Discovery of 5-(pyridin-3-yl)-1H-indole-4,7-diones as indoleamine 2,3-dioxygenase 1 (IDO1) inhibitors

Article abstract of DOI:10.1016/j.bmcl.2019.126901

Early studies demonstrated that over expression of indoleamine 2,3-dioxygenase (IDO1) in tumor microenvironment results in tumor immune escape. Herein, in order to simplify the structure of two kinds of IDO1 inhibitors from marine alkaloid, Exiguamine A and Tsitsikammamines, we designed, synthesized a series of 1H-indole-4,7-dione derivatives and evaluated their inhibitory activity in IDO1 enzyme and in IFN-γ stimulated Hela cells in vitro. The structure-activity relationship demonstrated that 5-(pyridin-3-yl)-1H-indole-4,7-dione is a promising scaffold for IDO1 inhibitors and most compounds with this core showed moderate inhibition potency at micromole level. Our further enzyme kinetics experiments reveal that these new developed compounds might act as reversible competitive inhibitors of IDO1.

Full text of DOI:10.1016/j.bmcl.2019.126901

Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of 5-(pyridin-3-yl)-1H-indole-4,7-diones as indoleamine 2,3-
dioxygenase 1 (IDO1) inhibitors
Kai-min Kong, Jia-wei Zhang, Bing-zhi Liu, Guang-rong Meng, Qian Zhang⁎
Department of Medicinal Chemistry, School of Pharmacy, Fudan University, Shanghai 201203, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Early studies demonstrated that over expression of indoleamine 2,3-dioxygenase (IDO1) in tumor micro-
environment results in tumor immune escape. Herein, in order to simplify the structure of two kinds of IDO1
inhibitors from marine alkaloid, Exiguamine A and Tsitsikammamines, we designed, synthesized a series of 1H-
indole-4,7-dione derivatives and evaluated their inhibitory activity in IDO1 enzyme and in IFN-γ stimulated Hela
cells in vitro. The structure-activity relationship demonstrated that 5-(pyridin-3-yl)-1H-indole-4,7-dione is a
promising scaffold for IDO1 inhibitors and most compounds with this core showed moderate inhibition potency
at micromole level. Our further enzyme kinetics experiments reveal that these new developed compounds might
act as reversible competitive inhibitors of IDO1.
Indoleamine 2,3-dioxygenase 1
Cancer immunotherapy
Marine alkaloid
1H-indole-4,7-diones
Reversible competitive inhibitor
Tryptophan, one of the essential amino acids in humans, can be
metabolized into a series of biologically active metabolites, such as
neurotransmitters serotonin and melatonin, kynurenic acid, as well as
the final production of nicotinamide adenine dinucleotide (NAD),
which can regulate human’s immune system, central nervous system,
gastrointestinal nervous system and intestinal microflora.1,2 In the
solely or combined with chemotherapeutic agents or established im-
munotherapeutic strategies, like anti-PD1/PD-L1 therapy for treating
several solid tumors.18
Exiguamine A, one classic type of marine alkaloid separated from
marine sponge Neopetrosia exigua, has been demonstrated as the most
potent IDO1 inhibitor among all natural inhibitors with a remarkable Ki
tryptophan-nicotinamide pathway, indoleamine 2,3-dioxygenase
1
of 41
3 nM.19 Meanwhile, some derivatives of Tsitsikammamine A,
(IDO1) participates in the first step and catalyzes the formation of N-
formylkynurnine by promoting the oxidative cleavage of indole 2,3-
double bonds. This step is believed to be the rate-determining step of
the whole catalytic cycle. In this regard, overexpression of IDO1 can
result in depletion of tryptophan and accumulation of different bioac-
tive metabolites, which are reported to lead to neurodegenerative dis-
orders (Alzheimer’s disease), age-related cataract, HIV encephalitis,
numerous tumor types, and especially tumor immunosuppression.3,4 It
has been considered that the depletion of tryptophan in micro-
environment can activate general control nonderepressible 2 kinase
(GCN2) pathway, which is closely related to both proliferation and
functional work of immune cells, and the production of kynurenine and
other metabolites, which can serve as aryl hydrocarbon receptor (AHR)
agonists, can result in T cell apoptosis.5–7
which was obtained from sponge Latrunculiidae, were reported to per-
form comparable IDO1 inhibitory activity as well20 (Fig. 1). Occasion-
ally, these two natural products share same core structure 3-ami-
noethyl-1H-indole-4,7-dione, when the carbon-nitrogen double bond of
Tsitsikammamines A is hydrolyzed. This interesting phenomenon and
our long research interest led us starting investigation on IDO1 in-
hibitors. In this context, we designed and synthesized a series of very
simple 3-aminoethyl-1H-indole-4,7-dione derivatives and conducted
extensive studies on their inhibitory activity in IDO1 enzyme.
Synthesis of designed compounds
As described in Scheme 1, the indole core was synthesized in four
steps employed 2,5-dimethoxybenzaldehyde (1) as starting material,
which is commercially available (CAS: 93-02-7). 1 was converted into
3,6-dimethoxy-2-nitrobenzaldehyde (2) under the condition of HNO3/
AcOH, then the nitro-group of 2 was reduced with ferric and hydro-
chloric acid into amino-group (3), which guided the following bromi-
nation that occurred at 5-position affording compound 4. The first three
Recent years have witnessed the continuous efforts to develop IDO1
inhibitors are ongoing in academia and pharmaceutical industries all
over the world.8–12 Inspiringly, several IDO1 inhibitors have advanced
into clinical trials, such as 1-MT,13 BMS-986205,14 PF-0684003,15 GDC-
091916 and INCB024360.17 These IDO1 inhibitors are being tested
⁎ Corresponding author.
E-mail address: zhangqian511@shmu.edu.cn (Q. Zhang).
https://doi.org/10.1016/j.bmcl.2019.126901
Received 5 September 2019; Received in revised form 21 November 2019; Accepted 9 December 2019
0960-894X/©2019ElsevierLtd.Allrightsreserved.
Pleasecitethisarticleas:Kai-minKong,etal.,Bioorganic&MedicinalChemistryLetters,https://doi.org/10.1016/j.bmcl.2019.126901

K.-m. Kong, et al.
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxxx
All of the target compounds were screened preliminarily for IDO1 in-
hibitory activity at the concentration of 10 μM with three duplicate
wells and Epacadostat was used as a reference compound. For the
compounds with percent inhibition more than 50% at 10 μM, their
median inhibition concentration values (IC50) were further measured.24
As shown in Table 1, the enzymatic assay results of 14a-f, 5-phenyl-
1H-indole-4,7-diones, indicated that the position of the substituents in
benzene ring greatly affected the inhibitory activity. The compounds
with a substituent at 4-position (14c, 14e and 14f), regardless of
electronic withdrawing or donating group, displayed percent inhibition
at 10 μM less than 50%. Meanwhile, those without any substituents
(14a) or with substituents at 2-position (14b and 14d) exhibited per-
cent inhibition over 50% obviously and IC50 values at 2–3 μM. These
data suggested that the binding pocket maybe not large enough to ac-
commodate a group at 4-position of phenyl moiety.
Fig. 1. Structure of Exiguamine A and Tsitsikammamine A.
steps are achieved with good to excellent yields (72%, 92% and 76%
respectively). Subsequently, 2-amino-5-bromo-3,6-dimethox-
ybenzaldehyde (4) reacted with (trimethylsilyl)diazomethane in MeOH
to construct the pyrrole ring of indole derivative (5) in a yield of 76%.21
By Vilsmeier reaction, a formyl-group was introduced into 3-position of
the indole-ring to afford compound 6, which further reacted with ni-
tromethane to give the compound with a nitrovinyl side chain (7). After
protection of free NeH in indole-ring of compound 7 by reacting with
tosyl chloride, the carbon-carbon double bond of the side chain was
reduced by sodium borohydride with a yield of 73% to provide com-
pound 9. Then the isolated nitro-group was then reduced under the
condition of zinc powder to give 1-tosyl-3-aminoethyl-5-bromo-4,7-di-
methoxy-1H-indole (10). Without purification, the amino group was
protected by di-tert-butyl bicarbonate affording the crucial and stable
intermediate (11) with a total yield of 71% for these two steps.
The methoxy groups of 11 were oxidized by ceric ammonium ni-
trate at 0 °C to produce the quinone compound (12), which further
reacted with various phenylboronic acid namely Suzuki reaction
smoothly to provide 5-substituted 1-tosyl-3-(tert-butoxycarbonyl)ami-
noethyl-1H-indole-4,7-diones (13a-f) with the yields range from 41% to
88%.22 Desulfonylation of 13a-f by tetrabutylammonium fluoride af-
forded the target compounds (14a-f) in good yields as shown in Scheme
1.
5-(3-Pyridinyl)-1H-indole-4,7-dione (14h), which contains pyr-
idinyl group rather than phenyl ring, showed potential enzymatic in-
hibition with a percent inhibition of 66% at 10 μM and IC50 of
1.90
0.96 μM. Dramatically, the inhibition of 5-(4-pyridinyl)-1H-
indole-4,7-dione (14g) was reduced with a percent inhibition rate of
only 12.42% at same concentration. These facts revealed that the po-
sition of nitrogen atom in pyridine ring is very critical for bioactivity as
well.
The primary structure activity relationship of N-alkylating ami-
noethyl 1H-indole-4,7-diones (16a-h) demonstrated that the smaller
alkyls are favorable to the inhibitory activity against IDO1 enzyme.
Compound 16a, bearing N-ethyl aminoethyl side chain, exhibited the
most potent inhibition among all the screened compounds with IC50
value of 0.16
0.02 μM. The inhibitory activity dropped slightly
while the bulk of the alkyls became larger. Furthermore, compared with
16a, the compound with N,N-diethyl moiety (16h) was demonstrated
about 14-fold less potency (IC50 value of 2.26
1.81 μM), which
implied that a single substituent at the aminoethyl is beneficial to the
activity (Table 1).
However, the Suzuki coupling of compound 12 with pyridin-4-yl or
pyridin-3-yl boronic acid resulted in very low yields with the similar
protocol, possibly due to the high reactivity of this kind of boronic
acids. Hence, we adjusted the synthetic strategy with removing the
coupling reaction before the oxidation. Suzuki couplings of compound
11 with pyridin-4-yl and pyridin-3-yl boronic acid respectively were
performed firstly to give compounds 12g and 12h with good yields. The
reaction of 11 with pyridin-2-yl boronic acid was also tried, but the
aimed product was not detected after several attempts. We deduced the
main reason for this result might ascribe to the instability of pyridin-2-
yl boronic acid under this condition. After oxidation of dimethoxy-
group and deprotection of 1-tosyl, two target compounds with 5-pyr-
idinyl substituted (14g and 14h) were obtained as well (Scheme 1).
In Scheme 2, we modified the side chain of 3-(2-(tert-butox-
ycarbonyl)aminoethyl)-5-(3-pyridyl)-1H-indole-4,7-dione (14h), which
expressed potent inhibition against IDO1 in enzymatic assay (see
Table 1). The reaction of 14h with trifluoroacetic acid gave the com-
pound with a side chain of 2-aminoethyl (15), which was utilized di-
rectly without further purification. Under condition of sodium triace-
toxyborohydride and various carbonyl compounds, the reductive
amination of compound 15 went smoothly and provided a series of N-
alkylation products (16a-h).23 Meanwhile, a new type of N-acylation
products (17a-d) were also obtained by reaction of compound 15 with
corresponding acyl chlorides or sulfonyl chlorides.
Effective activity in Hela cell stimulated by IFN-γ
The cellular IDO1 inhibitory activity (EC50) of all 5-(3-pyridinyl)-
1H-indole-4,7-diones (14h, 15 and 16a-h) was further tested in Hela
cell lines, of which the native human IDO1 is overexpressed by stimu-
lation of IFN-γ,25 and Epacadostat was also used as a reference com-
pound. As shown in Table 1, the result demonstrated that most of these
compounds show moderate IDO1 inhibition activity in this cellular
assay with EC50 values at micromole level, correlated with those data
that determined in enzyme assay. However, the potency of the tested
compounds in cellular assays displayed lower IDO1 inhibitory activity
than in enzymatic assay, which might result from the effect of mem-
brane permeability, or the complexity of kynurenine pathway.
Determination of inhibition type
In order to investigate the inhibition type of this kind of IDO1 in-
hibitors, enzyme kinetic experiment of compound 14h, which was
chosen as a representative, was conducted.
With the similar procedure of enzymatic assay, the reaction velocity
of conversion
ferent amountsL-otfryhpIDtoOp1harnanignetofroNm-fo0r.m15ylμkLyntuor2n.i5neμLca(cta=lyz0e.d40b4ymdgif/-
mL), was measured and calculated (see Supplementary material 1.3). In
this experiment, the concentration of
300 μM, and inhibitor was at 100 μM orLw-tirtyhpotuotphinahnibwitoars assetptolesditivaet
control group. According to the procedure described by Voet D et al.,26
compound 14h should be categorized as a reversible inhibitor based on
plotting a function of velocity (V) against enzyme amount (E) displayed
in Fig. 2A.
Bioactivity assay
Inhibitory activity against IDO1 enzyme
Human IDO1 was purchased from Nanjing Detai Biotechnology
Furthermore, it is suggested that 14h should act as a kind of com-
petitive inhibitor of IDO1 with the substrate of L-tryptophan according
Company, which was expressed in E. coli and purified to homogeneity.
2

K.-m. Kong, et al.
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxxx
Scheme 1. Reagents and conditions: (a) HNO3,
AcOH, 0 °C, 2 h, 72%; (b) Fe, HCl, EtOH, 80 °C, 86%;
(c) NBS, Acetone, 2 h, 0 °C, 92%; (d) TMSCHN2,
Cs2CO3, MeOH, 60 °C, 76%; (e) DMF, POCl3, 0 °C,
76%; (f) CH3NO2, CH3COONH4, 120 °C, 65%; (g)
TosCl, KOH, Acetonitrile, rt, 75%; (h) NaBH4, THF-
MeOH, 0 °C, 73%; (i) Zn, HCl, 1,4-Dioxane, 120 °C;
(j) Boc2O, DMAP, rt, 71% (two steps). (k) CAN,
Acetonitrile, 0 °C, 32%~78%; (l) Substituted phe-
nylboronic acid or pyridinyl boronic acid, Pd(PPh3)4,
K2CO3, 1,4-Dioxane, 120 °C, 41%–88%; (m) TBAF,
THF, rt, 45%–86%.
Table 1
IDO1 inhibitory activities of 14a-h, 15, 16a-h, 17a-d in vitro.
Compds.
IDO1 enzyme
Hela EC50 (μM)a
Inhibition@10 μM
IC50 (μM)a
14a
14b
14c
56.38%
63.23%
34.45%
67.30%
43.34%
25.52%
12.42%
66.51%
74.88%
81.67%
63.75%
79.17%
79.45%
64.95%
68.26%
71.26%
64.21%
57.47%
72.35%
65.72%
59.71%
97.59%
2.94
2.38
NTb
2.09
NT
0.54
NT
NT
NT
NT
NT
NT
NT
0.15
0.83
14d
14e
14f
NT
14g
14h
15
NT
1.90
0.51
0.16
0.51
0.88
0.56
1.50
4.00
0.50
2.26
1.36
0.77
2.08
3.51
0.097
0.96
0.01
0.02
0.31
0.50
0.27
0.30
0.66
0.14
1.81
0.38
0.20
0.25
0.41
5.40
6.62
3.58
2.60
2.72
4.53
2.54
> 10
4.22
> 10
> 10
4.60
5.09
4.92
0.013
1.70
0.86
1.31
0.20
0.69
0.58
0.46
16a
16b
16c
Scheme 2. Reagents and conditions: (a) TFA, CH2Cl2, 2 h, 81%; (b) Na
(OAc)3BH, carbonyl compounds, MeOH, 25%–38%; (c) sulfonyl chlorides or
acyl chlorides, Et3N, 30%–45%.
16d
16e
16f
16g
16h
17a
17b
17c
1.55
to the plot of [S]/[V] against the concentration of inhibitor (14h)
shown in Fig. 2B. The reaction rates (V) were obtained with the enzyme
at a concentration of 0.101 mg/mL, L-tryptophan (S) at 300 μM and
150 μM respectively, and compound 14h (I) at concentrations varied
from 0 μM to 4 μM.
0.11
0.77
0.50
17d
INCB024360
a
Mean value of at least three independent assays with three duplicates each
time;
Not test.
Interaction model simulation
b
Molecular docking models of compounds 14h binding with IDO1
were generated based on the co-crystal structure of IDO1 complexed
with Amg-1 (PDB code: 4PK5) by using Maestro suite, Schrodinger.
AMG-1 (Fig. 3A) was picked out by high throughput screening and
identified as a selective and reversible inhibitor of IDO1 with an IC50 of
3 μM.27 The crystal structure revealed that Amg-1 takes up two binding
pockets at catalytic domain of IDO1 protein, which are named as pocket
A and B (Fig. 3B).28 As illustrated in Fig. 3C and D, the scaffold of 14h,
5-(3-pyridyl)-1H-indole-4,7-dione, can fit into pocket A, with the
oxygen of quinone forming a hydrogen bond with Ser 167 and the NeH
of side chain at 3-position interacting with 7-propionate of heme. Upon
that, the nitrogen atom of 3-pyridyl and Cys129 might form an essential
H-bond interaction, which could lead to the activity difference of
compounds 14a-h.
However, instead of going into pocket B as Amg-1 does, the tert-
butoxycarbonyl moiety of 14h reaches into a small hole next to the
pocket B (Fig. 3B). Neither the small ethyl group of 16a nor the big
benzenesulfonyl one of 17d can fill the pocket B from their docking
models (see Fig. S3). This result inspired us attempting to change the
atom number of the side chain to adjust the direction of the substituted
amino-group into pocket B in next study.
In summary, a series of 1H-indole-4,7-dione derivatives as IDO1
inhibitors were designed and synthesized based on the structure and
bioactivity of Exiguamine A and Tsitsikammamines. Primary SAR study
suggested that introducing 3-pyridinyl at 5-position and a smaller N-
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K.-m. Kong, et al.
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxxx
A
B
5
4
3
2
1
0
L-tryptophan concentration
700
600
500
400
300
200
100
300 µM
150 µM
Control
14h
0.0
0.5
1.0
1.5
2.0
2.5
-1
0
1
2
3
4
hIDO1 (µL)
Conc. of 14h (µM)
Fig. 2. IDO1 enzyme kinetic data with L-tryptophan (S) and 14h (I). (A) Plot of reaction rate (V) against enzyme amount. (B) Plot of [S]/[V] against the concentration
of inhibitor.
Fig. 3. Proposed binding mode of 14h with IDO1
A
B
(PDB code: 4PK5). (A) Structure of Amg-1; (B)
Crystal structure of IDO1/Amg-1 complex; (C)
Modeling of 14h with IDO1 shown as cartoon and
(D) shown as surface. (Compound 14h: Green
stick; Amg-1: White line; Heme: Magenta stick).
(For interpretation of the references to colour in
this figure legend, the reader is referred to the
web version of this article.)
C
D
alkyl substituent in side chain at 3-position of 1H-indole-4,7-dione are
beneficial to the inhibitory activity. The scaffold of 5-(3-pyridinyl)-1H-
indole-4,7-dione is a promising pharmacophore for IDO1 inhibitor and
compound 16a was identified as a hit with a 50% inhibitory con-
doi.org/10.1016/j.bmcl.2019.126901.
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