Design, synthesis and structure-activity relationship study of novel naphthoindolizine and indolizinoquinoline-5,12-dione derivatives as IDO1 inhibitors

Article abstract of DOI:10.1016/j.bmc.2018.08.028

Indoleamine 2,3-dioxygenase 1 (IDO1) is regarded as a promising target for cancer immunotherapy. Many naphthoquinone derivatives have been reported as IDO1 inhibitors so far. Herein, two series of naphthoquinone derivatives, naphthoindolizine and indolizinoquinoline-5,12-dione derivatives, were synthesized and evaluated for their IDO1 inhibitory activity. Most of the target compounds showed significant inhibition potency and high selectivity for IDO1 over tryptophan 2,3-dioxygenase (TDO). The structure-activity relationship was also summarized. The most potent compounds 5c (IC₅₀ 23 nM, IDO1 enzyme), and 5b′ (IC₅₀ 372 nM, HeLa cell) were identified as promising lead compounds.

Full text of DOI:10.1016/j.bmc.2018.08.028

Accepted Manuscript
Design, synthesis and structure-activity relationship study of novel naphthoin-
dolizine and indolizinoquinoline-5,12-dione derivatives as IDO1 inhibitors
Rui Yang, Yu Chen, Liangkun Pan, Yanyan Yang, Qiang Zheng, Yue Hu, Yuxi
Wang, Liangren Zhang, Yang Sun, Zhongjun Li, Xiangbao Meng
PII:
S0968-0896(18)30756-9
https://doi.org/10.1016/j.bmc.2018.08.028
BMC 14515
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
23 April 2018
5 August 2018
21 August 2018
Please cite this article as: Yang, R., Chen, Y., Pan, L., Yang, Y., Zheng, Q., Hu, Y., Wang, Y., Zhang, L., Sun, Y.,
Li, Z., Meng, X., Design, synthesis and structure-activity relationship study of novel naphthoindolizine and
indolizinoquinoline-5,12-dione derivatives as IDO1 inhibitors, Bioorganic & Medicinal Chemistry (2018), doi:
https://doi.org/10.1016/j.bmc.2018.08.028
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Design, synthesis and structure-activity relationship study of novel naphthoindolizine and
indolizinoquinoline-5,12-dione derivatives as IDO1 inhibitors
1
1
1
1
1
2
1
Rui Yang , Yu Chen , Liangkun Pan , Yanyan Yang , Qiang Zheng , Yue Hu , Yuxi Wang , Liangren
1
2
1
1
Zhang , Yang Sun , Zhongjun Li , and Xiangbao Meng *
1
State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences,
Peking University, Beijing 100191, China.
2
State Key Laboratory of Pharmaceutical Biotechnology and Collaborative Innovation Center of
Chemistry for Life Sciences, School of Life Sciences, Nanjing University, Nanjing 210023, China.
Abstract
Indoleamine 2,3-dioxygenase 1 (IDO1) is regarded as a promising target for cancer
immunotherapy. Many naphthoquinone derivatives have been reported as IDO1 inhibitors so far.
Herein, two series of naphthoquinone derivatives, naphthoindolizine and indolizinoquinoline-
5,12-dione derivatives, were synthesized and evaluated for their IDO1 inhibitory activity. Most of
the target compounds showed significant inhibition potency and high selectivity for IDO1 over
tryptophan 2,3-dioxygenase (TDO). The structure-activity relationship was also summarized. The
most potent compounds 5c (IC50 23 nM, IDO1 enzyme), and 5b (IC50 372 nM, HeLa cell) were
identified as promising lead compounds.
Keywords
Indoleamine
2,3-dioxygenase
1;
cancer
immunotherapy;
Naphthoindolizine;
Indolizinoquinoline-5,12-dione derivatives.
1.
Introduction
L-tryptophan is one of the essential amino acids necessary for protein synthesis and nitrogen
balance in human body. Over 90% of L-tryptophan utilized by humans is processed through
kynurenine pathway [1]. The heme-containing enzyme indoleamine 2,3-dioxygenase 1 (IDO1)
catalyzes the first and rate-limiting step of kynurenine pathway, leading to the depletion of
tryptophan and the production of its metabolites, such as kynurenine, kynurenic acid, and
quinolinic acid [2]. IDO1 was first discovered in rabbit intestine in 1967 [3], and it is expressed
ubiquitously throughout human body except in liver.
IDO1 plays an essential role in the regulation of tryptophan homeostasis. Its activation
depletes the local tryptophan concentration and accumulates the metabolites of kynurenine
pathway. The deficit of tryptophan leads to starvation of cytotoxic T-cells in the
microenvironment and the metabolites of tryptophan can activate regulatory T-cells, which
further suppresses the immune response to tumors. Numerous studies have demonstrated that
1

the expression of IDO1 is up-regulated in many human cancers and associated with poor
prognosis [4]. IDO1 helps tumors to induce tolerance from the host’s immune system and it is
overexpressed in many human cancers, which suggest that it might be a promising target for
cancer immunotherapy.
Indeed, several groups have discovered that blockade of IDO1 can directly increase the ability
of tumor-bearing mice to reject tumors [5, 6]. Furthermore, IDO1 inhibitors combined with
chemotherapeutic agents or established immunotherapeutic strategies, like anti-PD1/PD-L1
therapy, seem to have better efficacy and produce superior response rates compared with single
agent therapy [4, 7-12]. These studies demonstrate that inhibition of IDO1 can reactivate the
immune system to break tumor-induced immune resistance.
Many naphthoquinone derivatives have been reported as IDO1 inhibitors. Andersen et al. [13]
reported that several natural products isolated from a marine invertebrate showed potent
inhibitory activities against IDO1 and the most potent one was Annulin B with a K
i
= 0.12 μM (Fig.
1). Since most of the marine natural product inhibitors contain a naphthoquinone core, Muller et
al [14] hypothesized that the naphthoquinone core was the relevant pharmacophore in Annulin B.
They screened commercially available compounds containing a quinone structure for IDO1
inhibitory activity and several compounds demonstrated micromolar levels of inhibitory potency,
such as menadione (IC50 = 0.72 μM) and Juglone (IC50 = 1.0 μM) (Fig. 1). Based on menadione’s
structure, they synthesized several pyranonaphthoquinone derivatives (PNQs) and the most
potent ones demonstrated nanomolar level of inhibition against IDO1, such as PNQ 1 (K
PNQ 2 (K = 61 nM), PNQ 3 (K = 66 nM) (Fig. 1).
i
= 70 nM),
i
i
Figure 1. Structure of naphthoquinone derivatives as IDO1 inhibitors.
Here, we designed and synthesized two new series of novel naphthoquinone derivatives,
2

naphthoindolizine (1-2) and indolizinoquinoline-5,12-dione derivatives (3-6) (Fig. 2). We tested
their inhibitory activity against IDO1 and found several compounds exhibited high inhibition
activity. The structure-activity relationship of the target molecules was summarized. The most
potent compound 5c showed nanomolar level of inhibition against IDO1 (IC50 = 23 nM) and low
cytotoxicity.
Figure 2. Structures of the naphthoindolizine derivatives (compounds 1-2) and
indolizinoquinoline-5,12-dione derivatives (compounds 3-6).
2.
Results and Discussion
2.1. Chemistry
The naphthoindolizine derivatives (NIs) were synthesized following the multi-step strategy
shown in Scheme 1. The core heterocyclic skeleton of 1a was synthesized through an one-pot
three-component reaction using 1,4-naphthoquinone, pyridine, and dimethyl butynedioate in
DMF in the presence of a catalytic amount of hydrated copper(II) chloride heated at 80°C for 16h
2
under 1 atm of O [15]. Compound 1a subsequently underwent hydrolysis to give the carboxylic
acid 1b. Finally, 1b was converted into amide 1c-f via reaction with amines by treatment with
EDCI and HOBT in dichloromethane. Ester 1g and 1h were obtained by the reaction of 1b and
ethanol or isopropanol by DCC and DMAP. Similarly, 2a-c were prepared using
5-hydroxy-1,4-naphthalenedione. No regioisomers were obtained and the hydroxyl group’s
position of 2a was characterized by NMR analysis. The H-H COSY spectrum indicated that the
signal at δ = 7.74 ppm could be assigned to H-4. Furthermore, the HMBC spectrum demonstrated
that the resonance at δ = 179.63 ppm was attributed to C-5 by long-range hetero-correlation with
H-4. The signal at δ = 162.10 ppm was attributed to C-1 by correlation with hydrogen of hydroxyl
(δ = 12.54 ppm) and the resonance at δ = 180.22 ppm was assigned to correlation between C-12
and OH (δ = 12.54 ppm) (Fig. 3).
3

Scheme 1. Synthesis of naphthoindolizine derivatives 1a-2c. Reagents and conditions: (i) DMF,
pyridine, dimethyl butynedioate, CuCl , 1atm O , refluxing; (ii) MeOH/H O, K CO ; (iii) DCM,
2
2
2
2
3
amines, EDCI, HOBt; (iv) DCM, DCC, DMAP, EtOH/isopropanol.
Figure 3. Structural characterization of 2a.
The indolizinoquinoline-5,12-dione derivatives (IQDs) were obtained as shown in Scheme 2-5.
Pyridine scaffolds were purchased or prepared as shown in Scheme 2.
6,7-Dichloroquinoline-5,8-dione (7) was obtained by oxidizing 8-hydroxyquinoline with sodium
chlorate in concentrated HCl solution [16]. The heterocyclic skeletons of 3a,a’-3j,j’ and 6e,e’ were
formed through a single-step reaction of substrate 7, pyridine derivatives and an active
methylene reagent (AMR). Two regioisomers (N,N-syn and N,N-anti isomers) were obtained in
the cyclization, which gave the same results as by Defant et al. [17]. Some of the isomers could be
isolated in pure form by chromatography, such as 3a/3a’, 3i/3i’, and 6e/6e’. But most of the
isomers were inseparable due to the similarity of their structure. 3a,a’-3f,f’ underwent hydrolysis
to give 4a,a’-4f,f’. The regioisomers 4a,a’-4e,e’ could be isolated readily by chromatography,
whereas 4f,f’ were not isolated due to their poor solubility. Finally, 4a,a’ and 4b,b’ were
converted into 5a,a’ and 5b,b’ by reaction with N,N-dimethyl-1,2-ethanediamine in the presence
2 2
of HBTU in CH Cl (Scheme 3). 6a,a’-6c,c’ were obtained through the reaction of 4a,a’ and aniline
derivatives as shown in Scheme 4. 6d,d’ were prepared by the reaction of
ethyl-m-aminobenzoate with 4a,a’ and subsequent hydrolysis of ester group (Scheme 4). 4a was
treated with N,N'-carbonyldiimidazole and thiosemicarbazide in DMF to afford 5c (Scheme 5)
[
18].
4

3
Scheme 2. Preparation of pyridine derivatives. Reagents and conditions: (i) MeOH, CH I, KOH; (ii)
DCM, Di-tert-butyl dicarbonate.
Scheme 3. Synthesis of indolizinoquinoline-5,12-dione derivatives 3a,a’-5b,b’ & 6e,e’. Reagents
and conditions: (i) NaClO , concd HCl, 50°C; (ii) EtOH, pyridine derivatives, AMR, refluxing; (iii)
MeOH/H O, K CO ; (iv) DCM, N,N-Dimethyl-1,2-ethanediamine, HBTU, DIPEA; (v) DCM, CF COOH.
3
2
2
3
3
Scheme 4. Synthesis of indolizinoquinoline-5,12-dione derivatives 6a,a’-6d,d’. Reagents and
conditions: (i) DCM, aniline derivatives, HBTU, DIPEA; (ii) DCM, ethyl-m-aminobenzoate, HBTU,
2
DIPEA; (iii) MeOH/H O, KOH.
5

Scheme 5. Synthesis of indolizinoquinoline-5,12-dione derivatives 5c. Reagents and conditions: (i)
DMF, thiosemicarbazide, N,N'-carbonyldiimidazole.
The structures of regioisomers 3a/3a’ and 3i/3i’ were confirmed by the NMR spectra, ESI-MS
spectrum, and melting point, in their pure form. The results were similar to those of Defant’s [17,
1
9]. The structures of regioisomers 4a,a’-4e,e’ were confirmed by R
value of 4a (N,N-syn) is larger than that of 4a’ (N,N-anti) in TLC (DCM/MeOH = 10: 1), we
hypothesized that the regioisomer with a larger R value was N,N-syn and the other regioisomer
with a smaller R value was N,N-anti. The NMR spectra of 4b and 4b’ confirmed our hypothesis.
f f
value in TLC. Since the R
f
f
The HMBC spectrum of 4b showed correlation of C-5 at δ = 185.45 ppm with H-4 at δ = 8.61 ppm
while the HMBC spectrum of 4b’ indicated correlation between C-5 at δ = 172.59 ppm and H-4 at
δ = 8.63 ppm. The structures of regioisomers 4c,c’-4e,e’ were assigned according to the pattern
f
of R value.
Figure 4. Structural assignments of the N,N-syn and N,N-anti regioisomers.
2.2. IDO1 Inhibitory Activities
The IDO1 inhibitory activities of 1-6 along with INCB024360 [20] were assessed by IDO1
inhibition assay. Under the conditions, the IC50 of INCB024360 was 0.073 μM. The results shown
in Table 1-4 indicated that many of our compounds strongly inhibited the catalytic activity of
IDO1.
As shown in Table 1, compounds 1a, 1b, 1g, 2a and 2b exhibited high inhibitory activity. Ester
side chains attached to the pyrrole ring had superior activity than carboxylic acids and amides.
Long and bulky ester side chains (1g and 1h) showed weaker inhibition. In addition, hydroxyl
group at C-1 in the A ring had no impact on the inhibition activity.
6

Table 1. IDO1 Inhibitory activities of 1a-h and 2a-c.
Inhibition Rate
Compd
R
IC50 (μM)
SD(μM)
(
% inhibition at 1 μM)
1
a
COOCH
COOH
3
77
61
27
24
22
15
68
13
77
60
29
-
0.156
0.714
ND
0.0297
1b
0.1209
1
c
CONH(CH
CONH(CH
2
)
)
2
N(CH
N(CH
3
)
)
2
-
1d
2
3
3
2
ND
-
1e
CONH(CH
CONH(CH
2
)
)
2
OH
OH
ND
-
1
f
2
3
ND
-
1
g
COOCH
2
CH
3
0.332
ND
0.0728
1h
COOCH(CH
3
)
2
-
2a
COOCH
COOH
3
0.087
ND
0.0099
2b
-
2
c
CONH(CH
2
)
2
N(CH
3
)
2
ND
-
INCB024360
-
0.073
0.0028
ND: Not detected.
Interestingly, the introduction of N atom in the A ring had dual effect. Compared with 1a, 1b
and 1c, the activities of 3i and 3i’ decreased, while the activities of 4a, 4a’, 5a, and 5a’
significantly increased (Table 2). The position of N atom in the A ring also showed variable effect.
For example, the inhibition rate of 3a is lower than its regioisomer 3a’ while that of 4a is higher
than 4a’, which were obtained respectively through hydrolysis of 3a and 3a’. But in general,
N,N-syn regioisomers were more potent IDO1 inhibitors. Modifications with smaller ester side
chains (3i and 3i’) led to stronger inhibition activities than the larger ones (3a and 3a’). But
modifications of IQD with carboxyl and amine side chains at the C ring demonstrated superior
inhibitory activity than ester group, which was different from NIs. Moreover, electron-donating
substituent groups at C-8, such as 3c,c’, 3d,d’, 3e,e’, 3h,h’, 4b,b’ and 5b,b’, generally showed
stronger inhibitory activity than electron-withdrawing groups (3f,f’ and 3g,g’). Compound 5b (IC50
=
3
50 nM) and 5b’ (IC50 = 39 nM) demonstrated strong inhibition, which suggested that SCH at C-8
and amine side chain at the pyrrole ring were beneficial to the activity. Compound 5c, carrying a
hydrophilic thiourea group as the side chain of C ring, showed the highest inhibition activity (IC50
=
23 nM).
7

Table 2. IDO1 Inhibitory activity of 3a,a’-5b,b’ and 5c.
Inhibition Rate
Compd
R
1
R
2
IC50 (μM) SD(μM)
(
% inhibition at 1 μM)
3
a
8-H
8-H
COOEt
COOEt
COOEt
COOEt
COOEt
COOEt
COOEt
COOEt
COOEt
COOMe
COOMe
COOEt
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
35
61
23
63
66
66
56
24
52
65
69
71
70
58
70.5
69
47
70
54
67
67
56
78
77
72.5
80.5
74.5
-
ND
0.977
ND
-
3a’
0.027
-
a
3
b,b’
8-SCH
3
a
3
c,c’
8-OCH
3
ND
-
a
3
d,d’
8-CH
8-I
3
ND
-
a
3
e,e’
ND
-
a
3
f,f’
8-COOMe
8-COOEt
ND
-
a
a
3
g,g’
ND
-
3
h,h’
8-NH
8-H
8-H
7-F
2
ND
-
3
i
0.438
0.448
0.314
0.288
1.192
ND
0.129
0.112
0.049
0.051
0.318
-
3i’
a
3j,j’
4a
8-H
8-H
4a’
a
4
b,b’
8-SCH
3
4
c
8-OCH
8-OCH
3
0.299
ND
0.064
-
4c’
3
4d
8-CH
8-CH
8-I
3
0.394
ND
0.028
-
4d’
3
4e
0.117
0.420
ND
0.020
0.105
-
4e’
8-I
a
4
f,f’
8-COOH
8-H
5a
CONH(CH
CONH(CH
CONH(CH
CONH(CH
2
2
2
2
)
2
)
2
)
2
)
2
N(CH
N(CH
N(CH
N(CH
3
3
3
3
)
)
)
)
2
2
2
2
0.127
0.188
0.050
0.039
0.023
0.073
0.032
0.013
0.001
0.003
0.006
0.003
5a’
8-H
5b
8-SCH
8-SCH
8-H
3
5b’
3
5
c
CONHNHC(=S)NH
-
2
INCB024360
-
a
ND: Not detected. The mixture was used to determine the inhibition rate.
When side chains at the C ring contained different anilines, the inhibition activities were
moderately increased (Table 3). Comparison of the two regioisomers, the N,N-syn isomers
generally showed more potent activity than the N,N-anti isomers. When the side chain was
benzyl group (6e and 6e’), the inhibition rate dropped significantly (Table 4), which suggested
8

that a flexible side chain might not be desirable and a conjugated system might be necessary.
Table 3. IDO1 Inhibitory activity of 6a,a’-6d,d’.
Inhibition Rate
Compd
R
IC50 (μM)
SD(μM)
(
% inhibition at 1 μM)
6
a
H
H
68
62
0.361
0.119
0.427
0.134
ND
0.075
0.020
0.110
0.026
-
6a’
6b
2-CH
2-CH
3-Cl
3-Cl
3
74
6b’
3
64
6
c
29.5
34
6c’
ND
-
-
+
+
6d
3-COO K
74.5
71.5
-
0.148
0.390
0.073
0.025
0.115
0.004
-
6
d’
INCB024360
ND: Not detected.
3-COO K
-
Table 4. IDO1 Inhibitory activity of 6e and 6e’.
Inhibition Rate
Compd
IC50 (μM)
(
% inhibition at 1 μM)
6
e
34
56
96
ND
ND
6
e’
INCB024360
ND: Not detected.
0.073
2.3. Selectivity over TDO
Tryptophan 2,3-dioxygenase (TDO), which is a distantly related tetrameric enzyme mainly
expressed in the liver, catalyzes the same reaction in kynurenine pathway. We also evaluated our
compounds for TDO inhibition [25] (Table 5). The results indicated that the compounds that
showed strong IDO1 inhibition activity also exhibited high selectivity for IDO over TDO.
9

Table 5. TDO Inhibition of several NIs and IQDs
Inhibition Rate
Compd
IC50 (μM) SD(μM)
(
% inhibition at 10 μM)
1
a
40
14
ND
ND
-
-
3a’
4a
28
ND
-
5
a
91
3.35
7.15
1.2
1.4
2.7
2.2
1.5
-
5a’
88
5
c
ND
ND
ND
ND
ND
ND
ND
6a
7.3
6a’
>100
>100
>100
41.6
>100
6b
-
6b’
-
6d
10.0
-
6d’
ND: Not detected.
2.4. Cytotoxicity Study
An et al [21] evaluated the cytotoxic activities of similar indolizinoquinoline-5,12-dione
derivatives and they found that most of their compounds showed significant cytotoxic activities
against a wide variety of tumor cell lines, such as lung adenocarcinoma cells, large-cell lung
carcinoma cells, promyelocytic leukemia cells and breast carcinoma cells. Considering the
similarity between the core structure of their compounds and ours, we also tested the
cytotoxicity of the most potent compound, 5c, by MTT using two human cancer cell lines, human
cervical carcinoma cells (HeLa), breast carcinoma cells (MCF-7), and embryonic kidney cells
(HEK293T). Doxorubicin was used as the positive control. According to Table 6, 5c showed weak
cytotoxic activities against the three human cell lines.
Table 6. Cytotoxic activities of 5c
Cytotoxic activities IC50 (μM)
Compd
HeLa
>20
MCF-7
>20
HEK293T
>20
5c
Doxorubicin 0.66±0.04 0.37±0.03 1.13±0.11
2.5. Cellular IDO-1 Inhibitory Activities of NIs and IQDs
To study the cellular IDO1 inhibitory activity, HeLa cells expressing human IDO1 were created
and used for assays with NIs and IQDs. Several compounds displayed significant IDO1 inhibitory
activity, such as 5a, 5a’, 5b and 5b’ (shown in Table 7). However, some compounds that showed
high inhibition against IDO1 in enzymatic assays did not demonstrated similar inhibition in
10

cellular assays, which might result from membrane permeability, or the complexity of kynurenine
pathway.
Table 7. Cellular IDO1 Inhibitory Activities
Inhibition Rate
IC50 (μM)
Compd
(
% inhibition at 1 μM)
HeLa
1
2
4
5
a
a
e
a
24
12
12
43
81
42
77
22
25
38
9
-
-
-
0.738
1.235
1.083
0.372
-
5a’
5b
5b’
5
c
6a’
-
6b’
2.662
-
6d
INCB024360
96
0.007
2.6. Molecular Docking study of 5c
Because of the large structural difference between 5c and the original ligand of 4PK5 [16],
there is no suitable binding conformation for docking 5c to 4PK5 directly. Alternatively, we used
Schrodinger software Induced Fit Docking (IFD) to expand the 4PK5 binding site gradually by
docking a series of smaller IDO inhibitors in sequence, during which the docking result of the
previous step was used to generate the next grid. In this manner, the IDO1 protein can be docked
with 5c by Schrodinger glide (Released Jan, 2017). Compound 5c was docked inside the IDO1
binding cleft (Fig. 5) by three hydrogen-bonds (amino acid L234, G236, and G263 with the side
chain, respectively),  interaction (amino acid F163 with pyridine moiety).
11

Figure 5. View of compound 5c docked inside the IDO1 binding cleft.
3.
Conclusions
Approximately fifty naphthoindolizine and indolizinoquinoline-5,12-dione derivatives were
synthesized and many of them demonstrated significant inhibitory activity against IDO1. For NIs,
introduction of small ester side chain at the C ring led to superior inhibitory activity and
introduction of a hydroxyl group at C-1 had no impact on inhibitory activity. For IQDs, carboxylic
acids and amides (generated from aliphatic amine or anilines) side chains exhibited higher
3
inhibitory activity than esters. Electron-donating groups at C-8, such as SCH , were beneficial to
the activity. Besides, N,N-syn isomers generally showed more potent inhibitory activity than
N,N-anti isomers. Several compounds that had strong IDO1 inhibitory activity also exhibited high
selectivity over TDO. 5c was identified as the most potent compound (IC50 = 23 nM) with weak
cytotoxicity, and 5b was the most potent one in cellular assay, which are promising leads for
developing potent and highly selective IDO1 inhibitors.
4.
Experimental protocols
4.1. General methods and materials
All starting materials and common laboratory chemicals were purchased from commercial
sources and used without further purification. All solvents were distilled and dried according to
standard procedures. Chemical reactions were monitored by thin layer chromatography using
silica gel 60 F254 aluminum supported plate (layer thickness 0.2 mm). Flash column
12

chromatography was performed with silica gel 200–300 meshes. Melting points were determined
1
13
using the X-5 apparatus. H and C NMR spectra were measured on a Bruker ARX400 instrument,
using TMS as internal standard [chemical shifts (δ) in ppm, coupling constants (J) in Hz]. High
resolution mass spectra were obtained by Electro Spray Ionization (ESI).
4
.2. General preparation of NIs
,4-Naphthaquionone or 5-hydroxyl-1,4-naphthalenedione (1.0 mmol), pyridine (3.0 mmol),
butynedioates (1.0 mmol) and hydrated copper(II) chloride (0.3 mmol) were mixed in 15 mL DMF
and stirred at 80 °C for 16 h under 1 atm of O [15]. After completion of the reaction, the reaction
1
2
mixture was cooled to room temperature and concentrated in vacuum. The residue was
subsequently subjected to flash column chromatography (ethyl acetate/petroleum ether, 1:6) to
give the corresponding target compound 1a or 2a. The methyl ester group of 1a and 2a was
hydrolyzed using K
2
CO
3
in MeOH/H
2
O. When the hydrolysis was complete, 1M HCl was added
Cl . The organic phase was dried with MgSO and
and the mixture was extracted with CH
2
2
4
concentrated in vacuum to give the product 1b and 2b. 1b or 2b (1.0 mmol) was dissolved in
DCM and EDCI (1.5 mmol), HOBT (1.5 mmol) were added to the solution. The mixture was stirred
at room temperature for 1h, followed by the addition of different amines (1.5 mmol) and the
reaction mixture was stirred for 5h. The solvent was evaporated under reduced pressure. The
crude product was purified by silica gel column chromatography to give the target compound
1c-1f and 2c. 1b or 2b (1.0 mmol) was dissolved in DCM and DMAP (1.5 mmol), DCC (1.5 mmol)
were added to the solution. The mixture was stirred at room temperature for 1h, followed by the
addition of different alcohols (1.5 mmol) and the reaction mixture was stirred for 5h at room
temperature. The solvent was evaporated under reduced pressure. The crude product was
purified by silica gel column chromatography (DCM/MeOH, 200:1) to give the target compound
1g and 1h.
4.2.1. Methyl 6,11-dioxo-6,11- dihydrobenzo[f]pyrido [1,2-a]indole-12-carboxylate (1a)
1
Orange solid; yield 43%; m.p. = 190-191°C; H NMR (400 MHz, CDCl
3
): δ = 9.84 (d, J = 7.0 Hz,
1
H), 8.33 (d, J = 9.1 Hz, 1H), 8.27–8.16 (m, 2H), 7.77–7.66 (m, 2H), 7.44 (ddd, J = 9.0, 6.8, 1.0 Hz,
H), 7.18 (td, J = 6.9, 1.2 Hz, 1H), 4.06 (s, 3H). Lit. [15]
1
4.2.2. 6,11-Dioxo-6,11-dihydrobenzo[f]pyrido[1,2-a]indole-12-carboxylic acid (1b)
1
Red solid; yield 93%; m.p. = 304-305°C; H NMR (400 MHz, DMSO-d
6
): δ = 13.60 (s, 1H),
9
.72–9.78 (m, 1H), 8.59–8.61 (m, 1H), 8.19–8.21 (m, 2H), 7.92 (d, J = 27.4 Hz, 2H), 7.72–7.73 (m,
H), 7.51–7.52 (m, 1H). Lit. [22]
1
4.2.3. N-(2-(Dimethylamino)ethyl)-6,11-dioxo-6,11-dihydrobenzo[f]pyrido[1,2-a]indole-12-carbox
amide (1c)
Red solid; yield 88%; m.p. = 142-143°C; H NMR (400 MHz, CDCl
1
3
): δ = 10.32 (s, 1H), 9.69 (d, J =
13

6.7 Hz, 1H), 8.94 (d, J = 9.0 Hz, 1H), 8.04 (dd, J = 14.4, 7.4 Hz, 2H), 7.60 (dt, J = 21.6, 7.1 Hz, 2H),
7.31 (dd, J = 16.0, 7.9 Hz, 1H), 7.08 (t, J = 6.5 Hz, 1H), 3.59 (d, J = 5.7 Hz, 2H), 2.65 (t, J = 6.4 Hz,
2H), 2.37 (s, 6H). Lit. [23]
4.2.4. N-(3-(dimethylamino)propyl)-6,11-dioxo-6,11-dihydrobenzo[f]pyrido[1,2-a]indole-12-carbo
xamide (1d)
1
Red solid; yield 90%; m.p. = 148-149°C; H NMR (400 MHz, MeOD): δ = 9.43 (d, J = 6.8 Hz, 1H),
.51 (d, J = 9.1 Hz, 1H), 7.83 (d, J = 7.5 Hz, 1H), 7.76 (d, J = 7.5 Hz, 1H), 7.62 (t, J = 7.4 Hz, 1H), 7.53
8
(t, J = 7.3 Hz, 1H), 7.37–7.27 (m, 1H), 7.11 (t, J = 6.9 Hz, 1H), 3.37 (t, J = 6.8 Hz, 2H), 2.73–2.59 (m,
1
3
2
1
1
H), 2.44 (s, 6H), 1.87–1.96 (m, 2H). C NMR (100 MHz, MeOD): δ = 182.50, 172.76, 162.38,
39.57, 133.96, 132.71, 132.65, 131.98, 127.70, 126.81, 126.38, 125.12, 123.71, 121.81, 120.59,
18.09, 108.08, 56.84, 44.14, 37.11, 26.35.Lit. [24]
4.2.5. N-(2-hydroxyethyl)-6,11-dioxo-6,11-dihydrobenzo[f]pyrido[1,2-a]indole-12-carboxamide
(1e)
1
Red solid; yield 43%; m.p. = 160-161°C; H NMR (400 MHz, DMSO-d
6
): δ = 10.05 (t, J = 5.1 Hz,
1
2
1
1
1
H), 9.57 (d, J = 6.8 Hz, 1H), 8.72 (d, J = 9.1 Hz, 1H), 7.96 (dd, J = 15.5, 7.3 Hz, 2H), 7.75 (dt, J =
0.1, 7.2 Hz, 2H), 7.57–7.41 (m, 1H), 7.31 (t, J = 6.8 Hz, 1H), 4.83 (t, J = 5.1 Hz, 1H), 3.61 (dd, J =
1
3
6
1.1, 5.6 Hz, 2H), 3.41 (dd, J = 11.1, 5.6 Hz, 2H). C NMR (100 MHz, DMSO-d ): δ = 183.50, 173.74,
62.40, 140.00, 135.00, 133.72, 133.63, 133.03, 128.60, 127.57, 127.43, 127.39, 126.00, 124.80,
22.59, 121.30, 119.15, 109.49, 60.67, 42.12. Lit. [25]
4.2.6. N-(3-hydroxypropyl)-6,11-dioxo-6,11-dihydrobenzo[f]pyrido[1,2-a]indole-12-carboxamide
(1f)
1
Red solid; yield 48%; m.p. = 225-226°C; H NMR (400 MHz, DMSO-d
6
): δ = 9.84 (t, J = 5.1 Hz,
1
H), 9.50 (d, J = 6.8 Hz, 1H), 8.65 (d, J = 9.1 Hz, 1H), 7.91 (dd, J = 11.7, 7.7 Hz, 2H), 7.71 (dt, J =
2.3, 7.2 Hz, 2H), 7.52–7.38 (m, 1H), 7.28 (t, J = 6.8 Hz, 1H), 4.55 (s, 1H), 3.57 (s, 2H), 3.32–3.36
2
1
3
(
m, 2H), 1.72–1.79 (m, 2H). C NMR (100 MHz, DMSO-d
6
): δ = 183.43, 173.48, 162.12, 139.82,
1
1
34.83, 133.57, 133.46, 132.84, 128.46, 127.41, 127.27, 125.84, 124.52, 122.49, 121.09, 119.08,
09.51, 58.99, 36.39, 32.77. Lit. [26]
4.2.7. Ethyl 6,11-dioxo-6,11- dihydrobenzo[f]pyrido [1,2-a]indole-12-carboxylate (1g)
1
Orange solid; yield 85%; m.p. = 157-158°C; H NMR (400 MHz, CDCl
3
): δ = 9.76 (d, J = 7.0 Hz,
1H), 8.25 (d, J = 9.1 Hz, 1H), 8.22–8.07 (m, 2H), 7.67 (p, J = 7.5 Hz, 2H), 7.45–7.32 (m, 1H), 7.12 (t,
J = 6.7 Hz, 1H), 4.50 (q, J = 7.1 Hz, 2H), 1.50 (t, J = 7.2 Hz, 3H). Lit. [23]
4.2.8. Isopropyl 6,11-dioxo-6,11- dihydrobenzo[f]pyrido[1,2-a]indole-12-carboxylate (1h)
1
Orange solid; yield 78%; m.p. = 131-133°C; H NMR (400 MHz, CDCl
3
): δ = 9.78 (d, J = 7.0 Hz,
1
H), 8.25 (d, J = 9.1 Hz, 1H), 8.22–8.11 (m, 2H), 7.68 (p, J = 7.6 Hz, 2H), 7.45–7.33 (m, 1H), 7.12 (t,
14

J = 6.9 Hz, 1H), 5.40 (dt, J = 12.5, 6.2 Hz, 1H), 1.49 (d, J = 6.3 Hz, 6H). Lit. [15]
4.2.9. Methyl
10-hydroxy-6,11-dioxo-6,11-dihydrobenzo[f]pyrido[1,2-a]indole-12-carboxylate
(2a)
1
Orange solid; yield 53%; m.p. = 240-241°C; H NMR (400 MHz, CDCl
3
): δ = 12.54 (s, 1H), 9.76 (d,
J = 7.1 Hz, 1H), 8.34 (d, J = 9.2 Hz, 1H), 7.74 (d, J = 7.5 Hz, 1H), 7.55 (t, J = 7.9 Hz, 1H), 7.50–7.42
1
3
(
m, 1H), 7.24–7.16 (m, 2H), 4.04 (s, 3H). C NMR (100 MHz, CDCl
3
): δ = 180.22, 179.63, 163.67,
1
1
62.10, 140.19, 135.48, 134.43, 129.13, 128.52, 128.28, 124.34, 121.61, 121.15, 120.08, 117.71,
+
15.90, 106.44, 52.13. HR-ESI-MS: Calcd for C18
H11NO
5
[M+Na] : 344.0535, found: 344.0543. The
structure was confirmed through H-H COSY and HMBC.
4.2.10. 10-Hydroxy-6,11-dioxo-6,11-dihydrobenzo[f]pyrido[1,2-a]indole-12-carboxylic acid (2b)
1
Red solid; yield 89%; m.p. >320°C; H NMR (400 MHz, CDCl
3
): δ = 13.99 (s, 1H), 12.54 (s, 1H),
9
7
1
.79 (d, J = 6.9 Hz, 1H), 8.98 (d, J = 8.9 Hz, 1H), 7.89 (d, J = 7.2 Hz, 1H), 7.61 (q, J = 8.9 Hz, 2H),
1
3
3
.37–7.30 (m, 2H). C NMR (150 MHz, CDCl ): δ = 185.44, 179.40, 162.72, 162.59, 141.80, 135.73,
32.39, 129.67, 128.37, 127.15, 126.44, 122.83, 121.56, 119.33, 115.71, 107.12. HR-ESI-MS:
+
Calcd for C17
H
9
NO
5
[M+H] : 308.0559, found: 308.0553.
4.2.11. N-(2-(dimethylamino)ethyl)-10-hydroxy-6,11-dioxo-6,11-dihydrobenzo[f]pyrido[1,2-a]indo
le-12-carboxamide (2c)
1
Red solid; yield 85%; m.p. = 185-186°C; H NMR (400 MHz, MeOD): δ = 9.79 (d, J = 5.4 Hz, 1H),
8
3
1
1
.90 (d, J = 10.1 Hz, 1H), 7.74 (s, 1H), 7.63 (d, J = 5.8 Hz, 2H), 7.40 (s, 1H), 7.30 (d, J = 8.3 Hz, 1H),
1
3
.92 (s, 2H), 3.52 (s, 2H), 3.08 (s, 6H). C NMR (100 MHz, MeOD): δ = 183.31, 179.26, 164.75,
61.96, 140.97, 135.33, 133.33, 128.84, 128.10, 125.70, 125.16, 122.14, 121.14, 120.12, 118.71,
+
15.86, 115.45, 108.47, 57.90, 42.72, 34.72. HR-ESI-MS: Calcd for C21
H
19
N
3
O
4
[M+H] : 378.1454,
found: 378.1450.
4
.3. Preparation of compound 7
,7-Dichloroquinoline-5,8-dinone (7) was prepared following Shaikh’s method [16] by oxidizing
-hydroxyquinoline with sodium chlorate in concentrated HCl solution at 40°C. The reaction was
6
8
diluted with water and the white precipitate was removed by filtration. The filtrate was extracted
with CH Cl and after the removal of CH Cl , the residue was recrystallized in methanol to obtain
a light yellow solid 7 with a yield of 29%.
2
2
2
2
4
.4. Preparation of pyridine derivatives
-Mercaptopyridine (9 mmol) was dissolved in 100 ml of 1% methanolic KOH solution and one
equivalent iodomethane was added. The reaction mixture was stirred at room temperature for
4h. After evaporating the solvent, the residue was extracted with CH Cl and concentrated
under vacuum to give 4-methylthiopyridine.
4
2
2
2
15

4
-Aminopyridine (10 mmol) was dissolved in 100 ml of CH
mmol) diluted with CH Cl was added dropwise to the solution. The reaction mixture was stirred
at room temperature for 24h. The solvent was removed to give
2 2
Cl and di-tert-butyl dicarbonate (12
2
2
4
4
7
-tert-butoxycarbonylaminopyridine.
.5. General preparation of IQDs
The heterocyclic skeleton was prepared according to the published methods [27, 28]. Substrate
(1.0 mmol) was added in 15 mL absolute ethanol and heated to dissolve. Then, the AMR (1.5
mmol) and pyridine derivative (3.0 mmol) were successively added to the solution. The reaction
mixture was refluxed for 16h at 80°C and concentrated in vacuum when the reaction was
complete. The residue was subsequently subjected to flash column chromatography to give the
corresponding target compound 3a,a’-3j,j’ and 6e,e’. The ethyl ester group of 3a,a’-3f,f’ was
hydrolyzed using K
2
CO
3
in MeOH/H
2
O. When the hydrolysis was complete, 1M HCl was added
Cl . The organic phase was dried with MgSO and
and the mixture was extracted with CH
2
2
4
concentrated in vacuum. The crude product was purified by silica gel column chromatography to
give the target compound 4a,a’-4f,f’ and the regioisomers of 4a-e and 4a’-e’ were separated in
the meantime. 4a-b or 4a’-b’ (1.0 mmol) was dissolved in CH
1.5 mmol) was added to the solution. The mixture was stirred at room temperature for 1h. Then
N,N-dimethyl-1,2-ethanediamine (1.5 mmol) was added and the reaction mixture was stirred for
h. The solvent was evaporated under reduced pressure. The crude product was purified by silica
gel column chromatography to give the target compound 5a-b and 5a’-b’.
2 2
Cl and HBTU (1.5 mmol), DIPEA
(
5
4.5.1. Ethyl 5,12-dioxo-5,12-dihydroindolizino[2,3-g]quinoline-6-carboxylate (3a)
1
Orange solid; yield 31%; m.p. = 225-226°C; H NMR (400 MHz, CDCl
3
): δ = 9.96 (d, J = 6.9 Hz,
1H), 9.04 (d, J = 3.7 Hz, 1H), 8.57 (d, J = 7.5 Hz, 1H), 8.37 (d, J = 9.0 Hz, 1H), 7.67 (dd, J = 7.6, 4.7
Hz, 1H), 7.62–7.42 (m, 1H), 7.33–7.18 (m, 2H), 4.54 (q, J = 7.1 Hz, 2H), 1.52 (t, J = 7.1 Hz, 4H). Lit.
17]
[
4.5.2. Ethyl 5,12-dioxo-5,12-dihydroindolizino[3,2-g]quinoline-11-carboxylate (3a’)
1
Orange solid; yield 12%; m.p. = 219-220°C; H NMR (400 MHz, CDCl
3
): δ = 9.74 (d, J = 6.7 Hz,
1H), 8.97 (s, 1H), 8.52 (d, J = 7.6 Hz, 1H), 8.34 (d, J = 9.0 Hz, 1H), 7.83–7.51 (m, 1H), 7.44 (t, J = 7.8
Hz, 1H), 7.18 (t, J = 6.5 Hz, 1H), 4.47 (q, J = 6.9 Hz, 2H), 1.50 (t, J = 7.0 Hz, 3H). Lit. [17]
4.5.3. Ethyl 8-(methylthio)-5,12-dioxo-5,12-dihydroindolizino[2,3-g]quinoline-6-carboxylate (3b)
and ethyl 9-(methylthio)-5,12-dioxo-5,12-dihydroindolizino[3,2-g]quinoline-11-carboxylate (3b’)
mixture (3b/3b’ = 86/14)
1
Purple solid; yield 38%; m.p. = 248-249°C; H NMR (400 MHz, CDCl
3
): δ = 9.65 (d, J = 7.4 Hz,
0.86H), 9.48 (d, J = 7.3 Hz, 0.14H), 8.99 (d, J = 4.7 Hz, 1H), 8.49 (t, J = 7.6 Hz, 1H), 7.94 (s, 1H), 7.63
(
dd, J = 7.8, 4.7 Hz, 1H), 6.96 (dd, J = 19.1, 7.4 Hz, 1H), 4.48 (q, J = 6.9 Hz, 2H), 2.58 (s, 3H), 1.50 (t,
16

1
3
J = 7.2 Hz, 3H). C NMR (100 MHz, CDCl
3
): δ = 178.95, 172.71, 163.08, 153.97, 149.38, 143.65,
1
2
40.31, 135.24, 134.09, 130.84, 128.37, 127.24, 126.82, 122.93, 117.00, 112.69, 103.99, 60.99,
+
9.70, 14.40. HR-ESI-MS: Calcd for C19
H
14
N
2
O
4
S [M+Na] : 389.0572, found: 389.0579.
4.5.4. Ethyl 8-methoxy-5,12-dioxo-5,12-dihydroindolizino[2,3-g]quinoline-6-carboxylate (3c) and
ethyl 9-methoxy-5,12-dioxo-5,12-dihydroindolizino[3,2-g]quinoline-11-carboxylate (3c’) mixture
(3c/3c’ = 56/44)
1
Red solid; yield 29%; m.p. = 195-196°C; H NMR (400 MHz, CDCl
3
): δ = 9.78 (d, J = 7.6 Hz,
0
.56H), 9.63 (d, J = 7.5 Hz, 0.44H), 9.05–8.87 (m, 1H), 8.52 (d, J = 7.7 Hz, 1H), 7.72 (d, J = 11.3 Hz,
H), 7.68–7.54 (m, 1H), 6.96–6.78 (m, 1H), 4.61–4.34 (m, 2H), 3.98 (s, 3H), 1.52 (q, J = 7.0 Hz, 3H).
1
1
3
3
C NMR (100 MHz, CDCl ): δ = 179.20, 178.35, 173.07, 172.59, 164.08, 163.62, 160.25, 160.17,
1
1
6
3
53.95, 153.40, 149.68, 149.52, 143.27, 143.04, 135.24, 134.06, 130.83, 130.42, 129.88, 129.43,
28.85, 127.03, 126.73, 122.73, 121.40, 112.51, 112.46, 104.49, 103.91, 98.46, 98.35, 61.03,
+
0.90, 56.01, 55.93, 14.37, 14.21. HR-ESI-MS: Calcd for C19
51.0980.
H
14
N
2
O
5
[M+H] : 351.0981, found:
4.5.5. Ethyl 8-methyl-5,12-dioxo-5,12-dihydroindolizino[2,3-g]quinoline-6-carboxylate (3d) and
ethyl 9-methyl-5,12-dioxo-5,12-dihydroindolizino[3,2-g]quinoline-11-carboxylate (3d’) mixture
(3d/3d’ = 46/54)
1
Red solid; yield 30%; m.p. = 210-211°C; H NMR (400 MHz, CDCl
3
): δ = 9.75 (d, J = 6.6 Hz,
0
.46H), 9.58 (d, J = 5.8 Hz, 0.54H), 8.98 (s, 1H), 8.51 (s, 1H), 8.10 (d, J = 8.2 Hz, 1H), 7.64 (s, 1H),
.16–6.85 (m, 1H), 4.49 (s, 2H), 2.47 (s, 3H), 1.51 (d, J = 6.5 Hz, 3H). Lit. [17]
7
4
9
4
.5.6. Ethyl 8-iodo-5,12-dioxo-5,12-dihydroindolizino[2,3-g]quinoline-6-carboxylate (3e) and ethyl
-iodo-5,12-dioxo-5,12-dihydroindolizino[3,2-g]quinoline-11-carboxylate (3e’) mixture (3e/3e’ =
6/53)
1
Brown solid; yield 27%; m.p. = 268-269°C; H NMR (400 MHz, CDCl
3
): δ = 9.64 (d, J = 7.3 Hz,
0.46H), 9.50 (d, J = 7.3 Hz, 0.53H), 9.03 (s, 1H), 8.82 (d, J = 14.6 Hz, 1H), 8.55 (d, J = 7.9 Hz, 1H),
1
3
7
.72–7.65 (m, 1H), 7.48–7.42 (m, 1H), 4.57–4.45 (m, 2H), 1.54–1.49 (m, 3H). C NMR (100 MHz,
): δ = 178.70, 177.90, 173.86, 173.24, 163.11, 162.65, 154.19, 153.86, 149.56, 149.24,
CDCl
3
1
1
9
40.40, 140.19, 135.51, 134.40, 130.95, 130.36, 130.12, 130.08, 129.02, 128.60, 128.42, 128.33,
28.00, 127.93, 127.30, 127.17, 126.75, 126.69, 123.08, 121.91, 120.10, 119.39, 105.89, 105.41,
+
4.35, 94.11, 61.47, 61.39, 14.31, 14.16. HR-ESI-MS: Calcd for C18
H11IN
2
O
4
[M+H] : 446.9842,
found: 446.9836.
4.5.7. 6-Ethyl 8-methyl 5,12-dioxo-5,12-dihydroindolizino[2,3-g]quinoline-6,8-dicarboxylate (3f)
and 11-ethyl 9-methyl 5,12-dioxo-5,12-dihydroindolizino[3,2-g]quinoline-9,11-dicarboxylate (3f’)
mixture (3f/3f’ = 57/43)
1
Orange solid; yield 35%; m.p. = 257-258°C; H NMR (400 MHz, CDCl
3
): δ = 9.92 (d, J = 7.3 Hz,
17

0
4
1
1
1
4
.57H), 9.78 (d, J = 7.3 Hz, 0.43H), 9.08–8.98 (m, 2H), 8.61–8.56 (m, 1H), 7.78–7.67 (m, 2H),
1
3
3
.60–4.49 (m, 2H), 4.01 (d, J = 4.2 Hz, 3H), 1.58–1.49 (m, 3H). C NMR (100 MHz, CDCl ): δ =
78.71, 177.81, 174.25, 173.55, 164.54, 163.00, 162.53, 154.32, 154.08, 149.54, 149.24, 138.72,
38.48, 135.60, 134.57, 130.95, 130.41, 129.19, 129.09, 128.79, 128.39, 128.10, 127.72, 127.33,
+
23.70, 123.40, 123.34, 116.78, 61.59, 53.04, 14.30. HR-ESI-MS: Calcd for C20
01.0750, found: 401.0749.
H
14
N
2
O
6
[M+Na] :
4.5.8. Diethyl 5,12-dioxo-5,12-dihydroindolizino[2,3-g]quinoline-6,8-dicarboxylate (3g) and
diethyl 5,12-dioxo-5,12-dihydroindolizino[3,2-g]quinoline-9,11-dicarboxylate (3g’) mixture (3g/3g’
=
59/41)
Orange solid; yield 35%; m.p. = 218-219°C; H NMR (400 MHz, CDCl
.59H), 9.77 (d, J = 7.3 Hz, 0.41H), 9.01 (d, J = 23.1 Hz, 2H), 8.57 (d, J = 7.8 Hz, 1H), 7.79–7.65 (m,
1
3
): δ = 9.91 (d, J = 7.2 Hz,
0
2
1
3
H), 4.59–4.51 (m, 2H), 4.51–4.43 (m, 2H), 1.53 (t, J = 7.1 Hz, 3H), 1.46 (t, J = 7.1 Hz, 3H). C NMR
): δ = 178.70, 177.79, 174.20, 173.52, 164.03, 162.95, 162.45, 154.31, 154.06,
(100 MHz, CDCl
3
1
1
6
49.54, 149.24, 138.74, 138.50, 135.57, 134.52, 130.93, 130.40, 129.57, 129.48, 128.80, 128.42,
28.05, 127.66, 127.30, 123.68, 123.26, 123.16, 122.51, 116.83, 109.63, 109.12, 62.20, 61.62,
+
1.54, 14.27. HR-ESI-MS: Calcd for C21
H
16
N
2
O
6
[M+Na] : 415.0906, found: 415.0904.
4.5.9. Ethyl 8-amino-5,12-dioxo-5,12-dihydroindolizino[2,3-g]quinoline-6-carboxylate (3h) and
ethyl 9-amino-5,12-dioxo-5,12-dihydroindolizino[3,2-g]quinoline-11-carboxylate (3h’) mixture
(3h/3h’ = 84/16)
1
Purple solid; yield 36%; m.p. = 275-276°C; H NMR (400 MHz, DMSO-d
6
) δ 9.48 (d, J = 7.5 Hz,
0
7
7
1
.84H), 9.39 (d, J = 7.6 Hz, 0.16H), 8.96–8.87 (m, 1H), 8.44–8.33 (m, 1H), 7.80–7.70 (m, 1H),
.24–7.18 (m, 1H), 6.87 (dd, J = 7.5, 2.4 Hz, 1H), 6.82 (s, 2H), 4.32 (q, J = 7.1 Hz, 2H), 1.37 (t, J =
1
3
.1 Hz, 3H). C NMR (100 MHz, DMSO-D6) δ 179.78, 170.73, 163.51, 154.02, 150.90, 150.04,
43.63, 134.79, 130.67, 129.85, 128.97, 127.11, 122.34, 111.24, 100.37, 96.46, 60.27, 14.73.
+
HR-ESI-MS: Calcd for C18
H
13
N
3
O
4
[M+H] : 336.0984, found: 336.0974.
4.5.10. Methyl 5,12-dioxo-5,12-dihydroindolizino[2,3-g]quinoline-6-carboxylate (3i)
1
3
Orange solid; yield 30%; m.p. = 235-236°C; H NMR (400 MHz, CDCl ): δ = 9.95 (d, J = 6.9 Hz,
1
H), 9.04 (d, J = 3.9 Hz, 1H), 8.56 (d, J = 7.7 Hz, 1H), 8.38 (d, J = 9.0 Hz, 1H), 7.72–7.59 (m, 1H),
.52 (t, J = 7.9 Hz, 1H), 7.30–7.22 (m, 1H), 4.05 (s, 3H). Lit. [19]
7
4
.5.11. Methyl 5,12-dioxo-5,12-dihydroindolizino[3,2-g]quinoline-11-carboxylate (3i’)
1
Orange solid; yield 11%; m.p. = 255-256°C; H NMR (400 MHz, CDCl
3
): δ = 9.79 (d, J = 7.0 Hz,
1
H), 9.01 (dd, J = 4.5, 1.4 Hz, 1H), 8.56 (dd, J = 7.8, 1.4 Hz, 1H), 8.38 (d, J = 9.1 Hz, 1H), 7.67 (dd, J
7.8, 4.6 Hz, 1H), 7.54–7.42 (m, 1H), 7.23 (t, J = 6.8 Hz, 1H), 4.04 (s, 3H). Lit. [19]
=
4
.5.12. Ethyl 7-fluoro-5,12-dioxo-5,12-dihydroindolizino[2,3-g]quinoline-6-carboxylate (3j) &
18

ethyl 10-fluoro-5,12-dioxo-5,12-dihydroindolizino[3,2-g]quinoline-11-carboxylate (3j’) mixture
(
3j/3j’ = 45/55)
1
Orange solid; yield 28%; m.p. = 220-221°C; H NMR (400 MHz, CDCl
3
): δ = 9.64 (d, J = 6.4 Hz,
0
.44H), 9.50 (d, J = 6.2 Hz, 0.55H), 9.04 (d, J = 9.2 Hz, 1H), 8.64–8.52 (m, 1H), 7.79–7.64 (m, 1H),
.20–7.05 (m, 1H), 4.63–4.46 (m, 2H), 1.58–1.42 (m, 3H). Lit. [21]
7
4.5.13. 6-benzylindolizino[2,3-g]quinoline-5,12-dione (6e)
1
Red solid; yield 23%; m.p. = 170-171°C; H NMR (400 MHz, CDCl
3
): δ = 9.81 (d, J = 7.1 Hz, 1H),
9
4
1
1
1
.00 (dd, J = 4.6, 1.5 Hz, 1H), 8.50 (dd, J = 7.8, 1.4 Hz, 1H), 7.65 (d, J = 9.0 Hz, 1H), 7.58 (dd, J = 7.8,
.7 Hz, 1H), 7.31 (d, J = 7.3 Hz, 2H), 7.28–7.21 (m, 3H), 7.17 (t, J = 7.2 Hz, 1H), 7.11 (t, J = 6.8 Hz,
1
3
3
H), 4.59 (s, 2H). C NMR (100 MHz, CDCl ): δ = 182.13, 171.02, 154.03, 151.25, 139.80, 138.03,
34.87, 130.71, 128.71, 128.60, 128.38, 126.41, 126.27, 125.81, 125.72, 121.31, 119.31, 118.44,
+
17.77, 29.73. HR-ESI-MS: Calcd for C22
H
14
N
2
O
2
[M+H] : 339.1134, found: 339.1127.
4.5.14. 11-benzylindolizino[3,2-g]quinoline-5,12-dione (6e’)
1
Red solid; yield 15%; m.p. = 201-202°C; H NMR (400 MHz, CDCl
3
): δ = 9.64 (d, J = 6.9 Hz, 1H),
8
.94 (d, J = 3.7 Hz, 1H), 8.55 (d, J = 7.4 Hz, 1H), 7.68 (d, J = 9.0 Hz, 1H), 7.63 (dd, J = 7.7, 4.6 Hz,
H), 7.39 (d, J = 7.5 Hz, 2H), 7.28–7.20 (m, 3H), 7.15 (t, J = 7.3 Hz, 1H), 7.08 (t, J = 6.8 Hz, 1H), 4.60
1
1
3
(
s, 2H). C NMR (100 MHz, CDCl
3
): δ = 181.27, 171.38, 154.91, 152.88, 149.63, 139.95, 138.03,
1
1
3
34.83, 134.45, 132.20, 128.66, 128.54, 128.31, 127.67, 127.19, 126.37, 126.23, 125.59, 120.05,
+
19.56, 119.46, 117.68, 29.72. HR-ESI-MS: Calcd for C22
39.1139.
H
14
N
2
O
2
[M+H] : 339.1134, found:
4.5.15. 5,12-Dioxo-5,12-dihydroindolizino[2,3-g]quinoline-6-carboxylic acid (4a)
1
Red solid; yield 89%; m.p. = 269.5-271.2°C; H NMR (400 MHz, DMSO-d
6
): δ = 9.81 (s, 1H), 9.07
(s, 1H), 8.55 (d, J = 6.8 Hz, 2H), 7.81 (d, J = 52.2 Hz, 2H), 7.53 (s, 1H). Lit. [29]
4
9
4
.5.16. 5,12-Dioxo-5,12-dihydroindolizino[3,2-g]quinoline-11-carboxylic acid (4a’)
1
Red solid; yield 89%; m.p. = 269.5-271.2°C; H NMR (400 MHz, DMSO-d
6
): δ = 13.44 (s, 1H),
.68 (s, 1H), 9.01 (s, 1H), 8.51 (s, 2H), 7.89 (s, 1H), 7.71 (s, 1H), 7.50 (s, 1H). Lit. [29]
.5.17. 8-(Methylthio)-5,12-dioxo-5,12-dihydroindolizino[2,3-g]quinoline-6-carboxylic acid (4b)
1
Blue solid; yield 89%; m.p. = 295-296°C; H NMR (600 MHz, CDCl
3
): δ = 13.54 (s, 1H), 9.71 (d, J =
7
=
1
.3 Hz, 1H), 9.11 (d, J = 4.6 Hz, 1H), 8.61 (d, J = 7.7 Hz, 2H), 7.71 (dd, J = 7.6, 4.9 Hz, 1H), 7.17 (d, J
1
3
3
8.2 Hz, 1H), 2.67 (s, 3H). C NMR (150 MHz, CDCl ): δ = 185.45, 171.94, 162.61, 155.72, 150.00,
46.08, 141.97, 135.64, 129.32, 127.04, 125.59, 122.22, 118.80, 113.95, 104.21, 14.59.
+
HR-ESI-MS: Calcd for C17
H
10
N
2
O
4
S [M+Na] : 361.0259, found: 361.0264. The structure was
confirmed through H-H COSY and HMBC.
19

4.5.18. 9-(Methylthio)-5,12-dioxo-5,12-dihydroindolizino[3,2-g]quinoline-11-carboxylic acid (4b’)
1
Purple solid; yield 89%; m.p. = 302-303°C; H NMR (600 MHz, CDCl
3
): δ = 13.63 (s, 1H), 9.60 (d,
J = 7.3 Hz, 1H), 9.06 (dd, J = 4.6, 1.6 Hz, 1H), 8.63 (d, J = 1.8 Hz, 1H), 8.59 (dd, J = 7.8, 1.6 Hz, 1H),
1
3
7
3
.77 (dd, J = 7.8, 4.6 Hz, 1H), 7.16 (dd, J = 7.3, 2.1 Hz, 1H), 2.67 (s, 3H). C NMR (150 MHz, CDCl ):
δ = 184.40, 172.59, 162.63, 153.88, 148.29, 145.93, 142.06, 134.63, 131.40, 128.70, 126.69,
+
126.22, 121.21, 118.83, 114.05, 104.84, 14.58. HR-ESI-MS: Calcd for C17
H
10
N
2
O
4
S [M+Na] :
361.0259, found: 361.0264. The structure was confirmed through H-H COSY and HMBC.
4.5.19. 8-Methoxy-5,12-dioxo-5,12-dihydroindolizino[2,3-g]quinoline-6-carboxylic acid (4c)
1
Red solid; yield 86%; m.p. >320°C; H NMR (400 MHz, CDCl
3
): δ = 13.53 (s, 1H), 9.77 (d, J = 7.6
Hz, 1H), 9.10 (dd, J = 4.7, 1.7 Hz, 1H), 8.60 (dd, J = 7.9, 1.7 Hz, 1H), 8.28 (d, J = 2.8 Hz, 1H), 7.69
1
3
(
dd, J = 7.9, 4.7 Hz, 1H), 7.03 (dd, J = 7.6, 2.7 Hz, 1H), 4.04 (s, 3H). C NMR (150 MHz, CDCl
3
): δ =
1
1
3
85.46, 171.74, 162.89, 161.29, 155.64, 150.03, 144.64, 135.58, 129.63, 129.23, 126.94, 125.95,
+
21.95, 114.20, 104.12, 99.80, 56.38. HR-ESI-MS: Calcd for C17
23.0652.
H
10
N
2
O
5
[M+H] : 323.0668, found:
4.5.20. 9-Methoxy-5,12-dioxo-5,12-dihydroindolizino[3,2-g]quinoline-11-carboxylic acid (4c’)
1
Red solid; yield 87%; m.p. >320°C; H NMR (400 MHz, CDCl
3
): δ = 13.62 (s, 1H), 9.66 (d, J = 7.6
Hz, 1H), 9.04 (dd, J = 4.6, 1.7 Hz, 1H), 8.59 (dd, J = 7.9, 1.6 Hz, 1H), 8.29 (d, J = 2.7 Hz, 1H), 7.76
1
3
(
dd, J = 8.0, 4.7 Hz, 1H), 7.03–6.99 (m, 1H), 4.04 (s, 3H). C NMR (150 MHz, CDCl
3
): δ = 184.42,
1
1
72.33, 162.90, 161.17, 153.77, 148.26, 144.72, 134.55, 131.37, 129.26, 128.64, 126.59, 114.23,
+
04.76, 99.91, 56.40. HR-ESI-MS: Calcd for C17
H
10
N
2
O
5
[M+H] : 323.0668, found: 323.0665.
4.5.21. 8-Methyl-5,12-dioxo-5,12-dihydroindolizino[2,3-g]quinoline-6-carboxylic acid (4d)
1
Red solid; yield 89%; m.p. >320°C; H NMR (400 MHz, CDCl
3
): δ = 13.58 (s, 1H), 9.81 (d, J = 6.9
Hz, 1H), 9.11 (d, J = 4.1 Hz, 1H), 8.77 (s, 1H), 8.62 (d, J = 7.5 Hz, 1H), 7.71 (dd, J = 7.6, 4.6 Hz, 1H),
1
3
7
1
1
3
.21 (d, J = 7.0 Hz, 1H), 2.57 (s, 3H). C NMR (100 MHz, CDCl ): δ = 185.51, 171.97, 162.53,
55.75, 150.06, 142.48, 142.13, 135.70, 129.33, 127.80, 127.05, 125.48, 122.30, 122.01, 121.24,
+
05.19, 21.97. HR-ESI-MS: Calcd for C17
H
10
N
2
O
4
[M+H] : 307.0719, found: 307.0726.
4.5.22. 9-Methyl-5,12-dioxo-5,12-dihydroindolizino[3,2-g]quinoline-11-carboxylic acid (4d’)
1
Red solid; yield 89%; m.p. >320°C; H NMR (400 MHz, CDCl
3
): δ = 13.67 (s, 1H), 9.71 (d, J = 7.4
Hz, 1H), 9.06 (d, J = 3.2 Hz, 1H), 8.79 (s, 1H), 8.61 (d, J = 7.0 Hz, 1H), 7.77 (dd, J = 7.5, 5.1 Hz, 1H),
1
3
7
1
2
3
.20 (d, J = 7.6 Hz, 1H), 2.56 (s, 3H). C NMR (150 MHz, CDCl ): δ = 184.47, 172.62, 162.55,
53.89, 148.31, 142.30, 134.68, 131.48, 128.70, 127.47, 126.12, 122.25, 121.42, 121.00, 105.85,
+
1.94. HR-ESI-MS: Calcd for C17
H
10
N
2
O
4
[M+H] : 307.0719, found: 307.0723.
4
.5.23. 8-Iodo-5,12-dioxo-5,12-dihydroindolizino[2,3-g]quinoline-6-carboxylic acid (4e)
1
Red solid; yield 89%; m.p. >320°C; H NMR (400 MHz, CDCl
3
): δ = 13.53 (s, 1H), 9.64 (dd, J = 7.2,
20

0
7
1
1
4
.7 Hz, 1H), 9.46 (d, J = 1.0 Hz, 1H), 9.14 (dd, J = 4.7, 1.6 Hz, 1H), 8.64 (dd, J = 7.9, 1.7 Hz, 1H),
1
3
3
.74 (dd, J = 7.9, 4.7 Hz, 1H), 7.61 (dd, J = 7.3, 1.8 Hz, 1H). C NMR (150 MHz, CDCl ): δ = 185.22,
72.38, 161.96, 155.95, 149.82, 141.71, 135.84, 131.55, 129.40, 128.64, 128.03, 127.33, 125.21,
+
22.38, 121.59, 121.20, 105.39, 96.51. HR-ESI-MS: Calcd for C16
18.9529.
H
7
N
2
O
4
I [M+H] : 418.9529, found:
4.5.24. 9-Iodo-5,12-dioxo-5,12-dihydroindolizino[3,2-g]quinoline-11-carboxylic acid (4e’)
1
Red solid; yield 89%; m.p. >320°C; H NMR (400 MHz, DMSO-d
6
): δ = 9.42 (d, J = 7.2 Hz, 1H),
9
1
1
.05 (dd, J = 4.6, 1.6 Hz, 1H), 8.87 (s, 1H), 8.53 (dd, J = 7.8, 1.4 Hz, 1H), 7.92 (dd, J = 7.8, 4.7 Hz,
1
3
6
H), 7.76 (d, J = 6.2 Hz, 1H). C NMR (100 MHz, DMSO-d ): δ = 181.61, 173.75, 163.20, 154.06,
49.12, 140.38, 134.60, 131.10, 129.55, 128.94, 128.54, 127.69, 127.42, 122.02, 97.32.
+
HR-ESI-MS: Calcd for C16
H
7
N
2
O
4
I [M+H] : 418.9529, found: 418.9530.
4.5.25. 5,12-Dioxo-5,12-dihydroindolizino[2,3-g]quinoline-6,8-dicarboxylic
acid
(4f)
&
5,12-dioxo-5,12-dihydroindolizino[3,2-g]quinoline-9,11-dicarboxylic acid (4f’)
1
Red solid; yield 78%; m.p. >320°C; H NMR (400 MHz, DMSO-d
6
): δ = 9.69 (d, J = 7.5 Hz, 0.24H),
9
2
1
.63 (d, J = 7.2 Hz, 0.76H), 9.07–8.99 (m, 1H), 8.89 (s, 1H), 8.53 (d, J = 7.7 Hz, 1H), 7.93–7.79 (m,
1
3
6
H). C NMR (150 MHz, DMSO-d ): δ = 181.99, 173.54, 163.30, 155.03, 153.90, 149.94, 149.18,
40.28, 135.50, 134.61, 131.36, 128.88, 127.88, 127.72, 127.34, 121.87, 121.26, 119.94.
-
HR-ESI-MS: Calcd for C17
H
8
N
2
O
6
[M-H] : 335.0304, found: 335.0298.
4.5.26. N-(2-(dimethylamino)ethyl)-5,12-dioxo-5,12-dihydroindolizino[2,3-g]quinoline-6-carboxa
mide (5a)
1
Red solid; yield 86%; m.p. = 169-170°C; H NMR (400 MHz, DMSO-d
6
): δ = 9.89 (t, J = 5.0 Hz,
1H), 9.77 (d, J = 6.9 Hz, 1H), 9.01 (d, J = 4.3 Hz, 1H), 8.76 (d, J = 9.1 Hz, 1H), 8.43 (d, J = 7.7 Hz, 1H),
7.80 (dd, J = 7.7, 4.7 Hz, 1H), 7.68–7.52 (m, 1H), 7.44 (t, J = 6.8 Hz, 1H), 3.55 (dd, J = 11.8, 5.9 Hz,
2H), 2.73 (s, 2H), 2.42 (s, 6H). Lit. [29]
4.5.27. N-(2-(dimethylamino)ethyl)-5,12-dioxo-5,12-dihydroindolizino[3,2-g]quinoline-11-carboxa
mide (5a’)
Red solid; yield 86%; m.p. = 169-170°C; H NMR (400 MHz, CDCl
1
3
): δ = 10.35 (s, 1H), 9.86 (d, J =
7.0 Hz, 1H), 9.10 (d, J = 9.2 Hz, 1H), 9.01 (d, J = 4.5 Hz, 1H), 8.57 (d, J = 7.8 Hz, 1H), 7.72 (dd, J =
7.7, 4.7 Hz, 1H), 7.59–7.45 (m, 1H), 7.25 (d, J = 6.8 Hz, 1H), 3.73 (dd, J = 12.4, 6.3 Hz, 2H), 2.81 (t, J
= 6.7 Hz, 2H), 2.45 (s, 6H). Lit. [29]
4.5.28. N-(2-(dimethylamino)ethyl)-8-(methylthio)-5,12-dioxo-5,12-dihydroindolizino[2,3-g]quino
line-6-carboxamide (5b)
1
Purple solid; yield 88%; m.p. = 241-242°C; H NMR (400 MHz, MeOD): δ = 9.39 (d, J = 6.4 Hz,
1
H), 8.95 (s, 1H), 8.21 (s, 1H), 7.93 (d, J = 7.9 Hz, 1H), 7.59–7.50 (m, 1H), 7.25 (d, J = 4.4 Hz, 1H),
21

1
3
3
1
1
4
.94 (s, 2H), 3.68–3.62 (m, 2H), 3.22 (s, 6H), 2.65 (s, 3H). C NMR (100 MHz, MeOD): δ = 181.00,
70.67, 162.70, 154.67, 148.41, 146.00, 140.60, 134.54, 128.71, 127.61, 126.62, 124.54, 120.71,
+
18.42, 114.15, 107.48, 58.01, 43.12, 34.25, 13.04. HR-ESI-MS: Calcd for C21
09.1334, found: 409.1328.
H
20
N
4
O
3
S [M+H] :
4.5.29. N-(2-(dimethylamino)ethyl)-9-(methylthio)-5,12-dioxo-5,12-dihydroindolizino[3,2-g]quino
line-11-carboxamide (5b’)
1
Purple solid; yield 88%; m.p. = 247-248°C; H NMR (400 MHz, MeOD): δ = 9.06 (d, J = 7.3 Hz,
1H), 8.90 (d, J = 3.3 Hz, 1H), 8.08 (d, J = 8.1 Hz, 1H), 7.81 (s, 1H), 7.76–7.67 (m, 1H), 7.03 (d, J = 8.4
1
3
Hz, 1H), 3.89 (s, 2H), 3.67–3.61 (m, 2H), 3.25 (s, 6H), 2.50 (s, 3H). C NMR (100 MHz, MeOD): δ =
181.25, 170.82, 163.16, 153.38, 147.20, 145.02, 140.18, 133.90, 129.65, 128.82, 125.93, 123.77,
20 4 3
120.14, 118.08, 113.31, 105.87, 58.60, 43.04, 34.19, 13.00. HR-ESI-MS: Calcd for C21H N O S
+
[
M+H] : 409.1334, found: 409.1336.
4.6. Synthesis of compound 6a,a’-6c,c’’
4a or 4a’ (1.0 mmol) was dissolved in DCM and HBTU (1.5 mmol), DIPEA (1.5 mmol) were
added to the solution. The mixture was stirred at room temperature for 1h. Then different
anilines (1.5 mmol) were added and the reaction mixture was stirred for 5h. The solvent was
evaporated under reduced pressure. The crude product was purified by silica gel column
chromatography to give the target compounds 6a-c and 6a’-c’.
4
.6.1. 5,12-Dioxo-N-phenyl-5,12-dihydroindolizino[2,3-g]quinoline-6-carboxamide (6a)
1
Red solid; yield 85%; m.p. >320°C; H NMR (400 MHz, CDCl
3
): δ = 12.35 (s, 1H), 10.06 (d, J = 7.7
Hz, 1H), 9.25 (d, J = 8.7 Hz, 1H), 9.09 (d, J = 5.0 Hz, 1H), 8.66 (d, J = 7.7 Hz, 1H), 7.93 (d, J = 8.5 Hz,
H), 7.73–7.68 (m, 1H), 7.56 (t, J = 8.4 Hz, 1H), 7.43 (t, J = 7.8 Hz, 2H), 7.33 (t, J = 6.7 Hz, 1H), 7.17
2
1
3
(
t, J = 7.5 Hz, 1H). C NMR (150 MHz, CDCl
3
): δ = 184.07, 172.55, 160.82, 155.16, 149.80, 141.66,
1
1
38.80, 135.75, 130.17, 129.05, 128.99, 128.28, 126.98, 124.39, 124.11, 123.70, 122.50, 120.51,
+
19.30, 110.96. HR-ESI-MS: Calcd for C22
H
13
N
3
O
3
[M+Na] : 390.0855, found: 390.0854.
4
.6.2. 5,12-Dioxo-N-phenyl-5,12-dihydroindolizino[3,2-g]quinoline-11-carboxamide (6a’)
1
Red solid; yield 86%; m.p. >320°C; H NMR (400 MHz, CDCl
3
): δ = 12.43 (s, 1H), 9.93 (d, J = 5.9
Hz, 1H), 9.25 (d, J = 8.9 Hz, 1H), 9.06 (d, J = 4.5 Hz, 1H), 8.61 (d, J = 8.7 Hz, 1H), 7.98 (d, J = 8.0 Hz,
H), 7.77–7.71 (m, 1H), 7.53 (d, J = 8.3 Hz, 1H), 7.39 (t, J = 7.1 Hz, 2H), 7.33–7.27 (m, 1H), 7.15 (t,
2
1
3
J = 7.5 Hz, 1H). C NMR (100 MHz, CDCl
3
): δ = 183.13, 173.08, 160.70, 153.82, 148.93, 141.74,
1
1
38.83, 134.56, 131.00, 128.96, 128.84, 128.20, 127.94, 124.93, 124.04, 123.92, 120.48, 119.25,
+
13 3 3
11.67. HR-ESI-MS: Calcd for C22H N O [M+Na] : 390.0855, found: 390.0848.
4
.6.3. 5,12-Dioxo-N-(o-tolyl)-5,12-dihydroindolizino[2,3-g]quinoline-6-carboxamide (6b)
1
Red solid; yield 90%; m.p. = 250-251°C; H NMR (400 MHz, CDCl
3
): δ = 11.73 (s, 1H), 10.00 (d, J
22

=
7.1 Hz, 1H), 9.17 (d, J = 8.9 Hz, 1H), 9.05 (d, J = 3.3 Hz, 1H), 8.58 (d, J = 7.8 Hz, 1H), 7.99 (d, J =
.4 Hz, 1H), 7.65 (dd, J = 7.5, 4.5 Hz, 1H), 7.51 (t, J = 7.4 Hz, 1H), 7.31–7.22 (m, 3H), 7.13 (t, J = 7.4
8
1
3
Hz, 1H), 2.52 (s, 3H). C NMR (100 MHz, CDCl
3
): δ = 183.53, 172.46, 161.09, 155.02, 149.76,
141.76, 136.30, 135.66, 130.64, 130.08, 128.91, 128.15, 126.88, 126.39, 125.18, 124.52, 124.37,
+
123.78, 122.37, 119.20, 110.54, 18.67. HR-ESI-MS: Calcd for C23
H
15
N
3
O
3
[M+Na] : 404.1011,
found: 404.1015.
4.6.4. 5,12-Dioxo-N-(o-tolyl)-5,12-dihydroindolizino[3,2-g]quinoline-11-carboxamide (6b’)
1
Red solid; yield 86%; m.p. = 242-243°C; H NMR (400 MHz, CDCl
3
): δ = 11.75 (s, 1H), 9.88 (d, J =
6.9 Hz, 1H), 9.18 (d, J = 9.2 Hz, 1H), 9.02 (d, J = 4.8 Hz, 1H), 8.57 (d, J = 7.8 Hz, 1H), 7.99 (d, J = 7.9
Hz, 1H), 7.69 (dd, J = 7.8, 4.7 Hz, 1H), 7.49 (t, J = 7.1 Hz, 1H), 7.30–7.22 (m, 3H), 7.12 (t, J = 7.4 Hz,
1
3
1
1
1
4
3
H), 2.58 (s, 3H). C NMR (100 MHz, CDCl ): δ = 182.55, 172.99, 161.07, 153.78, 148.89, 141.85,
36.23, 134.47, 130.97, 130.82, 130.62, 128.76, 128.04, 127.84, 126.23, 125.15, 124.36, 123.99,
+
21.28, 119.13, 111.21, 18.90. HR-ESI-MS: Calcd for C23
04.1011.
H
15
N
3
O
3
[M+Na] : 404.1011, found:
4.6.5. N-(3-chlorophenyl)-5,12-dioxo-5,12-dihydroindolizino[2,3-g]quinoline-6-carboxamide (6c)
1
Red solid; yield 83%; m.p. >320°C; H NMR (400 MHz, CDCl
3
): δ = 12.45 (s, 1H), 10.07 (d, J = 7.4
Hz, 1H), 9.23 (d, J = 9.1 Hz, 1H), 9.11 (dd, J = 4.9, 1.6 Hz, 1H), 8.67 (dd, J = 7.7, 1.9 Hz, 1H), 8.06 (t,
J = 1.8 Hz, 1H), 7.78 (d, J = 8.4 Hz, 1H), 7.72 (dd, J = 8.1, 4.6 Hz, 1H), 7.61–7.55 (m, 1H), 7.37–7.31
1
3
(
m, 2H), 7.14 (d, J = 8.0 Hz, 1H). C NMR (150 MHz, CDCl
3
): δ = 184.21, 172.59, 160.91, 155.26,
1
1
4
49.74, 141.68, 140.03, 135.78, 134.68, 130.11, 129.99, 129.22, 128.33, 127.05, 124.36, 124.05,
+
23.56, 122.58, 120.43, 119.38, 118.36, 110.44. HR-ESI-MS: Calcd for C22
24.0465, found: 424.0464.
H
12
N
3
O
3
Cl [M+Na] :
4.6.6. N-(3-chlorophenyl)-5,12-dioxo-5,12-dihydroindolizino[3,2-g]quinoline-11-carboxamide
(6c’)
1
Red solid; yield 81%; m.p. >320°C; H NMR (400 MHz, CDCl
3
): δ = 12.56 (s, 1H), 9.95 (d, J = 7.0
Hz, 1H), 9.24 (d, J = 9.5 Hz, 1H), 9.08 (dd, J = 4.5, 1.7 Hz, 1H), 8.63 (dd, J = 7.8, 1.7 Hz, 1H), 8.13 (t,
J = 2.1 Hz, 1H), 7.86 (d, J = 8.1 Hz, 1H), 7.77 (dd, J = 7.8, 4.6 Hz, 1H), 7.60–7.54 (m, 1H), 7.35–7.28
1
3
(
m, 2H), 7.12 (d, J = 7.4 Hz, 1H). C NMR (150 MHz, CDCl
3
): δ = 183.29, 173.14, 160.81, 153.90,
1
1
48.85, 141.77, 140.02, 134.68, 134.61, 130.97, 129.92, 129.10, 128.33, 128.01, 124.91, 124.01,
+
23.78, 121.51, 120.36, 119.36, 118.33, 111.16. HR-ESI-MS: Calcd for C22
H
12
N
3
O
3
Cl [M+Na] :
424.0465, found: 424.0464.
4.7. Synthesis of compound 6d and 6d’
4a or 4a’ (1.0 mmol) was dissolved in DCM and HBTU (1.5 mmol), DIPEA (1.5 mmol) were
added to the solution. The mixture was stirred at room temperature for 1h. Then
ethyl-m-aminobenzoate (1.5 mmol) was added and the reaction mixture was stirred for 5h. The
23

solvent was evaporated under reduced pressure. The crude product was purified by silica gel
column chromatography to give the target compound 8b and 8b’. The ethyl ester group of 8b and
8
b’ was hydrolyzed using KOH in MeOH/H
2
O. When the hydrolysis was complete, the reaction
mixture was filtrated and washed with CH
2
Cl and H O, followed by recrystallization in methanol
2
2
to obtain 6d and 6d’.
4.7.1. Ethyl 3-(5,12-dioxo-5,12-dihydroindolizino[2,3-g]quinoline-6-carboxamido)benzoate (8b)
1
Red solid; yield 90%; m.p. >320°C; H NMR (400 MHz, CDCl
3
): δ = 12.44 (s, 1H), 10.00 (d, J = 7.0
Hz, 1H), 9.18 (d, J = 9.2 Hz, 1H), 9.08 (dd, J = 4.5, 1.3 Hz, 1H), 8.64 (dd, J = 7.9, 1.3 Hz, 1H), 8.43 (s,
1
H), 8.19 (d, J = 8.5 Hz, 1H), 7.83 (d, J = 7.7 Hz, 1H), 7.69 (dd, J = 7.9, 4.7 Hz, 1H), 7.54 (t, J = 7.6 Hz,
H), 7.47 (t, J = 7.9 Hz, 1H), 7.31 (t, J = 6.6 Hz, 1H), 4.44 (q, J = 7.1 Hz, 2H), 1.45 (t, J = 7.1 Hz, 3H).
1
1
3
3
C NMR (100 MHz, CDCl ): δ = 184.02, 172.55, 166.48, 160.91, 155.18, 149.70, 141.61, 139.01,
1
1
4
35.81, 131.38, 130.11, 129.12, 129.06, 128.26, 127.04, 125.09, 124.76, 124.33, 123.57, 122.52,
+
21.29, 119.33, 110.44, 61.14, 14.44. HR-ESI-MS: Calcd for C25
62.4322.
H
17
N
3
O
5
[M+Na] : 462.4319, found:
4.7.2. Ethyl 3-(5,12-dioxo-5,12-dihydroindolizino[3,2-g]quinoline-11-carboxamido)benzoate (8b’)
1
Red solid; yield 91%; m.p. >320°C; H NMR (400 MHz, CDCl
3
): δ = 12.46 (s, 1H), 9.90 (d, J = 7.0
Hz, 1H), 9.21 (d, J = 9.2 Hz, 1H), 9.05 (dd, J = 4.6, 1.6 Hz, 1H), 8.60 (dd, J = 7.8, 1.6 Hz, 1H), 8.53 (s,
1H), 8.19 (d, J = 8.2 Hz, 1H), 7.83 (d, J = 7.7 Hz, 1H), 7.74 (dd, J = 7.8, 4.6 Hz, 1H), 7.53 (t, J = 7.4 Hz,
1H), 7.45 (t, J = 7.9 Hz, 1H), 7.30 (d, J = 6.9 Hz, 1H), 4.44 (q, J = 7.1 Hz, 2H), 1.45 (t, J = 7.1 Hz, 3H).
1
3
3
C NMR (100 MHz, CDCl ): δ = 183.16, 173.11, 166.46, 160.94, 153.84, 148.89, 141.80, 138.91,
1
1
4
34.63, 131.46, 131.00, 129.04, 128.92, 128.30, 127.99, 125.31, 124.94, 123.88, 121.47, 121.31,
+
19.33, 111.19, 61.08, 14.48. HR-ESI-MS: Calcd for C25
62.4324.
H
17
N
3
O
5
[M+Na] : 462.4319, found:
4.7.3. Potassium 3-(5,12-dioxo-5,12-dihydroindolizino[2,3-g]quinoline-6-carboxamido)benzoate
(6d)
1
Red solid; yield 82%; m.p. >320°C; H NMR (400 MHz, DMSO-d
6
): δ = 11.87 (s, 1H), 9.88 (d, J =
6
8
=
1
1
4
.8 Hz, 1H), 9.06 (d, J = 4.1 Hz, 1H), 8.87 (d, J = 7.6 Hz, 1H), 8.70 (d, J = 7.9 Hz, 1H), 8.07 (s, 1H),
.02 (d, J = 8.5 Hz, 1H), 7.89–7.82 (m, 1H), 7.69 (t, J = 8.0 Hz, 1H), 7.60 (d, J = 8.5 Hz, 1H), 7.51 (t, J
1
3
6
6.8 Hz, 1H), 7.28 (t, J = 8.0 Hz, 1H). C NMR (100 MHz, DMSO-d ): δ = 184.03, 172.89, 168.24,
60.74, 155.05, 149.78, 143.27, 140.40, 138.21, 135.96, 130.78, 129.59, 128.22, 127.93, 125.10,
-
22.77, 122.48, 121.00, 119.96, 119.78, 115.05, 110.11. HR-ESI-MS: Calcd for C23
10.0777, found: 410.0785.
H
12
N
3
O
5
K [M-K] :
4.7.4. Potassium 3-(5,12-dioxo-5,12-dihydroindolizino[3,2-g]quinoline-11-carboxamido)benzoate
(
6d’)
1
Red solid; yield 80%; m.p. >320°C; H NMR (400 MHz, DMSO-d
6
): δ = 11.86 (s, 1H), 9.89 (d, J =
24

7
8
7
1
1
.1 Hz, 1H), 9.06 (d, J = 5.2 Hz, 1H), 8.88 (d, J = 8.9 Hz, 1H), 8.70 (d, J = 7.7 Hz, 1H), 8.06 (s, 1H),
.02 (d, J = 8.1 Hz, 1H), 7.86 (dd, J = 7.9, 4.7 Hz, 1H), 7.69 (t, J = 8.3 Hz, 1H), 7.60 (d, J = 7.6 Hz, 1H),
1
3
6
.51 (t, J = 6.8 Hz, 1H), 7.27 (t, J = 7.6 Hz, 1H). C NMR (100 MHz, DMSO-d ): δ = 184.01, 172.87,
68.22, 160.71, 155.03, 149.76, 143.29, 140.37, 138.18, 135.93, 130.76, 127.90, 125.07, 122.75,
-
22.45, 120.98, 119.91, 119.77, 110.09. HR-ESI-MS: Calcd for C23
H
12
N
3
O
5
K [M-K] : 410.0777,
found: 410.0774.
4.8. Synthesis of compound 5c
A mixture of 4a (1.0 mmol) and N,N'-carbonyldiimidazole (2.0 mmol) in DMF was stirred at
room temperature for 1h. The reaction mixture was heated at 70°C for 40 minutes.
Thiosemicarbazide (3.0 mmol) was added and the reaction mixture was stirred at 70°C for
another 30 minutes. The solvent was evaporated under reduced pressure. 1M HCl was added and
the solid was filtrated and washed with water to give the target compound 5c. Lit. [18]
4
.8.1. 2-(5,12-Dioxo-5,12-dihydroindolizino[2,3-g]quinoline-6-carbonyl)hydrazine-1-carbothioami
de (5c)
Red solid; yield 55%; m.p. = 270-271°C; H NMR (400 MHz, DMSO-d
J = 6.9 Hz, 1H), 9.65 (s, 1H), 9.10 (d, J = 4.6 Hz, 1H), 8.58 (d, J = 7.9 Hz, 1H), 8.51 (s, 1H), 8.22 (s,
1
6
): δ = 11.19 (s, 1H), 9.86 (d,
1
3
1
H), 7.89 (dd, J = 7.8, 4.6 Hz, 1H), 7.83 (s, 1H), 7.72 (t, J = 7.3 Hz, 1H), 7.53 (t, J = 6.9 Hz, 1H). C
NMR (150 MHz, DMSO-d ): δ = 183.14, 182.85, 172.66, 162.08, 155.10, 150.19, 149.80, 139.76,
35.66, 130.58, 129.64, 128.29, 127.98, 125.68, 122.46, 121.48, 119.68, 107.11. HR-ESI-MS:
6
1
-
11 5 3
Calcd for C17H N O S [M-H] : 364.0504, found: 364.0496.
4.9. IDO1 inhibition assay
The assays were performed at room temperature as described in the literature using 20 nM
IDO and 2 mM D-Trp in the presence of 20 mM ascorbate, 3.5 μM methylene blue and 0.2 mg/mL
catalase in 50 mM potassium phosphate buffer (pH 6.5). The initial reaction rates were recorded
by continuously following the absorbance increase at 321 nm due to the formation of
N’-formlylkynurenine. The IC50 values were calculated using nonlinear regression with normalized
dose-response fit using Prism GraphPad [20].
4.10. TDO inhibition assay
The assay was performed by UV absorption using recombinant TDO and L-Tryptophan as
substrate. The UV absorption signal at 321 nm is correlated with the amount of
N-formylkynurenine reaction product of TDO. All reactions were conducted at room temperature.
The 100 µL reaction mixture in TDO Assay Buffer contained 50 mM TDO, the indicated amount of
the inhibitor, 200 µM tryptophan. The reaction mixture was incubated for 75 min prior to reading
the UV absorption signal. For the negative control (blank), 5 µL of the assay buffer was added
instead of the TDO. The initial reaction rates were recorded by continuously following the
25

absorbance increase at 321 nm due to the formation of N’-formylkynurenine. The IC50 values
were calculated using nonlinear regression with normalized dose−response fit using Prism
GraphPad [30].
4.11. MTT assay of cell viability
Human cancer cells [human cervical carcinoma cell (HeLa), breast carcinoma cell (MCF-7), and
3
embryonic kidney cells (HEK293T)] were seeded in each well of 96-well culture plate (3-4×10
per well). After overnight incubation, solution of compounds in DMSO (10 mM) was added to the
cells. Cells treated with DMSO 1% were used as negative control. After further incubation for 48 h,
MTT solution (5 mg/mL in PBS) was added to each well. After incubation for 4 h at 37 °C, MTT
solution was removed and 150 μL of DMSO was added to dissolve the crystals formed. Then,
absorbance at 570 nm was read using a microplate reader. The IC50 values were calculated using
Prism 5.0.
4.12. Cellular inhibition assay
HeLa Cells were seeded on 96-well plates (40,000 cells per well) and allowed to adhere for 24
hours. A final concentration of 100 ng/mL of human recombinant IFN-γ was added to the cells to
stimulate IDO1 expression. After 24 hours, cells were then treated with IDO inhibitor in DMSO at
a top dose of 10 μM (3-fold dilution all the way down to 0.1μM). Treated cells were then
incubated for 24 hours. At the end of the incubation, reactions were terminated by the addition
of 30% TCA to each well. Plate were incubated for 30 minutes at 50C. Cells were centrifuged 10
minutes at 4200 rpm. 100 μL of supernatants were transferred to new 96-well plates and mixed
with Ehrlich reagent. The resulting solution was incubated 10 minutes at room temperature.
Absorbance at 490 nm was read and the results were calculated using Prism GraphPad.
Acknowledgments
We gratefully acknowledge the National Natural Science Foundation of China (NSFC No.
81573272) and Beijing Natural Science Foundation (No. 7162109) for generous financial
supports.
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2
28

Highlights



48 naphthoindolizine and indolizinoquinoline-5,12-dione derivatives were
synthesized.
These compounds showed significant IDO1 inhibition and high selectivity over
TDO.
The most potent 5c (IDO1 IC50 23 nM) is a low cytotoxic, promising lead
compound.
29