Structure-activity study of brassinin derivatives as indoleamine 2,3-dioxygenase inhibitors

Article abstract of DOI:10.1021/jm0508888

A screen of indole-based structures revealed the natural product brassinin to be a moderate inhibitor of indoleamine 2,3-dioxygenase (IDO), a new cancer immunosuppression target. A structure-activity study was undertaken to determine which elements of the brassinin structure could be modified to enhance potency. Three important discoveries have been made, which will impact future IDO inhibitor development: (i) The dithiocarbamate portion of the brassinin lead is a crucial moiety, which may be binding to the heme iron of IDO; (ii) an indole ring is not necessary for IDO inhibition; and (iii) substitution of the S-methyl group of brassinin with large aromatic groups provides inhibitors that are three times more potent in vitro than the most commonly used IDO inhibitor, 1-methyl-tryptophan.

Full text of DOI:10.1021/jm0508888

684
J. Med. Chem. 2006, 49, 684-692
Structure-Activity Study of Brassinin Derivatives as Indoleamine 2,3-Dioxygenase Inhibitors
Paul Gaspari,† Tinku Banerjee,‡,§ William P. Malachowski,*,† Alexander J. Muller,*,‡ George C. Prendergast,‡
James DuHadaway,‡ Shauna Bennett,† and Ashley M. Donovan†
Department of Chemistry, Bryn Mawr College, Bryn Mawr, PennsylVania 19010-2899, and Lankenau Institute for Medical Research,
Wynnewood, PennsylVania 19096
ReceiVed September 8, 2005
A screen of indole-based structures revealed the natural product brassinin to be a moderate inhibitor of
indoleamine 2,3-dioxygenase (IDO), a new cancer immunosuppression target. A structure-activity study
was undertaken to determine which elements of the brassinin structure could be modified to enhance potency.
Three important discoveries have been made, which will impact future IDO inhibitor development: (i) The
dithiocarbamate portion of the brassinin lead is a crucial moiety, which may be binding to the heme iron of
IDO; (ii) an indole ring is not necessary for IDO inhibition; and (iii) substitution of the S-methyl group of
brassinin with large aromatic groups provides inhibitors that are three times more potent in vitro than the
most commonly used IDO inhibitor, 1-methyl-tryptophan.
Introduction
Understanding how tumors escape the host immune system
is a rapidly developing area in the field of cancer research.
Recently, the enzyme indoleamine 2,3-dioxygenase (IDO; EC
1.13.11.42) has been implicated in tumor immunosuppression.1
Several reports identify IDO as playing a role in undermining
Figure 1. IDO inhibitors.
a more vigorous immune response to tumor growth. Conse-
quently, we have been focused on identifying novel, potent IDO
inhibitors with the goal of developing a new therapeutic
approach to cancer treatment. IDO inhibitors alone or in
combination with other chemotherapeutics might provide an-
other tool in the oncology armamentarium.
IDO is an extrahepatic enzyme that catalyzes the initial and
rate-limiting step in the degradation of tryptophan (Trp) along
the kynurenine pathway that leads to the biosynthesis of
nicotinamide adenine dinucleotide.2 IDO does not, however,
handle dietary catabolism of Trp, which is instead the role of
the structurally unrelated liver enzyme Trp dioxygenase (EC
1.13.11.11). IDO is a monomeric 45 kDa heme-containing
Figure 2. Brassinin (1) structure.
â-carboline (Figure 1), a noncompetitive inhibitor with a Ki )
3.3 µM.5 However, the most commonly used IDO inhibitor is
1-methyl-Trp (Figure 1),6 a commercially available compound
that is a competitive inhibitor with a Ki ) 34 µM.
oxidase that is active with the heme iron in the ferrous (Fe2+
)
We undertook a screen of commercially available indole-
based molecules to find novel IDO inhibitors. Interestingly, the
natural product brassinin (1; Figure 2) was found to be a
moderately active competitive inhibitor, Ki ) 97.7 µM. Brassinin
is a phytoalexin in cruciferous plants7 and has demonstrated
some antifungal8 and anticancer activity.9 We undertook a
structure-activity relationship study of brassinin with the goal
of obtaining a more potent IDO inhibitor. We divided the
brassinin structure into four components: the indole core, the
alkane linker, the dithiocarbamate moiety, and the S-alkyl piece.
Analogues of brassinin that varied each of the four components
were synthesized. The study determined the significance and
flexibility of each of the four portions of the brassinin structure.
The experimentation led to more potent inhibitors and several
important developments in the field of IDO inhibition. Details
of this study and our findings are reported herein.
form. The ferric (Fe3+) form of IDO is inactive, and substrate
inhibition is believed to result from Trp binding to ferric IDO.3
The primary catalytic cycle of IDO does not involve redox
changes; nevertheless, IDO is prone to autoxidation; therefore,
a reductant is necessary to reactivate the enzyme. In vivo, IDO
purportedly relies on a flavin or tetrahydrobiopterin cofactor.
In vitro, methylene blue and ascorbic acid are believed to
substitute for the natural flavin or tetrahydrobiopterin cofactor.
Inhibition of IDO has previously been targeted for other
therapies, most notably neurological disorders.2b Several me-
tabolites of the kynurenine pathway are neurotoxic or are
implicated in neurodegeneration, e.g., quinolinic acid; therefore,
attention has focused on IDO. A recent review4 summarizes
the range of compounds that have been tested as IDO inhibitors.
Strikingly, almost all IDO inhibitors, whether competitive or
noncompetitive, retain the indole ring of the natural substrate.
Currently, the most potent IDO inhibitor reported is 3-butyl-
Results and Discussion
Chemistry. Brassinin dithiocarbamate analogues were syn-
thesized by adding an amine to carbon disulfide at 0 °C, stirring
for 1 h, and then adding an alkyl halide. Modification of the
indole core or alkane linker occurred by adding different amines.
Many amines were commercially available, although several
* To whom correspondence should be addressed. (W.P.M.) Tel: 610-
526-5016. Fax: 610-526-5086. E-mail: wmalacho@brynmawr.edu. (A.J.M.)
Tel: 610-645-8034. Fax: 610-645-2095. E-mail: mullera@mlhs.org.
† Bryn Mawr College.
‡ Lankenau Institute for Medical Research.
§ Current address: NewLink Genetics, Wynnewood, PA 19096.
10.1021/jm0508888 CCC: $33.50 © 2006 American Chemical Society
Published on Web 12/17/2005

Structure-ActiVity Study of Brassinin DeriVatiVes
Scheme 1. Dithiocarbamate Synthesis
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 2 685
Scheme 2
Scheme 3. Thiourea Synthesis
required synthesis. The indole-3-methanamine 25 of brassinin
1 was prepared through the reductive amination of indole-3-
carboxaldehyde. Although there are several different reductive
amination procedures reported in the literature,9,10 we found
Mehta’s procedure9a to be the most effective. Homotryptamine
26, the amine reagent for 4, was synthesized in three steps from
indole-3-propanoic acid following literature precedent.11 2-Ami-
nomethyl-naphthalene 28 was also synthesized in three steps
from 2-naphthoic acid (Scheme 2). Modifications of the S-alkyl
piece occurred by substituting various alkyl halides for io-
domethane, e.g., 12-18 (Scheme 1).
Scheme 4. Brassitin Synthesis
Modifications to the dithiocarbamate moiety included thio-
ureas (19 and 20; Scheme 3), S-alkyl thiocarbamates (21;
Scheme 4), thioamides (22; Scheme 5), and thiazoles (23 and
24; Scheme 6). The thioureas were synthesized by reacting
amines with methyl isothiocyanate (Scheme 3).35 The S-alkyl
thiocarbamate 21, a phytoalexin called brassitin, came from the
reaction of S-alkyl thiochloroformate with 25 (Scheme 4). The
thioamide 22 was synthesized by reaction of 25 with an acid
chloride and then treatment with Lawesson’s reagent (Scheme
5). Thiazoles were synthesized by reaction of thioformamide
or thioacetamide with R-bromoketones 31 (Scheme 6). The

686 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 2
Scheme 5. Thioamide Synthesis
Gaspari et al.
Figure 3. Dithiocarbamate analogues.
Scheme 6. Thiazole Synthesis
Figure 4. Molecular ESP mapped onto electron distribution for 32.
inhibitors that lack the indole core.4 The current demonstration
of inhibition with benzene and cycloalkyl-based structures
expands the range of structures that behave as IDO inhibitors.
Furthermore, benzene aromatic structures are more easily
derivatized with available synthetic methods than indole com-
pounds. Indole derivatives can also be a liability given the
neuroactivity of some indole-containing compounds, e.g., se-
rotonin and related indolealkylamines.
Variation of the Alkane Linker. Linker variation was
possible, and in the brassinin series, it was found that the longer
linker led to more potent compounds, cf. 1 vs 2 vs 4. However,
analogues that modified two brassinin components did not
replicate this trend (cf. 13 vs 15). Taken together, the results
with the alkane linker modifications and the indole core changes
indicate that the IDO active site is rather accommodating.
Variation of the Dithiocarbamate. The most interesting
results came from isosteric modifications of the dithiocarbamate.
The transformation of brassinin’s dithiocarbamate moiety to a
thiourea (19 and 20), thiocarbamate (21), thioamide (22), or
thiazole (23 and 24) led to weaker or no inhibition. Notably,
the S-methyl-thiocarbamate analogue 21 (brassitin),29 suffered
a complete loss in inhibitory activity with the substitution of a
carbonyl for the thiocarbonyl group. Given the recognized metal-
coordinating properties of dithiocarbamates,17,18 it is likely that
the dithiocarbamate moiety is chelating to the heme iron at the
active site of IDO.19 In fact, pyrrolidine dithiocarbamate
reportedly inhibits IDO,17a besides being a well-known anti-
oxidant and NF-κB inhibitor.20
Computational Experiments to Explore the Electronic
Nature of Dithiocarbamate. If the dithiocarbamate moiety is
binding to the heme iron, then the electronic nature or charge
of the group should be important to achieve optimum binding.
We performed several computational experiments to understand
the electronic nature of the dithiocarbamate sulfur vs the sulfur/
oxygen in the isosteric analogues. To reduce the computational
time, most of the experiments involved simplified analogues
32-36 (Figure 3), which lacked the indole ring. Figure 4 shows
the molecular electrostatic potential (ESP) mapped onto the
electron distribution of 32, the dithiocarbamate analogue.
Clearly, the sulfur of 32 projects the greatest electron density
and therefore is the richest and most available Lewis basic site
for iron binding.
Table 1. IDO Inhibition Data
compound Ki (µM) compound Ki (µM) compound Ki (µM)
1
2
3
4
5
6
7
8
97.7
82.54
40.95
33.97
42.06
179.6
47.57
72.41
9
62.36
149.35
1267
36.95
13.22
363.6
17.15
11.55
17
18
19
20
21
22
23
24
28.38
20.48
NIa
342.3
NI
202
10
11
12
13
14
15
16
1292
328.7
a NI, no inhibition detected.
R-bromoketones 31 were generated from the corresponding
R-diazoketone derivative 30,12 which was synthesized in
three steps from indole-3-acetic acid following a literature
procedure.13
Enzyme Studies. Brassinin analogues were analyzed for
inhibition of extracted and purified recombinant human IDO
produced in bacteria. The assay was conducted according to a
literature protocol,14 with ascorbic acid and methylene blue
serving the role of reductant.15 Catalase was added to prevent
IDO decomposition from peroxide side products.16 The enzyme
assay was monitored for formation of N-formylkynurenine by
hydrolyzing the formyl group and spectrophotometrically
analyzing for the conjugated imine generated from kynurenine
and 4-(dimethylamino)benzaldehyde. In all cases where inhibi-
tion was seen, the brassinin analogues demonstrated competitive
inhibition. The inhibitory constants shown are an average of
two or three trials. The array of analogues tested allowed for
an evaluation of the four components of the brassinin structure
and resulted in some important discoveries (Table 1).
Variation of the Indole Core. One of the most suprising
discoveries was the range of groups that could be substituted
for indole and still retain some IDO inhibitory activity. Indeed,
not only were flat aromatic structures (e.g., 5 and 7-10)
effective substitutes, but the adamantyl structure 6 could also
bind in the substrate pocket, based on the competitive inhibition
witnessed for all of these analogues. Although IDO is relatively
promiscuous, there are still very few reports of substrates or

Structure-ActiVity Study of Brassinin DeriVatiVes
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 2 687
Table 2. Charge Data for Dithiocarbamate Analogues
most commonly used IDO inhibitor. In addition, the structure-
activity relationship discoveries should greatly advance the
search for more potent IDO inhibitors, an exciting new cancer
target.
charge
Ki of related analogue
compound
ESP
NBO
(µM) (compound no.)
32
33
34
35
36
-0.466
-0.518
-0.550
-0.661
-0.133
-0.277
-0.318
-0.390
-0.711
0.290
97.7 (1)
202 (22)
NI (19)
NI (21)
1292 (23)
Experimental Section
Chemistry. All reactants and reagents were commercially
available and were used without further purification unless otherwise
indicated. Anhydrous THF was obtained by distillation from
benzophenone-sodium under argon immediately before use. An-
hydrous CH2Cl2 and Et3N were obtained by distillation from
calcium hydride under argon. Methanol was dried over Mg and
distilled under argon. A saturated solution of HCl in CH3OH was
made by bubbling HCl through a drying tube, filled with CaCl2,
into a cooled flask of anhydrous CH3OH under a stream of argon.
A saturated solution of NH3 in CH3OH was made by bubbling
anhydrous NH3 into an Erlenmyer flask with a predetermined
volume of CH3OH. Concentrated refers to the removal of solvent
with a rotary evaporator at normal water aspirator pressure followed
by further evacuation with a two-stage mechanical pump unless
otherwise indicated. Yields refer to chromatographically and
spectroscopically pure (>95%) compounds, except as otherwise
indicated. All new compounds were determined to be >95% pure
by nuclear magnetic resonance (NMR), high-performance liquid
chromatography (HPLC), and/or gas chromatography (GC) as
indicated (see Supporting Information). Melting points were
In Table 2, the ESP and natural bond order (NBO) charge
values were derived for 32-36. Elimination of the indole group
allowed for more rapid computational experiments and did not
affect the trend witnessed.21 For compounds 32-35, a clear trend
can be seen for both ESP and NBO charge calculations: the
smaller the charge on sulfur/oxygen, the better the inhibition.
Compound 36 breaks the trend; however, the conformationally
rigid thiazole ring may prevent an effective interaction between
the sulfur of the thiazole and the heme iron. The trend witnessed
with compounds 32-35 may seem counterintuitive, i.e., a more
electron rich sulfur/oxygen should be a better electron donor to
the Lewis acid heme iron. Nevertheless, optimum inhibition may
arise from a softer Lewis base coordinating with the softer active
ferrous form of the enzyme.22 Future inhibitor design will be
guided by the insights from these computational experiments.
Variation of the S-Alkyl Piece. The greatest increases in
potency were realized in modifications of the S-alkyl group.
Although alkyl groups that were longer than the methyl in
brassinin were less active, S-allyl brassinin 12 was two times
more potent than brassinin and the benzyl analogue 13 was
almost one order of magnitude more potent. Moreover, the
tryptamine/naphthyl analogue 16 was as potent as 13; pyridyl
analogues 17 and 18 also demonstrated modest inhibition.
Because all these compounds behaved as competitive inhibitors,
these analogues reveal a large additional pocket in the IDO
active site capable of accommodating flat, aromatic groups.
Sono has reported that â-carboline, a noncompetitive inhibitor,
binds to the heme iron at the active site but not in the same
space as the substrate.23 Moreover, the â-carboline reportedly
acts as a nitrogen donor ligand and competes with O2 for binding
to the heme iron. It is possible that the large aromatic S-alkyl
pieces are binding in the same pocket that accommodates the
tricyclic aromatic â-carboline structure. Nevertheless, the pyridyl
analogues 17 and 18 failed to demonstrate stronger inhibition
despite their similarity to the pyridyl ring of â-carboline.
1
determined using an open capillary and are uncorrected. H and
13C NMR spectra were recorded at 300 and 75 MHz, respectively.
Chemical shifts are reported in δ values (ppm) relative to an internal
reference (0.05% v/v) of tetramethylsilane (TMS) for 1H NMR and
the solvent peak in 13C NMR, except where noted. Peak splitting
patterns in the NMR are reported as follows: s, singlet; d, doublet;
t, triplet; q, quartet; m, multiplet; and br, broad. Rotamer peaks
(about 1/4 intensity) were seen for all dithiocarbamate structures.24
Normal phase HPLC (NP-HPLC) analysis was performed with UV
detection at 254 nm and a 5 µm silica gel column (250 mm × 4.6
mm), eluted with 90:10 n-hexane:IPA (or gradient) at 1 mL/min.
Reverse phase HPLC (RP-HPLC) analysis was performed with UV
detection at 254 nm and a C18 column (300 mm × 3.9 mm), eluted
with a gradient of H2O + 0.1% TFA and CH3CN + 0.1% TFA at
1 mL/min., unless otherwise indicated. GC analyses were performed
with an EI-MS detector fitted with a 30 m × 0.25 mm column
filled with cross-linked 5% PH ME siloxane (0.25 µm film
thickness); gas pressure 7.63 psi He. IR data were obtained with
an FT-IR spectrometer. Thin-layer chromatography (TLC) was
performed using silica gel 60 A precoated glass-backed plates (0.25
mm thickness) with fluorescent indicators, which were scored and
cut. Developed TLC plates were visualized with UV light (254 nm),
iodine, or KMnO4. Flash column chromatography was conducted
with the indicated solvent system using normal phase silica gel 60
A, 230-400 mesh. All reactions were carried out under an inert
atmosphere of argon or nitrogen unless otherwise indicated.
Indole-3-methanamine (25). Indole-3-carboxaldehyde (189 mg,
1.3 mmol) and NH4OH‚HCl (113 mg, 1.63 mmol) were dissolved
in a Parr flask with 15 mL of MeOH, which was previously
saturated with anhydrous ammonia. The flask was stoppered and
placed on a Parr shaker for 5 h. To the resulting solution was added
200 mg of Raney nickel (50% slurry in H2O), and the flask was
pressurized to 60 psi with H2 and allowed to shake overnight. The
next day, the resulting mixture was filtered through Celite and
volatiles were removed to yield 190 mg (100% yield) of a yellow
solid. The product was unstable, so it was used immediately in
subsequent reactions without further purification. 1H NMR (CDCl3/
CD3OD): δ 8.04 (br s, 1H, NH), 7.67 (d, 1H, ArH, J ) 7.8), 7.39
(d, 1H, ArH, J ) 7.1), 7.16 (3H, ArH), 4.07 (d, 2H, ArCH2, J )
7.4 Hz).
Conclusion
A systematic study of the IDO inhibitory activity of brassinin
has been undertaken, and three important discoveries have been
made. Contrary to most previously reported IDO inhibitors, an
indole ring was not necessary for inhibitory activity with the
dithiocarbamate analogues of brassinin. Although indole-
containing derivatives were still the most active inhibitors (i.e.,
13 and 16), the inhibitory activity retained by analogues, such
as 5 and 7-9, create new opportunities to further inhibitor
development. Importantly, new analogues might be possible that
avoid the pharmacological liabilities of the indole ring and
leverage the wealth of chemical methods for benzene substitu-
tion. The dithiocarbamate moiety is an optimum group for IDO
inhibition and probably chelates to the active site iron. Large
unsaturated groups on the dithiocarbamate sulfur can be
accommodated in the active site and lead to more potent
inhibitors of IDO. Although only small increases in potency
were achieved through the structure-activity study, the new
inhibitors (i.e., 13 and 16) are three times more potent than the
General Method for the Synthesis of Dithiocarbamates. The
amine (1.0 equiv) was dissolved in pyridine (2-3 mL), and the
solution was cooled to 0 °C. Triethylamine (1.0-1.1 equiv) and
carbon disulfide (1.1 equiv) were added, and the solution was stirred

688 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 2
Gaspari et al.
at 0 °C. After 30 min, iodomethane (1.0-1.2 equiv) was added
and the reaction was allowed to slowly warm to room temperature
overnight. The reaction was poured into 1 M H2SO4 and extracted
with EtOAc (3×). The organic layer was washed with brine, dried
with Na2SO4, and filtered. Concentration afforded a crude product
that was chromatographed as described.
124.9, 57.9, 39.4, 18.2 and signals due to a minor rotamer at 57.1,
39.8, 18.5. IR (KBr) νmax cm-1: 3226, 2948, 2916, 2088, 1483,
1371, 1337, 1070. NP-HPLC tR ) 4.5 min. RP-HPLC tR ) 12.2
min.
N-(Adamant-2-yl)-S-methyl-dithiocarbamate (6). The general
method was used with 2-adamantylamine HCl and 2 equiv of Et3N
1
Brassinin (1). Brassinin was formed from 25 according to the
general method. The crude yellow solid was chromatographed on
silica with EtOAc/hexanes (1:3), and the resulting yellow solid was
further purified by recrystallization from CH2Cl2/hexanes to yield
to afford a white solid (98% yield); mp 128-129 °C. H NMR
(CDCl3): δ 7.25 (br s, 1H, NH), 4.65 (t, 1H, CHNH, J ) 3.6 Hz),
2.63 (s, 3H, SCH3), 2.13 (m, 2H, CH2), 1.73 (m, 12H, CH2) and
signals due to a minor rotamer (ca. 36%) 4.08 (m), 2.68 (s). 13C
NMR (CDCl3): δ 197.8, 97.5, 61.1, 37.3, 32.7, 31.4, 27.4, 18.5
and signals due to a minor rotamer at 37.8, 32.0, 27.3, 19.3. IR
(KBr) νmax cm-1: 3351, 2918, 2852, 1497, 1384, 1117, 942. NP-
HPLC tR ) 4.1 min. RP-HPLC tR ) 13.2 min.
1
rose-colored crystals (43% yield). H and 13C NMR spectra, IR
spectrum, and mp matched a previous report for 1.25
N-[2-(Indol-3-yl)ethyl]-S-methyl-dithiocarbamate (2).26 The
general method was used with tryptamine and CH2Cl2 as the solvent.
The crude product was chromatographed with CH2Cl2/hexanes (2:
1) to afford a waxy yellow-white solid (87% yield); mp ) 59-64
N-[(Naphth-2-yl)methyl]-S-methyl-dithiocarbamate (7). The
general method was used with 28 and 2 equiv of Et3N and MeOH
as the solvents. The crude product was chromatographed on silica
with EtOAc/hexanes (15:85) to yield a yellow solid (54% yield);
1
°C. H NMR (CDCl3): δ 8.05 (br s, 1H, NH), 7.61 (d, 1H, ArH,
J ) 7.8 Hz), 7.36 (d, 1H, ArH, J ) 8.0 Hz), 7.22 (dt, 1H, ArH, J
) 8, 1 Hz), 7.14 (dt, 1H, ArH, J ) 8, 1 Hz), 7.01 (br s, 1H, NH),
4.05 (q, 2H, CH2NH, J ) 12.4, 6.6 Hz), 3.11 (t, 2H, ArCH2, J )
6.7 Hz), 2.56 (s, 3H, CH3) and signals due to a minor rotamer (ca.
29%) at 3.73 (m), 2.68 (s). 13C NMR (CDCl3): δ 198.8, 136.4,
127.1, 122.4, 122.1, 119.7, 118.7, 112.3, 111.3, 47.2, 23.9, 18.0,
and signals due to a minor rotamer at 201.6, 126.8, 118.4, 46.1,
24.6, 18.9. GC: tR ) 15.00 min. EI-MS m/z (%): 202 (27, M+-
SCH3), 143 (4), 130 (100). NP-HPLC tR ) 5.9 min.
1
mp 70-72 °C. H NMR, 13C NMR, and IR spectra matched a
previous report for 7.27
N-Benzyl-S-methyl-dithiocarbamate (8). The general method
was used with benzylamine, and the crude product was chromato-
graphed with EtOAc/hexanes (1:10) to yield an off-white oil (74%
1
yield). H NMR, 13C NMR, and IR spectra matched a previous
report for 8.28
N-Phenethyl-S-methyl-dithiocarbamate (9). The general method
was used with phenethylamine, and the crude product was chro-
matographed with EtOAc/hexanes (1:10) to yield an off-white solid
(85% yield); mp 50-51 °C. 1H NMR (CDCl3): δ 7.28 (5
overlapping H, ArH), 6.91 (br s, 1H, NH2), 4.02 (t, 2H, ArCH2CH2,
J ) 6.9), 2.98 (t, 2H, ArCH2CH2, J ) 7.0), 2.61 (s, 3H, SCH3)
and signals due to a minor rotamer (ca. 24%) at 3.71 (m), 2.69 (s).
13C NMR (CDCl3): δ 199.1, 138.2, 128.8, 128.7, 126.8, 48.0, 34.2,
18.1 and signals due to a minor rotamer at 47.3, 34.9, 18.5. IR
(KBr) νmax cm-1: 3340, 3240, 3026, 2918, 1946, 1496, 1337, 1095.
NP-HPLC tR ) 4.6 min. RP-HPLC tR ) 11.5 min.
N-4-Fluorophenethyl-S-methyl-dithiocarbamate (10). The gen-
eral method was used with 4-fluorophenethylamine, and the solvent
was CH2Cl2. The volatiles were removed, and the residue was
dissolved in EtOAc. The organic layer was washed with 1 M H2-
SO4 (40 mL), H2O (40 mL), and brine (30 mL). The resulting
organic solution was dried with Na2SO4 and filtered. The volatiles
were removed to yield a beige solid, which was chromatographed
with EtOAc/hexanes (8/92) to yield a white solid (89% yield); mp
59-60 °C. 1H NMR (CDCl3): δ 7.18 (m, 2H, ArH), 7.01 (m, 2H,
ArH), 3.96 (q, 2H, ArCH2CH2, J ) 7.0 Hz), 2.96 (t, 2H, ArCH2,
J ) 7.1 Hz), 2.62 (s, 3H, SCH3) and signals due to a minor rotamer
(ca. 24%) at 3.69 (q, J ) 6.6 Hz), 2.68 (s). 13C NMR (CDCl3): δ
199.4, 161.8 (d, J ) 243 Hz), 133.9, 130.2, 115.8, 48.0, 33.5, 18.1
and signals due to a minor rotamer at 130.1, 47.2, 30.9, 18.5. IR
(KBr) νmax cm-1: 3250, 3002, 2921, 1886, 1506, 1385, 1222, 940.8.
NP-HPLC tR ) 5.1 min. RP-HPLC tR ) 11.6 min.
N,S-Dimethyl-N-phenethyldithiocarbamate (11). The general
method was used with N-methylphenthylamine, and the solvent was
CH2Cl2. The crude product was chromatographed with EtOAc/
hexanes (1/19) to yield a white oil (85% yield). 1H NMR (CDCl3):
δ 7.29 (m, 5H, ArH), 4.25 (t, 2H, ArCH2CH2, J ) 6.9 Hz), 3.20
(s, 3H, NCH3), 3.01 (q, 2H, ArCH2, J ) 6.8 Hz), 2.66 (s, 3H SCH3)
and signals due to a minor rotamer (ca. 42%) at 3.89 (m), 3.47 (s).
13C NMR (CDCl3): δ 198.6, 138.9, 138.1, 129.3, 129.2, 129.1,
127.0, 59.5, 40.9, 32.9, 20.7 and signals due to a minor rotamer at
56.6, 44.6, 34.0. IR (KBr) νmax cm-1: 3025, 2917, 1949, 1808,
1485, 1386, 1292, 1185, 1100, 992.5. NP-HPLC tR ) 4.2 min. RP-
HPLC tR ) 12.7 min.
S-Allyl-brassinin (12). The general method was used with 25,
but allyl bromide was substituted for iodomethane. The crude
product was purified by chromatography on silica with EtOAc/
hexanes (3/7) to afford an orange oil (52% yield). 1H NMR
(CDCl3): δ 8.17 (br s, 1H, NH), 7.64 (d, 1H, ArH, J ) 7.8 Hz),
7.43 (d, 1H, ArH, J ) 8.1 Hz), 7.21 (3H, ArH), 7.03 (br s, 1H,
NH), 5.93 (m, 1H, SCH2CHdCH2), 5.22 (m, 2H, SCH2CHdCH2),
N-[2-(Benzo[b]thiophen-3-yl)ethyl]-S-methyl-dithiocarbam-
ate (3). The general method was used with 2-(benzo[b]thiophen-
3-yl)ethanamine and CH2Cl2 as the solvents. The crude product
was chromatographed with EtOAc/hexanes (1:9) to yield a light
amber oil, which slowly crystallized (22% yield); mp ) 81-84
1
°C. H NMR (CDCl3): δ 7.90 (m, 1H, ArH), 7.79 (m, 1H, ArH),
7.43 (m, 3H, ArH), 7.02 (br s, 1H, NH), 5.18 (d, 2H, ArCH2CH2,
J ) 4.17 Hz), 2.63 (m, 5H, SCH3 overlapping with ArCH2) and a
signal due to a minor rotamer (ca. 17%) at 4.91 (m). 13C NMR
(CDCl3): δ 199.2, 140.6, 137.7, 130.7, 125.9, 124.9, 124.7, 123.1,
121.7, 45.1, 18.3, 14.2 and signals due to minor rotamer peaks at
60.4, 21.0. IR (KBr) νmax cm-1: 3336, 3229, 3079, 2995, 2916,
1499, 1379, 1302, 1075, 926. NP-HPLC tR ) 4.8 min. RP-HPLC
tR ) 12.1 min.
N-[3-(Indol-3-yl)propyl]-S-methyl-dithiocarbamate (4). The
general method was used with 3-(indol-3-yl)-propan-1-amine, 2
equiv of Et3N, and MeOH as the solvents. After the reaction was
complete, the volatiles were removed and the residue was dissolved
in EtOAc (60 mL). The solution was washed with 0.5 M HCl (2 ×
30 mL), H2O (20 mL), and brine (20 mL). The organic solution
was dried with Na2SO4, filtered, and concentrated. The crude
product was chromatographed with EtOAc/hexanes (1:3) to yield
an off-white oil, which crystallized overnight (61% yield); mp )
54-56 °C. 1H NMR (CDCl3): δ 8.04 (br s, 1H, NH), 7.60 (d, 1H,
ArH, J ) 7.6), 7.35 (d, 1H, ArH, J ) 8.0), 7.20 (m, 1H, ArH),
7.12 (m, 1H, ArH), 7.04 (m, 1H, ArH), 6.91 (br s, 1H, NH), 3.82
(q, 2H, ArCH2CH2CH2, J ) 7.0 Hz), 2.86 (t, 2H, ArCH2CH2 J )
7.2 Hz), 2.52 (s, 3H, SCH3), 2.1 (m, 2H, ArCH2CH2) and signals
due to a minor rotamer (ca. 30%) 3.50 (q, J ) 6.3 Hz), 2.68 (s).
13C NMR (CDCl3): δ 198.7, 136.4, 127.1, 122.2, 121.6, 119.4,
118.7, 115.1, 111.3, 47.2, 28.3, 22.7, 18.0 and signals due to a
minor rotamer at 46.0, 28.9, 18.7. IR (KBr) νmax cm-1: 3410, 3321,
2919, 1888, 1504, 1337, 1094. EI-MS: m/z (%) 216 (57, M+-
SCH3), 183 (5), 156 (10), 131 (23). NP-HPLC tR ) 12.3 min. RP-
HPLC tR ) 11.9 min.
N-(Indan-2-yl)-S-methyl-dithiocarbamate (5). The general
method was used with 2-aminoindan HCl. The crude product in
EtOAc was decolorized with charcoal and filtered through Celite,
and the volatiles were removed to yield a clear oil. The oil was
chromatographed with EtOAc/hexanes (1:9) to yield an off-white
solid (74% yield); mp ) 106-108 °C. 1H NMR (CDCl3): δ 7.23
(4H, ArH), 7.10 (br s, 1H, NH), 5.31 (m, 1H, CH2CHCH2), 3.44
(m, 2H, CHCHCH), 2.98 (dd, 2H, CHCHCH, J ) 16.5 Hz, 3.7
Hz), 2.62 (s, 3H, SCH3) and signals due to a minor rotamer (ca.
38%) 4.78 (m), 2.70 (s). 13C NMR (CDCl3): δ 198.6, 140.5, 127.0,

Structure-ActiVity Study of Brassinin DeriVatiVes
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 2 689
5.05 (d, 2H, ArCH2, J ) 4.4 Hz), 3.92 (d, 2H, SCH2CHdCH2, J
) 7.7 Hz) and signals due to a minor rotamer (ca. 16%) at 4.69
(m), 4.09 (d, J ) 7 Hz). 13C NMR (CDCl3): δ 196.3, 136.2, 132.7,
126.4, 122.7, 120.2, 118.6, 118.5, 111.5, 110.3, 43.1, 38.3 and
due to a minor rotamer (ca. 20%) at δ 46.0, 42.0. IR (KBr) νmax
cm-1: 3436, 3191, 2914, 2837, 1592, 1515, 1451, 1387, 1358,
1326, 1300, 1204, 1089, 999, 935, 816, 736. EI-MS: m/z (%): 130
(100), 202 (24). GC tR ) 14.7 min. NP-HPLC tR ) 5.7 min. RP-
HPLC tR ) 13.2 min.
signals due to a minor rotamer at 41.0, 39.5. IR (KBr) νmax cm-1
:
3402, 2915, 1852, 1635, 1377, 1063. NP-HPLC tR ) 6.7 min. RP-
HPLC tR ) 11.6 min.
N-[2-(Indol-3-yl)ethyl]-S-[(pyrid-3-yl)methyl]dithiocarbam-
ate (17). The general method was used with tryptamine as the amine
and CH2Cl2 as the solvent. 3-(Bromomethyl)pyridine, HBr salt, was
used as the alkylating agent in place of iodomethane, and 2.0 equiv
of Et3N were used. The crude product was chromatographed with
EtOAc/hexanes (3:1) to afford a powdery tan solid (21% yield);
S-Benzyl-brassinin (13). The general method was used with 25,
but benzyl bromide was substituted for iodomethane and CH2Cl2
was used as the solvent. The crude product was chromatographed
on silica EtOAc/hexanes (3/7) to yield a translucent, yellow oil,
which slowly solidified. Recrystallization from CH2Cl2/hexanes
1
mp ) °C. H NMR (CDCl3): 8.5 (m, 2 H, ArH), δ 8.16 (br s, 1
1
yielded a bright yellow solid (50% yield); mp 101-102 °C. H
H), 7.69 (m, 1 H, ArH), 7.58 (t, 1 H, ArH, J ) 6.0 Hz), 7.37 (d,
1 H, ArH, J ) 6.0 Hz), 7.24-7.12 (m, 4 H, ArH), 7.03 (m, 1 H,
ArH), 4.52 (s, 2 H, SCH2), 4.08 (m, 2 H, ArCH2CH2), 3.12 (m,
2H, ArCH2), and signals due to a minor rotamer (ca. 25%) at 4.58
(s), 3.75 (m). 13C NMR (CDCl3): δ 197.0, 150.3, 148.8, 136.8,
133, 127, 123.6, 122.7, 122.4, 120, 118.9, 112.5, 111.6, 53.5, 47.7,
36.9, 24.2 and a signal due to a minor rotamer at 54.0. IR (KBr)
NMR (CDCl3): δ 8.22 (br s, 1H, NH), 7.62 (d, 1H, ArH, J ) 7.9
Hz), 7.28 (9H, ArH + PhH), 6.98 (br s, 1H, NH), 5.11 (d, 2H,
ArCH2, J ) 3.9 Hz), 4.55 (s, 2H, CH2Ph) and signals due to a
minor rotamer (ca. 19%) at 4.77 (d, J ) 4.5 Hz), 4.67 (s). 13C
NMR (CDCl3): δ 196.4, 136.6, 136.3, 129.0, 128.6, 127.5, 126.5,
124.0, 122.8, 120.3, 118.7, 111.4, 110.7, 43.2, 39.9. IR (KBr) νmax
cm-1: 3417, 3334, 3058, 1890, 1494, 1455, 1067. NP-HPLC tR )
6.8 min. RP-HPLC tR ) 12.5 min.
ν
max cm-1: 3403, 3306, 3164, 2917, 1724, 1619, 1500, 1455, 1421,
1392, 1332, 1257, 1089, 926, 851, 739. EI-MS: m/z (%): 130 (100),
202 (35). GC tR ) 14.8 min. NP-HPLC tR ) 27.9 min. RP-HPLC
tR ) 9.4 min.
S-Hexyl-brassinin (14). The general method was used with 25,
but 1-iodohexane was substituted for iodomethane. The crude
product was chromatographed on silica with EtOAc/hexanes (3/7)
to yield a golden oil (57% yield). 1H NMR (CDCl3): δ 8.18 (br s,
1H, NH), 7.65 (d, 1H, ArH, J ) 7.8 Hz), 7.43 (d, 1H, ArH, 8.1
Hz), 7.22 (3H, ArH), 6.99 (br s, 1H, NH), 5.06 (d, 2H, ArCH2, J
) 4.4 Hz), 3.26 (t, 2H, SCH2, J ) 7.5 Hz), 1.70 (m, 2H, SCH2CH2-
CH2CH2CH2CH3), 1.39 (6H, SCH2CH2CH2CH2CH2CH3), 0.88 (t,
3H, CH3, J ) 7.5 Hz) and signals due to a minor rotamer (ca.
19%) at 4.79 (d, J ) 4.8 Hz), 3.39 (t, J ) 7.5 Hz). 13C NMR
(CDCl3): δ 197.6, 136.2, 126.4, 124.0, 122.6, 120.1, 118.6, 111.5,
110.5, 43.0, 35.4, 29.0, 28.5, 22.5, 14.0 and signals due to a minor
rotamer at 42.0, 36.5. IR (KBr) νmax cm-1: 3409, 3328, 2955, 2927,
2855, 1620, 1494, 1456, 1379, 1094. NP-HPLC tR ) 6.0 min. RP-
HPLC tR ) 13.8 min.
N-[2-(Indol-3-yl)ethyl]-S-benzyl-dithiocarbamate (15). The
general method was used with tryptamine as the amine and CH2-
Cl2 as the solvent. Benzyl bromide was used as the alkylating agent
in place of iodomethane. The crude product was chromatographed
with EtOAc/hexanes (1:4) to yield white crystals (86% yield).
Further purification was accomplished by recrystallization in
EtOAc/hexanes to afford a 73% yield; mp ) 79-81 °C. 1H NMR
(CDCl3): δ 8.02 (br s, 1 H, NH), 7.58 (m, 1 H, ArH), 7.37 (m, 6
H, ArH), 7.32-7.18 (m, 1 H, ArH), 7.13 (t, 1 H, ArH, J ) 9.0
Hz), 6.99 (m, 2 H, ArH), 4.48 (s, 2 H, SCH2), 4.05 (q, J ) 6.0 Hz,
1 H, ArCH2CH2), 3.09 (m, 2 H, ArCH2) and signals due to a minor
rotamer (ca. 24%) at 4.59 (s), 3.74 (q, J ) 6.0 Hz). 13C NMR
(CDCl3): δ 197.2, 136.5, 136.3, 129.3, 128.9, 128.6, 127.6, 127.4,
127.1, 122.4, 122.1, 119.7, 118.7, 112.2, 111.3, 47.2, 39.8, 24.6,
23.9, and signals due to a minor rotamer at 135.7, 127.6, 118.4,
41.0, 24.6. IR (KBr) νmax cm-1: 3394, 3179, 1618, 1503, 1455,
1332, 1095, 936. EI-MS: m/z (%) 130 (100), 202 (37). GC tR )
14.8 min. NP-HPLC tR ) 7.6 min. RP-HPLC tR ) 12.9 min. Anal.
calcd for C18H18N2S2: C, 66.22; H, 5.56; N, 8.58; S, 19.64.
Found: C, 66.19; H, 5.43; N, 8.42; S, 19.87.
N-[2-(Indol-3-yl)ethyl]-S-[(pyrid-4-yl)methyl]dithiocarbam-
ate (18). The general method was used with tryptamine as the amine
and CH2Cl2 as the solvent. 4-(Bromomethyl)pyridine, HBr salt, was
used as the alkylating agent in place of iodomethane, and 2.0 equiv
of Et3N was used. The crude product was recrystallized with EtOAc/
hexanes (3:1) to afford tan crystals (50% yield); mp ) 125-127
°C. 1H NMR (CDCl3): 8.51 (m, 2 H, ArH), 8.08 (br s, 1 H), 7.59
(m, 1 H, ArH), 7.39 (d, 1 H, ArH, J ) 6.9 Hz), 7.28-7.00 (m, 6
H, ArH), 4.51 (s, 2 H, SCH2), 4.07 (m, ArCH2CH2, J ) 6.0 Hz),
3.14 (m, 2 H, ArCH2), and signals due to a minor rotamer (ca.
25%) at 4.60 (s), 3.75 (m). 13C NMR (CDCl3): δ 196.3, 150.1,
146.7, 136.7, 127.4, 124.3, 124.1, 122.7, 122.4, 120.0, 118.9, 112.5,
111.6, 47.9, 38.5, 24.2, 19.8. IR (KBr) νmax cm-1: 3404, 3299,
2917, 2851, 2178, 2099, 1600, 1508, 1455, 1416, 1337, 1225, 1091,
1002, 927, 743. EI-MS: m/z (%): 130 (100), 202 (29). GC tR )
14.7 min. NP-HPLC tR ) 28.9 min. RP-HPLC tR ) 9.4 min.
General Method for the Synthesis of Thioureas. The amine
was dissolved/suspended in CH2Cl2, cooled to 0 °C, and treated
with Et3N (2.1-2.2 equiv). Methyl isothiocyanate (1.1-1.5 equiv)
was added about 5 min later, and the reaction was allowed to slowly
warm to room temperature while stirring overnight.
N-[1-(Indol-3-yl)methyl]-N′-methyl-thiourea (19). The general
method was used with 25. The volatiles were removed from the
reaction, and the crude residue was recrystallized from EtOAc/
hexanes to yield a gold, crystalline solid (54% yield); mp 148-
1
150 °C. H NMR (DMSO-d6): δ 10.9 (br s, 1H, NH), 7.65 (m,
1H, ArH), 7.36 (m, 2H, ArH), 7.10 (t, 1H, ArH, J ) 7.2 Hz), 4.75
(br s, 2H, ArCH2), 2.85 (br s, 3H, NHCH3). 13C NMR (DMSO-
d6): δ 183.4, 137.1, 124.9, 124.4, 122.1, 119.4, 112.7, 111.9, 40.1
(overlapped with CDCl3), 31.5. IR (KBr) νmax cm-1: 3210, 1565,
1456, 1300, 1089. NP-HPLC (isocratic) tR ) 23.9 min. NP-HPLC
(gradient) tR ) 22.5 min.
N-[1-(Indol-3-yl)ethyl]-N′-methyl-thiourea (20). The general
method was used with tryptamine HCl. The crude product was
isolated by washing the reaction mixture with 1 M H2SO4 (2×),
saturated NaHCO3, and brine and drying with Na2SO4. After
concentration, the crude product was further purified by chroma-
tography with EtOAc/hexanes (gradient, 1/1 to 3/1) to afford an
oil that crystallizes on sitting to a light brown solid (92% yield);
N-[2-(Indol-3-yl)ethyl]-S-[(naphth-2-yl)methyl]dithiocarbam-
ate (16). The general method was used with tryptamine as the amine
and CH2Cl2 as the solvent. 2-(Bromomethyl)naphthalene was used
as the alkylating agent in place of iodomethane. The crude product
was chromatographed with EtOAc/hexanes (1:4) to afford the pure
product (59% yield). Further purification was accomplished by
recrystallization in EtOAc/hexanes to afford white crystals (29%
1
mp ) 102-106 °C. H NMR (CDCl3): δ 8.13 (s, 1H, NH), 7.60
1
yield); mp ) 158-160 °C. H NMR (CDCl3): δ 8.05 (br s, 1 H,
(d, 1H, ArH, J ) 7.8 Hz), 7.37 (d, 1H, ArH, J ) 8.1 Hz), 7.21 (t,
1H, ArH, J ) 7.0 Hz), 7.12 (t, 1H, ArH, J ) 7.0 Hz), 7.04 (s, 1H,
ArH), 5.75 (br s, 2H, NH-CdS), 3.79 (br d, 2H, ArCH2CH2, J )
5.4 Hz), 3.06 (t, 2H, ArCH2, J ) 6.6 Hz), 2.79 (br d, 3H, CH3, J
) 4.5 Hz). 13C NMR (CDCl3): δ 182.3, 136.3, 127.1, 122.4, 122.3,
NH), 7.90 (m, 1 H, ArH), 7.79 (t, J ) 9.4 Hz, 4 H, ArH), 7.46 (m,
5 H, ArH), 7.11-7.35 (m, 4 H, ArH), 6.97 (s, 1 H, ArH), 4.64 (s,
2 H, SCH2), 4.08 (q, ArCH2CH2, J ) 6.0 Hz), 3.12 (t, 2 H, ArCH ,
J ) 6.0 Hz), and signals due to a minor rotamer (ca. 25%) at 4.727
(s), 3.78 (q, J ) 6.0 Hz). 13C NMR (CDCl3): δ 197.3, 136.6, 134.2,
133.5, 132.9, 128.7, 128.0, 127.9, 127.3, 127.2, 126.5, 126.2, 122.6,
122.4, 119.9, 118.9, 112.5, 111.5, 47.4, 40.3, 24.1, 1.2 and signals
119.6, 118.5, 112.4, 111.4, 44.8, 30.5, 24.8. IR (KBr) νmax cm-1
:
3394, 3320, 3323, 3051, 1561, 1342. NP-HPLC tR ) 24.2 min.
RP-HPLC (1/1 MeOH/H2O) tR ) 6.1 min.

690 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 2
Gaspari et al.
Brassitin (21).29 Freshly made 25 (190 mg, 1.3 mmol) and Et3N
(271 µL, 1.95 mmol) were dissolved in anhydrous MeOH (10 mL).
The flask was cooled to 0 °C, and methyl chlorothiolformate (116
µL, 1.36 mmol) was added dropwise followed by stirring at room
temperature for 6 h. A few drops of H2O were added to quench
excess reagent, and the volatiles were evaporated. The residue was
dissolved in EtOAc (35 mL) and washed with 0.5 M HCl (2 × 20
mL), saturated NaHCO3 (20 mL), and brine (15 mL). The organic
solution was dried with Na2SO4, filtered, and concentrated to afford
a crude brownish-orange solid (270 mg). After recrystallization from
CH2Cl2/hexanes, beige crystals: 125 mg, 44% yield; mp 110-
2-Aminomethylnaphthalene (28). Compound 27 (1.00 g, 5.8
mmol) in THF (20 mL) was added slowly to a solution of LAH
(1.76 g, 46.4 mmol) in THF (45 mL) at 0 °C. The solution was
allowed to warm to room temperature, and the reaction was stirred
overnight. The reaction was cooled to 0 °C and quenched with H2O.
The solids were filtered from the solution through Celite and washed
with hot THF. The filtrate was concentrated, and the residue was
dissolved in EtOAc (80 mL) and washed with 1 M HCl (3 × 30
mL). The aqueous layer was basified with 6 M NaOH to a pH of
12, and the precipitate was extracted with EtOAc (3 × 30 mL).
The resulting organic solution was washed with brine (40 mL),
dried with Na2SO4, and filtered. Concentration afforded a slightly
yellow solid (510 mg, 56% yield); mp 55-56 °C. 1H NMR
(CDCl3): δ 7.80 (3H, ArH), 7.72 (s, 1H, ArH), 7.43 (m, 3H, ArH),
4.00 (s, 2H, ArCH2). 13C NMR (CDCl3): δ 140.6, 133.5, 132.5,
1
111 °C. H NMR (CDCl3): δ 8.15 (br s, 1H, NH), 7.64 (d, 1H,
ArH J ) 7.9), 7.39 (d, 1H, ArH J ) 7.1), 7.23 (m, 1H, ArH), 7.18
(m, 1H, ArH), 7.13 (m, 1H, ArH), 5.52 (br s, 1H, CH2NHC), 4.67
(d, 2H, ArCH2, J ) 5.1), 2.38 (s, 3H, SCH3). 13C NMR (CDCl3):
δ 167.6, 136.3, 126.3, 123.3, 122.5, 119.9, 118.7, 112.1, 111.3,
36.9, 12.4. EI-MS m/z (%): 220 (37, M+), 205 (9), 172 (12, M+-
SCH3), 130 (100). NP-HPLC tR ) 9.8 min. RP-HPLC (1/1 CH3-
CN/H2O + 0.1% TFA) tR ) 9.5 min.
128.2, 127.7, 126.1, 125.8, 125.5, 125.1, 46.6. IR (KBr) νmax cm-1
:
3362, 3291, 3050, 2915, 1950, 1596, 1507, 1358, 1273. GC tR )
9.0 min. EI-MS m/z (%): 157 (83, M+), 156 (100), 141 (15), 129
(49), 128 (40), 127 (24), 115 (10).
1-Bromo-3-(indol-3-yl)propanone (31). The diazoketone 30
(379 mg, 1.90 mmol) was dissolved in acetic acid (4 mL) and cooled
to 0 °C. HBr (48%, 0.51 mL) was added dropwise. Forty minutes
later, the reaction was diluted with H2O and then quenched at 5 °C
with saturated NaHCO3. The reaction mixture was extracted with
CH2Cl2 (2×), washed with saturated NaHCO3, H2O, and brine, dried
with Na2SO4, filtered, and concentrated to a brown oil (398 mg,
83% yield). The crude product was used immediately in the next
step. 1H NMR (CDCl3): δ 8.26 (br s, 1H, NH), 7.56 (d, 1H, ArH,
J ) 7.8 Hz), 7.39 (d, 1H, ArH, J ) 7.8 Hz), 7.27-7.13 (m, 3H,
ArH), 4.07 (s, 2H, CH2Br), 3.95 (s, 2H, ArCH2).
N-[(Indol-3-yl)methyl]propanamide (29). Compound 25 (1.00
g, 6.84 mmol) and Et3N (1.4 mL, 10.26 mmol) were dissolved in
MeOH and cooled to 0 °C. Propionyl chloride (633 mg, 6.84 mmol)
was added dropwise, and the reaction was stirred at room
temperature for 4 h. The volatiles were removed, and the residue
was taken up in CH2Cl2 (40 mL), washed with 10% citric acid (20
mL), saturated NaHCO3 (20 mL), and brine (20 mL). The organic
layer was dried with Na2SO4 and filtered, and the volatiles were
removed to yield 1.33 g of a white, crystalline solid (1.33 g, 96%
yield). An analytical sample was recrystallized form EtOAc/
hexanes; mp 91-92 °C. 1H NMR (CDCl3): δ 8.80 (br s, 1H, NH),
7.62 (d, 1H, ArH, J ) 7.85 Hz), 7.38 (d, 1H, ArH, J ) 7.2 Hz),
7.20 (3H, ArH), 5.80 (br s, 1H, NH), 4.60 (d, 2H, ArCH2, J ) 5.1
Hz), 2.20 (q, 2H, COCH2CH3, J ) 7.6 Hz), 1.14 (t, 3H,
COCH2CH3, J ) 7.6 Hz). 13C NMR (CDCl3): δ 173.6, 136.5,
126.6, 123.3, 122.5, 119.9, 118.8, 112.8, 111.4, 35.2, 29.7, 9.9. IR
(KBr) νmax cm-1: 3405, 1891, 1634, 1532, 1097.
General Method for the Synthesis of Thiazoles. R-Bromoke-
tone 31 was dissolved in EtOH and treated with thioamide (1.5
equiv) and NaHCO3 (1.5 equiv). The resulting mixture was heated
at reflux overnight. Upon cooling, the reaction material was
partitioned between EtOAc and half saturated NaHCO3. The
aqueous layer was extracted with EtOAc, and the combined organic
layers were washed with H2O and brine, dried with MgSO4, filtered,
and concentrated to a brown oily solid. The crude thiazole product
was purified by chromatography with EtOAc/hexanes (1:2).
4-[(Indol-3-yl)methyl]thiazole (23). The general method was
used with thioformamide30 to afford a 71% yield. 1H NMR
(CDCl3): δ 8.76 (d, 1H, SCHN, J ) 2.0 Hz), 8.11 (br s, 1H, NH),
7.52 (d, 1H, ArH, J ) 7.6 Hz), 7.36 (d, 1H, ArH, J ) 8.0 Hz),
7.18 (t, 1H, ArH, J ) 7.1 Hz), 7.08 (t, 2H, ArH, J ) 7.4 Hz), 6.90
(s, 1H, ArH), 4.34 (s, 2H, ArCH2). 13C NMR (CDCl3): δ 157.5,
152.5, 122.5, 122.1, 119.4, 119.1, 113.8, 113.5, 111.2, 27.6. IR
(CH2Cl2) νmax cm-1: 3626, 3470, 3051, 2987, 1420, 1264. GC tR
) 15.1 min. EI-MS m/z (%): 214 (100, M+), 213 (86), 186 (15),
154 (14), 130 (51). RP-HPLC (1/1 CH3CN/H2O + 0.1% TFA) tR
) 4.4 min.
N-[(Indol-3-yl)methyl]propanethioamide (22). Amide 29 (190
mg, 0.94 mmol) was dissolved in THF (20 mL). Lawesson reagent
(304 mg, 0.75 mmol) was added to the resulting solution, and the
reaction was stirred for 2 h at room temperature. The volatiles were
removed, and the residue was dissolved in CH2Cl2 (20 mL) and
washed with H2O (12 mL). The organic layer was dried with Na2-
SO4 and filtered. After standing, a white precipitate formed, which
was filtered, and the filtrate was concentrated. The resulting residue
(380 mg) was chromatographed with EtOAc/hexanes (1:1) to yield
a clear oil, which slowly crystallized (85 mg, 41% yield); mp 132-
134 °C. 1H NMR (CDCl3): δ 8.23 (br s, 1H, NH), 7.63 (1H, ArH),
7.41 (1H, ArH), 7.23 (3H, ArH), 4.98 (d, 2H, ArCH2, J ) 4.5 Hz),
2.68 (q, 2H, CSCH2CH3, J ) 7.5 Hz), 1.30 (t, 3H, CSCH2CH3, J
) 7.5 Hz). 13C NMR (CDCl3): δ 205.9, 136.3, 126.5, 124.0, 122.8,
120.3, 118.7, 111.5, 110.8, 42.2, 40.0, 13.5. IR (KBr) νmax cm-1
:
4-[(Indol-3-yl)methyl]-2-methyl-thiazole (24). The general
1
3331, 2975, 2931, 1523, 1413, 1090. EI-MS m/z (%): 218 (49,
M+), 163 (8), 131 (12), 130 (100). NP-HPLC tR ) 10.9 min. RP-
HPLC tR ) 10.4 min.
method was used with thioacetamide to afford a 56% yield. H
NMR (CDCl3): δ 8.17 (br s, 1H, NH), 7.53 (d, 1H, ArH, J ) 7.8
Hz), 7.34 (d, 1H, ArH, J ) 8.1 Hz), 7.17 (t, 1H, ArH, J ) 7.0 Hz),
7.10-7.04 (m, 2H, ArH), 6.63 (s, 1H, ArH), 4.23 (s, 2H, CH2),
2.69 (s, 3H, CH3). 13C NMR (CDCl3): δ 165.6, 162.3, 156.1, 136.4,
127.3, 122.6, 122.0, 119.3, 119.1, 113.4, 111.1, 27.7, 19.1. IR (KBr)
2-Naphthoyl Chloride. A 100 mL round-bottom flask was
charged with 2-naphthoic acid (2 g, 11.6 mmol) and SOCl2
(15 mL). The solution was refluxed for 4 h and then concen-
trated to yield a yellow solid, which was used without further
νmax cm-1: 3247, 3090, 2919, 1527, 1454, 1429, 1188. GC tR
)
15.4 min. EI-MS m/z (%): 228 (100, M+), 227 (71), 186 (22), 154
(23), 130 (39). RP-HPLC (1/1 MeOH/H2O) tR ) 18.6 min.
Computational Procedure. All electronic structure calculations
were carried out using the Gaussian 03 suite of programs.31 Natural
bond orbital (NBO) population analysis was done with NBO 3.1
as implemented in Gaussian 03.32 All compounds with terminal
methyl groups were optimized at the HF/6-31G*//HF/6-31G33 level.
ESP34 and NBO atomic charges were computed. The HF/6-31G*
molecular ESP surface was mapped onto the total density surface.
Inhibition Assays with IDO. The inhibition assays were
performed in a 96 well microtiter plate as described by Littlejohn
et al.14 with a small modification. Briefly, the reaction mixture
contained 50 mM potassium phosphate buffer (pH 6.5), 40 mM
1
purification (2.21 g, 100% yield). H NMR (CDCl3): δ 8.76 (s,
1H, ArH), 8.04 (2H, ArH), 7.93 (d, 2H, ArH, J ) 8.9 Hz), 7.66
(m, 2H, ArH).
2-Naphthamide (27). 2-Naphthoyl chloride (2.21 g, 11.6 mmol)
was dissolved in a MeOH/NH3 solution (2 M, 20 mL) and was
allowed to stir overnight. Volatiles were removed, and the resulting
white solid was triturated with EtOAc. The solid was filtered and
washed with cold EtOAc to yield a white solid, which was used
without further purification (1.98 g, 100% yield); mp 191-192 °C.
1H NMR (CDCl3): δ 8.39 (s, 1H, ArH), 7.90 (4H, ArH), 7.57 (m,
2H, ArH). 13C NMR (CDCl3): δ 169.3, 135.0, 132.6, 130.5, 129.0,
128.6, 128.1, 127.9, 127.8, 126.9, 123.7. IR (Nujol) νmax cm-1
3400, 3210, 1650, 1628, 1512, 1510.
:

Structure-ActiVity Study of Brassinin DeriVatiVes
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 2 691
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ascorbic acid, 400 µg/mL catalase, 20 µM methylene blue, and
purified recombinant IDO(1) optimized based on its activity. The
reaction mixture was added to the substrate, L-Trp, and the inhibitor.
The L-Trp was serially diluted from 200 to 25 µM, and the inhibitors
were tested at two concentrations, 200 and 400 µM. The reaction
was carried out at 37 °C for 60 min and stopped by adding 30%
(w/v) trichloroacetic acid. The plate was heated at 65 °C for 15
min to convert formylkynurenine to kynurenine and then was spun
at 6000g for 5 min. Finally, 100 µL of supernatant from each well
was transferred to a new 96 well plate and mixed with 2% (w/v)
p-(dimethylamino)benzaldehyde in acetic acid. The yellow color
generated from the reaction with kynurenine was measured at 490
nm using a Synergy HT microtiter plate reader (Bio-Tek, Winooski,
VT). The data were analyzed using Graph Pad Prism 4 software
(Graph Pad Software Inc., San Diego, CA).
Acknowledgment. We thank the National Institutes of
Health (NCI R01-CA 109542) and Bryn Mawr College (Faculty
Research Fund) for financial support of this work. We are
grateful to Prof. Michelle Francl of Bryn Mawr College for
assistance with the computational experimentation.
Supporting Information Available: Copies of 1H NMR spectra
for compounds 1-24, 27-29, and 31. Copies of 13C NMR spectra
for compounds 1-24 and 27-29. Copies of HPLC data for
compounds 1-24. Copies of GC data for compounds 15, 17, 18,
23, 24, and 28. Copies of MS data for compounds 15, 17, 18, 23,
24, and 28. This material is available free of charge via the Internet
at http://pubs.acs.org.
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