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The search for antinematodal agents from southern Australian marine sponges

Ed Hsiang Te Liu
School of Chemistry University of Melbourne, Australia Marine Natural Products Research Group Doctor of Philosophy Presentation Prof Rob Capon
E-mail: e.liu@chemistry.unimelb.edu.au URL: http://www.marinebioprospecting.net

Overview
Marine natural products research and the antinematodal agents discovery program. .
The isolation and structure elucidation of novel pyridine alkaloids. Synthesis of nematicidal marine lipid thiocyantins and structure activity relationship studies.

Australian marine environment

Australia is an island continent with mega biodiversity. Australia manages a vast marine Economic Exclusion Zone (EEZ): > 69,000 km coastline > 12,000 islands Australias EEZ includes: intertidal, shallow and deep water ecosystems spanning from tropical through temperate to antarctic regions.

Australian marine invertebrates

Australian marine environment is relatively unexplored, and therefore have attracted much attention as it is a rich source for novel agrochemicals. 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.

Great Australian Bight

Melbourne

Marine collection
Macro : invertebrates, algae > 3,000 Micro : bacteria, fungi > 50,000

Bioassay : Agrochemical

Active metabolites Basic Research Publish Applied Research Patent

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.

Aim of the project

From southern Australian marine sponges: 1. Isolate nematicidal metabolites by bioassay directed fractionations - Chromatography: SPE, Gel, HPLC, etc. 2. Determine structures of nematicidal metabolites - Spectroscopy: NMR, MS, IR, etc.

3. Structure activity relationship studies - Organic synthesis: analogues, model compounds.

Bioassay Directed Fractionation

Crude EtOH extract Bioassay

Solvent partitioning

Bioassay

SPE

Solid phase extraction (SPE)

Bioassay

HPLC

Bioassay

HPLC + PDA + ELSD

Marine metabolite

Bioassay

Bioassay Directed Fractionation

Waters 2700 Sample Manager

Microtitre plates

High throughput screening

Gel Chromatography

Centrifugal Evaporator

LC/MS

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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.
Present serious health risk to public: pets and humans are at risk. Growing levels of resistance to commercial anthelmintic drugs has been detected.

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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

+N

N+

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.

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Extraction and purification

Crude EtOH extract 3.04 g 50% total available crude extract solvent partitioning

CH2Cl2 soluble 686.5 mg 23% crude extract

n-butanol soluble 414.4 mg 13.9% crude extract sephadex gel

H2O soluble 1.88 g 63% crude extract

n-butanol soluble-1 25.6 mg 0.84 % crude extract

n-butanol soluble-2 176 mg 5.7 % crude extract SPE

n-butanol soluble-3 39.8 mg 1.31 % crude extract

fraction 5 pyridine alkaloid (impure) fraction 3 17.6 mg 11.2 mg 25.9 mg fraction 2 fraction 4 0.58 % crude extract 0.37 % crude extract 0.85 % crude extract 24.9 mg 14.2 mg 0.82 % crude extract 0.47 % crude extract

pyridine alkaloid 3.4 mg 0.11 % crude extract

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1H

NMR spectrum (CD3OD, 400 MHz)

C H

D B

A
G

A B

4.72

4.70

4.68

4.66

4.64

4.62

4.60

4.58

HI

J K

A
9.00 8.80

BC
8.60

D
8.40 8.20

F
E F
8.00

10

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COSY NMR spectrum

0

K
2

I
3

G
5

F
6

D C A B

0 p p m

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COSY NMR spectrum

E I F

X
G I

K J
E F J

X
G I

I H

C D

H K

B +

C D

H K

G F E
J

+N
G I

A E

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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

+N
I J

N+

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

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Thiocyanatin A-C
(Oceanapia sp)
1H

d 2.95, t (7.4 Hz)

OH
8

Thiocyanatin A NCS
1 1H d 1.85, quin (7.4 Hz)

16

SCN

LD99= 1.3

Thiocyanatin B

NCS

SCN

LD99= 0

Thiocyanatin C NCS

SCN

LD99= 0

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

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New thiocyanatins
(Oceanapia sp)
Me OH NCS m n m+n=11 SCN

O H2NCS

OH SCN m n m+n=11 LD99= 8.3

LD99= 4.2
Me NCS m m+n=9 n SCN
O H2NCS

m m+n=9 LD99= 17

SCN

LD99= 0

Dr Colin Skene unpublished results

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Synthesis of thiocyanatin A
NCS 1 2 OH 8 15 16 SCN

(Oceanapia sp)

OH HO OH

CO2Me CO2Me

Br

CO2H

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

RCH 2Br

PPh3

RCH 2P+Ph3Br-

Base Ph3P

RCHO fast

RCH=CHR

+ O=PPh3

PPh3

[O] slow

OPh3P+ R

Tetrahedron Lett. 1996, 37, 7703-7706

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Synthesis of thiocyanatin A
(Oceanapia sp)
H2SO4, MeOH
Br CO2H

PPh3, MeCN,
Br CO2Me

Reflux, 16 h NaHMDS THF/DMPU O2

Oxidant air air O2 O2 O2

Reflux, 16 h

CO2Me CO2Me

BrPh3P
60 C
NaHMDS 2.1 eqv 1 eqv 1 eqv 1 eqv 1 eqv

CO2Me

60 C
Temperature reflux 80 C 90 C 70 C 60 C Reaction Time 1h 1h 40 h 16 h 16 h

THF/DMPU 1:1 1:1 1:1 1:1 3:1

Yield % 0 30 35 49 72

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Synthesis of thiocyanatin A
(Oceanapia sp)
CO2Me CO2Me

m-CPBA

CO2Me O CO2Me

LiAlH4, ether

CH2Cl2
rt, 16 h

reflux, 20 h

OH HO OH

p-TsCl CH2Cl2 DMAP Et3N, rt.

TsO

OH OTs

KSCN,THF

Reflux, 16 h

NCS 1

OH 8 15

16 SCN

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Synthetic thiocyantin A
NCS 1 2 OH 8 15 16 SCN

(Oceanapia sp)
LD99= 0.85 (synthetic) LD99= 1.3 (natural product)

ppm

3.8

3.6

3.4

3.2

3.0

2.8

2.6

2.4

2.2

2.0

1.8

1.6

1.4

1.2

1.0

0.8

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Synthesis of thiocyanatin B & C

(Oceanapia sp)
OH TsO OTs

P-TsOH, toluene, reflux, 16 hh

TsO

OTs

KSCN, THF, reflux, 16 h.

NCS

SCN

LD99 = 0 (for both synthetic and natural material)

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Preparation of thiocarbamate functional group

1H

d 2.95, t

(Oceanapia sp)
O

O H2NCS

OH SCN m n

H2NCS
1H

m m+n=9 d 2.83, t LD = 17 99

n
1H

SCN

1H

m+n=11 d 2.83, t LD99= 8.3

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 the corresponding 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.

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Preparation of thiocarbamate functional group

(Oceanapia sp)
SCN

O SCNH2
Acid H2SO4 H2SO4 AcOH H2SO4 HCl(g) Solvent H2O AcOH MeOH Temperature 40 C 40 C 40 C 40 C 40 C Time 16 h 16 h 16 h 48 h 16 h Results no product no product no product no product product + impurity

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Preparation of thiocarbamate functional group

OH NCS
1H

(Oceanapia sp)
SCN

d 2.83, t
OH

1H

d 2.83, t
O SCNH2

O H2NCS

Exposure time of HCl(g) at 0 C 60 min 60 min 60 min 120 min 120 min

Temperature RT 40 C 40 C 40 C 40 C

Stirring time 16 h 16 h 24 h 16 h 24 h

Results product (63% yield) product + impurity product + impurity product + impurity product + impurity

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Structure activity relationship studies

OH NCS SCN

Me NCS

OH m n

(Oceanapia sp)
SCN

Thiocyanatin A LD99= 1.3

O H2NCS OH SCN m n m+n=11

m+n=11

LD99= 3.1
OH HO 1 8 16 OH

LD99= 44
1
16 O SCNH2

LD99= 8.3
O H2NCS 1 OH 8

OH SCN 9 18

NCS

LD99= 0
OH NCS 1 8

LD99= 8.3
CH3 O S O O NCS 1 8

16 OH

16 SCN

LD99= 3.2

LD99= 0

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Structure activity relationship studies

SCN NCS SCN

(Oceanapia sp)
NCS 1 SCN 16

LD99= 463
NCS SCN

LD99= 0
SCN

LD99= 0
O H2NCS m m+n=9 n SCN

LD99= 0
O SCN

LD99=17
HO

LD99= 0
16 SCN

NCS 1

O OH

SCN

LD99= 500

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 Joanne Ford & Dat Vuong & Shirley Dong Eric Mattsson & Michelle McNally (Technical Support) (Marine Invertebrates) (Marine Microbes)

Dr Michael Stewart & Ben Clark

(Terrestrial Microbes)

Industry Support
Dr Tom Friedel & Dr Kirstin Heiland Dr Ern Lacey & Dr Jenny Gill (Novartis) (MST)

Funding
Melbourne Research Scholarship Novartis Animal Health Australasia Pty Ltd Microbial Screening Technologies Pty Ltd