Penoxsulam-Structure-activity relationships of triazolopyrimidine sulfonamides

Article abstract of DOI:10.1016/j.bmc.2009.02.010

The discovery of the sulfonamide herbicides, which inhibit the enzyme acetolactate synthase (ALS), has resulted in many investigations to exploit their herbicidal activity. One area which proved particularly productive was the N-aryltriazolo[1,5-c]pyrimidine sulfonamides, providing three commercial herbicides, cloransulam-methyl, diclosulam and florasulam. Additional structure-activity investigations by reversing the sulfonamide linkage resulted in the discovery of triazolopyrimidine sulfonamides with cereal crop selectivity and high levels of grass and broadleaf weed control. Research efforts to exploit these high levels of weed activity ultimately led to the discovery of penoxsulam, a new herbicide developed for grass, sedge and broadleaf weed control in rice. Synthetic efforts and structure-activity relationships leading to the discovery of penoxsulam will be discussed.

Full text of DOI:10.1016/j.bmc.2009.02.010

Bioorganic & Medicinal Chemistry 17 (2009) 4230–4240
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Penoxsulam—Structure–activity relationships of triazolopyrimidine
sulfonamides
*
Timothy C. Johnson , Timothy P. Martin, Richard K. Mann, Mark A. Pobanz
Dow AgroSciences LLC, 9330 Zionsville Road, Indianapolis, IN 46268, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The discovery of the sulfonamide herbicides, which inhibit the enzyme acetolactate synthase (ALS), has
resulted in many investigations to exploit their herbicidal activity. One area which proved particularly
productive was the N-aryltriazolo[1,5-c]pyrimidine sulfonamides, providing three commercial herbi-
cides, cloransulam-methyl, diclosulam and florasulam. Additional structure–activity investigations by
reversing the sulfonamide linkage resulted in the discovery of triazolopyrimidine sulfonamides with cer-
eal crop selectivity and high levels of grass and broadleaf weed control. Research efforts to exploit these
high levels of weed activity ultimately led to the discovery of penoxsulam, a new herbicide developed for
grass, sedge and broadleaf weed control in rice. Synthetic efforts and structure–activity relationships
leading to the discovery of penoxsulam will be discussed.
Received 30 September 2008
Revised 3 February 2009
Accepted 9 February 2009
Available online 14 February 2009
Keywords:
Penoxsulam
Sulfonamide
Triazolopyrimidine
Herbicide
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
activity on grass and broadleaf weeds with grass crop selectivity
that was comparable to, or better than 2 or 3. One initiative to
The triazolopyrimidine sulfonamide class of herbicides are the
result of research efforts aimed at preparing bioisosteres of the sul-
fonyl urea herbicides.1 Initial efforts focused on the N-(triazolo[1,5-
a]pyrimidine) sulfonamides (1). Further investigations showed
that reversing the sulfonamide linkage (–SO2NH–) to give triazol-
o[1,5-a]pyrimidine sulfonanilides led to compounds with im-
proved herbicidal activity and eventually to the discovery of
flumetsulam (2).2 The triazolopyrimidine sulfonamides have dem-
onstrated competition with the amino acid leucine for binding to
acetolactate synthase (ALS) isolated from cotton (Gossypium
hirsutum).3
Since the discovery of flumetsulam, which was developed for
broadleaf weed control in maize and soybeans, many investiga-
tions were initiated to examine the effects of sulfonanilides con-
taining fused bicyclic heterocycles, and much of that work has
been reviewed.4,5 One area which proved to be quite productive
was the triazolo[1,5-c]pyrimidine class of sulfonanilides. Investiga-
tion of this class led to the discovery of florasulam (3), which was
commercialized for broadleaf weed control in cereals. All of the
triazolopyrimidines commercialized at that point in time were
developed for broadleaf weed control and all lacked commercial
levels of grass weed control.
expand the structure–activity relationship (SAR) around 3 and
related analogs focused on reversing the sulfonamide linkage
back to its original order.6 This modification afforded grass crop
selective sulfonamides which exhibited high levels of activity on
both grass and broadleaf weeds. Additional investigations even-
tually led to the discovery of penoxsulam (4), which exhibits
activity on both grass and broadleaf weeds with utility primarily
in rice (Fig. 1).
F
X
N
N
N
N
N
N
SO2NH
NHSO2
R
N
N
Reverse
Linkage
F
2
1
Flumetsulam ( )
Core
Heterocycle
Variation
O
F2HC
O
O
F
F
O
N
N
N
N
N
N
N
Subsequent research focused on the discovery of related sul-
fonamide analogs with a spectrum of activity that included
NHSO2
SO2NH
CF3
N
Reverse
Linkage
F
4
3
Florasulam ( )
Penoxsulam ( )
* Corresponding author. Tel.: +1 317 337 3060; fax: +1 317 337 3215.
E-mail address: tcjohnson@dow.com (T.C. Johnson).
Figure 1. Evolution of triazolopyrimidine herbicides leading to Penoxsualm.
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.02.010

T. C. Johnson et al. / Bioorg. Med. Chem. 17 (2009) 4230–4240
4231
2. Results and discussion
2.1. Synthetic chemistry
O
N
O
Cl
Cl
Cl
Cl
a
N
N
N
N
N
N
NHSO2
NHSO2
N
F
O
The majority of the target triazolo[1,5-c]pyrimidine sulfona-
mides, such as 4, were prepared via coupling 2-aminotriazol-
6g
7
o[1,5-c]pyrimidines
5
with
substituted
benzenesulfonyl
Scheme 2. Reagents: NaOMe, MeOH.
chlorides (Scheme 1).7–9 In these transformations, pyridine and
catalytic dimethylsulfoxide (DMSO) were essential for the forma-
tion of 6. It is believed that the reactive sulfilimine intermediate
was generated in situ, which allows for relatively non-nucleo-
philic aminotriazolopyrimidines to react with sulfonyl chlorides
under mild conditions.10–12 In addition to the coupling route,
some sulfonamides were prepared through further derivatization
of coupled targets as illustrated by the treatment of the 7-fluoro
analog 6g with sodium methoxide to afford the 7-methoxy analog
7 (Scheme 2). The substituted 2-aminotriazolo[1,5-c]pyrimidines
5 were prepared starting from 4-hydrazino-2-methylthiopyrimi-
2.2. SAR development
Initial efforts to determine the SAR of the triazolo[1,5-c]pyrim-
idines (6) focused on substitutions in the 7- and 8-positions of 6.
Extensive SAR work around florasulam (3) showed the highest lev-
els of activity were obtained with a methoxy in the 5-position, and
that varying substitution in the 7- and 8-positions resulted in dif-
ferent levels of activity on weeds and crops. Thus, analogs of 6
were prepared with a methoxy in the 5-position. A summary of
the herbicidal activity for compounds substituted in the 7- and
8-positions of 6 (R7 and R8, respectively, Fig. 2) is given in Table
1. In general compounds with substitution in the 8-position have
higher levels of activity than those with substitutions in the 7-po-
sition. Weak levels of activity are observed with no substitution in
either the 7- or 8-positions (6a). Both the 8-methoxy analog 6b and
8-chloro analog 6c have good levels of activity on both broadleaf
and grass weeds. Compared to 6c the 8-fluoro analog 6d causes a
decrease in activity. The 8-methyl analog 6e, like 6d, has much less
activity on grass and broadleaf weeds than either 6b or 6c. Increas-
ing the 8-alkoxy size from methoxy 6b to ethoxy 6f causes a de-
crease in activity on both broadleaf and grass weeds. The 7-
methoxy analog 7, unlike the 8-methoxy analog 6b, had very weak
activity on both grass and broadleaf weeds. The trends observed for
in vivo activity correlate with the trends observed for in vitro activ-
ity. The most active compounds, 6b and 6c, on grass and broadleaf
weeds also cause significant injury to rice (Oryza sativa).
Further SAR development focused on substitutions about the
phenyl ring. In these studies the highly active 5,8-dimethoxy sub-
stitution pattern on the triazolo[1,5-c]pyrimidine ring was re-
tained. The in vivo activity for compounds with various
substitutions on the phenyl ring (Fig. 3) is shown in Table 2. In gen-
eral, both mono and disubstitutions can impart good levels of
activity on both broadleaf and grass weeds. For mono substitutions
the methoxy analog 6h showed better activity on both grass and
broadleaf weeds than a trifluoromethyl 6i. By comparing the di-
chloro substituted sulfonamides 6b, 6j and 6k it can be seen that
2,6-disubstitution imparts the best activity on both grass and
broadleaf weeds. For disubstitutions, when one of the substituents
was methoxy good levels of activity are obtained when the 6-posi-
tion was substituted with either an electron donating substituent
(6l, 6-OMe) or an electron withdrawing substitutent (6m, 6-CF3).
Many of the highly active compounds were also very injurious to
rice. It was observed that increasing the size of the alkoxy from
methoxy to ethoxy and keeping a trifluoromethyl in the 6-position
(6n) resulted in loss of activity on most grass weeds. However, 6n
showed less injury to rice while maintaining good levels of activity
dines
8 8 were reacted with
(Scheme 3).13,14 Compounds
cyanogen bromide to give the 3-amino-5-methylthiotriazolo[4,3-
c]pyrimidines 9, usually as the hydrogen bromide salts. When
treated with sodium methoxide, 9 undergoes a Dimroth rear-
rangement with loss of the methylthio group to form the desired
2-amino-5-methoxytriazolo[1,5-c]pyrimidines 5. In this reaction,
the Michael acceptor ethyl acrylate was used to consume the
mercaptide by-product.
The arylsulfonyl chlorides used for the coupling reactions
were prepared via directed ortho metalation as outlined in
Schemes 4 and 5. When 10 was submitted to standard metala-
tion conditions a considerable amount of the undesired 1,2,5-tri-
substituted by-product was formed. However, when the lithiation
was conducted using conditions of thermodynamic control the
desired substitution pattern was obtained leading to 11 as the
only product. Thus, when 10 was treated with a catalytic amount
of diisopropylamine and less than 1 equiv of n-butyllithium, fol-
lowed by equilibration and treatment with propyl disulfide, 11
was isolated in >99% yield (Scheme 4).15 For sulfides 11 in which
R is methoxymethyl (MOM), standard MOM deprotection condi-
tions afforded the corresponding phenol (12), which was subse-
quently alkylated to give the appropriately substituted sulfide
(13). Conversion of the sulfides (11, 13) to their corresponding
sulfonyl chlorides 14 was carried out using chloro-oxidation con-
ditions, such as chlorine gas in aqueous acetic acid. It is notewor-
thy however, that attempts to prepare dialkoxybenzenesulfonyl
chlorides using the described chloro-oxidation conditions re-
sulted in ring chlorination prior to sulfonyl chloride formation.
In these reactions, standard metalation conditions afford the de-
sired regiochemistry and were therefore amenable to preparation
via
a simplified route. Thus, 1,3-dimethoxybenzene 15 was
sequentially treated with n-butyllithium and sulfur dioxide to
furnish the lithium benzenesulfinate.16–18 Subsequent oxidation
of the sulfinate with sulfuryl chloride afforded 2,6-di-
methoxybenzenesulfonyl chloride 16 (Scheme 5) in good yield.
In most cases the sulfonyl chlorides were reacted with the
aminotriazolo[1,5-c]pyrimidine immediately after workup with-
out further purification.
O
O
O
X
N
N
N
N
N
N
a
N
N
N
N
NH2
N S
NHSO2
N
R7
N
Y
R7
R7
R8
R8
R8
6
5
Sulfilimine
Scheme 1. Reagents: ArSO2Cl, pyridine, DMSO(catalytic), CH3CN.

4232
T. C. Johnson et al. / Bioorg. Med. Chem. 17 (2009) 4230–4240
R8
R8
O
N
O
O
H
X
R7
N NH2
R7
N
N
N
a
N
N
b
N
N
NHSO2
N
NH2
N
N
N
N
Y
R7
N
S
NH2
R8
S
8
9
5
Figure 3. Substitutions on the phenyl ring of the triazolopyrimidine sulfonamides.
Scheme 3. Reagents: (a) BrCN, i-PrOH; (b) NaOMe, MeOH, ethyl acrylate.
Table 2
Herbicidal activity for various mono- and disubstituted phenyl analogs (Fig. 3)
Compd
X
Y
BW
GW
Oryza
sativa
GR20
Echinochloa
crus-galli
GR80 (ppm)
OR
OR
OR
SPr
SO2Cl
CF3
a, b
e
GR80
(ppm)
GR80
(ppm)
(ppm)
CF3
CF3
6h
6i
6b
6j
6k
6l
6m
6n
6o
6p
2-OMe
2-CF3
2-Cl
2-Cl
3-Cl
2-OMe
2-OMe
2-OEt
2-O(CH2)2F
O(CH2)3F
H
H
1
13
2
19
126
0.5
<0.2
2
1
435
10
>1000
>1000
<0.1
1
191
66
<1
<1
8
<2
62
<0.5
2
250
250
>250
2
250
4
1000
>1000
<0.5
1
4
1
125
14
10
11
6-Cl
5-Cl
5-Cl
6-OMe
6-CF3
6-CF3
6-CF3
6-CF3
e
c
R = MOM
R = Alkyl
OR
OH
6
15
SPr
SPr
d
250
CF3
CF3
12
13
Based on the findings shown in Table 2, further investigations
were initiated into 2-fluoroalkoxy-6-trifluoromethyl substituted
phenyl analogs. Table 3 summarizes the activity on rice and key
rice weeds for specific 2-alkoxy-6-trifluoromethylphenyl substi-
tuted analogs (Fig. 4) when applied as a water-injected treatment
in the greenhouse to rice and weeds in the one to three leaf stage.
In these greenhouse trials, rates were expressed as gram active
ingredient per hectare (g ai/ha). In this test the ethoxy analog 6n
caused significant injury to rice. The 2-fluoroethoxy analog 6o
showed slightly less injury to rice with good control of key weeds.
The trifluoroethoxy analog 6q shows even less injury to rice with
some loss in weed control activity compared to 6o or 6n. The diflu-
oroethoxy analog 6r shows less injury to rice than 6q and better
activity on the key weeds. The difluoroisopropoxy analog 6s dem-
onstrated the least injury to rice while maintaining activity on key
weeds comparable to 6r. Based on these results 6o, 6r and 6s were
selected for further field testing in key rice countries.
Scheme 4. Reagents: (a) BuLi (0.95 equiv), TMEDA, (i-pr)2NH (10 mol %), THF or
Et2O; (b) (PrS)2; HCl, MeOH; (d) phenol alkylation; (e) Cl2, H2O, HOAc.
O
O
SO2Cl
O
a, b, c
O
16
15
Scheme 5. Reagents: (a) BuLi (1.2 equiv), TMEDA, THF or Et2O; (b) SO2, Et2O; (c)
SO2Cl2, hexane.
O
Cl
N
N
N
NHSO2
Cl
R7
N
R8
2.3. Field results—Screening
Figure 2. Substitutions in the 7- and 8-positions of the triazolopyrimidine ring.
Field testing of the three alkoxy trifluoromethylphenyl analogs
6o, 6r and 6s under different rice growing conditions in several rice
growing countries quickly sorted the analogs in order of perfor-
mance for weed control and rice. Table 4 summarizes the rice tol-
erance and weed control activity of the analogs when applied as
water injected treatments to transplanted japonica rice and weeds
at the three leaf stage under field conditions. These results demon-
strate the poor rice selectivity of 6o as compared to 6r and 6s, but
better weed control than 6s and slightly better weed control com-
pared to 6r. Based on rice selectivity and weed control, 6r was cho-
sen as the best analog in this study. Also, analog 6r appeared to
have significantly longer residual weed control activity than 6s,
which is an important criterion for a rice herbicide (data not
shown).
on broadleaf weeds and barnyard grass (Echinochloa crus-galli).
When the ethoxy group was substituted with terminal fluorine
(6o) the activity on broadleaf weeds was maintained and there
was slightly better activity on barnyard grass than was observed
for 6n. The 2-fluoropropoxy analog 6p shows a significant decrease
in activity on barnyard grass compared to either 6n or 6o.
Table 1
Dependence of herbicidal activity on substitutions in the 7- and 8-positions of the
triazolopyrimidine ring (Fig. 2)
Compd
R7
R8
BW
GW
Oryza sativa
ALS
GR80 (ppm)
GR80 (ppm)
GR20 (ppm)
I50 (nM)
Table 5 shows the results of testing the three analogs in direct
seeded indica rice as foliar applied treatments to rice and weeds
at the 2–3 leaf stage. Early greenhouse results had demonstrated
that indica rice had much better tolerance to this chemistry than
japonica rice. The results shown here demonstrated the excellent
indica rice tolerance to 6o, 6r and 6s. From a weed control perspec-
tive 6o had the highest level of weed control activity, 6s had the
lowest level of weed control activity, and 6r was intermediate
6a
6b
6c
6d
6e
6f
H
H
H
H
H
H
F
H
OMe
Cl
475
2
1
>2000
10
15
500
8
4
>125
250
250
8
1040
0.6
0.6
12
7
2
F
19
309
Me
OEt
H
<15
216
62
>250
>500
574
6g
7
27
195
OMe
H
>1000
>1000
>1000

T. C. Johnson et al. / Bioorg. Med. Chem. 17 (2009) 4230–4240
4233
Table 3
Herbicidal activity of alkoxy trifluoromethyl phenyl analogs (Fig. 4) when applied as a water injection treatment on transplanted paddy rice and selected weeds
Compd
Y
Oryza sativa
Echinochloa crus-galli
Monochoria vaginalis
Scirpus juncoides
Cyperus difformis
GR20 (g ai/ha)
GR80 (g ai/ha)
GR80 (g ai/ha)
GR80 (g ai/ha)
GR80 (g ai/ha)
6n
6o
6q
6r
OEt
1
1
1
5.2
5
<2
1
3
9
21
12
9
N A
8
36
14
31
O(CH2)2F
OCH2CF3
OCH2CHF2
OCH(CH2F)2
14
51
75
140
10
19
12
14
6s
Table 6
O
O
Y
NHSO2
F3C
Herbicidal activity of alkoxy trifluoromethyl phenyl analogs (Fig. 4) when applied as a
post-emergence foliar treatment to direct seeded japonica rice and selected weeds
N
N
N
N
Compd
Y
Oryza sativa
Echinochloa
crus-galli
GR80 (g ai/ha)
Sesbania
exaltata
GR80 (g ai/ha)
GR10 (g ai/ha)
Figure 4. 2-Alkoxy-6-trifluoromethylphenyltriazolo [1,5-c]pyrimidine sulfonamide
analogs.
6o
6r
O(CH2)2F
OCH2CF2H
12
65
12
9
10
7
China23 and as a water-injected application into flooded paddy rice
in Korea.24
As of 2008, penoxsulam has been registered as a rice herbicide
in 28 countries, being used for selective weed control in trans-
planted rice, direct-seeded japonica and indica rice, and in water-
seeded japonica and indica rice.
Table 7 is a global summary of weed control performance as
determined by many field trial evaluations of multiple rates of
penoxsulam under local rice growing conditions around the world
in many countries. We have seen that there are some biotype dif-
ferences within a weed species across geographies, so the follow-
ing table serves as a summary and guide to penoxsulam weed
control performance.
but close to 6o. Ratings at 30 and 60 days demonstrated the poor
residual weed control activity of 6s compared to 6o and 6r (data
not shown).
Table 6 shows results from testing 6o and 6r as post-emergence
foliar applications in direct seeded japonica rice at the 3–4 leaf
stage. The results in japonica rice were very compelling and
quickly showed the advantage of 6r over 6o from a rice selectivity
standpoint. The level of rice injury from 6o was not acceptable on
japonica rice at rates necessary for commercial levels of weed
control.
2.4. Field results—Global field trials
Based on the poor selectivity of 6o in japonica direct seeded
rice, as well as, the better foliar weed control and longer soil resid-
ual activity of 6r compared to 6s, 6r was chosen for development
as a rice herbicide with the common name ‘penoxsulam’.
3. Materials and methods
3.1. Synthesis
From 1998 to the end of 2007, over 2000 rice field trials have
been conducted testing 6r (penoxsulam) alone and in tank mixes
with many other products and multiple formulations. Penoxsulam
has been shown to provide effective weed control as a post-emer-
gence foliar application in direct-seeded rice globally;19–21 as a fo-
liar application in water-seeded japonica rice in Europe and
Eufrasia;22 as a post-emergence foliar application in Vietnam and
All melting points were recorded on a Thomas Hoover melting
point apparatus using the appropriate partial immersion ther-
mometer and are uncorrected. 1H NMR (300 MHz) and 13C NMR
(75 MHz) were obtained in either CDCl3 or DMSO-d6, used as pur-
chased, and were recorded on a Varian Gemini spectrometer and
referenced to an internal standard of tetramethylsilane (TMS 1H:
d 0.00). 1H–1H couplings are assumed to be first order and peak
multiplicity is reported as s (singlet), d (doublet), t (triplet), q
(quartet), sext (sextet), m (multiplet), or br (broad). Combustion
analyses were performed either in house or by Midwest Microlab,
Indianapolis, IN. Mass spectral data were obtained on a Hewlett-
Packard series 1100 mass selective detector, Hewlett-Packard
5890 series II with a Hewlett-Packard 5890A mass spectrometer,
or a Hewlett-Packard 6890 series GC system with a 5973 mass
selective detector. Flash or gravity chromatography was performed
using 230–400 mesh silica gel from EM Science, Darmstadt, Ger-
many using reagent grade solvents. Reaction were monitored by
Table 4
Herbicidal activity of specific alkoxy trifluoromethyl phenyl analogs (Fig. 4) when
applied as a water injection treatment on transplanted paddy rice and selected weeds
Compd
Y
Oryza
sativa
GR10
Echinochloa
crus-galli
GR80
Monochoria
vaginalis
GR80
Scirpus
juncoides
GR80
(g ai/ha)
(g ai/ha)
(g ai/ha)
(g ai/ha)
6o
6r
6s
O(CH2)2F
OCH2CHF2
OCH(CH2F)2
8
35
70
19
26
35
4.4
6
12
5
9
15
thin layer chromatography (TLC) on 250 lm precoated Analtech
Uniplate plates and visualized will ultraviolet light. Unless other-
wise noted, materials were obtained from commercial suppliers
and used as is.
Compounds 6a and 6c–s were prepared according to the proce-
dure for 6b below:
Table 5
Herbicidal activity of alkoxy trifluoromethyl phenyl analogs (Fig. 4) when applied as
post-emergence foliar treatment to direct seeded indica rice and selected weeds
Compd
Y
Oryza
sativa
Echinochloa
crus-galli
Scirpus
juncoides
Cyperus
difformis
GR10 (g ai/ha) GR80 (g ai/ha) GR80 (g ai/ha) GR80v
3.1.1. 2,6-Dichloro-N-(5,8-dimethoxy-1,2,4-triazolo[1,5-
c]pyrimidin-2-yl)benzene sulfonamide (6b)
A round bottom flask fitted with a drying tube (CaSO4) was
charged with 2-amino-5,8-dimethoxy-1,2,4-triazolo[1,5-c]pyrimi-
6o
6r
6s
O(CH2)2F
OCH2CHF2
>70
>70
5
7
4
6
4
7
OCH(CH2F)2 >70
15
20
12

4234
T. C. Johnson et al. / Bioorg. Med. Chem. 17 (2009) 4230–4240
Table 7
Global weed control performance summary of penoxsulam on rice weeds susceptible, moderately susceptible and tolerant are an indication of weed susceptibility to applications
of penoxsulam in rice on these plant species at local use rates across different geographies
Susceptible
Moderately susceptible
Tolerant
Acalypha australis
Aeschynomene spp.
Alisma plantago-aquatica
Alternanthera philoxeroides
Amaranthus spp.
Ammannia spp.
Bacopa spp.
Baltimora recta
Bergia capensis
Bidens tripartita
Boerhaavia diffusa
Caperonia spp.
Cardamine parviflora
Chenopodium spp.
Cleome viscosa
Commelina communis
Cyperus spp (annual)
Echinochloa spp. (annual)
Echinochloa polystachya
Eclipta alba
Heteranthera limosa
Kyllinga squamulate
Lindernia spp.
Lythrum junceum
Malachra alceifolia
Melochia spp.
Monochoria spp.
Nasturtium officinale
Nymphaea spp.
Oenanthe javanica
Phyllanthus niruri
Physalis angulata
Polygonum spp.
Butomus umbellatus
Commelina benghalensis
Corchorus spp.
Cyperus esculentus
Cyperus rotundus
Cyperus serotinus
Eriochloa spp.
Aneilema keisak
Brachiaria spp.
Cenchrus echinatus
Dactyloctenium spp.
Digitaria spp.
Eleusine indica
Eriochloa punctata
Euphorbia heterophylla
Leptochloa spp.
Murdannia nudiflora
Oryza sativa
Panicum spp.
Paspalum spp.
Rottboellia spp.
Scirpus planiculmis
Setaria spp.
Erucaria spp.
Euphorbia nutans
Glyceria declinata
Hyssopus spp.
Ipomoea spp.
Ischaemum rugosum
Jussiaea abyssinica
Ludwigia spp.
Portulaca oleracea
Rotala indica
Sagittaria spp.
Marsilea spp.
Scirpus juncoides
Scirpus mucronatus
Scleria pterota
Sesbania exaltata
Sphenoclea zeylanica
Spilanthes acmella
Tridax procumbens
Xanthium strumarium
Potamogeton distinctum
Scirpus maritimus
Sida spp.
Spigelia anthelmia
Elatine trianda
Eleocharis spp.
Fimbristylis spp.
dine (5a, 1.0 g, 5.1 mmol), 2,6-dichlorobenzenesulfonyl chloride
(1.26 g, 5.1 mmol), and anhydrous CH3CN (15 mL). To this mix-
ture was added anhydrous pyridine (0.82 mL, 10.2 mmol) and
2.06; N, 17.86; S, 8.18. Found: C, 36.77; H, 2.08; N, 17.72; S,
8.03.
anhydrous DMSO (72
l
L, 1.0 mmol). After 6 h, HPLC analysis
3.1.5. 2,6-Dichloro-N-(5-methoxy-8-methyl-1,2,4-triazolo[1,5-
c]pyrimidin-2-yl)benzenesulfonamide (6e)
of the reaction mixture indicated full consumption of the
amine. The CH3CN was removed in vacuo and the residue dis-
solved in CH2Cl2 (200 mL). The organic layer was washed with
2 N HCl (100 mL), H2O (2Â 100 mL), dried (MgSO4), filtered
and evaporated in vacuo to give a tan powder. The tan powder
was stirred with Et2O (100 mL) for 18 h. The solids were fil-
tered, rinsed with Et2O, and dried in a vacuum oven (50 °C,
high vacuum) to afford 6b as a tan powder (1.30 g, 63% yield):
mp 219–221 °C; 1H NMR (DMSO-d6) d 12.7 (br s, 1H), 7.66–7.52
(m, 4H), 4.11 (s, 3H), 3.88 (s, 3H). Anal. Calcd for
C13H11Cl2N5O4S: C, 38.63; H, 2.74; N, 17.33; S, 7.93. Found: C,
38.57; H, 2.81; N, 17.35; S, 8.13. In some cases it was necessary
to further purify the product by column chromatography (SiO2,
CH2Cl2/EtOH, 95:5).
The product was isolated as a white solid (86% yield): mp 218–
220 °C (d); 1H NMR (DMSO-d6) d 12.7 (br s, 1H), 7.77 (s, 1H), 7.66–
7.55 (m, 3H), 4.09 (s, 3H), 2.21 (s, 3H); 13C NMR (DMSO-d6) d 158.2,
154.1, 147.6, 142.0, 134.8, 134.7, 133.9, 131.6, 113.0, 55.9, 12.2;
MS-DIP (CI) m/z 388 ([M]+).
3.1.6. 2,6-Dichloro-N-(8-ethoxy-5-methoxy-1,2,4-triazolo[1,5-
c]pyrimidin-2-yl)benzenesulfonamide (6f)
The product was isolated as a white solid (36% yield): mp 217–
218 °C; 1H NMR (DMSO-d6) d 12.8 (br s, 1H), 7.67–7.54 (m, 4H),
4.16 (q, J = 7.0 Hz, 2H), 4.07 (s, 3H), 1.30 (t, J = 7.0 Hz, 3H); 13C
NMR (DMSO-d6) d 157.9, 148.4, 144.0, 138.0, 134.4, 133.8, 131.6,
126.3, 98.0, 65.8, 55.8, 14.5; MS-DIP (EI) m/z 417 ([M]+). Anal. Calcd
for C14H13Cl2N5O4S: C, 40.20; H, 3.13; N, 16.74; S, 7.67. Found: C,
40.10; H, 3.13; N, 16.78; S, 7.48.
3.1.2. 2,6-Dichloro-N-(5-methoxy-1,2,4-triazolo[1,5-
c]pyrimidin-2-yl)benzenesulfonamide (6a)
The product was isolated as a tan solid (14% yield): mp 225–
226 °C; 1H NMR (DMSO-d6) d 12.8 (br s, 1H), 7.99 (d, J = 6.2 Hz,
1H), 7.68–7.56 (m, 3H), 7.26 (d, J = 6.3 Hz, 1H), 4.17 (s, 3H).
3.1.7. 2,6-Dichloro-N-(7-fluoro-5-methoxy-1,2,4-triazolo[1,5-
c]pyrimidin-2-yl)benzenesulfonamide (6g)
The product was isolated as a tan powder (93% yield): mp
216–218 °C; 1H NMR (DMSO-d6) d 12.82 (br s, 1H), 7.67–7.57
(m, 3H), 6.99 (s, 1H), 4.14 (s, 3H); 13C (DMSO-d6) d 162.6,
160.2, 159.4, 156.1, 156.0, 148.7, 148.5, 134.7, 134.3, 133.9,
131.6, 85.0, 84.6, 57. 57.0. Anal. Calcd for C12H8Cl2FN5O3S: C,
36.75; H, 2.06; N, 17.86; S, 8.17. Found: C, 36.93; H, 1.88; N,
17.92; S, 8.32.
3.1.3. 2,6-Dichloro-N-(8-chloro-5-methoxy-1,2,4-triazolo[1,5-
c]pyrimidin-2-yl)benzenesulfonamide (6c)
The product was isolated as a white solid (61% yield): mp 214–
216 °C (d); 1H NMR (DMSO-d6) d 12.9 (br s, 1H), 8.10 (s, 1H), 7.67–
7.58 (m, 3H), 4.13 (s, 3H); 13C NMR (DMSO-d6) d 158.8, 151.9,
148.6, 142.7, 135.2, 134.9, 134.4, 132.1, 110.4, 57.0; MS-DIP (CI)
m/z 408 ([M]+). Anal. Calcd for C12H8Cl3N5O3S: C, 35.27; H, 1.97;
N, 17.14; S, 7.85. Found: C, 35.27; H, 1.93; N, 16.84; S, 7.47.
3.1.8. 2-Methoxy-N-(5,8-dimethoxy-1,2,4-triazolo[1,5-
c]pyrimidin-2-yl)benzenesulfonamide (6h)
The product was isolated as a tan solid (90% yield): mp 231–
233 °C; 1H NMR (DMSO-d6) d 11.8 (br s, 1H), 7.87 (dd, J = 1.5,
7.9 Hz, 1H), 7.57–7.54 (m, 2H), 7.15 (d, J = 8.2 Hz, 1H), 7.07 (t,
J = 7.6 Hz, 1H), 4.03 (s, 3H), 3.85 (s, 3H), 3.82 (s, 3H); 13C NMR
(DMSO-d6) d 158.4, 156.6, 148.0, 143.7, 139.3, 135.1, 130.6,
127.1, 123.4, 119.9, 112.9, 57.0, 56.1, 55.7. Anal. Calcd for
3.1.4. 2,6-Dichloro-N-(8-fluoro-5-methoxy-1,2,4-triazolo[1,5-
c]pyrimidin-2-yl)benzenesulfonamide (6d)
The product was isolated as a tan solid (50% yield): mp 211–
212 °C; 1H NMR (DMSO-d6) d 8.07 (d, J = 0.9 Hz, 1H), 7.52–7.65
(m, 3H), 4.10 (s, 3H). Anal. Calcd for C12H8Cl2N5O3S: C, 36.75; H,

T. C. Johnson et al. / Bioorg. Med. Chem. 17 (2009) 4230–4240
4235
C14H15N5O5S: C, 46.02; H, 4.14; N, 19.17; S 8.77. Found: C, 46.17; H,
4.00; N, 19.00; S, 8.99.
1H), 7.58–7.54 (m, 3H), 4.85–4.69 (dt, J = 3.8, 47.7 Hz, 2H), 4.44–
4.34 (dt, J = 3.8, 30.0 Hz, 2H), 4.02 (s, 3H), 3.82 (s, 3H); 13C NMR
(DMSO-d6), d 164.4, 158.3, 154.0, 147.9, 146.6, 146.1, 143.7,
141.5, 139.2, 131.1, 124.3, 123.9, 122.0, 118.3, 57.1, 55.8, 53.4;
MS-DIP (CI) m/z 465 ([M]+). Anal. Calcd for C16H15F4N5O5S: C
41.29; H, 3.25; N, 15.05; S, 6.89. Found: C, 41.35; H, 3.09; N,
14.89; S, 6.94.
3.1.9. 2-Trifluoromethyl-N-(5,8-dimethoxy-1,2,4-triazolo[1,5-
c]pyrimidin-2-yl)benzenesulfonamide (6i)
The product was isolated as a white powder (20% yield): mp
189–191 °C; 1H NMR (DMSO-d6) d 12.4 (br s, 1H), 8.32 (d,
J = 7.3 Hz, 1H), 8.00 (d, J = 7.9 Hz, 1H), 7.91–7.86 (m, 2H), 7.61 (s,
1 H), 4.07 (s, 3H), 3.88 (s, 3H).
3.1.16. 2-(3-Fluoropropoxy)-6-trifluoromethyl-N-(5,8-
dimethoxy-1,2,4-triazolo[1,5-c]pyrimidin-2-
3.1.10. 2,5-Dichloro-N-(5,8-dimethoxy-1,2,4-triazolo[1,5-c]
pyrimidin-2-yl)benzenesulfonamide (6j)
yl)benzenesulfonamide (6p)
The product was isolated as a tan solid (30% yield): mp 210–
212 °C; 1H NMR (DMSO-d6) d 11.79 (s, 1H), 7.72 (t, J = 8.1 Hz,
1H), 7.56–7.52 (m, 3H), 4.56 (dt, J = 5.8, 47.5 Hz, 2H), 4.19 (t,
J = 5.8 Hz, 2H), 4.02 (s, 3H), 3.83 (s, 3H), 2.12 (dp, J = 5.8, 26.1 Hz,
2H); LC–MS (ESI) m/z 480 ([M+H]+). Anal. Calcd for C17H17F4N5O5S:
C, 42.57; H, 3.57; N, 14.61; S, 6.69. Found: C, 42.48; H, 3.53; N,
14.42; S, 6.57.
The product was isolated as a white solid (62% yield): mp 219–
221 °C; 1H NMR (DMSO-d6) d 12.70 (br s, 1H), 8.16 (s, 1H), 7.79–
7.60 (m, 3H), 4.08 (s, 3H), 3.90 (s, 3H); 13C NMR (DMSO-d6) d
157.6, 148.1, 143.8, 139.3, 138.4, 134.5, 133.4, 131.8, 131.6,
129.6, 124.3, 57.2, 55.8; MS-DIP (CI) m/z 404 ([M]+). Anal. Calcd
for C13H11Cl2N5O4S: C, 38.63; H, 2.74; N, 17.33; S, 7.93. Found: C,
38.60; H, 2.85; N, 17.35; S, 8.13.
3.1.17. 2-(2,2,2-Trifluoroethoxy)-6-trifluoromethyl-N-(5,8-
dimethoxy-1,2,4-triazolo[1,5-c]pyrimidin-2-
yl)benzenesulfonamide (6q)
3.1.11. 3,5-Dichloro-N-(5,8-dimethoxy-1,2,4-triazolo[1,5-
c]pyrimidin-2-yl)benzenesulfonamide (6k)
The product was isolated as a white solid (34% yield): mp 195–
197 °C; 1H NMR (DMSO-d6) d 12.80 (br s, 1H), 8.01 (s, 1H), 7.99 (s,
2H), 7.65 (s, 1H), 4.12 (s, 3H), 3.93 (s, 3H); 13C NMR (DMSO-d6) d
157.9, 148.1, 144.0, 142.7, 139.4, 134.8, 133.0, 125.9, 124.2, 57.3,
55.9; MS-DIP (CI) m/z 404 ([M]+). Anal. Calcd for C13H11Cl2N5O4S:
C, 38.63; H, 2.74; N, 17.33; S, 7.93. Found: C, 38.80; H, 2.68; N,
17.09; S, 7.78.
The product was isolated as a white solid (30% yield): mp 211–
213 °C; 1H NMR (DMSO-d6) d 11.87 (s, 1H), 7.80 (t, J = 8.5 Hz, 1H),
7.67 (d, J = 7.9 Hz, 2H), 7.57 (s, 1H), 4.94 (q, J = 8.8 Hz, 2H), 4.04 (s,
3H), 3.83 (s, 3H); LC–MS (ESI) m/z 500 ([MÀH]À). Anal. Calcd for
C16H13F6N5O5S: C, 38.33; H, 2.61; N, 13.97; S, 6.39. Found: C,
38.26; H, 2.59; N, 13.89; S, 6.40.
3.1.18. 2-(2,2-Difluoroethoxy)-6-trifluoromethyl-N-(5,8-
dimethoxy-1,2,4-triazolo[1,5-c]pyrimidin-2-
yl)benzenesulfonamide (6r)
3.1.12. 2,6-Dimethoxy-N-(5,8-dimethoxy-1,2,4-triazolo[1,5-
c]pyrimidin-2-yl)-benzenesulfonamide (6l)
The product was isolated as an off-white solid (68% yield): mp
239–240 °C; 1H NMR (DMSO-d6) d 11.54 (s, 1H), 7.55 (s, 1H),
7.44 (t, J = 8.4 Hz, 1H), 6.74 (d, J = 8.4 Hz, 2H), 4.06 (s, 3H), 3.88
(s, 3H), 3.76 (s, 6H); 13C NMR (DMSO-d6) d 159.2, 158.4, 148.0,
143.7, 139.3, 134.1, 123.3, 117.3, 105.5, 57.0, 56.6, 55.7. Anal. Calcd
for C15H17N5O6S: C, 45.57; H, 4.33; N, 17.71; S, 8.11. Found: C,
45.47; H, 4.34; N, 17.54; S, 8.09.
The product was isolated as a tan solid (24% yield): mp 223–
224 °C; 1H NMR (DMSO-d6) d 11.89 (s, 1H), 7.76 (t, J = 8.2 Hz,
1H), 7.62 (m, 3H), 6.50 (tt, J = 4.2, 54.9 Hz, 1H), 4.48 (td, J =
3.8, 13.7 Hz, 2H), 4.03 (s, 3H), 3.83 (s, 3H); LC–MS (ESI) m/z
482 ([MÀH]À). Anal. Calcd for C16H14F5N5O5S: C, 39.76; H,
2.92; N, 14.49; S, 6.63. Found: C, 39.52; H, 2.74; N, 14.27; S,
6.82.
3.1.13. 2-Methoxy-6-trifluoromethyl-N-(5,8-dimethoxy-1,2,4-
triazolo[1,5-c]pyrimidin-2-yl)-benzenesulfonamide (6m)
The product was isolated as a white solid (29% yield): mp 238–
240 °C; 1H NMR (DMSO-d6) d 11.87 (s, 1H), 7.74 (t, J = 8.1, 8.4 Hz,
1H), 7.56–7.51 (m, 3H), 4.05 (s, 3H), 3.86 (s, 3H), 3.85 (s, 3H); 13C
NMR (DMSO-d6) d 158.3, 158.0, 148.1, 143.8, 139.3, 134.2, 129.6,
129.3, 129.0, 126.7, 126.8, 124.2, 123.9, 121.4, 119.54, 119.47,
118.2, 57.2, 55.8; MS-DIP (CI) m/z 434 ([M+1]+). Anal. Calcd for
C15H14F3N5O5S: C, 41.57; H, 3.26; N, 16.16; S, 7.40. Found: C,
41.46; H 3.20; N, 16.26; S, 7.34.
3.1.19. 1-[2-Fluoro-1-(fluoromethyl)ethoxy]-6-trifluoromethyl-
N-(5,8-dimethoxy-1,2,4-triazolo[1,5-c]pyrimidin-2-
yl)benzenesulfonamide (6s)
The product was isolated as a white solid (19% yield): mp 229–
231 °C; 1H NMR (DMSO-d6) d 11.81 (br s, 1H), 7.79–7.69 (m, 2H),
7.61–7.59 (m, 2H), 5.42–5.23 (m, 1H), 4.84–4.55 (m, 4H), 4.06 (s,
3H), 3.85 (s, 3H); LC–MS (ESI) m/z 498 ([MÀH]+). Anal. Calcd for
C17H16F5N5O5S: C, 41.05; H, 3.24; N, 14.08; S, 6.45. Found: C,
40.95; H, 3.12; N, 13.96; S, 6.57.
3.1.20. 2,6-Dichloro-N-(5,7-dimethoxy-1,2,4-triazolo[1,5-
c]pyrimidin-2-yl)benzenesulfonamide (7)
A round bottom flask was charged with 2,6-dichloro-N-(5-
methoxy-7-fluoro-1,2,4-triazolo[1,5-c]pyrimidin-2-yl)benzenesul-
fonamide (6g, 0.50 g, 1.3 mmol) and anhydrous MeOH (3 mL). To
3.1.14. 2-Ethoxy-6-trifluoromethyl-N-(5,8-dimethoxy-1,2,4-
triazolo[1,5-c]pyrimidin-2-yl)-benzenesulfonamide (6n)
The product was isolated as a white solid (29% yield): mp
232–234 °C (d); 1H NMR (DMSO-d6), d 11.8 (br s, 1H), 7.72 (t,
J = 8.3 Hz, 1H), 7.59 (s, 1H), 7.53 (d, J = 8.0 Hz, 2H), 4.23 (q,
J = 7.0 Hz, 2H), 4.06 (s, 3H), 3.86 (s, 3H), 1.20 (t, J = 7.0 Hz, 3H);
LC–MS (ESI) m/z 446 ([MÀH]À). Anal. Calcd for C16H16F3N5O5S:
C, 42.96; H, 3.60; N, 15.65; S, 7.17. Found: C, 42.85; H, 3.51;
N, 15.71; S, 7.24.
this mixture was added
a solution of sodium methoxide
(0.61 mL, 2.7 mmol, 25% in MeOH) at which time all the solids dis-
solved giving a dark solution. After 2 h, a solid had precipitated
from the solution. The mixture was stirred for 18 h, the reaction
quenched with HOAc (1 mL), and the methanol removed in vacuo
to give a tan solid. The crude material was purified by column
chromatography (SiO2, CH2Cl2/EtOH, 95:5) to afford 7 as a white
powder (0.30 g, 56% yield): mp 245–247 °C; 1H NMR (DMSO-d6)
d 12.6 (br s, 1H), 7.65–7.57 (m, 3H), 6.45 (s, 1H), 4.11 (s, 3H),
3.88 (s, 3H). Anal. Calcd for C13H11Cl2N5O4S: C, 38.63; H, 2.74; N,
17.33; S, 7.93. Found: C, 38.43; H, 2.75; N, 17.19; S, 8.09.
3.1.15. 2-(2-Fluoroethoxy)-6-trifluoromethyl-N-(5,8-
dimethoxy-1,2,4-triazolo[1,5-c]pyrimidin-2-
yl)benzenesulfonamide (6o)
The product was isolated as a white solid (52% yield): mp 233–
235 °C; 1H NMR (DMSO-d6) d 11.82 (br s, 1H), 7.72 (t, J = 8.0 Hz,

4236
T. C. Johnson et al. / Bioorg. Med. Chem. 17 (2009) 4230–4240
Compounds 5b–g were prepared according to the procedure for
5a below:
147.9, 143.1, 138.4, 113.2, 14.2. The product (5d) was isolated as
a tan solid (75% yield): mp >250 °C; 1H NMR (DMSO-d6) d 8.00 (s,
1H), 6.55 (s, 2H), 4.12 (s, 3H); 13C NMR (DMSO-d6), d 166.4,
151.6, 147.7, 140.9, 108.5, 56.1. Anal. Calcd for C6H6ClN5O: C,
36.11; H, 3.03; N, 35.09. Found: C, 36.11; H, 3.19; N, 34.82.
3.1.21. 2-Amino-5,8-dimethoxy-1,2,4-triazolo[1,5-c]pyrimidine
(5a)
4-Hydrazino-5-methoxy-2-methylthiopyrimidine 8a (5.0 g,
27 mmol)wasmixedwithisopropylalcohol(IPA,50 mL).Tothismix-
ture was added a solution of cyanogen bromide (10 mL, 30 mmol,
3 M, CH2Cl2). The mixture was stirred at room temperature for
30 min then warmed to 50 °C. After 1 h at 50 °C, there was no hydraz-
inopyrimidine detected by thin-layer chromatography (TLC, hexane/
EtOAc, 1:1). The reaction mixture was cooled to room temperature
and diluted with Et2O (150 mL). The solids were filtered, washed
with Et2O, and dried in a vacuum oven (50 °C, high vacuum) to afford
the crude 3-amino-8-methoxy-5-methylthio-1,2,4-triazolo[4,3-
c]pyrimidine hydrobromide as a tan solid (9a, 7.1 g, 90% yield): mp
180–182 °C; 1H NMR (DMSO-d6) d 7.6 (br s, 2H), 7.5 (s, 1H), 3.9 (s,
3H), 2.7 (s, 3H); 13C NMR (DMSO-d6) d 147.4, 141.8, 140.9, 120.3,
57.0, 14.2. The crude 3-amino-8-methoxy-5-methylthio-1,2,4-triaz-
olo[4,3-c]pyrimidine hydrobromide (55.7 g, 191 mmol) and methyl
acrylate (31 mL, 286 mmol) were mixed with CH3OH (200 mL). The
mixture was cooled in an ice bath. To the cold mixture was slowly
added a solution of sodium methoxide (60 mL, 286 mmol, 25%) in
CH3OH. After all the sodium methoxide was added, the mixture
was warmed to room temperature and stirred for 18 h. To the reac-
tion mixture was added HOAc (5 mL) and the solids were collected
by filtration. The solids were partitioned between Et2O (200 mL)
and H2O (200 mL). The insoluble material was collected by filtration
and dried in a vacuum oven (50 °C, high vacuum) to afford the crude
product as a tan solid (26.8 g, 72% yield). A sample of the crude mate-
rial (2.0 g) was recrystallized from DMF to afford 5a as a tan solid
(0.80 g): mp 201–203 °C; 1H NMR (DMSO-d6) d 7.5 (s, 1H), 6.3 (s,
2H), 4.1 (s, 3H), 3.9 (s, 3H); 13C NMR (DMSO-d6) d 166.0, 148.5,
143.7, 138.6, 123.1, 57.0, 55.4. Anal. Calcd for C7H9N5O2: C, 43.08;
H, 4.65; N, 35.88. Found: C, 43.24; H, 4.67; N, 35.59.
3.1.25. 2-Amino-8-fluoro-5-methoxy-1,2,4-triazolo[1,5-
c]pyrimidine (5e)
The intermediate 3-amino-8-fluoro-5-methylthio-1,2,4-triazol-
o[4,3-c]pyrimidine hydrobromide (9e) was isolated as a yellow
powder (82% yield): mp 168–170 °C; 1H NMR (DMSO-d6) d 7.78
(d, J = 1.7 Hz, 1H), 7.10 (br s, 3H), 2.64 (s, 3H); The product (5e)
was isolated as a tan solid (88% yield): mp >250 °C; 1H NMR
(DMSO-d6) d 7.95 (d, J = 2.4 Hz, 1H), 6.53 (s, 2H), 4.10 (s, 3H); 13C
NMR (DMSO-d6) d 166.4, 146.3, 145.4, 144.3, 127.5, 127.2, 56.0.
Anal. Calcd for C6H6FN5O: C, 39.35; H, 3.30; N, 38.24. Found: C,
39.33; H, 3.55; N, 37.89.
3.1.26. 2-Amino-5-methoxy-8-methyl-1,2,4-triazolo[1,5-
c]pyrimidine (5f)
The intermediate 3-amino-8-methyl-5-methylthio-1,2,4-triaz-
olo[4,3-c]pyrimidine hydrobromide (9f) was isolated as a yellow
solid (95% yield): mp 234–236 °C; 1H NMR (DMSO-d6) d 8.60 (s,
1H), 8.45 (br s, 3H), 3.53 (s, 3H), 3.10 (s, 3H); 13C NMR (DMSO-
d6), d 149.6, 147.9, 145.9, 113.8, 14.2, 12.0. The crude product
was purified by column chromatography (SiO2, CH2Cl2/EtOH,
95:5) to afford 5f as a tan solid (26% yield): mp >250 °C; 1H NMR
(DMSO-d6) d 7.63 (d, J = 1.1 Hz, 1H), 6.29 (s, 2H), 4.10 (s, 3H),
2.23 (d, J = 1.1 Hz, 3H); 13C NMR (DMSO-d6) d 166.3, 154.5, 147.3,
140.7, 111.6, 55.3, 12.6. Anal. Calcd for C7H9N5O: C, 46.92; H,
5.06; N, 39.08. Found: C, 46.70; H, 4.84; N, 39.09.
3.1.27. 2-Amino-7-fluoro-5-methoxy-1,2,4-triazolo[1,5-
c]pyrimidine (5g)
The intermediate 3-amino-7-fluoro-5-methylthio-1,2,4-triazol-
o[4,3-c]pyrimidine hydrobromide (9g) was isolated as a tan pow-
der (89% yield): mp 191–196 °C; 1H NMR (DMSO-d6) d 8.00 (br s,
3H), 7.23 (d, J = 1.2 Hz, 1H), 2.77 (s, 3H). The crude product (5g)
was isolated as a tan powder (84% yield): mp >250 °C; 1H NMR
(DMSO-d6) d 6.75 (d, J = 1.1 Hz, 1H), 6.39 (s, 2H), 4.12 (s, 3H). Anal.
Calcd for C6H6FN5O: C, 39.35; H, 3.30; N, 38.24. Found: C, 39.55; H,
3.31; N, 38.18.
3.1.22. 2-Amino-5-methoxy-1,2,4-triazolo[1,5-c]pyrimidine (5b)
The intermediate 2-amino-5-methylthio-1,2,4-triazolo[4,3-
c]pyrimidine (9b) was isolated as a pale yellow solid (0.65 g, 26%
yield): mp 232–234 °C (d); 1H NMR (DMSO-d6)
d 7.52 (d,
J = 6.6 Hz, 1H), 7.13 (d, J = 6.7 Hz, 1H), 6.08 (br s, 2H), 2.62 (s,
3H). The product (5b) was isolated as a tan powder (70% yield):
mp >250 °C; 1H NMR (DMSO-d6) d 7.85 (d, J = 6.1 Hz, 1H), 7.04 (d,
J = 6.1 Hz, 1H), 6.34 (s, 2H), 4.16 (s, 3H); 13C NMR (DMSO-d6), d
166.4, 154.3, 148.7, 143.25, 102.4, 55.6.
Compound 11b was prepared according to the procedure for
11a below:
3.1.28. 2-Propylthio-3-methoxymethoxy-trifluoromethylbenzene
(11a)
3.1.23. 2-Amino-8-ethoxy-5-methoxy-1,2,4-triazolo[1,5-
c]pyrimidine (5c)
A dry 2 L round bottom flask under N2 was charged with
anhydrous THF (500 mL), anhydrous TMEDA (67.8 mL,
449 mmol), anhydrous diisopropylamine (3.06 mL, 21.8 mmol),
and 1-methoxymethoxy-3-trifluoromethylbenzene (90.0 g, 436
mmol). The resulting light yellow solution was cooled in a dry-
ice/acetone bath (ca. À70 °C). To the cold solution was slowly
added a solution of n-butyllithium (174 mL, 435 mmol, 2.5 M
in hexane) such that the temperature remained between
À65 °C and À70 °C. During the addition of n-butyllithium the
solution turned a deep purple color. After all of the n-butyllith-
ium had been added, the cold bath was removed and the solu-
tion allowed to warm to 0 °C, and this temperature was
maintained for 2.5 h. The solution was again cooled to À70 °C
and propyl disulfide (75.2 mL, 480 mmol) was slowly added.
The resulting solution was warmed to room temperature, during
which time a brown solid precipitates. After 18 h at room tem-
perature, a portion of the volatiles (750 mL) was removed in
vacuo. The remaining residue was diluted with Et2O (700 mL)
and washed with water (3 Â 400 mL). The Et2O solution was
The intermediate 3-amino-8-ethoxy-5-methylthio-1,2,4-triaz-
olo[4,3-c]pyrimidine hydrobromide (9c) was isolated as pale yel-
low powder (90% yield): mp 160–163 °C; 1H NMR (DMSO-d6) d
7.57 (s, 1H), 4.24 (q, J = 7.1 Hz, 2H), 2.68 (s, 3H), 1.40 (t,
J = 7.1 Hz, 3H); 13C NMR (DMSO-d6) d 147.2, 141.8, 140.9, 139.3,
121.6, 65.6, 14.2. The crude product was purified by column chro-
matography (SiO2, acetone/hexanes, 60:40) to afford 5c as light tan
powder (55% yield): mp 190–191 °C; 1H NMR (DMSO-d6) d 7.48 (s,
1H), 6.27 (s, 2H), 4.18 (q, J = 7.1 Hz, 2H), 4.07 (s, 3H), 1.35 (t,
J = 7.1 Hz, 3H); 13C NMR (DMSO-d6,) d 166.0, 148.8, 143.9, 137.5,
124.8, 66.5, 55.4, 14.6.
3.1.24. 2-Amino-8-chloro-5-methoxy-1,2,4-triazolo[1,5-
c]pyrimidine (5d)
The intermediate 3-amino-8-chloro-5-methylthio-1,2,4-triazol-
o[4,3-c]pyrimidine hydrobromide (9d) was isolated as a yellow
powder (95% yield): mp >250 °C; 1H NMR (DMSO-d6), d 8.22 (br
s, 1H), 7.89 (s, 1H), 2.68 (s, 3H); 13C NMR (DMSO-d6) d 150.9,

T. C. Johnson et al. / Bioorg. Med. Chem. 17 (2009) 4230–4240
4237
dried (MgSO4), filtered, and the solvent removed in vacuo to af-
3.1.34. 1-[2-Fluoro-1-(fluoromethyl)ethoxy]-6-
trifluoromethylbenzene propyl sulfide (13d)
ford a crude gold oil. A GC analysis of the oil indicated a mixture
of starting material, propyl disulfide and 11a. The starting mate-
rial and propyl disulfide were removed by distillation at reduced
pressure (75 °C, 10 mm Hg) leaving the product as a tan oil
(119 g, 97% yield) that was used without purification: 1H NMR
The product was isolated as a colorless oil (49% yield): 1H NMR
(CDCl3) d 7.41 (dd, J = 2.0, 7.9 Hz, 1H), 7.36 (d, J = 8.0 Hz, 1H), 7.20
(dd, J = 1.9, 7.7 Hz, 1H), 4.82 (m, 2H), 4.76 (m, 1H), 4.67 (m, 2H),
2.87 (t, J = 7.4 Hz, 2H), 1.53 (m, 2H), 0.96 (t, J = 7.3 Hz, 3H); GC–
MS (EI) m/z 314 ([M]+). Anal. Calcd for C13H15F5OS: C, 49.68; H,
4.81. Found: C, 49.66; H, 4.79.
(CDCl3)
d 7.34 (m, 3H), 5.3 (s, 2H), 3.53 (s, 3H), 2.87 (t,
J = 7.4 Hz, 2H), 1.55 (m, 2H), 0.96 (t, J = 7.1 Hz, 3H); GC–MS (EI,
70 eV) m/z 280 ([M]+).
3.1.35. 1-(2,2-Difluoroethoxy)-6-trifluoromethylbenzene
propyl sulfide (13e)
3.1.29. 2-Propylthio-3-trifluoromethylanisole (11b)
Distillation of the crude product after workup gave 11b as a
clear oil (71% yield): bp 92 °C (ca. 1 mm Hg); 1H NMR (CDCl3) d
7.34–7.22 (m, 2H), 7.03 (d, J = 8.0 Hz, 1H), 2.80 (t, J = 7.5 Hz, 2H),
1.50 (s, J = 7.4 Hz, 2H), 0.93 (t, J = 7.5 Hz, 3H). Anal. Calcd for
C11H13F3OS: C, 52.79; H, 5.24; S, 12.81. Found: C, 52.65; H, 5.11;
S, 12.93.
The product was isolated as a colorless oil (78% yield): bp 100 °C
(0.5 mm Hg); 1H NMR (CDCl3) d 7.38 (m, 2H), 7.05 (m, 1H), 6.20 (tt,
J = 4.1, 54.9 Hz, 1H), 4.28 (td, J = 4.2, 12.9 Hz, 2H), 2.85 (t, J = 7.4 Hz,
2H), 1.54 (m, 2H), 0.96 (t, J = 7.4 Hz, 3H); GC–MS (EI) m/z 300
([M]+). Anal. Calcd for C12H13F5OS: C, 48.00; H, 4.36; S, 10.68.
Found: C, 47.92; H, 4.46; S, 10.76.
3.1.30. 2-Propylthio-3-trifluoromethylphenol (12)
3.1.36. 1-(3-Fluoropropoxy)-6-trifluoromethylbenzene propyl
sulfide (13f)
To a solution of 11a (119 g, 424 mmol) in methanol (400 mL)
was added a concentrated solution of HCl (5.0 mL, 37 wt %) and
the resulting solution was heated to 35 °C. After 3 d the solvent
was removed in vacuo to give a dark oil. The oil was dissolved in
CH2Cl2 (500 mL) and the solution washed with water (300 mL).
The organics were dried (MgSO4), filtered, and the solvent removed
in vacuo to afford the crude product as a brown oil. The oil was dis-
tilled at reduced pressure (Kugelrohr, bp 70 °C, ca. 1.0 mm Hg) to
afford 12 as a clear oil (84.5 g, 84% yield): 1H NMR (CDCl3) d 7.48
(s, 1H), 7.37 (t, J = 8.0 Hz, 1H), 7.27 (dd, J = 1.1, 8.2 Hz, 1H), 7.21
(dd, J = 1.1, 8.0 Hz, 1H), 2.69 (t, J = 7.4 Hz, 2H), 1.63 (s, J = 7.4 Hz,
2H), 1.0 (t, J = 7.4 Hz, 3H).
The product was isolated as a colorless oil (97% yield): 1H NMR
(CDCl3) d 7.39–7.30 (m, 2H), 7.08 (dd, J = 1.7, 6.0 Hz, 1H), 4.73 (dt,
J = 5.6, 47.0 Hz, 2H), 4.21 (t, J = 6.2 Hz, 2H), 2.82 (t, J = 7.3 Hz, 2H),
2.26 (dp, J = 5.9, 26.1 Hz, 2H), 1.54 (m, 2H), 0.96 (t, J = 7.4 Hz,
3H); GC–MS (EI) m/z 296 ([M]+).
Compounds 14b–h were prepared according to the procedure
for 14a below:
3.1.37. 2-Ethoxy-6-trifluoromethylbenzenesulfonyl chloride (14a)
Compound 13a (5.1 g, 19.3 mmol) was dissolved in a solution of
acetic acid (20 mL) and H2O (1.5 mL), and the resulting solution
was heated to 45 °C. To the warm solution was slowly added Cl2
(10.2 g, 144 mmol). During the addition of Cl2 the temperature of
the solution rose to 55 °C. After all of the Cl2 had been added, the
temperature of the solution was maintained at 55 °C (ca. 2 h).
The solution was then poured into ice water and the cold mixture
extracted with Et2O (150 mL). The Et2O layer was dried (MgSO4),
filtered, and the solvent removed in vacuo to afford a yellow oil.
The oil was purified by column chromatography (SiO2, 0?20%
EtOAc/hexane) to afford 14a as light yellow crystals (4.5 g, 81%
yield): mp 52–54 °C; 1H NMR (CDCl3) d 7.70 (t, J = 8.0 Hz, 1H),
7.45 (d, J = 8.0 Hz, 1H), 7.35 (d, J = 8.0 Hz, 1H), 4.34–4.27 (q,
J = 7.0 Hz, 2H), 1.55 (t, J = 7.0 Hz, 3H).
Compounds 13b–f were prepared according to the procedure
for 13a below:
3.1.31. 1-Ethoxy-6-trifluoromethylbenzene propyl sulfide (13a)
To a suspension of NaH (1.0 g, 25.4 mmol, 60% dispersion in
mineral oil) in anhydrous DMF (20 mL), under an N2 atmosphere,
was slowly added 12 (5.0 g, 21 mmol). To the resulting solution
was slowly added iodoethane (2.2 mL, 27 mmol). After a few min-
utes a solid began to precipitate and additional DMF (5 mL) was
added to dissolve the precipitate. The resulting solution was stirred
for 66 h. The mixture was diluted with H2O (100 mL) and extracted
with Et2O (200 mL). The Et2O solution was washed with H2O
(4 Â 200 mL), dried (MgSO4), filtered, and the solvent removed in
vacuo to afford the crude product as a tan oil. The oil was distilled
at reduced pressure (Kugelrohr, bp 90–100 °C, ca. 0.3 mm Hg) to
afford 13a as a colorless oil (5.1 g, 91% yield): 1H NMR (CDCl3) d
7.34–7.26 (m, 2H), 7.04 (d, J = 6.6 Hz, 1H), 4.15 (q, J = 7.0 Hz, 2H),
2.87 (t, J = 7.5 Hz, 2H), 1.58–1.50 (m, 5H), 0.97 (t, J = 7.3 Hz, 3H);
GC–MS (EI, 70 eV) m/z 264 ([M]+).
3.1.38. 2-Methoxy-6-fluorobenzenesulfonyl chloride (14b)
The product was isolated as a tan oil (76% yield): 1H NMR
(CDCl3) d 7.62–7.55 (m, 1H), 6.88–6.77 (m, 2H), 4.02 (s, 3H).
3.1.39. 2-Methoxy-6-trifluoromethylbenzenesulfonyl chloride
(14c)
The product was isolated as a white solid (54% yield): mp 86–
88 °C; 1H NMR (CDCl3) d 7.82 (m, 1H), 7.53 (d, J = 7.3 Hz, 1H),
7.46 (d, J = 8.6 Hz, 1H), 4.14 (s, 3H).
3.1.32. 1-(2-Fluoroethoxy)-6-trifluoromethylbenzene propyl
sulfide (13b)
The product was isolated as a colorless oil (95% yield): 1H
NMR (CDCl3) d 7.33–7.29 (m, 2H), 7.03 (dd, J = 3.8, 9.6 Hz, 1H),
4.80 (dt, J = 2.8, 47.3 Hz, 2H), 4.34–4.23 (m, 2H), 2.86 (t,
J = 7.3 Hz, 2H), 1.50 (m, 2H), 0.93 (t, J = 7.3 Hz, 3H); GC–MS (EI)
m/z 282 ([M]+).
3.1.40. 1-(2-Fluoroethoxy)-6-trifluoromethylbenzene sulfonyl
chloride (14d)
The product was isolated as a white solid (90% yield): mp 69–
72 °C; 1H NMR (CDCl3) d 7.74 (t, J = 8.7 Hz, 1H), 7.52 (d, J = 7.7 Hz,
1H), 7.42 (d, J = 8.6 Hz, 1H), 4.86 (dt, J = 4.04, 7.2 Hz, 2H), 4.48
(dt, J = 4.0, 27.0 Hz, 2H).
3.1.33. 1-(2,2,2-Trifluoroethoxy)-6-trifluoromethylbenzene
propyl sulfide (13c)
The product was isolated as a colorless oil (65% yield): bp 73–
76 °C (0.2 mm Hg); 1H NMR (CDCl3) d 7.40–7.35 (m, 2H), 7.05 (d,
J = 8.0 Hz, 1H), 4.43 (q, J = 8.1 Hz, 2H), 2.84 (t, J = 7.4 Hz, 2H), 1.51
(m, 2H), 0.93 (t, J = 7.2 Hz, 3H).
3.1.41. 1-(2,2,2-Trifluoroethoxy)-6-trifluoromethylbenzene
sulfonyl chloride (14e)
The product was isolated as a white solid (94% yield): mp 59–
63 °C; 1H NMR (CDCl3) d 7.80 (t, J = 7.7, 8.7 Hz, 1H), 7.64 (d,
J = 7.3 Hz, 1H), 7.39 (d, J = 8.3 Hz, 1H), 4.58 (q, J = 7.8 Hz, 2H).

4238
T. C. Johnson et al. / Bioorg. Med. Chem. 17 (2009) 4230–4240
3.1.42. 1-[2-Fluoro-1-(fluoromethyl)ethoxy]-6-
trifluoromethylbenzene sulfonyl chloride (14f)
application, plants were maintained in a greenhouse on a 23 °C
day/22 °C night thermoperiod and a 15 h photoperiod.
The product was isolated as a light yellow solid (100% yield):
mp 65–70 °C (d); 1H NMR (CDCl3) d 7.77 (td, J = 0.8, 8.5 Hz, 1H),
7.57 (t, J = 8.0 Hz, 2H), 5.07 (m, J = 5.2 Hz, 1H), 4.88 (m, 2H), 4.72
(m, 2H); MS-DIP (CI) m/z 338 ([M]+). Anal. Calcd for C10H8ClF5O3S:
C, 35.46; H, 2.38; S, 9.47. Found: C, 35.24; H, 2.53; S, 9.41.
Supplemental lighting for post-emergence tests was provided
with 1000 W metal halide overhead lamps which gave average
illumination of 500 uEMÀ2 sÀ1 PAR at the plant canopy. Plants trea-
ted post-emergence were watered by sub-irrigation to prevent
washing the chemical off the foliage. Plants were watered with fer-
tilizer solution 5–7 times per week to maintain good plant growth.
3.1.43. 1-(2,2-Difluoroethoxy)-6-trifluoromethylbenzene
sulfonyl chloride (14g)
3.2.1.2. Formulation and application of chemicals.
Stock
The product was isolated as a white solid (100% yield): mp 66–
68 °C; 1H NMR (CDCl3) d 7.81 (td, J = 0.5, 8.5 Hz, 1H), 7.62 (dd,
J = 0.5, 7.9 Hz, 1H), 7.42 (d, J = 8.5 Hz, 1H), 6.26 (tt, J = 4.1,
54.9 Hz, 1H), 4.45 (td, J = 4.1, 12.3 Hz, 2H). Anal. Calcd for
C9H6ClF5O3S: C, 48.00; H, 4.36; S, 10.68. Found: C, 47.92; H, 4.46;
S, 10.76.
solutions of sulfonamide compounds were formed by dissolving
samples in a solution of acetone/DMSO (97:3 v/v). Post-emergence
spray solutions were formulated by injecting aliquots of stock solu-
tion into spray solution comprised of acetone/water/isopropanol/
DMSO/Agridex/Triton X-155 (48.5:39:10:1.5:1.0:0.02 v/v). Appli-
cations were applied by spraying the foliage of test plants with a
DeVilbiss atomizer driven by compressed air at a pressure of 14–
28 kPa. Approximately 1.5 mL of spray solution was applied to
the plants in each pot. The volume of spray and adjuvant provided
thorough spray coverage.
3.1.44. 1-(3-Fluoropropoxy)-6-trifluoromethylbenzene sulfonyl
chloride (14h)
The product was isolated as a light yellow oil (81%): 1H NMR
(CDCl3) d 7.77 (t, J = 8.0, 8.5 Hz, 1H), 7.52 (d, J = 8.0 Hz, 1H), 7.42
(d, J = 8.5 Hz, 1H), 4.77 (dt, J = 5.5, 47.0 Hz, 2H), 4.38 (t, J = 6.0 Hz,
2H), 2.32 (dp, J = 5.8, 26.1 Hz, 2H); GC–MS (EI) m/z 320 ([M]+).
3.2.1.3. Assessment. Unless otherwise noted, the in vivo green-
house screening data were a tabulation of post-emergence foliar
applied results and expressed as a ‘percent in growth reduction’
(GR) for treated plants, where 0 is no injury and 100 is complete
kill, as visually compared to untreated plants. Assessments of phy-
totoxic effects were made two weeks after chemicals were applied
in post-emergence tests. The broadleaf weed activity (BW) is given
as an average of 80% reduction in growth at a given concentration,
expressed in parts per million (ppm), over five to eight broadleaf
weeds chosen from the following: Xanthium strumarium, Datura
stramonium, Chenopodium album, Helianthus spp., Ipomoea spp.,
Amaranthus retroflexus, Abutilon theophrasti, Veronica heteraefolia,
Ipomoea hederacea, Stellaria media and Polygonum convolvulus.
The grass weed activity (GW) is averaged over five weeds chosen
from Alopecurus spp., E. crus-galli, Setaria fabari, Sorghum halapense,
Digitaria sanguinalis and Avena fatua and expressed in a manner
similar to broadleaf weeds. The injury for a specified crop is ex-
pressed as a 20% reduction in growth compared to the untreated
crop.
3.1.45. 2,6-Dimethoxybenzenesulfonyl chloride (16)
A 1 L flask fitted with an addition funnel, thermometer and
mechanical stirrer was charged with 1,3-dimethoxybenzene (15,
14.2 mL, 108 mmol), anhydrous tetramethylethylenediamine
(TMEDA, 18 mL, 119 mmol) and petroleum ether (225 mL). The
resulting solution was cooled in an ice/water bath. A solution of
n-butyllithium (47.5 mL, 119 mmol, 2.5 M in hexanes) was slowly
added from the addition funnel such that the temperature did not
rise above 5 °C. The reaction mixture was stirred at 0 °C for 20 min
and then cooled in a dry-ice/acetone bath (À70 °C). To the resulting
mixture was added a cold (À65 °C) solution of SO2 (70 g, 1.08 mol)
in dry Et2O (100 mL) in portions such that the temperature did not
rise above À60 °C. The light yellow mixture was slowly warmed to
10 °C, and the solids were filtered and washed with dry Et2O. The
resulting solids were suspended in hexane (400 mL) and cooled
to 0 °C. To the cold suspension was added a solution of sulfuryl
chloride (14.5 g, 108 mmol) in hexane (200 mL) in portions such
that the temperature did not rise above 3 °C. The resulting mixture
was allowed to stir at 0 °C 1 h, and then filtered, and the resulting
filter cake washed with cold hexane. The solids were mixed with
Et2O (600 mL) and the Et2O mixture washed with H2O (600 mL).
The layers were separated, the aqueous phase extracted with addi-
tional Et2O, and the organics were combined, dried (MgSO4), fil-
tered and the solvent removed in vacuo to afford the crude
product as a light yellow solid (19.42 g, 76% yield): mp 89–91 °C;
1H NMR (CDCl3) d 7.51 (t, J = 8.5 Hz, 1H) 6.64 (d, J = 8.5 Hz, 2H)
3.92 (s, 6H); GC–MS (EI, 70 eV) m/z 236 ([M]+).
3.2.2. General procedure for water injection assay (Table 3)
In the field, puddled soil (saturated soil that has had its struc-
ture destroyed by constant mixing) provides a low-leaching, level,
and uniform soil layer which is essential to the root growth of the
young (usually 2–3 leaf) rice seedling transplants. In the
greenhouse, puddled soil was prepared to grow the different trans-
planted rice weeds to simulate a flooded rice paddy environment.
A 32 oz (960 mL, 10.5 cm diameter) disposable cup containing
650 mL of mud per One pot was used to grow transplanted rice
( O. sativa) and E. crus-galli. Other key rice weeds, Monochoria
vaginalis, Cyperus difformis, and Scirpus juncoides were all grown
in multiple 16 oz disposable cups with a volume of 250 mL of
mud per cup. All cups used in the transplanted paddy rice screen
are non-perforated to be able to maintain a constant depth of
water, preferably 3 cm deep. The rice herbicide program uses a
variety of non-sterilized mineral soils for its tests.
All weed seeds are either collected by field scientists or pur-
chased. Prior to filling the 16 and 32 oz cups with mud, a measured
amount of slow release fertilizer was added to the bottom of each
cup. The slow release fertilizer was the only fertilizer that the
plants received during the course of an experiment, since the addi-
tion of liquid fertilizer to the paddy flood will promote the growth
of algae, making test evaluation very difficult. The amount of mud
added to the cups allows for a 3 cm paddy flood depth to be
3.2. Biology data
3.2.1. General procedure for post-emergence tests (Tables 1 and
2)
3.2.1.1. Plant material. For post-emergence evaluations, seeds of
desired test species were planted in Grace-Sierra Metromix 306 (pH
6.0–6.8, O.M. 30%) in square plastic pots with a surface area of
64 cm2. Chemical or physical seed treatments were used on some
species to promote germination. Plants were grown for 7 to 21 days
in greenhouses typically maintained on a 23–29 °C day/22–28 °C
night thermoperiod and a 15 h photoperiod. Chemicals were applied
when plants were 2–12 cm in height and in the 1–4 true leaf stage of
growth. Exact height was species dependent. Following chemical

T. C. Johnson et al. / Bioorg. Med. Chem. 17 (2009) 4230–4240
4239
maintained above the mud surface during the course of green-
house testing.
layers of cheesecloth and centrifuged at 25,000g for 20 min.
The supernatant was filtered through layers of Miracloth
2
Rice seeds (O. sativa, japonica) were sown 3–8 seeds per cell in a
120 cell plastic plug tray (25 cm  50 cm  4.5 cm, cell dimen-
sion = 2.5 cm  2.5 cm) that had been filled with Metro Mix 360,
a soil-less planting medium consisting of peat moss, vermiculite
and fir bark with the top 1/4 in. of Metro Mix brushed off and
the tray sub-watered until the mix was completely saturated. After
the seeds were sown on the saturated Metro Mix, they were cov-
ered with approximately 0.5 cm Metro Mix. The covered seed
was lightly top watered and the trays were incubated at high tem-
perature and humidity (30 °C, 95% RH) for 48 h in the dark, rotating
the trays at 24 h to maintain uniformity. During this time the rice
seed should germinate and have approximately 1.5–2 cm of etio-
lated growth visible above the Metro Mix prior to removal from
the germination chamber and placed in the rice greenhouse. The
flats remain in the greenhouse an additional 88–96 h before being
transplanted into the deli cups. The day after they are moved into
the greenhouse, the flats were fertilized twice with a solution of 1/
4 tsp (2.5 g) ammonium sulfate + 1/4 tsp (1.8 g) iron chelate in 1
gallon (3.7 L) water. If not fertilized with this liquid fertilizer, rice
seedlings will grow poorly and be a nice shade of yellow instead
of a healthy green color. When the rice was at the 2–3 leaf stage,
typically at 6 d after seeding in the Metro Mix in the cell flat, it
was ready for transplanting. Cells of the plug trays were removed
intact and the remaining Metro Mix was washed off in water and
the seedlings grouped into hills or clusters of seedlings consisting
of 3 seedlings per hill or cluster, with a 32 oz deli cup planted with
2 hills. Each hill was pushed into the soil, submerging the roots and
seed hulls to a depth of about 2 cm from seed hull to soil surface.
After all cups have been transplanted, the cups were flooded to a
depth of 3 cm. The transplants were allowed to recover and devel-
op an additional 0.5–1 leaf prior to treatment. This usually requires
5 d. The entire process from seed soaking to treatment typically re-
quires 15 d.
and brought to 33% saturation with (NH4)2SO4 while stirring
for 40 min. After centrifugation at 25,000g for 20 min, the
supernatant was discarded and the pellet was re-suspended in
3 mL of extraction buffer. The re-suspended pellet could then
be frozen in liquid N2 and stored at À80 °C for up to 1 month
before use.
Enzyme activity was assayed colorimetrically by measuring the
amount of acetoin formed from acetolactate using the method of
Westerfeld.26 Standard reaction mixtures contained 20 mM HEPES
(pH 7.2), 5 mM MgCl2, 0.25 mM TPP, 3.3 lM FAD, 22.5 mM sodium
pyruvate and various concentrations of the inhibitors in a final
DMSO concentration of 0.7%. The reaction was initiated with the
enzyme and the mixture was incubated at 37 °C for 30 min. The
reaction was stopped with the addition of H2SO4 to produce a final
concentration of 0.5% H2SO4 and heated at 60 °C for 20 min to
decarboxylate the acetolactate. The acetoin concentration pro-
duced was determined by adding creatine and 1-naphthol to the
reaction mixture in final concentrations of 2.0 mg/mL and 20 mg/
mL, respectively. The mixture was made alkaline with the addition
of NaOH to a final concentration of 0.5 N and incubated at 37 °C for
40 min. The absorbency was measured at 530 nm to determine the
amount of acetoin produced. The reaction was determined to be
linear for at least 75 min. The in vitro inhibition of acetolactate
synthase is indicated by 50% inhibition of the enzyme (I50) in nano-
Molar units (nM).
References and notes
1. Kleschick, W. A.; Costales, M. J.; Dunbar, J. E.; Meikle, R. W.; Monte, W.
T.; Pearson, N. R.; Snider, S. W.; Vinogradoff, A. P. Pest. Sci. 1990, 29,
341.
2. Kleschick, W. A.; Gerwick, B. C.; Carson, C. M.; Monte, W. T.; Snider, S. W. J.
Agric. Food Chem. 1992, 40, 1083.
3. Subramanian, M. V.; Loney-Gallant, V.; Dias, J. M.; Mireles, L. C. Plant Physiol.
1991, 96, 341.
Application rates were calculated based on the surface area of
the treated pot. Application of each treatment was made by
injection of the treatment solution with an Eppendorf positive
displacement pipette. A serial dilution was used to apply the dif-
ferent rates tested for each active ingredient, achieved by chang-
ing the treatment volume delivered to each pot, where the high
rate receives 4 mL of the stock solution per pot, the second rate
2 mL per pot, etc. Following treatment, the flood depth in the
cups was held constant at 3 cm. It was very important not to
overfill the cups for 2 days after treatment, so as not to wash
out the treatment. The 3 cm depth was maintained by addition
of water at least twice a day. De-ionized water was used in
the rice greenhouse to prevent buildup of salts and dissolved
solids. This 3 cm depth was representative of commercial appli-
cations of actual herbicides in flooded transplanted paddy rice in
Japan and Korea.
4. Kleschick, W. A.. In Herbicides Inhibiting Branch Chain Amino Acid Biosynthesis;
Stetter, J., Ed.; Springer: Germany, 1994; Vol. 10, pp 119–143.
5. Johnson, T. C.; Mann, R. K.; Schmitzer, P. R.; Gast, R. E.; deBoer, G. J..
In Modern Crop Protection Compounds; Kramer, W., Schirmer, U., Eds.;
WILEY-VCH Verlag Gmbh
113.
& Co. KGaA: Weinheim, 2007; Vol. 2, pp 93–
6. Johnson, T. C.; Martin, T. P.; Mann, R. K. In Pesticide Chemistry; Ohkawa, H.,
Miyagawa, H., Lee, P. W., Eds.; WILEY-VCH Verlag Gmbh
Weinheim, 2007; pp 89–100.
& Co. KGaA:
7. Johnson, T. C.; Ehr, R. J.; Johnston, R. D.; Kleschick, W. A.; Martin, T. P.; Pobanz,
M. A.; Van Heertum, J. V.; Mann, R. K. U.S. Patent 5,858,924, 1999.
8. Johnson, T. C.; Ehr, R. J.; Johnston, R. D.; Kleschick, W. A.; Martin, T. P.; Pobanz,
M. A.; Van Heertum, J. V.; Mann, R. K. U.S. Patent 6,005,108, 1999.
9. Johnson, T. C.; Ehr, R. J.; Johnston, R. D.; Kleschick, W. A.; Martin, T. P.;
Pobanz, M. A.; Van Heertum, J. V.; Mann, R. K. U.S. Patent 6,303,814,
2001.
10. Johnson, T. C.; Nasutavicus, W. A. U.S. Patent 5,177,206, 1993.
11. Ringer, J. W.; Scott, A. C.; Pearson, D. L.; Wallin, A. P. U.S. Patent 5,973,148,
1999.
12. Hamilton, T. C. U.S. Patent 7,339,058(B2), 2008.
13. Miller, G. W.; Rose, F. L. J. Chem. Soc. 1963, 5642.
All tests were incubated in the greenhouse where conditions
were maintained on a 16 h day length with a daytime/nighttime
demand temperature of 29 °C (88 °F). Tests were typically evalu-
ated 3 weeks after application to allow sufficient time for weed
and rice to express maximum symptoms. Visual evaluations of test
results were made based on a scale of 0–100%, where 0% is no in-
jury or growth reduction and 100% is complete death.
14. Medwid, J. B.; Paul, R.; Baker, J. S.; Brockman, J. A.; Du, B. M.; Hallet, W. A.;
Hanifin, J. W.; Hardey, R. A.; Tarrant, M. E.; Torley, L. W.; Wrenn, S. J. Med. Chem.
1990, 33, 1230.
15. Smith, M. G.; Pobanz, M. A.; Roth, G. A.; Gonzales, M. A. U.S. Patent 6,462,240,
2002.
16. Hamada, T.; Yonemitsu, O. Synthesis 1986, 852. and references cited therein.
17. Jacob, P.; Shulgin, A. T. Synth. Commun. 1981, 11, 957.
18. Pinnick, H. W.; Reynolds, M. A. J. Org. Chem. 1979, 44, 160.
19. Mann, R. K.; Huang, Y.-H.; Larelle, D.; Mavrotas, C.; Min, Y. K.; Morell,
M.; Nonino, H.; Shiraishi, I. In Proceeding of the 3rd International
Temperate Rice Conference, Punta del Este, Uruguay, March 10–13,
3.2.3. General procedure for ALS enzyme extraction and assay
The following procedures were adapted from Muhitch et al.25
and were performed at 0–4 °C. The etiolated shoots of 5–6 d
old Hector Spring barley (100 g) were homogenized in a Waring
Blender for 2 min with 2.5 volumes of extraction buffer contain-
ing 20 mM HEPES (pH 7.2), 1 mM EDTA, and 5 mM DTT. The
homogenate was filtered through 2 layers of Miracloth and 4
2003, WD055,
p 68.
20. Mann, R. K.; Lassiter, R. B.; Haack, A. E.; Langston, V. B.; Simpson, D. M.;
Richburg, J. S.; Wright, T. R.; Gast, R. E.; Nolting, S. P. Abstracts of the 2003
Meeting of the Weed Science Society of America, Jacksonville, Florida, February
11–13, 2003; Kremer, R. J., Ed.; Weed Science Society of America: Lawrence,
Kansas; p 43.
21. Mann, R. K.; Mavrotas, C.; Huang, Y. H.; Larelle, D.; Patil, V.; Min, Y. K.; Shiraishi,
I.; Nguyen, L.; Nonino, H. L.; Morell, M. In Proceeding of the 20th Asian-Pacific

4240
T. C. Johnson et al. / Bioorg. Med. Chem. 17 (2009) 4230–4240
Weed Science Society; Chin, D. V., Ed.; Ho Chi Minh City, Vietnam, 2005, pp 289–
294.
22. Larelle, D.; Mann, R. K.; Cavanna, S.; Bernes, R.; Duriatti, A.; Mavrotas, C. In Crop
Science and Technology 2003, Congress Proceedings of the BCPC International
Congress, Glasgow, Scotland, November 10–12, 2003; British Crop Protection
Council, Bracknell, Berks, UK, 2003; Vol. 1, pp 75–80.
23. Wang, C. L.; Lee, M. S.; Li, Y. W.; Yao, Z. W.; Shieh, J. N.; Mann, R. K.; Huang, Y.
H.; Abstracts of the 15th International Plant Protection Congress, Beijing, China,
2004; p 598.
24. Min, Y. K.; Mann, R. K. Korean J. Weed Sci. 2004, 24, 192.
25. Muhitch, M. J.; Shaner, D. L.; Stidham, M. A. Plant Physiol. 1987, 83, 451.
26. Westerfeld, W. W. J. Biol. Chem. 1945, 161, 395.