Design, synthesis and bioevaluation of novel 6-substituted aminoindazole derivatives as anticancer agents

Article abstract of DOI:10.1039/d0ra09112j

In the present study, a series of 6-substituted aminoindazole derivatives were designed, synthesized, and evaluated for bio-activities. The compounds were initially designed as indoleamine 2,3-dioxygenase 1 (IDO1) inhibitors based on the structural feature of five IDO1 inhibitors, which are currently on clinical trials, and the important anticancer activity of the indazole scaffold. One of them, compound N-(4-fluorobenzyl)-1,3-dimethyl-1H-indazol-6-amine (36), exhibited a potent anti-proliferative activity with an IC50 value of 0.4 ± 0.3 μM in human colorectal cancer cells (HCT116). This compound also remarkably suppressed the IDO1 protein expression. In the cell-cycle studies, the suppressive activity of compound 36 in HCT116 cells was related to the G2/M cell cycle arrest. Altogether, the current findings demonstrate that compound 36 would be promising for further development as a potential anticancer agent.

Full text of DOI:10.1039/d0ra09112j

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PAPER
Design, synthesis and bioevaluation of novel 6-
substituted aminoindazole derivatives as
anticancer agents†
Cite this: RSC Adv., 2020, 10, 45199
de
Ngo Xuan Hoang,a Van-Hai Hoang,b Thi-Thu-Trang Luu,c Hung N. Luu,
Thien Ngo,f Duong Van Hieu,a Nguyen Huu Long,a Le Viet Anh,a Son Tung Ngo,
gh
Yen Thi Kim Nguyen,i Byung Woo Han,i Thanh Xuan Nguyen,j Dinh Thi Thanh Hai,a
Tran Thi Thu Hienk and Phuong-Thao Tran
*
a
In the present study, a series of 6-substituted aminoindazole derivatives were designed, synthesized, and
evaluated for bio-activities. The compounds were initially designed as indoleamine 2,3-dioxygenase 1
(IDO1) inhibitors based on the structural feature of five IDO1 inhibitors, which are currently on clinical
trials, and the important anticancer activity of the indazole scaffold. One of them, compound N-(4-
fluorobenzyl)-1,3-dimethyl-1H-indazol-6-amine (36), exhibited a potent anti-proliferative activity with an
IC50 value of 0.4 ꢀ 0.3 mM in human colorectal cancer cells (HCT116). This compound also remarkably
suppressed the IDO1 protein expression. In the cell-cycle studies, the suppressive activity of compound
36 in HCT116 cells was related to the G2/M cell cycle arrest. Altogether, the current findings
demonstrate that compound 36 would be promising for further development as a potential anticancer
agent.
Received 25th October 2020
Accepted 20th November 2020
DOI: 10.1039/d0ra09112j
rsc.li/rsc-advances
these approaches elicit advanced benets cancer patients, drug
resistance has become one of the big challenges in cancer
treatment.1–6 The resistance mechanisms are still under debate,
but most scientists support the immunosuppressive tumor
microenvironment concept.7–10
Introduction
Immunotherapy is a type of cancer treatment that exposes
cancer cells and/or enhances the immune system to ght cancer
cells along with chemotherapy, radiation and surgery. Although
Indoleamine 2,3-dioxygenase 1 (IDO1) is one of the heme-
containing enzymes involved in the immune system suppres-
sion process.11 It catalyzes the oxidative ring-opening of tryp-
tophan: the rst and rate-limiting step of the kynurenine
pathway.12 IDO1 suppresses the immune system via the kynur-
enine pathway by two mechanisms: (1) tryptophan depletion
and (2) toxicity of the metabolites in the kynurenine pathway.
Tryptophan depletion inhibits T-cell proliferation and induces
cell cycle arrest and the apoptosis of T lymphocytes, while
tryptophan metabolites promote the activity of regulatory T
cells, a types of T cell inhibits the maturation and cytotoxicity of
a T cell.12–14 IDO1 is an overexpression in different types of
cancer cells and tumors, such as prostate, colorectum,
pancreas, cervix, stomach, ovary and lung, and helps these
cancers escape the immune system.15,16 Prior studies have re-
ported that high IDO1 expression could be related to inade-
quate outcome of chemotherapy, radiotherapy17,18 and other
immunotherapies.10
aHanoi University of Pharmacy, 13-15 Le Thanh Tong, Hanoi 100000, Vietnam. E-mail:
thaotp119@gmail.com
bLaboratory of Medicinal Chemistry, Research Institute of Pharmaceutical Sciences,
College of Pharmacy, Seoul National University, Seoul 08826, Republic of Korea
cCollege of Pharmacy, Natural Products Research Institute, Seoul National University,
Seoul 08826, Republic of Korea
dDivision of Cancer Control and Population Sciences, UPMC Hillman Cancer Center,
University of Pittsburgh, Pittsburgh, PA 15232, USA
eDepartment of Epidemiology, Graduate School of Public Health, University of
Pittsburgh, PA 15261, USA
fFaculty of Pharmacy, Thai Binh University of Medicine and Pharmacy, Thai Binh City
410000, Vietnam
gLaboratory of Theoretical and Computational Biophysics, Ton Duc Thang University,
Ho Chi Minh City 700000, Vietnam
hFaculty of Applied Sciences, Ton Duc Thang University, Ho Chi Minh City 700000,
Vietnam
iCollege of Pharmacy, Seoul National University, Seoul 08826, Republic of Korea
jDepartment of Surgical Oncology, Viet-Duc University Hospital, Hanoi 100000,
Vietnam
During the last decades, thousands of compounds have been
identied as IDO1 inhibitors for cancer immunotherapy, but
none of these was approved and brought to the market.
Indoximod (D1MT, 1), PF-0684003 (2), novaximod (3), BMS-
986205 (4), and epacadostat (5) (Fig. 1A) are ve compounds
kVietnam University of Traditional Medicine, 2 Tran Phu, Ha Dong, Hanoi 100000,
Vietnam
† Electronic supplementary information (ESI) available: Experimental chemistry,
biology and molecular simulation docking. Additional data of 1H NMR, 13C NMR
& MS spectra of the compounds. See DOI: 10.1039/d0ra09112j
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Results and discussion
Chemistry
The synthesis of 6-substituted-indazole derivatives was per-
formed, as illustrated in Scheme 1. Briey, 6-nitro indazole
derivatives 9, 10 were methylated by iodomethane using
potassium carbonate in DMF to get two isomers: the major
products, N1-alkylindazoles 12, 13 and the minor products N2-
alkylindazoles 14, 15. The structures of two isomers were
conrmed by 2D-NMR before carrying out a reduction reaction
under hydrogen gas to afford corresponding 6-amino indazole
derivatives 11, 16–19. Next, the amines were reductively ami-
nated with a ketone or an aldehyde in the presence of a mild
and selective reductive agent, namely sodium cyanoborohy-
dride, to afford secondary amines 24–51. Moreover, acetamide
derivatives 20–23 were prepared from the corresponding amine
and acetic anhydride under basic conditions.
Fig. 1 (A) Clinical IDO1 inhibitors; (B) some IDO1 inhibitors bearing the
indazole structure; (C) novel designed indazole derivatives.
currently in clinical trials.19 They have been generally combined
with cytotoxic chemicals and immune checkpoint agents on
clinical trials.19,20 The combined therapies improved the efficacy
on the tumors of immune checkpoint and cytotoxic agents in
both preclinical-21,22 and clinical studies.23 The benets come
from the ability to restore immunosurveillance and blunt tumor
Anti-proliferative activity
neovascularization
via
re-programming
inammatory
The as-synthesized indazole derivatives were evaluated for the
in vitro anti-proliferative activity in ve different cancer cell
lines including HCT116, A549, SK-HEP-1, SNU-638, and MDA-
MB-231 using the sulforhodamine B method. Etoposide was
used as the positive control. The results are summarized in
Tables 1 and 2.
First, we examined the structure–activity relationship (SAR)
of the as-synthesized compounds on colon cancer cell (HCT116)
proliferation, in which required IDO activation increases the
expression of b-catenin and its targeting genes including cyclin
D and Axin2 for development.36 As demonstrated in Tables 1
and 2, the introduction of a methyl group at the C-3 position of
the indazole ring 33–37 caused a potential toxicity against
HCT116. Among them, the substitution of an aryl group in
processes.24 However, the combination of two drugs is able to
cause drawbacks, such as drug–drug interactions, different
pharmacokinetics and inconveniences in pharmaceutics and
treatment.25 Therefore, a new “multitarget” concept was enun-
ciated for complex diseases such as cancer.25,26 Based on this
new concept, a few X/IDO1 dual inhibitors were reported, such
as HDAC/IDO1 dual inhibitors27 and DNA/IDO1 dual targeting
agents.28
Indazole is an important scaffold in drug discovery, which is
present in different anticancer agents. Most of them are
receptor tyrosine kinase- (EGFR, FGFR, VEGFR) or serine/
threonine kinase (aurora kinase, cyclin-dependent kinase)
inhibitors.29–31 The diversity comes from the structure, in which
two nitrogen atoms can tautomerize and can be introduced to
different groups. In addition, indazole, a bioisostere of the
indole ring in tryptophan, was used as the skeletal structure for
design new IDO1 inhibitors.32–35 Indazoles bearing 3-hydrazide
groups (6)32 were rst studied in 2016 with IC50 values near the
nanomolar range. Next, some 4-amino (7)33,34 and 3-(hydrox-
ymethyl)35 (8) indazoles were also reported as IDO1 inhibitors at
the sub-nanomolar range (Fig. 1B). To date, all indazole deriv-
atives bear a huge group at position 4 and a small group (nor-
mally a halogen) at position 6.
indazoles 34–37 showed
a better cytotoxicity than the
cyclohexyl-substituted compound 33. A replacement of benzyl
by 3-pyridylmethyl 35 or uorobenzyl 36, 37 increased the toxic
potency from 2.7 to 18.8-fold as compared to 34. Of note,
compound 36 had a great anti-proliferative activity (IC50 ¼ 0.4 ꢀ
0.3 mM), which indicates the importance of the uoro group.
In this study, we chose indazole as the core structure for the
rational design of new IDO1- and cytotoxic agents. We simpli-
ed and considered the derivatization abilities of the indazole
structure, and then synthesized a library of 6-substituted ami-
noindazoles (Fig. 1C). Next, we evaluated their toxicities on ve
cancer cell lines and investigated the IDO1 inhibitory activities
in vitro. We further performed docking studies to calculate the
binding energy and analyzed the specic binding interactions
between the selected inhibitors and the IDO1 active site. Finally,
we examined the potential mechanisms of the most active
compound on the cell cycle arrest in human colorectal cancer
cells.
Scheme 1 Reagents and conditions: (a) CH3I (2.0 equiv.), excess
K2CO3, DMF, 60 ꢁC, 4 h, 95–98%; (b) H2, Pd/C (10 mol%), r.t., 4 h, 85–
96%; (c) ketone or aldehyde (1.0 equiv.), NaBH3CN (5.0 equiv.), AcOH,
MeOH, 40 ꢁC, 4 h, 73–96%; (d) excess Ac2O, TEA, DMC, r.t., 2 h, 89–
92%.
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Table 1 Cytotoxicity of N1-methyl indazole derivatives on five cell lines
R1 ¼ H (IC50a (mM))
R1 ¼ CH3 (IC50a (mM))
R2
Cpd A549
SK-HEP-1 SNU-638
MDA-MB-231 HCT116
Cpd A549
SK-HEP-1 SNU-638 MDA-MB-231 HCT116
24 >100
20 >100
>100
>100
>100
>100
>100
>100
>100
31 >100
>100
>100
>100
>100
>100
>100
>100
>100
26.7 ꢀ 15.7 21 >100
25 >100
26 >100
>100
>100
64.8 ꢀ 28.4 >100
>100
>100
32 >100
33 27.0 ꢀ 18.7 39.6 ꢀ 22.4 13.7 ꢀ 9.1 >100
34
5.1 ꢀ 23.3 28.1 ꢀ 20.0 1.9 ꢀ 2.0 >100
>100
36.8 ꢀ 11.2 3.8 ꢀ 3.9 >100
>100
>100
>100
>100
>100
>100
>100
14.5 ꢀ 16.3
27 >100
>100
>100
7.5 ꢀ 5.0
2.7 ꢀ 2.3
0.4 ꢀ 0.3
28 19.8 ꢀ 6.1 >100
29
0.7 ꢀ 0.1 >100
47.2 ꢀ 12.9 35 30.0 ꢀ 18.5 33.9 ꢀ 22.6 >100
1.2 ꢀ 1.2 >100
1.3 ꢀ 1.0 >100
34.9 ꢀ 21.3 36
6.8 ꢀ 2.4 50.1 ꢀ 23.4 1.8 ꢀ 2.2 47.5 ꢀ 8.4
42.2 ꢀ 15.0 2.5 ꢀ 2.6 81.7 ꢀ 25.2
30 19.9 ꢀ 10.6 >100
>100
37 >100
2.2 ꢀ 0.9
Etoposide
0.27 ꢀ 0.1 0.30 ꢀ 0.1 0.61 ꢀ 0.2 7.35 ꢀ 2.3
1.27 ꢀ 0.8
a
IC50: the concentration (mM) of compounds that produces a 50% reduction cell growth. All the data were calculated from the experiments
conducted in triplicate and repeated at least three times. Data represent mean ꢀ SD.
Changing the position of the uoro group from para to meta MDA-MB-231, A549, and SNU-638 cells with IC50 ¼ 1.7 ꢀ 1.1, 2.8
caused a 5.5 times activity drop. In contrast, introducing alkyl ꢀ 1.3, and 1.8 ꢀ 1.4 mM, respectively.
groups to 31, 32 resulted in non-toxic compounds. Similarly, the
In addition, the N-aromatic substitution of 6-aminoindazole
toxicity of acetamide derivative 21 was abolished. Removing the derivatives 29, 30, 34, 36, 37 exhibited considerable cytotoxicity
methyl group from the C-3 position of the indazole ring caused towards A549 and SNU-638 cancer cell lines with IC50 values
a reduction in the toxicity against HCT116, except for the acet- ranging from 0.7 to 10 mM. The cytotoxicity changed dramati-
ylated compound 20 (IC50 ¼ 26.7 ꢀ 15.7 mM). Besides, relocating cally among substituted groups. Moreover, there was no corre-
the methyl group from N1 to N2 decreased the anti-proliferative lation between the cytotoxicity and structure observed. In
activity of most of the compounds (Table 2), excluding iso- addition, the IC50 values of compounds 22, 34–37 on the normal
propyl- and acetyl compounds. In particular, compound 22 cell MRC5 were higher than the IC50 of them on HCT116 cancer
showed a potent activity with an IC50 value of 2.5 ꢀ 1.6 mM. The cell line. Specially, compound 36 demonstrated 29.6-fold
most effective groups, 3-pyridylmethyl 49, 4-uorobenzyl 50 and selectivity against HCT116 than MRC5 (Table S1 – ESI le†).
3-uorobenzyl 51, demonstrated less cytotoxicity of 5.8 to 34.5-
fold than the corresponding N1-methyl isomers. Overall, the
IDO1 inhibition assay
absence of the methyl group at the 3-position in the indazole
In order to examine the effects on IDO1 expression, which can
ring favored a small alkyl- or an acetyl-substituted group in
be well correlated to human colon cancer cell growth,37,38 we
displaying the anti-proliferative activity. In contrast, the
selected the compounds with the most anti-proliferative
substitution of an aryl group preferred 3-methylindazole
potency against HCT116 for the analysis of the IDO1-
compounds in presenting cytotoxic activities.
suppressive activity (Fig. 2). We found that at the concentra-
Next, the SAR of all as-synthesized compounds against the
tion of 10 mM, the expression of IDO1 in HCT16 cells seemed
other four cell lines including A549, SK-HEP-1, SNU-638, and
unaffected by compound 35 or 37 (Fig. 2); moreover, it was
MDA-MB-231 (Tables 1 and 2) was examined. Most of them
suppressed by a treatment with compound 22 or 36 (Fig. 2).
showed a mild suppressive effect on SK-HEP-1 and MDA-MB-
Furthermore, the effects of compound 36 on IDO1 expression
231 (IC50 > 20 mM) excluding compounds 39, 49, 50. We
were observed in a concentration-dependent manner (Fig. 3 and
observed that compound 39 had a great suppressive potency on
S2 – ESI le†).
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Table 2 Cytotoxicity of N2-methyl indazole derivatives on five cell lines
R1 ¼ H (IC50a (mM))
R1 ¼ CH3 (IC50 (mM))
a
MDA-MB-
MDA-MB-
231
R2
Cpd A549
SK-HEP-1 SNU-638
231
30.4 ꢀ 27.0 68.3 ꢀ 27.3 33.4 ꢀ 11.7 45 >100
>100 2.5 ꢀ 1.6 23 >100
20.6 ꢀ 1.7
2.8 ꢀ 1.3 1.7 ꢀ 1.1
HCT116
Cpd A549
SK-HEP-1 SNU-638
HCT116
38 38.9 ꢀ 13.2 >100
22 58.7 ꢀ 23.9 >100
>100
>100
>100
>100
>100
>100
>100
>100
39
1.8 ꢀ 1.4 >100
>100
>100
46 >100
>100
>100
>100
>100
40 37.4 ꢀ 10.2 >100
>100
>100
>100
>100
>100
>100
47 37.0 ꢀ 22.7 32.7 ꢀ 11.2 33.0 ꢀ 17.5 >100
26.1 ꢀ 13.8
>100
41 >100
42 >100
43 >100
>100
>100
>100
49.7 ꢀ 3.6 48 >100
>100
>100
>100
>100
>100
>100
>100
49 >100
19.4 ꢀ 17.0 19.1 ꢀ 13.5
13.8 ꢀ 6.5
63.3 ꢀ 13.8 >100
50 19.4 ꢀ 20.6 37.3 ꢀ 6.3 10.8 ꢀ 11.2 8.0 ꢀ 1.7
44 >100
>100
>100
>100
>100
51 >100
>100
>100
61.3 ꢀ 32.3 12.7 ꢀ 8.5
Etoposide
0.27 ꢀ 0.1 0.30 ꢀ 0.1 0.61 ꢀ 0.2 7.35 ꢀ 2.3
1.27 ꢀ 0.8
a
IC50: the concentration (mM) of compounds that produces a 50% reduction cell growth. All the data were calculated from the experiments
conducted in triplicate and repeated at least three times. Data represent mean ꢀ SD.
the active site region of IDO1. To validate the docking program
Autodock Vina, we performed a control docking experiment
with the 5PK compound in the IDO1 complex structure (PDB ID:
5EK3). 5PK was predicted to dock into the active site of IDO1 as
in the actual complex structure with a binding energy of
Fig. 2 Effect of 6-substituted aminoindazole derivatives on the IDO1
ꢂ9.5 kcal molꢂ1 (described in Fig. S1 of the ESI le†). We found
expression in human colorectal cancer cells. HCT116 were treated
that both compounds 35 and 36 bound to IDO1 in a comparable
way to that of the 5PK–IDO1 complex structure (Fig. 4A). In
with 10 mM of indicative compounds for 24 h. The immunoblotting
analysis was performed to determine IDO1 and b-actin expression.
addition, the binding energy of compound 35 (ꢂ8.1 kcal molꢂ1
)
was higher than that of compound 36 (ꢂ8.9 kcal molꢂ1), sug-
gesting a higher binding affinity towards IDO1 of compound 36
than that of compound 35. In the docking model, compounds
35 and 36 were recognized and/or stabilized by well-known
active site residues Tyr126, Phe163, Phe164, Ser167, Phe226,
Phe227, Arg231, Ser263, and His346,39 which play a similar role
in the 5PK–IDO1 complex structure (Fig. 4B). The docking
models showed that the N2 indazole group of compound 36
formed a hydrogen bond with the hydroxy group of Ser167, and
the urophenyl/pyridinyl ring formed a p–p interaction with
Phe226 (Fig. 4C). In addition, the 6-NH group in the compounds
also formed a hydrogen bond with the 7-propionate of the heme
ion (Fig. 4C).
Fig. 3 HCT116 cells were treated with compound 36 at various
concentrations. The immunoblotting analysis was performed to
determine IDO1 and b-actin protein levels.
Docking simulation analysis of IDO1 and its inhibitors
To gain insight into the interaction between inhibitors and
IDO1, we performed molecular docking simulation studies on
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Fig. 4 Docking model of IDO1 with 5PK and compounds. (A) The
docking models of 5PK, compound 35, and compound 36 are shown
in yellow green, light yellow, and pink models, respectively. (B) A close-
up view of docked compounds in the active site cavity of IDO1. (C) The
interaction mode of IDO1 with compound 36. The IDO1 residue
recognizing ligands are shown in the green stick model. The heme ion
is shown in a grey stick. Hydrogen bonds and p–p interactions are
shown in red and black dots, respectively.
Study mechanism of compound 36 on human colorectal
cancer cells
Fig. 6 Effect of compound 36 on the cell cycle distribution in human
colorectal cancer cells. (A and B) HCT116 cells were treated with
compound 36 for 24 h or 48 h. The cell cycle distribution was analyzed
via flow cytometry. (C) Effects of 36 on the expression of cell cycle-
related proteins in HCT116 cells. HCT116 cells were treated with 36 for
48 h. Subsequently, the protein expression levels of cell cycle-related
proteins were analyzed by the immunoblotting assay.
Compound 36 demonstrated
a concentration-dependent
suppressive effect on the activity of the HCT116 viability
(Fig. 5). On other cell lines, despite a modest effect on the IDO
expression, compound 36 still demonstrated a remarkable
inhibition of cell viability. This phenomenon suggested that
other mechanisms may also be responsible for the anti-
proliferative effect of compound 36. Therefore, to further
explore the possible mechanism of action of the anti-
proliferative activity of compound 36, the effect of compound
36 on the modulation of the cell cycle was determined.
A cell cycle analysis indicated that a treatment with 10 mM of
compound 36 stimulated the G2/M cell cycle arrest in HCT116
cells within 24 h of post-treatment (Fig. 6A). Furthermore,
a concentration-dependent concentration simulative effect on
the G2/M cell cycle arrest was observed at 48 h aer the
treatment (Fig. 6B). We further analyzed the expression of the
G2/M cell cycle regulatory proteins by immunoblot. We found
that the expression of p-CDC25C (Thr48), CDC25C, Chk1, and p-
cyclin B1 were down-regulated (Fig. 6C). These ndings sug-
gested that the anti-proliferative activity of compound 36 was
associated, at least partially, with the G2/M phase cell cycle
arrest through regulating cell cycle modulators in human
colorectal cancer cells.
Experimental section
Chemistry
All chemical reagents were commercially available and used
directly without any purication. Thin layer chromatography,
which was performed using Whatman® 250 mm Silica Gel GF
Uniplates and visualized under UV light at 254 nm, was used to
check the progress of reactions and preliminary evaluation of
the homogeneity of the compounds. In all cases, the
compounds achieved a purity of 97% or above as determined by
HPLC. Melting points were measured using a Gallenkamp
Melting Point Apparatus (LabMerchant, London, United
Kingdom) and were uncorrected. The purication of
Fig. 5 Anti-proliferative effect of compound 36 on HCT116 cells.
HCT116 cells were treated with compound 36 at various concentra-
tions for 72 h, and cell proliferation was determined by the SRB assay.
The data were presented as mean ꢀ SD. The experiment was per-
formed in triplicate (n ¼ 3).
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compounds was carried out using crystallization methods and/ Collection (Manassas, VA, USA). HCT116, SNU638, MDA-MB-
or open silica gel column ash chromatography employing 231, SK-HEP-1 were cultured, as described previously.40
Merck silica gel 60 (240 to 400 mesh) as the stationary phase.
Nuclear magnetic resonance spectra (1H NMR, 13C NMR) were
Sulforhodamine B assay
recorded on a JEOL JNM-LA 300 at 300 MHz and Bruker Ana-
lytik, DE/AVANCE Digital 500 at 500 and 125 MHz spectrome-
ters. Tetramethylsilane was used as the internal standard.
Chemical shis were reported in parts per million (ppm)
downelded from tetramethylsilane. Mass spectra with
different ionization modes including electron ionization (EI)
and electrospray ionization (ESI) were recorded using PE Bio-
systems API2000 (PerkinElmer, Palo Alto, CA, USA) and Mari-
The effect of the indicated compounds on cell proliferation was
calculated as described previously.40 In brief, HCT116 cells (2 ꢃ
104 cells per mL) were seeded in 96-well plates with different
compounds. Aer incubation, the cells were xed with a 50%
trichloroacetic acid (TCA) solution for 1 h, and cellular proteins
were stained with 0.4% sulforhodamine B (SRB) in 1% acetic
acid. The stained cells were dissolved in 10 mM Tris buffer (pH
10.0). The effect of the investigated compound on cell prolifer-
ner® (Azco Biotech, Inc. Oceanside, CA, USA) or LC-MSD-Trap-
ation was calculated as a percentage relative to a solvent-treated
SL (Agilent, CA, USA) mass spectrometers. All reagents and
control, and the IC50 values were evaluated via nonlinear
regression analysis (percent survival versus concentration).
Etoposide was used as the positive control.
solvents were purchased from Aldrich or Fluka Chemical Corp.
(Milwaukee, WI, USA) or Merck unless noted otherwise.
Solvents were used directly as purchased unless otherwise
indicated.
Flow cytometry for cell cycle analysis
General procedure for methylation (procedure 1). To
HCT116 cells (2 ꢃ 105 cells per mL) were plated in a 36 mm
a mixture of 6-nitroindazole derivative (1.0 equiv.) and excess
culture dish and incubated for 24 h. A fresh medium containing
the indicated concentration of compound 36 was added to
culture dishes. Following a 24 h or 48 h incubation, the cells
were harvested (via trypsinization and centrifugation), rinsed
twice with pre-cooled phosphate buffered saline (PBS), and
prepared for the cell cycle analysis as described previously.40
potassium carbonate in dry DMF was added iodomethane (2.0
ꢁ
equiv.) and stirred at 60 C for 4 h. The reaction mixture was
quenched with water and extracted with EA (3 times). The
organic layer was collected, washed with water and brine,
concentrated, and puried by silica gel column chromatog-
raphy using ethyl acetate/n-hexane (1/2) as the mobile phase to
get the two isomers: N1-methylation (major, less polar) product
and N2-methylation (minor, more polar) product.
Western blot analysis
HCT116 cells (2 ꢃ 105 cells per mL) were treated with various
concentrations of 36 for the indicated times. Western blot
analysis was carried out as described previously.41 The antibody
for IDO1 (ab55305) was purchased from Abcam (Abcam, Cam-
bridge, UK). Antibodies for CDC25C, phosphor-CDC25C (Thr48),
Chk1, Chk2, and phosphor-cyclin B1 (Ser147) were purchased
from Cell Signaling Technology (Danvers, MA, USA). Antibodies
for cyclin A (H-432), CDC2, cyclin B1 (H-433), and b-actin (C4)
were purchased from Santa Cruz Biotechnology (Santa Cruz, CA,
USA).
General procedure for reduction (procedure 2). The nitro-
indazole compound was dissolved in MeOH or THF and then
10% Pd/C was added. The mixture was stirred at room
temperature under hydrogen gas atmosphere for 4 h. The crude
mixture was ltered through Celite, and washed by MeOH. The
solution was concentrated and used for the next step without
any purication.
General procedure for reductive amination (procedure 3). A
mixture of amine (1.0 equiv.), aldehyde or ketone (1.0 equiv.)
and acetic acid (5.0 equiv.) in MeOH was stirred for 5 min, and
then NaBH3CN (5.0 equiv.) was added. The reaction mixture was
stirred at 40 ꢁC for 4 h, and then diluted with DCM. The organic
layer was washed using a bicarbonate solution and water, then
concentrated, and puried via silica gel column chromatog-
raphy using ethyl acetate/n-hexane (1/5 to 1/1) as the mobile
phase.
General procedure for acetylation (procedure 4). Excess
amount of acetic anhydride was added into a solution of amine
and TEA in DCM. The reaction mixture was stirred at room
temperature for 2 h and diluted with DCM. The organic solution
was washed with the bicarbonate solution and water, and then
puried via silica gel column chromatography using ethyl
acetate/n-hexane (1/3) as the mobile phase.
Statistical analysis
All experiments were repeated at least three times. The data are
shown as the means ꢀ standard deviation (SD) for the indicated
number of independently performed experiments. The
student's t-test was used for the determination of statistical
signicance, and IC50 values were determined by nonlinear
regression using the Sigma Plot (Systat Soware, Inc., San Jose,
CA, USA). The difference was considered statistically signicant
when p (*) < 0.05, (**) < 0.001, (***) < 0.0001.
Molecular simulation docking
The structure of IDO1 was acquired from the protein data bank
(PDB ID: 5EK3), and the structure of compound 35 and
compound 36 were drawn by the ChemDraw program (version
Cell culture
Human colorectal cancer cells (HCT116), gastric cancer cells Pro 16.0, Cambridge so, Cambridge, MA). The structure of the
(SNU638), breast cancer cells (MDA-MB-231), liver cancer cells control compound 5PK was extracted from the complex struc-
(SK-HEP-1) were purchased from American Type Culture ture of 5PK–IDO1 (PDB ID: 5EK3). The compounds were docked
45204 | RSC Adv., 2020, 10, 45199–45206
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RSC Advances
into the active site region of IDO1 using AutoDock Vina (version
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3
˚
20 A dimensions to contain the proposed active sites and hem,
which are essential for the enzyme activity.43 The conformations
of the ligands and receptors were xed. The program was run in 10 A. Botticelli, B. Cerbelli, L. Lionetto, I. Zizzari, M. Salati,
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Conclusions
¨
¨
In summary, four series of 6-substituted aminoindazoles were 12 S. Lob, A. Konigsrainer, H.-G. Rammensee, G. Opelz and
designed based on an anticancer privileged structure, indazole, P. Terness, Nat. Rev. Cancer, 2009, 9, 445.
targeting to inhibit the IDO1 activity, and the designed 13 M. Platten, N. von Knebel Doeberitz, I. Oezen, W. Wick and
compounds were synthesized through 3 steps. Of them, the K. Ochs, Front. Immunol., 2014, 5, 673.
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compounds (22, 35–37) showed potential cytotoxicity towards Cancer, 2015, 3, 51.
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indicated that compound 36 had a moderate inhibition activity Clin. Cancer Res., 2011, 17, 6985–6991.
of IDO1 towards the HCT116 cell line. The molecular modeling 16 C. Uyttenhove, L. Pilotte, I. Theate, V. Stroobant, D. Colau,
´
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site and created hydrogen bonds with Ser167 and 7-propionate
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in the heme structure. Further studies indicated that compound 17 B. C. Creelan, S. Antonia, G. Bepler, T. J. Garrett, G. R. Simon
36 arrested the G2/M cell cycle of HTC116 through regulating and H. H. Soliman, Oncoimmunology, 2013, 2, e23428.
the cell cycle modulators. Due to the good cytotoxicity on 18 H. Liu, Z. Shen, Z. Wang, X. Wang, H. Zhang, J. Qin, X. Qin,
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that compound 36 might be useful for the design of new anti- 19 T. Weng, X. Qiu, J. Wang, Z. Li and J. Bian, Eur. J. Med. Chem.,
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20 J. E. Cheong and L. Sun, Trends Pharmacol. Sci., 2018, 39,
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Conflicts of interest
21 A. J. Muller, J. B. DuHadaway, P. S. Donover, E. Sutanto-Ward
and G. C. Prendergast, Nat. Med., 2005, 11, 312.
22 M. Li, A. R. Bolduc, M. N. Hoda, D. N. Gamble, S.-B. Dolisca,
A. K. Bolduc, K. Hoang, C. Ashley, D. McCall and
A. M. Rojiani, J. Immunother. Cancer, 2014, 2, 21.
There are no conicts to declare.
Acknowledgements
This research was funded by the Vietnam National Foundation 23 M. Liu, X. Wang, L. Wang, X. Ma, Z. Gong, S. Zhang and Y. Li,
for Science and Technology Development (NAFOSTED) under
J. Hematol. Oncol., 2018, 11, 100.
grant number 108.05-2020.01. We would like to thank Professor 24 G. C. Prendergast, A. Mondal, S. Dey, L. D. Laury-Kleintop
Sang Kook Lee and Dr Duc-Hiep Bach (College of Pharmacy, and A. J. Muller, Trends Cancer, 2018, 4, 38–58.
Natural Products Research Institute, Seoul National University, 25 M. L. Bolognesi, Curr. Med. Chem., 2013, 20, 1639–1645.
Seoul 08826, Republic of Korea) for the biological part.
26 R. R. Ramsay, M. R. Popovic-Nikolic, K. Nikolic, E. Uliassi
and M. L. Bolognesi, Clin. Transl. Med., 2018, 7, 3.
27 K. Fang, G. Dong, Y. Li, S. He, Y. Wu, S. Wu, W. Wang and
C. Sheng, ACS Med. Chem. Lett., 2018, 9, 312–317.
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