Inhibitors of the detoxifying enzyme of the phytoalexin brassinin based on quinoline and isoquinoline scaffolds

Article abstract of DOI:10.3390/molecules22081345

The detoxification of the phytoalexin brassinin to indole-3-carboxaldehyde and S-methyl dithiocarbamate is catalyzed by brassinin oxidase (BOLm), an inducible fungal enzyme produced by the plant pathogen Leptosphaeria maculans. Twenty-six substituted quinolines and isoquinolines are synthesized and evaluated for antifungal activity against L. maculans and inhibition of BOLm. Eleven compounds that inhibit BOLm activity are reported, of which 3-ethyl-6-phenylquinoline displays the highest inhibitory effect. In general, substituted 3-phenylquinolines show significantly higher inhibitory activities than the corresponding 2-phenylquinolines. Overall, these results indicate that the quinoline scaffold is a good lead to design paldoxins (phytoalexin detoxification inhibitors) that inhibit the detoxification of brassinin by L. maculans.

Full text of DOI:10.3390/molecules22081345

molecules
Article
Inhibitors of the Detoxifying Enzyme of the
Phytoalexin Brassinin Based on Quinoline and
Isoquinoline Scaffolds
ID
M. Soledade C. Pedras * ID , Abbas Abdoli
and Vijay K. Sarma-Mamillapalle
Department of Chemistry, University of Saskatchewan, 110 Science Place, Saskatoon, SK S7N 5C9, Canada;
aba087@mail.usask.ca (A.A.); mas792@mail.usask.ca (V.K.S.-M.)
* Correspondence: s.pedras@usask.ca; Tel.: +1-306-966-4772
Received: 17 July 2017; Accepted: 8 August 2017; Published: 14 August 2017
Abstract: The detoxification of the phytoalexin brassinin to indole-3-carboxaldehyde and S-methyl
dithiocarbamate is catalyzed by brassinin oxidase (BOLm), an inducible fungal enzyme produced by
the plant pathogen Leptosphaeria maculans. Twenty-six substituted quinolines and isoquinolines are
synthesized and evaluated for antifungal activity against L. maculans and inhibition of BOLm. Eleven
compounds that inhibit BOLm activity are reported, of which 3-ethyl-6-phenylquinoline displays
the highest inhibitory effect. In general, substituted 3-phenylquinolines show significantly higher
inhibitory activities than the corresponding 2-phenylquinolines. Overall, these results indicate that
the quinoline scaffold is a good lead to design paldoxins (phytoalexin detoxification inhibitors) that
inhibit the detoxification of brassinin by L. maculans.
Keywords: antifungal; brassinin oxidase; camalexin; crucifer; Leptosphaeria maculans; paldoxin;
phenylquinoline; phytoalexin detoxification
1. Introduction
The conversion of the phytoalexin brassinin (
brassinin oxidase (BOLm), the only enzyme currently known to mediate the transformation of a
dithiocarbamate to an aldehyde [ ]. This transformation is a detoxification process that eliminates
the toxophore S-methyl dithiocarbamate of brassinin to afford non-toxic products (Scheme 1).
1) to indole-3-carboxaldehyde (2) is catalyzed by
1,2
Scheme 1. Detoxification of brassinin (1) by BOLm (Leptosphaeria maculans) and inhibitors camalexin
(3a), 5-methoxycamalexin (3b), brassilexin (4a) and 6-chlorobrassilexin (4b).
BOLm is an inducible phytoalexin detoxifying enzyme produced by a fungal pathogen of crucifer
crops (Leptosphaeria maculans (Desm.) Ces. et de Not., asexual stage Phoma lingam (Tode ex Fr.) Desm.)
that causes major epidemics worldwide. Brassinin (
1
) is a cruciferous phytoalexin produced by plants
Molecules 2017, 22, 1345; doi:10.3390/molecules22081345
www.mdpi.com/journal/molecules

Molecules 2017, 22, 1345
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of the family Brassicaceae (common name crucifer), which include globally cultivated crops belonging
to the Brassica genus [ ]. Brassica crops are of enormous importance worldwide as sources of oil,
food, feed, and fuel. Brassinin ( ) is an important phytoalexin because it functions as antimicrobial
plant defense and as biosynthetic precursor of several phytoalexins; depletion of brassinin ( ) through
detoxification is a pathogen’s strategy to weaken the defense system of brassicas [ ]. In principle,
) to build up in plant cells
3
1
1
3
inhibition of such a detoxification transformation could allow brassinin (
and stop pathogen growth.
1
As part of a research program to devise sustainable methods to protect plants against
fungal infections, we are particularly interested in the development of paldoxins, i.e., phytoalexin
detoxification inhibitors [
having a specific mechanism of action, the inhibition of brassinin detoxification by L. maculans [
4
]. Paldoxins of BOLm [
5
,
6
] are being considered as potential crop protectants
].
7
The attraction of this approach lies in the possibility of exploiting paldoxins as selective fungal enzyme
inhibitors. It is anticipated that such selective inhibitors will display lower toxicity levels to the
encompassing ecosystem and thus are less likely to have a negative environmental impact than
conventional fungicides.
Particularly because BOLm has not been expressed in heterologous systems and only relatively
small quantities can be obtained using classic chromatographic techniques, in depth structural studies
have not been carried out. Consequently, since the tertiary structure of BOLm remains unknown and no
relevant model systems have been reported, the design of inhibitors of BOLm is an ongoing challenge.
Preliminary screening of a library of more than 80 synthetic brassinin analogues and isosteres, designed
by replacement of the dithiocarbamate group of
sulfamide, sulfonamide, dithiocarbazate, amide, and ester functionalities, plus replacement of the
indolyl moiety with naphthalenyl and phenyl, did not identify BOLm inhibitors [ ]. Unexpectedly,
among several natural products, the phytoalexins camalexin (3a) [ ] and brassilexin (4a) [ ] were found
1 with carbamate, dithiocarbonate, urea, thiourea,
8
1
5
to inhibit BOLm. Upon optimization of both lead structures, inhibitors of BOLm more potent than
the parent compounds were obtained, 5-methoxycamalexin (3b) and 6-chlorobrassilexin (4b) became
the best competitive inhibitors of BOLm [7]. However, both 3b and 4b displayed stronger antifungal
activity, a characteristic less desirable in potential paldoxins due to potential toxicity to the plant and
surrounding living organisms. Hence, it is of interest to develop new scaffolds containing different
heterocyclic systems to establish structural correlations among BOLm inhibitors and their antifungal
activities against L. maculans. Herein, the inhibition of BOLm and the antifungal activities of a new
series of compounds having quinoline and isoquinoline skeletons are reported.
2. Results and Discussion
2.1. Design and Synthesis of Potential Inhibitors of BOLm
The most significant inhibitors of BOLm discovered to date are derived from indolyl containing
scaffolds, namely 5-methoxycamalexin (3b) and 6-chlorobrassilexin (4b) [7]. Quinolines are
monoazanaphthalenes that can be formally considered structural hybrids of indolyl and naphthalenyl
skeletons. For this reason, quinolines, and their structural isomers isoquinolines, are heterocyclic
scaffolds of interest to us as potential inhibitors of BOLm. A few quinoline derivatives currently
used as commercial agricultural fungicides include quinoxyfen and tebufloquin, which are known
to affect signal transduction pathways and the respiratory system, respectively [
polymerization [10,11].
9], and tubulin

Molecules 2017, 22, 1345
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Considering the above framework, the development of quinolines with a new mechanism of
action that targets specific fungal pathogens and offers selectivity is of great interest for crop protection.
Figure 1 shows the substituted quinolines and isoquinolines designed and synthesized for this purpose.
Figure 1. Quinolines 5a–8 and isoquinolines 9a–10b evaluated for inhibition of BOLm.
Syntheses of brassinin (
out as previously reported. Chemical syntheses of new quinolines 6e
of known quinolines 5a 5f 6a 6d 6f 7a
1
) [
8
], camalexin (3a) [12], and isoquinolines
8
[
13] and 9a
7e 7g, improved syntheses
9c and 10b
[13] were carried
,
7c
– ,
–
,
–
,
,
,
7b and 7f and syntheses of new isoquinolines 9b
,
were carried out as described below.
2.1.1. Quinolines
Quinolines can be synthesized using a number of classical methods including the Friedländer,
Skraup, Combes, and Doebner–von Miller syntheses from anilines and carbonyl compounds, as well as
metal-catalyzed dehydrogenative cyclization and other “greener” methods [14
quinolines 5a 5f 6a 6f and 7a 7g were synthesized using the Friedländer method by condensation of
the corresponding 2-aminobenzaldehydes, prepared from substituted 2-nitrobenzaldehydes [18], with
aldehydes or ketones. In short, 2-nitrobenzaldehydes 11a 11g were reduced with iron powder to the
corresponding amines by refluxing in EtOH/HCl for 40 min [19]. Aldehydes 12b 12d and 12f 12h or
ketones 12a 12e and 12i and KOH (1.2 eq.) were added to the reaction mixture that was kept at RT for
–17]. In the current work,
–
,
–
–
–
–
–
,
30 min, followed by heating under reflux for 3 h, to afford the corresponding quinolines 5a
and 7a–7g in reasonable yields (Table 1).
–5f, 6a–6f

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Table 1. Synthesis of substituted quinolines 5a–5f, 6a–6f and 7a–7g.
Starting Material
R1 (C#)
12
R2
R3
Product
%Yield (Reported Yield)
11a
11b
11c
11d
11e
11f
11a
11b
11c
11d
11e
11f
11g
11g
11g
11g
11g
11g
11g
H
6-Cl
5-Cl
12a
12a
12a
12a
12a
12a
12b
12b
12b
12b
12b
12b
12c
12d
12e
12f
H
H
H
H
H
Ph
Ph
Ph
Ph
Ph
Ph
H
H
H
H
H
H
H
H
Me
H
H
H
Ph
5a a
5b b
5c b
5d b
5e b
5f b
6a a
6b b
6c b
6d b
6e c
6f b
7a b
7b b
7c c
7d c
7e c
7f b
7g c
83 (99)
84
63
64
42
60
63 (87)
80
64
36
39
53
62
52
52
82
5-Br
5-OH
5-OMe
H
6-Cl
5-Cl
H
Ph
Ph
Ph
Ph
Ph
Ph
H
Me
H
Et
5-Br
5-OH
5-OMe
5-Ph
5-Ph
5-Ph
5-Ph
5-Ph
5-Ph
5-Ph
12g
12h
12i
i-Pro
Ph
H
69
quantitative
62
a
Previously synthesized using this method [19]; b Previously synthesized by different methods; c New compounds.
2.1.2. Isoquinolines
The best known classical approaches to isoquinoline scaffolds are the Bischler–Napieralski
synthesis, in which a -arylethylamide is converted into a 3,4-dihydroisoquinoline derivative,
β
the Pictet–Spengler reaction that involves an acid-catalyzed intramolecular cyclization, and the
Pomeranz–Fritsch reaction that uses benzylamino acetals [17]. In addition, a large variety of methods
that require less harsh conditions are available. In this work, substituted isoquinolines were synthesized
from their corresponding bromoisoquinolines 14a
the Pomeranz–Fritsch reaction [20 21]. Benzaldehydes 13a
dimethylacetal (Dean–Stark conditions) followed by cyclization in the presence of H2SO4/P2O5 to yield
isoquinolines 14a 14c. Thiazole substituted isoquinolines 9a 9c were prepared from isoquinolines
14a 14c by treatment with ethyl chloroformate followed by 2-trimethylsilylthiazole and deprotection
using o-chloranil (Scheme 2).
–
14c, which were prepared by a modification of
,
–
13c were treated with aminoacetaldehyde
–
–
–

Molecules 2017, 22, 1345
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Scheme 2. Synthesis of isoquinolines 9a–9c.
Syntheses of 1-phenylisoquinolines 10a and 10b were carried out by modifying a reported
procedure [22]; 1-chloroisoquinolines 15a and 15b were treated with iodine followed by PhMgCl
and Fe powder in THF (Scheme 3). Unlike in the reported procedure, none of the reactions proceeded
in the absence of iodine.
Scheme 3. Synthesis of isoquinolines 10a and 10b.
2.2. Antifungal Activity
The phytoalexins brassinin (
compare the antifungal activities of synthetic compounds with those of known phytoalexins.
The antifungal activity of 3a, quinolines 5a and isoquinolines 9a 10b against L. maculans
1) and camalexin (3a) were used as reference compounds to
1,
–
8
–
was determined employing the mycelial growth inhibition assay [23] described in Materials and
Methods. The mycelial growth of each plate was measured after incubation for five days and the
results were statistically analyzed (Table 2, results of six independent experiments conducted in
triplicate). In general, quinoline derivatives (0.50 mM) showed weaker antifungal activity than
camalexin (3a), except for 3-phenylquinoline (6a) and 6-methoxy-3-phenylquinoline (6f), whereas
5-chloro-3-phenylquinoline (6b) displayed the lowest growth inhibitory activity. 3-Phenylquinoline
(6a) showed stronger antifungal activity than its structural isomer 6-phenylquinoline (7a), whereas
structural isomers 6-methyl-3-phenylquinoline (6g) and 3-methyl-6-phenylquinoline (7c) caused
similar mycelial growth inhibition. Interestingly 3,6-diphenylquinoline (7g) was not growth inhibitory
and 1-(2-thiazolyl)isoquinoline (9a) was the most inhibitory of all tested compounds.

Molecules 2017, 22, 1345
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Table 2. Antifungal activity a of the phytoalexins brassinin (
1
), camalexin (3a), quinolines 5a
–8, and
isoquinolines 9a–10b against Leptosphaeria maculans.
Inhibition ± SD (%) a
Compound (#)
0.50 mM
0.20 mM
0.10 mM
50 ± 3 i,g
100 ± 0 c
54 ± 2 h
39 ± 2 l
26 ± 4 m
27 ± 4 m
68 ± 5 e,f
36 ± 4 l
100 ± 0 c
17 ± 3 n
28 ± 3 m
45 ± 2 k
73 ± 5 e
100 ± 0 c
69 ± 3 e,f
64 ± 0 g
68 ± 2 f
74 ± 2 e
67 ± 3 f
72 ± 3 e
49 ± 4 i,j,k
0 o
27 ± 0 l
41 ± 2 i,j
31 ± 4 k,l
18 ± 4 m,n
18 ± 5 m,n
14 ± 4 n,o
42 ± 4 h,i,j
20 ± 4 m
77 ± 4 d
11 ± 2 o
17 ± 3 m,n
34 ± 2 k
45 ± 4 h,i
68 ± 5 e
61 ± 2 f
55 ± 0 g
62 ± 3 f
67 ± 4 e
61 ± 3 f
66 ± 3 e
40 ± 0 j
14 ± 3 j
24 ± 3 i
16 ± 5 j
9 ± 2 k
Brassinin (1)
Camalexin (3a)
2-Phenylquinoline (5a)
5-Chloro-2-phenylquinoline (5b)
6-Chloro-2-phenylquinoline (5c)
6-Bromo-2-phenylquinoline (5d)
6-Hydroxy-2-phenylquinoline (5e)
6-Methoxy-2-phenylquinoline (5f)
3-Phenylquinoline (6a)
5-Chloro-3-phenylquinoline (6b)
6-Chloro-3-phenylquinoline (6c)
6-Bromo-3-phenylquinoline (6d)
6-Hydroxy-3-phenylquinoline (6e)
6-Methoxy-3-phenylquinoline (6f)
6-Methyl-3-phenylquinoline (6g)
6-Phenylquinoline (7a)
5 ± 2 l
0 m
32 ± 4 f,g,h
8 ± 2 k
39 ± 7 f
8 ± 4 k,l
5 ± 2 l
25 ± 0 i
30 ± 4 g,h
55 ± 5 c
47 ± 0 e
30 ± 0 h
48 ± 3 d,e
53 ± 0 c
48 ± 3 d,e
56 ± 3 c
33 ± 0 f,g
0 m
2-Methyl-6-phenylquinoline (7b)
3-Methyl-6-phenylquinoline (7c)
3-Ethyl-6-phenylquinoline (7d)
3-Isopropyl-6-phenylquinoline (7e)
2,6-Diphenylquinoline (7f)
3,6-Diphenylquinoline (7g)
0 p
54 ± 3 h
100 ± 0 c
75 ± 6 d,e
82 ± 5 d
54 ± 5 h,i
47 ± 3 j,k
46 ± 4 h
100 ± 0 c
54 ± 3 g
63 ± 3 e,f
32 ± 4 k
31 ± 4 k,l
8 ± 3 k,l
52 ± 4 c,d
15 ± 4 j
25 ± 6 i
30 ± 3 h
12 ± 2 j
2-(2-Thiazolyl)quinoline (8)
1-(2-Thiazolyl)isoquinoline (9a)
6-Bromo-1-(2-thiazolyl)isoquinoline (9b)
7-Bromo-1-(2-thiazolyl)isoquinoline (9c)
1-Phenylisoquinoline (10a)
7-Bromo-1-phenylisoquinoline (10b)
a The percentage of inhibition was calculated using the formula: % inhibition = 100
−
[(growth on amended/growth
b
in control)
×
100]; values are averages of six independent experiments conducted in triplicate; for statistical
analysis, one-way ANOVA tests were performed followed by Tukey’s test with adjusted
α set at 0.05; n = 6; different
letters in the same column (c–p) indicate significant differences (p < 0.05).
2.3. Inhibition of Brassinin Oxidase Activity
Cell-free protein extracts of mycelia of L. maculans containing BOLm activity induced by
3-phenylindole were employed (prepared as described in Section 3.4) [ ] to test the potential
paldoxin activity of quinolines 5a and isoquinolines 9a 10b. Compounds were evaluated at
concentrations (0.10 and 0.30 mM) close to the concentration of substrate required for half-maximal
activity (KM = 0.15 mM), in the presence of brassinin ( , 0.10 mM) and phenazine methosulfate (PMS)
as the electron acceptor cofactor (BOLm accepts a wide range of cofactors: PMS, small quinones or
flavin mononucleotide, FMN) [ ]. To compare the inhibitory activity of all new compounds with those
previously reported [ ], camalexin (3a) was used as a standard due to its established BOLm inhibitory
activity (ca. 53% at 0.30 mM) and chemical stability [ ]. Results of these assays are presented in Table 3.
1
–
8
–
1
1
7
6

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Table 3. Inhibitory effect of camalexin (3a) and quinolines 5a–8 and isoquinolines 9a–10b on
BOLm activity.
% Inhibition b
% Inhibition b
Potential Inhibitor (#) a
Potential Inhibitor (#) a
0.10 mM
0.30 mM
0.10 mM
0.30 mM
30 ± 4 c
n.i.
n.i.
53 ± 4 d
16 ± 2 d,e
n.i.
36 ± 2 e,f
n.i.
5a R = H
5b R = 5-Cl
5c R = 6-Cl
5d R = 6-Br
5e R = 6-OH
5f R = 6-OMe
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
7a R = H
7b R = 2-Me
7c R = 3-Me
7d R = 3-Et
19 ± 4 d
30 ± 4 c
11 ± 3 e
10 ± 2 e
12 ± 1 e
33 ± 6 e,f
64 ± 5 c
33 ± 5 e,f
19 ± 1 h
25 ± 1 g,h
n.i.
n.i.
11 ± 3 e
n.i.
7e R = 3-CH(CH3)2
7f R = 2-Ph
22 ± 1 h
n.i.
7g R = 3-Ph
14 ± 2 d,e
n.i.
30 ± 4 f,g
n.i.
6a R = H
6b R = 5-Cl
6c R = 6-Cl
6d R = 6-Br
6e R = 6-OH
6f R = 6-OMe
6g R = 6-Me
9a R = H
9b R = 6-Br
9c R = 7-Br
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
20 ± 4 d
19 ± 1 d
10 ± 3 e
34 ± 2 e,f
40 ± 3 e
32 ± 3 f
10a R = H
10b R = 7-Br
n.i.
n.i.
n.i.
n.i.
a
Brassinin (1) was used as substrate (0.10 mM) and PMS (0.10 mM) as cofactor in all experiments. BOLm
activity was measured under standard conditions (described in Section 3.4) in the presence of potential inhibitors
(0.10 and 0.30 mM). The specific activity of BOLm (24
1 nmol/mg/min) was comparable in all assays; b Inhibition
is expressed as a percentage of the BOLm activity (100%) of cell-free protein extracts containing brassinin ( ) and
±
1
inhibitor; results are means and standard deviations of experiments conducted in triplicate; n.i. = no inhibition. For
statistical analysis, one-way ANOVA tests were performed followed by Tukey’s test; n = 6; different superscript
letters within the same column (c–h) indicate significant differences (p < 0.05).
It is remarkable that of the 26 compounds tested, 11 inhibited BOLm activity, but none
of the halogenated quinolines or isoquinolines affected the activity of BOLm (Table 3). Except
for 2-methyl-6-phenyl-quinoline (7b), all substituted 6-phenylquinolines inhibited BOLm activity.
3-Ethyl-6-phenylquinoline (7d) showed the highest inhibitory effect (ca. 64% at 0.30 mM), somewhat
stronger than that of camalexin (3a), followed by 6-methoxy-3-phenylquinoline (6f) (ca. 40% at 0.30 mM),
whereas 6-hydroxy-3-phenylquinoline (6e), 6-phenylquinoline (7a), 3-methyl-6-phenylquinoline (7c), and
3-isopropyl-6-phenylquinoline (7e) showed comparable inhibitory effects. Substituted 3-phenylquinolines
showed significantly higher inhibitory activities than the corresponding 2-phenylquinolines. The number
of structures evaluated is still insufficient to make general conclusions regarding the structure–activity
correlations of quinolines and isoquinolines, compounds with substituents at C-4, C-7, and C-8 ought
to be considered. Nonetheless, it is apparent and relevant to note that halogenated compounds did not
show inhibitory activity against BOLm, contrary to results previously obtained with camalexin (3a
)

Molecules 2017, 22, 1345
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and brassilexin (4a) derivatives in which halogen-substituted compounds (e.g., 4b) were somewhat
more inhibitory of BOLm activity [7].
3. Materials and Methods
3.1. General
Chemicals were purchased from Alfa Aesar, Ward Hill, MA or Sigma-Aldrich Canada Ltd.,
Oakville, ON, Canada; solvents were HPLC grade and used as such. Flash column chromatography
(FCC) was carried out using silica gel grade 60, mesh size 230–400 Å or WP C18 prepscale bulk
packing 275 Å (J.T. Baker, NJ, USA). Organic extracts were dried over Na2SO4 and concentrated using
a rotary evaporator.
NMR spectra were recorded on Bruker 500 Avance spectrometers (Bruker Corporation, Billerica,
1
MA, USA), for H, 500.3 MHz and for 13C, 125.8 MHz; chemical shifts (
δ) are reported in parts
per million (ppm) relative to TMS; spectra were calibrated using the solvent peaks; spin coupling
constants (J) are reported to the nearest 0.5 Hz. FTIR data were recorded on a Bio-Rad FTS-40
spectrometer(Bio-Rad Laboratories, Inc., Hercules, CA, USA) and spectra were measured by the
diffuse reflectance method on samples dispersed in KBr. HREI-MS were obtained on a VG 70 SE mass
spectrometer(Waters Technologies Corporation, Milford, MA, USA) employing a solids probe or on a
Jeol AccuToF GCv 4G mass spectrometer (Jeol USA, Peabody, MA, USA) [field desorption (FD)] by
direct insertion.
HPLC analysis was carried out with Agilent high performance liquid chromatographs equipped
with quaternary pump, automatic injector, and photodiode array detector (DAD, wavelength range
190–600 nm), degasser, and a column Eclipse XDB-C18 (Agilent Technologies, Santa Clara, CA, USA)
(5
µ
m particle size silica, 4.6 mm i.d.
×
150 mm), having an in-line filter, using methods: A, mobile
phase 80% H2O—20% CH3CN for 45.0 min, linear gradient, at a flow rate of 0.40 mL/min; B, mobile
phase 50% H2O—50% MeOH to 100% MeOH for 25.0 min, linear gradient, at a flow rate 0.75 mL/min.
3.2. Synthesis
Syntheses of brassinin (1) [8], camalexin (3a) [12], and isoquinolines 8 [13] and 9a [13] were carried
out as previously reported. Satisfactory spectroscopic data identical to those previously reported were
obtained for all known compounds.
3.2.1. Phenylquinolines 5a–7g
General Procedure
Iron powder (0.4 eq.) was added to a solution of o-nitrobenzaldehyde (11a–11g, 1.0 eq.) in EtOH
(2.0 mL) followed by HCl (0.05 eq.) [19]. The reaction mixture was refluxed for 40 min followed
by cooling to room temperature. The corresponding aldehyde or ketone (1.2 eq.) was added to the
reaction mixture followed by KOH (1.2 eq.). The reaction mixture was stirred at RT for 30 min and
refluxed for an additional 3 h. The reaction mixture was filtered, the filtrate was neutralized (HCl),
extracted with EtOAc, and the combined extracts were dried and concentrated to dryness. The residue
was subjected to FCC (EtOAc–hexane, 3:7) to afford substituted quinolines 5a–7g.
2-Phenylquinoline (5a): Compound 5a was prepared from o-nitrobenzaldehyde 11a (151 mg, 1.00 mmol)
according to the above procedure in 83% yield (171 mg, 0.83 mmol), obtained as a white powder, m.p.
78–79 ◦C. Satisfactory spectroscopic data identical to those previously reported [24].
5-Chloro-2-phenylquinoline (5b): Compound 5b was prepared from o-nitrobenzaldehyde 11b (200 mg,
1.07 mmol) according to the above procedure in 84% yield (215 mg, 0.90 mmol), obtained as a white
◦
powder, m.p. 90–91 C. Although referenced in the SciFinder database as previously prepared (accessed

Molecules 2017, 22, 1345
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on 17 April 2017), structure 5b appears to be a new compound, since it was not published in the cited
reference [25].
1H-NMR (500 MHz, CDCl3):
1H), 7.71–7.59 (m, 2H), 7.56 (m, 2H), 7.50 (dd, J = 7, 7 Hz, 1H). 13C-NMR (125 MHz, CDCl3):
149.2, 139.2, 133.8, 131.4, 129.9, 129.5, 129.2, 129.1, 127.8, 126.5, 125.5, 119.9. HPLC tR = 24.4 ± 0.2 min
(method B). UV (HPLC, CH3OH-H2O) max (nm): 208, 262. FTIR (KBr)
max cm−1: 3058, 1611, 1593,
δ
8.63 (d, J = 9 Hz, 1H), 8.19 (m, 2H), 8.11 (d, J = 8 Hz, 1H), 7.99 (d, J = 9 Hz
,
δ
158.1,
λ
ν
1580, 1547, 1486, 1461, 1396, 1316, 1278, 1201, 1025, 960, 814, 775, 692, 671. HRMS-EI m/z (%): calc. for
C15H10NCl: 239.0502, measured 239.0507 (100), 204.08 (33).
6-Chloro-2-phenylquinoline (5c): Compound 5c was prepared from o-nitrobenzaldehyde 11c (200 mg,
1.07 mmol) according to the above procedure in 63% yield (161 mg, 0.670 mmol), obtained as a white
powder, m.p. 109–110 ◦C. Satisfactory spectroscopic data identical to those previously reported [26].
6-Bromo-2-phenylquinoline (5d): Compound 5d was prepared from o-nitrobenzaldehyde 11d (59 mg,
0.25 mmol) according to the above procedure in 64% yield (47 mg, 0.16 mmol), obtained as an off-white
powder, m.p. 123–125 ◦C. Satisfactory spectroscopic data identical to those previously reported [27].
6-Hydroxy-2-phenylquinoline (5e): Compound 5e was prepared from o-nitrobenzaldehyde 11e (200 mg,
1.19 mmol) according to the above procedure in 42% yield (112 mg, 0.51 mmol), obtained as a brown
powder, m.p. 220–223 ◦C. Satisfactory spectroscopic data identical to those previously reported [28].
6-Methoxy-2-phenylquinoline (5f): Compound 5f was prepared from o-nitrobenzaldehyde 11f (150 mg,
0.82 mmol) according to the above procedure in 60% yield (115 mg, 0.49 mmol), obtained as a white
powder, m.p. 135–137 ◦C. Satisfactory spectroscopic data identical to those previously reported [24].
3-Phenylquinoline (6a): Compound 6a was prepared from o-nitrobenzaldehyde 11a (200 mg, 1.32 mmol)
according to the above procedure in 63% yield (172 mg, 0.84 mmol), obtained as a light yellow powder,
m.p. 45–46 ◦C. Satisfactory spectroscopic data identical to those previously reported [19].
5-Chloro-3-phenylquinoline (6b): Compound 6b was prepared from o-nitrobenzaldehyde 11b (200 mg,
1.07 mmol) according to the above procedure in 80% yield (207 mg, 0.87 mmol), obtained as an off-white
powder, m.p. 115–116 ◦C. Compound 6b was previously synthesized using a different procedure but
no spectroscopic data was reported [29].
1H-NMR (500 MHz, CDCl3): δ 9.18 (d, J = 2 Hz, 1H), 8.22 (d, J = 2 Hz, 1H), 8.08 (d, J = 9 Hz, 1H), 7.88
(d, J = 2 Hz, 1H), 7.71 (m, 2H), 7.66 (dd, J = 9, 2 Hz, 1H), 7.55 (m, 2H), 7.47 (dd, J = 7.5, 7.5 Hz, 1H).
13C-NMR (125 MHz, CDCl3):
127.3, 126.4. HPLC tR = 22.9
FTIR (KBr)
max cm−1: 3053, 1487, 1456, 1182, 977, 900, 810, 758, 742, 694. HRMS-EI m/z (%): calc. for
δ
150.8, 148.1, 137.7, 135.0, 131.7, 130.2, 129.5, 129.2, 128.7, 128.6, 127.8,
±
0.2 min (method B). UV (HPLC, CH3OH-H2O) (nm): 208, 238, 257.
λ
max
ν
C15H10NCl: 239.0502, measured 239.0507 (100), 204.08 (15).
6-Chloro-3-phenylquinoline (6c): Compound 6c was prepared from o-nitrobenzaldehyde 11c (200 mg,
1.07 mmol) according to the above procedure in 64% yield (163 mg, 0.68 mmol), obtained as a white
powder, m.p. 105–107 ◦C. Satisfactory spectroscopic data identical to those previously reported [30].
6-Bromo-2-phenylquinoline (6d): Compound 6d was prepared from o-nitrobenzaldehyde 11d (200 mg,
0.869 mmol) according to the above procedure in 40% yield (100 mg, 0.35 mmol), obtained as a
light yellow powder, m.p. 116–118 ◦C. Satisfactory spectroscopic data identical to those previously
reported [31].
6-Hydroxy-3-phenylquinoline (6e): Compound 6e was prepared from o-nitrobenzaldehyde 11e (200 mg,
1.19 mmol) according to the above procedure in 39% yield (102 mg, 0.460 mmol), obtained as a light
brown powder, m.p. 223–225 ◦C.
1H-NMR (500 MHz, DMSO-d6):
7.84 (d, J = 7.5 Hz, 2H), 7.53 (dd, J = 7.5, 7.5 Hz, 2H), 7.44 (dd, J = 7.5, 7.5 Hz, 1H), 7.31 (dd, J = 2, 9 Hz,
δ 10.09 (s, 1H), 8.99 (d, J = 2 Hz, 1H), 8.41 (s, 1H), 7.89 (d, J = 9 Hz, 1H),

Molecules 2017, 22, 1345
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1H), 7.23 (d, J = 2 Hz, 1H). 13C-NMR (125 MHz, DMSO-d6):
δ
155.9, 146.1, 142.2, 137.4, 132.8, 131.1,
130.1, 129.2, 128.0, 127.1, 122.0, 108.7. HPLC tR = 14.0
±
0.2 min (method B). UV (HPLC, CH3OH-H2O)
λ
max
(nm): 206, 255, 342. FTIR (KBr)
ν
max
cm−1: 2952, 1620, 1496, 1457, 1376, 1246, 1217, 1166, 950, 898,
763, 701. HRMS-EI m/z (%): calc. for C15H11NO: 221.0841, measured 221.0838 (100).
6-Methoxy-3-phenylquinoline (6f): Compound 6f was prepared from o-nitrobenzaldehyde 11f (178 mg,
0.98 mmol) according to the above procedure in 53% yield (123 mg, 0.52 mmol), obtained as a yellow
powder, m.p. 127–129 ◦C. Satisfactory spectroscopic data identical to those previously reported [32].
6-Phenylquinoline (7a): Compound 7a was prepared from o-nitrobenzaldehyde 11g (20 mg, 0.09 mmol)
according to the above procedure in 62% yield (11 mg, 0.053 mmol), obtained as a light brown powder,
m.p. 105–106 ◦C. Satisfactory spectroscopic data identical to those previously reported [33].
2-Methyl-6-phenylquinoline (7b): Compound 7b was prepared from o-nitrobenzaldehyde 11g (50 mg,
0.25 mmol) according to the above procedure in 52% yield (25 mg, 0.11 mmol), obtained as a yellow
powder, m.p. 89–91 ◦C. Satisfactory spectroscopic data identical to those previously reported [34].
3-Methyl-6-phenylquinoline (7c): Compound 7c was prepared from o-nitrobenzaldehyde 11g (20 mg,
0.090 mmol) and propanal (50
obtained as a light yellow oil.
µL) according to the above procedure in 52% yield (10 mg, 0.050 mmol),
1H-NMR (500 MHz, CDCl3):
7.91 (d, J = 2 Hz, 1H), 7.73–7.71 (m, 2H), 7.51 (dd, J = 7.5, 8.0 Hz, 1H), 7.41 (dd, J = 8.5, 7.5 Hz, 1H), 2.55
(s, 3H). 13C-NMR (125 MHz, CDCl3):
152.6, 146.1, 140.7, 139.5, 135.1, 131.1, 129.8, 129.1, 128.5, 128.4,
127.9, 127.7, 125.1, 19.0. HPLC tR = 23.3 0.2 min (method A). UV (HPLC, CH3CN-H2O) max (nm):
210, 250. FTIR (KBr)
max cm−1: 2974, 1600, 1486, 1345, 1174, 900, 835, 759, 697, 570, 479. HRMS-FD
δ 8.78 (d, J = 1.5 Hz, 1H), 8.14 (d, J = 8.5 Hz, 1H), 7.97 (s, 1H), 7.93 (s, 1H),
δ
±
λ
ν
m/z (%): calc. for C16H13N: 219.1048, measured 219.1051 (100).
3-Ethyl-6-phenylquinoline (7d): Compound 7d was prepared from o-nitrobenzaldehyde 11g (60 mg,
0.27 mmol) according to the above procedure in 82% yield (50 mg, 0.21 mmol), obtained as a yellow oil.
1H-NMR (500 MHz, CDCl3):
J = 8.5 Hz, 1H), 7.73 (d, J = 7.5 Hz, 2H), 7.51 (dd, J = 7, 7.5 Hz, 2H), 7.41 (dd, J = 7.5, 7 Hz, 1H), 2.88 (q,
J = 8, 7.5, 7.5 Hz, 2H), 1.39 (t, J = 7.5, 7.5 Hz, 3H).13C-NMR (125 MHz, CDCl3):
151.9, 146.2, 140.7,
139.6, 137.3, 134.0, 129.6, 129.2, 128.6, 127.9, 127.7, 125.3, 26.5, 15.4. HPLC tR = 27.2 0.2 min (method
A). UV (HPLC, CH3CN-H2O) (nm): 210, 252. FTIR (KBr)
cm−1: 2965, 1599, 1487, 1348, 1179,
δ 8.80 (s, 1H), 8.15 (d, J = 8.5 Hz, 1H), 7.98 (d, J = 9.5 Hz, 2H), 7.93 (d,
δ
±
λ
max
ν
max
908, 837, 757, 697, 627, 570. HRMS-FD m/z (%): calc. for C17H15N: 233.1205, measured 233.1205 (100).
3-Isopropyl-6-phenyl-quinoline (7e): Compound 7e was prepared from o-nitrobenzaldehyde 11g (60 mg,
0.27 mmol according to the above procedure in 69% yield (45 mg, 0.18 mmol), obtained as a yellow oil.
1H-NMR (500 MHz, CDCl3):
J = 10 Hz, 1H), 7.74–7.72 (m, 1H), 7.51 (dd, J = 7.5, 8 Hz, 2H), 7.41 (dd, J = 7.5, 7.5 Hz, 1H), 3.19–3.13
(m, 1H), 1.41 (s, 3H), 1.40 (s, 3H).13C-NMR (125 MHz, CDCl3):
151.3, 146.4, 141.7, 140.7, 139.4, 132.3,
129.6, 129.1, 128.6, 128.5, 127.8, 127.6, 125.4, 32.1, 23.8. HPLC tR = 30.1 0.2 min (method A). UV
(HPLC, CH3CN-H2O) max (nm): 210, 252. FTIR (KBr)
max cm−1: 2960, 1599, 1486, 1351, 1178, 1077,
δ 8.85 (s, 1H), 8.17 (d, J = 8.5 Hz, 1H), 7.99 (d, J = 9.5 Hz, 2H), 7.93 (d,
δ
±
λ
ν
975, 909, 837, 757, 697, 473. HRMS-FD m/z (%): calc. for C18H17N: 247.1361, measured 247.1372 (100).
3,6-Diphenylquinoline (7f): Compound 7f was prepared from o-nitrobenzaldehyde 11g (40 mg,
0.18 mmol) according to the above procedure in 100% yield (50 mg, 0.18 mmol), obtained as a light
yellow powder, m.p. 131–133 ◦C.
1H-NMR (500 MHz, CDCl3):
δ 9.20 (s, 1H), 8.34 (s, 1H), 8.23 (d, J = 9 Hz, 1H), 8.10 (s, 1H), 8.00 (d,
J = 9 Hz, 1H), 7.76–7.74 (m, 4H), 7.57–7.51 (m, 4H), 7.48–7.42 (m, 2H).13C-NMR (125 MHz, CDCl3):
δ 150.0, 146.9, 140.4, 139.9, 138.0, 134.4, 133.6, 129.8, 129.4, 129.3, 129.2, 128.4, 128.3, 128.0, 127.6, 127.5,
125.8. HPLC tR = 30.1
±
0.2 min (method A). UV (HPLC, CH3CN-H2O)
λ
max
(nm): 210, 280. FTIR

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(KBr)
ν
cm−1: 3052, 1487, 1348, 1077, 910, 834, 758, 699, 571, 531, 497, 470. HRMS-FD m/z (%): calc.
max
for C21H15N: 281.1205, measured 281.1218 (100).
2,6-Diphenylquinoline (7g): Compound 7g was prepared from o-nitrobenzaldehyde 11g (20 mg,
0.090 mmol) according to the above procedure in 62% yield (15 mg, 0.053 mmol), obtained as a
light brown powder, m.p. 198–200 ◦C. Satisfactory spectroscopic data identical to those previously
reported [35].
3.2.2. Synthesis Isoquinolines 9b, 9c and 10b
Isoquinolines 9b and 9c
Aminoacetaldehyde dimethylacetal (3.0 eq.) was added to a solution of bromobenzaldehyde
13b or ◦13c (1.0 eq.) in toluene (30 mL). Each reaction mixture was refluxed (Dean–Stark apparatus)
at 120 C. After consumption of the starting material, each reaction mixture was concentrated to
dryness, then dissolved in conc. H2SO4 (2 mL) and added to a cold solution of P2O5 in conc. H2SO4
(0.5 mL). Each reaction mixture was heated at 160 ◦C for 30 min, allowed to cool to RT, neutralized
with NaOH (10 M), extracted with EtOAc, and concentrated to dryness. Each residue was subjected to
FCC to afford 6-bromoisoquinoline (14b, 30 mg, 0.14 mmol, 14%) and 7-bromoisoquinoline (14c, 99 mg,
0.47 mmol, 22%) [20,21]. Ethylchloroformate (1.0 eq.) was added to a solution of isoquinoline 14b or
14c (1.0 eq.) in DCM at 0 ◦C and stirred at the same temperature for 30 min, followed by addition
of 2-trimethylsilylthiazole (1.0 eq.). Each reaction mixture was stirred at RT for 3 h, concentrated
to dryness, and each residue was subjected to FCC. Each product was dissolved in benzene (5 mL),
o-chloranil (1.0 eq.) was added, and each reaction mixture was refluxed for 5 h. Each reaction mixture
was diluted with 5% NaOH (10 mL), extracted with DCM, and concentrated to dryness. Each reaction
mixture residue was subjected to FCC to afford the products 9b and 9c.
6-Bromo-1-(2-thiazolyl)isoquinoline (9b): 6-Bromoisoquinoline (14b, 30 mg, 0.14 mmol) was synthesized
starting from 4-bromobenzaldehyde (13c, 200 mg, 1.08 mmol) in 14% yield. Compound 9b was
synthesized starting from 6-bromoisoquinoline (14b, 100 mg, 0.48 mmol) in 15% yield over two steps
(21 mg, 0.07 mmol), obtained as an orange powder, m.p. 103–105 ◦C.
1H-NMR (500 MHz, CDCl3):
J = 2, 9 Hz, 1H), 7.61 (d, J = 5.5 Hz, 1H), 7.53 (d, J = 3 Hz, 1H). 13C-NMR (125 MHz, CDCl3):
149.9, 144.4, 143.0, 138.7, 132.3, 130.1, 129.1, 125.7, 124.0, 122.5, 121.2. HPLC tR = 22.4 0.2 min (method
B). UV (HPLC, CH3OH-H2O) (nm): 240, 345. FTIR (KBr)
cm−1: 2924, 2852, 1782, 1608, 1546,
δ
9.77 (d, J = 9 Hz, 1H), 8.58 (d, J = 5.5 Hz, 1H), 8.06 (m, 2H), 7.79 (dd,
170.6,
δ
±
λ
max
ν
max
1426, 1246, 1183, 947, 880, 810. HRMS-EI m/z (%): calc. for C12H7N2SBr: 291.9493, measured 291.9494
(100), 289.95 (95), 247.88 (53), 211.03 (21), 99.96 (39).
7-Bromo-1-(2-thiazolyl)isoquinoline (9c): 7-Bromoisoquinoline (14c, 99 mg, 0.47 mmol) was synthesized
starting from compound 13b in 22% yield [20]. Compound 9c was synthesized starting from
7-bromoisoquinoline (14c, 100 mg, 0.48 mmol) in 24% yield over two steps (35 mg, 0.12 mmol),
obtained as an off-white powder m.p. 110–112 ◦C.
1H-NMR (500 MHz, CDCl3):
(dd, J = 9, 2 Hz, 1H), 7.73 (d, J = 9 Hz, 1H), 7.67 (d, J = 5.5 Hz, 1H), 7.53 (d, J = 3 Hz, 1H). 13C-NMR
(500 MHz, CDCl3): 170.3, 148.6, 144.5, 142.3, 136.0, 134.2, 130.5, 128.6, 126.3, 123.1, 122.5, 122.1.
HPLC tR = 21.0 ± 0.2 min (method B). UV (HPLC, CH3OH-H2O) (nm): 240, 302, 350. FTIR (KBr)
δ 10.12 (s, 1H), 8.58 (d, J = 5.5 Hz, 1H), 8.08 (d, J = 3 Hz, 1H), 7.80
δ
λ
max
ν
cm−1: 3089, 1781, 1573, 1544, 1491, 1425, 1247, 1103, 947, 850, 813, 732. HRMS-EI m/z (%): calc. for
max
C12H7N2SBr: 291.9493, measured 291.9483 (100), 289.95 (99), 247.94 (34), 211.03 (32), 99.96 (24).
1-Phenylisoquinoline (10a): Iodine (18 mg, 0.073 mmol) was added to a solution of compound 15a (12 mg,
0.073 mmol) in THF (2 mL) and stirred at RT. After 30 min, Fe powder (16 mg, 0.29 mmol) followed by
PhMgCl (2 M in THF, 43
µL, 0.087 mmol) were added and stirred at RT. After 6 h, the reaction mixture
was diluted with ice-cold water (20 mL) and extracted with EtOAc. The combined organic extracts

Molecules 2017, 22, 1345
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were dried and concentrated to dryness to afford 10a in 67% yield (10 mg, 0.050 mmol) as an off-white
powder, m.p. 91–92 ◦C. Satisfactory spectroscopic data identical to those previously reported [36].
7-Bromo-1-phenylisoquinoline (10b): Iodine (18 mg, 0.073 mmol) was added to a solution of compound
15b (40 mg, 0.16 mmol) in THF (2 mL) and stirred at RT for 30 min followed by addition of Fe powder
(16 mg, 0.294 mmol) and PhMgCl (2 M in THF, 43 µL, 0.087 mmol). The reaction mixture was stirred
at RT for 6 h followed by dilution with ice-cold water, extraction with EtOAc, and concentration to
dryness to afford 10b in 47% yield (21 mg, 0.07 mmol) as yellow gum [36].
1H-NMR (500 MHz, CDCl3): δ 8.64 (d, J = 6 Hz, 1H), 8.07 (d, J = 2 Hz, 1H), 7.99 (d, J = 9 Hz, 1H), 7.68
(dd, J = 2, 7.5 Hz, 2H), 7.62 (dd, J = 2, 9 Hz, 1H), 7.58–7.53 (m, 4H). 13C-NMR (500 MHz, CDCl3):
δ
161.2,
143.6, 139.3, 138.2, 130.9, 130.1, 129.7, 129.3, 129.1, 128.7, 125.3, 125.1, 119.0. HPLC tR = 23.4 ± 0.2 min
(method B). UV (HPLC, CH3OH-H2O) (nm): 220, 258, 328. FTIR (KBr)
cm−1: 3061, 3033,
λ
max
ν
max
2923, 1480, 1429, 1261, 1091, 1042, 1008, 903, 800, 728, 697. HRMS-EI m/z (%): calc. for C15H10NBr:
284.9976, measured 284.9969 (10), 237.08 (26), 184.04 (41), 91.05 (100), 82.07 (86).
3.3. Antifungal Activity
The antifungal activity of brassinin (1), camalexin (3a), quinolines 5a–8, and isoquinolines 9a–10b
against L. maculans was evaluated using a mycelial radial growth assay carried out in PDA [23].
Cultures of L. maculans were grown on potato dextrose agar (PDA) plates (10 cm diameter) at 23 ± 1 ◦C
under constant light (fluorescent 32W T8 48" daylight) for seven days. DMSO solutions of each
compound (0.50, 0.20 and 0.10 mM) in PDA (1% DMSO concentration in PDA) were added to each
well (six-well plates, 1.5 mL per well) and inoculated with mycelia (4 mm diameter plugs cut from the
edges of 7-day-old solid cultures, placed upside down on the center of each well). Control cultures
containing 1% DMSO in PDA were prepared similarly. Plates were incubated under constant light and
the diameter of mycelial mat (mm) in each well was measured after five days of incubation. Each assay
was conducted in triplicate and repeated twice. The percentage of growth inhibition was calculated as
reported in Table 2.
3.4. Fungal Cultures, Preparation of Cell-Free Protein Extracts, and BOLm Activity
Liquid cultures of L. maculans (isolate UAHM 9410) were handled as described previously. In brief,
fungal spores were subcultured on V8 agar under continuous light at 23
±
1 ◦C; after 15 days, fungal
spores were collected aseptically and stored at
−
20 ◦C. Liquid cultures were initiated by inoculating
minimal media (100 mL) with fungal spores at 107/mL in Erlenmeyer flasks, followed by incubation
on a shaker under constant light at 23 ◦C for 48 h; 3-phenylindole (0.050 mM in DMSO) was added to
each flask to induce BOLm, and cultures were incubated for an additional 24 h. Cultures were gravity
filtered to separate mycelia from culture broth, the mycelia was washed with distilled water, wrung,
and stored at −20 ◦C.
Frozen mycelia (1.2 g) from L. maculans (obtained as reported above) were ground in ice-cold
◦
extraction buffer (5 mL, mortar and pestle) for 5 min at 4 C. The extraction buffer consisted of
diethanolamine (DEA, 25 mM, pH ~8.3), 10% (v/v) glycerol, D,L-dithiothreitol (DTT, 1 mM), and
1/200 (v/v) protease inhibitor cocktail (P-8215, Sigma-Aldrich Canada). The homogenate was
◦
centrifuged at 4 C for 30 min at 50,000 g. The resulting supernatant was dialyzed three times
(twice with 300 mL of dialyzing buffer for 3 h each time and then using 400 mL buffer for 12 h, using
dialyzing cassettes in buffer pH 8.3, diethanolamine, 25 mM, 5% glycerol, v/v, 10% triton X-100 in
deionized water). Dialyzed cell-free protein extracts were used for determination of the specific activity
of BOLm and for testing inhibitory activity of compounds.
Protein concentrations were determined as described by Bradford using the Coomassie Brilliant
Blue method with BSA as a standard (optical density (OD) was measured at 595 nm).

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Determination of BOLm Activity
The reaction mixture contained diethanolamine (20 mM, pH ~8.3), DTT (0.10 mM), 0.1% (v/v)
triton X-100, brassinin (1, 0.10 mM), PMS (0.10 mM), and cell-free protein extract (50.0 µL) in a total
volume of 1.0 mL. The reaction was carried out at 24 ◦C for 20 min. The product was extracted with
EtOAc (4 mL) and the extract was concentrated to dryness; the residue was dissolved in CH3CN
(200 µL) and analyzed by HPLC-DAD. The amount of indole-3-carboxaldehyde (2) in the reaction assay
was determined using a calibration curve built with pure indole-3-carboxaldehyde (λmax 220 nm).
4. Conclusions
Overall, our results indicate that at higher concentration (0.30 mM) 3-ethyl-6-phenylquinoline
(7d) inhibited BOLm activity to a larger extent than camalexin (3a) and displayed lower antifungal
activity against L. maculans, both very desirable characteristics. Furthermore, no direct correlation was
found between BOLm inhibitory activity of quinolines and their antifungal activity against L. maculans.
Nevertheless, considering the antifungal activity against L. maculans, compounds with a diversity
of scaffolds need to be synthesized and assayed before the desirable paldoxin activity of different
scaffolds can be predicted. To this end, availability of the crystal structure of BOLm would be of
great assistance.
The design of new compounds with new mechanisms of action to protect crops against fungi is
expensive and time consuming, providing rather unpredictable outcomes. Nonetheless, the discovery
of novel agrochemicals useful for sustainable crop treatments is crucial. It is expected that in the future,
broad range fungicides will be replaced with selective crop treatments and that paldoxins will be a
rational approach to treat specific fungal diseases such as that caused by L. maculans. Further work to
uncover paldoxins that can replace current fungicides and prevent crop infestations with L. maculans
is necessary.
1
Supplementary Materials: The following are available online. H- and 13C-NMR spectra of all new compounds:
5b, 6b, 6e, 7c, 7d, 7e, 7g, 9b, 9c and 10b.
Acknowledgments: Financial support for the authors’ work was obtained from the Natural Sciences and
Engineering Research Council of Canada (Discovery Grant to M.S.C.P.), the Canada Research Chairs program,
Canada Foundation for Innovation, the Saskatchewan Government, and the University of Saskatchewan (graduate
assistantship to A.A. and V.K.S.-M.). We acknowledge the technical assistance of K. Brown (NMR) and K. Thoms
(MS) from the Department of Chemistry and Saskatchewan Structural Sciences Centre.
Author Contributions: M.S.C.P., A.A. and V.K.S.-M. conceived and designed the experiments; A.A. and V.K.S.-M.
performed the experiments; M.S.C.P., A.A. and V.K.S.-M. analyzed the data; MSCP, A.A. and V.K.S.-M. wrote
the paper.
Conflicts of Interest: The authors declare no conflict of interest.
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