Synthesis and biological activity of 1-methyl-tryptophan-tirapazamine hybrids as hypoxia-targeting indoleamine 2,3-dioxygenase inhibitors

Article abstract of DOI:10.1016/j.bmc.2008.07.087

We have designed and synthesized new hypoxic-neoplastic cells-targeted indoleamine 2,3-dioxygenase (IDO) inhibitors. 1-Methyl-tryptophan (1MT)-tirapazamine (TPZ, 3-amino-1,2,4-benzotriazine 1,4-dioxide) hybrid inhibitors including 1 (TX-2236), 2 (TX-2235), 3 (TX-2228), and 4 (TX-2234) were prepared. All of these compounds were uncompetitive IDO inhibitors. TPZ-monoxide hybrids 1 and 3 showed higher IDO inhibitory activities than TPZ hybrids 2 and 4. Among these hybrids, hybrid 1 was the most potent IDO inhibitor. TPZ hybrids 2 and 4 showed stronger hypoxia-selective cytotoxicity than TPZ to EMT6/KU cells. These data suggest that TPZ hybrids 2 and 4 may act through their dual biological functions: first, they function as hypoxic cytotoxins in hypoxic cells, and then are metabolized to their TPZ-monoxide (3-amino-1,2,4-benzotriazine 1-oxide) hybrids, which function as IDO inhibitors.

Full text of DOI:10.1016/j.bmc.2008.07.087

Bioorganic & Medicinal Chemistry 16 (2008) 8661–8669
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological activity of 1-methyl-tryptophan-tirapazamine
hybrids as hypoxia-targeting indoleamine 2,3-dioxygenase inhibitors
a
a
a
b
a
a
Hitomi Nakashima , Yoshihiro Uto , Eiji Nakata , Hideko Nagasawa , Kazuhiro Ikkyu , Noriko Hiraoka ,
a
a
c
c
d
Kouichiro Nakashima , Yuki Sasaki , Hiroshi Sugimoto , Yoshitsugu Shiro , Toshihiro Hashimoto ,
Yasuko Okamoto , Yoshinori Asakawa , Hitoshi Hori
d
d
a,
*
a
Department of Life System, Institute of Technology and Science, Graduate School, The University of Tokushima, Minamijosanjimacho-2, Tokushima 770-8506, Japan
Laboratory of Pharmaceutical Chemistry, Gifu Pharmaceutical University, 5-6-1 Mitahara Higashi, Gifu 502-8585, Japan
Biometal Science Laboratory, RIKEN SPring-8 Center, Harima Institute, Hyogo 679-5148, Japan
b
c
d
Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Yamashiro-cho, Tokushima 770-8514, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
We have designed and synthesized new hypoxic-neoplastic cells-targeted indoleamine 2,3-dioxygen-
ase (IDO) inhibitors. 1-Methyl-tryptophan (1MT)-tirapazamine (TPZ, 3-amino-1,2,4-benzotriazine 1,4-
dioxide) hybrid inhibitors including 1 (TX-2236), 2 (TX-2235), 3 (TX-2228), and 4 (TX-2234) were pre-
pared. All of these compounds were uncompetitive IDO inhibitors. TPZ-monoxide hybrids 1 and 3
showed higher IDO inhibitory activities than TPZ hybrids 2 and 4. Among these hybrids, hybrid 1
was the most potent IDO inhibitor. TPZ hybrids 2 and 4 showed stronger hypoxia-selective cytotox-
icity than TPZ to EMT6/KU cells. These data suggest that TPZ hybrids 2 and 4 may act through their
dual biological functions: first, they function as hypoxic cytotoxins in hypoxic cells, and then are
metabolized to their TPZ-monoxide (3-amino-1,2,4-benzotriazine 1-oxide) hybrids, which function
as IDO inhibitors.
Received 7 July 2008
Revised 29 July 2008
Accepted 30 July 2008
Available online 5 August 2008
Keywords:
Indoleamine 2,3-dioxygenase
1
1
-Methyl-tryptophan (1MT)
MT-TPZ hybrids
Hypoxic-neoplastic cells
Ó 2008 Elsevier Ltd. All rights reserved.
9
1
. Introduction
expression was observed in some patients’ samples. IDO is associ-
ated with tumor immunosuppression.
Indoleamine 2,3-dioxygenase (IDO) is a monomeric 45 kDa
heme-containing dioxygenase that catalyzes the initial and rate-
limiting step of -tryptophan ( -Trp) catabolism in the kynurenine
1-Methyl-tryptophan (1MT; 5 in Fig. 1) is a competitive inhibi-
1
0
i
tor of IDO with a K = 19 lM. Hypoxic-neoplastic cells in a neo-
L
L
plasm indicate resistance to chemo-radiotherapy, probable tumor
1
,2
11–13
pathway. This step involves the oxidative cleavage of the 2,3
double bond in the indole moiety, resulting in the production of
N-formylkynurenine. Heme iron that exists in the active site of
metastasis, and an overall poor prognosis.
TPZ (6 in Fig. 1) is
a hypoxic cytotoxin, which is currently under phase II/III clinical
trials in combination with radiotherapy and cisplatin-based che-
2
+
3+
14
IDO is active as its ferrous (Fe ) form, whereas the ferric (Fe
)
motherapy. TPZ is reduced by one-electron reductases such as
3
,4
form is inactive. IDO-mediated tryptophan catabolism is shown
in Scheme 1. These kynurenine pathway metabolites, such as 3-
cytochrome P450 reductase to form a radical, which mediates
5
12
the induction of lethal double strand breaks in cellular DNA. Fur-
hydroxykynurenine, 3-hydroxyanthranilic acid, anthranilic acid,
and quinolinic acid, suppress T cell proliferation and differentia-
tion, and accelerate T cell apoptosis.6 Allogenic fetal rejection by
pregnant maternal mice is prevented by IDO-mediated tryptophan
ther metabolism produces the TPZ-monoxide (7 in Fig. 1). From the
above considerations, we reasoned that a hybrid molecule possess-
ing both IDO inhibitory activity and hypoxia-selective cytotoxicity
15
could be an effective and novel antineoplastic agent. Recently
7
16
17
catabolism. For this reason, IDO is associated with immunosup-
naphthoquinone analogues and exiguamine A were shown to
have higher IDO inhibitory activities than 1MT. They, however,
may affect other enzymes such as cytochrome P450 reductase.18
pression and immunotolerance. Expression of IDO has been re-
ported in various neoplastic cells, such as lung, prostatic,
8
pancreatic, and colorectal carcinomas. Especially in lung cancer,
To develop IDO-specific inhibitors, we chose the substrate (L-Trp)
IDO mRNA can be constitutively expressed, and the higher IDO
analogue 1MT, indoleamine as the IDO inhibitory unit for our de-
signed hybrids. We present here the synthesis and biological activ-
ity of 1MT-TPZ hybrids, which was defined as hybridization of 1MT
with TPZ or TPZ-monoxide 1 (TX-2236), 2 (TX-2235), 3 (TX-2228),
and 4 (TX-2234) as hypoxia-targeting IDO inhibitors.
*
Corresponding author. Tel.: +81 88 656 7514; fax: +81 88 656 9164.
E-mail address: hori@bio.tokushima-u.ac.jp (H. Hori).
0
968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.07.087

8
662
H. Nakashima et al. / Bioorg. Med. Chem. 16 (2008) 8661–8669
Scheme 1. IDO-mediated tryptophan catabolism.
Figure 1. Structure of 1MT-TPZ hybrids 1–4, 1MT 5, TPZ 6 and TPZ-monoxide 7.

H. Nakashima et al. / Bioorg. Med. Chem. 16 (2008) 8661–8669
8663
2
2
. Results
of Boc-N-1MT 8 with amines 16 and 17 were carried out using DIP-
CDI in the presence of HOBt and Et N to give the corresponding TPZ
hybrids 2 (TX-2235) and 4 (TX-2234) in good yields (98% and 93%,
3
.1. Molecular design
respectively).
The 1MT-TPZ hybrids were prepared by conjugation of the L-
isomer of 1MT with TPZ or TPZ-monoxide using flexible alkyl
chains as linking moieties. The amino group of 1MT was protected
with the tert-butyloxycarbonyl (Boc) group. The molecular orbitals
2.3. Biological activity
2.3.1. IDO inhibitory activity
(
MO) of all these newly constructed 1MT-TPZ hybrids were calcu-
The inhibition assays were performed in a 96-well microtiter
plate as described by Matin et al. and Takikawa et al. with a
minor modification. To quantify the concentration of kynurenine,
2
1
22
lated with WinMOPAC 3.0 (Fujitsu) by the PM3 method. The re-
sults including highest occupied molecular orbital (HOMO) and
Eigenvalue (EHOMO) and lowest unoccupied molecular orbital
a range of final concentrations of 0–500 lM kynurenine were mea-
(
LUMO) and Eigenvalue (ELUMO) are shown in Figure 2. The HOMO
sured in standard medium. The absorption data plotted against the
coefficient of hybrid 1 is localized at the 1MT moiety of the hybrid,
while the that of hybrid 2 localized at the TPZ moiety of the hybrid.
On the other hand, the LUMO coefficient of hybrids 1 and 2 are
localized at the TPZ moiety of the hybrid. The MO coefficients of
hybrids 3 and 4 (data not shown) are also localized in a similar re-
gion as found in hybrids 1 and 2.
corresponding concentrations of kynurenine showed good linearity
of Y = 0.0054X (r = 0.9999, data not shown). The production of
2
kynurenine was detected spectrophotometrically, and the K
m
and
K
i
values were determined using a Lineweaver–Burk plot. The K
m
value of
L-Trp was 207
l
M, the IDO inhibition constants K
i
of
1MT-TPZ hybrids are shown in Table 1. 1MT as a unit of the hybrid
9
i i
had an IDO inhibitory activity of K = 53.2 lM (lit. K = 19 lM),
2
.2. Synthesis
while the other units including TPZ 6, TPZ-monoxide 7, and Boc-
N-1MT 8 showed no inhibitory activities. As expected, the 1MT-
TPZ hybrids, including 1–4, displayed IDO inhibitory activities.
To conjugate 1MT and TPZ with amide bonds, we synthesized
the chloride 9 as previously described.19 The chloride 9 underwent
nucleophilic displacement with appropriate alkyl diamines to give
the corresponding 3-(aminoalkylamino) monoxides 10 and 11
The K
87.1, and 367
i
of these 1MT-TPZ hybrids 1, 2, 3, and 4, were 76.3, 197,
M, respectively. The TPZ-monoxide hybrids 1 and
l
3 showed stronger IDO inhibitory activities than the corresponding
TPZ hybrids 2 and 4. The length of alkyl linker chains of hybrids did
not influence their IDO inhibitory activities. The trifluoroacetamido
TPZ analogues without 1MT units, 12–15, also displayed no IDO
inhibitory activities. The combination of 1MT 5 with TPZ 6 gave
more effective inhibition than 1MT alone (1MT 5 + TPZ 6:
(
alkyl linker chain: n = 5 and 4, respectively) as shown in Scheme
2
0
2.
The amino group of 1MT was protected with a Boc group to
give Boc-N-1MT 8 quantitatively. The coupling reactions of Boc-
N-1MT 8 with amines 10 and 11 were carried out using DIPCDI
0
(N,N -diisopropylcarbodiimide) in the presence of HOBt (1-
hydroxybenzotriazole) to give the corresponding TPZ-monoxide
hybrids 1 (TX-2236) and 3 (TX-2228) in good yields (99% and
K
i
= 30.0
of 1MT 5 with TPZ-monoxide 7 did not enhance the inhibition of
1MT 5 (1MT 5 + TPZ-monoxide 7: K = 45.0; 1MT 5: K = 53.2 M).
lM; 1MT 5: 53.2 lM). On the other hand, the combination
8
9%, respectively). To synthesize TPZ hybrids, the amino groups
i
i
l
of 10 and 11 were protected with the trifluoroacetyl group to give
the corresponding trifluoroacetamides 12 (TX-2239) and 13 (TX-
2
The inhibition mode of these 1MT-TPZ hybrids were shown to be
uncompetitive, while 1MT was competitive as shown in Figure 3.
237) as shown in Scheme 3.20 Compounds 12 and 13 were then
2.3.2. Hypoxia-selective cytotoxicities23
oxidized with CF
acetamides 14 (TX-2240) and 15 (TX-2238) in moderate yields
50–52%). Compounds 14 and 15 were deprotected under basic
3 3
CO H to give the corresponding dioxide trifluoro-
Hypoxia-selective cytotoxicities of 1MT-TPZ hybrids 1–4 were
determined as shown in Table 2. The efficacies of the compounds
in killing aerobic or hypoxic EMT6/KU cells were determined by
clonogenic cell survival. The cytotoxicity was evaluated as the
(
conditions, and then treated with HCl to give the corresponding
dioxide amine hydrochlorides 16 and 17. The coupling reactions
Figure 2. Molecular orbital of 1MT-TPZ hybrids 1 (A) and 2 (B) calculated by WinMOPAC 3.0 (PM3).

8
664
H. Nakashima et al. / Bioorg. Med. Chem. 16 (2008) 8661–8669
2 2 3 2 2
Scheme 2. Synthesis of TPZ-monoxide hybrids 1 and 3. Reagents and conditions: (a) (Boc) O, 1 M NaOH aq, H O/1,4-dioxane = 1:2, rt; (b) alkyldiamine, Et N, CH Cl , rt; (c) 8,
DIPCDI, HOBt, CH Cl , rt.
2
2
Scheme 3. Synthesis of TPZ hybrids 2 and 4. Reagents and conditions: (a) (CF
HOBt, Et N, CH Cl , rt.
3 2 2 2 2 2 3 2 2 2 3
CO) O, CH Cl , rt; (b) 35% H O , (CF CO) O, CH Cl , rt; (c) 28% NH aq, MeOH, rt; (d) 8, DIPCDI,
3
2
2
IC50 value, and the hypoxic cytotoxicity ratio (HCR) was calcu-
lated as the HCR = IC50(Aero)/IC50(Hypo). TPZ 6 displayed hypoxic
centrations of more than 40
conditions.
lM, in both aerobic and hypoxic
cytotoxicity (IC50(Hypo) = 12
HCR = 3.2), whereas TPZ-monoxide 7 displayed no cytotoxicity
in either aerobic and hypoxic conditions (IC50(Aero) = >80 M;
IC50(hypo) = >40 M). TPZ hybrids 2 and 4 have higher hypoxic
cytotoxicity (2: IC50(Hypo) = 7.1 M; 4: IC50(Hypo) = 8.0 M) and
lM) and hypoxic selectivity
(
3. Discussion
l
l
IDO is activated only after its heme iron is reduced from the fer-
ric form to the ferrous form. This reduction takes place more easily
in reductive or hypoxic conditions. Ferrous form of heme iron is
easily oxidized to the inactive ferric form under aerobic conditions
while the reduction of IDO may not take place in aerobic condi-
l
l
hypoxic selectivity (2: HCR = 4.9; 4: HCR = >5.0) than TPZ, while
TPZ-monoxide hybrids 1 and 3 had lower hypoxic cytotoxicity
(1: IC50(Hypo) = 33 lM; 3: IC50(Hypo) = 33 lM) and hypoxic
selectivity (1: HCR = 1.3; 3: HCR = 1.1) than TPZ. TPZ-monoxide
hybrids 1 and 3, however, demonstrated cytotoxicity only at con-
5
tions. Maghzal et al. have reported that cytochrome b or cyto-
24
chrome P450 is involved in the reduction of IDO.
Thus,

H. Nakashima et al. / Bioorg. Med. Chem. 16 (2008) 8661–8669
8665
undergo one-electron reduction in aerobic conditions.12 We fo-
cused on this point to develop hypoxia-targeting IDO inhibitors.
To our knowledge, hypoxia-targeting IDO inhibitors have not
been previously reported. Our strategy involved the hybridization
of the hypoxic cytotoxin TPZ to the known IDO inhibitor 1MT to
develop hypoxia-targeting IDO inhibitors. The TPZ moiety involved
in the hybrids is also expected to work as a hypoxic cytotoxin. First,
we examined whether 1MT-TPZ hybrids have IDO inhibitory activ-
ity or not. Even though the 1MT-TPZ hybrids have the 1MT unit in
the molecules, they nonetheless showed uncompetitive inhibition,
different from that of 1MT (competitive inhibition, as shown in Fig. 3).
An uncompetitive inhibitor is able to bind to only an enzyme–sub-
Table 1
IDO inhibitory activity
Compound
K
i
value (
76.3
197
87.1
367
lM)
1
2
3
4
5
6
7
8
1
1
1
1
5
5
(TX-2236)
(TX-2235)
(TX-2228)
(TX-2234)
(1MT)
53.2
NI
NI
NI
NI
NI
NI
NI
c
(TPZ)
c
(TPZ-monoxide)
(Boc-N-1MT)
2 (TX-2239)
3 (TX-2237)
4 (TX-2240)
5 (TX-2238)
c
c
c
c
2
6
c
strate complex, not to an enzyme active site. Brastianos et al.
have reported that exiguamine A derived from a marine sponge
a
+ 6
+ 7
30.0
45.0
b
17
possesses strong IDO inhibitory activity. Furthermore, Carr et
al. have reported that analogues of exiguamine A, compounds that
are not indoleamine analogue, showed uncompetitive inhibition.27
1MT-TPZ hybrids have IDO inhibitory activities, and the TPZ-mon-
oxide hybrids are stronger IDO inhibitors. We found that the
HOMO coefficients of TPZ-monoxide hybrids 1 and 3 are localized
at their 1MT moieties, in contrast to the HOMO coefficients of TPZ
hybrids 2 and 4 that are localized at the TPZ moieties. The latter
have lower IDO inhibitory activities (Fig. 2A and B). Based on the
evidence that the combination of 1MT with other chemotherapeu-
tic agents, such as paclitaxel and cyclophosphamide, increased
Substrate (
.05 M.
(1MT) and 6 (TPZ) concn were each 200
(1MT) and 7 (TPZ-monoxide) concn were each 200
Not inhibited.
L-Trp) concn 0, 10, 20, 40, 80, 160 lM, inhibitor concn 400 lM, IDO concn
0
l
5
5
a
lM.
b
c
lM.
1
0,28
their therapeutic effects,
we examined the combinations of
1
MT 5 with TPZ 6 or that of 1MT 5 with TPZ-monoxide 7. The com-
bination of 1MT 5 with TPZ 6 was more effective than a single
treatment of 1MT 5, whereas the combination of 1MT with TPZ-
monoxide is the same as a single treatment of 1MT 5. Further in
vivo studies should be carried out to evaluate effects of the combi-
nation 1MT 5 with TPZ 6.
We also examined hypoxia-selective cytotoxicity of the 1MT-
TPZ hybrids by clonogenic cell survival. Hypoxic cytotoxicities of
TPZ hybrids 2 and 4 were up to about 1.5-fold higher compared
to their lead compound TPZ, and we observed preferential hypoxic
cytotoxicities. Nagasawa et al. have reported that hypoxic selectiv-
ity of TPZ to EMT6/KU cells at 10
lM concentration was 2.9, which
was estimated by the ratio of the colony number formed under the
Figure 3. Lineweaver–Burk plot of 1MT-TPZ hybrids 1, 2, and 1MT.
, 1 (TX-2236); , 2 (TX-2235); j, 1MT.
ꢁ
, no inhibitor;
2
3
ꢂ
N
aerobic and hypoxic conditions. Consistent with this, TPZ showed
similar hypoxic selectivity (HCR = 3.1). TPZ hybrids have higher hy-
poxia-selective cytotoxicity than their metabolite TPZ-monoxide
hybrids as shown in Table 2. The TPZ metabolite TPZ-monoxide 7
and TPZ-monoxide hybrids 1 and 3 did not display cytotoxicity.
However, hybrids 1 and 3 showed cytotoxicities at the higher drug
Table 2
Hypoxia-selective cytotoxicities
Compound
IC50(Aero)a
(lM)
IC50(Hypo)b
(
l
M)
HCR
3.2
6
7
1
2
3
4
(TPZ)
38
>80
42
35
36
12
>40
33
7.1
33
concentration of more than 40 lM. These cytotoxicities may be
(TPZ-monoxide)
(TX-2236)
(TX-2235)
(TX-2228)
(TX-2234)
due to the action of the N-methyl indole and benzotriazine moie-
1.3
4.9
1.1
29
ties as DNA intercalators. These data were consistent with their
molecular orbital calculations in that their LUMO coefficients were
localized at the TPZ moieties, as shown in Figure 2. In addition, ELU-
MO values of the TPZ hybrids were lower than those of the TPZ-
monoxide hybrids (2: ELUMO = ꢀ1.6422 eV; 1: ELUMO = ꢀ1.0791 eV).
For this reason, TPZ hybrids were more easily reduced to produce
their cytotoxic radicals, and these data suggest that 1MT-TPZ hy-
brids can work effectively not only as IDO inhibitors but also as
hypoxic cytotoxins. TPZ hybrids have stronger hypoxia-selective
cytotoxicities than TPZ itself. Based on the above observations
and considerations, we propose the following explanation for our
results, shown in Scheme 4: first, TPZ hybrids 2 are taken into hyp-
oxic cells and suffer one-electron reduction by reductases to gener-
ate hydroxyl radicals or TPZ radicals 2a that cause DNA double
strand breaks. At the same time, TPZ hybrids 2 are metabolized
to TPZ-monoxide hybrids 1, which are stronger IDO inhibitors
compared to the parent TPZ hybrids 2. TPZ hybrids 4 also function
in the same manner as 2. TPZ hybrids 2 and 4 can attack hypoxic
cells as dual antineoplastic agents functioning as hypoxic cytotox-
>40
8.0
>5.0
a
IC50 in aerobic conditions.
IC50 in hypoxic conditions (0% O , 5% CO , 95% N ).
2 2 2
b
activation of IDO is favored in hypoxic conditions, but IDO needs
molecular oxygen or superoxide for the catalytic reaction, concen-
trations of which are normally low in hypoxic conditions (hypoxia
pO
2
less than 5 mmHg).25 We reasoned that the hypoxic environ-
ment in neoplasm may stabilize the active ferrous form of IDO,
and catalyzes tryptophan degradation by using molecular oxygen
or superoxide existing in the hypoxic-neoplastic cells. Because of
O2
the oxygen affinity of rhIDO with Kd ¼ 54
lM (Sugimoto, unpub-
lished data), IDO still can function in hypoxic conditions. In anoxic
environment, this catalytic reaction does not take place because of
the lack of molecular oxygen. Also hypoxic cytotoxins undergo
one-electron reduction to produce cytotoxic radicals, but do not

8
666
H. Nakashima et al. / Bioorg. Med. Chem. 16 (2008) 8661–8669
Scheme 4. Proposed mechanism for IDO inhibition of 1MT-TPZ hybrid 2.
ins and IDO inhibitors. These 1MT-TPZ hybrids will be expected to
increase therapeutic benefit for cancer patients because of the de-
creased patient’s burden and increased effective and selective ther-
apeutic effects. In this study, to investigate our hypothesis we
evaluated the biological activities of 1MT-TPZ hybrids. Further
studies on their mode of action and their in vivo effects remain
to be studied. Many previous medicinal chemistry studies on hyp-
oxic cytotoxins have been focused only on the development of
more clinically effective TPZ analogues, especially Hay et al. have
reported pharmacokinetics and pharmacodynamics studies of
cytotoxicity than TPZ. We have developed hypoxia-targeting IDO
inhibitors with unique modes of action in that TPZ hybrids work
first as hypoxic cytotoxins and then their metabolites TPZ-monox-
ide hybrids function as IDO inhibitors.
4. Experimental
4.1. Chemistry
All chemicals and drugs were purchased from Wako Pure
Chemical industries, Ltd (Osaka, Japan) and Sigma–Aldrich Japan
(Tokyo, Japan). Melting points were determined on a Yanaco melt-
ing point apparatus (MP-S3 model) or Round Science RFS-10. NMR
1
2,13
TPZ analogues.
We suggest in our present studies that not only
TPZ and TPZ analogues, but also TPZ-monoxide analogues have po-
tential as promising antineoplastic agents. An additional important
consideration for developing hybrid drugs is the fact that our de-
signed 1MT-TPZ hybrids retained both IDO inhibitory activities
and hypoxic cytotoxicities. We expect that additional design of hy-
brid drugs such as these will increase in importance as a strategy
for development of antineoplastic agents.
In conclusion, 1MT-TPZ hybrids 1–4 were potent uncompetitive
IDO inhibitors. TPZ-monoxide hybrids 1 and 3 were stronger inhib-
itors than TPZ hybrids 2 and 4. Hybrid 1 was the most potent
among them. TPZ hybrids 2 and 4 have higher hypoxia-selective
spectra were obtained on a JEOL JNM-EX400 spectrometer at
1
400 MHz for H spectra. Spectra were obtained in CDCl
3
unless
otherwise specified, and are referenced to Me
4
Si. Chemical shifts
and coupling constants were recorded in units of ppm and Hz,
respectively. IR spectra were measured in KBr pellets on a Per-
kin-Elmer spectrum RX1-T1 spectrometer. Mass spectra were
determined on a JEOL JMS-AX-500 mass spectrometer using an
ionizing potential of 20 eV. Fast atom bombardment mass spectra
(FABMS) and high-resolution mass spectra (FABHRMS) were

H. Nakashima et al. / Bioorg. Med. Chem. 16 (2008) 8661–8669
8667
measured on a JEOL JMS-DX-303 instrument. Elemental analyses
4.1.4. 5-{(1-Oxido-1,2,4-benzotriazin-3-yl)aminopentyl}-2,2,2-
trifluoroacetamide (12)
Amine 10 (121.8 mg, 0.50 mmol) was suspended in CH
(3 mL), and (CF CO) O (690 L, 5.00 mmol) was added. The reac-
were performed with a Yanaco CHN Corder (MT-5). Solvents were
evaporated under reduced pressure on a rotary evaporator. Thin-
layer chromatography was carried out on aluminum-backed silica
gel (Merck 60 F254) with visualization of components by UV light
2 2
Cl
3
2
l
tion mixture was stirred at room temperature for 24 h, after which
time the solvent was evaporated. The residue was purified by rep-
recipitation to give 12 (165 mg, 98%) as a yellow solid: mp 183–
(
254 or 365 nm). Column chromatography was carried out on
KANTO Chemical silica gel 60 N (spherical, neutral) 40–50 m.
l
1
EtOAc refers to ethyl acetate; MeOH refers to methanol; DIPCDI re-
184 °C; H NMR d 8.30 (dq, J = 8.6, 0.5 Hz, 1H), 7.74 (ddd, J = 8.5,
0
fers to N,N -diisopropylcarbodiimide; HOBt refers to 1-hydroxy-
7.2, 1.2 Hz, 1H), 7.62 (dd, J = 8.5, 1.0 Hz, 1H), 7.33 (ddd, J = 8.5,
benzotriazole. All solvents were freshly distilled. Tirapazamine
7.2, 1.5 Hz, 1H), 6.44 (br s, 1H, NHCH
CH NHCO), 3.40 (q, J = 6.6 Hz, 2H, CH
CH ), 1.44–1.52 (m, 2H, CH ), 0.84–1.01 (m, 2H, CH
315, 2934, 1704, 1572, 1498, 1421, 1207, 1181, 760 cm ; Anal.
Calcd for C14 : C, 48.98; H, 4.70; N, 20.40. Found: C,
48.81; H, 4.92; N, 20.21.
2
), 3.55 (q, J = 6.8 Hz, 2H,
NH), 1.64–1.77 (m, 2H,
); IR (KBr)
3
0
11,31,32
19
6,
TPZ-monoxide 7,
3-Cl 9 were synthesized as previ-
2
2
ously described.
2
2
2
ꢀ1
3
4
.1.1. Boc-N-1-Methyl-
-Methyl- -trp 5 (54 mg, 0.25 mmol) was suspended in H
,4-dioxane = 1:2 (0.75 mL), and 1 M NaOH (250 L) and (Boc)
L, 0.20 mmol) were added. The reaction mixture was stirred
L
-tryptophan (8)
16 3 5 2
H F N O
1
L
2
O/
2
O
1
l
(
45
l
4.1.5. 4-{(1-Oxido-1,2,4-benzotriazin-3-yl)aminobutyl}-2,2,2-
trifluoroacetamide (13)
at room temperature for 4 h. The solvent was evaporated and the
residue was cooled in an ice-bath and acidified with 1 M HCl aq
Amine 11 (228.5 mg, 0.97 mmol) was suspended in CH
2 2
Cl
(
1
pH 2). The reaction mixture was extracted with EtOAc (3ꢃ
5 mL) and the organic layer was washed with brine, dried with
Na SO , and the solvent was evaporated. The residue was purified
by column chromatography, eluting with a gradient (5–10%) of
MeOH/CH Cl to give 8 (76.5 mg, 96%) as a pale purple powder:
mp 140–142 °C;
d, J = 8.3 Hz, 1H), 7.22 (ddd, J = 7.6, 7.0, 1.0 Hz, 1H), 7.11 (ddd,
J = 7.4, 7.1, 1.0 Hz, 1H), 6.91 (s, 1H), 3.74 (s, 3H, Me), 3.33 (m, 2H,
CH ), 1.42 (s, 9H, t-Bu); IR (KBr) 3326, 2985, 1726, 1660, 1480,
406, 1367, 1250, 1160, 743 cm ; EIMS m/z: M 318 (4.8), 245
2.0), 201 (1.6).
(5 mL) and (CF CO) O (1.35 mL, 9.70 mmol) was added. The reac-
tion mixture was stirred at room temperature for 46 h, after which
time the solvent was evaporated. The residue was purified by rep-
3
2
2
4
recipitation to give 13 (348.1 mg, quant.y) as a yellow solid: mp
1
2
2
199–203 °C; H NMR [CD
3
OD] d 8.21 (dq, J = 8.6, 0.5 Hz, 1H), 7.75
1
H
NMR
d
7.59 (d, J = 8.0 Hz, 1H), 7.33
(ddd, J = 8.5, 6.9, 1.5 Hz, 1H), 7.59 (d, J = 8.8 Hz, 1H), 7.32 (ddd,
J = 8.5, 7.3, 1.2 Hz, 1H), 3.47–3.52 (m, 2H, CH NHCO) 3.30–3.36
(m, 2H, CH ); IR (KBr) 3293, 2939,
NH), 1.66–1.73 (m, 4H, 2ꢃ CH
1700, 1587, 1497, 1420, 1182, 762 cm
: C, 47.42; H, 4.29; N, 21.27. Found: C, 47.21; H,
(
2
2
2
ꢀ
1
2
. Anal. Calcd for
ꢀ1
+
1
(
13 14 3 5 2
C H F N O
4.46; N, 21.06.
4
.1.2. [5-(1-Oxido-1,2,4-benzotriazine-3-yl)pentyl]-1,5-
4.1.6. 5-{(1,4-Dioxido-1,2,4-benzotriazin-3-yl)aminopentyl}-
2,2,2,-trifluoroacetamide (14)
pentanediamine (10)
Chloride 9 (20 mg, 0.11 mmol) was dissolved in CH
2
Cl
2
(2 mL).
L,
.40 mmol) were added. The reaction mixture was stirred at room
temperature for 2 days. The reaction mixture was partitioned be-
tween CH Cl and 0.1 M HCl aq, and the aqueous layer was washed
with CH Cl
(3ꢃ 30 mL). The aqueous layer was made basic with
M NaOH and extracted with CH Cl
(3ꢃ 20 mL). The organic
layer was washed with brine, dried with MgSO , and the solvent
was evaporated. The residue was dried in vacuo to give 1-oxide
0 (27.7 mg, quant.y) as a yellow solid: 1H NMR [CD
OD] d 8.22
Trifluoroacetamide 12 (52.6 mg, 0.15 mmol) was suspended in
Et
0
3
N
(46
lL, 0.33 mmol) and 1,5-pentanediamine (50
l
CH
The reaction mixture was stirred at 0 °C for 10 min, then 35%
(143 L, 1.50 mmol) was added dropwise. The reaction mix-
ture was stirred at room temperature for 24 h. The reaction mix-
ture was poured into brine and made basic with 28% aq NH , and
extracted with CH Cl
(3ꢃ 10 mL). The organic layer was dried
with Na SO and the solvent was evaporated. The residue was
2 2 3 2
Cl (3 mL), and (CF CO) O (209 lL, 1.50 mmol) was added.
H
2
O
2
l
2
2
2
2
3
2
2
2
2
2
4
2
4
purified by column chromatography eluting with a gradient
(0–10%) of MeOH/EtOAc to give dioxide 14 (28.4 mg, 52%) as a
red solid and starting material 12 (19.3 mg, 37%) as a yellow solid:
1
3
(
(
(
(
dd, J = 8.5, 0.7 Hz, 1H), 7.76 (ddd, J = 8.5, 6.9, 1.2 Hz, 1H), 7.59
d, J = 8.3 Hz, 1H), 7.34 (ddd, J = 8.4, 7.2, 1.2 Hz, 1H), 3.48
1
mp 197–199 °C; H NMR d 8.34 (dd, J = 8.8, 1.2 Hz, 1H), 8.27
t, J = 7.1 Hz, 2H, CH
2
NH
2
), 2.93 (t, J = 7.6 Hz, 2H, CH
2
NH), 1.72
(d, J = 8.8 Hz, 1H), 7.91 (ddd, J = 8.5, 7.2, 1.2 Hz, 1H), 7.54
m, 4H, CH
420, 1326, 761 cm
2
ꢃ 2), 1.51 (m, 2H, CH
2
); IR (KBr) 3308, 2934, 1571,
(t, J = 7.8 Hz, 1H), 6.56 (br s, 1H, NHCH
2
), 3.65 (q, J = 6.1 Hz, 2H,
NH), 1.75–1.87 (m, 2H,
), 1.45–1.53 (m, 2H, CH ); IR
ꢀ1
1
.
CH
CH
2
NHCO), 3.39 (q, J = 6.6 Hz, 2H, CH
), 1.69 (quin, J = 7.3 Hz, 2H, CH
2
2
2
2
ꢀ
1
4
.1.3. [4-(1-Oxido-1,2,4-benzotriazine-3-yl)butyl]-1,4-
butanediamine (11)
(KBr) 3277, 2936, 1702, 1636, 1497, 1412, 1184, 767 cm . EIMS
+
m/z 359 (M ); Anal. Calcd for C14
16 3 5 3
H F N O : C, 46.80; H, 4.49; N,
2 2
Chloride 9 (20 mg, 0.11 mmol) was dissolved in CH Cl (2 mL).
19.49. Found: C, 46.64; H, 4.33; N, 19.49.
Et
.33 mmol) were added. The reaction mixture was stirred at room
temperature for 14.5 h. The reaction mixture was partitioned be-
tween CH Cl and 0.1 M HCl aq, and the aqueous layer was washed
with CH Cl
(3ꢃ 20 mL). The aqueous layer was made basic with
M NaOH and extracted with CH Cl
(3ꢃ 25 mL). The organic layer
was washed with brine, dried with MgSO , and the solvent was
evaporated. The residue was dried in vacuo to give 1-oxide 11
25.9 mg, 100%) as a yellow solid: 1H NMR [CD
OD] d 8.20 (ddd,
J = 8.5, 1.5, 0.5 Hz, 1H), 7.75 (ddd, J = 8.5, 6.9, 1.5 Hz, 1H), 7.58
3
N
(46 lL, 0.33 mmol) and 1,4-butanediamine (34.6 mg,
0
4.1.7. 4-{(1,4-Dioxido-1,2,4-benzotriazin-3-yl)aminobutyl}-
2,2,2,-trifluoroacetamide (15)
2
2
Compound 13 (132.1 mg, 0.40 mmol) was suspended in CH
2
Cl
2
2
2
(4 mL), and (CF
tion mixture was stirred at 0 °C for 5 min, then 35% H
(329
was stirred at room temperature for 21.5 h. The reaction mixture
was poured into brine and made basic with 28% aq NH , and ex-
tracted with CH Cl
(3ꢃ 30 mL). The organic layer was dried with
Na SO and the solvent was evaporated. The residue was purified
3
2
CO) O (556
lL, 4.00 mmol) was added. The reac-
2
2
2
2 2
O
4
lL, 4.00 mmol) was added dropwise. The reaction mixture
(
3
3
2
2
(
(
1
d, J = 8.1 Hz, 1H), 7.32 (ddd, J = 8.5, 7.1, 1.2 Hz, 1H), 3.48
t, J = 7.1 Hz, 2H, CH NH ), 2.72 (t, J = 7.1 Hz, 2H, CH NH),
), 1.56–1.63 (m, 2H, CH ); IR (KBr) 3294,
2
4
2
2
2
by column chromatography eluting with a gradient (0–10%) of
MeOH/EtOAc to give dioxide 15 (69 mg, 50%) as a red solid and
starting material 13 (95 mg, crude) as a yellow solid: mp
.66–1.75 (m, 2H, CH
936, 1575, 1497, 1417, 1322, 761 cm
2
2
ꢀ1
2
.

8
668
H. Nakashima et al. / Bioorg. Med. Chem. 16 (2008) 8661–8669
1
93–196 °C; 1H NMR d 8.34 (dd, J = 8.8, 1.2 Hz, 1H), 8.28 (d,
J = 8.3 Hz, 1H), 7.91 (t, J = 7.3 Hz, 1H), 7.54 (t, J = 7.9 Hz, 1H), 7.33
br s, 1H, NHCO), 6.63 (br s, 1H, NHCH ), 3.68 (d, J = 5.4 Hz, 2H,
NH), 1.75–1.84 (m, 4H, 2ꢃ
); IR (KBr) 3269, 2940, 1703, 1627, 1496, 1415, 1363, 1183,
2 2
CH Cl to give 2 (105.4 mg, 98%) as a red powder: mp 105–
1
107 °C; H NMR d 8.33 (dd, J = 8.7, 1.2 Hz, 1H), 8.29 (d, J = 8.8 Hz,
1H), 7.87 (ddd, J = 8.5, 7.2, 1.2 Hz, 1H), 7.64 (d, J = 8.1 Hz, 1H),
7.50 (ddd, J = 8.7, 7.6, 1.5 Hz, 1H), 7.22 (ddd, J = 7.6, 6.9, 1.2 Hz,
1H), 7.11 (ddd, J = 8.0, 6.9, 1.0 Hz, 2H), 6.92 (s, 1H), 5.79 (br s,
(
CH
CH
2
2
NHCO), 3.45 (q, J = 6.6 Hz, 2H, CH
2
2
ꢀ
1
1
090, 722 cm ; Anal. Calcd for C13
H
14
F
3
N
5
O
3
: C, 45.22; H, 4.09;
1H, NHCH
(quin, J = 6.3, 6.6 Hz, 2H, CH
m, 2H, CH NH), 3.27 (d, J = 5.1 Hz, 1H), 3.07–3.21 (m, 2H, CH
1.59–1.70 (m, 6H, 3ꢃ CH ), 1.39 (s, 9H, t-Bu); IR (KBr) 3322,
2932, 1685, 1652, 1595, 1496, 1414, 1365, 1178, 766 cm ; HRMS
2
), 5.54 (br s, 1H, NHCO), 4.37 (br s, 1H, NHBoc), 3.84
NHCO), 3.74 (s, 3H, CH ), 3.50–3.56
),
N, 20.28. Found: C, 45.23; H, 4.07; N, 20.44.
2
3
(
2
2
4
.1.8. [5-(1,4-Dioxido-1,2,4-benzotriazine-3-yl)pentyl]-1,5-
2
ꢀ1
pentanediamine hydrochloride (16)
+
+
Compound 14 (68.2 mg, 0.19 mmoL) was suspended in MeOH
(FAB ) calcd for
564.2949; Anal. Calcd for C29
Found: C, 61.57; H, 6.63; N, 17.26.
C
29
H
38
N
7
O
5
(M+H) m/z 564.2937. Found
(
6.0 mL), and 28% aq NH
3
(6.0 mL) was added. The reaction mixture
H N O : C, 61.80; H, 6.62; N, 17.40.
37 7 5
was stirred at room temperature for 6 days, and the solvent was
then evaporated. The residue was acidified with 2 M HCl aq, and
the aqueous layer was washed with CH
3ꢃ 10 mL). The water was distilled off azeotropically at 40 °C to
give amine HCl salt 16 (57.5 mg, quant.y) as a yellow solid that
2
Cl
2
(3ꢃ 10 mL) and EtOAc
4.1.12. [4-(1-Oxido-1,2,4-benzotriazin-3-yl)aminobutyl]-(2S)-
N-tert-butoxycarbonyl-2-amino-(1-methyl-indole-3-yl)propana-
mide (3; TX-2228)
Boc-N-1MT 8 (41 mg, 0.13 mmol), HOBt (21.7 mg, 0.14 mmol),
and 11 (30 mg, 0.13 mmol) were dried in vacuo for 30 min. After
(
1
was used without further purification: H NMR [CD
3
OD] d 8.36
(
(
dd, J = 8.66, 1.22 Hz, 1H), 8.13 (t, J = 8.3 Hz, 1H), 8.02
d, J = 8.5 Hz, 1H), 7.69 (t, J = 7.6 Hz, 1H), 3.65 (t, J = 7.7 Hz, 2H,
2 2
CH Cl (2 mL) was added to the reaction mixture, DIPCDI (22 lL,
þ
ðCH
2
NH3 Þ, 2.95 (t, J = 7.3 Hz, 2H, CH
2
NH), 1.70–1.84 (m, 4H, 2ꢃ
0.14 mmol) was added and the reaction mixture was stirred at room
temperature for 2 h. The solvent was evaporated and the residue
was purified by column chromatography eluting with a gradient
(75–100%) of EtOAc/hexane to give 3 (61.5 mg, 89%) as a yellow
CH
2
2
), 1.49–1.57 (m, 2H, CH NH).
4
.1.9. [4-(1,4-Dioxido-1,2,4-benzotriazine-3-yl)butyl]-1,4-
1
butanediamine hydrochloride (17)
powder: mp 181–182 °C; H NMR d 8.25 (dd, J = 1.0, 8.5 Hz, 1H),
Compound 15 (94.9 mg, 0.27 mmol) was suspended in MeOH
7.69 (ddd, J = 1.5, 7.1, 8.5 Hz, 1H), 7.57–7.64 (m, 2H), 7.27–7.36 (m,
(
3
7.0 mL). 28% aq NH (7.0 mL) was added. The reaction mixture
2H), 7.20 (ddd, J = 1.0, 6.8, 7.6 Hz, 1H), 7.07 (ddd, J = 1.2, 7.0,
was stirred at room temperature for 4 days and the solvent was
then evaporated. The residue was acidified with 2 M HCl aq, and
7.9 Hz, 1H), 6.91 (s, 1H), 5.85 (br s, 1H, NHCH
4.39 (br s, 1H, NHBoc), 3.72 (s, 3H, CH
), 3.09–3.48 (m, 6H, 3ꢃ CH
1.76 (br, 2H, CH ), 1.41 (m, 11H, t-Bu, CH ); IR (KBr) 3331, 2937,
1688, 1655, 1360, 1172, 760 cm ; Anal. Calcd for C28 : C,
63.02; H, 6.61; N, 18.37. Found: C, 62.95; H, 6.52; N, 18.35.
2
), 5.30 (br s, 1H, NHCO),
3
2
),
the aqueous layer was washed with CH
3ꢃ 10 mL). The water was distilled off azeotropically at 40 °C to
give amine HCl salt 17 (79.5 mg, quant.y) as an orange powder that
2
Cl
2
(3ꢃ 10 mL) and EtOAc
2
2
ꢀ1
(
35 7 4
H N O
1
3
was used without further purification: H NMR [CD OD] d 8.34 (d,
J = 7.8 Hz, 1H), 8.20 (d, J = 8.5 Hz, 1H), 8.02 (ddd, J = 8.5, 7.0, 1.2 Hz,
4.1.13. [4-(1,4-Dioxido-1,2,4-benzotriazin-3-yl)aminobutyl]-(2S)-
N-tert-butoxycarbonyl-2-amino-(1-methyl-indole-3-yl)propana-
mide (4; TX-2234)
Boc-N-1MT 8 (84 mg, 0.26 mmol), HOBt (44 mg, 0.29 mmol)
and 17 (75 mg, 0.26 mmol) were dried in vacuo for 30 min. After
þ
1
H), 7.62 (t, J = 7.0 Hz, 1H), 3.62 (t, J = 6.3 Hz, 2H, ðCH
2
NH3 Þ, 3.00
(
t, J = 6.1 Hz, 2H, CH ).
2
NH), 1.71–1.84 (m, 4H, 2ꢃ CH
2
4
.1.10. [5-(1-Oxido-1,2,4-benzotriazin-3-yl)aminopentyl]-(2S)-
N-tert-butoxycarbonyl-2-amino-(1-methyl-indole-3-yl)propana-
mide (1; TX-2236)
Boc-N-1MT 8 (40.9 mg, 0.13 mmol), HOBt (20 mg, 0.13 mmol),
and 10 (29.6 mg, 0.12 mmol) were dried in vacuo for 30 min. After
CH
0.29 mmol) and Et
tion mixture was stirred at room temperature for 7 h. An additional
5 mL of CH Cl was added and stirring was continued for 2 h. The
solvent was evaporated and the residue was purified by column
chromatography eluting with a gradient (5–10%) of MeOH/CH Cl
The product was reprecipitated from CH Cl /hexane to give 4
(135.5 mg, 93%) as a red powder: mp 108–110 °C; H NMR d 8.33
(d, J = 8.1 Hz, 1H), 8.29 (d, J = 8.8 Hz, 1H), 7.88 (ddd, J = 8.5, 7.2,
1.2 Hz, 1H), 7.63 (d, J = 7.8 Hz, 1H), 7.52 (t, J = 8.5 Hz, 1H), 7.16 (t,
J = 8.1 Hz, 1H), 7.05–7.09 (m, 2H), 6.93 (s, 1H), 5.87 (br s, 1H,
2
Cl
2
(5 mL) was added to the reaction mixture, DIPCDI (45
lL,
3
N (92 L, 0.79 mmol) were added and the reac-
l
2
2
2
CH Cl
2
(3 mL) was added to the mixture, DIPCDI (21 lL,
0
.13 mmol) was added and the reaction mixture was stirred at
2
2
.
room temperature for 2.5 h. The solvent was evaporated and the
residue was purified by column chromatography eluting with a
2
2
1
gradient (75–100%) of EtOAc/hexane to give 1 (64.9 mg, 99%) as a
1
yellow powder: mp 180–183 °C; H NMR [CD
3
OD] d 8.28 (d,
J = 8.78 Hz, 1H), 8.14 (t, J = 9.02 Hz, 1H), 7.92–8.00 (m, 1H), 7.49–
7
7
3
.60 (m, 2H), 7.29 (d, J = 8.53 Hz, 1H), 7.11 (t, J = 7.31 Hz, 1H),
.00 (t, J = 7.31 Hz, 1H), 6.96 (s, 1H), 3.47–3.51 (m, 2H, CH NHCO),
.33–3.40 (m, 2H, CH ), 1.62–1.82
NH), 2.96–3.21 (m, 4H, 2ꢃ CH
), 1.22–2.00 (m, 9H, t-Bu); IR (KBr) 3338, 2936, 1685,
NHCH
J = 6.3, 6.6 Hz, 2H, CH
2H, CH NH), 3.28–3.33 (m, 1H), 3.09–3.18 (m, 2H, CH
4H, 2ꢃ CH ), 1.42 (s, 9H, t-Bu); IR 3343, 2970, 1618, 1593, 1496,
1416, 1364, 1171, 768 cm ; HRMS (FAB ) calcd for C28
2
), 5.26 (br s, 1H, NHCO), 4.41 (br s, 1H, NHBoc), 3.84 (quin,
NHCO), 3.74 (s, 3H, CH ), 3.45 (q, J = 6.3 Hz,
), 1.66 (br,
2
2
3
2
2
2
2
(
m, 2H, CH
2
2
ꢀ1
ꢀ1
+
1
653, 1363, 1173, 760 cm ; Anal. Calcd for C29
H
37
N
7
O
4
: C,
36 7 5
H N O
+
6
3.60; H, 6.81; N, 17.90. Found: C, 63.36; H, 6.75; N, 17.88.
(M+H) m/z 550.2781. Found 550.2802; Anal. Calcd for
: C, 61.19; H, 6.42; N, 17.84. Found: C, 60.92; H, 6.53;
28 35 7 5
C H N O
4.1.11. [5-(1,4-Dioxido-1,2,4-benzotriazin-3-yl)aminopentyl]-(2S)-
N, 17.98.
N-tert-butoxycarbonyl-2-amino-(1-methyl-indole-3-yl)propana-
mide (2; TX-2235)
4.2. Assay of IDO
Boc-N-1MT 8 (61 mg, 0.19 mmol), HOBt (32 mg, 0.21 mmol),
and amine 16 (57.5 mg, 0.19 mmol) were dried in vacuo for
4.2.1. Materials
3
0 min. After CH
l
2
Cl
2
(7 mL) was added to the mixture, DIPCDI
N (79 L, 0.57 mmol) were added, and
1-Methyl-
Sigma–Aldrich. L
L
-tryptophan and
L-kynurenine were purchased from
(
33 L, 0.21 mmol) and Et
3
l
-Tryptophan, ascorbic acid, methylene blue, bo-
the reaction mixture was stirred at room temperature for 7 h.
The solvent was evaporated and the residue was purified by col-
umn chromatography eluting with a gradient (5–10%) of MeOH/
vine liver catalase, and p-dimethylaminobenzaldehyde were pur-
chased from Wako chemicals (Osaka, JP). RhIDO was supplied by
Dr. Sugimoto.

H. Nakashima et al. / Bioorg. Med. Chem. 16 (2008) 8661–8669
8669
4
.2.2. Kynurenine standard curve
The standard medium (200 L) contained 1 M potassium phos-
phate buffer (20 L, pH 6.5) at a final concentration of 100 mM,
.4 M ascorbic acid (20 L, neutralized with 1 M NaOH aq) at a final
concentration of 40 mM, 1 mM methylene blue (4 L) at a final con-
centration of 20 M, 10 mg/mL catalase (4 L) at a final concentra-
tion of 200 g/mL, MilliQ water (112 L), and a series of kynurenine
solutions at final concentrations of 0, 10, 50, 100, 500 M. To the as-
say mixture was then added 40 L of 30% (w/v) of TCA. It was then
centrifuged (13,000 rpm, 4 °C, 15 min). Aliquots (125 L) of the
supernatant were treated with 2% (w/v) p-dimethylaminobenzal-
dehyde in acetic acid (125 L) in a 96-well microtiter plate. The yel-
were cultured to form colonies for 7–10 days. The colonies ob-
tained were fixed with methanol, stained with 5% Giemsa solu-
tions, and counted. Hypoxic cytotoxicity ratio (HCR) was
calculated as HCR = IC50 (Aero)/IC50 (Hypo).
l
l
0
l
l
l
l
Acknowledgements
l
l
l
The authors thank Drs. Kenneth L. Kirk, William A. Denny, and
Michael P. Hay for helpful advice. The authors also thank Ms. Maki
Nakamura, Ms. Kayoko Yamashita, Ms. Emiko Okayama, and the
staff of our Faculty for measurement of NMR, MS, and elemental
analyses.
l
l
l
low pigment derived from kynurenine was measured at 490 nm
using a Model 550 microplate reader (Bio-Rad, Tokyo, Japan).
References and notes
4
.2.3. IDO inhibition assay
1. Yamamoto, S.; Hayaishi, O. J. Biol. Chem. 1967, 242, 5260.
2. Sugimoto, H.; Oda, S.; Otsuki, T.; Hino, T.; Yoshida, T.; Shiro, Y. Proc. Natl. Acad.
Sci. U.S.A. 2006, 103, 2611.
3. 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.
The enzyme activity assay was carried out according to the
2
1
22
method of Matin and Takikawa with a minor modification.
All activity assays were carried out in aerobic condition. The stan-
dard medium (200
20 L, pH 6.5) at a final concentration of 100 mM, 0.4 M ascorbic
acid (20 L, neutralized with 1 M NaOH aq) at a final concentration
of 40 mM, 1 mM methylene blue (4 L) at a final concentration of
M, 10 mg/mL catalase (4 L) at a final concentration of 200 g/
-tryptophan (20 L) at a final concentration of 0, 10, 20, 40,
0, 160 M, with or without inhibitor solution in DMSO (10 L)
at a final concentration of 400 M, MilliQ water (112 L), and
.5 M rhIDO (20 L) at a final concentration of 0.05 M. The reac-
tion mixture was incubated at 20 °C for 20 min. To the assay mix-
ture was then added 40 L of 30% (w/v) TCA. The mixture was
lL) contained 1 M potassium phosphate buffer
4.
Taniguchi, T.; Sono, M.; Hirata, F.; Hayaishi, O.; Tamura, M.; Hayashi, K.; Iizuka,
(
l
T.; Ishimura, Y. J. Biol. Chem. 1979, 254, 3288.
5. Stone, T. W.; Darlington, L. G. Nat. Rev. Drug Disc. 2002, 1, 609.
6. Liu, Z.; Dai, H.; Wan, N.; Wang, T.; Bertera, S.; Trucco, M.; Dai, Z. J. Immunol.
l
l
2007, 178, 4260.
2
0
l
l
l
7
8
.
.
Munn, D. H. Science 1998, 281, 1191.
Uyttenhove, C.; Pilotte, L.; Theate, I.; Stroobant, V.; Colau, D.; Parmentier, N.;
Boon, T.; Eynde, B. Nat. Med. 2003, 9, 1269.
mL,
8
L
l
l
l
9.
Karanikas, V.; Zamanakou, M.; Kerenidi, T.; Dahabreh, J.; Hevas, A.; Nakou, M.;
Gourgoulianis, K. I.; Germenis, A. E. Cancer Biol. Ther. 2007, 6, 1258.
l
l
0
l
l
l
1
1
1
1
0. Hou, D. Y.; Muller, A. J.; Sharma, M. D.; DuHadaway, J.; Banerjee, T.; Johnson,
M.; Mellor, A. L.; Prendergast, G. C.; Munn, D. H. Cancer Res. 2007, 67, 792.
1. Hay, M. P.; Gamage, S. A.; Kovacs, M. S.; Pruijn, F. B.; Anderson, R. F.; Patterson,
A. V.; Wilson, W. R.; Brown, J. M.; Denny, W. A. J. Med. Chem. 2003, 46, 169.
2. Hay, M. P.; Hicks, K. O.; Pruijn, F. B.; Pchalek, K.; Siim, B. G.; Willson, W. R.;
Denny, W. A. J. Med. Chem. 2007, 50, 6392.
l
incubated at 65 °C for 20 min to hydrolyze N-formylkynurenine
to kynurenine. It was then centrifuged (13,000 rpm, 4 °C,
3. Hay, M. P.; Pchalek, K.; Pruijn, F. B.; Hicks, K. O.; Siim, B. G.; Anderson, R. F.;
Shinde, S. S.; Phillips, V.; Denny, W. A.; Wilson, W. R. J. Med. Chem. 2007, 50,
1
5 min). Aliquots (125
lL) of the supernatant were treated with
2
% (w/v) p-dimethylaminobenzaldehyde in acetic acid (125
l
L) in
6654.
a 96-well microtiter plate. The yellow pigment derived from kynu-
renine was measured at 490 nm using a Model 550 microplate
reader (Bio-Rad, Tokyo, Japan).
14. Covens, A.; Blessing, J.; Bender, D.; Mannel, R.; Morgan, M. Gynecol. Oncol. 2006,
100, 586.
1
1
5. Hanahan, D.; Weinberg, R. A. Cell 2000, 100, 57.
6. Kumar, S.; Malachowski, W. P.; DuHadaway, J. B.; LaLonde, J. M.; Carroll, P. J.;
Jaller, D.; Metz, R.; Predergast, G. C.; Muller, A. J. J. Med. Chem. 2008, 51, 1706.
4
.3. Hypoxia-selective cytotoxicity
17. Brastianos, H. C.; Vottero, E.; Patrick, B. O.; Van Soest, R.; Matainaho, T.; Mauk,
A. G.; Andersen, R. J. J. Am. Chem. Soc. 2006, 128, 16046.
1
1
8. Ishihara, Y.; Shiba, D.; Shibamoto, N. Toxicol. Appl. Pharmacol. 2006, 214, 109.
9. Shinde, S. S.; Anderson, R. F.; Hay, M. P.; Gamage, S. A.; Denny, W. A. J. Am.
Chem. Soc. 2004, 126, 7865.
EMT6/KU cells, a gift from Dr. Masunaga (Kyoto University, Kyo-
to), were cultured in Eagle minimum essential medium containing
2.5% (v/v) fetal bovine serum, and kanamycin (60 mg/L) at 37 °C
in 5% CO . The surviving cell fractions after drug treatment under
2
0. Hay, M. P.; Pruijn, F. B.; Gamage, S. A.; Sarath Liyanage, H. D.; Kovacs, M. S.;
1
Patterson, A. V.; Wilson, W. R.; Brown, J. M.; Denny, W. A. J. Med. Chem. 2004,
4
2
7, 475.
21. Matin, A.; Streete, I. M.; Jamie, I. M.; Truscott, R. J. W.; Jamie, J. F. Anal. Biochem.
006, 349, 96.
22. Takikawa, O.; Kuroiwa, T.; Yamazaki, F.; Kido, R. J. Biol. Chem. 1988, 263, 2041.
hypoxic or aerobic condition were determined in a clonogenic as-
2
say. Cells in late log phase were seeded in a 60-mm tissue culture
dish at a density of 103 cells per dish in 1 mL of medium, 5 ꢃ 102
2
2
2
3. Nagasawa, H.; Yamashita, M.; Mikamo, N.; Shimamura, M.; Oka, S.; Uto, Y.;
Hori, H. Comp. Biochem. Physiol. A 2002, 132, 33.
4. Maghzal, G. J.; Thomas, S. R.; Hunt, N. H.; Stocker, R. J. Biol. Chem. 2008, 283,
2
cells per dish in 0.5 mL of medium, 2 ꢃ 10 cells per dish in 1 mL
of medium. All compounds were dissolved in DMSO. Compound
solutions were diluted with culture medium at a concentration of
12014.
5. Brown, J. M.; Wilson, W. R. Nat. Rev. Cancer 2004, 4, 437.
0
5
.1, 1, 5, 10, 20, 40, and 80
lM for aerobic condition, and 0.1, 1, 2.5,
26. Cornish-Bowden, A. FEBS Lett. 1986, 203, 3.
27. Carr, G.; Chung, M. K. W.; Mauk, A. G.; Andersen, R. J. J. Med. Chem. 2008, 51,
, 10, 20, 40 M for hypoxic condition. After growth in a CO
l
2
incu-
2634.
bator for 24 h, culture medium was exchanged to drug containing
medium. The cells were then placed in a hypoxic chamber flushed
28. Muller, A. J.; DuHadaway, J. B.; Donover, P. S.; Sutanto-Ward, E.; Prendergast, G.
C. Nat. Med. 2005, 11, 312.
with 5% CO
min for hypoxic condition, for the aerobic condition were placed in
a CO incubator at 37 °C for 5 h. After the treatment, drugs were re-
moved by exchanging the medium for a fresh one and the cells
2
and 95% N
2
at 37 °C for 5 h at flow rates of 1.5–2.0 nL/
29. Anderson, R. F.; Harris, T. A.; Hay, M. P.; Denny, W. A. Chem. Res. Toxicol. 2003,
6, 1477.
1
3
3
0. Seng, F.; Ley, K. Angew. Chem., Int. Ed. Engl. 1972, 11, 1009.
1. Mason, J. C.; Tennant, G. J. Chem. Soc. B 1970, 911.
2
32. Fuchs, T.; Chowdhury, G.; Barnes, C. L.; Gates, K. S. J. Org. Chem. 2001, 66, 107.