Discovery and preliminary structure–activity relationship of 1H-indazoles with promising indoleamine-2,3-dioxygenase 1 (IDO1) inhibition properties

Article abstract of DOI:10.1016/j.bmc.2016.10.003

Indoleamine 2,3-dioxygenase 1 (IDO1)-mediated kynurenine pathway of tryptophan degradation is identified as an important immune effector pathway in the tumor cells to escape a potentially effective immune response. IDO1 is an attractive target for anticancer therapy and the discovery of IDO1 inhibitors has been intensely ongoing in both academic research laboratories and pharmaceutical organizations. Our study discovered that 1H-indazole was a novel key pharmacophore with potent IDO1 inhibitory activity. A series of new 1H-indazole derivatives were synthesized and determined the enzyme inhibitory activities, and the compound 2g exhibited the highest activity with an IC₅₀value of 5.3 μM. The structure–activity relationships (SARs) analysis of the 1H-indazole derivatives as novel IDO1 inhibitors indicated that the 1H-indazole scaffold is necessary for IDO1 inhibition, and the substituent groups at the both 4-position and 6-position largely affect inhibitory activity. The docking model exhibited that the effective interactions of 1H-indazoles with ferrous ion of heme and key residues of hydrophobic Pocket A and B ensured the IDO1 inhibitory activities. The study suggested that the 1H-indazole was a novel interesting scaffold for IDO inhibition for further development.

Full text of DOI:10.1016/j.bmc.2016.10.003

Accepted Manuscript
Discovery and preliminary structure-activity relationship of 1H-indazoles with
promising indolamine-2,3-dioxygenase 1(IDO1) inhibition properties
Shan Qian, Tao He, Wei Wang, Yanying He, Man Zhang, Lingling Yang, Guobo
Li, Zhouyu Wang
PII:
S0968-0896(16)30897-5
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.10.003
BMC 13330
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
4 August 2016
1 October 2016
5 October 2016
Please cite this article as: Qian, S., He, T., Wang, W., He, Y., Zhang, M., Yang, L., Li, G., Wang, Z., Discovery and
preliminary structure-activity relationship of 1H-indazoles with promising indolamine-2,3-dioxygenase 1(IDO1)
inhibition properties, Bioorganic & Medicinal Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.2016.10.003
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Discovery and preliminary structure-activity relationship of 1H-indazoles with
promising indolamine-2,3-dioxygenase 1(IDO1) inhibition properties
a
a
a
a
a
Shan Qian a, *, Tao He b, Wei Wang , Yanying He , Man Zhang , Lingling Yang , Guobo Li ,
Zhouyu Wang b,*
a Department of Pharmaceutical Engineering, Xihua University, Chengdu 610039, P.R. China.
b Department of Chemistry, Xihua University, Chengdu 610039, P.R. China.
*Address correspondence to these authors at Xihua University, Chengdu 610039, P.R. China;
E-mail: qians33@163.com; zhouyuwang77@gmail.com; Fax: +86-28-87720552.
ABSTRACT:
Indoleamine 2, 3-dioxygenase 1 (IDO1)-mediated kynurenine pathway of tryptophan degradation
is identified as an important immune effector pathway in the tumor cells to escape a potentially
effective immune response. IDO1 is an attractive target for anticancer therapy and the discovery of
IDO1 inhibitors has been intensely ongoing in both academic research laboratories and pharmaceutical
organizations. Our study discovered that 1H-indazole was a novel key pharmacophore with potent
IDO1 inhibitory activity. A series of new 1H-indazole derivatives were synthesized and determined the
enzyme inhibitory activities, and the compound 2g exhibited the highest activity with an IC50 value of
5.3 μM. The structure-activity relationships (SARs) analysis of the 1H-indazole derivatives as novel
IDO1 inhibitors indicated that the 1H-indazole scaffold is necessary for IDO1 inhibition, and the
substituent groups at the both 4-position and 6-position largely affect inhibitory activity. The docking
model exhibited that the effective interactions of 1H-indazoles with ferrous ion of heme and key
residues of hydrophobic Pocket A and B ensured the IDO1 inhibitory activities. The study suggested
that the 1H-indazole was a novel interesting scaffold for IDO inhibition for further development.
Keywords: Indoleamine 2,3-dioxygenase
1 (IDO1); 1H-Indazoles; Cancer immunotherapy;
Small-molecular inhibitors; Rational drug design.
1. Introduction
Immunotherapy is an exciting strategy of cancer therapy. It will be effective and critical to breach
immune suppressive mechanisms established by tumor cells in the treatment of cancer patients. Cancer
immunotherapy has been nominated for ‘Year Breakthrough of Cancer Research 2013’ by Science
magazine1. More than 50 phase III trials are currently on-going in cancer immunotherapy. Because of
distinguished effectiveness and novelty, cancer immunotherapies promise to be an innovation in the
treatment of cancer following operative treatment, chemotherapy, radiotherapy and targeted therapy.
Indoleamine 2, 3-dioxygenase 1 (IDO1) is an extrahepatic tryptophan catabolising enzyme encoded
by the IDO1 gene. IDO1 degrades the essential amino acid tryptophan (TRP) into kynurenine (KYN),
known as the ratelimiting and initial step of kynurenine pathway. IDO1 is silent in most tissues but
highly active in the placenta required for the maternal immune tolerance of the fetus. However, many
types of cancer cells and tumor environmental cells can express IDO1 in a constitutive manner or

strongly induced by inflammatory mediators such as interferon-gamma (IFN-γ) when host produces
inflammatory environment against the tumor 2, 3. IDO1-mediated catabolism of TRP is identified as an
important immune effector pathway to escape a potentially effective immune response in the tumor
cells 4-6. Thus, IDO is a pivotal target in cancer immunotherapy for small-molecule drugs via inhibiting
IDO enzyme or blocking its downstream signaling effects. IDO1 inhibitors might be also considerable
useful in combination with radiotherapy, chemotherapy or immunotherapy to trigger rapid regression
of aggressive tumors7, 8
Recently, the developments of IDO1 inhibitors are ongoing intensively in academia and
pharmaceutical companies, containing Pfizer Pharmaceuticals Ltd., Roche, Bristol-Myers Squibb9, 10
.
.
Several new IDO1 inhibitor scaffolds have been discovered by structure-based design, HTS, and
natural product screening11-25. However, the development of IDO1 pharmacological inhibitors is
proved to be a challenging work. Few structural classes of small molecules are known to be IDO1
inhibitors, and the most of them either showed low potency or was failed in vivo assay. Till now, only
three small-molecule compounds (Indoximod, Epacadostat13 and NLG91911, 26, 27) have entered clinical
trials. Hence, it is still of great interest to find new potent IDO1 inhibitors.
Structure-based in silico design of IDO inhibitors begin with the crystal structure of IDO bound to
the ligand PIM published by Sugimoto et al. in 2006 [Protein Data Bank (PDB) file"2D0T”28]. IDO1 is
a monomeric heme-containing enzyme. In the PIM-bound X-ray structure, the ligand is bound in a
catalytic site, in which nitrogen of imidazole ring is coordinated to Fe and phenyl ring is inside a
hydrophobic pocket (Pocket A). At the entrance of pocket A, there is another hydrophobic pocket
(pocket B) provided by the heme ring and multiple residues containing Phe226, Arg231, Ser235,
Phe291, Ile354, and Leu384. The larger ligands interact with residues located at both pocket A and B,
and thus may obtain better inhibitory activities (PDB file “4PK5”and “4PK6”29).
During our ongoing long-term project to discover potent IDO1 inhibitors for cancer
immunotherapies30, molecular docking technologies and pharmacophore models were performed to
design novel small-molecule IDO1 inhibitors using the valuable structural information revealed by
co-crystal structures of IDO1 with its ligands. Firstly, we performed key pharmacophore model to
design more than a dozen new small-molecule skeletons based on crystal structures of IDO1-ligands
complexes to identify potent IDO1 inhibitors. Secondly, the binding modes and affinities of small
molecules contained these skeletons were evaluated with molecular docking technology, which led to
the discovery of 1H-pyrazolo[3,4-d]pyrimidine and 1H-indazole derivatives. These two series were
likely to inhibit IDO1 enzyme via binding to the heme iron and occupying both Pocket A and B.
Therefore, a series of new 1H-pyrazolo[3,4-d]pyrimidine and 1H-indazole derivatives were synthesized
and determined IDO1 inhibitory activities. Interestingly, 1H-indazole was discovered to be a novel key
pharmacophore with potent IDO1 inhibitory activity. A series of new 1H-indazole derivatives were
synthesized and determined the enzyme inhibitory activities, and the compound 2g exhibited the
highest activity with an IC50 value of 5.3 μM (Fig. 1). In this paper, we report the chemical synthesis
and structure-activity relationships (SARs) analysis of the 1H-indazole derivatives as novel IDO1
inhibitors.

(A)
(B)
Fig.1. (A) Structure of compound 2g. (B) Schematic showing subgroups or atoms that were the focus
of structural modifications.
2. Chemistry
The N-benzyl-1H-pyrazolo[3,4-d]pyrimidin-4-amines (1a~d) were synthesized using the reaction
sequence highlighted in Scheme 1. The 1H-pyrazolo[3,4-d]pyrimidin-4(7H)-one (6a) was purchased
from
commercially
available
sources
(CAS:
315-30-0),
and
6-methyl-1H-pyrazolo[3,4-d]pyrimidin-4(7H)-one (6b) was readily synthesized by refluxing 5 in 1:1
polyphosphoric acid (PPA) and acetic acid. 1H-pyrazolo[3,4-d]pyrimidin-4(7H)-ones 6a~b were
reacted with thionyl chloride to produce 4-chloro-1H-pyrazolo[3,4-d]pyrimidines (7a~b), which on
interaction with substituted benzoic acids containing aminomethyl at the C3 or C4 position (8a and 8b)
afforded N-benzyl-1H-pyrazolo[3,4-d]pyrimidin-4-amines (1a~d) in 43-48% yield, respectively.
Scheme 1. Synthesis of N-benzyl-1H-pyrazolo[3,4-d]pyrimidin-4-amines 1a~d.
Scheme 2 showed the route of synthesizing (6-bromo-1H-indazol-4-yl)amino-acetamides 3a~b. The
starting materials 9a~b were reduced by iron powder in the presence of ammonium chloride to produce
amines 10a~b in quantitative yield. 6-bromo-1H-indazol-4-amine 10b was reacted with ethyl
bromoacetate in the presence of potassium iodide and potassium carbonate to produce ethyl
2-((6-bromo-1H-indazol-4-yl)amino)acetate 11, which was subsequently hydrolyzed in the presence of
NaOH to generate corresponding acetic acid 12. Combinations of acid 12 and amines (13 and 14) were
reacted in the presence of carbodiimide hydrochloride (EDCI), hydroxybenzotriazole (HOBT) and N,
N-diisopropylethylamine (DIEA) to yield the corresponding amides (3a and 15), respectively.
2-((6-Bromo-1H-indazol-4-yl)amino)-N-(4-ethoxycarbonylphenyl)acetamide 15 was subsequently
hydrolyzed in the presence of NaOH to generate corresponding acid 3b.

Scheme 2. Synthesis of (6-bromo-1H-indazol-4-yl)amino-N-acetamides 3a~b.
The 1H-indazole-4-amines with various substituent groups at C4 position (2a~m) were synthesized
using the reaction sequence highlighted in Scheme 3. N-Benzyl-1H-indazol-4-amines 2a~j were
prepared in 43%-82% yield by condensation of amines 9a~b and substituted benzaldehydes 16a-j
using Hantzsch ester 1,4-dihydropyridine (DHP) as the hydrogen source and trifluoroacetic acid as
acidic catalyst in dry CH2Cl2. Epoxidation of methyl 4-vinylbenzoate afforded methyl
4-(oxiran-2-yl)benzoate 17. Aminolysis of 1,2-epoxides 17 with arylamine 9b was performed in 1:1
ethanol/water to produce β-amino alcohols 18 and 19, which subsequently hydrolyzed in aq. NaOH to
afford 2k and 2m, respectively. All the synthesized compounds (2a~m) were fully confirmed on the
basis of their analytical and spectral studies. The 1H-NMR of 2a~m has shown characteristic peaks at
9.63-7.94, 6.83-6.64 and 6.01-5.89 ppm corresponds to C3-H, C7-H and C5-H of indazole, respectively.
It is also necessary to point out that the 1H-NMR of 2k has shown characteristic peaks at 6.97 (d), 5.13
(br), 4.68-4.63 (m) and 3.75-3.71 (m) ppm corresponding to NH, OH, NHCH and OHCH2, respectively.
1
However, the H-NMR of 2m has shown characteristic peaks at 6.71 (t), 5.78 (br), 4.91 (m) and 4.13
(m) ppm corresponding to NH, OH, OHCH and NHCH2, respectively. The 13C-NMR of the
synthesized compounds 2a~m have shown peaks at 173.2-167.7 and 156.7–98.1 ppm corresponding to
carboxylic acid and aromatic ring carbons, respectively.
Scheme 3. Synthesis of 1H-indazole-4-amines 2a~m.

The 6-bromo-1H-indazole-4-ethers 4a~b were synthesized using the reaction sequence highlighted
in Scheme 4. 4-Bromo-2,6-difluorobenzaldehyde 20 was reacted with hydrazine monohydrate to
quantitatively afford 6-bromo-4-fluoro-1H-indazole 21, the amino group of which was subsequently
protected with t-butyloxycarboryl (Boc) group in the present of (Boc)2O and dimethylaminopyridine
(DMAP). Methyl iodomethylbenzoates 23a~b were prepared by iodination of corresponding carbonyl
compounds using 1,1,3,3-tetramethyldisiloxane (TMDSO) and iodine. Compound 22 was hydrolyzed
in the presence of K2CO3 in water to generate corresponding phenol, which etherified with iodine
compounds 23a~b to afford ethers 24a~b. Ethers 24a~b were hydrolyzed in trifluoroacetic acid (TFA)
1
to afford amines 25a~b, which subsequently hydrolyzed in aq. NaOH to afford 4a~b. The H-NMR
spectra of 4a~b have shown characteristic peaks at 8.04-7.66, 7.51-7.50, 7.49 and 4.34 ppm
corresponding to C3-H, C7-H, C5-H of indazole and benzyl-CH2, respectively. The 13C-NMR of 4a~b
have shown peaks at 167.8-167.7 and 162.9–114.4 ppm corresponding to carboxylic acid and aromatic
ring carbons, respectively. High-resolution mass spectrometry is further evidence for the formation of
these products.
Scheme 4. Synthesis of 6-bromo-1H-indazole-4-ethers 4a~b.
3. Results and discussion
3.1. IDO1 inhibitory activities of 1H-pyrazolo[3,4-d]pyrimidine and 1H-indazole derivatives
To screen inhibitory activities of these 1H-pyrazolo[3,4-d]pyrimidine and 1H-indazole derivatives
against IDO1, we carried out enzyme assays using three inhibitor concentrations. The mixture
containing an aliquot of enzyme was incubated with a fixed substrate concentration (900 μM) and
different test sample concentrations (1 μM, 10 μM and 100 μM). Results were reported in Table 1.
Under the conditions, the IC50 value of Epacadostat was 67 nM, which was consistent with previous
value in Ref.9 (IC50= 72 nM). 1H-indazole derivatives (2a, 3a and 3b) were shown to have IDO1
inhibitory activities, and especially for compound 2a, the inhibitory ratios are 91% and 58% at the
concentrations of 100 μM and 10 μM, respectively. Compounds 1c and 1d with a methyl at the
6-position of 1H-pyrazolo[3,4-d]pyrimidine showed stronger IDO1 inhibitory activities than these with
1a and 1b, implying that the methyl at 6-position of 1H-pyrazolo[3,4-d]pyrimidine might be essential
to maintain the inhibitory activity through the interactions with hydrophobic residues of Pocket A.
Despite all this, 1H-pyrazolo[3,4-d]pyrimidines (1a~d) behaved as weak inhibitors. It revealed that
benzene ring of 1H-indazole preferred over nitrogen heterocycle of 1H-pyrazolo[3,4-d]pyrimidine to
have inhibitory potency, perhaps because of the hydrophobic property of Pocket A. Overall, our data

demonstrated that 1H-indazole might be a novel key pharmacophore with potent IDO1 inhibitory
activity.
Table 1.
The structures and activities of 1H-pyrazolo[3,4-d]pyrimidins 1 and 1H-indazoles 2~3.
Inhibition%
Compound
R1
H
R2
at 1μM
at 10μM
at 100μM
2
2
7
1a
1b
1c
1d
2a
3a
H
3
3
3
5
9
CH3
CH3
Br
32
37
94
34
3
5
19
4
49
8
Br
Br
-
3
12
37
-
3b
-
97
100
Epacadostat
3.2. Structural optimization of 1H-indazole derivatives
The purpose of the subsequent study was to perform structural modifications to optimize the potency
of 1H-indazole derivatives against IDO1. Structural hybridization of 1H-indazole derivatives was
carried out (as shown in Fig. 1b) with the view that (i) by defining 1H-indazole as the core skeleton,
which was kept fixed to optimize these substituent groups at both 4-position and 6-position; (ii) by
replacing the substituent (R1) at the 6-position with halogen-containing group; (iii) by replacing the
atom (Y) at the 4-position linker site with a nitrogen (-NH) and oxygen (-O), respectively, to largely
affect the location of the substituent groups at the 4-position; (iv) by increasing the structural diversity
of the substituent groups (R2) at the 4-position, in order to more preorganize for binding to IDO1.
Fourteen synthesized 1H-indazole derivatives with disubstituent groups at both 4-position and
6-position were subjected to IDO1 inhibition assay and their inhibitory ratios were shown in Table 2
and Fig. 2. As expected, some compounds displayed remarkable IDO1 inhibitory activities. Compound
2g exhibited the most IDO1 inhibitory activity with the inhibitory ratios of 100% and 70% at the
concentrations of 100 μM and 10 μM, respectively. Compound 2g was a good IDO inhibitor being
characterized with an IC50 value of 5.3 μM (Fig. 3).

Table 2.
The structures and activities of 1H-indazole-4-amines 2a~m and 1H-indazole-4-ethers 4a~b.
Inhibition ratio (%)
Comp.
2a
Y
R1
Br
H
R2
at 10 μM
at 100 μM
NH
NH
NH
49
94
1
24 a
2b
Br
37
100 b
2c
NH
Br
21
74 a
2d
NH
NH
NH
Br
Br
Br
20
42
70
84 b
100 a
100 b
2e
2f
2g
NH
NH
NH
Br
Br
Br
38
27
41
88
2h
2i
79 b
100 b
2j
NH
NH
O
Br
Br
Br
19
31
18
68
73
2k
2m
4a
69 b
O
Br
7
40
4b
(a) Visible precipitate present in intermediate dilutions; (b) These conditions demonstrated significant
background absorbance (greater than signal from positive control).

Fig. 2. The IDO1 inhibition ratio of 1H-indazoles.
-8.8
92
-8.3
91
-7.8
91
-7.3
88
-6.9
87
-6.4
82
-5.9
71
-5.4
51
-5
-4.5
9
log([2g]/M)
%Activity
26
Fig. 3. Data for the effect of 2g on IDO1 activity.
Molecular modeling was used to understand the binding mode of 1H-indazoles. Fig. 4 showed
compound 2g docked into IDO1 along with the X-ray structure of NLG919-IDO1 complex23. The
docking model (Fig. 4A) exhibited that the 2-position nitrogen atom of 1H-indazole provided
coordinate bond with ferrous ion of heme, similar to the imidazo[5,1-a]isoindole of NLG919 observed
in crystal structure of 5EK3 (Fig. 4B). Replacement of halogen atom at 6-position brought recognition
of increased hydrophobic interactions with TYR126, PHE164, LEU234 and PHE163 of Pocket A (Fig.
4A). These crucial interactions were not possible with 2b. Compound 2g may be showing the best
activity due to extra hydrogen bonding interactions between its phenolic hydroxyl group and ARG231
of IDO1, as well as amino of 4-position substitutuent and heme 7-propionate (Fig. 4A). As shown in
Fig. 4B, the binding mode of compound 2g is similar to that of NLG919. Therefore, the effective
interactions of 1H-indazoles with ferrous ion of heme and key residues of hydrophobic Pocket A and B
ensure the IDO1 inhibitory activities.

Fig. 4. (A) Binding mode of 2g and IDO1; (B) Superposition of the binding mode of 2g and the
X-ray structure of NLG919-IDO1 complex (PDB file “5EK3”).
3.3. SARs of 1H-indazole derivatives as IDO1 inhibitors
As shown as Table 2, compound 2b without any substituent (R1) at the 6-position showed poor
activity; while compound 2a with a bromine atom at the 6-position exhibited improved inhibitory
potency. Without considering any substituent on 1H-indazole ring, the data of compounds 2a and 2b
revealed that hydrophobic interactions of halogen atom at 6-position with residues of Pocket A
appeared to be very important for the IDO1 inhibitory activities of these 1H-indazoles.
There are two series of 1H-indazole derivatives, namely 4-amino-1H-indazoles and
4-oxo-1H-indazoles, which contain a nitrogen (-NH) and oxygen (-O) atom at the 4-position linker site,
respectively. The 4-amino-1H-indazoles 2a and 2d are more potent than the corresponding
4-oxo-1H-indazoles 5a and 5b, respectively. It revealed that the different bond angles between -NH
and -O at the 4-position linker site largely altered the location of substituent groups at the 4-position,
and thus affected the interaction between these compounds and enzyme.

We then explored the influence of different substituent groups (R2) at the 4-position of
1H-indazole. It was clear that the compounds with a polar carboxyl or hydroxyl group at the
para-position of benzene ring (2a, 2g and 2j) showed higher inhibitory potency relative to these
analogues bearing a methyl or halogen group at the para-position of benzene ring (2e and 2f).
Compared with the carboxyl compound 2a, the hydroxyl compound 2g exhibited higher IDO1
inhibitory activity. However, introduction of another carboxyl substituent at meta-position (2h) led to a
dramatical decrease of potency activity. It demonstrated that the interaction between polar groups and
ARG231 was important to maintain IDO1 inhibitory activity.
A comparison of inhibitory activities of these compounds 2a, 2d, 2g and 2j indicated that these
compounds with a polar carboxyl or hydroxyl group at the polar-position of benzene ring increased
their potency relative to those at either ortho- or meta-position. Introduction of carboxylmethyl
substituent at para-position (2i) led to a dramatical decrease of potency activity. Besides, the bridge
groups connecting Y atom at the 4-position and benzene ring of R2 substituent also could affect the
inhibitory activity. The data of compounds 2l, 2m, 4a and 4b revealed that replacement of the methyl
group by an extended linker led to decreased activities. The enzyme assay results above demonstrated
that the location of the polar group at the 4-position played a crucial role in the IDO1 inhibition.
In summary, these results of the SARs analysis (Fig. 5) indicated that 1H-indazole is necessary
for IDO1 inhibition, and the substituent groups at the both 4-position and 6-position largely affect
inhibitory activity.
Fig. 5. SARs of 1H-indazole derivatives as IDO1 inhibitors.
4. Conclusion
IDO1-mediated catabolism of TRP is identified as an important immune effector pathway in the
tumor cells to escape a potentially effective immune response. IDO is a pivotal target in cancer
immunotherapy for small-molecule drugs via inhibiting IDO enzyme or blocking its downstream
signaling effects. IDO1 inhibitors might also be considerable useful in combination with radiotherapy,
chemotherapy or immunotherapy to trigger rapid regression of aggressive tumors. Molecular docking
technologies and pharmacophore models were performed in our laboratory to design novel
small-molecule IDO1 inhibitors, which led to the discovery of 1H-pyrazolo[3,4-d]pyrimidine and

1H-indazole derivatives. We aimed at exploring the interest of the 1H-indazole scaffold, which shares
structural similarity with 1H-indole. SARs of 1H-indazole derivatives as IDO1 inhibitors showed: (i)
the halogen atom at 6-position was very important for the IDO1 inhibitory activities; (ii) the different
bond angles between -NH and -O at the 4-position linker site largely affected the interaction between
these compounds and enzyme; (iii) the polar carboxyl or hydroxyl group at the para-position of
benzene ring increased inhibitory potency; (iv) the location of the polar group at the 4-position played a
crucial role in the IDO1 inhibition. The docking model exhibited that the effective interactions of
1H-indazoles with ferrous ion of heme and key residues of hydrophobic Pocket A and B ensure the
IDO1 inhibitory activities. Given the exciting potential of these novel small-molecular inhibitors, we
believe that the 1H-indazoles will be a useful structural class of IDO1 inhibitors.
5. Experimental section
5.1. General chemistry information
All chemical reagents were purchased from commercial sources and used without further
purification. Reactions were performed using oven-dried glassware under an atmosphere of nitrogen
and in anhydrous conditions (as required). All reactions were monitored by thin layer chromatography
(TLC) precoated with 0.25 mm silica gel plates (HSGF254) and visualized with UV light (254 nm or
365 nm) or chromogenic agents. Most products were purified by column chromatography (silica gel
200 mesh). Proton and Carbon nuclear magnetic resonance (1H-NMR and 13C-NMR) were recorded in
designated solvents using a Bruker Avance 400 and Bruker DPX 300 spectrometer, respectively.
Chemical shifts (δ) are quoted in parts per million (ppm) with tetramethylsilane (TMS) as internal
standard and coupling constants (J values) were given in hertz (Hz). The splitting patterns were
described as s (singlet), d (doublet), dd (doublet doublet), t (triplet), q (quartet), m (multiplet), and br
(broad). Melting points were measured on an electrothermal melting point apparatus without correction.
ESI-MS was obtained on an Shimadzu-2010EV series liquid choromatograph mass spectrometer.
High-resolution mass spectrometry was performed by the micrOTOF-Q II 10203 mass spectrometer.
5.1.1. 6-Methyl-1H-pyrazolo[3,4-d]pyrimidin-4(7H)-one (6b)
5-amino-4,5-dihydro-1H-pyrazole-4-carboxamide 5 (commercially available, CAS: 5334-31-6, 1.0 g,
7.94 mmol) was taken into 1:1 PPA and acetic acid (12 mL) and the mixture was heated at 120C for
6-7 hours. The mixture was cooled to ambient temperature then taked into water (70 mL) and the pH
adjusted to approximately 5 by dropwise addition of 50% aqueous NaOH. The resulting white
precipitate was collected by filtration and washed with additional water. The solid was then suspended
in methanol and collected by filtration then washed with ethyl acetate. Drying in vacuo afforded 6b
1
(887.5 mg, 76%) as a colorless solid. H-NMR (400 MHz, d6-DMSO, ppm): δ 13.52 (s, 1H,
pyrazole-NH), 11.96 (s, 1H, pyrimidine-NH), 7.97 (s, 1H, pyrazole-CH), 2.34 (s, 1H, CH3). 13C-NMR
(100MHz, d6-DMSO, ppm): δ 158.6, 157.8, 154.6, 135.4, 103.8, 21.6. ESI-MS: 151.1 (M + H)+.
5.1.2. 3-(((1H-Pyrazolo[3,4-d]pyrimidin-4-yl)amino)methyl)benzoic acid (1a)
A suspension of 6a (0.5 g, 3.68 mmol) in thionyl chloride (15 mL) and DMF (1.5 mL) was refluxed
for 2 h under argon. The solution was concentrated, and the contents were poured into crushed ice
water (150 mL). The solution was extracted with ethyl actate (3 × 100 mL). The combined organic
layers were washed with brine, dried with anhydrous Na2SO4, and concentrated to give 7a (64%) as a
yellow solid.

To a solution of 7a (50.0 mg, 0.32 mmol) and aminomethyl-benzoic acids 8a (40.6 mg, 0.27 mmol) in
triethylamine (66.8 μL, 0.48 mmol), anhydrous DMF (1.5 mL) was added and the reaction mixture
was stirred at 80C for 4 h. The reaction mixture was diluted with ethyl acetate (8 mL) and was washed
with water (3 × 4 mL). The ethyl acetate layer was then dried with anhydrous Na2SO4, and
concentrated to give a residue that was subjected to purification through the column chromatography
on silica gel with DCM and MeOH (10:1) as eluent to afford the titled compounds 1a (31.1 mg, 43%)
1
as a yellow solid. mp: > 300C. H-NMR (400MHz, d6-DMSO, ppm): δ 13.44 (br, 2H, COOH and
pyrazole NH), 8.81 (m, 1H, amino), 8.23 (s, 1H, pyrimidine-CH), 8.17 (s, 1H, pyrazole-CH), 7.94 (s,
1H, Ar-H2), 7.83 (d, 1H, J = 7.6 Hz, Ar-H6), 7.46 (d, 1H, J = 7.6 Hz, Ar-H4), 7.46 (t, 1H, J = 7.6 Hz,
Ar-H5), 4.80 (d, 2H, J = 6.0 Hz, benzyl-CH2). HRMS (AP-ESI) Calcd. for C13H12N5O2: 269.0991 (M +
H)+. Found: 270.0982.
Compounds 1b~1d were synthesized following the procedure described above.
5.1.2.1. 4-(((1H-Pyrazolo[3,4-d]pyrimidin-4-yl)amino)methyl)benzoic acid (1b). Yield 41%;
yellow solid; mp: > 300C. 1H-NMR (400MHz, d6-DMSO, ppm): δ 14.50 (s, 1H, br, COOH), 13.41 (s,
1H, pyrazole NH), 8.79 (m, 1H, amino), 8.23 (s, 1H, pyrimidine CH), 8.17 (s, 1H, pyrazole CH), 7.91
(d, 2H, J = 7.6 Hz, Ar-H2 and Ar-H6), 7.46 (d, 2H, J = 7.6 Hz, Ar-H3 and Ar-H5), 4.82 (d, 2H, J = 5.2
Hz, benzyl-CH2). ESI-MS: 270.10 (M + H) +.
5.1.2.2. 3-(((6-Methyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)amino)methyl)benzoic acid (1c). Yield
36%; yellow solid; mp: > 300C. 1H-NMR (400MHz, d6-DMSO, ppm): δ 14.58 (s, 1H, pyrazole-NH),
12.92 (br, 1H, COOH), 11.43 (t, 1H, J = 6.0 Hz, amino), 8.52 (s, 1H, pyrazole-CH), 8.02 (s, 1H,
Ar-H2), 7.98 (d, 1H, J = 7.6 Hz, Ar-H6), 7.56 (d, 1H, J = 7.6 Hz, Ar-H4), 7.50 (t, 1H, J = 7.6 Hz,
Ar-H5), 5.02 (d, 2H, J = 6.0 Hz, benzyl-CH2), 2.33 (s, 1H, CH3). ESI-MS: 284.10 (M + H) +.
5.1.2.3. 4-(((6-Methyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)amino)methyl)benzoic acid (1d). Yield
39%; yellow solid; mp: > 300C. 1H-NMR (400MHz, d6-DMSO, ppm): δ 14.74 (s, 1H, pyrazole-NH),
12.94 (br, 1H, COOH), 11.39 (t, 1H, J = 6.0 Hz, amino), 8.55 (s, 1H, pyrazole-CH), 7.93 (d, 2H, J =
8.3 Hz, Ar-H2 and Ar-H6), 7.46 (d, 2H, J = 8.3 Hz, Ar-H3 and Ar-H5), 5.08 (d, 2H, J = 6.0 Hz,
benzyl-CH2), 2.34 (s, 1H, CH3). ESI-MS: 284.10 (M + H)+.
5.1.3. 1H-Indazol-4-amines (10a~b)
4-Nitro-1H-indazole 9a (commercially available, CAS: 2942-40-7, 1.4 g, 8.26 mmol) or
6-bromo-4-nitro-1H-indazole 9b (commercially available, CAS: 885518-46-7, 2.0 g, 8.26 mmol) was
taken in a round bottom flask having ethanol (20 mL) and water (10 mL) as solvents. Ammonium
chloride (221.5 mg, 4.13 mmol) was added and the solution was stirred for 5 min at ambient
temperature. To this content, a portion of iron powder (1.3 g, 23.46 mmol) was added slowly and
stirred for 5 min at 80C. Then another portion of iron powder (1.0 g, 17.86 mmol) was added and the
stirring was continued at 80C for 20 min. The mixture was immediately filtrated and the filter residue
was washed with ethanol (10 mL). The filtrate was concentrated under reduced pressure and then
dissolved in ethyl acetate (8 mL). The organics were washed with brine, dried with anhydrous Na2SO4
and concentrated. The solid produced was purified through the column chromatography on silica gel
with petroleum ether and ethyl acetate (8:1) as eluent to afford 10a (379.0 mg, 93%) or 10b (1.62 g,
92%) as a yellow solid.

5.1.4. Ethyl 2-((6-bromo-1H-indazol-4-yl)amino)acetate (11)
6-Bromo-1H-indazol-4-amine 10b (212.0 mg, 1.00 mmol), potassium iodide (14.9 mg, 0.09 mmol)
and potassium carbonate (345.0 mg, 2.50 mmol) were dissolved in DMF (5 mL). Ethyl bromoacetate
(167.0 μL, 1.50 mmol) was added and the mixture was stirred at 65C under argon for 12 h. The
solution was cooled to the ambient temperature and diluted with ethyl acetate (25 mL). The organics
were washed with water (3 × 20 mL) and brine, dried with anhydrous Na2SO4 and concentrated. The
solid produced was purified through the column chromatography on silica gel with and petroleum ether
and ethyl acetate (15:1) as eluent to afford 11 (206.9 mg, 70%) as a yellow solid.
5.1.5. 2-((6-Bromo-1H-indazol-4-yl)amino)acetic acid (12)
To the solution of ethyl 2-((6-bromo-1H-indazol-4-yl)amino)acetate 11 (80.0 mg, 0.27 mmol) in 1:1
ethanol/H2O (3 mL) added NaOH (32.2 mg, 0.81 mmol), and the mixture was stirred at ambient
temperature for 12 h. The solution was adjusted to pH 5 by addition of 1 mol/L aqueous HCl and then
concentrated under reduced pressure to afford 12 (72.0 mg, 100%) as a white solid.
5.1.6. 2-((6-Bromo-1H-indazol-4-yl)amino)-N-(tetrahydro-2H-pyran-4-yl)acetamide (3a)
To a solution of acid 12 (50.0 mg, 0.19 mmol) in CH2Cl2 (3 mL) was added
tetrahydro-2H-pyran-4-amine 13 (57 uL, 0.56 mmol), HOBT (32.4 mg, 0.22 mmol), DIEA (61 uL, 0.37
mmol) and EDCI (42.6 mg, 0.22 mmol) at 0C. The reaction mixture was stirred at 25C for 12 h, and
then concentrated under reduced pressure. The resulting mixture was diluted with CH2Cl2 (10 mL). The
organic layer was separated and washed with saturated aqueous NaHCO3 (5 mL) and brine (3 mL), and
dried over anhydrous Na2SO4. After removal of solvents under reduced pressure, the residue was
purified through column chromatography on silica gel with petroleum ether and ethyl acetate (10:1) as
eluent to give amide 3a (42.1 mg, 65%). mp: 292-294C. 1H-NMR (400 MHz, d6-DMSO, ppm): δ 8.22
(d, 1H, J = 7.5 Hz, amide), 8.07 (s, 1H, indazole-H3), 6.87 (s,1H, indazole-H7), 6.29 (d, 1H, J = 1.1 Hz,
indazole-NH), 6.14 (s, 1H, indazole-H5), 4.90 (s, 1H, CH2), 3.85-3.76 (m, 4H, pyran-H2 and
pyran-H6), 3.52-3.48 (m, 1H, pyran-H4), 3.42-3.34 (m, 4H, pyran-H3 and pyran-H5). HRMS (AP-ESI)
Calcd. for C1H16BrN4O2Na: 375.0433 (M + Na)+. Found: 375.0452.
5.1.7. 2-((6-Bromo-1H-indazol-4-yl)amino)-N-(4-carboxyphenyl)acetamide (3b)
2-((6-Bromo-1H-indazol-4-yl)amino)-N-(4-ethoxycarbonylphenyl)acetamide 15 (46.4 mg, 60%) was
synthesized from acid 12 (50.0 mg, 0.19 mmol) and ethyl 4-aminobenzoate 14 (91.5 mg, 0.56 mmol)
following the procedure of synthesizing 3a.
To the solution of amide 15 (40.0 mg, 0.10 mmol) in 1:1 ethanol/H2O (2 mL) added NaOH (11.5 mg,
0.29 mmol), and the mixture was stirred at ambient temperature for 12 h. The solution was adjusted to
pH 5 by addition of 1 mol/L aqueous HCl. The mixture was filtrated and the filter residue was washed
with water, dried under reduced pressure to afford 3b (15.6 mg, 42%) as a white solid. mp: > 300C.
1H-NMR (400 MHz, d6-DMSO, ppm): δ 12.80 (br, 1H, COOH), 10.67 (s, 1H, amide), 8.13 (s, 1H,
indazole-H3), 7.91 (d, 2H, J = 8.7 Hz, Ar-H2 and Ar-H6), 7.79 (d, 2H, J = 8.7 Hz, Ar-H3 and Ar-H5),
6.98 (s, 1H, indazole-H7), 6.32 (d, 1H, J = 1.2 Hz, indazole-NH), 6.18 (s, 1H, indazole-H5), 5.21 (s,
2H, benzyl-CH2). 13C-NMR (100MHz, d6-DMSO, ppm): δ 207.0, 167.3, 166.7, 143.9, 143.1, 132.9,
131.0, 126.0, 121.9, 118.9, 113.2, 104.7, 99.6, 52.2. ESI-MS: 389.02 (M + H)+.

5.1.8. 4-(((6-Bromo-1H-indazol-4-yl)amino)methyl)benzoic acid (2a)
To the solution of amines 9a (60 mg, 0.28 mmol) and substituted benzaldehydes 16a (36 mg, 0.24
mmol) in DCM (3 mL) added DHP (83.5 mg, 0.33 mmol) and molecular sieve (840.2 mg).
Trifluoroacetic acid (17.6 μL, 0.24 mmol) was added to the suspension dropwise and the mixture was
stirred at 40C for 12 h. The mixture was filtered and the filtrate was concentrated under reduced
pressure. The solid produced was purified through the column chromatography on silica gel to afford
1
the titled compound 2a (53 mg, 64%) as a brown solid. mp: 212-214C. H-NMR (400 MHz,
d6-DMSO, ppm): δ 12.80 (br, 2H, COOH and indazole-NH), 8.22 (s, 1H, indazole-H3), 7.91 (d, 2H, J
= 8.3 Hz, Ar-H2 and Ar-H6), 7.49 (d, 2H, J = 8.3 Hz, Ar-H3 and Ar-H5), 6.64 (s, 1H, indazole-H7),
6.01 (s, 1H, indazole-H5), 4.53 (s, 2H, benzyl-CH2). 13C-NMR (100MHz, d6-DMSO, ppm): δ 167.7,
145.3, 143.0, 142.2, 132.5, 130.0, 129.9, 127.5, 121.7, 112.6, 101.2, 100.6, 46.2. HRMS (AP-ESI)
Calcd. for C15H11BrN3O2: 344.0035 (M – H)-. Found: 344.0035.
Compounds 2b~2j were synthesized following the procedure described above.
5.1.8.1. 4-(((1H-Indazol-4-yl)amino)methyl)benzoic acid (2b). Yield 62%; brown solid; mp:
254-256C. 1H-NMR (400 MHz, d6-DMSO, ppm): δ 12.79 (br, 2H, COOH and indazole-NH), 8.22 (s,
1H, indazole-H3), 7.90 (d, 2H, J = 7.4 Hz, Ar-H2 and Ar-H6), 7.50 (d, 2H, J = 7.4 Hz, Ar-H3 and
Ar-H5), 6.99-6.96 (m, 1H, indazole-H6), 6.66 (d, 1H, J = 7.9 Hz, indazole-H7), 5.90 (d, 1H, J = 7.1 Hz,
indazole-H5), 4.52 (s, 2H, benzyl-CH2). 13C-NMR (100MHz, d6-DMSO, ppm): δ 167.7, 146.1, 142.0,
141.7, 132.1, 129.9, 129.7, 128.0, 127.5, 113.8, 98.2, 98.1, 46.5. ESI-MS: 266.10 (M – H)-.
5.1.8.2. N-Benzyl-6-bromo-1H-indazol-4-amine (2c). Yield 43%; brown solid; mp: 167-169C.
1H-NMR (400 MHz, d6-DMSO, ppm): δ 12.83 (s, 1H, indazole-NH), 8.23 (s, 1H, indazole-H3),
7.40-7.23 (m, 5H, Ar-H), 6.83 (s, 1H, indazole-H7), 6.06 (s, 1H, indazole-H5), 4.44 (d, 2H, J = 6.0 Hz,
benzyl-CH2). 13C-NMR (100MHz, d6-DMSO, ppm): δ 143.2, 142.1, 139.9, 132.7, 128.9, 127.5, 127.3,
121.7, 112.6, 101.2, 100.3, 46.4. ESI-MS: 302.02 (M + H)+.
5.1.8.3. 3-(((6-bromo-1H-indazol-4-yl)amino)methyl)benzoic acid (2d). Yield 60%; brown solid;
1
mp: 252-254C. H-NMR (400 MHz, d6-DMSO, ppm): δ 12.90 (br, 2H, COOH and indazole-NH),
8.22 (s, 1H, indazole-H3), 7.98 (s, 1H, Ar-H2), 7.83 (d, 1H, J = 7.6 Hz, Ar-H4), 7.64 (d, 1H, J = 7.6 Hz,
Ar-H6), 7.48 (t, 1H, J = 7.6 Hz, Ar-H5), 7.37 (t, 1H, J = 6.0 Hz, NH), 6.84 (s, 1H, indazole-H7), 6.05 (s,
1H, indazole-H5), 4.51 (d, 2H, J = 6.0 Hz, benzyl-CH2). 13C-NMR (100MHz, d6-DMSO, ppm): δ 167.8,
143.0, 142.2, 140.6, 132.5, 132.1, 131.4, 129.1, 128.3, 128.3, 121.7, 112.6, 101.2, 100.5, 46.0. ESI-MS:
344.02 (M – H)-.
5.1.8.4. 6-Bromo-N-(4-methylbenzyl)-1H-indazol-4-amine (2e). Yield 65%; yellow solid; mp:
1
154-156C. H-NMR (400 MHz, d6-DMSO, ppm): δ 12.82 (s, 1H, indazole-NH), 8.22 (s, 1H,
indazole-H3), 7.26 (d, 2H, J = 7.9 Hz, Ar-H2 and Ar-H6), 7.14 (d, 2H, J = 7.9 Hz, Ar-H3 and Ar-H5),
6.82 (s, 1H, indazole-H7), 6.04 (s, 1H, indazole-H5), 4.38 (d, 2H, J = 6.0 Hz, benzyl-CH2). 13C-NMR
(100MHz, d6-DMSO, ppm): δ 143.2, 141.2, 136.8, 136.3, 132.7, 129.4, 127.5, 121.7, 112.6, 101.2,
100.2, 46.2, 21.1. ESI-MS: 316.04 (M + H)+.
5.1.8.5. 6-Bromo-N-(4-chlorobenzyl)-1H-indazol-4-amine (2f). Yield 71%; yellow solid; mp:
1
158-160C. H-NMR (400 MHz, d6-DMSO, ppm): δ 12.85 (s, 1H, indazole-NH), 8.21 (s, 1H,

indazole-H3), 7.40 (s, 4H, Ar-H), 7.32 (t, 1H, J = 6.0 Hz, NH), 6.84 (s, 1H, indazole-H7), 6.03 (s, 1H,
indazole-H5), 4.43 (d, 2H, J = 6.0 Hz, benzyl-CH2). 13C-NMR (100MHz, d6-DMSO, ppm): δ 142.9,
142.1, 139.0, 132.6, 131.8, 129.3, 127.5, 121.7, 112.6, 101.3, 100.5, 45.7. ESI-MS: 335.98 (M + H)+.
5.1.8.6. 4-(((6-Bromo-1H-indazol-4-yl)amino)methyl)phenol (2g). Yield 59%; yellow solid; mp:
1
180-182C. H-NMR (400 MHz, d6-DMSO, ppm): δ 12.80 (s, 1H, indazole-NH), 9.29 (s, 1H,
indazole-H3), 8.21 (s, 1H, OH), 7.17 (d, 2H, J = 8.4 Hz, Ar-H3 and Ar-H5), 6.81 (s, 1H, indazole-H7),
6.72 (d, 2H, J = 8.4 Hz, Ar-H2 and Ar-H6), 6.06 (s, 1H, indazole-H5), 4.29 (d, 2H, J = 6.0 Hz,
benzyl-CH2). 13C-NMR (100MHz, d6-DMSO, ppm): δ 156.7, 143.2, 142.8, 132.7, 129.7, 128.8, 121.8,
115.6, 112.6, 101.2, 100.0, 46.1. ESI-MS: 318.01 (M + H)+.
5.1.8.7. 5-(((6-Bromo-1H-indazol-4-yl)amino)methyl)-2-hydroxybenzoic acid (2h). Yield 82%;
1
brown solid; mp: > 300C. H-NMR (400 MHz, d6-DMSO, ppm): δ 12.80 (br, 2H, COOH and
indazole-NH), 8.21 (s, 1H, indazole-H3), 7.82 (s, 1H, Ar-H6), 7.46 (d, 1H, J = 8.4 Hz, Ar-H4), 7.24 (t,
1H, J = 6.0 Hz, NH), 6.87 (d, 2H, J = 8.4 Hz, Ar-H3), 6.83 (s, 1H, indazole-H7), 6.06 (s, 1H,
indazole-H5), 4.35 (d, 2H, J = 6.0 Hz, benzyl-CH2). 13C-NMR (100MHz, d6-DMSO, ppm): δ 167.8,
143.0, 142.2, 140.6, 132.5, 132.1, 131.4, 129.2, 128.3, 121.7, 117.3, 112.6, 101.1, 100.4, 49.1. ESI-MS:
360.00 (M – H)-.
5.1.8.8.
2-(4-(((6-Bromo-1H-indazol-4-yl)amino)methyl)phenyl)acetic acid (2i). Yield 81%;
1
yellow solid; mp: 224-226C. H-NMR (400 MHz, d6-DMSO, ppm): δ 12.80 (br, 2H, COOH and
indazole-NH), 8.23 (s, 1H, indazole-H3), 7.33 (d, 2H, J = 8.0 Hz, Ar-H2 and Ar-H6), 7.23 (d, 2H, J =
8.0 Hz, Ar-H3 and Ar-H5), 6.83 (s, 1H, indazole-H7), 6.05 (s, 1H, indazole-H5), 4.40 (s, 2H, CH2NH),
3.18 (s, 2H, CH2COOH). 13C-NMR (100MHz, d6-DMSO, ppm): δ 173.2, 143.2, 142.2, 138.1, 134.0,
132.6, 129.9, 127.5, 121.8, 112.6, 101.2, 100.3, 49.1, 46.2. ESI-MS: 358.03 (M – H)-.
5.1.8.9. 2-(((6-Bromo-1H-indazol-4-yl)amino)methyl)phenol (2j). Yield 63%; yellow solid; mp:
1
158-160C. H-NMR (400 MHz, d6-DMSO, ppm): δ 12.81 (s, 1H, indazole-NH), 9.63 (s, 1H,
indazole-H3), 8.22 (s, 1H, indazole-H7), 7.19-7.05 (m, 2H, Ar-H4 and Ar-H6), 6.87-6.72 (m, 2H,
Ar-H3 and Ar-H5), 6.05 (s, 1H, indazole-H5), 4.34 (d, 2H, J = 5.8 Hz, benzyl-CH2). 13C-NMR
(100MHz, d6-DMSO, ppm): δ 155.4, 143.4, 142.1, 132.7, 128.5, 128.2, 125.3, 121.8, 119.4, 115.4,
112.5, 100.9, 100.0, 49.1. ESI-MS: 318.01 (M + H)+.
5.1.9. Methyl 4-(oxiran-2-yl)benzoate (17).
Methyl 4-vinylbenzoate (commercially available, CAS: 1076-96-6 , 1.0 g, 6.17 mmol) was dissolved
in DCM (30 mL). m-Chloroperbenzoic acid (2.1 g, 12.34 mmol) was added and the mixture was
refluxed for 2 h until the starting materiel was disappeared, then concentrated. The solid produced was
purified through the column chromatography on silica gel with petroleum ether and ethyl acetate (25:1)
as eluent to afford 17 (0.85 g, 77%) as a white solid.
5.1.10.
Methyl 4-(1-((6-bromo-1H-indazol-4-yl)amino)-2-hydroxyethyl)benzoate (18) and
methyl 4-(2-((6-bromo-1H-indazol-4-yl)amino)-1-hydroxyethyl)benzoate (19).
Aniline 9b (300.0 mg, 1.42 mmol) and 17 (229.2 mg, 1.42 mmol) were added in 1:1 ethanol/water
(20 mL) and the resulting mixture was refluxed for 12 h. The mixture was extracted with ethyl acetate

(3 ×15 mL). The combined organic layers were washed with brine, and then dried over anhydrous
Na2SO4. The solvent was evaporated and the crude product was charged on a silica gel column
chromatography with petroleum ether and ethyl acetate (6:1) as eluent to afford pure 18 and 19 (43%
overall yield).
Physical and spectral data for 18 and 19 is listed below.
1
18: Yield 20%; brown solid; H-NMR (400 MHz, d6-DMSO, ppm): δ 12.87 (s, 1H, indazole-NH),
8.33 (s, 1H, indazole-H3), 7.92 (d, 2H, J = 8.3 Hz, Ar-H2 and Ar-H6), 7.57 (d, 2H, J = 8.3 Hz, Ar-H3
and Ar-H5), 6.94 (d, 1H, J = 6.8 Hz, NH), 6.82 (s, 1H, indazole-H7), 5.88 (s, 1H, indazole-H5), 5.11 (t,
1H, J = 5.3 Hz, OH), 4.69-4.64 (m, 1H, NHCH), 3.83 (s, 3H, OCH3), 3.76-3.71 (m, 2H, OHCH2).
ESI-MS: 390.03 (M + H)+.
1
19: Yield 23%; brown solid; H-NMR (400 MHz, d6-DMSO, ppm): δ 12.81 (s, 1H, indazole-NH),
8.18 (s, 1H, indazole-H3), 7.94 (d, 2H, J = 8.3 Hz, Ar-H2 and Ar-H6), 7.56 (d, 2H, J = 8.3 Hz, Ar-H3
and Ar-H5), 6.83 (s, 1H, indazole-H7), 6.67 (t, 1H, J = 5.7 Hz, NH), 6.19 (s, 1H, indazole-H5), 5.77 (d,
1H, J = 4.4 Hz, OH), 4.93-4.89 (m, 1H, OHCH), 3.86 (s, 3H, OCH3), 3.34 (m, 2H, NHCH2). ESI-MS:
390.03 (M + H)+.
5.1.11. 4-(1-((6-Bromo-1H-indazol-4-yl)amino)-2-hydroxyethyl)benzoic acid (2k).
To the solution of ester 18 (45.0 mg, 0.11 mmol) in 1:1 ethanol/H2O (3 mL) added NaOH (13.2 mg,
0.33 mmol), and the mixture was stirred at ambient temperature for 12 h. The solution was adjusted to
pH 5 by addition of 1 mol/L aqueous HCl. The mixture was filtrated and the filter residue was washed
with water, dried under reduced pressure to afford 2k (38 mg, 88%) as a brown solid; mp: 181-184C.
1H-NMR (400 MHz, d6-DMSO, ppm): δ 12.87 (br, 2H, COOH and indazole-NH), 8.35 (s, 1H,
indazole-H3), 7.91 (d, 2H, J = 8.2 Hz, Ar-H2 and Ar-H6), 7.54 (d, 2H, J = 8.2 Hz, Ar-H3 and Ar-H5),
6.97 (d, 1H, J = 6.7 Hz, NH), 6.81 (s, 1H, indazole-H7), 5.89 (s, 1H, indazole-H5), 5.13 (br, 1H, OH),
4.68-4.63 (m, 1H, NHCH), 3.75-3.71 (m, 2H, OHCH2). 13C-NMR (100MHz, d6-DMSO, ppm): δ 167.7,
147.0, 142.5, 142.2, 132.7, 131.0, 129.9, 127.5, 121.5, 112.7, 101.9, 100.6, 65.8, 59.8, 49.1. HRMS
(AP-ESI) Calcd. for C16H15BrN3O3: 376.0297 (M + H)+. Found: 376.0299.
5.1.12. 4-(2-((6-Bromo-1H-indazol-4-yl)amino)-1-hydroxyethyl)benzoic acid (2m).
Compound 2m was synthesized following the procedure of 2k. Yield 87%, brown solid; mp:
165-168C. 1H-NMR (400 MHz, d6-DMSO, ppm): δ 12.85 (br, 2H, COOH and indazole-NH), 8.20 (s,
1H, indazole-H3), 7.92 (d, 2H, J = 8.3 Hz, Ar-H2 and Ar-H6), 7.53 (d, 2H, J = 8.3 Hz, Ar-H3 and
Ar-H5), 6.84 (s, 1H, indazole-H7), 6.71 (t, 1H, J = 5.7 Hz, NH), 6.19 (s, 1H, indazole-H5), 5.78 (br, 1H,
OH), 4.91 (m, 1H, OHCH), 4.13 (m, 1H, NHCH2). 13C-NMR (100MHz, d6-DMSO, ppm): δ 167.8,
149.4, 143.2, 142.2, 132.6, 129.6, 126.7, 121.9, 112.6, 100.8, 100.2, 70.6, 51.2. HRMS (AP-ESI) Calcd.
for C16H15BrN3O3: 376.0297 (M + H)+. Found: 376.0293.
5.1.13. 6-Bromo-4-fluoro-1H-indazole (21).
To a stirred suspension of 4-bromo-2,6-difluorobenzaldehyde 20 (commercially available, CAS
537013-51-7, 442.0 mg, 2.00 mmol) in ethylene glycol (10 mL) at ambient temperature was added
hydrazine monohydrate (0.19mL, 4.00 mmol). The resulting suspension was heated to 95C under
nitrogen for 2 h. The reaction was then cooled to 5C and diluted with water. The solution was
extracted with ethyl acetate, dried over anhydrous Na2SO4, and concentrated to yield
6-bromo-4-fluoro-1H-indazole 21 (430.0 mg, 100%) as a yellow solid.

5.1.14. t-Butyl 6-bromo-4-fluoro-1H-indazole-1-carboxylate (22).
To a solution of 6-bromo-4-fluoro-1H-indazole 21 (430.0 mg, 2.00 mmol) and DMAP (12.2 mg,
0.10 mmol) in DCM (10 mL) was added (Boc)2O (872.0 mg, 4.00mmol). The mixture was stirred at
ambient temperature for 2 h until the starting materiel was disappeared, then concentrated. The residue
produced was purified through the column chromatography on silica gel with petroleum ether and ethyl
acetate (60:1) as eluent to afford 22 (0.32 g, 50%) as colorless oil.
5.1.15. Methyl 4-(iodomethyl)benzoate (23a) and methyl 3-(iodomethyl)benzoate (23b).
To a solution of 4-formylbenzoic acid or 3-formylbenzoic acid (1.0 g, 6.67 mmol) in methanol (15
mL) was added SOCl2 (1.5 mL, 20.00 mmol) at 0C. The mixture was stirred at ambient temperature
for 12 h, and then concentrated. Saturated NaHCO3 solution was added to adjust the pH to 7-8. The
aqueous layer was extracted with ethyl acetate. The combined extracts was washed with brine, dried
over anhydrous Na2SO4 and evaporated in vacuo to give methyl 4-formylbenzoate and methyl
3-formylbenzoate (0.8 g, 76%), respectively.
A solution of TMDSO (1.08 mL, 6.09 mmol) in DCM (10 mL) was dropwise added to a cooled
mixture (-5C) of the corresponding carbonyl compounds (1.0 g, 6.09 mmol), iodine (933.0 mg, 3.67
mmol) in DCM (10 mL). The reaction was stirred at ambient temperature for 30 min until the starting
material was disappeared. Then, the mixture is taken up over DCM (50 mL) and washed with water
and saturated NaHCO3 solution. Drying and evaporation of the solvent yielded methyl
4-(iodomethyl)benzoate 23a (567.7 mg, 56%) and methyl 3-(iodomethyl)benzoate 23b (618.4 mg, 61%)
as a yellow solid, respectively.
5.1.16. t-Butyl 6-bromo-4-((4-(methoxycarbonyl)benzyl)oxy)-1H-indazole-1-carboxylate (24a)
and t-Butyl 6-bromo-4-((3-(methoxycarbonyl)benzyl)oxy)-1H-indazole-1-carboxylate (24b).
To a stirred solution of t-butyl 6-bromo-4-fluoro-1H-indazole-1-carboxylate 22 (100.0 mg,
0.32mmol) in DMF (5 mL) at ambient temperature was added K2CO3 (77.3 mg, 0.56mmol) followed
by water (28.8 μL, 1.60 mmol). The resulting suspension was heated at 90C. After 1 h, further water
(5.76 μL, 0.32 mmol) was added and the mixture was heated for another 1 h.
The reaction mixture was cooled to ambient temperature and K2CO3 (88.3 mg, 0.64 mmol) was
added with stirring followed by corresponding iodine compounds 23a~b (176.7 mg, 0.64 mmol). The
mixture was stirred at ambient temperature for 12 h. The reaction was diluted with water, and then
extracted with ethyl acetate. The combined extracts was washed with brine, dried over anhydrous
Na2SO4 and evaporated in vacuo. The crude product was charged on a silica gel column
chromatography with petroleum ether and ethyl acetate (40:1) as eluent to afford 24a (80.5 mg, 55%)
and 24b (60.0 mg, 41%) as a white solid, respectively.
5.1.17. Methyl 4-(((6-bromo-1H-indazol-4-yl)oxy)methyl)benzoate (25a) and methyl
3-(((6-bromo-1H-indazol-4-yl)oxy)methyl)benzoate (25b).
t-Butyl 6-bromo-1H-indazole-1-carboxylates 24a~b (60.0 mg，0.13mmol) was stirred in 20 v%
TFA in DCM (2 mL) for 2 hour and then concentrated under reduced pressure. Saturated NaHCO3
solution was added to adjust the pH to 7-8. The aqueous layer was extracted with ethyl acetate. The
combined extracts was washed with brine, dried over anhydrous Na2SO4 and evaporated in vacuo to
give 25a~b (46.0 mg, 98%) as yellow oil, respectively.

5.1.18. 4-(((6-Bromo-1H-indazol-4-yl)oxy)methyl)benzoic acid (4a).
To the solution of ester 25a (50.5 mg, 0.14 mmol) in 1:1 ethanol/H2O (6 mL) added NaOH (16.8 mg,
0.42 mmol), and the mixture was stirred at 100C for 12 h. The solution was cooled to ambient
temperature and then adjusted to pH 6-7 by addition of 1 mol/L aqueous HCl. The mixture was filtrated
and the filter residue was washed with water, dried under reduced pressure to afford 4a (39.8 mg, 82%)
1
as a colorless solid; mp: 165-167C. H-NMR (400 MHz, d6-DMSO, ppm): δ 12.89 (br, 1H, COOH),
7.93 (s, 1H, indazole-NH), 7.87 (d, 2H, J = 8.0 Hz, Ar-H2 and Ar-H6) , 7.66 (s, 1H, indazole-H3), 7.53
(d, 2H, J = 8.0 Hz, Ar-H3 and Ar-H5), 7.51 (s, 1H, indazole-H7), 7.49 (s, 1H, indazole-H5), 4.34 (s, 2H,
benzyl-CH2). 13C-NMR (100MHz, d6-DMSO, ppm): δ 167.7, 162.8, 160.4, 140.9, 134.2, 130.1, 129.9,
129.7, 128.2, 125.3, 121.3, 116.0, 115.7, 114.4. HRMS (AP-ESI) Calcd. for C12H15BrN2NaO5:
368.9851 (M + Na)+. Found: 369.0050.
5.1.19. 3-(((6-bromo-1H-indazol-4-yl)oxy)methyl)benzoic acid (4b).
Compound 4b (32.1 mg, 84%) was synthesized from acid 25b (39.7 mg, 0.11 mmol) following the
1
procedure of 4a. Colorless solid; mp: 140-142C. H-NMR (400 MHz, d6-DMSO, ppm): δ 12.85 (br,
1H, COOH), 8.04 (s, 1H, indazole-H3), 7.78 (d, 1H, J = 7.6 Hz, Ar-H6), 7.70 (s, 1H, Ar-H2), 7.69 (s,
1H, indazole-NH), 7.67 (d, 1H, J = 7.6 Hz, Ar-H4), 7.50 (s, 1H, indazole-H7), 7.49 (s, 1H,
indazole-H5), 7.44 (t, 1H, J = 7.6 Hz, Ar-H5), 4.33 (s, 2H, benzyl-CH2). 13C-NMR (100MHz,
d6-DMSO, ppm): δ 167.8, 162.9, 160.4, 137.1, 135.0, 131.8, 129.6, 129.3, 128.5, 126.3, 121.3, 116.0,
115.7, 114.5. ESI-MS: 369.00 [M + Na].
5.2. IDO1 inhibition assay
Human N-terminus IDO1 was expressed in E. coli and purified using Ni+ affinity chromatography as
described31. The assay was performed by UV absorption using recombinant IDO1 and TRY as substrate.
The UV absorption signal at 321 nm is correlated with the amount of N-formylkynurenine reaction
product of IDO1. The synthetic compounds were diluted in 10% DMSO and 5 L of the dilution was
added to a 100 L reaction so that the final concentration of DMSO is 0.5% in all reactions. All of the
reactions were conducted at room temperature. The 100 µL reaction mixture in IDO1 Assay Buffer
contains 40 nmol/L IDO1, the indicated amount of the inhibitors, 900 μmol/L TRY, and the coupled
reaction components. For the negative control (blank), 5 L of the assay buffer was added instead of
the IDO1, and 10% DMSO was added instead of the inhibitors. For the positive control, 10% DMSO
was added instead of the inhibitors. The reaction mixture incubated for 180 min prior to reading the UV
absorption signal. Absorption signals were measured using a Tecan Infinite M1000 plate reader.
Binding experiments were performed in duplicate at each concentration. The data were analyzed
using the computer software, Graphpad Prism. In the absence of the compound, the absorption signal
(At) in each data set was defined as 100% activity. In the absence of the IDO1, the absorption signal
(Ab) in each data set was defined as 0% activity. The percent activity in the presence of each compound
was calculated according to the following equation: % activity = [(A- Ab)/(At - Ab)]×100, where A= the
absorption signal in the presence of the compound. The percent inhibition was calculated according to
the following equation: % inhibition = 100 - % activity.
The values of % activity versus a series of compound concentrations were then plotted using
non-linear regression analysis of Sigmoidal dose-response curve generated with the equation
Y=B+(T-B)/1+10((LogIC50-X)×Hill Slope), where Y= percent activity, B=minimum percent activity,

T=maximum percent activity, X= logarithm of compound and Hill Slope=slope factor or Hill
coefficient. The IC50 value was determined by the concentration causing a half-maximal percent
activity.
5.3. Docking
The compounds were docked into IDO1 (PDB file “5EK3”) using DOCK6. Hydrogen was added to
all ligands and the receptor prior to performing the docking runs. The binding pocket was defined as
the binding site of the inhibitor NLG919 in the crystal structure. The number of docking runs was set to
10. All other parameters were left at their default values. The top-scored conformation was selected
asthe best docking result.
Acknowledgments
This work was supported by grants from the National Natural Science Foundation for the Youth of
China (81302647), applied basic research project of Sichuan Provincial Science and Technology
Department (2016JY0243), and innovation fund of post graduate from Xihua University. The authors
confirm that this article content has no conflicts of interest.
References and notes
1. Iversen, T. Z.; Andersen, M. H.; Svane, I. M. Basic Clin. Pharmacol. 2015, 116, 19.
2. Muller, A. J.; DuHadaway, J. B.; Chang, M. Y.; Ramalingam, A.; Sutanto-Ward, E.; Boulden, J.; Soler,
A. P.; Mandik-Nayak, L.; Gilmour, S. K.; Prendergast, G. C. Cancer Immunol. Immun. 2010, 59, 1655.
3. Muller, A. J.; Sharma, M. D.; Chandler, P. R.; DuHadaway, J. B.; Everhart, M. E.; Johnson, B. A.;
Kahler, D. J.; Pihkala, J.; Soler, A. P.; Munn, D. H.; Prendergast, G. C.; Mellor, A. L. P. Natl. Acad. Sci. USA
2008, 105, 17073.
4. Munn, D. H.; Sharma, M. D.; Baban, B.; Harding, H. P.; Zhang, Y. H.; Ron, D.; Mellor, A. L. Immunity
2005, 22, 633.
5. Metz, R.; Rust, S.; DuHadaway, J. B.; Mautino, M. R.; Munn, D. H.; Vahanian, N. N.; Link, C. J.;
Prendergast, G. C. Oncoimmunology 2012, 1, 1460.
6. Opitz, C. A.; Litzenburger, U. M.; Sahm, F.; Ott, M.; Tritschler, I.; Trump, S.; Schumacher, T.;
Jestaedt, L.; Schrenk, D.; Weller, M.; Jugold, M.; Guillemin, G. J.; Miller, C. L.; Lutz, C.; Radlwimmer, B.;
Lehmann, I.; von Deimling, A.; Wick, W.; Platten, M. Nature 2011, 478, 197.
7. Holmgaard, R. B.; Zamarin, D.; Munn, D. H.; Wolchok, J. D.; Allison, J. P. J. Exp. Med. 2013, 210,
1389.
8. Spranger, S.; Koblish, H. K.; Horton, B.; Scherle, P. A.; Newton, R.; Gajewski, T. F. J. Immun. Cancer
2014, 2, 3.
9. , U. F.; Majjigapu, S. R.; Vogel, P.; Zoete, V.; Michielin, O. J. Med. Chem. 2015, 58, 9421.
10. Jiang, T. Z.; Sun, Y. Y.; Yin, Z. C.; Feng, S.; Sun, L. P.; Li, Z. Y. Fut. Med. Chem. 2015, 7, 185.
11. Kumar, S.; Jaller, D.; Patel, B.; LaLonde, J. M.; DuHadaway, J. B.; Malachowski, W. P.; Prendergast,
G. C.; Muller, A. J. J. Med. Chem. 2008, 51, 4968.
12. Kumar, S.; Malachowski, W. P.; DuHadaway, J. B.; LaLonde, J. M.; Carroll, P. J.; Jaller, D.; Metz, R.;
Prendergast, G. C.; Muller, A. J. J. Med. Chem. 2008, 51, 1706.
13. Yue, E. W.; Douty, B.; Wayland, B.; Bower, M.; Liu, X. D.; Leffet, L.; Wang, Q.; Bowman, K. J.;
Hansbury, M. J.; Liu, C. N.; Wei, M.; Li, Y. L.; Wynn, R.; Burn, T. C.; Koblish, H. K.; Fridman, J. S.; Metcalf,
B.; Scherle, P. A.; Combs, A. P. J. Med. Chem. 2009, 52, 7364.

14. , U. F.; Awad, L.; Grosdidier, A.; Larrieu, P.; Stroobant, V.; Colau, D.; Cerundolo, V.; Simpson,
A. J. G.; Vogel, P.; Van den Eynde, B. J.; Zoete, V.; Michielin, O. J. Med. Chem. 2010, 53, 1172.
15. John, S.; Thangapandian, S.; Sakkiah, S.; Lee, K. W. Eur. J. Med. Chem. 2010, 45, 4004.
16. Huang, Q.; Zheng, M. F.; Yang, S. S.; Kuang, C. X.; Yu, C. J.; Yang, Q. Eur. J. Med. Chem. 2011, 46,
5680.
17. Dolusic, E.; Larrieu, P.; Blanc, S.; Sapunaric, F.; Pouyez, J.; Moineaux, L.; Colette, D.; Stroobant, V.;
Pilotte, L.; Colau, D.; Ferain, T.; Fraser, G.; Galleni, M.; Frere, J.-M.; Masereel, B.; Van den Eynde, B.;
Wouters, J.; Frederick, R. Eur. J. Med. Chem. 2011, 46, 3058.
18. 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.; Michielin, O. J. Med. Chem. 2012, 55, 5270.
19. Serra, S.; Moineaux, L.; Vancraeynest, C.; Masereel, B.; Wouters, J.; Pochet, L.; Frederick, R. Eur. J.
Med. Chem. 2012, 82, 96.
20. Yang, S. S.; Li, X. S.; Hu, F. F.; Li, Y. L.; Yang, Y. Y.; Yan, J. K.; Kuang, C. X.; Yang, Q. J. Med. Chem.
2013, 56, 8321.
21. , U. F.; Majjigapu, S. R.; Chambon, M.; Bron, S.; Pilotte, L.; Colau, D.; Van den Eynde, B. J.;
Turcatti, G.; Vogel, P.; Zoete, V.; Michielin, O. Eur. J. Med. Chem. 2014, 84, 284.
22. , U. F.; Majjigapu, S. R.; Vogel, P.; Zoete, V.; Michielin, O. J. Med. Chem. 2015, 58, 9421.
23. Peng, Y. H.; Ueng, S. H.; Tseng, C. T.; Hung, M. S.; Song, J. S.; Wu, J. S.; Liao, F. Y.; Fan, Y. S.; Wu, M.
H.; Hsiao, W. C.; Hsueh, C. C.; Lin, S. Y.; Cheng, C. Y.; Tu, C. H.; Lee, L. C.; Cheng, M. F.; Shia, K. S.; Shih,
C.; Wu, S. Y. J. Med. Chem. 2016, 59, 282.
24. Malachowski, W. P.; Winters, M.; DuHadaway, J. B.; Lewis-Ballester, A.; Badir, S.; Wai, J.; Rahman,
M.; Sheikh, E.; LaLonde, J. M.; Yeh, S. R.; Prendergast, G. C.; Muller, A. J. Eur. J. Med. Chem. 2016, 108,
564.
25. Lin, S. Y.; Yeh, T. K.; Kuo, C. C.; Song, J. S.; Cheng, M. F.; Liao, F. Y.; Chao, M. W.; Huang, H. L.; Chen,
Y. L.; Yang, C. Y.; Wu, M. H.; Hsieh, C. L.; Hsiao, W. C.; Peng, Y. H.; Wu, J. S.; Lin, L. M.; Sun, M. W.; Chao,
Y. S.; Shih, C.; Wu, S. Y.; Pan, S. L.; Hung, M. S.; Ueng, S. H. J. Med.Chem. 2016, 59, 419.
26. Li, M. H.; Bolduc, A. R.; Hoda, M. N.; Gamble, D. N.; Dolisca, S. B.; Bolduc, A. K.; Hoang, K.; Ashley,
C.; McCall, D.; Rojiani, A. M.; Maria, B. L.; Rixe, O.; MacDonald, T. J.; Heeger, P. S.; Mellor, A. L.; Munn,
D. H.; Johnson, T. S. J. Immun. Cancer 2014, 2, 21.
27. Nayak, A.; Hao, Z.; Sadek, R.; Dobbins, R.; Marshall, L.; Vahanian, N. N.; Ramsey, W. J.; Kennedy, E.;
Mautino, M.; Link, C.; Lin, R.; Royer-Joo, S.; Morrissey, K.; Mahrus, S.; McCall, B.; Pirzkall, A.; Munn, D.
H.; Janik, J. E.; Khleif, S. N. European Society for Medical Oncology (ESMO) 2015, 25.
28. Sugimoto, H.; Oda, S. I.; Otsuki, T.; Hino, T.; Yoshida, T.; Shiro, Y. P. Natl. Acad. Sci. USA 2006, 103,
2611.
29. Tojo, S.; Kohno, T.; Tanaka, T.; Kamioka, S.; Ota, Y.; Ishii, T.; Kamimoto, K.; Asano, S.; Isobe, Y. ACS
Med. Chem. Lett. 2014, 5, 1119.
30. Qian, S.; Zhang, M.; Chen, Q. L.; He, Y. Y.; Wang, W.; Wang, Z. Y. RSC Adv. 2016, 6, 7575.
31. Littlejohn, T. K.; Takikawa, O.; Skylas, D.; Jamie, J. F.; Walker, M. J.; Truscott, R. J. W. Protein Expres.
Purif. 2000, 19, 22.

Graphical abstract

Highlights




1H-indazole was a novel key pharmacophore with potent IDO1 inhibitory activity.
Compound 2g exhibited the highest activity with an IC50 value of 5.3 μM.
The interactions with heme ion and hydrophobic Pocket A and B are important.
The substituents at the both 4-position and 6-position largely affect activity.