Bifunctional Naphthoquinone Aromatic Amide-Oxime Derivatives Exert Combined Immunotherapeutic and Antitumor Effects through Simultaneous Targeting of Indoleamine-2,3-dioxygenase and Signal Transducer and Activator of Transcription 3

Article abstract of DOI:10.1021/acs.jmedchem.9b01386

Indoleamine-2,3-dioxygenase 1 (IDO1) and signal transducer and activator of transcription 3 (STAT3) are important targets in the tumor microenvironment for cancer therapy. In the present study, a set of naphthoquinone aromatic amide-oxime derivatives were designed, which stimulated the immune response via IDO1 inhibition and simultaneously displayed powerful antitumor activity against three selected cancer cell lines through suppressing STAT3 signaling. The representative compound 8u bound effectively to IDO1, with greater inhibitory activity relative to the commercial IDO1 inhibitor 4-amino-N-(3-chloro-4-fluorophenyl)-N′-hydroxy-1,2,5-oxadiazole-3-carboximidamide (IDO5L) in addition to the efficient suppression of nuclear translocation of STAT3. Consistently, in vivo assays demonstrated a higher antiproliferative activity of compound 8u in both wild-type B16-F10 isograft tumors and an athymic HepG2 xenograft model relative to 1-methyl-l-tryptophan (1-MT) and doxorubicin (DOX). This bifunctional compound with dual immunotherapeutic and anticancer efficacy may represent a new generation of highly efficacious drug candidates for cancer therapy.

Full text of DOI:10.1021/acs.jmedchem.9b01386

pubs.acs.org/jmc
Article
Bifunctional Naphthoquinone Aromatic Amide-Oxime Derivatives
Exert Combined Immunotherapeutic and Antitumor Effects through
Simultaneous Targeting of Indoleamine-2,3-dioxygenase and Signal
Transducer and Activator of Transcription 3
Rizhen Huang,∥ Xiaoteng Jing,∥ Xiaochao Huang, Yingming Pan, Yilin Fang, Guibin Liang, Zhixin Liao,
Hengshan Wang,* Zhenfeng Chen,* and Ye Zhang*
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ABSTRACT: Indoleamine-2,3-dioxygenase 1 (IDO1) and signal
transducer and activator of transcription 3 (STAT3) are important
targets in the tumor microenvironment for cancer therapy. In the
present study, a set of naphthoquinone aromatic amide-oxime
derivatives were designed, which stimulated the immune response
via IDO1 inhibition and simultaneously displayed powerful
antitumor activity against three selected cancer cell lines through
suppressing STAT3 signaling. The representative compound 8u
bound effectively to IDO1, with greater inhibitory activity relative
to the commercial IDO1 inhibitor 4-amino-N-(3-chloro-4-
fluorophenyl)-N′-hydroxy-1,2,5-oxadiazole-3-carboximidamide
(IDO5L) in addition to the efficient suppression of nuclear
translocation of STAT3. Consistently, in vivo assays demonstrated
a higher antiproliferative activity of compound 8u in both wild-type B16-F10 isograft tumors and an athymic HepG2 xenograft
model relative to 1-methyl-L-tryptophan (1-MT) and doxorubicin (DOX). This bifunctional compound with dual
immunotherapeutic and anticancer efficacy may represent a new generation of highly efficacious drug candidates for cancer therapy.
shrinkage but, more importantly, reactivate the dormant
immune response against malignancies to eliminate residual
cancer cells. Several immune-chemotherapeutic approaches
based on platinum (Pt) drugs incorporating IDO inhibitors
have achieved therapeutic synergy to date.9,10 However, the
clinical application of Pt-based drugs is inevitably limited by
severe toxicity, acute side effects, and inherent or acquired drug
resistance. Accordingly, the design and synthesis of novel
immune-chemotherapeutic monomers have attracted consid-
erable research attention in the field of medicinal chemistry.
Indoleamine-2,3-dioxygenase 1 (IDO1) is a heme-contain-
ing oxidoreductase that catalyzes tryptophan catabolism to
kynurenine through the kynurenine pathway.11 IDO-mediated
rapid consumption of tryptophan results in the suppression of
effector T-cell responses and enhancement of immunosup-
pression-modulated signals by T regulatory cells.12 IDO1-
induced immune tolerance is widely considered as one of the
INTRODUCTION
■
Cancer immunotherapy approaches have been a significant
focus of interest based on the hypothesis that progression of
spontaneous malignancies can be suppressed by the immune
system. Immune checkpoint therapy has been documented as a
breakthrough in cancer immunotherapy, with remarkable
therapeutic effects of anti-PDL1 (programmed death ligand
1), anti-PD1 (programmed cell death protein 1), and anti-
CTLA4 (cytotoxic T-lymphocyte-associated protein 4) agents
reported in multiple clinical trials as an alternative to
conventional chemotherapy following the FDA approval of
ipilumumab, pembrolizumab, and nivolumab.1−3 While
immune checkpoint inhibitors have shown clinical success as
cancer immunotherapy options, only a fraction of patients with
advanced cancers respond effectively to these agents, high-
lighting the continued requirement for improved therapeutic
strategies.4,5 Recent accumulating evidence suggests that
strategies combining immunotherapy and chemotherapy have
enormous potential to enhance clinical benefits due to
simultaneous targeting of different tumor cell survival path-
ways, which could aid in overcoming drug resistance in
advanced-stage cancers.6−8 Indeed, a multipronged immune-
chemotherapeutic approach would not only facilitate tumor
Received: August 22, 2019
Published: January 30, 2020
https://dx.doi.org/10.1021/acs.jmedchem.9b01386
© 2020 American Chemical Society
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Figure 1. Chemical structures of IDO1 and STAT3 inhibitors. (A) Structures of representative IDO1 inhibitors. (B) Naphthoquinone-based IDO1
and tryptophan 2,3-dioxygenase (TDO) inhibitors. (C) Naphthoquinone-based STAT3 inhibitors.
most critical mechanisms evolved by tumors to escape immune
surveillance.13 Furthermore, constitutive overexpression of
IDO1 in a variety of human tumors and host antigen-
presenting cells has been correlated with different tumor
progression parameters and poor prognosis.14−16 Preclinical
studies on mouse tumor models have shown that systemic
blockade of IDO1 activity with small-molecule inhibitors
successfully impairs outgrowth of tumors, delays metastasis
development, prolongs survival, and exerts synergistic ther-
apeutic effects with anticancer drugs to facilitate regression of
tumors that are otherwise recalcitrant to treatments. The
collective findings in the literature validate the utility of IDO1
as a key target for cancer immunotherapy.17,18 Several potent
small-molecule IDO1 inhibitors that effectively stimulate
antitumor immunity have been identified (Figure 1A), but
only two of these molecules, 4-amino-N-(3-chloro-4-fluoro-
phenyl)-N′-hydroxy-1,2,5-oxadiazole-3-carboximidamide
(IDO5L) and NLG919, have entered clinical trials to
date.19−23 A key limitation of checkpoint inhibitors is that
these molecules narrowly focus on modulating the immune
synapse but do not address the critical molecular determinants
primarily responsible for immune dysfunction in the tumor
microenvironment.24−26 Signal transducer and activator of
transcription 3 (STAT3), a point of convergence for numerous
oncogenic signaling pathways and a transcriptional regulator of
diverse tumor-promoting factors, is constitutively activated in
both tumor and immune cells within the tumor microenviron-
ment and therefore presents an attractive therapeutic
target.27,28 STAT3 regulates multiple genes crucial for tumor
cell survival, proliferation, migration, and angiogenesis,29 and
its targeting in tumors with constitutive STAT3 activation has
been shown to directly trigger tumor cell death and growth
inhibition in vivo.30 Additionally, constitutively activated
STAT3 inhibits the expression of mediators necessary for
immune activation and promotes the production of immuno-
suppressive factors that further stimulate STAT3 activity in
diverse immune cell subsets, altering gene expression programs
and thereby suppressing antitumor immune responses. Recent
studies have shown that inhibition of STAT3 signaling in the
hematopoietic system via ablation of STAT3 or treatment with
specific blockers elicits multicomponent antitumor immun-
ity.31 Intriguingly, antitumor effects are more marked when
tumor cells are sensitive to direct killing induced by STAT3
blockade,31 highlighting the enhanced therapeutic value of
approaches targeting both the tumor and its microenviron-
ment. Notably, STAT3 has been shown to maintain the
transcriptional expression of IDO and PD-L1 in human
cancers.26,32,33 In the current investigation, we aimed to
develop a type of novel small inhibitor molecule targeting both
IDO1 and STAT3.
Naphthoquinone structural motifs, such as 1,4-naphthoqui-
none and its derivatives, exhibit multiple pharmacological
activities and have been characterized as enzyme inhib-
itors.34−36 A recent study documented that IDO1 is an
essential target for naphthoquinone menadione displaying
antitumor activity (Figure 1B).37 Some substituted naphtho-
triazolediones were additionally identified as tryptophan 2,3-
dioxygenase (TDO) inhibitors using structure-based virtual
screening.38 On the other hand, 1,4-naphthoquinone-based
compounds are reported to bind directly to STAT3 and
effectively reduce cancer growth in vivo (Figure 1C).39,40 It is,
thus, reasonable to expect that structural modification of the
1,4-naphthoquinone backbone could facilitate both immuno-
modulatory and antitumor efficacies through simultaneous
targeting of IDO1 and STAT3. On the basis of the amide
moiety of Amg-1, which is located at the expanded
hydrophobic pocket,41 the aromatic amide was designed and
drawn into the naphthoquinone skeleton for binding to the
active pocket of IDO1, thus increasing the IDO1 inhibitory
activity and antitumor activity. In addition, as inspired by the
structure of IDO5L, the oxime moiety was also introduced into
the naphthoquinone skeleton for binding to the iron of heme
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Scheme 1. Synthetic Pathway to Target Compounds 7(7a−7z), 8(8a−8z), and 10(10a−10c)
a
Reagents and conditions: (a) phthalic anhydride, CH3COOH, 70 °C, 12 h; (b) oxalyl chloride, CH2Cl2, aromatic primary amines, Et3N, room
temperature (rt), 12 h; (c) hydrazine hydrate, CH3OH, rt, 8 h; (d) DMF, H2O, Et3N, rt, 18 h; (e) hydroxylamine, CH3CH2OH, 80 °C, 12 h; (f)
CH2Cl2, Et3N, 0 °C, 3 h.
a
Table 1. Structures and IDO1 Inhibitory Activities of 2-Amino-1,4-Naphthoquinone Derivatives 7a−7z, 8a−8z, and 10a−10c
compds.
R1
R2
rhIDO1 IC50 (μM)
7b
7c
7d
7e
3-CF3
4-CF3
2-F
3-F
4-F
2-Cl
3-Cl
4-Cl
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
>10
6.78 0.34
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
4.84 0.12
>10
>10
>10
>10
>10
9.60 0.36
>10
compds.
R1
R2
NOH
rhIDO1 IC50 (μM)
7f
7g
7h
7i
7j
7k
7l
7m
7n
7o
7p
7q
7r
7s
7t
7u
7v
7w
7x
7y
7z
10a
10b
10c
6
8a
8b
8c
8d
8e
8f
8g
8h
8i
8j
8k
8l
8m
8n
8o
8p
8q
8r
H
3.54 0.20
0.09 0.01
0.069 0.03
0.63 0.18
0.24 0.08
0.078 0.01
2.09 0.36
0.68 0.12
0.082 0.06
2.53 0.15
0.95 0.11
0.16 0.05
8.19 0.67
0.53 0.09
0.45 0.04
>10
1.51 0.24
3.21 0.13
8.27 0.34
0.074 0.02
0.046 0.01
0.097 0.02
0.25 0.08
2.22 0.14
6.11 0.15
>10
3-CF3
4-CF3
NOH
NOH
NOH
NOH
NOH
NOH
NOH
NOH
NOH
NOH
NOH
NOH
NOH
NOH
NOH
NOH
NOH
NOH
NOH
NOH
NOH
NOH
NOH
NOH
NOH
O
2-Br
3-Br
4-Br
2-F
3-F
4-F
2-Cl
3-Cl
4-Cl
2-Br
3-Br
2-OCH3
3-OCH3
4-OCH3
2-CH3
3-CH3
4-CH3
3,5-(CH3)2
3-Br-4-F
3,5-(OCH3)2
3-F-4-Br
3,5-(F)2
3-F-4-CH3
3-Cl-4-CH3
2-CH3-4-Br
3-CF3
4-Br
2-OCH3
3-OCH3
4-OCH3
2-CH3
3-CH3
4-CH3
3,5-(CH3)2
3-Br-4-F
3,5-(OCH3)2
3-F-4-Br
3,5-(F)2
3-F-4-CH3
3-Cl-4-CH3
2-CH3-4-Br
H
8s
8t
NOSO2Ph
NOSO2Ph
NOSO2Ph
8u
8v
8w
8x
8y
8z
7a
3-Br
3-OCH3
>10
>10
IDO5L
0.073 0.02
a
Data are mean SD values from three independent experiments.
>10
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and expected to act as a donor or acceptor of hydrogen bonds
when combined with STAT3. To our knowledge, the
combination of the aromatic amide portion, oxime group,
and naphthoquinone moiety has not been reported as a dual
inhibitor of IDO1 and STAT3 in the literature. Therefore, a
series of novel 1,4-naphthoquinone aromatic amide-oxime
derivatives co-targeting IDO1 and STAT3 were designed and
synthesized, with a view to developing effective anticancer
agents that exert synergistic effects through inhibition of
STAT3 signaling and simultaneous activation of the antitumor
immune response via IDO1 blockage. Our experimental data
clearly indicated that the most potent bifunctional inhibitor 8u
showed excellent in vivo antitumor efficacy in both wild-type
B16-F10 isograft tumors and athymic nude mice, thereby
presenting a more proficient immunochemotherapeutic agent
against cancers that fail to respond to current immune
checkpoint inhibitors or conventional chemotherapeutic
agents.
IDO5L and was, thus, chosen as the representative compound
for further investigation as described below.
Structure−activity relationship (SAR) studies were further
conducted with the aim of identifying the structural require-
ments for IDO1 inhibition. The importance of a single
substitution at the R1 position of the phenyl ring was initially
investigated. A strong preference for para-substitution was
demonstrated by the eightfold improvement in the IDO1
inhibition activity of p-fluoro 8f compared to that of o-fluoro
8d. Similarly, introduction of a fluorine group at the meta-
position of the phenyl ring (8e) improved the IDO1 inhibitory
activity relative to that of 8d, but the activity was slightly
weaker than that of 8f. Replacement of the fluorine group with
a trifluoromethyl group (8b, 8c) resulted in a twofold greater
inhibition of IDO1 than those of 8e and 8f. After replacement
of the fluorine in compound 8f with a chlorine (8i), bromine
(8l), or methoxy (8o) group at the para-position on the phenyl
ring, the presence of an electron-donating group on the phenyl
ring resulted in a progressive loss of activity. Complete loss of
activity was also achieved by introducing a methyl moiety at
the ortho-position of the phenyl ring, thus generating
compound 8p (IC50 > 10 μM). Regarding the effects of the
electronic properties of the chemical group at R1, the IDO1
inhibitory activity improved in the order methyl < bromine <
chlorine < fluorine < trifluoromethyl, thus highlighting an
important role of the electron-withdrawing group in the
inhibition of IDO1. Variations at R1 caused substantial
differences in inhibitory potency, presumably because of
different affinities to the binding pocket.
In addition, we investigated the influence of a second group
on the phenyl ring. The compound with a second methyl
group at position 2 (8z, IC50 > 50 μM), compared to the 4-Br
derivative (8l, IC50 = 0.16 μM), did not show improved
activity. In contrast, the addition of an m-fluoro substituent to
8l was tolerated and generated 8v, which showed strikingly
improved potency. Compounds 8w−8y had further substitu-
tions at the 3-position of the phenyl ring. Unfortunately, these
compounds displayed poor or retained inhibitory activity
against IDO1. The presence of a bromine atom at the 3-
position generated 8t (IC50 = 0.074 μM), which had a
relatively greater inhibitory activity comparable to that of 8f,
but 12-fold more potent than that of 8k. These results clearly
suggested that the electronic and steric properties at the 3-
position affect the IDO1 inhibitory activity. Intriguingly, the
3,5-dimethoxy substitution pattern produced the most potent
compound, 8u (IC50 = 0.046 μM), which was ∼11-fold more
active than the 3-methoxy derivative 8n (IC50 = 0.53 μM). The
same compound was 179- and 5-fold more active than the 3,5-
RESULTS AND DISCUSSION
■
Chemistry. The general procedures for the synthesis of 2-
amino-1,4-naphthoquinone aromatic amide-oxime derivatives
are described in Scheme 1. First, the reaction of commercially
available phthalic anhydride (1) with L-phenylalanine (2) in
the presence of acetic acid generating compound 3 was
performed as described in the literature.42 For the synthesis of
compound 4, compound 3 was treated with oxalyl chloride to
produce phthalic imide acid chloride, which was substituted
with aromatic primary amines to produce phthalic acid amide.
Compound 4 reacted with hydrazine hydrate in the presence
of ethanol at room temperature, thus generating aromatic
amide derivatives (5). 2-Amino-1,4-naphthoquinone aromatic
amide derivatives (7) were prepared, starting from 1,4-
naphthoquinone (6), which was treated with aromatic amide
derivatives (5) and triethylamine solution in water at room
temperature for 18 h, followed by hydroxylamine hydro-
chloride in ethyl alcohol at 80 °C for 12 h, to obtain the
corresponding 2-amino-1,4-naphthoquinone aromatic amide-
oxime derivatives (8). These derivatives were converted to 2-
amino-1,4-naphthoquinone oxime esters (10) through a
neutral nucleophilic substitution reaction with 4-tert-butylben-
zenesulfonyl chloride in dichloromethane in an ice bath. The
structures of target compounds 7, 8, and 10 were confirmed via
1H NMR, 13C NMR, and high-resolution mass spectrometry
(HRMS) (Supporting Information).
Inhibition of IDO1 Activity. A diverse library of 2-amino-
1,4-naphthoquinone aromatic amide-oxime derivatives was
synthesized and assayed in vitro against activated human IDO1
using a standard enzymatic assay according to previous
reports.43 The potent IDO5L, one of the earliest IDO
inhibitors characterized in the literature, was used as a positive
control.19 IC50 values obtained based on in vitro inhibition
activities of 2-amino-1,4-naphthoquinone aromatic amide-
oxime derivatives are summarized in Table 1.
As shown in Table 1, the newly synthesized 1,4-
naphthoquinone aromatic amide-oxime derivative 8 was
demonstrated to be a potent IDO1 inhibitor with the IC50
value mostly in the nanomolar range. The oxime derivative 8
(except for 8m, 8s, and 8y) was significantly more potent than
the original lead compound 6 and its derivatives 7 and 10.
Notably, compound 8u exhibited the best IDO1 inhibition
activity relative to that of the commercial IDO1 inhibitor
dimethyl (8s, IC50 = 8.27 μM) and 3,5-difluoro (8w, IC50
0.25 μM) derivatives, respectively.
=
SAR studies revealed the importance of the unique oxime
functional group in binding to IDO1. All modifications of the
oxime pharmacophore generated significantly less potent
derivatives (Table 1). Further modification of the carbonyl
group of 7c with an oxime group, thus generating 8c, resulted
in a 98-fold increase in the IDO1 inhibitory activity.
Esterification of the oxime group in 4-tert-butylbenzenesulfonyl
oxime esters (compound 10) revealed the requirement of a
free hydroxyl group for binding IDO1. Compounds 10b and
10c showed no activity. Only the trifluoromethyl substitution
derivative, 10a, showed measurable activity (albeit 106-fold
lower), compared to that of its oxime analogue, 8b, in the
IDO1 assay. This significant loss of activity may be explained
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Figure 2. (A) Proposed binding mode of compound 8u in the active site of IDO1 (PDB: 4PK5). Heme is reported as a cyan stick, IDO1 structure
is shown as a cartoon. (B) Key hydrogen bond interactions of compound 8u in the active site of IDO1. Compound 8u, IDO1 residues, and HEM
are shown as gray stick, yellow stick, and cyan stick, respectively. The red dashed lines indicate the H-bond interaction between 8u and IDO1. (C)
Biophysical binding data supports the interaction of compound 8u with human IDO1 protein. Dose−response curve determined by surface
plasmon resonance (SPR) for the binding of IDO1 with 8u is shown.
by additional esterification of the oxime moiety resulting in a
steric clash with the heme group in IDO1, thus preventing the
formation of the coordinate covalent bond with heme iron.
On the basis of the established SAR, the known binding
affinities of compounds containing the oxime moiety to iron
heme44 and data obtained with IDO1, we surmised that these
IDO1 inhibitors may form a dative bond with heme iron in the
ferrous state through the oxygen of the oxime moiety.
pocket (Figure S1A) and interacted with LEU230, ARG231,
PHE226, PHE227, and ILE354 (Figure S1B). Notably, π−π
interactions between the phenyl ring and side chain of PHE226
were observed, which may be essential for potent IDO1
inhibitory activity, consistent with earlier findings on the role
of PHE226.41 The NH of 8u was situated in the vicinity of 7-
propanoic acid group of the heme ring, where it formed a
strong hydrogen bond. Specifically, the methoxy group in the
R1 position as an acceptor established one hydrogen bond with
GLY236, which could also contribute to the improved potency
of 8u, and adopted a different rotamer, compared to previous
studies.41,45 The tight packing of the molecule within the
IDO1 active site (Figure S1C) clearly explained the lack of
activity of the analogues bearing a methyl substituent at the R1
position as well as the considerable challenges in modifying
oxime features.
UV−Visible Spectroscopy. UV−visible spectroscopy was
performed to further confirm binding of compound 8u with
heme-containing IDO1 protein. The unique UV absorption
properties of porphyrins are useful in heme protein studies.46
Heme is a porphyrin ring with an iron at its center, which
absorbs light at the UV−vis spectrum maximally at a
wavelength of ∼400 nm depending on the oxidation and
coordination states of its iron, characterized by the Soret band.
Absorbance spectra of the heme group are highly sensitive to
changes in the polarity of the heme environment upon ligand/
substrate binding, usually resulting in pronounced changes in
the spectral properties of heme.47 Therefore, changes in UV−
visible spectra induced by IDO1−ligand interactions could be
Molecular Docking. As revealed by SAR studies, 8u
exhibited the most potent inhibitory activity against IDO1. To
understand the mechanisms underlying the improved potency
of 8u at the molecular level, molecular docking studies of 8u in
the active site of IDO1 were performed using SYBYL-X 2.0
software. The docking scores are summarized in Table S1. The
docking model showed that 8u was strongly bound to the Trp-
binding site with its naphthoquinone oxime core almost
perpendicular to the heme plane (Figure 2A). The oxygen of
the oxime moiety coordinated with heme iron in the
catalytically active site, and the quinone part of 8u located
deep into the hydrophobic pocket (Figure 2B), where it was
appropriately positioned to facilitate π−π interactions with the
quinone aromatic core of several aromatic groups (TYR126,
PHE163, and PHE164). Additional lipophilic residues were in
close contact with the quinone group (VAL130, CYS129, and
LEU234). Remarkably, the hydroxyl hydrogen of the oxime
moiety formed additional hydrogen bonds with the porphyrin
ring of heme, which contributed to its inhibitory activity
against IDO1. Furthermore, the phenyl ring of the phenyl-
alanine moiety extended tightly into another hydrophobic
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Figure 3. In vivo anticancer activity of compound 8u. (A) Effects of the indicated treatments of 8u on B16-F10 melanoma tumor xenografts in
C57BL/6 mice. The images of the tumors after administering with compound 8u are also shown. (B) Tumor volumes were detected every 2 days
for 21 days of duration. (C) Inhibition of tumor growth for compound 8u and doxorubicin (DOX) in nude mice bearing HepG2 xenograft model.
(D) Tumor volumes were detected every 3 days. Data represent three individual experiments.
exploited to evaluate binding to IDO1. To obtain a more direct
indication of the binding site of compound 8u, UV−visible
spectra of ferric IDO1 were measured in the presence and
absence of the compound (Figure S2). In the absence of 8u,
the absorption spectrum of ferric IDO1 exhibited a Soret peak
at 403 nm, as described in the literature.46 The Soret peak
shifted to 413 nm in the presence of 8u, indicative of binding
to IDO1 and coordination with the iron of the heme group.23
Surface Plasmon Resonance Assays. To provide
definitive evidence of direct binding to IDO1, a surface
plasmon resonance (SPR)-based binding assay was performed
using a Biacore T200 instrument. This analytic technique
facilitates measurement of the kinetic and thermodynamic
parameters of ligand−protein complex formation and is widely
utilized to investigate enzyme/inhibitor interactions.48 Kinetic
association and dissociation measurements were obtained, and
the binding affinity of compound 8u for IDO1 was determined
with the aid of Biacore evaluation software. Compound 8u
efficiently interacted with the immobilized protein (Figure
2C), as demonstrated by the concentration-dependent
responses for association and dissociation, respectively, and
the clearly discernible exponential curves, indicative of binding
to and dissociation from the IDO1 protein, with an
equilibrium dissociation constant (KD, representing binding
affinity) of 0.08 μM. Notably, the kinetic dissociation constant
(Kd) between compound 8u and IDO1 was 0.0045 1/s,
suggesting that the 8u-IDO1 complexes formed were
extremely stable. Our data provided definitive evidence of
direct binding of derivatives of 2-amino-1,4-naphthoquinone
aromatic amide-oxime derivatives to IDO1.
but rather detected in antigen-presenting cells within tumor-
draining lymph nodes of the host animal.49 B16-F10 cells form
highly aggressive, poorly immunogenic tumors that are
resistant to a variety of immunotherapeutic strategies.
Nevertheless, growth of tumor isografts formed by these cells
can be suppressed significantly by direct inhibitors of
IDO1.17,37 After 21 days, 8u exhibited significantly stronger
tumor inhibition than the other two groups (Figures 3A and
S3A). Comparison of final mean tumor volumes between 8u-
treated and control animals at the 3-week endpoint revealed
marked suppression of tumor growth following 8u treatment of
B16-F10 tumor-challenged mice (Figure 3B) equating to a T/
C ratio of 29.47% (150 mg/kg). Specifically, 8u inhibited
70.53% of the tumor volume, while 1-MT reduced the tumor
volume by 57.21% (Figure 3B). The mice treated with 8u
showed marked reduction in total tumor weight, whereas the
effect on tumor degeneration was smaller in the 1-MT-treated
mice (Figure S3B). The body weights of the mice in each
group increased gradually, confirming the safety of 8u (Figure
S3C). Although 8u was incapable of eliciting tumor regression,
the compound effectively inhibited tumor growth, supporting
its potential as a single agent for tumor therapy.
Evaluation of Antiproliferative Activity. Menadione, an
anticancer agent with a naphthoquinone core, exhibits low
micromolar potency against IDO.50 Based on the finding that
compound 8u was an inhibitor of IDO1, we evaluated whether
8u and other naphthoquinone derivatives exerted antitumor
effects in Hct-116, SKOV3, A549, and HepG2 cancer cells via
the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bro-
mide (MTT) assay. Data are presented in Table 2, showing
potent cytotoxicity of several compounds in all of the cell lines
examined. Notably, compounds 8 exhibited significantly
greater antitumor activity relative to a number of other IDO
inhibitors examined, which displayed weak activity when
administered as single agents. There is growing consensus that
the clinical efficacy of some chemotherapeutic agents addi-
In Vivo Tumor Growth Inhibition. Compounds that
directly interfere with IDO-mediated tryptophan catalysis exert
antitumor effects. To establish whether IDO1 inhibition could
similarly reverse immune tolerance in vivo, we treated C57BL/
6 mice bearing B16-F10 with 8u or 1-methyl-l-tryptophan (1-
MT) for 21 days. IDO is not expressed in B16-F10 tumor cells,
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a
Table 2. Cytotoxic Activity of 2-Amino-1,4-Naphthoquinone Derivatives against Cancer Cell Lines
IC50 (μM)
compds.
R1
R2
NOH
Hct-116
SKOV3
A549
HepG2
8a
8b
8c
8d
8e
8f
8g
8h
8i
8j
8k
8l
8m
8n
8o
8p
8q
8r
H
1.54 0.43
0.13 0.03
0.083 0.02
0.19 0.10
0.89 0.28
0.11 0.05
1.09 0.39
0.56 0.26
0.071 0.05
3.19 0.47
1.22 0.33
0.31 0.07
1.41 0.44
0.66 0.19
0.58 0.28
9.72 0.70
1.62 0.12
3.86 0.53
3.39 0.23
0.097 0.04
0.037 0.01
0.16 0.08
0.78 0.13
2.55 0.41
6.53 0.76
9.89 0.55
>20
1.25 0.14
0.10 0.04
0.055 0.01
0.14 0.06
0.43 0.16
0.098 0.02
1.22 0.31
0.38 0.09
0.067 0.03
2.96 0.36
1.08 0.28
0.24 0.09
1.08 0.33
0.44 0.15
0.41 0.22
8.81 0.78
1.15 0.47
2.75 0.37
2.84 0.49
0.073 0.02
0.028 0.01
0.092 0.06
0.64 0.17
2.06 0.28
4.68 0.42
7.27 0.65
>20
1.47 0.39
0.11 0.09
0.074 0.03
0.16 0.08
0.81 0.21
0.10 0.07
1.18 0.65
0.53 0.17
0.069 0.02
3.05 0.46
1.21 0.23
0.28 0.15
1.23 0.26
0.63 0.11
0.49 0.21
9.08 0.37
1.46 0.36
3.54 0.28
3.17 0.34
0.088 0.03
0.033 0.01
0.12 0.08
0.69 0.29
2.34 0.32
6.50 0.62
9.48 0.41
>20
1.15 0.17
0.07 0.03
0.068 0.05
0.09 0.02
0.73 0.08
0.05 0.01
1.03 0.49
0.28 0.06
0.042 0.01
1.80 0.31
0.10 0.02
0.11 0.05
1.07 0.23
0.06 0.01
0.04 0.01
8.05 0.37
1.23 0.26
1.29 0.28
2.57 0.14
0.056 0.02
0.012 0.01
0.07 0.02
0.38 0.11
1.81 0.22
2.47 0.29
4.78 0.44
>20
3-CF3
4-CF3
NOH
NOH
NOH
NOH
NOH
NOH
NOH
NOH
NOH
NOH
NOH
NOH
NOH
NOH
NOH
NOH
NOH
NOH
NOH
NOH
NOH
NOH
NOH
NOH
NOH
O
2-F
3-F
4-F
2-Cl
3-Cl
4-Cl
2-Br
3-Br
4-Br
2-OCH3
3-OCH3
4-OCH3
2-CH3
3-CH3
4-CH3
3,5-(CH3)2
4-F-3-Br
3,5-(OCH3)2
3-F-4-Br
3,5-(F)2
4-CH3-3-F
3-Cl-4-CH3
2-CH3-4-Br
H
8s
8t
8u
8v
8w
8x
8y
8z
7a
7b
7c
7d
7e
7f
3-CF3
4-CF3
O
O
O
O
>20
17.23 0.38
>20
>20
>20
>20
16.52 0.59
>20
>20
>20
>20
>20
13.26 0.65
>20
>20
>20
18.03 0.51
>20
2-F
3-F
4-F
>20
>20
O
7g
7h
7i
2-Cl
3-Cl
4-Cl
O
O
O
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
7j
2-Br
O
>20
>20
>20
>20
7k
7l
3-Br
4-Br
O
O
>20
>20
>20
>20
>20
>20
>20
>20
7m
7n
7o
7p
7q
7r
2-OCH3
3-OCH3
4-OCH3
2-CH3
3-CH3
4-CH3
3,5-(CH3)2
4-F-3-Br
3,5-(OCH3)2
3-F-4-Br
3,5-(F)2
4-CH3-3-F
3-Cl-4-CH3
2-CH3-4-Br
3-CF3
O
O
O
O
O
O
O
O
O
O
O
O
O
O
>20
>20
>20
>20
>20
>20
>20
>20
15.38 0.76
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
13.69 0.83
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
16.43 0.92
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
11.44 0.24
>20
>20
>20
>20
>20
>20
>20
7s
7t
7u
7v
7w
7x
7y
7z
10a
10b
NOSO2Ph
NOSO2Ph
3-Br
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Table 2. continued
IC50 (μM)
compds.
R1
R2
Hct-116
>20
17.08 0.58
0.27 0.08
SKOV3
>20
16.27 0.62
0.16 0.17
A549
HepG2
>20
>20
0.069 0.02
10c
6
DOX
3-OCH3
NOSO2Ph
>20
>20
0.074 0.05
a
Data are mean SD values from three independent experiments.
Figure 4. Compound 8u targeted STAT3. (A) Western blot analysis of p-STAT3(Y705), p-STAT3(S727), and total STAT3 after treatment of
SKOV3 cells with 8u at the indicated concentrations and for the indicated times. (B) Western blots of STAT1 and STAT5 from whole-cell lysates
from cells were treated with 8u. β-actin antibody was used as reference control. (C) Effects of compound 8u (0.5 μM) on the nuclear translocation
of the p-STAT3 protein induced by cytokine IL-6. After serum-free overnight treatment, SKOV3 cells were pretreated with 8u for 8 h, followed by
IL-6 (50 ng/mL) for 30 min, and then processed for STAT3 nuclear translocation detection by immunofluorescence staining. (D) SPR analysis of
compound 8u with STAT3 protein. The equilibrium dissociation constant (KD) value is 0.53 μM.
tionally depends on their off-target immunomodulatory
effects.51,52 Indeed, the newly synthesized naphthoquinone
derivatives 8 were potent antitumor agents with IC50 values
mostly in the nanomolar range. Among these, 8u was the most
potent depending on the cell line, with IC50 values of 0.037
0.01, 0.028 0.01, 0.033 0.01 μM, and 0.012 0.01 against
Hct-116, SKOV3, A549, and HepG2, respectively. However,
the antitumor activity of compounds 7 that induced moderate
IDO1 inhibition was relatively weak. Based on the collective
kinetic and cell proliferation results, a window between IDO
inhibitory activity and general cytotoxicity of compound 7 was
hypothesized.
Antitumor Potency of 8u in Nude Mice in Vivo. To
validate whether IDO1 might present an alternative target for
compound 8u, a xenograft model was prepared by injecting
nude mice with human liver cancer HepG2 cells, followed by
administration of two doses of 8u once every 2 days. As
depicted in Figures 3C and S4A, intravenous administration of
8u at 10 or 20 mg/kg every 3 days for 21 consecutive days led
to significant suppression of tumor growth (53.4 and 59.0%),
compared to the vehicle group (Figure S4B). The antitumor
effect of 8u was additionally evident from the delayed increase
in xenograft volume, compared to doxorubicin (DOX) (Figure
3D). Notably, 8u was well tolerated and induced no significant
loss of body weight (Figure S4C), indicating that its antitumor
activity does not require T cell involvement. However, we
demonstrated in the previous IDO1 assay that 8u is a potential
IDO1 inhibitor that requires intact T-cell function to suppress
tumor outgrowth in wild-type C57BL/6 mice.17 These findings
suggested that 8u-induced tumor elimination may involve
alternative targets. The unique dual action of 8u was evidently
distinct from that of other known IDO1 inhibitors.
To our knowledge, some IDO1 inhibitors also have potential
in vitro antitumor activity. For example, menadione is a known
anticancer agent that exhibits low micromolar potency against
IDO.50 Moreover, Coluccia et al. reported that IDO1
inhibitors 5 and 21 induce dose-dependent growth inhibition
in the IDO1 expressing cancer cell lines HTC116 and HT29.20
β-lapachone is a naturally occurring 1,2-naphthoquinone-based
compound that has advanced into clinical trials, owing to its
tumor-selective cytotoxic properties. However, recent studies
have shown that IDO1 inhibitory activity is a previously
unrecognized attribute of the clinical candidate β-lapachone.53
The finding that β-lapachone shows IDO1 inhibitory activity
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Figure 5. Comparative antitumor potency of 8u and the combination of 1-MT and stattic in vivo in a B16-F10 tumor-challenged C57BL/6 mice
model. (A) Representative images in B16-F10 bearing mice. (B) Representative photos of the tumors at the end of treatment. (C) Average weight
of the tumors excised at the end of treatment. (D) Changes in tumor volume in different treatment groups measured every 2 days. (E) Changes in
body weight in different treatment groups measured every 2 days.
adds a new dimension distinct from its cytotoxic properties in
its potential utility as an anticancer agent, and a synergistic
benefit may be achieved through its combined cytotoxic and
immunologic effects. Another study has suggested that
pyranonaphthoquinone-based compounds possess dual IDO1
and topoisomerase II inhibitory activity, thus inhibiting cell
growth of human H460 cells.54,55 Compounds that integrate
tumoricidal activity along with IDO1 inhibitory activity may
produce substantially more robust single-agent antitumor
responses than IDO1 inhibitors that do not exert a cytotoxic
effect. Naphthoquinone-based compounds are reported to act
as direct small-molecule inhibitors of signal transducer and
activator of transcription 3 (STAT3).39,56 Recent studies have
identified STAT3 as a modulator of tumor-induced immune
suppression at many levels in the tumor microenvironment.57
Furthermore, PD-L1 expression in cancer cells is mediated
through the STAT3 pathway.58,59 Since STAT3 mediates
processes of carcinogenesis, such as tumor cell proliferation
and survival, angiogenesis, and invasion, the STAT3 pathway
may present a direct link between traditional oncogenesis and
immune suppression. Many studies have reported constitutive
expression of IDO1 and STAT3 in the four tested cancer cell
lines, except for IDO1 in HepG2 cells.12,14,20,60−62 The novel
compound 8u appears to regulate both IDO1 and STAT3 to
disrupt the tumor microenvironment, inducing a potent
synergistic antitumor effect. If so, this bifunctional inhibitor
exerting both immunomodulatory and conventional chemo-
therapy effects via IDO1 inhibition may hold promise for
cancers that respond poorly to conventional chemotherapeu-
tics. The potent immune-chemotherapeutic effects of 8u
prompted us to hypothesize that STAT3 may also be a
potential target of 8u.
activation of STAT3 is known to contribute to malignant
transformation and tumorigenesis, and its activity is ultimately
dependent on the phosphorylation of Y705.39 Treatment of
SKOV3 cells with different concentrations of 8u resulted in a
significant decrease in phosphorylated STAT3 (p-STAT3)
levels (Figure 4A). Specifically, the p-STAT3 (Y705) level was
reduced in a dose-dependent manner while levels of p-STAT3
(S727) were not influenced by 8u and total STAT3 expression
remained unchanged (Figure 4A). We further investigated the
levels of other STAT proteins, including STAT1 and STAT5.
Pretreatment with compound 8u had no effect on the levels of
other pSTAT proteins, as evident from western blot analyses
(Figure 4B). Subsequently, inhibition of STAT3 activation by
8u was evaluated using a dual luciferase assay. We observed a
reduction in luciferase expression in SKOV3 cells treated with
8u but no changes in STAT1 and STAT5 transcriptional
activity (Figure S5).
Mechanistically, STAT3 nuclear translocation was examined
via immunofluorescence staining. To this end, SKOV3 cells
were treated for 6 h with 0.5 μM 8u, followed by stimulation of
STAT3 translocation by adding IL-6 for 30 min. As shown in
Figure 4C, in SKOV3 cells treated with IL-6, intense nuclear
fluorescence was observed, indicative of nuclear translocation
of p-STAT3. Nuclear translocation of STAT3 in 8u-treated
cells was clearly inhibited relative to that in IL-6−stimulated
cells. Based on these results, we suggest that inhibition of
STAT3 phosphorylation by 8u may have impaired its
transcriptional activity by blocking nuclear translocation.
The SPR binding assay was performed to further examine
direct binding between 8u and STAT3. In the BIAcore
sensorgram, resonance curves exhibited strong binding
patterns between the two molecules in a dose-dependent
manner (Figure 4D). The kinetic parameter, dissociation
constant (KD), determined using BIAcore evaluation software,
Compound 8u Targets STAT3. To confirm our theory,
STAT3 expression was detected via western blotting. Aberrant
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was 0.53 μM, indicative of a high binding affinity.63 Our
collective data supported the hypothesis that 8u specifically
binds STAT3 protein with high affinity, thus indicating that
STAT3 was also the potential target of 8u.
least of 95% purity by reverse-phase high-performance liquid
chromatography (RP-HPLC) on ODS column (250 mm × 4.6 mm,
5 μm) with an eluent of methanol/water (80:20, V/V).
General Procedure for the Preparation of Compounds 7
and 8. Compound 3 (1 mmol) was added to dry CH2Cl2 (15 mL)
and stirred at 0 °C. Subsequently, oxalyl chloride (1.5 mmol) was
dripped into the mixture and stirred at room temperature for 6 h.
After the reaction, the solvent and excess oxalyl chloride were
evaporated under reduced pressure. Aromatic primary amines (1
mmol) and triethylamine (0.5 mmol) were added to the mixture and
stirred at room temperature for 0.5 h. After the reaction, the solvent
was evaporated under reduced pressure and the crude product was
purified by chromatography on silica gel eluted with petroleum ether/
ethyl acetate (V/V = 6:1), thus yielding compound 4. Compound 4
(1 mmol) and hydrazine hydrate (3 mmol) were added to ethanol (15
mL), and the mixture was stirred at room temperature for 8 h. After
the reaction was completed, the solvent was evaporated under
reduced pressure, and the crude product was purified with
chromatography on silica gel and eluted with petroleum ether/ethyl
acetate (V/V = 3:1) to obtain compound 5. Compound 5 (2 mmol)
and 1,4-naphthoquinone (3 mmol) were added to the mixture
containing triethylamine (1 mmol) and stirred at room temperature
for 18 h. After the reaction, the solvent was evaporated under reduced
pressure and the crude product was purified by chromatography on
silica gel and eluted with petroleum ether/ethyl acetate (V/V = 4:1),
thus yielding compounds 7a−7z. The structures were confirmed by
1H NMR, 13C NMR, and HR-MS.
2-((1,4-Dioxo-1,4-dihydronaphthalen-2-yl)amino)-N,3-diphenyl-
propanamide (7a). Yield: 70.2%, yellow solid, mp 101.7−103.2 °C.
[α]2D0 = −40 (c 0.1, AcOEt). 1H NMR (400 MHz, DMSO-d6) δ 10.17
(s, 1H, NH), 8.00−7.95 (m, 1H, NH), 7.90 (dd, J = 7.6, 0.7 Hz, 1H,
ArH), 7.79 (td, J = 7.5, 1.2 Hz, 1H, ArH), 7.71 (td, J = 7.5, 1.2 Hz,
1H, ArH), 7.55 (d, J = 7.7 Hz, 2H, ArH), 7.36−7.29 (m, 4H, ArH),
7.26 (t, J = 7.4 Hz, 2H, ArH), 7.19 (d, J = 7.6 Hz, 2H, ArH), 7.08 (t, J
= 7.4 Hz, 1H, ArH), 5.70 (s, 1H, CCH), 4.45 (q, J = 7.0 Hz, 1H,
CH), 3.27 (d, J = 6.8 Hz, 2H, CH2). 13C NMR (101 MHz, DMSO-
d6) δ 181.82, 181.00, 168.55, 147.50, 138.24, 136.92, 134.91, 132.68,
132.46, 130.19, 130.19, 129.24, 128.80, 128.80, 128.28,128.28,
126.68, 125.98, 125.37, 123.91, 119.74, 119.74,100.99, 57.55, 37.19.
HR-MS (m/z) (ESI): calcd for C25H21N2O3 [M + H]+: 397.1547;
found: 397. 1531. Purity: 97.66%.
To further support the favorable in vitro and in vivo data of
8u and to confirm that the 2-amino-1,4-naphthoquinone
derivatives are bifunctional inhibitors, we detected the KD
values of selected compounds toward STAT3 (8b−8f, 8h, 8q,
8s, and 7u) using SPR assays (Table S2). The 3-
trifluoromethyl and 4-trifluoromethyl derivatives 8b and 8c
both effectively bound STAT3 with an IC50 value of 0.99 and
0.64 μM, respectively, and were only 1- to 2-fold less active
than 8u. Compound 7u, in contrast, was 100-fold less active
than 8u, thus indicating that 8u was the best compound,
exhibiting the highest binding affinity toward STAT3 among
these derivatives.
We next evaluated the comparative antitumor efficacy of 8u
and the combination of 1-MT and stattic (STAT3 inhibitor)
using the B16-F10 tumor-bearing C57BL/6 mice model
(Figure 5A,B). The efficacy data showed that stattic at a
dose of 50 mg/kg had a reduced tumor volume and burden
during the treatment period with tumor growth inhibition of
59.3% (Figure 5C,D). Treatment of tumor-bearing mice with
the combination of 1-MT and stattic was slightly more
effective at inhibiting tumor progression compared to 8u alone.
Normal increase of body weight was observed for all mice
(Figure 5E), indicating that 8u efficiently suppressed tumor
growth in mice without obvious side effects.
CONCLUSIONS
■
In summary, we have presented a novel example of organic
compounds as multimodal anticancer agents that perform dual
targeting of IDO1 and STAT3, inducing greater resistance to
immune tolerance of the tumor microenvironment. SAR
analyses facilitated the identification of the main pharmaco-
phore groups of this new class of bifunctional inhibitors,
among which derivative 8u was determined as the most active
compound. Our SPR analysis provided evidence for the strong
binding affinity of 8u toward IDO1 and STAT3. The quinone
oxime core was critical for IDO1 inhibition, and the oxime
oxygen acted as the iron-binding group. Remarkably,
compound 8u reduced tumor growth to a significant extent
in both immunocompetent and nude mice, signifying dual
anticancer and immunomodulatory activity. As elevated
STAT3 is a common denominator of immune dysfunction in
multiple cancer types, our novel bifunctional small-molecule
immune modulator compound 8u provides an effective
cutting-edge immunochemotherapeutic agent for cancers that
fail to respond to currently available immune checkpoint
inhibitors by simultaneously disabling immune checkpoints
and counteracting STAT3-mediated signaling pathways.
2-((1,4-Dioxo-1,4-dihydronaphthalen-2-yl)amino)-3-phenyl-N-
(3-(trifluoromethyl)phenyl)propanamide (7b). Yield: 46.5%, yellow
solid, mp 163.1−164.5 °C. [α]2D0 = −29 (c 0.1, AcOEt). H NMR
1
(400 MHz, DMSO-d6) δ 10.47 (s, 1H, NH), 8.02−7.96 (m, 2H, NH,
ArH), 7.89 (dd, J = 7.6, 1.0 Hz, 1H, ArH), 7.83−7.76 (m, 2H, ArH),
7.71 (dd, J = 7.5, 1.3 Hz, 1H, ArH), 7.57 (t, J = 8.0 Hz, 1H, ArH),
7.44 (d, J = 7.8 Hz, 1H, ArH), 7.34−7.29 (m, 2H, ArH), 7.26 (dd, J =
8.1, 6.7 Hz, 3H, ArH), 7.19 (ddd, J = 7.1, 3.8, 1.3 Hz, 1H, ArH), 5.69
(s, 1H, CCH), 4.47 (dd, J = 14.8, 7.1 Hz, 1H, CH), 3.30 (d, J = 6.9
Hz, 2H, CH2). 13C NMR (101 MHz, DMSO-d6) δ 181.88, 180.99,
169.21, 147.63, 139.03, 136.91, 134.94, 132.67, 132.53, 130.21,
130.14, 129.24, 129.24, 128.32, 128.32, 126.73, 126.01, 125.39,
123.29, 120.28, 120.25, 115.81, 115.77, 101.07, 57.66, 37.01. HR-MS
(m/z) (ESI): calcd for C26H19F3N2O3Na [M + Na]+: 465.1421;
found: 465.1401. Purity: 99.38%.
2-((1,4-Dioxo-1,4-dihydronaphthalen-2-yl)amino)-3-phenyl-N-
EXPERIMENTAL SECTION
(4-(trifluoromethyl)phenyl)propanamide (7c). Yield: 45.8%, yellow
■
1
solid, mp 212.7−213.7 °C. [α]2D0 = −32 (c 0.1, AcOEt). H NMR
General Information. Compound 3 was synthesized according to
the literature.42 All reagents were purchased from commercial sources
and were used without further purification unless otherwise noted.
Melting points were recorded on a WRS-IA apparatus without
correction. Nuclear magnetic resonance (NMR) spectra were
recorded in DMSO-d6 on Bruker Advance (500 or 400 MHz) with
tetramethylsilane (TMS) as an internal standard. HRMS was
measured in FTMS EI or electrospray ionization (ESI) mode, and
the mass analyzer of the HRMS was time-of-flight (TOF). Flash
column chromatography was performed on silica gel (200−300
mesh). All of the compounds submitted for biological studies were at
(400 MHz, DMSO-d6) δ 10.50 (s, 1H, NH), 7.98 (d, J = 6.9 Hz, 1H,
NH), 7.92−7.86 (m, 1H, ArH), 7.80 (ddd, J = 8.6, 6.7, 2.8 Hz, 3H,
ArH), 7.74−7.67 (m, 3H, ArH), 7.35−7.24 (m, 5H, ArH), 7.18 (t, J =
7.1 Hz, 1H, ArH), 5.70 (s, 1H, CCH), 4.49 (q, J = 7.0 Hz, 1H,
CH), 3.30 (d, J = 6.8 Hz, 2H, CH2). 13C NMR (101 MHz, DMSO-
d6) δ 181.84, 180.98, 169.27, 147.57, 141.82, 136.83, 134.92, 132.65,
132.50, 130.19, 129.23, 129.23, 128.30, 128.30, 126.72, 126.16,
126.12, 126.00, 125.37, 123.75, 122.93, 119.61, 119.61, 101.06, 57.65,
37.01. HR-MS (m/z) (ESI): calcd for C26H19F3N2O3Na [M + Na]+:
487.1240; found: 487.1223. Purity: 96.05%.
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2-((1,4-Dioxo-1,4-dihydronaphthalen-2-yl)amino)-N-(2-fluoro-
phenyl)-3-phenylpropanamide (7d). Yield: 70.2%, yellow solid, mp
N-(4-Chlorophenyl)-2-((1,4-dioxo-1,4-dihydronaphthalen-2-yl)-
amino)-3-phenylpropanamide (7i). Yield: 71.3%, yellow solid, mp
182.5−183.9 °C. [α]2D0 = −36 (c 0.1, AcOEt). H NMR (400 MHz,
196.1−197.5 °C. [α]2D0 = −38 (c 0.1, AcOEt). H NMR (400 MHz,
1
1
DMSO-d6) δ 10.06 (s, 1H, NH), 7.97 (d, J = 7.4 Hz, 1H, NH), 7.90
(d, J = 6.9 Hz, 1H, ArH), 7.83−7.76 (m, 2H, ArH), 7.72 (td, J = 7.5,
1.1 Hz, 1H, ArH), 7.34 (d, J = 7.2 Hz, 2H, ArH), 7.27 (dd, J = 13.5,
6.0 Hz, 4H, ArH), 7.22−7.15 (m, 3H, ArH), 5.73 (s, 1H, CCH),
4.63 (dd, J = 14.0, 8.0 Hz, 1H, CH), 3.28 (t, J = 6.2 Hz, 2H, CH2).
13C NMR (101 MHz, DMSO-d6) δ 181.86, 181.03, 169.21, 155.17,
DMSO-d6) δ 10.29 (s, 1H, NH), 7.96 (d, J = 7.5 Hz, 1H, NH), 7.89
(d, J = 6.9 Hz, 1H, ArH), 7.79 (dd, J = 10.8, 4.1 Hz, 1H, ArH), 7.71
(t, J = 7.5 Hz, 1H, ArH), 7.59 (d, J = 8.9 Hz, 2H, ArH), 7.37 (d, J =
8.8 Hz, 2H, ArH), 7.31 (d, J = 7.0 Hz, 2H, ArH), 7.25 (dd, J = 12.7,
5.0 Hz, 3H, ArH), 7.18 (t, J = 7.1 Hz, 1H, ArH), 5.69 (s, 1H, C
CH), 4.44 (q, J = 7.0 Hz, 1H, CH), 3.27 (d, J = 6.8 Hz, 2H, CH2).
13C NMR (101 MHz, DMSO-d6) δ 181.84, 180.98, 168.76, 147.55,
137.20, 136.91, 134.91, 132.66, 132.48, 130.19, 129.24, 129.24,
128.74, 128.74, 128.30, 128.30, 127.54, 126.71, 125.99, 125.38,
121.28, 121.28, 101.01, 57.59, 37.09. HR-MS (m/z) (ESI): calcd for
C25H19ClN2O3Na [M + Na]+: 453.0976; found: 453.0958. Purity:
98.24%.
152.73, 147.59, 136.93, 134.96, 132.67, 132.52, 130.21, 129.30,
129.30, 128.38, 128.28, 126.69, 126.01, 125.39, 124.53, 124.47,
115.75, 115.56, 101.09, 57.17, 37.18. HR-MS (m/z) (ESI): calcd for
C25H20FN2O3 [M + H]+: 415.1452; found: 415.1437. Purity: 98.24%.
2-((1,4-Dioxo-1,4-dihydronaphthalen-2-yl)amino)-N-(3-fluoro-
phenyl)-3-phenylpropanamide (7e). Yield: 61.4%, yellow solid, mp
1
126.9−128.7 °C. [α]2D0 = −38 (c 0.1, AcOEt). H NMR (400 MHz,
N-(2-Bromophenyl)-2-((1,4-dioxo-1,4-dihydronaphthalen-2-yl)-
DMSO-d6) δ 10.42 (s, 1H, NH), 7.97 (d, J = 7.5 Hz, 1H, NH), 7.91−
7.85 (m, 1H, ArH), 7.85−7.77 (m, 1H, ArH), 7.72 (td, J = 7.5, 1.2
Hz, 1H, ArH), 7.54 (dd, J = 11.5, 1.9 Hz, 1H, ArH), 7.38−7.24 (m,
7H, ArH), 7.18 (t, J = 7.1 Hz, 1H, ArH), 6.92 (td, J = 8.4, 2.3 Hz, 1H,
ArH), 5.67 (s, 1H, CCH), 4.44 (dd, J = 14.7, 7.0 Hz, 1H, CH),
3.27 (d, J = 6.8 Hz, 2H, CH2). 13C NMR (101 MHz, DMSO-d6) δ
181.99, 181.08, 169.10, 163.37, 160.97, 147.70, 140.09, 139.99,
136.97, 135.08, 132.72, 132.66, 130.70, 130.28, 129.34, 128.42,
126.83, 126.13, 125.49, 115.51, 110.63, 106.67, 101.08, 57.69, 37.10.
HR-MS (m/z) (ESI): calcd for C25H20FN2O3 [M + H]+: 415.1452;
found: 415.1437. Purity: 96.84%.
amino)-3-phenylpropanamide (7j). Yield: 75.3%, yellow solid, mp
182.4−183.6 °C. [α]2D0 = −55 (c 0.1, AcOEt). H NMR (400 MHz,
1
DMSO-d6) δ 9.85 (s, 1H, NH), 7.98 (dd, J = 7.6, 1.1 Hz, 1H, NH),
7.91 (dd, J = 7.6, 1.1 Hz, 1H, ArH), 7.82 (td, J = 7.5, 1.3 Hz, 1H,
ArH), 7.76−7.71 (m, 1H, ArH), 7.66 (dd, J = 8.0, 1.3 Hz, 1H, ArH),
7.51 (dd, J = 8.0, 1.5 Hz, 1H, ArH), 7.40−7.35 (m, 3H, ArH), 7.32−
7.25 (m, 3H, ArH), 7.23−7.19 (m, 1H, ArH), 7.20−7.14 (m, 1H,
ArH), 5.81 (s, 1H, CCH), 4.60 (dd, J = 14.3, 7.7 Hz, 1H, CH),
3.31 (s, 1H, CH2). 13C NMR (101 MHz, DMSO-d6) δ 181.85,
181.02, 169.08, 147.58, 136.99, 135.45, 134.94, 132.73, 132.66,
132.52, 130.20, 129.30, 129.30, 128.33, 128.33, 128.11, 127.58,
127.23, 126.70, 125.99, 125.38, 118.15, 101.47, 57.28, 37.05. HR-MS
(m/z) (ESI): calcd for C25H20BrN2O3 [M + H]+: 475.0652; found:
475.0632. Purity: 98.13%.
2-((1,4-Dioxo-1,4-dihydronaphthalen-2-yl)amino)-N-(4-fluoro-
phenyl)-3-phenylpropanamide (7f). Yield: 76.6%, yellow solid, mp
192.4−194.2 °C. [α]2D0 = −33 (c 0.1, AcOEt). H NMR (400 MHz,
1
DMSO-d6) δ 10.22 (s, 1H, NH), 7.97 (dd, J = 7.6, 1.1 Hz, 1H, NH),
7.89 (dd, J = 7.6, 1.1 Hz, 1H, ArH), 7.80 (td, J = 7.5, 1.3 Hz, 1H,
ArH), 7.74−7.69 (m, 1H, ArH), 7.59−7.53 (m, 2H, ArH), 7.33−7.22
(m, 5H, ArH), 7.21−7.13 (m, 3H, ArH), 5.69 (s, 1H, CCH), 4.43
(dd, J = 14.8, 7.0 Hz, 1H, CH), 3.27 (d, J = 6.8 Hz, 2H, CH2). 13C
NMR (101 MHz, DMSO-d6) δ 181.83, 181.00, 168.50, 159.57,
157.18, 147.56, 136.96, 134.93, 132.68, 132.49, 130.20, 129.25,
129.25, 128.30, 128.30, 126.69, 126.00, 125.38, 121.64, 121.56,
115.53, 115.31, 100.98, 57.53, 37.13. HR-MS (m/z) (ESI): calcd for
C25H20FN2O3 [M + H]+: 415.1452; found: 415.1436. Purity: 98.52%.
N-(2-Chlorophenyl)-2-((1,4-dioxo-1,4-dihydronaphthalen-2-yl)-
amino)-3-phenylpropanamide (7g). Yield: 71.8%, yellow solid, mp
N-(3-Bromophenyl)-2-((1,4-dioxo-1,4-dihydronaphthalen-2-yl)-
amino)-3-phenylpropanamide (7k). Yield: 66.9%, yellow solid, mp
186.3−187.7 °C. [α]2D0 = −57 (c 0.1, AcOEt). H NMR (400 MHz,
1
DMSO-d6) δ 10.31 (s, 1H, NH), 7.98 (dd, J = 7.6, 1.0 Hz, 1H, NH),
7.90 (dd, J = 8.8, 1.3 Hz, 2H, ArH), 7.81 (td, J = 7.5, 1.4 Hz, 1H,
ArH), 7.72 (td, J = 7.5, 1.4 Hz, 1H, ArH), 7.49 (dt, J = 7.1, 2.2 Hz,
1H, ArH), 7.32−7.30 (m, 2H, ArH), 7.29−7.24 (m, 4H, ArH), 7.19
(ddd, J = 7.1, 3.9, 1.4 Hz, 1H, ArH), 5.68 (s, 1H, CCH), 4.44 (q, J
= 7.0 Hz, 1H, CH), 3.28 (d, J = 6.8 Hz, 2H, CH2). 13C NMR (101
MHz, DMSO-d6) δ 181.84, 180.97, 168.95, 147.58, 139.81, 136.88,
134.93, 132.65, 132.51, 130.84, 130.19, 129.22, 129.22, 128.30,
128.30, 126.70, 126.51, 126.00, 125.38, 122.02, 121.53, 118.45,
101.03, 57.62, 37.00. HR-MS (m/z) (ESI): calcd for C25H20BrN2O3
[M + H]+: 475.0652; found: 475.0632. Purity: 98.78%.
187.9−189.6 °C. [α]2D0 = −42 (c 0.1, AcOEt). H NMR (400 MHz,
1
DMSO-d6) δ 9.88 (s, 1H, NH), 8.00−7.95 (m, 1H, NH), 7.91 (dd, J
= 7.6, 1.0 Hz, 1H, ArH), 7.80 (td, J = 7.5, 1.3 Hz, 1H, ArH), 7.72 (td,
J = 7.5, 1.3 Hz, 1H, ArH), 7.59 (dd, J = 8.0, 1.4 Hz, 1H, ArH), 7.49
(dd, J = 8.0, 1.4 Hz, 1H, ArH), 7.36 (t, J = 5.6 Hz, 2H, ArH), 7.33−
7.26 (m, 4H, ArH), 7.25−7.18 (m, 2H, ArH), 5.81 (s, 1H, CCH),
4.64 (dd, J = 14.1, 7.9 Hz, 1H, CH), 3.34−3.26 (m, 2H, CH2). 13C
NMR (101 MHz, DMSO-d6) δ 181.86, 181.03, 169.19, 147.57,
136.94, 134.92, 134.11, 132.66, 132.50, 130.20, 129.55, 129.32,
129.32, 128.32, 128.32, 127.50, 127.11, 126.96, 126.71, 126.53,
125.98, 125.38, 101.41, 57.25, 37.14. HR-MS (m/z) (ESI): calcd for
C25H20ClN2O3 [M + H]+: 431.1157; found: 431.1142. Purity:
96.43%.
N-(4-Bromophenyl)-2-((1,4-dioxo-1,4-dihydronaphthalen-2-yl)-
amino)-3-phenylpropanamide (7l). Yield: 68.5%, yellow solid, mp
210.8−212.1 °C. [α]2D0 = −46 (c 0.1, AcOEt). H NMR (400 MHz,
1
DMSO-d6) δ 10.27 (s, 1H, NH), 7.98 (dd, J = 7.6, 1.0 Hz, 1H, NH),
7.89 (dt, J = 8.7, 4.4 Hz, 1H, ArH), 7.81 (td, J = 7.5, 1.3 Hz, 1H,
ArH), 7.73 (td, J = 7.5, 1.3 Hz, 1H, ArH), 7.52 (d, J = 2.1 Hz, 3H,
ArH), 7.30 (dd, J = 10.4, 3.6 Hz, 2H, ArH), 7.27−7.16 (m, 3H, ArH),
5.68 (s, 1H, CCH), 4.44 (q, J = 7.0 Hz, 1H, CH), 3.27 (d, J = 6.8
Hz, 2H, CH2). 13C NMR (101 MHz, DMSO-d6) δ 181.82, 180.99,
168.75, 147.57, 137.60, 136.89, 134.94, 132.66, 132.51, 131.65,
131.65, 130.20, 129.23, 129.23, 128.29, 128.29, 126.69, 126.01,
125.38, 121.64, 121.64, 115.56, 100.98, 57.59, 37.05. HR-MS (m/z)
(ESI): calcd for C25H20BrN2O3 [M + H]+: 475.0652; found:
475.0628. Purity: 98.87%.
N-(3-Chlorophenyl)-2-((1,4-dioxo-1,4-dihydronaphthalen-2-yl)-
amino)-3-phenylpropanamide (7h). Yield: 63.3%, yellow solid, mp
193.0−194.2 °C. [α]2D0 = −39 (c 0.1, AcOEt). H NMR (400 MHz,
1
DMSO-d6) δ 10.33 (s, 1H, NH), 7.97 (dd, J = 7.6, 1.0 Hz, 1H, NH),
7.89 (dd, J = 7.6, 1.0 Hz, 1H, ArH), 7.80 (td, J = 7.5, 1.3 Hz, 1H,
ArH), 7.75 (t, J = 2.0 Hz, 1H, ArH), 7.72 (td, J = 7.5, 1.4 Hz, 1H,
ArH), 7.44 (ddd, J = 8.2, 1.9, 1.0 Hz, 1H, ArH), 7.38−7.31 (m, 2H,
ArH), 7.31−7.24 (m, 4H, ArH), 7.21−7.12 (m, 2H, ArH), 5.69 (s,
1H, CCH), 4.44 (q, J = 7.0 Hz, 1H, CH), 3.28 (d, J = 6.8 Hz, 2H,
CH2). 13C NMR (101 MHz, DMSO-d6) δ 181.85, 180.97, 168.99,
147.58, 139.68, 136.88, 134.92, 133.11, 132.65, 132.50, 130.54,
130.20, 129.23, 129.23, 128.30, 128.30, 126.71, 126.00, 125.38,
123.62, 119.17, 118.07, 101.04, 57.63, 37.02. HR-MS (m/z) (ESI):
calcd for C25H20ClN2O3 [M + H]+: 431.1157; found: 431.1143.
Purity: 97.75%.
2-((1,4-Dioxo-1,4-dihydronaphthalen-2-yl)amino)-N-(2-methox-
yphenyl)-3-phenylpropanamide (7m). Yield: 72.9%, yellow solid,
mp 131.8−133.6 °C. [α]2D0 = −52 (c 0.1, AcOEt). H NMR (400
1
MHz, DMSO-d6) δ 9.54 (s, 1H, NH), 7.96 (d, J = 7.0 Hz, 1H, NH),
7.93−7.82 (m, 2H, ArH), 7.79 (td, J = 7.5, 1.0 Hz, 1H, ArH), 7.70
(td, J = 7.5, 1.1 Hz, 1H, ArH), 7.34 (d, J = 7.3 Hz, 2H, ArH), 7.30−
7.17 (m, 4H, ArH), 7.09 (dd, J = 11.3, 4.2 Hz, 1H, ArH), 7.03 (d, J =
7.2 Hz, 1H, ArH), 6.96−6.88 (m, 1H ArH,), 5.79 (s, 1H, CCH),
4.73 (dd, J = 14.0, 8.0 Hz, 1H, CH), 3.87−3.67 (m, 3H, CH3), 3.25
(t, J = 6.1 Hz, 2H, CH2). 13C NMR (101 MHz, DMSO-d6) δ 181.84,
181.07, 168.87, 150.05, 147.47, 137.02, 134.91, 132.68, 132.44,
130.16, 129.31, 129.31, 128.22, 128.22, 126.60, 126.45, 125.96,
1554
https://dx.doi.org/10.1021/acs.jmedchem.9b01386
J. Med. Chem. 2020, 63, 1544−1563

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
125.36, 125.13, 122.37, 120.27, 111.36, 101.26, 57.16, 55.77, 37.31.
HR-MS (m/z) (ESI): calcd for C26H23N2O4 [M + H]+: 427.1652;
found: 427.1638. Purity: 95.15%.
2.23 (s, 3H, CH3). 13C NMR (101 MHz, DMSO-d6) δ 181.79,
180.96, 168.31, 147.46, 136.97, 135.73, 134.84, 132.90, 132.67,
132.39, 130.16, 129.25, 129.25, 129.16, 129.16, 128.27, 128.27,
126.66, 125.94, 125.34, 119.75, 119.75, 100.97, 57.54, 37.25, 20.44.
HR-MS (m/z) (ESI): calcd for C26H22N2O3Na [M + Na]+: 433.1523;
found: 433.1505. Purity: 99.46%.
2-((1,4-Dioxo-1,4-dihydronaphthalen-2-yl)amino)-N-(3-methox-
yphenyl)-3-phenylpropanamide (7n). Yield: 68.9%, yellow solid, mp
115.3∼116.5 °C. [α]2D0 = −50 (c 0.1, AcOEt). H NMR (400 MHz,
1
DMSO-d6) δ 10.17 (s, 1H, NH), 7.96 (dd, J = 7.7, 1.1 Hz, 1H, NH),
7.89 (dd, J = 7.6, 1.0 Hz, 1H, ArH), 7.78 (td, J = 7.5, 1.3 Hz, 1H,
ArH), 7.70 (td, J = 7.5, 1.4 Hz, 1H, ArH), 7.34−7.30 (m, 2H, ArH),
7.29−7.17 (m, 6H ArH,), 7.14−7.10 (m, 1H, ArH), 6.66 (ddd, J =
8.2, 2.5, 0.7 Hz, 1H, ArH), 5.69 (s, 1H, CCH), 4.44 (dd, J = 14.8,
7.1 Hz, 1H, CH), 3.38 (s, 3H, CH3), 3.27 (d, J = 6.8 Hz, 2H, CH2).
13C NMR (101 MHz, DMSO-d6) δ 181.81, 180.98, 168.61, 159.52,
N-(3,5-Dimethylphenyl)-2-((1,4-dioxo-1,4-dihydronaphthalen-2-
yl)amino)-3-phenylpropanamide (7s). Yield: 63.7%, yellow solid,
mp 122.0−123.8 °C. [α]2D0 = −48 (c 0.1, AcOEt). H NMR (400
1
MHz, DMSO-d6) δ 10.03 (s, 1H, NH), 7.96 (dd, J = 7.6, 0.9 Hz, 1H,
NH), 7.89 (dd, J = 7.6, 1.0 Hz, 1H, ArH), 7.79 (td, J = 7.5, 1.2 Hz,
1H, ArH), 7.70 (td, J = 7.5, 1.3 Hz, 1H, ArH), 7.34−7.30 (m, 2H,
ArH), 7.27 (t, J = 7.5 Hz, 2H, ArH), 7.20 (q, J = 7.7 Hz, 4H, ArH),
6.71 (s, 1H, ArH), 5.68 (s, 1H, CCH), 4.42 (dd, J = 14.2, 7.6 Hz,
1H, CH), 3.30−3.17 (m, 2H, CH2), 2.22 (s, 6H, 2CH3). 13C NMR
(101 MHz, DMSO-d6) δ 181.80, 180.99, 168.43, 147.53, 138.14,
137.81, 137.81, 137.01, 134.90, 132.67, 132.45, 130.18, 130.18,
129.24, 129.24, 128.29, 128.29, 126.66, 125.97, 125.37, 125.37,
117.40, 100.97, 57.66, 37.17, 21.02, 21.02. HR-MS (m/z) (ESI):
calcd for C27H25N2O3 [M + H]+: 425.1860; found: 425.1843. Purity:
98.52%.
147.50, 139.44, 136.95, 134.89, 132.66, 132.44, 130.17, 129.63,
129.24, 128.28, 126.67, 125.97, 125.36, 111.90, 109.36, 105.43,
100.98, 57.61, 55.00, 37.16. HR-MS (m/z) (ESI): calcd for
C26H23N2O4 [M + H]+: 427.1652; found: 427.1637. Purity: 98.71%.
2-((1,4-Dioxo-1,4-dihydronaphthalen-2-yl)amino)-N-(4-methox-
yphenyl)-3-phenylpropanamide (7o). Yield: 78.9%, yellow solid, mp
1
149.8−151.7 °C. [α]2D0 = −45 (c 0.1, AcOEt). H NMR (400 MHz,
DMSO-d6) δ 10.06 (s, 1H, NH), 7.94 (dd, J = 7.6, 1.0 Hz, 1H, NH),
7.88 (dd, J = 7.6, 0.9 Hz, 1H, ArH), 7.76 (td, J = 7.5, 1.3 Hz, 1H,
ArH), 7.68 (td, J = 7.5, 1.3 Hz, 1H, ArH), 7.47−7.42 (m, 2H, ArH),
7.30 (dd, J = 12.3, 5.4 Hz, 2H, ArH), 7.26 (t, J = 7.4 Hz, 2H, ArH),
7.21−7.15 (m, 2H, ArH), 6.92−6.86 (m, 2H, ArH), 5.70 (s, 1H, C
CH), 4.42 (q, J = 7.0 Hz, 1H, CH), 3.70 (d, J = 3.7 Hz, 3H, CH3),
3.26 (d, J = 6.7 Hz, 2H, CH2). 13C NMR (101 MHz, DMSO-d6) δ
181.88, 181.03, 168.11, 155.77, 147.51, 137.00, 134.92, 132.73,
132.47, 131.33, 130.21, 129.31, 129.31, 128.33, 128.33, 126.73,
126.01, 125.41, 121.44, 121.44, 113.98, 113.98, 101.00, 57.50, 55.21,
37.31. HR-MS (m/z) (ESI): calcd for C26H23N2O4 [M + H]+:
427.1652; found: 427.1637. Purity: 98.91%.
N-(4-Bromo-3-fluorophenyl)-2-((1,4-dioxo-1,4-dihydronaphtha-
len-2-yl)amino)-3-phenylpropanamide (7t). Yield: 69.9%, yellow
1
solid, mp 242.5−244.2 °C. [α]2D0 = −39 (c 0.1, AcOEt). H NMR
(400 MHz, DMSO-d6) δ 10.45 (s, 1H, NH), 7.96 (dd, J = 7.6, 1.0 Hz,
1H, NH), 7.89 (dd, J = 7.6, 1.0 Hz, 1H, ArH), 7.79 (td, J = 7.5, 1.3
Hz, 1H, ArH), 7.71 (ddd, J = 9.4, 5.8, 1.7 Hz, 2H, ArH), 7.64 (t, J =
8.4 Hz, 1H, ArH), 7.29 (ddd, J = 18.4, 11.7, 4.6 Hz, 6H, ArH), 7.21−
7.15 (m, 1H, ArH), 5.68 (s, 1H, CCH), 4.45 (q, J = 7.0 Hz, 1H,
CH), 3.28 (d, J = 6.8 Hz, 2H, CH2). 13C NMR (101 MHz, DMSO-
d6) δ 181.83, 180.94, 169.15, 159.21, 156.79, 147.57, 139.45, 136.84,
134.90, 133.43, 132.62, 132.48, 130.17, 129.21, 129.21, 128.29,
128.29, 126.71, 125.98, 125.36, 116.99, 116.96, 107.79, 101.05, 57.63,
36.95. HR-MS (m/z) (ESI): calcd for C25H17BrFN2O3 [M-H]−:
491.0412; found: 491.0414. Purity: 97.99%.
2-((1,4-Dioxo-1,4-dihydronaphthalen-2-yl)amino)-3-phenyl-N-
(o-tolyl)propanamide (7p). Yield: 70.5%, yellow solid, mp 119.5−
120.7 °C. [α]2D0 = −61 (c 0.1, AcOEt). 1H NMR (400 MHz, DMSO-
d6) δ 9.54 (s, 1H, NH), 7.98 (d, J = 7.1 Hz, 1H, NH), 7.90 (d, J = 7.0
Hz, 1H, ArH), 7.86−7.78 (m, 2H, ArH), 7.72 (td, J = 7.6, 1.1 Hz, 1H,
ArH), 7.34 (d, J = 7.3 Hz, 2H, ArH), 7.26 (dd, J = 15.8, 8.0 Hz, 3H,
ArH), 7.18 (t, J = 7.2 Hz, 1H, ArH), 7.14−7.07 (m, 1H, ArH), 7.04
(d, J = 7.3 Hz, 1H, ArH), 6.91 (t, J = 7.6 Hz, 1H, ArH), 5.76 (d, J =
9.3 Hz, 1H, CCH), 4.72 (dd, J = 13.9, 8.1 Hz, 1H, CH), 3.81 (d, J
= 4.0 Hz, 3H, CH3), 3.32−3.18 (m, 2H, CH2). 13C NMR (101 MHz,
DMSO-d6) δ 181.85, 181.09, 168.88, 150.05, 147.51, 137.05, 134.97,
132.68, 132.50, 130.18, 129.32, 129.32, 128.23, 128.23, 126.61,
126.42, 126.00, 125.37, 125.15, 122.39, 120.28, 111.37, 101.22, 57.15,
55.78, 37.27. HR-MS (m/z) (ESI): calcd for C26H22N2O3Na [M +
Na]+: 433.1523; found: 433.150. Purity: 99.77%.
2-((1,4-Dioxo-1,4-dihydronaphthalen-2-yl)amino)-3-phenyl-N-
(m-tolyl)propanamide (7q). Yield: 60.2%, yellow solid, mp 147.5−
149.0 °C. [α]2D0 = −59 (c 0.1, AcOEt). 1H NMR (400 MHz, DMSO-
d6) δ 10.12 (s, 1H, NH), 7.98−7.92 (m, 1H, NH), 7.88 (d, J = 7.0 Hz,
1H, ArH), 7.76 (td, J = 7.5, 1.1 Hz, 1H, ArH), 7.67 (td, J = 7.5, 1.1
Hz, 1H, ArH), 7.43−7.36 (m, 2H, ArH), 7.33 (d, J = 7.2 Hz, 2H,
ArH), 7.30−7.16 (m, 5H, ArH), 6.89 (d, J = 7.5 Hz, 1H, ArH), 5.70
(s, 1H, CCH), 4.45 (dd, J = 14.4, 7.3 Hz, 1H, CH), 3.28 (d, J = 6.0
Hz, 2H, CH2), 2.26 (s, 3H, CH3). 13C NMR (101 MHz, DMSO-d6) δ
181.82, 180.99, 168.53, 147.52, 138.22, 138.06, 137.00, 134.87,
132.68, 132.42, 130.17, 129.27, 129.27, 128.66, 128.30, 128.30,
N-(3,5-Dimethoxyphenyl)-2-((1,4-dioxo-1,4-dihydronaphthalen-
2-yl)amino)-3-phenylpropanamide (7u). Yield: 73.0%, yellow solid,
mp 197.0−198.5 °C. [α]2D0 = −43 (c 0.1, AcOEt). H NMR (400
1
MHz, DMSO-d6) δ 10.04 (s, 1H, NH), 7.97 (dd, J = 7.6, 0.9 Hz, 1H,
NH), 7.90 (dd, J = 7.6, 1.0 Hz, 1H, ArH), 7.81 (td, J = 7.5, 1.3 Hz,
1H, ArH), 7.72 (td, J = 7.5, 1.3 Hz, 1H, ArH), 7.29 (dt, J = 14.9, 4.6
Hz, 4H, ArH), 7.23−7.16 (m, 3H, ArH), 7.08 (dd, J = 8.7, 2.3 Hz,
1H, ArH), 6.90 (d, J = 8.8 Hz, 1H, ArH), 5.68 (s, 1H, CCH), 4.41
(dd, J = 14.7, 7.0 Hz, 1H, CH), 3.72 (d, J = 4.7 Hz, 6H, 2CH3), 3.26
(d, J = 6.8 Hz, 2H, CH2). 13C NMR (101 MHz, DMSO-d6) δ 179.89,
169.77, 160.55, 160.55, 145.26, 140.46, 140.12, 137.17, 133.88,
132.95, 129.23, 129.23, 129.01, 128.45, 128.35, 128.35, 126.68,
125.86, 122.35, 97.89, 97.89, 95.82, 91.41, 57.89, 55.16, 55.16, 37.62.
HR-MS (m/z) (ESI): calcd for C27H25N2O5 [M + H]+: 457.1758;
found: 457.1740. Purity: 96.71%.
N-(4-Bromo-3-fluorophenyl)-2-((1,4-dioxo-1,4-dihydronaphtha-
len-2-yl)amino)-3-phenylpropanamide (7v). Yield: 55.3%, yellow
solid, mp 228.9−230.5 °C. [α]2D0 = −36 (c 0.1, AcOEt). H NMR
1
(400 MHz, DMSO-d6) δ 10.47 (s, 1H, NH), 7.96 (dd, J = 7.6, 1.0 Hz,
1H, NH), 7.89 (dd, J = 7.6, 1.0 Hz, 1H, ArH), 7.79 (td, J = 7.5, 1.3
Hz, 1H, ArH), 7.71 (ddd, J = 8.6, 4.9, 1.9 Hz, 2H, ArH), 7.63 (t, J =
8.4 Hz, 1H, ArH), 7.34−7.22 (m, 6H, ArH), 7.21−7.14 (m, 1H,
ArH), 5.68 (s, 1H, CCH), 4.45 (q, J = 7.0 Hz, 1H, CH), 3.28 (d, J
= 6.8 Hz, 2H, CH2). 13C NMR (101 MHz, DMSO-d6) δ 181.84,
180.95, 169.15, 159.21, 156.80, 147.55, 139.35, 136.81, 134.90,
133.42, 132.63, 132.48, 130.17, 129.22, 129.22, 128.30, 128.30,
126.72, 125.98, 125.37, 116.97, 107.54, 101.07, 57.62, 36.98. HR-MS
(m/z) (ESI): calcd for C25H19BrFN2O3 [M + H]+: 493.0558; found:
493.0592. Purity: 96.68%.
126.69, 125.96, 125.37, 124.59, 120.22, 116.88, 101.01, 57.65, 37.21,
+
21.12. HR-MS (m/z) (ESI): calcd for C26H23N2O3 [M + H]
:
411.1703; found: 411.1688. Purity: 98.92%.
2-((1,4-Dioxo-1,4-dihydronaphthalen-2-yl)amino)-3-phenyl-N-
(p-tolyl)propanamide (7r). Yield: 70.5%, yellow solid, mp 97.2−99.1
°C. [α]2D0 = −54 (c 0.1, AcOEt). H NMR (400 MHz, DMSO-d6) δ
1
10.10 (s, 1H, NH), 7.94 (dd, J = 7.6, 0.9 Hz, 1H, NH), 7.88 (dd, J =
7.6, 0.8 Hz, 1H, ArH), 7.76 (td, J = 7.5, 1.3 Hz, 1H, ArH), 7.67 (td, J
= 7.5, 1.3 Hz, 1H, ArH), 7.45 (d, J = 8.4 Hz, 2H, ArH), 7.32 (d, J =
7.1 Hz, 2H, ArH), 7.26 (t, J = 7.5 Hz, 2H, ArH), 7.18 (dt, J = 9.6, 6.5
Hz, 2H, ArH), 7.11 (d, J = 8.3 Hz, 2H, ArH), 5.70 (s, 1H,CCH),
4.44 (dd, J = 14.7, 7.1 Hz, 1H, CH), 3.27 (d, J = 6.8 Hz, 2H, CH2),
N-(3,5-Difluorophenyl)-2-((1,4-dioxo-1,4-dihydronaphthalen-2-
yl)amino)-3-phenylpropanamide (7w). Yield: 65.8%, yellow solid,
mp 171.2−173.5 °C. [α]2D0 = −35 (c 0.1, AcOEt). H NMR (400
1
MHz, DMSO-d6) δ 10.50 (s, 1H, NH), 7.98−7.94 (m, 1H, NH), 7.89
(dd, J = 7.6, 1.0 Hz, 1H, ArH), 7.78 (td, J = 7.5, 1.3 Hz, 1H, ArH),
7.70 (td, J = 7.5, 1.3 Hz, 1H, ArH), 7.33−7.24 (m, 7H, ArH), 7.22−
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https://dx.doi.org/10.1021/acs.jmedchem.9b01386
J. Med. Chem. 2020, 63, 1544−1563

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Article
7.16 (m, 1H, ArH), 6.96−6.89 (m, 1H, ArH), 5.68 (s, 1H, CCH),
4.45 (q, J = 7.0 Hz, 1H, CH), 3.28 (d, J = 6.8 Hz, 2H, CH2). 13C
NMR (101 MHz, DMSO-d6) δ 181.88, 180.95, 169.34, 163.56,
161.14, 147.57, 140.74, 136.80, 134.91, 132.63, 132.50, 130.18,
129.23, 129.23, 128.32, 128.32, 126.74, 125.99, 125.38, 102.69,
102.40, 101.12, 99.05, 57.67, 36.94. HR-MS (m/z) (ESI): calcd for
C25H19F2N2O3 [M + H]+: 433.1358; found: 433.1345. Purity:
98.99%.
MHz, DMSO-d6) δ 180.44, 170.28, 145.80, 140.88, 138.63, 137.39,
134.21, 133.59, 129.67, 129.67, 129.63, 129.38, 129.38, 128.89,
128.79, 128.79, 127.28, 126.36, 124.56, 122.83, 120.28, 120.28, 91.85,
58.27, 38.02. HR-MS (m/z) (ESI): calcd for C25H21N3O3 Na [M +
Na]+: 434.1475; found: 434.1469. Purity: 98.13%.
(Z)-2-((4-(Hydroxyimino)-1-oxo-1,4-dihydronaphthalen-2-yl)-
amino)-3-phenyl-N-(3-(trifluoromethyl)phenyl)propanamide (8b).
Yield: 38.9%, yellow-green solid, mp 122.2−123.5 °C. [α]2D0 = −26
1
(c 0.1, AcOEt). H NMR (400 MHz, DMSO-d6) δ 12.35 (s, 1H,
2-((1,4-Dioxo-1,4-dihydronaphthalen-2-yl)amino)-N-(2-fluoro-4-
OH), 10.54 (s, 1H, NH), 8.16 (d, J = 7.8 Hz, 1H, NH), 8.10−7.98
(m, 2H, ArH), 7.78 (d, J = 8.3 Hz, 1H, ArH), 7.71−7.64 (m, 1H,
ArH), 7.60−7.53 (m, 2H, ArH), 7.43 (d, J = 8.2 Hz, 1H, ArH), 7.35−
7.25 (m, 4H, ArH), 7.20 (t, J = 7.0 Hz, 1H, ArH), 6.60 (s, 1H, C
CH), 6.36 (d, J = 8.1 Hz, 1H, NH), 4.40 (dd, J = 14.4, 7.1 Hz, 1H,
CH), 3.24 (d, J = 6.8 Hz, 2H, CH2). 13C NMR (101 MHz, DMSO-
d6) δ 179.85, 170.32, 145.23, 140.48, 139.19, 137.08, 133.86, 132.95,
130.16, 129.23, 129.23, 129.01, 128.45, 128.37, 128.37, 126.72,
125.85, 125.42, 123.18, 122.34, 120.18, 115.69, 115.65, 91.42, 57.95,
37.50. HR-MS (m/z) (ESI): calcd for C26H20F3N3O3 Na [M + Na]+:
502.1349; found: 502.1341. Purity: 95.55%.
methylphenyl)-3-phenylpropanamide (7x). Yield: 70.5%, yellow
solid, mp 158.2−160.7 °C. [α]2D0 = −41 (c 0.1, AcOEt). H NMR
1
(400 MHz, DMSO-d6) δ 9.95 (s, 1H, NH), 7.96 (dd, J = 7.7, 1.0 Hz,
1H, NH), 7.90 (dd, J = 7.7, 1.0 Hz, 1H, ArH), 7.79 (td, J = 7.5, 1.3
Hz, 1H, ArH), 7.70 (td, J = 7.5, 1.3 Hz, 1H, ArH), 7.60 (t, J = 8.3 Hz,
1H, ArH), 7.39−7.32 (m, 2H, ArH), 7.27 (t, J = 7.5 Hz, 2H, ArH),
7.19 (ddd, J = 7.2, 6.6, 3.4 Hz, 2H, ArH), 7.08 (dd, J = 11.8, 1.1 Hz,
1H, ArH), 6.96 (d, J = 8.1 Hz, 1H, ArH), 5.73 (s, 1H, CCH), 4.60
(dt, J = 14.0, 7.1 Hz, 1H, CH), 3.34−3.20 (m, 2H, CH2), 2.27 (s, 3H,
CH3). 13C NMR (101 MHz, DMSO-d6) δ 181.83, 181.00, 169.03,
155.13, 152.70, 147.51, 136.92, 134.89, 132.67, 132.45, 130.18,
129.29, 129.29, 128.25, 128.25, 126.66, 125.96, 125.36, 124.80,
124.49, 122.55, 122.43, 101.07, 57.11, 37.22, 20.35. HR-MS (m/z)
(ESI): calcd for C26H22FN2O3Na [M + Na]+: 451.1428; found:
451.1410. Purity: 98.63%.
(Z)-2-((4-(Hydroxyimino)-1-oxo-1,4-dihydronaphthalen-2-yl)-
amino)-3-phenyl-N-(4-(trifluoromethyl)phenyl)propanamide (8c).
Yield: 48.2%, yellow-green solid, mp 197.1−198.7 °C. [α]2D0 = −28
1
(c 0.1, AcOEt). H NMR (400 MHz, DMSO-d6) δ 12.33 (s, 1H,
OH), 10.57 (s, 1H, NH), 8.16 (dd, J = 8.0, 0.6 Hz, 1H, ArH), 8.06
(dd, J = 7.9, 1.0 Hz, 1H, ArH), 7.78 (d, J = 8.6 Hz, 2H, ArH), 7.67
(dd, J = 11.2, 5.2 Hz, 3H, ArH), 7.60−7.54 (m, 1H, ArH), 7.35−7.24
(m, 4H ArH,), 7.20 (ddd, J = 6.7, 3.9, 1.7 Hz, 1H, ArH), 6.60 (s, 1H,
CCH), 6.36 (d, J = 8.2 Hz, 1H, NH), 4.43 (q, J = 7.0 Hz, 1H, CH),
3.24 (d, J = 6.7 Hz, 2H, CH2). 13C NMR (101 MHz, DMSO-d6) δ
179.84, 170.34, 145.21, 141.99, 140.42, 136.99, 133.85, 132.93,
129.21, 129.21, 128.99, 128.42, 128.33, 128.33, 126.69, 126.18,
126.14, 125.83, 123.94, 123.62, 122.32, 119.49, 119.49, 91.42, 57.88,
37.46. HR-MS (m/z) (ESI): calcd for C26H21F3N3O3 [M + H]+:
480.1530; found: 480.1521. Purity: 98.05%.
N-(3-Chloro-4-methylphenyl)-2-((1,4-dioxo-1,4-dihydronaphtha-
len-2-yl)amino)-3-phenylpropanamide (7y). Yield: 71.2%, yellow
solid, mp 99.8−101.4 °C. [α]2D0 = −70 (c 0.1, AcOEt). 1H NMR (400
MHz, DMSO-d6) δ 10.22 (s, 1H, NH), 7.97 (dd, J = 7.6, 0.9 Hz, 1H,
NH), 7.89 (dd, J = 7.6, 1.0 Hz, 1H, ArH), 7.80 (td, J = 7.5, 1.3 Hz,
1H, ArH), 7.72 (ddd, J = 8.9, 5.6, 1.3 Hz, 2H, ArH), 7.29 (tdd, J =
16.3, 8.6, 3.7 Hz, 7H, ArH), 7.18 (ddd, J = 7.0, 3.8, 1.4 Hz, 1H, ArH),
5.68 (s, 1H, CCH), 4.43 (q, J = 7.0 Hz, 1H, CH), 3.27 (d, J = 6.8
Hz, 2H, CH2), 2.26 (s, 3H, CH3). 13C NMR (101 MHz, DMSO-d6) δ
181.80, 180.95, 168.70, 147.56, 137.34, 136.91, 134.90, 132.99,
132.64, 132.47, 131.24, 130.58, 130.18, 129.22, 129.22, 128.28,
128.28, 126.67, 125.97, 125.36, 119.66, 118.30, 100.98, 57.57, 37.03,
(Z)-N-(2-Fluorophenyl)-2-((4-(hydroxyimino)-1-oxo-1,4-dihydro-
naphthalen-2-yl)amino)-3-phenylpropanamide (8d). Yield: 55.3%,
yellow-green solid, mp 226.5−228.2 °C. [α]2D0 = −33 (c 0.1, AcOEt).
1H NMR (400 MHz, DMSO-d6) δ 12.38 (s, 1H, OH), 10.09 (s, 1H,
+
18.94. HR-MS (m/z) (ESI): calcd for C26H22ClN2O3 [M + H] :
445.1313; found: 475.1306. Purity: 98.19%.
N-(4-Bromo-2-methylphenyl)-2-((1,4-dioxo-1,4-dihydronaph-
thalen-2-yl)amino)-3-phenylpropanamide (7z). Yield: 69.8%, yel-
NH), 8.16 (d, J = 7.8 Hz, 1H, ArH), 8.04 (d, J = 7.1 Hz, 1H, ArH),
7.77 (td, J = 7.8, 2.9 Hz, 1H, ArH), 7.70−7.65 (m, 1H, ArH), 7.60−
7.53 (m, 1H, ArH), 7.28 (dt, J = 9.5, 4.4 Hz, 5H, ArH), 7.18 (ddd, J =
9.7, 8.9, 3.2 Hz, 3H, ArH), 6.64 (s, 1H, CCH), 6.33 (d, J = 8.3 Hz,
1H, NH), 4.57 (dd, J = 14.6, 7.1 Hz, 1H, CH), 3.21 (d, J = 6.8 Hz,
2H, CH2). 13C NMR (101 MHz, DMSO-d6) δ 180.05, 170.36,
155.27, 152.83, 145.41, 140.57, 137.21, 133.99, 133.11, 129.41,
129.41, 129.16, 128.54, 128.45, 128.45, 126.82, 125.97, 125.50,
124.59, 122.48, 115.87, 115.68, 91.65, 57.42, 37.71. HR-MS (m/z)
(ESI): calcd for C25H20FN3O3 Na [M + Na]+: 452.1381; found:
452.1380. Purity: 99.66%.
low solid, mp 78.5−80.3 °C. [α]2D0 = −85 (c 0.1, AcOEt). H NMR
1
(400 MHz, DMSO-d6) δ 9.66 (s, 1H, NH), 8.18 (d, J = 8.0 Hz, 1H,
NH), 8.05 (d, J = 7.3 Hz, 1H, ArH), 7.73−7.64 (m, 1H, ArH), 7.62−
7.53 (m, 1H, ArH), 7.41 (d, J = 2.0 Hz, 1H, ArH), 7.32 (dt, J = 14.9,
4.9 Hz, 5H, ArH), 7.27−7.19 (m, 2H, ArH), 6.69 (s, 1H, CCH),
6.30 (d, J = 7.9 Hz, 1H, ArH), 4.49 (q, J = 7.2 Hz, 1H, CH), 3.28−
3.19 (m, 2H, CH2), 2.08 (s, 3H, CH3). 13C NMR (101 MHz, DMSO-
d6) δ 179.91, 169.88, 145.28, 140.51, 137.17, 135.14, 134.91, 133.92,
132.94, 132.80, 129.34, 129.34, 129.01, 128.82, 128.48, 128.40,
128.40, 127.06, 126.73, 125.86, 122.38, 117.80, 91.77, 57.44, 37.72,
17.46. HR-MS (m/z) (ESI): calcd for C26H21BrN2O3Na [M + Na]+:
511.0628; found: 511.0607. Purity: 97.39%.
(Z)-N-(3-Fluorophenyl)-2-((4-(hydroxyimino)-1-oxo-1,4-dihydro-
naphthalen-2-yl)amino)-3-phenylpropanamide (8e). Yield: 49.9%,
yellow-green solid, mp 201.8−203.5 °C. [α]2D0 = −30 (c 0.1, AcOEt).
1H NMR (400 MHz, DMSO-d6) δ 12.37 (s, 1H, OH), 10.45 (s, 1H,
General Procedure for the Preparation of Compounds 8. To
a solution of 1,4-naphthoquinone-2-aminoacylation-substituted ani-
lines (1 mmol) in absolute ethanol (25 mL) was added hydroxyl-
amine hydrochloride (1 mmol). The resulting mixture was refluxed at
80 °C for 12 h. Water (15 mL) was added, and the reaction mixture
was extracted with dichloromethane (3 × 20 mL), dried (Na2SO4),
filtered, and concentrated. The residue was purified by flash
chromatography (light petroleum/ethyl acetate, V/V = 4:1) to give
compounds 8 as a yellow-green solid.
NH), 8.19−8.14 (m, 1H, ArH), 8.05 (dd, J = 7.9, 0.8 Hz, 1H, ArH),
7.71−7.63 (m, 1H ArH,), 7.60−7.52 (m, 2H, ArH), 7.36 (dd, J =
11.4, 4.7 Hz, 1H, ArH), 7.33−7.25 (m, 6H, ArH), 7.23−7.16 (m, 1H,
ArH), 6.90 (td, J = 8.2, 1.8 Hz, 1H, ArH), 6.60 (s, 1H, CCH), 6.35
(d, J = 8.2 Hz, 1H, NH), 4.40 (q, J = 7.0 Hz, 1H, CH), 3.22 (d, J =
6.7 Hz, 2H, CH2). 13C NMR (101 MHz, DMSO-d6) δ 179.89,
170.13, 163.36, 160.96, 145.27, 140.48, 140.23, 140.12, 137.11,
133.89, 132.98, 130.61, 129.26, 129.03, 128.47, 128.39, 126.74,
125.89, 122.37, 115.38, 110.42, 106.55, 91.44, 57.90, 37.55. HR-MS
(m/z) (ESI): calcd for C25H20FN3O3 Na [M + Na] +: 452.1381;
found: 452.1377. Purity: 96.24%.
(Z)-2-((4-(Hydroxyimino)-1-oxo-1,4-dihydronaphthalen-2-yl)-
amino)-N,3-diphenylpropanamide (8a). Yield: 50.6%, yellow-green
solid, mp 230.0−231.2 °C. [α]2D0 = −36 (c 0.1, AcOEt). H NMR
1
(400 MHz, DMSO-d6) δ 10.27 (s, 1H, OH), 8.12 (d, J = 7.9 Hz, 1H,
NH), 8.05−7.97 (m, 1H, ArH), 7.70−7.60 (m, 1H, ArH), 7.61−7.52
(m, 1H, ArH), 7.50 (d, J = 7.7 Hz, 2H, ArH), 7.34−7.22 (m, 7H,
ArH), 7.18 (td, J = 5.7, 2.5 Hz, 1H, ArH), 7.06 (t, J = 7.4 Hz, 1H,
ArH), 6.58 (s, 1H, CCH), 6.25 (d, J = 8.0 Hz, 1H, NH), 4.35 (dd,
J = 13.6, 7.8 Hz, 1H, CH), 3.23−3.12 (m, 2H, CH2). 13C NMR (101
(Z)-N-(4-Fluorophenyl)-2-((4-(hydroxyimino)-1-oxo-1,4-dihydro-
naphthalen-2-yl)amino)-3-phenylpropanamide (8f). Yield: 56.6%,
yellow-green solid, mp 181.7−182.9 °C. [α]2D0 = −29 (c 0.1, AcOEt).
1H NMR (400 MHz, DMSO-d6) δ 12.36 (s, 1H, OH), 10.35 (s, 1H,
NH), 8.16 (d, J = 7.8 Hz, 1H, ArH), 8.05 (d, J = 7.1 Hz, 1H, ArH),
1556
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J. Med. Chem. 2020, 63, 1544−1563

Journal of Medicinal Chemistry
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Article
7.74−7.64 (m, 1H, ArH), 7.61−7.51 (m, 4H, ArH), 7.34−7.25 (m,
5H, ArH), 7.17 (dt, J = 17.7, 7.9 Hz, 4H, ArH), 6.61 (s, 1H, CCH),
6.32 (d, J = 5.4 Hz, 1H, NH), 4.39 (d, J = 4.7 Hz, 1H, CH), 3.22 (d, J
= 6.8 Hz, 2H, CH2). 13C NMR (101 MHz, DMSO-d6) δ 179.93,
169.64, 159.56, 157.17, 145.30, 140.48, 137.19, 134.88, 134.85,
133.93, 132.96, 129.29, 129.02, 128.49, 128.38, 126.72, 125.89,
122.39, 121.58, 121.50, 115.56, 115.34, 91.43, 57.79, 37.66. HR-MS
(m/z) (ESI): calcd for C25H20FN3O3 Na [M + Na]+: 452.1381;
found: 452.1381. Purity: 98.42%.
(Z)-N-(2-Chlorophenyl)-2-((4-(hydroxyimino)-1-oxo-1,4-dihydro-
naphthalen-2-yl)amino)-3-phenylpropanamide (8g). Yield: 55.9%,
yellow-green solid, mp 133.7−134.6 °C. [α]2D0 = −37 (c 0.1, AcOEt).
1H NMR (400 MHz, DMSO-d6) δ 12.36 (s, 1H, OH), 9.86 (s, 1H,
NH), 8.17 (d, J = 8.0 Hz, 1H, ArH), 8.05 (dd, J = 7.9, 0.9 Hz, 1H,
ArH), 7.71−7.65 (m, 1H, ArH), 7.62 (dd, J = 8.0, 1.4 Hz, 1H, ArH),
7.59−7.54 (m, 1H, ArH), 7.48 (dd, J = 8.0, 1.3 Hz, 1H, ArH), 7.32
(ddd, J = 9.9, 7.3, 5.4 Hz, 5H, ArH), 7.20 (ddd, J = 9.2, 3.5, 1.5 Hz,
2H, ArH), 6.68 (s, 1H, CCH), 6.39 (d, J = 8.1 Hz, 1H, NH), 4.55
(dd, J = 14.5, 7.1 Hz, 1H, CH), 3.27 (d, J = 6.8 Hz, 2H, CH2). 13C
NMR (101 MHz, DMSO-d6) δ 179.92, 170.18, 145.28, 140.51,
137.21, 134.30, 133.92, 132.97, 129.57, 129.33, 129.33, 129.03,
128.47, 128.39, 128.39, 127.54, 126.96, 126.83, 126.72, 126.29,
125.86, 122.38, 91.89, 57.52, 37.54. HR-MS (m/z) (ESI): calcd for
C25H21ClN3O3 Na [M + H]+: 446.1266; found: 446.1267. Purity:
95.24%.
37.48. HR-MS (m/z) (ESI): calcd for C25H21BrN3O3 [M + H]+:
490.0761; found: 490.0744. Purity: 98.33%.
(Z)-N-(3-Bromophenyl)-2-((4-(hydroxyimino)-1-oxo-1,4-dihydro-
naphthalen-2-yl)amino)-3-phenylpropanamide (8k). Yield: 51.1%,
yellow-green solid, mp 137.1−138.3 °C. [α]2D0 = −46 (c 0.1, AcOEt).
1H NMR (400 MHz, DMSO-d6) δ 12.35 (s, 1H, OH), 10.38 (s, 1H,
NH), 8.16 (d, J = 7.5 Hz, 1H, ArH), 8.05 (dd, J = 7.9, 1.0 Hz, 1H,
ArH), 7.88 (d, J = 1.8 Hz, 1H, ArH), 7.68 (td, J = 7.8, 1.4 Hz, 1H,
ArH), 7.62−7.53 (m, 1H, ArH), 7.47 (dt, J = 7.1, 2.1 Hz, 1H, ArH),
7.34−7.25 (m, 6H, ArH), 7.20 (ddt, J = 8.5, 5.5, 2.9 Hz, 1H, ArH),
6.58 (s, 1H, CCH), 6.33 (d, J = 8.1 Hz, 1H, NH), 4.37 (dd, J =
14.5, 7.1 Hz, 1H, CH), 3.22 (d, J = 6.8 Hz, 2H, CH2). 13C NMR (101
MHz, DMSO-d6) δ 179.88, 170.10, 145.25, 140.47, 140.01, 137.06,
133.87, 132.99, 130.91, 129.24, 129.24, 129.05, 128.45, 128.39,
128.39, 126.74, 126.45, 125.88, 122.36, 121.96, 121.60, 118.41, 91.44,
57.91, 37.51. HR-MS (m/z) (ESI): calcd for C25H21BrN3O3 [M +
H]+: 490.0761; found: 490.0760. Purity: 97.18%.
(Z)-N-(4-Bromophenyl)-2-((4-(hydroxyimino)-1-oxo-1,4-dihydro-
naphthalen-2-yl)amino)-3-phenylpropanamide (8l). Yield: 54.1%,
yellow-green solid, mp 180.9−182.4 °C. [α]2D0 = −42 (c 0.1, AcOEt).
1H NMR (400 MHz, DMSO-d6) δ 12.35 (s, 1H, OH), 10.35 (s, 1H,
NH), 8.16 (d, J = 7.9 Hz, 1H, ArH), 8.09−8.01 (m, 1H, ArH), 7.73−
7.61 (m, 1H, ArH), 7.62−7.47 (m, 5H, ArH), 7.28 (t, J = 7.3 Hz, 4H,
ArH), 7.19 (dt, J = 9.2, 4.2 Hz, 1H, ArH), 6.60 (s, 1H, CCH), 6.33
(d, J = 8.1 Hz, 1H, NH), 4.39 (q, J = 7.0 Hz, 1H, CH), 3.22 (d, J =
6.7 Hz, 2H, CH2). 13C NMR (101 MHz, DMSO-d6) δ 179.91,
169.90, 145.29, 140.46, 137.82, 137.09, 133.90, 132.97, 131.69,
131.69, 129.26, 129.26, 129.03, 128.47, 128.38, 128.38, 126.73,
125.89, 122.37, 121.60, 121.60, 115.49, 91.43, 57.88, 37.59. HR-MS
(m/z) (ESI): calcd for C25H20BrN3O3 Na [M + Na]+: 512.0580;
found: 512.0580. Purity: 96.35%.
(Z)-N-(3-Chlorophenyl)-2-((4-(hydroxyimino)-1-oxo-1,4-dihydro-
naphthalen-2-yl)amino)-3-phenylpropanamide (8h). Yield: 48.6%,
yellow-green solid, mp 160.2−161.9 °C. [α]2D0 = −35 (c 0.1, AcOEt).
1H NMR (400 MHz, DMSO-d6) δ 12.36 (s, 1H, OH), 10.42 (s, 1H,
NH), 8.16 (d, J = 7.9 Hz, 1H, ArH), 8.05 (d, J = 7.6 Hz, 1H, ArH),
7.75 (s, 1H, ArH), 7.67 (dd, J = 11.2, 4.0 Hz, 1H, ArH), 7.57 (t, J =
7.3 Hz, 1H, ArH), 7.43 (d, J = 8.5 Hz, 1H, ArH), 7.37−7.25 (m, 5H,
ArH), 7.19 (t, J = 6.6 Hz, 1H, ArH), 7.13 (dd, J = 7.9, 0.9 Hz, 1H,
ArH), 6.59 (s, 1H, CCH), 6.33 (d, J = 8.1 Hz, 1H, NH), 4.39 (dd,
J = 14.4, 7.0 Hz, 1H, CH), 3.22 (d, J = 6.7 Hz, 2H, CH2). 13C NMR
(101 MHz, DMSO-d6) δ 179.87, 170.11, 145.24, 140.45, 139.87,
137.06, 133.87, 133.14, 132.96, 130.59, 129.24, 129.24, 129.02,
128.45, 128.37, 128.37, 126.72, 125.86, 123.53, 122.35, 119.10,
118.02, 91.44, 57.89, 37.50. HR-MS (m/z) (ESI): calcd for
C25H21ClN3O3 [M + H]+: 446.1266; found: 446.1252. Purity:
95.42%.
(Z)-2-((4-(Hydroxyimino)-1-oxo-1,4-dihydronaphthalen-2-yl)-
amino)-N-(2-methoxyphenyl)-3-phenylpropanamide (8m). Yield:
54.8%, yellow solid, mp 183.3∼184.4 °C. [α]2D0 = −47 (c 0.1, AcOEt).
1H NMR (400 MHz, DMSO-d6) δ 12.35 (s, 1H, OH), 9.49 (s, 1H,
NH), 8.17 (dd, J = 8.0, 0.6 Hz, 1H, ArH), 8.06 (dd, J = 7.9, 1.0 Hz,
1H, ArH), 7.88 (dd, J = 8.0, 1.5 Hz, 1H, ArH), 7.71−7.64 (m, 1H,
ArH), 7.61−7.51 (m, 1H, ArH), 7.34−7.31 (m, 2H, ArH), 7.26 (t, J =
7.4 Hz, 2H, ArH), 7.21−7.16 (m, 1H, ArH), 7.11−7.05 (m, 1H,
ArH), 7.02 (dd, J = 8.2, 1.3 Hz, 1H, ArH), 6.93−6.88 (m, 1H, ArH),
6.65 (s, 1H, CCH), 6.41 (d, J = 8.3 Hz, 1H, NH), 4.63 (dd, J =
14.3, 7.4 Hz, 1H, CH), 3.76 (s, 3H, CH3), 3.21 (d, J = 6.9 Hz, 2H,
CH2). 13C NMR (101 MHz, DMSO-d6) δ 179.92, 169.74, 149.85,
145.27, 140.48, 137.34, 133.91, 132.92, 129.30, 129.30, 128.98,
128.44, 128.25, 128.25, 126.65, 126.56, 125.82, 124.91, 122.35,
122.00, 120.33, 111.38, 91.65, 57.43, 55.80, 37.58. HR-MS (m/z)
(ESI): calcd for C26H24N3O4 [M + H]+: 442.1761; found: 442.1748.
Purity: 98.43%.
(Z)-2-((4-(Hydroxyimino)-1-oxo-1,4-dihydronaphthalen-2-yl)-
amino)-N-(3-methoxyphenyl)-3-phenylpropanamide (8n). Yield:
50.6%, yellow-green solid, mp 115.4−117.2 °C. [α]2D0 = −45 (c 0.1,
AcOEt). 1H NMR (400 MHz, DMSO-d6) δ 12.34 (s, 1H, OH), 10.21
(s, 1H, NH), 8.16 (d, J = 7.5 Hz, 1H, ArH), 8.05 (dd, J = 7.9, 1.0 Hz,
1H, ArH), 7.71−7.65 (m, 1H, ArH), 7.60−7.54 (m, 1H, ArH), 7.32−
7.18 (m, 7H, ArH), 7.12−7.07 (m, 1H, ArH), 6.65 (dd, J = 8.2, 1.9
Hz, 1H, ArH), 6.60 (s, 1H, CCH), 6.30 (d, J = 8.2 Hz, 1H, NH),
4.38 (dd, J = 14.3, 7.3 Hz, 1H, CH), 3.72 (s, 3H, CH3), 3.20 (d, J =
6.8 Hz, 2H, CH2). 13C NMR (101 MHz, DMSO-d6) δ 179.90,
169.71, 159.56, 145.26, 140.45, 139.62, 137.17, 133.88, 132.96,
129.67, 129.24, 129.24, 129.02, 128.45, 128.35, 128.35, 126.68,
125.86, 122.35, 111.89, 109.29, 105.38, 91.40, 57.83, 55.05, 37.63.
HR-MS (m/z) (ESI): calcd for C26H24N3O4 [M + H]+: 442.1761;
found: 442.1760. Purity: 97.69%.
(Z)-N-(4-Chlorophenyl)-2-((4-(hydroxyimino)-1-oxo-1,4-dihydro-
naphthalen-2-yl)amino)-3-phenylpropanamide (8i). Yield: 53.2%,
yellow-green solid, mp 194.5−196.0 °C. [α]2D0 = −33 (c 0.1, AcOEt).
1H NMR (400 MHz, DMSO-d6) δ 12.35 (s, 1H, OH), 10.36 (s, 1H,
NH), 8.17 (d, J = 7.9 Hz, 1H, ArH), 8.05 (dd, J = 7.9, 0.9 Hz, 1H,
ArH), 7.70−7.63 (m, 1H, ArH), 7.61−7.54 (m, 3H, ArH), 7.40−7.34
(m, 2H, ArH), 7.32−7.24 (m, 4H, ArH), 7.19 (dt, J = 9.2, 4.2 Hz, 1H,
ArH), 6.61 (s, 1H, CCH), 6.33 (d, J = 8.1 Hz, 1H, NH), 4.40 (dd,
J = 14.5, 7.0 Hz, 1H, CH), 3.22 (d, J = 6.7 Hz, 2H, CH2). 13C NMR
(101 MHz, DMSO-d6) δ 179.89, 169.85, 145.27, 140.45, 137.39,
137.09, 133.89, 132.92, 129.24, 129.24, 128.98, 128.75, 128.75,
128.46, 128.35, 128.35, 127.43, 126.69, 125.85, 122.35, 121.21,
121.21, 91.43, 57.83, 37.60. HR-MS (m/z) (ESI): calcd for
C25H20ClN3O3 Na [M + Na]+: 468.1085; found: 468.1084. Purity:
6.41%.
(Z)-N-(2-Bromophenyl)-2-((4-(hydroxyimino)-1-oxo-1,4-dihydro-
naphthalen-2-yl)amino)-3-phenylpropanamide (8j). Yield: 57.2%,
yellow-green solid, mp 160.6−162.3 °C. [α]2D0 = −49 (c 0.1, AcOEt).
1H NMR (400 MHz, DMSO-d6) δ 12.30 (s, 1H, OH), 9.75 (s, 1H,
NH), 8.11 (dd, J = 8.0, 0.6 Hz, 1H, ArH), 7.99 (dd, J = 7.9, 1.0 Hz,
1H, ArH), 7.65−7.56 (m, 2H, ArH), 7.50 (ddd, J = 9.6, 7.0, 1.4 Hz,
2H, ArH), 7.32−7.27 (m, 3H, ArH), 7.23 (t, J = 7.5 Hz, 2H, ArH),
7.18−7.12 (m, 1H, ArH), 7.08 (td, J = 7.8, 1.6 Hz, 1H, ArH), 6.61 (s,
1H, CCH), 6.33 (d, J = 8.1 Hz, 1H, NH), 4.44 (dd, J = 13.8, 7.8
Hz, 1H, CH), 3.25−3.19 (m, 2H, CH2). 13C NMR (101 MHz,
DMSO-d6) δ 179.91, 170.10, 145.28, 140.55, 137.27, 135.63, 133.92,
132.98, 132.76, 129.32, 129.32, 129.04, 128.47, 128.42, 128.42,
128.17, 127.45, 126.94, 126.73, 125.86, 122.39, 117.95, 91.96, 57.60,
(Z)-2-((4-(Hydroxyimino)-1-oxo-1,4-dihydronaphthalen-2-yl)-
amino)-N-(4-methoxyphenyl)-3-phenylpropanamide (8o). Yield:
55.7%, yellow-green solid, mp 93.3−94.5 °C. [α]2D0 = −41 (c 0.1,
AcOEt). 1H NMR (400 MHz, DMSO-d6) δ 12.33 (s, 1H, OH), 10.06
(s, 1H, NH), 8.16 (d, J = 7.6 Hz, 1H, ArH), 8.05 (dd, J = 7.9, 1.0 Hz,
1H, ArH), 7.73−7.64 (m, 1H, ArH), 7.62−7.53 (m, 1H, ArH), 7.51−
7.38 (m, 2H, ArH), 7.33−7.26 (m, 4H, ArH), 7.20 (dd, J = 5.8, 2.8
1557
https://dx.doi.org/10.1021/acs.jmedchem.9b01386
J. Med. Chem. 2020, 63, 1544−1563

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Hz, 1H, ArH), 6.97−6.82 (m, 2H, ArH), 6.59 (s, 1H, CCH), 6.28
(d, J = 8.1 Hz, 1H, NH), 4.35 (dd, J = 14.4, 7.0 Hz, 1H, CH), 3.19 (d,
J = 6.7 Hz, 2H, CH2). 13C NMR (101 MHz, DMSO-d6) δ 179.91,
169.12, 155.64, 145.28, 140.44, 137.22, 133.90, 132.95, 131.50,
129.25, 129.25, 129.01, 128.46, 128.34, 128.34, 126.66, 125.86,
122.35, 121.30, 121.30, 113.96, 113.96, 91.35, 57.67, 55.23, 37.69.
HR-MS (m/z) (ESI): calcd for C26H24N3O4 [M + H]+: 442.1761;
found: 442.1761. Purity: 98.51%.
(Z)-2-((4-(Hydroxyimino)-1-oxo-1,4-dihydronaphthalen-2-yl)-
amino)-3-phenyl-N-(o-tolyl)propanamide (8p). Yield: 50.1%, yel-
low-green solid, mp 230.4−231.7 °C. [α]2D0 = −56 (c 0.1, AcOEt). 1H
NMR (400 MHz, DMSO-d6) δ 12.37 (s, 1H, OH), 9.62 (s, 1H, NH),
8.18 (d, J = 7.9 Hz, 1H, ArH), 8.06 (d, J = 7.1 Hz, 1H, ArH), 7.72−
7.66 (m, 1H, ArH), 7.60−7.54 (m, 1H, ArH), 7.38−7.22 (m, 6H,
ArH), 7.20−7.06 (m, 3H, ArH), 6.71 (s, 1H, CCH), 6.32 (d, J =
8.0 Hz, 1H, NH), 4.53−4.43 (m, 1H, CH), 3.28−3.21 (m, 2H, CH2),
2.09 (s, 3H, CH3). 13C NMR (101 MHz, DMSO-d6) δ 179.93,
169.68, 145.28, 140.53, 137.28, 135.68, 133.92, 132.94, 132.19,
130.37, 129.34, 129.34, 129.01, 128.49, 128.39, 128.39, 126.70,
126.00, 125.85, 125.65, 125.28, 122.37, 91.72, 57.46, 37.76, 17.76.
HR-MS (m/z) (ESI): calcd for C26H23N3O3 Na [M + Na]+:
448.1632; found: 448.1628. Purity: 99.28%.
(Z)-2-((4-(Hydroxyimino)-1-oxo-1,4-dihydronaphthalen-2-yl)-
amino)-3-phenyl-N-(m-tolyl)propanamide (8q). Yield: 49.0%, yel-
low-green solid, mp 124.5−125.6 °C. [α]2D0 = −54 (c 0.1, AcOEt). 1H
NMR (400 MHz, DMSO-d6) δ 12.35 (s, 1H, OH), 10.15 (s, 1H,
NH), 8.16 (d, J = 7.6 Hz, 1H, ArH), 8.05 (dd, J = 7.9, 0.9 Hz, 1H,
ArH), 7.73−7.63 (m, 1H, ArH), 7.64−7.48 (m, 1H, ArH), 7.42−7.25
(m, 6H, ArH), 7.24−7.14 (m, 2H, ArH), 6.88 (d, J = 7.5 Hz, 1H,
ArH), 6.60 (s, 1H,CCH), 6.31 (d, J = 8.2 Hz, 1H, NH), 4.38 (dd, J
= 14.5, 7.2 Hz, 1H, CH), 3.20 (d, J = 6.9 Hz, 2H, CH2), 2.26 (s, 3H,
CH3). 13C NMR (101 MHz, DMSO-d6) δ 179.94, 169.64, 145.31,
140.50, 138.42, 138.11, 137.23, 133.92, 133.00, 129.28, 129.28,
129.05, 128.73, 128.48, 128.39, 128.39, 126.71, 125.90, 124.53,
122.39, 120.19, 116.84, 91.41, 57.86, 37.67, 21.20. HR-MS (m/z)
(ESI): calcd for C26H24N3O3 [M + H]+: 426.1812; found: 426.1810.
Purity: 99.23%.
AcOEt). 1H NMR (400 MHz, DMSO-d6) δ 12.35 (s, 1H, OH), 10.53
(s, 1H, NH), 8.16 (d, J = 7.8 Hz, 1H, ArH), 8.05 (d, J = 7.0 Hz, 1H,
ArH), 7.72−7.55 (m, 4H, ArH), 7.28 (d, J = 6.8 Hz, 5H, ArH), 7.20
(dd, J = 5.9, 2.6 Hz, 1H, ArH), 6.58 (s, 1H, CCH), 6.35 (d, J = 8.1
Hz, 1H, NH), 4.38 (q, J = 7.0 Hz, 1H, CH), 3.22 (d, J = 6.7 Hz, 2H,
CH2). 13C NMR (101 MHz, DMSO-d6) δ 179.82, 170.25, 159.24,
156.83, 145.20, 140.42, 139.61, 136.98, 133.83, 133.46, 132.94,
129.21, 129.21, 129.00, 128.42, 128.35, 126.71, 125.84, 122.32,
116.92, 107.68, 101.47, 91.41, 57.90, 37.44. HR-MS (m/z) (ESI):
calcd for C25H19BrFN3O3 Na [M + Na]+: 530.0486; found: 530.0488.
Purity: 99.87%.
(Z)-N-(3,5-Dimethoxyphenyl)-2-((4-(hydroxyimino)-1-oxo-1,4-di-
hydronaphthalen-2-yl) amino)-3-phenylpropanamide (8u). Yield:
50.5%, yellow-green solid, mp 122.5∼123.6 °C. [α]2D0 = −39 (c 0.1,
AcOEt). 1H NMR (400 MHz, DMSO-d6) δ 12.35 (s, 1H, OH), 10.19
(s, 1H, NH), 8.16 (d, J = 7.9 Hz, 1H, ArH), 8.10−8.02 (m, 1H, ArH),
7.72−7.63 (m, 1H, ArH), 7.56 (dd, J = 11.1, 4.0 Hz, 1H, ArH), 7.38−
7.25 (m, 4H, ArH), 7.20 (dt, J = 9.1, 4.2 Hz, 1H, ArH), 6.81 (d, J =
2.2 Hz, 2H, ArH), 6.59 (s, 1H, CCH), 6.30 (d, J = 8.2 Hz, 1H,
NH), 6.24 (t, J = 2.2 Hz, 1H, ArH), 4.37 (dd, J = 14.1, 7.5 Hz, 1H,
CH), 3.71 (s, 6H,CH3, CH3), 3.26−3.14 (m, 2H, CH2). 13C NMR
(101 MHz, DMSO-d6) δ 179.89, 169.77, 160.55, 160.55, 145.26,
140.46, 140.12, 137.17, 133.88, 132.95, 129.23, 129.23, 129.01,
128.45, 128.35, 128.35, 126.68, 125.86, 122.35, 97.89, 97.89, 95.82,
91.41, 57.89, 55.16, 55.16, 37.62. HR-MS (m/z) (ESI): calcd for
C27H26N3O5 [M + H]+: 472.1867; found: 472.1851. Purity: 99.45%.
(Z)-N-(4-Bromo-3-fluorophenyl)-2-((4-(hydroxyimino)-1-oxo-1,4-
dihydronaphthalen-2-yl)amino)-3-phenylpropanamide (8v). Yield:
49.2%, yellow-green solid, mp 122.2∼124.2 °C. [α]2D0 = −33 (c 0.1,
AcOEt). 1H NMR (400 MHz, DMSO-d6) δ 10.47 (s, 1H, OH), 7.99
(dd, J = 7.6, 0.9 Hz, 1H, NH), 7.90 (dd, J = 7.6, 1.0 Hz, 1H, ArH),
7.82 (td, J = 7.5, 1.3 Hz, 1H, ArH), 7.76−7.62 (m, 3H, ArH), 7.36−
7.15 (m, 7H, ArH), 5.67 (s, 1H, CCH), 4.44 (q, J = 7.0 Hz, 1H,
CH), 3.27 (d, J = 6.8 Hz, 2H, CH2). 13C NMR (101 MHz, DMSO-
d6) δ 181.89, 181.00, 169.19, 147.63, 139.47, 139.37, 136.85, 135.01,
133.50, 132.66, 132.59, 130.22, 129.25, 128.34, 126.75, 126.06,
125.42, 117.05, 107.82, 107.55, 101.64, 101.44, 101.06, 57.64, 36.96.
HR-MS (m/z) (ESI): calcd for C25H19BrFN3O3Na [M + Na]+:
530.0486; found: 530.0471. Purity: 99.16%.
(Z)-N-(3,5-Difluorophenyl)-2-((4-(hydroxyimino)-1-oxo-1,4-dihy-
dronaphthalen-2-yl)amino)-3-phenylpropanamide (8w). Yield:
43.5%, yellow-green solid, mp 166.0−167.6 °C. [α]2D0 = −31 (c 0.1,
AcOEt). 1H NMR (400 MHz, DMSO-d6) δ 12.35 (s, 1H, OH), 10.57
(s, 1H, NH), 8.20−8.15 (m, 1H, ArH), 8.05 (dd, J = 7.9, 1.0 Hz, 1H,
ArH), 7.68 (td, J = 7.8, 1.4 Hz, 1H, ArH), 7.61−7.53 (m, 1H, ArH),
7.37−7.26 (m, 6H, ArH), 7.21 (dd, J = 5.9, 2.8 Hz, 1H, ArH), 6.93
(tt, J = 9.3, 2.3 Hz, 1H, ArH), 6.57 (s, 1H, CCH), 6.34 (d, J = 8.1
Hz, 1H, NH), 4.38 (dd, J = 14.5, 7.0 Hz, 1H, CH) 3.23 (d, J = 6.8 Hz,
2H, CH2). 13C NMR (101 MHz, DMSO-d6) δ 179.82, 170.43,
163.72, 161.30, 145.21, 140.87, 140.43, 136.93, 133.83, 132.95,
129.20, 129.20, 129.01, 128.42, 128.36, 128.36, 126.73, 125.84,
122.32, 102.59, 102.30, 98.96, 91.46, 57.95, 37.40. HR-MS (m/z)
(ESI): calcd for C25H19F2N3O3Na [M + Na]+: 470.1287; found:
470.1270. Purity: 98.14%.
(Z)-N-(2-Fluoro-4-methylphenyl)-2-((4-(hydroxyimino)-1-oxo-
1,4-dihydronaphthalen-2-yl)amino)-3-phenylpropanamide (8x).
Yield: 60.2%, yellow-green solid, mp 218.5−220.4 °C. [α]2D0 = −36
(c 0.1, AcOEt). 1H NMR (400 MHz, DMSO-d6) δ 9.96 (s, 1H, NH),
8.16 (dd, J = 8.0, 0.6 Hz, 1H, ArH), 8.05 (dd, J = 7.9, 1.0 Hz, 1H,
ArH), 7.72−7.65 (m, 1H, ArH), 7.58 (dt, J = 8.3, 4.7 Hz, 2H, ArH),
7.30 (dt, J = 14.9, 4.6 Hz, 4H, ArH), 7.23−7.17 (m, 1H, ArH), 7.08
(dd, J = 11.8, 1.1 Hz, 1H, ArH), 6.96 (d, J = 8.2 Hz, 1H, ArH), 6.63
(s, 1H, CCH), 6.32 (d, J = 8.3 Hz, 1H, NH), 4.55 (q, J = 6.9 Hz,
1H, CH), 3.21 (d, J = 6.7 Hz, 2H, CH2), 2.27 (s, 3H, CH3). 13C
NMR (101 MHz, DMSO-d6) δ 179.91, 170.03, 155.08, 152.64,
145.27, 140.44, 137.15, 136.01, 133.91, 132.94, 129.30, 128.99,
128.45, 128.30, 126.65, 125.84, 124.83, 124.80, 124.38, 122.63,
116.05, 115.86, 91.49, 57.24, 37.63, 20.41. HR-MS (m/z) (ESI):
calcd for C26H23FN3O3 [M + H]+: 444.1718; found: 444.1704. Purity:
99.34%.
(Z)-2-((4-(Hydroxyimino)-1-oxo-1,4-dihydronaphthalen-2-yl)-
amino)-3-phenyl-N-(p-tolyl)propanamide (8r). Yield: 50.5%, yellow-
green solid, mp 138.9−140.4 °C. [α]2D0 = −50 (c 0.1, AcOEt). H
1
NMR (400 MHz, DMSO-d6) δ 12.36 (s, 1H, OH), 10.24 (s, 1H,
NH), 8.16 (d, J = 7.6 Hz, 1H, ArH), 8.05 (dd, J = 7.9, 0.9 Hz, 1H,
ArH), 7.70−7.64 (m, 1H, ArH), 7.59−7.53 (m, 1H, ArH), 7.44 (d, J
= 8.4 Hz, 2H, ArH), 7.33−7.23 (m, 5H, ArH), 7.21−7.16 (m, 1H,
ArH), 7.10 (d, J = 8.3 Hz, 2H, ArH), 6.60 (s, 1H, CCH), 6.29 (d, J
= 8.2 Hz, 1H, NH), 4.40 (dd, J = 14.1, 7.4 Hz, 1H, CH), 3.29−3.12
(d, 2H, CH2), 2.24 (s, 3H, CH3). 13C NMR (101 MHz, DMSO-d6) δ
179.90, 169.39, 145.24, 140.41, 137.22, 135.98, 133.91, 132.92,
132.74, 129.26, 129.26, 129.17, 129.17, 128.97, 128.44, 128.31,
128.31, 126.63, 125.84, 122.35, 119.67, 119.67, 91.36, 57.68, 37.67,
20.50. HR-MS (m/z) (ESI): calcd for C26H23N3O3 Na [M + Na]+:
448.1632; found: 448.1619. Purity: 99.08%.
(Z)-N-(3,5-Dimethylphenyl)-2-((4-(hydroxyimino)-1-oxo-1,4-di-
hydronaphthalen-2-yl)amino)-3-phenylpropanamide (8s). Yield:
52.5%, yellow-green solid, mp 131.4−133.2 °C. [α]2D0 = −44 (c 0.1,
AcOEt). 1H NMR (400 MHz, DMSO-d6) δ 12.34 (s, 1H, OH), 10.06
(s, 1H, NH), 8.16 (d, J = 7.6 Hz, 1H, ArH), 8.05 (dd, J = 7.9, 1.0 Hz,
1H, ArH), 7.72−7.64 (m, 1H, ArH), 7.60−7.54 (m, 1H, ArH), 7.31−
7.25 (m, 4H, ArH), 7.21−7.17 (m, 3H, ArH), 6.64 (d, J = 49.4 Hz,
2H, ArH, CCH), 6.30 (d, J = 8.2 Hz, 1H, NH), 4.36 (dd, J = 14.2,
7.4 Hz, 1H, CH), 3.20−3.16 (m, 2H, CH2), 2.22 (s, 6H, 2CH3). 13
C
NMR (101 MHz, DMSO-d6) δ 179.94, 169.58, 145.30, 140.50,
138.35, 137.89, 137.89, 137.24, 133.91, 133.00, 129.27, 129.27,
129.06, 128.47, 128.39, 128.39, 126.71, 125.89, 125.33, 122.39,
117.37, 117.37, 91.40, 57.90, 37.67, 21.11, 21.11. HR-MS (m/z)
(ESI): calcd for C27H25N3O3 Na [M + Na]+: 462.1788; found:
462.1788. Purity: 97.32%.
(Z)-N-(3-Bromo-4-fluorophenyl)-2-((4-(hydroxyimino)-1-oxo-1,4-
dihydronaphthalen-2-yl)amino)-3-phenylpropanamide (8t). Yield:
55.5%, yellow-green solid, mp 170.2−171.6 °C. [α]2D0 = −34 (c 0.1,
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(Z)-N-(3-Chloro-4-methylphenyl)-2-((4-(hydroxyimino)-1-oxo-
1,4-dihydronaphthalen-2-yl)amino)-3-phenylpropanamide (8y).
Yield: 51.2%, yellow-green solid, mp 172.6−174.5 °C. [α]2D0 = −67
131.98, 131.28, 131.17, 130.98, 129.34, 129.34, 129.07, 128.40,
128.40, 128.33, 126.80, 126.66, 126.44, 126.44, 125.36, 124.39,
123.23, 122.04, 121.70, 118.51, 88.20, 57.85, 37.47, 35.09, 30.65,
30.65, 30.65. HR-MS (m/z) (ESI): calcd for C35H32BrN3O5S [M +
Na]+: 708.1138; found: 708.1109. Purity: 99.12%.
1
(c 0.1, AcOEt). H NMR (400 MHz, DMSO-d6) δ 12.28 (s, 1H,
OH), 10.30 (s, 1H, NH), 8.16 (d, J = 7.5 Hz, 1H, ArH), 8.05 (dd, J =
7.9, 1.0 Hz, 1H, ArH), 7.72 (d, J = 2.0 Hz, 1H, ArH), 7.70−7.65 (m,
1H, ArH), 7.60−7.54 (m, 1H, ArH), 7.33 (dd, J = 8.3, 2.1 Hz, 1H,
ArH), 7.30−7.26 (m, 4H, ArH), 7.20 (td, J = 5.7, 2.6 Hz, 1H, ArH),
6.65−6.64 (m, 1H, ArH), 6.58 (s, 1H,CCH), 6.31 (d, J = 8.1 Hz,
1H, NH), 4.36 (dd, J = 14.5, 7.0 Hz, 1H, CH), 3.21 (d, J = 6.7 Hz,
2H, CH2), 2.26 (s, 3H, CH3). 13C NMR (101 MHz, DMSO-d6) δ
179.85, 169.80, 145.22, 140.42, 137.52, 137.06, 133.86, 133.02,
132.94, 131.27, 130.49, 129.21, 129.21, 129.00, 128.43, 128.34,
128.34, 126.69, 125.84, 122.33, 119.59, 118.25, 91.40, 57.81, 37.52,
18.96. HR-MS (m/z) (ESI): calcd for C26H23ClN3O3 [M + H]+:
460.1422; found: 460.1405. Purity: 96.83%.
(Z)-N-(4-Bromo-2-methylphenyl)-2-((4-(hydroxyimino)-1-oxo-
1,4-dihydronaphthalen-2-yl)amino)-3-phenylpropanamide (8z).
Yield: 52.8%, yellow-green solid, mp 234.5−236.0 °C. [α]2D0 = −83
(c 0.1, AcOEt). 1H NMR (400 MHz, DMSO-d6) δ 9.66 (s, 1H, NH),
8.18 (d, J = 8.0 Hz, 1H, ArH), 8.05 (d, J = 7.3 Hz, 1H, ArH), 7.73−
7.63 (m, 1H, ArH), 7.61−7.52 (m, 1H, ArH), 7.41 (d, J = 2.0 Hz, 1H,
ArH), 7.35−7.28 (m, 4H, ArH), 7.27−7.20 (m, 2H, ArH), 6.69 (s,
1H, CCH), 6.30 (d, J = 7.9 Hz, 1H, NH), 4.49 (q, J = 7.2 Hz, 1H,
CH), 3.27−3.17 (m, 2H, CH2), 2.08 (s, 3H, CH3). 13C NMR (101
MHz, DMSO-d6) δ 179.91, 169.88, 145.28, 140.51, 137.17, 135.14,
134.91, 133.92, 132.94, 132.80, 129.34, 129.34, 129.01, 128.82,
128.48, 128.40, 128.40, 127.06, 126.73, 125.86, 122.38, 117.80, 91.77,
57.44, 37.72, 17.46. HR-MS (m/z) (ESI): calcd for C26H23BrN3O3
[M + H]+: 504.0917; found: 504.0903. Purity: 96.23%.
General Procedure for the Preparation of Compounds 10.
Under argon protection, a solution of compound 8b, 8k, or 8n (2
mmol) in dichloromethane (10 mL) was treated with 4-tert-
butylbenzenesulfonyl chloride (3 mmol), and the resulting mixture
was stirred for 30 min under an ice bath. Then, triethylamine (4
mmol) was added and the mixture was stirred for 10 min. The
reaction was quenched with ice water (20 mL), extracted with
dichloromethane (3 × 10 mL), dried (Na2SO4), filtered, and
concentrated. The residue was purified by flash chromatography
(light petroleum/ethyl acetate, V/V = 8:1) to give compounds 10 as a
yellow solid.
(E)-2-((4-((((4-(tert-Butyl)phenyl)sulfonyl)oxy)imino)-1-oxo-1,4-
dihydronaphthalen-2-yl)amino)-3-phenyl-N-(3-(trifluoromethyl)-
phenyl)propanamide (10a). Yield: 80.1%, yellow solid, mp 121.4−
122.6 °C. [α]2D0 = −25 (c 0.1, AcOEt). 1H NMR (400 MHz, DMSO-
d6) δ 10.71 (s, 1H, NH), 8.07 (s, 1H, NH), 7.98 (dd, J = 7.7, 1.3 Hz,
1H, ArH), 7.95−7.92 (m, 1H, ArH), 7.84 (dd, J = 15.3, 8.5 Hz, 3H,
ArH), 7.66−7.53 (m, 5H, ArH), 7.46 (d, J = 7.8 Hz, 1H, ArH), 7.34
(d, J = 7.0 Hz, 2H, ArH), 7.25 (t, J = 7.5 Hz, 2H, ArH), 7.16 (t, J =
7.3 Hz, 2H, ArH), 6.12 (s, 1H, CCH), 4.56 (dd, J = 13.8, 7.7 Hz,
1H, CH), 3.32−3.18 (m, 2H, CH2), 1.22 (d, J = 11.2 Hz, 9H, 3CH3).
13C NMR (101 MHz, DMSO-d6) δ 179.14, 169.46, 157.86, 151.72,
(E)-2-((4-((((4-(tert-Butyl)phenyl)sulfonyl)oxy)imino)-1-oxo-1,4-
dihydronaphthalen-2-yl)amino)-N-(3-methoxyphenyl)-3-phenyl-
propanamide (10c). Yield: 85.5%, yellow solid, mp 107.4−108.9 °C.
[α]2D0 = −18 (c 0.1, AcOEt). 1H NMR (400 MHz, DMSO-d6) δ 10.41
(s, 1H, NH), 7.95 (dd, J = 7.6, 1.4 Hz, 1H, NH), 7.89 (dd, J = 13.0,
5.0 Hz, 3H, ArH), 7.61−7.52 (m, 2H, ArH), 7.36−7.30 (m, 4H,
ArH), 7.26 (dt, J = 7.7, 6.1 Hz, 4H, ArH), 7.17 (d, J = 7.6 Hz, 2H,
ArH), 7.09 (d, J = 8.1 Hz, 1H, ArH), 6.70 (dd, J = 8.2, 1.8 Hz, 1H,
ArH), 6.11 (s, 1H, CCH), 4.54 (dd, J = 14.0, 7.5 Hz, 1H, CH),
3.74 (s, 3H, CH3), 3.30−3.21 (m, 2H, CH2), 1.22 (d, J = 11.7 Hz,
9H, 3CH3). 13C NMR (101 MHz, DMSO-d6) δ 179.15, 168.88,
159.60, 157.85, 151.76, 143.39, 139.53, 137.00, 133.99, 132.00,
131.13, 131.09, 129.73, 129.32, 129.32, 128.96, 128.31, 128.31,
126.69, 126.62, 126.37, 126.37, 125.35, 124.38, 123.15, 111.88,
109.36, 105.39, 88.10, 57.76, 55.01, 34.99, 31.05, 30.56, 30.56, 30.56.
HR-MS (m/z) (ESI): calcd for C36H36N3O6S [M + H]+: 638.2319;
found: 638.2294. Purity: 98.14%.
Enzymatic Assays. IDO1 inhibition assays were carried out
according to the manufacturers’ procedures, as described previously.43
The standard assay mixture (100 μL) contained 50 mM potassium
phosphate buffer (pH = 6.5), 10 mM ascorbate, 10 μM methylene
blue, 100 μg/mL catalase, 1 mM L-tryptophan, and 10 μg/mL of
rhIDO1. The plate was incubated at 37 °C in a dark environment, and
reactions were terminated after 60 min by the addition of 50 μL of
fluorogenic developer solution and further incubated at 45 °C in the
dark for 3 h. The plate was then allowed to cool to room temperature
for at least 1 h. After centrifugation at 1500 g for 5 min at 20 °C, the
fluorescence intensity (Ex/Em = 402/488 nm) was measured with a
Fluoroskan Ascent microplate reader (Infinite M1000 Pro, Tecan US,
Morrisville, NC) in endpoint mode. All determinations were carried
out in triplicate.
Cell Viability Assays. The cell lines Hct-116, HepG2, A549, and
SKOV3 were obtained from the Shanghai Cell Bank at the Chinese
Academy of Sciences. Hct-116, HepG2, and SKOV3 cell lines were
grown on 96-well microtiter plates at a density of 10 × 105 cells/well
in Dulbecco’s modified Eagle’s medium (DMEM) with 10% FBS.
DMEM and FBS were obtained from Gibco-Thermo (BRL Co. Ltd.).
The plates were incubated at 37 °C in a humidified atmosphere of 5%
CO2/95% air overnight. The cells were then exposed to different
concentrations of target compounds and DOX and incubated for
another 48 h. The cells were stained with 10 μL of MTT in an
incubator for approximately 4 h. The medium was discharged and
replaced by 100 mL of dimethyl sulfoxide (DMSO). The O.D. value
was read at 570/630 nm with a spectrophotometer.
Molecular Docking. All docking studies were carried out in
Sybyl-X 2.0 on a Windows workstation. The crystal structure of the
IDO1 proteins was retrieved from the RCSB Protein Data Bank
(IDO1: 4PK5).41 The synthetic analogues 7, 8, and 10 were selected
for the docking studies. The 3D structures of these selected
compounds were first built with Sybyl-X 2.0 sketch, and this was
followed by energy minimization with the MMFF94 force field and
Gasteiger-Marsili charges. We used Powell’s method for optimizing
the geometry with a distance-dependent dielectric constant and a
termination energy gradient of 0.005 kcal/mol. All selected
compounds were automatically docked into the binding pocket of
IDO1 through an empirical scoring function and a patented search
engine in the Surflex docking program. Before the docking process,
the natural ligand was extracted, and the water molecules were
removed from the crystal structure. Subsequently, the protein was
prepared with the Biopolymer module implemented in Sybyl. The
polar hydrogen atoms were added, and other parameters were
established by default to estimate the binding affinity characterized by
the Surflex-Dock scores in the software. The Surflex-Dock total scores,
which were expressed in −log 10 (Kd) units to represent binding
143.46, 139.06, 136.92, 134.02, 131.98, 131.12, 130.19, 129.30,
129.30, 129.00, 128.33, 128.33, 128.28, 128.28, 126.73, 126.32,
126.32, 125.34, 124.34, 123.16, 122.69, 120.30, 115.72, 88.16, 57.85,
37.45, 34.97, 31.04, 30.52, 30.52, 30.52, 26.32. HR-MS (m/z) (ESI):
calcd for C36H33N3O5S [M + H]+: 676.2088; found: 676.2062. Purity:
97.24%.
(E)-N-(3-Bromophenyl)-2-((4-((((4-(tert-butyl)phenyl)sulfonyl)-
oxy)imino)-1-oxo-1,4-dihydronaphthalen-2-yl)amino)-3-phenyl-
propanamide (10b). Yield: 85.2%, yellow solid, mp 121.0−122.6 °C.
[α]2D0 = −22 (c 0.1, AcOEt). 1H NMR (400 MHz, DMSO-d6) δ 10.54
(s, 1H, NH), 8.01 (dd, J = 7.7, 1.3 Hz, 1H, NH), 7.96−7.92 (m, 2H,
ArH), 7.88−7.80 (m, 2H, ArH), 7.69 (td, J = 7.6, 1.5 Hz, 1H, ArH),
7.63 (td, J = 7.6, 1.2 Hz, 1H, ArH), 7.60−7.56 (m, 2H, ArH), 7.49
(dt, J = 7.2, 2.0 Hz, 1H, ArH), 7.35−7.30 (m, 4H, ArH), 7.25 (t, J =
7.5 Hz, 2H, ArH), 7.15 (dd, J = 17.3, 7.7 Hz, 2H, ArH), 6.09 (s, 1H,
CCH), 4.51 (dd, J = 14.0, 7.5 Hz, 1H, CH), 3.32−3.15 (m, 2H,
CH2), 1.23 (s, 9H, 3CH3). 13C NMR (101 MHz, DMSO-d6) δ
179.21, 169.27, 157.98, 151.80, 143.49, 139.86, 136.90, 134.18,
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affinities, were applied to estimate the ligand−receptor interactions of
newly designed molecules.
lysate was subjected to a luciferase assay with a dual-luciferase assay
kit (Promega). STAT luciferase activity was measured with an
EnVision Mutilabel Reader (PerkinElmer). Relative luciferase activity
was calculated after the activity of STAT-dependent firefly luciferase
had been normalized to that of Renilla luciferase.
SPR Experiments. Surface plasmon resonance experiments were
performed with a Biacore T200 instrument at 25 °C. Sensor chips,
buffer stock solutions, and immobilization reagents were from GE
Healthcare. Recombinant human IDO1 was Novoprotein. Other
reagents were obtained from Sigma. For immobilization, PBS was
used as the running buffer. The four flow cells were treated in the
same manner to optimize throughput. In summary, by using a CM5
chip, spots 1 and 2 were activated with the coupling reagents EDC
and NHS for 10 min. IDO1 at a concentration of 20 μg/mL in 10
mM sodium acetate, pH 5, was injected onto the surface for 10 and 5
min at spots 1 and 2, respectively, to generate surfaces with high and
low densities. The immobilization levels ranged from 2302 to 1823
RU at spot 1 and from 948 to 1112 RU at spot 2. The unmodified
spot 3 was used as a reference. For kinetics and affinity measurements,
PBS (containing 0.05% DMSO) was used as the running buffer and
sample dilution buffer. Dose−response relationships were determined
with a twofold sample dilution from 0 to 500 μM and an injection
time of 60 s. For data processing, binding curves were corrected for
variations in DMSO concentration and normalized by molecular
weight. The KD values reported were derived from steady-state
binding responses and therefore correspond to the equilibrium
binding affinities of the compounds.
In Vivo Antitumor Efficacy in Nude Mice. The in vivo
antitumor activity of compound 8u was evaluated in the human
hepatocellular carcinoma cell line HepG2 in BALB/c nude mice. Five-
week-old male BALB/c nude mice were purchased from Shanghai
Ling Chang Biotechnology Co. Ltd. (China), and tumors were
induced through a subcutaneous injection of 1 × 107 cells in 100 μL
of sterile PBS into the dorsal region. The animals were divided into
four groups, starting on the second day. When the tumors reached a
volume of 100−150 mm3 in all mice on day 15, the first group was
injected via the tail vein with an equivalent volume of 5% dextrose as
the vehicle control. The second group was treated with doxorubicin at
a dose of 5 mg/kg body weight once every 3 days for 3 weeks. The
third and fourth groups were treated with complex 8u at doses of 10
mg/kg or 30 mg/kg body weight once every 3 days for 3 weeks,
respectively. All compounds were dissolved in vehicle. Tumor
volumes and body weights were recorded every other day after
drug treatment. All mice were sacrificed after 3 weeks of treatment,
and the tumor volumes were measured with electronic digital calipers
and examined by measurement of the length (A) and width (B) to
calculate the volume (V = AB2/2).
B16F10 melanoma was established in C57BL/6 mice through a
subcutaneous injection of 1 × 107 cultured cells. The animals were
divided into four groups starting on the first day. The four groups
were subsequently administered 5% dextrose, D-1-MT (200 mg/kg),
8u (100 mg/kg), and 8u (150 mg/kg), by tail vein injection once
every 2 days for 3 weeks. Tumor volumes and body weights were
recorded as described above.
Statistical Analysis. All of the results were expressed as mean
SD where applicable. GraphPad Prism 6 software (GraphPad
Software) was used for statistical analysis. All of the results are
expressed as mean S.D.
UV−Visible Spectra. UV−visible scans (200−700 nm) were
performed and recorded on a NanoDrop 2000 spectrophotometer
(Thermo Fisher Scientific, Inc.) with 100 μM IDO1 mixed with 2
mM tested compounds.
Western Blotting. Total cell lysates from cultured SKOV-3 cells
after compound 8u treatment, as described earlier, were obtained by
lysing the cells in ice-cold RIPA buffer with protease and phosphatase
inhibitors. Lysates were stored at −20 °C until use. The protein
concentrations were quantified by the Bradford method (BIO-RAD)
with a Multimode Varioscan instrument (Thermo Fisher Scientific).
Equal amounts of protein per lane were applied to a 12% sodium
dodecyl sulfate polyacrylamide gel for electrophoresis (SDS-PAGE)
and transferred to a poly(vinylidene difluoride) (PVDF) membrane
(Amersham Biosciences). After the membrane was blocked at room
temperature for 2 h in the blocking solution, primary antibody was
added and incubated at 4 °C overnight. Antibodies to STAT3 were
purchased from Abcam. After three TBST washes, the membrane was
incubated with corresponding horseradish peroxidase-labeled secon-
dary antibody (1:2000) (Santa Cruz Biotechnology) at room
temperature for 1 h. Membranes were washed with TBST three
times for 15 min, and the protein blots were detected with
chemiluminescent reagent (Thermo Fisher Scientific, Ltd.). The X-
ray films were developed with a developer and fixed with a fixer
solution.
Immunofluorescence Staining. Studies were performed as
previously described.64 Briefly, SKOV3 cells were cultured in six-
well plates (2 × 105 cells per well) for 24 h, pretreated with
compound 8u at 0.5 μM for 6 h, and then treated with IL-6 for
another 30 min. The cells were then washed and fixed with anhydrous
methanol for 15 min at room temperature, permeabilized with 0.1%
Triton X-100 for 15 min, and blocked with 1% bovine serum albumin
(BSA) for 10 min at room temperature. Next, the cells were incubated
with primary p-STAT3 antibody in PBS overnight at 4 °C followed by
FITC-conjugated antibody secondary antibodies. After being washed
with PBS three times, the cells were incubated in 10 μM 4′,6-
diamidino-2-phenylindole (DAPI) for 15 min in the dark. The
fluorescence of the p-STAT3 protein and nuclei was red and blue,
respectively, and live cell imaging was simultaneously accomplished
with a fluorescence microscope (Olympus BX-51, Tokyo, Japan).
Luciferase Assays. Cells were seeded in 24-well plates and
transiently transfected with STAT reporter plasmid (0.2 μg per well,
Addgene) and Renilla luciferase control reporter plasmid pRL-CMV
(0.1 μg per well, Promega), with Lipofectamine 2000 reagent
(Invitrogen). Then, the cells were incubated in complete medium
for 24 h with 8u. After the treatment, the cells were harvested in 100
μL of passive lysis buffer (Promega) per well. A 25 μL aliquot of cell
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.9b01386.
■
sı
Binding modes of 8u in complex with IDO1 (PDB ID:
4PK5); UV spectra of ferric IDO1 without and with
compound 8u; effects of the indicated treatments of 8u
on B16-F10 melanoma tumor xenografts in C57BL/6
mice; inhibition of tumor growth for compound 8u and
doxorubicin (DOX) in nude mice bearing HepG2
xenograft model; inhibitory effects of 8u on STATs;
table of docking scores (kcal/mol) for all studied
compounds; table of STAT3 binding affinities of
selected 2-amino-1,4-naphthoquinone derivatives; and
1H NMR, 13C NMR, HR-MS, and HPLC of target
compounds (PDF)
Molecular formula strings and some data (CSV)
AUTHOR INFORMATION
Corresponding Authors
■
Hengshan Wang − State Key Laboratory for the Chemistry and
Molecular Engineering of Medicinal Resources (Ministry of
Education of China), School of Chemistry and Pharmaceutical
Sciences of Guangxi Normal University, Guilin 541004, China;
orcid.org/0000-0001-6474-8323; Email: whengshan@
163.com
Zhenfeng Chen − State Key Laboratory for the Chemistry and
Molecular Engineering of Medicinal Resources (Ministry of
Education of China), School of Chemistry and Pharmaceutical
1560
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J. Med. Chem. 2020, 63, 1544−1563

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pubs.acs.org/jmc
Article
Sciences of Guangxi Normal University, Guilin 541004, China;
orcid.org/0000-0002-6548-6131; Email: chenzf@
gxnu.edu.cn
Ye Zhang − School of Pharmacy, Guilin Medical University,
Guilin 541004, China; State Key Laboratory for the Chemistry
and Molecular Engineering of Medicinal Resources (Ministry of
Education of China), School of Chemistry and Pharmaceutical
Sciences of Guangxi Normal University, Guilin 541004, China;
orcid.org/0000-0002-6903-5602; Email: zhangye81@
126.com
ABBREVIATIONS USED
■
IDO1, indoleamine-2,3-dioxygenase 1; STAT3, signal trans-
ducer and activator of transcription 3; IDO5L, 4-amino-N-(3-
chloro-4-fluorophenyl)-N′-hydroxy-1, 2, 5-oxadiazole-3-car-
boximidamide; 1-MT, 1-methyl-L-tryptophan; DOX, doxor-
ubicin; PDL1, programmed death ligand 1; PD1, programmed
cell death protein 1; CTLA4, cytotoxic T-lymphocyte-
associated protein 4; Pt, platinum; TDO, tryptophan 2,3-
dioxygenase; HRMS, high-resolution mass spectrometry;
NMR, nuclear magnetic resonance; DMEM, Dulbecco’s
modified Eagle’s medium; SDS-PAGE, sodium dodecyl sulfate
polyacrylamide gel electrophoresis; SPR, surface plasmon
resonance; MTT, 3-(4,5-dimethylthiazol-2yl)-2,5-diphenylte-
trazolium bromide; PVDF, poly(vinylidene difluoride)
Authors
Rizhen Huang − State Key Laboratory for the Chemistry and
Molecular Engineering of Medicinal Resources (Ministry of
Education of China), School of Chemistry and Pharmaceutical
Sciences of Guangxi Normal University, Guilin 541004, China;
School of Pharmacy, Guilin Medical University, Guilin 541004,
China; Jiangsu Province Hi-Tech Key Laboratory for
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Xiaoteng Jing − State Key Laboratory for the Chemistry and
Molecular Engineering of Medicinal Resources (Ministry of
Education of China), School of Chemistry and Pharmaceutical
Sciences of Guangxi Normal University, Guilin 541004, China
Xiaochao Huang − Jiangsu Province Hi-Tech Key Laboratory
for Biomedical Research and School of Chemistry and Chemical
Engineering, Southeast University, Nanjing 211189, China
Yingming Pan − State Key Laboratory for the Chemistry and
Molecular Engineering of Medicinal Resources (Ministry of
Education of China), School of Chemistry and Pharmaceutical
Sciences of Guangxi Normal University, Guilin 541004, China
Yilin Fang − State Key Laboratory for the Chemistry and
Molecular Engineering of Medicinal Resources (Ministry of
Education of China), School of Chemistry and Pharmaceutical
Sciences of Guangxi Normal University, Guilin 541004, China
Guibin Liang − State Key Laboratory for the Chemistry and
Molecular Engineering of Medicinal Resources (Ministry of
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Zhixin Liao − Jiangsu Province Hi-Tech Key Laboratory for
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Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.9b01386
Author Contributions
∥R.H. and X.J. contributed equally to this work.
̈
(11) Rohrig, U. F.; Majjigapu, S. R.; Vogel, P.; Zoete, V.; Michielin,
O. Challenges in the discovery of indoleamine 2,3-dioxygenase 1
Notes
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The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
■
The authors are grateful to the National Natural Science
Foundation of China (21431001, 21501032, and 81760626),
the Ministry of Education Innovation Team Fund
(IRT_16R15 and 2016GXNSFGA380005), the Innovative
Team & Outstanding Talent Program of Colleges and
Universities in Guangxi (2017-38), Guangxi New Century
Ten, Hundred and Thousand Talents Project ([2017]42),
Natural Science Foundation of Guangxi Province
(2016GXNSFAA380300 and AB17292075), and Guangxi
Funds for Distinguished Experts.
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