Discovery of oxathiapiprolin, a new oomycete fungicide that targets an oxysterol binding protein

Article abstract of DOI:10.1016/j.bmc.2015.07.064

Oxathiapiprolin is the first member of a new class of piperidinyl thiazole isoxazoline fungicides with exceptional activity against plant diseases caused by oomycete pathogens. It acts via inhibition of a novel fungal target - an oxysterol binding protein - resulting in excellent preventative, curative and residual efficacy against key diseases of grapes, potatoes and vegetables. Oxathiapiprolin is being developed globally as DuPont Zorvec disease control with first registration and sales anticipated in 2015. The discovery, synthesis, optimization and biological efficacy are presented.

Full text of DOI:10.1016/j.bmc.2015.07.064

Accepted Manuscript
Discovery of oxathiapiprolin, a new oomycete fungicide that targets an oxysterol
binding protein
Robert J. Pasteris, Mary Ann Hanagan, John J. Bisaha, Bruce L. Finkelstein,
Lisa E. Hoffman, Vann Gregory, John L. Andreassi, James A. Sweigard, Boris
A. Klyashchitsky, Yewande T. Henry, Richard A. Berger
PII:
S0968-0896(15)00648-3
DOI:
http://dx.doi.org/10.1016/j.bmc.2015.07.064
BMC 12493
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Accepted Date:
26 June 2015
28 July 2015
Please cite this article as: Pasteris, R.J., Hanagan, M.A., Bisaha, J.J., Finkelstein, B.L., Hoffman, L.E., Gregory, V.,
Andreassi, J.L., Sweigard, J.A., Klyashchitsky, B.A., Henry, Y.T., Berger, R.A., Discovery of oxathiapiprolin, a
new oomycete fungicide that targets an oxysterol binding protein, Bioorganic & Medicinal Chemistry (2015), doi:
http://dx.doi.org/10.1016/j.bmc.2015.07.064
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Discovery of oxathiapiprolin, a new oomycete fungicide that targets
an oxysterol binding protein
Robert J. Pasteris*, Mary Ann Hanagan, John J. Bisaha, Bruce L. Finkelstein, Lisa E. Hoffman, Vann
Gregory, John L. Andreassi, James A. Sweigard, Boris A. Klyashchitsky, Yewande T. Henry, and
Richard A. Berger
DuPont Crop Protection, Discovery Research, Stine-Haskell Research Center, P.O. Box 30, Newark, DE
19714, USA
* Corresponding author. Tel.: +1-302-366-5918; fax: +1-302-366-5738
E-mail address: robert.j.pasteris@dupont.com (R.J. Pasteris)
Abstract
Oxathiapiprolin is the first member of a new class of piperidinyl thiazole isoxazoline fungicides with
exceptional activity against plant diseases caused by oomycete pathogens. It acts via inhibition of a novel
fungal target - an oxysterol binding protein - resulting in excellent preventative, curative and residual
efficacy against key diseases of grapes, potatoes and vegetables. Oxathiapiprolin is being developed
globally as DuPont™ Zorvec™ disease control with first registration and sales anticipated in 2015. The
discovery, synthesis, optimization and biological efficacy are presented.
10,000 fold
Activity Improvement
F
S
S
Cl
N
N
N
N
N
N
N
O
O
F
N
O
O
CF3
Initial HTS Active
Oxathiapiprolin
1. Introduction
Enhancing world food production represents a significant challenge as the global population
increases by 2.4 billion people over the next 35 years.1 A key step in meeting this challenge is the
development of better tools to prevent the estimated 20% – 40% global crop losses caused by competition
with weeds, insects and plant diseases, despite the use of modern crop protection products.2 Plant
diseases caused by oomycete pathogens are particularly devastating for potato, grape and vegetable crops,
with the global crop losses due to Phytophthora infestans on potatoes (potato late blight; PLB) alone
estimated at $6.7 billion annually.3 The ability of plant pathogens to develop resistance to current control
technologies necessitates the search for novel chemical classes that can control diseases by acting against
new biochemical targets. Accordingly, one goal of our laboratory is to seek out new chemical classes
whose biological properties have not yet been well explored. In this paper, we will discuss the discovery,
synthesis, mode of action and biological efficacy of a new class of oomycete fungicides based on a novel
piperidinyl thiazole isoxazoline core and its optimization to oxathiapiprolin, a new tool to address key
grower disease control needs.

2. Results and discussion
2.1 Hit Identification and activity
The discovery of oxathiapiprolin began with the purchase of a diverse bis-amide compound
library from Tripos Associates built around a piperidine-thiazole-carbonyl core using a diverse set of acid
and amine moieties, as detailed in Figure 1. Compound 1 was the only member of this library where a
phenyl acetic acid moiety had been combined with a benzyl amine moiety and showed modest
preventative fungicidal activity against Phytophthora infestans on tomatoes (tomato late blight; TLB) and
Pseudoperonospora cubensis on cucumbers (cucumber downy mildew; CDM) and weak curative activity
against Plasmopara viticola on grapes (grape downy mildew; GDM) in early stage screens (Table 1).
This hint of curative activity, an important but rarely seen attribute for oomycete fungicides, caught our
attention and an optimization program was initiated.
R2
S
S
Cl
N
N
R3
N
N
R1
N
N
O
O
O
O
Tripos Library
1
Figure 1. Tripos bis-amide library details: R1 and R3 are aryl, heteroaryl and benzyl; R2 is hydrogen or
methyl. Compound 1 is a library member.
2.2 Chemistry and Initial SAR
The synthesis of compounds such as 1 is outlined in Scheme 1 and begins with condensation of
the commercially available Boc-protected piperidine thioamide 2 with ethyl bromopyruvate to give the
orthogonally protected piperidinyl thiazole 3. Amine deprotection, followed by acylation affords ester 4,
which upon base hydrolysis and amide coupling provides the bis-amides 5. These steps can be reversed
where ester hydrolysis and amide coupling precede amine deprotection and acylation.4
O
Br
OEt
S
O
S
1) TFA or HCl
OEt
Boc-N
Boc-N
NH2
N
EtOH
2) Amide Coupling
O
OH
R1
3
2
O
S
1) NaOH/THF/H2O
2) Amide Coupling
S
R2
N
R1
R1
OEt
N
N
N
N
R3
O
O
O
R3
R2
O
5
N
H
4
Scheme 1. The synthesis of bis-amides 5.

Using the method of Scheme 1, a positional scanning approach was used to assess the initial SAR
for the amine component by holding the 2-chlorophenyl acetic acid moiety constant. Numerous amine
variations were explored, but did not lead to significant improvement over the original N,α-dimethyl
benzyl amine moiety. Both the N-methyl and the α-substituent on the benzyl moiety were found to be
required for activity, with the (R)-enantiomer being preferred. An α-ethyl was slightly better than α-
methyl, but longer alkyl, alkenyl or halo alkyl groups reduced activity. Removing the α-substituent caused
loss of activity. Adding substituents to the phenyl ring did not appear to enhance activity, nor did
replacing the phenyl ring with pyridine or thiophene rings. Removal of the benzylic carbon or replacing it
with a nitrogen or oxygen atom eliminated activity. Simple anilides or alkyl amides were not active.
Variation of the acid moiety, holding the (R)-N,α-dimethyl benzyl amine moiety constant, was
more productive. Key observations were the requirement of a carbonyl linked to a 2,5-disubstituted
phenyl ring by either an unsubstituted methylene or nitrogen atom, with small alkyl, haloalkyl and
halogen substituents being optimal. The 2,5-dimethyl phenyl analog 6 (Figure 2), showed a 5-fold
increase in activity vs. the 2-chlorophenyl analog 1, with > 90% control of preventative and curative TLB
and curative GDM observed at an application rate of 40 ppm (Table 1).
Compound 6 showed rapid leaf penetration but did not move systemically in the plant, most
likely due to its high logP and low water solubility. The dimethyl phenyl ring was replaced with various
heterocycles in an effort to lower the logP. Appropriately substituted pyridines, thiophenes, imidazoles,
triazoles and thiazoles had similar activity as the phenyl, but a 3,5-disubstituted N-linked pyrazole gave a
significant boost in activity and systemicity. Thus, pyrazole acetamide 7 (Figure 2) showed a 20-fold
increase in activity vs. phenyl acetamide 1, with > 90% control of preventative and curative TLB and
curative GDM observed at an application rate of 10 ppm (Table 1).
S
S
N
N
N
N
N
N
N
N
CF3
O
O
O
O
7
6
Figure 2. Structures for bis-amides 6 and 7.
Modification of the piperidinyl thiazole core was carried out while keeping the acid and amine
moieties as shown in compound 7. A wide variety of chemical methods were needed to explore changes
to this portion of molecule and elaborating them here is beyond the scope of this review. The SAR
developed revealed that an unsubstituted piperidine ring was optimal. Replacing the piperidine ring with
a piperazine ring gave analogs with good activity, but poor photo stability, most likely due to extended
conjugation of the extra nitrogen lone pair. Replacing the piperidine ring with a homopiperidine ring or a
tetrahydropyridine ring lowered activity, while replacing the piperidine ring with a pyrrolidine ring or an
azetidine ring eliminated activity. Ring-opened analogs were not active.
High activity could be retained by replacing the thiazole ring with other 5-membered ring
heterocycles, as shown in Figure 3, with the original thiazole or corresponding oxazole being the best.
Substitution on the 5-position of the thiazole ring or replacing the thiazole ring with a phenyl, pyridine or
pyrimidine ring resulted in loss of activity.

S
N
N
N
N
N
CF3
O
O
N
O
N
S
S
S
High
Activity
N
N
N
N
N
N
N
N
N
N
O
O
O
O
O
O
O
O
O
O
O
N
O
N
O
N
N
S
N
N
N
High
Activity
H
O
N
N
N
O
O
H
N
N
N
N
O
N
Moderate
Activity
N
N
N
S
N
N
N
O
O
O
O
O
N
N
O
N
Low/no
Activity
O
N
N
N
N
N
N
N
O
O
O
O
O
Figure 3. Thiazole moiety SAR.
2.3 Restricting conformations
We had explored the three key regions of the molecule and an activity plateau appeared to have
been reached. Compound 7 had excellent preventative and curative oomycete control at 10 ppm, but
needed to be active at lower rates to be commercially viable. Inspection of 7 shows that it has eight
rotatable bonds allowing for a wide variety of accessible low energy conformations. Flexible molecules
may require more energy to adopt a needed binding conformation, reducing potency. If one could pre-
organize a molecule into the appropriate binding conformation, less energy would be required and
potency should increase. A program to conformationally restrict Compound 7 by forming new rings near
the chiral center region of the molecule was initiated by tying-back the N-methyl onto the α-methyl, the N-
methyl onto the phenyl and the α-methyl onto the phenyl as outlined in Figure 4. The first two
modifications were unproductive, but the third resulted in a remarkable boost in activity.

b
Tie-back α-methyl
onto N-methyl
S
S
N
N
N
N
N
N
N
N
N
N
a
a
c
CF3
O
CF3
O
O
O
7
8
Significant Activity Loss
Tie-back α-methyl
Tie-back N-methyl
onto phenyl ring
Cl
onto phenyl ring
b
c
S
S
N
N
N
N
N
N
N
N
N
N
CF3
O
CF3
O
O
O
10
9
Moderate Activity Loss
Significant Activity Gain
Figure 4. Restricting conformations near the chiral center.
The tetraline amide 10 showed a 1000-fold increase in activity vs. compound 1, with > 90%
control of preventative and curative TLB and curative GDM observed at an application rate of 0.4 ppm
(Table 1). Bis-amide 10 is the (R)-enantiomer; it’s corresponding (S)-enantiomer is significantly less
active. Field tests in the United States and Europe showed excellent preventative, curative and residual
efficacy against PLB and GDM at 100 gai/ha, outperforming commercial fungicides.5
With this approach validated, we turned our attention to the amide bond. Amide 7 exists as a
mixture of two rotamers, clearly observable in its NMR spectrum and requiring a temperature of 110 oC
for signal coalescence. We envisioned preparing the two isomeric imidazolines 11 and 12 as our best
probe compounds, but also prepared the phenyl isoxazoline 13 as a potential benzyl amide bioisostere
(see Figure 5). This was fortuitous, since both 11 and 12 were inactive, while isoxazoline 13 again
showed a remarkable boost in activity over compound 7.
Like tetraline amide 10, racemic isoxazoline 13 was 1000-fold more active than starting
Compound 1 (Table 1). The (R)-enantiomer of 13 was more active than the corresponding (S)-
enantiomer. Restricting the rotation of the phenyl ring by tying it back onto the 5-position of the
isoxazoline ring gave the spiro analog 14 (Figure 5) and another significant boost in activity. Spiro
compound 14 showed a 2500-fold increase in activity vs. Compound 1, with > 90% control of
preventative and curative TLB and curative GDM observed at an application rate of 0.08 ppm (Table 1).
Field tests demonstrated its superior performance versus the leading commercial oomycete fungicides.6,7

Compound 7
Amide bond rotation
S
S
N
N
N
N
N
N
N
N
N
N
CF3
O
CF3
O
O
O
7
7
Tie-back α-methyl
onto carbonyl
Tie-back N-methyl
onto carbonyl
S
S
N
N
N
N
N
N
N
N
N
N
CF3
CF3
O
O
N
N
12
11
S
S
N
N
N
N
N
N
N
N
CF3
CF3
O
O
N
N
13
14
O
O
Figure 5. Amide bond bioisosteres. Imidazolines 11 and 12 are not active. Isoxazolines 13 and 14 have
unbroken activity at 0.4 and 0.08 ppm, respectively.
2.4 Selecting the optimal candidate
An intensive effort looking at a wide range of alternate amide bioisosteres led to many active
chemotypes8,9 (Figure 6), but none were more active than the isoxazoline chemotype. The corresponding
isoxazole and the homologous dihydrooxazine were also quite active.
The piperidinyl thiazole isoxazolines can be readily prepared as shown in Scheme 2 for phenyl
isoxazoline 13. Nitrosation of dichloroacetone gives the bench stable dichloroketooxime 16 which
undergoes a [3+2] cycloaddition with styrene to form the chloroketoisoxazoline 17. Condensation with
the Boc-piperidine thioamide 2 forms the thiazole 18 followed by amine deprotection and coupling with
the pyrazole acetic acid 19 to afford 13 in high yield.

S
N
N
N
N
CF3
O
N
O
T
T
T
T
T
High
Activity
Ph
Ph
Ph
Ph
Ph
N
O
N
N
N
O
N
O
O
O
R
T
T
T
T
T
A
O
O
Moderate
Activity
Ph
Ph
Ph
Ph
N
N
N
O
N
O
N
Ph
R
T
T
T
T
T
T
N
Moderate
Activity
T
O
N
Ph
N
Tol
N
N
N
N
N
N
N
O
N
O
R
O
Ph
T
T
T
T
O
T
N
Low/no
Activity
Ph
Ph
Ph
Ph
N
N
N
N
O
Ph
Ph
Ph
T
Ph
A
T
O
N
N
Low/no
Activity
N
N
H
Ph
Figure 6. Isoxazoline moiety SAR.
S
NH2
Boc-N
Cl
Cl
Cl
2
t-BuONO
HCl
Styrene
NaHCO3
Cl
Cl
O
O
O
Pyridine/Ethanol
Ph
N
O
N
15
OH
16
17
S
1) HCl
S
N
Boc-N
N
N
N
2) EDC Coupling
Ph
N
N
Ph
CF3
O
O
N
18
O
13
N
OH
N
CF3
O
19
Scheme 2. Synthesis of compound 13.
Exploring the SAR of the isoxazoline moiety showed that 5-substitution was essential for high
activity, with a wide variety of aryl, heteroaryl and other groups being explored. A second 5-substituent
was also tolerated with 5,5-spiro analogs such as 14 being among the best. The phenyl ring of 14 is
twisted out of plane of the isoxazoline ring by the spiro ring fusion. This twist can also be forced by

adding substituents in the 2- and 6-positions of the isoxazoline phenyl ring of 13 to give analogs with
unprecedented levels of activity. For example, Figure 7 depicts the 2,6-difluorophenyl analog 20, which
showed a 10,000-fold increase in activity versus bis-amide 1, with > 90% control of preventative and
curative TLB and curative GDM observed at an application rate of 0.02 ppm (Table 1).6
S
F
N
N
N
N
CF3
O
N
O
20
F
Figure 7. Compound 20: unbroken activity at 0.02 ppm.
Table 1
Potency progression observed during optimization
Lowest rate
Relative
activityb
Compound
requireda
(ppm)
1
6
200
40
1
5
7
10
20
10
13
14
20c
0.4
1,000
1,000
2,500
10,000
0.4
0.08
0.02
aLowest compound application rate required to provide > 90 percent control of preventative and curative
b
TLB and curative GDM relative to the untreated control plants. Relative activity increase compared to
c
Compound 1. Oxathiapiprolin.
In parallel with the isoxazoline SAR development, work continued on exploring alternatives to
the pyrazole acetic acid moiety. Acyclic structures were explored and found to be highly active provided
that they were linear chains of 5-7 atoms preferably terminated with a trifluoromethyl group.10 The
piperidine could also be replaced with a 1,2-oxazinane, a hexahydropyridazine or a 1,5-
diazabicyclo[3.3.0]octane ring system to give compounds with high levels of activity.11
With so many chemotypes with high intrinsic activity, it soon became apparent that the key
challenge would be finding the best of the best to move forward into development. We had a wide
selection of optimized acid and isoxazoline moieties, the combination of which all produced viable

candidates. An added complication was the observation of polymorphism in many of these candidates,12
which necessitated finding the most stable polymorph for each one to assess formulation characteristics
and biological efficacy.
This effort led to the selection of a set of forty top analogs. These “final forty” underwent
extensive characterization and greenhouse testing to produce our “final four” candidates for broad scale
toxicological and field efficacy testing. All of the candidates performed well in the field with the 2,6-
difluorophenyl analog 20 (Figure 7) being selected as the commercial development candidate and has
been assigned the common name of oxathiapiprolin.
2.5 Biological activity
Compound 20 was evaluated in the field for control of the major oomycete diseases of grapes,
potatoes and vegetables. It demonstrated outstanding control of potato late blight at rates as low as 12-30
gai/ha in preventative and curative trials, along with residual control of 7-10 days. Control of grape
downy mildew was also impressive with both fruit and foliar protection at rates as low as 20-30 gai/ha.
Cucumber downy mildew and crown and root rot of peppers were also controlled at rates as low as 12-30
gai/ha. Compound 20 consistently outperformed all of the commercial disease control standards.
Compound 20 acts at multiple stages of the pathogen’s life cycle at extremely low concentrations.
Preventatively, it inhibits zoospore release and stops zoospore and sporangia germination. Curatively, it
stops mycelial growth within the host plant before visible lesions occur, offering protection at 1, 2 and 3
days. Post-infection, it stops mycelial growth and inhibits further lesion expansion, and as an
antisporulant, it inhibits spore production and viability.
Compound 20 is rapidly absorbed into the epicuticular waxy layer of the plant making it
extremely resistant to wash-off. Once inside the plant, it shows translaminar and acropetal systemic
movement, protecting treated leaves as they grow and expand and new leaves as they emerge and grow.
Compound 20 has an excellent toxicity profile, with low oral, neuro, developmental, dermal,
aquatic, avian and bee toxicity.
2.6 Mechanism of action
Initial work to determine the mechanism of action of the bis-amides focused on comparisons with
known fungicides and tests to determine if they acted via known mechanisms were all negative. Confocal
microscopy using a version of tetraline amide 10 tethered to a fluorescent tag showed localization to the
nucleus, Golgi and endoplasmic reticulum. Ligand localization could be prevented by high
concentrations of active analogs but not by other known fungal classes.
A bis-tritiated version of isoxazoline 13 was found to bind to a single, low abundance target
contained in a soluble protein extract from P. infestans. Displacement experiments with analogs of
varying potencies showed that the ability to bind to this target mirrored the observed biological activity.
At this point two parallel efforts were undertaken to identify the target protein using the model
oomycete Phytophthora capsici.13,14 A biochemical approach used a tethered version of isoxazoline 13 to
isolate binding proteins by affinity chromatography. One of these was identified by mass spectrometry as
an oxysterol binding protein (OSBP). A molecular genetics approach provided definitive data that OSBP
was a critical component in the mechanism of action. P. capsici zoospores were irradiated with

ultraviolet light and resistant mutants were selected with growth media infused with isoxazoline 14 or 20.
Numerous independent resistant mutants were isolated and were determined by sequencing to have a
single nucleotide polymorphism in the P. capsici OSBP gene. Changes at 9 different amino acid
positions, all located within the oxysterol binding domain, were observed . Additionally, transformation
of P. capsici cells with a plasmid encoding a mutated OSBP gene conferred resistance to compound 20.
OSBP represents a novel target for oomycete disease control. The cellular role of this protein
family is not well understood.15 Studies to provide definitive evidence for the cellular role of OSBP in
Phytophthora and the exact mechanism of compound 20’s cytotoxicity are still underway.
3. Conclusions
Compound 20 is the first member of a new class of highly-active oomycete fungicides, the
piperidinyl thiazole isoxazolines. It is effective at extremely low use rates and shows excellent
preventative and curative efficacy, excellent antisporulant properties and long residual control with
protection of new growth. It was discovered using a high throughput screening approach with diverse
structural classes and optimized to this high potency chemotype by conformationally restricting the
benzyl amide moiety using an isoxazoline ring as an amide bioisostere.
Compound 20 has a novel site of action, binding strongly to an oxysterol binding protein with an,
as yet, unknown cellular function. Compound 20 has the perfect combination of attributes to provide
outstanding oomycete disease control, has been assigned the common name of oxathiapiprolin and is
being developed globally as DuPont™ Zorvec™ disease control with first registration and sales
anticipated in 2015. We expect that products powered by oxathiapiprolin will become valuable tools for
growers in their efforts to combat these diseases.
4. Experimental Section
4.1 Chemistry
1
All new compounds were characterized by standard spectroscopic methods. H NMR spectra
were recorded on a Varian Unity 400 spectrometer at 400 MHz using deuterated solvent and
tetramethylsilane as an internal standard. Chemical shifts are reported in ppm downfield from the
standard (δ = 0.00). All reactions were carried out under anhydrous conditions under an inert atmosphere
(nitrogen or argon) with commercially available dry solvents. All the compounds described below are
known and these and alternate preparations for the compounds in Table 1 can be found in references 4
and 8. A crystal structure for form B of Compound 20 can be found in reference 12.
4.1.1. Preparation of 3-chloro-N-hydroxy-2-oxo-propanimidoyl chloride (16)
To a solution of 1,3-dichloroacetone (15, 150 g, 1.12 mol) in 2 M solution of hydrogen chloride in
diethyl ether (600 mL) was added t-butyl nitrite (126 g, 1.1 mol) over 45 minutes keeping the temperature
below 26 °C with a cold water bath. The reaction was allowed to stir overnight at ambient temperature
and the mixture was concentrated under reduced pressure. nBuCl was added and the mixture
concentrated under reduced pressure again. The resulting solid was slurried in nBuCl and collected under
filtration to give a 41.3 g of a white solid. The filtrate was concentrated under reduced pressure to about
half volume to give a second crop of white solid (25.8 g), and the filtrate was further concentrated to give

a third crop (26.5 g). The solids were combined to give 3-chloro-N-hydroxy-2-oxo-propanimidoyl
1
chloride (16, 93.6 g, 0.60 mol, 53%). H NMR (DMSO-d ) δ4.96 (s, 2H), 13.76 (s, 1H).
6
4.1.2. Preparation of 2-chloro-1-(4,5-dihydro-5-phenyl-3-isoxazolyl)ethanone (17)
To a mixture of styrene (6.79 g, 65.3 mmol) and powdered sodium bicarbonate (32.1 g 0.38 mol) in
acetonitrile (100 mL), 3-chloro-N-hydroxy-2-oxo-propanimidoyl chloride (16, 10 g, 64.1 mmol) was
added in 10 portions over 20 minutes. The reaction mixture was then stirred for additional 1 h and
filtered. The filtered solid was rinsed with acetonitrile, and the combined filtrates were concentrated under
reduced pressure to leave an oil, which was triturated first with hexanes and then with 1-chlorobutane to
give 2-chloro-1-(4,5-dihydro-5-phenyl-3-isoxazolyl)ethanone (17, 13.6 g, 61 mmol, 95%) as a white
1
solid. H NMR (CDCl ) δ3.13 (m, 1H), 3.66 (m, 1H), 4.96 (s, 2H), 5.83 (m, 1H), 7.34–7.44 (m, 5H).
3
4.1.3. Preparation of 1,1-dimethylethyl 4-[4-(4,5-dihydro-5-phenyl-3-isoxazolyl)-2 thiazolyl]-1-
piperidinecarboxylate (18)
A mixture of 1,1-dimethylethyl 4-(aminothioxomethyl)-1-tetrahydropyridine-carboxylate (2, 2.4
g, 10 mmol), 2-chloro-1-(4,5-dihydro-5-phenyl-3-isoxazolyl)ethanone (17, 2.3 g, 10 mmol), pyridine (1.6
g, 20 mmol) and tetrabutylammonium bromide (0.65 g, 2 mmol) in ethanol (20 mL) was refluxed for 2.5
h and cooled in an ice/water bath. The resulting precipitate was filtered, washed with cold ethanol and
air dried to give 1,1-dimethylethyl 4-[4-(4,5-dihydro-5-phenyl-3-isoxazolyl)-2 thiazolyl]-1-
piperidinecarboxylate (18, 2.85 g, 6.9 mmol, 69%) as a white solid. 1H NMR (CDCl3) δ1.47 (s, 9H), 1.7

(m, 2H), 2.1 (m, 2H), 2.85 (m, 2H), 3.2 (m, 1H), 3.45 (m, 1H), 3.84 (m, 1H) 4.2 (br s, 2H), 5.75 (m, 1H),
7.25–7.40 (m, 5H), 7.61 (s, 1H).
4.1.4. Preparation of 4-[4-[4,5-dihydro-5-phenyl-3-isoxazolyl]-2-thiazolyl]-1 [[5-methyl-3
(trifluoromethyl)-1H-pyrazol-1-yl]acetyl]piperindine (13)
To a solution of 1,1-dimethylethyl 4-[4-(4,5-dihydro-5-phenyl-3-isoxazolyl)-2 thiazolyl]-1-
piperidinecarboxylate (18, 0.815 g, 1.97 mmol) in dichloromethane (50 mL) was added a solution of
hydrogen chloride in diethyl ether (2 M, 10 mL, 20 mmol). The reaction mixture was stirred at room
temperature for 1 h to give a gummy precipitate. Methanol was added to dissolve the precipitate, and the
reaction mixture was stirred for an additional 1 h. The reaction mixture was concentrated under reduced
pressure and partitioned between ethyl acetate and saturated aqueous sodium bicarbonate solution, and the
organic layer was dried (MgSO4) and concentrated to give the free amine as a clear oil (0.31 g), which
solidified on standing.
A mixture of the resulting free amine (0.31 g, 1.0 mmol), 5-methyl-3-(trifluoromethyl)-1H-
pyrazole-1-acetic acid (19, 0.208 g, 1.0 mmol), 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide
hydrochloride (0.25 g, 1.3 mmol), triethylamine (150 uL, 1.08 mmol) and a catalytic amount of 1-
hydroxy-benzotriazole hydrate (~1 mg) in dichloromethane (5 mL) was swirled to form a vortex and held
at room temperature for 16 h. The reaction mixture was diluted with dichloromethane (10 mL), and
washed with 1 N aqueous hydrochloric acid and saturated aqueous sodium bicarbonate solution. The
organic layer was dried (MgSO4) and concentrated under reduced pressure to give 4-[4-[4,5-dihydro-
5-phenyl-3-isoxazolyl]-2-thiazolyl]-1 [[5-methyl-3-(trifluoromethyl)-1H-pyrazol-1-yl]acetyl]piperindine
(13, 0.47 g, 0.93 mmol, 93%) as a white foam. A sample crystallized from ethanol melted at 109 – 113
oC. 1H NMR (CDCl3) δ
1.8 (m, 2H), 2.2 (m, 2H), 2.32 (s, 3H), 2.9 (m, 1H), 3.3 (m, 2H), 3.42 (m, 1H),


3.85 (m, 1H) 4.05 (m, 1H), 4.55 (m, 1H), 4.98 (m, 2H), 5.75 (m, 1H), 6.33 (s, 1H), 7.25–7.42 (m, 5H),
7.63 (s, 1H).
4.2 Biology
Compounds were first dissolved in acetone in an amount equal to 3% of the final volume and then
suspended at the desired concentration (in ppm) in acetone and purified water (50/50 mix) containing
®
250 ppm of the surfactant Trem 014 (polyhydric alcohol esters). Spraying a 200 ppm test suspension
to the point of run-off on the test plants was the equivalent of a field rate of 500 g / hectare.
4.2.1 Phytophthora infestans on tomatoes (tomato late blight; TLB) preventative assay
The test suspension was sprayed to the point of run-off on tomato seedlings. The following day the
seedlings were inoculated with a spore suspension of Phytophthora infestans (the causal agent of tomato
late blight) and incubated in a saturated atmosphere at 20 °C for 24 h, and then moved to a growth
chamber at 20 °C for 5 days, after which disease ratings were made.
4.2.2 Phytophthora infestans on tomatoes (tomato late blight; TLB) curative assay
Tomato seedlings were inoculated with a spore suspension of Phytophthora infestans (the causal
agent of tomato late blight) and incubated in a saturated atmosphere at 20 °C for 17 h. After a short drying
period, the test suspension was sprayed to the point of run-off on the tomato seedlings and then moved to
a growth chamber at 20 °C for 4 days, after which disease ratings were made.
4.2.3 Plasmopara viticola on grapes (grape downy mildew; GDM) curative assay
Grape seedlings were inoculated with a spore suspension of Plasmopara viticola (the causal agent
of grape downy mildew) and incubated in a saturated atmosphere at 20 °C for 24 h. After a short drying
period, the test suspension was sprayed to the point of run-off on the grape seedlings and then moved to a
growth chamber at 20 °C for 5 days, after which the test units were placed back into a saturated
atmosphere at 20 °C for 24 h. Upon removal, disease ratings were made.
Acknowledgments
The authors wish to express their sincere gratitude to all those who made significant contributions to this
project, both in the discovery and commercial development of oxathiapiprolin. Special thanks are due to
James Bereznak, Lora Geist, Jeffrey Long, Eric Marshall, Pat Kovacs, Richard Crosswicks, Gilles
Seburyamo, Thomas Stevenson, Andy Liepa, Rafael Shapiro, Kingmo Sun, Balreddy Kamireddy, Martina
Crompton, Ghislaine Majeau, Sylvie Monette, Josh Willey, Jack Bruhn, Sarah Salas, Steven Pember,
Anne Carroll, David Nesnow, Clarence Hermanski, Renee Lett, Mei Dung, Wonpyo Hong, Charlene
Sternberg, Thomas Fuesler, and the DuPont Crop Protection Field Development Group.
References and notes
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world-population-projected-to-reach-9-6-billion-by-2050.html
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