Design, synthesis, and antifungal activity of inhibitors of brassilexin detoxification in the plant pathogenic fungus Leptosphaeria maculans

Article abstract of DOI:10.1016/j.bmc.2005.08.053

Potential inhibitors of Leptosphaeria maculans mediated detoxification of the phytoalexin brassilexin were designed and synthesized based on the planar heteroaromatic structure of isothiazolo[5,4-b]indole. Screening of these compounds for inhibition of br

Full text of DOI:10.1016/j.bmc.2005.08.053

Bioorganic & Medicinal Chemistry 14 (2006) 714–723
Design, synthesis, and antifungal activity of inhibitors
of brassilexin detoxification in the plant pathogenic fungus
Leptosphaeria maculans
M. Soledade. C. Pedras* and Mojmir Suchy
Department of Chemistry, University of Saskatchewan, 110 Science Place, Saskatoon, Canada SK S7N 5C9
Received 22 July 2005; revised 26 August 2005; accepted 26 August 2005
Available online 3 October 2005
Abstract—Potential inhibitors of Leptosphaeria maculans mediated detoxification of the phytoalexin brassilexin were designed and
synthesized based on the planar heteroaromatic structure of isothiazolo[5,4-b]indole. Screening of these compounds for inhibition of
brassilexin detoxification in cultures of L. maculans indicated that 4-(2-chlorophenyl)isothiazole had the largest effect on the rate of
brassilexin detoxification. However, the most antifungal compound among the potential inhibitors, isothiazolo[5,4-b]quinoline, did
not appear to affect the metabolism of brassilexin noticeably, suggesting that growth inhibition is not sufficient to slow down the rate
of brassilexin detoxification. Furthermore, it was determined that 4-arylisothiazoles as well as isothiazolo[5,4-b]thianaphthene
displayed antifungal activity against L. maculans.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
ward this end, a recent investigation of the metabolism
of brassilexin (1) in L. maculans demonstrated that the
first step of its detoxification involved reduction of the
isothiazole ring to 3-aminomethyleneindole-2-thione
(7), followed by hydrolytic and oxidative reactions to
yield 3-formylindolyl-2-sulfonic acid (9) via aldehyde 8
(Scheme 1). Because the enamine 7 showed a substan-
tially lower antifungal activity than brassilexin (1), these
findings suggested that potentially effective inhibitors or
modulators of brassilexin detoxification must slow down
or stop the reductive bioconversion of brassilexin (1)
into 3-aminomethyleneindole-2-thione (7).
The detoxification of phytoalexins by fungal plant
pathogens is of great concern as it can have a substan-
tially negative impact on the overall plant fitness.1 Phy-
toalexins are secondary metabolites produced de novo
by plants in response to diverse forms of stress, includ-
ing fungal infection. Brassilexin (1) is one of the most
potent antifungal phytoalexins produced by crucifer
plants, namely those of the genus Brassica.2,3 The fungal
pathogen Leptosphaeria maculans [(Desm.) Ces. et de
Not., asexual stage Phoma lingam (Tode ex Fr.) Desm.]
is able to overcome by detoxification some important
defenses of brassicas, including brassilexin (1),4 sinalexin
(2)4, brassinin (3), cyclobrassinin (4), brassicanal A (5),
and dioxibrassinin (6).1 Nonetheless, the strong in vitro
growth inhibitory activity of brassilexin (1) against L.
maculans suggests that an increase of its concentration
in the plant, through, for example, inhibition of the
detoxification pathway, could slow down or prevent
the spread of the pathogen. This prospect suggests that
potential inhibitors of brassilexin detoxification could be
used to protect the plant against the fungal invader. To-
NH
N
SCH
3
S
S
N
R
1 R=H
N
H
3
2 R=OCH
3
CHO
SCH
N
SCH
HO
3
S
N
3
N
H
5
H
4
NH
SCH
Keywords: Brassilexin; Detoxification inhibitor; Leptosphaeria macu-
lans; Phoma lingam; Phytoalexin.
3
S
O
*
Corresponding author. Tel.: +1 306 966 4772; fax: +1 306 966
4730; e-mail: s.pedras@usask.ca
N
H
6
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.08.053

M. Soledade et al. / Bioorg. Med. Chem. 14 (2006) 714–723
715
thiazoles 21 and 22 were synthesized and their effects
on the detoxification of brassilexin were examined.
Here, we describe results of these studies and show
that 4-(2-chlorophenyl)isothiazole (15) has the largest
effect on the rate of brassilexin detoxification. Howev-
er, the most antifungal compound among the poten-
tial inhibitors, isothiazolo[5,4-b]quinoline (10), did
not appear to affect the metabolism of brassilexin
noticeably. It is worth noting that the isothiazole ring
is a component of a great variety of compounds with
pharmacological activity, including antibacterial, anti-
viral, and insecticidal, but no antifungal activity ap-
pears to have been described to date.5
NH
2
N
S
S
N
H
1
N
H
7
CHO
CHO
SH
SO3H
N
H
N
H
9
8
Scheme 1. Metabolism of brassilexin (1) in Leptosphaeria maculans
(Phoma lingam).
2. Results
Considering the chemical structures of brassilexin (1,
isothiazolo[5,4-b]indole) and its first detoxification
intermediate, 3-aminomethyleneindole-2-thione (7), it
was of interest to evaluate the role of the indole–iso-
thiazole and isothiazole ring systems on the anti-
fungal activity and metabolism of 1. Although
previous studies with L. maculans indicate that the
indole ring is neither metabolized nor essential for
antifungal activity,1 the indole–isothiazole ring system
has not been examined. Non-degradable isothiazoles
might be effective inhibitors of brassilexin detoxifying
enzyme(s) produced in L. maculans. Hence, the in-
dole–isothiazole fused-ring system of 1 was replaced
with quinoline–isothiazole (10), benzothiophene–iso-
thiazole (11), indole–thiophene (12), and benzo-iso-
thiazole (13) to yield compounds 10–13, respectively
(Fig. 1). In addition, isothiazoles resulting from dis-
connecting the ꢀaꢁ bond of the indole ring and replac-
ing the NH group ortho to the isothiazole with
various substituents (H, Cl, CH3, NH2, NO2, OH,
and OCH3) were designed (Fig. 1). The resulting 2-
substituted-4-phenylisothiazoles 14–20, naphthyliso-
2.1. Syntheses
Compounds 106,7 and 128 were prepared similar to
previously described procedures, and 3-phenylthiophene
(23) was commercially available. New conditions for the
syntheses of compounds 11 and 13–20 are reported, and
the syntheses of compounds 21 and 22 are described
here for the first time.
Isothiazolo[5,4-b]thianaphthene (11) was previously pre-
pared from 2-methylsulfanylthianaphthene-3-carboxal-
dehyde oxime by heating in polyphosphoric acid
(PPA).9 Since the yield was low (18%), a new synthesis
of 11 was developed using less harsh conditions. 3-Bro-
mothianaphthene (24) was treated with t-BuLi and the
corresponding 3-lithiothianaphthene was quenched with
DMF, followed by addition of another equivalent of
t-BuLi. A solution of bromine in hexane was added to
the reaction mixture followed by quenching with HCl
to afford 2-bromothianaphthene-3-carboxaldehyde (25)
in 35% yield, based on 3-bromothianaphthene (24).
Heating 25 with ammonium thiocyanate10 in DMF
afforded compound 11 in 62% yield (Scheme 2).11
N
N
S
S
S
N
1,2-Benzoisothiazole (13) was prepared by cyclization
of 2-t-butylsulfanylbenzaldehyde oxime, obtained from
2-nitrobenzaldehyde (26),12 using acetic acid anhydride
in acetic acid to promote ring closure (Scheme 3).
PPA13 was previously used, however, we found that
isolation of 13 becomes rather difficult under these
conditions (repeated column chromatography), afford-
ing a lower yield than that obtained when the reaction
is carried out using acetic acid anhydride in acetic
acid.
S
10
11
N
N
H
S
13
12
N
N
N
S
S
S
N
i t-BuLi, Et O
2
ii DMF
a
R
Br
CHO
Br
H
14 R = H
15 R = Cl
16 R = CH
1
21, 1-naphthyl
22, 2-naphthyl
iii t-BuLi
S
24
S
25
iv Br , hexane,
2
3
S
35%
17 R = NO
2
18 R = NH
19 R = OH
20 R = OCH
2
NH SCN, DMF
4
N
62%
S
23
3
S
11
Figure 1. Chemical structures of potential inhibitors of brassilexin (1)
detoxification.
Scheme 2. Synthesis of isothiazolo[5,4-b]thianaphthene (11).

716
M. Soledade et al. / Bioorg. Med. Chem. 14 (2006) 714–723
i t-BuSH, K CO , DMF
S
S
2
3
N
N
ii NH OH.HCl, NaOH
2
CHO
SnCl .2H O
2
2
EtOH/H O
2
N
HCl, MeOH
71%
iii Ac O, AcOH,
2
S
NO
2
NO
NH
2
2
64%
26
13
17
18
NaNO
Scheme 3. Synthesis of 1,2-benzoisothiazole (13).
2
H SO , H O
2
4
2
44%
S
S
The syntheses of 4-arylisothiazoles 14–17, 21, and 22,
followed a modified multi-step synthesis,14 employing
aryl substituted acetic acids 27–32 as starting materials.
Thus, aryl-acetic acids 27–32 underwent double
Vilsmeier–Haack reaction, followed by basic hydrolysis
in boiling sodium hydroxide/ethanol/water15 and treat-
ment with thionyl chloride in dichloromethane to yield
chloroacroleins 33–38 (Scheme 4). Subsequent heating
of the chloroacroleins 33–38 with ammonium thiocya-
nate in DMF10 afforded 4-arylisothiazoles 14–17, 21,
and 22 in low to moderate yields (25–54%, Scheme 4).
When 2-nitrophenylacetic acid (30) was used, only very
low yields (less than 5%) of chloroacrolein 36 were ob-
tained. The Vilsmeyer–Haack reaction appeared to be
problematic as black tar was formed even at room tem-
perature. The problem was solved using commercially
available 2-nitrophenyl malondialdehyde which yielded,
upon reaction with thionyl chloride, chloroacrolein 36 in
97% yield. 4-(2-Aminophenyl)isothiazole (18, Scheme 5)
was prepared from 17 using SnCl2Æ2H2O in acidic meth-
anol16 in 71% yield. Diazotization of 18 and subsequent
hydrolysis16 afforded 4-(2-hydroxyphenyl)isothiazole
(19, Scheme 5) in 44% yield based on 17. Methylation
of 19 using ethereal diazomethane afforded 4-(2-meth-
oxyphenyl)isothiazole (20, Scheme 5) in 83% yield.
N
N
CH N , Et O
2
2
2
83%
OH
OCH
3
19
20
Scheme 5. Synthesis of compounds 18–20.
activity (22%, 5.0 · 10À4 M). Compounds 12, 22, and
23 were not sufficiently soluble in potato dextrose media
to reach the concentration of 5.0 · 10À4 M, however
using the concentration 2.0 · 10À4 M, moderate 12
(52%), 22 (53%) to weak 23 (27%) antifungal activity
was observed. This is the first time that isothiazolo[5,4-
b]thianaphthene (11), isothiazolo[5,4-b]quinoline (10),
and 4-arylisothiazoles 14–21 are shown to be antifungal.
2.3. Screening of potential inhibitors
Compounds 10–23 were screened for potential inhibi-
tion of the transformation of brassilexin (1) to 3-ami-
nomethyleneindole-2-thione (7) in cultures of L.
maculans over a 96 h period. The potential inhibitors
(at 1.0 · 10À4 and 2.0 · 10À4 M) were added to shake
cultures of L. maculans (48-h-old mycelia in minimal
media), cultures were incubated for 10 min (to allow
adsorption/transport of compounds into cells), and
brassilexin (1, 1.0 · 10À4 M) was then added to cultures
followed by additional incubation. Control cultures (48-
h-old mycelia in minimal media) prepared similarly but
containing only brassilexin (1) were incubated in paral-
lel. The stability of brassilexin and compounds 10–23
was determined by incubation in minimal media under
similar conditions. Samples were withdrawn from cul-
tures immediately after addition of brassilexin and at
24, 48, and 96 h, were extracted and the extracts were
analyzed by HPLC (photodiode array detector), to
determine the concentration of brassilexin (1) remaining
in the cultures at different times. Brassilexin (1) was sta-
ble in minimal medium for at least 10 days; compounds
10–13, and 18–20 were stable in minimal media but 14–
2.2. Antifungal bioassays
The antifungal activity of compounds 10–23 against L.
maculans was evaluated using a mycelial radial growth
assay, as described in the Experimental section. Results
of these assays (Table 1) showed that isothiazolo[5,4-
b]thianaphthene (11) inhibited completely the mycelial
growth of L. maculans at 5.0 · 10À4 M, whereas isothiaz-
olo[5,4-b]quinoline (10) showed complete inhibition of
mycelial growth even at 2.0 · 10À4 M. Antifungal activi-
ty (40–100%, 5.0 · 10À4 M) was observed with 4-aryliso-
thiazoles 14–21. The most antifungal compound among
the tested 4-arylisothiazoles appeared to be 4-(2-
hydroxyphenyl)isothiazole (19), where complete inhibi-
tion of mycelial growth was observed at 5.0 · 10À4 M.
1,2-Benzoisothiazole (13) exhibited weak antifungal
Cl
CHO
S
NH SCN
DMF
i POCl , DMF
3
ii NaOH, EtOH/H O
4
N
Ar
COOH
2
Ar
Ar
iii SOCl , CH Cl
2
2
2
27 Ar = Ph
28 Ar = 2-ClPh
14 Ar = Ph
15 Ar = 2-ClPh
16 Ar = 2-CH Ph
3
17 Ar = 2-NO Ph
21 Ar = 1-naphthyl
22 Ar = 2-naphthyl
33 Ar = Ph
34 Ar = 2-ClPh
35 Ar = 2-CH Ph
36 Ar = 2-NO Ph
37 Ar = 1-naphthyl
38 Ar = 2-naphthyl
29 Ar = 2-CH Ph
30 Ar = 2-NO Ph
31 Ar = 1-naphthyl
32 Ar = 2-naphthyl
3
3
2
2
2
Scheme 4. Synthesis of 4-arylisothiazoles 14–17, 21, and 22.

M. Soledade et al. / Bioorg. Med. Chem. 14 (2006) 714–723
717
Table 1. Inhibitory activity (%)a of compounds 10–23 against Leptosphaeria maculans (procedure is described in Section 4.3)
Compound
1.0 · 10À4 M
2.0 · 10À4 M
5.0 · 10À4 M
Brassilexin (1)
8
60 13
1
62
1
C.I.b
C.I.b
C.I.b
N.S.c
Isothiazolo[5,4-b]quinoline (10)
Isothiazolo[5,4-b]thianaphthene (11)
Thieno[2,3-b]indole (12)
C.I.b
60
49
7
8
5
33
52 12
1,2-Benzoisothiazole (13)
4-Phenylisothiazole (14)
N.I.d
32
14
46
56
51
44
25
44
59
60
53
27
2
6
7
9
6
6
3
5
8
4
5
22
69
79
65
7
4
5
6
5
5
5
7
4
7
9
4
8
4-(2-Chlorophenyl)isothiazole (15)
4-(2-Tolyl)isothiazole (16)
42
31
4-(2-Nitrophenyl)isothiazole (17)
4-(2-Aminophenyl)isothiazole (18)
4-(2-Hydroxyphenyl)isothiazole (19)
4-(2-Methoxyphenyl)isothiazole (20)
4-(1-Naphthyl)isothiazole (21)
4-(2-Naphthyl)isothiazole (22)
3-Phenylthiophene (23)
35
18
67 10
40
3
38
46
C.I.b
82
3
4
37
41
73
N.S.c
N.S.c
N.I.d
a % inhibition = 100 À [(growth on treated/growth on control) · 100)] SD; results are the means of at least three separate experiments.
b C.I. = complete inhibition.
c N.S. = not soluble.
d N.I. = no inhibition.
16 decomposed slowly (ca. 50% or more remaining in
solution after 96 h) to undetermined products; 3-phenyl-
thiophene (23) decomposed completely in 24 h, thus was
not used in further metabolism experiments. As shown
in Table 2, a few of the test compounds slowed down
the transformation of brassilexin (1) relative to control
cultures. For example, in the case of 4-arylisothiazoles
15 and 20 (2.0 · 10À4 M) 22% and 31%, respectively,
of brassilexin remained in the cultures after 48 h of incu-
bation, whereas almost complete transformation of
brassilexin (1) was observed in 24 h (<5% remaining)
in control cultures (containing only brassilexin). Fur-
thermore, brassilexin was present in the cultures incu-
bated with compounds 11, 15, and 20 (2.0 · 10À4 M)
even after 96 h of incubation. However, a few of these
compounds were metabolized by the fungus (Table 2),
slowly in the case of isothiazolo[5,4-b]thianaphthene
(11, 22% remaining after 96 h) and arylisothiazole 17
(33% remaining after 96 h), and faster in the case of 4-
(2-hydroxyphenyl)isothiazole (19), which was metabo-
lized completely in 48 h (no products of metabolism
could be detected). Because 4-naphthylisothiazoles 21
and 22 were not sufficiently soluble in minimal media
their stability in fungal culture or in minimal media
could not be determined at the highest concentration.
15–20 plus brassilexin (1) and cultures incubated with
brassilexin (1) only (Fig. 2 and Figs. 3–7 in Supplemen-
tary data). 4-(2-Chlorophenyl)isothiazole (15) was the
most active inhibitor of brassilexin detoxification among
the tested compounds (Fig. 2). When 15 was present at
3.0 · 10À4 M, 48% of brassilexin (1) remained in culture
after three hours of incubation, whereas with 15 at
10À4 M, 34% of brassilexin remained in culture, com-
pared to 24% in control flasks containing only brassilex-
in (1). The remaining progress curves indicated that 4-
(2-nitrophenyl)isothiazole (17) was the next best inhibi-
tor followed by isothiazolo[5,4-b]thianaphthene (11)
(Fig. 3 in Supplementary data). For example, when 17
was present at 3.0 · 10À4 M, 40% of brassilexin (1) re-
mained in culture after 3 h of incubation, whereas with
17 at 1.0 · 10À4 M, 28% of brassilexin remained in cul-
ture, compared to 24% in control flasks containing only
brassilexin (1). The remaining compounds 16, and 18–20
did not affect substantially the detoxification of brassi-
lexin over a 12-h period relative to control cultures con-
taining only brassilexin (1) (Figs. 3–7 in Supplementary
data).
3. Discussion and conclusion
Because compounds 11 and 15–20 appeared to have a
greater effect of on the rate of detoxification of brassilex-
in (1) to enamine 7 in cultures of L. maculans (Table 2),
additional time–course analyses were carried out. Thus,
brassilexin (1) was added to cultures after 30 min of
incubation with inhibitors 11 and 15–20 at
1.0 · 10À4 M and 3.0 · 10À4 M (instead of 10 min, as
reported in the first study to allow additional time for in-
ter/intracellular transport) and samples were withdrawn
at 3-h intervals. Extraction of the culture samples and
HPLC analyses of the extracts allowed the determina-
tion of the amounts of brassilexin (1) remaining in the
cultures at different times. Progress curves were con-
structed depicting the decrease of brassilexin (1) concen-
tration in cultures incubated with compounds 11 and
Potential inhibitors of L. maculans mediated transfor-
mation of brassilexin were designed and synthesized
based on the planar heteroaromatic structure of brassi-
lexin (1). It was determined that 4-arylisothiazoles 15–
20 as well as isothiazolo[5,4-b]thianaphthene (11) dis-
played antifungal activity against L. maculans (Table
1). Quinoline 10 and thianaphthene 11, both containing
an isothiazole moiety, displayed the strongest growth
inhibitory activity, whereas 3-phenylthiophene (23) was
the weakest inhibitor. Although 4-phenylisothiazole
(14) caused substantial growth inhibition, two of the
weakest inhibitors contained an isothiazole moiety as
well, suggesting that additional structural features other
than the isothiazole ring are necessary to inhibit the
growth of L. maculans. Furthermore, the 2-substituent

718
M. Soledade et al. / Bioorg. Med. Chem. 14 (2006) 714–723
Table 2. Concentrations of brassilexin (1) remaining in cultures of Leptosphaeria maculans incubated with compounds 10–22 over a 96-h period
(procedure is described in Sections 4.4)
Compound
Brassilexin (1) remaining in the cultures (molar %)a incubated with
compounds at
1.0 · 10À4 M
2.0 · 10À4
—
M
Control
24 h, <5%
Isothiazolo[5,4-b]quinoline (10)
24 h, <5%
48 h, N.D.b
24 h, <5%
48 h, N.D.b
Isothiazolo[5,4-b]thianaphthene (11)c
24 h, 16 3%
48 h, 19 1%
96 h, 8 5%
24 h, 23 2%
48 h, 27 1%
96 h, 19 1%
Thieno[2,3-b]indole (12)
24 h, <5%
48 h, N.D.b
24 h, <5%
48 h, N.D.b
1,2-Benzoisothiazole (13)
4-Phenylisothiazole (14)c
24 h, <5%
48 h, N.D.b
24 h, <5%
48 h, N.D.b
24 h, <5%
48 h, N.D.b
24 h, <5%
48 h, N.D.b
4-(2-Chlorophenyl)isothiazole (15)d
24 h, 23 1%
48 h, 23 1%
96 h, N.D.b
24 h, 23 5%
48 h, 22 5%
96 h, 12 10%
4-(2-Tolyl)isothiazole (16)c
24 h, 11 2%
48 h, < 5%%
96 h, N.D.b
24 h, 17 2%
48 h, 9 1%
96 h, N.D.b
4-(2-Nitrophenyl)isothiazole (17)c
24 h, 11 3%
48 h, < 5%
96 h, N.D.b
24 h, 17 2%
48 h, 9 1%
96 h, N.D.b
4-(2-Aminophenyl)isothiazole (18)
4-(2-Hydroxyphenyl)isothiazole (19)e
24 h, 6 1%
48 h, N.D.b
24 h, 8 1%
48 h, N.D.b
24 h, 8 2%
48 h, N.D.b
96 h, N.D.b
24 h, 13 1%
48 h, < 5%
96 h, N.D.b
4-(2-Methoxyphenyl)isothiazole (20)
4-(1-Naphthyl)isothiazole (21)
4-(2-Naphthyl)isothiazole (22)
24 h, 20 1%
48 h, < 5%
24 h, 28 1%
48 h, 31 3%
96 h, 20 8%
96 h, N.D.b
24 h, 31 8%
48 h, < 5%
96 h, N.D.b
24 h, 32 5%
48 h, < 5%
96 h, N.D.b
Not soluble
Not soluble
a Percentages were determined using a calibration curve and are averages of at least two independent experiments conducted in duplicate standard
deviation.
b N.D. = not detected.
c Compound is metabolized by the fungus (20–40% remaining after 96 h of incubation).
d Compound is metabolized by the fungus in 96 h.
e Compound is metabolized completely by the fungus after 48 h of incubation.
of the phenyl group of compounds 14–20 appeared to
affect substantially the inhibitory activity, as shown for
compounds 15–20 (strong inhibitors) and 14 (weaker
inhibitor). 4-Arylisothiazoles were shown previously to
exhibit a wide range of biological activities,5 however,
to the best of our knowledge, no reports on the anti-
fungal activity of compounds containing a 4-aryliso-
thiazole moiety have been published so far. This is the
first time that the antifungal activity of 4-arylisothiaz-
oles and isothiazolo[5,4-b]thianaphthene (11) has been
examined. Thus, these lead structures could guide the
search for new antifungal compounds useful for both
agrochemical and pharmaceutical industries.
Screening of compounds 10–22 for inhibition of brass-
ilexin detoxification in cultures of L. maculans indicat-
ed that isothiazolo[5,4-b]thianaphthene (11) and 4-
arylisothiazoles 15–20 slowed down the metabolism of
brassilexin (1). Further screening suggested that com-
pound 15 had the largest effect on the rate of brassilex-
in metabolism among the tested compounds (Fig. 2
and Figs. 3–7 in Supplementary data). That is, 15,
although slowly metabolized by the fungus, appeared
to inhibit the putative brassilexin reductase responsible
for the detoxification of brassilexin (1) to 3-aminome-
thyleneindole-2-thione (7, Scheme 1). Because the most
antifungal compound among the potential inhibitors

M. Soledade et al. / Bioorg. Med. Chem. 14 (2006) 714–723
719
0.12
0.1
0.12
[mM]
[mM]
0.1
0.08
0.06
0.04
0.02
0
0.08
0.06
0.04
0.02
0
0
3
6
9
12
0
3
6
9
12
time [h]
time [h]
brassilexin
brassilexin
brassilexin + 4-(2-chlorophenyl)isothiazole (0.1 mM)
brassilexin + 4-(2-chlorophenyl)isothiazole (0.3 mM)
brassilexin + 4-(2-nitrophenyl)isothiazole (0.1 mM)
brassilexin + 4-(2-nitrophenyl)isothiazole (0.3 mM)
Figure 2. Metabolism of brassilexin (1.0 · 10À4 M) in the presence of 4-(2-chlorophenyl)isothiazole (15) and 4-(2-nitrophenyl)isothiazole (17). All
points are averages of three independent experiments standard deviation.
(isothiazolo[5,4-b]quinoline (10)), did not affect the
metabolism of brassilexin noticeably (Table 2), we sug-
gest that the inhibitory effect of 15 on brassilexin
detoxification is likely due to its direct interaction with
the detoxification enzyme(s). Hence, it is concluded
that the isothiazole ring may be useful as a lead struc-
ture to further improve the design of this type of inhib-
itors. Nonetheless, only co-incubation of the potential
inhibitors and brassilexin (1) with the putative brassi-
lexin reductase, which still remains to be detected and
isolated, could confirm this hypothesis. It is expected
that isolation of the putative brassilexin reductase in-
volved in this detoxification reaction will assist the bio-
rational design of crop protection agents selective
against L. maculans.
plied Biosystems; fragments with relative intensity
lower than 10% are not reported.
4.2. Fungal cultures
Fungal cultures of L. maculans/P. lingam virulent isolate
BJ 125 were obtained from the IBCN collection, Agricul-
ture and Agri-Food Canada Research Station, Saskatoon
SK. Cultures were handled as described previously.17
4.3. Antifungal bioassays
Antifungal bioassays against L. maculans were carried
out as follows: a DMSO solution of the compound to
be tested (final concentration 5.0 · 10À4, 2.0 · 10À4
,
and 1.0 · 10À4 M, final DMSO concentration 1%) was
added to potato dextrose agar medium at ca. 50 ꢁC,
mixed quickly and poured onto six-well plates
(2.5 mL). An agar plug (8 mm diameter) cut from edges
of 7-day-old solid cultures was placed upside down on
the center of each plate and the plates were incubated
at 24 2 ꢁC under constant light for 5 days. The diam-
eter of the mycelia (in millimeter) was then measured
and compared with control plates containing only
DMSO. Each assay was conducted in triplicate and
repeated at least three times.
4. Experimental
4.1. General experimental procedures
All chemicals were purchased from Sigma–Aldrich Can-
ada Ltd., Oakville, ON. All solvents were HPLC grade
and used as such, except for CH2Cl2 and CHCl3 that
were redistilled. Organic extracts were dried over anhy-
drous Na2SO4 and solvents removed under reduced
pressure in a rotary evaporator.
4.4. Fungal metabolism
HPLC analysis was carried out with a high performance
liquid chromatograph equipped with quaternary pump,
automatic injector, and diode array detector (wave-
length range 190–600 nm), degasser, and a Hypersil
ODS column (5 lm particle size silica, 4.6 id · 200 mm),
equipped with an in-line filter. Mobile phase: 75% H2O/
25% CH3CN–100% CH3CN, for 35 min, linear gradient,
and a flow rate 1.0 mL/min. UV spectra were recorded
on Varian-Cary spectrophotometer in MeOH. Fourier
transform IR spectra were obtained on a Bio-Rad
FTS-40 spectrometer in KBr. NMR spectra were
recorded on Bruker Avance 500 spectrometers; d values
4.4.1. Time–course studies of metabolism. Solutions of
potential inhibitors 10–23 in acetonitrile (0.25 mL)
were added to liquid shake cultures (100 mL of mini-
mal media in 250 mL Erlenmeyer flasks, final concen-
trations 1.0 · 10À4, 2.0 · 10À4, or 3.0 · 10À4 M, Table
2 and Fig. 2 and Figs. 3–7 in Supplementary data) of
L. maculans (BJ-125, 44-h-old, 108/100 mL spores).
After 10 (Table 2) or 30 (Figs. 2–7) min of incubation
(shaker at 130 rpm, at 24 2 ꢁC), brassilexin (1) dis-
solved in acetonitrile (0.25 mL) was added and cultures
(final concentration 1.0 · 10À4 M) were further incubat-
ed for various periods of time. Samples (2.5 mL) were
withdrawn and either frozen or immediately extracted
with EtOAc (2 · 5 mL). The organic phases were
concentrated and analyzed by HPLC. Experiments
1
were referenced as follows: for H (500 MHz), CDCl3,
7.27 ppm; for 13C (125 MHz), CDCl3, 77.23 ppm. Mass
spectra (MS) were obtained on a VG 70 SE mass spec-
trometer using a solids probe or on a Q Star XL, Ap-

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M. Soledade et al. / Bioorg. Med. Chem. 14 (2006) 714–723
were performed in duplicate (Table 2) or triplicate
(Figs. 2–7); control flasks containing mycelia in mini-
mal media or compounds in minimal media were incu-
bated under similar conditions.
4.5.2. 1,2-Benzoisothiazole (13). 2-t-Butylsulfanylbenzal-
dehyde oxime12 (419 mg, 2 mmol) was dissolved in ace-
tic acid (0.8 mL) and acetic acid anhydride (0.4 mL),
and the mixture was heated with stirring for 20 min at
100 ꢁC. The reaction mixture was cooled to room tem-
perature, diluted with water (30 mL) and extracted with
dichloromethane. The combined organic extract was
washed with 0.5 M solution of NaOH (10 mL), and
dried, the solvent was evaporated and the residue was
separated by FCC (silica gel, 60 g, hexane/diethyl ether,
5:1) to afford 1,2-benzoisothiazole (13, 220 mg, 64%,
based on 2-nitrobenzaldehyde (26)) as a colorless solid,
4.5. Synthesis
4.5.1. Isothiazolo[5,4-b]thianaphthene (11). A solution of
t-BuLi in pentane (2 mL, 2.2 mmol) was added dropwise
to a solution of 3-bromothianaphthene (24, 426 mg,
2 mmol) in dry diethyl ether at À78 ꢁC (Ar atmosphere).
The mixture was stirred for 30 min at À78 ꢁC and DMF
(170 lL, 2.2 mmol) was then added, the cooling bath
was removed, and the reaction mixture was stirred for
further 30 min at room temperature. The reaction mix-
ture was cooled to À30 ꢁC, another portion of t-BuLi
in pentane (2 mL, 2.2 mmol) was added dropwise, the
cooling bath was removed and the mixture was stirred
for 30 min at room temperature. The reaction mixture
was cooled to À78 ꢁC, and a solution of bromine
(140 ll, 2.6 mmol) in hexane (2 mL) was added in one
portion. The temperature was allowed to gradually raise
to 0 ꢁC (2 h), the reaction mixture was diluted with 1 M
HCl (50 mL) and extracted with EtOAc. The combined
organic extract was dried, the solvent was evaporated,
and the residue was subjected to FCC (flash chromatog-
raphy) (15 g silica gel, hexane), to afford 2-bro-
mothianaphthene-3-carboxaldehyde (25) as a colorless
solid (170 mg, 35%), mp: 59–60 ꢁC (lit.11 74–76 ꢁC, ligr-
oin). HPLC: tR 25.6 min; 1H NMR d (500 MHz,
CDCl3): 10.24 (s, 1 H), 8.71 (d, J = 8 Hz, 1 H), 7.75 (d,
J = 8 Hz, 1 H), 7.48 (dd, J = 8, 8 Hz, 1 H), 7.45 (dd,
J = 8, 8 Hz, 1 H); 13C NMR d (125 MHz, CDCl3):
186.1 (s), 138.9 (s), 135.7 (s), 133.7 (s), 131.5 (s), 126.7
(d), 126.4 (d), 124.1 (d), 121.4 (d); HREIMS m/z: mea-
sured 241.9225, calcd for C9H5BrOS: 241.9224; EI-MS
m/z (relative int): 242 ([M+2]+, 100), 240 (M+, 100),
1
mp: 28–29 ꢁC, (lit.13 32–33 ꢁC). HPLC: tR 14.1 min; H
NMR d (500 MHz, CDCl3): 8.94 (s, 1 H), 8.09 (d,
J = 8 Hz, 1 H), 7.99 (d, J = 8 Hz, 1 H), 7.56 (dd, J = 8,
8 Hz, 1 H), 7.47 (dd, J = 8, 8 Hz, 1 H); 13C NMR d
(125 MHz, CDCl3): 155.2 (d), 151.9 (s), 136.3 (s),
128.0 (d), 125.1 (d), 124.3 (d), 119.8 (d); HREIMS m/
z: measured 135.0148, calcd for C7H5NS: 135.0143;
EI-MS m/z (rel int): 135 (M+, 100). FTIR mmax (cmÀ1
)
3060, 1594, 1481, 1250, 1211, 884, 750, 570, 489. UV
kmax nm (MeOH, e): 204 (31,700), 223 (19,400), 254
(3500), 298 (4100).
4.5.3. 4-Arylisothiazoles 14–16, 21, 22. NH4SCN
(304 mg, 4 mmol) was added to a solution of chloroac-
roleins 33–35, 37, and 38 (1 mmol) in DMF (1.5 mL),
and the reaction mixtures were heated for 16 h at
70 ꢁC with stirring (caution: in hood, NaOH trap for
HCN). The reaction mixture was diluted with brine
(20 mL) and extracted with ethyl acetate. The combined
extract was dried, the solvent evaporated, and the resi-
due subjected to FCC (silica gel) as follows: 33, chloro-
form/hexane (1:4); 34, dichloromethane/hexane (1:1); 35,
dichloromethane/hexane (1:1); 37, acetone/hexane (1:8);
38, diethyl ether/hexane (1:5).
213 (16), 211 (15), 132 (40), 89 (29). FTIR mmax (cmÀ1
)
4.5.4. 4-(2-Nitrophenyl)isothiazole (17). SOCl2 (1.3 mL,
18 mmol) was added to a suspension of 2-nitrophenyl
malondialdehyde (579 mg, 3 mmol) in dichlorometh-
ane (15 mL), and the mixture was stirred for 1 h at
room temperature. The solvent was evaporated and
the residue was subjected to FCC (silica gel, 15 g,
dichloromethane/hexane, 2:1). Evaporation of the sol-
vent afforded unstable chloroacrolein 36 as a colorless
oil (616 mg, 97%).18 NH4SCN (886 mg, 11.6 mmol)
was added to compound 36 dissolved in DMF
(5 mL) and the reaction mixture was stirred for 16 h
at 70 ꢁC (caution: in hood, NaOH trap for HCN).
The reaction mixture was cooled to room temperature,
diluted with brine (70 mL), and extracted with EtOAc,
the combined organic extract was dried, and the sol-
vent evaporated. The residue was subjected to FCC
(silica gel, 50 g, hexane/acetone, 5:1) to afford 4-(2-
3039, 1672, 1458, 1422, 1388, 751.
NH4SCN (187 mg, 2.5 mmol) was added to a solution of
aldehyde 25 (148 mg, 0.61 mmol) in DMF (1 mL) and
the mixture was stirred for 4 h at 70 ꢁC (caution: in
hood, NaOH trap for HCN). After cooling to room
temperature, the reaction mixture was diluted with brine
(50 mL) and extracted with diethyl ether. The combined
organic extract was washed with brine (50 mL) and
dried, the solvent was evaporated, and the residue was
subjected to FCC (20 g silica gel, dichloromethane/hex-
ane, 1:1) to afford isothiazolo[5,4-b]thianaphthene (11)
as a slightly orange oil, which was crystallized using
dichloromethane/hexane solution. Yield: 72 mg (62%),
mp: 79–80 ꢁC (lit.9 82–84 ꢁC, benzene/hexane). HPLC:
tR 21.2 min; 1H NMR d (500 MHz, CDCl3): 8.94 (s,
1 H), 8.05 (d, J = 8 Hz, 1 H), 7.83 (d, J = 8 Hz, 1 H),
7.50 (dd, J = 8, 8 Hz, 1 H), 7.43 (dd, J = 8, 8 Hz, 1 H);
13C NMR d (125 MHz, CDCl3): 158.8 (s), 148.9 (d),
146.2 (s), 142.6 (s), 130.3 (s), 125.7 (d), 125.6 (d), 123.7
(d), 122.3 (d); HREIMS m/z: measured 190.9862, calcd
for C9H5NS2: 190.9863; EI-MS m/z (relative int): 191
(M+, 100), 159 (20), 120 (26). FTIR mmax (cmÀ1) 3055,
1495, 1392, 1351, 1261, 762, 499. UV kmax nm (MeOH,
e): 226 (42,800).
nitrophenyl)isothiazole (17) as
(222 mg, 37%).
a
yellow solid
4.5.5. 4-(2-Aminophenyl)isothiazole (18) and 4-(2-
hydroxyphenyl)isothiazole (19). Compound 1816 was ob-
tained 71% yield, compound 1916 in 44% yield, based
on 4-(2-nitrophenyl)isothiazole (17). No spectroscopic
data was available, thus compounds were fully
characterized.

M. Soledade et al. / Bioorg. Med. Chem. 14 (2006) 714–723
721
4.5.6. 4-(2-Methoxyphenyl)isothiazole (20).19 4-(2-
Hydroxyphenyl)isothiazole (19, 44 mg, 0.25 mmol) was
dissolved in ethereal diazomethane solution (5 mL)
and the mixture was stirred for 8 h at room temperature.
The excess diazomethane was quenched with acetic acid,
the solvent was evaporated, and the residue was subject-
ed to FCC (silica gel, 10 g, hexane/acetone, 5:1) to afford
4-(2-methoxyphenyl)isothiazole (20) as a colorless oil
1 Hz, 1H); 13C NMR d (125 MHz, CDCl3): 170.5
(s), 154.6 (d), 148.9 (s), 133.1 (d), 132.3 (d), 129.5
(d), 129.0 (d), 127.3 (s), 126.4 (d), 125.2 (d); HREIMS
m/z: measured 186.0250, calculated for C10H6N2S:
186.0252; EI-MS m/z (rel int): 186 (M+, 100), 153
(14), 142 (11). FTIR mmax (cmÀ1) 3046, 1619, 1598,
1548, 1321, 1131, 927, 755. UV kmax nm (MeOH, e):
222 (16,100), 251 (59,200), 309 (4000), 323 (4900),
348 (2700).
(40 mg, 83%) HPLC: tR 18.6 min; 1H NMR
d
(500 MHz, CDCl3): 8.87 (s, 1 H), 8.86 (s, 1 H), 7.53 (d,
J = 7.5 Hz, 1 H), 7.36 (dd, J = 7.5, 7.5 Hz, 1 H), 7.04
(m, 2 H), 3.91 (s, 3H); 13C NMR d (125 MHz, CDCl3):
158.1 (d), 156.6 (s), 144.9 (d), 136.0 (s), 129.8 (d),
129.5 (d), 122.0 (s), 121.2 (d), 111.7 (d), 55.8 (q); HRE-
IMS m/z: measured 191.0407, calcd for C10H9NOS:
191.0405; EI-MS m/z (relative int): 191 (M+, 100), 176
4.5.9. Thieno[2,3-b]indole (12).8 Yield 29%, based on N-
1
Boc-2-chloroindole-3-carboxaldehyde.20 For H NMR
13
and C NMR.21 HREIMS m/z: measured 173.0297,
calcd for C10H7NS: 173.0299; EI-MS m/z (rel int): 173
(M+, 100), 100 (18). FTIR mmax (cmÀ1) 3393, 1437,
1379, 745, 706, 494. UV kmax nm (MeOH, e): 231
(47,100), 266 (8800).
(30), 148 (12), 121 (20), 77 (12). FTIR mmax (cmÀ1
)
2934, 1589, 1463, 1337, 1245, 1120, 1025, 751. UV kmax
nm (MeOH, e): 204 (45,800), 286 (10,400).
4.5.10. 4-Phenylisothiazole (14). Yield 54%, slightly yel-
low solid, mp: 32–33 ꢁC, (lit.22 35–36 ꢁC; 1it.23 33–
37 ꢁC). HPLC: tR 17.6 min; 1H NMR d (500 MHz,
CDCl3): 8.80 (s, 1H), 8.73 (s, 1H), 7.63 (dd, J = 7.5,
7.5 Hz, 2H), 7.47 (dd, J = 7.5, 7.5 Hz, 2H), 7.38 (dd,
J = 7.5, 7.5 Hz, 1H); 13C NMR d (125 MHz, CDCl3):
156.3 (d), 142.8 (d), 140.3 (s), 132.8 (s), 129.3 (d,
2 · C), 128.2 (d), 127.1 (d, 2· C). HREIMS m/z: mea-
sured 161.0303, calcd for C9H7NS: 161.0299; EI-MS
m/z (rel int): 161 (M+, 100), 134 (25). FTIR mmax
(cmÀ1) 3033, 1603, 1487, 1350, 1236, 860, 754. UV kmax
nm (MeOH, e): 203 (33,900), 243 (7300), 269 (8800).
4.5.7. 2-Aryl-3-chloroacroleins 33–35, 37, and 38. DMF
(0.7 mL) was added dropwise to POCl3 (0.7 mL,
7.5 mmol) and cooled to 0 ꢁC. Aryl acetic acids (27–
29, 31, and 32, 2.5 mmol) were then added and the
mixture was stirred at 85 ꢁC for 90 min (acid 31), 2 h
(acids 27–29) or 2.5 h (acid 32). The reaction mixtures
were cooled to room temperature and cracked ice was
added to adjust the volume to ca. 30 mL. The reaction
mixtures were extracted with dichloromethane, the
combined extracts were dried, and the solvents were evap-
orated to leave crude 2-phenyl-3-N,N-dimethylaminoac-
roleins as yellow oils. NaOH (25% aq soln, 5 mL) was
added to crude 2-phenyl-3-N,N-dimethylaminoacroleins
in EtOH (3.8 mL) and the reaction mixtures were refluxed
with stirring for 30 min (acids 28, 32), 45 min (acid 31) or
1 h (acids 27, 29). Ethanol was removed in vacuum, the
residue was diluted to ca. 30 mL by addition of cracked
ice and made acidic (pH < 3) using aqueous HCl (1:1).
The resulting mixtures were extracted with diethyl ether,
the combined extracts were dried, and the solvents were
evaporated to leave crude enolized 2-aryl malondialdehy-
des as yellow oils. SOCl2 (3.5 mL, 48 mmol) was added to
crude 2-aryl malondialdehydes in CH2Cl2 (5 mL) and
cooled to 0 ꢁC and the reaction mixtures were stirred at
0 ꢁC for 10 min (acid 29), 20 min (acids 28, 31) or 1 h
(acids 27, 32). The solvents were evaporated and the res-
idues were subjected to FCC (silica gel, 20 g, dichloro-
methane/hexane, 1:1, 33–35, 38; or silica gel 50 g,
dichloromethane/hexane, 2:1, 37). 2-Aryl-3-chloroac-
roleins18 (33, 310 mg, 75%; 34 267 mg, 53%; 35, 106 mg,
23%; 37, 187 mg, 35%; 38, 475 mg, 88%) were immediate-
ly used in the next step. All yields are based on starting
aryl acetic acids.
4.5.11. 4-(2-Chlorophenyl)isothiazole (15).19 Yield 30%,
slightly yellow oil. HPLC: tR 25.5 min; 1H NMR d
(500 MHz, CDCl3): 8.81 (s, 1H), 8.72 (s, 1H), 7.52 (m,
1H), 7.45 (m, 1H), 7.35 (m, 2H); 13C NMR d
(125 MHz, CDCl3): 158.0 (d), 146.5 (d), 136.9 (s),
132.9 (s), 132.0 (s), 131.2 (d), 130.6 (d), 129.5 (d),
127.3 (d); HREIMS (m/z: measured 194.9914, calcd for
C9H6ClNS: 194.9909); EI-MS m/z (rel int): 197 ([M+2]+,
37), 195 (M+, 100), 168 (24), 133 (11), 89 (10). FTIR
mmax (cmÀ1) 3064, 1691, 1531, 1470, 1228, 1074, 1038,
753. UV kmax nm (MeOH, e): 203 (50,000), 261 (7700).
4.5.12. 4-(2-Tolyl)isothiazole (16).19 Yield 25%, slightly
1
yellow oil. HPLC: tR 21.9 min; H NMR d (500 MHz,
CDCl3): 8.58 (s, 1H), 8.56 (s, 1H), 7.32 (m, 3H), 7.29
(m, 1H), 2.35 (s, 3H); 13C NMR d (125 MHz, CDCl3):
158.1 (d), 145.2 (d), 139.5 (s), 136.1 (s), 132.9 (s), 130.9
(d), 130.0 (d), 128.4 (d), 126.3 (d), 21.1 (q); HREIMS
m/z: measured 175.0459, calcd for C10H9NS: 175.0456;
MS-EI m/z (rel int): 175 (M+, 29), 149 (100), 105 (40),
57 (27). FTIR mmax (cmÀ1) 3062, 1602, 1531, 1480,
1227, 1117, 914, 755. UV kmax nm (MeOH, e): 202
(29,500), 260 (5100).
4.5.8. Isothiazolo[5,4-b]quinoline (10). This compound
was prepared following a literature procedure.6,7 The
overall process afforded a slightly yellow solid, iso-
thiazolo[5,4-b]quinoline (10, 80 mg, 15% yield based
on quinoline). Mp: 168–170 ꢁC, acetone/hexane (lit.7
169–170 ꢁC, ethyl acetate). HPLC: tR 13.6 min; 1H
NMR d (500 MHz, CDCl3): 9.13 (s, 1H), 8.90 (s,
1H), 8.21 (d, J = 8 Hz, 1H), 8.07 (d, J = 8 Hz, 1H),
7.91 (ddd, J = 8, 8, 1 Hz, 1H), 7.64 (ddd, J = 8, 8,
4.5.13. 4-(2-Nitrophenyl)isothiazole (17). Mp: 43–45 ꢁC
(lit.16 59.5–62.5 ꢁC, benzene/cyclohexane, containing
10% of 4-(4-nitrophenyl)isothiazole). HPLC: tR 15.9 min;
1H NMR d (500 MHz, CDCl3): 8.65 (s, 1H), 8.53 (s,
1H), 7.95 (dd, J = 8, 1 Hz, 1H), 7.67 (ddd, J = 8, 8,
1 Hz, 1H), 7.57 (ddd, J = 8, 8, 1 Hz, 1H), 7.48 (dd,
J = 8, 1 Hz, 1H); 13C NMR d (125 MHz, CDCl3):
156.9 (d), 149.2 (s), 146.2 (d), 134.7 (s), 132.9 (d),

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M. Soledade et al. / Bioorg. Med. Chem. 14 (2006) 714–723
132.2 (d), 129.4 (d), 127.7 (s), 124.7 (d); HREIMS
(m/z: measured 206.0144, calcd for C9H6N2O2 S:
206.0150); EI-MS m/z (rel int): 206 (M+, 100), 159
(21), 133 (17), 119 (25), 89 (44), 63 (16). FTIR mmax
(cmÀ1) 2916, 1609, 1525, 1348, 849, 783, 745. UV kmax
nm (MeOH, e): 202 (16,500), 253 (8900).
4.5.19. 2-(2-Chlorophenyl)-3-chloroacrolein (34). HPLC:
tR 22.9 min; 1H NMR d (500 MHz, CDCl3): 9.68 (s,
1H), 7.53 (s, 1H), 7.50 (d, J = 7 Hz, 1H), 7.37 (m, 2H),
7.18 (d, J = 7 Hz, 1H); 13C NMR d (125 MHz, CDCl3):
188.6 (d), 144.3 (s), 144.1 (d), 133.7 (s), 131.4 (d), 130.8
(d), 130.3 (s), 130.2 (d), 127.3 (d).
4.5.14. 4-(2-Aminophenyl)isothiazole (18). Mp: 46–48 ꢁC.
HPLC: tR 10.6 min; 1H NMR d (500 MHz, CDCl3):
8.74 (s, 1H), 8.69 (s, 1H), 7.21 (m, 2H), 6.85 (m, 2H),
3.85 (br s, 2H, D2O exch.); 13C NMR d (125 MHz,
CDCl3): 158.1 (d), 145.0 (d), 144.2 (s), 137.2 (s), 130.5
(d), 129.6 (d), 119.2 (d), 118.9 (s), 116.3 (d); HREIMS
m/z: measured 176.0406, calcd for C9H8N2S: 176.0408;
EI-MS m/z (rel int): 176 (M+, 100), 159 (10), 143 (42),
117 (26). FTIR mmax (cmÀ1) 3346, 3084, 1618, 1489,
1299, 750, 654. UV kmax nm (MeOH, e): 209 (25,200),
244 (10,700), 302 (2300).
4.5.20. 2-(2-Tolyl)-3-chloroacrolein (35). HPLC: tR
19.3 min; H NMR d (500 MHz, CDCl3): 9.70 (s, 1H),
7.48 (s, 1H), 7.29 (m, 3H), 7.05 (d, J = 7 Hz, 1H), 2.17
(s, 3H); 13C NMR d (125 MHz, CDCl3): 189.8 (d),
146.5 (s), 143.9 (d), 136.8 (s), 130.7 (d), 130.6 (s), 129.6
(d), 129.5 (d), 126.3 (d), 19.9 (q).
1
4.5.21. 2-(2-Nitrophenyl)-3-chloroacrolein (36). HPLC:
tR 15.3 min; 1H NMR d (500 MHz, CDCl3): 9.66 (s,
1H), 8.25 (d, J = 7 Hz, 1H), 7.75 (dd, J = 7, 7 Hz, 1H),
7.64 (dd, J = 7, 7 Hz, 1H), 7.50 (s, 1H), 7.35 (d,
J = 7 Hz, 1H); 13C NMR d (125 MHz, CDCl3): 188.2
(d), 148.4 (s), 144.5 (s), 141.9 (d), 134.2 (d), 132.4 (d),
130.6 (d), 126.5 (s), 125.5 (d).
4.5.15. 4-(2-Hydroxyphenyl)isothiazole (19). Mp: 138–
140 ꢁC (lit.16 136–138 ꢁC, methanol). HPLC: tR
1
11.0 min; H NMR d (500 MHz, CDCl3): 8.96 (s, 1H),
8.92 (s, 1H), 7.50 (dd, J = 7.5, 1.5 Hz, 1H), 7.25 (ddd,
J = 7.5, 7.5, 1.5 Hz, 1H), 7.00 (m, 2H), 6.84 (br s, 1H,
D2O exch.); 13C NMR d (125 MHz, CDCl3): 157.9 (d),
153.4 (s), 145.1 (d), 135.8 (s), 129.9 (d), 129.6 (d),
121.2 (d), 119.9 (s), 116.7 (d); HREIMS m/z: measured
177.0245, calcd for C9H7NOS: 177.0248; EI-MS m/z
4.5.22. 2-(1-Naphthyl)-3-chloroacrolein (37). HPLC: tR
22.1 min; H NMR d (500 MHz, CDCl3): 9.85 (s, 1H),
7.94 (m, 2H), 7.71 (s, 1H), 7.52 (m, 4H), 7.32 (d,
J = 7 Hz, 1H); 13C NMR d (125 MHz, CDCl3): 189.9
(d), 145. 2 (s), 144.5 (d), 134.0 (s), 131.0 (s), 129.9 (d),
129.1 (d), 128.7 (s), 127.9 (d), 127.0 (d), 126.6 (d),
125.7 (d), 125.0 (d).
1
(rel int): 177 (M+, 100), 118 (68). FTIR mmax (cmÀ1
)
3131, 1601, 1452, 1337, 1283, 751. UV kmax nm (MeOH,
e): 204 (29,000), 293 (5600).
4.5.23. 2-(2-Naphthyl)-3-chloroacrolein (38). HPLC: tR
1
23.8 min; H NMR d (500 MHz, CDCl3): 9.75 (s, 1H),
4.5.16. 4-(1-Naphthyl)isothiazole (21). Yield 34%, slightly
yellow solid, mp: 43–45 ꢁC. HPLC: tR 25.2 min; 1H NMR
d (500 MHz, CDCl3): 8.75 (s, 1H), 8.72 (s, 1H), 7.94 (m,
3H), 7.52 (m, 4H); 13C NMR d (125 MHz, CDCl3):
158.7 (d), 146.2 (d), 138.5 (s), 134.0 (s), 131.9 (s), 131.1
(s), 128.9 (d), 128.7 (d), 127.7 (d), 126.9 (d), 126.4 (d),
125.6 (d), 125.3 (d); HREIMS m/z: measured 211.0459,
calcd for C13H9NS: 211.0456; EI-MS m/z (rel int): 211
(M+, 100), 184 (13), 152 (33), 139 (13). FTIR mmax
(cmÀ1) 3058, 1592, 1506, 1397, 1021, 909, 776, 661. UV
7.91 (m, 4H), 7.55 (m, 2H), 7.43 (m, 2H); 13C NMR d
(125 MHz, CDCl3): 190.2 (d), 144. 9 (s), 143.3 (d),
133.7 (s), 133.4 (s), 129.7 (d), 128.8 (d), 128.4 (d),
128.2 (d), 127.7 (s), 127.3 (d), 126.9 (d), 126.8 (d).
Acknowledgments
Support for the authorsꢁ work was obtained in part from
the Natural Sciences and Engineering Research Council
of Canada (Discovery Grant to MSCP), NATO (fellow-
ship to MS) and the University of Saskatchewan. We
k
max nm(MeOH, e):204(48,400), 223(56,200), 290(9500).
´
4.5.17. 4-(2-Naphthyl)isothiazole (22). Yield 37%, slight-
ly yellow solid, mp: 72–73 ꢁC. HPLC: tR 25.5 min; H
would like to thank G. Seguin-Swartz, IBCN curator,
1
Agriculture and Agri-Food Canada Research Station,
Saskatoon SK, for kindly providing isolates of L. macu-
lans, and K. Brown and K. Thoms, Department of
Chemistry, University of Saskatchewan, for technical
assistance obtaining NMR and MS data, respectively.
NMR d (500 MHz, CDCl3): 8.94 (s, 1H), 8.83 (s,
1H), 8.08 (s, 1H), 7.91 (m, 3H), 7.72 (dd, J = 8.5,
1.5 Hz, 1H), 7.54 (m, 2H); 13C NMR d (125 MHz,
CDCl3): 156.4 (d), 143.0 (d), 140.1 (s), 133.8 (s),
133.0 (s), 130.1 (s), 129.1 (d), 128.3 (d), 128.0 (d),
126.9 (d), 126.6 (d), 125.8 (d), 125.2 (d); HREIMS m/
z: measured 211.0450, calcd for C13H9NS: 211.0456;
EI-MS m/z (rel int): 211 (M+, 100), 184 (12), 152
(12), 139 (11). FTIR mmax (cmÀ1) 3065, 1599, 1506,
1236, 863, 823, 749. UV kmax nm (MeOH, e): 204
(49,600), 241 (41,300), 267 (16,900).
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.bmc.
2005.08.053.
4.5.18. 2-Phenyl-3-chloroacrolein (33). HPLC: tR
1
17.6 min; H NMR d (500 MHz, CDCl3): 9.68 (s, 1H),
References and notes
7.45 (m, 3H), 7.36 (m, 3H); 13C NMR d (125 MHz,
CDCl3): 189.8 (d), 144.6 (s), 142.7 (d), 130.0 (s), 129.5
(2· d), 129.1 (d), 128.5 (2· d).
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