Rational design, synthesis and biological evaluation of ubiquinone derivatives as IDO1 inhibitors

Article abstract of DOI:10.1016/j.bioorg.2019.03.044

Indoleamine 2,3-dioxygenase 1 (IDO1) is an attractive therapeutic target for the treatment of cancer, chronic viral infections and neurological disorders characterized by pathological immune stimulation. Herein, a series of known metal-chelating ubiquinone derivatives were designed, synthesized and evaluated for the IDO1 inhibiting activities. The docking studies showed that the compounds 11, 16, 18 and coenzyme-Q1 exhibited different binding modes to IDO1 protein. Among these compounds, the most active compound is 16d with an IC₅₀ of 0.13 μM in enzymatic assay. The results reveal that a possible halogen bonding interaction between the bromine atom (3-Br) and Cys129 significantly enhances the inhibition activity against IDO1. This study provides structural insights of the interactions between ubiquinone analogues and IDO1 protein for the further modification and optimization.

Full text of DOI:10.1016/j.bioorg.2019.03.044

Accepted Manuscript
Rational design, synthesis and biological evaluation of ubiquinone derivatives
as IDO1 inhibitors
Yuyang Ding, Fei Tang, Xiaoqian Xue, Jinfeng Luo, Muzammal Hussain,
Yanhui Huang, Zhen Wang, Hao Jiang, Zhengchao Tu, Jiancun Zhang
PII:
S0045-2068(18)31375-0
DOI:
https://doi.org/10.1016/j.bioorg.2019.03.044
YBIOO 2870
Reference:
Bioorganic Chemistry
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Revised Date:
Accepted Date:
30 November 2018
15 March 2019
15 March 2019
Please cite this article as: Y. Ding, F. Tang, X. Xue, J. Luo, M. Hussain, Y. Huang, Z. Wang, H. Jiang, Z. Tu, J.
Zhang, Rational design, synthesis and biological evaluation of ubiquinone derivatives as IDO1 inhibitors,
Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.2019.03.044
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Rational design, synthesis and biological evaluation of ubiquinone
derivatives as IDO1 inhibitors
1
3
1
Yuyang Ding 1, 2, Fei Tang , Xiaoqian Xue , Jinfeng Luo , Muzammal Hussain 1, 2
,
Yanhui Huang 1, Zhen Wang 4, Hao Jiang 1*, Zhengchao Tu 1, 4* and Jiancun Zhang 1
1 State Key Laboratory of Respiratory Disease, Guangzhou Institutes of Biomedicine
and Health, Chinese Academy of Science, Guangzhou 510530, China.
2 University of Chinese Academy of Sciences, Beijing 100049, China.
3 Huizhou University, Huizhou 516007, China.
4 College of Pharmacy, Jinan University, Guangzhou 510632, China.
 Corresponding author:
E-mail: zhang_jiancun@gibh.ac.cn (J. Zhang), tu_zhengchao@gibh.ac.cn (Z. Tu),
jiang_hao@gibh.ac.cn (H. Jiang).

Abstract: Indoleamine 2,3-dioxygenase 1 (IDO1) is an attractive therapeutic target
for the treatment of cancer, chronic viral infections and neurological disorders
characterized by pathological immune stimulation. Herein, a series of known
metal-chelating ubiquinone derivatives were designed, synthesized and evaluated for
the IDO1 inhibiting activities. The docking studies showed that the compounds 11, 16,
18 and coenzyme-Q1 exhibited different binding modes to IDO1 protein. Among
these compounds, the most active compound is 16d with an IC50 of 0.13 μM in
enzymatic assay. The results reveal that a possible halogen bonding interaction
between the bromine atom (3-Br) and Cys129 significantly enhances the inhibition
activity against IDO1. This study provides structural insights of the interactions
between ubiquinone analogues and IDO1 protein for the further modification and
optimization.
Keywords: Indoleamine 2,3-dioxygenase 1; Inhibitor; Ubiquinone derivatives;
Docking; Halogen bonding.

1. Introduction
Indoleamine 2,3-dioxygenase 1 (IDO1) is a heme-containing non-secretory blood
enzyme, which degrades essential amino acid tryptophan in the kynurenine pathway
[1, 2]. It catalyzes the first and also the rate limiting step of kynurenine formation.
Depletion of tryptophan and the accumulation of the kynurenine pathway metabolites
can inhibit the immune response and induce T cell inactivation and apoptosis. In
addition, up-regulation of IDO1 can promote T cell differentiation into
immunosuppressive regulatory T cells [3-5]. The expression of IDO1 has been
identified as an important immune effector for tumor cells to escape effective innate
and adaptive immune responses. In addition, IDO1 up-regulation is associated with
poor prognosis of serous ovarian cancer, endometrial cancer, colorectal cancer and
acute myeloid leukemia [6-8]. Earlier studies have suggested that neurological
disorders, including Huntington’s disease, Alzheimer’s disease and Parkinson’s
disease are also associated with pathological tryptophan metabolism [9-11]. Therefore,
IDO1 has emerged as a valid drug target for treatment of various disease conditions.
In the past decade, several IDO1 inhibitors with different structural scaffolds have
been identified from rational structure design, high throughput screening, and natural
product screenings [12-17] (Figure 1). Currently, there are at least seven small
molecule IDO1 inhibitors under clinical development, being evaluated alone or in
combinations with other agents [18-24]. Unfortunately, Incyte’s new immunotherapy
drug Epacadostat [25-28] failed to demonstrate desired efficacy in conjunction with
Merck’s melanoma drug Keytruda in a recent trial. This Epacadostat-Keytruda trial
failure had cast a shadow over other programs [29, 30]. As IDO1 inhibitors offer
promising potential in immunotherapy and other fields, new chemical entities with
structural diversity and improved efficacy are greatly needed.

Figure 1. The representative IDO1 inhibitors
A number of natural product-derived IDO1 inhibitors containing quinone moiety have
been identified, which display moderate inhibition activities in enzymatic assays [31,
32]. Mechanistically, the quinone moiety was known to be able to coordinate with
heme iron metal. Some earlier studies have highlighted concerns on whether the
quinone moiety blocks the catalytic activity of IDO1 by a specific mechanism of
action or by an unspecific redox-cycling mechanism [14, 31, 33]. More recent studies
however have proved a direct binding interaction between quinone bearing
compounds and IDO1, sustaining argument in favor of a specific mechanism of action
for this class of inhibitors [31, 34, 35].
There are many approved drugs or diet supplement containing the quinone scaffold,
such as Idebenone and coenzyme Q10 (CoQ10). Idebenone and CoQ10 share the
same p-quinone headgroup (CoQ0) and both of them are treated as a drug or dietary
supplement in many countries [36-43]. CoQ10 is a main homologue of CoQ existing
in humans, and Idebenone, a synthetic quinone with similarities to coenzyme-Q1, had
been introduced to the market in 1986 under the name Avan in Japan [36-43].
Normally, if the quinone moiety has strong electron donating groups such as OR, NR2,
the quinone becomes less active toward reduction and nucleophilic additions. Based
on this fact, we wanted to examine if the CoQ0 scaffold can be an effective heme Fe
ion chelator for IDO1 enzyme. We first designed a series of new analogs and found
that coenzyme-Q1 demonstrated a moderately potent IDO1 inhibitor activity with an

IC50 value of 1.3 μM in enzyme assay. Using this as a lead molecule, we then
designed and synthesized a series of novel ubiquinone derivatives for IDO1 inhibitory
biological evaluation. Since several crystal structures of IDO1 in complex with
different inhibitors have been reported to date, we also conducted structure-guided
optimization of our designed inhibitors through a docking-based strategy (Figure 2).
Figure 2. Structure modification strategy
2. Chemistry
Scheme 1. Reagents and conditions: (a) CH3OCHCl2, TiCl4, 0 °C to rt, 4 h; (b) RMgBr, THF, 0 °C
to rt, 1 h; (c) CAN, CH3CN/H2O, rt, 0.5 h; (d) Pd/H2, EtOH, 90 °C, 24 h.
The general synthetic route for the target compounds 6a-d and 6f-j is shown in
Scheme 1. Initially, 2,3,4,5-tetramethoxytoluene
1
was reacted with
1,1-dichlordimethyl ether and titanium tetrachloride to afford an intermediatory
product 2,3,4,5-tetramethoxyl 6-methylbenzaldhyde 2 [44]. The product 2 was then
reacted with various Grignard reagents 5 through Grignard reaction to form
corresponding compounds 3, which were then oxidized with CAN to get the desired
compounds 6g-j [45]. Similarly, the dehydroxylation of compounds 3 in the presence
of catalytic Pd/C gave the corresponding compounds 4, which were further oxidized

with CAN to yield target compounds 6a-d and 6f.
Scheme 2. Reagents and conditions: (a) EtOH, rt, 2 h; (b) Br2, CCl4, rt, 12 h; (c) K2CO3, 9.2 mol%
Pd-Tetrakis (triphenylphosphine), CHCl3/H2O/MeOH, 60 °C, 12 h.
Scheme 2 illustrates the synthesis of compound 8 which was obtained by treatment of
commercially available 2,3-dimethoxy-5-methylbenzoquinone 7 with bromine in
presence of carbon tetrachloride [46], and then coupled with phenylboronic acid
through a Suzuki-Miyaura coupling to afford compound 6e [47]. Similarly,
compounds 11a-r were obtained by treatment of 7 with various amines in presence of
alcohol [48, 49].
Scheme 3. Reagents and conditions: (a) m-CPBA, DCM, 0 °C to rt, 3 h; (b) NaOH, MeOH/H2O,
0 °C to rt, 16 h; (c) CAN, CH3CN/H2O, rt, 1.5 h; (d) EtOH, RNH2, rt, 2 h.

As
shown
in
Scheme
3,
oxidation
of
commercially
available
2,3,4-tetramethoxybenzaldehyde 12 through Baeyer-Villiger oxidation with m-CPBA
produced 13. Compound 13 was then hydrolyzed into 2,3,4-trimethoxyphenol 14 with
sodium hydroxide, which was then oxidized with CAN to provide
2,3-dimethoxycyclohexa-2,5-diene-1,4-dione 15 [50]. Finally, the compound 15 was
reacted with various amines to yield target compounds 16a-j. In scheme 4,
compounds 18a-j were obtained with treatment of 11j with various isocyanates in
ethanol.
Scheme 4. Reagents and conditions: (a) EtOH, rt, 1 h.
3. Results and discussion
All the synthesized compounds were evaluated by in vitro IDO1 enzymatic inhibition
assay (Supplementary information), and the results are summarized in Tables 1-4.
Overall, the compounds 11a (0.7 μM), 16d (0.13 μM), and 18a (0.4 μM) were found
to be the most active ones among the synthesized series.
3.1 Design of target compounds and modeling studies
First, the chemical modification started with coenzyme-Q1 as the lead compound and
most of the modifications were carried out based on the published crystal structure of
IDO1 [23, 24, 51-53] in complex with INCB14943 (PDB ID: 5XE1). According to
our docking analysis, coenzyme-Q1 is situated ~3.5 Å above the plane of the heme,
coordinates with the heme iron and forms an edge-to-face π-π interaction with Phe163,
which was predicted to contribute to the inhibitory activity against IDO1. However,
the isoallyl of coenzyme-Q1 could not bind to the hydrophobic pocket A [53], which
is surrounded by residues Leu234, Val130 and Phe164 (Figure 3B).

Figure 3. (A) Binding details of IDO1 (cyan) with INCB14943 (yellow) (Glide SP: -7.599). (B)
Proposed binding mode of coenzyme-Q1 (orange) with IDO1 (Glide SP: -4.661). Residues
involved in interactions are shown as stick. The green dash line indicates π-π interaction, the
yellow dash line indicates halogen interaction, and the red dash line indicates hydrogen bond
interaction, respectively. (PDB: 5XE1) [46].
Recent studies [23, 26, 52, 53] have shown that the halogenated aromatic moieties can
efficiently bind into the pocket A of IDO1 binding site. Therefore, we used the
hybridization strategy to conjugate ubiquinone with aniline (aromatic moiety) to
afford compound 11a, which demonstrated an improved potency with an IC50 value of
0.70 μM. Through analysis of structure-activity relationship (SAR) and docking
results of 11a (Figure 4A and Table 2), we concluded that the aniline moiety was not
able to stretch itself into the pocket A. Most probably, the steric restriction of methyl
and the aniline moiety prevented its placement into the narrow entrance of pocket A.
Therefore, we decided to take advantage of ubiquinone skeleton and further modify it
by removing the methyl group, thereby obtaining demethylated derivatives 16. Finally,
this strategy worked for us and we obtained compound 16d with an IC50 value of 0.13
μM equivalent to Epacadostat.
Figure 4. (A) Proposed binding mode of 11a (green) (Glide SP: -5.404). (B) Proposed binding
mode of 16d (salmon) (Glide SP: -5.702). (C) Proposed binding mode of 18a (magenta) (Glide SP:
-5.676). The green dash lines indicate π-π interactions, the yellow dash line indicates halogen

interaction, the red dash lines indicate hydrogen bond interactions, and the blue dash lines indicate
interactions between heme iron and carbonyl, respectively.
The docking analysis of 16d (shown in Figure 4B) revealed that the interaction
between the bromine atom and the sulphur atom of Cys129 could be essential for
potent IDO1 inhibitory activity. Whereas, the binding mode of 18a suggested that the
hydrogen bonding interaction between the urea moiety and heme carboxylic acid may
contribute significantly to the inhibitory activity against IDO1. Interestingly, these
ubiquinone derivatives were predicted to have different binding modes to the IDO1
protein (Figures 3 and 4). Finally, the results of the SAR analysis and optimization
strategies were summarized in Figure 5.
Figure 5. The docking-guided optimization and SAR of ubiquinone derivatives
3.2 SAR of ubiquinone derivatives
Table 1. Structures and IDO1 inhibitory activities of the coenzyme-Q1 ubiquinone derivatives
IDO1 inhibition
IDO1 inhibition
Compd.
R
H
Compd.
6h
R
IC50 (μM)a
IC50 (μM) a
>10
>10
>10
coenzyme-Q0
coenzyme-Q1
1.30±0.05
6i
2.80±0.21
>10
6a
6j

3.54±0.17
0.70±0.09
6b
11a
4.52±0.31
5.11±0.38
1.12±0.21
3.92±0.47
6c
11n
11o
6d
>10
1.91±0.25
0.12±0.03
6e
11p
>10
>10
6f
Epacadostat
6g
a IC50 values are the means of more than two independent assays, presented as mean ± SD.
As shown in Table 1, 17 compounds were evaluated in IDO1 inhibition enzyme assay.
As we expected, some compounds displayed moderately potent IDO1 inhibitory
activities. To begin with the SAR, we first investigated the effect of linker region on
the potency of inhibitory activity. We found that the compounds 6b and 6d with a
linker of one or two carbon atoms displayed moderate inhibition of IDO1 with IC50
values 3.54 μM and 5.11 μM, respectively. However, a significant loss of inhibitory
potency was observed for compound 6e (IC50 > 10 μM), which possesses no linker
region. Further comparison of 6d with 11a (IC50 = 0.70 μM) and 6b with 11n (IC50 =
1.12 μM) revealed that NH group of the linker was essential to enhance the inhibitory
activity. Moreover, the compounds 6g-i with hydroxyl group at linker lost inhibitory
potency (IC50 > 10 μM) in comparison to their parent molecules 6a (IC50 > 2.8 μM),
6b (IC50 > 3.54 μM), and 6d (IC50 > 5.11 μM), respectively. On the other hand, the
compounds 11a, 11n and 11p possessing the same NH group on linker demonstrated
approximately five-fold increased inhibitory potency compared to their corresponding
parent compounds 6b, 6d and 6f, respectively. Through a series of further
comparisons (6b vs 6c, 6d vs 6f, 11a vs 11p, and 11a vs 11o), we concluded that the
substitution with aromatic group could improve inhibitory potency in comparison to
lipophilic substitutions. Overall, compound 11a emerged as the most potent
compound in this series, with an IC50 value of 0.70 μM in the in vitro enzymatic
assay.

The results from first SAR encouraged us to further study the effect of the substitution
groups on the aniline moiety, and compounds 11a-r were synthesized and evaluated
as shown in Table 2.
Table 2. Structures and IDO1 inhibitory activities of the NH-linker ubiquinone derivatives
Compd.
R1
H
R2
IDO1 inhibition IC50 (μM) a
0.70±0.09
11a
H
H
0.60±0.08
11b
11c
0.72±0.04
0.51±0.04
H
H
11d
11e
0.63±0.13
0.58±0.08
H
H
H
H
H
11f
11g
11h
11i
0.60±0.05
0.67±0.03
0.61±0.04
0.63±0.11
11j
H
0.78±0.13
11k

H
0.82±0.09
11l
H
H
H
1.08±0.07
1.12±0.21
3.92±0.47
11m
11n
11o
H
1.91±0.25
11p
Me
>10
>10
11q
11r
a IC50 values are the means of more than two independent assays, presented as mean ± SD.
Surprisingly, mono-substituted compounds 11b-m (IC50 = 0.51 – 1.08 μM) did not
show much improvement of inhibitory activity versus their parent compound 11a
(IC50 = 0.70 μM). Similarly, compounds with aliphatic amine group (11n-p) were also
substantially less potent than compounds with anilines. The influence of NH linker
was also investigated and the SAR results revealed that the tertiary amine moiety gave
inactive compounds (11q and 11r).
Table 3. Structures and IDO1 inhibitory activities of the de-methyl ubiquinone derivatives
Compd.
R
IDO1 inhibition IC50 (μM) a
0.60±0.21
16a
0.32±0.11
0.23±0.05
16b
16c

0.13±0.02
0.27±0.01
0.25±0.03
16d
16e
16f
0.16±0.02
16g
0.67±0.04
0.56±0.07
0.69±0.13
16h
16i
16j
a IC50 values are the means of more than two independent assays, presented as mean ± SD.
To further optimize compound 11a, modifications were performed to place the aniline
moiety into pocket A. For this purpose, we took advantage of structure-guided
optimization strategy while analyzing the binding mode of 11a after docking. Our
binding mode analysis revealed that methyl group of 11a was so big that the aniline
moiety could not stretch to the pocket A (Figure 4A).
Based on the insights from our modeling studies (discussed above), 10 compounds,
16a-j, were synthesized and evaluated (Table 3). The SAR of compounds 16b-g
accompanied by docking analysis of 16d (the most potent one) suggested that the
halogen bonding interaction between halogen atoms and the sulphur atom of Cys129
was beneficial to the IDO1 inhibition potency (Figure 4B). The analyses further
revealed that the weaker activity of fluorine and chlorine substitutions (16b and 16c)
versus that of bromine (16d) would be consistent with principles of halogen bonding
events, since fluorine and chlorine are electronegative and small, being unfavorable
for effective halogen binding compared to bromine. Further, disubstitued derivatives
16f and 16g were tolerated and showed no improvement in inhibitory activity
compared to monosubstituted derivatives (16c and 16d). Finally, replacement of the
aniline with benzylamine 16h-j resulted in a dramatic decrease in inhibitor potency

(Table 3).
Table 4. Structures and IDO1 inhibitory activities of the urea ubiquinone derivatives
Compd.
R
IDO1 inhibition IC50 (μM) a
0.40±0.08
18a
0.71±0.15
0.86±0.11
1.36±0.37
0.92±0.09
3.22±0.26
1.26±0.15
1.30±0.87
0.86±0.14
0.39±0.05
18b
18c
18d
18e
18f
18g
18h
18i
18j
a IC50 values are the means of more than two independent assays, presented as mean ± SD.
According to our docking analysis, the 18a and 11a showed similar binding mode to
the IDO1 protein. (Figure 4C and Figure 4A). For compound 18a, there was an
additional interaction between the two nitrogen atoms of urea and carboxylic acid of
heme, which possibly contribute to inhibitory potency for this series of compounds.
Among these compounds (Table 4), 18a and 18j exhibited higher inhibitory potency
with values of 0.40 μM and 0.39 μM, respectively. The SAR analyses of these urea

derivatives demonstrated that the substitution of aniline ring always led to decreased
IDO1 inhibitory activity. The benzylamine derivatives (18g and 18h) and
cyclohexylurea derivative (18i) exhibited slightly less inhibitory potency.
In this study, ubiquinone was discovered to be a novel key pharmacophore with
potent IDO1 inhibitory activity. At the beginning, the lead compound coenzyme-Q1, a
commercially available compound, exhibited moderate inhibition potency. Our
hybridization strategy to replace isoallyl with aniline (11a), improved the inhibitory
potency (IC50 = 0.70 μM) and prompted us to further investigate this series of
compounds. Based on the modeling predictions for 11a (discussed above), we
synthesized the demethylated series (16a-j) among which compound 16d was found
to be a most potent IDO1 inhibitor with IC50 of 0.13 μM. Further optimizations also
confirmed the critical interaction of the halogen atom and Cys129 in pocket A.
Similarly, in case of 18a, the interactions between urea moiety and hydroxyl of
Ser263 and carboxylic acid of heme were predicted essential to improve the inhibitory
activity. Overall, our computational and SAR analyses revealed that there are two
important factors affecting ubiquinone derivatives IDO1 inhibitory activity: the
formation of halogen bonding in pocket A and the interaction with carboxylic acid of
heme, which could provide further insights for future optimization.
Table 5. Cell-based Assay of ubiquinone derivatives
Cytotoxic activities
HEK293T, IC50 (μM)
Inhibitory activities
HeLa, IC50 (μM)
Compd.
18.9
28.0
72.1
3.1
19.9
11.2
11.1
11a
16d
18a
Doxorubicin
3. 3 Cellular IDO1 inhibitory activity assay
For the cell-based IDO1 inhibitory evaluation, we selected the compounds 11a, 16d,
and 18a which were the most potent ones among the synthesized series. The results
showed that 11a, 16d, and 18a, though having potent in vitro enzymatic inhibition,
did not demonstrate similar inhibition in cellular assay (Table 5), which might be
because of their weak membrane permeability or complexity of kynurenine pathway.

Our future studies will endeavor to improve the cell-activity of ubiquinone
derivatives.
3.4 Cell viability assay
In cell viability assay, 11a, 16d and 18a displayed significantly higher IC50 values as
compared to the standard drug doxorubicin (Table 5). The results showed ubiquinone
derivatives had weak cytotoxicity against the HEK293T cells, indicating possible
their high selectivity towards IDO1.
3.5 Determination of the inhibition type
The inhibition type was determined by dilution experiment. The results showed that
16d was a reversible inhibitor. (Supplementary material Figure S1).
4. Conclusion
In summary, a new structural class of IDO1 inhibitors, ubiquinone derivatives, has
been discovered. Among these, 27 compounds exhibited sub-micromolar potency in
the in vitro enzyme inhibition assay and the most active compound is 16d with an
IC50 of 0.13 μM. Docking studies have shown that coenzyme-Q1, 11, 16 and 18
adopted different binding modes to the IDO1. Halogenation of the aniline ring was
found particularly important in improving potency. SAR and modeling studies were
performed to understand the interactions of ubiquinone analogues to IDO1 protein,
which give us a direction for further lead optimization. We believe that the ubiquinone
derivatives may serve as novel and useful chemical probes for the design of more
effective IDO1 inhibitors.
Acknowledgements
We gratefully acknowledge financial support from the independent project of State
Key Laboratory of Respiratory Disease (Grant SKLRD-QN-201709), and the
“Personalized Medicines – Novel Target-based Antitumor Drug Discovery and
Development”, Strategic Priority Research Program of the Chinese Academy of
Sciences (Grant XDA12020336). M.H. is sponsored by CAS-TWAS President’s
Fellowship for international PhD students.

Appendix A. Supplementary date
Supplementary data related to this article (comprising methods and the NMR data of
synthesized compounds) can be found at
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Highlights:
 IDO1 is an attractive therapeutic target for the treatment of different disease
conditions.
 48 novel ubiquinone derivatives were designed and synthesized.
 Most of the compounds exhibited sub-micromolar potency in enzyme assay.
 The most active compound 16d exhibited an IC50 of 0.13 μM.
 The modeling studies revealed a halogen bonding interaction critical for IDO1
inhibition activity.

Graphical Abstract: