Using natural products to invent new chemicals for crop protectionp p

John Clough

Bioactive Natural Products: Translating Promise into PracticeOxford, 11-13 July 2016

1 Classification: PUBLIC

Crop protection chemicals are mostly herbicides, insecticides and fungicides

● The global market 28.0%Insecticides

● The global market for crop protection chemicals is large and growing 42 3%

Herbicides

28.0%

and growing.

● New products must b f hi h l t

42.3%

Fungicidesbe of high value tojustify the cost of their development ( $280 million)

2.9%Others26.8%

(~$280 million).2015 – Total sales value $51.2 billion

Source: Phillips McDougall

2 Classification: PUBLIC

A need for “sustainable intensification”

OxfordPopulation ~150kA city the size of

O f d 16 hOxford every 16 hours

3 Classification: PUBLIC

It is difficult to invent new crop protection chemicals!

● High potency – but selective.● Good chemical and metabolic stability –

but not too persistent in the environment.● Physical properties which allow● Physical properties which allow

movement to the target site.● Cost-effective.

Th di f h h i l iThe discovery of such chemicals requires good leads and then extensive synthesis

and biological screening

4 Classification: PUBLIC

Natural Products – uses in the crop protection industry

5 Classification: PUBLIC

Natural products – a proven source of innovation

Market for crop protection chemicals, showing relative values of NP derived classesrelative values of NP-derived classes

Total value 2015 = $51.2 billion

(Phillips McDougall)

Successes are infrequent, but rewards can be huge!

6 Classification: PUBLIC

Biology screening on target – lab to glasshouse to field

Robust, regular, reliable assays

7

g y

Classification: PUBLIC

Syngenta’s bioassay-directed discovery programmeWith partners at the Hubei Academy of Agricultural Sciences, Wuhan, China

● Aim – to isolate new bioactive natural products that can be used as products, or as leads for synthesis or biosynthesis.

● Source – actinomycetes and fungi collected in China.

“E l R iti ” id th di f k● “Early Recognition” avoids the re-discovery of known compounds.

● “Early Identification” allows only natural products of potential business interest to be taken forwardpotential business interest to be taken forward.

● Procedures under continuous improvement.

● Expectation – a small proportion of exciting natural products will make the whole exercise worthwhile.

8 Classification: PUBLIC

The herbicidal triketones

● Isolated from Callistemon citrinus ● Introduced by Syngenta in 2014 for the

BicyclopyroneLeptospermone

● Isolated from Callistemon citrinus, the red bottlebrush plant

● Moderate herbicidal activity, with bleaching symptomology

● Introduced by Syngenta in 2014 for the pre- and post-emergence control of grasses and large-seeded dicot weeds in maize (USA, Canada, South America).g y p gy

● No useful crop selectivity● New mode of action – inhibition of

hydroxyphenylpyruvate dioxygenase

( , , )● Sold as AcuronTM, a mixture with three

other herbicides + crop safener.● pKa = 3 06 and water-solubility =y yp y py yg ● pKa = 3.06 and water-solubility =

119g/litre (pH7.2, 25oC) – ideal properties for uptake and movement in plants.

9 Classification: PUBLIC

Viridiol and ash dieback

● The fungus Hymenoscyphus fraxineus causes dieback of the European ash and produces the the phytotoxin viridiol and related compounds.

● Viridiol produces necrotic lesions on the leaves of ash seedlings.p g- May play a role in the pathogenicity of the fungus.

● Viridiol and analogues showed herbicidal activity in our screens.● Electrophilic inhibitors of phosphatidylinositol 3-kinase and other

Viridiol

kinases.- Unattractive mode of action.

Demethoxyviridiol

Demethoxyviridin

10 Classification: PUBLIC

Sclerotiorin – a fungicidal natural product

O t b lit f i f i P i illi l ti● Orange metabolite of various fungi, e.g. Penicillium sclerotiorum.● First reported 1940; structure determined late 1950s.

● Weak fungicidal activity can it be improved? Selective?● Weak fungicidal activity – can it be improved? Selective?● Electrophilic – one of a large family of natural “azaphilones”.● Is activity due to non-specific electrophilicity?

RNH2

11 Classification: PUBLIC

Synthesis of analogues – key steps

PdCl2(PPh3)2

AgNO3, TFAthen

OHBrOH

Ph

CuI, Et3N, DMF

67%

Bu4NIO

IOOH

O H

OH

CHOO H

CHO

67% 81%O

O

OCl

O

O

Th k YANG G F WU Qi HUANG Sh i d LIN L

OAcO

OOH

● Thanks – YANG Guang-Fu, WU Qiongyu, HUANG Shaowei and LIN Long.● Lin Long et al., J. Agric. Food Chem., 2012, 60, 4480; Chem. Biol. Drug Design, 2012, 80, 682.● Compare John Porco et al., Org. Letts., 2006, 8, 5169.

12 Classification: PUBLIC

pH-Dependent equilibrium with a hydrate

H2O?O

ClO

Cl

O

OOH

O HOOH

O

pH 7Polar hydrate [M + 18]+

Low pH

● Equilibrium between parent and hydrated species in water.- Can be separated by HPLC.

● Hydrate forms slowly at pH7 but swiftly converts to parent at low pH.y y p y p p● Could this equilibrium lead to good transport properties in plants?

● In the end level of fungicidal activity too low – project closed● In the end, level of fungicidal activity too low project closed.

Torquil Fraser, Danielle Sayer

13 Classification: PUBLIC

Phosphonothrixin – introduction

● Herbicidal metabolite of the bacterium Saccharothrix sp. ST-888.

● First isolated and patented by Kureha● First isolated and patented by Kureha (JP 1993-181962, priority 28 June 93).

● Attractive polar natural product.● Can patent free analogues with E Takahashi et al J Antibiot 1995 48 1124● Can patent-free analogues with

improved activity be found?

I t t i l h bi id ft l k id

E Takahashi et al., J. Antibiot., 1995, 48, 1124 T Kimura et al., J. Antibiot., 1995, 48, 1130

Important commercial herbicides are often polar, weak acids(showing global sales 2014 – PhillipsMcDougall)

Glyphosate Glufosinate$425m$5720m $560m $670m

PinoxadenMesotrione

14 Classification: PUBLIC

Synthesis of phosphonothrixin

HCHO

DABCO THF

MeSO2Cl, Et3N

THF

HPO(OCH2Ph)2

N H THFDABCO, THF

~70% ~90%

THF NaH, THF

58%

OsO4

NMMO

H2 at 1 atm

Pd, MeOHNMMO Pd, MeOH

phosphonothrixinintermediate A

quantitative65%

● A robust and practical route:− 25g of the intermediate A prepared (free-flowing solid).− Enantiomers separated at intermediate A, then deprotected.

Earlier syntheses confirmed structure but are less useful for preparative purposes: J. Antibiot., 1995, 48, 1134; Tet. Letts., 1997, 38, 437; and Tet. Letts., 1998, 39, 6621.

15

, , , ; , , , ; , , ,

Classification: PUBLIC

Phosphonothrixin – herbicidal activity and physical properties

● Activity is post-emergence only.● Symptomology is bleaching, but is distinct

from that of herbicides with otherfrom that of herbicides with other bleaching modes of action.

● Mode of action not established – but believed to be novel.

(RS)-Phosphonothrixin

believed to be novel.

● Water-solubility >1000 ppm.● Hydrolytically stable pH4 to pH9 at 40oC● Hydrolytically-stable, pH4 to pH9, at 40oC.● Moderate photochemical half-life.● Short soil half-life (~1 week).

● Uptake into Amaranthus retroflexus is slow (~14% over 24 hours) but highly phloem mobile once in the plantphloem-mobile once in the plant.

Racemate, 500 ppm, Primary Profiling Screen, cool climate species

16 Classification: PUBLIC

Only one enantiomer is significantly activePrimary Profiling Screen – cool species

(RS)-PhosphonothrixinBleaching

symptomology(RS)-Phosphonothrixin symptomology

th l l

enantiomer 2enantiomer 1(RS)

ethyl analogue

17

enantiomer 2( )

Classification: PUBLIC

Formation of isomeric by-product

OsO4 H2 (1 atm), Pd

NMMO MeOH

RS-PhosphonothrixinCatalytic

NMM

H2 (1 atm), Pd

M OH

Product of -ketolrearrangement

MeOH

● A sample of dibenzyl-protected phosphonothrixin from a CRO contained ~10% of an isomeric product.

● It is the product of an α-ketol rearrangement – perhaps triggered by p g p p gg yresidual NMM from the osmylation step?

● The isomeric product, after debenzylation, shows herbicidal activity.

18 Classification: PUBLIC

Independent synthesis of the rearranged compound

● Made by an independent route to ensure no contamination with phosphonothrixin.

OsO4LiN(TMS)2

NMMO

71%

THF, 25oC

41%

H2 / Pd

MeOH

Quantitative

H2 / Pd

Quantitative

19 Classification: PUBLIC

Biological results – rearranged compoundPrimary Profiling Screeny gPost-emergence, 14 days

Both samples racemic (1) (2)

(1)

(2)

● Less active than phosphonothrixin but the same bleaching symptoms● Less active than phosphonothrixin, but the same bleaching symptoms.● New and readily accessible chemotype – promising lead, but activity

could not be improved and project closed.

20 Classification: PUBLIC

Protectant and systemic fungicides

• Older fungicides are “protectant”.Older fungicides are protectant .− Thinly cover the leaf surface,

protecting it from fungal spores which settle there.which settle there.

• Modern fungicides are mostly “systemic”.− Distribute themselves in plants

after spraying.p y g

21 Classification: PUBLIC

Advantages of systemic fungicides

• Move to new areas of growth after spraying.• Often move from the upper to the lower surface• Often move from the upper to the lower surface

of the leaf.• May be able to eradicate established fungal y g

infections.

• S t i it i f ti f h i l ti• Systemicity is a function of physical properties, e.g. octanol/water partition coefficient = Log P.

• Window of suitable Log P values for optimal g psystemicity.

• Log P can be estimated for target compounds.

22 Classification: PUBLIC

The strobilurins and oudemansins – introduction

OMe

Strobilurin A Oudemansin A

OMeMeO2C

OMeMeO2C

● Family of fungicidal natural products.CO Me

Oudemansiella mucida

● Inhibitors of mitochondrial respiration in fungi with a novel binding site on cytochrome b.

CO2Me

OMe

V Musilek et al., Folia Microbiol. (Prague), 1969, 377.

CONH2

OMe

N

W F Becker, G von Jagow, T Anke and W Steglich, FEBS

Letts., 1981, 132, 329.Myxothiazol A

CONH2MeOS

S

N

23 Classification: PUBLIC

Photostabilised analogues of strobilurin A

X

OMeMeO2C

Strobilurin A X

MeO2COMe

Strobilurin A

MeO2C

Many values of X give active compounds

24 Classification: PUBLIC

Evolution of ideas leading to azoxystrobin – summaryShowing octanol/water Log P values

N

OMeMeO2C

OOOMe

MeO2C

OOMe

MeO2C

O

Log P ~ 5Log P = 3.25 Log P = 1.80

NNThe orld’s biggest agric lt ral f ngicide

Azoxystrobin

OOMe

MeO2C

OCN

The world’s biggest agricultural fungicide

Sales 2014 ~$1200 million

“Reduced risk” pesticide – very favourable

Log P = 2.65

p yenvironmental and toxicological profile

combined with low use rates

25 Classification: PUBLIC

Soybean rust, Phakopsora pachyrhiziGood control with azoxystrobin

• Spread from Africa to South America in 1998South America in 1998 and then to the USA by 2004.

Treated

• Over-wintered in the southern states of the USA for the first time in 2004-05.

A t bi i hi hl• Azoxystrobin is highly active against this disease.

Untreated

26 Classification: PUBLIC

Solatenol

Succinate dehydrogenaseinhibitor (SDHI)

Amistar

27 Classification: PUBLIC

( )

Strobilurins – “Greening” and yield benefits

• Greening effect observed in various crops, e.g. cereals.• Prolongs the growing season and delivers additional yield increase.• Origin of effect – inhibition of ethylene biosynthesis (?).

28 Classification: PUBLIC

The strobilurins – final thoughts

• There is now a family of commercial strobilurinsstrobilurins.

• Originate from academic research conducted 30 years before the launch of the first commercial productscommercial products.

• The synthesis programme was successful because it was possible to vary structure

id l ith t l f ti itwidely without loss of activity.

• Huge industry effort.

− Many thousands of strobilurin analoguesMany thousands of strobilurin analogues synthesised.

− Exciting race to the patent office and >1000 patent applications now filed>1000 patent applications now filed.

29 Classification: PUBLIC

Natural products as chemicals for crop protectionSummary and outlooky

• Nature is an excellent source of biologically active compoundsbiologically-active compounds.

• Nature has optimised secondary metabolism for its own purposes, and these are different from ours.

• Natural product research is slow and uncertain, but can lead to noveland uncertain, but can lead to novel and highly successful products.

• The power of organic synthesisThe power of organic synthesis.

• Natural products are special because, by definition, nature has the “machinery” to make them.

30 Classification: PUBLIC

Acknowledgements

● Sclerotiorins – David Beattie, Torquil Fraser,

Yucheng Gu, Shaowei Huang, Dianne Irwin, LongYucheng Gu, Shaowei Huang, Dianne Irwin, Long

Lin, Danielle Sayer, Qiong Wu and Guang-Fu Yang

Ph h th i i J h Willi Ad B i● Phosphonothrixin – John Williams, Adam Burriss,

Neil Carter, John Paul Evans, Gavin Hall, Roger

Hall Anushka Howell Sophie Oliver TonyHall, Anushka Howell, Sophie Oliver, Tony

O’Sullivan, Anne Seville, Nic Smith and Steve Smith

● Strobilurins – Vivienne Anthony, Brian Baldwin,

Kevin Beautement, Paul de Fraine, Chris Godfrey,

Jeremy Godwin David Youle and many othersJeremy Godwin, David Youle and many others

31 Classification: PUBLIC

Thank youy

32 Classification: PUBLIC