N-Benzyl/Aryl Substituted Tryptanthrin as Dual Inhibitors of Indoleamine 2,3-Dioxygenase and Tryptophan 2,3-Dioxygenase

Article abstract of DOI:10.1021/acs.jmedchem.9b01079

Indoleamine 2,3-dioxygenase 1 (IDO1), which catalyzes the initial and rate-limiting step of the kynurenine pathway of tryptophan catabolism, has emerged as a key target in cancer immunotherapy because of its role in enabling cancers to evade the immune system. Tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase 2 (IDO2) catalyze the same reaction and play a potential role in cancer immunotherapy. Starting from our previously discovered tryptanthrin IDO1 inhibitor scaffold, we synthesized novel N-benzyl/aryl substituted tryptanthrin derivatives and evaluated their inhibitory efficacy on IDO1, TDO, and IDO2. Most compounds showed similar high inhibitory activities on both IDO1 and TDO, which were significantly superior over that of IDO2 with magnitude difference. We showed that N-benzyl/aryl substituted tryptanthrin directly interacted with IDO1, TDO, and IDO2, significantly augmented the proliferation of T cells in vitro, blocked the kynurenine pathway, and suppressed tumor growth when administered to LLC and H22 tumor-bearing mice.

Full text of DOI:10.1021/acs.jmedchem.9b01079

Article
pubs.acs.org/jmc
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N‑Benzyl/Aryl Substituted Tryptanthrin as Dual Inhibitors of
Indoleamine 2,3-Dioxygenase and Tryptophan 2,3-Dioxygenase
Dan Yang,† Shengnan Zhang,† Xin Fang,† Leilei Guo,† Nan Hu,† Zhanling Guo,† Xishuai Li,†
Shuangshuang Yang,† Jin Chao He,† Chunxiang Kuang,*,‡ and Qing Yang*,†
†State Key Laboratory of Genetic Engineering, Department of Biochemistry, School of Life Sciences, Fudan University, Songhu
Road 2005, Shanghai 200438, China
‡Department of Chemistry, Tongji University, Siping Road 1239, Shanghai 200092, China
S
* Supporting Information
ABSTRACT: Indoleamine 2,3-dioxygenase 1 (IDO1), which catalyzes the initial and rate-limiting step of the kynurenine
pathway of tryptophan catabolism, has emerged as a key target in cancer immunotherapy because of its role in enabling cancers
to evade the immune system. Tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase 2 (IDO2) catalyze the
same reaction and play a potential role in cancer immunotherapy. Starting from our previously discovered tryptanthrin IDO1
inhibitor scaffold, we synthesized novel N-benzyl/aryl substituted tryptanthrin derivatives and evaluated their inhibitory efficacy
on IDO1, TDO, and IDO2. Most compounds showed similar high inhibitory activities on both IDO1 and TDO, which were
significantly superior over that of IDO2 with magnitude difference. We showed that N-benzyl/aryl substituted tryptanthrin
directly interacted with IDO1, TDO, and IDO2, significantly augmented the proliferation of T cells in vitro, blocked the
kynurenine pathway, and suppressed tumor growth when administered to LLC and H22 tumor-bearing mice.
neurological diseases and cancer.5 The second enzyme is
tryptophan 2,3-dioxygenase (TDO, encoded by the TDO2
INTRODUCTION
■
Essential amino acid tryptophan (Trp) is metabolized along
gene), which is a tetrameric heme-containing enzyme. In
the kynurenine (Kyn) and serotonin (5-HT) pathways,
contrast, TDO constitutively expresses at high levels in the
producing an array of Kyn metabolites such as kynurenic
liver, where it regulates systemic Trp levels and plays a
acid, 3-hydroxykynurenine, quinolinic acid (QUIN), and
comparable role in cancer immunology.6,7 TDO is also
nicotinamide adenine dinucleotide, as well as a variety of
expressed in the brain and within the central nervous system
neuroactive substances including well-known neurotransmit-
ters serotonin and melatonin.1 The 5-HT pathway of Trp
(CNS), and TDO is involved in neurogenesis and can
modulate anxiety-like behaviors in mice.8 In 2007, a
metabolism is associated with psychiatric disorders including
major depressive disorder and schizophrenia.2 The kynurenine
monomeric heme-containing enzyme homologous to IDO1
was identified and named IDO2.9−11 IDO2 is primarily
pathway (KP) of Trp metabolism not only is implicated in
neurodegenerative diseases and neurological disorders but also
expressed in liver, kidney tubules, and reproductive tracts
plays a central role in tumor-induced immunosuppression.3
(e.g., epididymis, spermatozoa) apart from various cell lines
such as pancreatic, gastric, colon, and kidney carcinoma.9,10
The first and rate-limiting enzymes of the KP are
indoleamine 2,3-dioxygenase 1 (IDO1) (EC 1.13.11.52),
tryptophan 2,3-dioxygenase (TDO) (EC 1.13.11.11), and
indoleamine 2,3-dioxygenase 2 (IDO2) (EC 1.13.11). Three
IDO2 exhibits nonredundant roles in adaptive immunity12 and
autoimmunity,13 but its functions within the CNS are not
clear.
Overactivation of the three dioxygenases, which is mainly
dioxygenases have different tissue distribution and different
physiological roles.4 The first enzyme, IDO1, is a monomeric
associated with the upregulation of the KP and the production
heme-containing enzyme that is widely distributed in various
tissues of mammals. IDO1 has been linked to energy
homeostasis and immune defense that is relevant to both
Received: July 4, 2019
© XXXX American Chemical Society
A
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of excitotoxin QUIN, has been proved being implicated in the
pathogenesis of cancer immunoescape, neuroinflammatory
disorders, neurodegenerative disorders (Alzheimer’s disease),
and depression.14 The immunosuppressive effect of IDO1 is
attributed to the depletion of Trp and the increase in Kyn,
which involves three effector pathways of GCN2, mTOR, and
aryl hydrocarbon receptor (AhR),15 while the immunosup-
pressive effect imparted by TDO and IDO2 mainly is
attributed to AhR.16−18
Scheme 1. Synthetic Route To Generate N-Benzyl
Substituted Tryptanthrins Derivative 5
a
Since IDO1 plays a major role in this context, it has emerged
as a promising therapeutic target, prompting a search for highly
active inhibitors. Various chemical inhibitors of IDO1 have
been developed and tested for their immunostimulatory effects
in preclinical settings, including methylthiohydantoin-
tryptophan, brassinin, annulin B, exiguamine A and their
corresponding derivatives, INCB023843, 4-phenyl-1,2,3-tria-
zole, and tryptanthrin.19−29 1-Methyl-D-tryptophan (also
known as indoximod or NLG8189), epacadostat (also known
INCB24360), NLG919 (also known as GDC-0919), and
F001287 have entered clinical development.30−32 However,
the failure of clinical trials of several IDO1 inhibitors including
INCB24360, NLG919, and F001287 has been known since the
winter of 2017. Thus, discovery of new IDO1 inhibition
strategy has become extremely urgent. TDO inhibitors are also
being developed, the TDO-specific inhibitor LM10 has been
shown to mediate therapeutically relevant immunostimulatory
effects in mice with TDO-expressing tumors,33 although for
the moment only in preclinical settings. However, only a few
compounds are known to be IDO2 inhibitors;7,34,35 1-L-MT is
a more potent IDO2 inhibitor than D-1-MT.36,37 By screening
of a library of FDA-approved drugs in the HEK293T cells
transfected by mouse IDO2 gene, the proton pump drug
tenatoprazole was found to be an efficacious mIDO2 inhibitor
(with an IC50 value of 1.8 μM) but not for IDO1 or TDO
inhibition.38
Our previous studies have shown that natural product
tryptanthrin (indolo[2,1-b]quinazolin-6,12-dione) is a potent
IDO1 inhibitor scaffold; several tryptanthrin derivatives have
IDO1, TDO, and IDO2 inhibitory activities.29,39,40 Herein, we
designed and synthesized novel N-benzyl/aryl substituted
tryptanthrins and evaluated their IDO1, TDO, and IDO2
inhibitory potencies in both enzymatic and cellular levels.
Among these N-benzyl/aryl substituted tryptanthrin deriva-
tives, compound 5a was chosen for further in vitro and in vivo
studies because of its improved physical chemical properties.
We examined the interactions of 5a with IDO1, TDO, and
IDO2, the effects of 5a on the proliferation of T cells in vitro,
the blockade of KP in vivo, and the tumor growth in tumor-
bearing mice.
a
Reaction conditions: (i) m-CPBA, CH2Cl2, rt; (ii) 5-fluoroisatin or
isatin, Et3N, toluene, reflux; (iii) NBS, AIBN, CH2Cl2, reflux; (iv)
Et3N, KI, DMF, amine.
Pd(OAc)2 as catalyst owing to the worse reactivity of aryl
bromide compared to benzyl bromide.
Optimal Bioassay Systems of IDO1, TDO, and IDO2.
Table 1 and Table 2 respectively describe our enzymatic and
cellular assays of IDO1, TDO, and IDO2 inhibitory activity of
small molecules, which includes the cell line and the
composition of the assay solution.29,39−41
The bioassay of IDO1 has been well documented, including
several enzymatic assays by fluorescence method42−45 and the
cellular assay using interferon-γ (IFN-γ) stimulated HeLa cell
line.41 In contrast to IDO1, bioassays of TDO and IDO2 have
not been sufficiently studied; the preparation of active
recombinant hTDO and hIDO2, enzymatic assay conditions
such as pH value, the compositions of incubation medium, and
the specificity of cell line used in cellular assay have not been
addressed in detail.42−45 For example, the cellular TDO
inhibition assay has been routinely performed in A172 cell
line.42−44 Yet A172 is not a specific tool for cellular assay of
TDO because it expresses IDO1, IDO2, and TDO that all
contribute to the conversion of L-Trp to N-formyl kynurenine
(NFK). Recently, our group has established optimal hIDO2
and hTDO bioassay systems39,40 as summarized in Tables 1
and 2.
IDO1, IDO2, and TDO Inhibitory Activities of N-
Benzyl/Aryl Substituted Tryptanthrins. The known IDO1
selective inhibitors exhibit different inhibition types; for
example, 4PI46,47 and EOS20027148 are noncompetitive
IDO1 inhibitors, Amg-149 is competitive type, while BMS-
98620550 is irreversible type. Herein, the detailed kinetic
analyses of these N-benzyl/aryl substituted tryptanthrins were
performed based on plotting of the reciprocal of the reaction
velocity (1/V) against the inhibitor concentration ([I]). The
test compounds were categorized as reversible inhibitors.
Despite having the same structural skeleton, the test
compounds exhibited different types of inhibition. As shown
in Table 3, most compounds had a kinetic graphical mode that
suggested uncompetitive inhibition of IDO1, TDO, and IDO2.
The Ki values were evaluated by plotting [S]/V against [I],
where [S] represents the substrate concentration and V
represents the reaction velocity (Table 3).
RESULTS AND DISCUSSION
■
Chemistry. The synthesis of N-benzyl/aryl substituted
tryptanthrins derivatives is described in Schemes 1 and 2. 6-
Methylisatoic anhydride 2 was synthesized by Baeyer−Villiger
oxidation rearrangement of 5-methylisatin 1 utilizing 3-
chloroperbenzoic acid (mCPBA) as an oxygen donor.
Subsequently, compound 4 was achieved by an efficient two-
step process of condensation and NBS bromination. Then
nucleophilic substitution on benzyl bromide of 4 with various
amines in the presence of a catalytic quantity of KI provided
5a−f in 35−67% yield. Compound 8 was obtained by the
similar method with 3. It is noted that 9a−f was afforded with
As shown in Table 4, most N-benzyl/aryl substituted
tryptanthrins displayed similar IDO1 and TDO inhibitory
activities superior over IDO2 inhibitory activity with
magnitude difference. The IDO1 and TDO inhibitory activities
of most tryptanthrins were similar in both enzymatic and
cellular assays. From a structural point of view, the N-benzyl
substituted tryptanthrins showed higher IDO1, IDO2, and
B
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a
Scheme 2. Synthetic Route To Generate N-Aryl Substituted Tryptanthrins Derivative 9
a
Reaction conditions: (i) m-CPBA, CH2Cl2, rt; (ii) 5-fluoroisatin or isatin, Et3N, toluene, reflux; (iii) Pd(OAc)2, BINAP, amine, Cs2CO3, toluene,
reflux, 16 h.
(enzymatic IC50 = 71.8 nM, cellular IC50 = 7.1 nM),41 and 4-
Table 1. In Vitro Enzymatic IDO1, TDO, and IDO2
phenyl-1,2,3-triazole 1 (enzymatic IC50 = 83 μM, cellular IC50
= 8.9 μM).52 Similar tendency is observed in TDO inhibitors.
The enzymatic IC50 value of TDO inhibitor LM10 is 18.7
μM,40 which is 30-fold higher than its cellular IC50 value (0.62
μM).33 Furthermore, the enzymatic IC50 value of TDO
inhibitor 680C91 (1.3 μM)53 is nearly 5-fold higher than its
cellular IC50 value (0.28 μM).33 However, IDO1 inhibitor
EOS200271 displays better enzymatic IC50 value than its
cellular IC50 value (enzymatic IC50 = 0.41 or 0.15 μM, cellular
IC50 = 1.8 μM).48 BMS-986205 exhibits equal enzymatic and
cellular inhibitory activities (enzymatic IC50 = 1.1 nM, cellular
IC50 = 0.5 nM).54 The reason behind this remains unclear.
However, the reagents, pH value, and the activities of the
enzymes are different in the enzymatic assay and cellular
assays. In general, a good correlation between the enzymatic
assay and cellular assay is observed.
Under the same conditions, 1-L-MT showed weaker
inhibitory activities than N-benzyl/aryl substituted tryptan-
thrins on IDO1 and IDO2 in both enzymatic and cellular
assays but had no inhibitory activity on TDO. 1-L-MT was
reported to be a substrate rather than an inhibitor for
TDO.40,55 INCB24360 showed IDO2 and TDO enzymatic
inhibitory activities with IC50 values of 10.34 μM and 64.5 μM,
which were consistent with their previous report.56 The cellular
IC50 of INCB24360 to TDO was tested to be 0.261 μM.
Overall, our data demonstrate that N-benzyl/aryl substituted
tryptanthrins being tested are potent IDO1/TDO dual
inhibitors, while 1-L-MT and INCB24360 are IDO1 selective
inhibitors. To date, most known inhibitors are IDO1 selective,
IDO1/TDO dual inhibitor is rare, and RG-70099 (Roche/
CuraDev) has little information disclosed in the biomedical
literature.50
Interactions between 5a and hIDO1, hIDO2, hTDO.
To clarify the potent interaction between the N-benzyl/aryl
substituted tryptanthrins and IDO1, TDO, and IDO2, human
recombinant IDO1, TDO, and IDO2 were respectively
prepared and Carr−Purcell−Meiboom−Gill (CPMG) and
saturation transfer difference (STD) experiments were
operated. T1ρ NMR spectra for compound 5a, in the absence
(red) or in the presence of target protein (cyan), are shown in
Figure 1A; the difference between the red and cyan signals
indicates that the NMR signals for compound 5a are changed
due to the presence of the target protein, which means
compound 5a interacts with the target protein. The CPMG
experiments show that compound 5a directly interacts with
hIDO1, hTDO, and hIDO2. Additionally, STD spectrum of
compound 5a in the presence of hIDO1, hTDO, and hIDO2
shows clear signals, indicating that 5a directly interacts with
Inhibition Assays
final
assay
class
reagent
ascorbic acid
methylene chloride
catalase
concentration
a
IDO1
reducing agent
40 mM
20 μM
200 μg/mL
400 μM
50 mM
40 mM
20 μM
200 μg/mL
400 μM
50 mM
40 mM
20 μM
200 μg/mL
30 mM
50 mM
substrate
L-Trp
b
solvent
reducing agent
KPB buffer (pH = 6.5)
c
TDO
IDO2
ascorbic acid
methylene chloride
catalase
substrate
solvent
reducing agent
L-Trp
KPB buffer (pH = 7.0)
ascorbic acid
methylene chloride
catalase
d
substrate
solvent
L-Trp
KPB buffer (pH = 7.5)
a
In vitro enzymatic IDO1 inhibition assay was performed as in ref 29,
b
c
with some modifications. KPB: potassium phosphate buffer. In vitro
enzymatic TDO inhibition assay was performed as in ref 40, with
some modifications. In vitro enzymatic IDO2 inhibition assay was
performed as in ref 39, with some modifications.
d
Table 2. In Vitro Cellular IDO1, TDO, and IDO2 Inhibition
Assays
expression of IDO1, TDO,
assay
cell line
HeLa
IDO2
reagent
DMEM
a
IDO1
IDO1 expressed by IFN-γ
induction
b
TDO
IDO2
HEK293
TDO expressed by transient
transfection of
pcDNA3.1(+)-hTDO
DMEM
supplemented with
200 μM L-Trp
DMEM
supplemented with
200 μM L-Trp
c
U87 MG IDO2 expressed by transient
transfection of
pcDNA3.1(+)-hIDO2
a
In vitro cellular IDO1 inhibition assay was performed as in refs 29
b
and 41, with some modifications. In vitro cellular TDO inhibition
assay was performed as in ref 40, with some modifications. In vitro
cellular IDO2 inhibition assay was performed as in ref 39, with some
modifications.
c
TDO inhibitory activities when compared to their N-aryl
substituted analogues. All the tested N-benzyl/aryl substituted
tryptanthrins displayed much higher inhibitory efficiencies in
the cell-based assays than in the enzymatic assays. Usually,
cellular IC50 values of IDO1 inhibitors are all much lower than
the enzymatic IC50 values, for example, 1-L-MT (enzymatic
IC50 = 380 μM, cellular IC50 = 18.4 μM),51 INCB24360
C
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Table 3. IDO1, IDO2, and TDO Inhibitory Types and Ki Values of N-Benzyl/Aryl Substituted Tryptanthrins
a
b
c
d
e
f
g
U: uncompetitive; MC: mixed competitive. N: noncompetitive. MU: mixed uncompetitive. ND: not detected. NI: no inhibition. C:
h
i
competitive. Inhibitory type and Ki value according to ref 57. Inhibitory type and Ki value according to ref 39.
the target proteins. From a similar view, it is concluded that
compound 1-L-MT weakly interacts with hIDO1 but hardly
interacts with hTDO and hIDO2 (Figure 1B).
Subsequently, the heme binding study of 5a was conducted
by spectroscopic methods. Heme, a porphyrin ring with an
iron at its center, is a cofactor in the active site of IDO1, which
absorbs light in the UV−vis spectrum maximally at a
wavelength of around 400 nm depending on the oxidation
ing that 5a does not bind to the heme iron of IDO1 and has a
novel binding mode.
To date, only a few X-ray crystallography results of IDO1
inhibitor with IDO1 have been reported. The crystallization of
a weak noncompetitive IDO1 inhibitor 4PI with IDO1 was
first reported in 2006.47 Later in 2014, the crystallizations of
competitive IDO1 selective inhibitor Amg-1 with IDO1 were
reported.58 In 2015, a NLG919 analogue was shown to
coordinate with the heme iron and to occupy both pockets A
and B of IDO1.59 It was interesting to find that this compound
formed an extensive hydrogen bond network with IDO1, while
noncompetitive IDO1 inhibitor EOS200271 was recently
reported not to bind the heme group of IDO1.48 Similarly,
the irreversible IDO1 inhibitor BMS-986205 has been reported
to bind to the apo-form of the enzyme.60
The present study affords CPMG and STD to be a new
method besides X-ray crystallography and molecule docking to
confirm the interaction of IDO1 inhibitors to IDO1.
Effect of 5a on T Cell Proliferation. The upregulation of
KP can block T lymphocyte proliferation by depleting Trp
locally. It has been reported that 1-L-MT and INCB24360 can
and coordination states of its iron. Inhibitors that coordinate
46
the heme iron will shift the Soret band λmax
.
Inhibitors that
do not directly coordinate the iron will only modulate the
Soret band because the heme is highly sensitive to changes in
the polarity of its surroundings.46,48 As shown in Figure 2, a
clear redshift of the Soret band from 405 nm (ferric IDO1) to
410 nm was observed with INCB24360 (green curve), a
known as heme binding inhibitor of IDO1, while no apparent
shift was observed for 5a (blue curve). It was also noted that
the absorbance of Soret band at 405 nm significantly decreased
in the presence of 5a. This result was similar to the previous
report of IDO1 inhibitor EOS200271/PF-06840003,48 indicat-
D
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Table 4. IDO1, IDO2, and TDO Inhibitory Activities of N-Benzyl/Aryl Substituted Tryptanthrins
a
b
c
d
NI: no inhibition. IC50 value according to ref 57. IC50 value according to ref 39. ND: not detected.
augment T cell function that is stimulated by tumor cells.41,61
Here, to determine whether N-benzyl/aryl substituted
tryptanthrins could improve T cell proliferation, we evaluated
the efficacy of compound 5a on human T cell proliferation.
IFN-γ-treated IDO+ HeLa cells were cocultured with human T
cells in the presence of a soluble anti-CD3 antibody and
human recombinant IL-2. As shown in Figure 3, compound 5a
enhanced the proliferation of T cells stimulated with IDO+
HeLa cells, which is more effective than that of 1-L-MT. Our
results clearly demonstrate that N-benzyl/aryl substituted
tryptanthrins as highly potent IDO1/TDO dual inhibitors
can reverse the suppression of T lymphocyte, but 1-L-MT has
no significant effect on T cell proliferation at the same
concentration.
LPS, which indicated that the control mice took more Trp
than the modeling mice did because the mice were allowed ad
libitum access to food and water during the experiment. In
modeling mice, the KP was stimulated involving the decreased
Trp level, increased Kyn level, and the enhanced Kyn/Trp
ratio. The effect of 5a on KP was analyzed at 1 h, 6 h, 12 h, 24
and 30 h after dosing. It was found that 5a could significantly
increase the Trp level at 12 h and 24 h after dosing and yet not
affect the Kyn level and therefore markedly reversed the
enhanced Kyn/Trp ratio at 24 h after dosing. To date, IDO1
inhibitors with different inhibition types have been developed.
Structure, degree of similarity to the substrate L-Trp, and heme
binding ability of inhibitors make the mode of action disparate.
INCB24360 inhibits IDO1 activity in a fast and short mode,
while some inhibitors show an intriguingly slow onset of
inhibition.60 Our compound exhibited inhibition at 12 and 24
h.
Blockade Effect of 5a on LPS Stimulated and Basal KP
in Vivo. The blockade effect of 5a on overactivated KP was
first investigated using LPS-treated mice. As shown in Figure 4,
the Trp concentration in control mice at each sampling time
was generally higher than that in modeling mice treated with
Further, the blockade effect of 5a on basal KP was examined
using lung tissue of naive SD rats. As shown in Figure 4E, the
E
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Figure 1. CPMG and STD NMR studies on interactions between 5a and hIDO1, hTDO, and hIDO2. (A) Ligand observed T1ρ and STD spectra
indicate that compound 5a directly interacts with target proteins hIDO1, hTDO, and hTDO. (B) Ligand observed T1ρ and STD spectra indicate
that compound 1-L-MT weakly interacts with target protein (hIDO1 and hTDO) but does not interact with hIDO2. Left panel: T1ρ NMR spectra
for compound in the absence (red) or the presence of target protein (cyan). Right panel: STD spectrum of compound in the presence of target
protein.
block KP and exhibit therapeutic efficacy in vivo even if its
enzymatic IC50 is not so good. Both 5a and 1-L-MT retarded
the growth of tumors; specifically, tumor volume was reduced
56.2% with 5a and 33.6% with 1-L-MT (Figure 5A). 5a
significantly reduced tumor weight, while 1-L-MT showed less
effect on tumor weight (Figure 5B). Although 5a was incapable
of eliciting tumor regression, it did effectively inhibit tumor
growth.
Effect of 5a on Tumor Growth in H22 Tumor-Bearing
Mice. H22 tumor-bearing mice were treated with 5a or 1-L-
MT for 2 weeks. Tumor volume was reduced 47.3% by 5a and
15.8% by 1-L-MT (Figure 6A). 5a significantly reduced tumor
weight about 55.4%, while 1-L-MT showed less effect (Figure
6B). Together, the in vivo antitumor assays of 5a in LLC or
Figure 2. 5a was evaluated for heme binding. The Soret band for
H22 tumor-bearing mice reveal the therapeutic potential of 5a
ferric hIDO1 (λmax = 405 nm) was recorded with or without
as a single agent in the treatment of tumor.
inhibitors under normal atmospheric conditions in potassium
phosphate buffer using a UV−visible spectrophotometer (path length
of 1 mm).
The experiments with Ido1−/− mice would be of benefit to
clarify to what extent does the in vivo tumor growth of LLC or
H22 depend on IDO1-mediated immune suppression. It has
been shown that Ido1−/− mice have smaller tumors (about
50% reduction in tumor weight) and fewer nodules compared
with wild type mice on day 9 after LLC cells injection.63 LLC
growth was attenuated in Ido1−/− mice, and tumors were
smaller (50%, by tumor volume) than in wild type mice on day
20 after LLC tumor grafting.64 The treatment with IDO1
siRNA significantly delayed and suppressed the LLC growth
(69.78%, by tumor weight).65 These reports indicate that mice
lacking IDO1 genes were more resistant to LLC growth.
However, a similar study on H22 growth has not been found.
The importance of tumor-derived IDO1 in tumor immune
evasion has also been confirmed by the inhibition of PAN02
Trp concentration in the lung tissues of 5a-treated rats was
significantly higher than that in control rats. In addition, 5a
treatment markedly reduced the Kyn level and the Kyn/Trp
ratio.
Effect of 5a on Tumor Growth in LLC Tumor-Bearing
Mice. LLC tumor-bearing mice were treated with compound
5a or 1-L-MT for 2 weeks. 1-L-MT has been used as positive
control in many literature studies describing the antitumor
effects of IDO1 inhibitors, although it is still contentious how
1-L-MT acts in vivo; for example, 1-L-MT has been thought to
act as a Trp sufficient mimetic and reverse IDO1 immune
suppression in T cells.62 However, 1-L-MT can efficiently
F
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Figure 3. 5a enhanced the proliferation of T cells stimulated by IDO1-expressing HeLa cells. (A) Effect of 5a on T cell proliferation assayed by
flow cytometry. Appropriate numbers of HeLa cells and T cells were mixed in 96-well plates and treated with 150 U/mL IL-2 and 100 ng/mL anti-
CD3 antibody, as well as 60 ng/mL IFN-γ and various concentrations of 5a as indicated for 2 days. (B) Percent of T cell proliferation. Cell
proliferation was performed three times independently: (∗) P < 0.05, (∗∗) P < 0.01.
(murine pancreatic ductal adenocarcinoma cells) growth with
IDO1 inhibitor INCB023843 in both wild type and Ido1−/−
C57BL/6 mice.21
blockade of basal and upregulated KP by 5a in serum or tissues
was confirmed in naive SD rats, LPS-treated mice, and tumor
bearing mice. Taken together, these results led us to propose
that N-benzyl/aryl substituted tryptanthrin is an attractive
IDO1 and TDO dual inhibitor that may be used to resist the
immune tolerance.
Blockade Effects of 5a on KP in H22 or LLC Tumor-
Bearing Mice. To investigate if 5a could efficiently block KP,
the Kyn and Trp levels in the serum harvested from H22
tumor-bearing mice were examined by HPLC, and Kyn/Trp
ratios were calculated. As shown in Figure 7A, the
concentration of Kyn was lower in 5a or 1-L-MT treated
mice than that in control mice. In addition, mice treated with
5a had a significant increment of Trp level, while mice treated
with 1-L-MT had not (Figure 7B). As shown in Figure 7C, the
Kyn/Trp ratio was significantly lower in 5a-treated mice than
in control mice.
In addition, to investigate if 5a could efficiently block KP in
tumors, the Kyn and Trp levels in the tumors harvested from
LLC tumor-bearing mice were examined by LC−MS, and
Kyn/Trp ratios were calculated. As shown in Figure 7D−F, no
significant difference in the Trp concentrations was observed
between 5a-treated mice and control mice. Mice treated with
5a had a significant lower Kyn concentration and Kyn/Trp
ratio than control mice.
EXPERIMENTAL SECTION
■
1
General Chemistry Methods. H NMR and 13C NMR spectra
were recorded in DMSO-d6 or CDCl3 with TMS as the internal
standard by using a Bruker AV400 spectrometer. NMR data
1
processing was performed with MestReNova software. H chemical
shifts are reported in δ (ppm) as s (singlet), d (doublet), dd (doublet
of doublet), t (triplet), q (quartet), td (triplet of doublet), m
(multiplet) and are referenced to the residual solvent signal CDCl3
(7.26) or DMSO-d6 (2.50). Coupling constants (J) are expressed in
hertz (Hz). The protons of amino groups are not always indicated.
High-resolution electrospray ionization mass spectra (ESI) were
obtained on a VG Auto Spec 3000 or Finnigan MAT 90 instrument.
Unless otherwise noted, all reagents and solvents were purchased
from commercial suppliers such as Acros, Fluka, Sigma-Aldrich and
used without further purification. Dry solvents were purchased as
anhydrous reagents from commercial suppliers. All reactions were
monitored by TLC with Huanghai GF 254 silica gel-coated plates.
Column chromatography was carried out on 200−300 mesh silica gel
at medium pressure.
The purity of tested compounds was determined by high
performance liquid chromatography (HPLC, Shimadzu, Japan). The
detection wavelength was 260 nm. HPLC analysis of the test
compounds was performed using a Diamosil C18 column (150 mm ×
4.2 mm i.d., 5 μm, Japan) preceded by a C18 guard column (Dikma,
China). The column temperature was maintained at 30 °C at a flow
rate of 1 mL/min. The mobile phase was a mixture of acetonitrile and
0.1% formic acid (10:90, v/v). The purity of tested compounds was
confirmed as >95%.
CONCLUSIONS
■
Although the Keytruda (PD1 antibody)/epacadostat (Incyte
IDO1 inhibitor) combo crashed in the PhIII melanoma study,
continued interest in IDO1 remains high. Increasing findings
reinforce the need for inhibitors of IDO1, TDO, and IDO2
that may potentially be developed for therapeutic use. In this
study, we have identified N-benzyl/aryl substituted tryptan-
thrins as potent IDO1 and TDO dual inhibitors. With the
optimal assay method, both the enzymatic and cellular
inhibitory activities of these compounds against IDO1, TDO,
and IDO2 were investigated. The Ki values and inhibition
types were also evaluated. Further evaluation of antitumor
activity in vivo was performed with 5a, which directly
interacted with IDO1 and TDO and showed significant
immunoregulation activity of promoting T cell proliferation.
Testing in LLC and H22 tumor-bearing mice demonstrated
that 5a suppressed tumor growth evidently. Furthermore,
General Procedure for the Synthesis of N-Benzyl Sub-
stituted Tryptanthrin Derivatives (5a−f). 8-Fluoro-2-
methylindolo[2,1-b]quinazoline-6,12-dione or 2-methylindolo[2,1-b]-
quinazoline- 6,12-dione (1 mmol), NBS (267 mg, 1.5 mmol), and
AIBN (16 mg, 0.1 mmol) were added in 30 mL of carbon
tetrachloride, and the mixture was stirred at 80 °C for 16 h under
N2. The solvent was removed under vacuum, and the residue was
dissolved in dichloromethane (30 mL), washed with water (3 × 15
mL), dried over Na2SO4, and evaporated to give the compound 2-
G
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Figure 4. Blocking effects of 5a on KP. (A−D) 5a blocked the LPS stimulated KP in C57BL/6 mice. (A) For control and modeling, mice were
challenged with saline (ip) or LPS (5 mg/kg, ip), respectively. For dosing, at 24 h after modeling, mice were treated with either 0.5% CMC-Na (0.2
mL/18 g, ig) or 5a (60 mg/kg, ig). Sampling was 1 h, 6 h, 12 h, 24 h, 30 h after dosing, and the mice were sacrificed to harvest blood; n = 3−5
mice/group. (B−D) Kyn and Trp levels in serum were determined by HPLC, and the Kyn/Trp ratios were calculated. (E) 5a blocked the basal KP
in lung of naive SD rats. After administration of a single dose of 5a (30 mg/kg, ig) or 0.5% CMC-Na (control, 0.2 mL per rat, ig), the naive SD rats
were sacrificed to isolate lung tissues. Trp and Kyn levels were measured by LC−MS, and the Kyn/Trp ratios were calculated. (n = 3−4 rats/
group): (∗) P < 0.05, (∗∗) P < 0.01, (∗∗∗) P < 0.001.
Figure 5. 5a suppressed tumor growth effectively. LLC tumor-bearing C57BL/6 mice were treated orally once daily with 0.5% CMC-Na (control),
5a (45 mg/kg in 0.5% CMC-Na), or 1-L-MT (100 mg/kg in 0.5% CMC-Na). (A) Mean tumor volume of control (roundness), 5a-treated mice
(regular triangle), or 1-L-MT-treated mice (square) (n = 9−10 mice/group). The tumor size was measured in regular intervals. (B) Mean tumor
weight of each group (n = 9−10 mice/group): (∗) P < 0.05, (∗∗) P < 0.01.
(bromomethyl)-8-fluoroindolo[2,1-b]quinazoline-6,12-dione or 2-
(bromomethyl)indolo[2,1-b]quinazoline-6,12-dione as yellow solid,
which could be used directly in the next step without further
purification.
A suspension of 2-(bromomethyl)-8-fluoroindolo[2,1-b]-
quinazoline-6,12-dione or 2-(bromomethyl)indolo[2,1-b]quinazoline-
6,12-dione (1 mmol), amine (2 mmol), KI (5 mg), and triethylamine
(101 mg, 1 mmol) was dissolved in DMF (5 mL) and stirred for 2 h
H
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Figure 6. 5a suppressed tumor growth effectively. H22 tumor-bearing Kunming mice were treated orally once daily with 0.5% CMC-Na (control),
5a (45 mg/kg in 0.5% CMC-Na), or 1-L-MT (100 mg/kg in 0.5% CMC-Na). (A) Mean tumor volume of control (roundness), 5a-treated mice
(regular triangle) or 1-L-MT-treated mice (square) (n = 9−10 mice/group). The tumor size was measured in regular intervals. (B) Mean tumor
weight of each group (n = 9−10 mice/group): (∗) P < 0.05, (∗∗) P < 0.01.
Figure 7. 5a blocked KP in H22 and LLC tumor-bearing mice. (A−C) 5a blocked the KP in the serum of H22 tumor-bearing mice. After
administration of 5a, the serum of H22 tumor-bearing mice was harvested. Kyn and Trp levels were determined by HPLC and the Kyn/Trp ratios
were calculated. Control (roundness), 5a-treated mice (regular triangle), or 1-L-MT-treated mice (square) (n = 9−10 mice/group). (D−F) 5a
blocked the KP in the tumor of LLC tumor-bearing mice. After administration of 5a, the tumors of LLC tumor-bearing mice were separated. Kyn
and Trp levels were determined by LC−MS, and the Kyn/Trp ratios were calculated: control (roundness), 5a-treated mice (regular triangle), or 1-
L-MT-treated mice (square) (n = 4−6 mice/group); (∗) P < 0.05; (∗∗) P < 0.01; ns, not significant.
at room temperature. Then water (50 mL) was added and extracted
with dichloromethane (3 × 10 mL). The organic layer was combined,
washed with water (3 × 10 mL), dried over Na2SO4, and
concentrated in vacuum to afford the crude product, which was
purified by column chromatography on silica gel using dichloro-
methane/methanol (100:0 → 20:1) to give 5a−f.
I
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8-Fluoro-2-((4-methylpiperazin-1-yl)methyl)indolo[2,1-b]-
quinazoline-6,12-dione (5a). Yellow solid (246 mg, 65%). 1H
NMR (400 MHz, CDCl3) δ 8.65 (dd, J = 8.8, 4.0 Hz, 1H), 8.37 (d, J
= 1.5 Hz, 1H), 7.99 (d, J = 8.3 Hz, 1H), 7.88 (dd, J = 8.3, 1.8 Hz,
1H), 7.60 (dd, J = 6.5, 2.7 Hz, 1H), 7.48−7.58 (m, 1H), 3.72 (s, 2H),
2.62 (s, 8H), 2.40 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 181.71,
162.35, 159.87, 157.91, 145.62, 144.05, 142.30, 136.00, 130.83,
127.41, 124.79, 123.42, 119.68, 112.16, 111.92, 62.19, 55.02, 53.02,
45.93. HRMS (ESI): [M + H]+ m/z 379.1566 (calcd 379.1565 for
C21H20FN4O2).
(400 MHz, CDCl3) δ 8.59 (dd, J = 8.8, 4.1 Hz, 1H), 7.85 (d, J = 9.1
Hz, 1H), 7.70 (d, J = 2.9 Hz, 1H), 7.54 (dd, J = 6.6, 2.7 Hz, 1H), 7.43
(td, J = 8.7, 2.7 Hz, 1H), 7.35 (dd, J = 9.1, 2.9 Hz, 1H), 3.49 (t, 4H),
2.61 (t, 4H), 2.39 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 181.28,
162.22, 159.74, 157.87, 152.08, 142.00, 141.38, 138.08, 132.29,
124.95, 124.11, 121.59, 119.52, 111.63, 109.84, 54.61, 47.42, 45.10.
HRMS (ESI): [M + H]+ m/z 365.1407 (calcd 365.1408 for
C20H18N4O2).
8-Fluoro-2-(piperazin-1-yl)indolo[2,1-b]quinazoline-6,12-
dione (9b). Red solid (140 mg, 40%). 1H NMR (400 MHz, CDCl3)
δ 8.63 (dd, J = 8.8, 4.0 Hz, 1H), 7.89 (d, J = 9.0 Hz, 1H), 7.75 (d, J =
2.8 Hz, 1H), 7.57 (dd, J = 6.6, 2.5 Hz, 1H), 7.46 (td, J = 8.7, 2.6 Hz,
1H), 7.38 (dd, J = 9.1, 2.8 Hz, 1H), 3.45 (t, 4H), 3.09. 13C NMR
(100 MHz, CDCl3) δ 181.35, 162.25, 157.96, 152.53, 142.02, 141.29,
138.11, 132.31, 125.00, 124.25, 121.62, 119.55, 111.59, 111.54,
109.81, 48.61, 45.81. HRMS (ESI): [M + H]+ m/z 351.1250 (calcd
351.1252 for C19H16N4O2).
(8-Fluoro-6-oxo-2-(piperazin-1-ylmethyl)indolo[2,1-b]-
quinazolin-12(6H)-ylidene)oxonium (5b). Yellow solid (128 mg,
1
35%). H NMR (400 MHz, DMSO-d6) δ 8.47 (dd, J = 8.7, 4.0 Hz,
1H), 8.22 (d, J = 14.5 Hz, 1H), 7.88−7.93 (m, 2H), 7.78 (dd, J = 6.9,
2.3 Hz, 1H), 7.70−7.77 (m, 1H), 3.71 (s, 2H), 3.05 (s, 3H), 2.60 (s,
3H). 13C NMR (100 MHz, DMSO-d6) δ 182.00, 161.96, 159.52,
157.95, 145.99, 145.50, 142.71, 140.71, 136.26, 130.43, 127.19,
124.42, 123.48, 119.24, 112.03, 61.28, 50.35, 43.79. HRMS (ESI): [M
+ H]+ m/z 365.1407 (calcd 365.1408 for C20H18FN4O2).
8-Fluoro-2-(piperidin-1-yl)indolo[2,1-b]quinazoline-6,12-
1
dione (9c). Red solid (265 mg, 76%). H NMR (400 MHz, CDCl3)
(2-((4-(tert-Butoxycarbonyl)piperazin-1-yl)methyl)-8-fluoro-
δ 8.64 (dd, J = 8.8, 4.1 Hz, 1H), 7.87 (d, J = 9.1 Hz, 1H), 7.74 (d, J =
3.0 Hz, 1H), 7.57 (dd, J = 6.7, 2.7 Hz, 1H), 7.46 (td, J = 8.7, 2.7 Hz,
1H), 7.36 (dd, J = 9.1, 3.0 Hz, 1H), 3.51 (d, J = 5.6 Hz, 4H), 1.74 (s,
6H). 13C NMR (100 MHz, CDCl3) δ 181.22, 162.19, 159.71, 157.96,
152.28, 141.92, 140.97, 137.31, 132.38, 125.07, 124.08, 121.46,
119.53, 111.53, 109.52, 48.75, 25.35, 24.25. HRMS (ESI): [M + H]+
m/z 350.1301 (calcd 350.1299 for C20H17N3O2).
2-(4-Methylpiperazin-1-yl)indolo[2,1-b]quinazoline-6,12-
dione (9d). Red solid (197 mg, 57%). 1H NMR (400 MHz, CDCl3)
δ 8.58 (d, J = 8.1 Hz, 1H), 7.86 (t, J = 8.9 Hz, 2H), 7.73 (dd, J = 14.8,
5.6 Hz, 2H), 7.32−7.40 (m, 2H), 3.48 (d, J = 4.4 Hz, 4H), 2.61 (d, J
= 4.8 Hz, 4H), 2.38 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 182.26,
158.10, 151.93, 145.90, 141.48, 138.29, 137.61, 132.07, 126.86,
125.00, 124.92, 122.63, 121.70, 117.86, 109.95, 54.61, 47.48, 46.09.
HRMS (ESI): [M + H]+ m/z 347.1502 (calcd 347.1503 for
C20H19N4O2).
2-(Dimethylamino)-8-fluoroindolo[2,1-b]quinazoline-6,12-
dione (9e). Red solid (211 mg, 68%). 1H NMR (400 MHz, CDCl3)
δ 8.63 (dd, J = 8.8, 4.1 Hz, 1H), 7.87 (d, J = 9.1 Hz, 1H), 7.53−7.57
(m, 2H), 7.45 (td, J = 8.7, 2.7 Hz, 1H), 7.17 (dd, J = 9.1, 3.0 Hz, 1H),
3.20 (s, 6H). 13C NMR (100 MHz, CDCl3) δ 181.24, 161.78, 160.13,
158.08, 151.50, 141.91, 140.45, 136.46, 132.50, 125.04, 124.17,
119.51, 118.99, 111.54, 107.14, 40.43. HRMS (ESI): [M + H]+ m/z
310.0985 (calcd 310.0986 for C17H13N3O2).
6-oxoindolo[2,1-b]quinazolin-12(6H)-ylidene)oxonium (5c).
1
Yellow solid (246 mg, 53%). H NMR (400 MHz, CDCl3) δ 8.63
(dd, J = 8.8, 4.0 Hz, 1H), 8.35 (d, J = 1.1 Hz, 1H), 7.98 (d, J = 8.3 Hz,
1H), 7.87 (dd, J = 8.3, 1.6 Hz, 1H), 7.57 (dd, J = 6.5, 2.6 Hz, 1H),
7.48 (td, J = 8.6, 2.7 Hz, 1H), 3.69 (s, 2H), 3.45−3.48 (m, 4H), 2.46
(d, 4H), 1.46 (s, 9H). 13C NMR (100 MHz, CDCl3) δ 181.65,
162.36, 160.91, 159.87, 157.86, 154.74, 145.69, 144.11, 142.10,
135.91, 130.88, 127.39, 124.79, 123.43, 119.67, 112.03, 79.72, 62.28,
52.96, 42.71, 28.39. HRMS (ESI): [M + H]+ m/z 465.1935 (calcd
465.1933 for C25H26FN4O4).
2-((4-Methylpiperazin-1-yl)methyl)indolo[2,1-b]-
quinazoline-6,12-dione (5d). Yellow solid (223 mg, 62%). 1H
NMR (400 MHz, CDCl3) δ 8.65 (d, J = 8.1 Hz, 1H), 8.39 (s, 1H),
8.00 (d, J = 8.2 Hz, 1H), 7.93 (d, J = 7.5 Hz, 1H), 7.87 (d, J = 8.3 Hz,
1H), 7.81 (t, J = 7.8 Hz, 1H), 7.45 (t, J = 7.5 Hz, 1H), 3.71 (s, 2H),
2.60 (s, 8H), 2.37 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ
182.85, 158.11, 146.45, 146.23, 145.30, 140.53, 138.24, 136.18,
130.39, 127.41, 127.20, 125.20, 123.59, 122.72, 117.49, 60.79, 53.65,
50.61, 43.61. HRMS (ESI): [M + H]+ m/z 361.1657 (calcd 361.1659
for C21H21N4O2).
2-((Dimethylamino)methyl)-8-fluoroindolo[2,1-b]-
quinazoline-6,12-dione (5e). Yellow solid (217 mg, 67%). 1H
NMR (400 MHz, CDCl3) δ 8.64 (dd, J = 8.8, 4.0 Hz, 1H), 8.34 (s,
1H), 7.98 (d, J = 8.3 Hz, 1H), 7.87 (dd, J = 8.2, 1.3 Hz, 1H), 7.58
(dd, J = 6.5, 2.5 Hz, 1H), 7.49 (td, J = 8.6, 2.6 Hz, 1H), 3.62 (s, 2H),
2.31 (s, 6H). 13C NMR (100 MHz, CDCl3) δ 181.68, 162.34, 159.86,
157.89, 145.64, 144.08, 142.53, 136.05, 130.86, 127.44, 124.64,
123.39, 119.69, 112.01, 63.61, 45.41, 29.63. HRMS (ESI): [M + H]+
m/z 324.1146 (calcd 324.1143 for C18H15N3O2).
8-Fluoro-2-morpholinoindolo[2,1-b]quinazoline-6,12-dione
1
(9f). Red solid (214 mg, 61%). H NMR (400 MHz, CDCl3) δ 8.65
(dd, J = 8.8, 4.1 Hz, 1H), 7.93 (d, J = 9.0 Hz, 1H), 7.77 (d, J = 2.7 Hz,
1H), 7.58 (dd, J = 6.7, 2.7 Hz, 1H), 7.48 (td, J = 11.4, 5.7, 2.5 Hz,
1H), 7.39 (dd, J = 9.1, 3.0 Hz, 1H), 3.93 (t, 4H), 3.46 (t, 4H). 13C
NMR (100 MHz, CDCl3) δ 181.38, 160.24, 157.93, 152.28, 142.10,
141.70, 138.65, 132.34, 125.02, 124.32, 124.16, 121.54, 119.61,
111.75, 109.89, 66.47, 47.68. HRMS (ESI): [M + H]+ m/z 352.1093
(calcd 352.1092 for C19H15FN3O3).
CPMG and STD NMR Assays of Interactions between 5a and
hIDO1, hIDO2, hTDO. Samples used in the NMR experiments: 200
μM compound without or with the presence of 5 μM protein. Buffer
conditions: 20 mM sodium phosphate buffer, 100 mM NaCl, 5%
DMSO, pH 7.4. All NMR data for the compound without or with the
presence of target protein were collected on Bruker Avance III 600
MHz NMR spectrometer equipped with cryogenically cooled probe at
25 °C.
Heme Binding Studies. Optical absorption spectra of FeIII-
hIDO1 (187.5 μM) were recorded with or without inhibitor
treatment (375 μM) under normal atmospheric conditions in 50
mM KPB (pH 6.5) using a UV−visible spectrophotometer (Thermo
Fisher Scientific, USA, path length of 1 mm).
Enzymatic IDO1, TDO, and IDO2 Inhibition Assay Based on
Detection of Kynurenine.29,39,40 In vitro enzymatic IDO1, TDO,
and IDO2 inhibition assays were determined in a commonly used
system with respective modification, which are described in the Table
1. Briefly, a standard reaction mixture (0.5 mL) containing all reagents
8-Fluoro-2-(morpholinomethyl)indolo[2,1-b]quinazoline-
1
6,12-dione (5f). Yellow solid (171 mg, 47%). H NMR (400 MHz,
CDCl3) δ 8.63 (dd, J = 8.7, 4.0 Hz, 1H), 8.36 (s, 1H), 7.98 (d, J = 8.2
Hz, 1H), 7.88 (d, J = 8.2 Hz, 1H), 7.70−7.52 (m, 1H), 7.49 (td, J =
8.6, 2.4 Hz, 1H), 3.74−3.76 (m, 3H), 3.69 (s, 2H), 2.51 (s, 4H). 13C
NMR (100 MHz, CDCl3) δ 181.63, 162.36, 159.87, 157.87, 145.69,
144.11, 142.39, 141.63, 136.00, 130.85, 127.46, 124.79, 123.42,
119.67, 112.04, 66.92, 62.63, 53.62. HRMS (ESI): [M + H]+ m/z
366.1249 (calcd 366.1248 for C20H17N3O3).
General Procedure for the Synthesis of N-Aryl Substituted
Tryptanthrin Derivatives (9a−f). A suspension of 2-bromo-8-
fluoroindolo[2,1-b]quinazoline-6,12-dione or 2-bromoindolo[2,1-b]-
quinazoline- 6,12-dione (1 mmol), amine (2 mmol), Pd(OAc)2 (67
mg, 0.3 mmol), BINAP (311 mg, 0.5 mmol), and Cs2CO3 (650 mg, 2
mmol) in dried toluene (15 mL) was stirred at 110 °C for 16 h under
N2. After removal of the solvent in vacuum, the residue was dissolved
in dichloromethane (50 mL), washed with water (3 × 15 mL), dried
over Na2SO4, and concentrated in vacuum to provide the crude
product, which was purified by column chromatography on silica gel
using dichloromethane/methanol (20:1 → 10:1) to give 9a−f.
8-Fluoro-2-(4-methylpiperazin-1-yl)indolo[2,1-b]-
1
quinazoline-6,12-dione (9a). Red solid (266 mg, 73%). H NMR
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and hIDO1 (4−10 μg), hTDO (4−10 μg), or hIDO2 (350 μg) was
added to the solution containing the test sample at a determined
concentration. The reaction was carried out at 37 °C for 30 min and
stopped by adding 200 μL of 30% (w/v) trichloroacetic acid (TCA).
After heating at 65 °C for 15 min, the reaction mixture was
centrifuged at 12 000 rpm for 10 min. The supernatant (100 μL) was
transferred into a well of a 96-well microplate and mixed with 100 μL
of 0.3% (w/v) p-dimethylaminobenzaldehyde (DMAB) in acetic acid.
The yellow pigment derived from Kyn was measured at 492 nm using
a SPECTRAmax250 microplate reader (Molecular Devices, USA).
Enzymatic IC50 values were determined via nonlinear regression
analysis using GraphPad Prism 5.
Fudan University. Human peripheral blood mononuclear cells
(PBMCs) were isolated by centrifugation using Lymphoprep (Axis
Shield, Norway) following the manufacturer’s guidelines. Briefly,
blood was collected into a tube containing anticoagulant (15 mg/mL
ethylenediaminetetraacetic acid, EDTA), and the blood was diluted
by addition of an equal volume of 0.9% NaCl. The 6 mL of diluted
blood was carefully layered on the 3 mL of the prepared Lymphoprep
medium, then centrifugated at 800g for 20 min at room temperature.
After centrifugation the mononuclear cells form a distinct band at the
sample/medium interface. The cells are best removed from the
interface using a Pasteur pipet without removing the upper layer. The
harvested fraction was diluted with 0.9% NaCl to reduce the density
of the solution and pellet the cells by centrifugation at 250g for 10
min.
Cell Culture. HeLa, U87 MG, HEK293, H22, and LLC cells were
authenticated by short tandem repeat analysis and passaged for fewer
than 6 months before experiments. All cell lines were tested to be
negative for mycoplasma contamination. The cells were cultured in
DMEM (Gibco, USA) supplement with 10% (v/v) fetal bovine serum
(FBS, Gibco, USA), 1% (v/v) nonessential amino acid solution
(Gibco, USA), 100 μg/mL penicillin, and streptomycin (Gibco, USA)
at 37 °C in an atmosphere of 5% CO2 and 95% relative humidity.
Cellular IDO1 Inhibition Assay. In vitro cellular IDO1 inhibition
assay was performed as previously described29,41 with some
modifications, which are described in the Table 2. HeLa cells were
cultured in DMEM medium with 10% FBS, 1% pen/strep, 1%
nonessential amino acid, 1 mM Na-pyruvate. Cells were seeded at
25 000 cells per well into 96-well microplate in 100 μL of growth
medium and incubated at 37 °C and 5% CO2 overnight. The next day
100 μL per well of diluted inhibitor in growth medium was added at a
final concentration of 100 ng/mL human IFN-γ. Cells were incubated
at 37 °C in a CO2 incubator for 18 h. The next day 140 μL of medium
was removed into a new 96-well plate and 10 μL of 6.1 N TCA was
added. The plate was incubated at 50 °C for 30 min to hydrolyze
NFK produced by IDO1 to Kyn. The plate was then centrifuged at
2500 rpm for 10 min to remove sediments. 100 μL of supernatant per
well was transferred to another 96-well plate and mixed with 100 μL
of 2% (w/v) DMAB in acetic acid. The plate was incubated at rt for
10 min. The yellow color derived from Kyn was recorded by
measuring absorbance at 492 nm using a Multiscan spectrum Mk3
(Thermo Fisher Scientific, USA). Cellular IC50 values were
determined via nonlinear regression analysis using GraphPad Prism 5.
Cellular TDO and IDO2 Inhibition Assays. In vitro cellular
TDO and IDO2 inhibition assays were performed as previously
described39,40 that are shown in the Table 2. U87 MG and HEK293
cells were cultivated in DMEM containing 50 U/mL penicillin, 50
mg/mL streptomycin, 4500 mg/L glucose, and 10% inactivated FBS
(Gibco, USA) at 37 °C with 5% CO2 and 95% humidity. When 80%
confluence was reached, cells were transfected with pcDNA3.1(+)-
hIDO2 or pcDNA3.1(+)-hTDO using the transfection reagent
Lipofectamine 2000 according to the manufacturer’s instructions.
An empty pcDNA3.1(+) expression vector served as control. After 18
h of incubation, the transfected cells were seeded in 96-well culture
plates at a density of 2.5 × 104 cells/well and supplemented with 200
μM L-Trp in a final volume of 200 μL. A serial dilution of the tested
compounds was added to the culture medium after an additional 6 h
(IDO2) or 12 h (TDO) of incubation. The reaction was terminated
by addition of 30% (w/v) TCA (10 μL for 140 μL of the reaction
mixture) 24 h (IDO2) or 8 h (TDO) later. The plates were incubated
at 65 °C in water bath for 15 min to facilitate the transformation of
NFK to Kyn, followed by centrifugation at 13 000g for 10 min to
remove the sediments. An amount of 100 μL of the supernatant was
then transferred to another 96-well plate and mixed with a same
volume of 0.3% (w/v) DMAB in acetic acid. The percentages of
inhibition of Trp degradation or Kyn production by the compounds
were calculated by measuring the absorption at 492 nm using a
Multiscan spectrum Mk3 (Thermo Fisher Scientific, USA). Cellular
IC50 values were determined via nonlinear regression analysis using
GraphPad Prism 5.
Coculture of mixed lymphocyte and HeLa cells was performed as
previously described with some modifications.41 Briefly, the
lymphocyte was labeled with CFSE dye, according to CFSE cell
proliferation and cell tracking kit (Yeasen, China) instructions.
Stimulator ratio of 5: l (1 × 106 lymphocyte cells/well; 2 × 105 HeLa
cells/well) in culture medium consisted of 150 U/mL IL-2 (Sino
Biological, China), 100 ng/mL anti-CD3 (BD Biosciences, USA), and
60 ng/mL IFN-γ (Sino Biological, China) in 6-well cell culture plates
(Nunc, Denmark). Meanwhile, 1-L-MT (1 μM) or 5a of different
concentration gradients (40 nM, 200 nM, 500 nM, 1 μM) was added
to culture plates. After a two-day incubation, cell proliferation was
measured by flow cytometer (Beckman Gallios, USA) with FL1 and
FL2 channels.
Animal Model and Treatments. The 6- to 8-week-old female
C57BL/6, Kunming mice and SD rats used for the study were
purchased from Shanghai Laboratory Animal Center, CAS.
LLC tumor-bearing mice and H22 tumor-bearing mice were
constructed as described previously with some modifications.29,66 H22
cells (2 × 106 cells of each mouse) were inoculated into the abdomen
of Kunming mice and the ascites tumor cells were passaged two times
in the mice. After 1 week, the ascites tumor was collected and diluted
with normal saline. LLC cells and H22 ascites tumor cells were
injected sc into the right forelimb at 2 × 106 of each C57BL/6 and
Kunming mouse, respectively. After the implantation, the mice were
randomized into several groups: control, 5a, and 1-L-MT. Tumor
growth was monitored every 2 days. Perpendicular diameters of the
tumors were measured using vernier scale calipers, and the tumor
volume was calculated as follows: tumor size = long diameter × (short
diameter)2/2. When the primary tumor diameter had reached 5 mm,
therapy was initiated. The mice were treated orally once daily with
0.5% CMC-Na (control), 5a (45 mg/kg in 0.5% CMC-Na), or 1-L-
MT (100 mg/kg in 0.5% CMC-Na). After 2 weeks of treatment, the
mice were sacrificed, the tumors were dissected and weighed, and the
blood was harvested.
LPS stimulation of KP was performed in C57BL/6 mice as
previously described67,68 with some modifications. Briefly, mice were
injected ip with saline or Escherichia coli LPS (5 mg/kg). 24 h after
modeling (ip LPS), mice were treated with 0.5% CMC-Na (0.2 mL/
18 g, ig) or 5a (60 mg/kg, ig). After 0.5% CMC-Na or 5a treatment,
the mice were sacrificed and the blood was harvested at different
sampling times (1 h, 6 h, 12 h, 24 h, 30 h).
Naive SD rats were randomized into two groups of control and 5a.
The rats were treated with a single dose of 0.5% CMC-Na (control)
or 5a (30 mg/kg in 0.5% CMC-Na). The rats were sacrificed and the
lung tissues were dissected.
HPLC Analysis. In vivo PD (pharmacodynamics) efficacy was
evaluated by measuring the concentrations of Trp and Kyn in the
serum using an Agilent 1260 series HPLC system (Agilent Corp,
USA) equipped with a quad pump and a UV detector. The detection
wavelengths were 280 and 360 nm. HPLC analysis of the samples was
performed using an Agilent C18 column (5 μm particle size, L × i.d.
25 cm × 4.6 mm) preceded by a C18 guard column (Dikma, China).
The mobile phase was 15 mM sodium acetate (pH 3.6) containing
4% acetonitrile.
T Cell Proliferation Assay. Blood samples of healthy volunteers
were obtained from Changhai Hospital after informed consent. All
procedures were approved by the Medical Ethics Committee of
LC−MS Analysis. In vivo PD effect of 5a was evaluated by
measuring the concentrations of Trp and Kyn in the tumors using an
K
DOI: 10.1021/acs.jmedchem.9b01079
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Agilent 1100 LC−MS spectrometer. Agilent ZORBAX SB-C18 (2.1
mm × 150 mm, 5 μm) was used for separation. The precolumn was a
Waters Xterra MS C18 Guard column (2.1 mm × 10 mm). The
mobile phase was acetonitrile and 0.1% formic acid. The acetonitrile
ratio was 5−80% gradient elution. The analysis time was 5 min,
postcolumn equilibrium was performed for 3 min, the flow rate was
0.7 mL/min, the column temperature was 35 °C. The mass-to-charge
ratios of the Kyn, Trp, and internal standard metronidazole were
respectively 209, 205, and 172. The signal modes were positive ion
modes.
Ethics Approval and Consent To Participate. The 6- to 8-
week-old female C57BL/6, Kunming mice and SD rats used for the
study were purchased from Shanghai Laboratory Animal Center, CAS.
The use of animal was approved by the Animal Ethics Committee of
Fudan University, and experiments were performed in compliance
with ARRIVE guidelines. Blood samples of healthy volunteers were
obtained from Changhai Hospital after informed consent. All
procedures were approved by the Changhai Hospital Ethics
Committee.
Statistical Analysis. Data are expressed as mean values SD or
SEM. The one-way ANOVA method and two-way ANOVA method
were used to determine the statistical significance of the differences
observed between the control and the test-compound-treated groups
of mice. GraphPad Prism 5 was used to create the graphs and
implement the statistical analysis. Enzymatic and cellular IC50 values
were determined via nonlinear regression analysis using GraphPad
Prism 5. Significance values were set at (∗) P < 0.05, (∗∗) P < 0.01,
(∗∗∗) P < 0.001.
ACKNOWLEDGMENTS
■
We thank Changhui Su for providing constructive suggestions
on the writing. This work was supported by National Key
R&D Program of China (Grant 2016YFC1303503) and the
Key Biomedical Program of Shanghai (Grants 17431902200
and 18431902600).
ABBREVIATIONS USED
■
IDO1, indoleamine 2,3-dioxygenase 1; IDO2, indoleamine 2,3-
dioxygenase 2; TDO, tryptophan 2,3-dioxygenase; Trp,
tryptophan; Kyn, kynurenine; 5-HT, serotonin; KP, kynur-
enine pathway; CNS, central nervous system; QUIN,
quinolinic acid; AhR, aryl hydrocarbon receptor; Ki, inhibition
constant; IFN-γ, interferon-γ; NFK, N-formyl kynurenine;
KPB, potassium phosphate buffer; CPMG, Carr−Purcell−
Meiboom−Gill; STD, saturation transfer difference; FBS, fetal
bovine serum; EDTA, ethylenediaminetetraacetic acid; ESI,
electrospray ionization mass spectra; DMAB, p-
dimethylaminobenzaldehyde; TCA, trichloroacetic acid
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ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
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AUTHOR INFORMATION
Corresponding Authors
*C.K.: e-mail, kuangcx@tongji.edu.cn; telephone and fax, +86-
021-65982351.
*Q.Y.: e-mail, yangqing68@fudan.edu.cn; telephone and fax,
+86-021-31246641.
■
ORCID
Qing Yang: 0000-0003-4162-922X
Author Contributions
D.Y. performed most of the enzymatic, cellular, CPMG and
STD NMR and animal experiments. S.Z. partially contributed
to enzymatic, cellular, and animal experiments. X.F. partially
contributed to enzymatic, cellular, and CPMG and STD NMR
experiments. L.G., N.H., and Z.G. partially contributed to
animal experiments. S.Y., X.L., and J.C.H partially contributed
to enzymatic and cellular experiments. C.K. synthesized
tryptanthrins and analyzed chemistry and structure-related
results. Q.Y. and C.K. initiated the research, led the project
team, designed experiments, analyzed results, and wrote the
manuscript. All authors have given approval to the final version
of the manuscript.
Notes
The authors declare no competing financial interest.
L
DOI: 10.1021/acs.jmedchem.9b01079
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Journal of Medicinal Chemistry
Article
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DOI: 10.1021/acs.jmedchem.9b01079
J. Med. Chem. XXXX, XXX, XXX−XXX