Design and synthesis of indoleamine 2,3-dioxygenase 1 inhibitors and evaluation of their use as anti-tumor agents

Article abstract of DOI:10.3390/molecules24112124

Indoleamine 2,3-dioxygenase (IDO) 1 is the key enzyme for regulating tryptophan metabolism and is an important target for interrupting tumor immune escape. In this study, we designed four series of compounds as potential IDO1 inhibitors by attaching various fragments or ligands to indole or phenylimidazole scaffolds to improve binding to IDO1. The compounds were synthesized and their inhibitory activities against IDO1 and tryptophan 2,3-dioxygenase were evaluated. The cytotoxicities of the compounds against two tumor cell lines were also determined. Two compounds with a phenylimidazole scaffold (DX-03-12 and DX-03-13) showed potent IDO1 inhibition with IC₅₀ values of 0.3–0.5 μM. These two IDO1 inhibitors showed low cell cytotoxicity, which indicated that they may exert their anti-tumor effect via immune modulation. Compound DX-03-12 was investigated further by determining the in vivo pharmacokinetic profile and anti-tumor efficacy. The pharmacokinetic study revealed that DX-03-12 had satisfactory properties in mice, with rapid absorption, moderate plasma clearance (～36% of hepatic blood flow), acceptable half-life (～4.6 h), and high oral bioavailability (～96%). Daily oral administration of 60 mg/kg of compound DX-03-12 decreased tumor growth by 72.2% after 19 days in a mouse melanoma cell B16-F10 xenograft model compared with the untreated control. Moreover, there was no obvious weight loss in DX-03-12-treated mice. In conclusion, compound DX-03-12 is a potent lead compound for developing IDO1 inhibitors and anti-tumor agents.

Full text of DOI:10.3390/molecules24112124

molecules
Article
Design and Synthesis of Indoleamine 2,3-Dioxygenase 1
Inhibitors and Evaluation of Their Use as
Anti-Tumor Agents
Hui Wen † , Yuke Liu †, Shufang Wang, Ting Wang, Gang Zhang, Xiaoguang Chen, Yan Li,
Huaqing Cui , Fangfang Lai * and Li Sheng *
State Key Laboratory of Bioactive Substances and Function of Natural Medicine, Institute of Materia Medica,
Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing 100050, China;
wenhui@imm.ac.cn (H.W.); liuyuke@imm.ac.cn (Y.L.); wangshufang@imm.ac.cn (S.W.);
wangtingdlf@imm.ac.cn (T.W.); gzhang@imm.ac.cn (G.Z.); chxg@imm.ac.cn (X.C.);
yanli@imm.ac.cn (Y.L.); hcui@imm.ac.cn (H.C.)
* Correspondence: laifangfang@imm.ac.cn (F.L.); shengli@imm.ac.cn (L.S.);
Tel.: +86-10-63165185 (F.L.); Fax: +86-10-63017757 (F.L.)
† These two authors contributed equally to this paper.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editor: Qiao-Hong Chen
Received: 14 May 2019; Accepted: 4 June 2019; Published: 5 June 2019
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: Indoleamine 2,3-dioxygenase (IDO) 1 is the key enzyme for regulating tryptophan
metabolism and is an important target for interrupting tumor immune escape. In this study,
we designed four series of compounds as potential IDO1 inhibitors by attaching various fragments
or ligands to indole or phenylimidazole scaffolds to improve binding to IDO1. The compounds
were synthesized and their inhibitory activities against IDO1 and tryptophan 2,3-dioxygenase were
evaluated. The cytotoxicities of the compounds against two tumor cell lines were also determined.
Two compounds with a phenylimidazole scaffold (DX-03-12 and DX-03-13) showed potent IDO1
inhibition with IC50 values of 0.3–0.5 µM. These two IDO1 inhibitors showed low cell cytotoxicity,
which indicated that they may exert their anti-tumor effect via immune modulation. Compound
DX-03-12 was investigated further by determining the in vivo pharmacokinetic profile and anti-tumor
efficacy. The pharmacokinetic study revealed that DX-03-12 had satisfactory properties in mice, with
rapid absorption, moderate plasma clearance (
∼
36% of hepatic blood flow), acceptable half-life ( 4.6 h),
∼
and high oral bioavailability ( 96%). Daily oral administration of 60 mg/kg of compound DX-03-12
∼
decreased tumor growth by 72.2% after 19 days in a mouse melanoma cell B16-F10 xenograft model
compared with the untreated control. Moreover, there was no obvious weight loss in DX-03-12-treated
mice. In conclusion, compound DX-03-12 is a potent lead compound for developing IDO1 inhibitors
and anti-tumor agents.
Keywords:
indoleamine 2,3-dioxygenase; inhibitor; anti-tumor; immune modulation;
tryptophan metabolism
1. Introduction
Tryptophan metabolism is an important pathway for cancer immunotherapy [1–3]. The accumulation
of tryptophan can activate T cells, and a decrease in tryptophan concentration affects the regular function of
T cells and interferes with the differentiation of naïve T cells. The accumulation of tryptophan metabolites,
such as l-kynurenine and other downstream kynurenines (3-hydroxykynurenine and 3-hydroxyanthranilic
acid), is toxic to T cells and induces T cell apoptosis. Thus, increasing tryptophan degradation inhibits the
immune response [4–6].
Molecules 2019, 24, 2124; doi:10.3390/molecules24112124
www.mdpi.com/journal/molecules

Molecules 2019, 24, 2124
2 of 27
Indoleamine 2,3-dioxygenase (IDO; E.C. 1.12.11.17) 1 is a monomeric 41 kDa enzyme that
contains heme and converts l-tryptophan to N-formylkynurenine in non-hepatic tissues [ ].
Tryptophan 2,3-dioxygenase (TDO; E.C. 1.13.11.11) also catalyzes the degradation of l-tryptophan to
7
N-formylkynurenine [8]. However, these two enzymes are genetically distinct and have low sequence
homology [8].
IDO1 is an important enzyme in tryptophan metabolism and contributes to creating peripheral
immune tolerance by degrading l-tryptophan, which suppresses the function of T cells, and by
producing N-formylkynurenine, which inhibits T cells [8]. In addition, IDO1 can also trigger other
activators of anti-tumor immunity. Notably, IDO1 is overexpressed in a range of cancer tissues [
4–6].
Thus, blocking the activity of IDO1 is a potential strategy for tumor immunotherapy.
The crystal structures of recombinant human IDO1 have been resolved [
studies have revealed that the heme iron in the active site of IDO1 binding and forms adducts with
exogenous ligands including O2−, NO, CO, H2S, and CN− [
11 14]. IDO1 tolerates various substrates,
9,10], and biochemical
9
,
–
whereas TDO has strict substrate specificity. This difference can be used to develop selective IDO1
inhibitors [8].
During the development of anti-tumor drugs, potent IDO1 inhibitors were discovered, and
intensive efforts have been made to develop potent IDO1/TDO inhibitors with various structural
scaffolds. Some typical examples of IDO1/TDO inhibitors are shown in Figure 1 [1,15–21]. The indole
and phenylimidazole scaffolds have produced several potent IDO1 inhibitors. For example, IDO1
inhibitors indoximod (pathway IC50 = 450 nM) and PF-0684003 (IDO1 IC50 = 410 nM) are indole
derivatives [13], and GDC-0919 (IDO1 IC50 = 13 nM) and AMCL-17g (IDO1 IC50 = 77 nM) are
phenylimidazole derivatives [20].
Figure 1. Reported IDO1 inhibitors. Indoximod and PF-0684003 have an indole scaffold and GDC-0919
and AMCL-17g have a phenylimidazole scaffold.
In this study, in the early stages of searching for new IDO1 inhibitors, we used indole and
phenylimidazole as the core scaffolds. Four series of compounds were designed with different
strategies. The compounds were synthesized and the IDO1/TDO inhibitory activities were determined.
The in vivo pharmacokinetic profile and anti-tumor efficacy of a potent IDO1 inhibitor were evaluated
to explore its potential as an anti-tumor agent.
2. Results and Discussion
2.1. Design Strategy for the Four Compound Series
Four series of compounds were designed (Figure 2) to explore the structure–activity relationship
(SAR). The SAR information was used for further optimization. Indole is derived from tryptophan
and phenylimidazole is an IDO1 inhibitor [18]. Thus, tryptophan and phenylimidazole fit into the
active site of IDO1, and they were selected as the core scaffolds for designing the inhibitors. As we

Molecules 2019, 24, 2124
3 of 27
mentioned in the introduction, several exogenous ligands including hydroxylamine, O2−, NO, CO,
H2S, and CN− were able to bind with the heme iron in the active site of IDO1 [
11 14]. We attached
9
, –
various fragments and ligands to these scaffolds to strengthen the binding to the active site of IDO1.
In series 1, several ligands, such as hydroxylamine and SO, that interact strongly with heme iron
were linked to the double bond of the indole scaffold. We hoped to observe a synergistic effect between
the ligand and the indole ring in the IDO1 binding. Series 2 was based on compound PF-0684003
(Figure 1), which is an IDO1 inhibitor developed by Pfizer. The structure of PF-0684003 co-crystalized
with IDO1 revealed that there was no interaction between PF-0684003 and the heme iron, which is
usually observed between other IDO1 inhibitors and IDO1 [13]. Thus, several potential heme binding
ligands, such as hydroxylamine and SO, were added to the structure of PF-0684003 to enhance the
inhibitory activity.
Phenylimidazole is a weak IDO1 inhibitor. Crystal structures have revealed that the imidazole
ring binds with the heme iron [18,19]. Thus, in series 3 and 4, we used phenylimidazole as the scaffold,
and we attached fragments to either the imidazole ring (series 3) or the phenyl ring (series 4). These
fragments were intended to improve the binding in the active site of IDO1 through various interactions,
such as hydrogen bonds and π–π interactions.
Figure 2. Design strategies of the four series of inhibitors. Because the parent compounds of series 1
and 2, indole and PF-0684003, respectively, do not interact with the heme iron, various heme binding
ligands were attached to the indole scaffold. The imidazole ring interacts with the heme iron, and thus
various fragments were attached to the phenyl ring in series 3 and the imidazole ring in series 4 to
strengthen the binding to the active site of IDO1.
2.2. Synthesis of Selected Compounds
Although the compounds were classified into four series for examining the SAR, the structural
diversity meant that they required a variety of synthetic routes. We describe representative synthetic
schemes for four compounds. Syntheses for all the compounds are included in the Experimental section.
Scheme 1 shows the synthesis of DX-02-05. In the presence of a Lewis acid, ethyl
5-fluoro-1H-indole-2-carboxylate underwent a condensation reaction with 1H-pyrrole-2,5-dione to yield
compound DX-02-03. Alcohol
1
was obtained by reducing DX-02-03, and then
1 was directly oxidized
with MnO2 to produce aldehyde
2
. Aldehyde was condensed with hydroxylamine hydrochloride to
2

Molecules 2019, 24, 2124
4 of 27
obtain compound DX-02-04. Finally, the double bond in DX-02-04 was reduced with NaBH3CN to
obtain compound DX-02-05.
Scheme 1. Synthesis of DX-02-05. (a) BF3-Et2O, ClCH2CH2Cl, yield = 55%; (b) LiAlH4, CH3OH,
yield = 86%; (c) MnO2, dry dichloromethane, yield = 91%; (d) hydroxylamine hydrochloride, DIPEA,
CH3OH, yield = 66%; (e) NaBH3CN, 12 N HCl, CH3OH, yield = 43%.
Scheme 2 shows the synthesis of DX-02-07. Sulfur substitution of 5-fluoroindolin-2-one
was performed with P2S5 to produce compound
3
. Under basic conditions, compound
4
4
was
and
obtained through an SN2 substitution reaction with CH3I. A condensation reaction between
◦
1H-pyrrole-2,5-dione was performed at 120 C to obtain sulfide DX-02-06, which was oxidized to
racemic sulfoxide DX-02-07.
Scheme 2. Synthesis of DX-02-07. (a) P2S5, NaHCO3, dry tetrahydrofuran, yield = 75%; (b) CH3I,
K2CO3, acetone, yield = 83%; (c) 1H-pyrrole-2,5-dione, CH3CO2H, yield = 61%; (d) m-CPBA, dry
dichloromethane, yield = 35%.
Scheme 3 shows the synthesis of DX-03-12. First, the imidazole starting material was protected
with trityl chloride to avoid various side reactions. Compound
reaction with a boronic acid [22]. The methyl group was removed by BBr3 to expose the hydroxyl
group in compound . Compound was prepared in parallel by melting 3-chloropropylamine and
isobenzofuran-1,3-dione together. Compounds and were subjected to an SN2 nucleophilic reaction
to obtain compound under strong basic conditions. This step had a low yield, which was explained by
the high steric hindrance of the large molecules and the weak nucleophilicity of compound . The amine
6 was obtained by the Suzuki–Miyaura
7
8
7
8
9
7
protecting group was removed with hydrazine hydrate to obtain compound 10. Compound 10 was
reacted with trifluoromethanesulfonylchloride under basic conditions to give compound 11. Finally,
DX-03-12 was obtained by removing the protecting group on the imidazole with acetic acid.

Molecules 2019, 24, 2124
5 of 27
Scheme 3.
Synthesis of DX-03-12.
(a) Trityl Chloride, DIPEA, dry DMF, yield = 78%;
(b) (2-methoxyphenyl)boronic acid, K3PO4, 14% Pd(PPh3)4, DMF, inert atmosphere, yield = 63%;
(c) BBr3, inert atmosphere, dry dichloromethane, yield = 65%; (d) solvent-free melt, yield = 55%;
(e) KOH, dry DMF, yield = 71%; (f) hydrazine hydrate, CH3OH; (g) trifluoromethanesulfonylchloride,
DIPEA, dry dichloromethane, yield = 67%; (h) AcOH, CH3OH, yield = 86%.
Scheme 4 shows the synthesis of DX-04-02.
2-Iodoimidazole was substituted with
trifluoromethylbenzyl bromide to give compound 12. Compound 12 underwent a Suzuki–Miyaura
coupling reaction with (5-chloro-2-methoxyphenyl)boronic acid to obtain DX-04-01. Finally, the methyl
group was removed with BBr3 to obtain DX-04-02.
Scheme 4. Synthesis of DX-04-02. (a) DIPEA, dry DMF; (b) (5-chloro-2-methoxyphenyl)boronic acid,
K3PO4, Pd(PPh3)4, DMF, inert atmosphere, yield = 72%; (c) BBr3, inert atmosphere, yield = 53%.
2.3. In Vitro Biological Evaluation
We prepared the four series of compounds using the synthetic schemes in Section 2.2. Twelve
commercially available compounds were purchased (see Section 3.1). In total, we screened
50 compounds in vitro for their IDO1 and TDO inhibitory activities and cell cytotoxicities against
tumor cell lines H460 and MCF7 (Table 1). NLG919 was the reported potent IDO1/TDO inhibitor,
and Taxol are known commercial anti-cancer drug. They were used as the reference compounds in
this study.

Molecules 2019, 24, 2124
6 of 27
Table 1. In vitro IDO1/TDO inhibitory activities and cell cytotoxicities of series 1.
IDOa
IC50 or %
TDO a
IC50 or %
H460 a
IC50 or %
MCF7 a
IC50 or %
Compound
Structure
DX-01-01
9.7%
2.4%
8.9%
NA
NA
DX-01-02
DX-01-03
10.0%
39.2%
43.1 µM
10.3%
10.1%
26.3%
37.6%
DX-01-04
DX-01-05
3.3%
6.0%
15.1%
5.9%
24.3%
29.7%
10.2%
10.5%
DX-01-06
DX-01-07
6.2%
7.4%
8.4%
11.2%
22.2%
25.7%
34.6%
16.9%
DX-01-08
DX-01-09
DX-01-10
DX-01-11
DX-01-12
10.9%
2.6%
2.1%
-
3.2%
7.8%
8.9%
3.8%
33.9%
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
47.1%
DX-01-13
DX-01-14
19.7%
8.1%
23.1%
NA
10.1%
NA
11.1 µM
38.2%

Molecules 2019, 24, 2124
7 of 27
Table 1. Cont.
IDOa
IC50 or %
TDO a
IC50 or %
H460 a
IC50 or %
MCF7 a
IC50 or %
Compound
Structure
DX-01-15
21.3 µM
35.4%
NA
NA
DX-01-16
DX-01-17
23.2 µM
17.7 µM
19.7%
42.2%
44.5%
NA
20.9%
NA
DX-01-18
5.4%
-
NA
NA
DX-01-19
DX-01-20
DX-01-21
-
7.8%
3.8%
9.2%
NA
NA
NA
NA
NA
NA
15.7%
9.7%
DX-01-22
NLG919
13.2%
4.5%
NA
NA
0.064 µM
0.085 µM
NA
NA
Taxol
NA
NA
6 nM
15 nM
a
When the calculated IC50 of a compound is >50 µM, we show the inhibition rate (%) at 50 µM. -: no detectable
inhibition. NA: not tested.
The IDO1 and TDO inhibitory activities of the 22 compounds in series 1 were measured (Table 1).
The series 1 compounds contained heme binding fragments including CO, NO, CN, H2S, imidazole,
and hydroxylamine; however, the inhibition data showed that most compounds in series 1 exhibited
marginal IDO1/TDO inhibition. However, four compounds (DX-01-14, DX-01-15, DX-01-16, and
DX-01-17) containing hydroxylamine had moderate IDO1 inhibitory activities. The most active IDO1
inhibitor in series 1 was compound DX-01-14 with an IC50 of 11.1 µM. In addition, cell cytotoxicity
screening showed that most of the compounds were not toxic to mammalian cancer cell lines.
Generally speaking, these molecules have a rather low molecular weight, and the rather small
moiety causes them hard to bind tightly inside the active center of IDO1. Thus, a poor SAR was
observed for this series of compounds. Interestingly, for these four active compounds, they all contain
the fragment of hydroxylamine. This may suggest that hydroxylamine is the ideal ligand to attach to
the double bond of the indole scaffold, while still maintaining the binding with the heme iron.
Series 2 included seven compounds, which were all PF-0684003 derivatives (Table 2). However,
the in vitro IDO1/TDO inhibitory activities of all these PF-0684003 derivatives were lower than that of
the parent compound, and none were IDO1/TDO inhibitors. The cytotoxicity study demonstrated that
these PF-0684003 derivatives were not toxic to cancer cell lines.

Molecules 2019, 24, 2124
8 of 27
Table 2. In vitro IDO1/TDO inhibitory activities and cell cytotoxicities of series 2.
IDO a
IC50 or %
TDO a
IC50 or %
H460 a
IC50 or %
MCF7 a
IC50 or %
Compound
Structure
DX-02-01
6.3%
7.2%
-
NA
NA
NA
NA
DX-02-02
DX-02-03
2.3%
15.4%
21.6%
21.7%
22.1%
DX-02-04
DX-02-05
40.2%
12.5%
14.5%
16.3%
NA
NA
NA
NA
DX-02-06
22.6%
20.1%
19.6%
13.9%
30.1%
15.2%
19.9%
11.0%
DX-02-07
NLG919
0.064 µM
0.085 µM
NA
NA
Taxol
NA
NA
6 nM
15 nM
a
When the calculated IC50 of a compound is >50 µM, we show the inhibition rate (%) at 50 µM. -: no detectable
inhibition. NA: not tested.
The structure of PF-0684003 co-crystallized with IDO1 shows that PF-0684003 binds tightly in the
active site of IDO1 but does not interact with the heme iron [18]. In the present study, the addition of
the heme binding ligands [14] (SO and hydroxylamine in DX-02-07 and DX-02-05, respectively) to the
double bond of the indole ring abrogated the IDO1 inhibition, similar to the optimization reported in a
previous study.
Series 3 contains 13 phenylimidazole derivatives (Table 3). Compounds DX-03-01 to DX-03-10
showed marginal IDO1/TDO inhibition and weak cell cytotoxicities. However, compounds DX-03-11,
DX-03-12, and DX-03-13 are potent IDO1 inhibitors with IC50 values of 0.3–2.4 µM. Interestingly, these
three compounds show marginal TDO inhibition activity, and thus are IDO1/TDO selective inhibitors.
In addition, these compounds are not toxic to mammalian cancer cell lines.
Compound DX-03-01 is a known compound [18], which binds to the active site in IDO1.
The imidazole ring interacts with the heme iron, and the hydroxyl group binds to the nearby
Ser167 in the active site [7]. However, the substitution of the hydroxyl group with hydroxylamine
(DX-03-01 to DX-03-10) did not produce IDO1 inhibitors with higher activity. In DX-03-10 to DX-03-13,
the hydroxyl group was used to attach an alkyl chain bearing terminal groups used in other reported
IDO1 inhibitors [17
,19]. The terminal fragments, such as sulfamide and urea, are important to maintain
the binding [17,19].

Molecules 2019, 24, 2124
9 of 27
Table 3. In vitro IDO1/TDO inhibitory activities and cell cytotoxicity of series 3.
IDO a
IC50 or %
TDO a
IC50 or %
H460 a
IC50 or %
MCF7 a
IC50 or %
Compound
Structure
DX-03-01
26.3%
24.2%
19.4%
11.9%
31.3%
10.3%
13.9%
11.1%
20.3%
12.1%
NA
NA
NA
NA
DX-03-02
DX-03-03
DX-03-04
DX-03-05
5.0%
12.7%
NA
22.2%
34.7%
NA
DX-03-06
DX-03-07
25.6%
33.5%
12.9%
15.1%
NA
NA
NA
NA
DX-03-08
DX-03-09
DX-03-10
15.6%
5.3%
11.1%
6.4%
NA
40.5%
7.6%
NA
37.7%
24.7%
13.2%
21.5%
DX-03-11
DX-03-12
DX-03-13
2.4 µM
0.3 µM
0.5 µM
38.2%
36.4%
38.9%
NA
NA
6.6%
21.39
19.3%
13.79
NLG919
0.064 µM
0.085 µM
NA
NA
Taxol
NA
NA
6 nM
15 nM
a
When the calculated IC50 of a compound is >50 µM, we show the inhibition rate (%) at 50 µM. -: no detectable
inhibition. NA: not tested.

Molecules 2019, 24, 2124
10 of 27
Series 4 contains eight phenylimidazole derivatives (Table 4). Most compounds in this series
showed marginal IDO1 inhibition, but compound DX-04-05 showed moderate IDO1 inhibition.
However, compound DX-04-06, in which the hydroxyl group was methylated, showed decreased IDO1
inhibition. Most compounds in this series were toxic to the cancer cell lines H460 and MCF7 with an
IC50 of around 10−5 M, which indicates the compounds also interrupt the function of other targets in
the cells.
Table 4. In vitro IDO1/TDO inhibitory activities and cell cytotoxicities of series 4.
IDO a
IC50 or %
TDO a
IC50 or %
H460 a
IC50 or %
MCF7 a
IC50 or %
Compound
Structure
DX-04-01
46.2%
9.7%
26.5%
18.0%
11.96 µM
7.69 µM
8.53µM
DX-04-02
DX-04-03
35.72 µM
8.7%
17.3%
11.48 µM
15.44 µM
DX-04-04
DX-04-05
DX-04-06
11.9%
23 µM
13.5%
23.8%
22.4%
23.6%
5.49 µM
22.89 µM
48.37 µM
20.95 µM
42.55 µM
49.34 µM
DX-04-07
DX-04-08
38.2%
21.2%
21.4%
11.2%
27.4 µM
12.28 µM
7.5%
24.9%
NA
NLG919
Taxol
0.064 µM
0.085 µM
NA
NA
NA
6 nM
15 nM
a
When the calculated IC50 of a compound is >50
inhibition. NA: not tested.
µM, we show the inhibition rate (%) at 50
µM. -: no detectable
In series 4, the design strategy was to attach fragments to the imidazole ring instead of the phenyl
ring in series 3. However, this strategy does not maintain the IDO1 inhibitory activity. In addition,
the free hydroxyl group in series 4 appears to be important for forming a hydrogen bond with an
amino acid residue, such as Ser167 [
7], in the active site; methylating this hydroxyl group decreased
IDO1 inhibition (DX-04-05 versus DX-04-06).

Molecules 2019, 24, 2124
11 of 27
In summary, based on the SAR study, compounds DX-03-12 and DX-03-13 showed potent IDO1
inhibition activity, and compound DX-03-13 has some structure similarity as previous published
patent (WO2011/056652Al) [23]. Thus, the most potent IDO1 inhibitor DX-03-12 was used as the lead
compound for further study.
2.4. Pharmacokinetic Study of DX-03-12
The in vivo pharmacokinetic properties of compound DX-03-12 were evaluated in male ICR mice.
DX-03-12 was administered orally either as a bolus at a dose of 30 mg/kg or intravenously at a dose of
3 mg/kg. The plasma concentration-time profiles are shown in Figure 3. The pharmacokinetic parameters
were determined by non-compartmental analysis (Table 5). After intravenous administration (3 mg/kg),
compound DX-03-12 showed a moderate clearance of 33.3 mL/min/kg, which was approximately 36% of
mouse hepatic blood flow (90 mL/min/kg). The volume of distribution in the steady state was 10 L/kg,
suggesting that there was an extensive distribution of compound DX-03-12 in tissues. Compound DX-03-12
was rapidly absorbed after oral administration of 30 mg/kg. The plasma concentration was quantifiable at
the first sampling time point (5 min) and remained detectable at 24 h. The maximum plasma concentration
of compound DX-03-12 of 2963 ng/mL was observed 1 h post-dose. Compound DX-03-12 was eliminated
relatively slowly with a half-life of 4.6 h. The oral bioavailability of compound DX-03-12 was calculated to
be 96%.
The pharmacokinetic study revealed that compound DX-03-12 showed rapid absorption and
almost complete oral bioavailability in mice. After oral administration of 30 mg/kg in mice, it exhibited
a long half-life (4.6 h) due to moderate plasma clearance. The pharmacokinetic properties of compound
DX-03-12 in mice indicate that it has the potential to be a once-a-day drug.
Table 5. Pharmacokinetic parameters of DX-03-12 after a single oral or intravenous dose in male
ICR mice.
PO
30 mg/kg
iv
Parameters
Units
3 mg/kg
t1/2β
Tmax
Cmax
AUC(0-t)
AUC(0-∞)
Vss
h
h
4.61
3.49
0.033
2138
1346
1500
10.1
33.3
-
1
2963
12,902
13,094
-
ng/mL
h*ng/mL
h*ng/mL
L/kg
Cl
F%
mL/min/kg
-
96
Figure 3. Mean plasma concentration-time profile in male ICR mice after administration of a single
oral (30 mg/kg) or intravenous (3 mg/kg) dose of compound DX-03-12. PO stands for per os, which
means oral administration of drug; iv stands for intravenous administration of drug.

Molecules 2019, 24, 2124
12 of 27
2.5. In Vivo Anti-Tumor Efficacy Study of Compound DX-03-12
To determine whether the compound was effective against tumors in vivo, the anti-tumor effect of
DX-03-12 was evaluated in a B16F10 subcutaneous xenograft model in syngeneic mice (Figure 4). Oral
and intraperitoneal (i.p.) administration (30 or 60 mg/kg/day) of DX-03-12 significantly decreased the
growth of melanoma B16F10 xenograft tumors in a dose-dependent manner. DX-03-12 oral treatment
resulted in a 72.2% decrease in tumor weight and i.p. treatment resulted in a 72.3% decrease at a dose
of 60 mg/kg compared with the control tumor after 19 days treatment. DX-03-12 had no obvious effect
on the body weight of the mice and the peripheral white blood cells, which indicated that DX-03-12
had low toxicity in mice. In this study, the classic first line anti-cancer drug cyclophosphamide (CTX)
was used as the reference drug.
Figure 4. In vivo anti-tumor activity of DX-03-12 in B16F10 xenograft mice. (A,B) Tumors from each
group after 19 days of treatment. Mice bearing B16F10 xenografts were dosed orally or i.p. with
vehicle (0.5% methylcellulose), CTX (Cyclophosphamide, orally, 60 mg/kg twice a week), DX-03-12
(i.p., 30 mg/kg/day), DX-03-12 (i.p., 60 mg/kg/day), DX-03-12 (orally, 30 mg/kg/day), or DX-03-12 (orally,
60 mg/kg/day). Results are expressed as the mean
*** p < 0.001 versus vehicle. ( ) The body weight from each group after the treatment. There is no
obvious body weight difference among all of the groups. ( ) The white blood cells detected in the
±
SD (n = 5–7 for each group). * p < 0.05 and
C
D
blood of all groups of animals. There is also no obvious WBC difference among all of the groups.
3. Experiment Section
3.1. Chemistry
1
Reagents and solvents were obtained from commercial suppliers and used as received. H-NMR
spectra were obtained on an NMR spectrometer (Mercury, Varian, San Diego, CA, USA; 400 MHz).
Electrospray ionization (ESI) mass spectra and high-resolution mass spectroscopy (HRMS) were
performed with a liquid chromatograph/mass selective detector time-of-flight mass spectrometer

Molecules 2019, 24, 2124
13 of 27
(LC/MSD TOF, Agilent Technologies, Santa Clara, CA, USA). silica gel column chromatography was
performed with silica gel 60G (Qingdao Haiyang Chemical, Qingdao, China). Purity was determined
using HPLC, LC/MS and NMR spectroscopy. All of the synthesized compounds have the purity over
than 95%.
Several commercial available compounds were purchased from Beijing innochem Co. Ltd. (Beijing,
China). They are DX-01-01, DX-01-08, DX-01-09, DX-01-10, DX-01-11, DX-01-12, DX-01-18, DX-01-19,
DX-01-20, DX-01-21, DX-01-22, DX-03-01.
3.1.1. Preparation of (R/S) 3-(5-fluoro-2-((hydroxyamino)methyl)-1H-indol-3-yl)pyrrolidine-2,5-dione
(DX-02-05)
Preparation of (R/S) Ethyl 3-(2,5-dioxopyrrolidin-3-yl)-5-fluoro-1H-indole-2-carboxylate (DX-02-03)
Ethyl 5-fluoro-1H-indole-2-carboxylate (2 g, 9.7 mmol) and 1H-pyrrole-2,5-dione (1.40 g, 14.4 mmol)
were added to dry 1,2-dichloroethane (50 mL), and 46.5% BF3-Et2O (3.54 g, 11.6 mmol) was added to
reaction mixture at 20 ◦C. After the reaction mixture was stirred at 90 ◦C, until the starting material
disappeared in thin layer chromatography (TLC), the reaction mixture was distilled in vacuo, and
dichloromethane (50 mL) was added to the mixture. The organic extract was washed twice with
saturated aqueous NaHCO3 and NaCl (20 mL) respectively, and the organic extract was dried over
Na2SO4. After solvent was removed under vacuum, the product was purified as a white solid by
1
silica gel column chromatography, 1.62 g, yield 55%. H-NMR (400 MHz, DMSO-d6):
δ = 12.00 (s, 1H,
NH-indolyl), 11.31 (s, 1H, NH), 7.49–7.45 (m, 2H, H-indolyl), 7.18 (td, J = 9.2, 2.6 Hz, 1H, H-indolyl),
4.82 (dd, J = 9.6, 6.6 Hz, 1H, CH), 4.33–4.27 (m, 2H, CH2), 3.08 (dd, J = 17.7, 9.6 Hz, 1H, CH’H”), 2.70
(dd, J = 17.7, 6.6 Hz, 1H, CH’H”), 1.30 (t, J = 7.1 Hz, 3H, CH3). 13C-NMR (101 MHz, CD3OD):
δ =182.17,
180.15, 162.66, 160.66, 134.24, 129.01, 126.54, 119.11, 115.66, 114.82, 104.78, 62.19, 39.68, 39.37, 14.63.
HRMS (ESI): m/z [M + H]+ calculated for C15H14O4N2F: 305.09321; found: 305.09290.
Preparation of (R/S) 3-(5-fluoro-2-(hydroxymethyl)-1H-indol-3-yl) pyrrolidine-2,5-dione (1)
Ethyl 3-(2,5-dioxopyrrolidin-3-yl)-5-fluoro-1H-indole-2-carboxylate (DX-02-03, 1.3 g, 4.3 mmol)
were added to dry CH3OH (20 mL), LiAlH4 (0.13 g, 4.3 mmol) was added to reaction mixture gradually
at 20 ◦C. The reaction mixture was stirred at 20 ◦C until the starting material disappeared in TLC.
The reaction mixture was distilled in vacuo, and ethyl acetate (50 mL) was added to the mixture.
The organic extract was washed twice with saturated aqueous NaCl (20 mL), and the organic extract
was dried over Na2SO4. After solvent was removed under vacuum, the product was purified as a
1
white solid by silica gel column chromatography, 0.97 g, yield 86%. H-NMR (400 MHz, CD3OD):
δ = 7.30 (dd, J = 8.8, 4.4 Hz, 1H, H-phenyl), 6.93 (dd, J = 9.9, 2.4 Hz, 1H, H-phenyl), 6.89–6.83 (m,
1H, H-phenyl), 4.72 (d, J = 1.3 Hz, 2H, CH2), 4.46 (dd, J = 9.7, 5.6 Hz, 1H, CH), 3.20 (dd, J = 18.4,
9.7 Hz, 1H, CH’H”), 2.81 (dd, J = 18.4, 5.6 Hz, 1H, CH’H”). 13C-NMR (101 MHz, CD3OD):
δ = 182.67,
180.16, 160.22, 157.85, 139.57, 133.85, 127.82, 113.46, 110.70, 103.45, 56.67, 39.93, 39.00. HRMS (ESI): m/z
[M + H]+ calculated for C13H12N2O3F: 263.08265; found: 263.08160.
Preparation of (R/S) 3-(2,5-dioxopyrrolidin-3-yl)-5-fluoro-1H-indole-2-carbaldehyde (2)
3-(5-Fluoro-2-(hydroxymethyl)-1H-indol-3-yl)pyrrolidine-2,5-dione (1, 0.8 g, 3.1 mmol) was added
to dry dichloridemethane (20 mL), MnO2 (0.36 g, 4.1 mmol) was added to reaction mixture gradually
at 20 ◦C. The reaction mixture was stirred at 20 ◦C until the starting material disappeared in TLC.
The reaction mixture was filtered, the solvent was removed under vacuum, and the product was
1
purified as a white solid by silica gel column chromatography, 0.73g, yield 91%. H-NMR (400 MHz,
CD3OD):
1H, H-phenyl), 7.19 (td, J = 9.1, 2.4 Hz, 1H, H-phenyl), 4.91 (dd, J = 9.7, 6.0 Hz, 1H, CH), 3.29–3.21
(m, 1H, CH’H”), 2.84 (dd, J = 18.1, 6.0 Hz, 1H, CH’H”). 13C-NMR (101 MHz, CD3OD):
= 183.66,
δ = 10.00 (s, 1H, CHO), 7.50 (dd, J = 9.1, 4.3 Hz, 1H, H-phenyl), 7.27 (dd, J = 9.5, 2.3 Hz,
δ

Molecules 2019, 24, 2124
14 of 27
181.23, 179.66, 160.59, 158.30, 135.75, 135.36, 127.61, 117.26, 115.66, 105.59, 39.77, 39.35. HRMS (ESI):
m/z [M + H]+ calculated for C13H10N2O3F: 261.06700; found: 261.06638.
Preparation of (R/S) (E)-3-(2,5-dioxopyrrolidin-3-yl)-5-fluoro-1H-indole-2-carbaldehyde oxime
(DX-02-04)
3-(2,5-Dioxopyrrolidin-3-yl)-5-fluoro-1H-indole-2-carbaldehyde (0.5 g, 1.92 mmol) was added to
dry CH3OH (30 mL), then N,N-Diisopropylethylamine (0.5 g, 3.84 mmol) and hydroxylamine hydro
-chloride (0.13 g, 1.92 mmol) were added respectively at 20 ◦C. The reaction mixture was stirred at
◦
60 C until the starting material disappeared in TLC. After solvent was removed under vacuum,
the product was purified as a white solid by silica gel column chromatography, 0.35 g, yield 66%.
1H-NMR (400 MHz, CD3OD):
δ
= 8.22 (s, 1H, =CH), 7.32 (dd, J = 8.8, 4.4 Hz, 1H, H-indolyl), 7.06–6.86
(m, 2H, H-indolyl), 4.63 (dd, J = 9.7, 5.9 Hz, 1H, CH), 3.19 (dd, J = 18.3, 9.7 Hz, 1H, CH’H”), 2.81 (dd,
J = 18.7, 6.2 Hz, 1H, CH’H”). 13C-NMR (101 MHz, CD3OD):
δ =181.92, 179.89, 160.31, 157.98, 141.03,
136.51, 134.81, 132.30, 113.78, 112.58, 103.95, 39.83, 38.66. HRMS (ESI): m/z [M + H]+ calculated for
C13H11N3O3F: 276.07790; found: 276.07718.
Preparation of (R/S) 3-(5-fluoro-2-((hydroxyamino)methyl)-1H-indol-3-yl) pyrrolidine-2,5-dione
(DX-02-05)
3-(2,5-Dioxopyrrolidin-3-yl)-5-fluoro-1H-indole-2-carbaldehyde oxime (DX-02-04, 0.3 g, 1.1 mmol)
was added to CH3OH (20 mL), then NaBH3CN (0.07 g, 1.1 mmol) was added at 20 ◦C. 12N HCl was
added to reaction mixture continuously to keep acidic. The reaction mixture was stirred at 20 ◦C until
the starting material disappeared in TLC. After solvent was removed under vacuum, the product was
1
purified as a white solid by silica gel column chromatography, 0.13 g, yield 43%. H-NMR (400 MHz,
CD3OD):
H-indolyl), 4.50 (dd, J = 9.7, 5.5 Hz, 1H, CH), 4.12 (d, J = 3.6 Hz, 2H, CH2NH), 3.21 (dd, J = 18.5, 9.7
Hz, 1H, CH’H”), 2.85 (dd, J = 18.5, 5.5 Hz, 1H, CH’H”). 13C-NMR (101 MHz, CD3OD):
= 160.27,
δ = 7.30 (dd, J = 8.8, 4.5 Hz, 1H, H-indolyl), 6.93–6.88 (m, 1H, H-indolyl), 6.88–6.82 (m, 1H,
δ
157.92, 135.20, 130.85, 130.02, 113.00, 110.85, 110.54, 104.55, 104.31, 50.48, 38.34, 30.37. HRMS (ESI):
m/z [M + H]+ calculated for C13H13N3O3F: 278.09355; found: 278.09291.
3.1.2. Preparation of (R/S) 3-(5-fluoro-2-(methylsulfinyl)-1H-indol-3-yl)pyrrolidine-2,5-dione
(DX-02-07)
Preparation of 5-fluoroindoline-2-thione (3)
5-fluoroindolin-2-one (2 g, 13 mmol) and P2S5 (2.92 g, 13 mmol) were added to dry tetrahydrofuran
(30 mL). After the reaction mixture was stirred at room temperature for 1 h. Until the starting material
disappeared in thin layer chromatography (TLC), the NaHCO3 (3.43 g, 39 mmol) was added gradually.
After stirring at room temperature for 1 h, the reaction mixture was distilled in vacuo, dichloromethane
(50 mL) was added to the mixture. The organic extract was washed twice with saturated aqueous
NaCl (20 mL), and the organic extract was dried over Na2SO4. After solvent was removed under
vacuum, the product was obtained as yellow crystals by re-crystallization using ethanol, 1.62 g, yield
1
75%. H-NMR (400 MHz, CDCl3):
δ
= 10.14 (s, 1H, NH), 7.05–6.95 (m, 1H, H-phenyl), 6.94–6.89 (m, 1H,
= 203.39, 159.10, 140.24, 132.15, 114.87,
H-phenyl), 4.08 (s, 1H, CH2). 13C-NMR (101 MHz, CDCl3)):
112.47, 110.47, 49.15. HRMS (ESI): m/z [M + H]+ calculated for C8H7NFS: 168.02777; found: 168.02773.
δ
Preparation of 5-fluoro-2-(methylthio)-1H-indole (4)
5-fluoroindoline-2-thione (3, 1 g, 6 mmol), CH3I (0.99 g, 7 mmol) and K2CO3 (0.97 g, 7 mmol) were
added to acetone (30 mL), and the reaction mixture was stirred at room temperature until the starting
material disappeared in TLC. The reaction mixture was distilled in vacuo, ethyl acetate (50 mL) was
added to the mixture. The organic extract was washed twice with saturated aqueous NaCl (20 mL), and
the organic extract was dried over Na2SO4. After solvent was removed under vacuum, the product

Molecules 2019, 24, 2124
15 of 27
1
was purified as a white solid by silica gel column chromatography, 0.90 g, yield 83%. H-NMR (400
MHz, CDCl3): = 8.04 (s, 1H, NH), 7.23–7.13 (m, 2H, H-indolyl), 6.91 (s, 1H, H-indolyl), 6.49 (s, 1H,
δ
H-indolyl), 2.52 (s, 3H, CH3). 13C-NMR (101 MHz, CDCl3):
δ = 159.18, 156.85, 133.57, 129.02, 110.94,
110.47, 105.31, 104.73, 18.86. HRMS (ESI): m/z [M + H]+ calculated for C9H9NFS: 182.04342; found:
182.04425.
Preparation of (R/S) 3-(5-fluoro-2-(methylthio)-1H-indol-3-yl)pyrrolidine-2,5-dione (DX-02-06)
5-Fluoro-2-(methylthio)-1H-indole (4, 0.5 g, 2.7 mmol) and 1H-pyrrole-2,5-dione (0.54 g, 5.4 mmol)
were added to dry CH3CO2H (30 mL), and the reaction mixture was stirred at 120 ◦C until the starting
material disappeared in TLC. The reaction mixture was distilled in vacuo, ethyl acetate (100 mL) was
added to the mixture. The organic extract was washed twice with saturated aqueous NaCl (30 mL), and
the organic extract was dried over Na2SO4. After solvent was removed under vacuum, the product was
1
purified as a white solid by silica gel column chromatography, 0.47 g, yield 61%. H-NMR (400MHz,
CD3OD):
J = 9.2, 2.5 Hz, 1H, H-phenyl), 4.56 (dd, J = 9.8, 5.6 Hz, 1H, CH), 3.22 (dd, J = 18.3, 9.8 Hz, 1H, CH’H”),
2.77 (dd, J = 18.3, 5.6 Hz, 1H, CH’H”), 2.41 (s, 3H, CH3). 13C-NMR (101 MHz, CD3OD): δ = 182.38
δ = 7.28 (dd, J = 8.9, 4.5 Hz, 1H, H-phenyl), 7.00 (dd, J = 9.7, 2.4 Hz, 1H, H-phenyl), 6.91 (td,
,
180.11, 160.26, 135.05, 132.13, 127.67, 116.34, 113.18, 111.70, 103.25, 40.35, 39.10, 19.59. HRMS (ESI): m/z
[M + H]+ calculated for C13H12O2N2FS: 279.05980; found: 279.05930.
Preparation of (R/S) 3-(5-fluoro-2-(methylsulfinyl)-1H-indol-3-yl)pyrrolidine-2,5-dione (DX-02-07)
3-(5-Fluoro-2-(methylthio)-1H-indol-3-yl)pyrrolidine-2,5-dione (DX-02-06, 0.2 g, 0.7 mmol) was
added to dry dichloridemethane (20 mL), and 3-Chloroperoxybenzoic acid (0.15 g, 0.9 mmol) was
added gradually at 0 ◦C. The reaction mixture was stirred at 0 ◦C until the starting material disappeared
in TLC, and Na2S2O3 (0.11 g, 0.7 mmol) was added to the mixture. After reaction mixture was stirred
for 20 min, the reaction mixture was washed twice with saturated aqueous NaCl (20 mL), and the
organic extract was dried over Na2SO4. After solvent was removed under vacuum, the product was
1
purified as a white solid by silica gel column chromatography, 0.074 g, yield 35%. H-NMR (400MHz,
DMSO-d6):
δ = 12.28 (s, 1H, NH), 11.51 (s, 1H, NH), 7.51–7.48 (m, 1H, H-phenyl), 7.19–7.13 (m, 2H,
H-phenyl), 4.61 (ddd, J = 26.8, 9.7, 5.8 Hz, 1H, CH), 3.18 (ddd, J = 18.0, 9.7, 2.3 Hz, 1H, CH’H”), 2.98 (d,
J = 16.7 Hz, 3H, CH3), 2.76 (ddd, J = 41.8, 18.1, 5.8 Hz, 1H, CH’H”). 13C-NMR (101 MHz, DMSO-d6):
δ = 179.74, 177.94, 158.72, 138.29, 134.14, 125.83, 114.50, 113.93, 113.53, 104.46, 41.67, 38.62, 38.11. HRMS
(ESI): m/z [M + H]+ calculated for C13H12O3N2FS: 295.05472; found: 295.05508.
3.1.3. Preparation of N-(3-(2-(1H-imidazol-5-yl)phenoxy)propyl)-1,1,1-trifluoromethane sulfonamide
(DX-03-12)
Preparation of 4-iodo-1-trityl-1H-imidazole (5)
4-Iodo-1H-imidazole (5 g, 25.8 mmol) and N,N-diisopropylethylamine (6.65 g, 51.5 mmol) were
added to dry dimethylformamide (30 mL), and◦(chloromethanetriyl)tribenzene (7.89 g, 28.4 mm◦ol)
was added to reaction mixture gradually at 20 C. After the reaction mixture was stirred at 20 C
until the starting material disappeared in thin layer chromatography (TLC), the reaction mixture was
distilled in vacuo, ethyl acetate (200 mL) was added to the mixture. The organic extract was washed
twice with saturated aqueous NaCl (40 mL), and the organic extract was dried over Na2SO4. After
solvent was removed under vacuum, the product was purified as a white solid by silica gel column
1
chromatography, 8.77 g, yield 78%. H-NMR (400 MHz, CDCl3):
δ = 7.38–7.33 (m, 10H, H-phenyl,
H-imidazolyl), 7.13–7.09 (m, 6H, H-phenyl), 6.92 (d, J = 1.4 Hz, 1H, H-imidazolyl). 13C-NMR (101 MHz,
CDCl3): = 141.83, 140.56, 129.72 (6C), 128.29 (3C), 128.18 (6C), 127.91, 126.89 (3C), 75.84. HRMS (ESI):
m/z [M + H]+ calculated for C22H18N2I: 437.05092; found: 437.04934.
δ

Molecules 2019, 24, 2124
16 of 27
Preparation of 4-(2-methoxyphenyl)-1-trityl-1H-imidazole (6)
4-Iodo-1-trityl-1H-imidazole (5, 2 g, 4.58 mmol), (5-chloro-2-methoxyphenyl)boronic acid
(0.852 g, 4.58 mmol), K3PO4 (2.9 g, 13.7 mmol), Pd(PPh3)4 (0.74 g, 0.64 mmol) were added to
dry dimethylformamide (30 mL). The reaction mixture was stirred at 100 ◦C under inert atmosphere,
until the starting material disappeared in TLC. The reaction mixture was distilled in vacuo, and ethyl
acetate (100 mL) was added to the mixture. The organic extract was washed twice with saturated
aqueous NaCl (20 mL), and the organic extract was dried over Na2SO4. After solvent was removed
under vacuum, the product was purified as a white solid by silica gel column chromatography, 1.2 g,
1
yield 63%. H-NMR (400 MHz, CDCl3):
δ = 7.51–7.32 (m, 12H, H-phenyl, H-imidazolyl), 7.15–7.04 (m,
8H, H-phenyl, H-imidazolyl), 6.94 (d, J = 8.1 Hz, 1H, H-phenyl), 3.99 (s, 3H, CH3). HRMS (ESI): m/z
[M + H]+ calculated for C29H25N2O: 417.19614; found: 417.19647.
Preparation of 2-(1-trityl-1H-imidazol-4-yl)phenol (7)
4-(2-Methoxyphenyl)-1-trityl-1H-imidazole (
dichloridemethane (20 mL), BBr3 (0.71 g, 2.9 mmol) was added to reaction mixture gradually at
70 ◦C. The reaction mixture was stirred under inert atmosphere until the starting material disappeared
6,
1.0 g, 2.4 mmol) was added to dry
−
in TLC. The reaction mixture was diluted with dichloridemethane (50 mL), and was washed twice with
saturated aqueous NaCl (20 mL), then the organic extract was dried over Na2SO4. After solvent was
removed under vacuum, the product was purified as a white solid by silica gel column chromatography,
1
0.63 g, yield 65%. H-NMR (400 MHz, DMSO-d6):
δ = 11.19 (s, 1H, OH), 7.49–7.36 (m, 2H, H-phenyl),
7.33–7.10 (m, 16H, H-phenyl, H-imidazolyl), 6.84–6.70 (m, 2H, H-phenyl), 6.45 (s, 1H, H-phenyl).
13C-NMR (101 MHz, DMSO-d6):
δ = 147.24, 141.47, 137.70, 136.55, 128.71 (6C), 127.80 (6C), 127.56,
127.24 (3C), 126.99 (3C), 126.10, 118.39, 117.87, 115.68, 74.61. HRMS (ESI): m/z [M + H]+ calculated for
C28H23N2O: 403.18049; found: 403.18134.
Preparation of 2-(3-chloropropyl)isoindoline-1,3-dione (8)
3-Chloropropan-1-amine (3.77 g, 40.5 mmol) and isobenzofuran-1,3-dione (2 g, 13.5 mmol) were
mixed without solvent, and the reaction mixture was stirred at 140 ◦C (molten condition), until the
starting material disappeared in TLC. The reaction mixture was distilled in vacuo, ethyl acetate (100 mL)
was added to the mixture. The organic extract was washed twice with saturated aqueous NaCl (20 mL),
and the organic extract was dried over Na2SO4. After solvent was removed under vacuum, the product
1
was purified as a white solid by silica gel column chromatography, 1.66 g, yield 55%. H-NMR (400
MHz, DMSO-d6):
δ
= 7.85–7.78 (m, 4H, H-phenyl), 3.73–3.62 (m, 4H, CH2, CH2), 2.08–2.01(m, 2H,
= 167.38 (2C), 133.75 (2C), 131.19 (2C), 122.43 (2C), 42.31,
CH2). 13C-NMR (101 MHz, DMSO-d6):
δ
34.60, 30.39. HRMS (ESI): m/z [M + H]+ calculated for C11H11ClNO2: 224.04728; found: 224.04683.
Preparation of 2-(3-(2-(1-trityl-1H-imidazol-5-yl)phenoxy)propyl)isoindoline-1,3-dione (9)
2-(1-Trityl-1H-imidazol-4-yl)phenol (
to dry dimethylformamide (20 mL), after the reaction mixture was stirred at 20 C for 15 min,
2-(3-chloropropyl)isoindoline-1,3-dione (8
, 0.294 g, 1.32 mmol) was added to reaction mixture at 20 ◦C.
7
, 0.5 g, 1.2 mmol) and KOH (0.14 g, 2.5 mm◦ol) were added
After the reaction mixture was stirred at 60 ◦C until the starting material disappeared in thin layer
chromatography (TLC), the reaction mixture was distilled in vacuo, ethyl acetate (100 mL) was added
to the mixture. The organic extract was washed twice with saturated aqueous NaCl (20 mL), and the
organic extract was dried over Na2SO4. After solvent was removed under vacuum, the product was
1
purified as a white solid by silica gel column chromatography, 0.5 g, yield 71%. H-NMR (400 MHz,
DMSO-d6):
δ = 8.08 (dd, J = 7.7, 1.6 Hz, 1H, H-phenyl), 7.88–7.81 (m, 4H, H-phenyl, H-imidazolyl),
7.44–7.35 (m, 10H, H-phenyl), 7.17–7.13 (m, 7H, H-phenyl, H-imidazolyl), 7.12–7.08 (m, 1H, H-phenyl),
6.97 (t, J = 7.5 Hz, 1H, H-phenyl), 6.92 (d, J = 8.2 Hz, 1H, H-phenyl), 3.93 (t, J = 6.4 Hz, 2H, CH2), 3.43
(t, J = 6.5 Hz, 2H, CH2), 1.82–1.69 (m, 2H, CH2). 13C-NMR (101 MHz, DMSO-d6):
δ = 167.25, 154.09,

Molecules 2019, 24, 2124
17 of 27
141.77, 137.17, 134.72, 133.82, 131.11, 128.71 (6C), 128.65 (3C), 127.69, 127.66 (6C), 127.46, 127.36 (3C),
126.71, 125.92, 122.47, 121.86, 121.01, 120.60, 119.93, 111.33, 74.14, 64.42, 33.61, 27.54. HRMS (ESI): m/z
[M + H]+ calculated for C39H32N3O3: 590.24382; found: 590.24541.
Preparation of 3-(2-(1-trityl-1H-imidazol-4-yl)phenoxy)propan-1-amine (10)
2-(3-(2-(1-Trityl-1H-imidazol-4-yl)phenoxy)propyl)isoindoline-1,3-dione (9, 0.4 g, 0.68 mmol) and
35% hydrazine hydrate (0.12 g, 1.35 mmol) were added to CH3OH, and the reaction mixture was stirred
at 50 ◦C under inert atmosphere. After the starting material disappeared in thin layer chromatography
(TLC), the reaction mixture was distilled in vacuo, ethyl acetate (100 mL) was added to the mixture.
The organic extract was washed twice with saturated aqueous NaHCO3 and NaCl (20 mL) respectively,
and the organic extract was dried over Na2SO4. After solvent was removed under vacuum, the
unpurified product was added to the next step directly.
Preparation of 1,1,1-trifluoro-N-(3-(2-(1-trityl-1H-imidazol-4-yl)phenoxy)propyl) Methane
Sulfonamide (11)
3-(2-(1-Trityl-1H-imidazol-4-yl)phenoxy)propan-1-amine (crude 10, 0.3 g, 0.65 mmol) and
N,N-diisopropylethylamine (0.168 g, 1.3 mmol) were added to dry dichloridemethane (20 mL),
and trifluoromethanesulfonylchloride (0.12 g, 0.71 mmol) was added to reaction mixture gradually at
20 ◦C. After the starting material disappeared in thin layer chromatography (TLC), the reaction mixture
was distilled in vacuo, the reaction mixture was diluted with dichloridemethane (50 mL), and the
organic extract was washed twice with saturated aqueous NaCl (20 mL), and the organic extract was
dried over Na2SO4. After solvent was removed under vacuum, the product 11 was purified as a white
1
solid by silica gel column chromatography, 0.257 g, yield 67%. H-NMR (400 MHz, CDCl3):
δ = 7.80 (s,
1H, NH), 7.43–7.32 (m, 10H, H-phenyl, H-imidazolyl), 7.23–7.16 (m, 8H, H-phenyl, H-imidazol), 7.07 (d,
J = 1.5 Hz, 1H, H-phenyl), 6.97–6.86 (m, 2H, H-phenyl), 4.16 (t, J = 5.2 Hz, 2H, CH2), 3.57–3.46 (m, 2H,
CH2), 2.19–2.06 (m, 2H, CH2). 13C-NMR (101 MHz, CDCl3):
δ= 154.95, 142.08, 139.92, 138.22, 129.77
(6C), 128.56 (3C), 128.36, 128.15 (3C), 128.09 (6C), 121.68, 120.87, 119.04, 118.47, 111.86, 75.75, 67.83,
43.24, 29.80. HRMS (ESI): m/z [M + H]+ calculated for C32H29N3O3F3S: 592.18762; found: 592.18616.
Preparation of N-(3-(2-(1H-imidazol-5-yl)phenoxy)propyl)-1,1,1-trifluoromethane Sulfonamide
(DX-03-12)
1,1,1-Trifluoro-N-(3-(2-(1-trityl-1H-imidazol-4-yl)phenoxy)propyl)methanesulfonamide (11, 0.2 g,
0.34 mmol) and AcOH (0.5 mL) were added to CH3OH (10 mL), and the reaction mixture was stirred at
20 ◦C. After the starting material disappeared in thin layer chromatography (TLC), the reaction mixture
was distilled in vacuo, the product was purified as a white solid by silica gel column chromatography,
1
0.101 g, yield 86%. H-NMR (400 MHz, DMSO-d6):
δ =7.99 (d, J = 7.4 Hz, 1H, H-phenyl), 7.72 (s, 1H,
H-imidazolyl), 7.49 (s, 1H, H-imidazolyl), 7.18 (t, J = 7.8 Hz, 1H, H-phenyl), 7.03 (d, J = 8.1 Hz, 1H,
H-phenyl), 6.98 (t, J = 7.5 Hz, 1H, H-phenyl), 4.15 (t, J = 6.0 Hz, 2H, CH2), 3.40 (t, J = 6.8 Hz, 2H, CH2),
2.06–2.09 (m, 2H, CH2). 13C-NMR (101 MHz, DMSO-d6):
δ = 154.86, 135.47, 134.49, 127.46, 127.25,
124.99, 122.69, 120.97, 118.57, 112.34, 65.09, 41.39, 30.14. HRMS (ESI): m/z [M + H]+ calculated for
C13H15O3N3F3S: 350.07807; found: 350.07782.
3.1.4. Preparation of 4-chloro-2-(1-(4-(trifluoromethyl)benzyl)-1H-imidazol-4-yl) Phenol (DX-04-02)
Preparation of 4-iodo-1-(4-(trifluoromethyl)benzyl)-1H-imidazole (12)
4-Iodo-1H-imidazole (1 g, 5.2 mmol) and N,N-diisopropylethylamine (1.34 g, 10.4 mmol) were
added to dry dimethylformamide (20 mL), and 1-(bromomethyl)-4-(trifluoromethyl)benzene (1.48 g,
6.2 mmol) was added to reaction mixture at 20 ◦C. After the reaction mixture was stirred at 20 ◦C
until the starting material disappeared in thin layer chromatography (TLC), the reaction mixture was
distilled in vacuo, dichloromethane (50 mL) was added to the mixture. The organic extract was washed

Molecules 2019, 24, 2124
18 of 27
twice with saturated aqueous NaHCO3 and NaCl (20 mL) respectively, and the organic extract was
dried over Na2SO4. After solvent was removed under vacuum, the unpurified product was added to
the next step directly.
Preparation of 4-(5-chloro-2-methoxyphenyl)-1-(4-(trifluoromethyl)benzyl)-1H-imidazole (DX-04-01)
4-Iodo-1-(4-(trifluoromethyl)benzyl)-1H-imidazole (crude 12, 0.5 g, 1.4 mmol), (5-chloro-2-
methoxyphenyl)boronic acid (0.27 g, 1.4 mmol), K3PO4 (0.9 g, 4.3 mmol), Pd(PPh3)4 (0.23 g, 0.2 mmol)
were added to dry dimethylformamide (10 mL). The reaction mixture was stirred at 100 ◦C under
inert atmosphere, until the starting material disappeared in TLC. The reaction mixture was distilled in
vacuo, ethyl acetate (50 mL) was added to the mixture. The organic extract was washed twice with
saturated aqueous NaCl (20 mL), and the organic extract was dried over Na2SO4. After solvent was
removed under vacuum, DX-04-01 was purified as a white solid by silica gel column chromatography,
1
0.37 g, yield 72%. H-NMR (400 MHz, CD3OD):
δ = 7.94 (d, J = 2.5 Hz, 1H, H-phenyl), 7.81 (s, 1H,
H-imidazolyl), 7.65–7.63 (m, 3H, H-imidazolyl, H-phenyl), 7.40 (d, J = 8.0 Hz, 2H, H-phenyl), 7.15 (dd,
J = 8.7, 2.5 Hz, 1H, H-phenyl), 6.96 (d, J = 8.8 Hz, 1H, H-phenyl), 5.34 (s, 2H, CH2), 3.86 (s, 3H, CH3).
13C-NMR (101 MHz, CD3OD):
δ = 156.09, 142.76, 138.77, 137.13, 131.46, 129.05 (2C), 128.22, 127.44,
126.92 (2C), 124.85, 124.18, 121.78, 113.52, 112.48, 56.17, 51.22. HRMS (ESI): m/z [M + H]+ calculated
forC18H15ON2ClF3: 367.08195; found: 367.08374.
Preparation of 4-chloro-2-(1-(4-(trifluoromethyl)benzyl)-1H-imidazol-4-yl)phenol (DX-04-02)
4-(5-Chloro-2-methoxyphenyl)-1-(4-(trifluoromethyl)benzyl)-1H-imidazole (DX-04-01, 0.3 g,
0.82 mmol) was added to dry dichloridemethane (20 mL), BBr3 (0.24 g, 0.98 mmol) was added
to reaction mixture gradually at
70 ◦C. The reaction mixture was stirred under inert atmosphere until
−
the starting material disappeared in TLC. The reaction mixture was diluted with dichloridemethane
(50 mL), and was washed twice with saturated aqueous NaCl (20 mL), then the organic extract was
dried over Na2SO4. After solvent was removed under vacuum, DX-04-02 was purified as a white
1
solid by silica gel column chromatography, 0.15 g, yield 53%. H-NMR (400 MHz, CD3OD):
δ
= 7.85
(s, 1H, H-imidazolyl), 7.70–7.65 (m, 3H, H-imidazolyl, H-phenyl), 7.63 (d, J = 2.6 Hz, 1H, H-phenyl),
7.45 (d, J = 8.1 Hz, 2H, H-phenyl), 7.02 (dd, J = 8.7, 2.6 Hz, 1H, H-phenyl), 6.80 (d, J = 8.7 Hz, 1H,
H-phenyl), 5.37 (s, 2H, CH2). 13C-NMR (101 MHz, CD3OD):
δ = 155.21, 142.66, 140.49, 137.67, 129.87,
129.60, 129.12 (2C), 128.42, 126.89 (2C), 126.06, 125.01, 121.01, 118.79, 118.28, 51.17. HRMS (ESI): m/z
[M + H]+ calculated forC17H13ON2ClF3: 353.06630; found: 353.06635.
3.1.5. Preparation of 4-methyl-1H-indole-3-carboxylic Acid (DX-01-02)
Methyl 4-methyl-1H-indole-3-carboxylate (200 mg, 1.06 mmol) used hydrolysis reaction (2 N
NaOH water solution) to afford DX-01-02 (white solid, 151 mg, yield 82%). 1H-NMR (400 MHz,
CD3OD):
δ = 8.18 (dd, J = 3.9, 1.9 Hz, 1H, H-indolyl), 7.30 (d, J = 8.0 Hz, 1H, H-indolyl), 7.17 (t,
J = 7.7 Hz, 1H, H-indolyl), 7.01 (dd, J = 7.3, 0.7 Hz, 1H, H-indolyl), 2.77 (s, 3H, CH3). 13C-NMR
(101 MHz, CD3OD):
δ = 139.17, 133.89, 126.39, 125.96, 125.68, 120.61, 117.71, 112.19, 111.02, 23.30.
HRMS (ESI): m/z [M + H]+ calculated for C10H10NO2: 176.07061; found: 176.07129.
3.1.6. Preparation of 5-methyl-1H-indole-3-carboxylic Acid (DX-01-03)
Methyl 5-methyl-1H-indole-3-carboxylate (200 mg, 1.06 mmol) used hydrolysis reaction (2 N
NaOH water solution) to afford DX-01-03 (white solid, 150 mg, yield 81%). 1H-NMR (400 MHz,
CD3OD):
δ = 8.15 (d, J = 1.8 Hz, 1H, H-indolyl), 8.07 (s, 1H, H-indolyl), 7.39 (d, J = 8.3 Hz, 1H,
H-indolyl), 7.15 (dd, J = 8.3, 1.1 Hz, 1H, H-indolyl), 2.46 (s, 3H, CH3). 13C-NMR (101 MHz, CD3OD):
δ = 137.41, 134.35, 127.80, 127.00, 122.51, 120.09, 117.20, 113.08, 110.78, 21.77. HRMS (ESI): m/z [M + H]+
calculated for C10H10NO2: 176.07061; found: 176.07094.

Molecules 2019, 24, 2124
19 of 27
3.1.7. Preparation of 5-nitro-1H-indole-3-carboxylic Acid (DX-01-04)
Methyl 5-nitro-1H-indole-3-carboxylate (200 mg, 0.91 mmol) used hydrolysis reaction (2 N NaOH
1
water solution) to afford DX-01-04 (white solid, 172 mg, yield 92%). H-NMR (400 MHz, CD3OD):
δ = 8.99 (d, J = 2.2 Hz, 1H, H-indolyl), 8.14 (s, 1H, H-indolyl), 8.12 (dd, J = 9.0, 2.3 Hz, 1H, H-indolyl),
7.58 (d, J = 9.0 Hz, 1H, H-indolyl). 13C-NMR (101 MHz, CD3OD):
δ = 167.81, 144.32, 141.16, 136.51,
127.01, 118.93, 118.80, 113.45, 111.10, 49.00. HRMS (ESI): m/z [M + H]+ calculated for C9H7N2O4:
207.04003; found: 207.03940.
3.1.8. Preparation of 6-nitro-1H-indole-3-carboxylic Acid (DX-01-05)
Methyl 6-nitro-1H-indole-3-carboxylate (200 mg, 0.91 mmol) used hydrolysis reaction (2 N NaOH
1
water solution) to afford DX-01-05 (white solid, 168 mg, yield 90%). H-NMR (400 MHz, CD3OD):
δ = 8.38 (d, J = 1.5 Hz, 1H, H-indolyl), 8.22 (s, 1H, H-indolyl), 8.18 (d, J = 8.9 Hz, 1H, H-indolyl), 8.05
(dd, J = 8.8, 1.7 Hz, 1H, H-indolyl). 13C-NMR (101 MHz, CD3OD):
δ = 167.87, 144.97, 138.31, 136.78,
132.29, 122.19, 117.33, 109.89, 109.69. HRMS (ESI): m/z [M + H]+ calculated for C9H7N2O4: 207.04003;
found: 207.03949.
3.1.9. Preparation of 5-methoxy-1H-indole-3-carboxylic Acid (DX-01-06)
Methyl 5-methoxy-1H-indole-3-carboxylate (200 mg, 0.97 mmol) used hydrolysis reaction (2 N
NaOH water solution) to afford DX-01-06 (white solid, 140 mg, yield 75%). 1H-NMR (400 MHz,
CD3OD):
δ = 7.88 (s, 1H, H-indolyl), 7.57 (d, J = 2.4 Hz, 1H, H-indolyl), 7.31 (d, J = 8.8 Hz, 1H,
H-indolyl), 6.83 (dd, J = 8.8, 2.5 Hz, 1H, H-indolyl), 3.83 (s, 3H, CH3). 13C-NMR (101 MHz, CD3OD):
δ = 156.99, 133.58, 128.36, 115.37, 114.23, 113.88, 113.61, 104.51, 103.64, 56.06. HRMS (ESI): m/z [M + H]+
calculated for C10H10NO3: 192.06552; found: 192.06645.
3.1.10. Preparation of 7-bromo-1H-indole-3-carboxylic Acid (DX-01-07)
Methyl 7-bromo-1H-indole-3-carboxylate (200 mg, 0.79 mmol) used hydrolysis reaction (2 N
NaOH water solution) to afford DX-01-07 (white solid, 160 mg, yield 85%). 1H-NMR (400 MHz,
CD3OD): δ = 8.06 (dd, J = 8.0, 0.9 Hz, 1H, H-indolyl), 7.98 (s, 1H, H-indolyl), 7.36 (dd, J = 7.6, 0.7 Hz, 1H,
H-indolyl), 7.08 (t, J = 7.8 Hz, 1H, H-indolyl). 13C-NMR (101 MHz, CD3OD):
δ = 168.56, 138.29, 134.03,
129.07, 126.24, 123.59, 121.46, 117.02, 106.02. HRMS (ESI): m/z [M + H]+ calculated for C9H7NO2Br:
239.96547; found: 239.96452.
3.1.11. Preparation of (E) 1H-indole-3-carbaldehyde Oxime (DX-01-13)
1H-indole-3-carbaldehyde (200 mg, 1.38 mmol) following the similar procedure described for
1
the preparation of DX-02-04 afforded DX-01-13 (white solid, 158 mg, yield 72%). H-NMR (400 MHz,
DMSO-d6):
δ = 11.56 (s, 1H, OH), 11.17 (s, 1H, NH), 8.26 (s, 1H, =CH), 8.22 (d, J = 1.3 Hz, 1H, H-indolyl),
7.97 (d, J = 7.9 Hz, 1H, H-indolyl), 7.44 (d, J = 8.1 Hz, 1H, H-indolyl), 7.16 (t, J = 7.5 Hz, 2H, H-indolyl).
13C-NMR (101 MHz, CD3OD):
δ = 146.89, 138.66, 132.19, 127.98, 123.30, 121.40, 118.79, 112.59, 107.56.
HRMS (ESI): m/z [M + H]+ calculated for C9H9ON2: 161.07094; found: 161.07076.
3.1.12. Preparation of N-((1H-indol-3-yl)methyl)hydroxylamine (DX-01-14)
DX-01-13 (100 mg, 0.62 mmol) following the similar procedure described for the preparation of
1
DX-02-05 afforded DX-01-14 (white solid, 70 mg, yield 69%). H-NMR (400 MHz, DMSO-d6): δ = 10.87
(s, 1H, NH-indolyl), 7.58 (d, J = 7.8 Hz, 1H, H-indolyl), 7.34 (d, J = 8.1 Hz, 1H, H-indolyl), 7.29 (br, 1H,
NH), 7.24 (s, 1H, H-indolyl), 7.06 (t, J = 8.0 Hz, 1H, H-indolyl), 6.99–6.95 (m, 1H, H-indolyl), 5.66 (br, 1H,
OH), 4.04 (s, 2H, CH2). 13C-NMR (101 MHz, CD3OD):
δ
= 138.02, 128.61, 125.94, 122.58, 120.11, 119.41,
112.28, 110.24, 55.80. HRMS (ESI): m/z [M + H]+ calculated for C9H11ON2: 163.08659; found: 163.08611.

Molecules 2019, 24, 2124
20 of 27
3.1.13. Preparation of N-((5-(o-tolyl)-1H-indol-3-yl)methyl)hydroxylamine (DX-01-15)
5-Bromo-1H-indole-3-carbaldehyde (300 mg, 1.34 mmol) and o-tolylboronic acid (183 mg,
1.34 mmol) following the similar procedure described for the preparation of DX-03-12 and DX-02-05
1
afforded DX-01-15 (white solid, 145 mg, yield 43% from 5-bromo-1H-indole-3-carbaldehyde). H-NMR
(400 MHz, CD3OD):
δ
= 7.57–7.39 (m, 3H, H-phenyl, H-indolyl), 7.26–7.04 (m, 5H, H-phenyl, H-indolyl),
= 144.63, 137.29, 136.60, 135.38,
5.13 (s, 2H, CH2), 2.24 (s, 3H, CH3). 13C-NMR (101 MHz, CD3OD):
δ
131.19, 131.12, 128.56, 127.79, 126.62, 125.69, 124.87, 119.47, 112.33, 107.61, 62.12, 29.31. HRMS (ESI): m/z
[M + H]+ calculated for C16H17N2O: 253.13354; found: 253.13471.
3.1.14. Preparation of (E)-1H-indole-2-carbaldehyde Oxime (DX-01-16)
1H-indole-2-carbaldehyde (200 mg, 1.38 mmol) following the similar procedure described for
1
the preparation of DX-02-04 afforded DX-01-16 (white solid, 117 mg, yield 53%). H-NMR (400 MHz,
DMSO-d6):
δ = 11.29 (s, 1H, OH), 11.17 (s, 1H, NH), 8.14 (s, 1H, =CH), 7.52 (d, J = 8.0 Hz, 1H, H-indolyl),
7.37 (d, J = 8.2 Hz, 1H, H-indolyl), 7.12 (t, J = 7.6 Hz, 1H, H-indolyl), 6.98 (t, J = 7.5 Hz, 1H, H-indolyl),
6.56 (s, 1H, H-indolyl). 13C-NMR (101 MHz, CD3OD):
δ = 142.67, 138.93, 132.77, 129.48, 124.71, 122.20,
120.89, 112.84, 108.61. HRMS (ESI): m/z [M + H]+ calculated for C9H9ON2: 161.07094; found: 161.07077.
3.1.15. Preparation of N-((1H-indol-2-yl)methyl)hydroxylamine (DX-01-17)
DX-01-16 (60 mg, 0.37 mmol) following the similar procedure described for the preparation of
1
DX-02-05 afforded DX-01-17 (white solid, 39 mg, yield 65%). H-NMR (400 MHz, DMSO-d6):
δ = 10.88
(s, 1H, NH-indolyl), 7.46–7.39 (m, 2H, H-indolyl, NH), 7.32 (d, J = 8.3 Hz, 1H, H-indolyl), 7.03–6.97
(m, 1H, H-indolyl), 6.93 (t, J = 8.1 Hz, 1H), 6.28 (s, 1H, H-indolyl), 6.17 (br, 1H, OH), 4.00 (s, 2H, CH2).
13C-NMR (101 MHz, CD3OD):
δ = 137.98, 136.56, 122.09, 120.86, 120.84, 120.01, 111.84, 101.86, 51.98.
HRMS (ESI): m/z [M + H]+ calculated for C9H11ON2: 163.08659; found: 163.08636.
3.1.16. Preparation of (E)-3-((1H-indol-3-yl)methylene)pyrrolidine-2,5-dione (DX-02-01)
1H-indole-3-carbaldehyde (500 mg, 3.45 mmol) and 1H-pyrrole-2,5-dione (401 mg, 4.14 mmol)
following the similar procedure described for the preparation of DX-02-04 afforded DX-02-01 (white
1
solid, 398 mg, yield 51% from 1H-indole-3-carbaldehyde). H-NMR (400 MHz, DMSO-d6):
δ = 11.94 (s,
1H, NH-indolyl), 11.18 (s, 1H, NH), 7.81 (dd, J = 12.1, 5.1 Hz, 2H, H-indolyl, =CH), 7.70 (t, J = 2.1 Hz,
1H, H-indolyl), 7.54–7.41 (m, 1H, H-indolyl), 7.21–7.16 (m, 2H, H-indolyl), 3.52 (d, J = 2.2 Hz, 2H).
HRMS (ESI): m/z [M + H]+ calculated for C13H11O2N2: 227.08150; found: 227.08134.
3.1.17. Preparation of (R/S) 3-((1H-indol-3-yl)methyl)pyrrolidine-2,5-dione (DX-02-02)
DX-02-01 (200 mg, 3.45 mmol) following the similar procedure described for the preparation
of DX-02-04 afforded DX-02-02 (white solid, 65 mg, yield 32%). 1H-NMR (400 MHz, DMSO-d6):
δ = 11.06 (s, 1H, NH-indolyl), 10.91 (s, 1H, NH), 7.51 (d, J = 7.8 Hz, 1H, H-indolyl), 7.33 (d, J = 8.0 Hz
,
1H, H-indolyl), 7.16 (s, 1H, H-indolyl), 7.06 (t, J = 7.5 Hz, 1H, H-indolyl), 6.97 (t, J = 7.5 Hz, 1H,
H-indolyl), 3.20–3.12 (m, 1H, CH’H”), 2.95 (dd, J = 14.0, 8.5 Hz, 1H, CH’H”), 2.70–2.61 (m, 2H,
H-pyrrolidine-2,5-dione), 2.34 (dd, J = 18.0, 3.5 Hz, 1H, H-pyrrolidine-2,5-dione). HRMS (ESI): m/z
[M + H]+ calculated for C13H13N2O2: 229.09715; found: 229.09663
3.1.18. Preparation of (E) 2-(1H-imidazol-4-yl)benzaldehyde Oxime (DX-03-02)
Compound 5 (400 mg, 0.92 mmol) and (2-formylphenyl)boronic acid (138 mg, 0.92 mmol) following
the similar procedure described for the preparation of DX-03-12 and DX-02-04 afforded DX-03-02
(white solid, 98 mg, yield 57% from (2-formylphenyl)boronic acid). 1H-NMR (400 MHz, CD3OD):
δ
= 8.32 (s, 1H, =CH), 7.85–7.84 (m, 1H, H-phenyl), 7.79 (s, 1H, H-imidazolyl), 7.52 (d, J = 7.7 Hz,
1H, H-phenyl), 7.42–7.38 (m, 1H, H-phenyl), 7.34–7.30 (m, 1H, H-phenyl), 7.12 (s, 1H, H-imidazolyl).

Molecules 2019, 24, 2124
21 of 27
13C-NMR (100 MHz, DMSO-d6):
δ
= 148.70, 136.36, 133.83, 130.10, 129.50, 129.27, 128.72, 128.34, 127.09,
126.20. HRMS (ESI): m/z [M + H]+ calculated for C10H10ON3: 188.08184; found: 188.08191.
3.1.19. Preparation of N-(2-(1H-imidazol-4-yl)benzyl)hydroxylamine (DX-03-03)
Compound DX-03-02 (50 mg, 0.27 mmol) following the similar procedure described for the
preparation of DX-02-04 afforded DX-03-03 (white solid, 32 mg, yield 63%). 1H-NMR (400 MHz,
CD3OD): δ = 7.75 (s, 1H, H-imidazolyl), 7.53 (d, J = 1.7 Hz, 1H, H-phenyl), 7.47–7.44 (m, 1H, H-phenyl),
7.35–7.27 (m, 3H, H-imidazolyl, H-phenyl), 4.04 (s, 2H). HRMS (ESI): m/z [M + H]+ calculated for
C10H12ON3: 190.09749; found: 190.09737.
3.1.20. Preparation of (E) 2-(2H-tetrazol-5-yl)benzaldehyde Oxime (DX-03-04)
2-(2H-tetrazol-5-yl)benzaldehyde (200 mg, 1.15 mmol) following the similar procedure described
for the preparation of DX-03-12 and DX-02-04 afforded DX-03-04 (white solid, 78 mg, yield 36% from
1
2-(2H-tetrazol-5-yl)benzaldehyde). H-NMR (400 MHz, DMSO-d6):
δ = 11.23 (s, 1H, OH), 11.07 (s,
1H, H-tetrazolyl), 8.29 (s, 1H, =CH), 7.73–7.56 (m, 4H, H-phenyl). 13C-NMR (101 MHz, DMSO-d6):
δ = 156.17, 137.81, 132.14, 130.64, 129.27, 127.64, 116.84, 110.69. HRMS (ESI): m/z [M + H]+ calculated
for C8H8N5O: 190.07234; found: 190.07158.
3.1.21. Preparation of (E) 5-chloro-2-(1H-imidazol-4-yl)benzaldehyde Oxime (DX-03-05)
Compound 5 (300 mg, 0.69 mmol) and (4-chloro-2-formylphenyl)boronic acid (127 mg, 0.69 mmol)
following the similar procedure described for the preparation of DX-03-12 and DX-02-04 afforded
DX-03-05 (white solid, 70 mg, yield 46% from (4-chloro-2-formylphenyl)boronic acid). 1H-NMR
(400 MHz, DMSO-d6):
δ = 12.38 (s, 1H, OH), 11.32 (s, 1H, NH-imidazolyl), 8.77 (s, 1H, =CH), 7.80
(s, 1H, H-imidazolyl), 7.74 (s, 1H, H-imidazolyl), 7.63 (d, J = 2.4 Hz, 1H, H-phenyl), 7.44–7.42 (m,
2H, H-phenyl). 13C-NMR (101 MHz, DMSO-d6):
δ = 147.77, 138.56, 136.60, 133.12, 131.72, 131.48,
131.05, 129.25, 125.42, 116.28. HRMS (ESI): m/z [M + H]+ calculated for C10H9ClN3O: 222.04287;
found: 222.04330.
3.1.22. Preparation of (E) 2-fluoro-6-(1H-imidazol-4-yl)benzaldehyde Oxime (DX-03-06)
Compound 5 (300 mg, 0.69 mmol) and (3-fluoro-2-formylphenyl)boronic acid (116 mg, 0.69 mmol)
following the similar procedure described for the preparation of DX-03-12 and DX-02-04 afforded
DX-03-06 (white solid, 75 mg, yield 53% from (3-fluoro-2-formylphenyl)boronic acid). 1H-NMR
(400 MHz, DMSO-d6):
δ = 12.37 (s, 1H, OH), 11.24 (s, 1H, NH-imidazolyl), 8.37 (s, 1H, =CH), 7.76
(s, 1H, H-imidazolyl), 7.52 (s, 1H, H-imidazolyl), 7.36–7.13 (m, 3H, H-phenyl). 13C-NMR (101 MHz,
DMSO-d6):
δ = 161.48, 159.00, 144.68, 136.29, 130.27, 124.36, 122.95, 117.89, 116.35, 113.98. HRMS (ESI):
m/z [M + H]+ calculated for C10H9FN3O: 206.07242; found: 206.07250.
3.1.23. Preparation of (E) 2-(1H-imidazol-4-yl)-5-methoxybenzaldehyde Oxime (DX-03-07)
Compound
5 (300 mg, 0.69 mmol) and (2-formyl-4-methoxyphenyl)boronic acid (124 mg,
0.69 mmol) following the similar procedure described for the preparation of DX-03-12 and DX-02-04
afforded DX-03-07 (white solid, 113 mg, yield 76% from (2-formyl-4-methoxyphenyl)boronic acid).
1H-NMR (400 MHz, CD3OD):
δ
= 8.73 (s, 1H, H-imidazolyl), 7.78 (s, 1H, H-imidazolyl), 7.52–7.38
(m, 3H, H-phenyl), 7.15 (s, 1H, H-phenyl), 7.10–6.96 (m, 4H, H-phenyl), 3.85 (s, 3H, CH3). 13C-NMR
(101 MHz, CD3OD): = 141.55, 137.87, 133.07, 132.61, 121.44, 118.60, 117.79, 116.37, 113.90, 110.95,
55.90. HRMS (ESI): m/z [M + H]+ calculated for C11H12N3O2: 218.09240; found: 218.09206.
δ

Molecules 2019, 24, 2124
22 of 27
3.1.24. Preparation of (E) 4-(1H-imidazol-4-yl)-20-(trifluoromethyl)-[1,10-biphenyl]-3-carbaldehyde
Oxime (DX-03-08)
Compound
5
(300 mg, 0.69 mmol) and (3-formyl-20-(trifluoromethyl)-[1,10-biphenyl]-4-yl)
boronic acid (202 mg, 0.69 mmol) following the similar procedure described for the
preparation of DX-03-12 and DX-02-04 afforded DX-03-08 (white solid, 82mg, yield 36% from
(3-formyl-20-(trifluoromethyl)-[1,10-biphenyl]-4-yl)boronic acid). 1H-NMR (400 MHz, DMSO-d6):
δ
= 12.38 (s, 1H, OH), 11.14 (s, 1H, NH-imidazolyl), 8.85(s, 1H, H-imidazolyl), 7.86–7.79 (m, 2H,
H-imidazolyl, H-phenyl), 7.73–7.63 (m, 4H, H-phenyl), 7.46 (br, 2H, H-phenyl), 7.33 (s, 1H, H-phenyl).
13C-NMR (101 MHz, DMSO-d6):
δ = 148.54, 140.58, 137.83, 136.56, 132.89, 132.57, 130.11, 129.84, 129.53,
128.94, 128.64, 126.58, 126.32, 126.23, 126.04, 123.22, 116.04. HRMS (ESI): m/z [M + H]+ calculated for
C17H13F3N3O: 332.10052; found: 332.10123.
3.1.25. Preparation of (E) 3-(1H-imidazol-4-yl)-[1,10-biphenyl]-4-carbaldehyde Oxime (DX-03-09)
Compound
5
(300 mg, 0.69 mmol) and (4-formyl-[1,10-biphenyl]-3-yl)boronic acid (156 mg,
0.69 mmol) following the similar procedure described for the preparation of DX-03-12 and DX-02-04
afforded DX-03-09 (white solid, 81 mg, yield 45% from (4-formyl-[1,10-biphenyl]-3-yl)boronic acid).
1H-NMR (400 MHz, DMSO-d6):
δ = 11.01 (s, 1H, OH), 10.34 (s, 1H, NH-imidazolyl), 8.55 (s, 1H,
=CH), 8.39 (s, 1H, H-imidazolyl), 8.12 (s, 1H, H-imidazolyl), 7.58 (br, 1H, H-phenyl), 7.48–7.22 (m, 2H,
H-phenyl), 7.11–7.02 (m, 2H, H-phenyl), 6.87–6,81 (m, 2H, H-phenyl), 6.50 (s, 1H, H-phenyl). HRMS
(ESI): m/z [M + H]+ calculated for C16H14N3O: 264.11314; found: 264.11346.
3.1.26. Preparation of 4-(2-((4-(trifluoromethyl)benzyl)oxy)phenyl)-1,2,3-thiadiazole (DX-03-10)
2-(1,2,3-Thiadiazol-4-yl)phenol (100 mg, 0.56 mmol) and 1-(bromomethyl)-4-(trifluoromethyl)benzene
(134 mg, 0.56 mmol) following the similar procedure described for the preparation of 12 afforded DX-03-10
1
(white solid, 128 mg, yield 68%). H-NMR (400 MHz, DMSO-d6):
δ = 9.39 (s, 1H, H-thiadiazolyl), 8.26 (d, J
= 7.5 Hz, 1H, H-phenyl), 7.76 (d, J = 8.2 Hz, 2H, H-phenyl), 7.70 (d, J = 8.5 Hz, 2H, H-phenyl), 7.48–7.43
(m, 1H, H-phenyl), 7.31 (d, J = 7.7 Hz, 1H, H-phenyl), 7.17 (t, J = 7.5 Hz, 1H, H-phenyl), 5.44 (s, 2H, CH2).
HRMS (ESI): m/z [M + H]+ calculated for C16H12F3N2OS: 337.06169; found: 337.06116.
3.1.27. Preparation of N-(3-(2-(1H-imidazol-5-yl)phenoxy)propyl)-aminesulfonamide (DX-03-11)
Compound
9
(200 mg, 0.34 mmol) following the similar procedure described for the preparation
1
of DX-03-12 afforded DX-03-11 (white solid, 45 mg, yield 45% from compound
CD3OD):
9). H-NMR (400 MHz,
δ
= 8.67 (s, 1H, NH-imidazolyl), 8.08 (s, 1H, H-imidazolyl), 7.83 (s, 1H, H-imidazolyl), 7.69 (d,
J = 7.7 Hz, 1H, H-phenyl), 7.40 (t, J = 7.1 Hz, 1H, H-phenyl), 7.16 (d, J = 8.2 Hz, 1H, H-phenyl), 7.08 (t,
J = 7.4 Hz, 1H, H-phenyl), 4.20 (t, J = 6.0 Hz, 2H, CH2), 3.44 (t, J = 6.9 Hz, 2H, CH2), 2.11–2.03 (m, 2H,
CH2). HRMS (ESI): m/z [M + H]+ calculated for C12H17O3N4S: 297.10159; found: 297.10115.
3.1.28. Preparation of 1-(3-(2-(1H-imidazol-5-yl)phenoxy)propyl)-3-(4-(trifluoromethyl) phenyl)urea
(DX-03-13)
Compound
9
(200 mg, 0.34 mmol) following the similar procedure described for the preparation
1
of DX-03-12 afforded DX-03-13 (white solid, 77 mg, yield 56% from compound
DMSO-d6):
9). H-NMR (400 MHz,
δ
= 12.24 (s, 1H, NH-imidazolyl), 8.90 (s, 1H, H-imidazolyl), 8.01 (d, J = 7.4 Hz, 1H,
H-phenyl), 7.75 (s, 1H, H-imidazolyl), 7.61–7.54 (m, 5H, H-phenyl), 7.21–7.15 (m, 1H, H-phenyl), 7.06
(d, J = 7.8 Hz, 1H, H-phenyl), 7.02–6.94 (m, 1H, H-phenyl), 6.48 (t, J = 5.7 Hz, 1H, H-phenyl), 4.14 (t,
J = 6.1 Hz, 2H, CH2), 3.38–3.32 (m, 2H, CH2), 2.08–1.97 (m, 2H, CH2). 13C-NMR (101 MHz, DMSO-d6):
δ
= 155.42, 155.08, 144.71, 135.42, 129.17, 127.38, 127.13, 126.38 (2C), 123.78, 122.81, 121.53, 121.21,
120.86, 117.71 (2C), 112.33, 65.82, 36.89, 29.98. HRMS (ESI): m/z [M + H]+ calculated for C20H20O2N4F3:
405.15329; found: 405.15302.

Molecules 2019, 24, 2124
23 of 27
3.1.29. Preparation of 1-(3-bromo-4-fluorobenzyl)-4-(5-chloro-2-methoxyphenyl)-1H-imidazole
(DX-04-03)
2-Bromo-4-(bromomethyl)-1-fluorobenzene (500 mg, 1.88 mmol) following the similar procedure
described for the preparation of DX-04-02 afforded DX-04-03 (white solid, 480 mg, yield 65% from
1
2-bromo-4-(bromomethyl)-1-fluorobenzene). H-NMR (400 MHz, CD3OD):
δ = 7.95 (d, J = 2.7 Hz,
1H, H-phenyl), 7.78 (d, J = 1.3 Hz, 1H, H-imidazolyl), 7.64 (d, J = 1.3 Hz, 1H, H-imidazolyl), 7.57 (dd,
J = 6.5, 2.2 Hz, 1H, H-phenyl), 7.30–7.24 (m, 1H, H-phenyl), 7.20 (d, J = 8.5 Hz, 1H, H-phenyl), 7.16 (dd,
J = 8.8, 2.7 Hz, 1H, H-phenyl), 6.98 (d, J = 8.8 Hz, 1H, H-phenyl), 5.22 (s, 2H, CH2), 3.88 (s, 3H, CH3).
13C-NMR (101 MHz, CD3OD):
δ = 161.35, 158.96, 156.10, 138.33, 137.40, 133.91, 129.70, 128.04, 127.36,
126.81, 125.19, 121.53, 117.84, 113.47, 110.01, 56.13, 50.30. HRMS (ESI): m/z [M + H]+ calculated for
C17H14ON2BrClF: 394.99566; found: 394.99728.
3.1.30. Preparation of 2-(1-(3-bromo-4-fluorobenzyl)-1H-imidazol-4-yl)-4-chlorophenol (DX-04-04)
DX-04-03 (200 mg, 0.51 mmol) following the similar procedure described for the preparation
of DX-04-02 afforded DX-04-04 (white solid, 116 mg, yield 60%). 1H-NMR (400 MHz, DMSO-d6):
δ = 9.12 (s, 1H, H-imidazolyl), 8.40 (s, 1H, H-imidazolyl), 8.01 (s, 1H, H-imidazolyl), 7.89 (dd, J = 6.5,
1.7 Hz, 1H, H-phenyl), 7.56–7.49 (m, 1H, H-phenyl), 7.45 (t, J = 8.6 Hz, 1H, H-phenyl), 7.34 (dd, J =
8.8, 2.5 Hz, 1H, H-phenyl), 7.00 (d, J = 8.8 Hz, 1H, H-phenyl), 5.42 (s, 2H, CH2). 13C-NMR (101 MHz,
CD3OD): δ = 161.85, 159.35, 154.93, 136.62, 134.79, 130.61, 130.34, 126.90, 125.53, 119.70, 118.77, 118.32,
118.09, 117.92, 110.27, 51.71. HRMS (ESI): m/z [M + H]+ calculated for C16H12ON2BrClF: 380.98001;
found: 380.98065.
3.1.31. Preparation of 4-chloro-2-(1-(4-(trifluoromethyl)benzyl)-1H-1,2,3-triazol-4-yl)phenol (DX-04-05)
DX-04-06 (300 mg, 0.81 mmol) following the similar procedure described for the preparation
of DX-04-02 afforded DX-04-05 (white solid, 176 mg, yield 61%). 1H-NMR (400 MHz, DMSO-d6):
δ = 10.49 (s, 1H, OH), 7.85 (s, 1H, H-triazolyl), 7.64 (d, J = 8.1 Hz, 2H, H-phenyl), 7.34 (dd, J = 8.8, 2.7
Hz, 1H, H-phenyl), 7.20 (d, J = 8.1 Hz, 2H, H-phenyl), 7.16 (d, J = 2.7 Hz, 1H, H-phenyl), 6.98 (d, J = 8.8
Hz, 1H, H-phenyl), 5.62 (s, 2H, CH2). 13C-NMR (101 MHz, CD3OD):
δ = 155.39, 140.99, 136.49, 134.75,
132.46, 131.76, 131.11, 129.36 (2C), 126.49 (2C), 125.38, 124.09, 118.30, 116.15, 53.27. HRMS (ESI): m/z
[M + H]+ calculated for C16H12ON3ClF3: 354.06155; found: 354.06015.
3.1.32. Preparation of 4-(5-chloro-2-methoxyphenyl)-1-(4-(trifluoromethyl)benzyl)-1H-1,2,3-triazole
(DX-04-06)
4-chloro-2-ethynyl-1-methoxybenzene (500 mg, 3.01 mmol) and 1-(bromomethyl)-4-
(trifluoromethyl)benzene (717 mg, 3.01 mmol) following the similar procedure described
for the preparation of DX-04-02 afforded DX-04-06 (white solid, 675 mg, yield 61% from
1
4-chloro-2-ethynyl-1-methoxybenzene). H-NMR (400 MHz, CD3OD):
δ
= 7.71 (s, 1H, H-triazolyl), 7.50
(d, J = 8.1 Hz, 2H, H-phenyl), 7.40 (dd, J = 8.9, 2.7 Hz, 1H, H-phenyl), 7.14 (d, J = 8.1 Hz, 2H, H-phenyl),
7.09 (d, J = 2.6 Hz, 1H, H-phenyl), 7.03 (d, J = 8.9 Hz, 1H, H-phenyl), 5.55 (s, 2H, CH2), 3.64 (s, 3H,
CH3). 13C-NMR (101 MHz, CD3OD):
δ = 154.74, 143.61, 131.20, 130.91, 129.93 (2C), 129.81, 129.39,
127.59, 127.02 (2C), 125.98, 125.64, 118.63, 117.43, 55.31, 55.04. HRMS (ESI): m/z [M + H]+ calculated for
C17H14ON3ClF3: 368.07720; found: 368.07599.
3.1.33. Preparation of 4-chloro-2-(5-(4-(trifluoromethyl)phenyl)-1H-1,2,3-triazol-4-yl)phenol (DX-04-07)
DX-04-08 (200 mg, 0.57 mmol) following the similar procedure described for the preparation of
1
DX-04-02 afforded DX-04-07 (white solid, 144 mg, yield 75%). H-NMR (400 MHz, DMSO-d6):
δ = 15.28
(s, 1H, H-triazolyl), 9.91 (s, 1H, NH-triazolyl), 7.75–7.68 (m, 4H, H-phenyl), 7.37 (br, 2H, H-phenyl),
6.94 (s, 1H, H-phenyl). 13C-NMR (101 MHz, CD3OD):
= 155.58, 142.95, 137.77, 136.14, 131.62, 131.27,
δ

Molecules 2019, 24, 2124
24 of 27
130.86, 128.63 (2C), 126.43 (2C), 125.31, 124.28, 119.14, 118.53. HRMS (ESI): m/z [M + H]+ calculated for
C15H10ON3ClF3: 340.04590; found: 340.04453.
3.1.34. Preparation of 4-(5-chloro-2-methoxyphenyl)-5-(4-(trifluoromethyl)phenyl)-1H-1,2,3-triazole
(DX-04-08)
4-chloro-2-ethynyl-1-methoxybenzene (300 mg, 1.81 mmol) and 1-iodo-4-(trifluoromethyl)benzene
(491 mg, 1.81 mmol) following the similar procedure described for the preparation of DX-04-02 afforded
DX-04-08 (white solid, 338 mg, yield 53% from 4-chloro-2-ethynyl-1-methoxybenzene). 1H-NMR
(400 MHz, DMSO-d6):
δ = 15.41 (s, 1H, NH-triazolyl), 7.74 (d, J = 8.1 Hz, 2H, H-phenyl), 7.68–7.59
(m, 2H, H-phenyl), 7.54 (d, J = 8.9 Hz, 1H, H-phenyl), 7.48 (d, J = 2.7 Hz, 1H, H-phenyl), 7.15 (s, 1H,
H-phenyl), 3.42 (s, 3H, CH3). 13C-NMR (101 MHz, CD3OD):
δ = 157.20, 136.42, 131.71, 131.59, 131.14,
129.65, 128.37 (2C), 126.96, 126.75, 126.40 (2C), 124.27, 121.57, 114.17, 55.99. HRMS (ESI): m/z [M + H]+
calculated for C16H12ON3ClF3: 354.06155; found: 354.06079.
3.2. Pharmacological Method
3.2.1. Enzyme-Based IDO1s or TDOs Activity Assay
The compounds on IDO1 or TDO activity were determined as follows [16
reaction mixture (100 L) containing 100 mM potassium phosphate buffer (pH 6.5), 40 mM ascorbic acid
(neutralized with NaOH), 200 g/mL catalase, 20 M methylene blue and 0.05 M rhIDO1 or rhTDO
was added to the solution containing the substrate l-tryptophan and the test sample at a determined
concentration. The reaction was carried out at 37 ◦C for 45 min and stopped by adding 20
L of 30% (w/v)
trichloroacetic acid. After heating at 65 ◦C for 15 min, 100
L of 2% (w/v) p-dimethylaminobenzaldehyde
,24,25]: A standard
µ
µ
µ
µ
µ
µ
in acetic acid was added to each well. The yellow pigment derived from kynurenine was measured at
492 nm using a SYNERGY-H1 microplate reader (Biotek Instruments, Inc., Winooski, VT, USA). IC50
was analyzed using the GraphPad Prism 8.0 software (GraphPad Software, San Diego, CA, USA).
3.2.2. Cell Culture
B16-F10 mouse melanoma cells, NCI-H460 human lung cancer cell line and MCF7 were obtained
from American Type Culture Collection (ATCC). All the cell lines were cultured with Dulbecco’
modified Eagle’s medium (DMEM) (Invitrogen Corporation, Waltham, MA, USA) supplemented
with 10% fetal bovine serum (FBS) (Hyclone, Waltham, MA, USA) and 1% penicillin streptomycin
(Invitrogen) at 37 ◦C in 5% CO2.
3.2.3. Cell Viability Assay
The in vitro inhibitory effects of the compounds were measured by MTT assay [26]. Briefly,
different kinds of cells were plated in 96-well plates at a density of 2000/well. After attachment for
overnight at 37 ◦C, the cells were treated with the compounds at various concentrations. After 72 h
culture, the MTT reagent was added to each well, and plates were incubated for another 4 h at 37 ◦C.
Then, the blue-purple crystal formamidine was dissolved in DMSO after discarding the supernatant.
Samples were measured using microplate reader at a wavelength of 570 nm (Biotek Instruments, Inc.,
Winooski, VT, USA). IC50 values were calculated with GraphPad Prism 8.0 software.
3.2.4. Mice
C57BL/6 mice were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd.
(Beijing Vital River Laboratory Animal Technology Co., Ltd, Beijing, China). Studies involving mice
were approved by the Experimental Animal Management and Welfare Committee at the Institute of
Materia Medica, Peking Union Medical College.

Molecules 2019, 24, 2124
25 of 27
3.2.5. Pharmacokinetic Studies
The animal Care and Welfare Committee of Institute of Materia Medica, Chinese Academy of
Medical Sciences approved all animal care, housing, and laboratory procedures. Male ICR mice
were used in the single dose pharmacokinetic studies. Compound DX-03-12 was prepared as
3 mg/mL suspension with 0.5% CMC (containing 0.3% Tween 80) for oral use and was formulated
as 3 mg/mL solution with 5% DMSO in saline for intravenous injection. Twenty mice were divided
into two groups, 10 in each group. After fasting 12 h with free access to water, mice were treated
with a 3 mg/kg i.v. or 30 mg/kg oral dose of compound DX-03-12. Blood samples (50 µL) were
collected at 5, 15, 30 min, 1, 2, 4, 6, 8, 12 and 24 h after oral administration and 2, 5, 15, 30 min,
1, 2, 4, 6, 8, 12 and 24 h after intravenous injection. After centrifugation, the plasma samples
(15
chromatography/tandem mass spectrometry (Agilent Technologies, Santa Clara, CA, USA) with a
Zobax C18 column (50 mm 2.1 mm, 3.5 m). Compound detection was performed with the mass
spectrometer in MRM (multiple reaction monitoring, Agilent Technologies, Santa Clara, CA, USA)
negative ionization mode. The selected reaction monitoring transitions were m/z 348.5 160 for
µL) were precipitated by five volumes of acetonitrile. The supernatant were analyzed by liquid
×
µ
→
compound DX-03-12. The pharmacokinetic parameters were calculated with WinNonlin software V6.3
using non-compartmental analysis (Pharsight Corporation, Mountain View, CA, USA).
3.2.6. In Vivo Studies
The mouse melanoma cells B16F10 were cultured and harvested in saline. At day 0 of the
experiment, 1
×
106 cells were injected subcutaneously into mice, and treatment was initiated at
day 1 following the mice enrolled randomly in control and experimental groups [27]. For control
group, 0.5% CMCNa was orally administered every day. Compound DX-03-12 was dissolved in
0.5% CMC for oral treatment or dissolved in saline for intraperitoneal treatment. When the mice
were sacrificed, the tumors were stripped and weighted. The tumor growth inhibition (TGI) was
calculated as TGI = (1 – tumor weight treatment/tumor weight vehicle) × 100%. The statistical analysis
was performed with GraphPad Prism 8.0 software and the significance level was evaluated with a
one-way ANOVA model.
4. Conclusions
We designed four series of IDO1 inhibitors using indole and phenylimidazole as the scaffolds.
The design strategies were based on the mechanism of IDO1, focusing on the interaction between the
inhibitor and the heme iron. The four series of compounds were synthesized and their IDO1 and TDO
inhibition was evaluated. Most compounds showed marginal IDO1 inhibition, but eight compounds
showed moderate IDO1 inhibition. In particular, DX-03-12 and DX-03-13 showed IDO1 inhibition with
IC50s of 0.3 and 0.5 µM and low cell cytotoxicities against two cancer cell lines. A pharmacokinetic
study showed that DX-03-12 had satisfactory properties, with rapid absorption, moderate plasma
clearance, acceptable half-life, and high oral bioavailability. The in vivo anti-tumor efficacy in a B16F10
subcutaneous xenograft mouse model showed that DX-03-12 orally administered at a dose of 60 mg/kg
inhibited tumor growth by 72.2% compared with the control tumors. In summary, IDO1 inhibitor
DX-03-12 exhibits promising drug like properties, good pharmacokinetic properties, low cellular
toxicity, and impressive in vivo anti-tumor efficacy.
Author Contributions: L.S., F.L., and H.C. conceived and designed the experiments; H.W., Y.L. (Yuke Liu), S.W.,
T.W., and G.Z. performed the experiments, analyzed the data; Y.L. (Yan Li), and X.C. checked the data; L.S., F.L.,
and H.C. wrote the paper. Please contact L.S. for pharmacokinetic study, F.L. for pharmacology study and H.C. for
medicinal chemistry.
Funding: The research leading to these results has received funding from The CAMS Innovation Fund for
Medical Sciences (2017-I2M-1-010), The Drug Innovation Major Project (2018ZX09711001), National Natural
Science Foundation of China (81603190).

Molecules 2019, 24, 2124
26 of 27
Acknowledgments: We would like to express our appreciation to Jie Li of Beijing Chao-Yang Hospital for the
clinical issue discussion.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
Weng, T.; Qiu, X.; Wang, J.; Li, Z.; Bian, J. Recent discovery of indoleamine-2,3-dioxygenase 1 inhibitors
targeting cancer immunotherapy. Eur. J. Med. Chem. 2018, 143, 656–669. [CrossRef] [PubMed]
Muller, A.J.; DuHadaway, J.B.; Donover, P.S.; Sutanto-Ward, E.; Prendergast, G.C. Inhibition of indoleamine
2,3-dioxygenase, an immunoregulatory target of the cancer suppression gene Bin1, potentiates cancer
chemotherapy. Nat. Med. 2005, 11, 312–319. [CrossRef] [PubMed]
2.
3.
Platten, M.; Nollen, E.A.A.; Rohrig, U.F.; Fallarino, F.; Opitz, C.A. Tryptophan metabolism as a common
therapeutic target in cancer, neurodegeneration and beyond. Nat. Rev. Drug Discov 2019, 18, 379–401.
[CrossRef] [PubMed]
4.
5.
6.
Cervenka, I.; Agudelo, L.; Ruas, J. Kynurenines: Tryptophan‘s metabolites in exercise, inflammation, and
mental health. Science 2017, 357. [CrossRef] [PubMed]
Abdulla, A.-B.B. Kynurenine Pathway of Tryptophan Metabolism: Regulatory and Functional Aspects. Int. J.
Tryptophan. Res. 2017, 10. [CrossRef]
Zhai, L.; Spranger, S.; Binder, D.C.; Gritsina, G.; Lauing, K.L.; Giles, F.J.; Wainwright, D.A. Molecular
Pathways: Targeting IDO1 and Other Tryptophan Dioxygenases for Cancer Immunotherapy. Clin. Cancer
Res. 2015, 21, 5427–5433. [CrossRef] [PubMed]
7.
Chauhan, N.; Basran, J.; Efimov, I.; Svistunenko, D.A.; Seward, H.E.; Moody, P.C.; Raven, E.L. The role
of serine 167 in human indoleamine 2,3-dioxygenase: A comparison with tryptophan 2,3-dioxygenase.
Biochemistry 2008, 47, 4761–4769. [CrossRef] [PubMed]
8.
9.
Batabyal, D.; Yeh, S.R. Human tryptophan dioxygenase: A comparison to indoleamine 2,3-dioxygenase.
J. Am. Chem. Soc. 2007, 129, 15690–15701. [CrossRef]
Sugimoto, H.; Oda, S.; Otsuki, T.; Hino, T.; Yoshida, T.; Shiro, Y. Crystal structure of human indoleamine
2,3-dioxygenase: Catalytic mechanism of O2 incorporation by a heme-containing dioxygenase. Proc. Natl.
Acad. Sci. USA 2006, 103, 2611–2616. [CrossRef]
10. Rohrig, U.F.; Zoete, V.; Michielin, O. The Binding Mode of N-Hydroxyamidines to Indoleamine
2,3-Dioxygenase 1 (IDO1). Biochemistry 2017, 56, 4323–4325. [CrossRef]
11. Samelson-Jones, B.J.; Yeh, S.R. Interactions between nitric oxide and indoleamine 2,3-dioxygenase.
Biochemistry 2006, 45, 8527–8538. [CrossRef] [PubMed]
12. Yanagisawa, S.; Sugimoto, H.; Shiro, Y.; Ogura, T. A specific interaction of L-tryptophan with CO of
CO-bound indoleamine 2,3-dioxygenase identified by resonance Raman spectroscopy. Biochemistry 2010, 49,
10081–10088. [CrossRef] [PubMed]
13. Crosignani, S.; Bingham, P.; Bottemanne, P.; Cannelle, H.; Cauwenberghs, S.; Cordonnier, M.;
Dalvie, D.; Deroose, F.; Feng, J.L.; Gomes, B.; et al. Discovery of a Novel and Selective Indoleamine
2,3-Dioxygenase (IDO-1) Inhibitor 3-(5-Fluoro-1H-indol-3-yl)pyrrolidine-2,5-dione (EOS200271/PF-06840003)
and Its Characterization as a Potential Clinical Candidate. J. Med. Chem. 2017, 60, 9617–9629. [CrossRef]
[PubMed]
14. Yang, D.; Li, T.; Li, Y.; Zhang, S.; Li, W.; Liang, H.; Xing, Z.; Du, L.; He, J.; Kuang, C.; et al. H2S suppresses
indoleamine 2, 3-dioxygenase 1 and exhibits immunotherapeutic efficacy in murine hepatocellular carcinoma.
J. Exp. Clin. Cancer Res. 2019, 38, 88. [CrossRef] [PubMed]
15. Pham, K.N.; Yeh, S.R. Mapping the Binding Trajectory of a Suicide Inhibitor in Human Indoleamine
2,3-Dioxygenase 1. J. Am. Chem. Soc. 2018, 140, 14538–14541. [CrossRef] [PubMed]
16. Yang, S.; Li, X.; Hu, F.; Li, Y.; Yang, Y.; Yan, J.; Kuang, C.; Yang, Q. Discovery of tryptanthrin derivatives
as potent inhibitors of indoleamine 2,3-dioxygenase with therapeutic activity in Lewis lung cancer (LLC)
tumor-bearing mice. J. Med. Chem. 2013, 56, 8321–8331. [CrossRef] [PubMed]
17. Yue, E.W.; Douty, B.; Wayland, B.; Bower, M.; Liu, X.; Leffet, L.; Wang, Q.; Bowman, K.J.; Hansbury, M.J.;
Liu, C.; et al. Discovery of potent competitive inhibitors of indoleamine 2,3-dioxygenase with in vivo
pharmacodynamic activity and efficacy in a mouse melanoma model. J. Med. Chem. 2009, 52, 7364–7367.
[CrossRef] [PubMed]

Molecules 2019, 24, 2124
27 of 27
18. Kumar, S.; Jaller, D.; Patel, B.; LaLonde, J.M.; DuHadaway, J.B.; Malachowski, W.P.; Prendergast, G.C.;
Muller, A.J. Structure based development of phenylimidazole-derived inhibitors of indoleamine
2,3-dioxygenase. J. Med. Chem. 2008, 51, 4968–4977. [CrossRef]
19. Rohrig, U.F.; Majjigapu, S.R.; Grosdidier, A.; Bron, S.; Stroobant, V.; Pilotte, L.; Colau, D.; Vogel, P.; Van
den Eynde, B.J.; Zoete, V.; et al. Rational design of 4-aryl-1,2,3-triazoles for indoleamine 2,3-dioxygenase 1
inhibition. J. Med. Chem. 2012, 55, 5270–5290. [CrossRef]
20. Tojo, S.; Kohno, T.; Tanaka, T.; Kamioka, S.; Ota, Y.; Ishii, T.; Kamimoto, K.; Asano, S.; Isobe, Y. Crystal
Structures and Structure-Activity Relationships of Imidazothiazole Derivatives as IDO1 Inhibitors. ACS Med.
Chem. Lett. 2014, 5, 1119–1123. [CrossRef]
21. Zhu, M.M.T.; Dancsok, A.R.; Nielsen, T.O. Indoleamine Dioxygenase Inhibitors: Clinical Rationale and
Current Development. Curr. Oncol. Rep. 2019, 21, 2. [CrossRef]
22. Yan, N.; He, Y.; Wen, H.; Lai, F.; Yin, D.; Cui, H. A Suzuki-Miyaura method for labelling proliferating cells
containing incorporated BrdU. Analyst 2018, 143, 1224–1233. [CrossRef] [PubMed]
23. Mautino, M.; Kumar, S.; Jaipuri, F.; Kesharwani, T.; Zhang, X. Imidazole Derivatives as IDO inhibitors.
WO2011056652A1, 12 May 2011.
24. Austin, C.J.; Mizdrak, J.; Matin, A.; Sirijovski, N.; Kosim-Satyaputra, P.; Willows, R.D.; Roberts, T.H.;
Truscott, R.J.; Polekhina, G.; Parker, M.W.; et al. Optimised expression and purification of recombinant
human indoleamine 2,3-dioxygenase. Protein Expr. Purif. 2004, 37, 392–398. [CrossRef] [PubMed]
25. Littlejohn, T.K.; Takikawa, O.; Skylas, D.; Jamie, J.F.; Walker, M.J.; Truscott, R.J. Expression and purification of
recombinant human indoleamine 2, 3-dioxygenase. Protein Expr. Purif. 2000, 19, 22–29. [CrossRef]
26. Lai, F.; Shen, Z.; Wen, H.; Chen, J.; Zhang, X.; Lin, P.; Yin, D.; Cui, H.; Chen, X. A Morphological identification
cell cytotoxicity assay using cytoplasm-localized fluorescent probe (CLFP) to distinguish living and dead
cells. Biochem. Biophys. Res. Commun. 2017, 482, 257–263. [CrossRef] [PubMed]
27. He, Y.; Wen, J.; Cui, Q.; Lai, F.; Yin, D.; Cui, H. Quantitative Evaluation of in Vivo Target Efficacy of Anti-tumor
Agents via an Immunofluorescence and EdU Labeling Strategy. Front. Pharmacol. 2018, 9, 812. [CrossRef]
[PubMed]
Sample Availability: Samples of the compounds are available from the authors.
©
2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).