CN102516214B - Diterpenoid compounds with antifouling activities - Google Patents

Abstract

The invention relates to the antifouling agent field. The objective of the invention is to obtain new substances with the antifouling activities through carrying out extracting separation on marine sessile organisms, especially Acanthella cavernosa. The invention provides five diterpenoid compounds separated from the Acanthella cavernosa, the chemical structure of the five diterpenoid compounds is represented by formula (I), and the five diterpenoid compounds are named kalihinol O, kalihinol P, kalihinol Q, kalihinol R and kalihinol S respectively. The compounds undergo in vitro Balanus Amphitrite larva adsorbing activity tests, and results show that the compounds have obvious antifouling activities. A new source is provided for preparing environmentally-friendly high efficiency antifouling agents.

Description

A kind of diterpene-kind compound with anti-fouling activity

Technical field

The present invention relates to stain control agent field, be specifically related to a kind of extraction and separate 5 new diterpene-kind compounds with anti-fouling activity that obtain from the sponge Acanthella cavernosa of the South Sea.

Background technology

Marine fouling organism is the general name of marine animal, plant and microorganism to being grown in hull bottom and marine Artificial facilities surface.Fouling organism is multiplied in a large number, can form harm greatly to boats and ships and marine Artificial facilities, such as increasing ship resistance, blocking pipe, acceleration metallic corrosion, making instrument and engine failure, threat aquaculture etc. in ocean.Often be only expense for preventing and kill off marine Artificial facilities biodeterioration just up to 6,500,000,000 dollars.At present, use antifouling paint be prevent and kill off marime fouling the most economy be also the most widely used method, organic tin stain control agent, as tributyl tin (TBT), tributyltin oxide (TBTO) etc., it was once the most effective stain control agent, but because it is difficult to degraded, thereby ocean environment is formed to serious threat, forbidden by International Maritime Organizaton.And widely used organotin substitutes antifouling paint and also started to gather in coastal waters as Irgarol1051 and Sea-Nine211 etc., and likely become the threat that the marine eco-environment is new.Thereby from marine sessile organism, find anti-fouling activity material and then be developed to new and effective nontoxic antifouling paint and just becoming a large focus of global antifouling paint research.

It is clean that set invertebrates body surface in ocean usually keeps, and not by stained, chemical ecology research shows that the chemical defence mechanism of this and these marine organisms uniqueness is closely related.The meta-bolites with anti-fouling activity can be synthesized and discharge to these marine organisms in vivo, and the anti-fouling activity material that the unicellular lower eukaryote of their predations produces is also enriched in body surface, avoids fouling organism larva in its surface attachment; Recent research also finds that sponge can produce active substance, regulates and controls the variation of symbiosis in the microflora of sponge body surface, thereby prevents that fouling organism is in its surface attachment.At present, from marine organisms, obtained the multiple anti-fouling activity material with good DEVELOPMENT PROSPECT.The special ecotope in ocean makes to have had following four advantages from halobiontic anti-fouling activity material: (1) is efficient, and very low concentration just can reach and prevent stained effect; (2) nontoxic, can not work the mischief to people, air and marine organisms; (3) wide spectrum, all has fouling resistance effect to most fouling organisms; (4) easily degraded, the transformation period is short, can not endanger Marine ecosystems.Till 2009, the anti-fouling activity compound obtaining from marine organisms has exceeded 130 kinds, and wherein, about 52% obtains from sponge, and sponge becomes the main source of ocean anti-fouling activity material.

Belong to from Acanthella the diterpene-kind compound that sponge, separation obtains and conventionally contain the nitrogen-containing groups such as isonitrile base, lsothiocyanates, methane amide; as kalihinol A～L, they be verified there is antimalarial, pest-resistant, antimycotic, fouling resistance and (the Garson M.J. of cytotoxic activity to a certain degree; Simpson J.S.Marine isocyanides and related natural products – structure, biosynthesis and ecology.Nat.Prod.Rep.2004,21,164-179.)

Separate the report of other new diterpene-kind compounds with anti-fouling activity that obtain but so far there are no from sponge A.cavernosa.

Summary of the invention

The object of the invention is to particularly extract South Sea sponge Acanthella cavernosa to separate from marine sessile organism obtain the new material with anti-fouling activity.

The invention provides 5 diterpene-kind compounds that separation obtains from the sponge Acanthella cavernosa of the South Sea, their chemical structure is as shown in formula I:

The wherein R of each compound ₁, R ₂, R ₃replace respectively as follows:

Compound	R 1	R 2	R 3
				kalihinol?O	NCS	NC	β-Cl
kalihinol?P	NC	NCS	α-Cl
				kalihinol?Q	NC	NCS	β-Cl
kalihinol?R	NCS	NCS	β-Cl

kalihinol?S

NCS

NHCHO

α-Cl

The preparation method of diterpene-kind compound kalihinol O of the present invention, kalihinol P, kalihinol Q, kalihinol R, kalihinol S is specific as follows:

1. the total crude extract of preparation

South Sea sponge A.cavernosa is pulverized, and acetone seepage is extracted routinely, by extracting solution concentrating under reduced pressure, obtains crude extract; (natural organic-compound extracts and separates and Structural Identification, Beijing: Chemical Industry Press, 2004.8.)

2. separation and purification

Crude extract is dissolved in water, and with dichloromethane extraction three times, extract is dispersed in uses sherwood oil and dichloromethane extraction each three times successively in 60%～90% methyl alcohol, and decompression and solvent recovery, obtains petroleum ether part and methylene dichloride position medicinal extract; Methylene dichloride position medicinal extract is gone up to silica gel decompression column chromatography routinely, carry out gradient elution by sherwood oil-acetone system with 100:1-50:1-30:1-15:1-10:1-5:1-1:1, obtain altogether from small to large 14 part elutriant Fr1～Fr14 by the difference of polarity; Fr7 separates with reversed-phase silica gel column chromatography, with 70%～100% methanol/water gradient elution 4 hours, flow velocity 8ml/min, elutriant merges recovery and obtains altogether 5 part Fr7a～Fr7e,, Fr7a is prepared with RP-HPLC, preparation condition is 88% methanol/water, flow velocity 2ml/min, retention time is 29.8 points, obtains compound kalihinol O.Fr7c is prepared with RP-HPLC, and preparation condition is 90% methanol/water, flow velocity 2ml/min, and retention time is 27.9 points, obtains compound kalihinol P.Fr7d is prepared with RP-HPLC, and preparation condition is 93% methanol/water, flow velocity 2ml/min, and retention time is 20.7 points, obtains compound kalihinol Q.Fr7e is prepared with RP-HPLC, and preparation condition is 93% methanol/water, flow velocity 2ml/min, and retention time is 22.9 points, obtains compound kalihinol R.Fr8 separates with silica gel column chromatography, carries out gradient elution by methylene chloride-methanol system with 100:1-50:1-30:1-10:1, separates and has obtained compound kalihiol S.

Diterpene-kind compound kalihinol O of the present invention, kalihinol P, kalihinol Q, kalihinol R, kalihinol S test through In Vitro Anti barnacle Balanus amphitrite larva adsorption activity, they all can significantly suppress the absorption of barnacle B.amphitrite larva, show that compound of the present invention has obvious anti-fouling activity.

Therefore, compound of the present invention can be for the preparation of new stain control agent.The present invention provides new source for preparing environmentally friendly high-efficiency pollution-proof agent.

Accompanying drawing explanation

Fig. 1 is the experiment effect that the compounds of this invention kalihinol O～S suppresses the absorption of barnacle B.amphitrite larva

Embodiment

Now in conjunction with the embodiments and accompanying drawing, the present invention is described in detail, but enforcement of the present invention is not limited only to this.

Embodiment 1. prepares diterpene-kind compound kalihinol O～S of the present invention

1. prepare general extractive

Get 5.5 kilograms of sponge A.cavernosa weight in wet bases, with the acetone soln seepage extraction of 10 times of volumes, united extraction liquid, decompression and solvent recovery, obtains crude extract;

2. separation and purification

Crude extract is dissolved in water, and with dichloromethane extraction three times, extract is dispersed in uses sherwood oil and dichloromethane extraction each three times successively in 60%～90% methyl alcohol, and decompression and solvent recovery, obtains petroleum ether part (49g) and methylene dichloride position (30g) medicinal extract; Methylene dichloride medicinal extract is gone up to silica gel decompression column chromatography routinely, carry out gradient elution by sherwood oil-acetone system with 100:1-50:1-30:1-15:1-10:1-5:1-1:1, obtain altogether from small to large 14 part elutriant Fr1～Fr14 by the difference of polarity; Fr7(3.5g) separate (MeOH/H with reversed-phase silica gel column chromatography ₂o7:3to0:1), obtain altogether 5 part Fr7a～Fr7e; Fr7a is prepared with RP-HPLC, (MeOH/H ₂o9:1), obtained compound kalihinol O(45.6mg); Fr7a is prepared with RP-HPLC, and preparation condition is 88% methanol/water, flow velocity 2ml/min, and retention time is 29.8 points, obtains compound kalihinol O (43.7mg).Fr7c is prepared with RP-HPLC, and preparation condition is 90% methanol/water, flow velocity 2ml/min, and retention time is 27.9 points, obtains compound kalihinol P (27.9mg).Fr7d is prepared with RP-HPLC, and preparation condition is 93% methanol/water, flow velocity 2ml/min, and retention time is 20.7 points, obtains compound kalihinol Q (23.7mg).Fr7e is prepared with RP-HPLC, and preparation condition is 93% methanol/water, flow velocity 2ml/min, and retention time is 22.9 points, obtains compound kalihinol R.Fr8 separates with silica gel column chromatography, carries out gradient elution by methylene chloride-methanol system with 100:1-50:1-30:1-10:1, separates and has obtained compound kalihiol S (10.1mg).

Through magnetic resonance detection analysis, determine the chemical structure of the each compound of the present invention, their physico-chemical property and spectral data are respectively:

Kalihinol O: colourless crystallization, mp183.0-185.0 ℃. (c0.1,CHCl ₃)。UV λ _maxnm (log ε): 196 (3.64), 242 (sh, 2.70); IR (KBr) ν _maxcm ^-1: 3396,2975,2870,2164,2057,1448,1384; CD (CH ₃cN) λ _max(Δ ε) 184 (7.95); HRESIMS m/z447.1850[M+Na] ⁺(calcd for C ₂₂h ₃₃n ₂o ₂sClNa, 447.1849); ¹h and ¹³c NMR data are in table 1 and table 2.

Kalihinol P: colourless crystallization, mp179.0-182.0 ℃.

(c0.1,CHCl ₃)。UV λ _maxnm (log ε): 195 (3.34), 240 (sh, 2.38); IR (KBr) ν _maxcm ^-1: 3388,2982,2869,2154,2082,1473,1384; CD (CH ₃cN) λ _max(Δ ε) 187 (13.43); HRESIMSm/z447.1846[M+Na] ⁺(calcd for C ₂₂h ₃₃n ₂o ₂sClNa, 447.1849); ¹h and ¹³c NMR data are in table 1 and table 2.

Kalihinol Q: colorless oil.

(c0.2,CHCl ₃)。UV λ _maxnm (log ε): 196 (3.31), 240 (sh, 2.36); IR (KBr) ν _maxcm ^-1: 3424,2949,2873,2130,2074,1449,1384; CD (CH ₃cN) λ _max(Δ ε) 188 (13.26); HRESIMS m/z447.1847[M+Na] ⁺(calcd for C ₂₂h ₃₃n ₂o ₂sClNa, 447.1849); ¹h and ¹³c NMR data are in table 1 and table 2.

Kalihinol R: colorless oil.

(c0.1,CHCl ₃)。UV λ _maxnm (log ε): 197 (3.46), 244 (sh, 2.47); IR (KBr) ν _maxcm ^-1: 3531,2952,2885,2169,2104,1455,1383; CD (CH ₃cN) λ _max(Δ ε) 190 (10.41); HRESIMS m/z479.1571[M+Na] ⁺(calcd for C ₂₂h ₃₃n ₂o ₂s ₂clNa, 479.1570); ¹h and ¹³c NMR data are in table 1 and table 2.

Kalihinol S: colourless crystallization, mp173-175 ℃.

(c0.12,CHCl3)。

UV λ _maxnm (log ε): 198 (3.25), 242 (sh, 2.73); IR (KBr) ν _maxcm ^-1: 3425,2954,2872,2081,1682,1462,1379; CD (CH ₃cN) λ _max(Δ ε) 198 (12.13); HRESIMSm/z465.1958[M+Na] ⁺(calcd for C ₂₂h ₃₅n ₂o ₃sClNa, 465.1955); ¹h and ¹³c NMR data are in table 1 and table 2.

Table 1.Klihinol O～P's ¹h-NMR data (CDCl ₃, 500MHz)

Table 2.Klihinol O～P's ¹³c-NMR data (CDCl ₃, 125MHz)

Embodiment 2: the compounds of this invention anti-fouling activity test

Select the maximum fouling organism barnacle B.amphitrite larva of harm to set up anti-fouling activity screening model.Adult barnacle picks up from Hong Kong (22o19'N, 114o16'E) tideland, take Chaetoceros gracilis as food, by the nauplius larva of barnacle is cultured to cypris larva stage in the seawater filtering at 0.22-μ m, 28 ℃ of culture temperature, with the young Venus anti-fouling activity test of Venus stage.

Detection compound kalihinol O～P is dissolved in to dimethyl sulfoxide (DMSO) and is made into a series of concentration gradients (0.5～10 μ g/ml), take sterile filtration seawater as control group.Get approximately 20 kentrogons and put into 24 hole polystyrene plates, every hole adds 1ml test fluid, and 5 of each concentration repeat sample, 24 porocyte culture plates are placed in to 30 ℃ of incubators and place 24 hours.The larva number adhering at dissection Microscopic observation, EC ₅₀social science statistics program for value (SPSS) is analyzed.

Experimental result: as shown in Figure 1, the compounds of this invention kalihinol O～S suppresses the EC of barnacle B.amphitrite larva absorption ₅₀value is respectively 1.43,0.72, and 1.48,1.16,0.53 μ M shows that the compounds of this invention kalihinol O～S has significant anti-fouling activity, can be for the preparation of new stain control agent.

Claims (1)

1. the preparation method of diterpene-kind compound kalihinol O, kalihinol P, kalihinol Q, kalihinol R, kalihinol S, is characterized in that the method is specific as follows:

A, prepare total crude extract

South Sea sponge A.cavernosa is pulverized, and acetone seepage is extracted, and by extracting solution concentrating under reduced pressure, obtains crude extract;

B, separation and purification

Crude extract is dissolved in water, and with dichloromethane extraction three times, extract is dispersed in uses sherwood oil and dichloromethane extraction each three times successively in 60%～90% methyl alcohol, and decompression and solvent recovery, obtains petroleum ether part and methylene dichloride position medicinal extract; Methylene dichloride position medicinal extract is gone up to silica gel decompression column chromatography routinely, carry out gradient elution by sherwood oil-acetone system with 100:1-50:1-30:1-15:1-10:1-5:1-1:1, obtain altogether from small to large 14 part elutriant Fr1～Fr14 by the difference of polarity; Fr7 separates with reversed-phase silica gel column chromatography, with 70%～100% methanol/water gradient elution 4 hours, flow velocity 8ml/min, elutriant merges recovery and obtains altogether 5 part Fr7a～Fr7e, Fr7a is prepared with RP-HPLC, and preparation condition is 88% methanol/water, flow velocity 2ml/min, retention time is 29.8 points, obtains compound kalihinol O; Fr7c is prepared with RP-HPLC, and preparation condition is 90% methanol/water, flow velocity 2ml/min, and retention time is 27.9 points, obtains compound kalihinolP; Fr7d is prepared with RP-HPLC, and preparation condition is 93% methanol/water, flow velocity 2ml/min, and retention time is 20.7 points, obtains compound kalihinol Q; Fr7e is prepared with RP-HPLC, and preparation condition is 93% methanol/water, flow velocity 2ml/min, and retention time is 22.9 points, obtains compound kalihinol R; Fr8 separates with silica gel column chromatography, carries out gradient elution by methylene chloride-methanol system with 100:1-50:1-30:1-10:1, separates and has obtained compound kalihiol S;

Described diterpene-kind compound kalihinol O, kalihinol P, kalihinol Q, kalihinol R, kalihinol S, its chemical structure is as shown in formula I:

The wherein R of each compound ₁, R ₂, R ₃replace respectively as follows:

。

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