Application of camptothecin and derivatives thereof as antifouling agent

Abstract

The present invention provides application of camptothecin and derivatives thereof as antifouling agent, relating to camptothecin. Application of camptothecin and derivatives thereof as antifouling agent for facilities in water is provided, wherein the antifouling agent contains camptothecin and/or at least one of its derivatives. The application of camptothecin and derivatives thereof as antifouling agent for facilities in water can be used to prevent attachment of marine or freshwater micro-fouling organisms and/or large fouling organisms to the surfaces of facilities in the sea, lake, river or pool. The antifouling agent may also be used in mixture with other antifouling substance. Antifouling method for artificial facilities in water is provided. An antifouling paint for facilities in water is provided. Camptothecin and its derivatives have significant inhibitory activity to the attachment of fouling organisms, i.e. having antifouling activity, and can be used to prevent the attachment of fouling organisms on the surfaces of artificial facilities in water. Camptothecin and its derivatives have high antifouling activity, good antifouling efficiency, and broad-spectrum antifouling.

Claims

1. A method of antifouling, comprising administration of a compound having the structural formula: ##STR00002## wherein, R.sub.1 and R.sub.2 are each independently selected from a group consisting of H, amino, cyano, halogen, aldehyde, carboxyl, silyl, C.sub.1-C.sub.8 alkyl, C.sub.1-C.sub.8 alkylacyl, C.sub.1-C.sub.8 alkoxy and di(C.sub.1-C.sub.8 alkyl) amino; R.sub.3 and R.sub.4 are each independently selected from a group consisting of H, amino, cyano, halogen, aldehyde, carboxyl, silyl, C.sub.1-C.sub.8 alkyl, C.sub.1-C.sub.8 alkylacyl, C.sub.1-C.sub.8 alkoxy and di (C.sub.1-C.sub.8 alkyl) amino; or R.sub.3 and R.sub.4 form —OCH.sub.2O— or —OCH.sub.2CH.sub.2O— together; the compound being administered to a facility in water selected from the group consisting of ships, docks, mariculture netting, cages, offshore oil and gas platforms, buoys, wharves, piers, pipelines, stakes and submerged instruments and equipment, and combinations thereof.

2. The method according to claim 1, wherein the C.sub.1-C.sub.8 alkyl is unsubstituted or substituted by a group selected from a group consisting of halogen, cyano, nitro, hydroxyl, C.sub.1-C.sub.8 alkoxy, C.sub.1-C.sub.8 alkylamino, di (C.sub.1-C.sub.8 alkyl)amino and tri (C.sub.1-C.sub.8 alkyl) silyl.

3. The method according to claim 1, wherein the compound is selected from the listed compounds: TABLE-US-00005 NO. Compound Name R.sub.1 R.sub.2 R.sub.3 R.sub.4 1 Camptothecin (CPT) H H H H 2 10-hydroxyl-CPT H H —OH H 3 10-chloro-CPT H H —Cl H 4 10-bromo-CPT H H —Br H 5 10-cyano-CPT H H —CN H 6 10-nitro-CPT H H —NO.sub.2 H 7 10-carboxyl-CPT H H —COOH H 8 10-amino-CPT H H —NH.sub.2 H 9 10-methyl-CPT H H —CH.sub.3 H 10 10-aminomethyl-CPT H H —CH.sub.2NH.sub.2 H 11 7-methyl-CPT —CH.sub.3 H H H 12 7-ethyl-CPT —CH.sub.2CH.sub.3 H H H 13 7-hydroxymethyl-CPT —CH.sub.2OH H H H 14 7-(2-hydroxyl)ethyl-CPT —CH.sub.2CH.sub.2OH H H H 15 7-chloromethyl-CPT —CH.sub.2C1 H H H 16 7-(2-(methylamino)ethyl)-CPT —CH.sub.2CH.sub.2NHCH.sub.3 H H H 17 7-aldehyde-CPT —CHO H H H 18 7-(2-(trimethylsilyl)ethyl)-CPT —CH.sub.2CH.sub.2Si(CH.sub.3).sub.3 H H H 19 7-ethyl-10-hydroxyl-CPT —CH.sub.2CH.sub.3 H H H 20 9-methyl-CPT H —CH.sub.3 H H 21 9-hydroxyl-CPT H —OH H H 22 9-nitro-CPT H —NO.sub.2 H H 23 9-amino-CPT H —NH.sub.2 H H 24 9-acetylamino-CPT H —NHCOCH.sub.3 H H 25 9-dimethylaminomethyl-10-hydroxyl-CPT H —CH.sub.2N(CH.sub.3).sub.2 OH H 26 9-aminoethyl-10-hydroxyl-CPT H —CH.sub.2CH.sub.2NH.sub.2 OH H 27 10,11-methylenedioxy-CPT H H —OCH.sub.2O— 28 10,11-ethylenedioxy-CPT H H —OCH.sub.2CH.sub.2O— 29 7-chloromethyl-10,11-methylenedioxy-CPT —CH.sub.2Cl H —OCH.sub.2O— 30 7-chloromethyl-10,11-ethylenedioxy-CPT —CH.sub.2Cl H —OCH.sub.2CH.sub.2O— 31 9-amino-10,11-methylenedioxy-CPT H —NH.sub.2 —OCH.sub.2O—.

4. The method according to claim 1, wherein the antifouling agent is used to prevent the attachment of marine or freshwater large fouling organisms to the surfaces of the facility in the sea, lake, river or pool.

5. The method according to claim 1, wherein the antifouling agent is used in mixture with other antifouling substance, wherein the other antifouling substance is one member selected from a group consisting of coppery compound, zincous compound, isothiazolinone compound, triazine compound, N-2,4,6-trichlorophenyl maleimide, pyridinetriphenyl borane, 2-(p-chlorophenyl)-3-cyano-4-bromo-5-trifluoromethyl-pyrrole and N-(fluorodichloro methylthio)-phthalimide; wherein the coppery compound is one member selected from a group consisting of cuprous oxide, copper thiocyanate and copper pyrithione; and the zincous compound is one member selected from a group consisting of zinc oxide and zinc pyrithione; and the isothiazolinone compound is 4,5-dichloro-2-n-octyl-4-isothiazolin-3-tone; and the triazine compound is N-cyclopropyl-N′-(1,1-dimethylethyl)-6-(methylthio)-1,3,5-triazine-2,4-diamine.

6. The method according to claim 1, comprising the steps of: the compound of the structural formula: ##STR00003## being added into a paint, then the paint mixture being applied to the facility in water; or directly coated on the surfaces of the facility in water; or added into the components of the facility in water; or directly dissolved and released into the surrounding water environment of the facility in water.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows the inhibitory effect of CPT on the attachment of cypris larva of Balanus albicostatus.

(2) FIG. 2 shows the inhibitory effect of 10-hydroxyl-CPT on the attachment of cypris larva of Balanus albicostatus.

(3) FIG. 3 shows the inhibitory effect of CPT on the attachment of larva of Bugula neritina.

(4) FIG. 4 shows the inhibitory effect of 10-hydroxyl-CPT on the attachment of larva of Bugula neritina.

(5) FIG. 5 shows the inhibitory effect of CPT on the production of byssal threads of Perna viridis.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Example 1

Tests for Inhibitory Effects of CPT and Derivatives Thereof on the Attachment of Fouling Organisms

(6) (1) Test for Inhibitory Effect on the Attachment of Barnacles

(7) Barnacles are sessile Cirripedia with calcareous shells and widespread. They firmly attach to the surfaces of vessels and various offshore artificial facilities, and are one of the major marine fouling organisms as well as one of the major target organisms in the study of antifouling technology. Adults of Balanus albicostatus were collected from the rocks and piers at Baicheng Coast in Xiamen, and their cypris larvas were obtained by culturing in laboratory. It is the attachment of the cypris larvas that causes the barnacles to change from swimming life to attaching life. Thus, inhibition of the attachment of the cypris larvas can verify the antifouling activity of the compounds. CPT and 10-hydroxyl-CPT were dissolved in ethyl acetate respectively, and a series of concentration gradient were set according to the results of pre-experiment. 1 ml of each solution was taken to a petri dish respectively, 1 ml of ethyl acetate was taken to another blank petri dish serving as control group. After the solvents evaporated completely and the compounds were distributed evenly on the bottom surfaces and the side surfaces of the petri dishes, each petri dish was added with 10 ml of membrane filtrated seawater (filtrated by membrane with pore size of 0.22 μm, the same below). Each experimental group and control group had 3 paralleled cups, and each cup was added with about 30 cypris larvas of Balanus albicostatus. Stereomicroscope was used to observe the attachment of the cypris larvas 48 h after the addition, and the EC.sub.50 values of CPT and 10-hydroxyl-CPT, which are half inhibition concentrations for the attachment of cypris larvas of Balanus albicostatus, were determined. (EC.sub.50 refers to the effective concentration for inhibiting 50% of the attachment of the tested organisms. The lower the value is, the higher the antifouling activity will be, the same below.)

(8) The test results are showed in FIGS. 1 and 2. The results show that CPT and 10-hydroxyl-CPT can both significantly inhibit the attachment of cypris larvas of Balanus albicostatus at low concentration, showing efficient antifouling activity. The half inhibition concentrations (EC.sub.50) of CPT and 10-hydroxyl-CPT for the attachment were 1.73 μg ml.sup.−1 and 1.78 μg ml.sup.−1 respectively.

(9) (2) Test for Inhibitory Effect on the Attachment of Bugula

(10) Bugulas are marine bryozoan and widespread in various sea areas in the world. They often attach to the surfaces of marine artificial facilities like mariculture netting, cages, vessels, buoys, etc., and they are important marine fouling organisms as well. Bugula neritina, also called multicellular Bugula, its adults were collected from the fish culture net bins in Western sea area of Xiamen and put into an aquarium filled with fresh seawater after back to the laboratory to induce the release of their swimming larvas. It is the attachment of their swimming larvas that causes the Bugulas to change from swimming life to attaching life. Thus, inhibition of the attachment of the swimming larvas can verify the antifouling activity of the compounds. CPT and 10-hydroxyl-CPT were dissolved in ethyl acetate respectively, and a series of concentration gradient were set according to the results of pre-experiment. 1 ml of each solution was taken to a petri dish respectively; 1 ml of ethyl acetate was taken to another blank petri dish serving as control group. After the solvents evaporated completely and the compounds were distributed evenly on the bottom surfaces and the side surfaces of the petri dishes, each petri dish was added with 10 ml of membrane filtrated seawater. Each experimental group and control group had 3 paralleled cups, and each cup was added with about 30 larvas of Bugula neritina. Stereomicroscope was used to observe the attachment of the larvas of Bugula neritina 48 h after the addition, and the half inhibition concentrations for the attachment of larvas of Bugula neritina, EC.sub.50 values of CPT and 10-hydroxyl-CPT were determined.

(11) The test results are showed in FIGS. 3 and 4. The results show that CPT and 10-hydroxyl-CPT have significant inhibitory effect on the attachment of larvas of Bugula neritina, and the EC.sub.50 values were 15.02 μg ml.sup.−1 and 5.93 μg ml.sup.−1 respectively.

(12) (3) Test for Inhibitory Effect on the Attachment of Mussels by Produced Byssal Threads

(13) Mussels are common bivalve in marine fouling biocenoses, widespread, and are one of the major target organisms in the study of marine antifouling technology. Mussels explore surfaces by their feet, looking for attachment substrate. If proper surfaces for attaching were found, mussels would produce byssal threads to attach to them. Thus if the compounds inhibited mussels to produce byssal threads, the compounds could be proved to have antifouling activity. Perna viridis were collected from the fish net bins in offshore sea area of Zhangzhou, and in which those with shell length of 1.4˜2.4 cm were selected and washed with seawater, followed by gent cut-off of the byssal threads. And then the Perna viridis were washed with membrane filtrated seawater. CPT were first dissolved in a trace of dimethyl sulfoxide, and then mixed with membrane filtrated seawater to prepare a series of concentration gradient. 4 ml of each solution was taken into a 12-well plate, and each well was added with one Perna viridis. 4 ml of membrane filtrated seawater containing a trace of dimethyl sulfoxide and one Perna viridis were added into the control group. Each experimental group and control group had 8 paralleled groups. After 24-hours culture at room temperature, the amount of byssal threads produced by Perna viridis were observed, and the half inhibition concentration of CPT for the attachment of Perna viridis by produced byssal threads, EC.sub.50, were determined.

(14) The test results are showed in FIG. 5. The results show that CPT has significant inhibitory effect on the attachment of Perna viridis by produced byssal threads, the EC.sub.50 value was 10.78 μg ml.sup.−1.

(15) The other representative CPT derivatives listed in Table 1 were tested by the same above methods, and generally the values of EC.sub.50 were lower than 200 μg ml.sup.−1, which also had significant inhibitory effect on the attachment of fouling organisms, showing antifouling activity.

Example 2

Preparation of Antifouling Paint Containing CPT

(16) CPT was well mixed with acrylic resin, rosin, iron oxide red, thixotropic agent and organic solvent, and also glass beads were added. The mixture was stirred in high-speed dispersion machine until the fineness of the paints was around 80 μm, then the glass beads were removed by filtration with 100-mesh tulle, followed by discharge and obtaining the antifouling paint.

Example 3

Preparation of Antifouling Paints Containing Compound of CPT and Other Antifouling Agent

(17) Marine antifouling paints containing compound of CPT and another antifouling agent were prepared by the same method stated in Example 2. The types and weight ratios for the compound of CPT and another antifouling agent respectively were CPT:Cu.sub.2O (4:1), CPT:Cu.sub.2O (2:1), CPT:Cu.sub.2O (1:1), CPT:TCPM (1:1), CPT:ZnPt (1:1). Five different marine antifouling paints were prepared according to the types and weight ratios above, wherein TCPM is N-2,4,6-trichlorophenyl maleimide, ZnPt is Zinc pyrithione.

Example 4

Antifouling Efficiency in Sea Area Test for Antifouling Paint Containing CPT and Paints Containing Compound of CPT and Another Antifouling Agent

(18) As shown in Table 2, the tested antifouling agents were provided in three large groups: 1) CPT; 2) compound of CPT and another antifouling agent; 3) existing common antifouling agent (as positive control). All of the tested antifouling agents had a weight ratio of 20% to the marine antifouling paints.

(19) TABLE-US-00002 TABLE 2 Results of panel test in sea area antifouling agents coverage ratio of fouling organisms (%, Mean ± SE) in the paints after 3 months after 6 months after 9 months after 12 months 1) CPT 0.15 ± 0.12 11.6 ± 6.68 9.03 ± 2.64  39.67 ± 10.43 2) compound of CPT and another antifouling agent CPT:Cu.sub.2O (4:1) 0 5.87 ± 4.75 18.06 ± 7.87   61.05 ± 13.07 CPT:Cu.sub.2O (2:1) 0.84 ± 0.68 28.56 ± 14.79 62.77 ± 11.68  67.63 ± 13.07 CPT:Cu.sub.2O (1:1) 1.65 ± 0.90 54.24 ± 16.94 70.16 ± 16.43 90.85 ± 4.96 CPT:TCPM (1:1) 0.85 ± 0.14 30.12 ± 5.37  61.32 ± 23.09 86.93 ± 8.47 CPT:ZnPt (1:1) 0.44 ± 0.16 10.01 ± 4.32  35.10 ± 2.71  94.07 ± 3.71 3) existing common antifouling agent Cu.sub.2O 7.23 ± 2.02 91.75 ± 2.90  70.91 ± 13.40 98.88 ± 0.48 CuPt 13.09 ± 2.71  37.20 ± 11.69 43.11 ± 15.25 94.69 ± 1.01 paint with none 82.88 ± 7.48  90.16 ± 4.15  72.13 ± 10.16 95.41 ± 1.49 antifouling agent Note: TCPM is N-2,4,6-trichlorophenyl maleimide, ZnPt is Zinc pyrithione, CuPt is Copper pyrithione.

(20) The panel test in natural sea area was carried out referring to the national standard GB/T5370-2007 “Method for testing antifouling panels in shallow submergence”. The prepared antifouling paints each were evenly coated on the epoxy resin panels; paints with none antifouling agent and prepared by the same methods were provided as negative control; each coated sample had 6 paralleled groups. The tested panels were fixed in iron frames after the paints had dried, and they were hanged on the test buoyant rafts in the sea area near Dalipu Island in Xiamen in June, 2010. After the tested panels had been immersed in seawater up to 3, 6, 9 and 12 months, they were then photographed, and the coverage ratios of the fouling organisms in each coated sample areas were analyzed and counted. Herein, the coverage ratio of the fouling organisms is the ratio of surface area covered by marine large fouling organisms in the sample area divided by the whole surface area in the sample area (the same below), and the lower the value is, the higher the antifouling efficiency will be.

(21) The results of antifouling efficiency in sea area test on the prepared antifouling paints are shown in Table 2. During the test, the major large fouling organisms attaching to the tested panels were barnacles, mussels, oysters, sea squirts, sponges and bryozoans, etc. As can be seen from Table 2, the coverage ratios of the coated sample area with prepared antifouling agents containing CPT were far lower than the coverage ratios of the control coated sample area with none antifouling agent, which indicates that the paints prepared with CPT as antifouling agent have efficient antifouling performance, and the antifouling term is up to 12 months. As also can be seen from Table 2, among the antifouling agents which were used in the preparation of the paints with the same content of 20% (by weight), CPT had better antifouling efficiency in sea area than the existing common antifouling agents of cuprous oxide (Cu.sub.2O) and copper pyrithione (CuPt). On the other hand, the compounds of CPT and another antifouling agent of Cu.sub.2O, TCPM or ZnPt also showed certain antifouling efficiency, in which the CPT: Cu.sub.2O (4:1) had the best antifouling efficiency. However, both the antifouling efficiencies and the antifouling terms of the group of compounds were not as good as the group of pure CPT, which further verifies that CPT has efficient antifouling activity.

Example 5

Test for the Application of Antifouling Paint Containing CPT on the Mariculture Netting (an Artificial Facility in Water)

(22) The preparation method of antifouling paint containing CPT was the same as stated in Example 2, the weight ratio of CPT in the antifouling paint was also set to 20%. Dip coating method was used, and the nettings were immersed in each of the prepared paints respectively, and then taken out to dry in the air. The nettings were fixed on plastic frames by ribbons respectively and hanged in the mariculture area of nacre in Lingshui of Hainan in November, 2010. Paint with none antifouling agent and prepared by the same method was provided as negative control; the antifouling paint for wooden boats (chlorinated rubber as base material and cuprous oxide as major antifouling agent) which was bought from market was provided as positive control; the clean netting that had never immersed in any paint was provided as blank control. Each tested group had 3 paralleled groups. After the tested nettings had been immersed in seawater up to 3, 6, 9 and 12 months, they were then photographed, and the coverage ratios of the fouling organisms on the nettings respectively were analyzed and counted.

(23) The results of the test for application on the mariculture netting are shown in Table 3. During the test, the major large fouling organisms attaching to the nettings were bugulas, hydroides, sponges, sea squirts, oysters and seaweeds, etc. As can be seen from Table 3, the coverage ratios of fouling organisms on the nettings that coated with antifouling paint containing CPT were far lower than the nettings coated with none paint, which indicates that the antifouling paint containing CPT has good antifouling effect on mariculture netting, and the antifouling term is up to 12 months. On the other hand, the paint group with none antifouling agent did not show any antifouling effect, which indicates that the outstanding antifouling efficiency of marine antifouling paint containing CPT was derived from the antifouling activity of CPT. Besides, it also can be seen from Table 3 that the antifouling efficiency of marine antifouling paint containing CPT on the netting was better than the antifouling paint containing Cu.sub.2O as main antifouling agent. Generally, the results of the test indicate that the marine antifouling paint containing CPT has good antifouling application effect on mariculture netting.

(24) TABLE-US-00003 TABLE 3 The results of the test for application on mariculture netting coverage ratio of fouling organisms (%, Mean ± SE) tested groups after 3 months after 6 months after 9 months after 12 months marine antifouling 2.45 ± 0.42  2.19 ± 0.60 3.89 ± 1.07  9.64 ± 2.66 paint group with CPT antifouling paint with 5.23 ± 1.78 79.61 ± 4.27 64.60 ± 11.62 95.98 ± 2.55 Cu.sub.2O as main antifouling agent paint with none 85.00 ± 6.30  99.82 ± 0.05 69.08 ± 8.00  99.52 ± 0.24 antifouling agent blank control 71.16 ± 10.13 99.92 ± 0.03 54.83 ± 5.24  99.70 ± 0.21 with no paint

Example 6

Test for Application of Antifouling Paint Containing CPT on the Floating Bed (an Artificial Facility in Water)

(25) The preparation method of marine antifouling paint containing CPT was the same as stated in Example 2, the weight ratio of CPT in the marine antifouling paint was also set to 20%. The prepared paint was evenly coated on each floating bed component in the same specification (the floating bed component was a plastic foam board of 30×30 cm, coated with a plastic woven bag). After the paints had dried, the floating bed components were fixed on bamboo frames, and hanged in the Yundang Lake in Xiamen (previously a natural harbor, due to the construction of the dam, it has become substantially enclosed artificial lagoon and exchanges part of water with western sea area of Xiamen each day) in August, 2010. Paint with none antifouling agent and prepared by the same method was provided as negative control; the antifouling paint for wooden boats (chlorinated rubber as base material and cuprous oxide as major antifouling agent) which was bought from market was provided as positive control; clean floating bed components that had never coated with any paint were provided as blank control. Each tested group had 3 paralleled groups. After the tested floating bed components had been immersed in seawater up to 3, 6, 9 and 12 months, they were then photographed, and the coverage ratios of the fouling organisms on the floating bed component were analyzed and counted.

(26) The results of test for application on the floating bed are shown in Table 4. During the test, the major large fouling organisms attachting to the floating bed components were barnacles, bugulas, oysters, Mytilopsis salleis, sea squirts, and seaweeds, etc. As can be seen from Table 4, the coverage ratios of fouling organisms on the floating bed components that coated with antifouling paint containing CPT were far lower than the floating bed components coated with none paint, which indicates that the antifouling paint containing CPT has good antifouling effect on floating bed components, and the antifouling term is up to 12 months. On the other hand, the paint group with none antifouling agent did not show any antifouling effect, which indicates that the outstanding antifouling efficiency of marine antifouling paint containing CPT was derived from the antifouling activity of CPT. Besides, it also can be seen from Table 4 that the antifouling efficiency of marine antifouling paint containing CPT on the floating bed components was better than the antifouling paint containing Cu.sub.2O as main antifouling agent. Generally, the results of the test indicate that the marine antifouling paint containing CPT also has good antifouling application effect on floating bed.

(27) TABLE-US-00004 TABLE 4 The results of the test for application on floating bed coverage ratio of fouling organisms (%, Mean ± SE) tested groups after 3 months after 6 months after 9 months after 12 months marine antifouling 0.02 ± 0.01 0 29.32 ± 5.85  50.85 ± 15.11 paint group with CPT antifouling paint with 0.20 ± 0.10  31.78 ± 11.38 49.26 ± 8.32 98.27 ± 1.73 Cu.sub.2O as main antifouling agent paint with none 0.63 ± 0.29 50.87 ± 1.16 38.69 ± 3.64 99.97 ± 0.03 antifouling agent blank control group — 52.82 ± 5.56 50.89 ± 2.16 100 ± 0  with no paint Note: “—” means the photos of the group in that month were forgotten to taken, and the coverage ratio of fouling organisms of this group was not counted.