Synthesis and molecular modeling studies of N-Hydroxyindazolecarboximidamides as novel indoleamine 2,3-Dioxygenase 1 (IDO1) inhibitors

Article abstract of DOI:10.3390/molecules22111936

Indoleamine 2,3-dioxygenase 1 (IDO1) is an immunosuppressive enzyme that is highly overexpressed in various cancer cells and antigen-presenting cells. It has emerged as an attractive therapeutic target for cancer immunotherapy, which has prompted high interest in the development of small-molecule inhibitors. To discover novel IDO1 inhibitors, we designed and synthesized a series of N-hydroxyindazolecarboximidamides. Among the compounds synthesized, compound 8a inhibited both tryptophan depletion and kynurenine production through the IDO1 enzyme. Molecular docking studies revealed that 8a binds to IDO1 with the same binding mode as the analog, epacadostat (INCB24360). Here, we report the synthesis and biological evaluation of these hydroxyindazolecarboximidamides and present the molecular docking study of 8a with IDO1.

Full text of DOI:10.3390/molecules22111936

molecules
Article
Synthesis and Molecular Modeling Studies of
N0-Hydroxyindazolecarboximidamides as Novel
Indoleamine 2,3-Dioxygenase 1 (IDO1) Inhibitors
Dong-Ho Lee 1,2,† ID , Joo-Youn Lee 3,†, Jieun Jeong 1, Miok Kim 1,4, Kyung Won Lee 1,4
,
Eunseo Jang 1 ID , Sunjoo Ahn 1,5, Chang Hoon Lee 1,4,* and Jong Yeon Hwang 1,4,5,
*
1
Bio & Drug Discovery Division, Korea Research Institute of Chemical Technology, Daejeon 34114, Korea;
ldh1125@krict.re.kr (D.-H.L); chemworld@hanmail.net (J.J.); miok@krict.re.kr (M.K.);
ek00322@krict.re.kr (K.W.L.); eunseooo@krict.re.kr (E.J.); sahn@krict.re.kr (S.A.)
Department of Chemistry, Sogang University, Seoul 121-742, Korea
Korea Chemical Bank, Korea Research Institute of Chemical Technology, 141 Gajeong-ro, Yuseong-gu,
Daejeon 34114, Korea; leejy@krict.re.kr
Immunotherapy Convergence Research Center, Korea Research Institute of Bioscience and
Biotechnology (KRIBB), Daejeon 34141, Korea
Department of Medicinal Chemistry and Pharmacology, University of Science & Technology,
Daejeon 34113, Korea
2
3
4
5
*
†
Correspondence: lee2014@krict.re.kr (C.H.L.); jyhwang@krict.re.kr (J.Y.H.); Tel: +82-42-860-7045 (J.Y.H.)
These authors contributed equally to this work.
Received: 25 September 2017; Accepted: 8 November 2017; Published: 9 November 2017
Abstract: Indoleamine 2,3-dioxygenase 1 (IDO1) is an immunosuppressive enzyme that is highly
overexpressed in various cancer cells and antigen-presenting cells. It has emerged as an attractive
therapeutic target for cancer immunotherapy, which has prompted high interest in the development
of small-molecule inhibitors. To discover novel IDO1 inhibitors, we designed and synthesized a
series of N0-hydroxyindazolecarboximidamides. Among the compounds synthesized, compound
8a inhibited both tryptophan depletion and kynurenine production through the IDO1 enzyme.
Molecular docking studies revealed that 8a binds to IDO1 with the same binding mode as the
analog, epacadostat (INCB24360). Here, we report the synthesis and biological evaluation of these
hydroxyindazolecarboximidamides and present the molecular docking study of 8a with IDO1.
0
Keywords: indoleamine 2,3-dioxygenase 1; N -hydroxyindazolecarboximidamide; cancer immunotherapy;
tryptophan depletion; kynurenine production
1. Introduction
Indoleamine 2,3-dioxygenase 1 (IDO1) is a monomeric heme-containing enzyme that catalyzes
the oxidation of L-tryptophan to N-formylkynurenine [1,2]. N-Formylkynurenine is then converted
via the kynurenine pathway to a series of biologically-active metabolites including L-kynurenine,
kynurenic acid, 3-hydroxykynurenine, xanthurenic acid, anthranilic acid, and quinolinic acid.
These metabolites inhibit the proliferation and differentiation of T cells, leading to immune tolerance [
3–5].
Overexpression of IDO1 has been observed in various cancer cells and antigen-presenting cells,
and depletion of IDO1 has been shown to inhibit tumor growth and metastases [6–9]. Therefore, IDO1
has emerged as an attractive therapeutic target for cancer immunotherapy [10].
A number of IDO1 inhibitors have been reported in the literature and several of these compounds
are being investigated in clinical trials for use in combination therapy with other therapeutic
interventions (Figure 1) [11,12].
Molecules 2017, 22, 1936; doi:10.3390/molecules22111936
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Figure 1. Structure of IDO1 inhibitors in clinical trials.
Among the IDO1 inhibitors currently being studied, two advanced compounds, INCB24360
(epacadostat) [13] and GDC-0919 (Navoximod) [14 15], were found to be highly potent in both
,
enzymatic and cellular assays. The X-ray crystal structure of cmpd-24 (GDC-0919 analog) revealed
that the imidazoleisoindole core is located in pocket A, while the nitrogen atom of imidazole
coordinates with the heme iron [16]. The 1-cyclohexylethanol residue extends to pocket B and
forms hydrophobic interactions with Phe226, Ile354, and Leu384. Interestingly, compd-24 forms
two additional intramolecular hydrogen bonds with the IDO1 enzyme. The nitrogen atom in isoindole
binds to the hydroxyl group found in the cyclohexylethanol residue, while the hydroxyl group of
cmpd-24 forms an intramolecular hydrogen bond with the 7-propionic acid of the heme iron to increase
IDO1 binding affinity. In the case of INCB24360, its crystal structure and predicted model study suggest
that the hydroxyl group in the oxime moiety exists in the cis-conformation to bind to IDO1 and forms
the observed intramolecular hydrogen bonding network [13,17]. Recently, the X-ray crystal structure of
IDO1 with INCB14943 (epacadostat analog, PBD:5XE1) has also been reported. A nitrogen in the oxime
moiety of INCB14943 covalently coordinates with the heme iron, and a phenyl ring, situated in pocket
A, forms hydrophobic interactions with V130, F164, and L234. An intermolecular hydrogen bond
also forms between the NH2 in the furazan moiety and the side chain of S263 in IDO1. In addition,
the hydroxyl group in the oxime moiety is essential for intramolecular hydrogen bond networks
between the NH2 group in the furazan moiety and the NH moiety of INCB14943 [13,18].
As part of our efforts to identify a novel IDO1 inhibitor for immune-oncology drug discovery, we
designed novel N0-hydroxyindazolecarboximidamides
1, in which the indazole moiety is a surrogate
of furazan in epacadostat (Figure 2). Herein, we report the synthesis and anti-IDO1 activities of the
designed compounds and discuss the molecular docking study of the selected compound.
Figure 2. Rational design of novel IDO1 inhibitor (1) from the known molecules.
2. Results and Discussion
2.1. Chemistry
N0-Hydroxyindazolecarboximidamides were synthesized as described in Scheme 1, starting
from commercially-available 7-nitroindazole
Pd/C in a hydrogen atmosphere. The amino group in
of NaNO2 and KI [19]. Introduction of nitrile in was accomplished by treatment of Zn(CN)2,
Pd2(dba)3, 1,10-bis(diphenylphosphino)ferrocene (dppf), and Zn [19]. Treatment of hydroxylamine in
2. The nitro group in
2 was reduced to amine 3 using
3
was transformed to iodine by treatment
5

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5
afforded hydroxylamidine
6
, which was converted to N-hydroxyimidoyl chloride
7
by Sandmeyer
conditions with NaNO2 and HCl [13]. The N-hydroxyimidoyl chloride was substituted with the
desired hydroxyamidines by treatment of the corresponding amines. N-Methylindazole 12 was
synthesized from the commercially-available N-methylindazole carboxaldehyde , which was treated
8
9
with hydroxylamine to afford oxime 10. Oxime 10 was oxidized with N-chlorosuccinimide (NCS) to
afford N-hydroxyimidoyl chloride 11 [20], which was converted to the desired 12 by treatment of the
amine. 2-Hydroxyphenyl 16 was also prepared from 2-hydroxybenzaldehyde 13 by the same synthetic
method described above (for the creation of 12).
Scheme 1. Synthesis of compounds 8, 12 and 16. Reagents and conditions: (a) 10% Pd/C, H2, EtOH, r.t.;
(
b
) NaNO2, H2O, HCl, H2SO4, KI; (
c
) Zn(CN)2, Pd2(dba)3, dppf, Zn, DMA, reflux; (
d
) hydroxylamine
hydrogen chloride, NaHCO3, H2O, EtOH, 85 ◦ C; (
e
) (i) 6N HCl, H2O, AcOH, 45 C, (ii) HCl, NaNO2,
◦
◦
◦
H2O, 0 C, 3 h; (f) amines, NaHCO3, H2O, THF, 60 C; (g) NCS, DMF, r.t., 6 h.
2.2. Biological Evaluation
All synthesized compounds were evaluated for inhibitory activities against IDO1. For this
purpose, we generated human IDO1 gene-expressing HEK293 recombinant cells and used those
cells to analyze the inhibitory capacity of the compounds. Briefly, we induced the expression vector
system of IDO1 by treating HEK294 cells with doxycycline, and measured both the depletion of
L-tryptophan in culture media and the generation of kynurenine in IDO1-overexpressing HEK293 cells
by using the LC-MS system upon culture supernatants.
The synthesized compounds were tested at a concentration of 10
activities in cell-based assays measuring levels of tryptophan and kynurenine (Table 1). Among the
compounds, 8a displayed >50% inhibition against IDO1, whereas others were almost inactive at
10 M concentration. These selected active compounds were tested to determine IC50 values by
µM for IDO1-inhibitory
–
c
µ
dose-response curve (DRC) analysis (Table 1), and they exhibited moderate inhibitory activity ranges
in both tryptophan depletion and kynurine production. These actives possess 3-bromo or 3-chloro
substitution in the phenyl group, and the compounds with an additional 4-fluoro substitution showed
increased anti-IDO1 activity. 3-Bromo-4-fluorophenyl 8a was the most active in this series. Some active

Molecules 2017, 22, 1936
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compounds (8a–c) in the cell-based assay were further tested for hIDO1 inhibition by enzymatic assay
and displayed hIDO1 inhibitory activities. It was reported that the substituent of aniline in the A-pocket
is very important for IDO1 inhibition [13]. For example, fluoro- or no substituted compound in the
para position was active, while others were almost inactive. In addition, meta-halogen substituents in
aniline, including chloro- and bromo, were required for the activity. Thus, our results almost match
those of a previous report. The intra- and intermolecular hydrogen bonding network in the active site
of IDO1 plays an important role in their inhibitory activity, as mentioned earlier. We presumed that
the N-hydrogen in the indazole moiety could involve a hydrogen bonding complex in the active site of
IDO1. To confirm this hypothesis, N-methylindazole compound 12 was evaluated. As expected, it did
not inhibit IDO1 in both cell-based and enzymatic assays. Interestingly, phenol 16, which consists of
a bioisosteric replacement of indazole and bears a hydroxyl group for hydrogen bonding, showed
moderate inhibitory activity. This result strongly supports the hypothesis that a hydrogen bonding
network is critical for IDO1 inhibition.
Table 1. Inhibition of tryptophan depletion and kynurenine production through IDO1.
HEK293 IC50 (µM)
R1
hIDO1 Inhibition at 20 µM (%)
No.
Tryptophan Depletion
Kynurenine Production
8a
8b
8c
8d
8e
8f
3-Br-4-F
3-Cl-4-F
3-Cl
1.76
3.26
4.95
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
2.13
3.14
4.89
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
81
79
77
4-F
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
2,5-diF
3-Ac
8g
8h
8i
3-Me
4-Cl-3-Me
3-Cl-4-MeO
H
8j
8k
8l
4-Cl
4-MeO
4-Me
8m
12
16
>10
>10
15
7.76
7.91
n.t.
n.t. = not tested.

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2.3. Molecular Docking Study
To understand the binding mode of 8a for IDO1, docking studies were performed using the
IDO1 X-ray crystal structure in complex with Amg-1 (PBD code 4PK5) [21]. The proposed binding
model of 8a was very similar to that of epacadostat (Figure 3a) [17]. The hydroxyl group of the
hydroxyamidine moiety in 8a forms a covalent bond with the heme iron, and the 3-Br and 4-F
substituted phenyl groups bind into the hydrophobic pocket A, which consists of Tyr126, Cys129,
Val130, Phe163, Phe164, Leu234, Gly262, and Ala264. This proposed binding conformation of 8a
showed two intramolecular interactions: one being a hydrogen bond between the aniline NH and the
oxygen of the hydroxyamidine, and the other between the nitrogen of the hydroxyamidine and the
NH of indazole. In our docking model, we identified that halogen bonding between Cys129 and the
Tyr126 backbone improves the activity against IDO1. As a surface model of the active site, 8a does not
occupy the extended pocket B. We surmise that this could be an opportunity to further improve the
potency for 8a by structure-based design.
(a)
(b)
Figure 3. Docking model 8a against IDO1. (
a) Binding mode of 8a (pink, ball and stick style)
coordinated to heme; (b) The surface model of the active site bound to 8a is colored because of
hydrophobicity. Hydrogen bonds of inter- and intra-molecular interactions are shown as green dashed
lines and interaction residues are represented by the stick model.
3. Materials and Methods
3.1. Chemistry
3.1.1. General Chemistry
All materials were obtained from commercial suppliers and used without further purification.
Solvents in this study were dried using an aluminum oxide column. Thin-layer chromatography
was performed on pre-coated silica gel 60 F254 plates. Purification of intermediates was carried
out by normal phase column chromatography (MPLC, Silica gel 230-400 mesh). NMR spectra were
recorded on a Avance 300 MHz (Bruker, Billerica, MA, USA). LC/MS data were obtained using a
Waters 2695 LC and Micromass ZQ spectrometer (Waters, Milford, MA, USA). HR-MS was detected
on JMS-700 (JEOL, Tokyo, Japan). The identity of the final compounds was confirmed by proton NMR
and mass spectrometry.
3.1.2. General Procedure for the Preparation of Compounds
Synthesis of 1H-Indazol-7-amine 3
A mixture of 7-nitroindazole (3 g, 0.018 mol)) and 10% Pd/C (0.6 g) in EtOH (10 mL) was stirred
at rt under H2 atmosphere. The mixture was filtered through a pad of Celite and washed with EtOH.

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The filtrate was concentrated to give compound
3
(1.9 g, 78%, ivory solid).1H-NMR (300 MHz, CDCl3)
δ 8.09 (s, 1H), 7.24 (d, J = 8.2 Hz, 1H), 7.10–6.96 (m, 1H), 6.68 (d, J = 7.2 Hz, 1H), 4.12 (brs, 1H).
Synthesis of 7-Iodo-1H-indazole 4
To a solution of
3
(0.3 g, 2.25 mmol) in c-HCl (0.56 mL) and 98% H2SO4 (2 mL) was added
◦
dropwise a solution of NaNO2 (0.154 g, 2.23 mmol) in H2O (2 mL). The mixture was stirred at 0 C for
30 min. Then, a solution of KI (0.747 g, 4.5 mmol) in H2O (2 mL) was added dropwise and the mixture
was stirred at rt for 2 h. The mixture was diluted with EA, washed with sat. NaHCO3, water, and brine.
The organic layer was dried over MgSO4 and purified by column chromatography (MPLC) to give
1
compound
4
(0.17 g, 32%, dark ivory solid). H-NMR (300 MHz, CDCl3)
δ
10.14 (s, 1H), 8.26 (s, 1H),
7.82–7.71 (m, 2H), 7.04–6.92 (m, 1H).
Synthesis of 1H-Indazole-7-carbonitrile 5
A solution of
4 (0.3 g, 1.23 mmol), dppf (55 mg, 0.098 mmol), Pd2(dba)3 (28 mg, 0.049 mmol),
Zn (20 mg, 0.307 mmol), Zn(CN)2 (86.6 mg, 0.737 mmol) in DMA (30 mL, degassed) was heated at
120 ◦C for 5 h. The mixture was concentrated and diluted with EA. The organic layer was washed
with 2 N NH4OH and brine. The organic layer was dried over MgSO4 and concentrated to obtain the
crude mixture which was purified by column chromatography (MPLC) to give compound
5
(173 mg,
1
98%, white solid). H-NMR (300 MHz, CDCl3)
δ 11.85 (s, 1H), 8.41 (s, 1H), 8.09 (dd, J = 8.2, 0.9 Hz, 1H),
7.81 (dd, J = 7.3, 0.9 Hz, 1H), 7.36–7.30 (m, 1H).
Synthesis of (Z)-N0-Hydroxy-1H-indazole-7-carboximidamide 6
To a solution of
5
(0.1 g, 0.7 mmol) in H2O (1.5 mL)/EtOH (3 mL) were added hydroxylami◦ne
hydrogen chloride (0.073 g, 1.05 mmol) and sodium bicarbonate (0.118 g, 1.4 mmol) and heated at 85 C
for 8 h. The reaction mixture was diluted with EA, washed with water and brine. The organic layer was
dried over MgSO4 and concentrated to give compound
DMSO) 12.22 (s, 1H), 9.82 (s, 1H), 8.12 (s, 1H), 7.76 (dd, J = 20.6, 7.5 Hz, 2H), 7.17 (t, J = 7.5 Hz, 1H),
6.07 (s, 2H).
6
(84 mg, 68%, white solid) 1H-NMR (300 MHz,
δ
Synthesis of (E)-N-Hydroxy-1H-indazole-7-carbimidoyl chloride 7
To a suspension of 6 (74 mg, 0.42 mmol) in water (1 mL) were added acetic acid (0.5 mL) and
6 N HCl (1.26 mmol, 0.21 mL) and this suspension was stirred at 45 ◦C until complete solution was
achieved. Sodium chloride (0.074 g, 1.26 mmol) was added to the reaction mixture and stirred in an
ice/water/methanol bath. A solution of NaNO2 (0.029 g, 0.42 mmol) in water (1 mL) was added
◦
dropwise while maintaining the temperature below 0 C. After complete adding stirring was continued
◦
in the ice bath for 1.5 h and then the mixture was allowed to warm to 15 C. The precipitate was
collected by filtration and washed with cold water to give the desired compound
7 (65 mg, 79% ivory
solid) 1H-NMR (300 MHz, DMSO-d6)
δ
12.74 (s, 1H), 12.47 (s, 1H), 8.23 (s, 1H), 7.94 (d, J = 8.1 Hz, 1H),
7.82 (dd, J = 7.5, 0.6 Hz, 1H), 7.29 (t, J = 3.75 Hz, 1H).
General Procedure for the Synthesis of 8, 12 and 16
0
(Z)-N-(3-Bromo-4-fluorophenyl)-N -hydroxy-1H-indazole-7-carboximidamide (8a). To a solution of
7 (25 mg,
0.105 mmol) in THF (1 mL) at 60 ◦C was 3-bromo-4-fluoroaniline (20
µL, 0.105 mmol) was added and
stirred for 10 min. A solution of NaHCO3 (13 mg, 0.157 mmol) in water (1 mL) was added dropwise
and stirred at 60 ◦C for 3 h. The mixture was extracted with EA and washed with brine. The organic
layer was dried over MgSO4 and concentrated to obtain the crude mixture which was purified by
1
column chromatography (MPLC) to give compound 8a. H-NMR (300 MHz, CDCl3)
δ
12.01 (s, 1H),
8.14 (s, 1H), 7.73 (d, J = 8.0 Hz, 1H), 7.48 (s, 1H), 7.20 (d, J = 7.2 Hz, 1H), 7.01 (t, J = 7.7 Hz, 1H),

Molecules 2017, 22, 1936
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6.95 (dd, J = 5.7, 2.3 Hz, 1H), 6.78 (t, J = 8.4 Hz, 1H), 6.58–6.43 (m, 1H); LC/MS (ESI) m/z 349 [M + H]+;
HRMS (EI) m/z calcd. for C14H10BrFN4O [M+] 348.0022, found 348.0019.
(Z)-N-(3-Chloro-4-fluorophenyl)-N0-hydroxy-1H-indazole-7-carboximidamide (8b). 1H-NMR (300 MHz,
CDCl3)
δ 11.96 (brs, 1H), 8.15 (s, 1H), 7.74 (d, J = 8.0 Hz, 1H), 7.44 (s, 1H), 7.22 (d, J = 7.2 Hz,
1H), 7.06–6.94 (m, 1H), 6.85–6.75 (m, 2H), 6.53–6.39 (m, 1H); LC/MS (ESI) m/z 305 [M + H]+
HRMS (EI) m/z calcd. for C14H10ClFN4O [M+] 304.0527, found 304.0519.
;
(Z)-N-(3-Chlorophenyl)-N0-hydroxy-1H-indazole-7-carboximidamide (8c). 1H-NMR (300 MHz, CDCl3)
δ
11.91 (s, 1H), 8.16 (s, 1H), 7.74 (d, J = 8.0 Hz, 1H), 7.43 (s, 1H), 7.34–7.25 (m, 1H), 7.08–6.94 (m, 2H),
6.90 (d, J = 7.9 Hz, 1H), 6.77 (s, 1H), 6.51 (d, J = 7.8 Hz, 1H); LC/MS (ESI) m/z 287 [M + H]+; HRMS (EI)
m/z calcd. for C14H11ClN4O [M+] 286.0621, found 286.0627.
(Z)-N-(4-Fluorophenyl)-N0-hydroxy-1H-indazole-7-carboximidamide (8d). 1H-NMR (300 MHz, CDCl3)
δ
11.98 (s, 1H), 8.15 (s, 1H), 7.71 (dd, J = 8.0, 0.7 Hz, 1H), 7.43 (s, 1H), 7.21 (dd, J = 7.3, 0.7 Hz, 1H),
7.02–6.95 (m, 1H), 6.82–6.74 (m, 2H), 6.72–6.63 (m, 2H); LC/MS (ESI) m/z 271 [M + H]+.
1
(Z)-N-(2,5-Difluorophenyl)-N0-hydroxy-1H-indazole-7-carboximidamide (8e). H-NMR (300 MHz, CDCl3)
δ
8.21 (s, 1H), 7.83 (dd, J = 8.1, 0.8 Hz, 1H), 7.46–7.39 (m, 1H), 7.18 (s, 1H), 7.17–7.10 (m, 1H), 7.10–6.99
(m, 1H), 6.66–6.53 (m, 1H), 6.36–6.24 (m, 1H); LC/MS (ESI) m/z 289 [M + H]+.
(Z)-N-(3-Acetylphenyl)-N0-hydroxy-1H-indazole-7-carboximidamide (8f). 1H-NMR (300 MHz, DMSO)
δ
12.80 (s, 1H), 10.79 (s, 1H), 8.64 (s, 1H), 8.09 (s, 1H), 7.78 (d, J = 7.9 Hz, 1H), 7.35–7.27 (m, 3H),
7.16–7.04 (m, 2H), 6.88 (d, J = 9.5 Hz, 1H), 2.31 (s, 3H); LC/MS (ESI) m/z 295 [M + H]+.
1
(Z)-N0-Hydroxy-N-(m-tolyl)-1H-indazole-7-carboximidamide (8g). H-NMR (300 MHz, CDCl3)
δ 11.80
(s, 1H), 8.17 (s, 1H), 7.74 (dd, J = 8.1, 0.8 Hz, 1H), 7.37–7.27 (m, 2H), 7.11–6.95 (m, 2H), 6.79 (d, J = 7.5 Hz,
1H), 6.66 (s, 1H), 6.54 (d, J = 7.9 Hz, 1H), 2.21 (s, 3H); LC/MS (ESI) m/z 267 [M + H]+.
1
(Z)-N-(4-Chloro-3-methylphenyl)-N0-hydroxy-1H-indazole-7-carboximidamide (8h). H-NMR (300 MHz,
DMSO)
δ 12.77 (s, 1H), 10.75 (s, 1H), 8.44 (s, 1H), 8.10 (d, J = 1.3 Hz, 1H), 7.78 (d, J = 7.9 Hz, 1H),
7.26 (d, J = 6.3 Hz, 1H), 7.12–7.03 (m, 1H), 6.99 (d, J = 8.6 Hz, 1H), 6.74 (d, J = 2.6 Hz, 1H), 6.39 (dd,
J = 8.5, 2.7 Hz, 1H), 2.10 (s, 3H); LC/MS (ESI) m/z 301 [M + H]+.
1
(Z)-N-(3-Chloro-4-methoxyphenyl)-N0-hydroxy-1H-indazole-7-carboximidamide (8i). H-NMR (300 MHz,
CDCl3)
δ 12.0 (brs, 1H), 8.13 (s, 1H), 7.70 (d, J = 8.0 Hz, 1H), 7.40 (s, 1H), 7.20 (d, J = 7.3 Hz, 1H),
6.98 (t, J = 7.7 Hz, 1H), 6.86 (d, J = 2.6 Hz, 1H), 6.58 (d, J = 8.8 Hz, 1H), 6.49 (dd, J = 8.8, 2.6 Hz, 1H),
3.78 (s, 3H); LC/MS (ESI) m/z 317 [M + H]+.
(Z)-N0-Hydroxy-N-phenyl-1H-indazole-7-carboximidamide (8j). 1H-NMR (300 MHz, CDCl3)
δ 11.59
(brs, 1H), 8.18 (s, 1H), 7.76 (d, J = 8.0 Hz, 1H), 7.34 (d, J = 7.2 Hz, 2H), 7.16 (t, J = 7.6 Hz, 2H),
7.08–6.95 (m, 2H), 6.81 (d, J = 7.8 Hz, 2H); LC/MS (ESI) m/z 253 [M + H]+.
(Z)-N-(4-Chlorophenyl)-N0-hydroxy-1H-indazole-7-carboximidamide (8k). 1H-NMR (300 MHz, CDCl3)
δ
11.71 (brs, 1H), 8.18 (s, 1H), 7.77 (d, J = 8.0 Hz, 1H), 7.37–7.25 (m, 3H), 7.10 (d, J = 8.5 Hz, 2H),
7.06 (t, J = 7.7 Hz, 1H), 6.71 (d, J = 8.5 Hz, 2H); LC/MS (ESI) m/z 287 [M + H]+.
1
(Z)-N0-Hydroxy-N-(4-methoxyphenyl)-1H-indazole-7-carboximidamide (8l). H-NMR (300 MHz, CDCl3)
δ
11.95 (s, 1H), 8.14 (s, 1H), 7.70 (d, J = 7.9 Hz, 1H), 7.37 (s, 1H), 7.23 (d, J = 7.1 Hz, 1H), 6.97 (t, J = 7.7 Hz,
1H), 6.72 (d, J = 9.0 Hz, 2H), 6.65 (d, J = 8.9 Hz, 2H), 3.72 (s, 3H); LC/MS (ESI) m/z 283 [M + H]+.
1
(Z)-N0-Hydroxy-N-(p-tolyl)-1H-indazole-7-carboximidamide (8m). H-NMR (300 MHz, CDCl3)
δ 11.53
(s, 1H), 8.17 (s, 1H), 7.75 (d, J = 8.1 Hz, 1H), 7.32 (d, J = 6.6 Hz, 1H), 7.27 (s, 1H), 7.07–6.99 (m, 1H),
6.97 (d, J = 8.1 Hz, 2H), 6.73 (d, J = 8.3 Hz, 2H), 2.27 (s, 3H); LC/MS (ESI) m/z 267 [M + H]+.

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Synthesis of N-Hydroxy-1-methyl-1H-indazole-7-carbimidoyl chloride (11)
A solution of 1-methyl-1H-indazole-7-carbaldehyde oxime 10 (0.1 g, 0.57 mmol), NCS (0.076 g,
0.57 mmol) in DMF was stirred at rt for 6 h. The mixture was diluted with EA and washed with water
and brine. The organic layer was dried over MgSO4 and concentrated to give the crude product 11
1H-NMR (300 MHz, CDCl3)
10.11 (s, 1H), 8.08 (s, 1H), 7.84 (dd, J = 8.1, 0.9 Hz, 1H), 7.58 (dd, J = 7.2,
0.9 Hz, 1H), 7.24–7.19 (m, 1H), 4.15 (s, 3H).
.
δ
0
(Z)-N-(3-Bromo-4-fluorophenyl)-N -hydroxy-1-methyl-1H-indazole-7-carboximidamide (12). 1H-NMR (300 MHz,
CDCl3) 8.02 (s, 1H), 7.82 (dd, J = 8.1, 1.0 Hz, 1H), 7.60 (br, 1H), 7.35 (dd, J = 7.1, 1.0 Hz, 1H), 7.20–7.11
δ
(m, 1H), 6.82–6.75 (m, 1H), 6.73–6.64 (m, 1H), 6.38–6.29 (m, 1H), 4.09 (d, J = 6.2 Hz, 3H); LC/MS (ESI) m/z
363 [M + H]+.
(Z)-N-(3-Bromo-4-fluorophenyl)-N0,2-dihydroxybenzimidamide (16).
1H-NMR (300 MHz, CDCl3)
δ
7.27–7.18 (m, 1H), 7.13–7.07 (m, 1H), 7.07–6.98 (m, 2H), 6.98–6.89 (m, 1H), 6.76–6.65 (m, 2H); LC/MS
(ESI) m/z 325 [M + H]+.
3.2. Biology
3.2.1. Generation of Human Ido1 Gene Expressing Hek293 Recombinant Cells
cDNA of human IDO1 gene (provided from Korean UniGene, Daejeon, Korea) was inserted into
pcDNA5/FRT/TO expression vector (Invitrogen, Waltham, MA, USA) and transfected into Flp-In-Rex-
HEK293 cells (Invitrogen). After transfection, homogenous IDO1 expressing HEK293 cells were
selected using hygromycin and confirmed for their IDO1 expression using Western blot.
3.2.2. Cell Based Assay for Analysis of Anti-Ido1 Activity of Compounds by Determination of
Tryptophan and Kynurenine Using an LC-MS System
To analyze anti-IDO1 activity of compounds, human IDO1 expressing HEK293 recombinant cells
were seeded in 100 µL of complete MEM media containing 10% FBS in a 96-well plate. After incubation
for 48 hours, various concentrations of compounds with 0.2 ng/mL doxycyclin (Sigma Aldrich,
St. Louis, MO, USA) was treated and incubated for 24 h. Culture supernatants were collected and
prepared for analysis of tryptophan and kynurenine using LC-MS. For preparation of samples for
LC-MS analysis, 30
methanol. After centrifugation at 12,000 rpm for 10 min, 100
new 1.5 mL tubes. Distilled water (900 L) was added into 100
µ
L of culture supernatant was mixed with 2
L of supernatant was transferred into
L of supernatant and vortexed.
µM caffeine containing 270 µL
µ
µ
µ
The concentrations of tryptophan and kynurenine were determined by an LC-MS/MS system
consisting of an Agilent 1200 series HPLC system (Agilent Technologies, Santa Clara, CA, USA)
with an API 4000 QTRAP hybrid triple quadrupole/linear ion trap mass spectrometer (AB Sciex,
Foster City, CA, USA) equipped with a turbo electrospray interface. Chromatographic separation of
tryptophan and kynurenine was achieved on a Unison UK-C18 column (100 mm
×
2 mm i.d., 3
µm;
Imtakt, Kyoto, Japan) with a Securit◦y Guard C18 guard column (4 mm
×
20 mm i.d.; Phenomenex,
Torrance, CA, USA) maintained at 25 C. Isocratic mode was used to achieve separation of the analytes
using mobile phase (acetonitrile with 0.1 formic acid and water with 0.1% formic acid (20:80 v/v)
at a flow rate of 300
µL/min for 3 min. The multiple reaction monitoring (MRM) transition was
m/z 204.94 188.1 for tryptophan and m/z 208.97
→
→
192.00 for kynurenine under positive-ionization.
Optimized values for de-clustering potential, collision energy, entrance potential, and collision-exit
potential were 26 V, 15 eV, 9.5 V and 8 V and 21 V, 13 eV, 10 V and 8 V for tryptophan and kynurenine,
respectively. The o◦ptimized instrument conditions were as follows: ion spray voltage, 5500 V; source
temperature, 450 C; curtain gas, 25 psi; nebulizing gas (GS1), 30 psi; heating gas (GS2), 30 psi.
Data analysis was performed using Analyst Software version 1.4.2. (AB Sciex, Foster City, CA, USA).

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3.2.3. hIDO1 Enzymatic Assay
To analyze the hIDO1 inhibitory activities of compounds, we used IDO1 inhibitor screening assay
kit and followed the protocol provided from BPS Bioscience (San Diego, CA, USA). Briefly, 5 L of
L of compound in buffer at room
µ
IDO solution was aliquoted into a 384-well plate and treated with 5
µ
temperature for 3 h. The data was measured at λ = 320 to 325 nm.
3.3. Docking Study
Molecular modeling study of 8a against IDO1 was performed using the Schrödinger Suite 2017-2
(Schrödinger, LLC, New York, NY, USA) [22]. The X-ray crystal structure of IDO1 was obtained from the
Protein Data Bank [23]. The IDO1 protein preparation was revised using Protein Preparation Wizard in
Maestro v.11.2. The receptor grid box was generated 25
×
25
×
25 Å cubic size centered on complexed
heme. The compound was minimized using an OPLS_2005 force field with a dielectric constant value
80.0 in MacroModel v.11.6. The flexible docking of 8a was performed using the Standard Precision
method in Glide v.7.5 with positional constraints for covalent bonding with heme iron. The docking
model of 8a was visualized using Discovery Studio 2016 (Biovia, San Diego, CA, USA) [24].
4. Conclusions
In conclusion, we designed and synthesized a series of N0-hydroxyindazolecarboximidamides
for inhibition of IDO1. Among the compounds synthesized, compound 8a exhibited moderate IDO1
inhibitory activity in both tryptophan depletion and kynurenine production assays. In addition,
the molecular docking study with compound 8a suggested a structural basis for its IDO1 inhibition.
Although IDO1 inhibitory activities are relatively lower than those of known inhibitors, an indazole
scaffold might be a useful novel structure for further optimization study as an IDO1 inhibitor.
Acknowledgments: This work was supported by a research grant from National Research Council of Science and
Technology (NST) of the Korean government (CRC-15-KRIBB and CAP-15-11-KRICT).
Author Contributions: D.H.L., J.J., M.K., K.W.L. and E.J. performed the experiments; J.Y.L., S.A. and C.H.L.
contributed to analyze the data and to write the manuscript; J.Y.H. designed the study, analyzed the data,
and wrote the manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
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©
2017 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/).