Tubulin modulating antifungal and antiproliferative pyrazinone derivatives

Article abstract of DOI:10.1016/j.bmc.2015.08.038

A novel class of synthetic tubulin polymerization disruptors, based on a substituted pyrazin-2-one core, has been discovered. These molecules have proven to be potent broad spectrum fungicides, with activity on agriculturally important ascomycete and basidiomycete pathogens. They have also been found to be particularly potent against human rhabdomyosarcoma cells. Using an efficient synthetic route, the agricultural and medicinal activity was explored.

Full text of DOI:10.1016/j.bmc.2015.08.038

Bioorganic & Medicinal Chemistry 24 (2016) 435–443
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Tubulin modulating antifungal and antiproliferative pyrazinone
derivatives
a
a
a
b
Andrew E. Taggi a, , Thomas M. Stevenson , James F. Bereznak , Paula L. Sharpe , Steven Gutteridge ,
Robert Forman c, John J. Bisaha a, Daniel Cordova b, Martina Crompton c, Lora Geist a, Patrick Kovacs a,
Eric Marshall a, Ritesh Sheth a, Courtney Stavis a, Chi-Ping Tseng a
⇑
a DuPont Crop Protection, Discovery Chemistry, 1090 Elkton Road, Newark, DE 19714, USA
b DuPont Crop Protection, Chemical Genomics, 1090 Elkton Road, Newark, DE 19714, USA
c DuPont Crop Protection, Discovery Biology, 1090 Elkton Road, Newark, DE 19714, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel class of synthetic tubulin polymerization disruptors, based on a substituted pyrazin-2-one core,
has been discovered. These molecules have proven to be potent broad spectrum fungicides, with activity
on agriculturally important ascomycete and basidiomycete pathogens. They have also been found to be
particularly potent against human rhabdomyosarcoma cells. Using an efficient synthetic route, the
agricultural and medicinal activity was explored.
Received 23 June 2015
Revised 25 August 2015
Accepted 26 August 2015
Available online 28 August 2015
Ó 2015 Elsevier Ltd. All rights reserved.
Keywords:
Fungicide
Tubulin
Pathogen
Synthesis
Cell line
Agriculture
1. Introduction
ultimately into larger structures which govern critical cellular pro-
cesses, such as mitosis. Interference with the dynamic equilibrium
It is projected that the world will need to increase food produc-
tion by 60% in order to feed a global population expected to be in
excess of 9 billion people by 2050. At the same time, it is estimated
that every year global crop yields are reduced by 20–40% by agri-
cultural pests.1 Because of the ability of plant pathogens to rapidly
develop resistance, it is imperative that new classes of broad
spectrum disease control agents, with novel modes of action, be
discovered in order to prevent catastrophic yield losses, which
can approach 80% in some circumstances.2 In order to discover a
fungicide with activity on a broad range of taxonomic classes of
fungi, one must target an essential pathway that is fairly well con-
served across the pathogens of interest. Historically, some of the
most successful fungicides have inhibited critical processes such
as mitochondrial electron transport,3 sterol biosynthesis,4 and
tubulin polymerization.
between polymerization and depolymerization of tubulin has his-
torically been an effective strategy for treating diseases character-
ized by rapid cellular proliferation, such as cancer.5 Conceptually,
there is much similarity between the proliferation of cancer cells
and the rapid cellular propagation of a fungal infection. It is there-
fore not surprising that tubulin polymerization disruptors have
been successfully used in agricultural settings since the 1960s,
starting with methyl benzimidazole carbamates (MBCs)6, and
more recently with zoxamide7 and the quinolinyloxyacetamides8
(which have yet to be exploited commercially).
With tubulin polymerization modulators playing such an
important role in agriculture and medicine, it is imperative that
novel chemotypes of known classes of molecules, as well as mole-
cules that bind to completely different binding sites, be discovered
and studied. In this paper, we will discuss the synthesis and biolog-
ical evaluation of a new class of tubulin modulating molecules,
based on the pyrazin-2-one core, for both medicinal and agricul-
tural use.
Tubulin is a member of a superfamily of globular proteins which
exist in six subtypes, including the most common
a- and b-tubulin.
a-
and b-tubulin polymerize into heterodimers and then
⇑
Corresponding author.
E-mail address: andrew.e.taggi@dupont.com (A.E. Taggi).
http://dx.doi.org/10.1016/j.bmc.2015.08.038
0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.

436
A. E. Taggi et al. / Bioorg. Med. Chem. 24 (2016) 435–443
in the pyrido[2,3-b]pyrazines (3) suggested the possibility of a
2. Results and discussion
2.1. Chemistry
carbonyl to replace one of the pyrimidine nitrogens. To satisfy
the need for another sp2 center in the core, which would be the
attachment point for the alkyl side chain, the pyrimidine nitrogen
was conceptually moved over by one position, resulting in the pyr-
azin-2-one core (4).13
When we started our work in the area of tubulin polymerization
modulators, we were aware of the triazolopyrimidines (1, Fig. 1)
from American Cyanamid and eventually BASF9 as well as subse-
quent research programs at Bayer10 and Syngenta.11 In particular,
we were drawn to the 2-pyrazolyl-pyrimidines (2),12 as they had
opened a new vein of opportunity by moving away from the bicyc-
lic core, into a linear arrangement. Additionally, the internal imine
The optimization and evaluation of the pyrazin-2-ones was
greatly simplified by the early adoption of a modular synthesis,
which allowed us to specifically modify the four pendant function-
alities in order to explore their biological relevance (Scheme 1).
The synthesis usually began with a Strecker reaction between an
aldehyde, an amine, and cyanide to establish the functionalities
that would ultimately be in the 1 and 6 positions of the pyrazinone
core. If necessary, the Strecker reactions could be catalyzed with
sodium bisulfite or a Lewis acid such as indium(III) chloride.14 In
cases where the amine had poor nucleophilicity, such as anilines
or fluorinated alkyl groups, a two-step procedure was employed
where the imine was prepared under dehydrating conditions,
and then subsequently allowed to react with a cyanide source to
F
F
F
F
O
NH
NH
N
N
N
F
N
Cl
generate the a-aminonitrile.
5
cevipabulin TTI-237
Wyeth (2005)
The pyrazinone core was then formed by treating the
-aminonitrile (8) with oxalyl chloride at elevated temperatures.
a
This reaction often suffered from yields in the range of 40–60%,
with the remaining mass balance being composed of mono-chlori-
nated pyrazinone. It was subsequently found that a more efficient
conversion to the dichloro pyrazinone could be achieved by the
addition of a small amount of DMF to the reaction mixture after
F
F
F
F
F
A
F
1
F
N
N
N
N
N
the a-aminonitrile had reacted with the oxalyl chloride. This pro-
F
N
N
N
Cl
F
cedure resulted in increased product yields, approaching 90% in
some cases. Under the same conditions, use of oxalyl bromide
resulted in the 3,5-dibromo-pyrazin-2-one, which could be used
to explore alternate substitutions at the 5 position. Finally, the
3-chloro group could be displaced with a variety of nucleophiles,
including NH containing heterocycles, such as pyrazole to form
the final targets. The same chlorine could also be subjected to tran-
sition metal catalyzed cross-coupling reactions to yield targets
with C-linked heterocycles. Amides could also be prepared from
the same synthon by reaction with N-cyanomethyl-benzotriazoles
and base followed by oxidation with peracetic acid.15
While in related art, the alkylaminoalkoxy side chain of
cevipabulin (5) could be added through the nucleophilic displace-
ment of the 4-fluoro on a 2,4,6-trifluorophenyl,16 this was not
possible in the pyrazinone system due to the concomitant dis-
placement of the N-linked pyrazole. To overcome this difficulty,
N
Cl
2
BAS600
BASF
American Cyanamid / BASF (1998)
2a; A = N (2002)
2b; A = C (2004)
F
F
F
F
NH
N
N
O
N
N
N
F
N
F
Cl
N
F
3
4
Syngenta (2004)
DuPont (2004)
Figure 1. Different mono- and bicyclic tubulin modulating fungicides.
1) oxalyl chloride
H
N
F
X
R
R
chlorobenzene
100º C, 4h
X
X
F
F
X
7
F
N
R
N
NH2
O
N
N
2
3
R
6
1
O
H
HN
F
N
NaCN
NaHSO3
H2O/MeOH
2) DMF
N
Cl
5
K2CO3
DMF 50º C
F
O
F
CN
F
4
Cl
N
Cl
6a; X = H
6b; X = F
8a; X = H, R = i-butyl
10a; X = H, R = i-butyl
10b; X = F, R = CH2CF2CF3
11a; X = H, R = i-butyl
11b; X = F, R = CH2CF2CF3
8b; X = F, R = CH2CF2CF3
6c; X = MeO
8c; X = MeO, R = (S)-2-methylbutyl
8d; X = F, R = i-butyl
10c; X = MeO, R = (S)-2-methylbutyl
10d; X = F, R = i-butyl
11c; X = MeO, R = (S)-2-methylbutyl
11d; X = F, R = i-butyl
R
SnBu
N
N
N
3
TMS-CN
7
1) LHMDS, THF, rt
toluene, reflux
Dean-Stark
NH2
N
PdCl2(PPh3)2
toluene, reflux
ZnI2
2) NH3-dioxane
CH2Cl2, reflux
3) peracetic acid
CN
F
X
X
F
F
X
R
N
R
N
R
N
O
O
F
N
F
F
O
N
Cl
N
Cl
9b; X = F, R = CH2CF2CF3
NH2
13b; X = F, R = CH2CF2CF3
12a; X = H, R = i-butyl
Scheme 1. The general synthesis of pyrazin-2-one fungicides.

A. E. Taggi et al. / Bioorg. Med. Chem. 24 (2016) 435–443
437
O
F
OH
F
1) Cs2CO3,
DMF, 70° C
TBAI, 4Å sieves
BBr3
O
N
N
O
N
N
N
F
F
N
N
CH2Cl2, rt
N
Cl
Cl
2)
R
11c
N
15
Cl
16a; R = CH3
16b; R = Cbz
N
F
O
+
R
H2
Pd / C
O
NH2
F
O
N
N
N
–
O
N
Cl
F
N
HCl / CH3OH
Cl
F
N
N
N
Cl
17a; R = CH3
17b; R = Cbz
18b
Scheme 2. Further elaboration of the substitution on phenyl.
4-methoxy substituted analogs were prepared. The methyl group
was then cleaved using boron tribromide and the phenolic oxygen
was alkylated with the appropriate side chain. It was important
that the alkylation be conducted under strictly anhydrous condi-
tions as it was found that any hydroxide present in the reaction
would displace the N-linked pyrazole (Scheme 2).
bind at a location similar to the vinca alkaloids,18 which is consis-
tent with the related cevipabulin (5).16
2.3. Structure–activity relationships
Our chemical optimization program allowed us to explore the
structure-activity relationships for substitution in all four quad-
rants of the pyrazinone core. In Tables 1–5, we will discuss these
relationships in terms of the control of wheat glume blotch
(WGB, Stagonospora nodorum), wheat brown rust (WLR, Puccinia
recondita f. sp. Tritici), tomato gray mold (TBT, Botryotinia
fuckeliana) and ultimately a human rhabdomyosarcoma cell line
(RMS; ATCC CCL-136).
2.2. Mode of action
The effect of analogs 11a and 11d on the dynamics of tubulin
polymerization was investigated relative to paclitaxel, a known
tubulin polymerization enhancer, along with a DMSO control.
Figure 2 shows the effect on polymerization of bovine tubulin as
measured by the change in absorbance at 340 nm in the presence
and absence of 11a and 11d at the indicated concentrations. The
progress curve indicates an action similar to paclitaxel by dramat-
ically enhancing and stabilizing the polymerized state over the
course of the assay relative to the solvent control. The superior
potency of 11d over 11a in the bovine tubulin assay was consistent
with its superior antifungal activity on plants (Table 4, entries 1
and 2), supporting the idea that the antifungal activity is derived
from disrupting the dynamic equilibrium between polymerized
and depolymerized tubulin. Ultimately, using mutation studies,17
it was determined that while the compounds caused tubulin to
behave in a similar manner to paclitaxel, the pyrazinones actually
2.3.1. Influence of the nitrogen substituent on fungicidal
activity
Substitution at N1 tolerated the greatest structural diversity
including alkyl, cycloalkyl and aryl groups. While straight chain
alkyl and alkenyl groups (Table 1, entries 1 and 5) were potent in
our whole plant assays, we quickly found that branching, and in
particular branching on the beta carbon (entries 2–4), enhanced
potency and spectrum, as seen with iso-butyl, sec-butyl and cyclo-
propylmethyl. Ultimately, it was found that all of the activity
resided with the (S)-enantiomer of the sec-butyl side chain.
Branching could also be achieved using halogenated alkyl groups
(entries 6–8), which exhibited some improvement in activity over
the n-butyl analog (entry 1). Branching alpha to the pyrazinone
ring nitrogen was also tolerated, which allowed for the examina-
tion of cycloalkyl and aryl groups (entries 9–13). A variety of sub-
stituents in the ortho, meta and para positions of the aryl ring were
also acceptable.
2.3.2. Influence of substitution at the 3-positon of the
pyrazinone on fungicidal activity
Having established both iso-butyl and sec-butyl as excellent
nitrogen substituents, we chose to examine a broad range of sub-
stitution at the 3-position. Our initial hit in this area of chemistry
(11a) had established that an N-linked pyrazole (Table 2, entry 1)
resulted in excellent activity. We subsequently found that substi-
tution in the 3-position of the pyrazole was tolerated, though not
necessarily beneficial (entry 2). The addition of a methyl group in
the 5-position (entry 3) eliminated nearly all spectrum and
potency. Examination of additional N-linked azoles found that
1,2,4 and 1,2,3-triazoles were effective, although it appeared
preferable that there not be two nitrogens flanking the point of
connection as in the 1,2,3-triazol-2-yl (entries 4–6). An N-linked
imidazole was found to be completely inactive, suggesting that
Figure 2. The enhanced rate of polymerization of bovine tubulin by 11a (3
11d (3 M), relative to solvent (DMSO) control. The progress of tubulin polymer-
ization in the presence of paclitaxel (10 M) is shown for comparison.
lM) and
l
l

438
A. E. Taggi et al. / Bioorg. Med. Chem. 24 (2016) 435–443
Table 1
Influence of the nitrogen substituent on fungicidal activitya
F
F
R
N
O
N
F
N
N
Cl
WGB
200 40 10
100 98
100 100 90
100 100 98
WLR
200 40 10
100 88 26
100 100 100 92 100 100 100 100
100 100 100 89 100 100 100 100
TBT
Entry
1
R
n-butyl
2
2
200 40 10
2
0
-
0
0
0
0
0
0
0
0
-
-
-
100
0
2 (11d)
i-butyl
3 (4)
s-butyl
4
5
6
c-propyl-CH2-
allyl
-
-
-
-
-
100 100
100 97
99 78
100 89
-
-
-
-
-
100 100 80
99 89
-
-
-
-
-
99 98 98
99 99 70
100 99 38
100 100 99
99 99 99
0
CF3CF2CF2CH2-
CF3CF2CH2-
2-CF3-n-propyl
c-hexyl
99 96 68
100 99 91
100 100 92
7 (11b)
8
9
100
0
100 99 73
99 97 89
-
99 99 99
-
10
11
12
13
Ph
4-Cl-Ph
3-Cl-Ph
2-Cl-Ph
-
-
-
-
99 98 60
100 100 78
99 99 73
-
-
-
-
99 87 45
100 100 96
100 99 86
100 100 97
-
-
-
-
100 100 100
99 99 92
100 99 97
100 100 99
100 94
0
a
WGB, WLR and TBT results are given as percent control relative to the untreated control plants. Rates are given in
ppm. Gray shading represents control in excess of 70%.
Table 2
Influence of substitution at the 3-positon of the pyrazinone on fungicidal activitya
F
R
N
O
A
F
N
Cl
WGB
200 40 10
WLR
200 40
TBT
200 40
Entry
A
R
2
0
0
-
10
2
10
97 75
100 100 95
2
1 (11a)
pyrazol-1-yl
i-butyl
i-butyl
s-butyl
s-butyl
s-butyl
s-butyl
s-butyl
i-butyl
i-butyl
i-butyl
i-butyl
i-butyl
i-butyl
i-butyl
s-butyl
s-butyl
s-butyl
i-butyl
-
-
99 92
-
-
100 100 28
100 94 25
-
-
99
2
3-Me-pyrazol-1-yl
3,5-di-Me-pyrazol-1-yl
1,2,4-triazol-1-yl
1,2,3-triazol-1-yl
1,2,3-triazol-2-yl
imidazol-1-yl
1-Me-imidazole-4-yl
1-Me-imidazole-2-yl
pyrid-2-yl
95
0
0
0
-
3
0
73
98
73
86
0
22
98
67
19
0
0
-
-
-
0
0
0
-
-
-
4
100 94
-
100 100
5
73
0
0
0
0
-
-
67
-
-
98
97
0
81
0
33
-
-
6
-
-
-
7
8
0
0
0
0
0
-
-
0
0
0
-
-
0
0
0
0
-
-
97
0
67
0
0
-
99
0
97
0
0
-
100 99
0
-
9
0
0
10 (12a)
11
99
-
0
-
99
-
85
0
-
100 99
-
pyrid-3-yl
0
0
0
0
0
-
0
0
-
0
0
0
0
-
0
0
-
-
-
0
0
0
83
0
-
0
0
-
12
pyrid-4-yl
-
0
-
0
0
13
pyrazin-2-yl
98
-
0
99
-
89
0
98
-
98
0
14
thiazol-2-yl
0
0
-
0
-
0
-
15
CN
0
0
96
-
74
99
-
99
99
99
92
16
H2NC(=O)-
-
95
51
0
0
0
0
0
0
-
100 99 80
97 92
90 57
17
MeHNC(=O)-
-
-
99
74
0
-
18
MeC(=O)NH-
0
100 90
0
-
99
15
-
a
WGB, WLR and TBT results are given as percent control relative to the untreated control plants. Rates are given in ppm.
Gray shading represents control in excess of 70%.
having one nitrogen adjacent to the point of connection was possi-
bly required (entry 7). To further explore this, we turned our atten-
tion to C-linked azoles like 1-methyl-imidazole-4-yl (entry 8),
which returned a nitrogen to a flanking position, and restored
activity in the whole plant assays. While the alternate isomer
1-methyl-imidazole-2-yl has the required flanking nitrogen, it
has a methyl in the same location that was previously seen to elim-
inate nearly all activity (entries 3 and 9). The requirement that a
flanking nitrogen be present carried through to six membered
rings as evidenced by the activity of 2-pyridyl and 2-pyrazinyl
and the inactivity of 3- and 4-pyridyl (entries 10–13). It was sur-
prising, however, that the ‘pseudo’ six-membered 2-thiazole did
not show activity at the rates tested (entry 14). Perhaps a foreshad-
owing of future discoveries,19 it was found that the presence of a
pendant heterocycle was not a requirement, and it could be
replaced with simple groups such as cyano, and N- and C-linked

A. E. Taggi et al. / Bioorg. Med. Chem. 24 (2016) 435–443
439
Table 3
revealed that the methylene protons on the iso-butyl were clearly
diastereotopic, implying the presence of atropisomers due to
restricted rotation about the pyrazinone-phenyl bond.20
Subsequently, we were able to separate the two atropisomers by
chiral HPLC and determined that all of the fungicidal activity resided
with one atropisomer, while the other was completely inactive.
We also found that the phenyl ring could be replaced with lar-
ger cycloalkyl groups, such as cyclopentyl (entry 5), while smaller
rings such as cyclopropyl (entry 6) resulted in a complete loss of
activity. The cyclopropyl could be opened up to form an isopropyl
(entry 7), which results in very similar activity as the larger
cycloalkyl rings. These findings, plus the importance of 2-substitu-
tion on the phenyl ring, imply that this group needs to be out of
plane from the pyrazinone core in order to achieve significant
potency. Somewhat surprisingly, a t-butyl group was completely
inactive.
Influence of substitution at the 5-positon of the pyrazinone on fungicidal activitya
F
N
N
O
N
F
N
R
WGB
200 40 10
99 92
Br 100 100 86
WLR
200 40 10
100 100 28
TBT
200 40 10
99 97 75
100 100 99
Entry
1 (11a) Cl
R
2
0
-
0
-
2
2
-
-
-
2
3
4
100 99 88
-
0
-
-
0
-
Me
H
-
0
98
0
0
0
-
100 92
-
-
100 99
-
98
0
0
-
a
WGB, WLR and TBT results are given as percent control relative to the untreated
control plants. Rates are given in ppm. Gray shading represents control in excess of
70%.
2.3.5. Elaboration of the 6-phenyl: influence on fungicidal and
anti-proliferative activity
amides (entries 15–18). It remained true that heteroatoms were
required to be proximal to the linkage to the core, as simple alkyl
and phenyl groups were inactive.
From the time that we had first confirmed the tubulin-based
mode of action for the pyrazinone fungicides, we were interested
in determining whether they would also exhibit anti-proliferative
activity on cancer cell lines. We were pleased to find that against
rhabdomyosarcoma cells (RMS) our leading antifungal molecules
exhibited IC50 values in the 10–20 nM range (Table 5, entries 1
and 2), comparable to paclitaxel, which was run as a standard.
Knowing the relationship between our pyrazinones and the clinical
candidate cevipabulin (5), we chose to further elaborate the para
position of the phenyl ring on our leading antifungal candidates
with alkylamino-alkoxy chains.21 While a two carbon linker
proved to diminish the activity (entry 3), the analogs with a
three-carbon linker improved potency in excess of 10 fold (entries
4 and 5). This excellent potency was ultimately confirmed and
extended to a wide variety of cancer cell lines at an independent
laboratory. When examined in our traditional whole plant anti-
fungal assays, the most proficient compounds from the RMS assay
(entries 4 and 5) exhibited diminished potency as compared to the
parent antifungal compounds (entries 1 and 2). This is potentially
due to less-than-optimal physical properties, for whole plant
translation, imparted by the alkylamino-alkoxy side chain.
2.3.3. Influence of substitution at the 5-positon of the
pyrazinone on fungicidal activity
The least permissive portion of the molecule proved to be
5-chloro in our original hit compound 11a (Table 3, entry 1). We
found that it could be replaced with another halogen, such as bro-
mine, or a methyl group (entries 2 and 3) without significant
change in activity. Replacement of the chlorine with a proton
resulted in a near-complete loss of activity. Substitution with
larger groups was also detrimental. Due to the biological and syn-
thetic benefits of the 5-chloro, we did not examine additional
modifications.
2.3.4. Influence of substitution at the 6-positon of the
pyrazinone on fungicidal activity
Due to analogy with related areas already present in the litera-
ture, we knew the clear importance of an ortho halogen on the
6-phenyl, and thus our initial hit compound 11a possessed a
2,6-difluoro phenyl (Table 4, entry 1). Adding an extra fluorine in
the para position of the phenyl clearly improved the potency of
the molecule (entry 2), but it is debatable as to whether the addi-
tion of a third fluorine in the meta-position was as successful
(entry 3). 2,4-Disubstituted phenyls were also quite successful,
especially with a larger ortho-group such as chlorine (entry 4).
1H NMR analysis of the 2-chloro-4-fluoro-phenyl analog (entry 4)
3. Conclusions
A new class of pyrazinone fungicides has been demonstrated to
exhibit potent broad spectrum activity on a number of economi-
Table 4
Influence of substitution at the 6-positon of the pyrazinone on fungicidal activitya
N
N
R
O
N
N
Cl
WGB
200 40 10
99 92
WLR
200 40 10
100 100 28
100 100 100 92 100 100 100 100
100 99 32 100 98 94
100 100 100 83 100 100 100 100
TBT
Entry
1 (11a)
R
2
2
200 40 10
2
2,6-di-F-Ph
-
0
0
0
0
-
-
-
-
-
-
99 97 75
2 (11d) 2,4,6-tri-F-Ph 100 100 90
3
4
5
6
7
8
2,3,6-tri-F-Ph
2-Cl-4-F-Ph
c-pentyl
c-pro
i-pro
t-butyl
-
100 87
100 100 99
-
-
0
0
0
0
0
0
0
0
-
-
-
-
100 91
-
-
-
-
-
-
-
-
100 99
-
-
-
-
-
-
-
-
0
0
0
0
98 18
99 99
0
0
0
0
a
WGB, WLR and TBT results are given as percent control relative to the untreated control plants. Gray shading
represents control in excess of 70%.

440
A. E. Taggi et al. / Bioorg. Med. Chem. 24 (2016) 435–443
Table 5
Elaboration of the 6-phenyl and the influence on fungicidal and anti-proliferative activitya,b
O
O
OH
H
O
(s)
O
F
R
O
NH
O
N
N
O
O
O
OH
F
N
O
O
OH
Cl
N
O
Paclitaxel
WGB
WLR
TBT
Entry
1
2 (11c)
3
R
F
RMS 200 40 10
2
0
200 40 10
2
200 40 10
2
20
10
-
100 99
-
100 100 99
-
100 100 100
-OMe
100 100 100 69 100 100 100 99 99 99 99 99
-O(CH2)2NMe2
-O(CH2)3NMe2
210
1.3
-
-
-
-
0
0
-
100 91 55
0
99 68
0
0
0
4 (17a)
5 (18b)
Paclitaxel
94
0
0
100 99 89 68
-
99 94
-O(CH2)3NHMe-HCl 0.6
8.4
100 88 33
91
100 100 99 68 100 99 99 21
95
-
-
-
-
-
-
0
-
-
-
a
WGB, WLR and TBT results are given as percent control relative to the untreated control plants. Rates are given in ppm.
Gray shading represents control in excess of 70%.
b
RMS Cellular proliferation results are given as the IC50 (nM).
cally important plant pathogens including Stagonospora nodorum,
Puccinia recondita f. sp. tritici and Botryotinia fuckeliana. The mode
of action of these compounds was determined to be through inter-
ference with tubulin polymerization dynamics.
A highly-convergent synthesis allowed for the study of the
structure-activity relationships in all four quadrants of the pyrazi-
none core. Ultimately, it was found that branched alkyl groups and
an N-linked pyrazole in the 1 and 3 positions respectively, lead to
optimal activity. 2,4,6-Trisubstituted phenyl rings in the 6 position
of the pyrazinone core exhibited the best potency and spectrum,
although fungicidal activity was observed with other non-aromatic
and acyclic groups.
temperature remained below 35 °C. The reaction mixture was
stirred at room temperature for 18 h. The mixture was parti-
tioned between water (150 mL) and dichloromethane (150 mL).
The organic layer was washed with water (2 Â 50 mL). The
organic layer was dried (MgSO4) and evaporated under reduced
pressure to give an oil. Flash chromatographic purification on
silica gel with hexanes as eluant and pooling of appropriate
fractions gave 2,6-difluoro-a-[(2-methylpropyl)amino]benze-
neacetonitrile (8a, 4.92 g, 21 mmol, 55%) of the title compound
as an oil. 1H NMR (CDCl3): d 7.36 (m, 1 H), 6.98 (m, 2 H), 4.94
(bs, 1H), 2.75 (m, 1H), 2.52 (bs, 1 H), 1.84 (bs, 1 H), 1.78 (m,
1H), 0.95 (m, 6H).
While the optimization program was driven by the control of
agronomically-relevant plant pathogens, it was found that this
class of chemistry shows promise in pharmaceutical applications.
The best fungicides exhibited activity comparable to paclitaxel
against rhabdomyosarcoma cells, while a small number of analogs
optimized for pharmaceutical utility showed a ten-fold increase in
potency. Given the need for novel mode of action antifungals in
both agriculture and medicine, coupled with the potential for other
applications,22 this class of tubulin-modulating pyrazinones should
be the subject of further investigation.
4.1.2. 3,5-Dichloro-6-(2,6-difluorophenyl)-1-(2-methylpropyl)-2
(1H)-pyrazinone (10a)
A solution of oxalyl chloride (3.34 g, 26 mmol) in chlorobenzene
(35 mL) was stirred at 25 °C and 2,6-difluoro-a-[(2-methylpropyl)
amino]-benzeneacetonitrile (6a, 2.5 g, 9.0 mmol) was added via
an addition funnel. The resulting reaction mixture was heated at
70 °C for 18 h and at 90 °C for 24 h. The solvent was evaporated
under reduced pressure to leave an oil. This residue was subjected
to silica gel chromatographic purification using a gradient of ethyl
acetate/hexanes (1:9 to 2:3), and the appropriate fractions were
pooled to afford 3,5-dichloro-6-(2,6-difluorophenyl)-1-(2-methyl-
propyl)-2(1H)-pyrazinone (10a, 1.2 g, 3.6 mmol, 33%) as an oil
which solidified on standing. 1H NMR (CDCl3): d 7.6 (m, 1H), 7.1
(m, 1H), 7.0 (m, 1H), 3.7 (m, 2H), 1.9 (m, 1H), 0.9 (m, 3H), 0.7 (d, 3H).
4. Experimental section
4.1. Chemistry
All new compounds were characterized by standard spectro-
scopic methods. 1H NMR spectra were recorded on a Varian Unity
400 spectrometer at 400 MHz using deuterated solvent and
tetramethylsilane as an internal standard. Chemical shifts are
reported in ppm downfield from the standard (TMS, d = 0.00). All
reactions were carried out under anhydrous conditions under an
inert atmosphere (nitrogen or argon) with commercially available
dry solvents. Additional synthetic examples and compound charac-
terization can be found in the patent literature.13,21
4.1.3. 5-Chloro-6-(2,6-difluorophenyl)-1-(2-methylpropyl)-3-
(1H-pyrazol-1-yl)-2(1H)-pyrazinone (11a)
A mixture of 3,5-dichloro-6-(2,6-difluorophenyl)-1-(2-methyl-
propyl)-2(1H)-pyrazinone (10a, 0.20 g, 0.60 mmol), pyrazole
(45 mg, 0.66 mmol) and potassium carbonate (166 mg, 1.2 mmol)
dissolved in N,N-dimethylformamide (2 mL) was heated at 60 °C
for 18 h. The mixture was partitioned between ethyl acetate
(20 mL) and water (10 mL). The organic layer was washed with
water (3 Â 10 mL). The residue after evaporation was subjected
to silica gel chromatographic purification using a gradient of
hexanes/ethyl acetate (1:9 to 2:3) as eluant to give 5-chloro-6-
(2,6-difluorophenyl)-1-(2-methylpropyl)-3-(1H-pyrazol-1-yl)-2
(1H)-pyrazinone (11a, 0.060 g, 0.16 mmol, 27%). 1H NMR (CDCl3): d
9.1 (m, 1H), 7.9 (m, 1H), 7.5 (m, 1H), 7.1 (m, 2H), 6.5 (m, 1H), 3.8 (d,
2H), 2.0 (m, 1H), 0.8 (d, 6H). Mp = 118–119 °C.
4.1.1. 2,6-Difluoro-a-[(2-methylpropyl)amino]benzeneacetonitrile
(8a)
To a solution of isobutylamine (2.92 g, 40 mmol) and sodium
cyanide (1.94 g, 40 mmol) in water (40 mL) was added a solution
of 2,6-difluorobenzaldehyde (5.7 g, 40 mmol) in methanol
(40 mL). The addition was done at such a rate so that the

A. E. Taggi et al. / Bioorg. Med. Chem. 24 (2016) 435–443
441
4.1.4. 5-Chloro-6-(2,6-difluorophenyl)-1-(2-methylpropyl)-3-(2-
pyridinyl)-2(1H)-pyrazinone (12a)
mixture of 3,5-dichloro-6-(2,6-difluorophenyl)-1-(2-
methylpropyl)-2(1H)-pyrazinone (10a, 200 mg, 0.6 mmol),
4.1.8. 5-Chloro-1-(2,2,3,3,3-pentafluoropropyl)-3-(1H-pyrazol-
1-yl)-6-(2,4,6-trifluorophenyl)-2(1H)-pyrazinone (11b)
mixture of 3,5-dichloro-1-(2,2,3,3,3-pentafluoropropyl)-6-
A
A
(2,4,6-trifluorophenyl)-2(1H)-pyrazinone (10b, 0.47 g, 1.1 mmol)
and pyrazole (0.15 g, 2.2 mmol) in N,N-dimethylformamide
(5 mL) was heated to 60 °C overnight. The reaction mixture was
allowed to cool to room temperature and concentrated in vacuo.
The resulting residue was subjected to silica gel flash chromatogra-
phy (10% to 20% gradient of ethyl acetate in hexane as eluant) to
provide partially purified material. Trituration of this material with
a mixture of hexane and n-butyl chloride provided 5-chloro-1-
(2,2,3,3,3-pentafluoropropyl)-3-(1H-pyrazol-1-yl)-6-(2,4,6-trifluo-
rophenyl)-2(1H)-pyrazinone (11b, 0.30 g, 0.65 mmol, 59%) as a
white solid. 1H NMR (CDCl3): d 9.05 (d, 1H), 7.93 (d, 1H), 6.94–
6.88 (m, 2H), 6.55 (s, 1H), 4.75–4.50 (m, 2H). Mp = 147–149 °C.
tributylstannylpyridine (240 mg, 0.63 mmol) and bis(triph-
enylphosphino)palladium(II) chloride (20 mg, 0.03 mmol) was
heated in toluene at 110 °C for 18 h. The mixture was filtered
through a pad of CeliteÒ and rinsed with ethyl acetate. The solvent
was evaporated under reduced pressure. The residue after evapo-
ration was subjected to silica gel chromatographic purification
using a gradient of ethyl acetate/hexanes (1:9 to 2:3) to give
5-chloro-6-(2,6-difluorophenyl)-1-(2-methylpropyl)-3-(2-pyridi-
nyl)-2(1H)-pyrazinone (12a, 0.056 g, 0.15 mmol, 25%) as an oil.
1H NMR (CDCl3): d 8.86 (m, 1H), 8.43 (m, 1H), 7.83 (m, 1H), 7.59
(m, 1H), 7.38 (m, 1H), 7.12 (m, 2H), 3.79 (d, 2H), 2.00 (m, 1H),
0.79 (d, 6H).
4.1.9. 6-Chloro-3-oxo-4-(2,2,3,3,3-pentafluoropropyl)-5-(2,4,6-
trifluorophenyl)-pyrazine-2-carboxamide (13b)
4.1.5. 2,2,3,3,3-Pentafluoro-N-[(2,4,6-trifluorophenyl)methylene]-
1-propanamine (9b)
To a solution of 3,5-dichloro-1-(2,2,3,3,3-pentafluoropropyl)-6-
(2,4,6-trifluorophenyl)-2(1H)-pyrazinone (10b, 0.85 g, 2.0 mmol)
in tetrahydrofuran (10 mL) was added 1H-benzotriazole-1-ace-
tonitrile (0.48 g, 3.0 mmol) and lithium-bis(trimethylsilyl)amide
(1.0 M solution in tetrahydrofuran, 5.0 mL, 5 mmol). The resulting
mixture was stirred at room temperature for 1.5 h. A solution of
ammonia in dioxane (0.5 M, 12 mL, 6 mmol) was then added, and
the reaction mixture was stirred for an additional 10 min. Peracetic
acid (32 wt % solution in acetic acid), 1.68 mL) was added dropwise
to the reaction mixture, and the resulting mixture stirred at room
temperature for 3 h. Saturated aqueous sodium hydrogensulfite
was then added (50 mL) and the reaction mixture was extracted
with ethyl acetate (2 Â 50 mL). The combined organic extracts
were dried over MgSO4, filtered, and concentrated under reduced
pressure. The residue was purified by silica gel chromatography
(50–80% ethyl acetate in hexanes as eluant) to afford 6-chloro-3-
oxo-4-(2,2,3,3,3-pentafluoropropyl)-5-(2,4,6-trifluorophenyl)-pyr-
azine-2-carboxamide (13b, 0.150 g, 0.34 mmol, 17%). 1H NMR
(CDCl3): d 877 (br s, 1H), 6.94 (t, 2H), 6.17 (br s, 1H), 4.65 (m,
2H), Mp = 242–243 °C.
A mixture of 2,4,6-trifluorobenzaldehyde (4.5 g, 28 mmol) and
2,2,3,3,3-pentafluoropropylamine (4.2 g, 28 mmol) in toluene
(30 mL) was heated at reflux overnight using a Dean-Stark appara-
tus. The reaction mixture was allowed to cool to room temperature
and concentrated in vacuo to provide 2,2,3,3,3-pentafluoro-N-
[(2,4,6-trifluorophenyl)methylene]-1-propanamine (9b, 6.6 g,
23 mmol, 81%). This material was used directly in subsequent reac-
tions without further purification. 1H NMR (CDCl3): d 8.50 (s, 1H),
6.80–6.72 (m, 2H), 4.23–4.16 (m, 2H).
4.1.6. 2,4,6-Trifluoro-
benzeneacetonitrile (8b)
mixture of 2,2,3,3,3-pentafluoro-N-[(2,4,6-trifluorophenyl)
a-[(2,2,3,3,3-pentafluoropropyl)-amino]
A
methylene]-1-propanamine (9b, 6.6 g, 23 mmol), zinc iodide
(7.2 g, 23 mmol), and 5 Å molecular sieves (23 g) in dichloro-
methane (25 mL) was treated with trimethylsilyl cyanide (18 mL,
135 mmol) and the reaction mixture was heated at reflux over-
night. After cooling to room temperature, the reaction mixture
was filtered through CeliteÒ and concentrated in vacuo. The reac-
tion residue was treated with methanol (100 mL) and 10% aqueous
sodium bicarbonate solution (20 mL), and the resulting mixture
was extracted with diethyl ether (2 Â 50 mL). The ether phase
was separated, dried over MgSO4, and concentrated in vacuo. The
resulting crude residue was purified via silica gel flash
chromatography (5–10% gradient of ethyl acetate in hexane as
4.1.10. 2-(2,6-Difluoro-4-methoxy-phenyl)-2-((2S)-2-
methylbutylamino)-acetonitrile (8c)
To a solution of sodium bisulfite (1.71 g, 17 mmol) in a mixture
of deionized water (32 mL) and methanol (3.0 mL) at room tem-
perature was added 2,6-difluoro-4-methoxybenzaldehyde (2.7 g,
16 mmol). The reaction mixture was stirred for 15 min, and sodium
cyanide (0.81 g, 17 mmol) was added. The reaction mixture was
stirred for an additional 20 min and cooled using an ice water bath.
eluant) to provide 2,4,6-trifluoro-a-[(2,2,3,3,3-pentafluoro-
propyl)-amino]benzeneacetonitrile (8b, 1.0 g, 3.1 mmol, 14%).
1H NMR (CDCl3): d 6.86–6.74 (m, 2H), 5.04 (d, 1H), 3.55–3.30 (m,
2H), 2.27–2.21 (m, 1H).
A
solution of (S)-2-methylbutylamine (Sigma–Aldrich, 1.5 g,
17 mmol) in methanol (6.0 mL) was added over approximately
2 min, and the resulting reaction mixture was stirred at 0 °C for
15 min and then heated to 35 °C for 2 h. The resulting mixture
was then extracted with ethyl acetate (2 Â 40 mL), and the com-
bined organic layers were washed with brine, dried (MgSO4) and
concentrated to give 2-(2,6-difluoro-4-methoxy-phenyl)-2-((2S)-
2-methylbutylamino)-acetonitrile (8c, 3.9 g, 15 mmol, 93%) as an
oil. 1H NMR (CDCl3): d 6.51 (m, 2H), 4.83 (br s, 1H), 3.80 (s, 3H),
2.76 (m, 1H), 2.52 (m, 1H), 1.49 (m, 2H), 1.17 (m, 1H), 0.90 (m, 6H).
4.1.7. 3,5-Dichloro-1-(2,2,3,3,3-pentafluoropropyl)-6-(2,4,6-tri-
fluorophenyl)-2(1H)-pyrazinone (10b)
Oxalyl chloride (4.3 mL, 50 mmol) was added dropwise to a
mixture of 2,4,6-trifluoro-a-[(2,2,3,3,3-pentafluoropropyl)amino]
benzeneacetonitrile (8b, 3.2 g, 9.9 mmol) in chlorobenzene
(20 mL) at room temperature. The resulting mixture was heated
to 100 °C for 3 h, and then allowed to cool to room temperature.
One drop of N,N-dimethylformamide was then added. The reaction
mixture was reheated to 100 °C overnight. Then the reaction mix-
ture was again allowed to cool to room temperature and concen-
trated in vacuo to provide a crude residue, which was purified
via silica gel flash chromatography (10% ethyl acetate in hexane
as eluant) to provide 3,5-dichloro-1-(2,2,3,3,3-pentafluoro-
propyl)-6-(2,4,6-trifluorophenyl)-2(1H)-pyrazinone (10b, 0.47 g,
1.1 mmol, 11%).
4.1.11. 3,5-Dichloro-6-(2,6-difluoro-4-methoxyphenyl)-1-((2S)-
2-methylbutyl)-2(1H)-pyrazinone (10c)
A
solution of 2-(2,6-difluoro-4-methoxy-phenyl)-2-((2S)-2-
methylbutylamino)-acetonitrile (8c, 3.9 g, 15 mmol) in chloroben-
zene (16 mL) was added dropwise over 20 min to a solution of oxalyl
chloride (9.3 g, 73 mmol) in chlorobenzene (39 mL) at room temper-
ature. The reaction mixture was then heated to 100 °C overnight. N,
N-Dimethylformamide (0.5 mL) was then added, and the reaction
1H NMR (CDCl3): d 6.94–6.89 (m, 2H), 4.65–4.45 (m, 2H).

442
A. E. Taggi et al. / Bioorg. Med. Chem. 24 (2016) 435–443
mixture was heated for an additional 2 h. The reaction mixture was
then concentrated under reduced pressure and the resulting residue
was purified by MPLC (0–100% gradient of ethyl acetate in hexanes
as eluant) to give 2.9 g of 3,5-dichloro-6-(2,6-difluoro-4-methoxy-
phenyl)-1-((2S)-2-methylbutyl)-2(1H)-pyrazinone (10c, 3.8 g,
10 mmol, 69%) as an oil. 1H NMR (CDCl3): d 6.61 (m, 2H), 3.90 (s,
3H), 3.76 (m, 2H), 1.70 (m, 1H), 1.20 (m, 1H), 1.03 (m, 1H), 0.74 (m,
6H).
9.09 (d, 1H), 7.88 (d, 1H), 7.33 (m, 5H), 6.59 (m, 1H), 6.50 (m, 2H),
5.11 (s, 2H), 4.00 (m, 2H), 3.85 (m, 2H), 3.51 (t, 2H), 2.98 (s, 3H),
2.10 (m, 2H), 1.72 (m, 1H), 1.20 (m, 1H), 1.03 (m, 1H), 0.74 (m, 6H).
4.1.15. 5-Chloro-6-[2,6-difluoro-4-[3-(methylamino)propoxy]-
phenyl]-1-[(2S)-2-methylbutyl]-3-pyrazol-1-yl-pyrazin-2-one
hydrochloride (18b)
Phenylmethyl-N-[3-[4-[3-chloro-1,6-dihydro-1-[(2S)-2-
methylbutyl]-6-oxo-5-(1H-pyrazol-1-yl)-2-pyrazinyl]-3,5-difluo-
rophenoxy]propyl]-N-methylcarbamate (17b, 0.44 g, 0.73 mmol)
was dissolved in methanol (50 mL) and flushed with nitrogen.
Hydrogen chloride (1 M solution in diethyl ether, 4 mL) was added
followed by palladium on carbon (10% wt/wt, 0.12 g, 0.11 mmol)
and flushing with nitrogen was continued. A balloon containing
hydrogen gas was attached to the reaction mixture and the reac-
tion mixture was stirred at room temperature for 2 h. The reaction
mixture was filtered through CeliteÒ, diatomaceous filter aid, and
concentrated under reduced pressure. The reaction mixture was
redissolved in methanol, filtered, and then concentrated to give
5-chloro-6-[2,6-difluoro-4-[3-(methylamino)propoxy]-phenyl]-1-
[(2S)-2-methylbutyl]-3-pyrazol-1-yl-pyrazin-2-one hydrochloride
(18b, 0.34 g, 0.73 mmol, 99%). 1H NMR (methanol-d4) d 9.08 (d,
1H), 8.23 (m, 1H), 7.89 (d, 1H), 6.91 (m, 2H), 6.59 (s, 1H), 4.22 (t,
2H), 3.87 (m, 2H), 3.23 (m, 2H), 2.75 (s, 3H), 2.21 (m, 2H), 1.73
(m, 1H), 1.24 (m, 1H), 1.06 (m, 1H), 0.73 (m, 6H).
4.1.12. 5-Chloro-6-(2,6-difluoro-4-methoxyphenyl)-1-((2S)-2-
methylbutyl)-3-(1H-pyrazol-1-yl)-2(1H)-pyrazinone (11c)
A mixture of 3,5-dichloro-6-(2,6-difluoro-4-methoxyphenyl)-1-
((2S)-2-methylbutyl)-2(1H)-pyrazinone (10c, 3.8 g, 9.4 mmol),
pyrazole (0.69 g, 10 mmol) and potassium carbonate (2.6 g,
19 mmol) in N,N-dimethylformamide (40 mL) was heated to
60 °C overnight. The reaction mixture was concentrated under
reduced pressure and the residue was purified by MPLC (0–100%
gradient of ethyl acetate in hexanes as eluant) to give 5-chloro-
6-(2,6-difluoro-4-methoxyphenyl)-1-((2S)-2-methylbutyl)-3-(1H-
pyrazol-1-yl)-2(1H)-pyrazinone (11c, 2.5 g, 6.1 mmol, 67%). 1H
NMR (CDCl3): d 9.09 (m, 1H), 7.88 (m, 1H), 6.63 (m, 2H), 6.50 (m,
1H), 3.87 (m, 5H), 1.75 (m, 1H), 1.25 (m, 1H), 1.05 (m, 1H), 0.74
(m, 6H).
4.1.13. 5-Chloro-6-(2,6-difluoro-4-hydroxyphenyl)-1-((2S)-2-
methylbutyl)-3-(1H-pyrazol-1-yl)-2(1H)-pyrazinone (15)
To a solution of 5-chloro-6-(2,6-difluoro-4-methoxyphenyl)-1-
((2S)-2-methylbutyl)-3-(1H-pyrazol-1-yl)-2(1H)-pyrazinone (11c,
1.0 g, 2.4 mmol) in dichloromethane (24 mL) at À78 °C was slowly
added a solution of boron tribromide (1 M solution in dichloro-
methane, 9.8 mL, 9.8 mmol). The reaction mixture was allowed
to warm to room temperature overnight. Then the reaction mix-
ture was cooled to 0 °C and quenched with saturated aqueous
ammonium chloride solution. The reaction mixture was extracted
with dichloromethane (2 Â 40 mL) and ethyl acetate (2 Â 30 mL).
The organic layers were combined, dried over MgSO4 and concen-
trated. The crude residue was purified by MPLC (0–100% gradient
of ethyl acetate in hexanes as eluant) to yield 5-Chloro-6-(2,6-di-
fluoro-4-hydroxyphenyl)-1-((2S)-2-methylbutyl)-3-(1H-pyrazol-
1-yl)-2(1H)-pyrazinone (15, 0.51 g, 1.3 mmol, 53%).
4.1.16. 5-Chloro-6-[4-[3-(dimethylamino)propoxy]-2,6-
difluoro-phenyl]-1-[(2S)-2-methylbutyl]-3-pyrazol-1-yl-
pyrazin-2-one (17a)
A
solution of 5-chloro-6-(2,6-difluoro-4-hydroxyphenyl)-1-
[(2S)-2-methylbutyl]-3-(1H-pyrazol-1-yl)-2(1H)-pyrazinone (15,
0.2 g, 0.51 mmol) and Cs2CO3 (0.83 g, 2.5 mmol) in N,N-dimethyl-
formamide (10 mL) was heated to 70° C for 15 min. 3-Chloro-N,
N-dimethyl-1-propaneamine
hydrochloride
(16a,
0.24 g,
1.5 mmol) was then added and the reaction mixture was then
heated for an additional 1.5 h, and then cooled to room tempera-
ture. After the cesium carbonate was removed by filtering through
CeliteÒ, diatomaceous filter aid, the reaction mixture was
concentrated under reduced pressure. The crude oil was purified
by MPLC (0–30% gradient of methanol in methylene chloride as
eluant) to provide 5-chloro-6-[4-[3-(dimethylamino)propoxy]-
2,6-difluoro-phenyl]-1-[(2S)-2-methylbutyl]-3-pyrazol-1-yl-pyra-
zin-2-one (17a, 0.019 g, 0.040 mmol, 8%). 1H NMR (CDCl3) d 9.09
(m, 1H), 7.88 (m, 1H), 6.63 (m, 2H), 6.50 (m, 1H), 4.08 (m, 2H),
3.85 (m, 2H), 2.48 (m, 2H), 2.28 (s, 6H), 2.00 (m, 2H), 1.74 (m,
1H), 1.25 (m, 1H), 1.04 (m, 1H), 0.75 (m, 6H).
1H NMR (CDCl3): d 10.53 (br s, 1H), 9.24 (d, 1H), 7.94 (d, 1H),
6.74 (d, 2H), 6.57 (m, 1H), 3.90 (d, 2H), 1.76 (m, 1H), 1.26 (m,
1H), 1.05 (m, 1H), 0.77 (m, 6H).
4.1.14. Phenylmethyl N-[3-[4-[3-chloro-1,6-dihydro-1-[(2S)-2-
methylbutyl]-6-oxo-5-(1H-pyrazol-1-yl)-2-pyrazinyl]-3,5-
difluorophenoxy]propyl]-N-methylcarbamate (17b)
To a solution of 5-chloro-6-(2,6-difluoro-4-hydroxyphenyl)-1-
[(2S)-2-methylbutyl]-3-(1H-pyrazol-1-yl)-2(1H)-pyrazinone (15,
0.35 g, 0.89 mmol) in N,N-dimethylformamide (4 mL) was added
dry activated 4 Å molecular sieves (3.0 g). The reaction mixture
was stirred for 3 h at room temperature. Tetrabutylammonium
iodide (0.065 g, 0.18 mmol) and phenylmethyl N-(3-chloro-
propyl)-N-methylcarbamate (16b, 0.64 g, 2.7 mmol) in N,N-
dimethylformamide (1 mL), were added and the reaction mixture
was stirred for 15 min at room temperature. Cesium carbonate
(0.87 g, 2.7 mmol) was added and stirring was continued for
another 15 min. The reaction mixture was then heated to 75 °C
for 2 h and then cooled to room temperature. After the molecular
sieves and cesium carbonate were removed by filtering through
CeliteÒ the reaction mixture was concentrated under reduced
pressure. The crude oil was purified by MPLC (0–100% gradient
of ethyl acetate in hexanes as eluant) to provide phenylmethyl
N-[3-[4-[3-chloro-1,6-dihydro-1-[(2S)-2-methylbutyl]-6-oxo-5-
(1H-pyrazol-1-yl)-2-pyrazinyl]-3,5-difluorophenoxy]propyl]-N-
methylcarbamate (17b, 0.44 g, 0.73 mmol, 82%). 1H NMR (CDCl3): d
4.2. Biochemistry
Purified bovine tubulin was purchased from Cytoskeleton Inc.,
Denver, USA and the polymerization assay performed as recom-
mended by the manufacturer. The polymerization reaction con-
tained 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 in individual wells of
a 96-well microtiter plate. Polymerization was started by incuba-
tion at 37 °C and followed over time at 340 nm (Spectramax plate
reader). Paclitaxel (10 lM) and DMSO (0.5%) were used as positive
and negative controls, respectively. Experimental compounds were
added to different wells of the plate at the beginning of the reac-
tion over a range of concentrations in constant concentrations of
DMSO solution.
4.3. Biology
Compounds were first dissolved in acetone in an amount equal
to 3% of the final volume and then suspended at the desired

A. E. Taggi et al. / Bioorg. Med. Chem. 24 (2016) 435–443
443
concentration (in ppm) in acetone and purified water (50/50 mix)
containing 250 ppm of the surfactant TremÒ 014 (polyhydric
alcohol esters). Spraying a 200 ppm test suspension to the point
of run-off on the test plants was the equivalent of a field rate of
500 g/hectare. The biological evaluation for additional analogs
can be found in the patent literature.13,21
running the RMS assay. The authors are also grateful to Hugh
Brown, Michael E. Condon, Pat N. Confalone and Mark E. Thompson
for managerial and technical guidance.
References and notes
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The test suspension was sprayed to the point of run-off on
Stevens wheat seedlings. The following day the seedlings were
inoculated with a spore suspension of Stagonospora nodorum.
(the causal agent of wheat glume blotch) and incubated in a satu-
rated atmosphere at 20 °C for 48 h, and then moved to a growth
chamber at 22 °C for 5 days, after which disease ratings were
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and
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The test suspension was sprayed to the point of run-off on
Stevens wheat seedlings. The following day the seedlings were
inoculated with a spore suspension of Puccinia recondita f. sp. tritici.
(the causal agent of wheat leaf/brown rust) and incubated in a sat-
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chamber at 20 °C for 7 days, after which disease ratings were
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(the causal agent of gray mould on tomatoes) and incubated in sat-
urated atmosphere at 20 °C for 48 h, and then moved to a growth
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4.3.4. Rhabdomyosarcoma cell line (ATCC CCL-136)
Cells were dispensed into wells of an untreated 96-well micro-
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allowed to adhere for 1 –3 h before the media was removed and
replaced with media containing the test compound over a range
of concentrations. The plates were maintained at 37 °C in 5% CO2
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For determination of the proliferation IC50 value, a MTS tetra-
zolium compound was added as recommended by the manufac-
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the colored formazan adduct that forms after 3 –4 h at 37 °C by
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Acknowledgments
The authors gratefully acknowledge Mishelle Broomell for con-
ducting the tubulin polymerization assay and Matthew Sacher for