Synthesis and fungicidal activity of tubulin polymerisation promoters. Part 2: Pyridazines

Article abstract of DOI:10.1016/j.bmc.2012.03.035

Special tetrasubstituted pyridazines are potent fungicides by promoting the tubulin polymerisation, hereby disrupting the microtubule dynamics in the fungus. They are monocyclic analogs of similar substituted triazolopyrimidines and pyridopyrazines with the same mode of action. The fungicidal activity of these pyridazines was evaluated against the plant pathogens Botrytis cinerea (grey mould), Mycosphaerella graminicola (wheat leaf blotch) and Alternaria solani (potato and tomato early blight). Structure-activity relationship studies revealed the importance of a methyl and a chlorine substituent next to both ring nitrogen atoms and two aryl or heteroaryl groups in the other two pyridazine positions.

Full text of DOI:10.1016/j.bmc.2012.03.035

Bioorganic & Medicinal Chemistry 20 (2012) 2803–2810
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and fungicidal activity of tubulin polymerisation promoters. Part 2:
Pyridazines
⇑
Clemens Lamberth , Stephan Trah, Sebastian Wendeborn, Raphael Dumeunier, Mikael Courbot,
Jeremy Godwin, Peter Schneiter
Syngenta Crop Protection AG, Crop Protection Research, Schaffhauserstr. 101, CH-4332 Stein, Switzerland
a r t i c l e i n f o
a b s t r a c t
Article history:
Special tetrasubstituted pyridazines are potent fungicides by promoting the tubulin polymerisation,
hereby disrupting the microtubule dynamics in the fungus. They are monocyclic analogs of similar substi-
tuted triazolopyrimidines and pyridopyrazines with the same mode of action. The fungicidal activity of
these pyridazines was evaluated against the plant pathogens Botrytis cinerea (grey mould), Mycosphaerel-
la graminicola (wheat leaf blotch) and Alternaria solani (potato and tomato early blight). Structure–activ-
ity relationship studies revealed the importance of a methyl and a chlorine substituent next to both ring
nitrogen atoms and two aryl or heteroaryl groups in the other two pyridazine positions.
Ó 2012 Elsevier Ltd. All rights reserved.
Received 29 November 2011
Revised 12 March 2012
Accepted 15 March 2012
Available online 24 March 2012
Keywords:
Tubulin
Pyridazine
Heterocycle
Fungicide
Crop protection
1. Introduction
most of them being inhibitors of the tubulin polymerisation. How-
ever, promotion of tubulin polymerisation would still be a novel
Microtubules are hollow cylindrical tubes found in all eukaryotic
cell types. These essential cytoskeletal protein polymers play a
pivotal role in maintaining the growth, shape, division, motility
and functioning of the cell. Microtubules are key components of
the mitotic spindle, which enables the segregation of chromosomes
during the process of mitosis. They are built by polymerisation of
mode of action on the fungicide market.
[1,2,4]Triazolo[1,5-a]pyrimidines, such as BAS600F (1),17 have
been identified as promotores of tubulin polymerisation. This class
of experimental fungicides, which has been discovered by Shell in
the early 1990s,18 is highly active against a broad range of different
plant diseases.19 In addition, some [1,2,4]triazolo[1,5-a]pyrimidine
mimics, in which the five-membered triazole ring is replaced by a
six-membered heterocycle, have also been described. Such pyrido
[2,3-b]pyrazines,1 for example, 2, and pyrido[3,2-e][1,2,4]tria-
zines,20 such as 3, display a similar efficacy against phytopathogens
as the related triazolopyrimidines. Recently Sumitomo discovered,
that pyridazines, such as 4, are suitable monocyclic analogs of the
before mentioned bicylic tubulin polymerisation promoters.21 This
is not by surprise, because also in medicinal chemistry, pyridazines
have been able to mimic similar substituted bicyclic imidazopyrim-
idine and imidazotriazine GABAA agonists.22 Actually it is a closely
related substitution pattern, which is common to those 5,6-bicylic,
6,6-bicyclic and monocyclic promoters of tubulin polymerisation
shown in Figure 1. They all carry adjacent to a ring nitrogen atom
a halogen substituent, next to it there is a 2,4,6-trifluorophenyl ring,
which is followed by either an amine or another phenyl 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 persubstituted ring, is generally unsubstituted.
The pyridazine ring has a long-standing history as part of bio-
logically active compounds. Several highly efficacious pharmaceu-
two closely related heterodimers composed of
a- and b-tubulin
subunits. Interference with the microtubule homeostasis by dis-
rupting the dynamic equilibrium between the assembly of tubulin
into microtubules or, inversely, the depolymersiation of microtu-
bules into tubulin leads to arrested cell division and consequently
to apoptosis. Compounds which are able to inhibit one of these
two important processes are called antimitotics.2
Antimitotic drugs are a well-established class of chemothera-
peutic anti-cancer agents, as demonstrated by the two naturally
occurring tubulin polymerisation promoting families of taxanes,
for example, paclitaxel and docetaxel, and vinca alkaloids, such
as vinblastine and vincristine.3–9
In the meantime, compounds with this mode of action attract
more and more attention for their fungicidal efficacy. Antimitotics
are used already as agrochemical fungicides, such as the amides zox-
amide10,11 and ethaboxam,12,13 as well as the benzimidazoles beno-
myl,14,15 carbendazim,14 thiabendazole15,16 and fuberidazole,15
⇑
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.2012.03.035

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C. Lamberth et al. / Bioorg. Med. Chem. 20 (2012) 2803–2810
route is the cyclocondensation of the
a-bromoketone 7, which is
available in two steps from 5-chlorothiophenecarboxylic acid (5)
via the Weinreb amide 6, with 2,4,6-trifluorophenylacetic acid to
the hydroxyfuranone 8. This unique one-pot ring closure reaction
proceeeds via three different partial steps, which will be further
explained in Scheme 2.33,34 The subsequent ring enlargement of
the methyl- and hydroxy-substituted butenolide 8 to the pyridaz-
inone 9 has first been described by Caspi and Piatek.35,36 Finally 9 is
converted by chlorination to the fungicidally active pyridazine 10.
As worked out by researchers at Merck,33 the one-pot prepara-
tion of the hydroxyfuranone 8 starts with the substitution of the
bromine in 7 by the triethylammonium salt of 2,4,6-trifluorphenyl-
acetic acid to provide the ester 11, which after addition of DBU
undergoes an intramolecular aldol condensation to the furanone
12. Finally, bubbling air through the reaction mixture results in
oxidation to the desired hydroxyfuranone 8.
F
F
F
F
NH
N
N
N
N
N
F
F
N
F
N
F
N
Cl
1
2
BAS600F
Cl
F
F
F
F
F
F
NH
N
N
N
F
F
N
N
F
N
Cl
Using the general synthesis shown in Scheme 1, the pyridazine
substituents in positions 4, 5 and 6 can be easily varied by applica-
tion of different ketones and carboxyclic acids. Also several differ-
ent substituents can be introduced in position 3 by replacement of
the chloro function. The only limitation of this method is the fact,
that it is not possible to exchange the typical methyl group in posi-
tion 6 by non-carbon linked substituents, such as halogen, alkoxy,
amino etc. Because we wanted to test also such substituents in
pyridazine position 6, we envisaged a maleic anhydride instead
of the typically used methylfuranone as five-membered ring inter-
mediate. Therefore we started from the 4-chlorophenylglyoxylic
acid 14, which is available in two steps from chlorobenzene (13)
via Friedel–Crafts acylation and saponification (Scheme 3). After
transformation of 14 into its potassium salt, a Perkin-type conden-
sation37 with 2,4,6-trifluorophenylacetic acid delivered the diary-
lmaleic anhydride 15. This sensitive intermediate could be
transformed with hydrazine hydrate to the pyridazinedione 16,
which after dichlorination delivered the 3,6-dichloro-4,6-diarylpy-
ridazine 17. This compound is not only a close analog of Sumito-
mo’s pyridazine 4, but also a versatile building block for further
transformations. Its conversion with one equivalent of sodium
methoxide led regioselectively to 18, obviously because of sterical
hindrance in the attack of the other chloro group by the two
ortho-fluoro substituents in the adjacent phenyl ring. In a similar
manner, also the regioselective introduction of thioalkyl, different
amino groups and other halogens, such as fluoro and iodo into
pyridazine position 6 is possible.
3
4
Figure 1. Different mono- and bi-heterocyclic fungicidally active tubulin polymer-
isation promoters.
ticals23,24 and agrochemicals25–27 bear a pyridazine scaffold. Out-
side of the relatively narrow patent claims of Sumitomo covering
4, we have prepared many different pyridazines, for example,
derivatives of 4 with a fluorine or a cyano instead of the chloro sub-
stituent in position 3,28 with a heteroaryl ring instead of the triflu-
orophenyl in position 4,29 with a heteroaryl30 or an alkyl31 group
instead of the 4-chlorophenyl in position 5, and with a halogen
or an alkoxy group instead of the methyl group in position 6.32
In this article, we describe the synthesis and structure–activity
relationship of these fungicidal pyridazine tubulin polymerisation
promoters.
2. Results and discussion
2.1. Chemistry
Most of those diversely substituted pyridazines have been pre-
pared according to the original procedure, which researchers at
Sumitomo have used to approach such tetrasubstituted pyrida-
zines. This efficient process is demonstrated in Scheme 1 for the
synthesis of the chlorothienyl derivative 10. The key step of this
O
O
O
1. (COCl)2
2. MeONHMe.HCl
1. EtMgCl
2. Br2, HBr, AcOH
S
S
S
Cl
Cl
O
Cl
OH
N
Br
F
5
7
6
1.
OH, NEt3
O
F
F
2. DBU
3. air
Cl
Cl
F
F
S
N
S
N
F
F
F
F
H2NNH2.H2O
POCl3
Cl
S
F
F
F
HO
O
N
Cl
N
H
9
O
O
8
10
Scheme 1. General synthesis of pyridazine fungicides at the example of 10.

C. Lamberth et al. / Bioorg. Med. Chem. 20 (2012) 2803–2810
2805
F
F
F
F
O
OH,
O
O
O NEt3
S
S
F
Cl
F
Cl
O
Br
7
11
DBU
F
F
F
F
air
Cl
S
Cl
S
F
F
HO
O
O
O
O
8
12
Scheme 2. One-pot cyclocondensation of the hydroxyfuranone
bromoketone 7 and 2,4,6-trifluorophenylacetic acid.
8 from the a-
2.2. Mode of action
The highly active pyridazine fungicide 10 was submitted to a
polymerisation assay on pure porcine tubulin to check if also these
monocyclic compounds, as already confirmed for the mentioned
bicyclic subclasses, are disrupters of the microtubule dynamics. It
was compared against paclitaxel, which is a known tubulin poly-
merisation promoter and for which in the two highest concentra-
tions a clear increase in the OD340 value could be detected. A
very similar pattern was observed for 10, indicating a similar effect
on tubulin polymerization to paclitaxel. However, little effect of
10 has been observed at lower concentrations, which indicates a
lower promoting effect of this compound on pure porcine tubulin
compared to paclitaxel (Fig. 2).
Figure 2. Degree of polymerization of pure porcine tubulin in the presence of
paclitaxel and 10 at different concentrations in function of time.
activity relationship study of these positions revealed, that each
substituent exerts a distinct influence on the fungicidal efficacy.
2.3.1. Influence of the substituent in pyridazine position 3 on
the fungicidal activity
The best activity, especially against the Ascomycetes diseases
Mycosphaerella graminicola and Alternaria solani, was achieved with
3-chloropyridazines (Table 1, entry 3). The replacement of this
substituent with fluorine (entry 2) or methoxy (entry 5), easily
done by nucleophilic substitution reactions, leads to a significant
decrease of activity. The corresponding lactame (entry 1), which
is the required building block for the 3-chloropyridazine prepara-
tion, is completely inactive.
2.3. Structure–activity relationships
Tetrasubstituted pyridazine fungicides, such as 10 and 18, bear
four completely different functional groups. A thorough structure–
1. KOtBu
2.
F
OH,
F
Cl
F
O
1. MeO2CCOCl, AlCl
2. NaOH
3
O
Ac2O
OH
F
F
O
F
O
Cl
O
Cl
O
13
15
14
H2NNH .H2O,
2
NaOAC
Cl
Cl
Cl
F
F
F
F
F
F
Cl
O
HN
O
POCl3
NaOMe
F
F
N
F
N
N
Cl
N
H
16
O
N
Cl
17
18
Scheme 3. Synthesis of fungicidally active pyridazines with halogen, alkoxy, alkylthio and amino substituents in position 6.

2806
C. Lamberth et al. / Bioorg. Med. Chem. 20 (2012) 2803–2810
Table 1
Influence of the substituent R in pyridazine position 3 on the fungicidal activitya
Cl
F
F
N
F
N
R
Entry
R
Botrytis cinerea
Mycosphaerella graminicola
Alternaria solani
(tomato grey mould)
(wheat leaf blotch)
(tomato early blight)
1
2
3 (4)
OH
F
Cl
>200
8
6
>200
191
2
>200
17
2
4
5
CH3
OCH3
4
97
14
53
18
57
a
Results are given as the EC80 (mg LÀ1).
2.3.2. Influence of the substituent in pyridazine position 4 on
the fungicidal activity
of the two ortho-fluorine atoms is moved to the meta-position of
the phenyl ring (entry 3), if the para-methoxy group and one of
the ortho-fluorines change places (entry 4) or if the para-substitu-
ent is exchanged by hydrogen (entry 5), the efficacy clearly
decreases (Table 2). In principal it is possible to replace the haloge-
nated phenyl ring in pyridazine position 4 by a similar substituted
pyridine (entry 6).
As demonstrated by the very low EC80 values of entry 1, pyrid-
azines with a 2,4,6-trifluorophenyl moiety in position 4 seem to
possess the highest fungicidal activity, but the replacement of
the fluorine atom in the para-position of the phenyl ring by a
methoxy group also results in excellent activity (entry 2). If one
Table 2
Influence of the substituent R in pyridazine position 4 on the fungicidal activitya
Cl
N
R
N
N
Cl
Entry
1
R
Botrytis cinerea
(tomato grey mould)
Mycosphaerella graminicola
(wheat leaf blotch)
Alternaria solani
(tomato early blight)
F
F
1
1
1
3
2
1
F
F
CH
F
O
3
2
3
4
F
CH
O
3
23
46
95
19
17
F
F
F
F
>200
OCH3
5
6
78
15
60
9
54
33
F
Cl
N
Cl
a
Results are given as the EC80 (mg LÀ1).

C. Lamberth et al. / Bioorg. Med. Chem. 20 (2012) 2803–2810
2807
2.3.3. Influence of the substituent in pyridazine position 5 on
the fungicidal activity
clearly more active than the para-chlorophenyl lead compound 4
(entry 4).38 A benzyl group linked via the methylene group to the
pyridazine (entry 6) seems to possess a weaker activity than the al-
ready mentioned aryl or heteroaryl rings.
In principal this position of the pyridazine ring offers the great-
est flexibility, because several different substituents deliver a high
level of fungicidal activity (Table 3). Entries 2 and 3 demonstrate,
that 2-pyridyl as well as 3-pyridyl rings linked to pyridazine posi-
tion 5 offer very low EC80 values and therefore excellent efficacy.
Another successful substituent in this position are para-substituted
phenyl rings, here the 4-ethynyl-substituted derivative (entry 5) is
2.3.4. Influence of the substituent in pyridazine position 6 on
the fungicidal activity
It seems, that 6-methyl substituted pyridazines (entry 1) pos-
sess clearly the higher fungicidal activity compared to their
Table 3
Influence of the substituent R in pyridazine position 5 on the fungicidal activitya
F
F
R
N
F
N
Cl
Entry
R
Botrytis cinerea
Mycosphaerella graminicola
Alternaria solani
(tomato grey mould)
(wheat leaf blotch)
(tomato early blight)
Cl
1 (10)
25
41
5
49
4
S
2
1
1
6
N
Cl
N
3
1
2
2
2
Cl
4 (4)
CH
5
6
3
1
1
77
50
56
a
Results are given as the EC80 (mg LÀ1).
Table 4
Influence of the substituent R in pyridazine position 6 on the fungicidal activitya
Cl
F
F
R
N
F
N
Cl
Entry
R
Botrytis cinerea
Mycosphaerella graminicola
Alternaria solani
(tomato grey mould)
(wheat leaf blotch)
(tomato early blight)
1 (4)
2 (17)
3 (18)
4
CH3
Cl
OCH3
SCH3
N(CH3)2
6
2
2
127
58
124
40
96
14
90
6
134
92
42
16
5
a
Results are given as the EC80 (mg LÀ1).

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C. Lamberth et al. / Bioorg. Med. Chem. 20 (2012) 2803–2810
corresponding analogs with a chloro (entry 2), methoxy (entry 3),
methylthio (entry 4) or a dimethylamino group (entry 5) in this po-
sition (Table 4). Only the latter mentioned amine derivative
showed good activity against M. graminicola.
4.1.2. 2-Bromo-1-(5-chloro-thiophen-2-yl)-propan-1-one (7)
16 ml of a 1.5 M solution of ethylmagnesium chloride in tetra-
hydrofuran was added dropwise to a solution of 5-chloro-thio-
phene-2-carboxylic acid methoxy-methyl-amide (6, 6.2 g,
30 mmol) in 50 ml of tetrahydrofuran at 0 °C. The beige suspension
was stirred for 2 h at the same temperature. Subsequently 25 ml of
2 N hydrochloric acid were added and the reaction mixture was
poured into ethyl acetate. The organic layer was washed with
brine, dried over sodium sulfate and concentrated under reduced
pressure. The residue was purified by chromatography on silica
gel, using ethyl acetate/heptane 1:3 as eluent, delivering 1-(5-
chloro-thiophen-2-yl)-propan-1-one (4.0 g, 23 mmol) as a light
yellow solid (mp 51–53 °C). This intermediate was dissolved to-
gether with 0.1 ml of a 48% solution of hydrobromic acid in glacial
acetic acid in 60 ml of glacial acetic acid and cooled to 10 °C. Bro-
mine (3.9 g, 24 mol) was added dropwise at this temperature un-
der a nitrogen atmosphere. Subsequently the reaction mixture is
stirred for 1 h at room temperature. The resulting dark red solution
was concentrated under reduced pressure to deliver 2-bromo-1-
(5-chloro-thiophen-2-yl)-propan-1-one (7, 5.8 g, 23 mmol, 77%)
as a light brown oil, which could be used in the next step without
further purification. 1H NMR (CDCl3): d = 1.90 (d, 3H), 5.04 (q, 1H),
6.99 (d, 1H), 7.63 (d, 1H). MS (ESI): m/z = 254 (M+1).
3. Conclusions
Special tetrasubstituted pyridazines are highly active against
plant pathogens from the family of Ascomycetes, such as Botrytis
cinerea, M. graminicola and A. solani, but also against several Basid-
iomyetes and Deuteromycetes species. Their mode of action is the
promotion of tubulin polymerization, leading to the disruption of
microtubule dynamics. Such special tetrasubstituted pyridazines
can be prepared in only few steps, the key step is the condensation
of
with
sequently are ring-enlarged with hydrazine.
a
-unsubstituted carboxylic acids either with
a-bromoketones or
a-ketoacids to five-membered ring intermediates, which sub-
A careful analysis of the structure–activity relationships enabled
the identification of highly active fungicides. As it turned out, each
ring carbon atom of the pyridazine scaffold has to be specifically
substituted to reach the optimum efficacy. The most active pyrida-
zine fungicides bear next to both nitrogen ring atoms a chloro atom
and a methyl group. It is also important, that there is a bis-ortho hal-
ogen-sustituted phenyl ring with an additional para substituent
adjacent to the chlorine. Finally, we obtained the best fungicidal re-
sults with a para-substituted phenyl or pyridyl moiety between the
bis-ortho fluorophenyl ring and the methyl group.
4.1.3. 4-(5-Chloro-thiophen-2-yl)-5-hydroxy-5-methyl-3-(2,4,6-
trifluoro-phenyl)-5H-furan-2-one (8)
Triethylamine (1.2 g, 12 mmol) was added dropwise to
a
solution of 2-bromo-1-(5-chloro-thiophen-2-yl)-propan-1-one (7,
2.9 g, 11 mmol) and 2,4,6-trifluorophenylacetic acid (2.2 g,
12 mmol) in 40 ml of acetonitrile. The reaction mixture was stirred
for 16 h at room temperature, then 1,8-diazabicyclo[5,4,0]undec-7-
ene (4.1 g, 27 mmol) was added slowly, keeping the temperature
below 30 °C. The resulting dark brown solution was stirred 1 h at
room temperature, then air was bubbled through it for 3 h, during
which the colour of the solution changed to dark green. The reaction
mixture was poured on saturated aqueous ammonium chloride
solution and extracted with ethyl acetate. The organic layer was
washed with saturated aqueous sodium bicarbonate solution and
brine, dried over sodium sulfate and concentrated under reduced
pressure. The residue was purified by chromatography on silica
gel, using ethyl acetate/cyclohexane 1:5 as eluent, delivering
4-(5-chloro-thiophen-2-yl)-5-hydroxy-5-methyl-3-(2,4,6-trifluoro-
phenyl)-5H-furan-2-one (8, 2.2 g, 6.2 mmol, 54%) as a colourless
powder. Mp 155–156 °C. 1H NMR (CDCl3): d = 1.93 (s, 3H), 4.51
(s, 1H), 6.74–6.89 (m, 2H), 6.93 (d, 1H), 7.42 (d, 1H). MS (ESI):
m/z = 361 (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). Mass spectra were re-
corded on a Micromass LCT mass spectrometer. Melting points
were determined on a Büchi 535 melting point apparatus and are
uncorrected. Analytical thin-layer chromatography (TLC) was per-
formed using silica gel 60 F524 precoated plates. Preparative flash
chromatography was performed using silica gel 60 (40–63 lm, E.
Merck). All reactions were carried out under anhydrous conditions
in an inert atmosphere (nitrogen or argon) with dry solvents.
4.1.1. 5-Chloro-thiophene-2-carboxylic acid methoxy-methyl-
amide (6)
Oxalyl chloride (14.0 g, 0.11 mol) is added dropwise to a suspen-
sion of 5-chloro-thiophene-2-carboxylic acid (5, 15 g, 0.09 mol) and
catalytic amounts of N,N-dimethylformamide in 150 ml of dichlo-
romethane. The reaction mixture is stirred for 1 h at room temper-
ature, then heated to reflux for 1 h. After cooling to room
temperature, the reaction mixture is evaporated under reduced
pressure to deliver the intermediate 5-chloro-thiophene-2-carbox-
ylic acid chloride (16.7 g, 0.09 mol) as residue. This yellow oil was
dissolved in 180 ml of dichloromethane and N,O-dimethylamine
hydrochloride (9.9 g, 0.10 mol) was added at 5 °C. Subsequently
triethylamine (20.5 g, 0.20 mol) was added dropwise at 5 °C. The
resulting thick yellow suspension was diluted with 80 ml of dichlo-
romethane and stirred for 16 h at room temperature. It was washed
with water, the organic phase washed with brine, dried over
sodium sulfate and concentrated under reduced pressure to give
5-chloro-thiophene-2-carboxylic acid methoxy-methyl-amide (6,
18.8 g, 0.09 mol, 98%) as a yellow oil. 1H NMR (CDCl3): d = 3.38 (s,
3H), 3.80 (s, 3H), 6.96 (d, 1H), 7.79 (d, 1H). MS (ESI): m/z = 206
(M), 208 (M+2).
4.1.4. 5-(5-Chloro-thiophen-2-yl)-6-methyl-4-(2,4,6-trifluoro-
phenyl)-2H-pyridazin-3-one (9)
Hydrazine hydrate (0.3 g, 9.4 mmol) was added to a solution of
4-(5-chloro-thiophen-2-yl)-5-hydroxy-5-methyl-3-(2,4,6-trifluoro-
phenyl)-5H-furan-2-one (8, 2.0 g, 5.5 mmol) in 30 ml of 1-butanol.
The reaction mixture was heated to 120 °C for 16 h. Upon cooling
to room temperature a solid was obtained, which was filtered and
washed with hexane to deliver 5-(5-chloro-thiophen-2-yl)-
6-methyl-4-(2,4,6-trifluoro-phenyl)-2H-pyridazin-3-one (9, 1.3 g,
3.6 mmol, 66%) as light brown crystals. Mp 230–233 °C. 1H NMR
(CDCl3): d = 2.29 (s, 3H), 6.62–6.69 (m, 2H), 6.71 (d, 1H), 6.80 (d,
1H), 11.78 (br s, 1H). MS (ESI): m/z = 357 (M), 359 (M+2).
4.1.5. 3-Chloro-5-(5-chloro-thiophen-2-yl)-6-methyl-4-(2,4,6-
trifluoro-phenyl)-pyridazine (10)
A suspension of 5-(5-chloro-thiophen-2-yl)-6-methyl-4-(2,4,6-
trifluoro-phenyl)-2H-pyridazin-3-one (9, 890 mg, 2.5 mmol) and
5 ml of phosphorus oxychloride was heated to 110 °C for 1 h. After
cooling the reaction mixture was concentrated under reduced

C. Lamberth et al. / Bioorg. Med. Chem. 20 (2012) 2803–2810
2809
pressure. The remainder was taken up with ethyl acetate. The
4.1.9. 3,6-Dichloro-4-(4-chloro-phenyl)-5-(2,4,6-trifluoro-
phenyl)-pyridazine (17)
mixture of 4-(4-chloro-phenyl)-5-(2,4,6-trifluoro-phenyl)-
resulting solution was washed with water and brine, dried over so-
dium sulfate and evaporated. The residue was purified by chroma-
tography on silica gel, using ethyl acetate/cyclohexane 1:6 as
eluent, delivering 3-chloro-5-(5-chloro-thiophen-2-yl)-6-methyl-
4-(2,4,6-trifluoro-phenyl)-pyridazine (10, 880 mg, 2.3 mmol,
94%). Mp 102–103 °C. 1H NMR (CDCl3): d = 2.70 (s, 3H), 6.69–6.75
(m, 3H), 6.82 (d, 1H). MS (ESI): m/z = 375 (M), 377 (M+2).
A
1,2-dihydropyridazine-3,6-dione (16, 45 g, 0.13 mol) and 230 ml
of phosphorus oxychloride was heated to 120 °C for 2 h. The
reaction mixture was cooled and evaporated under reduced
pressure. The remainder was poured on water and extracted with
ethyl acetate. The organic layer was washed with brine, dried
over sodium sulfate and evaporated. The residue was purified by
chromatography on silica gel, using ethyl acetate/heptane 1:8
as eluent, delivering 3,6-dichloro-4-(4-chloro-phenyl)-5-(2,4,6-tri-
fluoro-phenyl)-pyridazine (17, 29 g, 75 mmol, 57%) as beige
crystals. 1H NMR (CDCl3): d = 6.59 (t, 2H), 7.01 (d, 2H), 7.26 (d, 2H).
MS (ESI): m/z = 390 (M), 393 (M+3).
4.1.6. 4-Chloro-phenylglyoxylic acid (14)
Methyl oxalyl chloride (265 g, 2.16 mol) was slowly added to a
suspension of aluminium chloride (200 g, 1.5 mol) in 750 ml of
chloroform at 0 °C. The reaction mixture was stirred for 1 h at
0 °C, then chlorobenzene (245 g, 2.19 mol) was slowly added at this
temperature. The resulting mixture was stirred for 16 h at room
temperature, then poured on water and diluted with dichlorometh-
ane. The phases were separated, the organic layer was washed with
water, dried over sodium sulfate and evaporated. The remainder
was purified by chromatography on silica gel, using ethyl acetate/
heptane 1:9 as eluent, delivering methyl 4-chloro-phenylglyoxy-
late (99 g, 0.5 mol). This intermediate was dissolved in 800 ml of
dioxane and 800 ml of 1 N sodium hydroxide were added. The mix-
ture was stirred for 2 h at room temperature, then acidified to pH 1
with 2 N hydrochloric acid and extracted with ethyl acetate. The or-
ganic layer was washed with brine, dried over sodium sulfate and
evaporated to deliver 4-chloro-phenylglyoxylic acid (14, 86 g,
0.47 mol 21%). 1H NMR (CDCl3): d = 7.53 (d, 2H), 8.10 (br s, 1H),
8.32 (d, 2H). MS (ESI): m/z = 186 (M+1), 165 (M–OH).
4.1.10. 3-Chloro-5-(4-chloro-phenyl)-6-methoxy-4-(2,4,6-
trifluoro-phenyl)-pyridazine (18)
0.25 g of a 30% solution of sodium methoxide in methanol was
added to a solution of 3,6-dichloro-4-(4-chloro-phenyl)-5-(2,4,6-
trifluoro-phenyl)-pyridazine (17, 0.48 g, 1.2 mmol) in 5 ml of
methanol. The reaction mixture was heated to reflux for 2 h, then
cooled, diluted with water and extracted with ethyl acetate. The
organic layer was washed with brine, dried over sodium sulfate
and evaporated. The remainder was purified by chromatography
on silica gel, using ethyl acetate/heptane 1:9 as eluent, delivering
3-chloro-5-(4-chloro-phenyl)-6-methoxy-4-(2,4,6-trifluoro-phe-
nyl)-pyridazine (18, 0.43 g, 1.1 mmol, 93%) as colourloss crystals.
1H NMR (CDCl3): d = 4.13 (s, 3H), 6.59–6.70 (m, 2H), 7.08 (d, 2H),
7.29 (d, 2H). MS (ESI): m/z = 385 (M), 387 (M+2)
4.1.7. 3-(4-Chloro-phenyl)-4-(2,4,6-trifluoro-phenyl)-furan-2,5-
dione (15)
4.2. Biochemistry
Potassium tert-butoxide (53 g, 0.47 mol) was added in portions
to a solution of 4-chloro-phenylglyoxylic acid (14, 86 g, 0.47 mol)
in 700 ml of methanol at room temperature. The mixture was stir-
red for 1 h at this temperature, then the white solid, which precip-
itated, was filtered, washed with cold methanol and dried in vacuo.
This potassium salt was taken up in 800 ml of acetic anhydride and
then 2,4,6-trifluorophenylacetic acid (73 g, 0.38 mol) was added.
The reaction mixture was heated first for 1 h to 80 °C, then for
1 h to 90 °C and finally for 1 h to 100 °C. Subsequently the mixture
was cooled to room temperature and the solvent was removed in
vacuo to obtain 3-(4-chloro-phenyl)-4-(2,4,6-trifluoro-phenyl)-
furan-2,5-dione (15, 148 g, 0.44 mol, 93%), which was directly
used in the next step without further purification. 1H NMR (CDCl3):
d = 6.76–6.83 (m, 2H), 7.30 (d, 2H), 7.82 (d, 2H). MS (ESI): m/z = 339
(M+1).
4.2.1. 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. 3-Chloro-5-(5-chloro-thiophen-2-yl)-6-
methyl-4-(2,4,6-trifluoro-phenyl)-pyridazine (10) was added in
different wells of a 96-well plate at the beginning of the reaction
at different concentrations (20, 2.2, 0.24 ppm) in a DMSO solution
(0.2 mM).
4.3. Biology
4.1.8. 4-(4-Chloro-phenyl)-5-(2,4,6-trifluoro-phenyl)-1,2-
dihydro-pyridazine-3,6-dione (16)
All tested compounds had a purity of at least 95%, the purifica-
tion has been either performed via chromatography (see Sections
4.1.5, 4.1.9 and 4.1.10) or by crystalisation.
120 g of a 2:1 mixture of hydrazine hydrate and water was
added dropwise to a mixture of 3-(4-chloro-phenyl)-4-(2,4,6-tri-
fluoro-phenyl)-furan-2,5-dione (15, 80 g, 0.24 mol) in 270 ml of
acetic acid. Sodium acetate anhydrous (22 g, 0.27 mol), was added
and the reaction mixture was heated to reflux for 2 h. Subsequently
the mixture was cooled, diluted with water and extracted with
ethyl acetate. The organic layer was washed with brine, dried over
sodium sulfate and evaporated under reduced pressure. The
residue was purified by chromatography on silica gel, using ethyl
acetate/heptane 1:3 as eluent, delivering 4-(4-chloro-phenyl)-5-
(2,4,6-trifluoro-phenyl)-1,2-dihydropyridazine-3,6-dione (16, 47 g,
0.13 mol, 57%) as colourless crystals. Mp 159–164 °C. 1H NMR
(CDCl3): d = 6.61 (t, 2H), 7.13 (d, 2H), 7.27 (d, 2H), 11.97 (br s,
1H), 13.83 (br s, 1H). MS (ESI): m/z = 353 (M), 355 (M+2).
4.3.1. Botryotinia fuckeliana (Botrytis cinerea)/tomato (action
against grey mould on tomato)
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).

2810
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spore suspension two days after application. The inoculated test
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