Synthesis and fungicidal activity of tubulin polymerisation promoters. Part 3: Imidazoles

Article abstract of DOI:10.1016/j.bmc.2012.10.052

A novel class of experimental fungicides has been discovered, which consists of special tetrasubstituted imidazoles. They are highly active against important phytopathogens, such as Botrytis cinerea (grey mould), Uncinula necator (grape powdery mildew), M

Full text of DOI:10.1016/j.bmc.2012.10.052

Bioorganic & Medicinal Chemistry 21 (2013) 127–134
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
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Synthesis and fungicidal activity of tubulin polymerisation promoters. Part 3:
Imidazoles
Clemens Lamberth a, , Raphael Dumeunier a, Stephan Trah a, Sebastian Wendeborn a, Jeremy Godwin a,
⇑
Peter Schneiter a, Andy Corran b
a Syngenta Crop Protection AG, Crop Protection Research, Schaffhauserstr. 101, CH-4332 Stein, Switzerland
b Syngenta Ltd, Jealott’s Hill International Research Centre, Bracknell, Berkshire RG42 6EY, United Kingdom
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 25 July 2012
Revised 29 October 2012
Accepted 31 October 2012
Available online 15 November 2012
A novel class of experimental fungicides has been discovered, which consists of special tetrasubstituted
imidazoles. They are highly active against important phytopathogens, such as Botrytis cinerea (grey
mould), Uncinula necator (grape powdery mildew), Mycosphaerella graminicola (wheat leaf blotch) and
Alternaria solani (potato and tomato early blight). Their fungicidal efficacy is due to their ability to pro-
mote fungal tubulin polymerization, which leads to a disruption of microtubule dynamics. These imida-
zoles are five-membered ring analogs of similar substituted triazolopyrimidines and pyridazines with the
same mode of action. A concise four-step synthesis route has been used to prepare them from commer-
cially available starting materials.
Keywords:
Tubulin
Imidazole
Heterocycle
Fungicide
Ó 2012 Elsevier Ltd. All rights reserved.
Crop protection
1. Introduction
either by another five-membered ring (as in 2) or by a six-mem-
bered heterocycle (as in 3 and 4). More recently monocyclic scaf-
The tubulin polymerization promoters undoubtedly belong,
together with the Succinate dehydrogenase inhibitors (SDHI’s),2,3
to those two fungicide classes, which attracted the most attention
in the research departments of the agrochemical industry during
the first decade of this century. The interest in a certain class of ac-
tive ingredients is easily measurable by the number of published
patents. Regarding the tubulin polymerization promoters, 303 pat-
ents have been published since 2000 by nine different companies.
In contrast to the SDHI’s, of which several examples, such as bixa-
fen,4 boscalid,5 fluopyram,6 fluxapyroxad,7 furametpyr,8 isopyra-
zam,9 penflufen,3 penthiopyrad10 and sedaxane2 have reached
the fungicide market recently, no tubulin polymerization promoter
has been commercialized so far. Although the members of this
class of experimental fungicides bear one of several different
mono- and biheterocyclic scaffolds, they all possess a similar
substitution pattern and the same mode of action, which leads to
the disruption of the microtubule dynamics. [1,2,4]Triazolo
[1,5-a]pyrimidines, such as BAS600F (1) have been the lead
structures for the tubulin polymerization promoters (Fig. 1).11
The pyrazolopyrimidine 2,12 the pyrido[2,3-b]pyrazine 313 and
the pyrido[3,2-e][1,2,4]triazine 414 are further examples, in which
the triazole ring of the triazolopyrimidines has been replaced
folds, such as the pyridine 5,15 the pyridazine 6,1 the pyrimidine
716 and the pyrazinone 8,17 have been in the focus of the research
activities. Obviously a closely related substitution pattern is com-
mon to those monocyclic, 5,6-bicyclic and 6,6-bicyclic promoters
of tubulin polymerization. Adjacent to a ring nitrogen atom they
all carry a halogen substituent, next to it there is a 2,4,6-trifluor-
ophenyl ring, which is followed by either an amine or an aryl/het-
eroaryl group. In the case of the heterobicyclic tubulin promoting
fungicides, the second ring, which is annelated between the amino
substituent and a ring nitrogen of the fully substituted ring, is gen-
erally unsubstituted, whereas all monocyclic compounds, with the
exception of the pyridine 5, are usually fully substituted.
2. Results and discussion
2.1. Chemistry
Inspired by the structural simplicity of the monocyclic tubulin
polymerization promoters and impressed by the high fungicidal
efficacy of the pyridazine 6, we decided to design five-membered
analogs of it. We planned to remove one of the two adjacent pyr-
idazine ring nitrogen atoms and to replace one of those two ring
carbon atoms of 6, which bear the aryl or heteroaryl substituents,
by a nitrogen atom, thus arriving by the imidazole 9 (Scheme 1).
Similar tetrasubstituted imidazoles with two different aryl
⇑
Corresponding author. Tel.: +41 62 8660224; fax: +41 62 8660860.
E-mail address: clemens.lamberth@syngenta.com (C. Lamberth).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.10.052

128
C. Lamberth et al. / Bioorg. Med. Chem. 21 (2013) 127–134
F
F
F
F
F
F
F
F
F
F
F
NH
NH
N
N
N
N
N
N
N
N
N
N
F
N
F
F
F
N
F
Cl
N
F
N
N
Cl
N
4
2
3
1
BAS600F
Cl
F
O
F
O
F
F
N
N
F
F
F
F
F
F
F
NH
O
N
N
F
F
F
F
N
Cl
N
Cl
N
Cl
N
N
Cl
N
N
5
6
7
8
Figure 1. Different mono- and biheterocyclic tubulin polymerization promoters.
Cl
compound was the imidazole 10, which is rather mimicking the
amino-substituted triazolopyrimidine BAS600F (1). Such fully
substituted imidazoles bearing alkyl, amino, aryl and halogen sub-
stituents have been completely unknown, therefore we had to de-
velop a novel synthesis route.
Cl
N
N
F
F
F
O
N
F
N
F
We chose to construct the heterocyclic scaffold of the first tar-
N
Cl
get compound 9 via a van Leusen imidazole synthesis.20–22 There-
Cl
6
9
fore
we
converted
4-chloroaniline
(11)
and
2,4,6-
trifluorobenzaldehyde (12) to the Schiff base 13. Its cyclisation
with the aid of TosMIC resulted in the diarylated imidazole 14,
which could be regioselectively brominated at the carbon atom be-
tween the two ring nitrogens. The exchange of the bromo substitu-
ent in 15 by a methyl group and the subsequent chlorination of
position 5 led to the fully substituted imidazole 17. Finally the
replacement of the fluoro atom in the para-phenyl position by a
F
F
F
O
N
N
N
N
N
F
N
F
N
N
Cl
Cl
methoxy function delivered the desired target compound
9
1
10
(Scheme 2).23
BAS600F
Because of the length of this six-step synthesis, we tried to facil-
itate the preparation of 9 in a much more elegant and efficient
fashion, in analogy to a method which had been recently described
by De Borggraeve et al.25 In this regard, we started from the same
starting materials 11 and 12, but this time converted them to-
gether with potassium cyanide in an indium(III) chloride catalyzed
one-pot three-component Strecker reaction into 18 (Scheme 3).24
Scheme 1. Imidazoles as five-membered ring mimics of fungicidally active
triazolopyrimidines and pyridazines.
substituents, a chloro and a methyl group have been evaluated as
cyclooxygenase-2 (COX-2) inhibitors.18,19 Another attractive target
Cl
F
F
F
F
O
F
F
Dean-
Stark
TosMIC,
K2CO3
NH2
N
N
H
+
F
100 %
52 %
N
F
Cl
F
Cl
14
12
11
13
NBS 61 %
Cl
Cl
N
Cl
Cl
F
F
F
F
F
O
F
F
AlMe3
N
N
N
Br
Pd(PPh3)4
NCS
57 %
NaOMe
74 %
N
F
N
N
F
N
F
F
62 %
Cl
Cl
16
15
17
9
Scheme 2. TosMIC-based (van Leusen-type) synthesis of the imidazole 9.

C. Lamberth et al. / Bioorg. Med. Chem. 21 (2013) 127–134
129
F
F
bears four completely different substituents, an alkyl group, an aryl
ring, an amine and a halogen atom.26
O
F
F
InCl3,
KCN
H
N
NH2
H
+
F
100 %
2.2. Structure–activity relationships
Cl
Cl
_
F
N
11
12
18
Quite soon we found out, that the diarylated imidazole 9 pos-
sesses a much higher fungicidal activity than the amino-substi-
+
tuted imidazole 10. Therefore we will focus on
9 and its
67 %
Cl Me2N=CHCl
derivatives in the forthcoming analysis of the structure–activity
relationship of the novel imidazole fungicides. Each of the four dif-
ferent ring positions of such imidazoles bear a specific substituent,
which exerts a distinct influence on the fungicidal efficacy. As it
was clear from the beginning, that the chlorine atom in position
4 is very important, we concentrated our efforts regarding the opti-
mization of the biological efficacy on the variation of the substitu-
ents in ring positions 1, 2 and 5.
Cl
Cl
Cl
F
F
F
iPr2NLi,
MeI
F
O
O
NaOMe
97 %
N
N
N
66 %
N
F
N
F
N
F
Cl
Cl
Cl
19
20
9
2.2.1. Influence of the substituent in imidazole position 1 on the
fungicidal activity
Scheme 3. Vilsmeier reagent-based synthesis of the imidazole 9.
In analogy to the structurally related pyridazine fungicides,1
a
4-chlorophenyl group between the 2,6-dihalophenyl and methyl
substituents seems to deliver the best broad-spectrum fungicidal
activity (Table 1, entry 1, 9). Moving this halogen substituent from
the para position to the meta position of the phenyl ring (entry 2)
or replacing it by an electron-donating methoxy group (entry 3) re-
duces the potency, especially against Mycosphaerella graminicola
and Alternaria solani. Only the exchange of the phenyl ring in imid-
azole position 1 by a pyridine under preservation of the position of
the chloro substituent (entry 4) leads to a equipotent efficacy, at
least in the control of Botrytis cinerea and A. solani. The replacement
of the N-phenyl to a N-benzylimidazole (entry 5) results in consid-
erably weaker fungicidal activity against all three important
phytopathogens.
This a-aminonitrile is then ring-closed with Vilsmeier reagent and
hydrochloric acid to the trisubstituted imidazole 19, which has
besides two aryl groups already the required chlorine atom in
place. The replacement of the 4-fluoro phenyl substituent in 19
by methoxy and the introduction of the methyl function by direct
lithiation of the imidazole and methylation deliver the fungicidally
active 9 in only four steps, compared to the six steps required by
the TosMIC approach.
A completely different synthesis pathway had to be designed
for the preparation of the target compound 10, intended to be a
five-membered monocyclic mimic of the triazolopyrimidine
BAS600F (1). The route starts from the commercially available
glycine amide 21, which is converted with 2,4,6-trifluorophenylis-
othiocyanate into the thiourea 22 (Scheme 4). Its alkylation to the
S-methylisothiourea 23 and subsequent ring-closure reaction with
Lawesson’s reagent delivers 24. This trisubstituted imidazole is
transformed by bromination of the remaining free imidazole ring
position and subsequent palladium-catalyzed bromine-methyl
exchange to the fully substituted imidazole 26. The next two steps
are the introduction of a methoxy function into the para-position
of the phenyl ring and the removal of the methylsulfanyl function
in ring position 2 with the aid of Raney-nickel, delivering 28.
Finally, the chlorination of the ring carbon atom between the
two heteroatoms yields the tetrasubstituted imidazole 10, which
2.2.2. Influence of the substituent in imidazole position 2 on the
fungicidal activity
As it is obvious from the synthesis routes described in Schemes
1 and 2, it is easily possible to vary the substituent at the ring car-
bon between the two imidazole nitrogen atoms broadly. A hydro-
gen atom (Table 2, entry 1) in this position leads to only weak
fungicidal activity, pointing out to the need of a substituted carbon
atom in imidazole position 2. It was possible to introduce three dif-
ferent halogen atoms into this position, a fluorine (entry 2), a chlo-
rine (entry 3) and a bromine atom (entry 4). From these halogens,
O
O
F
O
F
F
K2CO3,
MeI
H
N
H
N
H
N
Lawesson's
reagent
F
N
NH2
N
F
2,4,6-F3-PhNCS
69 %
N
N
N
S
S
N
39 %
96 %
F
F
F
N
F
S
22
21
23
24
NBS 60 %
F
F
F
F
F
F
O
F
O
O
Cs2CO3, Pd(dppf)Cl2,
trimethylboroxine
N
N
N
N
N
NCS
19 %
Raney-Ni
33 %
NaOMe
40 %
Br
N
N
N
N
N
Cl
42 %
N
F
N
N
F
F
N
N
F
F
S
S
S
26
28
25
27
10
Scheme 4. Synthesis of the imidazole 10.

130
C. Lamberth et al. / Bioorg. Med. Chem. 21 (2013) 127–134
Table 1
Table 3
Influence of the substituent R in imidazole position 1 on the fungicidal activitya
Influence of the substituent R in imidazole position 5 on the fungicidal activitya
O
Cl
F
R
N
N
F
Cl
N
R
N
Entry
R
Botrytis
cinerea
(tomato
grey
Uncinula
necator
(grape
powdery
mildew)
Mycosphaerella
graminicola
(wheat leaf
blotch)
Alternaria
solani
(tomato
early
Cl
Entry
R
Botrytis
cinerea
(tomato
grey
Uncinula
necator
(grape
powdery
mildew)
Mycosphaerella
graminicola
(wheat leaf
blotch)
Alternaria
solani
(tomato
early
mould)
blight)
Cl
mould)
blight)
1 (9)
2
1
1
1
2
2
20
63
1
4
F
F
F
F
1 (9)
8
2
2
1
6
2
15
1
F
F
F
F
2
3
OMe
OEt
OMe
Cl
2
17
12
3
4
5
32
72
82
1
16
11
67
61
65
4
35
37
N
4
5
2
3
6
1
OMe
F
F
F
Cl
41
16
76
59
OMe
a
Results are given as the EC80 (mg Lꢀ1).
Results are given as the EC80 (mg Lꢀ1).
a
Table 2
Influence of the substituent R in imidazole position 2 on the fungicidal activitya
2.2.3. Influence of the substituent in imidazole position 5 on the
fungicidal activity
Cl
The 2,4,6-trifluorophenyl ring, which is present in the lead
structures 1 to 8 (Fig. 1), delivers also in combination with the
imidazole scaffold strong fungicidal activity (Table 3, entry 1).
However, this substituent in imidazole position 5 is clearly outper-
formed by a 2,6-difluoro4-methoxy group, which gives the best re-
sults against the tested plant pathogens (entry 2). We tried to
further improve the fungicidal activity of this substituent, but sev-
eral different manipulations in the phenyl ring, for example, the
replacement of the para-methoxy function by an ethoxy group (en-
try 3), the exchange of one of the ortho-fluoro atoms by hydrogen
(entry 4) and the methoxy function changing places with one of
the fluorine atoms (entry 5) did not lead to any improvement.
F
F
N
R
N
F
Cl
entry
R
Botrytis
cinerea
(tomato
grey
Uncinula
necator
(grape
Mycosphaerella
graminicola
(wheat leaf
blotch)
Alternaria
solani
(tomato
early
powdery
mildew)
mould)
blight)
1
2
3
4
5
H
F
Cl
Br
33
180
54
45
(17)
59
78
1
67
65
44
83
8
>200
98
57
43
CH3
144
2
0.25
0.20
15
6
6
OCH3 173
65
42
71
0.15
0.10
0.05
0.00
Solvent control
Paclitaxel
9
17
a
Results are given as the EC80 (mg Lꢀ1).
the chloro function seems to have the highest fungicidal activity,
especially against Uncinula necator. Finally, we also checked the po-
tential of a methyl (entry 5, 17) and a methoxy group (entry 6). The
methyl substituent shows by far the best broad-spectrum activity
of all substituents tested in this position, as it is also the case in
the structurally related pyridazine fungicides, whereas the meth-
oxy function gave rather poor results.
0
100 200 300 400 500 600 700
Time, sec
Figure 2. Degree of polymerization of pure porcine tubulin in the presence of
paclitaxel and imidazoles 9 and 17 at 1.25 l
g mlꢀ1 in function of time (values
presented are a mean of two replicates).

C. Lamberth et al. / Bioorg. Med. Chem. 21 (2013) 127–134
131
2.3. Mode of action
(4-chloro-phenyl)-[1-(2,4,6-trifluoro-phenyl)-meth-(E)-yli-
dene]amine (13, 43.8 g, 0.16 mol, 100%). 1H NMR (CDCl3): d = 6.77
(t, 2H, J = 8.7), 7.08 (d, 2H, J = 8.6), 7.28 (d, 2H, J = 8.65), 8.49 (s, 1H).
LC–MS: tR = 5.99 min, m/z = 270 [M+1].
The highly active imidazole fungicides 9 and 17 (see Tables 1
and 2) were submitted to a polymerisation assay on pure porcine
tubulin to check if also these five-membered monocyclic com-
pounds, as already confirmed for the mentioned bicyclic pyrido-
pyrazine 313 and six-membered monocyclic pyridazine 6,1 are
disrupters of the microtubule dynamics. They were compared
against paclitaxel, which is a known tubulin polymerisation pro-
moter and for which a clear increase in the OD340 value could be
4.1.2. 1-(4-Chloro-phenyl)-5-(2,4,6-trifluoro-phenyl)-1H-
imidazole (14)
Toluenesulfonylmethyl isocyanide (TosMIC, 36.5 g, 0.18 mol)
and anhydrous potassium carbonate (34.4 g, 0.25 mol) are added
to
a solution of (4-chloro-phenyl)-[1-(2,4,6-trifluoro-phenyl)-
detected at 1.25
l
g mlꢀ1. A very similar pattern was observed in
meth-(E)-ylidene]amine (13, 33.6 g, 0.12 mol) in a mixture of
460 ml of N,N-dimethylformamide and 375 ml of 1,2-dimethoxy-
ethane. The reaction mixture is heated for 2 h at 100 °C, then
cooled to room temperature and filtered. The filtrate is evaporated,
the residue is absorbed on IsoluteÒ HM-N and purified by chroma-
tography on silica gel, using a mixture of heptane/t-butyl methyl
ether 3:1 as eluent, to obtain 1-(4-chloro-phenyl)-5-(2,4,6-tri-
fluoro-phenyl)-1H-imidazole (14, 19.9 g, 65 mmol, 52%). 1H NMR
(CDCl3): d = 6.59 (t, 2H, J = 7.3), 7.02 (d, 2H, J = 8.7), 7.22 (s, 1H),
7.28 (d, 2H, J = 8.6), 7.72 (s, 1H). LC–MS: tR = 1.80 min, m/z = 309
[M+1].
two different replicates for 9 and 17, indicating a similar effect
on tubulin polymerization (Fig. 2).
3. Conclusions
Novel tetrasubstituted imidazoles have been discovered as a
new class of fungicidally active compounds, which are able to con-
trol economically important plant diseases from the family of
Ascomycetes, such as B. cinerea, U. necator, M. graminicola and
A. solani. The most active compounds possess a similar substitution
pattern as related six-membered pyridazine fungicides. Their
mode of action is the promotion of tubulin polymerization, leading
to the disruption of microtubule dynamics. These fully substituted
imidazoles can be prepared in only four steps, the key step is the
4.1.3. 2-Bromo-1-(4-chloro-phenyl)-5-(2,4,6-trifluoro-phenyl)-
1H-imidazole (15)
A
mixture of 1-(4-chloro-phenyl)-5-(2,4,6-trifluoro-phenyl)-
1H-imidazole (14, 4.0 g, 13 mmol) and N-bromosuccinimide
(3.5 g, 20 mmol) in 40 ml of chloroform is heated for 1 h to 80 °C.
Subsequently the mixture is cooled to room temperature, then Iso-
luteÒ HM-N is added and the solvent is removed under reduced
pressure. The residue is purified by column chromatography on sil-
ica gel, using a mixture of heptane/ethyl acetate 4:1 as eluent, to
condensation of an
1,5-disubstituted-4-haloimidazole.
a-aminonitrile with Vilsmeier reagent to a
A structure–activity relationship study revealed the molecular
requirements for the best fungicidal activity. As it turned out, each
ring carbon of the imidazole scaffold as well as the substituted
nitrogen atom have to be specifically substituted to reach the opti-
mum efficacy. The most active imidazole fungicides bear a methyl
substituent in position 2, which means at the ring carbon between
the two heteroatoms. In addition, it is also important, that there is
a chlorine atom linked to the other carbon atom, which is adjacent
to the unsubstituted ring nitrogen. Furthermore, the third ring car-
bon typically bears a bis-ortho halogen-substituted phenyl ring
with an additional para-substituent. Finally, we obtained the best
fungicidal results with a para-substituted phenyl or pyridyl moiety
linked to the imidazole ring nitrogen.
obtain
2-bromo-1-(4-chloro-phenyl)-5-(2,4,6-trifluoro-phenyl)-
1H-imidazole (15, 3.1 g, 8.0 mmol, 61%) as a pale yellow-orange so-
lid. 1H NMR (CDCl3): d = 6.55 (t, 2H, J = 7.2), 7.05 (d, 2H, J = 8.5),
7.14 (s, 1H), 7.29 (d, 2H, J = 8.6). LC–MS: tR = 2.02 min, m/z = 389
[M+1], 391 [M+3].
4.1.4. 1-(4-chloro-phenyl)-2-methyl-5-(2,4,6-trifluoro-phenyl)-
1H-imidazole (16)
Tetrakis(triphenylphosphine)palladium (0.13 g, 0.11 mmol) is
added to as solution of 2-bromo-1-(4-chloro-phenyl)-5-(2,4,6-tri-
fluoro-phenyl)-1H-imidazole (15, 3.1 g, 8.0 mmol) in 162 ml of tet-
rahydrofuran. This mixture is heated to reflux for 10 min, then the
oil bath is removed and 12 ml of a 2 M solution of trimethylalumi-
num in toluene are added slowly. The reaction mixture is heated to
reflux for 7 h, then cooled to 0 °C. First 5 ml of methanol and then
IsoluteÒ HM-N is added and the solvents are removed under re-
duced pressure. The residue is purified by chromatography on sil-
ica gel, using a mixture of heptane/ethyl acetate 1:1 as eluent, to
obtain 1-(4-chloro-phenyl)-2-methyl-5-(2,4,6-trifluoro-phenyl)-
1H-imidazole (16, 1.6 g, 5.0 mmol, 62%) as a yellowish solid. 1H
NMR (CDCl3): d = 2.31 (s, 3H), 6.56 (t, 2H, J = 7.4), 7.07 (d, 2H,
J = 8.5), 7.13 (s, 1H), 7.28 (d, 2H, J = 8.6). LC–MS: tR = 1.36 min,
m/z = 323 [M+1].
4. Experimental section
4.1. Chemistry
All new compounds were characterized by standard spectro-
scopical methods. 1H NMR spectra were recorded on a Varian Unity
400 spectrometer at 400 MHz using CDCl3 as solvent and tetra-
methylsilane as internal standard. Chemical shifts are reported in
ppm downfield from the standard (d = 0.00), coupling constants
in Hz. Mass spectra were recorded on a Micromass LCT mass spec-
trometer. Melting points were determined on a Büchi 535 melting
point apparatus and are uncorrected. Analytical thin-layer chroma-
tography (TLC) was performed using silica gel 60 F524 precoated
plates. Preparative flash chromatography was performed using sil-
4.1.5. 4-Chloro-1-(4-chloro-phenyl)-2-methyl-5-(2,4,6-
trifluoro-phenyl)-1H-imidazole (17)
ica gel 60 (40–63 lm, E. Merck). All reactions were carried out un-
der anhydrous conditions in an inert atmosphere (nitrogen or
A mixture of 1-(4-chloro-phenyl)-2-methyl-5-(2,4,6-trifluoro-
phenyl)-1H-imidazole (16, 1.6 g, 5.0 mol) and N-chlorosuccinimide
(0.8 g, 6.2 mmol) in 32 ml of chloroform is heated for 16 h to 80 °C.
Subsequently the mixture is cooled to room temperature, then Iso-
luteÒ HM-N is added and the solvent is removed under reduced
pressure. The residue is purified by column chromatography on sil-
ica gel, using a mixture of heptane/ethyl acetate 4: 1 as eluent, to
obtain 4-chloro-1-(4-chloro-phenyl)-2-methyl-5-(2,4,6-trifluoro-
phenyl)-1H-imidazole (17, 1.0 g, 2.8 mmol, 57%) as a white solid.
argon) with dry solvents.
4.1.1. (4-Chloro-phenyl)-[1-(2,4,6-trifluoro-phenyl)-meth-(E)-
ylidene]amine (13)
A mixture of 4-chloro-aniline (11, 20.4 g, 0.16 mol) and 2,4,6-
trifluoro-benzaldehyde (12, 25.5 g, 0.16 mol) in 780 ml of toluene
is heated to reflux in a Dean-Stark apparatus for 4 d. Subsequently
the mixture is cooled, evaporated under reduced pressure to obtain

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C. Lamberth et al. / Bioorg. Med. Chem. 21 (2013) 127–134
1H NMR (CDCl3): d = 2.30 (s, 3H), 6.63 (t, 2H, J = 7.3), 7.07 (d, 2H,
J = 8.5), 7.36 (d, 2H, J = 8.6). LC–MS: tR = 1.96 min, m/z = 357 [M+1].
of tetrahydrofuran is added at – 20 °C 3.2 ml of a 1.8 M solution
of lithium diisopropylamide in tetrahydrofuran. The mixture is
stirred for 30 min at ꢀ20 °C, then methyl iodide (1.9 g, 13.4 mmol)
is added. The reaction mixture is stirred for further 30 min at
ꢀ20 °C, then for 30 min at room temperature. Subsequently Iso-
luteÒ HM-N is added to the reaction mixture and the solvent is
evaporated. The residue is purified by chromatography on silica
gel, using a mixture of heptane/ethyl acetate 2:1 as eluent, to ob-
tain delivering 4-chloro-1-(4-chloro-phenyl)-5-(2,6-difluoro-4-
methoxy-phenyl)-2-methyl-1H-imidazole (9, 1.1 g, 3.0 mmol,
66%). 1H NMR (CDCl3): d = 2.29 (s, 3H), 3.76 (s, 3H), 6.38 (d, 2H,
J = 9.1), 7.07 (d, 2H, J = 8.6), 7.34 (d, 2H, J = 8.7). LC-MS:
tR = 1.96 min, m/z = 371 [M+2].
4.1.6. 4-Chloro-1-(4-chloro-phenyl)-5-(2,6-difluoro-4-methoxy-
phenyl)-2-methyl-1H-imidazole (9)
A mixture of 4-chloro-1-(4-chloro-phenyl)-2-methyl-5-(2,4,6-
trifluoro-phenyl)-1H-imidazole (17, 0.4 g, 1.1 mmol), 0.48 ml of a
0.18 M solution of sodium methoxide in methanol and 5 ml of
methanol is stirred for 16 h at room temperature. Subsequently
the reaction mixture is poured on ice-water and extracted twice
with ethyl acetate. The combined organic layer is washed with
brine, dried over sodium sulfate and concentrated under reduced
pressure to deliver directly 4-chloro-1-(4-chloro-phenyl)-5-(2,6-
difluoro-4-methoxy-phenyl)-2-methyl-1H-imidazole (9, 0.3 g,
0.8 mol 74%). 1H NMR (CDCl3): d = 2.29 (s, 3H), 3.76 (s, 3H), 6.38
(d, 2H, J = 9.1), 7.07 (d, 2H, J = 8.6), 7.34 (d, 2H, J = 8.7). LC–MS:
tR = 1.95 min, m/z = 369 [M], 371 [M+2].
4.1.11. 1-[2-(4-methyl-piperidin-1-yl)-2-oxo-ethyl]-3-(2,4,6-
trifluoro-phenyl)-thiourea (22)
N,N-diisopropylethylamine (14.8 g, 0.11 mol) and 2,4,6-trifluor-
ophenyl isothiocyanate (11.1 g, 57 mol) are added successively to a
solution of 2-amino-1-(4-methyl-piperidin-1-yl)-ethanone hydro-
chloride salt (21, 11 g, 57 mol) in 385 ml of dichloromethane. The
reaction mixture is stirred for 1 h at room temperature and then
evaporated under reduced pressure. The residue is purified by col-
umn chromatography on silica gel, using a mixture of heptane/
ethyl acetate 1:1 as eluent, to obtain 1-[2-(4-methyl-piperidin-1-
yl)-2-oxo-ethyl]-3-(2,4,6-trifluoro-phenyl)-thiourea (22, 13.6 g,
39 mmol, 69%) as an orange solid. 1H NMR (CDCl3): d = 0.94 (d,
3H, J = 6.4), 0.95–1.14 (m, 2H), 1.52–1.68 (m, 2H), 1.74 (d, 1H,
J = 13.1), 2.60 (dt, 1H, J = 2.5, 13.0), 3.00 (dt, 1H, J = 2.5, 13.0),
3.75 (bd, 1H, J = 6.0, 13.0), 4.30 (bd, 1H, J = 13.2), 4.40 (bs, 2H),
6.78 (t, 2H, J = 7.8), 7.80 (br s, 1H), 8.10 (br s, 1H). LC-MS:
tR = 1.72 min, m/z = 346 [M+1].
4.1.7. (4-Chloro-phenylamino)-(2,4,6-trifluoro-phenyl)-
acetonitrile (18)
To a solution of 4-chloroaniline (11, 5.0 g, 39 mmol) in 64 ml of
tetrahydrofuran are added consecutively 2,4,6-trifluoro-benzalde-
hyde (12, 6.6 g, 41 mmol), indium(III) chloride (9.5 g, 43 mmol)
and potassium cyanide (10.2 g, 0.16 mol). The reaction mixture is
stirred for 16 h at room temperature, then diluted with water
and extracted with ethyl acetate. The combined organic layer is
dried over sodium sulfate and evaporated to deliver (4-chloro-phe-
nylamino)-(2,4,6-trifluorophenyl)-acetonitrile
(18,
11.7 g,
39 mmol, 100%). 1H NMR (CDCl3): d = 4.23 (d, 1H, J = 10.5), 5.56
(d, 1H, J = 9.0), 6.64 (d, 2H, J = 8.2), 6.72 (t, 2H, J = 8.3), 7.15–7.23
(m, 2H). LC–MS: tR = 1.91 min, m/z = 297 [M+1].
4.1.8. 4-Chloro-1-(4-chloro-phenyl)-5-(2,4,6-trifluoro-phenyl)-
1H-imidazole (19)
4.1.12. 2-Methyl-1-[2-(4-methyl-piperidin-1-yl)-2-oxo-ethyl]-3-
(2,4,6-trifluoro-phenyl)-isothiourea (23)
Chloromethylenedimethyliminium chloride (Vilsmeier reagent,
2.6 g, 20 mmol) and 1.7 ml of a 4 N solution of hydrochloric acid in
dioxane are added consecutively to a solution of (4-chloro-phenyla-
mino)-(2,4,6-trifluorophenyl)-acetonitrile (18, 2.0 g, 6.7 mmol) in
28 ml of dioxane. The reaction mixture is stirred for 2 h at 100 °C
and then cooled to room temperature. IsoluteÒ HM-N is added and
the solvent is removed under reduced pressure. The residue is puri-
fied by chromatography on silica gel, using a mixture of heptane/
ethyl acetate 3:2 as eluent, to deliver 4-chloro-1-(4-chloro-phenyl-
5-(2,4,6-trifluoro-phenyl)-1H-imidazole (19, 1.6 g, 4.5 mmol, 67%).
1H NMR (CDCl3): d = 6.69 (t, 2H, J = 7.4), 7.08 (d, 2H, J = 8.6), 7.37
(d, 2H, J = 8.5), 7.70 (s, 1H). LC-MS: tR = 1.93 min, m/z = 343 [M+1],
345 [M+3].
Anhydrous potassium carbonate (8.2 g, 59 mmol) and iodo-
methane (11.2 g, 78 mmol) are successively added to a solution
of 1-[2-(4-methyl-piperidin-1-yl)-2-oxo-ethyl]-3-(2,4,6-trifluoro-
phenyl)-thiourea (22, 13.6 g, 39 mmol) in 136 ml of acetonitrile.
The reaction mixture is stirred for 1 h at room temperature and
then concentrated under reduced pressure. The residue is taken
up in ethyl acetate and washed with water and brine. The organic
layer is dried over sodium sulfate and evaporated. The remainder is
purified by column chromatography on silica gel, using a mixture
of heptane/ethyl acetate 1:1 as eluent, to obtain 2-methyl-1-[2-
(4-methyl-piperidin-1-yl)-2-oxo-ethyl]-3-(2,4,6-trifluoro-phenyl)-
isothiourea (23, 13.6 g, 38 mmol, 96%). 1H NMR (CDCl3): d = 0.97 (d,
3H, J = 6.4), 1.11 (dq, 2H, J = 2.5, 13.2), 1.54–1.71 (m, 1H), 1.72 (d,
1H, J = 13.4), 2.44 (s, 3H), 2.67 (dt, 1H, J = 2.5, 13.0), 3.00 (dt, 1H,
J = 2.5, 13.0), 3.72 (bd, 1H, J = 13.6), 4.22 (s, 2H), 4.55 (d, 1H,
J = 13.0), 6.28 (s, 1H), 6.66 (t, 2H, J = 8.2). LC–MS: tR = 1.88 min,
m/z = 360 [M+1].
4.1.9. 4-Chloro-1-(4-chloro-phenyl)-5-(2,6-difluoro-4-methoxy-
phenyl)-1H-imidazole (20)
To a solution of 4-chloro-1-(4-chloro-phenyl)-5-(2,4,6-trifluoro-
phenyl)-1H-imidazole (19, 1.6 g, 4.6 mmol) in 28 ml of tetrahydro-
furan is added at 0 °C 1.7 ml of a 30% sodium methoxide solution
in methanol. The reaction mixture is stirred for 16 h at room temper-
ature. Subsequently it is diluted with aqueous ammonium chloride
solution and extracted with ethyl aceate. The organic layer is dried
over sodium sulfate and evaporated to deliver 4-chloro-1-(4-chloro-
phenyl)-5-(2,6-difluoro-4-methoxy-phenyl)-1H-imidazole (20, 1.6 g,
4.6 mmol, 97%). 1H NMR (CDCl3): d = 3.74 (s, 3H), 6.40 (d, 2H, J = 9.5),
7.03 (d, 2H, J = 8.7), 7.29 (d, 2H, J = 9.0), 7.60 (s, 1H). LC–MS:
tR = 1.93 min, m/z = 355 [M+1], 357 {M+3].
4.1.13. 4-methyl-1-[2-methylsulfanyl-3-(2,4,6-trifluoro-phenyl)-
3H-imidazol-4-yl]-piperidine (24)
2,4-Bis-(4-methoxyphenyl)-2,4-dithioxo-1,3,2,4-dithiadipho-
sphetane (Lawesson’s reagent, 3.6 g, 8.9 mol) is added to a solution
of 2-methyl-1-[2-(4-methyl-piperidin-1-yl)-2-oxo-ethyl]-3-(2,4,
6-trifluoro-phenyl)-isothiourea (23, 5.7 g, 15.9 mmol) in 57 ml of
1,2-dimethoxyethane. The reaction mixture, which changes its col-
our from green to red, is stirred for 16 h at room temperature and
then evaporated. The residual purple oil is diluted with tert-butyl
methyl ether, the precipitate is filtered and the filtrate is stirred
with 4 N sodium hydroxide solution, hereby the colour changes
from purple to blue. The aqueous phase is extracted with ethyl
acetate, the combined organic layer is washed with brine, dried
over sodium sulfate and evaporated under reduced pressure. The
4.1.10. 4-Chloro-1-(4-chloro-phenyl)-5-(2,6-difluoro-4-
methoxy-phenyl)-2-methyl-1H-imidazole (9)
To a solution of 4-chloro-1-(4-chloro-phenyl)-5-(2,6-difluoro-
4-methoxy-phenyl)-1H-imidazole (20, 1.6 g, 4.6 mmol) in 37 ml

C. Lamberth et al. / Bioorg. Med. Chem. 21 (2013) 127–134
133
remainder is purified by column chromatography on silica gel,
4.1.17. 1-[3-(2,6-difluoro-4-methoxy-phenyl)-5-methyl-3H-
using a mixture of heptane/ethyl acetate 7:3 as eluent, to obtain
4-methyl-1-[2-methylsulfanyl-3-(2,4,6-trifluoro-phenyl)-3H-imida-
zol-4-yl]-piperidine (24, 2.1 g, 6.3 mmol, 39%). 1H NMR (CDCl3):
d = 0.86 (d, 3H, J = 6.5), 0.99 (dq, 2H, J = 4.0, 12.0), 1.30–1.45 (m,
1H), 1.52 (d, 2H, J = 12.8), 2.48 (s, 3H), 2.59 (dt, 2H, J = 2.5, 11.9),
2.91 (d, 2H, J = 12.0), 6.72 (s, 1H), 6.85 (t, 2H, J = 7.9). LC–MS:
tR = 2.01 min, m/z = 342 [M+1].
imidazol-4-yl]-4-methyl-piperidine (28)
To
a
solution of 1-[3-(2,6-difluoro-4-methoxy-phenyl)-5-
methyl-2-methylsulfanyl-3H-imidazol-4-yl]-4-methyl-piperidine
(27, 0.23 g, 0.6 mmol) in 5 ml of ethanol are added 22 ml of an
aqueous Raney-Nickel suspension (100 g/l). The resulting black
suspension is heated to reflux for 90 min and then cooled to room
temperature. Celite is added to the mixture and the suspension is
filtered through a Celite pad. The Celite is rinsed with ethanol
and water, taking care that the pad always stays wet. The filtrate
is concentrated under reduced pressure, the residue taken up in
ethyl acetate and washed with water. The aqueous phase is ex-
tracted with ethyl acetate, the combined organic layer is washed
with brine, dried over sodium sulfate and evaporated under re-
duced pressure. The residue is purified by column chromatography
on silica gel, using a mixture of heptane/ethyl acetate 1:4 as eluent,
to obtain 1-[3-(2,6-difluoro-4-methoxy-phenyl)-5-methyl-3H-imi-
dazol-4-yl]-4-methyl-piperidine (28, 67 mg, 0.2 mmol, 33%) as a
white solid. 1H NMR (CDCl3): d = 0.86 (d, 3H, J = 6.5), 0.88–0.98
(m, 2H), 1.30–1.37 (m, 1H), 1.49 (d, 2H, J = 12.1), 2.26 (s, 3H),
2.83–2.91 (m, 4H), 3.85 (s, 3H), 6.56 (d, 2H, J = 8.9), 7.22 (s, 1H).
LC–MS: tR = 1.34 min, m/z = 322 [M+1].
4.1.14. 1-[5-bromo-2-methylsulfanyl-3-(2,4,6-trifluoro-phenyl)-
3H-imidazol-4-yl]-4-methyl-piperidine (25)
N-bromosuccinimide (1.4 g, 7.9 mmol) is added to a solution of
4-methyl-1-[2-methylsulfanyl-3-(2,4,6-trifluoro-phenyl)-3H-imida-
zol-4-yl]-piperidine (24, 2.1 g, 6.1 mmol) in 25 ml of chloroform.
The reaction mixture is stirred for 15 min at room temperature, then
diluted with dichloromethane and 1 N sodium hydroxide solution.
The phases are separated and the aqueous phase is extracted with
dichloromethane. The combined organic layer is washed with 1 N
sodium hydroxide solution and brine, dried over sodium sulfate
and evaporated under reduced pressure. The residue is purified by
column chromatography on silica gel, using a mixture of heptane/
ethyl acetate 9:1 as eluent, to obtain 1-[5-bromo-2-methylsulfanyl-
3-(2,4,6-trifluoro-phenyl)-3H-imidazol-4-yl]-4-methyl-piperidine
(25, 1.53 g, 3.6 mmol, 60%) as a yellowish solid. 1H NMR (CDCl3):
d = 0.85 (d, 3H, J = 6.4), 0.79–0.89 (m, 2H), 1.34–1.37 (m, 1H), 1.52
(d, 2H, J = 11.8), 2.53 (s, 3H), 2.84 (d, 2H, J = 11.6), 3.06 (dt, 2H,
J = 2.2, 11.7), 6.85 (t, 2H, J = 7.8). LC–MS: tR = 2.18 min, m/z = 422
[M+2].
4.1.18. 1-[2-Chloro-3-(2,6-difluoro-4-methoxy-phenyl)-5-
methyl-3H-imidazol-4-yl]-4-methyl-piperidine (10)
N-chlorosuccinimide (35 mg, 0.3 mmol) is added to a solution
of 1-[3-(2,6-difluoro-4-methoxy-phenyl)-5-methyl-3H-imidazol-
4-yl]-4-methyl-piperidine (28, 67 mg, 0.2 mmol) in 2 ml of chloro-
form. The reaction mixture is stirred for 1.5 h at 0 °C, then concen-
trated under reduced pressure and diluted with methanol. IsoluteÒ
HM-N is added and the solvent is removed under reduced pressure.
The residue is purified by column chromatography on silica gel,
using a mixture of heptane/ethyl acetate 4:1 as eluent, to obtain
1-[2-chloro-3-(2,6-difluoro-4-methoxy-phenyl)-5-methyl-3H-imi-
dazol-4-yl]-4-methyl-piperidine (10, 14 mg, 0.04 mmol, 19%) as a
yellow oil. 1H NMR (CDCl3): d = 0.85 (d, 3H, J = 6.6), 0.88–0.95 (m,
2H), 1.25–1.35 (m, 1H), 1.49 (d, 2H, J = 12.2), 2.21 (s, 3H), 2.82
(dt, 2H, J = 2.3, 11.5), 2.90 (d, 2H, J = 11.2), 3.87 (s, 3H), 6.59 (d,
2H, J = 8.9). LC–MS: tR = 2.16 min, m/z = 356 [M+1].
4.1.15. 4-Methyl-1-[5-methyl-2-methylsulfanyl-3-(2,4,6-trifluoro-
phenyl)-3H-imidazol-4-yl]-piperidine (26)
Cesium carbonate (4.7 g, 14 mmol), trimethylboroxine (0.7 g,
5.3 mmol) and [1,10-bis(diphenylphosphino)ferrocene] dichloro-
palladium(II) complex with dichloromethane (0.9 g, 1.1 mmol)
are added to a solution of 1-[5-bromo-2-methylsulfanyl-3-(2,4,
6-trifluoro-phenyl)-3H-imidazol-4-yl]-4-methyl-piperidine
(25,
1.5 g, 3.6 mmol) in 31 ml of dioxane. The reaction mixture is
heated to 90 °C for 16 h, then cooled to room temperature and
filtered. The filtrate is washed with water and brine, dried over
sodium sulfate and evaporated under reduced pressure. The
residue is purified by column chromatography on silica gel, using
a mixture of heptane/ethyl acetate 4:1 as eluent, to obtain 4-
methyl-1-[5-methyl-2-methylsulfanyl-3-(2,4,6-trifluoro-phenyl)-
3H-imidazol-4-yl]-piperidine (26, 0.55 g, 1.5 mmol, 42%) as an
orange solid. 1H NMR (CDCl3): d = 0.85 (d, 3H, J = 6.6), 0.81–0.91
(m, 2H), 1.24–1.36 (m, 1H), 1.49 (d, 2H, J = 12.0), 2.26 (s, 3H),
2.47 (s, 3H), 2.80–2.87 (m, 4H), 6.83 (t, 2H, J = 7.8). LC–MS:
tR = 1.99 min, m/z = 356 [M+1], 358 [M+3].
4.2. Biology
All tested compounds have been at least 95% pure, the purifica-
tion has been either performed via chromatography (see chapters
4.15, 4.19 and 4.10) or by crystalisation.
4.2.1. Botrytis cinerea (Botryotinia fuckeliana)/tomato (action
against grey mould on tomato)
4.1.16. 1-[3-(2,6-Difluoro-4-methoxy-phenyl)-5-methyl-2-
methylsulfanyl-3H-imidazol-4-yl]-4-methyl-piperidine (27)
Four-week old tomato plants cv. Roter Gnom were treated in a
spray chamber with the formulated test compound diluted in
water. The test plants were inoculated by spraying them with a
spore suspension two days after application. The inoculated test
plants were incubated at 20 °C and 95% rh in a greenhouse and
the percentage leaf area covered by disease was assessed when
an appropriate level of disease appeared on untreated check plants
(5–6 days after application).
To
a solution of 4-methyl-1-[5-methyl-2-methylsulfanyl-3-
(2,4,6-trifluoro-phenyl)-3H-imidazol-4-yl]-piperidine (26, 0.62 g,
1.8 mmol) in 6 ml of tetrahydrofuran is added 1.0 ml of a 5.4 M
solution of sodium methoxide in methanol. The reaction mixture
is heated for 30 min to reflux, then cooled to room temperature
and poured into cold 1 N hydrochloric acid. The resulting suspen-
sion is extracted twice with ethyl acetate and the combined
organic layer is washed with brine, dried over sodium sulfate
and evaporated under reduced pressure. The residue is purified
by column chromatography on silica gel, using a mixture of
heptane/ethyl acetate 3:2 as eluent, to obtain 1-[3-(2,6-difluoro-
4-methoxy-phenyl)-5-methyl-2-methylsulfanyl-3H-imidazol-4-yl]-
4-methyl-piperidine (27, 0.26 g, 0.7 mmol, 40%). 1H NMR (CDCl3):
d = 0.84 (d, 3H, J = 6.6), 0.87–0.94 (m, 2H), 1.25–1.34 (m, 1H), 1.49
(d, 2H, J = 12.1), 2.25 (s, 3H), 2.47 (s, 3H), 2.79–2.91 (m, 4H), 3.86
(s, 3H), 6.57 (d, 2H, J = 8.9). LC–MS: tR = 1.81 min, m/z = 368 [M+1].
4.2.2. Uncinula necator (Erysiphe necator)/grape (action against
powdery mildew on grape)
5-week old grape seedlings cv. Gutedel are sprayed in a spray
chamber with the formulated test compound diluted in water.
The test plants are inoculated by shaking plants infected with
grape powdery mildew above them 1 day after application. The
inoculated test plants are incubated at 24/22 °C (day/night) and
70% rh under a light regime of 14/10 h (light/dark) and the percent-
age leaf area covered by disease is assessed when an appropriate

134
C. Lamberth et al. / Bioorg. Med. Chem. 21 (2013) 127–134
level of disease appears on untreated check plants (7–9 days after
application).
References and notes
1. Part 2 Lamberth, C.; Trah, S.; Wendeborn, S.; Dumeunier, R.; Courbot, M.;
Godwin, J.; Schneiter, P. Bioorg. Med. Chem. 2012, 20, 2803.
2. Walter, H. In Bioactive Heterocyclic Compound Classes-Agrochemicals; Lamberth,
C., Dinges, J., Eds.; Wiley-VCH: Weinheim, 2012; pp 175–193.
3. Rheinheimer, J.; Rieck, H.; Coqueron, P.-Y. In Modern Crop Protection
Compounds; Krämer, W., Schirmer, U., Jeschke, P., Witschel, M., Eds.; Wiley-
VCH: Weinheim, 2012; pp 627–645.
4. Suty-Heinze, A.; Dunkel, R.; Krieg, U.; Rieck, H. In Modern Fungicides and
Antifungal Compounds VI; Dehne, H. W., Deising, H. B., Gisi, U., Kuck, K.-H.,
Russell, P.-E., Leyr, H., Eds.; DPG: Braunschweig, 2011; pp 69–73.
5. Stammler, G.; Brix, H. D.; Glaettli, A.; Semar, M.; Schoefl, U. Proceed. Int. Plant
Protect. Congr. Glasgow 2007, 1, 40.
6. Veloukas, T.; Karaoglanidis, G. S. Pest Manag. Sci. 2012, 68, 858.
7. Semar, M.; Strobel, D.; Strathmann, S.; Groeger, U. In Modern Fungicides and
Antifungal Compounds VI; Dehne, H. W., Deising, H. B., Gisi, U., Kuck, K.-H.,
Russell, P.-E., Leyr, H., Eds.; DPG: Braunschweig, 2011; pp 63–68.
8. Nagahori, H.; Yoshino, H.; Tomigahara, Y.; Isobe, N.; Kaneko, H.; Nakatsuka, I. J.
Agric. Food Chem. 2000, 48, 5754.
4.2.3. Mycosphaerella graminicola (Septoria tritici)/wheat
(action against leaf blotch on wheat)
Two-week old wheat plants cv. Riband were treated in a spray
chamber with the formulated test compound diluted in water.
The test plants were inoculated by spraying a spore suspension
on them one day after application. After an incubation period of
1 day at 22 °C/21 °C (day/night) and 95% rh, the inoculated test
plants were kept at 22 °C/21 °C (day/night) and 70% rh in a green-
house. Efficacy was assessed directly when an appropriate level of
disease appeared on untreated check plants (16–19 days after
application).
4.2.4. Alternaria solani/tomato (action against early blight on
tomato)
9. Harp, T. L.; Godwin, J. R.; Scalliet, G.; Walter, H.; Stalker, A. D.; Bartlett, D. L.;
Ranner, D. J. Aspects Appl. Biol. 2011, 106, 113.
Four-week old tomato plants cv. Roter Gnom were treated in a
spray chamber with the formulated test compound diluted in
water. The test plants were inoculated by spraying them with a
spore suspension two days after application. The inoculated test
plants were incubated at 22/18 °C (day/night) and 95% rh in a
greenhouse and the percentage leaf area covered by disease was
assessed when an appropriate level of disease appeared on un-
treated check plants (5–7 days after application).
10. Yanase, Y.; Yoshikawa, Y.; Kishi, J.; Katsuta, H. In Pesticide Chemistry; Ohkawa,
H., Miyagawa, H., Lee, P. W., Eds.; Wiley-VCH: Weinheim, 2007; p 295.
11. Montel, F.; Lamberth, C.; Jung, P. M. J. Tetrahedron 2008, 64, 6372.
12. Zeun, R.; Cederbaum, F.; Oostendorp, M. WO 2007/141010 (Syngenta). Chem.
Abstr. 2007, 148, 25706.
13. Crowley, P. J.; Lamberth, C.; Müller, U.; Wendeborn, S.; Nebel, K.; Williams, J.;
Sageot, O.-A.; Carter, N.; Mathie, T.; Kempf, H.-J.; Godwin, J.; Schneiter, P.;
Dobler, M. R. Pest Manage. Sci. 2010, 66, 178.
14. Crowley, P. J.; Lamberth, C.; Müller, U.; Wendeborn, S.; Sageot, O.-A.; Williams,
J.; Bartovic, A. Tetrahedron Lett. 2010, 51, 2652.
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16. Gypser, A.; Grote, T.; Schwoegler, A.; Rheinheimer, J. ; Schieweck, F.; Tormo i
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Ammermann, E.; Strathmann, S.; Lorenz, G.; Stierl, R. WO 2003/043993 (BASF).
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17. Bereznak, J. F.; Stevenson, T. M.; Sharpe, P. L.; Taggi, A. E. WO 2007/149448 (Du
Pont). Chem. Abstr. 2007, 148, 100635.
18. Almansa, C.; Alfon, J.; de Arriba, A. F.; Cavalcanti, F. L.; Escamilla, I.; Gomez, L.
A.; Miralles, A.; Soliva, R.; Bartroli, J.; Carceller, E.; Merlos, M.; Garcia-Rafanell,
J. J. Med. Chem. 2003, 46, 3463.
4.3. Biochemistry
4.3.1. Compounds testing on pure porcine tubulin
The HTS-tubulin polymerisation assay kit (Cytoskeleton Inc.,
Denver, USA) has been used following the manufacturer instruc-
tion. The standard polymerisation reaction contains 100 ll volume
of 4 mg/ml tubulin in 80 mM PIPES ph 6.9, 0.5 mM EGTA, 2 mM
MgCl2 and 1 mM GTP. The polymerisation was started by incuba-
tion at 37 °C and followed by absorption readings at 340 nm (Spec-
tramax). Paclitaxel and DMSO were used as positive and negative
controls, respectively. 4-Chloro-1-(4-chloro-phenyl)-5-(2,6-di-
fluoro-4-methoxy-phenyl)-2-methyl-1H-imidazole (9) and 4-
chloro-1-(4-chloro-phenyl)-2-methyl-5-(2,4,6-trifluoro-phenyl)-
1H-imidazole (17) were added in different wells of a 96-well plate
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at the beginning of the reaction at 1.25
(0.2 mM).
l
g.mlꢀ1 in a DMSO solution
26. Dumeunier, R.; Lamberth, C.; Trah, S. WO 2010/102866 (Syngenta). Chem.
Abstr. 2010, 153, 431359.