Synthesis and fungicidal activity of a series of novel aryloxylepidines

Article abstract of DOI:10.1002/ps.371

A series of novel (hetero)aryloxylepidine derivatives was devised as hybrid structures of the phenoxyquinoline and phenethoxyquin(az)oline fungicides. Synthesis of these targets required the development of several new routes to derivatised 4-hydroxymethylquinolines, and subsequent coupling with phenols or haloarenes. The aryloxylepidines generally showed moderate broad-spectrum fungicidal activity across several diseases of cereals. Substitution of the quinoline ring with chlorine at the 7- and/or 5-positions gave molecules with high levels of protectant activity against Erysiphe graminis f sp tritici (powdery mildew of wheat), but this did not improve the level of fungicidal activity against other diseases. In vitro activity against mitochondrial electron transport complex I (MET) derived from Ustilago maydis showed that 8-fluorolepidine analogues were moderately active at this target site, while the more fungicidally active 7- and 5,7-substituted compounds were inactive. This indicates that MET is not the primary target of these highly active powdery mildewicides.

Full text of DOI:10.1002/ps.371

Pest Management Science
Pest Manag Sci 57:844±851 2001)
DOI: 10.1002/ps.371
Synthesis and fungicidal activity of a series of
novel aryloxylepidines
Neil V Kirby,* John F Daeuble, L Navelle Davis, Anna C Hannum, Karin Hellwig,
Lori K Lawler, Marshall H Parker and Mary E Pieczko
Dow AgroSciences LLC, 9330 Zionsville Road, Indianapolis, IN 46268-1054, USA
Abstract: A series of novel ꢀhetero)aryloxylepidine derivatives was devised as hybrid structures of the
phenoxyquinoline and phenethoxyquinꢀaz)oline fungicides. Synthesis of these targets required the
development of several new routes to derivatised 4-hydroxymethylquinolines, and subsequent
coupling with phenols or haloarenes. The aryloxylepidines generally showed moderate broad-
spectrum fungicidal activity across several diseases of cereals. Substitution of the quinoline ring with
chlorine at the 7-and/or 5-positions gave molecules with high levels of protectant activity against
Erysiphe graminis f sp tritici ꢀpowdery mildew of wheat), but this did not improve the level of
fungicidal activity against other diseases. In vitro activity against mitochondrial electron transport
complex I ꢀMET) derived from Ustilago maydis showed that 8-¯uorolepidine analogues were
moderately active at this target site, while the more fungicidally active 7-and 5,7s-ubstituted
compounds were inactive. This indicates that MET is not the primary target of these highly active
powdery mildewicides.
# 2001 Society of Chemical Industry
Keywords: lepidines; fungicide; Erysiphe graminis; mitochondrial electron transport; quinoxyfen
1
INTRODUCTION
product mixtures that required lengthy separation.
Accordingly we sought more ef®cient routes to our
target structures.
The chemistry of nitrogen-containing heterocycles
remains a fertile area for the discovery of new
agricultural chemicals with a broad array of potential
applications. One structural motif that appears fre-
quently in the literature is illustrated in Fig 1Ða
quin az)oline ring substituted in the 4-position by a
linker group leading to an aromatic or heteroaromatic
ring. Some reported examples of this template include
nematicidal phenylacetamidoquinolines 1,1 fungicidal
benzyloxyquinazolines 2,2 the powdery mildewicide
5,7-dichloro-4- 4-¯uorophenoxy)quinoline, quinoxy-
fen,3 and the miticide 4-tert-butylphenethyl quinazo-
lin-4-yl ether, fenazaquin.4
Substituted 4-hydroxyquinolines are synthetically
accessible and were considered a viable starting point
for synthesis. Bromination of 7-chloro-4-hydroxyqui-
noline Fig 2, 3) to the 4-bromo-compound 4 was
achieved in good yield using triphenyl phosphite/
bromine,8 and this material was carbonylated in
ethanol to the corresponding quinoline carboxylate
5. Reduction with sodium borohydride in methanol
solution gave the desired 4-hydroxymethylquinoline
analogue 6.
This method proved successful for several substi-
tuent patterns but failed for the 5,7-dichloro-substi-
As part of a study to expand this interesting motif
and related diheteroaryl ethers,5 we chose aryloxyle-
pidines for examination.6
tuted
quinoline
system.
Bromination
and
carbonylation proceeded smoothly in this case, but
attempted reduction of the quinoline ester to the
corresponding alcohol was confounded by competitive
reduction of the quinoline ring. This problem was
circumvented by reaction of 4-bromo-5,7-dichloro-
quinoline Fig 3, 7) with styrene under Heck condi-
tions, ozonolysis of the aza-stilbene 8 to give the
corresponding aldehyde 9, and borohydride reduction
to the desired intermediate 10.
2
EXPERIMENTAL METHODS
2.1 Chemical synthesis
Lepidine chemistry is not widely explored in the
literature relative to other aspects of quinoline
chemistry. The standard methods of ring formation
via addition of a derivatised aniline hydrochloride to
methyl vinyl ketone, cyclisation of the resultant
aminoketone with a Lewis acid,7 and subsequent
benzylic bromination were low-yielding and gave
The Heck/ozonolysis method of converting bromo-
quinolines to the corresponding aldehydes gave a
convenient route to a-substituted quinolinemethanols.
* Correspondence to: Neil V Kirby, Dow AgroSciences LLC, 9330 Zionsville Road, Indianapolis, IN 46268-1054, USA
E-mail: nkirby@dowagro.com
(Received 19 October 2000; revised version received 18 May 2001; accepted 22 May 2001)
# 2001 Society of Chemical Industry. Pest Manag Sci 1526±498X/2001/$30.00
844

Synthesis and fungicidal activity of aryloxylepidines
Figure 1. Quin(az)oline products with fungicidal and insecticidal activity.
Reaction of aldehyde 9 Fig 3) with methylmagnesium
bromide in toluene solution gave the ethanol 11, while
reaction of aldehyde 13 Fig 4) with trimethyl
tri¯uoromethyl) silane and tetrabutylammonium
¯uoride in THF solution gave the a-tri¯uoromethyl
analogue 14.
50mmol),
bis triphenylphosphine)palladium II)
chloride 0.1g), and triethylamine 3ml) in ethanol
40ml) was sealed in a 200-ml stainless steel autoclave
and pressured to 1400kPa with carbon monoxide. The
reaction mixture was heated at 120°C for 12h, cooled
and vented. The reaction mixture was ®ltered through
The 4-hydroxymethyl quinolines were converted to
the target aryloxymethylquinolines, eg 15, by reaction
with a substituted phenol under Mitsunobu condi-
tions.9 Alternatively the hydroxymethylquinoline was
deprotonated with sodium hydride in THF solution
and alkylated with a halopyridine or other activated
heterocycle, eg 16 Fig 5).
2.1.1 ꢀ7-Chloroquinolin-4-yl)methanol ꢀFig 2, 6)
A
solution of 4-bromo-7-chloroquinoline 1.2g,
Figure 3. Preparation of (5,7-dichloroquinolin-4-yl)methanol and
derivatives.
Figure 2. Preparation of (7-chloroquinolin-4-yl)methanol.
Pest Manag Sci 57:844±851 2001)
845

NV Kirby et al
nitrile 1000ml) over three hours. The reaction
mixture was stirred at room temperature for 24h and
®ltered. The solid was suspended between water
1000ml) and dichloromethane 500ml) and basi®ed
with solid sodium hydrogen carbonate. The mixture
was extracted with dichloromethane 5Â500ml), the
organic extracts combined and dried over anhydrous
magnesium sulfate. The solution was ®ltered through
a plug of silica gel and concentrated under reduced
pressure to a volume of 1000ml. The mixture was
heated under re¯ux until the solid dissolved and
cooled to 0°C. Filtration gave the desired product 7 as
1
a white solid 60g, 85%). H NMR deuterochloro-
Figure 4. Preparation of a-substituted-(7-chloroquinolin-4-yl)methanol
derivatives.
form, dppm): 8.55 1H, d, J =6Hz), 8.05 1H, s),
7.78 1H, d, J =6Hz), 7.62 1H, s).
2.1.3 5,7-Dichloroquinoline-4-carboxaldehyde ꢀFig 3,
9)
Celite and concentrated under reduced pressure. The
residue was taken up in dichloromethane 50ml),
washed with water 50ml), and brine 50ml) and
dried over anhydrous sodium sulfate. Filtration and
evaporation of the solvent under reduced pressure
gave a tan-coloured oil, which was puri®ed by
chromatography over silica using acetonitrile‡di-
chloromethane as eluent to give a colourless syrup.
This was dissolved in methanol 50ml) and sodium
borohydride added over 2h until the reaction was
deemed complete using GC±MS. The reaction mix-
ture was diluted by water 50ml), acidi®ed with
hydrochloric acid 1M; 50ml), and then neutralised
with sodium hydrogen carbonate. The precipitate was
®ltered, dissolved in ethyl acetate 50ml), and dried
over anhydrous sodium sulfate. Filtration and eva-
poration of the solvent under reduced pressure gave
the desired product 6 as a cream-coloured solid
0.66g, 68%), mp: 160°C. 1H NMR deuterochloro-
form, dppm): 8.90 1H, d, J =4Hz), 8.10 2H, m),
7.65 1H, m), 7.60 1H, d, J =4Hz), 5.65 1H, t,
J =6Hz), 5.05 2H, d, J =6Hz).
A solution of 4-bromo-5,7-dichloroquinoline 7; 5.7g,
21mmol), palladium II) acetate 0.93g, 4.2mmol),
tri-o-tolylphosphine 2.5g, 8.2mmol), styrene 5ml,
26.2mmol), copper I) iodide 0.80g, 4.2mmol), and
triethylamine 4ml, 28mmol) in acetonitrile 41ml)
was heated at re¯ux for 3h. After cooling to ambient
temperature, the reaction was diluted with ethyl
acetate and ®ltered through a plug of silica gel. The
resulting solid was taken up in methanol‡dichloro-
methane solution 1‡1 by volume; 600ml) and
cooled to À78°C. Ozone was passed through the
system until the reaction was complete as determined
by GC±MS. Thiourea was added 5g, 600mmol) and
the mixture allowed to warm to ambient temperature,
®ltered through a plug of silica gel and rinsed through
with dichloromethane. The solvent was removed
under vacuum with slight heat until a white solid
precipitated. The solid was collected as several crops
to give the desired aldehyde 9 2.1g, 45% over two
steps), mp: 154°C. Analytical samples were obtained
by recrystallisation from ethyl acetate. 1H NMR
deuterochloroform, dppm): 11.10 1H, s), 9.15
1H, d, J =4Hz), 8.25 1H, d, J =3Hz), 8.15 1H,
d, J =3Hz), 7.65 1H, d, J =4Hz).
2.1.2 4-Bromo-5,7-dichloroquinoline ꢀFig 3, 7)
Bromine 15ml, 300mmol) was dissolved in aceto-
nitrile 200ml) and added dropwise to a suspension of
4-hydroxy-5,7-dichloroquinoline 60g, 280mmol)
and triphenyl phosphite 78ml, 300mmol) in aceto-
2.1.4 ꢀ5,7-Dichloroquinolin-4-yl)methanol ꢀFig 3, 10)
A solution of 4-bromo-5,7-dichloroquinoline 7; 5.7g,
21mmol), palladium II) acetate 0.93g, 4.2mmol),
tri-o-tolylphosphine 2.5g, 8.2mmol), styrene 5ml,
26.2mmol), copper I) iodide 0.80g, 4.2mmol), and
triethylamine 4ml, 28mmol) in acetonitrile 41ml)
was heated under re¯ux conditions for 3h. After
cooling to ambient temperature, the reaction mixture
was diluted with ethyl acetate 50ml) and ®ltered
through a plug of silica gel. The solvent was
evaporated under reduced pressure and the residual
solid dissolved in a mixture of methanol‡dichloro-
methane 1‡1 by volume; 600ml). The reaction
mixture was cooled to À78°C and ozone passed
through the solution until the reaction was determined
to be complete using GC±MS. Thiourea 5g, 60mol)
was added and the reaction allowed to warm to room
Figure 5. Preparation of lepidines.
846
Pest Manag Sci 57:844±851 2001)

Synthesis and fungicidal activity of aryloxylepidines
temperature. It was then ®ltered through a short silica
gel plug, eluting with methanol‡dichloromethane.
The solution was cooled to 0°C and sodium borohy-
dride 0.99g, 26mmol) added over 1h until the
reaction mixture was deemed complete using GC±
MS. The reaction mixture was quenched with hydro-
chloric acid 1M; 100ml), and extracted with hydro-
chloric acid 1M; 2Â100ml). The aqueous layers were
combined and neutralised with sodium hydrogen
carbonate. Filtration and recrystallisation of the solid
from ethyl acetate‡methanol gave the desired pro-
duct 10 as a white solid 2.8g, 58%), mp: 196°C. 1H
NMR deuterochloroform, dppm): 8.95 1H, d,
J =2Hz), 8.10 1H, d, J =4Hz), 7.90 1H, d,
J =4Hz), 7.85 1H, d, J =2Hz), 5.75 1H, m), 5.35
2H, m).
organic layers were dried over anhydrous magnesium
sulfate, ®ltered and concentrated to dryness. Puri®ca-
tion by ¯ash silica gel chromatography ethyl acet-
ate‡hexane) provided 14 1.32g, 96%) as a yellow
solid, mp: 161°C. 1H NMR deuterochloroform,
dppm): 8.96 d, 1H, J =4.5Hz), 8.15 d, 1H,
J =2.2Hz), 8.02 d, 1H, J =8.9Hz), 7.73 d, 1H,
J =4.5Hz), 7.58 dd, 1H, J =8.9, 2.2Hz), 5.83 m,
1H), 3.73 d, 1H, J =4.0Hz).
2.1.7 5,7-Dichloro-4-ꢀ4-fluorophenoxymethyl)quinoline
ꢀFig 5; 15)
A solution of 5,7-dichloroquinolin-4-yl)methanol
10; 1.37g, 6mmol), 4-¯uorophenol 0.78g, 7mmol),
and triphenylphosphine 3.14g, 12mmol) in dry THF
50ml) was stirred at room temperature and a solution
of diethyl azodicarboxylate 1.25g, 7mmol) in dry
THF 10ml) added slowly. The reaction mixture was
stirred at room temperature overnight, diluted with
ethyl acetate 50ml) and washed with hydrochloric
acid 1M; 50ml) and brine 50ml). The organic
solution was dried over anhydrous sodium sulfate,
®ltered and evaporated to dryness. Recrystallisation of
the solid from ethyl acetate‡hexane gave the desired
product 15 as a white solid 1.16g, 60%), mp: 105±
6°C. 1H NMR deuterochloroform, dppm): 9.00 1H,
d, J =4Hz), 8.15 1H, d, J =2Hz), 7.95 1H, d,
J =2Hz), 7.85 1H, d, J =4Hz), 7.15 4H, m), 5.85
2H, s).
2.1.5 1-ꢀ5,7-Dichloroquinolin-4-yl)ethanol ꢀFig 3; 11)
5,7-Dichloroquinoline-4-carboxaldehyde 9; 100mg,
0.44mmol) was dissolved in toluene 6ml) and cooled
to 0°C. Methylmagnesium bromide 1.4M in toluene/
tetrahydrofuran, 0.38ml, 0.53mmol) was dripped in
until the starting material was exhausted as judged by
TLC. The reaction was diluted with ethyl acetate
50ml) and hydrochloric acid 0.5M; 50ml) and
allowed to warm to ambient temperature. The organic
layer was additionally extracted thrice with hydro-
chloric acid 0.5M; 50ml). The aqueous layers were
combined and neutralised with sodium hydrogen
carbonate and extracted four times with ethyl acetate
50ml), the organic extracts were dried over
anhydrous magnesium sulfate, ®ltered and concen-
trated to give the desired product 11 101mg, 94%),
mp: 112°C. 1H NMR deuterochloroform, dppm):
8.95 1H, d, J =5Hz), 8.15 1H, d, J =2Hz), 8.00
1H, d, J =5Hz), 7.90 1H, d, J =2Hz), 6.25 1H,
m), 5.60 1H, d, J =4Hz), 1.40 1H, d, J =7Hz).
2.1.8 7-Chloro-4-ꢀ3-trifluoromethylpyridin-2-
yloxymethyl)quinoline ꢀFig 5; 16)
7-Chloroquinolin-4-yl)methanol 6; 0.6g, 3.12
mmol) was dissolved with stirring in dry THF
20ml) and 60% sodium hydride 0.15g, 3.75mmol)
added. The mixture was stirred at room temperature
for 1h and 2-chloro-3-tri¯uoromethylpyridine 0.62g,
3.42mmol) added. The reaction mixture was then
stirred overnight and quenched with water 50ml).
The mixture was extracted with ethyl acetate
2Â50ml), the combined organic extracts washed
with water and brine, and dried over anhydrous
sodium sulfate. Evaporation of the solvent under
reduced pressure and recrystallisation of the residual
solid from ethyl acetate‡hexane gave the desired
product 16 as a tan-coloured solid 0.90g, 84%), mp:
125±7°C. 1H NMR deuterochloroform, dppm): 8.92
1H, d, J =4Hz), 8.34 1H, s), 8.15 1H, d, J =2Hz),
8.00 1H, m), 7.94 1H, m), 7.61 1H, m), 7.56 1H,
dd, J =8Hz, 2Hz), 7.05 1H, m), 5.97 2H, s).
The materials prepared for this study are sum-
marised in Tables 1 and 2.
2.1.6 a-Trifluoromethyl-ꢀ7-chloroquinolin-4-
yl)methanol ꢀFig 4; 14)
7-Chloroquinoline-4-carboxaldehyde 13; 1.00g,
5.22mmol) was dissolved in a solution of trimethyl-
tri¯uoromethyl)silane in tetrahydrofuran 0.5M;
12.5ml) in an oven-dried, nitrogen-swept 100-ml
round-bottomed ¯ask, and the resulting solution was
cooled to 0°C under nitrogen. With stirring, tetra-n-
butylammonium
¯uoride
trihydrate
0.014g,
0.052mmol) was added as a solid and the mixture
allowed to warm slowly to room temperature over 2h
by which time thin-layer chromatography ethyl
acetate‡hexane) indicated complete consumption of
the starting material. The reaction mixture was cooled
to 0°C and 10% aqueous hydrochloric acid 3ml) was
added and the mixture stirred at room temperature
until cleavage of the trimethylsilyl ether was complete
as indicated by thin-layer chromatography 1h). The
reaction mixture was partitioned between saturated
sodium hydrogen carbonate and ethyl acetate, the
layers separated and the aqueous layer extracted with
an additional portion of ethyl acetate. The combined
2.2 Fungicidal activity
Plant materials wheat, Triticum aestivum L cv Monon;
rice, Oryza sativa L cv M-9) were grown in the
greenhouse from seed in a soil-less peat-based potting
mixture `Metromix'). The seedlings were used for
testing at the 1.5-leaf stage.
Compound formulation was accomplished by dis-
Pest Manag Sci 57:844±851 2001)
847

NV Kirby et al
Table 1. Aryloxylepidines analogues prepared in this study
Table 2. Heteroaryloxylepidine analogues prepared in this study
Compound
R1
R2
R3
Mp ꢀ°C)
Compound
R1
R2
R3
X
Mp ꢀ°C)
17
18
19
20
15
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
H
H
H
4-F
4-F
4-F
4-F
4-F
4-F
4-F
4-F
4-F
4-F
4-F
111±3
133±5
104±6
38±40
133±4
105±6
102±3
118±23
186±90
144±5
124±5
16
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
H
H
7-Cl
7-Cl
H
5-Cl
CÐCF3 125±7
7-Cl
C-F
CH
CH
CH
152±3
99±101
110±2
61±3
CH3
C6H13
H
7-Cl
H
7-Cl
4-CF3
5-CF3
6-CF3
H
7-Cl
H
7-Cl
5,7-Cl2
5,7-Cl2
5,7-Cl2
5,7-Cl2
H
7-Cl
H
5,7-Cl2
5,7-Cl2
5,7-Cl2
7-Cl
CÐCF3 176±8
CH3
C2H5
CN
H
4-CF3
6-CF3
5-F
CH
CH
N
135±6
108±10
150±2
81±3
H
CH=CH2 5,7-Cl2
H
H
H
H
H
H
H
H
H
H
H
H
H
8-Cl
8-F
H
CH3
CF3
H
7-Cl
5-F
N
7-Cl
5-F
N
91±2
2-Cl, 4-F 126±7
2-Cl, 4-F 116±7
5,7-Cl2
5-F
N
192±3
97±8
7-Cl
CH3 5,7-Cl2
5-F
N
5,7-Cl2 2-Cl, 4-F 142±3
H
H
H
H
7-Cl
5,7-Cl2
7-Cl
5-OCH3
5-OCH3
4,6-ꢀCH3)2
N
180±2
211±3
145±7
173±5
8-Cl
8-F
H
2-Cl, 4-F 132±5
2-Cl, 4-F 118±22
4-CꢀCH3)3 94±5
N
N
5,7-Cl2 4,6-ꢀCH3)2
N
7-Cl 4-CꢀCH3)3 105±6
5,7-Cl2 4-CꢀCH3)3 136±7
8-Cl 4-CꢀCH3)3 107±9
8-F 4-CꢀCH3)3 78±81
Test inoculum for rice blast Magnaporthe grisea
Hebert) Barr) was produced on potato dextrose agar
at 24°C under ¯uorescent lights. The conidia were
suspended in water and ®ltered through cheese cloth.
Tween 20 10 drops litreÀ1) was added and the test
plants were then inoculated by spraying to run off with
this conidia suspension at a concentration of 1Â106
solving technical materials in acetone, with serial
dilutions then made in acetone to obtain desired rates.
Final treatment volumes were obtained by adding nine
volumes of aqueous Triton X-100 0.11g litreÀ1),
resulting in solutions of 100ml litreÀ1 acetone and
0.1g litreÀ1 Triton X-100. Test rates were 400, 100,
conidia mlÀ1
.
Wheat and rice plants for glume blotch and rice
blast disease development were placed in a dew
chamber for 18h at 21°C and then moved to a
greenhouse for symptom development. Plants inocu-
lated with E graminis were maintained in the green-
house continuously. Symptoms developed in 7±10
days for all diseases on control plants. Disease
incidence on the leaves was assessed visually as the
percentage of leaf showing disease symptoms. Data
were expressed as percentage disease control on the
compound-treated plants relative to the non-fungi-
cide-treated controls.
25 and 6.25mg mlÀ1
.
In a high-volume foliar application, plants were
sprayed to run-off using two opposing Spraying
Systems 1/4JAUPM air atomisation nozzles operating
at approximately 138kPa).
Test inoculum for wheat powdery mildew Erysiphe
graminis DC f sp tritici Marchal) was produced in vivo
on stock plants in the greenhouse. The test plants were
inoculated by dusting spores from stock plants on test
plants 24h after spray application.
Test inoculum for glume blotch of wheat Stagono-
spora nodorum) was produced by culturing S nodorum
on potato dextrose agar at 18°C under long-wave UV
light for seven days to produce pycnidia. Pycnidio-
spores were suspended in water and ®ltered through
cheese cloth. Tween 20 30 drops litreÀ1) was added
and the test plants were then inoculated by spraying to
run-off with this inoculum suspension at a concentra-
2.3 Effects on mitochondrial activity
2.3.1 Isolation of submitochondrial particles from
Ustilago maydis
A liquid culture of Ustilago maydis DC Corda with a
turbidity of c 1.0 Abs absorbance units) at 450nm was
diluted 1:100 in potato dextrose broth Difco) and was
incubated at 30°C, 250rev minÀ1 for 18h. U maydis
sporidia were harvested by centrifugation at 3000g.
tion of 8Â106 pycnidiospores mlÀ1
.
848
Pest Manag Sci 57:844±851 2001)

Synthesis and fungicidal activity of aryloxylepidines
Sporidia were washed by suspension in Tris-HCl
buffer, 50mM; pH 7.5) and recentrifuged. Cells were
then resuspended in the above buffer ice cold) to form
a thick slurry and were broken with an ice-chilled
Bead-Beater2 containing 0.5-mm glass beads 1:1
ratio of glass beads to cells) for four 15-s cycles with
45-s cooling intervals. The homogenate was centri-
fuged at 3000g for 10min at 4°C. The supernatant
was then centrifuged at 100000g for 45min at 2°C.
The resulting pellets containing microsomes and
submitochondrial particles were homogenised in
assays were conducted in 96-well microtitre plates
with 100ml of assay buffer [Tris-HCl 30mM), sucrose
15mM), KCl 5mM), K2HPO4 1mM), MgCl2
0.5mM), Na2EDTA 0.2mM), pH 7.4] with or
without for background) 0.8mM NADH dipotas-
sium salt) and 100ml submitochondrial particle solu-
tion per well. The plates were read in
a
SPECTRAmax2 250 Molecular Devices, Sunnyvale,
CA) spectrophotometric plate reader at 340nm at
30°C. The concentrated submitochondrial particle
solutions were diluted in assay buffer without NADH
to give a rate, when combined 1:1 with assay buffer
with NADH, of approximately À30mAbs minÀ1 over
a period of 5min. I50 assays were done in duplicate
using half-logarithmic dilutions of each compound.
Tris-HCl
50mM;
pH
7.5)‡Triton
X-100
1.0g litreÀ1).
2.3.2 Assay of mitochondrial electron transport activity
Mitochondrial electron transport MET) activity was
monitored spectrophotometrically from the consump-
‡
tion of NADH conversion to NAD ) in submito-
chondrial membrane particles by measuring the
change in absorbance at 340nm over time. MET
3 RESULTS AND DISCUSSION
Excellent activity against E graminis was found for a
number of compounds Table 3) but activity against
E graminis
400mg mlÀ1 25mg mlÀ1
Snodorum
M grisea
Compound
400mg mlÀ1
25mg mlÀ1
400mg mlÀ1
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
100
100
97
100
100
0
0
0
0
0
0
75
0
90
0
0
100
100
90
97
100
0
0
0
75
40
75
75
0
0
0
20
0
0
100
90
100
20
100
100
0
60
20
0
60
0
100
100
0
50
0
0
75
0
0
75
0
50
0
0
0
0
0
0
0
100
90
97
75
0
75
0
0
75
0
0
0
0
0
0
0
0
75
0
75
0
0
0
0
50
20
60
0
75
0
50
0
40
0
0
0
0
0
0
0
90
40
100
50
97
100
20
100
75
100
97
97
100
100
97
75
97
0
60
0
60
0
75
0
100
100
100
100
20
0
0
0
40
50
60
75
90
0
0
0
0
75
0
60
0
100
97
50
40
60
40
90
50
50
40
0
90
0
100
100
97
90
75
0
0
0
100
100
100
97
0
0
0
0
0
90
0
0
100
0
0
50
0
Table 3. Fungicidal activity of the compounds
in this study (expressed as % disease control)
90
75
Pest Manag Sci 57:844±851 2001)
849

NV Kirby et al
Table 4. Biochemical activity for selected lepidines prepared in
this study
Small substituents on the aryl ring were tolerated, but
the larger lipophilic tert-butyl group substantially
reduced the fungicidal activity of these compounds
32±36). Within the series of pyridyl ethers 16, 37±
43), mildew activity was high and appeared to be
tolerant of the position of substitution with the notable
exception of 41, for which no explanation could be
formulated. Several of these compounds had activity
against S nodorum or M grisea, but this did not appear
to conform to a discernible pattern. The activity of the
7-chloroquinoline and 5,7-dichloroquinoline com-
pounds was generally very similar, but the 4,6-
dimethylpyrimidine analogues 51 and 52 departed
from this rule; the former compound being mildew
active, while the latter is devoid of activity against this
disease yet active against S nodorum.
One further area of substitution examined in this
study was the carbon atom alpha to the ether oxygen
atom. This was a position that had received little
attention in earlier studies or was impossible to
achieve with the phenoxyquinolines). Substitution
with a methyl group did not signi®cantly impact the
activity, but a larger alkyl group even an ethyl group)
was deleterious to the activity. That this appeared to
be a size constraint was reinforced by the activity of the
alpha-cyano and tri¯uoromethyl analogues 23 and 46.
These trends of activity with quinoline substitution
paralleled those seen in the SAR of previous series.
The SAR of phenoxyquinoline fungicides highlighted
7-chloro and 5,7-dichloro-quinoline analogues as
showing excellent protectant mildew activity. The
primary mode of action of quinoxyfen and related
materials has not been reported. In contrast, the mode
of action of phenethoxyquin az)olines, eg fenazaquin,
has been reported as inhibition of MET complex I,10
and this was tested for a number of the analogues
prepared in this study Table 4). 8-Fluoroquinolines
26, 31, 36 are better MET inhibitors than the
corresponding 7-chloro compounds 18, 28, 33 and
this may explain the broader spectrum of antifungal
activity seen with these compounds. However, even
the best example of a MET inhibitor in this series, 26,
is almost an order of magnitude less active than a
`known' MET active fenazaquin), which is only
moderately active as a fungicide.11 Accordingly, it
would appear that the aryloxylepidines contain ele-
ments of both of these earlier SARs, but optimisation
of both activities within one molecule would appear
impossible given the contradictions in requirements
for optimal substitution on the quinoline ring.
Compound
R1
R2
Iꢀ%) a I50 ꢀmmol) b
18
15
25
26
7-Cl
5,7-Cl2
8-Cl
8-F
4-F
4-F
4-F
4-F
32
33
Ð
1.0
0.73
10
27
H
2-Cl, 4-F 100
28
29
30
7-Cl 2-Cl, 4-F
5,7-Cl2 2-Cl, 4-F
8-Cl 2-Cl, 4-F
84
170
93
31
32
8-F
H
2-Cl, 4-F
1.6
4-CꢀCH3)3 77
33
34
35
7-Cl 4-CꢀCH3)3
5,7-Cl2 4-CꢀCH3)3
8-Cl 4-CꢀCH3)3
8-F 4-CꢀCH3)3
46
22
6.1
36
Fenazaquin
3.2
0.13
a Percentage inhibition of MET complex I from Ustilago maydis
at a concentration of 10mg mlÀ1
.
b I50 against MET complex I derived from Ustilago maydis.
other diseases was limited and failed to conform to a
readily discernible pattern. No single compound was
identi®ed that gave >50% control of all three diseases
at all rates tested. The compounds that came closest to
achieving this goal 36, 43) were insuf®ciently active
against one or more disease to be of further interest.
Substitution on the quinoline ring was examined
with a number of substitutions on the aryl ether
portion to determine trends within this series. Com-
pounds that were unsubstituted in the quinoline ring
17, 27, 32) had low levels of activity, particularly at
the lower rate. Introduction of a halogen atom on the
quinoline ring gave molecules with more diverse
activities. 8-Chloroquinolines 25, 30, 35) proved
completely inactive under our testing protocols, but
replacement of the chloro-substituent with a ¯uorine
atom 26, 31, 36) gave molecules with moderate
activity across the three diseases. Greatest fungicidal
activity, especially against E graminis, was found with
7-chloroquinoline eg 18±20), or 5,7-dichloroquino-
line 15, 21±24) analogues. Many of these molecules
gave >90% control of this disease at the lower rate, but
this increase in mildew activity was not re¯ected in
better disease control of the other diseases in this
study.
4
CONCLUSIONS
Lepidine analogues represent a further example of
fungicidal compounds based on the 4-substituted
quinoline template, with the compounds prepared in
this study showing activity against a range of patho-
gens. Examination of the in vivo and in vitro enzyme
data indicates that there are at least two classes of
materialsÐmoderately active inhibitors of mitochon-
Substitution on the hetero)aryl ether portion of the
molecule presented a more confusing SAR picture.
850
Pest Manag Sci 57:844±851 2001)

Synthesis and fungicidal activity of aryloxylepidines
Landbouwkd Toegepaste Biol Wet ꢀUniv Gent) 58 2b):485±90
1993).
drial electron transport complex I with broad-spec-
trum activity, and highly active powdery mildewicides
whose mode of action is unresolved. Attempts to
amalgamate these trends into a single molecule would
appear to be futile as the SAR trends for the two
activities lead in different directions.
5 Ishikawa K, Yoshikawa Y, Ishii T, Tanikawa H, Maeda S,
Kawashima H, Yanase Y, Shimotori
H and Mita R,
Pyrimidinyloxy thio)quinoline derivatives, preparation process
of the derivative, and agri-horticultural fungicide comprising
the derivative as active ingredient, European patent application
EP 497 371 1992).
6 Daeuble J, Davis LN, Hellwig K, Kirby N, Parker MH, Pieczko
M and Thomason LK, 4-Substituted quinoline derivatives
having fungicidal activity, US patent 6 117 884 2000).
7 Campbell KN and Shaffner IJ, Preparation of 4-methylquino-
lines. J Am Chem Soc 67:86 1945).
REFERENCES
1 Dreikorn BA, Edie RG, Hackler RE, Jourdan GP, Krumkalns
EV and Suhr RG, Nematocidal quinoline and quinazoline
derivatives, PCT patent application, WO 9 304 583 1993).
2 Yap MCH, Dreikorn BA, Davis LN, Suhr RG, Kaster SV, Kirby
NV, Paterson G, Graupner PR and Erickson WR, 4-
Arylalkoxyquinazolines with antifungal activity, in Synthesis
and Chemistry of Agrochemicals V, ed by Baker DR, Fenyes JG,
Basarab GS and Hunt DA, American Chemical Society,
Washington, DC, pp 258±272 1998).
8 Black DK, Landor SR, Patel AN and Whiter PF, Allenes. Part
XVI. The preparation of allenic and acetylenic bromides by the
triphenylphosphite dibromide method. J Chem Soc ꢀC), p 2260
1967).
9 Mitsunobu O, The use of diethyl azodicarboxylate and triphe-
nylphosphine in synthesis and transformation of natural
products. Synthesis 13:1±28 1981).
10 Hollingworth RM, Ahammadsahib KI, Gadelhak
G and
3 Longhurst C, Dixon K, Mayr A, Bernhard U, Prince K, Sellars J,
Prove P, Richard C, Arnold W et al, DE-795Ða novel
fungicide for the control of powdery mildew in cereals, in Proc
Brighton Crop Prot ConfÐPests Dis, BCPC, Farnham, Surrey,
UK, pp 27±32 1996).
McLaughlin JL, New inhibitors of complex I of the mitochon-
drial electron transport chain with activity as pesticides.
Biochem Soc Trans 22:230±233 1994).
11 Dreikorn BA, Suhr RG, Jourdan GP and Wright IG, Preparation
and testing of quinazoline derivatives as agrochemical fungi-
cides, European patent application, EP 326 329 1989).
4 Dutton R, Leonard P and Brow KC, FenazaquinÐa new,
selective acaricide for use in fruit crops. Meded
± Fac
Pest Manag Sci 57:844±851 2001)
851