Design, synthesis, and biological evaluation of 5-((8-methoxy-2-methylquinolin-4-yl)amino)-1H-indole-2-carbohydrazide derivatives as novel Nur77 modulators

Article abstract of DOI:10.1016/j.ejmech.2020.112608

Nur77 is a potential target for the treatment of cancer such as HCC. Herein, we detailed the discovery of a novel series of 5-((8-methoxy-2-methylquinolin-4-yl)amino)-1H-indole-2-carbohydrazide derivatives as potential Nur77 modulators. The studies of antiproliferative activity and Nur77-binding affinity of target compounds resulted in the discovery of a lead candidate (10g), which was a good Nur77 binder (K_D = 3.58 ± 0.16 μM) with a broad-spectrum antiproliferative activity against all tested hepatoma cells (IC₅₀ < 2.0 μM) and was low toxic to normal LO2 cells. 10g could up-regulate Nur77 expression and mediate sub-cellular localization of Nur77 to induce apoptosis in hepatocellular carcinoma cell lines, which relied on 10g inducing Nur77-dependent autophagy and endoplasmic reticulum stress as the upstream of apoptosis. Moreover, the in vivo assays verified that 10g significantly inhibited xenograft tumor growth. These results indicate that 10g has the potential to be developed as a novel Nur77-targeting anti-hepatoma drug.

Full text of DOI:10.1016/j.ejmech.2020.112608

Journal Pre-proof
Design, synthesis, and biological evaluation of 5-((8-methoxy-2-methylquinolin-4-
yl)amino)-1H-indole-2-carbohydrazide derivatives as novel Nur77 modulators
Baicun Li, Jie Yao, Kaiqiang Guo, Fengming He, Kun Chen, Zongxin Lin, Shunzhi Liu,
Jiangang Huang, Qiaoqiong Wu, Meijuan Fang, Jinzhang Zeng, Zhen Wu
PII:
S0223-5234(20)30580-8
DOI:
https://doi.org/10.1016/j.ejmech.2020.112608
EJMECH 112608
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 25 April 2020
Revised Date: 12 June 2020
Accepted Date: 19 June 2020
Please cite this article as: B. Li, J. Yao, K. Guo, F. He, K. Chen, Z. Lin, S. Liu, J. Huang, Q. Wu, M.
Fang, J. Zeng, Z. Wu, Design, Synthesis, and Biological Evaluation of 5-((8-Methoxy-2-Methylquinolin-4-
yl)Amino)-1H-Indole-2-Carbohydrazide Derivatives as Novel Nur77 Modulators, European Journal of
Medicinal Chemistry, https://doi.org/10.1016/j.ejmech.2020.112608.
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Synthesis, and Biological Evaluation of
5-((8-Methoxy-2-Methylquinolin-4-yl)Amino)-1H-Indole-2-Carbohyd
razide Derivatives as Novel Nur77 Modulators
Baicun Li1, 2, #, Jie Yao1, #, Kaiqiang Guo1,#, Fengming He1, Kun Chen1, Zongxin Lin1, Shunzhi
Liu1, Jiangang Huang1, Qiaoqiong Wu1, Meijuan Fang1,*, Jinzhang Zeng1,*, Zhen Wu1,*
Novel Nur77 Modulator 10g with anti-hepatoma activity, up-regulates Nur77 expression,
mediates sub-cellular localization of Nur77, induces Nur77-dependent ER stress and
autophagy, and results in cell apoptosis in HCC cells.

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Design, Synthesis, and Biological Evaluation of
5-((8-Methoxy-2-Methylquinolin-4-yl)Amino)-1H-Indole-2-Carbohyd
razide Derivatives as Novel Nur77 Modulators
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Baicun Li1, 2, #, Jie Yao1, #, Kaiqiang Guo1,#, Fengming He1, Kun Chen1, Zongxin Lin1,
Shunzhi Liu1, Jiangang Huang1, Qiaoqiong Wu1, Meijuan Fang1,*, Jinzhang Zeng1,*,
Zhen Wu1,*
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1 Fujian Provincial Key Laboratory of Innovative Drug Target Research and State Key Laboratory
of Cellular Stress Biology, School of Pharmaceutical Sciences, Xiamen University, Xiamen
361102, China
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2 State Key Laboratory of Medical Molecular Biology, Department of Physiology, Institute of
Basic Medical Sciences, Chinese Academy of Medical Sciences and School of Basic Medicine,
Peking Union Medical College, Beijing 100005, China
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#These authors contribute equally to this work
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*Corresponding author. Fujian Provincial Key Laboratory of Innovative Drug Target
Research and State Key Laboratory of Cellular Stress Biology, School of
Pharmaceutical Sciences, Xiamen University, Xiamen 361102, China. E-mail
addresses: fangmj@xmu.edu.cn (Meijuan Fang); jzzeng@xmu.edu.cn (Jinzhang
Zeng), and wuzhen@xmu.edu.cn (Zhen Wu).
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Abstract
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Nur77 is a potential target for the treatment of cancer such as HCC. Herein, we
detailed the discovery of novel series of
a
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5-((8-methoxy-2-methylquinolin-4-yl)amino)-1H-indole-2-carbohydrazide derivatives
as potential Nur77 modulators. The studies of antiproliferative activity and
Nur77-binding affinity of target compounds resulted in the discovery of a lead
candidate (10g), which was a good Nur77 binder (KD = 3.58±0.16 μM) with a
broad-spectrum antiproliferative activity against all tested hepatoma cells (IC50 < 2.0
μM) and was low toxic to normal LO2 cells. 10g could up-regulate Nur77 expression
and mediate sub-cellular localization of Nur77 to induce apoptosis in hepatocellular
carcinoma cell lines, which relied on 10g inducing Nur77-dependent autophagy and
endoplasmic reticulum stress as the upstream of apoptosis. Moreover, the in vivo
assays verified that 10g significantly inhibited xenograft tumor growth. These results
indicate that 10g has the potential to be developed as a novel Nur77-targeting
anti-hepatoma drug.
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Keywords: Nur77; quinoline; indole; antitumor; autophagy; ER stress.
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1. Introduction
Hepatocellular carcinoma (HCC) is one of the most common cancer worldwide,
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which affects more than 700,000 patients every year[1, 2]. As HCC is a highly
therapy-resistant cancer, the identification of new therapeutic targets and the
development of small molecule drugs with the new structure and novel mechanism
of action for HCC is necessary[3]. In a recent study, it was reported that Nur77 (also
known as NR4A1, TR3 or NGFI-B) interacted with phosphoenolpyruvate
carboxykinase (PEPCK1, the rate-limiting enzyme in gluconeogenesis) to increase
gluconeogenesis and suppress glycolysis, resulting in suppression of HCC
development[4]. Moreover, the UALCAN and Kalpen-Meier analyses based on the
TCGA dataset show that Nur77 is expressed at a significantly lower level in cancer
tissues than normal tissues in clinical samples (Figure 1A&1B). Kalpen-Meier
survival plot shows that the high expression of Nur77 is positively correlated with
overall survival (Figure 1C). Patients with high Nur77 expression tumors has
significantly longer overall survival (OS) than those with low Nur77 expression
tumors (Figure 1C). Additionally, it was widely reported that the nuclear receptor
Nur77 could serve as a tumor suppressor for HCC[4, 5]. Therefore, the identification
and optimization of small molecules which could upregulate the Nur77 level and
activate the Nur77 function have emerged as a promising therapeutic strategy for
HCC.
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Insert Figure 1 here.
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Figure 1. UALCAN and Kakaplan-meier analyses of Nur77 (NR4A1) in Hepatocellular
carcinoma (LIHC). Box plot analysis of the immunoreactive score (IRS) of Nur77 (NR4A1) in 50
normal samples and 340 HCC samples grouped into stage I-IV with TCGA LIHC
dataset(https://www.cancer.gov/). Nur77 expression is significantly down-regulated in LIHC.
There are statistically significant differences in the expression level of Nur77 between normal
tissues and tumor tissues of individual stages with a p-value less than 0.0001 (****) (A).
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Expression profiling of Nur77 in the Kakaplan-meier plotter database. Nur77 expression is
significantly lower in tumor tissues than in normal tissues. There are statistically significant
differences in the expression level of Nur77 between tumor tissues and normal tissues with a
p-value less than 0.05 (B). Kaplan-Meier survival curve shows the positive correlation between
overall survival of HCC patients and Nur77 expression levels in 60 months from Kaplan-Meier
Plotter dataset(http://kmplot.com/). Patients with Nur77 expression values below the 50th
percentile are classified as lower Nur77 levels, while above the 50th percentile are classified as
higher Nur77 levels. The median expression level was used as the cutoff. Survival information of
288 patients is available. HR (Hazards Ratio) =0.57, p(HR)=0.0022 (C).
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Orphan nuclear receptor Nur77 plays an vital role in cell metabolism[6, 7],
differentiation[8], proliferation, endoplasmic reticulum stress (ER stress)[9],
apoptosis[10], and autophagy[11]. Nur77 expression and function depend on tumor
type. As shown in Figure S1, Nur77 is overexpressed in a few types of tumors such
as DLBC (diffuse large B-cell lymphoma), PAAD (pancreatic adenocarcinoma), and
THYM (thymic carcinoma). However, Nur77 is notably underexpressed in the vast
majority of tumor tissues (Figure S1), e.g. LIHC (liver hepatocellular carcinoma),
BRCA (breast invasive carcinoma), LUAD (lung adenocarcinoma) and LUSC (lung
squamous cell carcinoma). In these Nur77-underexpressed tumors, Nur77 could act
as an anti-oncogene and display anti-tumor activity[4, 5]. The anti-tumor
mechanisms of Nur77 may be involved in many ways and are not fully understood.
During the past decades, many studies have demonstrated that Nur77 has the
anti-tumor activity by inducing apoptosis not only in HCC, but also in prostate, lung,
gastric, breast, and colon cancer cells[12-16]. Autophagy and endoplasmic-reticulum
(ER) stress are important biological mechanisms for cells to maintain homeostasis
and adapt to environmental stress[17]. It has been well documented that ER stress
and autophagy dysfunction contribute to the initiation and development of cancer[18,
19]. On the other hand, for tumor cells, incomplete autophagy flux and unrestrained
autophagy could result in cell death[20-22]. Nowadays, cancer therapies based on
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autophagy targeted agents have distinctly attracted much attention. Interestingly, it
has been found that Nur77 could interact with Nix to activate autophagy[11] or bind
to p62 to participate in autophagy[23]. Additionally, ER stress-mediated cell death in
a variety of diseases makes this process an attractive target for therapy[24]. ER stress
could induce apoptosis through both mediating autophagy and the eIF2/ATF4/CHOP
pathway[25]. It has also been proven that Nur77 can participate in ER stress to
induce cell death[9]. However, the mechanism of Nur77 transport to mitochondria
and endoplasmic reticulum participation in autophagy and endoplasmic reticulum
stress has still not been fully revealed.
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Due to the important effect of Nur77 in many cell biological processes, many
chemical entities of various scaffolds have been identified or developed as Nur77
modulators in recent years. For example, Nur77 modulators with potent antitumor
activity mainly include (Figure 2): 12-O-tetradecanoylphorbol-13-acetate (TPA)[26,
27],
6-(3-(1-adamantyl)-4-hydroxyphenyl)-2-naphthalene carboxylic acid (CD437 or
AHPN)[12], Celastrol[23] Cisplatin[28], Cytosporone B[29, 30], Fenretinide[31],
N-butylidenephthalide[32-34], methylene-substituted diindolylmethanes
(C-DIMs)[35], B-cell lymphoma-2 converting peptide (NuBCP-9)[36],
1-(2,4,6-trihydroxyphenyl)-1-nonanone (THPN)[11], paclitaxel[37], and
all-trans
retinoic
acid
(ATRA)[27],
,
Panobinostat[38], etc. Among them, THPN, Celastrol, and Cytosporon B can
specifically bind to the Nur77-LBD (the ligand-binding domain of Nur77) for
functional regulation[39]. However, there is no literature supporting on the direct
modulation on Nur77 of cisplatin, paclitaxel, 12-O-tetradecanoylphorbol-13-acetate
(TPA), Fenretinide, N-butylidene phthalide, Panobinostat, all-trans retinoic acid
(ATRA), and CD437. Additionally, it has been documented that TPA[40], ATRA[40],
N-butylidenephthalide[33], C-DIMs[35], NuBCP-9[36], and paclitaxel[37] can
induce Nur77-mediated apoptosis. THPN and Celastrol might induce autophagy via
Nur77 translocation to the mitochondria [11, 23]. CD437 and TPA can induce cancer
cell death through Nur77 transport to the mitochondria and endoplasmic reticulum
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(ER) [9, 40].
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Insert Figure 2 here.
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Figure 2. Chemical structures of representative Nur77 Regulators.
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Natural and synthetic compounds containing quinoline or/and indole motifs
boast a wide variety of biological activities such as anti-bacterial[41, 42],
anti-cancer[43-45], anticonvulsant[46, 47], anti-inflammatory[48, 49], and
cardiovascular activities[50, 51]. It has been implied that quinoline and indole are
two important bioactive moieties for drug design[52, 53]. Moreover, some identified
Nur77 modulators are with the structure of the indole ring (Panobinostat) or the
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quinoline
bioisosteres
such
as
naphthalene
Thus,
ring
(CD437)
series
and
of
3-hydroxynaphthalen-2(8H)-one
(Celastrol).
a
5-((8-methoxy-2-methylquinolin-4-yl)amino)-1H-indole-2-carbohydrazide
derivatives as new Nur77 modulators were designed, synthesized, and screened for
the antiproliferative activity and the direct binding ability with Nur77. The
structure−activity relationships (SARs) of synthesized compounds for
antiproliferative activity were elucidated. Compound 10g was identified as the most
potent binder of Nur77 which exhibited good anti-proliferative activity against liver
cancer cell lines (HepG2, QGY-7703, and SMMC-7721), and low cytotoxic property
in the liver normal cell line (LO2). Therefore, compound 10g was further
investigated for its molecular basis of anti-cancer activity as a good Nur77 binder.
The bioassay data indicated that 10g could upregulate the expression of Nur77 and
translocation to ER and mitochondria, where it participated in ER stress and
autophagy induction. Compound 10g could induce Nur77-dependent endoplasmic
reticulum stress in vitro, which triggered autophagy and apoptosis in hepatoma cells.
The anti-tumor effect of 10g was further verified in a nude mouse xenograft model.
Herein, we reported the design, synthesis, and subsequent biological evaluation of
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5-((8-methoxy-2-methylquinolin-4-yl)amino)-1H-indole-2-carbohydrazide
2
3
derivatives as a new class of Nur77 modulators.
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2. Results and Discussion
2.1. Chemistry
2.1.1 Structure-based drug design
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In the present study, the structure-based rational drug design was used to look
for a new class of Nur77-targeting compounds with potent antitumor activities. We
first used the SiteMap[54] suite in Schrödinger software to explore and analyze the
surface of Nur77-LBD (PDB ID: 4RE8), to identify all potential small-molecules
binding sites. Notably, the top-ranked site with a SiteScore and Dscore of 1.003 and
1.006, respectively, was composed of the co-crystal ligand (3mj) binding subsite (S’)
and a hydrophobic adjacent cavity (S’’), exhibiting a U-shape structure (Figure 3A
& 3B). The native ligand 3mj (a direct Nur77-modulator, Figure S2) was the initial
compound to design novel chemical entities targeting Nur77. Simultaneously, as
CD437 and Panobinostat could well bind to the pocket of Nur77-LBD in molecular
docking simulation (Figure S3 & Table S1), the two compounds were selected as the
leading compounds for the design of new Nur77-targeting compounds in the present
study. CD437 contains a naphthalene ring which is a quinoline bioisostere, while
Panobinostat has an indole ring. Herein, considering that the inclusion of the indole
and quinoline moieties might contribute to the anticancer potency and
Nur77-targeting binding potency, the benzene ring of 3mj was changed into a rigid
binary ring (the quinoline group), on which the indole moiety was further introduced.
Taken together, we used the scaffold replacement and molecular hybridization
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strategies,
leading
to
constructing
the
5-((8-methoxy-2-methylquinolin-4-yl)amino)-1H-indole core as the new Nur77
modulators’ scaffold. Subsequently, to obtain the “U-shape” molecules, together with
consideration to synthetic accessibility, the C-2 position of indole ring was extended
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4
via a linker (carbohydrazide) by another aromatic ring (R) holding or not holding
substituents (Figure 3C). The aromatic ring R with or without substituent groups are
described in Figure 3D, which were supposed to interact with the small hydrophobic
pocket (S’’ subsite).
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Insert Figure 3 here.
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Figure 3. Design strategies used in our present study to obtain novel Nur77 regulators with
5-((8-methoxy-2-methylquinolin-4-yl)amino)-1H-indole scaffold
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Next, to confirm the rationality of this structure-based design, the molecular
docking simulations between target compounds and Nur77-LBD were performed to
evaluate the binding affinities of protein-ligand complexes, by using SP Glide of
Schrödinger with default settings. The Glide docking scores of most target
compounds with Nur77-LBD were range from -4.799 to -7.788, while the native
ligand 3mj had a docking score of -5.021. The docking results indicated that most of
the target compounds had moderate binding affinities with Nur77-LBD. Among them,
10g (SP mode, docking score = -4.967) was selected as a representative compound
for further analysis (Figure S4 & S5). To gain more details on the interaction mode
of 10g and Nur77-LBD, we further performed Induced Fit docking simulations on
10g and Nur77-LBD (Figure S6). Figure 3E and 3F showed the docked pose of 10g
in the “U-shape” binding site of Nur77-LBD (docking score = -10.531, IFD Score =
-509.43).
As
depicted
in
Figure
3F,
5-((8-methoxy-2-methylquinolin-4-yl)amino)-1H-indole moiety of 10g was oriented
toward the larger hydrophobic pocket (S’ subsite), with an additional H-bond
occurring between the amine hydrogen atom and carboxyl moiety of Glu114 which
reinforced this binding conformation. The 4-(methylthio)phenyl moiety bound into
the small hydrophobic pocket (S’’ subsite) and formed hydrophobic interactions with
Leu204, Val208, Leu221, and Cys220. Beyond these, the oxygen atom of the
carbohydrazide linker also showed a hydrogen bond contact with the residue of
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Gln197. Moreover, we used MM-GBSA module integrated in Schrödinger to
calculate ∆ꢀꢁꢂꢃꢄvalues of Nur77-3mj complex and Nur77-10g complex obtained
from Induce Fit docking. Compared with the native ligand 3mj (∆ꢀꢁꢂꢃꢄ= -78.31
kcal/mol), 10g (∆ꢀꢁꢂꢃꢄ= -143.61 kcal/mol) showed a higher binding affinity to
Nur77. These results suggested that target compounds including 10g had the
potentials to be Nur77 binders.
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The
molecular
docking
study
demonstrated
that
5-((8-methoxy-2-methylquinolin-4-yl)amino)-1H-indole-2-carbohydrazide was the
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promising scaffold of novel Nur77 modulators for further examination. Therefore, a
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total
of
35
derivatives
of
5-((8-methoxy-2-methylquinolin-4-yl)amino)-1H-indole-2-carbohydrazide (Scheme
1 & Table 1) were synthesized accordingly. All compounds were evaluated for the
antiproliferative effects (Table 1) and Nur77-binding affinities (Figure S8). After a
preliminary screening, the most outstanding compound 10g was further investigated,
including the activities of the control of Nur77 expression, cell cycle arrest,
apoptosis-inducing, autophagy-inducing, and ER stress-inducing activities in
hepatoma carcinoma cells, and inhibition of tumor growth in a mouse hepatoma
xenograft model.
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2.1.2. Compound synthesis
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The target compounds 10a-10z and 10A-10I were synthesized according to the
synthetic route outlined in Scheme 1. (4-nitrophenyl) hydrazine 1 were refluxed with
ethyl pyruvate in ethanol to afford compound 2 and then underwent ring closure with
the presence of polyphosphoric acid to give ethyl 5-nitro-1H-indole-2-carboxylate 3.
Reduction of the nitro group of 3 with Fe in acetic acid aqueous obtained the key
intermediate, ethyl 5-amino-1H-indole-2-carboxylate 4. Meanwhile, the reaction of
2-methoxyaniline 5 with ethyl acetoacetate under polyphosphoric acid condition
provided 8-methoxy-2-methylquinolin-4-ol 6, followed by chlorination using POCl3
to get the other key intermediate 7 (4-chloro-8-methoxy-2-methylquinoline). Then,
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3
4
5
6
N-alkylation
reaction
of
4
with
7
gave
ethyl
5-((8-methoxy-2-methylquinolin-4-yl)amino)-1H-indole-2-carboxylate (8), followed
by
refluxing
with
hydrazine
hydrate
in
ethanol
to
get
5-((8-methoxy-2-methylquinolin-4-yl)amino)-1H-indole-2-carbohydrazide
(9).
Finally, target compounds (10a-10z and 10A-10I) were prepared via reactions
between 9 and corresponding aldehydes in refluxing ethanol.
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The chemical structures of target compounds were characterized by ESI-MS,
1
HRMS, H NMR, and 13C NMR. Moreover, these acylhydrazone compounds
(10a-10z and 10A-10I) were further identified as a single E-isomer according to our
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previous work[55] and other similar studies[56-59] .
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Insert Scheme 1 here.
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Scheme 1. Preparation of target compounds 10a-10z and 10A-10I. Reaction conditions: (a) Ethyl
pyruvate, ethanol, reflux; (b) Polyphosphoric acid, 100 , 2 h; (c) Fe, acetic acid aqueous, 70 , 2
h; (d) Polyphosphoric acid, ethyl acetoacetate, 120 ; (e) POCl3, 130 ; (f) 1-Butanol,
concentrated hydrochloric acid, reflux; (g) hydrazine hydrate, ethanol, reflux; (h) ethanol,
substituted aldehydes, trifluoroacetic acid, reflux.
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2.2. Biological evaluation
2.2.1. In vitro antiproliferative activity
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All the target compounds in this study were evaluated for the antiproliferative
activities against three human hepatoma cell lines (HepG2 cells, QGY-7703 cells,
and SMMC-7721 cells) and one normal hepatocyte line (LO2) using MTT assay with
Celastrol (PC1), CD437 (PC2), and Cisplatin (PC3) as references. As shown in Table
1, most target compounds with the different aromatic groups introduced at the
N′-methylene position, exhibited moderate antiproliferative activity against at least
one test cancer cell line, with IC50 value below 10.0 μM. According to these data, the
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preliminary
structure-activity
relationships
(SARs)
of
these
novel
5-((8-methoxy-2-methylquinolin-4-yl)amino)-1H-indole-2-carbohydrazide
derivatives were summarized.
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Insert Table 1 here.
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Table 1. In vitro antiproliferative activities of target compounds against liver cancer and normal
cells
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(i)The influence of substitutes of the phenyl ring
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When the moiety R was the ortho mono-substituted phenyl groups, compounds
with 2-fluoro (10a), 2-chloro (10b), and 2-methoxy (10c) substitutions, exhibited
excellent antiproliferative activities against all the four tested cell lines including
three cancer cell lines and one normal cancer cell line. When the moiety R was the
meta or para mono-substituted phenyl groups (10d-10j), the type of substituted
groups played a pivotal role in antiproliferative profiles. Compound 10d
(R=3-methylpheny) had a broad spectrum of antiproliferative activities against both
tumor and normal cells. However, compounds 10e-10j showed low antiproliferative
activity against at least one cell line. For example, the replacement of 3-CH3 with
3-OCH3 (10d vs 10e), resulted in significantly decreased antiproliferative activity in
both HepG2 and QGY-7703 cell lines with the IC50 value of higher than 20.00 μM.
The conversion of 3-CH3 to 3-CF3 (10d vs 10f) led to a sharp decreased
antiproliferative inhibition on SMMC-7721, but no obvious influence in HepG2 and
QGY-7703 cell lines. Inspiringly, compound 10g showed potent anti-proliferative
activities (IC50 < 5.0 µM) against all three liver cancer lines (HepG2, QGY-7703, and
SMMC-7721), and low cytotoxic property in liver normal cells (LO2). Interestingly,
changing the methoxy group (10h) to methylthio (10g) led to a significant difference
in activity (Table 1 & Figure S7), which might result from the different binding
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affinities of 10h and 10g with Nur77 (Table S2 & Figure S7).
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3
In the target compounds 10k-10z which contain di- or tri- substituted phenyl
ring (R), the types and substituted positions of substitutes displayed an important
relationship with the antiproliferative activity. Compounds with 2,3-dimethoxy (10k),
2,4-dimethoxy (10l), 2,3,4-trimethoxy (10x), and 4-methoxy-3-trifluoromethyl (10r)
substituted phenyl groups had good antiproliferative activities against all tested
cancer and normal cells. However, compounds with 2,5-dihydroxy (10m),
3,4-dimethoxy (10n), 3,5-dimethoxy (10w), and 3,4,5-trimethoxy (10y) substituted
phenyl groups showed poor cytotoxicity profiles against all the four cell lines. In
addition, substitution of nitro group (10p) and trifluoromethyl group (10t) at the C-4
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9
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position of phenyl ring had remarkably lowered the cytotoxicity (IC50＞20.0 μM),
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implying that substitution of electron-withdrawing groups at phenyl ring was
unhelpful for antiproliferative activity.
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(ii) The influence of different aromatic R groups
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Compounds
10B
and
10F
which
had
2-thiopheneyl
and
4-methoxynaphthalen-1-yl moieties, respectively, retained good antiproliferative
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activities for all tested cell lines (IC50, 1.0–17.0 μM). However, compounds with
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1H-pyrrole-2-yl (10A), 3-pyridyl (10D), and 4-pyridyl (10E) moieties displayed a
dramatic decrease in the cellular antiproliferative activity. Quinoline-4-yl (10G),
2-chloroquinoline-3-yl (10H), and 5-methoxy-2-methyl-1H-indole-3-yl (10I) groups
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introduced at the N′-methylene position had low toxic to LO2 cells but caused a
narrow spectrum antiproliferative activity against hepatoma cells.
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Taken together, cell proliferation assay demonstrated that most of the target
compounds exhibited moderate anticancer activity, preferably mono-substitution on
phenyl ring. Among them, 10g displayed potent and broad-spectrum antiproliferative
activity against all tested three hepatoma cell lines (HepG2, QGY-7703, and
SMMC-7721), and less cytotoxicity against LO2 cells (human normal liver cell line).
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These results suggested that 10g had the potential to become a safer candidate for
antitumor drugs.
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2.2.2. 10g can bind to Nur77 in vitro
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Nur77 is an attractive target for the treatment of HCC, we’d like to discover
new anticancer agents targeting Nur77. Thereby, all target compounds were
evaluated for the binding ability with Nur77-LBD using a single concentration
binding assay by surface plasmon resonance (SPR) technology-based experiment
(Figure S8). Importantly, the SPR experiment showed that the introduction of
mono-substituted phenyl ring at the carbon atom of hydrazine group (C=N) is more
beneficial for the binding activity with Nur77 compared with other substituted aryl
ring. When tested at 10.0 μM concentration, compounds 10h, 10g, and 10r could
well bind to Nur77 with RU values of 88.202, 91.587, and 90.948, respectively.
Among these three compounds, both 10g and 10r showed potent and broad-spectrum
antiproliferative activity against all tested hepatoma cells, with IC50 values of less
than 2.0 μM, but only 10g did not show significant cytotoxicity against liver normal
cells (LO2) at the tested concentrations (Table 1). The above assays showed that 10g
had low cytotoxic property, a broad spectrum antiproliferative activity against
hepatoma cells, and a potent binding affinity to Nur77. As the lack of selectivity
between normal and cancer cells is one of the main limitations of anticancer drugs,
10g was selected for further study.
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To confirm the physical binding ability of 10g with Nur77-LBD, the SPR assay,
and fluorescence ligand binding assay were performed. The SPR results showed that
10g dose-dependently bound to Nur77-LBD with a KD (equilibrium dissociation
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constant) value of 3.58±0.16 μM, and exhibited fast association/dissection kinetics to
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interact with Nur77-LBD (Figure 4A). The effect of 10g on Nur77-LBD
fluorescence intensity was shown in Figure 4B. There was a strong fluorescence
emission of Nur77-LBD peaked at 332 nm (excitation at 280 nm). When a fixed
concentration of Nur77-LBD was titrated with different concentrations of 10g, a
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remarkable intrinsic fluorescence decrease of Nur77-LBD was observed. The
calculated KD value from the fluorescence ligand binding assay was 1.34±0.19 μM.
Together, these results demonstrated that 10g could directly and efficiently bind to
Nur77-LBD in vitro.
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Insert Figure 4 here.
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Figure 4. 10g bound to Nur77-LBD in vitro. Characterization of the binding affinity between 10g
and Nur77-LBD by the SPR assay (A). Fluorescence titration curve of Nur77-LBD with 10g. The
concentration of 10g increased from 0.2 μM to 4 μM at an interval of 0.2 μM (B).
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2.2.3. 10g regulates Nur77 expression at the cellular level
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In all tested hepatocellular carcinoma cell lines, 10g could up-regulate the
expression of Nur77 in a dose-dependent manner (Figure 5A). In HepG2 cells, the
expression of Nur77 peaked after the treatment of 10g for 6 h, while the expression of
other nuclear receptors including RXRα and RARγ didn’t significantly change
(Figure 5B). This suggested that 10g might specifically regulate the expression of
Nur77. However, the mechanism by which Nur77 rapidly degraded after induction is
not well understood[60, 61]. It has been reported that the half-life of Nur77 is extremely
short, being about 20 to 40ꢀmin[62]. This may lead to a gradual decline in Nur77 levels
after 6 hours of 10g treatment.
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In recent years, various non-genomic actions of Nur77 have been discovered,
which are closely related to cell survival[63]. The antitumor activity of Nur77 was
largely based on its extranuclear non-genomic action, so we investigated whether 10g
caused the extranuclear transport of Nur77 by using immunofluorescence experiments.
As shown in Figure 5C, there were a large number of translocations of Nur77 into the
cytoplasm from the nucleus when HepG2 cells were treated with 10g for 6 h. The
results of western blotting analyses also showed that the amount of Nur77 in cytoplasm
increased, while that in the nucleus decreased with time increasing (Figure 5D).
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Furthermore, confocal microscopy analysis revealed that 10g promoted extensive
mitochondrial translocation of endogenous Nur77 (Figure 5E). Endogenous Nur77
was also recorded for its translocation to the endoplasmic reticulum from the cell
nucleus, when cells were treated with 10g (Figure 5F). These results demonstrated that
10g could promote the high expression and subcellular translocation of Nur77.
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Insert Figure 5 here.
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Figure 5. 10g regulated the expression and sub-cellular translocation of Nur77. HepG2,
QGY-7703, and SMMC-7721 cells treated with the indicated concentration of 10g for 6 hours were
analyzed for Nur77 by western blotting, GAPDH was used as an internal control (A). HepG2 cells
were treated with 2.0 μM 10g for the indicated time. The expression of Nur77, RXRα, and RARγ
were detected by western blotting (B). Fluorescent images of HepG2 cells treated with 2.0 μM 10g
for indicated time and detected by Nur77. The nuclei were stained with DAPI. Scale bar, 10 μm (C).
HepG2 cells were treated with 2.0 μM 10g for the indicated time. Nur77 was detected in the
cytoplasm and nuclear by western blotting. PARP was used as an internal control of the nucleus
and Tubulin was used as an internal control of cytoplasm (D). HepG2 cells were treated with 2.0
μM 10g for 6 hours and then incubated with the Nur77 antibody and Mito-tracker (E) or ER tracker
(F). Scale bar, 10 μm.
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2.2.4. Nur77-dependent antitumor mechanism of 10g in HCC cell lines
2.2.4.1. Growth inhibition of hepatoma carcinoma cells by 10g
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To evaluate the effect of 10g on cell viability and cell proliferation, MTT assay and
colony formation were performed. The results indicated that 10g reduced the viability
of HepG2, QGY-7703, and SMMC-7721 in a time-dependent manner (Figure 6A). The
inhibitory effect of 10g in HepG2, QGY-7703, or SMMC-7721 was also evaluated by
using the crystal violet exclusion assay. It showed that the colony formation units
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decreased significantly with the treatment of 10g (Figure 6B). In HepG2 cells,
phase-contrast micrograph experiments revealed that 10g induced cell morphology
alteration, such as cell shrinkage, vesicles accumulated, and reduced cell number
(Figure 6C). These data suggest that 10g has significant in vitro inhibitory effects on
hepatoma cells.
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Insert Figure 6 here.
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Figure 6. 10g inhibited the growth and proliferation of HCC. HepG2, QGY-7703, and
SMMC-7721 cells were treated with 2.0 μM 10g with the indicated time. Cell viability was
determined by MTT assays (A). The number of colonies was counted after the treatment of HepG2,
QGY-7703, and SMMC-7721 cells with DMSO or 2.0 μM 10g (B). HepG2 cells were treated with
10g at the indicated concentrations, and the morphological changes were observed by optical
microscopy at the indicated time (C).
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2.2.4.2. 10g induces Nur77-dependent apoptosis
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To examine whether the growth inhibition of cancer cells induced by 10g was
associated with apoptosis, we checked the level of the cleaved PARP (c-PARP, a
marker for apoptosis) in hepatoma cells treated with or without 10g. The level of the
cleaved PARP was increased with the increasing concentrations of 10g treatment in
HepG2, QGY-7703, and SMMC-7721 cells (Figure 7A). Additionally, Annexin
V-FITC/PI assay showed that early and late phase apoptosis cells increased
from 34.6% to 91.3% in HepG2, with an increase of 10g concentration from 1.0 to
2.0 μM (Figure 7B). To verify whether 10g could induce Nur77-dependent apoptosis,
the Nur77 knockdown (shNur77) HepG2 cells were employed. Firstly, the c-PARP
reversed obviously in shNur77 HepG2 cells treated with 10g (Figure 7C).
Furthermore, early and late phase apoptosis cells with 2.0 μM 10g treatment were
reduced from 80.5% to 40.0%, with Nur77 knocking down (Figure 7D). The
decrease of mitochondrial membrane potential is a landmark event in the early stage
of apoptosis. The decrease of cell membrane potential is easily detected by the
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transformation of JC-1 from red fluorescence to green fluorescence, which is being
used as a detection indicator of early apoptosis. So we further examined the effect of
10g on the cell membrane potential using JC-1 staining (Figure 7E). When HepG2
cells were treated with 2.0 μM 10g, the median intensity of green fluorescence of
JC-1 increased from 41669 to 71731 units, while the median intensity of red
fluorescence of JC-1 decreased from 23598 to 10298 units. However, there were no
significant changes in shNur77 HepG2 cells. Collectively, these results suggest that
the 10g inducing cell apoptosis is dependent on Nur77.
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To explore whether the apoptosis induced by 10g was caused by the nuclear
exudation of Nur77, HepG2 cells were pretreated with or without 20 nM leptomycin
B (LMB, an inhibitor of nuclear export) and then exposed to 10g for an additional 12
hours. Finally, we checked the translocation of Nur77 by fluorescent microscope and
examined the protein levels of PARP and Nur77 using Western blotting analysis. As
shown in Figure S9A, LMB could hinder Nur77 nuclear exudation induced by 10g.
Moreover, LMB inhibited the 10g induced apoptosis of HepG2 cells without
affecting the Nur77 expression (Figure S9B). On the other hand, overexpression of
Nur77 did not cause the death of HepG2 cells (Figure S9C). Collectively, these
results suggest that the 10g inducing cell apoptosis is dependent on Nur77
translocation.
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Insert Figure 7 here.
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Figure 7. 10g induced Nur77-dependent apoptosis. Western blot analysis of proteins involved in
apoptosis after treatment with 10g at the indicated concentrations for 12 hours in HepG2,
QGY-7703, and SMMC-7721 cells (A). Annexin V/PI staining of HepG2 cells treated with 10g at
the indicated concentrations for 12 hours and assessed by flow cytometry (B). HepG2 or shNur77
HepG2 cells were treated with 2.0 μM 10g for 12 hours. Cells were then subjected to Western
blotting for PARP cleavage and Nur77 detecting (C), Annexin V/PI staining for apoptosis
measurement (D), and JC-1 staining for mitochondrial membrane potential assay (E).
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2.2.4.3. 10g induces Nur77-dependent autophagy
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5
Autophagy plays a dual role in tumor cell metabolism. On one hand, under the
condition of nutrient deprivation, tumor cells maintain their survival through
autophagy. On the other hand, autophagy is a way to promote cell death. Some
anti-tumor drugs induce autophagy to cause the death of tumor cells. Beclin1 plays
an important role in the formation of phagocytes in the early stage of autophagy.
Meanwhile, the microtubule-associated protein 1 light chain 3 (LC3) is a widely
recognized biological marker of autophagy[64]. LC3 protein expression level will be
enhanced, and LC3-I will be transformed into LC3-II during autophagosome
maturation with the participation of various ATG proteins. P62/SQSTM1 is a
ligand-protein for LC3 binding, which is responsible for the degradation of
ubiquitinated aggregates in autophagosomes. Furthermore, p62 can induce complete
autophagy after binding to LC3 and is excessively expressed upon the activation of
autophagy[65].
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Nowadays, it has been reported that Nur77 can activate autophagy[11]. Herein,
LC3-II and Beclin1 protein levels were first determined in HepG2, QGY-7703 and
SMMC-7721 cells treated with or without 10g for 12 h. It was observed that LC3-II
and Beclin1 protein levels increased after 10g treatment in a dose-dependent manner
(Figure 8A). The most widely used method for autophagy detection is the
observation and quantification of GFP-LC3 puncta by fluorescence microscopy. To
verify 10g’s ability in autophagy induction, HepG2 cells transfected with the
GFP-LC3 were treated with or without 2.0 μM 10g for 6 h and GFP-LC3 puncta in
HepG2 cells was then checked by fluorescence microscopy. As depicted in Figure
8B, GFP-LC3 puncta were observed in cells treated with 10g but not in control cells.
However, GFP-LC3 puncta were not located in the vesicles. Surprisingly, 10g
treatment did not enhance p62 degradation, indicating that autophagy induced by 10g
was incomplete (Figure S10). These data imply that 10g can cause autophagy in
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HCC cells.
To further explore whether 10g induced autophagy was related to Nur77, LC3,
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3
and Beclin1 expression levels were checked in the scramble control group (scr) and
Nur77-knockdown group (shNur77) HepG2 cells treated with or without 2.0 μM 10g
for 6 or 12 h. Beclin1 expression level had no significant difference between normal
and Nur77-knockdown HepG2 cells, but LC3 expression level decreased in
Nur77-knockdown HepG2 cells (Figure 8C). Besides, HepG2 and shNur77 HepG2
cells both transfected with GFP-LC3 were treated 2.0 μM 10g for 6 h, and then
GFP-LC3 were examined by fluorescence microscopy. As shown in Figure 8D,
GFP-LC3 puncta in wild-type HepG2 cells treated with 10g displayed a dramatic
increase, while slightly increased in Nur77-knockdown HepG2 cells. We also found
that LC3-II expression slightly increased in Nur77 overexpressed HepG2 cells with
10g treatment (Figure S11). Taken together, these results suggest that 10g inducing
incomplete autophagy in HCC cells is dependent on Nur77 to some content.
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Insert Figure 8 here.
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Figure 8. Induction of autophagy by 10g in HCC. HepG2, QGY-7703 and SMMC-7721 were
treated with 10g at the indicated concentrations for 12 h, the levels of LC3 and Beclin1 were
detected by Western blotting (A). Phase-contrast and fluorescent images of HepG2 cells
transfected with GFP-LC3 and treated with 2.0 μM 10g for 6 hours. Scale bar, 10 μm (B). HepG2
or shNur77 HepG2 cells were treated with DMSO or 2.0 μM 10g for 6 hours or 12 hours. Cells
were then subjected to Western blotting for LC3, Beclin1, and Nur77 detecting (C). Fluorescent
images of HepG2 and shNur77 HepG2 cells transfected with GFP-LC3 and treated with 2.0 μM
10g for 6 hours. Scale bar, 10 μm (D).
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2.2.4.4. 10g induces Nur77-dependent ER stress
ER stress is caused by abnormal accumulation of proteins in the endoplasmic
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reticulum. ER stress might affect various stress responses of cells, such as injury,
autophagy, and apoptosis. Bip protein, as a member of heat shock protein 70, is
located in the endoplasmic reticulum cavity to assist the transfer of the newly formed
protein into the internal reticulum. As a target molecule of endoplasmic reticulum
stress, Bip accumulates when the endoplasmic reticulum occurs. CHOP and TRB3,
as response genes to ER stress, are highly expressed after ER stress, causing cell
biological behaviors such as apoptosis and autophagy. TRB3 is a downstream gene
of CHOP[66].
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Nur77 could participate in ER stress to induce cell death, so we checked
whether 10g caused ER stress in hepatocellular carcinoma cells. As shown in Figure
9A, the expression of endoplasmic reticulum stress-related proteins including Bip,
CHOP, and TRB3 was dose-dependently increased in all three HCC cell lines with
the treatment of 10g for 12 h. In line with this observation, immunostaining of
the ER luminal marker protein disulfide isomerase (PDI) showed a striking
dilation in the ER of 10g-treated HepG2 cells (Figure 9B). To clarify whether
10g mediate Nur77-dependent ER stress, HepG2 and shNur77 HepG2 cells were
treated with or without 2.0 μM 10g for 6 or 12 h, and then the expression levels of
Bip, CHOP, and TRB3 were analyzed by western blot (Figure 9C). It showed that
there was a significant decrease in the expression of Bip and CHOP in shNur77
HepG2 cells, compared to HepG2 cells treated with 10g. Meanwhile, we observed
that the expression levels of TRB3 both in HepG2 and shNur77 HepG2 cells were
decreased at 12 h, while Bip expression level was slightly increased with the
increased treated time of 10g. Simultaneously, immunostaining showed PDI dots in
shNur77 HepG2 cells treated with 10g were less than in wild-type HepG2 cells
treated with 10g (Figure 9D). These results reveal that 10g inducing ER stress is in
Nur77 dependent manner.
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Insert Figure 9 here.
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Figure 9. Induction of ER stress by 10g in HCC. HepG2, QGY-7703 and SMMC-7721 were
treated with 10g at the indicated concentrations for 12 h, the levels of Bip, TRB3, and CHOP
were detected by western blotting (A). Phase-contrast and fluorescent images of HepG2 cells and
treated with 2.0 μM 10g for 6 hours and then incubated with PDI antibody. Scale bar, 10 μm (B).
HepG2 or shNur77 HepG2 cells were treated with DMSO or 2.0 μM 10g for 6 hours and 12
hours. Cells were then subjected to Western blotting for TRB3, Bip, CHOP, and Nur77 detecting
(C). Fluorescent images of HepG2 and shNur77 HepG2 treated with 2.0 μM 10g for 6 hours and
then incubated with PDI antibody. Scale bar, 10 μm (D).
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2.2.4.5. 10g induces ER stress-mediated autophagy and autophagy-dependent cell
apoptosis
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We then investigated the relationship of autophagy, ER stress, and cell apoptosis,
which were induced by 10g. HepG2 cells were treated with 2.0 μM 10g for the
indicated time. The expression of Nur77 began to rise after 10g treatment for one
hour and reached the peak at 6 hours. The expression of Bip, p-EIF2a, and TRB3
increased at one to four hours. Subsequently, LC3 II expression began to increase in
4-6 hours. Finally, PARP was significantly cleaved after 6 hours (Figure 10A). To
further investigate whether autophagy and ER stress were contributed to 10g-induced
cell death, we combined 10g with the autophagy inhibitor 3-MA or the ER stress
inhibitor 4-PBA to treat HepG2 cells. Once autophagy and ER stress were inhibited,
PARP cleavage, Caspase-3 cleavage, and caspase-9 cleavage induced by 10g were
reduced and the ratio of Bax/Bcl2 was also decreased (Figure 10B and 10C).
Interestingly, when HepG2 cells were treated with 10g and 4-PBA at the same time,
the expression level of LC3-II was significantly weakened (Figure 10C), implying
that 10g-induced ER stress could promote autophagy.
Taken together, it suggests that 10g can induce cell apoptosis through ER stress
and autophagy pathways.
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Insert Figure 10 here.
Figure 10. 10g induced apoptosis-dependent autophagic cell death mediated by ER-stress.
HepG2 cells were treated with 2.0 μM 10g for indicated time. The levels of autophagy associated
proteins, ER stress-associated proteins, and Nur77 were detected by Western blotting (A). HepG2
cells were pretreated with 3-MA (2.0 mM) for 2 hours prior to exposure to 10g for an additional
12 hours. Western blotting was performed to examine the changes in protein levels of PARP,
cleaved-caspase-3, cleaved-caspase-9, Bcl-2, Bax, Bip, and LC3(B). HepG2 cells were pretreated
with 4-PBA (2.0 mM) for 2 hours prior to exposure to 10g for an additional 12 hours. Western
blotting was performed to examine the levels of PARP, cleaved-caspase-3, cleaved-caspase-9,
Bcl-2, Bax, Bip, EIF2a, TRB3, and LC3(C).
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2.2.5. The antitumor activities of 10g in vivo
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To evaluate the in vivo antitumor potency of 10g, we established a mice
hepatoma HepG2 xenograft model. 10g was administered once every day for 15 days
(10 mg/kg/day i.p. and 20 mg/kg/day i.p.), and the tumor sizes were measured every
day. After the experiment was completed, the mice were sacrificed to strip the tumors
and organ tissues, the body weight and stripped tumors were weighted, and the safety
and effectiveness of 10g were evaluated by hematoxylin and eosin (H&E) staining
and immunohistochemistry analysis.
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As shown in Figure 11, 10g treatment could lead to substantial suppression of
tumor growth (Figure 11A), the tumor growth inhibition (TGI) values at doses of 10
mg/kg/day and 20 mg/kg/day were 36.74 % and 62.38 %, respectively (Figure 11C).
The average tumor weights of drug treatment groups were 0.24 ± 0.13 g (20
mg/kg/day, i.p.) and 0.50 ± 0.15 g (10 mg/kg/day, i.p.), which were significantly
lower than control group (0.86 ± 0.18 g) (Figure 11B). The results shown in Figure
11C indicated that 10g exhibited almost no influence on the body weight of
experimental mice, while it significantly suppressed tumor weight, indicating its safe
usage. Furthermore, immunohistochemistry analysis showed that cells of the tumor
tissue in the drug groups were sparser and vacuolated than the control group (Figure
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11D). Immunostaining and Western blotting of tumor tissues prepared from
non-treated and treated mice revealed that the expression level of proliferation
marker PCNA was markedly reduced by 10g (Figure 11E&F). Together, these
results demonstrate that 10g inhibits tumor growth with good tolerability in vivo.
We also determined the autophagy and ER stress effect of 10g in animals.
Immunostaining and western blotting analyses of tumor tissues showed that
compared with the control group, the administration groups exhibited significantly
increased expressions of Nur77, autophagy (LC3, Beclin1) and ER stress (TRB3, Bip,
CHOP) markers (Figure 11E&F). Furthermore, the translocation of Nur77 in the
cytoplasm was also observed in the high dose treatment group (Figure 11F). These
results further suggest that 10g potently induces autophagy and ER stress in a
xenograft mouse model of HepG2 cells, which is consistent with the cellular results.
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Insert Figure 11 here.
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Figure 11. 10g exhibited antitumor efficacy in a xenograft mouse model of HepG2 cells. Nude
mice (n=15) injected with HepG2 (5×106 cells) were administrated with the indicated dose of 10g
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(10 mg/kg, i.p.; 20 mg/kg, i.p.) once a day and tumors were measured every day. 15 days after
administration of 10g, nude mice were sacrificed and tumors were removed, weighted and
showed (***P< 0.001, **P< 0.01, *P< 0.05) (A, B). Tumor volume and body weight of nude
mice during treatment (***P< 0.001, **P< 0.01, *P< 0.05, ·P< 0.05) (C). H&E staining in
tumors after 15 days of treatment with 10g (D). Micromagnification was 200, 400, and 1000
times respectively. Immunocytochemistry staining showing the expression of Nur77, PCNA, LC3,
Beclin1, TRB3, Bip, and CHOP in tumor tissues prepared from nude mice treated with or without
10g (10 mg/kg; 20 mg/kg) for 15 days (E). Western blot analysis of the expressions of Nur77,
PCNA, LC3, Beclin1, TRB3, Bip, and CHOP in tumor tissues prepared from nude mice treated
with or without 10g (10 mg/kg, i.p.; 20 mg/kg, i.p.) for 15 days (F).
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3. Conclusion
In summary, we presented the successful design and synthesis of a new class of
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5-((8-methoxy-2-methylquinolin-4-yl)amino)-1H-indole-2-carbohydrazide
derivatives as potent Nur77 modulators. One of the lead compounds of this class, 10g,
was identified as a Nur77-specific modulator with a broad-spectrum inhibition on
hepatoma cells and low toxic. 10g exhibited cellular Nur77-targeted anticancer
mechanism of action. The apoptosis, autophagy, and ER stress inductions of 10g were
in a Nur77-dependent manner. Moreover, further mechanistic experiments revealed
that 10g-induced ER stress led to the activation of incomplete autophagy and
promoted the apoptotic death of tumor cells. Taken altogether, 10g might be a
promising candidate for further development into a chemotherapeutic agent for the
treatment of HCC.
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4. Experimental section
4.1. Chemistry
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The commonly used reagents were purchased from Beijing Innochem Technology
Co., Ltd. and used without further purification. The melting points (mp) were
determined using a Shanghai Jingke SGW X-4 microscope melting point apparatus.
Thin-layer chromatography (TLC, silica gel HSGF254, Yantai Jiangyou Silicone
Development Co., Ltd. (Yantai, Shandong, China)) was used to monitor for
completeness of the reaction visualized by UV light (λ=254 nm or λ=365 nm). And
the obtained compounds were purified by silica gel column chromatography. 1H NMR
and 13C NMR were carried out using the Bruker Avance III spectrometer at 600 MHz
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for H NMR and 150 MHz for C NMR spectrophotometer, (Faculty of Pharmacy,
Xiamen University, Fujian, China), at 25 in DMSO-d6 using tetramethylsilane
(TMS) as an internal standard and chemical shifts were recorded in ppm on the δ scale
using DMSO-d6 (2.5) as a solvent. Coupling constant (J) values were estimated in
Hertz (Hz). Splitting patterns are designated as follows: s, singlet; br s, broad singlet;
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d, doublet; t, triplet; q, quartet; dd, doublet of doublet; m, multiplet. Mass spectra
were measured on an LCMS-2020 Single Quadrupole Mass Spectrometer (Shimadzu).
And the HR-ESI-MS analysis was performed on the Thermo Q-Exactive Mass
spectrometer (Thermo Fisher Scientific Corporation, Waltham, Massachusetts, USA)
equipped with electrospray ionization source (ESI).
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4.1.1. Ethyl (Z)-2-(2-(4-nitrophenyl)hydrazineylidene)propanoate 2
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8
To a stirred solution of (4-nitrophenyl)hydrazine 1 (10.0 g, 65.0 mmol) in
anhydrous ethanol (85 mL), ethyl pyruvate (8.1 g, 72.0 mmol) was added dropwise at
room temperature. After completion of the dropwise addition, the mixture was heated
at reflux for 2 hours. The reaction was cooled to room temperature, filtered under
suction, and washed with ethanol to give compound 2 (13.8 g). Yellow solid, yield
9
10
11
12
13
14
89.6%, mp: 197-199 ; 1H-NMR (600 MHz, CDCl3):
δ 8.21 (d, J=9.0 Hz, 2H), 8.09 (s,
1H), 7.28 (d, J= 9.0 Hz, 2H), 4.32-4.39 (m, 2H), 2.17(s, 3H), 1.40 (t, J= 7.1 Hz, 3H);
ESI-MS(+): m/z 252.1 [M+H]+.
15
4.1.2. Ethyl 5-nitro-1H-indole-2-carboxylate 3
16
17
18
19
20
21
22
23
Compound 2 (10.0 g, 42.0 mmol) and polyphosphoric acid (110.0 g) were added
into a dry 500 mL round-bottom flask. The reaction mixture was heated to 100°C
under stirring for 2 hours. The reaction solution was cooled to room temperature and
poured into 200 mL of ice water. Then, the mixture was filtered, and the filter cake
was collected and dried to give compound 3 (9.5 g). Green solid, yield: 90.2%;
1H-NMR (600 MHz, DMSO-d6):
δ 12.58 (br s, 1H), 8.71 (d, J= 2.0 Hz, 1H), 8.12 (dd,
J= 2.2, 9.2 Hz, 1H), 7.61 (d, J= 9.2 Hz, 1H), 7.42 (d, J= 1.0 Hz, 1H), 4.37 (q, J= 7.1
Hz, 2H), 1.35 (t, J= 7.2 Hz, 3H); ESI-MS(+): m/z 235.0 [M+H]+.
24
4.1.3. Ethyl 5-amino-1H-indole-2-carboxylate 4
25
26
27
Ethanol (80 mL), water (20 mL), acetic acid (10 mL), reduced iron powder (8.6 g,
154.0 mmol) were added in a dry 250 mL round bottom flask and stirred under 75°C
for half an hour to activate the iron powder. Then, compounds 3 (9.0 g, 39.0 mmol)
25

1
2
was added to the reaction mixture and heated to 75°C under stirring for 2 hours. The
reaction solution was filtered while hot, and the filtrate was removed under vacuum.
The resulting mixture was added with 80 mL of water and 80 mL of ethyl acetate. The
pH of the mixture was adjusted to around 7.5 with sodium bicarbonate solution and
then filtered. The organic layer was separated from the aqueous layer, dried with
anhydrous sodium sulfate (15 g) for 8 h, filtered, and concentrated in vacuo to provide
the crude product, which was purified by silica gel column chromatography
3
4
5
6
7
8
(petroleum ether: ethyl acetate = 3:1, v/v) to afford compound 4 (6.1 g). Yellowish
1
9
solid, yield: 77.7% ; H-NMR (600 MHz, DMSO-d6):
δ
11.38 (br s, 1H), 7.16-7.19
10
11
12
13
(m, 1H), 6.84 (dd, J = 0.8, 2.1 Hz, 1H), 6.69-6.73 (m, 2H), 4.63 (br s, 2H), 4.29 (q,
J=7.1Hz, 2H),1.31 (t, J=7.2 Hz, 3H); 13C-NMR (150 MHz, DMSO-d6):
161.9,142.5,
δ
132.0, 128.2, 127.1, 117.2, 113.2, 106.6, 103.6, 60.6, 14.8; ESI-MS(+): m/z 205.0
[M+H]+.
14
4.1.4. 8-methoxy-2-methylquinolin-4-ol 6
15
16
17
18
19
20
21
22
23
24
2-Methoxyaniline 5 (2.46 g, 20.0 mmol), ethyl acetoacetate (2.60 g, 20.0 mmol),
polyphosphoric acid (70 g) were added to a dry 100 mL three-necked flask,
respectively. The reaction was carried out at 120°C for 5 hours. The mixture was
poured into 100 mL of ice water, stirred until completely dissolved, and cooled to
room temperature. The pH of the mixture was adjusted to around 7.5 with the 30%
sodium hydroxide solution, cooled to room temperature, and filtered. The filter cake
was washed with water and dried to give compound 6 (3.70 g). White solid, yield
1
97.4%; H NMR (600 MHz, DMSO-d6):
δ 11.11 (br s, 1H), 7.60-7.62 (m, 1H), 7.22
(d, J = 4.2 Hz, 1H), 7.21 (s, 1H), 5.95 (s, 1H), 3.97 (s, 3H), 2.37 (s, 3H); ESI-MS(+):
m/z 190.0 [M+H]+.
25
4.1.5. 4-chloro-8-methoxy-2-methylquinoline 7
26
27
28
A mixture of 8-methoxy-2-methylquinolin-4-ol 6 (1.89 g, 10 mol) and
phosphorus oxychloride (20 mL) was added to the drying 100 mL pressure bottle. The
reaction was stirred at 130 for 4 hours. After the completion of the reaction, the
26

1
2
3
4
mixture was cooled to room temperature, removed the solvent under vacuum, added
50 mL of cold water, and decolorized by activated carbon. The filtrate was adjusted
pH to around 8.5 with 10% NaOH under ice bath, filtered to give compound 7 (1.26
g). White solid, yield 60.9%. ESI-MS(+): m/z 208.0 [M+H]+.
5
4.1.6.Ethyl 5-((8-methoxy-2-methylquinolin-4-yl)amino)-1H-indole-2-carboxylate 8
6
7
The mixture of 4-chloro-8-methoxy-2-methylquinoline 7 (41.0 mg, 20.0 mmol)
and intermediates 4 (1.1 eq) in a 25 mL round bottom flask was refluxed in 1-butanol
(8 mL) for 7 hours. The reaction was concentrated under vacuum to remove solvent to
give the crude product, which was purified by silica gel column chromatography
(DCM: MeOH = 20:1, v/v) to afford compound 8 (61 mg). Yellow solid, yield: 81.3%;
ESI-MS(+): m/z 376.1 [M+H]+.
8
9
10
11
12
4.1.7. 5-((8-methoxy-2-methylquinolin-4-yl)amino)-1H-indole-2-carbohydrazide 9
13
14
15
16
17
In a dry 100 mL round bottom flask, compound 8 (3.75 g, 10.0 mmol) and
hydrazine hydrate (20 mL) were added into ethanol (20 mL). The mixture was
refluxed for 10 hours. The reaction mixture was sufficiently cooled and filtered. The
filter cake was washed with 20 mL of ice water and dried to give compound 9 (2.20 g).
White solid, yield 61.1%; ESI-MS (+): m/z 362.5 [M+H]+.
18
4.1.8. General procedures for the synthesis of 10a-z and 10A-I
19
20
21
22
23
24
Compound 9 (72.0 mg, 0.2 mmol) was refluxed with different aldehyde (0.2 mol)
in absolute ethanol (5 mL) containing two drops of trifluoroacetic acid for 8 hours.
Then, the solvent was removed under reduced pressure at 40-45 to give the crude
product, which was purified by column chromatography to produce the corresponding
5-((8-methoxy-2-methylquinolin-4-yl)amino)-1H-indole-2-carbohydrazide derivatives
(10a-z and 10A-I).
25
26
27
4.1.8.1.(E)-N'-(2-fluorobenzylidene)-5-((8-methoxy-2-methylquinolin-4-yl)amino)-1H-
1
indole-2-carbohydrazide (10a). Orange solid (82.0 mg, 87.8%), mp: 228-230 . H
NMR (600 MHz, DMSO-d6):
δ
12.20 (br s, 1H), 12.16 (br s, 1H), 10.72 (s, 1H), 8.73
27

1
2
3
4
5
6
7
(br s, 1H), 8.21 (d, J = 8.4 Hz, 1H), 7.99 (t, J = 7.2 Hz, 1H), 7.81 (br s, 1H), 7.65-7.72
(m, 2H), 7.57 (d, J = 7.9 Hz, 1H), 7.49-7.55 (m, 1H), 7.46 (br s, 1H), 7.31-7.36 (m,
2H), 7.29 (dd, J = 2.0, 8.6 Hz, 1H), 6.57 (s, 1H), 4.12 (s, 3H), 2.59 (s, 3H); 13C NMR
(151 MHz, DMSO-d6):
δ 160.4, 158.4, 158.2, 157.9, 155.6, 154.7, 149.4, 136.4, 131.9,
130.0, 129.9, 128.0, 127.1, 126.8, 125.5, 123.0, 119.8, 117.2, 116.8, 116.6, 114.5,
114.4, 113.2, 104.6, 101.0, 57.2, 20.4; HRMS-ESI(+): m/z 468.1831 [M+H]+, calcd
+
for C27H23FN5O2 468.1830.
8
9
4.1.8.2.(E)-N'-(2-chlorobenzylidene)-5-((8-methoxy-2-methylquinolin-4-yl)amino)-1H
-indole-2-carbohydrazide (10b). Yellowish solid (67.0 mg, 69.4%), mp: 241-243 .
10
11
12
13
14
15
16
17
1H NMR (600 MHz, DMSO-d6):
δ 12.27 (br s, 1H), 12.17 (br s, 1H), 10.71 (s, 1H),
8.89 (br s, 1H), 8.21 (d, J = 8.4 Hz, 1H), 8.07 (d, J = 5.9 Hz, 1H), 7.81 (s, 1H),
7.68-7.72 (m, 1H), 7.66 (d, J = 8.4 Hz, 1H), 7.55-7.59 (m, 2H), 7.45-7.50 (m, 3H),
7.29 (dd, J = 2.0, 8.6 Hz, 1H), 6.57 (s, 1H), 4.12 (s, 3H), 2.58 (s, 3H); 13C NMR (151
MHz, DMSO-d6):
δ 158.3, 158.1, 157.9, 155.6, 154.7, 149.4, 143.7, 136.4, 133.7,
132.0, 130.5, 130.0, 129.9, 128.2, 127.4, 127.2, 123.0, 119.8, 118.9, 117.2, 114.5,
114.4, 113.2, 104.7, 101.1, 57.2, 20.4; HRMS-ESI(+): m/z 484.1536, 486.1507
[M+H]+, calcd for C27H23ClN5O2 484.1535, 486.1506.
+
18
19
20
21
22
23
24
25
26
27
4.1.8.3.(E)-5-((8-methoxy-2-methylquinolin-4-yl)amino)-N'-(2-methoxybenzylidene)-1
H-indole-2-carbohydrazide (10c). Yellowish solid (56.0 mg, 58.5%), mp: 243-245 .
1H NMR (600 MHz, DMSO-d6):
δ 12.11 (br s, 1H), 12.02 (s, 1H), 10.71 (s, 1H), 8.83
(s, 1H), 8.21 (d, J = 8.4 Hz, 1H), 7.91 (d, J = 7.3 Hz, 1H), 7.79 (s, 1H), 7.67-7.73 (m,
1H), 7.65 (d, J = 8.4 Hz, 1H), 7.57 (d, J = 7.9 Hz, 1H), 7.41-7.48 (m, 2H), 7.27 (dd, J
= 1.9, 8.7 Hz, 1H), 7.14 (d, J = 8.3 Hz, 1H), 7.05 (t, J = 7.5 Hz, 1H), 6.56 (s, 1H),
4.12 (s, 3H), 3.90 (s, 3H), 2.58 (s, 3H); 13C NMR (151 MHz, DMSO-d6):
δ 158.3,
158.2, 157.7, 155.6, 154.7, 149.4, 143.3, 136.3, 132.1, 130.0, 129.8, 128.1, 127.2,
126.0, 122.8, 122.7, 121.3, 119.7, 117.2, 114.5, 114.4, 113.2, 112.4, 104.3, 101.0, 57.2,
+
56.2, 20.4; HRMS-ESI(+): m/z 480.2032 [M+H]+, calcd for C28H26N5O3 480.2030.
28
4.1.8.4.(E)-5-((8-methoxy-2-methylquinolin-4-yl)amino)-N'-(3-methylbenzylidene)-1H
28