anti parasitic agents from australian marine environment

8/4/2019 Anti Parasitic Agents From Australian Marine Environment

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The search for antinematodal agents from southern

Australian marine sponges

Ed Hsiang Te Liu

School of ChemistryUniversity of Melbourne, Australia

Marine Natural Products Research Group

Doctor of Philosophy Presentation

Prof Rob Capon

E-mail: [email protected] URL: http://www.marinebioprospecting.net


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Overview

Marine natural products research and the antinematodal agents

discovery program.

.

Synthesis of nematicidal marine lipid thiocyantins and

structure activity relationship studies.

The isolation and structure elucidation of novel pyridine alkaloids.


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Australian marine environment

Australia is an island continent with mega biodiversity.

Australia manages a vast marine Economic Exclusion Zone (EEZ):

> 12,000 islands

> 69,000 km coastline

Australias EEZ includes: intertidal, shallow and deep water ecosystems

spanning from tropical through temperate to antarctic regions.


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Australian marine invertebrates

Great Australian

Bight

Melbourne

Australian marine invertebrate and algae rely on chemical defense system to

protect themselves from predators by excretion of toxins.

Marine toxins can display interesting biological activities such as:

paralytic effect, insecticidal activity, antiparasitic and anticancer properties.

Australian marine environment is relatively unexplored, and

therefore have attracted much attention as it is a rich sourcefor novel agrochemicals.


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Marine collection

Micro : bacteria, fungi

> 50,000

Macro : invertebrates, algae

> 3,000

Basic Research

Publish

Applied Research

Patent

Active metabolites

Bioassay :Agrochemical


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Agrochemicals from Marine Invertebrates

Biological testing :

Novartis Animal Health Pty Ltd, and

Microbial Screening Technologies Pty Ltd

Outcomes :

Over 100 sponge extracts identified as active, and numerous target

compounds under investigation.

Over 50,000 microbial isolates screened, and >100 identified as active.

Numerous target cultures & compounds under investigation.


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Aim of the project

From southern Australian marine sponges:

1. Isolate nematicidal metabolites by bioassay directed fractionations

3. Structure activity relationship studies

2. Determine structures of nematicidal metabolites

- Chromatography: SPE, Gel, HPLC, etc.

- Spectroscopy: NMR, MS, IR, etc.

- Organic synthesis: analogues, model compounds.


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Bioassay Directed Fractionation

Crude EtOH extract

Solvent partitioning

Bioassay

Solid phase extraction

(SPE)

HPLC

Marine metabolite

Bioassay

Bioassay

Bioassay

Bioassay

SPE

HPLC + PDA + ELSD


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Bioassay Directed Fractionation

Gel Chromatography LC/MS

Waters 2700

Sample Manager

Centrifugal Evaporator

Microtitre plates High throughput screening


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Why do we search for antinematodal agents?

Parasitic nematodes cause loss of production to the commercial

livestock industry many millions of dollars a year.

Growing levels of resistance to commercial anthelmintic drugs

has been detected.

Present serious health risk to public: pets and humans are at risk.


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What are nematodes?

Haemonchus contortus

(sheep, goats)

Ascaris lumbicoides(human)

Ascaris suum

(swine)

Globodera pallida

(potato)


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Mode of actions of commercial antinematodal agents

g- aminobutyric acid stimulator, which causes large flow of chloride ion into cells

results muscle paralysis and death of nematodes (avermactin structure class).

Microtubules formation inhibitor (benzimidazole structure class).


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Novel pyridine alkaloid

Isolated from the sponge Callyspongia spp.

Collected from Lonsdale Wall, Phillip Heads in Victoria.

Crude EtOH extract display nematicidal activity LD99 = 85 ppm.

n-BuOH soluble fraction from solvent partition increased

nematicidal activity to LD99 = 6.8 ppm.

NN

+

+


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Extraction and purification

Crude EtOH extract3.04 g

50% total available crude extract

CH2Cl2 soluble686.5 mg

23% crude extract

n-butanol soluble414.4 mg

13.9% crude extract

H2O soluble1.88 g

63% crude extract

n-butanol soluble-2176 mg

5.7 % crude extract

n-butanol soluble-339.8 mg

1.31 % crude extract

n-butanol soluble-125.6 mg


solvent partitioning

sephadex gel

SPE

fraction 3

25.9 mg0.85 % crude extract

fraction 224.9 mg


pyridine alkaloid (impure)


fraction 414.2 mg


fraction 5


pyridine alkaloid3.4 mg



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1H NMR spectrum (CD3OD, 400 MHz)

001122334455667788991010

A

BC D

E

F

GH

IJ K

............

A

BC D

NAB

CD

H

E F

X

G

.............7.7.7.7


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COSY NMR spectrum

p p m

00

p p m

0

1

2

3

4

5

6

7

8

9

1 0

AB

C

D

EF

G

HI

JK


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COSY NMR spectrum

1 . 0. 5. 0. 5. 0. 5. 0. 5. 0. 5. 0

EF

G

H

I

J

K

E F

I

E F

I

X

G

E F

I

X

GJ

NAB

CD

H

K

NAB

CD

H

K

G

I

E F

J

+

+


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Mass spectroscopy & 13C NMR spectrum

Mass spectrum shows two distinct peaks at 244 m/z (M2+), and 487 m/z (M+-H)

indicating that the molecule is a dimer.

13C NMR spectrum shows the chemical shifts of allylic carbons at:

35.5 ppm and 33.5 ppm. (E

32.6 ppm,Z

29.9 ppm)1

1. Sadtler Standard Carbon-13 Indexes; Sadtler Research Laboratories; Philadelphia, 1980.

NN

+

+

I J


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Thiocyanatin A-C

J. Org. Chem. 2001, 66, 7765-7769

SCNNCS

OH

SCNNCS

SCNNCS

18

16

1H d 2.95, t (7.4 Hz)

1H d 1.85, quin (7.4 Hz)

(Oceanapia sp)

LD99= 1.3

LD99= 0

LD99= 0

Thiocyanatin A

Thiocyanatin B

Thiocyanatin C


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New thiocyanatins

Dr Colin Skene unpublished results

OH

NCS SCN

Me

m n

m+n=11

NCS SCN

Me

m n

m+n=9

(Oceanapia sp)

LD99= 4.2

LD99= 0

LD99= 8.3

LD99= 17

OH

H2NCS SCNm n

m+n=11

O

H2NCS SCN

m n

m+n=9

O


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Synthesis of thiocyanatin A

NCS

OH

SCN81

2

15

16

HO

OH

OH

CO2Me

CO2Me

Br CO2H

(Oceanapia sp)


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Synthesis of thiocyanatin A(one-pot oxidation-Wittig coupling reaction)

(Oceanapia sp)

Tetrahedron Lett. 1996, 37, 7703-7706

RCH2BrPPh3

RCH2P+Ph3Br

-Base

Ph3P

Ph3P+

R

R

O-

RCHORCH=CHR O=PPh3+

[O] slow

fastPPh3+


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(Oceanapia sp)

Br CO2H Br CO2Me

BrPh3P CO2Me

CO2Me

CO2Me

H2SO4, MeOH

Reflux, 16 h

PPh3, MeCN,

Reflux, 16 h

NaHMDS

THF/DMPUO2

60 C

60 C

NaHMDS THF/DMPU Oxidant Temperature Reaction Time Yield %

2.1 eqv

1 eqv

1 eqv

1 eqv

1 eqv

1:1

1:1

1:1

1:1

3:1

air

air

O2

O2

O2

reflux

80 C

90 C

70 C

60 C

1 h

1 h

40 h

16 h

16 h

0

30

35

49

72


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(Oceanapia sp)

CO2Me

CO2Me

O

HO

OH

OHTsO

OH

OTs

NCS

OH

SCN81

2

15

16

m-CPBA

CH2Cl2

rt, 16 h

CO2Me

CO2Me

LiAlH4, ether

reflux, 20 h

p-TsCl

CH2Cl2

DMAP

Et3N, rt.

KSCN,THF

Reflux, 16 h


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Synthetic thiocyantin A

(Oceanapia sp)NCS

OH

SCN81

2

15

16

LD99= 0.85 (synthetic)

LD99= 1.3 (natural product)

ppm

26


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Synthesis of thiocyanatin B & C

(Oceanapia sp)

TsOOTs

OH

TsOOTs

NCSSCN

P-TsOH, toluene, reflux, 16 hh

KSCN, THF, reflux, 16 h.

LD99 = 0

(for both synthetic and natural material)

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Preparation of thiocarbamate

functional group

(Oceanapia sp)

LD99= 8.3LD99= 17

1H d 2.83, t 1H d 2.83, t 1H d 2.95, t

1H d 2.95, t

Two distinct triplet in 1H NMR spectrum, suggesting differing terminal

functional groups.

ESI(+)MS showed an intense ion at 397 m/z, 18 units higher than thecorresponding ion in thiocyanatin A.

This data is consistent with addition of H2O to one of the terminal

thiocyanate groups in thiocyanatin A to give a thiocarbamate.

H2NCS SCNm n

m+n=9

OOH

H2NCS SCNm n

m+n=11

O

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functional group

(Oceanapia sp)

Acid Solvent Temperature Time Results

H2SO4

H2SO4

AcOH

H2SO4

H2O

AcOH

-

-

40 C

40 C

40 C

40 C

16 h

16 h

16 h

48 h

no product

no product

no product

no product

HCl(g) MeOH 40 C 16 h product + impurity

SCNH2

O

SCN

29


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functional group

(Oceanapia sp)NCSSCN

OH

H2NCS SCNH2

OHO

O

Temperature Stirring t ime ResultsExposure time of HCl(g) at 0 C

60 min

60 min

60 min

120 min

120 min

RT

40 C

40 C

40 C

40 C

16 h

16 h

24 h

16 h

24 h

product (63% yield)

product + impurity

product + impurity

product + impurity

product + impurity

1H d 2.83, t1H d 2.83, t

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Structure activity relationship

studies

(Oceanapia sp)

SCNNCS

OH1

918

NCSOH

OH

18

16

H2NCS SCNH2

OHO

O

18

16

NCSSCN

OH

H2NCS SCN

OH

nm

O

m+n=11

HO

OH

OH

1 8

16

Thiocyanatin A

LD99= 1.3

LD99= 8.3

LD99= 0

LD99= 3.2

LD99= 44

NCSSCN

O

S

CH3O O

1 8

16

LD99= 8.3

LD99= 0

NCS SCN

OHMe

m n

m+n=11

LD99= 3.1

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Structure activity relationship

studies

(Oceanapia sp)

O

HO

SCN

OH

SCN

SCN

O

NCSSCN

NCSSCN

1

16

NCS SCN

1 16

H2NCS SCN

O

m n

m+n=9

NCSSCN

SCN

LD99= 463

LD99= 0

LD99=17

LD99= 500

LD99= 0

LD99= 0

LD99= 0

LD99= 28

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Conclusion

Novel pyridine alkaloids were isolated and structures were determined

by spectroscopic methods

Synthesis of marine natural products thiocyanatins A, B&C was completed.

The structure of new thiocyanatin containing a thiocarbamate functional group

was confirmed by preparation of bis-thiocarbamates.

Synthesis of thiocyantin analogues and structural activity relationship studies

indicated thiocyantin A was in fact the most active compound, in which the

secondary alcohol and both of the terminal thiocyanate groups are important

pharmacophor for nematicidal activity.

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Acknowledgments

MNP Research Group

Prof Robert Capon & Dr Colin Skene

Alicia Loveless, Lisa Goudie (Technical Support)

Joanne Ford & Dat Vuong & Shirley Dong (Marine Invertebrates)

Eric Mattsson & Michelle McNally (Marine Microbes)

Dr Michael Stewart & Ben Clark (Terrestrial Microbes)

Industry Support

Dr Tom Friedel & Dr Kirstin Heiland (Novartis)

Dr Ern Lacey & Dr Jenny Gill (MST)

Funding

Melbourne Research Scholarship

Novartis Animal Health Australasia Pty Ltd

Microbial Screening Technologies Pty Ltd

anti parasitic agents from australian marine environment

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