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Challenges inChallenges inAgrochemicals DesignAgrochemicals Design
K-J Schleifer
Computational Chemistry & BiologyBASF SE, Ludwigshafen
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BASFBASF –– TheThe Chemical CompanyChemical Company
104.779 employees worldwide, 33.000 at Ludwigshafen and > 8.000 in R&D
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BASFBASF´́ss PortfolioPortfolio
Oil & GasFunctionalSolutions
PerformanceProducts
AgriculturalSolutions
ConstructionChemicals
Chemicals
Inorganics
Petro-chemicals
Inter-mediates
PaperChemicals
Coatings
Dispersions& Pigments
Plastics
PerformancePolymers
Poly-urethanes
CropProtection
Oil & Gas
CareChemicals
Catalysts
PerformanceChemicals
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Insecticidesagainst harmful
insect pests
Herbicidesagainst weeds
Fungicidesagainst harmful
diseases
CropCrop protectionprotection
Otherse.g. growthregulators
Agricultural SolutionsAgricultural Solutions
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5
DrugsDrugs && AgrochemicalsAgrochemicals
Efficacy
&
Bioavailability
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Mode of Action ofMode of Action of AgrochemicalsAgrochemicals
HerbicidesLipid Synthesis- Acetyl CoA carboxylase
Branched Chain aa Synthase- Acetolactate synthase
Photosynthesis PS I / II
Protoporphyrinogen Oxidase
Pigment Synthesis- PDS- HPPD
EPSP Synthase (Glyphosate)
Microtubule Assembly
Cell Division
Cell Wall (cellulose) Synthesis
Auxin Transport
InsecticidesNervous SystemAcetylcholinesterase
Ion ChannelsGABA-gated Cl-channelsSodium channel modulatorsnAChR agonistsnAChR allosteric modulatorsnAChR channel blockerVGSC blockerRyanodine receptor modulatorsChloride channel activators
Respiration ChainMitochondrial cplx. I-V inhibitors
Growth RegulatorsChitin biosynthesis
Nuclear ReceptorEcdysone receptor agonists
FungicidesNucleic Acid Synthesis- RNA polymerase I- Adenosin-deaminase
Mitosis and Cell Devision- -tubulin assembly
Respiration- Succinate-dehydrogenase- Cytochrome bc1 (Qo & Qi)
AA and Protein Synthesis
Signal Transduction-MAP/Histidine kinase
Lipid and Membrane Synthesis- Methyltransferase
Sterol Biosynthesis- C14 demethylase- 14 reductase
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BioavailabilityBioavailability of Drugsof DrugsAbsorptionAbsorption
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BioavailabilityBioavailability ofof AgrochemicalsAgrochemicalsAbsorptionAbsorption –– PlantsPlants
Cuticula
Stomata
Roots
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BioavailabilityBioavailability ofof AgrochemicalsAgrochemicalsAbsorptionAbsorption –– Plants, Fungi and InsectsPlants, Fungi and Insects
Cell wallMembranes Oral
ChitinousExoskeleton
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BioavailabilityBioavailability of Drugsof DrugsDistributionDistribution
Blood
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BioavailabilityBioavailability ofof AgrochemicalsAgrochemicalsDistribution/TranslocationDistribution/Translocation -- PlantsPlants
Xylem
Phloem
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BioavailabilityBioavailability ofof AgrochemicalsAgrochemicalsDistributionDistribution -- InsectsInsects
dorsal artery
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BioavailabilityBioavailability of Drugsof DrugsMetabolismMetabolism
Blood
Skin
Phase I ReactionOxidation, Reduction, Hydrolysis
cytochrome P450 oxidases(Cyp P450)
Phase II ReactionConjugation of water-soluble groups
UDP-glucuronosyltransferasesglutathione S-transferases
Humans 57 genesDrosophila ~ 80 genesArabidopsis > 300 genes
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BioavailabilityBioavailability of Drugsof DrugsExcretionExcretion
Urine & Feces
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BioavailabilityBioavailability ofof AgrochemicalsAgrochemicalsExcretionExcretion -- PlantsPlants
inclusion of waste products
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ToxicologyToxicology ofof DrugsDrugs
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ToxicologyToxicology of an Insectizideof an Insectizide
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Regulatory relevant issuesRegulatory relevant issues ofof AgchemsAgchems
Enviromental Fate
Soil dissipation/accumulationBound residuesGround waterSurface waterAirRelevant metabolites
Residues
Residues levelToxic metabolites
Toxicology
CMR propertiesEndocrine disruptorsImmuno toxicityAcute/Chronic toxicityRisk for consumer, worker, residentSelected formulants
Biology
EfficacyCrop toleranceResistance riskImpact on food qualityImpact on succeeding & adjacent crops
Ecotoxicology
AquaticHoney bee, non-target arthropodsNon-target plantsSoil organismsWildlife (birds, mammals)Buffer zonesRecovery of biocoenoses
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BioavailabilityBioavailability ofof AgrochemicalsAgrochemicalsSpecificity of HerbicidesSpecificity of Herbicides
Crop Weed
ADMET
oo+o--
ADMET
oo--o+
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DevelopmentCandidate
1
Dossier
0 10
Lead Product
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ProductDevelopment
Years2 3 4 5 6 7 9
LeadIdentification Registration
Research Development
LeadOptimization
R&D cost per product around $ 250 million (industry average)
R&D ProcessR&D Process
Molecular ModellingSupport
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Compounds
Optimization
Target-basedScreening
Organism-basedScreening
New Lead Identification in Crop ProtectionNew Lead Identification in Crop Protection
Leads
DevelopmentCandidates
Output is complementary!
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Requirements of a Compound LibraryRequirements of a Compound Library
Compound Library TargetOrganism Compound Library
intrinsic activity at unknown target(s)
& bioavailability (ADME)
intrinsic activity at isolated target
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23Lemna (Duckweed)
OrganismOrganism--basedbased LeadLead IdentificationIdentification
PreScreen
Hit-validation
PreScreen
Leadfinder
Primary Screening
Greenhouse
Analog Syntheses
GreenhousePhysico-Chemistry
Lead-identification
HIT
TO
LE
AD
HIT
ID
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Hit-validation
PreScreen
Leadfinder
Primary Screening
Greenhouse
Analog Syntheses
GreenhousePhysico-Chemistry
Lead-identification
HIT
TO
LE
AD
HIT
ID
OrganismOrganism--basedbased LeadLead IdentificationIdentification
Leadfinder
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control a.i.
(Bleacher)
Hit-validation
PreScreen
Leadfinder
Primary ScreeningGreenhouse
Analog Syntheses
GreenhousePhysico-Chemistry
Lead-identification
HIT
TO
LE
AD
HIT
ID
OrganismOrganism--basedbased LeadLead IdentificationIdentification
Greenhouse
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1) Relevant chemical space
remove reactive & toxic fragments
choose only agro-like substances
2) Select a diverse subset for screening
3) Look for similar compounds closeto screening hits hit validation
Compound Selection for Random ScreeningCompound Selection for Random Screening
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1) remove reactive & toxic fragments
Descriptors for FilteringDescriptors for Filtering
substructures defined by chemist‘s knowledge
3) Select a diverse subset for screening
4) Look for similar compounds
MACCS keys and topological descriptors
2) choose only agro-like substances
1-D descriptors and pharmacophore points
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Molecular Weight
• 200 to 500 range (86%); < 200 (11%); > 500 (3%)
Melting Point (°C)
• 50 - 200 (60%); < 50 (30%); > 200 (10%)
pKa (acid)
• ~10% pKa < 5
pKa (base)
• ~1% pKa > 5
PhysChemPhysChem Properties of AgrochemicalsProperties of Agrochemicals
Pesticide Manual 11th Ed. 1997 (~ 700 cpds)
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LongLong--Distance TransportDistance Transport
Translocation in Xylem and Phloem
HAHA H+ + A-H+ + A-
pH 5 - 6pH 5 - 6
pH ~8pH ~8
“Intermediate Permeability”and “Weak Acid Theories”
“Intermediate Permeability”and “Weak Acid Theories”
Phloem Vessel
Xylem Vessel
modified, from D.A. Kleier et al. (1998)
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Bioavailability Rules: AgroBioavailability Rules: Agro vs.vs. DrugsDrugs
-< 200° C-< 300° Cmelting point
≤ 100.7 - 22-12 (H)
1-8 (I)
-HB-acceptors
≤ 50 - 1≤ 3 (H)
≤ 2 (I)
≤ 3HB-donors
≤ 51 – 5
7 (I)
≤ 3.5 (H)
0 - 5 (I)
≤ 3
logP < 3)
logPOW
≤ 500200 – 400
500 (I)
150 – 500~ 300molecularweight
LipinskyClarke-Delaney
TiceBriggsAuthors
Properties
“Rule of 5“„Guide of 2“I & post-emergence H
activity more likely
“Rule of 3“
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Screening StrategiesScreening Strategies
Compound Library TargetOrganism Compound Library
intrinsic activity at unknown target(s)
& bioavailability (ADME)
intrinsic activity at isolated target
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Target-Identification
Antisense Technology
ReduceGenexpression
Target Gen
Rateof Vitality
Target AntisenseAntisense--TechnologyTechnology
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Effect in the Plant (Phenotype)
TargetActivity
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Plant specific Targets?Plant specific Targets?
Example: HPPD InhibitorsExample: HPPD Inhibitors
Humans Plants
NH3
+
O
ONH
3
+
O
OH
O
O
O
OH
O
OH
OH
O
O
-Tocopherol
Co-factors forCarotenoid Biosynthesis
Maleylacetoacetate
Fumarylacetoacetate
Fumarate + Acetoacetate
Succinylacetoacetate
Succinylacetone
Plastoquinone-9
Phe Tyr HPP
Homogentisate
HPPD / Fe(II)
Tyrosinemia I Plant Death
Bleaching
FAAH
O2
CO2
heme biosynthesis
X
O
CF3
O
O
NO2
Nitisone (Orfadin®)
O
S
O
O
NO2
O
O
Mesotrione (Callisto®)
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Basis for Screening StrategyBasis for Screening Strategy
validated target
clear in vitro – in vivo correlation
biochemical assay
lots of active compounds
several X-ray co-crystallized structures
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StructureStructure--basedbased ScreeningScreening
H N
Fe
DOCKING
H N
Fe
O
S
Cl
O
O
N
N
O
Cl
Cl
O
S
Cl
O
O
N
N
O
Cl
Cl
1TG5.pdb
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StructureStructure--basedbased ScreeningScreening
H N
Fe
O
S
Cl
O
O
N
N
O
Cl
Cl
SCORING
Estimation of
• H-bonds
• Salt-bridges
• vdW-contacts
• electrostatic interactions
• etc.
relative binding affinities
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Activity
O
S
Cl
O
O
N
N
O
Cl
Cl
O
S
Cl
O
O
N
N
O
Cl
COOH
O
S
Cl
O
O
N
N
O
N
O
S
Cl
OH
O
O
N
N
O
O
SH
Cl
N
N
O
Cl
Cl
O
S
O
O
O
N
Cl
Cl
O
CF3
Cl
N
N
O
Br
O
S
O
O
N
N
O
ClA B C D
E F G H
Affinity/Activity
A B C D
E F G H
A B C D
E F G H
A B C D
E F G H
H N
Fe
O
S
Cl
O
O
N
N
O
Cl
Cl
Compound library
StructureStructure--basedbased virtualvirtual screeningscreening
O
S
Cl
O
O
N
N
O
Cl
Cl
O
S
F
O
O
N
N
O
Cl
Br
O
S
Cl
O
O
N
N
O
N
O
S
Cl
NH2
O
O
N
N
O
O
SH
Cl
N
N
O
Cl
Cl
O
S
O
O
O
N
Cl
Cl
O
CF3
Cl
N
N
O
Br
O
S
O
O
N
N
O
Cl
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StructureStructure--based Virtual Screeningbased Virtual Screening
Scoring with default ParametersScoring with default Parameters
0
10
20
30
40
50
60
70
80
90
100
0 100 200 300 400 500 600 700 800 900 1000
Anzahl gedockter Verbindungen (sortiert nach ComplOpt-Score)
An
zah
lX
XX
X-I
nh
ibit
ore
n
Default parameters
100 HPPD Inhibitors and 900 Chemicals from ACD
3.1375
500
VSbytested
chancebytestedEF %50
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100 HPPD Inhibitors and 900 Chemicals from ACD
0
10
20
30
40
50
60
70
80
90
100
0 100 200 300 400 500 600 700 800 900 1000Anzahl gedockter Verbindungen (sortiert nach ComplOpt-Score)
AnzahlXXXX-Inhib
itore
n Optimized parameters(e.g. clash factors)
5100
500
VSbytested
chancebytestedEF %50
StructureStructure--based Virtual Screeningbased Virtual Screening
Scoring with optimized ParametersScoring with optimized Parameters
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StructureStructure--based Virtual Screeningbased Virtual Screening
CrucialCrucial LigandLigand--Protein Interaction PatternProtein Interaction Pattern
Fe2+
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Default parametersEF50%= 1.3
Optimized parametersEF50%= 5
Optimized parameters +Binding modeEF50%= 6.3
0
10
20
30
40
50
60
70
80
90
100
0 100 200 300 400 500 600 700 800 900 1000
Anzahl gedockter Verbindungen (sortiert nach ComplOpt-Score)
An
zah
lak
tiver
Verb
ind
un
gen
You only have to test 8% of the library to find 50% of the actives!
100 HPPD Inhibitors and 900 Chemicals from ACD
StructureStructure--based Virtual Screeningbased Virtual Screening
Scoring withScoring with PharmacophorePharmacophore ConstraintsConstraints
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StructureStructure--based Virtual Screeningbased Virtual Screening
Test: 41 Actives and > 6.000 HTS nonTest: 41 Actives and > 6.000 HTS non--ActivesActives
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
0% 20% 40% 60% 80% 100%
Gedockte Verbindungen (sortiert nach "ComplOpt-Score")
Akti
ve
XX
XX
-In
hib
ito
ren
Enrichment Factor (EF50%) = 5.0
50%
100%
Score/sqrt(molwgt) Enrichment Factor = 7.1
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Kiefernzapfenrübling(Strobilurus tenacellus)
Buchenschleimrübling(Oudemansiella mucida)
OO
O
O
O
O
O
Strobilurin A
Oudemansin A
Defensive chemicals isolated from fungi (mid 70th):
Profs. Anke and Steglich
UniversityUniversity cooperationcooperation showedshowed thethe way toway to newnew leadlead structuresstructures
StrobilurinsStrobilurins --FungicidesFungicides fromfrom FungiFungi
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Biochemical site of action:respiratory chain
spore mitochondria
I
NADH
NAD+
Succinate
Fumarate
H+ H+
Cyt b *
III
Cyt c12e-
IV
H2O
1/2 O2
H+
UQpool
Cyt cATP
Synthase
H+ADP
ATP
III
NADH
NAD+
Succinate
Fumarate
H+ H+
Cyt b *
III
Cyt c12e-
IV
H2O
1/2 O2
H+
UQpool
Cyt cATP
Synthase
H+ADP
ATP
III
NADH
NAD+
Succinate
Fumarate
H+ H+
Cyt b *
III
Cyt c12e-
IV
H2O
1/2 O2
H+
UQpool
Cyt cATP
Synthase
H+ADP
ATP
II
respiratory chain
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Mode of ActionMode of Action
I
NADH
NAD+
Succinate
Fumarate
H+ H+
Cyt b *
III
Cyt c12e-
IV
H2O
1/2 O2
H+
UQpool
Cyt cATP
Synthase
H+ADP
ATP
III
NADH
NAD+
Succinate
Fumarate
H+ H+
Cyt b *
III
Cyt c12e-
IV
H2O
1/2 O2
H+
UQpool
Cyt cATP
Synthase
H+ADP
ATP
III
NADH
NAD+
Succinate
Fumarate
H+ H+
Cyt b *
III
Cyt c12e-
IV
H2O
1/2 O2
H+
UQpool
Cyt cATP
Synthase
H+ADP
ATP
II
Strobilurins block the fungal energy production by inhibition of thecomplex III of the respiratory chain.
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3D3D--QSAR ModelQSAR Model forfor StrobilurinsStrobilurins
Input:Chemically diverse ligandswith different activity levels
3D-conformationgeneration
structural alignment
very high activity (IC50 < 10-9)high activity (IC50 < 10-8)120 strobilurin analoguesbroad activity range ( 10-10 < IC50 < 10-5)
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3D3D--QSAR ModelQSAR Model forfor StrobilurinsStrobilurins
Input:Chemically diverse ligandswith different activity levels
3D-conformationgeneration
structural alignment
calculateproperty fields
property field: steric demand
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......E2
9.8120
...
E1Sn...
5.33
8.52
7.21
EnS2S1pIC50Cpd
Input:Chemically diverse ligandswith different activity levels
3D-conformationgeneration
structural alignment
calculateproperty fields
correlate propertyfields with activity
.........log 212150 nn EzEmEkShSbSayIC
3D3D--QSAR ModelQSAR Model forfor StrobilurinsStrobilurins
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Input:Chemically diverse ligandswith different activity levels
3D-conformationgeneration
structural alignment
calculateproperty fields
correlate propertyfields with activity
3D-QSAR model
Training set (120 cpds):reproduction of experimental data r2 = 0.95leave-one-out cross-validation q2 = 0.79
3D-QSAR Model for Strobilurins
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Input:Chemically diverse ligandswith different activity levels
3D-conformationgeneration
structural alignment
calculateproperty fields
correlate propertyfields with activity
3D-QSAR modelPrediction of independent test set: r2pred = 0.78
(32 compounds)
3D-QSAR Model for Strobilurins
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PDB code: 1SQB & 1SQP (2004)
XX--rayray StructureStructure solvedsolved!!TheThe keykey findsfinds itsits locklock……....
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StrobilurinStrobilurin History at BASFHistory at BASFFrom a Natural Product to Tailor-made Fungicides
Strobilurin A
O
O
O
1996 Kresoxim-methyl
O
O
ON
O Cereals
FVV
Dimoxystrobin
O
NH
ON
O
(invented by Shionogi)
Cereals
2004
2002
Pyraclostrobin
O
N
O
OO
NNCl
Cereals
FVV
Others
Target CropsTarget Crops
FVV
OthersCereals
Rice
Need for a rice fungicide to complete BASF’s strobilurin portfolio
!
from Strobilurus tenacellusAnke et al., 1977
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92 3 4 5 6 7 8logPow
Target test with isolatedyeast mitochondria
2
3
4
5
6
7
8
9
pl50
Targ
etA
ctivity
Lipophilicity
Biological Activity versus Lipophilicity
Optimum logPOW for in fungus activity = 3.5 ± 1
3.5
5.5
Fungal spore germinationtest with Botrytis cinerea
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log POW
2
3
4
5
6
1 2 3 4 5
pT50
Strong correlation betweendaphnia and fish
pI 5
0A
quatic
To
xicity
atD
aphnia
Lipophilicity
Aquatic Toxicity versus Lipophilicity
Optimum logPOW for acceptable aquatic tox profile = 0 ~ 3
logPOW correlates strongly
with daphnia toxicity
Strong regulatoryrestrictions for fish toxicityin Japan
Kresoxim-methyl (3,4)Dimoxystrobin (3,6)Pyraclostrobin (4,0)
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Lipophilicity Optimum
Preferred characteristic for a new rice fungicide: logPOW = 2 ~ 3
Lipophilicity OptimaLipophilicity Optima logPOW
Activity on Target Level 4.5 ~ 6.5
Activity in Whole Fungus 2.5 ~ 4.5
Low Aquatic Toxicity 0 ~ 3
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Optimisation ResultsOptimisation ResultsFour Potential Candidates Identified
N
O
NH
O O
NN
N
O
O
logPlogPOWOW
2.4
2.9
Orysastrobin
N
O
NH
O O
NN
O
N
O
O
N
O
NO
NH
O2.6
O
NO
N
NH
O
NO
2.7
IPIPRightsRights
Patent Restrictio
ns
(Ciba-Geigy)
+++
Biological ActivityBiological Activity
PYRIORPYRIOR RHIZSORHIZSO
+++
+++ +++
+++ +++
ResidualResidualEfficacyEfficacy
+++
Residual Efficacy
+
+++
Aquatic ToxicologyAquatic Toxicology
FishFish DaphniaDaphnia AlgaeAlgae
+++ +++ +++
AquaticToxicity
+ + +
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OrysastrobinLaunched as Arashi® in 2007
(E,E,E,E)-isomer
Successfully introduced in the Japanese and Korean markets
N
O
NH
O O
NN
N
O
O
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Acknowledgment
Thomas MietznerGerhard en-NaserKlaus Kreuz
Thomas GroteHubert SauterEgon HadenSiegfried StrathmannAkihide Watanabe
PhysChem
Orysastrobin
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Thank you !