Toward the control of Leptosphaeria maculans: Design, syntheses, biological activity, and metabolism of potential detoxification inhibitors of the crucifer phytoalexin brassinin

Article abstract of DOI:10.1016/j.bmc.2006.03.014

Brassinin (1), a crucial plant defense produced by crucifers, is detoxified by the phytopathogenic fungus Leptosphaeria maculans (Phoma lingam) to indole-3-carboxaldehyde using a putative brassinin oxidase. Potential inhibitors of brassinin detoxification

Full text of DOI:10.1016/j.bmc.2006.03.014

Bioorganic & Medicinal Chemistry 14 (2006) 4958–4979
Toward the control of Leptosphaeria maculans: Design, syntheses,
biological activity, and metabolism of potential detoxification
inhibitors of the crucifer phytoalexin brassinin
M. Soledade C. Pedras* and Mukund Jha
Department of Chemistry, University of Saskatchewan, 110 Science Place, Saskatoon, Canada SK S7N 5C9
Received 16 February 2006; revised 6 March 2006; accepted 7 March 2006
Available online 17 April 2006
Abstract—Brassinin (1), a crucial plant defense produced by crucifers, is detoxified by the phytopathogenic fungus Leptosphaeria
maculans (Phoma lingam) to indole-3-carboxaldehyde using a putative brassinin oxidase. Potential inhibitors of brassinin detoxifica-
tion were designed by replacement of its dithiocarbamate group (toxophore) with carbamate, dithiocarbonate, urea, thiourea, sulfa-
mide, sulfonamide, dithiocarbazate, amide, and ester functional groups. In addition, the indolyl moiety was substituted for
naphthalenyl and phenyl. The syntheses and chemical characterization of these potential detoxification inhibitors, along with their anti-
fungal and cytotoxic activity, as well as screening using cultures of L. maculans are reported. Overall, three types of interaction were
observed in cultures of L. maculans co-incubated with the potential inhibitors and brassinin: (1) a decrease on the rate of brassinin
detoxification due to the strong inhibitory activity of the compound on fungal growth, (2) a decrease on the rate of brassinin detoxi-
fication due to the inhibitory activity of the compound on the putative brassinin oxidase, and (3) a low to no detectable effect on the rate
of brassinin detoxification. A noticeable decrease in the rate of brassinin detoxification was observed in the presence of N0-methylbrass-
inin, methyl N-methyl-N-(naphthalen-2-ylmethyl) dithiocarbamate, tryptophol dithiocarbonate, and methyl 3-phenyldithiocarbazate.
Tryptophol dithiocarbonate appeared to be the best inhibitor among the designed compounds, representing the first inhibitor of brass-
inin detoxification and potentially the first selective protecting agent of oilseed crucifers against L. maculans infestation.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
chemical defenses.3 These detoxification reactions are
catalyzed by detoxifying enzymes, which are likely to
have evolved during the multiple life cycles of patho-
gens. Such phytoalexin detoxifying enzymes can be of
sufficient importance to determine virulence in a fungal
pathogen.3
Phytoalexins are low molecular weight secondary
metabolites produced de novo by plants in response to
various types of stress, including microbial attack.1
The crucifer phytoalexin brassinin (1) is of great interest
in the interaction of crucifers with their fungal patho-
gens due to both its biological activity and intermediacy
in the biosynthetic pathway of other relevant phytoalex-
ins (e.g., cyclobrassinin (2), brassilexin (3), rutalexin (4),
brassicanal A (5), and brassicanate A (6)).2 Crucifers are
cultivated around the world and comprise an extremely
valuable group of oilseeds and vegetables. Pathogens of
crucifers, similar to many plant pathogens, can metabo-
lize phytoalexins to less toxic compounds, a detrimental
process that deprives the plant of important induced
NH
S
N
SCH
3
SCH
3
S
N
H
N
H
1
2
O
NCH
3
N
S
O
S
N
H
N
H
3
4
COOCH
3
CHO
SCH
Keywords: Brassinin; Leptosphaeria maculans; Phytoalexins; Phoma
lingam; Dithiocarbamate; Brassica; Detoxifying enzyme; Brassinin
oxidase; Antifungal.
SCH
3
3
N
N
H
H
*
Corresponding author. Tel.: +1 306 966 4772; fax: +1 306 966
4730; e-mail: soledade.pedras@usask.ca
6
5
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.03.014

M. S. C Pedras, M. Jha / Bioorg. Med. Chem. 14 (2006) 4958–4979
4959
detoxification of phytoalexins,3 the inhibition of such en-
zymes may be a suitable strategy to control L. maculans.2
That is, enhancement of plant self-defenses through the
use of selective detoxification inhibitors, that is, paldox-
ins,2b could lead to a selective and environmentally safer
control of L. maculans. Selective inhibitors are less likely
to affect non-targeted organisms and thus are expected to
have lower impact on the cultivated ecosystem, that is, be
environmentally ‘neutral.’ Toward this end, we report
the design, syntheses, biological activity, screening,
and evaluation of potential brassinin detoxification
inhibitors.
The blackleg fungus [Leptosphaeria maculans (Desm.)
Ces. et de Not., asexual stage Phoma lingam (Tode ex
Fr) Desm.], an important pathogen of crucifer oilseeds
(rapeseed, canola, and mustard), can detoxify several
phytoalexins including brassinin (1) (Scheme 1),4 cyclo-
brassinin (2), brassilexin (3), and brassicanal A (5) via
different pathways.3 Considering that brassinin (1) is a
biosynthetic precursor of many other phytoalexins, this
metabolic detoxification can make plants particularly
vulnerable to further pathogen colonization. Because
fungal pathogens appear to use specific enzymes in the
NH
S
NH
O
ii
SCH
3
S
2. Results
SCH
3
N
H
1
N
H
The chemical structure of brassinin (1) contains a
dithiocarbamate group (toxophore, substructure A)
and an indole nucleus (substructure B) linked by a
methylene bridge located at the C-3 of indole (Fig. 1).
Brassinin (1) detoxification in mycelial cultures of L.
maculans involves the formation of brassinin sulfoxide
(7) and indole-3-carboxaldehyde (8) (Scheme 1) which
is oxidized slowly to indole-3-carboxylic acid.3 Howev-
er, it is not understood whether sulfoxide 7 is an inter-
mediate in the detoxification pathway, since it was not
detected in cell-free extracts containing brassinin
7
i
CHO
N
H
8
Scheme 1. Detoxification of brassinin (1) in the phytopathogenic
fungus Leptosphaeria maculans; (i) in mycelial cultures or cell-free
extracts (brassinin oxidase);4 (ii) in mycelial cultures.3
2'
NH
1'
4
5
6
3
1
SCH
3
3'
S
2
A
7
N
H
B
1
2'
NR
Ar
2'
2'
1'
1'
2'
SMe
1'
NR
NR
3'
NR
S
O
NHR'
NHR'
Ar
Ar
S
Ar
3'
1 Ar=3-InCH ; R=H
9 Ar=3-InCH ; R=Me
10 Ar=2-NpCH ; R=H
3'
S
2
O
O
Y
2
47 Ar=3-In; R=R'=H
22 Ar=3-In; R=R'=H
61 Ar=3-In; R=H; Y=NH
2
2
48 Ar=3-In; R=H; R'=Me
49 Ar=3-In; R=H; R'=Pr
50 Ar=3-In; R=H; R'=Ph,
51 Ar=3-In; R'=Me; R'=Pr
52 Ar=3-In; R'=Me; R'=Ph
53 Ar=2-Np; R=H; R'=Pr
54 Ar=2-Np; R=H; R'=Ph
55 Ar=2-Np; R=Me; R'=Pr
56 Ar=2-Np; R=Me; R'=Ph
57 Ar=1-Np; R=H; R'=Pr
58 Ar=1-Np; R=H; R'=Ph
59 Ar=1-Np; R=Me; R'=Pr
60 Ar=1-Np; R=Me; R'=Ph
11 Ar=2-NpCH ; R=Me 23 Ar=3-In; R=H; R'=Me
62 Ar=3-In; R=Me; Y=NH
2
2
24 Ar=3-In; R=H; R'=Et
25 Ar=3-In; R=H; R'=Pr
26 Ar=3-In; R=H; R'=Ph
27 Ar=3-In; R=Me; R'=H
28 Ar=3-In; R=Me; R'=Et
29 Ar=3-In; R=Me; R'=Pr
30 Ar=3-In; R=Me; R'=Ph
31 Ar=2-Np; R=R'=H
32 Ar=2-Np; R=H; R'=Et
33 Ar=2-Np; R=H; R'=Pr
34 Ar=2-Np; R=H; R'=Ph
35 Ar=2-Np; R=Me; R'=H
36 Ar=2-Np; R=Me; R'=Et
37 Ar=2-Np; R=Me; R'=Pr
38 Ar=2-Np; R=Me; R'=Ph
39 Ar=1-Np; R=R'=H
40 Ar=1-Np; R=H; R'=Et
41 Ar=1-Np; R=H; R'=Pr
42 Ar=1-Np; R=H; R'=Ph
43 Ar=1-Np; R=Me; R'=H
44 Ar=1-Np; R=Me; R'=Et
45 Ar=1-Np; R=Me; R'=Pr
46 Ar=1-Np; R=Me; R'=Ph
12 Ar=1-NpCH ; R=H
63 Ar=2-Np; R=H; Y=NH
2
2
13 Ar=1-NpCH ; R=Me
64 Ar=2-Np; R=Me; Y=NH
2
2
2
14 Ar=PhCH ; R=H
15 Ar=Ph; R=H
65 Ar=1-Np; R=H; Y=NH
2
2
66 Ar=1-Np; R=Me; Y=NH
67 Ar=3-In; R=H; Y=Me
68 Ar=3-In; R=Y=Me
69 Ar=2-Np; R=H; Y=Me
70 Ar=2-Np; R=Y=Me
71 Ar=1-Np; R=H; Y=Me
72 Ar=1-Np; R=Y=Me
2'
1'
O
SMe
Ar
3'
S
16 Ar=3-In
17 Ar=CH -3-In
2
2'
1'
2'
1'
NR
3'
XR
Ar
OMe
Ar
O
4
3
3'
O
5
73 Ar=3-In; XR=OCH
3
3-In =
18 Ar=3-In; R=H
19 Ar=3-In; R=Me
1
74 Ar=3-In; XR=NH
2
6
N
H
75 Ar=3-In; XR=NHCH
3
7
2'
Y
1'
X
1
SMe
1
2
2
Ar
S
2-Np =
1-Np =
3
3
20 Ar=Ph; X=Y=NH
4
21 Ar=Ph; X=CH NH; Y=NH
4
2
Figure 1. Potential inhibitors 9–75 of brassinin (1) detoxification.

4960
M.S.C Pedras, M. Jha / Bioorg. Med. Chem. 14 (2006) 4958–4979
oxidase activity.4 Notwithstanding the details of the
detoxification mechanism, it is clear that enzymatic
detoxification of brassinin (1) by a putative brassinin
oxidase4 takes place only on the side chain of brassinin
(1), while the indolyl component remains unaffected.
For this reason, to design selective inhibitors of brassi-
nin detoxification, the side chain of brassinin (1) was of
crucial importance. Alternatively, replacement of the
indolyl moiety with naphthalenyl (e.g., 10–13) or phenyl
(e.g., 14 and 15) would lead to structures in which the
contribution of the indolyl part (more polar) of brassi-
nin (1) to metabolism and toxicity could be directly
evaluated. In the case of naphthalene derivatives, both
C-1 and C-2 were chosen as points of substitution be-
cause previous work showed that a fungal pathogen
transformed these compounds differently.5 In addition,
since dithiocarbamates have strong biological activity,6
the design of compounds with a wider range of bioactiv-
ities was planned as well.
ethylamine (76), N0-methylindol-3-ylmethylamine (77),
naphthalen-2-ylmethylamine (78), N-methylnaphthalen-
2-ylmethylamine (79), naphthalen-1-ylmethylamine
(80), and N-methylnaphthalen-1-ylmethylamine (81)
were used in the syntheses of most of the designed com-
pounds. These key starting materials were synthesized
in good yields using known methodology.20–22
2.1.1. Syntheses of dithiocarbamates, dithiocarbonates,
carbamates, dithiocarbazates, ureas, and thioureas (1,
9–15, and 17–60). The syntheses of brassinin (1),2 N0-
methylbrassinin (9), and dithiocarbamates 10–155 were
carried out as reported earlier for brassinin by first
allowing the respective amines 76–83 to react with CS2
in pyridine and triethylamine followed by quenching
with MeI and acidic work-up (H2SO4). As expected, at-
tempts to transform indol-3-ylmethanol (86) to methyl
indol-3-ylmethyldithiocarbonate (16) under experimen-
tal conditions similar to those used for dithiocarbamates
were not successful as indol-3-ylmethanol (86) was
found to be stable under those conditions. When trieth-
ylamine was replaced with a stronger base such as NaH
for deprotonation of –(O)–H,23 followed by addition of
CS2, thioether 87 (30% yield) along with recovered start-
ing material (86, 40% yield) was obtained. Heating 3-
indolylmethanol (86) to 70 ꢁC in the presence of NaH
in THF resulted in extensive decomposition of the start-
ing material. Using a reagent such as 1-(methyldithio-
carbonyl)imidazole,24 a reagent developed for the mild
conversion of alcohols to S–Me dithiocarbonates, also
failed to give the desired compound 16 and instead
yielded thioether 87 (60% yield). By contrast, deproto-
nation of tryptophol (88), the CH2 homolog of 3-indol-
ylmethanol (86), with NaH in THF at 0 ꢁC followed by
addition of CS2 and quenching with MeI afforded the
corresponding dithiocarbonate 17 (15 min) in excellent
yield (95% yield). Methyl 3-phenyldithiocarbazate (20)
and methyl 3-benzyldithiocarbazate (21) were synthe-
sized from phenylhydrazine and benzylhydrazine,
respectively (Scheme 2).25
In most cases, isosteric replacement7 of the heteroa-
toms of the dithiocarbamate group of brassinin (1)
led to incorporation of carbamate, urea, thiourea, sul-
famide, sulfonamide, dithiocarbazate, amide, and ester
functional groups into the structure of potential inhib-
itors (16–75). Besides the potential role as detoxifica-
tion inhibitors, this wide range of functional groups
was designed to provide further insights into the
detoxification mechanism of brassinin as well as struc-
ture–activity correlations. Accordingly, replacement of
both sulfur atoms of brassinin (1) with oxygen atoms
afforded carbamates 18 and 19, whereas replacement
of the thiocarbonyl with carbonyl and the SCH3 with
NH2 or NHR (R = alkyl or phenyl) gave rise to
unsymmetrical urea derivatives 22–46 containing indo-
lyl or naphthalenyl (less polar) moieties. Similarly,
keeping the thiocarbonyl intact and replacing the
SCH3 group with NH2 or NHR (R = alkyl or phenyl)
resulted in unsymmetrical thioureas 47–60, whereas
dithiocarbazates 20 and 21 could be obtained by
replacing the (H)N-20 with (H)N–NH (hydrazine).
Sulfamides 61–66 and sulfonamides 67–72 were ob-
tained by replacing the thiocarbonyl group with a sul-
fone moiety and the SCH3 with NH2, NHCH3
(sulfamides), and CH3 (sulfonamides) containing indo-
lyl or naphthalenyl moieties as well. Amides 74 and 75
and ester 73 were obtained by replacing the (H)N-20
of brassinin (1) with CH2, the thiocarbonyl with a car-
bonyl, and SCH3 with either NH2/NHCH3 (amide) or
OCH3 (ester), respectively. Overall, it was anticipated
that some of these new compounds (Fig. 1) would
hinder brassinin (1) detoxification by inhibiting the
putative brassinin oxidase.
The syntheses of N-(indol-3-yl)methylurea (22),13 N-
indol-3-ylmethyl-N0-phenylurea (26),14 and N-(naphtha-
len-1-ylmethyl)-N0-phenylurea (44)26 were carried out as
previously described. Although urea 22 was prepared
quantitatively from gramine (89) and excess urea, a sim-
ilar attempt to make N-indol-3-ylmethyl-N0-methylurea
(23) by reacting gramine (89) and methylurea was unsuc-
cessful. Compound 23 could be obtained in good yield
by first reacting amine 76 with COCl2 to yield isocyanate
90, which was then reacted with CH3NH2 to give urea
23 (Scheme 3). Other unsymmetrical ureas (24–46) were
prepared in good yields by the reaction of primary or
secondary amines with alkyl isocyanates (Scheme 3).27
Similar to ureas 24–46, most of the unsymmetrical
thioureas 49–60 (Scheme 3) were synthesized by reacting
the corresponding primary or secondary amine with an
isothiocyanate (RNCS, R = Pr or Ph, Scheme 3).27
Methyl N-(indol-3-ylmethyl)-N-methylcarbamate (19)
was obtained in 85% yield by treatment of N-(indol-3-yl-
methyl)-N-methylamine (77) with ethylchloroformate
in the presence of N,O-bis(trimethylsilyl)acetamide
(Scheme 3).10
2.1. Syntheses of potential inhibitors and metabolism
Among the designed potential inhibitors 9–75,
compounds 9,8 10,5 12,5 14,9 15,9 18,10 20,11 21,12 22,13
26,14 48,15 72,16 73,17 74,18 and 7519 were reported previ-
ously. Of the designed sulfonamides 67–72, N-methyl-
N-(naphthalen-1-ylmethyl)methanesulfonamide (72) has
been used to study aldose reductase inhibitors, but its syn-
thesis was not described.16 Amines such as indol-3-ylm-

M. S. C Pedras, M. Jha / Bioorg. Med. Chem. 14 (2006) 4958–4979
4961
Ar
NHR
S
H
N
SR'
N
C X
i
Ar
NHR
NHR
i; ii
X
76 Ar=3-InCH ; R=H
1 Ar=3-InCH ; R=H
9 Ar=3-InCH ; R=Me
10 Ar=2-NpCH ; R=H
11 Ar=2-NpCH ; R=Me
12 Ar=1-NpCH ; R=H
13 Ar=1-NpCH ; R=Me
2
2
N
H
N
R
77 Ar=3-InCH ; R=Me
2
2
78 Ar=2-NpCH ; R=H
2
2
23 R=CH , X=O
47 R=H, X=S
48 R=CH , X=S
90 R=H; X=O
91 R=Boc; X=S
3
79 Ar=2-NpCH ; R=Me
2
2
80 Ar=1-NpCH ; R=H
2
2
3
81 Ar=1-NpCH ; R=Me
2
2
82 Ar=PhCH ; R=H
83 Ar=Ph; R=H
84 Ar=Ph-NH; R=H
14 Ar=PhCH ; R=H
15 Ar=Ph; R=H
20 Ar=PhNH; R=H
2
2
Scheme 4. Syntheses of urea 23 and thioureas 47 and 48. Reagents:
(i) RNH2 (R = H, Me), CH2Cl2; (ii) NaOMe, MeOH.
85 Ar=PhCH NH; R=H
21 Ar=PhCH NH; R=H
2
2
CH
2
O
ii
Ar
CH R
2
R
NH
R
N
N
2
Ar
i
SCH
NHR
ii
3
S
Me
S
Ar
86 Ar=3-In; R=OH
87 Ar=3-In; R=SCH
S
O
O
Ar
Ar
17 Ar=3-In
O
O
3
88 Ar=3-InCH ; R=OH
61 Ar=3-In; R=H
62 Ar=3-In; R=Me
63 Ar=2-Np; R=H
64 Ar=2-Np; R=Me
65 Ar=1-Np; R=H
66 Ar=1-Np; R=Me
2
67 Ar=3-In; R=H
68 Ar=3-In; R=Me
69 Ar=2-Np; R=H
70 Ar=2-Np; R=Me
71 Ar=1-Np; R=H
72 Ar=1-Np; R=Me
76Ar=3-In; R=H
77 Ar=3-In; R=Me
78 Ar=2-Np; R=H
79 Ar=2-Np; R=Me
80 Ar=1-Np; R=H
81 Ar=1-Np; R=Me
Scheme 2. Syntheses of dithiocarbamates 1, 9–15, and dithiocarbonate
17, and dithiocarbazates 20 and 21. Reagents: (i) for 9–15, CS2,
pyridine/Et3N; MeI; for 20 and 21, CS2, KOH, EtOH, MeI; (ii) NaH,
THF, MeI.
Scheme 5. Syntheses of sulfamides 61–66 and sulfonamides 67–72.
Reagents and conditions: (i) NH2SO2NH2, H2O, 100 ꢁC; (ii) ClSO2Me,
THF, rt.
Attempts to synthesize indol-3-ylmethylthiourea (47) by
condensing the salt of 76 with KSCN in THF28 were
unsuccessful. Thus, the syntheses of 47 and 48 involved
rather lengthy procedures starting with protection of
N-1 with Boc,21 followed by isothiocyanate formation,
which was then reacted with ammonia/methylamine to
give N-Boc-indolyl thioureas. The Boc group was
hydrolyzed using excess sodium methoxide to give 47
and 48 in moderate yields (Scheme 4).21
for 4 h and then cooling to rt, the precipitates of de-
sired sulfamides 61–66 were obtained in moderate
yields (Scheme 5).29 Sulfonamides 67–72 were synthe-
sized in good yields by reacting primary and second-
ary amines 76–81 with methane sulfonyl chloride in
THF (Scheme 5).30
2.1.2. Syntheses of sulfamides and sulfonamides (61–
72). The sulfamide functional group was conveniently
introduced into amines 76–81 upon treatment with
sulfamide at 100 ꢁC in aqueous solution. After heating
2.1.3. Syntheses of amides and ester (73–75). Amide 73
has been previously synthesized starting from 3-(indol-
3-yl)propanoic acid (92) and 3-(indol-3-yl)propanenitrile
R
R
N
ii
i
N
NHR
NHR'
NHR'
Ar
Ar
Ar
O
S
49 Ar=3-In; R=H; R'=Pr
50 Ar=3-In; R=H; R'=Ph,
51 Ar=3-In; R=Me; R'=Pr
52 Ar=3-In; R=Me; R'=Ph
53 Ar=2-Np; R=H; R'=Pr
54 Ar=2-Np; R=H; R'=Ph
55 Ar=2-Np; R=Me; R'=Pr
56 Ar=2-Np; R=Me; R'=Ph
57 Ar=1-Np; R=H; R'= Pr
58 Ar=1-Np; R=H; R'=Ph
59 Ar=1-Np; R=Me; R'=Pr
60 Ar=1-Np; R=Me; R'=Ph
24 Ar=3-In; R=H; R'=Et
25 Ar=3-In; R=H; R'=Pr
26 Ar=3-In; R=H; R'=Ph
27 Ar=3-In; R=Me; R'=H
28 Ar=3-In; R=Me; R'=Et
29 Ar=3-In; R=Me; R'=Pr
30 Ar=3-In; R=Me; R'=Ph
31 Ar=2-Np; R=R'=H
32 Ar=2-Np; R=H; R'=Et
33 Ar=2-Np; R=H; R'=Pr
34 Ar=2-Np; R=H; R'=Ph
35 Ar=2-Np; R=Me; R'=H
36 Ar=2-Np; R=Me; R'=Et
37 Ar=2-Np; R=Me; R'=Pr
38 Ar=2-Np; R=Me; R'=Ph
39 Ar=1-Np; R=R'=H
40 Ar=1-Np; R=H; R'=Et
41 Ar=1-Np; R=H; R'=Pr
42 Ar=1-Np; R=H; R'=Ph
43 Ar=1-Np; R=Me; R'=H
44 Ar=1-Np; R=Me; R'=Et
45Ar=1-Np; R=Me; R'=Pr
46 Ar=1-Np; R=Me; R'=Ph
76 Ar=3-In; R=H
77 Ar=3-In; R=Me
78 Ar=2-Np; R=H
79 Ar=2-Np; R=Me
80 Ar=1-Np; R=H
81 Ar=1-Np; R=Me
iii
R
N
OMe
Ar
O
NMe
2
18 Ar=3-In; R=H
19 Ar=3-In; R=Me
N
H
89
Scheme 3. Syntheses of ureas 24–46, thioureas 49–60, and carbamates 18–19. Reagents: (i) RNCO, CH2Cl2; (ii) RNCS, CH2Cl2; (iii) ClCOOMe,
N,O-bis(trimethylsilyl)acetamide.

4962
M.S.C Pedras, M. Jha / Bioorg. Med. Chem. 14 (2006) 4958–4979
increase in the activity with the increase in the size of al-
O
OH
kyl group substituent on the thiourea moiety. The activ-
ity of N-(indol-3-ylmethyl)-N-methyl-N-propylthiourea
(51) increased markedly with the replacement of (N)–
H with a CH3 group on thiourea moiety. The naphtha-
lenylthioureas 53–60 showed comparable antifungal
activity. Compounds containing sulfamide or sulfon-
amide functional groups (61–72) were found to have
mild antifungal activity, whereas carbamates 18 and
19, amides 73 and 74, and ester 75 showed little or no
toxicity against L. maculans at the tested concentrations.
In general, those compounds having substitution at C-2
were more active than the compounds with substitution
at C-1 of the naphthalene ring (Table 1).
i; ii
O
NHR
N
H
N
H
74 R = H
75 R = CH
92
3
Scheme 6. Syntheses of amides 74 and 75. Reagents and conditions:
(i) ClCOOEt, THF, Et3N, 0 ꢁC; (ii) NH3 for 74; MeNH2 for 75.
(93) in good yields.31,32 However, the synthesis of 73 and
74 was carried out using a milder procedure in which acid
92 was first reacted with ethylchloroformate in THF and
triethylamine, then ammonia/methyl amine was bubbled
through the solution for 30 min (Scheme 6).33 Ester 75
was prepared from acid 92 as published previously.31
2.2.2. Cytotoxic activity. Assays using brine shrimp lar-
vae (A. salina) were carried out for a preliminary evalu-
ation of cytotoxicity. Shrimp eggs were allowed to hatch
for 24 h, incubated with brassinin or designed com-
pounds at 0.5, 0.2, and 0.1 mM, and the number of sur-
viving shrimps was counted after 24 h. A control
experiment was carried out similarly without adding
any compound. The results summarized in Table 1 show
that compounds containing a dithiocarbamate group (1
and 9–13) were lethal at all the tested concentrations,
whereas carbamates (18 and 19) were found to be mod-
erately toxic. Among the ureas 22–46, naphthalen-2-
ylmethylurea (31) and naphthalen-1-ylmethylurea (39)
were found to be most potent. A decrease in lethality
was observed with the increase in the size of substituents
on the urea fragment of these compounds. For instance,
N-(indol-3-ylmethyl)-N-methyl-N-propylurea (29) and
N-methyl-N-(naphthalen-2-ylmethyl)-N-propylurea (37)
had no toxicity. Compounds having urea functional
group placed at the naphthalen-2-ylmethyl position were
found to be more active than the 1-substituted isomers.
Only N-(indol-3-ylmethyl)-N-methylthiourea (23) was
lethal at 0.5 and 0.2 mM, among thioureas 47–60. Other
compounds containing thiourea functionality were nota-
bly less toxic. Sulfamides, sulfonamides, and amides
were very mild or non-cytotoxic at the tested
concentrations.
2.2. Biological activity of potential inhibitors and
metabolism
The antifungal activity against L. maculans and cytotoxic
activity to Artemia salina (brine shrimp) of the synthe-
sized compounds (1 and 9–75) were determined using
established bioassay methods, as follows.
2.2.1. Antifungal activity. To establish the antifungal
activity, bioassays were carried out using a mycelial
radial growth assay, as described in Section 4. The
results summarized in Table 1 indicate that N0-methyl-
brassinin (9), naphthalen-2-ylmethylurea (31), and
N-(indol-3-ylmethyl)-N-methyl-N-phenylthiourea (52)
were the most potent antifungal compounds among all
tested compounds. All three compounds were complete-
ly inhibitory of mycelial growth at 0.2 mM and showed
a
substantial inhibition even at 0.1 mM (>70%,
0.1 mM). Interestingly, N0-methylbrassinin (9) was
found to be almost twice as antifungal as the naturally
occurring dithiocarbamate 1, suggesting that replace-
ment of the side chain (N)–H of brassinin with a CH3
group increased the toxicity toward L. maculans.
Among other dithiocarbamates, naphthalene-based
compounds 10–13 were significantly less inhibitory than
brassinin (1) and showed no pronounced effect when the
(N)–H was replaced with a CH3 group. Dithiocarbonate
17 showed substantially lower antifungal activity (41%,
0.5 mM) than brassinin (1), whereas dithiocarbazate
exhibited somewhat similar antifungal activity. In the
case of ureas 22–46, indole-based compounds 22–30
showed an increase in the activity with the increase of
the bulkiness of the alkyl substituent. For example, N-
(indol-3-ylmethyl)-N-propylurea (25) was twice as toxic
as N-(indol-3-ylmethyl)urea (22). However, introduction
of an additional CH3 group on the N of the urea moiety
(27–30) led to lower toxicity. Naphthalenyl ureas 31–46
were significantly more toxic than indolyl ureas 22–30
and showed a decrease in the antifungal activity with
the increase in bulkiness of the alkyl substituent on the
urea moiety. Methylated ureas 35–38 and 43–46 were
found to be substantially less toxic then their demethy-
lated analogs 31–34 and 39–42. While indol-3-
ylmethylthiourea (47) showed no toxicity to L. maculans
at the tested concentrations, other indolylthioureas (48–
60) were found to have antifungal activity, showing an
2.3. Screening of potential inhibitors and metabolism
The metabolism of brassinin (1) and potential detoxifi-
cation inhibitors 9–15 and 17–75 was investigated in cul-
tures of L. maculans over a period of 96 h. Initial
experiments were carried out to determine the time
required by L. maculans to completely metabolize brass-
inin (1) at three different concentrations (0.1, 0.2, and
0.3 mM). Next, the screening of potential inhibitors of
brassinin detoxification was performed by co-incubating
each compound with brassinin (1). Mycelial cultures
(48-h-old) in a chemically defined medium34 were incu-
bated with the potential inhibitors at 0.1 and 0.2 mM
for 10 min (to allow absorption/transport of compounds
into cells) before adding brassinin (1, 0.1 mM). Control
cultures prepared similarly but containing only brassinin
(1) or the potential inhibitor were incubated in parallel.
The stability of brassinin (1) and compounds 9–15 and
17–75 was determined by incubations in culture media
under similar conditions. Samples were withdrawn from

M. S. C Pedras, M. Jha / Bioorg. Med. Chem. 14 (2006) 4958–4979
4963
Table 1. Biological activity of brassinin (1) and potential detoxification inhibitors 9–15 and 17–75 against Leptosphaeria maculans and Artemia salina
Compound
Biological activity
Percentage of activity
0.2 mM
0.5 mM
0.1 mM
0
Brassinin (1)
Antifungal activitya
Cytotoxic activityb
100
100
0
0
45
100
6
0
100
0
N0-Methylbrassinin (9)
Antifungal activity
Cytotoxic activity
100
100
0
0
100
100
0
0
74
100
2
0
Methyl naphthalen-2-ylmethyldithiocarbamate (10)
Methyl N-methylnaphthalen-2-ylmethyldithiocarbamate (11)
Methyl naphthalen-1-ylmethyldithiocarbamate (12)
Methyl N-methylnaphthalen-1-ylmethyldithiocarbamate (13)
Methyl N-benzyldithiocarbamate (14)
Methyl N-phenyldithiocarbamate (15)
Tryptophol dithiocarbonate (17)
Antifungal activity
Cytotoxic activity
40
100
8
0
32
100
7
0
0
100
0
0
Antifungal activity
Cytotoxic activity
40
100
6
0
28
100
6
0
0
100
Antifungal activity
Cytotoxic activity
44
100
7
0
32
100
4
0
23
100
6
0
Antifungal activity
Cytotoxic activity
35
100
3
0
20
100
2
0
0
100
0
0
0
Antifungal activity
Cytotoxic activity
100
100
0
0
52
100
2
0
0
100
Antifungal activity
Cytotoxic activity
62
100
4
0
20
100
3
0
0
100
Antifungal activity
Cytotoxic activity
41
100
7
0
30
84 10
5
20
24
7
6
Methyl indol-3-ylmethylcarbamate (18)
Methyl 20-methylindol-3-ylmethylcarbamate (19)
Methyl phenyldithiocarbazate (20)
Antifungal activity
Cytotoxic activity
17
64
3
6
0
25 10
0
0
Antifungal activity
Cytotoxic activity
33
82
5
6
0
52 10
0
10
0
Antifungal activity
Cytotoxic activity
100
100
0
0
76
96
4
4
32
66
2
6
Methyl benzyldithiocarbazate (21)
Antifungal activity
Cytotoxic activity
84
100
5
0
26
82
6
7
0
56
6
Indol-3-ylmethylurea (22)
Antifungal activity
Cytotoxic activity
62
61
4
6
33
25 10
6
0
0
N-(Indol-3-ylmethyl)-N0-methylurea (23)
N-(Indol-3-ylmethyl)-N0-ethylurea (24)
N-(Indol-3-ylmethyl)-N0-propylurea (25)
N-(Indol-3-ylmethyl)-N0-phenylurea (26)
N-(Indol-3-ylmethyl)-N0-methylurea (27)
N-(Indol-3-ylmethyl)-N-methyl-N0-ethylurea (28)
N-(Indol-3-ylmethyl)-N-methyl-N0-propylurea (29)
N-(Indol-3-ylmethyl)-N-methyl-N0-phenylurea (30)
Naphthalen-2-ylmethylurea (31)
Antifungal activity
Cytotoxic activity
74
82 12
4
40
64
6
6
0
57
6
5
Antifungal activity
Cytotoxic activity
84
61 12
3
44
25
6
5
21
0
Antifungal activity
Cytotoxic activity
0
0
58
0
3
43
0
5
3
Antifungal activity
Cytotoxic activity
75
17
2
6
36
0
2
22
0
Antifungal activity
Cytotoxic activity
70
86
4
6
53
61
3
6
0
48 10
Antifungal activity
Cytotoxic activity
48
75
2
6
31
50
5
6
0
39
6
Antifungal activity
Cytotoxic activity
17
0
3
0
0
0
0
Antifungal activity
Cytotoxic activity
61
39
4
0
37
17
2
6
30
0
3
3
Antifungal activity
Cytotoxic activity
100
100
0
0
100
0
81
68 11
44
25 10
29 12
N0-Ethyl-N-(naphthalen-2-ylmethyl)urea (32)
N-(Naphthalen-2-ylmethyl)-N0-propylurea (33)
N0-Phenyl-N-(naphthalen-2-ylmethyl)urea (34)
Antifungal activity
Cytotoxic activity
64
68
3
6
5
23
0
3
3
2
Antifungal activity
Cytotoxic activity
74
48
5
0
48
14
6
6
24
0
Antifungal activity
Cytotoxic activity
61
36
3
6
60
0
2
58
0
(continued on next page)

4964
M.S.C Pedras, M. Jha / Bioorg. Med. Chem. 14 (2006) 4958–4979
Table 1 (continued)
Compound
Biological activity
Percentage of activity
0.2 mM
0.5 mM
0.1 mM
N-Methyl-N-(naphthalen-2-ylmethyl)urea (35)
Antifungal activity
Cytotoxic activity
100
100
0
0
66
64
6
6
24
32
2
6
N0-Ethyl-N-methyl-N-(naphthalen-2-ylmethyl)urea (36)
N-Methyl-N-(naphthalen-2-ylmethyl)-N0-propylurea (37)
N-Methyl-N-(naphthalen-2-ylmethyl)-N0-phenylurea (38) (low solubility)
Naphthalen-1-ylmethylurea (39)
Antifungal activity
Cytotoxic activity
60
71 10
4
41
25
5
6
18
0
4
4
2
Antifungal activity
Cytotoxic activity
76
0
6
38
0
3
3
4
21
0
Antifungal activity
Cytotoxic activity
64
14
4
5
30
0
15
0
Antifungal activity
Cytotoxic activity
100
0
0
46
0
36
72 11
6
N0-Ethyl-N-(naphthalen-1-ylmethyl)urea (40)
Antifungal activity
Cytotoxic activity
68
68 10
4
26
22
6
6
0
0
N-(Naphthalen-1-ylmethyl)-N0-propylurea (41)
Antifungal activity
Cytotoxic activity
54
0
6
4
0
36
0
4
0
0
N-(Naphthalen-1-ylmethyl)-N0-phenylurea (42) (low solubility)
N-Methyl-N-(naphthalen-1-ylmethyl)urea (43)
Antifungal activity
Cytotoxic activity
60
0
34
0
2
0
0
Antifungal activity
Cytotoxic activity
100
100
76
46
6
6
28
78
2
0
N0-Ethyl-N-methyl-N-(naphthalen-1-ylmethyl)urea (44)
N-Methyl-N-(naphthalen-1-ylmethyl)-N0-propylurea (45)
N-Methyl-N-(naphthalen-1-ylmethyl)-N0-phenylurea (46) (low solubility)
Indol-3-ylmethylthiourea (47)
Antifungal activity
Cytotoxic activity
50
64
5
6
31
32 10
4
0
0
Antifungal activity
Cytotoxic activity
53
14
4
6
21
0
3
0
0
Antifungal activity
Cytotoxic activity
54
32
4
6
18
0
6
0
0
Antifungal activity
Cytotoxic activity
0
79
0
29 12
0
0
0
3
N-(Indol-3-ylmethyl)-N0-methylthiourea (48)
Antifungal activity
Cytotoxic activity
73
100
38
100
2
0
75
6
3
N-(Indol-3-ylmethyl)-N0-propylthiourea (49)
Antifungal activity
Cytotoxic activity
64
78
4
6
29
24
3
6
0
0
N-(Indol-3-ylmethyl)-N0-phenylthiourea (50)
Antifungal activity
Cytotoxic activity
83
42 10
7
81
14
5
0
64
0
N-(Indol-3-ylmethyl)-N-methyl-N0-propylthiourea (51)
N-(Indol-3-ylmethyl)-N-methyl-N0-phenylthiourea (52)
N-(Naphthalen-2-ylmethyl)-N0-propylthiourea (53)
N-(Naphthalen-2-ylmethyl)-N0-phenylthiourea (54)
N-Methyl-N-(naphthalen-2-ylmethyl)-N0-propylthiourea (55)
N-Methyl-N-(naphthalen-2-ylmethyl)-N0-phenylthiourea (56)
N-(Naphthalen-1-ylmethyl)-N0-propylthiourea (57)
N-(Naphthalen-1-ylmethyl)-N0-phenylthiourea (58)
N-Methyl-N-(naphthalen-1-ylmethyl)-N0-propylthiourea (59)
Antifungal activity
Cytotoxic activity
35
38
3
6
30
14
4
6
0
0
Antifungal activity
Cytotoxic activity
100
0
0
100
0
0
1
4
7
4
4
6
5
73
0
3
Antifungal activity
Cytotoxic activity
100
0
0
62
0
0
0
Antifungal activity
Cytotoxic activity
81
14
4
6
39
0
0
0
Antifungal activity
Cytotoxic activity
89
0
6
6
4
3
32
0
0
0
Antifungal activity
Cytotoxic activity
68
0
30
0
0
0
Antifungal activity
Cytotoxic activity
90
0
34
0
0
0
Antifungal activity
Cytotoxic activity
86
0
46
0
0
0
Antifungal activity
Cytotoxic activity
76
14
4
6
31
0
0
0
(continued on next page)

M. S. C Pedras, M. Jha / Bioorg. Med. Chem. 14 (2006) 4958–4979
4965
Table 1 (continued)
Compound
Biological activity
0.5 mM
Percentage of activity
0.2 mM
0.1 mM
N-Methyl-N-(naphthalen-1-ylmethyl)-N0-phenylthiourea (60)
Indol-3-ylmethylsulfamide (61)
Antifungal activity
Cytotoxic activity
60
5
26
3
0
0
0
0
Antifungal activity
Cytotoxic activity
57
43
5
5
29
24
2
7
0
0
N-(Indol-3-ylmethyl)-N-methylsulfamide (62)
Naphthalen-2-ylmethylsulfamide (63)
Antifungal activity
Cytotoxic activity
43
32
5
6
20
14
7
6
0
0
Antifungal activity
Cytotoxic activity
48
29
4
6
37
0
8
0
0
N-Methyl-N-(naphthalen-2-ylmethyl)sulfamide (64)
Naphthalen-1-ylmethylsulfamide (65)
Antifungal activity
Cytotoxic activity
40
14
4
5
24
0
6
0
0
Antifungal activity
Cytotoxic activity
38
0
4
0
0
0
0
N-Methyl-N-(naphthalen-1-ylmethyl)sulfamide (66)
N-(Indol-3-ylmethyl)methanesulfonamide (67)
N-(Indol-3-ylmethyl)-N-methyl methanesulfonamide (68)
N-Methyl-N-(naphthalen-2-ylmethyl)methanesulfonamide (69)
N-Methyl-N-(naphthalen-2-ylmethyl)methanesulfonamide (70)
N-(Naphthalen-1-ylmethyl)methanesulfonamide (71)
N-Methyl-N-(naphthalen-1-ylmethyl)methanesulfonamide (72)
Indolyl-3-propanamide (73)
Antifungal activity
Cytotoxic activity
32
0
6
0
0
0
0
Antifungal activity
Cytotoxic activity
56
50
5
6
39
29
4
6
22
0
3
Antifungal activity
Cytotoxic activity
46
32
4
6
28
0
6
0
0
Antifungal activity
Cytotoxic activity
38
32 12
3
18
0
3
0
0
Antifungal activity
Cytotoxic activity
44
0
3
0
0
0
0
Antifungal activity
Cytotoxic activity
55
14
3
6
0
0
0
0
Antifungal activity
Cytotoxic activity
38
0
6
0
0
0
0
Antifungal activity
Cytotoxic activity
48 2
86 12
26
0
5
0
0
N0-Methyl-indolyl-3-propanamide (74)
Antifungal activity
Cytotoxic activity
0
64
0
0
0
0
6
a Percent of inhibition = 100 ꢀ [(growth on medium containing compound/growth on control medium) · 100)] standard deviation.
b Percent of mortality = 100 ꢀ [(number of surviving larvae in solution containing compound/number of surviving larvae in control solu-
tion) · 100)] standard deviation.
cultures immediately after addition of brassinin (1) and
at 24, 48, and 96 h, and were extracted with ethyl ace-
tate. The organic extracts were analyzed by HPLC (pho-
todiode array detection at 220 nm, brassinin
tR = 18.8 min) to determine the concentration of both
brassinin (1) (remaining in the cultures at different times)
and the indole-3-carboxaldehyde (8, tR = 6.8 min), the
product of brassinin biotransformation. Brassinin (1)
and potential inhibitors 9–15 and 17–75 were stable in
minimal media for at least 8 days. The rate of disappear-
ance of brassinin (1) in the presence of the potential
inhibitor was compared with that of control cultures
(mycelial culture containing only brassinin at 0.1, 0.2,
and 0.3 mM) by HPLC analyses of culture extracts.
HPLC analyses of culture extracts showed that brassinin
(1) at 0.1 mM was metabolized completely to indole-3-
carboxaldehyde (8) in ca. 16 h, while in cultures contain-
ing brassinin (1) at higher concentrations, 0.2 and
0.3 mM, complete metabolism to 8 took place in ca.
24 and 48 h, respectively. As shown in Table 2, some
of the compounds tested slowed down the metabolism
of brassinin (1) relative to control cultures. That is,
N0-methylbrassinin (9), methyl N-methyl-N-(naphtha-
len-2-ylmethyl)dithiocarbamate (11), tryptophol dithio-
carbonate (17), and methyl 3-phenyldithiocarbazate
(20) affected the rate of detoxification of brassinin (1)
noticeably (Table 2). In the presence of methylbrassinin
(9, 0.1 mM), brassinin (1, 0.1 mM) was completely
metabolized to 8 in ca. 48 h (vs 16 h for brassinin at
0.1 mM), whereas doubling the concentration of 9 slo-
wed down the transformation of brassinin (1) to 96 h
(vs 24 h for brassinin at 0.2 mM). Importantly, no bio-
transformation of product of 9 was detected in any of
the cultures. Less pronounced but similar results were
obtained when the cultures were incubated with methyl
N-methyl-N-(naphthalen-2-ylmethyl)dithiocarbamate (11)
and brassinin (1), as shown in Table 2. On the other
hand, methyl, N-benzyldithiocarbamate (14), and methyl
N-phenyldithiocarbamate (15) did not affect the metabo-
lism of brassinin (1).

4966
M.S.C Pedras, M. Jha / Bioorg. Med. Chem. 14 (2006) 4958–4979
Table 2. Compounds that were metabolized (9, 11, 14, 17, 20, 48, and 73–75 at 0.1 and 0.2 mM) or decreased the rate (9, 11, 17, and 20 at 0.1 and
0.2 mM) of brassinin detoxification in cultures of Leptosphaeria maculans co-incubated with brassinin (1, 0.1 mM) for different time periods (h)
Compounds at 0.1 mM and 0.2 mM
Biotransformation product (time required
for complete transformation)
Incubation time, remaining
brassinin (1) molar %a
0.1 mM
0.2 mM
Control (containing only brassinin (1))
N0-Methylbrassinin (9)c
Indole-3-carboxaldehyde (8)
No transformation
16 h, <5%
24 h, 25
48 h, n.d.b
72 h, n.d.
—
24 h, <5%
24 h, 94
48 h, 80
2
2
2
4
5
72 h, 20
96 h, n.d.
24 h, 72
Methyl N-methyl-N-(naphthalen-2-ylmethyl)
dithiocarbamate (11)c
No transformation
24 h, 11
48 h, n.d.
24 h, n.d.
24 h, 83
48 h, 75
72 h, 49
96 h, n.d.
24 h, 19
48 h, n.d.
24 h, n.d.
24 h, n.d.
24 h, n.d.
24 h, n.d.
4
48 h, <5%
24 h, n.d.
24 h, 97
Methyl N-benzyldithiocarbamate (14)
Tryptophol dithiocarbonate (17)
Benzoic acid
Tryptophol (120 h) (88)
4
4
5
3
4
4
48 h, 90
72 h, 68
96 h, <5%
24 h 68
Methyl 3-phenyldithiocarbazate (20)
Methyl 3-phenylthiocarbazate (48 h) (94)
3
4
48 h, <5%
24 h, n.d.
24 h, n.d.
24 h, n.d.
24 h, n.d.
N-(Indol-3-ylmethyl)-N0-methylthiourea (48)
3-(Indol-3-yl)propanamide (73)
N0-Methyl-3-(indol-3-yl)propanamide (74)
Methyl 3-(indol-3-yl)propanoate (75)
Indole-3-carboxaldehyde (30 h) (8)
3-(Indol-3-yl)propanoic acid (48 h) (92)
3-(Indol-3-yl)propanoic acid (168 h) (92)
3-(Indol-3-yl)propanoic acid (6 h) (92)
a Percentages were determined using calibration curves and are averages of at least two independent experiments conducted in duplicate standard
deviation.
b n.d., not detected.
c Compound not metabolized.
Complete detoxification of brassinin (1, 0.1 mM) in the
presence of tryptophol dithiocarbonate (17, 0.1 mM)
took place in 96 h (vs 16 h for brassinin alone); similar
results were obtained with 17 at 0.2 mM. Tryptophol
dithiocarbonate (17) was hydrolyzed to tryptophol
(88) in 120 h (enzymatic transformation). Methyl 3-
phenyldithiocarbazate (20) slowed down the transfor-
mation of brassinin (1, 0.1 mM) from 24 to 48 h,
although 20 was also metabolized to 94 within 48 h
of incubation (Table 2).
disease of crucifer crops. To design potential inhibi-
tors of brassinin detoxification, the structures of brass-
inin (1) and its metabolic products 7 and 8 were
modified to incorporate a wide range of functional
groups (9–75). The designed compounds were synthe-
sized (9–15 and 17–75) and their bioactivities and
metabolism in cultures of L. maculans were investigat-
ed. The antifungal and cytotoxic activity of these
compounds indicated that dithiocarbamates, dithiocar-
bazates, ureas, and thioureas were highly toxic to
L. maculans and A. salina, with dithiocarbamates
causing 100% death in larvae of A. salina even at
0.1 mM. By contrast, compounds having carbamate,
sulfamide, sulfonamide, amide or ester functional
groups had either very little or no activity against
L. maculans or A. salina. Interestingly, indolyl and
phenyl dithiocarbamates 1, 9, 14, and 15 were more
antifungal than their naphthalenyl analogs 10–13,
whereas in the case of ureas and thioureas, the napht-
halenyl substituted compounds (31–46) were more
active than the indolyl-substituted analogs (22–30).
Among the various naphthalenyl compounds, substitu-
tion at C-2 led to higher activity than substitution at
C-1 (Table 2). Compounds containing functional
groups identical or related to compounds 9–75 are
known to display pesticidal and/or other biological
activities.35
Of the remaining synthetic compounds that were
screened for the inhibitory activity, only compounds
48 and 73–75 were metabolized by L. maculans. The
thiourea 48 was biotransformed to 8 in 30 h whereas
72–75 were metabolized to 3-(indol-3-yl)propanoic acid
(92) in 48, 168, and 6 h, respectively, without affecting
the metabolism of brassinin (1). Compounds 10,
12–14, 18, 19, and 21–72 were not biotransformed by
L. maculans and did not appear to affect noticeably
the rate of metabolism of brassinin (1).
1'
2'
NH
H
N
SMe
Ar
O
94
3. Discussion and conclusion
Compounds 9–15 and 17–75 were screened for biotrans-
formation and inhibition of brassinin detoxification in
cultures of L. maculans by co-incubating these com-
pounds with brassinin (1). A noticeable decrease in the
rate of brassinin detoxification was observed in the pres-
ence of N0-methylbrassinin (9), dithiocarbamate 11,
Because brassinin (1) detoxification by L. maculans
can deprive plants of crucial natural chemical defens-
es, it is of great importance to understand and inhibit
this process. Selective inhibitors of brassinin detoxifi-
cation are potential protection agents against blackleg

M. S. C Pedras, M. Jha / Bioorg. Med. Chem. 14 (2006) 4958–4979
4967
0.35
dithiocarbonate 17, and dithiocarbazate 20. In agree-
ment with previous results,3 modification of the aromat-
ic substituent of brassinin (1) did not prevent metabolic
degradation; however, the presence of the –CH2–NH–
C@S– moiety on the side chain was essential for metab-
olism to occur. That is, replacement of the (N)H with a
(N)–CH3 group or deletion of the methylene bridge pre-
vented the degradation of dithiocarbamates 9, 11, and
15. This result is of great interest as it suggests that
the (N)H of the –NH–C@S– moiety might be playing
an important role in substrate recognition by the
putative brassinin oxidase. In addition, this result may
explain the substantially higher antifungal activity of
N0-methylbrassinin (9) relative to brassinin (1, Table 1)
against L. maculans. Furthermore, the slower transfor-
mation of dithiocarbamates 10 and 13 (to 2- and 1-
naphthoic acids, in 48 and 96 h, respectively) than that
of brassinin (1, 16 h) might be due to either the different
polarity of these naphthalenyl derivatives or/and the
selectivity of brassinin oxidase. Furthermore, carbamate
17, ureas 22–46, sulfamides 61–66, and sulfonamides
67–72 were not metabolized by L. maculans, whereas
among thioureas 47–60, only 48 was metabolized (to
aldehyde 8 in 30 h). Structurally, 48 is similar to brassi-
nin, having the S(CH3) atom replaced with NH(CH3);
however, the rate of metabolism of 48 was much slower
(30 vs 16 h for brassinin). Taken together, these meta-
bolic results point out the selectivity of brassinin oxi-
dase, although at this point the possibility that
somewhat more polar molecules are unable to reach
the metabolic cell site cannot be ruled out.
brassinin (0.1 mM)
0.3
0.25
0.2
brassinin (0.1 mM) + 17 (0.1 mM)
brassinin (0.1 mM) + 17 (0.2 mM)
brassinin (0.3 mM)
0.15
0.1
0.05
0
0
8
16 24 32 40 48 56 64 72 80 88 96
Time (h)
Figure 3. Progress curves for detoxification of brassinin (1, 0.1 mM) in
the presence of tryptophol dithiocarbonae (17, 0.1 and 0.2 mM) in
cultures of Leptosphaeria maculans.
co-incubating compounds with brassinin oxidase or
brassinin oxidase containing extracts. Therefore, further
testing of compounds 9, 11, 17, and 20 in brassinin oxi-
dase containing fractions is necessary. In addition, it is
necessary to understand the mechanistic details of brass-
inin oxidase mediated conversion of brassinin (1) to be
able to design more effective inhibitors. Although com-
pound 17 is a very good lead structure, compounds that
are not metabolized in culture are more likely to protect
oilseed crucifers against L. maculans infestation. It is
expected that isolation and characterization of the
brassinin oxidase involved in the detoxification of brass-
inin (1) will greatly facilitate the design of more effective
and selective inhibitors.
Overall, three types of interaction were observed in cul-
tures of L. maculans co-incubated with the compounds
under study (9–15 and 17–75) and brassinin (1): (1) a
decrease on the rate of brassinin detoxification due to
the strong inhibitory activity of the compound on myce-
lium growth (e.g., 9, Fig. 2); (2) a decrease on the rate of
brassinin detoxification due to the inhibitory activity of
the compound on brassinin oxidase (e.g., 17, Fig. 3); (3)
a low to no detectable effect on the rate of brassinin
detoxification (10, 12, 13, 15, 18, 19, and 21–75). It is
also possible that the decrease on the rate of brassinin
detoxification is due to strong inhibitory activity of the
compound on both mycelium growth and brassinin oxi-
dase; however, this hypothesis can be confirmed only by
4. Experimental
4.1. General experimental procedures
All chemicals were purchased from Sigma–Aldrich
Canada Ltd, Oakville, ON. All solvents were of HPLC
grade and used as such, except for CH2Cl2 and CHCl3
that were redistilled. Solvents used in syntheses were
dried over the following drying agents prior to use:
THF and diethyl ether over sodium/benzophenone,
CH2Cl2 and benzene over CaH2. Organic extracts were
dried over anhydrous Na2SO4 and solvents removed
under reduced pressure in a rotary evaporator.
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 to 100% CH3CN, for 35 min,
linear gradient, and a flow rate 1.0 mL/min. UV spectra
were recorded on a Varian-Cary spectrophotometer in
MeOH. NMR spectra were recorded on Bruker Avance
500 spectrometers; d values were referenced as follows:
for 1H (500 MHz), CDCl3 (CHCl3 at 7.27 ppm),
CD3CN (CD2HCN at 1.94 ppm), and (CD3)2SO
(CHD2SOCD3 at 2.50 ppm); for 13C (125.8 MHz),
CDCl3 (77.2 ppm), CD3CN (118.7 ppm), and (CD3)2SO
(39.5 ppm). Fourier transform infrared (FTIR) spectra
0.35
brassinin (0.1 mM)
0.3
brassinin (0.1 mM) + 9 (0.1 mM)
0.25
brassinin (0.1 mM) + 9 (0.2 mM)
0.2
brassinin (0.3 mM)
0.15
0.1
0.05
0
0
8
16 24 32 40 48 56 64 72 80 88 96
Time (h)
Figure 2. Progress curves for detoxification of brassinin (1, 0.1 mM) in
the presence of different concentrations of N0-methylbrassiin (9, 0.1
and 0.2 mM) in cultures of Leptosphaeria maculans.

4968
M.S.C Pedras, M. Jha / Bioorg. Med. Chem. 14 (2006) 4958–4979
were recorded on Bio-Rad FTS-40 spectrometers. Mass
spectra (MS) were obtained on a VG 70 SE mass spec-
trometer using a solid probe or on a Q Star XL, Applied
Biosystems.
100 mg, 0.68 mmol) and triethylamine (191 lL,
1.44 mmol) in pyridine (1 mL) at 0 ꢁC. After stirring
for 20 min, MeI (49 lL, 0.75 mmol) was added and the
reaction mixture was stirred for an additional 30 min.
The reaction mixture was acidified with H2SO4 (5 mL,
1.5 M), was extracted with Et2O, the organic phase
was dried (Na2SO4) and evaporated under reduced pres-
sure. Fractionation by FCC (silica gel, EtOAc/hexane,
50:50) afforded brassinin (1, 153 mg, 95% yield) as a
white solid. Mp: 132–133 ꢁC, CH2Cl2 (lit.38 132–
133 ꢁC). HPLC tR = 18.7 min. Spectroscopic data are
identical to published data.
4.2. Fungal cultures
Fungal cultures of L. maculans virulent isolate BJ 125
were obtained from the IBCN collection, Agriculture
and Agri-Food Canada Research Station, Saskatoon,
SK. Cultures were handled as described previously.36
4.3. Antifungal bioassays
4.6.2. N0-Methylbrassinin (9). Preparation and separa-
tion as reported above for brassinin (1) substituting 77
(100 mg, 0.63 mmol) for indol-3-ylmethylamine (76).
Red solid, 134 mg, 86% yield based on amine 77. Mp:
68–69 ꢁC, CH2Cl2. HPLC tR = 21.7 min. 1H NMR
(500 MHz, CD3CN) mixture of rotamers (1:2): d 9.32
(br s, 1H, D2O exchangeable), 7.73 (d, J = 7.5 Hz,
0.7H), 7.45 (d, J = 7.5 Hz, 1H), 7.37 (br s, 0.7H), 7.18
(dd, J = 7, 7 Hz, 1H), 7.08 (m, 1H), 5.53 (s, 1.3H),
3.22 (s, 2H), 2.66 (s, 3H); additional signals for minor
rotamer 7.63 (br s, 0.3H), 7.31 (br s, 0.3H), 5.16 (s,
0.7H), 3.42 (s, 1H). 13C NMR (500 MHz, CD3CN) mix-
ture of rotamers: d 197.9, 136.5, 126.6, 125.3, 122.0,
119.4, 119.2, 111.5, 110.1, 51.1, 37.6, 19.7; additional
signals for minor rotamer 197.1, 124.7, 119.6, 118.6,
111.7, 109.3, 49.4, 42.2. FTIR mmax (KBr): 3409, 480,
1388, 745 cmꢀ1. HREIMS: m/z measured 250.0604
(250.0598 calculated for C12H14N2S2). EIMS m/z (% rel-
ative abundance) 250 (M+, 35), 130 (100), 121 (15).
Virulent isolates of L. maculans were grown on potato
dextrose agar (PDA) plates at 24 1 ꢁC under constant
light for 7 days. The antifungal activity of compounds
was determined following a mycelial radial growth
bioassay, as described previously.37 All bioassays were
carried out in triplicate, at least two times.
4.4. Brine shrimp larvae bioassays
The cytotoxic activity of compounds was determined
using brine shrimp larvae (A. salina). Shrimp eggs
(1 scoop) were incubated in Petri dishes containing saline
water (3.8% NaCl solution) under constant light and eggs
were allowed to hatch for 24 h. Solutions of the com-
pounds to be tested were prepared by serial dilutions (fi-
nal concentrations 0.5, 0.2, and 0.1 mM in saline water
with 1% MeOH, prepared from 50 mM stock solutions
in MeOH) and dispensed (4 mL) into vials; 10 living
shrimps were then added to each vial and final volume
was made up to 5 mL using saline water. Control solu-
tions (1% MeOH in saline water) containing 10 living lar-
vae were prepared similarly. The vials were incubated for
24 h and the number of surviving larvae in each vial was
counted and compared with that in the control vial. The
experiments were performed in triplicate at least twice.
4.6.3. Methyl N-(naphthalen-2-ylmethyl)dithiocarbamate
(10). Preparation as reported above for brassinin (1),
substituting 78 (200 mg, 1.27 mmol) for indol-3-ylmeth-
ylamine (76). The crude reaction mixture was subjected
to FCC (silica gel, CH2Cl2/hexane, 80:20) to afford
methyl N-(naphthalen-2-ylmethyl)dithiocarbamate (10,
283 mg, 90% yield based on amine 78) as a white solid.
Mp: 72–73 ꢁC, CH2Cl2/hexane. HPLC tR = 24.2 min.
Spectroscopic data are identical to previously reported
data.5
4.5. Metabolism and screening of potential detoxification
inhibitors
Erlenmeyer flasks (125 mL, containing 50 mL of defined
media) were inoculated with spores (108 spores/100 mL)
of L. maculans and incubated at 24 1 ꢁC on shaker at
120 rpm under constant light for 48 h. The compound
(stock solution in DMSO) to be screened was added to
cultures (0.1 and 0.2 mM) and to uninoculated medium
(control), and flasks were then incubated for 10 min on
shaker at 120 rpm. Brassinin (final concentration
0.1 mM) was then added to the culture and cultures fur-
ther incubated. Control cultures containing brassinin (1)
or the compound only were incubated separately.
Samples (2 mL) were withdrawn at different times and
either frozen or immediately extracted with EtOAc (2·
4 mL). The organic phases were concentrated and ana-
lyzed by HPLC.
4.6.4. Methyl N-methyl-N-(naphthalen-2-ylmethyl)dithio-
carbamate (11). Preparation as reported above for brass-
inin (1), substituting 79 (100 mg, 0.58 mmol) for indol-3-
ylmethylamine (76). The crude reaction mixture was
subjected to FCC (silica gel, CH2Cl2/hexane, 80:20) to
afford methyl N-methyl-N-(naphthalen-2-ylmethyl)di-
thiocarbamate (11, 134 mg, 88% yield based on amine
79) as a white solid. Mp: 83–84 ꢁC, CH2Cl2/hexane.
1
HPLC tR = 28.6 min. H NMR (500 MHz, (CD3)2SO)
mixture of rotamers (1:2): d 7.90 (m, 3H), 7.62 (br s,
0.7), 7.51 (br s, 2H), 7.43 (d, J = 8 Hz, 0.7H), 5.51 (s,
1.4H), 3.33 (s, 2H), 2.62 (s, 2H); additional signals for
minor rotamer 7.71 (br s, 0.3H), 7.39 (d, J = 8 Hz,
0.3H), 5.22 (s, 0.7H), 3.50 (s, 1H), 2.58 (s, 1H). 13C
NMR (500 MHz, (CD3)2SO): d 199.3, 134.5, 133.7,
133.2, 129.2, 128.5, 128.4, 122.2, 126.9, 126.8, 126.4,
59.6, 21.0; additional signals for minor rotamer 192.2,
133.9, 129.4, 127.3, 127.0, 125.8, 57.8, 20.8. FTIR mmax
(KBr): 3054, 2918, 1481, 1382, 959, 752 cmꢀ1. HRE-
4.6. Syntheses
4.6.1. Brassinin (1). Carbon disulfide (45 lL, 0.75 mmol)
was added to a solution of indol-3-ylmethylamine (76,

M. S. C Pedras, M. Jha / Bioorg. Med. Chem. 14 (2006) 4958–4979
4969
IMS: m/z measured 261.0644 (261.0646 calculated for
C14H15NS2). EIMS m/z (% relative abundance) 225
(M+, 41), 141 (100), 115 (25).
1.5 mmol) in CH2Cl2 (1 mL) was added to a solution
of indol-3-ylmethylamine (76, 146 mg, 1.0 mmol) in sol-
vent CH2Cl2 (6 mL) and the mixture was stirred at rt for
30 min. The reaction was then cooled to 0 ꢁC and a solu-
tion of methyl chloroformate (115 lL, 1.5 mmol) in
CH2Cl2 (1.5 mL) was added. The reaction mixture was
allowed to stir at 0 ꢁC for an additional 60 min, was
quenched with H2O (10 mL), and was extracted with
EtOAc, and the organic extract was dried (Na2SO4)
and concentrated. The residue obtained was subjected
to FCC (silica gel, CH2Cl2) to give methyl N-(indol-3-
ylmethyl)carbamate (18) (163 mg, 80% yield from amine
76) as a white solid. Mp: 82–83 ꢁC, CH2Cl2. HPLC
tR = 9.2 min. Spectroscopic data are identical to previ-
ously reported data.10
4.6.5. Methyl N-(naphthalen-1-ylmethyl)dithiocarbamate
(12). Preparation as reported above for brassinin (1),
substituting 80 (150 mg, 0.95 mmol) for indol-3-ylmethyl-
amine (76). The crude reaction mixture was subjected to
FCC (silica gel, CH2Cl2/hexane, 80:20) to afford methyl
N-(naphthalen-1-ylmethyl)dithiocarbamate (12, 212 mg,
90% yield from amine 80) as a yellowish solid Mp: 85–
86 ꢁC, CH2Cl2/hexane. HPLC tR = 23.9 min. Spectro-
scopic data are identical to previously reported data.5
4.6.6. Methyl N-methyl-N-(naphthalen-1-ylmethyl)dithio-
carbamate (13). Preparation as reported above for
brassinin (1), substituting 81 (100 mg, 0.58 mmol) for in-
dol-3-ylmethylamine (76). The crude reaction mixture
was subjected to FCC (silica gel, CH2Cl2/hexane,
80:20) to afford methyl N-methyl-N-(naphthalen-1-ylm-
ethyl)dithiocarbamate (13, 130 mg, 85% yield from
amine 81) as a white solid. Mp: 132–135 ꢁC, CH2Cl2/
hexane. HPLC tR = 27.8 min. 1H NMR (500 MHz,
(CD3)2SO) mixture of rotamers (1:2): 1H NMR
(500 MHz, (CD3)2SO) mixture of rotamers (1:2): d
7.99 (m, 2H), 7.89 (d, J = 8 Hz, 1H), 7.57 (m, 3H),
7.49 (br s, 1H, D2O exchangeable), 7.21 (d, J = 6.5 Hz,
0.7H), 5.58 (s, 1.2H), 3.28 (s, 2H), 2.64 (s, 2H); addition-
al signals for minor rotamer 7.07 (d, J = 6.5 Hz, 0.3H),
5.56 (s, 0.7H), 3.53 (s, 1H), 2.55 (s, 1H). 13C NMR
(500 MHz, (CD3)2SO): d 199.1, 134.2, 132.0, 131.6,
129.5, 128.8, 127.4, 126.9, 126.4, 125.6, 124.0, 58.0,
44.8, 21.0; additional signals for minor rotamer 198.8,
131.4, 131.1, 127.0, 126.5, 123.7, 123.6, 55.9, 20.8. FTIR
4.6.9. Methyl N-(indol-3-ylmethyl)-N-methylcarbamate
(19). Preparation as reported above for methyl N-(in-
dol-3-ylmethyl)carbamate (18) substituting 77 for in-
dol-3-ylmethylamine (76). The crude reaction mixture
was subjected to FCC (silica gel, CH2Cl2) to afford
methyl N-(indol-3-ylmethyl)-N0-methylcarbamate (19)
as a light brown oil (185 mg, 85% yield). HPLC
1
tR = 12.4 min. H NMR (500 MHz, CD3CN) Mixture
of rotamers: d 9.22 (br s, 1H, D2O exchangeable), 7.63
(br s, 1H), 7.43 (d, J = 8 Hz, 1H), 7.23 (s, 1H), 7.16
(dd, J = 7, 8 Hz, 1H), 7.07 (dd, J = 7.5, 7 Hz, 1H),
4.61 (s, 2H), 3.74 (br s, 1H), 3.69 (br s, 2H), 3.77 (s,
3H). 13C NMR (500 MHz, CD3CN): d 156.7, 137.0,
127.2, 124.8, 122.1, 119.6, 119.4, 111.9, 111.8, 52.4,
43.7, 32.4. FTIR mmax (KBr): 3308, 1684, 1454,
741 cmꢀ1. HREIMS: m/z measured 218.1059 (218.1055
calculated for C12H14N2O2). EIMS m/z (% relative
abundance) 218 (M+, 67), 130 (100) 118 (11).
mmax (KBr): 2924, 1633, 1340, 1484, 1383, 800 cmꢀ1
.
HREIMS: m/z measured 261.0652 (261.0646 calculated
for C14H15NS2). EIMS m/z (% relative abundance) 261
(M+, 44), 141 (100), 115 (21).
4.6.10. Methyl 3-phenyldithiocarbazate (20). Carbon
disulfide (65 lL, 1.0 mmol) was added to a mixture of
phenylhydrazine (84, 100 lL, 0.93 mmol) and KOH
(156 mg, 2.80 mmol) in ethanol at 0 ꢁC. After stirring
the reaction mixture for 30 min, MeI (66 lL, 1.02 mmol)
was added and the reaction mixture was allowed to stir
for an additional 60 min at 0 ꢁC. The excess of solvent
was removed and the residue was diluted with H2O
(20 mL), was extracted with EtOAc, the extract was
dried (Na2SO4) and concentrated. The crude reaction
mixture was subjected to FCC (silica gel, CH2Cl2/hex-
ane, 80:20) to afford 20 (174 mg, 95% yield) as an off-
white solid. Mp: 133–134 ꢁC (lit.11 133 ꢁC), CH2Cl2/
hexane. HPLC tR = 17.5 min. Spectroscopic data are
identical to previously reported data.39
4.6.7. Tryptophol dithiocarbonate (17). Carbon disulfide
(82 lL, 1.36 mmol) was added to a mixture of trypto-
phol (88, 200 mg, 1.24 mmol) and NaH (95 mg, a 60%
suspension in oil, 2.48 mmol) in THF (5.0 mL) at 0 ꢁC.
After stirring the reaction mixture for 5 min, MeI
(150 lL, 1.36 mmol) was added and the reaction mixture
was allowed to stir for an additional 10 min at 0 ꢁC.
After dilution with H2O (20 mL) and extraction with
EtOAc, the extract was dried (Na2SO4) and concentrat-
ed to afford 17 (148 mg, 93% yield) as an off-white solid.
1
Mp: 54–55 ꢁC, EtOAc. HPLC tR = 28.0 min. H NMR
(500 MHz, CD3CN): d 9.15 (br s, 1H, D2O exchange-
able), 7.64 (d, J = 8 Hz, 1H), 7.42 (d, J = 8 Hz, 1H),
7.16 (m, 2H), 7.08 (dd, J = 7.5, 7.5 Hz, 1H), 4.87 (t,
J = 7 Hz, 2H), 3.26 (t, J = 7, 2 H), 2.53 (s, 3H). 13C
NMR (500 MHz, CD3CN): d 216.6, 136.8, 127.8,
123.5, 122.0, 119.3, 118.8, 111.8, 111.0, 74.5, 24.3,
4.6.11. Methyl 3-benzyldithiocarbazate (21). Preparation
as reported above for methyl 3-phenyldithiocarbazate
(20) substituting benzylhydrazine dihydrochloride (85,
150 mg, 0.77 mmol) for phenylhydrazine (84). The crude
reaction mixture was subjected to FCC (silica gel,
CH2Cl2/hexane, 80:20) to afford methyl 3-benzyldithioc-
arbazate (21) (94 mg, 87% yield) as a white solid. Mp:
76–77 ꢁC. HPLC tR = 20.3 min. Spectroscopic data are
identical to previously reported data.12
18.6. FTIR mmax (KBr): 3424, 1218, 1064, 744 cmꢀ1
.
HREIMS: m/z measured 251.0431 (251.0438 calculated
for C12H13NOS2). EIMS m/z (% relative abundance)
251 (M+, 5), 143 (100), 115 (10).
4.6.8. Methyl N-(indol-3-ylmethyl)carbamate (18). A
solution of N,O-bis(trimethylsilyl)acetamide (367 lL,
4.6.12. Indol-3-ylmethylurea (22). A mixture of urea
(2.0 g, 33.3 mmol), gramine (89, 500 mg, 2.87 mmol),

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M.S.C Pedras, M. Jha / Bioorg. Med. Chem. 14 (2006) 4958–4979
and crushed sodium hydroxide (100 mg, 2.5 mmol) was
heated at 134 ꢁC. After heating the reaction mixture
for 120 min, ice-cold water (50 mL) was added with vig-
orous stirring. The white precipitate formed was filtered
and crystallized to afford 22 (510 mg, 94% yield) as
white crystals. Mp: 149–151 ꢁC, water. HPLC
IMS: m/z measured 217.1214 (217.1215 calculated for
C12H15N3O). EIMS m/z (% relative abundance) 217
(M+, 90), 172 (8), 145 (80), 130 (100), 118 (30), 77 (12).
4.6.15. N-(Indol-3-ylmethyl)-N0-propylurea (25). Prepa-
ration as reported above for urea (24), starting from
76 (100 mg, 0.68 mmol) and substituting propylisocya-
nate for ethylisocyanate. The crude reaction mixture
was subjected to FCC (silica gel, CH2Cl2/MeOH, 98:2)
to afford 25 (138 mg, 87% yield) as a white solid Mp:
145–147 ꢁC, CH2Cl2/MeOH. HPLC tR = 9.8 min. 1H
NMR (500 MHz, CD3CN): d 9.18 (br s, 1H, D2O
exchangeable), 7.63 (d, J = 8 Hz, 1H), 7.41 (d,
J = 8 Hz, 1H), 7.15 (m, 2H), 7.06 (dd, J = 8, 7 Hz,
1H), 5.12 (br s, 1H D2O exchangeable), 4.94 (br s, 1H
D20 exchangeable), 4.44 (d, J = 5 Hz, 2H), 3.06 (m,
2H), 1.45 (m, 2H), 0.87 (t, J = 7 Hz, 3H). 13C NMR
(500 MHz, CD3CN): 158.7, 137.1, 127.1, 123.5, 122.0,
119.3, 119.2, 114.5, 111.7, 41.9, 35.6, 23.7, 11.0. FTIR
mmax (KBr): 3316, 1626, 1571, 742 cmꢀ1. HREIMS: m/z
1
tR = 4.8 min. H NMR (500 MHz, CD3CN): d 9.27 (br
s, 1H, D2O exchangeable), 7.63 (d, J = 8 Hz, 1H), 7.42
(d, J = 8 Hz, 1H), 7.16 (m, 2H), 7.07 (dd, J = 8, 8 Hz,
1H), 5.42 (br s, 1H, D2O exchangeable), 4.72 (br s,
2H, D2O exchangeable), 4.42 (d, J = 5 Hz, 2H). 13C
NMR (500 MHz, CD3CN): d 158.8, 136.6, 126.6,
123.2, 121.6, 119.0, 118.7, 113.7, 111.3, 35.2. FTIR mmax
(KBr): 3396, 1630, 1591, 742 cmꢀ1. HREIMS: m/z mea-
sured 189.0899 (189.0902 calculated for C10H11N3O).
EIMS m/z (% relative abundance) 189 (M+, 98), 145
(50), 130 (100), 118 (40).
4.6.13. N-(Indol-3-ylmethyl)-Nb-methylurea (23). A solu-
tion of indol-3-ylmethylamine (76, 100 mg, 0.63 mmol)
in CH2Cl2 (5 mL) was added dropwise to a vigorously
stirred mixture of COCl2 (49 lL, 0.69 mmol) and
CaCO3 (76 mg, 0.76 mmol) in CH2Cl2 (3 mL) and
H2O (5 mL). After stirring for 5 min at rt, the CH2Cl2
layer was separated and the aqueous layer was extracted
with additional CH2Cl2 (5 mL). The organic layer was
concentrated, and the residue was re-suspended into
CH2Cl2 (5 mL). A solution of methylamine (8.0 lL,
3.2 mmol) in THF was added to the reaction mixture
and the mixture was allowed to stir for 30 min, the sol-
vent was evaporated and the residue was subjected to
FCC (silica gel, CH2Cl2/MeOH, 98:2) to afford 23
(103 mg, 74% yield) as a white solid. Mp: 119–120 ꢁC,
CH2Cl2/MeOH. HPLC tR = 5.3 min. 1H NMR
(500 MHz, CD3CN): d 9.26 (br s, 1H, D2O exchange-
able), 7.63 (d, J = 8 Hz, 1H), 7.41 (d, J = 8 Hz, 1H),
7.16 (m, 2H), 7.06 (dd, J = 7, 7.5 Hz, 1H), 5.26 (br s,
1H, D2O exchangeable), 4.91 (br s, 1H, D2O exchange-
able), 4.44 (d, J = 5 Hz, 2H), 2.65 (d, J = 5 Hz, 3H). 13C
NMR (500 MHz, CD3CN): d 159.0, 136.6, 126.7, 123.1,
121.6, 118.9, 118.8, 114.0, 111.3, 35.2, 26.1. FTIR mmax
(KBr): 3405, 3308, 1629, 1572, 1256, 743 cmꢀ1. HRE-
IMS: m/z measured 203.1059 (203.1058 calculated for
C11H13N3O). EIMS m/z (% relative abundance) 203
(M+, 100), 145 (57), 130 (93), 118 (35).
measured
231.1369
(231.1371
calculated
for
C13H17N3O). EIMS m/z (% relative abundance) 231
(M+, 72), 145 (50), 130 (100), 102 (35), 73 (27).
4.6.16. N-(Indol-3-ylmethyl)-N0-phenylurea (26). Prepa-
ration as reported above for N-ethyl-N0-(indol-3-yl-
methyl)urea (24), starting from 76 (200 mg, 1.37 mmol)
and substituting phenylisocyanate for ethylisocyanate.
The crude reaction mixture was subjected to FCC (silica
gel, CH2Cl2/MeOH, 98:2) to afford 26 (344 mg, 95%
yield) as a white solid. Mp: 188–190 ꢁC, CH2Cl2/MeOH
(lit.14 195–196 ꢁC). HPCL tR = 15.3 min. 1H NMR
(500 MHz, CD3CN): d 9.18 (br s, 1 H, D2O exchange-
able), 7.68 (d, J = 8 Hz, 1H), 7.42 (m, 3H), 7.24 (m,
3H), 7.17 (m, 2H), 7.08 (dd, J = 8, 8 Hz, 1H), 6.97
(dd, J = 7.5, 7.5, 1H), 5.43 (br s, 2H, D2O exchange-
able), 4.54 (d, J = 5 Hz, 2H). 13C NMR (500 MHz,
CD3CN): 155.3, 140.3, 136.7, 128.7, 126.6, 123.4,
121.8, 121.7, 119.1, 118.8, 118.5, 113.5, 111.4, 35.0.
FTIR mmax (KBr): 3316, 1595, 1561, 738 cmꢀ1. HRE-
IMS: m/z measured 265.1214 (265.1215 calculated for
C16H15N3O). EIMS m/z (% relative abundance) 265
(M+, 52), 172 (12), 130 (100), 93 (64).
4.6.17. N-(Indol-3-ylmethyl)-N-methylurea (27). A mix-
ture of N-(indol-3-ylmethyl)-N-methylamine (77,
130 mg, 0.81 mmol) and KNCO (98 mg, 1.2 mmol) in
MeOH (5 mL) was allowed to reflux for 60 min. The
reaction mixture was cooled to rt and the solvent was
evaporated. The residue was suspended into H2O
(20 mL), was extracted with EtOAc, and the organic
extracts were combined, dried over Na2SO4, and con-
centrated to dryness. The crude reaction mixture was
subjected to FCC (silica gel, CH2Cl2/MeOH, 98:2) to af-
ford 27 (145 mg, 88% yield) as a white solid Mp: 141–
4.6.14. N-Ethyl-N0-(indol-3-ylmethyl)urea (24). Ethyliso-
cyanate (95 lL, 1.2 mmol) was added to a solution of in-
dol-3-ylmethylamine (76, 146 mg, 1.0 mmol) in CH2Cl2
(5 mL). After allowing the reaction mixture to stir for
15 min at rt, the solvent was evaporated and the crude
reaction mixture was subjected to FCC (silica gel,
CH2Cl2/MeOH, 98:2) to afford 24 (182 mg, 84% yield)
as a white solid. Mp: 170–172 ꢁC, CH2Cl2/MeOH.
1
HPLC tR = 7.4 min. H NMR (500 MHz, CD3CN): d
1
9.74 (br s, 1H, D2O exchangeable), 8.20 (d, J = 7.5 Hz,
1H), 7.98 (d, J = 8 Hz, 1H), 7.72 (m, 2H), 7.63 (dd,
J = 7, 8 Hz, 1H), 5.67 (br s, 1H, D2O exchangeable),
5.45 (br s, 1H, D20 exchangeable), 5.0 (d, J = 5, Hz,
2H), 3.68 (m, 2H), 01.61 (t, J = 7 Hz, 3H). 13C NMR
(500 MHz, CD3CN): 163.9, 142.3, 132.4, 128.9, 128.8,
127.3, 124.6, 124.5, 119.8, 117.0, 40.8, 40.3, 20.7. FTIR
mmax (KBr): 3440, 3234, 1580, 1352, 734 cmꢀ1. HRE-
142 ꢁC, CH2Cl2/MeOH. HPLC tR = 4.1 min. H NMR
(500 MHz,CD3CN): d 9.32 (br s, 1H, D2O exchange-
able), 7.66 (d, J = 8 Hz, 1H), 7.42 (d, J = 8 Hz, 1H),
7.21 (s, 1H), 7.15 (dd, J = 7.5, 7.5. Hz, 1H), 7.06 (dd,
J = 7.5, 7.5 Hz, 1H), 4.89 (br s, 2H, D2O exchangeable),
4.60 (s, 2H), 2.75 (s, 3H). 13C NMR (500 MHz,
CD3CN): d 159.2, 137.2, 127.2, 124.3, 122.1, 119.5,
119.4, 112.6, 111.7, 43.3, 33.4. FTIR mmax (KBr): 3234,

M. S. C Pedras, M. Jha / Bioorg. Med. Chem. 14 (2006) 4958–4979
4971
1642, 1593, 744 cmꢀ1
203.1053 (203.1059 calculated for C11H13N3O). EIMS
m/z (% relative abundance) 203 (M+, 81), 145 (48), 130
(100), 74 (86).
.
HREIMS: m/z measured
1526, 1444, 1238, 747 cmꢀ1. HREIMS: m/z measured
279.1374 (279.1372 calculated for C17H17N3O). EIMS
m/z (% relative abundance) 279 (M+, 14), 160 (16), 130
(48), 119 (100), 93 (48).
4.6.18.
N-Ethyl-N0-(indol-3-ylmethyl)-N0-methylurea
4.6.21. Naphthalen-2-ylmethylurea (31). Preparation as
reported for N-(indol-3-ylmethyl)-N-methylurea (27)
and substituting 78 (100 mg, 0.64 mmol) for 77. The
crude reaction mixture was subjected to FCC (silica gel,
CH2Cl2/MeOH, 98:2) to afford 31 (103 mg, 81% yield)
as a white solid. Mp: 193–194 ꢁC, CH2Cl2/MeOH. HPLC
tR = 8.3 min. 1H NMR (500 MHz, (CD3)2SO): d 7.86 (m,
3H), 7.72 (s, 1H), 7.48 (m, 2H), 7.42 (d, J = 8.5 Hz, 1H),
6.52 (br s, 1 H, D2O exchangeable), 5.57 (br s, 2H, D2O
exchangeable), 4.35 (d, J = 6 Hz, 2H). 13C NMR
(500 MHz, (CD3)2SO): d 159.1, 139.1, 133.4, 132.5,
128.2, 128.0, 127.9, 126.6, 126.3, 125.9, 125.3, 43.4. FTIR
(28). Ethylisocyanate (60 lL, 0.75 mmol) was added to
a solution of N-(indol-3-ylmethyl)-N-methylamine (77,
100 mg, 0.63 mmol) in CH2Cl2 (5 mL). After allowing
the reaction mixture to stir for 15 min at rt, the solvent
was evaporated and the crude reaction mixture was sub-
jected to FCC (silica gel, CH2Cl2/MeOH, 98:2) to afford
28 (124 mg, 86% yield) as a white solid. Mp: 118–
1
121 ꢁC, CH2Cl2/MeOH. HPLC tR = 9.2 min. H NMR
(500 MHz, CD3CN): d 9.19 (br s, 1H, D2O exchange-
able), 7.65 (d, J = 8 Hz, 1H), 7.41 (d, J = 8 Hz, 1H),
7.19 (d, J = 3.5 Hz, 1H), 7.15 (dd, J = 8, 8 Hz, 1H), 7.05
(dd, J = 8, 8 Hz, 1H), 5.13 (br s, 1H, D2O exchangable),
4.78 (s, 2H), 3.20 (m, 2H), 2.73 (s, 3H), 1.10 (t,
J = 7 Hz, 3H). 13C NMR (500 MHz, CD3CN): d 158.6,
137.1, 127.3, 124.2, 122.0, 119.6, 119.4, 112.9, 111.7,
43.2, 35.7, 33.1, 15.4. FTIR mmax (KBr): 3234, 1627,
1530, 743 cmꢀ1. HREIMS: m/z measured 231.1378
(231.1372 calculated for C13H17N3O). EIMS m/z (% rela-
tive abundance) 231 (M+, 76), 159 (18), 130 (100).
m
max (KBr): 3355, 1651, 1597, 15568 cmꢀ1. HREIMS: m/z
measured 200.0951 (200.0950 calculated for
C12H12N2O). EIMS m/z (% relative abundance) 200
(M+, 100), 156 (98), 141 (24), 129 (35).
4.6.22. N-Ethyl-N0-(naphthalen-2-ylmethyl)urea (32).
Preparation as reported for N-ethyl-N0-(indol-3-ylmeth-
yl)urea (24) and substituting 78 (100 mg, 0.64 mmol) for
76. The crude reaction mixture was subjected to FCC
(silica gel, CH2Cl2/MeOH, 98:2) to afford 32 (117 mg,
81% yield) as a white solid. Mp: 156–158 ꢁC, CH2Cl2/
MeOH. HPLC tR = 11.8 min. 1H NMR (500 MHz,
(CD3)2SO): d 7.85 (m, 3H), 7.71 (s, 1H), 7.48 (m, 2H),
7.42 (d, J = 8.5 Hz, 1H), 6.40 (br s, 1 H, D2O exchange-
able), 5.92 (br s, 1H, D2O exchangeable), 4.37 (d,
J = 6 Hz, 2H), 3.05 (m, 2H), 1.01 (t, J = 7 Hz, 3H).
13C NMR (500 MHz, (CD3)2SO): d 158.5, 139.1,
133.4, 132.5, 128.2, 128.0, 127.9, 126.6, 126.4, 125.9,
125.4, 43.5, 34.6, 16.2. FTIR mmax (KBr): 3340, 1622,
1572, 1264 cmꢀ1. HRESIMS: m/z measured 229.1341
(229.1340 calculated for C14H17N2O), ESIMS m/z
(% relative abundance) 229 (M+1+, 100), 114 (29).
4.6.19.
N-(Indol-3-ylmethyl)-N-methyl-N0-propylurea
(29). Preparation as reported above for N-ethyl-N0-(in-
dol-3-ylmethyl)-N0-methylurea (28), starting from 77
(220 mg, 1.37 mmol) and substituting propylisocyanate
for ethylisocyanate. The crude reaction mixture was
subjected to FCC (silica gel, CH2Cl2/MeOH, 98:2) to
afford 29 (269 mg, 80% yield) as a white solid. Mp:
1
116–118 ꢁC, CH2Cl2/MeOH. HPLC tR = 11.9 min. H
NMR (500 MHz, CD3CN): d 9.35 (br s, 1H, D2O
exchangeable), 7.65 (d, J = 8 Hz, 1H), 7.41 (d,
J = 8 Hz, 1H), 7.19 (s, 1H), 7.15 (dd, J = 7, 8 Hz, 1H),
7.05 (dd, J = 7, 7.5 Hz, 1H), 5.19 (br s, 1H, D2O
exchangeable), 4.62 (s, 2H), 3.14 (m, 2H), 2.74 (s, 3H),
1.51 (m, 2 H), 0.89 (t, J = 7 Hz, 3H). 13C NMR
(500 MHz, CD3CN): d 158.7, 137.2, 127.3, 124.2,
122.0, 119.6, 119.4, 112.9, 111.7, 43.3, 42.7, 33.0, 23.8,
11.1. FTIR mmax (KBr): 3245, 2963, 1628, 1531, 1351,
744 cmꢀ1. HREIMS: m/z measured 245.1538 (245.1541
calculated for C14H19N3S). EIMS m/z (% relative abun-
dance) 245 (M+, 55), 159 (24), 130 (100), 77 (7).
4.6.23. N-(Naphthalen-2-ylmethyl)-N0-propylurea (33).
Preparation as reported above for N-ethyl-N0-(indol-3-
ylmethyl)urea (24) and substituting 78 (150 mg,
0.96 mmol) for 76 and propylisothiocyanate for ethylis-
ocyanate. The crude reaction mixture was subjected to
FCC (silica gel, CH2Cl2/MeOH, 98:2) to afford 33
(190 mg, 80% yield) as a white solid. Mp: 166–168 ꢁC,
CH2Cl2/MeOH. HPLC tR = 14.2 min. 1H NMR
(500 MHz, (CD3)2SO): d 7.85 (m, 3H), 7.71 (s, 1H),
7.47 (m, 2H), 7.41 (d, J = 8.5 Hz, 1H), 6.40 (br s, 1H,
D2O exchangeable), 5.97 (br s, 1H, D2O exchangeable),
4.37 (d, J = 6 Hz, 2H), 2.99 (m, 2H), 1.39 (m, 3H), 0.84
(t, J = 7.5 Hz, 3H). 13C NMR (500 MHz, (CD3)2SO): d
158.6, 139.2, 133.4, 132.5, 128.2, 128.0, 127.9, 126.6,
126.4, 125.9, 125.3, 43.5, 41.7, 23.7, 11.8. FTIR mmax
(KBr): 3325, 1622, 1581, 1253 cmꢀ1. HRESIMS: m/z
4.6.20.
N-(Indol-3-ylmethyl)-N-methyl-N0-phenylurea
(30). Preparation as reported above for N-ethyl-N0-(in-
dol-3-ylmethyl)-N0-methylurea (28), starting from 77
(200 mg, 1.25 mmol) and substituting phenylisocyanate
for ethylisocyanate. The crude reaction mixture was
subjected to FCC (silica gel, CH2Cl2/MeOH, 98:2) to af-
ford 30 (293 mg, 84% yield) as a white solid. Mp: 147–
150 ꢁC, CH2Cl2/MeOH. HPLC tR = 16.8 min. 1H
NMR (500 MHz, CD3CN): d 9.29 (br s, 1H, D2O
exchangeable), 7.68 (d, J = 8 Hz, 1H), 7.47 (d,
J = 8 Hz, 2H), 7.43 (d, J = 8 Hz, 1H), 3H), 7.27 (m,
3H), 7.14 (m, 2H), 7.07 (dd, J = 7, 7.5 Hz, 1H), 7.02
(dd, J = 7.5, 7.5 Hz, 1H), 4.73 (s, 2H), 2.93 (s, 3H).
13C NMR (500 MHz, CD3CN): d 156.1, 140.8, 137.2,
129.1, 127.2, 124.5, 122.7, 122.1, 120.5, 119.6, 119.5,
112.4, 111.8, 49.3, 33.5. FTIR mmax (KBr): 3281, 1643,
measured
C15H19N2O). ESIMS m/z (% relative abundance) 243
243.1503
(243.1497
calculated
for
(M+1+, 100).
4.6.24. N-(Naphthalen-2-ylmethyl)-N0-phenylurea (34).
Preparation as reported for N-ethyl-N0-(indol-3-ylmeth-
yl)urea (24) and substituting 78 (180 mg, 1.15 mmol)

4972
M.S.C Pedras, M. Jha / Bioorg. Med. Chem. 14 (2006) 4958–4979
for 76 and phenylisothiocyanate for ethylisocyanate.
The crude reaction mixture was subjected to FCC (sil-
ica gel, CH2Cl2/MeOH, 98:2) to afford 34 (250 mg,
80% yield) as a white solid. Mp: 188–190 ꢁC, CH2Cl2/
MeOH. HPLC tR = 19.5 min. 1H NMR (500 MHz,
(CD3)2SO): d 8.59 (s, 1H, D2O exchangeable), 7.89
(m, 3H), 7.78 (s, 1H), 7.49 (m, 3H), 7.42 (d,
J = 7.5 Hz, 2H), 7.22 (t, J = 7.5, 2H), 6.89 (t, J = 7.5,
1H), 6.71 (t, J = 6 Hz, 1H, D2O exchangeable), 4.48
(d, J = 6 Hz, 2H). 13C NMR (500 MHz, (CD3)2SO): d
155.7, 140.9, 138.5, 133.4, 132.6, 129.1, 128.4, 128.0,
127.9, 126.6, 126.3, 126.0, 125.5, 121.6, 118.2, 43.4.
FTIR mmax (KBr): 3317, 1633, 1597, 1246 cmꢀ1. HRE-
SIMS: m/z measured 277.1331 (276.1335 calculated for
C18H17N2O). ESIMS m/z (% relative abundance) 277
(M+1+, 100), 149 (4).
1H), 7.48 (m, 2H), 7.35 (d, J = 7.5 Hz, 1H), 6.44 (br s,
1H, D2O exchangeable), 4.58 (s, 2H), 3.04 (m, 2H),
2,77 (s, 3H), 1.44 (m, 2H), 0.83 (t, J = 7.5 Hz, 3H).
13C NMR (500 MHz, (CD3)2SO): d 158.9, 137.5,
133.8, 133.0, 128.9, 128.4, 128.3, 127.0, 126.6, 126.5,
126.3, 52.0, 42.9, 34.6, 24.0, 12.2. FTIR mmax (KBr):
3344, 2962, 1630, 1534 cmꢀ1. HREIMS: m/z measured
256.1574 (256.1577 calculated for C16H20N2O). EIMS
m/z (% relative abundance) 256 (M+, 43), 170 (64), 141
(100), 115 (33).
4.6.28. N-Methyl-N-(naphthalen-2-ylmethyl)-N0-phenyl-
urea (38). Preparation as reported for N-ethyl-N0-(in-
dol-3-ylmethyl)-N0-methylurea (28), substituting 79
(120 mg, 0.70 mmol) for 77 and phenylisothiocyanate
for ethylisocyanate. The crude reaction mixture was
subjected to FCC (silica gel, CH2Cl2/MeOH, 98:2) to af-
ford 38 (152 mg, 91% yield) as a white solid. Mp: 133–
135 ꢁC, CH2Cl2/MeOH. HPLC tR = 18.7 min. 1H
NMR (500 MHz, (CD3)2SO): d 8.46 (s, 1H, D2O
exchangeable), 7.89 (m, 3H), 7.76 (s, 1H), 7.49 (m,
4H), 7.42 (d, J = 8.5 Hz, 1H), 7.24 (t, J = 8, 2 H), 6.89
(t, J = 7.5, 1H), 4.72 (s, 2H), 2.96 (s 3H). 13C NMR
(500 MHz, (CD3)2SO): d 156.6, 141.4, 137.0, 133.8,
133.1, 129.1, 129.0, 128.4, 128.4, 127.1, 126.7, 126.6,
126.5, 122.7, 120.1, 52.2, 35.2. FTIR mmax (KBr): 3326,
3055, 1642, 1532, 750 cmꢀ1. HREIMS: m/z measured
290.1421 (290.1419 calculated for C19H18N2O). EIMS
m/z (% relative abundance) 290 (M+, 30), 170 (19), 141
(100), 115 (15).
4.6.25. N-Methyl-N-(naphthalen-2-ylmethyl)urea (35).
Preparation as reported for N-(indol-3-ylmethyl)-N-
methylurea (27) and substituting 79 (100 mg,
0.58 mmol) for 77. The crude reaction mixture was sub-
jected to FCC (silica gel, CH2Cl2/MeOH, 98:2) to afford
35 (95 mg, 76% yield) as a white solid. Mp: 182–183 ꢁC,
CH2Cl2/MeOH. HPLC tR = 7.8 min. 1H NMR
(500 MHz, (CD3)2SO): d 7.87 (m, 3H), 7.68 (s, 1H),
7.48 (m, 2H), 7.37 (d, J = 8.5 Hz, 1H), 5.99 (s, 1H,
D2O exchangeable), 4.56 (s, 2H), 2.77 (s, 3H). 13C
NMR (500 MHz, (CD3)2SO): d 159.4, 137.1, 133.4,
132.6, 128.5, 128.0, 127.9, 126.6, 126.3, 126.0, 125.9,
51.6, 34.5. FTIR mmax (KBr): 3371, 1665, 1532,
746 cmꢀ1. HREIMS: m/z measured 214.1105 (214.1106
calculated for C13H14N2O). EIMS m/z (% relative
abundance) 214 (M+, 100), 197 (9), 170 (63), 141 (24),
115 (23).
4.6.29. Naphthalen-1-ylmethylurea (39). Preparation as
reported for N-(indol-3-ylmethyl)-N-methylurea (27)
and substituting 80 (100 mg, 0.63 mmol) for 77. The
crude reaction mixture was subjected to FCC (silica
gel, CH2Cl2/MeOH, 98:2) to afford 39 (97 mg, 74%
yield). Mp: 214–216 ꢁC, CH2Cl2/MeOH. HPLC
4.6.26.
N-Ethyl-N0-methyl-N0-(naphthalen-2-ylmeth-
yl)urea (36). Preparation as reported for N-ethyl-N0-(in-
dol-3-ylmethyl)-N0-methylurea (28) and substituting 79
(100 mg, 0.58 mmol) for 77. The crude reaction mixture
was subjected to FCC (silica gel, CH2Cl2/MeOH, 98:2)
to afford 36 (112 mg, 79% yield) as a white solid. Mp:
1
tR = 8.1 min H NMR (500 MHz, (CD3)2SO): d 8.10,
(d, J = 8 Hz, 1H), 7.94 (d, J = 8.5 Hz, 1H), 7.83 (d,
J = 8 Hz, 1H), 7.53 (m, 2H), 7.45 (m, 2H), 6.42 (br s,
1H, D2O exchangeable), 5.52 (s, 2H, D2O exchange-
able), 4.64 (d, J = 6 Hz, 2H). 13C NMR (500 MHz,
(CD3)2SO): d 158.9, 136.5, 133.8, 131.4, 128.9, 127.8,
126.6, 126.2, 125.9, 125.5, 124.05, 41.3. FTIR mmax
(KBr): 3430, 3339, 1649, 1600, 773 cmꢀ1. HRESIMS:
m/z measured 200.0950 (200.0949 calculated for
C12H12N2O), ESIMS m/z (% relative abundance) 200
(M+, 95), 141 (35), 129 (42).
1
150–151 ꢁC, CH2Cl2/MeOH. HPLC tR = 14.5 min. H
NMR (500 MHz, (CD3)2SO): d 7.87 (m, 3H), 7.67 (s,
1H), 7.48 (m, 2H), 7.35 (d, J = 8.5 Hz, 1H), 6.43 br s,
1 H, D2O exchangeable), 4.57 (s, 2H), 3.11 (m, 2H),
2,77 (s, 3H), 1.05 (t, J = 7 Hz, 3H). 13C NMR
(500 MHz, (CD3)2SO): d 158.8, 137.5, 133.8, 133.0,
128.9, 128.4, 128.3, 127.0, 126.5, 126.3, 52.0, 35.9,
34.5, 16.6. FTIR mmax (KBr): 3341, 2963, 1629,
1534 cmꢀ1
.
HREIMS: m/z measured 242.1411
4.6.30. N-Ethyl-N0-(naphthalen-1-ylmethyl)urea (40).
Preparation as reported for N-ethyl-N0-(indol-3-ylmeth-
yl)urea (24) and substituting 80 (164 mg, 1.04 mmol) for
76. The crude reaction mixture was subjected to FCC
(silica gel, CH2Cl2/MeOH, 98:2) to afford 40 (200 mg,
84% yield) as a white solid. Mp: 183–184 ꢁC, CH2Cl2/
MeOH. HPLC tR = 11.6 min. 1H NMR (500 MHz,
(242.1419 calculated for C15H18N2O). EIMS m/z (% rel-
ative abundance) 242 (M+, 74), 170 (94), 141 (100),
115 (29).
4.6.27. N-Methyl-N-(naphthalen-2-ylmethyl)-N0-propylu-
rea (37). Preparation as reported for N-ethyl-N0-(indol-
3-ylmethyl)-N0-methylurea
(28),
substituting
79
(CD3)2SO): d 8.09 (d, J = 8 Hz, 1H), 7.93 (d,
(100 mg, 0.58 mmol) for 77 and propylisothiocyanate
for ethylisocyanate. The crude reaction mixture was
subjected to FCC (silica gel, CH2Cl2/MeOH, 98:2) to af-
ford 37 (122 mg, 82% yield) as a white solid. Mp: 158–
159 ꢁC, CH2Cl2/MeOH. HPLC tR = 14.2 min. 1H
NMR (500 MHz, (CD3)2SO): d 7.86 (m, 3H), 7.67 (s,
J = 7.5 Hz, 1H), 7.82 (d, J = 8 Hz, 1H), 7.54 (m, 2H),
7.44 (m, 2H), 6.29 (br s, 1H, D2O exchangeable), 5.85
(br s, 1H, D2O exchangeable), 4.66 (d, J = 5.5 Hz,
2H), 3.04 (m, 2H), 0.99 (t, J = 7 Hz, 3H). 13C NMR
(500 MHz, (CD3)2SO): d 158.3, 136.6, 133.8, 131.4,
128.9, 127.7, 126.6, 126.2, 125.9, 125.5, 124.0, 41.4,

M. S. C Pedras, M. Jha / Bioorg. Med. Chem. 14 (2006) 4958–4979
4973
34.6, 16.2. FTIR mmax (KBr): 3330, 1618, 1580,
776 cmꢀ1. HREIMS: m/z measured 228.1260 (228.1262
calculated for C14H16N2O). EIMS m/z (% relative abun-
dance) 228 (M+, 60), 156 (100), 141 (58), 129 (21).
4.6.34.
N-Ethyl-N0-methyl-N0-(naphthalen-1-ylmeth-
yl)urea (44). Preparation as reported for N-ethyl-N0-(in-
dol-3-ylmethyl)-N0-methylurea (28) and substituting 81
(100 mg, 0.58 mmol) for 77. The crude reaction mixture
was subjected to FCC (silica gel, CH2Cl2/MeOH, 98:2)
to afford 44 (101 mg, 81% yield) as a white solid. Mp:
4.6.31. N-(Naphthalen-1-ylmethyl)-N0-propylurea (41).
Preparation as reported for N-ethyl-N0-(indol-3-ylmeth-
yl)urea (24) and substituting 80 (160 mg, 1.02 mmol) for
76 and propylisocyanate for ethylisocyanate. The crude
reaction mixture was subjected to FCC (silica gel,
CH2Cl2/MeOH, 98:2) to afford 41 (200 mg, 81% yield)
as a white solid. Mp: 136–137 ꢁC, CH2Cl2/MeOH.
174–175 ꢁC.
HPLC
tR = 14.1 min.
1H
NMR
(500 MHz, (CD3)2SO): d 8.13 (br s, 1H), 9.93 (br s,
1H), 7.83 (d, J = 8 Hz, 1H), 7.52 (br s, 2H), 7.47 (t,
J = 7.5 Hz, 1H), 7.27 (d, J = 7 Hz, 1H), 6.43 (br s, 1H,
D2O exchangeable), 4.89 (s, 2H), 3.11 (m, 2 H), 2.74
(s, 3H), 1.04(t, J = 7 Hz, 3H). 13C NMR (500 MHz,
(CD3)2SO): d 158.7, 134.9, 134.3, 132.0, 129.3, 128.3,
126.9, 126.6, 126.2, 125.8, 124.5, 49.8, 35.9, 34.3, 16.6.
FTIR mmax (KBr): 3347, 1630, 1532, 785 cmꢀ1. HRE-
IMS: m/z measured 242.1421(228.1419 calculated for
C15H18N2O). EIMS m/z (% relative abundance) 242
(M+, 93), 170 (82), 141 (100), 15 (54).
1
HPLC tR = 14.1 min. H NMR (500 MHz, (CD3)2SO):
d 8.09 (d, J = 8 Hz, 1H), 7.93 (d, J = 7.5 Hz, 1H), 7.82
(d, J = 8 Hz, 1H), 7.53 (m, 2H), 7.44 (m, 2H), 6.28 (br
s, 1H, D2O exchangeable), 5.90 (br s, 1H, D2O
exchangeable), 4.66 (d, J = 5.5 Hz, 2H), 2.98 (m, 2H),
1.38 (m, 2H), 0.83 (t, J = 7.5 Hz, 3H). 13C NMR
(500 MHz, (CD3)2SO): d 158.4, 136.6, 133.8, 131.4,
128.9, 127.7, 126.6, 126.2, 125.9, 125.5, 124.0, 41.6,
41.4, 23.7, 11.8. FTIR mmax (KBr): 3313, 1626, 1591,
4.6.35. N-Methyl-N-(naphthalen-1-ylmethyl)-N0-propyl-
urea (45). Preparation as reported for N-ethyl-N0-(in-
dol-3-ylmethyl)-N0-methylurea (28), substituting 81
(100 mg, 0.58 mmol) for 77 and propylisocyanate for
ethylisocyanate. The crude reaction mixture was subject-
ed to FCC (silica gel, CH2Cl2/MeOH, 98:2) to afford 45
(119 mg, 80% yield) as a white solid. Mp: 147–148 ꢁC.
792 cmꢀ1
.
HRESIMS: m/z measured 243.1497
(243.1491 calculated for C15H19N2O). ESIMS m/z (%
relative abundance) 243 (M+1+, 100), 114 (11).
4.6.32. N-(Naphthalen-1-ylmethyl)-N0-phenylurea (42).
Preparation as reported for N-ethyl-N0-(indol-3-ylmeth-
yl)urea (24), substituting 80 (160 mg, 1.02 mmol) for 76
and phenylisocyanate for ethylisocyanate. The crude
reaction mixture was subjected to FCC (silica gel,
CH2Cl2/MeOH, 98:2) to afford 42 (247 mg, 88% yield)
as a white solid. Mp: 221–223 ꢁC. HPLC tR = 19.5 min.
1H NMR (500 MHz, (CD3)2SO): d 8.51 (s, 1H, D2O
exchangeable), 8.13 (d, J = 8 Hz, 1H), 7.95 (d,
J = 8 Hz, 1H), 7.85 (d, J = 7.5 Hz, 1H), 7.56 (m, 2H),
7.49 (m, 2H), 7.41 (d, J = 8 Hz, 2H), 7.22 (t, J = 8 Hz,
2H), 6.89 (t, J = 7.5 Hz, 1H), 6.63 (br s, 1H, D2O
exchangeable), 4.77 (d, J = 5.5 Hz, 2H). 13C NMR
(500 MHz, (CD3)2SO): d 155.5, 140.9, 135.9, 133.8,
131.3, 121.1, 129.0, 128.0, 126.7, 126.3, 125.9, 125.8,
123.9, 122.6, 118.1, 41.2. FTIR mmax (KBr): 3323,
1628, 1571, 778 cmꢀ1 HREIMS: m/z measured
276.1260 (276.1262 calculated for C18H16N2O). EIMS
m/z (% relative abundance) 276 (M+, 49), 156 (10), 141
(100), 115 (15).
1
HPLC tR = 14.2 min. H NMR (500 MHz, (CD3)2SO):
d 8.12 (dd, J = 6, 6 Hz, 1H), 7.93 (dd, J = 6, 6 Hz,
1H), 7.83 (d, J = 8 Hz, 1H), 7.52 (dd, J = 6, 6 Hz, 2H),
7.47 (t, J = 7.5 Hz, 1H), 7.27 (t, J = 7 Hz, 1H), 6.41
(br s, 1H, D2O exchangeable), 4.89 (s, 2H), 3.05 (m,
2H), 2.74 (s, 3H), 1.44 (m, 2H), 0.84 (t, J = 7.5 Hz,
3H). 13C NMR (500 MHz, (CD3)2SO): d 158.7, 135.0,
134.3, 132.0, 129.3, 128.3, 126.9, 126.6, 126.4, 125.7,
124.5, 49.9, 42.9, 34.3, 24.0, 12.2. FTIR mmax (KBr):
3321, 1625, 1587 cmꢀ1
. HREIMS: m/z measured
256.1574 (256.1575 calculated for C16H20N2O). EIMS
m/z (% relative abundance) 256 (M+, 68), 170 (87), 141
(58), 15 (22).
4.6.36. N-Methyl-N-(naphthalen-1-ylmethyl)-N0-phenyl-
urea (46). Preparation as reported for N-ethyl-N0-(in-
dol-3-ylmethyl)-N0-methylurea (28), substituting 81
(100 mg, 0.58 mmol) for 77 and phenylisocyanate for
ethylisocyanate. The crude reaction mixture was subject-
ed to FCC (silica gel, CH2Cl2/MeOH, 98:2) to afford 46
(146 mg, 86% yield) as a white solid. Mp: 153–154 ꢁC,
CH2Cl2/MeOH. HPLC tR = 23.7 min. 1H NMR
(500 MHz, (CD3)2SO): d 8.43 (s, 1H, D2O exchange-
able), 8.13 (d, J = 7.5 Hz, 1H), 7.96 (d, J = 7 Hz, 1H),
7.86 (d, J = 8 Hz, 1H), 7.54 (m, 5H), 7.37 (d, J = 7 Hz,
1H), 7.24 (t, J = 8 Hz, 2 H), 6.95 (t, J = 7.5 Hz, 1H),
5.04 (s, 2H), 2.93 (s, 3H). 13C NMR (500 MHz,
(CD3)2SO): d 156.0, 141.0, 134.1, 133.9, 131.6, 129.0,
128.7, 128.0, 126.7, 126.3, 125.9, 125.5, 124.0, 122.3,
120.4, 49.6, 34.7. FTIR mmax (KBr): 3328, 1631, 1531,
4.6.33. N-Methyl-N-(naphthalene-1-ylmethyl)urea (43).
Preparation as reported for N-(indol-3-ylmethyl)-N-
methylurea (27) and substituting 81 (100 mg,
0.58 mmol) for 77. The crude reaction mixture was sub-
jected to FCC (silica gel, CH2Cl2/MeOH, 98:2) to afford
43 (98 mg, 78% yield) as a white solid. Mp: 193–194 ꢁC.
1
HPLC tR = 8.4 min. H NMR (500 MHz, (CD3)2SO): d
8.15, (br s, 1H), 7.93 (br s, 1H), 7.84 (d, J = 8 Hz, 1H),
7.52 (br s, 2H), 7.47 (t, J = 7 Hz, 1H), 7.31 (d, J = 7 Hz,
1H), 6.02 (br s, 1H, D2O exchangeable), 4.88 (s, 2H),
2.74 (s, 3 H). 13C NMR (500 MHz, (CD3)2SO): d
159.7, 134.9, 134.3, 132.2, 129.3, 128.9, 128.3, 126.9,
126.6, 126.5, 126.2, 125.7, 1245, 49.8, 34.6. FTIR mmax
(KBr): 3433, 3338, 1650, 1600, 774 cmꢀ1. HREIMS:
m/z measured 214.1112 (214.1106 calculated for
C13H14N2O). EIMS m/z (% relative abundance) 214
(M+, 21), 170 (88), 141 (100), 15 (19).
1245, 759 cmꢀ1
.
EIMS: m/z measured 290.1421
(290.1419 calculated for C19H18N2O). EIMS m/z (% rel-
ative abundance) 290 (M+, 38), 141 (100), 115 (11).
4.6.37. 3-Indolylmethylthiourea (47). A solution of 1-
Boc-indol-3-ylmethylamine (220 mg, 0.85 mmol) in
CH2Cl2 (5 mL) was added dropwise to a vigorously

4974
M.S.C Pedras, M. Jha / Bioorg. Med. Chem. 14 (2006) 4958–4979
stirred mixture of CSCl2 (71 lL, 0.94 mmol) and
CaCO3 (107 mg, 1.07 mmol) in CH2Cl2 (6 mL) and
H2O (10 mL). After stirring for 5 min at rt, the CH2Cl2
layer was separated and the aqueous layer was extract-
ed with additional CH2Cl2 (5 mL). The organic layers
were combined and concentrated, and the residue was
subjected to FCC (silica gel, CH2Cl2) to afford 1-Boc-
indol-3-ylmethylisothiocyanate (91) (196 mg, 71%
yield) as a light yellow solid. Ammonia gas was bub-
bled into a solution of isothiocyanate 91 in CH2Cl2
(10 mL). After 60 min, CH2Cl2 was evaporated, and
the residue was dissolved in MeOH, and Na (100 mg,
4.30 mmol cut in small pieces) was added to the solu-
tion. The mixture was stirred at rt for 60 min, the sol-
vent was evaporated, and the residue was dissolved in
H2O (20 mL) and extracted with EtOAc. The organic
layer was then evaporated and the residue was subject-
ed to FCC (silica gel, CH2Cl2/MeOH, 98:2) to afford
47 (59 mg, 40% yield) as an off-white solid. Mp: 132–
133 ꢁC, CH2Cl2/MeOH. HPLC tR =: 6.7 min. 1H
NMR (500 MHz, CD3CN) mixture of rotamers (1:3):
d 9.24 (br s, 1 H, D2O exchangeable), 7.67 (br s,
1H), 7.44 (d, J = 8 Hz, 1H), 7.27 (s, 1H), 7.18 (dd,
J = 7, 7.5 Hz, 1H), 7.09 (dd, J = 7.5, 7 Hz, 1H), 6.75
(br s, 1H, D2O exchangeable), 5.98 (br s, 1H, D2O
exchangeable), 4.85 (br s, 2H); Additional peaks of
minor rotamer 6.93 (br s, 0.5H, D2O exchangeable),
5.98 (br s, 1H, D2O exchangeable), 4.47 (br s, 0.5H).
13C NMR (500 MHz, CD3CN): d 183.1, 136.5, 126.6,
123.8, 121.7, 119.1, 118.7, 111.7, 111.5, 40.2. FTIR
yield) as a white solid. Mp: 114–116 ꢁC, CH2Cl2. HPLC
tR = 14.8 min. 1H NMR (500 MHz, CD3CN): d 9.21 (br
s, 1H, D2O exchangeable), 7.68 (d, J = 8 Hz, 1H), 7.43
(d, J = 8 Hz, 1H), 7.27 (d, J = 2 Hz, 1H), 7.18 (dd,
J = 8, 8, Hz, 1H), 7.08 (dd, J = 8, 8 Hz, 1H), 6.45 (br
s, 1H, D2O exchangeable), 6.31 (br s, 1H, D2O
exchangeable), 4.82 (br s, 2H), 3.37 (br s, 2H), 1.53
(m, 2H), 0.87 (t, J = 7 Hz, 3H). 13C NMR (500 MHz,
CD3CN): d 183.1, 136.6, 126.6, 123.8, 121.8, 119.2,
118.3, 112.1, 111.4, 45.5, 39.6, 22.0, 10.5. FTIR mmax
(KBr): 3251, 1548, 1458, 1135, 744 cmꢀ1. HREIMS:
m/z measured 247.1137 (247.1143 calculated for
C13H17N3S). EIMS m/z (% relative abundance) 247
(M+, 50), 145 (12), 130 (100), 77 (11), 61 (39).
4.6.40. N-(Indol-3-ylmethyl)-N0-phenylthiourea (50).
Preparation as reported for N-(indol-3-ylmethyl)-N0-
propylthiourea (49), starting from 76 (200 mg,
1.37 mmol) and substituting phenylisothiocyanate for
propylisothiocyanate. The crude reaction mixture was
subjected to FCC (silica gel, CH2Cl2) to afford 50
(345 mg, 90% yield) as a white solid. Mp: 153–155
(dec) ꢁC, CH2Cl2. HPLC tR = 18.0. 1H NMR
(500 MHz, CD3CN): d 9.21 (br s, 1H, D2O exchange-
able), 8.10(br s, 1H, D2O exchangeable), 7.70 (d,
J = 8 Hz, 1H), 7.43 (d, J = 8 Hz, 1H), 7.34 (dd, J = 8,
7.5 Hz, 2H), 7.28 (d, J = 7.5 Hz, 3H), 7.19 (m, 2H),
7.09 (dd, J = 7, 8 Hz, 1H), 6.71 (br s, 1H D2O exchange-
able), 4.94 (d, J = 5 Hz, 2H). 13C NMR (500 MHz,
CD3CN): d 181.3, 138.3, 136.5, 129.6, 127.1, 126.3,
125.2, 124.5, 122.2, 119.6, 119.2, 112.1, 111.9, 40.7.
FTIR mmax (KBr): 3261, 1530, 1498, 742 cmꢀ1. HRE-
IMS: m/z measured 281.0981 (281.0987 calculated for
C16H15N3S). EIMS m/z (% relative abundance) 281
(M+, 7), 152 (81), 135 (100), 93, (65).
mmax (KBr): 3295, 1611, 1539, 1457, 1346, 716 cmꢀ1
.
HREIMS: m/z measured 205.0672 (205.0673 calculated
for C10H11N3S). EIMS m/z (% relative abundance) 205
(M+, 45), 130 (100), 76 (25).
4.6.38. N-(Indol-3-ylmethyl)-N0-methylthiourea (48).
Preparation as reported for indol-3-ylmethylthiourea
(47) and substituting methylamine for ammonia. The
crude reaction mixture was subjected to FCC (silica
gel, CH2Cl2) to afford 48 (76 mg, 50% yield) as an off-
white solid. Mp: 110–112 ꢁC, CH2Cl2. HPLC
4.6.41. N-(Indol-3-ylmethyl)-N-methyl-N0-propylthiourea
(51). Propylisothiocyanate (124 lL, 1.20 mmol) was
added to a solution of N-(indol-3-ylmethyl)-N-methyl-
amine (77, 160 mg, 1.0 mmol) in CH2Cl2 (5 mL). After
allowing the reaction mixture to stir for 15 min at rt,
the solvent was evaporated and the crude reaction mix-
ture was subjected to FCC (silica gel, CH2Cl2) to afford
49 (219 mg, 84% yield) as a white solid. Mp: 129–131 ꢁC,
CH2Cl2. HPLC tR = 18.1 min. 1H NMR (500 MHz,
CD3CN): d 9.26 (br s, 1H, D2O exchangeable), 7.76
(d, J = 8 Hz, 1H), 7.42 (d, J = 8 Hz, 1H), 7.26 (s, 1H),
7.16 (dd, J = 7.5, 7.5 Hz, 1H), 7.05 (dd, J = 7.5,
7.5 Hz, 1H), 6.24 (br s, 1H, D2O exchangeable), 5.24
(s, 2H), 3.58 (m, 2H), 3.0 (s, 3H), 1.62 (m, 2H), 0.91
(t, J = 7.5 Hz, 3H). 13C NMR (500 MHz, CD3CN): d
182.1, 137.1, 127.1, 124.6, 122.2, 119.8, 119.5, 111.9,
111.8, 48.6, 47.7, 35.9, 22.7, 11.0. FTIR mmax (KBr):
3278 (br), 2962, 1532, 1381, 1343, 745 cmꢀ1. HREIMS:
m/z measured 261.1306 (261.1299 calculated for
C14H19N3S). EIMS m/z (% relative abundance) 261
(M+, 55), 160 (16), 132 (100), 130 (95), 102 (30).
1
tR = 8.5 min. H NMR (500 MHz, CD3CN): d 9.22 (br
s, 1H, D2O exchangeable), 7.68 (d, J = 8.5 Hz, 1H),
7.43 (d, J = 8 Hz, 1H), 7.27 (s, 1H), 7.71 (dd, J = 7.5,
7.5 Hz, 1H), 7.08 (dd, J = 7.5, 7, Hz, 1H), 6.51 (br s,
1H, D2O exchangeable), 6.32 (br s, 1H, D2O exchange-
able), 4.82 (br s, 2H), 2.89 (br s, 3H). 13C NMR
TM
(500 MHz, CD3CN) mixture of rotamers:
190.9,
136.4, 126.7, 124.2, 121.8, 119.2, 118.7, 111.4, 111.1,
56.5, 40.6; additional peaks of minor rotamer 190.2,
126.5, 124.2, 119.3, 118.4, 111.5, 111.3, 57.5, 38.0. FTIR
mmax (KBr): 3347, 1520, 1454, 1337, 1201, 1144,
745 cmꢀ1. HREIMS: m/z measured 219.0827 (219.0830
calculated for C11H13N3S). EIMS m/z (% relative abun-
dance) 219 (M+, 50), 130 (100), 90 (81).
4.6.39. N-(Indol-3-ylmethyl)-N0-propylthiourea (49).
Indol-3-ylmethylamine (76, 210 mg, 1.44 mmol) was dis-
solved in CH2Cl2 (5 mL) and propylisothiocyanate
(163 lL, 1.58 mmol) was added. After allowing the reac-
tion mixture to stir for 15 min at rt, the solvent was
evaporated and the crude reaction mixture was subject-
ed to FCC (silica gel, CH2Cl2) to afford 49 (305 mg, 86%
4.6.42. N-(Indol-3-ylmethyl)-N-methyl-N0-phenylthiourea
(52). Preparation as reported for N-(indol-3-ylmethyl)-
N-methyl-N0-propylthiourea (51), starting from 77
(200 mg, 1.25 mmol) and substituting phenylisothiocya-
nate for propylisothiocyanate. The crude reaction mix-

M. S. C Pedras, M. Jha / Bioorg. Med. Chem. 14 (2006) 4958–4979
4975
ture was subjected to FCC (silica gel, CH2Cl2) to afford
52 (291 mg, 83% yield) as a white solid. Mp: 159–
161 ꢁC, CH2Cl2. HPLC tR = 19.6 min. 1H NMR
(500 MHz, CD3CN): d 9.29 (br s, 1H, D2O exchange-
able), 7.81 (d, J = 8 Hz, 2H), 7.46 (d, J = 8 Hz, 1H),
7.36 (d, J = 6.5 Hz, 4H), 7.19 (m, 3H), 7.10 (m, 1H),
5.33 (s, 2H), 3.17 (s, 3H). 13C NMR (500 MHz,
CD3CN): d 182.2, 141.3, 137.1, 128.5, 127.1, 126.4,
125.4, 124.9, 122.3, 119.8, 119.7, 111.8, 111.4, 49.1,
37.0. FTIR mmax (KBr): 3361, 3240, 1520, 1382, 1329,
(d, J = 8.5, 1H), 5.23 (s, 2H), 3.49 (m, J = 7.5, 6 Hz,
2H), 3.03 (s, 3H), 1.57 (m, 2H), 0.85 (t, J = 7.5 Hz,
3H). 13C NMR (500 MHz, CD3CN): d 182.6, 136.5,
133.7, 133.1, 128.9, 128.4, 128.3, 127.1, 126.6, 126.4,
126.3, 56.7, 48.2, 37.6, 22.9, 12.1. FTIR mmax (KBr):
3325, 1581 1253 cmꢀ1
. HREIMS: m/z measured
272.1341 (272.1347 calculated for C16H20N2S). EIMS
m/z (% relative abundance) 272 (M+, 17), 202 (17), 171
(100), 155 (20), 127 (27).
753 cmꢀ1
.
HREIMS: m/z measured 295.1146
4.6.46. N-Methyl-N-(naphthalen-2-ylmethyl)-N0-phenyl-
thiourea (56). Preparation as reported for N-(indol-3-
ylmethyl)-N-methyl-N0-propylthiourea (51), substituting
79 (100 mg, 0.58 mmol) for 77 and phenylisothiocyanate
for propylisothiocyanate. The crude reaction mixture
was subjected to FCC (silica gel, CH2Cl2) to afford 57
(146 mg, 82% yield) as a white solid. Mp: 183–184 ꢁC,
CH2Cl2. HPLC tR = 23.9 min. 1H NMR (500 MHz,
(CD3)2SO): d 9.24 (s, 1H, D2O exchangeable), 7.91 (m,
3H), 7.79 (s, 1H), 7.52 (m, 3H), 7.37 (d, J = 8 Hz, 2H),
7.32 (t, J = 7.5 Hz, 2 H), 7.14 (t, J = 7 Hz, 1H), 5.34
(s, 2H), 3.22 (s, 3H). 13C NMR (500 MHz, (CD3)2SO):
d 182.4, 141.9, 136.0, 133.8, 132.2, 129.0, 128.4, 128.7,
128.5, 127.1, 127.0, 126.7, 126.6, 126.5, 125.5, 57.0,
38.6 FTIR mmax (KBr): 3239, 1526, 1337 cmꢀ1. HRE-
IMS: m/z measured 306.1184 (306.1191 calculated for
C19H18N3S). EIMS m/z (% relative abundance) 306
(M+, 7), 170 (70), 141 (100), 115 (38), 77 (86).
(279.1143 calculated for C17H17N3S). EIMS m/z (% rel-
ative abundance) 295 (M+, 3), 160(35), 135 (100), 130
(80), 77 (60).
4.6.43.
N-(Naphthalen-2-ylmethyl)-N0-propylthiourea
(53). Preparation as reported for N-(indol-3-ylmethyl)-
N-propylthiourea (49) and substituting 78 (150 mg,
0.95 mmol) for 76. The crude reaction mixture was sub-
jected to FCC (silica gel, CH2Cl2) to afford 53 (192 mg,
78% yield) as a white solid. Mp: 116–117 ꢁC, CH2Cl2.
1
HPLC tR = 19.3 min. H NMR (500 MHz, (CD3)2SO):
d 7.87 (m, 3H), 7.74 (s, 1H), 7.57 (br s, 1H, D2O
exchangeable), 7.48 (m, 3 H), 4.82 (br s, 2H), 3.34 (br
s, 2H), 1.51 (m 2H), .85 (t, J = 7 Hz, 3H). 13C NMR
(500 MHz, (CD3)2SO): d 138.0, 133.7, 133.0, 128.7,
128.4, 128.3, 127.0, 126.7, 126.5, 126.2, 47.9, 46.2,
40.5, 22.9, 12.2. FTIR mmax (KBr): 3257, 3073, 1556,
1359 cmꢀ1
.
HREIMS: m/z measured 258.1197
(258.1191 calculated for C15H18N2S). EIMS m/z (% rel-
ative abundance) 258 (M+, 50), 156 (25), 141 (100),
115 (20).
4.6.47.
N-(Naphthalen-1-ylmethyl)-N0-propylthiourea
(57). Preparation as reported for N-(indol-3-ylmethyl)-
N0-propylthiourea (49), substituting 80 (100 mg,
0.64 mmol) for 76. The crude reaction mixture was sub-
jected to FCC (silica gel, CH2Cl2) to afford 57 (133 mg,
81% yield) as a white solid. Mp: 114–115 ꢁC, CH2Cl2.
4.6.44.
N-(Naphthalen-2-ylmethyl)-N0-phenylthiourea
(54). Preparation as reported for N-(indol-3-ylmethyl)-
N0-propylthiourea (49), substituting 78 (200 mg,
1.27 mmol) for 76 and phenylisothiocyanate for propy-
lisothiocyanate. The crude reaction mixture was sub-
jected to FCC (silica gel, CH2Cl2) to afford 54
(327 mg, 88% yield) as a white solid. Mp: 172–
173 ꢁC, CH2Cl2. HPLC tR = 21.8 min. 1H NMR
(500 MHz, (CD3)2SO): d 9.66 (br s, 1H, D2O exchange-
able), 8.26 (br s, 1H, D2O exchangeable), 7.89 (d,
J = 8 Hz, 3H), 7.80 (s, 1H), 7.49, (m, 3H), 7.45 (d,
J = 8 Hz, 2H), 7.33 (dd, J = 7.5, 8 Hz, 2 H), 7.12 (dd,
J = 7, 7 Hz, 1H), 4.91 (d, J = 5 Hz, 2 H). 13C NMR
(500 MHz, (CD3)2SO): d 181.3, 139.5, 137.1, 133.3,
132.6, 129.1, 128.3, 127.9, 126.7, 126.4, 126.1, 125.9,
124.9, 123.9, 118.2, 47.8. FTIR mmax (KBr): 3382,
3174, 1544, 1588, 1270 cmꢀ1. HRESIMS: m/z measured
293.1116 (293.1106 calculated for C18H17N2S). ESIMS
m/z (% relative abundance) 293 (M+1+, 100), 163 (15),
114 (30).
1
HPLC tR = 19.5 min. H NMR (500 MHz, (CD3)2SO):
d 8.08 (d, J = 7.5 Hz, 1H), 7.95 (d, J = 8 Hz, 1H), 7.85
(d, J = 8 Hz, 1H), 7.76 (br s, 1H, D2O exchangeable),
7.55 (m, 2H), 7.47 (m, 2H), 5.08 (br s, 2H), 3.33 (br s,
2H), 1.48 (m, 2H), 0.85 (t, J = 7 Hz, 3H). 13C NMR
(500 MHz, (CD3)2SO): d 182.3, 135.4, 134.1, 131.8,
129.4, 128.4, 127.1, 126.7, 126.3, 124.4, 46.3, 40.5,
22.9, 12.1. FTIR mmax (KBr): 3257, 1550, 793 cmꢀ1
.
HREIMS: m/z measured 258.1200 (258.1191 calculated
for C15H18N2S). EIMS m/z (% relative abundance) 258
(M+, 35), 156 (18), 141 (100), 115 (21).
4.6.48.
N-(Naphthalen-1-ylmethyl)-N0-phenylthiourea
(58). Preparation as reported for N-(indol-3-ylmethyl)-
N0-propylthiourea (49), substituting 80 (100 mg, 0.64
mmol) for 76 and phenylisothiocyanate for propylisoth-
iocyanate. The crude reaction mixture was subjected to
FCC (silica gel, CH2Cl2) to afford 58 (156 mg, 84%
yield) as a white solid. Mp: 195–196 ꢁC, CH2Cl2. HPLC
1
4.6.45. N-Methyl-N-(naphthalen-2-ylmethyl)-N0-propyl-
thiourea (55). Preparation as reported for N-(indol-3-
ylmethyl)-N-methyl-N0-propylthiourea (51) and substi-
tuting 79 (100 mg, 0.58 mmol) for 77. The crude reaction
mixture was subjected to FCC (silica gel, CH2Cl2) to af-
ford 55 (126 mg, 79% yield) as a light yellow solid. Mp:
67–69 ꢁC, CH2Cl2. HPLC tR = 23.0 min. 1H NMR
(500 MHz, (CD3)2SO): d 7.87 (m, 3H), 7.69 (s, 1H),
7.59 (br s, 1H, D2O exchangeable), 7.50 (m, 2 H), 7.42
tR = 21.9 min. H NMR (500 MHz, (CD3)2SO): d 9.58
(br s, 1H, D2O exchangeable), 8.15 (br s, 1H, D2O
exchangeable), 7.88 (dd, J = 6.5, 6.5 Hz, 1H), 7.58 (m,
2H), 7.48 (m, 4H), 7.30 (dd, J = 7.5, 8 Hz, 2H), 7.10
(t, J = 7.5 Hz, 1H), 5.18 (d, J = 5 Hz, 2H). 13C NMR
(500 MHz, CD3CN): d 181.5, 140.2, 134.9, 134.2,
131.8, 129.4, 128.6, 127.2, 126.8, 126.6, 126.3, 125.0,
124.4, 123.8, 46.3. FTIR mmax (KBr): 3275, 1525,
1339 cmꢀ1
.
HREIMS: m/z measured 292.1036

4976
M.S.C Pedras, M. Jha / Bioorg. Med. Chem. 14 (2006) 4958–4979
(292.1034 calculated for C18H16N2S). EIMS m/z (% rel-
ative abundance) 292 (M+, 50), 160 (18), 141 (100), 115
(20), 93 (35).
4.6.52. N-(Indol-3-ylmethyl)-N0-methylsulfamide (62). A
mixture of N-(indol-3-ylmethyl)-N-methylamine (77,
220 mg, 2.0 mmol) and sulfamide (211 mg, 2.20 mmol)
in H2O (5 mL) was heated at 100 ꢁC for 120 min. The
reaction mixture was cooled to rt, was extracted with
EtOAc, the extract was dried, and the solvent was evap-
orated. The residue obtained was subjected to FCC
(CH2Cl2/MeOH, 98:2) to give 62 (115 mg, 35% yield)
as a, off-white solid. Mp: 142–144 ꢁC, CH2Cl2/MeOH.
HPLC tR = 5.8 min. 1H NMR (500 MHz, CD3CN):
7.69 (d, J = 8 Hz, 1H), 7.39 (d, J = 8 Hz, 1H, 7.24 (dd,
J = 8, 8 Hz, 1H), 7.19 (s, 1H), 7.12 (dd, J = 8, 8 Hz,
1H), 5.02 (br s, 1H, D2O exchangeable), 5.16 (br s,
2H, D2O exchangeable), 4.37 (d, J = 6 Hz, 2H), 3.78
(s, 3H). 13C NMR (500 MHz, CD3CN): d 137.2, 127.5,
125.5, 122.4, 119.8, 119.4, 112.0, 110.1, 46.22, 34.2.
FTIR mmax (KBr): 3409, 1341, 1161, 746 cmꢀ1. HRE-
IMS: m/z measured 239.0729 (239.0728 calculated for
C10H13N3O2S). EIMS m/z (% relative abundance) 239
(M+, 17), 147 (8), 130 (100).
4.6.49. N-Methyl-N-(naphthalen-1-ylmethyl)-N0-propyl-
thiourea (59). Preparation as reported for N-(indol-3-
ylmethyl)-N-methyl-N0-propylthiourea
(51)
and
substituting 81 (100 mg, 0.58 mmol) for 77. The crude
reaction mixture was subjected to FCC (silica gel,
CH2Cl2) to afford 59 (126 mg, 79% yield) as a white
1
solid. Mp: 81–82 ꢁC, CH2Cl2. HPLC tR = 22.7 min. H
NMR (500 MHz, (CD3)2SO): d 8.11 (d, J = 6 Hz, 1H),
7.94 (d J = 6 Hz, 1H), 7.84 (d, J = 8 Hz, 1H), 7.63 (br
s, 1H, D2O exchangeable), 7.54 (m, 2H), 7.47 (t,
J = 7.5 Hz, 1H), 7.19 (d, J = 7 Hz, 1H), 5.52 (s, 2H),
3.52 (m, 2H), 3.05 (s, 3H), 1.59 (m, 2H), 0.86 (t,
J = 7.5, 1H). 13C NMR (500 MHz, (CD3)2SO): d
182.5, 134.3, 133.8, 131.8, 129.4, 128.4, 127.0, 126.7,
126.3, 125.1, 124.4, 54.8, 48.2, 38.0, 22.9, 12.1. FTIR
mmax (KBr): 3361, 1530, 1544, 742 cmꢀ1. HREIMS: m/z
measured 272.1346 (272. 1347 calculated for
C16H20N2S). EIMS m/z (% relative abundance) 272
(M+, 100), 170 (52), 141 (55), 115 (13).
4.6.53. Naphthalen-2-ylmethylsulfamide (63). Prepara-
tion as reported for indol-3-ylmethylsulfamide (61) and
substituting 78 (200 mg, 1.27 mmol) for 76. The crude
reaction mixture was subjected to FCC (silica gel,
CH2Cl2/MeOH, 98:2) to afford 63 (228 mg, 76% yield)
as a white solid. Mp: 164–166 ꢁC, CH2Cl2/MeOH.
4.6.50. N-Methyl-N-(naphthalen-1-ylmethyl)-N0-phenyl-
thiourea (60). Preparation as reported for N-(indol-3-
ylmethyl)-N-methyl-N0-propylthiourea (51), substituting
81 (100 mg, 0.58 mmol) for 77 and phenylisothiocyanate
for propylisothiocyanate. The crude reaction mixture
was subjected to FCC (silica gel, CH2Cl2) to afford 60
(147 mg, 82% yield) as a white solid. Mp: 153–154 ꢁC,
CH2Cl2. HPLC tR = 23.7. 1H NMR (500 MHz,
(CD3)2SO): d 9.28 (br s, 1H, D2O exchangeable), 8.12
(d, J = 8 Hz, 1H), 7.97 (d, J = 8 Hz, 1H), 7.88 (d,
J = 8 Hz, 1H), 7.55 (m, 3H), 7.33 (m, 5H), 7.14 (t,
J = 7 Hz, 1H), 5.62 (s, 2H), 3.22 (s, 3H). 13C NMR
(500 MHz, (CD3)2SO): d 182.8, 141.9, 134.3, 133.4,
131.8, 129.4, 128.8, 128.4, 127.1, 126.9, 126.8, 126.4,
125.5, 125.2, 55.1, 39.0. FTIR mmax (KBr): 3257, 1538,
1341 cmꢀ1. HRMS: m/z measured 306.1181 (306.1190
calculated for C19H18N2S). EIMS m/z (% relative abun-
dance) 306 (M+, 51), 170 (31), 141 (100), 77 (46).
1
HPLC tR = 11.1 min. H NMR (500 MHz, (CD3)2SO):
d 7.91 (m, 4H), 7.52 (m, 3H), 5.57 (br s, 1H, D2O
exchangeable), 5.24 (br s, D2O exchangeable), 4.36 (d,
J = 5.5 Hz, 2H). 13C NMR (500 MHz, (CD3)2SO): d
134.2, 133.3, 131.8, 128.9, 128.8, 127.1, 126.7, 126.4,
125.8, 124.1, 45.5. FTIR mmax (KBr): 3272, 1329,
1154 cmꢀ1
.
HREIMS: m/z measured 236.0613
(236.0619 calculated for C11H12N2O2S). EIMS m/z (%
relative abundance) 236 (M+, 54), 155 (100), 141 (38),
128 (24).
4.6.54.
N-Methyl-N-(naphthalen-2-ylmethyl)sulfamide
(64). Preparation as reported for N-(indol-3-ylmethyl-
N0-methylsulfamide (62) and substituting 79 (100 mg,
0.58 mmol) for 77. The crude reaction mixture was sub-
jected to FCC (silica gel, CH2Cl2/MeOH, 98:2) to afford
64 (109 mg, 71% yield) as a white solid. Mp: 140–142 ꢁC,
CH2Cl2/MeOH. HPLC tR = 13.3 min. 1H NMR
(500 MHz, (CD3)2SO):TM 7.91 (m, 4H), 7.85 (s, 1H),
7.51 (m, 3H), 6.97 (s, 2H, D2O exchangeable), 4.25 (s,
2H), 2.56 (s, 3H). 13C NMR (500 MHz, (CD3)2SO): d
135.4, 133.7, 133.3, 129.0, 128.5, 128.4, 127.7, 127.2,
127.1, 126.8, 54.8, 35.6. FTIR mmax (KBr): 3269, 1341,
4.6.51. Indol-3-ylmethylsulfamide (61). A mixture of in-
dol-3-ylmethylamine (76, 200 mg, 1.37 mmol) and sul-
famide (147 mg, 1.50 mmol) in H2O (5 mL) was
heated at 100 ꢁC for 120 min. The reaction mixture
was cooled to rt, was extracted with EtOAc, the extract
was dried, and the solvent was evaporated. The residue
was subjected to FCC (CH2Cl2/MeOH, 98:2) to give 61
(135 mg, 44% yield) as a white solid. Mp: 132–134 ꢁC,
CH2Cl2/MeOH. HPLC tR = 8.6 min. 1H NMR
(500 MHz, CD3CN): d 9.24 (br s, 1H, D2O exchange-
able), 7.69 (d, J = 8 Hz, 1H), 7.44 (d, J = 8 Hz, 1H),
7.28 (d, J = 2.5 Hz, 1H), 7.18 (dd, J = 8, 8 Hz, 1H),
7.11 (dd, J = 8,8 Hz, 1H), 5.17 (br s, 3H D2O
exchangeable), 4.38 (d, J = 6 Hz, 2H). 13C NMR
(500 MHz, CD3CN): d 136.6, 126.7, 124.0, 121.8,
119.2, 118.7, 111.4, 111.1, 38.7. FTIR mmax (KBr):
3400, 3277, 1340, 1150, 744 cmꢀ1. HREIMS: m/z mea-
sured 225.0569 (225.0572 calculated for C9H11N3O2S).
EIMS m/z (% relative abundance) 225 (M+, 38), 144
(48), 130 (100), 118 (15).
1157 cmꢀ1
.
HREIMS: m/z measured 250.0777
(250.0776 calculated for C12H14N2O2S). EIMS m/z (%
relative abundance) 250 (M+, 43), 168 (54), 141 (100),
115 (11).
4.6.55. Naphthalen-1-ylmethylsulfamide (65). Prepara-
tion as reported for indol-3-ylmethylsulfamide (61) and
substituting 80 (200 mg, 1.27 mmol) for 76. The crude
reaction mixture was subjected to FCC (silica gel,
CH2Cl2/MeOH, 98:2) to afford 65 (231 mg, 77% yield)
as a white solid. Mp: 117–118 ꢁC, CH2Cl2/MeOH.
1
HPLC tR = 10.7 min. H NMR (500 MHz, (CD3)2SO):
d 8.15 (d, J = 8 Hz, 1H), 7.94 (d, J = 7.5 Hz, 1H), 7.86

M. S. C Pedras, M. Jha / Bioorg. Med. Chem. 14 (2006) 4958–4979
4977
(d, J = 8 Hz, 1H), 7.55 (m, 3H), 7.47 (t, J = 7.5 Hz, 1H),
7.06 (br s, 1H, D2O exchangeable), 6.74 (br s, D2O
exchangeable), 4.51 (d, J = 6 Hz, 2H). 13C NMR
(500 MHz, (CD3)2SO): d 134.4, 134.0, 131.9, 129.3,
128.6, 127.1, 127.0, 126.6, 126.2, 124.6, 45.2. FTIR mmax
(KBr): 3270, 1310, 1164, 795 cmꢀ1. HREIMS: m/z mea-
sured 236.0622 (236.0619 calculated for C11H12N2O2S).
EIMS m/z (% relative abundance) 236 (M+, 39), 154
(100), 141 (36), 115 (15).
3H), 2.68 (s, 3H). 13C NMR (500 MHz, CD3CN): d
137.2, 127.4, 125.6, 122.4, 119.9, 119.4, 112.0, 109.9,
45.6, 34.8, 33.9. FTIR mmax (KBr): 3406, 1322, 1148,
749 cmꢀ1. HREIMS: m/z measured 238.0774 (238.0776
calculated for C11H14N2O2S). EIMS m/z (% relative
abundance) 238 (M+, 29), 158 (23), 130 (100).
4.6.59. N-(Naphthalen-2-ylmethyl)methanesulfonamide
(69). Preparation as reported for indol-3-ylmethyl
methanesulfonamide (67) and substituting 78 (250 mg,
1.60 mmol) for 76. The crude reaction mixture was sub-
jected to FCC (silica gel, CH2Cl2) to afford 69 (303 mg,
81% yield) as a white solid. Mp: 132–134 ꢁC, CH2Cl2.
4.6.56.
N-Methyl-N-(naphthalen-1-ylmethyl)sulfamide
(66). Preparation as reported for N-(indol-3-ylmethyl-
N2-methylsulfamide (62) and substituting 81 (100 mg,
0.58 mmol) for 77. The crude reaction mixture was sub-
jected to FCC (silica gel, CH2Cl2/MeOH, 98:2) to afford
66 (99 mg, 68% yield) as a white solid. Mp: 145–146 ꢁC,
CH2Cl2/MeOH. HPLC tR = 14.6 min. 1H NMR
(500 MHz, (CD3)2SO): d 8.28 (d, J = 8 Hz, 1H), 7.96
(d, J = 8 Hz, 1H), 7.91 (d, J = 8 Hz, 1H), 7.53 (m, 4H),
7.50 (m, 1H), 7.02 (s, 2H, D2O exchangeable), 4.48 (s,
2H), 2.46 (s, 3H). 13C NMR (500 MHz, (CD3)2SO): d
134.4, 132.6, 132.4, 129.4, 129.3, 128.6, 127.1, 126.8,
126.2, 124.9, 53.3, 35.4. FTIR mmax (KBr): 3350, 3275,
1324, 1163, 782 cmꢀ1. HRMS: m/z measured 250.0768
(250.0776 calculated for C12H14N2O2S). EIMS m/z (%
relative abundance) 250 (M+, 47), 168 (60), 141 (100).
1
HPLC tR = 13.4 min. H NMR (500 MHz, (CD3)2SO):
d 7.90 (m, 3H), 7.84 (s, 1H), 7.67 (t, J = 6 Hz, 1H,
D2O exchangeable), 7.51 (m, 3H), 4.33 (d, J = 6 Hz,
2H), 2.88 (s, 3H). 13C NMR (500 MHz, (CD3)2SO): d
136.7, 133.7, 133.1, 128.7, 128.5, 128.4, 127.1, 126.9,
126.8, 126.7, 47.0, 40.8. FTIR mmax (KBr): 3275, 1312,
1151 cmꢀ1. HRMS: m/z measured 235.0671 (235.0667
calculated for C12H13NO2S). EIMS m/z (% relative
abundance) 235 (M+, 28), 155 (100), 141 (19), 128 (24).
4.6.60. N-Methyl-N-(naphthalen-2-ylmethyl)methanesulf-
onamide (70). Preparation as reported for N-(indol-3-
ylmethyl)-N-methyl methanesulfonamide (68) and
substituting 79 (100 mg, 0.58 mmol) for 77. The crude
reaction mixture was subjected to FCC (silica gel,
CH2Cl2) to afford 70 (113 mg, 78% yield) as a white sol-
4.6.57. Indol-3-ylmethylmethanesulfonamide (67). Metha-
nesulfonyl chloride (92 lL, 1.20 mmol) was added to a
solution of indol-3-ylmethylamine (76, 146 mg,
1.0 mmol) and triethylamine (265 lL, 2.0 mmol) in
THF (4 mL) at 0 ꢁC. The reaction mixture was allowed
to stir at rt for 60 min, the precipitate formed was fil-
tered off, the filtrate was concentrated, and the residue
was subjected to FCC (silica gel, CH2Cl2) to give in-
dol-3-ylmethyl methanesulfonamide (67) (146 mg, 65%
yield) as a light brown solid. Mp: 131–133 ꢁC, CH2Cl2.
1
id. Mp: 140–142 ꢁC, CH2Cl2. HPLC tR = 18.0 min. H
NMR (500 MHz, (CD3)2SO): d 7.93 (m, 3H), 7.84 (s,
1H), 7.52 (m, 2H), 7.48 (d, J = 8.5 Hz, 4.39 (s, 2H),
2.99 (s, 3H), 2.69 (s, 3H). 13C NMR (500 MHz,
(CD3)2SO): 134.9, 133.7, 133.3, 129.1, 128.5, 128.4,
127.6, 127.2, 127.0, 126.9, 54.1, 35.9, 35.2. FTIR mmax
(KBr): 3292, 2962, 1531, 1377 cmꢀ1. HRMS: m/z mea-
sured 249.0815 (249.0823 calculated for C13H15NO2S).
EIMS m/z (% relative abundance) 249 (M+, 48), 168
(95), 141 (100), 115 (27).
1
HPLC tR = 6.1 min. H NMR (500 MHz, CD3CN): d
9.29 (br s, 1H, D2O exchangeable), 7.71 (d, J = 8 Hz,
1H), 7.46 (d, J = 8 Hz, 1H), 7.29 (s, 1H), 7.20 (dd,
J = 7, 8 Hz, 1H), 7.13 (dd, J = 8, 8 Hz, 1H), 5.45 (br s,
1H, D2O exchangeable), 4.44 (d, J = 5 Hz, 1H), 2.83
(s, 3H). 13C NMR (500 MHz, CD3CN): d 137.0, 126.9,
124.5, 124.4 119.7, 119.1, 111.9, 111.6, 39.8, 38.9. FTIR
mmax (KBr): 3412, 1310, 1147, 749 cmꢀ1. HRMS: m/z
4.6.61. N-(Naphthalen-1-ylmethyl)methanesulfonamide
(71). Preparation as reported for indol-3-ylmethyl
methanesulfonamide (67) and substituting 80 (100 mg,
0.64 mmol) for 76. The crude reaction mixture was sub-
jected to FCC (silica gel, CH2Cl2) to afford 71 (119 mg,
80% yield) as a white solid. Mp: 130–131 ꢁC, CH2Cl2.
measured
224.0611
(224.0619
calculated
for
C10H12N2O2S). EIMS m/z (% relative abundance) 224
1
HPLC tR = 13.5 min. H NMR (500 MHz, (CD3)2SO):
(M+, 55), 144 (87), 130 (100), 80 (20).
d 8.17 (d, J = 8 Hz, 1H), 7.96 (d, J = 8 Hz, 1H), 7.88
(d, J = 8 Hz, 1H), 7.57 (m, 4H), 7.49 (dd, J = 8, 8 Hz,
1H), 4.62 (d, J = 6 Hz, 2H), 2.95 (s, 3H). 13C NMR
(500 MHz, (CD3)2SO): d 134.3, 134.2, 131.7, 129.4,
128.9, 127.1, 127.0, 126.7, 126.2, 124.5, 45.0, 40.8. FTIR
mmax (KBr): 3279, 1310, 1142, 773 cmꢀ1. HRMS: m/z
4.6.58.
N-(Indol-3-ylmethyl)-N-methylmethanesulfon-
amide (68). Methanesulfonyl chloride (79 lL, 1.02 mmol)
was added to a solution of indol-3-ylmethylamine (77,
150 mg, 0.93 mmol) and triethylamine (247 lL,
1.86 mmol) in THF (4 mL) at 0 ꢁC. The reaction mixture
was allowed to stir at rt for 60 min, the precipitate formed
was filtered off, the filtrate was concentrated, and the
residue was subjected to FCC (silica gel, CH2Cl2) to give
indol-3-ylmethyl methanesulfonamide (68) (156 mg, 70%
yield) as a white solid. Mp: 131–132 ꢁC, CH2Cl2. HPLC
measured
235.0666
(235.0667
calculated
for
C12H13NO2S). EIMS m/z (% relative abundance) 235
(M+, 36), 154 (100), 141 (19), 129 (22).
4.6.62. N-Methyl-N-(naphthalen-1-ylmethyl)methanesulf-
onamide (72). Preparation as reported for N-(indol-3-
1
tR = 8.3 min. H NMR (500 MHz, CD3CN): d 9.32 (br
ylmethyl)-N-methylmethanesulfonamide
(68)
and
s, 1H, D2O exchangeable), 7.73 (d, J = 8 Hz, 1H), 7.43
(d, J = 8 Hz, 1H), 7.28 (s, 1H), 7.17 (dd, J = 7, 8 Hz,
1H), 7.09 (dd, J = 7, 8 Hz, 1H), 4.45 (s, 2H), 2.78 (s,
substituting 81 (100 mg, 0.58 mmol) for 77. The crude
reaction mixture was subjected to FCC (silica gel,
CH2Cl2) to afford 72 (107 mg, 74% yield) as a white sol-

4978
M.S.C Pedras, M. Jha / Bioorg. Med. Chem. 14 (2006) 4958–4979
id. Mp: 98–99 ꢁC, CH2Cl2. HPLC tR = 14.1 min. 1H
NMR (500 MHz, (CD3)2SO): d 8.30 (d, J = 8 Hz, 1H),
7.96 (d, J = 8 Hz, 1H), 7.93 (d, J = 8.5 Hz, 1H), 7.53
(m, 4H), 4.68 (s, 2H), 3.06 (s, 3H), 2.60 (s, 3H). 13C
NMR (500 MHz, (CD3)2SO): 134.4, 132.2, 132.1,
129.5, 129.4, 128.3, 127.2, 126.8, 126.2, 124.5, 52.4,
60 min. The reaction mixture was neutralized using sat-
urated solution of Na2CO3 and extracted with EtOAc.
The organic extracts were combined, dried, and evapo-
rated to yield 75 (115 mg, 97% yield) as a white solid.
Mp: 80–82 ꢁC (lit.19 79–80 ꢁC), MeOH. 1H NMR
(300 MHz, CD3CN): d 7.99 (br s, 1H, D2O exchange-
able), 7.62 (dd, J = 8, 1 Hz, 1H), 7.36 (dd, J = 8 Hz, 1,
1H), 7.18 (m, 2H), 7.0 (s, 1H), 3.7 (s, 3H), 3.12 (t,
J = 8 Hz, 2H), 2.74 (t, J = 8 Hz, 2H).
35.0, 34.9. FTIR mmax (KBr): 1330, 1154, 790 cmꢀ1
.
HRMS: m/z measured 249.0822 (249.0823 calculated
for C13H15NO2S). EIMS m/z (% relative abundance)
249 (M+, 50), 168 (92), 141 (100), 115 (18).
4.7. Metabolism
4.6.63. 3-(Indolyl-3)propanamide (73). A solution of 3-
(indolyl-3)propanoic acid (92, 200 mg, 1.05 mmol) and
triethylamine (155 lL, 1.16 mmol) in THF (8 mL) was
cooled to 0 ꢁC on an ice bath. Methyl chloroformate
(92 lL, 1.16 mmol) was added dropwise to the cold solu-
tion. After allowing the reaction mixture to stir for
10 min at 0 ꢁC, dry ammonia gas was bubbled in for
60 min. The precipitate formed was filtered off, and the
filtrate was concentrated to give 73 (188 mg, 95% yield)
as a white solid. Mp: 134–136 ꢁC, THF. HPLC
4.7.1. Isolation of methyl 3-phenylthiocarbazate (94,
tR = 11.4 min), the biotransformation product of methyl
3-phenyldithiocarbazate (20). Five Erlenmeyer flasks
(250 mL) containing 100 mL of media inoculated with
spores (108 spores/100 mL) of L. maculans were incubat-
ed at 24 1 ꢁC on shaker at 120 rpm under constant
light. After 48 h, a solution of 20 (100 lL, 0.1 M) in
DMSO was added to the cultures. The flasks were then
incubated on shaker at 120 rpm for further 48 h. The
mycelia were filtered, the filtrates were combined, were
extracted with EtOAc to yield 9.3 mg of crude extract.
The crude extract was separated using prep TLC (silica
gel, CH2Cl2/MeOH, 98:2) to yield 6.2 mg of 94 as a
white solid. Mp: 151–152 ꢁC, CH2Cl2/MeOH. HPLC
tR = 11.4 min. 1H NMR (500 MHz, CD3CN): d 7.77
(br s, 1H, D2O exchangeable), 7.25 (t, J = 7 Hz, 2H),
6.89 (br s, 1H), 6.82 (d, J = 7.5 Hz, 2H), 6.56 (s, 1H,
D2O exchangeable), 2.18 (s, 3H). 13C NMR (500 MHz,
CD3CN): d 175.0, 147.9, 129.5, 121.0, 113.8, 11.2. FTIR
mmax (KBr): 3308, 1661, 1245, 753 cmꢀ1. HREIMS: m/z
1
tR = 6.1 min. H NMR (500 MHz, CD3CN): d 9.11 (br
s, 1H, D2O exchangeable), 7.57 (d, J = 8 Hz, 1 H),
7.37 (d, J = 8 Hz, 1H), 7.12 (dd, J = 8, 7 Hz, 1 H),
7.04 (m, 2H), 6.15 (br s, 1H, D2O exchangeable), 5.68
(br s, 1H, D2O exchangeable), 3.0 (t, J = 7 Hz, 2H,),
2.52 (t, J = 8 Hz, 2H). 13C NMR (500 MHz, CD3CN):
d 175.8, 137.6, 128.3, 123.1, 123.0, 122.5, 119.7, 119.5,
115.6, 112.4, 37.0, 21.8. FTIR mmax (KBr): 3396, 3177,
1648, 740 cmꢀ1. HREIMS: m/z measured 188.0949
(188.0950 calculated for C11H12N2O). EIMS m/z (% rel-
ative abundance) 188 (M+, 43), 144 (21), 130 (100).
measured
182.0515
(182.0514
calculated
for
C8H10N2OS). EIMS m/z (% relative abundance) 182
4.6.64. N-Methyl-3-(indolyl-3)propanamide (74). A solu-
tion of 3-(indolyl-3)propanoic acid (92, 50 mg,
0.26 mmol) and triethylamine (141 lL, 1.06 mmol) in
THF (3 mL) was cooled to 0 ꢁC on an ice bath. Methyl
chloroformate (23 lL, 0.29 mmol) was then added drop-
wise to the cold solution. After stirring for 10 min at
0 ꢁC, methylamine hydrochloride (270 mg, 4.0 mmol)
was added and the reaction mixture was stirred for
60 min at 0 ꢁC. The precipitate formed was filtered off,
the filtrate was concentrated, and the resulting yellow
oil was subjected to FCC (silica gel, CHCl3/MeOH,
95:5) to give 74 (46 mg, 85% yield) as an off-white solid.
Mp: 99–101 ꢁC (lit.19 97–99 ꢁC), CHCl3/MeOH. HPLC
(M+, 52), 107 (100), 77 (35).
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmc.2006.03.014.
References and notes
1
tR = 6.9 min. H NMR (500 MHz, CD3CN): d 9.30 (br
1. Bailey, J. A.; Mansfield, J. W., Eds.; Phytoalexins; Blackie
& Son: Glasgow, UK, 1982, pp 1–334.
s, 1H, D2O exchangeable), 7.58 (d, J = 8 Hz, 1H), 7.41
(d, J = 8 Hz, 1H), 7.17 (dd, J = 7, 8 Hz, 1H), 7.07 (m,
2H), 6.46 (br s, 1H, D2O exchangeable), 3.05 (t,
J = 7 Hz, 2 H,), 2.68 (d, J = 5 Hz, 3H), 2.51 (t,
J = 8 Hz, 3H). 13C NMR (500 MHz, CD3CN): d
173.6, 137.0, 127.7, 122.4, 121.8, 119.0, 118.9, 114.9,
111.7, 37.0, 26.7, 21.5. FTIR mmax (KBr): 3282, 1644,
1546, 743 cmꢀ1. HREIMS: m/z measured 202.1106
(202.1106 calculated for C12H14N2O). EIMS m/z (% rel-
ative abundance) 202 (M+, 44), 189 (6), 144 (25), 130
(100).
2. For recent reviews on cruciferous phytoalexins, see: (a)
Pedras, M. S. C.; Zaharia, I. L.; Khan, A. Q.
Phytochemistry 2000, 53, 161; (b) Pedras, M. S. C.;
Jha, M.; Ahiahonu, P. W. K. Curr. Org. Chem. 2003,
7, 1635.
3. For a recent review on metabolism and detoxification of
phytoalexins and analogs by phytopathogenic fungi, see:
Pedras, M. S. C.; Ahiahonu, P. W. K. Phytochemistry
2005, 66, 391.
4. Pedras, M. S. C.; Jha, M.; Okeola, O. G. Phytochemistry
2005, 66, 2609.
5. Pedras, M. S. C.; Ahiahonu, P. W. K.; Hossain, M.
Phytochemistry 2004, 65, 2685.
6. Thorn, G. D.; Ludwig, R. A. In The Dithiocarbamates
and Related Compounds; Elsevier: New York, 1962; pp
1–298.
4.6.65. Methyl 3-(indolyl-3)propanoate (75). H2SO4
(250 lL, concd) was added to a solution of 3-(indol-3-
yl)propanoic acid (92, 110 mg, 0.58 mmol) in MeOH
(2 mL) and the mixture was allowed to reflux for

M. S. C Pedras, M. Jha / Bioorg. Med. Chem. 14 (2006) 4958–4979
4979
7. For review of bioisosteres, see: Patani, G. A.; LaVoie, E. J.
Chem. Rev. 1996, 96, 3147.
23. Alajarin, M.; Vidal, A.; Ortin, M. M. Org. Biomol. Chem.
2003, 1, 4282.
8. Pedras, M. S. C.; Taylor, J. L. J. Nat. Prod. 1993, 56, 731.
9. Mohanta, P. K.; Dhar, S.; Samal, S. K.; Ila, H.; Junjappa,
H. Tetrahedron 2000, 56, 629.
24. Sun, W. Y.; Hu, J. Q.; Shi, Y. P. Synlett 1997, 1279.
25. Dubenko, R. G.; Bazavova, I. M.; Pel’kis, P. S. Zh. Org.
Khim. 1968, 4, 1057.
10. Pedras, M. S. C.; Okanga, F. I. Can. J. Chem. 2000, 78,
338.
11. Young, C. M. U.S. Pat. Appl. Publ., US 2003144309,
2003.
26. Kozhushko, B. N.; Lomakina, A. V.; Paliichuk, Yu. A.;
Shokol, V. A. Zh. Org. Khim. 1984, 20, 721.
27. Dimmock, J. R.; Vashishtha, S. C.; Stables, J. P. Phar-
mazie 2000, 55, 490.
12. Giuliani, A. M.; Trotta, E. Polyhedron 1988, 7, 1211.
13. Qureshi, S. K.; Rasheed, K.; Mekeri, A. K.; Hahn, G.
Pakistan J. Sci. Ind. Res. 1958, 1, 101.
14. Suvorov, N. N.; Velezheva, V. S.; Yarosh, A. V.; Erofeev,
Y. V.; Kozik, T. N. Khim. Geterotsikl. Soedin. 1975, 8,
1099.
28. Herr, R. J.; Kuhler, J. L.; Meckler, H.; Opalka, C. J.
Synthesis 2000, 1569.
29. Thompson, M. E. J. Org. Chem. 1984, 49, 1700.
30. Lee, C. H.; Kohn, H. J. Org. Chem. 1990, 55, 6098.
31. Dornyei, G.; Incze, M.; Kajtar-Peredy, M.; Szantay,
C. Collect. Czech. Chem. Commun. 2002, 67, 1669–
1680.
32. Kost, A. N.; Yudin, L. G.; Chernyshova, N. B.; Terenin,
V. I. Vestn. Mosk. Univ. Ser. 2: Khim. 1975, 16, 222.
33. Rao, A. V. R.; Chavan, S. P.; Sivadasan, L. Tetrahedron
1986, 42, 5065–5071.
34. Pedras, M. S. C.; Khan, A. Q.; Smith, K. C.; Stettner, S.
L. Can. J. Chem. 1997, 75, 825.
35. Goosey, M. W. Pestic. Sci. 1992, 34, 313.
36. Pedras, M. S. C.; Khan, A. Q. J. Agric. Food Chem. 1996,
44, 3403.
37. Pedras, M. S. C.; Suchy, M. Bioorg. Med. Chem. 2006, 14,
714.
38. Takasugi, M.; Monde, K.; Katsui, N.; Shirata, A. Bull
Chem. Soc. Jpn. 1988, 61, 285.
39. Pilkington, B. L.; Russell, S. E.; Whittle, A. J.; Mound, W.
R.; Turnbull, M. D.; Kozakiewicz, A. M.; Hughes, D. J.;
Whittingham, W. G. Br. UK Pat. Appl., GB 2329180,
1999.
15. Gaspari, P.; Banerjee, T.; Malachowski, W. P.; Muller, A.
J.; Prendergast, G. C.; DuHadaway, J.; Bennett, S.;
Donovan, A. M. J. Med. Chem. 2006, 49, 684.
16. Anon, U.K. Research Disclosure 1991, 331, 888.
17. Mewshaw, R. E.; Zhou, D.; Zhou, P.; Shi, X.; Hornby, G.;
Spangler, T.; Scerni, R.; Smith, D.; Schechter, L. E.;
Andree, T. H. J. Med. Chem. 2004, 47, 3823.
18. Kononova, V. V.; Vereshchagin, A. L.; Polyachenko, V.
M.; Semenov, A. A. Khim.-Farm. Zh. 1978, 12, 30.
19. Iqbal, Z.; Jackson, A. H.; Rao, K. R. N. Tetrahedron Lett.
1988, 29, 2577.
20. SanMartin, R.; Olivera, R.; Martinez de Marigorta, E.;
Dominguez, E. Tetrahedron 1995, 51, 5361.
21. Kutschy, P.; Dzurilla, M.; Takasugi, M.; Torok, M.;
Achbergerova, I.; Homzova, R.; Racova, M. Tetrahedron
1998, 54, 3549.
22. Borch, R. F.; Bernstein, M. D.; Durst, H. D. J. Am. Chem.
Soc. 1971, 93, 2897.