WO2018133114A1 - Novel natrural algicide with low toxicity to non-target organisms - Google Patents

Novel natrural algicide with low toxicity to non-target organisms Download PDF

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WO2018133114A1
WO2018133114A1 PCT/CN2017/072234 CN2017072234W WO2018133114A1 WO 2018133114 A1 WO2018133114 A1 WO 2018133114A1 CN 2017072234 W CN2017072234 W CN 2017072234W WO 2018133114 A1 WO2018133114 A1 WO 2018133114A1
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compound
kibdelomycin
composition
algicidal
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PCT/CN2017/072234
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French (fr)
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Ying Xu
Zhangli HU
Changyun Wang
Xiaoyan LIANG
Shuangfei Li
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Shenzhen University
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Publication of WO2018133114A1 publication Critical patent/WO2018133114A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D213/62—Oxygen or sulfur atoms
    • C07D213/70—Sulfur atoms
    • C07D213/71—Sulfur atoms to which a second hetero atom is attached
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
    • A01N43/34—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one nitrogen atom as the only ring hetero atom
    • A01N43/40—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one nitrogen atom as the only ring hetero atom six-membered rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D213/62—Oxygen or sulfur atoms
    • C07D213/63—One oxygen atom
    • C07D213/68—One oxygen atom attached in position 4
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/10—Nitrogen as only ring hetero atom
    • C12P17/12—Nitrogen as only ring hetero atom containing a six-membered hetero ring

Definitions

  • the present invention relates to a novel natural product isolated from the broth culture of a rare actinomycete strain Kibdelosporangium phytohabitans XY-R10 and its effect on controlling harmful algal blooms.
  • Harmful algal blooms (HABs) , a global problem threatening our environment, economy and health, are mainly evoked by two categories of algae (Glibert, P. M., et al. Oceanography, 2005, vol. 18 (2) , 135-147) .
  • One of them forming red tides in oceans is mainly caused by dinoflagellates.
  • Some notorious bloom forming dinoflagellates include Akashiwo sanguinea (Wardle, W. J., et al. In: National oceanic and atmospheric administration technical report national marine fisheries service, 1998, 143, 33-40; Bricelj, V. M., et al. J Shellfish Res. 1992, 11 (2) , 331-347. Friedman, C.
  • Toxic A. tamarense strains produce paralytic shellfish poisoning (PSP) toxins and have caused numerous illnesses and even deaths in humans after consumption of the contaminated shellfish.
  • PSP paralytic shellfish poisoning
  • Akashiwo sanguinea does not produce PSP toxins, yet its wide distribution and frequent outbreaks have been reported to be coincided with the mortality of other marine species, including crustaceans (Wardle, W. J., et al. In: National oceanic and atmospheric administration technical report national marine fisheries service, 1998, 143, 33-40. ) , oysters (Bricelj, V. M., et al. J Shellfish Res. 1992, 11 (2) , 331-347. Friedman, C.
  • Microcystis aeruginosa is of great economic and ecological importance as it produces the toxic microcystins, a type of pollutants often found in drinking water and causing public health problems and environmental issues in many countries, especially in heavily populated areas (Yen, H., et al. In Drinking Water Treatment, Supply and Management in Asia. 2006, 6, 161-167; Vasconcelos, V. M., Pereira, E. Water Res. 2001, 35 (5) , 1354-1357. Tencalla, F. G., et al. Aquat. Toxicol. 1994, 30 (3) , 215-224; Jochimsen, E. M., et al. N. Engl. J. Med. 1998, 338 (13) , 873-878; Carmichael, W. W. Hum. Ecol. Risk Assess. 2001, 7 (5) , 1393-1407) .
  • Kibdelosporangium is one of the rare actinomycetes genera and members of this genus are well known to produce novel antibiotics with glycopeptide, macrolides and polyketide structures, which have anticancer, antimicrobial and antiviral activities (Tiwari, K., et al. Crit. Rev. Biotechnol. 2012, 32 (2) , 108-132. ) .
  • reports on secondary metabolites are scarce from the recently sequenced K. phytohabitans which has a fairly large genome more than 10 MB (Xing, K., et al. Antonie Van Leeuwenhoek. 2012, 101, 433-441; Qin, S., et al. Appl. Soil Ecol. 2015, 93, 47-55) .
  • the members of the 2, 2’ -bipyridyl family of natural products including caerulomycins (Funk, A., et al. Canadian journal of microbiology. 1959, 5 (4) , 317-321; McInnes, A., et al. Canadian Journal of Chemistry, 1977, 55 (24) , 4159-4165) , collismycins (Shindo, K., et al. J. Antibiot. 1994, 47, 1072-1074. ) , SF2738B-F (Gomi, S., et al. J. Antibiot. 1994, 47, 1385-1394. ) , pyrisulfoxins (Tsuge, N., et al. J. Antibiot.
  • the present invention provides a compound of the formula I:
  • Y is optionally substituted C 1 to C 16 linear or branched chain alkyl
  • R 1 is H, C 1 to C 6 aliphatic hydrocarbon or cycloalkane or a halogen atom.
  • the R 1 is selected from the group consisting of methyl, ethyl, n-propyl, isopropyl, n-butyl and isobutyl.
  • the Y is selected from the group consisting of methyl, ethyl, n-propyl, isopropyl, n-butyl and isobutyl.
  • the present invention provides a composition comprising at least one preceding compound.
  • the present invention provides a algaecide having fungicidal and algicidal activity, characterized in containing the preceding compound or the preceding composition.
  • the present invention provides a algicidal agent comprising the preceding compound or the preceding composition, and acceptable salts thereof.
  • composition also comprising acceptable diluents, additives and/or carriers.
  • the present invention also provides the use of the preceding compound or the preceding agent or the preceding composition in lysing cells of eukaryotic or prokaryotic algae.
  • the algae is selected from the group consisting of Akashiwo sanguinea, Alexandrium tamarense, Prorocentrum micans, Chattonella marina, Heterosigma akashiwo, Microcystis aeruginosa.
  • the present invention provides a method for controlling harmful algal blooms using the preceding compound or the preceding agent or the preceding composition with effective concentration, wherein the effective concentration is 0.1 ⁇ g/mL to 100 ⁇ g/mL.
  • the effective concentration is 1-10 ⁇ g/mL.
  • the present invention also provides an iron chelator comprising the preceding compound or the preceding agent or the preceding composition.
  • the present invention also provides the use of the preceding compound or the preceding agent or the preceding composition in treating diseases associated with intracellular iron.
  • the diseases is selected from the group consisting of immunosuppressive disease, tumor, bacterial disease.
  • the present invention also provides a process for the isolation of the compound as defined in any one of the preceding claims useful as an algicidal compound from Kibdelosporangium phytohabitans XY-R10, the process comprises:
  • the bacterium is cultivated for a period of 6 days.
  • the bacterium is cultivated at 28°C.
  • the present invention also provides the use of a compound of the formula III in lysing cells of eukaryotic or prokaryotic algae
  • R is H, SCH 3 or SO 2 R 2 ;
  • R 2 is optionally substituted C 1 to C 16 linear or branched chain alkyl.
  • the R 2 is selected from the group consisting of methyl, ethyl, n-propyl, isopropyl, n-butyl and isobutyl.
  • the algae is selected from the group consisting of Akashiwo sanguinea, Alexandrium tamarense, Prorocentrum micans, Chattonella marina, Heterosigma akashiwo, Microcystis aeruginosa.
  • the present invention provides a novel algicidal compound belonging to the 2, 2’ -bipyridyl family of natural products from K. phytohabitans XY-R10.
  • the algicidal efficiencies of kibdelomycin A were evaluated on six species of harmful algae. The toxicity of kibdelomycin A towards five different species of aquatic organisms was also examined. Kibdelomycin A is a good algicidal agent. More importantly, it showed very low toxicity towards non-target organisms. Kibdelomycin A is a potential environmentally friendly algicide to replace CuSO 4 .
  • Figure 1 Chemical structures of compounds kibdelomycin A and its analogues caerulomycin A, collismysin A.
  • Figure 10 X-ray crystal structure of kibdelomycin A.
  • Figure 11 Algicidal effect of kibdelomycin A on A. sanguinea algal cells.
  • Light microscopic images ( ⁇ 10, ⁇ 40 magnification; a, d) showing intact algal cells in culture medium, while images ( ⁇ 10, ⁇ 40 magnification; b, c) showing immobilized, deformed cells, and images ( ⁇ 10, ⁇ 40 magnification; e, f) showing lysed algal cells.
  • Scale bars represent 100 ⁇ m in a, b, c and 50 ⁇ m in d, e, f.
  • FIG. 12 Time dependent antialgal efficiency of kibdelomycin A against M. aeruginosa. Two microliter of kibdelomycin A (10 ⁇ g/ ⁇ L) was added to the experimental group, and CuSO4 (2.5 ⁇ g/ ⁇ L) was added as the positive control. The values are mean values ⁇ SD of three experiments. Significant difference was reported when *P ⁇ 0.05; **P ⁇ 0.01.
  • FIG. 13 The effect of FeSO 4 on protecting the algal cells from lysing at the end of 36 h and 48 h. FeSO 4 was added to the algal cultures after they were treated by kibdelomycin A for different periods of time.
  • FIG. 14 (a) Aqueous FeSO 4 was added to pure kibdelomycin A dissolved in methanol to induce a violet color formation; (b) Molecular weight of the violet compound deduced form MS analysis suggested that the two molecules of kibdelomycin chelated one molecule of Fe (II) ; (c) Addition of FeSO 4 to kibdelomycin A at 1: 2 molar ratio resulted in a single peak in HPLC analysis further supported the complex was formed between two molecules of kibdelomycin A and one molecule of Fe (II) .
  • Kibdelomycin A refers to the compound isolated from the strain Kibdelosporangium phytohabitans XY-R10; the structure of kibdelomycin A is 4-methyloxyl-5-methylsulfonyl-2, 2’ -bipyridyl-6-carboxaldehyde oxime; and kibdelomycin A can be synthesized chemically or isolated from natural materials such as but not limited to Kibdelosporangium phytohabitans.
  • the strain Kibdelosporangium phytohabitans XY-R10 was isolated from the root sediments (3–5cm) of a mangrove plant Kandelia candel (L. ) Druce, collected from in Mai Po Inner Deep Bay Ramsar Site (E 114.05°, N22.49°) (Hong Kong, China) .
  • the bacterium was cultivated in multiple 250 mL Erlenmeyer flasks each containing 80 mL of SGTPY medium (5g starch, 5g glucose, 1g tryptone, 1g peptone, 1g yeast extract, 17g seasalts dissolved in 1 L of distilled water) at 28°C with agitation of 200 rpm for 6 days.
  • the culture broth (10 L) was extracted with a double volume of EtOAc three times.
  • the combined EtOAc layers were dried by an evaporator to give an EtOAc extract (1.5 g) , which was subjected to reverse phase silica chromatography using the eluent of water/methanol (7: 3, v/v) to provide five fractions.
  • the active fraction was further purified by using semi-preparative HPLC (phenomenex, 5 ⁇ m, 10 ⁇ 250 mm, 4 mL/min) eluting with 25%ACN-H 2 O to obtain the pure compound (5mg) .
  • Caerulomycin A (1mg) , collismysin A (1mg) were obtained from commercial vendors abcam (Shanghai, China) and Alfa Chemistry (New York, United States) , respectively.
  • Example 2 Identification of the algicidal compound from K. phytohabitans XY-R10
  • the structure of the active compound was established by extensive NMR and high-resolution mass spectroscopic data and confirmed by single-crystal X-ray diffraction analysis.
  • 1 H-and 13 C-NMR spectral data were obtained on a Bruker DRX-600 MHz in d 6 -DMSO with TMS as the internal standard instrument.
  • Mass spectrum data was analyzed by UPLC (Waters ACQUITY, USA) coupled with the Micro TOF-ESI-MS system (Bruker Daltonics GmbH, Bremen, Germany) .
  • Single-crystal data were measured on a on an Xcalibur, Atlas, Gemini ultra diffractometer.
  • kibdelomycin A belongs to the family of 2, 2’ -bipyridyl, and is most similar to pyrisulfoxin A, which was isolated as an antibiotic (Tsuge, N., et. al. J. Antibiot. 1999, 52, 505-507) .
  • the only significant differences in the NMR spectra between these two compounds were the chemical shifts of C-5 ( ⁇ C 124.5 in 1 vs ⁇ C 127.7 in pyrisulfoxin A) and C-9 ( ⁇ C 44.72 in 1 vs ⁇ C 39.4 in pyrisulfoxin A) .
  • the molecular weight of kibdelomycin A was determined to be 307.5143 deduced from the positive ion [M+H] + at m/z 308.0668 by HRESIMS data while pyrisulfoxin A has a molecular weight of 291.2546.
  • the 16 daltons more in molecular weight of kibdelomycin A than pyrisulfoxin A, the downfield shifts ofC-5 and the upfield shifts of C-9 indicate a sulfonyl group in kibdelomycin A to replace the sulfoxide group in pyrisulfoxin A.
  • HMBC analysis connected the methyl protons H-9 ( ⁇ H 3.41) to the quaternary carbon C-5 ( ⁇ C 124.5) of the aromatic ring ( Figure 1, 2) , for which the methyl sulfonyl group was allocated at the C-5 position.
  • the planar configuration of kibdelomycin A was also confirmed by the analysis of the X-ray single-crystal diffraction data ( Figure 3–10) .
  • Example 3 Algicidal/antialgal activities of kibdelomycin A
  • Kibdelomycin A was evaluated for its algicidal against five eukaryotic algal species including Akashiwo sanguinea, Alexandrium tamarense, Prorocentrum micans, Chattonella antiqua, Heterosigma akashiwo, as well as its antialgal activity against the notorious cyanobacterial bloom forming species Microcystis aeruginosa (These algal species were cultured using Guillard’s f/2 medium (Guillard, R. R. L., et. al. Dinoflagellates. 1984, 391-442) or BG-11 medium, under constant temperature (20 ⁇ 2°C or 25°C) and light (2000 lx, 12-h light/12-h dark cycle) conditions, Table 1) .
  • All tested compounds were dissolved in DMSO to make stock solutions of 50, 25, 10, 5, 2.5, 1.25, 0.625, 0.312, 0.156 mg/mL, respectively. Then 1 ⁇ L of the test solution was added to 1 ml of the algal culture in a well of a 24-well plate (Nunc, USA) . CuSO 4 was used as the positive control while DMSO was used in as the negative control.
  • A. sanguinea three dinoflagellates (A. sanguinea, A. tamarense, P. micans) and two raphidophytes (C. antiqua, H. akashiwo) were tested.
  • the algal strain was cultured to reach late exponential phase and added to the test solution.
  • the morphology of A. sanguinea cells was then examined under a light microscope at 0h, 6h, 12h, 24h, 36h, 48h, while the other algal species were examined daily during a 4-day period of incubation. Kibdelomycin A was tested on all 5 algal species while cearulomycin A and collismysin A were only tested on A. sanguinea due to their limited amount.
  • Microcystis aeruginosa was cultivated until the cell densities reached about 5 ⁇ 10 7 CFU/mL.
  • the chlorophyll a (Chl-a) contents of the cyanobacterial culture were determined for 0, 2, 4, 6, 8, 10 days according to the methods described by Chen et al (Chen, Y. W., et. al. J Lake Sci. 2006, 18, 550-552. ) .
  • the antialgal efficiency was calculated using the following equation:
  • Antialgal efficiency (%) (1–C t /C c ) ⁇ 100%
  • C c and C t are the Chl-a contents of the control and the sample treated by the antialgal compound, respectively.
  • kibdelomycin A was able to lyse all the treated cells of A. sanguinea, A. tamarense, P. micans, C. antiqua, H. akashiwo with minimal inhibitory concentrations (MIC values) of 1.25, 10, 5, 10, 5 ⁇ g/mL, respectively (Table 3) .
  • MIC values inhibitory concentrations
  • the MIC value here refers to the minimum concentration where 100%cells of the tested algae (except M. aeruginosa) population were lysed in comparison with the control, while MIC value refers to the minimum concentration at which algicidal efficiency on M. aeruginosa was up to 95%.
  • Embryos of Danio rerio (2 days after fertilization) juveniles of Daphnia magna newly hatched Artemia salina, adults of Hydra sinensis and Paramoecium caudatum were used as the test species.
  • the zebrafish scientific research community was adopted in fish water (pH 7.2–7.6, conductivity 500 mS, nitrates ⁇ 5 ppm) within a lighting cycle of 14 h light/10 h dark at 28.5°C.
  • Well-trained females produce fresh fertilized eggs.
  • Embryos can be raised in fish water and staged according to the description by Kimmel et al (Kimmel, C. B., et al. Dev. Dyn.
  • embryos at the shield stage were selected, and following, embryos were observed using the dissecting microscope and inverted compound microscope. 6–8 healthy embryos ( ⁇ 2dpf) were used and loaded into each well (200 ⁇ L fish water) of U-bottom 96-well plates, and kibdelomycin A, cearulomycin A, collismysin A and CuSO 4 were added to achieve the concentration of 0.31, 0.65, 1.25, 2.5, 5, 25, 75, 125 ⁇ g/mL, respectively. One percent of DMSO (v/v) in fish water serves as a negative control.
  • OECD guideline 202 D. magna was cultured in glass containers with Artificial Elendt M4 medium under a 16: 8 light-dark cycle at 20 ⁇ 1°C (OECD, 2004. OECD guideline 202. OECD Guidelines for Testing of Chemicals. Daphnia sp., acute immobilization test. Paris. Antkowiak, W.Z.; Gessner, W.P. Tetrahedron Lett. 1979, 21, 1931-1934. ) . The organisms were fed with Chlorella pyrenoidsa three times a week and the culture medium was refreshed. Ten healthy D. magna ( ⁇ 24 h) were placed in the 24 well plates, excess water was gently absorbed with a piece of soft absorbent paper and 1ml of the D. magna medium containing the test compounds was added immediately.
  • Hydra sinensis was cultivated in a rack and held vertically in a 15-litre glass aquarium filled with Hydra medium “M” at 22–24°C (Litchfield, J. D., et al. J. Pharm. Exp. Ther. 1949, 96, 399–408. ) , and the test organisms were fed with a few wheat grains.
  • P. caudatum was cultivated in a wheat culture medium at 22–24°C in the darkness.
  • the A. salina was cultivated according to the published protocols (Meyer, B. N., et al. Planta Medica 1982, 45, 31-34; Solis, P. N., et al. Planta Medica. 1993, 59, 250-252.
  • Kibdelomycin A belongs to the 2, 2’ -bipyridyl family of natural products, which have been reported to be associated with a variety of significant biological activities (Funk, A., et al. Canadian journal of microbiology. 1959, 5 (4) , 317-321; McInnes, A., et al. Canadian Journal of Chemistry, 1977, 55 (24) , 4159-4165; Shindo, K., et al. J. Antibiot. 1994, 47, 1072-1074; Gomi, S., et al. J. Antibiot. 1994, 47, 1385-1394; Tsuge, N., et al. J. Antibiot.
  • Kibdelomycin A, caerulomycin A and collismysin A lysed 100%algal cells of A. sanguinea with MIC values of 1.25 ⁇ g/mL, 1.25 ⁇ g/mL and 0.31 ⁇ g/mL, respectively (Table 4) .
  • Collismysin A was even more effective than CuSO 4 for lysing A. sanguinea.
  • the algicidal activity of kibdelomycin A, caerulomycin A and collismysin A might be due mainly to the skeleton of 2, 2’-bipyridine-6-carboxaldehyde oxime.
  • the LC 50 value ofkibdelomycin A to Danio rerio embryos is also greater than 125 ⁇ g/mL.
  • the large discrepancy between the LC and MIC values indicates that kibdelomycin A may be of low toxicities towards non-target organisms.
  • caerulomycin A and collismysin A were at least 150-fold more toxic to Danio rerio embryos than kibdelomycin A with LC 50 values of 0.873 ⁇ 0.05 ⁇ g/mL and 0.76 ⁇ 0.15 ⁇ g/mL, respectively. And they were also at least 20-fold more toxic to Daphnia magna than kibdelomycin A with LC 50 values of 2.05 ⁇ 0.10 ⁇ g/mL and 1.60 ⁇ 0.12 ⁇ g/mL, respectively. If the adequate quantities of caerulomycin A and collismysin A were added to reach the algicidal effects in the real environment, the safety of other aquatic organisms would be impaired.
  • Example 5 kibdelomycin A is a Fe (II) chelator
  • cearulomycin A exerts its immunosuppressive effect by depleting intracellular iron (Singla A. K., et al. Transplantation 2014, 97, 57-59. ) and collismycin A can act as an iron chelator to inhibit tumor cell growth (Kawatani, M., et al. Mol Cancer Ther 2013, 12, A243) .
  • kibdelomycin A, caerulomycin A and collismysin A killed the algal cells by hijacking intracellular irons.

Abstract

A novel 2, 2' -bipyridyl oxime, kibdelomycin A was isolated from the broth culture of a rare actinomycete strain Kibdelosporangium phytohabitans XY-R10. Its structure was established by extensive 1D and 2D NMR and high-resolution mass spectral analyses as well as single-crystal X-ray diffraction analysis. This natural product demonstrated a broad spectrum of algicidal activities. In addition, kibdelomycin A possesses lower toxicities compared with Copper sulfate Therefore, kibdelomycin A could potentially serve as an efficient and environmental-friendly algicide in controlling harmful algal blooms.

Description

A novel natural algicide with low toxicity to non-target organisms Technical Field
The present invention relates to a novel natural product isolated from the broth culture of a rare actinomycete strain Kibdelosporangium phytohabitans XY-R10 and its effect on controlling harmful algal blooms.
Technical Background
Harmful algal blooms (HABs) , a global problem threatening our environment, economy and health, are mainly evoked by two categories of algae (Glibert, P. M., et al. Oceanography, 2005, vol. 18 (2) , 135-147) . One of them forming red tides in oceans is mainly caused by dinoflagellates. Some notorious bloom forming dinoflagellates include Akashiwo sanguinea (Wardle, W. J., et al. In: National oceanic and atmospheric administration technical report national marine fisheries service, 1998, 143, 33-40； Bricelj, V. M., et al. J Shellfish Res. 1992, 11 (2) , 331-347. Friedman, C. S.； , et al. J Shellfish Res. 2002, 22 (2) , 603； Botes, L., et al. Harmful Algae. 2003, 2, 247-259； Jessup, D. A., et al. PLoS One. 2009, 4 (2) , e4550； Meyer, S. E., et al. Master thesis, San Diego State University, San Diego. 2012. ) , Alexandrium tamarense (Anderson, D., et al. Paralytic shellfish poisoning in southern China. Toxicon. 1996, 34 (5) , 579-590. ) , Prorocentrum micans (Zhou, M. J., et. al. Advance in Earth Science. 2006, 21 (7) , 673-679. ) . Toxic A. tamarense strains produce paralytic shellfish poisoning (PSP) toxins and have caused numerous illnesses and even deaths in humans after consumption of the contaminated shellfish. On the other hand, Akashiwo sanguinea does not produce PSP toxins, yet its wide distribution and frequent outbreaks have been reported to be coincided with the mortality of other marine species, including crustaceans (Wardle, W. J., et al. In: National oceanic and  atmospheric administration technical report national marine fisheries service, 1998, 143, 33-40. ) , oysters (Bricelj, V. M., et al. J Shellfish Res. 1992, 11 (2) , 331-347. Friedman, C. S.； , et al. JShellfish Res. 2002, 22 (2) , 603； Botes, L., et al. Harmful Algae. 2003, 2, 247-259. ) , abalones, sea urchins and sea birds (Jessup, D. A., et al. PLoS One. 2009, 4 (2) , e4550； Meyer, S. E., et al. Master thesis, San Diego State University, San Diego. 2012) . The other category forming green tides in freshwater bodies consists mainly of cyanobacteria. Among them, Microcystis aeruginosa is of great economic and ecological importance as it produces the toxic microcystins, a type of pollutants often found in drinking water and causing public health problems and environmental issues in many countries, especially in heavily populated areas (Yen, H., et al. In Drinking Water Treatment, Supply and Management in Asia. 2006, 6, 161-167； Vasconcelos, V. M., Pereira, E. Water Res. 2001, 35 (5) , 1354-1357. Tencalla, F. G., et al. Aquat. Toxicol. 1994, 30 (3) , 215-224； Jochimsen, E. M., et al. N. Engl. J. Med. 1998, 338 (13) , 873-878； Carmichael, W. W. Hum. Ecol. Risk Assess. 2001, 7 (5) , 1393-1407) .
To control HABs, physical or chemical methods have been adopted, but very few of them are applicable due to their high costs, secondary pollution, or impracticability (Anderson, D. M. Turning back the harmful red tides. Nature. 1997, 38, 513-514. Lee, Y. J., et al. HarmfulAlgae. 2008, 7, 154–162； Anderson, D. M. Ocean Coast Manag. 2009, 52, 342–347) . For example, copper sulphate (CuSO4) , the commonly used algicide, might stress or kill aquatic animals by inhibiting the entire phytoplankton community and cause subsequent water quality deterioration (Hrudey, S., et al. Toxic cyanobacteria in water. A guide to their public health consequences, monitoring and management. London, Routledge. 1999； Li, F. M., Hu, H. Y. Appl Environ Microb. 2005, 71, 6545-6553； Wang, B.； , et al. Harmful Algae. 2012, 13, 83-88) . Although biological methods are relatively effective in controlling HABs (Yoshinaga, I., et al. Mar. Ecol. Prog. Ser. 1998, 170, 33-44； Mayali,  X., et al. J. Eukaryot. Microbiol. 2004, 51 (2) , 139-144. ) , their mechanisms are usually complex and remain difficult to be resolved, posing obstacles to their development. Many genera of marine bacteria have been studied for their algicidal effects and important roles in regulating the growth of HABs (Mayali, X., et al. J. Eukaryot. Microbiol. 2004, 51 (2) , 139-144； Kodama, M., et al. Ecology of Harmful Algae Berlin. 2006, 243-255； Yang, C. Y., et al. Harmful Algae. 2012, 20, 132-141. ) , yet very few algicidal compounds have been isolated and identified. The relationships between algicidal bacteria and harmful algae are quite complex and have been investigated (Kodama, M., et al. Ecology of Harmful Algae Berlin. 2006, 243-255； Amin, S. A., et al. Microbiol. Mol. Biol. Rev. 2012, 76, 667-684)
In the course of finding more effective and environmentally friendly algicides, the outmost attention must be paid to investigating the toxicity of the active compounds towards non-toxic aqueous organisms. The small freshwater fish species Danio rerio serves as an important model organism in ecotoxicology (Hill, A. J., et al. Toxicol. Sci. 2005, 86, 6-19； Bopp, S. K., et al. Institute for Environment and Sustainability, Joint Research Center, European Commission, European Communities, Luxembourg. 2006) . Daphnia magna is a model organism widely used in freshwater eco-toxicological studies (Seda, J., Petrusek, A. J. Limnol. 2011, 70, 337. ) . Hydra sinensis (Beach, M. J. ； Pascoe, D. Water Res. 1998, 32, 101-106. ) , Paramecium caudatum (Takiguchi, N., et al. J. Biosci. Bioeng. 2002, 93, 416-420. ) , Artemia salina (P.K. Krishnakumar, et al. Fish. Technol. 2007, 44, 85-92； Awolola, G. V., et al. Int. J. Biol. Chem. Sci. 2010, 4, 633-641. ) are regarded as good representative organisms to investigate short-term toxicity of various compounds.
The genus Kibdelosporangium is one of the rare actinomycetes genera and members of this genus are well known to produce novel antibiotics with glycopeptide, macrolides and polyketide structures, which have anticancer,  antimicrobial and antiviral activities (Tiwari, K., et al. Crit. Rev. Biotechnol. 2012, 32 (2) , 108-132. ) . On the contrary, reports on secondary metabolites are scarce from the recently sequenced K. phytohabitans which has a fairly large genome more than 10 MB (Xing, K., et al. Antonie Van Leeuwenhoek. 2012, 101, 433-441； Qin, S., et al. Appl. Soil Ecol. 2015, 93, 47-55) .
The members of the 2, 2’ -bipyridyl family of natural products including caerulomycins (Funk, A., et al. Canadian journal of microbiology. 1959, 5 (4) , 317-321； McInnes, A., et al. Canadian Journal of Chemistry, 1977, 55 (24) , 4159-4165) , collismycins (Shindo, K., et al. J. Antibiot. 1994, 47, 1072-1074. ) , SF2738B-F (Gomi, S., et al. J. Antibiot. 1994, 47, 1385-1394. ) , pyrisulfoxins (Tsuge, N., et al. J. Antibiot. 1999, 52, 505-507. ) , et al., have been associated with various biologic activities, such as antibacterial, antifungal, antioxidant, anti-inflammatory and cytotoxic activities (Funk, A., et al. Canadian journal of microbiology. 1959, 5 (4) , 317-321； McInnes, A., et al. Canadian Journal of Chemistry, 1977, 55 (24) , 4159-4165； Shindo, K., et al. J. Antibiot. 1994, 47, 1072-1074； Gomi, S., et al. J. Antibiot. 1994, 47, 1385-1394； Tsuge, N., et al. J. Antibiot. 1999, 52, 505-507； Stadler, M. Arch. Pharm. (Weinheim) 2001, 334, 143-147； Singla A. K., et al. Transplantation 2014, 97, 57-59； Martinez, A., et al. WO2007017146 A3. 2007) . At the same time, the unique structures of caerulomycins and collismycins and their significant bioactivities have attracted many synthetic efforts, with accomplishments of the total synthesis of caerulomycins A, B, C, E (Trecourt, F., et al. J. Org. Chem. 1996, 61, 1673-1676； Mongin, F., et al. J. Org. Chem. 2002, 67, 3272-3276； Bobrov, D. N.； Tyvorskii, V. I. Tetrahedron 2010, 66, 5432-5434. ) , collismycins A and C (Trecourt, F., et al. J. Org. Chem. 1998, 63, 2892-2897) .
Summary of Invention
In one aspect, the present invention provides a compound of the formula I:
Figure PCTCN2017072234-appb-000001
wherein, X＝H, R1, OR1 or a halogen atom；
Y is optionally substituted C1 to C16 linear or branched chain alkyl；
R1 is H, C1 to C6 aliphatic hydrocarbon or cycloalkane or a halogen atom.
In some embodiment of the present invention, the R1 is selected from the group consisting of methyl, ethyl, n-propyl, isopropyl, n-butyl and isobutyl.
In some embodiment of the present invention, the Y is selected from the group consisting of methyl, ethyl, n-propyl, isopropyl, n-butyl and isobutyl.
In a specific embodiment of the present invention, wherein the compound is 4-methyloxyl-5-methylsulfonyl-2, 2'-bipyridyl-6-carboxaldehyde oxime of the formula II:
Figure PCTCN2017072234-appb-000002
In another aspect, the present invention provides a composition comprising at least one preceding compound.
In another aspect, the present invention provides a algaecide having fungicidal and algicidal activity, characterized in containing the preceding compound or the preceding composition.
In another aspect, the present invention provides a algicidal agent comprising the preceding compound or the preceding composition, and acceptable salts thereof.
In some embodiment of the present invention, wherein the composition also comprising acceptable diluents, additives and/or carriers.
In another aspect, the present invention also provides the use of the preceding compound or the preceding agent or the preceding composition in lysing cells of eukaryotic or prokaryotic algae.
In some embodiment of the present invention, wherein the algae is selected from the group consisting of Akashiwo sanguinea, Alexandrium tamarense, Prorocentrum micans, Chattonella marina, Heterosigma akashiwo, Microcystis aeruginosa.
In another aspect, the present invention provides a method for controlling harmful algal blooms using the preceding compound or the preceding agent or the preceding composition with effective concentration, wherein the effective concentration is 0.1μg/mL to 100μg/mL.
In some embodiment of the present invention, wherein the effective concentration is 1-10μg/mL.
In a further aspect, the present invention also provides an iron chelator comprising the preceding compound or the preceding agent or the preceding composition.
In a further aspect, the present invention also provides the use of the preceding compound or the preceding agent or the preceding composition in treating diseases associated with intracellular iron.
In some embodiment of the present invention, wherein the diseases is selected from the group consisting of immunosuppressive disease, tumor, bacterial disease.
In a further aspect, the present invention also provides a process for the isolation of the compound as defined in any one of the preceding claims useful as an algicidal compound from Kibdelosporangium phytohabitans XY-R10, the process comprises:
[a] cultivating the bacterium Kibdelosporangium phytohabitans XY-R10 in culture broth at 25-30℃ for 1-10 days；
[b] extracting the culture broth with EtOAc having a volume twice as that of the culture broth, and drying combined EtOAc layers；
[c] separating the active fraction using reverse phase silica chromatography with an eluent of water/methanol (7: 3, v/v) ；
[d] purifying the active fraction using semi-preparative HPLC with an eluent of 25％ACN-H2O to obtain the pure compound.
In some embodiment of the present invention, wherein the bacterium is cultivated for a period of 6 days.
In some embodiment of the present invention, wherein the bacterium is  cultivated at 28℃.
In a further aspect, the present invention also provides the use of a compound of the formula III in lysing cells of eukaryotic or prokaryotic algae
Figure PCTCN2017072234-appb-000003
wherein, R is H, SCH3 or SO2R2；
R2 is optionally substituted C1 to C16 linear or branched chain alkyl.
In some embodiment of the present invention, the R2 is selected from the group consisting of methyl, ethyl, n-propyl, isopropyl, n-butyl and isobutyl.
In some embodiment, wherein the algae is selected from the group consisting of Akashiwo sanguinea, Alexandrium tamarense, Prorocentrum micans, Chattonella marina, Heterosigma akashiwo, Microcystis aeruginosa.
Significance
The present invention provides a novel algicidal compound belonging to the 2, 2’ -bipyridyl family of natural products from K. phytohabitans XY-R10. The algicidal efficiencies of kibdelomycin A were evaluated on six species of harmful algae. The toxicity of kibdelomycin A towards five different species of aquatic organisms was also examined. Kibdelomycin A is a good algicidal agent. More importantly, it showed very low toxicity towards non-target organisms. Kibdelomycin A is a potential environmentally friendly algicide to replace CuSO4.
Brief Description of Drawings
Figure 1. Chemical structures of compounds kibdelomycin A and its analogues caerulomycin A, collismysin A.
Figure 2. Key HMBC correlations of kibdelomycin A.
Figure 3. 1H NMR (600 MHz, d6-DMSO) spectrum of kibdelomycin A.
Figure 4. 13C NMR (600 MHz, d6-DMSO) spectrum of kibdelomycin A.
Figure 5. 13C NMR spectrum (included DEPT 90, DEPT 135) of kibdelomycin A.
Figure 6. 1H–1H COSY (d6-DMSO) spectrum of kibdelomycin A.
Figure 7. HSQC (d6-DMSO) spectrum of kibdelomycin A.
Figure 8. HMBC (d6-DMSO) spectrum of kibdelomycin A.
Figure 9. HRESIMS spectrum of kibdelomycin A.
Figure 10. X-ray crystal structure of kibdelomycin A.
Figure 11. Algicidal effect of kibdelomycin A on A. sanguinea algal cells. Light microscopic images (×10, ×40 magnification； a, d) showing intact algal cells in culture medium, while images (×10, ×40 magnification； b, c) showing immobilized, deformed cells, and images (×10, ×40 magnification； e, f) showing lysed algal cells. Scale bars represent 100μm in a, b, c and 50μm in d, e, f.
Figure 12. Time dependent antialgal efficiency of kibdelomycin A against M. aeruginosa. Two microliter of kibdelomycin A (10μg/μL) was added to the experimental group, and CuSO4 (2.5 μg/μL) was added as the positive control. The values are mean values ±SD of three experiments. Significant difference was reported when *P<0.05； **P<0.01.
Figure 13. The effect of FeSO4 on protecting the algal cells from lysing at the end of 36 h and 48 h. FeSO4 was added to the algal cultures after they were treated by kibdelomycin A for different periods of time.
Figure 14. (a) Aqueous FeSO4 was added to pure kibdelomycin A dissolved in methanol to induce a violet color formation； (b) Molecular weight of the violet compound deduced form MS analysis suggested that the  two molecules of kibdelomycin chelated one molecule of Fe (II) ； (c) Addition of FeSO4 to kibdelomycin A at 1: 2 molar ratio resulted in a single peak in HPLC analysis further supported the complex was formed between two molecules of kibdelomycin A and one molecule of Fe (II) .
Figure 15. Algal culture treated by kibdelomyin A and the same peak corresponding to the Fe (II) -kibdelomycin A complex could be detected inside the algal cells.
Figure 16. Antibacterial activity of kibdelomycin A against Pseudomonas aeruginosa.
Table 1. List of algae used in this study.
Table 2. 1NMR Data (600 MHz, δin ppm, J in Hz) and 13C NMR Data (150 MHz, δin ppm) for kibdelomycin A.
Table 3. Algicidal/antialgal activities of kibdelomycin A.
Table 4. The algicidal effects of kibdelomycin A, caerulomycin A, collismysin A and CuSO4 on Akashiwo sanguinea, and toxicities against Danio rerio embryos and Daphnia magna.
Detailed Description
The present invention will be further illustrated below with reference to the specific examples. It should be understood that these examples are only used to describe the invention but not to limit the scope of the invention. The experimental methods with no specific conditions described in the following examples are generally performed under conventional conditions, and the materials used without specific description are purchased from common chemical reagents corporation.
Before describing the invention in detail, it is to be understood that this invention is not limited to particular biological systems or cell types. It is also to be understood that the terminology used herein is for the purpose of  describing particular embodiments only, and is not intended to be limiting. As used in this specification and the appended claims, the singular forms “a” , “an” and “the” include plural referents unless the content clearly dictates otherwise.
The term "kibdelomycin A" as used in this disclosure, refers to the compound isolated from the strain Kibdelosporangium phytohabitans XY-R10； the structure of kibdelomycin A is 4-methyloxyl-5-methylsulfonyl-2, 2’ -bipyridyl-6-carboxaldehyde oxime； and kibdelomycin A can be synthesized chemically or isolated from natural materials such as but not limited to Kibdelosporangium phytohabitans.
Examples
Example 1: Isolation of kibdelomycin A
The strain Kibdelosporangium phytohabitans XY-R10 was isolated from the root sediments (3–5cm) of a mangrove plant Kandelia candel (L. ) Druce, collected from in Mai Po Inner Deep Bay Ramsar Site (E 114.05°, N22.49°) (Hong Kong, China) . The bacterium was cultivated in multiple 250 mL Erlenmeyer flasks each containing 80 mL of SGTPY medium (5g starch, 5g glucose, 1g tryptone, 1g peptone, 1g yeast extract, 17g seasalts dissolved in 1 L of distilled water) at 28℃ with agitation of 200 rpm for 6 days. The culture broth (10 L) was extracted with a double volume of EtOAc three times. The combined EtOAc layers were dried by an evaporator to give an EtOAc extract (1.5 g) , which was subjected to reverse phase silica chromatography using the eluent of water/methanol (7: 3, v/v) to provide five fractions. Then the active fraction was further purified by using semi-preparative HPLC (phenomenex, 5μm, 10×250 mm, 4 mL/min) eluting with 25％ACN-H2O to obtain the pure compound (5mg) .
Caerulomycin A (1mg) , collismysin A (1mg) were obtained from commercial vendors abcam (Shanghai, China) and Alfa Chemistry (New York, United States) , respectively.
Example 2: Identification of the algicidal compound from K. phytohabitans XY-R10
Although a number of bacteria with agicidal effects have been isolated, identification of the active chemicals are challenging due to their wide variety of characteristics (Skerratt, J. H., et. al. Mar. Ecol. Prog. Ser. 2002, 244, 1-15. ) . Thus, only a handful of bacterial algicides have been purified and identified, including antibiotic-like substances (Nakashima, T., et. al. Appl. Microbiol. Biotechnol. 2006, 73, 684-690. ) , biosurfactants (Ahn, C. Y., et. al. Biotechnol. Lett. 2003, 25, 1137-1142； Wang, X., et. al. Harmful Algae 2005, 4, 433-443. ) , peptides (Jeong S. Y., et. al. Tetrahedron Lett. 2003 44, 8005-8007) , proteases (Lee, S. O., et. al. Biosci. Biotechnol. Biochem. 2002, 66, 1366-1369. ) , and other proteins (Mitsutani, A., et. al. Phycologia. 2001, 40, 286-291； Wang, B., et. al. Harmful Algae 2012, 13, 83–88. ) .
In this study, Akashiwo sanguinea (Table 1) were cultured using Guillard’s f/2 medium (Guillard, R. R. L., et. al. Dinoflagellates. 1984, 391-442) or BG-11 medium, under constant temperature (20±2℃ or 25℃) and light (2000 lx, 12-h light/12-h dark cycle) conditions； the algicidal activities of resultant fractions and the active compound were investigated and confirmed by the algicidal tests on Akashiwo sanguinea in 24 well plates. We found the crude extract of Kibdelosporangium phytohabitans XY-R10 were able to kill cells of different algal species. Following bioassay-guided fractionation, kibdelomycin A with strong algicidal activities was obtained.
Table 1 List of algae used in this study
Figure PCTCN2017072234-appb-000004
In addition, the structure of the active compound was established by extensive NMR and high-resolution mass spectroscopic data and confirmed by single-crystal X-ray diffraction analysis. 1H-and 13C-NMR spectral data were obtained on a Bruker DRX-600 MHz in d6-DMSO with TMS as the internal standard instrument. Mass spectrum data was analyzed by UPLC (Waters ACQUITY, USA) coupled with the Micro TOF-ESI-MS system (Bruker Daltonics GmbH, Bremen, Germany) . Single-crystal data were measured on a on an Xcalibur, Atlas, Gemini ultra diffractometer.
In particularly, to identify the chemical structure of kibdelomycin A, high-resolution mass and high-field NMR spectral analyses were carried out. The 1H NMR spectrum of kibdelomycin A showed two singlet methyl signals at δH 3.41, 4.16, five doublet olefinic or aromatic signals at δH 7.57, 8.02, 8.20, 8.42, 8.77, 8.84, one exchangeable proton signal at δH 11.73. The 13C NMR and DEPT data revealed that kibdelomycin A contained two methyl groups (δC 44.72, 57.25) , eleven olefinic or aromatic carbon atoms (Table 2) . These spectroscopic features suggested that kibdelomycin A belongs to the family of 2, 2’ -bipyridyl, and is most similar to pyrisulfoxin A, which was isolated as an antibiotic (Tsuge, N., et. al. J. Antibiot. 1999, 52, 505-507) . The only significant differences in the NMR spectra between these two compounds were the chemical shifts of C-5 (δC 124.5 in 1 vs δC 127.7 in  pyrisulfoxin A) and C-9 (δC 44.72 in 1 vs δC 39.4 in pyrisulfoxin A) . The molecular weight of kibdelomycin A was determined to be 307.5143 deduced from the positive ion [M+H] +at m/z 308.0668 by HRESIMS data while pyrisulfoxin A has a molecular weight of 291.2546. The 16 daltons more in molecular weight of kibdelomycin A than pyrisulfoxin A, the downfield shifts ofC-5 and the upfield shifts of C-9 indicate a sulfonyl group in kibdelomycin A to replace the sulfoxide group in pyrisulfoxin A. HMBC analysis connected the methyl protons H-9 (δH 3.41) to the quaternary carbon C-5 (δC 124.5) of the aromatic ring (Figure 1, 2) , for which the methyl sulfonyl group was allocated at the C-5 position. The planar configuration of kibdelomycin A was also confirmed by the analysis of the X-ray single-crystal diffraction data (Figure 3–10) . These data collectively suggest that the algicidal compound isolated from K. phytohabitans XY-R10 was 4-methyloxyl-5-methylsulfonyl-2, 2’ -bipyridyl-6-carboxaldehyde oxime, a novel compound which we named kibdelomycin A.
Table 2. 1NMR Data (600 MHz, δin ppm, J in Hz) and 13C NMR Data (150 MHz, δin ppm) for kibdelomycin A
Figure PCTCN2017072234-appb-000005
Figure PCTCN2017072234-appb-000006
aDMSO-d6. bCDCl3. bData were reported by Tsuge et al. 43
Example 3: Algicidal/antialgal activities of kibdelomycin A
Kibdelomycin A was evaluated for its algicidal against five eukaryotic algal species including Akashiwo sanguinea, Alexandrium tamarense, Prorocentrum micans, Chattonella antiqua, Heterosigma akashiwo, as well as its antialgal activity against the notorious cyanobacterial bloom forming species Microcystis aeruginosa (These algal species were cultured using Guillard’s f/2 medium (Guillard, R. R. L., et. al. Dinoflagellates. 1984, 391-442) or BG-11 medium, under constant temperature (20±2℃ or 25℃) and light (2000 lx, 12-h light/12-h dark cycle) conditions, Table 1) .
All tested compounds were dissolved in DMSO to make stock solutions of 50, 25, 10, 5, 2.5, 1.25, 0.625, 0.312, 0.156 mg/mL, respectively. Then 1 μL of the test solution was added to 1 ml of the algal culture in a well of a 24-well plate (Nunc, USA) . CuSO4 was used as the positive control while DMSO was used in as the negative control.
In particularly, for the algicidal bioassay, three dinoflagellates (A. sanguinea, A. tamarense, P. micans) and two raphidophytes (C. antiqua, H. akashiwo) were tested. The algal strain was cultured to reach late exponential  phase and added to the test solution. The morphology of A. sanguinea cells was then examined under a light microscope at 0h, 6h, 12h, 24h, 36h, 48h, while the other algal species were examined daily during a 4-day period of incubation. Kibdelomycin A was tested on all 5 algal species while cearulomycin A and collismysin A were only tested on A. sanguinea due to their limited amount.
For the antialgal bioassay, Microcystis aeruginosa was cultivated until the cell densities reached about 5×107 CFU/mL. The chlorophyll a (Chl-a) contents of the cyanobacterial culture were determined for 0, 2, 4, 6, 8, 10 days according to the methods described by Chen et al (Chen, Y. W., et. al. J Lake Sci. 2006, 18, 550-552. ) . The antialgal efficiency was calculated using the following equation:
Antialgal efficiency (％) ＝ (1–Ct/Cc) ×100％,
where Cc and Ct are the Chl-a contents of the control and the sample treated by the antialgal compound, respectively.
In this study, all the algicidal and antialgal bioassays were set up in triplicates and performed at least three times with different batches of algal cultures.
Results showed that kibdelomycin A was able to lyse all the treated cells of A. sanguinea, A. tamarense, P. micans, C. antiqua, H. akashiwo with minimal inhibitory concentrations (MIC values) of 1.25, 10, 5, 10, 5μg/mL, respectively (Table 3) . Thus kibdelomycin A is slightly less effective as the widely used algicide CuSO4 when MIC values were compared in mass concentrations. Moreover, the two algicides are almost the same effective if MICs are compared in molar concentrations (Table 3) . Within 48 hours, kibdelomycin A was already able to lyse all the treated cells of A. sanguinea and P. micans. For A. tamarense, H. akashiwo and C. marina, 3 to 4 days were  needed to lyse all of the algal cells. In addition, kibdelomycin A showed antialgal activity against M. aeruginosa with an MIC value of 20μg/mL (Table 3) and the inhibitory effect became evident on the 5th day of incubation.
Table 3. Algicidal/antialgal activities of kibdelomycin A
Figure PCTCN2017072234-appb-000007
The MIC value here refers to the minimum concentration where 100％cells of the tested algae (except M. aeruginosa) population were lysed in comparison with the control, while MIC value refers to the minimum concentration at which algicidal efficiency on M. aeruginosa was up to 95％.
In this study, the Akashiwo sanguinea was selected for more in-depth study due to its easily observed morphology. When under unfavorable culture conditions or exposed to exogenous substance stress, the cells of A. sanguinea often undergo severe morphological changes due to the lack of a rigid cellular structure. Within 24h of treatment by kibdelomycin A (1.25 μg/mL) , A. sanguinea cells slowed down their swimming speed. After 36h of treatment, most cells of A. sanguinea became darker, immobilized and slightly deformed (Figure 11； images b, e) . Eventually all cells were lysed  within 48h (Figure 11； images c, f) . As most A. sanguinea cells didn’t display obvious changes within the first 36 hours but they suddenly burst together within the following 12 hours or so, it was not practical to calculate the time-dependent algicidal efficiency of kibdelomycin A.
In recent decades, the cyanobacterium Microcystis aeruginosa has attracted increasing attention in research for posing threat to public health by producing toxins in global fresh waters (Reynolds, C. S., et al. Biol. Res. 1975, 50, 437-481. ) . In our study, kibdelomycin A showed antialgal activity on M. aeruginosa with an MIC value of 20μg/mL. When the algal culture of M. aeruginosa was treated with kibdelomycin A (20μg/mL) for 4 days, the algicidal efficiency against M. aeruginosa already reached 48.48±1.07％(Figure 12) . On the 10th day, the algicidal efficiency reached up to 96.54±0.27％ (Figure 12) , meaning that the growth of M. aeruginosa was almost fully inhibited. With a slightly higher concentration, kibdelomycin A could work as effective as CuSO4 in inhibiting the growth of M. aeruginosa. Example 4: Low-Toxicity of kibdelomycin A towards non-target organisms
Embryos of Danio rerio (2 days after fertilization) , juveniles of Daphnia magna newly hatched Artemia salina, adults of Hydra sinensis and Paramoecium caudatum were used as the test species. The zebrafish scientific research community was adopted in fish water (pH 7.2–7.6, conductivity 500 mS, nitrates<5 ppm) within a lighting cycle of 14 h light/10 h dark at 28.5℃. Well-trained females produce fresh fertilized eggs. Embryos can be raised in fish water and staged according to the description by Kimmel et al (Kimmel, C. B., et al. Dev. Dyn. 1995, 203, 253-310. ) . To minimize undesirable embryonic death and side effects, embryos at the shield stage (～2dpf) were selected, and following, embryos were observed using the dissecting microscope and inverted compound microscope. 6–8 healthy  embryos (～2dpf) were used and loaded into each well (200μL fish water) of U-bottom 96-well plates, and kibdelomycin A, cearulomycin A, collismysin A and CuSO4 were added to achieve the concentration of 0.31, 0.65, 1.25, 2.5, 5, 25, 75, 125μg/mL, respectively. One percent of DMSO (v/v) in fish water serves as a negative control.
According to the OECD guideline 202, D. magna was cultured in glass containers with Artificial Elendt M4 medium under a 16: 8 light-dark cycle at 20±1℃ (OECD, 2004. OECD guideline 202. OECD Guidelines for Testing of Chemicals. Daphnia sp., acute immobilization test. Paris. Antkowiak, W.Z.； Gessner, W.P. Tetrahedron Lett. 1979, 21, 1931-1934. ) . The organisms were fed with Chlorella pyrenoidsa three times a week and the culture medium was refreshed. Ten healthy D. magna (<24 h) were placed in the 24 well plates, excess water was gently absorbed with a piece of soft absorbent paper and 1ml of the D. magna medium containing the test compounds was added immediately.
Hydra sinensis was cultivated in a rack and held vertically in a 15-litre glass aquarium filled with Hydra medium “M” at 22–24℃ (Litchfield, J. D., et al. J. Pharm. Exp. Ther. 1949, 96, 399–408. ) , and the test organisms were fed with a few wheat grains. P. caudatum was cultivated in a wheat culture medium at 22–24℃ in the darkness. The A. salina was cultivated according to the published protocols (Meyer, B. N., et al. Planta Medica 1982, 45, 31-34； Solis, P. N., et al. Planta Medica. 1993, 59, 250-252. ) . A few drops of water containing desired individuals of P. caudatum, H. sinensis, A. salina were placed in the 24 well plates. The excess water was gently absorbed with a piece of soft absorbent paper and 1 mL of the P. caudatum, H. sinensis A. salina medium was added immediately, respectively. Kibdelomycin A was tested at the concentration of 25μg/mL, 50μg/mL, 75μg/mL, 100μg/mL, 125μg/mL. DMSO was added as the solvent control.
All the above toxicity tests had three replicates in one experiment and were repeated at least three times using different batches of organisms. Data was analyzed using SPSS15.0 software, and expressed as mean value±standard deviation (SD) of three replicates. The graph was plotted using the mean value, and all error bars indicated the SD of the three replicates. Statistical comparisons in all tests between treatments and controls were determined by one-way ANOVA followed by Tukey’s test. P<0.05 was considered statistically significant.
Kibdelomycin A belongs to the 2, 2’ -bipyridyl family of natural products, which have been reported to be associated with a variety of significant biological activities (Funk, A., et al. Canadian journal of microbiology. 1959, 5 (4) , 317-321； McInnes, A., et al. Canadian Journal of Chemistry, 1977, 55 (24) , 4159-4165； Shindo, K., et al. J. Antibiot. 1994, 47, 1072-1074； Gomi, S., et al. J. Antibiot. 1994, 47, 1385-1394； Tsuge, N., et al. J. Antibiot. 1999, 52, 505-507； Stadler, M., et al. Arch. Pharm. (Weinheim) 2001, 334, 143-147； Singla A. K., et al. Transplantation 2014, 97, 57-59； Martinez, A., et al. WO2007017146 A3. 2007) . Two structurally similar compounds, caerulomycin A and collismysin A demonstrate an astonishing range of biological activities including antibacterial and antifungal (Funk, A., et al. Canadian journal of microbiology. 1959, 5 (4) , 317-321) , anti-amoebic (Chatterjee D. K., et al. Z Parasitenkd 1984, 70, 569-573； Kaur S., et al. British Journal of Pharmacology 2015, 172, 2286-2299) , antiproliferative (Kawatani, M., et al. Mol Cancer Ther 2013, 12, A243. ) , immunosuppressive (Singla A. K. et al. Transplantation 2014, 97, 57-59. ) , and neuroprotectant activities (Sialera C. et al. Chem. Lett. 2013, 23, 5707-5709) . Due to their highly similar structures, it was therefore of interest to compare the effects of caerulomycin A and collismysin A on harmful algae with kibdelomycin A.
Kibdelomycin A, caerulomycin A and collismysin A lysed 100％algal cells of A. sanguinea with MIC values of 1.25μg/mL, 1.25μg/mL and 0.31μg/mL, respectively (Table 4) . Collismysin A was even more effective than CuSO4 for lysing A. sanguinea. The algicidal activity of kibdelomycin A, caerulomycin A and collismysin A might be due mainly to the skeleton of 2, 2’-bipyridine-6-carboxaldehyde oxime.
Table 4. The algicidal effects of kibdelomycin A, caerulomycin A, collismysin A and CuSO4 on Akashiwo sanguinea, and toxicities against Danio rerio embryos and Daphnia magna.
Figure PCTCN2017072234-appb-000008
Values are the mean±SD of three experiments
In the course of finding more effective and environmentally friendly algicides, the outmost attention should not be exclusively paid to activities to target organisms. Toxicity of the compounds is as important as their activities. In order to examine the toxicity of the algicides, we selected five representative aquatic species from the non-target organisms, including Danio rerio embryos, Hydra sinensis, Paramoecium caudatum, Daphnia magna and Artemia salina. Astonishingly, kibdelomycin A didn’t kill Hydra sinensis, Paramoecium caudatum or Artemia salina at a concentration of 125 μg/mL. Moreover, the LC50 value ofkibdelomycin A to Danio rerio embryos is also greater than 125μg/mL. Daphnia magna, which prey on algae as their main food source, is only slightly less tolerant to kibdelomycin A  (LC50＝47.24±5.4μg/mL) . The large discrepancy between the LC and MIC values indicates that kibdelomycin A may be of low toxicities towards non-target organisms.
By contrast, caerulomycin A and collismysin A were at least 150-fold more toxic to Danio rerio embryos than kibdelomycin A with LC50 values of 0.873±0.05μg/mL and 0.76±0.15μg/mL, respectively. And they were also at least 20-fold more toxic to Daphnia magna than kibdelomycin A with LC50 values of 2.05±0.10μg/mL and 1.60±0.12μg/mL, respectively. If the adequate quantities of caerulomycin A and collismysin A were added to reach the algicidal effects in the real environment, the safety of other aquatic organisms would be impaired. These data clearly demonstrate that even caerulomycin A and collismysin A are a bit more efficient than kibdelomycin A in inhibiting algal growth, yet their high toxicities rule out their probability as environmentally friendly algicides. One possible explanation of the significant discrepancy in toxicity of these three structurally similar compounds might be their different capabilities to translocate into the intracellular spaces of organisms other than algae. The presence of the methyl sulfonyl group in kibdelomycin A might be the major reason that decreased its toxicities significantly. In the aquatic organisms other than algae, kibdelomycin A might not get through the bio-membranes to work as an iron chelator, while caerulomycin A and collismysin A could reach inside the cells and severely interfere with certain essential biochemical functions involving the iron molecules. Yet the exact underlining mechanism awaits further investigation. From the same point of view, the algicide CuSO4 adopted worldwide currently is not an ideal algicide due to the small discrepancy between its MIC and LC values against target and non-target organisms, respectively (Table 4) .
Example 5: kibdelomycin A is a Fe (II) chelator
It has been reported that cearulomycin A exerts its immunosuppressive effect by depleting intracellular iron (Singla A. K., et al. Transplantation 2014, 97, 57-59. ) and collismycin A can act as an iron chelator to inhibit tumor cell growth (Kawatani, M., et al. Mol Cancer Ther 2013, 12, A243) . As both photosynthesis and nitrogen assimilation require irons in key enzymes, rapidly proliferating algal cells in a bloom must have a high demand for irons. It is therefore presumable that kibdelomycin A, caerulomycin A and collismysin A killed the algal cells by hijacking intracellular irons.
In order to prove our hypothesis, FeSO4 was added to the algal cultures after they were treated by kibdelomycin A for different periods of time. Figure 13 shows that FeSO4 could completely protect the algal cells from lysing at the end of 36h and 48h if they were only treated by the compound for the first 3 h. On the contrary, none of the algal cells remained alive after treated by the compound for 36 h. Ifthe algal cells were treated by 6 h and 12 h by the compound before addition of FeSO4, their surviving rates decreased a little bit only at the end of 48 h incubation. Moreover, addition of aqueous FeSO4 to pure kibdelomycin A dissolved in methanol would immediately induce a violet color formation (Figure 14a) . Molecular weight of the violet compound deduced form MS analysis suggested that the two molecules of kibdelomycin chelated one molecule of Fe (II) (Figure 14b) . Furthermore, addition of FeSO4 to kibdelomycin A at 1: 2 molar ratio resulted in a single peak in HPLC analysis further supported the complex was formed between two molecules of kibdelomycin A and one molecule of Fe (II) (Figure 14c) . Moreover, we analyzed the algal culture treated by kibdelomyin A and the same peak corresponding to the Fe (II) -kibdelomycin A complex could be detected inside the algal cells (Figure 15) . All the evidence piled up that kibdelomycin A inhibited algal growth by working as a Fe (II) chelator.
As iron chelators usually have antibacterial effects, we tested the antibacterial effects of kibdelomycin A and indeed found it could inhibit the growth of notorious opportunistic pathogen Pseudomonas aeruginosa with an MIC value around 100μg/ml (Figure 16) .
In conclusion, we have discovered a novel algicide kibdelomycin A whose mode-of-action may work as a selective iron chelator in algae. Preliminary data suggest it has very low toxicities towards non-target aquatic organisms, making it a promising candidate as an environmentally friendly algicide.

Claims (21)

  1. A compound of the formula I:
    Figure PCTCN2017072234-appb-100001
    wherein, X＝H, R1, OR1 or a halogen atom；
    Y is optionally substituted C1 to C16 linear or branched chain alkyl；
    R1 is H, C1 to C6 aliphatic hydrocarbon or cycloalkane or a halogen atom.
  2. The compound according to claim 1, characterized in that R1 is selected from the group consisting of methyl, ethyl, n-propyl, isopropyl, n-butyl and isobutyl.
  3. The compound according to claim 1, characterized in that Y is selected from the group consisting of methyl, ethyl, n-propyl, isopropyl, n-butyl and isobutyl.
  4. The compound according to claim 1, wherein the compound is 4-methyloxyl-5-methylsulfonyl-2, 2'-bipyridyl-6-carboxaldehyde oxime of the formula II:
    Figure PCTCN2017072234-appb-100002
  5. A composition comprising at least one compound as defined in any one of the preceding claims.
  6. A algaecide having fungicidal and algicidal activity, characterized in containing the compound or the composition as defined in any one of the preceding claims.
  7. A algicidal agent comprising the compound or the composition as defined in any one of the preceding claims and acceptable salts thereof.
  8. The algicidal agent according to claim 7, wherein the composition also comprising acceptable diluents, additives and/or carriers.
  9. Use of the compound or the agent or the composition as defined in any one of the preceding claims in lysing cells of eukaryotic or prokaryotic algae.
  10. The use according to claim 9, wherein the algae is selected from the group consisting of Akashiwo sanguinea, Alexandrium tamarense, Prorocentrum micans, Chattonella marina, Heterosigma akashiwo, Microcystis aeruginosa.
  11. A method for controlling harmful algal blooms using the compound  or the agent or the composition as defined in any one of claims 1-8 with effective concentration, wherein the effective concentration is 0.1μg/mL to 100μg/mL.
  12. The method according to claim 11, wherein the effective concentration is 1-10μg/mL.
  13. An iron chelator comprising the compound or the agent or the composition as defined in any one of the preceding claims.
  14. Use of the compound or the agent or the composition as defined in any one of the preceding claims in treating diseases associated with intracellular iron.
  15. The use according to claim 13, wherein the diseases is selected from the group consisting of immunosuppressive disease, tumor, bacterial disease.
  16. A process for the isolation of the compound as defined in any one of the preceding claims useful as an algicidal compound from Kibdelosporangium phytohabitans XY-R10, the process comprises:
    [a] cultivating the bacterium Kibdelosporangium phytohabitans XY-R10 in culture broth at 25-30℃ for 1-10 days；
    [b] extracting the culture broth with EtOAc having a volume twice as that of the culture broth, and drying combined EtOAc layers；
    [c] separating the active fraction using reverse phase silica chromatography with an eluent of water/methanol (7: 3, v/v) ；
    [d] purifying the active fraction using semi-preparative HPLC with an eluent of 25％ACN-H2O to obtain the pure compound.
  17. The process according to claim 16, wherein the bacterium is  cultivated for a period of 6 days.
  18. The process according to claim 16, wherein the bacterium is cultivated at 28℃.
  19. Use of a compound of the formula III in lysing cells of eukaryotic or prokaryotic algae
    Figure PCTCN2017072234-appb-100003
    wherein, R is H, SCH3 or SO2R2；
    R2 is optionally substituted C1 to C16 linear or branched chain alkyl.
  20. The use according to claim 19, characterized in that R2 is selected from the group consisting of methyl, ethyl, n-propyl, isopropyl, n-butyl and isobutyl.
  21. The use according to claim 19, wherein the algae is selected from the group consisting of Akashiwo sanguinea, Alexandrium tamarense, Prorocentrum micans, Chattonella marina, Heterosigma akashiwo, Microcystis aeruginosa.
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