the one obtained at the point (d) above. The yield on the total waste (that of the two wastes collected) ranges from 2-5% (w / w of dry biomass).
    g) Dissolve the total residue obtained in section (f) in the minimum amount of chloroform, in a pear-shaped flask (rotary evaporator flask), add silica gel for column chromatography (0.06-0.2 mm grain size) in the ratio 2 grams of gel per gram of extract, and evaporate the solvent in a rotary evaporator (3 mm Hg, 60 ° C) to obtain a "chromatographic head".
    h) Mount a chromatographic column with silica gel for column chromatography (0.040.06 mm grain size). The dimensions of the column are adapted to the amount of extract obtained in section (f), which is the same as saying that of the appropriate size to the amount of "chromatographic head" obtained in section (g). For reference, use the following ratio: 25 grams of silica gel for chromatographic column (0.04-0.06 mm) per gram of total residue obtained in (f).
    i) Elute the previous chromatographic column with hexane and mixtures of hexane and ethyl acetate (100: 0, 95: 5, 90:10, 85:15, 80:20, ... 0: 100), changing the polarity ( this is the proportion) each liter of eluent collected. The fractions collected will have a volume of 100 mL each.
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    权利要求:
    Claims (1)
    [1]
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