Discovery of inhibitors and substrates of brassinin hydrolase: Probing selectivity with dithiocarbamate bioisosteres

Article abstract of DOI:10.1016/j.bmc.2011.11.009

Brassinin hydrolase (BHAb), an inducible enzyme produced by the plant pathogen Alternaria brassicicola under stress conditions, catalyzes the hydrolysis of the methyl dithiocarbamate group of the phytoalexin brassinin, to indolyl-3-methanamine, methane thiol and carbonyl sulfide. Thirty four substrate inspired compounds, bioisosteres of brassinin and a range of related compounds, were evaluated as potential substrates and inhibitors of BHAb for the first time. While six compounds containing thiocarbamate, carbamate and carbonate groups displayed inhibitory activity against BHAb, only two were found to be substrates (thionecarbamate and dithiocarbamate). Methyl naphthalen-1-yl-methyl carbamate, the most potent inhibitor of the six, and methyl N′-(1-methyl- 3-indolylmethyl)carbamate inhibited BHAb through a reversible noncompetitive mechanism (K_i = 89 ± 9 and 695 ± 60 μM, respectively). Importantly, these carbamate inhibitors were resistant to degradation by A. brassicicola. Carbonates were also inhibitory of BHAb, but a quick degradation by A. brassicicola makes their potential use as crop protectants less likely. Overall, these results indicate that indolyl and naphthalenyl carbamates are excellent lead structures to design new paldoxins that could inhibit the detoxification of brassinin by A. brassicicola.

Full text of DOI:10.1016/j.bmc.2011.11.009

Bioorganic & Medicinal Chemistry 20 (2012) 225–233
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery of inhibitors and substrates of brassinin hydrolase:
Probing selectivity with dithiocarbamate bioisosteres
⇑
M. Soledade C. Pedras , Zoran Minic, Sajjad Hossain
Department of Chemistry, University of Saskatchewan, 110 Science Place, Saskatoon, Canada SK S7N 5C9
a r t i c l e i n f o
a b s t r a c t
Article history:
Brassinin hydrolase (BHAb), an inducible enzyme produced by the plant pathogen Alternaria brassicicola
under stress conditions, catalyzes the hydrolysis of the methyl dithiocarbamate group of the phytoalexin
brassinin, to indolyl-3-methanamine, methane thiol and carbonyl sulfide. Thirty four substrate inspired
compounds, bioisosteres of brassinin and a range of related compounds, were evaluated as potential sub-
strates and inhibitors of BHAb for the first time. While six compounds containing thiocarbamate, carba-
mate and carbonate groups displayed inhibitory activity against BHAb, only two were found to be
substrates (thionecarbamate and dithiocarbamate). Methyl naphthalen-1-yl-methyl carbamate, the most
Received 27 September 2011
Revised 3 November 2011
Accepted 5 November 2011
Available online 12 November 2011
Keywords:
Brassinin hydrolase
Detoxification
Naphthalenylmethyl carbamate
Naphthalenylmethyl carbonate
Naphthalenylmethyl dithiocarbamate
Paldoxin
0
potent inhibitor of the six, and methyl N -(1-methyl-3-indolylmethyl)carbamate inhibited BHAb through
a reversible noncompetitive mechanism (K = 89 ± 9 and 695 ± 60 lM, respectively). Importantly, these
i
carbamate inhibitors were resistant to degradation by A. brassicicola. Carbonates were also inhibitory
of BHAb, but a quick degradation by A. brassicicola makes their potential use as crop protectants less
likely. Overall, these results indicate that indolyl and naphthalenyl carbamates are excellent lead struc-
tures to design new paldoxins that could inhibit the detoxification of brassinin by A. brassicicola.
Ó 2011 Elsevier Ltd. All rights reserved.
Phytoalexin
Ser/Ser/Lys catalytic triad
1
. Introduction
Detoxification of the phytoalexin brassinin (1) by fungal plant
respectively (Scheme 1). Importantly, in every example investigated
hitherto, brassinin detoxifying enzymes appear to be inducible by
compounds that cause some sort of cell stress.
pathogens is mediated by different enzymes with catalytic activities
that depend on the fungal species. Brassinin hydrolases (BHs) cata-
lyze the hydrolysis of the methyl dithiocarbamate of brassinin (1),
to indolyl-3-methanamine (2), methane thiol and carbonyl sulfide
Native BHAb is a dimeric protein of 120 kDa, whereas native
BHLmL2 is a tetrameric protein with a molecular mass of 220 kDa.
1
–3
(Scheme 1).
Brassinin (1) is an antimicrobial plant metabolite
produced de novo by crucifers in response to fungal attack and other
4
–6
forms of stress.
BHAb is produced by Alternaria brassicicola
(
Schwein.) Wiltshire, whilst BHLmL2 is produced by Leptosphaeria
maculans (Desm.) [Ces. et de Not., asexual stage Phoma lingam (Tode
ex Fr.) Desm., isolate Laird 2 (LmL2), mustard virulent isolates]. BHs
were characterized using LC-ESI-MS/MS of tryptic digests followed
by sequence alignment analyses and chemical modifications, which
suggested that both enzymes belonged to the family of amidases
1
having the catalytic Ser/Ser/Lys triad. To date, no enzymes involved
in the catalytic hydrolysis of dithiocarbamate groups have been
reported, that is, BHs have unique substrate specificity and catalytic
activity designated as dithiocarbamate hydrolase. While both BHAb
and BHLmL2 catalyze the hydrolysis of brassinin (1),1 brassinin
7
oxidase (BOLm) from L. maculans (canola virulent isolates) and
brassinin glucosyl transferase (SsBGT1) from Sclerotinia sclerotiorum
8
Scheme 1. Enzyme mediated transformations of brassinin (1): BOLm, brassinin
oxidase from Leptosphaeria maculans (isolates virulent on canola) ; BHLmL2,
(
Lib.) de Bary catalyze oxidation and glucosylation detoxifications,
7
brassinin hydrolase from L. maculans (isolates virulent on brown mustard); BHAb,
⇑
Corresponding author. Tel.: +1 306 966 4772; fax: +1 306 966 4730.
E-mail address: soledade.pedras@usask.ca (M.S.C. Pedras).
1
brassinin hydrolase from Alternaria brassicicola ; SsBGT1, brassinin glucosyl trans-
ferase from Sclerotinia sclerotiorum.8
0
968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.11.009

2
26
M.S.C. Pedras et al. / Bioorg. Med. Chem. 20 (2012) 225–233
The purified enzymes showed hydrolytic activity toward brassinin
1), 1-methylbrassinin (1a, R = CH ), methyl tryptaminedithiocar-
bamate (6) and methyl tryptopholdithiocarbonate (7). Although
brassinin (1) was the best substrate for both BHLmL2 and BHAb,
BHAb exhibited relatively higher activity (about twofold) with
known cruciferous phytoalexin),13 thionecarbamate 13, and
carbamate 14. These compounds can provide insights into the effect
of toxophore size and polarity on the rate of enzymatic hydrolysis.
Similarly, replacement of the indolyl ring of brassinin with 1- and
2-naphthalenyl moieties and dithiocarbamate, carbamate and car-
bonate groups creates additional compounds (15–20) that probe
size and hydrophobic interactions in the active site of BHAb
(Scheme 2) (new syntheses and spectroscopic characterization de-
scribed in Section 4.3.3). The synthesis of 3-indolyl carbonate 11
could not be achieved, likely due to its high reactivity. For similar
purposes, other functional groups such as ureas, thioureas, sulfa-
mides, sulfonamides, amides, and esters were also incorporated,
amounting to a total of 23 compounds (Table S2, prepared according
to references cited in Supplementary data).
(
3
1
-methylbrassinin (1a) than BHLmL2. Conversely, the rates of
hydrolysis of methyl tryptaminedithiocarbamate (6) and methyl
tryptopholdithiocarbonate (7) catalysed by BHLmL2 were substan-
tially higher than those catalysed by BHAb.1 Investigation of the
effect of various cruciferous phytoalexins on the activities of BHAb
and BHLmL2 indicated that only the phytoalexin cyclobrassinin
1
3
(8) was a competitive inhibitor of both enzymes.
2
.1.1. Synthesis of naphthalenylmethyl carbamates 17 and 18
and carbonates 19 and 20
A variety of methods are available for the preparation of carba-
mates, as for example the Hofmann rearrangement of carboxamides
1
4
mediated by hypervalent iodine species, Curtius rearrangement of
1
5
acyl azides, trapping of carbamic acid species with (trimethyl-
1
6
silyl)diazomethane, or coupling of primary aliphatic amines and
Because phytoalexins are plant metabolites with defensive roles
1
7
dialkyl carbonates in supercritical carbon dioxide. Similarly, the
corresponding carbonates can be prepared by direct condensation
of carbon dioxide with alcohols using trisubstituted phosphine-car-
against pathogens, metabolic transformations that lead to a
decrease of their concentrations in disease stressed tissues are det-
rimental to plants. Within crucifers (family Brassicaceae), brassinin
1
8
19
bon tetrabromide or silver carbonate and alkyl halides. In this
work, we used a simpler procedure for preparation of both carba-
mates and carbonates; treatment of the corresponding amines
and alcohols with methyl chloroformate in the presence of TEA,11
or sodium hydride, respectively, afforded the desired carbamates
17 and 18 and carbonates 19 and 20 (Scheme 3). The preparation
of naphthalenylmethyl carbonates does not appear to have been
previously reported.
(
1) is of additional significance because it is inhibitory to many
fungal pathogens and is a biosynthetic precursor of several other
phytoalexins equally potent.6 For this reason, paldoxins (phytoa-
lexin detoxification inhibitors) are being developed and evaluated
as potential crop protectants against pathogens that detoxify
brassinin. Recently, paldoxins for BOLm were designed based on
4
different phytoalexin scaffolds and evaluated for inhibition of
brassinin oxidation. The best inhibitors of BOLm were based on
the structures of camalexin (9)9 and brassilexin (10) ; however,
to date no compounds other than cyclobrassinin (8) were found
to inhibit BHAb or BHLmL2 mediated hydrolysis of brassinin (1).
Considering that inhibitors of these hydrolytic enzymes are poten-
tial protectants of cruciferous crops against some of the most dam-
aging fungal pathogens, it is of great interest to develop such
compounds. Toward this end, bioisosteres of brassinin (1) and a
range of related compounds were evaluated as potential substrates
and inhibitors of BHAb and herein we describe results of this work.
10
2
.2. Antifungal bioassays and biotransformations by Alternaria
brassicicola
The antifungal activity of compounds 12–20 against A. brassici-
cola was determined employing the mycelial radial growth assay
described in the Section 4, using DMSO to dissolve each compound.
2
2
. Results and discussion
.1. Design and synthesis of potential substrates and inhibitors
of BHAb
Obviously, the chemical mechanism of transformation of brassi-
nin (1) mediated by BHAb1 is fundamentally different from that
7,11
mediated by BOLm (hydrolysis vs oxidation, Scheme 1).
None-
theless, because both BHAb and BOLm mediate transformations
1
2
that occur at the dithiocarbamate group, the design of bioisosteres
of brassinin (1) necessary to probe the substrate specificities of each
enzyme is based on similar principles. For example, replacement of
the sulfur atoms of the dithiocarbamate of 1 with oxygen atoms
generates three different isosteres, thiolcarbamate 12 (brassitin, a
Scheme 2. Bioisosteres of brassinin (1) and corresponding naphthalenyl derivatives
designed to probe the substrate specificity of BHAb.

M.S.C. Pedras et al. / Bioorg. Med. Chem. 20 (2012) 225–233
227
fungus did not produce enzymes able to transform 14. Further-
more, since naphthalenyl carbamates 17 and 18 were transformed
in culture, it was clear that the presence of a carbamate group was
not sufficient to prevent its enzyme-mediated hydrolysis.
2
.3. Substrates and inhibitors of BHAb
In preliminary assays, the hydrolase activity of BHAb was
Scheme 3. Synthesis of 1- and 2-naphthalenylmethyl carbamates 17 and 18 and
carbonates 19 and 20. Reagents and conditions: (i) ClCO Me, Et N, CH Cl , rt, 17
97%), 18 (96%); (ii) ClCO Et, NaH, THF, 0 °C, 19 (80%), 20 (51%).
screened using a range of indolyl and naphthalenyl synthetic
compounds to evaluate the substrate specificity (Tables 2 and
S2). In addition to 1-methylbrassinin (1a), only compound 13
was found to be a reasonable substrate, although the specific
activity of BHAb was three-fold lower relative to brassinin (1).
As summarized in Table 2, among the naphthalenyl containing
compounds, only dithiocarbamate 15 was enzymatically trans-
formed to the corresponding amine (ca. 5% transformation rela-
tive to 100% of brassinin), whereas neither indolyl carbamates
2
3
2
2
(
2
Assay control plates contained solvent without compound. After
days of incubation, the mycelial growth in each plate was
4
measured and the results were statistically analyzed (one-way AN-
OVA, Table 1). Methyl naththalen-2-ylmethyl carbamate 18 was
the most inhibitory of all compounds tested, causing complete
mycelial growth inhibition of A. brassicicola at 0.50 mM, whereas
1
3
-indolylmethyl carbamate 14 displayed the lowest activity. In
14, as previously established, nor 14a were hydrolyzed by
addition, the 2-substituted naphthalenyl derivatives were signifi-
cantly more inhibitory than the 1-substituted counterparts. Previ-
ously, compounds 15 and 16 were shown to display similar
inhibitory activities against L. maculans13 and S. sclerotiorum.
Cultures of A. brassicicola were incubated with each compound
and their transformations were monitored by HPLC (photodiode
array and ESI-MS detection). Samples were withdrawn from cul-
tures immediately after addition of each compound and then as
described for each case, up to five days. The samples were sub-
jected to neutral, acidic and basic extractions, and the extracts
were analyzed by HPLC, as reported in the Section 4. Media incu-
bated with each compound (control solutions) were analyzed sim-
ilarly to determine the chemical stability of each compound during
the incubation experiments. All compounds were stable in the con-
trol solutions for the duration of the experiment (up to 120 h).
As shown in Tables 1 and S1, except for 3-indolylmethyl carba-
mate 14, all compounds were hydrolyzed to the corresponding
amines or alcohols, however the transformation rates were quite
different. Specifically, it is of interest to compare the group of four
indolyl isosteres (dithio/thiocarbamates/carbamates) 1 and 12–14,
where substantial differences in transformation rates were found
BHAb. Of the 23 compounds listed in Table S2, only dithiocarba-
mate 26 was transformed (ca. 5% transformation relative to 100%
of brassinin) to the corresponding amine.
Next, the potential inhibitory effects of compounds 12, 14, 14a,
16–20 on BHAb (Table 2), together with those compounds 21–43
(Table S2), were tested at 0.10 and 0.30 mM (concentrations close
to the concentration of substrate required for half-maximal activity,
20
S0.5 = 0.24 mM) in the presence of brassinin (0.10 mM), as reported
in Section 4.5. Results of these assays are presented in Tables 2
(compounds 12–20) and S2 (compounds 21–43). Although most
of the compounds had no effect on BHAb (16, 18 and 21–43),
3-indolylmethyl carbamates 14 and 14a, 1-naththalenylmethyl
carbamate 17, and naphthalenylmethyl carbonates 19 and 20
displayed inhibitory activity. Methylation of the nitrogen of the
indole ring increased the inhibitory activity of 14a about twofold
relative to the non-methylated analogue 14; however, the inhibi-
tory effect observed with 17 was the strongest among the
i
compounds tested (K = 89 lM).
Additional experiments were carried out with compounds
known to covalently modify the active sites of Ser/Ser/Lys hydro-
lases. Since our previous work revealed that chemical modification
of the BHAb with PMSF (5.0 mM) resulted in loss of activity (51%),
to get additional information on the inhibitory effects of serine
modifying compounds, Pefabloc and phenylphosphorodiamidate
(0.50 and 1.0 mM) were incubated with BHAb followed by incuba-
tion with brassinin (0.10 mM). These experiments indicated that
Pefabloc (41 ± 4% at 0.50 mM; 55 ± 8% at 1.0 mM) was inhibitory,
but phenylphosphorodiamidate did not affect BHAb activity.
(Table 1). For example, while dithiocarbamate 1 and thiolcarba-
mate 12 showed t1/2 = 6 and 72 h, respectively, the transformation
rate of thionecarbamate 13 was in between, with a t1/2 = 18 h, and
carbamate 14 was not transformed. Purified BHAb (Table 2) med-
iated the transformation of 1 and 13, but did not transform 12 or
1
4. These results indicated that the transformations of 1 and 13
in cultures of A. brassicicola were mediated by BHAb, and that the
Table 1
Antifungal activity of brassinin (1) and compounds 12–20 against Alternaria brassicicola and t1/2 for biotransformation of each compound in liquid cultures
%
Inhibitiona
0.20 mM
Compound name/t1/2 h/r
t
(HPLC method A)
0.50 mM
59 (±0)m
0.10 mM
b
m
m
Brassinin (1)/6 h /12.1 min
Brassitin (12)/72 h/7.5 min
40 (±1)
30 (±2)
m
n
7 (±2)n
54 (±3)
54 (±3)
28 (±2)
49 (±1)
67 (±4)
66 (±1)
19 (±1)
30 (±1)
0
0
m
o
n
Methyl N -(3-indolylmethyl) thionecarbamate (13)/18 h/10.1 min
18 (±1)
n
m
o
n
8 (±1)n
Methyl N -(3-indolylmethyl) carbamate (14)/no biotransformation/6.2 min
15 (±1)
42 (±1)
m
m
Methyl N-(1-naphthalenylmethyl) dithiocarbamate (15)/12 h/17.8 min
Methyl N-(2-naphthalenylmethyl) dithiocarbamate (16)/12 h/17.8 min
Methyl N-(1-naphthalenylmethyl) carbamate (17)/60 h/11.8 min
Methyl N-(2-naphthalenylmethyl) carbamate (18)/60 h/11.9 min
Methyl N-(1-naphthalenylmethyl) carbonate (19)/12 h/16.2 min
Methyl N-(2-naphthalenylmethyl) carbonate (20)/12 h/ 16.7 min
33 (±1)
61 (±3)p
50 (±3)
o
o
n
2 (±1)n
16 (±4)
p
71 (±1)p
n
100 (±0)
17 (±1)
38 (±3)q
14 (±1)
n
3 (±1)
n
m
n
12 (±2)n
55 (±3)
21 (±3)
a
Percentage of growth inhibition calculated using the formula: % inhibition = 100 ꢀ [(growth on amended medium/growth in control medium) ꢁ 100]. Data are the
means ± SE; for statistical analysis, one-way ANOVA tests were performed followed by Tukey’s test with adjusted
a set at 0.05; n = 3; different letters in the same column (m–
q) indicate significant differences (P <0.05).
b
Data from Ref. 3.

2
28
M.S.C. Pedras et al. / Bioorg. Med. Chem. 20 (2012) 225–233
Table 2
Substrates and inhibitors of BHAb
Compound (#)/r
t
(HPLC method B)
Structure
Relative specific activity
%)
Inhibition (%)
0.10/0.30 mM
Type of inhibition/
i
K (lM)
(
Brassinin (1)/2.7 min
100
—
—
—
1
-Methylbrassinin (1a)a
50 ± 4b
—
a
Cyclobrassinin (8) /2.7 min
No transformation
No transformation
16 ± 2/26 ± 4
9 ± 3/18 ± 2
competitive/410 ± 80
Not determined
Brassitin (12)/1.6 min
0
Methyl N -(3-indolylmethyl) thionecarbamate (13)/2.1 min
32 ± 2
—
—
0
Methyl N -(3-indolylmethyl) carbamate (14)/1.5 min
No transformation
No transformation
10 ± 4/21 ± 4
28 ± 9/46 ± 3
Not determined
0
Methyl 1-methyl-N -(3-indolylmethyl) carbamate (14a)/2.2 min
Non-competitive/695 ± 60
Methyl N-(1-naphthalenylmethyl) dithiocarbamate (15)/5.7 min
Methyl N-(2-naphthalenylmethyl) dithiocarbamate (16)/5.7 min
Methyl N-(1-naphthalenylmethyl) carbamate (17)/3.1 min
5 ± 1
—
—
No transformation
No transformation
No inhibition
70 ± 3/89 ± 2
—
Non-competitive/89 ± 9
Methyl N-(2-naphthalenylmethyl) carbamate (18)/3.1 min
Methyl N-(1-naphthalenylmethyl) carbonate (19)/5.1 min
Methyl N-(2-naphthalenylmethyl) carbonate (20)/5.2 min
No transformation
No transformation
No transformation
No inhibition
38 ± 4/62 ± 4
29 ± 3/52 ± 3
—
Mixed/142 ± 14
Competitive/97 ± 22
a
Compounds previously tested as substrates.1
Data from Ref. 1.
b
2
.4. Determination of the types of inhibition of BHAb by com
of 1/Vmax values (decrease of Vmax of BHAb) in a dose-dependent
manner (Fig. 1A and B). The results showed that the intersection
points of all curves were on the 1/S axis indicating that the appar-
pounds 14a, 17, 19 and 20
To examine the kinetic mechanism by which the strongest
inhibitors (Table 2, compounds 14a, 17, 19 and 20) affected BHAb
activity, different concentrations of brassinin (1) were incubated
with various concentrations of each inhibitor, as describe in
Section 4.5. The kinetics of inhibition of BHAb is shown in the form
of Lineweaver–Burk double reciprocal plots (1/S vs 1/V) (Fig. 1).
These experiments revealed that incubation of brassinin (1) with
m
ent K values were essentially unchanged relative to the untreated
control. That is, these results indicated that inhibition by 14a and
17 could be attributable to either a reversible noncompetitive or
an irreversible mechanism.
0
To determine whether methyl N -(1-methyl-3-indolylmethyl)
carbamate (14a) and methylnaphthalen-1-yl-methylcarbamate (17)
inhibited BHAb reversibly, the purified enzyme was incubated with
0
0
methyl N -(1-methyl-3-indolylmethyl)carbamate (14a) and meth-
brassinin (1) in the presence of methyl N -(1-methyl-3-indolylmeth-
ylnaphthalen-1-yl-methylcarbamate (17) resulted in an increase
yl)carbamate (14a) (0.30 mM) and methylnaphthalen-1-yl-methylc-

M.S.C. Pedras et al. / Bioorg. Med. Chem. 20 (2012) 225–233
229
0
Figure 2. Characterization of the type of BHAb inhibition by methyl N -(1-methyl-
0
Figure 1. Lineweaver–Burk plots of BHAb activity. In the presence of (A) methyl N -
1-methyl-3-indolylmethyl)carbamate (14a) and (B) methylnaphthalen-1-yl-meth-
3
-indolylmethyl)carbamate (14a) (0.30 mM) and methyl naphthalen-1-yl-meth-
ylcarbamate (17). (A) Effects of methyl N -(1-methyl-3-indolylmethyl)carbamate
(
0
ylcarbamate (17).
(14a) (0.30 mM) or methyl naphthalen-1-yl-methylcarbamate (17) (0.1 mM) on
BHAb activity after dialysis. (B) Time- and concentration-dependent activity of
BHAb under increasing concentrations of methyl naphthalen-1-yl-methylcarba-
mate (17). Control experiments were performed in the absence of compounds
(dialysis of the purified enzyme). Results are expressed as mean ± standard
deviation of three separate experiments.
arbamate (17) (0.10 mM) and then subjected each mixture to exten-
sive dialysis (3 ꢁ 2 h , 4–8 °C). Dialysis restored the activity of BHAb
in the enzyme solutions treated with either compound 14a or 17
(Fig. 2A), indicating that these two compounds inhibited BHAb
through a reversible noncompetitive mechanism. The inhibitory con-
0
stant for methyl N -(1-methyl-3-indolylmethyl)carbamate (14a) and
of brassinin (1) incubated with various concentrations of each
inhibitor, as described in Section 4.5. Results of these experi-
ments are shown in the form of Lineweaver–Burk double
reciprocal plots (1/S vs 1/V) (Fig. 3A and B). These results
indicated that methyl naphthalen-1-yl-methylcarbonate (19)
exhibited mixed inhibition, as shown by the lines intersecting
at a common point. Kinetic analysis of methyl naphthalen-2-yl-
methylcarbonate (20) based on Lineweaver–Burk plots revealed
that the intersection points of all curves were on the 1/V axis,
indicating that this compound was a competitive inhibitor of
methylnaphthalen-1-yl-methylcarbamate (17) were
and 89 ± 9 M, respectively (Table 2). Further confirmation that the
i
K = 695 ± 60
l
inhibition was reversible was obtained as follows. Since one of the
characteristics of irreversible inhibitors is the time dependency of
the extent of inhibition, a set of experiments was carried out in the
presence of brassinin at 0.50 mM and various concentrations of
methyl naphthalen-1-yl-methylcarbamate (17). Aliquots were with-
drawn at different times (5–25 min) to measure the remaining
residual activity. It was apparent that methyl naphthalen-1-yl-meth-
ylcarbamate (17) did not inhibit BHAb in a time-dependent manner
indicating that the reaction was a reversible process (Fig. 2B). These
results indicated that both 14a and 17 inhibited brassinin hydrolysis
through a reversible noncompetitive process. That is, both 14a and 17
can bind to the enzyme and to the enzyme-substrate complex, which
suggests the presence of an additional site of interaction with BHAb.
Next, the kinetics of inhibition of BHAb by methyl naphtha-
len-1-yl-methylcarbonate (19) and methyl naphthalen-2-yl-meth-
ylcarbonate (20) was investigated using different concentrations
BHAb. The K values were determined to be 97 ± 22 lM for 20
i
and 142 ± 14 lM for 19 (Table 2).
Overall, these results suggested that substitution at positions 1
or 2 of the naphthalenyl ring affected the interaction of carbamates
and dithiocarbamates with BHAb. Specifically, substitution at C-1
yielded more potent inhibitors, for example 17 versus 18. By con-
trast, naphthalenyl carbonates 19 and 20 were similarly potent
inhibitors, albeit through different kinetic mechanisms, mixed
and competitive, respectively.

2
30
M.S.C. Pedras et al. / Bioorg. Med. Chem. 20 (2012) 225–233
3
. Conclusion
In previous work, among six natural plant defense products,
1
only cyclobrassinin (8) was found to inhibit competitively BHAb ;
in the work reported in this manuscript, among the 34 substrate
inspired compounds screened for the first time for substrate and
inhibitory activity of BHAb, six displayed inhibitory activity (12,
1
4, 14a, 17, 19 and 20), but only two were found to be substrates
(
13 and 15) of BHAb. Methyl naphthalen-1-yl-methylcarbamate
0
(17), the strongest inhibitor of the six, and methyl N -(1-methyl-
3
-indolylmethyl)carbamate (14a) inhibited BHAb through a
reversible noncompetitive mechanism. Most importantly, these
carbamate inhibitors 14a and 17 were resistant to degradation
by A. brassicicola (Table S1). Hence, these compounds are excellent
leads to design new paldoxins that inhibit the detoxification of
brassinin (1) by A. brassicicola. By contrast, although both carbon-
ates 19 and 20 inhibited BHAb, since these were quickly degraded
(Table 1 and S1) in cultures of A. brassicicola, their potential use as
lead structures to design paldoxins is of less interest. Further work
with additional fungal species needs to be carried out to determine
if the presence of carbonate hydrolases is common among plant
pathogens.
Fungicides containing dithiocarbamate and carbamate groups
2
1
have been used for decades to protect crops. While dithiocarba-
mates have multisite targets, carbamates like methyl benzimida
zol-2-yl-carbamate and other benzimidazole carbamates have
2
2
been shown to bind to b-tubulin assembly in mitosis. Carboxy-
lesterases (CES, EC 3.1.1.1) are multifunctional enzymes that
catalyze the hydrolysis of substrates containing ester, amide,
thioester, or carbamate groups and have a broad substrate specific-
ity in mammalian systems, partly attributed to a large active site
that allows entry of structurally diverse substrates including endo-
2
3
biotics and xenobiotics. However, some carbamate hydrolases
2
4,25
appear to have high substrate specificity.
Considering that
compounds 15–20 were transformed in cultures of A. brassicicola,
it is likely that, in addition to brassinin hydrolase, CES with broad
substrate range were also produced. For this reason, it was
puzzling to find that carbamates 14/14a were not transformed in
culture. Further work with other plant pathogens will be carried
out to establish the scope of this finding.
Figure 3. Lineweaver–Burk plots of BHAb activity. In the presence of (A) methyl
naphthalen-1-yl-methylcarbonate (19) and (B) methyl naphthalen-2-yl-methylcar-
bonate (20).
Due to sequence homology and presence of the Ser/Ser/Lys
triad, BHAb is placed in the amidase signature superfamily of ser-
ine hydrolases. These serine hydrolases include the well-studied
fatty acid amide hydrolase (FAAH) from mammalian systems.
Synthetic FAAH inhibitors that use a covalent irreversible mecha-
nism, as well as reversible FAAH inhibitors have been reported,
including N-aryl carbamates that act as reversible noncompetitive
2
.5. Enzymatic hydrolysis of naphthalenylcarbonates 19 and 20
During purification of BHAb and substrate specificity assays, it
wasobservedthatsemi-purifiedenzymaticfractionsofBHAbhydro-
lyzed methyl naphthalen-1-yl-methylcarbonate (19) and meth-
ylnaphthalen-2-yl-methylcarbonate (20), but did not hydrolyze
the corresponding dithiocarbamates 15 and 16, nor carbamates 17
or 18. By contrast, mycelial cultures of A. brassicicola biotransformed
compounds 15–20 to the corresponding amines or alcohols (Table
S1). These results suggested the presence of enzymes other than
BHAb in protein fractions obtained from mycelia. To determine the
presence of enzyme(s) with carbonate or carbamate specificities,
the cell-free extracts of the A. brassicicola mycelia were fractionated
by size exclusion chromatography on a Superdex 200 column and
protein fractions were analyzed for hydrolytic activity using carbon-
ates 19 and 20 and brassinin (1). The results obtained are given in
Figure S1, which showed a single peak of enzymatic activity using
brassinin (1) as substrate (ca. 120 kDa) and at least two peaks with
activity (ca. 150 and 350 kDa) obtained for methyl naphthalen-1-
yl-methylcarbonate (19) (Fig. S1). That is, these results indicated
the presence of different isoforms of hydrolytic enzymes with activ-
ity against naphthalenylcarbonates, but not against carbamates or
dithiocarbamates.
2
6–28
inhibitors of FAAH.
However, despite the number of studies
using FAAH, the role of the carbamate group remains unclear in
several cases. Similarly, although our carbamates 14a and 17 acted
as reversible noncompetitive inhibitors of BHAb, since carbamate
1
8 displayed no effect, it is not clear which additional structural
motifs of 14a and 17 contributed to their inhibitory effect.
Overall, our results indicated that the catalytic and potential
inhibitory sites of BHAb are highly selective in the interactions
with different compounds. This is a very desirable feature from
the perspective of designing selective inhibitors of BHAb. However,
to carry out BHAb structure-based design of inhibitors other
than substrate inspired inhibitors, it is important to determine
protein–substrate and protein–inhibitor structures. Because BHAb
is produced in very small amounts, is somewhat unstable, and no
heterologous expression systems are available to obtain recombi-
nant protein, crystallization and X-ray diffractometry studies using
BHAb are virtually impossible. Hence, the development of effective
expression systems for BHAb and other phytoalexin detoxifying
enzymes will be essential to design paldoxins that protect crucifers

M.S.C. Pedras et al. / Bioorg. Med. Chem. 20 (2012) 225–233
231
against black spot and other major fungal diseases of economically
important crops.
7.50 (m, 2H), 7.41 (m, 1H), 5.16 (br s, NH), 3.74 (s, 3H). 13C NMR
(125.8 MHz, CD CN): d 157.3, 136.1, 133.5, 133.0, 128.7, 127.9,
27.8, 126.4, 126.2, 126.1, 125.8, 52.4, 45.4. HRMS-EI: m/z (%)
215.0943 (100) (215.0946 calcd for C13H13NO ), 200 (62), 156 (40),
3
1
4
4
. Experimental
2
1
41 (41), 129 (46).
.1. Materials and general procedures
4
.2.3. Methyl (1-naphthalenylmethyl)carbonate (19)
Chemicals were purchased from Sigma-Aldrich Canada (Oak-
1-Naphthalenylmethanol (23, 100 mg, 0.63 mmol) in THF
ville, ON) and chromatography media and buffers from GE Health-
care. All operations regarding protein extraction, purification and
assays were carried out at 4 °C, except where noted otherwise.
All compounds were characterized using NMR spectral data (re-
corded on Bruker 500 MHz spectrometers) and HRMS-EI data.
HPLC analyses were carried out with Agilent systems equipped
with a quaternary pump, an automatic injector, a photodiode array
detector (wavelength range 190–600 nm) and a degasser. Elution
(1 mL) was added to a cold solution of NaH (75 mg suspension in
oil, 1.9 mmol) in THF (3 mL), the reaction mixture was stirred at
0 °C for 30 min, and then methyl chloroformate (94.5 lL,
0.82 mmol) was added. After stirring for 5 h at rt, the reaction mix-
ture was cooled to 0 °C, diluted with water, the solvent layer sep-
arated and the aqueous reextracted with EtOAc, the combined
organic extract was dried over Na SO and concentrated to dry-
2
4
ness. Purification of the crude product (SiO , EtOAc–hexanes) affor-
2
method A: an Eclipse XDB-C18 column (5
50 mm ꢁ 4.6 i.d.), mobile phase linear gradient of H
50:50 to 0:100) for 25.0 min and a flow rate 0.75 mL/min; elution
method B (for detection of amines) a Zorbax SB C18 column
3.5
m particle size silica, 100 ꢁ 3 mm i.d.), a linear gradient of
O–CH OH (each solvent containing 0.01% n-propylamine,
l
m particle size silica,
ded methyl 1-naphthalenylmethyl)carbamate (19) as a white
semi-solid (109 mg, 0.50 mmol, 80% yield). H NMR (500.3 MHz,
1
1
(
2
O–CH OH
3
CDCl ): d 8.10 (m, 1.6H), 7.90 (m, 3.4H), 7.63–7.45 (m, 6.4H),
3
13
5.70 (s, 0.6H), 5.68 (s, 2H), 3.86 (s, 0.6H), 3.84 (s, 3H). C NMR
(
H
l
(125.8 MHz, CDCl ): d 155.8, 133.7, 131.6, 130.8, 129.6, 128.7,
3
2
3
127.7, 126.8, 126.0, 125.2, 123.5, 68.1, 54.9. HRMS-EI: m/z mea-
4
0:60 to 0:100), for 10 min and a flow rate of 0.5 mL/min.
sured 216.0792 (216.0786 calcd for C13H12O3).
High-resolution mass spectral (HRMS) data were obtained using
an Agilent HPLC 1100 series directly connected to a QSTAR XL Mass
Spectrometer (Hybrid Quadrupole-TOF LC/MS/MS) with turbo
4.2.4. Methyl (2-naphthalenylmethyl)carbonate (20)
As reported above for 19, but using 2-naphthalenylmethanol
(24, 100 mg, 0.63 mmol). Purification of the crude product (SiO2,
EtOAc–hexanes) afforded methyl 2-naphthalenylmethyl)carba-
mate (20) as a white solid (34 mg, 0.16 mmol, 51% yield based on
recovered starting material, 50 mg; mp 51–52 °C).
spray ESI source. Samples were dissolved in CH
using a Hypersil ODS C-18 column (5 m particle size silica,
00 ꢁ 2.1 mm i.d.). The mobile phase consisted of a linear gradient
of H O–CH CN (each solvent containing 0.1% formic acid, 75:25 to
5:75 in 35 min to 0:100 in 5 min) and a flow rate of 1.0 mL/min.
3
CN and analyzed
l
2
1
2
3
H NMR
2
3
(500.3 MHz, CD CN): d 7.86 (m, 4H), 7.50 (m, 3H), 5.35 (s, 2H),
1
3
Data acquisition was carried out in either positive or negative
polarity mode per LC run. Data processing was carried out by Ana-
lyst QS Software. HRMS-EI spectral data were obtained using a VG
3.83 (s, 3H). C NMR (125.8 MHz, CD CN): d 156.0, 133.4, 133.3,
3
132.8, 128.6, 128.2, 127.9, 127.7, 126.6, 126.5, 125.9, 70.0, 55.1.
HRMS-EI: m/z measured 216.0781 (216.0786 calcd for C13H12O3).
7
0 SE mass spectrometer using a solids probe.
4
.3. Antifungal bioassays and biotransformations of compounds
4
.2. Synthesis
12–20 by Alternaria brassicicola
Compounds 1,13 1a, 12, 13, and 14–16 were prepared as pre-
6
6
13
Compounds 12–20 were used in antifungal bioassays, carried
out as previously described. In brief, 7-day-old cultures of A. bras-
3
viously reported, and their purity determined to be P98% by HPLC
1
and H NMR.
sicicola on PDA under constant light at 23 ± 1 °C were used for
mycelial radial growth assays. Plugs (4 mm) were cut from the
edges of mycelia and placed inverted onto 12-well agar plates
amended with test compounds (dissolved in DMSO). The final con-
centrations of each compound in agar varied from 0.10 to 0.50 mM,
with a DMSO concentration of 1%. The plates were allowed to grow
under constant light at 23 ± 1 °C; the diameter of the mycelial mat
was measured after 96 h and compared to control cultures grown
on plates containing DMSO only.
4
.2.1. Methyl N-[(1-naphthalenyl)methyl]carbamate (17)
Methyl chloroformate (58 L, 0.73 mmol) and triethylamine
183 L, 1.3 mmol) were added to a solution of 1-naphthalenylme-
thanamine (21, 100 mg, 0.64 mmol) in CH Cl (5 mL), and the reac-
tion mixture was stirred at rt for 2 h. The reaction mixture was
diluted with water, extracted with EtOAc, the combined organic
l
(
l
2
2
extract was dried over Na
2
SO
4
and concentrated to dryness. Purifi-
, EtOAc–hexanes) afforded methyl
-naphthalenylmethyl)carbamate (17) as a white solid (134 mg,
cation of the crude product (SiO
2
Liquid cultures of each pathogen were grown in 250 ml Erlen-
meyer flasks containing 100 mL of minimal medium inoculated
1
0
(
1
6
.62 mmol, 97% yield; mp 74–75 °C). H NMR (500.3 MHz, CD
two rotamers 0.2:0.8) d 8.09 (d, J = 8.2 Hz, 0.2H), 8.04 (d, J = 8.2 Hz,
3
OD):
fungal spores for a final concentration of 1 ꢁ 10 /100 mL. After
48 h at 23 ± 1 °C, under constant light a solution of each compound
0
5
.8H), 7.84 (d, J = 7.5 Hz, 1H), 7.81–7.75 (m, 1H), 7.53–7.38 (m, 4H),
3
(12–20) in CH CN (100–250 lL depending on solubility) was added
.06 (s, 0.4H), 4.72 (s, 1.6), 3.65 (br s, 3H).14 13C NMR (125.8 MHz,
to the cultures, for a final concentration of 0.10 mM. Similar control
solutions containing compounds only or mycelia were prepared.
The flasks were returned to the shaker, samples (5–10 mL) were
withdrawn at various times and immediately frozen or extracted
with EtOAc. The extracts were analyzed by HPLC using elution
method A for compounds 12–20.
3
CD OD): (two rotamers) d 159.6, 138.1, 135.6, 135.4, 132.8, 132.7,
1
1
29.8, 129.7, 129.2, 129.1, 127.4, 127.2, 126.9, 126.8, 126.5, 126.4,
26.2, 124.8, 124.4, 63.5, 52.7, 43.6. HRMS-EI: m/z (%) 215.0950
(
2
15) (215.0946 calcd for C13H13NO ), 158 (60), 141 (21), 129 (100).
4
.2.2. Methyl N-[(2-naphthalenyl)methyl]carbamate (18)
As reported above for 17, but using 2-naphthalenylmethanamine
4.4. Isolation, chromatographic purification and activity assay
of BHAb
(
22, 91 mg, 0.58 mmol). Purification of the crude product (SiO
EtOAc–hexanes) afforded methyl 2-naphthalenylmethyl)carbamate
18) as a white solid (120 mg, 0.56 mmol, 96% yield; mp 110–
2
,
(
1
A. brassicicola (isolate ATCC 96866) spores and liquid cultures
1
were obtained under the conditions described previously.1 For
3
11 °C). H NMR (500.3 MHz, CDCl ): d 7.83 (m, 3H), 7.72 (s, 1H),

2
32
M.S.C. Pedras et al. / Bioorg. Med. Chem. 20 (2012) 225–233
purification of BHAb, 0.6 L of liquid cultures were induced with 3-
phenylindole (0.20 mM). The cultures were incubated for an addi-
tional 24 h and then gravity filtered to separate mycelia from cul-
ture broth. Frozen mycelia (5 g) obtained from A. brassicicola (0.6 L)
were suspended in ice-cold extraction buffer (10 mL) and ground
inactivation studies were performed by preincubation of the
purified enzyme with 0.30 mM methyl (1-methyl-3-indolylmeth-
yl)carbamate (14a) or 0.10 mM methylnaphthalen-1-yl-methylc-
arbamate (17) at room temperature for 20 min (Fig. 2, 17 is a
stronger inhibitor than 14a). Samples were then placed in the
Slide-A-Lyzer membranes (molecular weight cutoff, 3500, Pierce
Chemical) and dialyzed tree times for 2 h at 4 °C against 1 L of a
buffer containing 20 mM DEA (pH 8.3), 0.1% Triton X-100, and 2%
glycerol. The dialyzed samples were collected and BHAb activity
was determined as described above. Control incubations lacked
inhibitors and the samples were compared with the controls. In
separate experiments, BHAb activities were determined in the
presence of inhibitor before and after dialysis.
(
mortar) for 5 min. The extraction buffer consisted of 25 mM dieth-
anolamine (DEA), pH 8.3, 5% (v/v) glycerol, 1 mM EDTA and 1:200
v/v) protease inhibitor cocktail (P-8215, Sigma). The suspension
(
was centrifuged for 60 min at 58,000g and the resulting superna-
tant (10 mL) was used for chromatographic separation, as previ-
1
ously described. Protein concentrations were determined as
described by Bradford using the Coomassie Brilliant Blue method
with bovine serum albumin (BSA) as a standard.
The reaction mixture contained 20 mM DEA (pH 8.3), 0.1%
Triton X-100, 0.05–1.0 mM brassinin, (in 5
lL acetonitrile), and
4
.8. Gel filtration on Superdex 200
5
0–100 L of purified protein in a total volume of 1.0 mL. The reac-
l
tion was carried out at 23 °C for 30 min and was stopped by the
The soluble protein extract from mycelia (0.3 ml, ca. 1 mg) was
addition of 0.1 mL solution of 30% ammonium sulfate (w/v) and
subjected to gel filtration on Superdex 200 (1 ꢁ 40 cm column
dimension). Proteins were eluted with 25 mM DEA buffer pH 8.3
containing 0.015% Triton X-100, 1% glycerol (v/v), and 50 mM NaCl.
The column was calibrated with protein markers consisting of blue
dextran (2000 kDa), apoferritin (443 kDa), b-amylase (200 kDa),
alcohol dehydrogenase (150 kDa), albumin (66 kDa) and carbonic
anhydrase (29 kDa). Fractions of 0.4 ml were collected at a flow
rate of 0.4 mL/min and used for testing the enzyme activity of dif-
ferent substrates.
1
1
5% ammonium hydroxide (w/v) as previously described. The
reaction assays were extracted with 2 mL of chloroform–methanol
95:5, v/v) and concentrated to dryness in a rotary evaporator. Ex-
tracts were dissolved in methanol (200 L) and analyzed by HPLC
(
l
using elution method B. The quantification was carried out using
a calibration curve of indolyl-3-methanamine.
4
.5. Inhibitory effects of compounds 12–20
To determine the inhibitors of BHAb, experiments were carried
Acknowledgments
out using brassinin at 0.10 mM (substrate) in the presence of 0.10
and 0.30 mM of each test compound. The reaction was initiated by
addition of purified BHAb. The standard deviations for each assay
were determined from four independent experiments. To deter-
mine the type of inhibition, experiments were carried using brass-
inin at 0.10–0.40 mM in the presence of 0.050–0.40 mM of each
We thank P. B. Chumala and K. Thoms from the Department of
Chemistry, University of Saskatchewan for technical assistance
obtaining LC–MS and HRMS data, respectively. Financial support
from Natural Sciences and Engineering Research Council of Canada
(
NSERC – Discovery Grant to M.S.C.P.), Canada Research Chairs Pro-
0
inhibitor (methyl N -(1-methyl-3-indolylmethyl)carbamate (14a)
gram (CRC – Tier I Grant to M.S.C.P.) is gratefully acknowledged.
and methyl naphthalen-1-yl-methylcarbamate (17)). The kinetics
of inhibition was transformed into Lineweaver–Burk double reci-
procal plots (1/S vs 1/V). To determine K
inhibitor, a secondary plot was constructed using the apparent 1/
i
for a noncompetitive
Supplementary data
V values obtained in presence and absence inhibitors versus inhib-
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.11.009.
itor concentration. K
i
for a competitive inhibitor was calculated on
values obtained in presence and absence
the basis of apparent K
m
of inhibitors versus inhibitor concentration. For both types of inhi-
bition, noncompetitive and competitive, linear regression was used
References and notes
to determine x-axis intercept value which is equal to negative K
The K value for the mixed type inhibitor was determined from Dix-
on plots of l/V versus [I].
i
.
1
2
.
.
Pedras, M. S. C.; Minic, Z.; Sarma-Mamillapalle, V. FEBS J. 2009, 276, 7412.
Pedras, M. S. C.; Gadagi, R. S.; Jha, M.; Sarma-Mamillapalle, V. K. Phytochemistry
2007, 68, 1572.
Pedras, M. S. C.; Chumala, P. B.; Jin, W.; Islam, M. S.; Hauck, D. W.
Phytochemistry 2009, 70, 394.
i
3
.
.
4
.6. Inhibitory effects of serine modifying reagents
Inhibitory studies of purified enzyme were done in 100 mM
4
Pedras, M. S. C.; Zheng, Q. A.; Sarma-Mamillapalle, V. K. Nat. Prod. Commun.
2007, 2, 319.
5
6
7
.
.
.
Pedras, M. S. C.; Yaya, E. E. Phytochemistry 2010, 71, 1191.
Pedras, M. S. C.; Yaya, E. E.; Glawischnig, E. Nat. Prod. Rep. 2011, 28, 1381.
Pedras, M. S. C.; Minic, Z.; Jha, M. FEBS J. 2008, 275, 3691.
phosphate buffer (pH 7.5) using brassinin 0.10 mM (substrate) in
presence of 0.50–1.0 mM of compound as phenylmethanesulfonyl
fluoride (PMSF), 4-(2-aminoethyl)benzene sulfonyl fluoride (Pefab-
loc) and phenylphosphorodiamide.
8. Sexton, A. C.; Minic, Z.; Cozijnsen, A. C.; Pedras, M. S. C.; Howlett, B. J. Fungal
Genet. Biol. 2009, 46, 201.
9
.
Pedras, M. S. C.; Minic, Z.; Sarma-Mamillapalle, V. K. J. Agric. Food Chem. 2009,
7, 2429.
5
1
1
0. Pedras, M. S. C.; Minic, Z.; Sarma-Mamillapalle, V. K.; Suchy, M. Bioorg. Med.
Chem. 2010, 18, 2456.
1. Pedras, M. S. C.; Minic, Z.; Sarma-Mamillapalle, V. K. Bioorg. Med. Chem. 2011,
4
.7. Mechanism-based inactivation studies
1
9, 1390.
The inactivation of BHAb was studied at 23 °C in buffer solu-
tions containing 20 mM DEA (pH 8.3), 0.1% Triton X-100,
12. Meanwell, N. A. J. Med. Chem. 2011, 54, 2529.
13. Pedras, M. S. C.; Jha, M. Bioorg. Med. Chem. 2006, 14, 4958.
1
4. Zagulyaeva, A. A.; Banek, C. T.; Yusubov, M. S.; Zhdankin, V. V. Org. Lett. 2010,
2, 4644.
5. Leathen, M. L.; Peterson, E. A. Tetrahedron Lett. 2010, 51, 2888.
0
.50 mM brassinin, 100
trations of methyl N -(1-methyl-3-indolylmethyl)carbamate (14a)
or methyl naphthalen-1-yl-methylcarbamate (17) (3.0 mL final
l
L of purified protein and various concen-
1
0
1
16. Ito, Y.; Ushitora, H. Tetrahedron 2006, 62, 226.
1
1
7. Selva, M.; Tundo, P.; Perosa, A. Tetrahedron Lett. 2002, 43, 1217.
8. Kadokawa, J.-i.; Hideyuki, H.; Fukamachi, S.; Karasu, M.; Tagaya, H.; Chiba, K. J.
Chem. Soc., Perkin Trans. 1 1999, 15, 2205.
volume). Portions (400
5, 10, 15, 20 and 25 min), the reaction was stopped and the resid-
ual enzymatic activities were determined. Additionally,
lL) were removed at suitable time intervals
(
19. Hoffman, W. A., III J. Org. Chem. 1982, 47, 5209.

M.S.C. Pedras et al. / Bioorg. Med. Chem. 20 (2012) 225–233
233
2
2
2
2
2
0. Pedras, M. S. C.; Ahiahonu, P. W. K.; Hossain, M. Phytochemistry 2004, 65, 2685.
1. Russell, P. E. J. Agric. Sci. 2005, 143, 11.
2. Casida, J. E. Chem. Res. Toxicol. 2009, 22, 609.
3. Ross, M. K.; Streit, T. M.; Herring, K. L.; Xie, S. J. Pestic. Sci. 2010, 35, 257.
4. Naqvia, T.; Cheesmanb, M. J.; Williamsb, M. R.; Campbellb, P. M.; Ahmeda, S.;
Russellb, R. J.; Scottb, C.; Oakeshottb, J. G. J. Biotechnol. 2009, 144, 89.
25. Chaudhry, G. R.; Mateen, A.; Kaskarã, B.; Bloda, M.; Riazuddin, S. Biotechnol.
Appl. Biochem. 2002, 36, 63.
26. Seierstad, M.; Breitenbucher, J. G. J. Med. Chem. 2008, 51, 2327.
27. Otrubova, K.; Ezzili, C.; Boger, D. L. Bioorg. Med. Chem. Lett. 2011, 21, 4674.
28. Deng, H. Expert Opin. Drug Discov. 2010, 5, 961.