Synthesis and biological evaluation of novel tryptoline derivatives as indoleamine 2,3-dioxygenase (IDO) inhibitors

Article abstract of DOI:10.1016/j.bmc.2012.12.028

Indoleamine 2,3-dioxygenase (IDO) plays a significant role in several disorders such as Alzheimer's disease, age-related cataracts and tumors. A series of novel tryptoline derivatives were synthesized and evaluated for their inhibitory activity against ID

Full text of DOI:10.1016/j.bmc.2012.12.028

Bioorganic & Medicinal Chemistry xxx (2013) xxx–xxx
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of novel tryptoline derivatives as
indoleamine 2,3-dioxygenase (IDO) inhibitors
Minoru Tanaka a, Xin Li a, Hidemasa Hikawa a, Takafumi Suzuki a, Katsuhiko Tsutsumi a, Masashi Sato a,
Osamu Takikawa b, Hideharu Suzuki a, Yuusaku Yokoyama a,
⇑
a Faculty of Pharmaceutical Sciences, Toho University, 2-2-1 Miyama, Funabashi, Chiba 274-8510, Japan
b National Institute for Longevity Science, 36-3 Gengo, Morioka-machi, Ohbu, Aichi 474-8511, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Indoleamine 2,3-dioxygenase (IDO) plays a significant role in several disorders such as Alzheimer’s dis-
ease, age-related cataracts and tumors. A series of novel tryptoline derivatives were synthesized and eval-
uated for their inhibitory activity against IDO. Substituted tryptoline derivatives (11a, 11c, 11e, 12b and
12c) were demonstrated to be more potent than known inhibitor MTH-Trp. Suzuki–Miyaura cross-cou-
pling reaction of 11a–d with phenylboronic acid proceeded in high yields. In most cases, C5 and C6 sub-
stitutions on the corresponding indole ring were well tolerated. The tryptoline derivative 11c is a
promising chemical lead for the discovery of novel IDO inhibitors.
Received 6 December 2012
Revised 23 December 2012
Accepted 24 December 2012
Available online xxxx
Keywords:
Indoleamine 2,3-dioxygenase
IDO
Ó 2013 Published by Elsevier Ltd.
Tryptoline derivatives
Tryptophan
Biomimetic synthesis
1. Introduction
studying biological roles of IDO. A more potent inhibitor, meth-
ylthiohydantoin-DL-tryptophan (MTH-Trp; 2), was discovered by
Catabolism of tryptophan, one of the least abundant of the
essential amino acids, is tightly controlled in the human body.1
Tryptophan is converted to kynurenine through the kynurenine
pathway, the major metabolic route.2 Kynurenine is further metab-
olized into neurotoxic quinolinic acid or neuroprotective kynuren-
ine acid (Scheme 1). Indoleamine 2,3-dioxygenase (IDO) catalyzes
the initial and rate-limiting step of the pathway.3 A large body of
evidence has shown that tryptophan and its metabolites play sig-
nificant roles in several biological processes4–7 and disorders,
including Alzheimer’s disease,8 age-related cataracts9 and cancer.10
Since tryptophan is essential for T-lymphocyte proliferation,
IDO is deeply related to immune system. IDO is constitutively ex-
pressed in various types of tumor cells, thus escaping from im-
mune detection and attack.11 High expression of IDO predicts
poor prognosis of serous ovarian cancer,12 endometrial cancer,13
colorectal cancer14 and acute myeloid leukemia,15 suggesting IDO
as an attractive therapeutic target for these diseases.11,16 The IDO
library screening in 2005.20 The same group also identified a mod-
erately active inhibitor brassinin (3) in a subsequent screening
O
O
NH2
O
OH
NH2
IDO
OH
NH
N
H
CHO
L
-Tryptophan
N-Formylkynurenine
OH
O
OH
N
O
O
NH2
O
Qunolinic acid
OH
inhibitors 1-methyl-D-tryptophan and INCB024360 (structure
OH
undisclosed) are currently being studied in phase 1 clinical trials
NH2
in patients with advanced malignancies.17,18
OH
1-Methyl-DL-tryptophan (1-MT; 1), the first IDO inhibitor
was reported in 199119 and has been used as a chemical tool for
L-Kynurenine
N
O
Kynurenic acid
Scheme 1. The kynurenine pathway of tryptophan metabolism.
⇑
Corresponding author. Tel.: +81 47 472 1589; fax: +81 47 472 1595.
E-mail address: yokoyama@phar.toho-u.ac.jp (Y. Yokoyama).
0968-0896/$ - see front matter Ó 2013 Published by Elsevier Ltd.
http://dx.doi.org/10.1016/j.bmc.2012.12.028
Please cite this article in press as: Tanaka, M.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2012.12.028

2
M. Tanaka et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
Me
S
protected with trimethylsilyldiazomethane to yield the corre-
sponding methyl esters 8.
O
O
S
Me
S
OH
NH2
N
NH
In general, the selective removal of amide protecting groups
such as acetyl groups is difficult, because strongly acidic or basic
conditions at high temperature are required to hydrolyze the
amide bond. This sometimes results in racemization and decompo-
sition of the indole ring. We have reported29 the conversion of opti-
cally active 4-bromo-N-acetyltryptophan to N-Boc-tryptophan
under mild conditions by using Boc2O. According to this method,
simultaneous deacetylation and protection with (Boc)2O were
accomplished to provide 9. Removal of the Boc-groups using triflu-
oroacetic acid in dichloromethane, followed by Pictet–Spengler
reaction gave tryptoline carboxylic acid methyl esters 10 as an
inseparable diastereomixture (cis-major).30 Cyclization of thiohyd-
antoin was accomplished by treatment with methyl isothiocyanate
and triethylamine in dichloromethane to afford MTH-benztrypto-
lines 6 as a more stable trans-racemate.31
N
H
N
N
H
N
H
Me
1-MT (1)
Ki = 30 μM
MTH-Trp (2)
Ki = 11.4 μM
Brassinin (3)
Ki = 98 μM
Me
Me
NH2
O
N
N
O
O
O
N
Me
N
H
N
H
5
3-Butyl-β-carboline (4)
Ki = 3 μM
Ki = 200 nM
2.2. Synthesis of substituted MTH-tryptoline derivatives
Figure 1. Structures of representative IDO inhibitors.
Next, we turned our attention to simplified analogues of 6 to
acquire synthetic tractability. Commercially available bromo- or
chloro-substituted indoles were converted to the corresponding
halogen-substituted tryptoline derivatives 11a–h in the same
way as for 6 (Scheme 3). Suzuki–Miyaura cross-coupling reaction
of bromo-substituted analogues 11a–d with phenylboronic acid
proceeded smoothly to give the corresponding adducts 12a–d in
high yield.
campaign.21 3-Butyl-b-carboline (4)22 and a synthetic analogue of
marine sponge natural product (5)23 were both discovered as
potent inhibitors during the lead optimization effort. All of these
inhibitors possess an indole moiety as a common structural feature
(Fig. 1). Very recently, hydroxyamidine inhibitors have also been
reported.24
The chiral analogues 15a and 15b were prepared to confirm the
Substituted tryptophans themselves also function as IDO inhib-
itors.25 We have developed biomimetic syntheses of tryptophan
derivatives utilizing direct coupling of indole units and serine
equivalent.26,27 The reaction of indole and serine in the presence
of acetic anhydride was the first practical synthesis of tryptophan
derivatives having substituents on the benzene ring. During the
course of this study, we developed a practical synthesis of benztry-
ptophans and attempted to synthesize analogues of these to find
new IDO inhibitors. As a result of this study, we discovered trypto-
line derivatives (MTH-benz[e]tryptoline, 6a; MTH-benz[f]trypto-
line, 6b; MTH-benz[g]tryptoline, 6c; Fig. 2) as novel IDO inhibitors.
Herein, we present the synthesis and biological evaluation
of these MTH-benztryptoline derivatives and MTH-tryptoline
analogues, which were more easily synthesized than the benz
analogues. These compounds should be promising chemical leads
in the discovery of novel IDO inhibitors.
importance of the absolute configuration (Scheme 4).30,31 Starting
from L-tryptophan, Pictet–Spengler reaction gave a mixture of cis
and trans compounds (4:1), which were separable to give the pure
cis-diastereomer 13 as the major product. Esterification of 13
followed by cyclization with methyl isothiocyanate proceeded
smoothly to afford the more stable trans-diastereomer 15a as a
single product by inverting the asymmetric center of the amino
acid to give the
prepared from
D-enantiomer. The enantiomer 15b (L-series) was
D
-tryptophan in the same manner. The stereochem-
istry was determined by NOE experiments and 13C NMR chemical
shifts,31 and the ee’s of 15a and 15b were determined to be
96.2% and 95.3%, respectively, by chiral HPLC analysis.
3. Measurement of IDO inhibitory activity
The synthetic compounds were evaluated for IDO inhibitory
activity using the literature method.32 Briefly, the reaction mixture
contained potassium phosphate buffer, ascorbic acid, methylene
2. Chemistry
blue, catalase, human recombinant IDO,33
L-tryptophan and testing
2.1. Synthesis of MTH-benztryptoline (6a–c)
materials diluted in DMSO. The reaction was conducted at 37 °C
and stopped by addition of trichloroacetic acid. After centrifuga-
tion, kynurenine in the supernatant was measured by HPLC and
ultraviolet absorbance at 360 nm. The IC50 values were the means
of more than two independent experiment sets calculated by linear
interpolation.
The synthesis of MTH-benztryptolines is shown in Scheme 2. As
we previously reported,26 DL-N-acetylserine reacted with acetic
anhydride to give an intermediate that reacted in one-pot with
benzindoles28 (7) to afford N-acetyl-DL-benztryptophans by
Michael addition. The resulting compounds were subsequently
4. Results and discussion
O
O
O
Me
S
Me
S
Me
S
H
H
H
The results are shown in Table 1. MTH-benztryptolines (6a–c)
showed comparable activity with 2.34 Simplified tryptoline ana-
logues (11a–h, 12a–d) retained comparable activity with 6a–c,
indicating that synthesis of benztryptoline derivatives were not
essential. All of these analogues were synthesized from commer-
cially available substituted indoles, demonstrating synthetic trac-
tability. Five of them (11a, 11c, 11e, 12b and 12c) were more
potent than 2 or 6a–c. Interestingly, substitution at the C40 position
N
N
N
N
N
N
Me
Me
Me
N
H
N
H
N
H
6a
6b
6c
Figure 2. Structures of MTH-benztryptolines (6a–c).
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M. Tanaka et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
3
O
O
OMe
OMe
i. a : 80 oC, 3 h
b : 50 oC, 4 h
c : 50 oC, 4 h
e
e
e
NHAc
iii, iv
NHBoc
v, vi
f
f
f
N
H
N
H
N
g
g
ii
g
Boc
7a-c
8a: 29%
8b: 43%
8c: 59%
9a: 82%
9b: 70%
9c: 70%
a, Benz[e]-; b, Benz[f]-; c, Benz[g]-
O
O
Me
S
OMe
H
H
N
e
e
NH
Me
N
vii
f
f
Me
N
H
N
H
g
g
10a-c (diastereo mixture)
6a: 71%
6b
6c
: 6%
: 19%
trans-form
Scheme 2. Reagents and conditions: (i) 2.0 mol equiv of DL-serine, 9.5 mol equiv of Ac2O in acetic acid; (ii) 2.6 mol equiv of trimethyl-silyldiazomethane, ethyl acetate/MeOH,
rt, 1 h; (iii) 5.0 mol equiv of (Boc)2O, 1.0 mol equiv of DMAP in THF, rt, 3 h; (iv) 11.0 mol equiv of H2NNH2ꢀH2O in dichloroethane/MeOH, rt, 3 h; (v) 10.0 mol equiv of
trifluoroacetic acid in dichloromethane, rt, 8 h; (vi) 10.0 mol equiv of acetaldehyde, rt, 5 h; (vii) 5.0 mol equiv of methyl isothiocyanate in triethylamine–dichloromethane
(3:1), rt, 3 h.
O
O
(S)
O
Me
S
4'
7'
OH
OH
NH2
H
H
N
5'
X
6'
i-vii
4'
7'
i
ii
(S)
(S)
5'
X
6'
N
NH
Me
N
H
y. 62%
crude
Me
N
H
N
H
N
H
y. 6-40%
11a h
- (trans-form)
X-indole (X = Br, Cl)
13
L
-Tryptophan
a
b
, X = 4'-Br; , X = 5'-Br;
c, X = 6'-Br; d, X = 7'-Br;
e, X = 4'-Cl; f, X = 5'-Cl;
g, X = 6'-Cl; h, X = 7'-Cl
O
O
Me
S
OMe
H
H
N
(S)
(S)
iii
(R)
NH
(S)N
Me
O
y. 69%
Me
S
Me
N
H
N
H
H
N
viii
4'
7'
5'
Ph
6'
N
y. 60-95%
14
15a
Me
N
H
O
O
(R)
12a d
-
(trans-form)
b
Me
OH
NH2
H
N
a
, 4'-Ph; , 5'-Ph;
i, ii, iii
y. 3
(S)
N
(R)
c, 6'-Ph; d, 7'-Ph
S
Me
N
N
H
Scheme 3. Reagents and conditions: (i) 2.0 mol equiv of DL-serine, 9.5 mol equiv of
Ac2O in acetic acid; (ii) 2.6 mol equiv of trimethyl-silyldiazomethane, ethyl acetate/
MeOH, rt, 1 h; (iii) 5.0 mol equiv of (Boc)2O, 1.0 mol equiv of DMAP in THF, rt, 3 h;
(iv) 11.0 mol equiv of H2NNH2ꢀH2O in dichloroethane/MeOH, rt, 3 h; (v) 10.0 mol e-
quiv of trifluoroacetic acid in dichloromethane, rt, 8 h; (vi) 10.0 mol equiv of
acetaldehyde, rt, 5 h; (vii) 5.0 mol equiv of methyl isothiocyanate in triethylamine–
dichloromethane (3:1), rt, 3 h; (viii) 1.5 mol equiv of phenylboronic acid,
0.10 mol equiv of PdCl2(PCy3)2, 1.5 mol equiv of K3PO4, 1,4-dioxane, reflux, 4 h.
H
15b
D
-Tryptophan
Scheme 4. Reagents and conditions: (i) 3.6 mol equiv of acetaldehyde, H2SO4, rt,
6 h; (ii) 1.5 mol equiv of thionyl chloride, MeOH, rt, 8 h; (iii) 5.0 mol equiv of methyl
isothiocyanate in triethylamine-dichloromethane (3:1), rt, 3 h.
of the corresponding indole was more favored than C60 in chloro
derivatives (11e vs 11g), whereas the opposite was true in the lar-
ger bromo substituents (11a vs 11c). In both cases, C70 substitution
was the least favored (11d and 11h), suggesting that the indole NH
well with the literature except for the phenyl derivative 12d. One
possibility is that the binding mode of 12d is different from those
of others because of steric clash between the phenyl substituent
and the heme iron. In contrast, the phenyl group at the C40 position
was more disfavored than at C70 (12a vs 12d), demonstrating
intolerance of bulky substituents at C40. This was in line with
MTH-benz[g]tryptoline 6c which showed stronger inhibitory
is essential. Recently, docking studies of L-Trp with IDO showed the
importance of the indolic nitrogen atom, which is involved in a ter-
nary complex with Fe(III) and superoxide.35 Our results accorded
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4
M. Tanaka et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
Table 1
Melting or decomposition points (Mp or Dp, respectively) were
IDO inhibitory activity of methylthiohydantoin derivatives.
obtained on a Büchi 535 melting point apparatus and are uncor-
rected. Elemental analyses were performed on a Perkin Elmer
2400 II CHN Analyzer. High performance liquid chromatography
(HPLC) was performed on a Waters 2795 Series chromatographs
using Chiralpak IC-3 column (15 cm) in mixture of hexane/etha-
nol/diethylamine (85:15:0.1).
Compound
Substituent
IC50 (lM)
MTH-Trp (2)
MTH-Benz[e]tryptoline (6a)
MTH-Benz[f]tryptoline (6b)
MTH-Benz[g]tryptoline (6c)
76.9
82.0
58.9
55.0
68.2
74.6
46.1
109.3
46.4
113.9
85.5
119.8
95.4
63.5
62.0
11a
11b
11c
11d
11e
11f
11g
11h
12a
12b
12c
12d
15a
15b
X = 40-Br
X = 50-Br
X = 60-Br
X = 70-Br
X = 40-Cl
X = 50-Cl
X = 60-Cl
X = 70-Cl
X = 40-Ph
X = 50-Ph
X = 60-Ph
X = 70-Ph
6.2. General procedure for the synthesis of N-acetyl-
benztryptophan methyl ester (8)
DL-Serine (2.0 mol equiv) was added to acetic acid (0.2 M) and
acetic anhydride (9.5 mol equiv), and the mixture was stirred for
3 h at 45 °C. To the reaction mixture, compound 7 (1.0 mol equiv)
was added and stirred under the conditions shown in Scheme 2.
After pouring into water, the mixture was concentrated in vacuo,
the residue was extracted with ethyl acetate. The combined organ-
ic layers were washed with 30% NaOH aq and brine, dried over so-
dium sulfate, and concentrated in vacuo. The residue was dissolved
in methanol (0.37 M) and ethyl acetate (0.16 M), 2.0 M trimethyl-
silyldiazomethane in toluene solution (2.6 mol equiv) was added
dropwise, and the mixture was stirred for 1 h. After the addition
of acetic acid (4.0 mol equiv), the reaction mixture was extracted
with ethyl acetate. The combined organic layers were washed with
brine, dried over sodium sulfate, and concentrated in vacuo.
The residue was purified by silica gel column chromatography
(hexane–ethyl acetate) to give the product.
81.1
39% Inhibition at 200
174.6
lM
activity than MTH-benz[e]tryptoline 6a. Phenyl substitutions at
both C50 and C60 were well tolerated (12b and 12c). According to
the docking model, substituents on the C50 or C60 positions are
directed toward the flexible loop. Consequently, the C60 position
is one of the most preferred by every substituent, predicting the
existence of chemical space in this area. The bromide 11c is one
of the best inhibitors and is being used as a chemical lead in further
discovery efforts.
6.2.1. N-Acetyl-benz[e]tryptophan methyl ester (8a)
Yield 29%. Pale yellow powder. Mp 178–183 °C. 1H NMR
(400 MHz, CDCl3): d = 8.42 (d, J = 8.4 Hz, 1H), 8.40 (br s, 1H), 7.90
(d, J = 8.0 Hz, 1H), 7.58 (d, J = 9.2 Hz, 1H), 7.55 (ddd, J = 8.4, 6.8,
1.6 Hz, 1H), 7.48 (d, J = 8.8 Hz, 1H), 7.40 (ddd, J = 8.0, 6.8, 1.2 Hz,
1H), 7.05 (d, J = 2.0 Hz, 1H), 5.98 (d, J = 8.0 Hz, 1H), 5.12 (dt,
J = 6.8, 5.2 Hz, 1H), 3.76 (dd, J = 15.2, 5.6 Hz, 1H), 3.69 (s, 3H),
3.59 (dd, J = 15.2, 6.4 Hz, 1H), 1.82 (s, 3H). 13C NMR (100 MHz,
CDCl3): d = 172.7, 169.9, 133.3, 129.9, 129.0, 128.9, 126.0, 123.9,
123.2, 122.9, 121.2, 120.2, 113.0, 112.9, 53.0, 52.3, 30.3, 23.2. MS
(EI) m/z 310 [M]+. Anal. Calcd for C18H18N2O3: C, 69.66; H, 5.85;
N, 9.03. Found: C, 69.86; H, 5.86; N, 8.71. IR (KBr): 3409, 3258,
The
than the
that the
L
-enantiomer 15b exhibited more potent inhibitory activity
-enantiomer 15a. This is well correlated with the fact
-enantiomer is the eutomer of 1-MT (1). We have recently
D
L
reported the convenient synthesis of chiral tryptophan derivatives
using Negishi cross-coupling between substituted indoles and iod-
oalanine derived from chiral serine.27 According to this method,
enantiomerically pure substituted tryptoline analogues could be
obtained.
5. Conclusion
A series of substituted tryptoline derivatives were synthesized
and evaluated for their inhibitory activity against IDO. Five com-
pounds revealed better activity than MTH-Trp (2), suggesting that
the template has sufficient chemical space. The most potent inhib-
itor 11c was easily reacted under cross-coupling conditions,
demonstrating synthetic tractability. It was shown that substitu-
tion at C50 or C60 was preferred. In the present study, we further
1749, 1644 cmꢁ1
.
6.2.2. N-Acetyl-benz[f]tryptophan methyl ester (8b)
Yield 43%. Colorless needles. Mp 191–194 °C. 1H NMR
(400 MHz, CDCl3): d = 8.38 (br s, 1H), 8.10 (s, 1H), 7.93 (d,
J = 7.6 Hz, 1H), 7.87 (d, J = 8.0 Hz, 1H), 7.81 (s, 1H), 7.35–7.42 (m,
2H), 7.20 (d, J = 2.4 Hz, 1H), 6.12 (d, J = 3.6 Hz, 1H), 5.03 (dd,
J = 5.2, 4.8 Hz, 1H), 3.70 (s, 3H), 3.47 (dd, J = 9.6, 5.6 Hz, 1H), 3.40
(dd, J = 14.8, 5.6 Hz, 1H), 1.99 (s, 3H). 13C NMR (100 MHz, DMSO-
d6): d = 172.5, 169.3, 136.9, 129.4, 129.3, 128.3, 127.8, 127.5,
127.2, 123.1, 122.0, 115.0, 108.5, 106.3, 52.8, 51.8, 27.2, 22.3. MS
(EI) m/z 310 [M]+. Anal. Calcd for C18H18N2O3: C, 69.66; H, 5.85;
N, 9.03. Found: C, 69.71; H, 5.89; N, 9.01. IR (KBr): 3392, 1752,
revealed that the L-enantiomer was the eutomer. We have already
established a synthetic method of chiral tryptophan analogues
having substituents on the benzene ring. Chiral substituted trypto-
line derivatives can be prepared according to the reported method.
In conclusion, tryptoline derivatives are considered to be prom-
ising chemical leads in the discovery of novel IDO inhibitors.
1642 cmꢁ1
.
6. Experimental
6.2.3. N-Acetyl-benz[g]tryptophan methyl ester (8c)
Yield 59%. Colorless needles. Mp 194–197 °C. 1H NMR
(400 MHz, DMSO-d6): d = 11.60 (br s, 1H), 8.23 (d, J = 8.0 Hz, 1H),
8.01 (d. J = 7.6 Hz, 1H), 7.89 (d, J = 8.0 Hz, 1H), 7.63 (d, J = 8.8 Hz,
1H), 7.50 (t, J = 6.8 Hz, 1H), 7.44 (d, J = 8.1 Hz, 1H), 7.37 (t,
J = 6.8 Hz, 1H), 7.19 (d, J = 1.6 Hz, 1H), 4.59 (dd, J = 9.2, 6.0 Hz,
1H), 3.59 (s, 3H), 3.23 (dd, J = 8.8, 6.0 Hz, 1H), 3.13–3.16 (m, 1H),
1.83 (s, 3H). 13C NMR (100 MHz, DMSO-d6): d = 172.1, 169.0,
130.2, 129.4, 128.0, 124.9, 123.2, 122.6, 121.8, 121.6, 120.3,
119.0, 118.5, 111.4, 53.4, 51.7, 27.2, 22.3. MS (EI) m/z 310 [M]+.
6.1. General experimental
Silica gel column chromatography was performed using Fuji
Silysia PSQ100B. 1H NMR spectra were recorded on a Bruker
Avance 400 (400 MHz) spectrometer. Chemical shifts are
expressed in ppm relative to TMS as an internal standard. Mass
spectra were measured in a combination with a Waters Acquity
UPLC system (0.05% trifluoroacetic acid in acetonitrile: 0.05%
trifluoroacetic acid in H2O) and a Micromass ZQ (ESI) spectrometer.
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M. Tanaka et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
5
Anal. Calcd for C18H18N2O3: C, 69.66; H, 5.85; N, 9.03. Found: C,
centrated in vacuo to give benztryptoline derivative (10), which
was used in the next step without purification.
69.62; H, 5.93; N, 8.87. IR (KBr): 3401, 3296, 1725, 1648 cmꢁ1
.
Compound 10 (1 mol equiv) was dissolved in dichloromethane
(0.17 M) and triethylamine (0.3 M). To a stirred solution, methyl iso-
thiocyanate (5.0 mol equiv) was added. The mixture was stirred for
3 h at 30 °C. After pouring into water, the mixture was concentrated
in vacuo, the residue was extracted with ethyl acetate. The com-
bined organic layers were washed with brine, dried over sodium sul-
fate, and concentratedin vacuo. The residue was purified by silica gel
columnchromatography(hexane–ethyl acetate) to give the product.
6.3. General procedure for the synthesis of N1,N2-di-Boc-
benztryptophan methyl ester (9)
Boc2O (5.0 mol equiv) was added to a stirred solution of com-
pound 8 (1.0 mol equiv) and DMAP (1.0 mol equiv) in tetrahydro-
furan (0.1 M), and the mixture was stirred for 3 h. After pouring
into water, the mixture was extracted with ethyl acetate. The com-
bined organic layers were washed with brine, dried over sodium
sulfate, and concentrated in vacuo, the residue was purified by sil-
ica gel column chromatography (hexane–ethyl acetate) to give a
yellow solid. The solid was dissolved in 1,2-dichloroethane
(0.24 M) and methanol (1 M). Hydrazine monohydrate (11 mol e-
quiv) was added dropwise to the solution. After stirring for 1 h,
water was added to the reaction mixture. The mixture was ex-
tracted with ethyl acetate, and the combined organic layers were
washed with brine, dried over sodium sulfate, and concentrated
in vacuo. The residue was purified by silica gel column chromatog-
raphy (hexane–ethyl acetate) to give the product.
6.4.1. trans-9,11-Dimethyl-10-thioxo-7,7a,9,10,11,12-
hexahydro-9,10a,12-triaza-benzo[c]cyclopenta[h]fluoren-8-one
(MTH-benz[e]tryptoline; 6a)
Yield 71%. Colorless powder. Dp 260–263 °C. 1H NMR (400 MHz,
CDCl3): d = 8.25 (s, 1H), 8.22 (d, J = 8.4 Hz, 1H), 7.90 (d, J = 8.0 Hz,
1H), 7.60 (d, J = 8.8 Hz, 1H), 7.54 (ddd, J = 8.4, 6.8, 1.6 Hz, 1H),
7.48 (d, J = 8.4 Hz, 1H), 7.42 (ddd, J = 8.0, 7.2, 0.8 Hz, 1H), 5.93 (q,
J = 6.4 Hz, 1H), 4.46 (dd, J = 10.8, 6.0 Hz, 1H), 3.92 (dd, J = 14.8,
5.6 Hz, 1H), 3.35 (s, 3H), 3.22 (dd, J = 15.2, 11.2 Hz, 1H), 1.69 (d,
J = 6.8 Hz, 3H). 13C NMR (100 MHz, DMSO-d6): d = 179.7, 173.4,
132.7, 132.2, 129.0, 128.6, 128.3, 125.7, 122.7, 122.3, 118.7,
113.5, 106.1, 55.1, 48.2, 27.4, 25.1, 19.2. MS (ESI) m/z 336
[M+H]+. HRMS (EI): C19H17N3OS requires m/z 335.1092, found m/
6.3.1. N1,N2-di-Boc-benz[e]tryptophan methyl ester (9a)
Yield 82%. Colorless solid. Mp 155–157 °C. 1H NMR (400 MHz,
acetone-d6): d = 8.53 (d, J = 8.4 Hz, 1H), 8.41 (d, J = 8.8 Hz, 1H),
8.01 (d, J = 7.6 Hz, 1H), 7.80 (d, J = 8.8 Hz, 1H), 7.70 (s, 1H), 7.63
(t, J = 8.4 Hz, 1H), 7.50 (t, J = 8.0 Hz, 1H), 6.45 (d, J = 8.1 Hz, 1H),
4.72 (s, 1H), 3.71–3.76 (m, 4H), 3.43 (dd, J = 15.2, 10.8 Hz, 1H),
1.70 (s, 9H), 1.33 (s, 9H). 13C NMR (100 MHz, DMSO-d6):
d = 172.5, 155.5, 148.9, 132.4, 130.2, 128.9, 127.6, 126.6, 125.3,
124.3, 123.7, 122.9, 117.8, 115.0, 84.1, 78.3, 53.0, 52.1, 29.2, 28.0,
27.6, 27.4. MS (EI) m/z 468 [M]+. Anal. Calcd For C26H32N2O6: C,
66.65; H, 6.88; N, 5.98. Found: C, 66.66; H, 6.94; N, 5.74. IR
z 335.1095. IR (KBr): 3380, 1722 cmꢁ1
.
6.4.2. trans-9,11-Dimethyl-10-thioxo-7,7a,9,10,11,12-
hexahydro-9,10a,12-triaza-benzo[b]cyclopenta[h]fluoren-8-one
(MTH-benz[f]tryptoline; 6b)
Yield 6%. Colorless powder. Dp 237–242 °C. 1H NMR (400 MHz,
CDCl3): d = 7.92 (d, J = 9.6 Hz, 1H), 7.91 (s, 1H), 7.86 (d, J = 8.8 Hz,
1H), 7.78 (br s, 1H), 7.73 (s, 1H), 7.33–7.40 (m, 2H), 5.90 (q,
J = 6.8 Hz, 1H), 4.42 (dd, J = 10.8, 5.6 Hz, 1H), 3.50 (dd, J = 15.2,
5.6 Hz, 1H), 3.34 (s, 3H), 2.89 (dd, J = 15.2, 10.8 Hz, 1H), 1.68 (d,
J = 6.8 Hz, 3H). HRMS (EI): C19H17N3OS requires m/z 335.1092,
(KBr): 3364, 1744, 1709 cmꢁ1
.
6.3.2. N1,N2-di-Boc-benz[f]tryptophan methyl ester (9b)
found m/z 335.1081. IR (KBr): 3355, 1726 cmꢁ1
.
Yield 70%. Colorless powder. Mp 158–165 °C. 1H NMR
(400 MHz, acetone-d6): d = 8.62 (s, 1H), 8.13 (s, 1H), 8.02 (d,
J = 7.2 Hz, 1H), 8.01 (d, J = 5.6 Hz, 1H), 7.71 (s, 1H), 7.45 (ddd,
J = 10.8, 6.0, 3.2 Hz, 2H), 6.34 (d, J = 7.6 Hz, 1H), 4.61 (q, J = 5.2 Hz,
1H), 3.71 (s, 3H), 3.38 (dd, J = 15.2, 5.2 Hz, 1H), 3.23 (dd, J = 15.2,
9.6 Hz, 1H), 1.71 (s, 9H), 1.33 (s, 9H). MS (EI) m/z 468 [M]+. Anal.
Calcd For C26H32N2O6: C, 66.65; H, 6.88; N, 5.98. Found: C, 66.83;
6.4.3. trans-9,11-Dimethyl-10-thioxo-7,7a,9,10,11,12-
hexahydro-9,10a,12-triaza-benzo[a]cyclopenta[h]fluoren-8-
one (MTH-benz[g]tryptoline; 6c)
Yield 19%. Colorless powder. Dp 247 °C. 1H NMR (400 MHz,
CDCl3): d = 8.58, (s, 1H), 7.98 (d, J = 8.0 Hz, 1H), 7.92 (d, J = 8.4 Hz,
1H), 7.52–7.58 (m, 3H), 7.44 (d, J = 8.0 Hz, 1H), 5.98 (q, J = 6.4 Hz,
1H), 4.41 (dd, J = 11.2, 5.6 Hz, 1H), 3.50 (dd, J = 14.8, 5.6 Hz, 1H),
3.33 (s, 3H), 2.90 (ddd, J = 14.8, 11.2, 2.0 Hz, 1H), 1.71 (d,
J = 6.8 Hz, 3H). HRMS (EI): C19H17N3OS requires m/z 335.1092,
H, 6.96; N, 5.78. IR (KBr): 3445, 1725 cmꢁ1
.
6.3.3. N1,N2-Di-Boc-benz[g]tryptophan methyl ester (9c)
Yield 70%. Colorless oil. 1H NMR (400 MHz, CDCl3): d = 8.82 (d,
J = 8.1 Hz, 1H), 7.90 (d, J = 8.0 Hz, 1H), 7.69 (d, J = 8.1 Hz, 1H),
7.51–7.61 (m, 2H), 7.43–7.46 (m, 2H), 3.69 (s, 3H), 3.32 (dd,
J = 6.4, 4.2 Hz, 1H), 3.26 (dd, J = 6.4, 4.2 Hz, 1H), 1.72 (s, 9H), 1.44
(s, 9H). 13C NMR (100 MHz, CDCl3): d = 172.3, 155.1, 150.3, 132.5,
130.9, 128.7, 126.0, 125.3, 125.2, 124.9, 124.5, 123.4, 117.6,
115.4, 84.1, 80.0, 53.8, 52.3, 29.7, 28.3, 28.1, 27.7. MS (ESI) m/z
491 [M+Na]+. HRMS (EI): C26H32N2O6 requires m/z 468.2260.
found m/z 335.1095. IR (KBr): 3354, 1726 cmꢁ1
.
6.4.4. trans-10-Bromo-2,5-dimethyl-1-oxo-5,6,11,11a-
tetrahydro-1H-imidazo[10,50:1,6]pydiro[3,4-b]indole-3(2H)-
thione (11a)
Yield 28%. Pale brown solid. Dp 285–288 °C. 1H NMR (400 MHz,
CDCl3): d = 11.56 (s, 1H), 7.38 (d, J = 8.0 Hz, 1H), 7.19 (d, J = 7.0 Hz,
1H), 7.02 (d, J = 8.0 Hz, 1H), 5.64 (q, J = 6.7 Hz, 1H), 4.81 (dd,
J = 10.8, 5.6 Hz, 1H), 3.73 (dd, J = 15.4, 6.1 Hz, 1H), 3.16 (s, 3H),
3.00–3.05 (m, 1H), 1.58 (d, J = 6.7 Hz, 3H). 13C NMR (100 MHz,
DMSO-d6): d = 179.7, 173.2, 137.4, 135.7, 124.3, 122.7, 112.5,
111.0, 104.3, 54.9, 48.1, 27.4, 24.0, 18.9. MS (ESI) m/z 364
[M+H]+. HRMS (EI): C15H14BrN3OS requires m/z 363.0041, found
Found m/z 468.2261. IR (neat): 1740, 1160 cmꢁ1
.
6.4. General procedure for the synthesis of MTH-tryptoline
derivatives (6, 11)
Trifluoroacetic acid (10.0 mol equiv) was added to a stirred
m/z 363.0043. IR (KBr): 3329, 1729, 1485 cmꢁ1
.
solution of compound
9 (1 mol equiv) in dichloromethane
(0.17 M). After stirring for 30 min, acetaldehyde (10.0 mol equiv)
was added to the reaction mixture. The mixture was stirred for an-
other 8 h, concentrated in vacuo, the residue was extracted with
ethyl acetate. The combined organic layers were washed with sat-
urated NaHCO3 aq and brine, dried over sodium sulfate, and con-
6.4.5. trans-9-Bromo-2,5-dimethyl-1-oxo-5,6,11,11a-
tetrahydro-1H-imidazo[10,50:1,6]pydiro[3,4-b]indole-3(2H)-
thione (11b)
Yield 32%. Pale brown solid. Mp 210–212 °C. 1H NMR (400 MHz,
CDCl3): d = 11.39 (s, 1H), 7.71 (d, J = 2.1 Hz, 1H), 7.33 (d, J = 8.7 Hz,
Please cite this article in press as: Tanaka, M.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2012.12.028

6
M. Tanaka et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
1H), 7.21 (dd, J = 8.4, 1.8 Hz, 1H), 5.67 (q, J = 6.7 Hz, 1H), 4.79 (dd,
J = 10.8, 5.6 Hz, 1H), 3.29 (dd, J = 10.2, 5.1 Hz, 1H), 3.16 (s, 3H),
2.69–2.85 (m, 1H), 1.57 (d, J = 6.7 Hz, 3H). 13C NMR (100 MHz,
DMSO-d6): d = 179.8, 173.2, 135.7, 135.0, 127.6, 124.0, 120.6,
113.2, 111.4, 104.0, 55.2, 48.3, 27.4, 22.3, 18.9. MS (ESI) m/z 364
[M+H]+. HRMS (EI): C15H14BrN3OS requires m/z 363.0041, found
1H), 7.03 (dd, J = 8.4, 1.8 Hz, 1H), 5.66 (q, J = 6.5 Hz, 1H), 4.79 (dd,
J = 11.3, 5.6 Hz, 1H), 3.29 (dd, J = 15.1, 5.9 Hz, 1H), 3.16 (s, 3H),
2.77–2.83 (m, 1H), 1.57 (d, J = 6.1 Hz, 3H). 13C NMR (100 MHz,
DMSO-d6): d = 179.8, 173.2, 136.7, 135.2, 126.1, 124.6, 119.6,
119.2, 110.9, 104.4, 55.2, 48.3, 27.4, 22.3, 18.9. MS (ESI) m/z 320
[M+H]+. Anal. Calcd for C15H14ClN3OSꢀ0.4H2O: C, 55.09; H, 4.56;
N, 12.85. Found: C, 55.29; H, 4.73; N, 12.64. IR (KBr): 3333, 1728,
m/z 363.0042. IR (KBr): 3345, 1739, 1476, 1312 cmꢁ1
.
1466, 1313 cmꢁ1
.
6.4.6. trans-8-Bromo-2,5-dimethyl-1-oxo-5,6,11,11a-
tetrahydro-1H-imidazo[10,50:1,6]pydiro[3,4-b]indole-3(2H)-
thione (11c)
6.4.11. trans-7-Chloro-2,5-dimethyl-1-oxo-5,6,11,11a-
tetrahydro-1H-imidazo[10,50:1,6]pydiro[3,4-b]indole-3(2H)-
thione (11h)
Yield 11%. Pale brown solid. Mp 254–256 °C. 1H NMR (400 MHz,
CDCl3): d = 11.35 (s, 1H), 7.55 (d, J = 1.5 Hz, 1H), 7.47 (d, J = 8.7 Hz,
1H), 7.14–7.16 (m, 1H), 5.66 (q, J = 6.3 Hz, 1H), 4.79 (dd, J = 11.0,
5.9 Hz, 1H), 3.28 (dd, J = 14.8, 5.6 Hz, 1H), 3.16 (s, 3H), 3.09 (dd,
J = 7.2, 4.6 Hz, 1H), 1.57 (d, J = 6.7 Hz, 3H). 13C NMR (100 MHz,
DMSO-d6): d = 179.8, 173.2, 137.1, 135.1, 124.9, 121.8, 120.0,
114.2, 113.8, 104.5, 55.2, 48.2, 27.4, 22.3, 18.8. MS (ESI) m/z 364
[M+H]+. HRMS (EI): C15H14BrN3OS requires m/z 363.0041, found
Yield 21%. Pale brown solid. Dp 283–285 °C. 1H NMR (400 MHz,
CDCl3): d = 11.44 (s, 1H), 7.49 (d, J = 7.7 Hz, 1H), 7.19 (d, J = 7.7 Hz,
1H), 7.03 (dd, J = 7.7 Hz, 1H), 5.70 (q, J = 6.1 Hz, 1H), 4.82 (dd,
J = 10.8, 5.6 Hz, 1H), 3.30 (dd, J = 15.1, 5.9 Hz, 1H), 3.17 (s, 3H),
2.83 (ddd, J = 15.0, 11.1, 1.5 Hz, 1H), 1.61 (d, J = 6.7 Hz, 3H). 13C
NMR (100 MHz, DMSO-d6): d = 179.7, 173.2, 135.6, 133.2, 127.7,
121.1, 120.0, 117.3, 115.6, 105.5, 54.9, 48.3, 27.4, 22.4, 19.0. MS
(ESI) m/z 320 [M+H]+. Anal. Calcd. for C15H14ClN3OSꢀ0.8H2O: C,
53.90; H, 4.70; N, 12.57. Found: C, 54.09; H, 4.57; N, 12.35. IR
m/z 363.0038. IR (KBr): 3331, 1727, 1468, 1313 cmꢁ1
.
6.4.7. trans-7-Bromo-2,5-dimethyl-1-oxo-5,6,11,11a-
tetrahydro-1H-imidazo[10,50:1,6]pydiro[3,4-b]indole-3(2H)-
thione (11d)
(KBr): 3367, 1725, 1480, 1308 cmꢁ1
.
Yield 6%. Pale brown solid. Mp 265–267 °C. 1H NMR (400 MHz,
CDCl3): d = 11.30 (s, 1H), 7.53 (d, J = 7.7 Hz, 1H), 7.33 (d, J = 7.7 Hz,
1H), 6.98 (t, J = 7.7 Hz, 1H), 5.71 (q, J = 6.7 Hz, 1H), 4.83 (dd, J = 11.0,
5.9 Hz, 1H), 3.29 (dd, J = 15.1, 5.9 Hz, 1H), 3.16 (s, 3H), 3.07–3.09
(m, 1H), 1.60 (d, J = 6.7 Hz, 3H). 13C NMR (100 MHz, DMSO-d6):
d = 179.7, 173.2, 135.6, 134.7, 127.5, 124.1, 120.4, 117.8, 105.6,
103.9, 54.9, 48.3, 27.4, 22.4, 19.1. MS (ESI) m/z 364 [M+H]+. HRMS
(EI): C15H14BrN3OS requires m/z 363.0041, found m/z 363.0040. IR
6.5. General procedure for the synthesis of MTH-
phenyltryptoline (12)
A mixture of MTH-bromotryptoline (1 mol equiv), phenylbo-
ronic acid (1.5 mol equiv), and PdCl2(PCy3)2 (0.1 mol equiv) in
1,4-dioxane (0.5 M) and 1.27 M K3PO4 aq (1.5 mol equiv) was re-
fluxed for 4 h. After pouring into water, the mixture was extracted
with ethyl acetate. The combined organic layers were washed with
brine, dried over sodium sulfate, and concentrated in vacuo. The
residue was purified by silica gel column chromatography (hex-
ane–ethyl acetate) to give the product.
(KBr): 3364, 1736, 1474, 1312 cmꢁ1
.
6.4.8. trans-10-Chloro-2,5-dimethyl-1-oxo-5,6,11,11a-
tetrahydro-1H-imidazo[10,50:1,6]pydiro[3,4-b]indole-3(2H)-
thione (11e)
6.5.1. trans-10-Phenyl-2,5-dimethyl-1-oxo-5,6,11,11a-
tetrahydro-1H-imidazo[10,50:1,6]pydiro[3,4-b]indole-3(2H)-
thione (12a)
Yield 28%. Colorless solid. Dp 260–263 °C. 1H NMR (400 MHz,
CDCl3): d = 11.54 (s, 1H), 7.34 (d, J = 7.7 Hz, 1H), 7.01–7.09 (m, 2H),
5.66 (q, J = 6.3 Hz, 1H), 4.82 (dd, J = 11.0, 5.9 Hz, 1H), 3.64 (dd,
J = 15.1, 5.9 Hz, 1H), 3.16 (s, 3H), 3.03 (ddd, J = 15.1, 11.0, 1.5 Hz,
1H), 1.58 (d, J = 6.7 Hz, 3H). 13C NMR (100 MHz, DMSO-d6):
d = 179.7, 173.2, 137.5, 135.5, 124.3, 122.8, 122.3, 119.4, 110.6,
103.7, 55.0, 48.1, 27.4, 24.0, 18.9. MS (ESI) m/z 320 [M+H]+. Anal.
Calcd For C15H14ClN3OSꢀ0.1H2O: C, 56.02; H, 4.45; N, 13.07. Found:
Yield 60%. Colorless solid. Dp 263–265 °C. 1H NMR (400 MHz,
CDCl3): d = 11.39 (s, 1H), 7.38–7.46 (m, 6H), 7.16 (t, J = 7.4 Hz,
1H), 6.88 (dd, J = 7.2 Hz, 1H), 5.66 (q, J = 6.7 Hz, 1H), 4.56 (dd,
J = 11.0, 5.4 Hz, 1H), 3.11 (s, 3H), 2.54–2.61 (m, 1H), 2.37 (dd,
J = 15.4, 5.6 Hz, 1H), 1.57 (d, J = 6.7 Hz, 3H). 13C NMR (100 MHz,
DMSO-d6): d = 179.5, 173.2, 140.9, 136.8, 134.8, 134.2, 129.2,
127.9, 127.0, 123.4, 121.4, 120.3, 110.6, 103.7, 55.2, 48.2, 27.3,
25.1, 18.8. 13C NMR (100 MHz, DMSO-d6): d = 179.5, 173.2, 140.9,
136.8, 134.8, 134.2, 129.2, 127.9, 127.0, 123.4, 121.4, 120.3,
110.6, 103.7, 55.2, 48.2, 27.3, 25.1, 18.8. MS (ESI) m/z 362
[M+H]+. HRMS (EI): C21H19N3OS requires m/z 361.1249, found m/
C, 55.89; H, 4.56; N, 12.86. IR (KBr): 3332, 1729, 1484, 1314 cmꢁ1
.
6.4.9. trans-9-Chloro-2,5-dimethyl-1-oxo-5,6,11,11a-
tetrahydro-1H-imidazo[10,50:1,6]pydiro[3,4-b]indole-3(2H)-
thione (11f)
Yield 32%. Pale brown solid. Dp 241–243 °C. 1H NMR (400 MHz,
CDCl3): d = 11.38 (s, 1H), 7.57 (d, J = 2.0 Hz, 1H), 7.37 (d, J = 8.7 Hz,
1H), 7.10 (dd, J = 8.4, 1.8 Hz, 1H), 5.67 (q, J = 6.7 Hz, 1H), 4.79 (dd,
J = 11.3, 5.6 Hz, 1H), 3.27–3.33 (m, 1H), 3.16 (s, 3H), 2.78 (dd,
J = 14.1, 11.0 Hz, 1H), 1.57 (d, J = 6.7 Hz, 3H). 13CNMR (100 MHz,
DMSO-d6): d = 179.8, 173.2, 135.9, 134.8, 127.0, 123.5, 121.4,
117.6, 112.7, 104.1, 55.2, 48.3, 27.4, 22.3, 18.9. MS (ESI) m/z 320
[M+H]+. Anal. Calcd for C15H14ClN3OSꢀ0.4H2O: C, 55.09; H, 4.56;
N, 12.85. Found: C, 54.95; H, 4.60; N, 12.58. IR (KBr): 3341, 1730,
z 361.1247. IR (KBr): 3342, 1735, 1478, 1318 cmꢁ1
.
6.5.2. trans-9-Phenyl-2,5-dimethyl-1-oxo-5,6,11,11a-
tetrahydro-1H-imidazo[10,50:1,6]pydiro[3,4-b]indole-3(2H)-
thione (12b)
Yield 92%. Colorless solid. Mp 180–182 °C. 1H NMR (400 MHz,
CDCl3): d = 11.23 (s, 1H), 7.80 (s, 1H), 7.69 (d, J = 7.7 Hz, 2H),
7.43–7.46 (m, 4H), 7.28–7.34 (m, 1H), 5.69 (q, J = 6.7 Hz, 1H),
4.82 (dd, J = 10.8, 5.6 Hz, 1H), 3.18 (s, 3H), 2.82–2.88 (m, 1H),
1.59 (d, J = 6.1 Hz, 3H). 13C NMR (100 MHz, DMSO-d6): d = 179.8,
173.3, 141.6, 135.9, 134.9, 131.4, 128.8, 126.6, 126.4, 126.2,
120.8, 116.4, 115.6, 111.6, 104.6, 67.3, 55.3, 48.4, 27.4, 22.8, 18.9.
MS (ESI) m/z 362 [M+H]+. HRMS (EI): C21H19N3OS requires m/z
361.1249, found m/z 361.1246. IR (KBr): 3361, 1737, 1471,
1475, 1311 cmꢁ1
.
6.4.10. trans-8-Chloro-2,5-dimethyl-1-oxo-5,6,11,11a-
tetrahydro-1H-imidazo[10,50:1,6]pydiro[3,4-b]indole-3(2H)-
thione (11g)
Yield 40%. Pale brown solid. Dp 237–239 °C. 1H NMR (400 MHz,
CDCl3): d = 11.35 (s, 1H), 7.52 (d, J = 8.2 Hz, 1H), 7.41 (d, J = 1.5 Hz,
1315 cmꢁ1
.
Please cite this article in press as: Tanaka, M.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2012.12.028

M. Tanaka et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
7
6.5.3. trans-8-Phenyl-2,5-dimethyl-1-oxo-5,6,11,11a-
tetrahydro-1H-imidazo[10,50:1,6]pydiro[3,4-b]indole-3(2H)-
thione (12c)
15 min to hydrolyze N-formylkynurenine produced by indole-
amine 2,3-dioxygenase to kynurenine. After centrifugation at
1500 ꢂ g for 5 min at 20 °C, kynurenine in the supernatant was
Yield 95%. Colorless solid. Mp 288–290 °C. 1H NMR (400 MHz,
CDCl3): d = 11.26 (s, 1H), 7.68 (d, J = 7.2 Hz, 2H), 7.57–7.59 (m,
2H), 7.46 (t, J = 7.7 Hz, 2H), 7.30–7.35 (m, 2H), 5.70 (q, J = 6.1 Hz,
1H), 4.82 (dd, J = 11.0, 5.9 Hz, 1H), 3.18 (s, 3H), 2.84 (dd, J = 14.3,
11.8 Hz, 1H), 1.60 (d, J = 6.7 Hz, 3H). 13C NMR (100 MHz, DMSO-
d6): d = 179.8, 173.3, 141.4, 137.0, 135.0, 134.0, 128.8, 126.7,
126.6, 125.3, 118.6, 118.3, 109.2, 104.1, 55.3, 48.4, 27.4, 22.5,
19.0. MS (ESI) m/z 362 [M+H]+. HRMS (EI): C21H19N3OS requires
m/z 361.1249, found m/z 361.1250. IR (KBr): 3319, 2927, 1730,
measured by HPLC with
a
reversed phase column
(4.6 mm ꢂ 15 cm) of TSK-100Z. The mobile phase was 10 mM
ammonium acetate containing 10% (w/v) methanol, and kynuren-
ine was detected by absorbance at 360 nm. All determinations
were carried out in triplicate. The data presented are average
values.
References and notes
1469, 1318 cmꢁ1
.
1. Macchiarulo, A.; Camaioni, E.; Nuti, R.; Pellicciari, R. Amino Acids 2009, 37, 219.
2. Peters, J. C. Adv. Exp. Med. Biol. 1991, 294, 345.
3. Takikawa, O.; Yoshida, R.; Kido, R.; Hayaishi, O. J. Biol. Chem. 1986, 261, 3648.
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Expert Rev. Mol. Med. 2006, 8, 1.
5. Munn, D. H.; Zhou, M.; Attwood, J. T.; Bondarev, I.; Conway, S. J.; Marshall, B.;
Brown, C.; Mellor, A. L. Science 1998, 281, 1191.
6.5.4. trans-7-Phenyl-2,5-dimethyl-1-oxo-5,6,11,11a-
tetrahydro-1H-imidazo[10,50:1,6]pydiro[3,4-b]indole-3(2H)-
thione (12d)
Yield 75%. Colorless solid. Mp 223–225 °C. 1H NMR (400 MHz,
CDCl3): d = 10.91 (s, 1H), 7.64 (d, J = 7.2 Hz, 2H), 7.55 (t, J = 7.7 Hz,
2H), 7.50 (t, J = 4.6 Hz, 1H), 7.42–7.45 (m, 1H), 7.12–7.13 (m, 1H),
5.72 (q, J = 6.5 Hz, 1H), 4.85 (dd, J = 11.0, 5.9 Hz, 1H), 3.17 (s, 3H),
2.85 (dd, J = 14.3, 10.8 Hz, 1H), 1.56 (d, J = 6.7 Hz, 3H). 13C NMR
(100 MHz, DMSO-d6): d = 179.6, 173.4, 138.6, 135.1, 133.6, 128.9,
128.3, 127.3, 126.7, 125.2, 121.9, 119.6, 117.5, 104.6, 54.9, 48.3,
29.0, 27.4, 22.4, 19.2. MS (ESI) m/z 362 [M+H]+. HRMS (EI):
6. Hwu, P.; Du, M. X.; Lapointe, R.; Do, M.; Taylor, M. W.; Young, H. A. J. Immunol.
2000, 164, 3596.
7. Liu, Z.; Dai, H.; Wan, N.; Wang, T.; Bertera, S.; Trucco, M.; Dai, Z. J. Immunol.
2007, 178, 4260.
8. Guillemin, G. J.; Brew, B. J.; Noonan, C. E.; Takikawa, O.; Cullen, K. M.
Neuropathol. Appl. Neurobiol. 2005, 31, 395.
9. Takikawa, O.; Littlejohn, T. K.; Truscott, R. J. Exp. Eye Res. 2001, 72, 271.
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11. Uyttenhove, C.; Pilotte, L.; Theate, I.; Stroobant, V.; Colau, D.; Parmentier, N.;
Boon, T.; Eynde, B. Nat. Med. 2003, 9, 1269.
12. Okamoto, A.; Nikaido, T.; Ochiai, K.; Takakura, S.; Saito, M.; Aoki, Y.; Ishii, N.;
Yanaihara, N.; Yamada, K.; Takikawa, O.; Kawaguchi, R.; Isonishi, S.; Tanaka, T.;
Urashima, M. Clin. Cancer Res. 2005, 11, 6030.
C
21H19N3OS requires m/z 361.1249, found m/z 361.1249. IR (KBr):
3376, 2925, 1730, 1474, 1308 cmꢁ1
.
13. Ino, K.; Yamamoto, E.; Shibata, K.; Kajiyama, H.; Yoshida, N.; Terauchi, M.;
Nawa, A.; Nagasaka, T.; Takikawa, O.; Kikkawa, F. Clin. Cancer Res. 2008, 14,
2310.
14. Brandacher, G.; Perathoner, A.; Ladurner, R.; Schneeberger, S.; Obrist, P.;
Winkler, C.; Werner, E. R.; Werner-Felmayer, G.; Weiss, H. G.; Göbel, G.;
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12, 1144.
15. Chamuleau, M. E.; Loosdrecht, A. A.; Hess, C. J.; Janssen, J. J.; Zevenbergen, A.;
Delwel, R.; Valk, P. J.; Löwenberg, B.; Ossenkoppele, G. J. Haematologica 2008,
93, 1894.
6.6. Synthesis of trans-2,5-dimethyl-1-oxo-5,6,11,11a-tetrahydro-
1H-imidazo[10,50:1,6]pyrido[3,4-b]indole-3(2H)-thione (15)
The title compounds were prepared according to the literature
method28,29 starting from
L- or D-tryptophan. The reaction condi-
tions and yields were shown in Scheme 4.
16. Yoshida, N.; Ino, K.; Ishida, Y.; Kajiyama, H.; Yamamoto, E.; Shibata, K.;
Terauchi, M.; Nawa, A.; Akimoto, H.; Takikawa, O.; Isobe, K.; Kikkawa, F. Clin.
Cancer Res. 2008, 14, 7251.
17. H. Lee Moffitt Cancer Center and Research Institute. ClinicalTrials.gov
[Internet]. Available from: http://clinicaltrials.gov/show/NCT00617422 NLM
Identifier: NCT00617422.
18. Incyte Corporation. ClinicalTrials.gov [Internet]. Available from: http://
clinicaltrials.gov/show/NCT01195311 NLM Identifier: NCT01195311.
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20. Muller, A. J.; DuHadaway, J. B.; Donover, P. S.; Sutanto-Ward, E.; Prendergast, G.
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21. Gaspari, P.; Banerjee, T.; Malachowski, W. P.; Muller, A. J.; Prendergast, G. C.;
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6.6.1. (5S,11aR)-2,5-Dimethyl-1-oxo-5,6,11,11a-tetrahydro-1H-
imidazo[10,50:1,6]pyrido[3,4-b]indole-3(2H)-thione (15a)
Colorless solid. Mp 284–287 °C. 1H NMR (400 MHz, CDCl3):
d = 11.14 (s, 1H), 7.49 (d, J = 7.7 Hz, 1H), 7.35 (d, J = 7.7 Hz, 1H),
7.10 (t, J = 7.7 Hz, 1H), 7.01 (t, J = 7.7 Hz, 1H), 5.67 (q, J = 6.5, 1H),
4.79 (dd, J = 11.0, 5.9 Hz, 1H), 3.28 (dd, J = 15.1, 5.9 Hz, 1H), 3.17
(s, 3H), 2.81 (m, 1H), 1.58 (d, J = 6.7 Hz, 3H). 13C NMR (100 MHz,
DMSO-d6): d = 179.7, 173.3, 136.3, 134.0, 125.8, 121.5, 118.9,
118.1, 111.2, 104.0, 55.3, 48.3, 27.3, 22.4, 18.9. MS (ESI) m/z 286
[M+H]+. HRMS (EI): C15H15N3OS requires m/z 285.0936, found m/
z 285.0935. IR (KBr): 3331, 1733, 1473, 1316 cmꢁ1. 96.2% ee.
6.6.2. (5R,11aS)-2,5-Dimethyl-1-oxo-5,6,11,11a-tetrahydro-1H-
imidazo[10,50:1,6]pyrido[3,4-b]indole-3(2H)-thione (15b)
Colorless solid. Mp 284–287 °C. 1H NMR (400 MHz, CDCl3):
d = 11.14 (s, 1H), 7.49 (d, J = 7.7 Hz, 1H), 7.35 (d, J = 8.2 Hz, 1H),
7.10 (t, J = 7.2 Hz, 1H), 7.01 (t, J = 7.2 Hz, 1H), 5.67 (q, J = 6.5, 1H),
4.79 (dd, J = 10.8, 5.6 Hz, 1H), 3.29 (m, 1H), 3.17 (s, 3H), 2.81 (m,
1H), 1.58 (d, J = 6.7 Hz, 3H). 13C NMR (100 MHz, DMSO-d6):
d = 180.0, 173.3, 136.3, 134.0, 125.8, 121.5, 118.9, 118.1, 111.2,
104.0, 55.3, 48.4, 27.3, 22.4, 18.9. MS (ESI) m/z 286 [M+H]+. HRMS
(EI): C15H15N3OS requires m/z 285.0936, found m/z 285.0935. IR
(KBr): 3332, 1733, 1475, 1316 cmꢁ1. 95.3% ee.
25. Southan, M. D.; Truscott, R. J. W.; Jamie, J. F.; Pelosi, L.; Walker, M. J.; Maeda, H.;
Iwamoto, Y.; Tone, S. Med. Chem. Res. 1996, 6, 343.
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Eur. J. Org. Chem. 2004, 6, 1244.
27. Tanaka, M.; Hikawa, H.; Yokoyama, Y. Tetrahedron 2011, 67, 5897.
28. Benz[e]indole (a) Watanabe, T.; Takahashi, H.; Kamakura, K.; Sakaguchi, S.;
Osaki, M.; Toyama, S.; Mizuma, Y.; Ueda, I.; Murakami, Y. Chem. Pharm. Bull.
1991, 39, 3145; Benz[f]indole (b) Watanabe, T.; Miyagi, C.; Murakami, Y. J.
Heterocycl. Chem. 1993, 30, 217; Benz[g]indole (c) Sakamoto, T.; Kondo, Y.;
Yamanaka, Y. Heterocycles 1986, 24, 1845.
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30. Brossi, A.; Focella, A.; Teitel, S. J. Med. Chem. 1973, 16, 418.
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Campa, J. G. J. Org. Chem. 1994, 59, 1583.
32. Takikawa, O.; Kuroiwa, T.; Yamazaki, F.; Kido, R. J. Biol. Chem. 1988, 263, 2041.
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Protein Expr. Purif. 2000, 19, 22.
34. MTH-Trp (2) was purchased from Sigma–Aldrich Co. LLC.
35. Macchiarulo, A.; Nuti, R.; Bellocchi, D.; Camaioni, E.; Pellicciari, R. Biochim.
Biophys. Acta 2007, 1774, 1058.
6.6.3. Enzymatic assay
The standard assay mixture (200
sium phosphate buffer (pH 6.5), 10 mM ascorbate, 10
lene blue, 100 g/ml catalase, 200 -tryptophan, and 10
mL rhIDO. The reaction, at 37 °C, was started by the addition of
the substrate and terminated after 60 min by adding 40 l of 30%
(w/v) trichloroacetic acid and further incubated at 50 °C for
l
L) contained 50 mM potas-
M methy-
g/
l
l
lM
L
l
l
Please cite this article in press as: Tanaka, M.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2012.12.028