CN103183666A - Cyclopiazonic acid compound, and preparation and application thereof - Google Patents

Abstract

The invention relates to the fields of medicines and novel pesticides, in particular to a cyclopiazonic acid compound and preparation and application thereof. The cyclopiazonic acid compound is 3-hydroxyl speradine A as described in formula (I) in the specification. The compound is used as a pesticide, a plant growth regulator or an antineoplastic drug. According to the invention, the novel cyclopiazonic acid compound with hydroxyl substitution at position 3 is obtained from marine sponge-associated fungi. The preparation of the compound provided by the invention is simple and efficient, and the prepared compound has high purity. A strain referred to in the invention has a reference value for further development and utilization of marine microbial resources.

Description

A kind of cyclopiazonic acid compounds and preparation and application

Technical field

The present invention relates to medicine and novel agrochemical field, be specially a kind of cyclopiazonic acid compounds and preparation and application.

Background technology

According to Food and Argriculture OrganizationFAO (FAO) report, can cause every year such as plant pest global grain drop in production to reach 31%-42%, directly threaten grain security.At present, the disease and pest problem of grain mainly relies on chemical synthetic pesticide and controls; But wherein there be low selectivity and the high toxicity of drug effect object in most of agricultural chemicals, also harmless or useful microorganism, insect and plant etc. is caused growth-inhibiting when killing target pest; Also can cause the problem of the aspects such as resistance, environmental pollution and food safety of insect.So searching low toxicity, environmentally friendly novel agrochemical are one and compel problem to be solved.

China is an ocean big country, has abundant Living marine resources; China also is a large agricultural country, has very strong dependency for the use of agricultural chemicals.The enforcement of this research will make the marine microorganism resource serve Agricultural Development better, and the novel low toxic and environment-friendly agricultural chemicals of development, reduce the toxicity agricultural chemicals usage quantity, ensure food safety and realize that the aspects such as sustainable development of agricultural have great importance.

The cyclopiazonic acid similar structures is very rare, has listed the natural product of all similar structures of present discovery in the accompanying drawing 1.Forefathers become to study to the intercrescence of α-cyclopiazonic acid, proved that it is to be developed by L-tryptophane, acetic acid, mevalonic acid, and its precursor compound are β-cyclopiazonic acid.It is to be derived by the D-tryptophane that the new compound iso-α that found in 2009-cyclopiazonic acid infers.Studies show that cyclopiazonic acid is by a part L-tryptophane, a part mevalonic acid, two molecule acetic acid synthesize, and β-cyclopiazonic acid is the precursor compound of cyclopiazonic acid.

Summary of the invention

The purpose of this invention is to provide a kind of cyclopiazonic acid compounds and preparation and application.

For achieving the above object, the present invention adopts technical scheme to be:

A kind of cyclopiazonic acid compounds, described cyclopiazonic acid compounds are 3-hydroxyl speradine A (3-hydroxyl speradine A), shown in (I):

There is the tautomer shown in figure (II) in E loop section shown in described (I) in the structural formula, and the E loop section can be expressed as following any structural unit;

Described E ring is (d) structure among the figure (II).

The preparation method of cyclopiazonic acid compounds:

1) aspergillus oryzae (Aspergillus oryzae) is inoculated in the liquid nutrient medium shake flask fermentation 6-10 days; Filter, filtrate is used absorption with macroporous adsorbent resin, and resin and filtrate mass volume ratio are for it than being 1: 10-30; After absorption is finished, use distilled water washing resin earlier, use the MeOH desorb again, get the cyclopiazonic acid crude extract behind the recovery methanol solvate;

2) the thick cyclopiazonic acid of gained separates with carrying out normal-phase chromatography in the step 1); At first removing non polar impurities with eluent ethyl acetate, is 90 with volume ratio: 10-80 again: ethyl acetate-methyl alcohol of 20 (v/v) wash-out, collect elution fraction, and namely get the chromatogram flow point that contains target compound;

3) above-mentioned gained chromatogram flow point adopts the anti-phase middle chromatogram of pressing to carry out purifying again, and adopting volume percent is the methanol aqueous solution wash-out of 50%-70%, collects elution fraction, namely gets the target compound of purity about 60%; Again the target compound of purity about 60% is carried out reversed-phase HPLC and carry out purifying, eluent is collected elution fraction for containing the methanol aqueous solution of trifluoroacetic acid (TFA), namely gets purity and is not less than 90% target compound.

Aspergillus oryzae (Aspergillus oryzae) was inoculated in the liquid nutrient medium shake flask fermentation 8 days; Fat is 20g/L with the ratio of filtrate.

The particle diameter of the silica filler that normal-phase chromatography separates described step 2) is 10-50 μ m.Described step 2) volume ratio of ethyl acetate-methyl alcohol is 85: 15 in.Pressing chromatograph packing material in anti-phase in the described step (3) is the ODS of particle diameter 40-60 μ m, and the ratio of ODS post wash-out methanol-water is 60%MeOH.Eluent is the methanol solution that contains the TFA of 0.03% concentration, wherein MeOH in the middle HPLC purifying of described step (3): the 0.03%TFA volume is=55-65: 45-35.Described MeOH: 0.03%TFA volume ratio=60: 40.

The application of cyclopiazonic acid compounds, described compound is as sterilant, plant-growth regulator or preparation anti-tumor drug.

Described compound is as the sterilant of control lepidoptera pest.The control lepidoptera pest is beet armyworm, Acariformes insect or carmine spider mite.

Described aspergillus oryzae is preserved in Chinese typical culture collection center (CCTCC) on December 26th, 2011, and preserving number is: CCTCC M 2011484, taxonomy called after: Aspergillus oryzae Hmp-F28.

Advantage of the present invention: the present invention obtains novel 3 cyclopiazonic acid compounds that exist hydroxyl to replace from coming from the ocean sponge fungi that grows nonparasitically upon another plant altogether.The effect that The compounds of this invention has excellent insecticidal activity, cytotoxic activity and promotes plant-growth.Preparation method provided by the present invention is simple, efficient simultaneously, products therefrom purity height.Bacterial strain involved in the present invention comes, and further development and use marine microorganism resource is had certain reference value.

Description of drawings

The compound that Fig. 1 provides for the embodiment of the invention ¹H NMR (Bruker AV 600, CDCl ₃) spectrogram.

The compound that Fig. 2 provides for the embodiment of the invention ¹³C NMR (Bruker AV 600, CDCl ₃) spectrogram.

The compound that Fig. 3 provides for the embodiment of the invention ₁H- ₁H COSY (Bruker AV 600, Pyridine-d ₅) spectrogram.

HSQC (Bruker AV 600, the CDCl of the compound that Fig. 4 provides for the embodiment of the invention ₃) spectrogram.

HMBC (Bruker AV 600, the CDCl of the compound that Fig. 5 provides for the embodiment of the invention ₃) spectrogram.

NOESY (Bruker AV 600, the Pyridine-d of the compound that Fig. 6 provides for the embodiment of the invention ₅) spectrogram.

The ESI Q-TOF MS spectrogram of the compound that Fig. 7 provides for the embodiment of the invention

The plant growth regulation of the romaine lettuce seedling of compound 3-hydroxyl speradine A (Lactuca sativa) that Fig. 8 provides for the embodiment of the invention (wherein, figure A sample concentration: 100 μ g/ml, figure B sample concentration: 50 μ g/ml, figure C sample concentration: 10 μ g/ml.)。

Embodiment

The following examples will give further instruction to the present invention, but not thereby limiting the invention.

The present invention relates to new cyclopiazonic acid is 3-hydroxyl speradine A (3-hydroxyl speradine A).Its general structure is shown in structure (I):

There is the tautomer shown in figure (II) in E ring in this structural general formula, and the E ring can be expressed as any structural unit in (II).

The preparation method may further comprise the steps:

(1), the preparation of cyclopiazonic acid crude extract: after aspergillus oryzae liquid state fermentation, filter, filtrate is used absorption with macroporous adsorbent resin, namely gets thick cyclopiazonic acid with the methyl alcohol desorption again.

(2), the rough segmentation of target compound: the thick cyclopiazonic acid of gained carries out the normal-phase chromatography separation with flash chromatography (flash chromatography) in the step (1); Filler is silica gel, adopt ethyl acetate-methyl alcohol (EtOAc-MeOH) solvent systems to carry out gradient elution, at first remove non polar impurities with eluent ethyl acetate, be 90 with volume ratio again: 10-80: ethyl acetate-methyl alcohol of 20 (v/v) wash-out namely gets the chromatogram flow point of target compound.

(3), the purifying of target compound: the flow point that the middle gained of step (2) contains target compound adopts the anti-phase middle chromatogram of pressing to carry out purifying again; Filler is ODS, and the employing volume percent is the target compound that the methanol aqueous solution wash-out of 50%-70% namely gets purity about 60%.Further carrying out purifying with reversed-phase HPLC namely gets purity and is not less than 90% target compound.

In aforesaid method: the described fermentation time of step (1) is 6-10 days, is preferably 8 days.The described macroporous adsorbent resin of step (1) can be HP20 series, HPD series, and D101 series, any in the AB-8 series, and be not restricted to cited model.The usage ratio of the described macroporous adsorbent resin of step (1) and fermented liquid is 10g/L-30g/L, is preferably 20g/L.The easy volume ratio of ethyl acetate-methanol mixed that the described wash-out of step (2) obtains the target compound flow point is 90: 10-80: 20, be preferably 85: 15 (v/v).The solvent of the described wash-out ODS of step (3) reverse-phase chromatographic column is that volume percent is the methanol aqueous solution of 50%-70%, and preferred concentration is the methanol aqueous solution of 60% volume percent.The described eluent for the HPLC purifying of step (3) is the methanol aqueous solution (volume percent) that contains the 55-65% of 0.03% trifluoroacetic acid (TFA), is preferably 60% the methanol aqueous solution (volume percent) that contains 0.03% trifluoroacetic acid (TFA).

Embodiment 1:

Substratum: 1. YGP substratum: yeast extract paste 5g, glucose 10g, peptone 5g, sea salt water 1L (sea salt concentration: 2.5%).2. solid Martin substratum: glucose 10g, peptone 5g, K ₂HPO ₄1g, MgSO ₄0.5g, agar 20g, sea salt water 1L (sea salt concentration: 2.5%).

The preparation of fermentation seed liquid: after streak inoculation on Martin's substratum, activation is 48 hours in 28 ℃ of incubators with aspergillus oryzae (Aspergillus oryzae Hmp-F28) bacterial strain.Spore inoculating after the picking activation is in the triangular flask of 3L in capacity, holds the YGP substratum of 1L in the triangular flask, places 28 ℃, cultivates in the 170rpm shaking table, waits to grow namely to can be used as fermentation seed liquid behind the bacterium ball and use about 48 hours.

The fermentation of bacterial strain Hmp-F28:

1. substratum: it is in the triangular flask of 3L that preparation YGP substratum 16L is sub-packed in 16 capacity, and every bottle of substratum is 1L, and 115 ℃ of autoclaving 30min cooling backs are standby.

2. inoculate and ferment: the seed liquor that makes previously with the pipette, extract after the sterilization is inoculated in the 16L fermented liquid, and inoculum size is 20ml/L.Then the 16L fermented liquid is placed 28 ℃, fermentation culture after 8 days in the 170rpm substratum, standby.

The preparation of target compound:

1. the preparation of cyclopiazonic acid crude extract: filtering fermentation liquor removed add HP20 type macroporous resin behind the thalline, resin demand is 20g/L, the fermented liquid that will add resin then jolt with 130rpm on the shaking table handle 1 hour after the elimination fermented liquid, then the gained resin is merged, and with after an amount of distilled water cleaning, add methyl alcohol and make methyl alcohol submergence resin, handle 20min at the 130rpm shaking table, meoh eluate is leached, use methanol-eluted fractions so repeatedly, and elutriant merged concentrating under reduced pressure, after the lyophilize meta-bolites crude extract 14.8g.

2. the rough segmentation of target compound: the particle diameter of silica filler is 10-50 μ m.The chromatographic column specification is: 70mm * 100mm.Adopt ethyl acetate-methyl alcohol (EtOAc-MeOH) solvent systems to carry out gradient elution, at first removing non polar impurities with eluent ethyl acetate, is 364.8 milligrams of the chromatogram flow points that ethyl acetate-methyl alcohol (v/v) wash-out of 85: 15 must contain target compound with volume ratio again.

3. the purifying of target compound: above-mentioned flow point adopted again press chromatogram to carry out purifying in anti-phase; Filler is ODS; The chromatographic column specification is 15mm * 70mm; ODS loading amount 4.0g.Adopting volume percent is that namely to get purity be 61.3% target compound for 60% methanol aqueous solution wash-out.Further carry out purifying with reversed-phase HPLC.The HPLC condition is: the chromatographic column specification is 10mm * 250mm; Flow velocity 2.5ml/min; Detect wavelength 220nm; Moving phase is the methanol aqueous solution that contains the volume percent 60% of 0.03%TFA; Type of elution: isocratic elution; Flow velocity is 2.5ml/min; The collection retention time is that the chromatographic peak of 13.7min namely gets target compound 3-hydroxyl speradine A.Its purity is 94%.

Structure with the greening compound is identified

The comprehensive 1D that adopts, 2D NMR, MS, spectrum means such as CD have been identified the chemical structure of this compound, and nuclear magnetic data has been given ownership.

ESI-Q-TOF MS m/z 405.1425[M+Na] ⁺(calcd.for C ₂₁H ₂₂N ₂O ₅Na, 405.1421), so the deduction molecular formula is C ₂₁H ₂₂N ₂O ₅

${\left[\mathrm{\&alpha;}\right]}_{\mathrm{<figure-callout\; id="20"\; label="D"\; filenames="HDA0000126005530000012.png,HDA0000126005530000022.png"\; state="\{\{state\}\}">D</figure-callout>}}^{20}=-233.5(c=0.5,{\mathrm{CHCl}}_3)$

Present negative cotton effect at the 220nm place on the CD spectrogram, the cotton effect appears in 290nm.Determine that 5 of compounds are the S configuration; Determine that in conjunction with NOESY spectrum this compound steric configuration is: 4S, 5S, 11R.

¹H-NMR(600MHz，CDCl ₃)δ2.60(1H，brd，J＝11.1Hz，H-5)，2.74(1H，d，J＝14.3Hz，H-12a)，6.91(1H，d，J＝7.4Hz，H-14)，7.30(1H，t，J＝7.4Hz，H-15)，6.71(1H，d，J＝7.4Hz，H-16)，2.31(3H，s，H-20)，1.11(3H，s，H-21)，1.70(3H，s，H-22)，3.25(3H，s，H-23)。

¹H-NMR(600MHz，pyridine-d ₅)δ3.54(1H，t，J＝8.16Hz，H-4)，3.30(1H，m，H-11)，3.36(1H，dd，J＝9.72，15.1Hz，H-12b)。

¹³C-NMR(150MHz，CDCl ₃)δ179.1(C-2)，73.3(C-3)，46.3(C-4)，68.3(C-5)，192.1(C-6)，104.7(C-7)，171.6(C-8)，62.5(C-10)，51.3(C-11)，25.8(C-12)，136.2(C-13)，121.9(C-14)，131.3(C-15)，106.4(C-16)，142.9(C-17)，124.8(C-18)，184.7(C-19)，19.5(C-20)，24.9(C-21)，27.6(C-22)，26.8(C-23)。

¹H NMR (Bruker AV 600, CDCl ₃) spectrogram is referring to Fig. 1.

¹³(Bruker AV 600, CDClx) spectrogram is referring to Fig. 2 for C NMR.

¹H- ¹H COSY (Bruker AV 600, Pyridine-d ₅) spectrogram is referring to Fig. 3.

HSQC (Bruker AV 600, CDCl ₃) spectrogram sees attached referring to Fig. 4

HMBC (Bruker AV 600, CDCl ₃) spectrogram sees attached referring to Fig. 5.

NOESY (Bruker AV 600, Pyridine-d ₅) spectrogram is referring to Fig. 6.

ESI Q-TOF MS spectrogram is referring to Fig. 7.

Embodiment 2:

Experimental technique:

1. the processing of sample: after the 3-hydroxyl speradine A taking-up that is chilled in before above-described embodiment gained compound in subzero 20 ℃ of refrigerators, be mixed with 1mg/mL, 0.5mg/mL, 0.25mg/mL, 0.125mg/mL, six kinds of concentration of 0.0625mg/mL by doubling dilution, sample number amounts to 36, and is standby.2. the hatching of halogen worm: halogen worm artemia hatching solution: see (Solis P N, Wright C W, Anderson M M et al. (1993) .A microwell cytotoxicity assay using Artemia salina (brine shrimp) .Planta Medica, 59 (3), 250-252. 3. halogen worm bioassay method: get the front with liquid-transfering gun and dilute good sample 100 μ L in the hole of 96 orifice plates, add 100 μ L halogen worm larva suspension (Halogen worm 15-20 only) again, sample concentration just has been diluted into 0.5mg/mL like this, 0.25mg/mL, 0.125mg/mL, 0.0625mg/mL, 0.03125mg/mL, each concentration repeats 3, do blank with halogen worm larva suspension, in 28 ℃ of illumination boxs, placed 24 hours, observe with the binocular anatomical lens, record halogen worm death toll, HCl with 4mol/L puts to death all halogen worms then, record halogen worm sum.And (1) calculation correction mortality ratio by formula.

Experimental result:

The deadly activity of the table 1. compound 3-hydroxyl halogen worm of speradine A (brine shrimp)

Calculate its LD ₅₀Be 39.7 μ g/mL.By this embodiment as can be seen the halogen worm of 3-hydroxyl speradine A have very strong deadly activity.Therefore has the good prospect that can be developed as sterilant.

Embodiment 3:

Employing is tested based on romaine lettuce (Lactuca sativa) growth of seedling of 24 orifice plates:

Romaine lettuce (Lactuca sativa) seed is that the big speed of the U.S. is given birth to.The seed of choosing full grains is put into the micropore of 24 orifice plates that are covered with filter paper, 10 seeds in every hole.Above-mentioned gained 3-hydroxyl speradine A is mixed with 10,50, the aqueous solution of 100 μ g/ml.Every hole adds the sample solution of 200 μ L.4 repetitions of each sample.Control group adopts the distilled water that adds 200 μ L.Cultivate in illumination box the sealing back.Culture condition is temperature: 19C, light application time 16 hours.Have growth of seedling and carefully take out the long and cauline leaf length of seedling measurement seedling root after 5 days.

Experimental result: as shown in Figure 8,3-hydroxyl speradine A just can show the growth-promoting effect to root and stem under the lower concentration of 10 μ g/ml.Growth table to root when concentration is 50 μ g/ml reveals the strongest promoter action.Yet along with the rising of concentration, when 100 μ g/ml, though the growth of stem is had certain promoter action, the growth table of root is revealed very obvious suppression effect.Experiment shows that 3-hydroxyl speradine A (＜50 μ g/ml) under lower concentration shows the promoter action to growth of seedling, and shows the restraining effect to growth of seedling when high density (0.1-2mg/ml).When 100 μ g/ml, show the obvious restraining effect to root, and not remarkable to the influence of stem.Along with the reduction of concentration, root and stem are all shown significant growth promoting function.This shows that above-described embodiment gained compound shows the growth regulating effect of the plant hormone sample that lower concentration promotes, high density suppresses.This may structurally show with indolylacetic acid (IAA) with above-described embodiment gained compound has the approximate hydrophilic segment of similar hydrophobic part and space length, thereby presents the similar hormone-like effect with IAA.

Embodiment 4:

The experiment of cell in vitro cytotoxic activity:

Cell cultures: the human body tumour cell in the vegetative period of taking the logarithm is 5 * 10 with the RPMI-1640 nutrient solution dilution that includes 10% (v/v) foetal calf serum (fetal bovine serum) ⁴The cell suspending liquid of/L is inoculated in 96 orifice plates, 100 μ L/ holes.Place 37 ℃, saturated humidity, 5%CO ₂Cultivate 24h in the incubator.

Add specimen: behind the compound sample with DMSO dissolving above-described embodiment institute, diluting with the RPMI-1640 nutrient solution that contains 10% (v/v) foetal calf serum is 7.6,38,76,152,380 μ mol/L.Above-mentioned pastille nutrient solution is added in 96 orifice plates, be positioned in the incubator with cell cultures the same terms and cultivate 48h.

The mtt assay result measures: add 20 μ L MTT solution (5mg/mL in RPMI-1640 nutrient solution) after adding specimen processing 48h.The centrifugal supernatant liquor of removing behind the dyeing 4h.Add DMSO, measure OD value under the 570nm with microplate reader.

Data analysis is calculated in accordance with the following methods:

(1). inhibitory rate of cell growth=[1-administration group OD value/control group OD value] * 100%.

(2). adopt the LOGIT method to calculate medicine half-inhibition concentration (IC ₅₀).

Test-results: the human melanoma cell of 3-hydroxyl speradine A (A375-S2) and human cervical carcinoma cell (HeLa) all show tangible cytotoxic activity, two kinds of cell strain inhibiting rates and IC ₅₀Value is respectively 43.3 μ mol/L and 29.6 μ mol/L.

This shows that 3-hydroxyl speradine A has potential anti-tumor activity, further is developed as the possibility of cancer therapy drug again.

Claims (12)

1. cyclopiazonic acid compounds, it is characterized in that: described cyclopiazonic acid compounds is 3-hydroxyl speradine A (3-hydroxyl speradine A), shown in (I):

2. by the described cyclopiazonic acid compounds of claim 1, it is characterized in that: there is the tautomer shown in figure (II) in the E loop section shown in described (I) in the structural formula, and the E loop section can be expressed as following any structural unit;

3. by the described cyclopiazonic acid compounds of claim 2, it is characterized in that: described E ring is (d) structure among the figure (II).

4. the preparation method of the described cyclopiazonic acid compounds of claim 1 is characterized in that:

1) aspergillus oryzae (Aspergillus oryzae) is inoculated in the liquid nutrient medium shake flask fermentation 6-10 days; Filter, filtrate is used absorption with macroporous adsorbent resin, and resin and filtrate mass volume ratio are for it than being 1: 10-30; After absorption is finished, use distilled water washing resin earlier, use the MeOH desorb again, get the cyclopiazonic acid crude extract behind the recovery methanol solvate; Described aspergillus oryzae is preserved in Chinese typical culture collection center (CCTCC) on December 27th, 2011, and preserving number is: CCTCC M 2011484, taxonomy called after: Aspergillus oryzae Hmp-F28;

2) the thick cyclopiazonic acid of gained separates with carrying out normal-phase chromatography in the step 1); At first removing non polar impurities with eluent ethyl acetate, is 90 with volume ratio: 10-80 again: ethyl acetate-methyl alcohol of 20 (v/v) wash-out, collect elution fraction, and namely get the chromatogram flow point that contains target compound;

3) above-mentioned gained chromatogram flow point adopts the anti-phase middle chromatogram of pressing to carry out purifying again, and adopting volume percent is the methanol aqueous solution wash-out of 50%-70%, collects elution fraction, namely gets the target compound of purity about 60%; Again the target compound of purity about 60% is carried out reversed-phase HPLC and carry out purifying, eluent is collected elution fraction for containing the methanol aqueous solution of trifluoroacetic acid (TFA), namely gets purity and is not less than 90% target compound.

5. the preparation method of the described cyclopiazonic acid compounds of claim 4 is characterized in that: aspergillus oryzae (Aspergillus oryzae) was inoculated in the liquid nutrient medium shake flask fermentation 8 days; Fat is 20g/L with the ratio of filtrate.

6. the preparation method of the described cyclopiazonic acid compounds of claim 4 is characterized in that: the particle diameter of the silica filler that normal-phase chromatography separates described step 2) is 10-50 μ m.

7. the preparation method of the described cyclopiazonic acid compounds of claim 4 is characterized in that: the volume ratio of ethyl acetate-methyl alcohol is 85: 15 described step 2).

8. the preparation method of the described cyclopiazonic acid compounds of claim 4 is characterized in that: pressing chromatograph packing material in anti-phase in the described step (3) is the ODS of particle diameter 40-60 μ m, and the ratio of ODS post wash-out methanol-water is 60%MeOH.

9. the preparation method of the described cyclopiazonic acid compounds of claim 4 is characterized in that: in the described step (3) in the HPLC purifying eluent be the methanol solution that contains the TFA of 0.03% concentration, wherein MeOH: the 0.03%TFA volume is=55-65: 45-35.

10. the preparation method of the described cyclopiazonic acid compounds of claim 9 is characterized in that: MeOH: 0.03%TFA volume ratio=60: 40.

11. the application of the described cyclopiazonic acid compounds of claim 1 is characterized in that: described compound is as sterilant, plant-growth regulator or preparation anti-tumor drug.

12. the application by the described cyclopiazonic acid compounds of claim 11 is characterized in that: described compound is as the sterilant of control lepidoptera pest.

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